Metabolic Syndrome: Underlying Mechanisms and Drug Therapies

Metabolic Syndrome Metabolic Syndrome Underlying Mechanisms and Drug Therapies Edited by Minghan Wang Amgen, Inc., T...

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Metabolic Syndrome

Metabolic Syndrome Underlying Mechanisms and Drug Therapies Edited by

Minghan Wang Amgen, Inc., Thousand Oaks, California, USA

Copyright Ó 2011 by John Wiley & Sons, Inc. All rights reserved Published by John Wiley & Sons, Inc., Hoboken, New Jersey Published simultaneously in Canada No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except as permitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, (978) 750-8400, fax (978) 750-4470, or on the web at www.copyright.com. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, (201) 748-6011, fax (201) 748-6008, or online at http://www.wiley.com/go/ permission. Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives or written sales materials. The advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor author shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. For general information on our other products and services or for technical support, please contact our Customer Care Department within the United States at (800) 762-2974, outside the United States at (317) 572-3993 or fax (317) 572-4002. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic formats. For more information about Wiley products, visit our web site at www.wiley.com.

Library of Congress Cataloging-in-Publication Data: Metabolic syndrome : underlying mechanisms and drug therapies / edited by Minghan Wang. p. ; cm. Includes bibliographical references and index. ISBN 978-0-470-34342-5 (cloth) 1. Metabolic syndrome–Pathophysiology. 2. Metabolic syndrome–Chemotherapy. I. Wang, Minghan, 1966[DNLM: 1. Metabolic Syndrome X–drug therapy. 2. Metabolic Syndrome X–physiopathology. WK 820 M58695 2011] RC662.4.M53 2011 616.3’99–dc22 2010019505 Printed in the United States of America 10 9 8

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Contents

Introduction

ix

Minghan Wang

Contributors

Part One

xi

The Physiology of Metabolic Tissues Under Normal and Disease States

1. Gut as an Endocrine Organ: the Role of Nutrient Sensing in Energy Metabolism

3

Minghan Wang

2. Central Glucose Sensing and Control of Food Intake and Energy Homeostasis

29

Lourdes Mounien and Bernard Thorens

3. Abnormalities in Insulin Secretion in Type 2 Diabetes Mellitus

53

Taly Meas and Pierre-Jean Guillausseau

4. Adipokine Production by Adipose Tissue: A Novel Target for Treating Metabolic Syndrome and its Sequelae

73

Vanessa DeClercq, Danielle Stringer, Ryan Hunt, Carla G. Taylor, and Peter Zahradka

5. Hepatic Metabolic Dysfunctions in Type 2 Diabetes: Insulin Resistance and Impaired Glucose Production and Lipid Synthesis

133

Ruojing Yang

v

vi

Contents

6. Energy Metabolism in Skeletal Muscle and its Link to Insulin Resistance

157

Minghan Wang

Part Two

Metabolic Diseases and Current Therapies

7. Mechanisms and Complications of Metabolic Syndrome

179

Minghan Wang

8. Emerging Therapeutic Approaches for Dyslipidemias Associated with High LDL and Low HDL

199

Margrit Schwarz and Jae B. Kim

9. Mechanism of Action of Niacin: Implications for Atherosclerosis and Drug Discovery

235

Devan Marar, Shobha H. Ganji, Vaijinath S. Kamanna, and Moti L. Kashyap

10. Current Antidiabetic Therapies and Mechanisms

253

Minghan Wang

Part Three

Drug Targets for Antidiabetic Therapies

11. GLP-1 Biology, Signaling Mechanisms, Physiology, and Clinical Studies

281

Remy Burcelin, Cendrine Cabou, Christophe Magnan, and Pierre Gourdy

12. Dipeptidyl Peptidase IV Inhibitors for Treatment of Diabetes

327

C.H.S. McIntosh, S.-J. Kim, R.A. Pederson, U. Heiser, and H.-U. Demuth

13. Sodium Glucose Cotransporter 2 Inhibitors

359

Margaret Ryan and Serge A. Jabbour

14. Fibroblast Growth Factor 21 as a Novel Metabolic Regulator

377

Radmila Micanovic, James D. Dunbar, and Alexei Kharitonenkov

15. Sirtuins as Potential Drug Targets for Metabolic Diseases Qiang Tong

391

Contents

16. 11b-Hydroxysteroid Dehydrogenase Type 1 as a Therapeutic Target for Type 2 Diabetes

vii

423

Clarence Hale and David J. St. Jean, Jr.

17. Monoclonal Antibodies for the Treatment of Type 2 Diabetes: A Case Study with Glucagon Receptor Blockade

459

Hai Yan, Wei Gu, and Murielle Veniant-Ellison

Part Four

Lessons Learned and Future Outlook

18. Drug Development for Metabolic Diseases: Past, Present and Future

471

Minghan Wang

Index

489

Introduction

It has been more than 20 years since Reaven first introduced the concept of syndrome X or insulin resistance syndrome to describe the clustering of several cardiovascular risk factors. The concept has evolved over the years and is now commonly referred to as metabolic syndrome, which covers the individual metabolic abnormalities of obesity, insulin resistance, hyperglycemia, dyslipidemia (high triglycerides and low HDL), and hypertension. Patients with metabolic syndrome have increased risk of developing cardiovascular disease (CVD) and type 2 diabetes mellitus (T2DM). Despite the debates surrounding the existence and definition of metabolic syndrome, the concept has been useful in understanding the interconnections of the various risk factors that are common in a large population of patients and thereby managing the overall disease risk. From the drug discovery standpoint, all the components of metabolic syndrome are therapeutic targets for the treatment of CVD and T2DM to reduce comorbidities and overall mortality. While there is a wealth of information concerning the clinical features and mechanisms of metabolic syndrome, putting them in the physiological context relevant to the development of therapeutics is essential for drug discovery. The goal of this book is to provide comprehensive understanding of the molecular and physiological abnormalities associated with metabolic syndrome and the therapeutic strategies for drug development. Part One is devoted to gaining an integrated understanding of the metabolic abnormalities at the tissue and pathway levels that are associated with disease states. In Part Two, metabolic syndrome is discussed at the physiological level and current therapies are summarized. These sections help lay the foundation to identify pathways and molecular targets for the development of antidiabetic therapies in Part Three. Since more than 80% type 2 diabetic patients have metabolic syndrome, a large portion of this book is devoted to antidiabetic therapies. Finally, the successes and failures in developing antidiabetic and cardiovascular drugs and lessons learned are discussed in Part Four. Although the chapters are contributed by different authors, the organization and the content of the book have been carefully designed so that the information is presented systematically. In the meantime, each chapter independently covers a subarea of metabolic or drug discovery topics, the reader has the flexibility to gain information on a specific tissue, pathway, or target in a time-efficient manner. Despite the exciting advances that have been made in developing antidiabetic and CVD therapies in the past several

ix

x

Introduction

decades, drug discovery in these areas continues to be a challenge. I hope this book will help the reader better understand the exciting science behind metabolic drug discovery and development and develop a greater appreciation of the complexity of metabolic syndrome as well as the treatment strategies.

MINGHAN WANG

Contributors

Remy Burcelin, Rangueil Institute of Molecular Medicine, INSERM U858, Toulouse, France Cendrine Cabou, Rangueil Institute of Molecular Medicine, INSERM U858, Toulouse, France Vanessa DeClercq, Department of Human Nutritional Sciences, University of Manitoba, Winnipeg, Canada; Canadian Centre for Agri-Food Research in Health and Medicine, St. Boniface Hospital Research Centre, Winnipeg, Canada H.-U. Demuth, Probiodrug AG, Biocenter, Halle (Saale), Germany James D. Dunbar, BioTechnology Discovery Research, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN, USA Shobha H. Ganji, Department of Veterans Affairs Healthcare System, Atherosclerosis Research Center, Long Beach, CA, USA; Department of Medicine, University of California, Irvine, CA, USA Pierre Gourdy, Rangueil Institute of Molecular Medicine, INSERM U858, Toulouse, France Wei Gu, Department of Metabolic Disorders, Amgen, Inc., Thousand Oaks, CA, USA Pierre-Jean Guillausseau, APHP, Department of Internal Medicine B, Hoˆpital Lariboisiere, Paris, France; Universite Paris 7, Paris, France Clarence Hale, Department of Metabolic Disorders, Amgen, Inc., Thousand Oaks, CA, USA U. Heiser, Probiodrug AG, Biocenter, Halle (Saale), Germany Ryan Hunt, Department of Human Nutritional Sciences, University of Manitoba, Winnipeg, Canada; Canadian Centre for Agri-Food Research in Health and Medicine, St. Boniface Hospital Research Centre, Winnipeg, Canada Serge A. Jabbour, Division of Endocrinology, Diabetes, and Metabolic Diseases, Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA, USA

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Contributors

Vaijinath S. Kamanna, Department of Veterans Affairs Healthcare System, Atherosclerosis Research Center, Long Beach, CA, USA; Department of Medicine, University of California, Irvine, CA, USA Moti L. Kashyap, Department of Veterans Affairs Healthcare System, Atherosclerosis Research Center, Long Beach, CA, USA; Department of Medicine, University of California, Irvine, CA, USA Alexei Kharitonenkov, BioTechnology Discovery Research, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN, USA Jae B. Kim, Global Development, Amgen, Inc., Thousand Oaks, CA, USA S.-J. Kim, Department of Cellular and Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, Canada; Diabetes Research Group, Life Sciences Institute, University of British Columbia, Vancouver, Canada Christophe Magnan, INSERM U858, Toulouse, France; University Paris Diderot, CNRS, Paris, France C.H.S. McIntosh, Department of Cellular and Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, Canada; Diabetes Research Group, Life Sciences Institute, University of British Columbia, Vancouver, Canada Devan Marar, Department of Veterans Affairs Healthcare System, Atherosclerosis Research Center, Long Beach, CA, USA; Department of Medicine, University of California, Irvine, CA, USA Radmila Micanovic, BioTechnology Discovery Research, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN, USA Taly Meas, APHP, Department of Internal Medicine B, Hoˆpital Lariboisiere, Paris, France; Universite Paris 7, Paris, France Lourdes Mounien, Department of Physiology, University of Lausanne, Lausanne, Switzerland; Center for Integrative Genomics, University of Lausanne, Lausanne, Switzerland R.A. Pederson, Department of Cellular and Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, Canada; Diabetes Research Group, Life Sciences Institute, University of British Columbia, Vancouver, Canada Margaret Ryan, Division of Endocrinology, Diabetes, and Metabolic Diseases, Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA, USA Margrit Schwarz, Department of Metabolic Disorders, Amgen, Inc., South San Francisco, CA, USA

Contributors

xiii

David J. St. Jean, Jr., Department of Medicinal Chemistry, Amgen, Inc., Thousand Oaks, CA, USA Danielle Stringer, Department of Human Nutritional Sciences, University of Manitoba, Winnipeg, Canada; Canadian Centre for Agri-Food Research in Health and Medicine, St. Boniface Hospital Research Centre, Winnipeg, Canada Carla G. Taylor, Department of Human Nutritional Sciences, University of Manitoba, Winnipeg, Canada; Canadian Centre for Agri-Food Research in Health and Medicine, St. Boniface Hospital Research Centre, Winnipeg, Canada Bernard Thorens, Department of Physiology, University of Lausanne, Lausanne, Switzerland; Center for Integrative Genomics, University of Lausanne, Lausanne, Switzerland Qiang Tong, USDA/ARS Children’s Nutrition Research Center, Baylor College of Medicine, Houston, TX, USA; Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA; Department of Medicine, Baylor College of Medicine, Houston, TX, USA; Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX, USA Murielle Veniant-Ellison, Department of Metabolic Disorders, Amgen, Inc., Thousand Oaks, CA, USA Minghan Wang, Metabolic Disorders Research, Amgen, Inc., Thousand Oaks, CA, USA Hai Yan, Department of Protein Science, Amgen, Inc., Thousand Oaks, CA, USA Ruojing Yang, Department of Metabolic Disorders – Diabetes, Merck Research Laboratories, Rahway, NJ, USA Peter Zahradka, Department of Physiology, University of Manitoba, Winnipeg, Canada; Department of Human Nutritional Sciences, University of Manitoba, Winnipeg, Canada; Canadian Centre for Agri-Food Research in Health and Medicine, St. Boniface Hospital Research Centre, Winnipeg, Canada

Part One

The Physiology of Metabolic Tissues Under Normal and Disease States

Chapter

1

Gut as an Endocrine Organ: the Role of Nutrient Sensing in Energy Metabolism MINGHAN WANG Department of Metabolic Disorders, Amgen, Inc., Thousand Oaks, CA, USA

INTRODUCTION Energy homeostasis is balanced by food intake and energy expenditure. Both events are controlled by complex sets of neuronal and hormonal actions. Food intake is driven by a central feeding drive, namely, the appetite, which is induced under the fasting state after energy consumption through physical activities. Following food digestion, the passage of nutrients through the gastrointestinal (GI) tract generates signals that produce sensations of fullness and satiation. In particular, nutrients interact with receptors in the small intestine and stimulate the release of peptide hormones, the actions of which mediate physiological adaptations in response to energy intake. The commonly known GI peptides include the incretins, glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide or gastric inhibitory peptide (GIP), as well as peptide tyrosine tyrosine (PYY), cholecystokinin (CCK), and oxyntomodulin. These peptide hormones are secreted from different regions of the small intestine. GLP-1, oxyntomodulin, and PYY are secreted from endocrine L cells that are mainly distributed in the distal small intestine (1, 2), whereas GIP is secreted from endocrine K cells primarily localized in the duodenum (3, 4). CCK is secreted from I cells in the duodenum (5). Nutrients released through the digestive tract induce secretion of GI peptide hormones, which subsequently bind to their respective receptors and trigger a cascade of physiological events. These receptors are expressed in tissues such as the central nervous system (CNS), the GI tract, and pancreas, and upon activation lead to suppression of appetite, Metabolic Syndrome: Underlying Mechanisms and Drug Therapies Edited by Minghan Wang Copyright 2011 John Wiley & Sons, Inc.

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Chapter 1

Gut as an Endocrine Organ

reduced gastric emptying, and assimilation of nutrients. Nutrients can also suppress the secretion of GI peptides. For example, ghrelin, a peptide hormone released by the stomach under the fasting state that stimulates food intake (6), is suppressed after food ingestion (6). GI peptides mediate two principal physiological events: (i) the feedback response on the CNS and the stomach to reduce food intake and slow gastric emptying, and (ii) the feedforward response, mediated particularly by the incretins, to prepare tissues for nutrient integration. In this regard, the small intestine is not only an organ for nutrient absorption but also a major site for providing hormonal regulation of energy intake and storage. GLP-1 and GIP are called incretins because they act on the pancreatic b-cells to increase insulin secretion at normal or elevated glucose levels. They also regulate glucagon secretion by pancreatic a-cells. These actions represent a critical step in preparing the body to switch from the fasting state to postprandial activities. By suppressing glucagon secretion, GLP-1 shut down hepatic gluconeogenesis and adipose lipolysis, two key biological pathways in maintaining energy homeostasis under the fasting state. In addition, GLP-1 can act directly on liver and muscle to regulate glucose metabolism independent of its incretin action (7). In the meantime, induction of insulin secretion by the incretins facilitates glucose uptake by the peripheral tissues. GLP-1 is also involved in the feedback response by acting on the CNS to suppress food intake. PYY and CCK exhibit a similar effect in the CNS underscoring the complexity of appetite regulation. The magnitude and potency of the feedback and feedforward responses depend on both the nutrient content and the length of small intestine exposed. Although both glucose and free fatty acids (FFAs) modulate the secretion of GI peptides, their actions are mediated by distinct mechanisms because they have different residence times in the small intestine and interact with different nutrient-sensing receptors. In fact, even the activity of FFAs varies with their chain length. Moreover, the intestinal length exposed to nutrients and the nutrient contact sites are important determinants in GI peptide secretion.

FOOD INTAKE AND NUTRIENT-SENSING SYSTEMS IN THE GI TRACT After ingestion, food chime is mixed with digestive juices in the stomach and propelled into the small intestine. The three segments of the small intestine, the duodenum, the jejunum, and the ileum, perform different digestive functions (Figure 1.1). Nutrients are generated from the digestion of carbohydrates, fat, protein, and other food components. The passage of nutrients through the small intestine not only facilitates absorption but also plays a role in regulating gastric emptying and satiety. The interaction of nutrients with the small intestine segments generates signals that regulate the rate of gastric emptying and food intake. The nutrient-sensing system consists of receptors, channels, and transporters in the open-type cells on the small intestine luminal surface. It responds to macronutrients and activates signaling pathwaysleading to the release of GI peptides, which subsequently act on the stomach and the CNS to

Food Intake and Nutrient-Sensing Systems in the GI Tract

5

Food (carbohydrates, fat, protein, etc.)

Stomach Target tissues

Ileum Duodenum

Neurons Circulation in bloodstream Colon

GI peptide

Jejunum Enterocyte Enteroendocrine cell

Figure 1.1 Localization of enteroendocrine cells in the GI tract. Enteroendocrine cells (exemplified by an L cell) are on the surface of the GI tract where their luminal sides detect nutrients passing in the lumen, leading to intracellular signals that stimulate the secretion of GI peptides. GI peptides exert biological effects by acting on their receptors in nearby neurons that transduce signals to target tissues. The peptides are carried to target tissues through the circulation and act locally on sites such as the CNS, the pancreas, and the stomach.

slow gastric emptying and suppress appetite, respectively (Figure 1.1). In addition, some peptides such as the incretins stimulate insulin secretion and regulate glucagon secretion to help integrate nutrients into tissues post absorption (Figure 1.1). Studies in pigs demonstrated that rapid injection of glucose into the duodenum during or immediately prior to feeding suppressed food intake (8, 9). The reduction in food intake far exceeded the energy content of the infused glucose (8, 9), suggesting that the effect of glucose on food intake is likely to be mediated by signaling events. In the meantime, hepatic portal or jugular infusion of glucose in pigs did not alter shortterm food intake (10). These data suggest that the regulatory effect of glucose on food intake is a preabsorptive event and the sites of regulation are in the GI tract. To further understand the mechanisms by which dietary carbohydrates regulate energy intake, glucose was infused into the stomach or different segments of the small intestine in pigs. The infusion started 30 min prior to the meal and continued until the pigs stopped eating (11). It was found that infusion of glucose into the stomach, duodenum, jejunum, or ileum each suppressed food intake (11). But comparatively, jejunal infusion caused more reduction in food intake than elsewhere (11). These data suggest that glucose may interact with receptors or other sensing components expressed in various parts of the small intestine to control short-term energy intake. In addition to glucose, FFAs released from fat digestion also play important albeit more complex roles in controlling energy intake. Healthy human volunteers receiving ileal infusion

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Chapter 1

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of lipids consumed a smaller amount of food and energy and had delayed gastric emptying (12). Ileal lipid infusion also accelerated the sensation of fullness during a meal (12). However, intravenous (i.v.) infusion of lipids did not affect food intake (12), suggesting that lipids may interact with ileal receptors to induce satiety and reduce food consumption. Further studies suggest that digestion is a prerequisite for the inhibitory effect of fat on gastric emptying and energy intake. For example, administration of a lipase inhibitor increased food intake in healthy subjects or type 2 diabetic patients receiving a high-fat meal (13, 14), suggesting that FFAs, the breakdown products of fat after ingestion, rather than triglycerides, are the active nutrients that exert the regulatory effects. Likewise, sugars from carbohydrate digestion, rather than carbohydrates themselves, are the active nutrients that induce intestinal signals. Although both glucose and FFAs can stimulate a set of GI peptides that regulate appetite, gastric emptying, and insulin and glucagon release, they have differential effects. For example, glucose stimulates robust secretion of both GLP-1 and GIP, whereas FFAs from a fat meal elicit only modest GLP-1 secretion despite equally robust GIP secretion (15). Further, not all FFAs are equally active since the stimulatory effect depends on their chain length. Although FFAs with a chain length of greater than C12 stimulate CCK release, further increase in chain length has no additional effect, and C11 or shorter FFAs are not active (16, 17). Like carbohydrate and fat meals, protein meals also activate the nutrient-sensing system but in different ways. In healthy human subjects, plasma GIP levels were elevated after both carbohydrate and fat meals but not a protein meal (15). However, intraduodenal amino acid perfusion in human subjects stimulated both GIP and insulin secretion (18, 19). Oral ingestion of mixed amino acids by healthy volunteers also increased plasma GLP-1 levels (20). These findings suggest that amino acids can function as nutrient-sensing agents, and a protein meal is likely to contribute to nutrient sensing in the GI tract. However, since mixed amino acids are not equivalent to a digested protein meal, GLP-1 secretion was studied in humans following a protein meal (15). A transient peak was observed at 30 min followed by a steady-state rise throughout the rest of the 3 h study period (15). The nutrients from the protein meal that stimulated GLP-1 secretion were a mixture of protein hydrolysates but not amino acids per se. It is important to carry out studies with protein hydrolysates that mimic the digested products in the GI tract. A protein hydrolysate (peptone) containing 31% free amino acids and 69% peptides induced the secretion of PYY and GLP-1 in the portal effluent of isolated vascularly perfused rat ileum after luminal administration (21). Peptones also induced CCK secretion and transcription in STC-1 cells, an established L cell line (22, 23). Peptones made from both albumin egg hydrolysate and meat hydrolysate stimulated the transcriptional expression of the proglucagon gene encoding GLP-1 in two L cell lines but not pancreatic glucagon-producing cell lines (24), suggesting that the signaling pathways mediating this effect are L cell/ small intestine specific. In STC-1 cells, the proglucagon promoter contains elements responsive to peptones (25). In contrast, the mixture of free amino acids is at best a weak stimulant (21, 24). These data suggest that free amino acids may have a limited role in protein meal-stimulated GLP-1 or PYY secretion. However, amino acids are indeed involved in nutrient sensing in the GI tract. Aromatic amino acids may play a

Molecular Mechanisms of Nutrient Sensing

7

role in gastrin secretion because they activate the calcium-sensing receptor (CaR) on gastrin-secreting antral cells (26, 27). In addition, amino acids also stimulate CCK release (28, 29) and gastric acid secretion (30). In addition to glucose, FFAs, amino acids, and digested peptides from proteins, other nutrients are also involved in the regulation of GI peptide secretion (21). At physiological concentrations, bile acids stimulate the secretion of PYY, GLP-1, and neurotensin (NT) (21). Interestingly, the threshold concentration of taurocholate for PYY and GLP-1 stimulation is about twofold that required for stimulating NT release (21), suggesting that there is a slight difference in the sensitivity of L cells and N cells to bile acids (21). In addition to the small intestine, the stomach plays an important role in terminating a meal. When rats were implanted with an extra stomach to which a liquid diet was infused, food intake was reduced regardless of whether food was allowed to empty into the small intestine or retained in the stomach (31). This effect is not likely to be mediated by neuronal mechanisms because the implanted stomach was completely denervated (31). This result suggests that the implanted stomach may have generated hormonal signals that affect food intake, and these hormonal signals may mediate the ability of the stomach to sense nutrient quality and quantity to alter the rate of gastric emptying and amount of food ingested (32, 33).

MOLECULAR MECHANISMS OF NUTRIENT SENSING It has been recognized that it is the monomeric nutrients that interact with luminal small intestinal receptors or other nutrient-sensing components and regulate the feedback and feedforward responses to food intake. What do we know about these receptors and their downstream pathways? The analogy between the intestinal nutrient sensing and taste reception by the tongue can shed new light on this question. Glucose sensing in taste buds is mediated by taste receptors expressed in the lingual epithelium (34). These receptors are G protein-coupled receptors (GPCRs) in the apical membranes of taste receptor cells (34). All the three members of the taste receptor family 1 (T1R) class of GPCRs are involved in this function by acting in combination to sense different tastes. The T1R2/T1R3 heterodimer senses sweet taste whereas the T1R1/T1R3 heterodimer senses amino acids and umami taste (35). These receptors activate a phospholipase C (PLC) b2-dependent pathway to increase intracellular Ca2 þ concentrations by coupling to the G proteins gustducin and/or transducin (34). The activated taste receptors may also stimulate the cAMP-dependent pathway (34). In an in vitro assay where T1R2/T1R3 were coupled to Ga15, a promiscuous G protein linked to PLC, T1R2/T1R3 responded to sweet taste stimuli, including glucose, fructose, lactose, and galactose, as well as synthetic sweeteners (35). The activity was inhibited by the sweet taste inhibitor lactisole (35). These data indicate that the T1R2/T1R3 complex mediates sweet sensation along with other components such as G proteins and PLC. Interestingly, the key components of the sweet taste transduction pathways are also expressed in the gut enteroendocrine cells (36), with the signaling events leading to GI peptide secretion by these cells (Figure 1.2). For example, the three members of

Carbohydrates

Fat

Protein

8 Glucose

FFAs

Amino acids and peptides

Glucokinase Taste receptors (i.e., T1R2/T1R3)

SGLT2

GPR40, GPR119, and GPR120

CaR, GPR93, and others

G proteins (Gs or Gq)

G proteins

ATP/ADP ratio Electrogenic activity G proteins (i.e., gustducin)

PLCβ2

Closure of KATP channels

Membrane depolarization

Intracellular [Ca2+ ]

Secondary messengers

or Intracellular cAMP Opening of voltagedependent Ca2+ channels

Secretion of GI peptides Secretion of GI peptides

Intracellular [Ca2+ ]

Secretion of GI peptides (i.e., GLP-1)

Figure 1.2

Potential signaling cascades that mediate GI nutrient sensing in response to main nutrients. Macronutrients, including sugars, FFAs, and amino acids/peptides, are derived through digestion from carbohydrates, fat, and protein. There are three potential pathways that can sense glucose: taste receptors, KATP channels, and SGLT1. FFAs and amino acids/peptides can activate GPCRs expressed in enteroendocrine cells. Activation of downstream signaling by these mechanisms triggers secretion of GI peptides.

Molecular Mechanisms of Nutrient Sensing

9

the T1R class of GPCRs are detected in brush cells, one form of solitary chemosensory cells (SCCs), in the apical membranes of rat jejunum (37). Also found in these cells are a-gustducin, transducin, and PLCb2 (37). In addition, a-gustducin is also expressed in brush cells of the stomach, the duodenum, and pancreatic ducts in rats (38, 39). Brush cells have a structure similar to lingual taste cells (39), suggesting that they may use similar nutrient-sensing pathways. Consistent with the findings in rats, T1R2, T1R3, and a-gustducin are expressed in mouse small intestine (40). Taste signaling elements, including the three subunits of gustducin (a-gustducin, Gb3, and Gg13), PLCb2, and taste receptors, were also found in human L cells (41). Taken together, these data suggest that the taste receptors and associated signaling components are present in gut cells and may be involved in nutrient sensing in a fashion similar to that by the lingual epithelium of the tongue. There are two functional consequences upon the activation of the taste receptor systems in the gut. The first is the release of GI peptides such as GLP-1, which mediates both feedback and feedforward responses to food intake as described above. Glucose induces GLP-1 secretion from enteroendocrine L cells by stimulating the taste receptors, the signal of which is mediated by the taste G protein gustducin. The role of gustducin in sugar sensing and glucose homeostasis was exemplified in a-gustducin null mice (41). In wild-type mice, ingestion of glucose induced a marked increase of GLP-1 secretion (41); in contrast, a-gustducin null mice exhibited defective GLP-1 secretion in response to glucose ingestion (41), suggesting that L cells of the gut sense glucose through similar mechanisms used by taste cells of the tongue. Thus, the gut cells can “taste” sugars and release mediators, such as the incretins, that in turn regulate food intake and nutrient assimilation. The second consequence of the taste receptor activation in the GI tract is elevated glucose transporter 2 (GLUT2) insertion on the apical membrane of the gut lumen to increase glucose absorption (37). The basal level of glucose absorption in the gut is mediated by sodium–glucose cotransporter 1 (SGLT1) and GLUT2 when glucose level is around 20 mM (37). At higher local glucose concentrations (30–100 mM), increased insertion of GLUT2 in the apical membrane occurs to facilitate additional glucose absorption (37). GLUT2 provides three to five times more capacity for glucose absorption than the SGLT1 pathway (37). Despite the above evidence that supports the role of the taste receptor system in mediating nutrient-sensing effects in the GI tract, several research groups have reported findings that dispute this notion. Although the artificial sweetener sucralose was shown to stimulate GLP-1 secretion from human L cells in vitro (41), it did not stimulate GLP-1 secretion in primary L cells (42). In addition, it did not stimulate GLP-1 or GIP release in healthy humans when delivered by intragastric infusion (43). This is in agreement with an earlier study in type 2 diabetic patients where the sweetener stevioside had no effect on GLP-1 or GIP release (44). Further, several sweeteners, including sucralose, were tested in Zucker diabetic fatty rats for their nutrient-sensing activity (45). Consistent with the previous reports, none of these sweeteners increased incretin secretion (45). Taken together, these data indicate that the role of the taste receptor system in GI nutrient sensing remains to be further clarified.

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Two additional signaling pathways in the GI tract have been proposed that could mediate GLP-1 secretion in response to glucose exposure. The first one is the classical glucose-sensing machinery employed by pancreatic b-cells for eliciting glucosedependent insulin secretion (46). This machinery includes components such as ATPsensitive potassium (KATP) channels and glucokinase (Figure 1.2). In this pathway, glucokinase serves as the rate-limiting step in glucose metabolism and therefore is also termed “glucose sensor.” Glucose metabolism increases the ATP/ADP ratio, which causes the closure of KATP channels and depolarization of the b-cell membrane. Next, membrane depolarization leads to opening of voltage-dependent Ca2 þ channels and accumulation of intracellular Ca2 þ , which triggers insulin release. Both the KATP channel subunits Kir6.2 and SUR1 and glucokinase were detected in GLUTag cells, an L cell line (46). In these cells, glucose concentrations between 0 and 20 mM decreased membrane conductance, caused membrane depolarization, and triggered action potentials (46). Tolbutamide also triggered action potentials in GLUTag cells (46), presumably by blocking the KATP channels. These data suggest that the classical glucose-sensing machinery involving glucokinase and KATP channels mediates glucose-induced GLP-1 release from L cells. However, if this notion is true, GLP-1 and GIP secretion following an oral glucose challenge should be lower in individuals with heterozygous glucokinase mutations that confer reduced activity. Unfortunately, when heterozygous glucokinase mutation carriers were subjected to oral glucose tolerance test (OGTT), they did not have altered GLP-1 or GIP secretion post oral glucose challenge compared to normal controls (47). This observation suggests that the glucokinase and KATP channel pathway does not mediate incretin secretion in the gut, or it is involved but there are other redundant pathways that can compensate for it. SGLT1 represents another novel glucose-sensing mechanism that triggers GLP-1 secretion (Figure 1.2). Both SGLT1 and SGLT3 are expressed in GLUTag cells (48), and GLP-1 secretion in response to glucose is inhibited by phlorizin, a SGLT inhibitor compound (48). Moreover, the EC50 value of glucose for glucose-induced GLP-1 secretion matches the Km of SGLT1 (49). These data suggest that SGLT1 could directly mediate glucose-induced GLP-1 release. This effect could be attributed to the electrogenic activity of SGLT1 because low glucose concentrations were shown to trigger small inward currents as they enter cells (48). This current could cause membrane depolarization, which could induce GLP-1 release (Figure 1.2). Like sugars, amino acids and FFAs also regulate endocrine response to food intake through activation of their respective GPCRs in enteroendocrine cells (Figure 1.2). L-Amino acids activate the T1R1/T1R3 heterodimer, which mediates umami taste in taste buds (35). These GPCRs are also expressed in the apical membranes of the gut (37) and couple to the G protein transducin to activate PLCb2 and stimulate Ca2 þ mobilization. Through this signaling system, amino acids may mediate GI peptide release and regulate food intake. In addition, the extracellular CaR may also act to sense amino acids released from protein digestion. CaR is abundantly expressed in epithelial cells and neurons of the stomach, the small intestine, and the large intestine (50). In the stomach, CaR is expressed on gastrin-releasing G cells and its activation stimulates intracellular Ca2 þ mobilization via the activation of PLC (51).

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CaR can be activated by aromatic amino acids (52), suggesting that it may act as a nutrient-sensing receptor in response to a protein diet. However, in the absence of Ca2 þ , aromatic amino acids had no effect on CaR-mediated signaling (52), suggesting that aromatic amino acids are not CaR agonists; rather, they may act as allosteric modulators to enhance the sensitivity of CaR to its agonist Ca2 þ . The proposed role of CaR in amino acid sensing has physiological support. Analysis of human jejunal content before and 3 h after ingestion of a protein-rich meal revealed that aromatic amino acids were more preferentially released than acidic, polar, and aliphatic amino acids (53). For example, the phenylalanine concentration in jejunum could reach 2 mM (53), a level similar to the EC50 value of phenylalanine in a Ca2 þ mobilization assay (52). In addition, L-phenylalanine can activate CCK secretion, presumably through CaR (54, 55). Further, protein hydrolysates directly activate GPR93 in enterocytes, suggesting that multiple GPCRs are involved in sensing of protein nutrients (23). The G protein species to which CaR and GPR93 are coupled are diverse; they depend on specific conditions in different cell types (56) and ligand species (23). As a result, these receptors stimulate the accumulation of a number of secondary messengers. Like glucose and amino acids, longer FFAs appear to interact directly with GPCRs in enteroendocrine cells. The FFA receptor GPR40 is a GPCR highly expressed in pancreatic b-cells mediating the FFA-stimulated glucose-dependent insulin secretion (57). GPR40 is activated by medium- and long-chain FFAs (57, 58). Interestingly, it is also expressed in endocrine L and K cells of the GI tract and mediates GLP-1 and GIP secretion (59). GPR120 is another GPCR expressed in the intestine especially in GLP-1 positive cells and acts as a receptor for unsaturated longchain FFAs (60). Activation of GPR120 both in vitro and in vivo led to increased GLP1 secretion (60), suggesting that GPR120 is a major intestinal FFA sensing receptor that mediates incretin release. Further, a recent study indicates that GPR120 also mediates the stimulation of CCK release by FFAs (61). GPR119, a receptor for endogenous ligands oleoyl-lysophosphatidylcholine (OLPC) and oleoylethanolamide (OEA) (62, 63), is expressed in pancreatic b-cells and upon activation enhances glucose-dependent insulin secretion (63). GPR119 is also localized in L cells and oral administration of a GPR119 agonist increased the release of both GLP-1 and GIP in normal but not GPR119 knockout mice (64), suggesting that GPR119 mediates longchain FFA-induced incretin release. The three GPCRs trigger different intracellular signaling pathways. GPR40 is coupled to the Gq-PLC pathway and upon activation increases the intracellular Ca2 þ accumulation (65), which leads to incretin secretion. Similarly, GPR120 also induces incretin release by triggering the accumulation of intracellular Ca2 þ (60). GPR119 is coupled to Gs and stimulates intracellular cAMP accumulation (66). In addition to enteroendocrine cells, the intestinal mucosa has two other types of sensory systems, neurons and immune cells (67). The sensory neurons are involved in the control of GI motility and signaling to the CNS that controls feeding behavior (67). The immune cells protect against harmful substances that may enter the GI tract. All the three sensing systems work in concert through direct contact with the intestinal contents.

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REGULATION OF INCRETIN SECRETION In 1902, Bayliss and Starling discovered that acid extracts of intestinal mucosa contained a hormone that could be carried to distal tissues via blood circulation and stimulate the exocrine secretion of the pancreas, and named this factor secretin (68). To test if this factor could be used to treat diabetes, Moore et al. administered duodenal mucosa extracts orally to several type 1 diabetics but did not see clear effects (69). The term “incretin” was first proposed by La Barre in 1932 to describe a hormone extracted from the upper gut mucosa with hypoglycemic effect (70). However, the existence of incretin was not proven until 1964, when two independent research groups discovered that an oral glucose load is associated with a significantly greater insulin response than intravenous administration of the same amount of glucose in human subjects (71, 72). The incretin activity was further evaluated by conducting i.v. glucose infusion isoglycemic to the profile generated from an oral glucose challenge. Despite the identical plasma glucose profiles generated by both the oral and the i.v. routes, the oral glucose challenge stimulated greater levels of insulin and C-peptide (73, 74), suggesting that intestinal factors may be released and involved in the stimulation of insulin secretion after oral glucose ingestion. This so-called “incretin effect” describes the important communication through enteroendocrine factors from the GI tract to pancreas in response to food ingestion. This response is a key part of the feedforward mechanism that increases insulin secretion in anticipation of rising blood glucose after food ingestion. There are two incretins, GLP-1 and GIP, both of which are rapidly released to the bloodstream after meal ingestion and stimulate glucose-dependent insulin secretion (GSIS) by pancreatic b-cells. In addition, GLP-1 also suppresses glucagon release by pancreatic a-cells, food intake, and gastric emptying, and is cardioprotective. In contrast, GIP does not exhibit these effects. GLP-1 is secreted from intestinal L cells, which are predominantly found in the distal jejunum, ileum, colon, and rectum (1). However, the distribution of L cells throughout the GI tract is somewhat species specific. The overall L cell density in rat or pig GI tract is greater than that in human gut (1), and higher levels are located in the distal jejunum, ileum, and rectum relative to other intestinal regions in humans (1). In dogs, L cells are predominantly concentrated in the jejunum and less so in the ileum (4). Recently, GLP-1 immunoreactive cells were detected in human duodenum (75), and GLP-1 and GIP were colocalized in a subset of endocrine cells in the small intestine (76). GIP is secreted from K cells located primarily in the duodenum (3), but they can be found in other parts of the small intestine (76). For instance, in dogs, GIP-secreting K cells are equally distributed in the duodenum and the jejunum (4). Both L and K cells are open-type endocrine cells that are in immediate contact with nutrients in the intestinal lumen, allowing nutrient-dependent regulation of incretin secretion. GIP is a 42-amino acid secreted peptide initially isolated from intestinal mucosa. It was named gastric inhibitory peptide but later renamed glucose-dependent insulinotropic peptide for its ability to stimulate insulin secretion (77). The secreted GIP from intestinal K cells is the active form GIP(1–42). GIP is rapidly cleaved at the

Regulation of Incretin Secretion

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N-terminus by dipeptidyl peptidase-4 (DPP-4) (also known as DPP IV, DP 4, CD26, and adenosine deaminase binding protein), an amino peptidase found in almost all organs and tissues (78), producing the inactive form GIP(3–42) (79). DPP-4 also processes other peptides such as GLP-1 (79), chemokines (80–82), and neuropeptides (83). GLP-1 is part of the proglucagon polypeptide that is expressed in both intestinal L cells and pancreatic a-cells. The proglucagon polypeptide is processed posttranslationally by prohormone convertases (PC) 1/3 and 2. PC1/3 is expressed in L cells whereas PC2 is expressed in a-cells. The tissue-specific expression of the convertase isoforms dictates which mature peptides are generated from the proglucagon polypeptides. In the small intestine, the posttranslational processing by PC1/3 produces GLP-1, GLP-2, glicentin, and oxyntomodulin (84, 85). In contrast, in pancreatic a-cells, PC2-mediated posttranslational processing generates glucagon, glicentin-related pancreatic peptide (GRPP), and the major proglucagon fragment (MPGF) that contains the GLP-1 and GLP-2 segments within its sequence (85). There are two equipotent active forms of GLP-1, GLP-1(7–36)amide and GLP-1(7–37). Both forms are prone to proteolytic cleavage by DPP-4 generating inactive GLP-1(9–36)amide and GLP-1(9–37), respectively (79). Carbohydrate, fat, and protein meals all stimulate GLP-1 secretion in human subjects with glucose being the strongest stimulant (15, 20). Unlike carbohydrates and fat that are also strong stimulants of GIP secretion, protein meals have no effect (15). The plasma concentrations of both hormones increase rapidly within 5–15 min after food ingestion (15, 20) but their actions are short lasting due to rapid proteolytic degradation by DPP-4 and other proteases. The plasma half-lives for intact GLP-1 and GIP are 1–2 and 7 min, respectively (86–88). DPP-4 is the main enzyme for incretin clearance as targeted disruption of the DDP-4 gene in mice led to improved stability of endogenous GLP-1 (89). The tissue distribution of DPP-4 plays an important role in GLP-1 degradation. There is a high level of DPP-4 in the endothelium of the capillaries surrounding L cells, and over 50% of newly secreted intact GLP-1 loses the N-terminal dipeptide and as a result is inactivated before entering the systemic circulation (90). The rapid rise of GLP-1 and GIP in the circulation ensures elevated GSIS in response to a meal, which is essential for the normalization of postprandial glucose. The disappearance of the incretins is in sync with the normalization of postprandial glucose. The first contributor of such a precise regulation is proteolytic degradation. In addition to DPP-4, the neutral endopeptidase 24.11 (NEP-24.11) is also involved in incretin degradation (91, 92). But DPP-4 is the main incretin degradation protease. Like DPP-4, NEP-24.11 is not selective against the incretins; it also processes other hormonal peptides (91). Its catalytic rates on vasoactive intestinal peptides (VIP) and glucagon are much faster than those on the incretins (91). The other factor that contributes to the rapid decline of plasma GLP-1 and GIP levels is a negative feedback mechanism, under which both hormones limit their own secretion by stimulating the somatostatin-mediated paracrine regulation. Somatostatin-positive D cells are located throughout the small intestine in close proximity to both L and K cells (4). In vitro, somatostatin inhibits GLP-1 secretion by L cells (93). In perfused porcine intestine, blocking somatostatin activity with a neutralizing monoclonal antibody increased GLP-1 secretion by 8–9-fold (94). Further,

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intravascular infusion of somatostain-28 strongly inhibited GLP-1 release in pigs (94). This finding is consistent with other somatostatin infusion studies in rats (95), sheep (96), and human subjects (97), where somatostatin inhibited GLP-1 or GIP secretion in vivo. These data suggest that GLP-1 secretion is tonically inhibited by the local release of somatostatin-28 from epithelial paracrine D cells. Compared to somatostatin-28, the enteric neuron-derived somatostatin-14 is much weaker in influencing GLP-1 secretion (94). The suppressive effect of somatostatin on GLP1 secretion is mediated by somatostatin receptor subtype 5 expressed in L cells (98). Since both GLP-1 and GIP stimulate somatostatin release (99, 100), somatostatin is believed to be a key player in a negative feedback loop that controls incretin release in the gut. The existence of the negative feedback loop on GLP-1 secretion is supported by further evidence in dogs and humans. Conscious dogs were orally given a DPP-4 inhibitor, which increased meal-induced active GLP-1 levels (101). However, the total GLP-1 levels in these dogs were reduced (101), presumably due to the inhibitory effect of elevated active GLP-1 on endogenous GLP-1 secretion. A similar result was observed in healthy human volunteers who received an oral dose of a different DPP-4 inhibitor (102). These data support the notion that GLP-1 can inhibit its own secretion in vivo as part of a negative feedback loop. In addition to direct stimulation by nutrients, GLP-1 secretion is also indirectly regulated by GIP released in the proximal intestine in rodents. After a meal, nutrients are expected to reach the distal L cells and stimulate GLP-1 release via direct contact. However, this does not explain the biphasic pattern of GLP-1 secretion after a meal, including a 15–30 min rapid rise after oral ingestion followed by a second minor peak at 90–120 min (15, 103). Since all the initial findings indicate that L cells are located in the distal intestine (ileum, colon, rectum), the rapid early rise of GLP-1 after food ingestion within 5–15 min is faster than the time required for unabsorbed nutrients to reach the L cells in the distal intestine. A proposed mechanism is the existence of a neuroendocrine loop that regulates GLP-1 secretion distally once the ingested nutrients reach the proximal intestine (duodenum). This regulatory mechanism is referred to as proximal–distal neuroendocrine loop or duodeno-ileal endocrine loop. Since high GIP levels can stimulate GLP-1 secretion (104, 105), it is possible that nutrient entry into the duodenum stimulates GIP release, which in turn stimulates GLP-1 secretion in the distal intestine even before the nutrients arrive. This notion is supported by several studies. First, intraarterial infusion of GIP into perfused rat colon strongly stimulated GLP-1 secretion (106). In another study, the flow of nutrients to the distal intestine was restrained in rats to prevent direct interaction of the luminal content with the distal L cells (107). Next, when fat or glucose was placed in the duodenal lumen of these animals, GLP-1 release was induced at a level comparable to that by directly placing nutrients into the ileum (107). In the meantime, a rapid rise in GIP was also observed (107). This finding suggests that GIP released from the proximal intestine may mediate the early secretion of GLP-1 in the distal intestine. The vagus nerve appears to play an important role in this regulation because bilateral subdiaphragmatic vagotomy abolished the GLP-1 secretion by fat placed into the duodenum (108). Further, GLP-1 secretion stimulated by physiological concentrations of infused GIP was completely abrogated with selective hepatic branch

Other GI Peptide Hormones or Neurotransmitters

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vagotomy (108). These data suggest that the vagus nerve mediates the GIP-stimulated GLP-1 response in the distal intestine in rats. The GIP-mediated regulation of GLP-1 release has not been validated in humans. Although there is an early rise of GLP-1 after oral ingestion in humans (15, 103), GIP does not play a role in mediating this response. Intraduodenal infusion of a small amount of glucose produced a rapid and short-lasting GLP-1 response but the GIP level did not change (20), suggesting that the GLP-1 response to the duodenal glucose infusion is not mediated by GIP. In a separate study, synthetic GIP was infused into both type 2 diabetic patients and normal subjects. The exogenously administered GIP increased insulin secretion but had no effect on circulating GLP-1 level in normal subjects (109, 110). A further study was carried out in patients with upper and lower gut resections (jejunal or ileal small intestinal resections and colectomy), and it was found that a clear and early (peak at 15–30 min) GLP-1 response after food ingestion was observed in the patients with gut resection as well as controls (111). These studies demonstrate that the early GLP-1 response to food ingestion is not mediated by GIP in humans. One proposed explanation is that the early rise in GLP-1 is also a direct effect of nutrients on L cells because in contrast to previous reports that L cells are primarily located in the distally lower jejunum, ileum, colon, and rectum (1), GLP-1 positive cells were also found in human duodenum in recent studies (75, 76). These data suggest that the early rise in GLP-1 in humans after a meal could be ascribed to GLP-1 secretion from L cells in the upper gut.

DISORDERS IN INCRETIN RESPONSE IN TYPE 2 DIABETES Meal- or oral glucose-induced GLP-1 response is decreased in type 2 diabetic patients as well as subjects with impaired glucose tolerance (IGT) (112, 113), but the GLP-1 response in IGT subjects trends higher than that in type 2 diabetics (112). This impairment could at least in part contribute to the disease pathogenesis. If this is one of the major causes of the defective glucose homeostasis in type 2 diabetes, administration of exogenous GLP-1 is expected to help normalize glucose control. It is encouraging that despite reduced GLP-1 secretion, type 2 diabetics still respond to GLP-1 infusion with augmented insulin release and improved glucose tolerance (109, 114). However, there are individual variations in response to exogenous GLP-1 administration among type 2 diabetics; glucose elimination is faster and lower glycemia was achieved in patients with lower baseline fasting plasma glucose (114). This finding suggests that GLP-1 treatment becomes less effective as the disease progresses. In contrast to GLP-1, GIP has diminished incretin effect in type 2 diabetic patients, suggesting that the GIP response is largely lost in the disease state (109). The underlying mechanism behind this observation is not clear. Based on these data, only GLP-1 is expected to have potential therapeutic value in treating type 2 diabetes.

OTHER GI PEPTIDE HORMONES OR NEUROTRANSMITTERS In addition to the incretins, other peptide hormones and neurotransmitters are also involved in the regulation of gastric emptying, food intake, and energy metabolism.

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There are many such peptides and some are yet to be assigned exact functional roles. They are secreted by different types of enteroendocrine cells distributed in different segments across the GI luminal surface. These cells sense luminal contents through direct interaction and secrete peptide hormones with regulatory effects. PYY and CCK are both involved in the regulation of food intake and gastric emptying. PYY-immunoreactive L cells are found in the distal small intestine, the colon, and the rectum (115). There are two forms of PYY, PYY(1–36) and PYY(3–36), in human blood (116), with PYY(3–36) derived from PYY(1–36) through DPP-4 proteolytic cleavage. Unlike GLP-1, both forms of PYY are bioactive. PYY(3–36) is the major form in human colonic mucosa. The plasma PYY level increases several fold after meal ingestion in humans. Compared to equivalent calories of protein and carbohydrate diets, fat is a more potent stimulus of PYY secretion (117). PYY can inhibit gastric acid and pepsin secretion and delay intestinal transit time (117), suggesting that PYY is a negative regulator of energy intake in response to food ingestion. PYY can interact with a family of Gi-coupled GPCRs, including Y1R, Y2R, Y4R, Y5R, and Y6R. Peripheral injection of PYY(3–36) was shown to inhibit food intake and reduce body weight in rats (118). PYY(3–36) also inhibited food intake in mice but not in Y2R-null mice (118), suggesting that the anorectic effects are mediated by Y2R. Consistent with the findings in animals, PYY(3–36) infusion significantly reduced appetite and food intake in human subjects of normal weight (118). Further, the circulating levels of PYY were significantly lower in obese subjects compared to lean controls, and like its effect in lean subjects, PYY infusion reduced food intake in obese individuals (119). These findings demonstrate that PYY is an anorectic agent and could be used to treat obesity. However, in contrast to peripheral administration, central administration of PYY increased food intake (120). Moreover, the anorectic effects of peripheral PYY(3–36) administration could not be reproduced by some research groups (121), although others have been successful in replicating the original findings (122, 123). These discrepancies remain to be resolved with further studies. Similarly, CCK is another gut peptide involved in the regulation of food intake and related physiological activities. CCK is expressed as a 115-amino acid peptide in cells and undergoes posttranslational proteolytic processing to generate CCK58 (124, 125), the main circulating form. CCK is secreted by both I cells in the proximal intestine and L cells in the distal intestine (5). CCK is also found in the brain (5). Further proteolytic cleavage of CCK-58 generates smaller but still biologically active CCKs, including CCK-39, CCK-33, CCK-22, CCK-12, and CCK-8 (126). CCK is secreted and released into the blood circulation upon food ingestion and induces satiety. Two CCK receptors mediate the CCK function: CCK-1 receptor, primarily expressed in the GI tract, and CCK-2 receptor, mainly expressed in the brain. CCK-1 receptor is also expressed in the hindbrain and hypothalamus. Part of the CCK action in the brain is mediated by suppressing the expression of orexins A and B, two peptides produced in the lateral hypothalamic areas that stimulate food intake (127). The suppression of food intake by CCK was demonstrated in animal models as well as humans. Rats deficient in CCK-1 receptor had increased meal size and developed obesity (128), suggesting that the satiation signal is mediated by CCK-1 receptor. CCK administration also decreased food intake in humans by

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shortening meals (129). The anorectic effects of CCK are weak because rats deficient in CCK-1 receptor developed only mild obesity (128), and CCK-1 receptor-null mice did not develop obesity (130). In addition, the anorectic effects were rapidly lost during repeated CCK administration (131), suggesting that behavioral tolerance may have developed under such a condition. These data question the suitability of CCK as an anti-obesity therapy. Other important GI peptides include oxyntomodulin, GLP-2, and ghrelin. Like GLP-1, oxyntomodulin and GLP-2 are proglucagon-derived peptides secreted from L cells (84, 85). Oxyntomodulin has been demonstrated to reduce food intake and body weight gain in rodents (132–134) and humans (135, 136). Interestingly, oxyntomodulin also increases energy expenditure in both animals and humans (133, 137). These effects are presumably mediated by GLP-1 receptor, although oxyntomodulin binds to it less avidly than GLP-1 (132). Oxyntomodulin also binds to glucagon receptor as its N-terminus contains the full glucagon sequence (138). However, it has a lower affinity than glucagon itself (138). The dual activation of both GLP-1 and glucagon receptors by oxyntomodulin might be a better explanation for the effects on food intake, body weight gain, and energy expenditure. Two independent studies demonstrated that dual activation of both GLP-1 and glucagon receptors with oxyntomodulin- or glucagon-derived peptides reduced food intake, body weight gain, body fat, hepatic steatosis, and blood glucose, and improved insulin sensitivity and lipid metabolism (138, 139). Although also derived from the proglucagon polypeptide and secreted from L cells, GLP-2 has no incretin effect. Rather, it is an intestinal growth factor. GLP-2 stimulates crypt cell proliferation and bowel growth in an ErbBdependent manner (140, 141). GLP-2 also increases intestinal lipid absorption through activation of CD36 (142), thereby mediating a key function in response to food intake. Ghrelin is secreted from the stomach (6, 143) and is the endogenous ligand of the growth hormone (GH) secretagogues receptor (143). There are acylated and unacylated forms of ghrelin and the acylation is essential for the activity (143). Unlike the incretins or PYY, it increases food intake and is involved in meal initiation marked by a pre-meal surge (144). Ghrelin is likely involved in the long-term regulation of body weight (145). Interestingly, ghrelin improved cardiac functions in rats with heart failure (146), suggesting that there may be a role of ghrelin in regulating cardiovascular function.

THE PHYSIOLOGICAL IMPORTANCE OF THE GUT: LESSONS LEARNED FROM GASTRIC BYPASS The metabolic role of the gut is further implicated in the fascinating findings from bariatric surgery, which produces dramatic and durable weight loss (147). Among many different types of bariatric surgical operations employed to treat severe obesity (147), the most commonly performed are laparoscopic adjustable gastric banding (LAGB), gastric bypass, and biliopancreatic bypass (147). Gastric bypass (or Roux-en-Y gastric bypass, RYGB) involves surgical reduction of the size of stomach and bypassing a portion of the proximal small intestine (Figure 1.3). The portion

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Food Duodenum The upper portion is separated from the main stomach

RYGB

Bypass

Bypassed section transports bile and pancreatic fluid into the gut

Figure 1.3

Illustration of Roux-en-Y gastric bypass.

bypassed is connected to the distal small intestine to allow the passage of pancreatic fluids and bile into the gut (Figure 1.3). This procedure causes dramatic weight loss and has been the most effective treatment of severe obesity. In a series of 608 patients with 95% follow-up for at least 16 years, the mean weight loss was 106 lb (148). Surprisingly, more than 80% of the patients with type 2 diabetes developed complete remission of the disease after the surgery (148, 149). Weight loss does not fully explain the remission of type 2 diabetes after gastric bypass because within days after surgery the hyperglycemia and hyperinsulinemia were totally normalized (148). Although the mechanisms behind the antidiabetic effect are not entirely clear, increased insulin secretion and improved b-cell function are likely involved. Lateonset hyperinsulinemic hypoglycemia has been observed in patients after the surgery (150–152), and some may even require partial or total pancreatectomy to prevent recurrent hypoglycemia (150, 152). This phenomenon underscores the robust improvement of pancreatic function achieved by RYGB. GLP-1 and PYY are two important gut hormones that are believed to mediate the more robust beneficial effects of RYGB compared to LAGB, a procedure that restricts food intake by banding the stomach but does not involve the bypass of the proximal intestine. The metabolic effects of LAGB are therefore results of reduced food intake and weight loss. The average reduction in body weight after LAGB is 28% compared to 40% after RYGB, and the remission of type 2 diabetes occurs in 48% relative to 84% in RYGB (153, 154). One of the key differences between these two different operations is the greater GLP-1 and PYY response post meal after RYGB surgery (155), suggesting that these peptide hormones may play an important role in promoting weight loss and improved insulin sensitivity. As mentioned above, RYGB results in improved insulin sensitivity before weight loss in the short term. This effect

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seems to persist even in the long run in a weight loss independent manner, although weight loss itself can lead to improved insulin sensitivity. When compared with a weight-matched group, the patients who underwent RYGB had lower fasting insulin and better insulin sensitivity (156), suggesting that in addition to weight loss something else leads to further improved insulin sensitivity in RYGB patients. In addition to suppressing appetite and weight loss after RYGB (157), the increased postprandial GLP-1 response could further improve insulin sensitivity by increasing b-cell mass and improving b-cell function. In fact, there is sustained elevation of GLP-1 secretion post meal in RYGB patients compared to normal controls (158). This may be counterintuitive because L cells are also found in human duodenum (75) and nutrient bypass of the proximal intestine is expected to cause reduction in GLP-1 release. It could be that this is a small loss relative to the robust increase in GLP-1 secretion by the distal intestine so that the total GLP-1 secretion is still elevated after RYGB. Two hypotheses have been proposed to explain the weight loss independent effect in RYGB based on the roles of the foregut and the hindgut. The hindgut hypothesis proposes that the beneficial effects result from the expedited delivery of nutrients to the distal small intestine and enhancement of physiologic signals that improve glucose homeostasis (159); the foregut hypothesis holds that the weight loss independent effect depends on the exclusion of the duodenum and proximal jejunum from nutrient passage, therefore preventing the secretion of a physiologic signal that promotes insulin resistance (159). Using nonobese diabetic GotoKakizaki (GK) rats, Rubino et al. demonstrated that duodenal–jejunal bypass (DJB), a stomach-preserving RYGB, improved oral glucose tolerance compared to a pair-fed sham-operated group (159). However, restoration of duodenal nutrient passage in the DJB rats reestablished impaired glucose tolerance (159), suggesting that the weight loss independent metabolic benefits in the DJB rats were likely to be driven by the nutrient bypass of the foregut. Why does the duodenal nutrient passage have a negative effect? These researchers proposed that a physiologic signal induced by duodenal nutrient passage might play a role. This negative signal could be an anti-incretin factor, which might be secreted from the proximal intestine in response to nutrient passage and stimulate insulin resistance (159). The anti-incretin factor may interfere with the incretin secretion and/or actions and ultimately inhibit insulin action (159). One of the possibilities is that the anti-incretin inhibits GLP-1 secretion and after nutrient bypass of the proximal intestine the suppression is relieved leading to elevated GLP-1 secretion. Although this hypothesis is consistent with the improved b-cell function in RYGB patients, it remains to be validated by identification of a factor with anti-incretin effect. While the anti-incretin concept helps explain the weight loss independent effects in RYGB patients, Rubino’s data do not exclude the involvement of the hindgut in the improvement of metabolic effects. In fact, a study in mouse models indicates that there is increased gluconeogenesis in the distal intestine post DJB but not gastric banding (160), and the increased local glucose concentration is detected by a GLUT2-dependent hepatoportal sensor, which leads to reduced food intake and body weight and improved insulin sensitivity (160). Thus, it seems that different sections of the small intestine

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play important roles via distinct mechanisms to achieve beneficial metabolic effects in RYGB.

SUMMARY In addition to its role in food intake and nutrient absorption, gut is also an endocrine organ for secreted GI peptides. The release of these peptides in response to food intake is mediated by the direct contact of macronutrients with enteroendocrine cells on the luminal side distributed throughout the GI tract. These GI peptides regulate a variety of physiological actions in response to food intake, including the feedback response to suppress food intake and the feedforward response for nutrient assimilation. The incretin GLP-1 plays important roles in both regulatory pathways. Different sets of GI peptides are stimulated in response to specific types of macronutrients. There are several potential nutrient-sensing mechanisms mediated by taste receptors, KATP channels, glucose transporters, and GPCRs. Further studies are required to clarify the relative contributions of these pathways. The robust metabolic benefits associated with RYGB suggest that changes in the secretion profiles of GI peptides may be beneficial, although the exact mechanism is still elusive. Further studies in gut biology will likely shed new light on the metabolic functions of GI peptides.

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Central Glucose Sensing and Control of Food Intake and Energy Homeostasis LOURDES MOUNIEN1,2 1 2

AND

BERNARD THORENS1,2

Department of Physiology, University of Lausanne, Lausanne, Switzerland Center for Integrative Genomics, University of Lausanne, Lausanne, Switzerland

INTRODUCTION Glucose plays an essential role in energy homeostasis by regulating the secretion of various hormones and the activation of neuronal circuits controlling feeding and energy expenditure (1, 2). Glucose-sensing systems are present at many anatomical sites including the mouth, the gastrointestinal tract, the hepatoportal vein, the liver, the endocrine pancreas, and the central nervous system (CNS). In the mouth, glucose activates taste receptors, which stimulate afferent fibers projecting to the brainstem and trigger the cephalic phase of insulin secretion (3–5). In the intestine, glucose stimulates the secretion of the gluco-incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), by a mechanism that may involve the sodium-dependent glucose transporter SGLT1 and the KATP channel (6) and possibly also the activation of sweet taste receptors (7–9). A local action of GLP-1 hormones is to increase the expression of the Naþ /glucose cotransporter SGLT1 and the translocation of the glucose transporter GLUT2 at the brush border of enterocytes, leading to increased glucose absorption (9). Glucose also activates autonomic and enteric neurons located in the gut mucosa (10, 11). Activation of the cholinergic neurons of the submucosal and myenteric plexus may be mediated by glucose binding to SGLT3 (12).

Metabolic Syndrome: Underlying Mechanisms and Drug Therapies Edited by Minghan Wang Copyright 2011 John Wiley & Sons, Inc.

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Glucose entering the portal vein activates vagal afferents that project to the CNS (13, 14) to control several adaptive responses such as stimulation of glucose storage by liver, soleus, heart, and brown adipose tissue; inhibition of counterregulation; termination of food intake; and stimulation of first-phase insulin secretion (10, 15, 16). In the liver, glucose stimulates glycogen synthesis (17) as well as the expression of genes involved in glycolysis and lipogenesis through transcriptional mechanisms mediated in large part by the glucose-sensitive transcription factor ChreBP (18, 19). In the pancreas, increase in plasma glucose triggers insulin secretion by b-cells (1, 20–22). In contrast, glucagon secretion by a-cells is triggered when glycemia falls below the euglycemic level (20, 23, 24). In the CNS, glucose modulates the activity of glucose-sensitive neurons located in the hypothalamus and brainstem. These belong to at least two classes: glucoseexcited (GE) neurons, whose firing activity is increased by rises in extracellular glucose concentrations, and glucose-inhibited (GI) neurons, which are activated when glucose concentrations decrease. These glucose-sensitive neurons control glucose homeostasis, feeding behavior, and energy homeostasis. The molecular basis for glucose monitoring and regulation of firing activity is being actively investigated. Present evidence indicates that there is a large diversity in the mechanisms of glucose sensing, which may define subpopulation of either glucose-excited or glucoseinhibited neurons. Recent experimental evidence also supports a role for glial cells in glucose sensing (25, 26). The complexity of these glucose-sensing mechanisms needs to be eventually completely understood to better manage pathologies caused by deregulated glucose and energy homeostasis. Here, we mainly focus on the mechanisms of glucose sensing by neurons.

BRAIN GLUCOSE SENSING Sites of Glucodetection Claude Bernard first implicated the brainstem in glucose homeostasis when he showed that puncturing the floor of the fourth cerebral ventricle of dogs rapidly induced diabetes (27). In 1953, Jean Mayer proposed that cells located in hypothalamus monitor plasma glucose levels by translating variations in glycemia into electrical or chemical signals to control feeding behavior (28). Several groups then identified GE neurons, whose electrical activity is increased by high glucose, and GI neurons activated by cellular glucoprivation (29–32). GE and GI neurons are mainly expressed in the hypothalamus, in particular in the arcuate (Arc), lateral (LHA), dorsomedial (DMH), ventromedial (VMH), and paraventricular (PVN) hypothalamic nuclei (32–38), and in the brainstem, in the area postrema (AP), the nucleus of the solitary tract (NTS), the dorsal motor nucleus of the vagus (DMNX), and the basolateral medulla (BLM) (14, 33, 34, 36, 39–41). High glucose excited (HGE) and high glucose inhibited (HGI) neurons, whose firing activity is regulated over glucose concentration ranges (5–20 mM), have also been identified in the Arc (35, 42).

Physiological Functions Modulated by Central Glucodetection

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Mechanisms of Glucodetection Glucose controls the electrical activity of GE neurons by a mechanism that shares similarities to glucose-induced insulin secretion by pancreatic b-cells (43, 44) (Figure 2.1a). Glucose signaling in these cells is initiated by the uptake of glucose by GLUT2 followed by its phosphorylation by glucokinase (hexokinase IV, Km for glucose 6 mM). Subsequent activation of mitochondrial metabolism and oxidative phosphorylation increases the ATP/ADP ratio. This leads to closure of ATP-sensitive potassium (KATP) channels, plasma membrane depolarization, and opening of voltage-sensitive Ca2þ channels. The influx of calcium then triggers insulin secretion. In GE neurons, rise in extracellular glucose also increases the cytosolic ATP/ADP ratio and induces closure of KATP channels (39, 45, 46), plasma membrane depolarization, Ca2þ entry, and neurotransmitter release (47, 48). However, recent studies suggest that some GE neurons may be activated by glucose in a KATP channel-independent manner (35, 49). Evidence also suggests that GK (50) and GLUT2 (51, 52) may not be required for activation of these neurons. The mechanisms through which GI neurons sense glucose are not well characterized (Figure 2.1b). However, the effect of glucose on GI neurons may be controlled by changes in Naþ /Kþ ATPase activity (30, 37) or by the opening of ATP-regulated chloride channels that leads to hyperpolarization (31, 50). In the LHA, inhibition by high glucose of the GI orexin neurons may depend on tandem-pore Kþ (TASK) or related channels (53–55).

PHYSIOLOGICAL FUNCTIONS MODULATED BY CENTRAL GLUCODETECTION Food Intake and Energy Expenditure The role of glucose in the control of food intake was demonstrated in many studies (2, 28). Particularly, it has been shown that initiation of feeding is preceded by a small drop in glycemia, and preventing it by infusing glucose suppresses initiation of feeding (56). In addition, peripheral or central administration of 2-deoxyglucose (2-DG), which induces neuroglycopenia, stimulates food intake (57–59). The modulation of feeding behavior and energy expenditure by CNS is a complex process that involves hypothalamic and brainstem neuronal circuits. In the hypothalamus, neurons integrate nutrient (lipid and glucose), hormonal (ghrelin, insulin, PYY3–36, leptin, CCK, GLP-1, and adiponectin), and nervous signals that convey information about food absorption and the levels of stored energy (10, 60–64). These signals are detected in large part by Arc neurons expressing the anorexigenic peptides POMC/CART or the orexigenic peptides NPY/AgRP. These neurons project to melanocortin 3 and 4 receptor-expressing neurons of the PVN and LHA (20, 65). Neurons in the PVN produce the anorexigenic neuropeptides TRH and CRF whereas neurons in the LH produce the orexigenic peptides MCH and orexin (62). Together these neurons form the melanocortin pathway and regulate peripheral metabolism

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(a) GLUCOSE GE neurons

Classical model

Alternative models GLUCOSE GLUT2 1,3

SGLT1,3

?

GK glucose

↑G6P VDCC

↑Ca2+

↑Ca2+

ATPase

↑ATP

↑ROS UCP2

Classical model

↑ROS ?

Alternative models GLUCOSE

ClC

C VD

GLUT2 1,3

Receptor

C

GK

↑Ca2+

TASK

?

↓pyruvate ↓ATP ATPase

↓ATP/ADP

Na+/K+ pump

Figure 2.1

?

↑H+

↓G6P

VDCC

VDCC

KC

GLUCOSE GI neurons

KC

UCP2 Ox. Ph.

KATP

(b)

↑Na+

↑pyruvate

↑ATP/ADP

TRP

GLUT2 1,3

KC

?

Ox. Ph. ↓H+

Models for the control of the electrical activity of GE and GI neurons by glucose. (a) Glucose activation of firing rate in GE neurons. In the b-cell model, glucose uptake through GLUT2, but in neurons possibly also by GLUT1 or GLUT3, is followed by its phosphorylation by GK. Pyruvate is then channeled into the mitochondria to eventually increase ATP production and induce a rise in the ATP/ADP ratio. This leads to the closure of KATP channel, membrane depolarization, and the entry of Ca2þ , which triggers the release of neurotransmitters. In an alternative mechanism, the activity of GE neurons is controlled by the electrogenic cotransport of Naþ and glucose by SGLT1, by activation of TRP channels, or by production of ROS following the activation of the oxidative phosphorylation. ROS could directly regulate the activity of Kþ channels or intracellular Ca2þ availability. UCP2 may serve as a regulator of ATP production through its decoupling activity or by reducing the production of ROS; both mechanisms lead to reduced glucose-induced neuronal activity. (b) Activation of GI neurons by a decrease in extracellular glucose concentration. A reduction in the ATP/ADP ratio leads to closure of Cl channels and/or a reduction in the activity of the Naþ /Kþ ATPase, plasma depolarization, and the entry of Ca2þ that triggers the secretion of neurotransmitters. Alternatively, a new pathway involving TASK channel may control the activity of GI neurons through a mechanism that does not involve glucose metabolism, possibly through interaction with a putative specific receptor. KC: K þ channel; VDCC: voltage-dependent calcium channel.

Multiplicity of Sensing Mechanisms

33

through activation of the autonomic nervous system and higher brain structures to control not only feeding behavior, but also arousal and reward (66–68). The hindbrain is also a site of glucodetection and may have a major role in regulating feeding in physiological conditions. Indeed, intracerebroventricular (i.c.v.) injection of 2-DG stimulates feeding only if it can have access to the brainstem (57, 69) and food uptake can be activated by injection of 5-thioglucose (5-TG) into NTS, DMNX, and BLM but not in hypothalamic nuclei (39–41, 70, 71). Glucose-sensitive neurons from the BLM are catecholaminergic and send projection to Arc and PVN (72). Destruction of these projections by immunotoxins suppresses the effect of 2-DG on food intake and on regulated expression of AgRP and NPY in the Arc (73, 74).

Counterregulation When blood glucose concentrations fall below the euglycemic level, a rapid counterregulatory response is activated to restore normoglycemia. This involves activation of the autonomic nervous system (75) that triggers glucagon secretion and the release of catecholamines from adrenal glands (76–78). The central sites of glucose detection that activate the autonomic nervous system are located in the hypothalamus and brainstem. In the hypothalamus, lesion studies as well as pharmacological and genetic approaches have provided evidence for an important role of the VMH in the control of glucagon secretion (79). For instance, glucagon release can be induced by direct injection of 2-DG in the VMH (80). In contrast, hypoglycemia-induced glucagon secretion can be suppressed by direct injection of glucose into this structure (81). Brainstem nuclei also play an important role in activating counterregulation. For instance, when the cerebral aqueduct is obstructed, 5-TG induces glucagon secretion when injected in the fourth but not in the third ventricle (69). In addition, injection of 5-TG directly in the NTS or the BLM nuclei containing the A1/C1 catecholaminergic neurons strongly stimulates glucagon secretion (70). In decerebrated rats, the hyperglycemic response to an i.p. injection of 2-DG is preserved (82) and c-fos staining revealed that the activated cells are present in the NTS, DMNX, and the catecholaminergic neurons of the BLM (72).

MULTIPLICITY OF SENSING MECHANISMS One important goal of current research is to identify each type of glucose-sensing neurons and to determine which physiological functions they control. One path to reach this goal is to identify the critical proteins that allow these neurons to respond to glucose and use these proteins as markers to identify the glucose-sensing neuron subpopulations, their topographical distribution, and the neuronal circuits they form. In recent years, many ion channels, transporters, or enzymes have been described to participate in central glucose sensing. Here, we review the list of these gene products and their role in glucose sensing.

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Glucose Transporters In most instances, glucose uptake and metabolism are required for glucose signaling in neurons. Glucose uptake may be catalyzed by either facilitated diffusion glucose transporters (Gluts) or Naþ -linked glucose transporters (SGLTs) (83–87), and both types of transporters have been associated with glucose sensing by hypothalamic and brainstem neurons. Glut1, Glut3, and Glut8 Glut1 is highly expressed in endothelial cells forming the blood–brain barrier and is required for glucose entry in the brain. Inactivating mutations of Glut1 reduce the concentration of glucose in the cerebrospinal fluid, which causes seizure and delayed development (88). Glut3 is a high-affinity glucose transporter expressed in neurons (89). Homozygous Glut3 knockout mice die during embryogenesis whereas heterozygous knockout mice have normal glucose homeostasis and feeding behavior (90), although they show defects in spatial learning and memory processing (91). Glut8 is a high-affinity glucose transporter expressed in neurons in different brain regions, including the hippocampus, the hypothalamus, and the brainstem (92, 93) but homozygous Glut8 knockout mice show no defect in glucose or energy homeostasis (94). Thus, Glut1, Glut3, and Glut8 play specific role in brain glucose metabolism unrelated to the glucose sensing and control of whole-body glucose and energy homeostasis. Glut2 Glut2 is a low-affinity glucose transporter, required for normal glucose sensing by pancreatic b-cells. In the brain, it is expressed in neurons, astrocytes, tanycytes, and epithelial cells lining the cerebral ventricles (95–99). The low level of expression of Glut2 makes its immunohistochemical detection challenging and its precise localization is still partly uncertain, although it is present in hypothalamic and brainstem nuclei (51, 95, 96, 100, 101). A role for central GLUT2 in glucose sensing has been suggested by i.c.v. or direct injection in the Arc of antisense oligonucleotides to reduce its expression (98, 102). This decreased feeding and body weight gain and suppressed 2-DG-induced feeding as well as the insulin response normally triggered by intracarotid glucose injection (98, 102). Studies with Glut2/ mice, which express a transgenic glucose transporter in their b-cells to restore normal glucosestimulated insulin secretion (ripglut1;glut2/ mice) (103), demonstrated a role for central Glut2 in the control of glucagon secretion in response to insulin-induced hypoglycemia or 2-DG-induced neuroglucopenia (26). Transgenic complementation studies revealed that Glut2 reexpression in astrocytes but not in neurons restored the counterregulatory response to hypoglycemia (26). The same mouse model was used to study the role of Glut2 in central glucose sensing and the control of feeding. It was demonstrated that in the absence of Glut2, the mouse presented defects in both feeding initiation and termination. This was

Multiplicity of Sensing Mechanisms

35

correlated with suppressed regulation of the hypothalamic anorexigenic (POMC, CART) and orexigenic (NPY, AGRP) neuropeptides during the fast to refed transition or following intracerebroventricular injection of glucose (104). In contrast to the impaired counterregulatory response, the abnormal feeding behavior of GLUT2-null mice did not rely on GLUT2 expression in astrocytes (105), suggesting that regulation of counterregulation and feeding behavior depends on Glut2 expression in different cell types, astrocytes and neurons, respectively. SGLT1 and SGLT3 SGLT1 is a Naþ –glucose symporter present in the brush border of intestinal epithelial cells, GLP-1 secreting L cells, and in the proximal straight tubule of the kidney nephrons (106–108). SGLT1 transports glucose and galactose as well as the nonmetabolizable analogues 3-O-methyl-D-glucose (3-O-MDG) and a-methyl-Dglucopyranoside (a-MDG) and is inhibited by phlorizin (107, 109). SGLT1 is also expressed in the hypothalamus and ependymal cells of the third and fourth ventricles (52, 110). A role for SGLT1 in L cell glucose sensing has been described (108, 111). As glucose uptake is electrogenic, it leads to membrane depolarization and GLP-1 secretion in a KATP-independent manner (112); GLP-1 secretion can also be triggered by the nonmetabolized substrates 3-O-MDG and a-MDG (113, 114). That SGLT1 may play a role in central glucose sensing is suggested by the finding that i.c.v. injection of phlorizin enhances food intake in rats (115) and inhibits activation of GE neurons in the VMH (44). On the other hand, hypothalamic GE neurons can be excited by a-MDG and 3-OMG (52). SGLT3 is expressed mainly in intestine, liver, kidney, and muscle (116). Pig SGLT3 transports glucose and a-MDG with relatively low affinity and, in contrast to SGLT1, does not transport galactose and 3-O-MDG (109). Human SGLT3 does not transport glucose when expressed in Xenopus laevis oocytes but may play a role as a glucose sensor in cholinergic neurons of the small intestine and at the neuromuscular junctions (12). In Xenopus oocytes expressing SGLT3, glucose produced a phlorizinsensitive inward current that depolarizes the membrane potential by up to 50 mV (12). SGLT3 mRNA is expressed in both cultured hypothalamic neurons and adult hypothalamus, suggesting that this transporter may also be involved in central glucose sensing (52).

Intracellular Mediators Glucokinase Following its uptake, glucose is phosphorylated by hexokinases. In pancreatic b-cells, glucokinase controls the flux of glucose metabolism and the dose response of glucosestimulated insulin secretion. In the brain, glucokinase is expressed in the Arc, LHA, DMH, VMH, and PVN, as well as in the brainstem and ependymocytes of the third and fourth ventricles (99, 101, 117, 118). Pharmacological inhibition of hypothalamic

36

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GK decreases the activity of GE neurons and increases that of GI neurons showing that GK is involved in both high glucose and glucopenia detection (32, 38, 44, 51, 119). Recently, GK has also been shown to be required for glucose sensing by GI and GE neurons in the NTS and DMNX (118). A role for central GK in glucagon secretion and feeding is evidenced by i.c.v. administration of the GK inhibitor alloxan that stimulates feeding (120). Reduction of GK activity in the VMH by injection of alloxan or by adenoviral-mediated transduction of a GK-specific shRNA showed that this enzyme is essential for the counterregulatory response to insulin-induced hypoglycemia (121) and for feeding (122). AMPK AMPK is a ubiquitous enzyme formed by a catalytic and b and g regulatory subunits (123, 124). AMPK is an intracellular fuel gauge activated by increased intracellular AMP/ATP ratio and by phosphorylation by the LKB1 and Ca2þ /Calmodulin-activated kinases (124, 125). AMPK turns on catabolic pathways such as fatty acid oxidation and turns off gluconeogenesis and lipogenesis (125). In pancreatic b-cells, activation of AMPK by low glucose suppresses glucose-induced glycolysis, mitochondrial oxidative metabolism, Ca2þ influx, and insulin secretion (126). A role for hypothalamic AMPK in metabolic regulation has been initially proposed based on studies indicating that its activity is inhibited by leptin, insulin, glucose, and refeeding. AMPK activity is regulated in these conditions only in the Arc and PVN but not in the VMH, DMH, and LHA nuclei (123). Adenoviral delivery of constitutively active or dominant negative forms of AMPK in medial hypothalamic nuclei activates or inhibits feeding, respectively (123), and i.c.v. administration or direct injection in the PVN of 5-amino-4-imidazolecarboxamide riboside (AICAR) stimulates feeding (127). Similarly, neuroglucopenia induced by i.c.v. injection of 2-DG increases hypothalamic AMPK activity and feeding, an effect that can be blocked by the AMPK inhibitor compound C (128). How AMPK activity in hypothalamic neurons controls feeding is not fully understood. In neuronal cell lines and on ex vivo hypothalamic explants, low glucose concentrations and AICAR increase AMPK activity and AgRP expression (129). In accordance with these observations, the specific deletion of the a-subunit of AMPK in POMC and AgRP neurons suppressed glucose sensing by these cells but preserved normal leptin or insulin action (130). AMPK may also be involved in the counterregulatory response to hypoglycemia or to neuroglucopenia (131–134). Stimulation of AMPK by microinjection of AICAR in the VMH increases endogenous glucose production whereas compound C or expression of a dominant negative form of AMPK in ARC and VMH impaired early counterregulation as evidenced by reduced glucagon and catecholamine responses to hypoglycemia (134). In contrast, overexpression of the dominant negative AMPK in the PVN attenuated late counterregulation and corticosterone responses (134).

Multiplicity of Sensing Mechanisms

37

Recently, it has been shown that the counterregulatory hormone response impaired by 2-DG-induced recurrent neuroglucopenia was partially restored by i.c.v. injection of AICAR (131). At the brainstem level, AMPK activity also contributes to energy homeostasis. For instance, AMPK activity is significantly increased in the NTS of fasted compared to ad libitum fed rats (135) and injection of compound C directly in the NTS induces a decrease in food intake and body weight (135). Regulation of AMPK in the brainstem may thus mediate the anorectic action of leptin, which is reversed by AICAR injections (135). mTOR The mammalian target of rapamycin (mTOR) is a conserved serine–threonine kinase that promotes anabolic pathways such as protein synthesis in response to growth factors, nutrients (amino acids and glucose), and stress (136). These mechanisms involve the regulatory proteins 70 kDa ribosomal protein S6 kinase (S6K1) and the eukaryotic initiation factor 4E-binding protein-1 (4EBP1), which are key regulators of protein synthesis (136, 137). mTOR exists in two distinct complexes. Target of rapamycin complex 1 (TORC1) is a functional association of mTOR with the scaffolding protein raptor, whereas TORC2 is the combination of mTOR with the protein rictor. mTORC1 functions as a nutrient/energy/redox sensor controlling protein synthesis and can be inhibited by rapamycin (138, 139). mTORC2 is an important regulator of the cytoskeleton and phosphorylates the serine/threonine protein kinase Akt/PKB at a serine residue S473 (140). TSC1 and TSC2 are tumor suppressors, and their gene products form a stable complex that inhibits mTORC1 activity (141). Glucose deprivation inhibits mTOR activity, and inhibition is abolished in TSC mutant cells. Interestingly, AMPK inhibits mTORC1 activity by phosphorylation of TSC2 as well as raptor (136). The group of McDaniel showed that glucose elevation activates mTOR/S6K1/ 4EBP1 and protein synthesis in an amino acid-dependent manner in both rodent and human islets (142–144). In rat brain, mTOR has been located in numerous brain structures including hypothalamus, thalamus, and cortex (145). In hypothalamus, mTOR is located in PVN and Arc (145). More precisely, 90% of NPY/AgRP neurons while 45% of POMC neurons also expressed mTOR protein (145). After fasting, there is a decrease in mTOR-positive cells in Arc suggesting that mTOR activity is low after glucose privation (145). In addition, leptin also stimulates mTOR activity and inhibition of mTOR with rapamycin blunts the anorexigenic effect of leptin (145). Recently, specific ablation of TSC1 in POMC neurons induced hyperphagic obesity and alteration of the morphology of POMC neurons (146). These phenotypes are reversed by treatment with rapamycin (146) suggesting an important role of POMC neurons in the control of metabolism. Interestingly, the activation of AMPK-dependent mechanism leads to the inhibition of mTOR activity (147). Thus, AMPK and mTOR may have reciprocal functions and interact in order to control energy homeostasis.

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UCP2/ROS Activation of the mitochondrial respiratory chain leads to Hþ extrusion from the mitochondria and establishment of an electrochemical gradient across the inner mitochondrial membrane. The transport of Hþ back into the mitochondrial matrix through the F0F1ATPase generates ATP. In pancreatic b-cells, the rise in intracellular ATP/ADP ratio is critical to link glucose metabolism to insulin secretion. The uncoupling protein UCP2, located in the inner mitochondrial membrane, can dissipate the electrochemical Hþ gradient, thereby decreasing the capacity of the cells to produce ATP (148). Accordingly, overexpression of UCP2 in islets or insulinoma cells blunts glucose-induced insulin secretion (149–151), and islets from UCP2/ mice have increased ATP levels and higher secretory response to glucose (152, 153). In brain, UCP2 is widely distributed and expressed at high levels in the hypothalamus, in particular in the Arc, VMH, PVN, and LHA, as well as in the brainstem (154) where it has been proposed to play a role in glucose sensing (155). For instance, increased expression of UCP2 in POMC neurons of mice fed a high-fat diet is associated with a loss of their glucose sensitivity, which can be prevented by genepin, an inhibitor of UCP2, or by UCP2 gene inactivation (156). UCP2 was also found to be critical for activation of NPY/AgRP neurons during fasting and in response to ghrelin (157, 158). Oxidative phosphorylation is also associated with the production of reactive oxygen species (ROS) and UCP2 may act as a negative regulator of ROS production (159–161). Indeed, initial studies of UCP2/ mice showed that their macrophages generated 80% more ROS than those from control mice (159). Importantly, ROS are also intracellular signaling molecules (162) that can regulate the activity of voltage-gated Kþ channels (163, 164) or Ca2þ influx (165–167). In b-cells, ROS may participate in the coupling between glucose metabolism and insulin secretion (168–174). In the hypothalamus, there is evidence that glucose sensing may involve ROS production (175). Exposure of hypothalamic slices to increase in glucose concentrations (from 5 to 20 mmol/L) stimulates ROS generation, which is reversed by addition of antioxidants. Intracarotid administration of antimycin or rotenone, which induces ROS formation, mimics the effect of glucose on Arc neurons’ activity and subsequent nervous-mediated insulin release (175).

Channels KATP Channel-Dependent Mechanisms The KATP channel plays a fundamental role in coupling changes in glucose metabolism to plasma membrane electrical activity (176). This channel is an octameric protein consisting of four copies of the pore-forming Kir6.2 channel (in pancreatic b-cells and in neurons) and four copies of the sulfonylurea receptor SUR1 (in pancreatic b-cells and brain) or SUR2B (in brain) (34, 177–180). The expression of these different subunits (Kir6.2, SUR1, and SUR2B) suggests a molecular diversity

Multiplicity of Sensing Mechanisms

39

of KATP in brain and especially in hypothalamus and brainstem (34, 46, 118, 156, 176, 177, 181–184). This channel is involved in central glucose sensing to control glucagon secretion and feeding. For instance, i.c.v. or intrahypothalamic injection of glibenclamide, a KATP channel inhibitor, blocks the counterregulatory response to a hypoglycemic clamp or induced by central administration of 5-TG (185). Genetic inactivation of Kir6.2 leads to impaired glucagon secretion in response to 2-DG administration and this is correlated with suppressed glucose-regulated firing of VMH neurons (46). In contrast, activation of this channel in the VMH amplifies counterregulatory hormone responses to hypoglycemia in normal and recurrently hypoglycemic rats (186). Kir6.2-null mice also have a smaller but significant feeding response than control mice to 2-DG injection (46). In addition, the acute regulation of membrane potential and firing of a subset of hypothalamic neurons by leptin and insulin is due to an action on the KATP channel that causes cell hyperpolarization (187–189). In a recent study, the role of the KATP channel in POMC neurons was addressed by transgenic expression in these neurons of a mutant Kir6.2 subunit that prevents ATP-mediated closure of the channel. This suppresses the response of these neurons to glucose without affecting feeding and induces mild glucose intolerance (156). KATP Channel-Independent Mechanisms Numerous studies suggest that the glucose-dependent firing activity of glucosesensing neurons is also controlled by mechanisms not involving the KATP channel. Table 2.1 lists these channels and their distribution in the hypothalamus and brainstem. Electrophysiological recording of neurons from Kir6.2/ mice showed firing activity triggered by increase in glucose concentrations from 5 to 20 mmol/L with a decrease in input resistance (35), suggesting that these HGE neurons used a KATP channel-independent mechanism to sense glucose. This response appears to depend on the opening of transient response potential (TRP) channels (35), which may play a role in maintaining intracellular Ca2þ concentration (190). The firing activity of GI neurons in response to decreased extracellular glucose may involve reduced activation of the Na þ /Kþ ATPase (30, 191), blockade of a Cl conductance (37, 192), or inhibition of acid-sensitive two-pore domain Kþ channels (TASK) (55). In the LHA, local application of glucose hyperpolarizes the GI orexin neurons, an effect that is prevented by ouabain (a blocker of the Na þ /K þ ATPase) and azide (an inhibitor of energy production), suggesting that glucose exerts its inhibitory effect through Naþ /Kþ ATPase (30). The response of the orexin neurons may involve a glucose-activated Kþ conductance (55), which based on the sensitivity to pH and halothane may be the K2p Twik1-related acid-sensitive Kþ channel subunit TASK3 (55). However, glucose-induced hyperpolarization of orexin neurons is unaffected not only in TASK3 knockout mice but also in TASK1 and TASK3/ TASK1-null mice suggesting that the exact mechanisms of activation of neurons by low glucose are still incompletely understood (53).

40

Table 2.1

Distribution in the Hypothalamus and Brainstem of the Proteins Involved in Central Glucose Sensing Hypothalamus

Brainstem

Arcuate Dorsomedial Lateral Paraventricular Ventromedial Ependymal Area Dorsal Nucleus Basolateral Ependymal nucleus nucleus hypothalamic nucleus nucleus layers postrema motor of the medulla layers of area of third nucleus of solitary (A1/C1) fourth ventricle the vagus tract ventricle Transporter Glut2 Glut1/3 SGLT1/3 Enzyme Glucokinase AMPK UCP UCP2/ROS Channels TRP CFTR/Cl Tandempore K þ Na/K ATPase KATP channels

? þ þ þ þ þ ? þ þ þ þ þ þ þ SGLT1/3 is detected in hypothalamic cultured neurons and in adult hypothalamus ND

þ þ ND

þ þ þ

þ þ ND

þ þ þ

(96–101) (51, 97, 99) (52, 110)

þ þ

þ þ

þ

þ

þ þ

þ ND

þ

þ þ

þ þ

þ þ

þ þ

(99, 117, 118) (130, 135)

þ

þ

þ

þ

þ

þ

þ

þ

ND

þ

(148, 156, 158, 175)

þ þ þ

ND þ

ND þ þ

þ þ þ

þ þ þ

ND ND þ

TRP1 is detected in brainstem tissue extract þ ND ND þ þ þ þ þ

(35) (191, 192) (55, 191)

þ

þ

þ

þ

þ

ND

þ

þ

þ

þ

ND

(30, 191)

þ

þ

þ

þ

þ

ND

þ

þ

þ

þ

ND

(34, 46, 118, 156, 177, 181)

þ , Positive; , negative; ND, undetermined; ?, conflictual data.

References

41

In Arc GI neurons, a role for Cl conductance has been evidenced for the response to low glucose concentrations (37, 192). As gemfibrozil, a cystic fibrosis transmembrane regulator (CFTR) blocker, prevents activation of GI neurons in both the Arc and VMH, the CFTR may be involved in this response (192). In the dorsal vagal complex, inhibition of Na þ /K þ ATPase by strophanthidin or ouabain suppressed the inward currents of GI neurons and a role for Cl channels can be excluded (191).

CONCLUSION Changes in glycemia are monitored by several systems located at different anatomical sites. GE and GI neurons, located in hypothalamus and brainstem, control many physiological responses such as counterregulation, feeding behavior, and energy expenditure. At the molecular level, present evidence indicates that a large number of mechanisms have evolved to tightly monitor increases or falls in glycemia. SGLT1, GLUT2, GK, and KATP channel appear to be associated with the response of GE neurons to increase in glucose concentrations. However, not all GE neurons do express GLUT2 or GK, suggesting a diversity in the function of the GE neurons. GI neurons increase their firing activity in response to fall in glucose concentrations by mechanisms that are still unclear but that do not involve the KATP channel but rather Na þ /Kþ ATPase pump, chloride channels, or TRP or TASK channels. Again, available evidence indicates that these pump and channels may be differently required by GI neurons present in different locations. AMPK, mTOR, UCP2, and the production of ROS are also involved in the response of neurons to changes in glycemia and it is so far not known whether they all contribute to the response of all GE or GI neurons or only to subpopulations of glucose-sensitive neurons. Although not fully discussed in this chapter, it has been recently suggested that some GE and GI neurons may sense glucose independently of glucose metabolism. For instance, hypothalamic GE neurons in culture are also excited by the nonmetabolizable glucose analogue a-MDG, which is a substrate of SGLT. In addition, in GI neurons such as orexin cells, glucose-induced hyperpolarization and inhibition are unaffected by GK inhibitors and mimicked by 2-DG. Therefore, the CNS, which critically depends on glucose for its function, has evolved many glucose-sensing mechanisms to monitor all aspects of energy supply and need and modulate glucose and energy homeostasis. Deciphering the complexity of glucose sensing by the CNS and the structure of the glucose-sensing neuronal circuits that control glucose and energy homeostasis still represents a formidable challenge.

REFERENCES 1. MARTY, N., M. DALLAPORTA, and B. THORENS. 2007. Brain glucose sensing, counterregulation, and energy homeostasis. Physiology (Bethesda) 22:241–251.

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Abnormalities in Insulin Secretion in Type 2 Diabetes Mellitus TALY MEAS1,2 1 2

AND

PIERRE-JEAN GUILLAUSSEAU1,2

APHP, Department of Internal Medicine B, Hoˆpital Lariboisiere, APHP Paris Universite Paris 7, Paris, France

INTRODUCTION According to the World Health Organization (WHO) and the American Diabetes Association (ADA), type 2 diabetes mellitus (T2DM) is defined as resulting from defects in both insulin secretion and insulin sensitivity. Since the discovery of plasma insulin radioimmunoassay by Salomon Berson and Rosalyn Yalow (1), evidence has been obtained that insulin secretion is severely impaired in T2DM. Numerous functional defects such as b-cell dysfunction and other pathological abnormalities have been described in T2DM patients. Functional alterations that lead to b-cell dysfunction, including abnormalities in the kinetics of insulin secretion and quantitative and qualitative defects, all progress with time. Pathological abnormalities then can at least in part explain functional alterations, which include b-cell loss and its progression and reduced b-cell mass.

NORMAL GLUCOSE HOMEOSTASIS Normal glucose homeostasis represents the balance between glucose appearance in systemic circulation (endogenous or meal-derived) and tissue glucose uptake and

Metabolic Syndrome: Underlying Mechanisms and Drug Therapies Edited by Minghan Wang Copyright 2011 John Wiley & Sons, Inc.

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utilization. This balance is tightly regulated and plasma glucose concentrations are maintained within a narrow range. Normal fasting and 2 h post-glucose load plasma glucose levels are defined as 60–110 mg/dL and 140 mg/dL, respectively. Glucose homeostasis is maintained by the highly coordinated interaction of three physiological processes: insulin secretion, tissue glucose uptake, and hepatic glucose production (HGP). In the fasting state, plasma glucose is almost exclusively provided by hepatic production via glycogenolysis and gluconeogenesis. Hepatic glucose appearance rate in the circulation is matched with tissue glucose uptake rate, which occurs mostly in tissues that require glucose such as the central nervous system. Fasting plasma glucose (FPG) concentration is maintained at constant levels by the matched regulation of glucose production and glucose uptake. Following a meal, rising plasma glucose levels promote hepatic glucose uptake and insulin-independent glucose disposal and stimulate the release and the production of insulin by pancreatic b-cells. Increased plasma insulin concentrations suppress HGP primarily by decreasing glycogenolysis and gluconeogenesis and increasing glucose disposal through stimulation of peripheral glucose uptake (mostly in the muscle). These responses minimize hyperglycemia and ensure the return of mealtime glycemic levels to pre-meal values (2–4).

INSULIN SECRETION AND EFFECTS ON TARGET TISSUES Glucose is the primary regulator of pancreatic b-cells by direct stimulation of insulin secretion and by modulating the insulin response to gut hormones and neural factors released during nutrient consumption. Insulin, like many hormones, displays rapid variations in plasma concentrations with frequent secretory peaks (periodicity 5–10 min), and less frequent larger oscillations (periodicity 60–120 min) (5). Normal insulin secretion in response to intravenous glucose follows a two-phase pattern. The first phase of insulin secretion (early or acute secretion) is rapid and sharp, reaching a maximum at 3–5 min and lasting for approximately 10 min. It represents mainly the release of stored granules. Second phase (late secretion) is gradual and persists for as long as glucose levels remain elevated. It stems from both stored secretory granules and de novo insulin synthesis. Early phase of insulin secretion is pivotal in the transition from the fasting state to the fed state with several different functions: to suppress HGP (6, 7), to suppress lipolysis (7), and to cross the endothelial barrier to prepare target cells for insulin action (8). Liver is a pivotal site in glucose metabolism regulation, and is responsive to minute changes in portal plasma insulin and glucagon concentrations. Early in the absorptive state during or after a meal, first-phase insulin secretion exerts an inhibitory effect on HGP by suppressing glycogenolysis and gluconeogenesis rate. In the fasting state, liver is almost the exclusive source of plasma glucose and therefore the most important site of insulin-mediated basal glucose release. Insulin’s first effect on skeletal muscle occurs during the absorptive state. Muscle is the major site of insulin-mediated dietary glucose uptake through stimulation of the insulin-sensitive glucose transport system. Insulin promotes conversion of glucose

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into glycogen by enhancing glycogen synthase activity through regulation of a cyclic adenosine monophosphate (cAMP)-mediated cascade. Free fatty acid (FFA) metabolism plays an important role in maintaining glucose homeostasis both in the postabsorptive and absorptive states. Adipose tissue is highly sensitive to insulin. The meal-stimulated increase in plasma insulin inhibits lipolysis and FFA release from adipose tissue. Physiologic elevation of FFAs enhances hepatic gluconeogenesis and inhibits glucose uptake and utilization in insulin-sensitive tissues. Therefore, insulin action on adipose tissue affects glucose metabolism in liver and muscle, and in the mealtime suppression of FFA release contributes to the increase in peripheral glucose uptake and utilization (9–11).

MEASURING INSULIN SECRETION AND b-CELL FUNCTION Measurement of Insulin Secretion Insulin secretion is markedly influenced by the route of glucose administration. When glucose is administered via the gastrointestinal tract, a much greater stimulation of insulin secretion is observed compared with similar hyperglycemia created with intravenous glucose. The difference in insulin secretion between intravenous versus oral glucose administration is referred to as the incretin effect and is mediated by glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) (12). Insulin secretion in response to intravenous glucose also differs from oral glucose in its temporal pattern. Following glucose ingestion, there is a gradual rise in plasma glucose concentration reflecting the slow rate of glucose absorption, and this is accompanied by a gradual increase in plasma insulin. The abrupt rise in plasma glucose following intravenous administration causes a rapid and transient increase in plasma insulin concentration (first-phase insulin secretion), which lasts for 10 min. This is followed by a slower, sustained rise in plasma insulin (second-phase insulin secretion), which persists as long as plasma glucose remains elevated (13). The hyperglycemic clamp is considered the gold standard for measuring first- and second-phase insulin secretion. Intravenous glucose tolerance test (IVGTT) has been widely used to assess insulin secretion. The acute insulin response (AIR) (0–10 min) correlates with first-phase insulin response during the hyperglycemic clamp (14). A disadvantage of IVGTT is that the plasma glucose concentration declines rapidly following glucose injection, precluding any second-phase insulin secretory response measurement. Indexes of insulin secretion derived from oral glucose tolerance test (OGTT) provide an estimate of insulin secretion during the more physiological route of glucose administration. The insulinogenic index (increment in plasma insulin increment in plasma glucose) during the first 30 min of the OGTT has been used widely in epidemiological studies as a surrogate measure of first-phase insulin secretion, although not extensively validated (15). In clinical studies, insulin secretion is evaluated by measuring plasma insulin or C-peptide response to oral or intravenous glucose. The amount of insulin secreted must be related to the increment in plasma glucose concentration, which provides the

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stimulus to b-cells. In normogluco-tolerant subjects, the amount of insulin secreted in response to glucose correlates inversely with peripheral insulin sensitivity (16). Reduced insulin sensitivity, through as-yet unidentified mechanisms, enhances plasma insulin response to any given glucose stimulus. Therefore, if one aims at comparing b-cell function between subjects with different insulin sensitivity, an insulin secretion/insulin resistance index (disposition index) should be used (17).

The Hyperbolic Sensitivity–Secretion Relationship To understand the role of b-cells, it has been useful to elucidate the quantitative relationship between insulin sensitivity and insulin action as it exists in nondiabetic individuals. Some years ago, R. Bergman postulated that, if b-cell function was normal, the sensitivity-secretion relationship could be expressed more efficiently as a rectangular hyperbola (18). The product of insulin sensitivity and insulin secretory response (insulin sensitivity index first-phase insulin response to glucose stimulation) would equal a constant, which was named the “disposition index (DI).” Based on a limited data set obtained in human volunteers, this author postulated that shifts in insulin sensitivity would be accompanied by compensatory alterations in b-cell sensitivity to glucose. The single parameter, DI, can thus be envisioned to predict the normal b-cell response adequate for any degree of insulin resistance. The DI is thus a measure of the ability of b-cells to compensate for insulin resistance. It can be considered a measure of pancreatic functionality in nondiabetic individuals (17). S. Kahn et al. were the first to confirm the hyperbolic relationship in a cohort of 96 nondiabetic subjects. They assessed the relationship between insulin response to intravenous stimuli and insulin sensitivity by quantifying these two variables in a large cohort of healthy subjects of less than 45 years of age (16). The nature of this relationship implies that the product of insulin sensitivity and insulin response is a constant for a given degree of glucose tolerance. This hyperbolic relationship exists whether the insulin response is examined following intravenous administration of glucose or nonglucose insulin secretagogues. Based on these analyses, it is apparent that the variations in insulin release in response to differences in insulin sensitivity are due to changes in the secretory capacity of b-cells rather than their sensitivity to glucose (19). Additional confirmations have emerged from studies with large cohorts (20). It appears that the proposed relationship provides a quantitative and convenient approach to expressing normal metabolic functionality in vivo.

ALTERATIONS IN INSULIN SECRETION KINETICS IN T2DM Alterations in Pulsatile Insulin Release In nondiabetic subjects, when endogenous insulin secretion is experimentally abolished by somatostatin infusion, pulsatile insulin administration is more effective in controlling glycemia than its continuous administration (21). Moreover, in T1DM patients, pulsatile insulin administration compared to continuous subcutaneous

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administration is associated with a 40% reduction in insulin doses needed to maintain normal glycemic control (22). The lower efficacy of continuous administration is related to the downregulation of insulin membrane receptors. Pulsatile insulin release is related to oscillations in intracellular Ca2 þ concentrations, which regulate exocytosis of insulin granules (5). Lack of oscillatory secretion can be caused by excessive intracellular Ca2 þ concentrations and may alter islet pattern (23). Prolonged exposure of islets to high Ca2 þ concentrations has been shown to be associated with apoptotic signals in b-cells (5). b-Cell “pace-maker” is severely altered in T2DM patients where reduction or absence of rapid secretory peaks is observed, and these abnormalities are present in the early phases of the disease (24–27).

First-Phase Insulin Secretion in Initial Stages of T2DM or FirstDegree Relatives At the time of diagnosis of T2DM, first-phase insulin secretion is abolished (9, 28–30), and the late phase is reduced and delayed. Reduction in first-phase insulin secretion takes place early in the course of the disease, as it has been reported in subjects with impaired glucose tolerance (IGT) and IFG (31) as well as normoglycemic first-degree relatives of patients with T2DM (32). The abolition of first-phase insulin secretion has been found not only in patients with overt T2DM but also at the initial stage of the disease such as IGT and impaired fasting glucose (IFG). The abolition of first-phase insulin secretion predicts further conversion of IGT or IFG to overt diabetes. Therefore, use of first-phase insulin secretion as a marker of T2DM has been proposed by some researchers. The decrease in first-phase insulin secretion after intravenous glucose in patients with mild abnormalities of glucose tolerance has been reported well before IGT received its definition by the World Health Organization (33). Long-term follow-up studies of patients with IGT have demonstrated conversion from IGT to T2DM in more than 50% of the cases. Thus, IGT should be considered as an at-high-risk state for development of T2DM. Most of the studies performed in patients with IGT revealed abolition or decrease in first-phase insulin secretion (32–35).

QUANTITATIVE AND QUALITATIVE ALTERATIONS IN INSULIN SECRETION In T2DM, a marked decrease in basal and glucose-stimulated plasma insulin concentration has been reported (36, 37) regardless of whether body mass index (BMI) is increased. Specific measurement of insulin and prohormones in T2DM patients by an immunoradiometric assay according to Hales coworkers (38) revealed true insulin deficiency. This defect is masked in T2DM patients by elevated circulating insulin propeptides equally represented by proinsulin 32–33 and 64–65. These peptides account for more than 40% of the circulating peptides compared to 5% in nondiabetic subjects (38, 39). Excess in proinsulin in T2DM is not a consequence of hyperstimulation of b-cells as it is devoid of the states of

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secondary hyperinsulinemia such as obesity (40) and liver diseases (41). It seems to indicate a diseased b-cell state rather than an altered functional state.

PROGRESSION OF ABNORMALITIES OF INSULIN SECRETION Progression of Abnormalities of Insulin Secretion When Progressing from Initial Steps to Overt T2DM Longitudinal studies evaluating both early phase insulin secretion and insulin sensitivity have shown that defects in both functions can predict the development of overt diabetes (42, 43). Longitudinal studies have shown that the transition from normal glucose tolerance (NGT) to diabetes is associated with a progressive deterioration in early insulin secretion. Pima Indians, progressing to IGT during a mean 5.1 year followup, were compared to subjects who remained NGT. Progression to IGT was accompanied by a 27% reduction in AIR. A further 51% reduction in AIR was observed during progression from IGT to diabetes. Increases in body weight and a 31% decrease in insulin sensitivity were also observed in patients progressing to diabetes. In contrast, in patients who remained NGT, while a similar decrease in insulin sensitivity was observed, AIR increased by 30%. This compensatory effect of insulin resistance by the b-cells explains the absence of progression of these subjects to diabetes. Similar conclusions can be drawn from a long-term (7–9 years) longitudinal study performed in normoglycemic relatives of patients with T2DM (44). b-Cell function, evaluated by determining DI values, decreased by 38% in subjects who progressed from NGT to IGT but by only 20% in subjects who remained NGT.

Progression of Abnormalities of Insulin Secretion in Overt T2DM Over Time Worsening of insulin secretion deficiency with time is a characteristic of overt T2DM. This gradual reduction is evidenced by longitudinal studies of large cohorts (45, 46). Studies in the control group of the UKPDS indicated that residual insulin secretory capacity was decreased by 50% at the time of diagnosis of diabetes with a further decrease of 15% 6 years later (45). This decrease was linear at least during the 6 year follow-up period. If one extends the line toward the left as a way to hypothesize disease progression in the past, the actual beginning of the disease may have happened 10 years ago. This extrapolation is consistent with the results drawn from the retinal status at the time of T2DM diagnosis according to Harris et al. (47). If one extends the line toward the right as a way to predict potential progression in the future, the line crosses the abscissas axis 10–12 years after the date of T2DM diagnosis. Thus, these data suggest that the natural history of progressive b-cell death has a length of 20–25 years. Different mechanisms have been proposed to explain the progressive reduction in insulin secretion, including glucotoxicity (48), lipotoxicity (49), and the effects of advanced glycation end products (AGEs) (50, 51). The deposition of an islet amyloid substance, also known as amylin (52), may also play a role.

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MECHANISMS OF b-CELL FAILURE Glucose-Stimulated Insulin Secretion and Glucotoxicity In pancreatic b-cells, glucose is transported across cytoplasmic membrane via specific transporters, glucose transporter 1 (GLUT1) and 2 (GLUT2), and is rapidly phosphorylated by a specific glucokinase with a high Km for glucose. The combination of transport and phosphorylation determines metabolic flux through glycolysis in b-cells. Increased glycolytic flux in b-cells results in a rapid increase in the production of reducing equivalents and increased electron transfer to the mitochondrial matrix, leading to increased ATP production in mitochondria and increased ATP/ADP ratio in the cytoplasm. This in turn results in several sequential events: the closure of the ATP-sensitive K þ (KATP) channels, depolarization of the cytoplasmic membrane, influx of extracellular Ca2 þ , a rapid increase in intracellular Ca2 þ , and activation of protein kinases, which then mediate exocytosis of insulin (53, 54). As glucose is the key physiological regulator of insulin secretion, it appears possible that it also regulates the long-term adaptation of insulin production by regulating b-cell turnover. However, it is important to stress that in human b-cells in vitro, graded increase in glucose from a physiological concentration of 5.6 to 11.2 mmol/L and above induces apoptosis. However, in rat islets, the same graded glucose increment decreases apoptosis, indicating that glucose affects the survival of islets differently in these species. This difference has created some confusion in the field (55). It also highlights the importance of genetic backgrounds in glucose sensitivity of the islets. Glucotoxicity of the islets can be defined as nonphysiological and potentially irreversible b-cell damage caused by chronic exposure to supraphysiological glucose concentrations along with the characteristic decreases in insulin synthesis and secretion caused by decreased insulin gene expression (56). Glucotoxicity, on the other hand, implies the gradual, time-dependent establishment of irreversible damage to cellular components of insulin production and consequently to insulin content and secretion (56). Glucose-induced apoptosis in b-cells is probably linked to the relative specificity of this toxicity toward b-cells but not to other islet or most nonislet cell types. The b-cell is extremely sensitive to small changes in ambient glucose. When these changes are of short duration and lie within the physiological range, such as after a meal, they lead to insulin secretion. When changes are of longer duration and more pronounced in magnitude, they could be translated by the b-cell glucose-sensing pathways into proapoptotic signals (57, 58).

Reactive Oxygen Species The toxic role of oxygen species, which are produced in excess in uncontrolled diabetes, is a pertinent explanation of the b-cell apoptosis (59). Long-term hyperglycemia also induces the generation of reactive oxygen species (ROS), leading to chronic oxidative stress because the islets express very low levels of antioxidant enzymes and activity. In b-cells, hyperglycemia induces mitochondrial production of

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superoxides that activates uncoupling protein 2 (UCP2), resulting in a decrease in intracellular ATP/ADP ratio and reduced glucose-stimulated insulin secretion (60). Diabetic islets are characterized by reduction in glucose-evoked insulin secretion, decreased cytosolic ATP and ATP/ADP ratio, abnormal hyperpolarization of the mitochondrial membrane, hyperexpression of UCP2 of complexes I and V of the respiratory chain, and high levels of a marker of oxidative stress, nitrotyrosine (61). These observations support the role of ROS in reduced b-cell function in T2DM. ROS, particularly hydroxyl radicals, interfere with normal processing of the mRNA of pancreas duodenum homeobox-1 (PDX-1), a transcription factor required for insulin gene expression and glucose-induced insulin secretion as well a critical regulator of b-cell survival (55, 56). The generation of ROS and reactive nitrogen species ultimately activates stress-induced pathways such as nuclear factor kB (NF-kB), stress kinases, and hexosamines. Del Guerra et al., using islets isolated from the pancreas of patients with T2DM and matched nondiabetic controls, demonstrated that several functional and molecular defects are present inT2DMislets(62). HeconfirmedthatT2DM isletsrelease less insulin than control islets. This perturbation is accompanied by altered expression of glucose transporters and glucokinase, reduced activation of AMP-activated protein kinase (AMPK) and alterations in some transcription factors regulating b-cell differentiation and function. The levels of oxidative stress markers,such as nitrotyrosine and 8hydroxy-20 -deoxyguanosine, were significantly higher in T2DM than in control islets, and correlated with the degree of impairment in glucose-stimulated insulin release. The addition of glutathione in the incubation medium caused reduction of oxidative stress (as suggested by diminished levels of nitrotyrosine), improved glucose-stimulated insulin secretion and increasedinsulin mRNA expression(62). Thus,Del Guerraetal. concluded that the functional impairment of T2DM islets could be, at least in part, reversible by reducing islet cell oxidative stress (62). It is important to emphasize that in this study the percentage of b-cells was only slightly (10%), although significantly, reduced in diabetic islets compared with control islets (62). As proposed by Robertson et al. (56), if the steady decline in b-cell function in T2DM is attributable in any significant manner to chronic oxidative stress-induced apoptosis but not deterioration in b-cell replication, interference with apoptosis by antioxidants or any other therapy might provide a much needed new treatment approach to stabilize b-cell. Excessive ROS not only damage cells directly by oxidizing DNA, protein, and lipids, but also indirectly by activating stress-sensitive intracellular signaling pathways such as NF-kB, p38 MAPK, JNK/SAPK, hexosamine, and others. Activation of these pathways results in the increased expression of numerous gene products that may cause cellular damage and play a major role in the etiology of the late complications of diabetes. In addition, recent in vitro and in vivo data suggest that activation of the same or similar stress pathways results in insulin resistance and impaired insulin secretion (63).

Lipotoxicity T2DM is associated with dyslipidemia characterized by an increase in circulating FFAs and changes in lipoprotein profile. Acute elevation of FFAs in healthy humans

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induced hyperinsulinemia; there is also an increase in glucose-stimulated insulin secretion after prolonged FFA infusion (48 and 96 h) (64, 65), but not in nondiabetic individuals genetically predisposed to developing T2DM (65). In healthy control subjects, the FFA-induced insulin resistance was compensated by the enhanced insulin secretion, whereas persistently elevated FFAs may contribute to progressive b-cell failure (b-cell lipotoxicity) in individuals genetically predisposed to T2DM. Santomauro et al. (66) demonstrated that overnight administration of the nicotinic acid analogue acipimox lowered plasma FFAs as well as fasting insulin and glucose levels, reduced insulin resistance, and improved OGTT in lean and obese nondiabetic subjects and in subjects with IGTand T2DM. The significant decrease in insulin levels suggested that plasma FFAs support between 30 and 50% of basal insulin levels. A sustained (7 day) reduction in plasma FFA concentrations in T2DM with acipimox was also associated with enhanced insulin-stimulated glucose disposal (reduced insulin resistance), decreased content of intramyocellular long-chain fatty acyl metabolites, improvement in OGTT with a slight decrease in mean plasma insulin levels (67). These data suggest that physiological increases in plasma FFA concentrations in humans enhance glucose-stimulated insulin secretion and are unlikely to be “lipotoxic” to b-cells (11) but may contribute to progressive b-cell failure in at least some individuals who are genetically predisposed to developing T2DM (65). For all studies reported in relation to the FFA–b-cell interaction, it is important to emphasize that the stimulatory effects on glucose-stimulated insulin secretion are physiological in nature, particularly during the fasted-to-fed transition. Circulating FFAs help maintain a basal rate of insulin secretion, keeping adipose tissue lipolysis in check. In rodent islets, increased FFAs have been shown to be proapoptotic in b-cells (68). Exposure of cultured human islets to saturated FFAs such as palmitate is highly toxic to b-cells, inducing b-cell apoptosis, decreased b-cell proliferation, and impaired b-cell function. In contrast, monounsaturated FFAs such as oleate are protective against both palmitate and glucose-induced apoptosis and induce b-cell proliferation. The deleterious effect of palmitic acid is mediated by ceramide-mitochondrial apoptotic pathways, whereas induction of the mitochondrial protein Bcl-2 by oleic acid may contribute to the protective effect of monounsaturated FFAs such as palmitoleic or oleic acids (69). The physiological or pathological significance of the effects of fatty acids and glucose on pancreatic b-cell function is a matter of debate. Although one can reasonably assert that fatty acid-induced b-cell death is clearly a toxic manifestation, their effects on functional parameters such as insulin secretion or gene expression are more difficult to categorize as either beneficial or deleterious responses in a short time frame, although they are clearly deleterious in the long run.

Islet Cell Amyloid The relevance of amyloid deposition in the deterioration of b-cell function has been the subject of debate for many years. Deposits composed mainly of islet amyloid polypeptide (IAPP), also known as amylin, have been reported in up to 90% of T2DM

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individuals compared with 10–13% of nondiabetic counterparts (70). IAPP is a 37-amino acid b-cell peptide that is costored and coreleased with insulin from b-cells in response to insulin secretagogues. Transgenic mice expressing human IAPP (hIAPP) in b-cells were obese and spontaneously developed diabetes characterized by islet amyloid deposition and decreased b-cell mass (71). Prospective studies in these mice support the hypothesis that the mechanism of the decreased b-cell mass is increased apoptosis (70). Alternatively, it is possible that IAPP formation is secondary to the onset of hyperglycemia and not of primary importance in the pathophysiology of T2DM (72). In a recently published review of islet amyloid (73), the authors concluded that in human T2DM, islet amyloidosis largely results from diabetes-related pathologies such as diabetes-associated abnormal proinsulin processing, which could contribute to the destabilization of granular IAPP, and therefore, it is not an etiological factor for hyperglycemia.

REDUCTION IN b-CELL MASS IN T2DM The normal pancreas contains approximately 1 million islets of Langerhans, and each islet includes b-cells (60–80%), a-cells (20–30%), somatostatin secreting d-cells (5–15%), and pancreatic polypeptide secreting cells (PP-cells). As mentioned above, b-cell mass is regulated by apoptosis, hypo- and hyperplasia, replication and neogenesis (74, 75). In other words, regulation of b-cell mass is a dynamic process where the actual mass represents the net balance between replication, growth, and neogenesis on one side and necrosis/apoptosis on the other. The phenomenon is also known as b-cell plasticity and allows adaptation to changes in demand of b-cell function (76). Such process is disrupted in T2DM where functional defects and decreased b-cell mass coexist. Both impaired proliferation and increased apoptosis may contribute to the loss of b-cell mass. Increased apoptosis has been observed in Zucker diabetic fatty (ZDF) rats, an animal model of T2DM (77). In these animals, expansion of b-cell mass in response to insulin resistance was shown to be inadequate. However, no defects in proliferation or neogenesis could be identified, suggesting that excessive rate of cell death by apoptosis could play a major role. It is difficult to distinguish the two mechanisms, cell formation and cell death, from each other in human tissue sections because dead cells are rapidly removed from the islet by macrophages and neighboring cells, making it hard to quantify cell death. Nonetheless, apoptosis is currently believed to represent the main cause for loss of b-cell mass in T2DM. This view is supported by necropsy data where pancreatic tissues from T2DM patients were compared to those from nondiabetic subjects (72). Moreover, elevated activities of apoptotic mediators caspase-3 and -8 have been found in b-cells from islets of T2DM patients (78). Most studies addressing the issue of b-cell loss have concluded that there is a marked reduction in the number of b-cells in postmortem specimens of pancreas obtained at necropsy of T2DM patients. In contrast to the adaptive increases in b-cell mass observed in rodent models of obesity (79) and obese human subjects (80),

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a marked reduction in b-cell mass in patients with T2DM has been reported by numerous groups. Recent data have provided new insights into islet pathology of T2DM and the mechanisms responsible for decreased b-cell mass (72). Pancreatic tissue samples from 124 autopsies have been examined with analysis stratified according to BMI (less or above 27 kg/m2). In this study, 91 obese (41 patients with T2DM, 15 subjects with IGT, and 35 nondiabetic subjects) and 33 lean subjects (16 patients with T2DM and 15 nondiabetic subjects) were included. Relative b-cell volume, frequency of b-cell apoptosis, b-cell replication, and neogenesis (new islet formation from exocrine ducts) were assessed. Compared to weight-matched controls, pancreas from overweight and lean T2DM patients presented with 63 and 41% deficits in relative b-cell volume, respectively. A similar decrease (41%) was observed in subjects with IGT. No difference was seen regardless of what previous T2DM treatments the patients received (diet, sulfonylureas, or insulin). Relative b-cell volume was increased in overweight patients compared to lean ones due to increased neogenesis. b-Cell replication was found to be low in all groups. Neogenesis, while increased in overweight patients, was not different between overweight T2DM patients and nondiabetic subjects, or between the lean T2DM patients and nondiabetic subjects. The most remarkable abnormality observed in islet samples from T2DM patients was increased b-cell apoptosis. Frequency of b-cell apoptosis was increased tenfold in normal weight patients and threefold in overweight patients compared to respective control groups. In this study, islet amyloid was present only in a minority of cases, around 10%, of patients with T2DM or IGT. There are two potential explanations for these results either small islet amyloid pancreatic peptide oligomers (nondetectable by light microscopy) are present and responsible for b-cell loss, or islet amyloid is not crucial in the pathogenesis of T2DM. The authors concluded that b-cell mass is decreased in T2DM due to increased b-cell apoptosis. A confirmation has been provided by recent in vitro data, indicating an increased rate of apoptosis in islets exposed to high glucose concentrations (81). Another recent study (82) quantified the b-cell mass in pancreas obtained at autopsy of 57 T2DM and 52 nondiabetic subjects of European origin. Sections from the body and tail of pancreas were immunostained for insulin. The b-cell mass was calculated from the volume density of b-cells (measured by point-counting methods) and the weight of the pancreas. The pancreatic insulin concentration was measured in some of the subjects. The main findings of this study were that b-cell mass slightly increases with BMI in both nondiabetic and T2DM subjects. On average, b-cell mass is 35–39% lower in T2DM than in nondiabetic subjects and so is the concentration of pancreatic insulin; but the variations of individual values are large. b-Cell mass does not correlate with the age of T2DM subjects at diagnosis of the disease, but decreases throughout the duration of clinical diabetes. Whether the loss of b-cell mass precedes and precipitates the clinical onset of the disease remains uncertain. It is also unclear if it accounts alone for the defects in insulin secretion. Prospective noninvasive studies measuring b-cell mass, insulin secretion, and insulin action in the same individuals are necessary to unequivocally address these questions (83).

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LINKAGE OF REDUCED b-CELL MASS AND DYSFUNCTION The role of reduced b-cell mass in the alterations of insulin secretion that characterizes T2DM has not yet fully elucidated. Assessing the possible contribution of a low b-cell mass to the development of T2DM is not easy. Does one become diabetic when the number of properly functioning b-cells has decreased, or when b-cell function has deteriorated beyond a threshold level needed to maintain normal glucose homeostasis, or both? Longitudinal in vivo measurements of b-cell mass, b-cell function, and insulin action in the same individuals would be needed to unequivocally address the issue. Still, clinical observations and experimental data support a close interrelationship between the two parameters. A large proportion of liver-related pancreatic donors who underwent a 50% pancreatectomy developed diabetes (84). Pharmacological or surgical reduction of b-cell mass in rodents results all in impaired insulin secretion (85). More recently, Matveyenko and Butler (86) carefully analyzed the effect of 50% pancreatectomy in normal dogs and showed that partial pancreatectomy resulted in IFG and IGT. Partial pancreas resection was associated with reduction of both basal and glucose-stimulated insulin secretion. Altogether, these data support a mechanistic role of reduced b-cell mass in the development of alterations in glucose homeostasis and progression toward T2DM. In conclusion, it seems that the major defect leading to decreased b-cell mass in T2DM is inappropriate apoptosis, while new islet formation and b-cell replication are normal. Therefore, therapeutic approaches designed to arrest apoptosis could have quite an impact on prevention and treatment of the disease.

THE COMPENSATION OF INSULIN RESISTANCE BY b-CELLS In nondiabetic controls, b-cell adapts its secretion rate to the level required by insulin sensitivity so that plasma glucose concentrations remain normal. A hyperbolic relation has thus been observed between insulin secretion and sensitivity in nondiabetic subjects (42). In uncomplicated obesity, insulin resistance is compensated by increased b-cell mass and insulin hypersecretion (19, 87). If compensation is absent or even incomplete, plasma glucose concentrations rise gradually defining the incipient stages IFG or IGT and then overt diabetes. Inability of the b-cell to adjust its secretion rate to increased insulin demand explains why glucose intolerance appears in the physiological setting of aging (88) and gestational diabetes.

ORIGIN OF b-CELL DYSFUNCTION As indicated above, b-cell dysfunction is present at the early stages of the disease, that is, IFG or IGT, and in normoglycemic first-degree relatives of patients with T2DM (32, 89, 90). These results rule out the hypothesis of a hyperinsulinemic state preceding T2DM, which was evoked from findings using nonspecific insulin assays (over-estimating “true” insulin concentrations), or from pseudo-longitudinal

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studies describing the “Starling curve of the pancreas.” But plasma glucose levels and insulin-sensitivity status were not taken into account in these studies.

Impacts of Genetics and/or Environment Studies of genetic susceptibility for T2DM in families revealed high concordance for T2DM in monozygotic twins compared with a lower occurrence in heterozygotic ones (80–90% compared to 40–50%). There is also a high frequency of T2DM in subjects with family history of diabetes (50% if both parents affected; 25–30% if only one firstdegree relative is affected). In monogenic subtypes of diabetes (MODY or MIDD), insulin deficiency is predominant. However, these subtypes represent only a minority of T2DM. In the recent years, multiple loci associated with T2DM have been discovered by gene candidate and/or genome scan strategies in large cohorts of patients and relatives (91). Presently, 18 susceptibility variants for T2DM have been described (92, 93), but their individual weight in the development of the disease is weak. Relative risk for developing T2DM in association with these variants ranges from 1.06 for ADAMTS9 to 1.37 for TCF7L2 (91). In a study aimed at evaluating the risk associated with the 18 loci in a large cohort of Scandinavian subjects with a mean follow-up period of 23.5 years, variants in 11 genes were associated with T2DM. These genes include TCF7L2 (Transcription factor 7-like 2), PPARG (peroxisome proliferator-activated receptor g), FTO (fat mass and obesity), KCNJ11 (ATPsensitive K þ channel), NOTCH2 [Notch homolog 2 (Drosophila, WFS1 for Wolfram syndrome 1; wolframin)], CDKAL1 (CDK5 regulatory subunit associated protein 1like 1), IGF2BP2 (insulin-like growth factor 2 mRNA binding protein 2), SLC30A8 [solute carrier family 30 (zinc transporter) member 8], JAZF1 (JAZF zinc finger 1), and HHEX (hematopoietically expressed homeobox) (94). Among these 11 variants, 8 were associated with alterations in insulin secretion. Recently, a major type 2 diabetes susceptibility gene, TCF7L2, which accounts for 20% of all cases, was identified by Grant et al. in Icelanders (95). Studies conducted in European Caucasian, Asian Indian, and Afro-Caribbean populations of both sexes have confirmed the ubiquitous distribution of the association (96). TCF7L2 is associated with alterations in insulin secretion. Genotype–phenotype relationship studies disclosed severely impaired insulin secretion in carriers of T2DM susceptibility variants (97). Nongenetic factors, particularly insufficient supply of nutrients during fetal development and the first years of life, may also be involved in a defective development of the islets. This defect may result in a reduced b-cell mass, and/or a reduction in the ability to compensate when insulin resistance is present in cases of pregnancy, overweight or obesity, low physical exercise levels, and aging. In this respect, Hales et al. have shown that subjects with birth weight in the lowest quintiles are more prone to IGT and T2DM in adulthood (98). Barker coworkers proposed that T2DM associated with a low birth weight could be the consequence of impaired b-cell function. This may result from in utero undernutrition during a critical period of fetal life and lead to abnormal development of the endocrine pancreas. This hypothesis has

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been supported by studies using animal models (99). If rodents are subjected to an overall reduction in maternal food intake (50% of the normal daily ration) during the last week of pregnancy and throughout the lactation, the offspring showed intra utero growth restriction. They were born with a reduced b-cell proliferation rate. Moreover, these alterations have consequences during adulthood. Inadequate pancreatic functions can develop in situations of increased insulin demand such as ageing or pregnancy. Further, fetal (or in utero) programming is associated with deterioration in glucose tolerance, insulinopenia, and b-cell mass reduction (100, 101). In humans, Barker reported that low birth weight was associated with defective insulin secretion in 21 year old adults during OGTT (102). But these data are not confirmed by other groups. A pathological study has shown that small gestational age does not alter fetal pancreas development and morphology in comparison to appropriate growth for gestational age (103). In a case study comparing young adults born SGA or appropriate for gestational age, subjects born SGA did not demonstrate any evidence of impairment of either the first- or the second-phase insulin secretion (104). Using another model, Flanagan et al. reached the same conclusion in a different adult cohort (103). In 8 year old Indian children, low birth weight is associated with insulin resistance without abnormality of insulin secretion (105).

CONCLUSIONS Substantial evidence supports the view that T2DM is a heterogeneous disorder. There is a progressive deterioration in b-cell function over time in T2DM. The UKPDS indicated that pancreatic islet function has been found to be at about 50% of normal capacity at the time of T2DM diagnosis regardless of the degree of insulin resistance. The decline of b-cell function is the limited capacity to compensate for insulin resistance. Both insulin resistance and b-cell dysfunction are usually present in classical T2DM as well as most individuals with IGT. The defect of insulin secretion in T2DM is related to two confounding components, insulin deficiency and b-cell secretory defect. On the other hand, there is an impaired glucose sensing in the b-cells. The reduction of b-cell mass is attributable to accelerated b-cell apoptosis. Identification of the factors conferring susceptibility (mainly genetic) and those that may accelerate such process (mainly environmental and metabolic) represents a major imperative, which could lead to new therapeutic strategies of slowing if not arresting b-cell loss, thus contributing to more robust glycemic control and reduction of the risk of developing long-term diabetic complications. Although difficult, with the new understanding of b-cell biology, it seems that we may be a little bit closer to a solution.

REFERENCES 1. YALOW, R.S., and S.A. BERSON. 1960. Immunoassay of endogenous plasma insulin in man. J Clin Invest 39:1157–1175. 2. DINNEEN, S., J. GERICH, and R. RIZZA. 1992. Carbohydrate metabolism in non-insulin-dependent diabetes mellitus. N Engl J Med 327:707–713.

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Adipokine Production by Adipose Tissue: A Novel Target for Treating Metabolic Syndrome and its Sequelae VANESSA DECLERCQ2,3, DANIELLE STRINGER2,3, RYAN HUNT2,3, CARLA G. TAYLOR2,3, AND PETER ZAHRADKA1,2,3 1

Department of Physiology, University of Manitoba, Winnipeg, Canada Department of Human Nutritional Sciences, University of Manitoba, Winnipeg, Canada 3 Canadian Centre for Agri-Food Research in Health and Medicine, St. Boniface Hospital Research Centre, Winnipeg, Canada 2

INTRODUCTION The topic of this chapter is adipokines, hormones produced and secreted by adipose tissue that have multiple effects on metabolism. In normal healthy adults, adipokines regulate the utilization and storage of lipids and help to coordinate their distribution throughout the body. Glucose metabolism is also modulated by various adipokines. Additionally, certain adipokines can influence the functions of specific target tissues, of which the heart, vasculature, brain, pancreas, and liver are included in this chapter. For instance, adipokines have a major role in maintaining the normal function of vascular tissues, independent of its metabolic state. Thus, when adipokine production is altered by obesity, a variety of changes can ensue, involving one or more of these tissues. For this reason, therapeutic strategies to correct adipokine imbalance will not only be useful for treating metabolic disorders, they will also help to minimize the end organ damage.

Metabolic Syndrome: Underlying Mechanisms and Drug Therapies Edited by Minghan Wang Copyright 2011 John Wiley & Sons, Inc.

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We have attempted to provide an overview of the role of adipokines and described the changes that occur in obesity as they relate to the metabolic syndrome (MetS). As well, we have outlined the state-of-the-art surgical, lifestyle, dietary, genetic, and pharmacological strategies available for manipulating adipokine levels. This provides a platform for understanding the impact that may be achieved by interventions designed to influence adipokine production, such as lifestyle, diet, surgery, and pharmaceuticals.

MetS AND OBESITY MetS refers to a cluster of metabolic abnormalities characterized by the coexistence of abdominal obesity, dyslipidemia, hypertension, insulin resistance, and glucose intolerance (1). It has been recently proposed that inflammation and nonalcoholic fatty liver disease (NAFLD) also be considered as elements of MetS (2). Although obesity and insulin resistance are generally recognized by different health organizations as central characteristics of MetS, the diagnostic criteria are variable (3). Several health organizations, including the World Health Organization, the National Cholesterol Education Program, the European Group for the Study of Insulin Resistance, and the American Association of Clinical Endocrinology have proposed their own diagnostic criteria for MetS; consequently, it has been difficult to estimate its true prevalence. Based on the criteria of the National Cholesterol Education Program and the results of the Third National Health and Nutrition Examination Survey, it was estimated that 47 million people in the United States have characteristics of MetS (4). In Canada, estimates reach as high as 26% of the population, or roughly 8 million people; however, the prevalence among ethnic subsets of the population range from 11% in the Inuit to as high as 45% in First Nations people (5). Furthermore, the prevalence of MetS among adolescents is increasing. Based on data collected from the Fourth National Health and Nutrition Examination Survey (1999–2000), the overall prevalence of MetS was 6.4%, or approximately 2 million adolescents, representing an increase of 2.2% from the period of 1988–1994. The rising prevalence of MetS is likely attributable to increasing obesity rates. Regardless of definition or diagnostic criteria, older age, reduced physical fitness, and higher percentage of body fat are associated with increased risk of MetS (5). In 1979, 13.8% of Canadian adults were obese. Results from the recent Canadian Community Health Survey reveal that, presently, 36.1% of adult Canadians have a body mass index (BMI) between 25 and 30, while 23.1% have a BMI greater than 30 (6). Globally, approximately 1.6 billion people are overweight, and 400 million people are obese (World Health Organization, 2005), and these individuals are at increased risk of developing MetS. In addition to obesity, MetS is considered a risk factor for diabetes, insulin resistance, and cardiovascular disease. Although all three conditions are interrelated, they are linked to distinct organ systems. The liver, pancreas, heart, and blood vessels, in particular, are affected by MetS and their failure is ultimately responsible for death (7). While it has long been recognized that obesity has a significant role in MetS,

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only in 1994 with the discovery of leptin, a hormone that is produced by adipose tissue (8), was it possible to begin to understand how adipose tissue exerts its effects on the metabolic state of other tissues.

MetS AND ADIPOSE TISSUE Adipose tissue contributes to both the onset and progression of MetS. Among the factors that influence the emergence of MetS are the total amount of adipose tissue and adipose tissue distribution, both of which affect the endocrine, inflammatory, and metabolic functions of adipose tissue. These changes in adipose properties coincide with adipocyte enlargement, which implicates cellular hypertrophy as the underlying cause. This state has been termed adipocyte dysfunction, and several excellent reviews on this topic have been written recently (9–12). White adipose tissue is the main type of adipose associated with obesity and related pathologies such as insulin resistance, hyperlipidemia, hypertension, coronary heart disease (CHD), and type 2 diabetes (13). Traditionally, adipose tissue was thought to provide cushioning, heat insulation, and energy storage. We now recognize that adipose tissue is an important endocrine organ, secreting adipokines that operate as hormones and paracrine factors that have key roles in modulating glucose and lipid metabolism (14, 15). White adipose tissue consists of adipocytes and adipocyte precursors, vascular tissue, and immune cells, primarily macrophages (13). Adipocytes, which represent the specialized cells of adipose tissue that store lipid, only account for approximately 50% of the cellular content of adipose tissue (9). Mature adipocytes are spherical in shape and vary greatly in size. During the first year of life, proliferation and differentiation of adipocytes is the highest, and these processes slow down considerably during adolescence (16). In adults that maintain energy balance, cell proliferation remains rather stable. However, when energy intake exceeds expenditure, expansion of adipose mass occurs (17, 18), initially by adipocyte hypertrophy and subsequently by hyperplasia (17, 18). Alterations in adipokine production in MetS occur primarily as a result of adipocyte hypertrophy. The metabolic effects of adipocyte dysfunction are manifested primarily through increased leptin secretion and decreased adiponectin secretion, however, secretion of adipokines such as monocyte chemoattractant protein-1 (MCP-1), which attracts macrophages to the adipose tissue (19, 20), is also elevated. Consequently, the increase in adipose tissue mass promotes macrophage infiltration of the adipose tissue, leading to twice as many macrophages in visceral adipose tissue compared to subcutaneous adipose tissue (21). The accumulation of macrophages in white adipose tissue in response to MCP-1 occurs through the CC chemokine receptor-2 (22), however, the extracellular matrix may contribute to macrophage-independent adipose inflammation due to the stress it places on adipocytes as they begin to enlarge (23). The increase in macrophages leads to chronic inflammation of the adipose tissue, since they are the source of many proinflammatory cytokines (20, 24). In fact, it now appears that the interaction between adipocytes

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and macrophages is one of the factors that leads to the hypersecretion of proatherogenic, proinflammatory, and prodiabetic adipokines, and the reduced secretion of protective, anti-inflammatory adipokines from adipose tissue that are characteristic of MetS (20, 24, 25). The blood vessels that permeate adipose tissue also secrete a number of cytokines, but their contribution to MetS has received limited attention. It is well established that vascular function can be altered in response to proinflammatory cytokines that originate from macrophages (26). Thus, adipose inflammation in MetS may be a factor in vascular dysfunction. However, at least one molecule released from vascular tissue, nitric oxide (NO), may also contribute to adipose tissue dysfunction in MetS.

Adipose Tissue: Visceral Versus Subcutaneous Dysregulation of adipokine production and secretion from site-specific adipose depots (subcutaneous and visceral) plays an important role in mediating the insulin resistance, diabetes and cardiovascular disease that accompany MetS (27). Subcutaneous adipose tissue mainly accumulates around the gluteal and femoral regions, whereas visceral adipose tissue is composed of omental and mesenteric adipose (28). However, intra-abdominal adipose appears to have the strongest association with MetS (29–31). The association between visceral adipose and MetS may be linked to an increase in free fatty acids (FFAs) entering the portal circulation (32, 33), and the resultant overabundance of circulating FFAs can in turn contribute to the development of insulin resistance (34, 35). Alternatively, the fact that subcutaneous and visceral adipose tissues demonstrate differences in the production of adipokines (36–40) suggests that they have different roles in modulating metabolism. For example, while leptin mRNA levels are higher in subcutaneous adipose, angiotensinogen mRNA levels are higher in visceral adipose, whereas tumor necrosis factor (TNF)-a mRNA levels are similar in both depots (38). These differences underscore how preferential expression of certain adipokines by different adipose depots can affect metabolism. Leptin Leptin regulates body weight and energy expenditure (41) as well as glucose and lipid metabolism, angiogenesis, immunity, and blood pressure homeostasis (27). Leptin has also been shown to cause vasodilatation in coronary arteries (42), but in obese individuals leptin-induced NO production is impaired due to leptin resistance (42, 43). Circulating levels of leptin are directly related to obesity, with increasing adipose mass associated with increases in serum leptin (44). Subcutaneous adipose produces approximately 80% of the circulating leptin (45), however, expression and secretion of leptin are correlated with cell size in both depots (46, 47). Adiponectin Adiponectin is expressed in adipose tissue. In the circulation, it exists in two forms full-length (primarily as trimers, hexamers, and multimeric complexes) and globular

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adiponectin (48, 49). Adiponectin has been shown to have antiatherogenic (50, 51) and anti-inflammatory (52) properties and is significantly reduced in patients with cardiovascular disease (53) and CHD (54, 55). Hypoadiponectinemia is also associated with the development of obesity-related hypertension (56) and type 2 diabetes mellitus (57). Unlike most other adipokines, circulating adiponectin concentrations are reduced in obesity (especially with increased visceral adipose tissue), type 2 diabetes mellitus, CHD, and MetS (58– 61). Likewise, secretion of adiponectin from adipose tissue is decreased with increased adipose mass (62), with omental adipose secreting more than subcutaneous adipose (63–65). Resistin Resistin is expressed by human adipocytes, but the majority is expressed by the macrophages embedded in adipose tissue (66). Data on levels of circulating resistin in obese humans are very inconsistent (67–69), however, there is good evidence that expression of resistin in adipose tissue is differentially regulated depending on the disease model (obesity, diabetes, and insulin resistance) (70–72). The visceral depot in mice appears to have the highest resistin expression (73). Likewise, Zucker diabetic fatty (ZDF) rats have higher resistin mRNA levels in visceral compared to subcutaneous adipose tissue (39). In humans, similar results have been observed, with abdominal adipose expressing and secreting more resistin than subcutaneous adipose tissue (74, 75). Inflammatory Cytokines Proinflammatory molecules such as TNF-a, C-reactive protein (CRP), and interleukin-6 (IL-6) are increased in the plasma of obese individuals (76–80). Adipose TNF-a levels are increased in obesity (81–83) and TNF-a appears to be produced equally by both subcutaneous and visceral adipose depots in humans (84). In contrast, about 30% of circulating IL-6 in obese individuals originates from adipose tissue, primarily from the visceral adipose (27, 37, 84). Renin-Angiotensin System Angiotensinogen is a major component of the renin-angiotensin system (RAS), and a fundamental regulator of systemic blood pressure. Angiotensinogen is the precursor to the vasoconstrictor angiotensin II (AngII), and thus plays an important role in hypertension (85) and vascular inflammation (86). Angiotensinogen is expressed in adipocytes (14), with higher levels in visceral adipose compared to subcutaneous adipose (38). Adipose tissue also has the ability to produce AngII due to the presence of a tissue-localized RAS (87). Interestingly, an increase in the activity of adipose tissue RAS is observed in individuals with obesity-related hypertension (88).

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Perivascular Adipose Perivascular adipose is the term that is applied to the adipose tissue located around vascular structures, including those present on the heart. A correlation has been shown between epicardial adipose tissue and some of the components of MetS such as waist circumference, diastolic blood pressure, and fasting insulin, but not circulating triglycerides or HDL (89). Rat aortic rings surrounded by perivascular adipose tissue display a lower contractile response compared to aortic rings without perivascular adipose (90). Similarly, vessels that have perivascular adipose such as the mesenteric arteries also show a reduced contractile response (91). Perivascular adipose has recently been shown to produce a variety of adipokines and be involved in vascular inflammation. For example, in epicardial adipose (includes surface of the heart especially around the coronary arteries), adiponectin mRNA levels are lower in patients with CHD (92, 93). In humans, the thickness of epicardial adipose tissue correlates with abdominal visceral adipose tissue and fasting insulin, and it is thought to behave like visceral adipose tissue (89). IL-6 and plasminogen activator inhibitor-1 (PAI-1) are higher in abdominal omental adipose compared to epicardial adipose, whereas leptin levels in subcutaneous abdominal adipose are higher than in epicardial adipose (93). More recently, Cheng et al. (94) found that adiponectin levels were lower in abdominal adipose compared to epicardial adipose, whereas TNF-a, IL-6, leptin, and visfatin were higher in abdominal compared to epicardial fat. In a recent study of subcutaneous, visceral, and perivascular adipose, Chatterjee et al. (95) reported that mice had higher levels of adiponectin and leptin in abdominal (epididymal) adipose compared to subcutaneous adipose, whereas levels of adiponectin and leptin were lower in perivascular adipose compared to subcutaneous adipose. When mice were fed a high fat (42% energy) diet for 2 weeks, leptin levels increased in all tissue depots but adiponectin levels were significantly reduced only in the perivascular adipose compared to chow fed animals (95). In primary human adipocytes, release of inflammatory mediators such as IL-8, IL-6, and MCP-1 was highest, and leptin and adiponectin secretion was lowest in cells from perivascular adipose relative to subcutaneous and visceral (peri-renal) adipose (95). Interestingly, perivascular adipocytes were smaller in size than adipocytes from subcutaneous or visceral adipose, a finding that correlated with a reduction in lipid droplet accumulation (95). While adipose tissue has a significant role in MetS, the morbidity associated with MetS is the result of changes in the properties of tissues that are targets for the actions of adipokines. Thus, while alterations in adipokine production may be a prime factor in the onset of MetS, the response of these target tissues is likely responsible for MetS progression.

MetS AND THE LIVER: NONALCOHOLIC FATTY LIVER DISEASE The term nonalcoholic fatty liver disease encompasses a spectrum of hepatic disorders, beginning with simple hepatic steatosis characterized by intracytoplasmic

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lipid droplets within hepatocytes (96). Inflammation and necrosis of hepatocytes marks the progression to the second stage, nonalcoholic steatohepatitis (NASH). Further inflammatory damage leads to fibrosis, with half of NASH patients progressing to this stage (97). Fifteen percent of patients with fibrosis advance to cirrhosis (97). If not detected and treated, cirrhosis can cause portal hypertension, hepatocellular carcinoma, and even liver failure (98, 99). It is estimated that 3% of patients with NAFLD develop liver failure or require liver transplantation (97). Insulin resistance is a precursor for the development of NAFLD. It is estimated that up to 75% of patients with type 2 diabetes mellitus have some form of NAFLD, and past history of type 2 diabetes mellitus is associated with a 26-fold increase in the risk of steatohepatitis (100, 101). Ninety-eight percent of patients with NASH are insulin resistant, and 87% exhibit attributes of MetS (102). Persons with type 2 diabetes mellitus and fatty liver have substantially higher insulin resistance than those with diabetes but without fatty liver (103). Studies have also shown that insulin resistance, elevated serum triacylglycerol (TAG) levels, and hyperinsulinemia are associated with NAFLD, regardless of body weight and BMI (104). Although there is strong evidence for an association among obesity, insulin resistance and NAFLD, nondiabetic and/or normal weight patients with NASH can also exhibit markers of insulin resistance (105). Obesity is also closely correlated with NAFLD, as the risk and severity of hepatic steatosis and steatohepatitis in obese patients is proportional to the degree of obesity (101). Although BMI and hepatic fat content are positively correlated, the relationship between waist circumference and hepatic fat content is stronger, highlighting a role for visceral adiposity in the development of hepatic steatosis (106– 108). It has been suggested that 30–40% of the variation in hepatic fat content can be explained by the variability in visceral adipose tissue (103). Hypertrophy of visceral adipose tissue and the resulting inflammatory response is a potential explanation for the strong relationship between visceral adipose tissue and fatty liver (109). The inflammatory response initiated by expanding visceral adipose tissue recruits macrophages which then secrete various proinflammatory cytokines. Prolonged exposure of adipocytes to these proinflammatory cytokines induces insulin resistance and leads to impaired insulin-mediated suppression of lipolysis. Consequently, there is an increased flux of FFAs from the visceral adipose tissue into the portal vein, resulting in direct delivery of fatty acids to the liver (110). Once they reach the liver, these FFAs can then be taken up by hepatocytes and bound to coenzyme A (CoA). The fatty acyl-CoAs can form hepatic TAG, but they can also interfere with insulin signaling and cause hepatocyte insulin resistance (111). In addition to increased release of FFAs, altered production and release of adipokines by hypertrophic adipose tissue represents another possible link between obesity and hepatic steatosis (112). In particular, increased production of IL-6 and TNF-a (20, 24, 113) can suppress the production of adiponectin, an anti-inflammatory adipokine that appears to have an important role in the development of hepatic steatosis (114). The relationship between adiponectin and hepatic steatosis is highlighted by the strong inverse relationship between circulating adiponectin and hepatic fat content (115, 116) as well as hepatic insulin resistance (116). In addition,

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genetic variability in the adiponectin receptor gene affects hepatic fat accumulation, supporting the important role of adiponectin signaling in the pathophysiology of hepatic steatosis in humans (117). However, the mechanism has yet to be elucidated.

Adipokines and NAFLD Much of our knowledge of the contribution of adipokines to the development of hepatic steatosis is derived from studies in obese and/or insulin resistant animal models, where it is evident that certain adipokines are prosteatotic, while others are antisteatotic. Expression of hepatic TNF-a is elevated in the ob/ob mouse, a model which develops obesity and insulin resistance due to a mutation in the leptin gene (118). Fatty liver, which is characteristic of this mouse model, is reversed upon treatment with a neutralizing TNF-a antibody (119), suggesting a likely role for TNFa in the development of hepatic steatosis. However, the actual therapeutic potential for modulating TNF-a expression may be limited, as circulating levels of this adipokine do not reflect expression in tissue (120). Conversely, ob/ob mice given adiponectin display reduced hepatomegaly, hepatic lipid content, serum alanine transaminase (ALT), TAG, and FFA after only 2 weeks of treatment (121). In humans (both adults and children), plasma concentrations of adiponectin are significantly lower in patients with NAFLD compared to both obese and healthy people (122–125). Furthermore, plasma adiponectin concentration is inversely associated with hepatic insulin sensitivity and hepatic lipid content (116). The amelioration of hepatic steatosis by adiponectin is hypothesized to occur via three possible mechanisms:stimulation of lipid oxidation via activation of AMP-activated protein kinase (AMPK), suppression of lipogenesis by decreasing activity of key lipogenic enzymes such as acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), and activation of peroxisome proliferator activated receptor-a (PPARa), a transcription factor involved in lipolytic gene expression (114, 121, 126). Leptin is considered another important regulator of hepatic fat, although the mechanisms responsible for the protective effect of this adipokine are not fully understood. The observation that leptin deficiency leads to hepatic steatosis, which is reversible upon leptin treatment, suggests a protective role for this adipokine against hepatic fat accumulation (127). Even more convincing evidence for a direct effect of leptin signaling in the process of hepatic lipid deposition comes from studies in the ZDF rat (128), which lacks a functional leptin receptor. When infused with a recombinant adenovirus containing the gene for a functional leptin receptor, hepatic steatosis is markedly decreased and, since almost all of the infused functional leptin receptor construct is taken up by the liver, the liver becomes the only leptin-responsive tissue (128). Therefore, any reduction in hepatic lipid content resulting from infection with the normal leptin receptor is due to the direct action of endogenous hyperleptinemia on the now leptin-responsive liver. Potential mechanisms responsible for this protective effect include stimulation of lipid oxidation by activating AMPK (129), reduced lipid synthesis, and increased lipid export (130). Development of leptin resistance due to chronic exposure of hepatocytes to high levels of circulating leptin is

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the likely explanation for hepatic steatosis despite elevated leptin in obesity. An interaction between resistin and leptin could also be responsible for the development of hepatic steatosis, since a cross between resistin-null and ob/ob mice does not exhibit fatty liver even though the degree of obesity is equivalent to that of ob/ob mice (131). Over time, hepatic steatosis will progress to steatohepatitis and finally fibrosis. The latter represents the point at which cirrhosis becomes evident. Hepatic stellate cells are normally found in a quiescent state in the healthy liver and become activated in response to liver injury. When this occurs, they begin to secrete collagen, which, if not stopped, is ultimately the cause of fibrosis. Evidence that adipokines influence fibrosis is now accumulating (132). Adiponectin, in particular, may have a protective role because it can suppress stellate cell activation by platelet-derived growth factor (PDGF) and connective tissue growth factor (CTGF) (133). In contrast, the proinflammatory effects of resistin may promote disease progression (134).

MetS AND THE PANCREAS The metabolic consequences of MetS on the pancreas are not as well defined as those of other organs such as liver and skeletal muscle. In one study of 104 adults with MetS, fatty pancreas, as detected by sonography, was present in 77% of the participants (135). Interestingly, coexistence of fatty pancreas and fatty liver were observed in 68% of the participants. After adjustment for age, BMI and lipid profile, fatty pancreas was independently related to insulin resistance (HOMA-IR), visceral adipose and ALT; furthermore, the number of MetS characteristics was significantly higher in the fatty pancreas group compared to the nonfatty pancreas group. Although increased pancreatic fat content has been negatively correlated with b-cell function (136), the relationship between pancreatic fat content and reduced b-cell function remains controversial (137), as other research has observed hypersecretion of insulin and no detrimental effects on functional characteristics of b-cells in the presence of pancreatic fat deposition (138). In humans, histological examination of fatty pancreas has revealed that the majority of fat is present in adipocytes within the exocrine tissue or in adipose tissue within the interlobular space, not within the islets themselves (139). This is contrary to what is observed in animal models such as the ZDF rat and high fat/high sucrose-fed swine, where higher pancreatic fat is associated with fibrotic, irregular, atrophied, and vacuolar islets containing lipid droplets and reduced insulin content (140). Adipokines likely influence pancreatic function, but few studies have examined this link. For instance, it was recently shown that the adiponectin type 1 receptor (AdipoR1) is reduced in the pancreas of obese mice (141). Leptin can inhibit glucagon release by a-cells in culture (142). Circulating leptin, but not adiponectin, levels also appear to be altered in acute pancreatitis, but a causal relationship has not been identified (143). Proinflammatory adipokines such as IL-6 and MCP-1 have also been reported to participate in the pathogenesis of pancreatitis (144).

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MetS AND THE BRAIN The effects of MetS on the brain result in changes in appetite, both in the fed and fasted states. The brain responds to short-term updates on food intake from gut-derived hormones such as cholecystokinin, peptide YY, and ghrelin while signals of long-term adiposity come from adipose tissue via the adipokine leptin (145, 146). Abdominal obesity, elevated serum leptin levels, and hypothalamic leptin resistance are common features of the MetS (147). Instead of the normal reduction in food intake and increase in energy expenditure that should accompany increased leptin secretion from adipose tissue, a leptin resistant state inhibits the amplified satiety message from reaching the hypothalamus. The leptin signal is only as strong as what is able to cross the blood–brain barrier, a process mediated by the short-form leptin receptor (ObRa). The leptin resistant state results in hypothalamic leptin insufficiency despite elevated blood levels (148). A decrease in central leptin levels affects pancreatic insulin secretion, energy expenditure and glucose metabolism as a result of impaired hypothalamic signaling (148, 149). These changes, in turn, can lead to obesity, hyperglycemia, hyperinsulinemia, and hypertriglyceridemia. This phenotype is mirrored in the db/db mouse model of type 2 diabetes mellitus, which lacks a functional leptin receptor. Impaired peripheral leptin signalling does not significantly alter insulin levels, energy expenditure, or adiposity when compared to impaired central leptin signaling or resistance (150). Hypothalamic neurons in the arcuate nucleus produce the appetite stimulating peptides neuropeptide Y (NPY) and agouti-regulated peptide (AGRP), both of which are inhibited by leptin. A third peptide that is stimulated by leptin, prepro-opionmelanocortin (POMC), is a precursor to a-melanocyte stimulating hormone (a-MHS), which suppresses appetite and leads to increased energy expenditure (151, 152). The disruption in signaling between leptin and NPY may be central to leptin resistance induced by energy rich diets (148). The interaction of the signal transduction and activator of transcription-3 (STAT-3) and the leptin receptor is important in the regulation of energy homeostasis in NPYand AGRP expressing neurons in the arcuate nucleus (153). Restoring the leptin-induced restraint on NPY release may be an important therapeutic target in future treatment of obesity and MetS. Dysregulation of the endocannabinoid system (ECS) along with leptin resistance is linked to abdominal obesity and may exacerbate key risk factors that lead to the development of cardiovascular disease and type 2 diabetes mellitus. Inflammation, an altered blood lipid profile, hepatic steatosis, and insulin and leptin resistance have all been associated with chronic endocannabinoid receptor stimulation. Endocannabinoids are lipid mediators, produced endogenously from membrane phospholipid precursors and triglycerides in response to elevated intracellular calcium (154). The majority of research has focused on two main endocannabinoids, arachidonoyl ethanolamide (or anandamide, AEA), and 2-arachidonoylglycerol (2-AG), which are derived from arachidonic acid (155). AEA and 2-AG are able to mimic the pharmacological effects of D9-tetrahydrocannabinol, the active compound of marijuana that stimulates appetite (156). Both AEA and 2-AG concentrations are elevated in the plasma and adipose tissue of obese humans (156). Diabetes also plays a role, as

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obese type 2 diabetic patients have greater 2-AG levels than nondiabetic weight matched controls (157). Two G protein-coupled ECS receptors are currently known. Cannabinoid receptor 1 (CB1) is highly abundant in the central nervous system, in particular the hypothalamus. CB1 receptors are also found peripherally in liver, gut, skeletal muscle, pancreas, and white adipose tissues. This receptor is linked to increased fat mass, decreased adipocyte proliferation, accelerated adipogenesis, elevated expression of hepatic sterol regulatory element binding protein-1 (SREBP-1), ACC and FAS, and decreased glucose uptake by various peripheral tissues (158). The CB2 receptor is expressed by immune and hematopoietic cells as well as in the pancreas and white adipose tissue. The effect of the CB1 receptor on metabolism, however, is not limited to appetite. Activation of CB1 can affect glucose, insulin, cholesterol, TAG, and leptin levels, all independent of food intake. Upregulation of PPARg mRNA mediated by CB1 activation leads to TAG accumulation in adipocytes (159). Cross talk exists between the leptin and ECS signaling systems, however, it seems to occur downstream of the leptin receptor. Both ob/ob (leptin deficient) and db/db mice, which have elevated levels of AEA and 2-AG in their hypothalamus, show reduced appetite following blockade of CB1 (160, 161). CB1-null mice remain sensitive to intracerebroventricular leptin injection while having relatively low fasting leptin levels when challenged with a high fat diet (162). CB1-null mice are also resistant to diet-induced obesity and weigh approximately 30% less than regular adult mice due to decreased food intake (163).

MetS AND THE CARDIOVASCULAR SYSTEM Numerous epidemiological studies have established a relationship between MetS and cardiovascular disease risk (164). Although the mechanisms responsible for promoting the progression of cardiovascular disease in MetS remain to be identified, a strong case can be made that the onset of insulin resistant and hyperglycemic states, which are exacerbated by obesity, underlies the pathophysiological changes that ensue (165). This concept is supported by the fact that accelerated atherosclerosis and restenosis are highly prevalent in diabetes, even in the absence of obesity (166). On the other hand, evidence is mounting that adipose tissue can influence vascular and cardiac tissues directly via adipokines (10). A clear understanding of the positive and negative actions of these hormones may assist in the development of interventional strategies that can be directed at the adipose tissue and/or the relevant target organs. Although this approach may seem paradoxical, altering adipokine production is now recognized as a plausible means of combating cardiovascular disease.

Diseases of the Heart and Vasculature The majority of cardiovascular disorders stem from either a reduction in the pumping efficiency of the heart or a diminution of blood flow through the vessels. The most

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common cardiovascular disorder associated with MetS is atherosclerosis, a progressive decrease in the diameter of the vessel lumen due to presence of plaque (167). Although the hyperlipidemia component of MetS may feature prominently in these circumstances, there is also evidence that MetS-induced alterations in the levels of various circulating factors may independently influence atherosclerosis. Regardless of the underlying cause, the resultant inability to provide adequate oxygen and nutrients eventually leads to organ failure. Whether this produces an acute (heart attack, stroke) or a chronic (heart failure, renal failure, neuropathy) response ultimately determines the degree of morbidity associated with the condition. Arterial narrowing and stiffening due to atherosclerosis can also affect cardiovascular hemodynamics, which in turn can cause hypertension and an increase in cardiac workload. These conditions promote cardiac hypertrophy, which in time develops into heart failure even in the absence of a heart attack. Certain features of hemodynamic disorders may also be attributed to MetS independent of atherosclerotic disease (167). On the other hand, genetic abnormalities and alterations in electrical conductance are forms of cardiovascular disease that typically are not a consequence of MetS (168), although dietary intake of long-chain omega-3 fatty acids has been shown to reduce the mortality attributable to arrhythmia (169).

Vascular Actions of Adipokines The vascular system serves as a conduit for blood, and thus provides the oxygen and nutrients needed for cells to function. A constant flow rate must therefore be maintained, and this requires tight control of blood pressure within a certain range. Likewise, vessels must be able to repair themselves if they are injured. The vascular response to these stimuli is mediated by various hormones, including adipokines. The incidence of cardiovascular disease in MetS, which is typically associated with higher circulating leptin and lower levels of circulating adiponectin, begs the question whether adipokines are causal factors. Certain studies have linked adipokines to endothelial dysfunction (170), whereas recent prospective studies by Sattar et al. (171, 172) have revealed that neither leptin nor adiponectin are strongly correlated with CHD risk. In fact, the association between leptin and CHD may be a consequence of the close correlation of leptin with BMI (172). These results are supported by an independent assessment of adipokine levels in elderly individuals (173). Indeed, leptin may more accurately predict diabetes than cardiovascular disease (174). The latter observation may clarify the association between leptin and cardiovascular disease, since accelerated atherosclerosis is a hallmark of the diabetic state. Regardless of these results with CHD, it remains plausible that adipokines contribute to other forms of cardiovascular disease. Adipokines have been reported to have multiple vascular effects. The contribution of specific adipokines is described in relation to normal and pathological blood vessel function. Various reviews have been written on this topic (12, 175–177) and we have therefore tried to emphasize the more recent publications and concepts.

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Leptin This hormone was originally identified by its ability to regulate food intake (178). However, evidence that leptin stimulated the sympathetic nervous system led to experiments that showed it was capable of increasing blood pressure (179). More recently, it was shown that leptin receptors are present on cells of the vasculature (180), which suggests that leptin may be able to directly influence their function. This has become an area of intense research, given that leptin levels are elevated in MetS. (a) Vascular Expression. Leptin is primarily secreted by adipose tissue. Other cells that have been shown to express leptin, albeit in small amounts, include fibroblasts and osteoblasts (181, 182). There is no evidence of leptin production by cells of the healthy vasculature, however, Reyes et al. (183) have shown it is produced by sinusoidal endothelial cells. The importance of this localized production has not been determined. Nevertheless, its presence may be sufficient to promote vascular disease progression. The leptin receptor (ObR) has been detected on coronary endothelial cells in culture (184) and on the endothelial and smooth muscle cells (SMCs) of normal vascular tissue (180). These data are consistent with leptin’s ability to directly affect these cells in culture (185, 186). Changes in ObR expression apparently occur during atherogenesis. Schroeter et al. (180) detected a decrease in ObR staining of SMCs of atherosclerotic lesions, whereas strong staining was associated with macrophages. There was no apparent change in endothelial ObR levels. A subsequent study by the same group confirmed that ObR levels in atherosclerotic plaque were associated with macrophage infiltration (187). (b) Vascular Tone. Blood pressure is maintained by a balance of vasoconstrictory and vasodilatory factors. Blood pressure is affected by MetS, as indicated by the prevalence of hypertension in this condition (188). A close association with obesity is evidenced by reports of improvements in blood pressure in conjunction with weight loss (189). The latter may be linked to leptin, which also declines with weight (190), since it has been shown that leptin can act both directly and indirectly to modulate vascular tone. The sympathetic nervous system is a key regulatory system for vascular tone. Leptin is known to act centrally via the hypothalamus to suppress appetite (191). As part of this process, leptin activates sympathetic neurons, and their activation may increase blood pressure (192, 193). The most direct experiment to demonstrate this relationship involved injection of leptin into the ventromedial hypothalamus of healthy rats, which resulted in an increase in blood pressure (194). At the same time, it has been difficult to clearly establish the link between these systems in the hypertensive state (195), and therefore it is not clear whether leptin contributes to hypertension via this mechanism. It is quite plausible that sympathetic activation of the kidney may be the mechanism by which leptin operates. Alternatively, leptin may function by affecting the vascular wall directly.

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Hypertension is closely associated with stiffening of the arterial wall and hypertrophy/hyperplasia of the medial smooth muscle layer. Leptin has been shown to stimulate SMC hypertrophy (196) and proliferation (197), and activation of either process is a prerequisite for medial enlargement. On the other hand, leptin has also been reported to block SMC proliferation (198). Inhibition of cell proliferation may be the result of a leptin-dependent increase in the production of the vasodilator NO via stimulation of endothelial nitric oxide synthase (eNOS) (199). As well, leptin has been shown to interfere with AngII-dependent vasoconstriction (200), a major factor in hypertension. Based on these data, it may therefore be presumed that leptin resistance could result in hypertension due to the lack of NO and loss of vasoconstrictor inhibition, although both processes may be connected (201). Alternatively, leptin may alter the balance between NO and peroxynitrite production (202), thus inducing endothelial dysfunction by increasing the levels of molecules associated with oxidative stress (203). Although more direct links between hypertension and leptin are lacking, there are nevertheless a number of studies that show a correlative relationship between circulating leptin levels and hypertension (190, 204, 205). It is also possible that these actions of leptin are indirect, and are the result of sympathetic activation by leptin (206, 207), which is a feature of MetS (208). Evidence that the vagal afferent nerves are targeted by leptin, thus interfering with baroreflex function and increasing blood pressure, has recently been reported (209). (c) Vascular Injury. The onset and progression of vascular disease is closely linked to the development of endothelial dysfunction, a consequence of injury to the vessel wall. Hyperleptinemia is associated with endothelial dysfunction and arterial stiffening (210). Leptin resistance, however, may serve as an adaptive mechanism to prevent this outcome (210). A major consequence of endothelial dysfunction is activation of the underlying SMCs. This process requires the modulation of SMC phenotype, which switches from the contractile state present in the healthy vessel to the synthetic state that is characterized by migration, proliferation, and secretion of extracellular matrix proteins (211). Similar events occur when the vascular wall is injured, either mechanically (e.g., bypass graft surgery) or by chemical mediators (e.g., hyperlipidemia). The resultant attempt to repair the vessel wall often leads to the formation of a lesion that can interfere with blood flow. Although both atherosclerotic (chronic injury) and restenotic (acute injury) lesions may have different constituents, it is multiplication of SMCs in the intimal space (neointimal hyperplasia) that underlies lesion formation. Leptin promotes neointimal hyperplasia (212), a fact which may explain the higher atherosclerosis rates in diabetes with hyperleptinemia. Leptin likely operates by stimulating SMC proliferation (197). Leptin could promote proliferation by increasing the responsiveness of cells to mitogenic agents. Juan et al. (71) reported that leptin increased endothelin-1 type A receptor

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expression, leading to enhanced proliferation in response to endothelin-1. Although indirect stimulation of SMC proliferation by leptin is supported by the lack of atherosclerosis in mice and rats that do not express leptin or lack a functional leptin receptor, a recent study by Lloyd et al. (213) has shown that atherosclerosis still occurs in a triple knockout mouse that does not express the LDL receptor, ApoE and leptin. This study indicates that a lack of leptin is insufficient to prevent atherosclerosis under conditions where extreme hyperlipidemic conditions exist. An alternate view has been proposed by Bohlen et al. (198) who found that leptin prevents vascular SMC proliferation in vitro. A similar finding was made by Nair et al. (214) with airway smooth muscle. Procopio et al. (199) recently reported that leptin stimulates eNOS expression by endothelial cells via AMPK, which could explain how leptin inhibits cell proliferation. These latter observations do not agree with the findings of Bodary et al. (215). These researchers compared neointimal formation in wild type, leptin deficient (ob/ob), and leptin receptor defective (db/db) mice and found that their inability to respond to leptin protection against formation of a neointimal lesion. Interestingly, lesion formation was equivalent to wild type in mice with a leptin receptor deficient in STAT-3 signalling (leptrs/s), although these animals were as obese as the db/db mice (215). These data suggest that exacerbation of vascular lesion formation by leptin is not a function of STAT-3-dependent signaling, but STAT-3 does mediate the effects of leptin on obesity. (d) Inflammation and Thrombosis. Vascular injury triggers an inflammatory response that results in attachment and infiltration of leukocytes as well as the differentiation of monocytes into macrophages. This process is driven by the release of chemoattractants from the injured endothelial cells and SMCs, and results in the release of inflammatory cytokines that further disturb endothelial function (216). Additionally, the altered surface properties of dysfunctional endothelial cells lead to greater adhesion of leukocytes. This in turn can precipitate formation of a thrombus or blood clot. Leptin can indirectly influence inflammation and thrombosis through an increase in the production of CRP (217). CRP is an acute phase protein that originates primarily from the liver, but sites of extrahepatic production include vascular SMCs and macrophages (218). Elevated levels of CRP in the circulation have been linked to increased thrombosis, possibly as a result of its ability to stimulate the expression of adhesion molecules by endothelial cells. Thus, the resultant increase in leukocyte attachment promotes both progression of atherosclerotic lesion formation and elevation of the risk of thrombosis. In parallel, innate production of CRP by the vascular and inflammatory cells may exacerbate the inflammatory state of adipose tissue and thus intensify the resultant dysfunction caused by inflammation. Leptin also enhances thrombosis by increasing platelet aggregation (219). The leptin receptor (ObRb) is present on platelets (220), and leptin binding

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results in release of intracellular calcium (221). Additionally, leptin enhances aggregation in response to ADP (221). Although leptin has no additional effect on platelet characteristics, and obesity does not trigger leptin resistance in platelets, the increased circulating levels of leptin may be sufficient to explain the increased platelet aggregation observed in obesity (222). (e) Angiogenesis. Tissue hypoxia results in the release of paracrine factors that promote the formation of new blood vessels to perfuse the region that is oxygen deficient. This process can thus enhance recovery after a heart attack by providing new blood vessels to the damaged region of the heart. At the same time, angiogenesis allows the enlargement of atherosclerotic plaques by providing oxygen to the cells that form the core of the lesion. Leptin appears to be a potent proangiogenic factor (223, 224) that operates by promoting endothelial dysfunction and cell proliferation (225). Leptin enhances the rate of angiogenic tube formation through the release of matrix metalloproteinases, enzymes that degrade the extracellular matrix and thus provide channels for elongation of the nascent capillaries (225). Thus, leptin supports the progression of MetS by assisting in the formation of vessels to carry nutrients during the expansion phase of obesity (226). Adiponectin Adiponectin is currently regarded as a potent vasoprotective hormone based on its ability to prevent atherosclerosis (227). Adiponectin likely operates through the endothelial cells since an inverse association exists between circulating adiponectin levels and endothelial dysfunction (228). As such, it is expected that adiponectin will affect a variety of vascular functions. But whether adiponectin functions directly on the vascular tissues or indirectly through induction of other cytokines remains unclear in many circumstances. (a) Vascular Expression. The abundant production of adiponectin by normal adipose tissue greatly exceeds that of other tissues. For this reason, production by other cell types has only recently been recognized. It was shown by Wolf et al. (229) that endothelial cells are capable of secreting adiponectin, at least in certain vascular beds. Interestingly, adiponectin is highly expressed in fetal SMCs (230), but is apparently not found in SMCs of the adult vasculature. Regardless, adiponectin secreted from periadventitial adipose tissue, the adipocytes found around blood vessels, may be more pertinent to its role in vascular function than production by cells of the vasculature itself, and possibly even with respect to circulating adiponectin (231). On the other hand, the primary adiponectin receptors, AdipoR1 and AdipoR2, are expressed by both vascular SMCs and endothelial cells (232). Their importance in vascular disease onset has been established by Zhang et al. (233), who showed that increased expression of the receptors increases the antiinflammatory actions of adiponectin.

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(b) Vascular Tone. An inverse correlation between hypertension severity and adiponectin levels has been identified (190), but a causal relationship remains to be proven. Cao et al. (234) have reported that blood pressure decreases when adiponectin levels increase. Fesus et al. (235) suggest that adiponectin functions as a vasodilator. This effect can certainly be linked to the fact that vascular tone is controlled in part by periadventitial adipose, a major source of local adiponectin (236), and the fact that changes in the properties of periadventitial adipose tissue have been linked to the onset of hypertension (237). The most compelling evidence of a link to blood pressure regulation is the fact that adiponectin induces expression of eNOS and can stimulate production of NO (56). Only one study, however, has examined directly the effect of introducing adiponectin into a hypertensive animal (238). These data suggest adiponectin can influence vascular tone, however, this may be mediated through the central nervous system rather than systemically. (c) Vascular Injury. The primary cause of most vascular disease is failure of the endothelial cell barrier, and this is especially true when blood vessels are injured. Several recent reviews of the effects of adiponectin on the vasculature as it relates to the development of atherosclerotic disease have been published (239–242). However, an interesting question has recently emerged: is a decrease in circulating adiponectin levels a cause of endothelial dysfunction? If this is the case, as suggested by Cao et al. (228), then changes in adiponectin production by adipose tissue may be the causal factor for the onset of cardiovascular disease in obesity. The consequence of the loss of a protective agent such as adiponectin may thus be disease progression. For instance, glucose-induced formation of reactive oxygen species is suppressed by adiponectin in endothelial cells (243). Likewise, secretion of adiponectin is linked to paraoxonase-1 (PON1) (244), a peroxidase that protects LDL from oxidation and is associated both with a reduction in atherosclerotic disease and increased longevity (245). Adiponectin also improves endothelial dysfunction, characterized as a decrease in the response of vessels to factors that trigger vasodilation (246), by activating the AMPKNOS pathway (247), and NO is a potent anti-proliferative agent (248). Although adiponectin may affect vascular remodeling in response to injury via this mechanism, there is also evidence that adiponectin can influence SMCs directly. Both SMC proliferation and migration are restricted in the presence of adiponectin (249, 250). These actions would likewise explain the inhibition of restenosis observed with adiponectin (251), which is supported by the negative association of adiponectin and restenosis (252). Interestingly, adiponectin also protects against arterial calcification (253). (d) Inflammation and Thrombosis. Production of adiponectin by macrophages may provide some positive benefits (229), particularly if accompanied by the release of anti-inflammatory cytokines. Increased NO release in response to adiponectin would also reduce inflammation (239). Additionally,

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adiponectin may suppress reactive oxygen species formation in endothelial cells, thereby reducing both oxidative and nitrative stress (254). The prevention of endothelial dysfunction by adiponectin reduces the expression of adhesion molecules, which reduces the risk of thrombosis by decreasing leukocyte adherence (61). The anti-thrombotic properties of adiponectin may also result from a decrease in platelet aggregation (255), although adiponectin was unable to block propylgallate-induced platelet aggregation in vitro when added to the blood of healthy humans (256). Interestingly, the CD40 ligand (CD40L) is elevated in MetS and has been shown to exacerbate inflammation (257). Since CD40L is a target of adiponectin, it has been proposed that the anti-inflammatory actions of adiponectin result from its ability to lower circulating levels of CD40L (257). The relationship between adiponectin and its truncated globular adiponectin version is not a topic of this review, but it has been shown that globular adiponectin can cause platelet activation through an interaction with the collagen receptor (258). (e) Angiogenesis. Low levels of circulating adiponectin are correlated with a decrease in collateral vessel formation in persons with occluded coronary arteries (259), while the converse is true when high levels are present (260). Although other factors affected by MetS may also be responsible, and addressing this point will only be possible by intervention studies, there is other experimental evidence that supports an inhibitory role for adiponectin in angiogenesis. Adiponectin blocks endothelial cell migration in response to vascular endothelial growth factor (261). Cyclooxygenase-2 (Cox-2) may mediate this process, since angiogenesis in response to adiponectin does not occur in Cox-2 deficient mice (262). Caloric restriction also promotes revascularization, and involves an adiponectin-dependent mechanism that relies on AMPK and eNOS (263). Interestingly, adiponectin promotes migration of endothelial progenitor cells (264), which may provide an additional explanation for its ability to block both atherosclerotic disease and restenosis.

Resistin Resistin is an adipokine that is primarily produced by adipocytes in rodents, but macrophages are the primary source of the resistin expressed by human adipose tissue. The infiltration of macrophages into adipose tissue likely explains the increase in circulating resistin seen in MetS (265). On the other hand, it has also been reported that circulating resistin levels are not correlated with MetS in humans (266). Interestingly, SMCs subjected to cyclical stretch or hypoxic conditions also produce resistin (267, 268), although the physiological relevance of this process has not been investigated. Resistin induces fatty acid binding protein in endothelial cells, possibly promoting hypertension via this mechanism (269). Alternatively, resistin blocks the effect of vasodilatory substances (270). Resistin is associated with inflammation (271), and can promote the release of proinflammatory cytokines from endothelial cells (272).

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Interestingly, resistin secretion is elevated in hyperhomocysteinemia and stimulation of SMC migration may lead to neointimal hyperplasia under these conditions (273). Visfatin Visfatin, also known as pre-B-cell colony-enhancing factor (PBEF) and nicotinamide phosphoribosyltransferase (Nampt), is specifically released by adipocytes (274). Visfatin is the secreted form of Nampt and likely has a different function than the intracellular protein, as indicated by the fact that these forms have different molecular masses (274). Visfatin is elevated in the proinflammatory state, and circulating levels increase in parallel with waist circumference (275). However, the available evidence suggests visfatin does not correlate with the presence of MetS (276). Rather, visfatin levels appear indicative solely of visceral fat accumulation (277). The lack of an association with MetS may be puzzling given that intracellular Nampt regulates Sirt1 activity, and this protein is closely linked with cell metabolic state and the positive actions of caloric restriction (278). Other Adipokines Vaspin (visceral adipose tissue-derived serine proteinase inhibitor) is secreted primarily by visceral adipose tissue and circulating levels vary with nutritional state (279). Furthermore, vaspin is associated both with endothelial dysfunction (280) and proatherogenic inflammation of smooth muscle (281). At this time, however, there is no evidence to link vaspin with atherosclerosis (282). Although apelin is secreted by white adipose tissue, it is also produced by many other cell types. Nevertheless, circulating apelin levels are increased in obesity, suggesting it may function as a hormone to influence other tissues (283). Apelin correlates with CHD, but not diabetes (284). Although it is claimed that there is a link between apelin and vascular injury, this view is not supported by the fact injury is also prevalent in diabetes (285). Apelin expression is induced by hypoxia, and subsequently promotes endothelial proliferation (286). A consequence of this interaction is the upregulation of angiogenesis (287), which would enable an increase in adipose mass and therefore obesity through the formation of blood vessels to oxygenate the new tissue (287). The localized production of apelin by other tissues may have a similar function and thereby ensure tissue perfusion under conditions when blood flow is reduced (288). This would be beneficial in the case of cardiac ischemia (289). However, whether secretion of apelin by adipose can influence these other tissues has not been determined.

Adipokines and the Heart The heart serves primarily as a pump to move nutrients, oxygen, and waste products to and from our tissues via the bloodstream. To accomplish this task, the heart has an efficient system for deriving energy from fatty acids. For this reason, alterations in

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metabolic state such as those that occur in MetS can significantly affect cardiac function. This altered metabolic state can have multiple effects on the cardiovascular system, from increasing the resting heart rate (290) to increasing the mortality of patients with heart failure (291, 292). While most heart disease can be attributed to changes in vascular function, MetS can also affect the heart muscle directly. Heart failure is the most serious consequence of reduced blood flow as well as increased workload due to hypertension or a valve disorder. Each of these conditions is associated with ischemia, a reduction in oxygen and nutrient access. As a result, the heart modifies its metabolism as a means of adapting to the lower oxygen levels. In parallel, the heart may begin a physical transformation depending upon the degree of damage inflicted on the muscle, especially if there is an acute loss of blood flow such as occurs during a heart attack. Ischemia also results in the secretion of cytokines from the heart that can promote the growth of new blood vessels and thus improve oxygen transport. What has become clear recently is that adipokines can influence progression of heart failure independent of their effects on blood vessels (293). And, as was seen with vascular disease, leptin and adiponectin have received the most attention with respect to heart failure. Leptin The LIPID (Long-Term Intervention with Pravastatin in Ischaemic Disease) study recently released results that indicate circulating leptin levels are predictive of recurrent cardiovascular events such as death, stroke, and heart attack in males who have experienced a heart attack or been hospitalized for angina (294). Interestingly, adiponectin was not associated with recurrences, which suggests leptin secretion is only elevated when the heart muscle itself is compromised. Currently, the source of this leptin is unknown, but it can be speculated that it is secreted from the pericardial adipose tissue. The leptin receptor is expressed by cardiomyocytes, which explains their ability to respond to leptin. Three distinct responses to leptin have been identified: (i) stimulation of fatty acid oxidation (295), (ii) decreased cardiac contractility (296), and (iii) cardiac hypertrophy (297). It is the latter response that likely explains the positive correlation between circulating leptin levels and poor prognosis for those with heart failure (298–300). Alternatively, leptin levels become elevated as a means of suppressing cardiac hypertrophy, and leptin resistance in MetS counters the benefits expected from this response (173, 293). Adiponectin Adiponectin is synthesized by cardiomyocytes (301). Furthermore, Skurk et al. (302) have suggested adiponectin production by cardiac tissue is controlled via a mechanism that is distinct from the adipose tissue. Their study also revealed that cardiac adiponectin production is suppressed in heart failure, while AdipoR1 and AdipoR2 remain unchanged. Interestingly, high levels of adiponectin are associated with poorer prognosis for persons with heart failure (303), although this is converse to the findings of Soderberg et al. (294). In part, these different conclusions may reflect different

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responses between healthy and obese individuals (304). As well, adiponectin might induce the expression of proinflammatory cytokines that negatively affect the heart tissue (305). Adiponectin may also directly stimulate enlargement of the heart and thus promote heart failure (306). Natriuretic peptides released by the heart stimulate secretion of adiponectin from adipocytes, even in patients with congestive heart failure (307), but whether this has positive or negative consequences on heart failure has yet to be determined. In contrast to its actions on heart failure, adiponectin provides protection against ischemia-reperfusion injury. Endothelial cells appear to have a significant role in this process. Reduction of oxidative and nitrative stress may represent the mechanism of action (254, 308). The AMPK-NOS pathway also contributes to the protective effects of adiponectin on this process (309, 310). Cardiac-specific production of adiponectin in response to ischemia is elevated through activation of hypoxia-inducible factor-1 (hif-1) (311). Adiponectin also is activated in the ischemic brain, and likely represents the underlying mechanism for its cerebroprotective actions (312). Other Adipokines There is evidence that high serum resistin levels are associated with a risk for heart failure (313, 314), possibly due to its relationship with cardiac injury (315). Both ischemic injury and heart failure will affect the heart’s pumping action, so the increase in resistin levels may be explained by the fact that mechanical stretch induces resistin expression by cardiomyocytes (316). A recent report indicates that resistin protects against myocardial infarction (317). It is therefore possible that resistin has a cardioprotective role in acute injury, but that it is ineffective with respect to longterm cardiac dysfunction such as heart failure. Apelin may protect against cardiac ischemia-reperfusion injury (289, 318), but whether adipose tissue, in particular epicardial adipose, plays a role in this process is uncertain. Visfatin also appears to have cardioprotective actions. In a recent study by Lim et al. (319), it was shown that administration of visfatin was capable of reducing cell death during an episode of ischemia-reperfusion. In contrast, fatty acid binding protein-4 (FABP4), an adipokine released in higher levels in MetS, has been shown to suppress cardiac contractility (320). This activity would have negative consequences for recovery after a heart attack and likely promote the development of heart failure in persons with MetS.

THERAPEUTIC INTERVENTIONS The ability to alter adipokine levels is expected to provide a means of improving health given the strong links between adipokines and certain disease states. On the other hand, implementing approaches that can successfully manipulate adipokine levels is fraught with difficulty, since little is yet known about the mechanisms that regulate adipokine synthesis and secretion. On the other hand, it is possible to propose

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that small molecule agonists or antagonists of adipokine receptors may be equally effective. At the same time, will the expected positive effects be realized? It is generally accepted that raising adiponectin levels will have numerous health benefits. Even so, there is the chance that negative outcomes may result, as was suggested by Wannamethee (321) who reported that elevated levels of adiponectin may increase the incidence of CHD. The following section will describe research studies designed to address these issues, and provide an overview of behavioral, nutritional, and pharmacological intervention strategies that have been tested in either animal models or humans.

Surgery Altered adipokine expression is tightly linked to adipocyte dysfunction, which is primarily due to adipocyte hypertrophy. Both animal and human studies have shown that weight loss restores the adipokine balance to one with fewer proinflammatory cytokines and more adiponectin (322, 323). These improvements are clearly linked to a reduction in adipocyte size (324). Bariatric surgery likewise improves adipocyte function according to the observed drop in circulating leptin levels and increase in circulating adiponectin (325). However, short- and long-term changes in glucose metabolism and insulin resistance due to caloric restriction and fat mass reduction that transiently affect adipokine production could influence interpretation of the results (326). Also noteworthy is the fact that removal of subcutaneous adipose exacerbates the inflammatory response, but this is followed by a reduction in proinflammatory adipokines (327, 328). Thus, surgical removal of adipose tissue appears generally beneficial to the subject, but whether this is the best treatment approach remains to be determined.

Lifestyle Greater caloric intake than utilization has long been recognized as the major cause of obesity. Consequently, increased exercise or decreased food consumption have been touted as the best means for managing this disease. This approach alone will not be successful for those individuals whose obesity is caused by genetic or endocrine abnormalities. Behavioral strategies (hypocaloric diets and exercise programs) that target weight loss have resulted in increased plasma adiponectin levels in adults with MetS (329, 330), diabetes (60, 323), and obesity (331), however, this does not seem to be the case in obese adolescent girls (332). Although few studies reported to date have addressed the question of adipocyte dysfunction directly, weight reduction has been confirmed to alter biomarkers of obesity. Choi et al. (333) have shown that levels of circulating adipocyte fatty acid binding protein (A-FABP) correlate with BMI. Furthermore, it was shown that A-FABP, which is higher in obese individuals, decreases when weight is lost. In dogs, weight loss is also associated with a reduction in proinflammatory adipokines such as TNF-a (322). Interestingly, some adipokines such as leptin and TNF-a are

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consistently improved with weight loss regimens, while others such as adiponectin are not (323). At the same time, adiponectin has been the focus of considerable study due to its association with metabolic and cardiovascular disease (334), as well as its status as a surrogate for adipocyte dysfunction (335). The concept that weight loss leads to improved adipose tissue function is strongly supported by the results of Pasarica et al. (336). In their study, the authors showed correlations among adipocyte size, adipokine production, and weight loss induced by lifestyle modification. Specifically, a 13% reduction in weight led to a decline in the number of large adipocytes, and this resulted in a 36% increase in circulating adiponectin levels. Interestingly, major proinflammatory adipokines (IL-6, TNF-a) did not change. Specific cardiovascular risk factors were not examined in this cohort, but significant improvements in glucose utilization were obtained. Similar data have been reported by Varady et al. (324), who found the level of improvement was dependent upon the amount of weight loss. Thus, it is reasonably clear that weight loss regimens can have a positive effect on the production of adipokines. Lifestyle interventions such as diet or physical activity that induce weight loss improve all factors of the MetS (337–339).

Dietary Components In relation to changes in dietary patterns, Bradley et al. (340) have recently shown that no significant changes in circulating adipokine levels occur when successful weight loss programs emphasize the reduction of specific macronutrients (e.g., carbohydrate or fat) from the diet. However, there may be other dietary constituents in our food that are capable of influencing adipokine levels. Some specific dietary components have been shown to increase plasma adiponectin levels. For instance, Decorde et al. (341) have shown that a melon extract provided to obese hamsters increased adiponectin levels by 61%. With the same animal model, Decorde et al. (341) showed that a diet enriched in grape phenolics could also positively modify adipokine levels. Additionally, resveratrol, a compound found in the skin of red grapes, has shown beneficial effects for reducing epididymal adipocyte size in mice (342) as well as increasing the circulating concentration of adiponectin, reducing TNF-a production and enhancing eNOS expression in visceral adipose tissue of obese Zucker rats (343). This study in rats also showed that resveratrol improved several parameters of MetS including dyslipidemia, hypertension, hyperinsulinemia, and inflammatory markers (343). Likewise, millet can increase adiponectin levels in mice with type 2 diabetes mellitus (344). Recently, Jobgen et al. (345) showed that dietary supplementation with L-arginine for 12 weeks reduces adipocyte size in diet-induced obese rats, and lowers serum concentrations of glucose, TAG, and leptin while increasing levels of NO metabolites. On the other hand, serum insulin and adiponectin levels were not affected by L-arginine supplementation (345). Oolong tea consumption for 1 month increased plasma adiponectin levels in patients with previous myocardial infarction and stable angina pectoris (346). Interestingly, omega-3 fatty acids from fish, specifically docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), also elevate circulating adiponectin in mice, and this occurs regardless of food intake and

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adiposity (347). Similar results have been reported with Rhizoma Dioscoreae Tokoronis extracts, Pollack (fish) protein, and mushroom-derived chitosan (348– 350), but none of these studies examined the relationship with adipocyte dysfunction. A definitive link between adipocyte size and the secretion of cardioprotective and proinflammatory cytokines has been identified (351, 352). While this association is being increasingly recognized, few studies have utilized this parameter to determine whether specific dietary components can improve adipocyte function. Pilvi et al. (353) showed recently that incorporation of lactalbumin into low-fat rodent chow resulted in smaller adipocytes after weight loss than the low-fat chow alone. Likewise, both a dietary herb and fiber combination (354) and persimmon leaf (355) have been shown to reduce adipocyte size in conjunction with improving adipocyte functional parameters. While the increase in smaller adipocytes and improved function are typically a product of the weight loss properties of these supplements, it has also been shown that improved function can be induced without a corresponding loss of weight. Noto et al. (356) conducted a study in which a mixture of conjugated linoleic acid (CLA) isomers was fed to obese (fa/fa) Zucker rats for 8 weeks. CLA has been proposed as a weight loss agent, but evidence of efficacy remains equivocal (357–360). In this case, the CLA regimen failed to reduce adipose mass, but there were significant improvements in the function of various organs, specifically the liver, kidneys, and pancreas (356, 361, 362). These changes in physiological parameters were associated with a decrease in adipocyte size, with a concomitant increase in cell number, and elevated circulating levels of adiponectin (363). As well, there were decreases in a number of proinflammatory mediators (361). These data and similar results from Nagao et al. (364) suggest it is possible to dissociate obesity from the end organ damage that typically occurs with increased weight. Interestingly, Lasa et al. (365) examined the effects of a single CLA isomer in the Syrian Golden hamster, a model of dietinduced obesity. Although many reports suggest trans-10,cis-12 CLA is the most biologically active isomer, no changes in adipose mass or adipocyte size were obtained. No other organs were examined, so it is not possible to ascertain whether this treatment had additional effects on the physiology of these animals. Likewise, there have been no beneficial effects observed with CLA treatment in most human studies on obesity management (366). However, the combination of CLAwith omega3 fatty acids over a 12-week period increased plasma adiponectin levels in young obese (BMI 30–36 kg/m2) men (367). Additional studies have been directed solely at adipocytes in culture based on the rationale that compounds that inhibit the differentiation of preadipocytes would interfere with weight gain (368). Whether these strategies would be effective in vivo has not been stringently tested. An argument can be made that maintaining cells in the preadipocyte state would not promote the production of protective adipokines such as adiponectin, although a reduction in proinflammatory cytokines might be expected. A number of phytochemicals have been tested for their ability to interfere with adipogenesis. These include xanthohumol, a prenylflavonoid present in hops and therefore found in small quantities in beer (369); genistein, quercetin, and resveratrol, polyphenols found in many plant species (370); epigallocatechin-3-gallate from

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tea (371); and guggulsterone, a compound present in tree gum that has been reported to exhibit cholesterol lowering properties (372). Of this brief list, resveratrol has also been reported to inhibit leptin release from adipocytes (373). An interesting alternative to utilizing a food product directly is to modify the item prior to ingestion. An example that has shown some success is biotransformation with bacteria. Vuong et al. (374) used this approach to modify blueberry juice. The resultant material was not only able to prevent adipocyte differentiation in vitro, its incorporation into the diet of obese mice also raised adiponectin levels, reduced weight by decreasing appetite, and was able to prevent onset of diabetes and obesity when provided to young animals. In all cases, however, none of these positive results have been translated to human studies, and concerns still exist regarding their implementation in humans (375). The data that have accumulated suggest there are a number of strategies that can be used to restore, at least in part, the normal functioning of adipose tissue in the absence of pharmacological intervention. However, it has to be noted that most studies have utilized animal models, and for this reason it is difficult to extrapolate to humans. Furthermore, few studies have examined the pharmacological aspects of using foodbased therapies for modulating the morbidities associated with obesity. In particular, the issue of concentration has received little consideration. While isolated compounds may be effective when used in vitro, delivering a similar dose to an animal or human may be very difficult, especially if it is at low concentrations in a product intended for oral consumption. As well, the form of the compound may be distinct from the pharmacological version. For instance, quercetin is typically found in a glucosidic form (rutin) in plants, and the activity of the conjugate may be different than the aglycoside. Within this context, passage through the gastrointestinal system or subsequently through the liver might also affect activity through the metabolic actions of these organs. Nevertheless, the indication that it is possible to improve overall health without a requirement to lose adipose mass, as was suggested by Noto et al. (363), does present a novel concept for the development of new therapeutic agents that can operate successfully without the need to induce weight loss.

Adipokine Therapy Pharmacological approaches to alter serum adipokine levels represent the state of the art in this field (242). However, before drugs with these adipokine modulatory properties were identified, direct delivery of adipokines was used in the first attempts to modulate obesity. The discovery of leptin was hailed as a breakthrough because it provided the first indication that obesity and appetite were controlled by biological factors. Numerous attempts were subsequently made to test the assumption that elevating leptin levels by direct infusion would lead to reduced weight as a consequence of a decrease in food intake. Leptin infusion into the mediobasal hypothalamus of rats both suppressed white adipose tissue lipogenesis and decreased vascular tone (376). Inhibition of lipogenesis occurs by reducing the expression of SREBP-1c and PPARg and their target genes FAS and ACC (163). Sympathetic

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innervations stimulate lipolysis through the activation of hormone-sensitive lipase along with increased expression of phosphoenol-pyruvate carboxykinase (PEPCK) and uncoupling protein-2 (UCP2). Independently of feeding behavior, leptin is able to simultaneously reduce de novo lipogenesis and FFA uptake in white adipose while preserving lean body mass through promotion of protein synthesis (376–378). While these data were encouraging, leptin infusion yielded few positive results in humans, and the best results were obtained in cases of leptin deficiency. The consequence of leptin replacement, therefore, is the amelioration of lipodystrophy, which is characterized as a marked loss of adipose tissue (379). The use of exogenous leptin as a method for treating MetS or obesity is not practical unless it is injected directly in to the hypothalamic circulation because most MetS patients already have highly elevated endogenous leptin production as well as leptin resistance. Treatment with recombinant leptin has minimal effect on adiposity in obese patients, even at supraphysiological doses (380). However, infusion of recombinant rat leptin into Fisher Brown Norway and Sprague-Dawley rats for seven days significantly reduced total intra-abdominal fat and caused a NO-dependent decrease in mean arterial pressure (381). Likewise, infusion of leptin and amylin in combination has been shown to restore leptin responsiveness in diet-induced obese rats (382). Increased responsiveness to leptin was determined by the increased phosphorylation of STAT-3 in the ventromedial hypothalamus (146). The improved sensitivity to leptin resulted in decreased caloric consumption and weight loss. The increased leptin sensitivity was not explained by the reduction in caloric intake alone (146). The same effect was demonstrated in humans, with combination therapy of pramlintide (amylin-analogue) and metreleptin (recombinant human leptin) resulting in a mean weight loss of 12.7 0.9% after 24 weeks, with the weight loss being significantly different from the control group after 4 weeks. In contrast, monotherapy of pramlintide and metreleptin yielded weight loss of 8.4 0.9% and 8.2 1.3%, respectively (146). Like leptin, adiponectin has been shown to have a wide range of physiological effects. In particular, increasing serum adiponectin levels is expected to influence both insulin sensitivity and vascular function. At this point, however, no direct infusion studies have been performed in humans, although numerous animal experiments have shown the validity of the basic premise (383). In contrast to employing the full-length protein, Lyzogubov et al. (384) describe a novel approach that uses a peptide containing sequence present in the globular domain of adiponectin that is responsible for AdipoR1 binding. Interperitoneal injection of this peptide prevented ocular neovascularization in an animal model of macular degeneration by blocking endothelial cell proliferation. Infusion of recombinant resistin in Sprague-Dawley rats worsened glucose homeostasis (385) and, in a similar way, intraperitoneal injections of resistin into mice impaired glucose homeostasis and insulin action (66). These results suggest that resistin may contribute to insulin resistance. Even so, longer studies using different animal models are needed. Clinical therapy with HIV protease inhibitors reduces plasma adiponectin concentrations and causes metabolic disorders such hyperlipidemia and atherosclerosis. HIV protease inhibitors reduce adiponectin secretion from 3T3-L1 adipocytes,

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however, infusion of adiponectin in mice ameliorates protease inhibitor-induced elevations of TAG and FFAs (386). Infusion of recombinant adiponectin significantly reduces insulin resistance by decreasing TAG content in muscle and liver in obese mice, thus ameliorating hyperglycemia and hyperinsulinemia (387). Infusion of peptides can also affect pancreatic function. Kapica et al. (388) have shown that leptin, apelin, and obestatin promote output of fluid and protein from the pancreas. As well, administration of exogenous resistin improved blood flow to the pancreas (389). Given these roles in pancreatic function, it is not surprising that circulating adipokine levels may also be linked to pancreatitis (143).

Gene Therapy Treatment of MetS may also be possible through the application of gene therapy. This field of research is in the early stages; however, some promising progress has been made. One type of gene therapy involves the use of adenoviruses, double-stranded DNA molecules carrying the genetic code for a particular gene. Some studies have used adenoviruses to transfer adipokine genes and study disease progression or treatment. For example, adenoviral transfer of the leptin gene into nonobese rats reverses streptozotocin-induced diabetes (390). Leptin insufficiency may be overcome by an intravenous injection of recombinant adeno-associated viral vectors that encode the leptin gene (rAAV-lep). A single injection leads to increased circulating leptin levels and normalizes body weight in obese rodents such as the ob/ob mouse. Experiments in genetically obese, diet-induced obese, or wild-type rodents given leptin either intracerebroventricularly or in specific hypothalamic sites results in leptin-induced downregulation of NPY signaling for the rodent’s lifetime (149). This restraint on NPY signaling leads to decreased levels of circulating triglycerides and FFAs and prevents insulin hypersecretion, a process that would normally precede weight gain (149). A single rAAV-lep intracerebroventricular injection normalizes blood glucose levels and prolongs life span of streptozotocin-induced diabetic mice and rats as well NOD (nonobese diabetic) mice by inhibiting the normal catabolic effects of a total lack of insulin (390). Apolipoprotein E-deficient mice treated with recombinant adenovirus expressing full-length adiponectin have a 30% reduction in aortic lesions (50). Furthermore, adiponectin-knockout (KO) mice developed hypertension; however, adenovirusdelivered adiponectin lowered elevated blood pressure in KO mice (56) and significantly decreased plasma TAG levels in normal mice (391). Based on these results, it would appear that viral delivery can successfully be used to elevate adipokine levels. Whether this approach will be feasible in the long term will depend upon development of vectors capable of constitutive, tissue-specific expression of these proteins.

Pharmacological Interventions The identification of drugs for weight control has received considerable interest. Drugs such as orlistat or sibutramine have been shown to reduce visceral obesity and

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improve parameters of MetS (392, 393). In general, it is assumed that weight reduction through pharmacological management will result in the same improvements in health as would be expected from lifestyle changes (394, 395). In particular, the changes would involve reductions in leptin and proinflammatory cytokines (e.g., IL-6, TNF-a) in conjunction with elevated adiponectin levels. As well, it is becoming evident that some of these pharmacological agents operate by modifying adipocyte dysfunction. The sections below describe the effect of both established and recently introduced pharmacological agents on adipokine production. It should be noted that compounds not currently employed may be more effective than those already available, but their utility as drugs has never been examined. For instance, it has been shown that induction of hypoxia inducible factor-1 (hif-1) or heme oxidase-1 (HO-1) under conditions of low oxygen levels will increase adiponectin expression (311). While activation of HO-1 can also be achieved with SnCl2 (234), it is unlikely that this compound will ever be used clinically since it is a strong irritant of mucosal membranes. However, the knowledge that HO-1 is a potential therapeutic target may lead to interventions that are feasible (396). Statins Statins are drugs developed to inhibit a key regulatory enzyme in the cholesterol synthesis pathway, HMG-CoA reductase. By reducing the ability to synthesize cholesterol in the liver, circulating cholesterol levels decrease. While this relationship is well recognized, statins also have pleiotropic effects that are independent of their cholesterol lowering actions. Targets of statins that have received considerable attention include RhoA and Rac1 (397). RhoA and Rac1 are key intracellular signaling proteins that regulate numerous cellular functions, among them proliferation and differentiation. It has recently been established that statins can prevent the differentiation of 3T3-L1 adipocytes in culture (398). This would explain their ability to modulate expression of leptin, resistin, and adiponectin in animals and humans (399–401). These data would also explain the weight loss attained with statin treatment in obese individuals with type 2 diabetes (402), although weight loss has not been reported in other statin trials. Given the relative safety profile of statins, there appears to be considerable potential for these drugs as weight-loss agents. On the other hand, if weight loss is not a major characteristic of statins, or it is limited to specific conditions such as type 2 diabetes mellitus, there is still substantial evidence that statins improve adipose function. In the latter case, increases in adiponectin levels could explain why statins ameliorate the vascular effects of obesity, and this improvement could explain the well-known cardioprotective actions of statins. For instance, in patients with CHD, 6 months of pravastatin treatment significantly increased plasma adiponectin levels along with improving other factors of MetS such as lowering CRP levels, total cholesterol, LDL-cholesterol, and improving hyperinsulinemia and hyperglycemia (403). Similarly, a recent study by Nomura et al. (404) demonstrated significant increases in serum adiponectin levels and reductions in total and LDL-cholesterol after 6 months of treatment with pravastatin. Likewise, hyperlipidemic patients with

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NASH show increased plasma adiponectin and reduced TNF-a levels after 24 months of atorvastatin treatment (405). However, 6 months of treatment with atorvastatin (406) or 3 months of rosuvastatin (407) in patients with type 2 diabetes mellitus did not alter plasma adiponectin levels but did improve, as expected, total and LDLcholesterol levels. The conflicting data may be due to the fact that different statins were used in these studies, as was indicated by Koh et al. (408). These investigators showed that a 2-month treatment with simvastatin significantly decreased plasma adiponectin levels and reduced insulin sensitivity while pravastatin significantly increased adiponectin levels and improved insulin sensitivity. Atorvastatin in patients with steatohepatitis and hyperlipidemia improved steatosis and steatohepatitis, and this was paralleled with a 25% increase in serum adiponectin; however, it is unknown if the improvement in steatohepatitis was related to the increase in circulating adiponectin (405). PPAR Agonists Both fibrates and thiazolidinediones (TZDs), like statins, were developed as agents for cholesterol lowering and increasing insulin sensitivity, respectively. These compounds operate by activating PPARs, key regulators of body metabolism. Fibrates are PPARa ligands, and therefore stimulate b-oxidation of fatty acids (409). Pathological weight loss is a side effect noted for the most commonly prescribed fibrate, fenofibrate, and these results are supported by evidence that this compound also prevents weight gain in animals (410). The increase in b-oxidation induced by fenofibrate may also explain its ability to reduce adipocyte size, and concomitantly decrease circulating levels of proinflammatory adipokines (411). In agreement with these data, it has been shown that fibrates are capable of increasing adiponectin levels in humans with hypertriglyceridemia (412). These results therefore support the concept that fibrates positively affect adipocyte function, and likely provide benefits that extend beyond their ability to lower serum lipids. The TZDs, in contrast to the fibrates, are ligands for PPARg and work as insulin sensitizers (413). PPARg is also an essential mediator of adipogenesis, and therefore agents such as rosiglitazone and pioglitazone are capable of affecting adipocyte function (414, 415). The latter likely explains why rosiglitazone increases plasma adiponectin levels and decreases resistin in obese persons with type 2 diabetes mellitus (416). However, the data showing effects of TZDs on resistin in these studies is not consistent (417–419). Even in animal studies, the data on TZDs and resistin are not as clear as that for adiponectin. Some studies have shown that TZDs upregulate resistin mRNA levels in adipose tissue of ob/ob mice and ZDF rats (420) whereas others report downregulated mRNA levels in adipose tissue of db/db mice and in 3T3L1 adipocytes (72, 421, 422). Both human and animal studies have shown that PPARg agonists increase plasma adiponectin levels (423–425). Many studies have shown the benefits of TZDs in increasing adiponectin levels and reducing proinflammatory mediators such as IL-6 and CRP in patients with MetS (328, 417–419). Work by Krzyzanowska et al. (426) suggests there is a relationship between adiponectin and FFA levels. The fact that rosiglitazone treatment promotes an increase in plasma

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adiponectin while decreasing FFA levels suggests this effect is mediated by adipocytes. The effect on adiponectin would also explain why rosiglitazone protects against endothelial dysfunction induced by FFAs (427). Randomized, placebo-controlled trials of pioglitazone treatment in patients with nonalcoholic steatohepatitis have shown that as little as 6 months of treatment can reduce hepatic lipid content, normalize liver function, and improve hepatic insulin sensitivity (428, 429). The reduction in liver lipid content was inversely associated with an increase in plasma adiponectin. Paradoxically, although patients given pioglitazone experienced an increase in percentage of body fat, plasma adiponectin increased. Adiponectinstimulated activation of AMPK is thought to be an important factor in mediating the metabolic effects of TZDs in the liver (429). Renin-Angiotensin System Inhibition AngII is a vasoactive molecule that is not only essential for normal cardiovascular function, but can also have detrimental effects if it is present at chronically high levels. The pathological effects of AngII include atherosclerosis and hypertension. Typically, angiotensinogen is secreted by the liver. It is then cleaved to angiotensin I by renin, which is produced by the kidney. AngII is then produced by cleavage with angiotensin converting enzyme (ACE), which is present on the luminal surface of endothelial cells. An important discovery was the identification of angiotensinogen, a precursor to AngII, as an adipokine (27). Furthermore, adipose tissue contains all of the components of the renin-angiotensin system necessary to convert angiotensinogen to AngII. Local production of AngII therefore does not require the circulating enzymes, but adipose-derived AngII can influence systemic levels. In this way, AngII production by adipocytes can promote the development of hypertension. Another way that AngII can impact health is through suppression of adiponectin production (430), which in turn can exacerbate endothelial dysfunction. The negative actions of AngII suggest that inhibitors of AngII production will have beneficial health effects. This has been seen with ACE inhibitors, a popular class of antihypertensive agents. These compounds function by lowering systemic AngII levels, and thus reduces blood pressure. In addition, they have been shown to lower body weight and increase plasma adiponectin levels in rats (431), presumably by decreasing adipocyte size (432). In humans, AngII receptor blockers (ARBs), as well as ACE inhibitors, increase plasma adiponectin concentrations (433–435). Specifically, ACE inhibitors such as ramipril and valsartan have been shown to increase adiponectin concentrations in patients with MetS (436). These results may explain the broad protective effects against renal, cardiovascular, and neural disease ascribed to ACE inhibitors (437). Interestingly, ARBs are not as effective as ACE inhibitors in raising adiponectin. The sole exception is telmisartan, an ARB that stimulates PPARg as well as blocks the AngII AT1 receptor (target of ARBs) (438, 439). The fact that these observations were made in human studies suggests this approach may have a great value in the treatment of various diseases linked to vascular dysfunction. While it is clear that elevating adiponectin may be a useful therapeutic approach for treating cardiovascular disease (440), some caution may be advised before

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implementing these therapies. This statement is based on the fact that some treatments designed to reduce blood pressure may negatively influence adiponectin. For instance, ACE inhibitors have been shown to increase circulating adiponectin, but in combination with a diuretic, adiponectin levels decrease (441). This may be due to the fact that diuretic monotherapy is also associated with a reduction of adiponectin (442). AMPK Activation AMPK plays an important role in regulating glucose and lipid metabolism. Activation of AMPK results in reduced deposition of lipids and enhances oxidation of stored fat, thus it may be a possible target for treatment of MetS (443). In humans, metformin, a member of the biguanide family of antidiabetic drugs that activate AMPK, has been shown to reduce serum leptin, insulin and glucose concentrations despite having no affect on body weight or adipose tissue mass (444). Interestingly, adiponectin also activates AMPK resulting in free fatty acid oxidation and glucose uptake by skeletal muscle (126, 445) and suppression of glucose production (126). Metformin also increases resistin protein levels in abdominal (epididymal) adipose tissue in db/db mice (446). Interestingly, metformin can also affect the expression of resistin in hepatic tissues and downregulation of resistin levels by metformin may lead to improved insulin sensitivity (447). Endocannabinoid Receptor Antagonists The ECS has been identified as an important modulator of metabolism (448), and it is now clear that the ECS plays a role in food intake and adipose accumulation in humans and animals (448–450). The discovery of two G protein-coupled receptors (CB1 and CB2) for D9-tetrahydrocannabinol has led to the identification of numerous endogenous cannabinoid receptor ligands and the development of various receptor agonists and antagonists. Activation of CB1 increases food intake, while blocking the CB1 receptor suppresses food intake. The CB2 receptor, in contrast, appears to modulate insulin secretion by the pancreas (451) and has been recently reported to influence hepatic steatosis, inflammation, and insulin resistance in obesity (452). Treatment with a CB1 receptor antagonist (rimonabant) increases plasma adiponectin in both obese humans and rats (453–455). Recently, Despres et al. (456) have shown that one year treatment with rimonabant, significantly reduces the ratio of intra-abdominal (visceral) adipose to subcutaneous adipose tissue while increasing serum adiponectin. While these studies show rimonabant is linked to an elevation of adiponectin levels, it appears this effect may be a consequence of weight loss (457). Five placebo-controlled randomized clinical trials have been completed using rimonabant, two of which included participants with type 2 diabetes mellitus (458). Rimonabant is recommended for patients with a BMI greater than 30 kg/ m2 and/or abnormal blood lipids. Pooled, one year data from the Rimonabant in Obesity (RIO) program showed that 20 mg/day of rimonabant resulted in significant weight loss as well as improvements in other end points such as HDL cholesterol,

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TAG, fasting glucose levels, as well as a 0.6% reduction in hemoglobin A1c (HbA1c) levels in diabetes (458, 459). With respect to other adipokines, a CB1 knockout mouse shows increased leptin resistance in association with hepatic steatosis, but little is known about the relationship between the ECS and leptin. It is possible that they operate as antagonizing systems in the hypothalamus in terms of satiety (460, 461), which agrees with evidence that leptin may regulate CB1 expression in the hypothalamus (160). Interestingly, resveratrol has been recently shown to bind to the CB1 receptor (462), which may explain its ability to affect leptin release. Further investigation will be necessary to fully understand the effects of the ECS on other adipokines. At the same time, the withdrawal of rimonabant from the market due to psychiatric side effects associated with depression (155) and an increase in death rate as a result of intensive treatment intended to lower HbA1c levels to <6.5% (463) suggests this class of drugs may not provide the panacea from obesity as it first appeared. Cyclooxygenase Inhibition Cox-2 inhibition has been recently examined as a mechanism of reducing inflammatory disease. Obesity is considered an inflammatory disease due to the high numbers of macrophages that infiltrate the adipose tissue (21). Lijnen et al. (464) recently examined the effect of the Cox-2 inhibitor rofecoxib (Vioxx) in an animal model of diet-induced obesity. These investigators found that the treatment attenuated weight gain and reduced adipocyte size. Furthermore, there were fewer macrophages present in the adipose tissue and 30% less leptin was produced. No differences in adiponectin or TNF-a were detected. Similar results were obtained with wild-type mice (465). Although some Cox-2 inhibitors have been withdrawn from the market, these compounds might represent an alternative route for modulating adipose function. The efficacy of these drugs may be due to the ability of adipocyte-derived prostaglandin E2 (PGE2) to stimulate the secretion of leptin by adipocytes (466). In retrospect, these results indicate PGE2 could also be classed as an adipokine. Inhibition of Monocyte Infiltration Weisberg et al. (467) showed that antagonists of the CC chemokine receptor-2, which is present on monocytes and binds MCP-1, increased adiponectin expression in adipose tissue of obese mice without altering weight or adipose mass. Although gross changes in body composition did not occur, there was a reduction in hepatic steatosis and improved insulin sensitivity. It is presumed that a reduction in monocyte infiltration into the adipose tissue is the primary cause of these positive changes. Interestingly, deletion of the MCP-1 gene did not produce similar effects (468), but this may be due to the fact that CC chemokine receptor-2 binds other members of the MCP family and thus there is sufficient redundancy to ensure that macrophages will still be drawn to adipose tissue as it enlarges due to excess caloric intake. This novel approach warrants further investigation given the significant contribution of macrophage infiltration to inflammation-mediated adipose dysfunction.

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Vitamin A All-trans-retinoic acid (RA) is the active metabolite of vitamin A and is a ligand of the retinoid X receptor, a cofactor of numerous nuclear hormone receptors including the PPARs. Several studies have investigated the contribution of RA to the actions of nuclear hormone receptors, and as a matter of course have identified RA-specific changes in cellular activity. One relevant example is a decrease in leptin expression in 3T3-L1 adipocytes and human adipose tissue explants with RA treatment in vitro (469). In agreement with this observation, both RA and vitamin A have been shown to lower plasma leptin levels in rats when given in pharmacological doses, but there were no changes in body weight or adipose mass (470). Interestingly, there was no effect of RA on adipokine levels in a human population except for a lowering of resistin (471). Of note, 13-cis retinoic acid, a compound considered to have antiinflammatory properties and is used for the treatment of acne, was shown to increase insulin resistance in adults. Paradoxically, it also increases plasma adiponectin levels (472, 473). Effects on TNF-a have also been reported (474). Ciliary Neurotrophic Factor Ciliary neurotrophic factor (CNTF) is a neurocytokine that suppresses AMPK in the hypothalamus (475) and thus exhibits similarities to leptin (476). At the same time, it can act peripherally to modulate metabolism by stimulating AMPK in skeletal muscle (477), and has been shown to induce weight loss in both animals and humans (478, 479). This weight reduction has been linked to changes in adiponectin production by the adipose tissue (480). It appears that remodeling of adipocytes by CNTF may represent the mechanism by which it achieves these results (481). These impressive results indicate there is strong potential for future development in this area.

SUMMARY The identification of leptin in 1994 indicated for the first time that adipose tissue plays a significant role in metabolic homeostasis, and its importance in this process is now well established. Adipokine production by adipose tissue has a regulatory role in the fasting/feeding cycle, with leptin contributing to satiation. Adipokines also link adipose tissue to various organs (liver, skeletal muscle) that are important for lipid and carbohydrate metabolism, but what has now become more accepted is the role of adipokines in modulating the function of other tissues, such as the pancreas, brain, heart, and blood vessels. At this point in time, exploration of the therapeutic potential of adipokines has revealed that it is possible to manipulate their production. The underlying mechanisms that influence adipokine levels in pathological conditions, however, have not been delineated to the point that specific drug targeting is possible. Until this transpires, careful investigation of the physiological effects of any intervention will require close attention, particularly from the standpoint that their manipulation has the potential to negatively affect a variety of metabolic pathways. But on a positive note, this approach appears to have great potential for treating

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metabolic diseases, even if weight loss is not achieved, by improving the function of adipose tissue.

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Hepatic Metabolic Dysfunctions in Type 2 Diabetes: Insulin Resistance and Impaired Glucose Production and Lipid Synthesis RUOJING YANG Department of Metabolic Disorders – Diabetes, Merck Research Laboratories, Rahway, NJ, USA

INTRODUCTION Insulin resistance is a condition in which liver, muscle, and adipose cells fail to respond to normal amount of circulating insulin to promote the storage of carbohydrates, lipids, and proteins. Insulin resistance is closely associated with type 2 diabetes mellitus (T2DM), central obesity, dyslipidemia, atherosclerosis, hypertension, and inflammation (1). The International Diabetes Federation estimates that more than 285 million people worldwide have diabetes and 438 million people will have this disease within 20 years. The American Diabetes Association estimated that the total annual cost of diabetes in the United States was $174 billion in 2007. There are two types of diabetes mellitus resulting in hyperglycemia. Type 1 diabetes mellitus (T1DM) is characterized by loss of the insulin-producing b-cells in the pancreas leading to insulin deficiency. T2DM accounts for more than 90% of all diabetes and is

Metabolic Syndrome: Underlying Mechanisms and Drug Therapies Edited by Minghan Wang Copyright 2011 John Wiley & Sons, Inc.

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characterized by insulin resistance combined with relatively reduced insulin secretion. Increased obesity, aging population, and sedentary lifestyles are the leading causes to the rising prevalence of T2DM (1). The pathogenesis of T2DM remains poorly understood. Current therapies for T2DM focus on strict glycemic control, which lowers the risk of diabetic complications including those in heart, kidney, eye, and nerves. T2DM develops from insulin resistance and is a progressive disease in the loss of insulin action (2, 3). In early stages, pancreatic b-cells can compensate for insulin resistance by secreting more insulin leading to increased circulating insulin levels to maintain normal glycemia (Figure 5.1). When b-cells can no longer secrete enough insulin to compensate for insulin resistance, glucose intolerance occurs leading to postprandial hyperglycemia. Insulin resistance occurs in liver with increased glucose production and impaired glycogen metabolism, in muscle with decreased glucose uptake, and in adipose tissue with increased lipolysis to increase circulating free fatty

Obesity Aging Lifestyle

Liver Increased glucose production Impaired glycogen metabolism

Skeletal muscle Decreased glucose uptake

Adipose tissue Increased lipolysis

Insulin resistance Increased free fatty acid

Pancreatic beta cell compensation

Hyperinsulinemia

Pancreatic beta cell decompensation

Glucose intolerance

Lipotoxicity Glucotoxicity

Diabetes

Pancreatic beta cell destruction

Figure 5.1 Progressive development of type 2 diabetes from insulin resistance. Obesity, aging, and sedentary lifestyles lead to insulin resistance in different tissues: (i) increased glucose production and impaired glycogen metabolism in liver; (ii) decreased glucose uptake in muscle and increased lipolysis in adipose tissue leading to increased plasma free fatty acid (FFA) levels. At early stage, pancreatic b-cells produce more insulin to compensate for insulin resistance and to maintain normal glycemia leading to hyperinsulinemia. When b-cells can no longer compensate for insulin resistance, glucose intolerance ensues leading to postprandial hyperglycemia. High plasma glucose and FFAs result in glucotoxicity and lipotoxicity to the cells, leading to more severe insulin resistance in muscle and liver and damaging b-cells to develop more severe diabetes (permission from The International Journal of Biochemistry & Cell Biology, 2008, Vol 40, Iss 12, page 5).

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acid (FFA) levels. Increased circulating glucose and FFAs cause lipotoxicity and glucotoxicity to cells, which further increase insulin resistance in muscle and liver and decrease insulin secretion by damaging b-cells, leading to more severe diabetes. Thus, T2DM occurs as a consequence of the failure of several regulatory systems: (i) reduced capacity of insulin to induce glucose uptake and to inhibit lipolysis in muscle and fat; (ii) dysregulated insulin release from b-cells; (iii) abnormally increased glucose production in liver. In T2DM, defects in suppression of hepatic glucose production and liver glycogen storage are observed and account for approximately one-third of the defect in total body glucose homeostasis (4, 5). This chapter focuses on the regulatory role of insulin in regulation of hepatic glucose and lipid metabolism and contribution of hepatic insulin resistance to the development of T2DM.

BALANCING HEPATIC GLUCOSE DISPOSAL AND PRODUCTION BY GLUCOSE-6-PHOSPHATE SYSTEM Liver senses changes in circulating glucose concentration and plays a major role to maintain glucose homeostasis: (i) it increases glucose disposal and storage at high glucose concentrations; (ii) it produces glucose via gluconeogenesis and glycogenolysis at low glucose concentrations. In T2DM, an imbalance between hepatic glucose production and disposal makes a major contribution to the development of hyperglycemia and other perturbations in fuel homeostasis (6). A membrane-bound glucose transporter, GLUT2, plays a major role to facilitate glucose diffusion in and out of the hepatocytes (7, 8). Hepatocytes with genetic disruption of GLUT2 had more than 95% reduction in glucose uptake (8). However, GLUT2 knockout mice had normal glucose production suggesting the existence of an alternative membrane traffic mechanism for glucose in this extreme condition (8). The balance between hepatic glucose production and glucose disposal and storage is ultimately determined by the relative rates of glucose phosphorylation and glucose-6-phosphate (G6P) hydrolysis (Figure 5.2). The hydrolysis of G6P to free glucose is catalyzed by the glucose-6-phosphatase (G6Pase) enzyme complex. The complex is comprised of a catalytic subunit sequestered within the endoplasmic reticulum (ER), a G6P translocase known as T1, and putative ER glucose and inorganic phosphate transporters (T2, T3) that move the reaction products back into the cytosol (6, 9, 10) (Figure 5.2). Overexpression of the G6Pase catalytic subunit or the T1 translocase in primary hepatocytes increased G6P hydrolysis and lowered intracellular G6P levels, which led to substantial decreases in glycolytic flux and glycogen deposition, and a parallel increase in gluconeogenesis (11, 12). Hepatic overexpression of G6Pase in rats exhibited several of the abnormalities associated with early-stage T2DM, including glucose intolerance, hyperinsulinemia, a marked decrease in hepatic glycogen content, and increased peripheral (muscle) triglyceride stores (13). These findings are consistent with the notion that increased activity of the G6Pase complex in liver can make a significant contribution to the development of T2DM, and clearly establish the importance of the tight control on the balance between glucose phosphorylation and G6P hydrolysis in the regulation of hepatic glucose metabolism.

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Phosphate

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Pyruvate

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Glycolysis FFA Triglyceride

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Figure 5.2

Glucose-6-phophate (G6P) system balances hepatic glucose disposal and production. Glucose enters cells via GLUT2 transporter and is phosphorylated by glucokinase to form G6P. Dissociation of glucokinase from the glucokinase regulatory protein (GKRP) in the nucleus releases the glucokinase into cytoplasm and activates its enzymatic activity. G6P can be stored into glycogen via activationofglycogen synthase (GS) and metabolizedthrough glycolysis promotinglipogenesis. When the plasma glucose levels are low, liver produces G6P through gluconeogenesis or glycogen breakdown via activation of glycogen phosphorylase (GPH) and its kinase (GPHK). G6P can be translocated into endoplasmic reticulum (ER) via glucose-6-phosphatase (G6Pase) translocase T1, where G6P is dephosphorylatedbyG6Paseandtheglucoseandphosphate istransportedout oftheERviaT2andT3translocase, respectively. Glycogen-targetingsubunit(GTS) ofproteinphosphatase1(PP1)isa scaffold proteinbinding to GS, GPH, GPHK, and glycogen to activate glycogen synthesis and suppress glycogen breakdown.

Glucose enters liver and is primarily phosphorylated by glucokinase (hexokinase IV) to form G6P (6) (Figure 5.2). This enzyme has a lower affinity for glucose and a higher catalytic capacity than other members of its gene family, and is limited in terms of its tissue distribution to liver, the islets of Langerhans, and certain specialized neuroendocrine cells in the pituitary and gastrointestinal tract (14, 15). In liver, glucokinase is sequestered in the nucleus bound to glucokinase regulatory protein (GKRP) at low glucose concentrations (16). Increased glucose concentrations trigger the translocation of glucokinase to the cytoplasm to generate G6P (Figure 5.2). Fructose-1-phosphate synergistically potentiates glucose-induced glucokinase translocation (17). Overexpression of glucokinase in primary hepatocytes led to profound increases in glycogen deposition and glycolytic rate, while overexpression of hexokinase I had a very limited impact on both variables (18). This difference in efficacy is likely explained by the fact that hexokinase I activity is strongly inhibited by the product of the reaction, G6P, while glucokinase is not subject to such regulation (19). Hepatic overexpression of glucokinase by sixfold increased liver glycogen content and robustly decreased blood glucose levels and insulin levels accompanied by a robust increase in circulating triglycerides and FFAs (20). Chronic increase in hepatic glucokinase activity by twofold decreased blood glucose levels

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without increases in circulating triglycerides and FFAs (21–23). In humans, loss-offunction mutations on glucokinase result in maturity onset diabetes of the young type 2 (MODY-2), while gain-of-function mutations lead to persistent hyperinsulinemic hypoglycemia of infancy (PHHI). Heterozygous glucokinase knockout in mice resulted in hyperglycemia and defective glucose-induced insulin secretion, while homozygous knockout was lethal (24–26). Pancreatic b-cell specific glucokinase knockout mice had profound hyperglycemia and died within three days of birth (27, 28). Liver-specific glucokinase knockout mice were mildly hyperglycemic when fasted with impaired glucose-induced insulin secretion (28). Thus, MODY-2 results from loss of glucokinase function in liver and b-cells, but the defect in b-cells plays a dominant role leading to hyperglycemia. Significant progress has been made to develop allosteric small molecule activators of glucokinase (29). Several glucokinase activators have been reported to lower blood glucose in insulin resistance animals or in T2DM patients (29–31). The activators stimulated glucokinase activity both in liver and pancreatic islets to enhance glucose disposal and increase insulin secretion.

REGULATION OF HEPATIC GLUCOSE METABOLISM BY INSULIN Insulin promotes glucose uptake in muscle and fat cells to increase circulating glucose disposal by stimulating the translocation of GLUT4 transporter to the plasma membrane (32). Insulin does not promote glucose uptake in liver since GLUT2 is the major glucose transporter and is located in the plasma membrane. Insulin increases circulating glucose disposal in liver by stimulating glucose utilization and storage as glycogen and lipids. Skeletal muscle is the major tissue for insulindependent glucose disposal (33). In the fasted state, liver is the main organ to produce glucose to maintain circulating glucose homeostasis. Liver produces glucose through two processes: (i) glycogenolysis that breaks down glycogen stores into glucose and (ii) gluconeogenesis that generates glucose using 3-carbon substrates. Nuclear magnetic resonance (NMR) studies in humans demonstrated that hepatic glycogenolysis and gluconeogenesis each contributed 50% to endogenous glucose production after 6–12 h fasting, but they account for 4 and 96% of glucose production, respectively, during prolonged fasting for 46–64 h (34). Insulin inhibits hepatic glucose production and promotes glucose disposal via several different mechanisms (32, 35), which will be described in more details below.

INSULIN-SIGNALING PATHWAY Insulin receptor belongs to a subfamily of receptor tyrosine kinases and consists of two a-subunits and two b-subunits (32). Insulin binds to the a-subunit to increase the kinase activity of the b-subunit, which autophosphorylates to further increase its tyrosine kinase activity. Activated insulin receptor phosphorylates tyrosine residues of its intracellular substrates, including four insulin receptor substrate (IRS-1 to -4) proteins (Figure 5.3). Genetic studies suggest that different IRS proteins play distinct

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Insulin R

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SREBP-1

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I P -Y

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mTOR p70

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Serine kinase

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Figure 5.3 Insulin regulates hepatic glucose and lipid metabolism. Insulin acts on its receptor to increase tyrosine phosphorylation of the insulin receptor and insulin receptor substrates (IRSs). Tyrosine phosphorylated IRS proteins lead to activation of the PI3 kinase pathway to increase Akt phosphorylation. Increased Akt phosphorylation regulates different metabolic pathways in liver. (i) Akt activates glycogen synthase to promote glycogen synthesis via inhibiting glycogen synthase kinase 3 (GSK3) and activation of glycogen-targeting subunit (GTS) of protein phosphatase 1 (PP1). Activation of GTS–PP1 complex also contributes to the suppression of hepatic glycogenolysis. (ii) Akt enhances hepatic glycolysis and lipogenesis via upregulation of genes in these pathways via sterol regulatory element binding protein-1 (SREBP-1). (iii) Akt inhibits gluconeogenesis via inhibition of several transcription factors, including forkhead-related proteins (FKHR), PPARg coactivator 1 (PGC-1), and hepatocytes nuclear factor (HNF). (iii) Akt also promotes protein synthesis via activation of mTOR/p70 (S6kinase). Activation of mTOR (mammalian target of rapamycin) signaling triggers IRS protein degradation, a feedback inhibition of insulin signaling, through kinases that increase serine/threonine phosphorylation of IRS. Protein tyrosine phosphatase 1B (PTP-1B) also inhibits insulin signaling through tyrosine dephosphorylation of insulin receptor and IRS proteins. Inflammatory signals and stress lead to insulin resistance via activation of those serine/threonine kinases to inhibit insulin signaling.

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roles in different tissues. Mice with genetic disruption of IRS-1 developed insulin resistance in peripheral tissues and impaired glucose tolerance (36, 37). IRS-2 knockout mice developed insulin resistance in both peripheral tissues and liver and impaired pancreatic b-cell function leading to the development of T2DM (38). IRS-3 or IRS-4 knockout mice displayed mild metabolic phenotypes (39). Several groups have proposed the model that IRS-1 regulates lipid metabolism in liver while IRS-2 regulates hepatic glucose production (40). However, recent studies demonstrated that double knockout of both IRS-1 and IRS-2 resulted in much more severe diabetes compared to either single knockout, suggesting that the two proteins are effectively interchangeable in relaying insulin action (41, 42). Tyrosine phosphorylated IRS proteins activate phosphatidylinositol 3-kinase (PI3K) pathway to increase Akt phosphorylation. Increased Akt phosphorylation regulates different metabolic pathways: (i) it promotes GLUT4 translocation to the membrane to increase glucose uptake in muscle and adipose tissue; (ii) it downregulates gluconeogenesis through forkhead-related protein (FKHR) in liver, such as FoxO1; (iii) it upregulates glycogen synthesis through phosphorylation of glycogen synthase kinase 3 (GSK3); (iv) it also increases protein synthesis through activation of mTOR/p70 (S6 kinase) (Figure 5.3). Activation of mTOR (mammalian target of rapamycin) signaling has been shown to be capable of triggering IRS-1 degradation, a feedback inhibition of insulin signaling, through kinases that increase phosphorylation of IRS-1 on serine-307 (43–46) (Figure 5.3). Several groups demonstrated that c-Jun N-terminal kinase (JNK), IkB kinase b (IKKb), and protein kinase C u (PKCu) inhibit insulin signaling via serine/ threonine phosphorylation of IRS-1 (47–49). These kinases can be activated by stress and inflammatory signals leading to the development of insulin resistance under these conditions (3, 50, 51). Insulin action can also be attenuated by tyrosine phosphatases. Cytoplasmic protein tyrosine phosphatase-1B (PTP1B) has been shown to play a crucial role in the negative regulation of insulin action via tyrosine dephosphorylation of insulin receptor or IRS proteins. Mice with genetic disruption of PTP1B were resistant to diet-induced obesity and had increased tyrosine phosphorylation of insulin receptor and IRS proteins and improved insulin sensitivity (52). Inhibition of PTP1B with antisense oligonucleotides (ASO) improved insulin sensitivity in insulin-resistant mice and in monkeys (53).

INSULIN REGULATES GLYCOGEN SYNTHESIS AND BREAKDOWN Insulin increases glycogen storage in cells through activation of glycogen synthesis and inhibition of glycogen degradation. In muscle and adipose tissue, insulin promotes GLUT4 translocation to increase glucose uptake, which further increases glycogen synthesis by increasing substrate availability and allosteric activation of glycogen synthesis enzymes (2, 6). In liver, elevated blood glucose promotes glucose uptake through GLUT2 to increase glycogen accumulation, which does not require insulin action. Although glucose plays a major role in glycogen synthesis in liver, insulin activates glycogen synthesis enzymes to further facilitate this process. Insulin

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promotes phosphorylation of GSK3 via Akt to inactivate its activity, which decreases the phosphorylation rate of glycogen synthase and increases its activity (54) (Figure 5.3). Glycogen synthase is the rate-limiting enzyme in glycogen synthesis and is activated when dephosphorylated by protein phosphatase 1 (PP1), which is dependent on insulin action (55, 56). Insulin does not promote activation of PP1 globally, but rather specifically activates PP1 localized in glycogen particles via physical association to glycogen-targeting subunits (GTS) (2, 32, 57) (Figure 5.3). Glycogen-targeting subunits of PP1 are scaffolding proteins that organize and regulate key enzymes of glycogen metabolism (57). Five members of GTS have been described with varying tissue distributions: the major skeletal muscle isoform, GM (also known as RGl or PPP1R3); a liver enriched form, GL (also known as PPP1R4); ubiquitously expressed protein targeting to glycogen (PTG or PPP1R5) and PPP1R6; and recently identified PPP1R3E with different tissue distributions in humans and rats (57, 58). All of these proteins share PP1 and glycogen-binding motifs and also, to varying degrees, bind to the glycogen-metabolizing enzymes glycogen synthase, glycogen phosphorylase, and phosphorylase kinase. Insulin administration restored glycogen-associated phosphatase activity in diabetic rats (59, 60). In insulin-deficient diabetic rats, hepatic GL and PTG expressions are reduced compared to wild-type rats and can be restored by insulin administration (61, 62). In liver, insulin acutely activates glycogen-bound PP1 through modulation of cAMP levels and reduction of phosphorylase a, which binds to the Cterminus of GL to inhibit PP1 activity (63–68). Inhibitors of the insulin-downstream target, PI3K, blocked insulin-induced activation of glycogen-associated PP1 (32). Glycogen breakdown requires glycogen phosphorylase and debranching enzymes. Glycogen phosphorylase controls the rate-limiting step of glycogenolysis to remove glucose from glycogen (69). Glycogen phosphorylase is regulated by phosphorylation as well as allosteric factors, including AMP, ATP, G6P, glucose, and caffeine (70). Catabolic hormones, such as epinephrine and glucagon, convert the inactive dephosphorylated glycogen phosphorylase b form to the active glycogen phosphorylase a form through cAMP-dependent protein kinase and phosphorylase kinase (68). In liver, glycogen phosphorylase is mainly activated by phosphorylation, whereas the allosteric factor AMP increases glycogen phosphorylase b activity by 10–20% and fails to activate glycogen phosphorylase a (71). Insulin indirectly inhibits activation of glycogen phosphorylase by suppressing the release of those catabolic hormones. Several groups demonstrated that insulin directly inhibited glycogen phosphorylase activity by promoting phosphorylase a to phosphorylase b, while other reports disputed their results (72–76). It has been shown that insulin activates GTS of PP1 to dephosphorylate glycogen phosphorylase and thereby inhibits glycogenolysis (55) (Figure 5.3).

EFFECT OF INSULIN ON GLUCONEOGENESIS One of the characteristics of insulin resistance is elevated hepatic glucose production leading to fasting hyperglycemia. Reduced suppression of hepatic glucose production by insulin also contributes to impaired glucose tolerance and postprandial

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hyperglycemia (77). Insulin has a direct role in the suppression of hepatic glycogenolysis and gluconeogenesis (78). Liver-specific insulin receptor knockout (LIRKO) mice completely blocked the insulin-signaling pathway in liver (79). In LIRKO mice, insulin failed to suppress hepatic glucose production, suggesting that an intact insulin-signaling system is required for insulin-mediated suppression of hepatic glucose production. Insulin suppresses the expression of several key genes in gluconeogenesis, including phosphoenolpyruvate carboxylase (PEPCK), glucose6-phosphatase, and fructose-1,6-bisphosphatase (35, 80). The factors involved in the insulin-mediated transcriptional regulation of gluconeogenesis genes have remained elusive. Intensive studies have indicated that the FKHR family of transcription factors, such as FoxO1, play a crucial role in insulin-mediated inhibition of gluconeogenic gene expression (35, 80). Several lines of evidence have shown that FKHR binds to the promoter region of several gluconeogenic genes to activate their transcription, and this effect can be blocked by insulin treatment (81–83). Insulin triggers phosphorylation of FKHR proteins via a PI3K-dependent pathway. Several groups demonstrated that phosphorylation of FKHR by activated Akt resulted in nuclear exclusion of FKHR proteins and consequently decreased the transcription of their target genes (84–86). However, there are conflicting reports indicating that Akt and FKHR are not required for insulin-mediated inhibition of gluconeogenic gene expression (87–90). The peroxisome proliferator-activated receptor gamma coactivator-1a (PGC-1a) functions as a master regulator of gluconeogenic gene expression in liver (91). Glucagon and glucocorticoids increase hepatic PGC-1a expression via cAMP response element binding protein (CREB) to induce gluconeogenic gene expression (92). PGC-1a binds to and activates FoxO1, glucocorticoid receptor, and HNF-4a, and fully activates the transcription of gluconeogenic genes (91, 93). PGC1a is strongly induced in several mouse models deficient in insulin action, whereas insulin does not have a direct effect on PGC-1a expression (91, 93). Recent studies demonstrated that insulin directly inhibited PGC-1a activity through Aktmediated phosphorylation of PGC-1a (94). Insulin also blocked PGC-1a activity on the expression of gluconeogenic genes by disrupting PGC-1a and FoxO1 interaction (93). Besides the direct effect of insulin on hepatic gluconeogenesis, several lines of evidence have demonstrated that insulin indirectly inhibits hepatic glucose production by limiting substrate availability for gluconeogenesis (95–100). Insulin acts on muscle and fat tissue to inhibit the release of gluconeogenic substrates and FFAs resulting in the suppression of hepatic glucose production (101–104). Insulin also indirectly inhibits hepatic glucose production by suppressing glucagon release since glucagon controls majority of basal glucose production (96, 97, 105–107). The flux of FFAs into liver plays a key role in controlling glucose production as supported by the following evidence: (i) a strong correlation between FFAs and hepatic glucose production; (ii) lack of insulin-mediated suppression of glucose production at basal FFA concentrations; (iii) reduction of FFAs leads to reduced glucose production regardless of insulin administration (108, 109). The flux of FFAs into liver provides energy and induction signals for gluconeogenesis through b-oxidation. During clamp study at well-controlled hormonal and metabolic

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conditions, increased plasma FFA levels within the physiological range robustly induced G6Pase expression in liver (110). Glycogenolysis and gluconeogenesis have similar contributions to the net hepatic glucose production except under the condition of prolonged fasting (more than 16 h) (111). In T2DM, the relative contribution of gluconeogenesis to hepatic glucose production is significantly increased after prolonged fasting based on NMR studies, which has not been confirmed by some studies (112–116). Either augmenting gluconeogenesis via infusion of gluconeogenic substrates or inhibition of gluconeogenesis did not alter circulating glucose levels in humans (117, 118). Inhibition of glycogenolysis by pharmacologic inhibitors of glycogen phosphorylase lowered blood glucose levels in diabetic animals (119–121). However, these inhibitors also significantly reduced gluconeogenic rate in diabetic animals through unknown mechanisms (121). These observations indicate that both gluconeogenesis and glycogenolysis have to be inhibited to reduce hepatic glucose production (68).

HEPATIC LIPID METABOLISM AND INSULIN RESISTANCE Insulin plays an essential role in the regulation of hepatic lipogenesis. Glucose enters liver cells via GLUT2 transporter and then is phosphorylated by glucokinase to produce G6P. G6P can be stored as glycogen or further metabolized through glycolysis and pentose pathways to generate substrates and NADPH for de novo FFA synthesis (35). The newly synthesized FFAs can then be esterified into triglycerides and packed and secreted in very low density lipoproteins (VLDL), which carry lipids to adipose tissue for storage. Insulin directly induces FFA and triglyceride synthesis through upregulation of genes in the lipogenic pathway, including glucokinase, acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), S14, liver pyruvate kinase (L-PK), and stearoyl-CoA desaturase (SCD) (35). The induction of lipogenic gene expression by insulin is mediated by the transcription factor sterol regulatory element binding protein-1C (SREBP-1C) (35) (Figure 5.3). Insulin not only induces SREBP-1C expression but also activates its activity by converting the inactive membrane-bound precursor to active nuclear form (122–125). The mechanism by which insulin regulates SREBP-1C expression is not clear. Recent studies have indicated a direct role of the liver X receptors (LXRs) in insulin-induced activation of SREBP-1C promoter activity (126). Insulin might directly activate LXRs, promote the production of LXR activating ligands such as oxygenated derivatives of cholesterol (127, 128), or activate LXR coactivators such as PGC-1 and ASC-2 (129–131). The transcription coactivator PGC-1b has recently been identified as a key activator of not only hepatic lipogenesis through coactivation of SREBP-1C but also lipoprotein secretion through coactivation of LXRa and Foxa2 (132, 133). Hepatic overexpression of PGC-1b significantly increased circulating triglyceride levels in rodents (132). Recent studies demonstrate an important role of PGC-1b in the development of fructose-induced insulin resistance (134, 135). Knockdown of PGC-1b by ASO normalized fructose-induced plasma triglyceride levels and reduced hepatic

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SREBP-1 and lipogenic gene expression (134, 135). Knockdown of PGC-1b prevented fructose-induced insulin resistance in liver and adipose tissue (134, 135). Insulin exerts effects on both hepatic glucose production and lipid metabolism: (i) suppressing glucose production through the FoxO1 pathway, (ii) promoting lipogenesis through SREBP-1C (35, 136). In T2DM, the FoxO1 pathway is resistant to insulin so that insulin fails to suppress hepatic glucose production, but the lipogenic pathway remains sensitive to insulin. This alteration in hepatic insulin action is named “selective” insulin resistance in T2DM (136). Hyperinsulinemia in T2DM contributes to the increased hepatic lipogenesis, leading to elevated circulating triglyceride levels and increased flux of triglycerides from liver to peripheral tissues (137, 138). Elevated circulating triglycerides increase lipid storage in various tissues and exert detrimental effects, namely “lipotoxicity,” to exacerbate insulin resistance in liver, muscle, and adipose tissue and damage pancreatic b-cells. Liver-specific insulin receptor knockout mice result in “total” insulin resistance in liver: hyperinsulinemia fails to suppress glucose production and also fails to activate SREBP-1C (78, 139). LIRKO mice were insulin resistant and developed hyperglycemia and hyperinsulinemia (78). However, LIRKO mice had low circulating triglycerides and liver triglyceride content (139). The hyperglycemia of LIRKO mice decreased with age, while hyperglycemia in “selective” insulin resistance mice progressed with age (78, 136). The less severe diabetes in LIRKO mice supports a long-term detrimental effect of hypertriglyceridemia on the progression of T2DM. This raises concerns about insulin therapy or insulin secretion-stimulating antidiabetic drugs over their potential of causing longterm detrimental effects via activation of hepatic lipogenesis (136). Given the unmet medical needs for T2DM patients, new drugs are preferred to improve whole body insulin sensitivity to normalize overall metabolic profiles. Hepatic steatosis, also known as fatty liver, is the most common cause of liver dysfunction and is associated with insulin resistance, T2DM, and other metabolic diseases (140, 141). Hepatic steatosis occurs as a result of increased de novo lipogenesis and excessive delivery of FFAs from adipose tissue, while lipid oxidation and export plays a minor role (142). In patients with nonalcoholic fatty liver disease (NAFLD), liver triglyceride content arises from different sources: 60% from FFAs, 30% from de novo lipogenesis, and 10% from the diet (143). Genetic disruption of hormone sensitive lipase (HSL) in mice inhibited lipolysis, reduced plasma FFAs, and prevented steatosis in liver (144, 145). These mice also had increased hepatic insulin sensitivity, suggesting that plasma FFAs play an important role in the development of hepatic steatosis and insulin resistance. Genetic studies to investigate the role of de novo lipogenesis in the development of liver steatosis and insulin resistance have been complicated by the effects on FFA oxidation. Inhibition of both ACC1 and ACC2 by ASO-inhibited lipogenesis, activated FFA oxidation, reduced liver triglyceride contents, and improved insulin sensitivity (146). Genetic disruption of SCD1 or inhibition of SCD1 by ASO prevented diet-induced hepatic steatosis and insulin resistance due to combined effects of reduced lipogenesis and increased FFA oxidation (147–150). Liver-specific knockout of FAS (FASKOL) significantly reduced circulating insulin levels with no effect on liver triglycerides in normal chowfed mice (151). When fed with zero-fat diet, FASKOL mice unexpectedly increased

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liver triglyceride content, similar to PPARa knockout mice, and can be corrected with a PPARa agonist. These led to the hypothesis that newly synthesized fat via FAS reaction serves as PPARa activators to suppress the fatty liver phenotype (151). Shulman’s group has demonstrated that metabolites from de novo lipogenesis, such as acyl-CoA, lysophosphatidic acid (LPA), and diacylglycerol (DAG), contribute to the development of insulin resistance (152). Despite substantial evidence for the association of hepatic steatosis and insulin resistance, several studies suggested an opposite role for hepatic steatosis with respect to insulin resistance (153–157). These investigators suggested that the accumulated triglycerides in liver are not toxic but rather protect cells from lipotoxicity presumably by alleviating FFA-triggered cytotoxicity (153, 154).

TARGETING HEPATIC INSULIN RESISTANCE FOR THE TREATMENT OF T2DM T2DM is a complex disorder of impaired metabolism, insulin resistance, and b-cell dysfunction. Substantial evidence indicates that strict glycemic control lowers the risk for diabetic complications, including cardiovascular disease, renal failure, and visual impairment. Current therapies achieve glycemic control in T2DM via different mechanisms. First, insulin secretagogues, such as sulfonylureas and meglitinides, increase circulating insulin levels by increasing insulin secretion from b-cells. Exogenous insulin therapy plays a major role in T2DM despite the side effects of hypoglycemia and weight gain. A high proportion of T2DM patients fail to respond to oral drugs (secondary failure) and ultimately require exogenous insulin to control hyperglycemia (158, 159). Although many physicians avoid exogenous insulin therapy in T2DM patients, some suggest that earlier use of insulin in carefully selected patients has long-term benefits (159–162). Second, thiazolidinediones (TZDs) and metformin enhance insulin action in insulin responsive tissues, such as liver, muscle, and adipose tissue. TZDs, such as rosiglitazone and pioglitazone, are agonists for the nuclear peroxisome proliferator-activated receptor gamma (PPARg) and mainly increase insulin sensitivity in peripheral tissues to promote glucose utilization. Consistent with previous reports, recent studies indicate that TZDs improve hepatic insulin sensitivity and suppress glucose production (163, 164). TZDs have become second-line therapies for T2DM despite several side effects, such as weight gain, edema, and increased risk of bone fractures. Recent studies have indicated that rosiglitazone therapy increases cardiovascular mortality in T2DM (165, 166). Others claim that the studies are not conclusive and more investigation is needed before considering rosiglitazone for removal from the market (167). Metformin is the only biguanide available for the treatment of T2DM. The principal action of metformin is to reduce hepatic glucose production and the mechanism is not fully understood (168–170). Metformin also improves insulin sensitivity, increases glucose uptake in skeletal muscle, and suppresses inflammation, which contributes to the glucose lowering effect in T2DM (158, 163). Recent studies have shown that metformin may act on adenosine-monophosphate-activated protein kinase (AMPK)

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to regulate cellular glucose and lipid metabolism (171). Third, a-glucosidase inhibitors, such as acarbose, decrease the rate of intestinal carbohydrate digestion and delay carbohydrate absorption to reduce postprandial hyperglycemia (158). Last, dipeptidyl peptidase-4 (DPP-4) inhibitors, such as Januvia, and glucagon-like peptide-1 (GLP-1) analogues, such as exenatide, increase the incretin effect of GLP-1 to reduce hyperglycemia. These drugs not only potentiate glucose-stimulated insulin secretion from b-cells but also suppress hepatic glucose production through direct action in liver and indirect effects by suppression of the glucagon release (172–175). Hepatic insulin resistance is characterized by excessive hepatic glucose production, which plays a major role in the development of fasting hyperglycemia in T2DM. As described above, insulin reduces hepatic glucose production via both direct and indirect means. A modest rise in plasma insulin leads to robust reduction in hepatic glucose production (98). Metformin reduces hyperglycemia in T2DM mainly through suppression of hepatic glucose production and this effect requires the presence of adequate insulin (159). This indicates that enhancing hepatic insulin sensitivity to suppress glucose production can reduce hyperglycemia in T2DM. Glucagon is the counter regulatory hormone of insulin, which binds to the glucagon receptor (GCGR) to promote hepatic gluconeogenesis and glycogenolysis leading to increased glucose production (176). Inhibition of glucagon action by glucagonneutralizing antibodies, antagonistic glucagon peptide analogues, or GCGR ASO inhibits hepatic glucose production and reduces blood glucose levels in diabetic animals (177–183). Substantial efforts have been made to identify small molecule inhibitors of GCGR. Several studies demonstrated that small molecule GCGR antagonists lower glucose in diabetic animals (184–192). An oral GCGR antagonist has been shown to inhibit glucagon-induced increase of blood glucose in humans (193). Suppression of hepatic gluconeogenesis or glycogenolysis enzymes leads to reduced hepatic glucose production and lowers glucose levels in animals. Inhibitors of G6Pase translocase or glycogen phosphorylase reduce blood glucose in animals (119, 194, 195). The major concerns about suppression of hepatic glucose production in T2DM are hypoglycemia and accumulation of hepatic glycogen, but neither side effect has been reported in T2DM patients treated with metformin or GCGR antagonists. However, mutations in G6Pase or G6Pase translocase in humans lead to glycogen storage diseases (196). It is important to understand the actions of future drug targets that are aimed at suppressing hepatic glucose production so that these potential side effects can be avoided.

SUMMARY This chapter describes our current understanding of how insulin regulates glucose and lipid metabolism in liver. Circulating glucose concentration plays a major role in activation of hepatic glycolysis, lipogenesis, and glycogen synthesis. Insulin further upregulates expression of genes in lipogenesis and glycolysis via SREBP-1 to potentiate these pathways. Insulin induces phosphorylation of Akt to activate glycogen synthase via inhibition of GSK3 and activation of GTS–PP1 complex. Insulin

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suppresses hepatic glucose production via inhibition of gluconeogenesis and glycogen breakdown. Insulin suppresses the release of catabolic hormones and inhibits lipolysis in adipose tissue, indirectly suppressing hepatic glucose production. Insulin signaling directly inhibits gluconeogenesis by downregulation of gluconeogenic gene expression via transcription factors, such as FoxO1, PGC-1a, and HNF-4. Insulin signaling also directly inhibits glycogenolysis via activation of GTS–PP1 complex. Elevated hepatic glucose production in T2DM is the major cause of fasting hyperglycemia and contributes to the development of postprandial hyperglycemia. T2DM develops from insulin resistance and normal levels of insulin fail to suppress hepatic glucose production in the disease state. However, the insulin-signaling pathway that promotes hepatic lipogenesis and glycolysis remains to be responsive to normal insulin levels. Elevated lipogenesis in the face of hyperinsulinemia leads to lipid accumulation in liver and muscle, which further increases insulin resistance in these tissues causing more severe diabetes. This suggests that targeting hepatic insulin resistance to achieve beneficial effects on both glucose and lipid metabolism will be more effective to control hyperglycemia in a long term in T2DM. Indeed, the most popular drug for T2DM, metformin, suppresses hepatic glucose production via activation of AMPK, which regulates both hepatic glucose and lipid metabolism. Recent progress in the development of glucagon receptor antagonists further supports that suppression of hepatic glucose production is clinically beneficial to control hyperglycemia in T2DM. The US Food and Drug Administration recently require evaluation of cardiovascular risks of new antidiabetic drugs. Impaired hepatic lipid metabolism contributes to multiple cardiovascular disease (CVD) risk factors, such as liver steatosis, elevated triglycerides, and decreased HDL. Thus, targeting genes that enhance hepatic insulin sensitivity to regulate both glucose and lipid metabolism will not only control hyperglycemia but also provide beneficial effects on the CVD risk factors in T2DM.

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Chapter

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Energy Metabolism in Skeletal Muscle and its Link to Insulin Resistance MINGHAN WANG Metabolic Disorders Research, Amgen, Inc., Thousand Oaks, CA, USA

INTRODUCTION Tissue insulin resistance is a main target for antidiabetic treatment. In patients with type 2 diabetes mellitus (T2DM), hepatic insulin resistance leads to increased glucose output. Further, insulin-stimulated glucose disposal in the peripheral tissues is impaired due to insulin resistance. Both abnormalities directly contribute to the hyperglycemic state in T2DM. The skeletal muscle accounts for 40% of body mass and about 75% of total insulin-stimulated glucose uptake and therefore, is a main tissue for insulin-dependent glucose utilization. In T2DM, insulin resistance in skeletal muscle is exemplified by the decreased ability of insulin to cause translocation of GLUT4, the main glucose transporter that mediates glucose uptake. Unlike in the insulin-sensitive state, GLUT4 in insulin-resistant cells is much less efficiently translocated to the plasma membrane, where it mediates glucose uptake into the cells. This is due to the impairment of intracellular insulin signaling in muscle cells of T2DM patients because signals downstream of insulin signaling trigger GLUT4 translocation under normal conditions. Several potential mechanisms are responsible for the development of insulin resistance in skeletal muscle in T2DM, including accumulation of intramyocellular fatty acid metabolites, mitochondrial dysfunction, and increased inflammatory state. Muscle insulin resistance also impacts the metabolism of other insulin-sensitive tissues, either by releasing secreted factors with metabolic functions

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or through altering whole-body energy homeostasis. The regulation of skeletal muscle growth is also critical in insulin sensitivity. Therefore, insulin resistance in skeletal muscle is a major pathologic factor in the development of hyperinsulinemia and hyperglycemia in metabolic syndrome and T2DM. Mitigation of skeletal muscle insulin resistance is critical in improving the systemic glycemic homeostasis.

GLUCOSE METABOLISM IN SKELETAL MUSCLE: THE IMPORTANCE OF GLUCOSE UPTAKE Glucose Transporters Glucose enters cells by facilitated diffusion and this process, namely glucose uptake, is mediated by glucose transporters (GLUTs). These transporters are encoded by genes that belong to the family of solute carriers 2A (SLC2A). The protein symbol for the family of the transporters is GLUT. To date, there are at least 14 members that have been described in this family (1–3). GLUT proteins share common structural characteristics, including 12-membrane helices, an N-linked glycosylation site, and intracellular N- and C-termini but exhibit significant differences in tissue distribution, substrate specificity, and kinetics of transport (4). They are located in the plasma membrane of cells and also found in intracellular vesicles. Based on sequence homology and other characteristics, the family members are divided into three classes. GLUT1–4 are categorized in class I (4). They are low-affinity and high-capacity glucose transporters and play important roles in glucose metabolism by mediating basal and insulinstimulated glucose uptake into tissues. GLUT1 is found in proliferating cells in the early developing embryo, the blood–brain barrier, human erythrocytes and astrocytes, and cardiac muscle (5). GLUT1 is also expressed in skeletal muscle and white adipose tissue (6, 7). GLUT1 expression is regulated at both the transcriptional and the posttranslational levels (8–11). GLUT2 mediates basal glucose transport in liver, small intestine, pancreas, kidney, and brain (12, 13) and its expression is subject to regulation by a variety of dietary and metabolic conditions (14–18). GLUT3 is primarily expressed in neuronal tissues (19–21). GLUT3 has higher affinity for glucose than the other class I glucose transporters and at least fivefold greater transport capacity than GLUT1 and GLUT4 (22). This is significant for its role in mediating glucose uptake by neurons since the glucose level surrounding neurons is only 1–2 mM compared with 5–6 mM in serum (22). GLUT4 is responsible for insulin-stimulated glucose uptake in skeletal muscle, adipose, and other insulin-sensitive tissues (23–28). The regulation of GLUT4 translocation by insulin signaling represents the major pathway for insulin-dependent glucose uptake in skeletal muscle.

The Important Role of GLUT4 in Glucose Uptake in Skeletal Muscle Glucose uptake by skeletal muscle cells is the rate-limiting step of glucose metabolism under normoglycemic conditions. Both GLUT1 and GLUT4 are expressed in skeletal

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muscle butGLUT4 is the main glucose transporter in this tissue. GLUT1 mediates basal glucose uptake, whereas GLUT4 mediates insulin-stimulated glucose uptake (29). Both GLUT4 protein level and glucose uptake are much higher in red oxidative muscle fibers than white glycolytic fibers in rat skeletal muscle (30, 31). This difference is smaller in human skeletal muscle (32, 33). GLUT4 is localized in intracellular membrane vesicles in the basal state. Upon insulin stimulation, GLUT4 is translocated from the intracellular pools to the plasma membrane, which was demonstrated in human skeletal muscle biopsies (34). In addition to insulin stimulation, exercise also induces glucose uptake in skeletal muscle (35, 36). Exercise and insulin appear to stimulate the translocation of two separate pools of intracellular glucose transporters to the plasma membrane (35), suggesting that exercise triggers GLUT4 translocation through a mechanism distinct from that by insulin (35). There are distinct intracellular GLUT4 pools that can be recruited by insulin and exercise, respectively (37). The muscle glucose uptake is also regulated by changes of GLUT4 expression. Muscle GLUT4 expression is induced by insulin, thyroid hormone, and exercise (38–41). One important link of insulin signaling to GLUT4 translocation is Akt substrate of 160 kDa (AS160), which is a Rab GTPase activating protein (GAP) (42–44). In fat cells, AS160 is phosphorylated by PKB/Akt upon insulin stimulation and inactivation of its GAP activity leads to GLUT4 vesicle translocation (42–44). Although the role of ASP160 in skeletal muscle GLUT4 translocation remains to be validated, it has been demonstrated in adipocytes that a dominant-inhibitory mutant of ASP160 blocked insulin-stimulated GLUT4 exocytosis (42). The exercise-stimulated GLUT4 translocation is believed to be mediated by adenosine 50 -monophosphate (AMP)activated protein kinase (AMPK), a key metabolic switch that regulates energy metabolism. It has been demonstrated that muscle contraction activates AMPK, which can stimulate glucose uptake in skeletal muscle in an insulin-independent manner (45). In T2DM patients, AMPK has the full capacity to stimulate glucose uptake in skeletal muscle (45). Therefore, exercise in T2DM induces AMPK activation in a way similar to normal insulin-sensitive individuals (46), although insulin-stimulated glucose uptake is impaired. The AMPK activation-stimulated glucose uptake is mediated by GLUT4 translocation to the plasma membrane (46), and muscle contraction stimulates GLUT4 translocation in an AMPK-dependent manner (5). Increased GLUT4 content in skeletal muscle after endurance training has been observed in both diabetic animals and humans (47, 48). In addition to induced translocation, transcription and translation of GLUT4 are increased in response to exercise, which is likely to be mediated by the activation of AMPK (49). Further, pharmacologic activation of AMPK by injection of adenosine analogue 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) increased GLUT4 content in muscle (50). The regulation of GLUT4 activity is not only by translocation but also through an unknown mechanism at the plasma membrane level. Overexpression of GLUT1 in mouse skeletal muscle increased basal glucose transport by several fold (51). In the GLUT1-overexpressing skeletal muscle of these animals, insulin-stimulated increase in cell surface GLUT4 content was identical to that in wild-type muscle (52, 53).

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However, there was no elevated GLUT4-mediated glucose uptake in the transgenic muscle upon stimulation with a number of stimuli, including insulin and those with glucose transport activation mechanisms distinct from insulin (51). This defect in GLUT4-mediated activation of glucose uptake does not involve the insulin receptor (51). These data demonstrate that the level of basal glucose uptake can impact insulin-stimulated GLUT4-mediated glucose transport, and this effect can be exerted without changing insulin-stimulated GLUT4 translocation to the plasma membrane. It is possible that the activity of the translocated GLUT4 in the plasma membrane is regulated by the basal rate of glucose transport. Under conditions of elevated basal glucose transport, a regulatory mechanism is activated so that the GLUT4 in the plasma membrane is not fully active. In GLUT4 knockout mice, there is decreased longevity associated with cardiac hypertrophy and severely reduced adipose tissue deposits (54). They have elevated postprandial hyperinsulinemia, suggesting insulin resistance in these animals. Compensation for this defect by increasing other GLUT members occurred in these animals, with GLUT2 elevated in liver and GLUT1 in heart, but not in skeletal muscle (54). These compensatory responses impaired the ability to conclusively determine the absolute role of GLUT4 in glucose homeostasis.

Mechanisms of GLUT4 Translocation GLUT4 recycles between the intracellular vesicular compartments and the plasma membrane; the process involves multiple steps such as endocytosis, sorting into vesicles, docking, and fusion with the plasma membrane. Insulin stimulates glucose uptake in skeletal muscle by enriching cell surface GLUT4 protein, which is associated with both elevated exocytosis and reduced endocytosis (55, 56). Insulin-stimulated GLUT4 translocation is rather a complex process and involves many proteins that mediate individual steps. It is believed that two separate and parallel pathways downstream of insulin signaling regulate this process. One pathway involves the activation of phosphoinositol 3-kinase (PI3K), which leads to Akt activation and phosphorylation of RabGAP AS160 (42). AS160 activity has been demonstrated to be required for GLUT4 translocation as a dominant negative mutant of AS160 impaired insulin-stimulated GLUT4 translocation (42). A second pathway that works in parallel to PI3K involves Cbl and TC10. Cbl is associated with Cbl-associated adapter protein (CAP) via interaction with the SH3 domain of CAP (57). Upon insulin receptor activation, Cbl is recruited along with CAP to the insulin receptor and becomes phosphorylated (57). After phosphorylation, the Cbl/CAP complex dissociates from insulin receptor and translocates to lipid rafts, where it triggers a cascade of signaling events eventually leading to the activation of small G proteins TC10a and TC10b (58, 59). Activated TC10 interacts with a number of potential effector proteins, including Exo70, a component of the exocyst complex (60). The exocyst complex plays an important role in docking of secretory vesicles containing GLUT4.

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INSULIN SIGNALING AND MECHANISMS OF INSULIN RESISTANCE IN SKELETAL MUSCLE Insulin receptor and other signaling components are expressed in skeletal muscle. The signaling cascade is similar to that in other tissue as described in other chapters in this book. Insulin binds to the a subunits of insulin receptor and induces phosphorylation of tyrosine residues on the b subunits, resulting in the increased receptor tyrosine kinase activity. The activated receptor then phosphorylates tyrosine residues on intracellular substrates including the family of insulin receptor substrates (IRSs). IRS-1 and -2 are both expressed in skeletal muscle and believed to mediate insulin signaling in this tissue. The tyrosine-phosphorylated IRS-1 and -2 help recruit PI3K to the plasma membrane. PI3K has two subunits, the 110 kDa regulatory subunit that binds to IRS and the 85 kDa kinase subunit that phosphorylates and activates downstream signaling molecules, including PDK-1 and -2. Subsequent phosphorylation and activation of Akt by PDKs leads to further activation of a signaling cascade that stimulates the translocation of GLUT4 to the plasma membrane. In type 2 diabetic patients, reduction in insulin-stimulated glucose uptake in the peripheral tissues is one of the main metabolic deficiencies. This is not caused by deficiency in GLUT4 expression in the cell because the total GLUT4 level does not change in insulin resistance cells in T2DM; rather, the trafficking and translocation of GLUT4 in response to insulin stimulation is impaired in the skeletal muscle of these patients (61–63). As a result, there is insufficient GLUT4 at the plasma membrane following insulin stimulation (61–63). These data suggest that defective insulin signaling impaired the ability of insulin to stimulate GLUT4 translocation from subcellular pools to the plasma membrane. Interestingly, physical training increases muscle GLUT4 translocation as well as expression (41), suggesting that AMPK activation can bypass the insulin-resistant state. Identification of the defective point in the insulin-signaling cascade in T2DM muscle is critical in understanding the mechanisms of insulin resistance. It has been reported that insulin receptor phosphorylation in skeletal muscle is reduced (64) or unaltered (62) in T2DM compared with normal subjects. However, IRS-1 tyrosine phosphorylation is reduced in skeletal muscle of T2DM patients while the IRS-1 protein level remains unchanged (62). The reduced IRS-1 phosphorylation upon insulin stimulation is expected to result in less PI3K activation and reduced downstream signaling. There are several mechanisms that may lead to reduced IRS-1 phosphorylation. Proinflammatory and stress signals are associated with macrophage infiltration into adipose tissue and skeletal muscle and can cause activation of JNK1 and IKKb, both of which can phosphorylate IRS-1 on its serine residues and impair its ability to be tyrosine-phosphorylated and activated. Therefore, macrophage-derived proinflammatory cytokines such as tumor necrosis factor a (TNFa), and interleukin (IL)-1b, -4, and -6 are likely involved in the development of skeletal insulin resistance. Alternatively, increased fatty acid uptake by muscle cells can lead to insulin resistance, causing intramyocellular accumulation of lipids (triglycerides) and fatty acyl metabolites. Two such metabolites, ceramide and diacylglycerol (DAG), play important

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roles in fatty acid-induced muscle insulin resistance. Ceramide inhibits insulindependent activation of Akt2/PKB (65), while DAG activates PKC isoforms, which promotes IRS-1 serine phosphorylation and inhibition of insulin signaling (66). Thus, hyperlipidemia is likely to contribute to the development of muscle insulin resistance. Since hepatic insulin resistance contributes to the development of hyperlipidemia with elevated free fatty acid (FFA) levels, insulin resistance in skeletal muscle could be at least in part a consequence of reduced insulin sensitivity in liver. On the other hand, skeletal muscle insulin resistance is likely to impact hepatic insulin sensitivity. In a study with young, lean, insulin-resistant individuals, it was found that decreased muscle glycogen synthesis due to insulin resistance increased carbohydrate flow to the liver and caused elevated lipogenesis (67), supporting the notion that insulin resistance in skeletal muscle may negatively impact hepatic insulin sensitivity. The intramyocellular lipid accumulation in insulin-resistant muscle results from an imbalance between FFA oxidation and uptake. Overwhelming evidence suggests that alterations in fatty acid oxidation in skeletal muscle play an important role in the development of muscle insulin resistance. There is reduced mitochondrial oxidation capacity in the muscle of insulin-resistant offspring of T2DM patients (68). Further, in obese, insulin-resistant individuals, there is reduced rate of fatty acid oxidation (69, 70). The overall activity of the mitochondrial respiratory chain is reduced in T2DM and obese individuals compared with lean controls and the mitochondria in skeletal muscle of T2DM and obese subjects is smaller than those from lean controls (71). While the fatty acid oxidation rate is reduced, fatty acid uptake by skeletal muscle is increased in obesity and insulin resistance. Fatty acid transporters in the plasma membrane of skeletal muscle cells from obese and diabetic humans are increased (72). Interestingly, high-fat diet caused a rapid increase of fatty acid transporter CD36 in the plasma membrane of skeletal muscle cells in rats with correlated increase in DAG and ceramide (73). This occurs at two weeks after on high-fat diet, which precedes the onset of insulin resistance (73), indicating that increased fatty acid uptake and intracellular accumulation of fatty acyl metabolites may be the cause of insulin resistance. Consistent with this, a hypothesis was proposed that incompletely oxidized products from fatty acid b-oxidation induce insulin resistance in cells. In summary, mitochondrial overload contributes to the development of insulin resistance (74).

KEY PATHWAYS THAT IMPACT MUSCLE METABOLISM PGC-1a and Biogenesis in Skeletal Muscle The peroxisome proliferator-activated receptor g (PPARg) coactivator 1a (PGC-1a) is a cold-inducible nuclear transcriptional activator that binds to PPARg and increases its transcriptional activity (75). PGC-1a is expressed in tissues with high metabolic activity, including brown adipose tissue, skeletal muscle, heart, liver, and brain (75–77). It stimulates mitochondrial biogenesis by increasing the expression of key enzymes of the respiratory chain and the cellular content of mitochondrial

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DNA (75, 78). In muscle cells, PGC-1a stimulates mitochondrial biogenesis and respiration via induction of uncoupling protein 2 (UCP-2) and the nuclear respiratory factors (NRFs), NRF-1 and NRF-2. (78). In addition, PGC-1a binds to and coactivates the transcriptional function of NRF-1 to increase mitochondrial DNA replication and transcription. PGC-1a also increases the expression of the genes involved in oxidative phosphorylation (OXPHOS) (79, 80), suggesting that it plays a key role in energy metabolism in tissues with high metabolic activity. Since increased intramyocellular accumulation of fatty acyl metabolites has been demonstrated as a cause of muscle insulin resistance, PGC-1a induced mitochondrial biogenesis is expected to increase b-oxidation and improve insulin sensitivity. Although it is not clear if the dysregulation of PGC-1a expression or activity is involved in the pathogenesis of type 2 diabetes, several lines of evidence suggest alterations in PGC-1a expression or function are associated with insulin resistance. A Gly482Ser polymorphism in PGC-1a increased the risk of type 2 diabetes in Danish and Japanese populations (81, 82). However, no association of this polymorphism with type 2 diabetes was noted in French or Australian populations or Pima Indians (83–85). The expression of PGC-1a and OXPHOS genes is reduced in muscle biopsies of type 2 diabetic patients, first-degree relatives of type 2 diabetics, and subjects with impaired glucose tolerance (79, 80) as well as animal models of insulin resistance and type 2 diabetes (86, 87), suggesting that the reduction in PGC1a expression could lead to decreased biogenesis and insulin resistance in the skeletal muscle of type 2 diabetic patients. Consistent with this notion, reduction in PGC-1a is associated with elevated fasting insulin levels and body mass index (BMI) in Pima Indians (88). However, caution should be taken when interpreting these results because according to a separate study, PGC-1a expression in the muscle of type 2 diabetic patents was unchanged despite reduced mitochondrial density and insulin signaling (89). The important role of PGC-1a in muscle metabolism is implicated in its ability to regulate glucose uptake and fiber-type switch. PGC-1a is likely to be one of the mediators of metabolic effects by muscle contraction during exercise. PGC-1a expression is induced by either a single bout of exercise (90, 91) or exercise training (87, 92, 93). AMPK is activated in skeletal muscle during contraction and stimulates PGC-1a expression (87, 94). In vitro, PGC-1a overexpression increased insulin-stimulated glucose uptake by stimulating GLUT4 expression in C2C12 cells (95). However, in vivo only when overexpressed modestly, PGC-1a induced GLUT4 expression and increased insulin-dependent glucose uptake (96). When PGC1a was overexpressed in very high levels, both GLUT4 expression and insulin sensitivity were reduced (97), probably due to indirect effect of increased fatty acid uptake. The physiological role of PGC-1a in determining muscle fiber type was revealed by genetic studies. In humans, PGC-1a expression is higher in red oxidative muscle fibers than white glycolytic muscle fibers (93). Transgenic overexpression of PGC-1a in the skeletal muscle of mice induced fiber-type conversion (98). In these animals, the glycolytic type II fibers were redder and had increased expression of genes of mitochondrial oxidative metabolism (98), which is characteristic of the oxidative type I fibers (98). Consistent with these findings, skeletal muscle-specific

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PGC-1a knockout mice had a shift from oxidative type I and type IIa fibers toward type IIx and IIb fibers (99). Further, these knockout animals had reduced endurance capacity (99), consistent with the observation of fiber-type switch. These findings suggest that PGC-1a is an important factor in regulating muscle fiber type determination. Paradoxically, despite the increased muscle mitochondrial density and correlated ATP synthesis in the animals with muscle-specific overexpression of PGC-1a, there was no change in whole-body energy expenditure (100). More surprisingly, the transgenic mice were more prone to fat-induced insulin resistance with decreased insulin-stimulated muscle glucose uptake (100). In these animals, PGC-1a overexpression was sixfold (100). Consistent with another study with 10- to 13-fold overexpression, both muscle GLUT4 expression and whole-body insulin sensitivity were reduced (97). These effects could be due to the elevated intramyocellular lipid accumulation caused by superphysiologic PGC-1a overexpression (100). With modest PGC-1a overexpression by 25%, there was increased muscle GLUT4 expression and insulin-stimulated glucose uptake (96). Taken together, these data support the role of PGC-1a as a metabolic regulator in skeletal muscle that mediates beneficial effects. Consistent with this notion, skeletal musclespecific PGC-1a knockout mice had reduced muscle GLUT4 expression and exhibited impaired glucose tolerance (101).

Myostatin Signaling Myostatin is a member of the TGFb superfamily of secreted factors. It exists in a protein complex including its propeptide and follistatin-like 3 where its activity is inhibited. Myostatin is a key secreted factor that regulates muscle mass (102). Loss of function in myostatin leads to strong muscle growth in mice and cattle (102, 103). Myostatin mutation is associated with muscle hypertrophy in a child (104). Inhibition of myostatin by neutralizing antibodies or antagonists resulted in increased muscle mass in adult normal mice or mice with muscular dystrophy (105–107). Both activin receptor type IIA (ActR-IIA) and IIB (ActR-IIB) mediates myostatin signaling, although myostatin has a higher affinity for ActR-IIB (108, 109). Further, both receptors bind multiple ligands (110). Activation of these receptors results in the activation of several Smad proteins, leading to their oligomerization and translocation of the complex to the nucleus, where it suppresses the transcription of genes important in muscle growth (111, 112). Myostatin-deficient mice have increased muscle growth and reduced fat mass, and corresponding increases in muscle strength (113). These changes are also associated with alterations in muscle fiber type distribution with a greater proportion of type IIb fibers (113), which explains short contraction and relaxation times in these animals. A separate study reported similar findings that there are more fast and glycolytic fibers in myostatin-deficient mice (114). Loss of myostatin function not only resulted in increased muscle mass but also improved insulin sensitivity (115). Myostatin knockout mice had increased carbohydrate utilization for energy but the overall lipid utilization per animal did not

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change (115). The knockout mice had improved glucose and insulin tolerance, and reduced blood glucose and insulin levels (115). Further, they had increased wholebody glucose utilization rate as revealed in a hyperinsulinemic-euglycemic clamp study (115), with correlated increases in glucose utilization by white and brown adipose tissues and a trend toward elevation in skeletal muscle (115). The mechanism of myostatin inhibition was investigated by overexpressing a dominant negative myostatin molecule in either muscle or fat to block myostatin signaling tissuespecifically. Muscle-specific blockade had profound effects, resulting in increased muscle mass and reduced fat mass; while fat-specific blockade had no effect on body composition, and glucose and insulin tolerance remained unchanged in these animals (115). These data suggest that the metabolic effects by myostatin inhibition are mainly mediated by blockade of signaling in skeletal muscle, and the decreased fat mass is an indirect result of metabolic changes in skeletal muscle (115). Further, these findings indicate that myostatin action in skeletal muscle is more relevant to insulin sensitivity. Other studies showed that genetic deficiency in myostatin led to reduced fat accumulation and increased muscle mass, and conferred resistance to diet-induced obesity (116–118), protected liver against obesity-induced insulin resistance (119). Consistent with these findings, transgenic overexpression of myostatin propeptide, which is expected to bind to myostatin and neutralize its activity, prevented dietinduced obesity and insulin resistance (120).

Adipokines and Myokines A number of adipokines from adipose tissue mediate metabolic effects in skeletal muscle and impacts its insulin sensitivity. Adiponectin and leptin are two important players. In addition, secreted factors from skeletal muscle with endocrine functions (commonly termed myokines) may also regulate skeletal muscle insulin sensitivity. Together, adipokines and myokines may be part of a network of secreted factors from tissues that work in concert to mediate tissue cross talk and ultimately whole-body metabolic homeostasis. Adiponectin improves muscle insulin sensitivity (121), at least in part by stimulating fatty acid oxidation in skeletal muscle and decreasing lipid accumulation (122, 123). This effect is mediated by the activation of AMPK (122, 123). There are two isoforms of adiponectin receptors, AdipoR1 and AdipoR2, although there is some controversy as to whether these receptors indeed mediate adiponectin signaling. AdipoR1 is the main isoform expressed in skeletal muscle. The skeletal muscle from obese and insulin-resistant humans confers adiponectin resistance, which is characterized with impaired AMPK activation and reduced stimulation of FA oxidation (124, 125). The onset of adiponectin resistance precedes lipid accumulation and the development of insulin resistance in skeletal muscle (73), suggesting that adiponectin response in skeletal muscle may be critical suppressor of lipid-derived signals that cause muscle insulin resistance. Like adiponectin, leptin also improves muscle insulin sensitivity (126, 127). Leptin stimulates fatty acid oxidation (128) and decreases fatty acid uptake (129). The leptin effect is at least partially mediated by AMPK activation (130). Under obese and insulin-resistant

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state, there is leptin resistance. The mechanism is not clear but at least impaired AMPK activation is involved. IL-6 is both an adipokine and a myokine in that it is secreted from both tissues under different metabolic states (131). In addition, IL-6 is also secreted by endothelial cells, fibroblasts, pancreatic b-cells, and osteoblasts as well as immune cells such as monocytes and macrophages at inflammatory sites (131). IL-6 level is increased in inflammatory state and serves as a key regulator of inflammation. In addition, IL-6 also has metabolic functions. IL-6 suppresses insulin action in both liver and adipose tissue (132); in contrast, it sensitizes insulin action in muscle (132). IL-6 release from muscle is elevated in response to exercise (133, 134), which may work in an autocrine fashion to stimulate glucose uptake and fatty acid oxidation in muscle (132). Given the complex metabolic functions of IL-6 in different tissues, its therapeutic value is likely to be limited. Fibroblast growth factor 21 (FGF-21) is metabolic regulator involved in the control of glucose and lipid metabolism (135, 136). FGF-21 is characterized as a myokine because in addition to other tissues, it is expressed in muscle and upon Akt activation its expression is elevated (137). In addition, FGF-21 expression is upregulated in cultured skeletal muscle cells by insulin stimulation (137). Interestingly, FGF-21 expression in human skeletal muscle is induced by hyperinsulinemia (138). These findings suggest that FGF-21 is regulated by insulin action in skeletal muscle and may play a role in mediating the cross talk between skeletal muscle and other metabolic tissues. The details of FGF-21 biology are covered separately in Chapter 14 of this book.

PPARb/d Peroxisome proliferator-activated receptor b or d (PPARb/d) is a nuclear receptor that controls the expression of genes involved in fatty acid oxidation. Transgenic overexpression of PPARd in skeletal muscle induced a muscle fiber type switch toward increased numbers of type I fibers (139, 140). These animals had increased exercise endurance and were resistant to high fat-induced obesity and insulin resistance (139, 140). These data suggest that PPARd activation in skeletal muscle may activate fatty acid metabolism and increase insulin sensitivity. In humans, the whole-body insulin sensitivity is positively correlated with the relative abundance of slow-twitch oxidative fibers and negatively with that of glycolytic type II fibers (141), suggesting that fatty acid metabolism in skeletal muscle is an important factor for insulin sensitivity. Exercise is a metabolic state where increased lipid oxidation occurs. Both short and endurance training increased PPARd expression in human and rodent skeletal muscle (142, 143). Since PPARd upregulates the expression of genes involved fatty acid uptake and oxidation (144, 145), this change is a key part of the regulatory mechanism to activate muscle lipid metabolism in response to exercise. In the meantime, PPARd either directly upregulates genes involved in mitochondrial biogenesis or increases PGC-1a expression (146, 147), which is a master regulator of mitochondrial function. These data demonstrate that PPARd is an important

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metabolic regulator of lipid oxidation and mitochondrial biogenesis in skeletal muscle and its activity is important in muscle insulin sensitivity.

MUSCLE TYPES AND ASSOCIATED METABOLIC DIFFERENCES The skeletal muscle consists of both oxidative and glycolytic fibers. These fibers have different preferences for metabolic substrates and play distinct roles in muscle energy metabolism. The red muscles (type I and type IIa fibers) are mitochondria-rich and oxidative using FFAs as substrates. They are involved in prolonged physical activities/ fasting state when FFA oxidation is the main supply of energy source. The major fibers in the red muscles are slow-twitch type I and fast-twitch type IIa. The white muscles are fast-twitch, glycolytic type IIx fibers in humans (IIx and IIb in rodents) (148). Fiber switch from fast-twitch to slow-twitch type I fibers has been observed in mice with skeletal muscle-specific transgenic overexpression of either PGC-1a (98) or PPARd (139), implicating the importance of these transcriptional regulators in muscle substrate utilization. Interestingly, insulin sensitivity is positively associated with the relative abundance of slow-twitch oxidative fibers and negatively with that of glycolytic type II fibers (141). Not only the mitochondrial density is higher (149), insulin-stimulated glucose uptake is also greater in slow-twitch muscle fibers than fast-twitch muscle fibers (32, 150). These observations support the notion that increased proportion of slow-twitch fibers may provide metabolic benefits such as increased energy expenditure and improved muscle insulin sensitivity. However, this is not to say that fast-twitch, glycolytic fibers are not metabolically beneficial. As a matter of fact, absolute increase in muscle mass, even in the form of glycolytic type IIb fibers, leads to improved insulin sensitivity. This has been exemplified in myostatindeficient mice (113, 115).

EFFECTS OF MUSCLE INSULIN RESISTANCE ON OTHER TISSUES The existence of adipokines and myokines suggest that there is metabolic cross talk between tissues. Muscle-specific transgenic overexpression of Akt induced the expression of myokines that mediate metabolic effects in distant tissues such as liver and adipose. Muscle-specific Akt transgenic mouse induced the expression of myokines that mediate metabolic effects in distant tissues such as liver and adipose (151). Further, improvement of insulin sensitivity in one tissue could indirectly leads to better insulin sensitivity in another tissue. This could be achieved through the beneficial effects on the improvement of the whole-body glucose homeostasis and insulin sensitivity. Overexpression of malonyl-CoA decarboxylase (MCD) in liver is expected to reduce malonyl-CoA level and activate FFA oxidation, which results in the improvement of hepatic insulin sensitivity. In the meantime, insulin sensitivity in skeletal muscle was also improved (152). This effect may be mediated directly by a secreted factor. Alternatively, it could be an indirect effect of improved overall

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metabolic homeostasis. Another example for tissue cross talk is that the impairment of metabolism in one tissue leads to the increased pressure for another tissue to take on more metabolic substrates. Insulin resistance in skeletal muscle is characterized with decreased muscle glycogen synthesis, which can change the ingested carbohydrate away from muscle glycogen synthesis to hepatic lipogenesis (67). In this case, skeletal muscle promotes the development of dyslipidemia, due to increased hepatic triglyceride synthesis and secretion (67). This notion is exemplified by increased hepatic lipogenesis following ingestion of high carbohydrate meals in young, lean, insulinresistant subjects compared with age–weight–BMI–activity-matched, insulinsensitive control subjects (67).

SUMMARY Skeletal muscle is one of the major carbohydrate and FFA utilizing tissues in the body, and its metabolic state plays an important role in glucose homeostasis. Insulin sensitivity and mitochondrial biogenesis in skeletal muscle are important factors in energy expenditure, glucose homeostasis, and the development of lipid disorders. Improvement of skeletal muscle insulin sensitivity by either increasing energy metabolism or directly improving insulin sensitivity will be major targets for the treatment of obesity and T2DM.

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100. CHOI, C.S., D.E. BEFROY, R. CODELLA, S. KIM, R.M. REZNICK, Y.J. HWANG, Z.X. LIU, H.Y. LEE, A. DISTEFANO, V.T. SAMUEL, D. ZHANG, G.W. CLINE, C. HANDSCHIN, J. LIN, K.F. PETERSEN, B.M. SPIEGELMAN, and G.I. SHULMAN. 2008. Paradoxical effects of increased expression of PGC-1alpha on muscle mitochondrial function and insulin-stimulated muscle glucose metabolism. Proc Natl Acad Sci USA 105:19926–19931. 101. HANDSCHIN, C., C.S. CHOI, S. CHIN, S. KIM, D. KAWAMORI, A.J. KURPAD, N. NEUBAUER, J. HU, V.K. MOOTHA, Y.B. KIM, R.N. KULKARNI, G.I. SHULMAN, and B.M. SPIEGELMAN. 2007. Abnormal glucose homeostasis in skeletal muscle-specific PGC-1alpha knockout mice reveals skeletal muscle-pancreatic beta cell crosstalk. J Clin Invest 117:3463–3474. 102. MCPHERRON, A.C., A.M. LAWLER, and S.J. LEE. 1997. Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member. Nature 387:83–90. 103. MCPHERRON, A.C., and S.J. LEE. 1997. Double muscling in cattle due to mutations in the myostatin gene. Proc Natl Acad Sci USA 94:12457–12461. 104. SCHUELKE, M., K.R. WAGNER, L.E. STOLZ, C. HUBNER, T. RIEBEL, W. KOMEN, T. BRAUN, J.F. TOBIN, and S. J. LEE. 2004. Myostatin mutation associated with gross muscle hypertrophy in a child. N Engl J Med 350:2682–2688. 105. WHITTEMORE, L.A., K. SONG, X. LI, J. AGHAJANIAN, M. DAVIES, S. GIRGENRATH, J.J. HILL, M. JALENAK, P. KELLEY, A. KNIGHT, R. MAYLOR, D. O’HARA, A. PEARSON, A. QUAZI, S. RYERSON, X.Y. TAN, K.N. TOMKINSON, G.M. VELDMAN, A. WIDOM, J.F. WRIGHT, S. WUDYKA, L. ZHAO, and N.M. WOLFMAN. 2003. Inhibition of myostatin in adult mice increases skeletal muscle mass and strength. Biochem Biophys Res Commun 300:965–971. 106. BOGDANOVICH, S., T.O. KRAG, E.R. BARTON, L.D. MORRIS, L.A. WHITTEMORE, R.S. AHIMA, and T.S. KHURANA. 2002. Functional improvement of dystrophic muscle by myostatin blockade. Nature 420:418–421. 107. BOGDANOVICH, S., E.M. MCNALLY, and T.S. KHURANA. 2008. Myostatin blockade improves function but not histopathology in a murine model of limb-girdle muscular dystrophy 2C. Muscle Nerve 37:308–316. 108. LEE, S.J., and A.C. MCPHERRON. 2001. Regulation of myostatin activity and muscle growth. Proc Natl Acad Sci USA 98:9306–9311. 109. REBBAPRAGADA, A., H. BENCHABANE, J.L. WRANA, A.J. CELESTE, and L. ATTISANO. 2003. Myostatin signals through a transforming growth factor beta-like signaling pathway to block adipogenesis. Mol Cell Biol 23:7230–7242. 110. de CAESTECKER, M. 2004. The transforming growth factor-beta superfamily of receptors. Cytokine Growth Factor Rev 15:1–11. 111. LIU, D., B.L. BLACK, and R. DERYNCK. 2001. TGF-beta inhibits muscle differentiation through functional repression of myogenic transcription factors by Smad3. Genes Dev 15:2950–2966. 112. RIOS, R., I. CARNEIRO, V.M. ARCE, and J. DEVESA. 2002. Myostatin is an inhibitor of myogenic differentiation. Am J Physiol Cell Physiol 282:C993–C999. 113. MENDIAS, C.L., J.E. MARCIN, D.R. CALERDON, and J.A. FAULKNER. 2006. Contractile properties of EDL and soleus muscles of myostatin-deficient mice. J Appl Physiol 101:898–905. 114. GIRGENRATH, S., K. SONG, and L.A. WHITTEMORE. 2005. Loss of myostatin expression alters fiber-type distribution and expression of myosin heavy chain isoforms in slow- and fast-type skeletal muscle. Muscle Nerve 31:34–40. 115. GUO, T., W. JOU, T. CHANTURIYA, J. PORTAS, O. GAVRILOVA, and A.C. MCPHERRON. 2009. Myostatin inhibition in muscle, but not adipose tissue, decreases fat mass and improves insulin sensitivity. PLoS One 4:e4937. 116. MCPHERRON, A.C., and S.J. LEE. 2002. Appendix A Suppression of body fat accumulation in myostatin-deficient mice. J Clin Invest 109:595–601. 117. LIN, J., H.B. ARNOLD, M.A. DELLA-FERA, M.J. AZAIN, D.L. HARTZELL, and C.A. BAILE. 2002. Myostatin knockout in mice increases myogenesis and decreases adipogenesis. Biochem Biophys Res Commun 291:701–706. 118. HAMRICK, M.W., C. PENNINGTON, C.N. WEBB, and C.M. ISALES. 2006. Resistance to body fat gain in ‘double-muscled’ mice fed a high-fat diet. Int J Obes (Lond) 30:868–870.

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137. IZUMIYA, Y., H.A. BINA, N. OUCHI, Y. AKASAKI, A. KHARITONENKOV, and K. WALSH. 2008. FGF21 is an Akt-regulated myokine. FEBS Lett 582:3805–3810. 138. HOJMAN, P., M. PEDERSEN, A.R. NIELSEN, R. KROGH-MADSEN, C. YFANTI, T. AKERSTROM, S. NIELSEN, and B.K. PEDERSEN. 2009. Fibroblast growth factor-21 is induced in human skeletal muscles by hyperinsulinemia. Diabetes 58:2797–2801. 139. WANG, Y.X., C.L. ZHANG, R.T. YU, H.K. CHO, M.C. NELSON, C.R. BAYUGA-OCAMPO, J. HAM, H. KANG, and R.M. EVANS. 2004. Regulation of muscle fiber type and running endurance by PPARdelta. PLoS Biol 2:e294. 140. LUQUET, S., J. LOPEZ-SORIANO, D. HOLST, A. FREDENRICH, J. MELKI, M. RASSOULZADEGAN, and P.A. GRIMALDI. 2003. Peroxisome proliferator-activated receptor delta controls muscle development and oxidative capability. FASEB J 17:2299–2301. 141. LILLIOJA, S., A.A. YOUNG, C.L. CULTER, J.L. IVY, W.G. ABBOTT, J.K. ZAWADZKI, H. YKI-JARVINEN, L. CHRISTIN, T.W. SECOMB, and C. BOGARDUS. 1987. Skeletal muscle capillary density and fiber type are possible determinants of in vivo insulin resistance in man. J Clin Invest 80:415–424. 142. MAHONEY, D.J., G. PARISE, S. MELOV, A. SAFDAR, and M.A. TARNOPOLSKY. 2005. Analysis of global mRNA expression in human skeletal muscle during recovery from endurance exercise. FASEB J 19:1498–1500. 143. FRITZ, T., D.K. KRAMER, H.K. KARLSSON, D. GALUSKA, P. ENGFELDT, J.R. ZIERATH, and A. KROOK. 2006. Low-intensity exercise increases skeletal muscle protein expression of PPARdelta and UCP3 in type 2 diabetic patients. Diabetes Metab Res Rev 22:492–498. 144. DRESSEL, U., T.L. ALLEN, J.B. PIPPAL, P.R. ROHDE, P. LAU, and G.E. MUSCAT. 2003. The peroxisome proliferator-activated receptor beta/delta agonist, GW501516, regulates the expression of genes involved in lipid catabolism and energy uncoupling in skeletal muscle cells. Mol Endocrinol 17:2477–2493. 145. SPRECHER, D.L., C. MASSIEN, G. PEARCE, A.N. BILLIN, I. PERLSTEIN, T.M. WILLSON, D.G. HASSALL, N. ANCELLIN, S.D. PATTERSON, D.C. LOBE, and T.G. JOHNSON. 2007. Triglyceride: high-density lipoprotein cholesterol effects in healthy subjects administered a peroxisome proliferator activated receptor delta agonist. Arterioscler Thromb Vasc Biol 27:359–365. 146. SCARPULLA, R.C. 2008. Transcriptional paradigms in mammalian mitochondrial biogenesis and function. Physiol Rev 88:611–638. 147. HANCOCK, C.R., D.H. HAN, M. CHEN, S. TERADA, T. YASUDA, D.C. WRIGHT, and J.O. HOLLOSZY. 2008. High-fat diets cause insulin resistance despite an increase in muscle mitochondria. Proc Natl Acad Sci USA 105:7815–7820. 148. SPANGENBURG, E.E., and F.W. BOOTH. 2003. Molecular regulation of individual skeletal muscle fibre types. Acta Physiol Scand 178:413–424. 149. SCHIAFFINO, S., and A. SERRANO. 2002. Calcineurin signaling and neural control of skeletal muscle fiber type and size. Trends Pharmacol Sci 23:569–575. 150. HENRIKSEN, E.J., R.E. BOUREY, K.J. RODNICK, L. KORANYI, M.A. PERMUTT, and J.O. HOLLOSZY. 1990. Glucose transporter protein content and glucose transport capacity in rat skeletal muscles. Am J Physiol 259:E593–E598. 151. WALSH, K. 2009. Adipokines, myokines and cardiovascular disease. Circ J 73:13–18. 152. AN, J., D.M. MUOIO, M. SHIOTA, Y. FUJIMOTO, G.W. CLINE, G.I. SHULMAN, T.R. KOVES, R. STEVENS, D. MILLINGTON, and C.B. NEWGARD. 2004. Hepatic expression of malonyl-CoA decarboxylase reverses muscle, liver and whole-animal insulin resistance. Nat Med 10:268–274.

Part Two

Metabolic Diseases and Current Therapies

Chapter

7

Mechanisms and Complications of Metabolic Syndrome MINGHAN WANG Department of Metabolic Disorders, Amgen, Inc., Thousand Oaks, CA, USA

INTRODUCTION Metabolic syndrome is a cluster of metabolic abnormalities that are usually found in individuals with high risk of non-insulin-dependent diabetes mellitus (NIDDM) or type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD). The abnormalities usually include obesity, insulin resistance (hyperinsulinemia), dyslipidemia (hypertriglyceridemia and low high-density lipoprotein (HDL)), hypertension, and hyperglycemia. Not every element of these disorders is found in the same patient. However, existence of three or more of these factors warrants diagnosis of the syndrome. Metabolic syndrome is a major risk factor to develop CVD and T2DM. For example, about 80% of T2DM patients have metabolic syndrome, underscoring the importance of disease awareness and the need of early treatment of high-risk populations. However, the concept of metabolic syndrome is debatable for a number of reasons. First, there is no single unifying mechanism for all the metabolic disorders involved, raising questions about the existence of the syndrome. Further, the criteria for the definition of the syndrome are different among various medical groups, making it difficult to have consistent diagnosis. Since the syndrome is not recognized as a disease by regulatory authorities, there is no clear clinical and regulatory path for drug development and approval. Despite these challenges, metabolic syndrome is still a useful concept to identify and manage risk factors in subjects with high risk of developing T2DM and CVD. It is also important to identify a high-risk T2DM and/or

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CVD population for prevention. Since each of the individual risk factors needs to be managed, understanding the etiology and interactions of these elements is important. Thus, drug discovery efforts to treat and minimize these risk factors could benefit from the concept of metabolic syndrome.

THE DEFINITION OF METABOLIC SYNDROME In 1988, Reaven first discovered that several cardiovascular risk factors tend to cluster together, including resistance to insulin-stimulated glucose uptake, glucose intolerance, hyperinsulinemia, increased plasma very-low-density lipoprotein (VLDL) triglyceride concentration, decreased HDL cholesterol concentration, and hypertension (1). At that time he defined this series of metabolic abnormalities as “syndrome X” (1). The insulin-stimulated glucose uptake in his observation was glucose disposal rate (Rd), which was measured by glucose clamp in human subjects, and therefore represents whole-body insulin sensitivity. Glucose intolerance refers to the reduced ability for the body to normalize plasma glucose in response to a 75 g oral glucose challenge. Since glucose normalization under such as a condition is driven by the insulin sensitivity of the peripheral tissues and the ability of pancreatic b-cells to secrete sufficient insulin, glucose intolerance is the result of insulin resistance or reduced glucose-dependent insulin secretion or combination of both. Reaven did not include obesity in the definition of syndrome X, but he indicated that the degree of obesity and sedentary lifestyle are correlated with the extent of insulin resistance regardless of genetic influences (1). The concept of syndrome X has evolved over the past 20 years and is more widely referred to as metabolic syndrome. In the meantime, the individual metabolic abnormalities are more closely defined as obesity (or central obesity), insulin resistance (hyperinsulinemia), hyperglycemia, dyslipidemia (high triglycerides and low HDL), and hypertension. The exact definition of metabolic syndrome varies among several professional organizations. However, the common features are similar. The National Cholesterol Education Program’s Adult Treatment Panel III report (ATPIII) definition includes the following risk factors: abdominal obesity (given as waist circumference), hypertriglyceridemia, low HDL, hypertension, and fasting hyperglycemia (2). While the World Health Organization (WHO) definition has more detailed criteria, it includes similar risk factors (2, 3). In addition, the WHO criteria include microalbuminuria (2, 3). Other risk factors such as family history of T2DM and high-risk ethnic group are included in the American Association of Clinical Endocrinologists (AACE) definition (4). Not all the metabolic abnormalities are found in the same individual; the diagnosis can be made if three or more characteristics are found in the same patient. In addition to the factors included in the variety of definitions, there are risk factors that are not apparent or easily measured but are critical in the development of CVD, such as endothelial dysfunction, increased vascular smooth muscle proliferation, vascular inflammation, and atherosclerosis. In obese individuals, there is increased inflammatory state in the adipose tissue characterized by macrophage accumulation (5), which is believed to be a major contributor to the

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development of insulin resistance. The main clinical outcomes of metabolic syndrome are CVD and T2DM. In the Kuopio Ischemic Heart Disease Risk Factor Study, 1209 Finnish men aged 42–60 years at baseline (1984–1989) who were initially without T2DM, CVD, or cancer were followed up through December 1998 (6). The study found that men with metabolic syndrome were several times more likely to die of CVD (6). Metabolic syndrome as defined by the WHO was associated with several times higher CVD mortality and about two times higher all-cause mortality (6). In the Framingham study, metabolic syndrome alone predicted about 25% of newly onset CVD (2). In the absence of diabetes, metabolic syndrome did not raise the 10-year risk of CVD to more than 20% (2). Almost half of the population-attributable risk for T2DM could be explained by the presence of metabolic syndrome (2). In addition, individuals with the syndrome are also susceptible to other conditions such as polycystic ovary syndrome, fatty liver, cholesterol gallstones, asthma, sleep disturbances, and cancer (2). Using the ATPIII definition and survey data collected between 1988 and 1994, a study found that the age-adjusted prevalence of metabolic syndrome among the U.S. adult population is more than 20% (7). The prevalence increases with age from 6.7% among people at 20–29 years of age to 43.5% among those at 60–69 years of age (7). Both men and women have similar age-adjusted prevalence (7). Mexican Americans have the highest prevalence and among both African and Mexican Americans, women have higher prevalence than men (7). In a study involving subjects from the European population between 1978 and 1987, the prevalence of metabolic syndrome was much lower (slightly over 10%) (8). A recent report using data from 1999–2000 indicates that the age-adjusted prevalence increased by 23.5% among women and 2.2% (not statistically significant) among men (9). Much of the increase is accounted for by increases in high blood pressure, waist circumference, and hypertriglyceridemia, particularly among women (9).

OBESITY AND INSULIN RESISTANCE Although decades of research efforts have been devoted to understanding the etiology of metabolic syndrome, there is no single causal mechanism that can explain the constellation of the risk factors in metabolic syndrome. Obesity and insulin resistance are believed to be central components but there is no concrete mechanistic evidence to support this notion. Insulin resistance is a common attribute in metabolic syndrome and T2DM. Tissue insulin resistance leads to glucose intolerance, which is compensated by increased insulin secretion by pancreatic b-cells to maintain normoglycemia. As a result, the patient develops hyperinsulinemia. Insulin resistance and the resultant hyperinsulinemia are believed to be involved in the development of other disorders in metabolic syndrome, such as hypertension and dyslipidemia (10). In this regard, some researchers even suggest that insulin resistance is likely the initiating factor of T2DM and other related complications. However, insulin resistance alone does not cause diabetes; b-cell failure is an important causal factor as well. Many insulin-resistant individuals do not develop T2DM because their b-cells can secrete

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sufficient insulin to counter insulin resistance. Nonetheless, insulin resistance is an important metabolic alteration that may set the stage for incremental loss of b-cell mass leading to b-cell failure. Insulin resistance in tissues that are important in metabolic response and energy homeostasis such as liver, adipose, and skeletal muscle is exemplified by distinct biological alterations. In liver, the ability of insulin to suppress glucose production is attenuated; in adipose tissue and skeletal muscle, there is reduced insulin-stimulated glucose uptake. These metabolic alterations result in both fasting and postprandial hyperglycemia. Impaired free fatty acid (FFA) metabolism could also contribute to increased hepatic glucose production and hyperglycemia (11). One of the normal functions of insulin is to suppress lipolysis in adipose tissue. Visceral fat is hyperlipolytic compared to subcutaneous fat and is resistant to the antilipolytic effect of insulin (12). Visceral obesity is associated with increased lipolysis due to its reduced sensitivity to insulin. This leads to increased FFA flux to liver promoting lipogenesis and hepatic insulin resistance. These disturbances further promote hyperglycemia and dyslipidemia (11). One important aspect of insulin resistance is the concomitant hyperinsulinemia, which occurs as a result of increased insulin release by pancreas to overcome the reduced insulin potency. Insulin resistance is believed to act through hyperinsulinemia to promote the development of other CVD risk factors (10). Although genetic defects and ethnic susceptibility are important contributors, the widespread obesity epidemic in the industrialized nations is primarily due to excessive energy intake and sedentary lifestyles. Compelling evidence indicates that Pima Indians have very high prevalence of obesity and T2DM. A study found that Pima Indians in Arizona are more obese than Mexican Pimas (13), and their prevalence of T2DM is several times higher than that in Mexican Pimas (13). This finding suggests that despite the genetic predisposition to these conditions, traditional lifestyles play a role in the development of obesity and T2DM. The link between obesity and insulin resistance has been well established. It is known that obesity, especially visceral (central) obesity, is correlated with insulin resistance and other cardiovascular risk factors (1). The prevalence of diabetes in an urban Asian Indian population is several times higher than that in the population living in a rural area with lower degree of obesity (14). This observation underscores the importance of obesity in the development of insulin resistance and diabetes. Obese people have postprandial hyperinsulinemia, which is characteristic of insulin resistance (15). Insulin sensitivity declines as body weight increases (16). The hyperinsulinemia in obese individuals correlates with the degree of insulin resistance but fails to fully compensate for insulin resistance (16). Therefore, hyperglycemia is often associated with insulin resistance. One potential underlying mechanism for obesity-induced insulin resistance is fat accumulation in liver and skeletal muscle. Elevated plasma FFA levels are observed in obese subjects and are associated with insulin resistance (17). This results in increased delivery of FFA into cells causing accumulation of fatty acyl metabolites, which can induce insulin resistance in liver and muscle (18). Thus, obesity plays an important role in the development of insulin resistance. However, obesity is not the absolute cause of insulin resistance because not every obese individual is insulin resistant (19). Rather, there are large variations in insulin

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sensitivity within the obese population (19). Some individuals are relatively insulin sensitive in the obese population (19), suggesting that there is a component of insulin sensitivity independent of obesity. Consistent with this notion, insulin sensitivity also varies in lean subjects due to differences in body fat distribution (20). These observations suggest that simple obesity per se does not necessarily induce insulin resistance. Obese individuals with only high subcutaneous fat depot are less likely to develop insulin resistance as those with central obesity (more fat distributed in the abdominal area). So when it comes to the link between insulin resistance and obesity, body fat distribution is as important as obesity itself. However, it is not clear if visceral fat is a causal factor or simply a better marker for insulin resistance. Compelling evidence indicates that insulin resistance is more closely associated with visceral adiposity. For this reason, the NCEP-ATPIII definition of metabolic syndrome identifies waist circumference rather than body mass index (BMI) as a risk factor to recognize the importance of central obesity (2). Certain ethnic groups are more prone to central obesity, such as Asians (21, 22), which may explain higher prevalence of T2DM at low BMI values. Less subcutaneous fat and more visceral adiposity are usually associated with more fat accumulation in liver, leading to elevated intracellular fatty acyl metabolites as mentioned above and greater hepatic insulin resistance (18). As a matter of fact, fatty liver alone is associated with strong insulin resistance. For instance, hepatic insulin resistance is found in patients with nonalcoholic steatohepatitis (NASH) or fatty liver disease (NAFLD) (23). Central obesity and reduced subcutaneous fat are found in patients with Cushing’s syndrome due to glucocorticoid excess (24). These patients develop severe insulin resistance (24), which at least can be explained by altered fat distribution. Similar findings were also made in some human immunodeficiency virus (HIV)-infected patients who were on combined highly active antiretroviral therapy (HAART) and developed a lipodystrophic syndrome. The condition is characterized with loss of subcutaneous fat, accumulation of abdominal fat, hypertriglyceridemia, and insulin resistance (25). This condition is also referred to as pseudo-Cushing’s syndrome because the fat distribution in these patients is similar to that in Cushing’s syndrome. Reducing hepatic lipid content is believed to improve insulin sensitivity. One of the beneficial effects of the thiazolidinediones (TZDs) is the reduction of hepatic fat accumulation (26), probably by increasing subcutaneous fat deposition (27).

HEPATIC INSULIN RESISTANCE AND DYSLIPIDEMIA Hypertriglyceridemia and low HDL are observed in individuals with metabolic syndrome and T2DM. Elevated low-density lipoprotein (LDL) levels are often seen in these patients. These are the characteristics of dyslipdemia that may have developed as a result of hepatic insulin resistance. Under normal conditions, plasma insulin has two hepatic actions. First, insulin suppresses gluconeogenesis by downregulating the expression of key gluconeogenic genes such as phosphoenolpyruvate

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carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase). This process is mainly mediated by insulin-stimulated phosphorylation of forkhead box O1 (FoxO1), a transcription factor that controls the expression of gluconeogenic genes (28). When phosphorylated, FoxO1 is retained in the cytosol and cannot enter the nucleus to induce gene expression (28). In addition, insulin also stimulates hepatic glycogen synthesis. As a result of these effects, hepatic glucose production is suppressed by insulin action. Second, insulin activates sterol regulatory element binding protein-1c (SREBP1c), a transcription factor that controls the expression of lipogenic genes (29, 30). Under the condition of hepatic insulin resistance, the FoxO1 pathway is not responsive to insulin suppression. Despite high insulin levels, the expression of hepatic gluconeogenic genes remains elevated. However, insulin is still capable of activating SREBP1c and inducing lipogenesis (30, 31). In the insulin-resistant state, there is compensatory hyperinsulinemia to overcome insulin resistance. The elevated plasma insulin is bound to increase lipogenesis at a level much higher than that under normal conditions, thereby causing elevated hepatic lipogenesis (31). The increased triglycerides are secreted in VLDL leading to hyperlipidemia. It is well known that HDL level is decreased in metabolic syndrome and T2DM patients. Although the precise mechanism remains to be elucidated, epidemiological studies have demonstrated an inverse relationship between plasma insulin and HDL levels (32–34). Consistent with these findings, HDL degradation rate is enhanced in T2DM patients (35). Thus, dyslipidemia is at least in part caused by insulin resistance and its resultant hyperinsulinemia. Dyslipidemia can further induce peripheral insulin resistance (36) since it leads to elevated lipid accumulation in muscle and adipose and impairment of insulin signaling (18). Although mounting evidence supports the notion that the gluconeogenic pathway is resistant to insulin action in T2DM and metabolic syndrome patients, a recent study indicates that the expression of hepatic PEPCK and G6Pase is not elevated in T2DM patients (37). This observation suggests that the failure of FoxO1 to respond to insulin action may not explain the increased gluconeogenesis under the insulin-resistant state. Rather, an alternative pathway may mediate hepatic insulin resistance. Recent work indicates that a transcription complex involving the cAMP response element binding protein (CREB), CREB binding protein (CBP), and transducer of regulated CREB activity 2 (TORC2) could play a role in connecting hepatic insulin resistance and increased gluconeogenesis (38). The CREB/TORC2/CBP complex controls the transcription of peroxisome proliferator-activated receptor g (PPARg) coactivator-1a (PGC-1a), which regulates the transcription of gluconeogenic genes. Since CREB is phosphorylated and constitutively occupies the PGC-1a promoter, the binding states of CBP and TORC2 determine the transcriptional activity of PGC-1a (38). Insulin negatively regulates the assembly of this complex. In the insulin-resistant state, the complex is more active leading to increased gluconeogenesis. This new finding helps further define the nature of hepatic insulin resistance and does not change the fact that augmented gluconeogenesis and dyslipdemia are the outcomes of hepatic insulin resistance. More details of hepatic insulin resistance are covered in Chapter 5.

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DEVELOPMENT OF HYPERTENSION Many studies have demonstrated the correlation of circulating insulin levels and blood pressure in obese individuals (39–41). In addition, healthy persons with hyperinsulinemia and normal glucose tolerance have increased blood pressure (42). Blood pressure elevation was not associated with body fat mass in obese women (43), and a decrease in blood pressure was not associated with a decrease in body fat (43). These findings suggest that insulin resistance is more likely related to hypertension than obesity, and hyperinsulinemia may be the link between hypertension and insulin resistance. After a period of chronic physical training, both systolic and diastolic blood pressure decreased in obese women (43), and these declines were associated with decreases in fasting insulin levels (43). Insulin resistance and glucose intolerance are not improved when hypertension is controlled using blood pressure lowering agents. However, improvement of insulin sensitivity and the resultant lowering of plasma insulin levels generally improve endothelial function and lower blood pressure. These observations suggest that hypertension may be a downstream disorder of insulin resistance, which may act through hyperinsulinemia and play a causal role in the development of hypertension. This, however, does not fully exclude the possibility that insulin resistance and hypertension can develop simultaneously under certain pathological conditions. And the progression of hypertension can be independent of insulin resistance and hyperinsulinemia. At the molecular level, insulin resistance can lead to hypertension through the regulatory effect on nitric oxide levels in endothelial cells. Nitric oxide is produced in vascular endothelial cells by endothelial nitric oxide synthase (eNOS) and regulates vasodilation. In insulin resistant state, there is reduced insulin-stimulated NO production in endothelial cells and increased NO destruction resulting in endothelial dysfunction and hypertension (44). A second mechanism for hypertension is also mediated by reduction of endothelial NO level. Mitochondrial dysfunction can occur in multiple tissues. In endothelial cells, it leads to elevated levels of reactive oxygen species (ROS), which decreases bioavailable NO resulting in endothelial dysfunction and hypertension (44). In the meantime, mitochondrial dysfunction in insulinsensitive tissues leads to insulin resistance (44, 45). In this case, hypertension develops in conjunction with insulin resistance but not through insulin resistance per se. Similarly, glucocorticoid excess can induce both insulin resistance and hypertension through separate mechanisms. Glucocorticoid excess induces insulin resistance by activating glucocorticoid receptor (GR) but it induces hypertension independently of insulin resistance by directly activating mineralocorticoid receptor (MR) (46).

MECHANISMS OF INSULIN RESISTANCE There are several potential mechanisms that may explain the development of insulin resistance. Although insulin resistance can occur in lean individuals, obesity, precisely central obesity, is closely correlated with insulin resistance in most subjects

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with metabolic syndrome. One hypothesis is that the elevated plasma FFAs in obese and T2DM patients are the primary cause of insulin resistance. First, increased FFAs can acutely induce insulin resistance (47). On the other hand, rapid reduction of FFAs by inhibiting lipolysis improves insulin sensitivity and glucose tolerance in lean and obese nondiabetic subjects and in obese patients with T2DM (48). These observations suggest that FFAs may contribute to the development of insulin resistance in subjects with metabolic syndrome. Compelling evidence suggests that increased circulating FFAs can induce insulin resistance in different ways. Intracellular accumulation of fatty acyl metabolites can induce insulin resistance by interfering with the insulin signaling pathway (18). Alternatively, the influx of FFAs from abdominal fat to liver can induce hepatic insulin resistance and augment glucose production (11). In a canine model with visceral adiposity promoted by high-fat diet, Bergman et al. demonstrated that hepatic insulin resistance occurred at a time when there was no peripheral insulin resistance (11, 49), suggesting that it is the portal FFAs from visceral fat that induced insulin resistance in liver before reaching the peripheral tissues. The peripheral insulin resistance is found mainly in adipose tissue and skeletal muscle. The common mechanism underlying insulin resistance in these tissues is the reduced ability of insulin to stimulate glucose uptake. Insulin-stimulated glucose uptake is mediated by glucose transporter 4 (GLUT4). In the insulin resistant state, there is a deficiency in insulin-stimulated GLUT4 translocation in the peripheral tissues. Shulman and colleagues demonstrated that this is caused by the impaired insulin signaling pathway in the cell (18). Elevated intracellular content of fatty acyl metabolites such as diacylglycerol (DAG) and fatty acyl-CoA due to hyperlipidemia and increased plasma FFAs activate a serine/threonine kinase pathway including protein kinase C (PKC) (18). These metabolites activate PKC leading to serine/ threonine phosphorylation of insulin receptor substrate 1 (IRS-1) and 2 (IRS-2), which inhibits insulin-stimulated tyrosine phosphorylation of these signaling molecules and reduces the ability of these molecules to activate phosphoinositide 3-kinase (PI3-kinase) (18). Insulin resistance can be induced by increased actions of proinflammatory cytokines and/or declined adipokine actions. It has been widely recognized that adipose tissue is not only a fat storage site but also an endocrine organ. Adipose tissue releases cytokines and adipokines that are involved in the regulation of insulin sensitivity. Recent research indicates that there is macrophage accumulation in adipose tissue as a result of increased fat cell apoptosis in obesity (5). In this regard, obesity is an inflammatory state where elevated cytokines such as TNFa and IL-6 promote insulin resistance. Activation of various inflammatory pathways leads to insulin resistance. This is supported by overwhelming evidence that inflammatory signals activate the c-Jun N-terminal protein kinase 1 (JNK1) and the inhibitor of NF-kB kinase-b (IKK-b), which interfere with the intracellular insulin signaling pathway (50, 51). In addition, increased adiposity is associated with reduced levels of adiponectin, an adipokine that increases insulin sensitivity by stimulating AMPK activation (52). Reduced adiponectin action in obesity may contribute to the development of insulin resistance. Other mechanisms that induce insulin resistance include

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glucocorticoid excess. In patients with Cushing’s syndrome, there is increased circulating cortisol level due to adrenal adenoma or elevated adrenocorticotropic hormone (ACTH) from pituitary tumors. Elevated circulating cortisol induces obesity and insulin resistance. As a result, patients with Cushing’s syndrome have central obesity, reduced subcutaneous fat, and hepatic steatosis. In addition, they exhibit severe insulin resistance along with hyperglycemia, hypertension, and dyslipidemia. These symptoms are typical of the constellation of the metabolic disturbances in metabolic syndrome. Given the remarkable resemblance of the set of disorders, these findings actually led to the hypothesis that metabolic syndrome could be caused by glucocorticoid excess. However, glucocorticoid excess is found only in a single percentage of T2DM patients, suggesting that glucocorticoid excess is not the primary cause of T2DM. Another mechanism that leads to insulin resistance is the age-related mitochondrial dysfunction. There is mounting evidence that as the body ages, the ability of mitochondria to oxidize substrates decreases. Increased lipid accumulation in cells leads to mitochondrial overload producing metabolites from incomplete FFA oxidation (53). As a result, partially oxidized products and reactive oxidative species are damaging to cells, which leads to insulin resistance (53).

DEVELOPMENT OF TYPE 2 DIABETES Although the development of diabetes is the result of incremental changes in glucose homeostasis, there are strict criteria for the definition of diabetes. Two tests can be used to diagnose diabetes. The blood glucose tests can be done under fasting condition. If an individual’s fasting glucose reaches 126 mg/dL or higher and confirmed thereafter, the individual is diagnosed to have diabetes. If the fasting glucose is between 100 and 125 mg/dL, it is considered impaired fasting glucose (IFG) or a prediabetes state. A second test is oral glucose tolerance test (OGTT). If the blood glucose level is between 140 and 199 mg/dL 2 h after taking 75 g oral glucose, the person has a form of prediabetes called impaired glucose tolerance (IGT). If the 2 h glucose level reaches 200 mg/dL or above and confirmed by repeating the test on another day, it means the person has diabetes. Both forms of prediabetic states implicate higher risk of developing diabetes. The manifestation of T2DM is marked by insulin resistance and b-cell failure. Before the onset of overt diabetes, individuals with obesity and insulin resistance have several risk factors for T2DM. The hepatic insulin resistance is characterized by increased gluconeogenesis and glucose output. In the meantime, the peripheral tissue insulin resistance impairs the ability of insulin to stimulate sufficient glucose uptake by these tissues, exemplified by decreased insulin-stimulated GLUT4 translocation. The insulin-resistant state is compensated by increased insulin secretion by the pancreatic b-cells. Increased insulin release can initially overcome the insulin resistance of peripheral tissues and sufficiently facilitate glucose transport. With the progression of insulin resistance, excessive insulin secretion cannot be maintained, at which point the insulin secretory capacity cannot keep up with the need to offset insulin resistance. Over time, b-cell exhaustion develops and further progresses into

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b-cell failure, at which stage hyperglycemia is further exacerbated to meet the criteria of diabetes. It is important to understand that the clinical course of the development of T2DM is progressive and the onset of the disease is a process of reduced b-cell mass. It is only at a point where b-cell failure leads to a certain degree of hyperglycemia that the diagnosis of T2DM can be made. At this point, the number of b-cells is reduced by about 50% (54), and the remaining cells operate at lower secretory capacity (55). IGT and IFG are two characteristics for the so-called prediabetic state, a condition in metabolic syndrome patients in transition to T2DM, depending on the state of b-cell health/failure. IGT and IFG are useful indicators/measures that may help predict the likelihood of T2DM manifestation. Prediabetes is an intermediate but alarming state prior to overt T2DM. It is associated with hyperglycemia but the glucose levels are not high enough to warrant diagnosis of diabetes. Compelling evidence suggests that increased cardiovascular risk factors at the prediabetic stage contribute to diabetic complications as the disease progresses into overt diabetes. Currently, there are 57 million people in the United States who have prediabetes. Most people with obesity and insulin resistance do not develop severe hyperglycemia or T2DM because the pancreatic b-cells increase insulin secretion sufficiently to overcome the reduced efficacy of insulin action. This observation underscores the importance of b-cell health in the pathogenesis of T2DM. Despite the fact that there are many proposed mechanisms for b-cell failure in T2DM patients, it is not clear what the major factor is. The decline in b-cell function is progressive just like the T2DM disease itself. Indeed, b-cell dysfunction exists in high-risk individuals even when their glucose levels are normal (56). It is widely accepted that in T2DM patients, glucotoxicity and lipotoxicity contribute significantly to b-cell dysfunction, loss of pancreatic b-cell mass, and eventually b-cell failure. Since insulin resistance is associated with hyperglycemia, elevated glucose levels could have toxic effects on b-cell function and these harmful effects can be mitigated by glucose lowering therapy (57). Under normal FFA levels, insulin secretion is not affected. However, chronic exposure of elevated FFAs to b-cells is associated with impaired insulin secretion and biosynthesis (58, 59). The lipotoxic effect is relevant because both hyperlipidemia and elevated FFA levels are observed in T2DM patients. Further, mitochondrial dysfunction in pancreatic b-cells may impair the capacity of b-cells to secrete insulin because of the importance of ATP/ADP ratio in insulin secretion (45).

MECHANISMS OF INCREASED CARDIOVASCULAR RISK IN T2DM Both T2DM patients and nondiabetic subjects with metabolic syndrome have increased risk of developing macrovascular complications, including coronary heart disease, stroke, and peripheral vascular disease, which may share the common pathogenic features of atherosclerosis, inflammation, and the prothrombotic state. In addition, microvascular complications, including retinopathy, nephropathy, and neuropathy, are commonly found in patients with late-stage T2DM. Hyperglycemia,

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hyperinsulinemia, and other metabolic disorders are major contributors to these metabolic consequences. There are several underlying mechanisms that are associated with the increased cardiovascular risk in individuals with T2DM and metabolic syndrome. These mechanisms are not only promoted directly by several elements of metabolic syndrome, such as hypertriglyceridemia, high LDL, low HDL, and hypertension, but also promoted by common metabolic disturbances such as oxidative stress, inflammation, endothelial dysfunction, vascular smooth muscle proliferation, and increased prothrombotic state. Some of these disturbances form the basis for the development of atherosclerosis. Hyperglycemia promotes vascular complications by inducing the expression of adhesion molecules in endothelial cells (60). Through the adhesion molecules, monocytes are recruited to the vascular wall and are involved in the pathogenesis of atherosclerotic lesions. Advanced glycation end products (AGEs) formed in the hyperglycemic state generate reactive oxygen species that can damage the vascular wall (61). In the meantime, both elevated glucose and AGE inhibit nitric oxide production by endothelial cells (62, 63), leading to impaired endothelial relaxation and vascular injury. Endothelial damage promotes the entry of activated monocytes into the subendothelial space, which secretes proinflammatory cytokines and differentiates into macrophages. Increased lipid uptake by macrophages results in the formation of foams cells, the hallmark of atherosclerosis. The development of atherosclerosis also involves proliferation and migration of vascular smooth muscle cells in concert with inflammation and apoptosis. In particular, the proinflammatory cytokines within atheroma direct leukocyte migration into the intima and induce lipid uptake by macrophages (64). C-reactive protein (CRP), an important biomarker and predictor for inflammation, is strongly correlated with the risk of atherosclerotic complications (64). The prothrombotic state in the atherosclerotic process involves several important molecules. PAI-1, fibrinogen, and von Willebrand factor (vWF), which are important markers of hemostasis and fibrinolysis, are associated with abdominal obesity and can be used as potential predictors for cardiovascular risk (65). Plasma PAI-1 levels are elevated in individuals with metabolic syndrome and are more closely related to liver steatosis than adipose tissue accumulation, although it is highly expressed in both visceral and subcutaneous adipose tissue (66). PAI-1 is also regulated by inflammatory signals. CRP increases PAI-1 expression in endothelial cells (67), suggesting that increased inflammation could induce the risk of thrombosis. Given the role of hyperglycemia in endothelial dysfunction, antidiabetic treatments may help reduce cardiovascular risks. In the UKPDS, metformin treatment was associated with a 39% reduction in cardiovascular disease (68). Insulin and SU treatments, although achieved similar glycemic control, exhibited trends of improvement of cardiovascular outcomes but statistical significance was not reached (68). Therapies that correct dyslipidemia, including LDL lowering and HDL raising agents, are effective approaches to improve cardiovascular outcomes. Typically used drugs are statins for lowering LDL, fibrates for reducing triglycerides, and niacin for raising HDL. In addition, antihypertensives and aspirin, which is used to reduce vascular inflammation, are commonly used. Weight loss has been an effective approach to mitigate multiple risk factors and decrease cardiovascular risk.

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ETHNIC VARIATIONS IN THE DEVELOPMENT OF METABOLIC ABNORMALITIES Studies involving individuals with different ethnic backgrounds indicate that certain ethnic groups are more susceptible to metabolic disorders than others. It is well known that Pima Indians have much higher risks of developing obesity and T2DM than other ethnic groups. They have inheritable genetic traits that predispose individuals to obesity and insulin resistance. In a study involving 384 Pima Indians with IGT, it was found that the cumulative incidence of T2DM was 25% and 61% at 5 and 10 years, respectively (69). While the high prevalence of obesity in Pima Indians is a risk factor for insulin resistance, other metabolic disturbances may also contribute to insulin resistance because Pima Indians were 17% more insulin resistant than Caucasians after accounting for differences in the degree of obesity (70). Pima Indians also have exaggerated early insulin response even after accounting for differences in insulin action, suggesting that the altered insulin secretion cannot be explained by insulin resistance (70). The enhanced b-cell sensitivity to glucose may play a role in the predisposition of this ethnic group to T2DM (70). However, decreased early insulin response could be a good predictor for the development of T2DM. For example, Mexican Americans have higher degree of insulin resistance and fasting hyperinsulinemia than Caucasians. In contrast to Pima Indians, they have decreased early insulin response to glucose excursion (71). It is not clear why opposite early insulin response patterns contribute to the risk of diabetes in these two high-risk ethnic groups. Other ethnic groups such as African-Americans and Hispanics have several fold elevated risk of developing T2DM compared to non-Hispanic Whites. Both nondiabetic African-Americans and Hispanics have increased insulin resistance and higher acute insulin response than nondiabetic non-Hispanic Whites (72). These alterations may be in large part responsible for the higher prevalence of T2DM in these minority groups (72). In a study comparing South Asians settled overseas with a European group, the South Asian group had several times higher prevalence of diabetes (19% versus 4%) than the European group (73). Moreover, they had higher blood pressures, higher fasting and post-glucose serum insulin concentrations, higher plasma triglyceride levels, and lower HDL concentrations (73). Ethnic differences are also associated with increased cardiovascular risk. Asian Indians have at least double the risk of coronary artery disease compared to Whites. This increased risk appears to be due to dyslipidemia characterized by plasma apolipoprotein B, lipoprotein(a), and triglycerides and low HDL levels. In addition, there is dysfunction in the small, dense HDL particles found in this population (74). In England and Wales, high mortality rate from CVD was found in all the migrant groups from South Asia, including Hindus and Sikhs from India and Muslims from Pakistan and Bangladesh (75, 76). Although Pima Indians have higher prevalence of developing T2DM, they have unique lipoprotein profiles. The plasma triglyceride levels in Pimas are higher than those in Europeans but cholesterol is lower (77). This could be due to differences in lipoprotein metabolism in Pimas. Interestingly, after accounting for the differences in the prevalence of T2DM, the mortality rates from

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CVD in Pimas are lower than those in Europeans (78). This could be due to the lower LDL concentrations in Pimas (79).

GENETIC MUTATIONS AS A RISK FACTOR In addition to the general risk factors such as excessive energy intake and sedentary lifestyles, genetics also plays a role in the development of metabolic abnormalities. Although T2DM in the general population is a polygenic disease, loss or gain of function in a single metabolic gene caused by mutations can increase the risks of insulin resistance and T2DM. The most common examples in which genetic mutations cause metabolic diseases are maturity onset diabetes of the young (MODY). There are eight types of MODYs corresponding to mutations in eight different metabolic genes (80). In MODY1, a loss-of-function mutation in HNF4a, a transcription factor that is involved in the regulation of pancreatic development, causes b-cell dysfunction (80). In MODY2, gain-of-function mutations in glucokinase activate the glucose sensor activity and increase insulin secretion, leading to hyperinsulinemic hypoglycemia (80). There are six additional types of MODYs involving other genes that may be important in the metabolic functions (80). Many studies have demonstrated that single nucleotide polymorphisms (SNPs) in certain genes predispose individuals to higher risks of obesity, insulin resistance, and T2DM. For example, Pima Indians are an ethnic group with increased risks of developing obesity and T2DM, and research efforts have identified a number of SNPs that may help explain the high prevalence of T2DM. These include SNPs in genes such as the Cav2.3 subunit of voltage-activated Ca2þ channels (81), 11b-hydroxysteroid dehydrogenase (11b-HSD1) (82), uncoupling protein-2 (UCP2) (83), and the activating transcription factor 6 gene (ATF6) (84). Using the same approach, many SNPs in a variety of other genes have been identified to be linked to risks of obesity and T2DM. The Pro12Ala polymorphism in PPARg is associated with decreased risk of T2DM (85). Strong linkage of the transcription factor 7-like 2 gene (TCF7L2) to susceptibility of T2DM has been demonstrated through SNP analysis. TCF7L2 is a transcription factor involved in the WNT signaling pathway and acts as a nuclear receptor for b-catenin (80). The strong association of TCF7L2 with T2DM has been demonstrated in populations of both European and Chinese ancestry (86, 87). Recent successes in genome-wide association (GWA) studies allow precise typing of a large number of SNPs and help capture many more genetic variants present in the human genome (80).

SUMMARY Metabolic syndrome is a group of metabolic disorders that are commonly found in subjects with increased T2DM and CVD risks. These disorders are associated with each other but the pathogenesis is not clear. Specifically, there is no single underlying mechanism that can explain the development of these metabolic abnormalities despite the fact that they cluster in the same patients. Recent evidence suggests that insulin

192

Excessive energy intake Sedentary lifestyles

Other unknown factors (important to β-cell health)

Glucotoxicity and lipotoxicity

Genetic predisposition and susceptibility

Metabolic syndrome • Central obesity • Insulin resistance • Dyslipidemia • Hypertension • Hyperglycemia Additional abnormalities: • Increased inflammatory state • Endothelial dysfunction • Prothrombotic state • Microalbuminuria • Atherosclerosis

β-cell failure

T2DM

Microvascular complications

Figure 7.1

CVD Stroke Peripheral vascular disease

Macrovascular complications

The development of metabolic syndrome, T2DM, and downstream complications. Among the variety of metabolic abnormalities discovered in metabolic syndrome, peripheral insulin resistance in combination with b-cell failure leads to T2DM. Both T2DM and metabolic syndrome lead to vascular diseases but T2DM is a more important and frequent cause than metabolic syndrome (highlighted by a thick arrow). Due to the lack of specific mechanisms, this figure represents only the general relationships of the disorders on the conceptual level.

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resistance might play a central and causal role in the development of dyslipidemia, hypertension, and hyperglycemia. Obesity is a major risk factor in the development of insulin resistance in general but insulin resistance can occur in lean individuals. Although some of the interrelationships between various metabolic dysfunctions have been proposed based on available evidence to date (Figure 7.1), mechanistic understanding of these links remains elusive. An important puzzle in understanding the development of T2DM is why some individuals with obesity and insulin resistance do not develop b-cell failure and hence do not have diabetes. Future studies should focus on dissecting molecular mechanisms that link the risk factors of metabolic syndrome and approaches to enhance the effectiveness in disease prevention, treatment, and management.

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37. SAMUEL, V.T., S.A. BEDDOW, T. IWASAKI, X.M. ZHANG, X. CHU, C.D. STILL, G.S. GERHARD, and G.I. SHULMAN. 2009. Fasting hyperglycemia is not associated with increased expression of PEPCK or G6Pc in patients with type 2 diabetes. Proc Natl Acad Sci USA 106:12121–12126. 38. HE, L., A. SABET, S. DJEDJOS, R. MILLER, X. SUN, M.A. HUSSAIN, S. RADOVICK, and F.E. WONDISFORD. 2009. Metformin and insulin suppress hepatic gluconeogenesis through phosphorylation of CREB binding protein. Cell 137:635–646. 39. BONORA, E., I. ZAVARONI, O. ALPI, A. PEZZAROSSA, F. BRUSCHI, E. DALL’AGLIO, L. GUERRA, C. COSCELLI, and U. BUTTURINI. 1987. Relationship between blood pressure and plasma insulin in non-obese and obese non-diabetic subjects. Diabetologia 30:719–723. 40. CHRISTLIEB, A.R., A.S. KROLEWSKI, J.H. WARRAM, and J.S. SOELDNER. 1985. Is insulin the link between hypertension and obesity? Hypertension 7:II54–II57. 41. LUCAS, C.P., J.A. ESTIGARRIBIA, L.L. DARGA, and G.M. REAVEN. 1985. Insulin and blood pressure in obesity. Hypertension 7:702–706. 42. ZAVARONI, I., E. BONORA, M. PAGLIARA, E. DALL’AGLIO, L. LUCHETTI, G. BUONANNO, P.A. BONATI, M. BERGONZANI, L. GNUDI, M. PASSERI, et al. 1989. Risk factors for coronary artery disease in healthy persons with hyperinsulinemia and normal glucose tolerance. N Engl J Med 320:702–706. 43. KROTKIEWSKI, M., K. MANDROUKAS, L. SJOSTROM, L. SULLIVAN, H. WETTERQVIST, and P. BJORNTORP. 1979. Effects of long-term physical training on body fat, metabolism, and blood pressure in obesity. Metabolism 28:650–658. 44. COOPER, S.A., A. WHALEY-CONNELL, J. HABIBI, Y. WEI, G. LASTRA, C. MANRIQUE, S. STAS, and J.R. SOWERS. 2007. Renin–angiotensin–aldosterone system and oxidative stress in cardiovascular insulin resistance. Am J Physiol Heart Circ Physiol 293:H2009–H2023. 45. KIM, J.A., Y. WEI, and J.R. SOWERS. 2008. Role of mitochondrial dysfunction in insulin resistance. Circ Res 102:401–414. 46. WANG, M. 2005. The role of glucocorticoid action in the pathophysiology of the metabolic syndrome. Nutr Metab (Lond) 2:3. 47. RODEN, M., T.B. PRICE, G. PERSEGHIN, K.F. PETERSEN, D.L. ROTHMAN, G.W. CLINE, and G.I. SHULMAN. 1996. Mechanism of free fatty acid-induced insulin resistance in humans. J Clin Invest 97:2859–2865. 48. SANTOMAURO, A.T., G. BODEN, M.E. SILVA, D.M. ROCHA, R.F. SANTOS, M.J. URSICH, P.G. STRASSMANN, and B.L. WAJCHENBERG. 1999. Overnight lowering of free fatty acids with Acipimox improves insulin resistance and glucose tolerance in obese diabetic and nondiabetic subjects. Diabetes 48:1836–1841. 49. KIM, S.P., M. ELLMERER, G.W. VAN CITTERS, and R.N. BERGMAN. 2003. Primacy of hepatic insulin resistance in the development of the metabolic syndrome induced by an isocaloric moderate-fat diet in the dog. Diabetes 52:2453–2460. 50. AGUIRRE, V., T. UCHIDA, L. YENUSH, R. DAVIS, and M.F. WHITE. 2000. The c-Jun NH(2)-terminal kinase promotes insulin resistance during association with insulin receptor substrate-1 and phosphorylation of Ser(307). J Biol Chem 275:9047–9054. 51. GAO, Z., D. HWANG, F. BATAILLE, M. LEFEVRE, D. YORK, M.J. QUON, and J. YE. 2002. Serine phosphorylation of insulin receptor substrate 1 by inhibitor kappa B kinase complex. J Biol Chem 277:48115–48121. 52. KADOWAKI, T., T. YAMAUCHI, N. KUBOTA, K. HARA, K. UEKI, and K. TOBE. 2006. Adiponectin and adiponectin receptors in insulin resistance, diabetes, and the metabolic syndrome. J Clin Invest 116: 1784–1792. 53. KOVES, T.R., J.R. USSHER, R.C. NOLAND, D. SLENTZ, M. MOSEDALE, O. ILKAYEVA, J. BAIN, R. STEVENS, J.R. DYCK, C.B. NEWGARD, G.D. LOPASCHUK, and D.M. MUOIO. 2008. Mitochondrial overload and incomplete fatty acid oxidation contribute to skeletal muscle insulin resistance. Cell Metab 7:45–56. 54. BUTLER, A.E., J. JANSON, S. BONNER-WEIR, R. RITZEL, R.A. RIZZA, and P.C. BUTLER. 2003. Beta-cell deficit and increased beta-cell apoptosis in humans with type 2 diabetes. Diabetes 52:102–110. 55. RODER, M.E., D. PORTE, JR., R.S. SCHWARTZ, and S.E. KAHN. 1998. Disproportionately elevated proinsulin levels reflect the degree of impaired B cell secretory capacity in patients with noninsulin-dependent diabetes mellitus. J Clin Endocrinol Metab 83:604–608. 56. KAHN, S.E. 2001. Clinical review 135: The importance of beta-cell failure in the development and progression of type 2 diabetes. J Clin Endocrinol Metab 86:4047–4058.

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57. GARVEY, W.T., J.M. OLEFSKY, J. GRIFFIN, R.F. HAMMAN, and O.G. KOLTERMAN. 1985. The effect of insulin treatment on insulin secretion and insulin action in type II diabetes mellitus. Diabetes 34:222–234. 58. SAKO, Y., and V.E. GRILL. 1990. A 48-hour lipid infusion in the rat time-dependently inhibits glucoseinduced insulin secretion and B cell oxidation through a process likely coupled to fatty acid oxidation. Endocrinology 127:1580–1589. 59. ZHOU, Y.P., and V.E. GRILL. 1994. Long-term exposure of rat pancreatic islets to fatty acids inhibits glucose-induced insulin secretion and biosynthesis through a glucose fatty acid cycle. J Clin Invest 93:870–876. 60. PIGA, R., Y. NAITO, S. KOKURA, O. HANDA, and T. YOSHIKAWA. 2007. Short-term high glucose exposure induces monocyte–endothelial cells adhesion and transmigration by increasing VCAM-1 and MCP-1 expression in human aortic endothelial cells. Atherosclerosis 193:328–334. 61. SCHLEICHER, E., and U. FRIESS. 2007. Oxidative stress, AGE, and atherosclerosis. Kidney Int Suppl S17–S26. 62. COHEN, R.A. 2005. Role of nitric oxide in diabetic complications. Am J Ther 12:499–502. 63. BUCALA, R., K.J. TRACEY, and A. CERAMI. 1991. Advanced glycosylation products quench nitric oxide and mediate defective endothelium-dependent vasodilatation in experimental diabetes. J Clin Invest 87:432–438. 64. LIBBY, P., P.M. RIDKER, and A. MASERI. 2002. Inflammation and atherosclerosis. Circulation 105:1135–1143. 65. MERTENS, I., and L.F. VAN GAAL. 2002. Obesity, haemostasis and the fibrinolytic system. Obes Rev 3:85–101. 66. ALESSI, M.C., D. BASTELICA, A. MAVRI, P. MORANGE, B. BERTHET, M. GRINO, and I. JUHAN-VAGUE. 2003. Plasma PAI-1 levels are more strongly related to liver steatosis than to adipose tissue accumulation. Arterioscler Thromb Vasc Biol 23:1262–1268. 67. DEVARAJ, S., D.Y. XU, and I. JIALAL. 2003. C-reactive protein increases plasminogen activator inhibitor1 expression and activity in human aortic endothelial cells: implications for the metabolic syndrome and atherothrombosis. Circulation 107:398–404. 68. UK, Prospective Diabetes Study Group. 1998. Effect of intensive blood-glucose control with metformin on complications in overweight patients with type 2 diabetes (UKPDS 34). UK Prospective Diabetes Study (UKPDS) Group. Lancet 352:854–865. 69. SAAD, M.F., W.C. KNOWLER, D.J. PETTITT, R.G. NELSON, D.M. MOTT, and P.H. BENNETT. 1988. The natural history of impaired glucose tolerance in the Pima Indians. N Engl J Med 319:1500–1506. 70. LILLIOJA, S., B.L. NYOMBA, M.F. SAAD, R. FERRARO, C. CASTILLO, P.H. BENNETT, and C. BOGARDUS. 1991. Exaggerated early insulin release and insulin resistance in a diabetes-prone population: a metabolic comparison of Pima Indians and Caucasians. J Clin Endocrinol Metab 73:866–876. 71. HAFFNER, S.M., H. MIETTINEN, S.P. GASKILL, and M.P. STERN. 1995. Decreased insulin secretion and increased insulin resistance are independently related to the 7-year risk of NIDDM in MexicanAmericans. Diabetes 44:1386–1391. 72. HAFFNER, S.M., R. D’AGOSTINO, M.F. SAAD, M. REWERS, L. MYKKANEN, J. SELBY, G. HOWARD, P.J. SAVAGE, R.F. HAMMAN, L.E. WAGENKNECHT, et al. 1996. Increased insulin resistance and insulin secretion in nondiabetic African-Americans and Hispanics compared with non-Hispanic whites. The Insulin Resistance Atherosclerosis Study. Diabetes 45:742–748. 73. MCKEIGUE, P.M., B. SHAH, and M.G. MARMOT. 1991. Relation of central obesity and insulin resistance with high diabetes prevalence and cardiovascular risk in South Asians. Lancet 337:382–386. 74. ENAS, E.A., V. MOHAN, M. DEEPA, S. FAROOQ, S. PAZHOOR, and H. CHENNIKKARA. 2007. The metabolic syndrome and dyslipidemia among Asian Indians: a population with high rates of diabetes and premature coronary artery disease. J Cardiometab Syndr 2:267–275. 75. MCKEIGUE, P.M., and M.G. MARMOT. 1988. Mortality from coronary heart disease in Asian communities in London. BMJ 297:903. 76. BALARAJAN, R., L. BULUSU, A.M. ADELSTEIN, and V. SHUKLA. 1984. Patterns of mortality among migrants to England and Wales from the Indian subcontinent. Br Med J (Clin Res Ed) 289:1185–1187. 77. HOWARD, B.V., M.P. DAVIS, D.J. PETTITT, W.C. KNOWLER, and P.H. BENNETT. 1983. Plasma and lipoprotein cholesterol and triglyceride concentrations in the Pima Indians: distributions differing from those of Caucasians. Circulation 68:714–724.

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78. NELSON, R.G., M.L. SIEVERS, W.C. KNOWLER, B.A. SWINBURN, D.J. PETTITT, M.F. SAAD, I.M. LIEBOW, B.V. HOWARD, and P.H. BENNETT. 1990. Low incidence of fatal coronary heart disease in Pima Indians despite high prevalence of non-insulin-dependent diabetes. Circulation 81:987–995. 79. HOWARD, B.V., G. EGUSA, W.F. BELTZ, Y.A. KESANIEMI, and S.M. GRUNDY. 1986. Compensatory mechanisms governing the concentration of plasma low density lipoprotein. J Lipid Res 27:11–20. 80. DORIA, A., M.E. PATTI, and C.R. KAHN. 2008. The emerging genetic architecture of type 2 diabetes. Cell Metab 8:186–200. 81. MULLER, Y.L., R.L. HANSON, C. ZIMMERMAN, I. HARPER, J. SUTHERLAND, S. KOBES, W.C. KNOWLER, C. BOGARDUS, and L.J. BAIER. 2007. Variants in the Ca V 2.3 (alpha 1E) subunit of voltage-activated Ca2 þ channels are associated with insulin resistance and type 2 diabetes in Pima Indians. Diabetes 56:3089–3094. 82. NAIR, S., Y.H. LEE, R.S. LINDSAY, B.R. WALKER, P.A. TATARANNI, C. BOGARDUS, L.J. BAIER, and P.A. PERMANA. 2004. 11beta-Hydroxysteroid dehydrogenase type 1: genetic polymorphisms are associated with type 2 diabetes in Pima Indians independently of obesity and expression in adipocyte and muscle. Diabetologia 47:1088–1095. 83. KOVACS, P., L. MA, R.L. HANSON, P. FRANKS, M. STUMVOLL, C. BOGARDUS, and L.J. BAIER. 2005. Genetic variation in UCP2 (uncoupling protein-2) is associated with energy metabolism in Pima Indians. Diabetologia 48:2292–2295. 84. THAMEEM, F., V.S. FAROOK, C. BOGARDUS, and M. PROCHAZKA. 2006. Association of amino acid variants in the activating transcription factor 6 gene (ATF6) on 1q21-q23 with type 2 diabetes in Pima Indians. Diabetes 55:839–842. 85. ALTSHULER, D., J.N. HIRSCHHORN, M. KLANNEMARK, C.M. LINDGREN, M.C. VOHL, J. NEMESH, C.R. LANE, S.F. SCHAFFNER, S. BOLK, C. BREWER, T. TUOMI, D. GAUDET, T.J. HUDSON, M. DALY, L. GROOP, and E.S. LANDER. 2000. The common PPARgamma Pro12Ala polymorphism is associated with decreased risk of type 2 diabetes. Nat Genet 26:76–80. 86. GRANT, S.F., G. THORLEIFSSON, I. REYNISDOTTIR, R. BENEDIKTSSON, A. MANOLESCU, J. SAINZ, A. HELGASON, H. STEFANSSON, V. EMILSSON, A. HELGADOTTIR, U. STYRKARSDOTTIR, K.P. MAGNUSSON, G.B. WALTERS, E. PALSDOTTIR, T. JONSDOTTIR, T. GUDMUNDSDOTTIR, A. GYLFASON, J. SAEMUNDSDOTTIR, R.L. WILENSKY, M.P. REILLY, D.J. RADER, Y. BAGGER, C. CHRISTIANSEN, V. GUDNASON, G. SIGURDSSON, U. THORSTEINSDOTTIR, J.R. GULCHER, A. KONG, and K. STEFANSSON. 2006. Variant of transcription factor 7-like 2 (TCF7L2) gene confers risk of type 2 diabetes. Nat Genet 38:320–323. 87. CHANG, Y.C., T.J. CHANG, Y.D. JIANG, S.S. KUO, K.C. LEE, K.C. CHIU, and L.M. CHUANG. 2007. Association study of the genetic polymorphisms of the transcription factor 7-like 2 (TCF7L2) gene and type 2 diabetes in the Chinese population. Diabetes 56:2631–2637.

Chapter

8

Emerging Therapeutic Approaches for Dyslipidemias Associated with High LDL and Low HDL MARGRIT SCHWARZ1 1 2

AND JAE

B. KIM2

Department of Metabolic Disorders, Amgen, Inc., South San Francisco, CA, USA Global Development, Amgen, Inc., Thousand Oaks, CA, USA

INTRODUCTION Dyslipidemia is defined as a disorder of lipoprotein metabolism and is a major independent risk factor for the development of atherosclerosis and cardiovascular disease (CVD). Perturbations of lipoprotein metabolism, including lipoprotein overproduction or deficiency, are also associated with other morbid components of metabolic syndrome such as type 2 diabetes and obesity. With the preponderance of data supporting a causal link between elevated levels of low-density lipoprotein cholesterol (LDL-C) and increased incidence of CVD, contemporary therapeutic interventions are primarily focused on lowering LDL-C. Decreasing cholesterol synthesis via inhibition of HMG-CoA reductase (statins) is the therapy of choice for lowering LDL-C, but the need for safely attaining ever more aggressive lipid management goals recommended in recent clinical guidelines has engendered several novel therapeutic approaches based on new and discrete mechanisms of action. Many of these approaches, as described in this chapter, are now at various stages of clinical development. Albeit most dyslipidemias are characterized by elevations of lipids carried by lipoproteins in the blood, the spectrum of lipoprotein disorders is far more diverse and may be perturbed by diet and lifestyle in addition to mutations in certain

Metabolic Syndrome: Underlying Mechanisms and Drug Therapies Edited by Minghan Wang Copyright 2011 John Wiley & Sons, Inc.

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genes involved in lipoprotein metabolism. Certain types of dyslipidemias are characterized by reduced levels of high-density lipoprotein cholesterol (HDL-C). An inverse correlation between CVD and the levels of cholesterol carried in HDL fractions has been observed in epidemiological studies; however, clinical proof of concept for a causal link has yet to be established. There is currently no marketed pharmacological intervention designed to specifically raise HDL-C, but the clinical research in this arena is highly active and provocative. This chapter attempts to briefly summarize derangements of lipoprotein metabolism and their metabolic consequences, as well as discuss emerging therapeutic strategies that target various components of lipoprotein production, transport, and clearance.

LIPOPROTEIN METABOLISM There have been numerous advances in the understanding of lipoprotein metabolism, function, and interactions over the past several decades. A large number of metabolic roles have been identified for apolipoproteins (apos), the principal protein moieties of lipoproteins, in addition to being cofactors that mediate clearance of lipids by their target tissues. A brief review of the major lipoprotein particles and their constituent molecules in this section is helpful in understanding the various lipid disorders, their relation to atherosclerotic disease, and potential therapeutic opportunities. Lipoproteins are biochemical entities carrying both lipids and proteins and have been classified by centrifugal separation characteristics (size and density), apo content, and the types of lipids they transport (1). A schematic overview of lipoprotein metabolism pathways is presented in Figure 8.1. Chylomicrons (density 0.95 g/mL) are very large particles that carry dietary (exogenous) cholesterol and triglycerides. They are formed in the enterocytes of the small intestine where they incorporate cholesterol and triglycerides absorbed from the gut with apoB48, and to a lesser extent apoC2 and apoE. The intestinal (apoB48) and hepatic isoforms (apoB100) of human apoB are encoded in a single gene and arise from differential splicing of the same primary APOB mRNA transcript, with apoB48 representing the amino-terminal 47% of apoB100 (2, 3). ApoB48 does not bind to the hepatic receptor for low-density lipoprotein (LDLR). Therefore, chylomicrons are not removed from the circulation where they are delivered via the lymphatic system; rather, they are acted upon by various lipoprotein lipases (LPLs) that hydrolyze the triglyceride core and release free fatty acids. The resulting chylomicron remnants are then cleared by the liver. Very-low-density lipoproteins (VLDLs; density 0.95–1.006 g/mL) are associated with apoB100, apoC2, and apoE and carry endogenous triglycerides and a small amount of cholesterol. VLDLs are synthesized and secreted by the liver, and their formation is controlled by the amount of lipids and apoB available in the hepatocyte, as well as by the enzymatic activities of acylCoA acyltransferase (ACAT) and microsomal triglyceride transfer protein (MTP). Once released into the plasma, VLDL can also serve as a substrate for LPLs, releasing free fatty acids and yielding intermediate-density lipoproteins (IDLs), which are either cleared by the liver or acted upon by hepatic lipase (HL) to form

B

B

B LPL

IDL HL

ox LDL

E

LPL

VLDL E

C2

C2

C2

LPL

LDL CD36 LOX1 SR-A

Remnant

B

B

E

Chylomicron CETP

LDL-R

Cholesterol pool

MTP ACAT

LRP

Liver

HDL LCAT ABCA1 ABCG1 SR-BI

Macrophage

HDL

A1 A1

Nascent HDL

Mature HDL

HDL

SR-BI

Small intestine

Fecal excretion

Figure 8.1 Overview of lipoprotein metabolism. Chylomicrons are formed in the enterocytes of the small intestine, incorporating cholesterol and triglycerides

201

absorbed from the gut with apoB. LPLs hydrolyze their triglyceride core and release free fatty acids. The resulting remnants are cleared by the liver. VLDLs are associated with apoB, apoC2, and apoE and carry endogenous triglycerides and cholesterol. Synthesized and secreted by the liver, their formation is regulated by MTP and ACAT. LPLs release free fatty acids from VLDLs, yielding IDLs, which are converted to LDLs by the action of HL. LDLs primarily transport cholesteryl esters and some triglycerides and are being cleared by the hepatic LDLR. A portion of LDL is converted to oxLDL and taken up by macrophages via CD36, LOX-1, or SR-A, contributing to the formation of atherogenic foam cells. HDLs contain apoA1 and phospholipids, and are formed in the liver and small intestine. Lipid-poor apoA1 interacts with ABCA1, ABCG1, and SR-B1 and accepts free cholesterol from peripheral tissues to form nascent HDL. These are remodeled by LCAT to yield mature, cholesteryl ester-rich HDL particles. Mature HDLs are converted to IDL and VLDL by CETP-mediated exchange of cholesteryl esters for triglycerides and cleared via the hepatic LDLR pathway or via SR-B1. The HDL-mediated flux of cholesterol from peripheral tissues into the plasma and to the liver for clearance is known as RCT and is thought to form the mechanistic basis for the antiatherogenic properties of HDL.

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low-density lipoproteins (LDLs; density 1.006–1.019 g/mL). Small molecule inhibitors of ACAT and MTP as well as antisense oligonucleotides targeting APOB are being developed as pharmacological interventions for decreasing plasma levels of LDL via this pathway. LDL particles contain apoB100, the main ligand for the LDLR, and primarily transport cholesteryl esters along with some triglycerides. Because the liver is the principal organ responsible for the clearance of LDL, plasma LDL-C levels are directly determined by the activity of the hepatic LDLR in most species including humans. Upon binding to the hepatic LDLR, LDL is endocytosed and transported to endosomes where it dissociates from the receptor and is subsequently catabolized in lysosomes. The endosomal LDLR, however, recycles back to the cell surface. Regulating the synthesis, expression, or recycling rate of the hepatic LDLR has been the primary target of contemporary pharmacological interventions such as statins, and more recently, inhibitors of proprotein convertase subtilisin-like kexin type 9 (PCSK9). Finally, high-density lipoproteins (HDLs; density 1.063–1.21 g/mL) are small particles containing apoA1 and phospholipids and are formed in the liver and small intestine. Lipid-poor apoA1 interacts with cell surface cholesterol transporters expressed on macrophages and other tissues, specifically ATP-binding cassette (ABC) transporters A1 (ABCA1) and G1 (ABCG1) and scavenger receptor class B, type 1 (SRB-1). ApoA1 accepts free cholesterol to form nascent HDL particles, which are subsequently remodeled by lecithin–cholesterol acyltransferase (LCAT) to yield mature, cholesteryl ester-rich HDL particles. Mature HDL particles are converted to IDL and VLDL by cholesterol ester transfer protein (CETP)-mediated exchange of cholesteryl esters for triglycerides, and then cleared via the hepatic LDLR pathway. HDL can also be taken up directly by the liver via hepatic SRB-1; however, the quantitative contribution of this pathway varies substantially from species to species, and it is generally believed that the CETP-mediated pathway is the main route of HDL clearance in humans. The HDL-mediated flux of cholesterol from peripheral tissues into the plasma and to the liver for clearance is known as reverse cholesterol transport (RCT) and is thought to form the mechanistic basis for the antiatherogenic properties of HDL. Pharmacological approaches aimed at inhibiting CETP, activating LCAT, or mimicking the activity of apoA1 seek to exploit this hypothesized link between RCT and reversal of atherosclerosis.

THE ROLE OF LIPOPROTEINS IN ATHEROGENESIS The metabolism of plasma lipoproteins is highly interrelated and forms a complex network designed to maintain lipid homeostasis and protect against vascular inflammation. The “lipid hypothesis” asserts that an imbalance in any aspect of this network attributable to dysfunctional formation, transport, or clearance of cholesterol-carrying lipoprotein particles is highly causal for the formation of atherosclerosis in vascular walls. Specifically, retention of LDL in the subendothelial space of the vasculature is thought to be the major initiating factor for atheromas. This was first proposed by Virchow in 1856 (4) and supported by early studies in rabbits (5). Since then, numerous

The Role of Lipoproteins in Atherogenesis

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studies have shown that the atherosclerosis process can be recapitulated in animal models by modifying the concentrations of lipids carried in lipoproteins either genetically or by increasing dietary cholesterol, in the absence of any other risk factor. In addition, numerous epidemiological, genetic, and pharmacological studies have successfully demonstrated a direct link between plasma LDL cholesterol levels and the incidence of atherosclerotic CVD in humans. Taken together, this research has resulted in the general acceptance of the lipid hypothesis by much of the scientific community (6). LDL is the major cholesterol-carrying lipoprotein and is the only lipoprotein fraction markedly elevated in the genetic disorder known as familial hypercholesterolemia (FH), the most thoroughly investigated lipoprotein disorder (7). As such, LDLC lowering has emerged as the primary goal for therapeutic intervention in the treatment of hyperlipidemias and CVD. It is difficult to argue against the overwhelming evidence provided by millions of patients who respond to LDL-C lowering agents with a decreased incidence of CVD. However, the “cholesterol controversy” (8) is still fueled by the fact that despite the wide use of statins, CVD remains the underlying cause of >35% of deaths in the United States (9). Clearly, some patients are exposed to residual CVD risk that is independent of their blood cholesterol levels. A growing body of evidence suggests that molecular events downstream of subendothelial LDL retention are also major contributors to plaque formation and growth, a hypothesis first put forward by Ross and Glomset in 1973 (10). The theory of atherosclerosis as an inflammatory disease has since gained support, and this does not necessarily contradict the lipid hypothesis (11). Endothelial injury and deposition of LDL in the subendothelial space both promote local inflammation, leading to the release of signaling molecules and the expression of endothelial cell adhesion molecules, which in turn recruit monocytes to the arterial wall (12). These monocytes differentiate into macrophages—a process driven by cytokines, interaction with the extracellular matrix, and the upregulation of scavenger receptors capable of binding and internalizing oxidized forms of LDL (oxLDL). Because these scavenger receptors are not subject to the same feedback regulations as the hepatic LDLR, oxLDL accumulates in macrophages and leads to the formation of foam cells (13, 14). However, oxLDL has also been shown to independently promote proinflammatory cytokine release and platelet aggregation, both of which further contribute to atherosclerotic lesion formation. Together, these processes result in the growth and destabilization of plaques, ultimately leading to plaque rupture and thrombosis, which in turn are directly linked to acute cardiovascular events such as stroke and myocardial infarction. In the context of LDL atherogenicity, Lp(a) lipoprotein, a macromolecule consisting of an LDL-like particle covalently linked to a specific apolipoprotein (a), may play an important role. Lp(a) was discovered in 1963 (15), and the human gene (LPA) encoding Lp(a) was cloned in 1987, revealing homology to plasminogen (16). LPA polymorphisms associated with high Lp(a) levels confer an increased risk of coronary artery disease (17, 18), but the physiological function of Lp(a) is still not well understood. Clinical studies have identified a strong correlation between Lp (a) levels and oxLDL, suggesting that the atherogenicity of Lp(a) may be mediated in part by associated proinflammatory oxidized phospholipids (19). In addition, its homology to plasminogen suggest a function of Lp(a) within the coagulation

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system (20). Because the causality of the link between Lp(a) function and heart disease has not been clearly proven, Lp(a) is not yet accepted as a well-established risk factor for cardiovascular disease. In contrast to LDL, HDL is thought to play an atheroprotective role, which was first postulated based on the observation that differences in HDL levels between men and premenopausal women correlate with differences in age-adjusted risk of CVD (21). These initial observations have been firmly validated in large epidemiological studies (22). HDL is thought to exert its atheroprotective effect by mediating RCT from vessel wall macrophages back to the plasma, and ultimately to the liver for catabolism and secretion, a hypothesis developed decades ago by Glomset based on his studies of LCAT (23). Today, the RCT hypothesis is generally well accepted, although the precise mechanism of in vivo RCT is still being debated (24, 25). HDL possesses other biological activities that may contribute to its antiatherogenic properties, such as the ability to prevent LDL oxidation and to inhibit inflammation (26, 27). Much of the recent research in the field has focused on understanding the heterogeneity of HDL particles and the functional role of its protein components. Certain subspecies of HDL acquire and carry proteins that may either protect or harm the artery wall, and oxidative damage inflicted by enzymes such as myeloperoxidase may generate “dysfunctional” HDL characterized by the loss of its antiatherogenic properties (28). Analyzing the HDL proteome from patients with established CVD and comparing it to that of healthy controls will help elucidate RCT-independent protective properties of HDL (29). In the meantime, the focus of therapeutic strategies that target HDL is shifting from a definition of efficacy based upon simply raising circulating HDL-C to a more comprehensive definition encompassing fundamental properties of HDL heterogeneity and functionality.

ETIOLOGY AND CLASSIFICATION OF DYSLIPIDEMIAS Dyslipidemia is a broad term that refers to a range of lipoprotein disorders. The original classification of dyslipidemias by Fredrickson is based on the pattern of lipoproteins obtained with electrophoresis or ultracentrifugation (30). The Fredricksen classification has been adopted by the World Health Organization (WHO) and remains a popular system mostly because of its historical relevance, but its prognostic value and clinical utility are limited. Conceptually, dyslipidemias can be classified as primary (i.e., caused by a genetic defect) or secondary, when they arise as a consequence of diet, metabolic or endocrine disorders, or medication (31, 32). The most common conditions associated with secondary dyslipidemia are elevated blood pressure, impaired glucose tolerance, and increased visceral fat—a combination of metabolic abnormalities seen in patients with the metabolic syndrome (33, 34). Genetic lipoprotein disorders are summarized in Table 8.1. They can affect any of the lipoprotein classes; however, LDL-dominant lipid disorders such as FH are the most important from a clinical perspective. FHs are autosomal dominant disorders that affect approximately 1 in 500 individuals (7). They are characterized by significantly elevated LDL-C, usually to levels well above 190 and up to 1000 mg/dL, and require

205

Etiology and Classification of Dyslipidemias Table 8.1

Genetic Lipoprotein Disorders

Disorder

Triglyceride-rich lipoproteins Lipoprotein lipase deficiency APOC2 deficiency Abetalipoproteinemia Remnant lipoproteins Dysbetalipoproteinemia III Hepatic lipase deficiency LDL particles Familial hypercholesterolemia Familial defective apoB100 Autosomal dominant hypercholesterolemia Autosomal recessive hypercholesterolemia Hypobetalipoproteinemia Familial sitosterolemia Familial Lp(a) hyperlipoproteinemia HDL particles ApoA1 deficiency Tangier disease Familial LCAT deficiency CETP deficiency

Gene

LPL APOC2 MTP

Effects on LDL-C

Effects on HDL-C

Effects on TG

#

## ## $

""" """ #

#

""

APOE HL LDLR APOB PCSK9

"" "" ""

ARH

"

APOB ABCG5/G8 APO(a)

# $ $

APOA1 ABCA1 LCAT CETP

## ## ## "

Adapted from Ref. 31.

aggressive treatment with LDL-C lowering drugs. FH can be caused by defects in the LDLR gene, a defective APOB gene, or missense mutations in PCSK9. In addition, a defect in the gene encoding the LDLR adapter protein 1 (ARH) has been linked to autosomal recessive forms of hypercholesterolemia (HC) (7). Triglyceride-dominant lipid disorders can arise as a consequence of defects in LPL, APOC2, APOA5, or MTP. These disorders are characterized by moderate to severe elevations of plasma triglycerides (up to 1000 mg/dL), and are usually associated with low levels of HDL-C. In addition to presenting an atherogenic risk, severe hypertriglyceridemias can lead to pancreatitis. Disorders characterized by low HDL (hypoalphalipoproteinemia) are caused by defects in APOA1, ABCA1 (Tangier disease), LCAT, or CETP. Affected individuals have HDL-C levels below 20 mg/dL and, in some cases, an increased risk of premature CVD. In addition, a large percentage of patients present with mixed dyslipidemia, characterized by elevated triglycerides, low HDL-C, and the presence of small, dense LDL particles. The causes can be primary or secondary, or both.

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EPIDEMIOLOGY The prevalence of dyslipidemia is very high and rising worldwide, with an estimated 50–70% of the U.S. adult population affected (9). Dyslipidemia is a major risk factor for developing CVD, and the WHO estimates that globally it is associated with more than half of all cases of ischemic heart disease, and more than 4 million deaths per year. Of the patients diagnosed with dyslipidemia, 26% are classified as having low risk for CVD, 46% as having moderate risk, and 28% as having high risk. Undertreatment for dyslipidemia is highly prevalent, due to factors that include insufficient detection, underprescribing, and low compliance. High LDL-C (>130 mg/dL) accounts for one-third of all specific dyslipidemia cases, and dyslipidemia associated with low HDL-C (<40 mg/dL) is present in 36% of U.S. adults. The total cost associated with cardiovascular diseases and stroke in the United States—many of which are related to dyslipidemia—is estimated to exceed $475 billion in 2009 (9). Treating lipoprotein disorders and their associated dyslipidemias is therefore a priority from both a public health and an economic perspective (35).

CURRENT TREATMENT GUIDELINES FOR DYSLIPIDEMIAS Therapeutic modalities for clinical management consist of lifestyle and dietary changes, treatment of secondary causes, and drug therapy. Based on accumulating evidence from clinical trials for the direct link between elevated LDL-C and CVD, the National Cholesterol Education Program’s Adult Treatment Panel III (NCEP ATPIII) continues to recommend lowering of LDL-C as the primary target of any therapy (36). LDL-C goals are tailored to different cardiovascular risk categories based upon identification of known coronary heart disease (CHD), CHD risk equivalents, and CV risk factors that include smoking, hypertension, low HDL (<40 mg/dL), family history of early CHD, and age. Importantly, ATPIII guidelines include diabetes as a high-risk CHD equivalent. Patients are classified as high-risk if they have manifest CHD, other forms of atherosclerotic disease, diabetes, or multiple risk factors that confer a 10-year risk for CHD of >20% as estimated from Framingham risk scores. In high-risk patients, the recommended LDL-C goal is <100 mg/dL, with acknowledgment that an LDL-C goal of <70 mg/dL is also an acceptable therapeutic option in high-risk patients. Data from the Get With The Guidelines program show that ATPIII guidelines may not be aggressive enough with LDL-C lowering, as the majority of hospitalized patients with CHD have LDL-C levels considered in the “normal” range. Further, nearly 50% of patients having first ACS events already have LDL levels below the target LDL-C level of 100 mg/dL (37).

CURRENTLY AVAILABLE DRUGS FOR THE TREATMENT OF DYSLIPIDEMIAS Currently available treatment options for dyslipidemias are summarized in Table 8.2 and will be discussed in more detail in the following sections.

Currently Available Drugs for the Treatment of Dyslipidemias Table 8.2

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Currently Available Dyslipidemia Therapies

Drug class

Effects on lipoproteins and triglycerides

Side effects

Clinical trial results

HMG-CoA reductase inhibitors (statins)a Bile acid sequestrantsb

LDL-C: # 18–55%; HDL-C: " 5–15%; TG: # 7–30% LDL-C: # 15–30%; HDL-C: " 3–5%; TG: no change LDL-C: # 5–25%; HDL-C: " 15–35%; TG: # 20–50%

Myopathy, liver enzyme elevation

Reduced major coronary events and total mortality Reduced major coronary events and CVD deaths Reduced major coronary events

Nicotinic acidc

Fibric acidsd

LDL-C: # 5–20%; HDL-C: " 10–20%; TG: # 20–50%

GI distress, constipation Flushing, hyperglycemia, hyperuricemia, GI distress, hepatotoxicity Dyspepsia, gallstones, myopathy

Reduced major coronary events

Adapted from Ref. 38 (NCEP guidelines). a

Pravastatin, simvastatin, fluvastatin, atorvastatin, and cerivastatin.

b

Cholestyramine, colestipol, and colesevelam. Immediate release nicotinic acid, extended release nicotinic acid (Niaspan), and sustained release nicotinic acid. c

d

Gemfibrozil, fenofibrate, and clofibrate.

HMG-CoA Reductase Inhibitors (Statins) Outcomes from large-scale trials conducted over many years support statins as the therapy of choice for nearly all dyslipidemia patients. A recent analysis of 10 randomized controlled trials and 70,388 patients without established CVD but with cardiovascular risk factors showed unambiguously that statins significantly reduced the risk of all-cause mortality, major coronary events, and major cerebrovascular events, and that there was no significant heterogeneity of the treatment effect in clinical subgroups (38). The recently concluded SPARCL study, a placebo-controlled, randomized trial designed to determine whether treatment with atorvastatin reduces strokes in 4731 subjects with recent stroke or transient ischemic attack, suggests that statins can be equally efficacious in preventing strokes as they are in preventing other CVD events (39). In addition, the ERASE trial demonstrated that newly initiated statin therapy was associated with rapid regression of coronary atherosclerosis in ACS patients, as assessed by coronary intravascular ultrasound (IVUS) (40). Statins function by inhibiting the rate-limiting step in cholesterol biosynthesis, the conversion of HMG-CoA to mevalonate. The resulting intracellular cholesterol depletion elicits a compensatory upregulation of LDLR gene transcription, resulting in increased LDL-C uptake by the liver and lower levels of circulating LDL-C (41–43).

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The treatment paradigm associated with statins is generally “lower is better” for LDLC, as placebo-controlled statin trials have consistently shown a significant reduction of CV events, even in patients with average or below average LDL-C levels (44, 45). However, individual statins do not all have the same potency, and their effects on LDL-C lowering vary considerably. A meta-analysis of 28 ACS trials demonstrated a clear dose–response relationship with statins in terms of LDL-C lowering, with rosuvastatin 40 mg/day achieving the greatest percentage reduction (56%) from baseline, followed by atorvastatin 80 mg/day (52%), simvastatin 80 mg/day (45%), and simvastatin 40 mg/day (37%) (46). Statins have multiple pleiotropic effects beyond LDL-C lowering (47) and may well improve the overall vascular inflammatory status, commonly measured by highsensitivity C-reactive protein (hsCRP) (48). There is ongoing debate in the scientific community on whether the reduction in CV events attributable to statins can be parsed from its anti-inflammatory versus LDL-C lowering effects. Prespecified analyses from the JUPITER study show that LDL-C and hsCRP are only weakly correlated in individual patients (r < 0.15); however, adjusted for changes in LDL-C, hsCRP is shown to be an independent predictor of future CV events, providing a potential rationale for dual goals for statin therapy in high-risk patients: LDL-C <70 mg/dL and hsCRP <2 mg/dL (49). Despite hsCRP’s potential as a biomarker for inflammation and CV risk, the preponderance of data thus far suggests that CRP is not a causal agent for CVD (50). Statins are generally well tolerated; however, up to 10% of patients develop muscle fatigue or muscle pain, which is a clinically important cause of statin intolerance or discontinuation. Severe myopathy or rhabdomyolysis, defined as life-threatening myopathy with renal involvement, is a rare complication (51). In general, the rate of adverse events associated with statins increases dose dependently and, in some cases, varies with the type of statin used. For example, simvastatin 80 mg/ day is associated with a rate of myopathy that is 26-fold higher than that observed with simvastatin 20 mg/day (46). The mechanisms of statin-related myopathy are not well understood, but findings such as these are bound to affect adherence and compliance levels in the clinical practice. In addition, some studies have found that statin treatment is associated with an increased risk of developing type 2 diabetes (52); however, this risk is far outweighed by the benefits of statins. Patients who do not tolerate statins do not easily reach their LDL-C target level, and combinations or alternative approaches for lowering LDL-C will need to be employed.

Cholesterol Absorption Inhibitors Cholesterol absorption inhibitors function by selectively interfering with the Niemann–Pick C1-like 1 protein (npc1l1) in the gut, resulting in a decreased rate of cholesterol uptake from the intestinal lumen into enterocytes (53, 54). The decreased flux of cholesterol from the gut to the liver triggers a compensatory upregulation of both LDLR protein expression and hepatic cholesterol biosynthesis;

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the net result is a decrease in circulating levels of LDL-C (55). Ezetimibe, the first drug approved in this class, presents an alternative therapeutic option for patients who do not reach LDL-C targets on maximally tolerated statin doses (56–61). It is also marketed in combination with simvastatin (vytorin), together generating annual sales of $3.5 billion in 2009 (62, 63). Although the beneficial effect of ezetimibe on LDLC lowering has been established both as monotherapy and in combination with statins, it is still unclear whether lowering LDL-C via cholesterol absorption inhibition will provide the same CV benefit as statins. The ENHANCE trial was designed to address this question (64). Vytorin was compared to simvastatin alone and was, as expected, more effective in lowering LDL-C (58% versus 41%). Surprisingly, however, the extent of atherosclerosis in the carotid arteries (measured as carotid intima-media thickness, cIMT) increased in both treatment groups, and the increase was more pronounced in the arm receiving vytorin. Few clinical trials have generated more publicity and controversial discussion than ENHANCE, as the long-standing paradigm of “the lower, the better” for LDL-C appeared to be challenged. Aspects of the trial design have been questioned along with the established mechanism of action of cholesterol absorption inhibitors (65–67). Does the mechanism by which LDL-C is being lowered matter? Does ezetimibe have unexpected effects on the vessel wall, or interactions with statins that interfere with their beneficial pleiotropic effects? The recently concluded SEAS trial further added to the controversy (68). SEAS was designed to test the hypothesis that aggressive lipid lowering with vytorin reduces CVD risk and the need for aortic valve replacement in patients with asymptomatic aortic stenosis. Despite LDL-C lowering of up to 61%, there was no difference in the primary end point; however, the incidence of cancer and cancer deaths were more frequent in the vytorin treatment group. Whether or not LDL-C lowering via cholesterol absorption inhibition will in fact translate into increased clinical benefit on cardiovascular outcomes relative to simvastatin monotherapy is currently being addressed in a large randomized control trial (IMPROVE-IT), which is recruiting up to 18,000 moderate- to high-risk patients stabilized after ACS (69). Early results are expected in 2012.

Bile Acid Sequestrants Bile acid sequestrants (BAS) inhibit reabsorption of bile acids from the gut and interrupt the enterohepatic circulation of bile acids (70, 71). A compensatory increase in cholesterol catabolism via hepatic bile acid synthesis leads to a depletion of intracellular cholesterol levels and an upregulation of LDLRs, resulting in lower plasma LDL-C (72, 73). BAS are among the oldest lipid lowering drugs. Two decades ago, BAS were the treatment of choice for HC because they have been shown to reduce CV risk as monotherapy (74, 75). However, their use was limited by their gastrointestinal side effects and they were eventually replaced by statins. Today, BAS are mainly used as an adjunctive therapy in patients with severe HC (76). Their tolerability was improved through the development of colesevelam, which was

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approved as a cholesterol lowering drug in 2000 (77). BAS have long been known to affect glucose metabolism and were recently evaluated in diabetic patients. Colesevelam significantly improved both plasma lipid levels and HbA1c (78), which led to a renewed consideration of BAS as a drug class (79).

Nicotinic Acid Nicotinic acid (niacin) interacts with gpr109A, a G protein-coupled receptor expressed in adipocytes, and reduces lipolysis and the release of free fatty acids (80–82). This results in decreased hepatic production of VLDL and lower levels of LDL-C. However, the LDL-C lowering effect of niacin is modest compared to its effect on HDL. Niacin raises HDL-C by reducing the transfer of lipids from HDL to VLDL and by delaying HDL clearance through the kidney. It is the most potent clinically used agent for increasing plasma HDL-C and the therapy of choice for mixed dyslipidemia (83). Niacin is also the only marketed antidyslipidemia drug that significantly lowers Lp(a) (84). Several clinical trials (Coronary Drug Project, ARBITER, and HATS) have demonstrated the beneficial effect of niacin on atherosclerosis progression, either as monotherapy or in combination with statins (85–88). Frequent side effects that limit its use include flushing, gastric irritation, liver toxicity, and altered glycemic control. Extended release formulations significantly reduce flushing, and the ability of such preparations to improve outcome is currently being evaluated in conjunction with statins in the AIM-HIGH and 2/THRIVE trials.

Fibric Acids Fibric acids, or fibrates, act on peroxisome proliferator-activated receptors (PPARs) and are structurally related to thiazolidinediones, a class of widely prescribed antidiabetic drugs. PPARs are transcription factors that, upon activation, regulate the expression of several target genes involved in lipid metabolism, including LPL, APOC2, and APOA1 (89). The effect of fibrates on HDL is associated with changes in apoA1 expression levels, whereas their hypotriglyceridemic effect is caused by enhanced catabolism of triglyceride-rich particles and reduced secretion of VLDL (90). The effect of fibrates on LDL-C is minimal. Fibrates (clofibrate, gemfibrozil, fenofibrate, and bezafibrate) are therefore mainly used for the treatment of hypertriglyceridemia and mixed dyslipidemia. Several clinical trials, such as the Helsinki Heart Study and VA-HIT, suggest that fibrate monotherapy reduces CV events, particularly in patients with insulin resistance and disorders of triglyceride metabolism (91, 92). However, fenofibrate failed to meet the primary end point of CVD death reduction in the recent FIELD trial, although the results may have been confounded by the uneven use of statins in the placebo versus fenofibrate arms (93). Adverse side effects of fibrates include gastrointestinal discomfort and hepatic and renal toxicity; further, gemfibrozil can affect the catabolism of statins and may increase the risk of myotoxicity in combination therapy settings (94).

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Combination Therapies A substantial number of high-risk patients do not achieve their LDL-C goals, partly due to poor compliance, but also because LDL-C goals of <70 mg/dL for veryhigh-risk patients are difficult to achieve with existing treatment options. This concern is validated by results from the Lipid Treatment Assessment Project 2 (L-TAP2), a survey conducted to determine the proportion of dyslipidemic patients receiving lipid lowering therapy who achieve their LDL-C goals (95). Approximately 10,000 patients were assessed in nine countries. Low-risk patients were more likely to attain their LDL-C goals (86%), whereas only 67% of high-risk patients reached their more aggressive LDL-C targets. In the United States, results were even clearer—only 35% of CVD patients with at least two risk factors reached their LDL-C goal of <70 mg/dL. These findings illustrate not only the high unmet need in antilipidemic therapy, but also the necessity for combination therapy with existing agents in the context of attaining aggressive LDL-C goals. Multidrug approaches include combinations of statins with absorption inhibitors (96) or bile acid sequestrants (76). For patients who do not respond adequately to high doses of statins and exhibit hypertriglyceridemia as the remaining lipid abnormality, a fibrate can be added to the statin therapy. An increased risk of myopathy is the major drawback associated with clinical use of this combination (97, 98). Patients who do not respond well to statins and have low HDL-C levels (<40 mg/dL) may benefit from an addition of niacin to their therapy (99). However, this approach is generally limited to nondiabetic patients as niacin therapy can adversely affect glycemic control in patients with type 2 diabetes or metabolic syndrome. As the prevalence of dyslipidemia associated with metabolic syndrome and diabetes is increasing worldwide, a demand for therapies that can address several consequences of these conditions simultaneously is rising as well. Most of the agents currently in clinical development pipelines are either single pill combinations or improved versions of already available medications. Pravafen (pravastatin þ fenofibrate), Cordaptive (laropiprant þ niacin ER), Simcor (simvastatin þ niacin ER), Fenoglide, and TriLipix (enhanced release fenofibrates) are examples for such combinations that are already marketed or in the late stage of clinical development. While developing and launching new single pill combinations and improved formulations may appear easier than discovering agents with new mechanisms of action, improved compliance or efficacy with the above strategies has yet to be demonstrated. Two recent developments will further impact the demand for combination therapies and/or novel dyslipidemia therapies. First, the recently completed JUPITER trial demonstrates that rosuvastatin treatment can dramatically reduce vascular events in apparently healthy men and women with normal LDL-C (<130 mg/dL) but who have elevated levels of hsCRP (>2 mg/L) (49). The unexpected magnitude of benefit in the treatment group compared with placebo (44% reduction of major CVD events) was associated with 50% reductions in both CRP and LDL-C. These findings are likely to impact new guidelines for cardiovascular disease prevention, particularly in light of the fact that a lower threshold beyond which LDL-C reduction ceases to be beneficial is not supported by existing data (100, 101). Second, the emerging concept

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of a lifetime risk index implies that the duration of exposure to LDL-C may be just as important as, if not more critical than, the absolute level of LDL-C at any given time (102, 103). Taking this concept one step further, it is conceivable that lowering cholesterol earlier in life by pharmacological intervention may produce an even greater impact on preventing CVD. Recommendations for a 50 mg/dL LDL-C goal for high-risk patients and preventive LDL-C lowering therapy for young, healthy adults are being discussed among experts in the field. However, statins and other existing therapies may not harbor the potential for consistently achieving such goals without clinical liabilities. Together, this underscores the necessity for developing more efficacious therapies for lowering LDL-C via novel mechanisms. Several promising new approaches are discussed below.

EMERGING THERAPIES FOR LOWERING LDL-C MTP Inhibitors MTP catalyzes the assembly of cholesterol, triglycerides, and apoB to VLDL or chylomicrons in hepatocytes and enterocytes. MTP is dysfunctional in patients with abetalipoproteinemia (Table 8.1), resulting in a complete lack of apoB-containing lipoproteins in the circulation (104). Consequently, inhibiting MTP may be a useful strategy for lowering LDL-C. The risk associated with this strategy is hepatic steatosis due to accumulation of triglycerides that cannot be incorporated into VLDL (105). Several MTP inhibitors have been tested in animal models; some of them are currently in clinical development. Implitapide (BAY 13-9952; AEGR-427) has been shown to lower LDL-C and attenuate atherosclerotic plaque progression in APOE knockout mice (106) and reduce fatty streak formation in rabbits fed a high-cholesterol diet. In healthy volunteers, implitapide was associated with a decrease in LDL-C and triglycerides (55% and 29%, respectively) and reduced postprandial lipemia. Gastrointestinal discomfort and abnormal liver function tests were reported as adverse events. In an effort to circumvent hepatic side effects, enterocyte-selective MTP inhibitors that are not absorbed systemically are also being developed. SLx-4090 has been evaluated in two phase I clinical trials and in a randomized, placebo-controlled phase II study in patients with dyslipidemia. The compound significantly reduced postprandial triglycerides and LDL-C, was undetectable in plasma, and caused no evident hepatic dysfunction (107). While MTP inhibitors as a class are likely to be effective in lowering chylomicrons, it remains to be seen whether their effect on LDL-C lowering is sufficient, and whether they are ultimately well tolerated in humans.

Apolipoprotein B Antisense Oligonucleotides and siRNA ApoB is the major component of LDL and plays a key role in its clearance by the liver. Mutations in APOB causing either enhanced clearance or decreased production of apoB are associated with lower plasma LDL-C and reduced CVD risk (108). Antisense oligonucleotides (ASOs) are single-stranded deoxyribonucleotides

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designed to induce degradation of their target mRNA, resulting in significant reduction of the encoded protein. ISIS 301012 (mipomersen) is an ASO that has been shown to effectively decrease the synthesis of apoB100 and to reduce plasma levels of apoB and LDL-C (109). In LDLR-deficient human APOB transgenic mice, ISIS 301012 reduced human APOB100 hepatic mRNA by 89% and plasma apoB100 levels by 68%. The compound also decreased circulating inflammatory cytokines and atheroma volume (110, 111). In phase I clinical studies, mipomersen caused a dosedependent and prolonged reduction of APOB100 and lowered LDL-C by up to 35% (112). Mipomersen has been shown to be equally effective when combined with a statin (113). In all clinical studies performed thus far, mipomersen has proven to be safe and well tolerated. Surprisingly, the compound was not associated with hepatic significant steatosis or liver function abnormalities, possibly due to a compensatory downregulation of fatty acid synthesis genes, as was observed in mice. Nevertheless, further development of this compound may be slowed by FDA requests for an outcome trial, possibly reflecting concerns around unproven modalities and novel mechanisms of action for lowering LDL-C. Small interfering RNAs (siRNA) may represent an alternative therapeutic modality for targeting APOB. siRNAs function by a fairly well-defined mechanism of action involving the RNA interference pathway, causing knockdown of target genes with a high degree of specificity. An APOB-specific siRNA has been evaluated in primates and was associated with dose-dependent silencing of APOB mRNA expression in the liver and up to 82% reduction in plasma LDL-C (114).

Anti-PCSK9 Antibodies and ASOs PCSK9 has recently been validated as a key regulator of LDL metabolism (115). PCSK9 is a secreted serine protease that interacts with the LDLR protein and accelerates its degradation. Figure 8.2 summarizes the current model for PCSK9 mode of action. LDL–LDLR complexes are internalized and transported to the acidic environment of the endosome, where the LDLR dissociates from LDL and recycles back to the cell surface. PCSK9 regulates this process and consequently the rate of LDL-C uptake by rerouting the LDLR to the lysosome for degradation (116). Human genetic studies provide strong validation for the role of PCSK9 in modulating LDL-C levels and the incidence of coronary heart disease. Gain-of-function (GOF) mutations in the PCSK9 gene are associated with elevated serum LDL-C levels (300 mg/dL) and premature CHD (117), whereas loss-of-function (LOF) mutations are associated with low serum LDL-C (100 mg/dL) (118). Strikingly, subjects harboring the heterozygous LOF mutations exhibited an 88% reduction in the incidence of CHD over a 15-year period relative to noncarriers of the mutations (119). Various approaches for inhibiting PCSK9 production, secretion, or its binding to the LDLR are currently assessed preclinically. Among these, the disruption of LDLR–PCSK9 interaction by antibodies (120) is noteworthy for two reasons. First, it provides evidence for the hypothesis that secreted, circulating PCSK9, as opposed to intracellular PCSK9, is indeed a major determinant of hepatic

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Chapter 8 Emerging Therapeutic Approaches LDL

LDL-R

Plasma

LDL-R

LDL

Hepatocyte Lysosome

Receptor recycling

Endocytosis

PCSK9

Secretion ER

LDL

LDLR and PCSK9 degradation

Selfprocessing

Endosome LDL PCSK9

Figure 8.2

Model for PCSK9 mechanism of action. PCSK9 is a serine protease that undergoes catalytic cleavage in the ER. The complex consisting of the prodomain and the catalytic fragment is secreted into plasma where it binds to cell surface LDLR. The LDLR/PCSK9 complex is internalized and prevents recycling of LDLRs from the endosomes to the cell surface by redirecting the LDLR to lysosomes for degradation. By regulating this process, PCSK9 directly affects the rate of LDL-C uptake from plasma.

LDLR regulation and LDL-C uptake. Second, fully human antibodies are validated therapeutic modalities that are currently being marketed for a variety of disease indications including cancer and inflammatory conditions. While the concept of an injectable therapeutic for treating a chronic disorder of lipoprotein metabolism is certainly novel, recently published preclinical data on anti-PCSK9 antibodies indicate that the approach holds promise. mAb1, a fully human monoclonal antibody against PCSK9, has been shown to effectively inhibit binding of PCSK9 to the LDLR, resulting in plasma cholesterol lowering in mice and nonhuman primates (121). A single injection of mAb1 caused rapid depletion of circulating plasma PCSK9 in cynomolgus monkeys and was associated with LDL-C lowering of up to 80% by day 10 post-injection. If the robust LDL-C lowering achieved with mAb1 is predictive for its efficacy in humans, an antibody against PCSK9 may represent a feasible alternative or add-on to statin therapy (122). Other strategies for inhibiting PCSK9 function include gene silencing via PCSK9-specific siRNAs or ASOs. PCSK9-specific siRNAs have been reported to produce a maximal plasma LDL-C decrease of 50% in cynomolgus monkeys (123) and PCSK9 ASOs have been shown to decrease plasma total cholesterol in hyperlipidemic mice (124). Irrespective of the approach for inhibiting PCSK9, combining it with a statin has the potential to lower LDL-C more robustly than either strategy alone, because statins generally increase the production and secretion of PCSK9. Based on descriptions of patients with LOF mutations in PCSK9, mechanism-related adverse events associated with PCSK9 inhibition are expected to be minimal. Therapies targeting PCSK9 may therefore hold substantial promise for the treatment of hypercholesterolemia (125).

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Thyroid Hormone Receptor Agonists Thyroid hormones (TH) T3 and T4 are important for normal development and cellular metabolism (126). Their effects are mediated via activation of specific thyroid hormone nuclear receptor (TR) isoforms that belong to the nuclear hormone receptor superfamily and act as transcription factors. TRs are ubiquitously expressed and regulate a variety of metabolic pathways, including many aspects of lipid metabolism. Thyroid hormones reduce body weight and increase metabolic rate, in part by increasing lipolysis. However, thyroid hormones can also have deleterious effects such as tachycardia, thus severely limiting their use in the treatment of metabolic disease. The TRb subtype appears to mediate lowering of LDL-C and possibly elevation of metabolic rate, whereas TRa is involved in the control of heart rate. Drug development efforts have therefore been focused on identifying a subtype-specific agonist (127). The hepatic LDLR promoter contains a thyroid-responsive element, and activation of the liver-specific TRb has been associated with increased LDLR expression in preclinical models. Furthermore, TRb activation has been shown to upregulate the mRNA encoding cyp7a1, the enzyme catalyzing the rate-limiting step of cholesterol conversion to bile acids. KB-141, a selective TRb agonist, caused significant LDL-C and body weight reduction in primates after 1 week of treatment, without adversely affecting heart rate (128). MB07811, a liver-targeted prodrug of the TRb agonist MB07344, lowered plasma total cholesterol in cynomolgus monkeys by 34% after 7 days of treatment, and the combination of MB07811 with atorvastatin resulted in a more pronounced reduction in plasma cholesterol than that observed with either treatment alone (129, 130). If the effects observed in primates persist in the clinic, selective thyromimetics have the potential to be developed for the treatment of hypercholesterolemia and the metabolic syndrome.

Phospholipase A2 Inhibitors Members of the phospholipase A2 superfamily are involved in lipoprotein modification and oxidation, and they trigger both vascular and systemic inflammatory responses. The subfamily of enzymes that warrant a more detailed discussion in the context of this chapter is that of secreted phospholipase A2 (sPLA2), a group of hydrolases that are functionally distinct from lipoprotein-associated phospholipase A2 (lp-PLA2), also known as platelet activating factor acetylhydrolase (paf-ah) (131, 132). The spla2 family contains more than 10 enzymes that hydrolyze phospholipids at the sn-2 position in a calcium-dependent manner, thereby releasing bioactive lipids such as arachidonic acid and lysophospholipids. Family members sPLA2-IIA, sPLA2-V, and sPLA2-X are highly expressed in human and mouse atherosclerotic lesions. These enzymes are thought to modify LDL by acting on its phospholipid components such that the resulting small, dense LDL particles have impaired binding affinity to the LDLR. The extended residence time of modified LDL in the circulation allows for their migration into the subendothelial space where they anchor to proteoglycans via apoB, and eventually lead to the formation of LDL aggregates

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and the generation of foam cells (133). The physiological relevance of this process for atherogenesis has been demonstrated using mice that overexpress sPLA-IIA and exhibit increased atherosclerotic lesion size and enhanced collagen deposition (134). sPLA2-IIA is the most extensively studied of the secretory phospholipases, and inhibitors of sPLA2-IIA are in clinical development. Varespladib, a small molecule inhibitor of sPLA2-IIA, had been originally studied as an anti-inflammatory agent in rheumatoid arthritis and as an antiseptic, but was later found to lower both CRP and LDL-C in patients with stable CVD (135). It was subsequently evaluated as an add-on to atorvastatin in ACS patients, where it met its primary end point of significant LDL-C reduction and resulted in a higher percentage of patients achieving and maintaining their target of 70 mg/dL LDL-C over a period of 16 weeks. While the exact mechanism underlying the observed LDL-C lowering remains unclear, these are promising clinical results and lay the foundation for further clinical evaluation of sPLA2 inhibitors as therapeutics for atherosclerosis and CVD (136).

EMERGING THERAPIES TARGETING HDL Multiple epidemiologic studies have identified low levels of HDL-C as an independent risk factor for CVD. The inverse relationship between HDL-C and the incidence of heart disease has been established in the Framingham study (137) and confirmed in the PROCAM (138) and the Quebec cardiovascular studies (139). Each 1 mg/dL decrease in plasma HDL-C was associated with a 2–3% increased risk for CVD. In the VA-HIT trial, in which CHD patients were treated with either gemfibrozil or placebo, lowering of HDL-C was associated with a decreased incidence of CV events; however, gemfibrozil also significantly affected plasma triglyceride levels, thus complicating the interpretation of the HDL-C–CHD correlation (92, 140). Similar results were obtained in the Helsinki Heart Study (141). In patients treated with statins, low HDL-C was established as an important residual risk factor for secondary CV events (142). A recent analysis of data from the Framingham Offspring Study from 1975 through 2003 indicates that raising HDL-C levels with commonly used lipid medications is an important determinant of the benefit associated with lipid therapy (143). Because of the inverse relation between HDL-C and CHD, therapeutic approaches targeting HDL were focused on simply raising plasma HDL-C until very recently. Based on the assumption that the primary antiatherogenic role of HDL occurs by promoting RCT, CETP has been a prominent target for drug development efforts. However, the failure of the CETP inhibitor torcetrapib in the ILLUMINATE trial has led to a critical reevaluation of both HDL-C raising as an appropriate antiatherogenic strategy and the role of CETP in RCTand atherogenesis (144, 145). In ILLUMINATE, patients treated with the CETP inhibitor torcetrapib had a paradoxical increase in CV events despite 70% HDL-C raising (146). Consequently, recent strategies targeting HDL have emphasized both HDL levels and HDL functionality, recognizing that particular HDL subspecies generated by specific interventions may have more favorable antiatherogenic properties than others (147), and that the HDL

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proteome may be functionally just as important as the HDL cholesterol content (148). With respect to RCT, it has become evident that the rate of movement of HDL-C through this pathway rather than absolute plasma HDL-C levels is important, and finally, the RCT pathway paradigm itself is being challenged (149). The following sections attempt to summarize current and novel therapeutic approaches targeting HDL in the “post-torcetrapid world.”

CETP Inhibitors CETP inhibition has long been discussed as a promising therapeutic approach in the atherosclerosis field (150, 151). CETP is secreted by the liver, is bound to HDL particles in the circulation, and facilitates the movement of cholesteryl ester from HDL to LDL and VLDL, in exchange for triglycerides (Figure 8.1). The antiatherogenic potential of CETP inhibition is based on its ability to increase the total pool of apoA1 and HDL by decreasing the rate of lipid exchange between HDL and LDL/ VLDL. However, since shuttling cholesteryl esters from HDL to apoB-containing lipoproteins represents the main route for hepatic clearance of cholesteryl esters via the LDLR, inhibition of CETP may lead to reduced clearance of lipoprotein particles and the accumulation of large, cholesteryl ester-rich HDL particles with unknown effects on atherogenesis. Rodents lack CETP activity and carry most of their cholesterol in HDL particles; however, they are still susceptible to diet-induced atherosclerosis. CETP transgenic mice treated with torcetrapib exhibit reduced lesion size, but develop more inflammatory lesions than mice treated with atorvastatin (152). In rabbits, CETP inhibition prevented atherogenesis in some, but not all, published studies (153–157). Human data linking CETP expression, HDL-C levels, and atherosclerosis are similarly inconclusive (158). While some patients with certain LOF mutations in their CETP gene had elevated levels of plasma HDL-C and fewer CV events (159, 160), patients with other CETP LOF variants appeared to have increased CVD risk, and this association was independent of HDL-C levels (161). Thus, the overall evidence for CETP inhibition as a viable and potent antiatherogenic strategy is conflicting at best. Torcetrapib was the first CETP inhibitor to reach clinical trials and showed a favorable profile in early human studies (162). The drug appeared to be well tolerated, and HDL-C was increased by up to 90% and LDL-C was lowered by up to 42% at high doses. Because the molecule was associated with increases in systolic and diastolic blood pressure in phase II trials, dosing was restricted to 60 mg/day for subsequent phase III trials. The ILLUMINATE trial was designed to compare the effect of combining torcetrapib with atorvastatin to that of atorvastatin and placebo in 15,067 individuals with high CVD risk. Despite a 72% increase in HDL-C and 25% decrease in LDL-C, patients treated with torcetrapib had a significantly higher rate of major CV events, including CVand non-CV deaths (146). The trial was terminated prematurely in December 2006. The mechanisms underlying the increase in CV morbidity and mortality associated with torcetrapib remain inconclusive. They clearly include offtarget effects that involve the activation of mineralocorticoid receptors by aldosterone

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and the subsequent induction of hypertension (146), but on-target adverse effects caused by proatherogenic properties of the particular HDL subspecies that accumulates as a result of CETP inhibition cannot be excluded. Additional studies evaluating the relation between torcetrapib-generated HDL and coronary atherosclerosis using IVUS (ILLUSTRATE) (163) or cIMT (RADIANCE) (164, 165) have generated conflicting results; furthermore, the exact role of CETP in the RCT pathway remains unclear. However, recent clinical studies indicate that small molecule CETP inhibitors that are structurally different from torcetrapib do not cause an increase in blood pressure (166–169), raising the possibility that CETP inhibition may still be a viable therapeutic strategy. Accordingly, the development of such molecules is still being pursued by Merck (anacetrapib) (166, 167) and Roche/Japan Tobacco (dalcetrapib/ JTT 705) (168, 169). In phase II trials, both molecules have been shown to be well tolerated, both as monotherapy and in combination with statins. Anacetrapib is being evaluated for long-term safety and tolerability (170) and separately in a double-blind, randomized, placebo-controlled phase III study to assess efficacy in combination with a statin in patients with CHD, hypercholesterolemia, or mixed hyperlipidemia (171). Results from these trials will likely determine the future therapeutic potential of small molecule CETP inhibitors.

ApoA1 and ApoA1 Mimetics ApoA1-based therapeutic approaches have received an equal amount of attention as those focused on CETP inhibition. ApoA1 is synthesized in hepatocytes and in small intestinal cells and is the major protein component of HDL. Mature apoA1 interacts with lipid transporters such as ABCA1, expressed on the surface of macrophages, hepatic, small intestinal, and endothelial cells, to form nascent preb-HDL particles containing free cholesterol. As an activator of LCAT, apoA1 enhances the esterification of free cholesterol and the maturation of HDL particles from nascent prebHDL to large, apoA1- and cholesteryl ester-rich HDL particles that are substrates for CETP. ApoA1 is therefore thought to be an important driving force behind HDL-C elevation and the flux of free cholesterol from peripheral tissues into the plasma. A variety of clinical and epidemiological studies have shown a direct correlation between plasma apoA1 and HDL levels (172, 173), and an inverse correlation between plasma apoA1 and susceptibility to atherosclerosis and CVD. Transgenic or adenoviral expression of human apoA1 in hyperlipidemic mice was associated with a twofold increase in HDL-C and a significant reduction in atherosclerotic lesions (174, 175). The antiatherosclerotic properties of apoA1 may in part be due to its ability to promote cholesterol efflux (176), but in addition to its role in RCT, the atheroprotective effect of apoA1 may be linked to its anti-inflammatory properties (177). There is experimental evidence suggesting that apoA1 may have the ability to remove proinflammatory oxidized phospholipids from LDL and from the artery wall. Mice and humans injected with human apoA1 generate LDL with a reduced effect on monocyte chemotactic activity in cell-based assays, when compared to LDL from respective controls. The investigators interpreted these findings as an indication

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that apoA1 infusion has the ability to generate LDL particles with an improved resistance to oxidation by factors such as lipid hydroperoxides released from artery wall cells (27, 178). Several approaches have attempted to exploit apoA1 atheroprotective properties for developing therapeutics, with mixed success. Studies using apoA1 Milano, a putatively atheroprotective variant of apoA1 found in long-lived individuals in rural Italy, produced a rapid regression of atherosclerosis in animal models. In clinical studies, infusion of apoA1 Milano formulated in phospholipid complexes (ETC216) was associated with a significant regression of coronary atherosclerosis as measured by IVUS after just five weekly infusions (179); however, the trial design left open the question of whether apoA1 Milano itself or the phospholipid component was the causative agent. These results have not been confirmed in subsequent studies and clinical development of ETC216 has since been abandoned. Finally, infusion of CSL-111, reconstituted HDL consisting of apoA1 from human plasma combined with soybean phosphatidylcholine, did not significantly reduce atheroma volume by IVUS in the ERASE trial (180). Using recombinant apoA1 as a therapeutic modality has become less appealing due to mixed trial results, and because large-scale manufacturing of a full-length apoA1 protein (243 amino acids) in a suitable and reproducible formulation has proven complex and expensive. Efforts to feasibly capture the potential benefits of apoA1 have evolved into utilizing apoA1-derived peptides (apoA1 mimetics) that preserve its lipid-binding properties. Peptides were designed of 18 amino acid residues in length that mimicked the secondary structure of apoA1 class A amphipathic helices, identified as the portion of apoA1 responsible for interacting with free cholesterol (181). Systematic studies were carried out to combine optimal physicochemical properties with biological activity as assessed in tissue culture models of the human artery wall. D-4F, a peptide containing four phenylalanine (F) blocking residues and protected from enzymatic degradation based on its content of only D-amino acids, was identified as a promising candidate and evaluated in vivo. Oral administration of D-4F to LDLR KO mice fed a Western diet was associated with a 79% reduction in atherosclerotic lesions. Similarly, D-4F reduced lesions by 75% in atherosclerotic APOE KO mice. In neither case was there a significant change in plasma HDL-C, but HDL particles appeared to have improved anti-inflammatory properties as determined by their ability to bind oxidized lipids in cell-based assays. D-4F, reverse D-4F, and several other apoA1 mimetics (5A, ATI-5261, LSI518P) were taken into clinical development by several pharmaceutical and biotechnology companies. Phase I clinical trial data for D-4F have been reported, suggesting a favorable effect on anti-inflammatory properties of HDL in high-risk CVD patients. It is unclear whether D-4F clinical development has progressed further, partly due to its low bioavailability. However, there continues to be promise in the field of apoA1 mimetics. A recent report describes an apoA1 mimetic peptide containing a proline residue that has greater in vivo HDL binding and anti-inflammatory ability than the 4F peptide (182). The authors hypothesize that apoA1 mimetic peptide structure influences lipoprotein association properties, thereby modifying peptide plasma kinetics as well as therapeutic efficacy.

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APOA1 Activators An alternative approach for exploiting apoA1 antiatherogenic properties is based on activating endogenous apoA1 production via transcriptional regulation. APOA1 gene expression is regulated by response elements present in the APOA1 promoter and enhancer, and by response elements found in the APOC3 enhancer, located in the same gene cluster (183). A variety of orphan and ligand-dependent nuclear receptors have been shown to activate APOA1 transcription via these elements, including PPARa and PPARd. Accordingly, PPARa and PPARd ligands such as GW501516 and T659 have been reported to increase serum apoA1 and HDL concentrations in various preclinical models. RVX-208 is an APOA1 transcriptional activator that increased apoA1 production and raised HDL-C in nonhuman primates; the compound is currently being evaluated in clinical trials. In a phase Ia study, RVX-208 was administered to healthy volunteers for 7 days and was associated with an 11% increase in apoA1 and a 42% increase in preb-HDL. Using plasma from treated subjects and controls, the investigators found a 10% increase in ABCA1dependent cholesterol efflux in a cell-based assay. These results were confirmed in a phase Ib/IIa study during which patients with low HDL were treated with RVX-208 for 28 days (184). While the overall changes in HDL quantity and functionality appear modest, transcriptional activation of apoA1 still holds promise as a therapeutic approach.

LXR Activators The therapeutic potential behind the activation of the liver X receptors a and b (LXRa and LXRb) is being actively pursued. LXRs are members of the nuclear receptor superfamily of ligand-activated transcription factors; they regulate gene expression by forming heterodimers with RXR and binding to target DNA sequences known as LXR-responsive elements (LXREs). (185). LXREs have been found in a number of genes that are critical for cholesterol absorption, transport, efflux, and excretion. To name a few, ABCG5 and ABCG8 along with NPC1L1 mediate LXR effects on intestinal cholesterol absorption; CYP7A1, encoding the rate-limiting enzyme in bile acid biosynthesis, regulates cholesterol catabolism and its excretion from the liver as bile acids; ABCA1 and ABCG1 mediate the initial steps in RCT and the maturation of HDL; and APOE, CETP, PLTP, and LPL play key roles in HDL remodeling. In addition, LXRs regulate the expression of a number of genes involved in lipogenesis via direct activation of SREBP1-c, which in turn leads to activation of FAS, ACC, and SCD-1. Finally, LXR regulates the transcription of ANGPTL3 and APOA5, genes involved in triglyceride metabolism (186, 187). Not surprisingly, therefore, activation of LXRs has the potential to result in beneficial effects on cholesterol transport and excretion, coupled with less favorable effects on hepatic lipid and plasma triglyceride metabolism. To address this dichotomy, considerable efforts have been invested in the identification of synthetic LXR ligands that are partial or gene-selective modulators, rather than pure agonists. Some approaches were designed to identify

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subtype-selective agonists, based on the assumption that activation of LXRb alone will raise plasma HDL-C and stimulate RCT without causing liver triglyceride accumulation. LXRb is expressed at low levels throughout the whole body, whereas LXRa is highly expressed in the liver, and at much lower levels in macrophages, intestine, adrenal glands, lungs, and kidneys. However, this approach has been hampered by the fact that LXRa and LXRb are very similar in structure and exhibit a high degree of homology between their ligand binding domains (188). The most potent LXR activators are the naturally occurring oxysterols: 22(R)-hydroxycholesterol, 20(S)-hydroxycholesterol, 24(S),25-epoxycholesterol, and 27-hydroxycholesterol. Synthetic activators developed and tested in preclinical models to date include the well-characterized nonsteroidal compounds T0901317 (189) and GW3965 (190). When administered to rodents, both compounds result in marked plasma HDL-C raising, but their effects on hepatic and plasma triglyceride metabolism were different. T0901317 was invariably associated with hypertriglyceridemia and steatosis, while the effect of GW3965 ranged from a mild elevation of triglycerides to no induction of steatosis. The steroidal LXR agonists ATI-829 (191) and DMHCA (192) showed promise as drug candidates based on their selective inhibition of genes involved in cholesterol metabolism. The hope associated with these and similar compounds is that their selective effects on macrophage and intestinal cholesterol transport will translate into antiatherogenic properties without upregulating hepatic lipogenic genes. Indeed, several interventional studies in murine models suggest that LXR activation may be beneficial in the treatment of atherosclerosis (193, 194). In addition to metabolic regulation, some of the observed effects on attenuation of plaque progression may well be mediated by a direct LXR protective action on the artery wall, such as repression of NF-kB and inhibition of its target genes IL-6 and MMP-9 (195). In summary, while LXR ligands may represent attractive therapeutic agents, considerable challenges remain to be addressed.

LCAT Activators LCAT activation is an emerging therapeutic strategy in the HDL field. LCAT is a plasma enzyme that catalyzes the esterification of free cholesterol and contributes to the maturation of HDL particles. The enzyme facilitates the transfer of 2-acyl groups from lecithin to free cholesterol, generating cholesteryl esters and lysolecithin. Newly formed cholesteryl esters are retained in the HDL core, forming large, mature HDL particles. LCAT plays a central role in promoting RCT based on its ability to maintain a free cholesterol gradient between peripheral tissues and plasma HDL. This hypothesis has been put forward decades ago by Glomset (23, 196) and is supported by observations in some, but not all, published studies in preclinical models. LCAT overexpression and LCAT gene therapy in monkeys and rabbits have been shown to generate an antiatherogenic lipoprotein profile and attenuate progression of atherosclerosis, respectively (197, 198). On the other hand, LCAT overexpression did not promote an increased rate of macrophage RCT in transgenic mice expressing human

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apoA1, even in the context of coexpression of SRB-1 or CETP, both of which facilitate the clearance of HDL particles by the liver. The impact of LCAT deficiency on atherosclerosis and CVD in humans remains similarly inconclusive. Complete lack of LCAT enzyme is associated with familial LCAT deficiency (FLD), a rare disease characterized by very low HDL-C, high plasma triglycerides, and reduced plasma apoA1 levels. Partial lack of enzyme activity leads to a syndrome known as fish-eye disease (FED) (199). A recent study surveying cIMT in 12 individuals with two LOF mutations in LCAT and 28 heterozygous carriers appears to indicate that genetic LCAT deficiency is not associated with enhanced preclinical atherosclerosis (200). On the other hand, heterozygosity for LCAT gene defects was associated with increased cIMT in 47 carriers versus 58 family controls in a different study, suggesting that LCAT protects against atherosclerosis (201). In both cases, the study populations were small and the relatively low plasma LDL-C levels typically found in LCATdeficient patients may have been a confounding factor. A synthetic small molecule activator of LCAT has been described; the compound was associated with a robust increase in HDL-C in rodent models, caused by the formation of large, apoA1-rich HDL particles (202). Whether or not LCAT activators have utility for reducing cholesterol accumulation in atherosclerotic plaques remains to be seen.

SUMMARY AND OUTLOOK Optimal treatment of dyslipidemias and their associated morbidities remains a formidable challenge, despite the efficacy of existing therapies such as statins, bile acid sequestrants, nicotinic acids, and fibrates. The prevalence of dyslipidemia worldwide remains high, and cardiovascular disease is still the main cause for mortality in the developed world. New drugs that could be used either alone or in combination with existing therapies are being developed, but face a variety of challenges, including a more stringent regulatory environment. Recent requests for cardiovascular outcome trials, partly triggered by the absence of a consistently robust relationship between surrogate markers and cardiovascular events, will add to the enormous costs of developing such drugs and will curb appetite in the biopharmaceutical industry for taking risks associated with novel mechanisms of action. Regardless, lowering of LDL-C remains a major target for intervention, as a large percentage of high-risk patients do not reach their LDL-C targets. Among the emerging therapies for lowering LDL-C, apoB and PCSK9 inhibitors hold substantial promise based on the strong validation of their molecular targets in human genetic disease and early preclinical and clinical findings. In addition, the development of therapies targeting HDL remains an active area of research despite recent setbacks associated with the development of CETP inhibitors. The focus in this area has shifted from simply raising HDL-C to providing evidence that newly generated HDL particles are “functional” and have antiatherogenic properties. The definition of “functional” continues to evolve as novel therapies such as apoA1 mimetics and activators are being assessed in new biological contexts. Further, there is a growing recognition that anti-inflammatory properties of HDL may clinically be as important

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127. MORKIN, E., P. LADENSON, S. GOLDMAN, and C. ADAMSON. 2004. Thyroid hormone analogs for treatment of hypercholesterolemia and heart failure: past, present and future prospects. J Mol Cell Cardiol 37:1137–1146. 128. GROVER, G.J., K. MELLSTROM, and J. MALM. 2005. Development of the thyroid hormone receptor betasubtype agonist KB-141: a strategy for body weight reduction and lipid lowering with minimal cardiac side effects. Cardiovasc Drug Rev 23:133–148. 129. ERION, M.D., E.E. CABLE, B.R. ITO, H. JIANG, J.M. FUJITAKI, P.D. FINN, B.H. ZHANG, J. HOU, S.H. BOYER, P.D. VAN POELJE, and D.L. LINEMEYER. 2007. Targeting thyroid hormone receptor-beta agonists to the liver reduces cholesterol and triglycerides and improves the therapeutic index. Proc Natl Acad Sci USA 104:15490–15495. 130. ITO, B.R., B.H. ZHANG, E.E. CABLE, X. SONG, J.M. FUJITAKI, D.A. MACKENNA, C.E. WILKER, B. CHI, P.D. VAN POELJE, D.L. LINEMEYER, and M.D. ERION. 2009. Thyroid hormone beta receptor activation has additive cholesterol lowering activity in combination with atorvastatin in rabbits, dogs and monkeys. Br J Pharmacol 156:454–465. 131. BOYANOVSKY, B.B., and N.R. WEBB. 2009. Biology of secretory phospholipase A2. Cardiovasc Drugs Ther 23:61–72. 132. ZALEWSKI, A., and C. MACPHEE. 2005. Role of lipoprotein-associated phospholipase A2 in atherosclerosis: biology, epidemiology, and possible therapeutic target. Arterioscler Thromb Vasc Biol 25:923–931. 133. KARABINA, S.A., I. BROCHERIOU, G. LE NAOUR, M. AGRAPART, H. DURAND, M. GELB, G. LAMBEAU, and E. NINIO. 2006. Atherogenic properties of LDL particles modified by human group X secreted phospholipase A2 on human endothelial cell function. FASEB J 20:2547–2549. 134. IVANDIC, B., L.W. CASTELLANI, X.P. WANG, J.H. QIAO, M. MEHRABIAN, M. NAVAB, A.M. FOGELMAN, D.S. GRASS, M.E. SWANSON, M.C. DE BEER, F. DE BEER, and A.J. LUSIS. 1999. Role of group II secretory phospholipase A2 in atherosclerosis. 1. Increased atherogenesis and altered lipoproteins in transgenic mice expressing group IIa phospholipase A2. Arterioscler Thromb Vasc Biol 19:1284–1290. 135. Anthera. 2009. A002 (Varespladib): short-term (16-week) treatment of acute coronary syndrome. http://www.anthera.com/products_a002.asp. 136. ROSENSON, R.S. 2009. Future role for selective phospholipase A2 inhibitors in the prevention of atherosclerotic cardiovascular disease. Cardiovasc Drugs Ther 23:93–101. 137. BODEN, W.E. 2000. High-density lipoprotein cholesterol as an independent risk factor in cardiovascular disease: assessing the data from Framingham to the Veterans Affairs High-Density Lipoprotein Intervention Trial. Am J Cardiol 86:19L–22L. 138. ASSMANN, G., H. SCHULTE, A. VON ECKARDSTEIN, and Y. HUANG. 1996. High-density lipoprotein cholesterol as a predictor of coronary heart disease risk. The PROCAM experience and pathophysiological implications for reverse cholesterol transport. Atherosclerosis 124(Suppl):S11–S20. 139. DESPRES, J.P., I. LEMIEUX, G.R. DAGENAIS, B. CANTIN, and B. LAMARCHE. 2000. HDL-cholesterol as a marker of coronary heart disease risk: the Quebec cardiovascular study. Atherosclerosis 153: 263–272. 140. RUBINS, H.B., and S.J. ROBINS. 2000. Conclusions from the VA-HIT study. Am J Cardiol 86:543–544. 141. HUTTUNEN, J.K., V. MANNINEN, M. MANTTARI, P. KOSKINEN, M. ROMO, L. TENKANEN, O.P. HEINONEN, and M.H. FRICK. 1991. The Helsinki Heart Study: central findings and clinical implications. Ann Med 23:155–159. 142. BARTER, P., A.M. GOTTO, J.C. LAROSA, J. MARONI, M. SZAREK, S.M. GRUNDY, J.J. KASTELEIN, V. BITTNER, and J.C. FRUCHART. 2007. HDL cholesterol, very low levels of LDL cholesterol, and cardiovascular events. N Engl J Med 357:1301–1310. 143. GROVER, S.A., M. KAOUACHE, L. JOSEPH, P. BARTER, and J. DAVIGNON. 2009. Evaluating the incremental benefits of raising high-density lipoprotein cholesterol levels during lipid therapy after adjustment for the reductions in other blood lipid levels. Arch Intern Med 169:1775–1780. 144. JOY, T.R., and R.A. HEGELE. 2008. The failure of torcetrapib: what have we learned? Br J Pharmacol 154:1379–1381. 145. BARTER, P. 2009. Lessons learned from the Investigation of Lipid Level Management to Understand its Impact in Atherosclerotic Events (ILLUMINATE) trial. Am J Cardiol 104: 10E–15E.

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Mechanism of Action of Niacin: Implications for Atherosclerosis and Drug Discovery DEVAN MARAR1,2, SHOBHA H. GANJI1,2, VAIJINATH S. KAMANNA1,2, 1,2 AND MOTI L. KASHYAP 1

Department of Veterans Affairs Healthcare System, Atherosclerosis Research Center, Long Beach, CA, USA 2 Department of Medicine, University of California, Irvine, CA, USA

INTRODUCTION Niacin, also known as nicotinic acid or vitamin B3, is a water-soluble vitamin B composed of a pyridine and a carboxyl group (Figure 9.1). In the human body, a supply of niacin is also derived from the essential amino acid tryptophan. The vitamin is converted in vivo to its amide counterpart nicotinamide and contributes to the formation of the coenzymes nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). NAD and NADP can be reduced to NADH and NADPH, respectively, and play a vital role in oxidation and reduction reactions, as well as participate in a myriad of metabolic processes. Niacin is found in many foods including dairy products, meats, nuts, and enriched cereals. It can also be obtained in over-the-counter supplements or as a prescribed medication. Deficiency in nicotinic acid leads to pellagra, a syndrome that is now rare in the industrialized world given increased availability of the vitamin. However, within the past 50 years significant discoveries have been made on the pharmacological

Metabolic Syndrome: Underlying Mechanisms and Drug Therapies Edited by Minghan Wang Copyright 2011 John Wiley & Sons, Inc.

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Figure 9.1 Niacin with molecular formula C6H4NO2.

properties of niacin at elevated dosages, in particular its beneficial effect on the lipid profile and its impact on cardiovascular disease. At pharmacological doses, niacin favorably affects circulating lipoproteins and impacts coronary heart disease (CHD) risk. After the discovery of its lipid altering properties by Altschul et al. in 1955 (1), it became one of the first widely used cholesterol modifying agents to affect cardiovascular disease outcomes. Low levels of high-density lipoprotein (HDL) are a well-recognized independent risk factor for CHD (2, 3). Studies such as the VA-HIT trial demonstrate that interventions to raise HDL reduce the risk of cardiovascular disease independent of their effect on serum low-density lipoprotein (LDL) levels (4). Furthermore, niacin remains the most effective agent available to raise HDL levels. Most healthcare providers are well aware of niacin’s effect on HDL. However, nicotinic acid also lowers apolipoprotein B-containing lipoproteins, as well as possesses antioxidant and anti-inflammatory properties. This chapter will discuss the mechanisms of action of niacin in relation to its effects on cholesterol and atherosclerosis, its use by the clinician, and the adverse properties that have limited the use of this older drug.

MECHANISMS OF ACTION Effect on HDL Niacin increases serum HDL levels largely by decreasing the catabolism of HDLapolipoprotein A-I (HDL-apoA-I), as opposed to increasing the synthetic rate (5, 6). The liver is a major source of apoA-I and HDL production in the human body. Research conducted using human hepatocytes (HepG2 cells) to assess in vitro niacin’s effect on HDL-apoA-I metabolism demonstrated a selective inhibition of the uptake of HDL-apoA-I but not HDL cholesterol ester (7). Niacin was observed to have no significant effect on the scavenger B1 receptor that is involved in cholesterol ester uptake. A proposed mechanism of niacin’s inhibition of HDL-apoA-I catabolism is its effect on the HDL holoparticle catabolism receptor pathway. b-Chain adenosine triphosphate synthase is a key component of the HDL holoparticle catabolic receptor pathway involved in HDL holoparticle uptake (proteins and lipids) (8). This mitochondrial protein acts as an HDL receptor on the cell surface. HepG2 cells preincubated with niacin demonstrate reduced surface expression of the b-chain in HepG2 cells and decreased uptake of radiolabeled HDL-apoA-I in hepatic cells (9). The above findings help explain niacin’s ability to raise serum HDL levels by

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limiting the catabolism of HDL-apoA-I without negatively impacting the clearance of cholesterol esters. Further investigation into the separated HDL subfractions, those containing lipoprotein A-I (Lp A-I) versus those with lipoprotein A-I and A-II (Lp A-I þ A-II), revealed that niacin preferentially increased Lp A-I levels relative to Lp A-I þ A-II. One study involving 139 patients with low HDL cholesterol treated with either niacin or the fibrate gemfibrozil demonstrated in the niacin arm a 24% increase in Lp A-I levels, with a more modest increase of 9% in Lp A-I þ A-II levels (10). No significant increase in Lp A-I levels was observed with gemfibrozil. Lp A-I particles were observed to be 70% more efficient than their Lp A-I þ A-II counterparts at delivering cholesterol esters in cultured HepG2 cells. These findings suggest that niacin has additional cardioprotective effects by preserving the HDL subfraction of Lp A-I, a lipoprotein more efficient at reverse cholesterol transport relative to Lp A-I þ A-II. There is emerging evidence that niacin may have some capacity to increase HDL particle production as well. In the liver, free cholesterol effluxes to apolipoprotein A-I by the ATP binding cassette transporter A1 (ABCA1) forming lipid-poor apoA-I particles with pre-beta mobility. The particles are released to the circulation where they pick up more free cholesterol from other sites, maturing to larger HDL particles that participate in reverse cholesterol transport via the scavenger B1 receptor and other reverse cholesterol transport pathways. Initial studies from our laboratory indicated that niacin stimulates the transcription of the ABCA1 gene as well as raises ABCA1 protein expression in HepG2 cells. Niacin also increases apoA-I-specific cholesterol efflux from HepG2 cells and results in amplified apoA-I lipidation by cholesterol and phospholipids. The overall result is increased HDL biogenesis and the formation of apoA-I lipid-poor particles that could potentially participate in reverse cholesterol transport. It should also be noted that niacin may not just increase serum HDL levels but perhaps the availability of cholesterol to be transferred to HDL particles in the periphery to be returned to the liver for clearance. Recent evidence suggests that niacin increases the expression of peroxisome proliferator-activated receptor-g and conserved domain 26 (CD36) and the transcription of ABCA1 transporters in monocytes (11). As ABCA1 is the major transporter involved in reverse cholesterol transport, niacin may have an additional role of enhancing clearance of peripheral cholesterol.

Effect on VLDL, LDL, and Triglycerides Besides its ability to elevate HDL levels, at therapeutic doses niacin also has the capacity to lower levels of apolipoprotein B-containing particles, such as very-lowdensity lipoprotein (VLDL) and LDL. The liver is the major site of production of apolipoprotein B, the main lipoprotein of VLDL and LDL. In hepatic cells, the rate of secretion and degradation of apolipoprotein B is strongly governed by the rate of synthesis of triglycerides to lipidate the apolipoprotein (12, 13). The addition of triglycerides to apolipoprotein B leads to the transfer of apolipoprotein B to the

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endoplasmic reticulum lumen and its subsequent secretion (14). Niacin is a direct, noncompetitive inhibitor of an important enzyme involved in the final stages of triglyceride synthesis: diacylglycerol acyltransferase-2 (DGAT2) (15). A 21% decrease in the synthesis rate of VLDL triglycerides has been observed in human subjects treated with niacin (16). In vitro experiments demonstrate a 20% reduction in apolipoprotein B secretion of HepG2 cells into their culture media compared to controls after treatment with nicotinic acid (17). By directly inhibiting DGAT2, decreased intrahepatic synthesis of triglycerides is achieved, and subsequently, decreased apolipoprotein B secretion and increased posttranslational degradation. This in turn contributes to a decline in the formation of VLDL, and consequently its eventual catabolic product, LDL. Different mechanisms of action pertaining to the influence of nicotinic acid on triglyceride metabolism and synthesis have been investigated. One potential mechanism that has been examined involves adipocyte lipolysis as a target of niacin’s beneficial effect on triglycerides (18). Peripheral adipocytes assist in the synthesis and storage of the body’s triglycerides, as well as their metabolism to free fatty acids and glycerol. Niacin has been observed to limit free fatty acid release from adipocytes by inhibiting triglyceride lipolysis (19). G protein-coupled receptors GPR109A and GPR109B (also known as HM74A and HM74, respectively) have been identified as nicotinic acid receptors found in adipocytes and immune cells, and GPR109A in particular demonstrates both specificity and high affinity for niacin (20–22). To strengthen evidence for GPR109A’s role in niacin-induced inhibition of adipose lipolysis, mice deficient in PUMA-G, a receptor analogous to GPR109A, do not demonstrate any alteration in plasma levels of free fatty acids or triglycerides after treatment with niacin (20). However, mice that do possess the PUMA-G receptor experienced a 30% decrease in their triglyceride levels with niacin therapy. Thus, it has been suggested that decreased levels of free fatty acids may cause less substrate availability for triglyceride synthesis and hence decreased serum levels of triglycerides. Despite the observed decrease in peripheral adipocyte lipolysis with niacin, this is unlikely the true underlying mechanism of nicotinic acid-induced decline in measured triglyceride levels in humans. Intuitively, decreased adipocyte lipolysis could result in increased adipose triglycerides, and consequently, obesity. However, the decline in plasma concentrations of free fatty acids by nicotinic acid is in fact a transient process, and levels rebound quickly within an hour (so much so that levels can actually be increased at 24 h) (23). It should also be noted that there is no evidence of decreased transport of nonesterified fatty acids with long-term therapy of niacin and that the drug does not significantly contribute to obesity. In addition, the required concentrations of nicotinic acid to affect the GPR109A receptor are a mere fraction of the elevated pharmacological concentrations needed to lower lipids in humans (21). These findings suggest that GPR109A and alterations in adipose lipolysis may not be the mechanism of action causing decreased serum triglycerides in humans. This effect may actually be the result of niacin’s inhibition of DGAT2 and decreased ability of the liver to produce triglycerides. This observation is reinforced by the fact that inhibiting DGAT2 in mice with antisense oligonucleotides results in decreased triglyceride production (24).

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239

Effect on Vascular Inflammation The cardioprotective properties of niacin are not just limited to its lipid lowering effects. In addition, it possesses antioxidant and anti-inflammatory properties that influence the development and the progression of atherosclerotic disease. As mentioned earlier, nicotinic acid is a precursor molecule of NAD and NADP. Studies with Jurkat cells, an immortalized cell line of human T-cell lymphoma, have demonstrated that niacin increases the cellular concentrations of NAD and also enhances expression of glucose 6-phosphate dehydrogenase (G6PD) (25). G6PD is the rate-limiting enzyme of the pentose phosphate pathway and participates in the production of the reduced form of NADP, NADPH (26). In turn, NADPH regulates reactive oxidative species (ROS) generating oxidases and preserves active forms of antioxidant enzymes such as catalase and glutathione reductase, leading to decreased cellular concentrations of ROS (27, 28). Further investigation by Ganji et al. involving in vitro studies of human aortic endothelial cells (HAECs) have also supported niacin’s antioxidant abilities. HAECs preincubated with nicotinic acid demonstrate elevation of NADPH levels by 54% (29). In addition, while total cellular levels of glutathione are not changed in HAECs treated with niacin, the reduced form of glutathione, GSH, was seen to increase by 98%, as was the ratio of GSH to oxidized glutathione (GSSG) (29). GSH is a well-known antioxidant and defends cells from oxidative stress from ROS. HAECs exposed to angiotensin II, a recognized inducer of oxidative stress and inflammation in endothelial cells, showed an eightfold increase in the number of ROS generated compared to control cells (29). However, HAEC counterparts preincubated with nicotinic acid and then angiotensin II demonstrate a dose-dependent reduction in ROS formation, with reductions as much as 86% (29). The oxidation of LDL has also been shown to be lowered with niacin treatment. Endothelial cell oxidation of LDL has been implicated as a factor in the development and progression of atherosclerotic disease (30). Oxidized LDL contributes to atherogenesis by transforming monocytes and macrophages into lipid-laden foam cells, as well as the chemotactic recruitment of monocytes to the endothelium (31, 32). Incubation of LDL in a HAEC mixture leads to the formation of endothelial cellinduced LDL oxidation. Mixtures of LDL, HAECs, and niacin result in markedly lower levels of measured oxidized LDL (29). This process is likely mediated by niacin increasing NADPH concentrations and decreasing production of oxidative species and the oxidation of LDL. The ability of nicotinic acid to decrease LDL oxidation may limit foam cell formation and the development of atherosclerosis in blood vessel. Impedance of monocyte chemotaxis into blood vessel walls may be another beneficial outcome of niacin therapy. Monocyte invasion into the arterial wall is an early step in the formation of an atherosclerotic lesion. Vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemotactic protein-1 (MCP-1) are key players involved in the adhesion and subsequent chemotaxis of immune cells into the vessel wall (33). The expression of both VCAM-1 and MCP-1 is increased by the inflammatory cytokine TNFa (29). With assessment of protein cell lysate by ELISA, HAECs preincubated with 1 mM of niacin demonstrate a 32% decline in VCAM-1 levels after stimulation with TNFa (29). A similar decrease of 34% in MCP-1

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secretion is observed in TNFa-treated HAECs preincubated with niacin (29). Furthermore, incubation of HAECs with 1 mM nicotinic acid subsequently treated with TNFa lowers TNFa-induced monocyte adhesion to endothelial cells by up to 54% (29). The reduction in VCAM-1 and MCP-1 expression by niacin is likely due to its inhibition of NF-kb activation, a promoter of inflammatory cytokine expression in TNFa-stimulated cells (29, 34).

Effect on Flushing A major side effect of nicotinic acid that has limited its use for dyslipidemia is its vasocutaneous flushing response. Prostanoids such as prostaglandin D2 and prostaglandin E2 have been implicated as mediators of this “niacin flush” (35). Furthermore, skin Langerhans cells express GPR109A and are the primary cells involved in niacininduced prostaglandin D2 release (36, 37). Studies with gene knockout mice reveal that PUMA-G (human orthologue GPR109A), cyclooxygenase type 1, and prostaglandin D2 and E2 receptors are involved in this adverse vasocutaneous reaction. To support these findings, mice lacking PUMA-G receptors do not demonstrate an increase in blood flow to the ears after treatment with niacin, a measure of niacin-induced flushing (38). The niacin flush is also absent in mice deficient in cyclooxygenase type 1 and diminished in mice without prostaglandin D2 and E2 receptors (39). These findings suggest that GPR109A/PUMA-G help mediate niacin-induced flushing with the production of prostaglandins D2 and E2 by Langerhans skin cells. Figure 9.2 summarizes evidence of mechanism of action of niacin. The data indicate that niacin by inhibiting hepatic DGAT2 decreases triglyceride synthesis and its availability for VLDL assembly resulting in increased posttranslational intrahepatic apoB degradation. Increased hepatocyte apoB degradation by niacin would decrease the number of VLDL and their catabolic product, LDL particles, which explains the lower apoB and LDL concentrations observed clinically after niacin treatment. In addition, niacin-mediated inhibition of TG synthesis may produce decreased concentrations of large TG-rich VLDL1 particles, which in turn may result in decreased formation of small, dense LDL particles. Niacin, through inhibiting surface expression of b-chain ATP synthase, may inhibit removal of HDL-apoA-I. These mechanisms of decreased HDL-apoA-I catabolism by niacin would increase HDL half-life and concentrations of Lp A-I HDL subfractions, thereby augmenting cholesterol efflux and reverse cholesterol transport. Increased residence time would also allow HDL size to increase (HDL2 > HDL3) from peripheral tissue cholesterol uptake. In addition, niacin inhibits vascular inflammation by decreasing endothelial ROS production, LDL oxidation, and subsequent VCAM-1 and MCP-1 expression resulting in decreased monocyte/macrophage adhesion and accumulation, key events in early atherogenesis (Figure 9.2). These in vitro studies describe a novel antiinflammatory mechanistic role for niacin in decreasing atherosclerosis beyond its conventional role as a lipid regulating agent. Figure 9.2 also indicates how niacin induces flushing.

Multiple Tissue/Cellular Target Sites of Action of Niacin

241

Figure 9.2

Summary of the proposed mechanisms of action of niacin on hepatic lipid/lipoprotein metabolism, aortic endothelial inflammation, and skin flush response. In hepatocytes, niacin directly inhibits DGAT2 enzyme resulting in decreased TG synthesis and subsequent increased intracellular apoB degradation and decreased secretion of VLDL and LDL particles. Niacin, through inhibiting the surface expression of hepatocyte HDL catabolism receptor b-chain ATP synthase, inhibits removal of HDL-apoA-I resulting in increased retention of HDL-apoA-I particles. In aortic endothelial cells, niacin directly increases NADPH and GSH levels resulting in decreased production of ROS and expression of oxidation-sensitive inflammatory VCAM-1 and MCP-1 genes associated with monocyte adhesion and chemotaxis, a key pathobiological initial step involved in atherogenesis. In skin Langerhans cells, niacin by activating GPR109A receptor increases the production of prostanoids including PGD2 and PGE2 resulting in skin vasodilatory flush adverse reactions. These new discoveries explain cellular mechanisms by which niacin confers its beneficial effects on atherosclerosis and adverse flush reaction. DGAT2, diacylglycerol acyltransferase-2; EC, endothelial cell; HDL, high-density lipoprotein; LDL, low-density lipoprotein; MCP-1, monocyte chemotactic protein-1; ROS, reactive oxygen species; PGD2, prostaglandin D2; PGE2, prostaglandin E2; TG, triglycerides; VCAM-1, vascular cell adhesion molecule-1; VLDL, very-low-density lipoprotein.

MULTIPLE TISSUE/CELLULAR TARGET SITES OF ACTION OF NIACIN As described above in “Mechanisms of Action” section and summarized in Figure 9.2, current evidence indicates that niacin acts on multiple tissues and targets to beneficially modulate lipid/lipoprotein profile, anti-inflammatory processes, and adverse flush reactions. Based on the current knowledge, liver appears to be the major target organ of niacin to increase HDL-apoA-I and decrease triglycerides and VLDL/LDL

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particles. The selective tissue distribution of GPR109A and GPR109B only in adipose tissue, spleen, and immune cells but not in other major tissues including liver, kidney, heart, intestine, and so on indicates that niacin receptor GPR109A may not be involved in niacin’s action on liver apoA-I/HDL catabolism, DGAT2 and apoB-bearing lipoprotein secretion, and vascular anti-inflammatory properties. In humans, physiologically PUMA-G/GPR109A-mediated adipocyte triglyceride lipolysis may be only a minor mechanism in explaining triglyceride lowering and other beneficial effects of niacin. However, PUMA-G/GPR109A receptor in Langerhans cells/macrophages is importantly involved in niacin-induced adverse flushing. The direct effect of niacin to increase aortic endothelial cell redox potential and its vascular anti-inflammatory properties may additionally account for its proven effects in atherosclerotic cardiovascular disease beyond its lipid regulation. The newly identified sites of action of niacin pave the way for potential drug discovery to yield agents superior to niacin but without its adverse effects. In addition, niacin’s mechanisms of action are unique and thus clinically form the rationale for combination therapy with other lipid regulators.

CLINICAL UTILIZATION OF NIACIN When assessing the lipid profile, much of the focus has been on LDL and its relation to coronary heart disease. The National Cholesterol Education Program’s Adult Treatment Panel III (NCEP-ATPIII) even concentrates on LDL as “the primary target of therapy” (40). However, it has become common knowledge among healthcare providers that low serum concentrations of HDL are also a risk factor for cardiovascular disease and that elevated levels of HDL actually confer a protective effect against both primary and secondary cardiovascular events. The Helsinki Heart Study, a 5-year randomized controlled trial investigating the effect of gemfibrozil on coronary artery disease (CAD) in patients with no known CAD, demonstrated that increasing the HDL in patients with low baseline levels was inversely proportional to the risk of CHD (41). Patients with lower levels of HDL at the start of the study appeared to benefit more from elevation of HDL than those with higher baseline levels. Multiple trials have illuminated the secondary prevention effects of raising low serum HDL in patients with known CAD. The previously mentioned VA-HIT trial, which was designed to assess the benefit of gemfibrozil in patients with CHD and both low HDL and LDL, revealed a significant reduction in major coronary events by raising the serum HDL independent of the intervention effect on LDL (4). This study showed an 11% fall in coronary events for every 5 mg/dL elevation in serum HDL. While the clinician does have varying options when confronted with a patient with low HDL, niacin remains the most potent tool to raise high-density lipoprotein. Niacin can be considered in patients with isolated low HDL, or those with elevated LDL and triglycerides, who are at risk for atherosclerotic disease and unable to manage their lipids with diet, exercise, or other pharmaceutical agents. Treatment with therapeutic doses of nicotinic acid can raise serum HDL from 15% to 35% (40).

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In addition, niacin is the only agent that targets all aspects of the lipid profile, including LDL, triglycerides, and lipoprotein(a), a risk factor for CAD (42). At high enough doses, nicotinic acid can decrease LDL up to 25% and triglycerides from 20% to 50% (40). Niacin is also the one antidyslipidemic drug that effectively lowers lipoprotein(a) and can decrease levels of the atherogenic particle by 30% (43). Treatment of dyslipidemia with niacin is available in two different FDAapproved formulations. Immediate release (IR) niacin has both quick absorption and excretion (44). The half-life of IR niacin is approximately 1 h and requires multiple daily dosing to achieve therapeutic effect (typically used thrice daily). The maximum recommended daily dose of IR niacin is 3 g, to be split up into multiple doses. Nicotinic acid is also available in an extended release (ER) formulation called Niaspan (Abbott Laboratories). ER niacin is absorbed slowly over 12 h, making once-daily dosing effective (44). The maximum recommended daily dose is 2 g. The metabolism of the various formulations of niacin plays a significant role in the adverse reactions observed with therapy. Nicotinic acid is metabolized via two different pathways (45). The conjugation pathway leads to the metabolism of nicotinic acid to nicoturinic acid and subsequent flushing. The nonconjugative pathway results in nicotinamide formation. Metabolites produced from this pathway are associated with increased risk of hepatotoxicity (45). IR niacin is metabolized largely by the conjugation pathway and thus leads to an increased incidence of flushing. ER niacin, however, is associated with a more intermediate absorption rate and balanced level of metabolism, resulting in less flushing and less hepatotoxic effects (44). It should also be noted that long-acting (LA) or time-release formulations of niacin are available over the counter as nutritional supplements. However, the timerelease niacin formulations are not FDA approved for the treatment of dyslipidemia and, at elevated dosages, may be associated with increased risk of hepatotoxicity compared to other approved formulations. Regardless of the formulation of niacin, it is best implemented at a lower dose and titrated upward to therapeutic levels to monitor how patients tolerate treatment.

CLINICAL OUTCOME TRIALS WITH NIACIN Several randomized controlled trials of subjects treated with nicotinic acid have found improved angiographic cardiovascular disease outcomes (Table 9.1). The Cholesterol Lowering Atherosclerosis Study I, a 2-year investigation of niacin and colestipol use in subjects with a history of coronary artery bypass, resulted in a 26% reduction in total cholesterol, a 43% reduction in LDL, and a 37% increase in HDL (46). In addition, angiographic regression of atherosclerotic lesions was noted to be more profound in the treatment group compared to placebo. In the Cholesterol Lowering Atherosclerosis Study II, following 4 years of therapy, more angiographic lesions either did not progress or actually regressed in the niacin group compared to placebo (47). Similar findings were echoed in the Familial Atherosclerosis Treatment Study (FATS) that showed combination therapy of either lovastatin or niacin with

244

Table 9.1

Angiographic Outcome Trials with Niacin

Study

Medication

Population/design

Change in lipids

Results 53% lack of progression; angiographic lesions in intervention group versus 15% for controls. Sixteen percent of subjects in the niacin and colestipol treatment group had regression of lesions Decreased progression and even regression of angiographic lesions in intensive treatment groups. Clinical cardiovascular events were lowered by 73%

Cholesterol Lowering Atherosclerosis Trial (37, 38)

Niacin and colestipol

Men with history of CABG and baseline angiograms. Two- and 4-year follow-up

Total cholesterol: # 26%; HDL: " 37%; LDL: # 43%; TG: # 21%

Familial Atherosclerosis Treatment Study (41)

Niacin/colestipol or lovastatin/colestipol combinations

Men <62 years of age with elevated apolipoprotein B. Baseline angiogram and at 2.5 years of study

TC: # 23%; HDL: " 43%; LDL: # 32%; TG: # 29%

Adverse Effects of Niacin

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colestipol decreased the incidence of coronary atherosclerotic lesion progression, and even decreased lesion burden in men with CAD at risk for recurrent events (48). Multiple trials have demonstrated improved clinical cardiovascular disease outcomes with niacin therapy (Table 9.2). The Coronary Drug Project was an early lipid altering investigation that displayed a 27% reduction in nonfatal myocardial infarction after 6 years of monotherapy with niacin compared to placebo (49). The incidence of stroke and transient ischemic attacks (TIAs) was also reduced by 26%. Total mortality declined by 11% with niacin monotherapy (42). Significant reduction in adverse coronary events has been particularly effective in patients treated with combination lipid lowering therapy with niacin, including bile acid sequestrants, statins, and fibrates. In the FATS trial, besides the aforementioned angiographic benefit, patients who were randomized to niacin combination therapy had a fall in clinical cardiovascular events by 80% over 2.5 years (48). The HDL Atherosclerosis Treatment Study (HATS) showed that patients treated with both niacin and simvastatin had a 60% decline in adverse cardiovascular events compared to placebo (50). Ischemic heart disease mortality and total mortality were decreased by 36% and 26%, respectively, compared to placebo in the Stockholm Ischemic Heart Disease Secondary Prevention Study, where subjects received combined niacin and clofibrate therapy (51).

ADVERSE EFFECTS OF NIACIN Niacin has many unique features that make it an ideal drug to combat abnormal levels of cholesterol. It targets all aspects of the lipid profile, remains the best agent to raise HDL, and is relatively inexpensive. However, the clinical use of nicotinic acid has been hindered by its side effects, particularly the vasocutaneous flushing response at elevated doses. The majority of patients initiated on high-dose niacin therapy experience flushing. It is often characterized as an unpleasant sensation of warmth, reddening, itching, and tingling. It can be so pronounced that the estimated rate of discontinuation of therapy is approximately 25% (52, 53). Niacin-induced flushing tends to decrease after a few weeks of use. With long-term use, many patients are able to tolerate the drug and only about 3% continue to experience severe flushing effects (54). Flushing is most pronounced with the IR formulation of nicotinic acid, which is also dosed multiple times daily. The ER formulation, which is associated with less flushing, can be taken once nightly to minimize symptoms. Taking a daily prostaglandin suppressing agent, such as aspirin or a nonsteroidal anti-inflammatory drug, 30 min prior to niacin can lessen the severity of the flushing response. Other significant side effects of niacin therapy include the previously mentioned hepatotoxic effects, hyperuricemia, hyperglycemia, dyspepsia, and activation of peptic ulcer disease. Patients started on niacin for dyslipidemia should have baseline liver function tests prior to initiation of therapy and periodic tests thereafter. The drug should be used with prudence in active or unexplained liver disease. Nicotinic acid should also be used with caution in patients with a history of gout or peptic ulcer disease and is contraindicated in severe or active disease episodes.

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Table 9.2

Clinical Trial Outcomes with Niacin

Study

Medication

Coronary Drug Project (42)

Niacin

HDL Atherosclerosis Treatment Study (43)

Niacin and simvastatin

Stockholm Ischemic Heart Disease Study (44)

Niacin and clofibrate

Population/design

Change in lipids

Results

Men with history of CAD. Mean baseline TC of 250 mg/dL and TG of 177 mg/dL Men and women with known CAD. Mean HDL of 31 mg/dL and mean LDL of 125 mg/dL

TC: # 10%; TG: # 26%

27% reduction in nonfatal MI. Strokes/TIAs decreased by 26%. Total mortality decreased by 11%

TC: # 29%; HDL: " 18%; LDL: # 40%; TG: # 34%

Men with history of MI followed for 5 years on therapy. Mean TC of 245 mg/dL and mean TG of 208 mg/dL

TC: # 13%; TG: # 19%

After 3 years of therapy, the niacin and simvastatin treatment group had 90% less major clinical events (fatal/nonfatal MI, CVA, revascularization, etc.). Regression of angiographic lesions also noted Ischemic heart disease mortality was decreased by 36%. All-cause mortality was lowered by 26% in the treatment group

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USE OF NIACIN IN DIABETES MELLITUS Controversy has surrounded the use of niacin for the treatment of dyslipidemia in patients with diabetes mellitus. Lipid abnormalities are common in non-insulindependent diabetes mellitus, in particular elevated triglycerides and low levels of HDL. Niacin appears to be an ideal drug in this disorder as it has the capability to both raise HDL and lower triglycerides. However, earlier studies of nicotinic acid in diabetic patients have raised concerns over worsening glycemic control as a side effect of therapy. For example, a smaller randomized study of 13 patients demonstrated a 16% increase in mean plasma glucose concentrations and a 21% increase in glycosylated hemoglobin (HbA1C) values in diabetic patients receiving therapeutic doses of immediate release niacin over 8 weeks (55). Many healthcare providers are still under the impression that nicotinic acid should not be readily used in patients with diabetes (56). More recent evidence indicates that niacin is indeed a useful tool in combating the dyslipidemia associated with diabetes mellitus. The Arterial Disease Multiple Intervention Trial (ADMIT), a multicenter randomized clinical control trial designed to assess the efficacy and safety of IR niacin in diabetic patients, concluded that nicotinic acid was nearly equally effective at modifying lipids in patients with diabetes compared to those without the disorder (57). Diabetic patients receiving niacin therapy had a 29% rise in HDL, a 23% decline in triglycerides, and an 8% decline in LDL (compared to 29%, 28%, and 9% changes in HDL, triglycerides, and LDL, respectively, in patients without diabetes). A modest increase in glucose levels of 8.7 mg/dL was noted in patients with diabetes on niacin, compared to 6.3 mg/dL increase with niacin use in nondiabetics (57). However, this worsened hyperglycemia in patients with diabetes appeared to be a temporary process with glucose levels at the end of the study near their pretherapy baseline values (58). No significant deviation of the HbA1c from baseline values to time of follow-up was found in diabetic patients treated with niacin (57). The Assessment of Diabetes Control and Evaluation of the Efficacy of Niaspan Trial (ADVENT), a study of once-daily ER niacin use in diabetic dyslipidemia, revealed that the medication appears well tolerated with effective elevation of HDL and lowering of triglycerides in patients with type 2 diabetes at doses less than 2000 mg (59). Again, a transient increase in fasting glucose levels was noted in patients on niacin during the study, with subsequent return to baseline levels by week 16 of the trial (58). Very mild but statistically significant elevation of less than 0.3% in the HbA1c was noted in patients taking 1.5 g of ER niacin. A higher percentage of patients did require the institution of new antiglycemic agents or adjustment of their current medication when treated with ER niacin, particularly at doses higher than 1000 mg daily (58). Post hoc analysis of the Coronary Drug Project data compared niacin monotherapy in patients with and without the metabolic syndrome (60). While there was a slight increase in fasting plasma glucose in patients treated with nicotinic acid versus placebo, individuals with the metabolic syndrome on niacin demonstrated comparable reduction in secondary adverse coronary events and overall mortality against those without the metabolic syndrome (60). Niacin use appears to be safe and efficacious in diabetic dyslipidemia and in those with impaired fasting glucose. It effectively targets derangements of the lipid profile

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specific for diabetes and clinical trials have demonstrated improved cardiovascular outcomes in patients on therapy compared to placebo. However, trials such as ADMIT and ADVENT were carried out in patients with HbA1c values of less than 9%. The clinician should be aware that niacin can be a powerful antidyslipidemic agent for his or her diabetic patients but caution should be exercised in implementing the drug in poorly controlled diabetes.

SUMMARY In summary, current evidence indicates that niacin acts on multiple tissues and targets to beneficially modulate lipoprotein profile, vascular inflammatory processes, and adverse skin flush reactions. In addition to its beneficial effects on lipoprotein profile, the direct effect of niacin to increase aortic endothelial cell redox potential and its vascular anti-inflammatory properties may additionally account for its proven effects in atherosclerotic cardiovascular disease beyond its lipid regulation. The new concepts on niacin’s mechanism of action and target sites pave the foundation for new drug discovery for atherosclerosis and cardiovascular disease.

ACKNOWLEDGMENTS This work has been supported, in part, by Veterans Affairs Merit Review Programs and Southern California Institute for Education and Research.

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8. MARTINEZ, L.O., S. JACQUET, J.P. ESTEVE, C. ROLLAND, E. CABEZON, E. CHAMPAGNE, T. PINEAU, V. GEORGEAUD, J.E. WALKER, F. TERCE, X. COLLET, B. PERRET, and R. BARBARAS. 2003. Ectopic beta-chain of ATP synthase is an apolipoprotein A-I receptor in hepatic HDL endocytosis. Nature 421:75–79. 9. ZHANG, L.H., V. KAMANNA, M. ZHANG, and M. KASHYAP. 2008. Niacin inhibits surface expression of ATP synthase beta chain in Hep G2 cells: implications for raising HDL. J Lipid Res 49:1195–1201. 10. SAKAI, T., V.S. KAMANNA, and M.L. KASHYAP. 2001. Niacin, but not gemfibrozil, selectively increases LP-AI, a cardioprotective subfraction of HDL, in patients with low HDL cholesterol. Arterioscler Thromb Vasc Biol 21:1783–1789. 11. RUBIC, T., M. TROTTMANN, and R.L. LORENZ. 2004. Stimulation of CD36 and the key effector of reverse cholesterol transport ATP-binding cassette A-1 in monocytoid cells by niacin. Biochem Pharmacol 67:11–419. 12. GINSBERG, H.N. 1995. Synthesis and secretion of apolipoprotein B from cultured liver cells. Curr Opin Lipidol 6:275–280. 13. DAVIS, R.A. 1999. Cell and molecular biology of the assembly and secretion of apolipoprotein B-containing lipoproteins by the liver. Biochim Biophys Acta 1440:1–31. 14. BOREN, J., L. GRAHAM, M. WETTESTEN, J. SCOTT, A. WHITE, and S.O. OLOFFSON. 1992. The assembly and secretion of apoB 100-containing lipoproteins in Hep G2 cells. ApoB 100 is cotranslationally integrated into lipoproteins. J Biol Chem 267:9858–9867. 15. GANJI, S., S. TAVINTHARAN, D. ZHU, Y. XING, V. KAMANNA, and M. KASHYAP. 2004. Niacin noncompetitively inhibits DGAT2 but not DGAT1 activity in HepG2 cells. J Lipid Res 45:1835–1845. 16. GRUNDY, S., H. MOK, L. ZEK, and M. BERMAN. 1981. Influence of nicotinic acid on metabolism of cholesterol and triglycerides in man. J Lipid Res 22:24–36. 17. JIN, F.Y, V.S. KAMANNA, and M.L. KASHYAP. 1999. Niacin accelerates intracellular apo B degradation by inhibiting triacylglycerol synthesis in human hepatoblastoma (HepG2) cells. Arterioscler Thromb Vasc Biol 19:1051–1059. 18. CARLSON, L.A. 2005. Nicotinic acid: the broad-spectrum lipid drug. A 50th anniversary review. J Intern Med 258:94–114. 19. CARLSON, L.A. 1963. Studies on the effect of nicotinic acid on catecholamine stimulated lipolysis in adipose tissue in vitro. Acta Med Scand 173:719–722. 20. TUNARU, S., J. KERO, A. SCHAUB, C. WUFKA, A. BLAUKAT, K. PFEFFER, and S. OFFERMANNS. 2003. PUMA-G and HM74 are receptors for nicotinic acid and mediate its anti-lypolytic effect. Nat Med 9:352–355. 21. WISE, A., S.M. FOORD, N.J. FRASER, A.A. BARNES, N. ELSHOURBAGY, M. EILERT, D.M. IGNAR, P.R. MURDOCK, K. STEPLEWSKI, A. GREEN, A.J. BROWN, S.J. DOWELL, P.G. SZEKERES, D.G. HASSALL, F.H. MARSHALL, S. WILSON, and N.B. PIKE. 2003. Molecular identification of high and low affinity receptors for nicotinic acid. J Biol Chem 278:9869–9874. 22. SOGA, T., M. KAMOHARA, J. TAKASAKI, S. MATSUMOTO, T. SAITO, T. OHISHI, H. HIYAMA, A. MATSUO, H. MATSUSHIME, and K. FURUICHI. 2003. Molecular identification of nicotinic acid receptor. Biochem Biophys Res Commun 303:364–369. 23. CARLSON, L.A., and L. ORO. 1962. The effect of nicotinic acid on the plasma free fatty acids: demonstration of a metabolic type of sympathicolysis. Acta Med Scand 172:641–645. 24. YU, X.X., S.F. MURRAY, S.K. PANDEY, S.L. BOOTEN, D. BAO, X.Z. SONG, S. KELLY, S. CHEN, R. MCKAY, B.P. MONIA, and S. BHANOT. 2005. Antisense oligonucleotide reduction of DGAT2 expression improves hepatic steatosis and hyperlipidemia in obese mice. Hepatology 42:362–371. 25. JACOBSON, E.L., and M.K. JACOBSON. 1993. A biomarker for the assessment of niacin nutriture as a potential preventive factor in carcinogenesis. J Intern Med 233:59–62. 26. YAN, Q., M. BRIEHL, C.L. CROWLEY, C.M. PAYNE, H. BERNSTEIN, and C. BERNSTEIN. 1999. The NAD þ precursors, nicotinic acid and nicotinamide upregulate glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase mRNA in Jurkat cells. Biochem Biophys Res Commun 255:133–136. 27. HARRIS, C.M., S.A. SANDERS, and V. MASSEY. 1999. Role of the flavin midpoint potential and NAD binding in determining NAD versus oxygen reactivity of xanthine oxidoreductase. J Biol Chem 274:4561–4569.

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28. CAO, Z., and Y. LI. 2002. Chemical induction of cellular oxidants affords marked protection against oxidative injury in vascular smooth muscle cells. Biochem Biophys Res Commun 292:50–57. 29. GANJI, S.H., Q. SHUCUN, Z. LINHUA, V.S. KAMANNA, and M.L. KASHYAP. 2009. Niacin inhibits vascular oxidative stress, redox-sensitive genes and monocyte adhesion to human aortic endothelial cells. Atherosclerosis 202:68–75. 30. STEINBERG, D. 2002. Atherosclerosis in perspective: hypercholesterolemia and inflammation as partners in crime. Nat Med 8:1211–1217. 31. STEINBRECHER, U.P., S. PARTHASRATHY, D.S. LEAKE, J.L. WITZTUM, and D. STEINBERG. 1984. Modification of low density lipoprotein by endothelial cells involves lipid peroxidation and degradation of low density lipoprotein phospholipids. Proc Nat Acad Sci USA 81:3883–3887. 32. QUINN, M.T., S. PARTHASARATHY, L.G. FONG, and D. STEINBERG. 1987. Oxidatively modified low density lipoproteins: a potential role in recruitment and retention of monocyte/macrophages during atherogenesis. Proc Nat Acad Sci USA 84:2995–2998. 33. LIBBY, P. 2002. Inflammation in atherosclerosis. Nature 420:868–874. 34. MARUI, N., M.K. OFFERMANN, R. SWERLICK, C. KUNSCH, C.A. ROSEN, M. AHMAD, R.W. ALEXANDER, and R.M. MEDFORD. 1993. Vascular cell adhesion molecule-1 (VCAM-1) gene transcription and expression are regulated through an antioxidant-sensitive mechanism in human vascular endothelial cells. J Clin Invest 92:1866–1874. 35. MORROW, J.D., W.G. PARSONS, and L.J. ROBERTS. 1989. Release of markedly increased quantities of prostaglandin D2 in vivo in humans following the administration of nicotinic acid. Prostaglandins 38:263–274. 36. KAMANNA, V.S., and M.L. KASHYAP. 2008. Mechanism of action of niacin. Am J Cardiol 101:20–26. 37. MACIEJEWSKI-LENOIR, D., J.G. RICHMAN, Y. HAKAK, I. GAIDAROV, D.P. BEHAN, and D.T. CONNOLLY. 2006. Langerhans cells release prostaglandin D2 in response to nicotinic acid. J Invest Dermatol 126:2637–2646. 38. BENYO, Z., A. GILLE, J. KERO, M. CSIKY, M.C. SUCHANKOVA, R.M. NUSING, A. MOERS, K. PFEFFER, and S. OFFERMANNS. 2005. GPR109A (PUMA-G/HM74A) mediates nicotinic acid-induced flushing. J Clin Invest 115:3634–3640. 39. CHENG, K., T.J. WU, K.K. WU, C. STURINO, K. METTERS, K. GOTTESDIENER, S.D. WRIGHT, Z. WANG, G. O’NEILL, E. LAI, and M.G. WATERS. 2006. Antagonism of the prostaglandin D2 receptor 1 suppresses nicotinic acid-induced vasodilation in mice and humans. Proc Natl Acad Sci USA 103:6682–6687. 40. NIH, 2001. Executive Summary of the National Cholesterol Education Program (NCEP) Expert Panel on the Detection, Evaluation and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). 41. MANNINEN, V., M.O. ELO, M.H. FRICK, K. HAAPA, O.P. HEINONEN, P. HEINSALMI, P. HELO, J.K. HUTTUNEN, P. KAITANIEMI, P. KOSKINEN, et al. 1988. Lipid alterations and decline in the incidence of coronary heart disease in the Helsinki Heart Study. JAMA 260:641–651. 42. MALIK, S., and M. KASHYAP. 2003. Niacin, lipids, and heart disease. Curr Cardiol Rep 5:470–476. 43. CARLSON, L.A., A. HAMSTEN, and A. ASPLUND. 1989. Pronounced lowering of serum levels lipoprotein Lp(a) in hyperlipidemic subjects treated with nicotinic acid. J Intern Med 226:271–276. 44. MCKENNEY, J. 2004. New perspectives on the use of niacin in the treatment of lipid disorders. Arch Intern Med 164:697–705. 45. PIEPHO, R.W. 2000. The pharmacokinetics and pharmacodynamics of agents proven to raise highdensity lipoprotein cholesterol. Am J Cardiol 86:35–40. 46. BLANKENHORN, D.H., S.A. NESSIM, R.L. JOHNSON, M.E. SANMARCO, S.P. AZEN, and L. CASHIN-HEMPHILL. 1987. Beneficial effects of combined colestipol–niacin therapy on coronary atherosclerosis and coronary venous bypass grafts. JAMA 257:3233–3240. 47. CASHIN-HEMPHILL, L., W.J. MACK, J.M. POGODA, M.E. SANMARCO, S.P. AZEN, and D.H. BLANKENHORN. 1990. Beneficial effects of colestipol–niacin on coronary atherosclerosis. A 4-year follow-up. JAMA 264:3013–3017. 48. BROWN, G., J.J. ALBERS, L.D. FISHER, S.M. SCHAEFER, J.T. LIN, C. KAPLAN, X.Q. ZHAO, B.D. BISSON, V.F. FITZPATRICK, and H.T. DODGE. 1990. Regression of coronary artery disease as a result of intensive lipidlowering therapy in men with high levels of apolipoprotein B. N Engl J Med 323:1289–1298.

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49. CORONARY DRUG PROJECT. 1975. Clofibrate and niacin in coronary heart disease. JAMA 231:360–381. 50. BROWN, B.G., X.Q. ZHAO, A. CHAIT, L.D. FISHER, M.C. CHEUNG, J.S. MORSE, A.A. DOWDY, E.K. MARINO, E.L. BOLSON, P. ALAUPOVIC, J. FROHLICH, and J.J. ALBERS. 2001. Simvastatin and niacin, antioxidant vitamins, or the combination for the prevention of coronary disease. N Engl J Med 345:1583–1592. 51. CARLSON, L.A., and G. ROSENHAMER. 1988. Reduction of mortality in the Stockholm Ischemic Heart Disease Secondary Prevention Study by combined treatment with clofibrate and nicotinic acid. Acta Med Scand 223:405–418. 52. MCKENNEY, J.M., J.D. PROCTOR, S. HARRIS, and V.M. CHINCHILI. 1994. A comparison of the efficacy and toxic effects of sustained- vs immediate-release niacin in hypercholesterolemic patients. JAMA 271:672–677. 53. KAMAL-BAHL, S., D. WATSON, and B. AMBEGAONKAR. 2009. Patients’ experiences of niacin-induced flushing in clinical practice: a structured telephone interview. Clin Ther 31:130–140. 54. BERGE, K.G. 1961. Side effects of nicotinic acid in treatment of hypercholesterolemia. Geriatrics 16:416–422. 55. GARG, A., and S.M. GRUNDY. 1990. Nicotinic acid as therapy for dyslipidemia in non-insulin-dependent diabetes mellitus. JAMA 264:2994–2996. 56. ADLER, E., and D. PAAUW. 2003. Medical myths involving diabetes. Prim Care 30:607–618. 57. ELAM, M.B., D.B. HUNNINGHAKE, K.B. DAVIS, R. GARG, C. JOHNSON, D. EGAN, J.B. KOSTIS, D.S. SHEPS, and E.A. BRINTON. 2000. Effect of niacin on lipid and lipoprotein levels and glycemic control in patients with diabetes and peripheral arterial disease: the ADMIT study. A randomized trial. Arterial Disease Multiple Intervention Trial. JAMA 284:1263–1270. 58. GOLDBERG, R.B., and T.A. JACOBSON. 2008. Effects of niacin on glucose control in patients with dyslipidemia. Mayo Clin Proc 83:470–478. 59. GRUNDY, S.M., G.L. VEGA, M.E. MCGOVERN, B.R. TULLOCH, D.M. KENDALL, D. FITZ-PATRICK, O.P. GANDA, R.S. ROSENSON, J.B. BUSE, D.D. ROBERTSON, and J.P. SHEEHAN. 2002. Efficacy, safety, and tolerability of once-daily niacin for the treatment of dyslipidemia associated with type 2 diabetes: results of the assessment of diabetes control and evaluation of the efficacy of niaspan trial. Arch Intern Med 162:1568–1576. 60. CANNER, P.L., C.D. FURBERG, and M.E. MCGOVERN. 2006. Benefits of niacin in patients with versus without the metabolic syndrome and healed myocardial infarction (from the Coronary Drug Project). Am J Cardiol 97:477–479.

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Current Antidiabetic Therapies and Mechanisms MINGHAN WANG Metabolic Disorders Research, Amgen, Inc., Thousand Oaks, CA, USA

INTRODUCTION Metabolic syndrome is a cluster of metabolic disorders, including obesity, insulin resistance, dyslipidemia (increased low-density lipoprotein (LDL), decreased highdensity lipoprotein (HDL), and high triglycerides (TG)), hyperglycemia, and hypertension. In addition, increased inflammation, endothelial dysfunction, and microalbuminuria are common comorbidities. Since metabolic syndrome is not recognized as a single disease by regulatory authorities, separate treatment of the individual disorders is a common practice. More importantly, to date, there is no single therapy that can treat all the individual abnormalities. Metabolic syndrome significantly increases the risks of type 2 diabetes mellitus (T2DM) and cardiovascular disease. As a matter of fact, more than 80% of T2DM patients have metabolic syndrome. There are generally two types of therapies, antidiabetics, which improve glycemic control and/or insulin sensitivity, and therapies that reduce cardiovascular risk factors, including antihypertensives, anti-inflammatory drugs, and drugs that treat lipid disorders. Most T2DM patients require both antidiabetic and cardiovascular therapies. Individuals with metabolic syndrome but without T2DM require treatments that reduce cardiovascular morbidity and mortality, although some antidiabetics have been demonstrated to prevent the onset of T2DM in this population. Further, additional therapies are required to treat microvascular and macrovascular complications in T2DM patients. This chapter will discuss established antidiabetic treatments, including lifestyle therapy, insulin, metformin, insulin secretagogues (sulfonylureas (SUs) and glinides), thiazolidinediones (TZDs), a-glucosidase inhibitors (AGIs),

Metabolic Syndrome: Underlying Mechanisms and Drug Therapies Edited by Minghan Wang Copyright 2011 John Wiley & Sons, Inc.

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dipeptidyl peptidase-4 (DPP-4) inhibitors, and exenatide. Weight loss drugs will be covered as well because they have the potential to improve insulin sensitivity. Therapies to reduce cardiovascular risk are discussed in other chapters. Treatments for diabetic complications will be discussed.

ANTIDIABETIC TREATMENTS Diet and Exercise Despite the availability of oral and injectable antidiabetic medications, the first treatment for newly diagnosed type 2 diabetics is diet and exercise. Sustained modification of lifestyles can have remarkable beneficial metabolic effects and play a critical role in the management of the disease. Healthy diets can help reduce body weight and cardiovascular risk factors. In addition, endurance exercise helps achieve weight loss, increases insulin sensitivity, and improves glucose homeostasis and lipid profiles. These benefits not only are important in slowing the progression of the disease but also could delay the onset of T2DM in individuals with obesity and insulin resistance. One major challenge with exercise is inadequate compliance. Moreover, many patients fail to achieve adequate glycemic control through exercise and antidiabetic medications have to be added to disease management. Exercise can improve glucose uptake and utilization in the skeletal muscle. Glucose entry into cells is mediated by a family of membrane-bound transporters. The GLUT4 transporter is the primary member that mediates insulin-stimulated glucose uptake into skeletal muscle and adipose tissue. One of the deficiencies in T2DM and insulin-resistant condition is reduced insulin-stimulated glucose uptake in these tissues. This is not a result of reduced total GLUT4 level, but rather due to the failure of insulin action to recruit sufficient GLUT4 protein to the cell surface (1, 2). Muscle contraction activates adenosine 50 -monophosphate (AMP)-activated protein kinase (AMPK), a key metabolic switch that regulates energy metabolism. AMPK mediates a number of beneficial effects, including stimulating glucose uptake in muscle in an insulin-independent manner (3). T2DM patients have normal exerciseinduced AMPK activity (4). Despite the impairment of insulin-stimulated glucose uptake, AMPK has the full capacity to stimulate glucose uptake in skeletal muscle of T2DM patients (3). AMPK activation during exercise is believed to mediate GLUT4 translocation to the plasma membrane leading to increased glucose uptake (4). It has been demonstrated that muscle contraction stimulates GLUT4 translocation in an AMPK-dependent manner (5). Endurance training also increases GLUT4 content in skeletal muscle in both diabetic animals and humans (6, 7). Both the transcription and translation of GLUT4 are increased in response to exercise, which is likely to be mediated by the activation of AMPK (8). In addition to the direct effect on glucose transport, exercise-stimulated AMPK activation can also improve insulin sensitivity. Activation of AMPK with a single dose of its activator 5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside (AICAR) increased insulin sensitivity in both muscle and liver in high-fat-fed rats (9). This effect may be achieved by modulating the

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insulin signaling pathway through transient AMPK activation, as the effect extends beyond AMPK activation (9). Consistent with this finding, endurance training increases insulin-stimulated phosphorylation of downstream signaling molecules such as IRS-1, IRS-2, PI3-kinase, and Akt-1/PKB (10), which is a hallmark of increased insulin sensitivity. One potential mechanism by which AMPK activation improves insulin sensitivity is the reduction of intracellular fatty acyl metabolites through activation of mitochondrial b-oxidation. Once activated, AMPK phosphorylates and inactivates acetyl-CoA carboxylase 2 (ACC2), an enzyme involved in the control of b-oxidation of the intracellular fatty acyl metabolites. ACC2 is located on the mitochondrial outer membrane and produces malonyl-CoA, which is a potent inhibitor of carnitine palmitoyltransferase 1 (CPT1), the rate-limiting enzyme in mitochondrial b-oxidation. ACC2 knockout mice have improved insulin sensitivity, reduced body weight, and increased mitochondrial b-oxidation (11). Likewise, the final consequence of AMPK activation is increased b-oxidation and improved insulin sensitivity. Increased b-oxidation depletes intramuscular content of fatty acyl metabolites, such as long-chain acyl-CoAs (12), which are known to mediate diet-induced insulin resistance by activating PKCu in skeletal muscle (13). Thus, through AMPK activation, exercise improves insulin signaling by directly increasing the capacity of insulin-stimulated IRS tyrosine phosphorylation. Increased substrate oxidation and mitochondrial uncoupling by endurance training have been demonstrated in humans (14), which could enhance muscle insulin sensitivity in the resting state and even reduce fat mass in the long run. The metabolic benefits of exercise have been examined in diabetes prevention trials. The Da Qing IGT and Diabetes Study demonstrated that exercise reduced the incidence of diabetes in prediabetic subjects with impaired glucose tolerance (IGT) from 68% to 41% over a period of 6 years (15). The Finnish Diabetes Prevention Study showed that exercise accompanied with diet reduced cardiovascular risks (16). The Diabetes Prevention Program compared the effects of metformin and intensive lifestyle intervention (diet and exercise) in individuals with IGT (17, 18). The intensive lifestyle intervention involved a low-calorie, low-fat diet and physical activity of moderate intensity, such as brisk walking for at least 150 min per week (17). Within an average follow-up period of 2.8 years, both intensive lifestyle intervention and metformin reduced the incidence of diabetes in individuals with IGT, but lifestyle intervention was more effective than metformin (17). Intensive lifestyle intervention improved cardiovascular risk and glucose tolerance profile better than metformin (18). These data strongly suggest that despite the available antidiabetic medications, lifestyle intervention is still the best course of action in preventing the development of T2DM.

Insulin T2DM is characterized by a combination of insulin resistance and progressive b-cell failure. On one hand, b-cell loss at various stages of T2DM impairs the capacity of the pancreas to secrete adequate insulin to normalize glucose; on the other hand, insulin

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resistance by the peripheral tissues requires more insulin to counter the diminished sensitivity to maintain glucose homeostasis. The increased hepatic glucose production in T2DM is also a result of insulin resistance, and to fully suppress hepatic gluconeogenesis, more insulin is needed. Thus, exogenous insulin therapy is an effective treatment to supplement endogenous insulin supply and overcome insulin resistance. Initially, the therapy was made possible with insulin derived from animals (bovine or porcine). Recombinant insulin or insulin analogues (insulin molecules with amino acid changes to improve certain properties) are currently available for use. Insulin is usually used as a second-line treatment if oral antidiabetic drugs fail to adequately control hyperglycemia. However, it could be introduced earlier for more aggressive glycemic control. Although insulin is a more effective antidiabetic therapy compared to oral antidiabetic drugs, insulin therapy is associated with hypoglycemia and weight gain. There are short-acting and long-acting insulin products. Short-acting insulin products, including regular insulin (Humulin R and Novolin R), aspart (Novolog), and lispro (Humalog), have rapid onset of action with short effective duration (19). They should be injected before meals to match postprandial glucose excursion. But even among the short-acting agents, there are key differences with respect to the timing of injection. For example, the onset of action for aspart is 5–10 min after injection; however, it takes regular insulin 30–60 min to reach maximal action (19). Longacting insulin has reduced dosing frequency, maintains peakless duration of action, and is intended to meet basal insulin requirements (19). One common long-acting product is glargine (Lantus) (19), a recombinant insulin analogue with long plasma half-life. Intensive insulin therapy has been examined for its effectiveness in glycemic control and consequential effects on microvascular complications. In the United Kingdom Prospective Diabetes Study (UKPDS), 3867 T2DM patients were enrolled with a 10-year follow-up to compare intensive insulin therapy with diet alone or SU treatments (20). The intensive insulin treatment group had lower glycosylated hemoglobin (hemoglobin A1c or HbA1c) level (7.1%) compared to the conventional diet treatment group (7.9%) (20). The intensive insulin treatment group also had 10% reduction in diabetes-related death and 25% risk reduction in microvascular end points compared to the conventional diet treatment group (20). The SU group had similar reduction in microvascular end points (20). However, intensive insulin therapy did not change macrovascular risks (20), suggesting that hyperglycemia is independent of cardiovascular risk factors, and to reduce these risks, therapies other than glycemic control agents need to be prescribed. Intensive insulin therapy was also assessed in the Kumamoto trial with Japanese T2DM patients. Compared to standard insulin therapy, intensive insulin treatment significantly slowed the onset and progression of diabetic microvascular complications (21). These clinical studies suggest that intensive glycemic control can reduce microvascular complications in T2DM patients. However, intensive insulin therapy is associated with a higher risk of hypoglycemia (20). The risk was not significant in the Kumamoto trial (21), probably due to the lower doses used in the study and glycemia-based dose adjustment strategy employed during the trial.

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Metformin In the 1920s, guanidine compounds with antidiabetic effects were found in the extract of a herbaceous plant Galega officinalis (also known as French lilac). By then, this plant had been used to treat diabetes for centuries. Studies of the guanidine compounds led to the discovery of biguanides. Biguanides were introduced in the 1950s for the treatment of diabetes (22). Two members of this drug class, phenformin and buformin, were withdrawn from the market due to their significant potential to cause lactic acidosis, a serious condition that is characterized by buildup of lactic acid in the body and acidification of the blood. Metformin is the only biguanide currently on the market and has a reduced risk of lactic acidosis if properly prescribed. It was introduced as Glucophage in the U.S. market in 1995 and is now available as a generic drug. Metformin can still cause lactic acidosis at a risk of 1 in every 30,000 patientyears, which is about 100 times lower than that by phenformin and manageable with the help of the vast clinical experience with this drug (23). Although the incidence is rare, the consequence is severe because lactic acidosis is associated with about 50% chance of fatality. For this reason, the drug is contraindicated in patients with increased risk of lactic acidosis, such as those with congestive heart failure, dehydration, and impaired hepatic function. Since the drug is mainly cleared through kidney, it must be avoided in patients with renal failure. Metformin does not work by stimulating insulin secretion and therefore does not cause hypoglycemia. Further, unlike several other antidiabetic drugs such as insulin, SUs, and TZDs that cause weight gain, metformin is weight neutral or even causes weight loss (22). Because of these attributes along with competitive efficacy and cost effectiveness, metformin is widely used as a first-line oral therapy for T2DM. Metformin reduces both fasting glucose and HbA1c levels and improves other metabolic end points in T2DM patients. The antidiabetic effect was demonstrated in the landmark UKPDS where metformin was compared with diet alone, insulin, or SUs in the treatment of overweight patients with T2DM over a mean duration of about 10 years (24). At the end of the study, the metformin group had a significantly reduced median HbA1c (7.4%) compared to the diet alone group (8.0%) (24). Further, metformin had risk reductions of 32% for any diabetes-related end point, 42% for diabetes-related death, and 36% for all-cause mortality compared to diet alone (24). Metformin showed a greater effect than insulin and SUs on any diabetes-related end point, all-cause mortality, and stroke (24). In this study, metformin was associated with less weight gain and lower incidence of hypoglycemia than insulin and SUs (24). These findings demonstrate that metformin is an efficacious antidiabetic drug with superior properties than insulin and SUs, and should be considered a more preferred therapy. Importantly, metformin reduced the risk of myocardial infarction by 39% and that of all macrovascular end points by 30% (24). This is a clear advantage because neither insulin nor SUs significantly decreased the risk of macrovascular complications in the UKPDS (20). Surprisingly, metformin did not significantly reduce microvascular end points in this study (24). This could be due to the smaller sample size. The antidiabetic effect of metformin was also demonstrated in many other clinical studies with monotherapy and combination therapies. While metformin is

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efficacious and safe in adult T2DM patients, its effectiveness and safety were also evaluated in pediatric patients with T2DM. In a randomized, double-blind, placebocontrolled trial involving 82 patients at 10–16 years of age, metformin at doses up to 1000 mg twice daily was administered for 16 weeks (25). Metformin significantly improved glycemic control and had no negative impact on body weight or lipid profile (25). This clinical study demonstrated both safety and effectiveness of the drug for the treatment of T2DM in pediatric patients (25). As mentioned above, metformin is associated with rare cases of lactic acidosis. It also causes gastrointestinal (GI) side effects such as abdominal discomfort, bloating, and diarrhea (26, 27), which is the primary reason for discontinuation. These GI side effects can be lessened or resolved by gradual dose titration and administration with food. To balance the benefit and risk, a dose range finding study was carried out to determine the lowest efficacious dose (26). In a double-blind, placebo-controlled study with 451 T2DM patients, metformin was administered at 500, 1000, 1500, 2000, or 2500 mg daily for 11 weeks. At week 7 or at the end of the study, the effects of reducing fasting glucose and HbA1c were significant at all tested doses compared to the placebo group (26). Further, the effect on fasting glucose and HbA1c was dose dependent (26). These data indicate that the metabolic benefits were observed at a dose of as low as 500 mg daily, while higher dose could be used for greater effects if tolerance and safety are ensured based on the conditions of individual patients. The mechanisms of the antidiabetic effects of metformin have been under investigation in both clinical and research studies. In a study with 10 obese T2DM patients, each subject initially received low doses and then stayed on high doses of metformin for 12 weeks (27). Both fasting glucose and HbA1c levels were significantly reduced in treated patients who also experienced weight loss (27). The rate of glucose production from lactate was decreased by 37% (27), suggesting that gluconeogenesis was inhibited by metformin treatment. To examine the mechanism by which metformin suppresses endogenous glucose production, 13C nuclear magnetic resonance (NMR) spectroscopy was used in combination with ingested 2H2O to measure hepatic glycogenolysis, gluconeogenesis, and endogenous glucose production (28). In poorly controlled T2DM patients, elevated glucose production was demonstrated with this method (28). After 3 months of metformin treatment, the gluconeogenesis rate was decreased by more than 30% (28). This study demonstrates that metformin reduces glucose production by suppressing the gluconeogenic pathway (28). In keeping with a previous study (27), these data establish the mechanistic basis for the hepatic effect by metformin. At the molecular level, metformin inhibits complex 1 of the mitochondrial respiratory chain (29), which is expected to result in increased uncoupling and consequently decreased ATP production, and thereby increase the AMP/ATP ratio and trigger AMPK activation. This notion is supported by direct evidence of AMPK activation in cultured primary hepatocytes treated with metformin (30). Further, in keeping with this proposed mechanism, metformin had no direct effect on partially purified AMPK enzyme in an in vitro kinase assay (30), indicating that the metformin-dependent AMPK activation is not mediated by a direct effect on AMPK; rather, metformin stimulates AMPK activation via an intermediate mechanism. AMPK activation has a number of downstream functional effects, one of

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which is suppression of hepatic gluconeogenesis. The expression of hepatic gluconeogenic genes such as phosphoenolpyruvate carboxykinase (PEPCK) and glucose6-phosphatase (G6Pase) is regulated by transcriptional coactivator, peroxisome proliferator-activated receptor g (PPARg) coactivator-1a (PGC-1a). The expression of PGC-1a itself is controlled by a transcriptional complex involving three transcription factors, cAMP response element binding protein (CREB), CREB binding protein (CBP), and transducer of regulated CREB activity 2 (TORC2). Since CREB is phosphorylated and constitutively occupies the promoter, the binding states of CBP and TORC2 determine the transcriptional activity of PGC-1a (31). Activated AMPK stimulated by metformin treatment increases the phosphorylation of CBP through protein kinase C (PKC) and triggers dissociation of both CBP and TORC2 from the transcriptional machinery (31). In addition, APMK directly phosphorylates TORC2, which limits its nuclear entry and blocks its activation (32). The final outcome of these AMPK actions is the reduced activity of the transcriptional complex CBP/CREB/ TORC2 and consequential decreased expression of the key gluconeogenic genes (31). Thus, the main mechanism of action of metformin is suppression of hepatic gluconeogenesis by decreasing the expression of gluconeogenic genes. Both insulin and glucagon also regulate the assembly of the same transcriptional complex. Metformin converges with the insulin signaling pathway on CBP phosphorylation (31). In addition to the hepatic effect, other beneficial effects of metformin have been proposed but remain controversial. For example, when incubated with skeletal muscle isolated from T2DM patients, metformin potentiated insulin-stimulated glucose transport (33). However, the concentration used in this study is higher than that could be achieved at clinical doses. It is not clear if the effect is physiologically relevant. The hepatic uptake of metformin is mediated by organic cation transporter 1 (OCT1) (34). Polymorphisms of OCT1 play an important role in the antidiabetic effect of metformin because it affects the hepatic accumulation of the drug. One such polymorphism, OCT1-420del, is found in about 20% of white Americans (34). In T2DM patients with this polymorphism, the antidiabetic effect of metformin is significantly reduced (34).

Thiazolindinediones (TZDs) TZDs are a class of antidiabetic compounds derived from clofibrate, a compound with a strong lipid lowering effect and a weak activity for glycemic control (35). Unlike clofibrate, TZDs have enhanced antidiabetic effects while maintaining some lipid lowering activity. TZDs increase insulin-stimulated glucose uptake in the peripheral tissues and suppress hepatic glucose production by improving tissue insulin sensitivity. Therefore, they are usually referred to as insulin sensitizers. Like metformin, TZDs do not induce insulin secretion or cause hypoglycemia. However, TZDs are associated with weight gain, fluid retention, and hemoglobin reduction. The first marketed TZD was troglitazone, which was withdrawn from Europe in 1998 and from the United States and Japan in 2000 due to idiosyncratic hepatotoxicity (36). The

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underlying mechanism of such toxicity is not clear but association with inflammatory response has been proposed (36). Whether this is a class effect or specific with troglitazone is debatable. With frequent monitoring of serum aminotransferases and symptoms, hepatotoxicity became a lessening concern with rosiglitazone (Avandia) and pioglitazone (Actos), the two TZDs that were approved later and are currently on the market. However, growing concerns about increased risk of heart failure in patients on rosiglitazone or pioglitazone became a major safety issue with this class of drugs. The increased incidence of heart failure is likely caused by fluid retention as a result of PPARg activation. Another important adverse effect of TZDs is increased risk of bone fracture in pre- and postmenopausal women (37), because PPARg inhibits osteoblastogenesis in bone marrow (38). Some notable differences exist between the two marketed TZDs. Unlike pioglitazone, which has an active metabolite and therefore a longer duration of action, rosiglitazone has no active metabolite and a shorter plasma half-life. Moreover, the two drugs have differential effects on lipoprotein profiles. The target of TZDs is PPARg, a nuclear receptor that controls the expression of genes involved in adipocyte differentiation and lipid metabolism. TZDs act as ligand of PPARg, which upon ligand binding is activated, induces adipocyte differentiation, and improves glucose utilization and insulin sensitivity in adipose tissue. Since PPARg is expressed at very low levels in liver and skeletal muscle, the main insulin sensitizing effects of TZDs in these tissues are believed to be mediated through their actions in adipocytes. It was proposed that secreted factors/adipokines from adipose tissue may play such roles. For example, adiponectin is an adipokine that exerts beneficial metabolic effects (39). TZDs increase the expression of adiponectin in adipose tissue (40), indicating that adiponectin may mediate some of the antidiabetic effects of TZDs. TZDs also regulate the expression of resistin and leptin in adipose tissue (41, 42). While adipose tissue is required for the antidiabetic effect of TZDs, the hypolipidemic effect appears to be mediated by other tissues (43). Robust clinical efficacy has been demonstrated with TZD monotherapy in major clinical studies. In a 26-week randomized, placebo-controlled study involving 493 T2DM patients, rosiglitazone administered at 2 or 4 mg twice daily decreased mean HbA1c relative to placebo by 1.2% and 1.5%, respectively (44). Rosiglitazone treatment also reduced fasting plasma glucose and fasting insulin levels, and improved insulin sensitivity and b-cell function (44). Fluid retention and weight gain were observed in patients treated with rosiglitazone (44). In the same study, rosiglitazone increased total cholesterol, LDL, and HDL with a neutral effect on TG levels (44). While increasing HDL is expected to be beneficial, elevated LDL by rosiglitazone is an unwanted effect. There was no significant incidence of elevated liver enzymes (44), suggesting that it may have a reduced risk of hepatotoxicity compared to troglitazone. The effectiveness of rosiglitazone demonstrated in this trial is consistent with that in another study involving 959 T2DM patients, where significant reduction in HbA1c was achieved with both once-daily and twice-daily treatments (45). Further, significant additional reduction in HbA1c was achieved when rosiglitazone was combined with metformin, insulin, or SU for 26 weeks (46). Like rosiglitazone, pioglitazone was shown to be efficacious in the treatment of

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T2DM. In a 6-month randomized, placebo-controlled dose response study, pioglitazone improved glycemic control in patients with T2DM (47). In addition to treatment of T2DM, TZDs are also effective in preventing the onset of T2DM in prediabetic populations. Pioglitazone treatment slowed the development of diabetes in women with a history of gestational diabetes in the Pioglitazone in the Prevention of Diabetes (PIPOD) study (48). Similar results were found with troglitazone treatment in the same population (49), suggesting that this is a class effect. In the Diabetes Reduction Assessment with Ramipril and Rosiglitazone Medication (DREAM) study, rosiglitazone administration at 8 mg daily for 3 years significantly reduced the incidence of T2DM and increased the likelihood of regression to normoglycemia in adults with impaired fasting glucose, impaired glucose tolerance, or both (50). Mechanistically, TZDs have multiple activities that may be cardioprotective. They decrease blood pressure (51, 52), improve endothelial function (53), and reduce vascular inflammation and vascular smooth muscle cell proliferation (54). There are also lipid benefits with TZD therapy but the exact effects have been variable in different studies. For example, there are differential effects on LDL level when different TZDs were used for treatment (47, 55, 56). This could be due to different study population characteristics or differential effects of individual TZDs. The latter seems to be more possible because the lipid profile generated by pioglitazone monotherapy in a number of studies appears to be more favorable than that by rosiglitazone (57–60). A summary analysis using data pooled from 19 clinical trials (11 with rosiglitazone and 8 with pioglitazone) indicated that rosiglitazone increases total cholesterol, HDL, and LDL with a neutral effect on TG (61). Pioglitazone, on the other hand, reduces TG and increases HDL with no effect on LDL or total cholesterol (61). This analysis suggests that pioglitazone has more beneficial effects on plasma lipids. The caveat of this analysis is that the conclusion is not based on a direct comparison study in the same study population; rather, there are significant differences in study population characteristics from the 19 trials that likely contribute to the differential lipid effects revealed in the analysis. Nonetheless, these beneficial lipid properties suggest that pioglitazone may have a greater beneficial effect in lipid-dependent cardiovascular risk (62), such as myocardial infraction and other atherosclerosis-related events. Further, pioglitazone more than rosiglitazone treatment results in a greater shift from small, dense LDL particles to larger, buoyant, and less atherogenic LDL particles (63). Separate from the atherosclerosis aspect, fluid retention associated with TZD treatment causes congestive heart failure, which became a growing concern. Given the potential benefits of lipid profiles and concerns of heart failure, understanding the overall cardiovascular risk associated with TZDs is important because the T2DM population has higher risk of cardiovascular disease. In this regard, both pioglitazone and rosiglitazone were evaluated in phase 4 clinical trials for cardiovascular safety. In the Prospective Pioglitazone Clinical Trial in Macrovascular Events (PROactive) study, the primary end point was the composite of all-cause mortality, nonfatal myocardial infarction (including silent myocardial infarction), stroke, acute coronary syndrome, endovascular or surgical intervention in the coronary or leg arteries, and amputation above the ankle (64). Although pioglitazone did not have a significant effect on the primary end point, it reduced the composite of all-cause mortality, nonfatal myocardial

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infarction, and stroke (64). There was a statistically significant increase in heart failure in the pioglitazone group (64). In the DREAM study that was designed to assess the potential of rosiglitazone in preventing the incidence of T2DM, there is increased incidence of heart failure in the rosiglitazone group (50), consistent with the heart failure concern and almost overall neutral effect on lipids. The Rosiglitazone Evaluated for Cardiac Outcomes and Regulation of Glycemia in Diabetes (RECORD) study was designed to evaluate the cardiovascular safety of rosiglitazone. But before the RECORD data were available, a meta-analysis of existing rosiglitazone clinical data was conducted and the results suggested that rosiglitazone treatment is associated with a significant increase in the risk of myocardial infarction and with an increase in the risk of death from cardiovascular causes that has borderline significance (65). A number of debates and criticisms followed on various aspects of the meta-analysis that led to the findings (66–69). The meta-analysis was based on small-scale, short-term trials that were not designed to assess cardiovascular outcomes. The statistical analysis method employed was also debatable. Although the results were not conclusive, it points to the importance of the cardiovascular safety issue with rosiglitazone. The later completed RECORD study was exactly designed to address this question. It was an open-label study involving 4447 patients with T2DM on metformin or SU monotherapy. Addition of rosiglitazone to glucose lowering therapy in T2DM patients increased the risk of heart failure and of some fracture, mainlyin women (70). Rosiglitazone did not increase the risk of overall cardiovascular morbidity or mortality, although it is inconclusive about any possible effect on myocardial infarction (70).

Sulfonylureas (SUs) and Glinides SUs are a group of insulin secretagogues. They are chemical compounds that bind to the SUR1 subunit of the voltage-dependent KATP channel and induce first-phase insulin secretion. The interaction of SUs with SUR1 leads to the closure of the KATP channel, resulting in the sequential events of reduction of potassium conductance, cell membrane depolarization, and calcium entry into the cell (71). The elevated intracellular calcium in turn induces insulin secretion by pancreatic b-cells (71). Since the insulin secretory effect is not glucose dependent, SUs cause hypoglycemia. Moreover, SUs increase weight gain. Although SUs have been demonstrated with antidiabetic efficacy in numerous clinical trials, there is a growing trend of decline in their use in recent years due to the availability of other treatment options. There are three generations of SUs. Glimepiride is the only third-generation agent with higher affinity for the target and therefore can control glucose levels at lower doses. The second-generation agents are short-acting but much more potent than the firstgeneration drugs. One potential safety issue with SUs is the maintenance of KATP channel-mediated ischemic myocardial preconditioning because the voltagedependent KATP channel is also expressed in the cardiac tissues. Glimepiride has less binding to the cardiac tissues, and it maintains myocardial preconditioning, while glibenclamide might be able to prevent it (72). This could be an advantage for glimepiride, although the clinical importance is unclear.

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An early clinical trial by the University Group Diabetes Program (UGDP) to compare the efficacy of oral agents and insulin found evidence of increased cardiovascular morbidity associated with SUs (73). The study was controversial and the method used in the study was widely criticized (74). The later UKPDS refuted the UGDP findings and demonstrated that no increased mortality was associated with T2DM patients treated with SUs (20). The antidiabetic effects of SUs have been demonstrated in multiple clinical trials with significant HbA1c reductions (20, 75, 76). In the UKPDS, T2DM patients were enrolled with a 10-year follow-up to compare intensive insulin therapy with diet alone or treatment with a sulfonylurea (20). Both the intensive insulin therapy and SU treatment reduced microvascular end points compared to the diet alone group (20). There was no significant difference between insulin-treated subjects and those on SU (20). These data demonstrate that SU therapy reduces microvascular complications. Since the mechanism of action of SUs is induction of insulin secretion by pancreatic b-cells and b-cell loss is part of disease progression in T2DM, the frequent loss of efficacy with SUs is common. One key question is whether this class of drugs exhausts b-cells and accelerates b-cell failure in T2DM patients. In the A Diabetes Outcome Progression Trial (ADOPT), rosiglitazone, metformin, and glyburide as initial treatment for recently diagnosed T2DM patients were evaluated in a doubleblind, randomized, controlled clinical trial involving 4360 patients over a median duration of 4 years (77). The primary outcome was the time to monotherapy failure. Glyburide had the shortest durability as a monotherapy and was associated with reduced b-cell function over time when compared with other therapies (77). This finding suggests that SUs (or generally insulin secretagogues) may exhaust b-cells over time and exacerbates T2DM. Glinides are non-SU insulin secretagogues. They bind to a different site of the KATP channel and induce insulin secretion by pancreatic b-cells in a way similar to SUs. Glinides have short half-lives, and rapid onset and short duration of action. Their main action is to target postprandial glucose excursion. There are two members of this class, repaglinide (a benzoic acid derivative) and nateglinide (a phenylalanine derivative). Comparative trials of repaglinide with SUs demonstrated that they have equivalent efficacy (78–80). The main side effects are hypoglycemia and weight gain, which may be less pronounced than SUs. One inconvenience with this class of drugs is the requirement of frequent dosing with meals.

a-Glucosidase Inhibitors (AGIs) The enzyme a-glucosidase is located in the brush border of the small intestine. It is involved in the final step of breakdown of complex carbohydrates to absorbable monosaccharides (81). Since T2DM patients have sluggish insulin response that cannot match the need to normalize postprandial glucose excursion, inhibition of a-glucosidase reduces the rate of carbohydrate digestion and thereby delays glucose absorption, which helps match the inadequate insulin response with the postprandial hyperglycemia in T2DM patients. AGIs are a class of nonabsorbable drugs.

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Compared to other antidiabetic drugs, AGIs have modest effects. They are used for older patients who have mild to moderate hyperglycemia and those who are prone to hypoglycemia and lactic acidosis. Since these drugs exert efficacy by blocking carbohydrate digestion, they should be taken at the start of meals and the effectiveness depends on the amount of complex carbohydrates in the meal. There are three AGIs for the treatment of T2DM: acarbose, miglitol, and voglibose. The antidiabetic effect of acarbose was evaluated as an add-on therapy to the following groups: diet alone, diet plus metformin, diet plus SU, and diet plus insulin, in a 1-year study (82). Acarbose was effective in blunting the rapid increase in plasma glucose after a carbohydrate meal (82), consistent with its mechanism of action. Acarbose improved long-term glycemic control in T2DM patients regardless of concomitant antidiabetic medication (82). AGIs do not cause hypoglycemia and weight gain. The main adverse effects of AGIs are abdominal pain, diarrhea, and flatulence (82, 83). This is because the unabsorbed complex carbohydrates are passed into the large intestine and metabolized by bacteria to produce gaseous waste products. It is generally recommended to start with a low dose and gradually increase the dosage to reduce GI side effects.

DPP-4 Inhibitors DPP-4 (also known as DPP IV, DP 4, CD26, adenosine deaminase binding protein), an amino peptidase found in almost all organs and tissues, is a protease (84) that is primarily responsible for GLP-1 inactivation through proteolytic cleavage at the N-terminus. Inhibition of DPP-4 increases plasma active GLP-1 level and potentiates incretin effects. Two DPP-4 inhibitors, Januvia and Onglyza, have been approved for the treatment of T2DM. Details of these drugs will be covered in Chapter 12. Comparison of these drugs with other antidiabetic drugs is shown in Table 10.1.

Exenatide Exenatide is a GLP-1 like peptide originally found in the saliva of the Gila monster living in the Arizona desert. It is more potent than GLP-1 itself in activating the GLP-1 receptor. Exenatide has robust antidiabetic effects presumably mediated by the GLP-1 receptor. Exenatide is currently on the market as an antidiabetic therapy known as Byetta. Several other GLP-1 analogues with longer plasma half-lives are under development for the treatment of T2DM. GLP-1 analogues as antidiabetic therapies will be discussed in Chapter 11. Attributes of exenatide are shown in Table 10.1.

Symlin Amylin is a peptide cosecreted with insulin from pancreatic b-cells and is deficient in insulin-treated patients with type 1 or 2 diabetes (85). Amylin has suppressive effects on the central signals that control food intake and thereby causes weight loss (86). It

Table 10.1

Summary of Current Antidiabetic Drugs for Monotherapy

Agents

MOA

Advantages

Disadvantages/side effects

Insulin Metformin

Exogenous insulin supplementation Suppression of glucose production; increased peripheral glucose uptake Increased insulin secretion

Robust efficacy No hypoglycemia; weight loss; effects on lipids

Hypoglycemia; weight gain GI side effects; lactic acidosis

Good efficacy

Hypoglycemia; weight gain; worsen beta-cell function Weight gain; edema; liver toxicity; heart failure GI discomfort; limited efficacy GI side effects; abdominal pain; no weight loss Nausea, vomiting, and diarrhea

SU and glinides TZDs a-GIs DPP-4 inhibitors (Januvia and Onglyza) Incretin mimetics (Byetta)

Symlin

Weight loss drugs (Meridia and Xenical)

Improved insulin sensitivity; increased peripheral glucose uptake Decreased gut glucose absorption Increased GLP-1 levels Increased glucose-dependent insulin secretion; direct metabolic effects on CNS and peripheral tissues Appetite suppression; slowing gastric emptying; inhibition of glucagon secretion Appetite suppression or reduced fat absorption

No hypoglycemia; insulin sensitizing No hypoglycemia No hypoglycemia No hypoglycemia; weight loss

Weight loss

Weight loss

Nausea; increased risk of insulin-induced hypoglycemia; limited efficacy Hypertension and tachycardia (Meridia); GI side effects (Xenical); limited efficacy

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Table 10.2

Currently Approved Antidiabetic Combination Therapies

Agents

Metformin SU TZDs Insulin Glinides a-GIs Byetta Januvia/Onglyza

Add-on agent Metformin

SU

TZDs

Yes Yes Yes Yes

Yes Yes

Yes (pioglitazone)

Yes Yes

Yes Yes Yes (glimepiride with Januvia; glyburide with Onglyza)

Yes Yes (pioglitazone only with Januvia; both pioglitazone and rosiglitazone with Onglyza)

Note: “Yes” means that the combination has been approved by the FDA at this time.

also suppresses inappropriate secretion of glucagon (such as postprandial glucagon secretion) and slows gastric emptying (87, 88). An amylin analogue, pramlintide (Symlin), has been approved as an antidiabetic therapy. Its approved use is for type 1 or type 2 diabetics who use insulin.

Combination Therapies Despite the robust effects of the current antidiabetic therapies, no single agent can provide adequate glycemic control. As the disease further progresses, the failure of monotherapy requires combination of several agents with complementary mechanisms to achieve stronger efficacy. Single-pill, fix-dose combination is more convenient than taking two separate pills. Since metformin is a generic drug and first-line antidiabetic therapy, add-on of other therapies to metformin has been a primary combination strategy. However, if the add-on agent works through a mechanism that could exacerbate the risk of lactic acidosis, caution should be taken. General issues concerning combination therapies such as drug–drug interaction should be considered as well. For example, if the add-on agent is cleared through a renal mechanism like metformin, the potential for drug–drug interaction should be evaluated to ensure that they do not inhibit each other’s clearance after combination. Among the current therapies, metformin is approved to be combined with most drugs except AGIs (Table 10.2).

WEIGHT LOSS DRUGS Given that the majority of T2DM patients are obese and robust weight loss may help control hyperglycemia, weight loss drugs could be used to treat T2DM. In this regard,

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a robust anti-obesity drug can be used as an antidiabetic therapy. The recent FDA guidelines on the development of antidiabetic drugs suggest that the agency may be open to this notion if adequate reduction of HbA1c can be demonstrated in T2DM with an anti-obesity therapy. Meridia and Xenical are two weight loss drugs. Meridia is a serotonin reuptake inhibitor and induces weight loss by suppressing appetite. Xenical inhibits fat breakdown in the GI tract and thereby decreases fat absorption. Both drugs cause weight loss, an attribute important in antidiabetic therapy.

TREATMENT OF DIABETIC COMPLICATIONS In addition to hyperglycemia, lipid disorders, hypertension, and obesity, T2DM patients have complications from these primary metabolic disorders. Macrovascular complications include coronary heart disease, stroke, and peripheral vascular diseases. Microvascular complications are metabolic consequences in the eye (retinopathy), nerves (neuropathy), and kidney (nephropathy). Treatment of the primary causes of these complications may help slow the onset of the complications but current therapies are not sufficient to prevent their development. Antidiabetic therapies and drugs that target hyperglycemia, hyperinsulinemia, hypertension, and lipid disorders are essential to reduce the incidence of these complications. In addition, direct interference of the complications is required with additional therapies.

Treatment of Macrovascular Complications Cardiovascular disease is the leading cause of morbidity and mortality in T2DM patients. Diabetic patients have two- to fourfold higher risk of cardiovascular disease than age-matched control subjects (89). The underlying causes of the macrovascular diseases are atherosclerosis, hypertension, vascular inflammation, and endothelial dysfunction. Because cardiovascular disease is largely responsible for mortality in T2DM patients, reduction of cardiovascular risk has been one of the primary treatment goals. All the antidiabetic drugs mentioned above are aimed at treating hyperglycemia and/or insulin resistance, although some of them may have modest beneficial effects on cardiovascular risk factors. For example, metformin reduced the risk of myocardial infarction by 39% and that of all macrovascular end points by 30% in the UKPDS (24). But this benefit alone is not sufficient to adequately minimize the cardiovascular risks in T2DM. Additional treatments targeting hypertension, dyslipidemia, and inflammation can further reduce cardiovascular risk. The widely employed treatments for macrovascular complications are medical treatments of the common comorbidities in T2DM such as dyslipidemia and hypertension to prevent cardiovascular events. Most T2DM patients have lipid disorders such as hypertriglyceridemia, low HDL, and sometimes elevated LDL. Hepatic very-low-density lipoprotein (VLDL) production is often elevated as a result of increased circulating free fatty acid levels. Therapies that improve the lipid profiles have beneficial cardiovascular effects. Fenofibrate is an oral medication that reduces

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triglyceride and modestly raises HDL. In the Diabetes Atherosclerosis Intervention Study (DAIS), the effect of correction of lipid abnormalities on coronary artery disease was examined (90). In this study, 418 patients were randomly assigned to fenofibrate or placebo treatment for at least 3 years. These patients were in good glycemic control with mild lipoprotein abnormalities and at least one visible coronary lesion. Half had no previous clinical coronary disease. At the end of the study, the fenofibrate group had much greater reduction in total plasma cholesterol, LDL, and TG concentrations than the placebo group (90). In addition, they had higher increases in HDL than the placebo group (90). Patients in the fenofibrate group had significantly smaller increases in stenosis than the placebo group (90). This study demonstrated that fenofibrate improved lipid profiles and reduced angiographic progression of coronary artery disease in T2DM patients (90). Statins are a class of LDL lowering drugs with slight HDL raising activity. This class of drugs has been demonstrated to improve cardiovascular risk end points. In the Collaborative Atorvastatin Diabetes Study (CARDS), T2DM patients received atorvastatin (10 mg daily) or placebo and were evaluated for cardiovascular and other outcomes over a median follow-up period of 3.9 years (91). Atorvastatin reduced relative risk of the composite primary end point (acute coronary heart disease events, coronary revascularization, or stroke) by 37% (91). Similar findings strongly support the notion that statins improve cardiovascular outcomes in T2DM patients (92). However, statins do not seem to correct endothelial dysfunction, a characteristic disorder of diabetes. In the Diabetes Atorvastatin Lipid Intervention (DALI) study, despite the reduction of LDL and TG and elevation of HDL in T2DM patients on atorvastatin, endothelial dysfunction was not reversed (93). The effect of the combination therapy using a statin and a fibrate was evaluated in the Diabetes and Combined Lipid Therapy Regimen (DIACOR) study in patients with T2DM and no history of coronary heart disease (94). The combination therapy was superior in changing cardiovascular risk factors such as high LDL and VLDL and low HDL (94), but was no more effective in reducing inflammatory biomarkers than either form of monotherapy (95). Control of hypertension limits morbidity and mortality more effectively than tight glycemic control in T2DM patients (96). Several classes of oral antihypertensives have been evaluated in patients with T2DM for their effects on cardiovascular end points. Angiotensin converting enzyme (ACE) inhibitors and calcium channel b-blockers are both effective. In the UKPDS, 1148 T2DM patients with hypertension were assigned to either tight or less tight blood pressure control with a median follow-up period of 8.4 years (97). The group with tight control of blood pressure had over 40% risk reduction in stroke, over 30% reduction in death of diseases related to diabetes, including myocardial infarction and stroke, and 24% reduction in diabetes-related end points (97). These data demonstrate that tight blood pressure control in T2DM patients can significantly reduce macrovascular risk factors. The type of antihypertensives used in the treatment is not as important as blood pressure reduction itself because an ACE inhibitor and a calcium channel b-blocker had similar effects in reducing diabetic complications (98). In addition to statins, fibrates, and antihypertensives, aspirin is an anti-inflammatory drug that can reduce serious vascular events but it increases the risk of gastrointestinal bleeding (99).

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In the UKPDS, metformin treatment achieved a mean HbA1c level of 7.4% relative to the 8% in a control group and was associated with a 39% reduction in cardiovascular disease (24). Insulin and SU treatments, although achieved similar glycemic control, exhibited trends of improvement of cardiovascular outcomes but statistical significance was not reached (24). These findings raised an important question: does aggressive glycemic control reduces cardiovascular risks in T2DM patients? Three landmark clinical trials, the Action to Control Cardiovascular Risk in Diabetes (ACCORD), the Action in Diabetes and Vascular Disease (ADVANCE), and the Veterans’ Administration Diabetes Trial (VADT), were designed to address this question. Although there are differences in patient populations, enrollment criteria, and treatment targets, all three studies were designed to evaluate aggressive glycemic control on cardiovascular outcomes. Unfortunately, the outcomes of the three studies are not consistent and raised a lot of questions and debates (100). It is hard to reach consensus in the interpretation of these results. In the ACCORD study, 10,251 T2DM patients with a median HbA1c level of 8.1% received either intensive therapy targeting <6.0% HbA1c or standard therapy targeting an HbA1c level of 7.0–7.9% (100). The patients had a mean age of 62 years and a mean duration of T2DM of 10 years, and 35% of them had previous cardiovascular events. Intensive glycemic control increased all-cause mortality by 22% and the study had to be stopped in 2008 (100). This study suggests that intensive treatment strategy is associated with increased risk of mortality. Although the intensive treatment group had more body weight gain and three times more episodes of severe hypoglycemia, it does not appear that the hypoglycemia was a cause of higher mortality in the intensive therapy group (100). The ADVANCE study involved 11,140 patients for a study period of 5 years. Randomization included standard glucose control regimen and intensive control regimen of 30–120 mg gliclazide modified release and any other glucose lowering agents needed to achieve the target HbA1c of 6.5% (100). Combined macro- and microvascular end points decreased by 10% (100). There was a trend toward reduction in cardiovascular mortality; microvascular mortality decreased by 14% (100). These results suggest that intensive glycemic control did not change all-cause mortality and reduced some of the serious diabetes complications such as renal events. In the VADT study, the goal was intensive glycemic control involving 1791 patients with a mean age of 60 years, an average body mass index of 31 kg/m2, and a mean duration of T2DM of 12 years. The average HbA1c was 9.4% and the mean follow-up period was 6 years (100). This patient population was considered in severe conditions. The primary outcome was the composite of cardiovascular deaths, including those caused by myocardial infarction, stroke, and congestive heart failure (100). The mean HbA1c at the end of the trial was 8.4% in the control group and 6.8% in the intensive glycemic treatment group. There was a nonsignificant 13% reduction in cardiovascular events with the intervention (100). Thus, in the “toughest patients,” there is no negative cardiovascular effect (if not positive) with intensive glycemic control (100). In all three studies, intensive glycemic control increased the risk of hypoglycemia. More analyses are required to fully understand these results and reconcile the

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findings in the three studies. Meta-analyses of past cardiovascular outcome trials with T2DM patients suggest that glycemic control either reduces cardiovascular risk or is risk neutral, but the outcome could depend on different patient populations [101, 102]. Possible mechanisms for increased mortality in ACCORD have been proposed (103). Patients who suffered from T2DM for a longer time often have autonomic neuropathy, which may impair counterregulation to hypoglycemia and cause decreased symptoms (103). Patients with autonomic neuropathy may have cardiac denervation that is often associated with myocardial ischemia and predisposition to arrhythmia (103). Under such conditions, unrecognized hypoglycemia caused by intensive therapy could trigger cardiac events (103).

Treatment of Microvascular Complications The microvascular complications in T2DM include metabolic consequences in the eye (retinopathy), nerves (neuropathy), and kidney (nephropathy). In addition to antidiabetic therapies, direct intervention of these complications is warranted. Laser photocoagulation can be used to reduce the risk of blindness from proliferative retinopathy but is associated with side effects. Control of blood pressure can reduce the progression of diabetic eye disease. An angiotensin receptor blocker, candesartan, was tested in T2DM patients with mild to modestly severe retinopathy. An overall change toward less severe retinopathy by the end of the trial was observed in the candesartan group (104). Diabetes accounts for approximately 40% of all patients beginning renal replacement therapy (105). Since diabetic nephropathy is associated with increased cardiovascular morbidity and mortality (105), slowing the progression of nephropathy is an important intervention point given that the complication cannot be eliminated. ACE inhibitors are effective in slowing the progression of diabetic nephropathy. For example, enalapril attenuated the decline in renal function and reduced the extent of albuminuria in normotensive, normoalbuminuric patients with T2DM (106). In the UKPDS involving 1148 T2DM patients with either tight or less tight blood pressure control, there was 37% reduction in microvascular complications in the group on tight blood pressure control (97). In the treatment of neuropathy, management of pain associated with diabetes is the main focus. Gabapentin is a drug for neuropathic pain and has been tested in clinical settings to treat diabetic peripheral neuropathy. In an 8-week study, T2DM patients on gabapentin had significantly lower mean pain scores than those in the placebo group (107). Gabapentin also improved quality of life measurements (107). Other pain medications are also used to treat diabetic neuropathy (108).

FUTURE STRATEGIES OF ANTIDIABETIC THERAPIES Monotherapy Versus Combination Therapy As mentioned above, there is no single drug that can fully restore normal glycemia. Over time, monotherapy tends to fail. The ADOPT study demonstrated that SUs tend

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to fail at a much faster pace than TZD or metformin (77). They also appear to accelerate b-cell failure (77). Metformin is the first-line therapy in the treatment of newly diagnosed T2DM patients. Over time, other antidiabetics need to be added to an existing monotherapy for adequate glycemic control. Combination with metformin is a certain path for a new drug, especially when the new medication has a complementary mechanism to metformin. The ideal profile for a new antidiabetic drug is that besides the robust hypoglycemic effect, it should have significant cardiovascular benefits. This can help reduce pill burden in a patient population that are known to be on polypharmacy.

Importance of Cardiovascular Morbidity and Mortality Regardless of what therapeutic strategies are employed to control hyperglycemia and cardiovascular complications, the combined outcome of morbidity and mortality in T2DM patients is a key measure of effectiveness of treatment strategies and disease management. The Skaraborg Hypertension and Diabetes Project concluded that poor glucose and lipid control and hypertension predicted all-cause mortality (109). Future drug development efforts should be directed at improving insulin sensitivity and glucose homeostasis, and in the meantime, reducing cardiovascular risk factors such as dyslipidemia and hypertension. These attributes should translate to improved cardiovascular end points in an outcome study. Further, robust body weight reduction with minimal side effects will continue to be an important approach.

Future Outlook on More Effective Therapies Since inflammation is involved in the development of both insulin resistance and atherosclerosis, targeting inflammatory pathways is likely to be an important future strategy that can both improve insulin sensitivity and reduce cardiovascular risk factors. Such therapies could represent added value to the existing treatments. It may help reduce the need for combination therapies with both antidiabetic and cardiovascular drugs. In addition to LDL lowering effect, the statins have anti-inflammatory activities as demonstrated in the JUPITER trial (110). Other areas of mechanism to impact are treatments of endothelial dysfunction, vascular smooth muscle cell proliferation, vascular inflammation, and the calcification and stability of atherosclerotic plaques to delay or prevent rupture. The new generations of drugs are long-acting GLP-1 analogues. These drugs are intended to take advantage of the incretin effect to boost glucose-dependent insulin secretion (GSIS). More endogenous insulin can be released to match the postprandial hyperglycemia in T2DM and counter insulin resistance. In addition, recent research demonstrated that they have incretin-independent beneficial effects (111). These drugs have no risk of hypoglycemia. Due to the intense interest in these drugs, they are discussed separately in a separate chapter in this book.

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SUMMARY In general, T2DM is a direct consequence of metabolic syndrome. Most individuals with metabolic syndrome are in the prediabetic state, which could progress into T2DM upon further exacerbation of insulin resistance and b-cell failure. This is implicated in the fact that most T2DM patients have metabolic syndrome. T2DM patients have not only hyperglycemia but also significantly increased cardiovascular risk factors. As a matter of fact, cardiovascular mortality accounts for most deaths in T2DM. A balanced strategy of both glycemic control and reducing cardiovascular risk is essential. Although physical exercise is metabolically beneficial, it is not sufficient to reach these treatment goals. Antidiabetic medications, initially as monotherapy and later in the form of combination therapies based on medical needs, are required for adequate glycemic control and reducing microvascular complications. Treatment of individual cardiovascular risk factors is equally important to decrease macrovascular complications. Additional therapies are used to treat microvascular and macrovascular complications. None of the current therapies alone or in combination can effectively control or mitigate the disease. More robust antidiabetic and cardiovascular therapies are targets for future drug discovery.

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83. CONIFF, R.F., J.A. SHAPIRO, D. ROBBINS, R. KLEINFIELD, T.B. SEATON, P. BEISSWENGER, and J.B. MCGILL. 1995. Reduction of glycosylated hemoglobin and postprandial hyperglycemia by acarbose in patients with NIDDM. A placebo-controlled dose-comparison study. Diabetes Care 18:817–824. 84. MENTLEIN, R. 1999. Dipeptidyl-peptidase IV (CD26)—role in the inactivation of regulatory peptides. Regul Pept 85:9–24. 85. WEYER, C., D.G. MAGGS, A.A. YOUNG, and O.G. KOLTERMAN. 2001. Amylin replacement with pramlintide as an adjunct to insulin therapy in type 1 and type 2 diabetes mellitus: a physiological approach toward improved metabolic control. Curr Pharm Des 7:1353–1373. 86. REDA, T.K., A. GELIEBTER, and F.X. PI-SUNYER. 2002. Amylin, food intake, and obesity. Obes Res 10:1087–1091. 87. GEDULIN, B.R., T.J. RINK, and A.A. YOUNG. 1997. Dose-response for glucagonostatic effect of amylin in rats. Metabolism 46:67–70. 88. YOUNG, A.A., B. GEDULIN, W. VINE, A. PERCY, and T.J. RINK. 1995. Gastric emptying is accelerated in diabetic BB rats and is slowed by subcutaneous injections of amylin. Diabetologia 38:642–648. 89. American Diabetes Association. 1998. Consensus development conference on the diagnosis of coronary heart disease in people with diabetes: 10–11 February 1998, Miami, Florida. American Diabetes Association. Diabetes Care 21:1551–1559. 90. Diabetes Atherosclerosis Intervention Study Group. 2001. Effect of fenofibrate on progression of coronary-artery disease in type 2 diabetes: the Diabetes Atherosclerosis Intervention Study, a randomised study. Lancet 357:905–910. 91. COLHOUN, H.M., D.J. BETTERIDGE, P.N. DURRINGTON, G.A. HITMAN, H.A. NEIL, S.J. LIVINGSTONE, M.J. THOMASON, and J.H. FULLER. 2005. Rapid emergence of effect of atorvastatin on cardiovascular outcomes in the Collaborative Atorvastatin Diabetes Study (CARDS). Diabetologia 48:2482–2485. 92. ARCA, M. 2007. Atorvastatin efficacy in the prevention of cardiovascular events in patients with diabetes mellitus and/or metabolic syndrome. Drugs 67(Suppl 1): 43–54. 93. VAN VENROOIJ, F.V., M.A. VAN DE REE, M.L. BOTS, R.P. STOLK, M.V. HUISMAN, and J.D. BANGA. 2002. Aggressive lipid lowering does not improve endothelial function in type 2 diabetes: the Diabetes Atorvastatin Lipid Intervention (DALI) Study: a randomized, double-blind, placebo-controlled trial. Diabetes Care 25:1211–1216. 94. MAY, H.T., J.L. ANDERSON, R.R. PEARSON, J.R. JENSEN, B.D. HORNE, F. LAVASANI, H.D. YANNICELLI, and J.B. MUHLESTEIN. 2008. Comparison of effects of simvastatin alone versus fenofibrate alone versus simvastatin plus fenofibrate on lipoprotein subparticle profiles in diabetic patients with mixed dyslipidemia (from the Diabetes and Combined Lipid Therapy Regimen study). Am J Cardiol 101:486–489. 95. MUHLESTEIN, J.B., H.T. MAY, J.R. JENSEN, B.D. HORNE, R.B. LANMAN, F. LAVASANI, R.L. WOLFERT, R.R. PEARSON, H.D. YANNICELLI, and J.L. ANDERSON. 2006. The reduction of inflammatory biomarkers by statin, fibrate, and combination therapy among diabetic patients with mixed dyslipidemia: the DIACOR (Diabetes and Combined Lipid Therapy Regimen) study. J Am Coll Cardiol 48:396–401. 96. BETTERIDGE, D.J. 2001. Lipid-lowering trials in diabetes. Curr Opin Lipidol 12:619–623. 97. UK, Prospective Diabetes Study, Group. 1998. Tight blood pressure control and risk of macrovascular and microvascular complications in type 2 diabetes: UKPDS 38. UK Prospective Diabetes Study Group. BMJ 317:703–713. 98. UK, Prospective Diabetes Study Group. 1998. Efficacy of atenolol and captopril in reducing risk of macrovascular and microvascular complications in type 2 diabetes: UKPDS 39. UK Prospective Diabetes Study Group. BMJ 317:713–720. 99. BAIGENT, C., L. BLACKWELL, R. COLLINS, J. EMBERSON, J. GODWIN, R. PETO, J. BURING, C. HENNEKENS, P. KEARNEY, T. MEADE, C. PATRONO, M.C. RONCAGLIONI, and A. ZANCHETTI. 2009. Aspirin in the primary and secondary prevention of vascular disease: collaborative meta-analysis of individual participant data from randomised trials. Lancet 373:1849–1860. 100. BLOOMGARDEN, Z.T. 2008. Glycemic control in diabetes: a tale of three studies. Diabetes Care 31:1913–1919. 101. RAY, K.K., S.R. SESHASAI, S. WIJESURIYA, R. SIVAKUMARAN, S. NETHERCOTT, D. PREISS, S. ERQOU, and N. SATTAR. 2009. Effect of intensive control of glucose on cardiovascular outcomes and death in

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Chapter 10 Current Antidiabetic Therapies and Mechanisms patients with diabetes mellitus: a meta-analysis of randomised controlled trials. Lancet 373:1765–1772. SELVIN, E., S. BOLEN, H.C. YEH, C. WILEY, L.M. WILSON, S.S. MARINOPOULOS, L. FELDMAN, J. VASSY, R. WILSON, E.B. BASS, and F.L. BRANCATI. 2008. Cardiovascular outcomes in trials of oral diabetes medications: a systematic review. Arch Intern Med 168:2070–2080. KAHN, S.E. 2009. Glucose control in type 2 diabetes: still worthwhile and worth pursuing. JAMA 301:1590–1592. SJOLIE, A.K., R. KLEIN, M. PORTA, T. ORCHARD, J. FULLER, H.H. PARVING, R. BILOUS, and N. CHATURVEDI. 2008. Effect of candesartan on progression and regression of retinopathy in type 2 diabetes (DIRECTProtect 2): a randomised placebo-controlled trial. Lancet 372:1385–1393. VORA, J.P., H.A. IBRAHIM, and G.L. BAKRIS. 2000. Responding to the challenge of diabetic nephropathy: the historic evolution of detection, prevention and management. J Hum Hypertens 14:667–685. RAVID, M., D. BROSH, Z. LEVI, Y. BAR-DAYAN, D. RAVID, and R. RACHMANI. 1998. Use of enalapril to attenuate decline in renal function in normotensive, normoalbuminuric patients with type 2 diabetes mellitus. A randomized, controlled trial. Ann Intern Med 128:982–988. BACKONJA, M., A. BEYDOUN, K.R. EDWARDS, S.L. SCHWARTZ, V. FONSECA, M. HES, L. LAMOREAUX, and E. GAROFALO. 1998. Gabapentin for the symptomatic treatment of painful neuropathy in patients with diabetes mellitus: a randomized controlled trial. JAMA 280:1831–1836. MAX, M.B., S.A. LYNCH, J. MUIR, S.E. SHOAF, B. SMOLLER, and R. DUBNER. 1992. Effects of desipramine, amitriptyline, and fluoxetine on pain in diabetic neuropathy. N Engl J Med 326:1250–1256. OSTGREN, C.J., U. LINDBLAD, A. MELANDER, and L. RASTAM. 2002. Survival in patients with type 2 diabetes in a Swedish community: Skaraborg Hypertension and Diabetes Project. Diabetes Care 25:1297–1302. RIDKER, P.M., E. DANIELSON, F.A. FONSECA, J. GENEST, A.M. GOTTO, JR., J.J. KASTELEIN, W. KOENIG, P. LIBBY, A.J. LORENZATTI, J.G. MACFADYEN, B.G. NORDESTGAARD, J. SHEPHERD, J.T. WILLERSON, and R.J. GLYNN. 2008. Rosuvastatin to prevent vascular events in men and women with elevated C-reactive protein. N Engl J Med 359:2195–2207. AYALA, J.E., D.P. BRACY, F.D. JAMES, B.M. JULIEN, D.H. WASSERMAN, and D.J. DRUCKER. 2009. The glucagon-like peptide-1 receptor regulates endogenous glucose production and muscle glucose uptake independent of its incretin action. Endocrinology 150:1155–1164.

Part Three

Drug Targets for Antidiabetic Therapies

Chapter

11

GLP-1 Biology, Signaling Mechanisms, Physiology, and Clinical Studies REMY BURCELIN1, CENDRINE CABOU1, CHRISTOPHE MAGNAN2,3, 1 AND PIERRE GOURDY 1

Rangueil Institute of Molecular Medicine, INSERM U858, Toulouse, France INSERM U858, Toulouse, France 3 University Paris Diderot, CNRS, Paris, France 2

INTRODUCTION In response to an oral glucose administration, insulin secretion is much more dramatically increased when compared to an intravenous injection of a similar glucose challenge. This effect is called the “incretin effect” and is due to the secretion of two peptides, the glucagon-like peptide 1 (GLP-1) and the glucose-dependent insulinotropic peptide (GIP). Over the past decade, both peptides have received a growing body of attention due to their exquisite capacity in stimulating insulin and reducing glucagon secretion in the presence of hyperglycemia only. Hence, this important pancreatic effect was at the dawn of new therapeutic strategies for the treatment of type 2 diabetes. GLP-1 has attracted the attention of the scientific, clinical, and industrial communities since it retains most of its insulinotropic activity in type 2 diabetic patients. However, both incretins are very rapidly degraded by the dipeptidyl peptidase IV (DPP-4), which is localized at the vicinity of the enteroendocrine cells secreting GLP-1 and GIP. Hence, DPP-4 resistant analogues to both peptides and inhibitors of DPP-4 have been generated and reduced hyperglycemia in type 2 diabetic patients. However, to date, the mechanisms through which both incretin peptides and DPP-4 inhibitors regulate glucose homeostasis have yet to be

Metabolic Syndrome: Underlying Mechanisms and Drug Therapies Edited by Minghan Wang Copyright 2011 John Wiley & Sons, Inc.

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described and fully explained. Numerous controversies exist regarding the direct or indirect role of GLP-1 in the stimulation of pancreatic secretion, the importance of GLP-1-regulated glucagon secretion, and the mechanisms through which DPP-4 inhibitors lower glycemia. Furthermore, over the years the physiological roles of GLP-1 have extended toward cardiovascular effects and the control of food intake and body weight gain. We will describe the physiology of GLP-1 and its therapeutic applications. In 1902, William M. Bayliss and Ernest H. Starling demonstrated that the acid extract of the intestinal mucosa stimulated exocrine pancreatic secretion, and this activity was mediated by a bloodstream-transported factor named secretin (1). Hence, they first invented the term “hormone.” Today GLP-1 and GIP are the two peptides enhancing stimulation of insulin secretion during a meal. The classical paradigm suggests that GLP-1 and GIP are released by L and K cells from the lower and upper small bowel, respectively. Upon binding to their corresponding receptors at the surface of the pancreatic insulin secreting b-cells, the hormone is released (Figure 11.1).

Nutrients

Muscle glucose utilization

↓

-Neural functions -Food intake -Whole-body glucose distribution -Neural protection -Cognition, memory

Hepatic glucose production ↓

GLP-1

Vagus nerve Indirect route ↓

GLP-1 G G G

Blood Flow

↓ Gastric emptying

GIP Glucose sensor system

Direct route

Pancreatic functions: - β-cell - α-cell

↑ insulin ↓ glucagon

Figure 11.1 Physiological regulation of nutrient-induced GLP-1 action. The nutrients are absorbed by the gut. The incretins, GLP-1 and GIP, are secreted after food ingestion. A first effect of the incretins is to bind to their corresponding receptors at the surface of the b-cell and to trigger glucose-induced insulin secretion. A second route involves detection by nutrient sensors of changes in mesenteric and portal concentrations of nutrients and incretins, after which a neural signal is sent through the vagus to the brain. The brain then redistributes the nutrient signal toward tissues including the endocrine pancreas, the blood vessels, and the gut itself. These physiological effects control consequently physiological functions such as hepatic glucose production and muscle glucose utilization.

Introduction

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GLP-1 DPP-4 inhibitors 78 His Ala Glu Gly x

DPP-4 degradation

Metabolites GIP (3-42) GLP-1 (9-36)

Renal GLP-1 Analogues & GLP-1 receptor agonists

clearance

Figure 11.2 GLP-1 and GIP are rapidly, over less than a minute, inactivated by DPP-4, which is ubiquitously distributed in the body. The enzyme cleaves the first two amino-terminal residues where a proline or an alanine is in the second position. The corresponding metabolites are then eliminated by the kidney.

A key feature of these peptides is that the insulinotropic efficacy requires the presence of the concomitant rise in plasma glucose concentration. Glycemia above a mean of 5 mM is mandatory for the insulinotropic effect (2, 3). Hence, this physiological characteristic represents a major breakthrough for the use of incretin-based therapies. Indeed, other insulin secretagogues, such as sulfonylureas, are glycemia independent and induce major hypoglycemia deleterious for the neural central system and the general physiology of the patients. Therefore, incretin-based therapies represent a preferred strategy for the treatment of diabetes. Buffering the enthusiasm for incretins for the treatment of hyperglycemic syndrome, it is important to note that both peptides are rapidly degraded by the enzyme DPP-4 (4, 5) (Figure 11.2). This enzyme is present at the much-closed vicinity of the GLP-1 secreting L cells and GIP secreting K cells from the intestine (6). Furthermore, DPP-4 circulates in the blood and further degrades the incretins into inactive peptides (6). It could be estimated that 50–80% of the incretins are degraded before reaching the systemic vena cava bloodstream and hence, less than 5–10% can reach the pancreatic artery to stimulate insulin secretion. This important discrepancy is supported by numerous observations, which showed that indirect routes for the regulation of pancreatic, that is, insulin and glucagon, secretions exist and could be considered as the most important mechanism involved in the control of endocrine pancreatic secretions (Figure 11.1). Despite the fact that more than 3000 original manuscripts and close to 700 reviews have been written so far about incretins, the

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precise molecular and physiological mechanisms through which incretins control glucose metabolism remain unclear. Certainly, incretins increase glucose-induced insulin secretion in vitro on isolated pancreatic islets and in vivo following a pharmacological injection of the peptides separately. However, upon a physiological meal, the sequence of events starting from the absorption of glucose to the stimulation of GLP-1 and GIP secretion into the portal vein, then to the binding to the receptors located at the surface of the insulin-secreting b-cells, and eventually the stimulation of insulin secretion, remains to be determined in this order and confirmed in humans. Consequently, it has been discussed that the physiological actions of GLP-1 could be mediated by the recruitment of the autonomic nervous system during an oral glucose load. Portal vein glucose sensors and the presence of the GLP-1 receptor (GLP-1R) in the brain, along with numerous lines of experimental evidence, support this concept (7–10). The contribution of the nervous versus the direct endocrine system to the physiological effect of GLP-1 actually remains a matter of debate. The initial understanding of the physiological functions of the incretins also concerned the reduction of food and water intake, as well as the slowing of gastric emptying. Hence, a deep effect on glucose metabolism was suggested. Over the past years, compelling evidence has showed that GLP-1 controls b-cell plasticity by preventing in vitro apoptosis and increasing cell proliferation. Hence, a new hope for the treatment of type 2 diabetic patients was generated. The reduced amount of pancreatic b-cells might be regenerated upon long-term GLP-1 treatment. However, there is so far no clear demonstration in humans of these mechanisms despite the evidence shown in mice and rats (11–13). Along the same line, discovery data showed promising cardioprotective and neuroprotective effects of GLP-1, suggesting that some cellular mechanisms could be envisioned for the treatment of degenerating diseases (14–17). Altogether, the discovery of the incretin concept has been around for some time, the mechanisms through which incretins control the physiology still deserve more work. Despite this, it is certain that GLP-1-based therapies can be used for the treatment of diabetes and, probably in the near future, some associated disorders such as cardiovascular or neurological diseases. Two classes of therapeutic agents have been generated (Figure 11.2). The first one consists of GLP-1 analogues such as Liraglutide and receptor agonists such as Exendin-4, which is derived from the venom of Gila monster (Holoderma suspectum), a lizard that lives in the Arizona desert. The peptide binds to the GLP-1R with an almost similar affinity to GLP-1 but is not degraded by the DPP-4 and remains stable and active in the blood for a longer period of time. GLP-1 analogues have been generated by either grafting free fatty acids at different positions of the molecule to ensure its binding to the albumin and hence slow release of the peptides, or fusing the peptide to carrier molecules to extend plasma half-life. However, all these peptides need to be injected. Some promising strategies are ongoing to transform the daily regimen into weekly injection or one with even longer acting duration. To overcome this problem, chemical compounds that boost plasma GLP-1 levels can be administered orally are being generated. A second class of compounds inhibits DPP-4 and prevents GLP-1 from being degraded. The DPP-4 inhibitors are currently on the market. Additional molecules

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(called gliptins) up to five could be released over the next years. Due to differences in their mode of action and type of inhibition, a large panel of molecules should be able to fit part of the needs linked to the large heterogeneity of diabetic patients. It is noteworthy that these new classes of therapeutic agents can be substitutes for the classical agents such as metformin and sulfonylureas. Indeed, a debate is ongoing regarding their efficacy, which is quite similar to metformin, the safety, which needs several more years on the market for a full assessment, and the physiological relevance as first-line use upon diagnosis of the diseases. The gliptins are also mainly used to overcome the therapeutic failure of the current pharmacological agents. This class of pharmaceutical molecules needs attention regarding their very recent use in clinic. A recent report from the FDA describes a few cases of pancreatitis following the treatment with DPP-4 inhibitors. However, whether these important side effects are directly linked to the gliptins remain to be demonstrated. So far no experimental evidence in vivo or in vitro can demonstrate any uncontrolled proliferating effect of these molecules, which are so far considered as safe. The therapeutic strategies are different between countries. As a result, different treatment approaches at times are used for Asian or Caucasian patients. This difference should generate numerous data that allows understanding how to improve the treatment of type 2 diabetes. Interestingly, some of the GLP-1-based molecules could be used in other diseases such as type 1 diabetes and Alzheimer’s disease due to their neuroprotective effects (18). Both diseases, at their very onset, are clearly associated with some extent of neuropathy (19, 20). Considering the important role of the autonomic nervous system in the control of glucose homeostasis, it is expected that molecules with neuroprotective effects, such as those derived from GLP-1-based therapies, should impact the extent of the impaired glucose tolerance by preserving from the development of dysautonomia.

ORIGIN OF THE INCRETIN CONCEPT Following the observation by Bayliss and Starling (1) and subsequent interpretation by Moore et al. (21), Zunz and La Barre demonstrated that oral administration of intestinal mucosa extract could reduce hyperglycemia in diabetic patients (22) (Figure 11.3). They hence described the incretin concept. Along with the discovery of insulin (23) around that time and the demonstration of glucose lowering characteristic of pancreatic extract, this finding suggests that the intestinal extract increases insulin secretion from the pancreas and reduced glycemia. However, this notion was only conclusively demonstrated 30 years later in 1963 by McIntyre. This conclusion was concomitant to the setup of the insulin radioimmunoassay by Yalow and Berson (24, 25). It is noteworthy that earlier than insulin the glucoregulatory hormone glucagon was discovered in 1923 as a hyperglycemic substance present in pancreatic extracts (26). Later on in 1969, Unger and Eisentraut described the gut to pancreas axis as a major regulatory mechanism of glucose metabolism. It was then showed that nutritional, nervous, and hormonal signals regulated the gut to pancreas axis (27). Still, it remains to be determined which molecule was responsible for triggering

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Cloning of proglucagon gene Experimental validation of incretins

Definition of incretins

1930

1970

1980

1990

Number of publications related to GLP-1 uppto to1992: 1992: Number of publications related to GLP-1 u 145 145

Figure 11.3

Incretin reduce glycemia of T2D

Cloning of the receptor

‘‘The enteropancreatic axis’’

1960

Development of GLP-1 analogues

2000 1993–2005: 1993–2005: 1892 1892

History of incretin concept and GLP-1.

insulin secretion in response to a meal or an oral glucose challenge. In 1979, Holst employed an improved gel-filtration purification method and other different biochemical techniques to identify a glucagon-like immunoreactivity in the porcine gastrointestinal tract (28). This gut-type glucagon was compared to the pancreatic type glucagon. It was described that the esophageal, the fundic, and the antropyloric parts of the gastric mucosa contained very small amounts of glucagon-like immunoreactivity, whereas the intestinal mucosa contained both types of glucagon with opposite gradients (28). Furthermore, pancreatic a-cell-like cells were reported to be present in the gastrointestinal mucosa (29). In 1980, GLP-1 was identified as a product of the same single gene that encodes glucagon and a GLP-1 analogue called GLP-2. Later on the GLP-1 receptor was then cloned (3, 30). Ever since the discovery of the insulinotropic effect of the incretins, it has been proposed to use the peptides for the treatment of diabetes. However, it was not known whether the magnitude of the incretin response was sufficient to normalize plasma glucose in type 2 diabetic patients with poor metabolic control. Numerous clinical trials were carried out and support the conclusion that GLP-1 mostly retains its insulinotropic activity in type 2 diabetic patients (31–39). The individual and combined contributions of GLP-1 and GIP to the incretin effect were studied in volunteers via infusion on separate occasions. The insulinotropic activity was then compared but the contribution of each molecule was still a matter of debate (40). The advent of molecular engineering in the mouse could demonstrate the additive effect of both incretin peptides (41). Following the incretin concept was the discovery of the enzyme, DPP-4, that prototypically inactivates the incretins (5, 39). Cleavage of the N-terminus of GLP-1 by DPP-4 converts the active GLP-1-(7–37) and -(7-36)-amide to the inactive GLP-1-(9-37) and -(9-36)-amide, respectively. The identification of the proteolytic GLP-1 fragments has been made possible by separation with HPLC and

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detection with ELISA and RIA using antibodies recognizing the N-terminus of GLP1. So far most available antibodies are directed toward the middle region or the Cterminus, and recognize both the intact GLP-1 and the proteolyzed, biologically inactive forms. Based on the identification and quantification of the intact and proteolyzed GLP-1 fragments, it was demonstrated using DPP-4 mutant mice (42) that the enzyme is the primary mechanism for GLP-1 degradation in human plasma in vitro and may have a key role in inactivating the peptide in vivo.

FROM THE PROGLUCAGON GENE TO PROTEIN AND SECRETION The gene encoding GLP-1 was first cloned in 1983 by G. Bell (43) (Figure 11.3). The author demonstrated that the preproglucagon gene originates from a gene that has evolved by duplication leading to closely related genes such as glucagon, GLP-1 and GLP-2 (Figure 11.4). Such coding sequences are driven by the same promoter. Hence, all peptides can putatively be produced in the same cell. However, there is an important posttranslational proteolytic processing mechanism, which provides the release of the proper peptides according to the specific endocrine cells. The processing is mediated by two different forms of prohormone convertases (PC), PC1/3 and PC2, which are expressed in the intestinal L cells and the pancreatic a-cells, respectively (44). The important role of the PCs on the maturation of the proglucagon 42 amino acids proGIP

S

N-terminal peptide

C-terminal peptide

GIP GIP Prohormone convertase 1/3 Posttranslational cleavage sites in endocrine K cells

DPP-4 cleavage site

GIP

1-YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ-42 42

Proglucagon

S GRPP

30 amino acids

Glucagon Glucagon

IPIP-1 1

GLP-1 GLP-1

33 amino acids IPIP-2 2

GLP-2 GLP-2

Prohormone convertase 1/3 Posttranslational cleavage sites in endocrine L cells

DPP-4 cleavage site

GLP-1

HAEGTFTSDVSSYLEGQAAKEFIAWLV 7-HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-36

Figure 11.4 Proglucagon and GIP peptides and processing. Glucagon, GLP-1 and GLP-2 are derived from a single gene, the proglucagon. Upon the action of prohormone convertase 2 (PC2), glucagon is produced in the pancreatic a-cells. Conversely, GLP-1 is produced in L cells through proteolytic processing of the proglucagon polypeptide by prohormone convertase 1/3 (PC1/3).

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polypeptide has been demonstrated using specific PC-null mice and in cell lines using adenovirus-mediated overexpression of the PC enzymes. Indeed, the 180-amino acid proglucagon polypeptide is processed by PC2 in the pancreatic a-cells to yield glucagon, whereas by PC1/3 in the intestinal L cells to yield GLP-1 and GLP-2, all of which are structurally related hormones with opposing metabolic actions (45, 46). The homology of the proglucagon genes between mammalian species is very high (greater than 90% amino acid sequence homology). Glucagon is encoded in exon 3, and GLP-1 and GLP-2 are encoded in exons 4 and 5, respectively (47). Importantly and for a yet unknown reason, the amino acid sequence of the GLP-1 is identical between all mammalian species. PC2 is responsible for generating the typical a-cell pattern of proglucagon processing, giving rise to glucagon and the entire C-terminal unprocessed half-molecule known as major proglucagon fragment (MPGF). In contrast, PC3, also identified as PC1, is the major neuroendocrine prohormone convertase in the intestinal L cells where MPGF is processed to release two glucagon-related peptides, GLP-1 and GLP-2, while the glucagon-containing N-terminal half-molecule (glicentin) is only partially processed to oxyntomodulin and small amounts of glucagon. GLP-1 and GLP-2 have approximately 50% amino acid homology to glucagon. The processing of GLP-1 also requires a step where the last dibasic amino acids are removed by carboxypeptidases (48). The peptides are separated from each other by intervening peptides IP1 and IP2. Importantly, GLP-2 does not have any insulinotropic activity but it is rather involved in intestinal permeability (49) and the stimulation of small intestinal growth through induction of intestinal epithelial proliferation (50–52). The two peptides GLP-1 and GLP-2 are hence produced in equal concentrations in the enteroendocrine L cells. Such cells are scattered among the enterocytes throughout the small bowel and ascending colon. Hence, a gradient of L cells is located along the small intestine in a direction opposite to the gradient of glucose into the intestine. This is supported by the observation that L cells are present in the colon where mostly no glucose is absorbed. Upon production in the cell, a set of posttranslational processing is required to generate mature GLP-1 (48, 53–56). The first 6 amino acids need to be removed to produce a fully active GLP-1 of 31 amino acids. In addition, GLP-1-(7-37) undergoes another posttranslational maturation since it can be amidated into GLP-1-(7-36) amide. In humans, the majority (at least 80%) of the circulating biologically active GLP-1 is the C-terminally amidated form.

THE GLP-1 RECEPTOR The GLP-1R is a G protein-coupled receptor (GPCR) cloned in 1992 by B. Thorens using transient expression of a rat pancreatic islet cDNA library (3). The receptor binds specifically to GLP-1 but not peptides of related structure and similar function, such as glucagon, GIP, vasoactive intestinal peptide, or secretin. The receptor is 463-amino acid long and contains seven transmembrane domains. Sequence homology is found only with the receptors for secretin, calcitonin, and parathyroid hormone, all of which are GPCRs. In a ligand-binding assay using [125I]GLP-1, GLP-1 was

The GLP-1 Receptor

289

GLP1 GLP1 R

P

Sur1 GS

PDE

AC ATP

Kir6.2

EPAC2 +

AKAP

EPAC2

cAMP-GEFII

P SIK2

Rim

Rab3

TORC2

P

Piccolo

RIM2 Ca++ Ca++ Ca++ Ca++ Ca++ Ca++ PKCs

+ Ser 133

K

+

cAMP

PKA

CREB Nucleus

Cell survival

Rab3A

Insulin secretion

Activated PKCs

Figure 11.5 Main molecular pathways for GLP-1 action involve insulin secretion and b-cell survival. GLP-1 increases adenylyl cyclase activity and cAMP production. This secondary messenger regulates separately the cAMP-GEFII, PKA, and EPAC pathways. Altogether, gene expression, insulin secretion, and b-cell survival are tightly regulated.

found to have a high affinity (Kd ¼ 0.5 nM) for its receptor, which activates adenylate cyclase with a GLP-1 EC50 ranging between 50 and 100 pM (57) (Figure 11.5). GLP-1 also induces the cAMP-regulated guanine nucleotide exchange factors of the Epac family (58). Interestingly, GLP-1 also elicits Ca2þ oscillations. Further, simultaneous measurements of intracellular Ca2þ concentration revealed that the two messengers cAMP and Ca2þ are interlinked and reinforce each other (58). Indeed, it has been shown that the Ca2þ influx alone can activate conventional protein kinase C (PKC) as well as novel PKC in insulin-secreting INS-1 cells (59). GLP-1 elicited the translocation of the endogenous PKCa and PKCe from the cytosol to the plasma membrane. Precisely, GLP-1 stimulated the translocation of myristoylated alanine-rich C kinase substrate (MARCKS)–GFP fusion from the plasma membrane to the cytosol in a Ca2þ -dependent manner, and the GLP-1-evoked translocation was blocked by PKC inhibitors, including a broad PKC inhibitor, bisindolylmaleimide I, a PKCe inhibitor peptide, antennapedia peptide-fused pseudosubstrate PKCe(149–164) (antp-PKCe) and a conventional PKC inhibitor, G€o-6976 (59). Upon binding to its receptor, GLP-1 activates all downstream molecular signaling pathways linked to cAMP production, through either cAMP-dependent protein kinase (PKA)dependent or -independent mechanisms (9), which are involved in the stimulation of gene expression and Ca2þ -dependent pathways such as PKCs. Such mechanisms involve the phosphorylation of the transcription factor cAMP responsible elementbinding protein (CREBP) and cAMP-regulated guanine nucleotide exchange factor

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(cAMP-GEFII), a cAMP sensor (60, 61). cAMP-GEFII binds to both Rim (Rab3interacting molecule; Rab3 being a small G protein) and its isoform, Rim2, both of which are putative regulators of fusion vesicles to the plasma membrane. cAMPGEFII, through its interaction with Rim2, mediates cAMP-induced, Ca2þ -dependent insulin secretion. This effect is not blocked by a PKA inhibitor, suggesting that it is going through a PKA-independent pathway. Accordingly, cAMP-GEFII is a direct target of cAMP-regulated exocytosis and is responsible for cAMP-dependent, PKAindependent exocytosis. GLP-1R signaling also increases proinsulin gene expression in a glucose-sensitive manner through a mechanism not clearly identified but independent of the cAMP pathway (61). These mechanisms have been recently described in insulin-secreting cell lines and might also be present in other cell types expressing the GLP-1R such as neuronal cells. However, only a few sets of data are available so far regarding the role of GLP-1 in the control of oxidative stress in the brain (62, 63) and hence its neuroprotection effect (14). In addition, two peptides from the Gila monster venom, Exendin-4 (4-39) and Exendin-9 (9-39) (64), displayed similar ligand-binding affinities to the human GLP1R (57). Whereas Exendin-4 acts as an agonist of the receptor inducing cAMP formation, Exendin-9 is an antagonist of the receptor, more precisely an inverse agonist, inhibiting GLP-1-induced cAMP production (65). Therefore, Exendin-4 is now used as a therapeutic agent for the treatment of type 2 diabetes. The GLP-1R, like many other cAMP-coupled receptors, can be desensitized. In in vitro assays using fibroblasts, the GLP-1R can be desensitized when incubated with GLP-1 over a few minutes (i.e., 5–10 min). The heterologous desensitization is mediated through phosphorylation by PKC of a 33-amino acid segment of the C-terminal cytoplasmic tail of the receptor (66, 67). Another proposed mechanism for the desensitization is that GLP-1 increases PKC activities leading in turn to its receptor inactivation (68). Excessive PKC activities have been described in response to glucose (69). Therefore, it is possible that hyperglycemia could reduce GLP-1 signaling and contribute to the diabetic phenotype.

PHYSIOLOGICAL ROLE OF GLP-1 Effects on Insulin Secretion The biological actions of GLP-1 in the b-cell include some main molecular mechanisms as follows. First, the secretion of insulin requires a mechanism involving cAMP production as described above and further detailed below. Second, GLP-1 stimulates insulin gene expression and insulin biosynthesis (70), presumably via increased expression by the b-cell specific transcription factor pancreatic and duodenal homeobox gene-1 (Pdx1) (61, 71, 72). Third, GLP-1 enhances the glucose competence of pancreatic b-cells. This involves a mechanism allowing the rapid equilibrium of glucose from either side of the plasma membrane. This is ensured by the glucose transporter GLUT2 and the glucokinase (73, 74). Eventually, GLP-1 stimulates b-cell mass expansion. GLP-1 acts as a growth factor for the b-cell in both experimental

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Figure 11.6

animal models and cultured b-cells by stimulating proliferation, survival, and differentiation. From a pathophysiological point of view regarding type 2 diabetes, GLP-1 addresses, at least as shown in animal models, both the defect in insulin secretion and the decline in b-cell mass that contribute to the deterioration of b-cell function in the etiology of type 2 diabetes. As depicted in Figure 11.6, GLP-1 acts at the molecular level through both PKA-dependent and -independent pathways as described above. Briefly, PKA-independent pathways are mainly involved in both GLP-1- and glucosestimulated insulin secretion and regulation of b-cell mass, whereas PKA-dependent pathways regulate GLP-1-induced expansion of b-cell mass, although indirect effects on insulin secretion could also be detected through PKC-dependent mechanisms (59, 68). Regulation of insulin secretion by GLP-1 is also mediated by a PKA-independent pathway. Increased intracellular cAMP concentration following activation of the GLP-1R leads to activation of cAMP-GEF, also named exchange protein activated by cAMP (EPAC) (75) (Figures 11.5 and 11.6). Both EPAC1 and 2 are expressed in pancreatic b-cells, but EPAC2 seems to play a major role in the regulation of GLP-1induced insulin secretion (75, 76). More precisely, EPAC2 has been demonstrated to be involved in distal steps of exocytosis most likely by controlling granule density near the plasma membrane (75). Through interaction with both Piccolo and Rim2, as well as the small GTPase Rap1, which is activated by cAMP specifically, EPAC2/ cAMP-GEFII may increase the docking of insulin-containing vesicles to the plasma

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membrane thus enhancing insulin granule dynamics (75). It is also noteworthy that sulfonylureas, a class of widely used antidiabetic drugs, interact directly with EPAC2 through a mechanism requiring Rap1 (76). Furthermore, EPAC2 is also an indirect activator of L-type voltage-dependent Ca2þ channels since it has been demonstrated to inhibit ATP-sensitive potassium channel activity in human pancreatic b-cells and rat INS-1 cells (77). It must be pointed out that pulsatility of cAMP production is also a key factor for an efficient GLP-1 response (78). This pulsatility is indirectly related to anchored kinase activator protein (AKAP18), which facilitates cAMP-responsive membrane events (79). Indeed, AKAP18 is responsible for the localization of PKA close to one of its substrates, phosphodiesterase 3B (PDE3B, Figure 11.5). Therefore, the activation of PKA by cAMP leads to the concomitant phosphorylation and activation of PDEs, which in turn hydrolyses cAMP. This loop of regulation can avoid the desensitization of the GLP-1R when cAMP is overproduced (66, 80, 81). However, silencing PDE3B had no effect on the GLP-1-potentiated insulin response in rat islets; in contrast, the depletion of PDE3B levels in rat islets increased insulin response to glucose by 70% (82). It was further increased by 23% when GLP-1 was added to glucose (82).

Effect on b-Cell Plasticity Studies showed that GLP-1 can promote b-cell replication in vitro (83, 84). This important information was also demonstrated in vivo in partially pancreatectomized rats, which become hyperglycemic in the fed state (85). The mechanisms proposed are numerous. They might be related to the growth promoting action of GLP-1. It has been shown that the transactivation of the EGF and IGF receptors was also required (11, 13). This was further associated with the subsequent activation of PI3K and its downstream effectors Akt, PKCz, and p38 MAPK (58, 83, 86–88). Furthermore, GLP-1 could mediate receptor signaling by inhibiting the forkhead transcription factor, FoxO1. This mechanism requires phosphorylation-dependent nuclear exclusion of FoxO1 in pancreatic b-cells (89). Furthermore, GLP-1 increases Pdx-1 and Foxa2 expression and stimulates Akt signaling as well. Interestingly, Exendin-4 increases FoxO1 nuclear translocation (90). These results indicate that FoxO1 mediates the effects of the incretin hormone on b-cell proliferation and survival. Eventually, another mechanism has been proposed through which Exendin-4 induces Wnt signaling in pancreatic b-cells, in both isolated islets and INS-1 cells under the basal and GLP-1-stimulated conditions (91). Both cyclin D1 and c-Myc, determinants of cell proliferation, are upregulated by Exendin-4 and are linked to Wnt signaling. The protein kinase Akt but neither PKA nor GSK3b is involved in this pathway (91). PKA-dependent pathway is also involved in the regulation of pancreatic b-cell mass (Figure 11.6). This could be related to enhanced b-cell proliferation as well as decreased b-cell apoptosis. Regarding proliferation, this effect is related to recruitment of CREB. Briefly, in the absence of GLP-1, there is low intracellular cAMP concentration and CREB is located within the cytosol in a complex involving transducer of regulated CREB activity 2 (TORC2) and 14-3-3

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protein (see Figure 11.6). The formation of this complex is dependent on the phosphorylation of the 14-3-3 protein by salt inducible kinase 2 (SIK2). In the presence of GLP-1, intracellular cAMP levels are increased leading to activation of PKA, which in turn phosphorylates and inactivates SIK2. The consequent dephosphorylation of the 14-3-3 protein results in its dissociation from the CREB/TORC2 complex. Finally, TORC2/CREB is targeted to the nucleus where CREB modulates the expression of genes involved in b-cell survival such as B-cell lymphoma2 (BCL2). PKA also directly phosphorylates CREB (on serine residue 133) thus also participating in its activation. In addition, it has been demonstrated that the GLP-1 effect can also partly involve the silencing of the negative regulators of the cAMP response element modulator-a and the dual specificity phosphatase DUSP14 (92). Studies demonstrate that the cAMP/PKA/CREB and the MAPK/ERK1/2 pathways can have additive effects in controlling b-cell proliferation (92). Another way to control b-cell mass is to regulate apoptosis. It has been recently demonstrated that GLP-1 agonists protect pancreatic b-cells (i.e., INS-1E or rat primary b-cells) from the lipotoxic endoplasmic reticulum (ER) stress pathway (93). GLP-1 activates the induction of the ER chaperone BiP and the antiapoptotic protein JunB that mediate b-cell survival under lipotoxic conditions. In addition, Exendin-4 and forskolin protected against synthetic ER stressors by inactivating caspase 12 and upregulating BCL-2 and XIAP, which altogether inhibit apoptosis (12).

Effect on Glucagon Secretion One of the major physiological actions of GLP-1 is to control plasma glucagon concentration. Glucagon is a small peptide hormone whose secretion is stimulated by hypoglycemia and inhibited by hyperglycemia. The control of glucagon secretion is multifactorial. First, although still controversial, it has been proposed that variations in plasma glucose concentrations can be directly detected by a-cells and regulate glucagon secretion (94–99). The corresponding mechanism is that pancreatic a-cells are electrically excitable and generate spontaneous Na þ - and Ca2þ -dependent action potentials (100, 101). Glucagon release is Ca2þ -dependent. It requires N-type Ca2þ channels at low glucose concentrations (102). Furthermore, evidence showed that a-cells are equipped with the same types of ATP-sensitive K þ channels that constitute the resting conductance in b-cells (103, 104). However, most data show that glucagon secretion is actually not reduced by glucose but rather increased in isolated a-cells (105). Therefore, other physiological mechanisms such as insulin are required to directly inhibit glucagon secretion (106). This implies that the inhibition of insulin secretion during hypoglycemia would consequently recover glucagon secretion since the concentration of insulin at the vicinity of the a-cells would be low (106). Another mechanism would be that in response to hyperglycemia, increased insulin secretion would directly inhibit glucagon production by activating insulin receptors at the surface of the a-cells (107, 108). Indeed, the intra-islet blood flow is such that secreted products from b-cells irrigate a-cells and hence could contribute to their regulation. This mechanism exposes the a-cells to high concentrations of insulin (109–111). This

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hypothesis also stipulates that more general b-cell released products could regulate glucagon secretion as well. Although this hypothesis is again still controversial (112). As a consequence, due to its insulinotropic effect, GLP-1 may contribute to the inhibitory effect of glucose on glucagon production. Eventually, it is important to consider that glucagon secretion is strongly regulated by the central nervous system and the autonomic output to a-cells (113). Pancreatic islets are richly innervated to enable autonomic regulation of endocrine cell hormone secretion. The most extensively studied are the sympathetic (adrenergic) and parasympathetic (cholinergic) nerves, which can project deeply into the islet, but other types of sensory neurons have also been detected, including GABAergic nerve bodies (114). Therefore, it is reasonable to consider that GLP-1 controls glucagon secretion through a multifactorial mechanism involving a putative effect on b-cells and via the central and autonomic nervous systems. Importantly, it is mostly accepted that there is almost no evidence of GLP-1R expression in a-cells. Therefore, it is unlikely that GLP-1 acts directly on a-cells to control glucagon secretion (115). Despite the wide acceptance, this concept is still a matter of debate and needs to be validated, because the expression of the GLP-1R has been detected by immunocytochemistry in a small subpopulation (20%) of glucagon-positive cells in dispersed rat islets (100, 101). Whether this has a relevant role remains to be determined since some biochemical evidence showed that GLP-1 even caused an increase in the rate of exocytosis in single rat a-cells (116, 117). Recently, it was shown that acute GLP-1R activation by Exendin-4 or DPP-4 inhibitor sitagliptin enhanced insulin action and suppressed hepatic glucose production in mice, as assessed by hyperinsulinemic-euglycemic clamp (118). No modification of whole-body glucose utilization was observed, suggesting that the effect on hepatic glucose production could be an indirect effect mediated by reduced plasma glucagon concentration. In vivo experiments using isolated pancreas showed that at 9 mM glucose, Exendin-4 reduced the glucagon response to arginine stimulation (119). This effect is thought to be paracrine-mediated through the concomitant increase in insulin and somatostatin concentrations (119). However, at low glucose concentrations, Exendin-4 did not affect insulin secretion but reduced glucagon release (119). The glucagonostatic effect of Exendin-4 was observed under conditions in which insulin and somatostatin were not affected, indicating that Exendin-4, per se, inhibits glucagon secretion by a-cells. It is noteworthy that the pancreas contains its own nervous system, which could have been responsible for the control of glucagon secretion (120, 121). Hence, the glucagonostatic effects of GLP-1 could be explained by the recruitment of the pancreatic autonomic nervous system. In humans, a series of clinical trials showed that GLP-1-based therapies control plasma glucagon concentrations. Exendin-4 has been commercialized as exenatide (Byetta). In clinical trials, exenatide lowers blood glucose through multiple mechanisms, including enhancement of glucose-dependent insulin secretion, reduction of food intake, and slowing of gastric emptying. In addition, a long-acting form of exenatide, which maintains activity for 1 week following a single injection, suppresses excessive glucagon secretion (122). Importantly, the glucagon lowering effect

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of exenatide was essentially observed in the postprandial state in type 2 diabetic patients. The other GLP-1-based therapeutic strategy that consists of inhibiting the GLP-1 degrading enzyme DPP-4, provides similar results on plasma glucagon concentration (123). The inhibition of glucagon secretion is certainly a major target for GLP-1-based therapies. This helps reducing fasted and absorptive hyperglycemia. However, it is also expected that these therapies could also increase glucagon secretion in hypoglycemic conditions. Indeed, the restoration of hypoglycemiainduced glucagon secretion is also a glucose-sensitive mechanism, which could be regulated by GLP-1. In response to hypoglycemia, DPP-4 inhibitors enhance hypoglycemia-induced glucagon secretion (124). In drug-naive patients with type 2 diabetes, the mean change in glucagon during hypoglycemic clamp was increased in patients receiving the DPP-4 inhibitor following a 4 week treatment. Conversely, following a meal test the glucagon secretion was reduced by 40% in the treated group (124). These data demonstrate that GLP-1-based therapies enhance the control of a-cell response to both hyperglycemia and hypoglycemia with respect to glucagon secretion.

Effect on Gastric Emptying The inhibition of gastric motility and hence emptying is among the first observed physiological actions of GLP-1 (125–127). The gut is indeed an important target for incretins since GIP and GLP-1 control acidic secretions by the stomach (128). However, solely GLP-1 enteric action can be blocked by vagotomy (128, 129). This was also demonstrated in rats where inhibition of gastric emptying could be mediated by brain delivery of GLP-1 (130). Furthermore, it has been proposed that the reduction of gastric emptying by GLP-1 is part of the hyperglycemia lowering effect of the peptide. This concept has been suggested in type 1 diabetic patients with no residual insulin secretion (131). Following a mixed meal, GLP-1 infusion reduced hyperglycemic episodes (131). However, hyperglycemic rebounds were observed when the GLP-1 infusion was stopped, which is consistent with the release of the meal stored in the stomach or the recovery of glucagon secretion. Hence, in type 2 diabetic patients, the reduced gastric emptying by GLP-1 could contribute to reduction in both hyperglycemia (34, 132) and postprandial hyperlipidemia (133). The cellular mechanisms behind the effect on gastric emptying are unknown but certainly linked to the neural effect of GLP-1. Therefore, one can hypothesize that other gut-released hormones such as cholecystokinin could be relaying on GLP-1 action.

ECTOPIC EFFECTS OF GLP-1 The proglucagon gene is also expressed in ectopic locations such as certain taste cells in the tongue, and some neurons in the brainstem, hypothalamus, and even in the intestinal tract (134–137). According to recent observation, the presence of GLP-1

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and GLP-1R in taste buds supports the original hypothesis. GLP-1 immunoreactivity was observed in spindle-shaped taste bud cells, with positive cells displaying a characteristic distribution that was confined to the cytosol (135). Furthermore, reverse transcription polymerase chain reaction assay showed that GLP-1 mRNA was expressed in circumvallate papillae (135). GLP-1 is produced in two distinct subsets of mammalian taste cells, while the GLP-1R is expressed on adjacent intragemmal afferent nerve fibers and likely to mediate taste response (136). GLP-1R knockout mice showed dramatically reduced taste responses to sweeteners in behavioral assays, indicating that GLP-1 signaling normally acts to maintain or enhance sweet taste sensitivity (136). In addition, a modest increase in citric acid taste sensitivity in these knockout mice suggests that GLP-1 signaling may modulate sour taste as well. Furthermore the GLP-1R knockout mice also exhibited an enhanced sensitivity to umami-tasting stimuli (134). In addition to the ectopic location of the GLP-1R in taste buds the receptor is also expressed in some enteroendocrine cells. Human duodenal L cells express sweet taste receptors (137, 138), the taste G protein gustducin, and several other taste transduction elements (139, 140). Double-labeling immunofluorescence and staining of serial sections of the small intestine demonstrated that a-gustducin is localized to enteroendocrine L cells that express peptide YY (PYY) and GLP-1 in the human colonic mucosa (139). Mouse intestinal L cells also express a-gustducin (141). Ingestion of glucose by a-gustducin null mice revealed deficiencies in secretion of GLP-1 and the regulation of plasma insulin and glucose (140). Isolated small bowel and intestinal villi from a-gustducin null mice showed markedly defective GLP-1 secretion in response to glucose (140). GLP-1 release from NCI-H716 cells was promoted by sugars and the noncaloric sweetener sucralose, and blocked by the sweet receptor antagonist lactisole or siRNA for a-gustducin. Therefore, it was concluded that L cells of the gut “taste” glucose through the same mechanisms used by taste cells of the tongue (141).

CENTRAL EFFECTS OF GLP-1 GLP-1, mostly the (7-36)-amide form, is produced in the brain where neurons from the brain stem projects to the hypothalamus (14, 142–146). The proglucagon mRNAexpressing entities were detected in the nucleus of the solitary tact and the dorsal and ventral medulla and olfactory bulb (147). GLP-1R has been found in many discrete areas of the brain including the hypocampus (14), the brainstem, and nuclei of the hypothalamus (148, 149). However, it could be expressed as well in numerous subtle regions such as the mitral cell layer of the olfactory bulb; temporal cortex; caudal hippocampus; lateral septum; amygdala; nucleus accumbens; ventral pallium; nucleus basalis Meynert; bed nucleus of the stria terminalis; preoptic area; paraventricular, supraoptic, arcuate, and dorsomedial nuclei of the hypothalamus; lateral habenula; zona incerta; substantia innominata; posterior thalamic nuclei; ventral tegmental area; dorsal tegmental, posterodorsal tegmental, and interpeduncular nuclei; substantia nigra, central gray; raphe nuclei; parabrachial nuclei; locus

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coeruleus, nucleus of the solitary tract; area postrema; dorsal nucleus of the vagus; lateral reticular nucleus; and spinal cord. The efferent connections from the nucleus of the solitary tract are widespread. When [125I]-labeled GLP-1 is injected intravenously into rats, it can be detected within 5 min in the postrema and subfornical area (150). These are regions of the circumventricular organs where circulating macromolecules are known to be able to pass across the blood–brain barrier. Over the past two decades, numerous studies have suggested a role for GLP-1 not only in insulin secretion but also in the control of food intake and body weight (151–155). Infusion of GLP-1 directly into the paraventricular nucleus of the rat suppresses feeding (156), suggesting that the receptor is indeed expressed in this area of the brain (157, 158). Its subtle anorexic role might be related to the regulation of leptin’s effect (154, 159). The inhibition of food intake by leptin could depend on the initial secretion of GLP-1. Hence, leptin could be anorectic following a meal where GLP-1 has previously increased the cAMP tone. It is noteworthy that GLP-1 can enter the brain from the blood (160), however, the central role of circulating GLP-1 on the control of brain function is most likely null since most of the gut-released GLP-1 is degraded by the DPP-4 within minutes (125). Therefore, a role of GLP-1 released directly by the brain is strongly implicated (161). Another possible mechanism would be through the expression of the GLP-1 receptor in the vagus nerve (9, 162) or the nodose ganglions (163). Indeed, a direct intraportal infusion of GLP-1 could inhibit food intake (164) and vagotomy prevented such effect (153). Furthermore, peripherally administered GLP-1R antagonist Exendin-9 prevented the central but not the peripheral inhibitory effect of GLP-1 on food intake (154). Hence, numerous GLP-1 dependent centers are most likely involved in the control of food intake. This high level of redundancy suggests the importance of the mechanism. The use of the commercial form of Exendin-4 has been associated with adverse effects such as nausea and vomiting. This could be due to excessive taste aversion or inhibition of gastric emptying (130) and food intake (165, 166). The central role of GLP-1 has now gained a wide range of effects including neuroprotection from neurotoxins and cognitive functions such as learning (14). Recent data showed that the murine GLP-1R plays an important role in the control of synaptic plasticity and in some forms of memory formation (18). Such effect could be involved in the development of Alzheimer’s disease. A recent study using positron emission tomography (PET) and 2-[F18]-deoxy-D-glucose (FDG) in young adult men and women showed that i.v. administration of GLP-1 affects glucose metabolism in the hypothalamus and brainstem, indicating that peripheral GLP-1 can access the CNS and modulate neuronal activity in humans (148). This study reinforces the taste aversion, anorectic, and nausea effect of exogenously administered GLP-1R agonists. Interestingly, the peak postprandial increase in plasma GLP-1 concentration is correlated with an increased regional cerebral blood flow in the left dorsolateral prefrontal cortex (167). The importance of this region has been previously implicated in PET studies of human satiation and the hypothalamus (167). Therefore, in physiological situation, and conversely to pharmacological states where large amount of GLP-1 are administered to dramatically increase the plasma GLP-1 concentration, almost no substantial amount of GLP-1 released by the gut following a meal can reach

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brain nuclei. This conclusion strongly supports the role of brain-released GLP-1 in the control of the above central effects. Altogether, this gut peptide could certainly be considered as a neurohormone and a brain hormone. So far no evidence suggests that brain GLP-1 could reach the systemic circulation and trigger metabolic effects. However, it is still suggested that terminal ends from the brain could project toward peripheral tissues and release this newly considered neurohormone at the vicinity of GLP-1R-expressing cells such as the b-cells. Numerous other centrally regulated effects of GLP-1 have been described such as on insulin secretion, vascular blood flow, hepatic glucose production, heart function, blood pressure, and peripheral lipid metabolism (8, 161, 168). This is included in the so-called gut-to-brain-to-periphery axis detailed below.

THE GLP-1 DEPENDENT GUT-TO-BRAIN-TO-PERIPHERY AXIS The gut-to-brain-to-periphery axis is defined as the mechanism through which nutrients, when absorbed by the intestine, generate a signal, which informs the peripheral tissues that energy is taken up (Figure 11.1) (8, 168). This involves a major role of the brain, which centralizes the information generated by the peripheral tissues. Before a meal, the body is considered in a fasting mode. The gut continuously informs the brain that no nutrients are available. In addition, endocrine, neural, and metabolic signals are being sent by different parts of the body also informing the brain that energy is being low. From all information the brain is, hence, directing these signals to generate new ones capable of using control, the energetic flux toward tissues for use and proper storage of energy. Following a meal the body switches from a catabolic to an anabolic state. The initiation of this switch is the role of the enteric nervous system. Several decades ago researchers reported that cellular units are activated by ascending impulses from the liver within the nucleus of the solitary tract (NTS) (169). The ascending branch from the vagus nerve was hence identified (170). This suggested that in the enteric area, which includes intestinal luminal cells and the mesenteric and hepatoportal veins, glucose is detected by specialized cells, which work as enteric glucose sensors. The latter transmits signals of endocrine and neuronal origin to peripheral tissues (171–175). Many of the neuronal signals are communicated via the vagus nerve to the brainstem (169, 176), which relays the glucose signal to hypothalamic nuclei, then to pertinent target cells. Recently, we demonstrated that the enteric glucose sensor system sends signals to peripheral tissues via a brain relay (10). In response to a low-rate intragastric glucose infusion, we quantified the c-Fos expression pattern in the brainstem and in the hypothalamus. We showed that the nucleus of the solitary tract was activated by the enteric glucose sensor. Interestingly, cells from the arcuate nucleus of the hypothalamus were switched to rest, suggesting that they might have been involved in maintaining the basal activity related to the fasting state. We could quantify that NPY-expressing cells were inhibited by the activation of the enteric glucose sensor suggesting that the basal tone was insured by NPY. Conversely, no colocalization with proopiomelanocortin-expressing cells could be detected. Importantly, the brain

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regulation was totally blunted in the GLP-1R knockout mice suggesting a role of the hormone. Hence, the endogenously secreted GLP-1 acts in the hepatic portal vein to increase the firing rate of the vagus nerve (177, 178) and regulate glucose metabolism (9, 179, 180). The role of the portal vein GLP-1 dependent glucose sensor on the control of glucose metabolism has been studied. We and others showed that enteric GLP-1 could control peripheral glucose metabolism (9, 162, 180, 181). However, this effect was strictly depending on the presence of a hepatoportal to periphery glucose gradient. Indeed, a physiological augmentation of GLP-1 in the hepatic portal vein does not increase whole-body glucose uptake when hyperglycemia is induced by peripheral glucose infusion (9). This indicates that a physiological increase in GLP-1 augments glucose utilization only when GLP-1 and glucose gradient conditions mimic the postprandial state (9, 182). Furthermore, the hepatic portal vein infusion of GLP-1 reduced the size of the ongoing meal compared with vehicle without affecting the subsequent intermeal interval, the size of subsequent meals, or cumulative food intake (164). The satiating effect of GLP-1 requires vagal afferent signaling, which might not originate from hepatic portal or hepatic GLP-1R but may act directly through the release of GLP-1 in the brain. The role of GLP-1 in the gut-to-brain axis involves two levels of regulation. First, in the portal vein the infusion of the GLP-1R antagonist blocked the glucose detection by the enteric sensor (9), which is activated by GLP-1. Second, GLP-1 is also secreted by neuronal cells from caudal region of the brainstem, the nucleus of the tractus solitarius (NTS) (183, 184). The specific role of brain GLP-1 signaling in the control of peripheral function and in the relay of the gut glucose signal has been addressed by infusing the GLP-1R antagonist directly into the brain lateral ventricle while glucose was infused into the gut or the systemic blood. In such a condition, we could show that in the presence of hyperglycemia insulin secretion was dramatically increased (185). In response to a gut infusion, brain GLP-1 signaling was necessary for insulin secretion since the blockade of the brain GLP-1R by Exendin-9 prevented gut glucose-induced insulin secretion (185). The signaling of GLP-1 in the brain controlled hepatic glucose utilization and storage (185, 186). This function is directly related to the physiological role of GLP-1 as a hormone secreted during the absorptive period. It is reasonable to hypothesize that GLP-1 triggers hepatic glucose storage, sparing glucose for further delivery during fasting. Indeed, we also showed that brain GLP-1 signaling sends a negative signal to the muscle to prevent their excessive utilization of glucose. This physiological mechanism might prevent an overt utilization of glucose by muscles during feeding to spare glucose for the liver. By this mean the large muscle mass might not predominate in front of the liver weight. The mechanisms through which brain GLP-1 controls peripheral glucose metabolism could be related to a change in arterial blood flow. Indeed, the autonomic nervous system largely innervates the arterial wall. We and others showed that the control of the arterial blood flow was tightly correlated with muscle glucose utilization (63, 187–191). Hence, a continuous 3 h brain infusion of a GLP-1R agonist decreased femoral arterial blood flow and whole-body glucose utilization in the conscious free-moving mouse clamped in a hyperinsulinemichyperglycemic condition. Both blood flow and glucose utilization were tightly

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correlated (62), which demonstrated the strict glucose dependency of brain GLP-1 action on glucose metabolism. Furthermore, hypothalamic nitric oxide synthase activity and the concentration of reactive oxygen species were also reduced in a GLP-1R-dependent manner, whereas the glutathione antioxidant capacity was increased (62). Central GLP-1 activated vagus nerve activity, and complementation with reactive oxygen species donor dose-dependently reversed the effect of brain GLP-1 signaling on peripheral blood flow (62).

CARDIOVASCULAR EFFECTS Over the past few years numerous data have been published showing a regulatory role of GLP-1 on the control of heart and vascular functions. However, such data are so far a matter of discrepancy and need to be clarified. This complexity is probably linked to the dual role of GLP-1 in the control of cardiovascular functions. Brain GLP-1 and peripheral GLP-1 signaling control directly heart and blood vessel functions. However, they do not always provide similar effects according to the studies considered. As an example, in most of the studies in rodents, GLP-1 increased blood pressure whereas in humans the hormone rather reduced blood pressure. So far this discrepancy is yet unexplained but could be related to the different pharmacology of the blood vessel adrenergic receptors. We will review some of these data below. In a first set of data, Yamamoto et al. showed that centrally and peripherally administered GLP-1R agonists dose-dependently increased blood pressure and heart rate in rats (184). This could be due to the fact that GLP-1R activation, as witnessed by the induction of c-fos expression, in the adrenal medulla and neurons in autonomic control sites in the rat brain, including medullary catecholamine neurons, provides input to sympathetic preganglionic neurons. Furthermore, GLP-1R agonists rapidly activated tyrosine hydroxylase transcription in brainstem catecholamine neurons. These findings suggest that the central GLP-1 system represents a regulator of sympathetic outflow leading to downstream activation of cardiovascular responses in vivo. Such vascular effects were similar to those observed by Barragan et al. (192, 193). The intracerebroventricular administration of GLP-1 produced an increased heart rate and blood pressure, which was blocked by previous administration of the GLP-1R antagonist Exendin-9 into the brain but not when it was intravenously injected. The regulatory role of GLP-1 on cardiovascular functions was also depending on peripheral signals since the intravenous administration of GLP-1 increased arterial blood pressure and heart rate. Importantly, these cardiovascular effects could be blocked by central administration of Exendin-9. This set of data demonstrates that brain GLP-1R signaling is central for the control of cardiovascular mechanisms (192, 193). This control seemed to depend on parasympathetic innervations since the bilateral vagotomy blocked the stimulating effect of intracerebroventricular GLP-1 (194). Indeed, the pretreatment with propranol, phentolamine, or reserpine did not prevent these effects, further indicating that they were not mediated by catecholamine receptors but rather through the parasympathetic nervous system (192, 193). There is most likely a role of GLP-1 which depends on the regional

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innervations since a recent study showed that GLP-1 increased muscle sympathetic nerve activity but did not affect cardiac sympathetic or parasympathetic indices, as assessed by spectral analysis (195). Specific adrenergic receptor agonist and antagonist have been used to depict the role of GLP-1 in the control of cardiovascular functions and innervations. First, yohimbine, an a2 adrenoreceptor antagonist, increased plasma catecholamines and the low-frequency component of heart rate power spectrum, suggesting increased cardiac sympathetic activity (195). In addition, the inhibition of nitric oxide (NO) synthase increased the blood pressure (BP) and reduced the heart rate but did not affect the balance between sympathetic and parasympathetic activity showing that many different regulatory pathways are involved in the control of heart function and blood pressure (195). Therefore, the role of GLP-1 is certainly a combination of action through these different pathways. In the rat, GLP-1 relaxed femoral artery rings in a dose–response manner. The relaxant effect from GLP-1 was completely inhibited by the specific GLP-1R antagonist, Exendin-9. Neither the specific nitric oxide synthase inhibitor, N-nitro-Larginine, nor removal of endothelium affected the GLP-1 relaxant effect (196). Therefore, GLP-1 has a direct vascular action relaxing conduit vessels in a way independent of NO and the endothelium. Hence, these first sets of data do demonstrate the discrepancies that exist regarding the systemic and the central roles of GLP-1 in the control of vascular functions. Our group also reported that brain GLP-1 signaling reduced the femoral arterial blood flow and increased the heart rate (62). Importantly, this effect was tightly correlated with the reduction of muscle glucose utilization (63) and was totally prevented in GLP-1R knockout mice or when Exendin-9 was infused into the brain. Interestingly, the original set of data showed that during high-fat diet-induced diabetes, the arterial blood flow was reduced. This feature could be overcome by the blockade of brain GLP-1R signaling in mice with genetic deletion of the coding genes or in mice infused into the brain ventricles with Exendin-9 (62, 63). This set of data suggests that the increased blood pressure, which characterizes most type 2 diabetic patients could be due to an excessive brain GLP-1 signaling. The mechanisms through which brain GLP-1 signaling affects cardiovascular function remain mostly unknown but could be related to the control of oxygen stress such as reactive oxygen species and endoplasmic reticulum stress (93). First, we showed that the production of reactive oxygen species could be dramatically reduced in the hypothalamus after a few hours of brain GLP-1 infusion. This effect was associated with the control of vascular blood flow and muscle glucose utilization (62). Second, it has been shown that GLP-1 treatment of diabetes, which is associated with the development of ER stress in b-cells, significantly reduced biochemical markers of islet ER stress in vivo (93). The GLP-1R agonist Exendin-4 improved the survival of purified rat b-cells with induced ER stress in vitro. It was suggested that these effects were associated with the induction of ATF-4 by ER stress and accelerated recovery from ER stress-mediated translational repression in b-cells in a PKA-dependent manner. The effect of brain GLP-1 seems to be affecting some vascular domains differently. Some of these effects were even opposing each other. The administration of GLP-1 reduced the mesenteric blood flow while vasorelaxed the hindquarter arteries (197). This could be related to the differences observed in the regional

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expression of cellular receptors, which have been demonstrated to have vasoregulatory functions. In addition, brain GLP-1 could also control the action of other hormones and regulates cardiovascular function. GLP-1 may play a role in the cardiovascular and other responses to vasopressin and oxytocin (198, 199). Furthermore, the intracerebroventricular injection of this peptide to rats with induced hypotensive hypovolemia results in elevation of their blood pressure (198). In conscious freely moving rats, the central injection of GLP-1 elevated the plasma concentration of vasopressin and corticosterone 15 and 30 min after administration. The animals given a central injection of GLP-1 developed transient hypoglycemia 20 min after the injection, which was fully restored to normal levels at 30 min. This was supposed to be associated with a change in the blood volume. These vascular effects were probably involving the magnocellular neurons of the paraventricular nuclei (PVN) and supraoptic nuclei (SON) and the parvicellular neurons of the medial parvicellular subregion of the PVN. This was concluded by studying c-fos immunocytochemistry as an index of stimulated neuronal activity. When the GLP-1R antagonist Exendin-9 was given before the GLP-1, c-fos expression in these neuroendocrine areas was almost completely abolished, suggesting that the effect of GLP-1 on c-fos expression is mediated via specific receptors. Therefore, GLP-1 triggers the activity of numerous hypothalamic neuroendocrine areas. Approximately 80% of the CRH-positive neurons in the hypophysiotropic medial parvicellular part of the PVN coexpressed c-fos after the i.c.v. administration of GLP-1. Hence, central administration of the neuropeptide GLP-1 activates the central CRH-containing neurons of the hypothalamo-pituitary-adrenocortical axis as well as oxytocinergic neurons of the hypothalamo-neurohypophysial tract. The authors concluded that GLP-1 activates the hypothalamo-pituitary-adrenocortical axis primarily through stimulation of CRH neurons. Administration via the same route was also shown to stimulate magnocellular hypothalamic neurons. This suggests that GLP-1 acts directly on these oxytocinergic nuclei, while indirectly stimulating release of vasopressin from the hypothalamus. The resultant of this neurohormonal balance is that GLP-1 indirectly regulates blood pressure. The consequences of this regulatory pathway could be numerous and are largely intuitively linked to the control of cardiovascular function. It is noteworthy that altogether the regulatory role of brain GLP-1 on the cardiovascular effect has to be interpreted within the frame of its physiological function. This hormone is produced during food intake and is hence considered as a hormone or a neuromediator of the absorptive state. Therefore, it should be involved in physiological mechanisms ensuring the control of glucose homeostasis during food intake. To that respect, the vasoconstrictor effect of brain GLP-1 could be considered necessary for a redistribution of the vascular blood beds in the whole body. This change in blood beds and in blood flow allows the distribution of glucose toward key organs for storage such as the liver. Indeed, following food intake, the liver should be the main organ responsible for glycogen storage allowing subsequent production of glucose the next morning during the fasting state. Hence, it is important to direct the flow of glucose toward the liver by reducing muscle blood flow and hence peripheral

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muscle glucose utilization. Certainly, reducing the femoral arterial blood flow by brain GLP-1 could be considered as a key physiological function indirectly controlling hepatic glucose metabolism. This could explain the surprising vasoconstrictive effect of GLP-1, which could be, intuitively, considered as negative within the frame of its therapeutic activity. On the basis of the above discussion regarding its cardiovascular regulatory action, there is some concern about the use of GLP-1 as an antidiabetic treatment drug in humans. However, the effects of long-term treatment of patients with GLP-1 differ from those on experimental animals. In humans, recent sets of data showed a beneficial role of GLP-1 in heart and vascular functions. Recently, 20 patients with coronary heart disease and preserved left ventricular function were treated with GLP-1 by continuous infusion beginning 12 h before and continuing after coronary artery bypass grafting for 48 h. Following surgery, the control group required greater use of inotropic and vasoactive infusions during the 48 h after the operation to achieve the same hemodynamic result. There were also more frequent arrhythmias requiring antiarrhythmic agents in the control group showing that GLP-1 improves heart function (200). The assessment of left ventricular ejection fraction in patients with chronic heart failure showed that GLP-1 treatment partly preserved from the heart failure (200). In patients, the systolic and diastolic blood pressure were reduced following 82 weeks of treatment with exenatide (201). Furthermore, it was also reported that 14 weeks of treatment with a longacting GLP-1 analogue improved glycemic control while decreasing systolic blood pressure (202). However, in many other studies GLP-1-based therapeutic strategies had no effect on blood pressure and cardiac functions. Again, it is still a matter of debate to determine whether its main control of the heart rate is directly mediated by the receptors located at the surface of the heart cells or whether brain GLP-1 signaling is the most important regulator. A direct effect of GLP-1 is to increase myocardial glucose uptake in dogs and to improve left ventricular performance. This effect was even observed in dogs with pacing-induced dilated cardiomyopathy (203). However, the authors proposed an original hypothesis. They suggested that since GLP-1-(7-36) is rapidly degraded in the plasma to GLP-1-(9-36) by DPP-4, an issue was that GLP-1(9-37), which originates from GLP-1-(7-37) degradation could still be the active moiety. Hence, the authors infused dogs with pacing-induced DCM. Although, the animals were characterized by a left ventricular myocardial insulin resistance under basal and insulin-stimulated conditions, the continuous intravenous infusion of GLP-1-(7-36) or GLP-1-(9-36) increased myocardial glucose uptake but without a significant increase in plasma insulin. In the sick dogs, GLP-1-(9-36) mimics the effects of GLP-1-(7-36) in stimulating myocardial glucose uptake and improving LV and systemic hemodynamics through insulinomimetic as opposed to insulinotropic effects (203). Other recent evidence showed that both GLP-1R-dependent and -independent pathways are required for the regulation of the cardiac function by GLP-1 (204). GLP-1 administration increased the glucose uptake, cAMP and cGMP release in the endothelium of cardiac and vascular myocytes. Furthermore, left ventricular pressure developed, and coronary flow in isolated mouse hearts was regulated by the peptide showing that these cardiac and vascular cells do express a functional GLP-1R (204).

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In addition to the regulatory role of GLP-1 in the cardiac function, GLP-1 has been shown to protect the heart from ischemia (15–17). Indeed, the peptide activates antiapoptotic-signaling pathways such as phosphoinositide 3-kinase and mitogenactivated protein kinase in pancreatic and insulinoma cells (93). Therefore, in both isolated perfused rat heart and in whole animal models of ischemia/reperfusion, the infarct size measured as the end point of injury was reduced by GLP-1 pretreatment. Again, this protection was abolished by the GLP-1R antagonist Exendin-9, the cAMP inhibitor Rp-cAMP, a PI3kinase inhibitor, and a p42/44 mitogen-activated protein kinase inhibitor. Western blot analysis demonstrated the involvement of numerous proapoptotic peptides and kinases currently involved in surviving. The protective effect of GLP-1 was inhibited by blocking the p70s6 kinase (17). The increased functional recovery and cardiomyocyte viability after ischemia reperfusion injury of isolated hearts and dilated preconstricted arteries from wild-type mice has been confirmed in a recent study (204). It was also observed in response to a 7 day treatment with GLP-1R agonist Liraglutide (205). The cardioprotective effect was associated with the regulation of the activity of Akt, GSK3b, PPARb/d, Nrf-2, and HO-1. The cardioprotective effects of Liraglutide remained detectable 4 days after cessation of therapy and may be partly direct, because Liraglutide increased cAMP formation and reduced the extent of caspase-3 activation in cardiomyocytes in a GLP-1R-dependent manner in vitro. In addition to the receptor dependent role of GLP-1, a striking observation was that unexpectedly, many of these actions of GLP-1 were also preserved in GLP-1R knockout mice. Furthermore, GLP-1-(9-36) administration during reperfusion reduced ischemic damage after ischemia-reperfusion and increased cGMP release, vasodilatation, and coronary flow in wild-type and the mutant mice. Studies using a DPP-4-resistant GLP-1R agonist, inhibitors of DPP-4, and nitric oxide synthase showed that the effects of GLP-1-(7-36) were partly mediated by GLP-1-(9-36) through a nitric oxide synthase-requiring mechanism that is independent of the known GLP-1R. The receptor to the GLP-1-(9-37) has yet not been identified.

EFFECTS ON DYSLIPIDEMIA Recent data from the literature suggest a role of GLP-1-based therapies in dyslipidemia (133, 206). First, the chronic treatment with Exenatide completely abolished the postprandial increase in triglyceride levels following a meal test in healthy patients. During GLP-1 infusion, plasma concentrations of nonesterified fatty acids (NEFAs) were suppressed by 39% in the fasting state and by 31 5% after meal ingestion (133). Second, 4 weeks of treatment of type 2 diabetes patients with vildagliptin, a DDP-4 inhibitor, decreased the total triglyceride by 85% and chylomicron triglyceride by 91%. There was a decrease in chylomicron apolipoprotein B-48 and chylomicron cholesterol. Third, GLP-1 infusion caused a dramatic and prompt decrease in lymph flow in response to a lipid infusion. In addition, GLP-1 also inhibited intestinal triolein absorption and lymphatic apoB and apoA-IV output. In conclusion, GLP-1 dramatically decreases intestinal lymph flow and reduces

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triglyceride absorption, and apoB and apoA-IV production. These findings suggest a novel role for GLP-1 in lipid absorption (207). So far no mechanistic data are available regarding DPP-4 inhibitors. Hence, this issue could be filed up with appropriate experiments. The impact of dyslipidemia on diabetes has been tremendously reported. Diabetic dyslipidemia contributes to the high morbidity and mortality in patients with type 2 diabetes. Therefore, it is of major importance to validate the therapeutic effect of DPP-4 inhibitors on lipid metabolism, and the mechanisms underlying these effects remain to be determined.

SECRETION The two peptides GLP-1 and GLP-2 are produced in equal concentrations in the enteroendocrine L cells. The L cells are scattered among the enterocytes throughout the small bowel and ascending colon. Hence, a gradient of L cells is along the gut and is opposite to the gradient of glucose into the intestine (125). This observation points out the role of L cells in the colon where mostly no glucose is absorbed. GLP-1 secretagogues are numerous (Figure 11.7). The peptides are secreted by nutrients and lipids and carbohydrates (208–212). Analogous to the insulin-secreting b-cells, glucokinase and the glucose transporter GLUT2 have been found in the mouse intestinal L cells (213). While the metabolism of carbohydrate might be important for the secretion of GLP-1, some evidence suggests that it is not considered essential. Luminal infusion of an isolated intestine with different carbohydrates, including glucose, fructose, and galactose, induce GLP-1 secretion (212). The mechanism could involve increased production of ATP from the metabolism of the carbohydrates by the endocrine cells. This hypothesis was confirmed by showing that inhibitors of glucose

Lipids Lipids

Non glucose Nonglucose Carbohydrate carbohydrates s

Cholecystoki Cholecystokinin nin

(Neuro)Hormon (Neuro) Hormone i.e. GIP) GIP (ei.e.,

Enteric L-cell

GLP-1/2 secretion Glucose

Neuromediators

Figure 11.7 Different factors involved in the control of GLP-1/2 secretion.

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metabolism such as 2-deoxyglucose and glucosamine inhibited GLP-1 secretion. In humans, circulating levels of the two hormones GIP and GLP-1 were measured over a 24 h period during which subjects consumed a mixed meal of carbohydrate, lipids, and proteins (214). The GLP-1 levels reached a maximum 30 min after the carbohydrate and 150 min after the fat load. However, ingestion of both carbohydrate and fat at the same time induced substantial rises in GLP-1 secretion, but the protein meal had no effect. Therefore, it remains difficult to precisely determine the fold increase of GLP-1 secretion. Altogether, the total plasma GLP-1 concentration increased within the range of 4- to 20-fold following a meal. This datum is approximate since it depends on a large set of conditions. First, it could be under the influence of the physiological conditions where the half-life of the peptides (following the stimulus considered such as meal or glucose) could vary. It was suggested that the mechanisms responsible for GLP-1 inactivation, such as DPP-4 mediated degradation and renal and hepatic clearance (215), might affect the plasma concentration of the peptides. The method for GLP-1 evaluation is also a factor. Several sets of antibodies that recognize either the N- or the C-terminal end of the peptide are available. However, the C-terminus of the peptide is amidated. So far, the percentage of amidation is not clearly known. In humans, it seems that most of the GLP-1 is amidated. Furthermore, this could vary according to the physiological situations. A preferred method would be to use a side-viewing antibody. However, this last peptide might not be absolutely specific for GLP-1 and could recognize glucagon and related molecules. Lipids provide a rather strong stimulus for GLP-1 and GIP. This could be linked to the expression of several FFA-binding receptors Gpr40 (216), GPR119 (217), and GPR120 (218), which are all GPCRs. Such receptors can bind different lipids such as the oleoylethanolamine involved in GLP-1 release (217, 218). Furthermore, hormones such as cholecystokinin (CCK), the GIP itself (219–225), miscellaneous neurohormones and neuromediators also play a role in GLP-1 secretion (226, 227). In addition, it is likely that both GIPR and GLP-1R are required for the full regulation of oral glucose-induced GLP-1 and GIP secretion (213). A recent original observation showed that the regulation of glucose-induced GLP-1 secretion was evidenced in taste buds from the tongue. The authors showed that taste of sucrose or its analogue, sucralose, could induce signaling through a mechanism similar to that described for the L cells (136, 228). To overcome the challenge of lack of primary L cells, progress has been made in studying incretin secretion by using an established L cell line designated GLUTag, which expresses the proglucagon gene and secretes immunoreactive GLP-1 in vitro (229). The enteroendocrine L cells could be purified and studied (230). The authors showed that single L cells are electrically excitable and glucose responsive. Sensitivity to tolbutamide and low-millimolar concentrations of glucose and a-methyl glucopyranoside, assessed in single L cells and by hormone secretion from primary cultures, suggested that GLP-1 release is regulated by the activity of sodium glucose cotransporter 1 and ATP-sensitive Kþ channels, consistent with their high expression levels in purified L cells by quantitative RT-PCR. In addition to nutrients and hormones, GLP-1 secretion and action are also dependent upon the integrity of the autonomic nervous system and essentially upon the signal

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sent by the vagus nerve (128, 129, 178, 231–234). Along the same line of evidence, a recent original observation was that sweet taste receptor subunit T1R3 and the taste G protein gustducin, expressed in enteroendocrine cells, underlie intestinal sugar sensing and regulation of SGLT1 mRNA and protein (140). TRPM5 and the T1R3 receptors seem to be also involved and are preferentially expressed in the proximal small intestine in humans, with the immunolabeling for G a-gustducin localized to solitary cells dispersed throughout the duodenal villous epithelium (235). The role of sugar receptors was implicated by the observation that dietary sugar and artificial sweeteners increased SGLT1 mRNA and protein expression, and glucose absorptive capacity in wild-type mice, but not in knockout mice lacking T1R3 or a-gustducin (140). Interestingly, mice deficient in the G protein gustducin were characterized by an impaired glucose-induced GLP-1 secretion (138). The rapid first phase release of both GLP-1 and insulin was totally blunted (138). The enteric L cells coexpress T1R3 and the G protein gustducin and hence, it was suggested that GLP-1 could be secreted by the same taste receptor mechanism on the tongue. This conclusion was challenged by the recent data from the purified L cells expressing green fluorescent protein, where almost no G protein gustducin, T1R3 and GLUT2 were observed (230). The reasons of this discrepancy are unknown and therefore, the precise molecular mechanism regulating gut GLP-1 secretion remains to be determined. Altogether, these data demonstrate that GLP-1 secretion certainly requires a complicated neuroendocrine loop involving mainly nutrient detectors, neural signals, the brain, and the autonomic nervous system (Figure 11.1). Furthermore, it is reasonable to suggest that GLP-1 action would require similar neuronal circuits.

CLINICAL STUDIES The potential of incretin-based therapies in diabetes was first demonstrated by encouraging results from preliminary clinical studies where GLP-1 was administered through either intravenous or subcutaneous route (236, 237). However, GLP-1 itself could not represent a suitable therapeutic approach because of its very short half-life due to a rapid degradation by the enzyme DPP-4. Thus, to circumvent this specific difficulty, two different strategies have been proposed to develop incretin-based antidiabetic drugs (238). .

.

GLP-1 mimetics or analogues are molecules that differ from the native GLP-1 in order to evade the DPP-4 inactivation, but conserve GLP-1R agonist activities. The molecular characteristics of these small peptides require subcutaneous administration for the treatment of diabetic patients. Inhibitors of DPP-4 extend the half-life of both endogenous GLP-1 and GIP and therefore enhance their metabolic action. In contrast to GLP-1R agonists, these drugs can be administered orally. An important characteristic is that the endogenous GLP-1 is released into the portal vein—its physiological site of secretion. Therefore, the mechanisms involved for the control of glycemia are certainly different.

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We provide thereafter a synthetic summary of the main clinical findings regarding the metabolic effects and the safety of the GLP-1R agonists and DPP-4 inhibitors, which are already, or will be soon, available for type 2 diabetes management.

GLP-1 Mimetics and Analogues Exenatide Exenatide was the first GLP-1 mimetic approved in 2005 by the US Food and Drug Administration for clinical use in type 2 diabetic patients. Exenatide is the synthetic version of Exendin-4, a natural peptide of 39 amino acids initially identified as a salivary product of the Gila monster, a lizard living in the Arizona desert (64, 239). Exendin-4 is coded by a gene distinct from that of GLP-1, but shows a 53% sequence homology with human GLP-1. The binding affinity of synthetic exenatide for GLP-1R is similar to that of native GLP-1, and this molecule is resistant to DPP-4 inactivation. Consequently, exenatide has a circulating half-life of 60–90 min, with increases in plasma exenatide concentrations lasting 4–6 h after subcutaneous administration (240). Due to these pharmacokinetic characteristics, exenatide is classically administered by subcutaneous injection twice daily. Phase III trials investigated the efficacy of adding exenatide to ongoing therapy in patients with insufficient glycemic control despite various oral antidiabetic regimens (metformin alone, sulphonylureas alone, a combination of both, or thiazolidinediones alone), with the usual starting dose of 5 mg twice daily for 4 weeks, followed by an increase to 10 mg twice daily (241–244). In these randomized studies, exenatide reduced HbA1c levels by 0.8–1.0% over 30 weeks. Furthermore, a significant weight loss of 1.5–3 kg was observed in exenatide-treated subjects, which was more pronounced during open-label extension, with a total weight loss reaching 4–5 kg after 80 weeks. The number of mild-to-moderate hypoglycemic events increased in patients treated with the combination of exenatide and sulphonylureas, but not in those receiving the combination of exenatide and metformin, despite a similar reduction in glycemia. Exenatide has also been compared with insulin glargine and biphasic aspart in noninferiority open-label add-on studies including diabetic subjects not achieving effective glucose control with the combination therapy of metformin and sulphonylurea (245, 246). In both trials, exenatide and insulin regimens reduced levels of HbA1c to a similar extent over 26 weeks. It is noteworthy that the respective effects of glargine and exenatide on glycemic profiles were really different, with the former preferentially reducing fasting glucose concentrations, while postprandial glucose reduction was greater with the latter, especially after breakfast and dinner. Finally, glycemic improvement was associated with a significant weight gain in patients receiving insulin, as expected, contrasting with a significant weight loss in exenatide-treated patients. Due to its inhibitory effect on gastric emptying, the most common adverse events of exenatide were gastrointestinal (nausea, or more rarely vomiting or diarrhea) (247). Most of these side effects are generally mild to moderate, peaked at the beginning of treatment and declined thereafter. Despite their high rate of incidence, gastrointestinal side effects are not considered major determinants of weight reduction observed

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during exenatide treatment. Importantly, due to only 50% homology with human GLP-1, up to 60% patients receiving exenatide develop antiexenatide antibodies with weak binding affinity. However, except in a few subjects harboring high titers, antibody formation has not been associated with impaired metabolic effectiveness of exenatide (238, 248). Liraglutide Liraglutide is a recombinant analogue of human GLP-1, which shares a 97% sequence homology with native GLP-1. The glutamic acid and 16-C free-fatty-acid addition to Lys26 enable the molecule to bind to albumin and the Arg34Lys substitution renders Liraglutide less susceptible to DPP-4 degradation, which contributes to a protracted circulatory half-life of about 10–14 h after subcutaneous administration in humans. Liraglutide can thus be given as a once-daily injection (249, 250). The clinical effects of Liraglutide treatment have been extensively investigated in a large program of Phase III studies, the Liraglutide Effect and Action in Diabetes (LEAD), including more than 4000 type 2 diabetic patients (250–254). Once-daily subcutaneous Liraglutide has been recently demonstrated to improve glycemic control (more than 1% decrease in HbA1c absolute values at the optimal dosage of 1.8 mg/day) compared with placebo or active comparator in adult patients with type 2 diabetes, both as monotherapy and in combination with one or two oral antidiabetic drugs such as metformin, sulfonylureas, or thiazolidinediones. Liraglutide also provided a better glycemic control than rosiglitazone or insulin glargine in add-on combination trials and, than glimepiride or glibenclamide in monotherapy trials. As reported with exenatide, Liraglutide administration led to progressive weight loss, and was associated with a low risk of hypoglycemia, especially in association with metformin. Here again, nausea, vomiting, and diarrhea were the most prominent adverse events but were generally mild and transient, and rarely caused discontinuation of Liraglutide treatment. In contrast to exenatide, exposure to Liraglutide has not been reported to induce antibody formation (250). Finally, the open-labeled LEAD 6 study recently reported that Liraglutide (1.8 mg once a day) provided significantly greater improvements in glycemic control than did exenatide (10 mg twice a day) in patients with inadequately controlled type 2 diabetes on maximally tolerated doses of metformin, sulphonylurea, or both (255). Indeed, mean HbA1c (1.1% versus 0.8%) and fasting plasma glucose (1.6 mmol/L versus 0.6 mmol/L) reduction was significantly greater with Liraglutide treatment than with exenatide. Accordingly, significantly more subjects achieved an HbA1c level of <7% with Liraglutide treatment than with exenatide (54% versus 43%). Both drugs were well tolerated, but nausea was less persistent and with minor hypoglycemia less frequent with Liraglutide than with exenatide. Long-Acting GLP-1R Agonists The development of several long-acting GLP-1R agonists that need less frequent parenteral administration is currently a matter of considerable interest (256).

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Two studies, evaluating a once weekly exenatide formulation (a polylactide– glycolide microsphere suspension containing 3% Exendin-4 peptide) for long-acting release (LAR), provided enthusiastic perspectives for the treatment of type 2 diabetes. First, in a 15 week placebo-controlled study, the administration of 2 mg once weekly exenatide (also termed Byetta LAR) significantly reduced HbA1c (2.1% versus placebo), fasting plasma glucose (3.2 mmol/L), and body weight (3.8 kg) in 45 subjects with type 2 diabetes suboptimally controlled with metformin (60%) and/or diet and exercise (40%) (257). HbA1C of less than 7% was achieved by 86% of subjects receiving once weekly exenatide, compared with 0% of subjects receiving placebo. Then, a 30 week, randomized, noninferiority study compared the same longacting release formulation of exenatide 2 mg administered once weekly to 10 mg exenatide administered twice a day (also termed Byetta), in 295 patients with type 2 diabetes, naive to drug therapy, or on one or more oral antidiabetic agents (258). At 30 weeks, the patients given exenatide once a week had significantly greater changes in HbA1c than those given exenatide twice a day (1.9% versus 1.5%; p ¼ 0.0023) and a significantly greater proportion of patients receiving treatment once a week versus twice a day achieved target HbA1c levels of 7.0% or less (77% versus 61% of evaluable patients, p ¼ 0.0039). This significant greater improvement in glycemic control was obtained with no increased risk of hypoglycemia and similar reductions in body weight. Interestingly, treatment-related nausea was reported in significantly fewer patients treated once a week than twice a day (258). DPP-4 Inhibitors Many DPP-4 inhibitors have been developed that specifically and potently inhibit DPP-4 activity after oral administration. Typically, these agents reduce serum DPP-4 activity by more than 80%, with some inhibition maintained for 24 h after one single dose or with once-daily treatment, resulting in a rise in postprandial levels of intact GLP-1. Sitagliptin, vildagliptin, and very recently saxagliptin, have been approved for clinical use, but a number of other molecules, including alogliptin, are currently under development (248, 256). Large meta-analyses recently considered the metabolic effects and the safety of DPP-4 inhibitors and provided very closed conclusions (247, 259, 260). The latest search for all available randomized controlled trials with duration of more than 12 weeks, either published or unpublished, was performed in type 2 diabetic patients (260). A total of 41 studies were included in the analysis, most of them investigating the sitagliptin or vildagliptin action. The results indicate that DPP-4 inhibitors induce a significant decrease in HbA1c level in comparison to placebo administration (0.7%) and a similar hypoglycemic efficacy for sitagliptin and vildagliptin (260). In active comparator studies, DPP-4 inhibitors showed a similar effect to that of glitazones, but metformin and sulfonylureas appeared to be more effective. The effect of DPP-4 inhibitors was neutral on body weight in placebocontrolled studies, while a modest but significant difference was detected in comparison with glitazones. The incidence of hypoglycemia with DPP-4 inhibitors was

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low and not significantly different from that observed with a placebo, even when these agents were used in combination with sulfonylureas or insulin. Administration of DPP-4 inhibitors was not associated with any increase in the overall risk of other adverse events as compared with placebo or glitazones, whereas the incidence was significantly lower than with metformin, sulfonylureas, and acarbose (260). Importantly, in striking contrast to GLP-1R agonists, no increase was found in the frequency of gastrointestinal events. In view of the widespread expression of DPP-4 on many cell types, including lymphocytes, there is a considerable interest in the long-term safety profile of DPP-4 inhibitors, especially about the infectious risk. A significant increase in the incidence of nasopharyngitis was observed with sitagliptin only, whereas an increase in the risk of urinary tract infection was found with vildagliptin only. The incidence of other infections was lower than in comparator groups (260).

CONCLUSIONS In conclusion, clinical data clearly demonstrate that agents that enhance or mimic incretin action exert promising metabolic effects for the treatment of type 2 diabetes, with a favorable safety profile in our current knowledge. Nevertheless, large clinical studies are now needed to compare these agents with existing oral therapies or insulin, or both, in terms of long-term safety and prevention of degenerative complications. Especially, the observation that GLP-1R agonists prevent myocardial function in pathophysiological animal models, as well as in pilot clinical studies, highlights the need for studies that assess cardiovascular endpoints in patients treated with DPP-4 inhibitors or GLP-1R agonists. Furthermore, determining whether chronic therapy with GLP-1R agonists or DPP-4 inhibitors could be associated with sustained longterm control of HbA1c and improvement in b-cell function beyond what’s achievable with existing agents represent a major challenge for future clinical studies. In addition, one should consider that, due to the physiological concept through which such strategies control glycemia, it is suggested that these drugs be used upon diagnosis of diabetes to better reduce the hyperglycemic and lipotoxic effects of the metabolic disease on other functions such as cardiovascular diseases.

REFERENCES 1. BAYLISS, W.M., and E.H. STARLING. 1902. The mechanism of pancreatic secretion. J Physiol 28:325–353. 2. WEIR, G.C., S. MOJSOV, G.K. HENDRICK, and J.F. HABENER. 1989. Glucagon like peptide I (7-37) actions on endocrine pancreas. Diabetes 38:338–342. 3. THORENS, B. 1992. Expression cloning of the pancreatic beta cell receptor for the gluco-incretin hormone glucagon-like peptide 1. Proc Natl Acad Sci USA 89:8641–8645. 4. DEACON, C.F. 2004. Circulation and degradation of GIP and GLP-1. Horm Metab Res 36:761–765. 5. DEACON, C.F., A.H. JOHNSEN, and J.J. HOLST. 1995. Degradation of glucagon-like peptide-1 by human plasma in vitro yields an N-terminally truncated peptide that is a major endogenous metabolite in vivo. J Clin Endocrinol Metab 80:952–957.

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200. SOKOS, G.G., L. NIKOLAIDIS, S. MANKAD, D. ELAHI, and R.P. SHANNON. 2006. Glucagon-like peptide-1 infusion improves left ventricular ejection fraction and functional status in patients with chronic heart failure. J Card Fail 12:694–699. 201. BLONDE, L., E. KLEIN, J. HAN, B. ZHANG, S. MAC, T. POON, K. TAYLOR, M. TRAUTMANN, D. KIM, and D. KENDALL. 2006. Interim analysis of the effects of exenatide treatment on A1C, weight and cardiovascular risk factors over 82 weeks in 314 overweight patients with type 2 diabetes. Diabetes Obes Metab 8:436–447. 202. VILSBØLL, T., M. ZDRAVKOVIC, T. LE-THI, T. KRARUP, O. SCHMITZ, J. COURRE`GES, R. VERHOEVEN, I. BUGA´NOVA´, and S. MADSBAD. 2007. Liraglutide, a long-acting human glucagon-like peptide-1 analog, given as monotherapy significantly improves glycemic control and lowers body weight without risk of hypoglycemia in patients with type 2 diabetes. Diabetes Care 30:1608–1610. 203. NIKOLAIDIS, L., D. ELAHI, Y. SHEN, and R. SHANNON. 2005. Active metabolite of GLP-1 mediates myocardial glucose uptake and improves left ventricular performance in conscious dogs with dilated cardiomyopathy. Am J Physiol Heart Circ Physiol 289:H2401–H2408. 204. BAN, K., H. NOYAN-ASHRAF, J. HOEFER, S. BOLZ, D. DRUCKER, and M. HUSAIN. 2008. Cardioprotective and vasodilatory actions of glucagon-like peptide 1 receptor are mediated through both glucagon-like peptide 1 receptor-dependent and -independent pathways. Circulation 117:2340–2350. 205. NOYAN-ASHRAF, M., A. MOMEN, K. BAN, A. SADI, Y. ZHOU, A. RIAZI, L. BAGGIO, M. HENKELMAN, M. HUSAIN, and D. DRUCKER. 2009. GLP-1R agonist liraglutide activates cytoprotective pathways and improves outcomes after experimental myocardial infarction in mice. Diabetes 58:975–983. 206. MATIKAINEN, N., S. MA¨NTTA¨RI, A. SCHWEIZER, A. ULVESTAD, D. MILLS, B. DUNNING, J. FOLEY, and M. TASKINEN. 2006. Vildagliptin therapy reduces postprandial intestinal triglyceride-rich lipoprotein particles in patients with type 2 diabetes. Diabetologia 49:2049–2057. 207. QIN, X., H. SHEN, M. LIU, Q. YANG, S. ZHENG, M. SABO, D.A. D’ALESSIO, and P. TSO. 2005. GLP-1 reduces intestinal lymph flow, triglyceride absorption, and apolipoprotein production in rats. Am J Physiol Gastrointest Liver Physiol 288:G943–G949. 208. KNAPPER, J.M., A. HEATH, J.M. FLETCHER, L.M. MORGAN, and V. MARKS. 1995. GIP and GLP-1(7-36) amide secretion in response to intraduodenal infusions of nutrients in pigs. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol 111:445–450. 209. KNAPPER, J.M., L.M. MORGAN, and J.M. FLETCHER. 1996. Nutrient-induced secretion and metabolic effects of glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1. Proc Nutr Soc 55:291–305. 210. KNAPPER, J.M., L.M. MORGAN, J.M. FLETCHER, and V. MARKS. 1995. Plasma and intestinal concentrations of GIP and GLP-1 (7-36) amide during suckling and after weaning in pigs. Horm Metab Res 27:485–490. 211. RITZEL, R., C. ORSKOV, J.J. HOLST, and M.A. NAUCK. 1995. Pharmacokinetic, insulinotropic, and glucagonostatic properties of GLP-1 [7-36 amide] after subcutaneous injection in healthy volunteers. Dose–response relationships. Diabetologia 38:720–725. 212. RITZEL, U., A. FROMME, M. OTTLEBEN, U. LEONHARDT, and G. RAMADORI. 1997. Release of glucagon-like peptide-1 (GLP-1) by carbohydrates in the perfused rat ileum. Acta Diabetologica 34:18–21. 213. CANI, P.D., J.J. HOLST, D.J. DRUCKER, N.M. DELZENNE, B. THORENS, R. BURCELIN, and C. KNAUF. 2007. GLUT2 and the incretin receptors are involved in glucose-induced incretin secretion. Mol Cell Endocrinol 276:18–23. 214. ELLIOTT, R.M., L.M. MORGAN, J.A. TREDGER, S. DEACON, J. WRIGHT, and V. MARKS. 1993. Glucagon-like peptide-1 (7-36)amide and glucose-dependent insulinotropic polypeptide secretion in response to nutrient ingestion in man: acute post-prandial and 24-h secretion patterns. J Endocrinol 138:159–166. 215. RUIZ-GRANDE, C., J. PINTADO, C. ALARCON, C. CASTILLA, I. VALVERDE, and J.M. LOPEZ-NOVOA. 1990. Renal catabolism of human glucagon-like peptides 1 and 2. Can J Physiol Pharmacol 68:1568–1573. 216. EDFALK, S., P. STENEBERG, and H. EDLUND. 2008. Gpr40 is expressed in enteroendocrine cells and mediates free fatty acid stimulation of incretin secretion. Diabetes 57:2280–2287. 217. OVERTON, H.A., M.C. FYFE, and C. REYNET. 2008. GPR119, a novel G protein-coupled receptor target for the treatment of type 2 diabetes and obesity. Br J Pharmacol 153(Suppl 1): S76–S81.

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238. DRUCKER, D.J., and M.A. NAUCK. 2006. The incretin system: glucagon-like peptide-1 receptor agonists and dipeptidyl peptidase-4 inhibitors in type 2 diabetes. Lancet 368:1696–1705. 239. ENG, J., W.A. KLEINMAN, L. SINGH, G. SINGH, and J.P. RAUFMAN. 1992. Isolation and characterization of exendin-4, an exendin-3 analogue, from Heloderma suspectum venom. Further evidence for an exendin receptor on dispersed acini from guinea pig pancreas. J Biol Chem 267:7402–7405. 240. NIELSEN, L.L., and A.D. BARON. 2003. Pharmacology of exenatide (synthetic exendin-4) for the treatment of type 2 diabetes. Curr Opin Investig Drugs 4:401–405. 241. BUSE, J.B., R.R. HENRY, J. HAN, D.D. KIM, M.S. FINEMAN, and A.D. BARON. 2004. Effects of exenatide (exendin-4) on glycemic control over 30 weeks in sulfonylurea-treated patients with type 2 diabetes. Diabetes Care 27:2628–2635. 242. DEFRONZO, R.A., R.E. RATNER, J. HAN, D.D. KIM, M.S. FINEMAN, and A.D. BARON. 2005. Effects of exenatide (exendin-4) on glycemic control and weight over 30 weeks in metformin-treated patients with type 2 diabetes. Diabetes Care 28:1092–1100. 243. KENDALL, D.M., M.C. RIDDLE, J. ROSENSTOCK, D. ZHUANG, D.D. KIM, M.S. FINEMAN, and A.D. BARON. 2005. Effects of exenatide (exendin-4) on glycemic control over 30 weeks in patients with type 2 diabetes treated with metformin and a sulfonylurea. Diabetes Care 28:1083–1091. 244. ZINMAN, B., B.J. HOOGWERF, S. DURAN GARCIA, D.R. MILTON, J.M. GIACONIA, D.D. KIM, M.E. TRAUTMANN, and R.G. BRODOWS. 2007. The effect of adding exenatide to a thiazolidinedione in suboptimally controlled type 2 diabetes: a randomized trial. Ann Intern Med 146:477–485. 245. HEINE, R.J., L.F. Van GAAL, D. JOHNS, M.J. MIHM, M.H. WIDEL, and R.G. BRODOWS. 2005. Exenatide versus insulin glargine in patients with suboptimally controlled type 2 diabetes: a randomized trial. Ann Intern Med 143:559–569. 246. NAUCK, M.A., S. DURAN, D. KIM, D. JOHNS, J. NORTHRUP, A. FESTA, R. BRODOWS, and M. TRAUTMANN. 2007. A comparison of twice-daily exenatide and biphasic insulin aspart in patients with type 2 diabetes who were suboptimally controlled with sulfonylurea and metformin: a non-inferiority study. Diabetologia 50:259–267. 247. AMORI, R.E., J. LAU, and A.G. PITTAS. 2007. Efficacy and safety of incretin therapy in type 2 diabetes: systematic review and meta-analysis. JAMA 298:194–206. 248. BOSI, E., P. LUCOTTI, E. SETOLA, L. MONTI, and P.M. PIATTI. 2008. Incretin-based therapies in type 2 diabetes: a review of clinical results. Diabetes Res Clin Pract 82(Suppl 2): S102–S107. 249. ELBROND, B., G. JAKOBSEN, S. LARSEN, H. AGERSO, L.B. JENSEN, P. ROLAN, J. STURIS, V. HATORP, and M. ZDRAVKOVIC. 2002. Pharmacokinetics, pharmacodynamics, safety, and tolerability of a single-dose of NN2211, a long-acting glucagon-like peptide 1 derivative, in healthy male subjects. Diabetes Care 25:1398–1404. 250. RUSSELL-JONES, D., A. VAAG, O. SCHMITZ, B.K. SETHI, N. LALIC, S. ANTIC, M. ZDRAVKOVIC, G.M. RAVN, and R. SIMO. 2009. Liraglutide vs. insulin glargine and placebo in combination with metformin and sulfonylurea therapy in type 2 diabetes mellitus (LEAD-5 met þ SU): a randomised controlled trial. Diabetologia 52:2046–2055. 251. GARBER, A., R. HENRY, R. RATNER, P.A. GARCIA-HERNANDEZ, H. RODRIGUEZ-PATTZI, I. OLVERA-ALVAREZ, P.M. HALE, M. ZDRAVKOVIC, and B. BODE. 2009. Liraglutide versus glimepiride monotherapy for type 2 diabetes (LEAD-3 Mono): a randomised, 52-week, phase III, double-blind, parallel-treatment trial. Lancet 373:473–481. 252. NAUCK, M., and M. MARRE. 2009. Adding liraglutide to oral antidiabetic drug monotherapy: efficacy and weight benefits. Postgrad Med 121:5–15. 253. ZINMAN, B., J. GERICH, J.B. BUSE, A. LEWIN, S. SCHWARTZ, P. RASKIN, P.M. HALE, M. ZDRAVKOVIC, and L. BLONDE. 2009. Efficacy and safety of the human glucagon-like peptide-1 analog liraglutide in combination with metformin and thiazolidinedione in patients with type 2 diabetes (LEAD-4 Met þ TZD). Diabetes Care 32:1224–1230. 254. MARRE, M., J. SHAW, M. BRANDLE, W.M. BEBAKAR, N.A. KAMARUDDIN, J. STRAND, M. ZDRAVKOVIC, T.D. Le THI, and S. COLAGIURI. 2009. Liraglutide, a once-daily human GLP-1 analogue, added to a sulphonylurea over 26 weeks produces greater improvements in glycaemic and weight control compared with adding rosiglitazone or placebo in subjects with Type 2 diabetes (LEAD-1 SU). Diabet Med 26:268–278.

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Dipeptidyl Peptidase IV Inhibitors for Treatment of Diabetes C.H.S. MCINTOSH1,2, S.-J. KIM1,2, R.A. PEDERSON1,2, U. HEISER3, 3 AND H.-U. DEMUTH 1

Department of Cellular and Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, Canada 2 Diabetes Research Group, Life Sciences Institute, University of British Columbia, Vancouver, Canada 3 Probiodrug AG, Biocenter, Halle (Saale), Germany

INTRODUCTION Approximately 246 million people worldwide were estimated to have diabetes mellitus in 2007, and it is predicted that a staggering 380 million will be afflicted with the disease by 2025 (1). This is a daunting prospect, both because of the longterm complications of the disease and the costs of treatment. Diabetes mellitus has been classified into two main groups: Type 1 diabetes (T1DM) that results from the autoimmune destruction of b-cells (2) and Type 2 (T2DM) that is characterized by deficient pancreatic b-cell function and insulin resistance (3). The majority of T2DM patients are obese and it is generally agreed that both genetics and environmental factors, particularly lack of exercise and excessive energy intake, are mainly responsible for development of obesity. Characterization of diabetogenic genes that contribute to both T1DM and T2DM is a fertile area of research (2, 4). The hyperglycemia that is characteristic of diabetes results mainly from increased hepatic glucose production and decreased peripheral tissue glucose uptake, resulting from the lack of insulin in T1DM and insulin resistance in the early stages of T2DM, with

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insulinopenia contributing in the later stages of the disease. Since insulin was first introduced, it has remained the main therapy for T1DM (5). However, in recent years a small percentage of patients have received either whole pancreas (6, 7) or islet (8–10) transplants. Treatment of T2DM involves lifestyle modification (diet and exercise) plus antidiabetic drug therapy (11, 12). Until 2006, the major drugs prescribed were those of the sulfonylurea or meglitinide families that act on pancreatic islet b-cells and stimulate insulin secretion (13, 14), agents that reduce insulin resistance, such as the biguanide metformin and the thiazolidinediones (15) and a-glucosidase inhibitors for lowering starch and sucrose digestion (12). During chronic treatment, measurements of glucose and hemoglobin A1c (HbA1c) levels have demonstrated that monotherapy frequently fails (16, 17) and that combination therapy, with a drug targeting the b-cells coupled with an insulin sensitizer, is more effective at reducing HbA1c levels to those recommended by professional diabetes organizations, such as the American Diabetes Association (ADA). Alternative therapies are therefore continually being developed, among the most recent of which are two classes of drugs, the “incretin mimetics” and “incretin enhancers” that take advantage of the physiological actions of gastrointestinal hormones, which stimulate insulin secretion in a glucose-dependent manner. The objectives of the current review are to introduce the incretin hormones, describe how basic science studies led to the introduction of dipeptidyl peptidase IV (DPP-4) inhibitors as T2DM therapeutics, to describe how they act, and discuss their potential for T1DM therapy.

THE INCRETIN CONCEPT AND DISCOVERY OF INCRETIN HORMONES The gastrointestinal (GI) tract is one of the largest endocrine organs of the body. It contains a multitude of regulatory peptides that act as signaling molecules within the gut and its associated organs, and for other organs, including the brain, bone, and cardiovascular system. Among these peptides are two, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) that are categorized as “incretin hormones.” The origin of this terminology dates back to the introduction of the term “hormone” by Starling (18), to describe the mode of action of secretin, and studies by Moore et al. (19) that led them to propose that “the internal secretion of the pancreas might be stimulated and initiated . . . by a substance of the nature of a hormone or secretin yielded by the duodenal mucous membrane.” Although numerous researchers examined the effects of injecting crude intestinal extracts on blood glucose levels, it was Jean La Barre, a Belgian physiologist, who provided the first strong evidence for the existence of factors released from the upper intestine that were capable of reducing circulating glucose levels, in the absence of any effect on the exocrine pancreas. He suggested the name incre´tine (20) for such factors. The significance of this concept became evident over 30 years later, when it was demonstrated that, due to a greater insulin response, glucose given via an intragastric or intrajejunal route was handled more efficiently than an equivalent amount administered intravenously (21, 22). The hormonal link between the gut and the

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endocrine pancreas was subsequently referred to as the enteroinsular axis (23) and Creutzfeldt reintroduced the term incre´tine in anglicized form (incretin) (24). It has been estimated that up to 60% of the total insulin secreted during a meal results from the incretin response (25). Importantly, Creutzfeldt emphasized the glucose-dependent nature of incretin action, as well as the potential importance of neural regulation in the enteroinsular axis. We now know that there is a strong interaction between GLP1 and autonomic reflexes involving the central nervous system (26, 27), but direct enteropancreatic neural connections (28) are probably also involved. The first incretin to be isolated was a 42 amino acid peptide (Figure 12.1) that exhibited acid inhibitory properties in dogs equipped with denervated gastric pouches, and was therefore named gastric inhibitory polypeptide (29, 30). It was subsequently demonstrated that intravenous GIP stimulated insulin secretion and increased glucose disposal in normal humans (31). Since a number of studies showed that the incretin effect was its more important action, definition of the GIP acronym was modified to glucose-dependent insulinotropic polypeptide (32). Evidence for additional incretin(s) later arose (Reviewed in Ref. 33) and, following discovery of the coding sequence for a glucagon-related peptide in an anglerfish proglucagon cDNA (34) several groups contributed to the identification of GLP-1(7-36)amide (Figure 12.1) and GLP-1(7-37) as intestinal products of mammalian proglucagon (35), the former being the major circulating species in blood. By convention the two peptides are now referred to collectively as “GLP-1” (36). GIP and GLP-1 belong to

Figure 12.1

Amino acid sequences of glucose-dependent insulinotropic polypeptide, glucagon-like peptide-1, and exendin-4. Regions of amino acid identity and the cleavage sites for DPP-4 are highlighted.

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the secretin/glucagon superfamily of peptides that share strong N-terminal regional homology (37). In humans, additional identified members of the family include GLP-2, growth hormone-releasing hormone (GHRH), vasoactive intestinal polypeptide (VIP), peptide histidine methionine (PHM), and pituitary adenylate cyclase-activating polypeptide (PACAP). GIP and GLP-1 are produced by gut enteroendocrine K- and L-cells, respectively. The two incretins are secreted during a meal in response to nutrients, with glucose and fat being the most potent stimuli in humans, and they stimulate insulin secretion in a glucose-dependent manner (36, 38, 39). For more detailed discussion of the characteristics of GIP and GLP-1 the reader is referred to a number of recent reviews (33, 40–42).

INCRETIN METABOLISM Shortly following their isolation, in vivo renal metabolism of GIP and GLP-1 was shown to occur (27, 43). Subsequently, infusion studies on both peptides demonstrated extremely short half-lives with respect to biological activity (43), and several approaches were taken to identify the enzyme(s) responsible for the rapid degradation that occurred. The amino-termini of GIP and GLP-1 consist of Tyr-Ala and His-Ala, respectively (Figure 12.1), dipeptides that are readily cleaved from oligopeptides by enzymes such as the cysteine peptidase dipeptidyl peptidase I (DPP-1; cathepsin C; E.C. 3.4.14.1) and the serine peptidase dipeptidyl peptidase IV (DPP-4; DPP-IV; CD26; E.C. 3.4.14.5). The metabolism of GIP and GLP-1 was first studied in vitro, by incubating native or 125I-labeled peptides with highly purified DPP-4, or in serum or plasma, and identifying products by HPLC (44, 45). Rapid N-terminal truncation was found to occur (44, 45) and MALDI-TOF mass spectroscopy (46) unequivocally demonstrated conversion of GIP1-42 and GLP-17-36 to the noninsulinotropic peptides, GIP3-42 and GLP-19-36 (Figures 12.1 and 12.2). The N-terminal degradation of GIP and GLP-1 was also shown to occur in vivo, following peptide administration to rodents (45, 46) and humans (47, 48). Confirmation of the in vivo generation of the truncation forms was achieved using region-selective antibodies and radioimmunoassay (RIA) (43, 49). Hepatic DPP-4 plays a major role in the N-terminal degradation of GIP, with GLP-1 degradation occurring in multiple sites, including the endothelium (43, 50). DPP-4 is believed to be the major enzyme involved in the metabolism of GLP-1, and it has been estimated that up to 75% of the intact biologically active peptide is degraded to GLP-19-36 by endothelial DPP-4 shortly after secretion (26, 51), prior to renal clearance of the N-terminally truncated peptide. It has been shown that neutral endopeptidase 24.11 also contributes significantly to GLP-1 degradation (52).

DPP-4: DISTRIBUTION, SUBSTRATE SPECIFICITY, AND STRUCTURE DPP-4 is an integral membrane glycoprotein that is a member of the large S9 family of prolyl oligopeptidases (53, 54). Interestingly, the DPP-4 gene in humans is localized

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Figure 12.2 Diagrammatic representations of the incretin response during a meal, termination of their actions by DPP-4, and the effect of inhibitor on insulin responses. (a) Incretins (GIP and GLP-1) are released in response to nutrients and transported through the blood to the endocrine pancreas, where they stimulate insulin secretion in a glucose-dependent manner. (b) DPP-4 terminates the incretin’s insulinotropic actions by degrading them at their N-termini. (c) DPP-4 inhibition results in a potentiation of incretin signaling, one consequence of which is a greater insulin response and more efficient glucose disposal.

adjacent to the proglucagon gene on chromosome 2q24 (55), although it is not known whether this has functional significance. The DPP-4 enzyme is ubiquitously expressed, with highest levels in humans in the bone marrow, the brush border of the small intestine and the proximal tubules of the kidney (56–58). At the cellular level, DPP-4 is highly expressed on the apical surfaces of epithelial and endothelial cells, including those of venules and capillaries (56, 58, 59). In keeping with a role in immune function, DPP-4 is also expressed on subsets of CD4 þ and CD8 þ T-cells and natural killer cells (56, 58, 60), where it is termed CD26. Soluble forms of DPP-4 are found in blood plasma (58, 61) and other secreted fluids (62), and the activity of enzyme present in blood plasma is used as a marker for the degree of inhibition achieved with administered inhibitors.

Substrate Specificity DPP-4 acts by selectively removing N-terminal dipeptides from oligopeptides, with preference for peptides having proline or alanine as the penultimate (P1) amino acid (44, 53, 56, 63–65); peptides with serine and threonine at P1 are cleaved less

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efficiently. Other peptides of the secretin/glucagon superfamily, including glucagon, GLP-2 and PACAP have also been shown to be DPP-4 substrates (66–68). Similarly, numerous additional peptides that act as hormones, neuropeptides, chemokines, and cytokines are metabolized by purified DPP-4 in vitro (69–71), although in most cases it is unclear as to whether such degradation has physiological significance. This is important since, although no major off-target effects have been observed in T2DM patients treated with DPP-4 inhibitors, the implications of chronically changing the activity of specific peptides is unknown. Since, depending on the peptide, N-terminal cleavage can reduce, ablate or increase their biological activity, or alter their receptor specificity; studies on the in vivo significance of such changes are still required.

DPP-4 Structure Over the past few years considerable structural information, that is critical for rational design of inhibitors, has been derived from physico-chemical techniques, X-ray crystallography, and cryo-transmission electron microscopy. The crystal structure of DPP-4 was first solved by two groups independently. Rasmussen et al. (72) determined the structure of recombinant human DPP-4 with the substrate-like inhibitor valine–pyrrolidide (Val–Pyr) bound to its active site, whereas Engel et al. crystallized the native enzyme, purified from porcine kidney, thus retaining its natural state of glycosylation (73). The catalytic serine residue and the shape of the active site were identified by cocrystallizing DPP-4 with a slow-tight binding inhibitor of the cyanopyrrolidine type or an irreversible inhibitor containing a boronic acid proline analogue (74, 75). Cocrystallization of DPP-4 with the slow-tight binding inhibitor also allowed the demonstration of an acyl-enzyme intermediate state, as well as nucleophilic attack of the active serine at the cyano-moiety, leading to production of an imidine intermediate. Similar conclusions were later drawn from cocrystallization studies on a slowly converted substrate, diprotein A (76). The postproline peptide bond was again shown to be attacked by the catalytic serine side chain leading to a carbonyl hydrate that then collapsed to an acyl-enzyme, in agreement with current theories of serine protease catalysis. The results from these X-ray crystallography experiments support previous data obtained from enzyme kinetic studies that led to the proposal for a two-step hydrolysis mechanism for catalysis with formation of an acyl-enzyme intermediate (77). DPP-4 exists as a dimer when in membrane-bound form (56, 60, 62). The 110 kDa monomeric subunit consists of a large globular extracellular domain that contains a glycosylated region, a central cysteine-rich region and a domain containing the catalytic triad. A hydrophobic helical region anchors the subunit to the plasma membrane and a six amino acid C-terminus constitutes the only internal segment of the protein (73). Studies on the native enzyme (72, 73, 78) and recombinant forms of DPP-4 (79) have shown that the catalytic triad is located in a large cavity formed by an a/b hydrolase fold and an 8-bladed b-propeller domain. The binding region for N-termini of peptide substrates contains a short helix, with a Glu205–Glu206 sequence motif (79). There is restricted entry to the active site, thus only allowing hydrolysis of

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small peptides. Two routes of entry have been proposed via the b-propeller, with products exiting via a side route (73), or through the side route (72, 80). A secondary binding site has been identified by Demuth and coworkers that exhibits differential binding to individual peptides of the secretin/glucagon superfamily (81), thus potentially allowing the development of more selective inhibitors (Ba¨r, K€uhn-Wache, Demuth et al., unpublished observations). In addition to its catalytic function, DPP-4/CD26 plays important structural and signaling roles, involving association with accessory proteins that include adenosine deaminase, fibronectin, and the protein tyrosine phosphatase CD45. These interactions contribute to immune function, including T-cell activation, regulation of cell binding to the extracellular matrix, and cell–cell signaling (56, 58, 65, 71, 82). In view of the importance of specificity for drug targeting, considerable attention has been directed at identifying enzymes with similar substrate specificity to DPP-4, as well as their possible presence in blood plasma. A number of enzymes that are structurally related to DPP-4 (53, 83) are found in multiple locations, including fibroblast activation protein a (FAP-a; Seprase; DP 5) (84, 85), DPP-8 (86, 87), and DPP-9 (87, 88), as well as the catalytically inactive DPP-6 (DPL1; DPX) (89) and DPP-10 (DPL2) (90). DPP II (DPP-2; quiescent cell proline dipeptidase (QPP); DPP-7) (61, 91) is a structurally unrelated enzyme that exhibits similar substrate specificity (92). FAP-a is a plasma membrane protein, whereas DP8, DP9, and DP II are normally found only in intracellular locations (53). Although these DPP-4-like enzymes could potentially contribute to a circulating pool, the majority of DPP-4-like activity in human plasma appears to be authentic DPP-4 (93). However, low levels of circulating DPP-4 activity can be detected in DPP-4/ rats, the identity of which has not been established. FAP-a has been identified in serum (94), but it is generally only expressed at remodeling sites, including the liver and tumors, but not normal tissues (85, 95). It now appears that the DPP-4-like activity in serum attributed to the human orthologue of mouse mahogany, attractin (DPPT-L) (96), was an artifact, since the purified recombinant enzyme exhibited no such activity (97).

CHEMICAL CLASSES OF INHIBITORS AND STRUCTURES The development of DPP-4 inhibitors has been reviewed in numerous publications and this section of the overview summarizes the cornerstones of inhibitor design in regard to the most important inhibitor classes (bold numbers in the following discussion refer to the structures presented in Figures 12.3–12.8).

Substrate-Like Inhibitors DPP-4 exhibits restricted substrate specificity, accepting glycine and hydrophobic amino acids in the P2-position, as well as facilitating the almost exclusive cleavage of substrates possessing proline at the P1-position. The first inhibitors were developed during early stages of the enzyme’s biochemical characterization by simply removing the carboxylate of the dipeptide hydrolysis products. This resulted in the production

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Figure 12.3

Structures of DPP-4 substrate-like inhibitors 1–10.

of aminoacyl pyrrolidine- and aminoacyl thiazolidine-containing proline-mimetics [1, 2]. These inhibitors were of moderate potency and the corresponding thiazolidines were subsequently found to be more effective inhibitors (98). The first molecule demonstrating therapeutic DPP-4-inhibitory efficacy in both animal and human studies was P32/98 [1] a molecule that possesses thiazolidine as a proline surrogate. A remarkable increase in inhibitory potency was achieved by introducing electrophiles

Figure 12.4

Structures of DPP-4 inhibitors 11–15.

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Figure 12.5 Structures of DPP-4 inhibitors 16–21.

such as cyanides [3], phosphonates [4], and boronic acids [5] in the position of the proline carbamide in the substrate structure. The cyano-pyrrolidide moiety is present in many DPP-4 inhibitors (for recent reviews, see Refs. 53, 99). The reaction of the catalytic serine with the electrophilic cyanide generates a covalently bound imidine intermediate, later hydrolyzed into an amide. These

Figure 12.6 Structures of DPP-4 inhibitors 22–24.

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Figure 12.7

Mode of prodrug DPP-4 inhibitor activation.

inhibitors act as slow-tight binders with low koff-rates leading to long-term inhibition of enzymatic activity. Boronic acid [4] and phosphonate-containing [5] inhibitors act in a similar way; in both cases the side chain of the catalytic serine becomes acetylated by the reactive inhibitors. In contrast to the corresponding cyanides, the esters formed are stable to hydrolysis, leading to an irreversible inhibition of DPP-4. A structural proof of this hypothesis was obtained with the crystallization of a boronic acid derivative in the active site of DPP-4. Dutogliptin [9] is an example of a boronic acid derivative currently in clinical development as an antidiabetic drug. A separate catalytic mechanism was taken advantage of in the development of irreversible hydroxamic acid derivative inhibitors [10]. With these inhibitors, substrate-like cleavage of the hydroxamic acid leads to the liberation of a reactive nitrene species that covalently binds to nucleophiles in the active site (100). In the course of DPP-4 inhibitor development over the last three decades, numerous proline analogues have been generated. The relatively rigid P1 binding

Figure 12.8

Structures of DPP-4 inhibitors 26–34.

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pocket, with its high specificity for proline residues results in a lower acceptance for larger rings, such as piperidine or the smaller azetidines. Also, only modifications by small substituents are tolerated. Elucidation of the protein structure of DPP-4 cocrystallized with different proline-containing surrogates revealed a hydrophobic niche within the active-site cleft, suggesting a better fit for asymmetric proline analogues and explaining the elevated potency of “asymmetric” proline analogues, such as the thiazolidines [1] or cyclopropane-fused derivatives [8, 13], over the more planar pyrrolidine derivatives. The metabolic instability of the thiazolidines and the lack of a sufficient potency of the pyrrolidines stimulated the development of corresponding fluorinated derivatives. These structural modifications at the proline ring were applied in combination with 2-cyano-substituents [8, 10, 11, 12] or derivatization by boronic acids [13]. A remarkable gain of selectivity toward the structurally closely related dipeptidyl peptidases DPP-8 and DPP-9 was achieved by the introduction of an acetylenyl group in position 5 of the pyrrolidinyl ring. This led to the successful development of the structure 14. Another pyrrolidine analogue is the 2,5-dicyano-pyrrolidine-based inhibitor [15]. Since C-terminally activated dipeptides spontaneously undergo cyclization to form diketopiperazines, inherent instability was abundant in DPP-4-inhibitors carrying reactive groups for interaction with the catalytic serine, such as cyanides or ketones. This problem was addressed by the introduction of conformationally restricted P2-groups [22, 23]. A similar approach for the enhancement of chemical stability was the introduction of bulky side chains such as those in the compounds [7] and [8]. Modification of the P2-position also led to increased selectivity over the DPP-4 related proteases DPP-8 and DPP-9 [24] (101, 102). There has been considerable discussion over the potential negative effects of long-term DPP-4 inhibition in patients with diabetes, particularly in regard to immune and cardiovascular functions. Since the metabolic actions of incretins influenced by DPP-4-inhibition are meal-associated, it has been argued that long-term DPP-4 inhibition is unnecessary and once-daily drug application is preferable. Although clinical trials on DPP-4 inhibitors have shown them to be generally safe (103–105), small increases in susceptibility to specific infections have been reported. Therefore, new approaches to the development of short-acting DPP-4 inhibitors are underway, among which are inhibitors based on the chemical instability of C-terminally activated dipeptides, with the inhibitor being stabilized in prodrug form [25]. Release of the prodrug by aminopeptidase activity in the gut and plasma, timedependently releases the active inhibitor and it is then inactivated by intramolecular cyclization (Figure 12.7). Again, the half-life of the active inhibitor can be influenced by the characteristics of the P2-side chain, with a preference for sterically bulky substituents (106, 107).

Nonsubstrate-Like DPP-4 Inhibitors Numerous chemical classes of DPP-4 inhibitors have resulted from high throughputand virtual-screening programs. Specific characteristics of the ligands often resulted

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in tighter binding and therefore in a higher inhibitory potency. In contrast to the substrate-like inhibitors, selectivity for DPP-8 and DPP-9 were seldom found to be critical with this type of inhibitors. The use of small substituents on aromatic rings was found to be an effective replacement for the P1-pyrrolidine present in the substratelike inhibitors, as in 26–29. An exception was found with the xanthine derivative 31, in which a 2-butyn-2-yl group occupies the proline-binding site. In contrast to substrate-related inhibitors, interaction with the P2-amide recognition site was accomplished using the ortho-substituents of the aromatic P1-fragments. Examples are the ortho-fluorine substituted b-phenylethylamines [26], aminopyrimidines [27], imidazoles [28], diphenylpropylamines [29], or pyrimidine 2,4-diones [32]. The essential interaction of Glu residues 205 and 206 with the N-terminus of the substrate is reflected in the presence of basic nitrogen containing amines in almost all inhibitory structures. In some cases, this feature led to problems in the course of the inhibitor development due to strong interactions with the hERG-channel. Exceptions from this rule are the carbamoyl triazoles [34] that lack a basic nitrogen. Additional binding sites, not addressed by the substrates are found with Phe 357, Tyr 547, and Arg 358 situated at longer distances from the Glu dyad and the P1 pocket. The H-bond between the trifluormethyl substituent in the triazolopyrazine part of Sitagliptin [26] and the Arg 358 side chain provides an illustrative example for this additional interaction leading to productive binding. A pi-stacking interaction with Tyr 547 interaction is found with the xanthine-type inhibitors [30, 31]. Compound 31 exhibits additional pi-stacking interactions with Trp 629. (See Refs. 108, 109, for example.)

DPP-4 INHIBITORS AS THERAPEUTICS FOR DIABETES The potential use of DPP-4 inhibitors as therapeutics arose from the demonstration that an intact N-terminus was critical for the insulinotropic activities of GIP and GLP-1. It was reasoned that if selective inhibition of DPP-4 could be achieved, then circulating levels of the bioactive peptides could be raised during a meal, thus potentiating insulin responses and improving glucose homeostasis in diabetic individuals (Figure 12.2) (45, 47, 53, 110–112). The first evidence for the exciting potential of DPP-4 inhibitors came from studies on their effects on glucose homeostasis and incretin activity in normal weight rodents and pigs. Gavage administration to Wistar rats of isoleucine thiazolidide (Ile-Thia; P32/98 [1]), at a dose resulting in 60–70% inhibition of plasma DPP-4 activity, increased integrated insulin responses to an oral glucose load by threefold and improved glucose tolerance (110, 113). Using a different experimental paradigm, the DPP-4 inhibitor valine–pyrrolidide was shown to augment glucose-induced insulin secretion and improve glucose disposal in response to GLP-1 infusion (114). Numerous additional studies, with a multitude of different DPP-4 inhibitors, provided support for the contention that prolongation of incretin action by inhibiting DPP-4 activity could form the basis of a viable therapy for T2DM. Preclinical assessment of the potential for DPP-4 inhibitors as therapeutics in T2DM was initially largely performed in rodents. In our laboratory, we study a Vancouver-bred strain of the Diabetic Fatty (VDF) Zucker rat (115–117) that

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develops a milder diabetes than the commercially available strains of Zucker Diabetic Fatty (ZDF) rats. The homozygous, obese fa/fa animals are hyperlipidemic and exhibit postprandial hyperglycemia, hyperinsulinemia, and insulin resistance. Following single oral doses of Ile-Thia, insulin responses in fa/fa rats were increased and integrated glucose profiles were reduced by 39% (118). While a graduate student, Andrew Pospisilik performed a series of chronic studies, in which Ile-Thia was administered to VDF rats (10 mg/kg bid orally) over 12 weeks, thus inhibiting the enzyme for 9 h per day (115). This resulted in reductions in fasting blood glucose from 8 to 6 mM by 12 weeks of inhibitor treatment, greatly improved glucose tolerance and increased early phase insulin secretion (115). Euglycemichyperinsulinemic clamp studies showed that inhibitor treatment induced a marked improvement in insulin sensitivity (116). Although there were no significant effects of inhibitor treatment on water or nutrient ingestion, body weight gain was reduced by 12.5%, mainly due to reduced fat deposition. A number of DPP-4 inhibitors from different chemical classes have shown similar improvements in glucose homeostasis in different diabetic animal models, including Zucker Fatty (ZF) (119) and ZDF (119–121) rats, and high fat-fed C57BL/6 (122, 123), ob/ob (121), and db/db (124) mice.

DPP-4 INHIBITORS IN TYPE 2 DIABETES The first reported studies on the administration of DPP-4 inhibitors to T2DM patients showed that P32/98 reduced glucose excursions and increased insulin responses during an OGTT (125) and, over a 4 week treatment period, the inhibitor NVP DPP728 reduced fasting and prandial glucose, and HbA1c levels (126). The clinical potential of a large number of inhibitors has subsequently been assessed in clinical trials (27, 127–129) (Table 12.1), and sitagliptin (MK0431; Merck & Co [26]), saxagliptin (BMS-477118; BMS/AstraZeneca [8]), and vildagliptin (LAF237; Novartis [7]) are now marketed as therapeutics. There have been numerous reviews of the development (130–134), pharmacokinetic and pharmacodynamic characteristics (103, 105, 135) and clinical trials (104, 105, 136, 137) of vildagliptin and sitagliptin and the reader is referred to the literature for detailed information. Recently, clinical trial data on alogliptin (Takeda [32]) (138, 139) and saxagliptin (121, 133) have also been presented. From the data available, it is evident that, although there are differences in dosage, biodistribution, and inhibition profiles between the various inhibitors, they are all rapidly absorbed and, at recommended clinical doses, achieve up to 90% inhibition of DPP-4 for 12–24 h following 1–2 tablets per day. Clinical results have demonstrated that the DPP-4 inhibitors constitute an important alternative T2DM therapy, with monotherapy resulting in decreases in both fasting and postprandial glucose, and sustained reductions in HbA1c levels of up to 1.5%, dependent upon the initial level. In combination with metformin, a thiazolidinedione or insulin, sitagliptin or vildagliptin treatment resulted in an additional 1% reduction in HbA1c when compared to placebo (103, 104, 140, 141). To date, the larger clinical trials with

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Table 12.1

DPP-4 Inhibitors Approved or in Clinical Trials

Compound MK-0431; Sitagliptin (Januvia) LAF237; Vildagliptin (Galvus) BMS-477118; Saxagliptin (Onglyza) SYR-322; Alogliptin ALS 2-0426/AMG 222 BI 1356 (Ondero) GW823093; Denagliptin GRC8200; Melogliptin PSN-9301 PHX1149 R1438 SSR-162369 TS-021 TA-6666

Company

Status

Merck Novartis BMS/AstraZeneca

Approved (FDA; EMEA) Approved (EMEA) Approved (FDA)

Takeda Amgen/Servier Boehringer Ingelheim GlaxoSmithKline Glenmark Prosidion (OSI) Phenomix Roche Sanofi-Aventis Taisho Tanabe

Phase Phase Phase Phase Phase Phase Phase Phase Phase Phase Phase

III (FDA Reviewed) I/II III II/III II/III II/III II II II II II

DPP-4 inhibitors have demonstrated that they are safe and well tolerated (103–105) with hypoglycemic episodes rarely occurring.

MODE OF ACTION OF DPP-4 INHIBITORS IN TYPE 2 DIABETES Effects of DPP-4 Inhibition on Circulating Incretins There has been considerable discussion in the literature on the mode of action of DPP-4 inhibitors in T2DM therapy (26, 27, 129, 142), and the differences in responses to those observed during treatment with the GLP-1 receptor agonist exenatide. There is strong evidence that administration of DPP-4 inhibitors to experimental animals and humans results in increased circulating levels of intact endogenous GLP-1 and GIP, and this led to the use of the term “incretin enhancer” to describe this class of drugs. Such in vivo enhancement of active peptide levels was greatly facilitated by the development of region-specific RIAs for GLP-1. In fasting dogs only 10% of total GLP-1 is normally in the active form, but DPP-4 inhibitor administration increases this fraction to 90% (49). The generality of this finding was confirmed with studies in rodents and pigs, with acute or chronic administration of DPP-4 inhibitors increasing levels of intact endogenous GLP-1 or GIP under basal or glucose-induced conditions (114, 120, 143). Interestingly, DPP-4 inhibitor treatment, while increasing levels of active GLP-1, has been shown to decrease total circulating GLP-1 levels, suggesting the presence of a feedback mechanism (49). Increases in the intact forms

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of both basal and food-stimulated GIP and GLP-1 levels have also been demonstrated in T2DM patients during DPP-4 inhibitor treatment (144–147).

Effects of DPP-4 Inhibition on Insulin and Glucagon Secretion In studies on diabetic animals, in which DPP-4 inhibitor treatment was shown to increase levels of active incretins and improve glucose disposal, concomitant increases in insulin secretion have frequently been reported (113, 118, 120, 148, 149), suggesting cause and effect. This conclusion was strongly supported by the finding that DPP-4 inhibitor administration produced minimal effects on glucose disposal in double GIP receptor and GLP-1 receptor (DIRKO) knockout mice (150). However, it is currently unclear as to the relative contribution of GLP-1 and GIP to mealstimulated insulin secretion in T2DM, or the effects of DPP-4 inhibition, due to blunting of the incretin response in these patients (39, 151). This has generally been attributed to reduced circulating GLP-1 levels (152, 153) and b-cell resistance to the insulinotropic action of GIP (33, 154–156), although resistance to GLP-1 has also been described in poorly controlled diabetics (157). Administration of exenatide or DPP-4 inhibitors can therefore be considered as partially correcting a GLP-1 deficit, whereas it is generally assumed that b-cell responses to GIP are absent. The origin of b-cell resistance to GIP in humans with diabetes is unknown (158), but similar resistance found in animal models, such as fa/fa VDF (159, 160) and 90% pancreatectomized (Px) hyperglycemic (161) rats, has been attributed to hyperglycemiaassociated downregulation of GIP receptor expression (159, 160). Despite the uncertainty, DPP-4 inhibitor treatment does result in augmented insulin secretion, although the changes observed in most human studies have been modest (126, 146, 162). Mathematical modeling of responses led Mari and coworkers to conclude that, when reductions in glucose were taken into consideration, 28 days of inhibitor treatment significantly increased insulin secretory patterns in humans (147). Further studies are needed to establish the level of potentiation obtained with inhibitor therapy. Recent studies have suggested that DPP-4 inhibitor therapy may improve overall b-cell function (163), although there appears to be a large disparity between the modest changes seen in diabetic humans and the markedly improved glucoseresponsiveness observed with chronic treatment of VDF rats (116) or C57BL/6J mice (122, 148). It is also unclear whether the positive effects of GLP-1 (164, 165) and GIP (166) on insulin biosynthesis seen in animal models can be replicated in humans with long-term therapy. Nevertheless, additional actions, apart from those on the b-cell, must contribute to the marked improvements in glucose homeostasis observed with DPP-4 inhibitor treatment (27, 142), of which suppression of glucagon secretion plays a major role. Elevated plasma glucagon in T2DM is an important contributor to both fasting and postprandial hyperglycemia. Administration of GLP-1 or GLP-1 analogues (167, 168) or exenatide (169, 170) lowers glucagon levels in type 2 diabetic patients, and suppression of glucagon during a meal-tolerance test was fivefold greater when T2DM patients were treated with a single dose of vildagliptin (129, 171). Increased insulin secretion and reduced glucagon secretion, therefore both appear to play important roles in DPP-4 inhibitor action.

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In preclinical studies, chronic Ile-Thia treatment was found to reduce hepatic glucose output and increase insulin-induced glucose uptake in VDF rats (115), and in vitro studies showed improvements in insulin-induced inhibition of isoproterenolstimulated lipolysis in abdominal adipocytes and insulin-stimulated skeletal muscle glucose uptake, suggesting improvements in insulin resistance. Although long-term GLP-1 infusion (6 week) has also been shown to modulate hepatic glucose output and reduce insulin resistance in type 2 diabetic subjects (172), incretin-mediated effects may not completely explain the improved glucose homeostasis with DPP-4 inhibitor treatment. Recently, DPP-4 inhibition was shown to increase hepatic glucose uptake in dogs (173) and to enhance insulin-mediated suppression of glucose production in mice (174), both through mechanisms that appeared to be incretin-independent. Whether such effects occur in humans is unknown. DPP-4 inhibitor administration has, however, been shown to also impact on fat metabolism in humans: vildagliptin treatment reduced fasting fatty acid flux from adipose tissue (163) and augmented postprandial mobilization and oxidation of lipids (175). Further studies are needed to establish whether these effects are incretin-mediated.

Potential Effects of DPP-4 Inhibitors on b-Cell Growth and Survival Apoptosis is central to the pathology of T2DM, with chronic hyperglycemia and hyperlipidemia, endoplasmic reticulum (ER)- and oxidative-stress and inflammation all contributing to its onset (176, 177). A major goal of drug development is therefore to promote b-cell survival and enhance the replenishment of b-cells and there has been significant interest in the potential application of GIP, GLP-1, and GLP-1 receptor agonists in this area (177–187). In vivo and in vitro studies have shown that GIP (183–185), GLP-1 (177, 182, 188), and exendin-4 (178, 182) all promote b-cell mitogenesis, and we are gradually gaining insight into the mechanisms underlying the antiapoptotic effects of GLP-1 (179–181, 189) and GIP (185–187) on the b-cell. There are, however, only two published studies on the b-cell protective effects of DPP-4 inhibitors in T2DM animal models. Des-fluoro-sitagliptin treatment of mice with diabetes, induced by high fat feeding plus injection plus streptozotocin treatment, increased the number of insulin-positive b-cells and normalized b-cell mass (190). Insulin content and glucose-induced insulin secretion were also greatly improved. Combined treatment of db/db mice with vildagliptin and an angiotensin II receptor antagonist for 8 weeks also resulted in increased islet b-cell area and this was associated with increased b-cell proliferation and reduced apoptosis (191). However, although these results are extremely encouraging, there are no direct data available to support a similar preservation of islet mass in humans receiving DPP-4 inhibitor treatment, although indirect estimates have suggested sustained increases in insulin responses, possibly through b-cell protection (162). Homeostasis model assessment (HOMA) of b-cell function and proinsulin-to-insulin ratio measurements also showed improvements with sitagliptin monotherapy and sitagliptin add-on-to metformin (192).

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Differences between Responses to Incretin Enhancers and Incretin Mimetics DPP-4 inhibitors and incretin mimetics, such as exenatide, exert similar actions on glucagon and insulin secretion, with equivalent reductions in HbA1c levels. By contrast, whereas DPP-4 inhibitors have been found to be weight neutral or to induce only small reductions in body weight (193), exenatide treatment results in sustained decreases in body weight (194–196). Since high doses of GLP-1 exert profound inhibitory effects on food intake in rodents (197, 198), a component of this reduction is likely due to reduced calorie intake (196, 199). A recent study on obese and diabetic humans supports this conclusion (200). However, exenatide also slows gastric emptying (169) and exerts additional effects on gastrointestinal function that induce nausea and vomiting. Although such responses have been reported to be transient in most patients, they could contribute to the reductions in body weight. Nevertheless, reduced gastric emptying probably contributes to improvements in glucose disposal. The differences in responses between incretin mimetics and incretin enhancers are thought to be at least partly due to the pharmacological levels of GLP-1 receptor agonist available, but since a significant component of GLP-1 action is mediated via autonomic reflexes and central nervous pathways (201, 202) it is also possible that exenatide gains greater access than the native hormone to the relevant receptors (26). Despite these clear distinctions in effects, there may well be additional long-term benefits of DPP-4 treatment of T2DM that have yet to be revealed.

DPP-4 INHIBITORS IN TYPE 1 DIABETES Type 1 diabetes is an autoimmune disorder (2) and insulin injection and pancreas or islet transplantation are the only treatments currently available. Interest in the potential for incretin therapy resulted from studies showing that GLP-1 infusion reduced fasting hyperglycemia (203), glycemic excursions (204, 205) and requirements for insulin. Although not definitively established, it is believed that such effects were due to reduced glucagon secretion and delayed gastric emptying (206). DPP-4 (CD26) is thought to play an important role in immune function, and DPP-4 inhibitors have been demonstrated to modulate these actions by suppressing T-lymphocyte (T-cell) proliferation, Th1 cytokine production, and trans-endothelial migration (56, 58, 60, 207). Reports of an association between autoimmune disease and elevated CD26 þ cells (208–210) suggested that treatment with DPP-4 inhibitors may benefit T1DM patients by targeting both the autoimmune process and the loss of islet mass. Streptozotocin-treated rats (117, 211) and mice (212) have been used to study the effects of DPP-4 inhibitor treatment in models that involve both apoptotic and necrotic mechanisms, but without a major autoimmune component. Rats treated with Ile-Thia (20 mg/kg daily po) prior to, and following, STZ treatment exhibited improved weight gain and nutrient intake, as well as marked reductions in fed blood glucose levels and increased insulin responses. Examination of the pancreas from the DPP-4

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inhibitor-treated rats revealed eightfold greater pancreatic insulin content than untreated STZ-animals and increased numbers of small islets. Although it was unclear as to whether DPP-4 inhibition induced neogenesis of b-cells, or protected a specific subpopulation of islets, the latter is more likely. In order to determine whether DPP-4 inhibitor treatment is capable of protecting islets in vivo, effects on transplanted islet survival have been examined. The DPP-4 inhibitor sitagliptin (MK0431; Merck) was administered orally to C57BL/6 mice that had been rendered diabetic with STZ and islets transplanted under their kidney capsule. Islet survival was assessed with intraperitoneal glucose tolerance tests (IPGTT) and imaging performed, using positron emission tomography (PET) (212). Following islet transplantation, mice fed a normal diet rapidly lost their ability to regulate blood glucose, reflecting the suboptimal islet transplant. By contrast, the MK0431 group fully regulated blood glucose throughout the study and PET imaging demonstrated a profound protective effect of MK0431 on islet graft size (212). The first model of spontaneous rodent type 1 diabetes to be examined with DPP-4 inhibitor treatment was the biobreeding (BB) rat (117). Treatment with Ile-Thia, beginning at 3 weeks of age, delayed onset of diabetes, reduced the incidence by 20%, and improved glucose tolerance in both prediabetic and diabetic rats (Pospisilik et al., unpublished observations). More recently, the effect of MK0431 on transplanted islet survival in nonobese diabetic (NOD) mice was determined with similar metabolic studies and PET imaging to those used in the STZ-diabetes study. When NOD mice received MK0431 in their chow both prior to and postislet transplantation, islet graft survival was greatly prolonged, whereas posttreatment alone showed much smaller beneficial effects. MK0431 pretreatment was also shown to result in decreased insulitis in diabetic NOD mice as well as reducing in vitro migration of isolated splenic CD4 þ T lymphocytes. It therefore appears that DPP-4 inhibitor treatment can reduce the effect of autoimmunity on graft survival partially by decreasing the homing of CD4 þ T cells into pancreatic b-cells. Preliminary studies indicate that DPP-4 inhibitors produce these beneficial effects via influencing direct action of soluble DPP-4 on CD4 þ T cells and potentiating the incretin response (Kim et al., unpublished observations). A number of centers are now examining whether DPP-4 inhibitor treatment can prolong islet graft survival in humans.

FUTURE TRENDS Introduction of the incretin enhancers has provided an additional approach to diabetes treatment. A number of new inhibitors will likely be introduced in the near future and, from the clinical data available, those currently in trials exhibit similar beneficial characteristics to sitagliptin, vildagliptin, and saxagliptin. It is difficult to predict whether major improvements in efficacy and specificity can be achieved, since all produce high levels of inhibition and exhibit very low potency with other members of the DPP-4 family. However, it would clearly be advantageous to achieve similar efficacy to the incretin mimetics with respect to gastric emptying and body weight, without the gastrointestinal side effects. One possible approach is to combine an

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incretin enhancer with an agent that stimulates incretin secretion. Recently, it was reported that agonists of the G protein-coupled receptor GPR119 stimulated secretion of both GLP-1 and GIP and potentiated sitagliptin effects on glucose tolerance in rodents (213), thus demonstrating proof of principle. The combination of DPP-4 inhibition with GPR119 agonist activity in a single compound could therefore prove to be a valuable addition to the incretin-based therapies for T2DM (214). It is also unclear as to whether there are further beneficial effects of DPP-4 inhibitor treatment that have yet to be revealed. Human studies have largely focused on the contribution made by GLP-1 during DPP-4 inhibition, while neglecting a possible role for GIP, due to the b-cell resistance to GIP discussed earlier. However, it is unknown whether such resistance exists in other target organs. Additionally, it was recently demonstrated that normalizing circulating glucose levels in ZDF (215) or Px (161) rats restored GIP receptor expression. Responsiveness of T2DM patients to both GIP and GLP-1 has also been shown to improve greatly following nearnormalization of glucose levels by intensive treatment with insulin (157) or a sulfonylurea drug (216). These findings indicate that, as circulating glucose stabilizes, further actions of the incretins may be potentiated, resulting in additional benefits from DPP-4 inhibitor therapy. Both GIP and GLP-1 have been shown to exert multiple effects on different body systems, suggesting that alternative therapeutic targets are available (Figure 12.9). This could be particularly important in the areas of bone metabolism and cardiovascular disease. Long-term use of thiazolidinediones has recently been linked to increased osteoclastic activity and bone loss, particularly in

Figure 12.9 Both GIP and GLP-1 exert a number of actions, in addition to those on insulin and glucagon secretion. Some of these are probably impacted upon by DPP-4 therapy.

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postmenopausal women (217). In preclinical studies, GIP was shown to preserve bone mass in rodents (218), probably via direct actions on both osteoblasts and osteoclasts (218, 219), whereas GLP-1 acts indirectly on bone via stimulation of calcitonin secretion (220). Whether such positive effects on bone metabolism occur in humans is currently under study. Cardiovascular disease is the most important complication of diabetes (221). Both incretins appear to contribute to regulation of the cardiovascular system (33, 222–224). GIP facilitates delivery of nutrients during a meal via effects on the hepato-portal system (33, 222). GLP-1 increases myocardial glucose uptake and coronary blood flow (224), and improves cardiac performance in humans (224, 225). In a number of animal models, GLP-1 has been shown to exert protective effects on the endothelium (224). The potential effects of DPP-4 inhibitor therapy on microvascular and macrovascular complications of diabetes are central to future long-term clinical trials. It is likely, therefore, that future research will reveal a number of beneficial effects of DPP-4 inhibitor treatment in addition to their effects on glucose homeostasis.

ACKNOWLEDGMENTS Studies on P32/98 described in the review were generously supported by funding to CMcI and RP from the Canadian Institutes of Health Research, the Canadian Diabetes Association, and the Canadian Foundation for Innovation, from the Michael Smith Research Foundation and Killam Trust Foundation (JAP) and the Federal Ministry of Education and Research, Germany and the Department of Science and Technology of Sachsen Anhalt (H-UD). Studies on MK0431 were generously supported by funding from Merck Frosst, Canada.

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190. MU, J., J. WOODS, Y.-P. ZHOU, R.S. ROY, Z. LI, E. ZYCBAND, Y. FENG, L. ZHU, C. LI, A.D. HOWARD, D.E. MOLLER, N.A. THORNBERRY, and B. ZHANG. 2006. Chronic inhibition of dipeptidyl peptidase-4 with a sitagliptin analog preserves pancreatic b-cell mass and function in a rodent model of type 2 diabetes. Diabetes 55:1695–1704. 191. CHENG, Q., P.K. LAW, M. DE GASPARO, P.S. LEUNG, Q. CHENG, P.K. LAW, M. DE GASPARO, and P.S. LEUNG. 2008. Combination of the dipeptidyl peptidase IV inhibitor LAF237 [(S)-1-[(3-hydroxy-1-adamantyl) ammo]acetyl-2-cyanopyrrolidine] with the angiotensin II type 1 receptor antagonist valsartan [N-(1oxopentyl)-N-[[2’-(1H-tetrazol-5-yl)-[1, 1’-biphenyl]-4-yl]methyl]-L-valine] enhances pancreatic islet morphology and function in a mouse model of type 2 diabetes. J Pharmacol Exp Ther 327:683–691. 192. SUAREZ-PINZON, W.L., J.R. LAKEY, A. RABINOVITCH, W.L. SUAREZ-PINZON, J.R.T. LAKEY, and A. RABINOVITCH. 2008. Combination therapy with glucagon-like peptide-1 and gastrin induces beta-cell neogenesis from pancreatic duct cells in human islets transplanted in immunodeficient diabetic mice. Cell Transplant 17:631–640. 193. VELLA, A., G. BOCK, P.D. GIESLER, D.B. BURTON, D.B. SERRA, M.L. SAYLAN, B.E. DUNNING, J.E. FOLEY, R.A. RIZZA, and M. CAMILLERI. 2007. Effects of dipeptidyl peptidase-4 inhibition on gastrointestinal function, meal appearance, and glucose metabolism in type 2 diabetes. Diabetes 56:1475–1480. 194. DEFRONZO, R.A., R.E. RATNER, J. HAN, D.D. KIM, M.S. FINEMAN, and A.D. BARON. 2005. Effects of exenatide (exendin-4) on glycemic control and weight over 30 weeks in metformin-treated patients with type 2 diabetes. Diabetes Care 28:1092–1100. 195. DRUCKER, D.J., J.B. BUSE, K. TAYLOR, D.M. KENDALL, M. TRAUTMANN, D. ZHUANG, L. PORTER, D.-S. GROUP, D.J. DRUCKER, J.B. BUSE, K. TAYLOR, D.M. KENDALL, M. TRAUTMANN, D. ZHUANG, and L. PORTER. 2008. Exenatide once weekly versus twice daily for the treatment of type 2 diabetes: a randomised, open-label, non-inferiority study. Lancet 372:1240–1250. 196. DEFRONZO, R.A., T. OKERSON, P. VISWANATHAN, X. GUAN, J.H. HOLCOMBE, L. MACCONELL, R.A. DEFRONZO, T. OKERSON, P. VISWANATHAN, X. GUAN, J.H. HOLCOMBE, and L. MACCONELL. 2008. Effects of exenatide versus sitagliptin on postprandial glucose, insulin and glucagon secretion, gastric emptying, and caloric intake: a randomized, cross-over study. Curr Med Res Opin 24:2943–2952. 197. TURTON, M.D., D. O’SHEA, I. GUNN, S.A. BEAK, C.M. EDWARDS, K. MEERAN, S.J. CHOI, G.M. TAYLOR, M.M. HEATH, P.D. LAMBERT, J.P. WILDING, D.M. SMITH, M.A. GHATEI, J. HERBERT, and S.R. BLOOM. 1996. A role for glucagon-like peptide-1 in the central regulation of feeding. Nature (London, UK) 379:69–72. 198. MORAN, T.H. 2006. Gut peptide signaling in the controls of food intake. Obesity 14(Suppl 5):250S–253S. 199. EDWARDS, C.M., S.A. STANLEY, R. DAVIS, A.E. BRYNES, G.S. FROST, L.J. SEAL, M.A. GHATEI, and S.R. BLOOM. 2001. Exendin-4 reduces fasting and postprandial glucose and decreases energy intake in healthy volunteers. Am J Physiol Endocrinol Metab 281:E155–E161. 200. GUTZWILLER, J.P., J. DREWE, B. GOKE, H. SCHMIDT, B. ROHRER, J. LAREIDA, and C. BEGLINGER. 1999. Glucagon-like peptide-1 promotes satiety and reduces food intake in patients with diabetes mellitus type 2. Am J Physiol 276:R1541–R1544. 201. IMERYUZ, N., B.C. YEGEN, A. BOZKURT, T. COSKUN, M.L. VILLANUEVA-PENACARRILLO, and N.B. ULUSOY. 1997. Glucagon-like peptide-1 inhibits gastric emptying via vagal afferent-mediated central mechanisms. Am J Physiol 273:G920–G927. 202. NAGELL, C.F., A. WETTERGREN, C. ORSKOV, and J.J. HOLST. 2006. Inhibitory effect of GLP-1 on gastric motility persists after vagal deafferentation in pigs. Scand J Gastroenterol 41:667–672. 203. CREUTZFELDT, W.O., N. KLEINE, B. WILLMS, C. ORSKOV, J.J. HOLST, and M.A. NAUCK. 1996. Glucagonostatic actions and reduction of fasting hyperglycemia by exogenous glucagon-like peptide I(7-36) amide in type I diabetic patients. Diabetes Care 19:580–586. 204. DUPRE, J., M.T. BEHME, I.M. HRAMIAK, P. MCFARLANE, M.P. WILLIAMSON, P. ZABEL, et al. 1995. Glucagon-like peptide I reduces postprandial glycemic excursions in IDDM. Diabetes 44:626–630. 205. GUTNIAK, M., C. ØRSKOV, J.J. HOLST, B. AHRE´N, and S. EFENDIC. 1992. Antidiabetogenic effect of glucagon-like peptide-1 (7-36) amide in normal subjects and patients with diabetes mellitus. New Engl J Med 326:1316–1322.

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223. SARACENI, C., T.L. BRODERICK, C. SARACENI, and T.L. BRODERICK. 2007. Effects of glucagon-like peptide-1 and long-acting analogues on cardiovascular and metabolic function. Drugs RD 8:145–153. 224. NYSTRO¨M, T. 2008. The potential beneficial role of glucagon-like peptide-1 in endothelial dysfunction and heart failure associated with insulin resistance. Horm Metab Res 40:593–606. 225. SOKOS, G.G., L.A. NIKOLAIDIS, S. MANKAD, D. ELAHI, R.P. SHANNON, G.G. SOKOS, L.A. NIKOLAIDIS, S. MANKAD, D. ELAHI, and R.P. SHANNON. 2006. Glucagon-like peptide-1 infusion improves left ventricular ejection fraction and functional status in patients with chronic heart failure. J Card Fail 12:694–699.

Chapter

13

Sodium Glucose Cotransporter 2 Inhibitors MARGARET RYAN AND SERGE A. JABBOUR Division of Endocrinology, Diabetes, and Metabolic Diseases, Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA, USA

INTRODUCTION In nondiabetic individuals, plasma glucose levels are tightly regulated to maintain a level of 80–110 mg/dL to ensure that adequate energy is provided, primarily to the brain and, secondarily, to the other organs of the body. This regulation of plasma glucose occurs via several mechanisms involving multiple different organ systems (1). Glucose homeostasis is affected by several major factors including glucose absorption from ingested nutrients via the small intestine, glucose production in the liver and glucose consumption by almost all tissues. Insulin, secreted from the b-cells of the pancreas, promotes the uptake of glucose by peripheral tissues for storage or consumption and inhibits glucose production by the liver (see Figure 13.1). The kidney plays a vital role in homeostasis by performing several functions, which are essential in maintaining fluid and electrolyte balance as well as glucose balance (2, 3). Glucose is the fuel-providing energy for the organs of the body and the major source of glucose is carbohydrate in food. The average human body has glucose stores of about 450 g and utilizes about 250 g of glucose daily under normal activity levels. Of the 250 g of glucose used per day, approximately half, or 125 g, is used by the brain alone, while the rest is utilized by the other organs and tissues of the body (4). The typical Western diet provides approximately 180 g of glucose per day, with the remainder of the daily requirements for the body coming from glucose stores as well as from gluconeogenesis in the liver and, to a lesser extent, the kidneys. In fact, humans can survive on a completely carbohydrate free diet, deriving all of their daily glucose requirements from gluconeogenesis alone. Despite wide fluctuations in

Metabolic Syndrome: Underlying Mechanisms and Drug Therapies Edited by Minghan Wang Copyright 2011 John Wiley & Sons, Inc.

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Chapter 13 Sodium Glucose Cotransporter 2 Inhibitors

Liver

Kidney

Insulin -

+ Gluconeogenesis

Pancreas [Glucose]

Intestine

Blood

+ Insulin

Peripheral Tissues

Figure 13.1 Glucose homeostasis: Insulin, secreted from the b-cells of the pancreas, promotes the uptake of glucose by peripheral tissues for storage or consumption and inhibits glucose production by the liver.

delivery (meals) and removal (exercise) from the circulation, serum glucose is maintained within a narrow range (5). Glucose homeostasis is important in order to prevent hyper- and hypoglycemia.

KIDNEY PHYSIOLOGY The functional unit of the kidney is the nephron; approximately 1,300,000 nephrons are found in each kidney (6). Each nephron has a glomerulus and a long tubule with three main components: proximal tubule, loop of Henle, and distal tubule. In addition to gluconeogenesis, the kidneys also play a major role in the body’s normal regulation of glucose by reabsorbing the approximately 180 g of glucose that are filtered through the glomeruli each day (7). Plasma glucose is neither protein bound nor complexed and is filtered freely at the glomerulus. The amount of glucose filtered is defined as the product of plasma glucose concentration and glomerular filtration rate (GFR) (2, 6). In nondiabetic patients with normal serum glucose levels, all of the glucose filtered through the kidneys is reabsorbed and none is excreted in the urine. However, as plasma glucose concentrations exceed 180–200 mg/dL, the kidney begins to excrete glucose into the urine (glucosuria) (2, 6–8). In normal renal physiology, 90% of the glucose is reabsorbed in the proximal convoluted tubule via

Glucose Transporters

361

the sodium glucose cotransporter type 2 (SGLT2). The remaining 10% of glucose is reabsorbed via the sodium glucose cotransporter type 1 (SGLT1) located in the descending limb of the proximal tubule (2, 6, 7).

GLUCOSE TRANSPORTERS The lipid bilayer that makes up the plasma membrane of all human cells is impermeable to hydrophilic molecules such as glucose. At all the levels of glucose transport mentioned above, glucose must therefore be transported across the lipid bilayer via carrier proteins. There are two main classes of glucose transporters in the body: The sodium-coupled glucose transporters (SGLTs) and the glucose transporter facilitators (GLUTs) (9, 10). The GLUTs are passive transporters, whereas the SGLTs are active cotransporters, or symporters, of glucose. The GLUT family of transporters facilitates the passage of glucose down concentration gradients across cell membranes. The SGLT family of transporters, by contrast, allow for active transport of glucose against concentration gradients (11). SGLTs are considered secondary active transporters as the energy for the overall process comes from ATP, which is consumed by sodium–potassium ATPase to maintain the intracellular–extracellular sodium gradient allowing the SGLTs to cotransport other molecules along with sodium as the sodium travels down its electrochemical gradient (9–11). In both the small intestine and the kidney, the SGLT glucose cotransporters work in tandem with GLUT2 to transport glucose into the bloodstream from either the gut lumen or the kidney filtrate (4, 9, 10). GLUT1 is a low-affinity transporter found mostly on erythrocytes and on endothelial cells of the brain, while GLUT2 is expressed on pancreatic b-cells, and in the liver, kidney, and small intestine (12). In addition to passive glucose transport, GLUT2 is involved with the sensing of plasma glucose levels by the pancreatic b-cells. As low-affinity glucose transporters, GLUT1 and GLUT2 cannot be saturated at physiologic glucose levels and thus the rate of glucose transport is dependant solely on the extracellular glucose concentration (11, 12). GLUT4 is a high-affinity passive glucose transporter found on insulin-sensitive cells such as those of skeletal muscle and adipose tissue and, in response to insulin, increased concentrations of GLUT4 receptors are translocated from intracellular membranes to the plasma membrane to increase glucose transport (12). In SGLTs, as previously mentioned, the transport of glucose is coupled to the energetically favored transport of sodium across the plasma membrane, down a concentration gradient and a membrane potential. In the steady state, the sodium is then transported back out of the cell via the sodium–potassium ATPase. The two SGLT molecules mostly involved with glucose transport are SGLT1 and SGLT2. SGLT2 is a member of a larger group of sodium substrate cotransporters, the solute carrier family 5A (SLC5A) gene family, of which there are 12 human genes expressed in tissues ranging from epithelia to the central nervous system (13). Six of these gene products, named SGLTs, actively transport sugars coupled to sodium ion transport as a driving force. This mechanism of action differentiates the SGLTs from the

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Chapter 13 Sodium Glucose Cotransporter 2 Inhibitors

facilitative-diffusion glucose transporters (GLUTs) that are involved in glucose uptake across the plasma membrane; for example, into skeletal muscle, adipose, and other tissues. SGLT2 is a 672-amino acid, high-capacity, low-affinity transporter expressed in the S1 segment of the proximal tubule, which is believed to mediate the majority of renal glucose reabsorption (10). The type 1 sodium–glucose cotransporter SGLT1, is primarily expressed in the small intestine but is also present in the S3 segment, or descending limb, of the proximal tubule. SGLT1 is a high-affinity, low-capacity glucose/galactose transporter that is thought to account for only a small proportion of renal glucose reabsorption. In addition, SGLT1 has a 10-fold greater affinity for galactose over glucose (1). Other members of the SLC5A family have been cloned and characterized (14) (see Table 13.1). Glucose digested from meals is absorbed via SGLT1 located in an apical site of the small intestinal epithelia. Blood glucose in the circulation is continuously filtered in the glomeruli of the kidneys, and then

Table 13.1

SLC5A Gene Products (14)

GENE

Protein

Substrates

Tissue Distribution

SLC5A1

SGLT1

Glucose and galactose

SLC5A2 SLC5A3

SGLT2 SMIT

Glucose Myo-inositol

SLC5A4

SGLT3

Glucose sensor

SLC5A5

NIS

Iodide

SLC5A6

SMVT

SLC5A7 SLC5A8

CHT SMCT1

SLC5A9

SGLT4

SLC5A10 SLC5A11

SGLT5 SGLT6

SLC5A12

SMCT2

Pantothenate, biotin, and lipoate Choline Short-chain fatty acids, lactate, and nicotinate Mannose, glucose, fructose, 1,5-AG, and galactose Glucose and galactose Myo-inositol, glucose, xylose, and chiro-inositol Short-chain fatty acids, lactate, and nicotinate

Small intestine, heart, trachea, and kidney Kidney Thyroid, testis, kidney, lung, and trachea Small intestine, uterus, lungs, thyroid, and testis Thyroid, salivary gland, and stomach Placenta, testis, skeletal muscle, liver, and small intestine Spinal cord Thyroid, trachea, kidney, and prostate Small intestine, kidney, liver, stomach, and lung Kidney Spinal cord, kidney, brain, and small intestine Kidney and small intestine

1,5-AG: 1,5-anhydro-D-glucitol; CHT: choline transporter; NIS: Na þ /iodide transporter; SMCT: sodium-coupled monocarboxylate transporter; SMIT: sodium-dependent myo-inositol transporters; SMVT: sodium-dependent multivitamin transporter.

Pathophysiology of Diabetes

K+

363

Na/K ATPase

Na+ Renal filtrate

Plasma Proximal convoluted tubule cell Glucose

Na+ Glucose

GLUT2 SGLT2

Figure 13.2 Proximal convoluted tubule cell showing the secondary active transport of glucose from the renal filtrate back into the plasma via SGLT2.

reabsorbed in the renal proximal tubules via SGLT2 and to a lesser extent via SGLT1 (see Figure 13.2).

PATHOPHYSIOLOGY OF DIABETES The normal physiology controlling glucose homeostasis has already been discussed. But, what pathophysiologic changes occur in diabetic patients? In persons with type 1 diabetes, there is loss of pancreatic b-cells with resultant absence of insulin secretion. In type 2 diabetes, there is a combination of impaired secretion of insulin from the pancreatic b-cells as well as a tissue resistance to the normal effects of insulin (15, 16). The net result of these disorders is impairment of glucose uptake by peripheral tissues and an increase in the circulating blood glucose levels. The increase in blood glucose levels in diabetic patients is seen in the kidneys as an increase in the filtered glucose load, as the filtered glucose load is directly linked to the plasma glucose concentration. The active transport and reabsorption of glucose from the kidney filtrate back into the blood, as previously mentioned, through SGLT2 and, to a lesser extent SGLT1, has a maximal rate. Once the maximal reabsorptive capacity of the kidney is exceeded, all glucose in excess of this maximal resorptive capacity will be excreted into the urine. The maximal resorptive capacity (Tm) of the kidney proximal tubule for glucose is variable between individuals, but in nondiabetic individuals it is approximately 375 mg/min (17) and, in healthy nondiabetic individuals, the normal filtered glucose load is much less than this maximum. Thus, all of the glucose that is filtered is reabsorbed and none appears in the urine. In persons with chronic hyperglycemia (i.e., diabetic patients), there is upregulation of the active glucose cotransporters SGLT2 as well as SGLT1 to allow for an increased maximal resorptive capacity of the kidney proximal tubule for glucose (4, 17). Despite this increased capacity, the severe hyperglycemia frequently encountered in diabetic patients often exceeds Tm and excess glucose that cannot be reabsorbed gets excreted into the urine. When the

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filtered glucose load exceeds the Tm, the glucose excretion rate rises linearly and parallels the filtered load (17, 18). Consistent with their physiological action to absorb glucose from the intestinal lumen or return to the bloodstream the glucose filtered into the nephron, SGLT1, and SGLT2 have been recognized as attractive targets for a new class of antidiabetic drugs (19, 20). One therapeutic approach is the suppression of intestinal glucose absorption via SGLT1 inhibition; this, however, has not yet been reported in clinical development. The other target is to suppress renal glucose reabsorption and effectively increase urinary glucose excretion via SGLT2 inhibition.

SGLT INHIBITION Reabsorption of as much of the filtered glucose load as possible may be adaptive in normal individuals to preserve energy; in diabetic individuals, however, this is maladaptive as it perpetuates the hyperglycemia in the plasma. One hypothesis would be that in persons with diabetics, who suffer from chronic hyperglycemia, it would be desirable to be able to excrete more of this excess glucose load in an effort to alleviate the hyperglycemia. When considering the possible implications of SGLT1 or SGLT2 inhibition, it is useful to look at the rare inherited disorders of SGLT dysfunction. Intestinal glucose–galactose malabsorption (GGM) is a rare autosomal recessive inherited disorder caused by mutations in the SGLT1 gene (21, 22). It was initially described in the 1960s (23, 24). Persons with GGM experience severe, even life-threatening, diarrhea with carbohydrate ingestion that can be seen as early as the neonatal period (21). The malabsorptive diarrhea can be ameliorated by removal of glucose, galactose, and lactose from the diet. In GGM, there is only mild glucosuria. Another rare disorder of sodium–glucose transport is termed familial renal glucosuria (FRG) caused by mutations in the SGLT2 gene (25, 26). In contrast to GGM, no diarrhea is seen in FRG and there is no intestinal glucose malabsorption. In FRG, however, the renal glucosuria may be quite pronounced, depending upon the type of mutation in the SGLT2 gene. Even in patients with severe FRG, with almost no ability to reabsorb glucose from the kidney, there have been no reports of impairment of kidney function or other side effects from the chronic glucosuria. FRG is thus considered a benign condition. In some individuals with severe forms of FRG, there is evidence for moderate volume depletion as evidenced by high plasma renin activity and serum aldosterone levels (4.2- and 2.7-fold the upper limit of normal) (27). Study of these inherited diseases of metabolism would indicate that the SGLT2 transporter, as opposed to SGLT1, appears to be the major transporter involved with renal glucose reuptake from the glomerular filtrate.

PHLORIZIN Given the potential for significant diarrheal side effects with SGLT1 inhibition, as well the unknown effects of blocking SGLT1 transporters on myocardial cells,

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researchers have focused less on SGLT1 inhibition and more on SGLT2 inhibition as a potential treatment for diabetes. SGLT2 is located exclusively in the kidneys and in the drugs affecting this molecule should thus have very little effect on other tissues. The proof of concept for the efficacy of SGLT inhibition came with a molecule called phlorizin. Phlorizin is a b-D-glucoside (see Table 13.2) found naturally in the bark of apple trees that acts as a nontransported competitive inhibitor of sodium/glucose cotransport at both SGLT1 and SGLT2 (4). Phlorizin was first isolated in 1835 from the root bark of the apple tree (14, 28) and has since been found to be a natural, potent SGLT specific inhibitor. It has been shown in several animal studies to lower both fasting and postprandial blood glucose levels without causing any hypoglycemia (29). It has also been shown to decrease insulin resistance at the peripheral tissue level (17, 29). Phlorizin, unfortunately, is poorly absorbed from the intestine and is easily hydrolyzed by lactase-phlorizin hydrolase (14). It is also nonspecific, inhibiting both SGLT1 and SGLT2 and thus has never been developed as a potential diabetes treatment despite its use as a proof of concept for SGLT inhibition. Phlorizin has, however, become the model upon which many SGLT inhibitors in development are based.

T-1095 T-1095 is a phlorizin analogue (see Table 13.2) developed by Tanabe Pharma Corp. as a prodrug that is more readily absorbable in oral form and is resistant to the hydrolization and degradation that phlorizin encounters in vivo (30). T-1095 is inactive in its ingested form and thus does not affect the SGLT1 molecules lining the small intestine. It is, however, readily absorbed from the intestine into the bloodstream where it is converted into its active form (31). The active form of T-1095 is then filtered by the kidneys where it acts to competitively inhibit both SGLT2 in the proximal convoluted tubule and SGLT1 in the descending limb, for a net effect of increased glucosuria and improved blood glucose levels (32). Animal studies in diabetic rats show that treatment with T-1095 leads to an increase in glucosuria accompanied by a decrease in plasma glucose levels and a decrease in hemoglobin A1c (HbA1c) over time (32, 33). Development of the T-1095 compound was discontinued after Phase II trials, perhaps due to the lack of selectivity of the T-1095 compound between SGLT1 and SGLT2. Several other SGLT2 specific SGLT inhibitors are, however, in various stages of active development and testing.

SERGLIFLOZIN Sergliflozin is a molecule developed by Kissei Pharmaceutical Co. Ltd based on benzylphenol glucoside (see Table 13.2). Its structural skeleton differs somewhat from that of phlorizin. Sergliflozin itself is an inactive prodrug. Similar to T-1095, sergliflozin is administered orally and then converted into an active metabolite in vivo after absorption. Unlike T-1095 and phlorizin, however, sergliflozin acts as a selective SGLT2 inhibitor, showing a 296-fold greater selectivity for the SGLT2 molecule

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Table 13.2

Structures of Different SGLT Inhibitors

Phlorizin

T-1095

Sergliflozin

Dapagliflozin

Remogliflozin

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367

versus the SGLT1 molecule (34). Animal studies in rodents and dogs reveal that oral administration of sergliflozin causes a dose-dependent glucosuria after an oral glucose load by lowering Tm of the kidney for glucose by greater than 60% (34). In hyperglycemic animal models, the administration of sergliflozin leads to lower blood glucose levels accompanied by lower circulating insulin levels, showing that sergliflozin has its effects on blood glucose management independent of insulin secretion. Also of note, in normoglycemic fasting animal models, there was no significant change in blood glucose levels after sergliflozin administration, indicating that the risk for hypoglycemia with serglifozin should be low (35). A study reported in abstract form showed that oral sergliflozin resulted in dosedependent urinary glucose excretion in animals and humans and may provide a new and unique approach to the treatment of diabetes mellitus (36). The study was conducted to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics of sergliflozin over 14 days of dosing in healthy overweight and obese human subjects. Eighteen subjects at 18–55 years of age with body mass index (BMI) of 25–35 kg/m2, were recruited in two cohorts. Subjects in cohort A received 500 mg of sergliflozin (n ¼ 6) or placebo (n ¼ 3) three times daily while subjects in cohort B received 1000 mg of sergliflozin (n ¼ 6) or placebo (n ¼ 3) three times daily for 14 days. Both dose regimens of sergliflozin were generally well tolerated. PK parameters were stable over 2 weeks of dosing with no accumulation of plasma concentrations of sergliflozin or its major metabolites. Sergliflozin caused a considerable dose-dependent increase in urinary glucose excretion (36). High doses of sergliflozin did not provoke hypoglycemia in nondiabetic subjects. Mean plasma glucagon-like peptide-1 (GLP-1) concentrations were increased by sergliflozin compared to placebo on days 1 and 14. There was a body weight reduction of 1.5 kg compared to placebo in sergliflozin-treated subjects from baseline to day 15 (36). Sergliflozin caused increases in urine electrolyte levels on day 1, which resolved by day 14 without any effect on serum electrolytes or on the calculated creatinine clearance. Sergliflozin caused a dose-dependent negative fluid balance on day 1, but this effect had resolved by day 14 in the 500 mg group and was less marked in the 1000 mg group (36). The most frequently reported adverse events were headache, dizziness, flatulence, and nausea (36). Sergliflozin administered over 2 weeks resulted in predictable pharmacokinetics, a urinary glucose excretion consistent with SGLT2 inhibition, and was generally well tolerated without symptoms or signs of hypoglycemia. Two additional clinical studies were conducted to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics of oral single doses of sergliflozin and reported in abstract form (37). Both studies were double-blind, randomized, placebocontrolled, single-dose escalation crossover studies. In study 1, sergliflozin doses ranged from 5 to 500 mg in 14 healthy males. In study 2, doses ranged from 50 to 500 mg in 8 subjects with type 2 diabetes. Dose proportionality was observed for pharmacokinetic parameters and no differences were observed between healthy and diabetic subjects. In both populations, there was a dose-dependent increase in urinary glucose excretion that plateaued, suggesting that glucose reuptake was maximally inhibited with the higher doses (37). The duration of urinary glucose excretion

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paralleled the plasma concentrations of the active drug moiety. Following an oral glucose tolerance test in subjects with type 2 diabetes, sergliflozin at 500 mg decreased plasma glucose concentrations over 4 h from 18.3 mmolh/L to 11.2 mmolh/L. There were small and transient increases in the urinary excretion of electrolytes, but no overall increase over 24 h (37). All doses were generally well tolerated. In healthy subjects, the most common adverse effects were headache and sore throat. In subjects with type 2 diabetes, the most common adverse events were headache and dyspepsia (37). In conclusion, sergliflozin was well tolerated, resulted in pharmacodynamic changes consistent with renal SGLT2 inhibition, and lowered plasma glucose concentrations in subjects with type 2 diabetes.

DAPAGLIFLOZIN Dapagliflozin, a drug under development by Bristol-Myers Squibb and AstraZeneca, is another SGLT2 selective inhibitor. In in vitro studies with various animal cells, dapagliflozin showed a 200–1200-fold selectivity for SGLT2 inhibition versus SGLT1 inhibition and appeared to be approximately 5-fold more potent at inhibiting the SGLT2 transporter as compared to sergliflozin (17, 38). Dapagliflozin is active in its ingested form; unlike sergliflozin and T-1095, it is not a prodrug. In rat models, dapagliflozin has been shown to have a bioavailability of 84% and a pharmacologic half-life of 4.6 h (17, 38). It circulates in the plasma largely bound to albumin, with a free fraction of approximately 4% in humans. Dapagliflozin has a c-glucoside chemical structure (see Table 13.2), which prologs its half-life and duration of action compared to phlorizin and sergliflozin. The longer half-life of dapagliflozin allows it to be developed as a possible once-daily oral antidiabetic drug. The effect of dapagliflozin on glucose homeostasis in normal and diabetic rats was reported in 2007 (39). In diabetic rats, dapagliflozin acutely induced renal glucose excretion at doses ranging from 0.01–1.0 mg/kg of body weight without inducing hypoglycemia. Additionally, as early as 2 h after a single oral dose, there was a statistically significant reduction in plasma glucose levels in diabetic rats treated with dapagliflozin compared to untreated diabetic rats of 101 and 128 mg/dL, at doses of 0.1 and 1.0 mg/kg, respectively (both p-values were less than 0.0001) (39). The safety, tolerability, pharmacokinetics, and pharmacodynamics of the drug were evaluated in single-ascending-dose (SAD; 2.5–500 mg) and multiple-ascending-dose (MAD; 2.5–100 mg daily for 14 days) studies in healthy subjects (40). Dapagliflozin exhibited dose-proportional plasma concentrations with a half-life of 17 h. The amount of glucosuria was also dose dependent. Cumulative amounts of glucose excreted on day 1, relating to doses from 2.5–100 mg (MAD), ranged from 18 to 62 g; day 14 values were comparable to day 1 values, with no apparent changes in glycemic parameters. Doses of 20–50 mg provided close-to-maximal SGLT2 inhibition for at least 24 h. Dapagliflozin demonstrated pharmacokinetic (PK) characteristics and dose-dependent glucosuria that are sustained over 24 h, which indicates that it is suitable for administration in once-daily doses (40). In a 14-day, Phase IIa clinical trial, the safety profile of multiple doses of dapagliflozin administered alone or concomitantly with metformin was evaluated in

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subjects with type 2 diabetes (41). This double-blind, placebo-controlled, randomized, parallel-group study was performed in 47 subjects (age 18–77) with established type 2 diabetes whowere either drug-na€ıve or on a stable dose of metformin for at least 4 weeks prior to randomization. At entry, HbA1c levels were between 6% and 10%, and the fasting serum glucose (FSG) was less than or equal to 240 mg/dL. Subjects were randomized to receive either placebo (n ¼ 8) or dapagliflozin at 5 mg (n ¼ 11), 25 mg (n ¼ 12), or 100 mg (n ¼ 16) once daily for 14 days in addition to their stable metformin dose and/or diet alone in an in-patient clinical research unit. The primary end point of the study was to assess both the safety and the tolerability profiles of multiple doses of dapagliflozin in subjects with type 2 diabetes. The secondary end points of the study included assessing the fasting serum glucose and postchallenge glucose excursion. On day 13, the FSG was significantly reduced in participants receiving dapagliflozin with or without metformin as compared to their FSG levels 2 days prior to first dose by 11.7% (p < 0.05), 13.3% (p < 0.05), and 21.8% (p < 0.001) for dapagliflozin at 5, 25, and 100 mg, respectively (41). In contrast, FSG was reduced by 6.3% in participants receiving placebo with or without metformin. There were no discontinuations due to adverse events and no serious adverse events occurred. Hypoglycemia was reported as adverse events in two subjects receiving dapagliflozin coadministered with metformin. There were two events of vulvovaginal infection in the study (one subject receiving dapagliflozin alone and one subject receiving dapagliflozin þ metformin). Adverse events occurred with similar frequency in subjects receiving dapagliflozin or placebo (41). The most frequently reported adverse events were constipation (n ¼ 7; 1/19 on dapagliflozin þ metformin, 3/20 on dapagliflozin alone, 2/6 on placebo þ metformin and 1/2 on placebo alone), nausea (n ¼ 5; 4/19 on dapagliflozin þ metformin and 1/6 on placebo þ metformin), and diarrhea (n ¼ 4; 3/19 on dapagliflozin þ metformin and 1/6 on placebo þ metformin) (41). A dose-ranging monotherapy study describes the efficacy, safety, and laboratory data for dapagliflozin treatment over 12 weeks (42). This was a prospective, 12-week, randomized, parallel-group, double-blind, placebo-controlled study, with a 2-week diet/exercise placebo lead-in, and 4-week follow-up. Three hundred and eighty-nine drug-na€ıve type 2 diabetic patients were equally randomized to once-daily dapagliflozin at 2.5, 5, 10, 20, or 50 mg, metformin XR at 750 mg force-titrated at week 2 to 1500 mg, or placebo. Patients were between 18 and 79 years of age and had baseline HbA1c of 7–10%. At week 12, all dapagliflozin groups achieved significant reductions in mean HbA1c change from baseline versus placebo. Adjusted mean reductions were from 0.55% to 0.90% for dapagliflozin, 0.18% for placebo, and 0.73% for metformin (42). FPG reductions were apparent by week 1 in all dapagliflozin groups. By week 12, adjusted mean FPG reductions were 16 to 31 mg/dL (dapagliflozin), 6 mg/dL (placebo), and 18 mg/dL (metformin), demonstrating dose-related FPG decreases and statistically significant reductions in the 5- to 50 mg dapagliflozin groups versus placebo. Adjusted mean postprandial plasma glucose (PPG) AUC reductions from baseline were 7053 to 10149 mgmin/dL (dapagliflozin), 3182 mgmin/dL (placebo), and 5891 mgmin/dL (metformin). Total body weight reductions occurred in all groups. Mean percent reductions at week 12 were 2.5% to 3.4% (dapagliflozin), 1.2% (placebo), and 1.7% (metformin).

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Generally, adverse events were reported at similar frequencies across all groups (42). No deaths or drug-related serious adverse events occurred. Hypoglycemic events were reported in 6–10% of dapagliflozin-treated patients with no dose relationship, 4% for placebo, and 9% for metformin. There were no symptomatic hypoglycemic events with a finger stick glucose 50 mg/dL. Infections of the urinary tract were seen in 5–12% of dapagliflozin-treated patients with no clear dose relationship, versus 6% for placebo and 9% for metformin. Genital infections were seen in 2–7% of dapagliflozin-treated patients, 0% for placebo, and 2% for metformin. There was no clinically meaningful change in estimated GFR in any group. This study demonstrated clinical efficacy of inhibiting renal glucose reabsorption with dapagliflozin in type 2 diabetic patients, and relative safety across numerous doses (42). Another trial was conducted to evaluate the efficacy and safety of dapagliflozin (Dapa) as an add-on to metformin (Met) over 24 weeks in type 2 diabetic patients inadequately controlled with Met alone (43). This randomized, double-blind, placebo-controlled, multicenter trial in North America and South America enrolled patients with type 2 diabetes, in the 18–77 years of age group. Eligible patients had inadequate glycemic control (HbA1c 7.0–10.0%) on stable dosing with Met 1500 mg/day. After a 2-week lead-in phase, 546 patients were equally randomized to oncedaily Dapa 2.5, 5, and 10 mg, or placebo (PBO), plus open-label Met. The primary end point was change in HbA1c at week 24. Other end points included changes in fasting plasma glucose (FPG) and % change in total body weight at week 24. Compared to add-on PBO at week 24, all add-on Dapa groups showed significant mean reductions from baseline in HbA1c (0.67%, 0.70%, and 084% for the doses of 2.5, 5, and 10 mg, respectively) (43). End point reductions in FPG were also significant for all Dapa groups versus PBO. Greater proportions of patients in all Dapa groups (33%, 37.5%, and 40.6% for 2.5, 5, and 10 mg, respectively) achieved HbA1c <7.0% at week 24 than patients on PBO (25.9%) (43). Weight loss with Dapa was continuous and progressive. More patients treated with Dapa achieved weight decreases 5% compared to PBO. Generally, adverse events were balanced across all groups. Compared with PBO, rates of urinary tract infections were similar or lower for Dapa (PBO, 8.0%; Dapa 2.5 mg, 4.4%; Dapa 5 mg, 7.3%; Dapa 10 mg, 8.1%), while rates of genital infections were higher (PBO, 5.1%; Dapa 2.5 mg, 8.0%; Dapa 5 mg, 13.1%; and Dapa 10 mg, 8.9%) (43). Laboratory monitoring revealed no clinically meaningful changes in markers for renal impairment or increases in mean serum creatinine. Changes in supine blood pressure at week 24 ranged from 3.1 to 5.9 systolic/2.1 to 2.7 diastolic mmHg with Dapa, compared to 0.3 systolic/0.4 diastolic mmHg with PBO. A similar proportion of patients across all four treatment groups, including PBO, had blood pressure measurements suggestive of orthostatic hypotension but without reported symptoms. Reports of hypoglycaemia were similar (PBO, 2.9%; Dapa 2.5 mg, 2.2%; Dapa 5 mg, 3.6%; and Dapa 10 mg, 3.7%), and none led to discontinuation of study medication. In conclusion, in type 2 diabetic patients who are inadequately controlled with Met alone, the addition of once-daily Dapa appears safe and is associated with significantly improved glycaemic control and clinically meaningful weight loss over 24 weeks compared to PBO.

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In a 12-week pilot study, the efficacy and safety of dapagliflozin (Dapa) was studied in type 2 diabetic patients who were poorly controlled with insulin (at least 50 units/day) plus other oral anti-diabetics (OADs) (metformin and/or thiazolidinediones) (44). The trial involved 26 study centers in the United States and in Canada. In order to initially monitor glycaemic parameters with reduced insulin dosing, a small preliminary cohort (n ¼ 4) received 20 mg single-blind Dapa, baseline OADs and 50% of baseline insulin dose. Afterward, the larger primary cohort (n ¼ 71) was randomized to double-blind placebo (PBO), 10 mg Dapa or 20 mg Dapa once daily, and baseline OADs and 50% of baseline insulin. At week 12, Dapa lowered HbA1c, postprandial glucose (PPG), and weight more than PBO (44). In both Dapa groups, 65.2% of patients showed decreases in HbA1c 0.5% versus 15.8% of patients in the PBO group. Dapagliflozin produced dose-dependent responses in both FPG and PPG. There were mean decreases in standing systolic/diastolic blood pressure in both Dapa groups (7.2/1.2 mmHg change from baseline of 130.7/78.9 mmHg (10 mg Dapa), -6.1/-3.9 mmHg change from baseline of 126.9/76.5 mmHg (20 mg Dapa)), compared to an increase in the PBO group ( þ 2.8/ þ 0.3 mmHg change from baseline of 128.9/ 76.9 mmHg). Adverse events (AEs) were balanced across all groups (44). Most frequently reported AEs (>5% in any group) included pollakiuria (urinary frequency), back pain, nasopharyngitis, nausea, headache, and upper respiratory tract infection. Three patients in the PBO group, seven patients in the 10 mg Dapa group, and six patients in the 20 mg Dapa group reported episodes of hypoglycaemia. Of these, one patient in the PBO group reported major hypoglycaemia. In insulin-resistant patients who had insulin reduced by 50%, Dapa was well tolerated, and improved glycaemic control and lowered weight more than PBO (44).

REMOGLIFLOZIN Remogliflozin etabonate is another SGLT2 inhibitor being developed by Kissei Pharmaceutical Co. Ltd. It is an inactive prodrug that is metabolized into its active form, called simply remogliflozin. Remogliflozin is based on the benzylpyrazole glucoside molecule (see Table 13.2) and thus has a slightly different structural scaffold than all the previously mentioned SGLT inhibitors (45). It has a 365-fold selectivity for SGLT2 versus SGLT1, making it slightly more discriminating for SGLT2 when compared to Kissei’s other investigational compound, sergliflozin. In vivo animal studies (45) found similar results to those seen with early trials of sergliflozin and dapagliflozin. In nondiabetic rats, use of remogliflozin led to an increase in urinary glucose excretion that followed a linear, dose-dependent pattern. Remogliflozin also caused a fall in plasma glucose levels after an oral glucose challenge, accompanied by a decrease in circulating insulin levels. When studies were performed in diabetic rats, remogliflozin showed much more pronounced antihyperglycemic effects after oral glucose loading, again preventing plasma glucose elevations and decreasing circulating insulin levels, with the difference as compared to placebo being much more marked (45). A 6-week study of remogliflozin in obese diabetic rats showed the use of remogliflozin led to lower average fasting plasma

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glucose as well as lower HbA1c levels as compared to the control group. In addition, at the end of the study period, the animals receiving remogliflozin had significantly lower circulating insulin levels, had less hypertriglyceridemia and had an overall loss of weight despite no observed change in food intake (45). It was inferred from this data that remogliflozin, through its effect of increasing urinary glucose excretion, was able to normalize plasma glucose levels and reverse many of the effects of glucose toxicity, specifically improving insulin sensitivity and decreasing insulin resistance at the level of the peripheral tissues.

SAFETY No long-term human trials are currently available to assess the safety of SGLT2 inhibitors, but in the longest running human trial published to date, a 24-week trial of dapagliflozin in type 2 diabetic patients (43), no serious adverse events were reported. Significantly, there was no excessive loss of electrolytes in the urine and urinary volume increased only marginally, with no changes in plasma electrolyte concentrations over the 12 weeks of therapy. Hypoglycemia would not be expected with SGLT2 inhibition, as the method of plasma glucose lowering is independent of pancreatic b-cell insulin secretion and is dependent on plasma glucose levels, thus at lower plasma glucose, less glucosuria occurs, decreasing the risk for hypoglycemia. One could postulate that significantly altering the rate of glucose reabsorption in the kidney could lead to deleterious long-term effects on that organ, but there has been no evidence to date, either in long-term animal or human studies, that shows any decrease in GFR, rise in creatinine or other negative renal effect. Given the selectivity of the SGLT2 inhibitors currently under investigation, no gastrointestinal (GI) side effects of diarrhea or malabsorption are expected as these molecules have minimal effects on SGLT1 (46). Consistent with, published studies to date, no GI effects have been reported.

SUMMARY If the SGLT2 inhibitors continue to prove safe and effective in Phase III clinical trials, we will have in hand an entirely new class of antidiabetic drugs against a new target in the fight against diabetes and comorbidities. SLGT2 inhibitors hold the promise of improving both fasting and postprandial glycemic control with minimal risk of hypoglycemia, GI side effects, weight gain or fluid retention, common drawbacks of other currently available antidiabetic medications. Type 2 diabetic patients are usually overweight or obese and have combined defects of insulin resistance and relative impairment of b-cell insulin secretion. Several of the current antidiabetic therapies, including sulfonylureas, insulin and thiazolidinediones, promote further weight gain (47, 48) as opposed to the SGLT2 inhibitors which, through glucosuria and caloric loss, could lead to weight loss. The weight loss effect can potentially improve many other parameters such as lipids, blood pressure, and inflammatory markers. SGLT2 inhibitors have been shown in early trials to improve both fasting and

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postprandial plasma glucose levels, and the use of SGLT2 inhibitors in type 2 diabetic patients could therefore not only improve glycemic control but also decrease insulin resistance and improve b-cell function. Furthermore, SGLT2 inhibitors work independently of insulin secretion, they are unlikely to cause hypoglycemia, as opposed to sulfonylureas (49, 50). For the same reason, they could be effective in improving glycemic control in both type 2 and type 1 diabetic patients. In type 1 diabetic population, SGLT2 inhibitors may prove effective at improving postprandial glycemic control as an adjunctive treatment to insulin, whereas in type 2 diabetic population, SGLT2 inhibitors could be used at any point in the treatment algorithm (51), either as monotherapy or in combination with other antidiabetic medications.

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19. ASANO, T., M. ANAI, H. SAKODA, M. FUJISHIRO, H. ONO, H. KURIHARA, and Y. UCHIJIMA. 2004. SGLT as a therapeutic target. Drugs Fut 29:461–466. 20. ASANO, T., T. OGIHARA, H. KATAGIRI, H. SAKODA, H. ONO, M. FUJISHIRO, M. ANAI, H. KURIHARA, and Y. UCHIJIMA. 2004. Glucose transporter and Na þ /glucose cotransporter as molecular targets of antidiabetic drugs. Curr Med Chem 11:2717–2724. 21. WRIGHT, E.M. 1998. Genetic disorders of membrane transport. Glucose glacatose malabsorption. Am J Physiol Gastrointest Liver Physiol 275:879–882. 22. TURK, E., B. ZABEL, S. MUNDLOS, J. DYER, and E.M. WRIGHT. 1991. Glucose/galactose malabsorption caused by a defect in the Na þ /glucose cotransporter. Nature 350:354–356. 23. LAPLANE, R., C. POLONOVSKI, M. ETIENNE, P. DEBRAY, J.C. LODS, and B. PISSARRO. 1962. L’intolerance aux sucres a transfert intestinal actif. Arch Fr Pediatr 19:895–944. 24. LINDQUIST, B., and G.W. MEEUWISSE. 1962. Chronic diarrhea caused by monosaccharide malabsorption. Acta Paediatr 51:674–685. 25. MAGEN, D., E. SPRECHER, I. ZELIKOVIC, and K. SKORECKI. 2005. A novel missense mutation in SLC5A2 encoding SGLT2 underlies autosomal-recessive renal glucosuria and aminoaciduria. Kidney Int 67:34–41. 26. FRANCIS, J., J. ZHANG, A. FARHI, H. CAREY, and D.S. GELLER. 2004. A novel SGLT2 mutation in a patient with autosomal recessive renal glucosuria. Nephrol Dial Transplant 19:2893–2895. 27. CALADO, J., Y. SZNAJER, D. METZGER, A. RITA, M.C. HOGAN, A. KATTAMIS, M. SCHARF, V. TASIC, J. GREIL, F. BRINKERT, M.J. KEMPER, and R. SANTER. 2008. Twenty-one additional cases of familial renal glucosuria: absence of genetic heterogeneity, high prevalence of private mutations and further evidence of volume depletion. Nephrol Dial Transplant 23:3874–3879. 28. EHRENKRANZ, R.R.L., N.G. LEWIS, C.R. KAHN, and J. ROTH. 2005. Phlorizin: a review. Diabetes Metab Res Rev 21:31–38. 29. ROSSETTI, L., G.I. SHULMAN, W. ZAWALICH, and R.A. DEFRONZO. 1987. Effect of chronic hyperglycemia on in vivo insulin secretion in partially pancreatectomized rats. J Clin Invest 80:1037–1044. 30. SAITO, A., T. SEIYAKU, and K. TSUJIHARA. 2002. SGLT inhibitor (T-1095). Nippon Rinsho 60:588–593. 31. TSUJIHARA, K., M. HONGU, K. SAITO, H. KAWANISHI, K. KURIYAMA, M. MATSUMOTO, A. OKU, K. UETA, M. TSUDA, and A. SAITO. 1999. Na þ -glucose cotransporter (SGLT) inhibitors as antidiabetic agents. 4. Synthesis and pharmacological properties of 40 -dehydroxyphlorizin derivatives substituted on the B ring. J Med Chem 42:5311–5324. 32. ADACHI, T., K. YASUDA, Y. OKAMOTO, N. SHIHARA, A. OKU, K. UETA, K. KITAMURA, A. SAITO, I. IWAKURA, Y. YAMADA, H. YANO, Y. SEINO, and K. TSUDA. 2000. T-1095, a renal Na þ -glucose transporter inhibitor, improves hyperglycemia in streptozotocin-induced diabetic rats. Metabolism 49:990–995. 33. YASUDA, K., Y. OKAMOTO, K. NUNOI, T. ADACHI, N. SHIHARA, A. TAMON, N. SUZUKI, E. MUKAI, S. FUJIMOTO, A. OKU, K. TSUDA, and Y. SEINO. 2002. Normalization of cytoplasmic calcium response in pancreatic beta-cells of spontaneously diabetic GK rat by the treatment with T-1095, a specific inhibitor of renal Na þ -glucose co-transporters. Horm Metab Res 34:217–221. 34. KATSUNO, K., Y. FUJIMORI, Y. TAKEMURA, M. HIRATOCHI, F. ITOH, Y. KOMATSU, H. FUJIKURA, and M. ISAJI. 2007. Sergliflozin, a novel selective inhibitor of low-affinity sodium glucose cotransporter (SGLT2), validates the critical role of SGLT2 in renal glucose reabsorption and modulates plasma glucose level. J Pharmacol Exp Ther 320:323–330. 35. FUJIMORI, Y., K. KATSUNO, K. OJIMA, I. NAKASHIMA, S. NAKANO, Y. ISHIKAWA-TAKEMURA, H. KUSAMA, and M. ISAJI. 2009. Sergliflozin etabonate, a selective SGLT2 inhibitor, improves glycemic control in streptozotocin-induced diabetic rats and Zucker fatty rats. Eur J Pharmacol 609:148–154. 36. HUSSEY, E.K., R.I. DOBBINS, R.R. STOLZ, N.L. STOCKMAN, R.L. O’CONNOR-SEMMES, A. KAPUR, S.C. MURRAY, and D.J. NUNEZ. A double-blind randomized repeat dose study to assess the safety, tolerability, pharmacokinetics and pharmacodynamics of three times daily dosing of sergliflozin, a novel inhibitor of renal glucose reabsorption, in healthy overweight and obese subjects. Presented at American Diabetes Association, 67th Annual Scientific Sessions. Chicago, USA, 2007, 0491-P. 37. HUSSEY, E.K., R.V. CLARK, D.M. AMIN, M.S. KIPNES, R.L. O’CONNOR-SEMMES, E.C. O’DRISCOLL, J. LEONG, S.C. MURRAY, R.L. DOBBINS, and D.J. NUNEZ. Early clinical studies to assess the safety, tolerability, pharmacokinetics and pharmacodynamics of single doses of sergliflozin, a novel inhibitor

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Chapter

14

Fibroblast Growth Factor 21 as a Novel Metabolic Regulator RADMILA MICANOVIC, JAMES D. DUNBAR,

AND

ALEXEI KHARITONENKOV

BioTechnology Discovery Research, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN, USA

INTRODUCTION Fibroblast growth factors (FGFs) function through four distinct subfamilies of FGF receptors (FGFRs), act on multiple tissues and organs and are primarily known to control processes of cell growth and differentiation, angiogenesis, and transformation. The mammalian FGF family currently consists of 22 members divided into 7 subfamilies based on their structural similarities and mode of action (1–3). Structurally, FGFs share a homologous core region consisting of 120–130 amino acids folded as 12 antiparallel b-strands. The core domain is bordered by flexible divergent amino and carboxyl termini that convey different biology to the ligands. Canonical FGFs require heparin sulfate glucosaminoglycans (HSGAG) from the extracellular matrix as cofactors for binding to their cognate receptors and to activate FGFR signaling efficiently. The HSGAG binding site is within the FGF core and is composed of a conserved, positively charged surface (4). Their actions are therefore of a local, autocrine/paracrine nature, where HSGAGs determine the radius of ligand diffusion (5). Over the past decade, new evidence has emerged indicating that FGF/FGFRmediated pathways play an important role in defining and controlling functions of endocrine tissues and organs, as well as regulating various metabolic processes as exemplified by the biology of FGF21, FGF19, and FGF23.

Metabolic Syndrome: Underlying Mechanisms and Drug Therapies Edited by Minghan Wang Copyright 2011 John Wiley & Sons, Inc.

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Fibroblast growth factor 21 (FGF21), a comparatively new structural member of the FGF family, was identified in 2000 (6). Mature human FGF21 is a secreted polypeptide of 181 amino acids, with 81% sequence identity to the mouse orthologue. As in the case of other FGFs, its core FGF homology region forms a globular domain composed of 12 antiparallel b-strands known as the b-trefoil motif. Within the FGF family, human FGF21 is most similar to FGF19 and FGF23 with 34% and 25% sequence identity at the amino acid level, respectively. Crystallographic analyses of FGF21, FGF19, and FGF23 have revealed that their heparin binding regions diverge significantly from the archetypal FGFs. This explains the weak to absent binding affinity of these FGFs for heparin (7–9), which enables them to avoid being trapped in the extracellular matrix, thus allowing their function in a humoral manner. Based on their structural relatedness, poor if any direct binding affinity for HSGAGs and FGFRs, presence in blood plasma, and ability to regulate metabolic processes, these three FGFs define a subfamily of “endocrine” FGFs (8–17). Furthermore, the members of this FGF subfamily require the presence of Klotho and bKlotho, which share 41% amino acid sequence identity (18–21), for efficient binding and activation of FGFRs. In spite of FGF21’s discovery only a decade ago, the understanding of FGF21 biology and pharmacology as a novel metabolic regulator is rapidly emerging. Furthermore, given its unmistakable ability to ameliorate disease phenotypes in preclinical studies without inducing any apparent adverse effects, FGF21 represents a novel and attractive therapeutic agent for treatment of type 2 diabetes mellitus, obesity, dyslipidemia, and liver steatosis. The specifics of FGF21 activities, in vitro and in vivo, under both normal and pathophysiological conditions and its utility as a therapeutic agent for metabolic diseases will be discussed in this chapter.

FGF21 EXPRESSION Initial identification of the tissue distribution of FGF21 expression has been done primarily in rodents and at the mRNA level. Endogenous FGF21 mRNA was originally detected in mice only in adult thymus and liver (6), but then in pancreas (22, 23), adipose tissue (24, 25), and muscle (26). In humans, FGF21 mRNA has been detected in the liver and adipose tissue (27, 28); however, the full spectrum of tissue specific localization of FGF21 is likely to be broader given that FGF21 expression is subject to an exceptionally dynamic and dramatic regulation (29). In cell cultures and tissues where FGF21 can be expressed, its levels are affected by various metabolic conditions such as starvation or fasting, high glucose or sucrose, and high fat ketogenic diets or chemical challenges (16, 30–36). Peroxisomeproliferator-activated receptor-a (PPARa) agonists induce a substantial increase of FGF21 in rodents, where they appear to directly regulate FGF21 gene expression (16, 34, 35). In adipocytes, PPARg activation leads to elevated FGF21 expression and function (20, 24, 37). Recent reports in man have shown a general increase in circulating FGF21 in conditions of metabolic disease. For example, serum levels of FGF21 are associated

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with obesity and increasing components of metabolic syndrome in humans, while FGF21 mRNA expression in subcutaneous fat correlated significantly with its circulating levels (27). FGF21 is also higher in type 2 diabetic patients, with or without ketosis (38, 39), and dyslipidemic individuals (40). Interestingly, FGF21 blood levels can be lowered following treatment with metabolic disease modifying agents such as insulin and fenofibrate, a PPARa agonist (28, 40). Elevated FGF21 levels have been found in subjects on hemodialysis with chronic renal failure, indicating that kidney function is another important determinant influencing FGF21 levels (41). By contrast, a study of anorexic women demonstrated that FGF21 circulating levels were significantly lower than in age-matched controls (42). While interesting, the caveats associated with experimentation reporting changing plasma FGF21 levels in man are that they are often assessed at a single time point and that the FGF21 immunoreactivity measured does not distinguish between full length/ active hormone and its clipped metabolic derivatives that may be of compromised activity (43, 44).

FGF21 IN VITRO EFFECTS Cells lacking bKlotho do not respond to FGF21 stimulation, while induction or introduction of bKlotho in these cells confers FGF21 responsiveness, as in the case of 3T3-L1 mouse fibroblasts (20) indicating that FGF21 activates its cognate FGFRs when it is associated with the adaptor protein bKlotho (Figure 14.1). Thus, the FGF21 receptor structurally consists of two precomplexed components, FGFR and bKlotho (20), both of which are necessary to form a functional FGF21 receptor. In a recent study with BaF3 cells (a murine pro-B-cell line), which do not express any endogenous FGFRs, overexpression of FGFR1c and FGFR3c, along with bKlotho, enabled FGF21 to initiate receptor activation and signaling events (21). In mouse 3T3L1 adipocytes and white adipose tissue, FGFR1 and FGFR2 are the most abundant receptors and they both can be activated by FGF21 (8, 20, 37). Given the unmistakable liver phenotype in FGF21 transgenic animals (8), the ability of FGF21 to directly act on liver (45, 46), where FGFR4 is the predominant FGFR (47), and evidence of FGFR4/ bKlotho interaction (19, 20), it is likely that this receptor may also support FGF21 action in the presence of bKlotho. Taken together, these results indicate that bKlotho may form the cognate FGF21 receptor complex with several if not all FGFR isotypes. Thus, bKlotho can be considered a primary determinant in mediating FGF21’s tissue specificity and actions rather than FGFRs as has been proposed earlier (48). An insight into how FGF21 interacts with the bKlotho/FGFR complex has been recently reported by two separate groups investigating the roles of the N- and C-termini of FGF21 in its functional activity (43, 44). By assessing ligand binding affinity for bKlotho and functional activity of consecutive N- and C-terminal truncation variants of FGF21, it was demonstrated that the carboxy-terminal region is important for bKlotho binding, thus determining the potency of the ligand, while the amino-terminus is involved in FGFR activation. The N-terminal deletions of FGF21 demonstrate partial agonism and could act as competitive antagonists of full-length

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Figure 14.1

Schematic representation of FGF21 receptor complex.

FGF21. Similar results were obtained in structure function studies pertaining to other members of the endocrine FGF family, FGF19 and FGF23 (49, 50). By analogy with other FGFs, FGF21 ligand binding, through the bKlotho subunit of the receptor complex, most likely causes receptor dimerization followed by the activation and phosphorylation of the intracellular tyrosine kinase domain. This in turn leads to the activation of a number of downstream signaling pathways including FRS2, MAPK, MEK1/2, RAF1, AKT, GSK3, p70S6K, STAT3, SHP2, and Ca2 þ flux (8, 22, 37). Activation of signaling pathways may vary in specific FGF21 target tissues and metabolic states (51). The fact that many of these signaling pathways can be activated in vitro by canonical FGFs such as FGF1 (2) is further evidence that endocrine FGF21 signals through activation of a conventional FGFR-mediated mechanism. The biological activity of FGF21 was first discovered in a cell-based highthroughput functional screen aimed at identifying novel secreted proteins with antidiabetic potential, where it stimulated glucose uptake in differentiated mouse 3T3-L1 and human primary adipocytes (8). In contrast to the rapid action of insulin, the effect of FGF21 on glucose uptake required several hours of treatment, pointing to transcriptional activation as a part of the FGF21 mechanism of action. The effect of FGF21 on glucose uptake was independent of insulin and additive to the activity of insulin upon cotreatment. The activity of FGF21 was not affected by the addition of exogenous heparin.

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Interestingly, treatment of 3T3-L1 adipocytes with a combination of FGF21 and the PPARg agonist rosiglitazone leads to a remarkable increase in glucose uptake compared to the administration of either agent independently (37). While rosiglitazone upregulates FGF21 expression in adipocytes (24), the mechanism of cell sensitization to FGF21 action is most likely due to a rosiglitazone-induced increase in expression of bKlotho (20), an FGF21 coreceptor, revealing that the FGF21 signaling pathway is under tight PPARg control. Several functional outcomes can be observed on cells that are responsive to FGF21. Insulin-independent glucose uptake in FGF21-treated adipocytes is mediated by de novo expression of the insulin-independent glucose transporter GLUT1, one of the FGF21 responsive genes in those cells (8, 37). While FGF21 ability to induce lipolysis in 3T3-L1 adipocytes has been suggested (16), an independent study conducted under similar experimental conditions yielded the opposite conclusion that FGF21 promotes lipid accumulation in adipocytes rather than inducing direct lipolytic activity (17). The latter is reasonable from a physiological perspective as both glucose uptake and lipid accumulation are parts of an overall energy sparing mechanism. When tested in isolated pancreatic islets and INS-1E cells, FGF21 suppressed glucose-mediated glucagon release from a-cells and stimulated insulin production from b-cells while providing protection from glucolipotoxicity and cytokine-induced apoptosis (8, 22). In addition, FGF21 activates signaling in pancreatic cells of exocrine origin, such as acinar cells (23), and in liver-derived cell cultures (46). As indicated in the introduction, growth-promoting activity is one of the most documented functions of classical FGFs. However, FGF21 did not induce proliferation when tested in several primary and immortalized cells otherwise sensitive to treatment with classical FGFs. In costimulation experiments, it did not block the mitogenic effect induced by those FGFs and is therefore not an antagonist of typical FGF action (8, 22).

FGF21 IN NORMAL PHYSIOLOGY In recent years, evidence has accumulated suggesting that FGF21 is a critical physiological regulator during the body’s adaptation to starvation. As mentioned earlier, FGF21 levels are significantly elevated in rodents upon fasting, when fed high fat, low carbohydrate, ketogenic diets, or when treated with PPARa agonists (16, 34, 35). The increase in serum ketone bodies in mice upon fasting correlated with the time of induction of FGF21 expression in the liver and the effect was significantly reduced in PPARa-deficient animals. Considering that ketogenesis is part of the physiological response to fasting, where PPARa is a critical component, a physiological role for FGF21 as a starvation, ketogenic or “Atkins” hormone was proposed (16, 34, 35). Supporting this conclusion is the finding that FGF21 deficiency in mice, achieved either by siRNA silencing (34) or by gene knockout (46), resulted in reduced serum ketone bodies, such as b-hydroxybutyrate, increased serum free fatty acids and cholesterol when animals were on a ketogenic diet, and development of

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hepatosteatosis, hypertriglyceridemia, and insulin resistance. It is then proposed that, during fasting, PPARa activation leads to increased circulating FGF21, which in turn promotes FGF21 actions in its responsive tissues. Using an enzyme-linked immunosorbent assay (ELISA), an assessment of FGF21 plasma levels in 76 normal weight subjects was recently conducted (40) that revealed considerable variation among individuals, 21–5300 pg/mL, with no correlation to age, gender, body mass index, blood glucose, serum lipids, or markers for bile acid and cholesterol synthesis. Additionally no relationship was found between FGF21 plasma levels and feeding, fasting, or circadian cycles in samples collected from subjects at 90 min intervals during the course of night. Short-term fasts, up to 48 h, produced a significant ketogenic response with an increase of serum b-hydroxybutyrate of up to 40-fold with no effect on FGF21 levels contrasting with what has been observed in mice (16, 34). Nevertheless, a strict 7-day fasting resulted in a 74% increase in circulating FGF21 levels accompanied by further pronounced ketogenesis. In addition, the treatment of hypertriglyceridemic patients with fenofibrate, which activates PPARa, lowered dyslipidemia and induced a small but significant elevation of FGF21 levels by 28%. No data was reported on the tissue expression of FGF21, either at the mRNA or at the protein level. Although the specific details of FGF21 regulation in the biology of fasting and starvation in humans may vary from those observed in rodents, these results demonstrate that in humans (1) FGF21 levels are highly variable and (2) ketogenesis can be induced independently of an increase in plasma FGF21, and (3) FGF21 expression can be increased by PPARa activation.

FGF21 IN PATHOPHYSIOLOGY OF METABOLIC DISEASE Experimental evidence reveals that circulating FGF21 levels differ in healthy versus diseased states in both rodents (16, 34) and humans (27, 28, 38, 40–42) indicating that the dysregulation of FGF21 activity may be involved in the pathophysiology of diseases. In humans, plasma FGF21 levels were elevated up to twofold in diabetic, obese, and hypertriglyceridemic patients compared to control subjects. In patients with type 2 diabetes, serum FGF21 levels correlated positively with fasting blood glucose, insulin levels, and insulin sensitivity while no such correlation was observed in healthy individuals (38, 40). In obese subjects and other populations with increased cardiovascular risk, such as patients exhibiting components of the metabolic syndrome, FGF21 levels were directly correlated to the degree of adiposity, insulin resistance, triglycerides, and cholesterol (27). In contrast, patients diagnosed with anorexia nervosa exhibited lower FGF21 levels than control subjects (42). Finally, patients with severely impaired renal function on chronic hemodialysis display median FGF21 levels that are 15-fold higher than in control subjects (41), although the physiological significance of this last observation remains to be elucidated. As systemic administration of FGF21 effectively ameliorates various metabolic abnormalities in animal models (51), it is tempting to speculate that the paradoxical increase in this protein’s levels is a compensatory mechanism to counter metabolic

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stress. Alternatively, FGF21 resistance might be present in metabolic disorders leading to the upregulation of this hormone. This mechanism would be reminiscent of the hyperinsulinemia and hyperleptinemia that occur in diabetes and obesity as a result of insulin- and leptin-resistance (52, 53). Supporting this view is a report indicating that healthy animals are more sensitive to the acute actions of exogenously administered FGF21 than metabolically compromised ones, such as ob/ob mice (45). Nevertheless, when tested in these animals in a chronic setting, FGF21 is perfectly efficacious in correcting multiple abnormalities of metabolic disease (51). Additional potential explanations for increased circulating FGF21 levels may include reduced plasma clearance in diseased states such as renal disease (41), ectopic FGF21 production from tissues other than liver (24), or circulation of terminally truncated inactive forms, which are indistinguishable from the full-length bioactive molecule by current detection methods (43). Clearly, the complete role of FGF21 in human pathophysiology remains elusive, and more studies are warranted.

FGF21 IN VIVO PHARMACOLOGY The effects of FGF21 forced expression in transgenic mice were described independently by three groups. In all cases, the animals appeared viable and healthy at birth (8, 16, 32). Notably, the FGF21 transgenic animals did not display liver neoplasia, tumors or any other abnormalities throughout their life span unlike animals expressing FGF19 (54). Further, FVB mice undergoing forced hepatic expression of FGF21 displayed delayed tumor initiation in the liver upon stimulation with chemical carcinogens (32). These results indicated that prolonged exposure to FGF21 did not lead to carcinogenesis in vivo. On the contrary, FGF21 appeared to act as a tumor suppressor in the early stages of cancer formation (32). Transgenic mice expressing human (8) or mouse FGF21 (16) in the liver exhibited an overall similar metabolic phenotype including reduced adipocyte size, glucose, insulin, cholesterol, and triglycerides and improved insulin sensitivity and glucose clearance as compared to control animals. However, some differences were observed. Mice expressing human FGF21 were resistant to weight gain and fat accumulation (8), while animals expressing mouse FGF21 surprisingly were reported to be obese despite being metabolically fit. They also entered the energy conserving state of torpor upon prolonged fasting (55), which has not been documented by others (8, 32). Given a profound FGF21 effect on energy expenditure (56) and the fact that in the latter study no torpor was observed in fed animals, the torpor phenotype in the aforementioned study (55), if confirmed, is unlikely to be a direct result of FGF21 action but rather can be attributed to a negative energy balance in fasted FGF21 transgenics. Systemic administration of FGF21 in mouse models of type 2 diabetes and obesity, such as ob/ob mice, lowered blood glucose and triglyceride levels (8), while in db/db mice administration of FGF21 led to the preservation of pancreatic b-cell function and mass (22). Diet-induced obesity (DIO) mice showed reversal of hepatic steatosis, ameliorated insulin resistance, and reduced body weight by 20% over the

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course of 2 weeks of FGF21 administration, but displayed no alteration in total caloric intake (56, 57). Further, loss of body weight was attributed predominantly to a reduction of fat mass consistent with observations of increased energy expenditure and reduced respiratory coefficient (RQ), which indicate a preferential utilization of fat as an energy source (56, 57). After FGF21 treatment, while in liver the expression of genes involved in fatty acid oxidation were increased and those involved in de novo lipogenesis were decreased, the genes involved in uncoupling and lipogenesis were elevated in adipose tissue prompting the hypothesis that coordinated changes in gene expression in liver and adipose tissue could result in futile cycling and increased energy expenditure (56). In addition to reducing insulin and leptin levels, suggesting an improvement in sensitivity to these hormones, FGF21 treatment elevated hypothalamic levels of AGRP and NPY, which are responsible for promoting appetite, raising the possibility that FGF21 may have central effects. The latter is consistent with the hyperphagia observed in FGF21-treated DIO mice (56, 57) when their food intake was normalized to body weight and the report that FGF21 can cross the blood–brain barrier (58). In diabetic Rhesus monkeys, a non-human primate model of type 2 diabetes, systemic administration of FGF21 effectively decreased plasma glucose and triglycerides to near normal levels, decreased LDL- while increasing HDL-cholesterol, improved insulin sensitivity, decreased markers of cardiovascular risk, such as C-reactive protein, RANTES, IL-8, PAI-1, and factor VII, and increased levels of adiponectin and ApoA1, markers of ameliorated cardiovascular function (59). These striking results further validated FGF21 as a potent metabolic regulator capable of correcting multiple abnormalities of compromised metabolic states. Indeed, FGF21 can induce a variety of pharmacological responses where specific functional outcomes can be achieved independently of one another (51, 56). While many outstanding questions about FGF21 biology still remain, the pharmacological effects of FGF21 are well documented in rodents and nonhuman primates and considerable progress has been made in the past few years in understanding the underlying mechanisms of FGF21 in vivo function (51). The identification of bKlotho as a critical subunit of the FGF21 receptor and its requirement for the initiation and propagation of the FGF21 signal provides a rational explanation for many, if not all, of the unique features of FGF21 biology. bKlotho serves as an adaptor molecule in the formation of an FGF21 ligand/receptor complex much as heparin does for canonical FGFs (4). This allows FGF21 to reach its target tissues while avoiding the body’s vast depot of heparansulphate proteoglycans. As FGF21 activates FGFRs but only in the context of bKlotho’s presence, bKlotho determines FGF21 tissue selectivity (51). Given the fact that FGFRs are expressed in a wide variety of cell and tissue types, but bKlotho is exclusively expressed in liver, pancreas, and adipose (60), these tissues were always primary candidates for FGF21 targeting (29). While FGF21 activities in adipose and pancreas were never in doubt (8, 22), it has been suggested that FGF21 has no direct hepatic action (55). Later work, however, firmly established that FGF21 can efficiently target liver consistent with the high levels of bKlotho expression in this tissue (45, 46, 56). Thus, much like insulin, FGF21 acts as a pleiotropic factor, utilizing the distinct molecular

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mechanisms that are determined by its receptor composition and the unique downstream signaling pathways present in the cells of its target organs. FGF21 exhibits the ability to influence metabolic parameters in “diseased” or “challenged” states, as during glucose tolerance tests or diet-induced obesity, without affecting them in “healthy” states as illustrated by the absence of adverse effects such as hypoglycemia (8, 56, 57, 59). Observations such as these suggest that, in part, the FGF21 mechanism of action is “sensitizing” in nature. Several lines of evidence indicate that FGF21 exerts its antihyperglycemic effects by functioning as an effective insulin sensitizer. FGF21 directly regulates insulin accumulation and secretion in vitro (22), effectively lowers insulin levels in vivo, improves peripheral tissue insulin sensitivity and reverses hepatic insulin resistance in diabetic animals (8, 45, 59). Its direct actions in pancreas (22), liver (46), and adipose (8) are thought to underlie these effects and an FGF21-dependent induction of insulin receptor transcripts in these tissues may provide a molecular basis for some of its mechanism of insulin sensitization (56). In addition, insulin may impact the FGF21 pathway, since it can induce FGF21 elevation in C2C12 myoblasts and 3T3-L1 adipocytes, and FGF21 levels are dramatically increased in the muscle of mice overexpressing AKT1, a key molecule within the insulin signaling cascade (26). Finally, tight associations between plasma FGF21 and insulin or insulin sensitivity in man (27, 28, 38) further suggest interplay between the insulin and the FGF21 pathways in vivo. FGF21 actions in liver and adipose are likely to underlie its lipid-lowering and anti-obesity effects (56, 57). In liver, FGF21 administration leads to gene expression changes consistent with increased b-oxidation of free fatty acids and suppression of de novo lipogenesis. In addition, results from a recent study indicated that stimulation of hepatic lipolysis and lipid utilization was at least partially mediated by FGF21 signaling under conditions that favor lipid metabolism, such as replacement of dietary carbohydrates with protein, and which result in weight loss, dyslipidemia, and hepatic steatosis (61). In adipose tissue exposed to FGF21, a parallel increase in both lipogenesis and lipid mobilization has been observed (8, 17, 37, 45). Together with the elevation in uncoupling proteins and PGC1a (56), which appears to be one of the main determinants of FGF21 action, these data suggest that FGF21 treatment induces a state of increased futile cycling and energy expenditure. Indeed, as measured by indirect calorimetry, FGF21-infused mice had a significantly higher energy expenditure rate than vehicle-treated animals (56, 57), which is indicative of FGF21’s ability to shift the balance between caloric intake and energy dissipation.

CONCLUSIONS The current review presented evidence that FGF21 is a metabolic regulator that exerts potent antidiabetic and lipid-lowering effects in rodent and monkey models of type 2 diabetes mellitus and obesity. This novel hormone-like factor also contributes to body weight regulation and is strongly involved in the physiological response to

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Chapter 14 Fibroblast Growth Factor 21 as a Novel Metabolic Regulator

metabolically challenged states such as nutritional deprivation, starvation, or ketogenic diet. The principal sites of action of FGF21 are liver, adipose tissue, and pancreas where it promotes various metabolic functions. From a pharmaceutical point of view, the beneficial metabolic effects of FGF21 are displayed without apparent side effects such are hypoglycemia, fluid retention, increased adiposity, pancreatitis, mitogenicity, or hyperplasia, most of which accompany current antidiabetic therapies, making this hormone a prime candidate for treatment of type 2 diabetes and related metabolic disorders.

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Sirtuins as Potential Drug Targets for Metabolic Diseases QIANG TONG USDA/ARS Children’s Nutrition Research Center, Baylor College of Medicine, Houston, TX, USA Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA Department of Medicine, Baylor College of Medicine, Houston, TX, USA Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX, USA

INTRODUCTION Recent studies of the sirtuin family of proteins, which possess NAD þ -dependent deacetylase and ADP-ribosyltransferase activities, indicate that they regulate many biological functions, such as longevity and metabolism (1–3). These findings also suggest that sirtuins might serve as valuable drug targets for the treatment of a variety of diseases, ranging from diabetes, cardiovascular diseases, neurodegenerative disorders, and cancer. This chapter reviews the current knowledge of sirtuins’ functions in metabolic regulation and their emerging roles as therapeutic targets for the metabolic syndrome.

CALORIC RESTRICTION AND SIRTUIN FAMILY OF GENES The sirtuin family of proteins is present in a wide range of organisms, from bacteria to mammals (4). The founding member of the sirtuin family, the yeast Sir2 (silent information regulator 2) gene was first identified as a regulator of the yeast mating type switch (5). In yeast, Sir2 maintains the silencing of the heterochromatin (6–8), and regulates many other functions (9–11). Interestingly, mutation of Sir2 shortens yeast life span, while an increased dosage of the Sir2 gene prolongs life span in Metabolic Syndrome: Underlying Mechanisms and Drug Therapies Edited by Minghan Wang Copyright 2011 John Wiley & Sons, Inc.

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yeast (12), Caenorhabditis elegans (13), and Drosophila (14). Sir2 encodes a nuclear protein, mainly localized in nucleoli and telomere region (15). Sir2 protein and its homologues belong to the class III deacetylases (16), which possesses NAD þ dependent protein deacetylation (17–19) and ADP-ribosylation (20) activities. The deacetylase activity, but not the ADP-ribosyltransferase activity, is essential for Sir2’s functions in mediating gene silencing and caloric restriction in yeast (17). Most importantly, mutation of Sir2 or blocking of NAD þ synthesis abolishes the effect of caloric restriction in yeast (21) or flies (14). Evidence does exist that in certain cases Sir2 does not mediate the effect of caloric restriction. For example, deletion of Sir2 further extends the chronological life span of yeast under caloric restriction (22) and yeasts with Sir2 and FOB1 double mutation still respond to dietary restriction to prolong replicative life span (23). However, at least the latter case might be explained by the compensatory action of Hst2, a Sir2 homologue (24). In higher organisms such as mammals, there are seven Sir2-like proteins, called sirtuins (SIRT1-SIRT7). They share a conserved core enzymatic domain with different subcellular localization (4). In general, SIRT1, SIRT6, and SIRT7 are in the nucleus. SIRT2 is in the cytoplasm, while SIRT3, SIRT4, and SIRT5 are mitochondrial proteins (Table 15.1). The substrates of these sirtuins are summarized in Table 15.2. It is certain that new substrates will be identified in future studies and new functions of sirtuins will be discovered. The possibility that sirtuin family genes may function as mediators for caloric restriction draws many interests, since caloric restriction, a dietary regimen low in calories without malnutrition, is the most potent nongenetic intervention to retard aging. Under dietary restriction, total calories derived from carbohydrate, fat, or protein are reduced to a level 25–60% below that of control animals fed ad libitum (25, 26). It was first discovered in the 1930s (27) that food restriction significantly increases the life span of rodents by as much as 50% (28). In addition, caloric restriction delays a variety of age-related diseases, including obesity (29), diabetes (30), tumors (31, 32), kidney diseases (33), and several types of neurodegenerative disorders (34–36). Caloric restriction decreases animal blood glucose and insulin levels and increases insulin sensitivity (37–39). Caloric restriction increases life span in a wide range of organisms, including yeast, rotifers, spiders, worms, fish, Table 15.1

Features of Mammalian Sirtuins

Sirtuins

Subcellular Localization

Enzymatic Activity

SIRT1 SIRT2 SIRT3 SIRT4 SIRT5 SIRT6 SIRT7

Nuclear Cytoplasmic, Nuclear (G2/M phase) Mitochondria Mitochondria Mitochondria Nuclear Nucleolar

Deacetylase Deacetylase, ADP-ribosyltransferase Deacetylase ADP-ribosyltransferase Deacetylase Deacetylase, ADP-ribosyltransferase Deacetylase

The Catalytic Actions of Sirtuins Table 15.2

393

Substrates of Sirtuins

Sirtuins

Substrates

SIRT1

Acetyl-CoA synthase1 (ACS1) (219), androgen receptor (259), Atg5,7,8 (114), BCL6 (260), BMAL (125), b-catenin (94), CFIm25 (261), cortactin (262), CRTC2 (131), E2F1 (79), FOXO1, 3, and 4 (263–267), HIC1 (268), HIF2a (103), histone H1, H3 and H4 (17, 269), histone H2A.z (151), HSF-1(102), IRS-2 (270), LKB1(118), LXR (136), Ku70 (101), MEF2D (142), NBS1 (271), MyoD (141), NF-kB (272), p53 (74–76), p73 (273), p300 (274), PCAF (141), PER2(126), PGC-1a (73, 109), PGC-1b (111), PTEN(171), Rb (275), Smad7 (276), STAT3 (133), SUV39H1 (113), TAFI68 (277), Tat (HIV) (278), Wrn (279), and zyxin (280).

SIRT2

CFIm25 (261), histone H4Lys16 (187) and H3Lys56 (188), FOXO1 (202, 203), FOXO3(127), p300 (196), and a-tubulin (185).

SIRT3

Acetyl CoA synthetase 2 (ACS2) (218, 219), glutamate dehydrogenase (207, 220), histone H4Lys16 (210) and H3Lys18 (196), isocitrate dehydrogenase 2 (220), Ku70 (222), NDUFA9 (223), and ATP synthase F1 complex alpha subunit 1 (224).

SIRT4

Glutamate dehydrogenase (GDH) (226, 227).

SIRT5

Cytochrome C (220) and carbamoyl phosphate synthetase 1 (CPS1) (221).

SIRT6

Histone H3 lysine 9 (H3K9)(233)

SIRT7

p53 (242)

SIRT4, ADP-ribosylates its substrate, GDH. All other substrates listed are modified by sirtuin deacetylases.

mice, and rats (40). This effect may also apply to nonhuman primates (39, 41–43) and even humans (44, 45). Studies have shown that long-term caloric restriction in humans resulted in reduced fasting serum insulin level, healthier lipid profile, and lower blood pressure with reduced atherosclerosis risk (45, 46).

THE CATALYTIC ACTIONS OF SIRTUINS The sirtuin deacetylation reaction is a two-step process (47). The first step is a reversible base-exchange reaction between NAD þ and acetyl group on the lysine of the polypeptide to generate 10 -O-a-peptidylamidate-ADP-ribose, with the release of nicotinamide. The second step is a transfer of acetyl group to the ADP-ribose moiety and the release of deacetylated polypeptide and the O-acetyl ADP-ribose (OAADPr). Nicotinamide is a potent sirtuin inhibitor, in a fashion of classical noncompetitive product inhibitor (47–51), while the acid form of nicotinamide, nicotinic acid, has very low affinity for sirtuins (51). Another product of the sirtuin enzymatic reaction, OAADPr, turns out to be bioactive on its own. It blocks starfish oocytes maturation when microinjected into oocytes (52). OAADPr

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also facilitates the assembly of yeast Sir2, Sir3, and Sir4 into chromatin silencing complexes (53).

THE NAD þ SYNTHESIS PATHWAYS Since NAD þ is a key cosubstrate for sirtuins, the regulation of NAD þ synthesis influences the action of sirtuins. The nicotinamide adenine dinucleotide (NAD) exists in oxidized (NAD þ ) or reduced form (NADH). The ratio between the oxidized and the reduced forms of NAD (NAD þ /NADH) is an important indicator of the redox state of the cell. NAD þ has long been known as critical electron carrier for energy metabolism. However, recent studies uncovered that NAD þ has a myriad of physiological functions, many of which were found to be mediated by sirtuins. Therefore, targeting the enzymes of the NAD þ synthesis pathways may also achieve some therapeutic benefits through indirect regulation of the activity of sirtuins. NAD þ is synthesized through two metabolic pathways, either from scratch (de novo pathway) using amino acids or recycled from the breakdown product of NAD þ (salvage pathway). The de novo synthesis of NAD þ starts with tryptophan (54) or aspartate in some bacteria and plants (55) to synthesize nicotinic acid mononucleotide (NaMN). Then, NaMN is converted to nicotinic acid adenine dinucleotide (NaAD) by nicotinamide/nicotinic acid mononucleotide adenylyltransferase (Nmnat). NAD þ synthetase catalyzes the last step of the synthesis of NAD þ from NaAD. Alternatively, through the salvage pathway, nicotinamide formed upon NAD þ turnover or dietary source can be used to form NAD þ (56). In yeast, nicotinamide is converted to nicotinic acid by nicotinamidase PNC1 (57). Then, nicotinic acid is catalyzed to form NaMN by nicotinic acid phosphoribosyltransferase (NPT1). In mammals, nicotinamide is converted to nicotinamide mononucleotide (NMN) by nicotinamide phosphoribosyltransferase (Nampt), also known as pre-B-cell colonyenhancing factor (PBEF) (58). NMN is then converted to NAD þ by Nmnat. Vitamin B3 (niacin), which consists of nicotinamide and its acid form, nicotinic acid, provides dietary source for NAD þ production. Diets also contain nicotinamide roboside, which can be phosphorylated to NMN by nicotinamide riboside kinase (Nrk) (59). Nampt/PBEF expression is upregulated in response to nutrient deprivation and stresses, such as fasting, serum deprivation, hypoxia, and DNA damaging agents (60). Nampt expression is also regulated by circadian clock (61, 62) and AMPK (63). Nampt protein and the downstream enzyme, Nmnat3 (64), are present in the mitochondria to increase the mitochondrial pool of NAD þ and offer protection against genotoxic stress induced cell death, in an SIRT3- and SIRT4-dependent manner (60). Nampt is also pivotal in the granulopoiesis process stimulated by granulocyte colony-stimulating factor (G-CSF) (65). Mice with Nampt haploinsufficiency have lower NAD þ levels in pancreatic islets and defects in glucose-stimulated insulin secretion (66). A Nampt inhibitor, FK-866, has been shown to promote cytotoxic agents-induced cancer cell death (67, 68). Nampt is also secreted into circulation (66, 69). Therefore, it is possible that Nampt may produce NMN from nicotinamide in plasma.

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SIRT1 SIRT1 is the mammalian orthologue of yeast Sir2. SIRT1 protein mainly resides in the nucleus outside of the heterochromatin region (70). SIRT1 protein actually contains two nuclear localization signals and two nuclear export signals and its subcellular localization could be affected by PI3K signaling (71). SIRT1 is also found in the cytoplasm of pancreatic cells (72), certain neurons and adult heart (71).

The Regulation of SIRT1 Transcription SIRT1 gene transcription is upregulated by nutrient deprivation (73). One of the underlying mechanisms is the increase of SIRT1 enzymatic activity under nutrient deprived condition leads to the deacetylation and activation of Forkhead box O3 (FOXO3), which activates SIRT1 gene expression by binding to p53 and relieving p53’s repression on the SIRT1 gene promoter (73). In a feedback loop, SIRT1 also deacetylates p53 to inhibit p53 activity (74–76). Another mechanism involves the protein called hypermethylated in cancer 1 (HIC1), which suppresses SIRT1 gene expression by forming a transcriptional repression complex with SIRT1 at the SIRT1 gene promoter (77). Nutrient deprivation such as the inhibition of glycolysis by 2deoxyglucose (2-DG) treatment leads to a decrease of HIC1 interaction with the redox sensing corepressor C-terminal binding protein (CtBP), resulting in an increase of SIRT1 expression (78). HIC transcription is also activated by p53, forming another feedback loop. SIRT1 transcription is also upregulated by E2F transcription factor 1 (E2F1) and cellular Myc (c-Myc); both bind directly to SIRT1 gene promoter (79, 80). DNA damage induces SIRT1 expression is E2F1 dependent (79). As a negative feedback, SIRT1 deacetylates E2F1 to repress its transcriptional activity (79). Similarly, SIRT1 deacetylates c-Myc to reduce c-Myc protein stability (80). On the other hand, SIRT1 expression is inhibited by BRCA1, through repressive binding of BRCA1 to the SIRT1 gene promoter (81). Posttranscriptionally, RNA binding protein human antigen R (HuR) recognizes the 30 - untranslated region of SIRT1 to mRNA stabilize the mRNA (82). Oxidative stress, as well as DNA damage, activates the checkpoint kinase 2 (CHK2) kinase to phosphorylate HuR to dislodge it from SIRT1 mRNA, resulting in a decrease of SIRT1 (82). Interestingly, SIRT1 translation is also suppressed by microRNA miR34a and miR-199a, both of which bind to the 30 -untranslated region of SIRT1 mRNA (83, 84). As another feedback, p53 activates miR34a gene expression (83, 84).

Posttranslational Modifications of SIRT1 Protein and Protein Regulators of SIRT1 Posttranslationally, SIRT1 protein is phosphorylated by cyclin B/cyclin-dependent kinase 1 (Cdk1) at Thr 530 and Ser 540 (85), by c-Jun N-terminal kinase 2 (JNK2) at Ser 27 (86), and by protein kinase CK2 at Ser 659 and Ser 661 (87). The

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phosphorylation of SIRT1 by cyclin B/Cdk1 is required for the deacetylase activity (85), while the SIRT1 phosphorylation by JNK2 stabilizes SIRT1 protein (86). In addition, SIRT1 is sumoylated at Lys 734. Stress-inducing agents increase SIRT1 interaction with SUMO1/sentrin specific peptidase 1(SENP1), leading to SIRT1 desumoylation and a reduction of SIRT1 deacetylase activity (88). SIRT1 is also regulated by its protein partners, which modulate its enzymatic activity either positively or negatively. For example, active regulator of SIRT1 (AROS) binds to SIRT1 and activates SIRT1 deacetylation of p53 (89). Necdin interacts with both SIRT1 and p53 to enhance SIRT1 deacetylation of p53 (90). On the other hand, deleted in breast cancer 1 (DBC1) protein interacts with SIRT1 to suppress SIRT1 activity (91, 92). Furthermore, the viral transactivator Tat produced by HIV can also bind to SIRT1 protein and inhibit SIRT1 deacetylase activity (93).

SIRT1 Substrates and Functions So far, more than 40 SIRT1 substrates have been identified (Table 2). SIRT1 has numerous physiological functions (2). It is also involved in diseases such as cancer (94–97) and neurodegeneration (98–100). In this section, the function of SIRT1 is discussed and SIRT1’s metabolic actions which are summarized in Figure 15.1, are then reviewed. SIRT1 protects cells against stress-induced apoptosis (74, 101). This may be mediated by the deacetylation and inhibition of p53 by SIRT1 (74–76). SIRT1 also suppresses E2F1-mediated transactivation and apoptosis (79). Stresses such as heat

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Figure 15.1

A myriad of metabolic regulatory actions of SIRT1 in various tissues.

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shock lead to acetylation of heat-shock factor 1 (HSF1), a major transcription factor activating stress response genes, especially chaperones, to facilitate the proper folding and function of proteins under stressed conditions. SIRT1 deacetylates HSF1 to increase its transcriptional activity (102). During hypoxia, SIRT1 also deacetylates and activates HIF-2a to mount a response against hypoxic stress (103). PPARg coactivator-1a (PGC-1a), a coactivator for many nuclear receptors and other transcription factors, regulates metabolism at multiple levels in various tissues (104, 105). For example, PGC-1a stimulates mitochondrial biogenesis (106), induces muscle fiber-type switch, and increases oxidative capacity in skeletal muscle (107). It also upregulates hepatic gluconeogenesis (108). SIRT1 is a PGC1a deacetylase (109). SIRT1 activates PGC-1a action in hepatocytes (109) and in muscle cells (110). SIRT1 also deacetylates and activates PGC-1b (111). Under energy-deficient conditions, SIRT1 and histone methyltransferase suppressor of variegation 3–9 homologue 1 (SUV39H1) are recruited to the energydependent nucleolar silencing complex (eNoSC), which contains nucleomethylin, to bind to the rDNA locus. This results in the deacetylation of histone H3 and dimethylation at Lys9 by SUV39H1 to silent chromatin at rDNA locus (112). SIRT1 also deacetylates SUV39H1 to increase SUV39H1 histone methyltransferase activity (113). The inhibition of the transcription of rRNA by eNoSC and SIRT1 saves the cell from the energy-consuming ribosome synthesis process. Nutrient deprivation also activates autophagy, a cellular self-digestion process. SIRT1 stimulates autophagy, by being part of the autophagy machinery and deacetylating other components of this machinery, such as autophagy proteins 5, 7, and 8 (Atg5, Atg7, and Atg8) (114). SIRT1 also impinges upon the AMP-activated protein kinase (AMPK) pathway. AMPK is a ubiquitous heterotrimeric serine/threonine protein kinase functions as cellular fuel sensor (115). AMPK is activated by AMP allosterically and by phosphorylation at Thr172 in the catalytic a-subunit by upstream AMPK kinases, such as LKB1 (116, 117). SIRT1 can deacetylate and activate LKB1 (118). Activated AMPK stimulates ATP-generating catabolic pathways, such as cellular glucose uptake and fatty acid b-oxidation. AMPK activation also represses ATP-consuming processes, such as lipogenesis, to restore intracellular energy balance (119, 120). AMPK not only increases PGC-1a expression (121, 122) but also activates PGC-1a by direct phosphorylation (123) and through the activation of SIRT1 by increasing Nampt expression and NAD þ synthesis (63, 124). The circadian clock plays an essential role in the regulation of organism function, including metabolism and behavior. Every cell has a molecular clock based on transcription–translation feedback loops with components such as CLOCK and brain and muscle Arnt-like protein-1 (BMAL1) transcription factor pair and their target genes, such as period homologue 2 (Per2) and cryptochrome (Cry), whose proteins suppress CLOCK-BMAL1 action in a negative feedback. The NAD þ biosynthesis enzyme Nampt’s expression, as well as the cellular NAD þ level, oscillate in circadian rhythm (61, 62). Therefore, SIRT1 protein level and activity also oscillate. SIRT1 deacetylates BMAL1 and Per2 to regulate the circadian clock (125, 126).

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SIRT1 Action in Liver In liver, fasting increases SIRT1 protein without any change of SIRT1 mRNA level (109). Caloric restriction also elevates SIRT1 expression in the liver (101, 127, 128). The protein level of SIRT1 in the liver is also upregulated by amino acid restriction (129). Conversely, high-fat diet (128) or chronic ethanol feeding (130) leads to a decrease of SIRT1 expression in rat liver, resulting in an increase in the acetylated active nuclear form of sterol regulatory element binding protein 1c (SREBP-1c), which is the key lipogenic transcription factor in liver (130). SIRT1 regulates hepatic gluconeogenesis in response to nutrient deprivation. During the early phase of fasting (6 h), the gluconeogenic program is driven by cAMP response element binding transcription factor (CREB) and CREB regulated transcription coactivator 2 (CRTC2). However, at a later stage of fasting (18 h), SIRT1 protein level is increased in hepatocytes to deacetylate CRTC2 at Lys 628 to allow E3 ubiquitin ligase COP1 to ubiquitinate CRTC2 for proteasomal degradation (131). In hepatocytes, SIRT1 deacetylates PGC-1a to activate the expression of gluconeogenic genes, such as PEPCK (109). SIRT1 also binds to Forkhead box O1 (FOXO1) through the LXXLL motif on FOXO1 to deacetylate and activate FOXO1 to stimulate gluconeogenesis (132). In addition, SIRT1 regulates liver glucose production through deacetylating and suppressing STAT3, which is a negative regulator of gluconeogenesis (133). Therefore, SIRT1 has anti-gluconeogenic function at the early stage of fasting by antagonizing CRTC2, but after prolonged fasting, SIRT1 activates PGC1a, STAT3 and FOXO1 to promote gluconeogenesis. Knockdown of SIRT1 in liver decreases hepatic glucose production and the mice are mildly hypoglycemic and insulin sensitive. Circulating cholesterol level is reduced, while hepatic free fatty acid and cholesterol content are increased, indicating that SIRT1 possibly increases liver glucose production and fatty acid oxidation and increases cholesterol efflux (134). SIRT1 regulates hepatic lipid metabolism. For instance, SIRT1 was reported to activate AMPK in hepatocytes, resulting in the suppression of lipid accumulation (135). SIRT1 also deacetylates and activates liver X receptor (LXR) nuclear receptor to regulate cholesterol and lipid homeostasis. SIRT1 activates the expression of LXR target genes, such as ABCA1, an ABC transporter for cholesterol efflux (136). In addition to LXR, SIRT1 also binds to and upregulates the action of another nuclear receptor, peroxisome proliferator-activated receptor a (PPARa), which is a critical transcription factor regulating fasting response and lipid oxidation in liver. Through PPARa, SIRT1 activates lipid metabolism. Mice with liver-specific knockout of SIRT1 develop fatty liver and hepatic inflammation when fed with a high-fat diet (137).

SIRT1 Action in Skeletal Muscle SIRT1, as well as PGC-1a, has a higher expression in the oxidative red muscles. Both acute endurance exercise and exercise training increase the expression of SIRT1 in the soleus and plantaris muscle (138). Caloric restriction increases skeletal muscle

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expression of SIRT1 and PGC-1a and is associated with increased mitochondrial biogenesis in humans (139). Functionally, SIRT1 stimulates skeletal muscle precursor cell proliferation (140). However, SIRT1 suppresses myocyte differentiation through suppression of the action of a key myogenic transcription factor, myoblast determination protein (MyoD) (141). This may also be achieved through SIRT1’s deacetylation of another myogenic transcription factor myocyte-specific enhancer factor 2D (MEF2D) at Lys 424 to suppress MEF2D activity (142). This action of SIRT1 is functional relevant in such that during nutrient deprivation such as low glucose condition, SIRT1 level is increased in the myocytes to inhibit myocyte differentiation (143). Mechanistically, low glucose activates AMPK to increase Nampt expression and NAD þ biosynthesis, leading to SIRT1 activation to suppress myogenesis (63). SIRT1 also regulates muscle metabolism. SIRT1 is induced by fasting to deacetylate and activate PGC-1a in skeletal muscle. This results in a simulation of fatty acid oxidation (110). In addition, SIRT1 deacetylates PGC-1b to promote Glut4 expression and insulin-stimulated glucose uptake (111). Therefore, SIRT1 plays a pivotal role in glucose utilization and fatty acid oxidation during nutrient deprivation and low glucose condition (110).

SIRT1 Action in the Cardiovascular System Aging significantly reduces SIRT1 activity in the heart, whereas exercise increases SIRT1 activity (144). Caloric restriction offers protection against myocardial ischemia in an AMPK-dependent fashion. Although caloric restriction does not alter the total level of SIRT1, the nuclear SIRT1 level is increased. This process appears to be mediated by nitric oxide production (145). Pressure overload and oxidative stress also stimulate SIRT1 expression in the heart (146). Cardiac expression of SIRT1 is also elevated in the Goto-Kakizaki rat, which has spontaneous diabetes and cardiac hypertrophy (147). During cardiac failure or in oxidative stressed cardiac myocytes, poly(ADP-ribose) polymerase-1 (PARP) is activated to deplete the cells of NAD þ , resulting in the inhibition of the enzymatic activity of SIRT1. Therefore, the upregulation of SIRT1 protein levels under these disease states might just be a compensatory feedback of the loss of SIRT1 activity. Activation of SIRT1 by supplying NAD þ to cells protects the cardiomyocytes against cell death (148). Fructose-enriched diet elevates cardiac NAD þ /NADH ratio and SIRT1 expression. SIRT1 mediates the stimulating effect of fructose on the expression of a-myosin heavy chain (a-MHC). This effect has been confirmed in a transgenic mouse model with cardiac-specific expression of SIRT1 (149). Cardiac-specific transgenic expression of SIRT1, when at a low to moderate level of overexpression (from 2.5- to 7.5-fold), protects the mouse heart against oxidative stress, age-dependent occurrence of cardiac hypertrophy and dysfunction. However, high level of cardiac SIRT1 expression (12.5-fold) induces oxidative stress and renders the mice more susceptible to cardiac hypertrophy and cardiomyopathy (146). One of the mechanisms for SIRT1 cardiac protective effects is through the activation

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of FOXO1-mediated production of antioxidant enzymes, such as catalase (146). SIRT1 inhibits p53-mediated cardiomyocyte apoptosis (150). Another possible mechanism is that SIRT1 deacetylates an essential histone variant H2A.z directly, resulting in the ubiquitination and degradation of H2A.z. Since H2A.z is upregulated during cardiac hypertrophy, downregulation of H2A.z by SIRT1 may protect cardiomyocytes against hypertrophy or apoptosis (151). In addition to the function in heart, SIRT1 also regulates vasculature. SIRT1 is highly expressed in endothelial cells and stimulates angiogenesis with upregulation of genes responsible for blood vessel development and vascular remodeling. Loss of SIRT1 in zebrafish or mice leads to abnormal angiogenesis during development or ischemia-induced angiogenesis. The angiogenic action of SIRT1 is mediated by SIRT1’s deacetylation FOXO1 to inhibit its antiangiogenic function (152). Furthermore, SIRT1 deacetylates and activates endothelial nitric oxide synthase (eNOS) to increase endothelium-dependent vascular relaxation (153). SIRT1 expression in mouse aorta is increased by caloric restriction but decreased by high-fat diet feeding. Treatment of endothelial cells with oxidized low-density lipoprotein (LDL) also elevates SIRT1 expression. SIRT1 transgenic mice with endothelial cell-specific expression have higher endothelial nitric oxide production and offers protection against atherosclerotic lesions when crossed with apolipoprotein E-deficient mice (154).

SIRT1 Action in Adipose Tissue SIRT1 plays an important role in adipose tissue. One single nucleotide polymorphism (SNP) of SIRT1 has been associated with visceral obesity in human (155). Fasting for 6 days elevates SIRT1 mRNA levels in subcutaneous adipose tissue of human, while obese women have significantly lower expression of SIRT1 in subcutaneous adipose tissue (156). SIRT1 attenuates adipogenesis and promotes lipolysis in the adipose tissue. This is mediated by the interaction of SIRT1 to PPARg to recruit corepressors NCoR (nuclear receptor corepressor) and SMRT (silencing mediator of retinoid and thyroid hormone receptors) to repress the action of PPARg. SIRT1 association to PPARg target gene promoters is greatly increased under the fasting condition, indicating that during nutrient deprivation SIRT1 inhibits adipogenesis and increases lipolysis to provide fuel for systemic needs (157). Since adipogenesis and osteogenesis share the same lineage in precursor cells, inhibition of adipogenesis promotes osteogenesis. This is the case for SIRT1 in such that SIRT1 binds to cartilage-specific transcription factor SRY-box 9 (Sox9) to increase the expression of cartilage-specific genes and chondrocyte formation (158). Accordingly, SIRT1 activating compounds promote lipolysis and osteoblast differentiation, while SIRT1 inhibiting compounds activate adipocyte differentiation (159, 160). SIRT1 activator resveratrol also increases epinephrine-stimulated lipolysis in human adipose tissue (156). SIRT1’s interaction with PPARg appears to specifically suppress a subset (group 2) of PPARg target genes, such as Fibroblast growth factor 21 (FGF21), but not the group 1 genes, such as fatty acid binding protein 4 (FABP4, also called aP2). Many of the group 2 genes regulated

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by SIRT1 are also the same ones activated by the treatment of PPARg ligands and antidiabetic drug thiazolidinediones (161). SIRT1 regulates adipocyte production of adiponectin and this regulation is complicated. On one hand, SIRT1 increases FOXO1 interaction with CCAAT/enhancer binding protein a (C/EBPa) to promote adiponectin transcription in adipocytes (162). On the other hand, SIRT1 inhibits the expression of endoplasmic reticulum oxidoreductin 1 (Ero1-La), an endoplasmic reticulum oxidoreductase, which is required for the secretion of high-molecularweight adiponectin complexes from adipocytes (163). Knockdown SIRT1 expression by RNA interference in adipocytes resulted in the suppression of insulin signaling, decreased glucose uptake and increased activation of JNK, which suppresses insulin signaling through phosphorylating IRS-1 at Ser307. Downregulation of SIRT1 also elicits the expression of inflammatory cytokines, such as TNFa, which may cause insulin resistance (164).

SIRT1 Action in Pancreatic b-cells SIRT1 also functions in the pancreatic b-cells to regulate insulin secretion (72, 165). In pancreatic b-cells, SIRT1 suppresses the expression of UCP2, which causes proton leak at the mitochondrial inner membrane and inhibits glucose-stimulated insulin secretion(72, 165). Consequently, SIRT1knockoutmice have higherb-cellUCP2expression and lower insulin secretion (165). On the other hand, b-cell-specific SIRT1-overexpressing (BESTO) transgenic mice have reduced UCP2 expression and increased insulinsecretionwhentriggeredbyglucose(72).Interestingly,whenthoseBESTO mice grew older (18–24months), thestimulating effect of SIRT1on insulin secretion was lost. It turns out that the circulating level of NMN, a precursor for NAD þ biosynthesis was greatly decreased in aged mice. Administration of NMN to the mice restored SIRT1 activity and SIRT1’s beneficial effect on glucose-stimulated insulin secretion (166). SIRT1 protects b-cells against oxidative stress by interacting with FOXO1 to increase the expression of NeuroD and MafA (v-maf musculoaponeurotic fibrosarcoma oncogene homologue A) transcription factors. For instance, hyperglycemia suppresses b-cell expression of MafA, while SIRT1 deacetylation and activation of FOXO1 antagonizes the downregulation of MafA (167). Cytokines such as interleukin-1b and interferon g cause pancreatic b-cell damage. This process is mediated by inducible nitric oxide synthase (iNOS) expression and nitric oxide production. In pancreatic b-cells, SIRT1 suppresses nuclear factor-kB (NFkB) to decrease iNOS expression, blunting the detrimental effects caused by cytokine treatment of b-cells (168). SIRT1 is also expressed at a high level in the glucagon-producing pancreatic a-cells (72), but the physiological significance of this has not been explored.

SIRT1 Regulation of the Insulin Signaling Pathway SIRT1 is an active regulator of the insulin signaling pathway. Insulin-resistant cells or tissues have decreased expression of SIRT1. SIRT1 regulates insulin signaling at

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multiple levels. SIRT1 inhibits the transcription of protein tyrosine phosphatase 1B (PTP1B), which is a negative regulator of the insulin signaling pathway (169). Therefore, activation of SIRT1 results in an enhancement of insulin signaling. SIRT1 interacts with IRS-1 and IRS-2. In fact, SIRT1 directly deacetylates IRS-2 to promote insulin stimulated IRS-2 tyrosine phosphorylation. Lysine to alanine mutation at four lysine residues on IRS-2 blunts insulin stimulated IRS-2 tyrosine phosphorylation, indicating the importance of the deacetylation of these residues for insulin signaling (170). SIRT1 also deacetylates PTEN (phosphatase and tensin homologue), another protein phosphatase known to antagonize the action of PI3K. However, the outcome of this action to the insulin signaling pathway has not been characterized (171).

SIRT1 Animal Models and Caloric Restriction One of the major interests of the field is to find out whether sirtuins mediate the action of caloric restriction. Caloric restriction increases SIRT1 protein levels in several tissues, including fat, brain, liver, and kidney (101, 127, 172). SIRT1 mRNA level is also increased in human peripheral blood mononuclear cells by 8 weeks of caloric restriction (173). However, a recent study indicated that the effects of dietary restriction on SIRT1 levels are more complex that SIRT1 protein amount and activity is actually downregulated in liver by caloric restriction (174). The discrepancy may be due to the possible circadian oscillation of SIRT1 protein levels in the liver (126) and that nutrient deprivation may shift the rhythm. Therefore, observations made at different times of the day may influence the outcome of up- or downregulation of SIRT1 in a particular tissue. SIRT1 knockout mice have been generated (96, 175, 176). These mice are generally unhealthy, with early lethality and small body size. They can survive when outbred. But they are sterile (175) and have impaired DNA damage response (96). Metabolically, SIRT1 null mice have higher food intake, high metabolic rate with increased physical activity and increased fatty acid oxidation (177). SIRT1 knockout mice do lose certain responses to caloric restriction. It was shown that mice deficient in SIRT1 are not able to increase physical activity in response to caloric restriction (178). In addition, while wild-type mice keep their metabolic rate during caloric restriction, SIRT1 null mice have a significantly decreased metabolic rate (177). Caloric restriction also fails to extend the life span of SIRT1 null mice (177). These findings indicate that SIRT1 at least partially mediates the effect of caloric restriction. The lack of requirement of SIRT1 for other effects of caloric restriction such as the increase of insulin sensitivity may be due to a compensatory action of other sirtuins. Transgenic mice with SIRT1 overexpression have also been generated. These animals present phenotypes that resemble effects of caloric restriction treatment. In one study, SIRT1 cDNA was knocked-in the b-actin locus. These mice have high SIRT1 protein levels in the adipose tissue and the brain. They are leaner with higher metabolic rate and are more insulin sensitive (179). They also have better performance in the rotarod test (179). Two other laboratories took another approach to

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generate SIRT1 transgenic mice, utilizing large genomic DNA fragments containing the SIRT1 gene (180, 181). Therefore, the SIRT1 transgene retains the natural regulatory mechanisms with an elevation of two- to threefold of SIRT1 protein in tissues where SIRT1 is normally expressed. Although there are discrepancies regarding food intake, energy expenditure, and adiponectin levels, both SIRT1 transgenic mouse models are more insulin sensitive without alteration of body weight under normal or high-fat diets. They are also protected from hepatic steatosis with less inflammatory response and increased expression of antioxidant genes such as manganese superoxide dismutase (MnSOD) and nuclear respiratory factor 1 (Nrf1) (180). Liver glucose production is repressed and adiponectin level is increased, even though glucose production from primary hepatocytes isolated from these transgenic mice have higher glucose production, indicating a nonautonomous action of SIRT1 regarding liver gluconeogenesis in vivo (181). All these SIRT1 overexpressing animal models consistently support the postulation that increasing SIRT1 action offers metabolic benefits against insulin resistance and fatty liver disease.

SIRT2 The SIRT2 gene is the mammalian orthologue of the yeast Hst2 gene, which has been shown to function in a complementary fashion to the yeast Sir2 gene in mediating life span extension by caloric restriction (24). SIRT2 is expressed ubiquitously in many tissues (182). We have reported that the expression of SIRT2 is induced by caloric restriction in several mouse tissues, such as the white adipose tissue and kidney (127). SIRT2 mRNA level is also increased in human peripheral blood mononuclear cells by 8 weeks of caloric restriction (173). SIRT2 is mainly a cytosolic protein (183, 184). It colocalizes with microtubules and functions as an a-tubulin deacetylase (185). SIRT2 actually shuttles between cytoplasm and nucleus (186) and transiently concentrate to the nucleus during mitosis to deacetylate histone H4 Lys16 (187) and H3 Lys56 (188). SIRT2 plays a role in the control of G2/M transition, with its expression and phosphorylation increased during G2/M phase (189). Cells overexpressing SIRT2 have an extended mitotic phase. SIRT2 is phosphorylated by cdk1 at Ser368 (190) and by cdk2 and cdk5 at Ser331 (191). SIRT2 is dephosphorylated by CDC14A (190) or CDC14B and subsequently degraded by 26S proteasome (189). SIRT2 acts as a mitotic checkpoint protein in the prophase to protect cells against chromosomal instability (192, 193). In addition, SIRT2 expression is downregulated in gliomas, while forced expression of SIRT2 in these cells causes growth arrest or apoptosis (194). Interestingly, a SIRT2 mutation disrupting its enzymatic activity was identified in a melanoma patient (195), in agreement with a possible tumor suppressor function of SIRT2. We have shown that SIRT2 level is elevated by oxidative stress and consequently deacetylates FOXO3a to activate FOXO3a-mediated antioxidative stress response (127). SIRT2 also deacetylates p300 and activates p300-mediated transcription (196). SIRT2 is expressed in oligodendroglial cells (197). It causes neurodegeneration and neuronal cell death (198–200). Inhibition of SIRT2 was shown to protect against a fruit fly model

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of Parkinson’s disease (201). ADP-ribosyltransferase activity of SIRT2 has also been reported (4). The metabolic actions of SIRT2 have not been fully elucidated. This may be partially due to the fact that SIRT2 shares some substrates with SIRT1 and SIRT2’s metabolic effect may be eclipsed by the compensatory action of SIRT1. We have demonstrated that 24 h fasting robustly induces SIRT2 mRNA and protein expression in the white adipose tissue and this is possibly mediated by b-adrenergic signaling (202). SIRT2 inhibits adipocyte differentiation via deacetylation of FOXO1 (203) to promote FOXO1’s inhibitory binding to the key adipogenic factor PPARg (202). Interestingly, SIRT2 expression in the brown adipose tissue is also elevated by cold exposure (202). Whether SIRT2 plays a role in adaptive thermogenesis is not known.

THE MITOCHONDRIAL SIRTUINS (SIRT3, SIRT4, AND SIRT5) SIRT3, SIRT4, and SIRT5 are mitochondrially localized sirtuin proteins. Mitochondrial membrane appears impermeable to NAD þ (60, 204). Therefore, the actions of mitochondrial sirtuins may be regulated by not only their own expression levels but also the local pool of NAD þ . SIRT3 was shown to reside in the mitochondrial matrix (205–208) or inner membrane (182, 209). SIRT3 appears to be the major mitochondrial protein deacetylase, since multiple hyperacetylated mitochondrial proteins have been detected only in mice deficient with SIRT3 but not in SIRT4 or SIRT5 null mice (207). SIRT3 has also been reported to enter nucleus (209, 210) and deacetylates histone H4 Lys16 (210) and H3 Lys18 (196). Genetic variations in SIRT3 gene have been linked to human longevity (211, 212). Interestingly, angiotensin II type 1 receptor (Agtr1a) deficient mice have extended life span with reduced oxidative damage, increased expression of Nampt and SIRT3 in the kidney (213). The anti-genotoxic stress effect of the NAD þ synthesis enzyme, Nampt, is mediated by SIRT3 and SIRT4 (60). SIRT3 may play an important role in adaptive thermogenesis in brown adipose (182), since its expression is upregulated by cold exposure but downregulated by elevated climate temperature. In addition, caloric restriction activates SIRT3 expression in both white and brown adipose. We found forced expression of SIRT3 in the HIB1B brown adipocytes enhances the expression of PGC-1a, UCP1, and a series of mitochondria-related genes. Functionally, sustained expression of SIRT3 decreases membrane potential and reactive oxygen species (ROS) production, while increasing cellular oxygen consumption (182). Resveratrol was found to increase SIRT3 expression in 3T3-L1 adipocytes (214). Leptin treatment also stimulates the expression of SIRT3 in adipose tissue (215). In human skeletal muscle, SIRT3 expression is decreased with age, while endurance training increases SIRT3 expression, and blunts age-associated decline of mitochondrial oxidative capacity (216). We have found that SIRT3 expression is higher in the slow-twitch muscle. SIRT3 level in rodent skeletal muscle is also elevated by caloric restriction, fasting and exercise training, and decreased by high-fat diet. In the muscle of SIRT3 knockout mice, we observed a downregulation of AMPK phosphorylation and PGC-1a expression (217).

The Mitochondrial Sirtuins (SIRT3, SIRT4, and SIRT5)

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SIRT3 has been found to deacetylate and activate the mitochondrial enzyme acetyl-CoA synthetase 2 (ACS2), which catalyzes the conversion of acetate to acetylCoA (218, 219). Glutamate dehydrogenase is also a substrate of SIRT3 (207, 220), although its enzymatic activity is not altered in the SIRT3 knockout mice (221). In addition, SIRT3 deacetylates and activates a TCA cycle enzyme, isocitrate dehydrogenase 2, which is also involved in antioxidant regeneration (220). SIRT3 protects cells, such as cardiomyocytes, against genotoxic and oxidative stress-mediated apoptosis, by interacting with and deacetylating Ku70 to increase its repression on the proapoptotic protein Bax (222). SIRT3 also deacetylates mitochondrial complex I subunit NDUFA9 to increase complex I activity (223). SIRT3 interaction with ATP synthase F1 complex alpha subunit 1 has also been reported (224). These actions of SIRT3 might be essential for the maintenance of a high cellular ATP production. SIRT3-deficient mice are phenotypically unremarkable in general (207), except a greatly decreased ATP level in liver, heart and kidney (223). This is similar to the mice lacking SIRT3 substrate ACS2, which also have 50% lower ATP. ACS2 null mice also have hypothermia and defective exercise capability under fasting condition (225). Therefore, SIRT3 null mice may display similar features when fasted. SIRT4 is a mitochondrial matrix protein (226). SIRT4 has ADP-ribosyltransferase activity (226, 227) but no detectable deacetylase activity. It was reported that SIRT4 ADP-ribosylates glutamate dehydrogenase to suppress its enzymatic activity (227). In pancreatic b-cells, SIRT4 expression is decreased by caloric restriction. SIRT4 inhibits amino acid-stimulated insulin secretion, via ADP-ribosylation and inhibition of glutamate dehydrogenase (226, 227). SIRT4 also binds to insulin degrading enzyme and the ADP/ATP carrier proteins, adenine nucleotide translocator 2 and 3 (ANT2 and ANT3), but the outcomes of these interactions are still uncharacterized (226). SIRT5 is also a mitochondrial matrix protein (221), although it was also reported to be localized in the mitochondrial intermembrane space (209). SIRT5 protects neuronal cell death when in cytoplasm and nucleus, while promoting neuronal apoptosis when residing in the mitochondria (200). Hepatic SIRT5 expression was shown to be decreased by alcohol consumption (228). The substrates and functions of SIRT5 have not been extensively characterized. There is a report that SIRT5 deacetylates cytochrome C (220). One recent finding has established SIRT5 as an important regulator of urea cycle. During fasting, the mitochondrial concentration of NAD þ is elevated to activate SIRT5. SIRT5 in turn deacetylates and activates carbamoyl phosphate synthetase 1 (CPS1); the enzyme catalyzes the first step of the urea cycle. Mice lacking SIRT5 are not able to activate CPS1 to detoxificate ammonia and exhibit elevated blood ammonia during fasting, caloric restriction, or high protein diet feeding, conditions associated with increased amino acid catabolism and generation of ammonia (221). CPS1 activity is not changed in mice deficient of SIRT3 or SIRT4 (221). A mass spectrometry analysis indicated that many mitochondrial proteins are acetylated (229). Although the acetyl transferase for mitochondrial proteins has not been identified, deacetylation of these proteins by mitochondrial sirtuins may regulate the function of these proteins and consequently a diverse array of metabolic pathways.

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SIRT6 AND SIRT7 SIRT6 is a nuclear protein associated mainly with heterochromatins (70, 230, 231). SIRT6 protein level is elevated by nutrient deprivation in cultured cells, in fasted mice, and in calorie restricted rats. This is due to an increase of SIRT6 protein stability without any change of SIRT6 mRNA level (232). SIRT6 deacetylates histone H3 Lys9 (H3K9). SIRT6 also binds to telomeres to maintain telomere structure and prevents premature cellular senescence (233). SIRT6 is involved in DNA repair (231). It enhances DNA double-strand break repair through interaction with the DNA doublestrand break repair factor DNA-PK (DNA-dependent protein kinase) (234). Inflammation in general and production of inflammation cytokines, such as TNFa, have been linked to insulin resistance and other metabolic disorders (235). SIRT6 interacts with the NFkB RelA subunit to repress NFkB transactivation and downregulation of the production of inflammation genes (236). However, it was recently reported that NAD þ produced by Nampt activates SIRT6 to stimulate immune cell synthesis of TNFa, but not other cytokines such as IL-2, IL-6, or RANTES (237). Therefore, SIRT6 may regulate TNFa production in an NFkBindependent manner. SIRT6-deficient mice display genomic instability, DNA damage repair defects and premature aging (231). These mice also exhibit severe metabolic defects, with hypoglycemia and very low levels of insulin-like growth factor 1 (IGF1) and insulin, which contribute to the early death (231, 238). Interestingly, haploinsufficiency of the RelA gene partially rescues the detrimental effects of SIRT6 knockout, indicating the unabated NFkB activation in the SIRT6 might be partially responsible for the lethality (236). SIRT7 is a nucleolar protein (70) associated with active rRNA genes (rDNA). SIRT7 binds to RNA polymerase I (Pol I) to stimulate Pol I-mediated transcription (239). SIRT7 is phosphorylated by cyclin B/CDK1 during mitosis and remains bound to RNA polymerase I and rDNA transcription factor UBF at the nucleolar organizer regions. SIRT7 gets dephosphorylated at the end of mitosis phase (240). SIRT7 has been shown to possess the capability to inhibit cell proliferation (241). Mice lacking SIRT7 have shorter mean and maximum life spans (242). SIRT7 null mice also develop cardiac hypertrophy and inflammatory cardiomyopathy, with extensive fibrosis and reduced oxidative stress resistance (242). The function of SIRT7 may be mediated by its deacetylation of p53 (242).

SIRTUIN MODULATING COMPOUNDS AND THEIR METABOLIC ACTIONS Due to the functional importance of sirtuins, efforts have been taken to screen for sirtuin regulators. Several methods have been developed (160, 243–245) and various SIRT1 or SIRT2 regulators have been reported in the literature (160, 201, 244, 246–249). Among them, resveratrol is the one that has attracted a lot of attention.

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Resveratrol, a polyphenol antioxidant present in grapes and red wine, has been reported as an activator of yeast Sir2 and mammalian SIRT1 (246). Resveratrol extends the life span of yeast (246), C. elegans (250), Drosophila (250), and fishes (251). Dietary supplementation of resveratrol protects mice against highfat-induced insulin resistance and fatty liver disease (252, 253). Although resveratrol does elicit a gene expression pattern reminiscent of caloric restriction, it does not extend the life span of mice fed on standard diet (254). It is worth noting that the activation of Sir2 or SIRT1 by resveratrol depends on the Fluor de Lys moiety of the peptide substrate. Without the fluorophore, resveratrol cannot activate SIRT1 (255, 256). Resveratrol activates many targets, including AMPK (257). Since AMPK activates SIRT1 (124), it is possible that resveratrol also activates SIRT1 indirectly. Efforts have been taken to screen for more specific and more potent sirtuin modulators than resveratrol. One of the newly identified SIRT1-specific activators, SRT1720, was shown to be more potent than resveratrol for SIRT1 activation (248). In addition, it is more specific without activation of AMPK (258). In animal studies, SRT1720 ameliorates the metabolic defects of the Zucker diabetic fatty (ZDF) rats (248). When given to high-fat-fed mice at a high dose, it blunted the development of obesity and fatty liver with increased energy expenditure and improved glucose homeostasis (248, 258). In addition, a SIRT2 inhibiting compound was shown to possess anti-Parkinson’s disease activity (201). All these results indicate a promising start for the development of sirtuin modulating drugs for the treatment of various diseases.

SUMMARY The sirtuin family of proteins is the mammalian homologues of yeast Sir2, which possess NAD þ -dependent deacetylase and ADP-ribosyltransferase activities. Sirtuins might mediate the effects of caloric restriction and regulate many biological functions, such as stress resistance, chromatin modification, cell differentiation, and metabolism. Sirtuins might serve as valuable drug targets for the treatment of a variety of diseases, ranging from diabetes, cardiovascular diseases, neurodegenerative disorders and cancer.

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11b-Hydroxysteroid Dehydrogenase Type 1 as a Therapeutic Target for Type 2 Diabetes CLARENCE HALE1 AND DAVID J. ST. JEAN, JR.2 1 2

Department of Metabolic Disorders, Amgen, Inc. Thousand Oaks, CA, USA Department of Medicinal Chemistry, Amgen, Inc. Thousand Oaks, CA, USA

INTRODUCTION It is becoming increasingly clear that type 2 diabetes mellitus (T2DM) is rapidly reaching the status of a global epidemic. As our society begins to cope with the large number of diagnosed cases, the strains on our economic and social architecture will drive the need for newer, more effective treatments. However, diabetes represents only one aspect of a more complex cluster of disorders commonly referred to as metabolic syndrome (MS) or Syndrome X (1). Metabolic syndrome represents features of central obesity, insulin resistance, dyslipidemia, and hypertension (2, 3). Though the overall pathogenesis of metabolic syndrome is not clear, insulin resistance does appear to play a central role (4). Although there is no universally accepted diagnosis criterion for MS, all definitions stress the importance of central obesity and insulin resistance. The prevalence of MS is high. It is estimated that at least one quarter of the world’s population is affected (5–8). This means that a significant portion of the worldwide human population is at risk of cardiovascular complications associated with the syndrome, such as atherosclerosis and stroke. Many of the features of MS are shared by patients who present with Cushing’s syndrome (CS) (9). These patients are characterized as having central obesity, hypertension, hyperlipidemia,

Metabolic Syndrome: Underlying Mechanisms and Drug Therapies Edited by Minghan Wang Copyright 2011 John Wiley & Sons, Inc.

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skeletal muscle wasting, and glucose intolerance. Cushing’s syndrome results from chronic exposure to excess glucocorticoid either through endogenous sources such as pituitary adenomas or exogenous sources such as oral corticosteroids. Specifically, CS is a result of hypercortisolism that is characterized by excess of the glucocorticoid hormone cortisol. However, circulating levels of cortisol are not consistently elevated in obese or insulin-resistant patients (10, 11), suggesting that glucocorticoid excess is unlikely accountable for the development of insulin resistance in T2DM patients. Glucocorticoids represent a class of molecules that play critical roles in inflammation and metabolism (Figure 16.1) (12, 13). These hormones are synthesized in the adrenal cortex and secreted from the adrenal gland, a process that in turn is regulated by signals from the hypothalamic–pituitary–adrenal (HPA) axis. Central to this effect is corticotrophin-releasing hormone (CRH) that acts on the pituitary causing it to release adrenocorticotropic hormone (ACTH), which in turn acts on the adrenal cortices inducing them to produce glucocorticoid hormones such as cortisol, the principal circulating glucocorticoid in man. Activation of the HPA axis and the subsequent release of glucocorticoids are thought to play a major role in the body’s response to stress or trauma (14–16). Glucocorticoids regulate many of the metabolic and cardiovascular responses to stress through activation of nuclear receptors. Binding of glucocorticoids with their receptors results in the formation of a complex that can then bind with glucocorticoid response elements (GREs) located in the promoter regions of target genes and thereby regulate transcription (17). This interaction could promote either elevation or repression of the transcription of downstream genes; the nature of the regulation dictates if the overall response influences metabolic or anti-inflammatory functions. For example, phosphoenolpyruvate carboxykinase (PEPCK) is a target gene for glucocorticoid regulation. Increased PEPCK expression induced by glucocorticoids can lead to elevated hepatic

Hepatic insulin resistance Dyslipidemia Hyperglycemia

Glucocorticoid target sissue

Liver

Peripheral insulin resistance

Adipose

Hepatic glucose output from gluconeogenesis Changes in lipid metabolism resulting in steatosis and insulin resistance

Formation of mature adipocytes resulting in increased body fat mass GLUT4 translocation resulting in insulin resistance

Peripheral insulin resistance

Skeletal muscle

Figure 16.1

GLUT4 translocation resulting in insulin resistance

Glucocorticoids play a key role in the pathways of glucose and lipid metabolism. Outlined in this figure are three key tissues that are sensitive to the effects of glucocorticoid action. Associated with each tissue are the outcomes that result from the insulin resistance induced by overproduction of active glucocorticoid.

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glucose output via increased gluconeogenesis. Glucocorticoids also directly alter the expression of key enzymes such as lipoprotein lipase (LPL) and hormone sensitive lipase (HSL), thereby regulating aspects of lipid metabolism (18). Perturbations of the pathways related to glucose or lipid metabolism are the foundation of MS pathogenesis. Glucocorticoids, due to their potent anti-inflammatory and immunosuppressive effects, have long been used as treatments for inflammatory conditions. However, their use is limited due to their potential of inducing obesity and insulin resistance. Glucocorticoid antagonism as an effective treatment for T2DM has not yielded much success either. Global antagonism of glucocorticoid action would lead to the activation of the HPA axis as well as diminish the important anti-inflammatory function of glucocorticoids (19). Direct regulation of local glucocorticoid levels in a given tissue, such as the liver or adipose tissue, would offer attractive therapeutic options. 11b-hydroxysteroid dehydrogenase type 1 (11b-HSD1) is primarily responsible for producing active glucocorticoids in tissues. Therefore, development of 11b-HSD1 inhibitors is a viable strategy to suppress tissue-specific glucocorticoid action.

BIOLOGICAL ROLE OF 11b-HSD1 Mineralocorticoids, such as aldosterone, and glucocorticoids, such as cortisol (humans and most mammals), or corticosterone (rodents and lower vertebrates) exert their effects through two receptor subtypes: glucocorticoid receptor (GR) and mineralocorticoid receptor (MR). MR is essentially nonselective for circulating corticosteroids; hence, it has similar binding affinities for aldosterone, cortisol, or corticosterone (20). This produces the paradox of why the activity of MR, which is mainly expressed in kidney, is essentially unaffected by high levels of circulating cortisol. It was hypothesized that a “gatekeeping” mechanism exists that regulates corticosteroid levels in a tissue-specific manner. Essentially, this mechanism maintains a low local glucocorticoid concentration that is different from that found in the general circulation. This mechanism came to light when 11b-hydroxysteroid dehydrogenases (11b-HSDs) were identified in microsomes. Two isozymes (type 1 and type 2; 11b-HSD1 and 11b-HSD2) catalyze the interconversion of the active 11-hydroxysteroid (cortisol) and the inactive 11-dehydro product (cortisone) (Figure 16.2). Cortisone has been shown to have a low affinity for GR, and therefore it has little or no transactivation activity toward GR, but the active cortisol can potentially activate MR. The existence of an interconversion enzyme helps to explain the MR paradox. Conversion of the active cortisol to the inactive cortisone would protect MR from activation. 11b-HSD2, which along with MR is mainly expressed in kidney, plays precisely such a role. Speculation that steroids undergo interconversion from active forms to inactive forms has been known for more than five decades (21). This was mainly supported by the observation that cortisone was ineffective when injected into arthritic joints; in contrast, orally administered cortisone had a pronounced antiarthritic effect (22, 23).

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Chapter 16 11b-Hydroxysteroid Dehydrogenase Type 1 O OH OH

O H H

O

H

11 β -HSD2

H

O

OH OH

HO

11 β -HSD1

H

Human

H

O Co rt iso ne

Co rt is ol

O

O OH

O

H

H H

H

O

OH

HO

11 β -HSD1

H

H

11 β -HSD2

H

Rodents

H

O

11-Dehydrocorticosterone

Corticosterone

Figure 16.2

11b-hydroxysteroids type 1 and type 2 catalyze the interconversion of inert cortisone and biologically active cortisol. Shown in this figure are the chemical structures of each glucocorticoid with subtle structural differences between the human and rodent chemical forms.

Other studies supported the conclusion that cortisone is biologically inactive and must be converted to its active form by some physiological process (24–26). Initial identification of an enzyme that catalyzed the conversion of cortisol to cortisone was also made in the 1950s (27). These studies were conducted in liver homogenates from rats, pigs, and bovines and demonstrated that the highest activity was from microsomal preparations (20). The authors concluded that the enzyme is associated with structural components of liver cells. Later this activity was described in multiple cell and tissue types, with the enzyme eventually being purified to homogeneity from rat liver preparations (28, 29). More recently, full-length recombinant human and rat 11b-HSD1 enzymes have been purified (30). These proteins retain both dehydrogenase and reductase activities. Both 11b-HSD1 and 11b-HSD2 belong to a large superfamily of short-chain alcohol dehydrogenase/reductase that use NAD(H) or NADP(H) as cosubstrates and contain a conserved catalytic domain (31, 32). The catalytic triad consists of S–Y–K, where the tyrosine residue is completely invariant. In the case of 11b-HSD1, the active site consists of a conserved YSASK motif where both serine residues are critical for catalysis but not necessary for substrate binding (33). Members of this family of proteins also share a common folding arrangement with a-helices and b-strands forming a Rossman fold for cofactor binding. The two 11b-HSD genes share little sequence homology, roughly 14%, and have distinct patterns of tissue expression as well as differences in regulatory and physiological functions. In mammals, 11b-HSD2 functions as a high-affinity NAD þ dependent dehydrogenase whose main function is to protect the MR from excess cortisol. Conversely, 11b-HSD1 differs from 11b-HSD2 by having a lower affinity for corticosteroid substrate as well as being a lower affinity NADPH-dependent enzyme. 11b-HSD1 in intact cells and tissues functions primarily as a reductase, converting

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inactive cortisone to biologically active cortisol, although isolated enzyme extract exhibit both reductase and dehydrogenase activities (Figure 16.2) (29, 34, 35). Though 11b-HSD1 mRNA can be found in many tissues, the highest abundance is found in glucocorticoid target tissues such as liver, adipose, and brain, while 11b-HSD2 is primarily expressed in mineralocorticoid target tissues such as kidney, colon, and placenta. Overall, the expression of 11b-HSD1 is highest in the liver. Areas of high expression are located around the hepatic central vein, while areas of low expression correspond to the hepatic artery, portal vein, and bile duct (36). Both these enzymes play key roles in prereceptor regulation of corticosteroid action (37). However, given the 11b-HSD1’s expression pattern fitting closely with glucocorticoid target tissues, this enzyme most likely contributes to regulation of glucose metabolism via GR at a step prior to ligand/receptor interaction. The gene for 11b-HSD1 has been identified in both humans (38) and rodents (39, 40). The human gene is located on chromosome 1q32.2, consists of 6 exons, and is over 30 kb in length (41), resulting in a 34 kD glycosylated protein. Initially these glycosylation sites were thought to be important for enzyme activity; however, later studies demonstrated this not to be the case (42, 43). The amino acid sequence identity of the human protein is 77% identical to the rat protein and 79% identical to the mouse protein. The two rodent proteins share roughly 87% amino acid homology. A single N-terminal transmembrane domain anchors 11b-HSD1 to the endoplasmic reticulum (ER) (44). The orientation is such that the catalytic domain resides in the luminal space of the ER (44, 45). This orientation may facilitate regulation of 11b-HSD1 catalytic activity through regeneration of cofactor NADPH by hexose-6-phosphate dehydrogenase (H6PD) (46). The significance of regeneration of NADPH is to continually drive 11b-HSD1 to function as a reductase. Thus, in the presence of significant levels of cortisone, there would be a preference for generation of cortisol. Hence, GR in the localized tissue area would have a continual source of ligand for activation. Such a mechanism would play an important role in tissues such as liver that modulate glucose metabolism in response to glucocorticoids, independent of the systemic glucocorticoid concentration. Support for this function comes from studies in H6PD knockout mice. Liver microsomal preparations from these mice exhibited a decrease in 11b-HSD1 reductase activity and an increase in dehydrogenase activity (47), suggesting that overall hepatic 11b-HSD1 activity has switched primarily from reductase to dehydrogenase in the presence of reduced NADPH regeneration. Recently solved crystal structures of guinea pig (48), human (49), and mouse (50) 11b-HSD1 enzymes provide a more detailed understanding of the substrate recognition and structural elements required for catalysis. From these structures it can be seen that murine and human 11b-HSD1 share common features, including virtually identical folds and dimeric organization. Though murine and human 11b-HSD1 enzymes do recognize slightly different natural substrates, with the difference of an additional hydroxyl group in the C17a position of cortisone, the active site residues are mostly conserved. The exception is at residue 177, which in humans is tyrosine and in rodents is glutamine (49, 50). This single amino acid change could help define substrate specificity and explain the subtle differences in substrate usage (51).

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Evidence suggests that both murine and human 11b-HSD1 adopt multimeric motifs, with murine being crystallized as a dimer and human adopting a tetrameric quaternary structure. Organization of the dimeric units is such that the C-terminal helix of one subunit faces the substrate binding pocket of the corresponding dimeric partner. It was postulated from the crystal structure that one tyrosine residue in particular (Y280) is involved in the stabilization of substrate (49). However, through more recent mutagenesis studies, this was shown not to be the case (52). This multimeric organization may be a result of 11b-HSD1’s localization to the ER lumen. The oxidizing environment of the ER lumen may promote the formation of disulfide bonds between 11b-HSD1 monomers or dimer pairs resulting in the stabilization of the entire complex. Overall implications of this structure would suggest a more efficient access to substrates that are located within the ER membrane. There are several factors that regulate the expression and the activity of 11b-HSD1 that have been identified and characterized. Tissue-specific regulation of expression appears to be driven by several different promoters that are independently active in liver and kidney (53). In the liver this transcriptional regulation has been proposed to be regulated by the C/EBP (CAAT/enhancer-binding protein) family of transcription factors, specifically C/EBPa (54). C/EBPa has been identified as a central regulator of energy metabolism, involved in the transcription of a number of metabolically important genes, including PEPCK and fatty acid synthase (FAS) (55, 56). This may imply that C/EBP indirectly regulates the level of active glucocorticoids in the liver through regulating the transcription of hepatic 11b-HSD1. Other transcription factors such as peroxisome proliferator-activated receptor g (PPARg) and liver X receptors (LXRs) repress 11b-HSD1 expression (57); however, the mechanism for this action is not clear. In addition to transcriptional regulation, other hormones and cytokines are able to regulate 11b-HSD1 activity. Factors that inhibit 11b-HSD1 activity include growth hormone and IGF-1 (58), whereas TNFa, IL-1b, and IL-6 increase its expression in adipose stromal cells (59). Elevated levels of serum and subcutaneous adipose tissue IL-6 have been noted in obese women, with these levels dropping during weight loss (60), which may help explain altered 11b-HSD1 expression in obesity. Notably, cortisol itself stimulates 11b-HSD1 expression in various cell types, including hepatocytes and adipocytes (34, 61). This has important implications regarding a feedback mechanism where 11b-HSD1 activity results in higher local levels of cortisol, which would stimulate more enzyme expression, thereby producing more active glucocorticoids. Eventually this cycle would lead to local excess glucocorticoid that would negatively impact cellular metabolism and insulin signaling. Both elevated IL-6 levels and enhanced cortisol generation are likely to contribute to an obese phenotype through increased induction of 11b-HSD1 expression.

11b-HSD1 MOUSE GENETICS 11b-HSD1 was initially thought to represent an arcane pathway for steroid metabolism. Once characterization of catalytic activity was determined using purified

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enzyme, it became increasingly clear that its function is to convert glucocorticoids from inactive to active forms. Later studies involving cloning and tissue expression further supported the notion that 11b-HSD1 functions to provide tissue-specific regulation of active glucocorticoid levels. The generation of mouse models that lack 11b-HSD1 or overexpress the enzyme in either adipose or liver led to another level of understanding with respect to the contribution of 11b-HSD1 to metabolic and cardiovascular homeostasis (Table 16.1). Mice that lack 11b-HSD1 develop normally and are viable and fertile (62, 63). These results suggest that at least in mice 11b-HSD1 does not play a significant role in fetal development or reproduction. There are, however, a number of significant physiological changes evident in the knockout mice. When exogenous 11-dehydrocorticosterone (11-DHC) was administered to adrenalectomized knockout mice, there was no detectable plasma corticosterone (62). The significance of this observation is that 11b-HSD1 appears to be the sole mechanism for converting inert corticosteroids into their active counterparts. Moreover, this mechanism appears to be robust enough to satisfy any local physiological needs. Mutant mice had enlarged adrenal glands and increased plasma corticosterone levels along with increased basal ACTH levels (62, 64), suggesting that there is HPA axis activation in these animals as a result of disruption of normal glucocorticoid feedback mechanisms. Indeed, 11b-HSD1 expression has been identified in subregions of the brain, including the hippocampus, hypothalamus, and, more important, the pituitary (65, 66). The adrenal hyperplasia and increased plasma corticosterone observed in the knockout mice is a compensatory response of the HPA axis to maintain normal corticosterone levels. These compensations are likely a direct result of loss of 11b-HSD1 reductase activity in brain. The most significant observations in the knockout mice dealt with glycemic control. Knockout mice had normal basal levels of PEPCK and glucose-6-phosphatase (G6Pase) mRNA expression. However, when these animals were challenged with fasting, they demonstrated impaired induction of these two important gluconeogenic enzymes. The knockout mice, when fed a highfat diet to induce obesity and insulin resistance, had significantly lower fasting plasma glucose levels compared to wild-type control animals that were on the same high-fat diet (62). Moreover, the null mice showed resistance to weight gain induced by high-fat diet despite consuming more calories (63). The reduced adiposity may be linked to reductions in visceral fat accumulation. Wild-type mice on high-fat diet had significantly higher amounts of mesenteric visceral fat, whereas mutant mice on the same high-fat diet accumulated more fat in the epididymal depot (63). Studies in women have demonstrated an association between body fat distribution and the presence of metabolic complications such as glucose intolerance and hyperinsulinemia (67). Another set of observations has demonstrated that surgical removal of visceral fat from Zucker diabetic fatty (ZDF) rats prevented the insulin resistance and glucose intolerance that accompany aging (68). These data are consistent with the fact that the reduced central obesity in the 11b-HSD1 knockout mice is metabolically beneficial. Other important features associated with the knockout phenotype are related to lipid and lipoprotein metabolism. Knockout mice fed ad libitum had lower triglycerides, probably due to a lower very-low-density

430

Table 16.1

Summary of Mouse Genetic Models of 11b-HSD1/2

Genetic model

Genetic manipulation of isozyme

Effect on glucocorticoid metabolism

Resulting effects on metabolism

References

Adipose-specific overexpression

aP2-HSD1 transgenic

apoE-HSD1 transgenic

Adipose-specific overexpression

aP2-HSD2 transgenic

Animals are insulin resistant with increased visceral adiposity and dyslipidemia; metabolic syndrome Normal glucose tolerance, body weights, and fat depots; mild insulin resistance at 24 weeks of age; hepatic steatosis; dyslipidemia Resistant to diet-induced obesity and insulin resistance

(71, 72)

Liver-specific overexpression

Increased adipose corticosterone as a result of elevated local 11b-HSD1 activity Increased liver corticosterone as a result of elevated local 11bHSD1 activity

Whole-body knockout

11b-HSD1 deficiency

Decreased adipose corticosterone as a result of elevated local 11b-HSD2 activity Decreased local corticosterone in target tissues as a result of deficient 11b-HSD1 activity

Animals are resistant to weight gain when fed a high-fat diet; reduction of visceral fat accumulation; hyperphagia; increased insulin sensitivity, HDL, apo-A1; adrenal hyperplasia; decreases in LDL and triglycerides

(73)

(70)

(62–64, 69)

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lipoprotein (VLDL) fraction, higher high-density lipoprotein (HDL) levels, and higher levels of apolipoprotein A1 (apoA1) (69). All these would indicate that the knockout animals have a phenotype with improved cardiovascular disease risk factors. As a compendium, these observations indicate that 11b-HSD1 has significant impacts on both gluconeogenesis and lipid metabolism, and that inhibition of this enzyme may lead to improved glycemic control in diabetes and possibly prevention of cardiovascular disease. Another interesting approach to study the tissue-specific effects of glucocorticoids on the development of MS was the generation of aP2-HSD2 transgenic mice. Through the use of the murine adipocyte fatty acid binding protein (aP2) promoter, 11b-HSD2 was overexpressed predominantly in adipose tissue (70). This model system capitalizes on the dehydrogenase activity of 11b-HSD2, which acts to inactivate corticosterone and thereby functions to lower the levels of the active glucocorticoid. These animals exhibited a phenotype that is strikingly similar to that of 11b-HSD1 knockout mice. Specifically, they were resistant to diet-induced obesity, had reduced accumulation of adipose tissue, and had increased insulin sensitivity (70). This study demonstrates that inactivation of glucocorticoid action in adipose tissue prevents the development of diet-induced obesity and insulin resistance. Several transgenic mouse models of 11b-HSD1 overexpression have been generated (71, 72). Once again the aP2 promoter system was used to selectively drive adipose tissue overexpression of 11b-HSD1. A modest two- to threefold increase in 11b-HSD1 activity was seen in subcutaneous and epididymal depots, with no increase of expression seen in brain, liver, skeletal muscle, or kidney (71). Overall these animals were hyperphagic and obese, with fat accumulation disproportionately in the visceral area, giving them the appearance of typical human central obesity. Corticosterone levels were elevated in adipose tissue but not in the overall systemic circulation. More important, these animals developed many of the classic features of metabolic syndrome, including glucose intolerance, insulin resistance, and hypertension (71). Here in the case of adipose tissue, it was demonstrated that increased levels of active corticosteroids through the actions of 11b-HSD1 can have a negative impact on systemic metabolism. To address the consequences of 11b-HSD1mediated elevation of corticosteroid levels in the liver, a mouse model overexpressing 11b-HSD1 in hepatocytes was developed (73). In this case, liver-specific expression was generated using hepatic regulatory sequences from the human apolipoprotein E (apoE) gene. The apoE-HSD1 mice had a two- to fivefold increase in liver 11b-HSD1 enzymatic activity, while there was no increase in enzymatic activity in adipose tissue. In this model, systemic plasma corticosteroid levels were also unaltered. In contrast to the aP2-HSD1 mice, the apoE-HSD1 mice had normal glucose tolerance, body weights, and fat depots. However, despite no alteration in body fat, the animals developed mild insulin resistance at 24 weeks of age (73). In addition, the apoE-HSD1 mice had other alterations in metabolism, including fatty liver, dyslipidemia, and hypertension (73). The subtle differences between the two phenotypes underscore the importance of tissue-specific activity of 11b-HSD1. These findings suggest that targeting the adipose 11b-HSD1 activity may be more important in treating metabolic diseases.

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Chapter 16 11b-Hydroxysteroid Dehydrogenase Type 1

ROLE OF 11b-HSD1 IN DISEASE PHYSIOLOGY As discussed in the previous section, mouse models have been developed to examine the metabolic effects of ablation or enhancement of 11b-HSD1 enzymatic activity. These models examined tissue-specific contributions of glucocorticoid action from adipose or liver to the development of obesity and insulin resistance. However, is the activity of 11b-HSD1 solely in adipose or liver tissue correlated with the insulin resistance and glucose intolerance associated with type 2 diabetes? The answer to this question may lie in the correlation between obesity in humans and altered regulation of 11b-HSD1. There have been studies from the mid-1960s linking obesity with increased cortisol secretion (74). However, later studies indicate that plasma cortisol concentrations do not appear to be increased in obese subjects (10, 11). Another approach to assess the impact of 11b-HSD1 activity on obesity is to measure the ratio of urinary cortisone to cortisol metabolites. These results have produced inconsistent results with positive, negative, or even neutral effects reported on obesity and MS (10, 75–77). However, since there are other pathways involved in cortisol metabolism (5a- and 5b-reductases, for example), the urinary ratio of cortisone and cortisol metabolites is not a specific readout of the endogenous 11b-HSD1 activity. The development of adult-onset obesity is a complex process that involves the formation of new fat cells (hyperplasia) as well as their subsequent enlargement (hypertrophy). Cortisol plays a significant role in this process by influencing the proliferation (inhibition) and differentiation (stimulation) of preadipocytes into adipocytes (78, 79). Several studies have demonstrated that glucocorticoid activity has an important role in the maturation of adipocytes from their precursor cells, including regulating the expression of genes that commit preadipocytes to differentiate into mature adipocytes (78, 80). The role of 11b-HSD1 activity in the development of visceral adiposity emerged from the observation that adipose stromal cells from human omental fat had higher oxoreductase activity than those from subcutaneous fat (81). This suggests that omental fat depots could be exposed to a constantly higher level of active glucocorticoid and hence exacerbating obesity and insulin resistance. From expression analysis, it was also clear that the 11b-HSD1 isozyme was responsible for this activity (81). From these observations, the same authors proposed that central obesity may result from excess adipose cortisol from increased 11b-HSD1 activity, which ultimately results in a state of “Cushing’s disease of the omentum.” Elevated 11b-HSD1 activity in visceral fat could account for the Cushing’s-like phenotype of central obesity and MS without observed increase in circulating corticosteroids. Studies linking 11b-HSD1 activity in adipose tissue with insulin resistance and diabetes are mixed at best. In fact, there is no correlation between 11b-HSD1 activity and T2DM, but only with obesity. For example, 11b-HSD1 activity has been shown to be increased in subcutaneous adipose tissue obtained from obese men. The increase in enzyme activity was associated with increases in body mass index (BMI) (75). Other studies have also demonstrated a correlation between increased 11b-HSD1 activity and obesity (82, 83). However, when comparisons were taken to the next logical step by investigating 11b-HSD1 activity in obese normal versus obese diabetic

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individuals, there was no obvious difference between the two groups (84). A more recent study does suggest that there may be increased levels of 11b-HSD1 mRNA in the subcutaneous and visceral adipose tissues of type 2 diabetic patients when compared to their BMI-matched controls (85). However, this group only looked at expression and not overall activity of the enzyme, which could still be the same as controls. The observations by Valsamakis et al. would support the notion that dysregulation of 11b-HSD1 is strongly associated with obesity and not necessarily being causal in the development of T2DM. The specific contributions that visceral or subcutaneous adipose tissue 11b-HSD1 make to the levels of systemic versus portal cortisol levels is not entirely clear. Since the enzyme activity in visceral adipose tissue is responsible for regulating the cortisol levels in the portal vein, it is conceivable that excess cortisol in the splanchnic circulation can contribute to hepatic insulin resistance. Supporting evidence for this notion comes from the overexpression of 11bHSD1 in mouse adipose tissue, where there is a two- to threefold increase in glucocorticoid levels in the portal vein (71). Using stable isotopic infusions of cortisol (d4-cortisol) in humans, others have shown that 11b-HSD1 does contribute to cortisol release from subcutaneous adipose tissue, but the contribution from the viscera is too low to influence portal vein cortisol concentrations (86). Therefore, there is no effect on hepatic glucocorticoid signaling. A caveat to this study is that the patients had chronic liver disease and were undergoing treatments that may influence cortisol secretion and metabolism. A clear conclusion from the study using radiotracer technique is that the liver is the primary source of splanchnic cortisol production, though the viscera do contribute substrate via delivery of cortisone into the portal vein (86, 87). Based on these findings, inhibition of liver 11b-HSD1 enzymatic activity would be a logical target for the treatment of T2DM because reducing the levels of active glucocorticoid in the liver would have the benefit of suppressing hepatic glucose output. Also, any drug taken through the oral route would have the benefit of first-pass liver exposure through the portal vein. Effects on adipose tissue should not, however, be dismissed, as the genetic rodent models do indicate a clear contribution of adipose 11b-HSD1 activity to metabolic functions. Also not to be overlooked is the potential role 11b-HSD1 plays in the pancreatic islet of Langerhans. 11b-HSD1 mRNA has been detected in the islets of humans and diabetic rodent models, and 11b-HSD1 enzymatic activity suppresses glucose-stimulated insulin secretion from islets isolated from ob/ob mice (88, 89). The effects of inhibiting islet 11b-HSD1 activity in the context of human T2DM are not as clear as those of liver and adipose tissue and require further study to define the true impact.

SMALL-MOLECULE THERAPEUTICS Suppression of 11b-HSD1 activity is expected to have the benefits of reducing hepatic glucose output, improve peripheral insulin sensitivity, and reduce adiposity through suppression of lipogenesis. These properties have made pharmacological

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Chapter 16 11b-Hydroxysteroid Dehydrogenase Type 1

inhibition of 11b-HSD1 activity a likely candidate for the treatment of T2DM. There is precedence that decreasing glucocorticoid activity has beneficial effects on glucose metabolism. For example, administration of the GR antagonist RU486 (mifepristone) to db/db mice resulted in a reduction of plasma glucose, probably through the suppression of PEPCK and hence lowering gluconeogenic output (90). Also, RU486 administration to patients with Cushing’s syndrome resulted in reduced plasma glucose as well as improvement of other metabolic parameters (91, 92). As can be imagined, this approach is limited because of the resulting consequences of systemic glucocorticoid deficiency, which leads to Addison’s disease. Prolonged glucocorticoid antagonism would also lead to the activation of the HPA axis resulting in adrenal hyperplasia (19). Thus, long-term direct antagonism of the GR does not represent a viable treatment option for T2DM. A number of compounds have been reported to be inhibitors of 11b-HSD1. Some are represented by synthetic steroids or are steroidal-like in overall structure, so they lack selectivity, potency, and general drug-like qualities (Figure 16.3) (93, 94). For example, the bile acid chenodeoxycholic acid is an inhibitor of 11b-HSD1, but it does not inhibit 11b-HSD2 (93). Moreover, its micromolar potency does not lend itself to being a very good drug candidate. Another weak inhibitor of 11b-HSD1 is metyrapone, which has been shown to be effective in decreasing levels of cortisol in man (95). However, this compound also inhibits the enzyme 11b-hydroxylase, which is another key enzyme in cortisol biosynthesis. Moreover, its millimolar potency makes it even less of a therapeutic option than the bile acids (93, 95). The licorice derivative glycyrrhetinic acid (GA) and its hemisuccinate ester carbenoxolone (CBX) are inhibitors of both 11b-HSD1 and 11b-HSD2 (93, 94). Both these compounds are significantly more potent inhibitors than that of bile acids or metyrapone, with reported IC50 values in the double-digit nanomolar range (93, 94). Even though CBX is not a selective inhibitor of 11b-HSD1, a clinical study involving T2DM subjects was conducted (96). Outcomes from this study do suggest that inhibition of 11b-HSD1 may have the potential to reduce glucose production rates through reduction of glycogenolysis (96). However, there was also a significant increase in

O

O

OH

OH O

O

N O HO

O

HO

N

O

GA

Figure 16.3

CBX

O

Metyrapone

Early nonselective inhibitors of 11b-HSD1 and 11b-HSD2 included glycyrrhetinic acid (GA), which is a derivative of licorice, and its hemisuccinate ester carbenoxolone (CBX). Other inhibitor molecules that are nonsteroidal in structure include metyrapone.

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blood pressure in the patients treated with CBX (96). It has been demonstrated that inhibition of 11b-HSD2 with licorice derivatives results in mineralocorticoid excess leading to hypertension (97, 98). The observed increase in blood pressure from this study also demonstrates that dual 11b-HSD1/11b-HSD2 inhibition have limited utility in treating a chronic condition such as T2DM. Since the initial report by Barf et al. (99), there has been an explosion of reports describing novel small-molecule inhibitors of 11b-HSD1. Recently, a number of manuscripts have appeared that provide a thorough review of the current patent literature (100–104). This chapter will therefore focus on reports that have appeared in peer-reviewed journals, with an emphasis on compounds that have published in vitro and in vivo data. Recently, Abbott Laboratories reported a variety of novel adamantane-based inhibitors of 11b-HSD1 (Figure 16.4) (105–110). An internal high-throughput screen (HTS) discovered amide Abbott-1. This compound exhibited excellent potency on mouse and human 11b-HSD1as well as significant selectivity over 11b-HSD2 (106). Although Abbott-1 was a promising starting point in terms of in vitro potency, this compound was rapidly metabolized by mouse liver microsomes (MLM). Identification of the major metabolites revealed multiple sites of reactivity, with oxidation primarily occurring on the adamantine ring. Attempts to systematically attenuate the metabolism of Abbott-1 eventually led to Abbott-2. This compound possessed better potency and was significantly more stable in MLM. Although the compound had a high in vivo clearance rate, it was suitable for a proof of concept in an ex vivo assay. In a typical ex vivo assay, animals are dosed with compounds and, after a prescribed time, selected tissues (typically liver, adipose, and/or brain) are collected, minced, and incubated with cortisone. The cortisol produced is quantified by LC/MS with the percent inhibition expressed as a percentage decrease relative to control animals. At 1 h, Abbott-2 showed modest inhibition (36, 51, 37% in liver, adipose and brain, respectively), although the effect had significantly declined or was absent at 7 h (106). Further attempts to improve the in vivo metabolic stability began from a structurally similar analogue Abbott-3 (105). Once again, although this molecule was potent and selective, it was plagued by high in vitro and in vivo clearance in mice. Investigation revealed that the primary amide was prone to hydrolysis. Analogue Abbott-4 proved to be one of the most potent analogues and displayed dramatically improved MLM stability. Compound Abbott-4 inhibited 11-bHSD1 activity in an ob/ob mouse ex vivo model, with liver 11b-HSD1 activity being significantly reduced out to 16 h (77%). Moderate inhibition in adipose was observed over the same period. Replacement of the liable primary amide group in Abbott-3 with a variety of functional groups, including heterocycles and alkyl carboxylic acids (108), has also been reported. Analogues Abbott-5 and Abbott-6 had improved pharmacokinetic (PK) profiles, although slight drops in potency were observed. Despite the loss in potency, both compounds showed significant reduction in 11b-HSD1 activity in an ob/ob mouse ex vivo model. Sulfone and sulfonamide replacements for the amide functionality in Abbott-3 have also been disclosed (107). Like the analogues described above, the metabolic

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Chapter 16 11b-Hydroxysteroid Dehydrogenase Type 1

stability was improved relative to Abbott-3. In this case, ex vivo activity was conducted in diet-induced obese (DIO) mice. Both compounds significantly reduced enzymatic activity out to 16 h in liver, adipose, and brain. Other analogues based around the adamantyl core of Abbott-1 have also been described. Abbott-9, which substituted a butyrolactam for the linear amide in H N

H N

N O

N

N

N O

HO

N

N CF3

NC Abbott-1 h-11 β-HSD1 IC 50 = 14 nM m-11 β-HSD1 IC 50 = 53 nM HEK IC 50 = 260 nM h-11β -HSD2 IC50 = >100,000 nM MLM CLint = 380 L/h/kg

Abbott-2 h-11 β-HSD1 IC 50 = 5 nM m-11 β-HSD1 IC 50 = 15 nM HEK IC 50 = 29 nM h-11 β-HSD2 IC 50 = >100,000 nM MLM CL int = 7 L/h/kg

Cl H N O

Cl H N

HO2 C

O

O

O

H 2N

O O

NH

Abbott-3 h-11β-HSD1 IC50 = 6 nM m-11β-HSD1 IC 50 = 3 nM HEK IC50 = 22 nM h-11 β-HSD2 IC 50 = 12,000 nM MLM (percent remaining af ter 30 min) = 43 Mouse in vivo CL = 4.2 L/h/kg

Abbott-4 h-11β-HSD1 IC50 = 15 nM m-11β-HSD1 IC 50 = 12 nM HEK IC 50 = 59 nM h-11β-HSD2 IC50 = 20,000 nM MLM (percent remaining after 30 min) = 94 Ex vivo dat a (ob/ ob mice, 30 mg/kg) % inhibition in liver (1 h/7 h/16 h) = 99/93/77 % inhibition in adipose (1 h/7 h/16 h) = 37/38/36

Cl

Cl N N N HN

H N

O

H N

O

HO O

O O

Abbott-5 h-11 β-HSD1 IC 50 =41 nM m-11 β-HSD1 IC 50 = 87 nM HEK IC 50 = 119 nM h-11β -HSD2 IC50 = >100,000 nM MLM (% percent remaining after 30 min) = 79 Ex vivo data (ob/ ob mice, 30 mg/kg) % inhibition in liver (7 h/16 h) = 62/37 % inhibition in adipose (7 h/16 h) =17/20

Figure 16.4

Abbott-6 h-11 β-HSD1 IC 50 =89 nM m-11 β-HSD1 IC 50 = 74 nM HEK IC 50 = 455 nM h-11β -HSD2 IC50 = 100,000 nM MLM (percent remaining after 30 min) = 78 Ex vivo data (ob/ ob mice, 30 mg/kg) % inhibition in liver (7 h/16 h) = 99/93 % inhibition in adipose (7 h/16 h) =50/37

Compounds reported in the literature by Abbott Laboratories to be inhibitors of 11b-HSD1. Included are data from biochemical- and cellular-based assays. Also included, where appropriate, are data from pharmacokinetic (PK) and pharmacodynamic (PD) studies as well as in vivo efficacy data from diabetic animal models. Human ¼ h, mouse ¼ m, and rat ¼ r for species forms of the enzyme used for assay.

437

Small-Molecule Therapeutics F H N

O O

S OO

F H N H2 N

Cl

S OO

Abbott-7 h-11β-HSD1 IC50 = 7 nM m-11β-HSD1 IC50 = 4 nM HEK IC50 = 98 nM h-11β-HSD2 IC 50 = 26,000 nM MLM (percent remaining af ter 30 min) = 57 Ex vivo Data (DIO mice, 30 mg/kg) % inhibition in liver (1 h/7 h/16 h) = 95/95/89 % inhibition in adipose (1 h/7 h/16 h) = 87/93/86 % inhibition in brain (1 h/7 h/16 h) = 90/90/77

O O

Cl

Abbott-8 h-11β-HSD1 IC50 = 6 nM m-11β-HSD1 IC50 = 3 nM HEK IC 50 = 125 nM h-11β-HSD2 IC50 = >100,000 nM MLM (percent remaining af ter 30 min) = 61 Ex vivo Data (DIO mice, 30 mg/kg) % inhibition in liver (1 h/7 h/16 h) = 70/78/82 % inhibition in adipose (1 h/7 h/16 h) = 62/88/93 % inhibition in brain (1 h/7 h/16 h) = 29/48/73

Cl

N

O

O

H N

CN

N H2 N

H 2N

O O

O

O Abbott-9 h-11β-HSD1 IC 50 = 3 nM m-11 β-HSD1 IC 50 = 2 nM HEK IC 50 = 45 nM h-11β -HSD2 IC50 = 32,000 nM MLM (percent remaining after 30 min) = 97 Ex vivo Data (DIO mice, 30 mg/kg) % inhibition in liver (1 h/7 h/16 h) = 99/94/67 % inhibition in adipose (1 h/7 h/16 h) = ND/71/46

Abbott-10 h-11β-HSD1 IC50 = 13 nM m-11β-HSD1 IC50 = 28 nM HEK IC50 = 450 nM h-11 β-HSD2 IC 50 = 100,000 nM MLM (percent remaining after 30 min) = 85 Ex vivo Data (DIO mice, 30 mg/kg) % inhibition in liver (7 h/16 h) = 80/70 % inhibition in adipose (7 h/16 h) = 40/20 % inhibition in brain (7 h/16 h) = 60/72

O Cl

N

NH2 Cl Abbott-11 h-11 β-HSD1 K i = 4 nM r-11β-HSD1 K i= 8 nM h- and r-11β-HSD2 IC 50 = >10,000 nM HEK IC 50 = 17 nM

Figure 16.4 (Continued ).

Abbott-3 (109), and Abbott-10, which contained a bridged replacement for the adamantyl core (110), were reported to have excellent potency and microsomal stability, and demonstrated a robust pharmacodynamic (PD) response in DIO mice. Scientists at Abbott have also disclosed another series of 11b-HSD1 inhibitors (111). The most promising of these analogues (Abbott-11) was active on both rat

438

Chapter 16 11b-Hydroxysteroid Dehydrogenase Type 1

and human 11b-HSD1, selective over 11b-HSD2, and potent in a HEK293 cellular assay. No PD data for these compounds was reported. Amgen has reported two different structural classes of 11b-HSD1 inhibitors: aminothiazolone (112–115) and amide/sulfonamide-based molecules (Figure 16.5) (116–120). In collaboration with Biovitrum, Amgen has disclosed a number of reports that describe a novel class of 11b-HSD1 inhibitors based on a thiazolone core. The first of these reports described the evolution of a HTS screening hit (Amgen-1) (114). Although the screening hit exhibited high in vivo clearance in rats, the in vitro potency and low molecular weight provided a promising starting point for optimization. Amgen-2 was potent, selective against 11b-HSD2, and demonstrated almost complete inhibition of 11b-HSD1 (88 and 91% at 2 and 6 h postdose, respectively) in adipose tissue of C57/Bl6 mice. Given the excellent profile seen with Amgen-2, a number of reports describing the PD effects of this compound have been published. Amgen-2 has been shown to effectively lower cortisol levels in adipose, as well as systemically, without activating the HPA axis (121). In a 12 week study, administration of Amgen-2 to Ldlr 3KO (Ldlr/Apob100/100Lepob/ob) mice was shown to be efficacious in improving glucose homeostasis at doses greater than 10 mg/kg (122). Compared to control animals, plasma insulin, blood glucose, and glucose tolerance were all improved, without a corresponding change in body weight or adipose/lean tissue masses. A 28 day study administering Amgen-2 to DIO mice resulted in improved glucose utilization in an intraperitoneal glucose tolerance test (IPGTT), along with statistically significant drops in cholesterol and nonesterified fatty acids (NEFA), without a decrease in body weight (123). A shorter 10 day study in KKAy mice showed an increase in adiponectin levels (124). Decreases in fasting blood glucose levels were also observed in this study, with drops comparable to rosiglitazone. Amgen later disclosed that the development of Amgen-2 was halted due to unacceptably high pregnane X receptor (PXR) activation that translated to increased cytochrome P450 3A4 (CYP3A4) activity in cryopreserved human hepatocytes (112). Incorporation of polar groups at the C-5 position of the thiazolone core was shown to reduce PXR activation. Amgen-3 showed potent biochemical and cellular activity as well as minimal PXR activation. This lower PXR activation led to only a minimal increase in CYP3A4 mRNA production in hepatocytes. Amgen-3 had a good PK profile across a variety of species (mouse, rat, dog, and monkey) and was shown to significantly reduce 11b-HSD1 at 3 and 30 mg/kg dose in a cynomolgus monkey ex vivo PD study. A similar analogue (Amgen-5) possessed excellent biochemical and cellular potency; however, the activity suffered from a dramatic protein shift in the presence of 3% human serum albumin (HSA) (115). It was postulated that the high protein binding caused this cell shift. Finally, Amgen and Biovitrum reported the evolution of another aminothiazolone Amgen-6 (113). Starting from Amgen-6, researchers at Amgen and Biovitrum optimized this scaffold replacing both the phenyl moiety and the substituents at the C-5 position. Amgen-7 had excellent potency and significantly decreased 11b-HSD1 activity in a C57/Bl6 mouse ex vivo PD assay.

Small-Molecule Therapeutics O

O N

N

CF3

Amgen-1 h-11 β-HSD1 Ki = 503 nM In vivo CL typically > 2.0 L/h/kg

CF3 S

N H CF3

S

N H

439

Amgen-2 h-11 β-HSD1 K i = 22 nM Whole-cell IC 50 = 33 nM Rat in vivo CL = 0.188 L/h/kg Ex vivo Data (C57/Bl6 mice, 30 mg/kg) % inhibition in adipose (2 h/6 h) = 88/91 PXR activity (% of positive control) = 45

O N N H

S

OH

F Amgen-3 h-11β-HSD1 K i = 35 nM Whole-cell IC 50 = 34 nM PXR activity (% of positive control) = 6 O

O N

N N H

S CF3

Amgen-4 h-11β-HSD1 K i = 110 nM

S

N H

Amgen-5 h-11 β-HSD1 K i = 23 nM Whole-cell IC 50 = 28 nM Rat in vivo CL = 0.760 L/h/kg %PPB = 99.8 Whole-cell IC50 with 3% HSA = 2000 nM

O N S N H Amgen-6 h-11β-HSD1 K i = 126 nM

O N S N H Amgen-7 h-11β-HSD1 K i = 3 nM h-11β-HSD2 IC 50 = 10,000 nM Ex vivo data (C57/Bl6 mice, 30 mg/kg) % inhibition in adipose (2 h/6 h) = 86/87 % inhibition in liver (2 h/6 h) = 89/79

Figure 16.5 Compounds reported in the literature by Amgen Inc. to be inhibitors of 11b-HSD1. Included are data from biochemical-and cellular-based assays. Also included, where appropriate, are data from pharmacokinetic (PK) and pharmacodynamic (PD) studies as well as in vivo efficacy data from diabetic animal models. Human ¼ h and mouse ¼ m for species forms of the enzyme used for assay.

440

Chapter 16 11b-Hydroxysteroid Dehydrogenase Type 1 NO2

N N N

N CF3

S O O

N

S O O

Amgen-8 h-11β-HSD1 IC 50 = 16 nM m-11β-HSD1 IC50 = 32 nM HEK293 IC 50 = 461 nM

HO

Amgen-9 h-11β-HSD1 IC 50 = 3 nM m-11β-HSD1 IC50 = 57 nM HEK293 IC50 = 53 nM

CF3

O NH 2

N N

S O O

Amgen-10 h-11β-HSD1 IC 50 = 0.7 nM HEK293 IC 50 = 14 nM Rat PK CL (iv, 0.5 mg/kg) = 1.4 L/h/kg %F (po, 2 mg/kg) = 41 Ex vivo Data (cynomolgous monkeys, 0.4, 2, 10 mg/kg) ED 50 in mesenteric fat at 2 h = 0.4 mg/kg

HO

HO

CF3

CF3

N N

O

O

N

Amgen-11 h-11β-HSD1 IC50 =1.7 nM HEK293 IC50 = 3.2 nM % remaining @ 30 min (HLM, RLM) = 5, <5

Amgen-12 h-11β-HSD1 IC 50 = 1.4 nM HEK293 IC 50 = 5.8 nM Rat PK CL (iv, 0.5 mg/kg) = 1.0 L/h/kg %F (po, 2 mg/kg) = 72 In vitro cytotoxicity (HeLa cells), IC 50 = 2.5 μM Ex vivo Data (cynomolgous monkeys, 2 and 10 mg/kg) ED 50 in mesenteric fat at 2 h = 2 mg/kg

Figure 16.5

(Continued ).

Amgen has also reported that substituted arylsulfonylpiperazines can be efficient inhibitors of 11b-HSD1 (120). Optimization of the initial HTS hit, Amgen-8, produced analogue Amgen-9. This compound showed excellent potency for both human and mouse enzymes, although no PD data was reported. Amgen-9 was cocrystallized with human 11-bHSD1. Analysis of the cocrystal structure revealed that the molecule binds in a “V” shape with the sulfonyl group engaged in a hydrogen bond with Ala172. Further optimization of this molecule led to the discovery of

Small-Molecule Therapeutics HO

CF 3

HO

441

CF3

N

N

O

O

N

NH 2

O Amgen-13 h-11 β-HSD1 IC 50 = 14 nM HEK293 IC 50 = 167 nM In vitro cytotoxicity (HeLa cells), IC 50 = >10 μM Rat PK CL (iv, 0.5 mg/kg)= 0.89 L/h/kg %F (po, 2 mg/kg) = 69

Amgen-12 h-11β -HSD1 IC 50 = 1.4 nM HEK293 IC50 = 5.8 nM In vitro cytotoxicity (HeLa cells), IC 50 = 2.5 μ M

HO

N

CF3

N O

OH

Amgen-14 h-11 β-HSD1 IC 50 = 14 nM HEK293 IC 50 = 52 nM In vitro cytotoxicity (HeLa cells), IC 50 = > 10 μM Rat PK CL (iv, 0.5 mg/kg) = 0.24 L/h/kg %F (po, 2 mg/kg) = 76 Ex vivo Data (Sprague Dawley Rats, 1, 3, 10, and 30 mg/kg, po) ED50 in epididymal fat at 2 h = 10 mg/kg

Figure 16.5 (Continued ).

Amgen-10, which possessed subnanomolar biochemical activity, moderate in vivo clearance, and was efficacious in a cynomolgus monkey ex vivo assay with an ED50 of 0.4 mg/kg (119). Amgen has also disclosed a report describing substituted benzamides as inhibitors of 11b-HSD1 (116). Starting from Amgen-11, optimization for metabolic stability delivered Amgen-12. This molecule demonstrated favorable PK profiles in three species (rat, cynomolgus monkey, and dog) and had an ED50 in a cynomolgus monkey ex vivo assay of 2.0 mg/kg. A cocrystal structure of Amgen-12 and 11bHSD1 was obtained. The conformations of Amgen-12 and Amgen-10 are similar, with both molecules adopting a “V” shape in the binding sites. Unfortunately, Amgen12 had an EC50 of 2.5 mM in an in vitro cytotoxicity assay using HeLa cells that prevented further development (118). Attempts to lower the cytotoxicity seen with Amgen-12 led to two structurally similar analogues (117, 118). Replacing the pyridine present in Amgen-12 with either a primary amide or a tertiary alcohol (Amgen-13 and Amgen-14) reduced the cytotoxic liability, albeit at the expense of both biochemical and cellular potency. In 2002, Biovitrum was the first company to describe “drug-like” inhibitors of 11b-HSD1 (99). This seminal publication described the in vitro potencies of the

442

Chapter 16 11b-Hydroxysteroid Dehydrogenase Type 1 Cl

S

O

H N

O O

N

N

N

HCl

S

Biovitrum-1 (BVT-2733) h-11 β-HSD1 IC 50 = 3341 nM m-11β-HSD1 IC50 = 96 nM h-11 β-HSD2 IC 50 = >10,000 nM

O N

O N

N

N H

O N H Biovitrum-2 h-11β-HSD1 K i = 100 nM

O

F3 CO Biovitrum-3 h-11β -HSD1 Ki = 19 nM Adipocyte IC 50 = 95 nM CL int (human/rat) = 6/6 μ L/min/mg

Figure 16.6

Compounds reported in the literature by Biovitrum to be inhibitors of 11b-HSD1. Included are data from biochemical- and cellular-based assays. Also included, where appropriate, are data from pharmacokinetic (PK) and pharmacodynamic (PD) studies as well as in vivo efficacy data from diabetic animal models. Human ¼ h and mouse ¼ m for species forms of the enzyme used for assay.

selective inhibitor Biovitrum-1 (Figure 16.6). Although only moderately potent (with poor activity on the human enzyme) on mouse 11b-HSD1 (IC50 ¼ 96 nM), this molecule lowered blood glucose levels in a dose-dependent manner without significant changes in liver and heart enzymes. After 1 week treatment in KKAy mice (167 mg/kg), there were decreases in mRNA levels of two key enzymes in hepatic glucose production (phosphoenolpyruvate (75% of control) and glucose-6-phosphatase (55% of control)) (125). Biovitrum-1 has been shown to improve the metabolic profile in a number of mouse diabetic models, including ob/ob and db/db (126). In hyperglycemic mice treated with Biovitrum-1, circulating glucose was significantly lowered (50–88% of control); however, no effect was seen in lean C57/Bl6 mice. This compound also improved glucose utilization in an oral glucose tolerance test (OGTT) in KKAy and ob/ob mice with glucose values ranging from 65–75% of control. In 2007, Biovitrum released two additional reports regarding their work toward selective 11b-HSD1 inhibitors. The first of these reports described a novel amide-based scaffold exemplified by Biovitrum-2 (127). Generally this series of compounds were only moderately potent on the human enzyme (100 to >10,000 nM). In the second communication, Biovitrum described the optimization of oxazolone-based inhibitors,

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443

O N

F

N

Figure 16.7 BMS-1 h-11β -HSD1 IC 50 = 0.1 nM

Compound reported in the literature by Bristol-Meyers Squibb to be an inhibitor of 11b-HSD1. Included are data from biochemical assays. Human ¼ h for species form of the enzyme used for assay.

which are structurally related analogues to the molecules codeveloped with Amgen. No PD data was reported for either series. Scientists at Bristol-Myers Squibb have published a single report regarding novel 11b-HSD1 inhibitors (Figure 16.7) (128). These compounds, exemplified by BMS-1, showed excellent biochemical potency. No PD data was reported for these compounds, although a cocrystal structure of BMS-1 and human 11b-HSD1 was released. Merck has released a number of publications describing triazole-based inhibitors of 11b-HSD1 (Figure 16.8) (129–134). The first of these reports, which appeared in

CF3

N N

N N

N

N

F

N

N O Merck-1 h-11β -HSD1 IC50 = 4 nM m-11β-HSD1 IC50 = 8 nM Mouse PD (10 mg/kg) % inhibition (1 h/4 h) = 43/44

Merck-2 h-11 β-HSD1 IC 50 = 2.2 nM m-11 β-HSD1 IC 50 = 1.9 nM Mouse PD (10 mg/kg) % inhibition (4 h/16 h) = 86/74 Cl

F3CO OMe N N Cl

N

N

N

Merck-3 h-11 β-HSD1 IC 50 = < 1 nM m-11β -HSD1 IC50 = <1 nM Mouse PD (10 mg/kg) % inhibition (4 h/16 h) = 70/74

Figure 16.8

N

F

Merck-4 h-11β-HSD1 IC50 = 5 nM m-11β -HSD1 IC50 = 16 nM Mouse PD (10 mg/kg) % inhibition (1 h/4 h) = 92/91

Compounds reported in the literature by Merck to be inhibitors of 11b-HSD1. Included are data from biochemical assays. Also included, where appropriate, are data from pharmacodynamic (PD) studies. Human ¼ h and mouse ¼ m for species form of the enzyme used for assay.

444

Chapter 16 11b-Hydroxysteroid Dehydrogenase Type 1

2005, describes the structure–activity relationship (SAR) around a series of fused triazole analogues (132). Analogue Merck-1 showed excellent potency for both mouse and human 11b-HSD1 and demonstrated a significant decrease in a systemic enzyme activity in a mouse PD model (43 and 44% reduction relative to control at 1 and 4 h postdose). In this assay, mice were dosed orally with the test compound. After a predetermined amount of time, [3H]-cortisone was injected intravenously via the tail vein. After an additional 2 min, blood was collected, and the steroids were extracted and analyzed by HPLC. In a separate report, Merck-1 was shown to lower body weight, insulin, fasting glucose, triglycerides, and cholesterol in DIO mice (131). This molecule was also shown to slow plague progression in a murine model of atherosclerosis (131). A structurally related analogue Merck-2 was reported to have a slight increase in potency, but improved PK profile (the adamantyl group in Merck-1 was identified to be a metabolic liability) of this compound led to a more robust effect in Merck’s PD assay, with enzyme activity significantly suppressed out to 16 h (130). In 2008, Merck described other triazole-based 11b-HSD1 inhibitors (exemplified by Merck-3 and Merck-4) (129, 133, 134). The majority of these compounds were shown to be exceedingly potent on both the mouse and human enzymes and showed a robust inhibition of 11b-HSD1 in Merck’s mouse PD assay. In 2009, researchers at Merck-Serono disclosed two series of 11b-HSD1 inhibitors (Figure 16.9) (135, 136). In general, the spirocyclic amide analogues possessed excellent potency for human 11-bHSD1 while exhibiting modest potency for the mouse isoform. The adamantyl-based inhibitors (see also Abbott’s 11b-HSD1 inhibitors) demonstrated good potency across three separate species (human, mouse, and rat). No PD data for either series was reported. In 2005, a single report from Novartis (in collaboration with Array BioPharma and Hoffman-La Roche) described perhydroquinolylbenzamides as inhibitors of 11b-HSD1 (Figure 16.10) (137). The most potent analogue Novartis-1 showed modest enzyme activity, but did show a reduction in liver corticosterone levels in adrenalectomized (ADX) mice.

O

H N

N O

H N O

O

H 2N Merck-Serono-1 h-11β-HSD1 IC 50 = 0.5 nM m-11β-HSD1 IC 50 = 377 nM h-11β -HSD2 IC50 = >100,000 nM

Figure 16.9

Merck-Serono-2 h-11 β-HSD1 IC 50 = 8 nM m-11 β-HSD1 IC 50 = 10 nM r-11 β-HSD1 IC 50 = 31 nM h-11β-HSD2 IC50 = >10,000 nM

Compounds reported in the literature by Merck-Serono to be inhibitors of 11b-HSD1. Included are data from biochemical assays. Human ¼ h, mouse ¼ m, and rat ¼ r for species form of the enzyme used for assay.

Small-Molecule Therapeutics

445

Cl H N O H

O

Cl

N

H

Novartis-1 h-11 β-HSD1 IC 50 = 100 nM h-11β -HSD2 (% inhibition at 10 μM) = 22 Rat primary hepatocytes (% inhibition at 1 μ M) = 59 ADX mouse liver corticosterone concentration (% change, 50 mg/kg) = –73

Figure 16.10 Compound reported in the literature by Novartis to be an inhibitor of 11b-HSD1. Included are data from biochemical and cellular assays. Human ¼ h for species form of the enzyme used for assay.

Pfizer has disclosed the optimization of an initial lead Pfizer-1 into the clinical candidate PF-915275 Pfizer-2 (Figure 16.11) (138). Although Pfizer-2 possessed excellent activity for human 11b-HSD1 and low in vivo clearance in rats, the weak potency on the rodent enzymes (mouse Ki ¼ 750 nM and rat IC50 ¼ 14.5 mM (hepatoma cells)) prevented evaluation in a rodent disease model. To circumvent this issue, in vivo efficacy was demonstrated in cynomolgus monkeys via 11b-HSD1mediated prednisone to prednisolone conversion (139). Maximal inhibition (87%) was observed with a 3 mg/kg oral dose. A single report has been published by Schering-Plough describing their discovery of azepane sulfonamides as 11b-HSD1 inhibitors (Figure 16.12). Optimization of the initial screening hit Schering-Plough-1 led to a variety of potent analogues, the most active of which was Schering-Plough-2. No in vivo PD data for these molecules was reported. Initial reports from Wyeth described their attempts to improve a Biovitrum analogue (Figure 16.13). It was thought that the corresponding oxazole derivatives

Cl

O O S N H

O O S N H

O N

N

N

NH2

NC Pfizer-1 h-11β-HSD1 Ki = 287 nM HLM = high CL

Pfizer-2 (PF-915275) h-11β-HSD1 IC 50 = <1 nM HEK293 EC50 = 5 nM m-11β-HSD1 K i = 750 nM

Figure 16.11 Compounds reported in the literature by Pfizer to be inhibitors of 11b-HSD1. Included are data from biochemical assays and cellular assays. Human ¼ h and mouse ¼ m for species form of the enzyme used for assay.

446

Chapter 16 11b-Hydroxysteroid Dehydrogenase Type 1 O O

N

O O N S

O Schering-Plough-1 h-11β-HSD1 IC50 = 111 nM m-11β-HSD1 IC 50 = 9000 nM

Schering-Plough-2 h-11β-HSD1 IC50 = 3 nM m-11β-HSD1 IC50 = 57 nM

Figure 16.12

Compounds reported in the literature by Schering-Plough to be inhibitors of 11b-HSD1. Included are data from biochemical assays. Human ¼ h and mouse ¼ m for species form of the enzyme used for assay.

Cl O

H N

S O O

H N N S O O O

N

N S

Biovitrum-14225 h-11β-HSD1 IC50 (cell) = 1.5 μ M h-11 β-HSD2 IC 50 = > 200 μ M

Wyeth-1 h-11β -HSD1 IC50 (cell) = 2.3 μ M h-11 β-HSD2 IC 50 = > 200 μ M

O O O S

O

O O S

CF3 Wyeth-2 h-11 β-HSD1 IC 50 = 0.19 μ M h-11 β-HSD2 IC 50 = > 200 μ M

O

Wyeth-3 h-11β -HSD1 IC50 = 0.06 μ M h-11 β-HSD2 IC 50 = > 200 μ M

N N

N

F3C

F

N N

S O O

Wyeth-4 h-11 β-HSD1 IC 50 = 26 nM m-11β -HSD1 IC50 = 10 nM Ex vivo Data (C57/Bl6, 10 mg/kg) % Inhibition in liver at 5 h = 44 Ex vivo Data (Sprague Dawley Rats, 30 mg/kg) % Inhibition in liver at 2 h = 59

Figure 16.13

F3 C

O

F

N

H2 N N

S Cl O O

Wyeth-5 h-11β-HSD1 IC 50 = 10 nM m-11 β-HSD1 IC 50 = 10 nM Ex vivo Data (C57/Bl6, 10 mg/kg) % Inhibition in epididymal fat at 5 h = 80

Compounds reported in the literature by Biovitrum and Wyeth to be inhibitors of 11b-HSD1. Included are data from biochemical assays and ex vivo assays. Human ¼ h and mouse ¼ m for species form of the enzyme used for assay.

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447

would maintain potency while improving certain physiochemical properties (140). Unfortunately, this approach failed to deliver compounds that were more potent, with the most active analogue being Wyeth-1. Interestingly, a side product Wyeth-2, derived from the formation of the desired oxazole, was isolated and tested. The O S

O O

NH

O

Shen and Leng-1 h-11 β-HSD1 IC 50 = 260 nM

O O S N H

OH

O

O

Cl

O

O

N

Shen and Leng-2 h-11 β-HSD1 IC 50 = 850 nM m-11β -HSD1 IC50 = 440 nM

Cl

S N

O O S N H

N

N

O

O Shen and Leng-3 h-11β-HSD1 IC 50 = 6 nM m-11β-HSD1 IC50 = 307 nM

Shen and Leng-4 h-11β-HSD1 IC 50 = 2 nM m-11β-HSD1 IC50 = 2 nM h- and m- 11 β-HSD2 IC50 = >1000 μM ob/ob mice PD data (50 mg/kg) % Reduction in HBA1c (at 23 d) = 0.57 % Reduction in serum insulin (at 23 d) = 28 OH

O OH

N OH

O O

OH

O HO

HO

Sterenin A h-11 β-HSD1 K i = 68 nM

Colletoic Acid h-11β-HSD1 IC50 = 13 nM m-11β -HSD1 IC50 = 460 nM

Figure 16.14 Compounds reported in the literature by miscellaneous authors to be inhibitors of 11bHSD1. Included are data from biochemical assays along with data from in vivo studies. Human ¼ h and mouse ¼ m for species form of the enzyme used for assay.

448

Chapter 16 11b-Hydroxysteroid Dehydrogenase Type 1 N

N N O Webster-1 h-11β-HSD1 IC50 = 80 nM h-11β-HSD2 (% inhibition at 10 μ M) = 0

Figure 16.14

N O Su-1 h-11β -HSD1 IC 50 = 113 nM

(Continued ).

keto-sulfone had an IC50 of 190 nM in an 11b-HSD1 whole-cell assay. In a separate report, Wyeth elaborated on the SAR with the most potent analogue shown in Figure 16.13 (Wyeth-3) (141). No PD data was reported for either structural class. Wyeth has also reported a series of piperazine-based inhibitors (see also Amgen’s 11b-HSD1 inhibitors). Wyeth-4 had an IC50 of 26 nM in an 11b-HSD1 cell-based assay and demonstrated significant activity in a mouse and rat ex vivo PD assay (142). After chronic dosing for 1 week, Wyeth-4 showed a 34% reduction in insulin in a cortisone-induced hyperinsulinemia model. A similar analogue to Wyeth-4, Wyeth-5 showed robust inhibition of 11b-HSD1 activity using an epididymal fat ex vivo assay (143). This analogue was also efficacious at lowering fed glucose and fasted insulin levels in DIO mice. In 2008 and 2009, Leng and coworkers published a number of reports describing their work toward a novel 11b-HSD1 inhibitor (Figure 16.14) (144–148). These reports, based mostly on molecular modeling and docking studies, described several potent inhibitors (Shen and Leng-1–3). Further optimization of one of these compounds produced Shen and Leng-4, which decreased levels of both hemoglobin Alc (HbA1c, 0.57%) and serum insulin (28%) in ob/ob mice after treatment for 23 days (148). Shen and Leng have also reported dual PPARg agonists/11b-HSD1 inhibitors (145). Takatsu and coworkers have described the inhibitory activity on 11b-HSD1 of two compounds isolated from fungal extracts, sterenin A and colletoic acid (149, 150). These molecules were reported as being potent and selective for human 11b-HSD1, with no observed inhibition of 11b-HSD2. Finally, two separate reports have appeared from academic research groups that describe adamantyl-based inhibitors of 11b-HSD1 (Figure 16.14) (151, 152).

SUMMARY Clearly, as the world population grows more obese, the healthcare system will be dealing with an avalanche of newly diagnosed T2DM. In response to this, the pharmaceutical industry is investing a significant amount of effort to develop new therapies for the treatment of this condition. Such new therapies are needed to replace medicines that have been in use for the past 50 years or to replace medicines that

References

449

have questions about long-term safety. Clearly, glucocorticoids such as cortisol play a critical role in the pathogenesis of T2DM. In addition, it is clear that 11b-HSD1 plays an equally critical role at the tissue level in the production of this powerful glucocorticoid. As has been described in this chapter, there are currently a myriad of potent and selective inhibitors of this enzyme with features that would make them useful pharmacological agents in the treatment of T2DM. Questions regarding the validity of targeting this mechanism for the treatment of T2DM will begin to be answered as these chemical entities reach clinical trials. Human studies will also uncover any potential liabilities associated with 11b-HSD1 inhibition. Liabilities such as dysregulation of the HPA axis can lead to consequences of increased ACTH, which may in turn result in abnormal adrenal responses. Overproduction of androgen hormones in females would be of particular concern, resulting in increased facial and body hair, pattern baldness, acne, and other enhanced male features. If inhibition of 11b-HSD1 has minimal effects on the HPA axis and if the effects of inhibition on glucose homeostasis are as robust as was observed in rodent genetic models, then drugs that inhibit 11b-HSD1 activity have the potential to be a valuable addition to the treatment options for T2DM.

REFERENCES 1. REAVEN, G.M. 1988. Banting lecture 1988. Role of insulin resistance in human disease. Diabetes 37:1595–1607. 2. GRUNDY, S.M., H.B. BREWER, Jr., J.I. CLEEMAN, S.C. SMITH, Jr., and C. LENFANT. 2004. Definition of metabolic syndrome: report of the National Heart, Lung, and Blood Institute/American Heart Association conference on scientific issues related to definition. Circulation 109:433–438. 3. REAVEN, G.M. 1991. Insulin resistance, hyperinsulinemia, hypertriglyceridemia, and hypertension: parallels between human disease and rodent models. Diabetes Care 14:195–202. 4. DEFRONZO, R.A., and E. FERRANNINI. 1991. Insulin resistance. A multifaceted syndrome responsible for NIDDM, obesity, hypertension, dyslipidemia, and atherosclerotic cardiovascular disease. Diabetes Care 14:173–194. 5. ATHYROS, V.G., E.S. GANOTAKIS, M. ELISAF, and D.P. MIKHAILIDIS. 2005. The prevalence of the metabolic syndrome using the National Cholesterol Educational Program and International Diabetes Federation definitions. Curr Med Res Opin 21:1157–1159. 6. ATHYROS, V.G., E.S. GANOTAKIS, M.S. ELISAF, E.N. LIBEROPOULOS, I.A. GOUDEVENOS, and A. KARAGIANNIS. 2007. Prevalence of vascular disease in metabolic syndrome using three proposed definitions. Int J Cardiol 117:204–210. 7. FORD, E.S., W.H. GILES, and W.H. DIETZ. 2002. Prevalence of the metabolic syndrome among US adults: findings from the third National Health and Nutrition Examination Survey. JAMA 287:356–359. 8. KOLOVOU, G.D., K.K. ANAGNOSTOPOULOU, K.D. SALPEA, and D.P. MIKHAILIDIS. 2007. The prevalence of metabolic syndrome in various populations. Am J Med Sci 333:362–371. 9. ARNALDI, G., A. ANGELI, A.B. ATKINSON, X. BERTAGNA, F. CAVAGNINI, G.P. CHROUSOS, G.A. FAVA, J.W. FINDLING, R.C. GAILLARD, A.B. GROSSMAN, B. KOLA, A. LACROIX, T. MANCINI, F. MANTERO, J. NEWELL-PRICE, L.K. NIEMAN, N. SONINO, M.L. VANCE, A. GIUSTINA, and M. BOSCARO. 2003. Diagnosis and complications of Cushing’s syndrome: a consensus statement. J Clin Endocrinol Metab 88:5593–5602. 10. FRASER, R., M.C. INGRAM, N.H. ANDERSON, C. MORRISON, E. DAVIES, and J.M. CONNELL. 1999. Cortisol effects on body mass, blood pressure, and cholesterol in the general population. Hypertension 33:1364–1368.

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Monoclonal Antibodies for the Treatment of Type 2 Diabetes: A Case Study with Glucagon Receptor Blockade HAI YAN1, WEI GU2, 1 2

AND

MURIELLE VENIANT-ELLISON2

Department of Protein Science, Amgen, Inc. Thousand Oaks, CA, USA Department of Metabolic Disorders, Amgen, Inc., Thousand Oaks, CA, USA

INTRODUCTION In the past two decades, monoclonal antibody (mAb)-based drugs have evolved into a successful modality of human therapeutics and constituted one of the most rapidly growing class of therapeutic agents in the industry. To date, more than 20 therapeutic mAbs have been approved in the United States for diverse disease indications such as oncology, chronic inflammation, and kidney transplantation. In the diabetes area, an aglycosylated humanized anti-CD3 antibody (ChAglyCD3) and an interleukin-1 beta (IL-1 beta) antibody (Xoma 053) are being tested for the treatment of type 1 diabetes mellitus (1, 2); however, no mAb-based therapy has been developed for the treatment of type 2 diabetes mellitus. The development of type 2 diabetes is linked to peripheral insulin resistance and incremental b-cell dysfunction. Hyperglycemia, impaired insulin secretion, and insulin resistance in target tissues such as liver are the key characteristics of type 2 diabetes (3, 4). Among the various metabolic and endocrine abnormalities, dysregulated glucagon secretion and action is a major factor. Glucagon is a 29-amino acid peptide hormone secreted by pancreatic a-cells in response to falling circulating glucose and plays an important role in maintaining glucose homeostasis in concert

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with insulin and other endocrine factors. Glucagon action is mediated by glucagon receptor (GCGR) (5), a member of the family B G-protein-coupled receptors (GPCRs). Glucagon stimulates glucose production by increasing glycogenolysis and gluconeogenesis while inhibiting glycogen synthesis in target tissues such as liver and kidney (6, 7). In patients with type 2 diabetes, the basal glucagon levels are elevated and the postprandial glucagon secretion is not suppressed by insulin, which is partially responsible for the increased hepatic glucose production (8–11). As a result, the ability of insulin to suppress gluconeogenesis and glycogenolysis is reduced in the postprandial state, leading to impaired glucose tolerance in these patients (12–14). Therefore, reducing glucagon levels and inhibiting GCGR activity are expected to reduce excess glucose production, which can lead to improved overall glycemic control in type 2 diabetes. It is believed that inhibition of the glucagon signaling pathway, particularly GCGR, represents promising therapeutic targets for the treatment of type 2 diabetes (15). In the past decade, extensive efforts have been devoted to the identification and development of small-molecule GCGR antagonists by a number of pharmaceutical companies (16, 17). However, the development of such compounds has been hindered by multiple impediments such as loss of efficacy due to elevated glucagon levels induced by compensatory response to the receptor blockade. The most advanced compound (Bay-27-9955) that was evaluated in early clinical trials significantly attenuated exogenous glucagon-stimulated glucose production in healthy volunteers (18). However, there is no report on whether this compound was tested in diabetic patients. The clinical development of this molecule was later terminated for unknown reasons. Given the fact that GCGR has the potential to be an efficacious therapeutic target for type 2 diabetes, and numerous challenges have been associated with the smallmolecule approach to block the receptor, we undertook a novel approach of antagonizing human GCGR (hGCGR) with a mAb. Developed by Kohler and Milstein (19) about 35 years ago, hybridoma technology has been extensively explored to target soluble proteins or single transmembrane cell-surface receptors for the development of human therapeutics (20, 21). Despite the successes with these targets, no significant progress has been made in targeting GPCRs, such as hGCGR, with mAbs due to various technical hurdles, including difficulties in expressing and purifying sufficient amounts of biologically active GPCR proteins as immunogens and lacking of proper screening tools, and the lack of robust detection systems to analyze hybridoma supernatants containing low concentrations of GPCR-specific antibodies. Although there are several reported clinical trials with anti-chemokine receptor mAbs, currently there are no therapeutic GPCR mAbs that have been approved as human therapeutics. In this chapter, we will discuss a strategy and process generating functional antagonizing GCGR antibodies. Moreover, we will summarize results from multiple in vitro and in vivo experiments supporting the concept of antagonizing hGCGR for the treatment of type 2 diabetes. We will also briefly discuss advantages as well as disadvantages of targeting GCGR with mAbs. The detailed process for generating and characterizing anti-GCGR mAbs has already been published (22–24).

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GCGR IMMUNOGEN GENERATION Identifying a proper hGCGR antigen source and generating a sufficient quantity of antigen were the first two critical steps for the antibody campaign. hGCGR is a member of the secretin GPCR receptor subfamily. It is 477-amino acid long and contains a relatively long N-terminal domain (116 amino acids), and three extracellular loops ranging from 12 to 32 amino acids in length (5, 25). Numerous experiments have demonstrated that the N-terminal domain and the extracellular loops are critical for ligand binding and subsequent receptor activation (26–28). The conventional expression and purification systems used for soluble antigens are not readily applicable to hGCGR antigen generation. Similar to other GPCRs, hGCGR requires proper lipid composition and membrane environment to retain its native structures and biological activities (29). To ascertain proper antigen sources for generating functional GCGR antibodies, we evaluated multiple approaches. Extracellular regions of receptors often contain antigens that can be used to raise antibodies. Consistent with this notion, functional rabbit polyclonal antibodies against GCGR were generated via immunization with extracellular domain-derived peptides (30). Multiple antigenic regions in the N-terminus and extracellular loops of hGCGR were identified and corresponding peptides were synthesized as potential antigens. To increase antigenicity, these peptides were further modified by adding T-cell epitopes at either the N-terminal or the C-terminal regions. In addition, the N-terminal region of hGCGR directly fused to mouse IgG1 Fc was also generated as a potential source of antigen. In the meantime, we reasoned that the full-length receptor could present not only the linear epitopes in the receptor-derived peptides but also the conformational epitopes unique to the topology of the receptor on the cell surface (31). Thus, major efforts were devoted to establish a recombinant stable cell line overexpressing hGCGR as an antigen source for generating functional antibodies. Multiple eukaryotic expression constructs encoding the full-length hGCGR with green fluorescent protein (GFP) tagged at C-termini were used. Stable pools of cells were first sorted based on GFP expression and then on binding with Cy5-labled glucagon (32). Single cell-derived stable cell lines from the sorted pools were expanded and assayed for hGCGR mRNA expression by bDNA analysis. In the cell line selected as the immunogen, receptors with two different affinities were detected with a total receptor expression level of about 45 pmol/mg cell membrane proteins, representing about 0.25% of the total membrane proteins.

IMMUNIZATION AND HYBRIDOMA GENERATION The hGCGR-derived peptides, the mouse Fc fusion protein with hGCGR N-terminal fragment, and the cell membrane fractions from the hGCGR-expressing cell line were used as antigen sources to generate mouse mAbs in C57BL/6 mice or human mAbs in XenoMouse (33). After multiple immunizations and immunization boosting regimes

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with the same antigens, the sera were titrated with original antigen as well as the fulllength hGCGR stable cell line. Although the serum samples from the peptideimmunized mice were immunoreactive to the original peptides, they did not demonstrate any specific binding to the hGCGR stable cell line, suggesting that the epitopes presented in the peptides are different from those in the full-length receptor. On the other hand, the serum samples from mice immunized with the cell membrane fractions from the hGCGR-expressing cell line induced specific FACS shift when assayed with the same cell line, indicating the presence of antibodies to cell-surface proteins, although not specifically to hGCGR as the sera also induced a similar FACS shift when assayed with the host cell line that does not express hGCGR. The inability to detect any significant difference in FACS shift profiles between the hGCGRexpressing and the host cell lines was somewhat expected given the fact that hGCGR only represents about 0.25% of the total membrane proteins in hGCGR stable cells. The sera from the mice immunized with hGCGR N-terminal fragment fusion protein demonstrated only a weak, but specific titer toward the full-length receptor. Based on these results, spleens from mice immunized with hGCGR cell membrane were harvested for hybridoma generation.

IDENTIFICATION OF GCGR BINDERS To identify hybridoma lines expressing hGCGR specific mAbs, a differential ELISA assay was developed. This assay requires running two concurrent ELISA assays for individual hybridoma supernatants: one with immunogen cells, expressing hGCGR, and another with the host cells, expressing no hGCGR. For each hybridoma supernatant, a ratio of ELISA signals was generated by dividing the signal obtained from the immunogen cells with that from the host cells. If the hybridoma supernatant contains an antibody against a cell-surface antigen common to both the host and the hGCGR cells, the expected signal ratio should be approximately around 1. A high ratio would indicate the presence of hGCGR specific antibodies in that particular hybridoma supernatant. The hybridoma lines with a high signal ratio were selected and expanded, and the supernatants were subjected to next round of differential ELISA assay until the hGCGR antibody-containing hybridoma lines were positively identified. In the end, about 50% of the binders from primary screen were eventually eliminated after three rounds of differential ELISA assays.

IDENTIFICATION OF GCGR mAbs WITH ANTAGONIZING ACTIVITY Establishing a reliable functional assay is critical for identifying functional antiGCGR antibodies. In reality, the ideal functional assay should be able to detect the antibody activity directly from crude hybridoma supernatants. More importantly, the assay should also be sensitive enough to detect the activity of the antibody at concentrations in the lower mg/mL (nM) range. Two functional assays were used to assess the presence of hGCGR-antagonizing antibodies in hybridoma supernatants.

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One assay was designed to measure the ability of the antibody to block glucagoninduced responses. Another assay employed an aequorin-based calcium readout in a cell line where hGCGR is coupled to Ga16. In the end, both functional assays produced similar results regarding the antagonizing activities of the GCGR antibodies. The hybridoma supernatants with hGCGR-antagonizing activities were also screened for activities toward GCGRs from other species. Although there is some degree of amino acid sequence diversity among the GCGRs from different species, the vast majority of the functional anti-hGCGR antibodies demonstrated crossreactivity toward GCGRs from mice, rats, and cynomolgus monkeys. Based on the results of competition assay with radiolabeled ligand and IC50 measurements in human and rodent functional cell lines, antibody cDNAs from several hybridoma lines were cloned and sequenced. Two antibodies (mAb A and mAb B) were recombinantly expressed as human IgG2 isotype to minimize antibody effector functions on target cells. In addition, mAb A was isotype-switched from human IgG2 to mouse IgG1 to generate mAb Ac for chronic studies in various mouse diabetes models. FACS analysis using Alexa-647 labeled antibodies demonstrated that both mAb A and mAb B specifically bind to recombinant cell lines expressing human, mouse, or cynomolgus monkeys GCGR. No specific binding was detected with the parental cell line or a cell line expressing recombinant human GLP-1R (34), the receptor with the highest sequence homology to hGCGR (5, 35). These results demonstrated the antigen specificity of the GCGR antibodies. In the functional assay, mAb B in the nanomolar range induced a rightward shift of the glucagon dose–response curve, confirming the specific antagonizing activity toward hGCGR. Similarly, the antibody also antagonized mouse and cynomolgus monkeys GCGRs in the functional assays. Schild analysis revealed that mAb B is a competitive antagonist since increasing concentrations of the antibody induced parallel rightward shifts of the glucagon dose–response curves, and such inhibition was surmountable with increasing concentrations of glucagon. Competition binding assays verified that mAb B is capable of displacing the binding of radiolabeled glucagon to hGCGR with an IC50 of around 0.5 nM, indicating the antibody is more potent than the cognate ligand in binding to the receptor.

IN VIVO EFFICACY STUDIES OF GCGR-ANTAGONIZING mAbs In the acute treatment setting, a single injection of mAb B at 1 or 3 mg/kg decreased blood glucose levels to a normal range in ob/ob mice fed ad libitum. Both doses achieved similar efficacy during the first 24 h while the 3 mg/kg dose had a longer duration of action (22), likely due to the favorable pharmacokinetic (PK) profile (24). The results also suggest that the 1 mg/kg dose of the antibody was sufficient to achieve maximum glucose-lowering efficacy in the experimental setting. In normal C57BL/6 mice, mAb B dose dependently improved glucose tolerance as demonstrated in an intraperitoneal glucose tolerance test (IPGTT) experiment (22).

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Chronic administration of mAb Ac for 34 days resulted in a sustained glucoselowering effect in diet-induced obesity (DIO) mice. Importantly, no treatment-related tachyphylaxis was observed in the treated animals. The sustained glucose-lowering effect was maintained even when one-tenth of the original dose was used for subsequent administrations (22). Similar to the acute treatment in C57BL/6 mice, the chronic treatment with the GCGR antibody significantly decreased fasting glucose levels in a dose-dependent manner without inducing hypoglycemia in DIO mice. The chronic treatment with the antibody led to overall improvement in metabolic profiles in DIO mice, including reduced insulin level and increased active GLP-1 level. As expected, the treatment with GCGR-antagonizing antibodies also led to hyperglucagonemia. Despite the elevated glucagon level in the treated animals, there was a sustained glucose-lowering effect throughout the treatment (22). Similar to the results obtained from GCGR knockout mice (36) and in vivo studies with GCGR antisense oligonucleotides (37), chronic treatment with a GCGRantagonizing antibody also induced a-cell hyperplasia in male C57BL/6 mice (23). Interestingly, the severity of the pancreatic a-cell hyperplasia in the treated mice reached a plateau during the first 3 weeks of the treatment period regardless of the doses tested, but the overall severity was far less than that displayed by GCGR KO mice (36). Importantly, the treatment-induced a-cell hyperplasia was reversed after a treatment-free recovery phase (washout period) in a dose-dependent manner (23). Despite the treatment-induced a-cell hyperplasia in the mice, b-cell function was preserved, and glucose clearance and excursion were significantly improved in the treated animals (22).

CONCLUSIONS Antagonizing the glucagon signaling pathway, including blocking glucagon receptor functions, has been recognized as a potential treatment for type 2 diabetes. Since the glucagon receptor is a GPCR, pharmaceutical companies have concentrated most of their efforts on developing small-molecule antagonists. Here, we applied the mAb approach to this traditional small-molecule target and demonstrated the feasibility of generating functional mAbs against GCGR (Figure 17.1). The antagonizing antibodies can achieve profound efficacy in lowering glucose levels in various animal models. No fasting hypoglycemia and tachyphylaxis in treated animals were observed. Despite the treatment-related pancreatic a-cell hyperplasia in the chronic treatment, b-cell function was well preserved. Although requiring intraperitoneal or intramuscular delivery in future clinical settings, the GCGR-antagonizing mAbs offer several advantages compared with GCGR small-molecule antagonists and other small-molecule antidiabetic drugs. The GCGR mAb has a long plasma half-life and allows for less frequent administration. The GCGR antibody would also provide high specificity toward hGCGR so that potential off-target side effects observed with small molecules could be avoided. In addition, antibody engineering of the GCGR antibody could also offer a

References Target validation

Proper antigen generation

Engineering and expressing antibodies

In vivo efficacy in animal models

Antibody source and immunization route selection Screening for cross-species reactivity

PKDM and toxicity studies

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Screening for binders

Screening for functional antibodies

Clinical candidates

Figure 17.1 The process for identification of functional GCGR antibodies.

wide range of PK profiles to accommodate therapeutic needs (38). In summary, GCGR-antagonizing mAbs could be an efficacious therapeutic for the treatment of type 2 diabetes. The process of generating functional GCGR antibody could be applicable to other GPCRs (Figure 17.1).

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10. SHAH, P., A. BASU, R. BASU, and R. RIZZA. 1999. Impact of lack of suppression of glucagon on glucose tolerance in humans. Am J Physiol 277:E283–E290. 11. MULLER, W.A., G.R. FALOONA, E. AGUILAR-PARADA, and R.H. UNGER. 1970. Abnormal alpha-cell function in diabetes. Response to carbohydrate and protein ingestion. N Engl J Med 283:109–115. 12. SHAH, P., A. VELLA, A. BASU, R. BASU, W.F. SCHWENK, and R.A. RIZZA. 2000. Lack of suppression of glucagon contributes to postprandial hyperglycemia in subjects with type 2 diabetes mellitus. J Clin Endocrinol Metab 85:4053–4059. 13. MITRAKOU, A., D. KELLEY, M. MOKAN, T. VENEMAN, T. PANGBURN, J. REILLY, and J. GERICH. 1992. Role of reduced suppression of glucose production and diminished early insulin release in impaired glucose tolerance. N Engl J Med 326:22–29. 14. SINGHAL, P., A. CAUMO, P.E. CAREY, C. COBELLI, and R. TAYLOR. 2002. Regulation of endogenous glucose production after a mixed meal in type 2 diabetes. Am J Physiol Endocrinol Metab 283:E275–E283. 15. JIANG, G., and B.B. ZHANG. 2003. Glucagon and regulation of glucose metabolism. Am J Physiol Endocrinol Metab 284:E671–E678. 16. DJURIC, S.W., N. GRIHALDE, and C.W. LIN. 2002. Glucagon receptor antagonists for the treatment of type II diabetes: current prospects. Curr Opin Investig Drugs 3:1617–1623. 17. SLOOP, K.W., M.D. MICHAEL, and J.S. MOYERS. 2005. Glucagon as a target for the treatment of Type 2 diabetes. Expert Opin Ther Targets 9:593–600. 18. PETERSEN, K.F., and J.T. SULLIVAN. 2001. Effects of a novel glucagon receptor antagonist (Bay 27-9955) on glucagon-stimulated glucose production in humans. Diabetologia 44:2018–2024. 19. KOHLER, G., and C. MILSTEIN. 1975. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495–497. 20. CARTER, P.J. 2006. Potent antibody therapeutics by design. Nat Rev Immunol 6:343–357. 21. AIRES DA SILVA, F., S. CORTE-REAL, and J. GONCALVES. 2008. Recombinant antibodies as therapeutic agents: pathways for modeling new biodrugs. BioDrugs 22:301–314. 22. YAN, H., W. GU, J. YANG, V. BI, Y. SHEN, E. LEE, K.A. WINTERS, R. KOMOROWSKI, C. ZHANG, J.J. PATEL, D. CAUGHEY, G.S. ELLIOTT, Y.Y. LAU, J. WANG, Y.S. LI, T. BOONE, R.A. LINDBERG, S. HU, and M.M. VENIANT. 2009. Fully human monoclonal antibodies antagonizing the glucagon receptor improve glucose homeostasis in mice and monkeys. J Pharmacol Exp Ther 329:102–111. 23. GU, W., H. YAN, K.A. WINTERS, R. KOMOROWSKI, S. VONDERFECHT, L. ATANGAN, G. SIVITS, D. HILL, J. YANG, V. BI, Y. SHEN, S. HU, T. BOONE, R.A. LINDBERG, and M.M. VENIANT. 2009. Long-term inhibition of the glucagon receptor with a monoclonal antibody in mice causes sustained improvement in glycemic control, with reversible alpha-cell hyperplasia and hyperglucagonemia. J Pharmacol Exp Ther 331:871–881. 24. LAU, Y.Y., P. MA, L. GIBIANSKY, R. KOMOROWSKI, J. WANG, G. WANG, H. YAN, M.M. VENIANT, and T. KAKKAR. 2009. Pharmacokinetic and pharmacodynamic modeling of a monoclonal antibody antagonist of glucagon receptor in male ob/ob mice. AAPS J 11:700–709. 25. VASSILATIS, D.K., J.G. HOHMANN, H. ZENG, F. LI, J.E. RANCHALIS, M.T. MORTRUD, A. BROWN, S.S. RODRIGUEZ, J.R. WELLER, A.C. WRIGHT, J.E. BERGMANN, and G.A. GAITANARIS. 2003. The G proteincoupled receptor repertoires of human and mouse. Proc Natl Acad Sci USA 100:4903–4908. 26. UNSON, C.G., C.R. WU, Y. JIANG, B. YOO, C. CHEUNG, T.P. SAKMAR, and R.B. MERRIFIELD. 2002. Roles of specific extracellular domains of the glucagon receptor in ligand binding and signaling. Biochemistry 41:11795–11803. 27. PARTHIER, C., M. KLEINSCHMIDT, P. NEUMANN, R. RUDOLPH, S. MANHART, D. SCHLENZIG, J. FANGHANEL, J.U. RAHFELD, H.U. DEMUTH, and M.T. STUBBS. 2007. Crystal structure of the incretin-bound extracellular domain of a G protein-coupled receptor. Proc Natl Acad Sci USA 104:13942–13947. 28. BOKOCH, M.P., Y. ZOU, S.G. RASMUSSEN, C.W. LIU, R. NYGAARD, D.M. ROSENBAUM, J.J. FUNG, H.J. CHOI, F.S. THIAN, T.S. KOBILKA, J.D. PUGLISI, W.I. WEIS, L. PARDO, R.S. PROSSER, L. MUELLER, and B.K. KOBILKA. 2009. Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor. Nature 463:108–112. 29. ROSENBAUM, D.M., S.G. RASMUSSEN, and B.K. KOBILKA. 2009. The structure and function of G-proteincoupled receptors. Nature 459:356–363.

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Part Four

Lessons Learned and Future Outlook

Chapter

18

Drug Development for Metabolic Diseases: Past, Present, and Future MINGHAN WANG Metabolic Disorders Research, Amgen, Inc., Thousand Oaks, CA, USA

INTRODUCTION Metabolic syndrome is associated with a variety of diseases and complications, which are direct or indirect consequences of its key components: obesity, insulin resistance, hypertension, dyslipidemia, hyperglycemia, prothrombotic state, and vascular inflammation. The clinical manifestation of these abnormalities is generally exemplified by conditions such as type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD), which embodies a broad spectrum of conditions such as myocardial infarction, acute coronary syndrome (ACS), and congestive heart failure. Stroke is another form of macrovascular complication associated with metabolic syndrome. Further, microvascular complications, including retinopathy, nephropathy, and neuropathy, are common in patients with late-stage T2DM. From the treatment point of view, T2DM and CVD are major targets for intervention due to the high prevalence of complications and death. In the past several decades, drug development efforts in the pharmaceutical and biotech industry have generated efficacious antidiabetic drugs and a variety of cardiovascular therapies. There is a lot to be learned from the successes with approved drugs and relevant experience. However, behind these successes are a greater number of failures from which important lessons can be learned, and the new learning has inspired scientists to seek more rigorous strategies and approaches. As a matter of fact, the discovery of many successful drugs is largely built on the lessons learned from the past failures. Further, both the drug discovery process and the regulatory landscape Metabolic Syndrome: Underlying Mechanisms and Drug Therapies Edited by Minghan Wang Copyright 2011 John Wiley & Sons, Inc.

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have been continuously evolving over the past several decades, and it is important to understand what was learned two decades ago may not be necessarily applicable to today’s drug discovery endeavors. One of the most noticeable changes in the past decade is the increased emphasis on safety by regulatory agencies. As drug development costs continue to increase substantially, new biomarkers that enable earlier efficacy and safety assessments are increasingly important in making development decisions. The tremendous knowledge built on past experience may help shorten development timelines and increase chances of success. However, none of the experience or lessons described below is meant to be the blueprint for success in metabolic drug discovery; rather, each drug discovery program has its unique path and objective use of the general principles is essential for success.

A HISTORICAL PERSPECTIVE WITH METABOLIC DRUG DISCOVERY The emergence of molecular biology in the 1970s greatly enabled modern drug discovery research. Prior to that, drug discovery was mainly driven by the assessment of pharmacological effects of chemical compounds in animals or humans. Metformin, thiazolidinediones (TZDs), and fibrates described below are typical products of such approaches. Although robust efficacy was demonstrated in animal models or even humans, the mechanism of actions (MOAs) for each of these compounds was not clear until a long time after their initial discovery. Metformin, today’s first-line antidiabetic therapy, is a product of trial and error. An herbaceous plant Galega officinalis (also known as French lilac) had been used to treat diabetes for centuries without a clear mechanism until early 1900s when guanidine compounds were identified in the extract of this plant that mediate the antidiabetic effect. Based on these compounds, biguanides were synthesized in the 1950s for the treatment of diabetes (1). The first members of this drug class, phenformin and buformin, were initially introduced to the market but were later withdrawn due to their potential of causing lactic acidosis, which led to death of some patients. Metformin was introduced on the US market in 1995 with reduced lactic acidosis risk at 1 in every 30,000 patient-years, which is about 100 times lower than that with phenformin (2). Today, the risk of lactic acidosis with metformin is manageable in clinic through careful monitoring and the drug is widely used around the world. If it was not for the courage of trial and error after the withdrawal of buformin and phenformin, it is not clear if metformin would have been made available to so many T2DM patients today. Retrospectively, improving upon the first two biguanides that resulted in the discovery of metformin turns out to be a courageous decision. Of course, this learning has to be put in the context of lack of effective oral antidiabetic therapies at the time. It could be argued that the need for an oral antidiabetic drug justified the risk that was taken to improve the biguanide class. Today, the landscape is completely different. Given the availability of other antidiabetic drugs, it would be hard to justify taking a similar risk. While we build on our own success in drug discovery, the tolerance of risk/benefit ratio is continuously being

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redefined, and it is possible that avoiding high-risk activities may have resulted in missed opportunities. Interestingly, the MOA of metformin is still not exactly clear today. Metformin is believed to exert its antidiabetic effect mainly by suppressing hepatic gluconeogenesis (3, 4). There is some evidence that metformin inhibits complex I of the mitochondrial respiratory chain (5), but the exact molecular target in vivo is still unclear. Another antidiabetic drug class, the TZDs, was discovered based on animal pharmacology. The original lead compound was clofibrate, which was discovered to have strong lipid-lowering but weak antidiabetic activity in humans (6). Through in vivo testing of synthetic analogues of clofibrate in rodents, the first TZD ciglitazone was identified with potent antihyperglycemic activity (7, 8). Following these efforts, continued chemical synthesis coupled with in vivo screening for antihyperglycemic activityled to the identification of otherTZDs (9). Bythen,the preclinical drug discovery was mainly enabled by chemical synthesis and direct in vivo testing. This obviously was not drug discovery at the molecular level; as a matter of fact, even when a compound was found to have desirable pharmacological effects at that time, the mechanism of action was largely unknown. The molecular target of TZDs was not identified until mid-1990s, when several research groups discovered that TZDs bind with and activate peroxisome proliferator-activated receptor g (PPARg) (10–12). The first TZD approved as an antidiabetic therapy was troglitazone (Resulin), which was later withdrawn from the market due to idiosyncratic liver toxicity. To date, two TZDs are in the market for the treatment of T2DM, rosiglitazone (Avandia) and pioglitazone (Actos). Although TZDs came from the derivatives of fibrates, the MOA of fibrates was not understood at an earlier time than that of TZDs. As a matter of fact, clofibrate was the first fibrate to be described in 1963 (13), and it was approved by the US FDA in 1967 as a cholesterol-lowering agent. Additional fibrates were subsequently developed, including gemfibrozil, fenofibrate, and bezafibrate (13). Amazingly, around the same time when TZDs were found to lower glucose by activating PPARg, fibrates were found to be synthetic ligands of PPARa and exert their pharmacological effect via activation of this nuclear receptor (14). These findings attracted a lot of attention in the late 1990s and early 2000s because it was (and still is) very rare that two members of a protein family are targets for known drugs. With the widely accepted notion that the PPARs were a golden target family, intense drug discovery efforts were focused on searching for activators of the third member, PPARb/d, as well as dual and pan PPAR agonists. While PPARg activation lowers glucose and PPARa activation reduces triglycerides, PPARb/d activation appeared to be a good strategy to raise HDL cholesterol (15). The enthusiasm toward this target family was dampened by later findings that linked PPAR activation to cardiovascular risks. Taking a different path from metformin, TZDs, or fibrates, the discovery of statins was initiated with a deliberate effort to find inhibitors of HMG-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis. The effort started in the early 1970s, when there was good understanding of enzymes in main metabolic pathways, although molecular biology was just starting to emerge. The research endeavor led by Dr. Akira Endo in Japan to search for fungal metabolites that inhibit HMG-CoA reductase led to the discovery of the statin class of compounds. After screening 6000

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microbial strains over a 2 year period (16), they identified the first statin, mevastatin (formerly, ML-236B or compactin), from Penicillium citrinum (17). Subsequently, it was demonstrated that mevastatin robustly lowered LDL cholesterol in animals and humans (18–20). These findings stimulated the worldwide development of other statin compounds in the 1980s and 1990s (16) and led to the launch of multiple statin drugs. Today, the statins are the most successful drug class in history in terms of wide clinical use and sales. The discovery of the first statin from fungal metabolites also stimulated intense search for microbial metabolites as drug-like molecules by major pharmaceutical companies. Often, pharmaceutical companies asked their employees traveling in different parts of the world to bring soil samples back to the central labs. With the explosion of molecular biology in the late 1970s, it started to change the way drug discovery was conducted. The discovery phase increasingly started with a specific protein target. Scientists gained better understanding of how a drug works and target selectivity. In subsequent years, high-throughput screening (HTS) was widely adopted to screen millions of compounds, where hits were selected and medicinal chemistry was used to modify compounds to improve physical and chemical properties, making them drug-like. More enzymes and receptors were cloned and expressed to facilitate selectivity. This totally changed the face of drug discovery in the throughput of testing compounds, scope of biological assays, the quantity and quality of leads, and the depth of understanding of MOAs. Using the new paradigm, more potent and selective molecules have been discovered. However, the new approach does not necessarily increase the success rate. Part of the reasons is that the initial discovery using pharmacological means is the result of decades or even centuries of accumulation of knowledge (in the case of biguanides). Further, it could be argued that the “lower-hanging fruits” have been “picked” and therefore similar successes are hard to repeat with the same approaches. Equally important is that the safety hurdles are getting higher due to the changes in the regulatory environment, which requires more studies and longer time to develop a drug. In the past decades, the biotech boom has positively impacted metabolic drug development. Protein engineering technologies coupled with emerging biology helped develop injectable drugs. Besides insulin, Byetta (exenatide) is another injectable antidiabetic drug from Mother Nature. It is the synthetic version of exendin-4, a peptide discovered in the saliva of Gila monster, a lizard that lives in the desert in Arizona (21). The success with Byetta as a therapy stimulated industrywide efforts of screening the so-called microproteins (small peptides from insects, lizards, and reptiles) for biological activities based on the notion that they have druglike properties.

LESSONS LEARNED FROM SUCCESSFUL STORIES There Is Always Room to Improve Once a class of drugs are discovered and established, there are usually obvious attributes and properties of these drugs that need to be improved for enhanced efficacy

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or reduced side effects/safety concerns or both. In addition, dosing convenience could be an attribute that makes a significant difference. One of the challenges in drug development is the lack of predictability of human efficacy in animal models. This limitation was typical in the development of different statin drugs. As discussed above, following the discovery of the first statin from fungal metabolites, industrial efforts searching for cholesterol-lowering statin drugs led to the introduction of several statins on the market. Since statins do not work in rodents, they were tested for cholesterol-lowering efficacy in dogs and monkeys. In the early 1990s, based on the experience with this class of compounds, medicinal chemistry efforts of developing better statins were widely applied in the pharmaceutical industry. Interestingly, the most successful statin, atorvastatin (Lipitor), is the so-called “me too” compound (22). By mid-1990s when atorvastatin entered phase 1 clinical trial, four statins were already on the market (22). But its LDL-lowering effect in human volunteers was much more robust than any other statin on the market at the time, partly due to good potency and longer plasma half-life in humans (22). Interestingly, the superior LDLlowering effect was not apparent in preclinical studies (22). This experience stresses the notion that there is always room to improve a drug and sometimes it is difficult to determine superiority in preclinical models if one is superior to another. Fully relying on preclinical models could be misleading and opportunities can be missed. To enable better decision making and ensure advancement of the best molecules to clinic, more predicative models and biomarkers are required.

Build Success on Failures The biguanide story offers interesting learning about managing risks. It is common that side effects are associated with drugs. Understanding the potential mechanism underlying a particular side effect is important in managing risks and seeking solutions to improve the drug’s properties. When it comes to safety, accumulating clinical experience is vital in the redesign to reduce side effects. The lactic acidosis caused by phenformin and buformin is likely related to their suppressive effect on gluconeogenesis, leading to the accumulation of gluconeogenic precursors that could increase lactate levels. This information helped with the redesign to reduce the drug’s activity, which led to the discovery of metformin. Obviously, the clinical experience with the first two members of the biguanide class provided the basis for discovering metformin. In a broad sense, it could be argued that the success of metformin was built on the failures of phenformin and buformin. Although there is overwhelming knowledge that had accumulated in the past, we still have to rely on trial and error in order to discover top-of-the-line products. Demanding an impeccable safety profile from the first drug of a novel class is next to impossible due to the lack of clinical experience. Once safety issues are identified with a new drug molecule, comprehensive risk assessment needs to be made to determine if the entire class should be abandoned. It is usually premature to terminate the entire class based on findings with one molecule, especially when the mechanisms underlying certain safety issues are not clear. At present, the industry has made substantial

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investment in developing predictable preclinical models and clinical biomarkers. These efforts, if successful, will surely help identify more efficacious and safer drugs and cut development costs. And it would be ideal if the predictability for human efficacy and safety is enhanced by these advancements. However, it remains to be seen if these efforts will be fruitful. In the end, it is possible that trial and error in the clinic cannot be bypassed by any imaginary shortcuts; taking calculated risks in the clinic may still be the most effective way to discover new drugs with robust efficacy and acceptable safety.

Dealing with Postmarket Issues Often, the entire safety profiles of a drug are not completely understood until years after it reaches the market. This is because certain rare safety signals cannot be detected in preclinical or clinical studies due to the small size of the study population. Certain safety issues can only come to light after the drug reaches the market when a large number of patients are exposed to it for a period long enough to reveal a rare signal. The first approved TZD, troglitazone (Resulin), did not show any issue with liver toxicity. Once it reached the market, there were some cases of idiosyncratic liver toxicity; in severe cases, it caused death (23). Neither of the two TZDs introduced later (rosiglitazone and pioglitazone) had the liver toxicity issue. However, they were linked to increased risk of congestive heart failure (24). A risk/benefit analysis is required to make a balanced decision in using these drugs on T2DM patients. After several years on the market, a small number of patients on Byetta developed pancreatitis (25). However, since T2DM patients generally have an increased risk of pancreatitis, it is not clear if these cases are associated with Byetta use. But similar pancreatitis cases were found in T2DM patients on Januvia, a DPP-4 inhibitor that works by increasing active endogenous GLP-1. Since both drugs work through activating the GLP-1 incretin axis, the rare but potentially deadly human pancreatitis cases may be associated with activation of this pathway. Despite the lack of causal evidence, the FDA and the drug manufacturers agreed to update the labels of both drugs to disclose potential pancreatitis risks associated with these drugs. This experience again strengthens the notion that in some cases, it may be impossible to identify safety signals in animal models or throughout clinical trials when the incidence is rare. This notion also stresses the importance of postmarket safety monitoring as part of the drug development process. Vytorin is a combination therapy consisting of simvastatin (Zocor) and ezetimibe (Zetia). In clinical studies, it was clearly demonstrated that Vytorin treatment resulted in much lower LDL cholesterol than simvastatin alone (26). When it comes to LDL cholesterol, there is a general belief of “the lower, the better.” This is consistent with the premise that statins reduce cardiovascular risks mainly by lowering LDL. However, it is not clear if further reduction of LDL by adding ezetimibe to statins can lead to further cardiovascular benefit. In the ENHANCE (Effect of Ezetimibe Plus Simvastatin Versus Simvastatin Alone on Atherosclerosis in the Carotid Artery) trial, Vytorin was compared with simvastatin for their effects on a surrogate for

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atherosclerosis progression (change in the mean carotid artery intima-media thickness (cIMT)) (27). At the end of the study, no difference in cIMT was observed between the two randomized patient groups, although there was a significant reduction in LDL cholesterol in the Vytorin group compared with the simvastatin alone group (27). This surprise finding raised issues about the potential benefit of further LDL lowering. However, since it is debatable whether cIMT is a valid surrogate marker for atherosclerosis progression in the coronary artery, an outcome trial comparing the effects of Vytorin and simvastatin alone is needed. The IMPROVE-IT (Examining Outcomes in Subjects with Acute Coronary Syndrome: Vytorin [Ezetimibe/Simvastatin] Versus Simvastatin) trial is designed to address this question (28). The issue surrounding the ENHANCE trial stresses the need for outcome studies, although there are overwhelming clinical and epidemiological data that suggest lowering LDL reduces cardiovascular risks. After all, LDL cholesterol is still a biomarker.

LESSONS LEARNED FROM FAILURES Risk and Benefit Ratio with New MOAs When considering therapeutic strategies, the risk/benefit ratio is a key factor for a given target or pathway. However, the real risk level usually remains unclear until in late clinical stage. This was revealed in the case of torcetrapib, a cholesterol ester transfer protein (CETP) inhibitor developed by Pfizer. CETP inhibitors like torcetrapib raise HDL and are postulated to reduce the incidence of coronary heart disease. In clinical studies, torcetrapib was efficacious in raising HDL, lowering LDL, and decreasing normalized atheroma volume, although there was no change in atheroma volume for the most diseased vessel segment (29). Further, torcetrapib treatment was associated with increased systolic blood pressure (29), which raised some concerns because higher blood pressure is a major cardiovascular risk. In a large phase 3 cardiovascular outcome study, torcetrapib/Lipitor combination was compared with Lipitor alone (30). The torcetrapib/Lipitor treatment was associated with an increase in cardiovascular events and an increase in total mortality, and the trial had to be halted prematurely (30). One potential explanation for the worsening cardiovascular outcome is that it was caused by the increased blood pressure, a likely off-target effect as other CETP inhibitors did not change blood pressure (31). Another off-target effect of torcetrapib is that it induced aldosterone and corticosterone levels in rats (31), which could increase cardiovascular risk. A second explanation is that the elevated HDL by torcetrapib is large and proatherogenic. However, several other CETP inhibitors are still in clinical cardiovascular outcome studies by other pharmaceutical companies. Whether CETP inhibitors improve cardiovascular outcomes remains unclear until additional clinical data are available. A second example for risk/benefit balance is Sanofi’s rimonabant, a CB1 antagonist. The main effect of this drug is to induce weight loss via blockade of the endocannabinoid system and thereby suppressing food intake. Clinical studies

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showed that rimonabant promoted body weight loss, reduced central obesity, and improved dyslipidemia (32). However, rimonabant is associated with psychiatric alterations such as depression and anxiety, and these side effects led the FDA to not approve this drug in the United States (32). Although it was initially approved in Europe, the increased incidence of depression and suicidal risks led to its later withdrawal. These safety findings are on-target effects because the CB1 receptor regulates food intake as well as mood (33). In this case, the mechanism is associated with safety risks that cannot be tolerated.

Disconnect Between Animal Models and Human Patients Like in other disease areas, there is often disconnect between animal and human data when developing metabolic drugs. For example, leptin was cloned in 1994 as an antiobesity hormone because in rodents, leptin or leptin receptor deficiency caused obesity. The discovery of leptin raised hopes of treating human obesity with this hormone. Unfortunately, leptin is only effective in inducing weight loss in rodents, including both leptin-deficient mice (ob/ob) and diet-induced obese mice (34). Obese humans do not respond to leptin treatment; they are leptin resistant (35). This example indicates that there are many physiological differences between animal models and human patients, and efficacy in animal models does not always translate in the clinical setting. In addition to efficacy, safety signals in animals are usually difficult to interpret with respect to the relevance to humans. This is especially true when a safety signal is not consistent across different animal species and there is no valid biomarker to reconcile the findings. The C cell tumor issue associated with long-acting GLP-1 analogues is a typical example. Although Byetta is efficacious in treating T2DM (36), its twice-daily injection regimen is not optimal. To reduce dosing frequency, GLP-1 analogues with longer plasma half-lives were developed. The first two agents are liraglutide (once daily; Victoza) and Byetta LAR (long-acting release) (once weekly). In animal studies, it appeared that sustained activation of the GLP-1 pathway induces thyroid C cell tumors in rodents (37). Liraglutide caused thyroid C cell tumors in both rats and mice at high doses (37). Byetta LAR caused C cell tumors only in female rats at the highest dose tested (38). At this time, liraglutide has been approved in both United States and Europe, and Byetta LAR is under regulatory review. Since there is a high background of spontaneous C cell cancer in rats and the condition is very rare in both mice and humans, it is still challenging to understand the level of tumor risk associated with long-acting GLP-1 analogues in humans (38). Careful monitoring and postmarket research are important to ensure patient safety. To do so, the FDA added a black box warning to the label of liraglutide that includes a potential increased risk of thyroid cancer. Further, first-line usage of the drug is not allowed. There are postapproval requirements that include cardiovascular safety study, a 5 year epidemiological study to evaluate thyroid cancer risks, a 15 year cancer registry to monitor thyroid cancer cases, and a Risk Evaluation and Mitigation Strategy (REMS) (39).

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ISSUES WITH EXISTING THERAPIES AND UNMET MEDICAL NEEDS The current therapies for T2DM and CVD described in previous chapters provide significant value to patients. The intensive efforts and enormous investments in metabolic drug discovery have led to multiple antidiabetic treatments. However, there is no single antidiabetic drug that can completely normalize hyperglycemia, mitigate complications, and eventually resolve T2DM. Rather, following a period of monotherapy, adequate glycemic control cannot be maintained in many patients and add-on of a different yet complementary antidiabetic therapy is required. The current standard of treatment for newly diagnosed T2DM is metformin because it has well-established efficacy and manageable safety profiles. Furthermore, metformin is a generic drug and has a cost advantage. To patients that fail metformin monotherapy, other antidiabetics such as thiazolidinediones (TZDs), sulfonylureas (SUs), and Januvia are added to the regimen. However, even combination therapies cannot completely resolve T2DM. Hence, the current unmet medical needs call for a robust monotherapy that can maintain glycemic control, preferably with additional beneficial effects such as inducing weight loss and eventually completely resolving diabetes. Since the long-term use of the drug is required, increased safety profiles are especially important. For CVD, there is an increasing need for more effective drugs, especially in areas such as heart failure where there is no effective therapy. In the lipid area, there has been tremendous success in lowering LDL cholesterol with the discovery and wide use of statins. HDL is considered good “cholesterol” based on its correlation with lower cardiovascular risks in epidemiological studies. Current HDL raising agents include fibrates and niacin, although the level of HDL elevation by the fibrate class is moderate. Niacin increases HDL cholesterol substantially, but it causes skin flushing in some patients. Moreover, there is evidence that long-term use of niacin may cause insulin resistance. Therefore, more effective and safer HDL-raising drugs are needed, and it should be demonstrated in late-stage trials that by pharmacologically raising HDL, these drugs improve cardiovascular outcomes. To decrease the morbidity and mortality associated with acute coronary syndrome and cardiovascular events, postmyocardial infarction, treatments for stabilization and regression of atherosclerotic plaques are needed. However, success in this area has been limited in the past decades. Overall, given the high morbidity and mortality associated with CVD, reducing cardiovascular risks has been and will continue to be the primary goal of novel therapies.

CURRENT APPROACHES AND CHALLENGES Improvement of Existing Therapies: Line Extension One of the current strategies for developing metabolic drugs is to build on the knowledge and clinical experience of existing therapies for improvement. For biologics, plasma half-life extension is usually an important attribute to focus on.

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Byetta is the first GLP-1 analogue approved for the treatment of T2DM (36). As described above, since Byetta requires twice-daily injections (36), long-acting GLP-1 analogues have been under development by multiple companies to reduce dosing frequency. Liraglutide is a GLP-1 analogue with one fatty acid conjugation that allows binding with serum albumin to extend plasma half-life, and thereby supporting oncedaily injection. Byetta has been reformulated as Byetta LAR to achieve once-weekly injection. Liraglutide has been approved in both the United States and Europe. Byetta LAR is currently under FDA review (38). Several other GLP-1 analogues are in clinical development (38), including taspoglutide from Roche, which requires onceweekly injection. DPP-4 inhibitors are one class of small-molecule drugs that increase the incretin effects by blocking the degradation of endogenous GLP-1. Januvia is the first DPP-4 inhibitor on the market. Januvia combined with metformin (Janumet) is also available as combination therapy. Januvia has limited efficacy with risk of pancreatitis added to the label. Despite these caveats, additional DPP-4 inhibitors are being advanced in clinic. Recently, saxagliptin (Onglyza) was the second DPP-4 inhibitor approved by the FDA.

Combination Therapies to Achieve Additive Efficacy Combination of existing therapies with different but complementary mechanisms continues to be another major focus. This is especially important if the components in the combined pill are generic drugs, given the increasing need to improve access to inexpensive and effective therapies in areas of the world where high costs are prohibitive. Most combinations such as the antilipidemic drugs described in Chapters 8 and 9 and the antidiabetic drugs described in Chapter 10 refer to one pill containing two active drugs. However, since multiple risk factors of cardiovascular disease and diabetes are found in patients with metabolic syndrome, mitigation of all these variables requires additional treatments. This unmet medical need inspired the strategy of combining multiple drugs in one single daily pill. The recent proof-of-concept phase 2 clinical trial, The Indian Polycap Study (TIPS), has attracted a lot of attention (40). This clinical trial was built on the concept that combination of several therapies that work through different mechanisms to reduce cardiovascular risks/events in a single daily pill can simultaneously reduce several cardiovascular risk factors and achieve a large effect in preventing cardiovascular disease with minimal adverse effects (41). The combination pill used in TIPS consists of five components: thiazide (a diuretic), atenolol (a b-blocker), ramipril (an ACE inhibitor), simvastatin (a statin), and aspirin. The three antihypertensive drugs in combination with a statin and aspirin are expected to reduce blood pressure, plasma LDL cholesterol, and prothrombotic state (blood clotting). The Polycap is therefore expected to substantially reduce stroke and myocardial infarction (40). The primary objective of the phase 2 study was to determine whether the effects of the Polycap are noninferior to those of its equivalent components (42). The subjects enrolled in the trial were middle-aged individuals without previous cardiovascular disease, but it is

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noteworthy that about one third of the subjects in the study had diabetes despite the fact that the other subjects had only one cardiovascular risk factor such as high blood pressure (40), suggesting that the study population is still present with increased cardiovascular risks rather than “average people” (43). The treatment period was 12 weeks (40). At the end of the study, the Polycap reduced blood pressure (both systolic and diastolic), platelet activation, and LDL cholesterol, although the LDL effect was smaller than that by simvastatin alone (40). Interestingly, the effect of blood pressure reduction was greater when more of the three antihypertensives were included (40), and the reduction by the combination of the three drugs was similar to that by the Polycap (40). Although it is not clear why the effect of simvastatin in the Polycap was smaller than simvastatin alone, the results from this trial support the feasibility of combination of multiple drugs in a single daily pill while maintaining most of the efficacious effects. However, the Polycap strategy is not a simple combination of multiple efficacies. Several important questions need to be asked: What is the value added to patients and clinical practice and what are the added risks? Clearly, additional work needs to be done to further understand the risk/benefit ratio of such an approach. For example, combining more drugs in one pill may increase the risks of side effects and drug–drug interaction either in the entire patient population or a select group of patients with distinct tolerance profiles. More studies on safety in outcome trials are likely to be required by regulatory authorities to fully demonstrate the risk/benefit balance of combination therapies (44). Further, when patients are denied the flexibility of taking a different drug in the same class, such as a statin different from the one in the combination pill, the value of the combination pill needs to be recalculated. According to the current guidelines for combination pills, regulatory authorities require the manufacturer to provide pills in every dose combination of each drug (44). To meet this requirement with a Polycap containing 5 drugs as in the TIPS study, approximately 100 pill strengths will have to be manufactured (44), which is literally unmanageable. Compared with the polypill strategy, combination of two drugs has achieved some degree of success. Niacin ER (extended release) has been used in combination pills for additive efficacy and improved safety profiles. Simcor (simvastatin þ niacin ER) combines the effects of LDL reduction of simvastatin and HDL elevation by niacin ER. Obviously, this combination is a success. Another successful case for combination therapy is Vytorin, which consists of simvastatin and ezetimibe (Zetia). Vytorin lowers hepatic cholesterol biosynthesis (simvastatin effect) and intestinal cholesterol absorption (ezetimibe effect), resulting in additive effect of LDL cholesterol reduction. But as discussed above, this drug suffered some setback in a recent study in which Vytorin did not impact a surrogate marker for atherosclerosis progression when compared with simvastatin alone (27). Although some combination pills that contain two active drugs have been approved by regulatory authorities, issues still exist for this approach. A different combination that also involved niacin was attempted by Merck to reduce a main side effect. Despite the robust effect of HDL elevation, niacin often causes skin flushing in some patients. It has been known that the flushing effect is mediated by prostaglandins. To reduce flushing, Merck attempted to combine niacin with a drug (laropiprant) that

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suppresses the effects of prostaglandins on blood vessels. However, the combination pill, Cordaptive, was rejected by the FDA (45). Although the agency did not reveal why it rejected the drug, one speculation is that Cordaptive had only modest improvement in flushing (53.3% had moderate or worse flushing) compared to niacin ER (78%) (46). Further, since laropiprant is a new chemical entity and with the lack of long-term data in humans, its safety profile is not clear. This adds significant risk to the combination pill. The combination of atorvastatin and ezetimibewas also pursued by Merck. However, as of November 2009, the FDA refused to file the NDA application. The FDA has identified additional manufacturing and stability data that need to be addressed (47).

Developing New Therapies Targeting Multiple Mechanisms To enhance therapeutic effects, efforts aimed at developing one drug molecule that hits two or more targets have been explored. This strategy has been used to target two or all the three PPARs to expand the pharmacological effects on both glycemic control and correction of dyslipidemia. As described above, there are three PPAR isoforms, PPARa, PPARd/b, and PPARg. While the antidiabetic effect of TZDs is mediated by PPARg, the triglyceride lowering effect of the fibrates is mediated by PPARa. To identify a drug with combined effect of lowering both blood glucose and lipid levels, dual PPARa/g agonists have been pursued in the industry. Multiple dual PPAR agonists targeting both hyperglycemia and hyperlipidemia in T2DM patients have been identified such as muraglitazar (48). In addition, PPARd/b activation has been shown to raise HDL cholesterol (15). This inspired the development of a PPAR pan molecule, which activates all three isoforms of PPARs and is expected to lower LDL cholesterol and triglycerides, raise HDL cholesterol, and normalize hyperglycemia (49). Although they are potent and efficacious, the cardiovascular risk inherently associated with PPAR agonists still exists (24, 50), and the safety concerns found in clinic have dampened the enthusiasm in this class of compounds. While targeting multiple mechanisms with one compound is an interesting strategy, one challenge is that it can hardly be applied to targets that do not belong to the same gene family due to the lack of structural homology. For this reason, there has been limited success with this strategy. However, if the therapeutic agent is a biologic, there is more feasibility because two individual protein fragments targeting two targets, respectively, can be fused or conjugated to form one molecule while maintaining both activities. Alternatively, a molecule can be designed based on biological peptides with dual action. One such example is oxyntomodulin, a peptide that activates both glucagon receptor and GLP-1 receptor. Recent reports suggest that metabolic therapeutic peptides with dual activities can be designed based on the oxyntomodulin sequence and MOA (51, 52).

Building on Innovative Mechanisms Drug discovery can take learning from both preclinical and clinical findings. In addition to the identification of genes that are involved in obesity and insulin

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resistance in genome-wide association studies (GWAS), emerging biology holds more promise for new drugs with robust efficacy. The increased understanding of calorie restriction and its mechanistic link to metabolic functions may help develop new ideas to fight obesity and insulin resistance. Another emerging area is potential MOAs associated with the robust effects of Roux-en-Y gastric bypass (RYGB) that can lead to remission of T2DM in more than 80% of patients that undergo the procedure as described in Chapter 1. Calorie restriction has been a fascinating area to understand how energy metabolism is regulated and its association with longevity. Many aspects of calorie restriction suggest that there are novel pathways that can be activated for increased energy metabolism, which could be employed to treat obesity and insulin resistance. In the meantime, activation of the biological systems for energy intake and integration bears resemblance to metabolic abnormalities in obesity and insulin resistance. If a drug can be developed to activate certain mechanisms to accelerate energy expenditure without increasing energy intake, weight loss can be induced and insulin sensitivity can be improved. Both obesity and T2DM can be treated with this approach. Sirtuins have been identified as central players in calorie restriction and as described in Chapter 15, further understanding of these proteins may open new drug discovery opportunities. There is a lot to be learned from RYGB. This procedure causes dramatic weight loss and has been the most effective treatment of severe obesity, Further, more than 80% of the patients with type 2 diabetes developed complete remission of the disease after the surgery (53, 54). Weight loss does not fully explain the remission of type 2 diabetes because within days after surgery, the hyperglycemia and hyperinsulinemia were totally normalized (53). As described in Chapter 1, changes in gut hormone secretion postsurgery have been observed. These gut peptides, including GLP-1, PYY, and maybe other yet to be identified members, are believed to mediate the robust beneficial effects. If novel endocrine factors can be identified, they could be used to develop drugs to treat obesity, insulin resistance, and T2DM. It has been a long-held view that adult humans do not have brown adipose until it was recently demonstrated that there is functional brown adipose tissue in adult humans (55–59). The amount of brown adipose tissue is induced by exposure to cold temperature (57–59). In addition, the activity of brown adipose tissue is negatively correlated with age and degree of obesity (56–59). These exciting findings open the possibility of pharmacologically increasing energy metabolism by stimulating the activity of brown adipose tissue, which is believed to be an effective approach to treat obesity and T2DM.

NEW INSIGHTS FROM CLINICAL RESEARCH Effect of Aggressive Glycemic Control on Cardiovascular Risks While T2DM is characterized by hyperglycemia, the major risk of death in T2DM patients is CVD. Diabetes is associated with comorbidities such as dyslipidemia,

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atherosclerosis, increased vascular inflammatory, and prothrombotic state, a collection of cardiovascular risk factors common in T2DM patients. Although many outcome trials have been conducted in T2DM patients with multiple cardiovascular risk factors, one fundamental question still remains unanswered: Does aggressive glycemic control reduce cardiovascular risks? In the UKPDS, metformin treatment reduced hyperglycemia and caused a 39% reduction in cardiovascular disease (60). Insulin and SU treatments did not lead to statistically significant improvement of cardiovascular outcomes, although they achieved glycemic control similar to metformin (60). These findings cannot conclusively determine the effects of glycemic control on cardiovascular risk. The recent data from three landmark clinical trials, the Action to Control Cardiovascular Risk in Diabetes (ACCORD), the Action in Diabetes and Vascular Disease (ADVANCE), and the Veterans’ Administration Diabetes Trial (VADT), shed new light on this question (61). Although there are differences in patient populations, enrollment criteria, and treatment targets, these studies generally suggest that aggressive glycemic control is unlikely to impact cardiovascular risk. It is noteworthy that in all three studies, intensive glycemic control increased the risk of hypoglycemia (61). More analyses are required to fully understand these results and reconcile the findings in the three studies. Meta-analyses of past cardiovascular outcome trials with T2DM patients suggest glycemic control either reduces cardiovascular risk or is risk neutral, but the outcome could be dependent on different patient populations (62, 63)

Mechanisms Versus the Time to Monotherapy Failure Among the different therapeutic mechanisms of treating diabetes, SUs target b-cells directly by enhancing insulin secretion. It works independently of glucose by blocking the KATP channel causing elevation of intracellular calcium. Although SUs are robust in lowering glucose by stimulating insulin secretion, they may have long-term liabilities. In the A Diabetes Outcome Progression Trial (ADOPT), rosiglitazone, metformin, and glyburide (a widely used SU) as initial treatment for newly diagnosed T2DM patients were evaluated in a double-blind, randomized, controlled clinical trial involving 4360 patients over a median duration of 4 years (64). In this study, glyburide had the shortest durability as monotherapy (64). Further, patients on glyburide had significantly reduced b-cell function compared with metformin or rosiglitazone at the end of the study period (64). This study suggests that SUs may cause b-cell exhaustion over time by inducing insulin secretion (65).

REGULATORY CHALLENGES AND FUTURE OPPORTUNITIES While therapeutic strategies are important in metabolic drug development, recent changes in the regulatory landscape will continue to shape the way drug discovery is conducted. Recently, both the US FDA and the European Medicines Agency (EMEA) have published new guidelines for antidiabetic drugs. One of the major emphases in

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developing antidiabetic drugs is the need for cardiovascular outcome studies, either in phase 3 or postmarket stage, depending on the result of risk analysis using the collection of available clinical data. At present, it is not possible to gain regulatory approval of drugs that target a cluster of risk factors because it is difficult to have a defined specific end point that covers multiple risk factors. Thus, a new drug has to have a main therapeutic indication for which there is a specific biomarker or/and outcome acceptable to regulatory agencies. After approval for the first indication, new clinical trials need to be conducted to demonstrate efficacy to gain approval for additional indications. Development of new biomarkers that help predict cardiovascular risks early in clinical trials is vital in reducing development risks. These include new imaging technologies for more precise readout.

SUMMARY Drug discovery in metabolic disease has undergone transition from traditional pharmacology to a new paradigm driven by molecular approaches. Although there are multiple antidiabetic drugs on the market, none of them is robust enough as monotherapy to fully control hyperglycemia. There is a clear need for safer and more efficacious antidiabetic drugs. Although there has been exciting success in developing lipid control drugs, the choices of therapies in treating cardiovascular disease as a whole are still limited. The lessons learned from past successes and failures are valuable in the discovery of more innovative drugs in the future. The development of new biomarkers will accelerate such endeavors.

REFERENCES 1. BAILEY, C.J. 1992. Biguanides and NIDDM. Diabetes Care 15:755–772. 2. MISBIN, R.I., L. GREEN, B.V. STADEL, J.L. GUERIGUIAN, A. GUBBI, and G.A. FLEMING. 1998. Lactic acidosis in patients with diabetes treated with metformin. N Engl J Med 338:265–266. 3. STUMVOLL, M., N. NURJHAN, G. PERRIELLO, G. DAILEY, and J.E. GERICH. 1995. Metabolic effects of metformin in non-insulin-dependent diabetes mellitus. N Engl J Med 333:550–554. 4. HUNDAL, R.S., M. KRSSAK, S. DUFOUR, D. LAURENT, V. LEBON, V. CHANDRAMOULI, S.E. INZUCCHI, W.C. SCHUMANN, K.F. PETERSEN, B.R. LANDAU, and G.I. SHULMAN. 2000. Mechanism by which metformin reduces glucose production in type 2 diabetes. Diabetes 49:2063–2069. 5. OWEN, M.R., E. DORAN, and A.P. HALESTRAP. 2000. Evidence that metformin exerts its anti-diabetic effects through inhibition of complex 1 of the mitochondrial respiratory chain. Biochem J 348(Part 3): 607–614. 6. BARNETT, D., J.G. CRAIG, D.S. ROBINSON, and M.P. ROGERS. 1977. Effect of clofibrate on glucose tolerance in maturity onset diabetes. Br J Clin Pharmacol 4:455–458. 7. KAWAMATSU, Y., T. SARAIE, E. IMAMIYA, K. NISHIKAWA, and Y. HAMURO. 1980. Studies on antihyperlipidemic agents. I. Synthesis and hypolipidemic activities of phenoxyphenyl alkanoic acid derivatives. Arzneimittelforschung 30:454–459. 8. SOHDA, T., K. MIZUNO, E. IMAMIYA, Y. SUGIYAMA, T. FUJITA, and Y. KAWAMATSU. 1982. Studies on antidiabetic agents. II. Synthesis of 5-[4-(1-methylcyclohexylmethoxy)-benzyl]thiazolidine-2,4-dione (ADD-3878) and its derivatives. Chem Pharm Bull (Tokyo) 30:3580–3600.

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Index

ABCA1 (ATP-binding cassette transporter A1), 201–202, 205, 218, 220, 237, 398 ABCG1 (ATP-binding cassette transporter G1), 201–202, 220 Absorptive period, 299 ACC2 (acetyl-CoA carboxylase 2), 143, 255 ACTH, 187, 424, 429, 449 Acute insulin response (AIR), 55, 190 Adipocyte hypertrophy, 75, 94 Adipokine action, 186 imbalance, 73 levels, 74 production, 73–76, 78, 83, 94–95, 100, 105 Adiponectin complexes, 401 levels, 78, 81, 88–89, 94–98, 100–103, 105, 403, 438 secretion, 75, 78, 98 transcription, 401 Agouti-regulated peptide (AGRP; AgRP), 31, 33, 35–38, 82, 384 AICAR (5-aminoimidazole-4-carboxamide ribonucleoside), 36–37, 159, 254 Akt, 37, 138–141, 145, 159–162, 166–167, 255, 192, 304, 380, 385 a-cell(s), 4, 12–13, 30, 62, 286–288, 293–295, 401, 459, 464 AGIs (a-glucosidase inhibitors), 254, 263–264, 266 AMPK (adenosine 50 -monophosphateactivated protein kinase) activity, 36–37, 254 in ARC, 36

in POMC, 36 inhibitor, 36 Antidiabetic therapies, 253, 264, 266–267, 270, 372, 386, 472 ApoA1 Milano, 219 Atherogenesis, 85, 202, 216–217, 239–241 ATP/ADP ratio, 8, 10, 31–32, 38, 59–60, 188 Basolateral medulla (BLM), 30, 40 b-cell apoptosis, 59, 61, 63, 66, 292 death, 58, 61 dysfunction, 53, 64, 66, 144, 188, 191, 459 failure, 59, 61, 181–182, 187–188, 192–193, 255, 263, 271–272 function, 18–19, 55–56, 58, 60–62, 64–66, 81, 139, 188, 260, 263, 291, 311, 327, 341–342, 373, 383, 464, 484 growth, 342 mass, 19, 53, 62–66, 182, 188, 290–293, 342 bKlotho, 378–381, 384 bKlotho/FGFR complex, 379 Biguanide, 103, 144, 257, 328, 472, 475 Bile acid sequestrants (BAS), 207, 209–211, 222, 245 Blood pressure diastolic, 78, 185, 217, 303, 371 systolic, 303, 477 Buformin, 257, 472, 475 Byetta LAR, 310, 478, 480 Carbenoxolone (CBX), 434 Cardiomyopathy, 303, 399, 406

Metabolic Syndrome: Underlying Mechanisms and Drug Therapies Edited by Minghan Wang Copyright Ó 2011 John Wiley & Sons, Inc.

489

490

Index

Cardiovascular disease (CVD), 146 Cardiovascular function(s), 17, 102, 301–302, 384 Cardiovascular morbidity and mortality, 253, 270–271 Carnitine palmitoyltransferase 1 (CPT1), 255 Cbl-associated adapter protein (CAP), 160 CD36 (conserved domain 36), 17, 162, 201, 237 Central glucodetection, 31 Central glucose sensing, 33–35, 39–40, Central nervous system (CNS), 3, 29, 54, 83, 89, 294, 329, 361 Central obesity, 133, 180, 182–183, 185, 187, 192, 423, 429, 431–432, 478 Ceramide, 61, 161–162 CETP (cholesterol ester transfer protein) inhibitor, 216–217, 477 Cholecystokinin (CCK), 3, 82, 295, 305–306 Cholesterol biosynthesis, 207, 209, 473, 481 Cholesterol synthesis pathway, 100 Chronic oxidative stress, 59–60 Chylomicrons, 200–201, 212 c-Jun N-terminal kinase (JNK), 139, 395 Clinical diabetes, 63 Clofibrate, 207, 210, 245–246, 259, 473 Congestive heart failure (CHF), 93, 257, 261, 269, 471, 476 Coronary heart disease (CHD), 236, 242, 267–268, 303, 477 C-peptide response, 55 C-reactive protein (CRP), 77, 189, 208, 384 CREB (cAMP response element binding protein), 141, 184, 259, 289, 292–293, 398 CREB binding protein (CBP), 184, 259 Cushing’s syndrome, 183, 187, 423–424, 434 Dapagliflozin, 366, 368–372 Diacylglycerol (DAG), 144, 161, 186, 238, 241 Diacylglycerol acyltransferase (DGAT), 238, 241 Disposition index (DI), 56

DPP-4 (dipeptidyl peptidase-4) inhibitor, 14, 294–295, 336, 338, 340–346, 476, 480 Duodenum, 3–5, 9, 12, 14–15, 18–19, 60 Dyslipidemia therapies, 207, 211 Ectopic effects, 295 Edema, 144, 265 Endocannabinoids, 82 Endocrine cells, 12, 287, 305 Endogenous glucose production, 36, 137, 258 Endothelial dysfunction, 84, 86, 88–91, 102, 180, 185, 189, 192, 253, 267–268, 271 Endothelial function, 87, 185, 261 Endothelial nitric oxide synthase (eNOS), 86, 185, 400 Energy balance, 75, 397 expenditure, 3, 17, 29, 31, 42, 76, 82, 164, 167–168, 383–385, 403, 407, 483 intake, 3–6, 16, 75, 182, 191–192, 327, 483 Enteric glucose sensor, 298 Enteric neurons, 29 Enterocytes, 11, 29, 200–201, 208, 212, 288, 305 Enteroendocrine cells, 5, 7–8, 10–11, 16, 20, 281, 296, 307 Epitopes, 461–462 Exenatide formulation, 310 Exendin-4, 284, 290, 292–294, 297, 301, 308, 310, 329, 342 Exendin-(9–39), 290, 299–310 Exocytosis, 57, 59, 159–160, 290–291, 294 Familial Atherosclerosis Treatment Study (FATS), 243–244 Fasting glucose, 57, 104, 187, 247, 257–258, 261, 308, 444, 464 Fatty acid oxidation in muscle, 166 Fatty acid uptake, 161–163, 165–166 Fatty acyl metabolites, 61, 161–163, 182–183, 186, 255 Feeding behavior, 11, 30–31, 33–35, 42, 98 Fenofibrate, 101, 207, 210–211, 267–278, 379, 382, 473

Index FGF21 (fibroblast growth factor 21) levels, 379, 381–383, 385 FGF receptor (FGFR), 377 FGFR1c, 379 FGFR3c, 379 FGFR4, 379 Fiber-type conversion, 163 Fibrate, 101, 210–211, 237, 268, 473, 479 First-phase insulin secretion, 30, 54–55, 57, 262 Food ingestion, 4, 12–16, 282 Free fatty acids (FFAs), 4, 76, 200–201, 210, 238, 284, 381, 385 Fully human monoclonal antibody, 214 Gastric emptying, 4–7, 12, 15–16, 265–266, 284, 294–295, 297, 308, 343–344 Gastrointestinal (GI) tract, 29, 55, 136, 286 Genetic susceptibility, 65 Ghrelin, 4, 17, 31, 38, 82 GI peptides, 3–6, 8–9, 17, 20. See also Gut peptides Glicentin, 13, 288 Glinides, 253, 262–263, 265–266 Glomeruli, 360, 362 GLP-1 (glucagon-like peptide 1) administration of, 297, 300–302, 341 analogues, 145, 264, 271, 283–284, 286, 341, 478, 480 brain, 298–303 central effects of, 296 ectopic effects of, 295 elevation of, 19 in heart, 303 infusion, 15, 295, 301, 304, 338, 342–343 receptor (GLP-1R) agonist, 340, 343 signaling, 290, 296, 299–301, 303 treatment, 15, 284, 301, 303 GLP-1-based therapies, 284–285, 294–295, 304 GLP-1-(7–36), 288, 303–304 GLP-1-(9–36), 303–304 GLP-1R antagonist, 297, 299–302, 304 Glucagon action, 145, 460 production, 293–294 receptor (GCGR) blockade, 459

491

release, 6, 12, 33, 81, 141, 145, 293–294, 381 Glucocorticoid action, 424–425, 431–432 Glucocorticoid excess, 183, 185, 187, 424 Glucokinase activators, 137 activity, 136–137 translocation, 136 Gluconeogenic substrates, 141–142 Glucoregulatory hormone, 285 Glucose disposal in the peripheral tissues, 157 intolerance, 39, 64, 74, 134–135, 180–181, 185, 424, 429, 431–432 output, 157, 187, 342, 424–425, 433 reabsorption, 362, 364, 370, 372 sensor, 10, 35, 191, 282, 298–299, 362 uptake rate, 54 Glucose-dependent insulin secretion (GSIS), 11–12, 180, 265, 271, 294 Glucose-dependent insulinotropic polypeptide or gastric inhibitory peptide (GIP), 3, 12, 29, 328–329 Glucose-induced apoptosis, 59, 61 Glucose-induced hyperpolarization, 39, 42 Glucose-6-phosphatase (G6Pase), 135–136, 141, 184, 259, 429, 442 Glucose 6-phosphate dehydrogenase (G6PD), 239 Glucotoxicity, 58–59, 134–135, 188, 192 GLUT4 expression, 159, 161, 163–164, 399 in the plasma membrane, 160 Glycogen breakdown, 136, 140, 146 metabolism, 134, 140 synthase, 55, 138–140, 145 Glycogen-targeting subunits (GTS), 140 Glycyrrhetinic acid (GA), 434 GPR119, 8, 11, 306, 345 GPR120, 8, 11, 306 Gustducin, 7–9, 296, 307 Gut peptides, 483. See also GI peptides Gut-to-brain-to-periphery axis, 298 GWAS (genome-wide association), 483 Hepatic gluconeogenesis, 4, 55, 141, 145, 256, 259, 397–398, 473 Hepatic glucose appearance rate, 54

492

Index

Hepatic insulin resistance, 79, 135, 144–146, 157, 162, 182–184, 186–187, 385, 424, 433 Hepatic insulin sensitivity, 80, 102, 143–146, 162, 167 Hepatic lipogenesis, 142–143, 146, 168, 184 Hexose-6-phosphate dehydrogenase (H6PD), 427 High-density lipoprotein (HDL), 179, 200, 236, 241–242, 431 High glucose excited (HGE) neurons, 30 High glucose inhibited (HGI) neurons, 30 HMG-CoA reductase, 100, 199, 207, 473 HM74, 238 HPA axis, 424–425, 429, 434, 438, 449 Human aortic endothelial cells (HAECs), 239 Hybridoma, 460–463 Hypercholesterolemia, 203, 205, 214–215, 218 Hyperglycemia chronic, 342, 363–364 fasting, 140, 145–146, 180, 343 postprandial, 134–135, 145–146, 182, 263, 271, 339, 341 Hyperglycemic clamp, 55 Hyperinsulinemia compensatory, 184 fasting, 190 postprandial, 160, 182 Hyperinsulinemic-euglycemic clamp, 165, 294 Hyperplasia a-cell, 464 neointimal, 86, 91 Hypoalphalipoproteinemia, 205 Hypothalamic neurons, 35–36, 39, 82, 302 Hypothalamic nuclei, 30, 33, 36, 298 i.c.v. injection, 35–37 Idiosyncratic hepatotoxicity, 259 IKKb (IkB kinase b), 139, 161 Ileum, 4–6, 12, 14–15 ILLUMINATE, 216–217 ILLUSTRATE, 211, 218 Immunogen generation, 461 Impaired fasting glucose (IFG), 57, 187, 247, 261 Impaired glucose homeostasis, 98

Impaired glucose production, 133 Incretin(s) circulating, 340 concept, 19, 284–286, 328 enhancers, 328, 343–344 metabolism, 330 mimetic(s), 265, 328, 343–344 secretion, 9–12, 19, 306, 345 Inflammatory cytokines, 77, 87, 89, 213, 401 Insulin receptor substrate (IRS), 137, 186 release, 10, 15, 38, 56–57, 60, 135, 182, 187 secretory capacity, 58, 187 Insulin-containing vesicles, 291 Insulin-dependent glucose uptake, 158, 163 Insulin-dependent glucose utilization, 157 Insulinotropic activity, 281, 286, 288 Intensive insulin therapy, 256, 263 Intracellular fatty acyl metabolites, 183, 255 Intramyocellular lipid accumulation, 162, 164 Intraperitoneal glucose tolerance test (IPGTT), 438, 463 Intravenous glucose tolerance test (IVGTT), 55 Ischemia-reperfusion, 93, 304 Islet amyloid polypeptide (IAPP), 61 Islet amyloidosis, 62 Januvia, 145, 264–266, 340, 476, 479–480 Jejunum, 4–5, 9, 11–12, 15, 19 JUPITER, 208, 211, 271 K cells, 3, 11–13, 282, 287 KATP channel inhibitor, 39 Kidney proximal tubule, 363 L cells, 3, 7, 9–17, 19, 35, 283, 287–288, 296, 305–307 Lactic acidosis, 257–258, 264–266, 472, 475 LCAT (lecithin–cholesterol acyltransferase) activators, 221, 222 deficiency, 205, 222 LDL oxidation, 204, 239–240

Index Leptin resistance, 76, 80, 82, 86, 88, 92, 98, 104, 166, 383 Lipid mobilization, 385 Lipoprotein metabolism, 190, 199–201, 214, 241, 429 Lipotoxicity, 58, 60–61, 134–135, 143–144, 188, 192 Liraglutide, 284, 304, 309, 478, 480 Liver-specific insulin receptor knockout, 141, 143 Liver weight, 299 Luminal administration, 6 LXR (liver X receptor) activators, 220–221 Macrovascular complications, 188, 192, 253, 257, 267, 272, 346 Malonyl-CoA decarboxylase (MCD), 167 Maturity onset diabetes of the young (MODY), 137, 191 Maximal resorptive capacity (Tm), 363 Metabolic phenotype, 383 Microsomal triglyceride transfer protein (MTP), 200 Microvascular complications, 188, 192, 256, 263, 267, 270, 272, 471 Mild insulin resistance, 430–431 Mipomersen, 213 Mitochondrial overload, 162, 187 Mitochondrial oxidation capacity, 162 Monoclonal antibodies (mAbs), 459 Monocyte adhesion, 240–241 Monocyte chemotactic protein-1 (MCP-1), 239, 241 Muscle glucose utilization, 282, 299, 301, 303 Muscle insulin resistance, 157–158, 162–163, 165, 167 Muscular dystrophy, 164 Myeloperoxidase, 204 Myocardial infarction, 93, 95, 203, 245, 257, 261–262, 267–269, 471, 480 Myokines, 165, 167 Myostatin, 164–165 Na þ /K þ ATPase activity, 31 NCEP-ATPIII (the National Cholesterol Education Program’s Adult Treatment Panel III), 183, 242

493

Neogenesis, 62–63, 344 Neuroglucopenia, 34, 36–37 Neuronal circuits, 29, 31, 33, 42, 307 Neuronal mechanisms, 7 Neuropathy, 84, 188, 267, 270, 285, 471 Neuropeptide Y (NPY), 82 Neuroprotective effects, 284–285 Neurotensin (NT), 7 Niaspan, 207, 243, 247 Nicotinamide phosphoribosyltransferase (Nampt), 91, 394 Nicotinic acid adenine dinucleotide (NaAD), 394 Nicotinic acid mononucleotide (NaMN), 394 Nitric oxide (NO) synthase, 86, 185, 300–301, 304, 400–401 Normal glucose tolerance (NGT), 58, 185, 430–431 Nuclear magnetic resonance (NMR) spectroscopy, 258 Nutrient absorption, 4, 20 Nutrient consumption, 54 O-acetyl ADP-ribose (OAADPr), 393 Oral glucose tolerance test (OGTT), 10, 55, 187, 368, 422 Organic cation transporter 1 (OCT1), 259 Oxidative phosphorylation (OXPHOS), 31–32, 38, 163 Oxyntomodulin, 3, 13, 17, 288, 482 Pancreatic and duodenal homeobox gene-1 (Pdx1), 290 Pancreatic development, 191 Pancreatic effect, 281 Pancreatic extract, 285 PCSK9 (proprotein convertase subtilisinlike kexin type 9) inhibitors, 222 Peptide tyrosine tyrosine (PYY), 3 Perfusion, 6, 91 Peripheral glucose metabolism, 299 Peripheral insulin resistance, 184, 186, 192, 424, 459 Peripheral insulin sensitivity, 56, 433 Perivascular adipose, 78 Peroxisome proliferator-activated receptor gamma (PPARc), 141, 144

494

Index

Peroxisome proliferator-activated receptor gamma coactivator-1a (PGC-1a), 141 Pharmacological agents, 100, 285, 449 Phenformin, 257, 472, 475 Phlorizin analoge, 365 Phosphatidylinositol 3-kinase (PI3K), 139 Phosphoenolpyruvate carboxykinase (PEPCK), 259, 424 Phospholipase C (PLC), 7 Pima Indians, 163, 182, 190–191 Plasma cortisol, 432 Plasma corticosterone, 429 Portal hypertension, 79 Positron emission tomography (PET), 297, 344 Postprandial hyperlipidemia, 295 Prepro-opionmelanocortin (POMC), 82 Proglucagon polypeptide, 13, 17, 287–288 Prohormone convertases (PC), 13, 287 Proinflammatory adipokines, 81, 94–95, 101 Protein kinase C (PKC), 139, 186, 259, 289 Protein tyrosine phosphatase-1B (PTP1B), 139 PVN, 30–31, 33, 35–38, 302

Roux-en-Y gastric bypass (RYGB), 17–18, 483

Rapamycin complex 1 (TORC1), 37 Reactive oxidative species (ROS), 187, 239 Red muscles, 167, 398 Renal failure, 84, 144, 257, 379 Renin-angiotensin system (RAS), 77, 102 Respiratory chain, 38, 60, 162, 258, 473 Reverse cholesterol transport (RCT), 202, 237, 240 Rimonabant, 103–104, 477–478 Risk Evaluation and Mitigation Strategy (REMS), 478

Vagal afferents, 30 Vagus nerve, 14–15, 282, 297–300, 307 Vascular cell adhesion molecule-1 (VCAM1), 239, 241 Very-low-density lipoproteins (VLDL), 200 Visceral adiposity, 79, 183, 186, 430, 432 Visceral obesity, 99, 182, 400

Saxagliptin, 310, 339–340, 344, 480 Second-phase insulin secretion, 55, 66 Secreted phospholipase A2 (sPLA2), 215 Sensing mechanisms, 20, 30, 33, 41 Severe insulin resistance, 134, 183, 187 Solitary chemosensory cells (SCCs), 9 SRB-1 (scavenger receptor class B, type 1), 202, 222 Steatohepatitis, 79, 81, 101–102, 183 Sterol regulatory element binding protein-1C (SREBP-1C), 142, 184 Synaptic plasticity, 297 Syndrome X, 180, 423 Systemic circulation, 13, 53, 298, 431 Thiazolidinediones (TZDs), 101, 144, 183, 210, 253, 308–309, 328, 345, 371–372, 401, 472, 479 Thyroid hormone nuclear receptor (TR), 215 Transient response potential (TRP), 39 Urinary cortisone,

White muscles,

432

167

Zucker Diabetic Fatty (ZDF) rats, 77, 339, 407, 429

9, 62,