Diabetes Research
Diabetes Research A Guide for Postgraduates Bernard Tuch Professor of Medicine, University of New South Wales and Director, Pancreas Transplant Unit, Prince of Wales Hospital, Sydney, Australia
Marjorie Dunlop Associate Professor, University of Melbourne and Principal Research Fellow, National Health and Medical Research Council, Australia
Joseph Proietto Associate Professor of Medicine, University of Melbourne and Head of the Metabolic Disorders Clinic, Royal Melbourne Hospital, Australia
harwood academic publishers Australia • Canada • France • Germany • India • Japan Luxembourg • Malaysia • The Netherlands • Russia • Singapore • Switzerland
Copyright © 2000 OPA (Overseas Publishers Association) N.V. Published by license under the Harwood Academic Publishers imprint, part of The Gordon and Breach Publishing Group. All rights reserved. No part of this book may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying and recording, or by any information storage or retrieval system, without permission in writing from the publisher. Amsteldijk 166 1st Floor 1079 LH Amsterdam The Netherlands This edition published in the Taylor & Francis e-Library, 2004.
British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library. ISBN 0-203-30500-0 Master e-book ISBN
ISBN 0-203-34312-3 (Adobe eReader Format) ISBN 90-5702-461-6 (Print Edition)
Contents
Foreword Acknowledgements 1 2 3 4 5 6
Diabetes–the Clinical Problem Normal Metabolic Physiology Type 1 Diabetes Type 2 Diabetes Aetiology of Complications in Type 1 and Type 2 Diabetes Critical Pathways to Establish a Career in Diabetes Research
Glossary Index
vii xi 1 13 43 61 75 105 115 125
This book is dedicated to all those graduate students who are entering the field of diabetes research and have the enthusiasm and the desire “to strive, to seek, to find and not to yield”.
Foreword
This book aims to guide the young investigator, who has just entered the field of diabetes, through the physiological and pathophysiological events which form the biological substrate of the disease, and to introduce some of the ‘bread-and-butter’ techniques used in this field. Also, and perhaps as importantly, it aims to attract the young, undecided investigator into diabetes research. Why diabetes, one among a myriad of diseases? It is estimated that at least 200 million people worldwide suffer from diabetes; in addition, the incidence of the disease is on the rise, especially (but not exclusively) in developing countries. Since the world population is increasingly aging, and diabetes incidence augments almost exponentially with age, expectations are that within a couple of decades the number of people with diabetes will double, if not triple. For a person attracted to biomedical research it is therefore important to know that his or her research deals with a problem that concerns a sizeable portion of humanity. In addition, diabetes is a chronic disease for which, as yet, there is no cure; a patient must live with this problem for decades, sometimes for a whole lifetime. The disease is not always easily controlled, and involves long-term complications which result from chronic hyperglycaemia; some are life-threatening, but all drastically reduce the quality of life, not only for the patient but also for the rest of their family. Whether one is a medical doctor or not, the knowledge that one’s research directly or indirectly may lead to measures that help alleviate the suffering of millions of human beings will be a source of immense satisfaction. Diabetes is a complex disease, with multiple forms and as many aetiologies, encompassing as differing medical specialities as endocrinology, immunology and ophthalmology, just to mention a few. Therefore, ‘diabetes research’ may be a synonym for research in basic immunology, in hormone action and signal transduction, in classical and not-so-classical endocrinology, in vascular biology, in metabolism at large, in several fields of molecular biology, and so on along an ever extending list of research disciplines. It is this trait of multidisciplinarity that makes modern diabetes research so attractive. And, in fact; this has always been true: it was with insulin that the first radioimmunoassay, the technique that revolutionised modern endocrinology, was established almost half a century ago, as it was with insulin that the first attempt at engineering a protein by molecular biology techniques succeeded. vii
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Thus you, the young investigator, whether you are attracted toresearch in autoimmunity, or processing of prohormones, or angiogenesis, or on the contrary antiangiogenesis, or in fact almost any topic of modern biology, your place is in diabetes research. Good research demands some IQ, a good deal of tenacity and perseverance, much work, some luck, but certainly a lot of money. Diabetes research is in the fortunate position that public as well as private granting agencies have acknowledged the impact that it may have for a disease with such extensive socio-economic consequences. Therefore, in many countries researchers in the diabetes field are comparatively better funded than in other research areas; this may indeed have contributed to some of the spectacular advances registered in the field over recent decades. Still another reason to opt for diabetes research. In this book, Bernard Tuch, Marjorie Dunlop and Joseph Proietto take you through a tour of the main highlights of diabetes research: Chapter 1 addresses itself to the non-medical investigator; in a nutshell it presents the main facts of diabetes to the scientist who has been estranged from health issues but having decided to enter diabetes research is nevertheless expected to have a minimal degree of knowledge about the disease. Diabetes being characterised by disorders of carbohydrate and lipid metabolism, its physiology is exposed in Chapter 2. This chapter is a reminder of the basics of metabolism: the enzymes and metabolic steps involved in the normal regulation of glucose homeostasis are described, the glucose transporter family is presented, the hormones that participate in maintaining blood glucose within tight limits and their mode of action are reviewed, and the metabolic fate of other nutrients such as lipids and proteins is briefly described. This abridged information is put into a practical context by describing in detail a concrete research exercise: how to plan an investigation that aims at discovering the mode of action of a new hypoglycaemic molecule. Such practical exercises, which form a recurrent theme in most of the following chapters, parachute us from eclectic theory to the living environment of the research laboratory. Chapter 3 deals with the autoimmune, insulin-dependent form of the disease, Type 1 diabetes. The main ideas regarding its pathogenic mechanisms, including the genes thought to be involved in this multigenic disease, are succinctly exposed, and the commonly used animal models of the disease described. The main part of the chapter is dedicated to advanced present and future treatments of Type 1 diabetes; these include pancreas and islet allotransplantation as well as xenotransplantation with and without immune isolation techniques, and gene therapy, which is still in its infancy. Some of the important issues that presently preoccupy the research community are briefly described; these serve as an excellent introduction towards the original questions that the young investigator is expected to formulate on his/her own. The overwhelming majority of people with
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diabetes suffer from non-insulin-dependent Type 2 diabetes. Even if this form of the disease may sometimes present itself as a disease milder than Type 1 diabetes, the huge number of patients justifies that the utmost effort be allocated to research on Type 2 diabetes. The main pathogenic mechanisms that lead to Type 2 diabetes, reduced action of insulin on its targets, as well as reduced production and release of insulin, are explained in Chapter 4. The heterogeneity of this disease is clearly exposed; this is apparent from the long list of specific animal models which, while assisting the scientist in his/her quest, also bring the confusion of the multiplicity, that nevertheless may indeed reflect reality for this form of diabetes. Both forms of diabetes bear the burden of the long-term vascular and neural complications of the disease. Accumulating experience teaches us that the differences between Type 1 and Type 2 diabetes regarding the types, intensities and incidences of these complications are marginal; they are therefore presented together in Chapter 5. This chapter gives a detailed account of the types of complications and their prevalence, exposes the present-day thinking on the mechanisms that lead to the complications, and describes the recent large clinical studies that have linked most of the complications to the degree of chronic blood glucose control. From such and other studies treatment strategies have emerged that help reduce, in some cases prevent; the appearance of the long-term diabetic complications; these, including the ones that are still at an experimental level, are described at some length. Finally, the experimental models that are at our disposal in this field are identified, as are the most pressing research questions. The number of publications in the field of diabetes, as in many other areas of biomedical research, has increased to such an extent that even the most seasoned scientist keeps track of advances with great difficulty. More serious for the neophyte diabetologist is his/her inability to distinguish the essential from the interesting but non-essential. A sine qua non condition for success when embarking on a meaningful research career is the ability to identify the sources of essential information. Chapter 6 provides an invaluable service to the young investigator by presenting such information sources for diabetes research. These include firstly a list of international and national learned societies in the fields of diabetes and endocrinology, complete with their internet addresses. These societies provide a variety of services, from publications covering basic and clinical research to grant and congress/ workshop information, which can introduce the young investigator to frontline diabetes research and diabetes researchers in the shortest possible way. A similar list is provided for journals, most of them with electronic online text, covering most areas of biology and medicine of relevance to diabetes research, with a short introductory note for each of them. The chapter also lists the major national and, more important, international funding sources in diabetes. Careful perusal of this list, and study of their web sites, is well spent effort even for the most senior researcher at this time of soaring research
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costs and perpetual chase after funding sources. Finally, the chapter presents several web sites related to diabetes/endocrinology, many of them also extremely useful for preparing teaching materials. This chapter thus summarises the critical expertise of senior diabetologists faced with our era’s information plethora, and tries to arm the young diabetes researcher with tools of survival in his/her quest for two essential items in research: information and money. Diabetes is one of the major chronic health hazards of modern times. Research must catch up with this disease, so that a cure is finally found and the disease eradicated. The more young unbiased brains join in this fight, the greater the possibility that a breakthrough will be forthcoming ‘in our time’. But remember also that diabetes is a vast, fascinating field where research of utmost sophistication is possible. It is the hope of the authors that many a young talent will be stimulated by this book to enter diabetes research. Be your motivation medical/humanitarian, or mainly ‘cold’ intellectual curiosity, you are all most welcome. Erol Cerasi
Acknowledgements
We gratefully acknowledge the artistic skills and support of Marcus Cremonese and Michael Oakey from the Medical Illustration Unit, Prince of Wales Hospital. They are responsible for the illustrations in this book. We also wish to thank Ms Angie Pinto, a medical student at The University of New South Wales, who proofread the chapters and helped make them suitable for the intended readership.
1. Diabetes: The Clinical Problem
Definition Diabetes mellitus, hereafter referred to as diabetes, is a disorder characterized by the presence of an excess of glucose in the blood and tissues of the body. The word diabetes is Greek for a siphon, referring to the discharge of an excess quantity of urine; and mellitus is Latin for honey. Thus diabetes mellitus means the passage of large amounts of sweet urine. This is derived from the fact that excess glucose in the blood spills over into the urine, absorbing fluids with it. Types of diabetes There are two main types of diabetes: type 1 diabetes, previously called insulin-dependent diabetes; and type 2 diabetes, previously called non-insulin dependent diabetes.
Type 1 diabetes Type 1 diabetes affects approximately 15% of all people with diabetes. It is rare in the first nine months of life and has peak incidences at 12, and between 20 and 35 years of age. It is caused by the destruction of the insulin-producing cells of the pancreas, called ß cells, which are located in islets or islands throughout that organ (Figure 1.1) See Chapter 3 for further details of this process. Clinically, a person with this disorder presents with numerous symptoms, including: • • • • •
frequent passage of urine (polyuria) drinking lots of water (polydipsia) muscle cramps blurred vision weight loss
Polyuria is due to the osmotic effect of excess glucose in the urine. The resultant fluid loss causes dehydration with the affected person trying to compensate for this by polydipsia. Muscle cramps are caused by electrolyte disturbances associated with the fluid loss. The blurred vision is due to excess 1
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Figure 1.1. An islet from the pancreas of (top) a non-diabetic mouse and (bottom) a mouse with diabetes caused by destruction of the ß cells in the islet Haematoxlin & eosin. Bar is 50 µm.
accumulation of glucose in the lens of the eye (see chapter 5 for further details). Weight loss is due to breakdown of protein and fat because of lack of insulin, as well as loss of water. The diagnosis of type 1 diabetes is made by taking a random blood sample and measuring the blood glucose level. This will be elevated, that is, >11.1
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mmol/L, with a value usually >20 mmol/L. Examination of the urine will show large amounts of glucose and may also show ketones, which are breakdown products of fats. Treatment of type 1 diabetes
Treatment is by: injection of insulin subcutaneously two to four times a day (Figure 1.2); and ensuring an even distribution of carbohydrates ingested throughout the day (Figure 1.3). The types of insulin used commonly are short and intermediate in action (Figure 1.4). The dynamics of insulin release for each of these types are:
Figure 1.2. A syringe and pen used for injection of insulin. Note the fine needle that is used to pierce the skin.
Figure 1.3. Typical distribution of carbohydrate throughout the day for a person with insulin-dependent diabetes who is consuming 1500 kilocalories a day.
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Figure 1.4. Types of insulin available for injection. Humalog is one type of short-acting insulin, protaphane is an example of intermediate-acting insulin, and mixtard 30/70 is a premixed version containing 30% short-acting and 70% intermediate-acting insulin.
It is quite common for the two types of insulin to be mixed before being injected. Premixed insulin is commercially available (Figure 1.4). The aim of treatment is to maintain blood glucose levels within the physiological range, that is, 4–8 mmol/L. Diabetic people commonly measure their own blood glucose levels using a drop of blood drawn from the fingertip. The blood is placed on a reagent strip which is inserted into a portable glucometer which gives a digital reading of the blood glucose level (Figure 1.5). It is also possible to estimate the degree of control of blood glucose levels over the past three months by measuring the level of glycated haemoglobin in the blood. This measures the amount of glucose bound to haemoglobin in the red cell, the half-life of which is three months. The average blood glucose level over the past month can be estimated from the serum level of
Figure 1.5. A glucometer for measuring blood glucose levels. Blood is placed on a disposable reagent strip, which can be seen already in the machine.
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fructosamine, which is an estimate of the amount of glucose bound to albumin. On occasion a person may present with or develop very high blood glucose levels and become sick. This is associated with the presence of ketones in the blood, urine and exhaled air. Treatment of this condition (ketoacidosis) requires: rehydration with intravenous fluids; intravenous infusion of insulin; and administration of antibiotics if necessary (an infection often precipitates ketoacidosis).
Type 2 diabetes Type 2 diabetes affects approximately 85% of all people with diabetes and usually occurs after the age of 40 years. It is caused by a combination of the following: • Resistance to the action of insulin in peripheral tissues such as muscle and fat cells. • Failure of the insulin-secreting cells of the pancreas to produce sufficient insulin. • Failure of insulin to inhibit the production of glucose in the liver. For further details of these causes see Chapter 4. Clinically, a person may have this disorder without being aware of it. Loss of vision, which usually takes years to develop, may be the first sign of problems. Risks for developing type 2 diabetes are: • • • • • •
family history of type 2 diabetes obesity high blood pressure high levels of cholesterol a sedentary lifestyle diabetes during a pregnancy.
Figure 1.6. Results of an oral glucose tolerance test for a person with diabetes (open circle) and a person who has normal blood glucose levels (filled circle).
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Figure 1.7. The contents of this bottle, containing 75 g glucose, is consumed to begin an oral glucose tolerance test.
Those at risk may reduce the chance that diabetes will develop by maintaining a normal body weight. If overweight, they should reduce their calorie intake. Keeping physically active is equally important. The diagnosis of type 2 diabetes is made by any of the following: • random blood glucose levels >11.1 mmol/L • fasting blood glucose levels >7 mmol/L • oral glucose tolerance test result >11.1 mmol/L (Figure 1.6), i.e., ingestion of 75 g glucose (Figure 1.7) after an overnight fast, with blood glucose level measured 2 hours later. Treatment of type 2 diabetes
The two major forms of treatment of type 2 diabetes are alterations in the diet and engaging in physical activity. If blood glucose levels cannot be maintained within normal limits by these means, oral hypoglycaemic agents can be used. There are three main types, based on their mode of action (Figure 1.8). • The sulphonylurea-types (e.g., glibenclamide) stimulate the pancreas to produce more insulin. • The biguanide-types (e.g., metformin) and the thiazolidinediones, (e.g., troglitazone) reduce insulin resistance and result in greater uptake of glucose by tissues. • Alpha glucosidase inhibitors (e.g., acarbose) inhibit the digestion of carbohydrates in food and hence reduce their absorption from the gut.
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Figure 1.8. The different types of oral hypoglycaemic agents available for treatment of non-insulin dependent diabetes. Diamicron is an example of an agent that stimulates the pancreas to produce more insulin, diabex is one that reduces insulin resistance, and glucobay is an agent that decreases absorption of carbohydrate from the gut.
A proportion of people with type 2 diabetes need insulin injections to control their glucose levels. Mostly this occurs five or more years after starting to use oral hypoglycaemic agents (when their potency wears off). The types of insulin used and the number of injections given per day are the same as for the treatment of type 1 diabetes. As with type 1 diabetes, the aim of treatment is to maintain blood glucose levels within the physiological range, that is, 4–8 mmol/L.
Hypoglycaemia This occurs when blood glucose levels drop, either because too much insulin is injected, or too many oral hypoglycaemic tablets are taken, or the person does not consume carbohydrates after taking these agents. This state occurs when the blood glucose level falls below 2.5 mmol/L, but in some people may occur between 3 and 4 mmol/L. The body usually recognizes these low levels and counter-regulatory mechanisms are activated to prevent the glucose level falling further and to ensure that it rises. Hormones released for this purpose include: glucagon from the a cells of pancreatic islets; cortisol from the cortex of the adrenal gland; growth hormone from the pituitary gland; and adrenaline from the medulla of the adrenal gland and the sympathetic nervous system.
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These hormones promote the generation of glucose from glycogen and amino acids, processes termed glycogenolysis and gluconeogenesis respectively. All cells require glucose for their metabolism. A very low blood glucose level for a prolonged period is not compatible with life. The symptoms and signs of hypoglycaemia are: • sweating, shaking, palpitations, pale skin and a feeling of anxiety. These are due to secretion of adrenalin • hunger and alteration in intellectual function ranging from difficulty in coping, inappropriate behaviour to coma and seizures. These are due to altered function of the brain, which is a heavy consumer of glucose • headache and abdominal pain, the causes of which are unknown. People with diabetes are trained to recognise the symptoms and signs of hypoglycaemia, and ingest sweets (e.g., jellybeans) containing rapidly absorbed sugar at their onset (Figure 1.9). If the person becomes unconscious, intramuscular injection of glucagon by a relative or friend, or intravenous injection of glucose by paramedical personnel, is required (Figure 1.9). Normalisation of blood glucose level and recovery of consciousness occur within minutes of these treatments.
Diabetic complications Most people who have had diabetes for many years will develop complications, to a large extent because their blood glucose levels are not maintained within the physiological range. It is the blood vessels of the
Figure 1.9. Agents used to overcome hypoglycaemia. Jelly beans are an example of glucose that can be taken orally. Glucagon and glucose 50% are given by injection to an unconscious person.
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Figure 1.10. The retina of a person with proliferative retinopathy and scarring. The white scar tissue can be seen across most of the picture.
body which primarily bear the brunt of these adverse effects. Both small blood vessels (termed microvascular) and large blood vessels (termed macrovascular) can be affected. Microvascular complications affect the: • retina of the eye, which can result in a decrease in vision and eventually blindness. Almost all people with diabetes will develop some retinopathy. Thus, 75% of people who have had diabetes for 15 years have this problem. The incidence continues to increase with duration of diabetes. About 25% of people develop vision-threatening retinopathy. Diabetes is the commonest cause of blindness in the western world (Figure 1.10). • nephrons of the kidney, resulting in leakage of protein and eventual decline in function. The incidence of this problem is decreasing, rates varying between 9% and 35%. People with type 1 diabetes are more likely to develop the disorder. The incidence of nephropathy peaks 15 years after the onset of diabetes and declines thereafter. Progression to chronic kidney failure with the need for dialysis and kidney transplantation may occur. • nerves of the peripheral nervous system, resulting in loss of feeling, affecting the feet in particular. It is possible to prevent or delay the progression of microvascular complications by maintaining blood glucose levels as normal as possible. Other strategies can also be used to slow the progression of complications.
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• Retinopathy: keep blood pressure and cholesterol levels as normal as possible and administer laser treatment to damaged blood vessels in the eye. This will prevent scarring of the retina. • Nephropathy: keep blood pressure as normal as possible and decrease leakage of protein into the urine (e.g., by daily administration of a drug which acts on the kidney to inhibit the metabolism of the presser agent angiotensin I). Macrovascular complications are due to a build-up of fat and other deposits on the walls of the large arteries. The process, which narrows the lumen of the artery and reduces blood flow through it; is called atherosclerosis. It affects most people, especially in western countries, as they become older. People with diabetes have a greater chance of developing significant atherosclerosis than do people without diabetes and various organs may be affected. Narrowing of the coronary arteries results in an increased incidence of heart attacks. Narrowing of the cerebral blood vessels results in an increased incidence of strokes. Narrowing of the arteries to the lower limbs can result in gangrene (Figure 1.11), necessitating amputation of part of a limb. It is possible to delay the development and progression of macrovascular complications by: • • • • •
maintaining blood pressure as normal as possible maintaining blood cholesterol levels as normal as possible keeping physically fit maintaining normal body weight not smoking.
Figure 1.11. The gangrenous toe of a person with long-standing diabetes who smoked cigarettes.
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Some people with diabetes will develop complications despite the these precautions. Many will have inherited genes predisposing them to conditions such as heart disease (e.g., several members of the family may have died from heart attacks at a young age). References and reviews Types of diabetes
Burge MR, Schade DS. Insulins. Endocrinol Metab Clin North Am 1997; 26:575–98 Goldstein DE, Little RR. Monitoring glycaemia in diabetes Short-term assessment Endocrinol Metab Clin North Am 1997; 26:475–86 Rifkin H, Porte D Jr (eds). Ellenbergand Rifkin’s Diabetes Mellitus Theory and Practice 4th edn. New York: Elsevier, 1990 Hypoglycaemia
Cryer PE, Gerich JE. Glucose counterregulation, hypoglycaemia, and intensive insulin therapy in diabetes mellitus. N Engl J Med 1985; 313:232–41 National Health & Medical Research Council of Australia Report of the Health Care Committee Expert Panel on Diabetes. Hypoglycaemia and diabetes. Canberra: Australian Government Publishing Service, 1991 Diabetic complications
Bojestig M, Arnqvist HJ, Hermansson G, Karleberg BE, Ludvigsson J. Declining incidence of nephropathy in insulin-dependent diabetes mellitus. New England Journal of Medicine 1994; 330:15–18 Clark CM, Lee DA. Prevention and treatment of the complications of diabetes mellitus. N Engl J Med 1995; 332:1210–17 Diabetes Control and Complications Trial Research Group. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. N Engl J Med 1993; 329:977–86 Parving H. Initiation and progression of diabetic nephropathy. N Engl J Med 1996; 335:1682–3
2. Normal Metabolic Physiology
Glucose The level of glucose in the blood is very tightly regulated, and in normal individuals is rarely found outside a range of 3.5–5.5 mmol/L (63–100 mg/ 100 mL). In the fasting resting state, glucose turnover (the rate of appearance and disappearance in the blood) is approximately 2.5–3.0 mg/kg/min. At this rate all glucose would disappear from the blood within ~100 minutes were it not continually replaced. Glucose concentration is tightly controlled because it is an essential source of energy, particularly for the brain, which cannot use other sources of energy. How is blood glucose maintained within a narrow range? The mechanisms used to maintain a normal blood glucose level depend on whether the body is fasting or in the immediate post-absorptive state. Both the rate of appearance and the rate of disappearance of glucose can be modified in order to maintain blood glucose within a narrow range.
Glucose appearance In the fasting state, when there is no glucose entering the blood from the intestine, glucose turnover is maintained by supply from the liver and the kidneys. It is thought that the liver contributes between 70% and 90% of the glucose while the kidneys contribute the remainder. Most of the glucose released by the liver comes from two sources— glycogen breakdown (glycogenolysis) and the synthesis of new glucose from three carbon fragments (gluconeogenesis). In the immediate post-absorptive state, most of the glucose is supplied by glycogenolysis, and as fasting progresses, gluconeogenesis becomes increasingly important Glycogenolysis
Glucose is stored in the form of glycogen, a branched polysaccharide (a 1→4)* of Dglucose with branches (α 1→6) occurring every 8–12 glucose residues (Figure 2.1). *This notation indicates how one molecule is linked to another, in this case carbon 1 of one molecule is linked to carbon 4 of the next. 13
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Figure 2.1. Chemical structure of glycogen showing α 1→4 bonds (a) and α 1→6 branch point (b).
The multiple branches mean that there are many non-reducing ends on which degrading enzymes can work, speeding up the process. Glycogen is abundant in the liver, where it can make up as much as 7% of wet weight, and is also present in skeletal muscle. Each glycogen molecule has an average molecular weight of several million and is stored in the form of tight clusters of granules that contain within them the enzymes required for its synthesis and breakdown. The enzyme glycogen phosphorylase catalyses the reaction that allows the splitting of the glycosidic linkage (α 1→4) joining the 2 terminal glucose molecules of a chain, releasing a-D-glucose-1-phosphate. Glycogen phosphorylase repeats the reaction until it reaches a point 4 glucose molecules from a branch point (α 1→6). To remove the last 4 molecules, a debranching enzyme, oligo (α 1→6) to (α 1→4) glucantransferase transfers 3 glucose molecules to the end of another chain (joining them via an α 1→4 bond) and removes the glucose molecule remaining at the branch point, releasing it as free glucose (as opposed to glucose-1-phosphate).
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Glucose-1-phosphate is converted reversibly to glucose-6-phosphate by the enzyme phosphoglucomutase. Glucose-6-phosphate is then converted to free glucose by the action of the enzyme glucose-6-phosphatase. This enzyme is present only in liver and kidney explaining why it is only these two tissues that can release glucose into the blood. Gluconeogenesis
Gluconeogenesis is the name given to the process whereby glucose is formed from 3 carbon non-hexose precursors (Figure 2.2). Precursors include lactate, pyruvate, glycerol and most of the amino acids. There are 10 enzymatic reactions required to convert pyruvate to glucose. Of these 7 are the reverse of the glycolytic reactions (see below). The other 3 (the conversion of: pyruvate to phosphoenol pyruvate; fructose 1,6bisphosphate to fructose 6-phosphate; and glucose-6-phosphate to glucose) cannot occur as a reversal of the reactions in glycolysis and require specific enzymes. Therefore it is at these three steps that regulation of gluconeogenesis can occur. The enzymes catalysing these irreversible steps are as follows. Pyruvate to phosphoenolpyruvate: A mitochondrial enzyme, pyruvate carboxylase converts pyruvate to oxaloacetate which is reduced to malate. Malate then leaves the mitochondria and in the cytosol it is reconverted to oxaloacetate. Phosphor nolpyruvate carboxykinase converts oxaloacetate to phosphoenolpyruvate. Fructose 1,6-bisphosphate to fructose 6-phosphate: This reaction is catalysed by the enzyme fructose 1,6-bisphosphatase. Glucose-6-phosphate to glucose: This is the final reaction of gluconeogenesis and is catalysed by the enzyme glucose-6-phosphatase.
Glucose disappearance Glucose is utilised by all tissues, but in the resting state, the brain uses approximately 65% of the available glucose. Glucose transport
Glucose enters cells via a family of specific glucose transporters. So far, 6 different glucose transporters have been isolated. They all share sequence homology, suggesting a common ancestral gene. Each has specific tissue distribution (Table 2.1). The general structure of these glucose transporters is shown in Figure 2.3. There are 12 transmembrane domains (M1 to M12) that are thought to form a pore through which glucose is transported.
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Figure 2.2. Outline of gluconeogenic and glycolytic pathways showing intermediates and key enzymes.
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GLUT1 is a widely expressed transporter that appears to mediate basal glucose transport in many tissues. There are particularly high levels in human erythrocytes, brain, blood-brain barrier and fetal tissues. Following synthesis in the rough endoplasmic reticulum, it is transported to the plasma membrane where it is inserted. Many studies have shown that there is a small intracellular pool of GLUT1 proteins since upon stimulation with insulin, GLUT1 levels increase modestly in the plasma membrane, at least in adipocytes. Table 2.1 The six different glucose transporters
*Concentration of substance giving half-maximal response.
GLUT2 is expressed predominantly in liver and pancreatic ß cells with some expression also in kidney and small intestine. This transporter resides entirely in the plasma membrane and is unique for having a high Km, thus transporting glucose proportionally to plasma glucose concentration over a wide range of glucose levels. This ability is of particular use in the two major organs where it is expressed; in liver to allow this organ to absorb glucose postprandially and in ß cells to allow this cell to respond to a wide range of glucose levels. It is of interest that these two tissues also express a unique hexokinase (glucokinase) which also has a high Km so that the liver and ß cell can transport and phosphorylate glucose over a wide range of plasma concentrations. GLUT3 is expressed predominantly in the brain and to a lesser extent in placenta and kidney—mRNA has been found throughout the brain and appears to be specific for neurones. GLUT4 is also known as the insulin-regulatable glucose transporter. GLUT4 is expressed in muscle, heart, white adipose tissue and brown fat tissue. Following synthesis two distinct targeting signals, one in the amino terminus and the other a dileucine motif in the carboxy terminus, target this transporter to specific intracellular vesicles. These transporters remain in these vesicles until the cell is stimulated by insulin, or exercise (in muscle
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Figure 2.3. Outline of the general structure of glucose transporters.
tissues). Following stimulation the GLUT4 moves rapidly to the plasma membrane, thus increasing the rate of glucose transport. GLUT5 is expressed mainly in the apical membrane of cells in the small intestine. This is predominantly a fructose transporter. GLUT6 is a pseudogene with no protein produced. All of these transporters mediate facilitative hexose transport This process does not require energy as transport occurs along a concentration gradient. A new transporter (GLUT8) with homology to GLUT4 has been presented in abstract form but its physiological role remains unknown. There are other transporters, which require Na+ to function. These sodium-dependent transporters are found in situations where glucose needs to be transported against a concentration gradient; such as the renal tubules and the intestine. This process is energy dependent.
Glucose metabolism Following entry into the cell, glucose can go into several pathways including synthesis of glycogen, breakdown to pyruvate, and entry into the pentose phosphate shunt or the hexosamine biosynthesis pathway. Each of these pathways will be briefly discussed. Glycogen synthesis
Glycogen synthesis occurs in nearly all tissues, but is especially significant in liver and muscle tissue. To start glycogen synthesis, glucose-6-phosphate is converted to Glucose-1-phosphate by the enzyme phosphoglucomutase. Glucose-1-phosphate is then converted to UDP-glucose by the action of
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the enzyme UDP-glucose pyrophosphorylase. The enzyme glycogen synthase transfers the glucosyl residue from UDP-glucose to a branch of the preexisting glycogen molecule. Thus glycogen synthase must have a polyglucose primer of at least 4 glucose residues. The puzzle as to how the initial glycogen was made was resolved with the discovery of the protein glycogenin which acts as the initial primer. Glycogen synthase is, however, not able to make the branch points (α1→6). These are formed by two other enzymes: amylo (1→4) to (1→6) transglycosylase or glycosyl (4→6) transferase. Glycogen synthase, like glycogen phosphorylase, is regulated by phosphorylation/dephosphorylation reactions (Figure 2.4). Glycolysis
Glycolysis is the process by which a series of chemical reactions is used to degrade one molecule of glucose into two molecules of pyruvate. During this process energy is released and is conserved as ATP.
Figure 2.4. Diagram illustrating how phospho-dephospho reactions regulate glycogen synthesis and degradation.
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The process involves 10 steps. In the first 5, energy is invested in the reaction in the form of two molecules of ATP. The steps are shown in the box below.
As mentioned previously, 7 of the 10 reactions are reversible and therefore are common to the gluconeogenic pathway (see above). The 3 non-reversible reactions (a, b & c) are mediated by three enzymes unique to glycolysis. These are: a. hexokinase, b. phosphofructo-1-kinase and c. pyruvate kinase. Pyruvate can have two fates. Under aerobic conditions it is oxidised to acetate which enters the citric acid (Krebs) cycle to be oxidised to CO2 and water (see below). In anaerobic conditions it is reduced to lactate by the enzyme lactate dehydrogenase. Citric acid (Krebs or tricarboxylic acid) cycle
The citric acid cycle is a mitochondrial pathway by which pyruvate is converted to CO2 and water with the release of energy in the form of ATP. The first step in the further metabolism of pyruvate is its oxidative decarboxylation by the enzyme complex pyruvate dehydrogenase to produce acetyl CoA. Pyruvate dehydrogenase is a complex of three distinct enzymes and five coenzymes. In the first step of the citric acid cycle, Acetyl CoA combines with oxaloacetate to form citrate. A series of eight subsequent steps (2–9) reforms oxaloacetate with the release of 2 molecules of CO2 and energy (Figure 2.5).
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A glucose molecule metabolised using aerobic glycolysis uses 2 ATP molecules but produces 40, giving a net production of 38 ATP molecules per glucose molecule.
Figure 2.5. Outline of the chemical reactions that constitute the tricataoxylic acid (Krebs) cycle.
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The pentose phosphate shunt (hexose monophosphate shunt)
The main functions of this pathway are to produce NADPH and essential pentoses such as D-ribose used in the biosynthesis of nucleic acids. NADPH is an essential cofactor in the biosynthesis of fatty acids and steroids. Therefore, the pentose phosphate pathway is particularly active in liver, adipose tissue, adrenal and mammary glands. In this pathway glucose-6-phosphate is converted in 4 steps to D-ribose5-phosphate with the production of 1 CO2 and 2 NADPH molecules. In tissues that require primarily NADPH, rather than ribose-5-phosphate, pentose phosphates can be recycled into glucoses-phosphate. Hexosamine biosynthesis pathway
The hexosamine biosynthesis pathway (HBP) branches from the glycolytic pathway at fructose-6-phosphate. Under normal circumstances only about 3% of glucose is diverted through this pathway. The first and rate-limiting step is catalysed by the enzyme glutamine:fructose-6-phosphate amidotransferase which joins a glutamine to fructose-6-phosphate to produce glucosamine-6-phosphate. This is further metabolised to UDP-N-acetylglucosamine which serves as a precursor to the synthesis of glycoproteins, glycolipids and proteoglycans.
Carbohydrate digestion The first step in carbohydrate absorption occurs when salivary a-amylase attacks dietary starch in the mouth, producing maltose, isomaltose and glucose. This reaction is stopped by the acidic conditions in the stomach. Following entry into the duodenum, pancreatic a-amylase continues the process which proceeds very rapidly. Within 10 minutes of entering the duodenum, most starch is converted into fragments containing on average 3 hexoses. Disaccharides are further split within the mucosal cells as they are absorbed. There are several disaccharidases, most of which reside in the duodenum and jejunum (Table 2.2). Two glucose transporters are involved in the transport of glucose from the gut lumen to the plasma. In the small intestine the energy-requiring, Na+-dependent glucose transporter SGLT1 is located in the apical plasma membrane of the absorptive epithelial cells and transports glucose from the gut lumen into the cell. The glucose then exits the cell from the baso-lateral side of the cell via GLUT2. Fructose enters the epithelial cell via GLUT5 located in the apical membrane.
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Table 2.2: Human intestinal disaccharidases
From the interstitial space around the basolateral aspect of the epithelial cells glucose makes its way into the portal venous system. Blood from the portal vein first passes through the liver where some is removed. The remainder is released into the systemic circulation via the hepatic vein. Glucose taken up by the liver is stored as glycogen. This occurs either by the direct route (that is, glucose→glucose-6-phosphate→glucose-1phosphate→UDP glucose→glycogen) or by the indirect route by which glucose first undergoes breakdown to pyruvate or lactate and is then reformed into glucose via gluconeogenesis before being stored as glycogen. Most glycogen synthesis occurs via the indirect pathway. Why this apparently inefficient way of glycogen deposition is preferred is not clear.
Regulation of glucose homeostasis Glucose levels are tightly regulated by a variety of mechanisms.
Insulin Insulin is produced in the ß cells of the islets of Langerhans. These islets are the endocrine part of the pancreas and are scattered throughout the exocrine pancreas whose main function is to release digestive enzymes into the gut Four main types of cells are present in the islet: the a cells produce the hormone glucagon, the ß cells produce insulin, the d cells produce somatostatin and the PP cells produce pancreatic polypeptide. Insulin is a protein molecule of 5700 molecular weight It is synthesised only in the ß cells of the pancreatic islets. The insulin gene produces an mRNA that codes for a larger protein preproinsulin. The amino terminus of this protein is a signal sequence that directs this precursor to secretory granules. This sequence is removed by proteolytic cleavage. This and the formation of 3 disulphide bonds results in the formation of proinsulin, which is stored in secretory granules. Before secretion, specific peptidases cleave 2 peptide bonds to produce a 2-chained hormone and a connecting peptide, C-peptide (Figure 2.6). Insulin and C-peptide are secreted at a 1:1 molar ratio. Measurement of C-peptide can therefore be used as a surrogate measure of endogenous insulin secretion. The secretion of insulin is stimulated primarily, but not exclusively, by glucose.
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Figure 2.6. Cleavage of preproinsulin and proinsulin to form insulin and Cpeptide
Glucose is transported into the ß cell by the glucose transporter GLUT2 and is subsequently phosphorylated by glucokinase. Glucose needs to be metabolised to produce ATP in order to stimulate insulin secretion. An increase in the ATP:ADP ratio closes the K+ channels, thus depolarising the membrane and opening Ca2+ channels (Figure 2.7).
Figure 2.7. Mechanism of glucose-mediated insulin secretion.
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Figure 2.8. Pattern of insulin secretion in the presence of sustained hyperglycaemia.
The influx of Ca2+ results in the movement of insulin storage vesicles to the surface where they fuse with the plasma membrane and release their contents of insulin, C-peptide and a small amount of intact proinsulin. When the islet is chronically stimulated by high glucose levels, the amount of unprocessed prohormone released is increased, possibly because of lack of time to convert it to insulin and C-peptide. Insulin secretion occurs in a biphasic manner with a rapid and large first phase and a longer second phase if hyperglycaemia persists (Figure 2.8). The amount of insulin secreted in response to a particular glucose stimulus is higher if the glucose is administered orally rather than intravenously. The increased secretion of insulin after oral glucose is due to the release from the gut of incretins which potentiate the effect of glucose on insulin secretion. There are 2 important incretins: gastric inhibitory polypeptide (GIP), also known as glucose-dependent insulinotropic peptide; and glucagon-like peptide-1 (GLP-1). Actions of insulin
Insulin has many actions. Insulin stimulates: • • • • • •
glucose transport in muscle and fat tissue glycogen synthesis lipid synthesis protein synthesis K+ entry into cells Na+ retention by renal tubules.
Insulin inhibits: • • • •
gluconeogenesis glycogenolysis lipolysis proteolysis
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Insulin stimulates glucose transport by causing the translocation of the glucose transporter GLUT4 from the intracellular compartment to the plasma membrane. Glycogen levels are regulated by the level of cAMP in the cell, which in turn is regulated by the relative levels of insulin and glucagon with the former hormone lowering and the latter increasing the levels of this second messenger. An increase in cAMP levels is brought about by a rise in glucagon and a fall in insulin as occurs during a fast This results in the activation of protein kinase A, which in turn phosphorylates phosphorylase b kinase (which phosphorylates and activates phosphorylase b) and glycogen synthase kinase (which phosphorylates and inactivates glycogen synthase a) thus resulting in net glycogen breakdown (Figure 2.9). Following a meal, insulin levels rise and glucagon levels fall, cAMP levels drop and the process described above is reversed. Protein kinase A also phosphorylates and activates phosphoprotein phosphatase inhibitor, which inhibits phosphoprotein phosphatase, thus assisting in maintaining glycogen phosphorylase and glycogen synthase phosphorylated (Figure 2.9).
Figure 2.9. Mechanism of hormonal regulation of glycogen synthesis and degradation.
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Insulin stimulates the biosynthesis of fatty acids and their esterification into triglycerides by a variety of different mechanisms. It activates lipoprotein lipase in capillaries, resulting in the release of free fatty acids (FFAs) from circulating chylomicrons and VLDL particles (see below). These FFAs are taken up by adipocytes and re-esterified into triglycerides. The stimulation of glucose transport by insulin results in glucose being metabolised through the pentose phosphate pathway, producing reducing equivalents in the form of NADPH which are necessary for lipid synthesis. Insulin inhibits hormone-sensitive lipase by reducing cAMP levels, preventing triglyceride breakdown. Insulin activates the enzymes acetyl coenzyme A carboxylase and fatty acid synthase, increasing fatty acid synthesis (see below). Insulin has a stimulatory effect on protein synthesis. It stimulates the uptake of amino acids and increases the conversion of t-RNA-bound amino acids into protein, possibly by increasing the phosphorylation of ribosomal S6. It also has an effect which inhibits protein breakdown.
Figure 2.10. Schematic diagram of the insulin receptor.
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Mechanism of insulin action Insulin mediates all of its effects by binding to a surface receptor. The insulin receptor is a 500 kilodalton glycoprotein composed of two identical a and two identical ß chains (Figure 2.10). The two α chains (130 kd) are joined together by a disulphide bond and each protrudes from the outer surface of the plasma membrane. These proteins bind insulin. The 95 kd ß subunits are joined to the α subunits by disulphide bonds and extend into the intracellular space. When insulin binds to the a subunit, a conformational change occurs, resulting in the activation of a tyrosine kinase which is an integral part of the ß chain. It is thought that each ß chain tyrosine phosphorylates the opposite ß chain, which results in a further increase in tyrosine kinase activity. The first downstream targets of the ß chain tyrosine kinase are the insulin receptor substrate (IRS) proteins. There is a family of these proteins, IRS1 to IRS4. IRS-1 was originally thought to be the main target of the insulin receptor. These proteins have multiple tyrosine phosphorylation sites (22) which, when phosphorylated, are able to bind to downstream adaptor proteins that then transduce the insulin signal. Anti-insulin hormones
Several hormones act against the effects of insulin on a variety of tissues. The action of these hormones is to increase blood glucose levels. These hormones are: glucagon; cortisol; adrenaline (epinephrine); and growth hormone.
Glucagon Glucagon is a 29 amino acid protein produced by the a cells of the islets of Langerhans. Its secretion is inhibited by high levels of glucose and stimulated by low levels of glucose, and high levels of amino acids. It increases blood glucose levels predominantly by increasing endogenous glucose production through the stimulation of glycogenolysis and gluconeogenesis. It also stimulates ketogenesis. Glucagon mediates its actions by binding to a specific receptor which is linked to adenylate cyclase, increasing the intracellular levels of cAMP.
Adrenaline (Epinephrine) Like glucagon, adrenaline can cause rapid increases in hepatic glucose production by increasing the intracellular levels of cAMP. cAMP increases glycogenolysis and the transcription of the rate-limiting enzyme in the gluconeogenic pathway, phosphoenolpyruvate carboxyltinase (PEPCK).
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In addition, adrenaline impairs glucose uptake in insulin-sensitive tissues and stimulates release of free fatty acids. Adrenaline also raises glucose levels by an inhibitory effect on insulin secretion.
Cortisol This hormone is secreted by the adrenal cortex with higher secretion in the early morning. Its secretion is greatly increased by many different types of stress. Cortisol is important in maintaining key enzymes in gluconeogenesis, especially PEPCK. The PEPCK gene has a cortisol response element in its promoter which increases transcription of the enzyme.
Growth hormone Release of growth hormone from the anterior pituitary increases significantly in response to stress. Growth hormone inhibits insulin-mediated suppression of hepatic glucose production and stimulation of glucose uptake. Reaction to hypoglycaemia
Hypoglycaemia, defined as a plasma glucose level <2.5 mmol/L (<45 mg%), is normally vigorously counteracted by the above anti-insulin hormones in an effort to protect the brain from glucopenia. Secretion of glucagon is the key acute response to hypoglycaemia. In the absence of glucagon, adrenaline becomes important Since cortisol and growth hormone rely on gene transcription for their actions, these two hormones are more important in response to more prolonged hypoglycaemia.
Other nutrients Lipids Lipids are water insoluble molecules that serve a variety of functions within the body. Cholesterol (Figure 2.11) is a precursor of all the steroid hormones (cortisol, aldosterone, testosterone, oestrogen etc.) and is an essential component of bile acid and cell membranes. Fatty acids are carboxylic acids made up of hydrocarbon chains of varying lengths. Some contain no double bonds and are said to be fully saturated. Others contain one or more double bonds. This allows the classification of fatty acids into families.
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Figure 2.11. Structure of cholesterol.
Figure 2.12. Structure of a fatty acid (oleic).
The general nomenclature for fatty acids is as follows: • The numbering of the carbon atoms starts from the ω end and proceeds to the carboxy terminus. • The accepted convention is to describe the fatty acid as, for example, 18:1, ω-9 (oleic acid). Under this system 18: refers to the fact that this fatty acid has a total of 18 carbons; 1 is the number of double bonds; and ω-9 means that the first double bond is between the ninth and tenth carbon (Figure 2.12). Table 2.3 illustrates some common fatty acids and their source. Fatty acids are an important energy source. Mammals cannot synthesise some fatty acids such as linoleic acid and these are known as essential fatty acids. These fatty acids are precursors of prostaglandins. Table 2.3. Common fatty acids and their dietary source.
Fatty acids can combine with molecules containing alcohol (OH) groups to form esters.
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Figure 2.13. Structure of triglyceride. Cholesterol ester
Most cholesterol in the plasma is esterified, the most common being cholesterol linolate and cholesterol oleate. Esterification of cholesterol converts it from being polar to non-polar. Glycerol esters
There are two types of glycerol esters. Triglycerides (triacylglycerol). The esterification of all three of hydroxyl groups of glycerol produces triglyceride (Figure 2.13), which serves as the storage form of fat in the body. Fatty acids can be released from triglycerides by specific enzymes when required. Phospholipids (phosphoglycerides). In these compounds, one of the two outer hydroxyl groups of glycerol is esterified with a phosphate containing molecule rather than fatty acids. An example is lecithin, in which choline phosphate is added to one hydroxyl group. This makes this end of the molecule hydrophilic while the other end, which is esterified with fatty acids, is hydrophobic. This structure allows phospholipids to be used in the construction of cell membranes and lipoproteins. Lipoprotein metabolism
The problem of moving hydrophobic fat molecules around the plasma was solved by nature using the lipoprotein particle (Figure 2.14). This complex structure consists of a hydrophobic core containing triglycerides and cholesterol esters and an outer hydrophilic shell containing phospholipids, free cholesterol, and proteins (apoproteins). Lipoproteins come in different sizes and densities, as shown in Table 2.4. Chylomicrons are manufactured in the intestine and carry the digested fat. The major apoproteins are the C proteins (I to III) and ApoB-48. As they circulate, the enzyme lipoprotein lipase (LPL) releases free fatty acid from the stored triglyceride. The fatty acids are taken up by muscle or fat cells where they are metabolised or reesterified into triglycerides.
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Figure 2.14. General structure of a lipoprotein particle.
Very low density lipoproteins (VLDL) are made in the liver and are released into the circulation. As triglyceride is removed, the particle becomes the more dense intermediate density lipoprotein (1DL). Low density lipoprotein (LDL) is formed from the metabolism of IDL and VLDL. It is rich in cholesterol. The major apoprotein is B-100. This particle can deliver cholesterol to the vascular tissues. It is cleared from the plasma by Table 2.4. Different types of lipoproteins
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the LDL receptor which is highly expressed in the liver. LDL levels correlate positively with vascular disease such as ischaemic heart disease. High density lipoprotein (HDL) is made in the liver and intestine. The major apoproteins in descending order are A-I, A-II, D, C-III, C-I and E. It transports cholesterol from peripheral tissues to the liver for disposal. HDL levels correlate inversely with ischaemic vascular disease. Apoproteins are essential to lipoprotein metabolism. Apart from an important structural role they can also act as enzyme activators and receptor ligands. Apo B-100 is recognised by the LDL receptor Apo C-II, which is present in chylomicrons and VLDL, activates LPL Apo E is the recognition protein for the liver chylomicron remnant receptor. There are 3 isoforms of ApoE, based on the amino acids that occupy positions 112 and 158 in the molecule E-2 (cys; cys), E-3 (cys; arg) and E-4 (arg; arg). The most common phenotype is E-3/E-3. E-2 has lower affinity for the chylomicron remnant receptor. Individuals homozygous for E-2 have hyperlipidemia and premature vascular disease. Apoprotein (a) is part of lipoprotein (a) in which it is linked by a disulphide bond to Apo-100 forming an LDL-like particle. There is a positive relationship between Lp(a) and vascular disease. Lipid synthesis
Cholesterol Most of the cholesterol in the body is synthesised internally from acetate in the liver. In the rate-limiting step, ß-hydroxy-ß-methylglutaryl-CoA (HMGCoA) is converted to mevalonate, a reaction catalysed by the rate-limiting enzyme HMG-CoA reductase. Cholesterol inhibits HMG-CoA reductase.
Fatty acids Fatty acid synthesis is a complex reaction in which the carbon chain is sequentially lengthened by 2 carbon units. The reaction is catalysed by a large (240,000 micron) enzyme called fatty acid synthase. This protein has 7 active sites and forms fatty acid from acetyl CoA and malonyl CoA. Throughout the lengthening procedure, the nascent fatty acid remains covalently bound to the enzyme. Malonyl CoA is formed from acetyl CoA and bicarbonate, a process catalysed by the enzyme acetyl CoA carboxylase. This reaction is ratelimiting and hence is a site of regulation. Palmitoyl-CoA, the end product, is an inhibitor of the enzyme; furthermore glucagon and adrenaline trigger phosphorylation and consequent inactivation of this enzyme. Citrate is a powerful allosteric stimulator.
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Proteins Metabolism of amino acids derived either from proteins in the diet or from breakdown of intracellular proteins can contribute energy. The amount of energy derived from amino acids depends on the metabolic situation of the animal. In carnivores as much as 90% of all energy needs are derived from amino acids, whereas in herbivores the fraction is very small. All amino acids contain an amino group which must be removed before the carbon component of the amino acid can be further used. In this process, which occurs in liver cytosol, the amino groups are first transferred to aketogluterate to form glutamate and a-keto acid. The a-keto acid can enter the citric acid cycle where it can be metabolised to CO2 and water or can enter the gluconeogenic pathway to be converted to glucose. The glutamate enters liver mitochondria where it undergoes oxidative deamination to produce a-ketogluterate and ammonia (NH4+). In liver mitochondria, ammonia is converted to urea in the urea cycle, the urea then being excreted by way of the kidney. Ammonia formed in other tissues is transported to the liver by L-glutamine (made by adding ammonia to L-glutamate) or as the amino group of alanine (made by adding ammonia to pyruvate). The deamination of alanine yields pyruvate, which enters the gluconeogenic pathway, completing a glucose-alanine cycle.
Practical exercises It is important for any scientist to have a clear understanding of the techniques used in his or her field of research. In this section, some commonly used methods in metabolic research are briefly described in sufficient detail to allow the student to grasp the important principles of the methods. References are provided to allow a more thorough exploration of a particular technique if required. To place these methods in a realistic context, a scientific problem is posed.
Y
ou are the scientist in charge of a laboratory owned by a pharmaceutical com pany that is searching for new hypoglycaemic drugs. A promising compound is brought to you to unravel its mode of action. You administer it orally to rats and blood glucose levels fall significantly. How can you work out its mode of action? You postulate that this compound has insulin-like activity and thus may stimulate glucose transport in insulin-sensitive tissues. You decide to test this by measuring glucose transport in adipose tissue in vitro.
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Measurement of glucose transport in vitro 1. Harvest epididymal fat pads from several 150–200 g male rats. 2. Isolate individual adipocytes using collagenase digestion. 3. After resting them for 30 minutes, divide isolated adipocytes into 10 aliquots taking care to use plastic containers or siliconised glass. 4. Divide the aliquots into two groups of five. 5. Pre-incubate for 30 minutes one group with 0, 0.1, 1.0, 10.0 and 100 nM insulin and the other set with 0, 0.1, 1.0, 10.0 and 100 ng/mL of the unknown compound. These concentrations were based on the dose that caused hypoglycaemia when injected in rats. 6. Add to each tube 0.25 µCi of 14C-2 deoxyglucose. (This compound is phosphorylated by hexokinase but cannot be further metabolised, thus trapping radioactively labelled glucose inside cells. 7. Incubate for a further 10 minutes. 8. Spin each aliquot through oil to separate fat cells from buffer. 9. Count the radioactivity present in the adipocytes floating on the oil in a ß counter. The result depicted in Figure 2.15 was obtained Insulin had the expected effect of stimulating 2-deoxyglucose entry into the cells in a dose-dependent manner, but the unknown compound had no such action. You are disappointed but figure that there are several possible explanations for the lack of apparent effect on glucose transport. 1. The compound needs to be processed in vivo before it becomes active. 2. The compound lowers blood glucose predominantly by lowering endogenous glucose production. 3. The compound does not work on adipose tissue but increases glucose transport in other tissues.
Figure 2.15. Glucose uptake into fat cells in response to insulin and to the unknown hypoglycaemic compound.
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How are you going to test these various possibilities? The first two can be tested by measuring the changes in flux rate of glucose (i.e., the rate of appearance and disappearance of glucose) in vivo.
Measurement of glucose flux Measurement of basal glucose kinetics
The ability to measure the rate of metabolite turnover is an important tool in understanding its metabolism. Radioactive and stable tracers have been used for many years to study the turnover of metabolites. There is general agreement that tracer methods give meaningrul results when employed during steady-state conditions. On the other hand, the difficulty in applying simplified mathematical models for the complex non-steady-state glucose system have led to the development of many different models. The use of non-steady-state kinetics will not be described here. Further information can be obtained from the References and Reviews. Steady-state kinetics
In 1950 Feller and colleagues attempted to measure glucose turnover in resting fasting rats using a single injection of 14C-glucose. However, to calculate glucose turnover with this method, it was necessary to assume instant mixing of the injected tracer, an inaccurate assumption which led to inaccurate results. In 1954 Searle and co-workers removed the need to assume instant mixing by combining the single injection with a constant infusion of 14C-glucose. This is the method (with some refinements) that is most widely used currently. In a resting, fasting rat (or human) glucose appearance is constant and is due to the release of glucose from the liver and to a lesser extent the kidney. Over a 2-hour period plasma glucose remains stable. Under these circumstances if a tracer of glucose is infused at a constant rate for a sufficient length of time, when steady-state levels of tracer are reached it is true that the ratio of the rate of infusion of tracer over the rate of endogenous glucose appearance equals the ratio of tracer versus tracee concentration or (1)
where
F is the rate of tracer infusion Ra is the rate of appearance of glucose G* is the concentration of glucose tracer G is the concentration of unlabelled glucose
The ratio of tracer to tracee is known as the specific activity (SA). Thus the formula can be rewritten as
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Ra = F/SA
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(2)
Of the above parameters F, G* and G can all be directly measured while Ra is the unknown quantity. If blood glucose level is constant then the rate of appearance must equal the rate of disappearance or Ra = Rd
(3)
Since glucose transport is by facilitative diffusion (see above), it occurs along a concentration gradient, thus Rd will be proportional to blood glucose. If it is necessary to compensate for the mass action of glucose, the metabolic clearance rate can be calculated by dividing the rate of glucose disappearance by the prevailing plasma glucose concentration or MCR = Rd/G
(4)
It is instructive to consider the units for each of the parameters calculated above. Thus in the second equation Ra=F/SA the units are
The units for Ra are therefore µmol/min. The units for glucose clearance are mL/min. Choice of tracer
An ideal tracer must be handled by the body in exactly the same way as the tracee. To prevent recycling of label, the ideal tracer is one in which the label is lost irreversibly. Tritium is such a tracer because glucose loses hydrogen to body water. While water can contribute hydrogen to glucose the dilution of tritiumlabelled water is so great that there is insignificant return of label to glucose. The tritium atom can be placed on different carbons. Generally carbon 3 or 6 are labelled. Recycling is least with the label on carbon 6, but charged labelled metabolites (lactate and pyruvate) are produced which must be removed prior to measuring specific activity as described below. To test the novel hypoglycaemic compound in rats, the following method is used. 1. 2. 3. 4.
Catheters are placed in the right jugular vein and left carotid artery of rats and exteriorised on the back of the neck. The rats are given one week to recover from surgery and are studied when they are gaining weight at a normal rate. On the day of the experiment, overnight fasted animals are attached via their catheters to an infusion and a sampling pump. After taking a basal blood sample, a primed constant infusion of [3H]-6 glucose is begun and maintained constant for the next 120 minutes.
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5. At 100, 110 and 120 minutes blood is taken to measure the concentration of [3H]-6 glucose and unlabelled glucose. This will allow calculation of basal rates of glucose turnover. 6. Immediately after the 120 minute sample an infusion of either insulin or the test compound is started at a constant rate. Glucose clamp procedure
7. Both compounds would cause blood glucose levels to fall with a consequent release of counter-regulatory hormones which would complicate the experiment To prevent this, glucose is infused at a rate sufficient to prevent glucose from falling below the basal level as determined by glucose measurements performed at 10-minute intervals. 8. When steady state is reached, three blood samples are taken at 10minute intervals for measurement of [3H]-6 glucose infusion rate. The catheters are then disconnected from the rat Tracer infusion rate is measured accurately by collecting in triplicate 3 five-minute samples in scintillation vials. In 3 preweighed tubes the flow rate of the glucose infusate is measured in triplicate to calculate the appearance rate of exogenous glucose. This completes the experiment. 9. The blood is centrifuged and plasma separated and stored at -20°C prior to assay. 10. To process the plasma for measurement of labelled glucose, 50µL of plasma from each sample is deproteinised with the addition of 50µL of 0.3 M BaOH and 50µL of 0.3M ZnSO4. 11. The mixture is vortexed and then centrifuged. An aliquot of the supernatant is passed down an anion exchange resin (Ag2X-8) to remove labelled metabolites of glucose such as lactate and pyruvate. 12. The eluent from the column is dehydrated to remove tritiated water. The dried residue is resuspended in distilled water and scintillant is added. The sample is counted in a ß counter, ensuring that the infusate and the samples are counted at the same time. We now have available the infusion rate of tracer [F], (dpm/min), the concentration of plasma glucose [G] (µmol/L), and the dpm of plasma [3H]6 glucose [G*] (dpm/mL). If this information is put in formula 2 above, the turnover rate of glucose can be calculated. It can easily be appreciated that if the compound lowers blood glucose by inhibiting endogenous glucose production, the replacement of blood glucose by exogenous glucose would mean that the ratio of [G*]/[G] would not change. Thus the measured total Ra would be the same as basal. If the infusion rate of unlabelled glucose is now subtracted from the measured total Ra, the calculated endogenous glucose release would be lower than basal by the amount of glucose infused exogenously.
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If on the other hand the compound lowers blood glucose by stimulating glucose uptake, the ratio of [G*]/[G] would fall. The uptake of both [G] and [G*] would be stimulated equally, but the infusion of unlabelled glucose would mean that the [G*] would fall whereas the [G] would not Thus specific activity would fell. Equation 2 (F/SA) would therefore give a large number indicating a higher turnover, that is, the disappearance rat of glucose (Rd) is high while the high Ra is due to endogenous glucose production plus the exogenously infused glucose. You perform the experiment and find that while insulin appears to lower blood glucose both by inhibiting endogenous glucose production and stimulating glucose uptake, the new compound only works by stimulating glucose uptake (Figure 2.16). The data from the two experiments could be interpreted as showing that the compound needs to be metabolised in vivo before it becomes active, or that it stimulates glucose uptake in muscle but not in fat tissue. This could be tested 2 ways; measuring glucose transport in muscle strips in vitro, using a procedure described above for fat cells, or glucose transport into individual cells can be measured in the intact animal in vivo. You decide on the second course of action because you want to investigate glucose transport in a variety of tissues. To perform this experiment the following procedure is followed. 1.
Rats are prepared as above. Instead of infusing [3H]-6 glucose, a bolus of either [3H] or [14C] labelled 2-deoxyglucose is given intravenously to the animal. Blood is taken at frequent intervals (2, 5, 10, 15, 30 & 45 minutes) to measure the tracer level in blood. Immediately after, the
Figure 2.16. Endogenous glucose production (left) and glucose uptake (right) measured in the basal state and in response to insulin or the unknown compound (UC).
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Figure 2.17. 2-Deoxyglucose uptake in fat, muscle and brain in response to insulin or the unknown compound administered in vivo.
2.
3.
animal is killed with an overdose of anaesthetic and various tissues are sampled. This technique relies on the fact that 2-deoxyglucose is trapped in the tissues as 2-deoxyglucose-6 phosphate with the exception of the liver and kidney which contain glucose-6 phosphatase and can dephosphorylate it, allowing it to be transported out of the cell again. To calculate tissue uptake, the disappearance rate of tracer from the blood is calculated as is the trapped 2-deoxyglucose in individual tissues (Kraegen, 1985). Figure 2.17 illustrates the data obtained from such an experiment
The conclusion is that the unknown compound stimulates glucose transport in muscle and fat tissues, but must be modified in vivo to function, thus explaining the negative result in the first experiment.
References and reviews Glucose homeostnsis
Mueckler M. Facilitative glucose transporters. EurJ Biochem 1994 219:713–25 Thorens B. Glucose transporters in the regulation of intestinal, renal, and liver glucose fluxes. Am J Physiol 1996; 270: G541–53
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Carbohydrate digestion
Lentze MJ. Molecular and cellular aspects of hydrolysis and absorption. Am J Clin Nutr 1995; 61(4 Suppl): 946–51S Regulation of glucose homeostasis
Kasuga M. Role of PI3-kinase and SH-PTP2 in insulin action. Diabet Med 1996; 13(Suppl6):S87–9 Rother KI, Imai Y, Caruso M, Beguinot F, Formisano P, Accili D. Evidence that IRS-2 phosphorylation is required for insulin action in hepatocytes. J Biol Chem 1998; 273:17491–7 Insulin action
White MF. The insulin signalling system and the IRS proteins. Diabetologia 1997; 40(Suppl2):S2–17 Techniques in metabolic research
Bergman RN, Phillips LS, Cobelli C. Physiologic evaluation of factors controlling glucose tolerance in man measurement of insulin sensitivity and beta-cell glucose sensitivity from the response to intravenous glucose. J Clin Invest 1981; 68: 1456–67 DeFronzo RA, Tobin JD, Andres R. Glucose clamp technique: a method for quantifying insulin secretion and resistance. Am J Physiol 1979; 237: E214–23 Feller DD, Strisower EH, Chaikoff IL. Turnover and oxidation of body glucose in normal and alloxan-diabetic rats. J Bibl Chem 1950; 187:571–88 Kraegen EW, James DE, Jenkins AB, Chisholm DJ. Dose-response curves for in vivo insulin sensitivity in individual tissues in rats. Am J Physiol 1985; 248: E353–62 Searle GL, Strisower EH, Chaikoff IL. Glucose pool and glucose space in the normal and diabetic dog. Am J Physiol 1954; 176:190–4
3. Type 1 Diabetes
Definition Type 1 diabetes mellitus is that form of diabetes caused by the destruction of the insulin producing (ß) cells of the pancreas, which are located in islets or islands throughout that organ (Figure 1.1). The process of destruction can occur over a period of several years, this phase being called the prediabetic phase (Figure 3.1). Eventually, when sufficient insulin-producing cells are destroyed, production of insulin is inadequate and this results in hyperglycaemia.
Pathogenesis What initiates the prediabetic phase is unknown, but is thought to be an environmental agent such as a toxin or virus. Thereafter, cells of the immune system, including lymphocytes and macrophages, are attracted to the islets and perpetuate the destructive process. Because these cells are the person’s own immune cells, the process is called an autoimmune one. The types of lymphocytes involved are CD4 and CD8, which are traditionally labelled as helper/inducer and suppressor/ cytotoxic cells respectively. The subset of CD4 lymphocytes involved is those that produce the cytokines interleukin 2, tumour necrosis factor and interferon ?, and this is called the Th (T helper) 1 subset The mechanism of destruction of ß cells by autoimmune cells is both direct and indirect. Direct, by cell to cell interaction: (a) granzyme and perform are released from the immune cell to lyse the ß cell membrane; (b) induction of the antigen FAS on the ß cell with attraction of toxic immune cells which express FAS ligand. Indirect, by production of toxic agents. These include: (a) the cytokines interleukin 1ß, tumour necrosis factor, and interferon g from immune cells;
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Figure 3.1. An average time course for autoimmune destruction of ß cells in Type 1 diabetes. The stage from commencement of loss of ß cells until the clinical onset of diabetes is called the prediabetic phase. During this time blood glucose levels remain normal.
(b) nitric oxide, from either immune cells, such as macrophages, or ß cells as a result of cytokine stimulation; (c) production of free radicles. It is possible for immune cells to be adjacent to islets for a long period of time before ß cell destruction begins. However, it is the ß cell and not other endocrine cells, which are sensitive to immune damage.
Prediabetes The most common markers of the prediabetic period are antibodies to: islet cells, the best being to a surface antigen (ICA 512); insulin; and glutamic acid decarboxykse. These antibodies do not cause destruction of the ß cells. Blood glucose levels are normal during this phase. Only when 80% or more of the islets are destroyed is there insufficient insulin production and resultant elevation of blood glucose levels. Several trials are on-going to prevent the progressive destruction of ß cells in the prediabetic phase, with the results expected to be available in this year. Methods being trialled include: • oral, parenteral and intranasal administration of insulin
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• administration of nicotinamide • vaccination with BCG (used for immunization against tuberculosis) or against the microbial agent which causes Q fever • refraining from giving cow’s milk to children
Genetics Type 1 diabetes is a genetic disorder and runs in families. There is a 5% chance of developing Type 1 diabetes if a first degree relative has the disorder. Type 1 diabetes is not the result of a single gene defect, rather there are at least 17 loci on 10 different chromosomes which are involved in humans. The genes most frequently associated with Type 1 diabetes in western countries are the class II histocompatibility genes DR3 and DR4, which are located on the short arm of chromosome 6. In Japan the association is with DR4 and DRw9 and in China with DR3 and DRw9. Another HLA class II molecule, DQß, is also involved with lack of aspartate on position 57 conferring susceptibility to Type 1 diabetes. The location of all genes responsible for type 1 diabetes appears in Table 3.1.
Transplantation Despite the best of endeavours it is often difficult to maintain blood glucose levels within the physiological range by the injection of insulin and, as a result, diabetic complications will develop. An alternative strategy to achieve the desired goal is to replace the missing ß cells with new ones from another Table 3.1. Human genes identified as being responsible for Type 1 diabetes. IDDM1 in the major histocompatibility complex on the short arm of chromosome 6 accounts for 35% of the observed familial clustering of type 1 diabetes. Each of the loci accounts for a smaller percentage.
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person. This can be achieved by transplanting: the whole pancreas with its blood supply; the islets of Langerhans; or 1 mm3 expiants of pancreatic tissue. It is possible to use expiants only from fetal pancreas in which the exocrine system containing enzymes, which digest tissue, is not fully developed.
Whole pancreas The pancreas is obtained with consent from young people who have died suddenly, for example, because of a motor vehicle accident Such transplants (Figure 3.2) have been carried out systematically since 1966. However, it was only in the middle of the 1980s that many of the initial problems associated with whole organ transplants were resolved. Today, this method of transplantation is no longer experimental, especially for those who are also receiving a kidney, with recipients being able to cease injections of insulin within days of receiving the graft. Between 1996 and 1999 1200 pancreatic transplants were carried out annually worldwide. Survival of pancreas transplants is now similar to that achieved with other solid organ transplants, such as kidney and heart. One year after transplantation, 76% of grafts are still functioning with recipients not requiring injections of insulin, while at the end of five years the percentage is 64%. Patient survival at these times is 91% and 80% respectively.
Figure 3.2. Pictorial representation of a whole organ pancreas transplant. Note the pancreatic duct is attached to the bladder via a patch of small bowel to allow drainage of exocrine secretions. The pancreatic artery and vein are anastomosed to the iliac vessels in the groin. A kidney transplant is also depicted. (Reproduced with permission of Dr R Allen, Westmead Hospital, Sydney.)
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A side effect of pancreatic transplantation, as with all forms of organ transplantation, is the need to take drugs to block activation of cells of the immune system and so prevent rejection of the transplant. The more traditional drugs that have been used are cyclosporin, azathioprine and prednisolone. More recently, these agents have been supplemented by FK506, mycophenolate mofetil, rapamycin, deoxyspergualin, anti-lymphocyte globulin, and monoclonal antibodies. Anti-CD3 is the main human antibody available; anti-CD4, anti-interleukin 2 receptor and most recently anti-CD40 ligand are also being trialled. All anti-rejection drugs have side effects, for example, increased incidence of infection and development of cancer. Attempts at transplanting the vascularized pancreas into patients without advanced renal complications have not been so successful in most centres, perhaps because it is difficult to detect pancreatic rejection in these recipients before it is too late to salvage the organ. As well, risk:benefit analysis of substituting immunosuppressive drugs for insulin for most people in this situation is questionable. Justification is easier in those people with debilitating autonomie neuropathy, for example, with frequent vomiting and/or diarrhoea, and those with frequent hypoglycaemic attacks that are not recognised or difficult to anticipate. Following transplantation of both pancreas and kidney, it can be expected that over the ensuing years, diabetic complications affecting the transplanted
Figure 3.3. Islets from adult rat pancreas. Their average diameter is 150µm.
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kidney will be less likely, and that there will be some, although minimal, improvement in loss of feeling in the lower limbs. Some improvement in the eyesight can be expected in a small number of people, especially in those who do not have advanced eye disease at the time of receiving a pancreatic transplant. The most dramatic improvement can be observed in those who receive a pancreas because of autonomie neuropathy or incapacitating hypoglycaemic episodes, with recipients becoming asymptomatic.
Islets of Langerhans Transplantation of the islets ofLangerhans (Figure 3.3), which comprise 1% of the organ, began in the 1970s, with successful normalization of blood glucose levels in diabetic rodents with as few as 500 islets. It was not until 1989 that the same success was achieved in humans with Type 1 diabetes, being mainly because of technical improvement in separating hundreds of thousands of islets from the donor human pancreas. The enzymes available to digest pancreatic tissue for separation of islets are collagenase, an impure preparation, and liberase, a purer preparation, recently produced (its usefulness is still being tested). Most diabetic recipients of islet cells have had advanced complications, previously having received a kidney transplant and are already taking antirejection drugs. The success rate with this form of transplantation in humans has been low, the major reasons for this being rejection of the graft both because of autoimmune lysis of ß cells and allograft destruction of the entire graft Between 1990 and 1998, 267 recipients in 25 institutions had been transplanted with islets, often from more than one pancreas. Of the recipients, 33 became insulin independent one week after transplantation, but only 20 still did not require exogenous insulin at 1 year. The longest duration of function has been 5.8 years. Most successful grafts were by injection into the liver. A better success rate has been achieved with adult islets allografted into recipients who became diabetic not because of autoimmune ß cell destruction but because of total pancreatectomy carried out as therapeutic treatment of a pancreatic malignancy. Nine of the 15 recipients became insulinindependent, with 6 still not requiring exogenous insulin at 1 year. One person still did not require injections of insulin at 5 years. The most successful form of adult human islet transplantation has been achieved with islets separated from the pancreas of the recipient; thus obviating the need for immunosuppressive agents. Pancreatectomy has been carried out in these people because of chronic pancreatitis. Insulin independence has been achieved in 77% of recipients with 67% still not requiring insulin at 1 year; one recipient still does not require exogenous insulin after 13 years.
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Figure 3.4. Islet-like cell clusters from fetal pig pancreas. Their average diameter is 100 µm.
Fetal pancreatic tissue Fetal pancreatic tissue has advantages over that from adults because the proportion of endocrine tissue is greater and the capacity of the tissue to proliferate is greater. Its disadvantages are that the ß cell is immature and is unable to secrete insulin when exposed to glucose, and that the number of ß cells and hence the amount of insulin produced is less than in the adult tissue. Although the immune system in the fetus is not fully developed, this does not mean that fetal pancreatic tissue is not immunogenic. Indeed, this tissue will be rejected when transplanted unless strategies are introduced to suppress the immune system. Fetal pancreatic tissue is capable of normalizing blood glucose levels when transplanted into diabetic rodents, but it takes much longer to achieve this goal than when adult islets are grafted: 1–5 months versus 1–3 days. The extra time is required for the tissue to increase in size and mature. Human fetal pancreatic tissue has been transplanted into diabetic humans taking anti-rejection drugs with survival of some of the graft for one year and some graft function. Insulin independence has not been achieved. Fetal pancreatic tissue is transplanted as: •
1 mm3 slices of tissue
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Figure 3.5. The three types of encapsulation devices for immunoprotecting islets and islet-like cell clusters when transplanted. They are (from top to bottom) microcapsules, a vascular device, and macrocapsules.
•
•
islet-like cell clusters (ICCs), also called proislets (Figure 3.4). They are formed by digesting the pancreas of humans as well as large animals, such as pigs and sheep, with the enzymes collagenase or liberase. The percentage of ß cells in these clusters may be as low as 5%, with undifferentiated cells being in the majority. After the clusters are transplanted, these precursor cells develop into ß cells islets, but this applies mainly to rodents. The dominant cell is the ß cell, just as it is in the adult pancreatic islet.
Encapsulation of Islets Attempts are being made to prevent rejection of non-vascularized pancreatic tissue by encapsulating it The membranes in such capsules have pores of a size sufficient to allow nutrients in and insulin out, but too small to allow entry of antibodies or cells of the immune system to destroy the encapsulated cells. There are three types of encapsulation devices (Figure 3.5). 1. 2.
Microencapsulation, with one islet surrounded by a capsule. These are usually made of some type of alginate. Vascular devices, which connect to an artery and vein, thus allowing blood to flow through the centre of the device. The islets are placed in a mesh around this central lumen.
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Macrocapsules, such as hollow fibres, macrobeads and diffusion chambers. These devices contain large numbers of islets.
Use of animal pancreatic tissue Lack of supply of human pancreatic tissue has limited the ability to carry out pancreatic transplants. Thus, for example, the average waiting time for a pancreas in Australia is 18 months from the time a person is accepted into the transplant program. One way of attempting to overcome this shortage is to use the pancreas of animals. The pig is the animal most favoured for this purpose because of physiological and biochemical similarities with humans. Similarities include the same range of blood glucose levels and a similar gastrointestinal tract with one stomach and fermentation in the hind gut. Pig insulin is as bioactive as human insulin, and is only one amino acid different. Transplantation of pig fetal pancreatic tissue will reverse diabetes in rodents, pigs and monkeys. Reversal of diabetes in humans has not yet been demonstrated although the tissue will survive if anti-rejection drugs are administered. Transplantation of animal tissue into immunosuppressed humans has disadvantages. It will be rejected. There are two types of rejection, acute and chronic. Acute rejection, with death of the graft within minutes to an hour, is caused by the presence of preformed antibodies, usually IgM in type, which react against endothelial cells in the graft and require complement to complete cell lysis. This form of rejection occurs only when tissue is transplanted across species (xenotransplantation); it does not occur when tissue is grafted between members of the same species (allotransplantation). This process particularly affects organ xenotransplants. Whether nonvascularized tissue, such as islets and tissue slices, which have few endothelial cells, are affected has yet to be firmly established. Transgenic pigs are being bred to overcome the problem of acute rejection of foreign organs. These animals are created by the insertion of the cDNA for inhibitors of human complement into the fertilized egg. Because of this strategy, lysis of endothelial cells cannot occur when organs from these animals are transplanted across species. The first modern human xenotransplant of a pig organ, whether pancreas or otherwise, has yet to be performed. Acute cellular rejection occurs during the first 3 days after transplantation and involves natural killer cells and macrophages. This form of rejection does not occur in allografts. Chronic rejection, over days and weeks, is caused by the infiltration of the graft by cells of the immune system, especially Th2 type lymphocytes. Cellular infiltration of grafts occurs equally in xenotransplantation and allotransplantation; in the latter case the type of lymphocytes involved are Th1.
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Infection of the recipient may also occur. If there is transmission of an agent from the animal, such as a retrovirus akin to the human immunodeficiency virus, it may cause a public health problem. At present there is no solid evidence that such an event is likely. Cancer in the recipient, because the immune system is suppressed, is another risk. Guidelines for the transplantation of animal tissues into humans are being developed. Some of the key points are: • pigs are acceptable as donors, but they should come from a closed colony and be serologically defined • tissue to be transplanted should be sterile • blood and tissue from the donor animal and blood from the recipient should be archived • the xenotransplant team should consist of an infectious diseases physician, veterinarian, transplant immunologist, hospital infection control specialist, and the director of the hospital clinical microbiology laboratory, in addition to a transplant surgeon • recipients should be advised of the possibility of transmission of unknown infectious agents and give informed consent • approval is required by the Human Ethics, Animal Care and Occupational Safety Committees of the institution where the transplant will take place
Gene therapy An alternative to using islets from dead people or animals is the genetic engineering of ß cells. There are three types. 1. Engineering of animal ß cell lines to act like normal ß cells. This requires insertion of genes for human insulin, glucokinase, and glucose transporter 2. Transplantation of these cells will most likely require their encapsulation to prevent rejection. Alternatively, they may be transfected with genes to prevent this, for example, to prevent transport of the class I major histocompatibility antigen from reaching the cell surface. 2. Engineering of non-ß cells to synthesize, store and secrete insulin. The hepatocyte is the cell most favoured at present for this role, since it possesses a number of features in common with ß cells (Figure 3.6). These include: (a) glucokinase, as compared to hexokinase, which all other cells possess, to phosphorylate glucose (b) glucose transporter 2 (c) ability to convert proinsulin to insulin.
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Figure 3.6. Pictorial representation of the genetic manipulation of liver cells to change their function and synthesize, store and secrete insulin. For this to be achieved the genes for insulin, under the control of a constitutive promoter (top), and glucose transporter 2 (bottom) are transfected.
The origin of the hepatocytes will be the patients with Type 1 diabetes; hence rejection of the cells should not occur. Other cell types being examined included muscle, haemopoietic, skin and neuroendocrine cells.
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3. Engineering of islet stem cells from the pancreas to become ß cells. The principle is to stimulate pancreatic duct cells, which are the precursors for ß and exocrine cells, to become ß cells. Such cells might be obtained from the pancreas of people with Type 1 diabetes. None of these engineering strategies is yet ready for use in humans. Insertion of genes into cells in order to modulate their function can be carried out using the following techniques: • viral infection, for example, retrovirus, adenovirus, adeno-associated virus • cationic liposomes containing encapsulated naked DNA • direct injection of naked DNA.
Animal models The study of Type 1 diabetes has benefited from there being available animal models of this disorder. The models most commonly used are the non-obese diabetic (NOD) mouse and the BioBreeding (BB) rat. The NOD mouse is more typical of what occurs in Type 1 diabetes in humans, with inflammatory cells destroying the ß cells of the pancreas. Administration of many agents, such as Freund’s adjuvant, oral insulin, vitamin D, and nicotinamide, will prevent the development of hyperglycaemia in these animals. How relevant such treatments are in preventing Type 1 diabetes in humans is being investigated (see prediabetes section in this Chapter). Type 1 diabetes does exist sporadically in other species, such as chimpanzees and cynomologus monkeys. The more common form of diabetes which exists in animals such as dogs and cats is, however, type 2 diabetes. Transgenic mice have been created examining the function of islets in the overexpression or absence of a variety of agents. Examples of mice overexpressing an antigen are FAS ligand, and interferon-?. Knockout mice have been created lacking key components of the ß cell including glucokinase, and the glucose transporter 2, as well as cytokines involved in autoimmune destruction of ß cells, such as interleukin 2.
Chemical models The most common mechanism of inducing Type 1 diabetes in animals for research purposes is by the administration of a ß cell toxin. Pancreatectomy can also be used, but this procedure also results in the removal of the exocrine tissue with its own morbidity and mortality because of the absence of digestive enzymes.
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The two ß cell toxins most widely used to induce Type 1 diabetes are streptozotocin and alloxan. Animals in which Type 1 diabetes has been induced by this means include mice, rats, pigs, monkeys, and baboons. Other agents which can kill ß cells, but are usually restricted to in vitro use, are pentamidine and vacor.
Research questions and directions The following is a list of some questions being addressed by the research community. They have been arranged according to the sections of this Chapter.
Pathogenesis • What is the initiating event that precipitates the autoimmune destruction of ß cells? • Why are human ß cells comparatively resistant to the destructive effect of autoimmune cytokines?
Prediabetes • What treatment strategy can be employed to prevent the progressive destruction of ß cells that occurs in the prediabetic state, so as to prevent the clinical onset of Type 1 diabetes?
Genetics • Full characterization of the genetic loci responsible for Type 1 diabetes.
Transplantation Whole pancreas
• What is the safest and most reliable means of draining exocrine secretions from the grafted pancreas? • Can whole pancreas be safely and reliably transplanted into people with Type 1 diabetes who do not have overt or incipient renal failure? Islets of Langerhans
• What non-immune mechanisms are responsible for failure of islets to function immediately after transplantation?
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• What strategies can be implemented to improve the function of islet grafts long term so as to match the success rates of whole pancreas transplants? Fetal pancreatic tissue
• What strategy needs to be implemented to allow fetal pancreatic tissue to function adequately to normalize blood glucose levels in a human? • What growth factors need to be applied to fetal ß cells so that they will rapidly mature in their ability to secrete insulin when exposed to glucose? Encapsulation of islets
• Which is the best form of encapsulation which will consistently and reliably allow survival and function long term of ß cells when transplanted? Use of animal pancreatic tissue
• Do antibody-mediated and acute cellular rejection of pig islets or ICCs occur when they are transplanted into humans? • Are pig retroviruses likely to be pathogenic if transmitted to immunosuppressed human recipients of pig tissue?
Gene therapy • Can genetic strategies be found to prevent the rejection of ß cell lines or islets when transplanted, for example, by their transfection with immunoprotective agents such as FAS ligand? • Can primary non-ß cells, such as hepatocytes, be genetically modified to store insulin once it is synthesized, and to release it in a physiological manner? • Can techniques be developed to characterize and isolate pancreatic stem cells from which ß cells develop?
Practical exercises Separation of rat islets (Figure 3.3)
1. 2. 3.
Anaesthetize an adult rat with, for example, 50 mg/kg pentobarbitone by intraperitoneal injection. Open the peritoneal cavity and insert a butterfly needle into the pancreatic duct Inject 1.5 mg/mL collagenase P (Boehringer Mannheim, Mannheim, Germany) in 10 mL Hank’s Balanced Salt Solution [HBSS] (without Ca2+ and Mg2+) into the pancreatic duct
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4. 5. 6.
7. 8. 9. 10. 11. 12. 13. 14.
15.
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Remove the pancreas and incubate it in a shaking water bath for 8 minutes at 37°C. Discard half the supernatant, leaving 5 mL covering the pancreatic digest Add 5 mL of 3 mg/mL collagenase P and incubate in the shaking water bath for 4–8 minutes at half the speed used for the initial digestion (step 4). Stop the digestion when islets appear to have formed by adding an excess volume of HBSS (with Ca2+ and Mg2+). Centrifuge at 1200 rpm for 4 minutes and discard the supernatant Wash twice with HBSS and centrifuge as in step 8. Add ice cold Kreb’s Ringer Phosphate Buffered Saline [KRPBS] to the pellet so that the total volume is 2 mL Add 14 mL of ice cold ficoll of density 1.107 g/mL and mix. Layer 5–10 mL of ice cold KRPBS on top of the ficoll. Centrifuge at 3000 rpm for 25 minutes at 4°C. Islets should be at the interphase. Suck these off and wash twice with HBSS (with Ca2+ and Mg2+) centrifuging at 1200 rpm for 4 minutes on each occasion. Wash islets twice with KRPBS. Islets are now ready for use.
Separation of pig fetal islet-like cell clusters (Figure 3.4) 1. 2. 2. 3. 4.
5. 6. 7. 8.
Obtain pig fetus from the uterus of pregnant sow that has been killed. Remove pancreas aseptically from pig fetus. Wash in 0.9% saline. Mince pancreas into fragments of =1 mm3 with scissors. Digest pancreatic fragments with 3 mg/mL collagenase P in 20 mL of Dulbecco’s Phosphate Buffered Saline supplemented with 2.24 g/L sodium bicarbonate and 4.77 g/LHEPES pH 7.4 [PBS] at 37°C for 14– 15 minutes. Add cold PBS to stop the process. Too long a digestion will result in the formation of single cells; too short, clumps of undigested tissue. Centrifuge at 1200 rpm and replace the supernatant with fresh PBS. Centrifuge again at 1200 rpm and replace supernatant with RPMI culture medium supplemented with 10% fetal calf or human serum. Place in non-attachable Petri dishes and allow to incubate at 5% CO2/ air for 3 days. During this time the ICCs round up and are suitable to be used for research purposes.
Measurement of human insulin by dioimmunoassay 1.
Prepare human insulin standards using the following concentrations: 800, 400, 200, 100, 50, 25, 12.5, 6.25 and 0 µU/mL.
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2.
Take 50 µL of each of these concentrations and place in plastic test tubes in triplicate. 3. Take 50 µL of each sample in duplicate 4. Place 100 µL of insulin assay buffer (8.18 g/LNaCl, 3.086 g Na2HPO4.2H2O, 18.62 g/Lethylene diaminetetraacetate, 0.1 g/LNaN3, 10 g/L bovine serum albumin; pH 7.6) in each of 3 tubes. These are for determining non-specific binding (NSB). 5. To the insulin standards and samples, but not the NSB tubes, add 50 µL of insulin antibody, the titre used being designed to give 40–50% binding. Shake and incubate at 4°C for 4 hours. 6. To all tubes add 50 µL of 125I-insulin, 10 000 counts per minute, shake and incubate overnight at 4°C. To 3 separate tubes add 50 µL of tracer in order to measure the total count (TC). 7. To all tubes except the TC tubes add 50 µL 15 g/L gamma globulins and shake. 8. To all tubes except the TC tubes then add 1 mL 150 g/L polyethylene glycol. 9. Vortex the tubes and incubate for 15 minutes at 4°C. 10. Centrifuge tubes except TC tubes at 3000 rpm for 20 minutes at 4°C and tip off supernatant. 11. Count the radioactivity in the pellet at the bottom of the tube. 12. Construct a standard curve comparing the concentration of insulin to the degree of binding for each concentration.
12. Read the concentration of insulin in each sample off the curve using the degree of binding for that sample.
References and reviews Pathogenesis
Atkinson MA, Maclaren NK. The pathogenesis of insulin-dependent diabetes mellitus. N Engl J Med 1994; 331:1428–36 Thai A-C, Eisenbarth GS. Natural history of IDDM. Diabetes Rev 1993; 1:1–14 Rossini AA, Greiner DL, Friedman HP, Mordes JP. Immunopathogenesis of diabetes mellitus. Diabetes Rev 1993; 1:43–75 Shehadeh NN, Lafferty KJ. The role of T-cells in the development of autoimmune diabetes. Diabetes Rev 1993; 1:141–51 Rabinovitch A. Roles of cytokines in IDDM pathogenesis and islet ß-cell destruction. Diabetes Rev 1993; 1:215–40
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Skyler JS, Marks JB. Immune intervention in type 1 diabetes mellitus. Diabetes Rev 1993; 1:15–42 Carel JC, Bougneres PF. Treatment of prediabetic patients with insulin: experience and future. European Prediabetes Study Group. Hormone Res. 1996; 45 Suppl 1:44–7 Gale EA. Molecular mechanisms of beta-cell destruction in IDDM: the role of nicotinamide. Hormone Res 1996; 45 Suppl 1:39–43 Genetics
Todd JA, Farrall M. Panning for gold: genomewide scanning in type 1 diabetes. Diabetes Rev 1997; 5:284–91 Aitman TJ. Todd JA. Molecular genetics of diabetes mellitus. Baillieres Clin Endocrinol Metab 1995; 9:631–56 Transplantation
Weir GC, Bonner-Weir S. Perspectives in diabetes: Scientific and political impediments to successful islet transplantation. Diabetes 1997; 46:1247–56 Ricordi C. Human islet cell transplantation: new perspectives for an old challenge. Diabetes Rev 1996; 4:356–69 Bland BJ (ed). International pancreas transplant registry newsletter. 1999; 11 Brendel MD, Hering BJ, Schultz AO, Schultz B, Bretzel RG. International Islet Transplant Registry 1999; 8 Groth CG, Korsgren O, Tibell A, Tollemar J, Möller E, Bolinder J, Östman J, Reinholt FP, Hellerström C, Andersson A. Transplantation of porcine fetal pancreas to diabetic patients. Lancet 1994; 344:1402–4 Tuch BE. Clinical results of transplanting fetal pancreas. In: Fetal tissue transplants in medicine, RG Edwards (ed). Cambridge, UK: Cambridge University Press, 1992, pp 215–37 Lanza RP, Chick WL Immunoisolation: at a turning point Immunology Today 197; 18:135–9 Gene therapy
Docherty K. Editorial review: Gene therapy for diabetes mellitus. Clin Sci 1997; 92: 321–30 Efrat S. Genetic engineering of ß-cells for cell therapy of diabetes: cell growth, function, and immunogenicity. Diabetes Rev 1996; 4:224–34 Simpson AM, Marshall GM, Tuch BE, Maxwell L, Szymanska B, Tu J, Beynon S, Swan MA. Gene therapy of diabetes: Glucose-stimulated insulin secretion in a human hepatoma cell line (HEP G2ins/g). Gene Therapy 1997;4:1202–15
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Animal models
Bowman MA, Leiter EH, Atkinson MA. Prevention of diabetes in the NOD mouse: implications for therapeutic intervention in human disease. Immunology Today l994; 15:115–20 Rohane PW, Fathman CG. Initiation of autoimmunity in NOD mice. Diabetes Rev 1993; 166–73 Jones EM, Sarvetnick NE. Transgenic animal models: insights into the regulation and maintenance of islet cell mass. Diabetes Rev 1996; 4:264–73 Marliss EB, Nakhooda EB, Poussier P, Sima AAF. The diabetic syndrome of the “B B” Wistar rat: possible relevance to type 1 (insulin-dependent) diabetes in man. Diabetologia 182; 22:225–32 Rabinovitch A. Immunoregulatory and cytokine imbalances in the pathogenesis of IDDM. Therapeutic intervention by immunostimulation? Diabetes 1994; 43: 613–21 Chemical models
Kolb H, Kröncke K-D. IDDM: Lessons from the low-dose streptozotocin model in mice. Diabetes Rev 1993; 1:116–26 Gale EA. Molecular mechanisms of beta-cell destruction in IDDM: the role of nicotinamide. Hormone Res 1996; 45 Suppl 1:39–43 Rerup CC. Drugs producing diabetes through damage of the insulin secreting cells. Pharmacol Rev 1970; 22:485–518
4. Type 2 Diabetes
Type 2 or non-insulin dependent diabetes is the most common form of diabetes, accounting for about 85% of all cases. In Type 1 diabetes, severe insulin deficiency (ß cell death) is the cause of the hyperglycaemia. In Type 2 diabetes, a defect in insulin action (insulin resistance) is combined with a more subtle defect in insulin secretion (ß cell glucose blindness) to produce hyperglycaemia. This chapter will discuss the pathophysiology of Type 2 diabetes.
Insulin resistance
Definition Insulin resistance can be said to exist when a normal concentration of insulin elicits a subnormal biological response.
Location Impaired insulin action in diabetes has been demonstrated in muscle, fat and liver tissues. How is insulin resistance measured? Abnormal insulin action has been documented in Type 2 diabetes using a variety of techniques. • Whole body glucose disposal using tracer kinetics and hyperinsulinaemic euglycaemic clamps (see Chapter 2). • Forearm perfusion techniques to measure muscle glucose uptake. • Insulin-stimulated glucose uptake in isolated fat cells (see Chapter 2). • Insulin-stimulated glucose uptake in isolated muscle strips.
Mechanisms Genetic causes
In chapter 2 the steps involved in insulin action were discussed. Many proteins are involved in mediating signal transduction, and malfunction of any is a potential cause of impaired insulin action. Furthermore, genetic defects within the pathways stimulated or inhibited by insulin could also 61
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cause insulin resistance. So far, mutations in the following genes have been reported to be associated with insulin resistance: • • • •
insulin receptor insulin receptor substrate 1 (IRS-1) Type 1 protein phosphatase Glycogen synthase
However, mutations in these genes appear to explain insulin resistance in only a small number of patients. It is becoming increasingly clear that in many cases of Type 2 diabetes, insulin resistance is an acquired defect. There are several mechanisms for secondary causes of insulin resistance. Secondary Causes
Glucose Induced Many previous studies have demonstrated that improving glucose control in diabetic patients by whatever means improves insulin action. These studies have been supplemented by in vitro studies in which it has been shown that skeletal muscle taken from type 2 diabetic patients and incubated with 8 mmol/L glucose shows impaired glucose transport, which can be partially reversed by incubation for 2 hours with 4 mmol/L glucose. Animal studies have confirmed that hyperglycaemia per se can cause defects in insulin action. Rats made hyperglycaemic by partial pancreatectomy develop insulin resistance which can be prevented by reversing the hyperglycaemia (by inducing glycosuria using phlorizin).
Figure 4.1. A schematic diagram of the hexosamine biosynthesis pathway.
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Adipocytes cultured in high glucose for 24 hours develop defects in basal and insulin-stimulated glucose transport if there is insulin in the medium to allow excess glucose entry into the cell. The hexosamine biosynthesis pathway (Figure 4.1) has been proposed as the mechanism by which glucose impairs basal and insulin stimulated glucose transport Glucose enters the glycolytic pathway and is largely converted to pyruvate. A small quantity of fructose-6-phosphate is converted to glucosamine-6-phosphate by the enzyme glutamine-fructose 6 phosphate amidotransferase (GFAT). Giucosamine -6-phosphate is in turn metabolised to UDP-N-acetylglucosamine which is a precursor for the synthesis of glycoproteins, glycolipids and proteoglycans. GFAT catalyses the ratelimiting step for this pathway. The effects of high glucose on glucose transport can be inhibited by azaserine, a competitive inhibitor of GFAT. Activation of protein kinase C (PKC) by hyperglycaemia has also been proposed as a mechanism for impaired insulin action. PKC is known to phosphorylate the insulin receptor, resulting in a decrease in tyrosine kinase activity. PKC activation also impairs basal glucose transport A link between the hexosamine biosynthesis pathway and PKC activation has been demonstrated. Activation of the hexosamine biosynthesis pathway has also been shown to impair insulin-mediated GLUT4 translocation.
Lipid induced Excess fat can cause insulin resistance in both liver and peripheral tissues (muscle). In 1963 Randle first proposed that increased free fatty acid oxidation inhibits glucose oxidation. More recently, it has been shown that free fatty acids induce insulin resistance in humans by reducing glucose transport/ phosphorylation and that this is followed by a reduction in both glycogen synthesis and glucose oxidation. It is possible that increased FFA metabolism also causes insulin resistance by increasing glucose flux through the hexosamine biosynthesis pathway (see above). Free fatty acids have been shown to cause hepatic insulin insensitivity. Studies in mice have shown that excess fat availability as a result of either obesity or a high fat diet increases gluconeogenesis by several mechanisms which include enhancing the activity of pyruvate carboxylase and the protein levels of fructose-1,6-bisphosphatase and increasing the glucose-6phosphatase/glucokinase ratio.
TNFα induced It has been shown in both rodents and man that adipocytes produce tumour necrosis factor alpha (TNF a) and that the amount of TNF a produced is proportional to the severity of the obesity. Reducing TNF a activity in a variety of ways, including TNF a gene deletion, administration of thiazolidinediones or dehydroepiandrosterone improves insulin sensitivity.
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Figure 4.2. Defect In insulin secretion in type 2 diabetes. There is impaired first phase insulin secretion but enhanced basal insulin levels (Left). The ß cells are capable of responding to non-glucose secretagogues, such as arginine (Right). (From Diabetes Care reproduced with permission)
It has been shown that TNF α induces serine phosphorylation of IRS1, converting it into an inhibitor of the IR tyrosine kinase. This effect is mediated via the P55 TNF α receptor, the activation of which is known to produce sphingomyelinase and ceramides. Exogenous addition of these compounds converts IRS1 into an inhibitor of the IR tyrosine kinase in vitro. Surprisingly, deletion of either the p55 and/or the p75 receptor does not ameliorate fat-induced insulin resistance.
Defects in insulin secretion Many subjects with insulin resistance never develop diabetes because the ß cells continue to hypersecrete insulin at a rate that is adequate to normalise blood glucose levels. For diabetes to appear, it is necessary for a defect in insulin secretion to be present in addition to defects in insulin action. The typical defect in Type 2 diabetes is shown in Figure 4.2. The ß cells appear to be blind to glucose but can secrete insulin in response to other secretagogues such as the amino acid arginine or to a sulphonylurea. In some types of diabetes known as maturity onset diabetes of youth (MODY) a genetic defect leading to an abnormalitiy of ß cell function will cause diabetes. The first gene to be identified was that of glucokinase (MODY 2). A defect in glucokinase function may be expected to result in a defect in insulin secretion and possibly in hepatic glucose uptake, since glucokinase is the isoform of hexokinase expressed in liver and ß cell.
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More recently, mutations in hepatic nuclear factors (HNF1a and HNF4 a) have been shown to lead to defects in insulin secretion. Mutations in mitochondrial DNA can cause diabetes due to abnormal insulin secretion. These patients may also have deafness. Defects in insulin secretion can be secondary to either excess fat or hyperglycemia. Both in vitro and in vivo studies have shown that hyperglycemia induces in ß cells the classical defect seen in Type 2 diabetes, i.e., an inability to respond to acute hyperglycemia but near normal response to other secretagogues. The hexosamine biosynthesis pathway has been implicated as the mechanism by which ß cell glucose toxicity occurs. Free fatty acids, that can acutely stimulate insulin secretion, have also been shown to cause a defect in insulin secretion when ß cells are exposed to it for longer periods.
Relative roles of insulin resistance and defects in insulin secretion While both experiments of nature and transgenic animals (see below) have demonstrated that a defect in insulin secretion can on its own cause hyperglycaemia, insulin resistance alone will not result in diabetes. So far, genetic defects that can explain the defect in insulin secretion in common forms of diabetes have not been described. It may be possible that there may be ß cell susceptibility genes that result in ß cell malfunction or even death only if the ß cell is placed under stress such as occurs in the presence of insulin resistance.
Figure 4.3. Positive feedback loop in diabetes. This diagram shows how the existence of glucose toxicity can set up a positive feed back loop and how one defect causing hyperglycemia can secondarily cause another that further elevates the glucose levels.
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The relationship between insulin resistance and defects in insulin secretion in the pathogenesis of Type 2 diabetes is complicated because each defect can cause or aggravate the other as shown in Figure 4.3. One possible scenario for the development of Type 2 diabetes is that obesity, resulting from a genetic predisposition combined with a sedentary lifestyle, causes insulin resistance which in those who inherit ß cell susceptibility genes leads to relative insulin deficiency. The combination of insulin resistance and insulin deficiency results in hyperglycemia.
Type 2 diabetes as a component of the metabolic syndrome (syndrome X) Typical type 2 diabetes is rarely found as an isolated abnormality. Obesity, hypertension, dyslipidaemia, and hyperuricaemia appear to cluster in the same individuals. Insulin resistance may be the underlying cause of the metabolic syndrome. The typical dyslipidaemia of type 2 diabetes is hypertriglyceridaemia and low HDL cholesterol. Mechanisms by which hyperinsulinaemia secondary to insulin resistance can lead to hypertension and hypertriglyceridaemia have been proposed (see DeFronzo 1991 in References and Reviews for details).
Lessons from animal models Animal models of type 2 diabetes have been used extensively to study the pathophysiology of insulin resistance and diabetes. Models can be divided into spontaneous, induced and transgenic (Tables 4.1–4.3). Table 4.1. Spontaneous animal models of type 2 diabetes.
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Table 4.2. Induced animal models of type 2 diabetes.
Table 4.3. Transgenic animal models of type 2 diabetes.
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Table 4.3. continued
Research questions and directions There remain many unanswered questions on the pathophysiology of type 2 diabetes. • • • • • •
What are the genes that confer susceptibility to type 2 diabetes under the appropriate environmental circumstances? How does increased flux through the hexosamine biosynthesis pathway cause defects in glucose transport and insulin action? How does excess glucose cause impaired ß cell function? How does excess fat availability cause insulin resistance in muscle? How does excess fat cause a defect in ß cell function? Is there a common pathway for fat and glucose mediated effects of muscle and ß cells?
Practical exercises
Glucose clamp with measurement of tracer kinetics in humans This technique is used to measure both hepatic and peripheral insulin action. 1. Patients are asked to fast overnight
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2. 3. 4. 5. 6. 7.
8.
9.
10.
11.
12. 13.
14.
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A long intravenous catheter (20 cm) is inserted into the antecubital vein for infusion. A butterfly needle (21 gauge) is inserted on the back of contracterai hand. This line is kept patent by a slow infusion of saline. Heparin is not used if free fatty acids need to be measured. The hand is kept warm with a heating lamp to allow sampling of arterialised venous blood. A basal blood sample is taken and stored on ice. A primed continuous infusion of 6,6, Dideuterated glucose [6,6 D2]glucose (or [6- 3H]-glucose) is commenced driven by a pump; infusion of tracers continues unchanged throughout the study. For [6,6 D2]glucose bolus is 0.7 g, constant infusion 7.0 mg/min; for or [6- 3H]glucose bolus is 15 (Ci and the constant infusion rate 10 (Ci/hour. At the same time, a primed continuous infusion of regular human insulin at ~ 30–40 mU/kg/hour is commenced (Bolus is the equivalent of 5–10 minutes insulin infusion). Insulin is infused in saline with 1OmL Haemaccel added to prevent insulin from sticking to tubing. Five minutes after the start of the insulin, an infusion of 25% glucose to which is added either or [6- 3H]-glucose or [6,6 D2]-glucose (hot GINF method), is commenced at a slow rate. Tracer and insulin are in the same bottle. AY connection is needed to allow all three reagents to be infused via the same intravenous line. Blood samples are taken every five minutes for measurement of glucose, and glucose infusion rate is varied in order to maintain euglycaemia. This process continues for 60–90 minutes after which the glucose infusion rate is fixed. 30 minutes after fixing the glucose infusion rate. Samples for measurement of glucose, insulin and either [6–3H]glucose or [6,6,D2]-glucose are taken at 10 minute intervals. At the end of the experiment insulin is ceased and glucose is continued for a further 10–20 minutes to avoid hypoglycaemia. The infusion rate of tracer is very carefully determined by collecting in triplicate 5 minutes of combined infusates. The Y connection is then disconnected and the glucose infusion rate alone is determined. The intravenous lines are removed and the patient is given food.
Processing
To calculate total glucose turnover, the samples are processed as follows. 1. Determine the percent enrichment of [6,6,D2.]-glucose or the specific activity of [6–3H]-glucose, by deproteinising 500 µL plasma by adding 500 µL 0.3 mol/L Ba(OH)2 and 500 µL 0.3 mol/LZnSO4.
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2. Pass the supernatant down an ion-exchange resin (Ag-2X8) to remove charged labelled metabolites of glucose. 3. Wash the columns with 4 mL distilled water and then dry the eluant in an oven to remove labelled water. 4. If using [6-3H]-glucose, redissolve the glucose with 1 mL distilled water, add 10 mL scintillant and count in a beta counter. 5. If using [6,6,D2.]-glucose, add to the dehydrated sample pyridine and acetic anhydride to derivatize the glucose. Assay the derivatized glucose in GasChromatograph Mass Spectrometer (GCMS) using the selected ion monitoring mode to determine the relative abundance of selected ions (e.g., m/z 98 and m/z 100). Calculations
Total glucose rate of appearance and disappearance is determined by the following formulae (as described in Chapter 2). Ra=F/SA where Ra=the rate of appearance of glucose. F=infusion rate of tracer. SA=the specific activity of 3H-6 glucose or % enrichment of 6,6 D2 glucose. Rd=Ra if blood glucose was constant where Rd=Rate of glucose disappearance EGP=Ra–infusion rate of glucose where EGP=endogenous glucose production. If the Specific activity is not stable and shows a trend either rising or decreasing, the following non-steady state formula can be used:
where F=infusion rate of tracer 0.65=the pool fraction that is involved in rapid change. Glucose space=volume of distribution of glucose (extracellular volume or 25% of body weight). While this one pool model is not perfect, major problems arise only when the rate of change of specific activity is large.
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Measurement of insulin secretion The intravenous glucose tolerance test (IVGTT) is a sensitive method for measuring first-phase insulin secretion. It is performed as follows. 1. Perform study in subjects who have fasted overnight 2. Insert catheters in a cubital vein and the dorsum of the opposite hand as described above. 3. After collecting two basal plasma samples 10 minutes apart, administer 2 mL/ kg of 25% glucose over a 3-minute period through the cubital vein. Flush with saline to minimise irritation of the vein by the hypertonic glucose solution. 4. Take blood samples at 1, 2, 3, 5, 7, 10, 12, 14, 16, 20, 25, 30 and 40 minutes for measurement of glucose and insulin. 5. First-phase insulin release is calculated as the insulin area above basal over 0–10 minutes.
Minimal model This technique can be used to simultaneously measure insulin sensitivity and insulin secretion. It has the advantage that it uses a modified IVGTT that can be performed in a large number of subjects, which would be difficult using the clamp technique. One disadvantage is that it requires that insulin be secreted in order for the model to function. This is a problem when studying subjects who have a defect in insulin secretion as occurs in many people with diabetes. This problem has been resolved by either infusing insulin to mimic endogenous insulin release or by infusing a sulphonylurea such as tolbutamide which will stimulate insulin secretion in people with Type 2 diabetes. Under these circumstances no comment can be made about insulin secretion. A second problem is that it cannot separate hepatic from peripheral insulin resistance. The technique is performed as described above for the IVGTT with the exception that sampling is more frequent The analysis is complex and will not be described here. A good introduction to the method can be found in Bregman, 1989 (see References and Reviews).
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References and reviews General reviews
DeFronzo RA. Pathogenesis of type 2 (non-insulin dependent) diabetes mellitus: a balanced overview. Diabetologia 1992; 35:389–7 Hattersley AT. Maturity-onset diabetes of the young: clinical heterogeneity explained by genetic heterogeneity. Diabet Med 1998; 15:15–24 Glucose toxicity
Marshall S, Bacote V, Traxinger RR. Discovery of a metabolic pathway mediating glucose-induced desensitization of the glucose transport system: Role of hexosamine biosynthesis in the induction of insulin resistance. J Biol Chem 1991; 266:4706–12 Fat toxicity
Paolisso G, Howard B.V. Role of non-esterfied fatty acids in the pathogenesis of Type 2 diabetes mellitus. Diabet Med 1998; 15:360–6 Metabolic syndrome (syndrome X)
Reaven GM. Syndrome X: 6 years later. J Intern Med 1994; 236 (supplement 736): 13–22 DeFronzo RA, Ferrannini E. Insulin resistance. A multifaceted syndrome resonsible for NIDDM, obesity, hypertension, dyslipidemia, and atherosclerotic cardiovascular disease. Diabetes Care 1991; 14:173–94 Genetic causes of diabetes
Velho G, Froguel P. Genetic, metabolic and clinical characteristics of maturity onset diabetes of the young. Eur J Endocrinol 1998; 138:233–9 Animal models
Lamothe B, Baudry A, Desbois P, Lamotte L, Bucchini D, Meyts PD, Joshi RL Genetic engineering in mice: impact on insulin signalling and action. Biochem J 1998 335:193–204 Techniques
Steele R, Wall J, DeBodo R, Altszuler N. Measurement of the size and turnover rate of body glucose pool by the isotope dilution method. Am J Physiol 1956; 187: 15–24
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Radziuk J, Norwich M, Vranic M. Experimental validation of measurements of glucose turnover in non-steady state. Am J Physiol 1978; 234: E84-E93 Bergman RN. Toward physiological understanding of glucose tolerance. Minimal model approach. Diabetes 1989; 38:1512–27
5. Aetiology of Complications in Type 1 and Type 2 Diabetes
The complications of diabetes, often called late complications, are damage and loss of function in organs that have glucose transporters which are not sensitive to insulin and thus do not require insulin for glucose entry. In this case the tissue glucose levels reflect circulating glucose levels which are determined by the degree of glycaemic control as achieved by insulin replacement or by therapy with oral hypoglycaemic agents. Diabetic complications, described in Chapter 1, are referred to as either microvascular or macrovascular. Microvascular complications include damage to the retina of the eye leading to loss of vision; the nephrons of the kidney leading to chronic renal failure; and the blood supply and cells of the peripheral nervous system with loss of nerve function. In macrovascular complications there are disorders of lipid metabolism and blood coagulation leading to narrowing of vessels and altered blood flow in the cardiovascular system leading to heart attack, stroke and limb amputations. The processes which contribute to these complications and the mechanisms which can be investigated are: • The progression of glomerular sclerosis leading to end stage renal failure (nephropathy) • The mechanisms of retinal capillary loss followed by angioneogenesis which causes loss of visual acuity (retinopathy) • The mechanisms causing reduction in nerve conduction velocity and loss of peripheral and autonomic nerve function (neuropathy) • Haemostatic abnormalities contributing to ischaemic heart disease • lipid abnormalities and the interactions of the cells of the vessel wall which lead to the formation of atheromatous plaque and narrowing of the artery in atherosclerosis Development of diabetic complications in type 1 and type 2 diabetes begins with repeated acute, but reversible, changes in cellular metabolism caused by increased levels of glucose. Alterations in target tissue function and structure follow and progress to the clinically recognised conditions referred to above as microvascular complications. Macrovascular complications are a result of atherosclerosis. Both microvascular and macrovascular disease can affect the coronary artery and cause cardiac dysfunction. 75
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In epidemiological studies the microvascular and macrovascular complications are linked. The slightly increased urinary albumin excretion (microalbuminuria), which is the first predictor of development of clinical nephropathy, is also a strong predictor of cardiovascular disease and of proliferative retinopathy.
Description and prevalence of complications The reported prevalence of microvascular and macrovascular complications varies widely. This is due in part to the criteria used for the detection of complications and some strikingly different incidences between different ethnicities and different lifestyle conditions.
Nephropathy There is a wide range in the severity and type of kidney damage experienced.
Figure 5.1. Thickening of the glomerular basement membrane in diabetes. Glomerular basement membrane increases with time after onset of diabetes with a slight thickening appearing after two years and an increase of 35% after five years. This is detected and measurements made using electron microscopy of glomerular biopsies. Panel (a) shows the thickened glomerular basement membrane in diabetes compared to a non-diabetic control in panel (b). (From Darmady, E.M. Renal Pathology. London: Butterworth Group, 1980. Reproduced with permission.)
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Figure 5.2. The appearance of the glomerulus in glomerulosclerosis. Nodular glomerulosclerosis is seen in advanced diabetic renal disease. The arrow shows the position of a nodule in a section of a glomerulus. A glomerulus may contain one or several of these acellular masses called KimmelstielWilson nodules. After 30 years of diabetes the thickening of the basement membrane (seen in Figure 5.1) and the formation of nodules may lead to the complete closure of many glomeruli and renal failure. (From Darmady, E.M. Renal Pathology. London: Butterworth Group, 1980. Reproduced with permission.)
Nephropathy is considered to be a progressive process, in which the rate of progression can be altered by interventions such as intensive therapy to improve glucose control or the treatment of raised blood pressure. The beginning of diabetic nephropathy is seen as microalbuminuria (levels of albumin excretion in the range 20–200mg/min, or 30–300 mg/24 hr, compared to levels < 15mg/mL in healthy non-diabetic controls). In type 1 and type 2 diabetes the prevalence rate is 20–30% in studies from Australia, Europe and the USA but epidemiological studies show this may be higher in type 2 diabetic people from some specific groups such as the Pima Indians, Mexican-Americans and Nauruans. In diabetic people with microalbuminuria the glomerular filtration rate may be increased or within the normal range. There are no symptoms at
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this early stage. The occurrence of microalbuminuria in type 1 diabetes indicates a 20-fold increase in the risk for the development of nephropathy. 70–80% of people who have type 1 diabetes and microalbuminuria will develop nephropathy, progressing through to macroalbuminuria (urinary albumin excretion >300mg/24hr). Thickening of the glomerular basement membrane (Figure 5.1) and expansion of the mesangial matrix of the glomerulus may eventually lead to closure of some or many glomeruli. This is termed glomerulosclerosis (Figure 5.2) and together with changes in the renal arteries and arterioles leads to a decline in glomerular filtration and subsequent renal failure. Diabetic nephropathy is a most common cause of end-stage renal failure accounting for approximately 25% of all cases. At this stage, dialysis and kidney transplantation are the only effective therapeutic options.
Retinopathy Within any diabetic population it can be anticipated that 30% will have some form of change to the retina associated with diabetes.
Figure 5.3. The appearance of human non-proliferative retinopathy. The retina can be viewed using an ophthalmoscope and photographed using a retinal camera. This photograph shows damage to the microvessels of the retina appearing as microaneurysms (a) and dot haemorrhages (b).
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Early retinal changes include background or non-proliferative retinopathy that manifest as bleeding (haemorrhages), leakage spots (microaneurysms) and some fatty exudates. 10% of diabetic people with retinal changes will progress to advanced retinopathy in which vision is threatened by swelling of the macula, the centre of vision on the retina. This thickening (macula oedema) can be stabilised and permanent damage avoided by laser treatment. Damage to the retinal vessels is characterised by loss of capillaries and their supporting cells called pericytes. The resulting loss of capillary circulation and nutrient and oxygen supply (ischaemia/hypoxia) provokes a response in which new fragile vessels grow (proliferative retinopathy) and haemorrhage readily (Figures 1.10 and 5.3). Retinal scarring can then follow and when this scar tissue contracts the retina may detach. Australian studies have shown that overall, each year retinopathy will develop in 8% of diabetic people who did not previously have any retinopathy. Duration of diabetes is the primary risk factor for retinopathy. Almost all those who have had diabetes for over twenty years will develop some form of retinopathy. Estimates from the USA show the overall mean age-adjusted prevalence of blindness due to diabetes increased by 28% between 1987 and 1994 due mainly to an increase in the number of diabetic people over the age of 65 years. An optimistic finding, however is that the prevalence of notifiable blindness had decreased by 17% in diabetic people under 44 years of age, indicating that methods for improving glucose control and the treatment of early diabetic eye changes may be having a positive effect. In addition to retinopathy in diabetic people, many prevalence studies have shown an association of cataract, in which the crystalline lens of the eye loses its transparency, and keratopathy, in which there is decreased sensation and ulceration of the cornea.
Neuropathy Estimates of the incidence of peripheral diabetic neuropathy range widely and are often dependent on how the diagnosis is made. There is a sophisticated machine for this called a biothesiometer, which tests the threshold at which vibrations can be perceived. Other less sensitive techniques measure the ability to sense a touch from a calibrated monofilament, piece of cotton wool, or pin prick. Many, if not all, of the changes are believed to follow a decrease in blood flow to and oxygenation of the nerve leading to its degeneration. Diabetic sensorimotor polyneuropathy can result in symptoms of changed sensation. There may be tingling or a feeling of pins-and-needles (paraesthesia) or burning or strong pain on touch (dysthesia). As well there
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may be sensory loss, often accompanied by a loss of power in the foot, lower limb and hand in a pattern called a stocking-glove distribution. Impaired sensation means that trauma, for example, a burn or cut, particularly to the extremities, is not recognised and infection may occur. This may lead to amputation. When the presence of pain, muscle weakness and sensory loss are used to diagnose peripheral neuropathy the incidence is around 40%. There is an increasing prevalence of peripheral neuropathy with duration of diabetes and with poor glycaemic control. In a study of 4398 cases of diabetes the prevalence was less than 10% at diagnosis of diabetes and 50% after diabetes of 25 years duration. Within the 25-year duration group the prevalence of neuropathy was 10–20% in those diabetic people with good glycaemic control and 60–70% in those with poor control. The prevalence of neuropathy that affects the nerves of the autonomie nervous system (autonomie neuropathy) is less easily established, as there are a wide variety of sites involved and varying degrees of overall severity and impairment Symptomatic autonomie neuropathy is usually found only in longstanding diabetes and can affect the cardiovascular, gastrointestinal and urogenital systems with symptoms of hypotension, episodic vomiting, diarrhoea, urinary incontinence and impotence.
Coronary artery disease and atherosclerosis The prevalence of coronary artery disease in people with diabetes ranges from 9.5% to 55%. Coronary artery disease is the most common cause of death in type 2 diabetes. It is also frequently found in people with type 1 diabetes, so that the mortality rate from cardiovascular disease in diabetic people approaches three times that of the general population. Multinational studies of coronary arteries in people with diabetes show an increased incidence of fatty streaks (early changes) and advanced atherosclerotic lesions. A change in diet and life style can alter the incidence of coronary artery disease in diabetes. This can be seen in diabetic Asians, who experienced a low incidence of coronary artery disease, until they moved to Japan or Hawaii, after which the incidence increased.
Unawareness of hypoglycaemia People with type 1 diabetes often lack the appropriate warning symptoms of hypoglycaemia and low brain glucose levels (neuroglucopaenia).
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The causes of unawareness of hypoglycaemia are unknown but are often found together with inadequate hypoglycaemic counterregulation. This is believed to be through a defect in central glucose regulation and may be related to impaired adrenaline secretion during hypoglycaemia but is not a direct result of autonomie neuropathy.
Contribution of genetics to complications Genetic factors may be important in the development of some diabetic complications as familial clustering of diabetic nephropathy has been described. The gene(s) or polymorphisms involved have not been identified but they are being extensively investigated. Analysis of discordant siblings has shown a major susceptibility locus on chromosome 3q. Even with high blood glucose levels for several years, nephropathy may be seen in only 40% of people with type 1 diabetes. By contrast over 90% of persons with poor glucose control for many years will develop retinopathy. From many studies it is clear that with or without a genetic component to these complications, the predominant factor in their initiation and progression is the extent to which blood glucose levels are regulated.
Long-term trials How can intervention alter the course of diabetic complications? Long term studies and trials have been undertaken in an attempt to identify those people with diabetes at high risk for developing diabetic complications and effective means of intervention. In these studies blood glucose control is determined by measuring either blood glucose levels or the degree of glycation of red blood cell haemoglobin. This is called the glycated haemoglobin, and is a measure of the exposure to glucose of the red cell over the preceding 2–3 months. The broad categories of studies and trials are: • Population studies to establish prevalence data, for example, The European Community Sponsored Concerted Action on the Epidemiology and Prevention of Diabetes (EURODIAB). This is a study that examines incidence of type 1 diabetes from 3 centres in Europe and the prevalence of both large and small vessel disease • Clinical trials to establish the effectiveness of interventions • Testing for genetic linkage between candidate genes and particular complications • Mathematical modelling of populations for evaluation of treatments Two large-scale trials currently providing information on type 1 and type 2 diabetes are: The Diabetes Control and Complications Trial, and The United Kingdom Prospective Diabetes Study.
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The Diabetes Control and Complications Trial (DCCT) This trial was conducted in multiple centres in the USA and compared the effects of intensive insulin therapy with those of conventional insulin therapy on the development and/or progression of long term complications of type 1 diabetes. The trial, which lasted 9 years, was completed in 1995 and has provided compelling evidence to link the average degree of control of glycaemia to the onset and progression of nephropathy, retinopathy and polyneuropathy. The DCCT cohort has recently provided evidence that there are familial (genetic) factors that influence the severity of diabetic retinopathy, independent of metabolic control, and that there may also be familial effects on the development of diabetic nephropathy. The cohort is continuing to be investigated in a new Epidemiology of Diabetes Interventions and Complications study.
The United Kingdom Prospective Diabetes Study (UKPDS) This trial commenced in 1977 with the primary aim to determine whether improved glycaemic control would prevent diabetic complications in people with type 2 diabetes. The Study has the secondary aim of comparing the efficacy of oral hypoglycaemic agents and dietary control in controlling hyperglycaemia. In the trial half of those enrolled, who had newly diagnosed diabetes, had early evidence of microvascular complications. The base-line prevalence of retinopathy and microalbuminuria was however not related to the age of the person. One of the final outcomes of the UKPDS, reported in 1998, showed that intensive glucose control during the first 10 years after diagnosis reduced the frequency of microvascular end-points but not diabetes-related mortality or myocardial infarction.
Predictive modelling studies Can complications be predicted? It is possible to model the complications of type 2 diabetes in probabilistic models that may be useful in evaluating the effect of preventative interventions. In one reported model the predicted incidence rates were: Microvascular (kidney)
• Microalbuminuria 53% • Proteinuria 40% • End-stage renal disease 17%
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Microvascular (eye)
• • • •
Non-proliferative retinopathy 79% Macular oedema 52% Proliferative retinopathy 19% Legal blindness 19%
Microvascular (peripheral neuropathy)
• Symptomatic sensorimotor neuropathy 31 % Macrovascular
• Cardiovascular disease 39% • Lower extremity amputation 17% The predicted life expectancy for a diagnosis of type 2 diabetes made prior to 75 years of age is 17 years. This modelling has not been described for type 1 diabetes.
Clinical studies Clinical studies for intervention in complications of diabetes can be multicentre, open, crossover, randomised, single-blind, double-blind, and/ or parallel in a number of combinations: The design of these often requires consultation with an epidemiologist or clinical statistician. There is a continual increase in the number of treatments under investigation and these are based on much previous in vitro research. Some of these will be introduced later in this Chapter (see Treatment Related To The Pathogenesis Of Complications). Future research may be expected to follow two main avenues: 1. The investigation of new-generation drugs that inhibit the mechanisms proposed to contribute to microvascular complications. 2. Specific hypothesis-driven investigations which will determine the underlying cause of a specific or distinctive feature of a particular complication. Many of the hypothesis-driven laboratory studies to investigate molecular and biochemical functions are relevant to pathogenesis and are expected to provide new treatment strategies.
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Pathogenesis How do diabetic complications begin? In the presence of raised glucose levels there is no single mechanism responsible for diabetic complications. It is increasingly apparent that the component cells of the microvasculature and macrovasculature (endothelial cells and smooth muscle cells) share a number of functional characteristics. When subjected to increased intracellular glucose levels during periods of poor metabolic control in diabetes, they respond by initiating changes that lead to damage. Both endothelial and smooth muscle cells contain insulin receptors but a common feature is insulin-independent glucose entry through insulinindependent glucose transporters, predominantly GLUT-1. The regulation of glucose transporters in microvascular cells and the possible contribution of local insulin concentrations and insulin resistance to microvascular cell function have not been widely investigated. It is know that insulin resistance contributes to the development of coronary artery disease; it may also be involved in the progression of microvascular disease. The cell types involved in microvascular dysfunction at specific sites are: • • • •
endothelial cells—in all organs mesangial cells—in the glomerulus pericytes—in the retina and cardiac microcirculation smooth muscle cells of arterioles and endoneurial capillaries—in all organs
Endothelial cells line both macrovessels and microvessels and maintain the integrity of the vasculature by acting as a barrier to transvascular flux. These cells have a regulatory role in adhesion of circulating cells, fibrinolysis and thrombosis, in extracellular matrix production and in maintaining vascular tone. Endothelial cells produce various antiproliferative and vasodilatory factors. A number of endothelial cell genes respond to flow and mechanical stimuli to provide antioxidant, antithrombotic and antiadhesive protection to the microvessel. Major vasoactive regulators produced by endothelial cells are the arachidonic acid products (eicosanoids). Of these, prostaglandin E2 and prostacyclin are believed to reduce arteriolar resistance and increase blood flow by an effect on the contraction of smooth muscle cells. A number of stimuli release an endothelium-derived relaxation factor identified as nitric oxide from endothelial cells. By action on smooth muscle cells nitric oxide is the major regulator of flow-dependent dilatation following increased arteriolar flow. Glomerular mesangial cells are smooth muscle-like pericytes that regulate intraglomemlar blood flow in the glomerulus to prevent distension in response to increased flow. Retinal capillary pericytes regulate blood flow within the retina and provide a structural support for the endothelial cells of the retinal capillaries. Pericytes provide trophic factors which maintain endothelial cells. However,
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Figure 5.4. The links between biochemical changes and the functional processes involved in the progression of microvascular complications.
it is possible that any decrease in blood flow, particularly in the retina, can cause hypoxia and induce the production of vascular endothelial growth factor (VEGF). This factor can cause alterations in permeability and cause microaneurysms.
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Vascular smooth muscle cells and related pericytes regulate vessel tone through a responsive calcium-dependent actin contractile system. In all blood vessels endothelial cells and smooth muscle cells are maintained within an extracellular matrix. Expansion of this matrix occurs in diabetes as an increase the thickness of the capillary basement membrane and general matrix expansion. These matrix changes can alter the barrier function of the endothelial cell and, by stiffening the vascular wall, may form a mechanical inhibition to vascular reactivity to flow changes. Vascular reactivity is altered in diabetes. In clinical studies microvascular perfusion is often monitored by use of laser Doppler which is a non-invasive technique to measure local tissue blood flow.
Cellular mechanisms causing complications How do cells respond to a diabetic environment to cause complications? There are several parallel mechanisms that may contribute to endothelial and smooth muscle cell dysfunction in diabetes. A number of these may be
Figure 5.5. Pathways for the use of glucose in cells under diabetic conditions. The metabolism of glucose can be by the enzymatic aldose reductase (AR) pathway under conditions of raised glucose to produce sorbitol. Sorbitol is converted to fructose by the enzyme sorbitol dehydrogenase (SDH) and in this process NADH is produced. Regeneration of NAD+ in the mitochondria can lead to the formation of reactive oxygen species (O -) as can the processes 2 of autoxidation and glycation (A/G). Antioxidant enzymes including superoxide dismutase (SDH) remove O -. The action of the glutathione pathway 2 (GR=glutathione reductase, GP=glutathione peroxidaase) and the pentose phosphate pathway (PPP) provide reduced pyridine nucleotide (NADPH) and glutathione (GSH) to remove O -. 2
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related to the production of reactive oxygen species and oxidative stress, a common feature of the cellular biochemical dysfunction in the presence of hyperglycaemia (Figure 5.4). Specific processes, which are increased when glucose is raised, are: • • • • •
glycolysis and glucose autoxidation de novo synthesis of diacylglycerol and activation of protein kinase C sorbitol pathway metabolism glycation of proteins oxidative changes to lipids and proteins
These processes are interrelated. Following the elevation of glucose levels there is an increase in the use of glucose through the pentose phosphate pathway (hexose monophosphate shunt), together with increased conversion of glucose to sorbitol by the enzyme aldose reductase. Sorbitol is subsequently metabolised to fructose by the enzyme sorbitol dehydrogenase. These pathways (shown in Figure 5.5) are interrelated in a manner that alters the intracellular balance of pyridine nucleotide cofactors so that there is an alteration in the capacity of the glutathione redox system and an hypoxia-like redox stress within the cell. The interaction of these pathways provides two glucose-derived triose phosphates that are both the precursors of diacylglycerol, a natural activator of protein kinase C. Triose phosphates, together with glucose, undergo autoxidation. In this process free radicals are produced which include superoxide, hydrogen peroxide and hydroxyl radicals. Advanced glycation end (AGE) products are formed by a non-enzymatic reactions between carbohydrates and tissue protein. These interactions, termed Maillard reactions, are accelerated by oxygen and oxidative reactions and the final product is a glycoxidation product. AGE products bind to a number of specific cell receptors (receptors for AGE) to induce a further oxidative stress which can alter DNA, lipids and proteins. Oxidatively modified lipi ds can be cytotoxic and contribute to apoptosis (programmed cell death) and loss of cells. Oxidatively modified lipids can also affect gene transcription. The consequences of raised glucose in target tissues are an alteration of: • • • •
signal transduction mechanisms growth factor production extracellular matrix formation and degradation cell-to-cell communication
Following metabolism of raised glucose or glycoxidative modification there are a number of intracellular processes that are altered in endothelial and smooth muscle cells.
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Figure 5.6. Pathways which can lead to increased intracellular metabolites, including increased prostaglandin production and some advance glycation products in microvascular tissue. The formation of those phosphates through the pentose phosphate pathway (PPP) and glycolysis (HK=hexokinase) provides the diacylglycerol (DG) cofactor for activation of protein kinase C (PKC) leading to the production of prostaglandins (PGs) [PLA2= phospholipase A2]. Methylglyoxal can also be formed from the those phosphates, which contributes to the formation of the lysine-crosslinked product of advanced glycation (AGE=advanced glycation end products).
Glucose-induced protein kinase C activation can regulate prostaglandin production. This is achieved through activation of cytosolic phospholipase A2, the enzyme supplying the substrate arachidonic acid for prostaglandin production (Figure 5.6) Glucose-induced protein kinase C activation can regulated the expression of mRNA for growth factors including vascular permeability factor/vascular endothelial growth factor, a cytokine that induces angiogenesis and increased endothelial permeability. Vascular endothelial growth factor may also contribute to increased blood flow. Protein kinase C, oxidative stress and/or advanced glycation end products can increase the expression of transforming growth factor beta 1 (TCF-ß1) and activation of latent TGF-ß1. This growth factor is antiproliferative but strongly supports matrix expansion and an increase of the basement membrane and matrix components fibronectin and collagen type IV. Under diabetic conditions the matrix formed contains proteoglycans with reduced anionic charge, particularly the main sulphated glycosaminoglycan heparan sulphate. Its loss in vascular tissue may reduce structural integrity and alter vessel wall selectivity, for example, promote microalbuminuria in
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Figure 5.7. Pathways that lead to functional changes in microvessels in diabetes may be linked through an increase in reactive oxygen species. O2are formed by the metabolism of glucose (see Figure 5.5).
the glomerulus, alter the anticoagulant activity of the vascular wall and/or contribute to retinal leakage. In glomerular mesangial cells nitric oxide can inhibit TGF-ß1 and thus suppress matrix synthesis. Reactive oxygen species may, however, modify this response, as superoxide will neutralise the effect of nitric oxide, by formation of peroxynitrite. Loss of nitric oxide may make a major contribution to the reduction of nutritive blood flow to peripheral nerves as well as be a factor in the loss of flow-dependent vasodilatation. These aspects are summarised in Figure 5.7. The cellular mechanisms that are activated or altered by glycoxidative stress may include any combination of the following processes. • Activation of protein kinase C. • Activation of mitogen-activated protein kinases including stress-activated and reactivating kinases and regulation of gene transcription and growth. • Activation of transcription factors AP-1 and NF?B. • Matrix interaction with cellular integrin receptors and tyrosine kinase activation. The interrelated molecular and biochemical effects of raised glucose have the following functional changes in diabetic tissues regardless of the type of diabetes. • Increased blood flow and failure of tissues to autoregulate blood flow (kidney and retina).
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• Increased vascular permeability (all microvascular sites) • Electrophysiologic dysfunction including reduced Na+/K+-ATPase (retina and peripheral nerve) • Impaired contractile function (heart, skeletal muscle, vascular smooth muscle and glomerular mesangial cell) • Impaired vasodilatation in response to decreased flow or decrease in perfusion pressure (peripheral and coronary microvessels) The interrelationship of the underlying biochemistries of vascular dysfunction can be seen in various investigations, including the following. 1. Increased blood flow in models of raised tissue glucose. The increase can be prevented by inhibitors of aldose reductase, sorbitol dehydrogenase, prostaglandin synthase, nitric oxide synthase and superoxide dismutase. 2. Microvascular cells exposed to raised glucose. Addition of inhibitors of protein kinase C, in particular an inhibitor of the ßII isoform of this enzyme, will prevent a reduction in Na+/K+-ATPase activity; an increase in arachidonic acid release, prostaglandin, fibronectin and collagen Type IV production; and expression of TGF-ß1 and vascular permeability factor/vascular endothelial growth factor. 3. Treatment of diabetic rats with antioxidants. This can prevent albuminuria, an increase in TGF-ß1, and activation of transcription factors by advanced glycation end products.
Specific considerations in the pathogenesis of macrovascular complications The biochemical alterations in endothelial cells and smooth muscle cells are common to the cells in both microvessels and macrovessels. However, there are a number of factors of specific relevance to macrovascular disease. These include: • Altered endothelium-dependent relaxation • An increase in procoagulant activity and a decrease in anticoagulant pathways leading to hypercoagulation • Depressed fibrinotysis due to alterations in the levels of tissue type plasminogen activator (tPA) and its fast acting inhibitor (PAI) The defects found in atherosclerosis are increased in diabetes. These include: • vascular AGE accumulation and oxidative damage • increased vascular matrix and vessel narrowing • increased mononuclear activation with cytokine and growth factor release and vascular cell proliferation • T cell stimulation
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• increased macrophage uptake of oxidised and AGE low density lipoproteins (LDL) and entry into the subendothelial space to create atheroma • an increase in glycoprotein apo(a)-containing lipoprotein (Lp(a)). This increase is observed in people with microvascular complications. The role of a number of factors is currently being examined. These are: • Insulin resistance in insulin-dependent glucose homeostasis. • AGEs and AGE-LDL in vascular expression of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). • Immune complexes formed with oxidised LDL and CD4+ Tcell-mediated immunity.
Models used in research of diabetic complications
In vitro models It is possible to model the raised glucose levels of diabetes in many cell types maintained in tissue culture. Models can include endothelial and smooth muscle cells isolated from microvessels of the peripheral circulation, endothelial cells and pericytes isolated from the retinal circulation and endothelial cells and mesangial cells from the renal glomerulus. Microvessels can be removed from organs for studies of reactivity and tissue and cell histomorphometric analysis.
In vivo models Cells and tissues are most often taken from rats made diabetic by destruction of pancreatic ß cells with streptozotocin (STZ). Rats are mostly used for this because microvascular complications are less severe in the mouse. Models for investigating diabetic complications that require a persistently raised level of glucose are the STZ-diabetic rat and the BB-Wistar diabetesprone rat Inbred strains with obesity, hyperglycaemia and insulin resistance are listed in Table 5.1. Table 5.1. Inbred strains with obesity, hyperglycaemia and insulin resistance
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At present these models are not widely used in research into complications, but have an emerging application in investigation of genetic associations. Reliable stem cell lines can be produced for the mouse so it is possible to genetically engineer gene knock-out in mice. Emerging models for investigation of specific microvascular aspects are: • Galactose feeding in rats and mice to investigate the involvement of the aldose reductase pathway • A transgenic mouse with overexpression of aldose reductase • A transgenic mouse with targeted myocardial overexpression of protein kinase C ßII isoform • Goto-Kakizaki rat >28 weeks of age with raised retinal VEGF immunoreactivity Investigations can involve: • Gene knockout (mouse models only) and overexpression studies of specific intracellular pathway components (rat and mouse models) • Pharmacological inhibitors • Blocking antibodies • Targeted tissue delivery of antisense nucleotides to prevent production of specific components of signal transduction cascades. Molecular techniques such as mRNA differential display can be used to identify new genes regulated by cellular conditions in diabetes, such as raised glucose and oxidative stress and tissue conditions such as increased laminar flow or increased extracellular matrix.
How To Implement These Models Many published papers contain condensed descriptions of the material and methods to successfully prepare these cell and animal models. It is recommended that a new investigator should, after choosing a model and a particular application, seek the advice of an investigator with prior experience or consult the author of the relevant publication. Simplified protocols for the production of diabetes in rats by administration of STZ and the preparation of microvascular endothelial cells and glomerular mesangial cells appear later in this chapter (see Practical Exercises).
Treatments related to the pathogenesis of complications Many trials, including the DCCT and UKPDS, highlight the importance of intensive insulin therapy and antihyperglycaemic therapies in halting or preventing complications. These therapies have been discussed in Chapter 1.
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A recent study has shown that in patients with type 1 diabetes with severe neuropathy, a simultaneous pancreas and kidney transplantation improved the defect in nerve conduction. Pancreatic islet transplants in experimental diabetic rats showed restoration to the non-diabetic condition of a number of factors implicated in the pathogenesis of renal complications. These included accumulation of AGE and extracellular matrix, and increases in TGF-ß1. Treatment needed to be instituted early in the course of diabetes for it to be effective; once renal abnormalities had occurred, they could not be reversed. Until ideal insulin replacement and glycaemic control can be achieved for all people with diabetes, a number of prophylactic interventions aimed at the pathogenesis of complications will be required. Some of the current treatments that are used in addition to glycaemic control include the following. Treatment to prevent hypertension
Treatments directed to the renin-angiotensin system, using angiotensin converting enzyme (ACE) inhibition, are considered the most effective. This treatment is aimed directly at a specific endothelial cell abnormality. The importance of linking biochemical and clinical studies can be seen in the recent description of an ACE gene polymorphism. One form confers an increased susceptibility to diabetic nephropathy and resistance to ACE inhibition in type 1 diabetes. This may mean that more appropriate treatment would be to concentrate on strict glucose control in these people with type 1 diabetes or to consider an alternative approach for altering the reninangiotensin system which does not involve angiotensin conversion, perhaps by angiotensin receptor blockade. Inhibitors of advanced glycation end products and their action
Advanced glycation end products (AGE) are modifications of proteins that are formed at a rate that is directly proportional to glucose concentration. AGEs are implicated in the pathogenesis of diabetic complications. Aminoguanidines are under investigation as inhibitors of AGE formation. Aminoguanidines interfere with the formation of early glycation products that are formed before irreversible modification of proteins has occurred. In clinical and animal studies, investigations with aminoguanidines show: • A decrease in microalbuminuria and retardation of the increasing fractional mesangial matrix volume and thus the expansion which is believed to ultimately obliterate glomerular capillary flow • A reduction in the number of acellular capillaries and pericyte loss in the retinal circulation
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• Prevention of reduced sciatic nerve endoneural blood flow and sensory nerve conduction velocity There are extensive investigations to describe the cellular receptors that recognise and respond to AGEs but these have not yet provided additional treatments. Inhibition of oxidative stress
Oxidative damage by free radicals has been implicated in the pathogenesis of microvascular and macrovascular complications in diabetes. There is increasing evidence that glycoxidation, a process that requires raised glucose levels, contributes to oxidative stress. The steady-state of reactive oxygen species is set by their production in cells of the microvasculature or in circulating cells. The balance is between superoxide, from which the further products hydrogen peroxide and hydroxyl radical can be formed, and the free-radical scavenging antioxidant activity of cells and serum. In type 1 and type 2 diabetes, even when uncomplicated by vascular disease, the antioxidant status is reduced, which may indicate a vulnerability to oxidative damage. Intracellular free radical defences include superoxide dismutase, catalase and the glutathione redox system together with a number of naturally occurring antioxidants including vitamin C and vitamin E. Antioxidants that have been administered in clinical trials include vitamin E, vitamin C, a-lipoic acid and troglitazone, an oral hypoglycaemic agent with antioxidant properties. Inhibition of aldose reductase
There is extensive biochemical evidence proposing that the enzyme aldose reductase and the polyol pathway are implicated in the pathogenesis of diabetic microvascular complications. Attempts to alter the course of microvascular dysfunction with a number of compounds designed to inhibit aldose reductase have not been outstanding. However, there is consensus from many trials that aldose reductase inhibition will slow the progression of microvascular complications. A number of structurally dissimilar aldose reductase inhibitors reduce sorbitol concentrations, prevent basement membrane thickening and reduce microalbuminuria in animal models. Clinical studies with aldose reductase inhibitors, in particular sorbinil, ponalrestat and tolrestat, in studies of retinopathy, neuropathy and cardiovascular performance show the importance of reaching effective and sustained concentrations of these agents in target organs. There are a number of aldose reductase inhibitors under development Of those inhibitors already trialed, tolrestat remains in therapeutic use.
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Research questions and directions The following is a list of some questions being addressed by the research community. They have been arranged according to the various sections in this Chapter.
Genetic causes • Can a major gene having a common disease allele be implicated in the genetic susceptibility to individual microvascular complications? • Can individuals at-risk for hyperglycaemia-related injury be identified?
Interventions • In long-term trials will the post-study review of UKPDS establish whether risk of diabetes-related mortality and myocardial infarction can be reduced in type 2 diabetes?
Cellular mechanisms causing complications • Can a common cause be assigned to hyperglycaemia-induced cellular changes? • Can the intracellular conditions in cells at various microvascular sites affected in diabetes be described in sufficient detail to link common pathways and exclude others? • Can the mechanisms of cell-cell and cell-matrix interaction be described in a manner that reflects the conditions at specific microvascular sites? • Can the specific proteins modified by glycoxidative reactions and which contribute to microvascular dysfunction be identified in vivo? • Can new biochemical and genetic markers be described and be of predictive value for future microvascular complications in children and adolescents? • Can specific differences be determined between cells at microvascular and macrovascular sites and their susceptibility to hyperglycaemia-induced damage?
Treatments related to the pathogenesis • Can pharmacological interventions be developed as an intensive treatment in preclinical microvascular complications? • Can pharmacological therapy be directed to specific microvascular locations?
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Practical exercises Each of these protocols requires that the investigator has obtained the appropriate approval from the Animal or Human Ethics Committees of their institution.
Streptozotocin (STZ) treatment of rats to induce insulin-dependent diabetes 1. 2. 3.
4. 5. 6. 7. 8. 9.
10.
11.
Rats should be between 180 and 200 g. Fast the rats overnight and weigh. Prepare sodium citrate buffer (100mM, pH 4.0) and STZ at a dose of 75 mg/ kg for each rat. This should be in an injection volume of 500 µL or less and be sterile filtered, using a 0.22 (µm Acrodisk, prior to use. Using a 25G needle attached to a 2 mL syringe, administer STZ by an intraperitoneal injection to an unanaesthetized rat. Control rats are injected with citrate buffer alone. Provide rats receiving a STZ injection with 5% sucrose in their overnight drinking water and food ad libitum. Remove 5% sucrose drinking water and replace with tap water and food ad libitum. 48 hours after injection test urine for the presence of glucose using a reagent strip for urinalysis (AMES, Bayer Diagnostic). Diabetic rats will have frequent passage of urine (poryuria) and will drink copiously (polydipsia) but may be housed under standard conditions, providing food and water ad libitum and frequent changes of bedding material for up to 21 days. If animals are to be kept longer, it is recommended that insulin be administered. Ultralente insulin 2U/rat, administered by intramuscular injection on alternate days, will maintain a diabetic rat in good health but with raised glucose levels. An insulin dose of 25U/kg can be given by intramuscular injection to restore glucose levels to control levels and provide an appropriate control group.
Preparation of glomeruli and culture of rat glomerular mesangial cells 1. 2. 3. 4.
Induce CO2 narcosis in rats and euthanase with sustained CO2 exposure. Remove kidneys aseptically from rat. Place in sterile Dulbecco’s Modified Eagles Medium (DMEM). Wash kidneys by 3 changes of medium.
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9. 10. 11. 12. 13.
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Place kidneys in a 10 cm sterile petri dish and cover with medium. Using forceps, place a kidney on sterile gauze held between thumb and forefinger so that the kidney can be cut in half using a scalpel blade. Using sterile scissors cut the outer 3–5 mm of cortex from each half kidney and place this in sterile DMEM. Assemble 3 sterile brass or stainless steel wire sieves 250 µm, 125 µm and 75 µm mesh sizes, 250 µm uppermost, on a shallow sterile tray and press the dissected kidney cortex fragments through the uppermost sieve using a spoon or rounded spatula. Note that cortex from STZ diabetic rats will be larger and more fibrous. Wash sieved fragments thoroughly onto the 125 µm sieve using DMEM. Repeat sieving through the 125 µm sieve as in steps 8 and 9. The glomeruli will be caught on the final sieve. Collect glomeruli from the surface of the final sieve using a plastic transfer pipette and washing with DMEM. Centrifuge the glomerular suspension at 800 rpm for 8 minutes. Remove supernatant DMEM and replace with fresh DMEM.
The glomeruli may be used for investigation at this stage, or further processed to obtain mesangial cells. 14.
15. 16.
17.
18. 19. 20.
Digest glomeruli with collagenase (Type V, Sigma) 5 mg/mL at 37°C for 10 minutes, inverting the digest each 2 minutes to produce glomerular cores. After 10 minutes centrifuge the digest at 800 rpm for 8 minutes. Discard supernatant medium and resuspend glomerular cores in growth medium (500mL DMEM, 120 mL fetal calf serum, 2 g glucose, 0.174 g glutamine) containing the antibiotics penicillin and streptomycin. Aliquot the glomerular core suspension into tissue culture plates at a concentration equivalent to the yield of 4 kidneys in 10 mL growth medium. Incubate at 5 % CO2/air for 3 days. Replace growth medium at 3-day intervals and subculture when the cells become confluent. On second and subsequent subcultures (up to passage 6) mesangial cells will be the predominant cell and suitable for research purposes.
Preparation of microvascular endothelial cells from rat heart 1. 2.
Anaesthetize an adult rat with, for example, 50 mg/kg pentobarbitone. Using aseptic technique, cannulate the aorta and remove heart from the thoracic cavity. Place in Ringer solution at 4°C.
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Briefly rinse the surface of the heart with 70% ethanol to devitalise endocardial cells. Perfuse the heart with calcium-free Krebs-Hepes buffer and with enzyme solution containing 0.074% collagenase II, 0.012% dispase (500 U/mL), 0.012% trypsin and 0.27% bovine serum albumin (Sigma) for 30 minutes. Incubate the perfused heart with frequent mixing for a further 20 minutes to complete disaggregation. Filter the digest through 200 µm nylon mesh to remove undigested tissue. Wash the filter surface with RPMI medium and centrifuge the washes to collect microvessels. Suspend the microvessels in growth medium, 500 mL RPMI, 120 mL fetal calf serum, 10 mg/mL endothelial cell growth factor, 50 mg/mL heparin (Sigma) and containing the antibiotics penicillin and streptomycin. Then seed into gelatin-T-75 (Falcon) coated tissue culture flasks at a concentration equivalent to the yield of 1 heart per 20 mL growth medium. Incubate at 5% CO2/air for 6 days. At this time endothelial cells will be present and can be subcultured. It is recommended that endothelial cells be separated by either fluorescence-activated cell sorting or by coupling to appropriately coated microbeads before using for research purposes.
References and reviews Description and prevalence of complications
Chiarelli F, Verotti A, Mohn A, Morgese G. The importance of microalbuminuria as an indicator of incipient diabetic nephropathy: therapeutic implications. Ann Med 1997; 29:439–45 Hodge AM, Dowse GK, Zimmet PZ. Microalbuminuria, cardiovascular risk factors, and insulin resistance in two populations with a high risk of type 2 diabetes mellitus. Diabet Med 1996; 13:441–9 [Wanigelas and Nauruan populations] Garza R, Medina R, Basu S, Pugh JA. Predictors of the rate of renal function decline in non-insulin-dependent diabetes mellitus. Am J Nephrol 1997:17: 59–67 [Mexican-American, African-American and non-Hispanic white renal patients] NHMRC. Clinical practice guidelines for the management of diabetic retinopathy. Canberra: Australian Government Publishing Service, 1997 Agardh E, Agardh C-D, Hansson-Lundblad C. The five-year incidence of blindness after introducing a screening program for early detection of treatable retinopathy. Diabet Medicine 1993; 10:555–9
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Dejgaard A. Pathophysiology and treatment of diabetic neuropathy. Diabet Med 1998;15:97–112 American Diabetes Association Clinical Practice Recommendations 1998. Diabetes Care 1998; 21 Suppl 1. Unawareness of hypoglycaemia
Meijer E, Hoekstra JB, Erkelens DW. Hypoglycaemia unawareness. Presse Med 1994; 23:623–7 Amiel SA. Hypoglycaemia in diabetes mellitus—protecting the brain. Diabetologia 1997; 40 Suppl 2:862–8 Genetic causes
The Diabetes Control and Complications Trial Research Group. Clustering of long-term complications in families with diabetes in the diabetes control and complications trial. Diabetes 1997; 46:1829–39 Parving HH, Jacobsen P, Tarnow L et al. Effects of deletion polymorphism of angiotensin converting enzyme gene on progression of diabetic nephropathy during inhibition of angiotensin converting enzyme. BMJ 1996; 313:591–4 Quinn M, Angelico MC, Warram JH et al. Familial factors determine the development of diabetic nephropathy in patients with IDDM. Diabetologia 1996; 39: 940–5 Moczulski DK, Rogue JJ, Antonellis A, Warran JH, Krolewski AS. Major susceptibility locus for nephropathy in type 1 diabetes on chromosome 3q: results of novel discordant sib-pair analysis. Diabetes 1998:47:1164–9 Long-term trials
The Diabetes Control and Complications Trial Research Group. Effect of intensive therapy on the development and progression of diabetic nephropathy in the Diabetes Control and Complications Trial. Kidney Int 1995; 47:1703–20 The Diabetes Control and Complications Trial Research Group. The relationship of glycaemic exposure (HbAlc) to the risk of development of progression of retinopathy in the diabetes control and complications trial. Diabetes 1995; 44: 968–83 The Diabetes Control and Complications Trial Research Group. The effect of intensive therapy on measures of autonomie nervous system function in the Diabetes Control and Complications Trial (DCCT) Diabetologia 1998; 41:416–23
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The Diabetes Control and Complications Trial Research Group. Effect of Intensive diabetes management on macrovascular events and risk factors in the Diabetes Control and Complications trial. Am J Cardiol 1995; 75:894–903 United Kingdom Prospective Diabetes Study Group. United Kingdom Prospective Diabetes Study (UKPDS) 33: Intensive blood glucose control with sulphonylurea or insulin compared with conventional treatment and the risk of complications in patients with type 2 diabetes. Lancet 1998; 352:837–53 Predictive modelling studies
Eastman RC, Javitt JC, Herman WH et al. Model of complications in NIDDM.I. Model construction and assumptions. Diabetes Care 1997; 20:725- 34 Insulin resistance and complications
Jaap AJ, Shore AC, Tooke JE. Relationship of insulin resistance to microvascular dysfunction in subjects with fasting hyperglycaemia. Diabetologia 1997:40:238–43 Haffner SM, Miettinen H. Insulin resistance implications for type II diabetes mellitus and coronary heart disease. Am J Med 1997; 103:152–63 Donnelly R, Qu X. Mechanisms of insulin resistance and new pharmacological approaches to metabolism and diabetic complications. Clin Exp Pharmacol Physiol 1998; 25:79–87 Cell types involved in microvascular complications
Heilig CW, Brosius FC III, Henry DN. Glucose transporters of the glomerulus and the implications for diabetic nephropathy. Kidney Int 1997; 60: Suppl S9–19 Mauer SM, Lane P, Zhu D, Fioretto P, Steffes MW. Renal structure and function in insulin-dependent diabetes in man. J Hypertens 1992; 10: Suppl S17–20 Hirchi KK, D’Amore PA. Control of angiogenesis by the pericyte: molecular mechanisms of significance. EXS 1997; 79:419–28 Ruggerio D, Lecomte M, Michoud E et al. Involvement of cell-cellinteractions in the pathogenesis of diabetic retinopathy. Diabetes Metab 1997; 23:30–42 Stehouwer CD, Lambert J, Dinker A J, van Hinsberg VW. Endothelial dysfunction and pathogenesis of diabetic angiopathy. Cardiovasc Res 1997; 34:55–68 Valensi P, Cohen-Boulakia F, Attali JR, Behar A. Changes in capillary permeability in diabetic patients. Clin Hemorheol Microcirc 1997; 17:389–94 Cellular mechanisms causing complications
Williamson JR, Chang K, Frangos M et al. Hyperglycemic pseudohypoxia and diabetic complications. Diabetes 1993; 42:801–13
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King GL, Brownlee M. The cellular and molecular mechanisms of diabetic complications. Endocrinol Metab Clin North Am 1996; 25:255–70 Chibber R, Molinatti PA, Rosatto N, Lambourne B, Kohner EM. Toxic action of advanced glycation end products on cultured retinal capillary pericytes and endothelial cells: relevance to diabetic retinopathy. Diabetologia 1997; 40:156–64 Koya D, King GL. Protein kinase C activation and the development of diabetic complications. Diabetes 1998; 47:859–66 Kennedy AL, Lyons TJ. Glycation, oxidation, and lipoxidation in the development of diabetic complications. Metabolism 1997; 46 (Suppl 1): 14–21 Nishimura C, Hotta Y, Gui T et al. The level of erythrocyte aldose reductase is associated with the severity of diabetic retinopathy. Diabetes Res Clin Pract l997; 37:173–7 Giugliano D, Ceriello A, Paolisso G. Oxidative stress and diabetic vascular complications. Diabetes Care 1996; 19:257–67 Haneda M, Araki S-I, Togawa M et al. Mitogen-activated protein kinase cascade is activated in glomeruli of diabetic rats and glomerular mesangial cells cultured under high glucose conditions. Diabetes 1997; 46:847–53 Macrovascular complications McMillan DE. Development of vascular complications in diabetes. Vasc Med 1997; 2:132–42 Tschoepe D, Roesen P. Heart disease in diabetes mellitus: a challenge for early diagnosis and intervention. Exp Clin Endocrinol Diabetes 1998; 106:16–24 Steiner G. Diabetes and atherosclerosis—a lipoprotein perspective. Diabetes Med 1997; 14 Suppl 3:538–44 Katsumori K, Wasada T, Kuroki H et al. Prevalence of macro- and microvascular diseases in non-insulin-dependent diabetic and borderline glucose-intolerant subjects with insulin resistance syndrome. Diabetes Res Clin Pract 1995; 29: 195–201 Ross R. Pathogenesis of atherosclerosis: a perspective for the 1990s Nature 1993; 263:801–10 Animal models used in research of diabetic of complications
Lindsay RM, Jameison NS, Walker SA et al. Tissue ascorbic acid and polyol pathway Metabolism in experimental diabetes. Diabetologia 1998; 41:516– 23 (STZ- diabetes and spontaneously diabetic BB rat) Vicario PP, Slater EE, Saperstein R. The effect of ponalrestat on sorbitol levels in the lens of obese and diabetic mice. Biochem Int 1989; 19:553– 61 [ob/ob mouse and db/db mouse]
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Reddi AS, Velasco CA, Reddy PR et al. Diabetic microangiopathy in KKmice. VI. Effect of glycemic control on renal glycoprotein metabolism and established glomerulosclerosis. Exp Mol Pathol 1990; 53:140–51 [KK mouse] Kowano K, Hirashima T Mori S et al. Spontaneously long-term hyperglycaemic rat with diabetic complications. Otsuka Long-Evans Tokushima Fatty (OLETF) strain. Diabetes 1992; 41:1422–8 [OLETF rat] O’ Donnell MP, Crary OS, Oda H et al. Irbesartan lowers blood pressure and ameliorates renal injury in experimental non-insulin-dependent diabetes mellitus. Kidney Int 1997; 63: Suppl S218–20 [Obese Zucker rat] Cerasi E, Kaiser N, Gross DJ. From sand rats to diabetic patients: is noninsulin -dependent diabetes mellitus a disease of the beta cell? Diabetes Metab 1997; 23 Suppl 2:47–51 [Israeli sand rat, Psammomys obesus] Agardh CD, Agardh E, Zhang H, Ostenson CG. Altered endothelial/pericyte ratio in Goto-Kakizaki rat retina. J Diabetic Complications 1997; 11:158– 62 [GKrat] Treatment related to the pathogenesis of complications
Pugliese G, Pricci F, Pesce C et al. Early, but not advanced, glomerulopathy is reversed by pancreatic islet transplants in experimental diabetic rats. Diabetes 1997;46:1198–1206 The Microalbuminuria Captopril Study Group. Captopril reduces the risk of nephropathy in IDDM patients with microalbuminuria. Diabetologia 1996; 39: 587- 93 Tribe RM, Poston L. Oxidative stress and lipids in diabetes: a role in endothelium vasodilator dysfunction? Vasc Med 1996; 1:195–206 Timimi FK, Ting HH, Haley EA et al. Vitamin C improves endotheliumdependent vasodilatation in patients with insulin-dependent diabetes mellitus. J Am Coll Cardiol 1998; 31:552–7 Zeigler D, Gries FA. Alpha-lipoic acid in the treatment of diabetic peripheral and cardiac autonomie neuropathy. Diabetes 1997; 46 Suppl 2: S62–6 Italian Study Group for the Implementation of the St Vincent Declaration, et al. A meta-analysis of trials on aldose reductase inhibitors in diabetic peripheral neuropathy. Diabetic Medicine 1996; 13:1017–26 Cooper M. Pathogenesis, prevention and treatment of diabetic nephropathy. Lancet 1998; 352:213–19
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Introduction to epidemiological and statistical design
Coggon D, Rose G, Barker DJP. Epidemiology for the uninitiated. Third Edition. London: BMJ Publishing Group, 1995 Michelson S, Schofield T. The Biostatistics Cookbook. The Most User-Friendly Guide for the Bio/Medical Scientist Dordrecht, Netherlands: Kluwer Academic Publishers, 1996
6. Establishing a Career in Diabetes Research
Getting started To build a career in any field of research requires a solid foundation, association with a respected research group and good collaborators. Here are some ways of getting started.
Membership in professional diabetes and endocrine societies Membership in professional diabetes-related societies can often require nomination by a member of the Society. Details for Membership of Societies with national and international members are included here.
Broad National and International Membership Further information on these societies is provided below. American Diabetes Association http://www.diabetes.org The Endocrine Society http://www.endo-society.org/ European Association for the Study of Diabetes http://www.uni-duesseldorf.de/ WWW/EASD/ International Diabetes Federation http://www.idf.org/
National Societies Australian Diabetes Society Canadian Diabetes Association Diabetes New Zealand The British Diabetic Association
http://www.racp.edu.au/ads http://www.diabetes.ca/ http://www.diabetes.org.nz/ http://www.diabetes.org.uk
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Diabetes Research
Researching the literature and publishing diabetesrelated research
Researching the literature There are a number of journals in which research specifically relating to diabetes is published. Many of the basic science Journals will publish investigations into molecular and cellular events common to normal and pathogenic cell function. These basic science journals include the following, and their content and policy for publication can be readily found with the following Internet links. The Journal of Clinical Investigation The Biochemical Journal The Journal of Biological Chemistry Endocrine EMBO Journal
http://www.jci.org/ http://www.portlandpress.co.uk/ bj.htm http://www.jbc.org/ http://humanapress.com http://www.emboj.org/?n18
A complete list of Journal with on-line text can be found at: http://www.ncbi.nlm.nih.gov/PubMed/fulltext.html
BioMedLink is an evaluated and annotated database of more than 4000 www resources for biological and medical researchers (http:// biomedlink.com). BioMedNet: http://biomednet.com is an internet community for biological and medical researchers and the BioMedNet Magazine HMS Beagle (http://biomednet.com/hmsbeagle) is a site which publishes frequent contemporary essays on grant application writing, funding and resources. Cell and Molecular Biology Online has an extensive listing of molecular bio-logic information and electronically published journals (http:// www.cellbio.com) A Dictionary of Cell Biology can be found at http://www.mblab.gla.ac.uk/ ~julian/Dict.html The Weizmann Institute of Science Genome and Bioinformatics publishes a data base of extensive information on protein and gene-related biological factors (http://bioinformatics.weizmann.ac.il/hotmolebase). Journals online from The Endocrine Society including: Endocrinology; The Journal of Clinical Endocrinology & Metabolism; Molecular Endocrinology and Endocrine Reviews can be found at http://endo.edoc.com.
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Publishing diabetes-related research The major journals publishing diabetes-related reports may have different aims and scope. It is strongly recommended that a recent edition of each journal is consulted for the Current Editor-in-Chief and for Advice to Authors. The journals and their contact information include the following.
Diabetes http://www.diabetes.org/diabetes
This is the journal of the American Diabetes Association (ADA). Diabetes publishes original research about the physiology and pathophysiology of diabetes and the reports can be on any aspect of laboratory, animal or human research from worldwide investigative groups. An emphasis is placed on “investigative reports focussing on areas such as the pathogenesis of diabetes and its complications, normal and pathologic pancreatic islet function and intermediary metabolism, pharmacological mechanisms of drug and hormone action, and biochemical and molecular aspects of normal and abnormal biological processes. Studies in the areas of diabetes education or the application of accepted therapeutic and diagnostic approaches to patients with diabetes mellitus are not published” (from the Journal’s Mission Statement). Diabetes also publishes organisational information for the ADA Membership and details of many related conferences and meetings.
Diabetes Care http://www.diabetes.org/diabetescare
This is another journal of the American Diabetes Association with a focus on clinical and applied research and education. “The journal publishes original articles on human studies in the following categories: 1) clinical care/education/nutrition, 2) epidemiology/health services/psychosocial research, 3) emerging treatments and technologies and 4) pathophysiology/ complications”. The ADA also publishes: • Diabetes Reviews, which summarises a specific topic in depth in each issue. Each review contains original material, describes basic and clinical investigations, discusses the clinical and physiological significance and places this in context of previously published information • Clinical Diabetes and Diabetes Spectrum, which cover latest treatment strategies and clinical management of diabetes.
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Diabetes Metabolism: Research and Reviews http://www.interscience.wiley.com/jpages/1520–7552/
This is a print and electronic journal considering articles on the etiology and pathogenesis of diabetes, together with issues of treatment and management related to patient care. It welcomes submissions in areas of controversy
Diabetes Nutrition and Metabolism: Clinical and Experimental. This is the official english language journal of the Italian Society of Diabetology and has a Regional Editor for Australasia. To date, the journal does not have a www address. The current Editor-in-Chief is: Professor Paolo Brunetti Diab Nutr Metab C.P. 1043 Ufficio A.P. 1–06124 Perugia Italy
Diabetes, Obesity and Metabolism This journal will consider for publication papers relevant to any aspect of clinical and experimental pharmacology in studies related to diabetes, obesity and metabolism. It contains original research papers, reviews and news and views and follows the evaluation and clinical application of drug discoveries. Information on the journal can be found at http://www.blackwellscience.com
Diabetes Research and Clinical Practice A full search Table of Contents can be found at http://www.elsevier.nl and an email service provides articles 3 weeks ahead of publication. This is the official journal of the International Diabetes Federation/Western Pacific region. This journal publishes research articles and reviews in all areas relevant to diabetes.
Diabetic Medicine This is the journal of the British Diabetic Association. It is a clinical research journal that publishes original research and reviews on all aspects of diabetes and its care. “The journal covers topics ranging from fundamental research
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to better health care delivery, to increase knowledge about the aetiology and pathogenesis of diabetes and its complications and promote new ideas about the disease management”. Information on the journal can be found at http://blackwell-science.com The current Editor is: Professor Stephanie A Amiel Department of Medicine King’s College School of Medicine and Dentistry Bessemer Road London, SES 9PJ United Kingdom
Diabetologia http://link.springer.de/link/service/journals/00125/index.htm
This is the Journal of the European Association for the Study of Diabetes (EASD) and publishes reports of clinical and experimental work on all aspects of diabetes research and related subjects. The journal also provides organisational information for the EASD membership and details of many related conferences and meetings.
Transplantation http://www.transplantjournal.com/
This is the official journal of the Transplantation Society and publishes articles of both a clinical and a basic research nature on all aspects of transplantation, both related and unrelated to diabetes.
Journal of Diabetes and its Complications http://www.elsevier.nl/inca/publications/store/5/0/5/7/7/0/index.htt
This journal has the aim to provide access to information which may allow the prevention of complications of diabetes. In addition to general articles on clinical aspects of diabetes, the journal publishes articles on basic research.
Diabetes Researc h Funding Most initial funding is obtained as mentor-based pre-doctoral and postdoctoral fellowships in which a newly qualified researcher will apply together with a more senior investigator. It is strongly recommended that you contact
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your Institution’s Office of Sponsored Research, or equivalent; which will be able to provide information on research training grants and the preparation of research proposals. Information and submission proposals for major funding can be obtained from the following sites.
International Juvenile Diabetes Foundation International [JDFI]
http://www.jdfcure.org/research/ Highly competitive national/international funding for diabetes research as Grants-in-Aid, Career Development Fellowships and Postgraduate Fellowships is provided by this organization. The web site also provides detailed descriptions and outcome statements on most currently funded JDFI research. Most countries have their own branches of the JDFI for researchers to work through.
Australia National Health and Medical Research Council [NHMRC]
http://www.health.gov.au/nhmrc/ Postdoctoral Fellowships include: CJ Martin Fellowship, Neil Hamilton Fairley Fellowship (tenable overseas and in Australia), Peter Doherty Fellowship, and Australian Clinical Research Postdoctoral Fellowship (tenable in Australia). Diabetes Australia Research Trust
This has a Research Grants Program which supports basic, clinical or applied research. Grants are used to support a particular program of work under the supervision of a responsible investigator. A Research Fellowship is also provided to undertake clinical or experimental research in an Australian university, affiliated teaching hospital, research institute or centre. Information is available through the research offices of universities. Juvenile Diabetes Foundation Australia
For details see International JDFI above. Contact details of the Australian branch of this organization are: JDF Australia 48 Atchison Street St Leonards NSW 2065 http://www.jdfa.org.au
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Juvenile Diabetes Foundation Australia also provides travel grants for young Australians researchers to travel to research institutions and scientific meetings for the purpose of gaining expertise in the study of type 1 diabetes.
Canada Medical Research Council of Canada
http://www.mrc.gc.ca/
Europe The European Science Foundation
This organisation coordinates Research Councils at the European level. http:/ /www.esf.org/ and associated links A European Community Research and Development Information Service is available on the European Community Research site and links to a Europewide network for industrial research and development http://www.cordis.lu/ http://www.cordis.lu/fp5/home.html http://www.eureka.be/ http://www.eurekalert.org/
Israel The United States-Israel Binational Science Foundation funds a wide range of investigations including diabetes-related research. Their contact address is: United States-Israel Binational Science Foundation 2 Alharizi St P.O.B 7677 Jerusalem 91076 e-mail:
[email protected] Fax: 972–2–5633287
New Zealand The Health Research Council of New Zealand
http://www.hrc.govt.nz/sites.htm
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South Africa The Medical Research Council
http://www.mrc.ac.za/
United Kingdom The Medical Research Council has information on Research Grant schemes and Guidance Notes at: http://www.mrc.ac.uk A data base containing over 400 descriptions of biomedical research funding, principally in the UK with a searchable data base and access to Science Policy Information News is provided by the Wellcome Trust Wisdom can be found at: http.//wisdom.wellcome.ac.uk/wisdom/fundhome.html
United States of America National Institutes of Health
National Institutes of Health Biomedical Research Training Resources and Opportunities can be found at the NIH Training web site. http:// www.nih.gov/ or at http://www.nih.gov/training/ American Diabetes Association
Competitive funding for research proposals (Research Awards, Career Development Awards, Clinical Research Awards, Lions SightFirst Retinopathy Awards, Medical Scholars Awards) and Training Awards (Mentor-Based Postdoctoral Fellowships) are available from the ADA. http://www.diabetes.org/diabetes or http://www.diabetes.org/research/default.asp The American Association for the Advancement of Science
The American Association for the Advancement of Science has funding opportunities for training in the biological and medical sciences http://www.grantsnet.org/ FEDIX
FEDIX is a free e-mail service that automatically delivers research and educational funding opportunities within specific areas of interest http://www.rams-fie.com/opportunity.htm
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Research funding opportunities and a US-wide listing of University Departmental and Sponsored Research Offices is available at http:// tram.rice.edu/TRAM/
Diabetes-related WWW sites There are a number of highly informative Internet sites with open access. Highly recommended sites with research-oriented information are as follows. The Academy for the Advancement of Diabetes Research and Treatment http://drinet.med.miami.edu/ Includes information on islet transplantation. The American Diabetes Association http://www.diabetes.org American Society of Transplantation formerly the American Society of Transplant Physicians http://www.a-s-t.org The American Society of Transplant Surgeons http://www.asts.org The Barbara Davis Center for Childhood Diabetes http://www.uchsc.edu.misc/diabetes/bdc.html Links to many other diabetes-related Web sites. Canadian Diabetes Association has a very good diabetes-related site of general interest with many further links. http://www.diabetes.ca/atoz/index.htm The Center for Disease Control Diabetes Home Page http://www.cdc.gov/nccdphp/ddt/ddthome.htm Diabcare http://www.diabcare.de/ The Diabetes Control and Complication Trial (DCCT) http://www.niddk.nih.gov/health/diabetes/pubs/dcctl/dcct.htm Diabetes Monitor http://www.diabetesmonitor.com/ Diabetes New Zealand http://www.diabetes.org.nz/ European Association for the Study of Diabetes (EASD) http://www.uni-duesseldorf.de/WWW/EASD/ Newsletter for the Immunology of Diabetes Society http://www.dem.it/IDSNEWS Insulin-Free World Foundation http://www.insulin-free.org
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International Diabetes Federation http://www.idf.org/ International Pancreatic Transplantation and Islet Association http://www.jr2.ox.ac.uk/ipita/ Has useful links related to pancreas and islet transplantation. The International Pancreatic Transplantation Registry A worldwide scientific database from institutes performing pancreas transplants. http://www. surg.umn.edu/iptr/ The Joslin Diabetes Center http://www.joslin.harvard.edu/ The Juvenile Diabetes Foundation International http://www.jdfcure.com/ The Karolinska Institute Library with diabetes and endocrine disease information http://www.mic.ki.se/Diseases/c19.html The National Institutes of Health NIDDK (Diabetes) http://www.niddk.nih.gov/ Transweb A site with information on transplantation and donation. http://transweb.org The UKPDS site with results and information slides which can be downloaded. http://www.drl.ox.ac.uk/ukpds/index.html The World Health Organisation site with links to the World Diabetes Newsletter of WHO http://www.who.int/ncd/dia/dia_home.htm
GLOSSARY
Simple definitions have been used for the terms in this Glossary. For more detailed information the reader is encouraged to access an internet service, such as Medline http://www.ncbi.nlm.nih.gov/PubMed/, using the term in this Glossary together with “and diabetes”. Further simple descriptions of general or diabetes-related terms may be found in one of the many www-based glossaries eg. http://www3.bc.sympatico.ca/me/patientsguide/index.htm http://www.graylab.ac.uk/omd/index.html http://www.childrenwithdiabetes.com/d_04_200.htm http://www.niddk.nih.gov/health/diabetes/pubs/dmdict/dmdict.htm Acellular capillary a segment of capillary with a tube-like structure where endothelial cells and pericytes are lost. Advanced glycation accelerated chemical modification of proteins by reducing sugars leading to cross-linked proteins. Aerobic requiring oxygen for respiration. Allograft/Allotransplantation transplantation of tissue between two members of the same species, but of different genetic makeup. Aminoguanidine a hydrazine compound which reacts with Amadori products to prevent further glycation of proteins. Anaerobic reaction occurring in the absence of molecular oxygen. Antecubital vein or structure located in front of the elbow joint Arterialised to transform venous blood into arterial blood. Apolipoprotein proteins on the surface of the lipoprotein complex that bind to specific enzymes or transport proteins. Arachidonic acid an unsaturatsed fatty acid which is the substrate for prostaglandin production. Arterialized venous blood is venous blood containing a large quantity of arterial blood from A/V shunts that open in the heat. Atheroslerosis progressive narrowing of the arteries with increased lipid deposits and overgrowth of smooth muscle cells.
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Autoimmune an ongoing self-destructive process by which one’s own immune cells, such as lymphocytes and macrophages, destroy specific cell types in the body. Autonomie neuropathy damage to the unconscious nervous system (sympathetic and parasympathetic nervous system). Autoxidation metal-catalysed oxidation. Basement membrane an organized extracellular matrix found around cells. It consists of two layers (lamina)—a basal lamina and a reticular lamina) containing collagens. BCG Bacille Calmette Guerin, an extract of Mycobacterium tuberculosis with no infectivity used in tuberculosis vaccination. Extracts stimulate lymphocytes and leukocytes and are used as adjuvant substances that stimulate or augment the immune response. Bioactive a substance which has an effect on living tissue. Biothesiometer an instrument which detects abnormal nerve function by testing the threshold of perception of a non-invasive vibration. Capillary vessels that connect the arterial and venous circulation forming a microvascular network. Capillary walls are selectively permeable to allow interchange between blood and tissue. Cardiovascular related to the heart and blood vessels (arteries, arterioles, capillaries, venules and veins). Catabolism destructive metabolic process by which organisms convert substances into excreted compounds to yield energy. Cataract an opacity on or in the lens or capsule of the eye which impairs vision. Cell-mediated immunity an immune response that involves effector Tlymphocytes. These cells originate from lymphoid stem cells which migrate from the bone marrow to the thymus and differentiate under the influence of thymic hormones. Chronic pancreatitis chronic inflammation of the pancreas. Collagens a superfamily of fibrillar extracellular matrix proteins. Contracterai on the opposite side. Coronary arteries the arteries which supply the heart muscle with oxygenated blood and which become narrowed or occluded in coronary artery disease. Cytokines small proteins (in the range of 5–20 kD) released by cells and which affect the function of other cells.
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Deproteinise removing all proteins from a solution. Derivatise to obtain one substance from another by addition of a chemical moiety. Dileucine motif two adjacent leucine amino acids within a protein that serve to guide the protein to a specific location within the cell. Disaccharides a class of sugars composed of two glycosidically linked single sugars. Discordant sib-pair analysis the use of siblings in which one is affected and the other unaffected by a disease to gain evidence for a major gene effect Investigation of the polymorphism of candidate genes is made stronger when both sibs have the same disease but differ in one aspect e.g., Sib pairs both with long-standing diabetes where only one is affected by diabetic nephropathy. Disulphide bond a linkage formed between the SH groups of two cysteine moieties either within or between peptide chains. Dyslipidaemia abnormal concentrations of any or all of the lipids in the plasma, such as cholesterol, triglycerides, and lipoprotein. Eluant the term for the fluid (such as a buffer solution) used in column chromatography to separate the components of a mixed sample. Encapsulation placing of cells inside a device which is transplanted so as to protect the cells from destruction by the immune system of the body. Endothelial the layer of cells that line the cavity of the heart and blood vessels. A number of endothelial factors act to modify vascular tone. End-stage renal failure the condition in which a patient has insufficient kidney function to support life and in which a new kidney or dialysis is needed for survival. Esterification the process of converting an acid into an alkyl or aryl derivative. In this case it consists of the reaction of a fatty acid with cholesterol. Exocrine pancreas the part of the pancreas that secretes its products through ducts or canals. Extracellular matrix material produced by cells and secreted into their surrounding. Many properties of the extracellular matrix determine the characteristics of tissue. Basement membranes are part of the extracellular matrix. Components of the extracellular matrix are fibres (of collagen and elastin), proteins (including fibronectin and laminin) and structural sulphated and non-sulphated polysaccharides. FAS a transmembrane protein (35 kD) on the cell surface that mediates apoptosis (programmed cell death).
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Fatty streaks a fatty patch on the artery wall caused by accumulation of cholesterol in foam cells (lipid-laden macrophages). Fibrinolysis the solubilisation of fibrin in a blood clot by the proteolytic action of plasmin. Insoluble fibrin is formed from fibrinogen by the proteolytic action of thrombin. Fibronectin a high molecular weight glycoprotein that occurs in an insoluble form in extracellular matrix or soluble form in plasma. Multiple domains in the protein allow interaction with other extracellular matrix components and with the surface of cells. Flow-dependent dilatation a mechanism for matching local blood flow to the requirements of a tissue. This involves the ability of cells lining the arterial blood vessels to respond to blood flow by the release of substances which dilate the vessel to increase blood flow. Gene transcription the synthesis of RNA by transcription from a gene DNA template by RNA polymerase. Glomerular filtration the filtration of substances through the glomerulus. The glomerular filtration rate is a measurement of the renal clearance of a marker that is freely filtered by the kidney and that does not undergo metabolism, or is secreted or absorbed by the kidney tubules. Markers may be radioactive or non-radioactive. Glomerulosclerosis the loss of functioning glomerulus by sclerosis. In diabetic glomerulosclerosis the mesangial region takes on a rounded nodular appearance and the basement membrane is thickened. Glomerulus the structures which form the functioning filtration unit of the kidney, comprised of capillary blood vessels, mesangial cells, epithelial cells (podocytes) and extracellular matrix within a capsule. Gluconeogenesis synthesis of glucose from non-carbohydrate precursors, such as pyruvate, amino acids and glycerol. Glucose turnover/Glucose flux rate of appearance and disappearance of glucose from the plasma. Glucose transporter protein that transports glucose across a plasma membrane. Glucopenia low glucose level in plasma. Glycated haemoglobin formed by the non-enzymatic interaction between glucose and the amino groups of the valine and lysine residues in haemoglobin. Glycogenorysis the enzymatic breakdown of glycogen into glucose and glucose-1-phosphate. Glycolysis the conversion of glucose to pyruvate. It generates ATP without consuming oxygen and is thus an anaerobic pathway.
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Glycoxidation oxidation of glucose in the presence of reactive oxygen species. Granzyme a family of proteases found in cells of the immune system involved in cell killing. Haemorrhage the escape of blood from blood vessels. HDL cholesterol cholesterol present in the High Density Lipoprotein fraction. Heterozygous containing two different alleles of the same gene. Hexose monophosphate shunt the series of biochemical reactions which convert glucose-6-phosphate to ribulose-5-phosphate with an associated generation of NADPH. Histocompatibility genes genes which encode cell-surface glycoproteins that bind processed antigens and export these peptides to the cell surface for presentation to T-cells. Homozygous containing two copies of the same allele. Hydrophilic water-loving, easily mixes with water. Hydrophobia water-fearing, does not mix with water. Hyperglycaemia greater than normal blood glucose levels. Human leucocyte antigen (HLA) a group of 4 genetic markers that are found on specific loci on chromosome 6 (in humans). Each locus contains several allelles corresponding to various diseases/conditions. The markers are also used to determine tissue compatibility. Hypertension persistently high arterial blood pressure. Hypertriglyceridaemia elevated triglyceride levels. Hyperuricaemia elevated uric acid levels in the bloodstream. Hypoglycaemia an abnormally low concentration of blood glucose. Hypoxia insufficient oxygen. Immunogenic capable of being recognized by the immune system. Immunosuppressive suppressing the immune system. Incretins hormones produced in the gut in response to food that enhance the effect of glucose to stimulate insulin secretion. Integrin superfamity of cell surface proteins that are involved in binding to extracellular matrix components. Interleukin 2 receptor binding site for interlekin 2, a cytokine released from activated T-lymphocytes which acts as a growth factor for other Tlymphocytes Intravenous Administered direcdy inside a vein.
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Ischaemia inadequate blood supply. Ischaemic heart disease Presence of partially blocked coronary arteries causing lack of nutient and oxygen supply to the cardiac muscle. Keratopathy a dysfunction of the cornea. Ketoacidosis excess production of ketones which lowers blood pH. Ketones acidic breakdown products of fat metabolism. Kinase an enzyme that is able to place a phosphate (PO 3–) group on a protein, 4 usually on a specific amino acid. Km concentration of substance giving half-maxinal response. Lipoprotein particles found in serum with a spherical hydrophobic core of triglycerides or cholesterol esters surrounded by a hydrophilic monolayer of phospholipids, cholesterol and apolipoproteins. Macroalbuminuria albumin excretion of greater than 300 mg/day in human subjects. Macrovascular relating to large vessels. Macula a small area of the retina where sharp images are perceived. This area provides central vision, reading vision and colour vision. Maillard reaction a non-enzymic reaction in which aldehydes, ketones or reducing sugars react with amino acids in peptides or proteins. Major histocompatibility complex (MHC) protein markers found on the outer cell membrane which are the product of a cluster of genes on chromosome 6 (human), concerned with antigen production and critical to transplantation. Major susceptibility locus a genomic region which is shown to be significantly linked to a specific trait. Mesangial matrix the extracellular matrix which supports the capillary loops in the glomerulus. Metabolic syndrome presence in the one individual of obesity, insulin resistance, glucose intolerance, hypertension, dyslipidaemia and hyperuricaemia. Microalbuminuria urinary levels of albumin between 30 and 300 mg/day. Microaneurysm swelling and distortion of a small blood vessel. Monofilament used as a neurofiinction test which measures the ability to perceive pressure from filaments of varying calibre which bend under a known force. Morbidity the amount of sickness caused by a disease. Mutation a permanent transmissible change in the genetic material, usually in a single gene.
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Nephropathy damage to the kidneys, especially the structural and functional unit called the nephron. Neuropathy damage to the nerves of the body. Nitric oxide a potent vessel relaxing compound produced from L-arginine by the enzyme nitric oxide synthase. Non-proliferative retinopathy also called early background retinopathy, which occurs when blood vessels in the retina are damaged and leak fluid or blood. This causes the retina to swell and form deposits or exudates. Depending on the location this damage may not affect vision. Oedema collection of fluid in the intercellular tissue spaces of the body, usually in a dependent part if one is standing. Oral glucose tolerance test test performed to determine if a person has diabetes. It requires the ingestion of 75 g glucose and the measurement of blood glucose levels 2 hours thereafter. Oxidative stress a highly oxidised environment within cells where chemically active free radicals are formed. Free radicals must receive or release electrons to achieve stable configuration and this process can damage large molecules of lipid and protein within the cell. Pancreatectomy surgical removal of the pancreas. Pancreatitis inflammation of the pancreas. Pathophysiology the abnormality in a physiological process that leads to disease. Pedal pulses the pulses in the foot which are examined by touch to evaluate patients with arterial disease. Pentose phosphate pathway see Hexose monophosphate shunt Perform molecule stored in granules of cytotoxic T lymphocytes which punches holes in the outer membrane of target cells. Pericyte a contractile smooth-muscle-like cell associated with microvessels. Polydipsia drinking large quantities of fluids. Polygenic a disease is polygenic when the abnormality results from the combined action of alleles of more than one gene. Polyuria passage of copious quantities of urine. Pre-diabetes the state before the onset of the symptoms of type 1 diabetes in which there is on-going destruction of ß cells Probabilistic model a statistical evaluation of likelihood. As used in the present context; a model to predict the natural course of a disease for comparison to the actual measured outcome.
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Prohormone the precursor of a protein hormone that requires removal of part of its sequence to make the active, mature form of the hormone. Proteoglycan a widely distributed class of proteins found on cell surfaces, within intracellular vessicles and in the extracellular matrix which are defined by the glycosaminoglycan polysaccharides which are linked to their protein core. Receptor for advanced glycation end products (RAGE) cell surface receptors which recognise chemically damaged proteins, including proteins with advanged glycation modification. These receptors are widely distributed amongst cells including endothelial cells, smooth muscle cells and macrophages. Reactive oxygen species formed by the transfer of one electron to oxygen to give superoxide anion radical which is catalysed further to hydrogen peroxide and then to highly reactive hydroxyl radicals. Reactive oxygen species react with biological macromolecules (lipids, proteins, nucleic acids and carbohydrates) to generate a second radical which can react with a second macromolecule in a chain reaction. Rough endoplasmic reticulum membrane organelle that forms sheets and tubules. It binds ribosomes engaged in translating mRNA for secreted proteins and the majority of transmembrane proteins. Secretagogue substance that induces secretion from cells such as insulin from pancreatic islet ß cells. Sensorimotor polyneuropathy nerve fibre damage with loss of both myelinated and unmyelinated fibres. Smooth muscle muscles of the body controlled by the autonomie nervous system. Starch form in which carbohydrates are stored in plants. Thrombosis formation of a solid mass, a thrombus, in the lumen of a blood vessel or the heart This is usually a result of damage to the vessel wall. Transcription factor a protein which binds to a regulatory site on a gene to activate transcription by RNA polymerase. Transforming Growth Factor-ß (TGF-ß) a family of cytokines which play a diverse role in control of growth, development and differentiation. Multiple forms signal through receptor complexes to interact with a set of evolutionary conserved proteins known as SMADs which translocate to the nucleus to initiate target gene transcription.
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Transgenic An organism that has had genes from another organism put into its genome through recombinant DNA techniques. Translocation as used in this book, it means the movement of a protein from one location to another within the cell. Vascular Endothelial cell Growth Factor (VEGF) a glycoprotein factor, originally described as vascular permeability factor due to its ability to increase microvascular permeability to plasma proteins. It stimulates endothelial cells and induces the sprouting of new bloodvessels (angiogenesis). Xenotransplantation transplantation of tissues from one species to another.
Index
a cell 23, 28 Acetyl CoA 20, 33 Acetyl coenzyme A carboxylase 27, 33 Adrenaline 28 Advanced glycation end product 87, 115 Albuminuria 78 Aldose reductase 90 inhibitor 94 Amino acids 15, 34 Aminoguanidine 93, 115 Ammonia 34 Amylo (1→4) to (1→6) transglycolase 19 Angiotensin converting enzyme 93 Animal models of Type 2 diabetes 66 spontaneous 66, 91 induced 67 transgenic 67 Anion exchange resin 38 Apoprotein 31, 115 Apoprotein (a) 33, 91 Apoptosis 87 Arginine 64 Arterialised venous blood 69 Atherosclerosis 75, 115 ATP 20 Autoimmune destruction 43–4, 48, 54–5, 116 Autonomie neuropathy 47, 80, 116 Autoxidation 87, 116 Azaserine 63 ß cells 1, 17, 23 ß counter 38 ß-hydroxy-ßmethylglutaryl-CoA (HMGCoA) 33 Brown fat 17 Biothesiometer 79, 116 BB rat 54
Calcium channel 24 CAMP 26 Capillary 79, 116 Carbohydrate digestion 22 Cardiovascular disease 80 Cataract 79, 116 Ceramide 64 CD4 lymphocytes 43 CD8 lymphocytes 43 Cholesterol 29 Cholestrol ester 31 Chylomicron 31 Citrate 33 Citric acid cycle see Tricarboxylic acid cycle Clinical trials DCCT 81, 82 EURODIAB 81 UKPDS 82 Collagenase 35, 48, 50, 57 Complications late 75 macrovascular 10–11, 75, 90 microvascular 9, 85, 89 Coronary arteries 10 Cortisol 29 C-peptide 23 δ cell 23 Dehydroepiandrosterone 63 Diabetes mellitus clinical onset 1 definition 1 types 1 Dileucine motif 17 Disaccharide 22 D-ribose 22 D-ribose 5-phosphate 22 Dyslipidaemia 66 Dysthesia 80 125
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Eicosapentaenoic acid 30 Encapsulation 50–1, 56, 117 Epinephrine—see adrenaline Essential fatty acid 30 Extracellular matrix 86, 117 Fatty acid synthase 27 FAS 43, 117 FAS ligand 43, 54 Fatty acid 29 Fatty acid synthase 33 Free radical 94 Fructosamine 5 Fructose 1,6 bisphosphate 15 Fructose 1,6 bisphosphatase 15 Fructose 6 phosphate 15, 22 Gangrene 10 Gastric inhibitory polypeptide (GIP) 25 Gene therapy 52–4 Genetic polymorphism 81 GFAT see Glutamine: fructose-6-phosphate amidotransferase Glomerular basement membrane 78 Glomerular filtration rate 77, 118 Glomerulosclerosis 78, 118 Glucagon 7, 23, 28 Glucagon-like peptide 1 (GLP-1) 25 Glucantransferase-oligo (α1→6) to (α1→4) 14 Glucokinase 17, 52, 54, 64 Glucometer 4 Gluconeogenesis 8, 13, 15, 118 Glucopenia 29 Glucosamine 6 phosphate 22 Glucose 13, appearance 13 disappearance 15 metabolism 18 transport 15, 18 turnover 13, 36 Glucose-alanine cycle 34 Glucose clamp 38, 68 Glucose 1 phosphate 14 Glucose 6 phosphate 15 Glucose 6 phosphatase 15 Glucose transporter 15, 52, 54, 118 sodium dependent 18, 22 Glutamic acid decarboxylase 44
Glutanine 22 Glutamine:fructose-6-phosphate amidotransferase 22, 63 GLUT1-GLUT6 17 Growth hormone 29 Glycated haemoglobin 4, 81, 118 Glycerol 15 Glycogen 13 synthesis 18 Glycogen phosphorylase 14, 19 Glycogen synthase 19, 26 Glycogen synthase kinase 26 Glycogenin 19 Glycogenolysis 8, 13, 118 Glycolysis 19, 118 Glycosyl (4®6) transferase 19 Glycoxidation 94 Granzyme 43, 119 Haemorrhage 78 Hexokinase 17, 20 Histocompatibility genes 45, 119 Hypoglycaemia 7–8, 29 Heart 17 Heart attacks 10 Hepatic nuclear factor 65 Hexosamine biosynthesis pathway 18, 22, 63 Hexose monophosphate shunt see Pentose phosphate shunt High density lipoprotein (HDL) 33 HMG-CoA see b-hydroxybmethylglutarylCoA HMG-CoA reductase 33 Hypertension 66 Hyperuricaemia 66 Hypoglycaemia 80 Immunosuppression 47 Incretin 25 Intestine 13 Insulin 3–4, 23, 57–8 action 24, 28 secretion 24, 64 synthesis 23 Insulin-producing hepatocytes 53 Insulin radioimmunoassay 57–8 Insulin receptor 28 Insulin receptor substrate (IRS) 28
Index
Insulin resistance 61 genetic 61 glucose-induced 62 lipid-induced 62 Integrin receptor 89 Interferon gamma 43, 54 Interleukin 1ß 43 Intermediate density lipoprotein (IDL) 32 Invertase 23 Islets 2, 23, 48, 50, 56–7 Islet cell antibodies 44 Islet-like cell clusters 48, 56, 57 Isomaltose 22 Intravenous glucose tolerance test (IVGTT) 71 Ketones 5, 120 Ketoacidosis 5, 120 Kidney 13, 17 Knockout mice 54, 92 Krebs cycle see Tricarboxylic acid cycle Lactase 23 Lactose 23 Lactate 15 Lecithin 31 LDL receptor 33 Liver 13, 17 Liberase 48, 50 Linoleic acid 30 Lipids 29 Lipid synthesis 33 Lipoprotein (a) 33 Lipoprotein lipase 31 Lipoprotein metabolism 31 Low density lipoprotein (LDL) 32 Malate 15 Malonyl CoA 33 Maltese 23 Maltose 22 Maturity Onset Diabetes of Youth (MODY) 84 Mesangial cell Mesangial matrix 78 Metabolic syndrome 66, 120 Microaneurism 78, 78, 85 Minimal model 71
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Mitochondria 15 Mitochondrial DNA 65 Mitogen-activated protein kinase 89 Muscle 17 NADPH 22 Nephropathy 9–10, 76–77, 121 Neuropathy 75, 79 Neuroglycopaenia 80 Nitric oxide 44, 121 NOD mouse 54 Non-insulin dependent diabetes see Type 2 diabetes Obesity 63, 66 Oleic acid 30 One pool model 70 Oral glucose tolerance test 6, 121 Oral hypoglycaemic agents 6–7, 76 Oxaloacetate 15 Oxidative stress 92 Palmitic acid 30 Palmitoyl CoA 33 Pancreas 23 Pancreatic a-amylase 22 Pancreatic polypeptide 23 Parathesia 79 Pentose phosphate shunt 18, 22, 86, 87 Pericyte 79 Perform 43, 121 Peripheral neuropathy 9, 121 Potassium channel 24 Prostaglandin 30 Protein kinase C 63 Phosphoenolpyruvate 15 Phosphoenolpyruvate carboxykinase 15 Phosphoglucomutase 15, 18 Phosphofructo—1 kinase 20 Phospholipid 31 Phosphoprotein phosphatase 19 Phosphoprotein phosphatase inhibitor 26 Phosphorylase a phosphatase 19 Phosphorylase b kinase 19, 26 Pig retroviruses 52, 56 Pigs 51–2 Plasma membrane 17 Polyuria 1, 121 Polydipsia 1
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Polysaccahride 13 PP cell 23 Prediabetic phase 43–5, 55, 121 Preproinsulin 23 Proinsulin 23 Proislets, see Islet-like cell clusters Protein 34 Protein kinase 19, 26 Protein synthesis 27 Pyruvate 15 Pyruvate carboxylase 15 Pyruvate dehydrogenase 20 Pyruvate kinase 20 Radioactive tracer 36 Retinopathy 9–10, 121 non-proliferative 78 proliferative 76, 79 Salivary α-amylase 22 Scintillant 38 Sensorimotor polyneuropathy 79 Smooth muscle cell 84 Somatostatin 23 Sphingomyelinase 64 Stable tracer 36 Streptozotocin 55, 91 Strokes 10 Sucrose 23 Sulphonylurea 64 Syndrome X see Metabolic syndrome Thiazolidinedione 63 Th1 lymphocytes 43, 51 Th2 lymphocytes 51 Transcription factors 89
Index
Transforming growth factor ß 88 Transgenic mice 54, 123 Transgenic pigs 51, 123 Transplantation 45–52, 55–6 whole pancreas 46–8, 55, 93 islets 48, 55 fetal pancreas 49–50, 56 animal pancreas 51–2 kidney 93 Trehalase 23 Trehalose 23 Tricarboxylic acid cycle 20, 34 Triglyceride 31 Triose phosphate 87 Tumour necrosis factor 43, 63 Type 1 diabetes 1, 43–60 definition 43 diagnosis 2 genetics 45, 55 treatment 3–5 Type 2 diabetes 1, 5, 61–74 diagnosis 6 treatment 6–7 UDP-glucose 19 UDP-glucose phosphorylase 19 UDP-N-Acetylglucosamine 22, 63 Urea cycle 34 Vasodilatory factors 84, 90 Very low density lipoprotein (VLDL) 32 White adipose tissue 17 Xenotransplantation 51–2, 123
