IGFs:Local Repair and Survival Factors Throughout Life Span (Research and Perspectives in Endocrine Interactions)

Research and Perspectives in Endocrine Interactions

For further volumes: http://www.springer.com/series/5241

David Clemmons Yves Christen

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Iain C.A.F. Robinson

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Editors

IGFs: Local Repair and Survival Factors Throughout Life Span

Editors Dr. David Clemmons University of North Carolina School of Medicine Div. of Endocrinology 8024 Burnett-Womack Hall Chapel Hill NC 27599-7170 USA [email protected]

Dr. Iain C.A.F. Robinson National Inst. Med. Research Lab. Endocrine Physiology The Ridgeway London Mill Hill United Kingdom NW7 1AA [email protected]

Dr. Yves Christen Fondation IPSEN pour la Recherche Therapeutique 65 quai George Gorse 92650 Boulogne Billancourt Cedex France [email protected]

ISSN: 1861-2253 ISBN: 978-3-642-04301-7 e-ISBN: 978-3-642-04302-4 DOI 10.1007/978-3-642-04302-4 Springer Heidelberg Dordrecht London New York Library of Congress Control Number: 2009935699 # Springer-Verlag Berlin Heidelberg 2010 This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer. Violations are liable to prosecution under the German Copyright Law. The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Cover design: WMXDesign GmbH, Heidelberg, Germany Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

Foreword

Insulin-like growth factors (IGFs), their binding proteins and their receptors play important roles in regulating growth, metabolism, proliferation and survival for many cells and tissues throughout lifespan in humans and other species. Circulating IGF1 is known to be an endocrine regulator, with metabolic effects related to, and partly convergent with, insulin signalling. IGF1 also mediates many of the growth promoting effects of GH, and there is an ongoing debate as to the relative contributions of endocrine-, vs locally-derived IGF1 for systemic growth. More recently however, it has become clear that IGFs may be key local growth and cellular survival factors for many different tissues, active from early in embryonic development, essential for normal maturation and growth during foetal life. IGFs continue to play important roles throughout adult life in many diverse processes such as tissue repair, cellular proliferation, tissue remodelling and metabolic regulation. IGF systems are tightly regulated; orderly control of cellular repair and metabolism is central to healthy ageing, whilst uncontrolled proliferation can lead to cancer. Mammalian IGF systems are complex, with distinct binding proteins that can have both IGF-dependent and independent actions, and a parallel complexity of numerous insulin-like peptides is evident in other species. However, common themes of growth, metabolism and lifespan regulation have emerged from studies in flies, worms, mice and humans. The IPSEN Foundation brought together a group of international experts to meet in Paris on December 1, 2008 during the 8th Colloque Me´decine et Recherche in Endocrinology, to share their latest research in IGF/ insulin-like peptides as local repair and survival factors and their system-wide roles as regulators of a long and healthy lifespan. lain C.A.F. Robinson

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Acknowlegements

The editors wish to express their gratitude to Mrs Jacqueline Mervaillie, Astrid de Ge´rard and Be´atrice Andre´ for the organization of the meeting and Mrs Mary Lynn Gage for her editorial assistance.

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Contents

GH & IGF1: Aspects of Global and Local Release and Actions . . . . . . . . . . . 1 Iain C.A.F. Robinson Hyperglycemia Regulates the Sensitivity of Vascular Cells to IGF-I Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 David R. Clemmons, Laura A Maile, Walker H Busby Jr, Timothy Nichols, Yan Ling, Jarkaslava Lieskovska, and Yashwanth Radhakrishnan IGFBP2 Supports ex Vivo Expansion of Hematopoietic Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 HoangDinh Huynh, Megan Kaba, Sonali Rudra, Junke Zheng, Catherine J. Wu, Harvey F. Lodish, and Cheng Cheng Zhang The Role of Insulin-like Growth Factor-I in Central Nervous System Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 A. Joseph D’Ercole and Ping Ye Stimulation of Proliferative Pathways by IGF-binding Proteins . . . . . . . . . . 59 Robert C. Baxter, Mike Lin, and Janet L. Martin Signaling Pathways that Regulate C. elegans Life Span . . . . . . . . . . . . . . . . . . . 69 Gary Ruvkun, Andrew V. Samuelson, Christopher E. Carr, Sean P. Curran, and David E. Shore IGF1 Regulation of Skeletal Muscle Hypertrophy and Atrophy . . . . . . . . . . 85 David J. Glass Growth Hormone, Insulin-like Growth Factor I and Insulin: their Relationship to Aging and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 Ruslan Novosyadlyy, Emily J. Gallagher, and Derek LeRoith ix

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The Functions of Insulin-like Peptides in Insects . . . . . . . . . . . . . . . . . . . . . . . . . . 105 Sebastian Gro¨nke and Linda Partridge IGF Receptors in the Adult Brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 Carlos De Magalhaes Filho and Martin Holzenberger The Role of the IGF-1 and its Partners in Central and Peripheral Metabolism: Considerations for Extending Healthy Life Span . . . . . . . . . . 143 Nir Barzilai, Derek M. Huffman, Pinchas Cohen, and Radhika H. Muzumdar Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155

Contributors

Nir Barzilai Departments of Medicine and Molecular Genetics, Institute for Aging Research, Belfer Building, Suite 701, The Albert Einstein College of Medicine, Bronx, NY, USA, [email protected] Robert C. Baxter Kolling Institute of Medical Research, Royal North Shore Hospital, University of Sydney (E25), St Leonards, NSW 2065, Australia, [email protected] Walker H. Busby Jr. Department of Medicine, UNC School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA David R. Clemmons CB# 7170, 8024 Burnett Womack, Division of Endocrinology, University of North Carolina, Chapel Hill, NC 27599-7170, USA, [email protected] Pinchas Cohen Department of Pediatrics, Mattel Children’s Hospital at the University of California, Los Angeles Geffen School of Medicine at UCLA, Los Angeles, CA, USA Carlos De Magalhaes Filho Inserm UMR 893 – Hoˆpital Saint Antoine, Baˆtiment Kourilsky, 184, rue du faubourg Saint Antoine, 75012 Paris, France Joseph D’Ercole Department of Pediatrics, University of North Carolina, Chapel Hill, NC, USA, [email protected] Emily J. Gallagher Division of Endocrinology, Diabetes and Bone Diseases, Department of Medicine, Mount Sinai School of Medicine, New York, NY, USA David Glass Novartis Institutes for BioMedical Research, 100 Technology Square, Room 4210, Cambridge, MA 02139, USA, [email protected]

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Sebastian Gro¨nke Department of Biology, University College of London, Darwin Building, Gower Street, London WC1E6BT, UK Martin Holzenberger Inserm UMR 893 – Hoˆpital Saint Antoine, Baˆtiment Kourilsky, 184, Rue du faubourg Saint Antoine 75012 Paris, France, martin. [email protected] Derek M. Huffman Departments of Medicine and Molecular Genetics, Institute for Aging Research, Belfer Building, Suite 701, The Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, USA HoangDinh Huynh Departments of Physiology and Developmental Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA Megan Kaba Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA, USA Derek LeRoith Division of Endocrinology, Diabetes and Bone Diseases, Department of Medicine, Mount Sinai School of Medicine, New York, NY, USA, derek. [email protected] Jarkaslava Lieskovska Department of Medicine, UNC School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Mike Lin Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, Sydney, Australia Yan Ling Department of Medicine, UNC School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Harvey F. Lodish Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA 02142, USA Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA Laura A. Maile Department of Medicine, UNC School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Janet L. Martin Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, Sydney, Australia Radhika H. Muzumdar Department of Pediatrics, The Institute for Aging Research, The Albert Einstein College of Medicine and Montefiore Medical Center, Bronx, NY, USA

Contributors

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Timothy Nichols Department of Medicine, UNC School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Ruslan Novosyadlyy Division of Endocrinology, Diabetes and Bone Diseases, Department of Medicine, Mount Sinai School of Medicine, New York, NY, USA Linda Partridge Department of Biology, University College of London, Darwin Building, Gower Street, London WC1E6BT, UK, [email protected] Yashwanth Radhakrishnan Department of Medicine, UNC School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Iain Robinson Division of Molecular Neuroendocrinology, MRC, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK, irobins@ nimr.mrc.ac.uk Sonali Rudra Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA Gary Ruvkun Department of Molecular Biology, Massachusetts General Hospital, Simches Res. Blg, 185 Cambridge St, 7th Floor, CPZN-7250, Boston, MA 02114, USA, [email protected] Catherine J. Wu Dana-Farber Cancer Institute, 77 Avenue Louis Pasteur, Boston, MA 02115, USA Ping Ye Department of Pediatrics, University of North Carolina, Chapel Hill, NC, USA Cheng Cheng Zhang Departments of Physiology and Developmental Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA, USA, [email protected] Junke Zheng Departments of Physiology and Developmental Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA

GH & IGF1: Aspects of Global and Local Release and Actions Iain C.A.F. Robinson

1 Introductory Remarks When David Clemmons, Yves Christen and I planned a meeting in Paris on new aspects of the insulin-like growth factor (IGF) axis, we sought to bring together speakers from a diverse range of disciplines and research areas, which truly reflects the involvement of IGFs, insulin-like peptides and their common signaling mechanisms in a wide variety of biological processes across large evolutionary distances from worms and flies to man. Although the relative role of IGFs and growth hormone (GH) in mammalian growth continues to be a matter of some debate, the IGF system is clearly an important regulator of this process. However, this area is the subject of numerous meetings and reviews, so in this meeting we aimed to discuss the pleiotropic roles of the IGF system regulating other aspects of cellular processes, particularly focusing on IGFs and insulin-like peptides as local repair, proliferative and survival factors throughout the life span in multiple species. There are few tissues that do not utilize some form of IGF/IGF receptor signaling, and recent research insights were presented at this meeting on the role of IGFs in many different systems, including the nervous, musculoskeletal, metabolic, haematopoietic and vascular systems. Given their near ubiquitous presence, it is unsurprising that IGFs and their binding proteins are also deeply implicated in dysregulated cell growth pathologies, including proliferation and cancer of different types (Samani et al. 2007). Whilst their exact roles in different cancers remain undefined, new signaling pathways are being defined that may alter our focus from systemic to more local effects of IGFs, and the circulating protein partners of IGFs, initially thought of as passive binding proteins (BPs), clearly have independent biological activities in their own right (e.g. Valentinis et al. 1995; Schedlich et al.

I.C.A.F. Robinson Division of Molecular Neuroendocrinology MRC, National Institute for Medical Research, Mill Hill, London, NW7 1AA, UK e-mail: [email protected]

D. Clemmons et al. (eds.), IGFs: Local Repair and Survival Factors Throughout Life Span, Research and Perspectives in Endocrine Interactions, DOI 10.1007/978-3-642-04302-4_1, # Springer-Verlag Berlin Heidelberg 2010

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2007). Not least, these findings raise the enticing prospect of novel targets in the IGF system, which is already a major focus for therapeutic intervention in human cancers (Clemmons 2007). An accumulating body of evidence about the role of insulin-like peptide genes and longevity has been obtained in a variety of model species, from genetically tractable worms, flies and mice to dogs and human observational studies (Wolkow et al. 2000; Kenyon 2005; Giannakou et al. 2007; Sutter et al. 2007; Vigh and Suh 2005; Suh et al. 2008; Selman et al. 2008). This meeting provided an opportunity to bring together experts using different model systems, and some clearly unifying concepts emerged around the conserved insulin/IGF signaling mechanisms that appear to be key players regulating aging, life span, and health span across a wide evolutionary landscape (Piper et al. 2008; Berryman et al. 2008; Broughton and Partridge 2009). In this brief introduction I will discuss some aspects of local vs global signaling affecting secreted peptide growth factors such as IGFs. The remaining chapters in this book illustrate how the latest research in such diverse fields is giving rise to new ideas about the local physiological roles of this system in the growth, repair and survival of the organism.

2 IGF1: Global and Local Growth Factor Figure 1 illustrates the rapid increase in the published IGF research literature over the last three decades. A few time points are indicated, ranging from the elucidation of IGF primary sequences (Rinderknecht and Humbel 1978a,b) to experimental studies with recombinant IGF1 (Skottner et al. 1987), gene deletion experiments highlighting the role of IGFs in fetal growth and development (Baker et al. 1993; Liu et al. 1993), and the final widespread availability of IGF1 for clinical therapies (Chernausek et al. 2007), illustrating the long time scale between discovery and therapeutic application. Numerous other landmarks could also have been selected on this publication trajectory, many establishing the wide range of IGFs biological effects for which no therapeutic exploitation is yet established. Whilst this literature analysis necessarily begins with references to IGF1, it builds on a large body of work relating to the role of several somatomedin activities prior to their recognition and redefinition as IGFs (Daughaday et al. 1987), following the seminal formulation of the somatomedin hypothesis of Salmon and Daughaday (1957). Whilst this insightful concept recognized that many of the actions of GH are mediated indirectly via a local growth factor, the somatomedin hypothesis needed to be modified with the recognition of additional direct actions of GH in many tissues, including the growth plate (Isaksson et al. 1987) and the adipocyte (Flint et al. 2003), and in many other tissues that express the GH receptor in fetal and adult life (Waters and Kaye 2002). Nevertheless, the concept that global regulators of systemic growth invoke locally produced regulators of autocrine or paracrine growth and metabolism continues to broaden (Kaplan and Cohen 2007) and remains relevant across a wide range of signaling systems.

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IGF Publications 1978-2008 IGF1 approved for human use 2000

Mouse knockouts of IGF/R & embryonic growth

1800 1600

Number

1400 1200

Recombinant IGF1 for experimental use

1000 800 600

IGF1+2 sequenced

400 200 0 1978

1988

1998

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Year Pubmed Nov 2008

Fig. 1 Publications on IGF1 over three decades. Arrows track the studies reported on IGF1 in PubMed to November 2008, increasing relatively slowly after its sequencing, cloning and production of recombinant material for experimental use but then expanding as the phenotypes of gene deletion and transgenic experiments became clear. Note that IGF1 was only finally approved for human use three decades following its discovery, despite its implication in mediating statural growth a decade before that (Salmon and Daughaday 1957)

Over the last two decades, most of the main players in the insulin/IGF signaling systems have been identified and their functions unraveled in much detail, both extracellularly [IGF splice variants, binding proteins, acid labile subunit (ALS)] and intracellularly (IGF receptors, tyrosine kinase cascades, targets and modulators), and specific tools to measure, mimic, or interfere with IGF signaling have been developed. We are thus in a much better position to assess the somatomedin hypothesis at a local tissue or cellular level. However, it is also worth reflecting on what could be described as the reverse of the somatomedin concept. With a myriad of local growth factor circuits capable of playing many roles, and the complex cross-talk of intracellular pathways affecting IGF sensitivity in different tissues (Maile et al. 2008), it may be equally pertinent to ask which global factors coordinate quantitatively the actions of these multiple local growth factors, so that appropriate inter-organ signaling is maintained for the benefit of the whole organism. The increasingly sophisticated analytical studies at the cell and tissue levels demand a better understanding of the systems biology or integrative physiology of this system (Piper et al. 2008), which is somewhat lagging behind the “express train” of molecular genetics and will be required to translate cellular discoveries to effective systemic therapeutics.

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Despite the availability for some years of small amounts of clinical grade recombinant IGF1 for rare applications in human subjects (Schoenle et al. 1991), it is only relatively recently that this growth factor has become established and available internationally to treat IGF-deficient, GH-resistant short stature (Chernausek et al. 2007) and insulin resistance syndromes (McDonald et al. 2007). New indications in patients often only become recognized some time after the free availability of material for clinical research by a wide variety of clinical investigators, often in disciplines beyond those for which approval was originally obtained. However, IGF1 remains largely a systemic therapeutic (subcutaneous injection) delivered via an endocrine route to treat a global condition (short stature), and broadening the use of this powerful local growth factor to other conditions needs to be approached cautiously in well-defined studies (Rosenbloom 2008). With my colleague Ross Clark, sadly no longer with us, we had the opportunity to assess the local effects of systemic administration of recombinant IGF1 infusions in GH- and IGF-1-deficient rodents (Skottner et al. 1989). Apart from the primary goal of assessing systemic growth responses, Ross noted what has been evident in other studies in animals (Schoenle et al. 1982) and more recently in man (Chernausek et al. 2007), namely that different tissues showed differential responsiveness to endocrine-supplied IGF (Fig. 2). It is reasonable to predict that local replacements with IGF1 will be required to capitalize on IGF’s paracrine activities; replacement therapies could prove beneficial in mimicking local deficits, but this will likely require developments in local, rather than global, delivery mechanisms. Obvious target tissues in which local IGF1 might play useful therapeutic roles include the skeleton (Giustina et al. 2008), cartilage (Gaissmaier et al. 2008), skin (Semenova et al. 2008) and eye (Smith 2005). However, we will need a better understanding of paracrine growth factor physiology, and more thought about how IGF1 infusions in dwarf rats induce differential organ growth 160

IGF–1

% CONTROL

hGH 140

120 100

80 Liver

Lung

Testis

Brain Kidney Adrenal Spleen

Fig. 2 Differential organ growth promotion by systemic IGF1. This figure shows the relative organ weights in dwarf growth hormone (GH)-deficient rats stimulated to grow to similar extents by intravenous infusions of GH or recombinant IGF1. All tissues grew in response to both agents, but note the marked extra growth response of kidney and spleen in response to IGF1 vs GH. (From Skottner et al. 1989, with permission)

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to avoid rather than exploit excess growth factor exposure via an endocrine route, in order to minimize unwanted systemic actions on other tissues of locally delivered IGF1. Here too we may perhaps benefit from studies of paracrine actions of locally delivered insulin-like signaling peptides from simpler organisms such as worms and flies, which rely less on endocrine communication and more on specific local delivery or expression. Progress will require development of more sophisticated microphysiological methods in those species to accompany the powerful new genetic approaches that these organisms offer (Samuelson et al. 2007).

3 Access all Areas? The IGF system shares both endocrine and paracrine modes of delivery, and in mammals at least there is a major component of extracellular regulation of IGF1 activity that both endocrine and paracrine secretory mechanisms engage. The classical endocrine mechanisms are illustrated in the top panel of Figure 3. Protein hormones such as insulin or GH are stored in specific secretory vesicles in a Gland store pulsatile release

a Endocrine

Bloodstream

Target tissues

transmission

blood store BPs + ALS

b continuous release

blood buffer BPs + ALS

c autocrine/paracrine

Fig. 3 Endocrine vs paracrine secretory systems. a) The classical endocrine system, with a gland reserve of hormone and transmission of bursts of hormone release in the bloodstream to stimulate target tissues that selectively express the relevant hormone receptor. b) The endocrine IGF1 system has no primary gland store, but the peptide is constitutively produced from many tissues, with liver being a major source. The bloodstream serves as the IGF reservoir, retaining IGF1 complexed with binding proteins (BPs) and acid labile subunit (ALS) to prevent rapid elimination. A small proportion of free IGF dissociated from these complexes can bind IGF receptors in the target tissues. c) IGF1 is also generated locally in many tissues which are also targets for its action. They also produce binding proteins which may block or enhance IGF1 local action. BP and ALS in the circulation now serve to capture and buffer any locally produced IGF1 escaping to signal elsewhere

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specialized cell or tissue factory. Circulating hormone levels are low under unstimulated conditions until boosted rapidly by exocytotic release and entry to the circulation. The signal level is thus primarily regulated by controlling entry rate into the circulation, which acts simply to transmit the signal to responsive tissues and also ensure a steady clearance of the hormone once secretion is terminated. Regulation of synthesis needs not be rapid and is rarely the rate–determining step in circulating protein hormone concentrations, except in long-term deficit situations where replenishment of stores occurs more slowly than their depletion. Selective action of endocrine hormones requires target cells to express specific receptors for the hormone that capture and signal rapid changes in hormone concentration. Contrast this with the endocrine regulation of IGF1 (Fig. 3, middle panel). Here there is no large “gland” store of peptide, with many tissues producing the peptide (though the liver is quantitatively the major source in mammals). Without storage in regulated secretory vesicles, secretion occurs via a constitutive pathway. This secretion is not necessarily constant, but changes are slow and largely dependent on synthesis and trafficking rates (Burgess and Kelly 1987). The circulation becomes the extracellular “store” of endocrine IGF1, retaining it in binary complexes with binding proteins or in ternary complexes with ALS in slowly to very slowly cleared virtual “compartments.” Total serum IGF1 levels thus depend not only on IGF1 synthesis rates but also on the capacity, avidity and stability of these protein complex buffers. Finally, the lower panel in Figure 3 illustrates a more typical autocrine or paracrine IGF1 system. Here, the local signaling is highly dependent on the local synthesis and release of IGF acting on the producer cell, or its neighbours, by local diffusion. Local production of specific binding proteins may attract or sequestrate IGFs, either localizing them to target cells or retaining them in the producer tissue. The same processes that affect storage in the circulation, e.g., proteolysis of BPs to release IGF1, would also be specifically operative in local tissues (e.g., protease release or activation following tissue damage) either to accumulate a local pool of IGF1 or, conversely, to allow it to escape rapidly from the local environment. In this context, intact BPs in the circulation would act more as a buffer to prevent the local activity of IGF generated in one paracrine site from spreading to other sites in an active form. Such mechanisms are likely to affect the efficacy of locally delivered (or generated) IGF1, and we have few non-invasive tools that enable us to monitor active IGF1 concentrations at the tissue level in vivo. Differentiating endocrine and paracrine sources of IGF1 is important for two reasons. One is in interpreting the relative importance of endocrine, liver-derived IGF1 vs paracrine, tissue-derived IGF1 to systemic effects of IGF1 on growth (LeRoith 2008). There are methods (Frystyk 2007) for measuring “free” IGF1 existing in the circulation in dynamic equilibrium with complexed forms of IGF1 with binding proteins, but unfortunately we understand very little about the dynamics of how, and in what form, IGF1 is “delivered” to the tissues from an endocrine route, and indeed whether it acts entirely as free IGF1 or acts together with binding proteins or fragments thereof on membranes of their target cells.

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A second practical implication is the pragmatic use of measurements of total serum IGF1 in titrating GH therapies. Many factors other than GH determine a “serum IGF1” value, including binding proteins which are themselves subject to endocrine and nutritional regulation. Surprisingly good growth can be seen in some models with low total serum IGF1 levels following deletion of hepatic IGF1, and recent multiple deletion studies of IGF1, BP3 and ALS demonstrate that metabolic or growth consequences are not straightforwardly predictable from serum IGF1 levels (Yakar et al. 2009) and that local effects on bone are affected by different components of the IGF/BP3/ALS complex (Yakar et al. 2006). It is well recognized that spot serum IGF1 values need to be interpreted with care in evaluating therapies intended to raise endocrine IGF1, as they are equivalent to measuring storage reserve rather than active levels of IGF1. They will be even more difficult to interpret with therapies intended to increase local delivery of this peptide to specific tissue targets. New methods that could dynamically monitor IGF1 release across different circulatory beds would help discriminate between endocrine and paracrine contributions to local IGF1 signaling in different tissues. At least six binding proteins play roles in regulating IGF activity, not simply as complexes in the circulation but also as inhibitors or promoters of IGF1 effects, either directly or by altering the pharmacokinetic environment and availability for receptor binding in the extracellular space (Holly and Perks 2006). We now know that binding proteins have activities in their own right (Baxter 2000; Huynh et al.2008) and are altered significantly by extracellular modifications such as proteolysis or phosphorylation (Graham et al. 2007; Boldt and Conover 2007). Indeed, one can also ask whether in some tissues the binding of IGF1 peptides regulates the bioactivities of binding proteins, rather than the other way around. IGF1 analogs and peptide fragments that either do not bind or bind and displace IGF1 from specific binding proteins (Lowman et al. 1998) could be complementary to genetic deletion experiments in addressing the specific roles of some of these binding proteins. This binding protein complexity adds to different storage mechanisms in distinguishing insulin from IGF activities in mammals but has not been thought to play a major role in lower organisms, where greater complexity appears to be present instead in the number of insulin-like peptides which have different cellular origins, subcellular storage compartments and differential, overlapping or complementary functions on growth, proliferation and metabolism (Guarente and Kenyon 2000; Broughton et al. 2008). However, the recent identification of protein orthologs that may relate to BPs and ALS (Arquier et al. 2008; Honegger et al. 2008) in the fly raises the question of whether a similar extracellular regulation of activities in local tissues also operates on insulin-like peptide systems across many species. These studies could also shed light on the IGF-independent activities of these binding protein homologs, particularly in identifying the receptor or other target mechanisms through which they operate. There are good reasons to study local IGF1 actions, given the potential benefits of IGF1 for survival in tissues where cell loss is difficult to replace effectively. The

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obvious example is the central nervous system. There is strong circumstantial evidence that IGF1 and its receptors are an important factor for neuronal survival, neurogenesis and longevity in rodents or humans (Sullivan et al. 2008; Zhang et al. 2007; Kappeler et al. 2008; Suh et al. 2008), though attempts to harness this finding directly or indirectly to ameliorate degenerative damage for clinical benefit have not yet proved successful. A better understanding of the pathophysiological role of the IGF1/BP systems in brain damage, and delivery systems capable of overcoming or circumventing the blood-brain barrier could open up new opportunities for therapeutic exploitation of local IGF1 actions, truly addressing the theme of this meeting: to promote local repair and survival throughout life span. Acknowledgement I would like to take this opportunity to record the fond memories and contributions, practical and intellectual, in this field, of Ross Clark, my long-term friend and colleague.

References Arquier N, Ge´minard C, Bourouis M, Jarretou G, Honegger B, Paix A, Le´opold P. Drosophila ALS regulates growth and metabolism through functional interaction with insulin-like peptides. Cell Metab 7:333–338 Baker J, Liu JP, Robertson EJ, Efstratiadis A (1993) Role of insulin-like growth factors in embryonic and postnatal growth. Cell 75:73–82 Baxter RC (2000) Insulin-like growth factor (IGF)-binding proteins: interactions with IGFs and intrinsic bioactivities. Am J Physiol Endocrinol Metab 278:E967–976 Berryman DE, Christiansen JS, Johannsson G, Thorner MO, Kopchick JJ (2008) Role of the GH/ IGF-1 axis in lifespan and healthspan: lessons from animal models. Growth Horm IGF Res 18:455–471 Boldt HB, Conover CA (2007) Pregnancy-associated plasma protein-A (PAPP-A): a local regulator of IGF bioavailability through cleavage of IGFBPs. Growth Horm IGF Res 17:10–18 Broughton S, Partridge L (2009) Insulin/IGF-like signaling, the central nervous system and aging. Biochem J 418:1–12 Broughton S, Alic N, Slack C, Bass T, Ikeya T, Vinti G, Tommasi AM, Driege Y, Hafen E, Partridge L (2008) Reduction of DILP2 in Drosophila triages a metabolic phenotype from lifespan revealing redundancy and compensation among DILPs. PLoS One 3:e3721 Burgess TL, Kelly RB (1987) Constitutive and regulated secretion of proteins.Annu Rev Cell Biol 3:243–293 Chernausek SD, Backeljauw PF, Frane J, Kuntze J, Underwood LE; GH Insensitivity Syndrome Collaborative Group (2007) Long-term treatment with recombinant insulin-like growth factor (IGF-1) in children with severe IGF-1 deficiency due to growth hormone insensitivity. J Clin Endocrinol Metab 92:902–910 Clemmons DR (2007) Modifying IGF1 activity: an approach to treat endocrine disorders, atherosclerosis and cancer. Nature Rev Drug Discov 6:821–833. Daughaday WH, Hall K, Salmon WD Jr, Van den Brande JL, Van Wyk JJ (1987) On the nomenclature of the somatomedins and insulin-like growth factors. Mol Endocrinol 1:862–863. Flint DJ, Binart N, Kopchick J, Kelly P (2003) Effects of growth hormone and prolactin on adipose tissue development and function. Pituitary 6:97–102 Frystyk J (2007) Utility of free IGF-1 measurements. Pituitary 10:181–187 Gaissmaier C, Koh JL, Weise K (2008) Growth and differentiation factors for cartilage healing and repair. Injury 39 Suppl 1:S88–96

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Giannakou ME, Partridge L (2007) Role of insulin-like signaling in Drosophila lifespan. Trends Biochem Sci 32:180–188 Giustina A, Mazziotti G, Canalis E (2008) Growth hormone, insulin like growth factors and the skeleton. Endocr Rev 29:535–559 Graham ME, Kilby DM, Firth SM, Robinson PJ, Baxter RC (2007) The in vivo phophorylation and glycosylation of human insulin-like growth factor-binding protein-5. Mol Cell Proteomics 6:1392–1405 Guarente L, Kenyon C (2000) Genetic pathways that regulate ageing in model organisms. Nature 408:255–262 Holly J, Perks C (2006) The role of insulin-like growth factor binding proteins. Neuroendocrinology 83:154–160 Honegger B, Galic M, Ko¨hler K, Wittwer F, Brogiolo W, Hafen E, Stocker H (2008) Imp-L2, a putative homolog of vertebrate IGF-binding protein 7, counteracts insulin signaling in Drosophila and is essential for starvation resistance. J Biol 7:10 Huynh H, Iizuka S, Kaba M, Kirak O, Zheng J, Lodish HF, Zhang CC (2008) Insulin-like growth factor-binding protein 2 secreted by a tumorigenic cell line supports ex vivo expansion of mouse hematopoietic stem cells. Stem Cells 26:1628–1635 Isaksson OGP, Lindahl A, Nilsson A, Isgaard J (1987) Mechanism of the stimulatory effect of growth hormone on longitudinal bone growth. Endocr Rev 8:426–438 Kaplan SA, Cohen P (2007) The somatomedin hypothesis 2007: 50 years later. J Clin Endocrinol Metab 92; 4529–4535 Kappeler L, De Magalhaes Filho C, Dupont J, Leneuve P, Cervera P, Pe´rin L, Loudes C, Blaise A, Klein R, Epelbaum J, Le Bouc Y, Holzenberger M (2008) Brain IGF-1 receptors control mammalian growth and lifespan through a neuroendocrine mechanism. PLoS Biol 6:e254 Kenyon C (2005) The plasticity of aging: insights from long-lived mutants. Cell 120:449–460 LeRoith D (2008) Clinical relevance of systemic and local IGF-1: lessons from animal models. Pediatr Endocrinol Rev 5 Suppl 2:739–743 Liu JP, Baker J, Perkins AS, Robertson EJ, Efstratiadis A (1993) Mice carrying null mutations of the genes encoding insulin-like growth factor 1 (Igf-1) and type 1 IGF receptor (Igf1r). Cell 75:59–72 Lowman HB, Chen YM, Skelton NJ, Mortensen DL, Tomlinson EE, Sadick MD, Robinson IC, Clark RG (1998) Molecular mimics of insulin-like growth factor 1(IGF-1) for inhibiting IGF-1: IGF-binding protein interactions. Biochemistry 37:8870–8878 Maile LA, Capps BE, Miller EC, Aday AW, Clemmons DR (2008) Integrin-associated protein association with SRC homology 2 domain containing tyrosine phosphatase substrate 1 regulates igf1 signaling in vivo. Diabetes 57: 2637–2643 McDonald A, Williams RM, Regan FM, Semple RK, Dunger DB (2007) IGF1 treatment of insulin resistance. Eur J Endocrinol 157 Suppl 1:S51–56 Piper MD, Selman C, McElwee JJ, Partridge L (2008) Separating cause from effect: how does insulin/IGF signaling control lifespan in worms, flies and mice? J Intern Med 263: 179–191 Rinderknecht E, Humbel RE (1978a) The amino acid sequence of human insulin-like growth factor 1 and its structural homology with proinsulin. J Biol Chem 253:2769–2776 Rinderknecht E, Humbel RE (1978b) Primary structure of human insulin-like growth factor II. FEBS Lett 89:283–286 Rosenbloom AL (2008) Insulin-like growth factor-1 (rhIGF-1) therapy of short stature. J Pediatr Endocrinol Metab 21:301–315 Salmon WD Jr, Daughaday WH (1957) A hormonally controlled serum factor which stimulates sulfate incorporation by cartilage in vitro. J Lab Clin Med 49:825–836 Samani AA, Yakar S, LeRoith D, Brodt P (2007) The role of the IGF system in cancer growth and metastasis: overview and recent insights. Endocr Rev 28:20–47 Samuelson AV, Klimczak RR, Thompson DB, Carr CE, Ruvkun G (2007) Identification of Caenorhabditis elegans genes regulating longevity using enhanced RNAi-sensitive strains. Cold Spring Harb Symp Quant Biol 72:489–497

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Schedlich LJ, Muthukaruppan A, O’Han MK, Baxter RC (2007) Insulin-like growth factor binding protein-5 interacts with the vitamin D receptor and modulates the vitamin D response in osteoblasts. Mol Endocrinol 21:2378–2390 Schoenle E, Zapf J, Humbel RE, Froesch ER (1982) Insulin-like growth factor 1 stimulates growth in hypophysectomized rats. Nature 296:252–253 Schoenle EJ, Zenobi PD, Torresani T, Werder EA, Zachmann M, Froesch ER (1991) Recombinant human insulin-like growth factor 1 (rhIGF1) reduces hyperglycaemia in patients with extreme insulin resistance. Diabetologia 34:675–679 Selman C, Lingard S, Choudhury AI, Batterham RL, Claret M, Clements M, Ramadani F, Okkenhaug K, Schuster E, Blanc E, Piper MD, Al-Qassab H, Speakman JR, Carmignac D, Robinson IC, Thornton JM, Gems D, Partridge L, Withers DJ (2008) Evidence for lifespan extension and delayed age-related biomarkers in insulin receptor substrate 1 null mice. FASEB J 22:807–818 Semenova E, Koegel H, Hasse S, Klatte JE, Slonimsky E, Bilbao D, Paus R, Werner S, Rosenthal N (2008) Overexpression of mIGF-1 in keratinocytes improves wound healing and accelerates hair follicle formation and cycling in mice. Am J Pathol 173:1295–1310 Skottner A, Clark RG, Robinson IC, Fryklund L (1987) Recombinant human insulin-like growth factor: testing the somatomedin hypothesis in hypophysectomized rats. J Endocrinol 112:123–132 Skottner A, Clark RG, Fryklund L, Robinson IC (1989) Growth responses in a mutant dwarf rat to human growth hormone and recombinant human insulin-like growth factor 1. Endocrinology 124:2519–2526 Smith LE (2005) IGF-1 and retinopathy of prematurity in the preterm infant. Biol Neonate 88:237–244 Suh Y, Atzmon G, Cho MO, Hwang D, Liu B, Leahy DJ, Barzilai N, Cohen P (2008) Functionally significant insulin-like growth factor 1 receptor mutations in centenarians. Proc Natl Acad Sci USA 105:3438–3442 Sullivan KA, Kim B, Feldman EL (2008) Insulin-like growth factors in the peripheral nervous system. Endocrinology 149:5963–5971 Sutter NB, Bustamante CD, Chase K, Gray MM, Zhao K, Zhu L, Padhukasahasram B, Karlins E, Davis S, Jones PG, Quignon P, Johnson GS, Parker HG, Fretwell N, Mosher DS, Lawler DF, Satyaraj E, Nordborg M, Lark KG, Wayne RK, Ostrander EA (2007) A single IGF1 allele is a major determinant of small size in dogs. Science 316:112–115 Valentinis B, Bhala A, DeAngelis T, Baserga R, Cohen P (1995). The human insulin-like growth factor (IGF) binding protein-3 inhibits the growth of fibroblasts with a targeted disruption of the IGF-1 receptor gene. Mol Endocrinol 9:361–367 Vijg J, Suh Y (2005) Genetics of longevity and aging. Annu Rev Med 56:193–212 Waters MJ, Kaye PL (2002) The role of growth hormone in fetal development. Growth Horm IGF Res 12:137–146 Wolkow CA, Kimura KD, Lee MS, Ruvkun G (2000) Regulation of C. Elegans life-span by insulinlike signaling in the nervous system. Science 290:147–150 Yakar S, Bouxsein ML, Canalis E, Sun H, Glatt V, Gundberg C, Cohen P, Hwang D, Boisclair Y, Leroith D, Rosen CJ (2006) The ternary IGF complex influences postnatal bone acquisition and the skeletal response to intermittent parathyroid hormone. J Endocrinol 189:289–299 Yakar S, Rosen CJ, Bouxsein ML, Sun H, Mejia W, Kawashima Y, Wu Y, Emerton K, Williams V, Jepsen K, Schaffler MB, Majeska RJ, Gavrilova O, Gutierrez M, Hwang D, Pennisi P, Frystyk J, Boisclair Y, Pintar J, Jasper H, Domene H, Cohen P, Clemmons D, Leroith D (2009) Serum complexes of insulin-like growth factor-1 modulate skeletal integrity and carbohydrate metabolism. FASEB J 23:709–719 Zhang J, Moats-StaatsBM, Ye P, D’Ercole AJ (2007) Expression of insulin-like growth factor system genes during the early postnatal neurogenesis in the mouse hippocampus. J Neurosci Res 85:1618–1627

Hyperglycemia Regulates the Sensitivity of Vascular Cells to IGF-I Stimulation David R. Clemmons, Laura A Maile, Walker H Busby Jr, Timothy Nichols, Yan Ling, Jarkaslava Lieskovska, and Yashwanth Radhakrishnan

Abstract Insulin-like growth factor-I (IGF-I) stimulates a coordinated growth response in all tissues. However, following injury, increases in IGF-I locally can stimulate regional changes in cell growth that occur during the repair process. Atherosclerosis is a disease in which local increases in IGF-I synthesis have been shown to stimulate arterial smooth muscle cell proliferation, and this is believed to be an important component of early proliferative lesion development. In addition to stimuli that enhance atherosclerotic lesion progression by increasing IGF-I concentrations, such as increases in oxidized LDL and advanced end glycosylation products, changes in IGF-I sensitivity occur in response to variables that are known to increase the risk of coronary artery disease, such as hyperglycemia. Following induction of hyperglycemic stress, smooth muscle cells become sensitized to stimulation of cell proliferation by IGF-I. The mechanism for this increased sensitivity involves cooperative signaling through the aVb3 integrin receptor. Specifically, when smooth muscle cells are exposed to hyperglycemic conditions, they markedly increase their synthesis and secretion of aVb3 ligands such as thrombospondin, osteopontin and vitronectin. These proteins in turn activate aVb3 signaling, which then functions in coordination with IGF-I receptor-linked signaling mechanisms to enhance cell migration and proliferation. The pathway that mediates this effect requires Shc phosphorylation, which leads to MAP kinase activation. Inhibition of either of these processes will retard the smooth muscle cell response to IGF-I. For Shc to be phosphorylated in response to IGF-I requires activation of Src kinase, which directly phosphorylates Shc. Correspondingly, Src kinase activation requires ligand occupancy of the aVb3 integrin; therefore, blocking ligand occupancy results in an inability to generate an increase in phosphoShc and hence no induction of MAP kinase. Having discovered the components of this mechanism,

D.R. Clemmons (*) Department of Medicine, UNC School of Medicine, University of North Carolina, Chapel Hill, NC 27599-7170, USA e-mail: [email protected]

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we wished to determine whether inhibiting ligand occupancy of aVb3 during hyperglycemia would result in attenuation of MAP kinase activation and lesion proliferation. We prepared a monoclonal antibody to the active site on aVb3 that was the principle binding site for aVb3 ligands. This antibody disrupted not only ligand occupancy but also activation of the aVb3- linked signaling pathway and inhibited IGF-I stimulated smooth muscle replication in cells cultured in hyperglycemic conditions. To determine if this mechanism was operative in vivo, pigs were made diabetic and received an infusion of this antibody for three months. The antibody resulted in a 68% reduction in lesion area in the diabetic animals, whereas lesions that received a control antibody had no change. Importantly, the antibody also inhibited activation of the aVb3-linked signaling pathway as well as IGF-I stimulated Shc and MAP kinase activation. These studies clearly indicate that, under hyperglycemic conditions, there is cooperative signaling between the aVb3 and the IGF-I receptor, leading to enhanced SMC proliferation. The findings in experimental animals suggest that blocking activation of this pathway may be a successful therapeutic approach for inhibiting the atherosclerosis that occurs in diabetes.

1 Introduction IGF-I receptors are ubiquitously distributed in tissues, and therefore chronic stimulation by IGF-I leads to a generalized growth response (Liu et al. 1991). However, in addition to its role as a systemic anabolic factor, IGF-I is an important mediator of the cellular proliferative response that occurs in tissues in response to injury. Specifically following mechanical or thermal injury, the cells that repair the wound express increased IGF-I concentrations that, following secretion, bind to other cells within the local microenvironment and stimulates a local increase in cell proliferation (Edwall et al. 1989). This autocrine/paracrine activity of IGF-I has been shown to be an important component of the response to injury. One injury model that has been studied extensively is atherosclerotic lesion formation. In response to balloon denudation injury, a wave of IGF-I synthesis occurs in arterial smooth muscle cells (SMC), and this increase occurs at the time that the repair process is activated (Cercek et al. 1990). Furthermore, inhibiting the ability of the locally produced IGF-I to bind to receptors results in attenuation of SMC division (Hayry et al 1995). Conversely, overexpression of IGF-I in arteries of mice was shown to lead to an enhanced proliferative response following balloon denudation injury (Zhu et al. 2001). In addition to balloon injury within blood vessels, chemical injuries result in an increase in IGF-I synthesis. Specifically, exposure of macrophages (a cell type that is abundantly present in atherosclerotic lesions) to either oxidized LDL or advanced end glycosylation products results in a major increase in IGF-I synthesis (Kirstein et al. 1992). Similarly, when human lesions have been examined by immunohistochemistry, abundant IGF-I is present in macrophages within the

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subendothelial space (Grant et al. 1994). Finally, administration of IGF-I antibodies following induction of injury in rodents has been shown to attenuate SMC proliferation, suggesting it is the locally secreted IGF-I that is important for regulating the proliferative phase of atherosclerotic lesion development. A second stimulus for atherosclerotic lesion development during the proliferative phase is ligand-induced activation of the aVb3 integrin. During the development of atherosclerotic lesions, aVb3 is activated, and blocking ligand occupancy using peptides that inhibit the ability of the arg-gly-asp (RGD) sequence, containing mostly aVb3 ligands, to bind to aVb3 results in attenuation of lesion formation (Nichols et al. 1999). This process has been demonstrated in both rodents and large animal models of atherosclerosis. However, development of drugs that mimic RGD peptide antagonists has been complicated by the fact that they all possess agonist activity. Additionally, antibodies and other types of RGD-binding site inhibitors have not been uniformly effective in prohibiting proliferative lesion formation. However, because of the known role of aVb3 in lesion formation, attempts to determine whether these complex ligands might bind to other sites on this integrin offer the possibility of alternative approaches to inhibiting its activation. Following ligand binding to aVb3, it is phosphorylated by an unknown tyrosine kinase on two phosphotyrosines in its intracellular domain (Ling et al. 2003). When activated these phosphotyrosines form a binding site for intracellular signaling molecules that contain PTB domains. Calderwood and coworkers clearly showed that phosphorylation of these sites results in the localization of an important signaling molecule, DOK1 to phosphorylated b3 (Calderwood et al. 2003). However, the significance of DOK1 localization was not determined by these investigators. Therefore, the signaling pathway that is activated by aVb3 that might be linked to SMC proliferation had not been definitively determined. Activation of the IGF-I signaling pathway generally results in stimulation of receptor autophosphorylation followed by binding of IRS-1 to phosphotyrosines on the IGF-I receptor cytoplasmic domain (Samani et al 2007). IRS-1 then acts as a docking protein for several intracellular signaling molecules, including the p85 subunit of PI-3 kinase and in some systems the cell signaling molecule Shc. However, in SMC cultured under normoglycemic conditions, Shc is not activated in response to IGF-I stimulation (Maile et al. 2007). The PI-3 kinase pathway is activated to some extent and this leads to an increase in protein synthesis. However, without Shc activation, there is no subsequent activation of MAP kinase and no increase in cell proliferation. In contrast, if ligand occupancy of the aVb3 integrin is simulated by adding vitronectin into SMC cultures, then IGF-I can stimulate Shc activation and MAP kinase activation and a full proliferative response. Based on this observation, we undertook studies to determine the role of exposure to hyperglycemia in altering the aVb3 activation. To determine the site on aVb3 that was activated by ligands, we developed specific reagents to inhibit ligand binding, then tested the hypothesis that inhibiting this interaction would inhibit the effect of hyperglycemia on IGF-I signaling.

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2 Role of Ligand Occupancy of aVb3 in Stimulating the Response to IGF-I When SMC are cultured under normoglycemic conditions, stimulation of the IGF-I receptor results in activation of PI-3 kinase and stimulation of protein synthesis. However, there is no stimulation of cell proliferation and no activation of Shc and/ or MAP kinase. In contrast, if aVb3 ligands are added to normoglycemic medium followed by the addition of IGF-I, there is robust stimulation of Shc tyrosine phosphorylation activation of the MAP kinase pathway and a 2.4-fold increase in cell proliferation (Maile et al. 2008). To discern the mechanism by which ligand occupancy of aVb3 was enhancing IGF-I signaling, several studies were undertaken. Initially we demonstrated that there was no constitutive phosphorylation of two critical tyrosines on the b3 subunit. In contrast, addition of vitronectin resulted in stimulation of phosphorylation of these tyrosines. Importantly, the signaling protein DOK1, which contains a PTB domain, localized to these tyrosines (Ling et al 2005a), as proven by mutating both tyrosines to phenylalanine and showing that DOK1 no longer bound to activated aVb3. Furthermore, we were able to demonstrate that addition of vitronectin to quiescent cultures resulted in not only stimulation of b3 phosphorylation but also stimulation of DOK1 recruitment. DOK1 contains not only a PTB domain but also two tyrosines that are contained in YXXL motifs. Further analysis of our studies showed that these tyrosines were phosphorylated in response to IGF-I receptor stimulation and that, when phosphorylated, they recruited an SH2 domain containing tyrosine phosphatase, termed SHP-2. SHP-2 is normally present in cells that are in an inactive state in cytoskeleton. However, following DOK1 phosphorylation and DOK1 recruitment to b3, SHP-2 was recruited from the cytoskeleton to DOK1 and then subsequently the DOK1/ SHP-2 complex was recruited to the b3 subunit. Thus ligand occupancy of aVb3 mediated this DOK-1-SHP-2 recruitment to the plasma membrane. This plasma membrane-associated reservoir of SHP-2 turned out to be critical for IGF-Istimulated signaling. Although SHP-2 is a phosphatase, its phosphatase activity appears to be of minimal importance in mediating this signaling response. In contrast, SHP-2 contains other domains that are critical for mediating the mitogenic signal. Specifically following ligand occupancy of the IGF-I receptor, the tyrosine kinase activity on the receptor directly phosphorylates a transmembrane protein termed SHPS-1 (Maile and Clemmons 2002). SHPS-1 is an important signaling protein that is involved in cellular self recognition. It contains three immunoglobulin domains in its extracellular domain and a cytoplasmic tail with 4 YXXL/I/V motifs (Oshima et al. 2002). These four tyrosines are all phosphorylated in response to IGF-I receptor stimulation. Following their phosphorylation, two of these phosphotyrosines recruit SHP-2 to SHPS-1. To prove that these phosphotyrosines had to be activated in order to recruit SHP-2, we mutated them to phenylalanines and showed that the stimulation of cells by IGF-I did not result in SHP-2 recruitment to SHPS-1. To further bolster this hypothesis, we expressed a cytoplasmic domain deletion

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mutant and showed that SHP-2 could not be recruited to SHPS-1 under these conditions (Ling et al. 2005). Failure to recruit SHP-2 to SHPS-1 led to an inability to activate MAP kinase. Additionally, we developed a second strategy to disrupt SHPS-1 phosphorylation (see below), and, using this strategy, we were able to confirm the importance of SHPS-1 phosphorylation in IGF-I signaling. To discern how SHP-2 recruitment to SHPS-1 resulted in Shc activation, we probed the functional significance of other domains contained within SHP-2. We determined that a polyproline sequence present in SHP-2 resulted in recruitment of Src kinase to SHPS-1 (Lieskovska et al. 2003). This recruitment occurred through a polyproline-SH3 domain interaction and required SHP-2 localization on SHPS-1 for this interaction to occur. Once Src kinase was recruited to SHPS-1, we were able to demonstrate that, if it became activated when it bound to SHP-2, Src activation was achieved by phosphorylation of tyrosine 418, resulting in removal of autoinhibition. Once this activation has occurred, Src can then autophosphorylate two C-terminal tyrosines that are contained in YXXL/I/V motifs. When these tyrosines have been autophosphorylated Src kinase, is capable of recruiting Shc through a YXXL/I/V-SH-2 domain interaction. We demonstrated through mutagenesis that inhibition of Src/SHP-2 association resulted in an inability to activate Src and an inability of Src to recruit Shc. To further determine that Src was actually phosphorylating Shc, we used a kinase-dead mutant of Src and showed that, although it bound to the SHPS-1/SHP-2 complex, it could not recruit Shc and could not activate Shc phosphorylation. To further prove the importance of Src tyrosine autophosphorylation, we mutated the tyrosines that were necessary for Shc recruitment and showed that, when this mutant was expressed, it autoactivated but did not recruit Shc and subsequently Shc was not phosphorylated. In summary, these studies demonstrated that the scaffolding protein SHPS-1 is critical for recruitment of a SHP-2/Src/Shc signaling complex. When SHPS-1 is tyrosine phosphorylated, this complex is recruited to SHPS-1 through simultaneous activation of the aVb3 integrin and the IGF-I receptor. It is fully capable of activating the MAP kinase pathway, leading to enhancement of cell proliferation and migration. Furthermore assembly of this complex and its activation is absolutely required for SMC to proliferate in response to IGF-I (Clemmons and Maile 2005).

3 Augmentation of PI-3 Kinase Activation Although we were able to demonstrate that, under normoglycemic conditions, the SHPS-1 signaling complex is not activated and there is minimal ligand occupancy of aVb3 integrin, the PI-3 kinase pathway is activated, resulting in P70S6 kinasemediated increases in protein synthesis in response to IGF-I. However, activation of this pathway appears suboptimal. Therefore, we explored the possibility that optimal activation of this pathway also required aVb3 ligand occupancy. We were able to demonstrate that, in the presence of vitronectin, assembly of the SHPS-1 signaling complex resulted in recruitment of Grb-2 to this complex following Shc

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tyrosine phosphorylation (Radhakrishnan et al. 2008). Grb-2 recruitment is known to be essential for Ras/MAP kinase activation. An earlier report had shown that activated Grb-2 could under certain circumstances recruit the p85 subunit of PI-3 kinase (Pleiman et al. 1994). This recruitment is believed to have occurred through an interaction between two polyproline domains contained in the p85 subunit and two SH3 domains contained in Grb-2. To definitively determine if this interaction took place, we prepared mutants of p85 in which the prolines within each of the two polyproline domains were substituted with alanines. Substitution for the prolines in either region or the combination resulted in marked attenuation of the ability of p85 to bind to Grb-2. More importantly, this disassociation resulted in a marked decreased in p85/p110 association and a major decrease in PI-3 kinase enzymatic activity in response to IGF-I. To confirm this finding, we prepared a Grb-2 mutant in which the ability of the SH3 domains to bind to p85 was attenuated. This mutant also showed marked reduction in p85/Grb-2 association and PI-3 kinase activation in response to IGF-I. Expression of either type mutant resulted in decreased AKT activation and downstream signaling in response to IGF-I. Therefore, it appears that assembly of this SHPS-1 complex is not only important for MAP kinase pathway activation but is also optimal for PI-3 kinase pathway activation and ultimately cell migration as well as cell proliferation in response to IGF-I.

4 Role of Hyperglycemia in Modulating aVb3/IGF-I Receptor-linked Signaling Hyperglycemic stress is known to activate simple intracellular signaling mechanisms, including release of reactive oxygen species and activation of protein kinase C; however, exactly how each of these mechanisms leads to enhanced cell proliferation in atherosclerosis has not been determined. We reasoned that, because there was cooperation between the aVb3 and IGF-I receptor-linked signaling pathways, hyperglycemia might be activating aVb3. Initially we analyzed SMC cultures that had been maintained in normoglycemic (5 mM glucose) or hyperglycemic (25 mM glucose) conditions. We determined that, in the presence of glucose concentrations as low as 12 mM, synthesis and secretion of three important aVb3 ligands was markedly stimulated (Maile et al. 2007). When we analyzed the abundance of the ligands on the cell surface, we were able to show that there was a major increase in the amount of thrombospondin, osteopontin and vitronectin associated with aVb3 when cells were exposed to glucose concentrations of 12 mM or greater. In contrast, in the presence of 5 mM glucose, there was minimal binding of these ligands and no increase in b3 phosphorylation. Subsequently, we determined that there was no assembly of the signaling molecules on aVb3, that is, no recruitment of DOK1/SHP-2 complex and no recruitment of SHP-2 to SHPS-1 if cells were maintained in 5 mM glucose. Consequently, the Shc/MAP kinase pathways were not activated and cell growth could not be stimulated.

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To confirm the importance of the aVb3 receptor, we added pure vitrorectin to cultures maintained in 5 mM glucose. The cultures could be stimulated to replicate in the presence of IGF-I. Similarly MAP kinase and Shc phosphorylation could be easily induced. To further determine the significance of exposure to high glucose with its subsequent increase in aVb3 ligands, we wished to determine how blocking ligand occupancy of aVb3 would alter these biochemical reactions and the cellular response to IGF-I. Since competitive antagonists based on the RGD binding site had failed to inhibit these interactions due to partial agonist activity, we searched for a second binding domain that might be mediating the enhancement of aVb3 phosphorylation. A separate binding site within vitronectin for the aVb5 integrin had been reported previously. Therefore we reasoned that it was possible that this sequence contained adequate activity to mimic these effects. We prepared an11amino acid synthetic peptide that contained the sequence within this region. This peptide was as active as native vitronectin in enhancing b3 phosphorylation and the Shc phosphorylation in response to IGF-I (Maile et al. 2006a). To determine the site on aVb3 that bound to this peptide, we again reviewed the literature and found that there was a study that had substituted a specific sequence contained in b3 integrin with a sequence from the b1 integrin. They had shown that, following expression of this mutant, aVb3 no longer bound to vitronectin. Since this sequence was distinct from the known sequence that bound to the RGD domain of aVb3 ligands, it was a reasonable candidate for binding to a secondary binding site (Maile et al. 2006b). To prove this, we prepared an antibody to this domain, hereafter referred to as the C-loop region because it is a six-amino acid sequence contained between two cysteines at positions 177 and 183 within the b3 subunit. We demonstrated that this antibody was capable of inhibiting vitronectin, thrombospondin or osteopontin binding to aVb3 and, most importantly, it inhibited activation by the synthetic peptides. Incubation with this antibody with cells cultured in 12 mM glucose or higher showed that the antibody also inhibited cell proliferation in response to IGFI even in cells cultured in 25 mM glucose. Therefore, it appeared that direct interaction between this heparin binding domain in aVb3 ligands and the C-loop domain of b3 was responsible for aVb3 activation and subsequent cooperative signaling between the IGF-I receptor and aVb3 during hyperglycemic conditions.

5 In Vivo Validation that Cooperative Signaling Between aVb3 Integrin and the IGF-I Receptor Acceretes Atherosclerosis in Diabetes To determine if activation of the signaling pathway occurred in vivo and if it would result in vascular smooth muscle cell proliferation, we prepared a monoclonal antibody and purified it to homogeneity and tested its efficacy in an animal model of diabetic atherosclerosis. Gerrity and coworkers (2001) had validated that pigs that are made diabetic and receive a high fat diet not only have proliferative

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atherosclerosis but also have an accelerated rate of lesion progression compared to animals that are not diabetic. Furthermore, they demonstrated that there was increased production of reactive oxygen species and activation of signaling pathways within these lesions that were similar to those that occurred in diabetic humans. Therefore, we used the Gerrity model to test our hypothesis. Following induction of diabetes with streptozotacin, the animals were made hypercholesterolemic by being fed a high fat diet. After a one-month equilibration period, Alzet mini pumps were placed in the femoral arteries of eight pigs. Each pig received an infusion of the active antibody into one femoral artery and an irrelevant control antibody into a contralateral femoral artery. Therefore, each animal served as its own control. Following three months of continuous infusion, the animals were sacrificed and the lesions analyzed morphometrically and biochemically. Morphometric analysis of 12 sections per lesion showed there was a 68% reduction in neointimal area. Importantly, these lesions appeared typical of the proliferative phase of atherosclerosis, with foam cell formation, macrophage infiltration and a typical neointima composed of both form cells and a fibrous cap. Biochemical analysis of the lesions showed that the b3 subunit of aVb3 integrin was constitutively activated by the presence of diabetes with increased b3 phosphorylation. Importantly infusion of the antibody markedly inhibited b3 phosphorylation. Additionally, there was diminished Shc binding to SHPS-1 and Shc phosphorylation as well as MAP kinase activation. The abundance of IGFBP-5 (a gene whose transcription is stimulated by IGF-I) was significantly reduced. Assessment of Ki67 labeling showed that the mitotic index was reduced from 31% to 9% in the antibody-treated lesions. In summary, it appears that both the IGF-I and aVb3 signaling pathways are constitutively activated in a porcine model of diabetic atherosclerosis. Infusion of the antibody with disruption of ligand occupancy of b3 results in attenuation of IGF-I signaling, as evidenced by decreased IGFBP-5 induction and reduced atherosclerotic lesion formation. The findings suggest that development of this antibody for testing in humans is a worthwhile objective. These studies have described the assembly of a signaling complex that forms on the cytoplasmic tail of a cell surface signaling proteins SHPS-1 in response to hyperglycemic stress. The importance of assembly of these types of signaling complexes for cellular response to other types of stresses is likely to provide important and useful information about the cellular response to stress. Acknowledgments The authors wish to thank Ms. Laura Lindsey for her help in preparing the manuscript. This work was supported by a grant from the National Institutes of Health, AG02331.

References Cereck B, Fishbein MC, Forrester JS, Helfant RH, Fagin JA (1990) Induction of insulin-like growth factor I messenger RNA in rat aorta after balloon denudation. Circ Res 66:1755–1760 Calderwood DA (2003) Integrin beta cytoplasmic domain interactions with phosphotyrosinebinding domains: a structural prototype for diversity in integrin signaling. Proc Natl Acad Sci USA 100:2272–2277

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Clemmons DR, Maile LA (2005) Interaction between insulin-like growth factor-I receptor and alphaVbeta3 integrin linked signaling pathways: cellular responses to changes in multiple signaling inputs. Mol Endocrinol 19:1–11 Edwall D, Schalling M, Jennische E, Norstedt G (1989) Induction of insulin-like growth factor I messenger ribonucleic acid during regeneration of rat skeletal muscle. Endocrinology 124:820–825 Gerrity RG, Natarajan R, Nadler JL, Kimsey T (2001) Diabetes-induced accelerated atherosclerosis in swine. Diabetes 50:1654–1665 Grant MB, Wargovich TJ, Ellis EA, Caballero S, Mansour M, Pepine CJ (1994) Localization of insulin-like growth factor I and inhibition of coronary smooth muscle cell growth by somatostatin analogues in human coronary smooth muscle cells. A potential treatment for restenosis? Circulation 89:1511–1517 Hayry P, Mylla¨rniemi M, Aavik E, Alatalo S, Aho P, Yilmaz S, Ra¨isa¨nen-Sokolowski A, Cozzone G, Jameson BA, Baserga R (1995) Stabile D-peptide analog of insulin-like growth factor-1 inhibits smooth muscle cell proliferation after carotid ballooning injury in the rat. FASEB J 9:1336–1344 Kirstein M, Aston C, Hintz R, Vlassara H (1992) Receptor-specific induction of insulin-like growth factor 1 in human monocytes by advanced glycosylation end product-modified proteins. J Clin Invest 90:439–446 Lieskovska J , Ling Y, Badley-Clarke J, Clemmons DR (2006) The role of Src kinase in insulinlike growth factor-dependent mitogenic signaling in vascular smooth muscle cells. J Biol Chem 281:25041–25053 Ling Y, Maile LA, Clemmons DR (2003) Tyrosine phosphorylation of the b3-subunit of the aVb3 integrin is required for membrane association of the tyrosine phosphatase SHP-2 and its further recruitment to the insulin-like growth factor I receptor. Mol Endocrinology 17:1824–1833 Ling Y, Maile LA, Badley-Clarke J, Clemmons DR (2005a) DOK1 mediates SHP-2 binding to the alphaVbeta3 integrin and thereby regulates insulin-like growth factor I signaling in cultured vascular smooth muscle cells. J Biol Chem 280:3151–3158 Ling Y, Maile LA, Lieskovska J, Badley-Clarke J, Clemmons DR (2005b) Role of SHPS-1 in the regulation of insulin-like growth factor I-stimulated SHC and mitogen-activated protein kinase activation in vascular smooth muscle cells. Mol Biol Cell 16:3353–3364 Liu JP , Baker J, Perkins AS, Robertson EJ, Efstratiadis A (1993) Mice carrying null mutations of the genes encoding insulin-like growth factor I (Igf-1) and type 1 IGF receptor (Igf1r). Cell 75:59–72 Maile LA, Clemmons DR (2002) Regulation of insulin-like growth factor I receptor dephosphorylation by SHPS-1 and the tyrosine phosphatase SHP-2. J Biol Chem 277:8955–8960 Maile LA, Busby WH, Sitko K, Capps BE, Sergent T, Badley-Clarke J, Ling Y, Clemmons DR (2006a) The heparin-binding domain of vitronectin is the region that is required to enhance insulin-like growth factor-I signaling. Mol Endocrinol 20:881–892 Maile LA, Busby WH, Sitko K, Capps BE, Sergent T, Badley-Clarke J, Clemmons DR (2006b) Insulin-like growth factor-I signaling in smooth muscle cells is regulated by ligand binding to the 177CYDMKTTC184 sequence of the beta3-subunit of the alphaVbeta3. Mol Endocrinol 20:405–413 Maile LA, Capps BE, Ling Y, Xi G, Clemmons DR (2007) Hyperglycemia alters the responsiveness of smooth muscle cells to insulin-like growth factor-I. Endocrinology 148:2435–2443 Maile LA, Capps BE, Miller EC, Allen LB, Veluvolu U, Aday AW, Clemmons DR (2008) Glucose regulation of integrin-associated protein cleavage controls the response of vascular smooth muscle cells to insulin-like growth factor-I. Mol Endocrinol 22:1226–1237 Nichols TC, du Laney T, Zheng B, Bellinger DA, Nickols GA, Engleman W, Clemmons DR (1999) Reduction in atherosclerotic lesion size in pigs by aVb3 inhibitors is associated with inhibition of insulin-like growth factor-I mediated signaling. Circ Res 85:1040–1045 Oshima K, Ruhul Amin AR, Suzuki A, Hamaguchi M, Matsuda S (2002) SHPS-1, a multifunctional transmembrane glycoprotein. FEBS Lett 519:1–7

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Pleiman CM, Hertz WM, Cambier JC (1994) Activation of phosphatidylinositol-3’ kinase by Srcfamily kinase SH3 binding to the p85 subunit. Science 263:1609–1612 Radhakrishnan Y, Maile LA, Ling Y, Graves LM, Clemmons DR (2008) Insulin-like growth factor-I stimulates Shc-dependent phosphatidylinositol 3-kinase activation in Grb2-associated p85 in vascular smooth muscle cells. J Biol Chem 283:16320–16331 Samani AA, Yakar S, LeRoith D, Brodt P (2007) The role of IGF system in cancer growth and metastasis: overview and recent insights. Endocrine Rev 28:20–47 Zhu B, Zhao G, Witte DP, Hui DY, Fagin JA (2001) Targeted overexpression of the IGF-I in smooth muscle cells of transgenic mice enhances neointimal formation through increased proliferation and cell migration after intraarterial injury. Endocrinology 142:3598–3606

IGFBP2 Supports ex vivo Expansion of Hematopoietic Stem Cells HoangDinh Huynh, Megan Kaba, Sonali Rudra, Junke Zheng, Catherine J. Wu, Harvey F. Lodish, and Cheng Cheng Zhang

Abstract The hematopoietic stem cell (HSC), through proliferation and differentiation, gives rise to all lymphoid, myeloid, and erythroid cells. Successful HSC transplantation is often limited by the numbers of HSCs, and robust methods to expand HSCs ex vivo are needed (Bryder et al. 2006; Tse et al. 2008). We demonstrated that insulin-like growth factor binding protein 2 (IGFBP2), secreted by a tumorigenic cell line, enhanced ex vivo expansion of mouse and human HSCs (Huynh et al. 2008; Zhang et al. 2008). We established a completely defined, serum-free culture system for mouse HSCs containing SCF, TPO, FGF-1, Angptl3, and IGFBP2. As measured by competitive repopulation analyses, there was a 48fold increase in numbers of long-term repopulating mouse HSCs after 21 days of culture. We sought to use a similar condition to culture human cord blood HSCs. We measured the activity of multipotent human SCID-repopulating cells (SRCs) by transplantation into non-obese, diabetic severe combined immunodeficiency (NOD/SCID) mice; secondary transplantation was performed to evaluate the selfrenewal potential of SRCs. We showed that a serum-free culture containing SCF, TPO, FGF-1, Angiopoietin-like 5, and IGFBP2 supports a
20 fold net expansion of repopulating human cord blood HSCs, a number potentially applicable to several clinical processes, including HSC transplantation. This was the first demonstration that IGFBP2 stimulates expansion or proliferation of stem cells. We are studying whether the role of IGFBP2 in regulation of HSCs is IGF-dependent, and whether IGFBP2 regulates the self-renewal and differentiation of HSCs in vivo.

C.C. Zhang (*) Departments of Physiology and Developmental Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390, USA Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge Massachusetts 02142, USA e-mail: [email protected]

D. Clemmons et al. (ed.), IGFs: Local Repair and Survival Factors Throughout Life Span, Research and Perspectives in Endocrine Interactions, DOI 10.1007/978-3-642-04302-4_3, # Springer-Verlag Berlin Heidelberg 2010

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1 Introduction Hematopoietic stem cells (HSCs) are defined by their abilities to self-renew and to differentiate into all blood-cell types (Abramson et al. 1977; Jordan et al. 1990; Morrison et al. 1995; Till and McCulloch 1961). HSCs form the basis of bone marrow transplantation and are also a promising cell target for gene therapies. The difficulty of ex vivo expansion of HSCs has severely hampered the clinical uses and biological studies of these cells (Bryder et al. 2006; Sorrentino 2004). Numerous attempts have been made to increase the number of mouse and human HSCs in culture (for review, see Bryder et al. 2006; Domen and Weissman 1999; Sauvageau et al. 2004; Sorrentino 2004; North et al. 2007; Kirouac and Zandstra 2006; Sauvageau et al. 2004). However, the proper mixture of growth factors and cytokines to use in culture conditions has not yet been determined and new growth factors for HSCs need to be identified. We previously showed that IGF-2 as well as several angiopoietin-like proteins (Angptls), a group of growth factors secreted by a fetal liver HSC - supportive cell population, supported ex vivo expansion of murine HSCs (Zhang et al, 2006a; Zhang and Lodish 2004, 2005). Here we demonstrate that insulin-like growth factor binding protein 2 (IGFBP2), secreted by a cultured tumorigenic cell line, stimulated ex vivo expansion of mouse HSCs.Based on these findings, we established a completely defined, serum-free culture system for murine HSCs that includes the growth factors SCF, TPO, FGF-1, Angptl3, and IGFBP2. As measured by competitive repopulation analyses, there was a 48-fold increase in numbers of long-term, repopulating mouse HSCs after three weeks of culture. A similar defined, serum-free medium containing SCF, TPO, FGF-1 (or Flt3-L), Angptl5, and IGFBP2 supported a 20-fold increase in human SCID repopulating cells (SRCs), as measured by transplantation in NOD/SCID mice. To our knowledge, this is the highest level ofex vivo expansion of HSCs yet achieved in a defined culture and is also the first demonstration that IGFBP2 stimulates the expansion of murine stem cells. Our findings suggest that other cancer cells may secrete IGFBP2 or other HSC–stimulating proteins.

2 Materials and Methods 2.1

Mice

C57BL/6 CD45.2, CD45.1, and NOD/SCID (NOD.CB17-Prkdcscid/J) mice were purchased from the Jackson Laboratory or the National Cancer Institute and were maintained at the Whitehead Institute or the University of Texas Southwestern Medical Center animal facility. All animal experiments were performed with the approval of M.I.T. or UT Southwestern Committee on Animal Care.

IGFBP2 Supports ex Vivo Expansion of Hematopoietic Stem Cells

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3 Culture Medium STIF medium is defined as StemSpan serum-free medium (StemCell Technologies) supplemented with 10 mg/ml heparin (Sigma), 10 ng/ml mouse SCF, 20 ng/ml mouse TPO, 20 ng/ml mouse IGF-2 (all from R&D Systems), and 10 ng/ml human FGF-1 (Invitrogen). STF medium was the same medium but without IGF-2. When medium contained Flt3-L, we used 50 ng/ml human SCF, 10 ng/ml human TPO, and 50 ng/ml human Flt3-L. The indicated amounts of purified mouse Angptl3 (a gift from R&D Systems), human Angptl5 (Abnova, Taiwan), or human IGFBP2 (R&D Systems) were added. Conditioned medium was collected from confluent 293T or 3T3 cells after overnight culture.

4 Mouse HSC Culture Twenty BM SP Sca-1þ CD45þ cells isolated from 8- to 10-week-old C57BL/6 CD45.2 mice were plated in one well of a U-bottom 96-well plate (3799; Corning) with 160 ml of the indicated medium. Cells were cultured at 37 C in 5% CO2 and indicated levels of O2. For the purpose of competitive transplantation, we pooled cells from 12 culture wells and mixed them with competitor/supportive cells before the indicated numbers of cells were transplanted into each mouse. For Western blotting, bone marrow Lin- cells isolated by AutoMacs or by FACS were cultured overnight in STF medium, followed by starvation for 4 h in serum-free medium (containing 0.5% BSA), and treatment with 500 ng/ml IGFBP2. For quantitative RT-PCR, Lin-Sca-1þKitþFlk-2 cells were cultured in STF medium for three days, followed by replacement with fresh STF medium. One hour later, 500 ng/ml IGFBP2 was added as indicated.

5 Human Cell Culture Fresh and cryopreserved human cord blood cells were purchased from Cambrex, StemCell Technologies Inc., and AllCells. All of the cells were from pooled donors. CD34þ or CD133þ purity checked by flow cytometry was higher than 90%. After thawing, the cell viability tested by trypan blue exclusion was higher than 72%. The thawed cells were centrifuged and resuspended with StemSpan medium before being aliquoted for immediate transplantation or culture. In Figure 1, total human cord blood mononuclear cells were plated at 1  106 cells/ml of STIF medium, with 100 ng/ml Angptl3 or Angptl5. Medium volume was increased by adding fresh medium at day 5, 8, 12, 15, and 18 to maintain cell densities at 5  105–1.5  106 cells/ml. Cells were cultured at 37 C in 5% CO2 and normal O2. Fresh human cord

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Fig. 1 Purified IGFBP2 stimulates ex vivo expansion of HSCs. (a) Shown are competitive repopulations at 4 months post-transplantation from 20 freshly isolated SP Sca-1þ CD45þ cells (bar 1), and the progenies of the same number of cells cultured for 10 days in STF medium (bar 2), in 293T conditioned medium supplemented with SCF, TPO and FGF-1 (293T conditioned STF medium) (bar 3), in STF medium with 100 ng/ml IGFBP2 (bar 4), in 293T conditioned STF medium pretreated with 10 mg/ml anti-IGFBP2 antibody (bar 5), and in 293T conditioned STF medium pretreated with 10 mg/ml control antibody (bar 6), respectively (n ¼ 5). * or ** Significantly different from the value of bar 1 or bars 2 and 5, respectively; p < 0.05. (b) Twenty CD45.2 bone marrow SP Sca-1þ CD45þ cells were cultured for 10 days in serum-free medium with 10 ng/ml SCF, 20 ng/ml TPO, 10 ng/ml FGF-1 (STF medium) (bars 1 and 4), in STF medium containing 500 ng/ml IGFBP2 (bars 2 and 5), and in STF medium containing both 500 ng/ml IGFBP2 and 100 ng/ml Angptl3 (bars 3 and 6). The cells were then cotransplanted with 1  105 CD45.1 total bone marrow cells into CD45.1 recipients (n ¼ 6–7). Engraftments at 1 month or 4 months post-transplantation are shown. * or ** Significantly different from bar 4 or bar 5 value, respectively. Student’s t-test, p < 0.05. (c–d) Bone marrow of three mice in conditions represented by bar 6 of Figure 3C was pooled at four months post-transplantation and transplanted into secondary recipients. Total hematopoietic and multilineage engraftments of secondary transplanted mice (n ¼ 5) are shown. (The type on the word Repopulation on the y axis is clipped in all 4 parts of the figure)

blood CD34þ cells and cryopreserved CD133þ cells used in the experiments in Figures 2–5 were plated at 1  104 cells/well in one well of a U-bottom 96-well plate (3799; Corning) with 200 ml of the indicated medium for two days. On day 3, cells were pooled from individual wells and transferred to six-well plates at 5  104 cells/ml. Fresh medium was added at days 4 and 7 to keep the cell density at 2  105 cells/ml (day 4) or 7  105/ml (day 7). Cells were cultured at 37 C in 5% CO2, and normal O2 or 5% O2 (low O2) levels. For transplantation, we pooled cells from all the culture wells before the indicated numbers of cells were transplanted into each mouse.

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Fig. 2 Limiting dilution analysis of the repopulating ability of HSCs before and after culture. Adult BM SP CD45þSca-1þ cells were directly transplanted or cultured for 21 days in serum-free conditioned STF medium containing 100 ng/ml of purified Angptl3 and 500 ng/ml IGFBP2. Irradiated CD45.1 congenic mice were injected with 1  105 CD45.1 BM competitor cells and 1, 5, 25, or 100 freshly isolated SP CD45þSca-1þ cells (left; n ¼ 24) or the cultured progenies of 0.2, 1, 5, or 10 SP CD45þSca-1þ cells (right; n ¼ 26). One hundred freshly isolated SP Sca-1þ CD45þ cells and the cultured progeny of 5 or 10 input cells repopulated all recipients and these data points are not plotted. Plotted is the percentage of recipient mice containing more than 1% CD45.2 myeloid and lymphoid cells in nucleated peripheral blood cells four months after transplant versus the number of input-equivalent cells injected

6 Flow Cytometry For analyzing repopulation of mouse HSCs, peripheral blood cells of recipient CD45.1 mice were collected by retro-orbital bleeding, followed by lysis of red blood cells and staining with anti-CD45.2-FITC, and anti-CD45.1-PE, and antiThy1.2-PE (for T-lymphoid lineage), anti-B220-PE (for B-lymphoid lineage), antiMac-1-PE, anti-Gr-1-PE (cells costaining with anti-Mac-1 and anti-Gr-1 were deemed to be of the myeloid lineage), or anti-Ter119-PE (for erythroid lineage) monoclonal antibodies (BD Pharmingen). The “percent repopulation” shown in all figures was based on the staining results of anti-CD45.2-FITC and anti-CD45.1-PE. In all cases, FACS analysis of the above-listed lineages was also performed to confirm multilineage reconstitution. For analyzing human hematopoietic engraftment in NOD/SCID mice, we followed a published protocol (Cashman et al. 2002). Briefly, bone marrow cells from recipient NOD/SCID mice were stained with anti-human CD45-PE, CD71PE, CD15-FITC, and CD66b-FITC to quantify the total human hematopoietic (CD45/71þ) cell population as well as the subset of exclusively granulopoietic (CD15/66bþ) cells within this population. Cells were stained with anti-human CD34-FITC and anti–human CD19-PE and CD20-PE to quantify human progenitor (CD34þ) and B-lineage (CD34- CD19/20þ) populations. In the experiment in Figure 1, only total human hematopoietic (CD45/71þ) engraftment was measured. Anti-human CD34-FITC was used to quantitate CD34þ cells in culture. All antihuman antibodies were purchased from Becton Dickinson.

IGFBP2 Supports ex Vivo Expansion of Hematopoietic Stem Cells

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Fig. 3 Culture of human cord blood CD133þ cells in the presence of IGFBP2 or Angptl5 stimulates ex vivo expansion of SRCs. (a) Culture of 1  104 cryopreserved human cord blood CD133þ cells was initiated in serum-free Stem Span medium supplemented with 50 ng/ml human SCF, 10 ng/ml human TPO, and 50 ng/ml human Flt3-L, together with 100 ng/ml human IGFBP2. Total cell numbers were counted during the 10 days of culture period. (b) Multilineage engraftment in NOD/SCID recipients transplanted with cultured progenies from 8,000 initial CD133þ cells (n ¼ 6–9). Lanes 1–4, cells cultured in SCF, TPO, and Flt3-L. Lanes 5–8, cells cultured in SCF, TPO, Flt3-L, and IGFBP2. *Value is significantly different from the value of the lane 1 cells. Student’s t-test, p < 0.05

7 Competitive Reconstitution Analysis The indicated numbers of mouse CD45.2 donor cells were mixed with 1  105 freshly isolated CD45.1 competitor bone marrow cells, and the mixture was injected intravenously via the retro-orbital route into each of a group of 6- to

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Fig. 4 Culture of human cord blood CD133 cells in the presence of Angptl5 and IGFBP2 stimulates ex vivo expansion of SRCs. (a) Culture of 1  104 cryopreserved human cord blood CD133þ cells was initiated in serum-free STF medium (containing SCF, TPO, and FGF-1) or in STF medium supplemented with 500 ng/ml human Angptl5 and 500 ng/ml human IGFBP2, at 5% O2. Total cell numbers were counted during the 11 days of culture period. (b) Extent of human chimerism in the bone marrow of NOD/SCID mice transplanted with 8,000 or 15,000 uncultured human cord blood CD133þ cells or in the progenies of 8,000 initial CD133þ cells cultured in STF medium with or without Angptl5 and IGFBP2 for 11 days. Each symbol represents the engraftment of a single transplanted mouse assayed at two months post-transplantation (n ¼ 7–8). *Significantly different from lanes 1-3. Student’s t-test, p < 0.05. (c) Representative FACS plots of bone marrow cells from one mouse at the condition represented by lane 1 of (b) (Post-thaw) or at the condition represented by lane 4 of panel (b) (Cultured in STF medium containing IGFBP2 and Angptl5) at twp months post-transplantatiom. Percentages of cells in each quadrant are listed.

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9-week-old CD45.1 mice previously irradiated with a total dose of 10 Gy. 106 bone marrow cells collected from primary recipients were used for secondary transplantation. To measure reconstitution of transplanted mice, peripheral blood was collected at the indicated times post-transplant and the presence of CD45.1þ and CD45.2þ cells in lymphoid and myeloid compartments were measured as described (Zhang et al. 2006a, b; Zhang and Lodish 2004, 2005). Calculation of CRUs in limiting dilution experiments was conducted using L-Calc software (StemCell Technologies; Zhang et al. 2006b).

8 NOD/SCID Transplant Uncultured or cultured progenies of human total cord blood mononuclear cells or CD133þ or CD34þ cells at indicated days were pooled together and the indicated portions were injected intravenously via the retro-orbital route into sub-lethally irradiated (350 rad) 8- to 10-week-old NOD/SCID mice. Eight weeks after transplantation, bone marrow nucleated cells from transplanted animals were analyzed by flow cytometry for the presence of human cells. For secondary transplantations, bone marrow aspirates from one hind leg of a primary recipient were used to transplant two secondary recipients, as described (Hogan et al. 2002). Calculation of CRUs in limiting dilution experiments was conducted using L-Calc software (StemCell Technologies; Zhang et al. 2006a, b; Zhang and Lodish 2005). For limiting dilution analysis, mice were considered to be positive for human HSC engraftment when at least 1% (for primary transplantation) or 0.1% (for secondary transplantation) CD45/71þ human cells were detected among the mouse bone marrow cells, unless otherwise indicated.

9 Mass Spectrometry The conditioned medium was resolved by SDS-PAGE. Protein “bands” ranging from 10 –70 kD were excised and samples analyzed by the MIT mass spectrometry core facility. Trypsin digestion was performed, and peptide mixtures were loaded onto a triphasic LC/LC column and tandem mass spectra were analyzed.

◂Fig. 4 (continued) (d) Summary of multilineage reconstitution from mice in lanes 2 and 4 of (b) (n ¼ 8). Some mice transplanted with uncultured cells had zero percent donor repopulation and these data points are not plotted. *Values are significantly different from values of the uncultured cells. Student’s t-test, p < 0.05. (e) Bone marrow cells collected from mice represented by lane 4 of (b) were transplanted into secondary recipients; bone marrow aspirate from one hind leg from a primary recipient was used to transplant two secondary recipients. Multilineage engraftment in secondary NOD/SCID recipients was assayed at five to eight weeks post-transplantation (n ¼ 12 mice transplanted)

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Quantitative RT-PCR

Total RNA was isolated from bone marrow Lin-Sca-1þKitþFlk-2 cells. Firststrand cDNA was synthesized using SuperScript II RT (Invitrogen). Samples were analyzed in triplicate 25 ml reactions (300 nM of primers, 12.5 ml of Master mix), which was adapted from the standard protocol provided in SyBR Green PCR Master Mix and RT-PCR Protocols provided by Applied Biosystems. Primers were purchased from Qiagen or Sigma. The default PCR protocol was used on an Applied Biosystems Prism 7000 Sequence Detection System. The mRNA level in each population was normalized to the level of beta-actin RNA transcripts present in the same sample as described (Liao et al. 2007; Zhang et al. 2006a).

11 11.1

Results Purified Recombinant IGFBP-2 Stimulates Ex Vivo Expansion of HSCs

In an attempt to search for new HSC stimulating factors, we found that serum-free conditioned medium collected from 293T cells supported expansion of HSCs. We identified IGFBP 2 as the factor in the 293T conditioned medium that supports HSC expansion (data not shown). We tried to add purified IGFBP2 to the HSC culture medium, in the absence or presence of other factors. As with other known HSC growth factors, IGFBP2 alone

◂

Fig. 5 Limiting dilution analysis of human cord blood CD133þ cells transplanted into NOD/SCID mice after culture at normal or low oxygen levels. (a–b) Culture of 2  105 cryopreserved human cord blood CD133þ cells was initiated in serum-free STF medium supplemented with 500 ng/ml human Angptl5 and human 100 ng/ml IGFBP2. The numbers of total cells (a) and CD34þ cells (b) were counted. (c) Amount of human chimerism in the bone marrow of NOD/SCID mice transplanted with the indicated numbers (5,000, 10,000, 20,000) of post-thaw uncultured human cord blood CD133þ cells or in the progenies of 1,667, 5,000, or 10,000 CD133þ cells cultured in STF medium with Angptl5 and IGFBP2 in ambient oxygen (lanes 4-6) or 5% oxygen (lanes 7-9) for 10 days. Each symbol represents the engraftment of a single transplanted mouse assayed at two months post-transplantation. Horizontal dotted lines represent arbitrary cut-offs of 0.1% and 1% reconstitution. (d–e) Limiting dilution analysis of the repopulating ability of cells before culture (d) and after culture for 10 days in serum-free STF medium containing 500 ng/ml of Angptl5 and 100 ng/ml IGFBP2 at normal or low O2 levels. (e) Plotted is the percentage of recipient mice containing less than 1% human hematopoietic populations in recipient mouse bone marrow six to eight weeks after transplant versus the number of input or input-equivalent cells injected. The progenies of 10,000 input cells cultured at normal or low O2 repopulated all recipients and these data points (zero percent negative mice) are not plotted. (f) Multilineage engraftment in NOD/SCID recipients transplanted with 20,000 post-thaw uncultured CD133þ cells (n ¼ 8) or cultured progenies of 5,000 initial CD133þ cells at normal O2 (n ¼ 10). Some mice transplanted with uncultured cells had zero percent donor repopulation and these data points are not plotted

Fold of untreated samplee xpression

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H. Huynh et al. 2.5 n=3

n=7

n=8

n=7

2

n=2

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1

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0 hoxB1

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hoxB4

hoxA5

hoxC6

hoxB8

hoxC8 hoxD8

hoxA9

etv6

gfi1b

myc

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p21

Fig. 6 IGFBP2 induced several Hox gene expression. Bone marrow Lin-Sca-1þKitþFlk-2 cells were cultured in STF medium, treated with or without 100 ng/ml IGFBP2 for 3 h before being collected for analysis. The gene expression in samples untreated by IGFBP2 was normalized to 1. The numbers of replicated experiments are shown. Each experiment contained three real-time PCR reactions. Shown are the results of averages of all real-time PCR reactions. *Significantly different from the values of untreated samples, p < 0.05. (fix type in y axis – currently the “e” from expression appears at the end of “sample”)

could not support HSC expansion; the inclusion of IGFBP2 in our serum-free medium supplemented with SCF, TPO, FGF-1, and IGF-2 (STIF medium) supported expansion of HSCs (data not shown). We found that, in serum-free STIF medium, doses of recombinant IGFBP2 that were equal to or higher than 100 ng/ mL supported HSC expansion. Considering that 100 ng/mL of IGFBP2 is approximately 3 nM, IGFBP2 stimulated HSC expansion at the nanomolar level. Because IGFBP2 can bind and modulate the biological effects of IGFs (Ranke and Elmlinger 1997), we next tested whether IGFBP2 stimulated HSC expansion if we did not add IGF-2 to the culture. Figure 1 shows that culture of HSCs in the presence of SCF, TPO, and FGF-1 (STF medium) supplemented with IGFBP2 results in dramatically increased repopulating activities compared to freshly isolated HSCs. In one experiment (Fig. 1A), 20 freshly isolated SP Sca-1þ CD45þ cells supported an average of 1.0% engraftment (bar 1). The cultured progenies of the same number of cells after 10 days in STF medium had an increased engraftment of 9.8% (bar 2). Again, culture of the same number of purified HSCs in 293T conditioned medium supplemented with SCF, TPO, and FGF-1 (293T conditioned STF medium) resulted in a dramatic increase of repopulating HSC activity, to 39.3% (bar 3). The addition of 100 ng/ml recombinant IGFBP2 to STF medium also resulted in a large increase in HSC expansion (bar 4) relative to freshly isolated cells or cells cultured in STF medium (bars 1 and 2). Importantly, treatment of 293T conditioned STF medium with 10 mg/ml anti-IGFBP2 neutralizing antibody again abrogated the HSC stimulating effect of IGFBP2 (bar 5); the control isotype antibody did not show any inhibitory effect (bar 6). We next tested whether IGFBP2 had HSC stimulatory effects that were additive to those of Angptl3, an HSC growth factor we recently identified (Zhang et al. 2006a). When 20 CD45.2 bone marrow SP Sca-1þ CD45þ cells were cultured for 10 days in STF medium, a modest engraftment of 7.9  3.6% was observed at four months post-transplantation (Fig. 1B, bar 4). The inclusion of IGFBP2 in the culture

IGFBP2 Supports ex Vivo Expansion of Hematopoietic Stem Cells

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significantly increased the engraftment to 24.4  7.3% (bar 5). The addition of Angptl3 further increased the engraftment to 49.2  11.3% (bar 6). The numbers of total cells after 10 days of culture were on average 5,000 and did not differ significantly among the different conditions. In particular, the numbers of Lin-Sca-1þKitþ cells or Lin-Sca-1þIGF2-hFcþPrP-CD62L- cells, populations enriched for cultured mouse hematopoietic HSCs and progenitors (Zhang and Lodish 2005), were also not significantly different. This finding attests to the notion that bone marrow transplantation is still the only reliable method to measure HSC activity; surface phenotypes are unreliable metrics of actual numbers of HSCs. Thus, in the absence of IGF-2, both IGFBP2 and Angptl3 stimulate the expansion of long-term repopulating murine HSCs. To ensure that the LT-HSCs indeed were expanded during culture, we pooled the bone marrow from primary recipients and transplanted them into secondary recipients. The originally cultured donor cells repopulated lymphoid and myeloid lineages at 1.5, 4, and 7 months after the secondary transplant (Fig. 1C and 1D).

12

A Cocktail Including IGFBP2 Supports an Approximately 48-fold Increase in Numbers of Repopulating Mouse HSCs

We then developed a defined culture condition that included IGFBP2 for expansion of HSCs. Figure 2 shows that a cocktail containing IGFBP2 dramatically increased the numbers of HSCs in culture after 21 days of culture. Freshly isolated BM SP CD45þSca-1þ cells were either directly transplanted or cultured for 21 days at normal O2 in serum-free STF medium supplemented with Angptl3 and IGFBP2. The frequency of repopulating cells (competitive repopulating units or CRU frequency) for freshly isolated SP CD45þSca-1þ cells was 1 per 34 (95% confidence interval for mean: 1/17 to 1/68, n ¼ 24). After culture, the number of cells was too few to be counted accurately. Therefore we normalized the CRU frequency to the number of cells added to the culture. After culture the CRU frequency increased to 1/0.7 input equivalent cells (95% confidence interval for mean: 1/0.3 to 1/1.4, n ¼ 26), representing a more than 48-fold increase in the number of functional LT-HSCs (increase ¼ 34/0.7; P < 0.05, Student’s t-test). To our knowledge, this is the highest level of ex vivo expansion of HSCs yet achieved in a defined culture.

13

IGFBP2 Stimulates Ex Vivo Expansion of Cultured Human Cord Blood CD133þ Cells

To test the role of IGFBP2 on human HSCs, we used a serum-free medium containing human SCF, TPO, and Flt3-L, a commonly used cocktail for human cell culture, as the basal medium to culture cryopreserved CD133þ cord blood cells. The total number of cells increased more than 210- and 162-fold in the basal medium, and basal medium supplemented with IGFBP2, respectively, over the

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10-day culture (Fig. 3A). Next, 8,000 uncultured CD133þ cells, or their cultured progenies, were transplanted into NOD/SCID mice. IGFBP2 significantly enhanced ex vivo expansion of SRCs (Fig. 3B). Transplantation of cells cultured in the presence of SCF, TPO, and Flt3-L generated mice that had an average of 0.8% human cells in the bone marrow. Addition of IGFBP2 to the culture increased the chimerism to 11.3% (Fig. 3B, compare lanes 1 and 5). IGFBP2 (lanes 6-8) led to increases in the formation of human myeloid cells (CD15/66bþ), B-lymphoid cells (CD34CD19/20þ), and primitive (CD34þ) human cells in the bone marrow (Fig. 3B). Thus IGFBP2, when added together with SCF, TPO, and Flt3-L, each stimulated expansion of human cord blood SRCs.

14

IGFBP2 and Angptl5 Together Stimulate Extensive Ex Vivo Expansion of SRCs of Cultured Human Cord Blood CD133þ Cells

In the experiment described in Figure 4, representing three independent experiments, we added IGFBP2 and Angptl5, together with SCF, TPO, and FGF-1, to our serum- free culture. A recent report suggested that hypoxia improves expansion of human SRCs (Danet et al. 2003) and thus, in parallel, we sought to use a low O2 pressure (5% O2) to expand human HSCs. As shown in Figure 4A, we plated 1  104 cryopreserved human cord blood CD133þ cells; after 11 days of culture in 5% O2, the total numbers of cells increased greater than 200-fold either in STF medium or STF medium containing Angptl5 and IGFBP2. We transplanted cells before and after culture into sublethally irradiated NOD/ SCID mice. Eight thousand uncultured CD133þ cells had an average chimerism of 0.2% at two months post-transplantation (Fig. 4B, lane1) and 15,000 uncultured CD133þ cells showed an increased but still modest engraftment – an average chimerism of 2.0% (Fig. 4B, lane 2). In striking contrast, after 11 days of culture with SCF, TPO, FGF-1, Angptl5, and IGFBP2, 2.1  106 cells – that is, the progenies of 8,000 initial cells – engrafted all recipient mice and showed significantly increased chimerism relative to that from 8,000 or 15,000 uncultured cells (average 39.5%; lane 4; p < 0.05, student’s t-test). In contrast, the cultured progenies of the same 8,000 initial cells cultured in STF medium without Angptl5 and IGFBP2 (now 1.6  106 cells) exhibited poor engraftment, similar to that of their uncultured counterparts (lane 3). Figure 4C shows human hematopoietic engraftment at two months in representative mice that were transplanted with uncultured or cultured human cord blood CD133þ cells. A mouse that was transplanted with cells cultured in STF medium containing both Angptl5 and IGFBP2 (lane 4 of Fig. 4B) displayed a much higher engraftment of total hematopoietic (CD45/71þ 58%), myeloid (CD15/66bþ 8.1%), B-lymphoid (CD34-CD19/20þ 37%), and primitive (CD34þ 4%) human cells than the mouse transplanted with uncultured cells (lane 1 of Fig. 4B: 0.19%, 0.03%, 0.1%, and 0.02%, respectively). A summary of multi-lineage engraftment of mice

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transplanted with uncultured cells (Fig. 4B, lane 2) and cells cultured in STF medium containing Angptl5 and IGFBP2 (Fig. 4B, lane 4) is shown in Figure 4D. The progenies of 8,000 cells, after culture, repopulated myeloid and lymphoid lineages two months post-transplantation, attesting to the expansion of human stem cell activity. To measure the self-renewal potential of SRCs after culture in medium containing IGFBP2 and Angptl5, we collected bone marrow from the primary mice transplanted with uncultured cells (lane 2 of Fig. 4B) or cells cultured in STF medium containing Angptl5 and IGFBP2 (lane 4 of Fig. 4B) and transplanted them into sublethally irradiated secondary recipients (Guenechea et al. 2001; Hogan et al. 2002). While uncultured cells could not engraft secondary recipients (not shown), the cultured cells showed positive engraftment of myeloid, B-lymphoid, and primitive cells after the secondary transplantation (Fig. 4E). These data indicate a net expansion of HSCs during the initial culture period, and we thus conclude that Angptl5 and IGFBP2 together support extensive ex vivo expansion of human SRCs. We confirmed that we were able to dramatically expand human SRCs in our culture system in two additional independent experiments that also directly tested the response of HSCs to culture in low and ambient oxygen. In a representative study, we cultured 2  105 cryopreserved human cord blood CD133þ cells in STF medium containing Angptl5 and IGFBP2 under normal or low O2 conditions. After 10 days of culture, the number of total cells in both cultures had increased more than 200-fold (Fig. 5A). We did observe a slightly higher number of CD34þ primitive cells after five days of culture at normal versus low O2 (Fig. 5B). As part of the limiting dilution assay to quantitate the SRC frequencies before and after culture, we measured the engraftment by 5,000, 10,000, and 20,000 uncultured CD133þ cells and the progenies of 1,667, 5,000, and 10,000 cells after culture. Figure 5C shows that all mice transplanted with the cultured progenies of 1,667 CD133þ cells engrafted at a level greater than 0.1%. In a limiting dilution assay, recipient mice are often considered to be positive for human HSC engraftment when at least 0.1% human cells are detected among the mouse bone marrow cells (Amsellem et al. 2003). If we use this standard, then the CRU for cultured cells cannot be measured, as at the lowest dose all recipient mice are positive (Fig. 5C). To make a comparison possible between cultured and uncultured cells, we arbitrarily chose to use 1% chimerism as the cut-off for positive engraftment. Based on this standard, Figure 5D shows that the frequency of repopulating cells (CRU) for this particular sample of uncultured CD133þ cells is 1 per 64,075 cells (95% confidence interval for mean: 1/23,919 to 1/171,643, n ¼ 25). That is, as calculated from Poisson statistics, injection of an average of 64,075 cells from this lot of uncultured human CD133þ cells is sufficient to repopulate 63% (¼ 1-1/e) of transplanted mice. Figure 5E shows there was an 8- or 20-fold increase in the number of SRCs after cultured in STF medium containing Angptl5 and IGFBP2 at low or normal O2, respectively. Specifically, when cells were cultured in STF medium containing Angptl5 and IGFBP2 at low O2, the CRU frequency was 1/7,871 normalized to the number of input cells (95% confidence interval for mean: 1/3,460 to 1/17,903, n = 16),
8 fold greater than that of the uncultured

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cells. Strikingly, when we cultured the cells at normal O2, the CRU frequency increased to 1/3,235 input equivalent cells (95% confidence interval for mean: 1/1,908 to 1/5,486, n = 27). These cultured cells had much greater levels of multi-lineage engraftment than uncultured cells (Fig. 5F).

15

IGFBP2 Upregulates HoxB Gene Expression

To identify the intracellular factors that are induced by IGFBP2 in HSCs, a set of transcripts important for HSC function was assessed by quantitative RT-PCR. Treatment by IGFBP2 led to induction in the levels of several Hox mRNAs in bone marrow Lin-Sca-1þKitþFlk-2 cells (Christensen and Weissman 2001), including HoxA3, HoxB3, HoxB4, and HoxC6 (Fig. 6). We are studying whether the role of IGFBP2 in regulation of HSCs is IGF-dependent and whether IGFBP2 regulates the self-renewal and differentiation of HSCs in vivo.

16

Discussion

Previously we showed that IGF-2 and Angptls independently stimulated expansion ex vivo of hematopoietic stem cells (Zhang et al. 2006a; Zhang and Lodish 2004, 2005). Here we establish that IGFBP2 is an additional HSC-supportive factor secreted by tumorigenic 293T cells. IGFBPs are a family of six circulating proteins that bind IGF-1 and IGF-2 with an affinity equal or greater than that of the three IGF receptors. IGFBPs modulate the biological effects of IGFs by controlling IGF distribution, function, and activity (Ranke and Elmlinger 1997). IGFBP2 preferentially binds IGF-2 over IGF-1. IGFBP2 is expressed in the fetus and in a number of adult tissues and biological fluids (Rajaram et al.1997). It is overexpressed in many tumors and in some cases its expression level correlates with the grade of malignancy (Hoeflich et al. 2001; Moore et al. 2003; Schutt et al. 2004). The expression of IGFBP2 is controlled by a number of hormones, growth factors, and transcription factors, including growth hormone, insulin, IGF-1, IGF-2, TGF-b, IL-1, chorionic gonadotropin, follicle-stimulating hormone, estrogen, glucocorticoids, SP1, activating enhancer binding protein 4, and NFkB (reviewed in reference Hoeflich et al. 2001), as well as p53 (Grimberg et al. 2006). Our finding that IGFBP2 promotes the expansion of HSCs was unexpected in light of the IGF-dependent inhibitory effects that IGFBP2 has on normal somatic growth (Hoeflich et al. 1999). Nevertheless, consistent with our result, IGFBP2deficient mice showed decreased spleen weight and total splenic lymphocyte numbers (Wood et al. 2000). Recently, several studies suggested that, in addition to modulating IGF activities, IGFBP2 has intrinsic bioactivities that are independent of IGF1 or IGF2. For example, IGFBP2 binds to the cell surface (Russo et al. 1995; Schutt et al. 2004) and its binding to integrin alpha 5 (Dunlap et al. 2007; Schutt et al. 2004; Wang et al. 2006), or alpha v (Pereira et al. 2004) influences cell

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mobility (Dunlap et al. 2007; Schutt et al. 2004; Wang et al. 2006) and proliferation (Hoeflich et al. 2001; Moore et al. 2003). IGFBP2 was shown to stimulate telomerase activity (Moore et al. 2003), modulate MAPK and PI3K activities (Moore et al. 2003), and activate MMP-2 (Wang et al. 2003) and the Akt pathway (Dunlap et al. 2007). In addition, it was shown that oxidative stress led to the uptake of IGFBP2 into the cell cytosol after 12-24 h (Besnard et al. 2001; Hoeflich et al. 2001). Here we showed that IGFBP2 stimulated ex vivo expansion of HSCs even when IGF-2 was not included in the culture medium. It also has additive or synergistic effects with Angptl3 and other HSC cytokines, including SCF, TPO, FGF-1. Future studies will be conducted to clarify whether IGFBP2 directly stimulates signal transduction pathways in purified HSCs and whether IGF-initiated signaling is involved. We also showed that, within 3 h of treatment, IGFBP2 induced expression of several Hox genes in bone marrow Lin-Sca-1þKitþFlk2cells, a cell population highly enriched in HSCs. Hox proteins are an evolutionary preserved family characterized by a 60-amino acid DNA-binding homeodomain. Hox genes of the A, B, and C, but not the D, clusters are transcribed during normal hematopoiesis (Abramovich et al. 2005). Importantly, ectopic expression of HoxB4 supports HSC self-renewal in culture (Antonchuk et al. 2002). These results support our preliminary hypothesis that IGFBP2 supports HSC expansion partially through upregulation of the expression of several Hox genes. Further studies are needed to investigate the detailed mechanism for IGFBP2’s function on HSCs. It is surprising that IGFBP2 was identified as a protein secreted by a tumorigenic cell line that supports stem cells. It is known that bone marrow hematopoietic progenitors can be recruited to solid tumor sites in vivo (Kaplan et al. 2007). We plan to test whether other cancer cells are enriched in HSC-stimulating activities such as IGFBP2, which may open a new avenue for the study of stem cell niches in pathological conditions. It will also be interesting to determine whether the role of IGFBP2 in solid cancer development has any connection with its ability to expand HSCs in the tumor microenvironment. Our finding that IGFBP2 stimulates the expansion of HSCs suggests that IGFBP2 may be a growth promoter for certain normal and cancer stem cells. Acknowledgments Support to H.F.L. is from NIH grant R01 DK 067356. Support to C.C.Z. is from a NIH grant K01 CA 120099-01, American Cancer Society grant ACS-IRG-02-196, American Society of Hematology Junior Faculty Scholar Award, Welch Foundation grant I-1701, and the Michael. L. Rosenberg Endowed Scholar Fund from University of Texas Southwestern Medical Center.

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Amsellem S, Pflumio F, Bardinet D, Izac B, Charneau P, Romeo PH, Dubart-Kupperschmitt A, Fichelson S (2003) Ex vivo expansion of human hematopoietic stem cells by direct delivery of the HOXB4 homeoprotein. Nature Med 9:1423–1427 Antonchuk J, Sauvageau G, Humphries RK (2002) HOXB4-induced expansion of adult hematopoietic stem cells ex vivo. Cell 109:39–45 Besnard V, Corroyer S, Trugnan G, Chadelat,K, Nabeyrat E, Cazals V, Clement A (2001) Distinct patterns of insulin-like growth factor binding protein (IGFBP)-2 and IGFBP-3 expression in oxidant exposed lung epithelial cells. Biochim Biophys Acta 1538:47–58 Bryder D, Rossi DJ, Weissman IL (2006) Hematopoietic stem cells: the paradigmatic tissuespecific stem cell. Am J Pathol 169:338–346 Cashman J, Dykstra B, Clark-Lewis I, Eaves A, Eaves C (2002) Changes in the proliferative activity of human hematopoietic stem cells in NOD/SCID mice and enhancement of their transplantability after in vivo treatment with cell cycle inhibitors. J Exp Med 196:1141–1149 Christensen JL, Weissman IL (2001).Flk-2 is a marker in hematopoietic stem cell differentiation: a simple method to isolate long-term stem cells. Proc Natl Acad Sci USA 98:14541–14546 Danet GH, Pan Y, Luongo JL, Bonnet DA, Simon MC (2003) Expansion of human SCIDrepopulating cells under hypoxic conditions. J Clin Invest 112:126–135 Domen J, Weissman IL (1999) Self-renewal, differentiation or death: regulation and manipulation of hematopoietic stem cell fate. Mol Med Today 5:201–208 Dunlap SM, Celestino J, Wang H, Jiang R, Holland EC, Fuller GN, Zhang W (2007) Insulin-like growth factor binding protein 2 promotes glioma development and progression. Proc Natl Acad Sci USA 104:11736–11741 Grimberg A, Coleman CM, Shi Z, Burns TF, MacLachlan,TK, Wang W,El-Deiry WS (2006) Insulin-like growth factor factor binding protein-2 is a novel mediator of p53 inhibition of insulin-like growth factor signaling. Cancer Biol Ther 5:1408–1414 Guenechea G, Gan OI, Dorrell C, Dick JE (2001) Distinct classes of human stem cells that differ in proliferative and self-renewal potential. Nature Immunol 2:75–82 Hoeflich A, Wu,M, Mohan S, Foll J, Wanke R, Froehlich T, Arnold G.J, Lahm H., Kolb HJ, Wolf E (1999) Overexpression of insulin-like growth factor-binding protein-2 in transgenic mice reduces postnatal body weight gain. Endocrinology 140:5488–5496 Hoeflich A, Reisinger R, Lahm H, Kiess W, Blum WF, Kolb HJ, Weber, MM, Wolf E (2001) Insulin-like growth factor-binding protein 2 in tumorigenesis: protector or promoter? Cancer Res 61:8601–8610 Hogan CJ, Shpall EJ, Keller G (2002) Differential long-term and multilineage engraftment potential from subfractions of human CD34þ cord blood cells transplanted into NOD/SCID mice. Proc Natl Acad Sci USA 99:413–418 Huynh H, LIizuka S, Kaba M, Kirak O, Zheng J, Lodish HF, Zhang CC (2008) IGFBP2 secreted by a tumorigenic cell line supports ex vivo expansion of mouse hematopoietic stem cells. Stem Cells 26:1628–1635 Jordan CT, McKearn JP, Lemischka IR (1990) Cellular and developmental properties of fetal hematopoietic stem cells. Cell 61:953–963 Kaplan RN, Psaila B, Lyden D (2007) Niche-to-niche migration of bone-marrow-derived cells. Trends Mol Med 13:72–81 Kirouac DC,Zandstra PW (2006) Understanding cellular networks to improve hematopoietic stem cell expansion cultures. Curr Opin Biotechnol 17:538–547 Liao MJ, Zhang CC, Zhou B, Zimonjic DB, Mani SA, Kaba M, Gifford A, Reinhardt F, Popescu NC, Guo W, Eaton EN, Lodish HF, Weinberg RA (2007) Enrichment of a population of mammary gland cells that form mammospheres and have in vivo repopulating activity. Cancer Res 67:8131–8138 Moore MG, Wetterau LA, Francis MJ, Peehl DM, Cohen P (2003) Novel stimulatory role for insulin-like growth factor binding protein-2 in prostate cancer cells. Int J Cancer 105:14–19 Morrison SJ, Uchida N, Weissman IL (1995) The biology of hematopoietic stem cells. Annu Rev Cell Dev Biol 11:35–71

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The Role of Insulin-like Growth Factor-I in Central Nervous System Development A. Joseph D’Ercole and Ping Ye

Abstract Insulin-like growth factor-I (IGF-I) signaling through its interaction with the type 1 IGF receptor (IGF1R) is necessary for normal brain growth. IGF-I/ IGF1R signaling exerts actions on cells of each major neural lineage. Among these actions are 1) stimulating proliferation in neural progenitors and possibly in pluripotent neural stem cells, 2) augmentation of neuron and oligodendrocyte survival, and 3) promotion of neuron and oligodendrocyte differentiation, such as synaptogenesis and myelination. These pleiotropic actions suggest that IGF-I/ IGF1R signaling facilitates the actions of other factors that provide primary instructions for specific cellular events. During development, IGF-I stimulated actions are effected in an autocrine/paracrine fashion in brain. Consequently in the mouse, brain IGF-I overexpression leads to brain overgrowth, whereas blunting of either brain IGF-I or IGF1R expression leads to brain growth retardation. While few humans have been found to have IGF-I or IGF1R gene mutations, studies of these individuals indicate that IGF-I/IGF1R signaling serves similar roles in man.

1 Introduction The expression and interaction of insulin-like growth factor-I (IGF-I) and its principle cognate receptor, the type 1 IGF receptor (IGF1R), is required for normal central nervous system (CNS) development (see reviews: Anlar et al. 1999; Bondy and Cheng 2004; Russo et al. 2005; Popken et al. 2005). When alterations are made in the mouse genome that lead to changes in the expression of either IGF-I or IGF1R, there are significant changes in brain growth (see review in D’Ercole et al. 2002). IGF-I overexpression results in brain overgrowth, whereas null mutations in A.J. D’Ercole (*) Department of Pediatrics, University of North Carolina CB7039, Chapel Hill, NC 27599-7039, USA e-mail: [email protected]

D. Clemmons et al. (eds.), IGFs: Local Repair and Survival Factors Throughout Life Span, Research and Perspectives in Endocrine Interactions, DOI 10.1007/978-3-642-04302-4_4, # Springer-Verlag Berlin Heidelberg 2010

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either IGF-I or the IGF1R cause brain growth retardation, as does overexpression of inhibitory IGF binding proteins. While IGF-II likely also influences CNS development and/or function, in the mouse its overexpression does not appear to increase brain size, and its deletion does not appear to alter the rate of brain growth, at least during postnatal life. IGF-I signaling through the IGF1R influences each major neural cell lineage by stimulating 1) proliferation of neural progenitors and possibly pluripotent neural stem cells, 2) survival of neurons and oligodendrocytes, 3) differentiation of neurons, including neuritic outgrowth and synaptogenesis, 4) differentiation of oligodendrocytes, including expression of myelin gene proteins and myelination, and 5) multiple metabolic and functional activities (Russo et al. 2005). The wideranging nature of IGF-I actions appear to be largely determined by the signaling mechanisms in place in specific neural cells at precise times in development. For example, IGF-I stimulates the proliferation of neuron progenitors, and later in postmitotic neurons it promotes survival and differentiation, such as neuritic outgrowth and synapse formation. These pleiotropic activities of IGF-I also make it unlikely that IGF-I provides the primary instructive signals for these biologic events. In other words, IGF-I does not appear to initiate signals that trigger a fundamental change or changes intrinsic to developmental programs, such as directing stem cell differentiation towards a single lineage. Rather it appears that IGF-I acts in concert with other agents that provide instructive signals and either facilitates or augments specific processes. The findings that all major neural cell types are present in IGF-I or IGF1R null mutant mice support this view. Likewise, although IGF-I overexpression increases the number of neural cells in each lineage during development, the changes it induces in cytoarchitecture and pace of development are often subtle.

2 Overview of IGF-I Effects on Brain Growth Multiple lines of evidence indicate that IGF-I actions on brain development are exerted in an autocrine/paracrine fashion. IGF-I expression is widespread in the brain during development and in the adult and occurs in a temporal, region-specific pattern (Russo et al. 2005; Popken et al. 2005). While neurons are the major cells of IGF-I expression, IGF-I is expressed by all major neural cell types, including astrocytes, oligodendrocytes and microglia. The expression of the IGF1R, as well as IGF binding proteins (IGFBP), is also developmentally regulated and widespread. The latter finding argues that all the proteins necessary for regulated IGFI action are present in specific areas at precise developmental times. Perhaps the strongest argument for IGF-I acting locally in brain development comes from experiments in which manipulations of the mouse genome lead to changes in brain expression of IGF-I and to significantly altered brain growth (see review in D’Ercole et al. 2002). Brain IGF-I overexpression, regardless of whether it is accompanied by widespread somatic IGF-I overexpression, results in an increased brain size. The magnitude of the overgrowth depends on the magnitude

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Brain weight (% Littermate Controls)

180 MT/IGF-1 Tg Nestin/IGF-1 Tg

160 140 120 100 E18 0

20 40 Time (Days)

60

80

Fig. 1 Time course of brain weight growth in IGF-I-overexpressing mice. Brain weights (expressed as a percentage of littermate controls) are shown for two transgenic lines of mice in which IGF-I expression is controlled either by the metallothionein-I promoter (MT/IGF-I) or by nestin genomic regulatory elements (nestin/IGF-I)

and the developmental time of IGF-I overexpression. When nestin genomic regulatory elements direct brain IGF-I overexpression in nestin/IGF-I mice, transgene expression occurs from early in embryonic brain development, and brain weight is increased by 10–20% from late in gestation onward (Popken et al. 2004; see Fig. 1). IGF-I transgene expression directed by the metallothionein-I (MT-1) promoter begins in late gestation, increases postnatally and continues at a high level throughout life (Ye et al. 1995a). As a consequence, brain overgrowth is first significant at about two weeks of life and increases asymptotically throughout life (Fig. 1). The magnitude appears to depend upon the degree of transgene overexpression, with some lines of mice exhibiting brain weights as much as 80% of normal weight. Ablation of IGF-I expression in null mutant mice results in a dramatic reduction in both body and brain size, such that, in young adulthood, body and brain weights are reduced by 65% to 75% and 29% to 38%, respectively (Beck et al. 1995; Ye et al. 2002). In these mice, the hippocampus is most retarded in weight (by 46%), followed by the cerebellum (by 35%), cerebral cortex (by 30%), diencephalon (by 22%) and brain stem (by 20%). The degree of hippocampus weight retardation, relative to that in normal mice, became greater during the course of postnatal growth, suggesting that IGF-I signaling influences postnatal hippocampal growth and maintenance of hippocampal integrity. Ablation of IGF1R expression also results in body and brain growth retardation. At the time of birth when these mice die, body weights are about 45% of normal (Liu et al. 1993; Baker et al. 1993). The degree of brain weight retardation, however, was not quantified. In other mice in which IGF1R expression is blunted in neural progenitors (using a nestin-driven Cre transgene and a loxed IGF1R allele), brain weights are reduced by 60% at 90 days of age in mice homozygous for the deletion and by 40% in mice heterozygous for the deletion. Brain weight retardation was not accompanied by body growth retardation (Wen et al., unpublished data). In the latter, mouse hippocampus also exhibited the most dramatic retardation (Fig. 2). In another mouse line in which one IGF1R allele was mutated

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Fig. 2 Photomicrographs of the hippocampus in a P60 heterozygous Nestin/IGF1R conditional knockout mouse (IGF1R -/WT) and a littermate control. The volume of the IGF1R -/WT hippocampus is about 35% of the control mouse. Coronal sections of paraffin-embedded tissues were stained with Cresyl Violet. Scale bars = 50 mm

such that IGF1R abundance was reduced by about 40%, brain size was decreased by 10% at ten weeks of postnatal age (Holzenberger et al. 2003). Despite the apparently dominant role of locally produced IGF-I on brain development, circulating IGF-I also likely influences the brain. IGFs are transported across the blood-brain barrier (e.g., Pulford et al. 1999) and peripherally administered IGF-I exerts actions in the brain (Mackay et al. 2003). It seems certain, therefore, that blood-born IGF-I influences brain biology, at least at later developmental stages when IGF-I plasma levels are high and brain IGF-I expression is lower.

3 Neural Stem and Progenitor Cells There is substantial evidence that IGF-I stimulates the proliferation of progenitors that become neurons, oligodendrocytes, and astroglia. Whether IGF-I influences pluripotent neural stem cells (NSC) is not certain. NSC are generally defined as progenitors having the capacity to divide, whether in culture or in vivo, and to differentiate into each of three major neural lineages (Kennea and Mehmet 2002). In the embryo, NSC reside in the neuroepithelium, a layer of cells lining the lateral ventricles. In later development and in the adult, proliferating NSC persist in the subventricular zone (SVZ), a remnant of embryonic ventricular zones (VZ) and in the subgranular layer (SGL) of the dentate gyrus of the hippocampus. Whether embryonic NSC and those remaining in the adult represent the same population of cells is not clear, and differences between these populations may explain the conflicting data (see below). Multiple studies have demonstrated expression of IGF-I and the IGF1R, as well as some IGFBPs, in cultured NSC derived from both embryo and adult (see review: Popken et al. 2005); IGF-I actions in NSC, thus, are possible. Cultured neuroepithelial cells from embryonic day (E) 10 mouse are dependent on IGF-I for proliferation and survival (Drago et al. 1991). These cells synthesize IGF-I and proliferate in response to basic fibroblast growth factor (bFGF), but this response is completely inhibited by co-incubation with antibodies to IGF-I,

The Role of Insulinike Growth Factor- in Central Nervous System Development Fig. 3 IGF-I shortens the duration of the cell cycle. The schematic shows that IGF-I shortens the cell cycle in embryonic neuron progenitors by decreasing the duration of G1 phase. IGF-I also increases the number of cells remaining in the cell cycle (see Hodge et al. 2004)

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Promotes Cell Cycle Entry

G0

Shortens G1 and as a result the cell cycle G1

M G2 S

indicating that IGF-I mediates, at least in part, bFGF actions. IGF-I also has been shown to increase the number of putative NSC derived from E14 mouse striatum (Arsenijevic et al. 2001) and in cultured embryonic rat hypothalamic cells (TorresAleman et al. 1990a). In the former study, IGF-I stimulated proliferation and the development of cell aggregates that subsequently developed into multiple lineages (Arsenijevic et al., 2001). A differing result was obtained from studies of cultured NSC derived from E12.5-14.5 mouse olfactory bulb (Vicario-Abejon et al. 2003). These NSC did not appear to require IGF-I for proliferation but were dependent on IGF-I for lineage development. Studies of adult-derived NSC also yielded similarly conflicting results, with some indicating IGF-I promotion of the neuron lineage (Brooker et al. 2000) and others of the oligodendrocyte lineage (Hsieh et al. 2004). These studies likely reflect differences in the origin and/or developmental stages of the NSC studied and in culture conditions. They also may show that the in vivo milieu is unlikely to be replicated in culture. The lack of a unique marker for NSC makes it difficult to distinguish between IGF-I in vivo stimulation of proliferation of NSC or of fate-committed neural progenitors. Analysis of transgenic mice that overexpress IGF-I under the control of nestin genomic regulatory elements suggests that IGF-I could contribute to the embryonic development of multiple neural lineages in a sequence that mimics normal development. Nestin, an intermediate filament protein, is expressed in NSC, radial glia and neuron progenitors. This IGF-I overexpression results in an increased portion of proliferating progenitors in the VZ and SVZ (Popken et al. 2004). These IGF-I stimulated events clearly result in an increased number of neuron progenitors in the cerebral cortex intermediate zone and neurons in the cortical plate in E14 telencephalon. Detailed evaluations of proliferation in these mice show that IGF-I shortens the duration of the cell cycle (due solely to a decrease in G1 phase) and increases the number of cells remaining in the cell cycle (Hodge et al. 2004; Fig. 3). Whether the proliferating VZ cells are pluripotent NSC, fate-restricted precursors or both remains uncertain. Similarly, in vivo administration of IGF-I increases neuron number in the adult dentate gyrus, but again whether this represents NSC or neuron progenitor proliferation is not certain. The finding that IGF1R signaling appears to be required for the proliferation of pluripotent human embryonic stem cells (Bendall et al. 2007) argues that IGF-I also may expand NSC. Intriguingly, with advancing development not only is the number of

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neurons markedly increased in the cerebral cortex, but the number of glial cells is similarly increased. This finding suggests that IGF-I stimulates the proliferation of NSC, some of which initially differentiate into neurons and some of which become glial cells at a later time in development.

4 Neurogenesis The full course of neurogenesis, beginning with the proliferation of neuron progenitors and extending through neuritic growth, synaptogenesis and neuron elimination by apoptosis, is influenced by IGF-I. A number of in vitro studies demonstrated that IGF-I promotes neuron precursor proliferation (Lenoir and Honegger 1983; Drago et al. 1991; D’Ercole et al. 1996). Similar results have been obtained in adult-derived neuron progenitors (Anderson et al. 2002). When it was evaluated in mutant mice, neuron number was found to be increased in all IGF-I overexpressing mice (D’Ercole et al. 2002). The developmental time, as well as the location, of IGF-I overexpression appears to be key in determining whether promotion of proliferation or survival is the predominant mechanism for these increases. When IGF-I is overexpressed during the time of peak neurogenesis (E11 to E17) in nestin/IGF-I mice, there is a dramatic increase in the number of bromodesoxyuridine (BrdU)-labeled proliferating cells and neurons (Popken et al. 2004), such that at E16 the volume and cell number in the cortical plate are increased by more than 50%. At this embryonic time in cortical plate, neurons are the predominant cell type, and apoptosis is rare (Hodge et al. 2007); thus, the increase in cell number likely reflects IGF-I-stimulated proliferation of neuronal precursors. By early in postnatal life, the total number of neurons in multiple brain regions is increased, with the dentate gyrus exhibiting the greatest increase (69%). Inhibition of apoptosis contributes to the postnatal increases in neuron number (Hodge et al. 2007; Ye et al. 1996), but direct measure of the number of proliferating cells indicates that increased neuron progenitor proliferation is the predominant mechanism during development (Popken et al. 2004; Hodge et al. 2004; Hodge et al. 2007). IGF-I also appears to differentially influence specific brain regions (Hodge et al. 2005). In all mutant mice evaluated, the neuron numbers in the hippocampus and the dentate gyrus, as well as their respective sizes, are most influenced by alterations in IGF-I signaling. Other differential effects have been observed in the cerebral cortex. For example, in nestin/IGF-I mice, both the volume and neuron number in the motor cortex are relatively more increased than in the somatosensory cortex. Neuron number in cortical layers also is disproportionally increased, with the greatest increases being in layer 1, followed by layer V > II/III > VI > IV. Consistently, IGF-I null mutant mice and those also not expressing leukemia inhibitory factor (LIF) show deficiencies in medullary motor neurons (Vicario-Abejon et al. 2004). While the reason(s) for these effects is not known, a greater responsiveness of specific progenitor populations to IGF-I seems a reasonable possibility.

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Neuron Apoptosis

IGF-I anti-apoptotic actions are well documented in studies of cultured cells, including in neurons (Aizeman and De Vellis 1987; Takadera et al. 1999; Yamada et al. 2001; Torres-Aleman et al. 1990a,b). The pro-survival effects of IGF-I also are well documented in vivo. In a line of transgenic mice in which IGF-I is highly overexpressed in the cerebellum during postnatal development, there is a near doubling of granule neurons, yet evaluations indicate only modest increases in proliferation (Ye et al. 1996). As judged by TUNEL, these IGF-I overexpressing mice had about half the number of apoptotic cells during the normal peak times of postnatal apoptosis (Chrysis et al. 2001). Similarly, when apoptotic cell number was evaluated in the cerebral cortex of nestin/IGF-I mice using immunocytochemistry for activated caspase-3, the number of apoptotic neurons was significantly decreased (by 31–46%) at all times studied (Hodge et al. 2007). Because the number of apoptotic neurons in the embryonic cortex is very small, promotion of survival does not contribute significantly to the total neuron number at this time. During postnatal life, however, IGF-I anti-apoptotic effects likely are important in maintaining the increased number of cerebral cortical neurons that persist through life in these mice.

4.2

Differentiation of neurons

Both in vitro and in vivo studies have demonstrated the capacity of IGF-I to stimulate neuronal differentiation. Studies demonstrating IGF-I stimulation of neuritic outgrowth were among the first to show IGF-I actions on neural cells (e.g., Aizeman and De Vellis 1987; Ang et al. 1993). Later IGF-I was shown to increase dendrite growth in cultured neonatal Purkinje cells (Fukudome et al. 2003) and the assembly of axonal growth cones in cultured hippocampal neurons (Pfenninger et al. 2003). In the latter cultures, IGF-I actions were mediated by its interaction with high-density expression of the IGF1R in the growth cone (Laurino et al. 2005). Addition of IGF-I to rat cortical slices results in increases in dendritic branching and in the number of apical and basal dendrites in pyramidal cells in somatosensory cortex (Niblock et al. 2000). Studies of transgenic and mutant mice are consistent with the findings in cultured neurons, and often demonstrate that IGF-I influences the size of neuron cell bodies as well as their neuritic outgrowth. A reduction in pyramidal neuron soma size has been observed in IGF-I null mutant mice (Cheng et al. 2003), accompanied by an increase in cell density, and thus, a decrease in neuropil – that is, the area not occupied by neuron cell bodies or soma. By inference, the decrease in neuropil suggests that neuritic outgrowth is diminished. A marked increase in cell density, necessarily accompanied by decreased neuropil, also has been observed in the embryonic brain of IGF1R null mutant mice (Baker et al. 1993).

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In contrast, the somatosensory cortex of IGF-I overexpressing mice exhibits increased neuron cell size, decreased neuron density, and an increase in the area occupied by neuropil (Gutie´rrez-Ospina et al. 1996), indicating increases in neuritic outgrowth. When medullar nuclei were evaluated in a different line of IGF-I overexpressing mice, evidence of increased neuritic outgrowth was found in some, but not all, nuclei, despite the finding that all nuclei exhibited increased volumes. Similarly, increases in the cell sizes of motor neurons were observed in some nuclei (Dentremont et al. 1999). These findings suggest specificity in IGF-I neurite growth promoting actions. More detailed studies in IGF-I overexpressing mice have directly demonstrated that IGF-I stimulates increases in the number of synapses, and thus, by inference an increase in neurites. In the hippocampus during development there is an increase in synapse number that is greater than the increase in neuron number (O’Kusky et al. 2000). The natural process of synapse elimination, however, is not altered by IGF-I overexpression, and thus, the synapse-to-neuron ratio is normal in the adult, although both are increased. In the hypoglossal nuclei, IGF-I also increases synapse number, as well as the length of myelinated axons (O’Kusky et al. 2003). Again the normal synapse-to-neuron ratio is retained. Consistently, IGF-I null mutants demonstrate a decrease in dendrite length and complexity in portions of the cortex (Cheng et al. 2003). The findings that IGF-I null mutants have reduced peripheral nerve conduction velocities (Gao et al. 1999) and sensorineural hearing loss (Cediel et al. 2006) could result from such alterations in axon growth and dendritic arborization, in addition to a loss of neurons. The slower conduction velocities also have been associated with a greater portion of peripheral nerve axons with reduced diameters and a decrease in myelination. Importantly, sensorineural hearing loss has been described in humans with IGF-I or IGF1R gene mutations (Abuzzahab et al. 2003; Walenkamp and Wit 2007).

5 Glia Development Signalling through the IGF1R plays a key role in glia development and is essential for normal oligodendrocyte development and myelination. Glia lineage development occurs in the absence of IGF1R signalling but IGF-I signaling is important for achieving a normal number of oligodendrocytes and astrocytes and to normal myelination.

5.1

Oligodendrocytes

Multiple studies of cultured oligodendrocyte progenitor cells (OPC) demonstrate that IGF-I signaling, mediated by the IGF1R, promotes survival and augments differentiation and myelin formation (Barres et al. 1993; McMorris et al. 1993;

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Ye and D’Ercole 1999; Ness et al. 2002; Ness and Wood 2002). While there is evidence that IGF-I augments proliferation, the increase in cell number stimulated by IGF-I is primarily mediated by inhibition of apoptosis. For example, in the absence of FGF or PDGF, IGF-I only minimally increases BrdU labeling (Barres et al. 1992). Studies of mixed mouse glial cultures and a non-transformed OPC line, called OL-1, show that exposure to caspase inhibitors produces an increased cell number, but to a lesser degree than IGF-I (Ye and D’Ercole 1999; Lagarde et al. 2007). Concurrent treatment with caspase inhibitors and IGF-I also produces increased OL-1 cell numbers similar to those observed with IGF-I alone. Because IGF-I only stimulates a modest increase in BrdU uptake, these studies suggest that IGF-I is primarily a survival factor for OPC. IGF-I also stimulates oligodendrocyte lineage progression in OL-1 cells, as manifested by an increased number of cell processes and the expression of myelin-associated protein genes. The conclusions drawn from studies of cultured OPC are supported by analysis of mutant mice. The most direct evidence comes from studies of mutant mice with blunted IGF1R expression, specifically in oligodendrocyte precursors (IGF1Rpreoligo-ko mice) or in mature oligodendrocytes (IGF1Roligo-ko mice; Zeger et al. 2007). Studies of mice with a global deletion of the IGF1R gene provide limited information because these mutants die perinatally prior to the time of oligodendrocyte maturation (Liu et al. 1993). Nonetheless, a decreased number of cultured oligodendrocyte precursors derived from IGF1R null mutant embryos has been observed. In the IGF1Rpre-oligo-ko mice, the promoter for Olig1, a helix-loop-helix transcription factor that is expressed in early OPC and a key to oligodendrocyte progenitor differentiation, was used to direct Cre expression and the subsequent excision of an exon coding for most of the IGF binding domain of the IGF1R. In IGF1Roligo-ko mice, the promoter for proteolipid protein, a major myelin-protein gene expressed in mature oligodendrocytes, directed Cre expression specifically in mature oligodendrocytes. Evaluation of IGF1Rpre-oligo-ko mouse brain regions composed primarily of oligodendrocyte lineage cells, such as corpus callosum (CC) and anterior commissure (AC), showed a marked decrease in their volume (by 35% to 55%) and cell number (by 54% to 70%) at two and six weeks of postnatal age, respectively. IGF1Roligo-ko mice exhibited fewer marked reductions that were most apparent at older ages, consistent with the later ablation of IGF1R signaling in mature oligodendrocytes. The number of NG2 positive oligodendrocyte precursors was decreased by 60% in IGF1Rpre-oligo-ko mice, resulting in a 56% decrease in mature oligodendrocytes. As assessed by immunostaining for Ki67, a cell cycle marker, precursor proliferation was reduced. An increase in the number of apoptotic cells also was observed, but the magnitude of such increases is difficult to assess because apoptotic cells do not usually retain the antigens that allow their identification and are rapidly removed. Taken together, studies of IGF1R-deficient mice make it clear that IGF1R signaling is required for normal oligodendrocyte development and myelination. Given the above finding, it is not surprising that IGF-I null mutants exhibit alterations of oligodendrocyte development and myelination. The alterations in myelination, however, are ameliorated through the course of postnatal development.

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Fig. 4 Myelin basic protein (MBP) staining of the cerebral cortex (CTX) and brainstem (BS) from IGF-I null mutant and littermate control mice at two weeks of age. Brains of an IGF-I null mutant mouse (panels B and D) and a littermate control (panels A and C) were fixed with 4% paraformaldehyde, sectioned and stained using an anti-MBP antibody. MBP immunoreactivity is shown as black. CC = corpus callosum. Scale bars = 50 mm (see Ye et al. 2002)

IGF-I null mutant mice exhibit much greater retardation of oligodendrocyte-rich brain regions (about 70% decreases in the CC and AC, compared to 38% reduction in brain size) with comparable reductions in oligodendrocytes and an increase in the percentage of unmyelinated axons (Beck et al. 1995). Despite a reduced total number of oligodendrocytes and their progenitors, the concentrations of myelinassociated proteins and their mRNAs, while low during early development (Fig. 4), become normal in early adulthood (Ye et al. 2002; Cheng et al. 1998), suggesting that in the absence of IGF-1 signaling some OPC and oligodendrocytes survive but develop more slowly. IGF-II expression is increased in the brains of IGF-I null mutants and likely exerts a compensatory stimulation of myelin production in the existing oligodendrocytes (Ye et al. 2002). Consistently, myelination also is reduced when IGFBP-1, an inhibitor of both IGF-I and IGF-II actions, is overexpressed (Ye et al. 1995a; Silha and Murphy 2002). IGFBP-1 transgenic mice exhibit a smaller percentage of myelinated axons with few myelin wraps (Ye et al. 1995a), findings similar to those observed in IGF-I null mutant mice (Beck et al. 1995). A deficit in myelination might contribute to the reduced nerve conduction velocities (Gao et al. 1999) and the sensorineural hearing loss (Cediel et al. 2006) observed in IGF-I null mutants. To date no deficits in myelination have been described in humans with IGF-I or IGF1R gene defects (Walenkamp and Wit 2007; Abuzzahab et al. 2003; Okubo et al. 2003). Detailed studies aimed at myelination in these rare patients, however, have not been performed. Because few of these patients were evaluated early in postnatal life, myelination deficits may have been ameliorated over time, as they are in IGF-I null mutant mice (Ye et al. 2002;Cheng et al. 1998).

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IGF-I overexpression has dramatic effects on myelination. In transgenic mice carrying a metallothionein-I-directed IGF-I transgene, total brain myelin content is elevated four-fold (Carson et al. 1993). This elevation results from a modestly increased number of oligodendrocytes and a greater increase in the expression of myelin associated gene proteins (Ye et al. 1995a). The latter finding indicates that IGF-I directly influences the expression of these genes, rather than simply increasing the number of oligodendrocytes. As a result of these IGF-I actions axons with smaller diameters are myelinated and the number of myelin layers wrapped around each axon is greater (Ye et al. 1995b). Transgenic mice overexpressing IGF-I under the control of a myelin basic protein promoter (MBP) exhibit a similar phenotype (Luzi et al. 2004).

5.2

Astrocytes

A number of lines of evidence point to the involvement of IGF-I in astrocyte development. Cultured neonatal rat astroglia proliferate in response to IGF-I and EGF (Han et al. 1992; Chernausek 1993), and a combination of both appears to elicit a synergistic proliferative response (Han et al. 1992). IGF-I is synthesized by astrocytes and this expression is augmented by EGF and FGF. As is the case with other types of cultured neural cells, antibodies against IGF-I block the proliferative response to both IGF-I and EGF, indicating that EGF actions are at least in part mediated by stimulating IGF-I synthesis. In nestin/IGF-I-overexpressing mice, total glia number is increased in the postnatal cerebral cortex to a magnitude similar to that of neurons, suggesting that the increased progenitor proliferation during embryonic development ultimately facilitates glial, as well as neuronal, differentiation (Popken et al. 2004). When IGF-I overexpression is driven in astrocytes under the control of an astrocyte-specific protein promoter, glial fibrillary acidic protein (GFAP) promoter, astrocyte number is increased by 56% when assessed in the dentate gyrus (Ye et al. 2004a). The astrocyte cell body size, as well as expression of GFAP, is also increased, indicating that IGF-I can act on glia in an autocrine fashion. This astrocyte-specific IGF-I overexpression also leads to increased neuron and oligodendrocyte number, indicating paracrine IGF-I actions.

6 Conclusions A significant amount of data and multiple lines of evidence make it apparent that normal mammalian brain growth and development are dependent on IGF1R signaling, predominately through its interaction with IGF-I. While information in man comparable to that derived from studies of cultured neural cells and from animal studies is lacking, rare individuals with IGF-I gene deletions or mutations that result in severe deficits in IGF-I expression have been reported, and each exhibits

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microcephaly and severe mental retardation (see review in Walenkamp and Wit 2007). There have been no reports of humans born with mutations leading to absent IGF1R expression, but two children with heterozygous IGF1R mutations leading to reduced IGF1R expression exhibited either microcephaly and mild mental retardation or learning disorders, and another with an IGF1R mutation leading to reduced IGF-I affinity exhibited altered behavioral characteristics (Abuzzahab et al. 2003; Okubo et al. 2003). The site of IGF-I expression appears to influence its actions. When overexpressed in neurons prenatally, neuron and glia numbers are most influenced (Popken et al. 2004), whereas postnatal neuron expression has a major effect on oligodendrocyte number and myelination (Carson et al. 1993; Ye et al. 1995a) and more modest, yet very significant, effects on neurons (Gutie´rrez-Ospina et al. 1996). When overexpressed in astrocytes, the site of increased IGF-I following a wide variety of injuries, astrocytes appear to be a main target (Ye et al. 2004b). These data strongly support the hypothesis that IGF-I acts locally near its sites of expression in an autocrine and/or paracrine fashion during brain development. The IGF1R modulates multiple neural events in concert with signaling from many other sources. The cross talk elicited by the interaction of IGF-I with multiple other signals determines the responses of the target cells. For the most part, alterations in IGF1R signaling influence the magnitude of developmental events, rather than specifying them. One of the many remaining challenges is elucidating the mechanisms that govern cell responses to IGF-I. More important is the utilization of our knowledge of IGF-I/IGF1R signaling to develop strategies to treat diseases of brain. A wide variety of injuries and developmental disorders involving neuronal apoptosis and demyelination would appear to be amenable to IGF-I treatment. Acknowledgments The research reported from the authors’ laboratories was supported by NIH R01 grant HD008299, NS038891, and NS048868, and an NIH training grant T32 DK07129.

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O’Kusky JR, Ye P, D’Ercole AJ (2000) Insulin-like growth factor-I promotes neurogenesis and synaptogenesis in the hippocampal dentate gyrus during postnatal development. J Neurosci 20:8435–8442 O’Kusky JR, Ye P, D’Ercole AJ (2003) Increased expression of insulin-like growth factor I augments the progressive phase of synaptogenesis without preventing synapse elimination in the hypoglossal nucleus. J Comp Neurol 464:382–391 Okubo Y, Siddle K, Firth H, O’Rahilly S, Wilson LC, Willatt L, Fukushima T, Takahashi S, Petry CJ, Saukkonen T, Stanhope R, Dunger DB (2003) Cell proliferation activities on skin fibroblasts from a short child with absence of one copy of the type 1 insulin-like growth factor receptor (IGF1R) gene and a tall child with three copies of the IGF1R gene. J Clin Endocrinol Metab 88:5981–5988 Pfenninger KH, Laurino L, Peretti D, Wang X, Rosso S, Morfini G, Caceres A, Quiroga S (2003) Regulation of membrane expansion at the nerve growth cone. J Cell Sci 116:1209–1217 Popken GJ, Hodge RD, Ye P, Zhang J, Ng W, O’Kusky JR, D’Ercole AJ (2004) In vivo effects of insulin-like growth factor-I (IGF-I) on prenatal and early postnatal development of the central nervous system. Eur J Neurosci 19:2056–2068 Popken GJ, Dechert-Zeger M, Ye P, D’Ercole AJ (2005) Brain development. Adv Exp Med Biol 567:187–220 Pulford BE, Whalen LR, Ishii DN (1999) Peripherally administered insulin-like growth factor-I preserves hindlimb reflex and spinal cord noradrenergic circuitry following a central nervous system lesion in rats. Exp Neurol 159:114–123 Russo VC, Gluckman PD, Feldman EL, Werther GA (2005) The insulin-like growth factor system and its pleiotropic functions in brain. Endocr Rev 26:916–943 Silha JV, Murphy LJ (2002) Minireview: insights from insulin-like growth factor binding protein transgenic mice. Endocrinology 143:3711–3714 Takadera T, Matsuda I, Ohyashiki T (1999) Apoptotic cell death and caspase-3 activation induced by N- methyl-D-aspartate receptor antagonists and their prevention by insulin-like growth factor I. JNeurochem 73:548–556 Torres-Aleman I, Naftolin F, Robbins RJ (1990a) Trophic effects of basic fibroblast growth factor on fetal rat hypothalamic cells: Interactions with insulin-like growth factor I. Dev Brain Res 52:253–257 Torres-Aleman I, Naftolin F, Robbins RJ (1990b) Trophic effects of insulin-like growth factor-I on fetal rat hypothalamic cells in culture. Neuroscience 35:601–608 Vicario-Abejon C, Yusta-Boyo MJ, Fernandez-Moreno C, de Pablo F (2003) Locally born olfactory bulb stem cells proliferate in response to insulin-related factors and require endogenous insulin-like growth factor-I for differentiation into neurons and glia. J Neurosci 23:895–906 Vicario-Abejon C, Fernandez-Moreno C, Pichel JG, De Pablo F (2004) Mice lacking IGF-I and LIF have motoneuron deficits in brain stem nuclei. Neuroreport 15:2769–2772 Walenkamp MJ, Wit JM (2007) Genetic disorders in the GH IGF-I axis in mouse and man. Eur J Endocrinol 157 Suppl 1:S15-S26 Yamada M, Tanabe K, Wada K, Shimoke K, Ishikawa Y, Ikeuchi T, Koizumi S, Hatanaka H (2001) Differences in survival-promoting effects and intracellular signaling properties of BDNF and IGF-1 in cultured cerebral cortical neurons. J Neurochem 78:940–951 Ye P, Carson J, D’Ercole AJ (1995a) In vivo actions of Insulin-like Growth Factor-I (IGF-I) on brain myelination: Studies of IGF-I and IGF Binding Protein-1 (IGFBP-1) transgenic mice. J Neurosci 15:7344–7356 Ye P, Carson J, D’Ercole AJ (1995b) Insulin-like growth factor-I influences the initiation of myelination: Studies of the anterior commissure of transgenic mice. Neurosci Lett 201:235–238 Ye P, D’Ercole AJ (1999) Insulin-like growth factor I protects oligodendrocytes from tumor necrosis factor--induced injury. Endocrinology 140:3063–3072

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Stimulation of Proliferative Pathways by IGF-binding Proteins Robert C. Baxter, Mike Lin, and Janet L. Martin

Abstract The diverse roles of the insulin-like growth factors (IGFs) in stimulating cell mitogenesis, survival, motility and differentiation have been extensively documented. IGF actions are modulated by IGF-binding proteins (IGFBPs), which can affect IGF signaling through the type 1 IGF receptor (IGFR1) but may also have independent actions on cell proliferation, migration and apoptosis. How these actions are initiated is not well understood, because evidence for unique IGFBP receptors is limited, but it is clear that in some cases recognized signal-transduction mechanisms (e.g., integrins, nuclear receptors) are responsive to IGFBPs. IGFBP-3, which is the major IGF-transport protein in the adult circulation, has cellular actions that go beyond the blocking of IGFR1 activation by sequestering IGFs. These include the induction of apoptosis through nuclear receptors and inhibition of proliferation by activating cell-surface TGF-b receptors. In some cases, however, these IGFR1-independent actions of IGFBP-3 are stimulatory rather than inhibitory to cell growth or motility. This characteristic is of particular interest because there are many cancers in which a high tissue level of IGFBP-3 expression — possibly induced by hypoxia — is associated with high-grade, rapidly-growing tumors. IGFBP-3 has been shown to potentiate signaling through the EGF receptor (EGFR), which is overexpressed in almost half of basal-type breast cancers, and EGFR kinase inhibition can prevent IGFBP-3-stimulated breast cancer cell growth. A report that IGFBP-3 activates sphingosine kinase 1 (SphK1), which may transactivate both EGFR and IGFR1 through sphingosine 1-phosphate (S1P) generation, prompted an investigation of this system in breast epithelial cells. IGFBP-3 induced SphK1 in MCF-10A cells, and the potentiation of EGFR activation by IGFBP-3 was lost when SphK1 was either inhibited or downregulated by siRNA, which is consistent with S1P generation being an essential step in the activation. IGFR1 activation by an IGF-I analog was also potentiated by IGFBP-3, and this effect R.C. Baxter (*) Kolling Institute of Medical Research, University of Sydney (E25), Royal North Shore Hospital, St Leonards NSW 2065, Australia e-mail: [email protected]

D. Clemmons et al. (eds.), IGFs: Local Repair and Survival Factors Throughout Life Span, Research and Perspectives in Endocrine Interactions, DOI 10.1007/978-3-642-04302-4_5, # Springer-Verlag Berlin Heidelberg 2010

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could also be blocked by SphK1 inhibition or downregulation, or by EGFR kinase inhibition. This finding suggests that the transactivation of IGFR1 signaling by IGFBP-3 requires both SphK and EGFR. These data might explain how high tissue IGFBP-3 levels are associated with rapid growth of some tumors and suggest that blockade of the SphK/S1P system may be therapeutically effective in selected cancers.

1 Introduction : The Insulin-like Growth Factors and Their Binding Proteins The insulin-like growth factors, IGF-I and IGF-II, influence cell proliferation, survival, differentiation and motility by activating multiple signalling pathways initiated by the type 1 IGF receptor (IGFR1). The diverse effects of these peptides play intimate roles in both normal somatic growth and survival, from the neonate to the elderly (Blum and Baumrucker 2008, Berryman et al. 2008), and in the aberrant cell growth characteristic of neoplasia (Yuen and Macaulay 2008). The tissue availability of IGFs and their access to cell receptors are regulated by IGF binding proteins (IGFBPs). The six members of the IGFBP family, IGFBP-1 to -6, share considerable structural homology and bind IGFs with high affinity. IGFBPs limit IGF efflux from the circulation by forming binary IGF-IGFBP complexes and, in the case of IGFBP-3 and IGFBP-5, ternary complexes together with the acidlabile subunit (Baxter 2000). In the cellular environment, high affinity IGF binding by IGFBPs typically inhibits IGFR1 activation by IGF-I or IGF-II, since IGFs bind to the IGFBPs with higher affinity than their binding to the receptor. Although the IGFBPs are named for their binding to IGFs, they have a wide variety of ligands, including cell-surface molecules such as integrins (Firth and Baxter 2002). Both IGFBP-1 (Matsumoto et al. 2008) and IGFBP-2 (Wang et al. 2006) sequences contain an Arg-Gly-Asp motif known to affect cell functions including motility through interaction with a5b1 integrin. These effects are believed to occur independently of IGF binding by the IGFBPs. Other IGFBPs including IGFBP-3 and IGFBP-5 have basic, heparin-binding motifs that have the potential to affect cell function by interacting with cell-surface proteoglycans (Booth et al. 1995). IGFBP-5 also binds to the matrix protein thrombospondin-1 to influence IGF-I function in a manner that does not depend on the IGF-IGFBP interaction (Moralez et al. 2005). These examples are just a few of the many cases in the literature in which IGFBPs appear to act extracellularly in an IGF-independent manner, that is, without modulating signalling through IGFR1 (Firth and Baxter 2002). Best recognized as secreted poteins, IGFBPs have also been shown to interact with intracellular ligands (Firth and Baxter 2002). Of particular interest are interactions with the class 2 nuclear hormone receptors, including the retinoid X receptor (RXR), the retinoic acid receptor (RAR) and the vitamin D receptor (VDR) (Liu et al. 2000, Schedlich et al. 2007a, b). Both IGFBP-3 and IGFBP-5

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have been reported to translocate to the cell nucleus (Schedlich et al. 2000) and can modulate ligand-activated transcriptional activity mediated through these nuclear receptors. It is unclear, however, how the IGFBPs traverse the plasma membrane or escape from their normal secretory pathway to interact with cytoplasmic or nuclear ligands.

2 Growth Inhibition by IGFBP-3 As previously reviewed (Firth and Baxter 2002, Ali et al. 2003), numerous studies have shown that IGFBP-3, the predominant IGFBP in the adult human circulation, can inhibit the growth of many cell types. The IGFBP3 gene is a target of the tumor suppressor p53 and may mediate some of its antiproliferative and pro-apoptotic effects (Buckbinder et al. 1995). In cells in which IGF-I or IGF-II can stimulate proliferation or survival through IGFR1 activation, IGFBP-3 typically inhibits these processes by preventing access of the IGF to its receptor (Firth and Baxter 2002). IGFBP-3 can also inhibit growth by mechanisms that do not depend on IGF binding, as illustrated by studies in cells with a disrupted IGFR1 gene (Valentinis et al. 1995), cells that for other reasons lack IGF sensitivity (Gill et al. 1997), or the use of IGFBP-3 mutants with very low IGF affinity (Kim et al. 2004). In some cells, IGFBP-3-mediated apoptosis may require its presence in the cell nucleus and interaction with RXRa (Cobb et al. 2006) but may also be induced by an IGFBP-3 analog that does not translocate to the nucleus (Butt et al. 2002).

3 Growth Stimulation by IGFBP-3 In contrast to the considerable body of literature supporting a growth-inhibitory and pro-apoptotic role for IGFBP-3, there is also evidence that it can stimulate cell proliferation. Two decades ago, IGFBP-3 was shown to potentiate IGF-I-stimulated DNA synthesis in human fibroblasts (De Mellow and Baxter 1988) and breast cancer cells (Chen et al. 1994), and amino acid uptake in bovine fibroblasts (Conover 1992). At that time, no definitive mechanism was elucidated, but the effect was seen even when an IGF-I analog with very low affinity for IGFBP-3 was used; that is, the potentiation of IGF action does not require interaction between IGF-I and IGFBP-3. This rules out the suggested explanation that IGFBP-3 acts by binding to the cell surface and concentrating IGF-I near its receptor, perhaps to be released by controlled proteolysis. More recently, the authors have also demonstrated that IGFBP-3 potentiates EGF action in breast epithelial cells (Martin et al. 2003). Like the potentiation of IGF action, this effect required the preincubation of IGFBP-3 with the cells. IGFBP-3 was shown to stimulate EGF receptor (EGFR) phosphorylation at

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Tyr1068 and to increase ERK signalling downstream of EGFR. This phenomenon was proposed to explain the observation that IGFBP-3-producing breast cancer cells, initially growth-inhibited in vitro, grew larger and more aggressive tumors in nude mice than their non-IGFBP-3-secreting counterparts (Butt et al. 2004). EGFR was implicated because the IGFBP-3-producing tumors expressed increased EGFR by immunohistochemistry, and IGFBP-3-secreting cells showed enhanced EGF sensitivity, and selective inhibition by an EGFR kinase inhibitor, when compared in vitro to non-IGFBP-3-producing cells (Butt et al. 2004). The finding that IGFBP-3-expressing breast cancer cells grow larger tumors in mice than non-IGFBP-3-expressing cells is consistent with the observation in women with breast cancer that larger, more aggressive cancers with poor prognoses express higher tissue IGFBP-3 mRNA and protein than their smaller, less aggressive counterparts (Rocha et al. 1996). Similarly, some other cancers show a positive association between high IGFBP-3 expression and more aggressive, or later-stage, cancer (Chuang et al. 2008, Xue et al. 2008), although this is not seen universally.

4 Investigations into the Mechanism of Growth Stimulation by IGFBP-3 A possible mechanism linking IGFBP-3 with enhanced activation of growth factor receptors was suggested by a report that, in human endothelial cells, a decrease in the pro-apoptotic activity of IGFBP-3 favoring cell growth and survival was associated with increased activation of sphingosine kinase (SphK) and production of sphingosine 1-phosphate (S1P) (Granata et al. 2004). This finding raised the possibility that SphK might be involved in the potentiation of IGF- and EGFstimulated growth responses by IGFBP-3 in cancer cells. The SphK system plays an important role in tumorigenesis. SphK1 has oncogenic properties (Xia et al. 2000) and is overexpressed in a variety of human tumors relative to adjacent normal tissue (French et al. 2003). S1P modulates diverse cellular functions including proliferation, survival and angiogenesis and while some of its cellular effects derive from intracellular activity as a second messenger, S1P is now known to also act extracellularly as a ligand for a family of G protein-coupled receptors (GPCR) originally known as endothelial differentiation gene (EDG)-1, -5 and -3 and now known as the S1P receptors S1P1, S1P2 and S1P3, respectively (Spiegel and Milstien 2003). The significance of this finding, in the context of IGFBP-3 potentiation of growth factor receptor signalling, lies in the fact that transactivation of growth factor receptors by GPCR, including S1P receptors, is well-documented and increasingly recognized as an important mechanism for enhancing signalling through both EGF and IGF receptors. EGFR transactivation by S1P was one of the first such interactions identified (Daub et al. 1996), and IGFR1- and plateletderived growth factor receptor (PDGFR)-mediated signalling and cellular effects

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Fig. 1 Time course of the change in the ratio of SphK1 mRNA to Sphk2 mRNA in MCF-10A breast epithelial cells following exposure to 100 ng/ml recombinant human IGFBP-3. Values shown are mean  SD from three experiments. mRNA levels were measured by quantitative RT-PCR, using hydroxymethylbilane synthase expression as an internal control

have also now been shown to be modulated by transactivation involving members of the S1P receptor family. To test the hypothesis that SphK might be upregulated by IGFBP-3, and might therefore be involved in mediating some IGFBP-3 effects, MCF-10A human breast epithelial cells were incubated with IGFBP-3, and SphK1 and SphK2 mRNA levels were measured by quantitative RT-PCR. SphK1 gene expression increased rapidly, peaking 3 h after exposure to 100 ng/ml IGFBP-3. Concomitantly there was a reciprocal decrease in SphK2 gene expression, also maximal at 3 h. Figure 1 shows the time course of change in the ratio of SphK1:SphK2 expression in response to IGFBP-3, reaching almost 3-fold above the control ratio in the absence of IGFBP-3. While SphK1 is known to mediate growth stimulation, SphK2 has been associated with growth inhibition and the induction of apoptosis (Spiegel and Milstien 2007). The reciprocal regulation of these two kinases, and the marked increase in their ratio in response to IGFBP-3, are consistent with an increase in cell growth and survival, and a concomitant decrease in apoptosis, in response to IGFBP-3. If growth stimulation by IGFBP-3 involves SphK1, it should be blocked by Sphk inhibitors. Figure 2 illustrates the previously reported potentiation of EGF-stimulated EGFR phosphorylation by IGFBP-3 (Martin et al. 2003) and demonstrates that the SphK inhibitor, N,N-dimethylsphingosine (DMS, 10 mM) completely blocks the effect. Similar inhibition of IGFBP-3 action was seen with a second SphK inhibitor, 2-(p-hydroxyanilino)-4-p-chlorophenylthiazole (SKI, 10 mM), or by downregulation of SphK1 using specific siRNA sequences. SphK2 downregulation by siRNA did not inhibit IGFBP-3 action (data not shown). In contrast, pertussis toxin, which uncouples some GPCR-mediated processes by catalyzing the ADP-ribosylation of the a subunits of the trimeric G proteins Gi, Go, and Gt, failed to prevent potentiation by IGFBP-3, indicating that these G proteins are not involved. Similar to the potentiation of ligand-stimulated EGFR activation by IGFBP-3, ligand-stimulated IGFR1 phosphorylation is also further increased by exogenous

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Fig. 2 Potentiation of EGFR phosphorylation by IGFBP-3. MCF-10A breast epithelial cells were exposed to IGFBP-3 at 10 ng/ml for 24 h prior to stimulation (stim) with 1 ng/ml EGF. The degree of EGFR phosphorylation on Tyr1068, detected by immunoblot in the upper panel, is quantitated in the lower panel. Results are expressed relative to the EGFR phosphorylation in the presence of EGF alone. Exposure to IGFBP-3 increased receptor phosphorylation, an effect blocked by the SphK inhibitor DMS but not by pertussis toxin (PTX)

IGFBP-3, providing an explanation for the early reports that, in some circumstances, preincubation with IGFBP-3 could enhance IGF-I-stimulated DNA synthesis in fibroblasts and breast cancer cells (Chen et al. 1994, De Mellow and Baxter 1988). In the present studies, long Arg3-IGF-I has been used as the receptor ligand instead of IGF-I, as it has extremely low affinity for IGFBP-3 but activates the receptor normally. The SphK inhibitors DMS and SKI, or SphK1 downregulation using siRNA, were found to prevent IGFBP-3 from potentiating ligand-stimulated IGFR1 activation, similar to their effect on EGFR activation (Martin et al. 2009). The requirement for SphK1 activity in the growth-stimulatory actions of IGFBP3 suggests that S1P mediates these actions. In MCF-10A cells, acute (5 min) exposure to S1P alone, between 10 and 1000 nM, had little effect on the basal IGFR1 or EGFR phosphorylation state but, similar to preincubation with IGFBP-3, potentiated ligand-activated IGFR1 and EGFR phosphorylation. However, longer exposure to S1P, in a 24-h thymidine incorporation experiment, was found to stimulate DNA synthesis, as shown in Figure 3. This effect involved EGFR transactivation, since it was prevented by coincubation with the EGFR kinase inhibitor AG1478 (1 mM). What is the connection between the potentiation of IGFR1 and EGFR signalling by IGFBP-3? Further studies in the authors’ laboratory have established that the EGFR inhibitor AG1478 prevents the transactivation of IGFR1 by IGFBP-3 (Martin et al. 2009), implying that EGFR activation by IGFBP-3 is an obligatory step in the pathway to IGFR1 activation. Finally, while the effects described above all involve the addition of exogenous recombinant human IGFBP-3, endogenous IGFBP-3 in MCF-10A cells may exert a tonic stimulatory effect on IGFR1 and EGFR activation by their respective ligands. This possibility is demonstrated by the

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Fig. 3 Stimulation of DNA synthesis by S1P is blocked by EGFR inhibition (inhib). Thymidine incorporation was measured over 24 h in MCF10A cells treated with exogenous S1P as indicated, in the absence or presence of the EGFR kinase inhibitor AG1478 (1 mM)

observation that downregulation of endogenous IGFBP-3 using siRNA decreased ligand-stimulated phosphorylation of both receptors by about 50% (Martin et al. 2009) and suggests a previously unrecognized role for IGFBP-3 in the regulation of these signalling pathways.

5 Conclusion and Therapeutic Implications In these studies we are beginning to elucidate the mechanisms underlying the dichotomous activity of IGFBP-3, whereby it is inhibitory and pro-apoptotic in some situations but can also act to stimulate cell growth and survival. We hypothesize that the key elements to this stimulatory activity are the transactivation of EGFR by IGFBP-3 and the intermediary role of SphK1 and the generation of S1P in this process. An important question that remains unanswered is the nature of the initial step in the process, i.e., the mechanism by which SphK1 is activated by IGFBP-3. We have demonstrated that this activation involves regulation at the mRNA level, but whether SphK1 translocation is also necessary is unknown. More importantly, the initial signalling system through which IGFBP-3 effects the upregulation of SphK1 remains to be discovered. The specific steps involved in EGFR transactivation by S1P are also unclear. In MCF-10A cells it does not appear to involve the release of a soluble EGFR ligand, as the process was not inhibited by matrix metalloprotease inhibitors (data not shown). Once activated, EGFR appears to be responsible for the transactivation of IGFR1, leading to a concerted activation of two major growth stimulatory signalling pathways. The implications for cancer therapy are two-fold. First, we have established that EGFR activation is involved in IGFBP-3 action (Martin et al. 2003) and have shown experimentally in breast cancer cell models that cells producing IGFBP-3 can be growth inhibited by EGFR kinase inhibition, whereas cells not producing

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IGFBP-3 cannot (Butt et al. 2004). This finding implies that, by screening for IGFBP-3 expression, it may be possible to distinguish cancers that will respond to EGFR inhibition therapy from those that will not respond. Interestingly, consistent with this hypothesis, a recent study suggests that loss of IGFBP expression by A431 human squamous carcinoma cell tumors may be involved in the development of insensitivity to EGFR kinase inhibition (Guix et al. 2008). Second, we have now demonstrated the obligatory involvement of SphK1 in this process, opening the possibility that IGFBP-3-producing cancers may be amenable to pharmacological intervention at the level of SphK inhibition or S1P receptor inhibition. It will be important to conduct extensive further experimentation to validate this model in many other conditions and to test whether its potential for targeting cancer therapy based on IGFBP-3 status offers any opportunities for implementation in clinical practice.

References Ali O, Cohen P, Lee KW (2003) Epidemiology and biology of insulin-like growth factor binding protein-3 (IGFBP-3) as an anti-cancer molecule. Horm Metab Res 35:726–733 Baxter RC (2000) Insulin-like growth factor (IGF) binding proteins: Interactions with IGFs and intrinsic bioactivities. Am J Physiol 278:E967–E976 Booth BA, Boes M, Andress DL, Dake BL, Kiefer MC, Maack C, Linhardt RJ, Bar K, Caldwell EEO, Weiler J, Bar RS (1995) IGFBP-3 and IGFBP-5 association with endothelial cells: Role of C-terminal heparin binding domain. Growth Reg 5:1–17 Buckbinder L, Talbott R, Velasco-Miguel S, Takenaka I, Faha B, Seizinger BR, Kley N (1995) Induction of the growth inhibitor IGF-binding protein 3 by p53. Nature 377:646–649 Butt AJ, Fraley KA, Firth SM, Baxter RC (2002) IGF-binding protein-3-induced growth inhibition and apoptosis do not require cell surface binding and nuclear translocation in human breast cancer cells. Endocrinology 143:2693–2699 Butt AJ, Martin JL, Dickson KA, McDougall F, Firth SM, Baxter RC (2004) Insulin-like growth factor binding protein-3 expression is associated with growth stimulation of T47D human breast cancer cells: the role of altered epidermal growth factor signaling. J Clin Endocrinol Metab 89:1950–1956 Chen J-C, Shao Z-M, Sheikh MS, Hussain A, LeRoith D, Roberts CT, Fontana JA (1994) Insulinlike growth factor-binding protein enhancement of insulin-like growth factor-I (IGF-I)mediated DNA synthesis and IGF-I binding in a human breast carcinoma cell line. J Cell Physiol 158:69–78 Chuang ST, Patton KT, Schafernak KT, Papavero V, Lin F, Baxter RC, Teh BT, Yang XJ (2008) Over expression of insulin-like growth factor binding protein 3 in clear cell renal cell carcinoma. J Urol 179:445–449 Cobb LJ, Liu B, Lee KW, Cohen P (2006) Phosphorylation by DNA-dependent protein kinase is critical for apoptosis induction by insulin-like growth factor binding protein-3. Cancer Res 66:10878–10884 Conover CA (1992) Potentiation of insulin-like growth factor (IGF) action by IGF-binding protein-3: Studies of underlying mechanism. Endocrinology 130:3191–3199 Daub H, Weiss FU, Wallasch C, Ullrich A (1996) Role of transactivation of the EGF receptor in signalling by G-protein-coupled receptors. Nature 379:557–560

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De Mellow JS, Baxter RC (1988) Growth hormone-dependent insulin-like growth factor (IGF) binding protein both inhibits and potentiates IGF-I-stimulated DNA synthesis in human skin fibroblasts. Biochem Biophys Res Commun 156:199–204 Firth SM, Baxter RC (2002) Cellular actions of the insulin-like growth factor binding proteins. Endocr Rev 23:824–854 French KJ, Schrecengost RS, Lee BD, Zhuang Y, Smith SN, Eberly JL, Yun JK, Smith CD (2003) Discovery and evaluation of inhibitors of human sphingosine kinase. Cancer Res 63:5962–5969 Gill ZP, Perks CM, Newcomb PV, Holly JM (1997) Insulin-like growth factor-binding protein (IGFBP-3) predisposes breast cancer cells to programmed cell death in a non-IGF-dependent manner. J Biol Chem 272:25602–25607 Granata R, Trovato L, Garbarino G, Taliano M, Ponti R, Sala G, Ghidoni R, Ghigo E (2004) Dual effects of IGFBP-3 on endothelial cell apoptosis and survival: involvement of the sphingolipid signaling pathways. FASEB J 18:1456–1458 Guix M, Faber AC, Wang SE, Olivares MG, Song Y, Qu S, Rinehart C, Seidel B, Yee D, Arteaga CL, Engelman JA (2008) Acquired resistance to EGFR tyrosine kinase inhibitors in cancer cells is mediated by loss of IGF-binding proteins. J Clin Invest 118:2609–2619 Kim HS, Ingermann AR, Tsubaki J, Twigg SM, Walker GE, Oh Y (2004) Insulin-like growth factor-binding protein 3 induces caspase-dependent apoptosis through a death receptormediated pathway in MCF-7 human breast cancer cells. Cancer Res 64:2229–2237 Liu B, Lee HY, Weinzimer SA, Powell DR, Clifford JL, Kurie JM, Cohen P (2000) Direct functional interactions between insulin-like growth factor-binding protein-3 and retinoid X receptor-alpha regulate transcriptional signaling and apoptosis. J Biol Chem 275: 33607–33613 Martin JL, Weenink SM, Baxter RC (2003) Insulin-like growth factor-binding protein-3 potentiates epidermal growth factor action in MCF-10A mammary epithelial cells. Involvement of p44/42 and p38 mitogen-activated protein kinases. J Biol Chem 278:2969–2976 Martin JL, Lin MZ, McGowan EM, Baxter RC (2009) Potentiation of growth factor signaling by insulin-like growth factor-binding protein-3 in breast epithelial cells requires sphingosine kinase activity. J Biol Chem 284:25542–25552 Matsumoto H, Sakai K, Iwashita M (2008) Insulin-like growth factor binding protein-1 induces decidualization of human endometrial stromal cells via alpha5beta1 integrin. Mol Hum Reprod 14:485–489 Moralez AM, Maile LA, Clarke J, Busby WH, Jr., Clemmons DR (2005) Insulin-like growth factor binding protein-5 (IGFBP-5) interacts with thrombospondin-1 to induce negative regulatory effects on IGF-I actions. J Cell Physiol 203:328–334 Rocha RL, Hilsenbeck SG, Jackson JG, Lee AV, Figueroa JA, Yee D (1996) Correlation of insulin-like growth factor-binding protein-3 messenger RNA with protein expression in primary breast cancer tissues: detection of higher levels in tumors with poor prognostic features. J Natl Cancer Inst 88:601–606 Schedlich LJ, Le Page SL, Firth SM, Briggs LJ, Jans DA, Baxter RC (2000) Nuclear import of insulin-like growth factor-binding protein-3 and -5 is mediated by the importin beta subunit. J Biol Chem 275:23462–23470 Schedlich LJ, Graham LD, O’Han MK, Muthukaruppan A, Yan X, Firth SM, Baxter RC (2007a) Molecular basis of the interaction between IGFBP-3 and retinoid X receptor: role in modulation of RAR-signaling. Arch Biochem Biophys 465:359–369 Schedlich LJ, Muthukaruppan A, O’Han MK, Baxter RC (2007b) Insulin-like growth factor binding protein-5 interacts with the vitamin D receptor and modulates the vitamin D response in osteoblasts. Mol Endocrinol 21:2378–2390 Spiegel S, Milstien S (2003) Exogenous and intracellularly generated sphingosine 1-phosphate can regulate cellular processes by divergent pathways. Biochem Soc Trans 31:1216–1219 Spiegel S, Milstien S (2007) Functions of the multifaceted family of sphingosine kinases and some close relatives. J Biol Chem 282:2125–2129

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Valentinis B, Bhala A, DeAngelis T, Baserga R, Cohen P (1995) The human insulin-like growth factor (IGF) binding protein-3 inhibits the growth of fibroblasts with a targeted disruption of the IGF-I receptor gene. Mol Endocrinol 9:361–367 Wang GK, Hu L, Fuller GN, Zhang W (2006) An interaction between insulin-like growth factorbinding protein 2 (IGFBP2) and integrin alpha5 is essential for IGFBP2-induced cell mobility. J Biol Chem 281:14085–14091 Xia P, Gamble JR, Wang L, Pitson SM, Moretti PA, Wattenberg BW, D’Andrea RJ, Vadas MA. (2000) An oncogenic role of sphingosine kinase. Curr Biol 10:1527–1530 Xue A, Scarlett CJ, Jackson CJ, Allen BJ, Smith RC. (2008) Prognostic significance of growth factors and the urokinase-type plasminogen activator system in pancreatic ductal adenocarcinoma. Pancreas 36:160–167 Yuen JS, Macaulay VM (2008) Targeting the type 1 insulin-like growth factor receptor as a treatment for cancer. Expert Opin Ther Targets 12:589–603

Signaling Pathways that Regulate C. elegans Life Span Gary Ruvkun, Andrew V. Samuelson, Christopher E. Carr, Sean P. Curran, and David E. Shore

Abstract Insulin-like growth factor signaling and other endocrine axes contribute to stress and longevity regulation in C. elegans and other animals. By genetic and functional genomic analysis, we have identified players in this pathway. Many of these genes are conserved and likely to mediate endocrine regulation of longevity across the animal kingdom. The life span of an organism is regulated by both genetic and environmental influences in many species (Finch and Austad 2001; Finch and Ruvkun 2001). During the last 15 years, major progress on the genetics of life span has been realized through the study of long-lived mutants in the nematode, Caenorhabditis elegans. In C. elegans, mutations in the insulin/IGF-1/daf-2 signaling pathway can more than double the life span of an animal (Kenyon et al. 1993; Morris et al. 1996, 2003, 2005; Kawano et al. 2005; Paradis et al. 1999; Paradis and Tuvkun 1998). Decreased DAF-2 signaling also causes increased fat storage and dauer arrest (Kenyon et al. 1993; Kimura and Hirano 1997; Kimura et al. 1997; Wadsworth and Riddle 1989). Signaling from DAF-2 is mediated through the AGE-1 phosphatidylinositol 3-kinase (PI3K), PDK-1, and AKT-1/2 kinases to antagonize DAF-16, orthologous to human FOXO, a forkhead transcription factor. The function of this pathway in mediating longevity and metabolism is conserved in C. elegans, Drosophila, and mammals (Finch and Ruvkun 2001; Garofalo 2002; Nakae et al. 2002; Holzenberger et al. 2003; Bluher et al. 2003). The daf-2 insulin/IGF1 pathway also regulates the expression of free radical detoxifying enzymes, consistent with free radical theories of aging and the involvement of mitochondria and reactive oxygen-species (ROS) in aging (Larsen 1993; Vanfleteren and De Vreese 1995; Honda and Yasuda 1999; Honda and Honda 1999). These phenotypes are

G. Ruvkun (*) Department of Molecular Biology, Massachusetts General Hospital, Simches Research Building, 185 Cambridge Street, CPZN-7250, MA 02114, Boston, USA e-mail: [email protected]

D. Clemmons et al. (eds.), IGFs: Local Repair and Survival Factors Throughout Life Span, Research and Perspectives in Endocrine Interactions, DOI 10.1007/978-3-642-04302-4_6, # Springer-Verlag Berlin Heidelberg 2010

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suppressed by daf-16 loss-of-function mutations, suggesting that daf-16 is negatively regulated by the daf-2 pathway and is the major downstream mediator of genes that extend life span (Ogg et al. 1997; Lin et al. 1997). Gene microarray analyses of daf-2 mutants have revealed thousands of transcripts that change in low daf-2 signaling but do not detect directly regulated genes (Golden and Melov 2004, McElwee et al. 2003; Murphy et al. 2003).) daf-2 signaling from both the nervous system and intestine regulates life span (Apfeld and Kenyon 1998; Wolkow et al. 2000; Libina et al. 2003). Using RNA interference to screen for defects in daf-2 pathway-mediated longevity enhancement, we identified a comprehensive genetic network necessary for long life span induction in a daf-2 insulin/IGF1 signalingdeficient mutant. Recent genome-wide RNAi screens for increased longevity have identified
100 potential regulators of life span in C. elegans from diverse cellular pathways, many of which are evolutionarily conserved (Libina et al. 2003; Hamilton et al. 2005). By far the most potent regulator of life span revealed by these screens is the daf-2 insulin signaling pathway. In many organisms, the rate of aging is tied to reproduction. In C. elegans, germline proliferation produces a DAF-16- and KRI-1mediated signal that negatively regulates life span, whereas the somatic gonad promotes life span extension (Hsin and Kenyon 1999; Arantes-Oliveira et al. 2002, 2003; Berman and Kenyon 2006). Caloric restriction (CR) also extends life span across species, including yeast, worms, flies, and mice (Lin et al. 2000; Partridge et al. 2005; Howitz et al. 2003; Wood et al. 2004; Lakowski and Hekimi 1996, 1998; Houthoofd et al. 2002a, b, c, 2003, 2005a, b, 2007). The sir-2.1 and let-363 genes in C. elegans regulate life span via CR (Vellai et al. 2003; Wolff and Dillin 2006; Wolff et al. 2006). Finally, perturbations in mitochondrial function increase life span (Feng et al. 2001; Dillin et al. 2002a, b; Larsen 1993, 2001; Larsen et al. 1995a, b; Larsen and Clarke 2002). The mechanism of longevity induced by defective mitochondria is thought to occur during development, as previous attempts to use RNAi to inhibit mitochondrial function in adults have not been shown to increase life span (Dillin et al. 2002a, b). Life span is dramatically increased in dwarf mice with defects in growth hormone signaling and decreased IGF-I signaling, as well as in mice with defects in insulin signaling within fat and neurons. Importantly, we have found that it is insulin signaling in the nervous system that is key for longevity control. Similar results have emerged from an analysis of insulin and IGF-I signaling in the mammalian nervous system. This insulin-like signaling pathway is part of a global endocrine system that controls whether the animals grow reproductively or arrest at the dauer diapause stage. The connection between longevity and diapause control may not be parochial to C. elegans. Diapause arrest is an essential feature of many vertebrate and invertebrate life cycles, especially in regions with seasonal temperature and humidity extremes. Animals in diapause arrest slow their metabolism and their rates of aging and can survive for periods much longer than their reproductive life span. We have discovered that other arrest points induced by gene inactivations may be as highly regulated as the dauer arrest point. In common with the dauer arrest

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point, these other arrest points are induced by environmental inputs, for example, starvation or natural products that target conserved cellular components such as the mitochondrion or the ribosome. Our analysis suggests that the integrity of these core cellular components is assessed, either in cells that tend to be the most exposed to the environment or in all cells, and that a signaling pathway to endocrine control of development and reproduction may operate. This signaling pathway appears to be distinct from insulin signaling and we are exploring it. In addition to their possible roles in longevity control, the insulin signaling genes identified by C. elegans genetics may reveal components of insulin signaling in mammals that are important for the understanding and eventual treatment of diabetes. Diabetes is a common disease that affects the production or response to insulin, causing devastating metabolic dysregulations. The molecular basis of the defective insulin response in the more common adult onset or type II diabetes is unknown. It is at least in part a genetic disease: the disease shows autosomal dominant transmission but is likely to be multifactorial based on pedigree analysis. In addition, it is clear that both genetically and environmentally induced obesity is a major modulator of diabetes symptoms.

1 Gene Activities that Mediate Increased Life Span of C. Elegans Insulin-like Signaling Mutants Genetic and RNA interference screens for life span regulatory genes have revealed that the daf-2 insulin-like signaling pathway plays a major role in C. elegans longevity. This pathway converges on the DAF-16 transcription factor and may regulate life span by controlling the expression of a large number of genes, including free radical detoxifying genes, stress resistance genes, and pathogen resistance genes. We conducted a genome-wide RNA interference screen to identify genes necessary for the extended life span of daf-2 mutants and identified approximately 200 gene inactivations that shorten daf-2 life span. daf-2 mutant animals were screened for increased mortality using an RNAi library containing 15,718 C. elegans genes to inactivate each of these genes. E. coli strains expressing double-stranded RNAs derived from each of these C. elegans genes were fed to daf-2(e1370) mutant animals, allowing systematic inactivation of C. elegans genes by feeding dsRNA. About 500 RNAi clones from the primary screen were reexamined in a more detailed longitudinal life span analysis: 159 gene inactivations caused a significantly shorter life span than control daf-2 animals, and this decrease in longevity was reproducible in multiple independent populations. Forty-one gene inactivations functioned specifically within the daf-2 pathway to shorten life span, but not decreasing the life span of daf-2;daf-16 animals. Six gene inactivations shortened life span in both daf-2 and daf-2;daf-16 strains; these genes regulate life span independently of insulin/IGF1 signaling. Fifty-seven gene inactivations more dramatically shortened the life span of daf-2

R elative lifespan daf-2(e1370); daf-16(mgDf47)

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animals compared to daf-2;daf-16, but still shortened the life span of daf-2;daf-16 animals, suggesting that they function in a parallel/converging pathway to insulin/ IGF1 signaling. Many of these potential progeric genes also shortened wild type life span. The gene inactivations that are decreasing daf-2 life span more than wild type or daf-2;daf-16 life span are likely to be the genes regulated by insulin/IGF1 signaling to, in turn, protect organisms from the ravages of aging. We assessed these progeric gene inactivations to see whether they show molecular and demographic evidence of faster aging. At the 26th day of adulthood, on average
90% of daf-2(e1370) animals were alive but
40% actively thrashed in water. The transition from active to lethargic movement was measured longitudinally and compared to mortality to examine whether a particular gene inactivation altered the active span. Many organisms, including C. elegans, have an exponential increase in the force of mortality with chronological age that can be approximated by the Gompertz model reviewed in (Pletcher et al. 2000). Characterization of the mortality phenotype for each gene inactivation distinguishes whether life span is shortened due to an increased initial mortality rate (IMR) or a decrease in the mortality rate doubling time (MRDT). Seventy-seven gene inactivations that shorten daf-2 mutant life span produced a significant increase in the rate of daf-2 aging. Eleven gene inactivations produced an increased rate of aging on a par with loss of daf-16: Y65B4A.3, hsf-1, cel-1, F28D1.9, C06A5.1, pnk-1, CD4.4, F55B12.4, rab-7, ccr-4, and ufd-1.

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Aging C. elegans show intestinal autofluorescence partially as a result of lysosomal deposits of the pigment lipofuscin. Aging tissues accumulate this pigment in secondary lysosomes due to the oxidative degradation and autophagocytosis of cellular components (Klaas 1977; Russell and Nurse 1987a,b; Russell and Seppa 1987; Brunk and Terman 2002). daf-2 mutant animals show delayed onset of lipofuscin accumulation (Garigan et al. 2002) and loss of daf-16 causes earlier accumulation of lipofuscin in daf-2 animals (Gerstbrein et al. 2005). Of 39 strictly daf-2 pathway-specific clones, 17 gene inactivations caused strong or moderate acceleration in age pigment accumulation, which was a much higher rate of progeria than control RNAi clones. These data suggest that the genes we have identified are truly progeric. One of the responses to a decline in daf-2 is dramatic upregulation of the DAF16 target gene sod-3 (Honda and Yasuda 1999; Honda and Honda 1999). sod-3 is a manganese superoxide dismutase that functions to protect cells from oxidative stress and whose function may promote normal life span. Thirty-four gene inactivations suppressed the daf-2-dependent induction of sod-3 expression in non-neuronal cells, including C29F9.1, C29F9.2, smk-1, mag-1, F28D1.9, and cua-1. Of the progeric gene inactivations identified in our screen, the most enriched genes were those annotated to mediate vesicle sorting. For example, compared to loss of daf-16, inactivation of Y65B4A.3 caused the greatest acceleration of daf2 aging. Y65B4A.3 is homologous to human charged multivesicular body protein 6 and the myristolyated subunit of yeast ESCRT-III, the endosomal sorting complex required for transport of transmembrane proteins into the multivesicular body pathway to the lysosomal/vacuolar lumen (Babst et al. 2002). Sixteen other endocytosis/vesicular trafficking-related genes also shortened daf-2 life span and increased the rate of aging when inactivated, nine of which strongly increased the Gene Inactivated

Relative Lifespan

T23F2.1, tag-249 Y80D3A.5, cyp-42A1 C03D6.3, cel-1 Y53C10A.12, hsf-1 W04G3.2 Y76A2A.2, cua-1 C06A5.1 Y65B4A.3 F28D1.9 F19B6.2, ufd-1 C33H5.18 Y61A9LA.5 Y39C12A.2 Y6D11A.2, arx-4 BE10.2 Y59A8B.2 F28B12.3, vrk-1 F30A10.10 T07D3.7, alg-2 F39H11.2, tlf-7 W03C9.3, rab-7 CD4.4 F52C6.2 C40H5.6 M05B5.2

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Glycosyltransferase Cytochrome P450 mRNA capping enzyme Heat shock transcription factor unknown ATP7A/MNK, Cu transport, Hailey-Hailey disease unknown ESCRT-3 subunit, endosomal vesicular trafficking FAT4 homolog, linked to insulin resistance Ubiquitin fusion-degradation protein CDP-diglyceride synthetase, phosphatidylinositol lipid signaling pseudogene? Blast e-92 ? vps-32, ESCRT-3 component pseudogene Actin-related protein Arp2/3 complex, subunit ARPC2 Sterol C5 desaturase Ubiquitin C-terminal hydrolase protein kinase, endocytosis? Ubiquitin C-terminal hydrolase posttranscriptional gene silencing TBP-like transcription factor small GTPase, endocytosis ESCRT-I subunit, endosomal protein sorting Ubiquitin-like proteins pseudogene? Blast e-138 ? npl-4.2, Ub ER proteins proteasome degradation unknown

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rate of aging without increasing IMR: C31H2.1, T27F7.1, gdi-1, B0303.9, rab-7, CD4.4, Y47G6A.18, F30A10.6, and wwp-1. One model for the role of endocytosis in aging is recycling of damaged cellular components. However, the age pigment accumulation varies between these gene inactivations: inactivation of some endocytosis genes caused accelerated accumulation of age pigment, whereas other inactivations caused less pigment accumulation. Thus, the accumulation of aging pigments during aging is a marker of the endocytotic decline, but some sorting defects also lead to less pigment accumulation. These data point to the endocytosis defects as key in aging, not age pigment accumulation per se. Many of the genes involved in vesicular trafficking to lysosomes that are necessary for decreased insulin/IGF1 signaling to extend C. elegans life span are also required for induction of sod-3. Specifically, all five subunits in the ESCRT complexes - CD4.4 (I), C27F2.5 (II), F17C11.8 (II), Y65B4A.3 (III), and T27F7.1 (III) - were required for elevation of sod-3 levels. Given that many miRNAs are regulated late in the adult life span, it may be significant that genes annotated to act in miRNA and siRNA pathways are essential for a long life span in both daf-2 and wild type animals (Boehm and Slack 2005; Ibanez-Ventoso et al. 2006). This finding suggests that these small RNAs are important in the aging process. Comparing the 104 genes identified in this screen against three other RNAi screens - enhancement of the mg279 weak allele of let-7 to identify factors required for miRNA activity, and two independent screens to identify genes required for siRNA - showed a significant overlap with our screen. These annotations appeared at two to three times the frequency expected by chance.

2 Life Span Regulation by Evolutionarily Conserved Genes Essential for Viability In addition to its regulation of life span, the daf-2 insulin-like signaling pathway is a key component of a global endocrine system that controls whether the animals grow reproductively or arrest at the dauer diapause stage. Other arrest points induced by gene inactivations may be as highly regulated and as key to life span regulation as the dauer arrest point. These other arrest points may be induced by natural products that target conserved cellular components such as the mitochondrion or the ribosome. We interpret the developmental arrest differently than most who work on essential genes. There are thousands of gene inactivations in C. elegans (and other organisms) that cause severe developmental arrest. Usually such arrests are considered the consequence of a loss of an essential cellular component and that the animal cannot develop past a developmental milestone in the absence of the particular gene product. However, another view of such developmental arrests is that they are “programmed” responses to a deficiency in a key function and that active signaling pathways mediate the arrest point as a sort of “developmental

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Fig. 3 Relationships between protein synthesis and lifespan

checkpoint.” This view is motivated by the finding that arrest at the dauer stage is an active process and can be abrogated by mutations in dauer defective genes, such as the Foxo factor DAF-16 or the Smad factor DAF-3 or the nuclear hormone receptor DAF-12. To reveal this essential pathway signaling system, we screened the 2,700 gene inactivations that cause embryonic or larval arrest in C. elegans for increased adult life span by initiating the gene knockdown once the animal had reached adulthood, thus bypassing any developmental abnormalities. We identified 64 genes that can extend life span when inactivated post-developmentally. More than 90% of the genes we identified were conserved from yeast to humans. Our yield of 64 gene inactivations out of 2,700 tested (
2.4%) was four-fold higher than that from the previous 89 gene inactivations out of 16,000 screened (
0.6%), and a higher proportion of the gene inactivations caused large increases in longevity. Sorting by life span increase, inactivation of genes involved in protein synthesis caused the most potent life span increase. We identified several RNAi clones that target components of the translation initiation factor (eIF) complex - egl-45(eIF3, 52%), eif-3.F(eIF3, 32%), eif-3.B(eIF3, 51%), ifg-1(eIF4G, 55%), T27F7.3(eIF1, 25%) - and two clones targeting inf-1(eIF4A, 46% and 28%). Inhibition of these genes increased life span up to 50% longer than with control RNAi. Although components of the 80S ribosome complex were represented in the RNAi library that we screened, only components of the 43S complex, predominantly translation initiation factors, emerged as regulators of life span. Under conditions of ER stress, a transient block in protein synthesis occurs. Inactivation of the ER oxidoreductase ero-1(32%) also increased life span.

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Signals from proliferating germ cells negatively regulate C. elegans aging, via the insulin-signaling pathway (Kenyon et al. 1993; Kenyon 1996, 2001, 2004, 2005; Kenyon and Gerson 2007). We identified glp-1(33%), which codes for a DSL family ligand receptor in the germline that controls germ cell proliferation and negatively regulates life span (Arantes-Oliveira et al. 2002, 2003). Vacuolar Hþ-ATPases were another potent life span regulator to emerge from the screen. These proteins acidify intracellular compartments and act in synaptic transmission and cell death signaling cascades (Syntichaki et al. 2005). UNC-32, a subunit of the vacuolar ATPase, regulates male longevity (Gems 2000; Gems and Riddle 2000a, b). The vacuolar ATPase is comprised of a V0/V1 heteromultimer. We identified one of each type of subunit from our screen; vha-6(V0, 24%) and tag300(V1, 23%). We were surprised to identify an RNAi clone targeting ced-3 from our screen since ced-3(0) mutants fail to undergo programmed cell death but are not lethal (Ellis and Horvitz 1986). However, we noted synthetic lethality between ced3(717) and daf-2(e1370), indicating a genetic interaction between these longevitypromoting pathways. In support of this finding, the increased life span phenotype of ced-3(RNAi) was dependent upon daf-16. To classify the pathways represented by these new genes, we performed secondary assays: DAF-16 localization, sod-3 expression, arrested larval survival, suppression of polyglutamine aggregation, and aberrant fat metabolism, and we clustered the genes by the phenotypes observed. Because loss-of-function daf-16 alleles are epistatic to many longevity-promoting mutations, we inactivated the 64 candidate longevity genes in a strain also carrying a null daf-16 (mgDf47) mutation and scored for life span extension. Gene inactivations targeting mitochondrial genes that increase adult life span did not depend on DAF-16 (Lee and Amon 2003; Lee et al. 2003a, b; Dillin et al. 2002a, b). The gene inactivations targeting the protein synthesis machinery also increased life span in the absence of DAF-16. Thus, our analysis placed some of these longevity genes within the insulin-signaling pathway, whereas others were independent of this pathway. Many of the 2,700 gene inactivations initiated by feeding RNAi from the L1 stage cause highly penetrant developmental arrest at stages ranging from early larval to sterile adult stage. A subset of these gene inactivations caused arrested larvae to survive longer than control animals and induced DAF-16 nuclear localization at the arrested stage, suggesting that the larval arrest induced uses homologous pathways to dauer arrest. We tested if the larval arrest and increased survival of the arrested larvae induced by these gene inactivations depended on DAF-16 activity by inactivating the same gene in a daf-16(mgDf47);eri-1(mg366) double mutant. The absence of DAF-16 abrogated the long-lived phenotype for most of the arrested larvae. For most gene inactivations, daf-16 mutant animals still arrested when fed the RNAi clone; however, they did not survive as long in the arrested state. But daf-16(mgDf47) weakly suppressed larval arrest of inf-1 or spg-7 inactivation. These data suggest that, at almost any stage, stress pathways requiring daf16 may be triggered by gene inactivations to ensure the survival of the arrested larvae.

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Many of the gene inactivations that cause increased survival of arrested larvae encode translation factors. Translation is a major target of antibiotics, which are produced by a wide range of fungi and microbes that nematodes encounter in the environment. As a larvae or adult enters an environment with an antibiotic, there may be signaling pathways that detect ribosomal deficiency to trigger cessation of reproductive developmental trajectory, arrest at a particular developmental point, as well as xenobiotic protective pathways. The induced stress adaptation and survival pathways would ensure that the animal could live long enough to escape the antibiotic and resume reproductive development. Inhibition of translation by RNAi of translation factors may mimic the ribosomal function deficiency induced by antibiotics in the normal C. elegans ecosystem and trigger this physiological response, developmental arrest and cessation of aging. Because it is a pre-reproductive arrest, this response may be under natural selection to trigger longevity-enhancing pathways (Tercero et al. 1996). Strikingly, other gene inactivations with potent, arrested larval life span increases, a vacuolar ATPase and the mitochondrial ATP synthase are also targets of natural antibiotics (Huss et al. 2002; Breen et al. 1986).

3 Stress Response Systems Activated in Long-lived C. elegans Screens performed by our lab and others have identified over 200 genes that extend the longevity of C. elegans when inactivated by RNAi or mutation (Curran and Ruvkun 2007; Kim et al. 2007; Kim and Sun 2007; Hamilton et al. 2005; Hansen et al. 2005; Lee and Amon 2003; Lee et al. 2003a, b; Johnson et al. 2002). These studies highlighted roles for mitochondrial function, ER function, environmental sensing, insulin signaling, protein synthesis and reproduction in the regulation of longevity (Hamilton et al. 2005; Curran and Ruvkun 2007; Tatar 2005; Viswanathan et al. 2005; Berdichevsky and Guarente 2006; Berdichevsky et al. 2006). Stress resistance may contribute directly to extended longevity and is undoubtedly co-regulated with longevity (Yu and Chung 2001; Yu and Larsen 2001; Yu et al. 2001; Tatar 2005; Honda and Honda 2002; Johnson et al. 1996; 2000, 2002; McElwee et al. 2004; Garsin et al. 2003; Kenyon 2005). Selection of stressresistant mutants in yeast, fly and worms is a powerful prescreen for mutants with long-lived phenotypes (Cypser and Johnson 2003). The degree of tolerance to secondary stress is directly related to the degree of longevity extension (Cypser and Johnson 2003). Furthermore, over-expression of stress resistance genes (sod, catalase, hsp-16 or hsp-70) leads to increased life span in both worms and flies (Cypser and Johnson 2003). Hsu et al. (2003a, b) explored the contribution of heat shock proteins to the longevity of daf-2(lf) mutant worms, finding that the heat shock transcription factor hsf-1 and downstream small heat shock proteins hsp-16.1, hsp-16.49, hsp-12.6 and sip-1 contribute significantly to the daf-2(lf) mutant’s extended life span. The extension of longevity by some treatments may be analogous to hormesis. Hormesis is a coordinated response to sub-lethal environmental stress, during

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which diverse stress response elements are activated. The response entails enhancement of stress resistance phenotypes and concomitant longevity extension (Cypser and Johnson 2003; Cypser et al. 2006). Further, daf-16 plays an essential role in at least some examples of both responses, including the extension of longevity induced by hormetic exposure to heat and the extension of longevity mediated by insulin signaling mutants such as daf-2 (Johnson et al. 2002; Cypser et al. 2006). We have observed a panel of a dozen stress-responsive GFP fusions in strains experiencing gene inactivations corresponding to a library of
200 life spanextending RNAi clones as well as across another 230 progeric gene inactivations.

4 The Cherry-picked Longevity RNAi Library A major prediction from the longevity-enhancing gene inactivation work and from the dauer arrest longevity work is that essential gene inactivation will induce an arrest program that includes detoxification pathways and longevity-enhancing pathways. We tested a heat-induced GFP fusion, hsp-16::GFP, an ER stress-induced gene, hsp-4::GFP, and two mitochondrial stress-induced genes, hsp-6::GFP and gst-4::GFP. hsp-6::GFP and hsp-60::GFP activation was observed only in response to mitochondrial RNAi clones. hsp-6 was activated by almost all mitochondrial events, whereas hsp-60::GFP expression was only induced in a subset of these. hsp-4 was strongly activated by only two clones, both targeting genes with ERrelated annotations (ero-1 and sams-1). sod-3 and gst-4 reporters predominantly respond to RNAi clones with mitochondrial targets, suggesting that an oxidative response is linked to mitochondrial dysfunction and not to the extension of longevity in general. This finding is consistent with the possible increased production of reactive oxygen species and caused by disruption of the ETC (Anson and Hasford 2004; Rea et al. 2007). We also tested the idea that the essential gene inactivations mimic the stress with drugs or environmental stresses. Tunicamycin induces HSP-4 in wild type, a component of the ER UPR. Heat induces HSP-16.2. HSP-16.2 is upregulated in response to heat and other environmental stresses, presumably by the presence of misfolded proteins. Sodium azide induces gst-4. Antimycin inhibits the function of complex 3 by binding the cytochrome b subunit. Antimycin induces hsp-6, a component of MT UPR. The drug treatments, like the gene inactivations, induce a variety of stress-sensing GFP fusions. For example, drugs such as antimycin or Table 1 The cherry-picked longevity RNAi library Source Primary phenotype Hansen et al. 2005 Longevity Hamilton et al. 2005 Longevity Lee et al. 2003a, b; Lee and Amon 2003 Longevity Curran and Ruvkun 2007 Longevity Kim and Sun 2007; Kim et al. 2007 Stress Resistance Samuelson et al. 2007 Progeric in daf-2

# Genes identified 29 89 17 64 84 230

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azide that affect the mitochondria induce the mitochondrial GFP fusions such as GST-4::GFP or hsp-6::GFP.

5 Identification of C. elegans Genes Regulating Longevity using Enhanced RNAi-sensitive Strains Full genome RNAi screens for increased longevity have identified 120 gene inactivations that increase life span. Insulin-like signaling emerged from these comprehensive screens as the most potent regulator of life span, with mitochondrial and ribosomal components also figuring large. Neuronal insulin signaling is key in the regulation of life span and neurons are generally refractory to RNAi, suggesting that more profound increases in life span might be detected in a strain with enhanced neuronal RNAi (Wolkow et al. 2000; Kennedy et al. 2004). Mutations have been identified in C. elegans that enhance the efficacy of both neuronal and global RNAi (eri mutation). A strain carrying a combination of two eri mutants, lin-15b(n744) and eri-1(mg366), is most sensitive to RNAi and most enhanced for neuronal RNAi. In fact, this strain has been used in RNAi screens for synaptic components (Sieburth et al. 2005). A feeding-based RNAi library was used to systematically inactivate 16,757 annotated C. elegans genes in lin-15b(n744);eri-1(mg366) animals. This screen revealed 115 gene inactivations that induce extended survival at a point when mortality of control animals approaches 100%. These gene inactivations were tested in a longitudinal life span analysis (Samuelson et al. 2007a, b). Eighteen gene inactivations extending life span were characterized in more detail. The majority of gene inactivations reduced the relative rate of aging of lin-15b(n744); eri-1(mg366) animals. Metabolism genes were the largest annotated class of genes identified, reaffirming the link between energy production and longevity. Gene inactivations impairing mitochondrial function extended life span largely independently of daf-16 function.

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IGF-1 Regulation of Skeletal Muscle Hypertrophy and Atrophy David J. Glass

Abstract Insulin-like growth factor 1 (IGF-1) can induce skeletal muscle hypertrophy, defined as an increase in skeletal muscle mass. Hypertrophy occurs as a result of an increase in the size, as opposed to the number, of pre-existing skeletal muscle fibers. IGF-1’s pro-hypertrophy activity comes predominantly through its ability to activate the Phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. Akt is a serine-threonine protein kinase that can induce protein synthesis and can block the transcriptional upregulation of key mediators of skeletal muscle atrophy, the E3 ubiquitin ligases MuRF1 and MAFbx (also called Atrogin-1), by phosphorylating and thereby inhibiting the nuclear translocation of the FOXO (also called “forkhead”) family of transcription factors. Once phosphorylated by Akt, the FOXOs are excluded from the nucleus, and upregulation of MuRF1 and MAFbx is blocked. MuRF1 and MAFbx mediate atrophy by ubiquitinating particular protein substrates, causing them to undergo degradation by the proteasome. MuRF1’s substrates include several components of the sarcomeric thick filament, including Myosin Heavy Chain (MyHC). Thus, by blocking MuRF1 activation, IGF-1 helps prevent the breakdown of the thick filament under atrophy conditions. IGF-1 also can dominantly inhibit the effects of a secreted protein called myostatin, which is a member of the TGFb family of proteins. Deletion or inhibition of myostatin causes an increase in skeletal muscle size, because myostatin acts to both inhibit myoblast differentiation and block the Akt pathway. Thus by blocking myostatin, IGF-1 stimulates differentiation and protein synthesis by this distinct mechanism. Myostatin induces the phosphorylation and activation of the transcription factors of Smad2 and Smad3, downstream of the ActRII (Activin Receptor type II)/Alk (Activin Receptor-like kinase) receptor complex. Other TGFb-like molecules can also block differentiation, including TGF-b1, GDF-11, activinA, BMP-2 and BMP-7. As mentioned, myostatin also downregulates the D.J. Glass Novartis Institutes for BioMedical Research Inc., 100 Technology Square room 4210, Cambridge, MA 02139, USA e-mail: [email protected]

D. Clemmons et al. (eds.), IGFs: Local Repair and Survival Factors Throughout Life Span, Research and Perspectives in Endocrine Interactions, DOI 10.1007/978-3-642-04302-4_7, # Springer-Verlag Berlin Heidelberg 2010

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Akt/mTOR/p70S6 protein synthesis pathway, which mediates both differentiation in myoblasts and hypertrophy in myotubes. Blockade of the Akt/mTOR pathway, using siRNA to RAPTOR, a component of “TORC1” (TOR signaling Complex 1), increases myostatin-induced phosphorylation of Smad2, which establishes a “feedforward mechanism”, since myostatin can downregulate TORC1, and this downregulation in turn amplifies myostatin signaling. Blockade of RAPTOR also facilitates myostatin’s inhibition of muscle differentiation. When added to post-differentiated myotubes, myostatin causes a decrease in their diameter; however, this decrease does not happen through the normal “atrophy pathway.” Rather than causing upregulation of the E3 ubiquitin ligases MuRF1 and MAFbx, which have previously been shown to mediate skeletal muscle atrophy, myostatin decreases expression of these atrophy markers in differentiated myotubes, as well as other genes normally upregulated during differentiation, such as MyoD and myogenin. These findings demonstrate that myostatin signaling acts by blocking genes induced during differentiation, even in a myotube, as opposed to activating the distinct “atrophy program.”

1 Protein Synthesis Pathways Downstream of IGF-1 IGF-1 is a protein growth factor that can increase muscle mass in part by stimulating the Phosphatidylinositol-3 Kinase (PI3K)/Akt pathway, resulting in the downstream activation of targets that induce protein synthesis (Bodine et al. 2001b; Rommel et al. 2001; Fig. 1). Weight-bearing or anabolic exercise leads to activation of the PI3K/Akt pathway by directly inducing muscle expression of IGF-1 (DeVol et al. 1990; Yan et al. 1993), which is sufficient to induce hypertrophy of skeletal muscle (Vandenburgh et al. 1991), as was demonstrated in transgenic mice in which IGF-1 is overexpressed in skeletal muscle (Coleman et al. 1995; Musaro et al. 2001). Activation of Akt is sufficient to induce hypertrophy in vivo, as was shown by the production of transgenic mice in which a mutant, constitutively active, form of Akt is conditionally expressed in adult skeletal muscle (Izumiya et al. 2008; Lai et al. 2004; Mammucari et al. 2007). In that setting, acute activation of Akt for two to three weeks is sufficient to induce a doubling in the size of skeletal muscle; this increase occurs via an increase in the average cross-sectional area of individual muscle fibers, caused by an increase in TORC1/p70S6K protein synthesis pathways (Lai et al. 2004). Conversely, in settings of skeletal muscle atrophy, Akt activation is downregulated (Sugita et al. 2004).

2 Hypertrophy Mediators Downstream of PI3K and Akt: the Akt/TORC1/p70S6K and Akt/GSK3 Pathways Genetic experiments using Drosophila originally helped to define a pathway that included PI3K and Akt that can control cell size (Fig. 1). This is the pathway that is recruited by IGF-1 in mammals. In mammals, IGF-1 induces phosphorylation of the

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Fig. 1 Hypertrophy signaling dominantly regulates atrophy signaling. On the left, the IGF-1 signaling pathways relevant to hypertrophy are presented. Signaling molecules that have been shown to have a negative effect on hypertrophy are colored red. Proteins whose activation induces hypertrophy are shown in green. Selected abbreviations: GSK3b - glycogen synthase kinase 3 beta; mTOR - mammalian target of rapamycin; PI3K - phosphatidylinositol-3 kinase. On the right, signaling pathways relevant to atrophy are illustrated. Multiple different perturbations can induce skeletal muscle atrophy: pictured is TNFalpha signaling, since it induces NF-kB, a transcription factor whose activation is required for maximal atrophy. NF-kB activation is the trigger that induces transcriptional upregulation of the E3 ubiquitin ligase MuRF1. A second ligase, MAFbx, is also upregulated in all physiologic settings of atrophy studied; however, its trigger has not been determined. Activation of Akt inhibits the transcriptional upregulation of MAFbx and MuRF1 via the inhibtion of the FOXO family of transcriptional factors and also via a second mechanism downstream of mTOR

IGF-1 receptor, a tyrosine kinase that directly activates the insulin receptor substrate (IRS; Bohni et al. 1999), resulting in the activation of PI3K (Leevers et al. 1996), the mammalian target of rapamycin (mTOR, also known as FRAP or RAFT-1; Zhang et al. 2000), and p70 S6 Kinase (p70S6K; Montagne et al. 1999) (p70S6K); each resulted in decreases in cell size. Although IGF-1 activates mTOR and p70S6K downstream of PI3K/Akt activation, amino acids can activate mTOR directly, causing a subsequent stimulation of p70S6K activity (Burnett et al. 1998; Hara et al. 1998). Thus mTOR appears to have an important and central function in integrating a variety of growth signals, from simple nutritional stimulation to activation by protein growth factors, resulting in protein synthesis.

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Rapamycin is a pharmacologic agent that binds to mTOR and inhibits its function (Lorenz and Heitman 1995; Pallafacchina et al. 2002; Sabers et al. 1995). In vitro, when applied to myotube cultures, rapamycin blocks activation of p70S6K downstream of either activated Akt or IGF-1 stimulation (Pallafacchina et al. 2002; Rommel et al. 1999, 2001; Fig. 1). However, rapamycin does not completely block IGF-1 mediated hypertrophy in vitro, which indicates that other pathways downstream of Akt but independent of mTOR play a role in some settings of hypertrophy. There are two distinct mTOR complexes, TORC1 and TORC2 (Bhaskar and Hay 2007). TORC1 includes a protein called RAPTOR; this complex can be inhibited by rapamycin (Schalm et al. 2003). In a second, rapamycinindependent complex, TORC2, mTOR is complexed with a protein called RICTOR, which has been characterized as being able to phosphorylate Akt on serine 473 in a positive feedback mechanism (Sarbassov et al. 2005). In addition to stimulating p70S6 kinase, activation of mTOR via TORC1 inhibits PHAS-1 (also known as 4E-BP), which is a negative regulator of the protein initiation factor eIF-4E (Hara et al. 1997; Proud 2004). PHAS-1 can directly bind raptor (Hara et al. 2002; Kim do et al. 2002). Mutations in PHAS-1 that inhibit interaction with raptor also inhibit mTOR-mediated phosphorylation of PHAS-1 (Choi et al. 2003). Finally, overexpression of raptor can enhance the phosphorylation of PHAS-1 by mTOR in vitro (Choi et al. 2003; Schalm et al. 2003). mTOR binds PHAS-1 by a TOR signaling (TOS) motif; this same motif is found in p70S6K (Schalm et al. 2003), demonstrating both a mechanism for mTOR’s interaction with its downstream signaling components and the possibility that there may be some selective hierarchy in signaling (since the same motif binds mTOR, one might wonder if there is competition for the same mTOR interaction site). In summary, mTOR can increase protein synthesis by modulating two distinct pathways, the p70S6K pathway and the PHAS-1 pathway (Fig. 1).

3 A Second Hypertrophy Mediator downstream of PI3K and Akt: GSK3b Glycogen synthase kinase 3 beta, GSK3b, is a distinct substrate of Akt that can modulate hypertrophy. Phosphorylation by Akt inhibits GSK3b activity (Cross et al. 1995). Expression of a dominant-negative kinase inactive form of GSK3b induces significant hypertrophy in myotubes (Rommel et al. 2001), as does inhibition of GSK3 with Lithium (Vyas et al. 2002). GSK3b blocks protein translation initiated by the eIF2B protein (Hardt and Sadoshima 2002) and blocks differentiation induced by a transcription factor called NFAT (Rommel et al. 2001; van der Velden et al. 2008). Inhibition or loss of GSK-3b protein activity results in enhanced myotube formation and muscle-specific gene expression during differentiation (van der Velden et al. 2008). In addition, GSK-3b inhibition restores myogenic differentiation following calcineurin blockade, which further implicates the involvement of the transcription factor NFAT in myoblast differentiation

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downstream of IGF-1 activation (van der Velden et al. 2008). Furthermore, GSK3b-deficient myoblasts demonstrated enhanced nuclear translocation of NFATc3 and elevated NFAT-sensitive promoter transactivation (van der Velden et al. 2008). IGF-1-mediated inhibition of GSK-3b is therefore a distinct mechanism for inducing hypertrophy and promoting myoblast differentiation.

4 Skeletal Muscle Atrophy Occurs via Induction of Distinct E3 Ubiquitin Ligases, Whose Expression can be Inhibited by IGF-1 Skeletal muscle atrophy occurs in a variety of settings, including disuse, denervation, cancer cachexia, renal failure, and burns. There is a distinct set of genes that are inversely regulated under IGF-1-induced hypertrophy conditions vs. Dexamethasone (DEX)-induced atrophy (Latres et al. 2005); these include the gene MAFbx (Muscle Atrophy F-box; also called Atrogin-1; Bodine et al. 2001a; Gomes et al. 2001). A second gene, MuRF1 (Muscle Ring Finger1; Bodine et al. 2001a), is significantly upregulated under atrophy conditions (Bodine et al. 2001a). Both MuRF1 and MAFbx/Atrogin were shown to encode E3 ubiquitin ligases (Bodine et al. 2001a). Expression of MuRF1 and MAFbx is stimulated in 13 distinct models of skeletal muscle atrophy (Bodine et al. 2001a; Dehoux et al. 2003; Deruisseau et al. 2004; Gomes et al. 2001; Li et al. 2003). Mice that are null for MuRF1 (MuRF1-/-) and mice that are null for MAFbx (MAFbx-/-) appear phenotypically normal. However, under atrophy conditions, significantly less muscle mass is lost in either MuRF1-/- or MAFbx-/- animals in comparison to control littermates (Bodine et al. 2001a). MuRF1 encodes a protein that contains three domains: a RING-finger domain (Borden and Freemont 1996), which is required for ubiquitin ligase activity (Kamura et al. 1999); a “B-box,” whose function is unclear; and a “coiled-coil domain,” which may be required for the formation of heterodimers between MuRF1 and a related protein, MuRF2 (Centner et al. 2001). Proteins that have these three domains have been called “RBCC” proteins (RING, B-BOX, CoiledCoil domain; Saurin et al. 1996), or “TRIM” proteins (tripartite motif; Reymond et al. 2001). MuRF1 has been demonstrated to have ubiquitin ligase activity that depends on the presence of the RING domain for that activity (Bodine et al. 2001a). MuRF1 has been shown to bind to the myofibrillar protein titin, at the M line (Centner et al. 2001; McElhinny et al. 2002; Pizon et al. 2002). MuRF1 and Myosin Heavy Chain (MyHC) physically interact, as demonstrated by immune-precipitation of epitope-tagged MuRF1 protein, which co-immunoprecipitated MyHC protein; this finding led to the discovery that MyCH was a substrate of MuRF1 (Clarke et al. 2007). Subsequently, it was shown that several other proteins in the thick filament of muscle were also degraded by MuRF1, including Myosin light chain and Myosin binding protein C (Cohen et al. 2009). MAFbx/Atrogin-1 contains an F-box domain, a characteristic motif seen in a family of E3 ubiquitin ligases called SCFs (for Skp1, Cullin, F-box; Jackson and

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Eldridge 2002). F-box containing E3 ligases usually bind a substrate only after that substrate has first been post-translationally modified, for example by phopshorylation (Jackson and Eldridge 2002), suggesting the possibility of a signaling pathway in which a potential substrate is first phosphorylated as a response to an atrophyinduced stimulus and then degraded via MAFbx. Substrates have been suggested for MAFbx, including MyoD (Tintignac et al. 2004) and calcineurin (Li et al. 2004). However, it has not yet been demonstrated if either protein is ubiquitinated by MAFbx in skeletal muscle or during atrophy conditions. In cardiac muscle, MAFbx has no effect on Akt activation in response to IGF-1 or insulin challenge in cardiomyocytes, nevertheless it can repress Aktdependent hypertrophy by activating the Forkhead transcription factors via a distinct type of ubiquitination - i.e., ubiquitination using Lysine 63, which perturbs transcriptional activity (in this case that of the FOXO transcription factors) rather than inducing proteasomal degradation (Li 2007). FOXO activitation was shown to be required to activate the atrophy transcriptional program (Sandri et al. 2004; Stitt et al. 2004), as will be discussed further in the next section. Because FOXO proteins regulate MAFbx expression in skeletal and cardiac muscle, these findings indicated the presence of a feed-forward mechanism in which MAFbx is activated by, and in turn coactivates, FOXO3a and FOXO1 (Li 2007), making it clear why IGF-1’s ability to inhibit FOXO via activation of Akt is necessary to inhibit uncontrolled atrophy in skeletal muscle.

5 IGF-1/PI3K/Akt Inhibition of FOXO Transcription Factors Blocks Upregulation of MuRF1 and MAFbx Studies of differentiated myotube cultures demonstrated that treatment of myotubes with the cachectic glucocorticoid dexamethasone promotes enhanced protein breakdown and increased expression of genes broadly involved in the ubiquitinproteasome proteolytic pathway (Du et al. 2000; Hong and Forsberg 1995; Wang et al. 1998). In vitro treatment of myotubes with dexamethasone induces atrophy, accompanied by the specific increased expression of MAFbx and MuRF1 (Sandri et al. 2004; Stitt et al. 2004). The upregulation of MAFbx and MuRF1 was antagonized by simultaneous treatment with IGF-1 (Sacheck et al. 2004; Sandri et al. 2004; Stitt et al. 2004), acting through the PI3K/Akt pathway; this finding demonstrated a novel role for Akt: in addition to stimulating skeletal muscle hypertrophy, Akt stimulation could dominantly inhibit the induction of atrophy signaling (Fig. 1). Similarly, MuRF1 and MAFbx were activated in a separate model of atrophy, diabetes, and here too IGF-1 blocked the transcriptional upregulation (Lee et al. 2004). Genetic activation of Akt was shown to be sufficient to block the atrophy-associated increases in MAFbx and MuRF1 transcription (Stitt et al. 2004). The mechanism by which Akt inhibited MAFbx and MuRF1 upregulation was demonstrated to involve the FOXO family of transcription factors (Lee et al.

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2004; Sandri et al. 2004; Stitt et al. 2004). In myotubes, FOXO transcription factors are excluded from the nucleus when phosphorylated by Akt and translocate to the nucleus upon dephosphorylation. The translocation and activity of FOXO transcription factors are required for upregulation of MuRF1 and MAFbx; in the case of FOXO3, activation was demonstrated to be sufficient to induce atrophy (Sandri et al. 2004), a finding that was subsequently supported by the transgenic expression of FOXO1, which resulted in atrophic phenotype (Kamei et al. 2004).

6 IGF-1 Regulation of Myostatin In addition to IGF-1, other secreted proteins have been demonstrated to perturb skeletal muscle size. Myostatin, also called GDF-8, is a TGF-b family member that is a negative regulator of muscle mass (Lee et al. 2004). Myostatin’s effect was demonstrated in studies with mice that were made null for the myostatin gene (McPherron et al.1997) and also by correlating increases in muscle mass that were observed in strains of cattle with a loss of myostatin (Grobet et al. 1997; Kambadur et al. 1997; McPherron and Lee 1997). The loss of myostatin resulted in a more than doubling in muscle mass. It has been suggested that other TGF-b superfamily molecules, distinct from myostatin, play a role in modulating skeletal muscle size, since Myostatin-/- mice that are mated with mice that are transgenic for follistatin (TGfollistatin), which is capable of inhibiting not only myostatin but also its close relative GDF-11, and other TGF-b molecules, such as the activins, resulted in an even greater increase in muscle size (Lee 2007). In vitro studies with myostatin have been performed on rodent cells and have shown that myostatin can block the differentiation of myoblasts into myotubes (Langley et al. 2002; McFarlane et al. 2006; Rios et al. 2004; Yang et al. 2007). Experiments both in vitro and in vivo have demonstrated that myostatin signals by first binding the type II activin receptor, IIb, which then allows for interaction with type I receptors ALK4 or ALK5 (Tsuchida et al. 2008). The binding of myostatin to these receptor complexes results in the phosphorylation and activation of the transcription factors Smad2 and Smad3, which translocate to the nucleus upon phosphorylation (Rebbapragada et al. 2003). In a study of myostatin and other TGF-b molecules on human skeletal myoblasts (HuSkMC) and myotubes, HuSkMCs responded to myostatin at physiologic concentrations, 0.1–300 ng/ml, resulting in a decrease in fusion index, myotube diameter, creatine kinase (CK) activity and expression of MyoD and myogenin (Trendelenburg et al. 2009). Follistatin, a more general inhibitor of TGFb molecules, can induce an additive increase in muscle mass when combined with myostatin (Lee 2007). A range of other TGFb molecules are able to block muscle differentiation, including the more distantly related activins and BMP-2 (Trendelenburg et al. 2009). Myostatin inhibits activation of Akt in both myoblasts and myotubes (Trendelenburg et al. 2009). It was recently reported that muscle-specific ablation of TORC1 (by ablating RAPTOR) results in a dystrophic phenotype (Bentzinger et al. 2008). Inhibition

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of RAPTOR, and thus TORC1, does not by itself block muscle differentiation but does contribute to myostatin’s inhibitory effects by resulting in an increase in myostatin-induced Smad phosphorylation, establishing a feed-forward mechanism: myostatin activates Smad2, which inhibits Akt, inhibiting TORC1, which in turn potentiates myostatin’s activation of Smad2. These findings are outlined in Figure 1. Addition of IGF-1 dominantly blocks the effects of myostatin when applied to either myoblasts or myotubes (Trendelenburg et al. 2009). The precise intersection between the two pathways may be multifold, but it is clear that Akt is a particular nexus and that IGF-1 can rescue the activation of Akt that is blunted by myostatin. The demonstration that IGF-1 can dominantly overcome myostatin inhibition adds to the rationale for IGF-1-based treatment regimens in clinical settings where myostatin is active.

7 Conclusion A considerable amount of recent progress has been made in the understanding of the signaling pathways that mediate skeletal muscle hypertrophy and atrophy. Whereas it was appreciated many years ago that hypertrophy comes about via an increase in the rate of protein synthesis, and atrophy through an increase in protein degradation, only now can specific signaling pathways be drawn, since the particular molecular mediators of hypertrophy and atrophy in skeletal muscle have only recently been determined. Furthermore, it is only through recent studies that hypertrophy pathways have been shown to be dominant over the induction of atrophy mediators. These findings help to give hope that novel drug targets may be found to block skeletal muscle atrophy seen in a variety of clinical conditions, from the cachexia of AIDS, sepsis and cancer to the gradual loss of muscle mass observed during normal aging. Acknowledgments Thank you to Drs. M. Fishman, B. Richardson, A. Mackenzie, as well as the rest of the Novartis Community, for their enthusiastic support and input. This work, in particular referenced studies, was performed in large part by A.U. Trendelenburg, B. Clarke, and E. Latres.

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Growth Hormone, Insulin-like Growth Factor I and Insulin: their Relationship to Aging and Cancer Ruslan Novosyadlyy, Emily J. Gallagher, and Derek LeRoith

Abstract Over the past decade, it has become apparent that growth hormone (GH), insulin-like growth factor (IGF-I) and insulin play an important role in the regulation of life span and aging, in addition to their well-established functions in controlling somatic growth and metabolism. Cancer is one of the age-related diseases that shorten life span. Growing evidence indicates that the aforementioned hormones are important modulators of tumor growth and progression. Enhanced signaling through the GH/IGF-I axis is observed in different types of cancer, whereas a reduction results in extended life span. Similarly, insulin resistance and hyperinsulinemia promote aging and tumor growth, whereas improved insulin sensitivity extends life span. Therefore, GH/IGF-I and insulin signaling can be a molecular link between the two processes, coupling cancer and longevity mechanistically. In the current article, we have highlighted some data available in invertebrates and mammals supporting this hypothesis.

1 Introduction Aging is a naturally occurring physiological process, and reaching an old age is achieved by healthy attributes transcending disease states. Age-related death results from disorders including cardiovascular and neurodegenerative diseases, cancer and diabetes. Medical interventions have led to a substantial reduction in cardiovascular morbidity and mortality, causing a significant increase in average life expectancy during the last few decades. Therefore, understanding the mechanisms underlying healthy aging, as well as the pathogenesis of age-related disorders, will eventually help to reduce mortality and extend the healthy life span. D. LeRoith (*) Division of Endocrinology, Diabetes and Bone Diseases, Department of Medicine, Mount Sinai School of Medicine, New York, USA e-mail: [email protected]

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In this review we will discuss those factors involved in aging, focusing particularly on the growth hormone/insulin-like growth factor I (GH/IGF-I) axis and insulin. We examine the effect of these hormones on cancer, an important cause of mortality in the older population. Maintaining a balance within the GH/IGF-I/insulin axis is a crucial determinant of normal aging, while alterations in this system may underlie the development of cancer and other age-related disorders. Studying the interplay of these factors in aging and cancer is both an important area of research and may lead to potential therapeutic implications.

2 GH/IGF-I/insulin axis in the aging process An important age-related process is the somatopause, during which there is an agerelated reduction in circulating GH and IGF-I levels (Lamberts et al. 1997). Moreover, IGF-I and/or insulin signaling has been identified as one of the major pathways controlling life span in many species, and suppression of this signaling promotes longevity in different animals, including worms, insects and mammals (Tatar et al. 2003). The following sections will expand on these concepts.

2.1

Insects and worms

Insulin-like peptides as well as their cognate receptor (daf-2) are major regulators of the dauer state in C. elegans, whereby C. elegans larvae undergo dauer diapause (a physiological state with low metabolic and reproductive activity) in response to food deprivation, which normally extends life span. Mutations in the genes encoding key components of the insulin/IGF-I signaling pathways, including daf-2, AGE-1 (PI3K homolog), pdk-1, Akt1/2 and sgk, result in reduced signaling and increase life span (Taguchi and White 2008). On the other hand, mutations in daf-16 and daf-18 genes (FOXO and PTEN orthologs, respectively) increase insulin/IGF-I receptor signaling and reduce life span (Larsen et al. 1995). Similar to C. elegans, depression of IGF/insulin signaling in D. melanogaster, by mutating genes encoding the drosophila insulin receptor and Chico (an IRS-like molecule), induces diapause, which is accompanied by an extended life span (Taguchi and White 2008). Taken together, these data provide compelling evidence that in both C. elegans and D. melanogaster, inhibition of IGF/insulin signaling has a longevity-promoting effect.

2.2 2.2.1

Mammals Humans

While physiological aging is associated with a state of relative GH and IGF-I deficiency (Lamberts et al. 1997), recent studies suggest that relative IGF-I

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resistance (as demonstrated by reduced insulin signaling in cultured cells) may also extend life span in humans. In a cohort of Ashkenazi Jewish centenarians, Suh et al. (2008) found increased serum IGF-I levels and reduced in vitro cellular IGF-IR activity, suggesting IGF-I resistance that presumably promoted longevity in this cohort.

2.3

Rodents

The role of the GH/IGF-1/insulin axis in controlling mammalian life span has been extensively addressed in numerous animal models. Experimental attenuation of individual components within the GH/IGF-1/insulin axis suggests their importance, though not always consistently, due to secondary aging-related changes.

3 Lessons from dwarf mice Snell dwarf mice that possess a mutation in the Pit-1 gene, demonstrate defects in the development of the pituitary gland, resulting in a dramatic reduction of body size (33% of wild-type (WT) controls), stunted growth, late onset obesity and infertility (Flurkey et al. 2001). Ames dwarf mice (Prop-1mutant) that do not produce GH, prolactin (PRL) and TSH, have markedly reduced circulating levels of insulin, IGF-I and glucose (Brown-Borg et al. 1996). Intriguingly, despite all these abnormalities, Snell and Ames dwarf mice show significantly extended life spans. The “little” (Ghrhrlit/lit) mice with a missense mutation in the growth hormone releasing hormone receptor (Ghrhr) gene are GH deficient. These mice are fertile but have reduced body size (67% of WT), stunted growth and obesity in later life. Their lifespan is extended by 25% (Lin et al. 1993). Mice with a targeted disruption of the Ghr gene develop signs resembling human Laron syndrome and therefore, are also known as Laron mice. Ghr / mice demonstrate severe postnatal growth retardation, proportionate dwarfism, GHR deficiency, reduction of circulating IGF-I levels and marked elevation of serum GH concentrations, suggesting a severe GH resistance. The body weight of Ghr knockout mice is about one-third that of WT and they have an increased life span (Zhou et al. 1997; Coschigano et al. 2003). GH transgenic mice, on the other hand, have markedly increased body size but have an average life span less than 50% that of controls (Pendergrass et al. 1993). Mice with liver-specific IGF-I deficiency (LID mice) have a 75–85% reduction in circulating IGF-I levels but visually demonstrate no overt phenotype (Yakar et al. 1999). LID mice however, have moderately increased body adiposity, significantly elevated serum GH and marked insulin resistance (LeRoith and Yakar 2007). They demonstrate a significantly shortened life span (Adamo,

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unpublished data). In contrast to Igf1r / mice, Igf1r+/ mice are viable and do not display dwarfism. Moreover, their energy metabolism, nutrient intake, physical activity and reproductive function are unaffected (Holzenberger et al. 2003). Igf1r+/ mice also displayed increased resistance to oxidative stress, an important pathophysiological component of aging. Hence, Igf1r+/ females seemed to live longer than WT females; their life spans were increased by 33%. Changes in life span in males were not statistically significant. Importantly, in contrast with Igf1r+/ males, which developed marked glucose intolerance, long-lived Igf1r+/ female mice were more tolerant to glucose than respective WT controls. Taken together, these data thus support the hypothesis that improved insulin sensitivity is a major determinant of mammalian longevity.

4 Insulin receptor and insulin receptor substrates and life span In contrast to Ir null mice, fat-specific IR knockout (FIRKO) mice are viable and long-lived. Life span is extended by 18%. FIRKO mice demonstrate decreased fat mass, reduced circulating triglyceride levels and resistance to age-related obesity and obesity-induced glucose intolerance (Bluher et al. 2002, 2003). Moreover, FIRKO mice also display enhanced mitochondrial gene expression and augmented oxidative metabolism, resulting in an increased metabolic rate and respiratory exchange (Katic et al. 2007). Thus, suppression of IR signaling in adipocytes may extend life span indirectly in a cell non-autonomous manner, by enhancing whole body insulin sensitivity. Female Irs1 / mice are also long-lived (32% increase in life span). Irs1 / females develop persistent insulin resistance with preserved b cell function but preserved glucose tolerance, suggesting that factors other than insulin sensitivity are involved in increased lifespan (Selman et al. 2008).

5 The role of GH/IGF/insulin axis in cancer promotion Cancer, which is one of the age-related disorders, is often accompanied by enhanced signaling though the GH/IGF-I/insulin axis (Pollak et al. 2004). It also became apparent that obesity and/or T2D promote tumor growth and progression and therefore may significantly worsen age-related mortality, which may be associated or even caused by insulin resistance and/or hyperinsulinemia (Calle and Kaaks 2004; Wolf et al. 2005). In tumor cells, the IGF-I receptor (IGF-IR) is often amplified, upregulated and/or hyperactivated. Furthermore, increased circulating IGF-I levels are considered a

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significant risk factor for the development of various types of cancers, including breast, prostate, colon, and lung cancer (Pollak et al. 2004; Yakar et al. 2005). Several lines of evidence also suggest that insulin and its receptor may promote tumor growth (Frasca et al. 2008). Although the oncogenic role of insulin, IGFs and their receptors (i.e. their ability to initiate carcinogenesis) has been demonstrated in cell culture systems, most studies indicate that they are primarily enhancers of tumor growth and progression, rather than real oncogenic proteins. Indeed, the IGF gene signature is associated with numerous poor prognostic markers in breast cancer and seems to be one of the strongest indicators of disease outcome (Creighton et al. 2008). LID mice with reduced circulating IGF-I levels demonstrate significant reductions in tumor development and growth and metastases, even in the face of elevated GH and insulin levels, suggesting a more powerful effect of the IGF-I levels on these processes. On the other hand, elevated GH levels in GH transgenic mice demonstrate increased tumor burden, presumably due to the elevated IGF-I levels. Interestingly, LID mice crossed with TRAP mice that develop prostate cancer, have no reduction in tumor burden (Anzo et al. 2008), suggesting that GH or insulin may have IGF-I-independent effects on certain tumors.

6 Insulin receptor (IR) and insulin receptor substrates (IRS) and cancer IRS proteins are apparently indispensable for the transforming activity of many cellular and viral oncogenes. Similar to the IGF-IR, expression and activity of these adaptor proteins are increased in many tumors (Dearth et al. 2007; Gibson et al. 2007). In addition, the IR has been reported to play a tumor-promoting role (Frasca et al. 2008). Taken together, these data suggest that hyperactivity of the IR, IGF-IR and IRS proteins enhances tumor growth; moderate suppression of the IR/IGF-IR/ IRS signaling extends life span.

7 The importance of calorie restriction in aging and cancer Calorie restriction (CR) has been shown to increase life span in D. melanogaster, C. elegans and many other animal models. However, the data in humans are limited, although epidemiological and observational studies do suggest a possible effect in humans. For example, the Okinawan population in the isolated island prefecture of Japan, consume fewer calories than the rest of the Japanese population, and it was proposed that CR contributed to the long and healthy lives of Okinawan adults (Willcox et al. 2006). Possible mechanisms underlying the effects of CR include the mammalian target of rapamycin (mTOR) pathway. Genetic disruption of the target of rapamycin

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(TOR) pathway has been shown to extend the life span of Drosophila, and analogous mutations in C. elegans have similar effects (Kapahi et al. 2004; Hansen et al. 2007). Glucose and amino acids activate the mTOR pathway, resulting in the initiation of protein synthesis. This process is inhibited by a lack of available nutrients. Insulin/IGF-I signaling also promotes growth through the mTOR pathway (Taguchi and White. 2008). This pathway therefore integrates insulin/IGF-I signaling with nutrient sensing, protein synthesis and cell growth (Levine et al. 2006). In contrast, deregulation of the mTOR pathway, resulting in its increased activity, is observed in many types of cancer (Petroulakis et al. 2006). CR also results in decreased IGF-I levels in rodents and humans (Thissen et al. 1994). As mentioned above, dwarf mice deficient in GH and IGF-I as well as mice lacking GHR have an extended life span. Therefore, the effect of CR on life span and cancer can be mediated, at least in part, by circulating IGF-I. In addition, CR prevents the formation of reactive oxygen species or attenuates the physiological age-related decline in the protective enzymes, such as catalase, superoxide dismutase and glutathione peroxidase (Masoro 2005). CR also improves the ability to repair damaged DNA, renew proteins and increase non-enzymatic antioxidant processes. Thus, there are a number of possible mechanisms whereby CR may promote aging.

8 Summary and Conclusions There is growing evidence for the role of the GH/IGF-I axis and insulin in controlling life span. On one hand, they affect cellular events directly, while on the other hand, they promote the growth of cancer cells; all these effects are involved in reducing longevity. In this review we have outlined some of the possible mechanisms playing a role. We have not covered some exciting new developments in the field, including Klotho, FOXO proteins and sirtuins, which have been shown to affect both aging and cancer. Studies on these pathways, while exciting, are nevertheless not well defined, though they may prove to be critical in mediating the insulin and IGF-I effects.

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The Functions of Insulin-like Peptides in Insects Sebastian Gro¨nke and Linda Partridge

Abstract The insulin/Igf-like signalling pathway plays key biological roles in diverse organisms, in growth and development, fecundity, stress resistance, metabolism and lifespan. The identity and functions of insulin-like peptides in mammals are well documented. Although genes encoding related peptides are present in the genomes of invertebrates, often in multiple copies, their biological roles are less well understood. Seven such genes are present in the fruit fly Drosophila melanogaster, they show higher sequence homology to insulin than to other mammalian peptides. Evolutionary conservation of different regions of the peptides among Drosophila species suggests that they are cleaved, like insulin. Each Drosophila Insulin Like Peptide (DILP) is expressed in a characteristic tissue-and stagespecific manner, suggesting that they may have unique biological functions. Ablation of neurosecretory cells in the brain that produce 3 of the DILPs results in an array of phenotypes, including developmental delay, reduced body size, stress resistance, metabolic phenotypes, reduced fecundity and increased lifespan, but assignment of these phenotypes to individual DILPs awaits genetic analysis. ILPs are also present in other insects, including the silk moth (38 genes), the honey bee (2 genes), and two mosquito species (7–8 genes), and insulin binding proteins have also been found in insects. Given the diverse and central functions of insulin/Igflike signalling, deeper understanding of the roles of these invertebrate insulin-like peptides and the mechanisms by which they achieve them will throw deeper light on the functioning of this system in mammals.

L. Partridge (*) Department of Biology, University College London, Gower Street, London, WC1E 6BT, UK and Max Planck Institute for Biology of Ageing, Gleueler Straße 50a, 50931 Ko¨ln, Germany e-mail: [email protected]

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1 Introduction The insulin/insulin-like growth factor signalling (IIS) pathway has an evolutionarily conserved role in nutrient sensing. In addition, this pathway plays a key role in determination of growth rate and size, metabolic traits, various forms of stress resistance and fecundity. Furthermore, reduction in IIS activity can extend life span in the nematode worm Caenorhabditis elegans, the fruit fly Drosophila melanogaster and the mouse. This increase in life span is a result of increased health during ageing (Kenyon 2005; Grotewiel et al. 2005; Wessells and Bodmer 2007; Selman et al. 2008) and is associated with resistance to the pathology associated with specific ageing-related diseases (Cohen et al. 2006; Pinkston et al. 2006). There is hence considerable interest in understanding how this pathway achieves these diverse functions and the ways in which they are regulated in response to external and internal conditions. In mammals, the ligands of the IIS pathway are peptide hormones, including insulin, the insulin-like growth factors (IGF) and relaxins, which are synthesized as pre-propeptides consisting of a signal peptide and contiguous B-C-A peptides (Fig. 1). In insulin and relaxin, the C-peptide is clipped out by a convertase enzyme targeting basic amino acid cleavage sites to produce a heterodimeric peptide consisting of A- and B-chain linked by two to three disulfide bridges (Fig. 1A). In contrast, IGFs contain a shortened C-peptide, which is not removed, resulting in a single chain peptide hormone (Fig. 1B). Insulin and IGFs activate receptor tyrosine kinases, insulin receptor (InR) and IGF-receptor, respectively. In contrast, relaxins function through the activation of leucine-rich G-protein coupled relaxin receptors. In mammals, insulin secretion from pancreatic b-cells in the gut is regulated in response to blood sugar levels and controls carbohydrate metabolism. IGFs are secreted in a developmentally regulated manner and control growth. Relaxins are mainly produced by the ovary and have multiple functions (Sherwood 2004). Insulin-like peptides (ILP) are not restricted to vertebrates but have been identified across a broad range of invertebrate phyla, including molluscs, the nematode C. elegans and several insect species. The first insect ILPs (Bombyxins) were purified from the silkmoth Bombyx mori in the 1980s, but only with the recent sequencing of several insect genomes, including that of the fruit fly Drosophila melanogaster, the honey bee Apis mellifera, the red-flour beetle Tribolium castaneum and two mosquitos, Anopheles gambiae and Aedes aegypti, could ILPs be identified in all of these species. Most insect genomes encode several ILPs, ranging from two identified in Apis mellifera to 39 in Bombyx mori. The identification of seven ILPs in Drosophila (termed dilp1-7), one of the best-established genetic model organisms, has greatly promoted research into ILP function in insects. The ease of genetic manipulation in Drosophila together with the relative simplicity of the intracellular IIS pathway mean that we can hope for rapid progress in understanding the roles and molecular mode of action of the Drosophila ILPs (DILPs).

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Fig. 1 Proposed structure and protein alignments of ILPs from the fruit fly Drosophila melanogaster (Dmel), the yellow fever mosquito Aedes aegypti (Aaeg), the malaria mosquito Anopheles gambiae (Agam), the honeybee Apis mellifera (Amp), the silk moth Bombyx mori (Bmor), the desert locust Schistocerca gregaria (Scg) and the red flour-beetle Tribolium castaneum (Tcas). a) Most insect ILPs resemble the insulin/relaxin-like structure, consisting of a heterodimeric peptide of A and B chain linked by two disulfide bridges. Sequence homology between most insect ILP is restricted to a few conserved amino acids in the A and B chain including cysteine residues involved in disulfide bridge formation. b) Putative insect IGF-like peptides, including BIGFLP from Bombyx mori, which has been suggested to be a single chain peptide hormone. c) Unusual high amino acid sequence conservation in dILP7-like peptides. For details see textly. A and B chains in the protein alignments are marked with grey bars, the C-peptide with black bars. Conserved cysteine residues are indicated by an asterisk (*) putative basic cleavage sites by a triangle (□)

In this review we will discuss recent progress in Drosophila, investigating how DILPs function in the control of the growth, metabolism, life span, reproduction and behaviour of the fly. We shall also summarise results from studies of ILP function in other insects that suggest that ILPs are involved in caste determination

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and queen longevity in honeybees and in control of egg production in response to blood feeding in mosquitoes. We shall describe the discovery of a novel IGF-like protein in the silkmoth Bombyx mori. Finally we shall describe the identification and functional characterisation of novel insect insulin-like binding proteins (IGFBP) in Drosophila and locusts, which may suggest the existence of an ancient, insulin-like binding protein as a common ancestor for insect and mammalian IGFBPS.

2 The ILPs of Drosophila Although the presence of a single insulin/Igf receptor ortholog InR in Drosophila has been known since the 1980s (Thompson et al. 1985), it was not until the sequencing of the Drosophila melanogaster genome that seven Drosophila insulin-like peptide genes (dilp1-7) were identified (Brogiolo et al. 2001). The genes for dilp1-4 are located in a cluster on chromosome III, separated by approximately 20 kb from dilp5. dilp6 and dilp7 are both located on the X chromosome at two different loci (Brogiolo et al. 2001). The dilp genes encode putative prepropeptides of 107-156 amino acids that contain a signal peptide, B chain, C-peptide and A chain (Fig. 1). Basic amino acid consensus cleavage sites between the A and the B chain in all seven dilps suggest that the active peptides consist of separate A and B chains, resembling insulin and relaxin (Fig. 1A; Brogiolo et al. 2001). However, the presence of basic cleavage sites may not always result in the removal of the C-peptide, as has been recently suggested for an IGF-like Bombyx mori protein (Okamoto et al. 2009, see below). DILP6 contains a shortened C-peptide similar to the Bombyx protein, suggesting that DILP6 may resemble IGF rather than insulin (Fig. 1B). Sequence comparison based on the A and B chains of the Drosophila peptides with insulin, relaxin and IGF in humans revealed a higher sequence similarity with insulin. DILP2 is the most closely related Drosophila ILP, with 35% identity to mature human insulin (Brogiolo et al. 2001). The expression pattern of dilps during development and in adult flies has been extensively studied by RNA in situ hybridisation and immunocytochemistry. Each dilp gene is expressed in a tissue- and stage-specific manner. In embryos, expression only of dilp2 and dilp4 in midgut and mesoderm and ubiquitous expression of dilp7 was detected (Brogiolo et al. 2001). During the larval stages, dilp1, 2, 3 and 5 are expressed in median neurosecretory cells (MNC) of the brain (Brogiolo et al. 2001; Ikeya et al. 2002; Rulifson et al. 2002). dilp2, 3 and 5 continue to be expressed in the MNC of adult flies whereas dilp1 expression could not be detected in adult MNC (Broughton et al. 2005). The MNC extend processes to the lateral protocerebrum, suboesophageal ganglion, corpora cardiaca and the heart. The heart surface has been suggested to be the site of DILP2 release into the hemolymph (Rulifson et al. 2002). Interestingly, corpora cardiaca cells of the ring gland, which express the fly analogue of glucagon, the Adipokinetic hormone (AKH), contain DILP2 peptide. dilp2 mRNA expression could not be detected in these cells,

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suggesting that DILP2 protein moves from the MNCs to the corpora cardiaca (Rulifson et al. 2002). Although dilp2, 3 and 5 are co-expressed in MNCs, each gene has its own MNC-specific enhancer that is independently regulated during development and in response to stress (Ikeya et al. 2002). dilp2 is expressed from the first instar larval stage on, whereas dilp5 expression starts in the second instar and dilp3 expression in the mid-late third larval stage. Starvation reduces expression of dilp3 and dilp5 transcript levels in larvae, but dilp2 expression is not changed (Ikeya et al. 2002). In addition to MNC expression, dilp2 is also expressed in imaginal discs and salivary glands. dilp5 is expressed in follicle cells of the female ovary (Brogiolo et al. 2001). Upon dietary restriction of adult flies, dilp5 expression is downregulated, but dilp2 and dilp3 expression levels are not changed (Min et al. 2008). Expression of dilp4 in adult flies has not been examined yet. dilp6 is predominantly expressed in the fat body and the expression is strongly up-regulated during the transition from larval to pupal stage (Okamoto et al. 2009). dilp7 is expressed in specific neurons (dMP2) of the ventral nerve cord and in a few neurons in the brain (Miguel-Aliaga et al. 2008; Yang et al. 2008). Interestingly, dilp7-positive neurites project close to the MNC (Miguel-Aliaga et al. 2008). In addition, dilp7 neurons innervate the hindgut, the suboesophageal ganglion and the female reproductive tract (Yang et al. 2008). In male flies, many dilp7-positive neurons in the brain also express fruitless, a master regulator of courtship behaviour in Drosophila (Yang et al. 2008). The seven IIS ligands of Drosophila are thus expressed both at different stages in the life cycle and in different tissues, suggesting that there is also diversification of their functions. Three of the Drosophila dilps (2, 3 and 5) are expressed in the MNC, both in larvae and adults. An important role of these cells in development, metabolism and life span regulation in Drosophila has been demonstrated by genetic cell ablation experiments. The expression of a proapoptotic gene under the control of MNC-specific enhancers results in specific ablation of MNC, at a developmental stage that depends upon the enhancer used. Ablation of the cells during the early first larval instar results in severe developmental delay and adults that are approximately 40% smaller, as a reduction of both cell size and cell number (Rulifson et al. 2002). MNC-ablated larvae also have increased hemolymph sugar levels, suggesting a diabetic-like phenotype. The growth and blood sugar phenotypes were partially rescued by overexpression of dilp2, suggesting that loss of dilp expression by cell ablation produced these phenotypes (Rulifson et al. 2002). Interestingly, these MNCablated flies are protected against the normal, age-related decline in cardiac function seen in wild type flies (Wessells et al. 2004). Ablation of the MNC later in development, during the late third larval stage, caused only minor delays in development and slightly reduced body weight (Ikeya et al. 2002). The adult flies had lower fecundity and increased storage of lipids and carbohydrate, were more resistant against starvation and oxidative stress and were long lived (Broughton et al. 2005). Flies in which MNC were ablated in post larval stages were long-lived, but they had normal fecundity (Buch et al. 2008), suggesting both that reduced fecundity is not necessary for the extension of life span and that

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the fecundity defects of MNC-ablated flies are caused by developmental defects. Life span extension in these flies was diet-dependent and present only on proteinrich food (Buch et al. 2008). The results of ablation of the dilp-producing MNCs are strongly suggestive of a role of dilps in the resulting phenotypes. However, rescue experiments by overexpression of the dilps present in the MNCs have not yet been performed, and it is thus yet to be demonstrated that the phenotypes seen in MNC-ablated flies are caused by loss of one or more of their IIS ligands. Neither is it yet known if dilp2, 3 and 5 have specific functions or act redundantly. Several studies have suggested a role for dilp2 in determination of life span because of its transcriptional down-regulation in mutant, long-lived flies (Hwangbo et al. 2004; Wang et al. 2005; Bauer et al. 2007; Lee et al. 2008). Over-expression of the forkhead transcription factor dFoxo in the adult fat body extends life span in flies (Hwangbo et al. 2004; Giannakou et al. 2004) and specifically reduces expression of dilp2, but not of dilp3 or dilp5, in MNC (Hwangbo et al. 2004). dilp2 expression also responds to activation of the stress-sensing JNK pathway. Increased JNK activity in MNCs reduces dilp2 levels and extends life span (Wang et al. 2007). In addition, expression of a dominant negative form of p53 in MNCs reduces dilp2 expression, which was suggested to be responsible for the increased life span of these flies (Bauer et al. 2007). Reduction of dilp2 expression in hypomorphic mutants of the Drosophila NPY ortholog NPF, neuropeptides involved in the regulation of food consumption in mammals and flies, was also suggested to be causal for their increased life span (Lee et al. 2008). However, the view that dilp2 is an important determinant of adult life span has been challenged recently by the finding that double-stranded RNA interference (RNAi)-mediated knock-down of dilp2 is not sufficient to extend life span in flies (Broughton et al. 2008). Interestingly, reduction in dilp2 causes up-regulation of dilp3 and dilp5 transcription, which, in case of dilp3, appears to occur via autocrine regulation through the insulin signalling pathway within the MNC (Broughton et al. 2008). Assignment of specific functions to individual dilps awaits gene-specific mutants. Weak, ubiquitous over-expression of all seven dilp during development causes an increase in body and organ size (Ikeya et al. 2002). dilp2 was found to be the most potent growth regulator and strong dilp2 over-expression causes lethality (Ikeya et al. 2002; Honegger et al. 2008). These gain-of-function experiments show that all dilps possess the ability to promote growth and therefore probably act as InR agonists (Ikeya et al. 2002). RNAi-mediated knock-down of dilp1 and dilp2 during development results in slightly smaller flies (Lee et al. 2008). dilp2 knockdown also increases trehalose storage in adult flies, suggesting that dilp2 is involved in the regulation of carbohydrate mechanism (Broughton et al. 2008). In addition, over-expression of dilp2 supresses germ line stem cell loss in ageing females, probably through action in the stem cell niche (Hsu and DrummondBarbosa 2009). Recently, dilp7 has been associated with egg-laying behaviour. Female flies with genetic hyperpolarisation of neurons producing dilp7 are sterile and display an "egg-jamming" phenotype in their reproductive tract and do not show the sucrose-avoidance normally seen in egg-laying females (Yang et al.

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2008). Whether these phenotypes are a direct consequence of reduced dilp7 secretion remains to been shown. Inspection of the 12 fully sequenced Drosophila genomes shows that the seven dilp genes have been evolutionarily conserved during the more than 40 million years of evolution of the Drosophilidae flies. This finding implies differentiation of function of individual dilps. For future research it will be important to generate dilp-specific mutant flies, analyse their phenotypes and determine how specificity in dilp signalling is achieved, because flies have seven dilps but only one InR. The recent discovery of an insect ILP-binding protein is important, because it could regulate dilp-specific interaction with the InR, similar to IGFBP in mammals (see below).

3 ILPs in Other Insects ILPs are increasingly being studied in non-model organisms, and many clues to the origin and evolutionary diversification of the functions of IIS are being revealed. As in Drosophila, ILPs are expressed in MNCs in the brain of other insect species including Bombyx mori (Mizoguchi et al. 1987), Aedes aegypti (Riehle et al. 2006) and Locusta migratoria (Goltzene et al. 1992), suggesting an important and evolutionary conserved function for these cells and their ligands. Similar developmental programs are involved in the specification of MNCs in Drosophila and pancreatic ß-cells in mammals, suggesting that insulin-producing cells of invertebrates and vertebrates may be derived from a common ancestry, despite the great differences in their anatomical location and appearance (Wang et al. 2007). A similar conclusion has been drawn from the analysis of the developmental program specifying dilp7-producing cells in Drosophila (Miguel-Aliaga et al. 2008). Work on ILPs in several insect species has thrown further light on their evolution.

3.1

Silk moth Bombyx mori (Lepidoptera)

The first insect ILP was purified from adult heads of the silk moth Bombyx mori as a peptide that stimulated ecdysone production in another moth species. It was termed Bombyxin (Nagasawa et al. 1984). Similar to the C. elegans genome (Li and Kim 2008), the Bombyx genome encodes multiple copies of bombyxin genes, 38 identified so far, which can be classified according to sequence homology into seven subfamilies (A-G) (Ishizaki 2004). The reasons for the large size of the gene family are not understood. All bombyxin genes lack introns and five of them appear to be pseudogenes. The large size of the family may therefore be a consequence of a high rate of gene duplication, which is plausible since the genes are arranged in three major clusters in the genome, coupled with a high rate of loss (Ishizaki 2004).

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An analysis of the rate of molecular evolution of this gene family would throw light on this possibility. Alternatively, these ligands may be stably differentiated in function, a possibility that cannot yet be evaluated, because the functions of the individual Bombyxin peptides are not well characterised. Similar to insulin secretion in mammals, glucose stimulates the release of Bombyxin into the hemolymph, and hemolymph Bombyxin titers are decreased under starvation conditions (Masumura et al. 2000). Injection of synthetic Bombyxin II into neck-ligated larvae resulted in a dose-dependent reduction of hemolymph sugar (trehalose) levels accompanied by elevated trehalase activity in muscle and midgut tissue. Increased trehalase activity has been suggested to facilitate trehalose uptake into tissues and thereby to be causal for the observed reduction of hemolymph trehalose levels (Satake et al. 1997). In addition, glycogen storage in the fat body was lowered and glycogen phosphorylase was activated in the fat body tissue after Bombyxin II injection (Satake et al. 1997). Thus, the function of Bombyxin is to promote the consumption of carbohydrate stores and not the accumulation of reserves, contrary to insulin function in mammals. Injection of synthetic Bombyxin II into adult animals had no effect on hemolymph sugar or lipid levels (Satake et al. 1999). Bombyxin also shows growth-promoting activity in vitro on imaginal discs from the butterfly Precis coenia (Nijhout and Grunert 2002). The growth-promoting activity was observed both with larval hemolymph from Bombyx and when brain extracts were used (Nijhout and Grunert 2002), the latter being consistent with the endogenous expression pattern of Bombyxins, which are expressed in MNCs in the larval brain (Mizoguchi et al. 1987). So far the structure of the mature ligands in Drosophila has not been directly characterised, and it is therefore not known if they are cleaved into A and B chains, like insulins, or function as single peptides, like IGF. Amino acid sequencing of purified peptides has shown that Bombyxins consist of a heterodimer of A and B chains linked by two disulfide bridges (Ishizaki 2004). Bombyxin II was the first insect ILP for which the three-dimensional structure was resolved by NMR, and this structure resembles relaxin more than insulin (Nagata et al. 1995). Remarkably, in addition to the Bombyxins, a Bombyx IGF-like peptide, termed Bommo-IGFLP (BIGFLP), has very recently been purified from the hemolymph of adult moths and shown to possess growth-promoting activity in vitro (Okamoto et al. 2009). BIGFLP was initially identified as an 8 kDa band on Western blots cross-reacting with a Bombyxin-II antibody. The corresponding peptide was subsequently purified and shown to consist of a single-chain peptide, based both on sequencing and on its molecular mass as determined by Maldi-Tof MS analysis (Okamoto et al. 2009). BIGFLP is predominantly expressed in the fat body, the functional equivalent of the mammalian liver and fat tissue, at low levels during larval stages, and expression and secretion strongly increase during the pupal stage (Okamoto et al. 2009). Purified BIGFLP peptide specifically promotes growth of adult tissues but not of larval tissue in vitro, suggesting that Bombyxins promote growth during larval stages whereas BIGFLP functions during pupal stages to induce growth during metamorphosis (Okamoto et al. 2009). Phylogenetic comparisons showed that BIGFLP is not a member of the Bombyxin family and, based on

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sequence homology, no clear orthologs in other species could be identified. However, ILPs with a shortened C-peptide are also present in Drosophila (Dilp6), the red flour-beetle Tribolium castaneum (ILP3), the honeybee Apis mellifera (AmelILP1) and the mosquito Aedes aegypti (Aaeg Ilp6), suggesting that IGF-like peptides could be more widespread in insects than previously thought (Fig. 1B, Okamoto et al. 2009). Whether these peptides act through the known insect InR or through an IGFR-like receptor still remains to be shown.

3.2

Honey Bee Apis mellifera (Hymenoptera)

Because of their eusocial status, with three castes and a reproductive queen largely freed from the other activities of daily living, the roles of the IIS ligands in the honeybee are of particular interest. The first evidence for the presence of an ILP was material in head extracts that was immunoreactive with an antibody to porcine insulin. The head extract also stimulated insulin-like activity in rat adipocytes and was able to displace porcine insulin from insulin receptors (O’Connor and Baxter 1985). The completion of the honeybee genome in 2006 led to the identification of two bee ILPs (AmILP1 and AmILP2) and two insulin receptors (AmIR1 and AmIR2; Wheeler et al. 2006). Subsequent studies have linked expression levels of IIS pathway components with caste determination (Wheeler et al. 2006; de Azevedo and Hartfelder 2008), worker foraging behaviour (Hunt et al. 2007; Ament et al. 2008) and honeybee queen longevity (Corona et al. 2007). Female bees have two castes, reproductive queens and sterile workers that forage and care for the hive and brood. Workers and queens develop from the same genome and developmental fate is determined by the larval diet. Only queen larvae are fed with royal jelly secreted by worker bees, and both the quantity and quality of the royal jelly are important for initiating and maintaining the queen developmental path (Wheeler et al. 2006). Interestingly, royal jelly exhibits insulinlike bioactivity (Dixit and Patel 1964; Kramer et al. 1977), later attributed to specific fatty acids (Wu and Brown 2006). During the critical phase of honeybee caste determination, AmILP-1, AmILP-2 and AmIR-2 differ in expression between worker and queen larvae (Wheeler et al. 2006; de Azevedo and Hartfelder 2008). AmIlp1 is specifically enriched in queen larvae and higher expression levels are dependent on royal jelly feeding. In contrast, AmIlp2 is more highly expressed in worker larvae and the pattern of its expression is better correlated with growth rate than with caste determination (Wheeler et al. 2006). The exact functional significance of difference in ILP expression for caste determination is not yet known, but the data suggest that IIS may regulate larval fate and growth, dependent on queenand worker-specific larval diets. A striking feature of honeybee queens and the queens of other eusocial insect species is that they are long-lived (up to five years in the case of honeybees) relative to workers (Keller and Genoud 1997) despite their high fecundity (up to 2,000 eggs/ day). Non-reproductive worker bees on the other hand have a maximum life span of

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10 months but typically survive just four to six weeks. Shorter life span is linked to environmental conditions. Worker bees under favourable conditions switch from nest activities to foraging and only survive one to three weeks of active foraging (Seehuus et al. 2006). Old queens have lower head expression levels of AmIlp1 and of the two insulin receptors AmIR1 and AmIR2 compared to old workers. In addition, AmIlp1 expression decreases with age in queens and increases in old workers (Corona et al. 2007), suggesting that reduced IIS contributes to honeybee queen longevity. The differential expression of ILPs between queens and workers may be evolutionary conserved, because it also occurs in the distantly related wasp Polistes metricus (Toth et al. 2007). Age-related division of labour in workers and foraging behaviour have also been linked to insulin signalling in honeybees. IIS components are overrepresented in quantitative trait loci that influence pollen foraging specialisation (Hunt et al. 2007). Brain AmIlp1 and abdomen AmIR1 and AmIR2 are more highly expressed in bees that forage compared to nest-based nurse bees. These expression differences are probably caused by a decline in nutritional status associated with behavioral maturation. Poor nutrition also increases brain AmIlp1 expression in field colonies in association with season and colony size (Ament et al. 2008).

3.3

Mosquito species (Diptera)

In blood-feeding mosquitoes such as the yellow fever mosquito, Aedes aegypti, or the African malaria mosquito, Anopheles gambiae, ILPs play an important role in egg production following a blood meal. Sucking blood is essential for egg production and stimulates the release of peptide hormones from MNCs in the mosquito brain. These neuropeptides then stimulate production of ecdysteroid hormones from the ovary, which in turn induce the secretion of yolk protein from the fat body. Bovine insulin has a direct stimulatory effect on ovarian ecdysteroid production. A mosquito insulin receptor homologue (MInR) is expressed in ovaries (Graf et al. 1997), implying that ILPs are involved in the control of egg production in response to blood feeding. Sequencing of mosquito genomes revealed the presence of seven ILP genes in Anopheles gambiae (AgamILP1-7; Riehle et al. 2002; Krieger et al. 2004) and eight in Aedes aegypti (AaegILP 1-8; Riehle et al. 2006). Similar to the genomic organisation of dilp genes in Drosophila, the Anopheles genes Agam1-4 are located in a cluster on chromosome III separated from Agam6 and 7 by an approximately 23 kb intervening sequence. AgamIlp5 is located on chromosome II (Krieger et al. 2004). AgamIlp1, 3, 6 and 7 are arrayed in tandem and are probably the result of a recent gene duplication, because nucleotide and protein sequences are very similar between the duplicated genes (98.7% for 3/6, 95.5% for 1/7). Evolutionary rate analysis suggests that both duplicated gene pairs are under purifying selection (Krieger et al. 2004).

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Similar gene duplication is not found in Aedes aegypti, but the putative orthologs Aaeg1 (AgamIlp1/7) and Aaeg3 (AgamIlp3/6) are also located next to each other. Interestingly, AaegIlp8, 1 and 3 are in very close proximity, suggesting that these genes might form a eukaryotic operon, transcribed as polycistronic pre-mRNA and subsequently processed to individual transcripts (Riehle et al. 2006). All eight Aedes aegypti ILP genes encode peptides containing a signal peptide, B, C and A chain characteristic of members of the insulin superfamily (Fig. 1). AaegILP6 has a shortened C-peptide and a C-terminal extension characteristic of IGFs (Fig. 1B). The B-peptides of AaegILP1, 2, 5 and 7 have amino-terminal extensions of 20-25 amino acids that contain basic amino acids that could be potential cleavage sites to generate additional bioactive peptides, as has been shown for the glycogenolysisinhibiting peptide of the locust ILP (Riehle et al. 2006; Clynen et al. 2003). However, the amino acid sequence of these potential bioactive peptides is not conserved between mosquitoes and locusts (Riehle et al. 2006). Except for AgamILP5 and AegILP5, there is only very limited amino acid sequence conservation between mosquito and fly ILPs (Fig. 1A, B; Krieger et al. 2004; Riehle et al. 2006). The similarities are mostly restricted to the key cysteines and leucines and the mono- and dibasic proteolytic cleavage sites, and clear orthologs cannot be assigned (Riehle et al. 2006). Remarkable, however, is the high degree of conservation between the mosquito AgamILP5 and AegILP5 and their fly ortholog DILP7 (Fig. 1C). AgamILP5 and AaegILP5 exhibit 81% amino acid sequence similarity to each other and 64-66% to DILP7 (48% amino acid identity in the full length protein, 64% in the A and B chains; Krieger et al. 2004; Riehle et al. 2006). The reason for this exceptionally high evolutionary conservation of DILP7/AgamILP5/ AegILP5 is not known, but it might suggest that these genes have an essential function that is different from the other ILPs and can therefore not be compensated by them. Expression of most AaegIlps is enriched in the head (AaegIlp1,3,4,6,7,8), consistent with their proposed function as brain-derived neuropeptides. The IGF-like AaegIlp6 is expressed throughout development in all body regions except the ovaries. AaegIlp5 is mostly expressed in the abdomen and the posterior midgut (Riehle et al. 2006), similar to the expression pattern of its orthologs AgamIlp5 in Anopheles (Krieger et al. 2004) and dilp7 in Drosophila (Miguel-Aliaga et al. 2008). AaegIlp4 shows female-specific expression in heads and also in the ovary, as does AaegIlp7. There are no specific antibodies for mosquito ILPs, but immunocytochemistry with Locust ILP or Bombyxin antibodies detected ILPs in the MNCs together with axons in the stomatogastric nervous system, endocrine cells in the midgut, cells in the suboesophageal ganglion and axons in the abdominal ganglion (Riehle et al. 2006). Recently, the head-specific AaegILP3 has been suggested to be involved in the control of egg production and metabolism in the yellow fever mosquito (Brown et al. 2008). In mosquitoes that are first blood fed and then decapitated to exclude the action of brain-derived peptide hormones, injection of synthetic AaegILP3 stimulates egg maturation, as measured by yolk uptake into oocytes. In addition, AaegILP3 in vitro stimulates ecdysteroid secretion from ovaries. Injection of

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AaegILP3 into sugar-fed decapitated females results in reduced circulating sugar levels and increased glycogen and lipid stores (Brown et al. 2008). Cross-linking experiments with radiolabeled 125I-AaegILP3 suggest that AaegILP3 binds to ovary membranes. RNAi-mediated silencing of the MInR in ovaries greatly reduces yolk uptake and ecdysteroid production and the in vitro ecdysteroid secretion induced by AaegILP3 is dependent on the expression of MInR (Brown et al. 2008). Taken together, these results suggest that AaegILP3 could be a brain-derived ILP that is released into the hemolymph in response to blood feeding and directly regulates egg maturation through its action on the MInR in the ovary. There are still several open questions. There is no evidence for blood-induced secretion of AaegILP3 from the mosquito brain. In addition, because no other AaegILPs have been tested, it is not clear if this is a specific effect mediated by AaegILP3 only or if other AaegILPs would have similar effects. The observation that bovine insulin can induce a similar response in the mosquito ovary, although with lower specific activity, demonstrates that injection experiments only show the capability of a molecule to induce a phenotype but do not necessarily address the endogenous function of the gene. Loss-of-function analysis with gene-specific mutants would be necessary to address these questions. During blood feeding, mosquitoes ingest human insulin, and it was recently suggested that the exogenous insulin could mimic the endogenous hormonal control of ageing in mosquitoes (Kang et al. 2008). In their study, Kang et al. fed mosquitoes (Anopheles stephensi) with sucrose solutions or an artificial blood meal (washed erythrocytes in saline) with and without added human insulin. Life span of insulin-fed mosquitoes was decreased by approximately 9% compared to the buffer-fed controls, suggesting that dietary human insulin can shorten the life span of mosquitoes under laboratory conditions (Kang et al. 2008). Whether this has any relevance for survival of mosquitoes in the wild still remains to be shown, but life span shortening would act to reduce transmission of the malaria parasite.

4 ILP-binding Proteins Insulin-like growth factor binding proteins (IGFBP) are important regulators of insulin/IGF function in mammals. Binding to IGFBP prolongs the half-life of circulating IGFs and modulates IGF availability and activity. IGF in circulation is found preponderantly in a ternary complex of two IGFs, 1 IGFBP and the protein Acid Labile Subunit (ALS). ALS is essential for the stabilisation of circulating IGF (Domene et al. 2005, 2007). In mammals, the IGFBP superfamily consists of IGFBP1-6, which bind IGF with high affinity, and the low-affinity IGFBP related proteins (IGFBP-rP; Hwa et al. 1999). Mammalian IGFBPs consist of highly conserved and cysteine-rich N- and C-terminal domains joined by a variable linker domain. Both the N- and

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C-terminal domains are required for high affinity IGF binding. The C-terminal domain is essential for the interaction with ALS (in IGFBP-3; Baxter and Firth 1995). Among the members of the IGFBP superfamily, only the N-terminal domain is conserved (Hwa et al. 1999), and it contains an IGF binding pocket involved in IGF and insulin binding. IGFBPs can also have important biological functions that are independent of their ability to bind insulin/IGFs. In insects, two types of proteins are known to bind ILP: the Imaginal-morphogenesis protein Late-2 (Imp-L2; Andersen et al. 2000; Alic and Partridge 2008) and Neuroparsins (Badisco et al. 2007, 2008).

4.1

Imp-L2

The insulin binding ability of Imp-L2 was first described for an orthologous protein IBP in the fall armyworm Spodoptera (Andersen et al. 2000). IBP acts as an inhibitor of insulin action in vitro. This finding was recently extended by the analysis of loss- and gain-of-function phenotypes of Imp-L2 mutants in Drosophila (Honegger et al. 2008). A genetic modifier screen for the hyperplasia produced in the eye of InR-over-expressing flies identified Imp-L2 as a potent antagonist of InRinduced growth. Increased expression of Imp-L2 inhibits growth in a cell nonautonomous way and results in developmentally delayed, smaller flies. Imp-L2 function is not essential under standard conditions, but its removal results in flies with an increased body size (Honegger et al. 2008). Consistent with its proposed function as a negative regulator of IIS antagonising the function of endogenous Drosophila ligands, Imp-L2 binds DILP2 in vitro, genetically interacts with dilp2 in vivo and functions as an inhibitor of IIS under stress conditions. Imp-L2 is a member of the immunoglobulin superfamily and contains two Ig C2-like domains. DILP2 binding activity is dependent on the presence of the second Ig C2-like domain (Honegger et al. 2008). Orthologs of Imp-L2 are present in other insects (e.g., Apis mellifera, Anopheles gambiae) and in the nematode C. elegans (ZIG-4). Importantly, the second Ig C2-like domain of Imp-L2 also shows homology to the C-terminal region of the human IGFBP-rP1 (also known as IGFBP7). Like IGFBP in mammals, Imp-L2 was recently shown to directly interact with a Drosophila homolog of ALS (dALS; Colombani et al. 2003; Arquier et al. 2008). In vitro co-precipitation studies demonstrated that Imp-L2, dALS and DILP2 are part of a trimeric complex that could also be immunoprecipitated from larval hemolymph in vivo. Fat body-specific, RNAi-mediated silencing of dALS resulted in increased body mass, reduction in hemolymph sugar levels and increased lipid storage, whereas the over-expression of dALS resulted in the opposite phenotypes, demonstrating an in vivo antagonistic function of dALS and IIS in Drosophila (Arquier et al. 2008). Interestingly, very recently it was shown that dALS is allelic to convoluted (conv), an essential gene required for epithelial morphogenesis in the

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Drosophila tracheal system, and at least some of the functions of dALS/conv are independent of IIS pathway activity (Swanson et al. 2009). DILP2 has been found to bind directly to Imp-L2 but not to ALS. In contrast, DILP5 could specifically be co-precipitated with ALS, and Imp-L2 suppressed this interaction, which might suggest the existence of other proteins with the ability to bind ILPs in flies (Arquier et al. 2008). In this context it is interesting to note that the Drosophila Twisted gastrulation protein (TSG) shows around 40% similarity to the N-terminal domain of mammalian IGFBPs (Mason et al. 1994). Tsg is a secreted 25 kDa protein involved in embryonic development that functions with SOG/ Chordin to antagonize BMP signalling (Ross et al. 2001). Its potential insulin binding activity has not yet been addressed. The apparently relative paucity of proteins potentially involved with interactions with DILPs in Drosophila, together with the potential for genetic analysis, provides a promising system in which to investigate the mechanisms and functions of these interactions, which have so far proved difficult to elucidate in the more complex mammalian system.

4.2

Neuroparsins

Neuroparsins are a family of neuropeptides that were initially discovered in the MNCs of the migratory locust, Locusta migratoria (Girardie et al. 1987; Boureme et al. 1987). The subsequent cloning of the neuroparsin A cDNA (Lagueux et al. 1992) and analysis of the secondary structure revealed that Neuroparsin A is an 83 amino acid long (22 AA signal peptide) secreted cysteine-rich peptide that contains six intramolecular disulfide bridges (Hietter et al. 1991). Neuroparsin-like peptides are present in diverse insects (Badisco et al. 2007), including three NP-like peptides in Locusta migratoria (NP A, NPP 2, 3) and four in the desert locust Schistocerca gregaria (NP 1-4), the Ovary Ecdysteroidogenic Hormone (OEH; Brown et al. 1998) in the mosquitoes Anopheles gambiae and Aedes aegypti (Brown et al. 1998; Riehle et al. 2002), and the queen brain-selective protein in the honeybee Apis mellifera. Remarkably, no Neuroparsin-like peptide could be identified in the genomes of the Drosophila species, suggesting that Neuroparsins were lost from this genus (Badisco et al. 2007). The first insight that Neuroparsin-like peptides could be involved in the binding of insulin-like peptides came from the observation that they show sequence homology to the N-terminal domain of mammalian IGFBPs, with the highest similarity to IGFBP-rP1 (Janssen et al. 2001; Claeys et al. 2003). Recently, the first experimental evidence for a direct interaction between Neuroparsins and insulin-like peptides has been found in Schistocerca gregaria (Badisco et al. 2008). Purified endogenous ScG-ILP binds recombinant Scg-NP4 in vitro. Whether this interaction also occurs in vivo remains to be seen. Injection of either purified Neuroparsin-like peptides or inhibitory a-Neuroparsin antiserum has revealed Neuroparsin to have pleiotropic functions in locusts.

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Injection of NP-A increased hemolymph trehalose and lipid levels (Moreau et al. 1988), had antidiueretic effects (Fournier and Girardie 1988) and delayed vitellogenesis (Girardie et al. 1987). Injection of a-Neuroparsin antiserum caused precocious vitellogenesis and disturbed metamorphosis (Girardie et al. 1987). Neuroparsins have also been associated with the transition from the solitary to the gregarious stage in locusts, which eventually results in the formation of locusts swarms, and is associated with changes in behaviour, colour patterns, size, weight, development and reproduction (Badisco et al. 2007). In Schistocerca gregaria, a phase transition-dependent regulation of neuroparsin transcript levels has been observed (Claeys et al. 2005), suggesting that neuroparsin function could be causal to some of the physiological changes associated with phase transition (Badisco et al. 2007). In the honeybee Apis mellifera a Neuroparsin-like protein has been described as queen brain-selective protein 1 (GenBank accession no. Q1T786). Although the function of this gene has not yet been analysed, queen-specific expression could imply a potential role of Neuroparsins in caste determination or in queen longevity, possibly by modulating IIS. The mosquito Neuroparsin OEH was originally identified in the yellow fever mosquito Aedes aegypti as a neuropeptide released from MNCs in the mosquito brain in response to a blood meal (Brown et al. 1998). The bioactive peptide consists of 83 amino acids, which shares 29% similarity to NP-A from Locusta migratoria. OEH was shown to stimulate secretion of ecdysteroid hormones from ovaries. Interestingly, the same gonadotropic effect is seen when bovine insulin or the endogenous AaegILP3 is injected into female mosquitoes (Riehle and Brown 1999; Brown et al. 2008), suggesting that OEH and ILPs might act in parallel. A direct binding of OEH to an insulin-like ligand has not yet been demonstrated, but it is tempting to speculate that, in contrast to Imp-L2 in Drosophila, which acts as an inhibitor of ILP function, OEH could be an ILP-binding protein that stimulates their function. As mosquitoes contain both the Neuroparsin OEH and an ImpL2 homolog, it would be interesting to see if these proteins act antagonistically in the regulation of ILP function. Expression of both Neuroparsin and Imp-L2 is under hormonal control (Claeys et al. 2006; Osterbur et al. 1988; Natzle et al. 1986). Injection of activated ecdysone (20HE) into Schistocerca gregaria resulted in ectopic Neuroparsin expression in the gonads (Claeys et al. 2006). Similarly, Imp-L2 expression is induced in response to ecdysone treatment in Drosophila (Osterbur et al. 1988; Natzle et al. 1986). Remarkably, both Neuroparsin and Imp-L2 show sequence homology to the mammalian IGFBP-rP1, but the homology is restricted to different parts of the IGFBP-rP1 protein. Neuroparsins show sequence homology with the supposed IGF-binding N-terminal domain of IGFBP-rP1, whereas Imp-L2 shows homology to the C-terminal immunoglobulin-like domain of IGFBP-rP1. Although Imp-L2 and Neuroparsins do not show any obvious sequence homology to each other, they might both have evolved from a common ancestral insulin-binding protein, which was also the ancestor of the mammalian IGFBP-rP1.

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5 Conclusions The insect ILPs and their binding proteins provide insights into the evolutionary origins and diversification of these versatile gene families. The IIS system has diverse biological functions and has been co-opted to the control of novel functions in specific lineages, such as caste determination in honeybees and phase transition in locusts. The relative simplicity of the genes families in Drosophila melanogaster, a model organism, provides an ideal opportunity to analyse interactions between ligands and their bindings proteins, which have proved a challenge in the more complex mammalian system. In addition, the importance of many insects as pests, disease vectors and producers of commodities such as honey and silk make elucidation of the role of their IIS of considerable potential, applied significance.

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IGF Receptors in the Adult Brain Carlos De Magalhaes Filho and Martin Holzenberger

Abstract IGF-I action in the brain is not limited to its growth-promoting effects during fetal and neonatal development. Significant effects of IGF-I in the adult brain have been demonstrated in various contexts. The potent neuroprotective action of IGF was discovered in vitro and has been confirmed in vivo. The proposed mechanisms of IGF-I neuroprotection depend on age, and very interesting implications of IGF signaling in neurodegeneration, notably in amyotrophic lateral sclerosis and Alzheimer’s disease, have been observed. The rescue of neurons following IGF-I administration has been extensively studied in rodent models in which a cerebrovascular accident is caused by experimental ischemia/hypoxia. Discrepancies have arisen in the effects of IGF-I observed in vivo, possibly reflecting the different actions of exogenous and endogenous IGF-I. Further studies are needed to elucidate the intracellular mechanisms of neuronal death following hypoxia/ ischemia and the role of IGF-I in this process. Growing evidence demonstrates the selective action of IGF-I on glial cells. However, few experimental models can address the specific roles of astrocytes and microglia in the neuronal rescue mediated by IGF signaling. Endothelial cells also respond to IGF-I, possibly mediating neuroprotective effects of IGF-I through the control of edema following CNS damage. Recent studies on longevity and aging suggest that decreased insulin/ insulin-like signaling increases life span and survival. Similarly, caloric restriction reduces IGF-I levels in the circulation and in the brain, at the same time increasing the longevity of the organism. Paradoxically, excess IGF-I promotes neuronal survival in the brain and reduces the longevity of the organism.

M. Holzenberger (*) Inserm et Universite´ Pierre et Marie Curie - UMR 893, Hoˆpital Saint-Antoine, Baˆt. Kourilsky 184 rue du Faubourg Saint-Antoine, 75012, Paris, France, e-mail: [email protected]

D. Clemmons et al. (eds.), IGFs: Local Repair and Survival Factors Throughout Life Span, 125 Research and Perspectives in Endocrine Interactions, DOI 10.1007/978-3-642-04302-4_10, # Springer-Verlag Berlin Heidelberg 2010

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1 Introduction IGF-I action in the brain is not limited to growth-promoting effects during fetal life and the neonatal period. IGF-I has been shown to have significant effects on various processes in the adult brain. Difficulties in the interpretation of IGF ligand or receptor knockout models come from the fact that developmental effects are strongly cumulative and may not be easily distinguished from effects in the adult. Transgenic models overexpressing IGF-I in the brain pose similar problems; indeed, most data on the effects of IGF-I on the adult brain have been obtained from models in which IGF-I was administered centrally or peripherally. These studies provide a detailed, yet in many areas still incomplete, understanding of the role of IGF-I signaling in the adult brain. IGF-I is implicated in adult neurogenesis, energy homeostasis of the CNS, and certain aspects of cognition. Mammalian neurogenesis occurs mainly during embryogenesis and is completed during the neonatal period. IGF-I levels diminish with this decreasing level of neurogenesis, with much lower expression levels being observed in adults than in neonates (Rotwein et al. 1988). Similarly, IGF1R expression diminishes rapidly after birth. However, all adult neurons show significant levels of IGF-1R, with very high levels of IGF-1R also found in the adult choroid plexus, meninges and blood vessels (Marks et al. 1991). Certain areas of the mammalian brain, for instance the dentate gyrus of the hippocampus, continue to produce neurons throughout adult life (Gage et al. 1998). Peripheral administration of IGF-I increases the number of newly formed neurons in the hippocampus of hypophysectomized rats (Aberg et al. 2000) through IGF-I action on neural stem cell differentiation (Brooker et al. 2000). Moreover, the olfactory bulb mitral cells, which undergo constant life-long renewal, express elevated levels of IGF-I (Werther et al. 1990). It is now well established that IGF-I promotes neurogenesis in the adult mammalian brain. There is also evidence that IGF-I plays a functional role in cognitive processes. IGF-I modulates neuronal excitability through modulation of membrane ion channels and glutamate receptors and through regulation of the size of synapses (Torres-Aleman 2000). IGF-I also promotes synaptic plasticity by increasing brain-derived neurotrophic factor (BDNF) production and activating c-fos in the hippocampus (Ding et al. 2006). Moreover, several studies have shown changes in IGF-I action in the brain of aged rats (Sonntag et al. 1999; Poe et al. 2001). These changes are correlated with the decline of cognitive function during aging (Rollero et al. 1998; van Dam and Aleman 2004). Administration of IGF-I in aged rats diminishes the age-related decline of memory (Markowska et al. 1998). Finally, several studies suggest that IGF-I has a homeostatic role, maintaining the integrity of brain microvasculature (Sonntag et al. 1997).

2 The Neuroprotective Action of IGF In Vitro and In Vivo There has been considerable interest in the neuroprotective potential of IGF-1R signaling in the adult brain for a number of years. IGF signaling is thought to play a predominantly homeostatic role in the brain under physiological conditions, getting

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involved in neuronal protection and cell repair when the nervous tissue is exposed to damage. The ability of IGF-I to act as a survival factor has been well established in cultured neurons. In vitro, IGF-I protects neurons from cell death induced by various sorts of damage, including hypoglycemia (Cheng and Mattson 1992), hyperglycemia (Russell and Feldman 1999), ß-amyloid toxicity (Dore et al. 1997), osmotic stress (Matthews and Feldman 1996; Matthews et al. 1997), deprivation of specific growth and survival factors (Russell et al. 1998), low potassium (D’Mello et al. 1993) and ceramide (Willaime-Morawek et al. 2005). The neuroprotective action of IGF-I has been observed in several neuronal cell types, including cortical, septal and hippocampal neurons, dopaminergic neurons and neurons in the granular cell layer of the cerebellum. The increase in survival due to IGF-I is essentially mediated by the anti-apoptotic effects of IGF-1R signaling on neurons (Gluckman et al. 1992; Breese et al. 1996; Sandberg Nordqvist et al. 1996; Singleton et al. 1996; D’Mello et al. 1997; Tagami et al. 1997; Walter et al. 1997; Beilharz et al. 1998). For instance, activation of IGF-1R in cultured neurons maintains BCL-2 levels and prevents ICE induction during apoptosis induced by osmotic stress (Singleton et al. 1996). IGF-1R-dependent pathways also regulate Ca2þ homeostasis, inhibiting neuronal apoptosis triggered by a lack of potassium (Galli et al. 1995). In addition to its function as a survival factor in vitro, IGF-I is a potent mitogen for several other CNS cell types implicated in neuroprotection, namely astrocytes (Ballotti et al. 1987; Torres-Aleman et al. 1990; Chernausek 1993), macrophages (Scheven and Hamilton 1991; Mueller et al. 1994; Li et al. 1996; Long et al. 1998) and microglial cells (O’Donnell et al. 2002). Given the multiple roles of IGF-I in cell survival, proliferation and differentiation of neurons and glial cells in vitro, recent studies have addressed the role of IGF-I in neuroprotection in vivo. Several studies have investigated the neuroprotective effects of IGF-I in experimental models of perinatal CNS lesion. Whereas certain neuroprotective mechanisms are similar in the developing and adult brain, it is becoming increasingly clear that the brain of young rodents often reacts differently from that in the adult. For instance, whereas neuronal cell death under hypoxic/ischemic (H/I) conditions occurs mainly by apoptosis in the young brain, neuronal death in the adult brain occurs mainly through necrosis in the center of the infarct tissue, suggesting different age-dependent mechanisms. Similarly, whereas injection of a glutamate receptor antagonist causes apoptosis in the neonatal brain, it leads to necrosis in the adult brain (Portera-Cailliau et al. 1997). In this model, BDNF injection provided notable neuroprotection in young mice but was much less efficient in adults (Cheng et al. 1997). The following chapter will focus on studies carried out in animal models at the adult stage.

3 A Role for IGF Signaling in Neurodegenerative Disease There is also considerable interest in the therapeutic potential of IGF signaling in neurodegenerative diseases. Amyotrophic lateral sclerosis (ALS, or Charcot’s disease) is a neurodegenerative disease characterized by skeletal muscle atrophy,

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generally occurring in adults after the age of 40. It progresses rapidly, leading to death within a few years of the appearance of the symptoms. Circulating IGF-I is low in ALS patients and may contribute to the pathogenesis of this disease (TorresAleman et al. 1998; Wilczak et al. 2003). IGF-1R levels, in contrast, are increased in the spinal cord, possibly due to a compensatory increase in IGF-1R production (Adem et al. 1994). Several studies over recent years have suggested that IGF-I has therapeutic potential in ALS treatment. This growth factor is unique in that it stimulates the regeneration of sensory and motor neurons in adult animals (Anlar et al. 1999). IGF-I prevents apoptosis in motoneurons (Kaspar et al. 2003; Vincent et al. 2004), glial cells (Delaney et al. 1999) and muscle cells (Singleton and Feldman 2001), all of which are involved in ALS pathogenesis (Cleveland and Rothstein 2001). One clinical study showed that ALS patients receiving daily injections of IGF-I for nine months had a slower disease progression and better quality of life than placebo-treated patients (Lai et al. 1997). However, the secondary effects of this high-dose therapy remain problematic. In Alzheimer’s disease, patients show changes in circulating insulin and IGF-I that are often associated with abnormal responses to insulin (Tham et al. 1993; Mustafa et al. 1999). There is now substantial evidence for a direct role of insulin and IGF-I in the clearance and metabolism of the amyloid ß peptide (Aß). Injection of IGF-I into the cerebral parenchyma of old rats reduces Aß levels to levels usually found in young rats (Carro et al. 2002; Carro and Torres-Aleman 2004). IGF-I facilitates the clearance of Aß by promoting the transport of proteins carrying Aß from the CNS to the choroid plexus (Carro, Trejo et al. 2002). Moreover, IGF-I reduces tau phosphorylation and promotes tau binding to the microtubules of neuronal cells (Hong and Lee 1997). Disruption of IGF-I signaling, in contrast, increases tau phosphorylation (Schubert et al. 2003). These data demonstrate a potential role for IGF-I in the regulation of the tau protein. Chronic IGF-I administration has therefore been proposed as a potential therapeutic strategy in Alzheimer’s disease (Dore et al. 2000; Carro and Torres-Aleman 2004). Furthermore, IGF-I protects neurological functions and preserves normal histology in a model of encephalomyelitis (Yao et al. 1996; Liu et al. 1997). The capacity of IGF-I to stimulate neurite outgrowth also makes it a good candidate for the treatment of diabetic neuropathies (Schmidt et al. 1999). In conclusion, studies over recent years have demonstrated beneficial effects of IGF-I in certain CNS diseases. Additionally, IGF-I may be a suitable candidate molecule for treating the consequences of cerebrovascular accidents (CVAs), a domain receiving significant investments from pharmaceutical research trying to find efficient neuroprotective substances.

4 Experimental Ischemia/hypoxia as a Model for CVA In humans, CVAs occur at a mean age of 70 years, but they can occur at any age. CVA is the major cause of hemiplegia: in France alone, about 100,000 individuals are affected by CVAs each year. Mortality six months after the first incident is

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between 30 and 40%. CVA is the second most frequent cause of death worldwide, and is the sixth most frequent cause of disability in adult men and women. In France, CVAs are the third most frequent cause of death after cancer and cardiopathies and the first cause of acquired disability. About 75% of CVAs are caused by ischemia (Thrift et al. 2001), i.e., by lack of blood supply to the brain, most often due to atheroma or an embolus obstructing the cerebral arteries. Ischemia reduces oxygenation in the CNS tissue, which, if it persists, triggers cell death. The most common rodent model of CVA uses surgical H/I. This model was developed by Levine and Vanucci in the rat (Levine 1960; Rice et al. 1981; Vannucci and Vannucci 2005) and has recently been adapted to the adult mouse (Vannucci et al. 2001). The technique combines unilateral ligature of the common carotid artery followed several hours later by a defined period of respiration in an atmosphere of reduced oxygen concentration. It has been shown that infarction of brain tissue occurs when the cerebral blood flow (CBF) falls under 30% of its normal level. Ischemia by ligation of the common carotid artery decreases the CBF by half, while hypoxia reduces oxygen flow by a further 20%. Thus, combined hypoxia and ischemia decreases the oxygen flow to below the critical threshold in the middle cerebral artery, causing cerebral damage similar to CVA. Experimental H/I techniques mostly damage the areas of terminal vascularization, i.e., areas only irrigated by the artery that was disrupted. H/I can also damage areas of the brain in which blood circulation occurs through anastomosis of the Willis circle from different arteries. In these areas, it is the degree of hypoxia that determines the point at which collateral irrigation becomes insufficient. Under conditions of H/I, cells in the ischemic zone can increase the oxygen fraction that they extract from the blood to compensate for the reduced CBF and to maintain cell metabolism (Baron 2001). However, the extent of compensation is limited. H/I leads to infarction of the brain tissue, characterized by massive cell death due to lack of oxygen. The infarct develops into a macroscopically visible area of necrotic tissue. This area is called the ischemic center, in which the oxygen supply has attained a critical threshold. The area surrounding this is the penumbra. Histological analysis reveals the ischemic center to be a pan-necrotic zone with generalized cell death of neurons, glia and blood vessel endothelial cells (Garcia et al. 1997). The only viable cells that can be observed in this zone are the infiltrating inflammatory cells, notably the macrophages. In the penumbra, neurons stop functioning, but their structure is generally preserved. Their survival depends on the nature of the blood circulation. Their ability to increase local oxygen extraction plays a major role in the development of the lesion in this zone (Sobesky et al. 2005). In the absence of rapid reperfusion or sufficient neuroprotection, the ischemic center spreads into the penumbra zone. With sufficient collateral reperfusion, histological examination of this zone reveals an area with late (so-called selected) neuronal death. This late neuronal death is characterized by irreversible damage to the neuronal cell populations that are most sensitive to H/I. Thus, late neuronal death is associated with structural changes in astrocytes and microglia. After H/I, the infarct starts in the region initially irrigated by the middle cerebral artery and extends to the areas irrigated by the anterior cerebral artery, as well as to some

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areas irrigated by the posterior cerebral artery. In humans, the zones lying close to the midline are more protected from lesions, because they have a better collateral blood supply through the circle of Willis.

5 Exogenous Versus Endogenous IGF-I IGF-I can act as a neuroprotective agent in cerebral H/I. Intraventricular administration of IGF-I can prevent up to 80% of the neuronal loss following H/I in the rat brain (Gluckman et al. 1992; Guan et al. 1993; Guan et al. 1996). Intranasal application of IGF-I reduces neuronal infarct induced by closure of the middle cerebral artery (Liu et al. 2001a, b). Similarly, subcutaneous or intravenous injections of IGF-I following various types of neuronal damage can prevent or reduce subsequent brain damage (Hatton et al. 1997; Saatman et al. 1997; Fernandez et al. 1998). The time window within which IGF-I exerts an effective neuronal protection is limited to two hours following neuronal damage in the rat. Nevertheless, this interval can be prolonged by several hours if it is combined with postischemic hypothermia. Hypothermia reduces the extent of late cell death after H/I (Guan et al. 1993, 2000). IGF-I injection during the acute post-ischemic phase prevents cortical infarct, suggesting that glia and neurons are protected. Moreover, IGF-I can reduce selective neuronal death and improve functional recovery after H/I (Guan et al. 2001). Finally, the neuroprotection mediated by IGF-I is dosedependent (Gluckman et al. 1992; Guan et al. 1993). Whereas most studies report that administration of IGF-I protects neurons, a recent study shows that high levels of serum IGF-I correlate with increased size of cerebral infarct and low serum IGF-I correlates with smaller lesions (Endres et al. 2007). If confirmed, these results suggest that injection of exogenous IGF-I into the peripheral blood stream or directly into the brain ventricle system produces effects different from those of endogenous, circulating or locally produced IGF-I. It will therefore be important to further investigate the role of endogenous IGF-I in response to ischemia. Several studies have shown induction of IGF system components in response to experimental H/I, suggesting a particular role for endogenous, brain-derived IGF-I in this process (Gluckman et al. 1992; Beilharz et al. 1995; Breese et al. 1996; Sandberg Nordqvist et al. 1996; Walter et al. 1997; O’Donnell et al. 2002). H/I triggers survival responses in regions of the brain that are stressed but still viable. After the lesion, a wave of IGF-I production is triggered, stimulating the proliferation of repair cells. The increase in IGF-I in the mouse and the rat can be detected from three days after H/I and takes place only in the infarct hemisphere (Gluckman et al. 1992; O’Donnell et al. 2002). This IGF response occurs following a CNS lesion (O’Donnell et al. 2002) but also in other tissues secondary to peripheral lesions, e.g., in blood vessels (Cercek et al. 1990), skeletal muscle (Edwall et al. 1989) and cartilage (Chu et al. 2004). IGF-I action depends strongly on other components of the IGF system. IGF binding proteins are also induced in response to H/I (Gluckman et al. 1992; Beilharz et al. 1995; Breese et al. 1996; Sandberg

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Nordqvist et al. 1996; Walter et al. 1997). The genes encoding IGFBP-2, IGFBP-3 and IGFBP-5 are differentially expressed in specific brain regions after H/I. The corresponding proteins may be involved in spatio-temporal regulation of IGF-I actions. The production of IGF-I and IGFBP-2, -3, -4 and -5 by microglia, oligodendrocytes, reactive astrocytes and the surviving neurons also suggests a role for the locally produced IGFBPs in the transport of IGF-I from the site of production towards its site of action. High levels of IGFBP-2 are found in the penumbra, produced by reactive astrocytes that lie adjacent to the surviving neurons. These observations suggest a specific role for IGFBP-2 in modulating the action of IGF-I (Beilharz et al. 1998; O’Donnell et al. 2002). Other growth factors implicated in local repair, in particular PDGF, FGF-2 and EGF, are capable of stimulating local synthesis of IGF-I. IGF-I may also interact with estrogens (Garcia-Segura et al. 2006) and with the neurotrophic factors NT-4, NT-3, CNTF, BDNF and GDNF (Lindholm et al. 1996).

6 Intracellular Mechanisms of Neuronal Death after H/I The intracellular mechanisms of neuronal death following H/I remain unclear (Olson and McKeon 2004). Several potential causes of cell death have been proposed, depending on whether the studies were based on histological criteria or specific intracellular changes during cell death. Potential mechanisms thus include oxidative stress, excitotoxicity, apoptosis, autophagy, para-apoptosis, oncosis and programmed or accidental necrosis. Discrepancies in these findings may also be due to the rapid progress in research on the mechanisms of cell death or the particular methods used to identify apoptotic cells (Charriaut-Marlangue and Ben-Ari 1995). Cell death after H/I occurs in two phases: the acute phase corresponds to the hypoxic period and a period immediately afterwards that is characterized by a cascade of events initiated by an acute lack of cellular energy. The brain is unique in that it has a high metabolic activity and very limited stocks of energy, its neurons being highly dependent on aerobic glucose metabolism. If oxygen availability is too low, ATP production decreases, severely disrupting neuronal energy metabolism. The main consequences of this disruption are increased production of reactive oxygen species (ROS), loss of ionic gradients, the release of large amounts of glutamate, and a decrease in cell pH. These events together lead to the cell death of neurons and other cell types. The PARP protein may also play an important role in cell death following H/I. This protein mediates DNA base excision repair in response to DNA damage. H/I activates PARP, which can then promote neuronal death through NAD depletion and a lack of energy substrates in neurons (Endres et al. 1997). The second phase of neuronal death extends the initial damage; this phase may continue for several days, or weeks, after the initial attack (Clark et al. 1993; Li et al. 1995; Chalmers-Redman et al. 1997). This late cell death is the consequence of the combined effects of acute phase events, inflammation, and activation of glial cells. A large proportion of neuronal death is apoptotic

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(Linnik et al. 1993; Beilharz et al. 1995; Hara et al. 1997; Rosenblum 1997; Namura et al. 1998). ROS resulting from the dysfunction of energy metabolism, and increased levels of pro-inflammatory cytokines IL-1ß, IL-6, TNF-a and TGF-ß, are pro-apoptotic stimuli (Chan 1996). Thus, cortical infarcts induced by H/I are smaller in transgenic mice overexpressing Bcl2 than in control mice (Kitagawa et al. 1998). In contrast, disruption of Bcl2 increases the size of the infarct and impairs the functional recovery of the animal (Hata et al. 1999). The precise mechanisms of the late cell death remain unclear; however, the involvement of caspase-independent pathways seems to have been established (Cho and Toledo-Pereyra 2008). Several studies have investigated the role of IGF-I in neuronal survival after H/I in the adult. However, few have elucidated which mechanisms of cell death are regulated by IGF-I in H/I. Some studies performed in immature animals suggest that IGF-I prevents post-H/I apoptosis in the brain (Gluckman et al. 1992; Beilharz et al. 1998; Cao et al. 2003). In vitro, IGF-I inhibits several mechanisms of neuronal death after H/I in the adult brain. It protects neurons from the NMDA-dependent apoptosis implicated in post-H/I excitotoxicity (Tagami et al. 1997). IGF-I activates transcription of molecules that mediate resistance to oxidative stress through the IGF-1R/PI3K/NF-B pathway. IGF-I has also been shown to promote FKHRL1 phosphorylation in cortical and hippocampal neurons. FKHRL1 is a FOXO transcription factor that activates apoptosis in its unphosphorylated form (Zheng et al. 2002). These findings strongly suggest that IGF-I is neuroprotective through antiapoptotic activity. Additionally, IGF-I increases hypoxin-inducible factor 1 (HIF-1) levels in neurons and glia. HIF, which is mainly active under hypoxic conditions, stimulates the expression of genes encoding proteins such as erythropoietin, which increases the availability of oxygen to the cells (Zelzer et al. 1998). IGF-I also increases cellular glucose metabolism, which is essential for the survival of neurons after severe insults. This occurs through increased activity of glucose transporter and modulation of glycolytic enzymes (Cheng et al. 2000). Thus, IGF-I is neuroprotective, but the mode of action in vivo after H/I is not fully understood, partly due to the fact that many studies on the neuroprotective roles of IGF-I have focused on the direct effects on neurons, with few studies investigating the potential intermediate role of IGF-I in astrocytes and glia. Indeed, IGF-I may act through IGF-1R on glial cells to regulate the production of survival factors, which may in turn act on the surrounding neurons. A major role for glial cells in neuropathologies has been clearly established (Seifert et al. 2006). To understand the effects of IGF-I in the brain, its action on astroglial cells needs to be considered.

7 IGF-I Action on Glial Cells Astrocytes represent the largest population of cells in the brain, with a ratio of 10 astrocytes to one neuron in the human brain. The cell soma of neurons, with the exception of synaptic contacts, is covered by astrocytic membranes. This close anatomical relationship specific functions, e.g., the compartmentalization

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of neuroprotective factors released from astrocytes. After neuronal damage, astrocytes close to the infarct zone become hypertrophic and increase their production of GFAP. This astrocytic response is associated with the production of several neuroprotective molecules. The functional units formed between astrocytes and neurons constitute a neuroprotective cellular network (Albrecht et al. 2002; Chen and Swanson 2003; Pekny and Nilsson 2005). Astrocytes, as observed for other peripheral cells, produce IGF-1R (Baron-Van Evercooren et al. 1991). These receptors are also present on parts of the astrocytic cell membrane that are in contact with cerebral blood vessels (Garcia-Segura et al. 1997). In the adult brain, the major role of astrocyte IGF-1R signaling seems to be to modulate the response to cell damage. IGF-I exerts pleiotropic effects on astrocytes, stimulating astrocyte proliferation. In their reactive form, these astrocytes themselves produce IGF-I, which then acts as an autocrine signal to stimulate cell growth (Antoniades et al. 1992). IGF-I also regulates glucose and glutamate capture from the extracellular space by astrocytes (Masters et al. 1991; Suzuki et al. 2001). This increase in glutamate capture reduces its excitotoxicity and seems to be an important mechanism enhancing neuronal survival in the penumbra of the infarct (Swanson et al. 2004). This astrocytic response is beneficial for the CNS but, beyond a certain level, may rather increase the damage. Previous findings have demonstrated that overshooting the proliferation of astrocytes may generate a physical and biochemical barrier that hinders, in particular, the essential post-traumatic axonal regeneration (Zhu et al. 2007). Hyper-reactive astrocytes can be activated by pro-inflammatory cytokines like TNF-a and may themselves secrete pro-inflammatory and cytotoxic cytokines, potentially leading to neuronal damage (Ridet et al. 1997; Fawcett and Asher 1999; Dong and Benveniste 2001). However, in the presence of IGF-I, these reactive astrocytes may become anti-inflammatory. Under such conditions, proinflammatory molecules from astrocytes, such as Cox2, iNOS2, and TNF-a, are no longer produced, but neurotrophic and neuroprotective mediators, namely SOD, are secreted. Together, these and other data suggest that IGF-I can rescue neurons after H/I not only directly but also indirectly, by stimulating the neuroprotective properties of reactive astrocytes. Microglial cells can be considered as the endogenous immune system of the CNS. These cells, representing 20% of the total glial cell population of the brain, are constantly moving, inspecting neurons for damage, plaques or infectious agents in the CNS. During infection, for instance, microglial cells react quickly to increase inflammation and destroy the infectious agent. After H/I damage to the brain, the combined microglial and macrophage cell population of the brain comprises the endogenous activated microglia and the peripheral macrophages that migrate into the brain following rupture of the blood-brain barrier (BBB; Stoll and Jander 1999). Within the zones of damage, the microglia/macrophages are located in the ischemic center, where they are often the only viable cells remaining. Reactive astrocytes, in contrast, are not present in the ischemic center but are located in the penumbra, where neuronal death is more selective (O’Donnell et al. 2002). These microglia/macrophages in areas of cell death may simultaneously exert both inhibitory and stimulatory effects, both slowing down the process of cell death and

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at the same time promoting recovery (Streit 2000). In one study, transgenic mice overproducing IGF-I in the brain were challenged with cuprizone, a neurotoxin that increases microglia/macrophages in the corpus callosum. These mice displayed greater increases in the numbers of microglia/macrophages than in control mice (Mason et al. 2000). Another study, led by Terry Wood, has shown that in mice, from three days after H/I, the expression of IGF-I was induced selectively in strongly proliferating microglia/macrophages in the infarct area but not in neurons and astrocytes (O’Donnell et al. 2002). These studies suggest that IGF-I produced by microglia/macrophages acts as an autocrine and paracrine factor, promoting cell proliferation in the infarct (O’Donnell et al. 2002). Microglial proliferation is beneficial because these cells clear cellular debris after apoptosis. Microglial cells also secrete numerous trophic factors capable of protecting neurons (Stoll and Jander 1999; Streit 2000). Interestingly, outside the brain, monocytes/macrophages also play a stimulatory role in scarring, eliminating cell debris and promoting functional recovery (Winston et al. 1999). Induction of IGF-I in microglial cells may also have inhibitory effects on cell survival in the brain after H/I. The microglia/macrophages secrete cytotoxic proteases, hydrogen superoxide, NO and excitotoxins that can damage viable cells and thereby contribute to neuronal and glial cell death (Colton and Gilbert 1987; Banati et al. 1993). Microglia can also damage neurons by releasing glutamate and aspartate. Such cytotoxic action allows infected cells or bacteria to be destroyed but also causes collateral damage to neurons. In vitro, IGF-I stimulates the production of TNF-a in monocytes and macrophages (Renier et al. 1996). In neurons, however, TNF-a promotes resistance to IGF-I by inhibiting the phosphorylation of the IGF-1R substrate IRS-2 and thereby reducing the activity of PI3K (Venters et al. 1999). IGF-I secreted by microglia/macrophages may thus increase TNF-a production, which in turn inhibits survival signals initiated by IGF-I in the neighboring neurons. Additionally, the suppression of microglial/macrophage activation reduces the infarct volume after cerebral ischemia, providing further evidence of a role for microglial cells in cell death (Giulian and Vaca 1993; Yrjanheikki et al. 1998; Tikka et al. 2001). Microglia/macrophages seem to have different functions, depending on the degree of damage to the brain tissue (Gehrmann et al. 1995a, b; Streit 2000). In areas of limited damage, with a significant number of surviving cells, microglia/macrophages rescue cells, including the neurons, and eliminate cell debris. However, microglial activity following a very strong inflammatory response can be destructive in the adult brain (Gehrmann et al. 1995a).

8 The Role of Edema in H/I Damage and Neuronal Death Following CNS Damage For the evaluation of the neuroprotective action of a molecule, not only must the direct effects on neurons and indirect effects on glia be considered but its effects on brain edema are also important. Cytotoxic edema develops in brain tissue

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approximately six hours after H/I injury. This edema is caused by the release of a multitude of intracellular substances after initial cell death. Moreover, responses of the cerebral parenchyma and the H/I lesion itself damage the BBB. Damage to the BBB allows substances such as cytokines and inflammatory cells to pass from the vascular compartment to the cerebral parenchyma, leading to vasogenic edema at about 24 hours after H/I. This edema increases the intracranial pressure, magnifying the effects of H/I, which can compress areas of the brain with vital functions and create secondary ischemia, further compromising blood flow in the brain (Wang and Shuaib 2007). The protection of the BBB endothelial cells is therefore itself a neuroprotective mechanism that has only recently received more attention (Takahashi and Macdonald 2004; Wang and Shuaib 2007). Thus, after H/I, IGF-I could exert neuroprotective effects through the stimulation of angiogenesis and the production of vascular-endothelial growth factor (VEGF) by endothelial cells (Dunn 2000). Indeed, angiogenesis in the brain increases after trauma, in particular through the production of IGF-I (Lopez-Lopez et al. 2004). Moreover, injection of IGF-I after brain lesion accelerates vascular remodeling and increases the density of blood vessels in the brain. It is thus becoming increasingly clear that the neurotrophic and neuroprotective actions of growth factors such as IGF-I are not directed only at neurons but rather affect the entire microenvironment, including glial cell populations and cells forming the blood vessels of the brain.

9 Conclusion IGF-I signaling in the adult brain is thought to maintain homeostasis of the CNS under physiological conditions. IGF-I plays a role in energy homeostasis of the brain, cell-to-cell communication, adult neurogenesis and cognition. Clinically, the most important aspect seems to be the neuroprotective activity of IGF-I. IGF-I deficiency or resistance occurs in several diseases of the CNS associated with neuronal cell death, whether induced by oxidative stress, inflammation or excitotoxicity. Despite numerous studies demonstrating the anti-apoptotic and neuroprotective effects of IGF-I in vivo, the underlying mechanisms remain unclear, in particular the corresponding roles of glial cells and neurons. This area thus requires further investigation for the development of new therapeutic approaches based on the use of IGF-I or related molecules. IGF-1R signaling plays a key role in brain development and its consideration is of prime importance in brain injury and degenerative diseases of the CNS. Recent studies into longevity suggest that decreased insulin/insulin-like signaling increases life span and survival. Similarly, caloric restriction reduces IGF-I levels in the circulation and in the brain and increases the longevity of the organism. Paradoxically, excess IGF-I in the brain promotes neuronal survival but seems to reduce the longevity of the organism. More studies are now needed to improve our understanding and to clarify these issues.

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The Role of the IGF-1 and its Partners in Central and Peripheral Metabolism: Considerations for Extending Healthy Life Span Nir Barzilai, Derek M. Huffman, Pinchas Cohen, and Radhika H. Muzumdar

Abstract There is growing evidence to suggest a role for GH/IGF system in regulation of life span, or rate of aging. The role of IGF-1 in aging and disease is very complex. High IGF-1 levels is associated with cancers and low IGF-1 has been implicated in the pathogenesis of a wide range of conditions including diabetes and glucose intolerance, osteoporosis, poor cognitive function and coronary heart disease, thus highlighting the complex role of IGF axis in humans. In addition, we have shown that members of GH/IGF axis, IGF-1, IGFBP-3 as well as a novel binding partner for IGFBP-3 termed HN influence glucose metabolism through the hypothalamus. This suggests that acute regulation of peripheral insulin action is probably elicited in the hypothalamus and the integrity of GH/IGF system is probably needed to ensure normal metabolism with aging. Therefore, it is intriguing to speculate that an ability to ameliorate the age-associated decline in CSF IGF-1 levels without increasing circulating IGF-1 levels would be beneficial in potentially preventing or treating two scourges with enormous morbidity and mortality, diabetes and cancer. Potentially, administration of IGF-1 receptor antagonists that does not cross the blood brain barrier, will protect peripheral tissues from cancer, yet will allow IGF-1 to act in the brain to allow the beneficial effects related to other diseases such as cognition and glucose tolerance. In addition, the newest link to IGF system, HN could offer exciting patho-physiologic links as well as potential treatment options for neuro-degeneration and diabetes.

N. Barzilai (*) Institute for Aging Research, Departments of Medicine and Molecular Genetics, Belfer Building, Suite 701, The Albert Einstein College of Medicine, Bronx, USA e-mail: [email protected]

D. Clemmons et al. (eds.), IGFs: Local Repair and Survival Factors Throughout Life Span, 143 Research and Perspectives in Endocrine Interactions, DOI 10.1007/978-3-642-04302-4_11, # Springer-Verlag Berlin Heidelberg 2010

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1 IGF axis, age-related disease and longevity There is growing evidence to suggest an important role for the growth hormone/ insulin-like growth factor (GH/IGF) system in the regulation of life span and agerelated diseases. Disruption of IGF/insulin signaling in lower organisms such as nematodes, yeasts and flies has been associated with prolongation of life span. In mammals, the insulin-signaling pathway is considerably more complex and is separated from the IGF-axis system, which includes IGF-I and IGF-II, six IGF binding proteins (IGFBP-1 to -6), and nine IGFBP-related proteins (IGFBP-rPs; LeRoith 1997; Rosenfeld et al. 2000). As in lower species, spontaneous genetic alterations to the GH/IGF axis in rodents, such as in Snell and Ames mice, and heterozygous disruption of the IGF-1 receptor (IGF-1R) in a genetic model have been shown to prolong life span. In humans, there is tremendous evidence to suggest that high IGF-1 levels are a risk factor for many types of cancers. Therefore, since the discovery of the IGF-1R in the 1980s, there has been great interest in developing therapeutic strategies to inhibit IGF-1-R signaling, including tyrosine kinase inhibitors and IGF monoclonal antibodies to treat various cancers (Rodon et al. 2008). However, just as high IGF-1 levels are associated with cancers (2004), a low IGF-1 level has been implicated in the pathogenesis of a wide range of conditions, including type 2 diabetes and glucose intolerance (Sandhu et al. 2002), osteoporosis (Zofkova 2003), poor cognitive function (Trejo et al. 2007) and coronary heart disease (Juul et al. 2002; Janssen and Lamberts 2002), thus highlighting the complex role of the IGF axis in humans. Because of the associations of IGF-1 with disease risk in humans and its involvement in life span determination of model systems, we recently examined whether there was an association between the GH/IGF-1 system and human longevity. In a genetic analysis of IGF-1R in exceptionally long-lived individuals, we identified alterations in the IGF-1R gene that were overrepresented in our female centenarians versus controls. These mutations were functional, with carriers having a lower maximal height and greater IGF-1 levels compared to those without these mutations. Furthermore, a lower number of IGF-1R as well as lower activation of AKT by IGF-1 were observed in transformed lymphoblasts obtained from these subjects with IGF-1 R mutations (Suh 35 al. 2008). While this finding suggests a role for this pathway in the modulation of human life span and highlights the complex role of the IGF system in various biological functions (Fig. 1), it also raises a more relevant clinical question: how to best modulate the IGF signal for optimal benefit against many age-related diseases? In this review, we suggest that some peripheral actions of IGF-1 are elicited in part through the hypothalamus. Furthermore, we propose that a better understanding of these mechanisms may lead to strategies for directing IGF-1 to the brain to enhance its favorable actions on metabolism, while avoiding potentially deleterious effects in the periphery.

Role of IGF‐1 in age‐related diseases and longevity

N eural protec tion (A D *) C ardio protec tion (IH D *) Ins ulin s ens itiz ation (D M *) P rotec tion from O s teoporos is

Longevity

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C anc er

Longevity

Fig. 1 Summary of the risk of increased IGF-1 levels on diseases in relation to longevity. Longevity risks are increased with high IGF-1 in relationship to some diseases whereas they are decreased in relationship to cancer. Several of the favorable effects of IGF-1 may be exerted through the brain, whereas risks for cancer are probably peripheral. Here we suggest models wherein IGF-1 will be administered together with a peripheral blocker of IGF-1 receptor in the hopes of inducing overall healthy aging and longevity. AD: Alzheimer’s disease; IHD: ischemic heart disease; DM: diabetes mellitus

2 IGF-1 and Glucose Metabolism IGF-1 originally got its name based upon insulin-like activity demonstrated by this peptide in vitro, not surprising considering the structural homology between IGF-1 and its receptor to insulin and its receptor. The effects of IGF-1 on insulin sensitivity are, in part, related to its ability to suppress GH, which has an insulin antagonistic effect. However, IGF-1 exerts additional effects on insulin sensitivity that are not mediated through suppression of GH action (O’Connell and Clemmons 2002). For instance, IGF-I has insulin-like effects on peripheral uptake of glucose and fatty acids (Moxley et al. 1990) and subjects treated with IGF-1 commonly report hypoglycemia. Administration of IGF-1 has been shown to increase glucose uptake and inhibit hepatic glucose production in healthy subjects, in insulin resistance states and in both type 1 and type 2 diabetes (Boulware et al. 1994; Moses et al. 1996). Animal models associated with decreased IGF-1 levels or function have been shown to have impaired glucose tolerance including IGF-1R knock out heterozygotes and homozygotes, underscoring the metabolic role of IGF-1 on glucose homeostasis (Berryman et al. 2008). While these models suggest a role for the IGF-1 axis in modulating peripheral glucose metabolism, the interpretation of these studies is limited in that these studies do not delineate whether the effects could be mediated through the insulin receptor or the IGF-1/insulin hybrid receptor or identify sites of action of IGF-1.

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3 Central Regulation of Peripheral Glucose Metabolism A potential role for hypothalamic nuclei in influencing glucose metabolism was suggested as early as 19th century. Direct stimulation of various hypothalamic nuclei has been shown to either increase or decrease blood glucose levels through the autonomic nervous system (Sudo et al. 1991). Recently, research from our lab and from others has identified a central role for hypothalamic nuclei in regulating glucose homeostasis. In particular, the arcuate nucleus within the mediobasal hypothalamus (MBH) has been recognized as a major regulatory center for determining nutrient availability, glucose homeostasis and overall energy balance (Halaas et al. 1995). The neurons in the arcuate nucleus have the ability to respond to hormonal and nutrient stimuli, thereby regulating food intake, energy expenditure, glucose sensing, and insulin action. Leptin, which is a peptide secreted by adipocytes, has been shown to modulate body weight by decreasing food intake and increasing energy expenditure, as well as to regulate peripheral insulin action by activating leptin receptors in the MBH (Muzumdar et al. 2003). In addition, centrally administered leptin has been shown to decrease glucose-stimulated insulin secretion via central melanocortin receptors (Muzumdar et al. 2003) and to selectively decrease visceral fat (VF) (Boghossian S et al. 2007). Similarly, insulin acts through the MBH to decrease appetite, increase energy expenditure, decrease VF and inhibit hepatic glucose production irrespective of peripheral insulin levels (Obici et al. 2002b). This process has been elegantly studied by selectively abolishing central insulin receptors by intra-cerebroventricular (ICV) infusion of insulin receptor antisense (Obici et al. 2002a). Furthermore, a downstream pathway of PI3 kinase has been described in the central actions of both insulin and leptin with the use of PI3 kinase inhibitors (Lam et al. 2005). Nutrients also have a direct effect on the central regulation of glucose metabolism. Hypothalamic infusions of glucose, free fatty acids, and long chain fatty acid CoAs, such as oleic acid, have been shown to reduce endogenous glucose levels through inhibition of hepatic glucose production (Obici et al. 2002c). Taken together, these findings highlight the crucial role of the MBH in controlling energy balance and glucose homeostasis.

4 Regulation of Peripheral Glucose Metabolism by Hypothalamic IGF-1 and its Receptor IGF -1 is made and secreted by the liver and found primarily in the circulation whereas IGF-1R is expressed in many tissues. Remarkably, IGF-1R levels are paradoxically low in liver (Frick et al. 2000) and IGF-1 fails to stimulate primary hepatocytes. In spite of no effect on liver, several studies have shown that IGF-1 can modulate hepatic insulin action, raising questions regarding how these effects are mediated.

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The suppression of glucose output from liver (HGP) with IGF-1 peripheral administration in several studies (Moxley et al. 1990; Shojaee-Moradie et al. 1995; Jacob et al. 1991), coupled with the relative scarcity of IGF-1R in liver, supports the possibility of effects through the CNS similar to leptin and insulin. Indeed, IGF-1 has been shown to have multiple biologic roles in the CNS. Though circulating IGF-1 is primarily derived from the liver, it has been shown to work in a paracrine fashion through local production in the brain. IGF-1 has also been demonstrated to be transported across the blood-brain barrier and is present in cerebrospinal fluid (CFS; Heinze et al. 1998). Furthermore, IGF-1R (both mRNA and protein), similar to insulin receptors, are abundantly expressed in various areas of the brain, including the hypothalamus (Garcia-Segura et al. 1997; FernandezGalaz et al. 1999). To specifically demonstrate a central role for IGF-1 through the hypothalamus on glucose metabolism, we infused IGF-1 into the third ventricle of conscious, unstressed catheterized young rats and studied peripheral glucose metabolism using hyperinsulinemic-euglycemic clamps, which is the gold standard technique. Insulin, leptin and IGF-1 levels in circulation were comparable between the IGF-1-infused and control animals. The infusion of IGF-1 into the third ventricle was continuous, and the dose used was small and comparable (in molar quantities) to the doses of (ICV) insulin that demonstrate a physiological effect (Kovacs et al. 2002). Infusion of IGF-1 into the third ventricle was able to affect peripheral hepatic insulin action even in the absence of demonstrable leak of human IGF-1 into the periphery or change in peripheral GH levels. These studies demonstrate that IGF-1 influences glucose metabolism through the hypothalamus and helps explain the apparent puzzle of significant metabolic effects of IGF-1 at the level of the liver in the absence of substantial amounts of hepatic IGF-1R.

5 IGFBP-3 and Glucose Metabolism: We and others have shown that IGFBP-3 plays a significant role in glucose metabolism (Muzumdar et al. 2006; Kim et al. 2007). IGFBP-3 accounts for 85% of IGF-1 binding by the binding proteins and circulates in serum as a ternary complex with IGF-1 and acid labile subunit (ALS). Its well-characterized role is to serve as a binding protein for IGF-1 and increase the half-life of IGF-1 from just a few minutes to several hours. Recent evidence has shown that, in addition to serving as a binding protein for IGF-1, IGFBP-3 has a direct, nonIGF-1 dependent biological role in many tumor cell lines in vitro, by inhibiting cellular proliferation and inducing apoptosis through a yet unidentified receptor (Rajah et al. 1997). A relationship between elevated circulating IGFBP-3 levels and hyperglycemia is suggested in various physiological and pathological states associated with insulin resistance, such as puberty (Lofqvist et al. 2005), acromegaly (Wass et al. 1982)

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and treatment with recombinant human growth hormone (Cutfield et al. 2000). Transgenic mice overexpressing IGFBP-3 demonstrate impaired glucose tolerance and decreased glucose uptake in both liver and muscle (Silha et al. 2002). We demonstrate that an acute increase in circulating IGFBP-3 to levels seen in physiologic states of insulin resistance like puberty leads to impairment of insulin action. The impaired insulin action under euglycemic-hyperinsulinemic clamp is both at the level of the liver (increased HGP) and the skeletal muscle, as evidenced by decreased glucose uptake (Muzumdar et al. 2006). Infusion of IGFBP-3 over seven days using subcutaneous osmotic pumps also impaired insulin action and increased visceral fat (Kim et al. 2007). Effects of IGFBP-3 on glucose metabolism could be secondary to decreased availability of IGF-1. However, evidence is accumulating that IGFBP-3 can directly affect glucose metabolism and insulin action, independent of IGF-1 binding. IGF-independent effects of IGFBP-3 on Akt have been described in adipocytes (Shim et al. 2001) and endothelial cells (Franklin et al. 2003). IGFBP-3 impairs the GLUT-4 translocation in response to insulin in cultured adipocytes. IGFBP-3 has been shown to activate tyrosine phosphatases and bind RXR in the nucleus after cellular internalization (Schedlich et al. 2007). This binding of IGFBP-3 to RXR-a can impair PPAR-g signaling (Dello Russo et al. 2003), offering another potential mechanism by which IGFBP-3 impairs insulin action.

6 Role of Central IGFBP-3 on Glucose Metabolism We had earlier demonstrated that IGF-1 regulates hepatic glucose metabolism through the hypothalamus, highlighting its critical role in mediating peripheral glucose metabolism. Since IGFBP-3 has been detected in the CSF and brain in normal and in pathologic states like Alzheimer’s disease (Rensink et al. 2002), in which insulin resistance plays a prominent role, we examined if some of the physiological effects of IGFBP-3 on glucose metabolism are elicited through the brain. We acutely modulated IGFBP-3 levels in the CNS by infusing IGFBP-3 into the third ventricle and carefully monitored the peripheral effects on insulin action. We found that IGFBP-3 influences overall insulin action through decreases in insulin-induced suppression of HGP, decreased glucose disposal and impaired suppression of free fatty acids (FFA) levels (Muzumdar et al. 2006). The effects of central IGFBP-3 on overall systemic insulin action were in contrast to central IGF-1 administration, where the predominant effect was on the liver. The effects of IGFBP-3 on insulin action through the hypothalamus are opposite those of IGF-1, which raises the question as to whether effects of IGFBP-3 are IGF-1dependent (secondary to IGF-1 binding and thus decreased availability of IGF-1) or IGF-1-independent. IGFBP-3 mutants with altered interactions with its protein partners serve as useful tools to evaluate the specific mechanism. The nuclear localization sequence (NLS) mutant of IGFBP-3 protein binds IGF-1 normally but fails to bind to

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other IGFBP-3 ligands, including cell-surface proteins (preventing cell binding and internalization), importin-beta (preventing nuclear transport), various extracellular matrix proteins, such as transferrin and type-1 collagen, and various soluble, intracellular, extracellular and circulating proteins (Muzumdar et al. 2006). We have previously characterized the mutant and showed that it fails to internalize into cells and does not mediate nuclear actions of IGFBP-3 in spite of normal IGF-1 binding and equal inhibition of IGF-dependent effects. We infused this mutant into the third ventricle (targeting the hypothalamus) and studied its effects on insulin action in the liver and muscle using hyperinsulinemiceuglycemic clamps. In contrast to IGFBP-3, this mutant minimally inhibited hepatic and peripheral insulin action in vivo. The decreased activity of the NLSmutant IGFBP-3, compared to IGFBP-3, demonstrates that IGF-I binding alone is insufficient to mediate the central effects of IGFBP-3 we observed. The partial, but not significant effects of the NLS mutant are compatible with some inhibition of local IGF action as a component of the effect of IGFBP-3 in the CNS. Thus, it appears that IGFBP-3 antagonizes insulin action through mechanisms in the CNS involving both IGF-dependent and IGF-independent pathways. The effects of peripheral infusion of IGFBP-3 on glucose metabolism could also be centrally mediated as the amount of human IGFBP-3 detected in the CSF during a peripheral infusion of this protein can not accurately reflect its availability near the hypothalamus, its internalization, as well as local production in the hypothalamus. Since IGFBP-3 is expressed in brain tissue and is elevated in the brains of patients with Alzheimer’s disease, it is intriguing to speculate that IGFBP-3 may be involved in the well-recognized insulin resistance in this condition. The extent of the individual contribution of IGF-1-dependent vs. non-IGF-1-dependent effects of a peripheral IGFBP-3 infusion can be discerned by studying the effects of peripheral infusions with a non-IGF-1 binding mutant of IGFBP-3 in vivo.

7 Novel IGFBP-3 Binding Partner Humanin as a Regulator of Glucose Metabolism Humanin (HN) is a recently identified, 24-amino acid polypeptide that has a welldescribed role in neuroprotection against cell death associated with Alzheimer’s disease (Hashimoto et al. 2001), from Alzheimer’s disease-specific insults (Sponne et al. 2004), prion-induced apoptosis (Sponne et al. 2004) and chemically induced neuronal damage (Krejcova et al. 2004). HN directly interacts with a variety of proapoptotic proteins, including Bax-related proteins (Guo et al. 2003) and insulin-like growth factor binding protein-3 (IGFBP-3; Ikonen et al. 2003). Recently, it was demonstrated that binding of HN with IGFBP-3 prevents the activation of caspases (Liu et al. 2000). HN and IGFBP-3 have opposing roles on cell survival; HN protects against apoptosis whereas IGFBP-3 induces cell death (Ikonen et al. 2003). Based upon the molecular interaction between HN and IGFBP-3, and the emerging link between Alzheimer’s disease and insulin resistance (Craft 2005), we

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examined whether HN, in addition to its neuroprotective roles, may serve as a centrally acting regulator of glucose homeostasis. Infusion of HN ICV significantly improved overall insulin sensitivity, both in the liver and skeletal muscle. Thus HN appears to mediate peripheral glucose metabolism through the MBH like leptin, insulin and IGF-1. The central effects of HN on insulin action are associated with activation of hypothalamic STAT-3 signaling, effects that are negated by co-inhibition of hypothalamic STAT-3 in vivo. Inhibition of hypothalamic STAT-3 attenuated the robust insulin-sensitizing effects of peripherally administered potent analogs of HN, highlighting the role of MBH in mediating the insulin-sensitizing effects of HN (Muzumdar et al. 2009). This study provides evidence that HN and IGFBP-3 have opposing roles in peripheral insulin action via the hypothalamus. Thus, endogenous IGFBP-3 in the hypothalamus may temper the effects of HN on peripheral glucose metabolism in vivo. Furthermore, HN analogues that do not bind IGFBP-3 have greater insulinsensitizing effects than native HN. In summary, HN is a novel peptide that is linked to the IGF system and has many similarities to IGF-1, including anti-apoptotic effects, the ability to bind IGFBP-3 and centrally mediated insulin- sensitizing effects.

8 Conclusions The GH/IGF axis is an important modulator of insulin signaling and glucose metabolism. Evidence that insulin signaling is involved in the longevity of lower organisms and rodents has raised the possibility that this conserved pathway may be an important determinant of human life span. In this paper, we highlight the unique role of the hypothalamus in mediating effects of the IGF axis, IGF-1 and IGFBP-3 on peripheral glucose metabolism and energy homeostasis. Since IGFBP-3 is up-regulated in Alzheimer’s disease brains, it could play a role in the insulin resistance that is well associated with this condition. The physiologic role of IGF-1 on glucose metabolism through the hypothalamus is especially fascinating considering that 1) aging increases the risk for both diabetes and cancer, two pathological process that are affected by IGF-1 system in opposite manner: an increase in IGF-1 predisposing to cancer and a decrease in IGF-1 increasing the risk of diabetes; and 2) aging is associated with a decrease in both circulating and CNS IGF-1 levels. Therefore, it is intriguing to speculate that, from a therapeutic standpoint, an ability to ameliorate a decline in CSF IGF-1 levels without increasing circulating IGF-1 levels would work out beneficially in potentially preventing or treating two scourges with enormous morbidity and mortality: diabetes and cancer. A second approach might be the administration of IGF-1 receptor antagonists that do not cross the blood-brain barrier, thus reducing the cancer risk of peripheral tissues while enhancing the beneficial actions of IGF-1 to help prevent other diseases such as diabetes. Along these lines, the newest link in the IGF system, HN, could offer another potential approach to the treatment of both neurodegeneration and type 2 diabetes.

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Acknowledgments This work was supported by grants from the National Institutes of Health (K08 AG027462 to R.H.M and AG21654 and AG18381 to N.B.) and by the Core laboratories of the Albert Einstein Diabetes Research and Training Center (DK 20541).

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Index

A

D

Aging, 97–102 Alzheimer, 128 Apoptosis, 48, 49, 51, 54, 127, 128, 131, 132, 134 Astrocytes, 44, 50, 53, 54, 127, 129, 131–134 Atherosclerosis, 17–18

daf-2 mutants, 70–73, 77, 78 Dentate gyrus, 46–48, 53 Drosophila melanogaster, 98, 101

B

F

Binding proteins, 1, 3, 5–7 Brain and brain development, 43–46, 48, 49, 51–54

Flow cytometry, 26–27 Free IGF1, 4–6

E Edema, 134–135 EGF receptor (EGFR), 60–66

G C Caenorhabditis elegans, 98, 101, 102 genes regulating longevity, 79 stress response systems, 77–78 Caloric restriction (CR), 101–102 Cancer growth hormone, 97–102 insulin, 97–102 insulin receptor (IR), 98, 100, 101 mouse model, Cell cycle, 47, 51 Cerebral cortex, 45, 47–49, 52, 53 Culture, 22–27, 29, 30, 33–39 Cytokine, 132, 133, 135

Global endocrine system, 74 Glutamate, 126, 127, 131, 133, 134 G proteins, 63 Growth hormone (GH) cancer, 97–102 deficiency, receptor, 99

H Hematopoietic stem cell H(SC), 21–39 Hippocampus , 45, 46, 48, 50 Humanin, 149, 180 Hyperglycemia, 11–18 Hypothalamus, 144, 146–150

155

156

I IGF, 22, 23, 34, 35, 38, 39 cancer, 97–102 deficiency, lifespan, 99, 100 publications, 2, 3 receptor, 62 IGF-1 clinical recombinant, 2–4 and glucose metabolism, 145–147 and hypothalamus, 144, 147, 149 local replacement therapy, 4 and longevity, 144–145, 150 serum, 6, 7 IGF binding protein (IGFBP), 44, 46, 52 IGFBP-1, 60 IGFBP-2, 21–39 IGFBP-3, 60–66 and glucose metabolism, 147–150 and hypothalamus, 148–150 IGF-II, 44, 52 IGF-IR, 99–101 Infarct, 127, 129, 130, 132–134 Initial mortality rate (IMR), 72, 74 Insulin, 97–102, 128, 135 Insulin-like growth factor-I (IGF-I), 43–54 Insulin-like signaling mutants, 71–74 Insulin receptor (IR), 98, 100, 101 Insulin receptor substrate, mouse model,

L Life span, 69–79, 99, 100 Limiting dilution analysis, 26, 30, 33 Lipofuscin, 73 Longevity, 135 Longevity control, 70, 71, 75–79 Lysosomes, 73, 74

M Macrophages, 127, 129, 133, 134 MAP kinase, 13–18 Mice null mutant, 44, 45, 48, 49, 52 transgenic, 45, 47–49, 52, 53 Microglia, 129, 131, 133, 134

Index

Model species, 2 Myelination , 44, 50–54

N Neurogenesis, 126, 135 Neuron, 44, 46–50, 54 Neuroprotection, 127, 129, 130 NOD/SCID, 22, 26, 27, 29, 30, 33, 36 Nuclear receptors, 60, 61

O Oligodendrocyte, 44, 46, 47, 50–54

P PARP, 131 Pigment accumulation, 73, 74 PI-3 kinase, 13–16 Progenitors, 44–48, 50–53 Proliferation, 44, 46–49, 51, 53

R Repopulation, 22, 25, 26, 30, 33 RNAi, 71, 73–79

S Shc, 13–18 SHPS-1, 14–16, 18 Signaling pathways, 69–79 Smooth muscle cell, 12, 17 Somatomedin, 2, 3 Sphingosine kinase (SphK), 60, 62, 63, 66 Sphingosine-1-phosphate (S1P), 60, 62–66 Storage, 6, 7 Stress, 77–79, 127, 131, 132, 135 Stress response systems, 77–78 Survival, 127, 129, 130, 132–135 Synapses, 50

T Target of rapamycin (mTOR), 101

Index

Ternary complex, 6 TNF, 132–134 Transactivation, 60, 62–65 Transplantation, 22, 23, 25, 29, 30, 33–37

157

Type 1 IGF receptor (IGF1R), 43–47, 49–54

V aVb3 integrin, 13, 15, 17, 18