Role of R-Spondin-1 in the Regulation of Pancreatic Β-Cell Behaviour

ROLE OF R-SPONDIN-1 IN THE REGULATION OF PANCREATIC β-CELL BEHAVIOUR

Victor Shing Chi Wong

A thesis submitted in conformity with the requirements

for the degree of Doctor of Philosophy

Graduate Department of Physiology

University of Toronto

ROLE OF R-SPONDIN-1 IN THE REGULATION OF PANCREATIC β-CELL

BEHAVIOUR

Victor Shing Chi Wong Doctor of Philosophy Department of Physiology University of Toronto 2011

GENERAL ABSTRACT

R-spondin-1 (Rspo1) is an intestinal growth factor known to exert its effects through activation of the canonical Wnt (cWnt) pathway, but its function in the β-cell had not been explored. In Chapter 2, Rspo1 mRNA was found to be expressed in murine islets and the murine MIN6 and TC -cell lines, and Rspo1 protein was detected in MIN6 -cells. Rspo1 activated cWnt signaling and induced insulin mRNA expression in MIN6 -cells. Analysis of

MIN6 and mouse -cell proliferation revealed that Rspo1 stimulated cell growth and significantly abolished cytokine-induced cellular apoptosis. Rspo1 also stimulated insulin secretion in a glucose-independent fashion. Chapter 2 further demonstrated that the glucagon- like peptide-1 receptor agonist, exendin-4 (EX4), stimulated Rspo1 mRNA transcript levels in

MIN6 cells in a glucose-, time-, dose- and PI3-kinase-dependent fashion. Together, these studies demonstrate that Rspo1 is a novel -cell growth factor and insulin secretagogue that is regulated by EX4. In Chapter 3, the role of Rspo1 in -cells in vivo was explored using Rspo1 knock-out (Rspo1-/-) mice. Rspo1-/- mice had normal fasting glycemia but an improved glycemic control after an oral glucose challenge compared to Rspo1+/+ mice, with no difference in insulin sensitivity but an enhanced insulin response over 30 min; glucagon responses were

normal. Rspo1 deficiency also resulted in an increase in -cell mass in association with an increase in Ki67-positive -cells, a marker of proliferation, relative to Rspo1+/+ mice. Rspo1-/- pancreatic tissues also demonstrated a significant increase in the number of insulin-positive ductal cells, suggestive of -cell neogenesis. Rspo1-/- islets displayed no changes in glucose- induced insulin secretion but showed a complete absence of glucose-induced suppression glucagon secretion. Treatment of Rspo1-/- mice for 2 wk with EX4 resulted in a similar glycemic profile to EX4-treated Rspo1+/+ mice after an oral glucose challenge, with no changes in insulin sensitivity. Interestingly, EX4 administration to Rspo1-/- normalized -cell mass to a level comparable to that in Rspo1+/+ mice. Although further studies are required, the findings in this thesis reveal a novel role for Rspo1 as a regulator of -cell behaviour in vivo, and suggest novel roles for Rspo1 in both - and ductal-cells.

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ACKNOWLEDGEMENTS

“It is not the critic who counts, not the man who points out how the strong man stumbled, or where the doer of deeds could have done better. The credit belongs to the man who is actually in the arena; whose face is marred by the dust and sweat and blood; who strives valiantly; who errs and comes short again and again... who at the best knows in the end the triumph of high achievement, and who, at worst, if he fails, at least fails while daring greatly; so that his place shall never be with those cold and timid souls who know neither victory nor defeat." -Theodore Roosevelt

Graduate school as a Doctor of Philosophy student is, without a doubt, a large part of my life. I have one true ambition that is a juggernaut in its own right and it is a race with endless nights and weekends clocked in the laboratory. But I remained resilient, especially knowing and befriended so many people during my journey; they have become my lasting inspiration to move forward with optimism and confidence! There are so many I wish to give thanks to and so much to say, but I will save that for my autobiography in the future. There are, however, honorable mentions that rightfully deserve their place in this precious little liberty from the rest of these dull and unsentimental scientific jargons.

I have to firstly thank my family for their unconditional and relentless support. My parents and my sister are my constant reminder that all things are possible through bringing up a deaf child into a hearing and verbal world, rather than the common path of the deaf culture limited to visual language that is sign. It inspires me to never live in the fear of challenging on-going dogmas and beliefs. It inspires me to take the road less taken.

It goes without saying that I am in a large debt to my supervisor, Dr. Patricia Brubaker. I look upon her in awe and amazement ever since I started as an undergraduate project student. Her teaching skills are extremely influential and stimulating, and her patience and understanding with me ad infinitum! I am apologetic for failing to be a “role model” PhD student and wake up for the lab at 10 A.M. the latest every day. Nevertheless, I hope she realized that the motivation to become a great scientist is not lost in me; after all she is someone I aspire to be and I know it in my heart that I will surprise and make her proud one day.

Although he had absolutely no reason to do so, Dr. Gary Lewis, who was my Master of Science supervisor, became my PhD mentor offering me invaluable advices during the frustrating and uncertain times. He constantly reminds me that life is much more than just laboratory work and publications, that although they are important career-wise, it is crucial to have balance in order to see things more objectively. For those heart-to-heart talks, I am eternally indebt to him as well.

There were so many post-doctoral fellows that came and went but they are definitely not forgotten. Dr. Younnes Anini was the first among those silent teachers with a big heart. If there was an award for the Best Teaching in post-doctoral fellows, I will make sure he will win that at iv

any cost. Drs. Roman Iakoubov and Lina Lauffer came along and I am speechless even to this day about their intellect. They are walking medical encyclopedias and like Dr. Anini, they are incredibly generous and I am grateful for all of their help and entertaining company.

Five years of PhD is a long time, but they flew by so quickly because it was one wild and fun adventure with friends who filled it with much laughter and joy. When I first started out in Dr. Brubaker‟s laboratory, I came to know Drs. Philip Dubé and Eric Shin. These two are, in my opinion, the epitome of what it takes to be the “role model” graduate student: you can be successful without sacrificing anything to help others in need. Their sincere altruism, modesty and willingness to share their intellect made a lasting impression on me even to this day, and are ones I vow to carry on. I lift my glass in gratitude also to Katherine Rowland as I have to thank her immensely for being such an incredible friend. Although it seems redundant from afar the fact that we have shared insurmountable amounts of coffee breaks but I treasure them more than anything else in the world. Because in those few precious minutes, they are memories of a kind, courageous and intelligent colleague who would spend the time to listen and provide support that turned my angry fist into a celebratory high-five hand gesture. It goes without saying then, that I look forward to see what this friendship will continue to bring as we advance to new levels of our scientific careers.

I am also indebt to my sidekicks Andrew Mulherin, Monika Poreba, and Shivangi Trivedi. I have never been in a better laboratory of friends. Thank you for being such an amazing person you are. I would also like to thank Andrea Yeung, Will Schultz and Amy Oh, all of whom are dedicated and committed undergraduate students that I had a pleasure to work closely with. Thank you for all your hardwork and it was nice to have someone to blame on a just-in-case basis. And to Charlotte Dong, thank you for all the giggles and Stuart Wiber, where have you been all my life?! From the moment we met, we knew that being as crazy as we are is the only way to live. As a lab, we usually create so many evil laboratory schemes in hopes to take over the world, but Angelo Izzo, our lab technician, would never agree to any of it. For that, he also has my sincere gratitude for preventing us in doing something we will later regret. Thank you for all your help, your ideas and opinions that are often controversial and provocative, but they make for very entertaining memories.

Lastly but not the least, I have to thank my coach and friend, Tara Norton. She is one of the best professional triathletes Canada ever gazed, and the opportunity to train under her wing in past 2 years, had allowed me to grow as an athlete in leaps and bounds. Although she had nothing to do with my PhD studies, she inadvertently helped me develop a mental toughness that extends above and beyond the race course. As a result, I am never more excited and focused to meet every obstacle that most people mistaken my composure for ease. As one curtain falls, another rises and I am never more motivated to take on the world. But before I do, I want to take a humble bow and say to all my friends and family: I love you all, thank you for being in my life. v

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ...... IV TABLE OF CONTENTS ...... VI LIST OF FIGURES ...... IX LIST OF TABLES ...... X LIST OF ABBREVIATIONS ...... XI 1 INTRODUCTION ...... 2 1.1 Type 2 Diabetes Mellitus: The Problem ...... 3 1.2 Pancreatic β-Cell And Its Physiologic Regulation ...... 5 1.2.1 Pre- and post-natal β-cell growth and function ...... 7 1.2.2 Adaptive β-cell growth and function in physiological and pathophysiological states ...... 10 1.2.3 Pancreatic dynamics in response to growth factors ...... 22 1.3 Canonical Wnt Signaling ...... 34 1.3.1 cWnt signaling in pancreatic development ...... 37 1.3.2 cWnt signaling in mature -cells ...... 38 1.3.3 Activation of cWnt signaling in pancreatic β-cells ...... 40 1.3.4 cWnt signaling in T2DM: the TCF7L2 paradox ...... 44 1.4 R-spondin: a new player in the Wnt game...... 45 1.4.1 Function of R-spondin proteins ...... 47 1.4.2 R-spondin proteins in human diseases ...... 50 1.4.3 R-spondin proteins and the canonical Wnt signaling pathway ...... 51 1.5 Rationale and Hypothesis ...... 54 2 R-SPONDIN-1 IS A NOVEL -CELL GROWTH FACTOR AND INSULIN SECRETAGOGUE IN VITRO ...... 56 2.1 Abstract ...... 56 2.2 Introduction...... 57 2.3 Experimental Procedures ...... 60 2.3.1 Cell culture...... 60 2.3.2 Isolation and culture of intact and dispersed mouse islets...... 60 2.3.3 RNA isolation...... 60 2.3.4 RT-PCR...... 60 2.3.5 Real-Time PCR...... 61 2.3.6 Protein extraction, cell fractionation and immunoblotting...... 62 2.3.7 Cell proliferation assays...... 63 2.3.8 Apoptosis assays...... 64 2.3.9 Insulin secretion assay...... 65 vi

2.3.10 Statistical Analysis...... 66 2.4 Results ...... 67 2.4.1 Expression of Rspo1 and cWnt signaling molecules in murine -cells...... 67 2.4.2 Rspo1 stimulates cWnt signaling and insulin mRNA expression in MIN6 -cells. . 67 2.4.3 Rspo1 stimulates -cell proliferation...... 68 2.4.4 Rspo1 prevents cytokine-induced apoptosis in -cells...... 69 2.4.5 Rspo1 stimulates -cell insulin secretion...... 69 2.4.6 EX4 stimulates Rspo1 expression in a glucose-, dose-, time- and PI3-kinase- dependent manner...... 70 2.5 Discussion ...... 84 2.6 Acknowledgements...... 89 3 R-SPONDIN-1 DEFICIENCY IN MICE IN VIVO IMPROVES GLYCEMIC CONTROL AND INCREASES -CELL MASS...... 91 3.1 Abstract ...... 91 3.2 Introduction...... 92 3.3 Experimental Procedures ...... 95 3.3.1 Animals...... 95 3.3.2 Metabolic Tests...... 95 3.3.3 Immunological and morphometric analyses...... 96 3.3.4 Immunoblotting...... 96 3.3.5 qRT-PCR...... 97 3.3.6 In vitro secretion assays...... 97 3.3.7 Statistical Analysis...... 98 3.4 Results ...... 99 3.4.1 Rspo1-/- mouse pancreas are phenotypically indistinguishable from their wild-type counterparts...... 99 3.4.2 Rspo1-/- mice display better glucose handling without changes in insulin sensitivity...... 99 3.4.3 Rspo1-/- mice have increased β-cell mass due to increases in β-cell proliferation and neogenesis...... 100 3.4.4 Rspo1-/- mouse islets display normal insulin release but abnormal glucagon secretion...... 101 3.4.5 Rspo1 may be required for Exendin-4-regulation of β-cell mass...... 101 3.5 Discussion ...... 110 3.6 Acknowledgements...... 114 4 SUMMARY OF RESULTS AND GENERAL DISCUSSION ...... 116 4.1 Summary of Results ...... 116 4.2 General Discussion ...... 119 4.3 Limitations of the present study and future directions ...... 125 4.4 Conclusions ...... 130 vii

5 APPENDIX ...... 134 6 REFERENCE LIST ...... 136

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LIST OF FIGURES

Chapter 1: Introduction

Figure 1.1. Overview of the Canonical Wnt (cWnt) signaling network……………………...35

Chapter 2: R-spondin-1 is a novel β-cell growth factor and insulin secretagogue in vitro

Figure 2.1. Rspo1 and cWnt signaling molecules are expressed in murine β-cells…………..73

Figure 2.2. Rspo1 activates cWnt signaling and increases insulin mRNA levels in MIN6 β-cells……………………………………………………………....…...... 75

Figure 2.3. Rspo1 stimulates β-cell proliferation…………………………………………….77

Figure 2.4. Rspo1 inhibits cytokine-inudced β-cell apoptosis……………………………….78

Figure 2.5. Rspo1 stimulates insulin secretion in MIN6 β-cells and isolated mouse islets...... 80

Figure 2.6. Rspo1 is regulated by EX4 in the β-cell………………………………………….82

Chapter 3: The role of R-spondin-1in the β-cell in vivo

Figure 3.1. Rspo1-/- mice are phenotypically indistinguishable from their wild-type counterparts……………………………………………………………………...103

Figure 3.2. Rspo1-/- mice have improved glycemic control…………………………………105

Figure 3.3. Rspo1-/- mice have an increase in BCM……………………………………...... 106

Figure 3.4. Rspo1-/- have normal insulin secretion but an abnormal glucagon response to high glucose……………………………………………………………………….…..108

Figure 3.5. Treatment with EX4 normalizes glucose homeostasis and BCM in Rspo1-/- mice……………………………………………………………………………...109

Chapter 4: Summary of results and general discussion

Figure 4.1. Proposed working model………………………………………………………..132

LIST OF TABLES

Chapter 1: Introduction

Table 1.1. Growth factors and their effect on β-cell behaviour………………………………...23

Table 1.2. Impact of R-spondin deficiency in xenopus, mouse and humans………………..…50

Chapter 2: R-spondin-1 is a novel β-cell growth factor and insulin secretagogue in vitro

Table 2.1. RT-PCR primers………………………………………………..…………………..72

Chapter 4: Summary of results and general discussion

Table 4.1. Summary of results………………………………………………………..………118

LIST OF ABBREVIATIONS

ADAMTS A Disintegrin-like And Metalloprotease with Thrombospondin ADP Adenosine diphosphate APC Adenopolyposis Coli ATP Adenosine Tri-Phosphate β-TrCP β-Transducin repeat containing protein bcl-2 B-cell CLL/lymphoma 2 BCM β-cell mass BMP Bone Morphogenetic Protein BrdU 5-bromo-2-deoxyuridine cAMP Cyclic adenosine monophosphate CK1 Casein-kinase-1 CRD Cysteine rich domain Cre Cyclization recombination CREB cAMP response element-binding cWnt Canonical Wnt CXCR4 CXC motif chemokine receptor 4 Dkk Dickkopf DNA Deoxyribonucleic Acid DPIV Dipeptidyl peptidase IV Dsh Dishevelled EGF Epidermal growth factor ER Endoplasmic reticulum ERK Extracellular Signal-Regulated Kinase EX4 Exendin-4 FFA Free fatty acid FGF Fibroblast growth factor Foxa Forkhead box subfamily A FOXO Forkhead box subfamily O Frz Frizzled Gcgr Glucagon receptor GIP Gastric inhibitory peptide/glucose-dependent insulinotropic polypeptide GIPR Gastric inhibitory peptide/glucose-dependent insulinotropic polypeptide receptor GK Glucokinase GK Goto-Kakizaki GLP-1 Glucagon-like peptide-1 GLP-1R Glucagon-like peptide-1 receptor GLUT2 Glucose transporter 2 GLUT4 Glucose transporter 4 GPR40 G-Protein-coupled Receptor 40 GRB2 Growth Factor Receptor-Bound 2 GSIS Glucose-stimulated insulin secretion GSK3 Glycogen synthase kinase 3 HbA1c Haemoglobin A1c (glycated) HGF Hepatocyte growth factor xi

HNF Hepatocyte nuclear factor IAPP Islet Amyloid Polypeptide IFNγ Interferon γ IGF-1 Insulin-like growth factor-1 IκB Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor IL-1β Interleukin-1β IL-5 Interleukin-5 iNOS Inflammatory nitric oxide synthase ins2 Insulin 2 IP Intraperitoneal IR Insulin receptor IRS Insulin receptor substrate ITT Insulin tolerance test JAK Janus kinase JNK Jun N-terminal kinase + K ATP ATP-sensitive K+ channel KGF Keratinocyte growth factor Kv Voltage-gated potassium channels LEF Lymphocyte enhancer factor LRP Lipoprotein receptor-related protein MafA v-maf musculoaponeurotic fibrosarcoma oncogene homolog A MAPK Mitogen-activated protein kinase MEK MAPK/ERK kinase mRNA Messenger ribonucleic acid mTOR Mammalian target of rapamycin myc Myelocytomatosis oncogene NFκB Nuclear factor of kappa light polypeptide gene enhancer in B-cells NeuroD Neurogenic differentiation Ngn3 Neurogenin 3 OGTT Oral glucose tolerance test PARP Poly ADP (adenosine diphosphate)-ribose polymerase PCR Polymerase chain reaction PDGF Platelet-derived growth factor PDX Pancreatic and duodenal homeobox PEA3 Polyomavirus enhancer activator 3 PI3-kinase Phosphoinositide-3 kinase PIP3 Phosphatidylinositol-3,4,5-phosphate PKA Protein kinase A PKB Protein kinase B PP Pancreatic Polypeptide PPARγ Peroxisome proliferator-activated receptor γ qRT-PCR Quantitative real time polymerase chain reaction ROS Reactive oxygen species Rspo Roof-plate spondin RT-PCR Reverse transcriptase-polymerase chain reaction SCF Skp1-Cullin-F-box xii

SCO-spondin Subcommisural organ-spondin SDF-1 Stromal-cell dervied factor-1 siRNA Small interfering RNA SNARE Soluble N-ethylmaleimide-sensitive factor attachment protein receptor SNPs Single nucleotide polymorphisms STAT Signal transducer and activator of transcription STZ Streptozotocin T1DM Type 1 diabetes mellitus T2DM Type 2 diabetes mellitus TCF4 T-cell factor-4 TCF7l2 Transcription factor 7-like 2 TGFβ Transforming growth factor β TNF Tumor necrosis factor TSR-1 Thrombospondin type 1 repeats TUNEL Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling UKPDS United Kingdom Prospective Diabetes Study VDCC Voltage-dependent Ca2+ channel VEGF Vascular endothelial cell growth factor Wnt Wg (wingless) + Int1 (chromosomal integration site of mouse mammary tumor virus on mouse chromosome 15) WTX Wilms‟ Tumor Suppressor on X chromosome

Methodological Abbreviations

% percent º C degrees Celsius g gram hr hour(s) l litres M molar (moles/l) min minute(s) mol moles s second(s) wk week

Prefixes k kilo- (x 103) c centi- (x 10-2) m milli- (x 10-3) μ micro- (x 10-6) n nano- (x 10-9) p pico- (x 10-12)

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CHAPTER 1:

INTRODUCTION

Some of the text in this chapter is reproduced with permission from:

Wong V.S., Brubaker P.L. Minerva Endocrinologica 2006 Jun;31(2): 107-124.

Author contribution:

V.S.C. Wong produced all text and figures presented in this chapter.

1 INTRODUCTION

Type 2 diabetes mellitus (T2DM) and its main clinical antecedents including impaired fasting blood glucose, obesity and insulin resistance, represent perhaps the largest public health problem in North America, with the prevalence of these metabolic conditions increasing every decade. According to the World Health Organization, the current global estimate of 170 million people diagnosed with T2DM is set to rise to 366 million by 2030 (1;2). It will be inevitable that within the next decade, health-care systems world-wide will face the prospect of overwhelming demands given the ever-accelerating number of individuals diagnosed with

T2DM (3). The number of diabetes cases in developing countries such as India and China is swelling particularly rapidly, mainly due the change towards urbanization and sedentary lifestyles (2). There is growing evidence showing that β-cell failure occurs much earlier than originally proposed during the development of T2DM. Given the staggering financial and human suffering costs incurred by diabetes and its co-morbid conditions, any safe new therapeutic interventions that prove to have a beneficial effect in slowing or delaying the progression of β-cell failure can lead to more durable glycemic control, and thus would have subsequent major public health benefits. This thesis aimed to characterize a novel player that has regulatory effects on the β-cell, thereby providing a potential therapeutic target for the treatment of T2DM.

1.1 Type 2 Diabetes Mellitus: The Problem

Glucose serves as a primary fuel for all cells, ensuring proper function and survival.

Glucose levels in the plasma are maintained within a narrow range, since hypoglycemia produces cellular death and prolonged hyperglycemia also results in cellular damage. Thus, the maintenance of glucose homeostasis in the narrow physiological range of 4 - 7 mM is stringently regulated, primarily by two opposing hormones: insulin and glucagon. Following a meal, glucose stimulates secretion of insulin from the -cells of the pancreatic islets of

Langerhans. The resulting elevated level of insulin promotes glucose uptake by peripheral tissues such as skeletal muscles, while simultaneously acting on the liver to suppress hepatic glucose production. When blood glucose levels fall, glucagon is secreted from the -cells which are localized together with the -cell within the islets. Glucagon stimulates the breakdown of glycogen to release glucose from the liver, thereby promoting elevation of blood glucose. The dynamic actions of these hormones ultimately determine glycemia.

T2DM is primarily viewed as a metabolic disease whereby glucose metabolism is improperly regulated by insulin, although a more correct view of diabetes is that there are perturbations of many metabolic growth, and inflammatory pathways. T2DM is a complex condition that is not attributable to a single pathophysiological mechanism (4). However, fasting hyperglycemia remains the principle hallmark of T2DM. The development of T2DM usually requires the presence of both insulin resistance and impaired -cell function (5).

Insulin binds to the insulin receptor which contains instrinsic tyrosine kinase activity, resulting in the intracellular auto-phosphorylation of tyrosine residues. The activated receptor, in turn, recruits and phosphorylates a number of substrate molecules such as insulin receptor substrate (IRS) 1/2, which are adapter molecules playing a major role in the coupling to PI3- kinase-protein kinase B (PKB, also known as Akt) and mitogen-activated protein kinases

(MAPK). Recruitment of PI3-kinase to the plasma membrane produce the lipid secondary messenger (phosphatidylinositol (PIP3)) which, in turn, activates a serine/threonine phosphorylation cascade of pleckstrin homology domain-containing proteins including phosphoinositide-dependent kinase-1, PKB/Akt, and the atypical protein kinases C δ and λ isoforms. Subsequent downstream events include stimulation of glucose uptake via GLUT4 translocation, glycogen synthesis, inhibition of lipolysis, and altered gene expression, depending on the tissue or cell. Activation of the MAPK pathway occurs via the guanine nucleotide exchange factor Son-of-sevenless, and GRB2 that are crucial to the mitogenic effects of insulin

(reviewed in (6)). Specifically, with respect to carbohydrate metabolism, insulin resistance implies the impairment of insulin-stimulated glucose uptake by cells, the two major tissues involved being the skeletal muscle and adipose tissue, and overproduction of glucose by the liver (5;7).

In the pre-diabetic state, insulin-resistant individuals manifest hyperinsulinemia but they do not develop hyperglycemia as long as their -cells are able to compensate by maintaining a level of insulin output that is sufficient to overcome their insulin resistance. A decline in functional -cell mass, therefore, contributes to the development and maintenance of hyperglycemia as the individual becomes overtly diabetic. Indeed, a convincing study from

United Kingdom Prospective Diabetes Study (UKPDS) has shown that a decline in -cell function parallels the progression of diabetes (8). Furthermore, analyses conducted by Butler et al found decreases in -cell mass in obese and lean humans with T2DM (9). It must be borne in mind that the dysfunction can be as a result of a decline in -cell mass and/or intrinsic defects in insulin secretion. Although these two are distinct pathophysiological states with a complex relationship, a loss of -cells will nevertheless influence insulin output from the pancreas.

Given that T2DM represents a serious and growing epidemic that poses a major public health threat in the 21st century, innovative therapeutic strategies are required to reduce the incidence of diabetes in susceptible individuals (such as those with impaired glucose tolerance and features of the insulin resistance or metabolic syndrome). The effectiveness of dietary modification, an increase in physical activity, and the use of metformin, acarbose and thiazolidinediones (activators of peroxisome proliferator-activated receptors γ) have recently been shown to be beneficial (10). In addition and more importantly, new therapeutic strategies have targeted -cell replacement and regeneration in the hope of restoring sufficient -cell mass, thereby preventing or reversing diabetes. Therefore, the stimulation of -cell mass expansion represents an exciting arsenal against diabetes. This will nonetheless require significant understanding of the mechanisms that regulate -cell behaviour.

1.2 Pancreatic β-Cell And Its Physiologic Regulation

The pancreas is comprised of three major cell types (in addition to the supporting, endothelial and neuronal cells): endocrine, exocrine and ductal cells. The endocrine cells which constitute only 1-2% of the parenchyma are organized into clusters called islets of Langerhans, and these micro-organs are scattered throughout the exocrine parenchyma. The pancreatic islets of Langerhans are inhabited by four main endocrine cell types: , , , and pancreatic polypeptide (PP) cells, which produce the hormones glucagon, insulin, somatostatin, and pancreatic polypeptide, respectively. A fifth cell type that was recently discovered is the ε cell that secretes ghrelin (11). The quantitative composition of these endocrine cells in islets has been reported in humans and rodents. In rodents, β-cells represent the predominant (> 50%) cell type with -cells and δ-cells representing 10% to 20% and 3% to 10% respectively. However, the composition in human islets is varied and shows more heterogeneity than in rodents, where in human β-cells have been reported to range from 28% to 75% with α-cells ranging from 10%

6 to 65%, and δ-cells ranging from 1% to 22% (12). Moreover, the cytoarchitecture of the islets also differs between species (elegantly reviewed in further depth by R. Scott Heller (13)). In rodent islets, the β-cell population is clustered at the core surrounded by a mantle of other endocrine cell types. Interestingly in humans (and non-human primates), Meier et al recently reported that human islets at 13-25 post-natal display a similar architecture (14). In mature human islets, however, islet cell types are dispersed without a clear pattern of cellular subdivisions (15). This reported cellular topography of human islets has functional implications. As an example, the β-cells in rodent islets are electrically coupled to each other via cell-cell contacts (e.g. gap junctions, connexins, cadherins, ephrins), resulting in synchronous

Ca2+ oscillations that lead to pulsatile insulin secretion (discussed further below). Ca2+ recordings from whole human islets do not show the typical coordinated oscillatory patterns

(16;17); therefore, human β-cell activities may be functionally independent. Moreover, given the larger proportion of α-cells in humans, it has been suggested that α-cells may play a more integral role in the overall activity of the human islet than in rodent islets (15).

Intercellular communication between endocrine cells can occur via cell-cell contacts, vascular circulation and/or autocrine/paracrine mechanisms (e.g. insulin regulates the -cell as an example of autocrine action, and insulin inhibits glucagon secretion, and somatostatin inhibits both insulin and glucagon secretion as examples of paracrine actions). It has been proposed that the direction of the microcirculation establishes an intra-islet signaling order but this is based on the assumption of the mantle-core arrangement of endocrine cells as found in rodents. Non-specific localization of endocrine cells in the human islet, where cells are randomly ordered along the blood vessels, argues against the anatomical basis for an order in paracrine signaling driven by the direction of intra-islet blood flow. This observation, however, does not diminish the importance of the islet vasculature. Afterall, it is critical, not only for

7 delivery of oxygen (especially for the high oxygen demands from the β-cells) and other nutritional (e.g. glucose, fatty acids) and humoral factors (e.g. glucagon-like peptide-1 (GLP-1), insulin-like growth factor-1 (IGF-1)) that regulate islet function to produce timely responses to changes in plasma glucose concentration via release of hormones into the circulation. Although it is crucial to dissect the regulators of islet functions towards better understanding of integrated physiology as a whole, this review will narrow its focus to β-cell biology.

1.2.1 Pre- and post-natal β-cell growth and function

Rather than being static throughout life, total -cell mass fluctuates in response to various physiological and pathological challenges. These changes are achieved by regulation of cell number (e.g. via -cell proliferation, -cell apoptosis, and/or islet neogenesis) and/or by changes in cell size. The contribution of each of these determinants is dependent on the specific condition, such as the presence of metabolic demand, growth factors, inflammatory cytokines, and age. Although direct determination of -cell mass is difficult in humans, due to an inability to image the -cell in vivo, changes in -cell mass have clearly been demonstrated in rodents, under a variety of physiological, pathophysiological and experimental conditions.

A morphometric study by McEvoy and Madson discovered that the number of -cells increases rapidly after day e20 in rats (18), doubling during the last 2 days of gestation (19). These changes occur in parallel with changes in expression of the insulin-like growth factors (IGF) and their receptors (20-24), and both gain- and loss-of-function analyses in rodent models have implicated IGF signaling in the expansion of -cell mass during fetal life. However, other peptide growth factors (e.g. vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF-7 and FGF-10)) also contribute to endocrine cell formation and islet expansion (25-27). Moreover, the rapid pre-natal increase in -cell mass is ascribed not only to high mitotic activity, which contributes 10-20% of total -cell growth, but

8 also to differentiation of putative precursor cells to -cells (neogenesis), representing the remaining 80% (19). In human fetal pancreas during 20 weeks of gestation, the contribution of

-cell replication to the rapid expansion of -cell mass is also relatively low (28-30). Although it is apparent that differentiation from precursor cells is one mechanism of increasing -cell mass in fetal life, another source of -cells is from a pool of proliferating “stem cells” that express the cytoskeletal protein keratin; these cells are restricted to during fetal life and generate protodifferentiated cells that co-express insulin and cytokeratin during islet morphogenesis

(31;32). Such observations indicate the possibility of conversion of ductal cells into -cells.

Interestingly, this observation is not limited to rodents; a similar conclusion is also drawn in human fetal pancreas where immature -cells also express the ductal cell marker, cytokeratin 19

(33). There are no studies to date that quantify the relative contribution of each of the aforementioned pools of progenitor cells to the expansion of -cell mass in fetal development/life.

The expansion of -cell mass continues post-natally until weaning, but at a reduced rate compared to the fetal stage (18;34). The new cells derive not only from -cell replication but also from the recruitment of undifferentiated -cell precursors (35). There is also a substantial remodeling of the pancreas that occurs in the neonatal rodent, with a transient wave of apoptosis occurring in the -cells between 1-2 weeks of age (36;37); however, this does not reduce -cell mass as new -cells compensate for the loss. This process is thought to be critical for replacement of „immature‟ -cells that exhibit slow glucose-stimulated insulin release by

„mature‟ -cells that respond more appropriately to changes in glycemia.

The rate of -cell replication in rodents is approximately 18% new cells per day during the perinatal period, and this drops to ~2-3% new cells per day in the adult (36). Thus, as -cell

9 mass increases linearly with age and body weight in rodents, this is due to an initial increase in cell number (hyperplasia) that is followed by an increase in cell size (hypertrophy) (38-40).

Nevertheless, if the rate of mitosis in adult pancreatic -cells continued without cell loss, it has been estimated that -cell mass would double every month (41). Therefore, cellular apoptosis is required to maintain -cell mass in steady-state. Indeed, -cells have been determined to have a finite life span of ~60 days, and they undergo apoptosis at a frequency of 0.5% for every 6 hrs in the 3-month old rat (36;38). Finally, although it has been suggested that -cell replication is the only source of new -cells in the adult rodent (42), other studies have demonstrated the existence of potential -cell „precursors‟ or „stem cells‟ in the pancreas, including the ductal epithelium (43;44). Further studies will clearly be required to resolve this issue.

What of -cell function with respect to its ability to secrete insulin appropriately in response to different glycemic challenges? It is well-established that in mature -cells, glucose enters via GLUT2 (45) and is quickly phosphorylated by the high Km rate-limiting enzyme glucokinase (GK) (46-48). Glucose metabolism in the β-cell results in a series of intricate intracellular events involving an increase in the ATP/ADP ratio, depolarization of the plasma

+ 2+ membrane by closure of the ATP-sensitive K (KATP) channels, and influx of Ca via voltage- dependent Ca2+ channels. It is generally accepted that this increase in cytosolic Ca2+ triggers exocytotic fusion of insulin granules via soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) machinery involving the formation of a protein-protein complexes

(49), although the precise mechanism(s) by which Ca2+ triggers granule fusion remains somewhat unresolved. This represents the mechanism underlying the first phase of insulin release; however, the cause of the second phase of insulin release is relatively unclear. It is generally accepted that this is modulated/mediated by KATP channel-independent pathways (50-

52). In fact, KCl and other nonnutrient secretagogues can induce a first-phase type release,

10 while only fuel-type secretagogues, such as glucose, can produce a sustained second-phase insulin release (53). Moreover, the first- and second-phase events also differ in their requisite

SNARE machinery proteins (49). Nevertheless, this signature biphasic secretion pattern does not appear until after birth, as a low and „sluggish‟ response to glucose was reported in rat islets at days 19.5-20 of gestation (54-64). In mice, Rozzo et al found that β-cells at birth are not responsive to stimulatory levels of glucose and they show a high basal insulin release (65). This observation coincides with a depolarized membrane potential in the -cells. At postnatal day 2, the basal insulin secretion returned to levels seen in adults, coinciding with gradually hyperpolarized membrane potentials, primarily as a result of increased expression of KATP channels.

Pancreatic β-cells were once viewed as terminally differentiated cells that were incapable of proliferating further. However, differentiated -cells display remarkable plasticity throughout adult life depending on physiological or pathophysiological states with varied metabolic demands. Hence, -cell mass can change in response to a number of stimuli, including pregnancy, insulin resistance, hyperglycemia, hypercaloric feeding, and pancreatectomy, through alterations in the rates of proliferation, apoptosis and/or neogenesis.

1.2.2 Adaptive β-cell growth and function in physiological and pathophysiological states

Pregnancy. Increases in -cell mass during pregnancy are required to facilitate maternal nutrient supply to the fetus, and failure to compensate can lead to gestational diabetes.

In rats, -cell mass increases by 3-fold during pregnancy, due to increases in both hyperplasia and hypertrophy of the -cell (66;67). Similar observations have also been made in pancreatic autopsy samples from pregnant humans (66). It currently remains unclear as to whether -cell neogenesis also contributes to these increases in mass. Pregnancy also causes functional changes in the -cell, including increased glucose sensitivity, insulin biosynthesis, and glucose

11 metabolism (68), which have been attributed to increased circulating levels of placental lactogen and/or prolactin (69-71). -cell mass returns to normal levels in the post-partum period (72), in association with decreases in -cell replication and size, as well as an increase in -cell apoptosis (73;74). It is well established that exposure of rat islets in vitro to prolactin or lactogen stimulates DNA synthesis and insulin production (69). Mitogenic effects of prolactin and placental lactogen have also been demonstrated in cultured neonatal rodent and human islets

(70;71). Overexpression of placental lactogen in the murine β-cell similarly causes a dramatic increase in β-cell proliferation and β-cell mass, even resulting in hypoglycemia (75). Similarly, global deletion of the prolactin receptor in mice reduces β-cell mass and impairs insulin secretion (76). Moreover, pregnant mice heterozygous for the prolactin receptor null mutation exhibited reduced β-cell proliferation, decreased β-cell size and mass, and impaired glucose tolerance (77). In this case, an interesting observation arose, such that the maternal genotype had a significant effect on the phenotype of the female offspring, suggesting that in utero exposure to impaired glucose homeostasis alters the epigenetic memory of the offspring‟s β- cells (78;79). These studies provide evidence for a direct regulation of β-cell growth by prolactin and placental lactogen. Although the mechanisms underlying these processes are not fully understood, one recent study pointed out that serotonin signaling is critical in driving the changes in β-cell mass during pregnancy. Lactogenic signaling increases the expression of the serotonin synthetic enzyme, tryptophan hydroxylase-1, and serotonin production rises sharply in the β-cell during pregnancy or after treatment with lactogens in vitro. Loss or pharmacological blockage of the serotonin receptor in the β-cell of pregnant female mice also prevents pregnancy-induced increases in β-cell replication and expansion of β-cell mass (80). Recently,

Butler et al reported that there is adaptive increase in β-cell numbers in pancreata collected from autopsies of pregnant humans. Although the magnitude of this increase is limited relative to

12 rodents, the authors observed a significant increase in small islets and insulin-positive ductal cells, without changes in β-cell replication or apoptosis. Such observations suggest that in humans, unlike rodents, the adaptive β-cell response in pregnancy is achieved via an increase in

β-cell number and this is associated with an increase in β-cell „neogenesis‟ (81).

β-cell function is shown to be enhanced during pregnancy in rodents since the increase in insulin secretion cannot be explained by β-cell mass expansion alone (82). Moreover, Butler et al reported an increase in β-cell mass from autopsies of pregnant women (81), but such expansion is insufficient to meet the reported two-fold elevation in insulin secretion reported by another study (83), suggesting that enhanced β-cell function is responsible. Indeed, Nielsen et al observed an increase in circulating levels of C-peptide during pregnancy in T1DM women, and this increase was associated with improvement in glycemic control during pregnancy, suggesting improved β-cell function during pregnancy (84). Although the mechanism behind this functional adaptation is unclear, these studies suggest that both an increase in β-cell mass and function are necessary for metabolic adaptation during pregnancy.

Insulin Resistance. Insulin resistance represents a second adaptive condition that is associated with alterations in -cell mass, due to increased metabolic demand for biologically effective insulin. Mice with ablation of one allele of the insulin receptor, IRS-1, or both of these genes have compensatory growth of -cell mass (85). These mice have normal body weights and maintain normal glucose levels in the face of marked insulin resistance up to 6 months of age (85). However, plasma insulin levels are increased by 400-fold compared to those of wild- type animals. Furthermore, mice with double heterozygosity of the null genes for the insulin receptor and IRS-1 demonstrate that a 40-fold increase in -cell mass, most notably due to increased islet size rather than number (85).

Although the studies mentioned above show the important role of insulin receptor signaling in -cell growth and homeostasis, the precise role of insulin in -cell mass expansion is unclear as insulin can exert direct effect on -cell mass but may also function indirectly through an alteration in glycemia (see below). One study clarified this issue by infusing insulin while maintaining normal glucose levels. In these euglycemic-hyperinsulinemic rats, a 50% increase in -cell mass was observed (86), providing evidence of a direct effect of insulin to promote -cell expansion in vivo independently of its modulating effect on plasma glucose concentrations. Interestingly, in these hyperinsulinemic rats, activation of the neogenic process contributed more to the expansion of -cell mass, as the rate of -cell replication was dramatically decreased to 90% of that in control rats.

What are the mechanisms underlying the increase in β-cell mass in response to the hyperinsulinemia that accompanies the insulin resistant state? To answer this, Kulkarni et al explored one possible candidate protein: PDX-1 (87). Based on in vivo and in vitro studies, this homeobox gene has been ascribed several fundamental roles in adult β-cells, including glucose sensing, insulin biosynthesis, and insulin exocytosis (88). Interestingly, while Pdx-1+/– isolated islets and dispersed β-cells have normal GSIS, Pdx-1 haploinsufficiency results in significant β- cell apoptosis (89). When PDX-1-heterozygous knockout mice were crossed with insulin receptor and IRS-1 double heterozygous knockout mice; the compensatory increase of -cell mass was completely abolished in association with increased -cell apoptosis (87). This is a strong indication that the PDX-1 transcription factor is not only important in -cell development but also in regulation of the adaptive response to hyperinsulinemia. This is, thus far, the only study demonstrating a mechanistic link between insulin resistance and subsequent changes in - cell mass. It can be hypothesized that the involvement of PDX-1 in adult -cell adaptation might indicate recapitulation of the embryonic -cell development to produce new -cells.

Hyperglycemia. Glucose is a stimulator of -cell growth in vitro and in vivo (90-92).

When rats are infused with glucose to achieve hyperglycemia, -cell numbers increase by approximately 50% within 24 hours but return to basal levels 7 days after cancellation of the glucose infusion (86). The -cell mass of high glucose-infused rats was increased by 65% compared with that of saline controls without changes in -cell size. Since the rate of -cell replication decreased, neogenesis from a pool of precursor cells must be responsible for the expansion of -cell mass (86). However, in another study, Topp et al saw a doubling of -cell mass over the 6 days of glucose infusion and the authors attributed this expansion to three stages of adaptation: neogenesis; hypertrophy and hyperplasia; and further hyperplasia coupled with a second wave of neogenesis (93). Furthermore, -cell function was 4-6 times higher than in control rats throughout the experiment. Therefore, the adaptation of -cell mass to metabolic perturbations can also be witnessed during chronic hyperglycemia.

What of apoptosis of the -cells in the presence of high glucose? It has been demonstrated that, in rat islets in vitro, incubation with glucose for one week promotes -cell survival by reducing cell death (94). This protection is dependent on protein synthesis as blockage leads to -cell apoptosis. These findings suggested that the normal protein synthetic activity of -cells suppresses a constitutively-expressed apoptotic program, and that the survival of -cells depends on their production of factors which inhibit an endogenous suicide program

(94). However, high glucose concentrations have also been reported to stimulate -cell apoptosis. The sand rat, Psammonmys obesus, is considered a useful example of the detrimental effects of glucose on -cells. Through selective breeding, two strains of this rodents have been developed: one that is diabetes-prone and develops overt diabetes, and another strain that is diabetes-resistant with normoglycemia but hyperinsulinemia (95;96). In the diabetes-prone

15 animals, hyperglycemia as a result of a high calorie diet leads to an acute increase in -cell proliferation as measured by the presence of the proliferative marker, Ki67 (97). This compensation quickly dissipates after 7 days, as a progressive increase in the rate of -cell apoptosis takes over (97). Isolated islets from diabetes-prone sand rats demonstrates a linear increase in -cell apoptosis as glucose is increased from 5.5 to 33 mM (97). A similar trend is also observed in human islets: 1) -cell proliferation was reduced by 42% and 61% in presence of 11.1 mmol/L and 33.3 mmol/L glucose, respectively, and 2) -cell apoptosis showed a 2.4- and 3.5-fold increase at 11.1 mmol/L and 33.3 mmol/L glucose, respectively (98;99).

The detrimental effect of excessive glucose concentrations is referred to as

'glucotoxicity' although the mechanism behind this effect on the -cell remains unresolved. A few possibilities have been proposed, including the involvement of cytokines such as interleukin

(IL)-1 (100). Another recently proposed mechanism involves chronic activation of the nutrient-sensing serine/threonine protein kinase, mammalian Target of Rapamycin (mTOR) in

-cells where its activation triggers serine/threonine phosphorylation of IRS-2. Serine/threonine phosphorylation of IRS-2 marks it for degradation and this may lead to increased ß-cell apoptosis (101). The most popular hypothesis, however, is the oxidative stress concept wherein the generation of reactive-oxygen species (ROS) as a consequence of chronically-increased glucose metabolism in -cells causes β-cell dysfunction and apoptosis (102). Of equal importance, Porte and Kahn argue for the role of glucose-induced pro-insulin overproduction/misfolding and islet amyloid-associated peptide (IAPP; or amylin), in promoting

ER stress in the -cells (103).

Obesity. Obesity represents one of the major risk factors for development of T2DM and is thought to confer this increased risk via its strong association with insulin resistance

(104;105). Therefore, similar to the discussion pertaining to insulin resistant states, the

16 proposed concept also applies here: pancreatic β-cells adapt, by increasing function and mass, to compensate for the peripheral insulin resistance that accompanies obesity. Free fatty acids

(FFAs) may also play a crucial role. When pancreatic islets from non-diabetic non-obese rats were cultured for 7 days with high amounts of long-chain free fatty acids (FFAs), islet hyperplasia was observed (106). Other studies argue for a crucial role of leptin in β-cell growth in obesity, as plasma leptin levels are found to be high in obese subjects, and the leptin receptor is present in islet cells (107). Moreover, leptin treatment in RINm5F and MIN6 β-cell lines, which express the leptin receptor, stimulates proliferation at low concentrations (1-5 nM), levels that are comparable to that found in obese subjects (108;109). However, leptin is probably not the main factor of β-cell expansion in obesity since Zucker fatty rats, an animal model of obesity due to hyperphagia as a result of genetic defect in the leptin receptor (110;111), display a dramatic increase in β-cell mass. Moreover, β-cell hyperplasia has also been demonstrated in db/db mice which similarly have a leptin receptor mutation (112;113). Leptin mutation in mice

(ob/ob) also causes obesity due to hyperphagia and insulin resistance, while two independent studies demonstrated that the β-cell mass expansion seen in ob/ob mice is due to increased islet volume but not number (114;115). Finally, normal rats placed on a high-fat diet for six weeks have a modest increase in body weight, mild insulin resistance and glucose intolerance.

Furthermore, β-cell size significantly increases by 30–40% in these animals (116). The above mentioned animal models of insulin resistance due to obesity display the incredible flexibility of the β-cell mass to adapt to changes in metabolic status whereby an increase in functional β-cell mass allows sufficient insulin release to maintain glucose homeostasis.

What of the β-cell mass in non-diabetic but obese humans? Such a study warrants investigation, however, it is extremely difficult. A recent and impressive article reported a careful analysis in post-mortem specimens from subjects with or without diabetes. Among the

17 non-diabetic obese individuals, the β-cell volume was found to be increased by approximately

50% in comparison with the lean subjects (117).

What of β-cell function under the condition of obesity? Pancreatic islets from rats treated with FFA display increased insulin secretion in response to glucose (106). In fact, fatty acids are required for normal β-cell function and are essential for both glucose- and non- glucose-induced insulin secretion (118;119). The role of fatty acids in insulin secretion has been exemplified by the use of genetically engineered mice with deletion of the fatty acid receptor GPR40, which exhibit a significant reduction in nutrient-induced insulin secretion

(120-123). Conversely, overexpression of GPR40 in the β-cells caused an increased insulin secretory response to both glucose and fat (124). Although these observations are not consistent, they suggest that fatty acids are required for an appropriate insulin secretory response to nutrients (125).

Experimental Diabetes. Insulin hyper-secretion has been found to occur in obesity, glucose intolerance and in response to fat infusion in humans as well as mice (126-130), and this is associated with increased β-cell mass. Due to such compensation, 70%-75% of obese individuals do not develop T2DM in response to the increased metabolic demand. The question remains, however, what of the remaining 25%-30% of the obese individuals who do develop

T2DM? There are limited data available on β-cell mass in humans, derived primarily from autopsy studies. A number of studies on T2DM subjects indicate a progressive decline in β-cell function preceding onset of diabetes (131-133). However, there is no means to establish whether this decline is due to impaired β-cell mass and/or declining function. Studies in T2DM subjects have revealed a 0-65% loss of β-cell mass (134-138), as well as β-cell apoptosis

(138;139). Butler et al demonstrated three critical observations: 1) obese individuals with

T2DM have a 63% deficit in relative β-cell volume compared with obese non-diabetics, 2)

18 obese individuals with impaired fasting glucose, a group at high risk of developing diabetes, have a 40% deficit in β-cell volume compared to obese control subjects with normal fasting glucose, and 3) the frequency of β-cell apoptosis is increased 3-fold in obese diabetics compared to obese non-diabetic subjects (138). These observations imply that the deficit in β-cell mass

(as reflected by β-cell volume) is an early and primary pathophysiological process in the development of T2DM.

Although it is impossible to establish the role of β-cell deficiency during the development of T2DM in humans, animal studies have provided invaluable insights. In addition to the „non-diabetes-prone‟ Zucker fatty rats, breeding has generated a second colony of

„diabetes-prone‟ Zucker fatty rats that develop diabetes due to marked β-cell apoptosis (140-

142). Hence, at 12 weeks of age, male diabetic Zucker rats increase their β-cell mass by two- fold compared to their lean counterparts (fa+/fa-) whereas non-diabetic Zucker fatty rats increase their β-cell mass by four-fold (143). The failure of adequate β-cell mass expansion in the diabetic Zucker rats does not seem to be attributable to alterations in β-cell replication and size as these variables remain unchanged; therefore, this observation implies that the increased rate of β-cell apoptosis must be the principle mechanism (144).

While the precise mechanisms that are responsible for the development of T2DM are not fully understood, it is generally accepted that multiple genetic defects under specific environmental conditions (e.g. diet) are required (145). Moreover, it is now becoming appreciated that different inbred murine strains harbor different susceptibilities for either obesity or diabetes. For instance, C57BL/6J mouse strain exhibits defective glucose tolerance and when fed with high-fat diets, they develop insulin resistance, increased fasting plasma glucose levels and, subsequently, diabetes (146-152). These changes are associated with defects at the level of the β-cell since C57BL/6J mice are actually more insulin-sensitive when compared with other

19 strains (i.e. AKR/J, DBA/2 or 129X1 mice) (129;153-156). Toye et al identified three genetic loci responsible for glucose intolerance in this strain of mice, one of which encoded the glucokinase enzyme (157). Furthermore, when we consider obesity and diabetes, expression of either the leptin gene (ob/ob) or the leptin receptor gene (db/db) mutation on the C57BL/6 strain results in massive obesity accompanied by insulin resistance with only transient diabetes, while the same mutations in the C57BL/KsJ strain produces initial obesity and insulin resistance followed by life-shortening diabetes (158-161). Together, these studies indicate the importance of the mouse strain on the resultant phenotype of a dietary challenge and/or particular genetic manipulation.

There is a growing interest in the role of the β-cell in the link between obesity and the development of T2DM which is pertinent to this discussion. Similar to the paradoxically unfavourable effects of chronic hyperglycemia on the -cell, fatty acids are toxic when chronically present in excessive levels. Lipotoxicity refers to such deleterious effects of fatty acids whereby metabolic products such as generation of ceramides from palmitate and other by- products that can induce oxidative stress, leading to β-cell dysfunction and apoptosis (162-166).

Alternatively, fatty acids can activate novel protein kinase C isoforms by production of intracellular long chain acyl-CoA, which can lead to serine/threonine phosphorylation of IRS molecules, in particular IRS-2 which is critical for β-cell survival (167-170). It must also be borne in mind that the current view of obesity as a inflammatory disorder is also garnering popularity (171). Hence, in obesity, adipokines are elevated in the circulation, including leptin, tumor necrosis factor (TNF ), and IL-6. Some of these cytokines can induce β-cell apoptosis through induction of signaling pathways that activate the transcription factor NF B through protein kinase I B or via the Janus Kinase-2/Signal Transducer and Activator of Transcription

(JAK/STAT) signaling pathway (172-174). Therefore, it is likely that obesity and increased fatty acid levels are detrimental to genetically susceptible individuals.

Experimental reduction of β-cell mass has also been a useful tool in understanding β-cell dynamics. This thesis will limit attention to two experimental approaches that provide excellent demonstrations of β-cell plasticity: non-surgical and surgical means to decrease β-cell mass.

Non-surgical methods of damaging the β-cell include the administration of toxins such as streptozotocin (175) and alloxan (176). Streptozotocin-induced destruction of β-cells in rats given intravenously on the day of birth reduces β-cell mass by approximately 90% in 48 h.

Intriguingly, twenty days later, ~40% of the normal β-cell mass is restored (177). Despite the restoration of a significant amount β-cell mass, these animals still develop glucose intolerance at six weeks of age. Therefore, in this model, β-cell replication is insufficient to regenerate functional mass to maintain normoglycemia (178;179). Moreover, the ability to regenerate β- cells declines during the first 5 days of life in rats and this is probably due to the lack of progenitor pools for neogenesis to produce more β-cells (180). Alloxan has also been used to create a model of β-cell damage. One study demonstrated an interesting β-cell dynamic by perfusing a part of the pancreas in the rat with alloxan, leaving the remaining portion spared of its toxic effects (181). The spared β-cells proliferate, whereas neogenesis of β-cells from duct cells leads to regeneration in the perfused part of the pancreas. Non-surgical means of pancreatic damage also provide an opportunity to study various external stimuli to induce changes in β-cell mass. For instance, one recent study used alloxan-induced β-cell destruction followed by application of gastrin and EGF. The authors found these hormones restore glycemic control in mice, with a β-cell growth rate of more than 30% per day, leading to a doubling of the β-cell population within 3 days (182). Regenerative growth induced by the

21 gastrin and EGF treatment led to the restoration of 30–40% of the normal beta-cell mass within 7 days and this regenerative effect was due to proliferating precursor ductal cells (183).

Partial pancreatectomy represents another model of tissue injury wherein β-cell regeneration has been studied in rodents. Surgical removal of part of the pancreas is followed by only limited regenerative growth, although the regenerative response is proportional to the amount of pancreas removed (184). Nevertheless, a β-cell mass compensatory mechanism occurs in pancreatectomized rodents. Hence, 60% pancreatectomy results in normoglycemia due to an increased β-cell mass several weeks after the procedure (185). Dor et al used a transgenic cell labeling approach to show that after two-thirds pancreatectomy, there is no evidence for formation of new β-cells by differentiation from insulin-negative progenitor or stem cells (186). In contrast, Bonner-Weir et al reported the presence of β-cell neogenesis from proliferating ducts after 90% pancreatectomy (187). Perhaps, the discrepancy is due to the extent of pancreatectomy (i.e., between 70 and 90% pancreatectomy). Indeed, such differences in β-cell compensatory responses have been noted by others: there is a doubling of β-cell mass within one week after surgery in 90% pancreatectomized rats, whereas only a 30–40% increase over four-week period is observed following 60% pancreatectomy (188;189). Therefore, the regenerative response of the pancreas may be dependent on the amount of tissue damage and/or the requirement for metabolic compensation.

Most studies in relation to diabetes discussed thus far have been carried out in rodents, and in contrast to humans, they have high capacity for pancreas regeneration after partial pancreatectomy and a higher β-cell turnover. Does partial pancreatectomy provoke new β-cell formation and increased β-cell mass in humans? Donors with 50% pancreatectomy are associated with 25% risk of developing abnormal glucose tolerance or diabetes in the year after the procedure (190-193). Indeed, 43% of healthy humans who underwent hemipancreatectomy

22 have impaired fasting glucose, impaired glucose tolerance, or diabetes on follow-up. This strongly argues against compensatory β-cell expansion in response to experimental diabetes.

Indeed, Menge and colleagues reported two fundamental insights in humans: 1) β-cell mass and new β-cell formation are not increased after a 50% partial pancreatectomy, and 2) β-cell turnover is unchanged by a 50% partial pancreatectomy (194). It appears that humans demonstrate a restricted β-cell capacity to regenerate, and differences in β-cell behaviour between rodents and humans should be considered when evaluating new theories and treatment options to restore β-cell mass in patients with diabetes.

1.2.3 Pancreatic dynamics in response to growth factors

In addition to changes in β-cell mass in response to physiologic and pathophysiologic stimuli, rates of growth and death of the β-cells can also be manipulated by a number of growth factors (Table 1.1). Several factors have been identified as stimulators of β-cell growth, including hepatocyte growth factor (HGF), epidermal growth factor (EGF), insulin-like growth factors 1 and 2 (IGF-1, IGF-2), prolactin, gastric inhibitory peptide (GIP), parathyroid hormone- related protein, and glucagon-like peptide-1 (GLP-1). A full discussion of the effects of these different growth factors is beyond the scope of the present review, which is focused instead on

GLP-1. However, the reader is referred to several excellent reviews for more information (195-

197).

Table 1.1. Growth factors and their effects on β-cell behaviour.

Growth factor Model Species Proliferation Apoptosis Neogenesis Function Ref in vitro Activin islet rat n/a n/a n/a ↑ (198-200) human n/a n/a n/a ↑ (201) islet rat ↓ ↑ n/a n/a (201) Ad ALK7 INS1 rat ↓ ↑ n/a n/a (202) Ad ALK7 AR42J rat n/a ↑ ↑ n/a (203;204) Betacellulin islet fetal human n/a n/a n/a ↑ (205) AR42J rat n/a n/a ↑ n/a (203) INS1 rat ↑ n/a n/a n/a (206) RINm5F rat ↔ n/a n/a n/a (206) Betacellulin delta AR42J rat n/a ↑ ↑ n/a (207) 4 EGF islet mouse n/a n/a n/a ↑ (208) canine n/a ↓## n/a n/a (209) human ↑ n/a ↑# ↑# (210) βTC mouse n/a ↓## n/a n/a (209) INS1 rat ↑ n/a n/a ↑ (211) RINm5F rat ↑ n/a n/a ↑ (211) FGF islet GK rat n/a n/a n/a ↑ (212) db/db mouse n/a n/a n/a ↑ (212) rat n/a ↓** n/a n/a (212) INS1 rat n/a ↓** n/a n/a (212) HGF islet human ↑ n/a n/a n/a (213-217) human ↔ n/a n/a n/a (218) human n/a ↓** n/a n/a (219) INS1 rat ↔ n/a n/a n/a (206) rat ↑ n/a n/a n/a (220;221) RINm5F rat n/a ↓* n/a n/a (222) Duct rat n/a n/a ↑ n/a (223) AR42J rat n/a n/a ↑ ↑ (224;225) Gastrin islet rat n/a n/a n/a ↔ (226) Growth hormone islet human ↑ n/a n/a n/a (227) rat ↑ n/a n/a n/a (227;228) RINm5F rat ↑ n/a n/a ↑ (226;229) Growth hormone- islet rat ↑ ↓ n/a n/a (230) releasing hormone INS1 rat ↑ n/a n/a ↑ (230) Insulin/IGFs pancreas rat n/a n/a n/a ↓/↑ (231) islet rat n/a n/a n/a ↑ (231) rat ↑ ↓ n/a n/a (232;233) human n/a ↓** n/a ↑ (234;235) MIN6 mouse ↓ ↑ n/a n/a (236) IR KO INS1 rat ↑ ↓** n/a n/a (237-239) Lactogens islet rat ↑ n/a n/a ↑ (69;70;240;241) human ↑ ↓ n/a n/a (70) INS1 rat ↑ n/a n/a n/a (242;243) KGF fetal human ↑ n/a ↑ ↑ (244) pancreas Parathyroid islet rat ↑ n/a n/a ↑ (mRNA) (245) hormone-related MIN6 mouse ↑ n/a n/a ↑ (mRNA) (246) protein INS1 rat ↑ n/a n/a ↑ (mRNA) (247) SDF-1 MIN6 mouse n/a ↓**,# n/a n/a (248) INS1 rat ↔ ↓**,# n/a n/a (248) TGFα islet rat ↔ n/a n/a ↔ (249;250)

INS1 rat ↔ n/a n/a n/a (206;239) RINm5F rat ↔ n/a n/a n/a (206) TGFβ islet rat n/a n/a n/a ↔** (251) rat ↔ n/a n/a ↑ (250;252) rat n/a ↓**,^ n/a n/a (253;254) islet monkey/human n/a n/a n/a ↑ (255) DN Smad3 islet human n/a ↓ ↓ ↓ (256) MIN6 mouse n/a n/a n/a ↑ (257) INS1 rat n/a n/a n/a ↑ (mRNA) (258) RINm5F rat n/a ↓** n/a ↑** (259) VEGF islet mouse n/a n/a n/a ↔ (260) mouse n/a n/a n/a ↑ (261) islet human n/a ↓** n/a ↑ (262) AD hVEGF

Growth factor Model Species Proliferation Apoptosis Neogenesis Function Ref in vivo Activin FSTL3 KO mouse ↑ na ↑ na (263) tTA-PDX1- mouse n/a n/a n/a ↓ (264) Smad7 ALK7 KO mouse ↑ ↔ n/a ↑ (265) Pancreatic rat ↑ n/a ↑ ↑ (266) duct cells in STZ rats STZ rat ↑ ↔ ↑ n/a (267) betacellulin STZ rat ↑ ↔ ↑ n/a (267) STZ rat ↔ n/a ↔ ↑ (insulin (268) production) Alloxan mouse ↔ n/a ↑ n/a (269) 90% PT rat ↑ ↔ ↑ n/a (270) STZ mouse ↑ ↔ ↑ ↔ (271) Ad hBTC mouse ↑ n/a ↑ n/a (272;273) ICR/STZ Ad mBTC mouse ↑ n/a ↑ ↑ (274) STZ BTC Tg mouse ↑ ↔ n/a ↑ (275) betacellulin delta4 STZ rat ↑ ↔ ↑ ↑ (207) EGF PDX-1-DN mouse ↓ ↔ n/a ↓ (276) EGFR Heparin- mouse ↑ n/a ↑ n/a (277) binding EGF gene injection ins-EGF mouse ↑ n/a n/a ↑ (278) FGF PDX-1- mouse ↓ ↔ n/a ↓ (gradual (279) FGFR1c loss of (FRID1) GLUT2) FGF21 mouse (db/db) ↑ ↔ n/a ↑ (212) injection HGF RIP-HGF Tg mouse ↑ ↓*** n/a ↑ (280;281) RIP-HGF Tg mouse/HFD ↔ ↑ n/a n/a (282) HGF cDNA mouse/STZ ↑ ↓ na ↑ (283) injection Ad HGF ↔ ↓ na ↑ (mRNA) (284) injection RIP-Cre c- mouse ↔ n/a ↑ ↓ (221) Met KO RIP-Cre c- mouse ↓ n/a n/a ↓ (285) Met KO Ad HGF IT mouse/STZ ↑ ↓ n/a ↔ (285) Ad HGF IT rat/STZ ↑ ↓ n/a ↑ (286) Ad HGF IT monkey ↔ ↓ n/a ↑ (287) Gastrin Gastrinomas human ↑ n/a n/a ↔ (288-291) Diabetic mouse ↔ ↓ ↑ ↑ (210;292-295) NOD

(combination with GLP-1 or EGF) Alloxan- mouse ↑ n/a ↑ n/a (296) treated (combination with EGF) Pancreatic rat ↔ ↔ ↑ n/a (297;298) duct ligation TGF /ins- mouse n/a n/a n/a ↑ (299) Gastrin Growth Hormone GH- rat ↑ n/a n/a n/a (300;301) secreting tumors JI-36 agonist mouse/STZ n/a n/a n/a ↑ (230) treated IT GHR KO mouse ↓ n/a n/a ↓ (302) Insulin/IGFs IR-/- mouse ↔ n/a n/a ↑ (303-305) IGF-1R-/- mouse ↓/↔ n/a n/a (305;306) IRR-/- mouse ↔ n/a n/a ↔ (307) IR-/-/IGF-1R-/- mouse ↔ n/a n/a n/a (305) ins1-/-/ins2-/- mouse ↑ n/a n/a n/a (308;309) IGF-1-/-/IGF-2-/- mouse ↔ n/a n/a n/a (305) IGF-1-/- mouse ↔ n/a n/a ↔ (310) βIRKO mouse ↓ n/a n/a ↓ (311;312) βIGF-1R-/- mouse ↔ n/a n/a ↓ (313;314) βDKO mouse ↓ n/a n/a ↓ (315) LID mouse ↑ n/a n/a ↑ (316-318) PID mouse ↑ n/a n/a ↔ (319) βIGF-1 mouse ↔ n/a ↔ ↔ (320;321) overexpress βIGF-2 mouse ↑ n/a n/a ↑ (322) overexpress IGF-2 Tg mouse ↑ ↓ ↑ (323) Lactogens RIP-PL1 Tg mouse ↑ n/a n/a ↑ (75) PRLR KO mouse ↓ ↔ n/a ↓ (76) KGF ins-KGF mouse ↑ n/a n/a ↑ (278;324) elastase- mouse ↑ n/a ↑ ↓ (325) KGF Parathyroid RIP-PTHrP mouse ↑/↔ ↔ n/a n/a (326) hormone-related Tg protein TGFα Pancreatic rat n/a n/a ↑ n/a (298) duct ligation TGFβ tTA-PDX-1- mouse n/a n/a n/a ↓ (264) Smad7 elastase- mouse ↑ n/a ↑ n/a (327) Smad4 DN Smad3 KO mouse ↔ n/a n/a ↑ (255) VEGF PDX-1-Cre mouse ↑ n/a n/a n/a (328) VEGF PDX-1-Cre mouse ↔ n/a n/a ↓ (329) VEGF fl/fl RIP-Cre mouse ↔ ↔ n/a ↓ (260) VEGF fl/fl RIP-Cre mouse ↔ n/a n/a ↔ (330) VEGF fl/fl HFD

* challenged with FFA, ** challenged with TNF , IL-1β, IFNγ, *** challenged with STZ, # challenged with thapsigargin, ^ challenged with spleen cells from diabetic BB rats.

Abbreviations used: Ad = Adenoviral-mediated, DN = dominant negative, IT = islet transplantation, KO = knockout, PT = pancreatectomy, STZ = streptozotocin, Tg = transgenic.

GLP-1 is a peptide hormone secreted by the intestinal L-cell in response to nutrient ingestion (331). Synthesis of GLP-1 occurs via tissue-specific post-translational processing of proglucagon in the L-cells of the distal small intestine and colon (332-334). Although this gene also encodes for several additional hormones, including the related peptides glucagon and GLP-

2 (335), none of these peptides is known to affect β-cell mass: for this reasion, the focus of this review will therefore remain on GLP-1. Several factors that regulate proglucagon gene expression in the intestine have now been elucidated (336). However, a very recent study has demonstrated a link between risk for T2DM and a variant in the gene for the transcription factor,

TCF4 (TCF7L2) (337), a demonstrated regulator of proglucagon gene expression in the L-cell

(338). This finding has suggested a possible causative relationship between proglucagon gene expression and the development of this disease, although no relationship between TCF7L2 polymorphisms and circulating levels of GLP-1 have been identified to date (339). However, circulating levels of GLP-1 are reduced in patients with T2DM, and this has been ascribed to impaired secretion from the intestinal L-cell (340). The biological consequences of such a reduction in GLP-1 levels are discussed below.

The physiologic role of GLP-1 has been mainly elucidated through the administration of

GLP-1 receptor antagonists to normal subjects (341), as well as through studies on mice lacking the GLP-1 receptor (342). Together, these actions have been found to be largely anti-diabetic in nature, and include but are not limited to, stimulation of glucose-dependent insulin secretion, enhancement of proinsulin gene expression, suppression of glucagon release, and inhibition of both gastrointestinal motility and further food intake [reviewed in (343;344)]. These diverse biological effects of GLP-1 are mediated through the seven-transmembrane G-protein coupled

GLP-1 receptor, which has a tissue distribution consistent with the biological actions of its ligand (345-348). When taken together, therefore, the biological actions of GLP-1 provide a

28 physiological „brake‟ to glycemic excursions following nutrient ingestion. Consistent with this notion, loss of GLP-1 receptor signaling is associated with reduced glucose tolerance following a meal (341;342).

Because of its physiological role in the regulation of post-prandial glycemia, GLP-1 actions have also been examined following exogenous administration. Since the first demonstration that GLP-1 stimulates insulin release in normal humans (349), many groups have shown that GLP-1 treatment acutely (350-352) and chronically (353;354) normalizes plasma glucose concentrations in patients with T2DM. However, one of the major drawbacks of GLP-1 administration is its short biological half-life, which is less than 2 min, due to rapid proteolytic cleavage by the protease, dipeptidylpeptidase IV (DP IV) (340;355). This has led to the development of long-acting GLP-1 analogs, degradation-resistant GLP-1 receptor agonists, and

DP IV inhibitors for use in patients with T2DM (356). One example of such an agent is exendin-

4 (EX4), a DP IV resistant GLP-1 receptor agonist that was originally isolated from the salivary glands of the lizard Heloderma suspectum (357;358). Excitingly, long-term administration of

EX4 to patients with T2DM reduces both HbA1c levels and body weight (359), and the FDA has approved the use of EX4 (exenatide; ByettaTM) for use in T2DM (360).

GLP-1 increases β-cell function and growth. The first identified biological actions of

GLP-1 were the enhancement of insulin biosynthesis as well as glucose-dependent insulin release. This characteristic has been crucial in the development of incretin-based therapeutics, as the glucose-dependency greatly reduces the risk of hypoglycemia. The cellular mechanisms of GLP-1‟s glucose-dependent insulintropic effects include the following:

+ 1. K ATP channels: GLP-1 binds to the GLP-1 G protein-coupled receptor and activates

adenylate cyclase, which generates the intracellular second messenger 3′–5′-cyclic

adenosine monophosphate (cAMP). A number of studies have shown that GLP-1 causes

+ closure of K ATP channels and thereby facilitates membrane depolarization which

+ induces insulin release (361-365). The mechanism underlying the effect on K ATP

channels is thought to involve cAMP-dependent activation of protein kinaseA (PKA) as

+ inhibition of PKA reverses the effects of GLP-1 on K ATP channels (361;363;366).

+ Furthermore, in mice engineered to lack K ATP channels, GLP-1 -induced insulin

secretion is diminished (367;368).

2. Intracellular Ca2+ levels: A handful of studies have demonstrated that activation of PKA

leads to a GLP-1-induced increase in voltage-dependent Ca2+ channel (VDCC) activity

resulting in increased Ca2+ entry into β-cells (364;369-371). Additional Ca2+ is released

from intracellular stores of endoplasmic reticulum through GLP-1-stimulated PKA and

cAMP-regulated guanine nucleotide exchange factor-II (Epac2, also termed cAMP-

GEFII) to sensitize Ca2+ channels (ryanodine receptors) in the ER (372-374). The

process of intracellular Ca2+ release is thus initiated by the transient increase in calcium

entering the cell through VDCCs, resulting in further increases in intracellular Ca2+

through Ca2+-induced Ca2+ release (373-376). This process has also been shown to

+ affect mitochondrial ATP production, leading to further effects on K ATP channels in a

positive feedforward direction (377;378). Moreover, opening of the VDCC allows

exocytosis of the “readily releasable pool” of insulin granules (which comprise only

<1% of the insulin-containing granules) (369;379;380). The remaining fraction of

insulin-containing granules must be primed and mobilized, and GLP-1 has been

demonstrated to influence these steps in a PKA- and Epac2-dependent fashion

(369;381;382). The increased availability of insulin-containing granules for exocytosis

has been estimated to account for as much as 70% of the insulinotropic activity of GLP-

1 (364;383).

+ + 3. K v channels: K v channels are essential for restoration of the cell membrane potential

following depolarization, thereby limiting Ca2+ entry and subsequent exocytosis of

+ insulin (384). GLP-1 receptor activation has been shown to inhibit K v channel currents

in rat pancreatic β-cells and this effect appears to be both PKA- and PI3-kinase-

dependent (385;386).

4. Gene expression: GLP-1 also acts synergistically with glucose to promote insulin gene

transcription, mRNA stability, and biosynthesis via activation of cAMP/PKA-dependent

and -independent signaling pathways (387-390). Nuclear factor of activated T-cell

(NFAT) may also be an important mediator of GLP-1-induced insulin gene transcription

(391;392). Furthermore, GLP-1 increases Pdx-1 gene transcription and binding of PDX-

1 to the insulin gene promoter (393). β-cell-specific inactivation of the Pdx-1 gene in

vivo or in vitro results in loss of the GLP-1-dependent effects on pancreatic β-cell

function (388;394).

One of the more recent and exciting areas of research on the biological actions of GLP-1 is based on observations that GLP-1 and EX4 increase β-cell mass via stimulation of β-cell neogenesis and proliferation, and suppression of β-cell apoptosis. Although most such studies to date have been conducted in rodents models and in human islets, the findings lend some hope to the notion that long-term GLP-1 therapy may prevent or even reverse the progressive loss of

β-cell mass that occurs in patients with T2DM (138).

A wide variety of studies have demonstrated that administration of GLP-1, its analogs or its receptor agonists enhances β-cell mass, in normal rats and mice (388;395-397). Furthermore,

GLP-1-induced increases in β-cell mass have been shown in animals under metabolic stress, including aged, glucose-intolerant rats (398), rats undergoing adaptation due to partial pancreatectomy (396), obese ob/ob and db/db mice, as well as fa/fa, Goto-Kakizaki (GK) and

Sand rats (397;399-403), and neonatal streptozotocin-treated diabetic rats (404;405).

Immunohistochemical examination of the pancreata from these animals has revealed that the trophic effects of GLP-1 are mediated through enhancement of β-cell proliferation and neogenesis, as well as through suppression of β-cell apoptosis.

A number of different approaches have been taken to elucidate the mechanisms by which GLP-1 enhances β-cell mass. In studies conducted in vivo, GLP-1 has been shown to augment the increased levels of various pro-survival proteins, such as Akt/PKB and p44 MAPK

(400), and to reduce the activation of pro-apoptotic proteins, including caspase-3 (400;401).

Investigations using IRS-2 knockout mice also implicated a role for this protein in the cell survival effects of EX4, as EX4 treatment failed to arrest the progressive β-cell loss that occurs in IRS-2-/- mice (406). However, elucidation of the intracellular mechanisms underlying these effects requires more detailed in vitro approaches using isolated β-cells, islets and/or insulin- producing β-cell lines.

Treatment of β-cell lines with GLP-1 or its receptor agonists increases proliferation (407-

409). A number of different signaling pathways have been implicated in this effect of GLP-1 on

β-cells, including the atypical PKC isozyme, PKC (410), the epidermal growth factor receptor

(409), the PI3-kinase/Akt signaling pathway (400;407), FOXO1(411), and CREB/IRS-2 (412).

It remains to be established whether these pathways function in concert or independently to mediate these beneficial actions of GLP-1 on the β-cell.

The anti-apoptotic effects of GLP-1 have recently been explored in some detail. EX4 treatment directly reduces the extent of cellular apoptosis in purified rat islets exposed to cytotoxic cytokines (405), while INS-1 cells are protected from apoptosis induced by either staurosporine or cytokines (400;413). Treatment of MIN6 mouse β-cells with GLP-1 similarly reduces apoptosis caused by hydrogen peroxide (414), while GLP-1 reduces palmitate-induced

32 apoptosis in RINm5F cells (415). Finally, GLP-1 can also prevent apoptosis that is induced by activation of the calcium/ryanodine receptor pathways (416). The mechanism(s) underlying these effects has been shown to involve a large number of different signaling molecules, including cAMP (414;415), PI3-kinase/Akt (413;414) and p38 mitogen-activated protein kinase

(MAPK) (417), while the downstream effects of these pathways include inhibition of the proapoptotic caspase-3 (413;417) and/or calpain (416) pathways. Interestingly, GLP-1 treatment also reduces cytokine-induced necrosis in the INS-1E cells (413). This effect occurs in association with reduced expression of iNOS, and also requires the actions of Akt.

The effects of GLP-1 on β-cell neogenesis are very poorly understood. In vitro studies using undifferentiated pluripotential cells, such as pancreatic AR42J cells, fetal pig islet-like clusters, and undifferentiated human pancreatic progenitor or ductal cells, have demonstrated that GLP-1R agonists induce differentiation toward a β-cell- or islet-like phenotype (418-424).

One recent study has further demonstrated that GLP-1 enhances the level of expression of insulin in glucose-responsive, insulin-producing cells derived from mouse embryonic stem cells

(425). The precise mechanisms underlying these effects remain largely a mystery. However, the trophic actions of GLP-1 do appear to require expression of PDX-1, as EX4 was unable to increase β-cell mass in β-cellPdx-1–/– mice, as compared to β-cellPdx-1+/+ animals (388).

Furthermore, differentiation of pancreatic ductal/epithelial cells into β-cells by GLP-1 is facilitated by Pdx-1 expression (423;426). Although GLP-1 has been demonstrated to increase

Pdx-1 levels in vitro (407), Pdx-1 expression has variably been reported to be increased (398) or not affected (427) by EX4 treatment in vivo, making interpretation of these findings difficult.

Furthermore, whether the effects of GLP-1 to enhance β-cell mass are mediated through a recapitulation of β-cell development or alternative routes remains unclear, as a very recent study has indicated that GLP-1 does not enhance expression of pancreatic

BETA2/NeuroD/Neurogenin3 (Ngn3) in mice undergoing adaptation to partial pancreatectomy

(428).

Finally, of particular importance, the trophic actions of GLP-1 have also been demonstrated using human cells. In human islet cells treated for 5 days with GLP-1, enhanced preservation of islet morphology and reduced β-cell apoptosis were observed, in association with decreased expression of pro-apoptotic caspase-3 and increased levels of the pro-survival protein, bcl-2 (429). GLP-1 treatment also enhanced the conversion of human pancreatic ductal cells into β-like cells (424). Whether GLP-1 receptor signaling will promote cell survival in the scenario of islet transplantation in humans remains to be established. However, a more rapid reversal of hyperglycemia was found when mice were transplanted with islets that had been pre- treated with EX4 as compared to untreated islets (430).

It must be cautioned that not all growth factors confer the same straight-forward beneficial effects on the pancreatic β-cells. One prime example is HGF which is a potent β-cell mitogen and an insulinotropic agent in vivo and in vitro. HGF promotes β-cell survival against streptozotocin and in the hypoxic and nutrient-deprived environment present in the early hours after islet transplantation (281;283;287;431;432). Recently, Santangelo et al have shown that

HGF also protects rat insulinoma RINm5F cells from FFA-induced apoptosis (222). However, when HGF is overexpressed in the β-cell under the control of the rat insulin promoter, it facilitates β-cell death under high fat diet conditions (282). This detrimental effect of HGF was further confirmed in in vitro experiments in mouse and human primary β-cells where it exacerbates the apoptotic effect of palmitate in β-cells. Although this proapoptotic effect of HGF for the β-cell may appear paradoxical, HGF has been reported to induce or facilitate apoptosis in other cell types, although the exact mechanisms are unclear (433). In the light of this new

34 finding, HGF can act as an antiapoptotic or proapoptotic agent in the β-cell and is context dependent (i.e. cellular condition).

1.3 Canonical Wnt Signaling

Recently, several studies have implicated a link between Wnt mutations and the development of T2DM (337;434-436). Wnts are secreted lipid-modified glycoproteins (437) that serve as ligands for receptor-mediated signaling, and are well known for their roles during embryonic development including, but not limited to, cell fate determination, proliferation, and motility. These functions control the establishment of the primary axis and generation of the body plan during embryogenesis (438). In addition to regulating development, defects in Wnt signaling are implicated in tumourigenesis and human birth defects including spina bifida (438).

There are three known independent pathways of Wnt signaling, namely the „canonical‟ (cWnt), and the two „non-canonical‟, „Planar Cell Polarity‟ and „Wnt/Ca2+‟ pathways. These independent Wnt signaling pathways do not negate the possibility of cross-talk between each other and, in fact, there is evidence that non-canonical Wnt pathways antagonize cWnt signaling

(439). Finally, there are currently 19 known members of the Wnt ligand family and 10 Frizzled

(Frz) receptor isoforms with additional associated receptors, and there is currently no clear-cut answer to which isoforms contributes to the „canonical‟ and/or „non-canonical‟ components of the Wnt signaling paradigm. For simplicity, therefore, this thesis will focus on the more established „canonical‟ Wnt pathway (Figure 1.1).

Figure 1.1. Overview of the Canonical Wnt (cWnt) signaling network. The “OFF” state involves the absence of a ligand, and the degradation complex of Axin, APC, and GSK3β among other co-factors target β-catenin for ubiquitin-mediated degradation. Wnt binding to the

Frizzled receptor and LRP co-receptors (“ON” state) induces phosphorylation of LRP and recruitment of Axin, thus preventing the formation of the degradation complex. Dsh is also phosphorylated, and the degradation complex is inhibited, leading to accumulation of cytosolic

β-catenin. Accumulated β-catenin then translocates to the nucleus to interact with TCF/LEF family of transcription factors to initiate a set of genes, cWnt target genes.

In the absence of cWnt signaling, β-catenin is phosphorylated by a complex made up of the tumor suppressors axin and adenomatous polyposis coli (APC), and the enzymes glycogen synthase kinase 3 (GSK3), casein kinase 1 α (CK1α) and the recently identified Wilms Tumor

Suppressor (WTX) protein (440;441). Phosphorylated β-catenin is then recognized by the F-box protein β-TrCP, a component of an SCF (Skp1-Cullin-F-box) E3 ubiquitin ligase and is subsequently poly-ubiquitinated and degraded by the proteasome complex (Figure 1.1.). When a cWnt ligand binds to its cell surface receptor comprising the seven-pass transmembrane protein Frizzled (Frz) and low-density-lipoprotein-related protein 5/6 (LRP 5/6), the signal is transduced by two concurrent or sequential events: 1) Frz/Dishevelled (Dsh): the intracellular protein Dsh interacts with Frz and, through an unknown mechanism, becomes activated and blocks the degradation of β-catenin by bringing the cellular GSK3 inhibitor, GBP/Frat, into the degradation complex (442-444). Moreover, studies have also provided evidence for the recruitment of Axin and GSK3 away from the degradation complex, thereby allowing β-catenin stablization (445); and 2) LRP/Axin: recent studies have established that LRP6 is dually phosphorylated by CK1γ and GSK3, which promotes the binding of Axin. This interaction allows axin to be recruited away from the degradation complex to the membrane (446;447). β- catenin thus accumulates in response to cWnt signaling and translocates to the nucleus, where it complexes with lymphocyte enhancer factor/T-cell factor (LEF/TCF) and activates expression of Wnt target genes that include cell cycle kinase activator cyclin D1 and the transcription factors MYC and PEA3 (448-450). For a comprehensive list of Wnt target genes, please refer to http://www.stanford.edu/rnusse/pathways/targets.html. In pancreatic β-cells, the TCF4 transcription factor (encoded by the TCF7L2 gene) is a major form of TCF involved in downstream cWnt signaling responsible for the activation of growth-promoting genes in response to glucagon-like peptide-1 (GLP-1) receptor agonists (451).

1.3.1 cWnt signaling in pancreatic development

Expression of components of the cWnt signaling pathway such as Wnt ligand family members and various Frz receptors is documented in the developing mouse pancreas. Their expression as well as other modulators of Wnt signaling, LRP co-receptors and secreted

Dickkopf (Dkk) proteins also extends to mature mouse, rat, chicken, fish and human pancreas, as well as to human islets and rodent β-cell lines (452-455). TCF7L2 is also reported to be expressed in human and mouse islets and β-cell lines (338;451;456). Although not revealing biological significance, these obversations nonetheless imply that Wnt signaling is present and perhaps active during the pancreatic developmental process and in mature tissues. Nonetheless, several studies have established that a functional cWnt signaling pathway is active in islets during development through the use of a cWnt reporter mouse that expresses β-galactosidase under the control of the cWnt target gene, conductin/Axin2; β-galactosidase expression in the islets of these animals persists for at least 6 weeks after birth (457).

The role of cWnt signaling specifically in the β-cell is controversial as the majority of studies have established a role for cWnt signaling in exocrine pancreatic development, where the disruption of cWnt signaling results in an almost complete lack of the exocrine compartment. Mice that overexpress Wnt1 and Wnt5a under the control of the PDX-1 promoter show perturbed patterning of the foregut, including the pancreatic domain (452), causing a subsequent reduction in pancreatic size but also lack of islet formation. This is consistent with the observation that Wnt5a is required for islet cell migration in the developing mouse embryo (458). The impact of Wnt signaling on pancreatic endocrine development is less clear. However, although Murtaugh et al and Wells et al demonstrated that the endocrine pancreas of mice with conditional β-catenin knockout develops normally and is functionally intact (459;460), Dessimoz et al used a different β-catenin knockout approach and found a

38 reduction in endocrine islet numbers (457). The discrepencies in these observations cannot be readily explained, but interpretation must be exercised with caution since the use of PDX-1- driven Cre-recombinase system to knock out β-catenin in different strains of mice may result in different recombination efficiencies and expression times during pancreatic development. In support of this theory, Heiser et al demonstrated that expression of constitutively active β- catenin early in development prevents pancreatic development, but later expression results in a hyperplastic pancreas (461). Such observations strongly suggest a temporal role for cWnt signaling in regulating proper pancreatic maturation. It is interesting to note that β-catenin pancreas-specific knockout mice also have dramatic upregulation of a related protein, plakoglobin/γ-catenin (459). Although plakoglobin has not been shown to directly compensate for β-catenin in cWnt signaling, recent studies have suggested it can function independently to regulate the cWnt signaling pathway (462).

1.3.2 cWnt signaling in mature -cells

In addition to its role in pancreas development, the cWnt pathway is involved in β-cell growth and survival in the adult. Hence, activation of cWnt signaling via Wnt3A induces the proliferation of mouse islet and MIN6 cells in vitro, in association with up-regulation of pro- proliferative genes, including cyclin D1 and D2 (451). Furthermore, overexpression of degradation-resistant β-catenin in the mouse β-cell leads to normal pancreatic development with a significant increase in β-cell mass and function (463). Conversely, increasing the expression of axin, a negative regulator of cWnt signaling, impairs β-cell expansion with a corresponding decrease in cWnt-stimulated gene expression (463). Likewise, overexpression of a constitutively active form of GSK3β, also a negative regulator of the cWnt pathway, in the β- cells of mice decreases β-cell proliferation and mass, resulting in impaired glucose tolerance

(464). Consistent with these findings, Shu et al. showed that depletion of TCF7L2 mRNA in

39 human islets causes a decrease in β-cell proliferation, an increase in levels of apoptosis, and a decline in the levels of active Akt, an important β-cell survival factor (456). Similarly, expression of dominant-negative TCF7L2 in INS1 cells decreases proliferation rates while overexpression of TCF7L2 in mouse and human islets protects β-cells against glucotoxicity or cytokine-induced apoptosis (451;456).

Surprisingly, several studies have found that cWnt signaling may play a role in regulating the secretory function of mature β-cells. Mice lacking LRP5 showed impaired glucose tolerance due to reduced GSIS, while pretreatment of isolated islets with Wnt- conditioned media resulted in enhanced GSIS from wild-type but not LRP5-/- islets (465).

Consistent with the impaired GSIS, the steady-state levels of mRNAs for several important molecules [transcription factors (e.g. Tcf1, Tcf2, Foxa1, and HNF-4 ); glucose-sensing protein

(glucokinase); insulin-signaling proteins (insulin receptor, IGF-1 receptor, and IRS-2)] were profoundly decreased in the LRP5-/- islets (465). In contrast, overexpression of a soluble Frz8- cysteine rich domain (CRD)-IgG fusion protein, which functions as a Wnt signaling antagonist by inhibiting the binding of Wnt proteins to the Frz receptors (466), in the developing pancreatic epithelium of mice leads to grossly reduced pancreatic mass, but does not affect adult -cell function, suggesting that Wnt signaling is not critical for normal glucose metabolism and insulin secretion (466). The reason for the discrepancies between LRP5-/- and Ipf1/Frz8CRD mice is unknown, but further studies support the notion that cWnt signaling can enhance -cell function.

Hence, the observation that islets from animal models of diabetes have lower levels of inactive

GSK3β, indicating that an increase in GSK3β activity may be detrimental to β-cell function

(464). Schinner et al also reported that activating cWnt signaling via adipocyte-derived Wnt molecules increases insulin secretion in primary mouse islets and activates transcription of the glucokinase gene in both islets and INS1 cells (467). It is interesting to note that this effect was

40 found to occur only in the presence of PPARγ, indicating that there might be interplay between cWnt signaling and PPARγ in pancreatic β-cells (467). Finally, reducing levels of TCF7L2 by siRNA in isolated mouse and human islets decreases glucose-stimulated insulin secretion, expression of insulin and PDX-1, and insulin content (456) . When taken together, therefore, cWnt signaling appears to be generally beneficial for β-cell health.

1.3.3 Activation of cWnt signaling in pancreatic β-cells

Activation of the cWnt pathway in β-cells is not limited to cWnt ligands, as there are other non-Wnt related ligands or signaling pathways with crosstalk and protein-protein interactions that add further complexity to Figure 1.1. Numerous studies have demonstrated that growth factors such as insulin (468), IGF-1 (469), FGF (470), EGF (471), HGF (472), PDGF

(473), and parathyroid hormone (474) can activate cWnt signaling pathways in non-pancreatic tissues. Evidence for crosstalk in pancreatic β-cells has also been clearly defined by Liu and

Habener, who demonstrated activation of the cWnt signaling pathway by the incretin hormone,

GLP-1 in INS1 β-cells and isolated islets, such that GLP-1 and EX4 enhanced cWnt signaling and increased expression of the cWnt target genes, c-myc and cyclin D1 (451). The GLP-1- induced activation of cWnt signaling was found to be mediated through PKA, Akt and

MEK/ERK but was independent of GSK3 (451). Indeed, EX4 causes cAMP-activation of PKA, which could potentially directly activate β-catenin via direct phosphorylation of β-catenin to prevent its degradation (475). Moreover, EX4 enhances the interaction of TCF7L2 and β- catenin with the cyclin D1 promoter. The importance of this finding is further emphasized by the demonstration that inhibition of cWnt signaling via knockdown of β-catenin or overexpression of dominant-negative TCF7L2 blunts EX4-induced β-cell proliferation.

Moreover, transfection of dominant-negative TCF7L2 in INS1 β-cells inhibits the basal proliferation rate by 50% as compared to control cells expressing an empty vector (451). It is

41 interesting to note that INS1 β-cells express high basal levels of cWnt signaling activity, possibly due to high secretion of endogenous cWnt ligands and/or high expression levels of Frz receptors, and this is independent of PKA activity (451). Nonetheless, these observations clearly imply that both basal and GLP-1-mediated β-cell proliferation is dependent on active cWnt signaling.

There are several components of the c Wnt-signaling pathway that serve as potential modes of crosstalk with other signaling pathways in β-cell. For example, insulin signaling phosphorylates GSK3 via a PI3-kinase/Akt-dependent mechanism (476). A recent study has further demonstrated that treatment of gut endocrine cells with insulin increases levels of nuclear β-catenin and enhances TCF binding, indicating cross-talk between insulin and cWnt signaling (477), and confirming a mechanism demonstrated in several other cell types

(478;479). Since insulin is a β-cell growth factor, and inhibitors of GSK3 stimulate proliferation of INS1 β-cells and isolated rat islets (480), the promotion of β-cell proliferation by insulin could occur by regulating GSK3 activity.

Nuclear interaction of β-catenin with transcription factors is not limited to the LEF/TCF family. β-catenin can also bind to the FOXO family of transcription factors that are known to play a critical role in regulating β-cell behaviour (481). For instance, haploinsufficiency of

FOXO1 reverses diabetic the phenotype in heterozygous insulin receptor:Irs2-/- mice (482).

Conversely, transgenic expression of constitutively nuclear FOXO1 in β-cells exacerbates the phenotype of the insulin receptor heterozygous mice, in part by preventing β-cell proliferation

(483). Moreover, several studies have shown that FOXO1 and PDX-1 in the pancreatic β-cell appears to have a mutually exclusive relationship, where in transgenic expression of constitutively nuclear FOXO1 decreases Pdx-1 expression via FOXO1‟s repression of the Pdx-1 transcription factor FOXA2. Conversely, haploinsufficiency of FOXO1 increases Pdx-1

42 expression (482). In addition to its deleterious effects on β-cell growth, FOXO1 appears to be essential for β-cell function under oxidative stress conditions. Kawamori et al demonstrated that reactive oxygen species activate c-Jun N-terminal kinase (JNK), decrease Pdx-1 expression and increase the nuclear localization of FOXO1 where it binds to the promoters of in2 transcription factors, NeuroD and MafA (484;485). In an attempt to explain the inverse relationship between

β-catenin and FOXO1, given that cWnt signaling promotes (463), while FOXO1 prevents (486), the proliferation of β-cells, Hoogeboom et al demonstrated competition for β-catenin between the FOXO and TCF transcription factors (487). Thus, ectopic expression of FOXO3a/4 or oxidative stress reduces binding of β-catenin to TCF, therefore decreasing cWnt target gene transcription, whereas siRNA-mediated knockdown of FOXO4 facilitates β-catenin interaction with TCF (487). Although these observations were not investigated in pancreatic β-cells, it nonetheless raises the potential of cWnt signaling to FOXO through β-catenin.

In addition to transcriptional activation, β-catenin also interacts with other cytosolic proteins such as the cell-adhesion system. In combination with α-catenin, β-catenin forms an essential link between E-cadherin and the actin cytoskeleton (488). Carvell et al. demonstrated that overexpression of E-cadherin decreases β-cell proliferation, while reducing E-cadherin levels has the opposite effect without affecting insulin secretory function (489). Although not directly examined, this finding of β-cell growth regulation by E-cadherin may occur via altered

β-catenin availability for cWnt signaling. Furthermore, β-catenin also forms a complex with the

HGF receptor, c-met and, as mentioned above, HGF is a known activator of β-cell proliferation

(472). In hepatocytes, c-met activation causes tyrosine phosphorylation of β-catenin and subsequent translocation to the nucleus in a cWnt-independent manner, leading to induction of proliferation (472;490;491). Moreover, HGF was found to increase c-myc mRNA through activation of MAPK and PI3-kinase leading to inhibition of GSK3, which also causes

43 translocation of β-catenin to the nucleus and increased TCF transcriptional activity (492).

Although these studies were not conducted in the pancreatic β-cell, they provide potential mechanisms whereby HGF-mediated β-cell proliferation could occur through stabilization of β- catenin.

Finally, Kayali et al reported expression of a chemokine, stromal-cell derived factor-1

(SDF-1), and its associated receptor (CXCR4) in both the fetal mouse pancreas and the proliferating duct epithelium of the nonobese diabetic mouse (493). The cross-talk between the

SDF-1-CXCR4 and cWnt signaling pathways was first demonstrated by Luo et al. in studies of rat neural progenitor cells (494). However, transgenic mice expressing SDF-1 specifically in the β-cell are protected against streptozotocin-induced diabetes and this was shown to be dependent on Akt and its downstream pro-survival, anti-apoptotic signaling pathways (495).

Recently, Liu and Habener reported that SDF-1 also activates cWnt signaling in INS1 cells and isolated mouse islets via a Gαi/o-PI3K-Akt cascade, suppression of GSK3, and stabilization of β- catenin (248). Interestingly, SDF-1 signaling in INS1 β-cells increases the levels of β-catenin mRNA. Finally, activated cWnt signaling is also required for the cytoprotective, survival actions of SDF-1 on β-cells (248). Thus, there appear to be differences in the mechanisms of the interactions of SDF-1/CXCR4 and GLP-1/GLP-1R pathways with cWnt signaling in the β- cells. Although both SDF-1 and GLP-1 activate β-catenin/TCF7L2-mediated gene expression, they have different pathways of interaction with upstream components of the cWnt signaling pathway: 1) SDF-1 inhibits formation of the destruction complex via inhibition of GSK3β and casein kinase-1 by Akt (248), whereas 2) GLP-1 signaling leads to phosphorylation of β-catenin on serine-675 by PKA (451). These proposed different pathways by SDF-1 and GLP-1to stabilize β-catenin suggests the potential for synergistic effects on downstream cWnt signaling leading to β-cell growth and survival.

1.3.4 cWnt signaling in T2DM: the TCF7L2 paradox

Studies to delineate the role of cWnt signaling in β-cells are of particular importance since Grant et al reported that single nucleotide polymorphisms (SNPs) in TCF7L2 are associated with the development of T2DM (337). In fact, TCF7L2 polymorphism is considered a stronger indicator than any other genetic marker for this metabolic disorder (496), in several populations including Icelandic, Danish, US, and Asian cohorts. Since the majority of these

SNPs reside in the non-coding regions of the TCF7L2 gene (i.e. introns 3, 4 or 5), there is no clear explanation for the effects on TCF7L2 function and/or activity. However, Mondal et al provided evidence that non-coding SNPs affect alternative splicing of TCF7L2, such that isoforms containing alternative exons 12, 13, and particularly the islet-specific predominant transcript containing exon 13a have distinct properties, are associated with obesity, and their levels in adipocytes are associated with T2DM risk (497). Functional analyses further demonstrated that these isoforms are translated in adipocytes and are targeted to the nucleus where they bind β-catenin (497). Although not studied in pancreatic β-cells, the observations suggest different physiological roles for and regulation of the splice isoforms. Nevertheless, it remains unknown as to how an intron 3 SNP might alter splicing of TCF7L2, whether the changes observed in adipocytes also occur in β-cells, and whether altered TCF7L2 splicing can affect cWnt target genes and ultimately, whole-body glucose homeostasis.

The incretin hormone, GLP-1, appears to be one link between TCF7L2 function and development of T2DM. Glucose clamp studies on carriers of the TCF7L2 polymorphism revealed two crucial abnormalities: 1) reduced insulin secretion during oral, but not intravenous, glucose tolerance tests , and 2) impaired GLP-1-induced insulin secretion (498). It had been speculated that the defect in oral glucose tolerance in patients with the TCF7L2 variants is a result of impaired GLP-1 secretion from gut endocrine cells, as Ni et al reported that TCF7L2 is

45 essential for proglucagon transcription and, thus, GLP-1 synthesis in a gut endocrine cell line

(GLUTag) (499). However, Schaufer et al found that non-diabetic carriers of the risk-associated

TCF7L2 SNPs do not have defects in GLP-1 secretion (498). However, an alternative mechanism may involve decreased expression of the receptors for GLP-1 (GLP-1R) and GIP

(GIPR), as Shu et al reported that GLP-1R and GIPR expression is decreased in islets from humans with T2DM, as well as in isolated human islets treated with TCF7L2 siRNA (500).

Furthermore, knockdown of TCF7L2 also reduces GSIS from rodent β-cells (501;502).

Therefore, the defect in the enteroinsular axis in individuals with TCF7L2 polymorphisms appear to be at the level of impaired incretin responses in the β-cell, rather than due to decreased production of GLP-1 by intestinal L-cells.

The studies described above correlate altered cWnt signaling and/or reduced TCF7L2 levels with decreased β-cell function. However, such a proposal is met with conflicting reports.

Hence, Lyssenko et al. demonstrated that levels of TCF7L2 mRNA are increased in the islets of diabetic patients and that TCF7L2 expression in islets negatively correlates with insulin secretion (503). This suggests that increased levels of TCF7L2 in islets increase the risk of diabetes by decreasing β-cell function. It must be stressed, however, that an increase in TCF7L2 mRNA levels in human islets does not necessarily equate to an increase in functional TCF7L2 protein. Additional studies that unlock the relationship between TCF7L2 expression, mRNA splicing and functional isoforms are clearly required to fully understand the role of this transcription factor in T2DM.

1.4 R-spondin: a new player in the Wnt game

The R-Spondin (roof plate-specific spondin; Rspo) family of secreted proteins has recently been implicated in the regulation of the cWnt signaling pathway. It was first identified by Kamata et al via genetic screening of the neuroblastoma/spinal cord-19 cell line in vitro and

46 in vivo in the developing roof plate of neural tube and the dorsal part of telencephalon; as a result, sequencing analysis identified Rspo1 and revealed it as a novel gene of the TSP family

(504). The mammalian family of Rspo proteins include four independent gene products with

40–60% amino acid sequence identity and substantial structural similaries (505). The N- terminal signal peptide leader sequences, which indicate that Rspo is secreted, display the least similarity amongst the isoforms. The C-terminal domain varies in length and is a region of positively-charged amino acids that can potentially serve as a nuclear localization signal. There are three highly conserved regions; two adjacent cysteine-rich furin-like domains, followed by a common thrombospondin type 1 repeats (TSR-1) domain. Each of these domains is encoded by discrete exons. Due to this TSR-1 domain, Rspo proteins are categorized as part of the TSR-1- containing superfamily of proteins that includes other „spondins‟ such as floor plate (F)-spondin, subcommisural organ (SCO)-spondin, and the ADAMTS family of proteases and protease inhibitors. Many of the activites of these proteins, including angiogenesis, wound healing, cell adhesion and migration, are localized to the TSR-1 domain. However, it is interesting to note that the Rspo family is relatively small compared to the rest of TSR-1-containing proteins, with the largest being Rspo3 with 273 amino acids. Although published reports to-date suggest that the furin-like domains are sufficient for inducing β-catenin stabilization in vitro, and that the

TSR-1 and C-terminal domains are dispensable, these findings do not negate the possibility that

Rspo proteins can participate in other yet-to-be identified signaling pathways (505).

The Rspo protein family has been conserved throughout evolution. Human Rspo1

(hRspo1) shows identities of 98%, 88%, 67%, 60% and 51% compared to orthologs from chimp, mouse, chicken, frog and zebrafish, respectively (506). Expression of Rspo is not limited to vertebrates, however, as a distinctive Rspo-type protein is encoded in the purple sea urchin (Strongylocentrotus purpuratus) genome that shares a similar structural organization to

47 that seen in vertebrates. It is therefore possible that Rspo proteins activate a common receptor or class of receptors to exert a conserved set biological functions.

1.4.1 Function of R-spondin proteins

Upon the discovery of Rspo1, Kamata and colleagues demonstrated by in situ hybridization that Rspo1 expression was upregulated in the dorsal part of the neural tube on 10 and 12 days post-conception, especially in the boundary region between the roof plate and the neuroepithelium, suggesting that Rspo1 might be a novel marker and regulator of this region

(504). To examine the function of the function of Rspo1, transfection of an epitope-tagged

Rspo1 into COS7 and 293HEK cells showed its localization in both the nucleus and cell medium in vitro, suggesting that Rspo1 can be retained in the nucleus or secreted (504). There are no studies to date to examine Rspo1‟s role in the nucleus. However, in an examination of the Wnt1/3a double knockout mouse, expression of Rspo1 was found to be reduced, suggesting for the first time, a relationship between Rspo1 and cWnt signaling (504).

Kazanskaya et al demonstrated that Rspo2 and 3 are co-expressed with the cWnt ligands

Wnt8 and Wnt3a, respectively, again an indication of coupling between the two systems (505).

Furthermore, experimental upregulation of cWnt ligands induces upregulation of Rspo2 and

Rspo3, indicating that the observed coexpression during Xenopus development in Kazanskaya‟s study is due to regulation of Rspo2 and Rspo3 by Wnt8 and Wnt3a, respectively (505). Rspo2 is of physiological relevance since it upregulates myogenic genes during Xenopus muscle development. In addition, the myogenic effects of Rspo2 are repressed by expression of dominant-negative forms of dishevelled, dkk1, or GSK-3β, all of which block cWnt signaling,

(505). Taken together, therefore, these results indicate that Rspo2 regulates Xenopus myogenesis in a cWnt-dependent manner.

A fascinating study in 2005 demonstrated a role for Rspo1 as a potent mitogen for gastrointestinal epithelial cells, thus providing for the first time evidence that Rspo1 is a growth factor. Transgenic mice expressing human Rspo1 exhibited a profound increase in proliferation of the intestinal crypt epithelial cells (507). These proliferative effects of Rspo1 correlated with the activation of β-catenin and subsequent transcriptional activation of cWnt target genes, and were reproduced in mice injected with recombinant Rspo1 protein (507). Moreover, adenoviral mediated Rspo2-4 transfection into mice also induces gastrointestinal proliferation and β- catenin activation (506). The potency of Rspo1 as an intestinal growth factor also implies that it has therapeutic potential. Hence, in acute and chronic experimental colitis, treatment of

Rspo1 improves mucosal integrity in the small intestine and colon by stimulating crypt cell growth and mucosal regeneration (508). Moreover, Rspo1 significantly reduces overproduction of proinflammatory cytokines and preserved mucosal barrier function. Rspo1 treatment also alleviated mucositis in the oral cavity of mice receiving concomitant 5-fluorouracil and x-ray radiation (509). Together, these studies strongly support the potential use of Rspo1 as a novel treatment for colitis or oral mucosal damage induced by intensive chemotherapy and/or radiotherapy. Interestingly, and of key importance to the present series of studies, Kim et al reported the expression of Rspo1 in the human pancreas but its role was not investigated (507).

Chassot et al demonstrated that Rspo1 is a critical regulator of sexual development

(510). Male reproductive development has been studied to some detail and it involves Sox9- induced regression of the Mullerian duct, the precursor of the uterus, oviduct and part of the vagina; testosterone secreted by Leydig cells induces Wolffian duct development into epididymis, vas deferens and seminal vesicles. In contrast, very little is known about the molecular pathways governing ovarian differentiation. However, Chassot et al showed in Rspo1 knockout female mice, that there is a masculinization of the reproductive system (510). Rspo1

49 knockout XX gonads are not only smaller than normal mice, but also contain clear seminiferous tubules in addition to some less-developed cord structures albeit with few gonocytes.

Interestingly, some gonocytes were also detected outside of the cord structures that resembled quiescent G1 gonocytes typical of male gonads (510). Female Rspo1 null mice also demonstrate external masculinized genitalia with an increased distance from the vagina to the anus, and this masculinization of reproductive organs persists throughout adulthood (10 weeks) (510). These observations have been confirmed by another group (511). Moreover, Chassot et al showed that cWnt signaling is required for the regulation of ovary development, as ectopic expression of constitutively-activate β-catenin rescues the abnormal masculinization in XX Rspo1 knockout gonads (510). Therefore, these data shows that Rspo1 is essential for the activation of the cWnt signaling pathway in female gonadal differentiation.

Table 1.2. Impact of R-spondin deficiencies in xenopus, mouse and humans.

Xenopus Mouse Human

Rspo1 Female-to-male sex reversal Female-to-male sex reversal

(510;512) (hermaphroditism) (513;514)

Palmoplantar hyperkeratosis

(513)

Rspo2 Defective Craniofacial malformation

myogenesis (505) (515)

Distal limb loss (515)

Lung hypoplasia (515)

Rspo3 Defective angiogensis (516)

Defects in placental

development (517)

Rspo4 Anonychia/Hyponychia

congenita (518-524)

1.4.2 R-spondin proteins in human diseases

As shown in Table 1.2, mutations of Rspo protein family members have been reported in humans. Although these often lead to a severe phenotype, they have a significant impact on our understanding of Rspo‟s biological actions. For instance, Parma and colleagues describe a recessive mutation in the gene encoding Rspo1 with a single nucleotide insertion, leading to frameshift and a new stop condon and resulting in the abolition of Rspo1 (513). Consistent with the phenotype seen in Rspo1 knockout mice, the lack of Rspo1 in humans leads to a complete female-to-male sex reversal (513). Moreover, the mutant carriers display palmoplantar

51 hyperkeratosis and predisposition to squamous cell carcinoma of the skin due to defective adhesion properties of keratinocytes. It is interesting to note that Rspo1 was not found in keratinocytes but in the underlying fibroblastic cells (513). Therefore, this suggests that Rspo1 is secreted from fibroblasts to act as a paracrine modulator of keratinocytes.

Rspo4 expression has been specifically localized to the mouse nail mesenchyme at embryonic day 15.5, suggesting a crucial role in nail morphogenesis (520;523;524). Consistent with this finding, a rare autosomal recessive mutation in which there is an absence or severe hypoplasia of all fingernails and toenails (Anonychia and hyponychia congenita respectively) has been reported in individuals with homozygous or compound heterozygous mutations in the gene encoding Rspo4 (523). Several mutations were identified in exons 2 and 3 which encode the cysteine-rich furin-like domain that is required for cWnt signaling (521;522;524).

Moreover, mutations are also reported to reside in the 5' and 3' ends of introns, leading to inappropriate exon skipping or intron inclusion in the mature mRNA transcript, respectively

(520).

1.4.3 R-spondin proteins and the canonical Wnt signaling pathway

The precise mechanism of Rspo and cWnt signaling interaction remains a mystery.

A series of findings in Xenopus confirms that Rspo2 activates the cWnt signaling pathway: 1)

Rspo2 is co-expressed with and induced by Wnts; 2) Rspo2 induces cWnt signaling and strongly synergizes with Wnt3a ligands; 3) Rspo2 is a secreted activator and is required for cWnt signaling in in vitro and in vivo; 4) Rspo2-induced cWnt signaling is blocked by Dkk1, a soluble inhibitor of LRP, and GSK3; and 5) overexpression of Rspo2 blocks signaling of Activin,

Nodal, and BMP4 - although not physiologically relevant, this finding suggests that Rspo2 not only has TGF-β inhibiting effects, but also interacts with and regulates other signaling pathways

(505). However, a reduction of Rspo2 protein has no effect on lithium-activated cWnt

52 signaling, indicating that Rspo2 acts upstream of the Axin/APC/GSK3 degradation complex

(505).

In contrast to these findings, Kim et al observed marginal inhibition of Rspo1 protein activity by DKK1 in HEK293 cells and have found cell lines, such as the mouse fibroblast L- cell line, that stabilize β-catenin in response to Wnt3A, but not to Rspo1 (507). These results suggest that Rspo1-mediated effects may be independent of Frz receptors. However, it must be cautioned that although L-cells express Frz receptors for cWnt signaling, they may lack a specific component required for Rspo-mediated signal transduction. One elegant study provided several lines of evidence that Rspo family members function as Frz and LRP receptor ligands in vitro: 1) Rspo is a secreted protein; 2) unlike Wnt ligands that form a ternary complex with Frz and LRP5/6 receptors, Rspo proteins failed to form a ternary complex but can nonetheless bind to both receptors; and 3) there is a positive modulation of Wnt ligand activity by Rspo via direct interaction between the two ligands (525). As a result of these interactions, Rspo induces the cWnt signaling pathway, initiating cWnt target gene expression (525). In contrast, Binnerts et al reported that Rspo1 does not directly bind to the LRP6 co-receptor (526). Instead, they found that Rspo1 interacts with one additional transmembrane component, Kremen1 (526). DKK1 inhibits LRP6 by coupling LRP6 with Kremen1, subsequently targeting it for internalization

(527-530). Therefore, Rspo1 interferes with DKK binding to Kremen1 and the authors thereby proposed a model in which Rspo1 regulates cWnt signaling by antagonizing Kremen/DKK- dependent LRP6 internalization. Although there are no explanations to date to explain two different mechanisms by which Rspo1 can activate cWnt signaling, it is important to note that the two studies are not mutually exclusive. However, Rspo proteins may also act independently of cWnt, through a novel receptor/signaling pathway that: 1) impacts β-catenin without utilizing the Frz/LRP receptor complex; and/or 2) amplifies the expression of cWnt proteins, leading to a

53 secondary activation of the cWnt pathway. Given the complex interplay with cWnt pathways

(and possibly, other Wnt pathways), understanding the precise mode of action of Rspo proteins in physiology and development is of utmost importance.

1.5 Rationale and Hypothesis

Rspo1 is potent gastrointestinal growth factor known to activate the cWnt pathway, while cWnt signaling plays a role in pancreatic development, and -cell growth and function.

The finding that Rspo1 is expressed in human islets (507), mandates further investigation. The specific aims of this thesis are intended to answer one fundamental question: what is the role of

Rspo1 in mature pancreatic -cells? Using a two-pronged approach of in vitro -cell models

(Chapter 2) and in vivo Rspo1 knockout mice (Chapter 3), I have therefore examined the general hypothesis that Rspo1 is a -cell growth factor and secretagogue.

CHAPTER 2:

R-SPONDIN-1 IS A NOVEL -CELL GROWTH FACTOR AND INSULIN SECRETAGOGUE IN VITRO

The work presented in this chapter corresponds to the following publication, reproduced with permission:

Wong V.S., Yeung A., Schultz W., Brubaker P.L. J Biol Chem 2010 Jul 9;285(28):21292-302.

Author contributions:

A. Yeung and W. Schultz were 4th year undergraduate students working directly under my supervision. A. Yeung contributed the examination of cWnt pathway mRNA expression in the

MIN6 β-cell line (Figures 2.1B and 2.1D). W. Schultz contributed to analyses of nuclear β- catenin in the MIN6 β-cell line in response to various treatments (Figure 2.2A).

2 R-spondin-1 is a novel -cell growth factor and insulin secretagogue in vitro

2.1 Abstract

R-spondin-1 (Rspo1) is an intestinal growth factor known to exert its effects through activation of the canonical Wnt (cWnt) signaling pathway and subsequent expression of cWnt target genes.

We have detected Rspo1 mRNA in murine islets and the murine MIN6 and TC -cell lines, and Rspo1 protein in MIN6 -cells. Rspo1 activated cWnt signaling in MIN6 -cells by increasing nuclear -catenin and c-myc, a cWnt target gene. Rspo1 also induced insulin mRNA expression in MIN6 cells. Analysis of MIN6 and mouse -cell proliferation by 3H-thymidine and BrdU incorporation respectively revealed that Rspo1 stimulated cell growth. Incubation of

MIN6 and mouse -cells with cytokines (IL-1 /TNFα/interferon-γ) significantly increased cellular apoptosis; this increase was abolished by pre-treatment with Rspo1. Rspo1 also stimulated insulin secretion in a glucose-independent fashion. We further demonstrated that the glucagon-like peptide-1 receptor agonist, exendin-4 (EX4), stimulated Rspo1 mRNA transcript levels in MIN6 cells in a glucose-, time-, dose- and PI3-kinase-dependent fashion. This effect was not limited to this -cell line, as similar time-dependent increases in Rspo1 were also observed in the TC -cell line and mouse islets in response to EX4 treatment. Together, these studies demonstrate that Rspo1 is a novel -cell growth factor and insulin secretagogue that is regulated by EX4. These findings suggest that Rspo1 and the cWnt signaling pathway may serve as a novel target to enhance -cell growth and function in patients with type 2 diabetes.

2.2 Introduction

Type 2 diabetes mellitus (T2DM) represents a serious and growing epidemic that poses a major public health threat in the 21st century. The development of T2DM usually requires the presence of both insulin resistance and impaired -cell function, but also involves the loss of - cells (138). Moreover, Type 1 diabetes mellitus (T1DM) is characterized by the autoimmune- mediated destruction of -cells. Therefore, novel therapeutic approaches that enhance -cell mass expansion, as well as -cell function, represent an exciting arsenal against diabetes.

Wnt signaling has been demonstrate to play important roles in development as well as in the pathogenesis of a variety of diseases, including diabetes (434). Activation of this pathway requires interaction between a secreted glycoprotein, Wnt, and a seven-transmembrane receptor protein, Frizzled (Frz). There are at least three distinct intracellular Wnt pathways, including, most notably, the canonical Wnt (cWnt) cascade that leads to changes in intracellular -catenin levels and is thought to be involved in cell fate specification and proliferation. -catenin is normally phosphorylated and targeted for proteolysis by a complex of proteins, including adenomatosis polyposis coli (APC), axin and the serine/threonine kinase glycogen synthase kinase-3 (GSK3 ) (Figure 2.1A). cWnt activation of the Frz and low density lipoprotein receptor-related protein (LRP) co-receptors results in dissociation of this degradation complex, permitting entry of -catenin into the nucleus to activate cWnt target genes in conjunction with

TCF/LEF family transcription factors and, possibly, other DNA-binding partners (531). cWnt target genes have been identified in different models and these include, but are not limited to, the cell-cycling genes, c-myc and cyclinD1

(http://www.stanford.edu/~rnusse/pathways/targets.html).

Interestingly, mice that lack the gene encoding the LRP5 show impaired glucose tolerance due to perturbed glucose-stimulated insulin secretion (GSIS) (465). Furthermore,

58 adipocyte-secreted Wnts have been shown to stimulate insulin secretion and glucokinase gene transcription in INS1 cells in vitro through the activation of cWnt signaling (467). In contrast, transgenic mice that over-express a dominant-negative form of mouse Frz8 under the Ipf-1/Pdx-

1 promoter are normoglycemic and display normal GSIS (466). However, the -cells of these mice produce four times more and secrete twice as much insulin as those of wild-type littermates, suggesting the presence of compensatory mechanisms to achieve and maintain normoglycemia (466). Finally, Rulifson et al demonstrated that conditional pancreatic -cell specific expression of degradation-resistant -catenin leads to -cell expansion, increased insulin production and serum levels, and enhanced glucose handling (463). This observation is further strengthened by a recent study from Liu and Habener showing that exendin4 (EX4), a glucagon- like peptide-1 (GLP-1) receptor agonist, stimulates -cell proliferation via activation of the cWnt signaling pathway (451).

The roof plate-specific spondin (R-spondin; Rspo) protein family consists of four structurally related members (Rspo1-4), with conserved cysteine-rich furin-like and thrombospondin domains. Several lines of evidence indicate that Rspo family members function as Frz and/or LRP receptor ligands in vitro: 1) Rspo is a secreted protein (507); 2) unlike Wnt ligands that form a ternary complex with Frz and LRP receptors, Rspo proteins failed to form a ternary complex but can nonetheless bind to both receptors (525); 3) there is a positive modulation of Wnt ligand activity by Rspo via direct interaction between the two ligands (532); and 4) Rspo prevents LRP6 internalization (533). Furthermore, transgenic mice expressing human Rspo1 exhibit a profound increase in proliferation of intestinal crypt epithelial cells, which correlates with the activation of -catenin (507). Adenoviral-mediated transfection of each isoform of Rspo into mice also induces gastrointestinal proliferation in association with -catenin activation (506). Unexpectedly, although expressed at high levels in

59 the gut, Rspo1 has also been detected in human pancreatic islets by immunohistochemisty (507).

However, no studies to-date have examined the role of Rspo1 in -cell physiology. Therefore, in the present study, we have determined the role of Rspo1 in the mature pancreatic -cell in vitro, though analysis of the effects of Rspo1 on -cell proliferation, apoptosis and insulin secretion, as well as through determination of the effects of known -cell regulatory factors (i.e. glucose and GLP-1) on Rspo1 expression.

2.3 Experimental Procedures

2.3.1 Cell culture.

MIN6 -cells (mouse insulinoma cell line, a kind gift from Drs. J. Miyazaki, University of Tokyo and D.F. Steiner, University of Chicago) were maintained in Dulbecco‟s modified

Eagle‟s medium (DMEM; Gibco BRL/Invitrogen, Burlington, ON, Canada) containing 25 mM glucose and supplemented with 2 mM L-glutamine, 10% heat-inactivated fetal bovine serum

(FBS), penicillin (100 U/ml), streptomycin (100 mg/L), and 71 μM 2-mercaptoethanol in humidified 5% CO2, 95% air at 37 C. βTC β-cell line were maintained in DMEM containing

25 mM glucose, 2 mM L-glutamine, 10% heat-inactivated FBS, penicillin (100 U/ml), and streptomycin (100 µg/ml).

2.3.2 Isolation and culture of intact and dispersed mouse islets.

Islets were isolated from 20-30 g CD1 mice (Charles River, St. Constant, QC) by collagenase digestion, as previously described (413) and were cultured in RPMI

1640 containing 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin (Gibco

BRL/Invitrogen) for two days after isolation. Mouse islet cells were dispersed by incubation with Dispase II (Roche Laboratories, Mississauga, ON) as previously described (534) and were plated on 35 mm petri-dishes (for Live-Cell Analyses; ibidi, Ingersoll, ON). Cells were then cultured overnight.

2.3.3 RNA isolation.

Animal tissues or cells grown to approximately 80-90% confluence were lysed for preparation of RNA using either the RNeasy or RNeasy Micro Kit according to the manufacturer‟s instructions (Qiagen Inc., Mississauga, ON). RNA was quantified by spectrophotometry (absorbance at 260 nm) and stored at -80 C until use.

2.3.4 RT-PCR.

Equal amounts of RNA isolated from animal tissues, cells or islets were analyzed by RT-

PCR using a One-Step kit (Qiagen Inc.). RT-PCR primers and conditions have been reported previously (452;535-543) and are listed in Table 2.1. All primers were further verified using positive control samples selected based on previous reports listed in the expression database

(http://www.informatics.jax.org/; data not shown). Negative control reactions were performed using RNase-free water without template.

2.3.5 Real-Time PCR.

MIN6, TC and islets were serum-starved overnight and then incubated with media alone (containing the appropriate vehicle; PBS or DMSO), recombinant Wnt3a (641 pM; R&D

Systems, Minneapolis, MN), recombinant mouse Rspo1 (various doses ranging from 34.5 pM -

34.5 nM; R&D Systems), or EX4 (1 - 100 nM; Bachem, Torrance, CA) with or without high glucose (25 mM) or inhibitors (LY294002 (50 μM; Sigma-Aldrich, Oakville, ON), wortmannin

(100 nM; Sigma-Aldrich), H89 (10 μM; Sigma-Aldrich), SB239063 (10 μM; Calbiochem,

Mississauga, ON, Canada), PD98059 (20 μM; Sigma-Aldrich), or U0126 (1 μM; New England

Biolabs, Mississauga, ON)) for the indicated amount of time, ranging from 30 min to 24 hr.

Wnt3a, Rspo1 and EX4 concentrations were selected based upon previous reports

(465;507;544).

Five μg of total RNA from samples were reverse-transcribed with Superscript II Reverse

Transcriptase (Invitrogen). Semi-quantitative RT-PCR (qRT-PCR) was performed in a

Chromo4 Continuous Fluorescence Detection unit with Opticon Monitor 3 software (Bio-Rad

Laboratories, Mississauga, ON) using Taqman Gene Expression Assays for specific primers

(Applied Biosystems, Foster City, CA). All reactions were performed in duplicate, and control reactions were performed without RT enzyme and/or without template. The linearity of

62 amplification of the Taqman primer-probe sets was verified over nine orders of magnitude (data not shown).

Ribosomal protein 18S RNA (no. Hs99999901_sl) was used as the endogenous control for all quantitative analyses of mRNA expression and was not found to change in response to any of the experimental treatments tested (data not shown). Relative quantification of Rspo1 (2 sets of primers used; set 1 no. Mm00507076_m1 and set 2 no. Mm00507077_m1), c-myc (no.

Mm00487803_m1), cyclinD1 (no. Mm00432359_m1) , and Insulin2 (no. Mm00731595_gH) mRNA expression was calculated using the cycle threshold [ C(t)] method (545).

2.3.6 Protein extraction, cell fractionation and immunoblotting.

Cells and islets were lysed with RIPA buffer (50 mM glycerol phosphate, 10 mM

HEPES (pH 7.4), 1% Triton X-100, 70 mM NaCl, 2 mM EGTA, 1 mM Na3VO4, 1 mM NaF, and EDTA-free protease inhibitors (Roche)). Proteins of interest were detected with primary antibodies targeted against mouse Rspo1 (goat IgG, 1:1000; R&D Systems), cleaved caspase3

(rabbit IgG, 1:1000; New England Biolabs), or pan-actin (rabbit IgG, 1:1000; Sigma Aldrich).

Immunoblotted membranes were then probed with the appropriate secondary antibodies (HRP- linked anti-rabbit and HRP-linked (1:2000; New England Biolabs), and HRP-linked anti-goat

(1:2000; Jackson Immunoresearch Laboratories, West Grove, PA)) and visualized by electrochemical luminescence detection system (Amersham Pharmacia Biotech, Baie d Urfe,

QC). Membranes were subsequently treated at 50 °C for 30 min with stripping buffer (62.5 mM

Tris-HCl, pH 6.8, 2% SDS, and 100 mM -mercaptoethanol) for a second round of immunoblotting.

To determine protein levels of nuclear -catenin, MIN6 cells were grown to 80 – 90% confluency in 10 cm dishes and serum-starved overnight, followed by treatment with either media alone (control), EX4 (10 nM), LiCl (20 mM), Wnt3a (641 pM) or Rspo1 (34.5 pM - 34.5

63 nM) for 30 min or 8 hr. Lysates were then centrifuged at 2,000 X g for 5 min. The pellets were resuspended in 350 μl of Buffer A (20 mM HEPES (pH 7.5), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol and protease and phosphatase inhibitors (Roche Laboratories) and incubated on ice for 15 min. Cells were further lysed by addition of 10% NP-40 (to a final concentration of 1%; Sigma Aldrich) and vortexed for 1 min. Nuclei were pelleted by centrifugation for 10 min at 1,600 X g at 4°C. The pellets were washed once with 400 μl of buffer A and the nuclear fraction was further pelleted for 10 min at 1600 X g at 4 C. Nuclei were solubilized by addition of one pellet volume of NE buffer (20 mM Tris (pH 8.0), 420 mM

NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol, and protease and phosphatase inhibitors).

One-fourth pellet volume of 5 M NaCl and one pellet volume of NE buffer were further added to the resulting pellet. Nuclei were then homogenized by sonication for 5 sec and all samples

(e.g. nuclei and supernatants) were stored at -80 C until used (546). Fifty μg of nuclear protein, as measured by Bradford protein assay (Bio-Rad Laboratories, Mississauga, ON), was used for immunoblotting using antibodies against -catenin (mouse IgG, 1:1000; BD Transduction

Laboratories, Franklin Lakes, NJ) and poly-ADP-ribose polymerase (PARP; mouse IgG,

1:1000; nuclear fraction protein loading control (547), BD Transduction Laboratories) as described.

2.3.7 Cell proliferation assays.

MIN6 cells were grown to 80 - 90% confluence in 24-well plates, serum-starved overnight and then treated with media alone (control), EX4 (10 nM) or various doses of recombinant Rspo1 (34.5 pM - 34.5 nM) overnight in the presence of serum-free media with 25 mM glucose. Cell proliferation was measured as described (400). Briefly, cells were incubated with 37 kBq/ml 3H-methylthymidine (specific activity: 3000 GBq/mmol; Amersham Pharmacia

Biotech, Pittsburgh, PA) for 4 hr. Cells were then washed twice in cold PBS and incubated for

30 min in 1 ml of 5% trichloroacetic acid at 4 C to precipitate the DNA. The liquid layer was removed by aspiration and 500 µl of 0.1 M sodium hydroxide was added to the cells for an additional 30 min at room temperature with gentle shaking. The solubilized material was then transferred to 4 ml of scintillant, and radioactive counts were determined by liquid scintillation counting.

For measurement of murine β-cell proliferation, dispersed islet cells were treated for

48hr with media alone (control), EX4 (10 nM) or recombinant Rspo1 (34.5 nM) in the presence of serum-free media with 20 mM glucose and, for the last 24 hr, 5‟-bromo-2‟-deoxyuridine

(BrdU, 10 µM) was added. Cells were washed with PBS and fixed in 10% formalin and incubated with mouse anti-BrdU (1:200; Sigma-Aldrich) and guinea pig anti-insulin (1:200;

Dako Diagnostics, Mississauga, ON) antibodies. Cells were then gently washed with PBS and incubated for 30 min with appropriate secondary antibodies (Texas Red conjugated anti-mouse

(1:200) and Cy2-conjugated anti-guinea pig (1:200); Jackson Immunolaboratories, West Grove,

PA) and then mounted with mounting medium for fluorescence containing DAPI (VectaShield;

Vector Laboratories, Inc., Burlingame, CA). Proliferative index is expressed as a percentage of

BrdU- and insulin-positive cells over total insulin-positive cells analyzed under Zeiss Axioplan microscope with Axiovision software (Carl Zeiss Canada, Don Mills, ON). A minimum of 100

β-cells was counted per treatment.

2.3.8 Apoptosis assays.

MIN6 cells were grown to 80 - 90% confluency and apoptosis assay was performed as previously described (413). Briefly, cells were seeded in 12-well cell culture dishes for 24 hr and subsequently pre-incubated with either media alone (control), EX4 (10 nM), Wnt3a (641 pM), or Rspo1 (34.5 pM - 34.5 nM) for 18 hr. The cells were then incubated with a mixture of cytokines (10 ng/ml IL-1 , 50 ng/ml TNFα, 50 ng/ml IFNγ; Sigma Chemical Company, St

Louis, MO) in the absence or presence of treatment, as described above, for another 18 hr. This incubation time was based on the results of (413). Apoptosis was measured by immunoblotting for cleaved caspase3 (413).

For measurement of murine β-cell apoptosis, after overnight serum starvation, cells were pre-incubated with either media alone (control), EX4 (10 nM), or Rspo1 (34.5 nM) for 18 hr.

The cells were then incubated with a mixture of cytokines, as above, in the absence or presence of treatment, as described, for an additional overnight incubation. Dispersed cells were then washed and fixed in 10% formalin, and stained for insulin as above, with apoptosis detection performed using a TUNEL detection kit (Roche). β-cell apoptosis is expressed as a percentage of TUNEL- and insulin-positive cells over total number of insulin-positive cells, analyzed using a Zeiss system as above.

2.3.9 Insulin secretion assay.

MIN6 cells were grown to 90% confluence in 24-well plates, and serum-starved overnight. Cells were then treated for 2 hr with high glucose (25 mM) medium containing media alone (control), EX4 (10 nM), Wnt3a (641 pM), or Rspo1 (34.5 pM - 34.5 nM) for an additional 2 hr with serum-free medium. The medium was then transferred and spun at 2000 X g at 4°C for 1 min, and the supernatant was collected and placed on ice. Insulin secretion studies on isolated mouse islets were performed as previously described (548). Briefly, islets were cultured overnight in 2 mM glucose RPMI1640 (Gibco BRL/Invitrogen) with 10% FBS and penicillin/streptomycin. Islets were then washed and incubated with experimental media that consisted of either low (2 mM) or high glucose (20 mM) RPMI 1640 with or without Rspo1

(34.5 nM) for 2 hr. A total of 10 islets of approximately the same size were used per treatment group. Media samples were taken and centrifuged at 700 X g at 4°C for 1 min and the

66 supernatant was collected. Total islet DNA was measured using a spectrophotometer (A260 nm) after extraction in 75% ethanol and 0.09 N hydrochloric acid.

Samples were diluted into the assay buffer and assayed for insulin using an insulin RIA kit according to the manufacturer‟s instructions (Linco Research, St. Louis, MO). Cell protein content was determined by Bradford assay.

2.3.10 Statistical Analysis.

All data are expressed as mean ± SEM. In some experiments, data were log10 transformed to normalize variance for statistical analysis. Data were analyzed by Student‟s t-test or by one- or two-way ANOVA, followed by appropriate post-hoc testing using Statistical

Analysis System software (SAS v 9.1.3, Cary, NC). Statistical significance was assumed at p<0.05.

2.4 Results

2.4.1 Expression of Rspo1 and cWnt signaling molecules in murine -cells.

Conventional RT-PCR demonstrated that Rspo1 mRNA is expressed in whole mouse pancreas as well as in isolated murine islets (Figure 2.1B). Rspo1 mRNA was also detected in the murine MIN6 and TC -cell lines. Moreover, the Rspo3 and Rspo4, but not Rspo2, isoforms were detected in both mouse islets and MIN6 -cells (Figure 2.1B). Examination of the relative expression levels of Rspo1 by qRT-PCR using 2 different Rspo1 primer sets revealed that, although expression was lower in mouse islets and TC -cells, Rspo1 was highly expressed in the MIN6 -cell line (Figure 2.1C). Therefore, the MIN6 -cell line was used as our main in vitro model to study Rspo1.

RT-PCR of total RNA from MIN6 -cells was conducted to determine the expression of essential components of the cWnt pathway, including specific isoforms of Wnt ligands, Frizzled and LRP receptors, and intracellular cWnt signaling molecules such as Axin, dishevelled, APC, and GSK3 (Figure 2.1A). As shown in Fig. 2.1D, MIN6 -cells expressed mRNA transcripts for the majority of Wnt ligands (except Wnt2b, Wnt5b, and Wnt9b) and Frz receptors (except

Frz9 and Frz10). Both isoforms of the LRP co-receptors and all tested intracellular cWnt signalling molecules were also detected in MIN6 -cells. Collectively, these observations implied that MIN6 -cells are capable of a functional cWnt signaling response.

2.4.2 Rspo1 stimulates cWnt signaling and insulin mRNA expression in MIN6 -cells.

Activation of cWnt signaling involves the stabilization of -catenin and its subsequent translocation to the nucleus where it interacts with the TCF/LEF family of transcription factors to initiate transcription of cWnt target genes. To determine whether Rspo1 induces cWnt signaling in MIN6 -cells, cells were incubated for 30 min with media alone or Rspo1 at 34.5 pM, 345 pM and 3.45 nM, as well as with EX4 (10 nM) and Wnt3a (641 pM), positive controls,

68 and nuclear lysates were used to immunoblot for -catenin (Figure 2.2A). Rspo1 at the 345 pM and 3.45 nM doses, but not Wnt3a, was found to significantly enhance nuclear -catenin levels

(p<0.05). Although EX4 did not increase nuclear -catenin within this time frame, we found that EX4 (and LiCl; positive control) significantly enhanced nuclear -catenin after 8 hr of incubation, by 1.5-fold (p<0.05, Figure 2.2A inset).

To determine if Rspo1-induced increases in nuclear -catenin translate to transcriptional output, two cWnt target genes, c-myc and cyclinD1, were analyzed by qRT-PCR. Rspo1, at concentrations of 345 pM and 3.45 nM, significantly, increased c-myc, but not cyclinD1 mRNA, after 12 hr of incubation (Figure 2.2B and C; p<0.05 - 0.01), at which time, there was no effect of Wnt3a (641 pM). However, incubation with Wnt3a for 4 hr stimulated a 3.5-fold increase in c-myc and a 3-fold increase in cyclinD1 mRNA levels in the MIN6 cells (p<0.05 for both; Figure 2.2B inset and C inset). Together, these findings established that Rspo1 induces cWnt signaling in MIN6 -cells by increasing nuclear -catenin levels, resulting in a subsequent elevation of c-myc mRNA levels, and that the timing and effects of Rspo1 on MIN6 -cells differ from those of both EX4 and Wnt3a. Moreover, qRT-PCR revealed that, at all concentrations tested, Rspo1 also enhanced insulin2 mRNA levels after 12 hr (Figure 2.2D; p<0.05 - 0.01). Interestingly, treatment with EX4 stimulated insulin2 mRNA levels only after

24 hr incubation by 2-fold (p<0.01; data not shown), indicating that Rspo1 and EX4 may regulate insulin2 mRNA expression via different pathways.

2.4.3 Rspo1 stimulates -cell proliferation.

Cell proliferation assay using 3H-methylthymidine incorporation revealed that EX4 and

Wnt3a (positive controls) stimulated MIN6 -cell proliferation by nearly two-fold compared to the control group (Figure 2.3A, p<0.05 - 0.01), consistent with prior reports (451;463).

Treatment with recombinant mouse Rspo1 at doses of 345 pM and 3.45 nM also stimulated

MIN6 -cell proliferation, reaching a maximum of 2.2 fold (p<0.01) of controls. The highest dose of Rspo1 tested (34.5 nM) did not stimulate further proliferation. To assess whether Rspo1 can stimulate -cell proliferation, dispersed mouse islet cells were incubated with EX4 (10 nM, positive control) and Rspo1 (34.5 nM) for 48 hr and BrdU was added for the last 24 hr. Figure

2.3B shows that Rspo1 at 34.5 nM induced a 2.5-fold increase in BrdU incorporation in insulin- positive cells (p<0.01), while EX4 enhanced -cell proliferation by 2.8-fold (p<0.01).

2.4.4 Rspo1 prevents cytokine-induced apoptosis in -cells.

In addition to the enhancement of cell growth, inhibition of apoptosis is another important variable in the -cell growth equation. As shown in Figure 2.4A, the level of activated, cleaved caspase3 was significantly increased by 7-fold (p<0.05) following treatment of the MIN6 cells with a mixture of cytokines for 18 hr, and this increase was completely prevented by pre-treatment with EX4 (p<0.05) or Wnt3a (641 pM), as well as by all doses of

Rspo1 (Figure 2.4A, p<0.01). The level of activated caspase3 in the presence of cytokines was not further reduced when MIN6 cells were co-treated with both Wnt3a and Rspo1 (data not shown). A similar observation was observed in dispersed murine -cells, such that treatment with cytokines for 18 hr significantly increased the number of TUNEL-positive -cells by 6-fold

(p<0.01); however pre-treatment with either EX4 (10 nM) or Rspo1 (34.5 nM) significantly reduced cytokine-induced apoptosis (p<0.05; Figure 4B).

2.4.5 Rspo1 stimulates -cell insulin secretion.

It is well established via knockout of LRP5 that the manipulation of cWnt signaling induces changes in -cell function (465). MIN6 -cells were therefore treated for 2 hr with either media alone (control), EX4 (10 nM; positive control), Wnt3a (641 pM) or Rspo1 (34.5 pM - 34.5 nM; Figure 2.5A) in the presence of high (25 mM) glucose. Not only EX4, but also

Wnt3a stimulated insulin secretion under these conditions (p<0.001). Furthermore, while no

70 changes were seen with Rspo1 at the low doses tested (34.5 pM and 345 pM), insulin secretion from MIN6 -cells treated with Rspo1 at higher doses (3.45 nM and 34.5 nM) was increased to

2- and 5-fold of control, respectively, in a dose-dependent fashion (Figure 2.5A; p<0.001 vs. control; p<0.001 for 34.5 nM vs. 3.45 nM). Rspo1-stimulated insulin secretion in MIN6 -cells was not glucose dependent as difference in secretion was not seen between low and high glucose in the presence of Rspo1 (Figure 2.5A inset). We next evaluated whether Rspo1 can regulate insulin secretion in mouse islets. Static incubation of islets with Rspo1 at 34.5 nM for

2 hr induced a significant increase in insulin secretion and this effect was also glucose- independent (Figure 2.5B).

2.4.6 EX4 stimulates Rspo1 expression in a glucose-, dose-, time- and PI3-kinase-dependent

manner.

Finally, since β-cell behaviour is regulated by both glucose and GLP-1, we determined whether Rspo1 is affected by these factors. Rspo1 mRNA levels were therefore examined in

MIN6 cells treated for various times with either media alone (control) or incremental doses of

EX4 (1 - 100 nM) at either low (5 mM) or high (25 nM) glucose. Treatment with high glucose alone increased Rspo1 mRNA levels by 2-fold, while EX4 at 10 nM for 8 hr induced a further increase in Rspo1 mRNA levels, an effect that was seen only under high-glucose conditions

(Figure 2.6A; p<0.05). A time-course study demonstrated that Rspo1 mRNA levels peaked at

3-fold of control levels following 8 hr of EX4 (10 nM) treatment with high glucose (Figure

2.6B; p<0.05) and returned back to basal levels at 12 - 24 hr (Figure 2.6B). Consistent with the mRNA findings, changes in Rspo1 protein levels were observed in response to EX4 treatment at

12 hr but not at 8 hr (Figure 2.6C; p<0.01). To determine if the observed effect of EX4 on

Rspo1 mRNA is restricted to the MIN6 -cells, the murine TC -cell line as well as isolated mouse islets were also tested. Treatment with EX4 at 10 nM stimulated Rspo1 mRNA by 4-fold

71 at 8 hr in the βTC cells (Figure 2.6D; p<0.05). A similar induction of Rspo1 mRNA was also observed in the isolated mouse islets (Figure 2.6E; p<0.05), albeit only at an earlier (i.e. 4 hr) time point (preliminary 8 hr and 12 hr data, not shown).

To delineate the mechanism of action whereby EX4 regulates Rspo1 mRNA expression,

MIN6 cells were co-treated with EX4 and various inhibitors of the known GLP-1 receptor signaling pathway. Consistent with previous observations, EX4 treatment increased Rspo1 mRNA levels by 2-fold (Figure 2.6F). Co-treatment with LY294002 (a PI3-kinase inhibitor) significantly attenuated Rspo1 mRNA expression (p<0.01 vs. EX4 alone), and a similar reduction was seen in cells co-treated with wortmannin (data not shown). In contrast, H89 (a

PKA inhibitor), SB203580 (p38 MAPK inhibitor), and PD98059 and U0126 (MEK inhibitors) had no effect on EX4-induced Rspo1 mRNA levels. Treatment with each of these inhibitors alone did not alter Rspo1 mRNA levels (data not shown). These findings indicate that EX4 regulates Rspo1 mRNA expression in a PI3-kinase-dependent fashion.

Table 2.1. RT-PCR primers. Primers and conditions used to detect multiple cWnt signaling molecules as depicted Figure 2.1A. The RT-PCR primers were designed to recognize mouse sequences.

Figure 2.1. Rspo1 and cWnt signaling molecules are expressed in murine -cells. A. A simplified schematic of cWnt and Rspo1 signaling showing cWnt ligand and Rspo1 binding to the Frz receptor and LRP5/6 co-receptor, as well as the intracellular protein Dishevelled and the

β-catenin degradation complex consisting of APC, Axin and GSK3β. B. RT-PCR analysis of

Rspo1 - 4 mRNA in murine pancreas, islets, and MIN6 and TC -cell lines. A 100 -1000 bp ladder was used. No RNA was used in the negative (-ve) control. C. Relative qRT-PCR quantification of Rspo1 mRNA transcripts in murine islets, and MIN6 and TC -cells. Primer

74 set 1 = exons 2 - 3 and primer set 2 = exons 3 - 4. Relative expression levels of Rspo1 were normalized to 18S rRNA expression. (n = 5 – 30). D. RT-PCR for mRNA transcripts of various cWnt signaling molecules in MIN6 -cells. A 100 -1000 bp ladder was used.

Figure. 2.2. Rspo1 activates cWnt signaling and increases insulin2 mRNA levels in MIN6

-cells. A. Ratio of nuclear -catenin to nuclear PARP in MIN6 cells treated with EX4, Wnt3a, and increasing doses of Rspo1 for 30 min. (Inset: ratio of nuclear β-catenin to nuclear PARP in

MIN6 β-cells treated with LiCl and EX4 for 8 hr). A representative blot is shown. All values are expressed as fold-relative to the control (media alone; n = 4 – 6). B, C and D. Relative expression analysis of c-myc (B), cyclinD1 (C) and insulin2 (D) mRNA levels by qRT-PCR in

MIN6 -cells treated with media alone (control), Wnt3a or increasing doses of Rspo1 for 12 hr.

(Insets B and C: relative expression analyses of c-myc and cyclinD1 after 4 hr incubation with

76 media alone (control) or Wnt3a). Data were normalized to the housekeeping gene 18S rRNA (n

= 9 – 11) and are displayed relative to vehicle-treated controls. * p < 0.05 and ** p < 0.01.

Figure 2.3. Rspo1 stimulates -cell proliferation. A. MIN6 -cells were treated with either media alone (control), EX4, Wnt3a or increasing doses of Rspo1 overnight, and their proliferation index was determined by 3H-thymidine incorporation assay (n = 14 – 33). B.

Dispersed murine islet cells were treated with media alone (control), EX4 or Rspo1for 48 hr and

BrdU was added for the last 24 hr. Cells were then fixed and co-stained for insulin and BrdU.

Proliferative index was determined as the number of BrdU- and insulin-positive cells over total insulin-positive cells and data is presented as fold of control (n = 4). * p < 0.05, ** p < 0.01.

Figure 2.4. Rspo1 inhibits cytokine-induced -cell apoptosis. A. and B. Effects of Rspo1 on activated, cleaved caspase-3 in MIN6 -cells (A) or TUNEL in dispersed murine -cells (B).

Cells were incubated in serum-free media overnight, pre-treated with media alone (control),

EX4, Wnt3a or the specified doses of Rspo1 for 18 hr, and then incubated without (basal) or with a combined cytokine cocktail for a further 18 hr. MIN6 -cells was analyzed by immunoblotting for cleaved caspase-3 and pan-actin (n = 4 – 8). A representative blot is shown.

Dispersed islet cells were fixed, and then co-stained for insulin and TUNEL (n = 6). Apoptotic index was expressed as fold change relative to the basal control group. * p < 0.05 and ** p <

0.01 when compared with control (basal); # p < 0.05 and ## p < 0.01 when compared with control + cytokines.

Figure 2.5. Rspo1 stimulates insulin secretion in MIN6 β-cells and isolated mouse islets. A.

Insulin secretory response to Rspo1 in MIN6 -cells (n = 5 - 12) was tested by static incubation of media containing media alone (control), EX4, Wnt3a or indicated doses of Rspo1 for 2 hr with low or high glucose Insulin in the media was measured by radioimmunoassay, and the

81 results were normalized to total protein content. (inset: MIN6 -cells was treated with or without Rspo1 (34.5 nM) under low (2 mM) or high glucose (25 mM) conditions (n = 6 - 12).

Data were normalized to total protein content and expressed as fold of low glucose alone).

Insulin secretion is expressed as fold of low glucose control). B. Insulin secretion in isolated mouse islets was determined after 2 hr incubation with low or high glucose and with or without

Rspo1. The results were normalized to total protein content and expressed as fold of low glucose alone. ** p < 0.01, *** p < 0.001 compared to control values, @@@ p < 0.001 for 34.5 nM compared to 3.45 nM Rspo1, and # p < 0.05 and ### p < 0.001 for high glucose compared to high glucose in presence of 34.5 nM Rspo1.

Figure 2.6. Rspo1 is regulated by EX4 in the -cell. A. MIN6 -cells were treated with EX4 at indicated concentrations under low or high glucose conditions for 8 hr. mRNA levels of

Rspo1 were examined by relative qRT-PCR using 18S as the internal control and then

83 normalized to control (5 mM glucose without EX4). B. MIN6 -cells were incubated in high glucose conditions with media alone (control) or EX4 for the indicated times. C. Protein levels of Rspo1 and actin were determined by immunoblot of MIN6 -cells treated with media alone

(control) or EX4 for 8 or 12 hr. Optical densities of Rspo1 were normalized to that of pan-actin and were further normalized to their appropriate controls. A representative blot is shown for the

12 hr time point. D. qRT-PCR for Rspo1 mRNA expression in TC -cells after incubation with media alone (control) or EX4 for the indicated times. Relative expression values were normalized 18S rRNA and then to the 4 hr control group. E. qRT-PCR for Rspo1 mRNA levels in mouse islets after incubation with media alone (control) or EX4 for 4 hr. Relative expression values were normalized to the control group. F. MIN6 -cells were treated with or without EX4 and with various inhibitors, as indicated, for 8 hr. Relative expression values for

Rspo1 were normalized to 18S rRNA and then to the control treatment. * p < 0.05, ** p < 0.01.

2.5 Discussion

Previous studies have demonstrated that the cWnt signaling pathway plays a crucial role in the maintenance of -cell behaviour (248;451;463;465). Recently, Rspo has been established as a novel family of secreted activators of cWnt signaling (506). Although Rspo1 has been detected in human pancreas (507), the effects of Rspo1 on the -cell have not been explored.

We now provide evidence that murine islets, MIN6 and βTC -cells express Rspo1. We further found that Rspo1 activates cWnt signaling in the MIN6 -cells, and that Rspo1 not only enhances -cell growth and survival, but is also an insulin secretagogue.

In the present study, we have found expression of Rspo1 in multiple -cell models. It is interesting to note that the MIN6 and βTC -cells as well as murine islets also expressed two other isoforms of Rspo (i.e. Rspo3 and Rspo4). However, TC -cells also expressed Rspo2, whereby this isoform was undetectable in the other models, raising the possibility that the TC

-cell line may not be directly comparable to murine islets in vivo. Previous studies have demonstrated that MIN6 -cell line retains normal regulation of GSIS, similar to isolated mouse islets, whereby other -cell lines such as RIN, HIT and TC cells do not exhibit physiological

GSIS (549). Moreover, MIN6 -cells were demonstrated to express high levels of Rspo1 mRNA, as well as Rspo1 protein, therefore serving as a useful model for the present investigation. The MIN6 -cells were further found to express functional cWnt signaling, as indicated by expression of essential cWnt signaling molecules, as well as by nuclear -catenin translocation and cWnt target gene expression (i.e. c-myc and cyclinD1) in response to LiCl and

Wnt3a. In line with previous reports that Rspo1 can activate cWnt signaling

(506;507;526;532;533), we also found that Rspo1 increased nuclear -catenin as well as the cWnt target gene, c-myc in MIN6 -cells. In contrast, we did not see any changes in expression

85 levels of cyclinD1 after 12 hr treatment of Rspo1. This observation gives rise to the possibility that these genes exhibit differential responsiveness to or temporal regulation by Wnt3a and

Rspo1 in the MIN6 -cells, such as reported for their responses to other growth factors/hormones (e.g. estradiol vs. insulin) (550).

-cell growth in vivo is determined by the rates of replication and apoptosis, as well as neogenesis (551). Several lines of evidence have established cWnt signaling as a pathway that regulates -cell growth: 1) conditional pancreatic -cell specific expression of degradation- resistant -catenin leads to -cell expansion, increased insulin production and serum levels, and enhanced glucose handling (463), and 2) endogenous Wnt3a is required for basal proliferation of INS-1 cells (451). Consistent with these findings, we found an enhancement of MIN6 -cell proliferation in response to treatment with Wnt3a. Furthermore, Rspo1 was also found to induce significant growth of both the MIN6 cells and dispersed murine -cells in vitro.

Interestingly, the highest dose of Rspo1 tested did not stimulate any further proliferation in the

MIN6 -cells, and it remains possible that this cell line became desensitized by this recombinant protein. Further studies to examine the potential regulatory mechanisms induced by Rspo1 are crucial in understanding its role in cWnt signaling. Nevertheless, these findings are consistent with studies demonstrating that Rspo1 enhances intestinal growth through a cWnt-dependent pathway (506;507).

Our finding that Rspo1 exerts proliferative effects on the -cell prompted the question as to whether Rspo1 also protects the MIN6 -cells from apoptosis. The cytotoxic effects of cytokines on -cells have been demonstrated to include apoptosis, with caspase3 as the enzyme responsible for the features of cell death in this model (552). Consistent with previous results in INS-1E -cells (413), we found that cytokine treatment increased cleaved caspase3 activity in the MIN6 -cells, whereas treatment with EX4 decreased cytokine-induced caspase3 levels.

However, in addition to proliferation, several downstream mediators of cWnt signaling have been found to regulate apoptosis in a variety of cell types, including -cells (248;553-559). The present study shows, for the first time, that Rspo1 inhibits cytokine-induced apoptosis in the

MIN6 -cells. Consistent with this observation, we also report a parallel anti-apoptotic effect of

Rspo1 in dispersed mouse -cells treated with cytokines. Moreover, we found that the anti- apoptotic effect of Rspo1 in MIN6 -cells was not further enhanced by the addition of the cWnt ligand, Wnt3a. However, given the possibility that one mechanism of action of Rspo1 involves enhanced Wnt ligand activity through stabilization of the Frz and LRP5/6 receptor complex

(526), this observation does not preclude a requirement for endogenously-secreted Wnt ligands for the actions of Rspo1. It remains possible that the MIN6 -cells, like INS-1E -cells (451), secrete endogenous Wnt ligands, in which case, further addition of the Wnt ligand may not be required for the anti-apoptotic effect of Rspo1.

To further establish the functional role of Rspo1 in regulating -cell behaviour, the effect of Rspo1 on insulin secretion was investigated. Although cWnt signaling molecules have been found to enhance insulin secretion from INS-1 -cells (465;467), the mechanism of action is unclear. Nonetheless, Fujino et al found impaired glucose-stimulated insulin secretion in LRP5 knockout mice, in association with decreased expression of glucokinase (465). In this study, we demonstrated that Rspo1 enhances insulin secretion in MIN6 and dispersed mouse -cells under acute conditions. Interestingly, Rspo1-induced insulin secretion in both MIN6 and dispersed -cells was independent of glucose levels as also reported for Wnt3a (465).

Moreover, we also found that Rspo1 upregulates insulin mRNA expression in vitro. In line with this observation, Loder et al reported that silencing of TCF7L2, a crucial transcription factor in the cWnt signaling pathway, results in reduced levels of insulin mRNA (501). A recent study by da Silva Xavier et al has further demonstrated that the TCF7L2 gene is required for

87 maintenance of -cell genes regulating secretory granule fusion (502). It therefore remains possible that Rspo1 and the cWnt signaling pathway can also regulate insulin secretion chronically, at the level of insulin secretory granules. Given the importance of the TCF7L2 gene as a strong predictor for the development of T2DM (337), the regulation of insulin secretion and gene expression by Rspo1 warrants more detailed investigation.

-cell behaviour is determined by many factors including glucose as a major regulator of insulin synthesis and release, as well as of -cell mass (195;560;561). Furthermore, GLP-1 and its long-acting receptor agonist, EX4, have been well-characterized as both glucose-dependent insulin secretagogues and -cell growth factors. We have now demonstrated that EX4 increases

Rspo1 expression in the MIN6 -cells in a dose- and time-dependent fashion and that this occurs only under high glucose conditions. This novel finding was not restricted to the MIN6 - cell line, as similar effects of EX4 on Rspo1 mRNA were observed in mouse TC cells, as well as in murine islets. Although interesting to note that glucose levels altered EX4-induced Rspo1 mRNA levels, this observation was not entirely surprising. Glucose regulation of -cell behaviour has been well-established and numerous studies have shown this nutrient can operate as a facilitator to enhance the actions of -cell growth factors. Most notably, glucose confers - cell responsivity by co-regulation with cAMP-increasing incretins such as GLP-1 that is especially vital for their mitogenic/anti-apoptotic actions (412;562;563).

Finally, the binding of GLP-1 or EX4 to the GLP-1R is known to stimulate adenylyl cyclase, leading to an increase in intracellular cAMP levels and activation of PKA (393;564-

566). However, treatment with the PKA inhibitor H89 did not change basal or EX4-stimulated levels of Rspo1 mRNA, indicating that PKA is not required for this effect. GLP-1 has also been reported to stimulate a number of MAPK signaling pathways, including ERK1/2 (406;567-570) and p38 MAPK (571;572), to regulate -cell behaviour. Nonetheless, we found that inhibition

88 of ERK1/2 with either PD98059 or U0126, or of p38 MAPK with SB203580, did not affect basal or EX4-induced changes in Rspo1 mRNA levels. In contrast, co-incubation of MIN6 - cells with EX4 and the PI3-kinase inhibitors, LY294002 and wortmannin, abolished the EX4- induced increase in Rspo1 transcript levels. It is interesting to note that the PI3-kinase/Akt pathway appears to be involved in many pathways regulating -cell behaviour. Most notably,

GLP-1 has been shown to exert its proliferative and anti-apoptotic effects in INS-1E cells via

PI3-kinase/Akt, as these beneficial effects were abolished in the presence of wortmannin and by overexpression of a kinase-dead Akt construct (400;413). Moreover, PI3-kinase gamma knockout mice demonstrate abnormal β-cell secretory responses that may involve downstream glucose-sensing pathways (544;573). Our findings that EX4 regulates Rspo1 mRNA levels via a PI3-kinase-dependent pathway, therefore adds further evidence for a role of PI3-kinase in

GLP-1 signaling in the -cell.

Numerous investigations of transgenic mice expressing cWnt signaling molecules have provided clear evidence for the impact of cWnt signaling pathway in regulating -cell biology.

Our present data provide further support for this view by demonstrating, for the first time, the growth, survival and functional effects of Rspo1 on the -cell. Studies of cWnt signaling in - cells from insulin resistant or diabetic models have only been recently reported. Krützfeldt et al observed a relative increase in Wnt4, a specific inhibitor of cWnt signaling, in islets of insulin- resistant mice (574). Alternatively, Lee et al reported upregulation of several cWnt signaling molecules, including -catenin, TCF7L2, and the cWnt ligand Wnt2b in islets from subjects with T2DM (575). The results of these studies suggest that cWnt signaling can be altered in insulin resistant and/or diabetic states. There are currently no reports to-date to examining the

-cell responses to and/or secretion of Rspo1 under such pathophysiological conditions. Future

89 research in this area will therefore be important if Rspo1 is to be considered as a novel target for the therapeutic treatment of patients with T2DM.

2.6 Acknowledgements

The authors are grateful to Drs. J. Miyazaki (University of Tokyo) and D.F. Steiner

(University of Chicago) for the gift of MIN6 -cell, and to Angelo Izzo (University of Toronto) for his technical expertise in mouse islet preparation. This work was supported by an operating grant from the Canadian Diabetes Association (#2374) and by an equipment grant from the

Banting and Best Diabetes Centre (BBDC), University of Toronto. V.S.C.W. was supported by a Doctoral Research Award from the Canadian Institutes of Health Research, W.S. and A.Y. by the BBDC Summer Studentship program, University of Toronto, and P.L.B. by the Canada

Research Chairs program.

CHAPTER 3

THE NOVEL ROLE OF R-SPONDIN-1 IN THE -CELL IN VIVO

The work presented in this chapter corresponds to the following manuscript:

Wong V.S., Oh A., Chassot A., Chaboissier C.M., Brubaker P.L. Submitted for publication.

Author contributions:

A. Oh was a 4th year undergraduate student working directly under my supervision and contributed to the examination of β-cell apoptosis via IHC for cleaved caspase3 as well as to the determination of β-cell size (Figure 3.3C).

Drs. A. Chassot and C.M. Chaboisser generated and provided the French colony of Rspo1-/- mice.

3 R-spondin-1 deficiency in mice in vivo improves glycemic control and increases -cell

mass.

3.1 Abstract

R-spondin1 (Rspo1) is a gut growth factor that acts through canonical Wnt (cWnt) signaling, leading to induction of cWnt target genes (i.e. c-myc). We previously established that Rspo1 stimulates proliferation and insulin secretion, and inhibits cytokine-induced apoptosis, in MIN6 and murine β-cells in vitro. We also demonstrated that Exendin-4 (EX4), a glucagon-like peptide-1 receptor agonist, stimulates Rspo1 production in vitro. We thus investigated the role of Rspo1 in -cells in vivo using Rspo1 knock-out (Rspo1-/-) mice. Rspo1-/- mice had normal fasting glycemia and demonstrated no differences in body and pancreatic weights compared to wild-type (Rspo1+/+) mice. However, unexpectedly, Rspo1-/- mice had improved glycemic control after an oral glucose challenge compared to Rspo1+/+ mice, with no difference in insulin sensitivity but an enhanced insulin response over 30 min; glucagon responses were normal.

Rspo1 deficiency also resulted in a 2.3-fold increase in -cell mass in association with a 2.3-fold increase in Ki67-positive -cells, a marker of proliferation, relative to Rspo1+/+ mice.

Unexpectedly, Rspo1-/- pancreatic tissues also demonstrated a significant increase in the number of insulin-positive ductal cells, suggestive of -cell neogenesis. In contrast to the in vivo findings, Rspo1-/- islets displayed no changes in glucose-induced insulin secretion but showed a complete absence of glucose-induced suppression of glucagon secretion. Treatment of Rspo1-/- mice for 2 wk with EX4 resulted in a similar glycemic profile to EX4-treated Rspo1+/+ mice after an oral glucose challenge, with no changes in insulin sensitivity. Interestingly, EX4 administration to Rspo1-/- normalized -cell mass to a level comparable to that in Rspo1+/+ mice.

The present study therefore reveals a novel role for Rspo1 as a regulator of -cell behaviour in vivo, and suggests novel roles for Rspo1 in both - and ductal cells.

3.2 Introduction

Type 1 (T1DM) and Type 2 Diabetes (T2DM) are complex metabolic disorders rooted in a single cause: loss of functional β-cell mass. Thus, there is a continual growth in the number of studies that are focused on developing strategies to maintain and/or promote β-cell growth and function. The canonical Wnt (cWnt) signaling pathway has recently been implicated in β- cell development and function. Initial studies have identified multiple Wnt ligands, Frizzled

(Frz) receptors and other modulators of the cWnt signaling pathway in the developing and mature murine pancreas (338;451-456;507;576). Further studies revealed that cWnt pathway is involved in regulating mature β-cell growth: Rulifson et al demonstrated that overexpression of constitutively active β-catenin in mouse β-cell leads to activation of cWnt pathway with concomitant increase in cWnt target genes and a significant increase in β-cell mass and function

(463). In contrast, ectopic expression of negative regulators of cWnt signaling, such as axin or

GSK3β, decreases β-cell mass and proliferation (463;464). Moreover, cWnt signaling is also implicated in regulating β-cell function. Studies using LRP5-/- mice revealed an impaired glucose tolerance due to reduced glucose-stimulated insulin secretion (GSIS) in association with a significant decrease in mRNA levels of β-cell transcription factors (e.g. Tcf1, Tcf2, Foxa1,

HNF-4α), glucokinase, and insulin-signaling proteins in the LRP5-/- islets (465). In line with this observation, Schinner et al found that incubation of primary mouse islets and INS1 cells in vitro with adipocyte-derived Wnt molecules increases insulin secretion and transcription of glucokinase gene (467).

The impact of cWnt signaling in β-cells is highlighted by the report that single nucleotide polymorphisms in TCF7L2 gene are associated with the development of T2DM and are currently the strongest genetic indicator for this metabolic disorder. Although it remains unclear as to how polymorphisms in TCF7L2 translate to functional defects in β-cells, in vitro

93 expression of dominant-negative or siRNA knockdown of TCF7L2 in β-cells or islets decreases

β-cell proliferation and GSIS (451;456;501;502) whereas overexpression of this transcription factor in mouse and human islets protects β-cell death from glucotoxicity or cytokine-induced apopotsis (456). Interestingly, Liu and Habener reported that a β-cell growth factor, GLP-1, requires an active cWnt signaling pathway to elicit its beneficial effects on β-cell proliferation

(451). Moreover, they also reported that a chemokine, stromal-cell derived factor-1 (SDF-1), also activates and requires the cWnt pathway for its cytoprotective actions on β-cells (248).

Regardless of the proposed differential pathways utilized by GLP-1 and SDF-1, these observations strongly suggest that cWnt signaling in mature β-cells is a promising therapeutic revenue.

The roof plate-specific spondin (R-spondin; Rspo) protein family consists of four related members (namely Rspo1-4) that have structural similarities with conserved cysteine-rich furin- like and thrombospondin (TSP) domains (506). Numerous studies have demonstrated that

Rspo1 is a regulator of the cWnt signaling pathway, both in development (504;505;543) and in the adult mouse (504). Although the precise mechanism by which Rspo1 activates cWnt signaling remains unclear, several recent studies have demonstrated that Rspo family members function as ligands and/or modulators of the cWnt co-receptors, Frz and LRP (532) (526). As a result of these interactions, Rspo induces the cWnt signaling pathway and initiates Wnt target gene expression (532).

We have recently demonstrated the presence of Rspo1 mRNA transcripts in murine pancreas and islets, as well as in the murine MIN6 and βTC β-cell lines (576). We have further used these in vitro models to establish that Rspo1 is a -cell growth factor, stimulating -cell proliferation and inhibiting -cell cytokine-induced apoptosis (576). Rspo1 also enhances insulin secretion in a glucose-independent fashion in these cells (576). Moreover, treatment of

-cells with EX4 increases Rspo1 expression in a glucose-, time- ,dose-and PI3-kinase- dependent manner (576). However, although Rspo1 has been recently examined for its role in reproductive development using the Rspo1 knock-out (Rspo1-/-) mouse (510), its effects on glucose metabolism in vivo have not been explored. We have therefore now examined whole body glucose homeostasis and markers of β-cell growth and function using the Rspo1-/- mouse.

3.3 Experimental Procedures

3.3.1 Animals.

Rspo1-/- mice were generated by insertion of the LacZ gene followed by a neomycin resistant cassette into exon3 of Rspo1; mice were genotyped as previously described (510).

Unless otherwise indicated, animals were given ad libitum access to water and standard rodent chow with a 12 hr light/dark cycle. All animal protocols were approved by the Animal Care

Committee of the University of Toronto and by the Université de Nice-Sophia Antipolis

(France), and all in vivo experiments were performed using mice at both sites (Nice, France and

Toronto, Canada). The Rspo1-/- mice housed in France were on a C57Bl/6 and FVBN mixed strain and while the Rspo1-/- mice in Toronto were crossed onto a CD1 background

(C57Bl/6/FVBN/CD1). Preliminary comparison between two strains show no differences body weights, and metabolic responses. Therefore, metabolic data are shown as combined data from two mouse strains. In vitro studies were performed using mice from the Toronto colony only.

Wild-type (Rspo1+/+) and Rspo1-/- mice were age- (6-12 wk) and sex-matched; some animals were injected with either PBS (vehicle) or EX4 (10 nmol/kg, ip; Bachem, Torrance, CA) daily for 14 d, as previously described (544).

3.3.2 Metabolic Tests.

Mice were fasted overnight or for 6 hr for oral glucose tolerance tests (OGTT) and insulin tolerance tests (ITT), respectively. Basal blood samples were collected from a tail vein at t = 0 min for measurement of glucose using the One Touch Basic glucose meter (a kind gift from Lifescan Canada, Burnaby, BC). For OGTT, mice were gavaged with glucose (1.5 mg/g) and additional blood samples were collected at t = 10, 20, 30, 60, 90, and 120 min for glucose measurements, and at t = 0 and 30 min for determination of plasma insulin and glucagon concentrations using an Insulin ELISA for small sample volumes (Crystal Chem, Chicago, IL)

96 and a glucagon radioimmunoassay kit (Linco Research, St. Louis, MO), respectively. For ITT, mice were injected (ip) with human biosynthetic insulin (0.3 U/kg; Novo Nordisk

Pharmaceutical Industries, Toronto, ON), and additional blood samples were collected at t = 10,

20, 30, 60, 90, and 120 min for glucose measurements.

3.3.3 Immunological and morphometric analyses.

Mouse tissues (pancreas (cut into 6-8 pieces), liver, adipose, muscle and small intestine) were weighed, fixed in formalin (Sigma-Aldrich, Oakville, ON), paraffin-embedded, sectioned and stained with hematoxylin & eosin for gross morphometric analyses. For determination of β- cell mass (BCM), pancreatic sections were dewaxed, hydrated, and incubated overnight at 4˚C with a guinea pig anti-insulin antibody (Dako Diagnostics, Mississauga, ON). The sections were then incubated for 1 hr at room temperature with biotinylated anti-guinea pig antibody (Vector

Laboratories, Burlington, ON), and subsequently treated for 1 hr with avidin/biotin complex

(Vectastain Elite ABC Kit; Vector Laboratories, Burlingame, CA). Slides were stained with

3,3'-diaminobenzidine tetrahydrochloride (Sigma-Aldrich) for 5 min, washed with tap water, and counterstained with hematoxylin. Pancreatic slides were then scanned at the Advanced

Optical Microscopy Facility (Princess Margaret Hospital, Toronto, ON) and β-cell and total pancreatic area per section were measured using Aperio ImageScope software (Aperio

Technologies, Vista, CA). Total BCM for each pancreas was determined as the product of the total cross-sectional β-cell area over the total pancreatic area times the weight of the pancreas.

3.3.4 Immunoblotting.

Proteins were collected from pancreas and immunoblotted, as previously described in

Chapter 2, using primary antibodies targeted against mouse Rspo1 (goat IgG, 1:1000; R&D

Systems, Minneapolis, MN), or pan-actin (rabbit IgG, 1:1000; Sigma Aldrich), followed by incubation with HRP-linked anti-goat (1:2000; Jackson Immunoresearch Laboratories, West

Grove, PA) and anti-rabbit (1:2000; New England Biolabs, Pickering, ON) secondary antisera and visualization using electrochemical luminescence (Amersham Pharmacia Biotech, Baie d Urfe, QC).

3.3.5 qRT-PCR.

Murine islets were isolated by collagenase digestion and maintained for 2 d, as previously described in Chapter 2. Islets were then lysed using the RNeasy Micro Kit, according to the manufacturer‟s instructions (Qiagen Inc., Mississauga, ON). Semi-quantitative

RT-PCR (qRT-PCR) was performed in a Chromo4 Continuous Fluorescence Detection unit with

Opticon Monitor 3 software (Bio-Rad Laboratories, Mississauga, ON, Canada) using Taqman

Gene Expression Assays for specific primers (Applied Biosystems, Foster City, CA). All reactions were performed in duplicate, and control reactions were performed without RT enzyme and/or without template. The linearity of amplification of the Taqman primer-probe sets was verified over nine orders of magnitude (data not shown). Ribosomal protein 18S RNA (no.

Hs99999901_sl) was used as the endogenous control for all quantitative analyses of mRNA expression and was not found to change in response to any of the experimental treatments tested

(data not shown). Relative quantification of glucokinase (Gck; no. Mm00439129_m1), Glut2

(no. Mm00446224_m1), insulin2 (no. Mm00731595_gH), Pdx-1 (no. Mm00435565_m1),

Rspo2 (no. Mm00555790_m1), 3 (no. Mm00661105_m1) and 4 (no. Mm00615419_m1) mRNA expression was calculated using the cycle threshold [ C(t)] method (545).

3.3.6 In vitro secretion assays.

Secretion studies using isolated mouse islets were performed as previously described in

Chapter 2. Briefly, islets were cultured overnight in 20 mM glucose RPMI1640 (Gibco

BRL/Invitrogen) with 10% FBS and penicillin/streptomycin. Islets were then washed and incubated with experimental media that consisted of either low (2 mM) or high glucose (20 mM)

RPMI 1640 with or without EX4 (10 nM) for 2 hr. A total of 10 islets of approximately the same size were used per treatment group. Media samples were centrifuged at 700 X g at 4°C for

1 min and the supernatant was collected. Samples were then radioimmunoassayed for insulin and glucagon using insulin and glucagon kits from Linco Research. Islet DNA was collected by extraction in extraction solution containing 75% ethanol and 0.09N hydrochloric acid and was determined by spectrophotometry.

3.3.7 Statistical Analysis.

Analysis System software (SAS v 9.1.3, Cary, NC). Statistical significance was assumed at p<0.05.

3.4 Results

3.4.1 Rspo1-/- mouse pancreas are phenotypically indistinguishable from their wild-type

counterparts.

Rspo1-/- mice were phenotypically normal, consistent with previously published results

(510). They shared similar body weights to their wild-type counterparts with no differences in their pancreatic weights (Figure 3.1A and B). qRT-PCR for the other isoforms of Rspo (Rspo2-

4) revealed no significant changes in islets from Rspo1-/- mice relative to wild-type islets, indicating no compensatory responses (Figure 3.1C). To confirm the results of the genotyping, pancreatic lysates from Rspo1+/+ and Rspo1-/- animals were examined by Western blot for Rspo1 protein. As shown in Figure 3.1D, Rspo1+/+ pancreas was positive for Rspo1, whereas its expression was minimal in pancreata from Rspo1-/- mice.

Gross morphological analyses of insulin-responsive tissues from wild-type and knockout mice revealed no remarkable changes in adipose tissue, liver or skeletal muscle (Figure 3.1E).

Since Rspo1 is also a potent gastrointestinal growth factor (507), we also sought to see if there were changes in the small intestine. Figure 3.1E demonstrates no remarkable differences between the jejunal architecture of Rspo1+/+ and Rspo1-/- mice.

3.4.2 Rspo1-/- mice display better glucose handling without changes in insulin sensitivity.

We next analyzed the effects of Rspo1 deficiency on whole-body glucose metabolism.

Fasting glycemia was in the normal range for wild-type mice (6.3 ± 0.5 mM), and was not significantly different in knockout animals (5.2 ± 0.5 mM; Figure 3.2A). However, Rspo1-/- mice had significantly better glycemic control after an oral glucose challenge (Figure 3.2A).

Consistent with this observation, the area-under-the-curve (AUC) for the glycemic response to oral glucose was significantly lower in the knockout animals relatively to the wild-type mice

100

(p<0.05, Figure 3.2A inset). In contrast, the glycemic response to an insulin tolerance test was not different between wild-type and knockout mice (Figure 3.2B).

In line with the improved glycemic control in Rspo1-/- mice, there was a significant increase in plasma insulin levels in response to oral glucose in the Rspo1-/- mice as compared to the muted response observed in the wild-type animals (p<0.05, Figure 3.2C). Plasma glucagon concentrations were not different between the animals in either the fasting state (e.g. at t = 0 min) or at t = 30 min following the oral glucose challenge (Figure 3.2D).

3.4.3 Rspo1-/- mice have increased β-cell mass due to increases in β-cell proliferation and

neogenesis.

One possible mechanism underlying the better glycemic handling in Rspo1-/- mice could involve changes in the number of β-cells. Therefore, we stained pancreatic sections for insulin to determine total BCM. Islet architecture and appearance were normal; however, Rspo1-/- mice were found to have significantly increased BCM, by 2.3-fold relative to wild-type mice (p<0.05,

Figure 3.3A). To determine whether the changes in BCM were a result of increased numbers of islets and/or β-cells, stained sections were subjected to morphometric analyses. The average number of islets per pancreatic section, and their distribution by size (arbitrarily set as 1, 2-100 or greater than 100 β-cells) were not different between wild-type and knockout mice (Figure

3.3B). However, examination of pancreatic sections that were co-stained for insulin and the proliferative marker, Ki67, demonstrated that β-cell proliferation was significantly increased, by

2.3-fold (p<0.01) in knockout mice relative to wild-type animals, mirroring the changes observed in BCM (p<0.01, Figure 3.3C). The number of apoptotic β-cells, detected by insulin and cleaved-caspase3 co-staining, was extremely low in all pancreata (~1-2 apoptotic β-cells per

~1500 β-cells); nonetheless, no changes in β-cell apoptosis could be detected in the Rspo1-/- mice (data not shown). Moreover, we saw no changes in β-cell size as determined by the

101 number of insulin-positive cells within a fixed circumference (data not shown). However, there were significantly more insulin-positive ductal cells in the Rspo1-/- mice, an indication of β-cell neogenesis, whereas such cells were almost completely absent in the Rspo1+/+ animals (p<0.05,

Figure 3.3D).

In keeping with the observed increase in BCM, qRT-PCR demonstrated that ins2, Pdx-1, gck and glut2 mRNA levels were increased in islets isolated from Rspo1-/- mice compared with those obtained from wild-type animals (p<0.05-0.01, Figure 3.3E).

3.4.4 Rspo1-/- mouse islets display normal insulin release but abnormal glucagon secretion.

To better understand the islet responses to increasing glucose concentrations, we next examined hormone secretion from isolated islets. Incubation with high glucose (20 mM) for 2 hr stimulated insulin secretion (GSIS) in islets from wild-type mice (p<0.05, Figure 3.4A).

Moreover, both basal insulin secretion and GSIS were normal in islets from Rspo1-/- mice

(p<0.05, Figure 3.4A). In contrast, although glucagon release was not different between the two groups of islets under low glucose conditions, Rspo1-/- islets failed to demonstrate normal glucose-induced suppression of glucagon release, as was observed in Rspo+/+ islets (p<0.05;

Figure 3.4B).

3.4.5 Rspo1 may be required for Exendin-4-regulation of β-cell mass.

Finally, since we have previously shown that Rspo1 is regulated by EX4 treatment in murine β-cells in vitro, we determined whether the response to chronic EX4 treatment is affected by the loss of Rspo1. Following treatment of the mice for 2 weeks with EX4 (10 nmol/kg i.p.), an OGTT revealed that Rspo1-/- retained the trend of a lower glycemic profile relative to the wild-type animals, but that this difference was no longer statistically significant

(Figure 3.5A). ITT also revealed a trend towards reduced insulin sensitivity in the EX4-treated

Rspo1-/- mice relative to their wild-type counterparts but this change did not reach statistical

102 significance (Figure 3.5B). However, in contrast to the significant differences in BCM seen between Rspo1+/+ and Rspo1-/- mice in the basal state, 2-wk of EX4 treatment completely abolished these differences, such that BCM was not different between the two groups of animals

(Figure 3.5C). Interestingly, the loss of difference between the wildtype and knockout mice for both glucose tolerance and BCM appeared to be due to a reduced response of the Rspo1-/- mice to EX4 treatment, as islets from wild-type and Rspo1-/- mice treated with EX4 for 2 wk did not demonstrate any differences in insulin secretion under basal conditions or in response to stimulation with high glucose (p<0.05; Figure 3.5D).

103

Figure 3.1. Rspo1-/- mice are phenotypically indistinguishable from their wild-type counterparts. A. Body weights of Rspo1+/+ and Rspo1-/- over the course of 16 days (n = 11-

12). B. Pancreatic weights normalized to body weights (n = 11-12). C. qRT-PCR analysis of

Rspo2-4 mRNA in isolated murine islets. Relative expression levels of Rspo2-4 were normalized to 18S rRNA expression (n = 4-10). D. Immunoblotting analysis of Rspo1 from

104 pancreatic lysates of Rspo1+/+ and Rspo1-/- mice. Relative protein levels of Rspo1 were normalized to total actin (n = 5-6; representative blot is shown). E. H&E stained sections of adipose, liver, skeletal muscle and jejunal tissues from Rspo1+/+ and Rspo1-/- mice (n = 11-12, representative figures are shown).

105

Figure 3.2. Rspo1-/- mice have improved glycemic control. A. Glycemic profiles in Rspo1+/+ and Rspo1-/- mice after an oral glucose challenge (OGTT). Inset: Area-under-the-curve (AUC) of the glucose excursions (n = 11-12). B. Glycemic responses in Rspo1+/+ and Rspo1-/- to an intraperitoneal insulin tolerance test (ITT). Inset: AUC of the glucose excursions (n = 11-12).

C. and D. Plasma insulin (C) and glucagon (D) levels at t = 0 and 30 min after administration of an OGTT (n = 3-7). * p < 0.05.

106

107

Figure 3.3. Rspo1-/- mice have an increase in BCM. A. and B. BCM (A) and total number of islets per section (B) were determined in insulin-stained pancreatic sections from Rspo1+/+ and

Rspo1-/- mice (n = 11-12). Islets were arbitrarily distributed based on size, as either single insulin-positive cells, between 2 to 100 insulin positive-cells or more than 100 β-cells. C. The number of β‐cells within a fixed circular area of 700μm2 was counted. The approximate size of a β‐cell was then determined by dividing area of circle by number of β‐cells within the fixed area. D. Proliferating β-cells as determined by co-staining for insulin and Ki67. The number of proliferating β-cells was normalized to the total number of β-cells. Arrows indicate positive cells. E. β-cell neogenesis was determined by the number of insulin-positive cells in the pancreatic ducts. Arrows indicate positive cells. (n = 11-12). F. qRT-PCR analyses of ins2,

Pdx-1, gck, and glut2 mRNA in isolated murine islets (n = 4-10). Relative expression levels of each transcript were normalized to 18S rRNA expression. *, P < 0.05, **, P < 0.01.

108

Figure 3.4. Rspo1-/- have normal insulin secretion but an abnormal glucagon response to high glucose. A. and B. Effects of low and high glucose on insulin (A) and glucagon secretion

(B) by isolated murine islets were determined by radioimmunoassay. Islets were incubated in serum-free media overnight, pre-treated with low glucose, and then incubated low or high glucose concentrations as indicated. Results were normalized to total DNA content and data are expressed as fold of wild-type islets under low glucose conditions (n = 4-5). * p < 0.05.

109

Figure 3.5. Treatment with EX4 normalizes glucose homeostasis and BCM in Rspo1-/- mice. A. and B. OGTT (A) and ITT (B) were performed in Rspo1+/+ and Rspo1-/- mice after 2 wk of EX4 treatment (n = 10). C. BCM was determined in insulin-stained pancreatic sections from EX4-treated mice (n = 10). D. Islets were collected from EX4-treated Rspo1+/+ and

Rspo1-/- mice for GSIS analyses. Insulin secretion was analyzed by radioimmunoassay and results were normalized to total DNA content. Data were expressed as fold of untreated wild- type mouse islets under low glucose conditions (n = 4-5).

110

3.5 Discussion

Recent studies have established the importance of cWnt signaling in the regulation of - cell behaviour (577). Rspo1 has recently been established as a novel regulator of cWnt signaling in the β-cell (506), Hence, in MIN6 -cells, Rspo1 increases nuclear β-catenin translocation in association with increased c-myc and ins2 mRNA transcript levels. The effects of Rspo1 on both MIN6 and primary murine -cells in vitro also include enhanced proliferation and protection from cytokine-induced apoptosis, as well as stimulation of glucose-independent insulin secretion (576). We now provide novel findings that Rspo1 is also a regulator of whole- body glucose homeostasis via changes in -cell behaviour in vivo.

In the present study, we have confirmed using immunoblotting the knockout of Rspo1 protein in the pancreatic lysate of Rspo1-/- mouse. The detection of lower levels of Rspo1 protein is consistent with the positional interruption in exon 3 of the Rspo1 gene. The unaffected region of exon 1 to 2 may still be transcribed and translated, and given that our anti-

Rspo1 antibody was raised against the near-entirety of Rspo1 sequence (amino acids 21-209), the presence of detectable Rspo1 in knockout mice may represent non-specific binding of the antibody to other isoforms of Rspo which share 40-60% pair-wise amino acid sequence identity as well as having similar molecular weights to Rspo1 (505). We have found that Rspo1 deficiency does not impact overall body and pancreatic weights, and that there are no gross morphological differences in three principle insulin-sensitive tissues, namely, adipose tissue, liver and skeletal muscle. Given that Rspo1 is a potent gastrointestinal mitogen (507;578), it is interesting to note that there were also no remarkable morphological changes in the small intestine of knockout mice. Together these findings indicate that, while Rspo1 is required for normal reproductive system development (510), it may be dispensable for development of these major nutrient-handling tissues.

111

We have previously shown that Rspo1 is expressed in murine islets (576). Here we show by qRT-PCR that the other known isoforms of Rspo (i.e. Rspo2-4) are also expressed in murine islets. Previous studies have shown that many transgenic mouse models demonstrated a compensatory response by upregulating other isoforms of the deficient protein (579-581).

However, we were unable to detect any upregulation of the other isoforms of Rspo in the Rspo1-

/- animals, at least in the isolated murine islets. This finding suggests that, despite the known similar actions of these 4 isoproteins in the stimulation of gastrointestinal growth (506), they do not exhibit compensatory actions in the islet.

Somewhat surprisingly, the glycemic profile during an OGTT was found to be significantly reduced in the Rspo1-/- mice relative to Rspo1+/+ mice, indicating improved glucose handling in the absence of Rspo1. This phenomenon was associated with a greater change in plasma insulin from 0 to 30 min in the Rspo1-/- mice, in the absence of any difference in glucose response to insulin between Rspo1+/+ and Rspo1-/- animals. Together these findings suggested that the differences resided at the level of the pancreatic -cell. Consistent with this possibility, we found a significant increase in BCM in Rspo1-/- mice, by 2.3-fold relative to the Rspo1+/+ mice.

-cell growth in vivo is determined by the rates of proliferation and apoptosis of existing

-cells, as well as by islet neogenesis. We found that the increase in BCM in Rspo1-/- animals was not due to changes in -cell apoptosis or in the total number or size distribution of the islets but, rather, was due mainly to an increased number of proliferating -cells. These findings stand in marked contrast to our previous report that treatment of MIN6 and primary murine -cells with Rspo1 in vitro increases -cell proliferation (576). This discrepancy between the in vitro and in vivo findings is difficult to reconcile. However, the hormone secretion studies using isolated islets may provide some insight, wherein GSIS was normal, but there was a complete absence of the normal suppression of glucagon secretion seen in response to high glucose. This

112 observation implies that local release of glucagon within the islets may lead to -cell expansion, resulting in improved glucose homeostasis in the Rspo1-/- animals. Consistent with this possibility, -cell-specific overexpression of the glucagon receptor results in improved glucose tolerance in an OGTT and increased BCM, with no changes in insulin sensitivity (582), similar to the findings of the present study. Although it is unknown as to how Rspo1 may functions in the α-cell, preliminary studies in our laboratory indicate that Rpso1 mRNA transcripts are detectable in the TC murine -cell line (data not shown).

Interestingly, we also found an increased number of insulin-positive ductal cells in the

Rspo1-/- mouse pancreas. Such observations have been characterized by others as -cell neogenesis, whereby ductal cells differentiate to give rise to new -cells (583). Whether increased neogenesis contributes to the enhance BCM observed in Rspo1 null mice remains unclear at the present time. Nonetheless, collectively, these findings suggest that the actions of

Rspo1 in the endocrine pancreas may not be restricted to the -cell.

The rate of -cell apoptosis in the wild-type mice in the present study was extremely low, at ~0.1%, and ~0.2% in both wild-type and Rspo1-/- mice. This is consistent with the reported apoptotic rate of <0.1% hr in normal C57Bl/6 mice (405). Although we have previously reported that treatment of MIN6 and primary murine -cells with Rspo1 in vitro prevents cytokine-induced apoptosis (576), this observation is similar to findings made with epidermal growth factor (EGF) wherein EGF protects -cells from hydrogen peroxide-induced apoptosis in vitro, but overexpression of a dominant-negative EGF receptor in the mouse pancreas did not alter -cell apoptosis in vivo (276). Future studies using metabolically challenged animals may be necessary to reveal the role of Rspo1 in -cell survival and/or adaptation, as recently reported for the conditional β-cell specific GSK3β (β-GSK3β) knockout mice. When fed on a high-fat diet, these mice exhibit improved glucose tolerance and expanded

113

BCM with increased proliferation, in association with increased levels of islet IRS1, IRS2 and

PDX-1 proteins levels, as well as activation of Akt/PKB (584).

We previously established a relationship between Rspo1 and the GLP-1 receptor agonist,

EX4 in vitro, such that acute treatment with EX4 was found to increase the levels of Rpso1 mRNA transcripts in multiple cell models, as well as to enhance Rspo1 protein levels in MIN6 cells (576). It was therefore interesting that treatment with EX4 for 2 weeks eliminated the improved glycemic profile observed in Rspo1-/- mice after an oral glucose challenge.

Furthermore, the increased BCM was normalized in the Rspo1-/- mice in response to EX4 treatment. The loss of differences in both glycemic profile and BCM in Rspo1-/- mice with EX4 treatment parallels that reported for pre-diabetic obese Zucker fatty rats treated with the GLP-1 analog, liraglutide, such that BCM was reduced with concomitant decrease in -cell proliferation (585). Chronic treatment with EX4 in PI3-kinase γ knockout mice improves -cell function and again, reduces the abnormally high BCM in these animals to a level comparable to that seen in wild-type mice (544). Moreover, the loss of difference between wildtype and

Rspo1-/- mice appeared to be concomitent to a reduced effect of EX4 in the Rspo1-/- animals.

One possible explanation for such a change could be altered in GLP-1R expression. Indeed,

Shu et al reported that siRNA-mediated depletion of TCF7L2 in human islets downregulation

GLP-1R expression with the associated loss of incretin response (500). It remains to be demonstrated whether the lack of EX4 response in Rspo1-/- mice is due to the dysregulation of

GLP-1R expression, although a preliminary analysis of GLP-1R mRNA transcript levels in whole pancreatic extracts did not reveal any such changes (data not shown). Finally, although our findings did not show any changes in GSIS between islets isolated from EX4-treated

Rspo1+/+ and Rspo1-/- mice, it remains possible that chronic EX4 treatment suppressed α-cell function (586), thereby reducing the stimulus for -cell proliferation.

114

In conclusion, our studies using Rspo1-/- mice have provided novel insights into the role of Rspo1 in the -cell, and suggest possible roles for this protein in α-cells and ductal cells.

Further studies are clearly warranted if Rspo1 is to be considered as a novel target for the therapeutic treatment of patients with type 2 diabetes.

3.6 Acknowledgements

The authors are grateful to Dr. J. Wysolmerski (Yale University, CT) for the gift of a breeding pair of Rspo1-/- mice to establish the Toronto colony. This work was supported by an operating grant from the Canadian Diabetes Association (#2374) and by an equipment grant from the Banting and Best Diabetes Centre (BBDC), University of Toronto. V.S.C.W. was supported by a Doctoral Research Award from the Canadian Institutes of Health Research,

A.H.O. by the BBDC Summer Studentship program, and P.L.B. by the Canada Research Chairs program.

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CHAPTER 4

SUMMARY OF RESULTS AND GENERAL DISCUSSION

116

4 Summary of results and general discussion

4.1 Summary of Results

This thesis identified a novel regulator of β-cell behaviour. In Chapter 2, we first identified the presence of Rspo1 mRNA and protein in the MIN6 β-cell line and isolated mouse islets and confirmed that MIN6 β-cells respond to Rspo1 via activation of cWnt signaling in vitro. We further demonstrated that Rspo1 stimulates β-cell proliferation and inhibits cytokine- induced β-cell apoptosis, and it also increases insulin secretion in a glucose-dependent fashion.

Surprisingly, we also found that there is a relationship between GLP-1 and Rspo1 in MIN6 cells: EX4 stimulated Rspo1 mRNA expression in a glucose-, time-, dose- and PI3-kinase- dependent manner. Chapter 3 attempts to extend the in vitro observations to an in vivo setting by using Rspo1-/- mice. Deficiency in Rspo1 in vivo did not cause any impairment in pancreatic or body weights and these mice displayed normal fasting glycemia. Metabolic analyses of

Rspo1-/- mice demonstrated better glycemic control after an oral glucose challenge with no changes in insulin sensitivity. This change in glycemic profile in the Rspo1-/- animal is associated with a marked increase in BCM, and this is due to increase β-cell proliferation and neogenesis. Although there is no change in GSIS in isolated islets from Rspo1-/- mice, insulin- stimulated suppression of glucagon release was absent in Rspo1-/- islets, thus suggesting a possible role of Rspo1 in the α-cell. Interestingly, chronic administration of EX4 in Rspo1-/- mice normalized all parameters in Rspo1 deficient mice so that they are comparable in glycemic excursion and BCM to the wild-type mice.

Initial studies on cWnt signaling in relation to diabetes have fostered substantial efforts and interests towards dissection of the role of this pathway in multiple tissues involved in glucose homeostasis, especially the pancreatic β-cells. However, the majority of these studies described the impact of cWnt signaling on pancreatic development via gain- and loss-of-

117 function studies, and only a handful have thoroughly investigated cWnt signaling in governing adult β-cell behaviour, namely growth and function. Furthermore, given the complexity of cWnt signaling, which is compounded by multiple isoforms of ligands, receptors, co-receptors and intracellular signaling molecules, the precise mechanism of action of this pathway in the β- cell has remained elusive. Moreover, a newly-identified secreted cWnt signaling ligand, Rspo1, has also been detected by immunohistochemistry in human islets (507). However, at the time that the present studies were initiated, nothing further was known about Rspo1 and its possible function(s) in regulating glucose homeostasis. This thesis therefore addressed the role of Rspo1 in the murine β-cell in vitro and in vivo. In Chapter 2, I delineated the effects of recombinant mouse Rspo1 on murine β-cells in vitro using MIN6 and βTC cell lines and dispersed mouse β- cell, demonstrating that Rspo1 enhances β-cell proliferation, survival and insulin secretion. I further defined a stimulatory relationship between the well- established β-cell growth factor and secretagogue, EX4, a GLP-1 receptor agonist, and Rspo1. In Chapter 3, I sought to identify the role of Rspo1 in the β-cell in vivo, using Rspo1-/- mice, and determined that while Rspo1 deficiency has a positive impact on whole-body glucose homeostasis, enhancing β-cell growth and secretion, it has a negative effect on -cell function in vitro. Together, these studies demonstrate for the first time the importance of Rspo1 for regulating β-cell biology. A summary of my results is listed in Table 4.1.

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Murine β-cells Rspo1-/- mice (in vitro) (in vivo)

Proliferation ↑ ↑

* Apoptosis ↓ -

Insulin secretion ↑ -

Table 4.1. Summary of results. Chapter 2 of this thesis explored the effects of Rspo1 on

MIN6 -cells in vitro including the stimulation of proliferation, inhibition of cytokine-induced apoptosis and increase insulin secretion. In Chapter 3, impact of Rspo1 deficiency leads to a rather surprising series of results with an increase -cell proliferation leading to -cell mass expansion, but no changes in apoptosis (asterisk indicates basal condition) and glucose- stimulated insulin secretion from isolated islets of Rspo1-/- animals. These seemingly contradictory results between in vitro and in vivo are discussed in the following section.

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4.2 General Discussion

In 2005, Kim et al reported that systemic administration of human Rspo1 in mice leads to massive intestinal proliferation in vivo, and this is associated with its activation of the cWnt signaling pathway in vitro (507). In this same study, the authors also reported that Rspo1 is expressed in the human „islet‟, although they did not specify the localization of this protein to any distinct cell type(s). In Chapter 2, I have shown that Rspo1 mRNA transcripts are expressed in murine islets, MIN6 and βTC -cells, while Rspo1 protein was also shown to be present in the MIN6 cells. I further found that MIN6 -cells express several Wnt ligands and Frz receptors, LRP5 and 6 co-receptors and intracellular components necessary for a functional cWnt signaling cascade. Although the ability of Rspo1 to activate cWnt signaling has reported previously in other cell lines, such as HEK293 cells (507), I have now extended this phenomenon to mouse MIN6 -cells, with the demonstration that treatment with Rspo1 activates cWnt signaling as detected by nuclear accumulation of -catenin and stimulation of mRNA transcript levels for the cWnt target genes, c-myc and cyclinD1. It was interesting to note that in addition to the cWnt target genes, Rspo1 also increased ins2 mRNA levels in vitro.

However, it is unclear whether the increase in ins2 mRNA was due to direct effect of Rspo1 through the cWnt or another pathway, or was a direct result of the concomintant increase in - cell proliferation. Although the latter possibility is strengthened by the finding that ins2 mRNA levels were increased in parallel with BCM in vivo, even in the absence of Rspo1, other studies have demonstrated that the ins2 gene may be a direct cWnt target through the actions of the cWnt transcription factor, TCF4 (501;587).

Since Rspo1 is an established gastrointestinal growth factor and cWnt signaling has been previously shown to stimulate -cell proliferation, I first delineated whether exogenous administration of recombinant mouse Rspo1 impacts -cell growth. I demonstrated that

120 overnight administration of Rspo1 in MIN6 cells produced ~2-fold increase in proliferation, similar to the effects of EX4 and Wnt3a. Moreover, I extended this observation to dispersed murine -cells, thus confirming that the proliferative effect of Rspo1 was not cell-line specific.

However, in marked contrast to these in vitro findings, the absence of Rpso1 in vivo led to a paradoxical increase in BCM that was mediated, in large part through an increase in -cell proliferation. As discussed in the Introduction and Chapter 3, stimulatory influences to BCM may include insulin resistance and increased glucagon production, of which only the latter possibility is consistent with my observations in the Rspo1-/- mice (as discussed in more detail below). Indeed, previous literature has shown that glucagon does play a role in regulating -cell growth. For instance, ablation of the glucagon receptor (Gcgr-/-) decreased the number of β-cells per islet compared with control animals (588). Further analyses revealed that the level of expression of PDX-1, GLUT2 and MafA (a transcription factor proposed to be involved in glucose-stimulated insulin gene transcription) was lower in β-cells of Gcgr–/– animals relative to wild-type mice (588). Moreover, Gelling et al showed that increased glucagon action specifically in the -cell yields a better glycemic excursion to oral glucose challenge in association with an increase in BCM (582). These studies together strongly support a role for glucagon in the regulation of β-cell growth. Furthermore, it is possible that Rspo1 functions negatively in the α-cell; therefore, the absence of Rspo1 relieves this inhibition whereby aberrant glucagon secretion promotes the β-cell growth seen in the Rspo1-/- mice. Further study to determine which, if any of these suggestions is valid, is clearly required.

-cell growth is also dictated by the rate of apoptosis. -cell loss, partially caused by inflammatory cytokines, is well known in T1DM and T2DM (589;590). Thus, one mechanism to preserve overall -cell viability, growth, and function, is to reduce apoptosis. Chapter 2 showed that Rspo1 prevented cytokine-induced apoptosis in both MIN6 and dispersed murine -

121 cells, as determined by activated/cleaved caspase3 and TUNEL staining, respectively.

However, again, this in vitro finding was not recapitulated my in vivo studies, in which no difference between wild-type and Rspo1-/- mice could be detected. However, such a finding does not preclude a role for Rspo1 in regulating -cell apoptosis in vivo. In fact, these observations are limited, as my Rspo1-/- mice were metabolically-unchallenged and apoptosis rates were extremely low. This may therefore represent a poor in vivo model to examine the effect of Rspo1 on -cell apoptosis. Indeed, many previous studies demonstrating anti-apoptotic properties of β-cell growth factors required the presence of β-cell-specific toxins, such as streptozotocin. For instance, Garcia-Ocana et al reported that there is a significant reduction in

β-cell apoptosis in mice overexpressing HGF in the β-cells when the animals are challenged with streptozotocin (280;431). Therefore, it is important to differentiate between basal and induced β-cell apoptosis, and I therefore cannot currently answer if Rspo1 is a pro-survival factor in vivo.

Finally, with respect to -cell growth, I also found an increased number of insulin- positive ductal cells in the Rspo1-/- mouse pancreas, indicative of a positive effect of Rspo1 deficiency on -cell neogenesis. Interestingly, previous studies have similarly implicated the cWnt pathway as an inhibitor of differentiation. Thus, it has been shown that inhibition of the cWnt pathway leads to spontaneous adipogenesis, while prevention of basal cWnt-mediated signaling prevents preadipocyte differentiation (591). While this scenario may be extrapolated to pancreatic ductal cells, characterization of this phenomenon has been difficult due to issues related to identification of duct-like progenitors. Several studies have shown that a population of duct-like epithelial cells expressing Ngn3 represent endocrine precursor cells (592-595). It was further demonstrated that Ngn3 was required for the differentiation of these cells in the embryo but that Ngn3 expression is off postnatally. Lineage tracing experiments during

122 embryonic development using inducible Ngn3-Cre mice confirmed these findings (596). More importantly, Heimberg‟s group attributed the increase in BCM after ductal ligation to replication of pre-existing β-cells as well as the differentiation of a subset of ductal cells into islet cells, including glucose responsive β-cells (597). The authors proposed that ductal ligation could recapitulate the expression of Ngn3 in rare ductal cells that are similar to embryonic precursors

(597). Using specific lentiviruses, they further documented a consistent role for Ngn3 in the increase in BCM and showed that neogenesis played an important in β-cell formation after ductal ligation (597). These results support the theory of β-cell neogenesis; however, Ngn3 may not be a good marker for β-cell neogenesis of ductal origin, as other studies have identified a few Ngn3-positive cells within adult islets, suggesting that Ngn3-positive progenitor non-ductal cells could reside within the islets (598;599). The possibility that ductal progenitor cells can migrate and incorporate themselves into islets was not investigated in previous studies on Ngn3

(596). Cytokeratin 19 and carbonic anhydrase II are other commonly used to detect duct cells, but these markers may also be expressed in additional pancreatic cell types. Inada et al demonstrated that carbonic anhydrase II expressing pancreatic cells give rise to new islets in neonates as well as in adults after ductal ligation; however, the authors pointed out that carbonic anhydrase II is also expressed in neuronal cells (600). Therefore, future analyses with ductal specific markers will be required to determine whether Rspo1 deficiency leads to a recapitulation of the β-cell developmental program.

Since it has been demonstrated that cWnt signaling can also regulate -cell function

(465), I also examined the effect of Rspo1 on insulin secretion. In Chapter 2, treatment with

Rspo1 acutely enhances insulin secretion by MIN6 -cells as well as murine islets, in a glucose- independent fashion. This is in line with previously reported effect of Wnt3a on glucose- independent insulin secretion in murine islets (465). Together, these observations suggest that

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Rspo1 or Wnt3a can regulate insulin secretion independently of the glucose-sensing machinery

+ 2+ of the -cell (e.g. K ATP and/or voltage-dependent Ca channels) and/or may regulate the exocytosis pathway. However, in contrast to the in vitro setting, Rspo1-/- mice have a better glycemic control after an oral glucose challenge, with no changes in insulin sensitivity. This enhanced glucose tolerance was likely due to a greater change in plasma insulin after oral glucose challenge, possibly consequent to the enhanced BCM observed in the Rspo1-/- mice. As

I also discovered that Rspo1-/- islets have abnormal suppression of glucagon release by glucose, this could provide a possible explanation for the discrepancy between the in vitro and in vivo observations. Hence, a number of studies have indicated glucagon signaling is required for normal β-cell function. For example, GSIS was significantly reduced in isolated islets from

Gcgr-/- mice (588). Trimble et al also reported that glucagon-rich islets from the dorsal pancreas of rats secrete more insulin in response to glucose than islets with lower glucagon content, as found in the more ventral lobes of the pancreas (601). Impaired GSIS from rat β-cells can also be reversed by the addition of nanomolar concentrations of glucagon (602). Conversely,

Huypens et al demonstrated that the inhibition of the glucagon receptor by an antagonist, des-

His1-[Glu9]glucagon-amide, suppressed GSIS in dispersed human islets (603). Gelling et al showed that increased glucagon action specifically in the -cell yield better glycemic excursion to oral glucose challenge and these mice displayed increased BCM (582). There is currently no literature to-date examining the role of Rspo1 or cWnt signaling in the α-cell, however, two studies provided some tantalizing insights. By using an intestinal GLUTag cell line, Ni et al and

Yi et al reported that activation of cWnt signaling via lithium treatment leads to TCF4 transcription factor binding to proglucagon gene promoter and a subsequent increase in proglucagon mRNA levels. Given that the proglucagon gene is also present in the pancreatic α- cell, it remains possible that Rspo1 can activate cWnt signaling to regulate proglucagon gene

124 expression, and thereby glucagon secretion (338;499). Our unpublished data reveal that Rspo1 is expressed in the αTC α-cell line. This indicates that Rspo1 may play a role in regulating α- cell function, possibly in an autocrine fashion, although further studies are required to establish this hypothesis.

The absence in changes of insulin sensitivity in Rspo1-/- mice is also somewhat surprising, and is contradictory to the observation reported by Abiola et al (604). The authors found that activation of cWnt signaling by administration of the cWnt ligand, Wnt10b, or by inhibition of GSK3, can stimulate muscle glucose uptake under basal and glucose-induced insulin-resistant conditions. Nonetheless, my finding that Rspo1 deficiency does not change the gross morphology of several insulin-sensitive tissues may indicate that Rspo1 is dispensable for cWnt-mediated glucose uptake and, therefore, these studies may not be mutually exclusive.

Finally, in Chapter 2, there are similarities between the effects of Rspo1 and EX4 on - cell behaviour, including proliferation, apoptosis and insulin secretion. I therefore examined whether there is a relationship between these two factors My results demonstrated that EX4 upregulates Rspo1 mRNA and protein levels in MIN6 cells, and Rspo1 mRNA transcripts in both the TC cell line and isolated mouse islets; this effect was PI3-kinase-dependent in the

MIN6 cells. PI3-kinase mediates diverse cellular pathways such as apoptosis, differentiation, metabolism and proliferation, and both transcriptional and post-transcriptional mechanisms are involved. Several laboratories have demonstrated that the growth and survival effects of GLP-1 on islet cells are mediated by the PI3-kinase/Akt pathway (400;407;413;544); however, the exact downstream mechanisms in -cells remained unclear. My findings demonstrate that this pathway could potentially involve Rspo1. Given that EX4 induced Rspo1 mRNA at 8 hr and

Rspo1 protein expression at 12 hr, whereas Rspo1-induced -cell proliferation and survival were observed after an overnight incubation, it is possible that Rspo1 acts a downstream

125 secreted auto/paracrine factor to promote GLP-1-induced -cell proliferation and survival.

Unfortunately, I was unable to test this possible EX4-Rspo1 relationship in functional in vitro studies due to technical difficulties with the Rspo1 siRNA; nevertheless, our studies suggest an additional pathway whereby GLP-1 can exert its beneficial effects on -cell biology. However, in Chapter 3, the relationship between Rspo1 and EX4 in vivo was addressed. Treatment with

EX4 in mice for 2 weeks in Rspo1+/+ and Rspo1-/- mice normalized all metabolic parameters; namely, there was a loss of the improvement in glycemic tolerance after an oral glucose challenge and a loss of the enhanced BCM in Rspo1-/- mice. Similar losses of phenotypic differences have been reported for prediabetic obese Zucker fatty rats and for PI3-kinase γ mice treated with liraglutide (GLP-1 analog) or with EX4 respectively (544;585).

These studies indicate that GLP-1-induced expansion of BCM in vivo is dependent on the prevailing metabolic conditions. Although the exact relationship between GLP-1 and Rspo1 in vivo is unclear, and until further studies are performed, it remains possible GLP-1 and Rspo1 interplay involves a complex series of autocrine/paracrine effects, particularly since GLP-1 is known to stimulate somatostatin release (605), which in turn has been shown to inhibit proliferation of rat RINm5F insulinoma cells (408). Moreover, chronic treatment of EX4 in

Rspo1-/- mice resulted in an unexpected „normalization‟ glycemic excursion and BCM that is comparable to wild-type controls.

4.3 Limitations of the present study and future directions

As in any study, there are a number of limitations that must be acknowledged, as well as potential future studies that could be conducted to resolve the issues arising.

(1) In my in vitro studies, the demonstration of effects of Rspo1 on -cell behaviour lacked mechanistic detail. Hence, although I showed that Rspo1 is functional by demonstrating its ability to increase nuclear -catenin and cWnt target genes mRNA levels, I did not

126 demonstrate whether the cWnt pathway is required for the effects of Rspo1 on -cell proliferation, apoptosis or function. This will require additional studies using, for example, sFRP to inhibit binding of Wnt ligands to Frz receptors, overexpression of GSK3 or Axin, or of dominant-negative TCF4. Such studies will yield invaluable insight not only into the mechanism of action of Rspo1, but also on the importance of cWnt signaling in the -cell.

Furthermore, Chapter 2 reported that there was a temporal differential effects between Rspo1 and Wnt3a in the activation of cWnt signaling (i.e. nuclear β-catenin, and mRNA of c-myc, cyclinD1 and ins2). These obversations suggest that Rspo1 can act through a cWnt- independent, yet-to-be-identified pathway in the -cell. Moreover, although Rspo1 acutely enhances insulin secretion in MIN6 -cells as well as murine islets in a glucose-independent fashion, the mechanism behind this effect is unknown. As mentioned earlier, this observation suggests that Rspo1 can regulate insulin secretion independently of the glucose-sensing

+ 2+ machinary of the -cell (e.g. K ATP and/or voltage-dependent Ca channels) and this is in accord with previously reported effects of Wnt3a. Moreover, Da Silva Xavier et al have demonstrated that the TCF7L2 gene is required for maintenance of expression of -cell genes important for secretory granule fusion, such as syntaxin-1A and Munc18-1 (502). As such studies of gene expression were performed under chronic conditions, it remains to be seen whether Rspo1 can acutely regulate secretion at the level of exocytosis. Further studies to determine granules dynamics, using electron microscopy and electrical capacitance, are required.

(2) My reported anti-apoptotic effect of Rspo1 was observed under cytokine-treated conditions and, thus, was more a recapitulation of T1DM that of T2DM; e.g. I did not examine whether Rspo1 can rescue -cells under other apoptosis-inducing conditions, such as

127 glucotoxicity, lipotoxicity, and amyloid formation, all of which have been implicated to play a role in the loss of -cells in T2DM.

(3) Although it was previously reported that other isoforms of Rspo also display growth effects in the small intestine, the present study on -cells was limited to Rspo1. It would be interesting to examine further whether the Rspo2-4 also elicit the same effects on the -cell as

Rspo1.

(4) It is always possible that in vitro cell lines may not accurately reflect in vivo settings.

Indeed, this was clearly demonstrated in the present study by the discrepancies observed between Chapters 2 and 3. To answer this, future studies will require examinion of -cell behaviour following administration of recombinant Rspo1 at different doses and for different durations into both normal and metabolically-challenged mice, such as streptozotocin-treated mice, and rodent models of T2DM (e.g. high fat diet). However, even such an endeavour will not demonstrate direct effects of Rspo1 on the -cell and, thus, my work on primary mouse - cells may represent the most ideal condition whereby Rspo1 was found to directly regulates - cell proliferation and apoptosis.

(5) Although I showed that Rspo1 induces -cell proliferation and inhibits -cell apoptosis at several concentrations, it is unknown how these concentrations relate to physiological levels of Rspo1. Future studies will be required to determine the physiological levels of Rspo1 in vivo via enzyme-linked immunoabsorbant assay or radioimmunoassay.

(6) An important unanswered question required to translate my findings into the clinical setting is whether Rspo1 has identical effects on human islets. It is well-known that rodent - cells inherit a greater capacity for replication relative to human -cells (606). For instance, following partial pancreatectomy in humans, Menge and colleagues reported that the fractional

-cell area of the pancreas remained unchanged with no induction of proliferation or neogenesis

128

(ductal transdifferentiation), which is in stark contrast to the significant enhancement of BCM,

-cell proliferation and neogenesis that is seen in mice following a 60% pancreatectomy (607).

Therefore, it is critical to determine whether and how the human -cell responds to Rspo1 before it can be consider for any therapeutic relevance.

(7) Liu et al reported that there is basal endogenous cWnt signaling activity in INS1 cells mediated by endogenous cWnt signaling components (451). In my study, the addition of

Wnt3a ligand did not further reduce Rspo1‟s anti-apoptotic effects. Thus, it is possible that

MIN6 cells can produce endogenous cWnt ligands (as also suggested by my findings of expression of mRNA for multiple cWnt ligands) whereby further addition of a cWnt ligand does not elicit any additional anti-apoptotic effect; it is also noted that I did not examine the effect of

Rspo1 and Wnt3a co-treatment on -cell proliferation and insulin secretion. As I did not measure endogenous Rspo1 (or cWnt ligands such as Wnt3a) levels in the -cells in vitro and, unfortunately, my attempts to produce an in vitro knockdown of Rspo1 via siRNA were unsuccessful, the impact of endogenous Rspo1 and/or cWnt activity on regulating basal -cell behaviour is currently lacking.

(8) Although Nakashima et al reported that MIN6 cells are not pure -cells, as they secrete other hormones such as glucagon, somatostatin and ghrelin (608), there is currently no

-cell line that perfectly mimics primary -cell physiology. However, several factors justify my use of MIN6 -cells as an appropriate in vitro model for our studies. Firstly, Poitout et al compared various rodent -cell lines and reported that murine MIN6 along with rat INS1 cells retain normal regulation of glucose-induced insulin secretion (549). Secondly, I showed that

MIN6 cells display a functional cWnt signaling response not only to Rspo1, but also to LiCl, the cWnt ligand, Wnt3a, and EX4. Furthermore, my finding that murine TC -cells express

Rspo2, whereas this isoform was undetectable in the MIN6 cells and murine islets, raises the

129 possibility that the TC -cell line may not be directly comparable to murine islets. Finally, my studies on the MIN6 cells are are consistent with previous observations. Hence, Schinner and colleagues reported that incubation of rat INS1 cells and mouse islets with adipocyte-derived

Wnt molecules can activate cWnt-driven luciferase reporter activity and transcription of the cWnt target gene, cyclinD1 (467). Furthermore, Liu et al established that Wnt3a increases cWnt-driven luciferase reporter activity in rat INS1 cells and murine islets (451).

Collectively, therefore, these data suggest that the murine MIN6 cell line may be appropriate model for the study of Rspo1 in the -cell. Nonetheless, my observation that levels of Rspo1 mRNA were markedly higher in the MIN6 cells as compared to murine islets does indicate the possibility of differences between these models and, hence, the need for caution in direct extrapolation. As a consequence of this, all of my key studies on the MIN6 cells (Chapter 2) were also recapitulated in isolated mouse islets.

(9) The use of global Rspo1-/- mice has limitations, as whole-body knockouts may yield too many variables to isolate the cause of abnormal -cell behaviour; namely, whether the changes in BCM was due to changes in development or function in the Rspo1-/- animals.

Moreover, other determinant of metabolism such as nutrient absorption across the intestinal barrier, and hormonal- and/or neuronal-regulation of food intake may need to be taken into consideration. Hence, -, α- and ductal-cell specific knockout animal models using proinsulin-, proglucagon- or carbonic anhydrase II-driven Cre recombinase, respectively, to excise the floxed Rspo1 gene will be required to bypass any developmental changes consequent to Rspo1 deficiency. These animals can then be challenged by OGTT and ITT to examine their general glucose homeostasis, followed by - and α-cell analyses in vivo, as well as in isolated islets to examine their behaviour in vitro. Chronic treatment with EX4 in these animals will further our understanding of a functional outcome of the EX4-Rspo1 relationship in the - and (possibly) α-

130 cell. Moreover, in Chapter 3, I did not examine α-cell or δ-cell mass and these parameters will provide further insights as to whether Rspo1 deficiency induces changes in endocrine function independently or in addition to any role in development.

(10) Although I showed that there are no compensatory responses to the loss of Rspo1 by other isoforms (i.e. Rspo2-4) in the isolated islets from Rspo1-/- animals, I did not examine whether these isoforms are expressed and/or altered in other pancreatic endocrine cells such as

δ-cells or the exocrine tissue. There are currently no ideal antibodies against Rspo1-4 for immunohistochemical analyses to localize these proteins in endocrine cells of the islet. Hence, it remains possible that compensatory responses occurred there and that these isoforms may act in a paracrine fashion on the -cell within the islet.

(11) Finally, one surprising finding of Rspo1-/- mice includes the significant increase in insulin-positive ductal cells. To further delineate the role of Rspo1 in neogenesis in this animal model, a ductal-ligation injury could be induced in Rspo1-/- mice to examine whether Rspo1 is required for the formation of those ductal progenitors. Moreover, the use of an inducible tissue- specific knockout of Rspo1 in the pancreatic ductal epithelium via carbonic anhydrase II-driven

Cre recombinase will also allow determination of the role of Rspo1 in regulating -cell neogenesis.

4.4 Conclusions

My in vitro and in vivo studies provide novel and important insights to Rspo1 as crucial regulator of -cell behaviour. In Chapter 2, I showed that Rspo1 is a -cell growth factor and secretagogue. I also found that EX4 regulates Rspo1 in vitro in a dose-, time-, glucose- and PI3- kinase-dependent fashion. In Chapter 3, I demonstrated that whole-body knockout of Rspo1 in vivo impacts -cell behaviour in an unexpected manner as compared to my in vitro findings.

Hence, Rspo1 deficiency in mice impacts glucose homeostasis through better glycemic

131 tolerance and increased BCM. Islets from Rspo1-/- mice have normal GSIS but abnormal suppression of glucagon release by glucose, at least in vitro, therefore implying that Rspo1 also has a role in regulating α-cell behaviour. Although this thesis has numerous limitations, as discussed in the previous section, I have proposed several potential mechanisms in attempt to explain the observed phenomena, as illustrated in Figure 4.1. Although these are tantalizing possibilities, they will nonetheless require future studies, as discussed above. Nonetheless, the results of these studies in this thesis suggest that Rspo1 as a novel mediator of -cell behaviour, and should be considered as a potential therapeutical strategy in promoting the maintenance of functional β-cell mass in diabetes.

132

Figure 4.1. Proposed working model. Chapter 2 demonstrated that Rspo1 is a novel β-cell growth factor with anti-apoptotic effects and insulin secretory activity. Moreover, activation of

GLP-1R signaling leads to an increase in Rspo1 expression. In Chapter 3, Rspo1 deficiency led to a unexpected increase in BCM due to increase in β-cell proliferation and neogenesis (not shown in figure). In addition, Rspo1 deficiency yielded a defect in glucose-mediated suppression of islet glucagon secretion, suggestion a role for Rspo1 in the -cell whereby aberrant glucagon release can lead to enhanced β-cell proliferation.

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APPENDIX:

PERMISSION TO REPRODUCE PREVIOUSLY PUBLISHED MATERIAL

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5 Appendix

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6 Reference list

1. Amos,AF, McCarty,DJ, Zimmet,P: The rising global burden of diabetes and its complications: estimates and projections to the year 2010. Diabet Med 14 Suppl 5:S1-85, 1997

2. King,H, Aubert,RE, Herman,WH: Global burden of diabetes, 1995-2025: prevalence, numerical estimates, and projections. Diabetes Care 21:1414-1431, 1998

3. Zimmet,P, Alberti,KG, Shaw,J: Global and societal implications of the diabetes epidemic. Nature 414:782-787, 2001

4. Reaven,GM: Role of insulin resistance in the pathophysiology of non-insulin dependent diabetes mellitus. Diabetes Metab Rev 9 Suppl 1:5S-12S, 1993

5. DeFronzo,RA: Lilly lecture 1987. The triumvirate: beta-cell, muscle, liver. A collusion responsible for NIDDM. Diabetes 37:667-687, 1988

6. Frojdo,S, Vidal,H, Pirola,L: Alterations of insulin signaling in type 2 diabetes: a review of the current evidence from humans. Biochim Biophys Acta 1792:83-92, 2009

7. DeFronzo,RA, Banadonna,RC, Ferrannini,E: Pathogenesis of NIDDM: A balanced overview. Diabetes Care 15:318-368, 1992

8. Matthews,DR, Cull,CA, Stratton,IM, Holman,RR, Turner,RC: UKPDS 26: Sulphonylurea failure in non-insulin-dependent diabetic patients over six years. UK Prospective Diabetes Study (UKPDS) Group. Diabet Med 15:297-303, 1998

9. Butler,AE, Janson,J, Bonner-Weir,S, Ritzel,R, Rizza,RA, Butler,PC: Beta-cell deficit and increased beta-cell apoptosis in humans with type 2 diabetes. Diabetes 52:102-110, 2003

10. Mohler,ML, He,Y, Wu,Z, Hwang,DJ, Miller,DD: Recent and emerging anti-diabetes targets. Med Res Rev 29:125-195, 2009

11. Prado,CL, Pugh-Bernard,AE, Elghazi,L, Sosa-Pineda,B, Sussel,L: Ghrelin cells replace insulin-producing beta cells in two mouse models of pancreas development. Proc Natl Acad Sci U S A 101:2924-2929, 2004

137

12. Brissova,M, Fowler,MJ, Nicholson,WE, Chu,A, Hirshberg,B, Harlan,DM, Powers,AC: Assessment of human pancreatic islet architecture and composition by laser scanning confocal microscopy. J Histochem Cytochem 53:1087-1097, 2005

13. Heller,RS: The comparative anatomy of islets. Adv Exp Med Biol 654:21-37, 2010

14. Meier,JJ, Kohler,CU, Alkhatib,B, Sergi,C, Junker,T, Klein,HH, Schmidt,WE, Fritsch,H: Beta-cell development and turnover during prenatal life in humans. Eur J Endocrinol 162:559-568, 2010

15. Cabrera,O, Berman,DM, Kenyon,NS, Ricordi,C, Berggren,PO, Caicedo,A: The unique cytoarchitecture of human pancreatic islets has implications for islet cell function. Proc Natl Acad Sci U S A 103:2334-2339, 2006

16. Santos,RM, Rosario,LM, Nadal,A, Garcia-Sancho,J, Soria,B, Valdeolmillos,M: Widespread synchronous [Ca2+]i oscillations due to bursting electrical activity in single pancreatic islets. Pflugers Arch 418:417-422, 1991

17. Valdeolmillos,M, Santos,RM, Contreras,D, Soria,B, Rosario,LM: Glucose-induced oscillations of intracellular Ca2+ concentration resembling bursting electrical activity in single mouse islets of Langerhans. FEBS Lett 259:19-23, 1989

18. McEvoy,RC, Madson,KL: Pancreatic insulikn-, glucagon-, and somatostatin-positive islet cell populatins during the perinatal development of the rat. I. Morphometric quantitation. Biol Neonate 38:248-254, 1980

19. Eriksson,U, Swenne,I: Diabetes in pregnancy: growth of the fetal pancreatic B cells in the rat. Biol Neonate 42:239-248, 1982

20. Hogg,J, Hill,DJ, Han,VK: The ontogeny of insulin-like growth factor (IGF) and IGF- binding protein gene expression in the rat pancreas. J Mol Endocrinol 13:49-58, 1994

21. Hill,DJ, Hogg,J, Petrik,J, Arany,E, Han,VK: Cellular distribution and ontogeny of insulin-like growth factors (IGFs) and IGF binding protein messenger RNAs and peptides in developing rat pancreas. J Endocrinol 160:305-317, 1999

22. Han,VK, Lund,PK, Lee,DC, D'Ercole,AJ: Expression of somatomedin/insulin-like growth factor messenger ribonucleic acids in the human fetus: identification, characterization, and tissue distribution. J Clin Endocrinol Metab 66:422-429, 1988

138

23. Van Schravendijk,CF, Foriers,A, Van den Brande,JL, Pipeleers,DG: Evidence for the presence of type I insulin-like growth factor receptors on rat pancreatic A and B cells. Endocrinology 121:1784-1788, 1987

24. Fehmann,HC, Jehle,P, Markus,U, Goke,B: Functional active receptors for insulin-like growth factors-I (IGF-I) and IGF-II on insulin-, glucagon-, and somatostatin-producing cells. Metabolism 45:759-766, 1996

25. Scharfmann,R: Control of early development of the pancreas in rodents and humans: implications of signals from the mesenchyme. Diabetologia 43:1083-1092, 2000

26. Murtaugh,LC, Melton,DA: Genes, signals, and lineages in pancreas development. Annu Rev Cell Dev Biol 19:71-89, 2003

27. Lammert,E, Cleaver,O, Melton,D: Induction of pancreatic differentiation by signals from blood vessels. Science 294:564-567, 2001

28. Stefan,Y, Grasso,S, Perrelet,A, Orci,L: A quantitative immunofluorescent study of the endocrine cell populations in the developing human pancreas. Diabetes 32:293-301, 1983

29. Bouwens,L, Lu,WG, De Krijger,R: Proliferation and differentiation in the human fetal endocrine pancreas. Diabetologia 40:398-404, 1997

30. Polak,M, Bouchareb-Banaei,L, Scharfmann,R, Czernichow,P: Early pattern of differentiation in the human pancreas. Diabetes 49:225-232, 2000

31. Bouwens,L, Wang,RN, De Blay,E, Pipeleers,DG, Kloppel,G: Cytokeratins as markers of ductal cell differentiation and islet neogenesis in the neonatal rat pancreas. Diabetes 43:1279-1283, 1994

32. Bouwens,L, De Blay,E: Islet morphogenesis and stem cell markers in rat pancreas. J Histochem Cytochem 44:947-951, 1996

33. Bouwens,L, Lu,WG, De Krijger,R: Proliferation and differentiation in the human fetal endocrine pancreas. Diabetologia 40:398-404, 1997

34. Kaung,HL: Growth dynamics of pancreatic islet cell populations during fetal and neonatal development of the rat. Dev Dyn 200:163-175, 1994

139

35. Kaung,HL: Growth dynamics of pancreatic islet cell populations during fetal and neonatal development of the rat. Dev Dyn 200:163-175, 1994

36. Scaglia,L, Cahill,CJ, Finegood,DT, Bonner-Weir,S: Apoptosis participates in the remodeling of the endocrine pancreas in the neonatal rat. Endocrinology 138:1736-1741, 1997

37. Petrik,J, Arany,E, McDonald,TJ, Hill,DJ: Apoptosis in the pancreatic islet cells of the neonatal rat is associated with a reduced expression of insulin-like growth factor II that may act as a survival factor. Endocrinology 139:2994-3004, 1998

38. Finegood,DT, Scaglia,L, Bonner-Weir,S: Dynamics of beta-cell mass in the growing rat pancreas. Estimation with a simple mathematical model. Diabetes 44:249-256, 1995

39. Montanya,E, Nacher,V, Biarnes,M, Soler,J: Linear correlation between beta-cell mass and body weight throughout the lifespan in Lewis rats: role of beta-cell hyperplasia and hypertrophy. Diabetes 49:1341-1346, 2000

40. Bonner-Weir,S: Islet growth and development in the adult. J Mol Endocrinol 24:297- 302, 2000

41. Hellerstrom,C, Andersson,A, Groth,CG, Sandler,S, Jansson,L, Korsgren,O, Swenne,I, Petersson,B, Tollemar,J, Tyden,G: Experimental pancreatic transplantation in diabetes. Diabetes Care 11 Suppl 1:45-53, 1988

42. Dor,Y, Brown,J, Martinez,OI, Melton,DA: Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation. Nature 429:41-46, 2004

43. Xu,G, Kaneto,H, Lopez-Avalos,MD, Weir,GC, Bonner-Weir,S: GLP-1/exendin-4 facilitates beta-cell neogenesis in rat and human pancreatic ducts. Diabetes Res Clin Pract 2006

44. Seaberg,RM, Smukler,SR, Kieffer,TJ, Enikolopov,G, Asghar,Z, Wheeler,MB, Korbutt,G, van der,KD: Clonal identification of multipotent precursors from adult mouse pancreas that generate neural and pancreatic lineages. Nat Biotechnol 22:1115-1124, 2004

45. Bell,GI, Kayano,T, Buse,JB, Burant,CF, Takeda,J, Lin,D, Fukumoto,H, Seino,S: Molecular biology of mammalian glucose transporters. Diabetes Care 13:198-208, 1990

140

46. Matschinsky,FM, Ghosh,AK, Meglasson,MD, Prentki,M, June,V, von Allman,D: Metabolic concomitants in pure, pancreatic beta cells during glucose-stimulated insulin secretion. J Biol Chem 261:14057-14061, 1986

47. Iynedjian,PB, Jotterand,D, Nouspikel,T, Asfari,M, Pilot,P-R: Transcriptional induction of glucokinase gene by insulin in cultured liver cells and its repression by the glucagon- cAMP system. J Biol Chem 264:21824-21829, 1989

48. Meglasson,MD, Manning,CD, Najafi,H, Matschinsky,FM: Glucose transport by radiation-induced insulinoma and clonal pancreatic beta-cells. Diabetes 35:1340-1344, 1986

49. Wang,Z, Thurmond,DC: Mechanisms of biphasic insulin-granule exocytosis - roles of the cytoskeleton, small GTPases and SNARE proteins. J Cell Sci 122:893-903, 2009

50. Aizawa,T, Komatsu,M, Asanuma,N, Sato,Y, Sharp,GW: Glucose action 'beyond ionic events' in the pancreatic beta cell. Trends Pharmacol Sci 19:496-499, 1998

51. Taguchi,N, Aizawa,T, Sato,Y, Ishihara,F, Hashizume,K: Mechanism of glucose-induced biphasic insulin release: Physiological role of adenosine triphosphate-sensitive K+ channel-independent glucose action. Endocrinology 136:3942-3948, 1995

52. Straub,SG, Sharp,GW: Glucose-stimulated signaling pathways in biphasic insulin secretion. Diabetes Metab Res Rev 18:451-463, 2002

53. Gembal,M, Gilon,P, Henquin,JC: Evidence that glucose can control insulin release independently from its action on ATP-sensitive K+ channels in mouse B cells. J Clin Invest 89:1288-1295, 1992

54. Asplund,K, Westman,S, Hellerstrom,C: Glucose stimulation of insulin secretion from the isolated pancreas of foetal and newborn rats. Diabetologia 5:260-262, 1969

55. Asplund,K: Effects of intermittent glucose infusions in pregnant rats on the functional development of the foetal pancreatic B-cells. J Endocrinol 59:285-293, 1973

56. Asplund,K: Dynamics of insulin release from the foetal and neonatal rat pancreas. Eur J Clin Invest 3:338-344, 1973

57. Sodoyez-Goffaux,F, Sodoyez,JC, De Vos,CJ, Foa,PP: Insulin and glucagon secretion by islets isolated from fetal and neonatal rats. Diabetologia 16:121-123, 1979

141

58. Rhoten,WB: Insulin secretory dynamics during development of rat pancreas. Am J Physiol 239:E57-E63, 1980

59. Grill,V, Lake,W, Freinkel,N: Generalized diminution in the response to nutrients as insulin-releasing agents during the early neonatal period in the rat. Diabetes 30:56-63, 1981

60. Freinkel,N, Lewis,NJ, Johnson,R, Swenne,I, Bone,A, Hellerstrom,C: Differential effects of age versus glycemic stimulation on the maturation of insulin stimulus-secretion coupling during culture of fetal rat islets. Diabetes 33:1028-1038, 1984

61. Hole,RL, Pian-Smith,MC, Sharp,GW: Development of the biphasic response to glucose in fetal and neonatal rat pancreas. Am J Physiol 254:E167-E174, 1988

62. Hughes,SJ: The role of reduced glucose transporter content and glucose metabolism in the immature secretory responses of fetal rat pancreatic islets. Diabetologia 37:134-140, 1994

63. Bliss,CR, Sharp,GWG: A critical period in the development of the insulin secretory response to glucose in fetal rat pancreas. Life Sci 55:423-427, 1994

64. Bergsten,P, Aoyagi,K, Persson,E, Eriksson,UJ, Hellerstrom,C: Appearance of glucose- induced insulin release in fetal rat beta-cells. J Endocrinol 158:115-120, 1998

65. Rozzo,A, Meneghel-Rozzo,T, Delakorda,SL, Yang,SB, Rupnik,M: Exocytosis of insulin: in vivo maturation of mouse endocrine pancreas. Ann N Y Acad Sci 1152:53-62, 2009

66. Van Assche,FA, Aerts,L, de,PF: A morphological study of the endocrine pancreas in human pregnancy. Br J Obstet Gynaecol 85:818-820, 1978

67. Van Assche,FA, Gepts,W, Aerts,L: Immunocytochemical study of the endocrine pancreas in the rat during normal pregnancy and during experimental diabetic pregnancy. Diabetologia 18:487-491, 1980

68. Sorenson,RL, Brelje,TC: Adaptation of islets of Langerhans to pregnancy: beta-cell growth, enhanced insulin secretion and the role of lactogenic hormones. Horm Metab Res 29:301-307, 1997

142

69. Nielsen,JH: Effects of growth hormone, prolactin, and placental lactogen on insulin content and release, and deoxyribonucleic acid synthesis in cultured pancreatic islets. Endocrinology 110:600-606, 1982

70. Brelje,TC, Scharp,DW, Lacy,PE, Ogren,L, Talamantes,F, Robertson,M, Friesen,HG, Sorenson,RL: Effect of homologous placental lactogens, prolactins, and growth hormones on islet B-cell division and insulin secretion in rat, mouse, and human islets: implication for placental lactogen regulation of islet function during pregnancy. Endocrinology 132:879-887, 1993

71. Brelje,TC, Parsons,JA, Sorenson,RL: Regulation of islet beta-cell proliferation by prolactin in rat islets. Diabetes 43:263-273, 1994

72. Marynissen,G, Aerts,L, Van Assche,FA: The endocrine pancreas during pregnancy and lactation in the rat. J Dev Physiol 5:373-381, 1983

73. Scaglia,L, Smith,FE, Bonner-Weir,S: Apoptosis contributes to the involution of beta cell mass in the post partum rat pancreas. Endocrinology 136:5461-5468, 1995

74. Marynissen,G, Aerts,L, Van Assche,FA: The endocrine pancreas during pregnancy and lactation in the rat. J Dev Physiol 5:373-381, 1983

75. Vasavada,RC, Garcia-Ocana,A, Zawalich,WS, Sorenson,RL, Dann,P, Syed,M, Ogren,L, Talamantes,F, Stewart,AF: Targeted expression of placental lactogen in the beta cells of transgenic mice results in beta cell proliferation, islet mass augmentation, and hypoglycemia. J Biol Chem 275:15399-15406, 2000

76. Freemark,M, Avril,I, Fleenor,D, Driscoll,P, Petro,A, Opara,E, Kendall,W, Oden,J, Bridges,S, Binart,N, Breant,B, Kelly,PA: Targeted deletion of the PRL receptor: effects on islet development, insulin production, and glucose tolerance. Endocrinology 143:1378-1385, 2002

77. Huang,C, Snider,F, Cross,JC: Prolactin receptor is required for normal glucose homeostasis and modulation of beta-cell mass during pregnancy. Endocrinology 150:1618-1626, 2009

78. Aerts,L, Van,BR, Van Assche,FA: PROLACTIN-deficiency in adult offspring of diabetic mothers. Int J Exp Diabetes Res 1:31-38, 2000

79. Boloker,J, Gertz,SJ, Simmons,RA: Gestational diabetes leads to the development of diabetes in adulthood in the rat. Diabetes 51:1499-1506, 2002

143

80. Kim,H, Toyofuku,Y, Lynn,FC, Chak,E, Uchida,T, Mizukami,H, Fujitani,Y, Kawamori,R, Miyatsuka,T, Kosaka,Y, Yang,K, Honig,G, van der,HM, Kishimoto,N, Wang,J, Yagihashi,S, Tecott,LH, Watada,H, German,MS: Serotonin regulates pancreatic beta cell mass during pregnancy. Nat Med 16:804-808, 2010

81. Butler,AE, Cao-Minh,L, Galasso,R, Rizza,RA, Corradin,A, Cobelli,C, Butler,PC: Adaptive changes in pancreatic beta cell fractional area and beta cell turnover in human pregnancy. Diabetologia 53:2167-2176, 2010

82. Sorenson,RL, Brelje,TC: Adaptation of islets of Langerhans to pregnancy: beta-cell growth, enhanced insulin secretion and the role of lactogenic hormones. Horm Metab Res 29:301-307, 1997

83. Homko,C, Sivan,E, Chen,X, Reece,EA, Boden,G: Insulin secretion during and after pregnancy in patients with gestational diabetes mellitus. J Clin Endocrinol Metab 86:568-573, 2001

84. Nielsen,LR, Rehfeld,JF, Pedersen-Bjergaard,U, Damm,P, Mathiesen,ER: Pregnancy- induced rise in serum C-peptide concentrations in women with type 1 diabetes. Diabetes Care 32:1052-1057, 2009

85. Bruning,JC, Winnay,J, Bonner-Weir,S, Taylor,SI, Accili,D, Kahn,CR: Development of a novel polygenic model of NIDDM in mice heterozygous for IR and IRS-1 null alleles. Cell 88:561-572, 1997

86. Paris,M, Bernard-Kargar,C, Berthault,MF, Bouwens,L, Ktorza,A: Specific and combined effects of insulin and glucose on functional pancreatic beta-cell mass in vivo in adult rats. Endocrinology 144:2717-2727, 2003

87. Kulkarni,RN, Jhala,US, Winnay,JN, Krajewski,S, Montminy,M, Kahn,CR: PDX-1 haploinsufficiency limits the compensatory islet hyperplasia that occurs in response to insulin resistance. J Clin Invest 114:828-836, 2004

88. Ahlgren,U, Jonsson,J, Jonsson,L, Simu,K, Edlund,H: beta-cell-specific inactivation of the mouse Ipf1/Pdx1 gene results in loss of the beta-cell phenotype and maturity onset diabetes. Genes Dev 12:1763-1768, 1998

89. Johnson,JD, Ahmed,NT, Luciani,DS, Han,Z, Tran,H, Fujita,J, Misler,S, Edlund,H, Polonsky,KS: Increased islet apoptosis in Pdx1+/- mice. J Clin Invest 111:1147-1160, 2003

144

90. Swenne,I: The role of glucose in the in vitro regulation of cell cycle kinetics and proliferation of fetal pancreatic B-cells. Diabetes 31:754-760, 1982

91. Bonner-Weir,S, Deery,D, Leahy,JL, Weir,GC: Compensatory growth of pancreatic beta- cells in adult rats after short-term glucose infusion. Diabetes 38:49-53, 1989

92. Bernard,C, Berthault,MF, Saulnier,C, Ktorza,A: Neogenesis vs. apoptosis As main components of pancreatic beta cell ass changes in glucose-infused normal and mildly diabetic adult rats. FASEB J 13:1195-1205, 1999

93. Topp,BG, McArthur,MD, Finegood,DT: Metabolic adaptations to chronic glucose infusion in rats. Diabetologia 47:1602-1610, 2004

94. Hoorens,A, Van de,CM, Kloppel,G, Pipeleers,D: Glucose promotes survival of rat pancreatic beta cells by activating synthesis of proteins which suppress a constitutive apoptotic program. J Clin Invest 98:1568-1574, 1996

95. Kalderon,B, Gutman,A, Levy,E, Shafrir,E, Adler,JH: Characterization of stages in development of obesity-diabetes syndrome in sand rat (Psammomys obesus). Diabetes 35:717-724, 1986

96. Nesher,R, Gross,DJ, Donath,MY, Cerasi,E, Kaiser,N: Interaction between genetic and dietary factors determines beta-cell function in Psammomys obesus, an animal model of type 2 diabetes. Diabetes 48:731-737, 1999

97. Donath,MY, Gross,DJ, Cerasi,E, Kaiser,N: Hyperglycemia-induced beta-cell apoptosis in pancreatic islets of Psammomys obesus during development of diabetes. Diabetes 48:738-744, 1999

98. Maedler,K, Spinas,GA, Lehmann,R, Sergeev,P, Weber,M, Fontana,A, Kaiser,N, Donath,MY: Glucose induces beta-cell apoptosis via upregulation of the Fas receptor in human islets. Diabetes 50:1683-1690, 2001

99. Federici,M, Hribal,M, Perego,L, Ranalli,M, Caradonna,Z, Perego,C, Usellini,L, Nano,R, Bonini,P, Bertuzzi,F, Marlier,LN, Davalli,AM, Carandente,O, Pontiroli,AE, Melino,G, Marchetti,P, Lauro,R, Sesti,G, Folli,F: High glucose causes apoptosis in cultured human pancreatic islets of Langerhans: a potential role for regulation of specific Bcl family genes toward an apoptotic cell death program. Diabetes 50:1290-1301, 2001

145

100. Maedler,K, Sergeev,P, Ris,F, Oberholzer,J, Joller-Jemelka,HI, Spinas,GA, Kaiser,N, Halban,PA, Donath,MY: Glucose-induced beta cell production of IL-1beta contributes to glucotoxicity in human pancreatic islets. J Clin Invest 110:851-860, 2002

101. Briaud,I, Dickson,LM, Lingohr,Mk, McCuaig,JF, Lawrence,JC, Rhodes,CJ: Insulin receptor substrate-2 proteasomal degradation mediated by a mammalian target of rapamycin (mTOR)-induced negative feedback down-regulates protein kinase B- mediated signaling pathway in beta-cells. J Biol Chem 280:2282-2293, 2005

102. Tanaka,Y, Gleason,CE, Tran,PO, Harmon,JS, Robertson,RP: Prevention of glucose toxicity in HIT-T15 cells and Zucker diabetic fatty rats by antioxidants. Proc Natl Acad Sci U S A 96:10857-10862, 1999

103. Porte,D, Jr., Kahn,SE: beta-cell dysfunction and failure in type 2 diabetes: potential mechanisms. Diabetes 50 Suppl 1:S160-S163, 2001

104. Ludvik,B, Nolan,JJ, Baloga,J, Sacks,D, Olefsky,J: Effect of obesity on insulin resistance in normal subjects and patients with NIDDM. Diabetes 44:1121-1125, 1995

105. Burke,JP, Williams,K, Gaskill,SP, Hazuda,HP, Haffner,SM, Stern,MP: Rapid rise in the incidence of type 2 diabetes from 1987 to 1996: results from the San Antonio Heart Study. Arch Intern Med 159:1450-1456, 1999

106. Milburn,JL, Jr., Hirose,H, Lee,YH, Nagasawa,Y, Ogawa,A, Ohneda,M, Beltrandelrio,H, Newgard,CB, Johnson,JH, Unger,RH: Pancreatic beta-cells in obesity. Evidence for induction of functional, morphologic, and metabolic abnormalities by increased long chain fatty acids. J Biol Chem 270:1295-1299, 1995

107. Emilsson,V, Liu,YL, Cawthorne,MA, Morton,NM, Davenport,M: Expression of the functional leptin receptor mRNA in pancreatic islets and direct inhibitory action of leptin on insulin secretion. Diabetes 46:313-316, 1997

108. Islam,MS, Morton,NM, Hansson,A, Emilsson,V: Rat insulinoma-derived pancreatic beta-cells express a functional leptin receptor that mediates a proliferative response. Biochem Biophys Res Commun 238:851-855, 1997

109. Tanabe,K, Okuya,S, Tanizawa,Y, Matsutani,A, Oka,Y: Leptin induces proliferation of pancreatic beta cell line MIN6 through activation of mitogen-activated protein kinase. Biochem Biophys Res Commun 241:765-768, 1997

146

110. Tanabe,K, Okuya,S, Tanizawa,Y, Matsutani,A, Oka,Y: Leptin induces proliferation of pancreatic beta cell line MIN6 through activation of mitogen-activated protein kinase. Biochem Biophys Res Commun 241:765-768, 1997

111. Phillips,MS, Liu,Q, Hammond,HA, Dugan,V, Hey,PJ, Caskey,CJ, Hess,JF: Leptin receptor missense mutation in the fatty Zucker rat. Nat Genet 13:18-19, 1996

112. Takaya,K, Ogawa,Y, Isse,N, Okazaki,T, Satoh,N, Masuzaki,H, Mori,K, Tamura,N, Hosoda,K, Nakao,K: Molecular cloning of rat leptin receptor isoform complementary DNAs--identification of a missense mutation in Zucker fatty (fa/fa) rats. Biochem Biophys Res Commun 225:75-83, 1996

113. Gapp,DA, Leiter,EH, Coleman,DL, Schwizer,RW: Temporal changes in pancreatic islet composition in C57BL/6J-db/db (diabetes) mice. Diabetologia 25:439-443, 1983

114. Wang,Q, Brubaker,PL: Glucagon-like peptide-1 treatment delays the onset of diabetes in 8 week-old db/db mice. Diabetologia 45:1263-1273, 2002

115. Tomita,T, Doull,V, Pollock,HG, Krizsan,D: Pancreatic islets of obese hyperglycemic mice (ob/ob). Pancreas 7:367-375, 1992

116. Bock,T, Pakkenberg,B, Buschard,K: Increased islet volume but unchanged islet number in ob/ob mice. Diabetes 52:1716-1722, 2003

117. Lingohr,MK, Buettner,R, Rhodes,CJ: Pancreatic beta-cell growth and survival--a role in obesity-linked type 2 diabetes? Trends Mol Med 8:375-384, 2002

118. Stein,DT, Stevenson,BE, Chester,MW, Basit,M, Daniels,MB, Turley,SD, McGarry,JD: The insulinotropic potency of fatty acids is influenced profoundly by their chain length and degree of saturation. J Clin Invest 100:398-403, 1997

119. Dobbins,RL, Chester,MW, Stevenson,BE, Daniels,MB, Stein,DT, McGarry,JD: A fatty acid- dependent step is critically important for both glucose- and non-glucose-stimulated insulin secretion. J Clin Invest 101:2370-2376, 1998

120. Latour,MG, Alquier,T, Oseid,E, Tremblay,C, Jetton,TL, Luo,J, Lin,DC, Poitout,V: GPR40 is necessary but not sufficient for fatty acid stimulation of insulin secretion in vivo. Diabetes 56:1087-1094, 2007

147

121. Lan,H, Hoos,LM, Liu,L, Tetzloff,G, Hu,W, Abbondanzo,SJ, Vassileva,G, Gustafson,EL, Hedrick,JA, Davis,HR: Lack of FFAR1/GPR40 does not protect mice from high-fat diet- induced metabolic disease. Diabetes 57:2999-3006, 2008

122. Alquier,T, Peyot,ML, Latour,MG, Kebede,M, Sorensen,CM, Gesta,S, Ronald,KC, Smith,RD, Jetton,TL, Metz,TO, Prentki,M, Poitout,V: Deletion of GPR40 impairs glucose-induced insulin secretion in vivo in mice without affecting intracellular fuel metabolism in islets. Diabetes 58:2607-2615, 2009

123. Kebede,M, Alquier,T, Latour,MG, Semache,M, Tremblay,C, Poitout,V: The fatty acid receptor GPR40 plays a role in insulin secretion in vivo after high-fat feeding. Diabetes 57:2432-2437, 2008

124. Nagasumi,K, Esaki,R, Iwachidow,K, Yasuhara,Y, Ogi,K, Tanaka,H, Nakata,M, Yano,T, Shimakawa,K, Taketomi,S, Takeuchi,K, Odaka,H, Kaisho,Y: Overexpression of GPR40 in pancreatic beta-cells augments glucose-stimulated insulin secretion and improves glucose tolerance in normal and diabetic mice. Diabetes 58:1067-1076, 2009

125. Brownlie,R, Mayers,RM, Pierce,JA, Marley,AE, Smith,DM: The long-chain fatty acid receptor, GPR40, and glucolipotoxicity: investigations using GPR40-knockout mice. Biochem Soc Trans 36:950-954, 2008

126. Boden,G, Chen,X, Rosner,J, Barton,M: Effects of a 48-h fat infusion on insulin secretion and glucose utilization. Diabetes 44:1239-1242, 1995

127. Ferrannini,E, Natali,A, Bell,P, Cavallo-Perin,P, Lalic,N, Mingrone,G: Insulin resistance and hypersecretion in obesity. European Group for the Study of Insulin Resistance (EGIR). J Clin Invest 100:1166-1173, 1997

128. Weyer,C, Hanson,RL, Tataranni,PA, Bogardus,C, Pratley,RE: A high fasting plasma insulin concentration predicts type 2 diabetes independent of insulin resistance: evidence for a pathogenic role of relative hyperinsulinemia. Diabetes 49:2094-2101, 2000

129. Andrikopoulos,S, Massa,CM, ston-Mourney,K, Funkat,A, Fam,BC, Hull,RL, Kahn,SE, Proietto,J: Differential effect of inbred mouse strain (C57BL/6, DBA/2, 129T2) on insulin secretory function in response to a high fat diet. J Endocrinol 187:45-53, 2005

130. Andrikopoulos,S, Blair,AR, Deluca,N, Fam,BC, Proietto,J: Evaluating the glucose tolerance test in mice. Am J Physiol Endocrinol Metab 295:E1323-E1332, 2008

148

131. Gerich,JE: The genetic basis of type 2 diabetes mellitus: impaired insulin secretion versus impaired insulin sensitivity. Endocr Rev 19:491-503, 1998

132. Mason,CC, Hanson,RL, Knowler,WC: Progression to type 2 diabetes characterized by moderate then rapid glucose increases. Diabetes 56:2054-2061, 2007

133. Xiang,AH, Wang,C, Peters,RK, Trigo,E, Kjos,SL, Buchanan,TA: Coordinate changes in plasma glucose and pancreatic beta-cell function in Latino women at high risk for type 2 diabetes. Diabetes 55:1074-1079, 2006

134. Rahier,J, Goebbels,RM, Henquin,JC: Cellular composition of the human diabetic pancreas. Diabetologia 24:366-371, 1983

135. Kloppel,G, Lohr,M, Habich,K, Oberholzer,M, Heitz,PU: Islet pathology and the pathogenesis of type 1 and type 2 diabetes mellitus revisited. Surv Synth Pathol Res 4:110-125, 1985

136. Sakuraba,H, Mizukami,H, Yagihashi,N, Wada,R, Hanyu,C, Yagihashi,S: Reduced beta- cell mass and expression of oxidative stress-related DNA damage in the islet of Japanese Type II diabetic patients. Diabetologia 45:85-96, 2002

137. Yoon,KH, Ko,SH, Cho,JH, Lee,JM, Ahn,YB, Song,KH, Yoo,SJ, Kang,MI, Cha,BY, Lee,KW, Son,HY, Kang,SK, Kim,HS, Lee,IK, Bonner-Weir,S: Selective beta-cell loss and alpha-cell expansion in patients with type 2 diabetes mellitus in Korea. J Clin Endocrinol Metab 88:2300-2308, 2003

138. Butler,AE, Janson,J, Bonner-Weir,S, Ritzel,R, Rizza,RA, Butler,PC: Beta-cell deficit and increased beta-cell apoptosis in humans with type 2 diabetes. Diabetes 52:102-110, 2003

139. Marchetti,P, Del,GS, Marselli,L, Lupi,R, Masini,M, Pollera,M, Bugliani,M, Boggi,U, Vistoli,F, Mosca,F, Del,PS: Pancreatic islets from type 2 diabetic patients have functional defects and increased apoptosis that are ameliorated by metformin. J Clin Endocrinol Metab 89:5535-5541, 2004

140. Butler,AE, Janson,J, Bonner-Weir,S, Ritzel,R, Rizza,RA, Butler,PC: Beta-cell deficit and increased beta-cell apoptosis in humans with type 2 diabetes. Diabetes 52:102-110, 2003

149

141. Tokuyama,Y, Sturis,J, DePaoli,AM, Takeda,J, Stoffel,M, Tang,J, Sun,X, Polonsky,KS, Bell,GI: Evolution of beta-cell dysfunction in the male Zucker diabetic fatty rat. Diabetes 44:1447-1457, 1995

142. Pick,A, Clark,J, Kubstrup,C, Levisetti,M, Pugh,W, Bonner-Weir,S, Polonsky,KS: Role of apoptosis in failure of beta-cell mass compensation for insulin resistance and beta-cell defects in the male Zucker diabetic fatty rat. Diabetes 47:358-364, 1998

143. Finegood,DT, McArthur,MD, Kojwang,D, Thomas,MJ, Topp,BG, Leonard,T, Buckingham,RE: Beta-cell mass dynamics in Zucker diabetic fatty rats. Rosiglitazone prevents the rise in net cell death. Diabetes 50:1021-1029, 2001

144. Pick,A, Clark,J, Kubstrup,C, Levisetti,M, Pugh,W, Bonner-Weir,S, Polonsky,KS: Role of apoptosis in failure of beta-cell mass compensation for insulin resistance and beta-cell defects in the male Zucker diabetic fatty rat. Diabetes 47:358-364, 1998

145. Kahn,SE: The relative contributions of insulin resistance and beta-cell dysfunction to the pathophysiology of Type 2 diabetes. Diabetologia 46:3-19, 2003

146. Surwit,RS, Kuhn,CM, Cochrane,C, McCubbin,JA, Feinglos,MN: Diet-induced type II diabetes in C57BL/6J mice. Diabetes 37:1163-1167, 1988

147. Surwit,RS, Seldin,MF, Kuhn,CM, Cochrane,C, Feinglos,MN: Control of expression of insulin resistance and hyperglycemia by different genetic factors in diabetic C57BL/6J mice. Diabetes 40:82-87, 1991

148. Burcelin,R, Crivelli,V, Dacosta,A, Roy-Tirelli,A, Thorens,B: Heterogeneous metabolic adaptation of C57BL/6J mice to high-fat diet. Am J Physiol Endocrinol Metab 282:E834-E842, 2002

149. Fueger,PT, Bracy,DP, Malabanan,CM, Pencek,RR, Granner,DK, Wasserman,DH: Hexokinase II overexpression improves exercise-stimulated but not insulin-stimulated muscle glucose uptake in high-fat-fed C57BL/6J mice. Diabetes 53:306-314, 2004

150. Surwit,RS, Feinglos,MN, Rodin,J, Sutherland,A, Petro,AE, Opara,EC, Kuhn,CM, Rebuffe-Scrive,M: Differential effects of fat and sucrose on the development of obesity and diabetes in C57BL/6J and A/J mice. Metabolism 44:645-651, 1995

151. Black,BL, Croom,J, Eisen,EJ, Petro,AE, Edwards,CL, Surwit,RS: Differential effects of fat and sucrose on body composition in A/J and C57BL/6 mice. Metabolism 47:1354- 1359, 1998

150

152. Petro,AE, Cotter,J, Cooper,DA, Peters,JC, Surwit,SJ, Surwit,RS: Fat, carbohydrate, and calories in the development of diabetes and obesity in the C57BL/6J mouse. Metabolism 53:454-457, 2004

153. Rossmeisl,M, Rim,JS, Koza,RA, Kozak,LP: Variation in type 2 diabetes--related traits in mouse strains susceptible to diet-induced obesity. Diabetes 52:1958-1966, 2003

154. Goren,HJ, Kulkarni,RN, Kahn,CR: Glucose homeostasis and tissue transcript content of insulin signaling intermediates in four inbred strains of mice: C57BL/6, C57BLKS/6, DBA/2, and 129X1. Endocrinology 145:3307-3323, 2004

155. Kayo,T, Fujita,H, Nozaki,J, E X, Koizumi,A: Identification of two chromosomal loci determining glucose intolerance in a C57BL/6 mouse strain. Comp Med 50:296-302, 2000

156. Ahren,B, Pacini,G: Insufficient islet compensation to insulin resistance vs. reduced glucose effectiveness in glucose-intolerant mice. Am J Physiol Endocrinol Metab 283:E738-E744, 2002

157. Toye,AA, Lippiat,JD, Proks,P, Shimomura,K, Bentley,L, Hugill,A, Mijat,V, Goldsworthy,M, Moir,L, Haynes,A, Quarterman,J, Freeman,HC, Ashcroft,FM, Cox,RD: A genetic and physiological study of impaired glucose homeostasis control in C57BL/6J mice. Diabetologia 48:675-686, 2005

158. Coleman,DL, Hummel,KP: Symposium IV: Diabetic syndrome in animals. Influence of genetic background on the expression of mutations at the diabetes locus in the mouse. II. Studies on background modifiers. Isr J Med Sci 11:708-713, 1975

159. Coleman,DL, Hummel,KP: The influence of genetic background on the expression of the obese (Ob) gene in the mouse. Diabetologia 9:287-293, 1973

160. Hummel,KP, Coleman,DL, Lane,PW: The influence of genetic background on expression of mutations at the diabetes locus in the mouse. I. C57BL-KsJ and C57BL-6J strains. Biochem Genet 7:1-13, 1972

161. Leiter,EH, Coleman,DL, Hummel,KP: The influence of genetic background on the expression of mutations at the diabetes locus in the mouse. III. Effect of H-2 haplotype and sex. Diabetes 30:1029-1034, 1981

151

162. Leiter,EH, Coleman,DL, Hummel,KP: The influence of genetic background on the expression of mutations at the diabetes locus in the mouse. III. Effect of H-2 haplotype and sex. Diabetes 30:1029-1034, 1981

163. McGarry,JD, Dobbins,RL: Fatty acids, lipotoxicity and insulin secretion. Diabetologia 42:128-138, 1999

164. Unger,RH, Zhou,YT: Lipotoxicity of beta-cells in obesity and in other causes of fatty acid spillover. Diabetes 50 Suppl 1:S118-S121, 2001

165. Wrede,CE, Dickson,LM, Lingohr,MK, Briaud,I, Rhodes,CJ: Protein kinase B/Akt prevents fatty acid-induced apoptosis in pancreatic beta-cells (INS-1). J Biol Chem 277:49676-49684, 2002

166. Willaime-Morawek,S, Brami-Cherrier,K, Mariani,J, Caboche,J, Brugg,B: C-Jun N- terminal kinases/c-Jun and p38 pathways cooperate in ceramide-induced neuronal apoptosis. Neuroscience 119:387-397, 2003

167. Robertson,RP, Harmon,J, Tran,PO, Poitout,V: Beta-cell glucose toxicity, lipotoxicity, and chronic oxidative stress in type 2 diabetes. Diabetes 53 Suppl 1:S119-S124, 2004

168. Zick,Y: Insulin resistance: a phosphorylation-based uncoupling of insulin signaling. Trends Cell Biol 11:437-441, 2001

169. White,MF: Insulin signaling in health and disease. Science 302:1710-1711, 2003

170. Wrede,CE, Dickson,LM, Lingohr,MK, Briaud,I, Rhodes,CJ: Fatty acid and phorbol ester-mediated interference of mitogenic signaling via novel protein kinase C isoforms in pancreatic beta-cells (INS-1). J Mol Endocrinol 30:271-286, 2003

171. Greene,MW, Morrice,N, Garofalo,RS, Roth,RA: Modulation of human insulin receptor substrate-1 tyrosine phosphorylation by protein kinase Cdelta. Biochem J 378:105-116, 2004

172. Finegood,DT: Obesity, inflammation and type II diabetes. Int J Obes Relat Metab Disord 27 Suppl 3:S4-S5, 2003

173. Donath,MY, Storling,J, Maedler,K, Mandrup-Poulsen,T: Inflammatory mediators and islet beta-cell failure: a link between type 1 and type 2 diabetes. J Mol Med 81:455-470, 2003

152

174. Rui,L, Yuan,M, Frantz,D, Shoelson,S, White,MF: SOCS-1 and SOCS-3 block insulin signaling by ubiquitin-mediated degradation of IRS1 and IRS2. J Biol Chem 277:42394- 42398, 2002

175. Ueki,K, Kondo,T, Kahn,CR: Suppressor of cytokine signaling 1 (SOCS-1) and SOCS-3 cause insulin resistance through inhibition of tyrosine phosphorylation of insulin receptor substrate proteins by discrete mechanisms. Mol Cell Biol 24:5434-5446, 2004

176. Junod,A, Lambert,AE, Stauffacher,W, Renold,AE: Diabetogenic action of streptozotocin: relationship of dose to metabolic response. J Clin Invest 48:2129-2139, 1969

177. Lenzen,S, Panten,U: Alloxan: history and mechanism of action. Diabetologia 31:337- 342, 1988

178. Wang,RN, Bouwens,L, Kloppel,G: Beta-cell proliferation in normal and streptozotocin- treated newborn rats: site, dynamics and capacity. Diabetologia 37:1088-1096, 1994

179. Bonner-Weir,S, Trent,DF, Honey,RN, Weir,GC: Responses of neonatal rat islets to streptozotocin: limited B-cell regeneration and hyperglycemia. Diabetes 30:64-69, 1981

180. Cantenys,D, Portha,B, Dutrillaux,MC, Hollande,E, Roze,C, Picon,L: Histogenesis of the endocrine pancreas in newborn rats after destruction by streptozotocin. An immunocytochemical study. Virchows Arch B Cell Pathol Incl Mol Pathol 35:109-122, 1981

181. Wang,RN, Bouwens,L, Kloppel,G: Beta-cell growth in adolescent and adult rats treated with streptozotocin during the neonatal period. Diabetologia 39:548-557, 1996

182. Waguri,M, Yamamoto,K, Miyagawa,JI, Tochino,Y, Yamamori,K, Kajimoto,Y, Nakajima,H, Watada,H, Yoshiuchi,I, Itoh,N, Imagawa,A, Namba,M, Kuwajima,M, Yamasaki,Y, Hanafusa,T, Matsuzawa,Y: Demonstration of two different processes of beta-cell regeneration in a new diabetic mouse model induced by selective perfusion of alloxan. Diabetes 46:1281-1290, 1997

183. Rooman,I, Bouwens,L: Combined gastrin and epidermal growth factor treatment induces islet regeneration and restores normoglycaemia in C57Bl6/J mice treated with alloxan. Diabetologia 47:259-265, 2004

153

184. Rooman,I, Bouwens,L: Combined gastrin and epidermal growth factor treatment induces islet regeneration and restores normoglycaemia in C57Bl6/J mice treated with alloxan. Diabetologia 47:259-265, 2004

185. Pearson,KW, Scott,D, Torrance,B: Effects of partial surgical pancreatectomy in rats. I. Pancreatic regeneration. Gastroenterology 72:469-473, 1977

186. Leahy,JL, Bonner-Weir,S, Weir,GC: Minimal chronic hyperglycemia is a critical determinant of impaired insulin secretion after an incomplete pancreatectomy. J Clin Invest 81:1407-1414, 1988

187. Dor,Y, Brown,J, Martinez,OI, Melton,DA: Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation. Nature 429:41-46, 2004

188. Bonner-Weir,S, Baxter,LA, Schuppin,GT, Smith,FE: A second pathway for regeneration of adult exocrine and endocrine pancreas. A possible recapitulation of embryonic development. Diabetes 42:1715-1720, 1993

189. Leahy,JL, Bonner-Weir,S, Weir,GC: Minimal chronic hyperglycemia is a critical determinant of impaired insulin secretion after an incomplete pancreatectomy. J Clin Invest 81:1407-1414, 1988

190. Kendall,DM, Sutherland,DE, Najarian,JS, Goetz,FC, Robertson,RP: Effects of hemipancreatectomy on insulin secretion and glucose tolerance in healthy humans. N Engl J Med 322:898-903, 1990

191. Seaquist,ER, Kahn,SE, Clark,PM, Hales,CN, Porte,D, Jr., Robertson,RP: Hyperproinsulinemia is associated with increased beta cell demand after hemipancreatectomy in humans. J Clin Invest 97:455-460, 1996

192. Seaquist,ER, Robertson,RP: Effects of hemipancreatectomy on pancreatic alpha and beta cell function in healthy human donors. J Clin Invest 89:1761-1766, 1992

193. Robertson,RP, Lanz,KJ, Sutherland,DE, Seaquist,ER: Relationship between diabetes and obesity 9 to 18 years after hemipancreatectomy and transplantation in donors and recipients. Transplantation 73:736-741, 2002

194. Menge,BA, Tannapfel,A, Belyaev,O, Drescher,R, Muller,C, Uhl,W, Schmidt,WE, Meier,JJ: Partial pancreatectomy in adult humans does not provoke beta-cell regeneration. Diabetes 57:142-149, 2008

154

195. Vasavada,RC, Gonzalez-Pertusa,JA, Fujinaka,Y, Fiaschi-Taesch,N, Cozar-Castellano,I, Garcia-Ocana,A: Growth factors and beta cell replication. Int J Biochem Cell Biol 38:931-950, 2006

196. Ackermann,AM, Gannon,M: Molecular regulation of pancreatic beta-cell mass development, maintenance, and expansion. J Mol Endocrinol 38:193-206, 2007

197. Assmann,A, Hinault,C, Kulkarni,RN: Growth factor control of pancreatic islet regeneration and function. Pediatr Diabetes 10:14-32, 2009

198. Totsuka,Y, Tabuchi,M, Kojima,I, Shibai,H, Ogata,E: A novel action of activin A: stimulation of insulin secretion in rat pancreatic islets. Biochem Biophys Res Commun 156:335-339, 1988

199. Shibata,H, Yasuda,H, Sekine,N, Mine,T, Totsuka,Y, Kojima,I: Activin A increases intracellular free calcium concentrations in rat pancreatic islets. FEBS Lett 329:194-198, 1993

200. Verspohl,EJ, Ammon,HP, Wahl,MA: Activin A: its effects on rat pancreatic islets and the mechanism of action involved. Life Sci 53:1069-1078, 1993

201. Zhang,N, Kumar,M, Xu,G, Ju,W, Yoon,T, Xu,E, Huang,X, Gaisano,H, Peng,C, Wang,Q: Activin receptor-like kinase 7 induces apoptosis of pancreatic beta cells and beta cell lines. Diabetologia 49:506-518, 2006

202. Florio,P, Luisi,S, Marchetti,P, Lupi,R, Cobellis,L, Falaschi,C, Sugino,H, Navalesi,R, Genazzani,AR, Petraglia,F: Activin A stimulates insulin secretion in cultured human pancreatic islets. J Endocrinol Invest 23:231-234, 2000

203. Mashima,H, Ohnishi,H, Wakabayashi,K, Mine,T, Miyagawa,J, Hanafusa,T, Seno,M, Yamada,H, Kojima,I: Betacellulin and activin A coordinately convert amylase-secreting pancreatic AR42J cells into insulin-secreting cells. J Clin Invest 97:1647-1654, 1996

204. Ishiyama,N, Kanzaki,M, Seno,M, Yamada,H, Kobayashi,I, Kojima,I: Studies on the betacellulin receptor in pancreatic AR42J cells. Diabetologia 41:623-628, 1998

205. Demeterco,C, Beattie,GM, Dib,SA, Lopez,AD, Hayek,A: A role for activin A and betacellulin in human fetal pancreatic cell differentiation and growth. J Clin Endocrinol Metab 85:3892-3897, 2000

155

206. Huotari,MA, Palgi,J, Otonkoski,T: Growth factor-mediated proliferation and differentiation of insulin-producing INS-1 and RINm5F cells: identification of betacellulin as a novel beta-cell mitogen. Endocrinology 139:1494-1499, 1998

207. Ogata,T, Dunbar,AJ, Yamamoto,Y, Tanaka,Y, Seno,M, Kojima,I: Betacellulin-delta4, a novel differentiation factor for pancreatic beta-cells, ameliorates glucose intolerance in streptozotocin-treated rats. Endocrinology 146:4673-4681, 2005

208. Lee,HY, Jung,H, Jang,IH, Suh,PG, Ryu,SH: Cdk5 phosphorylates PLD2 to mediate EGF-dependent insulin secretion. Cell Signal 20:1787-1794, 2008

209. Maeda,H, Rajesh,KG, Maeda,H, Suzuki,R, Sasaguri,S: Epidermal growth factor and insulin inhibit cell death in pancreatic beta cells by activation of PI3-kinase/AKT signaling pathway under oxidative stress. Transplant Proc 36:1163-1165, 2004

210. Suarez-Pinzon,WL, Lakey,JR, Brand,SJ, Rabinovitch,A: Combination therapy with epidermal growth factor and gastrin induces neogenesis of human islet {beta}-cells from pancreatic duct cells and an increase in functional {beta}-cell mass. J Clin Endocrinol Metab 90:3401-3409, 2005

211. Kuntz,E, Broca,C, Komurasaki,T, Kaltenbacher,MC, Gross,R, Pinget,M, Damge,C: Effect of epiregulin on pancreatic beta cell growth and insulin secretion. Growth Factors 23:285-293, 2005

212. Wente,W, Efanov,AM, Brenner,M, Kharitonenkov,A, Koster,A, Sandusky,GE, Sewing,S, Treinies,I, Zitzer,H, Gromada,J: Fibroblast growth factor-21 improves pancreatic beta-cell function and survival by activation of extracellular signal-regulated kinase 1/2 and Akt signaling pathways. Diabetes 55:2470-2478, 2006

213. Otonkoski,T, Beattie,GM, Rubin,JS, Lopez,AD, Baird,A, Hayek,A: Hepatocyte growth factor/scatter factor has insulinotropic activity in human fetal pancreatic cells. Diabetes 43:947-953, 1994

214. Hayek,A, Beattie,GM, Cirulli,V, Lopez,AD, Ricordi,C, Rubin,JS: Growth factor/matrix- induced proliferation of human adult beta-cells. Diabetes 44:1458-1460, 1995

215. Otonkoski,T, Cirulli,V, Beattie,M, Mally,MI, Soto,G, Rubin,JS, Hayek,A: A role for hepatocyte growth factor/scatter factor in fetal mesenchyme-induced pancreatic beta-cell growth. Endocrinology 137:3131-3139, 1996

156

216. Beattie,GM, Montgomery,AM, Lopez,AD, Hao,E, Perez,B, Just,ML, Lakey,JR, Hart,ME, Hayek,A: A novel approach to increase human islet cell mass while preserving beta-cell function. Diabetes 51:3435-3439, 2002

217. Pais,E, Park,J, Alexy,T, Nikolian,V, Ge,S, Shaw,K, Senadheera,S, Hardee,CL, Skelton,D, Hollis,R, Crooks,GM, Kohn,DB: Regulated expansion of human pancreatic beta-cells. Mol Ther 18:1389-1396, 2010

218. Lefebvre,VH, Otonkoski,T, Ustinov,J, Huotari,MA, Pipeleers,DG, Bouwens,L: Culture of adult human islet preparations with hepatocyte growth factor and 804G matrix is mitogenic for duct cells but not for beta-cells. Diabetes 47:134-137, 1998

219. Panakanti,R, Mahato,RI: Bipartite adenoviral vector encoding hHGF and hIL-1Ra for improved human islet transplantation. Pharm Res 26:587-596, 2009

220. Gahr,S, Merger,M, Bollheimer,LC, Hammerschmied,CG, Scholmerich,J, Hugl,SR: Hepatocyte growth factor stimulates proliferation of pancreatic beta-cells particularly in the presence of subphysiological glucose concentrations. J Mol Endocrinol 28:99-110, 2002

221. Roccisana,J, Reddy,V, Vasavada,RC, Gonzalez-Pertusa,JA, Magnuson,MA, Garcia- Ocana,A: Targeted inactivation of hepatocyte growth factor receptor c-met in beta-cells leads to defective insulin secretion and GLUT-2 downregulation without alteration of beta-cell mass. Diabetes 54:2090-2102, 2005

222. Santangelo,C, Matarrese,P, Masella,R, Di Carlo,MC, Di,LA, Scazzocchio,B, Vecci,E, Malorni,W, Perfetti,R, Anastasi,E: Hepatocyte growth factor protects rat RINm5F cell line against free fatty acid-induced apoptosis by counteracting oxidative stress. J Mol Endocrinol 38:147-158, 2007

223. Li,XY, Zhan,XR, Lu,C, Liu,XM, Wang,XC: Mechanisms of hepatocyte growth factor- mediated signaling in differentiation of pancreatic ductal epithelial cells into insulin- producing cells. Biochem Biophys Res Commun 398:389-394, 2010

224. Mashima,H, Shibata,H, Mine,T, Kojima,I: Formation of insulin-producing cells from pancreatic acinar AR42J cells by hepatocyte growth factor. Endocrinology 137:3969- 3976, 1996

225. Mashima,H, Yamada,S, Tajima,T, Seno,M, Yamada,H, Takeda,J, Kojima,I: Genes expressed during the differentiation of pancreatic AR42J cells into insulin-secreting cells. Diabetes 48:304-309, 1999

157

226. Sjoholm,A, Sandberg,E, Ostenson,CG, Efendic,S: Peptidergic regulation of maturation of the stimulus-secretion coupling in fetal islet beta cells. Pancreas 20:282-289, 2000

227. Nielsen,JH, Linde,S, Welinder,BS, Billestrup,N, Madsen,OD: Growth hormone is a growth factor for the differentiated pancreatic beta-cell. Mol Endocrinol 3:165-173, 1989

228. Hellerstrom,C, Sjoholm,A, Swenne,I: Effects of growth hormone and related growth factors on DNA replication and insulin production in pancreatic islet beta-cells. Acta Paediatr Scand Suppl 377:55-62, 1991

229. Zhang,F, Zhang,Q, Tengholm,A, Sjoholm,A: Involvement of JAK2 and Src kinase tyrosine phosphorylation in human growth hormone-stimulated increases in cytosolic free Ca2+ and insulin secretion. Am J Physiol Cell Physiol 291:C466-C475, 2006

230. Ludwig,B, Ziegler,CG, Schally,AV, Richter,C, Steffen,A, Jabs,N, Funk,RH, Brendel,MD, Block,NL, Ehrhart-Bornstein,M, Bornstein,SR: Agonist of growth hormone-releasing hormone as a potential effector for survival and proliferation of pancreatic islets. Proc Natl Acad Sci U S A 107:12623-12628, 2010

231. Leahy,JL, Vandekerkhove,KM: Insulin-like growth factor-I at physiological concentrations is a potent inhibitor of insulin secretion. Endocrinology 126:1593-1598, 1990

232. Hill,DJ, Hogg,J, Petrik,J, Arany,E, Han,VK: Cellular distribution and ontogeny of insulin-like growth factors (IGFs) and IGF binding protein messenger RNAs and peptides in developing rat pancreas. J Endocrinol 160:305-317, 1999

233. Robitaille,R, Dusseault,J, Henley,N, Rosenberg,L, Halle,JP: Insulin-like growth factor II allows prolonged blood glucose normalization with a reduced islet cell mass transplantation. Endocrinology 144:3037-3045, 2003

234. Eizirik,DL, Skottner,A, Hellerstrom,C: Insulin-like growth factor I does not inhibit insulin secretion in adult human pancreatic islets in tissue culture. Eur J Endocrinol 133:248-250, 1995

235. Giannoukakis,N, Mi,Z, Rudert,WA, Gambotto,A, Trucco,M, Robbins,P: Prevention of beta cell dysfunction and apoptosis activation in human islets by adenoviral gene transfer of the insulin-like growth factor I. Gene Ther 7:2015-2022, 2000

236. Ohsugi,M, Cras-Meneur,C, Zhou,Y, Bernal-Mizrachi,E, Johnson,JD, Luciani,DS, Polonsky,KS, Permutt,MA: Reduced expression of the insulin receptor in mouse

158

insulinoma (MIN6) cells reveals multiple roles of insulin signaling in gene expression, proliferation, insulin content, and secretion. J Biol Chem 280:4992-5003, 2005

237. Dickson,LM, Lingohr,Mk, McCuaig,J, Hugl,SR, Snow,L, Kahn,BB, Myers,MGJr, Rhodes,CJ: Differential activation of protein kinase B and p70(S6)K by glucose and insulin-like growth factor 1 in pancreatic beta-cells (INS-1). J Biol Chem 276:21110- 21120, 2001

238. Hugl,SR, White,MF, Rhodes,CJ: Insulin-like growth factor I (IGF-I)-stimulated pancreatic beta-cell growth is glucose-dependent. Synergistic activation of insulin receptor substrate-mediated signal transduction pathways by glucose and IGF-I in INS-1 cells. J Biol Chem 273:17771-17779, 1998

239. Lingohr,Mk, Dickson,LM, McCuaig,JF, Hugl,SR, Twardzik,DR, Rhodes,CJ: Activation of IRS-2-mediated signal transduction by IGF-1, but not TGF-alpha or EGF, augments pancreatic beta-cell proliferation. Diabetes 51:966-976, 2002

240. Billestrup,N, Nielsen,JH: The stimulatory effect of growth hormone, prolactin, and placental lactogen on beta-cell proliferation is not mediated by insulin-like growth factor-I. Endocrinology 129:883-888, 1991

241. Brelje,TC, Sorenson,RL: Role of prolactin versus growth hormone on islet B-cell proliferation in vitro: implications for pregnancy. Endocrinology 128:45-57, 1991

242. Friedrichsen,BN, Richter,HE, Hansen,JA, Rhodes,CJ, Nielsen,JH, Billestrup,N, Moldrup,A: Signal transducer and activator of transcription 5 activation is sufficient to drive transcriptional induction of cyclin D2 gene and proliferation of rat pancreatic beta- cells. Mol Endocrinol 17:945-958, 2003

243. Friedrichsen,BN, Galsgaard,ED, Nielsen,JH, Moldrup,A: Growth hormone- and prolactin-induced proliferation of insulinoma cells, INS-1, depends on activation of STAT5 (signal transducer and activator of transcription 5). Mol Endocrinol 15:136-148, 2001

244. Movassat,J, Beattie,GM, Lopez,AD, Portha,B, Hayek,A: Keratinocyte growth factor and beta-cell differentiation in human fetal pancreatic endocrine precursor cells. Diabetologia 46:822-829, 2003

245. Villanueva-Penacarrillo,ML, Cancelas,J, de,MF, Redondo,A, Valin,A, Valverde,I, Esbrit,P: Parathyroid hormone-related peptide stimulates DNA synthesis and insulin secretion in pancreatic islets. J Endocrinol 163:403-408, 1999

159

246. Sawada,Y, Zhang,B, Okajima,F, Izumi,T, Takeuchi,T: PTHrP increases pancreatic beta- cell-specific functions in well-differentiated cells. Mol Cell Endocrinol 182:265-275, 2001

247. Garcia-Ocana,A, Vasavada,RC, Takane,KK, Cebrian,A, Lopez-Talavera,JC, Stewart,AF: Using beta-cell growth factors to enhance human pancreatic Islet transplantation. J Clin Endocrinol Metab 86:984-988, 2001

248. Liu,Z, Habener,JF: Stromal cell-derived factor-1 promotes survival of pancreatic beta cells by the stabilisation of beta-catenin and activation of transcription factor 7-like 2 (TCF7L2). Diabetologia 52:1589-1598, 2009

249. Sjoholm,A, Kindmark,H: Short- and long-term effects of beta-cellulin and transforming growth factor-alpha on beta-cell function in cultured fetal rat pancreatic islets. Endocrine 11:189-193, 1999

250. Sjoholm,A: Effects of transforming growth factor beta, tumor necrosis factor alpha and interferon gamma on pancreatic islet beta-cell responsiveness to transforming growth factor alpha. Biosci Rep 16:415-423, 1996

251. Hao,W, Palmer,JP: Recombinant human transforming growth factor beta does not inhibit the effects of interleukin-1 beta on pancreatic islet cells. J Interferon Cytokine Res 15:1075-1081, 1995

252. Sjoholm,A, Hellerstrom,C: TGF-beta stimulates insulin secretion and blocks mitogenic response of pancreatic beta-cells to glucose. Am J Physiol 260:C1046-C1051, 1991

253. Koevary,SB: Effects of incubation of islet cells with transforming growth factor beta (TGF-beta) on their susceptibility to diabetic lymphocyte mediated destruction in the rat, in vitro. Diabetes Res 18:123-127, 1991

254. Koevary,SB: Effects of transforming growth factor beta (TGF-beta) on the lymphocyte- and cytokine-mediated destruction of islet cells in the BB/Wor rat. Lymphokine Cytokine Res 10:337-342, 1991

255. Lin,HM, Lee,JH, Yadav,H, Kamaraju,AK, Liu,E, Zhigang,D, Vieira,A, Kim,SJ, Collins,H, Matschinsky,F, Harlan,DM, Roberts,AB, Rane,SG: Transforming growth factor-beta/Smad3 signaling regulates insulin gene transcription and pancreatic islet beta-cell function. J Biol Chem 284:12246-12257, 2009

160

256. Hanley,S, Rosenberg,L: Transforming growth factor beta is a critical regulator of adult human islet plasticity. Mol Endocrinol 21:1467-1477, 2007

257. Sekine,N, Yamashita,N, Kojima,I, Miyazaki,J, Ogata,E: Bimodal effect of transforming growth factor-beta on insulin secretion in MIN6 cells. Diabetes Res Clin Pract 26:7-14, 1994

258. Sayo,Y, Hosokawa,H, Imachi,H, Murao,K, Sato,M, Wong,NC, Ishida,T, Takahara,J: Transforming growth factor beta induction of insulin gene expression is mediated by pancreatic and duodenal homeobox gene-1 in rat insulinoma cells. Eur J Biochem 267:971-978, 2000

259. Mabley,JG, Cunningham,JM, John,N, Di Matteo,MA, Green,IC: Transforming growth factor beta 1 prevents cytokine-mediated inhibitory effects and induction of nitric oxide synthase in the RINm5F insulin-containing beta-cell line. J Endocrinol 155:567-575, 1997

260. Brissova,M, Shostak,A, Shiota,M, Wiebe,PO, Poffenberger,G, Kantz,J, Chen,Z, Carr,C, Jerome,WG, Chen,J, Baldwin,HS, Nicholson,W, Bader,DM, Jetton,T, Gannon,M, Powers,AC: Pancreatic islet production of vascular endothelial growth factor--a is essential for islet vascularization, revascularization, and function. Diabetes 55:2974- 2985, 2006

261. Iwashita,N, Uchida,T, Choi,JB, Azuma,K, Ogihara,T, Ferrara,N, Gerber,H, Kawamori,R, Inoue,M, Watada,H: Impaired insulin secretion in vivo but enhanced insulin secretion from isolated islets in pancreatic beta cell-specific vascular endothelial growth factor-A knock-out mice. Diabetologia 50:380-389, 2007

262. Narang,AS, Sabek,O, Gaber,AO, Mahato,RI: Co-expression of vascular endothelial growth factor and interleukin-1 receptor antagonist improves human islet survival and function. Pharm Res 23:1970-1982, 2006

263. Mukherjee,A, Sidis,Y, Mahan,A, Raher,MJ, Xia,Y, Rosen,ED, Bloch,KD, Thomas,MK, Schneyer,AL: FSTL3 deletion reveals roles for TGF-beta family ligands in glucose and fat homeostasis in adults. Proc Natl Acad Sci U S A 104:1348-1353, 2007

264. Smart,NG, Apelqvist,AA, Gu,X, Harmon,EB, Topper,JN, MacDonald,RJ, Kim,SK: Conditional expression of Smad7 in pancreatic beta cells disrupts TGF-beta signaling and induces reversible diabetes mellitus. PLoS Biol 4:e39, 2006

161

265. Bertolino,P, Holmberg,R, Reissmann,E, Andersson,O, Berggren,PO, Ibanez,CF: Activin B receptor ALK7 is a negative regulator of pancreatic beta-cell function. Proc Natl Acad Sci U S A 105:7246-7251, 2008

266. Park,MK, Han,C, Lee,KH, Hong,SH, Kim,HS, Lee,YJ, Jeong,IK, Noh,JH, Yang,TY, Lee,MS, Kim,KW, Lee,MK: Effects of activin A on pancreatic ductal cells in streptozotocin-induced diabetic rats. Transplantation 83:925-930, 2007

267. Li,L, Yi,Z, Seno,M, Kojima,I: Activin A and betacellulin: effect on regeneration of pancreatic beta-cells in neonatal streptozotocin-treated rats. Diabetes 53:608-615, 2004

268. Chen,S, Ding,J, Yu,C, Yang,B, Wood,DR, Grayburn,PA: Reversal of streptozotocin- induced diabetes in rats by gene therapy with betacellulin and pancreatic duodenal homeobox-1. Gene Ther 14:1102-1110, 2007

269. Yamamoto,K, Miyagawa,J, Waguri,M, Sasada,R, Igarashi,K, Li,M, Nammo,T, Moriwaki,M, Imagawa,A, Yamagata,K, Nakajima,H, Namba,M, Tochino,Y, Hanafusa,T, Matsuzawa,Y: Recombinant human betacellulin promotes the neogenesis of beta-cells and ameliorates glucose intolerance in mice with diabetes induced by selective alloxan perfusion. Diabetes 49:2021-2027, 2000

270. Li,L, Seno,M, Yamada,H, Kojima,I: Promotion of beta-cell regeneration by betacellulin in ninety percent-pancreatectomized rats. Endocrinology 142:5379-5385, 2001

271. Li,L, Seno,M, Yamada,H, Kojima,I: Betacellulin improves glucose metabolism by promoting conversion of intraislet precursor cells to beta-cells in streptozotocin-treated mice. Am J Physiol Endocrinol Metab 285:E577-E583, 2003

272. Tokui,Y, Kozawa,J, Yamagata,K, Zhang,J, Ohmoto,H, Tochino,Y, Okita,K, Iwahashi,H, Namba,M, Shimomura,I, Miyagawa,J: Neogenesis and proliferation of beta-cells induced by human betacellulin gene transduction via retrograde pancreatic duct injection of an adenovirus vector. Biochem Biophys Res Commun 350:987-993, 2006

273. Shin,S, Li,N, Kobayashi,N, Yoon,JW, Jun,HS: Remission of diabetes by beta-cell regeneration in diabetic mice treated with a recombinant adenovirus expressing betacellulin. Mol Ther 16:854-861, 2008

274. Yamamoto,Y, Yamada,S, Kodera,T, Hara,A, Motoyoshi,K, Tanaka,Y, Nagaoka,T, Seno,M, Kojima,I: Reversal of streptozotocin-induced hyperglycemia by continuous supply of betacellulin in mice. Growth Factors1, 2008

162

275. Dahlhoff,M, Algul,H, Siveke,JT, Lesina,M, Wanke,R, Wartmann,T, Halangk,W, Schmid,RM, Wolf,E, Schneider,MR: Betacellulin protects from pancreatitis by activating stress-activated protein kinase. Gastroenterology 138:1585-94, 1594, 2010

276. Miettinen,PJ, Ustinov,J, Ormio,P, Gao,R, Palgi,J, Hakonen,E, Juntti-Berggren,L, Berggren,PO, Otonkoski,T: Downregulation of EGF receptor signaling in pancreatic islets causes diabetes due to impaired postnatal beta-cell growth. Diabetes 55:3299- 3308, 2006

277. Kozawa,J, Tokui,Y, Moriwaki,M, Li,M, Ohmoto,H, Yuan,M, Zhang,J, Iwahashi,H, Imagawa,A, Yamagata,K, Tochino,Y, Shimomura,I, Higashiyama,S, Miyagawa,J: Regenerative and therapeutic effects of heparin-binding epidermal growth factor-like growth factor on diabetes by gene transduction through retrograde pancreatic duct injection of adenovirus vector. Pancreas 31:32-42, 2005

278. Krakowski,ML, Kritzik,MR, Jones,EM, Krahl,T, Lee,J, Arnush,M, Gu,D, Mroczkowski,B, Sarvetnick,N: Transgenic expression of epidermal growth factor and keratinocyte growth factor in beta-cells results in substantial morphological changes. J Endocrinol 162:167-175, 1999

279. Hart,AW, Baeza,N, Apelqvist,A, Edlund,H: Attenuation of FGF signalling in mouse beta-cells leads to diabetes. Nature 408:864-868, 2000

280. Garcia-Ocana,A, Takane,KK, Syed,MA, Philbrick,WM, Vasavada,RC, Stewart,AF: Hepatocyte growth factor overexpression in the islet of transgenic mice increases beta cell proliferation, enhances islet mass, and induces mild hypoglycemia. J Biol Chem 275:1226-1232, 2000

281. Garcia-Ocana,A, Vasavada,RC, Cebrian,A, Reddy,V, Takane,KK, Lopez-Talavera,JC, Stewart,AF: Transgenic overexpression of hepatocyte growth factor in the beta-cell markedly improves islet function and islet transplant outcomes in mice. Diabetes 50:2752-2762, 2001

282. Gonzalez-Pertusa,JA, Dube,J, Valle,SR, Rosa,TC, Takane,KK, Mellado-Gil,JM, Perdomo,G, Vasavada,RC, Garcia-Ocana,A: Novel proapoptotic effect of hepatocyte growth factor: synergy with palmitate to cause pancreatic {beta}-cell apoptosis. Endocrinology 151:1487-1498, 2010

283. Dai,C, Li,Y, Yang,J, Liu,Y: Hepatocyte growth factor preserves beta cell mass and mitigates hyperglycemia in streptozotocin-induced diabetic mice. J Biol Chem 278:27080-27087, 2003

163

284. Park,YH, Ryu,HS, Choi,DS, Chang,KH, Park,DW, Min,CK: Effects of hepatocyte growth factor on the expression of matrix metalloproteinases and their tissue inhibitors during the endometrial cancer invasion in a three-dimensional coculture. Int J Gynecol Cancer 13:53-60, 2003

285. Dai,C, Huh,CG, Thorgeirsson,SS, Liu,Y: Beta-cell-specific ablation of the hepatocyte growth factor receptor results in reduced islet size, impaired insulin secretion, and glucose intolerance. Am J Pathol 167:429-436, 2005

286. Lopez-Talavera,JC, Garcia-Ocana,A, Sipula,I, Takane,KK, Cozar-Castellano,I, Stewart,AF: Hepatocyte growth factor gene therapy for pancreatic islets in diabetes: reducing the minimal islet transplant mass required in a glucocorticoid-free rat model of allogeneic portal vein islet transplantation. Endocrinology 145:467-474, 2004

287. Fiaschi-Taesch,NM, Berman,DM, Sicari,BM, Takane,KK, Garcia-Ocana,A, Ricordi,C, Kenyon,NS, Stewart,AF: Hepatocyte growth factor enhances engraftment and function of nonhuman primate islets. Diabetes 57:2745-2754, 2008

288. ZOLLINGER,RM, Ellison,EH: Primary peptic ulcerations of the jejunum associated with islet cell tumors of the pancreas. Ann Surg 142:709-723, 1955

289. Creutzfeldt,W, Arnold,R, Creutzfeldt,C, Track,NS: Pathomorphologic, biochemical, and diagnostic aspects of gastrinomas (Zollinger-Ellison syndrome). Hum Pathol 6:47-76, 1975

290. Larsson,LI, Sundler,F, Hakanson,R, Rehfeld,JF, Stadil,F: Immunofluorescent localization of gastrin in rabbit antropyloric mucosa to argyrophil cells exhibiting formaldehyde-ozone-induced fluorescence. Histochemie 37:81-87, 1973

291. Meier,JJ, Butler,AE, Galasso,R, Rizza,RA, Butler,PC: Increased islet beta cell replication adjacent to intrapancreatic gastrinomas in humans. Diabetologia 49:2689- 2696, 2006

292. Suarez-Pinzon,WL, Power,RF, Yan,Y, Wasserfall,C, Atkinson,M, Rabinovitch,A: Combination therapy with glucagon-like peptide-1 and gastrin restores normoglycemia in diabetic NOD mice. Diabetes 57:3281-3288, 2008

293. Suarez-Pinzon,WL, Lakey,JR, Rabinovitch,A: Combination therapy with glucagon-like peptide-1 and gastrin induces beta-cell neogenesis from pancreatic duct cells in human islets transplanted in immunodeficient diabetic mice. Cell Transplant 17:631-640, 2008

164

294. Suarez-Pinzon,WL, Rabinovitch,A: Combination therapy with epidermal growth factor and gastrin delays autoimmune diabetes recurrence in nonobese diabetic mice transplanted with syngeneic islets. Transplant Proc 40:529-532, 2008

295. Suarez-Pinzon,WL, Yan,Y, Power,R, Brand,SJ, Rabinovitch,A: Combination therapy with epidermal growth factor and gastrin increases beta-cell mass and reverses hyperglycemia in diabetic NOD mice. Diabetes 54:2596-2601, 2005

296. Rooman,I, Bouwens,L: Combined gastrin and epidermal growth factor treatment induces islet regeneration and restores normoglycaemia in C57Bl6/J mice treated with alloxan. Diabetologia 47:259-265, 2004

297. Rooman,I, Lardon,J, Bouwens,L: Gastrin stimulates beta-cell neogenesis and increases islet mass from transdifferentiated but not from normal exocrine pancreas tissue. Diabetes 51:686-690, 2002

298. Wang,RN, Rehfeld,JF, Nielsen,FC, Kloppel,G: Expression of gastrin and transforming growth factor-alpha during duct to islet cell differentiation in the pancreas of duct-ligated adult rats. Diabetologia 40:887-893, 1997

299. Wang,TC, Bonner-Weir,S, Oates,PS, Chulak,M, Simon,B, Merlino,GT, Schmidt,EV, Brand,SJ: Pancreatic gastrin stimulates islet differentiation of transforming growth factor alpha-induced ductular precursor cells. J Clin Invest 92:1349-1356, 1993

300. Garay,GL, Akerblom,HK, Martin,JM: Experimental hypersomatotropism: serum growth hormone and insulin, and pituitary and pancreatic changes in MtT-W15 tumor-bearing rats before and after tumor removal. Horm Metab Res 3:82-89, 1971

301. Parsons,JA, Hartfel,MA, Hegre,OD, McEvoy,RC: Effect of MtTW15 mammosomatotropic tumors on pancreatic islet hormones. Diabetes 32:67-74, 1983

302. Liu,JL, Coschigano,KT, Robertson,K, Lipsett,M, Guo,Y, Kopchick,JJ, Kumar,U, Liu,YL: Disruption of growth hormone receptor gene causes diminished pancreatic islet size and increased insulin sensitivity in mice. Am J Physiol Endocrinol Metab 287:E405- E413, 2004

303. Accili,D, Drago,J, Lee,EJ, Johnson,MD, Cool,MH, Salvatore,P, Asico,LD, Jose,PA, Taylor,SI, Westphal,H: Early neonatal death in mice homozygous for a null allele of the insulin receptor gene. Nat Genet 12:106-109, 1996

165

304. Joshi,RL, Lamothe,B, Cordonnier,N, Mesbah,K, Monthioux,E, Jami,J, Bucchini,D: Targeted disruption of the insulin receptor gene in the mouse results in neonatal lethality. EMBO J 15:1542-1547, 1996

305. Kido,Y, Nakae,J, Hribal,ML, Xuan,S, Efstratiadis,A, Accili,D: Effects of mutations in the insulin-like growth factor signaling system on embryonic pancreas development and beta-cell compensation to insulin resistance. J Biol Chem 277:36740-36747, 2002

306. Withers,DJ, Burks,DJ, Towery,HH, Altamuro,SL, Flint,CL, White,MF: Irs-2 coordinates Igf-1 receptor-mediated beta-cell development and peripheral insulin signalling. Nat Genet 23:32-40, 1999

307. Kitamura,T, Kido,Y, Nef,S, Merenmies,J, Parada,LF, Accili,D: Preserved pancreatic beta-cell development and function in mice lacking the insulin receptor-related receptor. Mol Cell Biol 21:5624-5630, 2001

308. Duvillie,B, Currie,C, Chrones,T, Bucchini,D, Jami,J, Joshi,RL, Hill,DJ: Increased islet cell proliferation, decreased apoptosis, and greater vascularization leading to beta-cell hyperplasia in mutant mice lacking insulin. Endocrinology 143:1530-1537, 2002

309. Duvillie,B, Cordonnier,N, Deltour,L, ndoy-Dron,F, Itier,JM, Monthioux,E, Jami,J, Joshi,RL, Bucchini,D: Phenotypic alterations in insulin-deficient mutant mice. Proc Natl Acad Sci U S A 94:5137-5140, 1997

310. Liu,JL, LeRoith,D: Insulin-like growth factor I is essential for postnatal growth in response to growth hormone. Endocrinology 140:5178-5184, 1999

311. Otani,K, Kulkarni,RN, Baldwin,AC, Krutzfeldt,J, Ueki,K, Stoffel,M, Kahn,CR, Polonsky,KS: Reduced beta-cell mass and altered glucose sensing impair insulin- secretory function in betaIRKO mice. Am J Physiol Endocrinol Metab 286:E41-E49, 2004

312. Kulkarni,RN, Bruning,JC, Winnay,JN, Postic,C, Magnuson,MA, Kahn,CR: Tissue- specific knockout of the insulin receptor in pancreatic beta cells creates an insulin secretory defect similar to that in type 2 diabetes. Cell 96:329-339, 1999

313. Xuan,S, Kitamura,T, Nakae,J, Politi,K, Kido,Y, Fisher,PE, Morroni,M, Cinti,S, White,MF, Herrera,PL, Accili,D, Efstratiadis,A: Defective insulin secretion in pancreatic beta cells lacking type 1 IGF receptor. J Clin Invest 110:1011-1019, 2002

166

314. Kulkarni,RN, Holzenberger,M, Shih,DQ, Ozcan,U, Stoffel,M, Magnuson,MA, Kahn,CR: beta-cell-specific deletion of the Igf1 receptor leads to hyperinsulinemia and glucose intolerance but does not alter beta-cell mass. Nat Genet 31:111-115, 2002

315. Ueki,K, Okada,T, Hu,J, Liew,CW, Assmann,A, Dahlgren,GM, Peters,JL, Shackman,JG, Zhang,M, Artner,I, Satin,LS, Stein,R, Holzenberger,M, Kennedy,RT, Kahn,CR, Kulkarni,RN: Total insulin and IGF-I resistance in pancreatic beta cells causes overt diabetes. Nat Genet 38:583-588, 2006

316. Yu,R, Yakar,S, Liu,YL, Lu,Y, LeRoith,D, Miao,D, Liu,JL: Liver-specific IGF-I gene deficient mice exhibit accelerated diabetes in response to streptozotocin, associated with early onset of insulin resistance. Mol Cell Endocrinol 204:31-42, 2003

317. Yakar,S, Liu,JL, Fernandez,AM, Wu,Y, Schally,AV, Frystyk,J, Chernausek,SD, Mejia,W, Le,RD: Liver-specific igf-1 gene deletion leads to muscle insulin insensitivity. Diabetes 50:1110-1118, 2001

318. Yakar,S, Setser,J, Zhao,H, Stannard,B, Haluzik,M, Glatt,V, Bouxsein,ML, Kopchick,JJ, LeRoith,D: Inhibition of growth hormone action improves insulin sensitivity in liver IGF-1-deficient mice. J Clin Invest 113:96-105, 2004

319. Lu,Y, Herrera,PL, Guo,Y, Sun,D, Tang,Z, LeRoith,D, Liu,JL: Pancreatic-specific inactivation of IGF-I gene causes enlarged pancreatic islets and significant resistance to diabetes. Diabetes 53:3131-3141, 2004

320. George,M, Ayuso,E, Casellas,A, Costa,C, Devedjian,JC, Bosch,F: Beta cell expression of IGF-I leads to recovery from type 1 diabetes. J Clin Invest 109:1153-1163, 2002

321. Guo,Y, Lu,Y, Houle,D, Robertson,K, Tang,Z, Kopchick,JJ, Liu,YL, Liu,JL: Pancreatic islet-specific expression of an insulin-like growth factor-I transgene compensates islet cell growth in growth hormone receptor gene-deficient mice. Endocrinology 146:2602- 2609, 2005

322. Devedjian,JC, George,M, Casellas,A, Pujol,A, Visa,J, Pelegrin,M, Gros,L, Bosch,F: Transgenic mice overexpressing insulin-like growth factor-II in beta cells develop type 2 diabetes. J Clin Invest 105:731-740, 2000

323. Petrik,J, Pell,JM, Arany,E, McDonald,TJ, Dean,WL, Reik,W, Hill,DJ: Overexpression of insulin-like growth factor-II in transgenic mice is associated with pancreatic islet cell hyperplasia. Endocrinology 140:2353-2363, 1999

167

324. Krakowski,ML, Kritzik,MR, Jones,EM, Krahl,T, Lee,J, Arnush,M, Gu,D, Sarvetnick,N: Pancreatic expression of keratinocyte growth factor leads to differentiation of islet hepatocytes and proliferation of duct cells. Am J Pathol 154:683-691, 1999

325. Wagner,M, Koschnick,S, Beilke,S, Frey,M, Adler,G, Schmid,RM: Selective expansion of the beta-cell compartment in the pancreas of keratinocyte growth factor transgenic mice. Am J Physiol Gastrointest Liver Physiol 294:G1139-G1147, 2008

326. Porter,SE, Sorenson,RL, Dann,P, Garcia-Ocana,A, Stewart,AF, Vasavada,RC: Progressive pancreatic islet hyperplasia in the islet-targeted, parathyroid hormone- related protein-overexpressing mouse. Endocrinology 139:3743-3751, 1998

327. Simeone,DM, Zhang,L, Treutelaar,MK, Zhang,L, Graziano,K, Logsdon,CD, Burant,CF: Islet hypertrophy following pancreatic disruption of Smad4 signaling. Am J Physiol Endocrinol Metab 291:E1305-E1316, 2006

328. Lammert,E, Gu,G, McLaughlin,M, Brown,D, Brekken,R, Murtaugh,LC, Gerber,HP, Ferrara,N, Melton,DA: Role of VEGF-A in vascularization of pancreatic islets. Curr Biol 13:1070-1074, 2003

329. Jabs,N, Franklin,I, Brenner,MB, Gromada,J, Ferrara,N, Wollheim,CB, Lammert,E: Reduced insulin secretion and content in VEGF-a deficient mouse pancreatic islets. Exp Clin Endocrinol Diabetes 116 Suppl 1:S46-S49, 2008

330. Toyofuku,Y, Uchida,T, Nakayama,S, Hirose,T, Kawamori,R, Fujitani,Y, Inoue,M, Watada,H: Normal islet vascularization is dispensable for expansion of beta-cell mass in response to high-fat diet induced insulin resistance. Biochem Biophys Res Commun 383:303-307, 2009

331. Dube,PE, Brubaker,PL: Nutrient, neural and endocrine control of glucagon-like peptide secretion. Horm Metab Res 36:755-760, 2004

332. Rothenberg,ME, Eilertson,CD, Klein,K, Zhou,Y, Lindberg,I, McDonald,JK, Mackin,RB, Noe,BD: Processing of mouse proglucagon by recombinant prohormone convertase 1 and immunopurified prohormone convertase 2 in vitro. J Biol Chem 270:10136-10146, 1995

333. Rouille,Y, Martin,S, Steiner,DF: Differential processing of proglucagon by the subtilisin-like prohormone convertases PC2 and PC3 to generate either glucagon or glucagon-like peptide. J Biol Chem 270:26488-26496, 1995

168

334. Dhanvantari,S, Seidah,NG, Brubaker,PL: Role of prohormone convertases in the tissue- specific processing of proglucagon. Mol Endocrinol 10:342-355, 1996

335. White,JW, Saunders,GF: Structure of the human glucagon gene. Nucleic Acids Res 14:4719-4730, 1986

336. Trinh,DK, Zhang,K, Hossain,M, Brubaker,PL, Drucker,DJ: Pax-6 activates endogenous proglucagon gene expression in the rodent gastrointestinal epithelium. Diabetes 52:425- 433, 2003

337. Grant,SF, Thorleifsson,G, Reynisdottir,I, Benediktsson,R, Manolescu,A, Sainz,J, Helgason,A, Stefansson,H, Emilsson,V, Helgadottir,A, Styrkarsdottir,U, Magnusson,KP, Walters,GB, Palsdottir,E, Jonsdottir,T, Gudmundsdottir,T, Gylfason,A, Saemundsdottir,J, Wilensky,RL, Reilly,MP, Rader,DJ, Bagger,Y, Christiansen,C, Gudnason,V, Sigurdsson,G, Thorsteinsdottir,U, Gulcher,JR, Kong,A, Stefansson,K: Variant of transcription factor 7-like 2 (TCF7L2) gene confers risk of type 2 diabetes. Nat Genet ePub: 2006

338. Yi,F, Brubaker,PL, Jin,T: TCF-4 mediates cell type-specific regulation of proglucagon gene expression by beta-catenin and glycogen synthase kinase-3beta. J Biol Chem 280:1457-1464, 2005

339. Pilgaard,K, Jensen,CB, Schou,JH, Lyssenko,V, Wegner,L, Brons,C, Vilsboll,T, Hansen,T, Madsbad,S, Holst,JJ, Volund,A, Poulsen,P, Groop,L, Pedersen,O, Vaag,AA: The T allele of rs7903146 TCF7L2 is associated with impaired insulinotropic action of incretin hormones, reduced 24 h profiles of plasma insulin and glucagon, and increased hepatic glucose production in young healthy men. Diabetologia 52:1298-1307, 2009

340. Vilsboll,T, Agerso,H, Krarup,T, Holst,JJ: Similar elimination rates of glucagon-like peptide-1 in obese type 2 diabetic patients and healthy subjects. J Clin Endocrinol Metab 88:220-224, 2003

341. Edwards,CM, Todd,JF, Mahmoudi,M, Wang,Z, Wang,RM, Ghatei,MA, Bloom,SR: Glucagon-like peptide 1 has a physiological role in the control of postprandial glucose in humans: studies with the antagonist exendin 9-39. Diabetes 48:86-93, 1999

342. Hansotia,T, Drucker,DJ: GIP and GLP-1 as incretin hormones: lessons from single and double incretin receptor knockout mice. Regul Pept 128:125-134, 2005

343. Drucker,DJ: Minireview: The glucagon-like peptides. Endocrinology 142:521-527, 2001

169

344. Meier,JJ, Nauck,MA: Glucagon-like peptide 1(GLP-1) in biology and pathology. Diabetes Metab Res Rev 21:91-117, 2005

345. Raufman,JP, Singh,L, Eng,J: Exendin-3, a novel peptide from Heloderma horridum venom, interacts with vasoactive intestinal peptide receptors and a newly described receptor on dispersed acini from guinea pig pancreas. Description of exendin-3(9-39) amide, a specific exendin receptor antagonist. J Biol Chem 266:2897-2902, 1991

346. Thorens,B: Expression cloning of the pancreatic beta cell receptor for the gluco-incretin hormone glucagon-like peptide 1. Proc Natl Acad Sci U S A 89:8641-8645, 1992

347. Raufman,JP, Singh,L, Singh,G, Eng,J: Truncated glucagon-like peptide-1 interacts with exendin receptors on dispersed acini from guinea pig pancreas. Identification of a mammalian analogue of the reptilian peptide exendin-4. J Biol Chem 267:21432-21437, 1992

348. Brubaker,PL, Drucker,DJ: Structure-function of the glucagon receptor family of G protein-coupled receptors: The glucagon, GIP, GLP-1 and GLP-2 receptors. Recept Chann 8:179-188, 2002

349. Kreymann,B, Ghatei,MA, Williams,G, Bloom,SR: Glucagon-like peptide-1 7-36: A physiological incretin in man. Lancet 2:1300-1304, 1987

350. Gutniak,M, Orskov,C, Holst,JJ, Ahrén,B, Efendic,S: Antidiabetogenic effect of glucagon-like peptide-1 (7-36)amide in normal subjects and patients with diabetes mellitus. New Engl J Med 326:1316-1322, 1992

351. Nauck,MA, Kleine,N, Orskov,C, Holst,JJ, Willms,B, Creutzfeldt,W: Normalization of fasting hyperglycaemia by exogenous glucagon- like peptide 1(7-36)amide in type 2 (non-insulin-dependent) diabetic patients. Digestion 54:389, 1993

352. Ahren,B, Larsson,H, Holst,JJ: Effects of glucagon-like peptide-1 on islet function and insulin sensitivity in noninsulin-dependent diabetes mellitus. J Clin Endocrinol Metab 82:473-478, 1997

353. Todd,JF, Wilding,JPH, Edwards,CMB, Khan,FA, Ghatei,MA, Bloom,SR: Glucagon-like peptide-1 (GLP-1): A trial of treatment in noninsulin-dependent diabetes mellitus. Eur J Clin Invest 27:533-536, 1997

170

354. Zander,M, Madsbad,S, Madsen,JL, Holst,JJ: Effect of 6-week course of glucagon-like peptide 1 on glycaemic control, insulin sensitivity, and beta-cell function in type 2 diabetes: a parallel-group study. Lancet 359:824-830, 2002

355. Kieffer,TJ, McIntosh,CHS, Pederson,RA: Degradation of glucose-dependent insulinotropic polypeptide and truncated glucagon-like peptide 1 in vitro and in vivo by dipeptidyl peptidase IV. Endocrinology 136:3585-3596, 1995

356. Drucker,DJ: Enhancing incretin action for the treatment of type 2 diabetes. Diabetes Care 26:2929-2940, 2003

357. Eng,J: Exendin peptides. Mt Sinai J Med 59:147-149, 1992

358. Nielsen,LL, Baron,AD: Pharmacology of exenatide (synthetic exendin-4) for the treatment of type 2 diabetes. Curr Opin Investig Drugs 4:401-405, 2003

359. DeFronzo,RA, Ratner,RE, Han,J, Kim,DD, Fineman,MS, Baron,AD: Effects of exenatide (exendin-4) on glycemic control and weight over 30 weeks in metformin- treated patients with type 2 diabetes. Diabetes Care 28:1092-1100, 2005

360. Exenatide (Byetta) for type 2 diabetes. Med Lett Drugs Ther 47:45-46, 2005

361. Holz,GG, Kühtreiber,WM, Habener,JF: Pancreatic beta-cells are rendered glucose- competent by the insulinotropic hormone glucagon-like peptide-1(7-37). Nature 361:362-365, 1993

362. Light,PE, Manning Fox,JE, Riedel,MJ, Wheeler,MB: Glucagon-like peptide-1 inhibits pancreatic ATP-sensitive potassium channels via a protein kinase A- and ADP- dependent mechanism. Mol Endocrinol 16:2135-2144, 2002

363. Suga,S, Kanno,T, Ogawa,Y, Takeo,T, Kamimura,N, Wakui,M: cAMP-independent decrease of ATP-sensitive K+ channel activity by GLP-1 in rat pancreatic beta-cells. Pflugers Arch 440:566-572, 2000

364. Gromada,J, Ding,WG, Barg,S, Renström,E, Rorsman,P: Multisite regulation of insulin secretion by cAMP-increasing agonists: Evidence that glucagon-like peptide 1 and glucagon act via distinct receptors. Pflugers Arch 434:515-524, 1997

171

365. Gromada,J, Anker,C, Bokvist,K, Knudsen,LB, Wahl,P: Glucagon-like peptide-1 receptor expression in Xenopus oocytes stimulates inositol trisphosphate-dependent intracellular Ca2+ mobilization. FEBS Lett 425:277-280, 1998

366. Ding,WG, Gromada,J: Protein kinase A-dependent stimulation of exocytosis in mouse pancreatic -cells by glucose-dependent insulinotropic polypeptide. Diabetes 46:615- 621, 1997

367. Nakazaki,M, Crane,A, Hu,M, Seghers,V, Ullrich,S, guilar-Bryan,L, Bryan,J: cAMP- activated protein kinase-independent potentiation of insulin secretion by cAMP is impaired in SUR1 null islets. Diabetes 51:3440-3449, 2002

368. Shiota,C, Larsson,O, Shelton,KD, Shiota,M, Efanov,AM, Hoy,M, Lindner,J, Kooptiwut,S, Juntti-Berggren,L, Gromada,J, Berggren,PO, Magnuson,MA: Sulfonylurea receptor type 1 knock-out mice have intact feeding-stimulated insulin secretion despite marked impairment in their response to glucose. J Biol Chem 277:37176-37183, 2002

369. Gromada,J, Bokvist,K, Ding,WG, Holst,JJ, Nielsen,JH, Rorsman,P: Glucagon-like peptide 1(7-36) amide stimulates exocytosis in human pancreatic -cells by both proximal and distal regulatory steps in stimulus-secretion coupling. Diabetes 47:57-65, 1998

370. Ammala,C, Ashcroft,FM, Rorsman,P: Calcium-independent potentiation of insulin release by cyclic AMP in single B-cells. Nature 363:356-358, 1993

371. Holz,GG, Leech,CA, Habener,JF: Activation of a cAMP-regulated Ca2+-signaling pathway in pancreatic -cells by the insulinotropic hormone glucagon- like peptide-1. J Biol Chem 270:17749-17757, 1995

372. Yada,T, Itoh,K, Kakei,M, Tanaka,H: Glucose metabolism by rat pancreatic beta-cells produces dual change in cytosolic Ca2+. Jpn J Physiol 43 Suppl 1:S115-S118, 1993

373. Kang,G, Chepurny,OG, Holz,GG: cAMP-regulated guanine nucleotide exchange factor II (Epac2) mediates Ca2+-induced Ca2+ release in INS-1 pancreatic beta-cells. J Physiol 536:375-385, 2001

374. Islam,MS, Leibiger,I, Leibiger,B, Rossi,D, Sorrentino,V, Ekstrom,TJ, Westerblad,H, Andrade,FH, Berggren,PO: In situ activation of the type 2 ryanodine receptor in pancreatic beta cells requires cAMP-dependent phosphorylation. Proc Natl Acad Sci U S A 95:6145-6150, 1998

172

375. Kang,G, Joseph,JW, Chepurny,OG, Monaco,M, Wheeler,MB, Bos,JL, Schwede,F, Genieser,HG, Holz,GG: Epac-selective cAMP analog 8-pCPT-2'-O-Me-cAMP as a stimulus for Ca2+-induced Ca2+ release and exocytosis in pancreatic beta-cells. J Biol Chem 278:8279-8285, 2003

376. Gromada,J, Dissing,S, Bokvist,K, Renström,E, Frokjær-Jensen,J, Wulff,BS, Rorsman,P: Glucagon-like peptide I increases cytoplasmic calcium in insulin-secreting TC3-cells by enhancement of intracellular calcium mobilization. Diabetes 44:767-774, 1995

377. Holz,GG: New insights concerning the glucose-dependent insulin secretagogue action of glucagon-like peptide-1 in pancreatic beta-cells. Horm Metab Res 36:787-794, 2004

378. Tsuboi,T, da,S, X, Holz,GG, Jouaville,LS, Thomas,AP, Rutter,GA: Glucagon-like peptide-1 mobilizes intracellular Ca2+ and stimulates mitochondrial ATP synthesis in pancreatic MIN6 beta-cells. Biochem J 369:287-299, 2003

379. Eliasson,L, Ma,X, Renstrom,E, Barg,S, Berggren,PO, Galvanovskis,J, Gromada,J, Jing,X, Lundquist,I, Salehi,A, Sewing,S, Rorsman,P: SUR1 regulates PKA-independent cAMP-induced granule priming in mouse pancreatic B-cells. J Gen Physiol 121:181- 197, 2003

380. Eliasson,L, Renstrom,E, Ding,WG, Proks,P, Rorsman,P: Rapid ATP-dependent priming of secretory granules precedes Ca(2+)-induced exocytosis in mouse pancreatic B-cells. J Physiol 503 ( Pt 2):399-412, 1997

381. Renstrom,E, Eliasson,L, Rorsman,P: Protein kinase A-dependent and -independent stimulation of exocytosis by cAMP in mouse pancreatic B-cells. J Physiol 502 ( Pt 1):105-118, 1997

382. Hisatomi,M, Hidaka,H, Niki,I: Ca2+/calmodulin and cyclic 3,5' adenosine monophosphate control movement of secretory granules through protein phosphorylation/dephosphor ylation in the pancreatic -cell. Endocrinology 137:4644- 4649, 1996

383. Gromada,J, Holst,JJ, Rorsman,P: Cellular regulation of islet hormone secretion by the incretin hormone glucagon-like peptide 1. Pflugers Arch 435:583-594, 1998

384. MacDonald,PE, Wheeler,MB: Voltage-dependent K(+) channels in pancreatic beta cells: role, regulation and potential as therapeutic targets. Diabetologia 46:1046-1062, 2003

173

385. MacDonald,PE, Wang,X, Xia,F, El-Kholy,W, Targonsky,ED, Tsushima,RG, Wheeler,MB: Antagonism of rat beta-cell voltage-dependent K+ currents by exendin 4 requires dual activation of the cAMP/protein kinase A and phosphatidylinositol 3-kinase signaling pathways. J Biol Chem 278:52446-52453, 2003

386. MacDonald,PE, Salapatek,AM, Wheeler,MB: Glucagon-like peptide-1 receptor activation antagonizes voltage-dependent repolarizing K(+) currents in beta-cells: a possible glucose-dependent insulinotropic mechanism. Diabetes 51 Suppl 3:S443-S447, 2002

387. Drucker,DJ, Philippe,J, Mojsov,S, Chick,WL, Habener,JF: Glucagon-like peptide I stimulates insulin gene expression and increases cyclic AMP levels in a rat islet cell line. Proc Natl Acad Sci USA 84:3434-3438, 1987

388. Li,Y, Cao,X, Li,LX, Brubaker,PL, Edlund,H, Drucker,DJ: {beta}-Cell Pdx1 Expression Is Essential for the Glucoregulatory, Proliferative, and Cytoprotective Actions of Glucagon-Like Peptide-1. Diabetes 54:482-491, 2005

389. Alarcon,C, Wicksteed,B, Rhodes,CJ: Exendin 4 controls insulin production in rat islet beta cells predominantly by potentiation of glucose-stimulated proinsulin biosynthesis at the translational level. Diabetologia 49:2920-2929, 2006

390. Fehmann,H-C, Habener,JF: Insulinotropic hormone glucagon-like peptide-I(7-37) stimulation of proinsulin gene expression and proinsulin biosynthesis in insulinoma TC-1 cells. Endocrinology 130:159-166, 1992

391. Lawrence,MC, Bhatt,HS, Easom,RA: NFAT regulates insulin gene promoter activity in response to synergistic pathways induced by glucose and glucagon-like peptide-1. Diabetes 51:691-698, 2002

392. Lawrence,MC, Bhatt,HS, Watterson,JM, Easom,RA: Regulation of insulin gene transcription by a Ca(2+)-responsive pathway involving calcineurin and nuclear factor of activated T cells. Mol Endocrinol 15:1758-1767, 2001

393. Wang,X, Zhou,J, Doyle,ME, Egan,JM: Glucagon-like peptide-1 causes pancreatic duodenal homeobox-1 protein translocation from the cytoplasm to the nucleus of pancreatic beta-cells by a cyclic adenosine monophosphate/protein kinase A-dependent mechanism. Endocrinology 142:1820-1827, 2001

174

394. Wang,H, Iezzi,M, Theander,S, Antinozzi,PA, Gauthier,BR, Halban,PA, Wollheim,CB: Suppression of Pdx-1 perturbs proinsulin processing, insulin secretion and GLP-1 signalling in INS-1 cells. Diabetologia 48:720-731, 2005

395. Edvell,A, Lindström,P: Initiation of increased pancreatic islet growth in young normoglycemic mice (Umeå +/?). Endocrinology 140:778-783, 1999

396. Xu,G, Stoffers,DA, Habener,JF, Bonner-Weir,S: Exendin-4 stimulates both -cell replication and neogenesis, resulting in increased -cell mass and improved glucose tolerance in diabetic rats. Diabetes 48:2270-2276, 1999

397. Stoffers,DA, Kieffer,TJ, Hussain,MA, Drucker,DJ, Bonner-Weir,S, Habener,J, Egan,JM: Insulinotropic glucagon-like peptide 1 agonists stimulate expression of homeodomain protein IDX-1 and increase islet size in mouse pancreas. Diabetes 49:741-748, 2000

398. Perfetti,R, Zhou,J, Doyle,ME, Egan,JM: Glucagon-like peptide-1 induces cell proliferation and pancreatic-duodenum homeobox-1 expression and increases endocrine cell mass in the pancreas of old, glucose-intolerant rats. Endocrinology 141:4600-4605, 2000

399. Rolin,B, Larsen,MO, Gotfredsen,CF, Deacon,CF, Carr,RD, Wilken,M, Knudsen,LB: The long-acting GLP-1 derivative NN2211 ameliorates glycemia and increases beta-cell mass in diabetic mice. Am J Physiol Endocrinol Metab 283:E745-E752, 2002

400. Wang,Q, Li,L, Xu,E, Wong,V, Rhodes,C, Brubaker,PL: Glucagon-like peptide-1 regulates proliferation and apoptosis via activation of PKB in pancreatic (INS-1) beta- cells. Diabetologia 47:478-487, 2004

401. Farilla,L, Hui,H, Bertolotto,C, Kang,E, Bulotta,A, Di Mario,U, Perfetti,R: Glucagon-like peptide-1 promotes islet cell growth and inhibits apoptosis in Zucker diabetic rats. Endocrinology 143:4397-4408, 2002

402. Tourrel,C, Bailbe,D, Lacorne,M, Meile,MJ, Kergoat,M, Portha,B: Persistent improvement of type 2 diabetes in the Goto-Kakizaki rat model by expansion of the beta-cell mass during the prediabetic period with glucagon-like peptide-1 or exendin-4. Diabetes 51:1443-1452, 2002

403. Uckaya,G, Delagrange,P, Chavanieu,A, Grassy,G, Berthault,MF, Ktorza,A, Cerasi,E, Leibowitz,G, Kaiser,N: Improvement of metabolic state in an animal model of nutrition- dependent type 2 diabetes following treatment with S 23521, a new glucagon-like peptide 1 (GLP-1) analogue. J Endocrinol 184:505-513, 2005

175

404. Tourrel,C, Bailbe,D, Meile,MJ, Portha,B: Glucagon-like peptide-1 and exendin-4 stimulate beta-cell neogenesis in streptozotocin-treated newborn rats resulting in persistantly improved glucose homeostasis at adult age. Diabetes 50:1562-1570, 2001

405. Li,Y, Hansotia,T, Yusta,B, Ris,F, Halban,PA, Drucker,DJ: Glucagon-like peptide-1 receptor signaling modulates beta cell apoptosis. J Biol Chem 278:471-478, 2003

406. Park,S, Dong,X, Fisher,TL, Dunn,S, Omer,AK, Weir,G, White,MF: Exendin-4 uses Irs2 signaling to mediate pancreatic beta cell growth and function. J Biol Chem 281:1159- 1168, 2006

407. Buteau,J, Roduit,R, Susini,S, Prentki,M: Glucagon-like peptide-1 promotes DNA synthesis, activates phosphatidylinositol 3-kinase and increases transcription factor pancreatic and duodenal homeobox gene 1 (PDX-1) DNA binding activity in (INS-1)- cells. Diabetologia 42:856-864, 1999

408. Stark,A, Mentlein,R: Somatostatin inhibits glucagon-like peptide-1-induced insulin secretion and proliferation of RINm5F insulinoma cells. Regul Pept 108:97-102, 2002

409. Buteau,J, Foisy,S, Joly,E, Prentki,M: Glucagon-like peptide 1 induces pancreatic beta- cell proliferation via transactivation of the epidermal growth factor receptor. Diabetes 52:124-132, 2003

410. Buteau,J, Foisy,S, Rhodes,CJ, Carpenter,L, Biden,TJ, Prentki,M: Protein kinase Czeta activation mediates glucagon-like peptide-1- induced pancreatic beta-cell proliferation. Diabetes 50:2237-2243, 2001

411. Buteau,J, Accili,D: Regulation of pancreatic beta-cell function by the forkhead protein FoxO1. Diabetes Obes Metab 9 Suppl 2:140-146, 2007

412. Jhala,US, Canettieri,G, Screaton,RA, Kulkarni,RN, Krajewski,S, Reed,J, Walker,J, Lin,X, White,M, Montminy,M: cAMP promotes pancreatic beta-cell survival via CREB- mediated induction of IRS2. Genes Dev 17:1575-1580, 2003

413. Li,L, El Kholy,W, Rhodes,CJ, Brubaker,PL: Glucagon-like peptide-1 protects beta cells from cytokine-induced apoptosis and necrosis: Role of protein kinase B. Diabetologia 48:1339-1349, 2005

414. Hui,H, Nourparvar,A, Zhao,X, Perfetti,R: Glucagon-like peptide-1 inhibits apoptosis of insulin-secreting cells via a cyclic 5'-adenosine monophosphate-dependent protein

176

kinase A- and a phosphatidylinositol 3-kinase-dependent pathway. Endocrinology 144:1444-1455, 2003

415. Kwon,G, Pappan,KL, Marshall,CA, Schaffer,JE, McDaniel,ML: cAMP Dose- dependently prevents palmitate-induced apoptosis by both protein kinase A- and cAMP- guanine nucleotide exchange factor-dependent pathways in beta-cells. J Biol Chem 279:8938-8945, 2004

416. Johnson,JD, Han,Z, Otani,K, Ye,H, Zhang,Y, Wu,H, Horikawa,Y, Misler,S, Bell,GI, Polonsky,KS: RyR2 and calpain-10 delineate a novel apoptosis pathway in pancreatic islets. J Biol Chem 279:24794-24802, 2004

417. Ehses,JA, Casilla,VR, doty,T, Pospisilik,JA, Winter,KD, Demuth,HU, Pederson,RA, McIntosh,CH: Glucose-dependent insulinotropic polypeptide promotes beta-(INS-1) cell survival via cyclic adenosine monophosphate-mediated caspase-3 inhibition and regulation of p38 mitogen-activated protein kinase. Endocrinology 144:4433-4445, 2003

418. Movassat,J, Portha,B: Beta-cell growth in the neonatal Goto-Kakisaki rat and regeneration after treatment with streptozotocin at birth. Diabetologia 42:1098-1106, 1999

419. Zhou,J, Wang,XL, Pineyro,MA, Egan,JM: Glucagon-like peptide 1 and exendin-4 convert pancreatic AR42J cells into glucagon- and insulin-producing cells. Diabetes 48:2358-2366, 1999

420. Abraham,EJ, Leech,CA, Lin,JC, Zulewski,H, Habener,JF: Insulinotropic hormone glucagon-like peptide-1 differentiation of human pancreatic islet-derived progenitor cells into insulin-producing cells. Endocrinology 143:3152-3161, 2002

421. Bulotta,A, Hui,H, Anastasi,E, Bertolotto,C, Boros,LG, Di,MU, Perfetti,R: Cultured pancreatic ductal cells undergo cell cycle re-distribution and beta-cell-like differentiation in response to glucagon-like peptide-1. J Mol Endocrinol 29:347-360, 2002

422. Hardikar,AA, Wang,XY, Williams,LJ, Kwok,J, Wong,R, Yao,M, Tuch,BE: Functional maturation of fetal porcine beta-cells by glucagon-like peptide 1 and cholecystokinin. Endocrinology 143:3505-3514, 2002

423. Zhou,J, Pineyro,MA, Wang,X, Doyle,ME, Egan,JM: Exendin-4 differentiation of a human pancreatic duct cell line into endocrine cells: involvement of PDX-1 and HNF3beta transcription factors. J Cell Physiol 192:304-314, 2002

177

424. Xu,G, Kaneto,H, Lopez-Avalos,MD, Weir,GC, Bonner-Weir,S: GLP-1/exendin-4 facilitates beta-cell neogenesis in rat and human pancreatic ducts. Diabetes Res Clin Pract 73:107-110, 2006

425. Bai,L, Meredith,G, Tuch,BE: Glucagon-like peptide-1 enhances production of insulin in insulin-producing cells derived from mouse embryonic stem cells. J Endocrinol 186:343-352, 2005

426. Koizumi,M, Doi,R, Fujimoto,K, Ito,D, Toyoda,E, Mori,T, Kami,K, Kawaguchi,Y, Gittes,GK, Imamura,M: Pancreatic epithelial cells can be converted into insulin- producing cells by GLP-1 in conjunction with virus-mediated gene transfer of pdx-1. Surgery 138:125-133, 2005

427. De Leon,DD, Farzad,C, Crutchlow,MF, Brestelli,J, Tobias,J, Kaestner,KH, Stoffers,DA: Identification of transcriptional targets during pancreatic growth after partial pancreatectomy and exendin-4 treatment. Physiol Genomics 24:133-143, 2006

428. Lee,CS, De Leon,DD, Kaestner,KH, Stoffers,DA: Regeneration of pancreatic islets after partial pancreatectomy in mice does not involve the reactivation of neurogenin-3. Diabetes 55:269-272, 2006

429. Farilla,L, Bulotta,A, Hirshberg,B, Li,CS, Khoury,N, Noushmehr,H, Bertolotto,C, Di Mario,U, Harlan,DM, Perfetti,R: Glucagon-like peptide 1 inhibits cell apoptosis and improves glucose responsiveness of freshly isolated human islets. Endocrinology 144:5149-5158, 2003

430. King,A, Lock,J, Xu,G, Bonner-Weir,S, Weir,GC: Islet transplantation outcomes in mice are better with fresh islets and exendin-4 treatment. Diabetologia 48:2074-2079, 2005

431. Garcia-Ocana,A, Takane,KK, Reddy,VT, Lopez-Talavera,JC, Vasavada,RC, Stewart,AF: Adenovirus-mediated hepatocyte growth factor expression in mouse islets improves pancreatic islet transplant performance and reduces beta cell death. J Biol Chem 278:343-351, 2003

432. Nakano,M, Yasunami,Y, Maki,T, Kodama,S, Ikehara,Y, Nakamura,T, Tanaka,M, Ikeda,S: Hepatocyte growth factor is essential for amelioration of hyperglycemia in streptozotocin-induced diabetic mice receiving a marginal mass of intrahepatic islet grafts. Transplantation 69:214-221, 2000

433. Higashio,K, Shima,N, Goto,M, Itagaki,Y, Nagao,M, Yasuda,H, Morinaga,T: Identity of a tumor cytotoxic factor from human fibroblasts and hepatocyte growth factor. Biochem Biophys Res Commun 170:397-404, 1990

178

434. Welters,HJ, Kulkarni,RN: Wnt signaling: relevance to beta-cell biology and diabetes. Trends Endocrinol Metab 19:349-355, 2008

435. Guo,YF, Xiong,DH, Shen,H, Zhao,LJ, Xiao,P, Guo,Y, Wang,W, Yang,TL, Recker,RR, Deng,HW: Polymorphisms of the low-density lipoprotein receptor-related protein 5 (LRP5) gene are associated with obesity phenotypes in a large family-based association study. J Med Genet 43:798-803, 2006

436. Kanazawa,A, Tsukada,S, Sekine,A, Tsunoda,T, Takahashi,A, Kashiwagi,A, Tanaka,Y, Babazono,T, Matsuda,M, Kaku,K, Iwamoto,Y, Kawamori,R, Kikkawa,R, Nakamura,Y, Maeda,S: Association of the gene encoding wingless-type mammary tumor virus integration-site family member 5B (WNT5B) with type 2 diabetes. Am J Hum Genet 75:832-843, 2004

437. Willert,K, Brown,JD, Danenberg,E, Duncan,AW, Weissman,IL, Reya,T, Yates,JR, III, Nusse,R: Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 423:448-452, 2003

438. Logan,CY, Nusse,R: The Wnt signaling pathway in development and disease. Annu Rev Cell Dev Biol 20:781-810, 2004

439. Torres,MA, Yang-Snyder,JA, Purcell,SM, DeMarais,AA, McGrew,LL, Moon,RT: Activities of the Wnt-1 class of secreted signaling factors are antagonized by the Wnt- 5A class and by a dominant negative cadherin in early Xenopus development. J Cell Biol 133:1123-1137, 1996

440. Major,MB, Camp,ND, Berndt,JD, Yi,X, Goldenberg,SJ, Hubbert,C, Biechele,TL, Gingras,AC, Zheng,N, Maccoss,MJ, Angers,S, Moon,RT: Wilms tumor suppressor WTX negatively regulates WNT/beta-catenin signaling. Science 316:1043-1046, 2007

441. Angers,S, Moon,RT: Proximal events in Wnt signal transduction. Nat Rev Mol Cell Biol 10:468-477, 2009

442. van,AR, Berns,A: Re-evaluating the role of Frat in Wnt-signal transduction. Cell Cycle 4:1065-1072, 2005

443. van,AR, Nawijn,MC, Lambooij,JP, Proost,N, Jonkers,J, Berns,A: Frat oncoproteins act at the crossroad of canonical and noncanonical Wnt-signaling pathways. Oncogene 29:93-104, 2010

179

444. van,AR, Nawijn,M, Franca-Koh,J, Zevenhoven,J, van der,GH, Jonkers,J, Berns,A: Frat is dispensable for canonical Wnt signaling in mammals. Genes Dev 19:425-430, 2005

445. MacDonald,BT, Tamai,K, He,X: Wnt/beta-catenin signaling: components, mechanisms, and diseases. Dev Cell 17:9-26, 2009

446. Cadigan,KM, Liu,YI: Wnt signaling: complexity at the surface. J Cell Sci 119:395-402, 2006

447. He,X, Semenov,M, Tamai,K, Zeng,X: LDL receptor-related proteins 5 and 6 in Wnt/beta-catenin signaling: arrows point the way. Development 131:1663-1677, 2004

448. Tetsu,O, McCormick,F: Beta-catenin regulates expression of cyclin D1 in colon carcinoma cells. Nature 398:422-426, 1999

449. He,TC, Sparks,AB, Rago,C, Hermeking,H, Zawel,L, da Costa,LT, Morin,PJ, Vogelstein,B, Kinzler,KW: Identification of c-MYC as a target of the APC pathway. Science 281:1509-1512, 1998

450. Howe,LR, Crawford,HC, Subbaramaiah,K, Hassell,JA, Dannenberg,AJ, Brown,AM: PEA3 is up-regulated in response to Wnt1 and activates the expression of cyclooxygenase-2. J Biol Chem 276:20108-20115, 2001

451. Liu,Z, Habener,JF: Glucagon-like peptide-1 activation of TCF7L2-dependent Wnt signaling enhances pancreatic beta cell proliferation. J Biol Chem 283:8723-8735, 2008

452. Heller,RS, Dichmann,DS, Jensen,J, Miller,C, Wong,G, Madsen,OD, Serup,P: Expression patterns of Wnts, Frizzleds, sFRPs, and misexpression in transgenic mice suggesting a role for Wnts in pancreas and foregut pattern formation. Dev Dyn 225:260- 270, 2002

453. Heller,RS, Klein,T, Ling,Z, Heimberg,H, Katoh,M, Madsen,OD, Serup,P: Expression of Wnt, Frizzled, sFRP, and DKK genes in adult human pancreas. Gene Expr 11:141-147, 2003

454. Pedersen,AH, Heller,RS: A possible role for the canonical Wnt pathway in endocrine cell development in chicks. Biochem Biophys Res Commun 333:961-968, 2005

180

455. Wang,QM, Zhang,Y, Yang,KM, Zhou,HY, Yang,HJ: Wnt/beta-catenin signaling pathway is active in pancreatic development of rat embryo. World J Gastroenterol 12:2615-2619, 2006

456. Shu,L, Sauter,NS, Schulthess,FT, Matveyenko,AV, Oberholzer,J, Maedler,K: Transcription factor 7-like 2 regulates beta-cell survival and function in human pancreatic islets. Diabetes 57:645-653, 2008

457. Dessimoz,J, Bonnard,C, Huelsken,J, Grapin-Botton,A: Pancreas-specific deletion of beta-catenin reveals Wnt-dependent and Wnt-independent functions during development. Curr Biol 15:1677-1683, 2005

458. Kim,HJ, Schleiffarth,JR, Jessurun,J, Sumanas,S, Petryk,A, Lin,S, Ekker,SC: Wnt5 signaling in vertebrate pancreas development. BMC Biol 3:23, 2005

459. Murtaugh,LC, Law,AC, Dor,Y, Melton,DA: Beta-catenin is essential for pancreatic acinar but not islet development. Development 132:4663-4674, 2005

460. Wells,JM, Esni,F, Boivin,GP, Aronow,BJ, Stuart,W, Combs,C, Sklenka,A, Leach,SD, Lowy,AM: Wnt/beta-catenin signaling is required for development of the exocrine pancreas. BMC Dev Biol 7:4, 2007

461. Heiser,PW, Lau,J, Taketo,MM, Herrera,PL, Hebrok,M: Stabilization of beta-catenin impacts pancreas growth. Development 133:2023-2032, 2006

462. Zhurinsky,J, Shtutman,M, Ben-Ze'ev,A: Differential mechanisms of LEF/TCF family- dependent transcriptional activation by beta-catenin and plakoglobin. Mol Cell Biol 20:4238-4252, 2000

463. Rulifson,IC, Karnik,SK, Heiser,PW, Ten,BD, Chen,H, Gu,X, Taketo,MM, Nusse,R, Hebrok,M, Kim,SK: Wnt signaling regulates pancreatic beta cell proliferation. Proc Natl Acad Sci U S A 104:6247-6252, 2007

464. Liu,Z, Tanabe,K, Bernal-Mizrachi,E, Permutt,MA: Mice with beta cell overexpression of glycogen synthase kinase-3beta have reduced beta cell mass and proliferation. Diabetologia 51:623-631, 2008

465. Fujino,T, Asaba,H, Kang,MJ, Ikeda,Y, Sone,H, Takada,S, Kim,DH, Ioka,RX, Ono,M, Tomoyori,H, Okubo,M, Murase,T, Kamataki,A, Yamamoto,J, Magoori,K, Takahashi,S, Miyamoto,Y, Oishi,H, Nose,M, Okazaki,M, Usui,S, Imaizumi,K, Yanagisawa,M, Sakai,J, Yamamoto,TT: Low-density lipoprotein receptor-related protein 5 (LRP5) is

181

essential for normal cholesterol metabolism and glucose-induced insulin secretion. Proc Natl Acad Sci U S A 100:229-234, 2003

466. Papadopoulou,S, Edlund,H: Attenuated Wnt signaling perturbs pancreatic growth but not pancreatic function. Diabetes 54:2844-2851, 2005

467. Schinner,S, Ulgen,F, Papewalis,C, Schott,M, Woelk,A, Vidal-Puig,A, Scherbaum,WA: Regulation of insulin secretion, glucokinase gene transcription and beta cell proliferation by adipocyte-derived Wnt signalling molecules. Diabetologia 51:147-154, 2008

468. Yi,F, Sun,J, Lim,GE, Fantus,IG, Brubaker,PL, Jin,T: Cross talk between the insulin and Wnt signaling pathways: evidence from intestinal endocrine L cells. Endocrinology 149:2341-2351, 2008

469. Playford,MP, Bicknell,D, Bodmer,WF, Macaulay,VM: Insulin-like growth factor 1 regulates the location, stability, and transcriptional activity of beta-catenin. Proc Natl Acad Sci U S A 97:12103-12108, 2000

470. Ladher,RK, Anakwe,KU, Gurney,AL, Schoenwolf,GC, Francis-West,PH: Identification of synergistic signals initiating inner ear development. Science 290:1965-1967, 2000

471. Bonvini,P, An,WG, Rosolen,A, Nguyen,P, Trepel,J, Garcia de,HA, Dunach,M, Neckers,LM: Geldanamycin abrogates ErbB2 association with proteasome-resistant beta-catenin in melanoma cells, increases beta-catenin-E-cadherin association, and decreases beta-catenin-sensitive transcription. Cancer Res 61:1671-1677, 2001

472. Monga,SP, Mars,WM, Pediaditakis,P, Bell,A, Mule,K, Bowen,WC, Wang,X, Zarnegar,R, Michalopoulos,GK: Hepatocyte growth factor induces Wnt-independent nuclear translocation of beta-catenin after Met-beta-catenin dissociation in hepatocytes. Cancer Res 62:2064-2071, 2002

473. Fischer,AN, Fuchs,E, Mikula,M, Huber,H, Beug,H, Mikulits,W: PDGF essentially links TGF-beta signaling to nuclear beta-catenin accumulation in hepatocellular carcinoma progression. Oncogene 26:3395-3405, 2007

474. Kulkarni,NH, Halladay,DL, Miles,RR, Gilbert,LM, Frolik,CA, Galvin,RJ, Martin,TJ, Gillespie,MT, Onyia,JE: Effects of parathyroid hormone on Wnt signaling pathway in bone. J Cell Biochem 95:1178-1190, 2005

182

475. Hino,S, Tanji,C, Nakayama,KI, Kikuchi,A: Phosphorylation of beta-catenin by cyclic AMP-dependent protein kinase stabilizes beta-catenin through inhibition of its ubiquitination. Mol Cell Biol 25:9063-9072, 2005

476. Patel,S, Doble,B, Woodgett,JR: Glycogen synthase kinase-3 in insulin and Wnt signalling: a double-edged sword? Biochem Soc Trans 32:803-808, 2004

477. Patel,S, Doble,B, Woodgett,JR: Glycogen synthase kinase-3 in insulin and Wnt signalling: a double-edged sword? Biochem Soc Trans 32:803-808, 2004

478. Sun,J, Jin,T: Both Wnt and mTOR signaling pathways are involved in insulin-stimulated proto-oncogene expression in intestinal cells. Cell Signal 20:219-229, 2008

479. sbois-Mouthon,C, Cadoret,A, Blivet-Van Eggelpoel,MJ, Bertrand,F, Cherqui,G, Perret,C, Capeau,J: Insulin and IGF-1 stimulate the beta-catenin pathway through two signalling cascades involving GSK-3beta inhibition and Ras activation. Oncogene 20:252-259, 2001

480. Mussmann,R, Geese,M, Harder,F, Kegel,S, Andag,U, Lomow,A, Burk,U, Onichtchouk,D, Dohrmann,C, Austen,M: Inhibition of GSK3 promotes replication and survival of pancreatic beta cells. J Biol Chem 282:12030-12037, 2007

481. Essers,MA, de Vries-Smits,LM, Barker,N, Polderman,PE, Burgering,BM, Korswagen,HC: Functional interaction between beta-catenin and FOXO in oxidative stress signaling. Science 308:1181-1184, 2005

482. Kitamura,T, Nakae,J, Kitamura,Y, Kido,Y, Biggs,WH, III, Wright,CV, White,MF, Arden,KC, Accili,D: The forkhead transcription factor Foxo1 links insulin signaling to Pdx1 regulation of pancreatic beta cell growth. J Clin Invest 110:1839-1847, 2002

483. Okamoto,H, Hribal,ML, Lin,HV, Bennett,WR, Ward,A, Accili,D: Role of the forkhead protein FoxO1 in beta cell compensation to insulin resistance. J Clin Invest 116:775-782, 2006

484. Kawamori,D, Kaneto,H, Nakatani,Y, Matsuoka,TA, Matsuhisa,M, Hori,M, Yamasaki,Y: The forkhead transcription factor Foxo1 bridges the JNK pathway and the transcription factor PDX-1 through its intracellular translocation. J Biol Chem 281:1091-1098, 2006

485. Kitamura,YI, Kitamura,T, Kruse,JP, Raum,JC, Stein,R, Gu,W, Accili,D: FoxO1 protects against pancreatic beta cell failure through NeuroD and MafA induction. Cell Metab 2:153-163, 2005

183

486. Nakae,J, Biggs,WH, III, Kitamura,T, Cavenee,WK, Wright,CV, Arden,KC, Accili,D: Regulation of insulin action and pancreatic beta-cell function by mutated alleles of the gene encoding forkhead transcription factor Foxo1. Nat Genet 32:245-253, 2002

487. Hoogeboom,D, Essers,MA, Polderman,PE, Voets,E, Smits,LM, Burgering,BM: Interaction of FOXO with beta-catenin inhibits beta-catenin/T cell factor activity. J Biol Chem 283:9224-9230, 2008

488. Brembeck,FH, Rosario,M, Birchmeier,W: Balancing cell adhesion and Wnt signaling, the key role of beta-catenin. Curr Opin Genet Dev 16:51-59, 2006

489. Carvell,MJ, Marsh,PJ, Persaud,SJ, Jones,PM: E-cadherin interactions regulate beta-cell proliferation in islet-like structures. Cell Physiol Biochem 20:617-626, 2007

490. Apte,U, Zeng,G, Muller,P, Tan,X, Micsenyi,A, Cieply,B, Dai,C, Liu,Y, Kaestner,KH, Monga,SP: Activation of Wnt/beta-catenin pathway during hepatocyte growth factor- induced hepatomegaly in mice. Hepatology 44:992-1002, 2006

491. Zeng,G, Apte,U, Micsenyi,A, Bell,A, Monga,SP: Tyrosine residues 654 and 670 in beta- catenin are crucial in regulation of Met-beta-catenin interactions. Exp Cell Res 312:3620-3630, 2006

492. Li,Y, Guessous,F, Johnson,EB, Eberhart,CG, Li,XN, Shu,Q, Fan,S, Lal,B, Laterra,J, Schiff,D, Abounader,R: Functional and molecular interactions between the HGF/c-Met pathway and c-Myc in large-cell medulloblastoma. Lab Invest 88:98-111, 2008

493. Kayali,AG, Van,GK, Campbell,IL, Stotland,A, Kritzik,M, Liu,G, Flodstrom- Tullberg,M, Zhang,YQ, Sarvetnick,N: The stromal cell-derived factor-1alpha/CXCR4 ligand-receptor axis is critical for progenitor survival and migration in the pancreas. J Cell Biol 163:859-869, 2003

494. Luo,Y, Cai,J, Xue,H, Mattson,MP, Rao,MS: SDF1alpha/CXCR4 signaling stimulates beta-catenin transcriptional activity in rat neural progenitors. Neurosci Lett 398:291-295, 2006

495. Yano,T, Liu,Z, Donovan,J, Thomas,MK, Habener,JF: Stromal cell derived factor-1 (SDF-1)/CXCL12 attenuates diabetes in mice and promotes pancreatic beta-cell survival by activation of the prosurvival kinase Akt. Diabetes 56:2946-2957, 2007

496. Nauck,MA, Meier,JJ: The enteroinsular axis may mediate the diabetogenic effects of TCF7L2 polymorphisms. Diabetologia 50:2413-2416, 2007

184

497. Mondal,AK, Das,SK, Baldini,G, Chu,WS, Sharma,NK, Hackney,OG, Zhao,J, Grant,SF, Elbein,SC: Genotype and tissue-specific effects on alternative splicing of the transcription factor 7-like 2 gene in humans. J Clin Endocrinol Metab 95:1450-1457, 2010

498. Schafer,SA, Tschritter,O, Machicao,F, Thamer,C, Stefan,N, Gallwitz,B, Holst,JJ, Dekker,JM, T'hart,LM, Nijpels,G, van Haeften,TW, Haring,HU, Fritsche,A: Impaired glucagon-like peptide-1-induced insulin secretion in carriers of transcription factor 7-like 2 (TCF7L2) gene polymorphisms. Diabetologia 50:2443-2450, 2007

499. Ni,Z, Anini,Y, Fang,X, Mills,G, Brubaker,PL, Jin,T: Transcriptional activation of the proglucagon gene by lithium and beta-catenin in intestinal endocrine L cells. J Biol Chem 278:1380-1387, 2003

500. Shu,L, Matveyenko,AV, Kerr-Conte,J, Cho,JH, McIntosh,CH, Maedler,K: Decreased TCF7L2 protein levels in type 2 diabetes mellitus correlate with downregulation of GIP- and GLP-1 receptors and impaired beta-cell function. Hum Mol Genet 18:2388-2399, 2009

501. Loder,MK, da,S, X, McDonald,A, Rutter,GA: TCF7L2 controls insulin gene expression and insulin secretion in mature pancreatic beta-cells. Biochem Soc Trans 36:357-359, 2008

502. da,S, X, Loder,MK, McDonald,A, Tarasov,AI, Carzaniga,R, Kronenberger,K, Barg,S, Rutter,GA: TCF7L2 regulates late events in insulin secretion from pancreatic islet beta- cells. Diabetes 58:894-905, 2009

503. Lyssenko,V: The transcription factor 7-like 2 gene and increased risk of type 2 diabetes: an update. Curr Opin Clin Nutr Metab Care 11:385-392, 2008

504. Kamata,T, Katsube,K, Michikawa,M, Yamada,M, Takada,S, Mizusawa,H: R-spondin, a novel gene with thrombospondin type 1 domain, was expressed in the dorsal neural tube and affected in Wnts mutants. Biochim Biophys Acta 1676:51-62, 2004

505. Kazanskaya,O, Glinka,A, del,BB, I, Stannek,P, Niehrs,C, Wu,W: R-Spondin2 is a secreted activator of Wnt/beta-catenin signaling and is required for Xenopus myogenesis. Dev Cell 7:525-534, 2004

506. Kim,KA, Zhao,J, Andarmani,S, Kakitani,M, Oshima,T, Binnerts,ME, Abo,A, Tomizuka,K, Funk,WD: R-Spondin proteins: a novel link to beta-catenin activation. Cell Cycle 5:23-26, 2006

185

507. Kim,KA, Kakitani,M, Zhao,J, Oshima,T, Tang,T, Binnerts,M, Liu,Y, Boyle,B, Park,E, Emtage,P, Funk,WD, Tomizuka,K: Mitogenic influence of human R-spondin1 on the intestinal epithelium. Science 309:1256-1259, 2005

508. Zhao,J, De,VJ, Narushima,S, Beck,EX, Palencia,S, Shinkawa,P, Kim,KA, Liu,Y, Levy,MD, Berg,DJ, Abo,A, Funk,WD: R-spondin1, A Novel Intestinotrophic Mitogen, Ameliorates Experimental Colitis in Mice. Gastroenterology 2007

509. Zhao,J, Kim,KA, De,VJ, Palencia,S, Wagle,M, Abo,A: R-Spondin1 protects mice from chemotherapy or radiation-induced oral mucositis through the canonical Wnt/beta- catenin pathway. Proc Natl Acad Sci U S A 106:2331-2336, 2009

510. Chassot,AA, Ranc,F, Gregoire,EP, Roepers-Gajadien,HL, Taketo,MM, Camerino,G, de Rooij,DG, Schedl,A, Chaboissier,MC: Activation of beta-catenin signaling by Rspo1 controls differentiation of the mammalian ovary. Hum Mol Genet 17:1264-1277, 2008

511. Tomizuka,K, Horikoshi,K, Kitada,R, Sugawara,Y, Iba,Y, Kojima,A, Yoshitome,A, Yamawaki,K, Amagai,M, Inoue,A, Oshima,T, Kakitani,M: R-spondin1 plays an essential role in ovarian development through positively regulating Wnt-4 signaling. Hum Mol Genet 17:1278-1291, 2008

512. Maatouk,DM, DiNapoli,L, Alvers,A, Parker,KL, Taketo,MM, Capel,B: Stabilization of beta-catenin in XY gonads causes male-to-female sex-reversal. Hum Mol Genet 17:2949-2955, 2008

513. Parma,P, Radi,O, Vidal,V, Chaboissier,MC, Dellambra,E, Valentini,S, Guerra,L, Schedl,A, Camerino,G: R-spondin1 is essential in sex determination, skin differentiation and malignancy. Nat Genet 38:1304-1309, 2006

514. Tomaselli,S, Megiorni,F, De,BC, Felici,A, Marrocco,G, Maggiulli,G, Grammatico,B, Remotti,D, Saccucci,P, Valentini,F, Mazzilli,MC, Majore,S, Grammatico,P: Syndromic true hermaphroditism due to an R-spondin1 (RSPO1) homozygous mutation. Hum Mutat 29:220-226, 2008

515. Yamada,W, Nagao,K, Horikoshi,K, Fujikura,A, Ikeda,E, Inagaki,Y, Kakitani,M, Tomizuka,K, Miyazaki,H, Suda,T, Takubo,K: Craniofacial malformation in R-spondin2 knockout mice. Biochem Biophys Res Commun 381:453-458, 2009

516. Kazanskaya,O, Ohkawara,B, Heroult,M, Wu,W, Maltry,N, Augustin,HG, Niehrs,C: The Wnt signaling regulator R-spondin 3 promotes angioblast and vascular development. Development 135:3655-3664, 2008

186

517. Aoki,M, Mieda,M, Ikeda,T, Hamada,Y, Nakamura,H, Okamoto,H: R-spondin3 is required for mouse placental development. Dev Biol 301:218-226, 2007

518. Seitz,CS, van,SM, Frank,J, Senderek,J, Zerres,K, Hamm,H, Bergmann,C: The Wnt signalling ligand RSPO4, causing inherited anonychia, is not mutated in a patient with congenital nail hypoplasia/aplasia with underlying skeletal defects. Br J Dermatol 157:801-802, 2007

519. Nakamura,M, Miyachi,Y: Congenital hyponychia without RSPO4 mutation. Acta Derm Venereol 88:511-512, 2008

520. Ishii,Y, Wajid,M, Bazzi,H, Fantauzzo,KA, Barber,AG, Blaydon,DC, Nam,JS, Yoon,JK, Kelsell,DP, Christiano,AM: Mutations in R-spondin 4 (RSPO4) underlie inherited anonychia. J Invest Dermatol 128:867-870, 2008

521. Chishti,MS, Kausar,N, Rafiq,MA, Amin,M, Ahmad,W: A novel missense mutation in RSPO4 gene underlies autosomal recessive congenital anonychia in a consanguineous Pakistani family. Br J Dermatol 158:621-623, 2008

522. Bruchle,NO, Frank,J, Frank,V, Senderek,J, Akar,A, Koc,E, Rigopoulos,D, van,SM, Zerres,K, Bergmann,C: RSPO4 is the major gene in autosomal-recessive anonychia and mutations cluster in the furin-like cysteine-rich domains of the Wnt signaling ligand R- spondin 4. J Invest Dermatol 128:791-796, 2008

523. Blaydon,DC, Ishii,Y, O'Toole,EA, Unsworth,HC, Teh,MT, Ruschendorf,F, Sinclair,C, Hopsu-Havu,VK, Tidman,N, Moss,C, Watson,R, de,BD, Wajid,M, Christiano,AM, Kelsell,DP: The gene encoding R-spondin 4 (RSPO4), a secreted protein implicated in Wnt signaling, is mutated in inherited anonychia. Nat Genet 38:1245-1247, 2006

524. Bergmann,C, Senderek,J, Anhuf,D, Thiel,CT, Ekici,AB, Poblete-Gutierrez,P, van,SM, Seelow,D, Nurnberg,G, Schild,HH, Nurnberg,P, Reis,A, Frank,J, Zerres,K: Mutations in the gene encoding the Wnt-signaling component R-spondin 4 (RSPO4) cause autosomal recessive anonychia. Am J Hum Genet 79:1105-1109, 2006

525. Nam,JS, Turcotte,TJ, Smith,PF, Choi,S, Yoon,JK: Mouse cristin/R-spondin family proteins are novel ligands for the Frizzled 8 and LRP6 receptors and activate beta- catenin-dependent gene expression. J Biol Chem 281:13247-13257, 2006

526. Binnerts,ME, Kim,KA, Bright,JM, Patel,SM, Tran,K, Zhou,M, Leung,JM, Liu,Y, Lomas,WE, III, Dixon,M, Hazell,SA, Wagle,M, Nie,WS, Tomasevic,N, Williams,J, Zhan,X, Levy,MD, Funk,WD, Abo,A: R-Spondin1 regulates Wnt signaling by inhibiting internalization of LRP6. Proc Natl Acad Sci U S A 104:14700-14705, 2007

187

527. Glinka,A, Wu,W, Delius,H, Monaghan,AP, Blumenstock,C, Niehrs,C: Dickkopf-1 is a member of a new family of secreted proteins and functions in head induction. Nature 391:357-362, 1998

528. Mao,B, Wu,W, Li,Y, Hoppe,D, Stannek,P, Glinka,A, Niehrs,C: LDL-receptor-related protein 6 is a receptor for Dickkopf proteins. Nature 411:321-325, 2001

529. Mao,B, Wu,W, Davidson,G, Marhold,J, Li,M, Mechler,BM, Delius,H, Hoppe,D, Stannek,P, Walter,C, Glinka,A, Niehrs,C: Kremen proteins are Dickkopf receptors that regulate Wnt/beta-catenin signalling. Nature 417:664-667, 2002

530. Semenov,MV, Tamai,K, Brott,BK, Kuhl,M, Sokol,S, He,X: Head inducer Dickkopf-1 is a ligand for Wnt coreceptor LRP6. Curr Biol 11:951-961, 2001

531. Welters,HJ, Kulkarni,RN: Wnt signaling: relevance to beta-cell biology and diabetes. Trends Endocrinol Metab 19:349-355, 2008

532. Wei,Q, Yokota,C, Semenov,MV, Doble,B, Woodgett,J, He,X: R-spondin1 is a high affinity ligand for LRP6 and induces LRP6 phosphorylation and beta-catenin signaling. J Biol Chem 282:15903-15911, 2007

533. Kim,KA, Wagle,M, Tran,K, Zhan,X, Dixon,MA, Liu,S, Gros,D, Korver,W, Yonkovich,S, Tomasevic,N, Binnerts,M, Abo,A: R-Spondin Family Members Regulate the Wnt Pathway by a Common Mechanism. Mol Biol Cell 2008

534. Li,L, El Kholy,W, Rhodes,CJ, Brubaker,PL: Glucagon-like peptide-1 protects beta cells from cytokine-induced apoptosis and necrosis: Role of protein kinase B. Diabetologia 48:1339-1349, 2005

535. Mohamed,OA, Dufort,D, Clarke,HJ: Expression and estradiol regulation of Wnt genes in the mouse blastocyst identify a candidate pathway for embryo-maternal signaling at implantation. Biol Reprod 71:417-424, 2004

536. Rhee,CS, Sen,M, Lu,D, Wu,C, Leoni,L, Rubin,J, Corr,M, Carson,DA: Wnt and frizzled receptors as potential targets for immunotherapy in head and neck squamous cell carcinomas. Oncogene 21:6598-6605, 2002

537. Chen,Y, Stump,RJ, Lovicu,FJ, McAvoy,JW: Expression of Frizzleds and secreted frizzled-related proteins (Sfrps) during mammalian lens development. Int J Dev Biol 48:867-877, 2004

188

538. Ranheim,EA, Kwan,HC, Reya,T, Wang,YK, Weissman,IL, Francke,U: Frizzled 9 knock-out mice have abnormal B-cell development. Blood 105:2487-2494, 2005

539. Stump,RJ, Ang,S, Chen,Y, von,BT, Lovicu,FJ, Pinson,K, de Iongh,RU, Yamaguchi,TP, Sassoon,DA, McAvoy,JW: A role for Wnt/beta-catenin signaling in lens epithelial differentiation. Dev Biol 259:48-61, 2003

540. Lyu,J, Costantini,F, Jho,EH, Joo,CK: Ectopic expression of Axin blocks neuronal differentiation of embryonic carcinoma P19 cells. J Biol Chem 278:13487-13495, 2003

541. Jho,EH, Zhang,T, Domon,C, Joo,CK, Freund,JN, Costantini,F: Wnt/beta-catenin/Tcf signaling induces the transcription of Axin2, a negative regulator of the signaling pathway. Mol Cell Biol 22:1172-1183, 2002

542. Etheridge,SL, Spencer,GJ, Heath,DJ, Genever,PG: Expression profiling and functional analysis of wnt signaling mechanisms in mesenchymal stem cells. Stem Cells 22:849- 860, 2004

543. Nam,JS, Turcotte,TJ, Yoon,JK: Dynamic expression of R-spondin family genes in mouse development. Gene Expr Patterns 7:306-312, 2007

544. Li,LX, MacDonald,PE, Ahn,DS, Oudit,GY, Backx,PH, Brubaker,PL: Role of phosphatidylinositol 3-kinasegamma in the beta-cell: interactions with glucagon-like peptide-1. Endocrinology 147:3318-3325, 2006

545. Pfaffl,MW: A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45, 2001

546. Everly,DN, Jr., Kusano,S, Raab-Traub,N: Accumulation of cytoplasmic beta-catenin and nuclear glycogen synthase kinase 3beta in Epstein-Barr virus-infected cells. J Virol 78:11648-11655, 2004

547. Meares,GP, Jope,RS: Resolution of the nuclear localization mechanism of glycogen synthase kinase-3: functional effects in apoptosis. J Biol Chem 282:16989-17001, 2007

548. Li,L, El Kholy,W, Rhodes,CJ, Brubaker,PL: Glucagon-like peptide-1 protects beta cells from cytokine-induced apoptosis and necrosis: Role of protein kinase B. Diabetologia 48:1339-1349, 2005

189

549. Poitout,V, Olson,LK, Robertson,RP: Insulin-secreting cell lines: classification, characteristics and potential applications. Diabetes Metab 22:7-14, 1996

550. Mawson,A, Lai,A, Carroll,JS, Sergio,CM, Mitchell,CJ, Sarcevic,B: Estrogen and insulin/IGF-1 cooperatively stimulate cell cycle progression in MCF-7 breast cancer cells through differential regulation of c-Myc and cyclin D1. Mol Cell Endocrinol 229:161-173, 2005

551. Bonner-Weir,S: Perspective: Postnatal pancreatic cell growth. Endocrinology 141:1926-1929, 2000

552. Hengartner,MO: The biochemistry of apoptosis. Nature 407:770-776, 2000

553. Morin,PJ, Vogelstein,B, Kinzler,KW: Apoptosis and APC in colorectal tumorigenesis. Proc Natl Acad Sci U S A 93:7950-7954, 1996

554. He,TC, Chan,TA, Vogelstein,B, Kinzler,KW: PPARdelta is an APC-regulated target of nonsteroidal anti-inflammatory drugs. Cell 99:335-345, 1999

555. Orford,K, Orford,CC, Byers,SW: Exogenous expression of beta-catenin regulates contact inhibition, anchorage-independent growth, anoikis, and radiation-induced cell cycle arrest. J Cell Biol 146:855-868, 1999

556. Strovel,ET, Sussman,DJ: Transient overexpression of murine dishevelled genes results in apoptotic cell death. Exp Cell Res 253:637-648, 1999

557. Yun,SI, Yoon,HY, Chung,YS: Glycogen synthase kinase-3beta regulates etoposide- induced apoptosis via Bcl-2 mediated caspase-3 activation in C3H10T1/2 cells. Apoptosis 14:771-777, 2009

558. Torii,K, Nishizawa,K, Kawasaki,A, Yamashita,Y, Katada,M, Ito,M, Nishimoto,I, Terashita,K, Aiso,S, Matsuoka,M: Anti-apoptotic action of Wnt5a in dermal fibroblasts is mediated by the PKA signaling pathways. Cell Signal 20:1256-1266, 2008

559. Vuga,LJ, Ben-Yehudah,A, Kovkarova-Naumovski,E, Oriss,T, Gibson,KF, Feghali- Bostwick,C, Kaminski,N: WNT5A is a Regulator of Fibroblast Proliferation and Resistance to Apoptosis. Am J Respir Cell Mol Biol 2009

560. Bouwens,L, Rooman,I: Regulation of pancreatic beta-cell mass. Physiol Rev 85:1255- 1270, 2005

190

561. Martens,GA, Pipeleers,D: Glucose, regulator of survival and phenotype of pancreatic beta cells. Vitam Horm 80:507-539, 2009

562. Costes,S, Broca,C, Bertrand,G, Lajoix,AD, Bataille,D, Bockaert,J, Dalle,S: ERK1/2 control phosphorylation and protein level of cAMP-responsive element-binding protein: a key role in glucose-mediated pancreatic beta-cell survival. Diabetes 55:2220-2230, 2006

563. Hussain,MA, Porras,DL, Rowe,MH, West,JR, Song,WJ, Schreiber,WE, Wondisford,FE: Increased pancreatic beta-cell proliferation mediated by CREB binding protein gene activation. Mol Cell Biol 26:7747-7759, 2006

564. Ding,WG, Renström,E, Rorsman,P, Buschard,K, Gromada,J: Glucagon-like peptide I and glucose-dependent insulinotropic polypeptide stimulate Ca2+-induced secretion in rat - cells by a protein kinase A-mediated mechanism. Diabetes 46:792-800, 1997

565. Lester,LB, Langeberg,LK, Scott,JD: Anchoring of protein kinase A facilitates hormone- mediated insulin secretion. Proc Natl Acad Sci U S A 94:14942-14947, 1997

566. Dyachok,O, Isakov,Y, Sagetorp,J, Tengholm,A: Oscillations of cyclic AMP in hormone- stimulated insulin-secreting beta-cells. Nature 439:349-352, 2006

567. Briaud,I, Lingohr,Mk, Dickson,LM, Wrede,CE, Rhodes,CJ: Differential activation mechanisms of Erk-1/2 and p70(S6K) by glucose in pancreatic beta-cells. Diabetes 52:974-983, 2003

568. Arnette,D, Gibson,TB, Lawrence,MC, January,B, Khoo,S, McGlynn,K, Vanderbilt,CA, Cobb,MH: Regulation of ERK1 and ERK2 by glucose and peptide hormones in pancreatic beta cells. J Biol Chem 278:32517-32525, 2003

569. Gomez,E, Pritchard,C, Herbert,TP: cAMP-dependent protein kinase and Ca2+ influx through L-type voltage-gated calcium channels mediate Raf-independent activation of extracellular regulated kinase in response to glucagon-like peptide-1 in pancreatic beta- cells. J Biol Chem 277:48146-48151, 2002

570. Trumper,J, Ross,D, Jahr,H, Brendel,MD, Goke,R, Horsch,D: The Rap-B-Raf signalling pathway is activated by glucose and glucagon-like peptide-1 in human islet cells. Diabetologia 48:1534-1540, 2005

571. Friedrichsen,BN, Neubauer,N, Lee,YC, Gram,VK, Blume,N, Petersen,JS, Nielsen,JH, Moldrup,A: Stimulation of pancreatic beta-cell replication by incretins involves

191

transcriptional induction of cyclin D1 via multiple signalling pathways. J Endocrinol 188:481-492, 2006

572. Kemp,DM, Habener,JF: Insulinotropic hormone glucagon-like peptide 1 (GLP-1) activation of insulin gene promoter inhibited by p38 mitogen-activated protein kinase. Endocrinology 142:1179-1187, 2001

573. Pigeau,GM, Kolic,J, Ball,BJ, Hoppa,MB, Wang,YW, Ruckle,T, Woo,M, Manning Fox,JE, MacDonald,PE: Insulin granule recruitment and exocytosis is dependent on p110{gamma} in insulinoma and human {beta}-cells. Diabetes 2009

574. Pigeau,GM, Kolic,J, Ball,BJ, Hoppa,MB, Wang,YW, Ruckle,T, Woo,M, Manning Fox,JE, MacDonald,PE: Insulin granule recruitment and exocytosis is dependent on p110{gamma} in insulinoma and human {beta}-cells. Diabetes 2009

575. Lee,SH, Demeterco,C, Geron,I, Abrahamsson,A, Levine,F, Itkin-Ansari,P: Islet specific Wnt activation in human type II diabetes. Exp Diabetes Res 2008:728763, 2008

576. Wong,VS, Yeung,A, Schultz,W, Brubaker,PL: R-spondin-1 is a novel beta-cell growth factor and insulin secretagogue. J Biol Chem 285:21292-21302, 2010

577. Liu,Z, Habener,JF: Wnt signaling in pancreatic islets. Adv Exp Med Biol 654:391-419, 2010

578. Dube,PE, Forse,CL, Bahrami,J, Brubaker,PL: The essential role of insulin-like growth factor-1 in the intestinal tropic effects of glucagon-like peptide-2 in mice. Gastroenterology 131:589-605, 2006

579. Mao,C, Tili,EG, Dose,M, Haks,MC, Bear,SE, Maroulakou,I, Horie,K, Gaitanaris,GA, Fidanza,V, Ludwig,T, Wiest,DL, Gounari,F, Tsichlis,PN: Unequal contribution of Akt isoforms in the double-negative to double-positive thymocyte transition. J Immunol 178:5443-5453, 2007

580. Terauchi,Y, Tsuji,Y, Satoh,S, Minoura,H, Murakami,K, Okuno,A, Inukai,K, Asano,T, Kaburagi,Y, Ueki,K, Nakajima,H, Hanafusa,T, Matsuzawa,Y, Sekihara,H, Yin,Y, Barrett,JC, Oda,H, Ishikawa,T, Akanuma,Y, Komuro,I, Suzuki,M, Yamamura,K, Kodama,T, Suzuki,H, Yamamura,K, Kodama,T, Suzuki,H, Koyasu,S, Aizawa,S, Tobe,K, Fukui,Y, Yazaki,Y, Kadowaki,T: Increased insulin sensitivity and hypoglycaemia in mice lacking the p85 alpha subunit of phosphoinositide 3-kinase. Nat Genet 21:230-235, 1999

192

581. Mashimo,H, Goyal,RK: Lessons from genetically engineered animal models. IV. Nitric oxide synthase gene knockout mice. Am J Physiol 277:G745-G750, 1999

582. Gelling,RW, Vuguin,PM, Du,XQ, Cui,L, Romer,J, Pederson,RA, Leiser,M, Sorensen,H, Holst,JJ, Fledelius,C, Johansen,PB, Fleischer,N, McIntosh,CH, Nishimura,E, Charron,MJ: Pancreatic beta-cell overexpression of the glucagon receptor gene results in enhanced beta-cell function and mass. Am J Physiol Endocrinol Metab 297:E695-E707, 2009

583. Juhl,K, Bonner-Weir,S, Sharma,A: Regenerating pancreatic beta-cells: plasticity of adult pancreatic cells and the feasibility of in-vivo neogenesis. Curr Opin Organ Transplant 15:79-85, 2010

584. Liu,Y, Tanabe,K, Baronnier,D, Patel,S, Woodgett,J, Cras-Meneur,C, Permutt,MA: Conditional ablation of Gsk-3beta in islet beta cells results in expanded mass and resistance to fat feeding-induced diabetes in mice. Diabetologia 2010

585. Sturis,J, Gotfredsen,CF, Romer,J, Rolin,B, Ribel,U, Brand,CL, Wilken,M, Wassermann,K, Deacon,CF, Carr,RD, Knudsen,LB: GLP-1 derivative liraglutide in rats with beta-cell deficiencies: influence of metabolic state on beta-cell mass dynamics. Br J Pharmacol 140:123-132, 2003

586. Dunning,BE, Foley,JE, Ahren,B: Alpha cell function in health and disease: influence of glucagon-like peptide-1. Diabetologia 48:1700-1713, 2005

587. Lyssenko,V, Lupi,R, Marchetti,P, Del,GS, Orho-Melander,M, Almgren,P, Sjogren,M, Ling,C, Eriksson,KF, Lethagen,AL, Mancarella,R, Berglund,G, Tuomi,T, Nilsson,P, Del,PS, Groop,L: Mechanisms by which common variants in the TCF7L2 gene increase risk of type 2 diabetes. J Clin Invest 117:2155-2163, 2007

588. Vuguin,PM, Kedees,MH, Cui,L, Guz,Y, Gelling,RW, Nejathaim,M, Charron,MJ, Teitelman,G: Ablation of the glucagon receptor gene increases fetal lethality and produces alterations in islet development and maturation. Endocrinology 147:3995-4006, 2006

589. Donath,MY, Storling,J, Maedler,K, Mandrup-Poulsen,T: Inflammatory mediators and islet beta-cell failure: a link between type 1 and type 2 diabetes. J Mol Med 81:455-470, 2003

590. Donath,MY, Halban,PA: Decreased beta-cell mass in diabetes: significance, mechanisms and therapeutic implications. Diabetologia 47:581-589, 2004

193

591. Bennett,CN, Ross,SE, Longo,KA, Bajnok,L, Hemati,N, Johnson,KW, Harrison,SD, Macdougald,OA: Regulation of Wnt signaling during adipogenesis. J Biol Chem 277:30998-31004, 2002

592. Apelqvist,A, Li,H, Sommer,L, Beatus,P, Anderson,DJ, Honjo,T, Hrabe de,AM, Lendahl,U, Edlund,H: Notch signalling controls pancreatic cell differentiation. Nature 400:877-881, 1999

593. Jensen,J, Heller,RS, Funder-Nielsen,T, Pedersen,EE, Lindsell,C, Weinmaster,G, Madsen,OD, Serup,P: Independent development of pancreatic - and -cells from Neurogenin3-expressing precursors: A role for the Notch pathway in repression of premature differentiation. Diabetes 49:163-176, 2000

594. Gradwohl,G, Dierich,A, LeMeur,M, Guillemot,F: neurogenin3 is required for the development of the four endocrine cell lineages of the pancreas. Proc Natl Acad Sci U S A 97:1607-1611, 2000

595. Schwitzgebel,VM, Scheel,DW, Conners,JR, Kalamaras,J, Lee,JE, Anderson,DJ, Sussel,L, Johnson,JD, German,MS: Expression of neurogenin3 reveals an islet cell precursor population in the pancreas. Development 127:3533-3542, 2000

596. Gu,G, Dubauskaite,J, Melton,DA: Direct evidence for the pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors. Development 129:2447- 2457, 2002

597. Xu,X, D'Hoker,J, Stange,G, Bonne,S, De,LN, Xiao,X, Van de,CM, Mellitzer,G, Ling,Z, Pipeleers,D, Bouwens,L, Scharfmann,R, Gradwohl,G, Heimberg,H: Beta cells can be generated from endogenous progenitors in injured adult mouse pancreas. Cell 132:197- 207, 2008

598. Lejonklou,MH, Edfeldt,K, Johansson,TA, Stalberg,P, Skogseid,B: Neurogenin 3 and neurogenic differentiation 1 are retained in the cytoplasm of multiple endocrine neoplasia type 1 islet and pancreatic endocrine tumor cells. Pancreas 38:259-266, 2009

599. Dror,V, Nguyen,V, Walia,P, Kalynyak,TB, Hill,JA, Johnson,JD: Notch signalling suppresses apoptosis in adult human and mouse pancreatic islet cells. Diabetologia 50:2504-2515, 2007

600. Inada,A, Nienaber,C, Katsuta,H, Fujitani,Y, Levine,J, Morita,R, Sharma,A, Bonner- Weir,S: Carbonic anhydrase II-positive pancreatic cells are progenitors for both

194

endocrine and exocrine pancreas after birth. Proc Natl Acad Sci U S A 105:19915-19919, 2008

601. Trimble,ER, Halban,PA, Wollheim,CB, Renold,AE: Functional differences between rat islets of ventral and dorsal pancreatic origin. J Clin Invest 69:405-413, 1982

602. Pipeleers,DG, In't Veld,PA, Van de Winkel,M, Maes,E, Schuit,FC, Gepts,W: A new in vitro model for the study of pancreatic A and B cells. Endocrinology 117:806-816, 1985

603. Huypens,P, Ling,Z, Pipeleers,D, Schuit,F: Glucagon receptors on human islet cells contribute to glucose competence of insulin release. Diabetologia 43:1012-1019, 2000

604. Abiola,M, Favier,M, Christodoulou-Vafeiadou,E, Pichard,AL, Martelly,I, Guillet- Deniau,I: Activation of Wnt/beta-catenin signaling increases insulin sensitivity through a reciprocal regulation of Wnt10b and SREBP-1c in skeletal muscle cells. PLoS One 4:e8509, 2009

605. D'Alessio,DA, Fujimoto,WY, Ensinck,JW: Effects of glucagonlike peptide I-(7-36) on release of insulin, glucagon, and somatostatin by rat pancreatic islet cell monolayer cultures. Diabetes 38:1534-1538, 1989

606. Menge,BA, Schrader,H, Breuer,TG, Dabrowski,Y, Uhl,W, Schmidt,WE, Meier,JJ: Metabolic consequences of a 50% partial pancreatectomy in humans. Diabetologia 52:306-317, 2009

607. Peshavaria,M, Larmie,BL, Lausier,J, Satish,B, Habibovic,A, Roskens,V, Larock,K, Everill,B, Leahy,JL, Jetton,TL: Regulation of pancreatic beta-cell regeneration in the normoglycemic 60% partial-pancreatectomy mouse. Diabetes 55:3289-3298, 2006

608. Nakashima,K, Kanda,Y, Hirokawa,Y, Kawasaki,F, Matsuki,M, Kaku,K: MIN6 is not a pure beta cell line but a mixed cell line with other pancreatic endocrine hormones. Endocr J 56:45-53, 2009