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

β-CELL BEHAVIOUR

By

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

© Copyright by Victor Shing Chi Wong (2011)

ii

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

iii

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.

iv

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

v

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.

vi

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

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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

viii

5 APPENDIX ......................................................................................................................... 134

6 REFERENCE LIST .......................................................................................................... 136

ix

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

x

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

xi

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

xii

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

xiii

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

)

1

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.

2

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.

3

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

4

(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.

5

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

membrane by closure of the ATP-sensitive K+ (KATP) channels, and influx of Ca

2+ 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 Ca

2+ 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).

13

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.

14

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

20

(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).

23

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

Growth factor Model

in vitro

Species Proliferation Apoptosis Neogenesis Function Ref

Activin islet rat n/a n/a n/a ↑ (198-200)

human n/a n/a n/a ↑ (201)

islet

Ad ALK7

rat ↓ ↑ n/a n/a (201)

INS1

Ad ALK7

rat ↓ ↑ n/a n/a (202)

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

4

AR42J rat n/a ↑ ↑ n/a (207)

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-

releasing hormone

islet rat ↑ ↓ n/a n/a (230)

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

IR KO

mouse ↓ ↑ n/a n/a (236)

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

pancreas

human ↑ n/a ↑ ↑ (244)

Parathyroid

hormone-related

protein

islet rat ↑ n/a n/a ↑ (mRNA) (245)

MIN6 mouse ↑ n/a n/a ↑ (mRNA) (246)

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)

24

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

DN Smad3

monkey/human n/a n/a n/a ↑ (255)

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

AD hVEGF

human n/a ↓** n/a ↑ (262)

Growth factor Model

in vivo

Species Proliferation Apoptosis Neogenesis Function Ref

Activin FSTL3 KO mouse ↑ na ↑ na (263)

tTA-PDX1-Smad7

mouse n/a n/a n/a ↓ (264)

ALK7 KO mouse ↑ ↔ n/a ↑ (265)

Pancreatic

duct cells in STZ rats

rat ↑ n/a ↑ ↑ (266)

STZ rat ↑ ↔ ↑ n/a (267)

betacellulin STZ rat ↑ ↔ ↑ n/a (267)

STZ rat ↔ n/a ↔ ↑ (insulin

production)

(268)

Alloxan mouse ↔ n/a ↑ n/a (269)

90% PT rat ↑ ↔ ↑ n/a (270)

STZ mouse ↑ ↔ ↑ ↔ (271)

Ad hBTC

ICR/STZ

mouse ↑ n/a ↑ n/a (272;273)

Ad mBTC

STZ

mouse ↑ n/a ↑ ↑ (274)

BTC Tg mouse ↑ ↔ n/a ↑ (275)

betacellulin delta4 STZ rat ↑ ↔ ↑ ↑ (207)

EGF PDX-1-DN EGFR

mouse ↓ ↔ n/a ↓ (276)

Heparin-

binding EGF gene

injection

mouse ↑ n/a ↑ n/a (277)

ins-EGF mouse ↑ n/a n/a ↑ (278)

FGF PDX-1-FGFR1c

(FRID1)

mouse ↓ ↔ n/a ↓ (gradual loss of

GLUT2)

(279)

FGF21 injection

mouse (db/db) ↑ ↔ n/a ↑ (212)

HGF RIP-HGF Tg mouse ↑ ↓*** n/a ↑ (280;281)

RIP-HGF Tg mouse/HFD ↔ ↑ n/a n/a (282)

HGF cDNA

injection

mouse/STZ ↑ ↓ na ↑ (283)

Ad HGF

injection

↔ ↓ na ↑ (mRNA) (284)

RIP-Cre c-

Met KO

mouse ↔ n/a ↑ ↓ (221)

RIP-Cre c-

Met KO

mouse ↓ n/a n/a ↓ (285)

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

NOD

mouse ↔ ↓ ↑ ↑ (210;292-295)

25

(combination

with GLP-1

or EGF)

Alloxan-

treated

(combination with EGF)

mouse ↑ n/a ↑ n/a (296)

Pancreatic

duct ligation

rat ↔ ↔ ↑ n/a (297;298)

TGF /ins-Gastrin

mouse n/a n/a n/a ↑ (299)

Growth Hormone GH-

secreting tumors

rat ↑ n/a n/a n/a (300;301)

JI-36 agonist

treated IT

mouse/STZ n/a n/a n/a ↑ (230)

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

overexpress

mouse ↔ n/a ↔ ↔ (320;321)

βIGF-2

overexpress

mouse ↑ n/a n/a ↑ (322)

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-

KGF

mouse ↑ n/a ↑ ↓ (325)

Parathyroid

hormone-related

protein

RIP-PTHrP

Tg

mouse ↑/↔ ↔ n/a n/a (326)

TGFα Pancreatic

duct ligation

rat n/a n/a ↑ n/a (298)

TGFβ tTA-PDX-1-

Smad7

mouse n/a n/a n/a ↓ (264)

elastase-Smad4 DN

mouse ↑ n/a ↑ n/a (327)

Smad3 KO mouse ↔ n/a n/a ↑ (255)

VEGF PDX-1-Cre

VEGF

mouse ↑ n/a n/a n/a (328)

PDX-1-Cre

VEGF fl/fl

mouse ↔ n/a n/a ↓ (329)

RIP-Cre

VEGF fl/fl

mouse ↔ ↔ n/a ↓ (260)

RIP-Cre

VEGF fl/fl

HFD

mouse ↔ n/a n/a ↔ (330)

26

* 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.

27

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

29

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).

30

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

31

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 pro-

apoptotic 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

33

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).

35

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.

36

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).

37

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.

44

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.

48

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.

50

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

Xenopus Mouse Human

Rspo1 Female-to-male sex reversal

(510;512)

Female-to-male sex reversal

(hermaphroditism) (513;514)

Palmoplantar hyperkeratosis

(513)

Rspo2 Defective

myogenesis (505)

Craniofacial malformation

(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.

54

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.

55

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).

56

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.

57

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.

60

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.

61

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

64

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

65

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.

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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

69

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.

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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.

73

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.

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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.

77

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.

78

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),

79

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.

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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.

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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.

84

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.

86

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.

90

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.

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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.

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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

94

-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.

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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

97

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)

98

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.

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.

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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.

115

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.

118

Murine β-cells

(in vitro)

Rspo1-/-

mice

(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.

119

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

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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

123

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

of the -cell (e.g. K+

ATP and/or voltage-dependent Ca2+

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-[Glu

9]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

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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

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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

machinary of the -cell (e.g. K+

ATP and/or voltage-dependent Ca2+

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.

133

APPENDIX:

PERMISSION TO REPRODUCE PREVIOUSLY PUBLISHED MATERIAL

134

5 Appendix

135

136

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