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The role of the transcription factor Pax6 in the development and function of pancreatic α- and β- cells

KATZ, Liora

Abstract

The transcription factor paired-box-6 (Pax6) plays a key role in the endocrine differentiation cascade of the pancreas. Mutations in Pax6 are associated with anomalies including diabetes.

This study elucidates the impact of Pax6 in pancreatic a- and ß-cell function and differentiation. In a-cells, we found Pax6 to be critical for glucagon biosynthesis and processing, by directly and indirectly activating the glucagon gene through the transcription factors cMaf and NeuroD1, and the processing enzyme of proglucagon in a-cells PC2 and its molecular chaperon 7B2. In regard to ß-cells, the study foundsPax6 to control the cell function and differentiation through the control of genes coding for insulin-1, insulin-2, glucose-transporter-2, glucokinase, GLP-1-receptor, FFA receptor GPR40 as well as the transcription factors Pdx1, MafA and Nkx6-1. The study maps the complex network in which Pax6 regulates transcription of genes important for differentiation of the endocrine pancreas and plays a major role in the islet function.

KATZ, Liora. The role of the transcription factor Pax6 in the development and function of pancreatic α- and β- cells. Thèse de doctorat : Univ. Genève, 2010, no. Sc. 4197

URN : urn:nbn:ch:unige-59997

DOI : 10.13097/archive-ouverte/unige:5999

Available at:

http://archive-ouverte.unige.ch/unige:5999

Disclaimer: layout of this document may differ from the published version.

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UNIVERSITÉ DE GENÈVE

Département de Biologie cellulaire FACULTE DES SCIENCES

Professeur Didier Picard

Département de médecine interne, Service d'endocrinologie, diabétologie et nutrition

FACULTE DE MEDECINE

Professeur Jacques Philippe

The role of the transcription factor Pax6 in the development and function of pancreatic α- and β- cells

THÈSE

Présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie

par

Liora S. Katz (-Spiter) de

Jérusalem (Israël) Thèse N° 4197

GENEVE

Atelier de reprographie ReproMail 2010

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Dedicated to Jonathan,

my dearly-loved son.

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Table of Contents

Acknowledgments ... 11

Résumé en Français ... 12

English Summary ... 14

Abbreviations ... 16

Introduction ... 18

Pancreas ... 18

1.1 Functionality and morphology ... 18

Diabetes Mellitus ... 22

3. Pancreatic development-Morphogenesis ... 28

3.1 Transcription factors regulating pancreatic development ... 28

3.2 Specification of endocrine progenitors ... 33

Pax family of transcription factors and Pax6 ... 39

4.1 Pax Family ... 39

4.2 Pax6 in detail ... 39

Objectives ... 43

Chapter 1- Pax6 Regulates the Proglucagon Processing Enzyme PC2 and Its Chaperone 7B2 44 Abstract ... 45

Introduction ... 46

Materials and Methods ... 48

Cell culture... 48

RNA preparation and RT-PCR analysis ... 48

Transient transfection assays ... 48

Promoter analysis ... 49

EMSA ... 49

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Chromatin immunoprecipitation (ChIP) assay ... 50

siRNAs ... 50

Total cell extract and Western blot assays ... 50

Plasmids ... 51

Site-directed mutagenesis ... 51

Results ... 52

Both PC2 and 7B2 are decreased in cells deficient in Pax6 activity ... 52

Transcription factor binding to the PC2 and 7B2 promoters ... 56

Pax6, cMaf, MafB, and Beta2/NeuroD1 activate the PC2 and 7B2 gene promoters ... 57

Direct binding of transcription factors to the PC2 and 7B2 promoters and their relative contributions to promoter activation ... 59

Specific silencing of cMaf and Beta2/NeuroD1 results in decreased transcription of the PC2 and 7B2 genes ... 63

The cMaf and Beta2/NeuroD1 transcription factors are able to rescue the low transcription levels of PC2 and 7B2 in Pax6-DN306 clones ... 66

Discussion ... 67

Acknowledgments ... 72

Supplemental material ... 73

Chapter 2- Pax6 regulates genes important for glucose stimulated insulin biosynthesis and secretion ... 74

Abstract ... 75

Introduction ... 76

Materials and Methods ... 78

Primary cells ... 78

Cell culture... 78

RNA preparation and RT-PCR analysis ... 78

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RNA interference ... 79

Western blot analyses ... 79

Immunofluorescence ... 80

Plasmids ... 80

Promoter analysis ... 80

Electrophoretic mobility shift assays (EMSAs) ... 80

Chromatin immunoprecipitation assay (ChIP) ... 81

Insulin secretion test ... 81

Results ... 82

Knock-down of Pax6 in primary β-cells from rat ... 82

Pax6 regulates the transcription of Insulin1 and Insulin2, GLUT2, GK, MafA, Pdx1 and Nkx6.1 ... 84

Pax6-silencing in β-cell-lines results in decreased insulin, GK, MafA, Pdx1 and Nkx6.1 gene transcription ... 86

Binding of Pax6 to the GK, Pdx1, MafA and Nkx6.1 promoters ... 89

Pax6 can activate transcription from the insulin, GK, Pdx1, MafA and Nkx6.1 gene promoters in a heterolgous cell line ... 92

Silencing of Pax6 in primary rat β-cells results in decreased insulin secretion in response to glucose ... 92

Discussion ... 94

Chapter 3- Pax6 regulates the β-cell response to incretins and free fatty acids ... 99

Abstract ... 100

Introduction ... 100

Materials and Methods ... 102

Primary cells ... 102

Cell culture... 102

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RNA preparation and RT-PCR analysis ... 102

RNA interference ... 103

Western blot analyses ... 103

Promoter analysis ... 104

Electrophoretic mobility shift assays (EMSAs) ... 104

Chromatin immunoprecipitation assay (ChIP) ... 104

Plasmids ... 105

Insulin secretion test ... 105

Results ... 106

Silencing of Pax6 transcription and expression in β-cells ... 106

Pax6 regulates transcription of GLP1-R and GPR40 ... 106

GLP1-R and GPR40 promoters contain binding sites to Pax6 and Pdx1 ... 108

Pax6 and Pdx1 activate GLP1-R and GPR40 promoters ... 110

Silencing of Pax6 in primary rat β-cells results in no amplification of insulin secretion in response to glucose ... 111

Discussion ... 112

Perspectives... 114

General Conclusions and Perspectives... 115

The role of Pax6 in α-cells ... 116

The role of Pax6 in β-cells ... 117

Pax6 in pancreatic somatostatin producing cells, eye, and intestine ... 117

Pax6 and Diabetes ... 118

Pax6 and pancreas development ... 119

Integration of the results in α- and β- cells ... 120

Modulation of Pax6 gene expression by nutrients ... 121

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Perspectives... 122

Annex- Pax6 controls the expression of critical genes involved in pancreatic -cell development ... 123

Abstract ... 124

Introduction ... 125

Materials and methods ... 127

Cell culture... 127

Materials and plasmids ... 127

RNA interference ... 127

Pax6-DN306 stable InR1G9 clones ... 128

Western blot analyses ... 128

RNA preparation and RT-PCR analysis ... 129

Total cell count, BrdU labeling and apoptosis measurement ... 129

Transient transfection assays ... 129

Promoter analysis ... 129

Electrophoretic mobility shift assays (EMSAs) ... 130

Chromatin immunoprecipitation assay (ChIP) ... 131

Site directed-mutagenesis ... 131

Results ... 132

Identification of Pax6 target genes with specific inhibition of the Pax6 gene by siRNA in InR1G9 cells ... 132

Effects of the dominant-negative form of Pax6 (Pax6-DN306) on the glucagon gene promoter ... 134

Generation and characterization of Pax6-DN306 InR1G9 stable clones ... 136

Overexpression of Pax6-DN306 does not affect the apoptosis and cell proliferation rate ... 138

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Effects of constitutive expression of Pax6-DN306 in InR1G9 clones ... 139

Identification and characterization of Pax6 binding sites on Isl1, c-Maf, Beta2/NeuroD1 and PC2 gene promoters ... 142

Analysis of Isl1, c-Maf and Beta2/NeuroD1 gene promoter activities in BHK-21 cell and InR1G9 cells ... 146

Discussion ... 151

Acknowledgments ... 157

Supplemental material ... 158

References ... 161

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Acknowledgments

The first person I would like to thank is my thesis supervisor, Prof. Jacques Philippe. Thank you for the long and high quality guidance in the mysteries of science. Thank you for the fruitful ideas and for sharing your huge pool of knowledge. I also would like to express my sincere appreciation for your incredible knowledge in the field of endocrinology and diabetes. Despite the enormous work load you are dealing with on a daily basis, I always felt you are interested in my work and you always found time to discuss science with me.

Some people are involved in the work presented on the pages of this thesis and must be mentioned for their specific contribution: Yvan Gosmain, Claire Cheyssac, Audrey Guerardel, Aline Mamin, Charna Dibner, Valerie Schwitzgebel and Eric Marthinet. A big thank to each one of you for contributing to the work presented here, for the fabulous working environment you have created, and for sharing your experience and knowledge.

A special thanks to Sara Deakin for proofreading parts of this thesis.

I would like to thank Prof. Richard James and Prof. Walter Reith for undertaking the task as scientific advisers of my Ph.D. thesis.

I am and will always be grateful to my parents back in Israel for the tremendous support and encouragement during this period of PhD and for teaching me the meaning of wisdom, curiosity and love.

Last, but not least, a gigantic thanks to my beloved husband, Itamar, for supporting me all along this path, giving ideas, asking questions, reading abstract, manuscripts, PhD drafts but most of all, thank you Itamar for being you!

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Résumé en Français

Le pancréas est une glande amphicrine des systèmes digestif et endocrinien. La partie exocrine, constituant 95-99% de l'organe, est formée par une arborescence de conduits terminés par des grappes de cellules acineuses qui synthétisent et sécrètent des enzymes digestives.

Les îlots de Langerhans, la partie endocrine du pancréas représentant 1-5% du pancréas, sont composés de groupes de cellules disséminés dans le parenchyme de l'organe. Les îlots contiennent cinq types cellulaires différents: des cellules β, α, δ, PP et ε sécrétrices respectivement d'insuline, de glucagon, de somatostatine, de polypeptide pancréatique et de ghréline.Un dysfonctionnement endocrine du pancréas conduit au diabète, une maladie qui affecte 170 millions de personnes dans le monde.

Le facteur de transcription Paired box 6 (Pax6) joue un rôle clé dans la cascade de différenciation du pancréas endocrine.Il a été démontré qu’il lie et régule les promoteurs de l'insuline, du glucagon et de la somatostatine. Des mutations dans Pax6 sont associées à un diabète, une aniridie, des malformations cérébrales et d’autres anomalies. Les souris ayant une mutation homozygote de Pax6 présentent une malformation des îlots, une diminution du nombre de cellules α, β, δ et PP et une augmentation du nombre de cellules ε, sans aucune modification de la masse du pancréas endocrine.

Cette thèse vise à élucider l'impact de Pax6 sur la fonction et la différenciation des cellules α et β.

Le premier chapitre de la section des résultats ainsi que l’article lié en annexe présentent l’étude des gènes-cibles de Pax6 dans les cellules α. Nous avons utilisé la lignée cellulaire InR1G9 (cellules α de hamster) transfectée avec un petit ARN interférent (siRNA) dirigé contre Pax6 ainsi que des clones InR1G9 exprimant une forme dominante-négative de Pax6.

Nous avons montré que Pax6 régule la transcription et l'expression du proglucagon; de la prohormone convertase 2 (PC2) l’enzyme de transformation du proglucagon dans les cellules α, de 7B2 le chaperon moléculaire de PC2, ainsi que les facteurs de transcription cMaf, Beta2/NeuroD1 et Isl1. Ces facteurs de transcription ont été démontrés précédemment

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jouer un rôle important dans la régulation de la transcription du gène du glucagon et le développement des cellules α. Nous avons également caractérisé les promoteurs de ces gènes et montré que Pax6 lie directement et régule la transcription des gènes du glucagon, de 7B2, de cMaf, de Beta2/NeuroD1 et Isl1.En outre, nous avons caractérisé les promoteurs des gènes cibles de Pax6 et avons déterminé les sites de liaison fonctionnels de cMaf et Beta2/NeuroD1 sur les promoteurs de PC2 et 7B2. Nous concluons que Pax6 est essentiel pour la biosynthèse du glucagon et pour sa régulation directe ou indirecte via cMaf, Beta2/NeuroD1 et Isl1, ou PC2 et 7B2.

Les deux autres chapitres des résultats explorent les gènes cibles de Pax6 dans les cellules β.

Nous avons développé trois modèles de déficience en Pax6 à l'aide d’un siRNA dirigé contre Pax6 dans des cellules β primaires de rat et dans deux lignées cellulaires productrices d’insuline -βTC3 et HIT-T15. Nous avons montré que Pax6 régule les gènes de l'insuline 1, l'insuline 2, du transporteur de glucose 2 (GLUT2), de la glucokinase (GK), du récepteur GLP- 1, du récepteur aux acides gras libres (AGL) GPR40, ainsi que des facteurs de transcription Pdx1, MafA et Nkx6.1.Tous les gènes mentionnés ci-dessus sont impliqués dans la sécrétion d'insuline en réponse au glucose, ou dans sa potentialisation en réponse aux incrétines et aux AGL. En outre, les facteurs de transcription Pdx1, MafA et Nkx6.1 sont impliqués dans la transcription de l'insuline et la spécification des cellules β au cours du développement.Nous avons constaté la présence de sites de liaison Pax6 sur les promoteurs des gènes de l‘insuline 1, GK, Pdx1, MafA, Nkx6.1, GLP1-R, et de GPR40 et démontré que Pax6 était capable de transactiver ces promoteurs. Grâce à ces résultats, nous avons pu établir la carte du réseau complexe dans lequel Pax6 régule la transcription de gènes importants pour la fonction des cellules β et la différenciation.

La thèse montre que Pax6 est un facteur de transcription clé dans la différenciation du pancréas endocrine et joue un rôle majeur dans le fonctionnement normal du pancréas. La thèse contribue à la compréhension du rôle de Pax6 dans le développement du diabète de type 2.

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English Summary

The pancreas is an amphicrine gland of the digestive and endocrine systems. The exocrine part constitutes 95-99% of the organ, and is formed by a tree of ducts terminated by clusters of acinar cells synthesizing and secreting digestive enzymes.

The islets of Langerhans, the endocrine part of the pancreas representing 1-5% of the organ, are composed by group of cells scattered throughout the organ’s parenchyma. The islets contain five different cell types: , , , PP and ε cells, each producing a different hormone:

insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin, respectively. Pancreatic dysfunction leads to diabetes, a disease affecting 170 million of people worldwide.

The transcription factor paired box 6 (Pax6) plays a key role in the endocrine differentiation cascade of the pancreas. It has been demonstrated to bind and regulate the insulin, glucagon and somatostatin gene promoters. Mutations in Pax6 are associated with diabetes mellitus, aniridia, brain malformation, and other anomalies. Pax6 homozygous mutant mice manifest the following: a failure to form islets; a decrease in the number of β, δ, and pancreatic polypeptide-producing cells; a lack of α-cells; and an increase in ghrelin-positive ε cells without any change in endocrine pancreatic mass.

This thesis aims to elucidate the impact of Pax6 in α- and β-cell function and differentiation.

The first chapter of the results section and an interlinked study (presented in the annex) investigated the target genes regulated by Pax6 in α-cells. We used InR1G9 α-cells transfected with Pax6 small interfering RNA (siRNA) and InR1G9 clones expressing a dominant-negative form of Pax6. We found Pax6 to regulate the transcription and expression of the following: proglucagon; the processing enzyme of proglucagon in α cells- prohormone convertase 2 (PC2); the molecular chaperon of PC2- 7B2; as well as the transcription factors cMaf, Beta2/NeuroD1 and Isl1. These transcription factors were previously shown to play an important role in the regulation of glucagon transcription and α- cell development. We further characterized the promoters of these genes and found Pax6 to directly bind and regulate the transcription of glucagon, 7B2, cMaf, Beta2/NeuroD1 and Isl1.

Additionally, we characterized the promoters of Pax6-target genes and found functional

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binding sites of cMaf and Beta2/NeuroD1 on the promoters of PC2 and 7B2. We conclude that Pax6 is critical for glucagon biosynthesis and processing by directly and indirectly activating the glucagon gene through cMaf, Beta2/NeuroD1 and Isl1, as well as the PC2 and 7B2 genes.

The remaining two chapters of the results section explore the target genes of Pax6 in β-cells.

We developed three Pax6-deficient models using partial knock-down with siRNA in primary rat β cells and in two cell-lines- βTC3 and HIT-T15 insulin-producing cells. We found Pax6 to regulate the genes coding for insulin 1, insulin 2, glucose transporter 2 (GLUT2), glucokinase (GK), the GLP-1 receptor, the free fatty acids (FFA) receptor GPR40 as well as the transcription factors Pdx1, MafA and Nkx6.1. All the genes mentioned above are implicated in the secretion of insulin in response to glucose, or in its amplification in response to incretins and FFA. Furthermore the transcription factors Pdx1, MafA, and Nkx6.1 are implicated in insulin transcription and β-cell specification during development. We further find Pax6 binding sites on the insulin 1, GK, Pdx1, MafA, Nkx6.1, GLP1-R and GPR40 gene promoters, and demonstrate that Pax6 is able to transactivate transcription from these promoters. Through these results we manage to map the complex network in which Pax6 regulates transcription of genes important for β-cell function and differentiation.

The thesis demonstrates that Pax6 is a key transcription factor in the differentiation of the endocrine pancreas and plays a major role in the islet function. The thesis contributes to the understanding of the role of Pax6 in the development of type 2 diabetes mellitus.

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Abbreviations

ATP- adenosine triphosphate BHK- babyhamster kidney bp- base pair(s)

bHLH- basic helix-loop-helix BSA- bovine serum albumin Ca2+- calcium

cAMP- cyclic adenosine 3’,5’-monophosphate ChIP- chromatin Immunoprecipitation

DN- dominant negative DNA- deoxyribonucleic acid

dNTP- deoxynucleoside triphosphate EMSA- electrophoretic mobility shift assay ER- endoplasmic reticulum

eYFP- enhanced yellow fluorescent protein FFA- Free fatty acids

G protein- guanine nucleotide binding protein GDP- guanosine 5’-diphosphate

GFP- green fluorescent protein GIP- gastric inhibitory polypeptide GK- glucokinase

GLP1 (-R)- glucagon-like peptide-1 (receptor) GLUT2- facilitated glucose transporter 2 GPCR- G protein-coupled receptor GPR/GPCR- G-protein coupled receptor GSIS- glucose stimulated insulin secretion GTP- guanosine 5’-triphosphate

HEK- human embryonic kidney IP3- inositol 1,4,5 trisphosphate

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17 LB- medium Luria-Bertani medium

LUC- luciferase

MODY- maturity onset diabetes of the young NIDDM- non-insulin-dependent diabetes mellitus PAP- placental alkaline phosphatase

Pax- paired box

PCR- polymerase chain reaction

Pdx1- pancreatic duodenal homobox-1

PPAR- peroxisome proliferator activated receptor PST- proline-serine-threonine

RNA- ribonucleic acid

RT-PCR- Reverse transcription polymerase chain reaction.

SDS- sodium dodecyl sulfate

SDS-PAGE- sodium dodecyl sulfate polyacrylamide gel electrophoresis SEM- standard error of the mean

siRNA- small interfering RNA T1D- Type 1 diabetes mellitus T2D- Type 2 diabetes mellitus TM- transmembrane

WT- wild type

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Introduction

Pancreas

1.1 Functionality and morphology

The pancreas is divided into three main compartments: exocrine acinar tissue (corresponds to approximately 95% of the organ) that produces digestive enzymes, the endocrine tissue (around 2% of the organ) composed of the islets of Langerhans and the duct tissue through which the digestive enzymes are driven into the digestive tube [1, 2].

The pancreas is located across the back of the abdomen, behind the stomach (Fig. 1). The head of the pancreas is on the right side of the abdomen and is connected to the duodenum (the first section of the small intestine) through a small tube called the pancreatic duct. The narrow end of the pancreas, called the tail, extends to the left side of the body.

FIG. 1: The pancreatic structure, location and organization. Adapted from [3].

1.1.1 The endocrine pancreas

The endocrine pancreas is composed of scattered islets of Langerhans within the exocrine tissue, representing 1-5% of the pancreatic mass. The islets are round, compact, highly vascularized with scanty connective tissue and are from endodermal origin. An adult islet is composed of five different cell types characterized by their specific hormone secretion- , ,

, PP and ε cells, each producing a different hormone: insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin, respectively [4-6]. The proportion of the different cells

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within the islets is rather constant:  cells account for 65-80% of the islet cells,  15-20%,  3-10%, PP 3-5% and ε cells represent less than 1% [7-9].

In both humans and rodents the islets form a spherical structure. In rodents insulin producing cells are central, surrounded by a rim of α, δ and PP cells, while in humans the islets are less organized (Fig. 2) [8].

FIG. 2: Islets of Langerhans show striking interspecies differences. Confocal micrographs (1- μm optical sections), showing representative immunostained pancreatic sections containing islets of Langerhans from human (A), and (B) mouse Insulin-immunoreactive (red), glucagon- immunoreactive (green), and somatostatin-immunoreactive (blue) cells are all found randomly distributed in human islets. By contrast, insulin-containing cells are located in the core, and glucagon- and somatostatin-containing cells in the mantle of mouse islets (Scale bar, 50 μm). Adapted from [8].

Insulin

Insulin is secreted in response to increased blood glucose (concentrations), to trigger glucose uptake in the liver, muscles and adipocytes. Insulin consists of 51 amino acids, forming two chains A and B linked by disulfic bridges. Insulin is synthesized as a precursor of 11.5 kDa, preproinsulin; the first 25 amino acids are hydrophobic and allow rapid penetration to the

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endoplasmic reticulum (ER) during synthesis. Preproinsulin is then cleaved by a sequence specific peptidase followed by structural changes in the Golgi. Insulin is then enclosed in clathrin vesicles where the acid milieu will activate Prohormone convertase 2 and 1/3 (PC2 and PC1/3) that will in turn cleave proinsulin into insulin and C-peptide. In parallel, the vesicles mature into granules that are then ready to release insulin and C-peptide in equimolar quantities [10, 11]. Insulin stimulates liver and muscle to synthesize glycogen and proteins and the adipose cells to synthesize triacylglycerols. Insulin represses glycogenolysis and neoglucogenesis and promotes glucose entry and processing into muscle cells and adipocytes. In addition, it stimulates acinar degranulation.

Glucagon

Glucagon is the antagonist hormone to insulin effects, and is secreted in response to hypoglycemia; it is inhibited by hyperglycemia, insulin, and somatostatin. It stimulates the release of glucose and fatty acids by increasing hepatic glycogenolysis and neoglucogenesis to restore normoglycemia. Additionally, it stimulates insulin secretion by the β-cells and represses acinar secretion [12, 13]. Glucagon consists of 29 amino-acids. The protein is derived from a larger precursor, preproglucagon, [14, 15], which in the intestines is cleaved to produce, in contrast to the pancreas, glucagon-like-peptide 1 and 2 (GLP1 and GLP2).

Somatostatin

Somatostatin is a cyclic tetradecapeptide hormone and neurotransmitter that inhibits the release of peptide hormones in many tissues. It is also released from the hypothalamus to inhibit growth hormone (GH, somatotropin) and thyroid-stimulating hormone (TSH) secretion from the anterior pituitary. It is secreted by δ-cells of the islets of Langerhans in the pancreas to inhibit the release of glucagon and insulin and by similar D cells in the gastrointestinal tract, the principal source of circulating somatostatin [4].

Pancreatic Polypeptide

Pancreatic polypeptide (PP) is an orexigenic hormone secreted during hyperglycemia, which inhibits gallbladder contraction, gut motility and pancreatic secretion while promoting gastric emptying by repressing hypothalamic feeding-regulatory peptides and by acting on the vago-vagal and vago-sypathetic reflex arcs [16].

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21 Ghrelin

Ghrelin expressed ε-cells of the pancreas and represent 1% of the embryonic and adult endocrine pancreas, [17, 18]. Ghrelin levels increase before meals and decrease after meals.

It is considered the counterpart of the hormone leptin, produced by adipose tissue, which induces satiation when present at higher levels. Ghrelin is synthesized as a preprohormone, which is proteolytically processed to yield a 28-amino acid peptide. An interesting and unique modification is imposed on the hormone during synthesis in the form of the addition of an n-octanoic acid bound to one of its amino acids; this modification is necessary for biologic activity.

The predominant source of circulating ghrelin is the gastrointestinal tract, primarily the stomach, but also in smaller amounts the intestine. The hypothalamus in the brain is another significant source of ghrelin; smaller amounts are produced in the placenta, kidney, and pituitary gland. In the fetus,in contrast to the adult, the pancreas and not the stomach isa major source of circulating ghrelin. Furthermore, high ghrelin and ghrelin receptor gene expression in the fetal pancreas is intriguing and suggeststhat ghrelin may play an important role in islet-celldevelopment [19].

1.2 The exocrine pancreas

The exocrine tissue represents 95-99% of the pancreatic mass and consists of serous acini of highly polarized tall cells producing the digestive enzymes (amylase, lipase and phospholipase) as well as pro-enzymes (elastase, procarboxypeptidase, trypsinogen, pepsinogen, deoxyribonuclease and ribonuclease) stored in zymogen granules in the apical pole. Once secreted and activated in the lumen of the acinus, they are forwarded through the ductal network to the duodenum, allowing the intestinal digestion of nutrients. The ductal tree begins within the acini with a small duct lined by centro-acinar cells, followed by the intercalated ducts, lined by a single cubic epithelium. The intercalated ducts are followed by the intralobular ducts and finally by the interlobular ducts, often lined by a bistratified epithelium. The main pancreatic duct is joined by the common bile duct and finally ends up in the duodenum. Before entering the duodenum, pancreatic and hepatic secretion accumulate with the ampulla of Vater, closed by the sphincter which opens in response to nitric oxide and stimulation of the autonomous system [20-22].

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22 Diabetes Mellitus

Diabetes mellitus is a group of metabolic disorders characterized by chronic high blood glucose levels (hyperglycemia). This is a result of impaired insulin secretion and/or impaired action of insulin on its target tissues: muscle, liver and adipose tissue. Diabetes Mellitus has several risk factors and has multiple etiologies. In the long term, hyperglycemia causes degenerative complications at the micro and macro vascular level with severe consequences for the diabetic patient.

2.1 Diagnosis

Diabetes is diagnosed by measurement of blood glucose levels. The current recommendations of the ADA (American Diabetes Association) for the diagnosis of diabetes are: Symptoms of diabetes and a casual plasma glucose ≥200 mg/dl (11.1 mmol/l). Casual is defined as any time of day without regard to time since last meal. The classic symptoms of diabetes include polyuria, polydipsia, and unexplained weight loss. More specifically, diagnostic criteria are fasting plasma glucose levels (FPG) ≥126 mg/dl (7.0 mmol/l) (fasting is defined as no caloric intake for at least 8 h). Or a 2-h plasma glucose ≥200 mg/dl (11.1 mmol/l) during an OGTT (oral glucose tolerance test). The test should be performed as described by the World Health Organization, using a glucose load containing the equivalent of 75-g anhydrous glucose dissolved in water [23].

2.2 Worldwide Prevalence

In 1985 there were an estimated 30 million people with diabetes. Today diabetes affects more than 230 million people, almost 6% of the world's adult population. In many countries in Asia, the Middle East, Oceania and the Caribbean, diabetes affects 12-20% of the adult population.

2.3 Classification

The classification of diabetes includes four clinical classes:

1. Type 1 diabetes (T1D), results from β-cell destruction, usually leading to absolute insulin deficiency.

2. Type 2 diabetes (T2D), results from a progressive insulin secretory defect on the background of insulin resistance.

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3. Other specific types of diabetes due to other causes, e.g., genetic defects of β-cell function, genetic defects in insulin action, diseases of the exocrine pancreas (such as in cystic fibrosis), endocrinopathies, drug- or chemical-induced diabetes (such as in the treatment of AIDS or after organ transplantation), infections, uncommon forms of immune-mediated diabetes, other genetic syndromes sometimes associated with diabetes.

4. Gestational diabetes mellitus (GDM) (diagnosed during pregnancy).

2.3.1 Type 1 Diabetes Mellitus

T1D is the form of diabetes due primarily to β-cell destruction. T1D accounts for 5-10% of diabetic patients worldwide, though its prevalence varies greatly between populations. It is sub-classified into two main categories - type 1A and 1B. Type 1A is diagnosed by the presence of antibodies against proteins of pancreatic islets such as anti-insulin, anti- glutamate decarboxylase, anti-tyrosine phosphatase IA-2 and anti IA-2β antibodies.

However, especially in nonwhites, T1D can occur in the absence of these autoimmune antibodies and without evidence of any autoimmune disorder. Nevertheless, it is characterized by low insulin and C-peptide levels similar to type 1A, individuals with this type of diabetes are prone to ketoacidosis but the pathogenetic basis for their insulinopenia remains obscure. This is called type 1B diabetes [24].

Typically T1D affects young individuals and is diagnosed before the age of 20. The primary symptoms are often referred to as “cardinal syndrome”- polyuria, polydipsia and polyphagia accompanied by asthenia [24].

Causes

In Diabetes type 1A, hyperglycemia is the result of an attack of the immune system which causes the reduction in β-cell number. Pancreas biopsies from T1D patients show inflammation of T lymphocytes, B lymphocytes and macrophages. This immune attack causes a deficit in insulin production (insulinopenia) [25].

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24 Treatments

Classically, T1D is a type of diabetes in which insulin is required for survival. This involves injecting insulin under the skin -in the fat - for it to get absorbed into the blood stream where it can then access all the cells of the body that require it.

2.3.2 Type 2 Diabetes Mellitus

T2D is the most common form of diabetes (90-95%). 80% of type 2 diabetes are preventable by changing diet, increasing physical activity and improving lifestyle. It is a chronic, complex disorder of a rapidly growing global importance. It is characterized by concomitant defects in both insulin secretion (from the β-cells in the pancreatic islets) and insulin action (in fat, muscle, liver and elsewhere), the latter being typically associated with obesity [26].

Causes

The diabetes pandemic, which consists primarily of type 2 diabetes, has evolved in association with rapid cultural changes, aging populations, increasing urbanization, dietary changes, decreased physical activity and other unhealthy lifestyles and behavioral patterns.

Without effective prevention and control programs, the incidence of diabetes is likely to continue rising globally.

Usually T2D develops after the age of 40. 80% of T2D patients are overweight or have abdominal obesity. T2D is frequently not diagnosed until complications appear, and approximately one-third of all people with diabetes may be undiagnosed. In contrast to T1D, it is characterized by a relative, rather than absolute, insulin insufficiency due to a defect in β-cell production and peripheral insulin resistance.

Several genome associations have been found to be linked to T2D revealing an important role of genetic inheritance in the development of T2D.

Treatment

People with type 2 diabetes may require oral hypoglycemic drugs to lower their blood glucose and some may need insulin injections at some point. Drugs for type 2 diabetes come in various classes — biguanides, α-glucosidase inhibitors, amylin agonists, dipeptidyl- peptidase 4 (DPP-4) inhibitors, meglitinides, sulfonylureas and thiazolidinediones. Each class

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contains one or more specific drugs. Some of these drugs are taken orally, others must be injected.

2.3.3 Other specific types of diabetes MODY-Maturity onset Diabetes of the Young

MODY corresponds to about 5% of diabetes cases. It is caused by mutations in a single gene.

The following genes have been identified: HNF4α, GCK, TCF1/HNF1α, Pdx1/Ipf, TCF2/HNF1β and Beta2/NeuroD1.

It superficially resembles T2D, but it is characterized by impaired insulin secretion with minimal or no defects in insulin action. MODY appears in childhood or young adulthood, before the age of 25 years, and in most cases, at least two other members of the immediate family are affected. It is believed to account for 2% to 3% of all cases of diabetes.

Causes

MODY is associated with specific monogenic defects of β-cell function. Most of these are characterized by a dominant pattern of inheritance.

MODY1- (hepatic nuclear factor 4α, HNF4α) MODY1 was the first gene defect discovered [27], it is an extremely rare form of MODY. The transcription factor HNF4α has an important role in pancreatic development and maintenance of β-cell function. It has similar effects to MODY3, usually respond very well to oral sulfonylurea drugs. Due to progressive β-cell failure, a large proportion of the patients will eventually require insulin therapy.

MODY2- (pancreatic glucokinase gene, GK). Glucokinase is the rate limiting enzyme for glucose metabolism and is critical to regulate insulin secretion in response to glucose.

MODY2 patients normally do not show signs or symptoms. Diagnosis is made during routine blood testing. Generally MODY2 has an excellent prognosis. It is non-progressive, rarely requires drug or insulin therapy, and can usually be managed by exercise and diet alone. It is most commonly diagnosed in childhood or during pregnancy.

MODY3- (hepatic transcription factor 1α, HNF1α). MODY3 occurs more frequently than other types of MODY. It is normally diagnosed later in life. MODY3 can cause progressive

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diabetes, which may result in diabetic complications. People with MODY3 may be at an elevated risk of developing diabetic retinopathy. Patients are often responsive to sulfoylureas, a class of antidiabetic agents. MODY3 may also cause a severe form of diabetes that may need to be controlled with insulin. HNF1α is a transcription factor known to control a regulatory network important for differentiation of β-cells.

MODY4- (insulin promoter factor1/pancreas duodenum homeobox1, IPF1/PDX1) MODY4 is a rare form of MODY that often is associated with a mild form of diabetes. Pdx1 is a transcription factor vital to the development of the embryonic pancreas and in adults it continues to play a role in the regulation and expression of genes coding for insulin, facilitated glucose transporter 2 (GLUT2), glucokinase, and somatostatin.

MODY5- (hepatic nuclear factor 1β, HNF1β/TCF2). This rare form of MODY is often associated with renal disease, including kidney dysfunction or cysts, which are frequently diagnosed before diabetes. It may require several different treatments because it leads to multiple organ abnormalities. TCF2 is involved in the early stages of embryonic development of several organs, including the pancreas, where it contributes to differentiation of pancreatic endocrine Ngn3+ cell progenitors from non-endocrine embryonic duct cells.

MODY6- (Beta2/NeuroD1 gene). This is an extremely rare form of MODY. Little is known about the severity of diabetes associated with MODY6. Beta2/NeuroD1 promotes transcription of the insulin gene as well as some genes involved in formation of β-cells and parts of the nervous system.

Other genes were recently identified as MODY genes coding for- KLF11 (a pancreatic transcription factor induced by glucose to regulate insulin) , CEL (carboxyl ester lipase, a major component of pancreatic juice and responsible for the duodenal hydrolysis of cholesterol esters), and BLK (a nonreceptor tyrosine-kinase of the src family, expressed in β- cells where it enhances insulin synthesis and secretion in response to glucose by up- regulating the transcription factors Pdx1 and Nkx6.1) [28-30]. However, mutations in these factors are extremely rare and only lately characterized.

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27 MIDD-Maternity Inherited Diabetes and Deafness

A rare form of diabetes corresponding to 1-3% of diabetes cases is caused by mutations in the mitochondrial DNA. A point mutation in the gene coding for the tRNA of leucine was identified [31]. The molecular mechanism standing behind this mutation is yet to be demonstrated, but it has been shown that as a consequence of this point mutation, there is β-cell dysfunction evident by a defect in insulin secretion in response to glucose [32].

Neonatal diabetes

A rare form of diabetes, in Europe its prevalence is estimated to be 1:50000 births. It is characterized by severe hyperglycemia accompanied by low insulin levels, polyuria, dehydration, and insufficient weight gain in the first ten days after birth. This diabetes exists in two distinct clinical forms:

1. Permanent neonatal diabetes (PND) where there is a requirement for insulin therapy for life. PND has been associated with mutations in Pdx1, GCK, FOXP3 and KCNJ11 (Kir6.2) [33-35].

2. Transient neonatal diabetes (TND) where there is remission after insulin therapy with a higher risk of developing diabetes at the adulthood [34]. Genetic analyses have demonstrated a genetic transmission of TND. Two genes where identified to be in association with TND; ZAC, which regulates exit from the cell cycle, apoptosis and also regulates the protein PACAP1 (pituitary adenylate cyclase activating polypeptide receptor 1) which is involved in insulin secretion and the gene HYMAI which has an unknown function [36].

2.3.4 Gestational diabetes

Gestational diabetes is defined as hyperglycemia occurring during pregnancy. The causes have not yet been clearly defined. However, the strong decrease in insulin resistance after birth suggests a role of the placental hormones [37]. In the USA, gestational diabetes is responsible for about 7% of complications during pregnancy. A woman that suffered from gestational diabetes has a 70% risk of developing T2D in the next 10 years. Several genes were found to be associated with this kind of diabetes-e.g. Sur1, Capn10 [37].

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3. Pancreatic development-Morphogenesis

3.1 Transcription factors regulating pancreatic development

The mouse pancreas develops as evaginations from dorsal and ventral regions of the developing foregut destined to become duodenum [38]. Its developmental program is initiated by factors released by the mesoderm at E9.0. Later, the epithelium of the foregut undergoes branching morphogenesis into a ductal tree and results in the formation of two primordial pancreas organs consisting primarily of undifferentiated ductal epithelium. At E13.5 the gut rotates clockwise, bringing the two primordial pancreas buds together;

eventually, they fuse into a single organ. The exocrine pancreas differentiates from the ductal epithelium and afterwards acini are clearly visible. The endocrine cells appear from the very beginning of the epithelium and undergo extensive proliferation after the fusion of the dorsal and ventral buds, a stage often referred to as the second developmental transition. The islet cells then begin to organize into islet-like clusters, a process that continues maturation of the islets cells and still carries on until shortly after birth, this is the third developmental transition [39] (Fig. 3).

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FIG. 3: Events of pancreatic organogenesis. Development of the dorsal rudiment only is depicted after E8.5. Yellow shading indicates the extent of Ipf1/Pdx1 expression and gray indicates the early-differentiated endocrine cell clusters. The double arrow indicates the orthogonal directions of cell division of islet versus acinar/ductal precursors. Adapted from [39].

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30 Pdx1

The pancreas develops as evaginations from dorsal and ventral regions of the developing foregut that express the homeodomain transcription factor Pdx1 [40-43]. Pancreatic and duodenal homeobox 1 (Pdx1) is essential for the normal proliferation and differentiation of embryonic pancreatic precursors [40, 44, 45]. Epithelial cells in these buds proliferate and form branching ductules that differentiate and give rise to mature ductal, endocrine and acinar elements [39, 46-48]. Pdx1 is expressed early in all pancreatic progenitor cells and is later restricted to adult β-cells plus a fraction of δ-cells. Lineage tracing studies have shown that Pdx1-expressing cells at E8.5 are multipotent progenitors, giving rise to all pancreatic lineages [49, 50].

Pdx1 knockout mice display pancreatic agenesis [51]. Interestingly, in those knockout mice, some Insulin and Glucagon-expressing cells develop in the dorsal bud region. Mice heterozygous for Pdx1 have elevated blood glucose levels and impaired glucose tolerance [52]. Similarly, humans with heterozygous mutations in Ipf1/Pdx1 are highly predisposed to MODY (subtype 4) or adult-onset type 2 diabetes while homozygous mutations result in pancreatic aplasia [53, 54].

Interestingly, although the pancreatic epithelium of Pdx1 null-mice fails to develop, the surrounding mesenchyme grows and develops normally indicating that development of the pancreatic mesenchyme does not rely on the epithelium [55].

Pbx1

Pre B-cell leukemia transcription factor 1 (Pbx1) is expressed at E10.5 in mesenchymal and Pdx1-expressing epithelial cells of the developing pancreas. In the adult it is widely expressed in ductal, acinar and islet cells. Pbx1-/- mice exhibit agenesis of both the dorsal and ventral pancreas due to a strong (35-40%) reduction of epithelial cell proliferation suggesting that Pbx1 is essential for pancreatic epithelial cell proliferation [56]. Islet1 (isl1) and neurogenin3 (ngn3) two critical genes for the development of the endocrine cells of the pancreas are absent in Pbx1 mutant mice and accordingly no endocrine cells are present [56]. In addition to controlling the proliferation of pancreatic progenitor cells, Pbx1 seems to also play a role in the differentiation of endocrine cells.

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31 Isl1

The LIM homeodomain protein Isl1 is expressed in the developingpancreas and the central nervous system [57, 58]. In adult animals,Isl1 is expressed in all of the hormone-producing cells of pancreaticislets, but its low level of expression in ß-cellsargues against it having an important function to regulate insulin gene transcription. Targeted disruption of the isl1 gene resultsin an early arrest of embryonic development on approximately E9.5 [46]. Isl1 is required for the formation of dorsal, butnot ventral, pancreatic mesenchyme. Mice in which Isl1 expressionis disrupted by gene knockout fail to develop a dorsal pancreas,but partially execute the differentiation of the ventral exocrine pancreas [59]. The dorsal pancreatic endoderm obtained fromIsl1-null mice and studied ex vivo responds to signals transmitted from co-cultured wild-type mesenchyme by differentiating into pancreatic tissue. These findings reinforce the critical requirement of mesenchymal signaling to the endoderm in allowing normal pancreatic development. The elucidation of additional signalingpathways that are critical for development of the pancreas requiresadditional investigation.

Ptf1 complex

Pancreas specific transcription factor-1 (Ptf1) is a trimeric transcription factor essential to the development of the pancreas and to the maintenance of the differentiated state of the adult exocrine pancreas. It is composed of three members of the basic helix-loop-helix (bHLH) family of proteins. The first member, p75, is essential for the nuclear localization of the complex [60]. The two other members, p64 and Ptf1a-p48 are the DNA-binding subunits [61]. Ptf1-p48 (Ptf1a) is expressed and restricted to all pancreatic progenitors being more specific than Pdx1 which in addition to the developing pancreas is also expressed in the caudal stomach and duodenum. The coexpression of both Pdx1 and Ptf1a is necessary and sufficient to trigger the pancreatic differentiation cascade.

During the second developmental transition (around E12.5) Ptf1a expression becomes restricted to developing acinar cells [62]. Functional binding sites for the Ptf1 complex have been found in the promoters of all acinar digestive enzymes examined [61, 63]. In Ptf1a- deficient embryos, pancreas development is severely impaired; exocrine progenitors are reprogrammed into a duodenal fate, there is no acini nor duct formation and endocrine cells were found to be relocated into the spleen [64].

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32 Foxa1, Foxa2, Foxa3 (HNF3α, HNF3β, HNF3γ)

The Foxa group of the Forkhead family comprising Foxa1, Foxa2 and Foxa3 is essential for the regulation of hepatocyte-specific expression of several target genes [65]. They are expressed in embryonic endoderm, the germ layer that gives rise to the digestive system, and contribute to the specification of the pancreas and the regulation of glucose homoeostasis [66].

Foxa2 is expressed as early as E6.5 in the mesoderm and its expression is maintained high in the pancreas of adult mice. Foxa2 null-mice die at E10-11 [67, 68], but the formation of a dorsal pancreatic bulge is still possible [38]. Foxa2 null embryos do not survive long enough to study the role of Foxa2 in pancreatic development. In order to dissect the function of Foxa2 specifically in the pancreas, a β-cell-specific deletion was generated. These mice survived to birth and died around P10 from hypoglycemia. The specific β-cell knockout demonstrated islet disorganization, but cell lineages were present in appropriate proportions [69], indicating a possible role for Foxa2 in islet formation. Ablation of Foxa2 only after E14.5 results in an absence of α-cells [70], suggesting Foxa2 has a critical role in the regulation of the terminal differentiation steps and maturation of glucagon-producing α- cells.

The expression of Foxa1 initiates by E9 in the entire gut region including the liver primordium and the floorplate of the neural tube. The expression of Foxa1 is maintained in organs of endoderm lineage in the adult [71, 72]. Foxa1 null-mice die shortly after birth from hypoglycemia [73, 74]. The Foxa1 knockout mice have low levels of circulating plasma glucagon, but a normal pancreatic cell proportion [73, 74]. Foxa1 was demonstrated to activate the proglucagon gene promoter [73].

Foxa3 is first detected in the extra-embryonic endoderm before E8.0 [75] and more strongly in cells of the invaginating hindgut near E9.0. Foxa3 gene seems to plays a crucial role in maintenance of glucose homoeostasis in the adult [76]. Fox3 knockout mice are viable, with normal blood glucose levels [77].

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33 Ngn3

Neurogenin3 (Ngn3), a basic helix-loop-helix (bHLH) transcription factor, expressed transiently in endocrine precursors [50, 78], is the earliest known marker for specific endocrine precursor cells [79, 80]. These Ngn3-expressing cells are a subset of the entire Pdx1+ endodermal epithelial cells, and they first appear at embryonic day E8.5, peak at E15.5 and are undetectable at birth. Ngn3 is never coexpressed with hormones or other markers of differentiated cells.

Ngn3 has been shown to regulate the expression of Beta2/NeuroD1 concurrently with Isl1 and Pax6 to give rise to endocrine cells during development and in adults [78, 79, 81-84]. As a consequence the pancreatic hormones are expressed, followed by switching off Ngn3 expression [83]. The specification of different islet cell types and the completion of the differentiationprocess require the activation of transcription factors thatare downstream of Ngn3 [85].

Mice with homozygous mutations in both ngn3 alleles lack differentiated endocrine cells but have nearly normal acini and ducts [79]. In addition to these major transcription factors several other transcription factors and signaling molecules play critical roles in pancreatic development and endocrine differentiation (for reviews see [39, 46, 47, 86-90]).

3.2 Specification of endocrine progenitors

Expression of Ngn3 in pancreatic precursors converts them into endocrine precursors.

However, expression of additional transcription factors is needed to trigger differentiation (specification) of these cells into the different endocrine lineages. This is illustrated by the importance of the timing of Ngn3 expression in pancreatic progenitors. Different subsets of endocrine lineages are specified at different stages of development, with glucagon+ cells induced first, followed by insulin+, PP+ and somatostatin+ cells [91]. In addition, pancreatic endocrine cell differentiation has shown to be plastic and it is possible to convert one endocrine cell type to another by the misexpression or knockout of one or more transcription factors (Fig. 4).

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FIG. 4: Schematic model representing the transcription factors implicated in the specification of the endocrine and exocrine pancreas [92].

3.3 Transcription factors directing pancreatic development and function of α- and β- cells

Several transcription factors are activated during early pancreatic development to ensure normal organogenesis and the subsequent differentiation of the different endocrine cell types.

Beta2/NeuroD1

Beta2/NeuroD1 is class B bHLH. It is a key transcription factor required for pancreatic development and endocrine cell differentiation. It is first expressed at E9, in a subset of epithelial cells most of which co-express glucagon. Later, Beta2/Neurod1 is expressed in all endocrine cells of the developing and adult pancreas indicating that, unlike Ngn3, Beta2/Neurod1 expression is maintained in differentiated endocrine cells [93].

The ubiquitous E2A family of proteins, including E12 and E47, function either as homo- or heterodimers with Beta2/NeuroD1 to bind and transactivate promoters via conserved

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sequence elements known as E-boxes [94-97]. Beta2/NeuroD1is, expressed in pancreatic endocrinecells, the intestine, and brain [93, 95, 98].

The heterodimer Beta2/NeuroD1-E47 represents an islet-specific transcription factor that controls both insulin [93, 99-102] and glucagon gene transcription [100]. ß2/E47 specifically has been reported to bind and activate the glucokinase promoter in the islet [103].

Beta2/NeuroD1binds and activates the promoter of Sur1 [104], which playsa major role in the activation of insulin secretion upon glucose stimulation [105], and may be controlled at the transcriptional and post-transcriptional level by an increase in intracellular Ca2+

concentration [104]. The principal role of Beta2/NeuroD1 may be in the maintenance of insulinexpression in mature ß-cells by active repressionof the somatostatin promoter [106].

Mutations in Beta2/NeuroD1are linked to MODY6 in humans and are associated withtype 2 diabetes in the heterozygous state [107]. Mice knockout for Beta2/Neurod1 die after or several months after birth of severe diabetes, depending on their genetic background [93, 108]. They have a 60% reduction of endocrine cells and fail to develop morphologically distinct islets [93].

Arx and Pax4

The transcription factor aristaless related homeobox (Arx), demonstrates plasticity in the specification of endocrine lineages [87, 109, 110]. Arx is detected in the pancreatic epithelium, in endocrine precursor cells along with Pax4 from E10.5 to E18.5, when Arx becomes α-cell specific.

The paired box 4 (Pax4) transcription factor, is first expressed in some cells of the ventral spinal cord and pancreas at E9.5. Like Ngn3, Pax4 is transiently expressed in pancreatic progenitors during development and is downregulated shortly after birth. In the developing pancreas, its expression is restricted to β-cells and some δ-cells but it is absent from α-cells [87].

The analysis of Pax4-, Arx- and Pax4-Arx- double knockout mice revealed that the choice between α- β- and δ- cell fate in the endocrine progenitors is regulated by the activity of these factors. Arx promotes the α-cell fate while Pax4 regulates specification of β-, and δ- cells. Both factors negatively regulate each other's expression, thereby regulating the

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proportion of these cells. Interestingly, over-expression of Arx in early pancreatic epithelium, embryonic endocrine cells or post-natal insulin+ cells resulted in conversion of β-cells into δ- cells under all conditions. This observation demonstrates a significant plasticity in endocrine lineages and opens new possibilities of converting the fate of other endocrine cells into insulin+ cells. Interestingly, insulin producing cells specified during development do undergo maturation steps before they become glucose-responsive β-cells [111-114].

NK Family

NK-family members and homeodomain protein Nkx2.2 and Nkx6.1 are expressed in pancreatic endocrine cells and involved in their differentiation.

During pancreas development, Nkx2.2 is first expressed in the whole epithelium from E9.5 to E12.5. Then, as the pancreatic program resolves into endocrine and exocrine cells, Nkx2.2 expression persists only in the endocrine cells. In the adult pancreas, Nkx2.2 is expressed in α-, β- and PP-cells but not in δ-cells [115]. Nkx2.2 null-mice have a large number of endocrine cell precursors without hormone production. Nkx2.2 is necessary for the maturation of ß-cells [115], whereas its distant homologue Nkx6.1 controls their expansion [82].

The expression pattern of Nkx6.1 is similar to that of Nkx2.2. At E10, it is expressed in pancreatic progenitor cells in combination with Pdx1 and Ptf1a [116, 117]. Interestingly, Insulin+/Glucagon+ early hormone expressing cells do not express either Nkx6.1 or Pdx1 [118]. At the beginning of the second developmental transition, Nkx6.1 is expressed in ductal and periductal cells expressing Pdx1 or Ngn3 and in Insulin-expressing cells [82]. At E15.5, all Nkx6.1 expressing cells also express Nkx2.2, but many Nkx2.2 expressing cells do not express Nkx6.1. Finally, at later developmental stages and postnatally, Nkx6.1-expression is restricted to β-cells [82]. Interestingly the phenotype of Nkx2.2 and Nkx6.1 double mutant is similar to that of Nkx2.2, indicating that Nkx6.1 acts downstream of Nkx2.2 [82]. Nkx6.1 knockout mice have a selective reduction of β-cells with a normal amount of other endocrine cell types. Nkx2.2 is co-expressed with insulin [115] and Ngn3 [80, 115] during pancreatic development. Foxa2 and Ngn3 are proposed to lie upstream fromNkx2.2 in the hierarchy of

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ß-cell differentiation transcription factors [119]. Prado et al. suggest that high expression levels of both Nkx2.2 and Pax4 are independentlyrequired to specify or maintain ß-cell fate [120].The absence of either transcription factor resulted in ß-cellsfailing to form, with the ß- cells replaced by ghrelin-producingε-cells [120]. Pax6 expression depends on Nkx2.2 [121], thusit is probable that Nkx2.2 acts upstream of Pax6 in the samepathway to regulate ß- and ε-cell fates [120].

Pax6

The paired box 6 (Pax6) is a pairedomain-homeodomain transcription factor. In the pancreas, it is expressed in both ventral and dorsal buds from E9. In the endocrine differentiation cascade, Pax6 stands downstream of Ngn3 [18]. At E10.5, Pax6 is detected in glucagon-expressing cells and later at E15.5, Pax6-positive cells express either Insulin or Glucagon. Finally, in newborns, Pax6 is restricted to mature islet cells [122, 123].

Mutations in Pax6 are related to diabetes mellitus, aniridia, brain malformation, and other anomalies [124, 125]. Pax6 homozygous mutant mice fail to form islets; show a decreased number of β-, δ-, and pancreatic polypeptide-producing cells; a lack of α- cells; and an increase in ghrelin-positive ε-cells without any change in endocrine pancreatic mass [18, 126].

This thesis is aimed at better defining the role of Pax6 in α- and β- cells function. Therefore, the last sub-chapter of the introduction will further focus on the Pax family of transcription factors and in particular Pax6.

Sox4

Sox4 is a member of the Sry/hydroxymethylglutaryl (HMG) box family of transcription factors. Sox4 is expressed in all cells of the endocrine pancreas and the developing pancreas of mutant mice for Sox4 is similar to wild type up to E12.5 but die at die at E14 due to cardiac defects. However, when put in culture explants have 50% less α- and β- cells compared to normal mice [127, 128]. Furthermore, α-cell differentiation is drastically reduced in zebrafish mutants for Sox4 [129].

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38 Large MAFs

The cloning of the insulin gene transcription factor RIPE3b1 as MafA [130] initiated systematic examination of the function of Maf factors in the endocrine pancreas. Maf proteins are subdivided into two classes, “large” (236-370 AA; c-Maf, MafB, NRL and MafA/L-Maf) and “small” (149-162 AA; MafF, MafG and MafK) [131-134]. The leucine zipper domain allows the Maf proteins to dimerize with themselves or other basic leucine zipper (bZIP) factors and thereby influence the DNA binding specificity of these factors [132, 135, 136].

In the pancreas, MafA, MafB, and cMaf are expressed in pancreatic endocrine cells [111].

MafA, which is -cell specific, is a glucose-responsive insulin gene transcription factor that binds to the insulin Maf Responsive Element (MARE) and activates insulin gene expression [130, 137-140]. MafA expression is restricted to Insulin-expressing cells from E13.5 to adults.

Its absence in Nkx6.1 mutant mice suggests that it lies downstream in the β-cell differentiation cascade [141]. Interestingly, mice deficient in MafA have normal pancreatic islets at birth, but the ratio of  to  cells is gradually reduced, resulting in glucose intolerance by 8-12 weeks, and development of diabetes with age [142]. Identification of downstream targets of MafA as critical regulators of insulin synthesis and secretion [143, 144] (such as, insulin, Pdx1, Nkx6.1, GLUT2, GK, GLP1 receptor, prohormone convertase 1/3 (PC1/3), pyruvate carboxylase and granuphilin [145]) suggests a mechanism by which MafA may regulate the maturation process.

MafB is restricted to α-cells in adults. During pancreatic development, at E10.5, MafB is expressed in glucagon+ cells and at E15.5, although all glucagon+ cells express MafB, a significant proportion of cells express only MafB. These early MafB+/Glucagon- cells express the panendocrine marker Synaptophysin and some even express Insulin [111, 140], suggesting that MafB could play a role in the differentiation of β-cells. In addition, MafB was also shown to regulate the expression of genes specific for β-cell differentiation such as Pdx1, Nkx6.1 and GLUT2 [146, 147].

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cMaf is expressed in both α- and β- cells during embryonic development and in adults. cMaf knock out mice die shortly after birth and they have a decreased amount of insulin- and glucagon- producing cells.

Pax family of transcription factors and Pax6 4.1 Pax Family

Paired box (Pax) genes are a family of tissue specific transcription factors containing a paired domain and usually a partial or complete homeodomain. Pax proteins are important in early animal development and formation of several tissues from all germ layers in the mammalian embryo. This family is composed of nine genes, Pax1-9.

The original finding that the paired domain conferred a sequence-specific DNA-binding function, together with a Proline–Serine–Threonine-rich (PST) carboxy terminus that is characteristic of other transcriptional activators, led to the supposition that Pax proteins would be involved in transcriptional regulation [148, 149] (Fig. 5).

FIG. 5: Functional domains of Pax6. Structure of the human full length protein. PD, paired domain; G, Gap; HD, homeodomain; PST, Proline-serine-threonine (transactivating domain).

4.2 Pax6 in detail

Pax6 is a Paired homeodomain transcription factor expressed in the eye, nose, pancreas, and central nervous system from the early stages of embryonic development [150].

Pax6 appears to be necessary for the correct execution of islets’ endocrine cell differentiation [151, 152]. It is expressed early in the developing pancreas (E 9.0) in cells destined to an endocrine cell fate and does not seem to be required for cell type specification [122, 126]. However, Pax6 is essential for the normal expression of final differentiation markers such as insulin, glucagon and somatostatin [153]. Pax6 regulates the C2 element of the insulin gene [122], the G1 and G3 elements of the glucagon gene [154, 155] and somatostatin gene expression [156].

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Pax6 Heterozygous mutations in humans cause congenital eye anomalies such as aniridia.

Heterozygous mutations in Pax6 might also lead to glucose intolerance characterized by impaired insulin secretion [157]. Therefore, haploinsufficiency of this gene is sufficient to obtain a phenotype, illustrating that regulation of transcription factor levels are of great importance.

Previous studies have shown that various forms of Pax6 with different molecular weights exist, and at least four variants of Pax6 (p46, p48, p43, and p32) were detected in cellular extracts (Fig. 6) [158, 159]. The various forms are the result of alternative splicing [158]. All forms of Pax6 bear a conserved C-terminal transactivation domain, which contains relatively rich proline, PST residues. Several phosphorylation sites have been identified in this region of the human and zebrafish Pax6. It has been shown that phosphorylation of these sites in Pax6 is carried out by p38, ERK [160], and homeodomain-interacting protein kinase 2 [150]. On the other hand, dephosphorylation of Pax6 remains largely unknown.

FIG. 6: Summary of multiple forms of Pax6 and its conserved PST domain. A, diagram of four different variants of Pax6 (p33/32, p43, p46, and p48). PST, proline-, serine- and threonine- enriched activation domain; PD, paired domain; HD, homeodomain. B, phosphorylation sites identified in the PST domain from zebrafish or human Pax6. Adapted from [161].

4.2.1 Pax6 knockout models

There are several Pax6 knock out models, including cortex- and eye- specific knockout [162, 163]. The general Pax6 knockout mice as well as the endocrine specific knockout mice develop and die from diabetes few days after birth.

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