The role of homologous cell-cell contacts in insulin secretion

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Thesis

Reference

The role of homologous cell-cell contacts in insulin secretion

JACQUES, Fabienne

Abstract

Dans ce travail, nous avons étudié comment est-ce que les contacts cellules-cellules influencent la sécrétion d'insuline des cellules β-pancréatiques. Cette étude nous a permis de mettre en évidence le double rôle des contacts intercellulaires dans la régulation de la sécrétion d'insuline puisque les cellules privées de contacts entre elles présentent une sécrétion basale trop élevée ainsi qu'une sécrétion stimulée trop faible en comparaison des cellules en contact les unes avec les autres. Nous montrons que les contacts physiques intercellulaires permettent de maintenir un niveau de calcium cytosolique stable, ce qui est indispensable pour une sécrétion basale adéquate et que, plus spécifiquement, l'engagement des molécules d'adhésion E-cadhérine entre cellules adjacentes permet d'optimiser la sécrétion d'insuline suite à une stimulation au glucose. Finalement, nous montrons que les cellules isolées restent capables de répondre au glucose une fois que leur sécrétion basale a été normalisée.

JACQUES, Fabienne. The role of homologous cell-cell contacts in insulin secretion. Thèse de doctorat : Univ. Genève, 2009, no. Sc. 4055

URN : urn:nbn:ch:unige-16438

DOI : 10.13097/archive-ouverte/unige:1643

Available at:

http://archive-ouverte.unige.ch/unige:1643

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

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UNIVERSITE DE GENEVE

Département de Biologie Cellulaire FACULTE DES SCIENCES Professeur D.Picard

Département de Médecine FACULTE DE MEDECINE

Génétique et Développement Professeur P.A. Halban

The Role of Homologous Cell-Cell Contacts in Insulin Secretion

THESE

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

par

Fabienne JAQUES de Lutry et Epesses (VD)

Thèse N° 4055

GENEVE

Centre d’édition des HUG 2009

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TABLE OF CONTENTS

AKNOWLEDGMENTS………... -

ABBREVIATIONS………... 4

RESUME EN FRANÇAIS………... 6

ENGLISH SUMMARY... 10

GENERAL INTRODUCTION...……... 13

1. Pancreatic ββββ-Cells..………... 15

1.1 Glucose Sensing Mechanisms : From Glucose Sensing to Insulin Release 16 1.1.1 Triggering and Amplifying Pathways... 16

1.1.2 Different Secretagogues... 18

1.1.2.1 Nutrient Secretagogues...………... 18

1.1.2.2 Non-Nutrient Secretagogues………... 19

1.2 Insulin Synthesis and Processing………... 20

1.2.1 Insulin Biosynthetic Pathway... 20

1.2.2 Regulation of Insulin Synthesis…... 21

1.3 Exocytotic Machinery………... 22

1.4 Vesicle Recycling…………..………... 26

1.5 Constitutive vs. Regulated Secretory Pathways………... 27

1.6 Biphasic Insulin Secretion………... 29

2. The ββββ-Cell Environment………... 31

2.1 Indirect Communication………... 32

2.2 Direct Communication………... 33

2.2.1 Cell-ECM Interactions in β-Cells... 33

2.2.2 Cell-Cell Interactions in β-Cells... 34

2.2.2.1 Adherens Junctions... 35

2.2.2.2 Gap Junctions... 37

3. Calcium Homeostasis………... 39

3.1 Calcium Homeostasis in Excitable Cells ………... 40

3.2 L-Type Channels in Pancreatic ββββ-Cells………... 41

3.3 Other Ca²⁺-Channels in Pancreatic ββββ-Cells………... 42

AIMS OF THE PROJECT... 44

RESULTS... 46

1. PART I: Role of Cell-Cell Interaction in Insulin Secretion………... 46

1.1 Paper………... 46

1.2 Major Findings and Perspectives………... 59

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2. PART II: The Role of E-cadherin in Insulin Secretion………... 61

2.1 Introduction………... 61

2.1.1 WNT Signalling Pathway... 63

2.1.2 Rho Family of small GTPases... 64

2.1.3 Protein Kinases Activation... 65

2.1.4 Impact on Other Adhesive Structures... 65

2.2 Material and Methods………... 67

2.2.1 Cell Culture... 67

2.2.2 Mouse Islets Isolation... 67

2.2.3 Insulin Secretion Assay... 68

2.2.4 RNA Isolation and Quantitative RT-PCR... 68

2.2.5 Proteins Extraction and Western Blotting... 68

2.2.6 Transient Transfection of MIN6B1 Cells and hGH Secretion Assay... 69

2.2.7 Generation of Stable Cell Line Expressing E-cadherin siRNA... 69

2.2.7.1 pSUPERIOR.puro... 69

a) Construction and Production of Tetracycline-Inducible pSUPERIOR.puro Vector Carrying shRNA Directed Against E-cadherin mRNAs... 69

b) Generation of a Stable Tetracycline-Inducible E-cadherin siRNA-expressing Subline69 2.2.7.2 Lentivirus... 69

a) Construction and Production of Lentiviral Vector Carrying shRNA directed against E-cadherin mRNAs... 69

b) Generation of Stable Cell Lines Expressing E-cadherin siRNA... 70

2.3 Results………... 71

2.3.1 Transient Transfection of MIN6B1 Cells... 71

2.3.2 Generation of a Stable Tetracycline-Inducible E-cadherin siRNA-expressing Subclone... 72

2.3.3 Generation of Stable Cell Lines Expressing E-cadherin siRNA Using Lentiviruses... 74

2.3.4 Inhibition of E-cadherin using DECMA-1 Antibody... 76

2.3.5 Activation of E-cadherin using E-cadherin/Fc Proteins... 79

2.4 Conclusions and Perspectives... 80

GENERAL CONCLUSIONS AND PERSPECTIVES... 86

REFERENCES... 96

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AKNOWLEDGEMENTS

This thesis owes its existence to the help and support of many people. In the first place, I wish to express my sincere gratitude to Professor Philippe Halban, for giving me the opportunity to work in his lab and carry out my research project. I would like to thank him for his guidance as well as his support and encouragement during the four years of this thesis. Furthermore I particularly would like to thank him for his humanity.

Many thanks to Professor Alain Ktorza and Professor Didier Picard for accepting to be members of the jury during my thesis defense with a special thank to Professor Alain Ktorza for the interesting and helpful discussions.

I would like to thank all staff members of Professor Halban’s group for their assistance and support: Domenico Bosco, Maryse Girard, Carmen Gonelle-Gispert, Esperanza Gattuso, Valérie Lilla, Marine Maiullari, Géraldine Parnaud, Nadja Perriraz, Caroline Rouget, Barbara Yermen and Eve Zeender. More specifically I would like to thank Alejandra Tomas Catala for the discussions and cooperations that substantially contributed to this work, Eva Hammar and Pascale Ribaux for their kindness and availability in the laboratory, Anne-Lise Prost, who has been a source of enthusiasm and inspiration and Jean-Claude Irminger, who constantly offered invaluable assistance in critical situations.

I would also like to thank Mélanie Cornut, Stéphane Dupuis, Katarina Rickenbach and Dieter Rondas for their precious friendship. I will miss our animated coffee break...

I am also very grateful to all members of Professor Nicolas Demaurex’s group, in particular to Helène Jousset, for their very useful assistance and generosity.

Finally, I would like to take this opportunity to express my love and gratitude to my family, for their understanding and endless love throughout my studies, with a particular thank you to Dan, for his moral support and his patience during these last four years and simply for being there.

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ABBREVIATIONS Ach : Acetylcholine

ADP/ATP : Adenosine diphosphate/Adenosine triphosphate cAMP : cyclic AMP

CCK : cholecystokinin Cx : Connexin

DAG : diacylglycerol DHP : dihydropyridine

DMEM : Dulbecco's Modified Eagle's Medium ECM : extracellular matrix

ER : endoplasmic reticulum

FACS : Fluorescence-activated cell sorting FCS : fetal calf serum

FFA : free fatty acids GD : gestational diabetes

GEYS : Gey’s Balanced Salt Solution GFP : green fluorescent protein

GIP : glucose-dependent insulinotropic polypeptide GK : glucokinase

GLP : glucagon-like peptides GLUT-2: glucose transporter-2

GSIS : Glucose stimulated insulin secretion

HEPES : 4-(2-HydroxyEthyl)-1-PiperazineEthaneSulfonic acid hGH : human growth hormone

HVA/LVA :high/low voltage activated channels IP3R : inositol triphosphate receptors VDCC KATP Channel : ATP-sensitive K⁺ channel KRBH : Krebs-Ringer Bicarbonate HEPES LC-CoA : long-chain acyl-CoA

MAPK : Mitogen Activated Protein Kinase MEK : map-erk kinase

NAD/NADP: nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate NCX : Na⁺/Ca²⁺ exchangers

OMS : Organisation Mondiale de la Santé

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PACAP : pituitary adenylate cyclase-activating polypeptide PCR : Polymerase Chain Reaction

PIs : pseudo-islets

PI3K : Phosphoinositide-3 kinase PKA : protein kinase A

PKC : protein kinase C PLC : protein kinase C

PMCA : plasma membrane Ca²⁺ pumps RER : rough endoplasmic reticulum ROC : receptor-operated channels RNAi : RNA interference RRP : readily releasable pool RTK : receptor tyrosine kinase RyR : ryanodine receptors

SDS-PAGE : sodium dodecyl sulfate polyacrylamide gel electrophoresis SERCA : Sarco(Endo)plasmic Reticulum Ca²⁺ -ATPase

ShRNA : small hairpin RNA SiRNA : small interfering RNA SM proteins : Sec1/Munc18 proteins

SNARE : soluble N-ethyl maleimide sensitive factor attachment protein receptor SOC : store-operated channels

TCA : tricarboxylic acid cycle TetR :tetracycline receptor TGN : trans region of the Golgi T1D : type 1 diabetes mellitus T2D : type 2 diabetes mellitus

VDCC : voltage-dependant calcium channel WHO : World Health Organization

[Ca²⁺]c : cytosolic Ca²⁺ concentration [Ca²⁺]ER : Ca²⁺ concentration in the ER

[Ca²⁺]m : Ca²⁺ concentration of the mitochondrial matrix

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RESUME EN FRANCAIS

Le Diabète est une maladie chronique caractérisée par un taux de glycémie élevé à jeun et postprandial. Si le diabète n’est pas diagnostiqué ou s’il n’est pas correctement traité, il peut mener à des complications sévères, allant jusqu’au décès. Le diabète se manifeste lorsque l’insuline, qui est la principale hormone responsable de réguler la glycémie, n’est plus produite en quantité suffisante ou lorsque les tissus-cibles (en particulier les muscles) se désensibilisent et ne réagissent plus correctement malgré la présence de l’hormone. Dans ce cas on parle de résistance à l’insuline. Il existe différents types de diabète, mais le diabète le plus répandu est le diabète de type 2, représentant 90% de tous les diabètes. Il est typiquement caractérisé par une résistance à l’insuline ainsi que par une diminution de l’insuline sécrétée, due à une diminution du nombre des cellules β pancréatiques (cellules productrices d’insuline) ainsi que par un dysfonctionnement de ces dernières. Au vu du nombre croissant de personnes atteintes par cette pathologie (l’OMS prévoit une prévalence de 6% de la population mondiale d’ici à 2025), il est capital de comprendre les différents mécanismes qui mènent à ces troubles.

Les cellules β pancréatiques, qui sont les seules cellules du corps à produire et à sécréter de l’insuline, font partie de micro-organes nommés îlots de Langerhans. Ces îlots sont formés de 4 types de cellules majoritaires (les cellules α, β, δ and PP) qui produisent chacune des hormones distinctes impliquées dans le métabolisme. Ces îlots constituent la partie endocrine du pancréas et sont disséminés dans le tissu exocrine du pancréas qui est largement majoritaire (>98% du pancréas total). A l’intérieur de ces îlots, les cellules β sont en contact direct avec d’autres cellules β, d’autres cellules endocrines et la matrice extracellulaire (déposée par les cellules endothéliales des vaisseaux traversant l’îlot). A travers ces contacts physiques, ces différents éléments peuvent communiquer et s’influencer mutuellement. De plus, les îlots sont richement

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irrigués et innervés, permettant l’arrivée de molécules comme les nutriments ou les neurotransmetteurs qui peuvent agir sur la cellule β. Toutes ces interactions confèrent un environnement adéquat à la cellule β et toute perturbation de la structure bien définie d’un îlot peut mener à un dysfonctionnement majeur. A l’heure actuelle, il est largement accepté que l’intégrité de cet environnement est essentielle pour la survie et le bon fonctionnement des cellules β mais les mécanismes moléculaires reliant ces différentes interactions et la sécrétion d’insuline restent peu connus. Puisque le diabète de type 2 se caractérise par une diminution de la sécrétion d’insuline et par une désorganisation de la structure des îlots, il semblait particulièrement intéressant d’étudier plus en détail le lien entre ces interactions directes ou indirectes entre les différents composants de l’îlot et la sécrétion d’insuline. Pour ce projet, nous avons décidé de nous focaliser sur les contacts homologues entre les cellules β. Le but du projet était de mieux caractériser comment les contacts cellule-β-cellule-β mènent à une meilleure régulation de la sécrétion d’insuline.

Dans la première partie du projet, nous avons commencé par mettre au point un modèle pour étudier les contacts entre cellules : les cellules étaient cultivées soit de manière éparse, avec une densité très faible permettant aux cellules d’être isolées et de n’avoir pas ou très peu de contacts les unes avec les autres, soit de manière confluente, où le même nombre de cellules était réparti sur une surface beaucoup plus restreinte, obligeant les cellules à former de nombreux contacts les unes avec les autres. Ceci nous a permis de mettre en évidence deux problèmes majeurs chez les cellules éparses : une forte élévation de la sécrétion basale d’insuline et une diminution de l’insuline sécrétée suite à une stimulation au glucose comparé aux cellules confluentes. Ces deux défauts couplés avaient pour résultat une stimulation quasiment nulle en réponse au glucose chez les cellules éparses. Grâce à de nombreuses techniques, nous avons pu établir que ces deux problèmes avaient des origines différentes et

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que la faiblesse de la réponse au glucose était due, du moins en partie, au désengagement de la molécule d’adhésion E-cadhérine, qui permet de relier deux cellules adjacentes et de transmettre différents signaux à l’intérieur du cytoplasme. Nous avons également montré que la sécrétion basale élevée des cellules isolées était due à une activité calcique spontanée de ces cellules qui menait à l’exocytose des granules d’insuline même en absence de stimulation. A l’heure actuelle, nous n’avons toujours pas pu identifier le ou les canaux calciques responsables de cette entrée de calcium mais nous avons établi que ce calcium venait de l’extérieur de la cellule et non pas des stocks calciques intracellulaires. Nous avons également démontré que ces pics de calciums spontanés finissaient par disparaître avec le temps lorsque les cellules étaient maintenues en bas glucose. De manière très intéressante, nous avons constaté que lorsque cette activité calcique spontanée avait suffisamment chuté (de manière naturelle suite à une incubation prolongée en bas glucose ou de manière provoquée par suppression du calcium extracellulaire), la sécrétion basale d’insuline diminuait significativement et permettait aux cellules isolées de répondre au glucose de manière tout à fait satisfaisante. Cela nous montre que les cellules éparses ne perdent pas la capacité de répondre au glucose mais que cette réponse est tout simplement masquée par leur activité basale excessive. Ces résultats ont été publiés dans le journal « Endocrinology » (Endocrinology 149(5):2494-2505 (2008)).

Dans la suite du projet, nous avons voulu mieux définir comment la liaison des E-cadhérines se trouvant sur des cellules adjacentes pouvait mener à une amélioration de la sécrétion d’insuline, puisque l’E-cadhérine est connue pour activer de nombreuses voies de signalisation dans d’autres types cellulaires. Malheureusement, malgré un nombre élevé de tentatives détaillées dans la partie « Résultats », nous n’avons pas réussi à mettre au point un système suffisamment robuste et reproductible pour étudier en détail les voies de signalisation activées par l’engagement des E-cadhérines chez les cellules β.

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En conclusion, nous avons démontré que les contacts entre cellules β sont très importants pour maintenir une sécrétion basale d’insuline à un niveau adéquat. Ceci est d’importance majeure puisque chez les cellules isolées, du moment que nous rétablissons la sécrétion basale les cellules se retrouvent capable de répondre à une stimulation au glucose de manière totalement convenable. Tout en gardant en tête que notre modèle est très éloigné de la situation réelle, nous pensons que ces résultats peuvent être utiles pour la compréhension du dysfonctionnement de la sécrétion d’insuline observée chez les patients diabétiques de type 2.

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ENGLISH SUMMARY

Diabetes is a chronic disease characterized by abnormally high blood sugar levels (hyperglycemia). If diabetes is not diagnosed or if not properly treated it can lead to severe complications, with increased mortality. Diabetes occurs when there is absolute insulin deficiency (type 1) or relative insulin deficiency in the face of insulin resistance (type 2). Type 2 diabetes represents >90% of all diabetes and is typically characterized by insulin resistance and a deficient insulin secretion due to a decrease in both the number and function of pancreatic β- cells (that produce insulin). Given the increasing number of people affected by this pathology (WHO estimates a prevalence of 6% of the world population by 2025), it is crucial to understand the mechanisms leading to these disorders.

Pancreatic β-cells, which are the only cells in the body to produce and secrete insulin, are part of micro-organs called the islets of Langerhans. These islets are made up of 4 main types of cells (α-, β-, δ- and PP cells) which each produce different hormones involved in metabolism. Islets, the so-called endocrine pancreas are scattered throughout the exocrine pancreas and compose just 1-2% of the total tissue. Within islets, β-cells are in direct contact with other β-cells, other endocrine cells and/or extracellular matrix. These various elements of the physical environment of the islet are interactive and, moreover, islets are richly irrigated and innervated, allowing entry of molecules such as nutrients and neurotransmitters that may also influence β-cell function and survival. All these interactions provide a suitable environment to the β-cell and any disruption of this well defined structure can lead to a major dysfunction. At the moment, it is widely accepted that the integrity of this environment is essential for the survival and proper functioning of the β- cells but the molecular mechanisms linking these different interactions and insulin secretion remain unknown. Since type 2 diabetes is characterized by a decrease in insulin secretion and a

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disruption of islet structure, it seemed particularly interesting to explore further the link between these direct or indirect interactions between different islet components and insulin secretion. For this project, we decided to focus on homologous contacts between β-cells. The aim of this project was to better characterize how β-cell to cell-β contacts lead to improved regulation of insulin secretion.

In the first part of the project, we developed a model to study the contacts between cells: the cells were grown either in a dispersed manner, with a very low density allowing cells to be isolated with no or very few contacts between each other or in a confluent manner, where the same number of cells was plated over much smaller area, forcing the cells to form contacts between each other. This has allowed us to highlight two major problems in dispersed cells: a strong increase in basal insulin secretion and reduced secretion in response to glucose stimulation as compared to confluent cells. This results in nearly no stimulation in response to glucose in dispersed cells. Using several techniques, we showed that these problems had different origins and that the weakness of the response to glucose was due, at least partially, to the disengagement of the adhesion molecule E-cadherin, which allows two connecting cells to communicate together. We also showed that elevated basal secretion of dispersed cells was due to a spontaneous calcium activity of these cells leading to exocytosis of insulin granules even in the absence of stimulation. At the moment, we still have not been able to identify the calcium channels responsible for the entry of calcium but we have shown that the calcium came from outside the cell and not from intracellular calcium stocks. We have also demonstrated that these spontaneous calcium peaks ultimately disappear with time when cells were kept in low glucose for a prolonged period (6h). Very interestingly, we found that when this spontaneous calcium activity had dropped sufficiently (after a prolonged incubation in low glucose or after removal of extracellular calcium), the basal insulin release decreased significantly and allowed

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not lose the ability to respond to glucose but that the glucose response is simply masked by their excessive basal secretion. These results are published in the journal "Endocrinology"

(Endocrinology 149 (5) :2494-2505 (2008)).

In the second part of the project, we attempted to better define how the engagement of E- cadherin located in adjacent cells could lead to an improvement in insulin secretion, as E- cadherin is known to activate many signaling pathways in other cell types. Unfortunately, despite many attempts, we failed to develop a system sufficiently robust and reproducible to study in detail signaling pathways that are activated by the engagement of E-cadherin in β-cells.

In conclusion, we show that contacts between β-cells are very important to maintain basal insulin secretion at an appropriately low level. This was shown to be the result of low spontaneous calcium activity in cells with well established intercellular contacts. This is of major importance since once basal secretion has been restored, dispersed cells are able to respond to glucose stimulation in a suitable manner. We believe that these results may be important for understanding disturbed regulation of glucose-stimulated insulin secretion in type 2 diabetes.

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GENERAL INTRODUCTION

Pancreatic beta cells (β-cells) are particularly important because they are central in the regulation of blood glucose level (glycemia). They produce and secrete insulin in response to glucose as well as other metabolic and hormonal secretagogues. Insulin regulates glycemia by promoting glucose uptake in muscle and adipocytes through activation of a complex cascade of signalling events (1; 2) and suppressing hepatic glucose production while favouring glycogen deposition (3). These various biological endpoints of insulin action are reached via different signaling cascades. When the liver is saturated with glycogen, any additional glucose taken up by hepatocytes is shunted into pathways leading to synthesis of fatty acids, which are exported from the liver as lipoproteins and provide free fatty acids for use in other tissues, including adipocytes, which use them to synthesize triglycerides. Insulin is also an anti-lipolytic hormone thereby favouring fat deposition in time of abundance (4-6).

When β-cells do not produce enough insulin or when the body cannot efficiently respond to the insulin it produces, blood glucose levels rise leading in some instances to the metabolic disorder diabetes mellitus. This chronic metabolic disease can result in severe complications such as blindness, renal failure and nerve damages and can even lead to early death due to macrovascular complications (7-10). Leaving aside the various (rare) monogenic forms, the three main forms of diabetes mellitus are type 1 (T1D), type 2 (T2D), and gestational diabetes (GD). These three forms have different causes and population distributions. T1D is characterized by massive T-cell mediated autoimmune destruction of β-cells, leading to an absolute deficiency of insulin (11). In contrast, T2D is first characterized by insulin resistance (or reduced insulin sensitivity) even if it is inevitably combined with reduced insulin secretion resulting in relative insulin insufficiency (12; 13). Obesity or overweight are found in 80% of

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patients diagnosed with T2D. Aside from central obesity (= abdominal fat), aging and genetic heredity are known to predispose individuals for insulin resistance (14). Finally, GD occurs during pregnancies (2-5%) and is similar to T2D in several aspects. Even if it typically disappears after delivery, about 20-50% of affected women will develop T2D later in life. GD can be deleterious for the health of the baby or mother and thus needs to be correctly treated.

Diabetes is a major Public Health issue since it is assumed that in the year 2025 at least 6% of the world population will suffer from diabetes. This explains why the β-cell and its secretory product insulin are gaining increasing attention both in public and industrial research.

Today there is no cure for Diabetes. Individuals with T1D must inject insulin several times each day in order to survive. Those with T2D could in many cases treat their condition by altering their lifestyle but lack of compliance typically results in the use of hypoglycemic oral agents (sulphonylureas, metformin, glitazones and the more recent DPP-4 inhibitors), while insulin therapy for T2D is becoming an increasingly favored last line of therapy. Although the results of two major studies (UKPDS for T2D and DCCT for T1D) indicate clearly that hyperglycemia is responsible for most diabetic complications, even modern therapy and patient care cannot normalize this effectively in the long term.

Although changes in β-cell mass underlie both T1D and T2D, driven by altered balance between proliferation and apoptosis, this will not be the major focus of this thesis.

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1. Pancreatic ββββ-Cells

β-cells are located in the islets of Langerhans, which constitute the endocrine part of the pancreas. These islets represent only 1 % of the total gland and are embedded within the exocrine tissue. There are four main cell types in the islets that are classified by their secretion:

α-cells secrete glucagon, β-cells secrete insulin, δ-cells secrete somatostatin and PP cells secrete pancreatic polypeptide. Glucagon and insulin are both involved in carbohydrate metabolism while somatostatin and pancreatic polypeptide are more involved in endocrine and/or exocrine system regulation. The proportion of α-, β-, δ- and PP cells in the islets represents 20-25, 65-80, 2-5 and 1-2% respectively. Islets are richly vascularized and contain 500-5000 cells depending on their size. Rodent islets, unlike the human ones, show the characteristic β-cells core with other cells in the islet periphery (15; 16)(Fig 1).

Figure 1: Typical architecture of rodent islets

In rodent islets, the vastly predominating β-cells are clustered in the core of a generally round islet, surrounded by a mantle of non β-cells (α-, δ- and PP-cells). Islets are richly vascularized and innervated.

In vivo, β-cells are in close contact with other β- and non β-cells and with extracellular matrix (ECM), mainly deposited by endothelial cells. Adapted from (17).

Blood vessel

Nerve

ECM

β-cells Non

β-cells

Blood vessel

Nerve

ECM

β-cells Non

β-cells

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To maintain normoglycemia, it is crucial for the β cells to accurately sense blood glucose elevation and respond quickly by releasing stored insulin while simultaneously producing more.

Glucose sensing, insulin biosynthesis as well as insulin secretion are finely tuned mechanisms and will thus be further described in the next sections.

1.1 Glucose Sensing Mechanisms: From Glucose Sensing to Insulin Release

Pancreatic β-cells act as glucose sensors and this glucose sensing mechanism is essential for maintenance of glucose homeostasis. Insulin secretion is directly proportional to glycemia thanks to the high capacity, low affinity glucose transporter-2 (GLUT-2), which allows glucose to be rapidly equilibrated across the β-cell membrane (18-20). After glucose has entered the β-cell, it is phosphorylated to glucose-6-phosphate by the high KM glucokinase (GK), which is considered as the glucose sensor in the pancreatic β-cell (21). After its phosphorylation, glucose is metabolized by glycolysis to produce pyruvate, NADH and ATP. The resultant increase in ATP: ADP ratio causes the closure of the ATP-sensitive K⁺ channels (KATP) and subsequent depolarization of the plasma membrane (22; 23). This depolarization then leads to opening of the voltage-gated Ca²⁺ channels (24). Finally, the resultant elevation in cytosolic Ca²⁺ triggers insulin exocytosis as described later (Fig 2) (25; 26).

1.1.1 Triggering and Amplifying Pathways

This cascade of events, from glucose sensing to Ca²⁺-elevation and subsequent exocytosis, is called the triggering pathway or KATP channel-dependant pathway (27). In fact, besides its role in this pathway, glucose also produces signals that potentiate the action of Ca²⁺ on the exocytotic

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process. This is the amplifying pathway. This pathway does not increase [Ca²⁺]i but needs elevated [Ca²⁺]i to be effective. In contrast with the triggering pathway, the amplifying pathway does not need a threshold glucose concentration to be activated and is directly proportional to glucose level, even at low glucose (27). The fact that the amplifying pathway remains silent as long as [Ca²⁺]i has not been raised by the first pathway ensures that insulin is not inappropriately secreted in the presence of low glucose concentrations. There is thus a clear hierarchy between the two pathways: at first the triggering pathway determines if insulin is secreted or not (through Ca²⁺-elevation) and then the amplifying pathway serves to optimize the secretory response induced by the triggering signal (28)(Fig 2).

Figure 2: Pathways leading to insulin secretion

Glucose, the main stimulus for insulin secretion, typically activates both the triggering and the amplifying pathway. In both pathways, metabolism of glucose is needed. In the triggering pathways, glucose uptake via GLUT2 is followed by generation of pyruvate, whose oxidation by mitochondria leads to increases in intracellular ATP-to-ADP ratio and closure of K_ATP-channels. This is followed by cell depolarization, opening of VDCC, Ca²⁺ influx and subsequent exocytosis. In the amplifying pathway, cellular messenger and effectors are still not precisely known, but NADPH could represent a major key player.

Beside glucose, many other nutrient or non-nutrient secretagogues can stimulate insulin secretion, mainly GLUT 2

Glucose

X

VDCC K_ATP

TCA cycle ATP

FFA

G

G AC PLC

(Ca²⁺stock)

PKC PKA

G GPR40

Amino acids

FFA

DAG IP₃

cAMP

Ca

²⁺ ^G

Triggering

Amplifying

pathway

Exocytosis

Ach, CCK

PACAP, GLP-1 Glucagon, GIP

NADPH?

ER pathway GLUT 2

Glucose

X

VDCC K_ATP

TCA cycle ATP

FFA

G

G AC AC PLC

PLC

(Ca²⁺stock)

PKC PKC PKA PKA

G GPR40

Amino acids

FFA

DAG IP₃

cAMP

Ca

²⁺ ^G

Triggering

Amplifying

pathway

Exocytosis

Ach, CCK

PACAP, GLP-1 Glucagon, GIP

NADPH?

ER pathway

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The amplifying pathway requires glucose metabolism but currently, intracellular messenger or effectors of this pathway are still unknown, in contrast with the triggering pathway where the key players are well identified (see 1.1 and Fig 2). However, recent studies point out the possible role for byproducts of the pyruvate/isocitrate cycle, in particular NADPH and α-ketoglutarate, in potentiating the glucose-induced Ca²⁺-mediated signaling pathway (29-31), while the role of glutamate (32) has been challenged (30).

1.1.2 Different Secretagogues

Although glucose provides the primary stimulus for insulin secretion, several agents can increase or inhibit insulin secretion in vitro and in vivo (33). The cell will then integrate all these ambient signals into an appropriate insulin secretory rate in order to maintain normal glucose homeostasis (34-38).

These secretagogues can be categorized as initiator or potentiator. Initiator agents are able to produce a sufficient increase in [Ca²⁺]i leading to exocytosis, while potentiator agents have no effect on insulin secretion when used alone but can increase glucose-induced insulin secretion (28). Only nutrient secretagogues can act as initiators (39).

1.1.2.1 Nutrient Secretagogues

Nutrients (or fuel secretagogues) include many sugars and amino acids as well as free fatty acids (FFA). Amino acids like L-glutamine, L-alanine or L-leucine individually are poor secretagogues, but there is evidence that they activate both triggering and amplification processes (40). Like sugars, they are metabolized to acetyl-Co-A, which enters the TCA cycle

(21)

and results in an increase of ATP production with subsequent Ca²⁺-elevation and exocytosis (41;

42) (Fig 2). Arginine, which can also initiate insulin secretion is not strictly speaking a nutrient secretagogue since it acts by another mechanism, presumably by cell membrane depolarization leading to extracellular calcium entry (43). In contrast to amino acids, FFAs do not stimulate insulin secretion in the absence of glucose but they can significantly increase glucose-induced insulin secretion (GSIS) (44). The mechanism by which FFAs act on insulin secretion is still not fully understood but many hypotheses have been proposed. In β-cells, FFAs are converted to long-chain acyl-CoAs (LC-CoAs). One recent hypothesis is that LC-CoAs act by activating G protein-coupled receptors such as GPR40 (45; 46), leading to [(Ca²⁺)i] elevation through activation of IP3R and VDCC (47). A second hypothesis is that Lc-CoAs directly stimulate insulin secretion through the formation of complex lipids such as DAG leading to PKC activation (37;

48; 49) (Fig 2).

1.1.2.2 Non-Nutrient Secretagogues

Although β-cells are specialized in fuel sensing, they are also subject to a major neurohormonal control, such as intestinal hormones (incretins) and neurotransmitters (acetylcholine) (35; 50- 52). In contrast with most of the fuel secretagogues, non-nutrients act only as potentiators of secretion and are recognized by β-cells through conventional receptors, rather than by their metabolism. Most of them act through a specific G protein-coupled receptor and activation of protein kinases. For example acetylcholine (Ach) and cholecystokinin (CCK) activate receptors coupled to phospholipid-dependent protein kinase C (PLC). This leads to generation of IP3 and DAG, favoring Ca²⁺-release from intracellular store (by IP3) and protein kinase C (PKC) activation (by DAG) (35). Other secretagogues such as glucagon, glucagon-like peptides (GLP), glucose-dependent insulinotropic polypeptide (GIP) and pituitary adenylate cyclase-activating

(22)

and increase intracellular cyclic AMP (cAMP) levels, which in turn activates protein kinase A (PKA) (51; 53; 54). Then, PKC as well as PKA signal pathways potentiate signals generated by fuel secretagogues to induce full biphasic insulin response (see 1.6) (55; 56)(Fig 2).

1.2 Insulin Synthesis and Processing

1.2.1 Insulin Biosynthetic Pathway

Insulin is initially synthesized as a single polypeptide-chain precursor molecule called preproinsulin. This molecule contains an N-terminal 24-residue extension signal peptide that serves to facilitate the translocation of the newly forming preproinsulin from the cytosol to the rough endoplasmic reticulum (RER) lumen. Shortly after its translocation, the signal peptide is cleaved by a peptidase, giving rise to the proinsulin molecule (57). Proinsulin then forms intramolecular disulphide bonds to gain its adequate tertiary structure. At this stage, proinsulin is transported from the RER to the cis region of the Golgi apparatus, and then from one cisterna to the next until it reaches the trans region of the Golgi (TGN) (Fig 3, left panel). Proinsulin is concentrated in the TGN, where it combines with Zn²⁺ to form hexameric multimers within a newly forming β-granule (58; 59). It is in this immature granule (also called progranule) that proinsulin conversion to insulin will take place, thanks to different proteins that are packaged into β-granules with proinsulin. In particular two endopeptidases (PC 1/3 and PC 2) and one carboxypeptidase (CP H/E) are required for processing of proinsulin in insulin within the β- granules (60; 61). Correct cleavage of proinsulin gives rise to mature insulin with its A- and B- chains properly aligned as well as C-peptide (Fig 3, right panel). Mature insulin and C-peptide are stored in the secretion granules with only small amounts of residual proinsulin. Once insulin is liberated from proinsulin, it crystallizes with Zn²⁺ that is concentrated in β-granules. The C-

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peptide remains in solution, while the mature insulin forms a large dense core in the granule (62). This large dense core, which consists of hexameric units of crystalline zinc insulin, is estimated to contain up to 250’000 insulin molecules per granules (63).

Figure 3: Insulin biosynthesis and processing

Left panel: insulin biosynthesis from nucleus to secretion. Insulin is originally synthesized as a precursor and need to be processed to become a mature protein. In β-cells, more than 99% of the proinsulin is targeted to the regulated pathway and is converted to mature insulin. Adapted from (64). Right panel:

NUCLEUS

Insulin gene

Insulin mRNA

RER

Preproinsulin synthesis

GOLGI

cis trans

Proinsulin synthesis

PLASMA MEMBRANE

Constitutive pathway

Regulated pathway

Immature

granule Mature granule containing

insulin Membrane recycling

proinsulin

insulin

40 min 10-15 min 30-40 min 2-4h 1-5d

40 min

> 99%

< 1%

NUCLEUS

RER

GOLGI

cis trans

Immature

proinsulin

insulin

40 min 10-15 min 30-40 min 2-4h 1-5d

40 min

> 99%

< 1%

NUCLEUS

RER

GOLGI

cis trans

Immature

proinsulin

insulin

40 min 10-15 min 30-40 min 2-4h 1-5d

40 min

NUCLEUS

RER

GOLGI

cis trans

Immature

proinsulin

insulin

40 min 10-15 min 30-40 min 2-4h 1-5d

40 min

> 99%

< 1%

(24)

1.2.2 Regulation of Insulin Synthesis

To ensure that β-granule numbers are continually maintained at an optimal level in order to retain its capacity to secrete, the β-cell coordinates a rapid and specific stimulation of proinsulin biosynthesis for the duration of insulin secretion. Glucose, and in general most nutrients that stimulate insulin secretion, act at various levels on the biosynthetic pathway (63): they increase transcription and translocation of the preproinsulin mRNA to the RER (66-70) and they decrease mRNA degradation (71). Beside their role at the mRNA level, nutrients also regulate insulin synthesis at the translational level: glucose has been shown to increase proinsulin biosynthesis to an impressive ≥ 10fold within 1h. Furthermore, biosynthesis of the two proinsulin processing endopeptidases PC1/3 and PC2 is also increased following stimulation by nutrients, allowing a coordinately increased in insulin processing (72; 73).

All these regulations do not occur at the same moment: for period ≤ 4h, glucose-induced insulin biosynthesis is totally mediated at the translational level. For longer period, ≥ 12h, this translational level is supplemented by increased insulin gene transcription. In the even longer term, ≥ 24h, insulin biosynthesis is further amplified by increasing mRNA stability (63).

Furthermore, a group has revealed recently a direct link between exocytosis and control of insulin gene expression through ICA512, an intrinsic granule membrane protein. Following granule exocytosis, the cytoplasmic domain of ICA512 is cleaved. The cleaved fragment is then targeted to the nucleus, where it activates insulin gene expression, allowing a direct coordination between granule production and consumption (74; 75).

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1.3 Exocytotic Machinery

Following production at the TGN and subsequent maturation, each β-granule needs to be transported to the cell surface. This is facilitated by interaction first with microtubules and then the microfilamentous network of the cytoskeleton. β-granule translocation along the microtubules is dependant on the motor protein kinesin-1, while their movement along the cortical network of microfilaments is driven by the motor protein myosin Va (76-80). Interestingly, the actin cytoskeleton seems to play a dual role in granule secretion: beside its role in granule transport, it appears also to act as a physical barrier to avoid granule release (81). Actually, the recruitment of granules would require the physical disintegration, or at least rearrangement, of this barrier (82). It is evident that both mechanisms must be spatially and temporally distinct. This could reflect a role for the cortical actin network in both arrival and eventually docking of vesicles at the plasma membrane.

Once β-granules have reached the plasma membrane area, some steps are still required before the final release of the vesicle content into the extracellular space. These steps include vesicle docking at the plasma membrane, vesicle priming (=granule preparation for fusion) and ultimately fusion between that vesicle and the plasma membrane. All these successive steps are very similar in all secretory cells and involve a complex interplay between numerous proteins such as Rabs, Rab effectors, Sec1/Munc18 (SM) proteins, synaptotagmin and SNAREs (83).

SNARE (soluble N-ethyl maleimide sensitive factor attachment protein receptor) proteins operate at the very last step of this sequence and represent the minimal machinery for membrane fusion: each membrane destined to fuse contains at least one SNARE with a membrane anchor (84-87). They include syntaxin 1A and SNAP-25 at the plasma membrane

(26)

opposed membranes form a complex in “trans” that progressively assembles from the N-terminal tips toward the C-terminal membrane anchors, thus clamping the two membranes together. In the “trans” complex, all SNARE motifs adopt an alpha-helical structure and are aligned in parallel, forming a twisted coiled-coil (88; 89). As formation of the SNARE complex progresses, the opposed membranes are pulled tightly together imposing the fusion reaction. During fusion, the complex reorients from “trans” to “cis” (84). Energy released during assembly is certainly used for surmounting the fusion barrier.

According to current concepts, specificity in membrane traffic is completed by successive layers of regulation operating upstream of SNARE assembly. It involves members of conserved protein families implicated in docking and priming. More and more evidence designates Rab proteins and Sec1/Munc18 (SM) proteins as master regulators of membrane docking and priming (90- 92). Vesicle docking represents an initial reversible interaction of β-granules with the plasma membrane and constitutes a pool of unprimed granules in close proximity to the exocytotic sites.

Rabs are thought to orchestrate this initial contact between membranes fated to fuse and to assure that only appropriate organelles are tethered. More precisely, Rab proteins, localized on vesicle membranes, are generally believed to promote vesicle docking through interaction with a specific effector molecule. In β-cells, Rab 27a forms an endogenous complex with granuphilin, its effector (93). Granuphilin directly binds to the H3 domain of syntaxin 1A, allowing tethering of insulin granules to the plasma membrane by an interaction with both Rab 27a and syntaxin 1A (94; 95). The current hypothesis is that Munc-18-1, by interacting with syntaxin 1A and granuphilin, stabilizes syntaxin 1A in a “closed” conformation, thereby preventing syntaxin from binding to its partner SNAP-25 and VAMP-2. This fusion incompetent state could represent a temporal brake for exocytosis to prevent incoming vesicles from being constitutively fused (Fig 4) (96), allowing syntaxin 1a to bind to its partner SNAP-25 and VAMP-2 to form the SNARE- complex.

(27)

After docking, β-granules have to be primed to become competent for fusion in response to a Ca²⁺ trigger. Indeed, SM proteins are involved in preparing and proofreading SNARE proteins for trans-complex formation, a mechanism known as vesicle priming. In β-cells, Munc 13-1 is thought to mediate vesicle priming by activating syntaxin and promoting SNARE complex formation: it would interact with syntaxin 1A to unfold the “closed” conformation to an “open”

conformation (Fig 4) (97).

Figure 4: Exocytosis

Before fusion, granules need to be transported near to the plasma membrane, docked and primed. The docking process implicates proteins such as granuphilin and Munc 18-1 and leads to the stabilization of a complex including syntaxin 1A, in a “fusion incompetent” state, which prevent binding of syntaxin 1a with SNAP 25 and VAMP 2. Participation of the Munc 13-1 for the priming step allow the preparation of the granule to become fusion-competent and allowing the SNARE-pairing (association of the three SNAREs:

+

Munc 18-1

Granuphillin SNAP 25

Munc 13-1 Syntaxin 1A

VAMP 2 -

Docking Priming

+

Munc 18-1

VAMP 2 Munc 18-1

VAMP 2 -

ATP Granule transport

Granule fusion Granule priming

Granule docking

Actin microfilament

+

Munc 18-1

VAMP 2 -

+

Munc 18-1

VAMP 2 Munc 18-1

VAMP 2 -

Granule docking

+

Munc 18-1

VAMP 2 -

+

Munc 18-1

VAMP 2 Munc 18-1

VAMP 2 -

Granule docking ATP Granule transport

Granule docking

Actin microfilament

(28)

Another essential protein implicated in vesicle exocytosis is synaptotagmin, a 65 KD protein, localized to the β-granules via a transmembrane domain. Exocytosis occurs in response to an elevation of cytosolic calcium and synaptotagmin represents the best characterized exocytotic Ca²⁺-sensor in β-cells. There are 16 isoforms of synaptotagmin and several of them have been detected in β-cells but with many variations between primary and clonal β-cells lines. It seems however that synaptotagmin 7 and 9 are both expressed in insulin-producing cell lines and primary β-cells and that they play a crucial role in GSIS (98). An hypothesis would be that synaptotagmin 9 constitutes a low-affinity Ca²⁺-sensor, localized on and responsible for the release of low Ca²⁺-sensitive pool of granules and synaptotagmin 7 constitutes a high-affinity Ca²⁺-sensors, localized on and responsible for the release of high Ca²⁺-sensitive pool of granules (99) (Ca²⁺-sensitive pools are further mentioned in 3.2).

1.4 Vesicle Recycling

Recently, a controversy about the fusion mechanism has emerged. It has been known for a long time that exocytosis is coupled to endocytosis in β-cells (100), however it is not clear yet if exocytosis proceeds by full fusion of vesicle, followed by classical endocytosis, or by kiss-and- run fusion, where vesicle integrity is maintained (101). In the first situation, exocytosis occurs via the initial opening of a fusion pore, which expands to incorporate the vesicle membrane to the plasma membrane (Fig 5a). To maintain cell size, this is followed by conventional endocytosis, where clathrin and other adaptor proteins initiate an inward curvature of the plasma membrane and dynamin-2 is responsible for the separation of the newly formed vesicle from the plasma membrane (102). Vesicles can then be recycled to the TGN. In β-cells, this classical endocytotic pathway is regulated by Ca²⁺ and glucose, allowing the maintenance of membrane homeostasis even in case of sustained exocytosis (103). However, it has been proposed that not all granules

(29)

follow this conventional exocytosis/endocytosis pathway but rather fuse to the plasma membrane by a kiss-and-run mechanism. This means that vesicles fuse with the plasma membrane only transiently and are reinternalized intact (Fig 5b). In this pathway, exocytosis occurs through the opening and closure of a very small fusion pore. Dynamin-1 seems to be responsible for the restriction of the pore extension to avoid the full fusion of the granule with the plasma membrane (104). The exact size of the fusion pore is still a matter of debate but if we assume that the insulin crystal is able to partially dissolve when the fusion pore forms, then the release of monomeric/dimeric insulin during kiss-and-run is totally imaginable (105). Another controversy remains about the recycling of such vesicles. In other cell types, after kiss-and-run exocytosis, vesicles are refilled directly in the cytoplasm. In the β-cell context, it is less clear since these must return to the TGN to be refilled with peptide cargo. However, it has been suggested that insulin granules could stack and communicate with each other. This may allow transmission of granule content through transient membrane pores from one granule to the next (106). This would form a chain from TGN to the plasma membrane, allowing the replenishment of empty vesicles. For the moment, kiss-and-run as a mechanism for β-granules exocytosis and recycling and, the real contribution of this pathway to total exocytosis, is still under debate and needs to be further studied.

Figure 5: Vesicle recycling

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endocytosis.

B: Kiss-and-run mechanism, with transient opening of the fusion pore and conservation of the vesicle membrane integrity. Adapted from (101).

1.5 Constitutive vs. Regulated Secretory Pathways

In general, two classical secretory pathways are described in eukaryotic cells: one pattern is called constitutive secretion and the other regulated secretion. In the first ubiquitous pathway, proteins are introduced into small secretory vesicles and continuously secreted from the cell regardless of environmental factors. Indeed, no external signals are needed to initiate this process. The secretion is thus closely linked to the level of protein biosynthesis and occurs rapidly after synthesis of the protein. In regulated secretion, a process unique to certain cell types including β-cells, proteins are packaged into secretory vesicles distinct from small constitutive granules and remain in an intracellular storage pool until a proper stimulus activates the exocytotic process. In this more specialized pathway, proteins are only secreted in response to a specific signal, allowing for precise and rapid secretion in response to secretagogues (107).

In pancreatic β-cells, both pathways are known to exist and the TGN seems to represent the most distal compartment common to both pathways (Fig 2). Currently, it is still not very clear how proinsulin is targeted to one or the other pathway (108). However, in β-cells, this sorting event is very efficient since more than 99% of proinsulin is targeted to the regulated secretory pathway (109). It is well accepted that proinsulin conversion to insulin occurs in β-granules (58).

However, it has been shown that conversion of proinsulin to insulin can also occur in the constitutive pathway (110). Nevertheless, proinsulin conversion in the constitutive pathway is clearly less efficient as compared to the regulated pathway and the major constituents of constitutive secretion are proinsulin and/or conversion intermediate rather than mature insulin

(31)

(111). This is in contrast with regulated secretion where prohormone conversion is highly efficient and where granules contain only 1 to 2 percent of intact proinsulin (62)(Fig 2). Recently, other secretory pathways have been described, including the constitutive-like secretory pathway.

However, the reel contribution of this pathway to the β-cell function remains controversial and will not be discussed in detail here (for details (65)).

1.6 Biphasic Insulin Secretion

Glucose-stimulated insulin secretion in many mammalian species including man typically follows a biphasic time course (112; 113). The first phase occurs shortly after the glucose load and typically lasts 4-10 min. It is followed by a slower, sustained second secretion phase (99) (Fig 6a). The first phase is thought to represent the rapid release of a fusion-competent pool of granules, called readily releasable pool (RRP) (114). Secretion of these granules is Ca²⁺- dependant but does not require ATP since these granules have progressed past the ATP- dependant priming step (83). In contrast, exocytosis of granules in the slower second phase is dependant upon both Ca²⁺ and ATP since granules have to be recruited into the RRP from the reserve pool and require maturation, via priming, to become fusion-competent (115). The rate of second-phase insulin exocytosis is directed by the rate at which granules can be primed.

Microscopic and biochemical experiments have shown that the secretory granules exist in distinct functional pools. It has been estimated that β-cells contain 10’000-13’000 β-granules (115-117). Electron microscopy suggests that around 10% of the granules are physically docked to the plasma membrane and around 30% of the granules are located within one granule diameter from the plasma membrane. It has been established that these physically docked granules represent a releasable pool, which can be further divided in two subgroups: a morphologically docked pool, which contains ~1200 granules that are located at the plasma

(32)

membrane but need additional modifications to become fusion-competent, and a RRP, which contains 50-100 granules ready for fusioning that are released during the first phase of insulin secretion (114; 118; 119) (Fig 6b). Even if it is now well accepted that the first phase is due to exocytosis of pre-primed granules, the origin of granules secreted during the second phase is still not very clear. The second phase could be due to the replenishment of the RRP, which may depend on physical translocation of granules from the reserve pool (since blockage of motor protein kinesin abolishes the second phase (78)) or on the chemical modification of pre-docked granules from the morphologically docked pool (120). Another theory has emerged recently, proposing a new exocytotic pathway involving fusion of undocked vesicles functioning in parallel with the conventional pathway of pre-docked vesicles (90). However, the mechanism responsible for this new pathway is unknown and needs to be further studied.

Fig 6: Biphasic insulin secretion and different granule pools

a) In response to a glucose challenge, insulin secretion is characterized by a first phase that occurs promptly and by a prolonged second phase. Adapted from (120).

b) In β-cell, granules are divided in distinct granule pools, according to their localization and their state of

“readiness” for fusion (primed or non-primed). Granules from the readily releasable pool are supposed to be ready for fusion and to generate the first phase of secretion following stimulation. Adapted from (118).

a. b.

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2. The ββββ-Cell Environment

In tissues, cells reside in a highly organized three-dimensional microenvironment formed by other cells and extracellular matrix (ECM). Under normal conditions, intra-tissular communication arises through a complex network of interactions: physically, through direct cell-cell contact or through the intervening extracellular matrix, and biochemically, through soluble signaling molecules. In combination, these interactions provide the information that is necessary to maintain cellular differentiation and function. It has become increasingly evident over the last years that the extracellular environment determines important aspects of cell physiology, including in islets of Langerhans, which have a well defined cellular architecture (Fig 1).

In the context of pancreatic β-cells, numerous studies have shown that a suitable environment is essential for proper insulin secretion (121-126). It has been shown already that when β-cells are attached on an appropriate ECM they are protected against apoptosis and respond better to secretagogues than cells attached on plastic (127; 128). Regarding cell-cell interactions, it is known that E-cadherin, which is one of the most important adhesion molecules on pancreatic β- cells (129; 130), plays a major role in insulin secretion (131). It is also well recognized that Connexin 36 (Cx36), the protein that forms gap junctions in β-cells, is required for appropriate insulin secretion (132). Those physical interactions thus deserve to be further described. Finally, indirect communication (through signaling molecules) has also been shown to strongly impact on β-cells functions in many physiological and pathological situations (35-37; 133).