The role of glutamate synthesis and release mechanisms in hormone secretion of pancreatic islet cells
Le régulateur principal de la sécrétion hormonale par les cellules des îlots pancréatiques est le glucose. Cependant, plusieurs autres nutriments, hormones et neurotransmetteurs ont été suggérés comme ayant un rôle dans le contrôle de la sécrétion hormonale. Un candidat hypothétique est le neurotransmetteur excitatoire glutamate. Le fait que les îlots pancréatiques possèdent tous les composants de l'appareil de signalisation du glutamate (transporteur vésiculaire du glutamate, récepteurs du glutamate et système de capture du glutamate) a conduit à émettre l'hypothèse selon laquelle cet acide aminé fonctionne comme molécule de signalisation auto- et paracrine au sein de l'îlot. En revanche, d'autres études suggèrent que le glutamate agit comme facteur de couplage entre métabolisme et sécrétion dans les cellules pancréatiques ß. Le métabolisme mitochondrial est crucial pour lier la reconnaissance du glucose à la sécrétion de l'insuline. Les facteurs de couplage sont générés pendant l'activation du cycle des acides tricarboxyliques (TCA).
BURKHARDT, Nicole. The role of glutamate synthesis and release mechanisms in hormone secretion of pancreatic islet cells . Thèse de doctorat : Univ. Genève, 2009, no.
URN : urn:nbn:ch:unige-13332
DOI : 10.13097/archive-ouverte/unige:1333
UNIVERSITE DE GENEVE
Département de Biologie Cellulaire FACULTE DES SCIENCES Professeur Jean-Claude Martinou
Département de Physiologie Cellulaire et FACULTE DE MEDECINE
Métabolisme Professeur Claes B. Wollheim
The role of glutamate synthesis and release mechanisms in hormone secretion of pancreatic islet cells
Présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologique
Nicole Burkhardt (-Feldmann) de
Thèse N° 4057
First of all, I would like to thank Prof. Claes B. Wollheim not only for giving me the opportunity of doing my Ph.D. thesis in his laboratory and on an exciting project but also for the freedom he offered me in managing my project. I also would like to thank him for his trust and continuous support. He taught and encouraged me to never give up even when it seemed hopeless. I also would like to express my sincere appreciation for his incredible knowledge not only in the field of diabetes but also on many other topics. I was fascinated by his amazing memory and passion for science.
My special thanks go to Dr. Andreas Wiederkehr for his continuous support, for the infinite discussions we had about my projects and for sharing his scientific excitement with me. He stood by my side at critical moments of my thesis, always providing me with help and technical support.
I wish to thank Prof. Jean-Claude Martinou and Prof. Philip Newsholme for accepting to be members of my jury, the latter also for coming from far away.
I would like to thank Prof. Nicolas Demaurex and Prof. Dominique Müller for undertaking the task as godfathers of my Ph.D. thesis.
I would like to thank our collaborators Prof. Jorge Tamarit-Rodriguez and Dr. Rafael Martin del Rio for providing their technical and scientific support.
I wish to thank all past and current members not only of the laboratory of Prof. Claes B.
Wollheim but also the group of Dr. Benoît Gauthier. I have had 4.5 years of beautiful memories with great people who always offered me their help. The pleasant time I spent in the lab would not have been possible without some exceptional individuals. I wish to thank Nicole Aebischer and Asllan Gjinovci for having initiated me into the secrets of pancreatic islet preparation and pancreas surgery. Furthermore, I would like to thank Olivier Dupont for his kindness, support and technical advice. I also wish to thank Clare Kirkpatrick for her patience in answering all scientific and technical questions. I would especially like to thank Kai Hui Hu He. I am so happy to share a friendship with her. She knew how to make me laugh and forget the stress and problems of everyday life.
I also would like to thank our secretaries Catherine Hugard and Geneviève Ruckstuhl for their kindness, receptiveness and enormous work in all administrative questions.
I would like to thank my family for having believed in me and supported me throughout my studies.
Last but not least, I would like to thank my wonderful husband and three lovely kids.
Without them this thesis work would not have been possible. They helped me overcome all the worries and problems and to continue even in difficult situations. I would like to especially thank Karim for his help, his investment, his advice and continuous support but above all for his love and his patience.
This work has been supported by the Swiss National Science Foundation as well as the Merck Foundation.
TABLE OF CONTENTS
Table of contents III
Résumé (en français) VII
Chapter 1 – Introduction 1
1.1 Pancreas 1
1.2 Endocrine pancreas & islets of Langerhans 1
1.3 Regulation of hormone secretion 2
1.3.1 Neuronal regulation of hormone secretion 2
1.3.2 Hormonal regulation of islet secretion 3
1.3.3 Nutritional regulation of islet secretion 3
1.3.4 Islet microcirculation 4
1.3.5 Junctional communication 5
1.4 Diabetes mellitus 6
1.4.1 Type 1 diabetes 6
1.4.2 Type 2 diabetes 7
1.4.3 Maturity onset diabetes of the young (MODY) 7
1.5 Mitochondria 7
1.5.1 Structure & function of mitochondria 7
1.5.2 Cell respiration & mitochondrial metabolism 8
1.5.3 Anaplerosis & Cataplerosis 11
1.6 Mitochondrial metabolism and insulin secretion 12
1.6.1 Biphasic insulin secretion 12
1.6.2 KATP-dependent pathway of glucose-stimulated insulin secretion 13 1.6.3 KATP-independent pathways of insulin secretion 14
1.7 Regulation of glucagon secretion 23
1.7.1 Insulin 24
1.7.2 Zinc 24
1.7.3 GABA 25
1.7.4 Glutamate 25
1.7.5 Somatostatin 25
1.7.6 GLP-1 26
1.7.8 Autonomic regulation of glucagon secretion 27
1.7.9 α-cell stimulus secretion coupling 28
1.8 Glutamate 29
1.8.1 Glutamate system 29
1.8.2 Extracellular messenger function (autocrine and paracrine signalling) 30
1.8.3 Intracellular messenger function 31
1.8.4 Glutamate biosynthesis 32
1.8.5 Aspartate aminotransferases 37
Chapter 2 – Objectives 39
Chapter 3 – Results 40
3.1 Mechanism of glucose-mediated glutamate production in α- and β-cells: Impact on insulin but not glucagon secretion’
3.1.1 Context 40
3.1.2 Objectives 40
3.1.3 Strategy of experiments & methods 40
3.1.4 Results & Conclusion 41
3.1.5 Submitted article in Endocrinology 43
3.2 Implication of cytoplasmic aspartate aminotransferase in glutamate generation and its impact on glucose-stimulated insulin secretion (GSIS)’
3.2.1 Context 66
3.2.2 Objectives 66
3.2.3 Recombinant adenovirus construction 66
3.2.4 Repression of cAAT protein expression in INS-1E cells 75 3.2.5 Effects of recombinant adenovirus (AdshRNAcAAT3) infection on intra-
cellular metabolite production and insulin secretion 76
Chapter 4 – Discussion & Perspectives 81
Chapter 5 – Conclusion 90
Chapter 6 – Material & Methods 91
6.1 Cell Culture 91
6.2 Bacteria growth media and transformation 91
6.3 RNA interference (RNAi) 91
6.3.1 Short hairpin RNA (shRNA) cloning 92
6.3.2 INS-1E cell transient transfection 93
6.3.3 RNA isolation & reverse transcription 94
6.3.4 Quantitative real-time PCR (QT-PCR) 95
6.3.5 Construction of recombinant adenoviral DNA 96
6.3.6 Production of recombinant infectious Adenovirus 98
6.3.7 Adenovirus purification 99
6.3.8 Adenovirus titer determination 100
6.3.9 Efficiency of adenovirus 100
6.3.10 Adenoviral infection of INS-1E cells 100
6.4 Western Blot analysis 101
6.5 Hormone & metabolite assay 102
6.6 Insulin immunoassay (EIA) 103
6.7 Glucagon radioimmunoassay (RIA) 103
6.8 α-ketoglutarate assay 104
6.9 Glutamate assay 105
6.10 Rat pancreatic islet isolation 107
6.11 Islet cell isolation 107
Chapter 7 – References 109
c/mAAT Cytoplasmic or mitochondrial aspartate aminotransferase
AMPA α-amino-3-3hydroxy-5-methyl-4-isoxozole propionic acid
cAMP Cyclic AMP
DC Dicarboxylate carrier
EAAT Excitatory amino acid transporters GAD Glutamate decarboxylase
GABA γ-aminobutyric acid
GDH Glutamate dehydrogenase
GLP-1 Glucagon-like peptide 1
GSIS Glucose-stimulated insulin secretion
i/mGluR Ionotropic or metabotropic glutamate receptors
KATP-channel ATP-sensitive potassium-channel αKG α-ketoglutarate
OMM/IMM Outer/inner mitochondrial membrane
PC Pyruvate carboxylase
Trans-PDC L-trans-pyrrolidine-2,4-dicarboxylate PDH Pyruvate dehydrogenase
PKA Proteinkinase A
SSA Succinate semialdehyde
SSADH Succinate semialdehyde dehydrogenase
TCA Tricarboxylate acid
VGLUT Vesicular glutamate transporters
ΔΨm/c Mitochondrial or plasma membrane potential
Le régulateur principal de la sécrétion hormonale par les cellules des îlots pancréatiques est le glucose. Cependant, plusieurs autres nutriments, hormones et neurotransmetteurs ont été suggérés comme ayant un rôle dans le contrôle de la sécrétion hormonale. Un candidat hypothétique est le neurotransmetteur excitatoire glutamate. Le fait que les îlots pancréatiques possèdent tous les composants de l’appareil de signalisation du glutamate (transporteur vésiculaire du glutamate, récepteurs du glutamate et système de capture du glutamate) a conduit à émettre l’hypothèse selon laquelle cet acide aminé fonctionne comme molécule de signalisation auto- et paracrine au sein de l’îlot. En revanche, d’autres études suggèrent que le glutamate agit comme facteur de couplage entre métabolisme et sécrétion dans les cellules pancréatiques β. Le métabolisme mitochondrial est crucial pour lier la reconnaissance du glucose à la sécrétion de l’insuline. Les facteurs de couplage sont générés pendant l’activation du cycle des acides tricarboxyliques (TCA). Plusieurs groupes ont rapporté une augmentation au niveau cellulaire du glutamate en réponse au glucose, tandis que d’autres équipes n’ont pas observé de changement. Le glutamate peut être produit par l’intermédiaire du TCA α- ketoglutarate (αKG) par des réactions de transamination. Les enzymes impliquées n’ont pas été identifiées, cependant deux voies principales ont été proposées. Le glutamate pourrait être généré à partir du αKG soit par l’action de l’enzyme mitochondriale glutamate déhydrogenase, soit par l’aspartate aminotransférase cytoplasmique (cAAT) lors de l’activation de la navette malate-aspartate.
La constatation que la sécrétion d’insuline glucose-dépendante (SIGD) est potentialisée par l’addition à des cellules clonales β perméabilisées du glutamate ou d’un analogue membrane-perméant indique un rôle messager intracellulaire du glutamate. En outre, la surexpression de glutamate décarboxylase (GAD), qui réduit le niveau intracellulaire de glutamate, diminue la SIGD. Par contre, l’augmentation en continue de glutamate induite par la glutamine n’a pas d’effet sur la sécrétion d’insuline.
Le but de ce travail de thèse était d’identifier la voie métabolique dans la biosynthèse du glutamate médiée par le glucose. En particulier, l’impact de la suppression de cAAT sur la genèse du glutamate a été investigué. De plus, la SIGD a été analysée. Nous confirmons l’existence d’une génération de glutamate glucose-dépendante dans les îlots pancréatiques de rats ainsi que dans les cellules α et β d’îlots de rat. L’inhibition de cAAT et de l’activité de la navette malate-aspartate par l’emploi de bloqueurs pharmacologiques a provoqué une diminution du glutamate total. La SIGD et non la
spécifité de l’aminooxyacétate (AOA) sur la répression de cAAT est remise en question, l’activité enzymatique a été spécifiquement sous-régulée à l’aide de la technique de short hairpin ARN (shARN). La suppression enzymatique a provoqué une réduction du niveau cellulaire de glutamate, alors que les quantités de αKG augmentaient. Il est à noter que la SIGD était également diminuée. Ces données suggèrent un rôle-clé du glutamate intracellulaire dans la régulation de SIGD. Cette hypothèse est soutenue par le fait qu’une diminution du relâchement de glutamate cause une potentialisation de SIGD dans les îlots et les cellules β isolées.
Enfin, les mécanismes de relâchement du glutamate comme messager extracellulaire dans la sécrétion hormonale ont été analysés. Des études précédentes avaient démontré la présence de composants de l’appareillage de signalisation du glutamate dans les cellules d’îlots pancréatiques. Ces données indiquent la possibilité d’une co-sécrétion de glutamate et d’hormones par les granules sécrétoires. Cette co-libération a été analysée dans les cellules α et β isolées d’îlots de rats afin de différencier les mécanismes opérant dans les principaux types cellulaires. Les cellules d’îlots ont été exposées à une variété de stimuli hormonaux. Le relâchement de glutamate et la sécrétion hormonale ont été suivis simultanément et comparés. Les résultats montrent que la libération de glutamate par l’exocytose granulaire paraît peu probable car l’acide aminé est relâché de manière élevée et non-régulée. En revanche, nos résultats suggèrent que les transporteurs d’acides aminés excitatoires (EAAT) jouent un rôle-clé dans le relâchement de glutamate dans les îlots entiers ainsi que dans les cellules d’îlots purifiées. L’inhibition du système de transport de glutamate sodium-dépendant a causé une diminution du relâchement de glutamate dans les deux systèmes céliaques. Les EAATs qui captent habituellement le glutamate de l’espace extracellulaire sont également connus pour relâcher des acides aminés par captage inversé.
En résumé, l’étude présente suggère que le glutamate fonctionne plutôt comme facteur de métabolisme et de sécrétion intracellulaire plutôt que comme molécule de
signalisation auto- et paracrine. Cependant, il faut noter que ces expériences ont été réalisées en absence d’acides aminés ou autres nutriments extracellulaires, alors que dans le pancréas les îlots sont exposés à une variété de facteurs du flux sanguin ainsi qu’à des stimuli neuronaux.
Glucose is the primary regulator of hormone secretion from pancreatic islet cells.
However, several other nutrients, hormones and neurotransmitters have been suggested to participate in the control of hormone release. The excitatory neurotransmitter glutamate is a putative candidate. The finding that pancreatic islet cells possess all components of the glutamate signalling machinery (vesicular glutamate transporter, glutamate receptors and glutamate uptake system) led to the hypothesis that this amino acid functions as auto- or paracrine signalling molecule within the islet. In contrast, other studies suggest that glutamate acts as a metabolism-secretion coupling factor in pancreatic β-cells. Mitochondrial metabolism is crucial for linking glucose recognition to insulin secretion. Coupling factors are generated during TCA cycle activation. Several groups reported a rise in cellular glutamate level in response to glucose whereas others did not observe any change. Glutamate can be produced from the TCA cycle intermediate α-ketoglutarate (αKG) by transamination reactions. However, the implicated enzymes in the β-cell have not been identified. Two main pathways have been suggested. Glutamate might be generated from αKG either through the action of the mitochondrial enzyme glutamate dehydrogenase (GDH) or by cytoplasmic aspartate aminotransferase (cAAT) during malate-aspartate shuttle activity.
The findings that either a membrane-permeant glutamate analogue or glutamate added to permeabilized clonal β-cells potentiated glucose-stimulated insulin secretion (GSIS) substantiate the putative intracellular messenger role of glutamate. Furthermore, overexpression of glutamate decarboxylase (GAD) which reduces intracellular glutamate levels decreased GSIS. In contrast, glutamine-induced increase in glutamate content had no effect on insulin secretion.
The aim of this thesis work was to identify the pathway implicated in glucose-mediated glutamate biosynthesis. In particular, the impact of cAAT suppression on glutamate generation and GSIS was investigated. We confirm a glucose-stimulated glutamate generation in rat pancreatic islets as well as in isolated rat islet α- and β-cells. Inhibition of cAAT and malate-aspartate shuttle activity using pharmacological blockers caused a decrease in total glutamate. GSIS but not glucagon secretion was impaired in isolated islet cells. As the specificity of aminooxyacetate (AOA) on cAAT activity is questioned, the enzyme was specifically downregulated using short hairpin RNA (shRNA) technology.
Enzyme suppression caused a reduction in cellular glutamate level whereas αKG amounts increased. Remarkably, GSIS was also reduced. These data suggest a pivotal role of
finding that a decrease in glutamate release caused a potentiation of GSIS in islets and isolated β-cells.
Finally, glutamate release mechanisms and the role of glutamate as extracellular messenger in hormone secretion were investigated. Previous studies demonstrated the presence of components of the glutamate signalling machinery in pancreatic islet cells.
These findings were taken to indicate glutamate and hormone co-secretion from
secretory granules. Glutamate release was analysed in isolated rat islet α- and β-cells in order to differentiate release mechanism between the main islet cell types. Islet cells were exposed to various hormone stimuli. Glutamate release and hormone secretion were monitored simultaneously and compared. The results indicate that glutamate release through vesicle exocytosis seems unlikely as the amino acid is released in a high and unregulated manner. In contrast, our results suggest that excitatory amino acid transporters (EAATs) play a key role in glutamate release in whole islets as well as in purified islet cells. Inhibition of the sodium-dependent glutamate transport system caused a decrease in glutamate release in both cell systems. EAATs which usually take up glutamate from the extracellular space are also known to release amino acids by reversal uptake.
On balance, the present study suggests that glutamate functions rather as an intracellular factor in metabolism-secretion coupling than as an auto-or paracrine
signalling molecule. However, it should be noted that these experiments were performed in the absence of extracellular amino acids or other nutrients whereas in the pancreas the islets are exposed to various factors in the blood flow as well as to neuronal stimuli.
CHAPTER 1 – INTRODUCTION
The pancreas is a gland that is divided into two functionally different compartments: the exocrine and the endocrine pancreas. The exocrine pancreas which represents ~98% of pancreatic tissue secretes pancreatic juice into the small intestine through a system of exocrine ducts. The pancreatic juice contains digestive enzymes and an alkaline fluid for proper digestion. Digestive enzymes are synthesized and secreted from exocrine acinar cells whereas the bicarbonate- and salt-rich alkaline fluid is released from epithelial cells lining pancreatic ducts. Digestive enzymes participate in the breakdown of carbohydrates, proteins and fat in the chyme (partly digested food expelled by the stomach into the duodenum). Three major groups of enzymes can be distinguished for efficient digestion:
1. Proteases trypsin and chymotrypsin digest proteins into peptides, but cannot digest proteins and peptides to single amino acids in contrast to carboxypeptidase which has this ability.
2. Pancreatic lipase hydrolyses dietary triglyceride moieties into a 2-monoglyceride and two free fatty acids.
3. Amylase hydrolyses the dietary carbohydrate starch to maltose.
The bicarbonate- and salt-rich alkaline solution neutralizes the acid coming into the duodenum from the stomach. The mechanism underlying bicarbonate secretion depends on the enzyme carbonic anhydrase. In pancreatic duct cells, the bicarbonate is secreted into the lumen of the duct and hence into pancreatic juice.
1.2 Endocrine pancreas & islets of Langerhans
The endocrine pancreas is composed of micro-organs called islets of Langerhans which produce and secrete hormones. The islets of Langerhans constitute approximately 1 to 2% of the pancreas mass. Approximately one million islets can be found in the adult human pancreas. The islet size is variable. It ranges from 40 to 900µm . Each islet contains ~2000 cells divided into five different types of endocrine cells [2, 3]. The most abundant are β-cells (~50-80%), which produce insulin, and α-cells, which secrete glucagon (~15-20%) [4, 5]. The other islet cell types are somatostatin-producing δ-cells (3-10%), pancreatic polypeptide-secreting PP-cells (3-5%) and ghrelin-producing ε-cells (<1%).
Figure 1.1: Representation of the endocrine and exocrine pancreas. (A) Representation of the pancreas and its localization in the digestive system
(www.gopetsamerica.com/anatomy/pancreas.aspx). (B) Exocrine pancreas. (C) Schematic presentation of exo- and endocrine tissue. ((B) & (C) ) (D) Histological analysis of exocrine (D1) and endocrine (D1, D2) pancreas. Insulin is located in the islet core and is labelled in green. Glucagon-containing α-cells in the islet mantle are shown in blue and somatostatin is labelled in red.
1.3 Regulation of hormone secretion
The secretion of hormones from pancreatic islets is regulated by neuronal, hormonal and nutritional stimuli as well as by the microcirculation.
1.3.1 Neuronal regulation of hormone secretion
The endocrine pancreas receives regulatory innervation from the autonomic nervous system. Two types of innervation can be distinguished: (1) the adrenergic sympathetic and (2) the muscarinic parasympathetic input. It has been demonstrated that in human pancreas activation of α-adrenergic fibers caused a suppression of insulin, glucagon, somatostatin, and PP secretion. In contrast, activation of β-adrenergic fibers strongly increased somatostatin secretion whereas the insulin, glucagon and PP release was only mildly affected. Cholinergic innervation of the islets via muscarinic acetylcholine receptors strongly stimulated insulin, glucagon and PP release but only mildly inhibited somatostatin secretion [7-9].
1.3.2 Hormonal regulation of islet secretion
Islet hormone secretion can be affected by paracrine and autocrine actions within the islet. Insulin activates β-cells but inhibits glucagon release from α-cells . In contrast, glucagon secretion from α-cells activates hormone release from β- and δ-cells .
Somatostatin inhibits both, α- and β-cells [11, 12]. Ghrelin, a hormone that stimulates appetite and thereby food intake, is primarily produced in rat or human stomach during fasting. However, ghrelin-positive cells have also been detected in islets . It has been suggested that ghrelin acts in a paracrine stimulatory fashion on α-cell glucagon release during hypoglycaemia . Furthermore, exogenous ghrelin was shown to increase cytosolic Ca2+-concentrations in β-cells and to stimulate insulin secretion in isolated rat pancreatic islets . In contrast, endogenous Ghrelin attenuated intracellular Ca2+- oscillations and glucose-stimulated insulin secretion .
1.3.3 Nutritional regulation of islet secretion
Many nutrients in the blood flow regulate islet hormone secretion but the main regulator is glucose. Blood glucose homeostasis is maintained by the interplay of islet hormone secretion. The rise in blood glucose level (hyperglycemia) after food intake causes an increase in β-cell glucose metabolism and thereby insulin secretion. Subsequently, liver muscle and fat tissue cells will take up glucose from the blood, to store it as glycogen in the liver and muscle, and stop the use of fat as an energy source. Glucose transport is stimulated by insulin in myocytes and adipocytes, but not in hepatocytes. The major counterhormone of insulin, glucagon, is released from α-cells during hypoglycaemia.
During fasting conditions or exercise, when blood glucose concentrations are low, glucagon is secreted from α-cells. Glucagon binds to its receptor on hepatocytes, resulting in the release of glucose from the liver. Two different mechanisms cause an elevation of blood sugar level. During glycogenolysis stored hepatic glycogen is converted to glucose. If glycogen stores are depleted, glucagon stimulates glucose synthesis in the liver in a process called gluconeogenesis. Not only insulin and glucagon are implicated in the regulation of blood glucose levels. High blood glucose concentrations also stimulate somatostatin secretion and inhibit PP release [7, 8].
Figure 1.2: Glucose homeostasis. Regulation of blood glucose level by insulin and glucagon.
Even though glucose is the primary regulator of hormone secretion, other nutrients are known to influence hormone release. For instance, the amino acid glutamate has been shown to stimulate insulin secretion in the presence of stimulatory glucose concentrations [16-20]. In contrast, in the presence of low glucose conditions (fasting conditions), the amino acid caused a stimulation of glucagon, but not insulin secretion .
1.3.4 Islet microcirculation
Islets are highly vascularized [1, 21, 22]. They are all connected to the arterial circulation and respond to changes in arterial content simultaneously. Every islet receives
arterial blood via afferent vessels (arterioles and capillaries). Nutrients and hormones cross the capillary endothelium to regulate islet hormone secretion. Paracrine signalling depends on the direction of the microcirculation. In the rat, it has been demonstrated that the blood passes first the islet core consisting of β-cells. Then, the intra-islet blood flows to the α- and δ-cell containing islet mantle [10, 23-27]. Anterograde and retrograde perfusion experiments using antibodies against islet hormones in several species have provided significant evidence for a B-to A to D-cell order. However, other models of microcirculation have been suggested. It has been proposed that afferent arterioles reach first islet cells located in the periphery. Secretory products of non-β-cells would affect insulin secretion from β-cells but not vice versa [2, 28-30]. A third model that has been described suggest that the blood enters through an arteriole on one side of the islet, branches into capillaries to perfuse all types of endocrine cells on this side before reaching islet cells on the other side of the micro-organ. In this model the blood flow is controlled by external and internal gates that close or open to regulate blood supply to a specific islet region consisting of endocrine cells of the same type [31-34]. In this model interactions between all islet cell types are possible. Thus, the inhibitory action of somatostatin on insulin and glucagon secretion can be mediated not only by paracrine signalling but also by the microcirculation .
1.3.5 Junctional communication
Cell-to cell contacts play an important role in hormone secretion. These junctional communications are required for normal basal hormone secretion. Isolated, non- aggregated β- and α-cells have been shown to display increased basal hormone secretion rates [36-39]. However, re-aggregation of the isolated α-cell fraction caused a 10-fold reduction of basal glucagon secretion rate , demonstrating the importance of intercellular contacts. Increasing the density of single α-cells per well did not affect basal glucagon release. Therefore, it can be concluded that the normalized basal glucagon secretion rate in re-aggregated a-cells was caused by the formation of cell-cell contacts rather than by a local accumulation of secreted autocrine factors. Furthermore, it has been demonstrated that gap junctions are required for normal glucose-stimulated insulin secretion (GSIS) by rat β-cells [40, 41]. Furthermore, repression of the cell adhesion molecule E-cadherin in confluent clonal β-cells reduced GSIS in a similar fashion as observed in isolated cells .
1.4 Diabetes mellitus
Diabetes mellitus or diabetes denotes a syndrome of disordered metabolism resulting in abnormal high blood glucose level (hyperglycemia). According to the World Health Organization, in the year 2000 171 million people worldwide suffered from diabetes. This figure is expected to double by the year 2030 . Especially Type 2 diabetes (or non insulin-dependent diabetes mellitus NIDDM or adult-onset diabetes), is increasing rapidly. The greatest increase in the prevalence (total number of cases at a certain time) has occurred in Asia and Africa due to urbanization, life style changes and a more
‘Western-style’ diet (leading to obesity).
Diabetes mellitus refers to a group of diseases. All forms have hyperglycemia and glucose intolerance in common, caused by either insulin depletion, disturbed insulin efficiency or both. Type 1 and Type 2 diabetes are the most common forms of the metabolic disease. Less common types, like Maturity Onset Diabetes of the Young (MODY) affect only 1-5% of all cases (National Diabetes Information Clearinghouse (NDIC); National Institute of Diabetes and Digestive and Kidney Diseases NIH).
Gestational diabetes resembles Type 2 diabetes and occurs in 2-5% of all pregnancies, improving or disappearing after delivery.
Typical diabetic symptoms comprise excessive urine production, causing compensatory thirst and increased fluid intake, blurred vision, unexplained weight loss, lethargy, and changes in energy metabolism. Diabetes and an inappropriate treatment can cause various symptoms. Acute complications like hypoglycaemia, ketoacidosis, and nonketotic hyperosmolar coma occur when diabetes is inadequately treated. Long-term complications are manifest in heart, brain, kidneys and peripheral nerves. Average life expectancy is thus shortened by cardiovascular and cerebrovascular disease as well as chronic renal failure. Microvascular damage leads to peripheral nerve disease and retinopathy, often resulting in blindness.
1.4.1 Type 1 diabetes
Type 1 diabetes is characterized by a complete loss of insulin-producing β-cells due to a T-cell-mediated autoimmune destruction. This type of diabetes represents 10% of all chronic diabetes cases. Patients with type 1 diabetes depend on regular insulin injections in order to replace to lacking hormone. Other therapy approaches are under investigation. Pancreas and islet transplantation is one possibility to replace β-cells whereby the latter seems to be less invasive. Other non-insulin treatments include monoclonal antibodies or stem cell based therapy. These approaches have been tested in
animals but clinical trials are still to be accomplished  (see also www.cliniclog.com./type_1_diabetes_trials.php).
1.4.2 Type 2 diabetes
Type 2 diabetes patients usually show both insulin resistance and reduced insulin secretion. Impaired signalling through the insulin receptor underlies the defective hormone action. The main cause of Type 2 diabetes is obesity. 55% of all patients diagnosed with Type 2 diabetes show increased body mass index (BMI). Other factors that seem to play an important role are aging and a family history. Several predisposing gene polymorphisms have been identified by genome-wide association studies of this polygenic disease . Nowadays, more and more children are affected due to an increase in childhood obesity in the last decade. Increasing physical activity, a carbohydrate-poor diet as well as weight loss improve diabetic symptoms. Initially, when insulin production and resistance are still moderately impaired, oral medication can improve symptoms (e.g. sulfonylurea). If disease progresses insulin therapy becomes necessary.
1.4.3 Maturity onset diabetes of the young (MODY)
MODY appears before the age of 25 and is characterized by a disruption of insulin production or release by the pancreatic β-cells. This monogenic disease is autosomal dominantly inherited. Most of the cases are caused by mutations in transcription factor genes (e.g. hepatocyte nuclear factor, HNF). One form of MODY (MODY2) shows mutations in the glucokinase gene .
Mitochondria are membrane-enclosed organelles found in most eukaryotic cells. The most prominent role of mitochondria is the production of ATP and the regulation of cell metabolism. Other functions include signalling, cell differentiation, cell death as well as the control of the cell cycle and cell growth.
1.5.1 Structure & function of mitochondria
Mitochondria are composed of different compartments each of which has a specialized function. These compartments comprise:
(1) Outer mitochondrial membrane (OMM) (2) Intermembrane space
(3) Inner mitochondrial membrane (IMM) (4) Cristae
Figure 1.3: Schematic representation of mitochondrial compartments.
Both the inner and the outer mitochondrial membrane are composed of a phospholipid bilayer and specific proteins. The OMM encloses the organelle. OMM-proteins, so-called porins, allow the free diffusion of molecules less than 5kD. As small molecules like sugar and ions can pass the OMM easily, their concentration in the intermembrane space is usually close to that in the cytosol. Larger proteins like cytochrome C need specific signalling sequences to be transported across the OMM. The IMM contains four types of proteins with different functions: (A) proteins of the electron transport chain that perform redox reactions during oxidative phosphorylation, (B) the ATP-generating ATP-synthase, (C) transport proteins that regulate metabolite passage, and (D) protein import machinery. Infoldings of the IMM – called cristae – enhance the ability of the IMM to produce ATP. The space that is enclosed by the IMM is called the matrix. It contains high concentrations of various enzymes implicated in pyruvate and fatty acid oxidation or in the tricarboxylic acid cycle (TCA cycle).
1.5.2 Cell respiration & mitochondrial metabolism
Cell respiration refers to metabolic reactions that convert biochemical energy from nutrients (glucose, amino acids, and fatty acids) into ATP. The generation of ATP from
glucose comprises four different steps, which take place in different cellular compartments (cytosol and mitochondria):
(2) Oxidative decarboxylation of pyruvate (3) TCA cycle activity
(4) Oxidative phosphorylation (electron transport chain)
Glucose enters the β-cell by facilitated diffusion via the glucose transporter 2 (GLUT2).
The hexose equilibrates across the plasma membrane and is phosphorylated by glucokinase, a high Km hexokinase. The phosphorylation is irreversible and glucose-6-P is retained in the cell. Subsequently, glycolysis takes place in the cytoplasm. In a sequence of enzymatic reactions glucose is converted into two molecules of pyruvate. Thereby, two molecules of ATP as well as two reducing equivalents (nicotinamide adenine dinucleotide (NADH)) are generated. Glycolysis requires no oxygen and is also called anaerobic metabolism. The overall reaction of glycolysis is:
Glucose + 2 NAD+ + 2 Pi + 2 ADP → 2 pyruvate + 2 NADH + 2 ATP + 2H+ + 2H2O
Pyruvate is transported into the mitochondria where it flows into mitochondrial pathways through either anaplerotic (pyruvate carboxylase, PC) or oxidative (pyruvate dehydrogenase, PDH) reactions. Pyruvate is either oxidized by PDH to form acetyl-CoA (aerobic respiration) or carboxylated to generate oxalacetate by PC (anaplerotic pathway). In β-cells, pyruvate enters mitochondrial metabolism through PC and PDH in approximately equal proportion [46-50]. During aerobic respiration, one reducing equivalent NADH and CO2 are released. Acetyl-CoA enters the TCA cycle, also known as Krebs cycle or citric acid cycle. Each step of the cycle is catalyzed by a specific enzyme.
Throughout the TCA cycle, acetyl-CoA is converted into citrate, isocitrate, α- ketoglutarate, succinyl-CoA, succinate, fumarate, malate, and finally, oxalacetate. One complete turn of the cycle results in the formation of two molecules of CO2 and one molecule GTP as well as three reducing equivalents NADH and one FADH2.
Figure 1.4: Enzymatic reactions and the generation of reducing equivalents during the TCA cycle activity.
The final step of aerobic respiration is called oxidative phosphorylation. During this process, electrons from the generated reducing equivalents NADH and FADH2 enter the respiratory chain (also known as electron transport chain). The electron transport chain comprises a series of protein complexes named complex I-IV. Electrons are transferred from electron donors (NADH, FADH2) to electron acceptors (complex I-IV). The final electron acceptor is oxygen and H20 is formed in the presence of two protons. While electrons are transported from one complex to another, protons (H+) are pumped into the intermembrane space, and a proton gradient is generated. This proton gradient is essential for ATP generation. The accumulated protons re-enter the mitochondrial matrix through the ATP-synthase which uses the energy stored in the gradient to produce ATP from ADP and inorganic phosphate (Pi). This final step links respiration to ATP generation.
Figure 1.5: The electron transport chain and ATP generation.
1.5.3 Anaplerosis & Cataplerosis
The oxidation of acetyl-CoA to CO2 by the TCA cycle is the central process in energy metabolism. However, TCA cycle intermediates also function in biosynthetic pathways.
Intermediates can be removed from the TCA cycle to be converted to fatty acids or amino acids. This necessitates anaplerotic reactions that replenish TCA cycle intermediates to ensure its continued function. Pyruvate carboxylase (PC), which generates oxalacetate from pyruvate, is the major anaplerotic enzyme.
Conversely, it is equally important that intermediates are removed from the TCA cycle.
During amino acid catabolism, TCA cycle intermediates are generated which can enter the Krebs cycle. Excess of these molecules must be removed by a process called cataplerosis. Cataplerotic enzymes include phosphoenolpyruvate carboxykinase (PEPCK), glutamate dehydrogenase, and aspartate aminotransferase and citrate lyase. Each of these enzymes uses a TCA cycle intermediate as substrate and converts it to a product that is removed from the TCA cycle.
The regulation of anaplerosis and cataplerosis depends on the metabolic and physiological state of the cell. The following figure illustrates intermediates that enter or leave the TCA cycle during anaplerotic and cataplerotic reactions :
Figure 1.6: Anaplerotic and cataplerotic intermediates entering and exiting the TCA cycle .
1.6 Mitochondrial metabolism and insulin secretion
1.6.1 Biphasic insulin secretion
Two phases of insulin secretion can be distinguished. The first phase takes place within seconds after glucose stimulation. This phase is also called the triggering phase. Insulin is released as a sharp peak probably from the ready- releasable pool of insulin secretory granules. This step is caused by an increase in intracellular calcium concentrations [Ca2+]i. The second phase of insulin secretion is characterized by a gradually increasing insulin secretion rate which reaches its plateau approximately 30min after glucose stimulation . It also requires Ca2+ and ATP but other calcium and ATP-independent pathways are activated during this second phase of insulin secretion. Therefore, this phase is also known as the amplification phase [53, 54].
Figure 1.7: Biphasic insulin secretion after glucose stimulation.
Insulin secretagogues that do not affect mitochondrial metabolism, such as arginine or potassium, can only trigger the first phase of insulin release and raise intracellular calcium concentration but cannot cause the second phase of insulin secretion. In contrast, fuel secretagogues, such as leucine, can trigger both the first and second phase of insulin exocytosis.
1.6.2 KATP-dependent pathway of glucose-stimulated insulin secretion (GSIS)
Insulin secretion from pancreatic β-cells is regulated by blood glucose level. Mitochondrial metabolism is essential for the coupling of glucose recognition to insulin exocytosis.
Glucose enters the β-cell through the glucose transporter Glut2 . In the cytoplasm, glucose is immediately phosphorylated to glucose-6-phosphate by glucokinase and thereby retained in the cell. This phosphorylation initiates its conversion to pyruvate during glycolysis. Pyruvate is taken up by the mitochondria together with protons and serves as substrate for either pyruvate dehydrogenase (PDH) or pyruvate carboxylase (PC), depending on allosteric regulators like ATP, NADH or acetyl-CoA . These enzymes ensure the formation of acetyl-CoA (PDH) or oxalacetate (PC). PC provides anaplerotic input to the TCA cycle (see chapter 1.5.3). During TCA activity reducing equivalents (NADH) are produced and transferred to the respiratory chain, leading to hyperpolarization of the mitochondrial membrane (ΔΨm). Cytosolic glycolytic produced NADH is coupled to the mitochondrial electron transport chain through mainly two redox shuttle mechanisms (malate-aspartate shuttle & glycerol-3-phosphate shuttle). The hyperpolarization of the mitochondrial membrane is caused by protons pumped into the intermembrane space. The energy of the proton gradient is used to generate ATP. ATP is transferred into the cytosol, raising the ATP/ADP ratio. Subsequently, the ATP-sensitive potassium channels (KATP –channel) close and the plasma membrane depolarizes (ΔΨc).
Voltage-gated Ca2+-channels open followed by Ca2+-influx. The increase in intracellular calcium concentrations [Ca2+]i triggers insulin secretory granule exocytosis .
Figure 1.8: Model for coupling glucose metabolism to insulin secretion in the β-cell.
Glucose equilibrates across the plasma membrane and is phosphorylated by glucokinase (GK). Pyruvate (Pyr) is generated during glycolysis and enters the mitochondrion where it activates the TCA cycle. Reducing equivalents (red.equ.) are generated and transferred to the electron transport chain. During oxidative phosphorylation the mitochondrial membrane hyperpolarizes (ΔΨm) and ATP is generated. ATP is transported into the cytosol raising the ATP/ADP ratio. Subsequently, KATP-channel close and the plasma membrane depolarize (ΔΨc). Voltage-gated Ca2+-channels open increasing intracellular calcium concentrations ([Ca2+]c) which triggers insulin secretion .
1.6.3 KATP-independent pathways of insulin secretion
The increase in [Ca2+]i in response to glucose stimulation is necessary but not sufficient for a full development of biphasic insulin secretion. KATP-independent pathways have been suggested to potentiate the second phase of GSIS. This hypothesis was supported by the finding that in the absence of functional KATP-channels, insulin secretion is reduced, but not completely abolished [59, 60]. Furthermore, there is evidence that glucose can control insulin release independently from its action on ATP-sensitive K+- channels [61, 62]. Several studies suggest that KATP-independent insulin secretion is also controlled by nucleotides (ATP, GTP, cAMP), metabolites, hormones (GLP-1 & GIP) neurotransmitters (e.g. acetylcholine) and amino acids (e.g. glutamate, glutamine, leucine, arginine, alanine). The following illustration demonstrates putative metabolism- secretion factors involved in the amplification of GSIS.
Figure 1.9: Summary of proposed additive signals involved in the amplifying pathway of glucose-stimulated insulin secretion .
22.214.171.124 Nucleotide-mediated insulin secretion
Several putative messengers, or additive signals, have been proposed to participate in metabolism-secretion coupling. Among them, nucleotides are possible candidates.
Intracellular ATP has two functions. On the one hand, an increase in the ATP/ADP ratio is necessary for KATP-channel closure. On the other hand, ATP has a role in priming granules for fusion with the plasma membrane and the exocytotic release of insulin. However, at non-stimulatory Ca2+-concentrations, ATP does not induce insulin release in permeabilized cells . In contrast, in the presence of stimulatory Ca2+-concentrations, ATP potentiates exocytosis [64, 65]. In the absence of extracellular calcium glucose- stimulated ATP generation did not promote insulin secretion . However, when the KATP-channel is kept in an open state using the inhibitor diazoxide and [Ca2+]i is raised
independently of KATP-channel activation was observed. These results were taken to suggest that ATP stimulates insulin exocytosis in a KATP-independent manner .
The nucleotide GTP is formed in the mitochondria during TCA cycle activity. It was proposed that GTP triggers insulin exocytosis via GTPases [67, 68]. Evidence in support of a role for mitochondrial GTP production in GSIS involves experiments with siRNA- mediated suppression of the GTP-generating form of succinyl-CoA synthetase (SCS-GTP).
Suppression of this enzyme in clonal β-cells and rat islets resulted in impaired GSIS and reduced GTP levels . In contrast, repression of the ATP-generating form of this enzyme (SCS-ATP) enhanced insulin release at stimulatory glucose concentrations .
Furthermore, the same study demonstrated that suppression of SCS-GTP impairs glucose-stimulated increases in [Ca2+]i whereas downregulation of SCS-ATP had the opposite effect. In general, GTP is not leaving the mitochondria, but the nucleotide can also be generated from ATP in the cytosol through the action of nucleoside diphosphate kinase. In contrast to ATP, GTP is able to initiate insulin secretion in a Ca2+-independent manner in native [68, 70] and in clonal β-cells [64, 68, 71]. This pathway is completely and selectively blocked by reducing GTP levels in the islet [72, 73]. It is not known if GTP acts via monomeric or heteromeric G-proteins to control insulin exocytosis [71, 74].
It is well established that cyclic AMP (cAMP) potentiates Ca2+-dependent GSIS. Several hormones, including glucagon, GLP-1 (glucagon-like peptide 1) and glucose-dependent insulinotropic polypeptide (GIP), are known to increase intracellular cAMP levels. The action of these hormones involves the activation of G-protein coupled receptors (GPCR), followed by activation of adenylate cyclase (AC) and a rise in cAMP concentrations [75, 76]. Subsequently, cAMP can activate either proteinkinase A (PKA)–dependent or PKA- independent pathways in order to potentiate GSIS .
Figure 1.10: Protein kinase-dependent and –independent pathways implicated in the regulation of GSIS .
Several PKA-dependent pathways have been suggested by which cAMP increases [Ca2+]i. The phosphorylation of the KATP-channel subunit Kir6.2 (pore-forming subunit) and SUR1 (regulatory subunit) by PKA has been demonstrated to decrease KATP-channel activity . Furthermore, PKA-mediated activation of voltage-dependent L-type Ca2+-channels (VDCC) caused an increase in Ca2+-influx and thereby a potentiation of insulin granule exocytosis . The phosphorylation of the glucose transporter GLUT2 by PKA has also been shown to decrease glucose transport activity by GLP-1 in purified β-cells and thus affect GSIS . Another PKA-dependent pathway that has been suggested is the mobilization of Ca2+-ER-stores [81, 82] and the activation of Ca2+-activated nonselective ion channels .
Cyclic AMP can also stimulate insulin exocytosis via PKA-independent mechanisms. It enhances the translocation of granules to the plasma membrane (active zone) and increases the size of the ready releasable pool as well as the rate of replenishment [84, 85]. The protein cAMP-GEFII (GTP-exchange factor) has been described as direct target of cAMP in the regulation of insulin exocytosis . Cyclic AMP-GEFII-mediated exocytosis of insulin secretory granules requires direct interaction of the factor with the (active zone) protein Rim2. It also interacts with the active zone protein piccolo 
The exact mechanism by which cAMP binding to cAMP-GEFII affects insulin exocytosis has still to be elucidated.
There is strong evidence in support of an important role of the pyridine nucleotide NADPH in the regulation of insulin secretion. NADPH is generated during glucose metabolism (pentose phosphate pathway, malic enzyme & isocitrate dehydrogenase). Experiments from toadfish islet cells propose that NADPH stimulates insulin release from secretory granules . Generation of NADPH is an early event in β-cell activation preceding the rise in [Ca2+]i, a prerequisite for triggering nutrient-mediated insulin release [89, 90].
Several studies demonstrated an increase in the NADP/NADP ratio in proportion to glucose concentrations and GSIS, whereas this relationship does not exist for the NADH/NAD+ ration and GSIS [91, 92]. The elevation of NADPH levels occurs more rapidly in the cytosol than in the mitochondria . Conversely, if the NADPH/NADP ratio was decreased through molecular manipulations GSIS was impaired [92, 94, 95].
Furthermore, addition of NADPH but not NADH to patch-clamped β-cells increased cell capacitance and thereby insulin exocytosis . Two targets have been proposed for NADPH. The NADPH-dependent glutathione reductase has been suggested which catalyzes the formation of reduced glutathione from glutathione. Reduced glutathione is then converted to glutaredoxin (GRX), which participates in posttranslational modifications of proteins, including exocytosis-regulating t-SNARE proteins . The administration of GRX to patch-clamped β-cells caused a potentiation of NADPH-induced exocytotic activity . The second potential target of NADPH that has been proposed is the voltage-dependent K+-channel (Kv-channel) . Kv-channels might serve as negative regulators of insulin secretion as they repolarize glucose-stimulated action potentials and inhibit Ca2+-influx through voltage-gated calcium channels. It has been shown that an increase in the cytosolic NADPH/NADP ratio in patch-clamped β-cells was associated with an increased rate of Kv-channel inactivation . Additonally, NADPH oxidase seems also play an important role in the regulation of insulin secretion through the generation of reactive oxygen species (ROS) .
126.96.36.199 Pyruvate cycling and other metabolism-secretion coupling factors
The generation of second messengers other than ATP during glucose metabolism has been suggested to play a major role in the amplification phase in GSIS. In particular, pyruvate cycling seems to represent an important pathway. Islet β-cells express more or less equal amounts of pyruvate carboxylase (PC) and pyruvate dehydrogenase (PDH) in a high manner [50, 100]. Only low levels of PC were observed in non-β cells . Pyruvate
flows into mitochondrial metabolism pathways either through anaplerotic (PC) and oxidative (PDH) in approximately equal proportions [46-48, 50].
There is considerable evidence linking PC-catalyzed anaplerotic influx into the TCA cycle and pyruvate cycling to the control of GSIS [47, 101-103]. Three major pathways can be considered which have the generation of oxalacetate from pyruvate in common.
The pyruvate/malate pathway involves the conversion of oxalacetate to malate by mitochondrial malate dehydrogenase (mMDH), the export of malate to the cytoplasm via the dicarboxylate carrier (DC) and the formation of pyruvate from malate by the cytosolic malic enzyme (MEc). Evidence in support of the role of malate in insulin secretion showed elevated malate level after glucose stimulation in clonal β-cells [103, 104] and rodent islets . Furthermore, suppression of MEc in clonal β-cells by siRNA technique caused a ~40% reduction of GSIS [106, 107]. In contrast, these findings were not confirmed in physiologically more relevant primary islets .
An alternative pathway of pyruvate cycling involves the generation of citrate from oxalacetate and acetyl-CoA. Subsequently, citrate is exported from the mitochondria to the cytosol via the citrate/isocitrate carrier (CIC) and cleaved by ATP-citrate lyase (CL) to oxalacetate and acetyl-CoA. Pyruvate is recycled via mMDH and MEc. By-products of this pathway are malonyl-CoA and long chain acyl CoAs (LC-CoA) [104, 108, 109]. It has been demonstrated that glucose stimulation of β-cells caused a rise in malonyl-CoA during insulin secretion [108, 109]. Furthermore, LC-CoA stimulated insulin granule exocytosis in permeabilized β-cells while opening KATP-channels in patch-clamped β-cells [110, 111]. However, inhibition of glucose-stimulated malonyl-CoA or LC-CoA generation had no impact on GSIS [112-114]. Furthermore, acute inhibition of acetyl-CoA carboxylase 1 (ACC), which catalyzes the conversion of acetyl-CoA to malonyl-CoA, by pharmacological tools did not affect GSIS . Considering this converging evidence, it is unlikely that malonyl-CoA is a coupling factor. Additionally, neither siRNA-mediated silencing of CL nor CL knockdown affects GSIS [94, 103].
During the pyruvate/isocitrate cycle, citrate and isocitrate exit the mitochondria via CIC.
In the cytosol, citrate can be converted to isocitrate by cytosolic aconitase.
Subsequently, α-ketoglutarate is generated by cytosolic NAD(P)-dependent isocitrate dehydrogenase (ICDs) which can be recycled to pyruvate or oxalacetate by several
The importance of the citrate/isocitrate export in GSIS was demonstrated in a recent study. Inhibition of CIC activity in clonal β-cells or primary rat islets caused a marked reduction of GSIS . The same results were obtained when CIC expression was repressed in clonal β-cells and islets using siRNA techniques. The downregulation of the shuttle also prevented glucose-stimulated citrate accumulation in the cytosol.
Conversely, overexpression of the transporter enhanced GSIS and raised cytosolic citrate level.
The formation of α-ketoglutarate from isocitrate by ICDc also seems to play an important role in the amplifying pathway of insulin secretion. SiRNA-mediated suppression of this enzyme caused a marked impairment of GSIS in clonal β-cells as well as in rat islets . The increment of pyruvate cycling activity and NADPH level were also attenuated.
In summary, of the three pyruvate cycling pathways the pyruvate/isocitrate cycle seems to be the most important process in the control of GSIS.
Generation of secretion coupling factors may not require pyruvate cycling. The TCA cycle intermediate α-ketoglutarate itself has been demonstrated to stimulate insulin secretion . Furthermore, another Krebs cycle byproduct succinate was shown to directly promote insulin exocytosis in permeabilized pancreatic β-cells . The membrane- permeable methyl-succinate was also a potent secretagogue .
188.8.131.52 Amino acid-dependent stimulation of insulin secretion
Several amino acids have been proposed to participate in the amplifying pathway of glucose-stimulated insulin secretion. Among them, L-glutamine, L-glutamate, L-alanine, L-arginine and L-leucine have been suggested [57, 118-122]. Their physiological levels in the plasma vary depending on the amino acid (glutamine: 390-650 µmol/L; glutamate:
18-98 µmol/L; alanine: 230-510 µmol/L; arginine: 13-64 µmol/L; leucine: 66-170 µmol/L; www.nlm.nih.gov/medlineplus/ency/article/003361.html). Mitochondrial metabolism is crucial for the coupling of amino acid and glucose recognition to insulin exocytosis. Consequently, key enzymes and transporters (e.g. aspartate aminotransferase, malate-aspartate shuttle, and glutamate dehydrogenase) play an important role in the control of insulin secretion.
Figure 1.11: Amino acids implicated in the amplifying pathway of glucose stimulated insulin secretion (modified from Newsholme et al 2007 ).
Glutamine is the most abundant free amino acid in the body and it plays an important role in promoting and maintaining function of various organs (e.g. liver, neurons). It is implicated in several cell specific processes such as metabolism (e.g., oxidative fuel), cell integrity (apoptosis, cell proliferation), protein synthesis, and degradation, insulin resistance and insulin secretion . Furthermore, glutamine has been demonstrated to regulate the expression of many genes related to metabolism, signal transduction, cell defence and repair, and to activate intracellular signalling pathways. Thus, glutamine is involved in a broad spectrum of cellular processes.
Previous studies have shown that glutamine is consumed at high rates by both islets and clonal β-cells . The amino acid is rapidly taken up and metabolized by islets but glutamine alone fails to stimulate insulin secretion both in the absence and presence of glucose. However, a marked stimulation of insulin release by L-glutamine occurs in the presence of leucine . This action was attributed to activation of the mitochondrial enzyme glutamate dehydrogenase (GDH) by leucine. GDH catalyses the reversible generation of α-ketoglutarate from glutamate. The proposed model for the role of
cycle, hence increasing the ATP/ADP ratio and stimulation of insulin release. In this model, glutamate is generated from glutamine through the action of phosphate-activated glutaminase whereas leucine acts as allosteric activator of GDH resulting in α- ketoglutarate generation . However, at stimulatory glucose concentrations glutaminolysis is inhibited, presumably via GTP-mediated allosteric inhibition of GDH .
Paradoxically glutamine alone fails to stimulate insulin secretion. It has been suggested that glutamine is metabolized to GABA and aspartate which accumulates in islets [127, 128]. In the absence of leucine, there was no oxidation of glutamine via the TCA cycle.
These results were taken to explain the poor ability of glutamine to stimulate insulin secretion. In contrast, a NMR study performed in clonal β-cells demonstrated that the main products of glutamine metabolism are glutamate and aspartate . There was no production of GABA. However, glutamine-derived glutamate increased the production of glutathione. Addition of glucose stimulated glutamate generation but did not affect glutamine consumption [126, 129]. Furthermore, glutamine increased the flux through GDH without affecting insulin secretion . Interestingly, SUR1-/- mice mice revealed an increased glucose-stimulated glutamate production compared to control .
However, GSIS was dramatically decreased compared to control whereas basal insulin secretion rates were increased 6-fold . The transgenic mice showed a suppression of glutamate decarboxylase (GAD) expression which catalyzes the conversion of glutamate to GABA. Concomitantly, GABA levels were decreased 10-fold. The reduction of GAD level was caused by an increase in intracellular Ca2+-concentration, explaining also elevated basal insulin secretion rates of SUR1-/- mice and the reduced GSIS compared to controls [130, 131].
Leucine can stimulate insulin secretion through two different mechanisms, both leading to increased mitochondrial metabolism: (1) Allosteric activation of GDH and (2) Deamination of leucine to α-ketoisocaproate (αKIC) followed by entry into the TCA cycle via acetyl-CoA [121, 132, 133]. However, leucine-induced insulin secretion is inhibited in the presence of high glucose concentrations due to allosteric inhibitory effects of GTP and ATP on the activity of GDH. Thus, GDH flux towards oxidative deamination of glutamate to aKG is inhibited at elevated glucose concentrations [134, 135] (see also glutamine paragraph). Interestingly, patients with hyperinsulinism (increased insulin secretion rates) were shown to have mutations in the inhibitory allosteric GTP-binding site of GDH.
Affected patients are more responsive to leucine and develop hypoglycaemia [135-137].
The role of glutamate in insulin secretion is debated. Extracellular and intracellular messenger functions have been proposed for this amino acid. The participation of glutamate in metabolism secretion coupling will be discussed in chapter 1.8.3.
Evidence suggests that L-alanine promotes insulin secretion in clonal β-cells and islets at basal glucose concentrations by a mechanism requiring oxidative metabolism . This stimulatory effect was caused by electrogenic Na+ co-transport, followed by membrane depolarization, generation of Ca2+ spike potentials and an increase in [Ca2+]i [138, 139].
Furthermore, metabolism and oxidation of alanine is important for its insulinotropic effects [140, 141]. It has been demonstrated that alanine is metabolised to glutamate, aspartate and lactate in clonal β-cells . Additonally, alanine stimulates glucose oxidation and thereby insulin secretion. In contrast to clonal cells, alanine is a weak secretagogue in native β-cells.
The stimulation of insulin secretion in response to this cationic amino acid is caused by membrane depolarization [122, 142-144]. Arginine is transported into the β-cells via the electrogenic transporter mCAT2A. Membrane depolarization, activation of voltage-gated Ca2+-channels and Ca2+-influx result in stimulated insulin release.
In contrast to the other amino acids, homocystein dose-dependently inhibited insulin secretion at moderate and stimulatory glucose concentrations from clonal β-cells .
In the presence of homocystein TCA cycle-dependent glucose metabolism was reduced . Similar effects were observed when insulin release was stimulated with alanine, arginine or α-KIC. Homocystein might play a role in Type 2 diabetes as elevated homocystein levels have been reported in these patients .
1.7 Regulation of glucagon secretion
The regulation of glucagon secretion from α-cells is still a matter of debate. The most favoured hypothesis suggests that paracrine or autocrine mechanisms are the most relevant. Thus, β-cell secretory products during hyperglycemia modulate α-cell activity . The most important regulators are summarized in the following illustration:
Figure 1.12: Summary of main physiological regulators of α-cell stimulus secretion coupling .
Insulin has been well characterized as an inhibitor of glucagon secretion from α-cells. It is secreted during hyperglycemia from pancreatic β-cells . Several studies have demonstrated the impact of insulin on glucagon secretion. Inhibition of insulin action by administration of insulin antibody in in vitro studies in rats resulted in an increase in glucagon release [36, 149, 150]. Furthermore, in mice lacking GSIS, insulin-mediated inhibition of glucagon secretion was absent [151, 152]. Conversely, administration of exogenous insulin to isolated rat α-cells or rodent islets inhibited glucagon secretion at basal but not at elevated glucose concentrations [36, 153]. Furthermore, several studies demonstrate the impact of intra-islet insulin concentrations on glucagon release [154, 155]. Therefore, the β-cell “switch-off” hypothesis was postulated suggesting that glucagon secretion during hypoglycaemia requires the cessation of insulin release from β- cells. The discovery of insulin receptor expression on α−cells supports this hypothesis . It was also demonstrated that insulin inhibits electrical activity and glucagon secretion from isolated rat α-cells . This inhibition is probably caused by KATP-channel activation and membrane hyperpolarization. Insulin has also been reported to activate GABAA-receptors on α-cells and inhibit glucagon secretion .
Another putative paracrine signalling molecule is the metal ion zinc (Zn2+). Zinc co- crystallizes with insulin in β-cell secretory granules and is co-released from rat and