Type 1 Diabetes: Cellular, Molecular & Clinical Immunology

Chapter 2 - The Pancreatic Beta-Cell
Suparna A. Sarkar, 3/1/2012 Update of chapter by Kirstine Juhl, John C. Hutton and George S. Eisenbarth

Cell Therapy of Diabetes PowerPoint slide set - Updated 7/06
Proprotein Processing and Pancreatic Islet Function PowerPoint slide set - Updated 11/06
Stimulus-Secretion Coupling in the Pancreatic Beta-Cell PowerPoint slide set - Updated 7/08

Human Fetal Pancreas Development slide set Updated 03/12

Introduction
The two most common forms of diabetes in man (Type 1A and Type 2) have very different etiologies and different clinical presentation¹. Nevertheless, the underlying loss of islet beta cell function has similar consequences in terms of glycemic control and the emergence of long-term complications. type 1 Diabetes (T1D) is a polygenic T-cell dependent autoimmune disease, characterized by the selective destruction of the ß-cells of the islets of Langerhans^1-6 and that susceptible individuals have inherent defects in critical immunomodulatory mechanisms⁷ that increase the risk of a pathogenic rather than protective immune response to self^{6, 8, 9}. Type 2 Diabetes is typically linked to dysmetabolism or metabolic syndrome and the presence of insulin resistance, however a large subset of T1D patients routinely exhibits insulin resistance^10-13 contributing to the metabolic distress in islets.  With the rising incidence of T1D and T2D, it is now being argued that both T1D and T2D are essentially disorders of altered insulin resistance set against the backdrop of genetic susceptibility¹⁴ and the inflammatory process; in T1D brought about by the autoimmune component of the disease process¹⁵.

In type 1A (autoimmune diabetes) the loss of beta cells is often close to absolute with less than 1% of beta cells remaining in patients with long-term diabetes^16-18 with prolonged C peptide production¹⁹.  In most patients with almost no remaining beta cells, essentially all of the islets are devoid of beta cells while islets contain cells expressing glucagon and somatostatin. Such islets are termed pseudoatrophic islets²⁰. Nevertheless, some beta cells remain often as scattered single cells in the parenchyma and ducts.  In a small subset of patients, even with long-term type 1A diabetes, significant C-peptide is present and lobules of pancreas remain where all the islets contain beta cells and appear essentially normal in terms of expression of insulin while the rest of the pancreas is devoid of beta cells in islets^20-22. The figure below illustrates a section of one such pancreas from the nPOD collection (jdrfnpod.org) where the pancreatic lobule on the right is stained dark and at higher power one can observe that all of the islets in this lobule lack insulin²³.  In contrast to the lobule on the left, all of the islets contain insulin.  The dark staining of the right lobule likely results from pancreatic acinar atrophy that occurs with severe loss of pancreatic insulin.  Shrinkage in overall pancreatic mass in patients with type 1 diabetes has long been noted^24-26. Analysis of decreased pancreatic volume was recently combined with imaging of iron particle pancreatic accumulation to help distinguish patients with type 1 diabetes from normal controls^27-29.

 In fact, C-peptide secretion in long-standing diabetic patients has now been explained by two different patterns of beta cell survival, which possibly reflect different subsets of type 1 diabetes.  In a recent study²⁰ associated Pattern A with type 1A diabetes that histologically had lobular retention of islet areas with “abnormal’ beta cells producing the apoptosis inhibitor BIRC5 (survivin) and HLA class I. In pattern B, 100% of all islets contained normal-appearing but quantitatively reduced beta cells without survivin or HLA class I. Baculoviral IAP repeat 5 BiRC5³⁰ is an apoptosis inhibitor that is produced in the beta cells of fetal human pancreas³¹, but not in adult islets. It is also found in the beta cells in areas of pancreatitis³². The presence of survivin, in all surviving islet beta cells Pattern A patients, may result from 1) inflammatory changes that did not result in beta cell destruction of a subset of islets or 2) be protected from destruction and further lymphocytic infiltration. An alternative hypothesis extended by the authors is that lobular regions with beta cells of Pattern A pancreas represent areas of beta cell regeneration. Although the sudden onset of type 1A belies the fact that the underlying loss of beta cell mass is the culmination of many years of gradual and progressive loss of beta cells in the face of autoimmune attack which is first evident with the appearance of autoantibodies to islet proteins in the preceding years (see other chapters)^33-38. In the NOD mouse the infiltration of the islets with immune and inflammatory cells that initiates the disease first appears in the islets of the pancreatic periphery, affects a subpopulation of islets and is possibly benign or at least kept in check by the presence of regulatory T cells^39-42. The invasive insulitis seen in NOD mice closer to disease onset may reflect a change in the balance of destructive and protective responses in favor of the former. The histological changes in man are comparatively mild and may reflect the slower progression of the disease or possibly a different immune process. The islet tends to be viewed as the source of autoantigen that supports or initiates the immune attack and ultimately the victim of the crime.

Histopathological examination of pancreata from diabetic organ donors procured from nPOD was examined with the goal to provide a foundation for the informed selection of potential therapeutic targets within the chemokine/receptor family⁴³. CCL5, CCL8, CCL22, CXCL9, CXCL10 and CX3CL1 were the major chemokines transcribed and translated by human islet cells in response to in vitro inflammatory stimuli. CXCL10 was identified as the dominant chemokine expressed in vivo in the islet environment of prediabetic animals and T1D patients, while CCL5, CCL8, CXCL9 and CX3CL1 proteins were present at lower levels in the islets of both species. Importantly, additional expression of the same chemokines in human acinar tissues emphasized an underappreciated involvement of the exocrine pancreas in the natural course of T1D that will require consideration for further T1D pathogenesis and immune intervention studies.

Undoubtedly, much more needs to be learned about the reaction of the islet to cytokine mediators of the immune response and about how the beta cell manages to survive so long or replenish its population from progenitor cells in the pancreas. Since the mechanism of autoimmune destruction by effector cells may be mediated by CD4+ cells, and thus indirect, there is also the question of whether the beta cell is uniquely susceptible to oxygen and nitrogen free radicals or cytokine mediators of cell death which may account for the fact that other islet cells exposed to same molecules survive while the beta cell dies.

The focus of the following review is to discuss the wealth of information regarding the physiological and pathophysiological responses of the islet to nutrient secretagogues and pharmacological agents and to emphasize how the beta cell differs from its neighbors and from other endocrine tissues and how it may participate in its own demise in type 1 diabetes. The review also illustrates the challenges faced by investigators wishing to genetically engineer non-b cells for cellular therapy of type 1 diabetes or wanting to introduce specific genes into the beta cell population to afford it greater protection from autoimmune attack.

Development of the Human Pancreas

Similar to the mouse pancreas, the human pancreas develops from two endodermal diverticula, the dorsal and ventral⁴⁴, which fuses around 56 days post coitum of development⁴⁵ .  The pancreas comprises of 3 important cell lineages: Endocrine, acinar and ductal (which together make up the exocrine pancreas). The morphogenesis of the endocrine tissue, however, is unlikely to be equivalent given the differences in gestation (260 vs 20 days) and the larger relative volume of the human pancreas⁴⁶. Human fetal pancreases obtained at gestational ages 9–23 weeks were processed in parallel for immunohistochemistry and gene expression profiling by Affymetrix microarray⁴⁷. At 9–11 weeks, the pancreas was made up principally of mesenchymal tissue interspersed with PDX1 positive branched epithelial structures containing scattered hormone-negative neurogenin3-positive endocrine cells. Protoacinar structures marked by carboxy esterase lipase (CEL) expression were noted by 15–19 weeks, along with clusters of endocrine cells producing either glucagon or insulin. By 20–23 weeks, vascularized islet-like structures appeared. Analysis of Ki67 immunoreactivity showed that the replicative rate of endocrine cells was low and suggested that the endocrine expansion was derived from hormone-negative precursors. Insulin, glucagon, somatostatin, ghrelin and pancreatic polypeptide transcripts were present at 9–10 weeks as confirmed by quantitative PCR and increased progressively, commensurate with the expansion of endocrine cell volume. The human equivalent of a mouse endocrine secondary transition was not evident, neither in terms of morphology nor in dramatic changes in endocrine-specific transcriptional regulators. By contrast, exocrine genes showed a marked transition at around 11 weeks, associated with a greater than six-fold increase in exocrine gene transcripts.  The terminal differentiation of human endocrine tissue into late gestation and the presence of NEUROG3 are in contrast with findings in the mouse, where neurog3 is transiently expressed from e12.5–e15.5. This indicates that the human fetal pancreas could provide an abundant islet precursor cell population that could be expanded ex vivo for therapeutic transplantation for the treatment of brittle and unstable type 1 diabetes. The ductal cells also develop from the PDX1 expressing primordial pancreatic epithelium and expresses Cytokeratin 19 (CK19), cystic fibrosis transmembrane receptor, DBA lectin, Carbonic anhydrase 2⁴⁸. (For images of the human fetal pancreas development, please refer to power-point slides).

Physiology of the islet of Langerhans

The endocrine pancreas is arranged in clusters of secretory cells the Islets of Langerhans scattered throughout the exocrine glandular tissue (Fig. 1)^{49, 50}. In man, the pancreas contains around one million islets that comprise 1-2% of the total mass of the gland. The islets are separated from the exocrine tissue by a capsule made up of connective tissue fibers and by glial like cells and human islets vary in size from less than 50 up to several thousand cells. Four different endocrine cell types are contained in the islets; beta cells, which produce insulin and constitute 60-80% of the endocrine cell mass, glucagon secreting a-cells (10-20%), somatostatin producing d-cells (~5%) and pancreatic polypeptide secreting PP-cells (<1%)⁴⁴.   The cells within islets are critically dependent upon a series of transcription factors during embryonic development and of note, in knockouts of the homeodomain protein nkx2.2, almost all islet cells are replaced with ghrelin producing cells⁵¹.  In parallel, in the human pancreas, the endocrine genes insulin, glucagon and somatostatin steadily increase from 9–15 weeks, tapering off by 18–23 weeks⁴⁷.  This was in tandem with other dense core secretory granule markers such as PCSK1, PCSK1N, PTPRN, SLC30A8, IAPP, CHGA and CHGB that peak in the later phase of development. Components of the stimulus secretion coupling machinery such as GCK and GLUT2 (SLC2A4) are however more variable. The high signal intensity observed for insulin, glucagon and somatostatin correspond to the abundance of immunopositive cells detected by immunohistochemistry. Low signals for ghrelin (GHRL) and pancreatic polypeptide correspond to the lower frequency of these cells in the histological analysis.

Figure 1. The pancreas is located close to the duodenum. The enlarged part of the pancreas shows the exocrine acinar cells next to the islet endocrine cells. The islets consist of four different cell types, which secrete different peptide hormones. The majority of the islets are beta cells that secrete insulin. The a-cells secrete glucagon, d-cells secrete somatostatin and PP cells secrete pancreatic polypeptide.

Most mammalian islets have a beta cell rich core surrounded by a mantle of alpha-, delta- and PP-cells with some controversy as to applicability to human islets. Afferent arterioles enter the beta cell rich medulla where the arterioles split into a branching system of capillaries that traverse the beta cell mass before reaching the mantle of a- and d-cells. Thus, the beta cells are the first endocrine islet cell type to sense blood-borne metabolic changes. Insulin is known to inhibit glucagon secretion and glucagon to stimulate insulin secretion. Somatostatin will inhibit the secretion of both insulin and glucagons. Somatostatin's inhibition of insulin secretion is reported to result from its inhibition of glucose metabolism of the beta cell (14) and glucagon; hence the anatomical relationship of the islet cells is critical to the function of the organ.  The islets are innervated by autonomic nerve fibers, containing both parasympathetic and sympathetic nerves that enter the islets and terminate in proximity to the endocrine cells.

The architecture of the human islets is however more heterogeneous. A progressive increase in the endocrine cell population is evident from 11–23 weeks, reflecting both the progressive expansion of the pancreatic epithelium and an increase in the density of endocrine cells within the epithelium⁴⁷. With increasing fetal age evidence of formation of endocrine cell clusters especially from 15 weeks onwards is noted, although solitary endocrine cells were still observed at all time-points. The endocrine cell clusters at early time-points displayed a significant homotypic association of either insulin- or glucagon-positive cells. At later developmental time-points, heterotypic endocrine cell clustering and the appearance of typical islet-like structures is a feature.

The Beta Cell as a Neuron-like Cell

Though islet beta cells are derived from endoderm and not ectoderm they share many properties with neurons that probably relate to their function in terms of secreting a peptide hormone stored in dense core secretory granules and having microvesicles with "neurotransmitters".   The neuron-like properties include presence of microvesicles containing molecules such as GABA gamma-Aminobutyric acid (GABA)⁵², an inhibitory neurotransmitter, expression of enzymes such as Glutamic acid decarboxylase (GAD, both GAD65 and GAD67), IA-2 (ICA512)^{53, 54}, IA-2beta (phogrin)⁵⁵. Surface expression of complex neuronal/Oligodendrocyte gangliosides such as GQ gangliosides (e.g. target of monoclonal A2B5) and the targets of monoclonals 3G5⁵⁶ and R2D6⁵⁷, and expression of type 2 monoamine vesicular transporters(VMAT2)^{58, 59} is also seen.  These shared neuronal/neuroendocrine properties are of importance in considering the pathophysiology of autoimmune beta cell destruction (e.g. though GAD65 autoantibodies are characteristic of type 1 diabetes of man, only beta cells and not the many other cell types that express GAD65 are destroyed in type 1A diabetes and in contrast in Stiff Man Syndrome neurologic disease predominates in association with very high levels of GAD65 autoantibodies) and recently for the in vivo detection of islets, potentially for determining beta cell mass.  PAM peptidylglycine alpha-amidating monooxygenase gene encodes a multifunctional protein with two enzymatically active domains with catalytic activities; peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL)^60-62. These catalytic domains work sequentially to catalyze neuroendocrine peptides to active alpha-amidated products is also found in hypothalamic magnocellular neurons, the hippocampal formation, and olfactory cortex⁶³ and human endocrine pancreas⁶⁴. PAM is localized primarily in the secretory granules of alpha cells not in the beta, PP and Delta cells. Alpha cells secrete GLP1, which possess a carboxy terminal amide group⁶⁵ while beta and delta cells that secrete insulin and somatostatin respectively which are not terminally amidated. Investigators have utilized [(11)C]Dihydrotetrabenazine ([(11)C]DTBZ) that binds specifically to VMAT2, with PET scanning to estimate beta cell mass in the BB type 1 diabetes rat⁵⁹.   It is possible that many of the technologies developed to interrogate the brain (e.g. MRI spectroscopy) will be applicable to studies of islet beta cells, if the goal is determination of approximate mass rather than direct visualization of the relatively small islets, given the shared biochemistry of neurons and islets.

The Biosynthesis of Insulin

 
The islet beta cell is the principal cell in the adult mammal able to transcribe the insulin gene. The insulin receptor by comparison is widely distributed even on cells that are not thought to be insulin responsive and in the case not even exposed to significant concentrations of the hormone. This may reflect the evolution of the molecule from primarily a neurotransmitter to an endocrine function⁶⁶.  Of note a series of rare mutations which result in misfolded insulin and neonatal diabetes have now been identified⁶⁷.  Beta cell death may be the result of ER stress^68-70.
Insulin mRNA is translated to a pre-pro-insulin peptide, which is rapidly processed in the rough endoplasmic reticulum to pro-insulin by removal of the N-term signal peptide. Proinsulin contains the A and B chains of insulin linked by the C peptide, a structure that helps to align the disulfide bridges that are generated between the A and B chain. Proinsulin is transported through the Golgi apparatus and thereafter further processed in the maturing granule to insulin by excision of the C peptide by the endopeptidases, PC1 and PC2 (prohormone convertases), and carboxypeptidase H. The bioactive insulin molecule consists of an A and B chain (21 and 30 amino acids, respectively), linked intramolecularly by disulfide bridges^71-77.

Role of Zinc in insulin biosynthesis (Leah Sheridan, PhD)

Insulin secretion is tightly regulated by extracellular secretagogues/inhibitors, as well as intracellular signaling pathways.  It is packaged in dense core vesicles (DCVs) as a hexamer bound to two Zn²⁺ ions and remains stable in this configuration at pH5.5 until fusion of the DCV and plasma membranes during exocytosis^{78, 79} . The complex is then released into the pH neutral circulation, and dissociates, allowing both insulin and Zn²⁺ to act independently.

The Zn²⁺ content of pancreatic b-cells is one of the highest in the body⁸⁰.  This is evidently due to its additional role in insulin crystal formation, beyond its various fundamental roles in protein stability and function, DNA replication, metabolic enzyme activity, and cellular protection against apoptosis and oxidative stress^{81, 82}.  In addition, co-secreted Zn²⁺ is believed to play both an autocrine and paracrine role by activating K_ATP channels, thereby inhibiting the secretory process^{83, 84}, and regulating alpha cell glucagon secretion^{84, 85}. Zn²⁺ is also implicated in b-cell death via a paracrine mechanism⁸⁶.  Although it is apparent that mammalian cells require Zn²⁺ for many cellular processes, it can also be toxic. The regulation of intracellular Zn²⁺ is therefore of great importance to maintain homeostatic cellular physiology. One such regulatory mechanism involves controlling the influx and efflux of Zn²⁺ across membranes, both cellular and subcellular, by Zn²⁺ transporters. 

Mammalian Zn²⁺ transporters were first discovered in the mid-1990s, and were shown to transport Zn²⁺ and other metal ions into the cytoplasm either from the extracellular space, or from the organellar lumen. These were members of the SLC39 (also known as ZIP) family of Zn²⁺ transporters. Later, the SLC30 (also known as ZnT) family of Zn²⁺ transporters were identified that worked counter to ZIPs, in that they transport Zn²⁺ out of the cytoplasm, either out of the cell into the extracellular space, or into the lumen of organelles^{87, 88}. Mammals carry genes for 10 homologous Zn²⁺ export proteins (ZnT1 through 10). The overall homology of ZnT9, however, to other members of the ZnT family is very low⁸⁹, and may not function in Zn²⁺ transport, but potentially in DNA excision-repair^{90, 91}.  ZnT8 and ZnT10 were the latest members to be identified, and akin to ZnT1-7, share the same predicted structure of six transmembrane (TM)-spanning domains with cytoplasmic amino (N) and carboxy (C) terminals (see above cartoon), and contain a histidine-rich intracellular loop between TM IV and V which may act as the Zn²⁺ binding region. ZnT6 is an exception, in that it retains the prokaryotic serine-rich loop. Despite the similar topological structure of the ZnT family, the overall sequence similarities are relatively low, with the highest being 53.5% between ZnT2 and ZnT8⁸⁹. This sequence divergence may reflect the different localizations of each transporter (i.e. ZnT1-plasma membrane, ZnT2-endosome/lysosome, ZnT3-synaptic vesicles, etc)^{92, 93} and correspondingly the different tasks and means of regulation in which each transporter partakes; a combined bioinformatics/molecular engineering strategy to identify potential molecular targets of circulating type 1 diabetic autoantibodies⁹⁴.  In such, they identified 51 candidate islet autoantigens from microarray mRNA expression profiling experiments on isolated mouse and human pancreatic islets, FACS sorted b-cells and mouse pancreatic tumor cell lines. Among the candidates, this study identified the Zn²⁺ transporter ZnT8 as a novel type 1 diabetes (T1D) autoantigen, and furthermore, localized the epitope of immunoreactivity to the C-terminus of the protein. Among the other major known targets of diabetes autoantibodies (insulin, GAD, IA2, and phogrin) identified by the study, ZnT8 was found to be b-cell specific, displayed moderate to abundant islet expression, and is associated with the regulated secretory pathway. 

The identification of ZnT8 with roots in the pathogenesis of T1D creates opportunities for the development of improved diagnostic capabilities as well as provides an additional strategy for the creation of therapeutic interventions. Like the other four main autoantigens associated with T1D studied to date (insulin, GAD, IA2, and phogrin), ZnT8 has been shown to be associated with the regulated pathway of secretion, having been localized to intracellular vesicles in HeLa cells, and co-localized with insulin granules in INS-1 cells and insulin in human islets^{95, 96}. The importance of such an association with the secretion pathway remains to be elucidated; however, one can speculate that trafficking of these antigens to and from the surface membrane may play a role in the diabetic pathology. Due to the specific function of insulin secretion by pancreatic b-cells, proteins specifically related to that function are potentially more likely to be targeted by the immune system. Several possible mechanisms come to mind:  1) Proteins associated with DCVs (i.e. insulin, phogrin) that get trafficked to and from the surface in response to insulin secretagogues, expose extracellular epitopes that are recognized by T Cell receptors or circulating antibodies secreted by B cells; 2) Epitopes may be exposed to the immune system during normal tissue turnover, especially for intracellular proteins (i.e. GAD and IA2) that would not expose surface epitopes; 3) Recycling of secretory pathway associated proteins enables access to post-golgi compartments where the protein could be cut up by proteosomes and presented on the cell surface by MHC I for recognition by T Cells.  These are only a few of many potential cellular mechanisms by which a protein may become antigenic.  In light of the recent discovery of ZnT8 and its identification as a T1D autoantigen, it is essential to investigate its role in pancreatic b-cells to gain insight into its mechanism of antigen presentation, and furthermore, to generally learn why many other autoantigens are associated with the DCV (i.e. insulin, CPE and amylin). One study has been conducted on the functionality of ZnT8 to date, which showed that its overexpression does not play a role in Zn²⁺ toxicity, and protected INS-1 cells, an insulin-secreting cell line, from death during Zn²⁺ depletion (86).  ZnT8 therefore appears to be a functional channel that actively participates in intracellular Zn²⁺ transport. However, much remains to be discovered about this transporter. It has been shown to co-localize with insulin⁹⁵, but whether ZnT8 is trafficked to the surface membrane with insulin upon stimulation, and therefore truly associated with DCVs remains to be shown.  In addition, if ZnT8 is trafficked to the surface membrane, how long it lingers there before being re-endocytosed, and where it is trafficked to following its retrieval is unknown. The processes of DCV exocytosis and endocytosis are well studied. DCVs undergo regulated exocytosis in response to signals that elevate cytosolic Ca²⁺, a process that must ultimately be coupled to endocytosis of the granule membrane to avoid significant increases in cell surface area.  The concept that exocytosis can occur by two related, but mechanistically distinct mechanisms (“kiss-and-run” vs. “complete-collapse”)^97-99; has its endocytic counterpart that “kiss-and-run” events are associated with rapid clathrin-independent retrieval, and “complete-collapse” with a slower clathrin-dependent mechanism^{97, 98, 100}. In neuroendocrine tissues the type of event that occurs is dictated by the strength and duration of the stimulus¹⁰¹, and possibly related to the prevailing cytosolic Ca²⁺ level^{97, 102}. Thus clathrin-independent endocytosis predominates in the initial phases of the secretory response, while chronic stimulation (> 5 min) leads to a shift to a mainly clathrin-dependent mechanism and full fusion of DCVs¹⁰¹. Insulin secretion in vivo is biphasic, which may reflect the release of docked vs. recruited DCVs, changes in the stimulus intensity as reflected by plasma membrane potential and cytosolic Ca²⁺, or indeed the mechanism of granule fusion and retrieval as discussed above^103-105. 

Most mammalian insulin binds Zn co-ordinately forming hexamers and can form large crystalline arrays which promote condensation and aggregation of the hormone in the acidic core of the secretory granule. Proinsulin although forming Zn hexamers is prevented from further aggregation by the highly charged C-peptide, a physical property that may be important in retaining its solubility in the proximal elements of the secretory pathway. In the mature secretory granule C peptide exists in equimolar amounts with insulin in the granule and is co-secreted thus making it a useful marker for beta cell function in diabetics receiving insulin therapy. Transcription of the insulin gene is modulated by nutrient secretagogues, a variety of cytokines and by insulin itself. Insulin signal transduction enhances transcription of the insulin gene through phosphorylation of insulin receptor substrate (IRS)-1 and -2, and activation of phosphatidylinositol-3-kinase (PI3K) and it has been postulated that insulin plays a feed forward role in insulin biosynthesis ensuring that the stores of insulin are always replenished¹⁰⁶. Exogenous insulin is also shown to hyperpolarize beta cell plasma membrane in mice by insulin-induced activation of the KATP channels possible through the PI3K thereby turning off the islet insulin secretion¹⁰⁷. Glucose is also shown to stimulate the production of proinsulin through rapid activation of translation of a pre-formed pool of mRNA¹⁰⁸. Given the high rates of proinsulin synthesis and the need for proper folding of proinsulin within the endoplasmic reticulum it has been hypothesized that pancreatic beta cells are particularly susceptible to apoptosis induced by endoplasmic reticulum (ER) stress¹⁰⁹. Wolfram syndrome (DIDMOAD: Diabetes Insipidus/Diabetes Mellitus/Optic atrophy/Nerve Deafness) results from mutations of the WFS1 gene which is a transmembrane protein of the ER regulated by the unfolded protein response and with a role in ER calcium transport¹⁰⁹.  

The proinflammatory cytokines IL-1b, TNFa and IFNg modulate transcription of the insulin gene. Culturing human beta cells 48 or 72 h with single cytokines does not affect the cellular content in insulin or proinsulin whereas the combination of several cytokines (IL-1β + IFNγ) disproportionally elevates medium proinsulin levels¹¹⁰. This is caused by a conserved proinsulin synthesis and a slower hormone conversion possibly contributed to lower expression of PC1 and PC2 convertases. These cytokines mediates signal transduction involving binding to specific receptors, through MAPK and SAPK and mobilization of diverse transcription factors: NFkB, AP-1 and STAT-1 involved in beta cell apoptosis ^{111, 112}

Physiological regulation of islet hormone secretion

 
The blood glucose level is maintained within a narrow range around 5 to 7 mM in the fasting state mainly by the combined and reciprocal action of insulin and glucagon^{113, 114}. Essentially, when the blood glucose concentration is elevated after a meal, the beta cells are stimulated to secrete insulin. Conversely, when blood glucose levels are low the a-cells are stimulated to secrete glucagon, which leads to glycogenolysis and gluconeogenesis in the liver and therefore release of glucose to the blood. Between meals, when the plasma glucose decreases, the release of the neurotransmitter norepinephrine and the neuropeptide galanin from the sympathetic nerves will directly activate glucagon release and inhibit insulin release¹¹⁵. During a meal or the postprandial state, the parasympathetic nerves potentiate glucose-induced insulin release from islet beta cells. This is achieved by the release of the neurotransmitter acetylcholine and the neuropeptides pituitary adenylate cyclase activating polypeptide (PACAP), vasoactive intestinal polypeptide (VIP), GLP-1 ^116-119 and GIP¹¹³. The innervation of the islet and its microanatomy is probably important in the orchestration of these responses. Somatostatin inhibits both insulin and glucagon secretion and potentially serves as a modulator of islet hormone release¹²⁰. However, it is unclear whether it has a physiological role given the vascular anatomy of the islet in which the secreted products from the a- and d-cell enter the blood circulation without reaching the beta cells. The physiological role of islet PP is unknown, but appears not to affect the secretion of the other islet hormones except a possible inhibition of islet insulin release^121-123. Rare cells within islets secrete ghrelin, with ghrelin primarily produced in the gastrointestinal tract and ghrelin under experimental conditions can influence insulin secretion and is reported to increase IA-2 beta but not IA-2 message in islets and inhibition of IA-2 beta with RNAi decreased ghrelin inhibition of insulin secretion^{124, 125}.  Much of the physiological regulation of insulin secretion can be reproduced in vitro and a wealth of information has been generated from the study of isolated islets, which appear to behave as autonomous organs. Such studies have led to the classification of insulin secretagogues into three groups, initiators, potentiators and inhibitors. Glucose is the most important initiator in monogastric animals, but other carbohydrates, amino acids and fatty acids alone can also initiate insulin secretion. Pharmacological agents like the clinically useful sulphonylureas tolbutamide and glibenclamide also initiate insulin secretion in a glucose-independent manner. Arginine initiates insulin secretion by depolarizing the plasma membrane because of its transport in a positive charge form. It triggers insulin release by increasing [Ca2+]i through a KATP channel-independent pathway^{126, 127}. Potentiators of insulin secretion do not initiate insulin secretion by themselves but enhance stimulation of secretion in the presence of an initiator. Examples of potentiators are the intestinal glucoincretin hormones glucagon-like peptide 1 (GLP-1)¹²⁸ and gastric insulinotropic polypeptide (GIP), which provide fine regulators of insulin secretion in the post-prandial state along with neurotransmitters. Other neurotransmitters and hormones such as somatostatin, galanin, adrenalin and even endocannabinoids (or potentially increase through CB2 receptors ¹²⁹ reduce insulin secretion (Fig. 2).

Figure 2. Examples of potentiators, initiators and inhibitors of insulin secretion from the pancreatic beta cell. Abbreviations used: GIP, gastric insulinotropic polypeptide; VIP, vasoactive intestinal polypeptide; GLP-1, glucagon-like peptide 1.

Islet Pathophysiology in type 1 diabetes

 
Type 1 diabetes is characterized by the appearance of circulating antibodies targeted at beta cell proteins, which appear many years before the onset of clinical disease. Proteins such as insulin, GAD, IA2, IA-2 beta (phogrin) and CPH (carboxy-peptidase H) are specifically targeted but as yet it is not settled if any one (or unknown targets) of these molecules is primary or dominant in the autoimmune response. In the NOD mouse model there is accumulating evidence that insulin may be a primary autoantigen with knockouts or alterations of specific insulin sequences preventing diabetes, while knockouts of GAD65, IA-2, IA-2 beta (and even both IA-2s) do not change the course of development of NOD diabetes^130-132. A recent study of lymphocytes from pancreatic lymph node of patients with type 1 diabetes also implicated insulin¹³³. Insulin is the only molecule in this group that is specific to the beta cell though the others have the common feature of being associated with regulated pathway of secretion. Insulin and CPH are secreted molecules and to some extent remain on the cell surface following exocytosis. By contrast the dominant epitopes seen by antibodies reactive to GAD, IA2 and phogrin are intracellular. It is presently unknown whether the beta cell is targeted directly or indirectly, whether the antigens are presented by the living cell or through death or antigen shedding and why the a-cell survives in spite of sharing many autoantigens with the beta cells and being bathed in the same milieu of soluble immune effector molecules. One hypothesis is that the beta cell participates in its own destruction by increased presentation of antigens in response to glucose, a situation that worsens as beta cell mass decreases and the remaining cells compensate by increased transcription and translation of secretory pathway proteins¹³⁴. Beta and alpha-cells have a differential sensitivity to cytokines in that inhibition of glucose-stimulated insulin release by IL-1 is reversible, whereas the effect on glucose-modulated glucagon release is not¹³⁵. The beta cells are more sensitive to cytokines than the other three endocrine cell types in the islets. Cytokine-induced free radicals in beta cells such as NO catalyzed by the inducible nitric oxide synthase¹³⁶ may be involved in beta cell-specific destruction in type 1 diabetes¹³⁷. NO is an important mediator but not the sole mediator of cytokine-induced cytotoxicity in beta cells as shown by the fact that inhibition of NO production in human islets did not prevent cytokine-induced apoptosis, a-cells do not express NO in response to cytokines. Beta cells but not a-cells induce the expression of heat shock protein 70 (hsp70), heme oxygenase and MnSOD (manganese superoxide dismutase) upon exposure to IL-1¹³⁸. Native beta cells have been shown to possess low scavenging potential for oxygen-derived free radicals and over expression in beta cells of scavenger or antioxidant enzymes such as MnSOD, catalase and glutathione peroxidase has been shown to increase their survival after exposure to NO and reactive oxygen species¹³⁷. Production of oxygen free radicals mediated by macrophages can damage beta cells directly resulting in type 1 diabetes in NOD mice¹³⁹. Superoxide dismutase and catalase protected isolated beta cells against alloxan-induced diabetes in vivo indicating a role for superoxide radicals and hydrogen peroxide in the toxicity of alloxan¹⁴⁰.
Studies with isolated islets have provided models of how mediators of the immune response may damage the islet in the autoimmune response in type 1 diabetes. The macrophage cytokine IL-1b induces an initial phase of functional stimulation in rodent pancreatic islets, which is followed after 4-7 hours by a progressive inhibition of (glucose-induced) insulin release and eventually overt damage to the beta cell caused by production of NO^{136, 141}. The two cytokines, TNFa and INFg potentiate the IL-1b-induced production of toxic NO and oxygen free radicals which inhibits insulin secretion^{142, 143}. This has led to the concept that IL-1b in combination with INFg and TNFa plays an important role for beta cell dysfunction and death. The general process of cytokine-induced beta cell 'de-differentiation' with impairment of some of the most differentiated functions of beta cells, such as the preferential mitochondrial metabolism of glucose and the biosynthesis and release of insulin is paralleled by the activation of proteins related to cell survival, such as heat shock proteins and antioxidant enzymes¹⁴⁴. There are important species differences in beta cell response to IL-1. IL-1 has stimulatory effects on human islets with no formation of NO compared to rodent islets, which produces NO when exposed to IL-1. Human islets cultured in the presence of IL-1b alone exhibit a more prolonged stimulatory phase, which may last up to 48 hours¹³⁶. But again, with prolonged exposure of human islets to IL-1b + TNFa + INFg an inhibition of human islet function is also observed. This difference between species is probably due to better capacity of human islets to scavenge oxygen free radicals and to the higher content of hsp70, catalase and superoxide dismutase in these cells¹¹². Hsp70 has a direct anti-apoptotic role by inhibiting protein aggregation, decreasing formation of oxygen free radicals and blocking effector caspases and it could also decrease necrosis by preventing cellular ATP depletion. The cytokine-induced beta cell de-differentiation is probably associated to cytokine-induced down-regulation of pancreas duodenum homeobox gene-1 (PDX-1) and Isl-1. PDX-1 is a crucial gene for beta cell development and for the maintenance of its differentiated phenotype (see chapter with Jensen). The decreased expression of PDX-1 together with the inhibition of Isl-1 expression could contribute to the observed decreased expression of insulin and glucokinase mRNA which depend on PDX-1 for their regulated transcription. While IL-1b inhibits PDX-1 and Isl-1 expression and up-regulate c-myc it does not trigger beta cell apoptosis¹¹².Plasma type II secreted phospholipase A2 (PLA2) may be closely involved in the pathophysiology of acute pancreatitis. The serum levels of type II PLA2 has been shown to correlate with the severity of the disease¹⁴⁵. Also, an inhibitor of type II PLA2 protects the pancreas against tissue damage when pancreatitis is induced in vitro¹⁴⁶. Interestingly PLA2 enzyme isoforms are also produced by inflammatory cells and by the islets themselves (see below). As in the case of the soluble immune effector molecules we have little information on the actual concentrations to which the beta cells are exposed and the actual in vivo response in terms of regulation of receptors and signaling pathways.

The Stimulus-Secretion Coupling of the Pancreatic beta cell
The pancreatic beta cell is unusual from the biochemical standpoint in that it has a high rate of glycolytic and oxidative metabolism and that its glucose metabolism is largely insulin-independent. Most-strikingly it responds to extracellular glucose concentrations by increased metabolic flux even at concentrations of the sugar as high as 25 mM. The latter provides a unique mechanism that couples the availability of metabolizable nutrients to the control of secretory, translational, and transcriptional processes without the mediation of ligand-specific cell surface receptors. It may also be the Achilles heel of the cell in that intracellular proteins are exposed to higher concentrations of glucose and reactive metabolites than would otherwise occur in a typical cell that restricts glucose entry and its metabolism to meet its energetic demands. Within the secretory granule insulin is stored complexes with zinc and the beta cell has mechanisms to take up zinc¹⁴⁷ and transport it into the secretory granule (ZnT-8[expressed only in beta cells], a cation diffusion facilitator)¹⁴⁸. Two other transport systems that are abundant in islet beta cells and important for islet physiology are GPR40 (G protein-coupled receptor 40 a free fatty acid receptor) and 4F2 coupled amino acid transporters. GPR40 mediates the insulin secretory stimulation of fatty acids such as linoleic acid reportedly through cAMP and protein kinase A activity, and inhibition of delayed rectifying voltage gated K+ channels¹⁴⁹. The 4F2 heavy chain (CD98 heavy chain) is a component of heterodimeric amino acid transporters that is highly represented on the surface of islet beta cells¹⁵⁰. Of note this molecule is also an "activation" antigen and present at high levels on malignant cells^{151, 152}.

The beta cell Glucose Sensor

 
The beta cell glucose sensor consists of the combination of two molecules, which have a restricted tissue distribution, namely Glut2 and glucokinase (GK). Glucose enters the beta cell through the glucose transporter Glut2 (Km ~17 mM) and is quickly phosphorylated by GK to glucose 6 phosphate (G6P) (Fig. 3)¹¹⁴. Glut2 expression changes in diabetes and hyperglycemic states, which seems to underline its importance in the response of the beta cell, however there is great species variation in the expression of this molecule and little evidence per se that glucose entry is rate limiting to glucose metabolism in the beta cell. GK on the other hand, although present in other tissues such as the liver and kidney appears to be driven by a pancreatic specific promoter^{106, 153, 154}. Its high expression and accompanying downregulation of low Km forms of hexokinases^155-157 create a situation where kinetics of the enzyme dictates the kinetics of the generation of G6P in a cellular context. There has been considerable debate as to whether the beta cell has a specific glucose 6 phosphatase (G6Pase) and whether it participates with GK in a regulatory substrate cycling activity^{158, 159}. The beta cell expresses islet G6Pase related protein (IGRP) a beta cell specific homolog of the liver G6Pase catalytic subunit (55% identity) but the molecule is apparently not catalytically active¹⁶⁰. Of note IGRP is a major target of CD8 T lymphocytes of the NOD mouse¹³³. Other biochemical properties of the beta cell which have been implicated in its ability to sense metabolic fuels include the absence of lactate dehydrogenase and thus an inability to re-oxidize cytoplasmically generated NADH¹⁶¹ and the enhanced expression of the mitochondrial glycerophosphate shuttle^{162, 163} which may be involved in the transport of reducing equivalents in to and out of the mitochondria. Anaplerosis (the refilling of Krebs cycle intermediates) is shown to be implicated in the KATP-independent pathways of insulin secretion perhaps via malonyl-CoA formation and lipid esterification processes or through a pyruvate/malate shuttle generating NADPH¹⁶⁴. Anaplerosis requires conversion of pyruvate into oxaloacetate by pyruvate carboxylase an enzyme that is abundant in islet tissue¹¹⁵. Consistent with the view that anaplerosis is an essential component of beta cell signaling is the fact that the dose dependence of insulin release highly correlates with the accumulation of citrate, malate, and citrate-derived malonyl-CoA in the cell¹⁶⁴. The fact that the association of rise in citrate and a-ketoglutarate and initiation of insulin secretion does not require Ca2+ further indicates that anaplerosis is an early event of beta cell activation¹³¹. A net result of this biochemical machinery is that rates of glucose metabolism are strongly correlated to changes in the redox state of pyridine nucleotides (particularly NADPH^{165, 166} and the ratio of ATP/ADP in the cell both which may act as intracellular signals for ionic and other events in the cell (Fig. 3)¹⁶⁷. The beta cell is electrically excitable and when stimulated with glucose it shows oscillations in membrane potential characterized by bursts of 5-15 sec duration. Increasing glucose lengthen the burst and shortens the interval leading to continuous spiking over a base of DY <-40 mV^{168, 169}. The beta cell resting membrane potential of about -70 mV is mainly determined by the activity of ATP-sensitive K+ channels (KATP)⁶⁶. When the ATP/ADP ratio increases as a result of glucose metabolism, the KATP channels in the plasma membrane close^{170, 170-176}. This causes the cell membrane to depolarize due to the presence of an inwardly directed cation current that is dominant in the absence of a reduced KATP-conductance. The nature of this current is currently unknown but may be a member of the trp family that carries Na+ and Ca2+ ions¹⁷⁷. The resulting membrane depolarization leads to opening of the voltage-dependent L-type Ca2+ channels in the plasma membrane. The resultant Ca2+ influx elevates the intracellular free Ca2+ concentration [Ca2+]i and initiates exocytosis of insulin-containing secretory granules (Fig. 3).

Figure 3. Stimulus-secretion coupling in the pancreatic beta cell. Glucose is transported across the plasma membrane through the GLUT2 transporter. Glucose is metabolized through the glycolysis and Krebs (TCA) cycle. The increased ATP/ADP ratio leads to closure of the ATP-sensitive K+ channel in the plasma membrane, membrane depolarization and opening of the voltage-dependent Ca2+ channels. The resulting Ca2+ influx stimulates release of the insulin-containing granules by exocytosis. [Note: Trp is the transient receptor potential gene family that first was cloned from Drosophila where it function in photoreception, but which seems to include Ca2+ store-operated channels and inositol 1,4,5-trisphosphate-activated channels.]

Besides the effect of glucose on the KATP channels glucose is also shown to affect the stimulus-secretion coupling independently of the KATP channels thereby amplifying the pathway of glucose-induced insulin secretion¹⁷⁸. This has been shown by the fact that glucose dose-dependently increases insulin secretion when the cell is depolarized and KATP channels cannot be closed (diazoxide plus high K+)¹⁷⁹. Under conditions when the KATP channels are closed by sulfonylureas and therefore the beta cell plasma membrane is depolarized and insulin secretion is increased, glucose is shown to be able to further amplify insulin secretion dose-dependently. This KATP channel-independent (amplifying) effect of glucose on insulin secretion is Ca2+-dependent but not mediated by any further rise in [Ca2+]i¹⁷⁹. The amplifying pathway serves to augment the secretory response induced by the triggering signal and creates dose-dependent secretory response in the 5-25 mM concentration range. Glucose may also regulate insulin secretion by a Ca2+-independent mechanism^180-182. Under stringent Ca2+-deprived conditions when protein kinases A and C are activated simultaneously, glucose stimulates insulin release in the absence of an elevation of [Ca2+]i. This Ca2+-independent amplification of glucose-induced insulin release may require GTP late in stimulus-secretion coupling^{183, 184}. This has been interpreted in terms of two different mechanism of exocytosis, one driven by Ca2+ and the other by GTP. Others argue that glucose-induced insulin release is mainly regulated by the two Ca2+-dependent pathways (rise in beta cell [Ca2+]i and increase in Ca2+ efficacy) without significant contribution of a Ca2+-independent mechanism. In the following, some of the key molecular components in the beta cell stimulus-secretion coupling will be described.

The ATP-Sensitive K+ Channel Complex

KATP channels are expressed in different cell types including islets, heart, muscles and ventromedial hypothalamus (VMH) and serve to couple cell metabolism to membrane excitability. They are composed of a pore-forming complex consisting of subunits, a specific K+ channel (Kir6.2) surrounded by regulatory sulphonylurea (SUR) binding subunits. See reviews^185-187. SUR (Mw 140 kDa) is a member of the ABC-transporter superfamily and Kir6.2 (390 amino acids, 35 kDa) a member of the inward rectifier K+ channel superfamily. Four subunits of Kir6.2 and SUR1 each constitute the functional channel complex in islets^{188, 189}. KATP channel activity is modulated by a range of compounds and intracellular metabolites. Channel activity is reduced by Mg-ATP and sulphonylureas, the most commonly used class of compounds in the treatment of type 2 diabetes. ADP and diazoxide conversely activate KATP channel activity. Changes in the beta cell ATP/ADP ratio are an important physiological regulator of channel activity with an increase in the blood glucose concentration leading to an enhanced ATP/ADP ratio and consequently channel closure. Recently, KATP channels have also been detected in a- and d-cells^189-191, whereas the functional consequence of channel closure in the d-cell is similar to that in the beta cell (increased hormone release), inhibition of channel activity in the a-cell leads to inhibition of glucagon release¹⁹². The physiologic importance of this channel in man has become abundantly clear in the past several years with the discovery that approximately ½ of neonatal diabetes results from activating mutations of the Kir6.2 molecule. There is a hierarchy of disease dependent on the severity of the functional effect   of the mutations leading to transient neonatal diabetes, permanent neonatal diabetes, and finally a syndrome with mental retardation and neonatal diabetes (DEND syndrome: developmental delay, epilepsy, and neonatal diabetes^193-195. Paternal inactivating mutations of the genes for Kir6.2 and SUR1 combined with loss of the corresponding maternal chromosomal (11p15) region in focal areas of the pancreas leads to focal islet lesions, while autosomal inheritance of mutations results in diffuse pancreatic lesions, and both cause hyperinsulinemic hypoglycemia^{185, 196-201}. Currently the genetic bassis of congential hyperinsulinemia have been recognized due to associations and mutations in eight genes: ABCC8, KCNJ11, HADH1, GCK, GLUD1, SLC16A1, UPC2 and HNF4a^202-205.

 Mutations in HADH1 which encodes fatty acid oxidation enzyme  SCHAD causes recessive hyperinsulinemia²⁰⁶. Dominant mutations of GCK cause hyperinsulinemia that is resistant to therapy^{207, 208}. Dominant mutations of GLUD1 which encodes mitochondrial glutamate dehydrogenase is characterized by hyperammonemia²⁰⁹. It is thought that glutamate dehydrogenase mutations lead to increased ATP in islet beta cells, closing the K sensitive ATP channel and thus inappropriate insulin secretion and hypoglycemia. Abnormalities of mitochondria are increasingly recognized as central to beta cell abnormalities and in particular there are a series of mitochondrial mutations that lead to diabetes and more recently reports that mitochondrial "stress" and abnormalities related to mitochondrial "uncoupling" are important for insulin secretion defects in type 2 diabetes^{210, 211}.

The Beta Cell Calcium Channels
Membrane depolarization, resulting from KATP channel closure leads to elevation of cytosolic Ca2+ via voltage-gated Ca2+ channels in the plasma. The influx of Ca2+ in turn regulates several steps in exocytosis, such as the size of the readily releasable vesicle pool, the fusion event, and expansion of the fusion pore²¹². The Ca2+ concentration required for initiating insulin secretion in different experimental systems has been estimated in the range of 10-30 mM^{213, 214}. The resting level is in the submicromolar range.
Pancreatic beta cells are found to express N-, P/Q- and L-type Ca2+ channels, with the major contribution to Ca2+ influx mediated by the L-type channel (large and long-lasting)⁶⁶. The L-type channels are characterized by the requirement of a strong depolarization to activate. They inactivate slowly, which is seen in a patch-clamp experiment during a voltage-clamp depolarization to 0 mV where channel inactivation is maximal (Fig. 4)²¹⁵. The L-type channels are by themselves inactivated by Ca2+ ions at the inner side of the membrane, providing a negative feedback mechanism that limits Ca2+ entry into the cell²¹⁶. There is also a voltage-dependent component in the inactivation The beta cell L-type Ca2+ channel activity is blocked by dihydropyridines e.g. nifedipine and stimulated by the cyclic AMP/protein kinase A pathway. Elevated cAMP levels lead to a reduced rate of Ca2+ channel inactivation whereas the peak current is only moderately increased²¹⁷.

Figure 4. Ca2+ current in a mouse beta cell is mediated by L-type Ca2+ channels. The peak amplitude and the integrated Ca2+ current are depicted resulting from a membrane depolarization to 0 mV during a voltage-clamp experiment.

Molecular Motors and SNARES

When the Ca2+ channels in the plasma membrane open and Ca2+ flows in the cell, the onset of exocytosis follows less than 50 ms later. This latency is shorter than the time required for Ca2+ to equilibrate in the cytosol, which suggests that the granules are located in the vicinity of the Ca2+ channels and thus sensitive to local [Ca2+]i changes. Indeed, areas with a high density of Ca2+ channels are found to co-localize with the granules and represent 'hot spots' of secretion¹⁵⁸. A mouse beta cell contains about 13,000 secretory granules²¹⁸ but only a small fraction (50-75) granules²¹⁹ in the readily releasable pool (RRP) are available for immediately release^{220, 221}. The remainder referred to as the reserve pool needs to be mobilized into the RRP before they can undergo exocytosis. The number of granules released seems to be dependent on the phosphorylation state of key proteins. Activation of protein kinase A and C (PKA and PKC) or inhibition of protein phosphatases is required for a large exocytotic response²¹⁷. The effects of these kinases are additive, which suggests that phosphorylation of different regulatory proteins are involved. Glucose-stimulated insulin secretion in vivo is typically biphasic and characterized by a steep transient first phase is followed by a gradually developing second phase (Fig. 5). In one model it is explained as the first phase represents release of granules in RRP, triggered by Ca2+ influx, and the second phase most likely reflects the ATP-dependent recruitment and release of granules from the reserve pool²²¹. The electrophysiological response is also biphasic so this conclusion is open to debate.

Figure 5. Glucose-induced insulin secretion is characterized by a rapid first phase and a slower second phase insulin released to the bloodstream. Abbreviation used: iv, intra venous.

Mobilization of granules may involve physical translocation within the beta cell and/or chemical modification²²² and docking of granules near the plasma membrane. Docked granules need to be primed in order to enter the RRP. The insulin granules are transported from the reserve pool to the plasma membrane initially along microtubules and then along the microfilament networks of the cytoskeleton associated with the plasma membrane²²³. An ATP-dependent motor protein such as kinesin is thought to provide the necessary motive force for transportation. However, the exact mechanism and its regulation involved in the transport of granules towards the plasma membrane remains largely unknown²²⁴.
The processes by which granules in close proximity to the plasma membrane bind and fuse to it can be divided into three phases docking, priming and fusion based on electrophysiological and secretion studies performed on various neurosecretory tissues and in combination with biochemical and molecular genetic analysis. Docking describes the initial reversible interaction of granules with the plasma membrane and constitutes a pool of unprimed granules in close proximity to the exocytotic site. There is little morphological evidence of the existence of granule docking, but the presence of this pool in the beta cell is suggested from the speed by which RRP is replenished following addition of ATP in the cytosol. The next priming step may involve ATP-hydrolysis mediated in part by the ATPase N-ethylmaleimide-sensitive factor (NSF), which is activated by the soluble NSF-attachment factor (a-SNAP)²²⁵. NSF functions as a molecular chaperone to activate SNAp REceptor (SNARE) proteins destined for the fusion complex (Fig. 6)²²⁴. Another possible ATP-dependent event in priming is the synthesis of phosphatidylinositol (4,5)-bisphosphate (PIP2), which is thought to be important in the recruitment and modulation of proteins implicated in the fusion apparatus - namely the Ca2+ binding synaptotagmin and calcium-dependent activator protein for secretion (CAPS)^{212, 226}. Multiple different molecules influence insulin granule exocytosis. The transcription factor HNF-1 when mutated is a cause of a severe form of MODY (Maturity Onset Diabetes of Youth) and in addition to its many effects upon islets and insulin secretrion, the molecule collectrin is regulated downstream of HNF-1, and collectrin under or over expression leads to decreased or enhanced insulin secretion²²⁷. Insulin secretion is deficient in a mouse with a mutant Akt/PKB kinase and this protein regulates exocytosis in a model system²²⁸.

Figure 6. Docking, priming and fusion of insulin-containing granules. Granules from the reserve pool needs to be mobilized in order to enter RRP prior to release. The insert illustrates the three primary stages of insulin exocytosis at the plasma membrane, docking, priming and fusion. The granule is tethered at the plasma membrane during docking. Thereafter the a-SNAP activates NSF, which hydrolyses ATP thereby activating the SNARE proteins. A second ATP-dependent step in the priming process could be PI-4kinase phosphorylation in the generation of PIP2 (after PI-5kinase phosphorylation). Synaptotagmin is proposed to act as a Ca2+ sensor and a fusion clamp preventing fusion until a Ca2+ signal arises. When the granule is primed, Ca2+ is the only requirement for granule fusion with the plasma membrane.

Fusion of granules

Conserved protein families involved in fusion include the SNAREs, the Rab GTPases, and the Sec1/munc18-related proteins (also referred to as SM proteins)²²⁹. Granuphilin is reported to be in dense core vesicles of beta cells and binds to GTP-bound Rab27a²³⁰. Rab27a, on the granule membrane is involved in the regulation of exocytosis of secretory granules. Granuphilin also directly interacts with plasma membrane bound SNARE protens²³⁰. Insulin granules docked to the membrane are reduced in granuphilin deficient beta cells²³¹ despite granuphilin null mice having augmented insulin secretion²³¹. The SNARE proteins were originally identified as synaptic proteins, but are now generally accepted as universally involved in the core machinery in all intracellular fusion events. The primed granules are ready to fuse with the plasma membrane in an ATP-independent process when the intracellular Ca2+ concentration [Ca2+]i am sufficiently elevated.
Synaptobrevin (VAMP-2, synaptic vesicle-associated membrane protein) is located on the beta cell granule as a v-SNARE, and syntaxin 1 and SNAP-25 (synaptosome-associated protein of 25 kDa) are located on the plasma membrane as t-SNARE (Fig. 6)^{232, 233}. Synaptobrevin, SNAP-25 and syntaxin 1 assemble in a 1:1:1 stoichiometry to form a complex that drives membrane fusion. Helical portions in the SNARE complex bundle together (two helical domains from SNAP-25 and one from both syntaxin 1 and synaptobrevin) and form an extended coiled-coil rod-like structure (see note), which brings the granule into close proximity with the plasma membrane, thereby overcoming the free energy barrier for fusion (Fig. 6)²³⁴.
[Note: Coiled-coil structure is composed of several a-helices that interact through hydrophobic residues and stabilizing electrostatic interactions between the side chains. This results in a very stable structure resembling a strong rope.]It has been postulated that munc18 functions in a late stage in the fusion process in chromaffin cells, where its dissociation from syntaxin 1 determines the kinetics of postfusion events²³⁵. However, a munc18 null mutation leads to a selective defect in the docking of LDCVs at the target membrane and overexpression of munc18 leads to an increase in number of fusion-competent vesicles without affecting the kinetics of vesicle fusion. This suggests that munc18 is also important in the docking step before trans-SNARE complexes have assembled. The essential role of these proteins in neurosecretion is illustrated by the potent inhibition of insulin secretion by various botulinum and tetanus toxins that specifically cleave the different SNARE proteins. Studies with botulinum neurotoxin A which cleaves VAMP-2 and SNAP-25 demonstrate that VAMP-2 is absolutely necessary for insulin exocytosis whereas the action of SNAP-25 could be partially reversed by higher levels of Ca2+ or cAMP potentiation²³⁶. Botulinum neurotoxin C1 mainly cleaves syntaxin and reduces K+-induced insulin release by 95% but glucose stimulated insulin release only by 25% pointing to further complexity in the exocytotic mechanism²³⁷. The fusion event is highly Ca2+-dependent (>10 mM), probably due to regulatory proteins that function as Ca2+ sensors. Synaptotagmin is the best-characterized exocytotic Ca2+ sensor with two Ca2+-binding regions, the C2 domains (see note) C2A and C2B, each sharing homology with the C2 regulatory domain of protein kinase C (PKC). The C2A domain is responsible for the Ca2+-dependent insertion of synaptotagmin into the beta cell granule membrane at low micromolar (5 mM) free Ca2+ and the binding of synaptotagmin to the core complex protein syntaxin 1^{224, 238}. CAPS is another Ca2+-binding protein thought to be important for Ca2+-triggered fusion from neuroendocrine cells, including the beta cell²²⁴. It contains a pleckstrin homology domain (see note) that exhibits binding specificity towards PIP2, which makes it a specific target for phospholipid mediators formed during priming. The lipid specificity of CAPS is switched at elevated Ca2+ concentrations and is thought to help disrupt the plasma membrane bilayer to permit fusion²³⁹. Synaptotagmin is also found to have an inhibitory function in exocytosis. It has been suggested to lock the fusion core complex by preventing a-SNAP from binding until a stimulatory Ca2+ signal arises^{225, 233}. Other components of the fusion process include syntaphilin that acts as a syntaxin clamp in regulating assembly of the SNARE complex during membrane fusion events²³⁴ and NSF that may be necessary to dissociate SNARE complexes formed within a single membrane in favor of complexes between membranes (trans-SNARE complex)^{240, 241}. [Note: A C2 domain contains about 120-140 amino acids and has been found in single or multiple copies in more than 60 proteins. It was first discovered in PKC as a Ca2+-dependent, phospholipid-binding domain, but now several other C2 domains have been shown to mediate protein-protein interactions and Ca2+-independent binding to proteins and phospholipids^{242, 243}. Pleckstrin homology domain mediates membrane association of kinases with their target molecules, in some cases by interaction with the headgroup of a phosphoinositide lipid²⁴³.
Rab GTPases represent a large family of homologous Ras-like GTP-binding proteins that direct the vectorial movement of secretory vesicles. Four isoforms of Rab3 (Rab3A, -B, -C, and -D) have been identified so far. Rab3A is associated with dense-core insulin-containing secretory granules and thought to play a negative role in the fusion process by preventing Ca2+-dependent exocytosis²⁴⁴. Rab3A activation is shown to inhibit Ca2+-evoked exocytosis in beta cells by possibly binding to calmodulin at low stimulatory [Ca2+]i²⁴⁵. Calmodulin is a key Ca2+ sensing protein and this may be a way for Ca2+ to act in concert with GTP in the insulin exocytotic mechanism (provides a downstream connection between an intracellular secondary messenger (Ca2+) and the exocytotic machinery of the pancreatic beta cell). It is postulated ²⁴⁶ that it is a way to direct calmodulin to the cytoplasmic face of a b-granule membrane. When [Ca2+]i increases, calmodulin binds CaMKII more readily than Rab3A and this activates CaMKII to phosphorylate effector exocytotic molecules and enhance either the transport of a b-granule to the plasma membrane and/or the mechanism of insulin exocytosis¹⁸⁷.
Ca2+ plays another essential role in triggering endocytosis following secretion. This may be controlled by the Ca2+/calmodulin-dependent serine/threonine protein phosphatase calcineurin, which is postulated to be a calcium sensor for endocytosis in synaptosomes. Calcineurin is activated by calcium and thereby leads to dephosphorylation of proteins involved in endocytosis like dynamin, amphiphysin 1, amphiphysin 2, and synaptojanin^{247, 248}.

Signaling inside the beta cell
Metabolizable secretagogues and extracellular molecules such as hormones, neurotransmitter that act by binding to receptors on the beta cell plasma membrane appear to mediate their effects through a common set of second messengers. These effects include 1) modulation of adenylate cyclase-cAMP system and activation of protein kinase A (PKA) and recent evidence that cAMP may influence the ATP-sensitive Potassium channel by signaling through Epacs (Exchange Proteins Activated by Cyclic AMP)²⁴⁹, 2) generation of inositol 1,4,5-triphosphate (IP3) and sn-1,2-diacylglycerol (DAG) resulting in activation of the PKC and finally 3) changes in intracellular free Ca2+ ions that function through Ca2+-dependent regulatory proteins such as calmodulin and PKC. Phosphoinositide signaling appears to also be important for insulin exocytosis with recent evidence using RNAi technology of effect through phospholipase D1, Ca2+ dependent activator protein for secretion 1, and Munc-18-interacting protein 1²⁵⁰.
Hormones binding to G-protein coupled receptors on the cell surface signal to a heterotrimeric seven transmembrane G-protein (inhibitory G-protein, Gi or stimulatory G-protein, Gs), which convey the signal to effector systems such as adenylate cyclase. When a stimulatory G-protein is activated (e.g. glucagon stimulation of liver cells) the adenylate cyclase, which resides on the inner plasma membrane, converts ATP to the second messenger cyclic AMP (cAMP) that activates the cAMP-dependent serine/threonine kinase PKA. PKA phosphorylates several proteins at their serine or threonine residues causing a change in either enzyme activity or protein structure. An example is the activation of glycogen mobilization by activating glycogen phosphorylase.
cAMP has a short half-life in the cytoplasm and it is quickly hydrolyzed to AMP by the action of cAMP phosphodiesterases which is important for shutting down the stimulation by the second messenger. cAMP also potentiates Ca2+-dependent insulin secretion^251-253. Besides acting on the Ca2+ channels, the major action of cAMP on insulin release is by a direct effect on the secretory machinery itself^{251, 254}. This is thought to be a result of an increased Ca2+ sensitivity of exocytosis by cAMP via an extension of the distance over which the Ca2+ channels can recruit the secretory granules for release²⁵¹. It has been suggested that cAMP acts by increasing the release probability of granules already in RRP (PKA-independent) and by accelerating granule mobilization thereby refilling the RRP (PKA-dependent)²⁵³. A recent study has reported that Epac-selective cAMP analogs, but not PKA-selective analogs influence K ATP channels. Epac 1 and 2 bind to SUR1 of the ATP K+ channel, and thus cAMP may stimulate insulin secretion through a novel Epac mediated mechanism²⁴⁹. Other G-protein receptors (inhibitory and stimulatory G, Go; stimulatory G, Gq) are coupled to phospholipase C (PLC). Acetylcholine stimulates exocytosis by activating the cholinergic muscarinic receptor that acts via a stimulating G-protein (Gq), which is bound to the Ca2+-dependent membrane-bound PLC. PLC hydrolyses PIP2 in the membrane to yield the two second messengers IP3 and DAG. IP3 diffuses to the endoplasmic reticulum (ER) where it binds to and opens a Ca2+ channel in the ER membrane thereby allowing Ca2+ to exit from the ER stores into the cytosol resulting in increased [Ca2+]i. The membrane bound DAG stimulates protein kinase C (PKC) by lowering its requirement for Ca2+. DAG is also produced when PLC is activated following increases in [Ca2+]i, thus PKC will be activated under all conditions in which intracellular Ca2+ is elevated²⁵⁴. PKC is shown to modulate Ca2+-dependent insulin secretion^{217, 251}. However, glucose-stimulated insulin secretion is unaffected in PKC-depleted islets, indicating that this enzyme is not essential for glucose stimulated insulin secretion. Rather, PKC appears to be involved in the stimulation of insulin secretion by potentiators of release e.g. acetylcholine and carbamylcholine by sensitizing the secretory machinery to Ca2+²¹⁷. Glucose-induced insulin secretion is associated with inhibition of free fatty acid (FFA) oxidation, increased esterification and complex lipid formation by pancreatic beta cells. Increases in the mass of DAG, triglyceride and phosphatidic acid are observed. Exogenous FFA also potentiate glucose-stimulated secretion of insulin, possibly by providing additional acyl groups for long chain-CoA formation or complex lipid synthesis. FFA does not stimulate insulin secretion in the absence of glucose probably due to their rapid entry into the mitochondria when malonyl-CoA levels are low. 116 LC-CoA opens the ATP-sensitive K+ channel whereas the corresponding FFA closes the channel. Long-chain fatty acids stimulate PKC, but the saturated ones are ineffective ²⁵⁴ or much less effective than the unsaturated ones²⁵⁵.

Phospholipase A2 Superfamily and Pancreatic beta cell Function
The phospholipase A2 family plays an important role in the inflammatory response seen in IDDM by generating inflammatory molecules from lipids substrate. Phospholipase A2 (PLA2) constitutes a growing superfamily of enzymes that hydrolyze membrane phospholipids at the sn-2 position generating lipid second messengers as well as structural modulations of cellular membranes (Fig. 7). The fatty acid released from sn-2 is in most cases arachidonic acid.

Figure 7. Illustration of cleavage sites for phospholipase A2 in the glycerol backbone of a phospholipid. Phospholipase A2 cuts the phospholipids in the sn-2 position resulting in formation of a fatty acid and a lysophospholipid, which can be further metabolized to eicosanoids and PAF, respectively. Abbreviations used: PAF, platelet-activating factor (modified from (171).

Several different types of PLA2 have been identified. They include: 1) cytosolic PLA2 (cPLA2), 2) secreted PLA2 (sPLA2), 3) Ca2+-independent PLA2 (iPLA2) and 4) platelet-activating factor acetylhydrolase (PAF-AH). These groups are further subdivided according to structure, molecular weight, substrate specificity, cellular localization, and Ca2+ dependency of the enzyme²⁵⁶. The cytosolic forms of PLA2 (group IV PLA2) are high molecular weight interfacial PLA2 enzymes (see note). They show a preference for phosphatidylcholine (PC) with arachidonic acid (AA) in the sn-2 position and are thought to play an important role in providing free AA for eicosaniod biosynthesis^{242, 243, 257}. Type IV knock-out mice (IV cPLA2a-/- mice) show deficient inflammatory response ²⁵⁸ and peritoneal macrophages from IV cPLA2a-/- mice fail to produce prostaglandins and leucotrienes after stimulation²⁵⁹. [Note: Interfacial PLA2 enzymes need to bind to an aggregated surface in order to access the phospholipid substrates.]
Group I, IIs (IIA, IIC, IID, IIE, IIF), III, V, IX, X and XI PLA2 comprise a group of low molecular weight PLA2 (13-18 kDa) interfacial enzymes which are either secreted or extracellular PLA2 (sPLA2). These enzymes have been implicated in various physiological and pathological functions, including lipid digestion, lipid mediator generation, cell proliferation, exocytosis, antibacterial defense, inflammatory diseases and cancer²⁶⁰.
The Ca2+-independent PLA2 (iPLA2) is a group of cytosolic PLA2 of 85-88 kDa whose major cellular function is the mediation of phospholipid remodeling and homeostasis^{246, 261, 262}. This enzyme seems to regulate the incorporation of AA and other fatty acids into membrane phospholipids and as such may be a limiting factor for eicosanoid biosynthesis though it has been questioned in the case of the rat pancreatic islet group VIA enzyme²⁶³.
Platelet-activating factor acetylhydrolase (PAF-AH) is another group of Ca2+-independent PLA2's²⁶⁴. All of them have restricted substrate specificity and are selective for PAF (see note) and for phospholipids with a short (or intermediate) acyl chain at the sn-2 position that protects normal membrane lipids from hydrolysis by PAF-AH. A second group of substrates is oxidatively fragmented phospholipids. The main function of PAF-AH is to act as scavenger of bioactive phospholipids especially to regulate the level of PAF²⁶⁵. (Note: PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine), a bioactive phospholipid involved in asthma. It is a mediator of a wide range of immune and allergic reactions. It is synthesized in a regulated pathway and activates inflammatory cells. Lysophospholipid can be converted to PAF.)
Different types of PLA2 in islets and pancreatic beta cells
Pancreatic islets contain at least five different types of PLA2 (Table 2). Among the secreted form, both type IB and IIA are found with type IB more abundant. Type IB has been localized to the secretory granules of beta cells and stimulation with insulin secretagogues results in the co-secretion of insulin and the enzyme²⁶³. Type IB also appears to be regulated by fasting and ambient glucose^{263, 266, 267}. Furthermore, mRNA for a sPLA2 membrane receptor is found in rat pancreatic islets.

Table 1. PLA2 groups in the pancreatic islet.

By use of different inhibitors of PLA2, it has been demonstrated that Ca2+-dependent PLA2 may participate in acetylcholine-induced activation of the granule movement in pancreatic beta cells²⁶⁸. It is proposed that the type IV cPLA2 couples the secretagogue induced increase in intracellular Ca2+ to the liberation of AA and the subsequent release of insulin²⁶⁹. Both exogenous sPLA2 and products of its action have also been shown to suppress KATP channel activity in excised and cell-attached patches of plasma membranes from HIT insulinoma cells²⁷⁰.
In glucose-stimulated islets, hydrolysis of AA from membrane phospholipids appears in part to be mediated by a PLA2 that is catalytically active in the absence of Ca2+ and inactivated by the iPLA2 inhibitor HELSS (haloenollactone suicide substrate)²²⁴. However, iPLA2 is not required for AA incorporation or phospholipid remodeling in beta cells, suggesting that iPLA2 plays another role in these cells. iPLA2 in rat and human beta cells is implicated as a putative glucose sensor, which can couple alterations in glycolytic metabolism to the generation of biologically active eicosanoids and thereby facilitate glucose-induced insulin secretion²⁷¹. iPLA2 is activated by ATP and other nucleotides and it has been postulated that it may respond to the increase in the ATP/ADP ratio after glucose stimulation, however this function has been questioned.
Besides playing an important role in the stimulus-secretion coupling in the beta cell different types of PLA2 may be involved in inflammatory responses. Type II sPLA2 and type IV cPLA2 mRNA is upregulated in areas with histologic damage in pancreatitis suggesting that these isoforms might contribute to the morphologic changes that occur²⁷². In addition, several proinflammatory cytokines such as IL-1, IL-6 and TNFa have been shown to induce transcription of the type IIA sPLA2 gene in a variety of cells. TNF priming of human neutrophils (PMN) caused marked increase in fMLP stimulated AA release in parallel to enhanced activity and secretion of sPLA2 and activity of cPLA2²⁷³. Type I sPLA2, on the contrary, is non-toxic to acinar cells in both severe and mild pancreatitis. This may indicate that type II but not type I sPLA2 have pathophysiologic importance in severe acute pancreatitis associated with systemic inflammatory response syndrome.

Arachidonic acid in cell signaling

 
Arachidonic acid is a major constituent of islet phospholipids comprising about 34-39% of the sn-2 fatty acyl mass in islet glycerophospholipids ^{271, 274, 275} compared to 13% in rat brain and heart and 1% in rat liver²⁷⁶. The intracellular level of free AA in islets is normally in the low micromolar range, but rises to a level of 50-200 mM during glucose stimulation^{277, 278}. Such changes could result by the action of PLA2, but also by the action of diacylglycerol lipase and monoacylglycerol lipase on diacylglycerol (DAG), generated from the action of phospholipase C (Fig. 10)^{279, 280}. Phosphatidic acid (PA), generated by phospholipase D hydrolysis of PL, is also a potential source of AA²⁷⁹.

Figure 8. Arachidonic acid can be made in different ways, but also degraded in several ways. AA can be reesterified into membranes or metabolized by the lipoxygenase pathway to leucotrienes, by the cyclooxygenase pathway to prostaglandins and thromboxanes or by the cytochrome P450 monooxygenase enzyme to epoxyeicosatrienoic acid. Abbreviations used: PAPH, phosphatidic acid phosphohydrolase.

Inhibition of AA release prevents insulin secretion²⁸¹. Increases in the availability of AA (or its metabolites) by itself, however is probably insufficient to initiate insulin release, instead it seems that AA potentiates the action of other secretagogue²⁸². In different cell types, AA has been associated with several specific cellular processes such as regulation of PKC and PLC and modulation of intracellular Ca2+ transients^283-286. In beta cells, it has been suggested that both Ca2+-dependent and Ca2+-independent components are involved in PLA2-induced insulin secretion. The Ca2+-independent component could be a result of AA or a lipoxygenase product affecting late events in stimulus-secretion coupling^{287, 288}. The AA-induced Ca2+-dependent event is biphasic with an early rise probably reflecting release of Ca2+ from intracellular stores and a sustained phase, which could reflect extracellular Ca2+ entry possible mediated by voltage-sensitive Ca2+ channels ²⁸⁹

Role of arachidonic acid metabolites in cell signaling
When AA is generated, it can be reesterified into phospholipids by acyltransferases or metabolized by the cyclooxygenase (COX), lipoxygenase (LPX) or cytochrome P450 epoxygenase pathways generating prostaglandins and thromboxanes, leukotrienes and lipoxins, or epoxyeicosatrienoic acids²⁹⁰.  Prostaglandins made by the cyclooxygenase-pathway acting on AA have been demonstrated, in cell-attached patches on a beta cell line, to be involved in insulin secretion by increasing KATP channel activity²⁷⁰. E Prostaglandins inhibit insulin secretion and a pertussis toxin insensitive G alpha i family member G alpha z apparently mediates this effect²⁹¹.  The lipoxygenase metabolites of AA are potential mediators of insulin secretion. During nutrient-induced insulin secretion 12-hydroxyeicosatetraenoic acid (12-HETE; a product of 12-lipoxygenase metabolized AA) is augmented²⁸² and a lipoxygenase inhibitor (nordihydroguaiaretic acid, NDGA) inhibits glucose-induced insulin secretion²⁹². Products of the lipoxygenase pathway have also been demonstrated to play a role in apoptosis induced by Ca2+ store depletion in beta cells by using the inhibitor NDGA²⁹³.
The cytokine IL-1 also augments islet production of 12-HETE and PGE2²⁷⁶. This is not caused by an increased production of AA but by suppression of reesterification of AA into islet phospholipids. This might occur as a result the IL-1-induced production of NO and inhibition of ATP-dependent conversion of the fatty acid to a coenzyme A thioester intermediate²⁷⁶. 12-HPETE, a precursor of 12-HETE, has been shown to induce apoptosis of cells with reduced levels of glutathione peroxidase. Thus the effects of IL-1 to increase substrate flux through the lipoxygenase pathway and to augment NO production may interact cooperatively to inflict injury on beta cells, and at the same time stimulate insulin release in the short term.

 Pathophysiogical role of lipid metabolites and lysophospholipids on beta cell signaling

Inflammatory response in the pancreas not only plays an important role in the destruction of islet tissue in autoimmune diabetes (see chapter by Mandrup Poulsen), but also in the ability of the pancreatic beta cell to recover from such insult. Pancreatitis induced by cellophane wrapping (CW), surgery, coxsackievirus B4, cerulein treatment or with other chemicals^294-296 for example can lead to islet regeneration. Locally produced INFg causes lymphocyte infiltration and islet cell destruction and while IFN-g gene linked to an insulin promoter produces diabetes new cells are formed from duct epithelial cells and they differentiate into endocrine cells²⁹⁷. The proinflammatory cytokines, IL-1, TNF and perhaps IL-6 is shown to induce PLA2 expression and release²⁹⁸. The proinflammatory action of TNF depends in part on activation of PLA2²⁹⁹. In humans with acute pancreatitis, there is a correlation between elevated serum PLA2 levels and inflammation and lipid metabolites are also potential mediators in these events. In this regard it will be of interest to see whether selective inhibition of PLA2 can be used as a therapeutic approach against type 1 diabetes.  Recently, components of the sphingolipid pathways have been thought to be important contributors to the pathophysiology of diabetes^300-302. PLA2 hydrolysis of membrane phospholipids also generates a lysophospholipid (lysoPL). Several types of lysoPL's exist in the islet membrane: lysophosphatidylcholine (lysoPC), -ethanolamine (lysoPE), -serine (lysoPS) and -inositol (lysoPI). LysoPL's and phosphatidic acid (PA) are shown to promote insulin release when the cellular content of either lipid increases²³⁴. LysoPL's affect stimulus-secretion coupling at a number of points^{247, 248, 303, 304}. They have been observed to inhibit directly the KATP channel activity in the pancreas³⁰⁵. LysoPC and lysophosphatidylglycerol can also effectively mobilize Ca2+ from intracellular stores³⁰⁴. LysoPAF has been demonstrated to circumvent the inhibition of glucose-induced insulin release caused by phospholipase inhibitors³⁰⁶.

Micro RNA  and Diabetes

There may be as many as 1,000 different micro RNAs in the human genome, Micro RNAs, following production in the nucleus by the enzyme Drosha are exported to the cytoplasm, and processed by the enzye DICER.  They then are incorporated into the RNA-induced silencing complex¹⁵⁰ and function to either silence by binding to messenger RNA or by directing the cleavage of mRNA. It is estimated that there are 68 miRNAs in beta cells. MiRNAs play essential roles in many biological processes in mammals, including insulin secretion³⁰⁷, beta-cell development^308-311, and adipocyte differentiation³¹². The function of one islet specific miRNA has recently been elucidated. MiRNA-375 is reported to inhibit insulin secretion by inhibiting the production of the protein myotrophin and thereby decreasing granule exocytosis³¹².  In fact more MiRNAs have been recently reported to be enriched in the islets (miR-127-3p, miR-184, miR-195 and miR-493) of which many of them are differentially expressed in healthy and glucose intolerant subjects³¹³.  Further support also comes from MiRna that have been identified to play a role in insulin  biosynthesis and signaling^314-319.

Concluding remarks
Despite considerable progress in defining the molecular physiology of insulin secretion there remain many unanswered questions. It is also important to recognize that some current techniques may introduce complexity, such as the recent report that the RIP-Cre transgene itself produces mice with impaired glucose tolerance³²⁰. Nevertheless given our current knowledge it is clear that the pancreatic beta cell is an excitable cell that is critically dependent upon oxidative metabolism ³²¹ and dependent on the influx of Ca2+ through gated channels and its subsequent removal from the cell by intracellular sequestration or plasma membrane pumping leaves it in a precarious position when faced with agents that disrupt Ca2+ homeostasis in the cell. In a similar way, its susceptibility to drugs such as the DNA alkylating agent streptozotocin or the free radical generator alloxan may be the consequence of the mechanism of sugar transport, which can carry these agents into the cell. Such phenomenon may explain in part why so many of the agents that one associates with pancreatic beta cell damage and death turn out to be specific secretagogues at least in the short-term. The challenge is to discover which interactions are part of the normal responsiveness of the cell to metabolizable substrates, peptides and neurotransmitters and which are truly deleterious and thus a legitimate target for pharmacological intervention. It is likely that the concepts that there are "good" or a "bad" cytokines in the context of autoimmune diabetes is no more valid than that of glucose being "good" or "bad" in the context of glucotoxicity and type 2 diabetes. The short-term effects of secretagogues on islet function have been extensively documented but there is also the need to consider the broader perspective of how the cell responds in the longer term to inflammatory stimuli or physiological changes in energy homeostasis such as accompany pregnancy and starvation (Fig. 9).

Figure 9. Yin and Yan representation of the pancreatic beta cell in terms of induction of either cell death or secretory response to secretagogues such as inflammatory cytokines and PLA2.

Reference List

      1.   Eisenbarth GS. Type 1 diabetes: molecular, cellular and clinical immunology. Adv Exp Med Biol 2004;552.

      2.   Wucherpfennig KW. Insights into autoimmunity gained from structural analysis of MHC-peptide complexes. Curr Opin Immunol 2001;13(6).

      3.   Yoon JW, Jun HS. Cellular and molecular pathogenic mechanisms of insulin-dependent diabetes mellitus. Ann N Y Acad Sci 2001;928.

      4.   Rosmalen JG, Leenen PJ, Pelegri C, Drexhage HA, Homo-Delarche F. Islet abnormalities in the pathogenesis of autoimmune diabetes. Trends Endocrinol Metab 2002;13(5).

      5.   Dib SA, Gomes MB. Etiopathogenesis of type 1 diabetes mellitus: prognostic factors for the evolution of residual beta cell function. Diabetol Metab Syndr 2009;1(1).

      6.   Zhang L, Gianani R, Nakayama M et al. Type 1 diabetes: chronic progressive autoimmune disease. Novartis Found Symp 2008;292.

      7.   Rossini AA. Autoimmune diabetes and the circle of tolerance. Diabetes 2004;53(2).

      8.   Arif S, Tree TI, Astill TP et al. Autoreactive T cell responses show proinflammatory polarization in diabetes but a regulatory phenotype in health. J Clin Invest 2004;113(3).

      9.   Bluestone JA, Herold K, Eisenbarth G. Genetics, pathogenesis and clinical interventions in type 1 diabetes. Nature 2010;464(7293).

    10.   Llaurado G, Gallart L, Tirado R et al. Insulin resistance, low-grade inflammation and type 1 diabetes mellitus. Acta Diabetol 2012.

    11.   Nadeau KJ, Regensteiner JG, Bauer TA et al. Insulin resistance in adolescents with type 1 diabetes and its relationship to cardiovascular function. J Clin Endocrinol Metab 2010;95(2).

    12.   Rodrigues TC, Biavatti K, Almeida FK, Gross JL. Coronary artery calcification is associated with insulin resistance index in patients with type 1 diabetes. Braz J Med Biol Res 2010;43(11).

    13.   Wilkin TJ. Insulin resistance and progression to type 1 diabetes in the European Nicotinamide Diabetes Intervention Trial (ENDIT): response to Bingley et al. Diabetes Care 2008;31(4).

    14.   Wilkin TJ. The accelerator hypothesis: a review of the evidence for insulin resistance as the basis for type I as well as type II diabetes. Int J Obes (Lond) 2009;33(7).

    15.   Atkinson MA, Eisenbarth GS. Type 1 diabetes: new perspectives on disease pathogenesis and treatment. Lancet 2001;358(9277).

    16.   Butler AE, Galasso R, Meier JJ, Basu R, Rizza RA, Butler PC. Modestly increased beta cell apoptosis but no increased beta cell replication in recent-onset type 1 diabetic patients who died of diabetic ketoacidosis. Diabetologia 2007;50(11):2323-2331.

    17.   Meier JJ, Lin JC, Butler AE, Galasso R, Martinez DS, Butler PC. Direct evidence of attempted beta cell regeneration in an 89-year-old patient with recent-onset type 1 diabetes. Diabetologia 2006;49(8):1838-1844.

    18.   Atkinson MA, Gianani R. The pancreas in human type 1 diabetes: providing new answers to age-old questions. Curr Opin Endocrinol Diabetes Obes 2009;16(4):279-285.

    19.   Wang L, Lovejoy NF, Faustman DL. Persistence of Prolonged C-peptide Production in Type 1 Diabetes as Measured With an Ultrasensitive C-peptide Assay. Diabetes Care 2012;35(3).

    20.   Gianani R, Campbell-Thompson M, Sarkar SA et al. Dimorphic histopathology of long-standing childhood-onset diabetes. Diabetologia 2010;53(4).

    21.   Keenan HA, Sun JK, Levine J et al. Residual insulin production and pancreatic ss-cell turnover after 50 years of diabetes: Joslin Medalist Study. Diabetes 2010;59(11):2846-2853.

    22.   Foulis AK, Stewart JA. The pancreas in recent-onset type 1 (insulin-dependent) diabetes mellitus: insulin content of islets, insulitis and associated changes in the exocrine acinar tissue. Diabetologia 1984;26(6):456-461.

    23.   Eisenbarth GS. Banting Lecture 2009: An Unfinished Journey: Molecular Pathogenesis to Prevention of Type 1A Diabetes. Diabetes 2010;59(4):759-774.

    24.   Henderson JR. Why are the islet of Langerhans? Lancet 1969;2(7618):469-470.

    25.   Williams JA, Goldfine ID. The insulin-pancreatic acinar axis. Diabetes 1985;34(10):980-986.

    26.   Williams AJ, Chau W, Callaway MP, Dayan CM. Magnetic resonance imaging: a reliable method for measuring pancreatic volume in Type 1 diabetes. Diabet Med 2007;24(1):35-40.

    27.   Gaglia JL, Guimaraes AR, Harisinghani M et al. Noninvasive imaging of pancreatic islet inflammation in type 1A diabetes patients. J Clin Invest 2011;121(1):442-445.

    28.   Jiao Y, Peng ZH, Xing TH, Qin J, Zhong CP. Assessment of islet graft survival using a 3.0-Tesla magnetic resonance scanner. Anat Rec (Hoboken) 2008;291(12).

    29.   Wang P, Yigit MV, Medarova Z et al. Combined small interfering RNA therapy and in vivo magnetic resonance imaging in islet transplantation. Diabetes 2011;60(2).

    30.   Altieri DC. Molecular cloning of effector cell protease receptor-1, a novel cell surface receptor for the protease factor Xa. J Biol Chem 1994;269(5).

    31.   Liggins C, Orlicky DJ, Bloomquist LA, Gianani R. Developmentally regulated expression of Survivin in human pancreatic islets. Pediatr Dev Pathol 2003;6(5).

    32.   Hasel C, Bhanot UK, Heydrich R, Strater J, Moller P. Parenchymal regression in chronic pancreatitis spares islets reprogrammed for the expression of NFkappaB and IAPs. Lab Invest 2005;85(10).

    33.   Achenbach P, Bonifacio E, Koczwara K, Ziegler AG. Natural history of type 1 diabetes. Diabetes 2005;54 Suppl 2.

    34.   Achenbach P, Bonifacio E, Ziegler AG. Predicting type 1 diabetes. Curr Diab Rep 2005;5(2).

    35.   Bingley PJ, Gale EA. Progression to type 1 diabetes in islet cell antibody-positive relatives in the European Nicotinamide Diabetes Intervention Trial: the role of additional immune, genetic and metabolic markers of risk. diabetol 2006.

    36.   Bottazzo GF, Bosi E, Bonifacio E, Mirakian R, Todd I, Pujol-Borrell R. Pathogenesis of type I (insulin-dependent) diabetes: possible mechanisms of autoimmune damage. Br Med Bull 1989;45(1).

    37.   Eisenbarth GS. Italian Society of Diabetology Mentor Award. The stages of Type 1A diabetes: retrospective and prospective. Diabetes Nutr Metab 2004;17(6).

    38.   Palmer JP, Fleming GA, Greenbaum CJ et al. C-Peptide Is the Appropriate Outcome Measure for Type 1 Diabetes Clinical Trials to Preserve beta-Cell Function: Report of an ADA Workshop, 21-22 October 2001. diab 2004;53(1).

    39.   Andre I, Gonzalez A, Wang B, Katz J, Benoist C, Mathis D. Checkpoints in the progression of autoimmune disease: lessons from diabetes models. Proc Natl Acad Sci U S A 1996;93(6).

    40.   Bresson D, Togher L, Rodrigo E et al. Anti-CD3 and nasal proinsulin combination therapy enhances remission from recent-onset autoimmune diabetes by inducing Tregs. Journal of Clinical Investigation 2006;116(5).

    41.   Dai YD, Jensen KP, Lehuen A et al. A peptide of glutamic acid decarboxylase 65 can recruit and expand a diabetogenic T cell clone, BDC2.5, in the pancreas. J Immunol 2005;175(6).

    42.   Liu W, Putnam AL, Xu-Yu Z et al. CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells. J Exp Med 2006;203(7).

    43.   Sarkar SA, Lee CE, Victorino F et al. Expression and regulation of chemokines in murine and human type 1 diabetes. Diabetes 2012;61(2).

    44.   Slack JM. Developmental biology of the pancreas. Development 1995;121(6).

    45.   Piper K, Brickwood S, Turnpenny LW et al. Beta cell differentiation during early human pancreas development. J Endocrinol 2004;181(1).

    46.   Richardson MK, Hanken J, Gooneratne ML et al. There is no highly conserved embryonic stage in the vertebrates: implications for current theories of evolution and development. Anat Embryol (Berl) 1997;196(2).

    47.   Sarkar SA, Kobberup S, Wong R et al. Global gene expression profiling and histochemical analysis of the developing human fetal pancreas. Diabetologia 2008;51(2).

    48.   Reichert M, Rustgi AK. Pancreatic ductal cells in development, regeneration, and neoplasia. J Clin Invest 2011;121(12).

    49.   Iburi T, Izumiyama H, Hirata Y. [Endocrine glands of pancreas]. Nihon Rinsho69 Suppl 2.

    50.   Youos JG. The role of alpha-, delta- and F cells in insulin secretion and action. Diabetes Res Clin Pract93 Suppl 1.

    51.   Prado CL, Pugh-Bernard AE, Elghazi L, Sosa-Pineda B, Sussel L. Ghrelin cells replace insulin-producing beta cells in two mouse models of pancreas development. Proc Natl Acad Sci U S A 2004;101(9).

    52.   Sorenson RL, Garry DG, Brelje TC. Structural and functional considerations of GABA in islets of Langerhans. Beta-cells and nerves. Diabetes 1991;40(11).

    53.   Wiest-Ladenburger U, Hartmann R, Hartmann U, Berling K, Bohm BO, Richter W. Combined analysis and single-step detection of GAD65 and IA2 autoantibodies in IDDM can replace the histochemical islet cell antibody test. Diabetes 1997;46(4).

    54.   Aanstoot HJ, Kang SM, Kim J et al. Identification and characterization of glima 38, a glycosylated islet cell membrane antigen, which together with GAD65 and IA2 marks the early phases of autoimmune response in type 1 diabetes. J Clin Invest 1996;97(12).

    55.   Hawkes CJ, Wasmeier C, Christie MR, Hutton JC. Identification of the 37-kDa antigen in IDDM as a tyrosine phosphatase-like protein (phogrin) related to IA-2. diab 1996;45(9).

    56.   Bartholomeusz RK, Campbell IL, Harrison LC. Pancreatic islet A2B5- and 3G5-reactive gangliosides are markers of differentiation in rat insulinoma cells. Endocrinology 1989;124(6).

    57.   Alejandro R, Shienvold FL, Hajek SAV, Pierce M, Paul R, Mintz DH. A ganglioside antigen on the rat pancreatic B cell surface identified by monoclonal antibody R2D6. J Clin Invest 1984;74.

    58.   Anlauf M, Eissele R, Schafer MK et al. Expression of the two isoforms of the vesicular monoamine transporter (VMAT1 and VMAT2) in the endocrine pancreas and pancreatic endocrine tumors. J Histochem Cytochem 2003;51(8).

    59.   Harris PE, Ferrara C, Barba P, Polito T, Freeby M, Maffei A. VMAT2 gene expression and function as it applies to imaging beta-cell mass. J Mol Med (Berl) 2008;86(1).

    60.   Eipper BA, Green CB, Mains RE. Expression of prohormone processing enzymes in neuroendocrine and non-neuroendocrine cells. J Natl Cancer Inst Monogr 1992;(13).

    61.   Eipper BA, Milgram SL, Husten EJ, Yun HY, Mains RE. Peptidylglycine alpha-amidating monooxygenase: a multifunctional protein with catalytic, processing, and routing domains. Protein Sci 1993;2(4).

    62.   Eipper BA, Perkins SN, Husten EJ, Johnson RC, Keutmann HT, Mains RE. Peptidyl-alpha-hydroxyglycine alpha-amidating lyase. Purification, characterization, and expression. J Biol Chem 1991;266(12).

    63.   Schafer MK, Stoffers DA, Eipper BA, Watson SJ. Expression of peptidylglycine alpha-amidating monooxygenase (EC 1.14.17.3) in the rat central nervous system. J Neurosci 1992;12(1).

    64.   Martinez A, Montuenga LM, Springall DR, Treston A, Cuttitta F, Polak JM. Immunocytochemical localization of peptidylglycine alpha-amidating monooxygenase enzymes (PAM) in human endocrine pancreas. J Histochem Cytochem 1993;41(3).

    65.   Orskov C, Bersani M, Johnsen AH, Hojrup P, Holst JJ. Complete sequences of glucagon-like peptide-1 from human and pig small intestine. J Biol Chem 1989;264(22).

    66.   Ashcroft FM, Rorsman P. Electrophysiology of the pancreatic beta-cell. Prog Biophys Mol Biol 1989;54(2).

    67.   Colombo C, Porzio O, Liu M et al. Seven mutations in the human insulin gene linked to permanent neonatal/infancy-onset diabetes mellitus. J Clin Invest 2008;118(6).

    68.   Hodish I, Liu M, Rajpal G et al. Misfolded proinsulin affects bystander proinsulin in neonatal diabetes. J Biol Chem 2011;285(1).

    69.   Hodish I, Absood A, Liu L et al. In vivo misfolding of proinsulin below the threshold of frank diabetes. Diabetes 2011;60(8).

    70.   Atkinson MA, Bluestone JA, Eisenbarth GS et al. How does type 1 diabetes develop?: the notion of homicide or beta-cell suicide revisited. Diabetes 2011;60(5).

    71.   Grimaldi KA, Siddle K, Hutton JC. Biosynthesis of insulin secretory granule membrane proteins. Control by glucose. Biochem J 1987;245(2).

    72.   Orci L, Ravazzola M, Storch MJ, Anderson RG, Vassalli JD, Perrelet A. Proteolytic maturation of insulin is a post-Golgi event which occurs in acidifying clathrin-coated secretory vesicles. Cell 1987;49(6).

    73.   Hutton JC, Davidson HW, Peshavaria M. Proteolytic processing of chromogranin A in purified insulin granules. Formation of a 20 kDa N-terminal fragment (betagranin) by the concerted action of a Ca2+-dependent endopeptidase and carboxypeptidase H (EC 3.4.17.10). Biochem J 1987;244(2).

    74.   Orci L, Ravazzola M, Amherdt M et al. Conversion of proinsulin to insulin occurs coordinately with acidification of maturing secretory vesicles. J Cell Biol 1986;103(6 Pt 1).

    75.   Orci L, Ravazzola M, Perrelet A. (Pro)insulin associates with Golgi membranes of pancreatic B cells. Proc Natl Acad Sci U S A 1984;81(21).

    76.   Lazure C, Seidah NG, Pelaprat D, Chretien M. Proteases and posttranslational processing of prohormones: a review. Can J Biochem Cell Biol 1983;61(7).

    77.   Lernmark A, Chan SJ, Choy R et al. Biosynthesis of insulin and glucagon: a view of the current state of the art. Ciba Found Symp 1976;41.

    78.   Dodson G, Steiner D. The role of assembly in insulin's biosynthesis. Curr Opin Struct Biol 1998;8(2).

    79.   Emdin SO, Dodson GG, Cutfield JM, Cutfield SM. Role of zinc in insulin biosynthesis. Some possible zinc-insulin interactions in the pancreatic B-cell. Diabetologia 1980;19(3).

    80.   Clifford KS, MacDonald MJ. Survey of mRNAs encoding zinc transporters and other metal complexing proteins in pancreatic islets of rats from birth to adulthood: similar patterns in the Sprague-Dawley and Wistar BB strains. Diabetes Res Clin Pract 2000;49(2-3).

    81.   Vallee BL, Falchuk KH. The biochemical basis of zinc physiology. Physiol Rev 1993;73(1).

    82.   Chimienti F, Aouffen M, Favier A, Seve M. Zinc homeostasis-regulating proteins: new drug targets for triggering cell fate. Curr Drug Targets 2003;4(4).

    83.   Bloc A, Cens T, Cruz H, Dunant Y. Zinc-induced changes in ionic currents of clonal rat pancreatic -cells: activation of ATP-sensitive K+ channels. J Physiol 2000;529 Pt 3.

    84.   Franklin I, Gromada J, Gjinovci A, Theander S, Wollheim CB. Beta-cell secretory products activate alpha-cell ATP-dependent potassium channels to inhibit glucagon release. Diabetes 2005;54(6).

    85.   Ishihara H, Maechler P, Gjinovci A, Herrera PL, Wollheim CB. Islet beta-cell secretion determines glucagon release from neighbouring alpha-cells. Nat Cell Biol 2003;5(4).

    86.   Kim BJ, Kim YH, Kim S et al. Zinc as a paracrine effector in pancreatic islet cell death. Diabetes 2000;49(3).

    87.   Kambe T, Narita H, Yamaguchi-Iwai Y et al. Cloning and characterization of a novel mammalian zinc transporter, zinc transporter 5, abundantly expressed in pancreatic beta cells. J Biol Chem 2002;277(21).

    88.   Eide DJ. Metal ion transport in eukaryotic microorganisms: insights from Saccharomyces cerevisiae. Adv Microb Physiol 2000;43.

    89.   Seve M, Chimienti F, Devergnas S, Favier A. In silico identification and expression of SLC30 family genes: an expressed sequence tag data mining strategy for the characterization of zinc transporters' tissue expression. BMC Genomics 2004;5(1).

    90.   Sim BK, Fogler WE, Zhou XH et al. Zinc ligand-disrupted recombinant human Endostatin: potent inhibition of tumor growth, safety and pharmacokinetic profile. Angiogenesis 1999;3(1).

    91.   Sim DL, Yeo WM, Chow VT. The novel human HUEL (C4orf1) protein shares homology with the DNA-binding domain of the XPA DNA repair protein and displays nuclear translocation in a cell cycle-dependent manner. Int J Biochem Cell Biol 2002;34(5).

    92.   Palmiter RD, Findley SD. Cloning and functional characterization of a mammalian zinc transporter that confers resistance to zinc. EMBO J 1995;14(4).

    93.   Wenzel HJ, Cole TB, Born DE, Schwartzkroin PA, Palmiter RD. Ultrastructural localization of zinc transporter-3 (ZnT-3) to synaptic vesicle membranes within mossy fiber boutons in the hippocampus of mouse and monkey. Proc Natl Acad Sci U S A 1997;94(23).

    94.   Wenzlau JM, Juhl K, Yu L et al. The cation efflux transporter ZnT8 (Slc30A8) is a major autoantigen in human type 1 diabetes. Proc Natl Acad Sci U S A 2007;104(43).

    95.   Chimienti F, Devergnas S, Favier A, Seve M. Identification and cloning of a beta-cell-specific zinc transporter, ZnT-8, localized into insulin secretory granules. Diabetes 2004;53(9).

    96.   Chimienti F, Devergnas S, Pattou F et al. In vivo expression and functional characterization of the zinc transporter ZnT8 in glucose-induced insulin secretion. J Cell Sci 2006;119(Pt 20).

    97.   An S, Zenisek D. Regulation of exocytosis in neurons and neuroendocrine cells. Curr Opin Neurobiol 2004;14(5).

    98.   Tsuboi T, McMahon HT, Rutter GA. Mechanisms of dense core vesicle recapture following "kiss and run" ("cavicapture") exocytosis in insulin-secreting cells. J Biol Chem 2004;279(45).

    99.   Artalejo CR, Elhamdani A, Palfrey HC. Secretion: dense-core vesicles can kiss-and-run too. Curr Biol 1998;8(2).

100.   Wu LG. Kinetic regulation of vesicle endocytosis at synapses. Trends Neurosci 2004;27(9).

101.   Artalejo CR, Elhamdani A, Palfrey HC. Sustained stimulation shifts the mechanism of endocytosis from dynamin-1-dependent rapid endocytosis to clathrin- and dynamin-2-mediated slow endocytosis in chromaffin cells. Proc Natl Acad Sci U S A 2002;99(9).

102.   Artalejo CR, Henley JR, McNiven MA, Palfrey HC. Rapid endocytosis coupled to exocytosis in adrenal chromaffin cells involves Ca2+, GTP, and dynamin but not clathrin. Proc Natl Acad Sci U S A 1995;92(18).

103.   Straub SG, Shanmugam G, Sharp GW. Stimulation of insulin release by glucose is associated with an increase in the number of docked granules in the beta-cells of rat pancreatic islets. Diabetes 2004;53(12).

104.   Straub SG, Sharp GW. Hypothesis: one rate-limiting step controls the magnitude of both phases of glucose-stimulated insulin secretion. Am J Physiol Cell Physiol 2004;287(3).

105.   Daniel S, Noda M, Straub SG, Sharp GW. Identification of the docked granule pool responsible for the first phase of glucose-stimulated insulin secretion. Diabetes 1999;48(9).

106.   Leibiger B, Leibiger IB, Moede T et al. Selective insulin signaling through A and B insulin receptors regulates transcription of insulin and glucokinase genes in pancreatic beta cells. Mol Cell 2001;7(3).

107.   Leibiger IB, Leibiger B, Moede T, Berggren PO. Exocytosis of insulin promotes insulin gene transcription via the insulin receptor/PI-3 kinase/p70 s6 kinase and CaM kinase pathways. Mol Cell 1998;1(6).

108.   Schuit FC, In't Veld PA, Pipeleers DG. Glucose stimulates proinsulin biosynthesis by a dose-dependent recruitment of pancreatic beta cells. Proc Natl Acad Sci U S A 1988;85(11).

109.   Fonseca SG, Fukuma M, Lipson KL et al. WFS1 is a novel component of the unfolded protein response and maintains homeostasis of the endoplasmic reticulum in pancreatic beta-cells. J Biol Chem 2005;280(47).

110.   Hostens K, Pavlovic D, Zambre Y et al. Exposure of human islets to cytokines can result in disproportionately elevated proinsulin release. J Clin Invest 1999;104(1).

111.   Cnop M, Welsh N, Jonas JC, Jorns A, Lenzen S, Eizirik DL. Mechanisms of Pancreatic {beta}-Cell Death in Type 1 and Type 2 Diabetes: Many Differences, Few Similarities. Diabetes 2005;54 Suppl 2:S97-S107.

112.   Eizirik DL, Mandrup-Poulsen T. A choice of death - the signal-transduction of immune-mediated beta-cell apoptosis. diabetol 2001;44(12).

113.   Schuit FC, Huypens P, Heimberg H, Pipeleers DG. Glucose sensing in pancreatic beta-cells: a model for the study of other glucose-regulated cells in gut, pancreas, and hypothalamus. Diabetes 2001;50(1).

114.   Matschinsky FM. Banting Lecture 1995. A lesson in metabolic regulation inspired by the glucokinase glucose sensor paradigm. Diabetes 1996;45(2).

115.   Schuit F, De Vos A, Farfari S et al. Metabolic fate of glucose in purified islet cells. Glucose-regulated anaplerosis in beta cells. J Biol Chem 1997;272(30).

116.   Drucker DJ. The biology of incretin hormones. Cell Metab 2006;3(3).

117.   Hay CW, Sinclair EM, Bermano G, Durward E, Tadayyon M, Docherty K. Glucagon-like peptide-1 stimulates human insulin promoter activity in part through cAMP-responsive elements that lie upstream and downstream of the transcription start site. J Endocrinol 2005;186(2).

118.   Park S, Dong X, Fisher TL et al. Exendin-4 uses Irs2 signaling to mediate pancreatic beta cell growth and function. J Biol Chem 2006;281(2).

119.   Triplitt C, Wright A, Chiquette E. Incretin mimetics and dipeptidyl peptidase-IV inhibitors: potential new therapies for type 2 diabetes mellitus. Pharmacotherapy 2006;26(3).

120.   de Sa SV, Correa-Giannella ML, Machado MC et al. Somatostatin receptor subtype 5 (SSTR5) mRNA expression is related to histopathological features of cell proliferation in insulinomas. Endocr Relat Cancer 2006;13(1).

121.   Degano P, Peiro E, Miralles P, Silvestre RA, Marco J. Effects of rat pancreatic polypeptide on islet-cell secretion in the perfused rat pancreas. Metabolism 1992;41(3).

122.   Lundquist I, Sundler F, Ahren B, Alumets J, Hakanson R. Somatostatin, pancreatic polypeptide, substance P, and neurotensin: cellular distribution and effects on stimulated insulin secretion in the mouse. Endocrinology 1979;104(3).

123.   Szecowka J, Tatemoto K, Rajamaki G, Efendic S. Effects of PYY and PP on endocrine pancreas. Acta Physiol Scand 1983;119(2).

124.   Doi A, Shono T, Nishi M, Furuta H, Sasaki H, Nanjo K. IA-2beta, but not IA-2, is induced by ghrelin and inhibits glucose-stimulated insulin secretion. Proc Natl Acad Sci U S A 2006;103(4).

125.   Takahashi H, Kurose Y, Kobayashi S et al. Ghrelin enhances glucose-induced insulin secretion in scheduled meal-fed sheep. J Endocrinol 2006;189(1).

126.   Henquin JC. Triggering and amplifying pathways of regulation of insulin secretion by glucose. Diabetes 2000;49(11).

127.   Stiernet P, Guiot Y, Gilon P, Henquin JC. Glucose acutely decreases pH of secretory granules in mouse pancreatic islets: mechanisms and influence on insulin secretion. J Biol Chem 2006;.

128.   Johnson KH, O'Brien TD, Betsholtz C, Westermark P. Islet amyloid polypeptide: mechanisms of amyloidogenesis in the pancreatic islets and potential roles in diabetes mellitus. Lab Invest 1992;66.

129.   Juan-Pico P, Fuentes E, Javier Bermudez-Silva F et al. Cannabinoid receptors regulate Ca(2+) signals and insulin secretion in pancreatic beta-cell. Cell Calcium 2006;39(2).

130.   Kubosaki A, Nakamura S, Notkins AL. Dense Core Vesicle Proteins IA-2 and IA-2{beta}: Metabolic Alterations in Double Knockout Mice. diab 2005;54 Suppl 2.

131.   Moriyama H, Abiru N, Paronen J et al. Evidence for a primary islet autoantigen (preproinsulin 1) for insulitis and diabetes in the nonobese diabetic mouse. Proc Natl Acad Sci U S A 2003;100(18).

132.   Nakayama M, Abiru N, Moriyama H et al. Prime role for an insulin epitope in the development of type 1 diabetes in NOD mice. Nature 2005;435(7039).

133.   Han B, Serra P, Amrani A et al. Prevention of diabetes by manipulation of anti-IGRP autoimmunity: high efficiency of a low-affinity peptide. Nat Med 2005;11(6).

134.   Rotig A, Cormier V, Chatelain P, et al. Deletion of mitochondrial DNA in a case of early-onset diabetes mellitus, optic atrophy, and deafness (Wolfram syndrome, MIM 222300). J Clin Invest 1993;91.

135.   Rabinovitch A, Pukel C, Baquerizo H. Interleukin-1 inhibits glucose-modulated insulin and glucagon secretion in rat islet monolayer cultures. Endocrinology 1988;122(6).

136.   Chen MC, Paez-Espinosa V, Welsh N, Eizirik DL. Interleukin-1beta regulates phospholipase D-1 expression in rat pancreatic beta-cells. Endocrinology 2000;141(8).

137.   Nielsen K, Karlsen AE, Deckert M et al. Beta-cell maturation leads to in vitro sensitivity to cytotoxins. Diabetes 1999;48(12).

138. Strandell E, Buschard K, Saldeen J, Welsh N. Interleukin-1 beta induces the expression of hsp70, heme oxygenase and Mn-SOD in FACS-purified rat islet beta-cells, but not in alpha-cells. Immunol Lett 1995;48(2).

139.   Horio F, Fukuda M, Katoh H et al. Reactive oxygen intermediates in autoimmune islet cell destruction of the NOD mouse induced by peritoneal exudate cells (rich in macrophages) but not T cells. Diabetologia 1994;37(1).

140.   Jorns A, Tiedge M, Lenzen S, Munday R. Effect of superoxide dismutase, catalase, chelating agents, and free radical scavengers on the toxicity of alloxan to isolated pancreatic islets in vitro. Free Radic Biol Med 1999;26(9-10).

141.   Eizirik DL, Sandler S, Welsh N, Juntti-Berggren L, Berggren PO. Interleukin-1 beta-induced stimulation of insulin release in mouse pancreatic islets is related to diacylglycerol production and protein kinase C activation. Mol Cell Endocrinol 1995;111(2).

142.   Karlsen AE, Sparre T, Nielsen K, Nerup J, Pociot F. Proteome analysis--a novel approach to understand the pathogenesis of Type 1 diabetes mellitus. Dis Markers 2001;17(4).

143.   Karlsen AE, Storling ZM, Sparre T et al. Immune-mediated beta-cell destruction in vitro and in vivo-A pivotal role for galectin-3. Biochem Biophys Res Commun 2006;344(1).

144.   Eizirik DL. Interleukin-1 induced impairment in pancreatic islet oxidative metabolism of glucose is potentiated by tumor necrosis factor. Acta Endocrinol (Copenh) 1988;119(3).

145.   Miura M, Endo S, Kaku LL et al. Plasma type II phospholipase A2 levels in patients with acute pancreatitis. Res Commun Mol Pathol Pharmacol 2001;109(3-4).

146.   Uhl W, Schrag HJ, Schmitter N, Aufenanger J, Nevalainen TJ, Buchler MW. Experimental study of a novel phospholipase A2 inhibitor in acute pancreatitis. Br J Surg 1998;85(5).

147.   Gyulkhandanyan AV, Lee SC, Bikopoulos G, Dai F, Wheeler MB. The Zn2+-transporting Pathways in Pancreatic beta-Cells: A ROLE FOR THE L-TYPE VOLTAGE-GATED Ca2+ CHANNEL. J Biol Chem 2006;281(14).

148.   Chimienti F, Favier A, Seve M. ZnT-8, a pancreatic beta-cell-specific zinc transporter. Biometals 2005;18(4).

149.   Itoh Y, Hinuma S. GPR40, a free fatty acid receptor on pancreatic beta cells, regulates insulin secretion. Hepatol Res 2005.

150.   Srikanta S, Telen M, Posillico JT et al. Monoclonal antibodies to a human islet cell surface glycoprotein: 4F2 and LC7-2. Endocrinology 1987;120(6).

151.   Kanai Y, Endou H. Heterodimeric amino acid transporters: molecular biology and pathological and pharmacological relevance. Curr Drug Metab 2001;2(4).

152.   Kim dK, Ahn SG, Park JC, Kanai Y, Endou H, Yoon JH. Expression of L-type amino acid transporter 1 (LAT1) and 4F2 heavy chain (4F2hc) in oral squamous cell carcinoma and its precusor lesions. Anticancer Res 2004;24(3a).

153.   Magnuson MA. Tissue-specific regulation of glucokinase gene expression. J Cell Biochem 1992;48(2).

154.   Watada H, Kajimoto Y, Miyagawa J et al. PDX-1 induces insulin and glucokinase gene expressions in alphaTC1 clone 6 cells in the presence of betacellulin. Diabetes 1996;45(12).

155.   Ferber S, BeltrandelRio H, Johnson JH et al. GLUT-2 gene transfer into insulinoma cells confers both low and high affinity glucose-stimulated insulin release. Relationship to glucokinase activity. J Biol Chem 1994;269(15).

156.   German MS. Glucose sensing in pancreatic islet beta cells: the key role of glucokinase and the glycolytic intermediates. Proc Natl Acad Sci U S A 1993;90(5).

157.   Halban PA, Praz GA, Wollheim CB. Abnormal glucose metabolism accompanies failure of glucose to stimulate insulin release from a rat pancreatic cell line (RINm5F). Biochem J 1983;212(2).

158.   Khan A, Chandramouli V, Ostenson CG et al. Glucose cycling is markedly enhanced in pancreatic islets of obese hyperglycemic mice. Endocrinology 1990;126(5).

159.   Ling ZC, Khan A, Delauny F et al. Increased glucocorticoid sensitivity in islet beta-cells: effects on glucose 6-phosphatase, glucose cycling and insulin release. Diabetologia 1998;41(6).

160.   Ebert DH, Bischof LJ, Streeper RS et al. Structure and promoter activity of an islet-specific glucose-6-phosphatase catalytic subunit-related gene. Diabetes 1999;48(3).

161.   Sekine N, Cirulli V, Regazzi R et al. Low lactate dehydrogenase and high mitochondrial glycerol phosphate dehydrogenase in pancreatic beta-cells. Potential role in nutrient sensing. J Biol Chem 1994;269(7).

162.   Giroix MH, Rasschaert J, Sener A et al. Study of hexose transport, glycerol phosphate shuttle and Krebs cycle in islets of adult rats injected with streptozotocin during the neonatal period. Mol Cell Endocrinol 1992;83(2-3).

163.   Sener A, Malaisse WJ. Hexose metabolism in pancreatic islets. Ca(2+)-dependent activation of the glycerol phosphate shuttle by nutrient secretagogues. J Biol Chem 1992;267(19).

164.   Farfari S, Schulz V, Corkey B, Prentki M. Glucose-regulated anaplerosis and cataplerosis in pancreatic beta-cells: possible implication of a pyruvate/citrate shuttle in insulin secretion. Diabetes 2000;49(5).

165.   Malaisse WJ, Hutton JC, Kawazu S, Herchuelz A, Valverde I, Sener A. The stimulus-secretion coupling of glucose-induced insulin release. XXXV. The links between metabolic and cationic events. Diabetologia 1979;16(5).

166.   Newgard CB, McGarry JD. Metabolic coupling factors in pancreatic beta-cell signal transduction. Annu Rev Biochem 1995;64:689-719.

167.   DeFronzo RA, Prato SD. Insulin resistance and diabetes mellitus. J Diabetes Complications 1996;10(5).

168.   Rorsman P, Bokvist K, Ammala C, Eliasson L, Renstrom E, Gabel J. Ion channels, electrical activity and insulin secretion. Diabete Metab 1994;20(2).

169.   Williams JA. Electrical correlates of secretion in endocrine and exocrine cells. Fed Proc 1981;40(2).

170.   Ashcroft FM. K(ATP) channels and insulin secretion: a key role in health and disease. Biochem Soc Trans 2006;34(Pt 2).

171.   Ashcroft FM, Rorsman P. ATP-sensitive K+ channels: a link between B-cell metabolism and insulin secretion. Biochem Soc Trans 1990;18(1).

172.   Ashcroft SJ, Ashcroft FM. Properties and functions of ATP-sensitive K-channels. Cell Signal 1990;2(3).

173.   Bryan J, Aguilar-Bryan L. The ABCs of ATP-sensitive potassium channels: more pieces of the puzzle. Curr Opin Cell Biol 1997;9(4).

174.   Cook DL, Satin LS, Ashford ML, Hales CN. ATP-sensitive K+ channels in pancreatic beta-cells. Spare-channel hypothesis. Diabetes 1988;37(5).

175.   Loussouarn G, Pike LJ, Ashcroft FM, Makhina EN, Nichols CG. Dynamic sensitivity of ATP-sensitive K(+) channels to ATP. J Biol Chem 2001;276(31).

176.   Song DK, Ashcroft FM. ATP modulation of ATP-sensitive potassium channel ATP sensitivity varies with the type of SUR subunit. J Biol Chem 2001;276(10).

177.   Sakura H, Ashcroft FM. Identification of four trp1 gene variants murine pancreatic beta-cells. Diabetologia 1997;40(5).

178.   Gembal M, Detimary P, Gilon P, Gao ZY, Henquin JC. Mechanisms by which glucose can control insulin release independently from its action on adenosine triphosphate-sensitive K+ channels in mouse B cells. J Clin Invest 1993;91(3).

179.   Gembal M, Gilon P, Henquin JC. Evidence that glucose can control insulin release independently from its action on ATP-sensitive K+ channels in mouse B cells. J Clin Invest 1992;89(4).

180.   Komatsu M, Noda M, Sharp GW. Nutrient augmentation of Ca2+-dependent and Ca2+-independent pathways in stimulus-coupling to insulin secretion can be distinguished by their guanosine triphosphate requirements: studies on rat pancreatic islets. Endocrinology 1998;139(3).

181.   Komatsu M, Schermerhorn T, Noda M, Straub SG, Aizawa T, Sharp GW. Augmentation of insulin release by glucose in the absence of extracellular Ca2+: new insights into stimulus-secretion coupling. Diabetes 1997;46(12).

182.   Komatsu M, Sharp GW, Aizawa T, Hashizume K. Glucose stimulation of insulin release without an increase in cytosolic free Ca2+ concentration: a possible involvement of GTP. Jpn J Physiol 1997;47 Suppl 1:S22-4.

183.   Li GD, Luo RH, Metz SA. Effects of inhibitors of guanine nucleotide synthesis on membrane potential and cytosolic free Ca2+ levels in insulin-secreting cells. Biochem Pharmacol 2000;59(5).

184.   Regazzi R, Li G, Ullrich S, Jaggi C, Wollheim CB. Different requirements for protein kinase C activation and Ca2+-independent insulin secretion in response to guanine nucleotides. Endogenously generated diacylglycerol requires elevated Ca2+ for kinase C insertion into membranes. J Biol Chem 1989;264(17).

185.   Ashcroft FM, Gribble FM. ATP-sensitive K+ channels and insulin secretion: their role in health and disease. Diabetologia 1999;42(8).

186.   Babenko AP, Aguilar-Bryan L, Bryan J. A view of sur/KIR6.X, KATP channels. Annu Rev Physiol 1998;60:667-87.

187.   Polak M, Shield J. Neonatal Diabetes Mellitus -- genetic aspects 2004. Pediatr Endocrinol Rev 2004;2(2).

188.   Miki T, Nagashima K, Seino S. The structure and function of the ATP-sensitive K+ channel in insulin-secreting pancreatic beta-cells. J Mol Endocrinol 1999;22(2).

189.   Suzuki M, Fujikura K, Inagaki N, Seino S, Takata K. Localization of the ATP-sensitive K+ channel subunit Kir6.2 in mouse pancreas. Diabetes 1997;46(9).

190.   Bokvist K, Olsen HL, Hoy M et al. Characterisation of sulphonylurea and ATP-regulated K+ channels in rat pancreatic A-cells. Pflugers Arch 1999;438(4).

191.   Gopel SO, Kanno T, Barg S, Rorsman P. Patch-clamp characterisation of somatostatin-secreting -cells in intact mouse pancreatic islets. J Physiol 2000;528(Pt 3).

192.   Gopel SO, Kanno T, Barg S, Weng XG, Gromada J, Rorsman P. Regulation of glucagon release in mouse -cells by KATP channels and inactivation of TTX-sensitive Na+ channels. J Physiol 2000;528(Pt 3).

193.   Hattersley AT, Ashcroft FM. Activating mutations in Kir6.2 and neonatal diabetes: new clinical syndromes, new scientific insights, and new therapy. diab 2005;54(9).

194.   Koster JC, Remedi MS, Dao C, Nichols CG. ATP and sulfonylurea sensitivity of mutant ATP-sensitive K+ channels in neonatal diabetes: implications for pharmacogenomic therapy. diab 2005;54(9).

195.   Proks P, Girard C, Haider S et al. A gating mutation at the internal mouth of the Kir6.2 pore is associated with DEND syndrome. EMBO Rep 2005;6(5).

196.   Nestorowicz A, Glaser B, Wilson BA et al. Genetic heterogeneity in familial hyperinsulinism [published erratum appears in Hum Mol Genet 1998 Sep;7(9):1527]. Hum Mol Genet 1998;7(7).

197.   Flanagan S, Damhuis A, Banerjee I et al. Partial ABCC8 gene deletion mutations causing diazoxide-unresponsive hyperinsulinaemic hypoglycaemia. Pediatr Diabetes 2011.

198.   Flanagan SE, Kapoor RR, Banerjee I et al. Dominantly acting ABCC8 mutations in patients with medically unresponsive hyperinsulinaemic hypoglycaemia. Clin Genet 2011;79(6).

199.   Ismail D, Smith VV, de Lonlay P et al. Familial focal congenital hyperinsulinism. J Clin Endocrinol Metab 2011;96(1).

200.   Kapoor RR, Flanagan SE, James CT et al. Hyperinsulinaemic hypoglycaemia and diabetes mellitus due to dominant ABCC8/KCNJ11 mutations. Diabetologia 2011;54(10).

201.   Kumaran A, Kapoor RR, Flanagan SE, Ellard S, Hussain K. Congenital hyperinsulinism due to a compound heterozygous ABCC8 mutation with spontaneous resolution at eight weeks. Horm Res Paediatr 2010;73(4).

202.   James C, Kapoor RR, Ismail D, Hussain K. The genetic basis of congenital hyperinsulinism. J Med Genet 2009;46(5).

203.   Kapoor RR, Flanagan SE, James C, Shield J, Ellard S, Hussain K. Hyperinsulinaemic hypoglycaemia. Arch Dis Child 2009;94(6).

204.   Kapoor RR, James C, Hussain K. Hyperinsulinism in developmental syndromes. Endocr Dev 2009;14.

205.   Poon M, Hussain K. Postprandial hyperinsulinaemic hypoglycaemia and type 1 diabetes mellitus. BMJ Case Rep 2009;2009.

206.   Kapoor RR, James C, Flanagan SE, Ellard S, Eaton S, Hussain K. 3-Hydroxyacyl-coenzyme A dehydrogenase deficiency and hyperinsulinemic hypoglycemia: characterization of a novel mutation and severe dietary protein sensitivity. J Clin Endocrinol Metab 2009;94(7).

207.   Cuesta-Munoz AL, Huopio H, Otonkoski T et al. Severe persistent hyperinsulinemic hypoglycemia due to a de novo glucokinase mutation. Diabetes 2004;53(8).

208.   Osbak KK, Colclough K, Saint-Martin C et al. Update on mutations in glucokinase (GCK), which cause maturity-onset diabetes of the young, permanent neonatal diabetes, and hyperinsulinemic hypoglycemia. Hum Mutat 2009;30(11).

209.   Kawajiri M, Okano Y, Kuno M et al. Unregulated insulin secretion by pancreatic beta cells in hyperinsulinism/hyperammonemia syndrome: role of glutamate dehydrogenase, ATP-sensitive potassium channel, and nonselective cation channel. Pediatr Res 2006;59(3).

210.   Chan CB, Saleh MC, Koshkin V, Wheeler MB. Uncoupling protein 2 and islet function. diab 2004;53 Suppl 1.

211.   Joseph JW, Koshkin V, Saleh MC et al. Free fatty acid-induced beta-cell defects are dependent on uncoupling protein 2 expression. J Biol Chem 2004;279(49).

212.   Ann K, Kowalchyk JA, Loyet KM, Martin TF. Novel Ca2+-binding protein (CAPS) related to UNC-31 required for Ca2+-activated exocytosis. J Biol Chem 1997;272(32).

213.   Bokvist K, Eliasson L, Ammala C, Renstrom E, Rorsman P. Co-localization of L-type Ca2+ channels and insulin-containing secretory granules and its significance for the initiation of exocytosis in mouse pancreatic B-cells. EMBO J 1995;14(1).

214.   Wiser O, Trus M, Hernandez A et al. The voltage sensitive Lc-type Ca2+ channel is functionally coupled to the exocytotic machinery. Proc Natl Acad Sci U S A 1999;96(1).

215.   Nilius B, Hess P, Lansman JB, Tsien RW. A novel type of cardiac calcium channel in ventricular cells. Nature 1985;316(6027).

216.   Gutnick MJ, Lux HD, Swandulla D, Zucker H. Voltage-dependent and calcium-dependent inactivation of calcium channel current in identified snail neurones. J Physiol 1989;412:197-220.

217.   Ammala C, Eliasson L, Bokvist K et al. Activation of protein kinases and inhibition of protein phosphatases play a central role in the regulation of exocytosis in mouse pancreatic beta cells. Proc Natl Acad Sci U S A 1994;91(10).

218.   Dean PM. Ultrastructural morphometry of the pancreatic -cell. Diabetologia 1973;9(2).

219.   Eliasson L, Renstrom E, Ding WG, Proks P, Rorsman P. Rapid ATP-dependent priming of secretory granules precedes Ca(2+)-induced exocytosis in mouse pancreatic B-cells. J Physiol 1997;503(Pt 2).

220.   Gromada J, Hoy M, Renstrom E et al. CaM kinase II-dependent mobilization of secretory granules underlies acetylcholine-induced stimulation of exocytosis in mouse pancreatic B-cells. J Physiol 1999;518(Pt 3).

221.   Rorsman P, Eliasson L, Renstrom E, Gromada J, Barg S, Gopel S. The Cell Physiology of Biphasic Insulin Secretion. News Physiol Sci 2000;15:72-77.

222.   Rorsman P. The pancreatic beta-cell as a fuel sensor: an electrophysiologist's viewpoint. Diabetologia 1997;40(5).

223.   Huang JD, Brady ST, Richards BW et al. Direct interaction of microtubule- and actin-based transport motors. Nature 1999;397(6716).

224.   Easom RA. Beta-granule transport and exocytosis. Semin Cell Dev Biol 2000;11(4).

225.   Sollner T, Bennett MK, Whiteheart SW, Scheller RH, Rothman JE. A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion. Cell 1993;75(3).

226.   Rupnik M, Kreft M, Sikdar SK et al. Rapid regulated dense-core vesicle exocytosis requires the CAPS protein. Proc Natl Acad Sci U S A 2000;97(10).

227.   Fukui K, Yang Q, Cao Y et al. The HNF-1 target collectrin controls insulin exocytosis by SNARE complex formation. Cell Metab 2005;2(6).

228.   Evans GJ, Barclay JW, Prescott GR et al. Protein kinase B/Akt is a novel cysteine string protein kinase that regulates exocytosis release kinetics and quantal size. J Biol Chem 2006;281(3).

229.   Jahn R, Sudhof TC. Membrane fusion and exocytosis. Annu Rev Biochem 1999;68:863-911.

230.   Izumi T, Gomi H, Torii S. Functional analysis of Rab27a effector granuphilin in insulin exocytosis. Methods Enzymol 2005;403.

231.   Gomi H, Mizutani S, Kasai K, Itohara S, Izumi T. Granuphilin molecularly docks insulin granules to the fusion machinery. J Cell Biol 2005;171(1).

232.   Jacobsson G, Bean AJ, Scheller RH et al. Identification of synaptic proteins and their isoform mRNAs in compartments of pancreatic endocrine cells. Proc Natl Acad Sci U S A 1994;91(26).

233.   Lang J. Molecular mechanisms and regulation of insulin exocytosis as a paradigm of endocrine secretion. Eur J Biochem 1999;259(1-2).

234.   Lao G, Scheuss V, Gerwin CM et al. Syntaphilin: a syntaxin-1 clamp that controls SNARE assembly. Neuron 2000;25(1).

235.   Fisher RJ, Pevsner J, Burgoyne RD. Control of fusion pore dynamics during exocytosis by Munc18. Science 2001;291(5505).

236.   Huang X, Kang YH, Pasyk EA et al. Ca(2+) influx and cAMP elevation overcame botulinum toxin A but not tetanus toxin inhibition of insulin exocytosis. Am J Physiol Cell Physiol 2001;281(3).

237.   Land J, Zhang H, Vaidyanathan VV, Sadoul K, Niemann H, Wollheim CB. Transient expression of botulinum neurotoxin C1 light chain differentially inhibits calcium and glucose induced insulin secretion in clonal beta-cells. FEBS Lett 1997;419(1).

238.   Li C, Ullrich B, Zhang JZ, Anderson RG, Brose N, Sudhof TC. Ca(2+)-dependent and -independent activities of neural and non-neural synaptotagmins. Nature 1995;375(6532).

239.   Loyet KM, Kowalchyk JA, Chaudhary A, Chen J, Prestwich GD, Martin TF. Specific binding of phosphatidylinositol 4,5-bisphosphate to calcium-dependent activator protein for secretion (CAPS), a potential phosphoinositide effector protein for regulated exocytosis. J Biol Chem 1998;273(14).

240.   Broer A, Friedrich B, Wagner CA et al. Association of 4F2hc with light chains LAT1, LAT2 or y+LAT2 requires different domains. Biochem J 2001;355(Pt 3).

241.   Xu T, Ashery U, Burgoyne RD, Neher E. Early requirement for alpha-SNAP and NSF in the secretory cascade in chromaffin cells. EMBO J 1999;18(12).

242.   Clark JD, Lin LL, Kriz RW et al. A novel arachidonic acid-selective cytosolic PLA2 contains a Ca(2+)-dependent translocation domain with homology to PKC and GAP. Cell 1991;65(6).

243.   Sharp JD, White DL, Chiou XG et al. Molecular cloning and expression of human Ca(2+)-sensitive cytosolic phospholipase A2. J Biol Chem 1991;266(23).

244.   Iezzi M, Escher G, Meda P et al. Subcellular distribution and function of Rab3A, B, C, and D isoforms in insulin-secreting cells. Mol Endocrinol 1999;13(2).

245.   Kajio H, Olszewski S, Rosner PJ, Donelan MJ, Geoghegan KF, Rhodes CJ. A low-affinity Ca2+-dependent association of calmodulin with the Rab3A effector domain inversely correlates with insulin exocytosis. Diabetes 2001;50(9).

246.   Balsinde J, Balboa MA, Insel PA, Dennis EA. Regulation and inhibition of phospholipase A2. Annu Rev Pharmacol Toxicol 1999;39:175-89.

247.   Bauerfeind R, Takei K, De Camilli P. Amphiphysin I is associated with coated endocytic intermediates and undergoes stimulation-dependent dephosphorylation in nerve terminals. J Biol Chem 1997;272(49).

248.   Lai MM, Hong JJ, Ruggiero AM et al. The calcineurin-dynamin 1 complex as a calcium sensor for synaptic vesicle endocytosis. J Biol Chem 1999;274(37).

249.   Kang G, Chepurny OG, Malester B et al. cAMP Sensor Epac As A Determinant Of ATP-Sensitive Potassium Channel Activity In Human Pancreatic Beta Cells And Rat INS-1 Cells. J Physiol 2006.

250.   Waselle L, Gerona RR, Vitale N, Martin TF, Bader MF, Regazzi R. Role of phosphoinositide signaling in the control of insulin exocytosis. Mol Endocrinol 2005;19(12).

251.   Ammala C, Ashcroft FM, Rorsman P. Calcium-independent potentiation of insulin release by cyclic AMP in single beta-cells. Nature 1993;363(6427).

252.   Hamilton PB, Stevens JR, Gidley J, Holz P, Gibson WC. A new lineage of trypanosomes from Australian vertebrates and terrestrial bloodsucking leeches (Haemadipsidae). Int J Parasitol 2005;35(4).

253.   Renstrom E, Eliasson L, Rorsman P. Protein kinase A-dependent and -independent stimulation of exocytosis by cAMP in mouse pancreatic B-cells. J Physiol 1997;502(Pt 1).

254.   Ashcroft FM, Proks P, Smith PA, Ammala C, Bokvist K, Rorsman P. Stimulus-secretion coupling in pancreatic beta cells. J Cell Biochem 1994;55 Suppl:54-65.

255.   Murakami K, Chan SY, Routtenberg A. Protein kinase C activation by cis-fatty acid in the absence of Ca2+ and phospholipids. J Biol Chem 1986;261(33).

256.   Tischfield JA. A reassessment of the low molecular weight phospholipase A2 gene family in mammals. J Biol Chem 1997;272(28).

257.   Leslie CC. Properties and regulation of cytosolic phospholipase A2. J Biol Chem 1997;272(27).

258.   Sapirstein A, Bonventre JV. Specific physiological roles of cytosolic phospholipase A(2) as defined by gene knockouts. Biochim Biophys Acta 2000;1488(1-2).

259.   Bonventre JV, Huang Z, Taheri MR et al. Reduced fertility and postischaemic brain injury in mice deficient in cytosolic phospholipase A2. Nature 1997;390(6660).

260.   Murakami M, Nakatani Y, Kuwata H, Kudo I. Cellular components that functionally interact with signaling phospholipase A(2)s. Biochim Biophys Acta 2000;1488(1-2).

261.   Balsinde J, Dennis EA. Function and inhibition of intracellular calcium-independent phospholipase A2. J Biol Chem 1997;272(26).

262.   Winstead MV, Balsinde J, Dennis EA. Calcium-independent phospholipase A(2): structure and function. Biochim Biophys Acta 2000;1488(1-2).

263.   Ramanadham S, Ma Z, Arita H, Zhang S, Turk J. Type IB secretory phospholipase A2 is contained in insulin secretory granules of pancreatic islet beta-cells and is co-secreted with insulin from glucose-stimulated islets. Biochim Biophys Acta 1998;1390(3).

264.   Dennis EA. The growing phospholipase A2 superfamily of signal transduction enzymes. Trends Biochem Sci 1997;22(1).

265.   Stafforini DM, McIntyre TM, Zimmerman GA, Prescott SM. Platelet-activating factor acetylhydrolases. J Biol Chem 1997;272(29).

266.   Loweth AC, Scarpello JH, Morgan NG. Phospholipase A2 expression in human and rodent insulin-secreting cells. Mol Cell Endocrinol 1995;112(2).

267.   Metz S, Holmes D, Robertson RP, Leitner W, Draznin B. Gene expression of type I phospholipase A2 in pancreatic beta cells. Regulation of mRNA levels by starvation or glucose excess. FEBS Lett 1991;295(1-3).

268.   Niwa T, Matsukawa Y, Senda T, Nimura Y, Hidaka H, Niki I. Acetylcholine activates intracellular movement of insulin granules in pancreatic beta-cells via inositol trisphosphate-dependent [correction of triphosphate-dependent] mobilization of intracellular Ca2+. Diabetes 1998;47(11).

269.   Parker KJ, Jones PM, Hunton CH, Persaud SJ, Taylor CG, Howell SL. Identification and localisation of a type IV cytosolic phospholipase A2 in rat pancreatic beta-cells. J Mol Endocrinol 1996;17(1).

270.   Eddlestone GT. ATP-sensitive K channel modulation by products of PLA2 action in the insulin-secreting HIT cell line. Am J Physiol 1995;268(1 Pt 1).

271.   Gross RW, Ramanadham S, Kruszka KK, Han X, Turk J. Rat and human pancreatic islet cells contain a calcium ion independent phospholipase A2 activity selective for hydrolysis of arachidonate which is stimulated by adenosine triphosphate and is specifically localized to islet beta-cells. Biochemistry 1993;32(1).

272. Kashiwagi M, Friess H, Uhl W et al. Phospholipase A2 isoforms are altered in chronic pancreatitis. Ann Surg 1998;227(2).

273.   Seeds MC, Jones DF, Chilton FH, Bass DA. Secretory and cytosolic phospholipases A2 are activated during TNF priming of human neutrophils. Biochim Biophys Acta 1998;1389(3).

274.   Ma Z, Ramanadham S, Kempe K, Chi XS, Ladenson J, Turk J. Pancreatic islets express a Ca2+-independent phospholipase A2 enzyme that contains a repeated structural motif homologous to the integral membrane protein binding domain of ankyrin. J Biol Chem 1997;272(17).

275.   Turk J, Wolf BA, Lefkowith JB, Stump WT, McDaniel ML. Glucose-induced phospholipid hydrolysis in isolated pancreatic islets: quantitative effects on the phospholipid content of arachidonate and other fatty acids. Biochim Biophys Acta 1986;879(3).

276.   Ma Z, Zhang S, Turk J, Ramanadham S. Stimulation of insulin secretion and associated nuclear accumulation of iPLA(2)beta in INS-1 insulinoma cells. Am J Physiol Endocrinol Metab 2002;282(4).

277.   Wolf BA, Pasquale SM, Turk J. Free fatty acid accumulation in secretagogue-stimulated pancreatic islets and effects of arachidonate on depolarization-induced insulin secretion. Biochemistry 1991;30(26).

278.   Wolf BA, Turk J, Sherman WR, McDaniel ML. Intracellular Ca2+ mobilization by arachidonic acid. Comparison with myo-inositol 1,4,5-trisphosphate in isolated pancreatic islets. J Biol Chem 1986;261(8).

279.   Bell RL, Stanford N, Kennerly DA, Majerus PW. Diglyceride lipase: a pathway for arachidonate release from human platelets. Adv Prostaglandin Thromboxane Res 1980;6:219-24.

280.   Konrad RJ, Major CD, Wolf BA. Diacylglycerol hydrolysis to arachidonic acid is necessary for insulin secretion from isolated pancreatic islets: sequential actions of diacylglycerol and monoacylglycerol lipases. Biochemistry 1994;33(45).

281.   Konrad RJ, Jolly YC, Major C, Wolf BA. Inhibition of phospholipase A2 and insulin secretion in pancreatic islets. Biochim Biophys Acta 1992;1135(2).

282.   Metz SA. Pancreatic islet phospholipase A2: differential, Ca2+-dependent effects of lysophospholipids, arachidonic acid, and its lipoxygenase-derived metabolites on insulin release. Adv Prostaglandin Thromboxane Leukot Res 1987;17B:668-76.

283.   Damron DS, Van Wagoner DR, Moravec CS, Bond M. Arachidonic acid and endothelin potentiate Ca2+ transients in rat cardiac myocytes via inhibition of distinct K+ channels. J Biol Chem 1993;268(36).

284.   Graber MN, Alfonso A, Gill DL. Ca2+ pools and cell growth: arachidonic acid induces recovery of cells growth-arrested by Ca2+ pool depletion. J Biol Chem 1996;271(2).

285.   McPhail LC, Clayton CC, Snyderman R. A potential second messenger role for unsaturated fatty acids: activation of Ca2+-dependent protein kinase. Science 1984;224(4649).

286.   Shuttleworth TJ. Arachidonic acid activates the noncapacitative entry of Ca2+ during [Ca2+]i oscillations. J Biol Chem 1996;271(36).

287.   Metz SA. Exogenous arachidonic acid promotes insulin release from intact or permeabilized rat islets by dual mechanisms. Putative activation of Ca2+ mobilization and protein kinase C. Diabetes 1988;37(11).

288.   Metz SA. The pancreatic islet as Rubik's Cube. Is phospholipid hydrolysis a piece of the puzzle? Diabetes 1991;40(12).

289.   Ramanadham S, Gross R, Turk J. Arachidonic acid induces an increase in the cytosolic calcium concentration in single pancreatic islet beta cells. Biochem Biophys Res Commun 1992;184(2).

290.   Hirabayashi T, Shimizu T. Localization and regulation of cytosolic phospholipase A(2). Biochim Biophys Acta 2000;1488(1-2).

291.   Kimple ME, Nixon AB, Kelly P et al. A role for G(z) in pancreatic islet beta-cell biology. J Biol Chem 2005;280(36).

292.   Kato R, Yamamoto S, Nakadate T, Nakaki T. Possible involvement of phospholipase A2 activation and lipoxygenase product(s) in the mechanism of insulin secretion. Adv Prostaglandin Thromboxane Leukot Res 1983;12:265-70.

293.   Zhou YP, Teng D, Dralyuk F et al. Apoptosis in insulin-secreting cells. Evidence for the role of intracellular Ca2+ stores and arachidonic acid metabolism. J Clin Invest 1998;101(8).

294.   Frossard JL, Bhagat L, Lee HS et al. Both thermal and non-thermal stress protect against caerulein induced pancreatitis and prevent trypsinogen activation in the pancreas. Gut 2002;50(1).

295.   Ramsingh AI, Chapman N, Tracy S. Coxsackieviruses and diabetes. Bioessays 1997;19(9).

296.   Sevilla N, Homann D, von Herrath M et al. Virus-induced diabetes in a transgenic model: role of cross-reacting viruses and quantitation of effector T cells needed to cause disease. J Virol 2000;74(7).

297.   Gu D, Sarvetnick N. Epithelial cell proliferation and islet neogenesis in IFN-g transgenic mice. Development 1993;118(1).

298.   Pruzanski W, Vadas P. Phospholipase A2--a mediator between proximal and distal effectors of inflammation. Immunol Today 1991;12(5).

299.   van Puijenbroek AA, Wissink S, van der Saag PT, Peppelenbosch MP. Phospholipase A2 inhibitors and leukotriene synthesis inhibitors block TNF-induced NF-kappaB activation. Cytokine 1999;11(2).

300.   Deevska GM, Nikolova-Karakashian MN. The twists and turns of sphingolipid pathway in glucose regulation. Biochimie 2011;93(1).

301.   Fox TE, Bewley MC, Unrath KA et al. Circulating sphingolipid biomarkers in models of type 1 diabetes. J Lipid Res 2011;52(3).

302.   Ortsater H. Arachidonic acid fights palmitate: new insights into fatty acid toxicity in beta-cells. Clin Sci (Lond) 2011;120(5).

303.   Metz SA. Mobilization of cellular Ca2+ by lysophospholipids in rat islets of Langerhans. Biochim Biophys Acta 1988;968(2).

304.   Rustenbeck I, Lenzen S. Effects of lysophosphatidylcholine and arachidonic acid on the regulation of intracellular Ca2+ transport. Naunyn Schmiedebergs Arch Pharmacol 1989;339(1-2).

305.   Gasser KW, Holda JR. ATP-sensitive potassium transport by pancreatic secretory granule membrane. Am J Physiol 1993;264(1 Pt 1).

306.   Metz SA. Ether-linked lysophospholipids initiate insulin secretion. Lysophospholipids may mediate effects of phospholipase A2 activation on hormone release. Diabetes 1986;35(7).

307.   Fernandez-Valverde SL, Taft RJ, Mattick JS. MicroRNAs in beta-cell biology, insulin resistance, diabetes and its complications. Diabetes 2012;60(7).

308.   Joglekar MV, Parekh VS, Hardikar AA. New pancreas from old: microregulators of pancreas regeneration. Trends Endocrinol Metab 2007;18(10).

309.   Joglekar MV, Parekh VS, Mehta S, Bhonde RR, Hardikar AA. MicroRNA profiling of developing and regenerating pancreas reveal post-transcriptional regulation of neurogenin3. Dev Biol 2007;311(2).

310.   Joglekar MV, Joglekar VM, Hardikar AA. Expression of islet-specific microRNAs during human pancreatic development. Gene Expr Patterns 2009;9(2).

311.   Joglekar MV, Parekh VS, Hardikar AA. Islet-specific microRNAs in pancreas development, regeneration and diabetes. Indian J Exp Biol 2011;49(6).

312.   Cuellar TL, McManus MT. MicroRNAs and endocrine biology. J Endocrinol 2005;187(3).

313.   Bolmeson C, Esguerra JL, Salehi A, Speidel D, Eliasson L, Cilio CM. Differences in islet-enriched miRNAs in healthy and glucose intolerant human subjects. Biochem Biophys Res Commun404(1).

314.   Davalos A, Goedeke L, Smibert P et al. miR-33a/b contribute to the regulation of fatty acid metabolism and insulin signaling. Proc Natl Acad Sci U S A108(22).

315.   Fred RG, Bang-Berthelsen CH, Mandrup-Poulsen T, Grunnet LG, Welsh N. High glucose suppresses human islet insulin biosynthesis by inducing miR-133a leading to decreased polypyrimidine tract binding protein-expression. PLoS One5(5).

316.   Frost RJ, Olson EN. Control of glucose homeostasis and insulin sensitivity by the Let-7 family of microRNAs. Proc Natl Acad Sci U S A108(52).

317.   Gallagher IJ, Scheele C, Keller P et al. Integration of microRNA changes in vivo identifies novel molecular features of muscle insulin resistance in type 2 diabetes. Genome Med2(2).

318.   Hennessy E, Clynes M, Jeppesen PB, O'Driscoll L. Identification of microRNAs with a role in glucose stimulated insulin secretion by expression profiling of MIN6 cells. Biochem Biophys Res Commun396(2).

319.   Kalis M, Bolmeson C, Esguerra JL et al. Beta-cell specific deletion of Dicer1 leads to defective insulin secretion and diabetes mellitus. PLoS One6(12).

320.   Lee JY, Ristow M, Lin X, White MF, Magnuson MA, Hennighausen L. RIP-Cre revisited, evidence for impairments of pancreatic beta-cell function. J Biol Chem 2006;281(5).

321.   Wiederkehr A, Wollheim CB. Implication of mitochondria in insulin secretion and action. Endocrinology 2006.