Insulin Secretory Granules Research Articles

Characterization of Phospholipids in Insulin Secretory Granules and Mitochondria in Pancreatic Beta Cells and Their Changes with Glucose Stimulation

The lipid composition of insulin secretory granules (ISG) has never previously been thoroughly characterized. We characterized the phospholipid composition of ISG and mitochondria in pancreatic beta cells without and with glucose stimulation. The phospholipid/protein ratios of most phospholipids containing unsaturated fatty acids were higher in ISG than in whole cells and in mitochondria. The concentrations of negatively charged phospholipids, phosphatidylserine, and phosphatidylinositol in ISG were 5-fold higher than in the whole cell. In ISG phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin, fatty acids 12:0 and 14:0 were high, as were phosphatidylserine and phosphatidylinositol containing 18-carbon unsaturated FA. With glucose stimulation, the concentration of many ISG phosphatidylserines and phosphatidylinositols increased; unsaturated fatty acids in phosphatidylserine increased; and most phosphatidylethanolamines, phosphatidylcholines, sphingomyelins, and lysophosphatidylcholines were unchanged. Unsaturation and shorter fatty acid length in phospholipids facilitate curvature and fluidity of membranes, which favors fusion of membranes. Recent evidence suggests that negatively charged phospholipids, such as phosphatidylserine, act as coupling factors enhancing the interaction of positively charged regions in SNARE proteins in synaptic or secretory vesicle membrane lipid bilayers with positively charged regions in SNARE proteins in the plasma membrane lipid bilayer to facilitate docking of vesicles to the plasma membrane during exocytosis. The results indicate that ISG phospholipids are in a dynamic state and are consistent with the idea that changes in ISG phospholipids facilitate fusion of ISG with the plasma membrane-enhancing glucose-stimulated insulin exocytosis.

Autocrine effect of Zn²⁺ on the glucose-stimulated insulin secretion.

It is well known that zinc (Zn(2+)) is required for the process of insulin biosynthesis and the maturation of insulin secretory granules in pancreatic beta (β)-cells, and that changes in Zn(2+) levels in the pancreas have been found to be associated with diabetes. Glucose-stimulation causes a rapid co-secretion of Zn(2+) and insulin with similar kinetics. However, we do not know whether Zn(2+) regulates insulin availability and secretion. Here we investigated the effect of Zn(2+) on glucose-stimulated insulin secretion (GSIS) in isolated mouse pancreatic islets. Whereas Zn(2+) alone (control) had no effect on the basal secretion of insulin, it significantly inhibited GSIS. The application of CaEDTA, by removing the secreted Zn(2+) from the extracellular milieu of the islets, resulted in significantly increased GSIS, suggesting an overall inhibitory role of secreted Zn(2+) on GSIS. The inhibitory action of Zn(2+) was mostly mediated through the activities of KATP/Ca(2+) channels. Furthermore, during brief paired-pulse glucose-stimulated Zn(2+) secretion (GSZS), Zn(2+) secretion following the second pulse was significantly attenuated, probably by the secreted endogenous Zn(2+) after the first pulse. Such an inhibition on Zn(2+) secretion following the second pulse was completely reversed by Zn(2+) chelation, suggesting a negative feedback mechanism, in which the initial glucose-stimulated Zn(2+) release inhibits subsequent Zn(2+) secretion, subsequently inhibiting insulin co-secretion as well. Taken together, these data suggest a negative feedback mechanism on GSZS and GSIS by Zn(2+) secreted from β-cells, and the co-secreted Zn(2+) may act as an autocrine inhibitory modulator.

Stability of proICA512/IA-2 and its targeting to insulin secretory granules require β4-sheet-mediated dimerization of its ectodomain in the endoplasmic reticulum.

The type 1 diabetes autoantigen ICA512/IA-2/RPTPN is a receptor protein tyrosine phosphatase of the insulin secretory granules (SGs) which regulates the size of granule stores, possibly via cleavage/signaling of its cytosolic tail. The role of its extracellular region remains unknown. Structural studies indicated that β2- or β4-strands in the mature ectodomain (ME ICA512) form dimers in vitro. Here we show that ME ICA512 prompts proICA512 dimerization in the endoplasmic reticulum. Perturbation of ME ICA512 β2-strand N-glycosylation upon S508A replacement allows for proICA512 dimerization, O-glycosylation, targeting to granules, and conversion, which are instead precluded upon G553D replacement in the ME ICA512 β4-strand. S508A/G553D and N506A/G553D double mutants dimerize but remain in the endoplasmic reticulum. Removal of the N-terminal fragment (ICA512-NTF) preceding ME ICA512 allows an ICA512-ΔNTF G553D mutant to exit the endoplasmic reticulum, and ICA512-ΔNTF is constitutively delivered to the cell surface. The signal for SG sorting is located within the NTF RESP18 homology domain (RESP18-HD), whereas soluble NTF is retained in the endoplasmic reticulum. Hence, we propose that the ME ICA512 β2-strand fosters proICA512 dimerization until NTF prevents N506 glycosylation. Removal of this constraint allows for proICA512 β4-strand-induced dimerization, exit from the endoplasmic reticulum, O-glycosylation, and RESP18-HD-mediated targeting to granules.

Chronic effects of neuroendocrine regulatory peptide (NERP-1 and -2) on insulin secretion and gene expression in pancreatic β-cells

Neuroendocrine regulatory peptides (NERP-1 and -2) are novel amidated peptides derived from VGF, a polypeptide secreted from neurons and endocrine cells through a regulated pathway. Dr. Nakazato Masamitsu reported that NERP-1 and -2 may have a local modulator function on the human endocrine system, and clearly showed expression of NERP-1 and -2 in human pancreas islets. Based on these data, we investigated the alteration of insulin secretion, insulin granule-related protein, and pancreas-specific transcription factors in response to NERPs expression. We confirmed the expression of NERP-1 and -2 in the pancreas of a human diabetes patient, in addition to diabetic animal models. When INS1 cells and primary rat islets were incubated with 10nM NERPs for 3days, glucose-stimulated insulin secretion levels were blunted by NERP-1 and -2. The number of insulin granules released from the readily releasable pool, which is associated with the first phase of glucose-stimulated insulin release, was decreased by NERP-1 and -2. Insulin granule-related proteins and mRNAs were down-regulated by NERP-2 treatment. NERP-2 decreased the expression of BETA2/NeuroD and insulin and controlled the nucleo-cytoplasmic translocation of FOXO1 and Pdx-1. We observed that NERP-2 levels were dramatically increased in diabetic pancreas. In conclusion, NERP-2 may play an important role in insulin secretion through the regulation of insulin secretory granules and β-cell transcription factors. In addition, NERP-2 expression is increased in diabetic conditions. Therefore, we suggest that NERPs may be potent endogenous suppressors of glucose-dependent insulin secretion.

Trophic actions of oral vanadium and improved glycemia on the pancreatic beta-cell ultrastructure of streptozotocin-induced diabetic rats.

Oral vanadyl sulfate (vanadium) has potent hypoglycemic effects in diabetes animals, but data about its actions on pancreatic beta-cells (BC) ultrastructure is limited. Partial diabetic rats were treated with vanadium and insulin injection and their effects on BC ultrastructure are studied. Male rats were made diabetic with intravenous streptozotocin injection (STZ, 40 mg/kg). Animals were randomly divided to control (CD), vanadium-treated (1 mg/mL VOSO4 + 5H2O in base solution, VTD) and insulin-treated (80 U/kg/day NPH insulin injection, ITD) diabetic groups. Treatments started 10 days after STZ injection and terminated after 2 months. Intermittent tail blood samples were taken for measurements of blood glucose (BG) and plasma insulin (PI). Finally animals were sacrificed and pancreata prepared for assessments of BC ultrastructure, islets histology and insulin immunoreactivity (IIR). Vanadium decreased BG (P<0.0001), elevated the reduced PI (P<0.001), prevented islet atrophy and restored BC ultrastructure. Low BG seen during treatment in VTD and ITD only persisted in VTD after vanadium withdrawal. Hyperglycemia worsened in CD and repaired in ITD shortly after insulin withdrawal. CD islets were atrophied, had scattered IIR spots. BC had pyknotic nuclei, vacuolated cytoplasm and few tiny insulin secretory granules. VTD islets looked normal with compact centered IIR spots. BC had well-developed endoplasmic reticulum, many insulin secretory granules and mitochondria. ITD islet structure was slightly better than CD and BC had some immature insulin secretory granules. The trophic actions of vanadium in diabetic rats effectively renovated beta cell ultrastructure and prevented pancreatic islets atrophy, whereas the relief of glucotoxicity seen with insulin treatment could repair some beta cells and partially prevented islet atrophy.

SORCS1 is necessary for normal insulin secretory granule biogenesis in metabolically stressed β cells.

We previously positionally cloned Sorcs1 as a diabetes quantitative trait locus. Sorcs1 belongs to the Vacuolar protein sorting-10 (Vps10) gene family. In yeast, Vps10 transports enzymes from the trans-Golgi network (TGN) to the vacuole. Whole-body Sorcs1 KO mice, when made obese with the leptin(ob) mutation (ob/ob), developed diabetes. β Cells from these mice had a severe deficiency of secretory granules (SGs) and insulin. Interestingly, a single secretagogue challenge failed to consistently elicit an insulin secretory dysfunction. However, multiple challenges of the Sorcs1 KO ob/ob islets consistently revealed an insulin secretion defect. The luminal domain of SORCS1 (Lum-Sorcs1), when expressed in a β cell line, acted as a dominant-negative, leading to SG and insulin deficiency. Using syncollin-dsRed5TIMER adenovirus, we found that the loss of Sorcs1 function greatly impairs the rapid replenishment of SGs following secretagogue challenge. Chronic exposure of islets from lean Sorcs1 KO mice to high glucose and palmitate depleted insulin content and evoked an insulin secretion defect. Thus, in metabolically stressed mice, Sorcs1 is important for SG replenishment, and under chronic challenge by insulin secretagogues, loss of Sorcs1 leads to diabetes. Overexpression of full-length SORCS1 led to a 2-fold increase in SG content, suggesting that SORCS1 is sufficient to promote SG biogenesis.

Ultrastructural and immunohistochemical analysis of the 8-20 week human fetal pancreas

Development of the human pancreas is well-known to involve tightly controlled differentiation of pancreatic precursors to mature cells that express endocrine- or exocrine-specific protein products. However, details of human pancreatic development at the ultrastructural level are limited. The present study analyzed 8–20 week fetal age human pancreata using scanning and transmission electron microscopy (TEM), TEM immunogold and double or triple immunofluorescence staining. Primary organization of islets and acini occurred during the developmental period examined. Differentiating endocrine and exocrine cells developed from the ductal tubules and subsequently formed isolated small clusters. Extracellular matrix fibers and proteins accumulated around newly differentiated cells during their migration and cluster formation. Glycogen expression was robust in ductal cells of the pancreas from 8–15 weeks of fetal age; however, this became markedly reduced at 20 weeks, with a concomitant increase in acinar cell glycogen content. Insulin secretory granules transformed from being dense and round at 8 weeks to distinct geometric (multilobular, crystalline) structures by 14–20 weeks. Initially many of the differentiating endocrine cells were multihormonal and contained polyhormonal granules; by 20 weeks, monohormonal cells were in the majority. Interestingly, certain secretory granules in the early human fetal pancreatic cells showed positivity for both exocrine (amylase) and endocrine proteins. This combined ultrastructural and immunohistochemical study showed that, during early developmental stages, the human pancreas contains differentiating epithelial cells that associate closely with the extracellular matrix, have dynamic glycogen expression patterns and contain polyhormonal as well as mixed endocrine/exocrine granules.

YES, a Src Family Kinase, Is a Proximal Glucose-specific Activator of Cell Division Cycle Control Protein 42 (Cdc42) in Pancreatic Islet β Cells

Second-phase insulin secretion sustains insulin release in the face of hyperglycemia associated with insulin resistance, requiring the continued mobilization of insulin secretory granules to the plasma membrane. Cdc42, the small Rho family GTPase recognized as the proximal glucose-specific trigger to elicit second-phase insulin secretion, signals downstream to activate the p21-activated kinase (PAK1), which then signals to Raf-1/MEK/ERK to induce filamentous actin (F-actin) remodeling, to ultimately mobilize insulin granules to the plasma membrane. However, the steps required to initiate Cdc42 activation in a glucose-specific manner in β cells have remained elusive. Toward this, we identified the involvement of the Src family kinases (SFKs), based upon the ability of SFK inhibitors to block glucose-stimulated Cdc42 and PAK1 activation events as well as the amplifying pathway of glucose-stimulated insulin release, in MIN6 β cells. Indeed, subsequent studies performed in human islets revealed that SFK phosphorylation was induced only by glucose and within 1 min of stimulation before the activation of Cdc42 at 3 min. Furthermore, pervanadate treatment validated the phosphorylation event to be tyrosine-specific. Although RT-PCR showed β cells to express five different SFK proteins, only two of these, YES and Fyn kinases, were found localized to the plasma membrane, and of these two, only YES kinase underwent glucose-stimulated tyrosine phosphorylation. Immunodetection and RNAi analyses further established YES kinase as a proximal glucose-specific signal in the Cdc42-signaling cascade. Identification of YES kinase provides new insight into the mechanisms underlying the sustainment of insulin secretion via granule mobilization/replenishment and F-actin remodeling.

Actin regulates the turnover of insulin secretory granules

Introduction: Insulin secretion is required for regulation of glucose homeostasis and its impairment leads to diabetes. Insulin is produced by the pancreatic b-cells, it is stored within insulin secretory granules (SGs) and released upon glucose stimulation. Newly synthesized insulin is preferentially released and mobile insulin SGs are more prone to undergo exocytosis. This suggests that SG age and mobility are key factors affecting insulin secretion.

Translation of diabetogenic viruses and secretory granule proteins in beta cells

Introduction: Glucose entry in pancreatic β-cells triggers insulin secretion and the biosynthesis of insulin secretory granules (SGs). Previously we showed that glucose and cAMP independently promote the binding of PTBP1 to mRNAs for proteins, thus enhancing their stability and translation. PTBP1 is also key for IRES-mediated translation of picornaviruses, including coxsackieviruses B (CVB). It has been suggested that CVB infection may trigger or precipitate type 1 diabetes. Since PTBP1 is required for translation of both picornaviruses and most T1D autoantigens we asked if and how CVB infection of insulin-expressing cells affects the expression of SG proteins.

Proteome Analysis and Conditional Deletion of the EAAT2 Glutamate Transporter Provide Evidence against a Role of EAAT2 in Pancreatic Insulin Secretion in Mice

Islet function is incompletely understood in part because key steps in glutamate handling remain undetermined. The glutamate (excitatory amino acid) transporter 2 (EAAT2; Slc1a2) has been hypothesized to (a) provide islet cells with glutamate, (b) protect islet cells against high extracellular glutamate concentrations, (c) mediate glutamate release, or (d) control the pH inside insulin secretory granules. Here we floxed the EAAT2 gene to produce the first conditional EAAT2 knock-out mice. Crossing with Nestin-cyclization recombinase (Cre) eliminated EAAT2 from the brain, resulting in epilepsy and premature death, confirming the importance of EAAT2 for brain function and validating the genetic construction. Crossing with insulin-Cre lines (RIP-Cre and IPF1-Cre) to obtain pancreas-selective deletion did not appear to affect survival, growth, glucose tolerance, or β-cell number. We found (using TaqMan RT-PCR, immunoblotting, immunocytochemistry, and proteome analysis) that the EAAT2 levels were too low to support any of the four hypothesized functions. The proteome analysis detected more than 7,000 islet proteins of which more than 100 were transporters. Although mitochondrial glutamate transporters and transporters for neutral amino acids were present at high levels, all other transporters with known ability to transport glutamate were strikingly absent. Glutamate-metabolizing enzymes were abundant. The level of glutamine synthetase was 2 orders of magnitude higher than that of glutaminase. Taken together this suggests that the uptake of glutamate by islets from the extracellular fluid is insignificant and that glutamate is intracellularly produced. Glutamine synthetase may be more important for islets than assumed previously.

Lipoproteins and β-Cell Functions: From Basic to Clinical Data.

Lipoproteins and β-Cell Functions: From Basic to Clinical Data.

Effect of fluoroquinolones on mitochondrial function in pancreatic beta cells

Hyper- and hypoglycaemias are known side effects of fluoroquinolone antibiotics, resulting in a number of fatalities. Fluoroquinolone-induced hypoglycaemias are due to stimulated insulin release by the inhibition of the KATP channel activity of the beta cell. Recently, it was found that fluoroquinolones were much less effective on metabolically intact beta cells than on open cell preparations. Thus the intracellular effects of gatifloxacin, moxifloxacin and ciprofloxacin were investigated by measuring NAD(P)H- and FAD-autofluorescence, the mitochondrial membrane potential, and the adenine nucleotide content of isolated pancreatic islets and beta cells. 100μM of moxifloxacin abolished the NAD(P)H increase elicited by 20mM glucose, while gatifloxacin diminished it and ciprofloxacin had no significant effect. This pattern was also seen with islets from SUR1 Ko mice, which have no functional KATP channels. Moxifloxacin also diminished the glucose-induced decrease of FAD-fluorescence, which reflects the intramitochondrial production of reducing equivalents. Moxifloxacin, but not ciprofloxacin or gatifloxacin significantly reduced the effect of 20mM glucose on the ATP/ADP ratio. The mitochondrial hyperpolarization caused by 20mM glucose was partially antagonized by moxifloxacin, but not by ciprofloxacin or gatifloxacin. Ultrastructural analyses after 20h tissue culture showed that all three compounds (at 10 and 100μM) diminished the number of insulin secretory granules and that gatifloxacin and ciprofloxacin, but not moxifloxacin induced fission/fusion configurations of the beta cell mitochondria. In conclusion, fluoroquinolones affect the function of the mitochondria in pancreatic beta cells which may diminish the insulinotropic effect of KATP channel closure and contribute to the hyperglycaemic episodes.

Regulation of β-cell function by RNA-binding proteins

β-cells of the pancreatic islets are highly specialized and high-throughput units for the production of insulin, the key hormone for maintenance of glucose homeostasis. Elevation of extracellular glucose and/or GLP-1 levels triggers a rapid upregulation of insulin biosynthesis through the activation of post-transcriptional mechanisms. RNA-binding proteins are emerging as key factors in the regulation of these mechanisms as well as in other aspects of β-cell function and glucose homeostasis at large, and thus may be implicated in the pathogenesis of diabetes. Here we review current research in the field, with a major emphasis on RNA-binding proteins that control biosynthesis of insulin and other components of the insulin secretory granules by modulating the stability and translation of their mRNAs.

Liraglutide ameliorates glycometabolism and insulin resistance through the upregulation of GLUT4 in diabetic KKAy mice

Liraglutide, a long-lasting glucagon‑like peptide‑1 analogue, has been used for the treatment of patients with type 2 diabetes mellitus since 2009. In this study, we investigated the anti-diabetic effects and mechanisms of action of liraglutide in a spontaneous diabetic animal model, using KK/Upj-Ay/J (KKAy) mice. The KKAy mice were divided into 2 groups, the liraglutide group (mice were treated with 250 µg/kg/day liraglutide) and the model group (treated with an equivalent amount of normal saline). C57BL/6J mice were used as the controls (treated with an equivalent amount of normal saline). The treatment period lasted 6 weeks. During this treatment period, fasting blood glucose (FBG) levels and the body weight of the mice were measured on a weekly basis. Our results revealed that liraglutide significantly decreased FBG levels, the area under the curve following a oral glucose tolerance test and insulin tolerance test, increased serum insulin levels, reduced homeostasis model assessment of insulin resistance and increased the insulin sensitivity index. Furthermore, liraglutide ameliorated glycometabolism dysfunction by increasing glycolysis via hexokinase and glycogenesis via pyruvate kinase activation. An ultrastructural examination of the pancreas revealed that liraglutide improved the damaged state of islet β cells and increased the number of insulin secretory granules. The real-time PCR results revealed that the gene expression of glucose transporter 4 (GLUT4) increased following treatment with liraglutide. Liraglutide also upregulated the protein expression of GLUT4 in liver tissue and skeletal muscle. Our results suggest that liraglutide ameliorates glycometabolism and insulin resistance in diabetic KKAy mice by stimulating insulin secretion, increasing glycogenesis and glycolysis and upregulating the expression of GLUT4.

Exocyst Sec5 Regulates Exocytosis of Newcomer Insulin Granules Underlying Biphasic Insulin Secretion

The exocyst complex subunit Sec5 is a downstream effector of RalA-GTPase which promotes RalA-exocyst interactions and exocyst assembly, serving to tether secretory granules to docking sites on the plasma membrane. We recently reported that RalA regulates biphasic insulin secretion in pancreatic islet β cells in part by tethering insulin secretory granules to Ca2+ channels to assist excitosome assembly. Here, we assessed β cell exocytosis by patch clamp membrane capacitance measurement and total internal reflection fluorescence microscopy to investigate the role of Sec5 in regulating insulin secretion. Sec5 is present in human and rodent islet β cells, localized to insulin granules. Sec5 protein depletion in rat INS-1 cells inhibited depolarization-induced release of primed insulin granules from both readily-releasable pool and mobilization from the reserve pool. This reduction in insulin exocytosis was attributed mainly to reduction in recruitment and exocytosis of newcomer insulin granules that undergo minimal docking time at the plasma membrane, but which encompassed a larger portion of biphasic glucose stimulated insulin secretion. Sec5 protein knockdown had little effect on predocked granules, unless vigorously stimulated by KCl depolarization. Taken together, newcomer insulin granules in β cells are more sensitive than predocked granules to Sec5 regulation.

Impaired Glucose Tolerance in a Mouse Model of Sidt2 Deficiency

Sidt2 was identified as a novel integral lysosomal membrane protein recently. We generated global Sidt2 knockout mice by gene targeting. These mice have a comparatively higher random and fasting glucose concentration. Intraperitoneal and oral glucose tolerance tests in Sidt2 knockout mice indicated glucose intolerance and decreased serum insulin level. Notably, the Sidt2−/− mice had hypertrophic islets compared with control mice. By Western blot and immunofluorescence, Sidt2−/− mouse islets were shown to have increased insulin protein, which actually contained more insulin secretory granules than their controls, demonstrated by electromicroscopy. Consistent with the in vivo study, isolated islet culture from the Sidt2−/− mice produced less insulin when stimulated by a high concentration of glucose or a depolarizing concentration of KCl. Under electromicroscope less empty vesicles and more mature ones in Sidt2−/− mice islets were observed, supporting impaired insulin secretory granule release. In conclusion, Sidt2 may play a critical role in the regulation of mouse insulin secretory granule secretion.

Fibroblast Growth Factor Receptor Like-1 (FGFRL1) Interacts with SHP-1 Phosphatase at Insulin Secretory Granules and Induces Beta-cell ERK1/2 Protein Activation

FGFRL1 is a newly identified member of the fibroblast growth factor receptor (FGFR) family expressed in adult pancreas. Unlike canonical FGFRs that initiate signaling via tyrosine kinase domains, the short intracellular sequence of FGFRL1 consists of a putative Src homology domain-2 (SH2)-binding motif adjacent to a histidine-rich C terminus. As a consequence of nonexistent kinase domains, FGFRL1 has been postulated to act as a decoy receptor to inhibit canonical FGFR ligand-induced signaling. In pancreatic islet beta-cells, canonical FGFR1 signaling affects metabolism and insulin processing. This study determined beta-cell expression of FGFRL1 as well as consequent effects on FGFR1 signaling and biological responses. We confirmed FGFRL1 expression at the plasma membrane and within distinct intracellular granules of both primary beta-cells and βTC3 cells. Fluorescent protein-tagged FGFRL1 (RL1) induced a significant ligand-independent increase in MAPK signaling. Removal of the histidine-rich domain (RL1-ΔHis) or entire intracellular sequence (RL1-ΔC) resulted in greater retention at the plasma membrane and significantly reduced ligand-independent ERK1/2 responses. The SHP-1 phosphatase was identified as an RL1-binding substrate. Point mutation of the SH2-binding motif reduced the ability of FGFRL1 to bind SHP-1 and activate ERK1/2 but did not affect receptor localization to insulin secretory granules. Finally, overexpression of RL1 increased cellular insulin content and matrix adhesion. Overall, these data suggest that FGFRL1 does not function as a decoy receptor in beta-cells, but rather it enhances ERK1/2 signaling through association of SHP-1 with the receptor's intracellular SH2-binding motif.

Inhibitory effect of zinc on glucose‐stimulated zinc/insulin secretion in an insulin‐secreting β‐cell line

Diminished or inappropriate secretion of insulin is associated with type II diabetes. The cellular/molecular mechanism coupled with the regulation of insulin secretion is still under intense investigation. Divalent ion zinc (Zn(2+)) is co-packaged and co-secreted with insulin and is intimately involved in the process of insulin biosynthesis and the maturation of insulin secretory granules. The study reported here investigated glucose-stimulated zinc secretion (GSZS) and the effect of zinc on glucose-stimulated insulin secretion (GSIS) in the HIT-T15 pancreatic β-cell line. Zinc secretion was measured using a newly developed fluorescent zinc imaging approach, and the insulin secretion was measured using an enzyme-linked immunosorbent assay. There was apparent granular-like zinc staining in β-cells. The application of glucose induced detectable zinc secretion or GSZS. Like GSIS, GSZS was dependent on the glucose concentration (5-20 mm) and the presence of extracellular calcium. The application of a zinc chelator enhanced GSZS. When brief paired-pulse glucose stimulations, which involve the initial glucose stimulation followed by a second round of glucose stimulation, were applied, zinc secretion or GSZS that followed the first pulse was inhibited. This inhibition was reversed by zinc chelation, suggesting a feedback mechanism on GSZS by zinc secreted from β-cells. Finally, the application of zinc (50 μm) strongly inhibited GSIS as measured by enzyme-linked immunosorbent assay. The present study suggests that insulin secretion is regulated by co-secreted zinc that may act as an autocrine inhibitory modulator.

Permanent Neonatal Diabetes inINSC94Y Transgenic Pigs

Mutations in the insulin (INS) gene may cause permanent neonatal diabetes mellitus (PNDM). Ins2 mutant mouse models provided important insights into the disease mechanisms of PNDM but have limitations for translational research. To establish a large animal model of PNDM, we generated INSC94Y transgenic pigs. A line expressing high levels of INSC94Y mRNA (70–86% of wild-type INS transcripts) exhibited elevated blood glucose soon after birth but unaltered β-cell mass at the age of 8 days. At 4.5 months, INSC94Y transgenic pigs exhibited 41% reduced body weight, 72% decreased β-cell mass (−53% relative to body weight), and 60% lower fasting insulin levels compared with littermate controls. β-cells of INSC94Y transgenic pigs showed a marked reduction of insulin secretory granules and severe dilation of the endoplasmic reticulum. Cataract development was already visible in 8-day-old INSC94Y transgenic pigs and became more severe with increasing age. Diabetes-associated pathological alterations of kidney and nervous tissue were not detected during the observation period of 1 year. The stable diabetic phenotype and its rescue by insulin treatment make the INSC94Y transgenic pig an attractive model for insulin supplementation and islet transplantation trials, and for studying developmental consequences of maternal diabetes mellitus.