KCl-stimulated Insulin Secretion Research Articles

BCAS2 Participates in Insulin Synthesis and Secretion via mRNA Alternative Splicing in Mice.

Insulin secreted by pancreatic β cells is essential for maintaining blood glucose levels. Diabetes is caused primarily by a loss of β cells or impairment of β-cell function. A previous whole-transcriptome analysis of islets from a type 2 diabetes group and a control group showed that a splicing disorder occurred in approximately 25% of splicing events. Breast carcinoma amplified sequence 2 (BCAS2) is a spliceosome component whose function in islet β cells is unclear. Here, we report that knockdown of Bcas2 decreased glucose- and KCl-stimulated insulin secretion in the NIT-1 cell line. Pancreas weight, glucose tolerance, and insulin sensitivity were measured in normal chow-fed Bcas2 f/f-βKO mice, and β-cell mass and islet size were analyzed by immunohistochemistry. Glucose intolerance developed in Bcas2 f/f-βKO mice, but there were no significant differences in pancreas weight, insulin sensitivity, β-cell mass, or islet size. Furthermore, observation of glucose-stimulated insulin secretion and insulin secretion granules in normal chow-fed mice revealed that the insulin level in serum and the number of insulin secretion granules were decreased in Bcas2 f/f-βKO mice. These differences were related to abnormal splicing of Syt7 and Tcf7l2 pre-mRNA. Taken together, these results demonstrate that BCAS2 is involved in alternative splicing during insulin synthesis and secretion.

317-OR: The Novel Glucokinase GCK (Q26L) Gene Mutation Site Causes Diabetes

Glucokinase (GCK) mainly exists in pancreatic β-cell, hepatocytes and brain, and is the first-rate limiting enzyme in glucose metabolism pathway. The heterozygous inactivation mutation of GCK leads to Maturity-onset diabetes of the young (MODY2) , an autosomal dominant mild fasting hyperglycemia. In the monogenic diabetes registration project initiated by our research group, we found a novel inactivation mutation site of GCK gene, c.77A>T (p.Q26L) . We sought to explore the pathogenic mechanism with a newly constructed mouse model.The proband was first diagnosed with diabetes at 11. Her father was diagnosed with diabetes at 32, and his mother was healthy. Genetic testing showed there was a novel heterozygous missense mutation site c.77A >T in exon 1 of the GCK of the proband, which resulted in the change of the 26th amino acid from glutamine to leucine. The pedigree indicated the mutation of proband was inherited from her father, not her mother. A global knock-in GCK (Q26L) mice (GCKKI/-) model was successfully established. From 3-16 weeks, substantial group differences in fasting blood glucose, other than body weight, was observed in GCKKI/-. At 3-week, impaired glucose tolerance and lower serum insulin level of GCKKI/- mice were detected. There was no difference in insulin sensitivity and content of proinsulin and insulin in the islet of GCKKI/-. In vitro experiment, decreased glucose-stimulated insulin secretion was observed in the GCKKI/-, but no significant changes in KCl stimulated insulin secretion. Furthermore, the results of different glucose concentration stimulated insulin secretion and islet perfusion showed the insulin secretion threshold increased and the insulin secretion ability decreased in the GCKKI/-. In the GCKKI/- liver, there were no changes in the morphology, but glycogen content was reduced. The level of plasma HDL significantly increased, plasma TC and LDL levels decreased, and TG levels did not change. Disclosure S.Ji: None. H.Shu: None. W.Feng: None. J.Zhen: None. J.Qiao: None. Y.Huang: None. S.Chen: None. M.Liu: None. Funding National Natural ScienceFoundation of China (81830025,81620108004,81800733,and 81900720) , the Ministry of Science and Technologyof China (2019YFA0802502) , the Tianjin Municipal Science andTechnology Bureau (17ZXMFSY00150)

314-OR: Synaptotagmin-9 Regulates Early Phase Insulin Secretion by Regulating Tomosyn-1

Insulin secretion from pancreatic β-cells occurs in a biphasic manner. The loss of insulin secretion, entailing the inability of insulin granules to fuse with the plasma membrane (PM) via the formation of the SNARE complex, is a key determinant in impaired glucose tolerance and development of type 2 diabetes (T2D) . Synaptotagmins (Syt) are calcium sensors that regulate insulin-containing granule fusion to the PM for insulin secretion. Syt9 is highly expressed in β-cells; however, its role in insulin secretion is not well-characterized. Our goal is to determine how Syt9 affects insulin secretion from pancreatic β-cells. Mice lacking Syt9 (Syt9-/-) have improved glucose clearance with no change in insulin action than wild-type control mice. Interestingly, Syt9-/- mice showed increased plasma insulin levels at 5 min and 15 min post-glucose challenge. Similarly, islets isolated from Syt9-/- mice demonstrate increased stimulation of insulin secretion in response to the early phase insulin secretagogues (KCl and tolbutamide) compared to WT control islets. Similarly, siRNA-mediated knockdown of Syt9 in INS1 cells increased KCL-stimulated insulin secretion at 5 min and min. Altogether, these data suggest that Syt9 regulates the early phase of insulin secretion from β-cells. Strikingly, Syt9-/- mouse islets have reduced Tomosyn-1 levels (∼50%) without altering the levels of other key SNARE proteins (Stx1A, Stx4A, SNAP25) . Tomosyn-1, an inhibitor of insulin secretion whose increased protein abundance is associated with reduced insulin secretion, binds, cofractionates, and colocalizes with Syt9 and Stx1A. The knockdown of Tomosyn-1 in INS1 β-cells treated with KCL prevents the localization of Syt9 with Stx1A. Further, the knockdown of Syt9 or Tomosyn-1 increases the SNARE complex formation. In sum, these data demonstrate the identification of a novel Stx1A-Tomosyn-1-Syt9 molecular complex that regulates the fusion of insulin granules in the early phase. Disclosure M.Rahman: None. H.Alsharif: n/a. S.Bhatnagar: None. Funding R01DK120684-01

A human pancreatic ECM hydrogel optimized for 3-D modeling of the islet microenvironment

Extracellular matrix (ECM) plays a multitude of roles, including supporting cells through structural and biochemical interactions. ECM is damaged in the process of isolating human islets for clinical transplantation and basic research. A platform in which islets can be cultured in contact with natural pancreatic ECM is desirable to better understand and support islet health, and to recapitulate the native islet environment. Our study demonstrates the derivation of a practical and durable hydrogel from decellularized human pancreas that supports human islet survival and function. Islets embedded in this hydrogel show increased glucose- and KCl-stimulated insulin secretion, and improved mitochondrial function compared to islets cultured without pancreatic matrix. In extended culture, hydrogel co-culture significantly reduced levels of apoptosis compared to suspension culture and preserved controlled glucose-responsive function. Isolated islets displayed altered endocrine and non-endocrine cell arrangement compared to in situ islets; hydrogel preserved an islet architecture more similar to that observed in situ. RNA sequencing confirmed that gene expression differences between islets cultured in suspension and hydrogel largely fell within gene ontology terms related to extracellular signaling and adhesion. Natural pancreatic ECM improves the survival and physiology of isolated human islets.

79 - Differential Effects of Voclosporin and Tacrolimus on Insulin Secretion From Human Islets

The incidence of diabetes after organ transplantation (NODAT) has increased, in part due to calcineurin inhibitors, particularly tacrolimus (TAC). Voclosporin (VCS) is a next generation calcineurin inhibitor that causes less NODAT in clinical trials, but its effects on human beta cells are unknown. We compared the effects of TAC and VCS on the dynamics of insulin secretory function, the programmed cell death rate and the transcriptomic profile of isolated human islets. We studied 2 clinically relevant doses of TAC (10 ng/ml, 30 ng/ml) and VCS (20 ng/ml, 60 ng/ml), meant to approximate the trough and peak concentrations of each drug. We found that the TAC, but not VCS, caused a significant impairment of both 15 mM glucose-stimulated insulin secretion and 30 mM KCl-stimulated insulin secretion, pointing to a possible molecular defect in the distal stages of exocytosis after Ca2+ entry via voltage-gated Ca2+ channels. Both TAC and VCS inhibited insulin secretion at high concentrations. No effects on insulin contents were identified. RNA sequencing showed that TAC, and to a lesser extent VCS, decreased the expression of genes that regulate exocytosis, including synaptotagmins (SYT16, SYT5), ER-to-Golgi traffic (PITPNM1) and hormone processing (PAM). Pathway analysis showed a significant enrichment of Gene Ontology terms, including ‘syntaxin binding.’ No significant deleterious effects on cell survival were observed with either drug in this model. Collectively, these data suggest that calcineurin inhibitors can directly inhibit insulin exocytosis. These data also support prior clinical evidence demonstrating the favourable glucose homeostasis profile of VCS when compared to TAC.

Differential Effects of Voclosporin and Tacrolimus on Insulin Secretion From Human Islets.

The incidence of new onset diabetes after transplant (NODAT) has increased over the past decade, likely due to calcineurin inhibitor–based immunosuppressants, including tacrolimus (TAC) and cyclosporin. Voclosporin (VCS), a next-generation calcineurin inhibitor, is reported to cause fewer incidences of NODAT but the reason is unclear. While calcineurin signaling plays important roles in pancreatic β-cell survival, proliferation, and function, its effects on human β-cells remain understudied. In particular, we do not understand why some calcineurin inhibitors have more profound effects on the incidence of NODAT. We compared the effects of TAC and VCS on the dynamics of insulin secretory function, programmed cell death rate, and the transcriptomic profile of human islets. We studied 2 clinically relevant doses of TAC (10 ng/mL, 30 ng/mL) and VCS (20 ng/mL, 60 ng/mL), meant to approximate the clinical trough and peak concentrations. TAC, but not VCS, caused a significant impairment of 15 mM glucose-stimulated and 30 mM KCl-stimulated insulin secretion. This points to molecular defects in the distal stages of exocytosis after voltage-gated Ca2+ entry. No significant effects on islet cell survival or total insulin content were identified. RNA sequencing showed that TAC significantly decreased the expression of 17 genes, including direct and indirect regulators of exocytosis (SYT16, TBC1D30, PCK1, SMOC1, SYT5, PDK4, and CREM), whereas VCS has less broad, and milder, effects on gene expression. Clinically relevant doses of TAC, but not VCS, directly inhibit insulin secretion from human islets, likely via transcriptional control of exocytosis machinery.

Identification and Analysis of a Novel Glucose Toxicity–Sensitive Gene in Pancreatic ß Cell

Chronic hyperglycemia in type 2 diabetes (T2D) causes pancreatic β-cell dysfunction, which is well known as glucose toxicity (GT), whereas the treatment of T2D improves that dysfunction. However, its underlying molecular mechanism has not yet been clarified. In this study, we aimed to identify and analyze a novel factor whose gene expression was altered by GT (GT-sensitive) and involved in β-cell function. For this purpose, type 2 diabetic db/db mice were treated with empagliflozin to selectively alleviate GT, as we recently reported (Shimo N, et al., 2015). Using the islet RNA from empagliflozin-treated (E) and untreated (U) mice, we performed DNA microarray analysis, and only 43 factors out of 33,779 probes showed more than two times as high signal levels in E as U. Among them, we focused on Tmem163, which is reported to be associated with T2D risk in GWAS and involved in the regulation of intracellular zinc levels. The Tmem163 mRNA expression levels in islets were significantly lower in U than normal control mice, and higher in E than U, which indicated that Tmem163 was GT-sensitive. The tissue distribution analysis by immunohistochemistry and immunoelectron microscopy revealed that Tmem163 was highly localized in pancreatic β-cells and accumulated in the membrane of insulin granule. Next, we found that Tmem163 knockdown significantly reduced glucose-stimulated and KCl-stimulated insulin secretion in MIN6 cells. In vivo, Tmem163 null mice (N) were comparable in body weight and blood glucose levels, but showed decreased glucose tolerance and insulin secretion in intraperitoneal glucose tolerance test, compared with wild type mice. Furthermore, the zinc content of islets in N tended to be lower than a hetero knockout mouse, suggesting a mechanism for β-cell dysfunction. These in vitro and in vivo findings suggest that the decreased expression of the novel GT-sensitive gene Tmem163 in islets causes the insufficient insulin secretion from pancreatic β-cells induced by chronic hyperglycemia. Disclosure N. Shimo: Speaker's Bureau; Self; Daiichi Sankyo Company, Limited. T. Matsuoka: None. D. Kawamori: Research Support; Self; Japan Society for the Promotion of Science, Japan Diabetes Society. Speaker's Bureau; Self; Ajinomoto Co., Inc., Astellas, K.K., Daiichi Sankyo Company, Limited, Dai-nippon Sumitomo Pharmaceutials, MSD K.K., Sanofi K.K., Sanwa Kagaku Kenkyusho Co., Ltd., Takeda Pharmaceuticals, Kowa Pharmaceuticals, Eli Lilly and Company. S. Takebe: None. T. Miyatsuka: Research Support; Self; Eli Lilly and Company, Japan Society for the Promotion of Science, Daiichi Sankyo Company, Limited, Novartis Pharma K.K., Astellas Pharma Inc., MSD K.K., Takeda Pharmaceutical Company Limited. I. Shimomura: Research Support; Self; Appliance Company, Panasonic Corporation. Speaker's Bureau; Self; Astellas Pharma Inc., AstraZeneca, Covidien Japan Inc.. Research Support; Self; Daiichi Sankyo Company, Limited. Speaker's Bureau; Self; Daiichi Sankyo Company, Limited. Research Support; Self; Eli Lilly Japan K.K., Eli Lilly Japan K.K., Kaken Pharmaceutical Co., Ltd.. Speaker's Bureau; Self; Kowa Pharmaceutical Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Mochida Pharmaceutical Co., Ltd., MSD K.K., Nippon Boehringer Ingelheim Co. Ltd., Novartis Pharma K.K.. Research Support; Self; Novo Nordisk Pharma Ltd.. Speaker's Bureau; Self; Novo Nordisk Pharma Ltd., Ono Pharmaceutical Co., Ltd.. Research Support; Self; Roche DC Japan K.K., Rohto Pharmaceutical Co., Ltd. Speaker's Bureau; Self; Rohto Pharmaceutical Co., Ltd, Sanwa Kagaku Kenkyusho Co., Ltd.. Research Support; Self; Sanofi K.K.. Speaker's Bureau; Self; Sanofi K.K.. Research Support; Self; Shionogi & Co., Ltd.. Speaker's Bureau; Self; Sumitomo Dainippon Pharma Co., Ltd.. Research Support; Self; Taisho Toyama Pharmaceutical Co., Ltd.. Speaker's Bureau; Self; Taisho Toyama Pharmaceutical Co., Ltd.. Research Support; Self; Takeda Pharmaceutical Company Limited. Speaker's Bureau; Self; Takeda Pharmaceutical Company Limited. Research Support; Self; Teijin Pharma Limited. Speaker's Bureau; Self; Teijin Pharma Limited.

Differential Insulin Secretion of High-Fat Diet-Fed C57BL/6NN and C57BL/6NJ Mice: Implications of Mixed Genetic Background in Metabolic Studies.

Many metabolic studies employ tissue-specific gene knockout mice, which requires breeding of floxed gene mice, available mostly on C57BL/6N (NN) genetic background, with cre or Flp recombinase-expressing mice, available on C57BL/6J (JJ) background, resulting in the generation of mixed C57BL/6NJ (NJ) genetic background mice. Recent awareness of many genetic differences between NN and JJ strains including the deletion of nicotinamide nucleotide transhydrogenase (nnt), necessitates examination of the consequence of mixed NJ background on glucose tolerance, beta cell function and other metabolic parameters. Male mice with NN and NJ genetic background were fed with normal or high fat diets (HFD) for 12 weeks and glucose and insulin homeostasis were studied. Genotype had no effect on body weight and food intake in mice fed normal or high fat diets. Insulinemia in the fed and fasted states and after a glucose challenge was lower in HFD-fed NJ mice, even though their glycemia and insulin sensitivity were similar to NN mice. NJ mice showed mild glucose intolerance. Moreover, glucose- but not KCl-stimulated insulin secretion in isolated islets was decreased in HFD-fed NJ vs NN mice without changes in insulin content and beta cell mass. Under normal diet, besides reduced fed insulinemia, NN and NJ mice presented similar metabolic parameters. However, HFD-fed NJ mice displayed lower fed and fasted insulinemia and glucose-induced insulin secretion in vivo and ex vivo, as compared to NN mice. These results strongly caution against using unmatched mixed genetic background C57BL/6 mice for comparisons, particularly under HFD conditions.

Functional Analysis of Novel Candidate Regulators of Insulin Secretion in the MIN6 Mouse Pancreatic β Cell Line.

Elucidating the regulation of glucose-stimulated insulin secretion (GSIS) in pancreatic β cells is important for understanding and treating diabetes. The pancreatic β cell line, MIN6, retains GSIS but gradually loses it in long-term culture. The MIN6 subclone, MIN6c4, exhibits well-regulated GSIS even after prolonged culture. We previously used DNA microarray analysis to compare gene expression in the parental MIN6 cells and MIN6c4 cells and identified several differentially regulated genes that may be involved in maintaining GSIS. Here we investigated the potential roles of six of these genes in GSIS: Tmem59l (Transmembrane protein 59 like), Scgn (Secretagogin), Gucy2c (Guanylate cyclase 2c), Slc29a4 (Solute carrier family 29, member 4), Cdhr1 (Cadherin-related family member 1), and Celsr2 (Cadherin EGF LAG seven-pass G-type receptor 2). These genes were knocked down in MIN6c4 cells using lentivirus vectors expressing gene-specific short hairpin RNAs (shRNAs), and the effects of the knockdown on insulin expression and secretion were analyzed. Suppression of Tmem59l, Scgn, and Gucy2c expression resulted in significantly decreased glucose- and/or KCl-stimulated insulin secretion from MIN6c4 cells, while the suppression of Slc29a4 expression resulted in increased insulin secretion. Tmem59l overexpression rescued the phenotype of the Tmem59l knockdown MIN6c4 cells, and immunostaining analysis indicated that the TMEM59L protein colocalized with insulin and GM130, a Golgi complex marker, in MIN6 cells. Collectively, our findings suggested that the proteins encoded by Tmem59l, Scgn, Gucy2c, and Slc29a4 play important roles in regulating GSIS. Detailed studies of these proteins and their functions are expected to provide new insights into the molecular mechanisms involved in insulin secretion.

Expression and regulation of a two-pore domain K+ channel KCNK16 in rat islets

Objective To observe the expression of a two-pore domain K+ channel KCNK16 in rat islets, and to explore its regulation by insulin secretagogues. Methods Real time-PCR and immunohistochemistry were employed to observe the expression of KCNK16 in rat islets and other tissues. Rat islets were exposed to glucose, prolactin, palmitate, and trichostatin A(TSA)for 24 h and KCNK16 expression were determined by realtime-PCR and Western blot. Glucose- and KCl-stimulated insulin secretion were assayed by ELISA after islets were exposed to TSA for 24 h. The dose- and time-dependent effects of TSA on KCNK16 expression in INS1-E cells were determined by RT-PCR. Results KCNK16 was highly expressed in rat islets. Glucose, prolactin, and palmitate did not affect the expression of KCNK16 in rat islets. TSA treatment significantly potentiated glucose- and KCl-stimulated insulin secretion in isolated rat islets(P<0.05), along with decreased KCNK16 mRNA and protein expressions(P<0.01). TSA inhibited KCNK16 expression in a dose- and time-dependent manner in INS1-E cells(P<0.05). Conclusion KCNK16 is exclusively and highly expressed in rat islets and its expression can be down-regulated by TSA treatment. It is possible that TSA enhances insulin secretion via decreasing KCNK16 expression. (Chin J Endocrinol Metab, 2015, 31: 452-456) Key words: KCNK16; Islet; Insulin secretion; Trichostatin A

Characterization of Pancreatic Islets in Two Selectively Bred Mouse Lines with Different Susceptibilities to High-Fat Diet-Induced Glucose Intolerance

Hereditary predisposition to diet-induced type 2 diabetes has not yet been fully elucidated. We recently established 2 mouse lines with different susceptibilities (resistant and prone) to high-fat diet (HFD)-induced glucose intolerance by selective breeding (designated selectively bred diet-induced glucose intolerance-resistant [SDG-R] and -prone [SDG-P], respectively). To investigate the predisposition to HFD-induced glucose intolerance in pancreatic islets, we examined the islet morphological features and functions in these novel mouse lines. Male SDG-P and SDG-R mice were fed a HFD for 5 weeks. Before and after HFD feeding, glucose tolerance was evaluated by oral glucose tolerance test (OGTT). Morphometry and functional analyses of the pancreatic islets were also performed before and after the feeding period. Before HFD feeding, SDG-P mice showed modestly higher postchallenge blood glucose levels and lower insulin increments in OGTT than SDG-R mice. Although SDG-P mice showed greater β cell proliferation than SDG-R mice under HFD feeding, SDG-P mice developed overt glucose intolerance, whereas SDG-R mice maintained normal glucose tolerance. Regardless of whether it was before or after HFD feeding, the isolated islets from SDG-P mice showed impaired glucose- and KCl-stimulated insulin secretion relative to those from SDG-R mice; accordingly, the expression levels of the insulin secretion-related genes in SDG-P islets were significantly lower than those in SDG-R islets. These findings suggest that the innate predispositions in pancreatic islets may determine the susceptibility to diet-induced diabetes. SDG-R and SDG-P mice may therefore be useful polygenic animal models to study the gene–environment interactions in the development of type 2 diabetes.

Identification of a pathway by which glucose regulates β-catenin signalling via the cAMP/protein kinase A pathway in β-cell models

Pancreatic β-cells are highly responsive to changes in glucose, but the mechanisms involved are only partially understood. There is increasing evidence that the β-catenin signalling pathway plays an important role in regulating β-cell function, but the mechanisms regulating β-catenin signalling in these cells is not well understood. In the present study we show that β-catenin levels and downstream signalling are regulated by changes in glucose levels in INS-1E and β-TC6-F7 β-cell models. We found a glucose-dependent increase in levels of β-catenin in the cytoplasm and nucleus of INS-1E cells. Expression of cyclin D1 also increased with glucose and required the presence of β-catenin. This was associated with an increase in phosphorylation of β-catenin on Ser552, which is known to stabilize the molecule and increase its transcriptional activity. In a search for possible signalling intermediates we found forskolin and cell-permeable cAMP analogues recapitulated the glucose effects, suggesting a role for cAMP and PKA (cAMP-dependent protein kinase/protein kinase A) downstream of glucose. Furthermore, glucose caused sustained increases in cAMP. Two different inhibitors of adenylate cyclase and PKA signalling blocked the effects of glucose, whereas siRNA (small interfering RNA) knockdown of PKA blocked the effects of glucose on β-catenin signalling. Finally, reducing β-catenin levels with either siRNA or pyrvinium impaired glucose- and KCl-stimulated insulin secretion. Taken together the results of the present study define a pathway by which changes in glucose levels can regulate β-catenin using a mechanism which involves cAMP production and the activation of PKA. This identifies a pathway that may be important in glucose-dependent regulation of gene expression and insulin secretion in β-cells.

Gelsolin Associates with the N Terminus of Syntaxin 4 to Regulate Insulin Granule Exocytosis

The plasma membrane soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) protein syntaxin (Syn)4 is required for biphasic insulin secretion, although how it regulates each phase remains unclear. In a screen to identify new Syn4-interacting factors, the calcium-activated F-actin-severing protein gelsolin was revealed. Gelsolin has been previously implicated as a positive effector of insulin secretion, although a molecular mechanism to underlie this function is lacking. Toward this, our in vitro binding studies showed the Syn4-gelsolin interaction to be direct and mediated by the N-terminal Ha domain (amino acid residues 39-70) of Syn4. Syn4-gelsolin complexes formed under basal conditions and dissociated upon acute glucose or KCl stimulation; nifedipine blocked dissociation. The dissociating action of secretagogues could be mimicked by expression of the N-terminal Ha domain of Syn4 fused to green fluorescent protein (GFP) (GFP-39-70). Furthermore, GFP-39-70 expression in isolated mouse islet and clonal MIN6 β-cells initiated insulin release in the absence of appropriate stimuli. Consistent with this, the inhibitory GFP-39-70 peptide also initiated Syn4 activation in the absence of stimuli. Moreover, although MIN6 β-cells expressing the GFP-39-70 peptide maintained normal calcium influx in response to KCl, KCl-stimulated insulin secretion and the triggering pathway of insulin secretion were significantly impaired. Taken together, these data support a mechanistic model for gelsolin's role in insulin exocytosis: gelsolin clamps unsolicited soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE)-regulated exocytosis through direct association with Syn4 in the absence of appropriate stimuli, which is relieved upon stimulus-induced calcium influx to activate gelsolin and induce its dissociation from Syn4 to facilitate insulin exocytosis.

Isoprenylcysteine carboxyl methyltransferase facilitates glucose-induced Rac1 activation, ROS generation and insulin secretion in INS 832/13 β-cells

Isoprenylcysteine carboxyl methyltransferase (ICMT) catalyzes the post-translational methylation of C-terminal cysteines of isoprenylated proteins, including small G-proteins and the γ-subunits of heterotrimeric G-proteins. It is widely felt that carboxymethylation promotes efficient membrane association of the methylated proteins and specific protein-protein interactions. In the current study, we tested the hypothesis that ICMT-mediated carboxymethylation of specific proteins (e.g., Rac1) plays a regulatory role in glucose-stimulated insulin secretion (GSIS). Western blot analysis indicated that lCMT is expressed and predominantly membrane associated in INS 832/13 β-cells. siRNA-mediated knockdown of endogenous expression of ICMT markedly attenuated glucose, but not KCl-induced insulin secretion. These findings were further supported by pharmacological observations, which suggested a marked reduction in glucose-, but not KCl-stimulated insulin secretion by acetyl farnesyl cysteine (AFC), a selective inhibitor of ICMT. In addition, glucose-induced Rac1 activation, a hallmark signaling step involved in glucose-stimulated insulin secretion, was markedly inhibited following pharmacological (AFC) or molecular biological (siRNA-ICMT) inhibition of ICMT. Lastly, we also noticed a marked reduction in glucose-induced acute increase in the generation of reactive oxygen species in INS 832/13 cells pre-treated with AFC or transfected with siRNA-ICMT. Together, these data suggest that ICMT regulates glucose-induced Rac1 activation, generation of reactive oxygen species and insulin secretion in pancreatic β-cells.

Defects in beta cell Ca2+ signalling, glucose metabolism and insulin secretion in a murine model of KATP channel-induced neonatal diabetes mellitus

Mutations that render ATP-sensitive potassium (K(ATP)) channels insensitive to ATP inhibition cause neonatal diabetes mellitus. In mice, these mutations cause insulin secretion to be lost initially and, as the disease progresses, beta cell mass and insulin content also disappear. We investigated whether defects in calcium signalling alone are sufficient to explain short-term and long-term islet dysfunction. We examined the metabolic, electrical and insulin secretion response in islets from mice that become diabetic after induction of ATP-insensitive Kir6.2 expression. To separate direct effects of K(ATP) overactivity on beta cell function from indirect effects of prolonged hyperglycaemia, normal glycaemia was maintained by protective exogenous islet transplantation. In endogenous islets from protected animals, glucose-dependent elevations of intracellular free-calcium activity ([Ca(2+)](i)) were severely blunted. Insulin content of these islets was normal, and sulfonylureas and KCl stimulated increased [Ca(2+)](i). In the absence of transplant protection, [Ca(2+)](i) responses were similar, but glucose metabolism and redox state were dramatically altered; sulfonylurea- and KCl-stimulated insulin secretion was also lost, because of systemic effects induced by long-term hyperglycaemia and/or hypoinsulinaemia. In both cases, [Ca(2+)](i) dynamics were synchronous across the islet. After reduction of gap-junction coupling, glucose-dependent [Ca(2+)](i) and insulin secretion was partially restored, indicating that excitability of weakly expressing cells is suppressed by cells expressing mutants, via gap-junctions. The primary defect in K(ATP)-induced neonatal diabetes mellitus is failure of glucose metabolism to elevate [Ca(2+)](i), which suppresses insulin secretion and mildly alters islet glucose metabolism. Loss of insulin content and mitochondrial dysfunction are secondary to the long-term hyperglycaemia and/or hypoinsulinaemia that result from the absence of glucose-dependent insulin secretion.

The Mitochondrial 2-Oxoglutarate Carrier Is Part of a Metabolic Pathway That Mediates Glucose- and Glutamine-stimulated Insulin Secretion

Glucose-stimulated insulin secretion from pancreatic islet beta-cells is dependent in part on pyruvate cycling through the pyruvate/isocitrate pathway, which generates cytosolic alpha-ketoglutarate, also known as 2-oxoglutarate (2OG). Here, we have investigated if mitochondrial transport of 2OG through the 2-oxoglutarate carrier (OGC) participates in control of nutrient-stimulated insulin secretion. Suppression of OGC in clonal pancreatic beta-cells (832/13 cells) and isolated rat islets by adenovirus-mediated delivery of small interfering RNA significantly decreased glucose-stimulated insulin secretion. OGC suppression also reduced insulin secretion in response to glutamine plus the glutamate dehydrogenase activator 2-amino-2-norbornane carboxylic acid. Nutrient-stimulated increases in glucose usage, glucose oxidation, glutamine oxidation, or ATP:ADP ratio were not affected by OGC knockdown, whereas suppression of OGC resulted in a significant decrease in the NADPH:NADP(+) ratio during stimulation with glucose but not glutamine + 2-amino-2-norbornane carboxylic acid. Finally, OGC suppression reduced insulin secretion in response to a membrane-permeant 2OG analog, dimethyl-2OG. These data reveal that the OGC is part of a mechanism of fuel-stimulated insulin secretion that is common to glucose, amino acid, and organic acid secretagogues, involving flux through the pyruvate/isocitrate cycling pathway. Although the components of this pathway must remain intact for appropriate stimulus-secretion coupling, production of NADPH does not appear to be the universal second messenger signal generated by these reactions.

Glucose-stimulated Cdc42 Signaling Is Essential for the Second Phase of Insulin Secretion

The small Rho family GTPases Cdc42 and Rac1 have each been shown to function in insulin exocytosis and are presumed to function in actin remodeling and insulin granule mobilization. However, whether either GTPase is required for the mobilization phase of insulin release (second phase) and are linked in a common signaling pathway has remained unknown. Here we demonstrate that small interfering RNA-mediated depletion of Cdc42 from isolated islets results in the selective loss of second phase insulin release. Consistent with a role in this nutrient-dependent phase, Cdc42 activation was detected exclusively in response to D-glucose and was unresponsive to KCl or non-metabolizable glucose analogs in MIN6 beta-cells. Cdc42 activation occurred early in secretion (3 min), whereas Rac1 activation required approximately 15-20 min, suggesting Cdc42 as proximal and Rac1 as distal regulators of second-phase secretion. Importantly, Rac1 activation and function was linked in a common pathway downstream of Cdc42; Cdc42 depletion ablated glucose-induced Rac1 activation, and expression of constitutively active Rac1 in Cdc42-depleted cells functionally restored glucose-stimulated insulin secretion. Occurring at a time midway between Cdc42 and Rac1 activations was the phosphorylation of p21-activated-kinase 1 (Pak1), and this phosphorylation event required Cdc42. Moreover, small interfering RNA-mediated Pak1 depletion abolished Rac1 activation and glucose-stimulated insulin release, suggesting that Pak1 may mediate the link between Cdc42 and Rac1 in this pathway. Taken together, these data substantiate the existence of a novel signaling pathway in the islet beta-cell whereby Cdc42 functions as a key proximal transmitter of the glucose signal early in stimulus-secretion coupling to support the later stage of insulin release.

Cell death and impairment of glucose-stimulated insulin secretion induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the β-cell line INS-1E

The aim of this research was to characterize 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity on the insulin-secreting beta-cell line INS-1E. A sharp decline of cell survival (below 20%) was observed after 1 h exposure to TCDD concentrations between 12.5 and 25 nM. Ultrastructurally, beta-cell death was characterized by extensive degranulation, appearance of autophagic vacuoles, and peripheral nuclear condensation. Cytotoxic concentrations of TCDD rapidly induced a dose-dependent increase in intracellular calcium concentration. Blocking calcium entry by EGTA significantly decreased TCDD cytotoxicity. TCDD was also able to rapidly induce mitochondrial depolarization. Interestingly, 1 h exposition of INS-1E cells to very low TCDD concentrations (0.05-1 nM) dramatically impaired glucose-stimulated but not KCl-stimulated insulin secretion. In conclusion, our results clearly show that TCDD exerts a direct beta-cell cytotoxic effect at concentrations of 15-25 nM, but also markedly impairs glucose-stimulated insulin secretion at concentrations 20 times lower than these. On the basis of this latter observation we suggest that pancreatic beta-cells could be considered a specific and sensitive target for dioxin toxicity.

Protein Kinase Cδ Plays a Non-redundant Role in Insulin Secretion in Pancreatic β Cells

Protein kinase C (PKC) is considered to modulate glucose-stimulated insulin secretion. Pancreatic beta cells express multiple isoforms of PKCs; however, the role of each isoform in glucose-stimulated insulin secretion remains controversial. In this study we investigated the role of PKCdelta, a major isoform expressed in pancreatic beta cells on beta cell function. Here, we showed that PKCdelta null mice manifested glucose intolerance with impaired insulin secretion. Insulin tolerance test showed no decrease in insulin sensitivity in PKCdelta null mice. Studies using islets isolated from these mice demonstrated decreased glucose- and KCl-stimulated insulin secretion. Perifusion studies indicated that mainly the second phase of insulin secretion was decreased. On the other hand, glucose-induced influx of Ca2+ into beta cells was not altered. Immunohistochemistry using total internal reflection fluorescence microscopy and electron microscopic analysis showed an increased number of insulin granules close to the plasma membrane in beta cells of PKCdelta null mice. Although PKC is thought to phosphorylate Munc18-1 and facilitate soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors complex formation, the phosphorylation of Munc18-1 by glucose stimulation was decreased in islets of PKCdelta null mice. We conclude that PKCdelta plays a non-redundant role in glucose-stimulated insulin secretion. The impaired insulin secretion in PKCdelta null mice is associated with reduced phosphorylation of Munc18-1.

The Mitochondrial Citrate/Isocitrate Carrier Plays a Regulatory Role in Glucose-stimulated Insulin Secretion

Glucose-stimulated insulin secretion (GSIS) is mediated in part by glucose metabolism-driven increases in ATP/ADP ratio, but by-products of mitochondrial glucose metabolism also play an important role. Here we investigate the role of the mitochondrial citrate/isocitrate carrier (CIC) in regulation of GSIS. Inhibition of CIC activity in INS-1-derived 832/13 cells or primary rat islets by the substrate analogue 1,2,3-benzenetricarboxylate (BTC) resulted in potent inhibition of GSIS, involving both first and second phase secretion. A recombinant adenovirus containing a CIC-specific siRNA (Ad-siCIC) dose-dependently reduced CIC expression in 832/13 cells and caused parallel inhibitory effects on citrate accumulation in the cytosol. Ad-siCIC treatment did not affect glucose utilization, glucose oxidation, or ATP/ADP ratio but did inhibit glucose incorporation into fatty acids and glucose-induced increases in NADPH/NADP+ ratio relative to cells treated with a control siRNA virus (Ad-siControl). Ad-siCIC also inhibited GSIS in 832/13 cells, whereas overexpression of CIC enhanced GSIS and raised cytosolic citrate levels. In normal rat islets, Ad-siCIC treatment also suppressed CIC mRNA levels and inhibited GSIS. We conclude that export of citrate and/or isocitrate from the mitochondria to the cytosol is an important step in control of GSIS.