Chronic Exposure to Excess Nutrients Left-shifts the Concentration Dependence of Glucose-stimulated Insulin Secretion in Pancreatic β-Cells

Hyperinsulinemia (HI) is elevated plasma insulin at basal glucose. Impaired glucose tolerance is associated with HI, although the exact cause and effect relationship remains poorly defined. We tested the hypothesis that HI can result from an intrinsic response of the β-cell to chronic exposure to excess nutrients, involving a shift in the concentration dependence of glucose-stimulated insulin secretion. INS-1 (832/13) cells were cultured in either a physiological (4 mm) or high (11 mm) glucose concentration with or without concomitant exposure to oleate. Isolated rat islets were also cultured with or without oleate. A clear hypersensitivity to submaximal glucose concentrations was evident in INS-1 cells cultured in excess nutrients such that the 25% of maximal (S_0.25) glucose-stimulated insulin secretion was significantly reduced in cells cultured in 11 mm glucose (S_0.25 = 3.5 mm) and 4 mm glucose with oleate (S_0.25 = 4.5 mm) compared with 4 mm glucose alone (S_0.25 = 5.7 mm). The magnitude of the left shift was linearly correlated with intracellular lipid stores in INS-1 cells (r² = 0.97). We observed no significant differences in the dose responses for glucose stimulation of respiration, NAD(P)H autofluorescence, or Ca²⁺ responses between left- and right-shifted β-cells. However, a left shift in the sensitivity of exocytosis to Ca²⁺ was documented in permeabilized INS-1 cells cultured in 11 versus 4 mm glucose (S_0.25 = 1.1 and 1.7 μm, respectively). Our results suggest that the sensitivity of exocytosis to triggering is modulated by a lipid component, the levels of which are influenced by the culture nutrient environment.     

Intracellular Serotonin Modulates Insulin Secretion from Pancreatic β-Cells by Protein Serotonylation

While serotonin (5-HT) co-localization with insulin in granules of pancreatic β-cells was demonstrated more than three decades ago, its physiological role in the etiology of diabetes is still unclear. We combined biochemical and electrophysiological analyses of mice selectively deficient in peripheral tryptophan hydroxylase (Tph1−/−) and 5-HT to show that intracellular 5-HT regulates insulin secretion. We found that these mice are diabetic and have an impaired insulin secretion due to the lack of 5-HT in the pancreas. The pharmacological restoration of peripheral 5-HT levels rescued the impaired insulin secretion in vivo. These findings were further evidenced by patch clamp experiments with isolated Tph1−/− β-cells, which clearly showed that the secretory defect is downstream of Ca²⁺-signaling and can be rescued by direct intracellular application of 5-HT via the clamp pipette. In elucidating the underlying mechanism further, we demonstrate the covalent coupling of 5-HT by transglutaminases during insulin exocytosis to two key players in insulin secretion, the small GTPases Rab3a and Rab27a. This renders them constitutively active in a receptor-independent signaling mechanism we have recently termed serotonylation. Concordantly, an inhibition of such activating serotonylation in β-cells abates insulin secretion. We also observed inactivation of serotonylated Rab3a by enhanced proteasomal degradation, which is in line with the inactivation of other serotonylated GTPases. Our results demonstrate that 5-HT regulates insulin secretion by serotonylation of GTPases within pancreatic β-cells and suggest that intracellular 5-HT functions in various microenvironments via this mechanism in concert with the known receptor-mediated signaling.     

Brain-selective Kinase 2 (BRSK2) Phosphorylation on PCTAIRE1 Negatively Regulates Glucose-stimulated Insulin Secretion in Pancreatic β-Cells

Brain-selective kinase 2 (BRSK2) has been shown to play an essential role in neuronal polarization. In the present study, we show that BRSK2 is also abundantly expressed in pancreatic islets and MIN6 β-cell line. Yeast two-hybrid screening, GST fusion protein pull-down, and co-immunoprecipitation assays reveal that BRSK2 interacts with CDK-related protein kinase PCTAIRE1, a kinase involved in neurite outgrowth and neurotransmitter release. In MIN6 cells, BRSK2 co-localizes with PCTAIRE1 in the cytoplasm and phosphorylates one of its serine residues, Ser-12. Phosphorylation of PCTAIRE1 by BRSK2 reduces glucose-stimulated insulin secretion (GSIS) in MIN6 cells. Conversely, knockdown of BRSK2 by siRNA increases serum insulin levels in mice. Our results reveal a novel function of BRSK2 in the regulation of GSIS in β-cells via a PCTAIRE1-dependent mechanism and suggest that BRSK2 is an attractive target for developing novel diabetic drugs.     

Acute Inhibition of PI3K-PDK1-Akt Pathway Potentiates Insulin Secretion through Upregulation of Newcomer Granule Fusions in Pancreatic β-Cells

In glucose-induced insulin secretion from pancreatic β-cells, a population of insulin granules fuses with the plasma membrane without the typical docking process (newcomer granule fusions), however, its mechanism is unclear. In this study, we investigated the PI3K signaling pathways involved in the upregulation of newcomer granule fusions. Acute treatment with the class IA-selective PI3K inhibitors, PIK-75 and PI-103, enhanced the glucose-induced insulin secretion. Total internal reflection fluorescent microscopy revealed that the PI3K inhibitors increased the fusion events from newcomer granules. We developed a new system for transfection into pancreatic islets and demonstrated the usefulness of this system in order for evaluating the effect of transfected genes on the glucose-induced secretion in primary cultured pancreatic islets. Using this transfection system together with a series of constitutive active mutants, we showed that the PI3K-3-phosphoinositide dependent kinase-1 (PDK1)-Akt pathway mediated the potentiation of insulin secretion. The Akt inhibitor also enhanced the glucose-induced insulin secretion in parallel with the upregulation of newcomer granule fusions, probably via increased motility of intracellular insulin granules. These data suggest that the PI3K-PDK1-Akt pathway plays a significant role in newcomer granule fusions, probably through an alteration of the dynamics of the intracellular insulin granules.     

Insulin Signaling Regulates Mitochondrial Function in Pancreatic β-Cells

Insulin/IGF-I signaling regulates the metabolism of most mammalian tissues including pancreatic islets. To dissect the mechanisms linking insulin signaling with mitochondrial function, we first identified a mitochondria-tethering complex in β-cells that included glucokinase (GK), and the pro-apoptotic protein, BAD_S. Mitochondria isolated from β-cells derived from β-cell specific insulin receptor knockout (βIRKO) mice exhibited reduced BAD_S, GK and protein kinase A in the complex, and attenuated function. Similar alterations were evident in islets from patients with type 2 diabetes. Decreased mitochondrial GK activity in βIRKOs could be explained, in part, by reduced expression and altered phosphorylation of BAD_S. The elevated phosphorylation of p70S6K and JNK1 was likely due to compensatory increase in IGF-1 receptor expression. Re-expression of insulin receptors in βIRKO cells partially restored the stoichiometry of the complex and mitochondrial function. These data indicate that insulin signaling regulates mitochondrial function and have implications for β-cell dysfunction in type 2 diabetes.     

Insulin Secretion Induced by Glucose-dependent Insulinotropic Polypeptide Requires Phosphatidylinositol 3-Kinase γ in Rodent and Human β-Cells

PI3Kγ, a G-protein-coupled type 1B phosphoinositol 3-kinase, exhibits a basal glucose-independent activity in β-cells and can be activated by the glucose-dependent insulinotropic polypeptide (GIP). We therefore investigated the role of the PI3Kγ catalytic subunit (p110γ) in insulin secretion and β-cell exocytosis stimulated by GIP. We inhibited p110γ with AS604850 (1 μmol/liter) or knocked it down using an shRNA adenovirus or siRNA duplex in mouse and human islets and β-cells. Inhibition of PI3Kγ blunted the exocytotic and insulinotropic response to GIP receptor activation, whereas responses to the glucagon-like peptide-1 or the glucagon-like peptide-1 receptor agonist exendin-4 were unchanged. Downstream, we find that GIP, much like glucose stimulation, activates the small GTPase protein Rac1 to induce actin remodeling. Inhibition of PI3Kγ blocked these effects of GIP. Although exendin-4 could also stimulate actin remodeling, this was not prevented by p110γ inhibition. Finally, forced actin depolymerization with latrunculin B restored the exocytotic and secretory responses to GIP during PI3Kγ inhibition, demonstrating that the loss of GIP-induced actin depolymerization was indeed limiting insulin exocytosis.     

Inhibition of Pancreatic β-Cell Ca2+/Calmodulin-dependent Protein Kinase II Reduces Glucose-stimulated Calcium Influx and Insulin Secretion,Impairing Glucose Tolerance

Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells is caused by Ca²⁺ entry via voltage-dependent Ca²⁺ channels. CaMKII is a key mediator and feedback regulator of Ca²⁺ signaling in many tissues, but its role in β-cells is poorly understood, especially in vivo. Here, we report that mice with conditional inhibition of CaMKII in β-cells show significantly impaired glucose tolerance due to decreased GSIS. Moreover, β-cell CaMKII inhibition dramatically exacerbates glucose intolerance following exposure to a high fat diet. The impairment of islet GSIS by β-cell CaMKII inhibition is not accompanied by changes in either glucose metabolism or the activities of K_ATP and voltage-gated potassium channels. However, glucose-stimulated Ca²⁺ entry via voltage-dependent Ca²⁺ channels is reduced in islet β-cells with CaMKII inhibition, as well as in primary wild-type β-cells treated with a peptide inhibitor of CaMKII. The levels of basal β-cell cytoplasmic Ca²⁺ and of endoplasmic reticulum Ca²⁺ stores are also decreased by CaMKII inhibition. In addition, CaMKII inhibition suppresses glucose-stimulated action potential firing frequency. These results reveal that CaMKII is a Ca²⁺ sensor with a key role as a feed-forward stimulator of β-cell Ca²⁺ signals that enhance GSIS under physiological and pathological conditions.     

Store-operated Ca2+ Entry Mediated by Orai1 and TRPC1 Participates to Insulin Secretion in Rat β-Cells

Store-operated Ca²⁺ channels (SOCs) are voltage-independent Ca²⁺ channels activated upon depletion of the endoplasmic reticulum Ca²⁺ stores. Early studies suggest the contribution of such channels to Ca²⁺ homeostasis in insulin-secreting pancreatic β-cells. However, their composition and contribution to glucose-stimulated insulin secretion (GSIS) remains unclear. In this study, endoplasmic reticulum Ca²⁺ depletion triggered by acetylcholine (ACh) or thapsigargin stimulated the formation of a ternary complex composed of Orai1, TRPC1, and STIM1, the key proteins involved in the formation of SOCs. Ca²⁺ imaging further revealed that Orai1 and TRPC1 are required to form functional SOCs and that these channels are activated by STIM1 in response to thapsigargin or ACh. Pharmacological SOCs inhibition or dominant negative blockade of Orai1 or TRPC1 using the specific pore mutants Orai1-E106D and TRPC1-F562A impaired GSIS in rat β-cells and fully blocked the potentiating effect of ACh on secretion. In contrast, pharmacological or dominant negative blockade of TRPC3 had no effect on extracellular Ca²⁺ entry and GSIS. Finally, we observed that prolonged exposure to supraphysiological glucose concentration impaired SOCs function without altering the expression levels of STIM1, Orai1, and TRPC1. We conclude that Orai1 and TRPC1, which form SOCs regulated by STIM1, play a key role in the effect of ACh on GSIS, a process that may be impaired in type 2 diabetes.     

The Role of IRE1α in the Degradation of Insulin mRNA in Pancreatic β-Cells

Background

The endoplasmic reticulum (ER) is a cellular compartment for the biosynthesis and folding of newly synthesized secretory proteins such as insulin. Perturbations to ER homeostasis cause ER stress and subsequently activate cell signaling pathways, collectively known as the Unfolded Protein Response (UPR). IRE1α is a central component of the UPR. In pancreatic β-cells, IRE1α also functions in the regulation of insulin biosynthesis.

Principal Findings

Here we report that hyperactivation of IRE1α caused by chronic high glucose treatment or IRE1α overexpression leads to insulin mRNA degradation in pancreatic β-cells. Inhibition of IRE1α signaling using its dominant negative form prevents insulin mRNA degradation. Islets from mice heterozygous for IRE1α retain expression of more insulin mRNA after chronic high glucose treatment than do their wild-type littermates.

Conclusions/Significance

These results reveal a role of IRE1α in insulin mRNA expression under ER stress conditions caused by chronic high glucose. The rapid degradation of insulin mRNA could provide immediate relief for the ER and free up the translocation machinery. Thus, this mechanism would preserve ER homeostasis and help ensure that the insulin already inside the ER can be properly folded and secreted. This adaptation may be crucial for the maintenance of β-cell homeostasis and may explain why the β-cells of type 2 diabetic patients with chronic hyperglycemia stop producing insulin in the absence of apoptosis. This mechanism may also be involved in suppression of the autoimmune type 1 diabetes by reducing the amount of misfolded insulin, which could be a source of “neo-autoantigens.”     

Glucagon-Like Peptide-1 Induced Signaling and Insulin Secretion Do Not Drive Fuel and Energy Metabolism in Primary Rodent Pancreatic β-Cells

Background

Glucagon like peptide-1 (GLP-1) and its analogue exendin-4 (Ex-4) enhance glucose stimulated insulin secretion (GSIS) and activate various signaling pathways in pancreatic β-cells, in particular cAMP, Ca²⁺ and protein kinase-B (PKB/Akt). In many cells these signals activate intermediary metabolism. However, it is not clear whether the acute amplification of GSIS by GLP-1 involves in part metabolic alterations and the production of metabolic coupling factors.

Methodology/Prinicipal Findings

GLP-1 or Ex-4 at high glucose caused release (∼20%) of the total rat islet insulin content over 1 h. While both GLP-1 and Ex-4 markedly potentiated GSIS in isolated rat and mouse islets, neither had an effect on β-cell fuel and energy metabolism over a 5 min to 3 h time period. GLP-1 activated PKB without changing glucose usage and oxidation, fatty acid oxidation, lipolysis or esterification into various lipids in rat islets. Ex-4 caused a rise in [Ca²⁺]_i and cAMP but did not enhance energy utilization, as neither oxygen consumption nor mitochondrial ATP levels were altered.

Conclusions/Significance

The results indicate that GLP-1 barely affects β-cell intermediary metabolism and that metabolic signaling does not significantly contribute to GLP-1 potentiation of GSIS. The data also indicate that insulin secretion is a minor energy consuming process in the β-cell, and that the β-cell is different from most cell types in that its metabolic activation appears to be primarily governed by a “push” (fuel substrate driven) process, rather than a “pull” mechanism secondary to enhanced insulin release as well as to Ca²⁺, cAMP and PKB signaling.     

A Role for Cytosolic Isocitrate Dehydrogenase as a Negative Regulator of Glucose Signaling for Insulin Secretion in Pancreatic ?-Cells

Cytosolic NADPH may act as one of the signals that couple glucose metabolism to insulin secretion in the pancreatic ß-cell. NADPH levels in the cytoplasm are largely controlled by the cytosolic isoforms of malic enzyme and isocitrate dehydrogenase (IDHc). Some studies have provided evidence for a role of malic enzyme in glucose-induced insulin secretion (GIIS) via pyruvate cycling, but the role of IDHc in ß-cell signaling is unsettled. IDHc is an established component of the isocitrate/α–ketoglutarate shuttle that transfers reducing equivalents (NADPH) from the mitochondrion to the cytosol. This shuttle is energy consuming since it is coupled to nicotinamide nucleotide transhydrogenase that uses the mitochondrial proton gradient to produce mitochondrial NADPH and NAD⁺ from NADP⁺ and NADH. To determine whether flux through IDHc is positively or negatively linked to GIIS, we performed RNAi knockdown experiments in ß-cells. Reduced IDHc expression in INS 832/13 cells and isolated rat islet ß-cells resulted in enhanced GIIS. This effect was mediated at least in part via the K_ATP-independent amplification arm of GIIS. IDHc knockdown in INS 832/13 cells did not alter glucose oxidation but it reduced fatty acid oxidation and increased lipogenesis from glucose. Metabolome profiling in INS 832/13 cells showed that IDHc knockdown increased isocitrate and NADP⁺ levels. It also increased the cellular contents of several metabolites linked to GIIS, in particular some Krebs cycle intermediates, acetyl-CoA, glutamate, cAMP and ATP. The results identify IDHc as a component of the emerging pathways that negatively regulate GIIS.     

Insulin Receptor-Overexpressing β-Cells Ameliorate Hyperglycemia in Diabetic Rats through Wnt Signaling Activation

To investigate the therapeutic efficacy and mechanism of β-cells with insulin receptor (IR) overexpression on diabetes mellitus (DM), rat insulinoma (INS-1) cells were engineered to stably express human insulin receptor (INS-IR cells), and subsequently transplanted into streptozotocin- induced diabetic rats. Compared with INS-1 cells, INS-IR cells showed improved β-cell function, including the increase in glucose utilization, calcium mobilization, and insulin secretion, and exhibited a higher rate of cell proliferation, and maintained lower levels of blood glucose in diabetic rats. These results were attributed to the increase of β-catenin/PPARγ complex bindings to peroxisome proliferator response elements in rat glucokinase (GK) promoter and the prolongation of S-phase of cell cycle by cyclin D1. These events resulted from more rapid and higher phosphorylation levels of insulin-signaling intermediates, including insulin receptor substrate (IRS)-1/IRS-2/phosphotylinositol 3 kinase/v-akt murine thymoma viral oncogene homolog (AKT) 1, and the consequent enhancement of β-catenin nuclear translocation and Wnt responsive genes including GK and cyclin D1. Indeed, the higher functionality and proliferation shown in INS-IR cells were offset by β-catenin, cyclin D1, GK, AKT1, and IRS-2 gene depletion. In addition, the promotion of cell proliferation and insulin secretion by Wnt signaling activation was shown by 100 nM insulin treatment, and to a similar degree, was shown in INS-IR cells. In this regard, this study suggests that transferring INS-IR cells into diabetic animals is an effective and feasible DM treatment. Accordingly, the method might be a promising alternative strategy for treatment of DM given the adverse effects of insulin among patients, including the increased risk of modest weight gain and hypoglycemia. Additionally, this study demonstrates that the novel mechanism of cross-talk between insulin and Wnt signaling plays a primary role in the higher therapeutic efficacy of IR-overexpressing β-cells.     

Secretion of β-glucosidase by Ochromonas danica

-Glucosidase released by the phytoflagellate Ochromonas danica was the result of secretion; this was adduced from the following: (1) The enzyme was released during growth, including early log phase. (2) The amount released was calculated to be much more than could be attributed to cell lysis. (3) -Glucosidase was released by cells during short term incubation in a dilute salt solution; this release was nearly linear for at least 24 h. (4) Release occurred while cell counts remained nearly constant and cells remained viable. (5) Control experiments excluded cell damage resulting from incubation and cell manipulation as a source of the exoenzyme. (6) No alkaline phosphatase was released and 5 times less phosphoglucose isomerase than glucosidase was released while the cells contained 7 times more phosphoglucose isomerase. (7) The kinetics of release of nonspecific protein and UV absorbing material was markedly different from glucosidase release. (8) Glucosidase release was temperature and energy dependent; anaerobiosis decreased enzyme release. (9) Release was inhibited by cycloheximide. (10) Cells incubated with ³H-leucine synthesized labeled protein which was secreted linearly for at least 24h. Cycloheximide inhibited incorporation of ³H-leucine into protein and the secretion of the labeled protein.Non-Standard Abbreviations CHI cycloheximide - DNP 2,4-dinitrophenol - IAA iodoacetic acid - PGI phosphoglucose isomerase - SIS salt incubation solution     

Silencing Mitogen-activated Protein 4 Kinase 4 (MAP4K4) Protects Beta Cells from Tumor Necrosis Factor-��-induced Decrease of IRS-2 and Inhibition of Glucose-stimulated Insulin Secretion

Obesity and type 2 diabetes present partially overlapping phenotypes with systemic inflammation as a common feature, raising the hypothesis that elevated cytokine levels may contribute to peripheral insulin resistance as well as the decreased beta cell functional mass observed in type 2 diabetes. In healthy humans, TNF-α infusion induces skeletal muscle insulin resistance. We now explore the impact of TNF-α on primary beta cell function and the underlying signaling pathways. Human and rat primary beta cells were sorted by FACS and cultured for 24 h ± 20 ng/ml TNF-α to explore the impact on apoptosis, proliferation, and short-term insulin secretion (1 h, 2.8 mm glucose followed by 1 h, 16.7 mm glucose at the end of the 24-h culture period) as well as key signaling protein phosphorylation and expression. Prior exposure to TNF-α for 24 h inhibits glucose-stimulated insulin secretion from primary beta cells. This is associated with a decrease in glucose-stimulated phosphorylation of key proteins in the insulin signaling pathway including Akt, AS160, and other Akt substrates, ERK as well as the insulin receptor. Strikingly, TNF-α treatment decreased IRS-2 protein level by 46 ± 7% versus control, although mRNA expression was unchanged. While TNF-α treatment increased MAP4K4 mRNA expression by 33 ± 5%, knockdown of MAP4K4 by siRNA-protected beta cells against the detrimental effects of TNF-α on both insulin secretion and signaling. We thus identify MAP4K4 as a key upstream mediator of TNF-α action on the beta cell, making it a potential therapeutic target for preservation of beta cell function in type 2 diabetes.Obesity and type 2 diabetes mellitus are widespread metabolic disorders characterized by an overlapping phenotype of insulin resistance with a relative deficiency in insulin secretion underlying hyperglycemia in type 2 diabetes. Systemic inflammation is also a feature of obesity and type 2 diabetes, raising the hypothesis that elevated cytokine levels may contribute to peripheral insulin resistance as well as decreased beta cell function and mass. Correlative studies provide evidence that type 2 diabetes is associated with elevated levels of the proinflammatory cytokine tumor necrosis factor-α (TNF-α)² in adipose tissue (1), skeletal muscle (2), and plasma (3–5). In beta cells, TNF-α has been shown to induce insulin resistance mediated by nitric oxide (6) and a reduction of glucose-stimulated calcium influx (7). The impact of TNF-α on the beta cell insulin signaling pathway has not been studied even though this pathway is known to be implicated in various aspects of beta cell function. Glucose effects on beta cell growth and survival thus require activation of the insulin receptor (IR) and insulin receptor substrate-2 (IRS-2) (8). Previously, we showed that activation of this pathway by glucose was essential for insulin secretion and that the Akt substrate 160 (AS160) was involved in glucose-stimulated insulin secretion (GSIS) (9). IR/IRS-2/Akt signaling is known to enhance beta cell replication and function (8–16) and an IGF-2/IGF-1 receptor autocrine loop underlies the glucose effect on insulin signaling via IRS-2 (17).TNF-α induces insulin resistance in insulin-sensitive tissues like adipose tissue and skeletal muscle by excessive phosphorylation of extracellular signal-regulated kinase-1/2 (ERK-1/2) and c-Jun N-terminal kinase (JNK), concomitant with increased serine and reduced tyrosine phosphorylation of IRS-1 (18–20). However, this can be prevented by silencing mitogenic-activated protein kinase kinase kinase kinase isoform 4 (MAP4K4) (18, 19). MAP4K4 is a member of the germinal center kinase group related to Saccharomyces cerevisiae MAP4K, Sterile 20 (21, 22). A recent study has shown that its silencing in macrophages suppresses systemic inflammation thereby preventing diabetes (23). We now explore the action of TNF-α on primary beta cell function and its impact on beta cell insulin signaling after glucose stimulation, showing for the first time that MAP4K4 acts as a key upstream mediator of TNF-α action on GSIS.     

A Variant of GJD2, Encoding for Connexin 36, Alters the Function of Insulin Producing β-Cells

Signalling through gap junctions contributes to control insulin secretion and, thus, blood glucose levels. Gap junctions of the insulin-producing β-cells are made of connexin 36 (Cx36), which is encoded by the GJD2 gene. Cx36-null mice feature alterations mimicking those observed in type 2 diabetes (T2D). GJD2 is also expressed in neurons, which share a number of common features with pancreatic β-cells. Given that a synonymous exonic single nucleotide polymorphism of human Cx36 (SNP rs3743123) associates with altered function of central neurons in a subset of epileptic patients, we investigated whether this SNP also caused alterations of β-cell function. Transfection of rs3743123 cDNA in connexin-lacking HeLa cells resulted in altered formation of gap junction plaques and cell coupling, as compared to those induced by wild type (WT) GJD2 cDNA. Transgenic mice expressing the very same cDNAs under an insulin promoter revealed that SNP rs3743123 expression consistently lead to a post-natal reduction of islet Cx36 levels and β-cell survival, resulting in hyperglycemia in selected lines. These changes were not observed in sex- and age-matched controls expressing WT hCx36. The variant GJD2 only marginally associated to heterogeneous populations of diabetic patients. The data document that a silent polymorphism of GJD2 is associated with altered β-cell function, presumably contributing to T2D pathogenesis.     

Tight Coupling between Glucose and Mitochondrial Metabolism in Clonal ��-Cells Is Required for Robust Insulin Secretion

The biochemical mechanisms underlying glucose-stimulated insulin secretion from pancreatic β-cells are not completely understood. To identify metabolic disturbances in β-cells that impair glucose-stimulated insulin secretion, we compared two INS-1-derived clonal β-cell lines, which are glucose-responsive (832/13 cells) or glucose-unresponsive (832/2 cells). To this end, we analyzed a number of parameters in glycolytic and mitochondrial metabolism, including mRNA expression of genes involved in cellular energy metabolism. We found that despite a marked impairment of glucose-stimulated insulin secretion, 832/2 cells exhibited a higher rate of glycolysis. Still, no glucose-induced increases in respiratory rate, ATP production, or respiratory chain complex I, III, and IV activities were seen in the 832/2 cells. Instead, 832/2 cells, which expressed lactate dehydrogenase A, released lactate regardless of ambient glucose concentrations. In contrast, the glucose-responsive 832/13 line lacked lactate dehydrogenase and did not produce lactate. Accordingly, in 832/2 cells mRNA expression of genes for glycolytic enzymes were up-regulated, whereas mitochondria-related genes were down-regulated. This could account for a Warburg-like effect in the 832/2 cell clone, lacking in 832/13 cells as well as primary β-cells. In human islets, mRNA expression of genes such as lactate dehydrogenase A and hexokinase I correlated positively with HbA_1c levels, reflecting perturbed long term glucose homeostasis, whereas that of Slc2a2 (glucose transporter 2) correlated negatively with HbA_1c and thus better metabolic control. We conclude that tight metabolic regulation enhancing mitochondrial metabolism and restricting glycolysis in 832/13 cells is required for clonal β-cells to secrete insulin robustly in response to glucose. Moreover, a similar expression pattern of genes controlling glycolytic and mitochondrial metabolism in clonal β-cells and human islets was observed, suggesting that a similar prioritization of mitochondrial metabolism is required in healthy human β-cells. The 832 β-cell lines may be helpful tools to resolve metabolic perturbations occurring in Type 2 diabetes.     

Insulin Secretion and Ca2+ Dynamics in β-Cells Are Regulated by PERK (EIF2AK3) in Concert with Calcineurin

Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) (EIF2AK3) is essential for normal development and function of the insulin-secreting β-cell. Although genetic ablation of PERK in β-cells results in permanent neonatal diabetes in humans and mice, the underlying mechanisms remain unclear. Here, we used a newly developed and highly specific inhibitor of PERK to determine the immediate effects of acute ablation of PERK activity. We found that inhibition of PERK in human and rodent β-cells causes a rapid inhibition of secretagogue-stimulated subcellular Ca²⁺ signaling and insulin secretion. These dysfunctions stem from alterations in store-operated Ca²⁺ entry and sarcoplasmic endoplasmic reticulum Ca²⁺-ATPase activity. We also found that PERK regulates calcineurin, and pharmacological inhibition of calcineurin results in similar defects on stimulus-secretion coupling. Our findings suggest that interplay between calcineurin and PERK regulates β-cell Ca²⁺ signaling and insulin secretion, and that loss of this interaction may have profound implications in insulin secretion defects associated with diabetes.     

Protection of Pancreatic β-Cells from Various Stress Conditions Is Mediated by DJ-1

Pancreatic β-cells are vulnerable to multiple stresses, leading to dysfunction and apoptotic death. Deterioration in β-cells function and mass is associated with type 2 diabetes. Comparative two-dimensional gel electrophoresis from pancreatic MIN6 cells that were maintained at varying glucose concentrations was carried out. An induced expression of a protein spot, detected in MIN6 cells experiencing high glucose concentration, was identified by mass spectrometry as the oxidized form of DJ-1. DJ-1 (park7) is a multifunctional protein implicated in familial Parkinsonism and neuroprotection in response to oxidative damage. The DJ-1 protein and its oxidized form were also induced following exposure to oxidative and endoplasmic reticulum stress in MIN6 and βTC-6 cells and also in mouse pancreatic islets. Suppression of DJ-1 levels by small interfering RNA led to an accelerated cell death, whereas an increase in DJ-1 levels by adenovirus-based infection attenuated cell death induced by H₂O₂ and thapsigargin in β-cell lines and mouse pancreatic islets. Furthermore, DJ-1 improved regulated insulin secretion under basal as well as oxidative and endoplasmic reticulum stress conditions in a dose-dependent manner. We identified TFII-I (Gtf2i) as DJ-1 partner in the cytosol, whereas the binding of TFII-I to DJ-1 prevented TFII-I translocation to the nucleus. The outcome was attenuation of the stress response. Our results suggest that DJ-1 together with TFII-I operate in concert to cope with various insults and to sustain pancreatic β-cell function.