Modeling K,ATP-Dependent Excitability in Pancreatic Islets

In pancreatic β-cells, K,ATP channels respond to changes in glucose to regulate cell excitability and insulin release. Confirming a high sensitivity of electrical activity to K,ATP activity, mutations that cause gain of K,ATP function cause neonatal diabetes. Our aim was to quantitatively assess the contribution of K,ATP current to the regulation of glucose-dependent bursting by reproducing experimentally observed changes in excitability when K,ATP conductance is altered by genetic manipulation. A recent detailed computational model of single cell pancreatic β-cell excitability reproduces the β-cell response to varying glucose concentrations. However, initial simulations showed that the model underrepresents the significance of K,ATP activity and was unable to reproduce K,ATP conductance-dependent changes in excitability. By altering the ATP and glucose dependence of the L-type Ca²⁺ channel and the Na-K ATPase to better fit experiment, appropriate dependence of excitability on K,ATP conductance was reproduced. Because experiments were conducted in islets, which contain cell-to-cell variability, we extended the model from a single cell to a three-dimensional model (10×10×10 cell) islet with 1000 cells. For each cell, the conductance of the major currents was allowed to vary as was the gap junction conductance between cells. This showed that single cell glucose-dependent behavior was then highly variable, but was uniform in coupled islets. The study highlights the importance of parameterization of detailed models of β-cell excitability and suggests future experiments that will lead to improved characterization of β-cell excitability and the control of insulin secretion.     

A Syntenic Cross Species Aneuploidy Genetic Screen Links RCAN1 Expression to β-Cell Mitochondrial Dysfunction in Type 2 Diabetes

Type 2 diabetes (T2D) is a complex metabolic disease associated with obesity, insulin resistance and hypoinsulinemia due to pancreatic β-cell dysfunction. Reduced mitochondrial function is thought to be central to β-cell dysfunction. Mitochondrial dysfunction and reduced insulin secretion are also observed in β-cells of humans with the most common human genetic disorder, Down syndrome (DS, Trisomy 21). To identify regions of chromosome 21 that may be associated with perturbed glucose homeostasis we profiled the glycaemic status of different DS mouse models. The Ts65Dn and Dp16 DS mouse lines were hyperglycemic, while Tc1 and Ts1Rhr mice were not, providing us with a region of chromosome 21 containing genes that cause hyperglycemia. We then examined whether any of these genes were upregulated in a set of ~5,000 gene expression changes we had identified in a large gene expression analysis of human T2D β-cells. This approach produced a single gene, RCAN1, as a candidate gene linking hyperglycemia and functional changes in T2D β-cells. Further investigations demonstrated that RCAN1 methylation is reduced in human T2D islets at multiple sites, correlating with increased expression. RCAN1 protein expression was also increased in db/db mouse islets and in human and mouse islets exposed to high glucose. Mice overexpressing RCAN1 had reduced in vivo glucose-stimulated insulin secretion and their β-cells displayed mitochondrial dysfunction including hyperpolarised membrane potential, reduced oxidative phosphorylation and low ATP production. This lack of β-cell ATP had functional consequences by negatively affecting both glucose-stimulated membrane depolarisation and ATP-dependent insulin granule exocytosis. Thus, from amongst the myriad of gene expression changes occurring in T2D β-cells where we had little knowledge of which changes cause β-cell dysfunction, we applied a trisomy 21 screening approach which linked RCAN1 to β-cell mitochondrial dysfunction in T2D.     

Deletion of the Mitochondrial Flavoprotein Apoptosis Inducing Factor (AIF) Induces β-Cell Apoptosis and Impairs β-Cell Mass

Background

Apoptosis is a hallmark of β-cell death in both type 1 and type 2 diabetes mellitus. Understanding how apoptosis contributes to β-cell turnover may lead to strategies to prevent progression of diabetes. A key mediator of apoptosis, mitochondrial function, and cell survival is apoptosis inducing factor (AIF). In the present study, we investigated the role of AIF on β-cell mass and survival using the Harlequin (Hq) mutant mice, which are hypomorphic for AIF.

Methodology/Principal Findings

Immunohistochemical evaluation of pancreata from Hq mutant mice displayed much smaller islets compared to wild-type mice (WT). Analysis of β-cell mass in these mice revealed a greater than 4-fold reduction in β-cell mass together with an 8-fold increase in β-cell apoptosis. Analysis of cell cycle dynamics, using BrdU pulse as a marker for cells in S-phase, did not detect significant differences in the frequency of β-cells in S-phase. In contrast, double staining for phosphorylated Histone H3 and insulin showed a 3-fold increase in β-cells in the G2 phase in Hq mutant mice, but no differences in M-phase compared to WT mice. This suggests that the β-cells from Hq mutant mice are arrested in the G2 phase and are unlikely to complete the cell cycle. β-cells from Hq mutant mice display increased sensitivity to hydrogen peroxide-induced apoptosis, which was confirmed in human islets in which AIF was depleted by siRNA. AIF deficiency had no effect on glucose stimulated insulin secretion, but the impaired effect of hydrogen peroxide on β-cell function was potentiated.

Conclusions/Significance

Our results indicate that AIF is essential for maintaining β-cell mass and for oxidative stress response. A decrease in the oxidative phosphorylation capacity may counteract the development of diabetes, despite its deleterious effects on β-cell survival.     

Pancreatic Alpha-Cell Dysfunction Contributes to the Disruption of Glucose Homeostasis and Compensatory Insulin Hypersecretion in Glucocorticoid-Treated Rats

Glucocorticoid (GC)-based therapies can cause insulin resistance (IR), glucose intolerance, hyperglycemia and, occasionally, overt diabetes. Understanding the mechanisms behind these metabolic disorders could improve the management of glucose homeostasis in patients undergoing GC treatment. For this purpose, adult rats were treated with a daily injection of dexamethasone (1 mg/kg b.w., i.p.) (DEX) or saline as a control for 5 consecutive days. The DEX rats developed IR, augmented glycemia, hyperinsulinemia and hyperglucagonemia. Treatment of the DEX rats with a glucagon receptor antagonist normalized their blood glucose level. The characteristic inhibitory effect of glucose on glucagon secretion was impaired in the islets of the DEX rats, while no direct effects were found on α-cells in islets that were incubated with DEX in vitro. A higher proportion of docked secretory granules was found in the DEX α-cells as well as a trend towards increased α-cell mass. Additionally, insulin secretion in the presence of glucagon was augmented in the islets of the DEX rats, which was most likely due to their higher glucagon receptor content. We also found that the enzyme 11βHSD-1, which participates in GC metabolism, contributed to the insulin hypersecretion in the DEX rats under basal glucose conditions. Altogether, we showed that GC treatment induces hyperglucagonemia, which contributes to an imbalance in glucose homeostasis and compensatory β-cell hypersecretion. This hyperglucagonemia may result from altered α-cell function and, likely, α-cell mass. Additionally, blockage of the glucagon receptor seems to be effective in preventing the elevation in blood glucose levels induced by GC administration.     

β-cell Smad2 null mice have improved β-cell function and are protected from diet-induced hyperglycemia

Understanding signaling pathways that regulate pancreatic β-cell function to produce, store, and release insulin, as well as pathways that control β-cell proliferation, is vital to find new treatments for diabetes mellitus. Transforming growth factor-beta (TGF-β) signaling is involved in a broad range of β-cell functions. The canonical TGF-β signaling pathway functions through intracellular smads, including smad2 and smad3, to regulate cell development, proliferation, differentiation, and function in many organs. Here, we demonstrate the role of TGF-β/smad2 signaling in regulating mature β-cell proliferation and function using β-cell-specific smad2 null mutant mice. β-cell-specific smad2-deficient mice exhibited improved glucose clearance as demonstrated by glucose tolerance testing, enhanced in vivo and ex vivo glucose-stimulated insulin secretion, and increased β-cell mass and proliferation. Furthermore, when these mice were fed a high-fat diet to induce hyperglycemia, they again showed improved glucose tolerance, insulin secretion, and insulin sensitivity. In addition, ex vivo analysis of smad2-deficient islets showed that they displayed increased glucose-stimulated insulin secretion and upregulation of genes involved in insulin synthesis and insulin secretion. Thus, we conclude that smad2 could represent an attractive therapeutic target for type 2 diabetes mellitus.     

EGFR Signaling Promotes β-Cell Proliferation and Survivin Expression during Pregnancy

Placental lactogen (PL) induced serotonergic signaling is essential for gestational β-cell mass expansion. We have previously shown that intact Epidermal growth factor –receptor (EGFR) function is a crucial component of this pathway. We now explored more specifically the link between EGFR and pregnancy-induced β-cell mass compensation. Islets were isolated from wild-type and β-cell-specific EGFR-dominant negative mice (E1-DN), stimulated with PL and analyzed for β-cell proliferation and expression of genes involved in gestational β-cell growth. β-cell mass dynamics were analyzed both with traditional morphometrical methods and three-dimensional optical projection tomography (OPT) of whole-mount insulin-stained pancreata. Insulin-positive volume analyzed with OPT increased 1.4-fold at gestational day 18.5 (GD18.5) when compared to non-pregnant mice. Number of islets peaked by GD13.5 (680 vs 1134 islets per pancreas, non-pregnant vs. GD13.5). PL stimulated beta cell proliferation in the wild-type islets, whereas the proliferative response was absent in the E1-DN mouse islets. Serotonin synthesizing enzymes were upregulated similarly in both the wild-type and E1-DN mice. However, while survivin (Birc5) mRNA was upregulated 5.5-fold during pregnancy in the wild-type islets, no change was seen in the E1-DN pregnant islets. PL induced survivin expression also in isolated islets and this was blocked by EGFR inhibitor gefitinib, mTOR inhibitor rapamycin and MEK inhibitor PD0325901. Our 3D-volumetric analysis of β-cell mass expansion during murine pregnancy revealed that islet number increases during pregnancy. In addition, our results suggest that EGFR signaling is required for lactogen-induced survivin expression via MAPK and mTOR pathways.     

Interleukin-12 (IL-12)/STAT4 Axis Is an Important Element for β-Cell Dysfunction Induced by Inflammatory Cytokines

Pathology driving β-cell loss in diabetes is poorly defined. Chronic subclinical inflammation is associated with β-cell dysfunction. Acute in vitro exposure of islets and β-cells to an inflammatory cytokine cocktail (IL-1β/TNF-α/IFN-γ) results in loss of cell function and viability. The contribution of each cytokine alone or in combination has been evaluated in homogeneous mouse β-cell lines and primary mouse islets. Cytokine cooperation is required for β-cell apoptosis with the most potent combinations including IL-1β. Single cytokine exposure did not induce β-cell apoptosis. Expression of endogenous interleukin-12 in β-cells correlated with inflammatory cytokine combinations that induced β-cell apoptosis. Uncoupling of the IL-12 axis by a block of IL-12 production, inhibition of IL-12 receptor/ligand interaction or disruption of IL-12 receptor signaling conferred protection to β-cells from apoptosis induced by inflammatory cytokine stimulation. Signaling through STAT4 is indicated since disruption of IL-12 concomitantly reduced inflammatory cytokine stimulation of endogenous IFN-γ expression. Primary mouse islets isolated from mice deficient in STAT4 show resistance to inflammatory-cytokine-induced cell death when compared to islets isolated from wild type mice. Collectively, the data identify IL-12 as an important mediator of inflammation induced β-cell apoptosis. Modulation of IL-12/STAT4 signaling may be a valuable therapeutic strategy to preserve islet/β-cell viability in established diabetes.     

Glucose-Dependent Insulin Secretion in Pancreatic β-Cell Islets from Male Rats Requires Ca2+ Release via ROS-Stimulated Ryanodine Receptors

Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells requires an increase in intracellular free Ca²⁺ concentration ([Ca²⁺]). Glucose uptake into β-cells promotes Ca²⁺ influx and reactive oxygen species (ROS) generation. In other cell types, Ca²⁺ and ROS jointly induce Ca²⁺ release mediated by ryanodine receptor (RyR) channels. Therefore, we explored here if RyR-mediated Ca²⁺ release contributes to GSIS in β-cell islets isolated from male rats. Stimulatory glucose increased islet insulin secretion, and promoted ROS generation in islets and dissociated β-cells. Conventional PCR assays and immunostaining confirmed that β-cells express RyR2, the cardiac RyR isoform. Extended incubation of β-cell islets with inhibitory ryanodine suppressed GSIS; so did the antioxidant N-acetyl cysteine (NAC), which also decreased insulin secretion induced by glucose plus caffeine. Inhibitory ryanodine or NAC did not affect insulin secretion induced by glucose plus carbachol, which engages inositol 1,4,5-trisphosphate receptors. Incubation of islets with H₂O₂ in basal glucose increased insulin secretion 2-fold. Inhibitory ryanodine significantly decreased H₂O₂-stimulated insulin secretion and prevented the 4.5-fold increase of cytoplasmic [Ca²⁺] produced by incubation of dissociated β-cells with H₂O₂. Addition of stimulatory glucose or H₂O₂ (in basal glucose) to β-cells disaggregated from islets increased RyR2 S-glutathionylation to similar levels, measured by a proximity ligation assay; in contrast, NAC significantly reduced the RyR2 S-glutathionylation increase produced by stimulatory glucose. We propose that RyR2-mediated Ca²⁺ release, induced by the concomitant increases in [Ca²⁺] and ROS produced by stimulatory glucose, is an essential step in GSIS.     

Chronic Antidiabetic Sulfonylureas In Vivo: Reversible Effects on Mouse Pancreatic β-Cells

Background

Pancreatic β-cell ATP-sensitive potassium (K_ATP) channels are critical links between nutrient metabolism and insulin secretion. In humans, reduced or absent β-cell K_ATP channel activity resulting from loss-of-function K_ATP mutations induces insulin hypersecretion. Mice with reduced K_ATP channel activity also demonstrate hyperinsulinism, but mice with complete loss of K_ATP channels (K_ATP knockout mice) show an unexpected insulin undersecretory phenotype. Therefore we have proposed an “inverse U” hypothesis to explain the response to enhanced excitability, in which excessive hyperexcitability drives β-cells to insulin secretory failure without cell death. Many patients with type 2 diabetes treated with antidiabetic sulfonylureas (which inhibit K_ATP activity and thereby enhance insulin secretion) show long-term insulin secretory failure, which we further suggest might reflect a similar progression.

Methods and Findings

To test the above hypotheses, and to mechanistically investigate the consequences of prolonged hyperexcitability in vivo, we used a novel approach of implanting mice with slow-release sulfonylurea (glibenclamide) pellets, to chronically inhibit β-cell K_ATP channels. Glibenclamide-implanted wild-type mice became progressively and consistently diabetic, with significantly (p < 0.05) reduced insulin secretion in response to glucose. After 1 wk of treatment, these mice were as glucose intolerant as adult K_ATP knockout mice, and reduction of secretory capacity in freshly isolated islets from implanted animals was as significant (p < 0.05) as those from K_ATP knockout animals. However, secretory capacity was fully restored in islets from sulfonylurea-treated mice within hours of drug washout and in vivo within 1 mo after glibenclamide treatment was terminated. Pancreatic immunostaining showed normal islet size and α-/β-cell distribution within the islet, and TUNEL staining showed no evidence of apoptosis.

Conclusions

These results demonstrate that chronic glibenclamide treatment in vivo causes loss of insulin secretory capacity due to β-cell hyperexcitability, but also reveal rapid reversibility of this secretory failure, arguing against β-cell apoptosis or other cell death induced by sulfonylureas. These in vivo studies may help to explain why patients with type 2 diabetes can show long-term secondary failure to secrete insulin in response to sulfonylureas, but experience restoration of insulin secretion after a drug resting period, without permanent damage to β-cells. This finding suggests that novel treatment regimens may succeed in prolonging pharmacological therapies in susceptible individuals.     

Genetic Deficiency of Glycogen Synthase Kinase-3β Corrects Diabetes in Mouse Models of Insulin Resistance

Despite treatment with agents that enhance β-cell function and insulin action, reduction in β-cell mass is relentless in patients with insulin resistance and type 2 diabetes mellitus. Insulin resistance is characterized by impaired signaling through the insulin/insulin receptor/insulin receptor substrate/PI-3K/Akt pathway, leading to elevation of negatively regulated substrates such as glycogen synthase kinase-3β (Gsk-3β). When elevated, this enzyme has antiproliferative and proapoptotic properties. In these studies, we designed experiments to determine the contribution of Gsk-3β to regulation of β-cell mass in two mouse models of insulin resistance. Mice lacking one allele of the insulin receptor (Ir^+/−) exhibit insulin resistance and a doubling of β-cell mass. Crossing these mice with those having haploinsufficiency for Gsk-3β (Gsk-3β^+/−) reduced insulin resistance by augmenting whole-body glucose disposal, and significantly reduced β-cell mass. In the second model, mice missing two alleles of the insulin receptor substrate 2 (Irs2^−/−), like the Ir^+/− mice, are insulin resistant, but develop profound β-cell loss, resulting in early diabetes. We found that islets from these mice had a 4-fold elevation of Gsk-3β activity associated with a marked reduction of β-cell proliferation and increased apoptosis. Irs2^−/− mice crossed with Gsk-3β^+/− mice preserved β-cell mass by reversing the negative effects on proliferation and apoptosis, preventing onset of diabetes. Previous studies had shown that islets of Irs2^−/− mice had increased cyclin-dependent kinase inhibitor p27^kip1 that was limiting for β-cell replication, and reduced Pdx1 levels associated with increased cell death. Preservation of β-cell mass in Gsk-3β^+/−Irs2^−/− mice was accompanied by suppressed p27^kip1 levels and increased Pdx1 levels. To separate peripheral versus β-cell–specific effects of reduction of Gsk3β activity on preservation of β-cell mass, mice homozygous for a floxed Gsk-3β allele (Gsk-3^F/F) were then crossed with rat insulin promoter-Cre (RIP-Cre) mice to produce β-cell–specific knockout of Gsk-3β (βGsk-3β^−/−). Like Gsk-3β^+/− mice, βGsk-3β^−/− mice also prevented the diabetes of the Irs2^−/− mice. The results of these studies now define a new, negatively regulated substrate of the insulin signaling pathway specifically within β-cells that when elevated, can impair replication and increase apoptosis, resulting in loss of β-cells and diabetes. These results thus form the rationale for developing agents to inhibit this enzyme in obese insulin-resistant individuals to preserve β-cells and prevent diabetes onset.     

Intrinsic Islet Heterogeneity and Gap Junction Coupling Determine Spatiotemporal Ca2+ Wave Dynamics

Insulin is released from the islets of Langerhans in discrete pulses that are linked to synchronized oscillations of intracellular free calcium ([Ca²⁺]_i). Associated with each synchronized oscillation is a propagating calcium wave mediated by Connexin36 (Cx36) gap junctions. A computational islet model predicted that waves emerge due to heterogeneity in β-cell function throughout the islet. To test this, we applied defined patterns of glucose stimulation across the islet using a microfluidic device and measured how these perturbations affect calcium wave propagation. We further investigated how gap junction coupling regulates spatiotemporal [Ca²⁺]_i dynamics in the face of heterogeneous glucose stimulation. Calcium waves were found to originate in regions of the islet having elevated excitability, and this heterogeneity is an intrinsic property of islet β-cells. The extent of [Ca²⁺]_i elevation across the islet in the presence of heterogeneity is gap-junction dependent, which reveals a glucose dependence of gap junction coupling. To better describe these observations, we had to modify the computational islet model to consider the electrochemical gradient between neighboring β-cells. These results reveal how the spatiotemporal [Ca²⁺]_i dynamics of the islet depend on β-cell heterogeneity and cell-cell coupling, and are important for understanding the regulation of coordinated insulin release across the islet.     

Protective Antioxidant and Antiapoptotic Effects of ZnCl2 in Rat Pancreatic Islets Cultured in Low and High Glucose Concentrations

Aim/Hypothesis

Rat pancreatic islet cell apoptosis is minimal after prolonged culture in 10 mmol/l glucose (G10), largely increased in 5 mmol/l glucose (G5) and moderately increased in 30 mmol/l glucose (G30). This glucose-dependent asymmetric V-shaped profile is preceded by parallel changes in the mRNA levels of oxidative stress-response genes like Metallothionein 1a (Mt1a). In this study, we tested the effect of ZnCl₂, a potent inducer of Mt1a, on apoptosis, mitochondrial oxidative stress and alterations of glucose-induced insulin secretion (GSIS) induced by prolonged exposure to low and high vs. intermediate glucose concentrations.

Methods

Male Wistar rat islets were cultured in RPMI medium. Islet gene mRNA levels were measured by RTq-PCR. Apoptosis was quantified by measuring islet cytosolic histone-associated DNA fragments and the percentage of TUNEL-positive β-cells. Mitochondrial thiol oxidation was measured in rat islet cell clusters expressing “redox sensitive GFP” targeted to the mitochondria (mt-roGFP1). Insulin secretion was measured by RIA.

Results

As observed for Mt1a mRNA levels, β-cell apoptosis and loss of GSIS, culture in either G5 or G30 vs. G10 significantly increased mt-roGFP1 oxidation. While TPEN decreased Mt1a/2a mRNA induction by G5, addition of 50–100 µM ZnCl₂ to the culture medium strongly increased Mt1a/2a mRNA and protein levels, reduced early mt-roGFP oxidation and significantly decreased late β-cell apoptosis after prolonged culture in G5 or G30 vs. G10. It did not, however, prevent the loss of GSIS under these culture conditions.

Conclusion

ZnCl₂ reduces mitochondrial oxidative stress and improves rat β-cell survival during culture in the presence of low and high vs. intermediate glucose concentrations without improving their acute GSIS.     

ATP-independent glucose stimulation of sphingosine kinase in rat pancreatic islets

Sphingosine kinase (SPHK) catalyzes sphingosine 1-phosphate production, promoting cell survival and reducing apoptosis in isolated rat pancreatic islets. Glucose, the primary islet β-cell growth factor and insulin secretagogue, increased islet SPHK activity by 3- to 5-fold following acute (1 h) or prolonged (7 days) stimulation. Prolonged stimulation of islets with glucose induced SPHK1a and SPHK2 mRNA levels; there were no changes in SPHK protein expression. To isolate the metabolic effects of glucose on SPHK activation, islets were stimulated with glucose analogs or metabolites. 2-deoxy-D-glucose (2-DG), an analog phosphorylated by glucokinase but not an effective energy source, activated SPHK similarly to glucose. In contrast, 3-o-methylglucose (3-oMeG), which is transported but neither phosphorylated nor metabolized, did not increase islet SPHK activity. Glyceraldehyde and α-ketoisocaproic acid (KIC), metabolites that stimulate glycolysis and the citric acid cycle, respectively, did not activate islet SPHK. Moreover, inorganic phosphate blocked glucose-induced SPHK activation. A role for SPHK activity in β-cell growth was confirmed when small interfering (si)SPHK2 RNA transfection reduced rat insulinoma INS-1e cell SPHK levels and activity and cell growth. Glucose induced an early and sustained increase in islet SPHK activity that was dependent on glucose phosphorylation, but independent of ATP generation or new protein biosynthesis. Glucose-supported β-cell growth appears to be in part mediated by SPHK activity.     

Moderate Hypoxia Induces β-Cell Dysfunction with HIF-1–Independent Gene Expression Changes

Pancreatic β-cell failure is central to the development and progression of type 2 diabetes. We recently demonstrated that β-cells become hypoxic under high glucose conditions due to increased oxygen consumption and that the pancreatic islets of diabetic mice but not those of control mice are moderately hypoxic. However, the impact of moderate hypoxia on β-cell number and function is unknown. In the present study, moderate hypoxia induced a hypoxic response in MIN6 cells, as evidenced by increased levels of HIF-1α protein and target genes. Under these conditions, a selective downregulation of Mafa, Pdx1, Slc2a2, Ndufa5, Kcnj11, Ins1, Wfs1, Foxa2, and Neurod1, which play important roles in β-cells, was also observed in both MIN6 cells and isolated pancreatic islets. Consistent with the altered expression of these genes, abnormal insulin secretion was detected in hypoxic MIN6 cells. Most of the hypoxia-induced gene downregulation in MIN6 cells was not affected by the suppression of HIF-1α, suggesting a HIF-1–independent mechanism. Moderate hypoxia also induced apoptosis in MIN6 cells. These results suggest that hypoxia is a novel stressor of β-cells and that hypoxic stress may play a role in the deterioration of β-cell function.     

Pancreatic β-Cell Death due to Pdx-1 Deficiency Requires Multi-BH Domain Protein Bax but Not Bak

Diabetes develops in Pdx1-haploinsufficient mice due to an increase in β-cell death leading to reduced β-cell mass and decreased insulin secretion. Knockdown of Pdx1 gene expression in mouse MIN6 insulinoma cells induced apoptotic cell death with an increase in Bax activation and knockdown of Bax reduced apoptotic β-cell death. In Pdx1 haploinsufficient mice, Bax ablation in β-cells increased β-cell mass, decreased the number of TUNEL positive cells and improved glucose tolerance after glucose challenge. These changes were not observed with Bak ablation in Pdx1-haploinsufficient mice. These results suggest that Bax mediates β-cell apoptosis in Pdx1-deficient 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.