A role for trans-caryophyllene in the moderation of insulin secretion

Glucose-stimulated insulin secretion (GSIS) is essential for the control of metabolic fuel homeostasis and its impairment is a key element in the failure of β-cells in type 2 diabetes. Trans-caryophyllene (TC), an important constituent of the essential oil of several species of plants, has been reported to activate the type 2 cannabinoid receptor (CB2R). The effects of TC on GSIS are still unknown. Our results demonstrate that administration of TC in MIN6 cells promotes GSIS in a dose dependent manner. However, inhibition of CB2R by a specific inhibitor or specific RNA interference abolished the effects of TC on GSIS, which suggests that the effects of TC on GSIS are dependent on activation of CB2R. Further study demonstrated that treatment with TC leads to the activation of small G protein Arf6 as well as Rac1 and Cdc42. Importantly, Arf6 silencing abolished the effects of TC on GSIS, which suggests that Arf6 participates in mediating the effects of TC on GSIS. We conclude from these data that TC has a novel role in regulating GSIS in pancreatic β-cells.     

Prkar1a in the regulation of insulin secretion

The incidence of type 2 diabetes mellitus (T2DM) is rapidly increasing worldwide with significant consequences on individual quality of life as well as economic burden on states' healthcare costs. While origins of the pathogenesis of T2DM are poorly understood, an early defect in glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells is considered a hallmark of T2DM 1.Upon a glucose stimulus, insulin is secreted in a biphasic manner with an early first-phase burst of insulin, which is followed by a second, more sustained phase of insulin output 2. First phase insulin secretion is diminished early in T2DM as well is in subjects who are at risk of developing T2DM 3 4 5 6.An effective treatment of T2DM with incretin hormone glucagon-like peptide-1 (GLP-1) or its long acting peptide analogue exendin-4 (E4), restores first-phase and augments second-phase glucose stimulated insulin secretion. This effect of incretin action occurs within minutes of GLP-1/E4 infusion in T2DM humans. An additional important consideration is that incretin hormones augment GSIS only above a certain glucose threshold, which is slightly above the normal glucose range. This ensures that incretin hormones stimulate GSIS only when glucose levels are high, while they are ineffective when insulin levels are below a certain threshold 7 8.Activation of the GLP-1 receptor, which is highly expressed on pancreatic β-cells, stimulates 2 -distinct intracellular signaling pathways: a) the cAMP-protein kinase A branch and b) the cAMP-EPAC2 (EPAC=exchange protein activated by cAMP) branch. While the EPAC2 branch is considered to mediate GLP-1 effects on first-phase GSIS, the PKA branch is necessary for the former branch to be active 9 10. However, how these 2 branches interplay and converge and how their effects on insulin secretion and insulin vesicle exocytosis are coordinated is poorly understood.Thus, at the outset of our studies we have a poorly understood intracellular interplay of cAMP-dependent signaling pathways, which - when stimulated - restore glucose-dependent first phase and augment second phase insulin secretion in the ailing β-cells of T2DM.     

Identification of a novel GPR119 agonist, AS1269574, with in vitro and in vivo glucose-stimulated insulin secretion

The G protein-coupled receptor 119 (GPR119) is highly expressed in pancreatic β-cells. On activation, this receptor enhances the effect of glucose-stimulated insulin secretion (GSIS) via the elevation of intracellular cAMP concentrations. Although GPR119 agonists represent promising oral antidiabetic agents for the treatment of type 2 diabetes therapy, they suffer from the inability to adequately directly preserve β-cell function. To identify a new structural class of small-molecule GPR119 agonists with both GSIS and the potential to preserve β-cell function, we screened a library of synthetic compounds and identified a candidate molecule, AS1269574, with a 2,4,6-tri-substituted pyrimidine core. Here, we examined the preliminary in vitro and in vivo effects of AS1269574 on insulin secretion and glucose tolerance. AS1269574 had an EC₅₀ value of 2.5 μM in HEK293 cells transiently expressing human GPR119 and enhanced insulin secretion in the mouse pancreatic β-cell line MIN-6 only under high-glucose (16.8 mM) conditions. This contrasted with the action of the sulfonylurea glibenclamide, which also induced insulin secretion under low-glucose conditions (2.8 mM). In in vivo studies, a single administration of AS1269574 to normal mice reduced blood glucose levels after oral glucose loading based on the observed insulin secretion profiles. Significantly, AS1269574 did not affect fed and fasting plasma glucose levels in normal mice. Taken together, these results suggest that AS1269574 represents a novel structural class of small molecule, orally administrable GPR119 agonists with GSIS and promising potential for the treatment of type 2 diabetes.     

Exendin-4, a glucagon-like peptide-1 receptor agonist, inhibits cell apoptosis induced by lipotoxicity in pancreatic β-cell line

Lipotoxicity plays an important role in the underlying mechanism of type 2 diabetes mellitus. Prolonged exposure of pancreatic β-cells to elevated concentrations of fatty acid is associated with β-cell apoptosis. Recently, glucagon-like peptide-1 (GLP-1) receptor agonists have been reported to have direct beneficial effects on β-cells, such as anti-apoptotic effects, increased β-cell mass, and improvement of β-cell function. The mechanism of GLP-1 receptor agonists' protection of pancreatic β-cells against lipotoxicity is not completely understood. We investigated whether the GLP-1 receptor agonist exendin-4 promoted cell survival and attenuated palmitate-induced apoptosis in murine pancreatic β-cells (MIN6). Exposure of MIN6 cells to palmitate (0.4mM) for 24h caused a significant increase in cell apoptosis, which was inhibited by exendin-4. Exposure of MIN6 cells to exendin-4 caused rapid activation of protein kinase B (PKB) under lipotoxic conditions. Furthermore, LY294002, a PI3K inhibitor, abolished the anti-lipotoxic effect of exendin-4 on MIN6 cells. Exendin-4 also inhibited the mitochondrial pathway of apoptosis and down-regulated Bax in MIN6 cells. Exendin-4 enhanced glucose-stimulated insulin secretion in the presence of palmitate. Our findings suggest that exendin-4 may prevent lipotoxicity-induced apoptosis in MIN6 cells through activation of PKB and inhibition of the mitochondrial pathway.     

AS1907417, a novel GPR119 agonist, as an insulinotropic and β-cell preservative agent for the treatment of type 2 diabetes

G-protein-coupled receptor (GPR) 119 is involved in glucose-stimulated insulin secretion (GSIS) and represents a promising target for the treatment of type 2 diabetes as it is highly expressed in pancreatic β-cells. Although a number of oral GPR119 agonists have been developed, their inability to adequately directly preserve β-cell function limits their effectiveness. Here, we evaluated the therapeutic potential of a novel small-molecule GPR119 agonist, AS1907417, which represents a modified form of a 2,4,6-tri-substituted pyrimidine core agonist, AS1269574, we previously identified. The exposure of HEK293 cells expressing human GPR119, NIT-1 cells expressing human insulin promoter, and the pancreatic β-cell line MIN-6-B1 to AS1907417, enhanced intracellular cAMP, GSIS, and human insulin promoter activity, respectively. In in vivo experiments involving fasted normal mice, a single dose of AS1907417 improved glucose tolerance, but did not affect plasma glucose or insulin levels. Twice-daily doses of AS1907417 for 4 weeks in diabetic db/db, aged db/db mice, ob/ob mice, and Zucker diabetic fatty rats reduced hemoglobin A1c levels by 1.6%, 0.8%, 1.5%, and 0.9%, respectively. In db/db mice, AS1907417 improved plasma glucose, plasma insulin, pancreatic insulin content, lipid profiles, and increased pancreatic insulin and pancreatic and duodenal homeobox 1 (PDX-1) mRNA levels. These data demonstrate that novel GPR119 agonist AS1907417 not only effectively controls glucose levels, but also preserves pancreatic β-cell function. We therefore propose that AS1907417 represents a new type of antihyperglycemic agent with promising potential for the effective treatment of type 2 diabetes.     

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.     

Sphingosine 1-phosphate (S1P) regulates glucose-stimulated insulin secretion in pancreatic beta cells

Recent studies suggest that sphingolipid metabolism is altered during type 2 diabetes. Increased levels of the sphingolipid ceramide are associated with insulin resistance. However, a role for sphingolipids in pancreatic beta cell function, or insulin production, and release remains to be established. Our studies in MIN6 cells and mouse pancreatic islets demonstrate that glucose stimulates an intracellular rise in the sphingolipid, sphingosine 1-phosphate (S1P), whereas the levels of ceramide and sphingomyelin remain unchanged. The increase in S1P levels by glucose is due to activation of sphingosine kinase 2 (SphK2). Interestingly, rises in S1P correlate with increased glucose-stimulated insulin secretion (GSIS). Decreasing S1P levels by treatment of MIN6 cells or primary islets with the sphingosine kinase inhibitor reduces GSIS. Moreover, knockdown of SphK2 alone results in decreased GSIS, whereas knockdown of the S1P phosphatase, Sgpp1, leads to a rise in GSIS. Treatment of mice with the sphingosine kinase inhibitor impairs glucose disposal due to decreased plasma insulin levels. Altogether, our data suggest that glucose activates SphK2 in pancreatic beta cells leading to a rise in S1P levels, which is important for GSIS.     

Voltage-gated K channel KCNQ1 regulates insulin secretion in MIN6 β-cell line

KCNQ1, located on 11p15.5, encodes a voltage-gated K⁺ channel with six transmembrane regions, and loss-of-function mutations in the KCNQ1 gene cause hereditary long QT syndrome. Recent genetic studies have identified that single nucleotide polymorphisms located in intron 15 of the KCNQ1 gene are strongly associated with type 2 diabetes and impaired insulin secretion. In order to understand the role of KCNQ1 in insulin secretion, we introduced KCNQ1 into the MIN6 mouse β-cell line using a retrovirus-mediated gene transfer system. In KCNQ1 transferred MIN6 cells, both the density of the KCNQ1 current and the density of the total K⁺ current were significantly increased. In addition, insulin secretion by glucose, pyruvate, or tolbutamide was significantly impaired by KCNQ1-overexpressing MIN6 cells. These results suggest that increased KCNQ1 protein expression limits insulin secretion from pancreatic β-cells by regulating the potassium channel current.     

The relationship between GPR40 and lipotoxicity of the pancreatic β-cells as well as the effect of pioglitazone

Free fatty acids (FFAs) acutely stimulate insulin secretion from pancreatic β-cells, whereas impair β-cell function following long term exposure. GPR40, a FFAs receptor, has been demonstrated to be activated by both medium and long chain FFAs and played an important role in insulin release. This study was performed to determine the contribution of GPR40 to short- and/or long-term effects of FFAs on glucose-stimulated insulin secretion (GSIS) and the expression of PDX-1 and GLUT2 in pancreatic β-cells, as well as the intervenient effects of pioglitazone on lipotoxicity of β-cells. βTC6 cell line stably expressing GPR40shRNA were established and the intervention of FFAs and pioglitazone on GSIS and expression of PDX-1 and GLUT2 in βTC6 cells was investigated. Results showed that 1-h exposure to FFAs significantly enhanced GSIS and increased expression of PDX-1 and GLUT2 in pSilencer-control transfected cells, but not in cells transfected with GPR40shRNA. While 48-h exposure to FFAs significantly impaired GSIS in pSilencer-control transfected cells as well as cells transfected with GPR40shRNA. Furthermore, pioglitazone enhanced insulin secretion in pSilencer-control transfected cells exposed to FFAs for 48 h, but not in cells transfected with GPR40shRNA. These results indicate that GPR40 mediates the short-term effects of FFAs on GSIS, but does not mediate the chronic lipotoxicity on β-cells. The reverse role of pioglitazone on lipotoxicity of β-cells may be related to GPR40.     

Inhibition of the receptor for advanced glycation endproducts (RAGE) protects pancreatic β-cells

Advanced glycation endproducts (AGEs) and the receptor for AGEs (RAGE) have been linked to the pathogenesis of diabetic complications, such as retinopathy, neuropathy, and nephropathy. AGEs may induce β-cell dysfunction and apoptosis, another complication of diabetes. However, the role of AGE-RAGE interaction in AGE-induced pancreatic β-cell failure has not been fully elucidated. In this study, we investigated whether AGE–RAGE interaction could mediate β-cell failure. We explored the potential mechanisms in insulin secreting (INS-1) cells from a pancreatic β-cell line, as well as primary rat islets. We found that glycated serum (GS) induced apoptosis in pancreatic β-cells in a dose- and time-dependent manner. Treatment with GS increased RAGE protein production in cultured INS-1 cells. GS treatment also decreased bcl-2 gene expression, followed by mitochondrial swelling, increased cytochrome c release, and caspase activation. RAGE antibody and knockdown of RAGE reversed the β-cell apoptosis and bcl-2 expression. Inhibition of RAGE prevented AGE-induced pancreatic β-cell apoptosis, but could not restore the function of glucose stimulated insulin secretion (GSIS) in rat islets. In summary, the results of the present study demonstrate that AGEs are integrally involved in RAGE-mediated apoptosis and impaired GSIS dysfunction in pancreatic β-cells. Inhibition of RAGE can effectively protect β-cells against AGE-induced apoptosis, but cannot reverse islet dysfunction in GSIS.     

NF-κB p65 represses β-catenin-activated transcription of cyclin D1

We have previously reported that obesity-induced diabetes developed in high-fat diet (HFD)-fed BDF1 mice. This is caused by insufficient insulin response to an excess glucose load. In this study, we have shown that the enhanced expression of retinaldehyde dehydrogenase 3 (Raldh3) causes functional disorders of pancreatic islets in diabetic mouse models. In the pancreatic islets of HFD-induced diabetic BDF1 mice and spontaneously diabetic C57BL/KsJ^db/db mice, gene expression analysis with oligonucleotide microarray revealed a significant increase in Raldh3 expression. Exposure to a culture medium containing a higher glucose concentration (25 mM) significantly increased Raldh3 expression in murine MIN6 and alphaTC1 clone 9 cells, which derived from the α and β-cells of pancreatic islets, respectively. Overexpression of Raldh3 reduced the insulin secretion in MIN6 cells, and surprisingly, increased the glucagon secretion in alphaTC1 clone 9 cells. Furthermore, the knockdown of Raldh3 expression with siRNA decreased the glucagon secretion in alphaTC1 clone 9 cells. Raldh3 catalyzes the conversion of 13-cis retinal to 13-cis retinoic acid and we revealed that 13-cis retinoic acid significantly reduces cell viability in MIN6 and alphaTC1 clone 9 cells, but not in cells of H4IIEC3, 3T3-L1, and COS-1 cell lines. These findings suggest that an increasing expression of Raldh3 deregulates the balanced mechanisms of insulin and glucagon secretion in the pancreatic islets and may induce β-cell dysfunction leading to the development of type 2 diabetes.     

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.     

Involvement of RhoA/ROCK in insulin secretion of pancreatic β-cells in 3D culture

Cell-cell contacts and interactions between pancreatic β-cells and/or other cell populations within islets are essential for cell survival, insulin secretion, and functional synchronization. Three-dimensional (3D) culture systems supply the ideal microenvironment for islet-like cluster formation and functional maintenance. However, the underlying mechanisms remain unclear. In this study, mouse insulinoma 6 (MIN6) cells were cultured in a rotating 3D culture system to form islet-like aggregates. Glucose-stimulated insulin secretion (GSIS) and the RhoA/ROCK pathway were investigated. In the 3D-cultured MIN6 cells, more endocrine-specific genes were up-regulated, and GSIS was increased to a greater extent than in cells grown in monolayers. RhoA/ROCK inactivation led to F-actin remodeling in the MIN6 cell aggregates and greater insulin exocytosis. The gap junction protein, connexin 36 (Cx36), was up-regulated in MIN6 cell aggregates and RhoA/ROCK-inactivated monolayer cells. GSIS dramatically decreased when Cx36 was knocked down by short interfering RNA and could not be reversed by RhoA/ROCK inactivation. Thus, the RhoA/ROCK signaling pathway is involved in insulin release through the up-regulation of Cx36 expression in 3D-cultured MIN6 cells.     

High passage MIN6 cells have impaired insulin secretion with impaired glucose and lipid oxidation

Type 2 diabetes is a metabolic disorder characterized by the inability of beta-cells to secrete enough insulin to maintain glucose homeostasis. MIN6 cells secrete insulin in response to glucose and other secretagogues, but high passage (HP) MIN6 cells lose their ability to secrete insulin in response to glucose. We hypothesized that metabolism of glucose and lipids were defective in HP MIN6 cells causing impaired glucose stimulated insulin secretion (GSIS). HP MIN6 cells had no first phase and impaired second phase GSIS indicative of global functional impairment. This was coupled with a markedly reduced ATP content at basal and glucose stimulated states. Glucose uptake and oxidation were higher at basal glucose but ATP content failed to increase with glucose. HP MIN6 cells had decreased basal lipid oxidation. This was accompanied by reduced expressions of Glut1, Gck, Pfk, Srebp1c, Ucp2, Sirt3, Nampt. MIN6 cells represent an important model of beta cells which, as passage numbers increased lost first phase but retained partial second phase GSIS, similar to patients early in type 2 diabetes onset. We believe a number of gene expression changes occurred to produce this defect, with emphasis on Sirt3 and Nampt, two genes that have been implicated in maintenance of glucose homeostasis.     

Novel GPR119 agonist AS1535907 contributes to first-phase insulin secretion in rat perfused pancreas and diabetic db/db mice

G protein-coupled receptor (GPR) 119 is highly expressed in pancreatic β-cells and enhances the effect of glucose-stimulated insulin secretion (GSIS) on activation. The development of an oral GPR119 agonist that specifically targets the first phase of GSIS represents a promising strategy for the treatment of type 2 diabetes. In the present study, we evaluated the therapeutic potential of a novel small molecule GPR119 agonist, AS1535907, which was modified from the previously identified 2,4,6-tri-substituted pyrimidine core agonist AS1269574. AS1535907 displayed an EC₅₀ value of 4.8 μM in HEK293 cells stably expressing human GPR119 and stimulated insulin secretion in rat islets only under high-glucose (16.8 mM) conditions. In isolated perfused pancreata from normal rats, AS1535907 enhanced the first phase of insulin secretion at 16.8 mM glucose, but had no effect at 2.8 mM glucose. In contrast, the sulfonylurea glibenclamide predominantly induced insulin release in the second phase at 16.8 mM glucose and also markedly stimulated insulin secretion at 2.8 mM glucose. In in vivo studies, a single 10 μM administration of AS1535907 to diabetic db/db mice reduced blood glucose levels due to the rapid secretion of insulin secretion following oral glucose loading. These results demonstrate that GPR119 agonist AS1535907 has the ability to stimulate the first phase of GSIS, which is important for preventing the development of postprandial hypoglycemia. In conclusion, the GPR119 agonist AS1535907 induces a more rapid and physiological pattern of insulin release than glibenclamide, and represents a novel strategy for the treatment of type 2 diabetes.     

Ultra-structural study of insulin granules in pancreatic β-cells of db/db mouse by scanning transmission electron microscopy tomography

Insulin granule trafficking is a key step in the secretion of glucose-stimulated insulin from pancreatic β-cells. The main feature of type 2 diabetes (T2D) is the failure of pancreatic β-cells to secrete sufficient amounts of insulin to maintain normal blood glucose levels. In this work, we developed and applied tomography based on scanning transmission electron microscopy (STEM) to image intact insulin granules in the β-cells of mouse pancreatic islets. Using three-dimensional (3D) reconstruction, we found decreases in both the number and the grey level of insulin granules in db/db mouse pancreatic β-cells. Moreover, insulin granules were closer to the plasma membrane in diabetic β-cells than in control cells. Thus, 3D ultra-structural tomography may provide new insights into the pathology of insulin secretion in T2D.     

Group X Secretory Phospholipase A2 Regulates Insulin Secretion through a Cyclooxygenase-2-dependent Mechanism

Group X secretory phospholipase A₂ (GX sPLA₂) potently hydrolyzes membrane phospholipids to release arachidonic acid (AA). While AA is an activator of glucose-stimulated insulin secretion (GSIS), its metabolite prostaglandin E2 (PGE2) is a known inhibitor. In this study, we determined that GX sPLA₂ is expressed in insulin-producing cells of mouse pancreatic islets and investigated its role in beta cell function. GSIS was measured in vivo in wild-type (WT) and GX sPLA₂-deficient (GX KO) mice and ex vivo using pancreatic islets isolated from WT and GX KO mice. GSIS was also assessed in vitro using mouse MIN6 pancreatic beta cells with or without GX sPLA₂ overexpression or exogenous addition. GSIS was significantly higher in islets isolated from GX KO mice compared with islets from WT mice. Conversely, GSIS was lower in MIN6 cells overexpressing GX sPLA₂ (MIN6-GX) compared with control (MIN6-C) cells. PGE2 production was significantly higher in MIN6-GX cells compared with MIN6-C cells and this was associated with significantly reduced cellular cAMP. The effect of GX sPLA₂ on GSIS was abolished when cells were treated with NS398 (a COX-2 inhibitor) or L-798,106 (a PGE2-EP3 receptor antagonist). Consistent with enhanced beta cell function, GX KO mice showed significantly increased plasma insulin levels following glucose challenge and were protected from age-related reductions in GSIS and glucose tolerance compared with WT mice. We conclude that GX sPLA₂ plays a previously unrecognized role in negatively regulating pancreatic insulin secretion by augmenting COX-2-dependent PGE2 production.     

Suppression of NADPH oxidase 2 substantially restores glucose-induced dysfunction of pancreatic NIT-1 cells

Defects in insulin secretion by pancreatic cells and/or decreased sensitivity of target tissues to insulin action are the key features of type 2 diabetes. It has been shown that excessive generation of reactive oxygen species (ROS) is linked to glucose-induced β-cell dysfunction. However, cellular mechanisms involved in ROS generation in β-cells and the link between ROS and glucose-induced β-cell dysfunction are poorly understood. Here, we demonstrate a key role of NADPH oxidase 2 (NOX2)-derived ROS in the deterioration of β-cell function induced by a high concentration of glucose. Sprague-Dawley rats were fed a high-fat diet for 24 weeks to induce diabetes. Diabetic rats showed increased glucose levels and elevated ROS generation in blood, but decreased insulin content in pancreatic β-cells. In vitro, increased ROS levels in pancreatic NIT-1 cells exposed to high concentrations of glucose (33.3 mmol·L(-1)) were associated with elevated expression of NOX2. Importantly, decreased glucose-induced insulin expression and secretion in NIT-1 cells could be rescued via siRNA-mediated NOX2 reduction. Furthermore, high glucose concentrations led to apoptosis of β-cells by activation of p38MAPK and p53, and dysfunction of β-cells through phosphatase and tensih homolog (PTEN)-dependent Jun N-terminal kinase (JNK) activation and protein kinase B (AKT/PKB) inhibition, which induced the translocation of forkhead box O1 and pancreatic duodenal homeobox-1, followed by reduced insulin expression and secretion. In conclusion, NOX2-derived ROS could play a critical role in high glucose-induced β-cell dysfunction through PTEN-dependent JNK activation and AKT inhibition.