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Objective: To determine the subunit expression and functional activation of phagocyte-like NADPH oxidase (Nox), reactive oxygen species (ROS) generation and caspase-3 activation in the Zucker diabetic fatty (ZDF) rat and diabetic human islets., Research Design and Methods: Expression of core components of Nox was quantitated by Western blotting and densitometry. ROS levels were quantitated by the 2',7'-dichlorofluorescein diacetate method. Rac1 activation was quantitated using the gold-labeled immunosorbent assay kit., Results: Levels of phosphorylated p47(phox), active Rac1, Nox activity, ROS generation, Jun NH(2)-terminal kinase (JNK) 1/2 phosphorylation, and caspase-3 activity were significantly higher in the ZDF islets than the lean control rat islets. Chronic exposure of INS 832/13 cells to glucolipotoxic conditions resulted in increased JNK1/2 phosphorylation and caspase-3 activity; such effects were largely reversed by SP600125, a selective inhibitor of JNK. Incubation of normal human islets with high glucose also increased the activation of Rac1 and Nox. Lastly, in a manner akin to the ZDF diabetic rat islets, Rac1 expression, JNK1/2, and caspase-3 activation were also significantly increased in diabetic human islets., Conclusions: We provide the first in vitro and in vivo evidence in support of an accelerated Rac1-Nox-ROS-JNK1/2 signaling pathway in the islet β-cell leading to the onset of mitochondrial dysregulation in diabetes.

Objective: Reactive oxygen species (ROS) is one of most important factors in impaired metabolism secretion coupling in pancreatic β-cells. We recently reported that elevated ROS production and impaired ATP production at high glucose in diabetic Goto-Kakizaki (GK) rat islets are effectively ameliorated by Src inhibition, suggesting that Src activity is upregulated. In the present study, we investigated whether the glucagon-like peptide-1 signal regulates Src activity and ameliorates endogenous ROS production and ATP production in GK islets using exendin-4., Research Design and Methods: Isolated islets from GK and control Wistar rats were used for immunoblotting analyses and measurements of ROS production and ATP content. Src activity was examined by immunoprecipitation of islet lysates followed by immunoblotting. ROS production was measured with a fluorescent probe using dispersed islet cells., Results: Exendin-4 significantly decreased phosphorylation of Src Tyr416, which indicates Src activation, in GK islets under 16.7 mmol/l glucose exposure. Glucose-induced ROS production (16.7 mmol/l) in GK islet cells was significantly decreased by coexposure of exendin-4 as well as PP2, a Src inhibitor. The Src kinase-negative mutant expression in GK islets significantly decreased ROS production induced by high glucose. Exendin-4, as well as PP2, significantly increased impaired ATP elevation by high glucose in GK islets. The decrease in ROS production by exendin-4 was not affected by H-89, a PKA inhibitor, and an Epac-specific cAMP analog (8CPT-2Me-cAMP) significantly decreased Src Tyr416 phosphorylation and ROS production., Conclusions: Exendin-4 decreases endogenous ROS production and increases ATP production in diabetic GK rat islets through suppression of Src activation, dependently on Epac.

Objective: Our previous studies demonstrated that nutrient regulation of mammalian target of rapamycin (mTOR) signaling promotes regenerative processes in rodent islets but rarely in human islets. Our objective was to extend these findings by using therapeutic agents to determine whether the regulation of glycogen synthase kinase-3 (GSK-3)/beta-catenin and mTOR signaling represent key components necessary for effecting a positive impact on human beta-cell mass relevant to type 1 and 2 diabetes., Research Design and Methods: Primary adult human and rat islets were treated with the GSK-3 inhibitors, LiCl and the highly potent 1-azakenpaullone (1-Akp), and with nutrients. DNA synthesis, cell cycle progression, and proliferation of beta-cells were assessed. Measurement of insulin secretion and content and Western blot analysis of GSK-3 and mTOR signaling components were performed., Results: Human islets treated for 4 days with LiCl or 1-Akp exhibited significant increases in DNA synthesis, cell cycle progression, and proliferation of beta-cells that displayed varying degrees of sensitivity to rapamycin. Intermediate glucose (8 mmol/l) produced a striking degree of synergism in combination with GSK-3 inhibition to enhance bromodeoxyuridine (BrdU) incorporation and Ki-67 expression in human beta-cells. Nuclear translocation of beta-catenin responsible for cell proliferation was found to be particularly sensitive to rapamycin., Conclusions: A combination of GSK-3 inhibition and nutrient activation of mTOR contributes to enhanced DNA synthesis, cell cycle progression, and proliferation of human beta-cells. Identification of therapeutic agents that appropriately regulate GSK-3 and mTOR signaling may provide a feasible and available approach to enhance human islet growth and proliferation.

Objective: The transcription factor nuclear factor-kappaB (NF-kappaB) and the mitogen-activated protein kinases (MAPKs) c-Jun NH(2)-terminal kinase (JNK) 1/2 are known to play decisive roles in cytokine-induced damage of rodent beta-cells. The upstream events by which these factors are activated in response to cytokines are, however, uncharacterized. The aim of the present investigation was to elucidate a putative role of the MAPK kinase kinase-1 (MEKK-1) in cytokine-induced signaling., Research Design and Methods: To establish a functional role of MEKK-1, the effects of transient MEKK-1 overexpression in betaTC-6 cells, achieved by lipofection and cell sorting, and MEKK-1 downregulation in betaTC-6 cells and human islet cells, achieved by diced-small interfering RNA treatment, were studied., Results: We observed that overexpression of wild-type MEKK-1, but not of a kinase dead MEKK-1 mutant, resulted in potentiation of cytokine-induced JNK activation, inhibitor of kappaB (IkappaB) degradation, and cell death. Downregulation of MEKK-1 in human islet cells provoked opposite effects, i.e., attenuation of cytokine-induced JNK and MKK4 activation, IkappaB stability, and a less pronounced NF-kappaB translocation. betaTC-6 cells with a downregulated MEKK-1 expression displayed also a weaker cytokine-induced iNOS expression and lower cell death rates. Also primary mouse islet cells with downregulated MEKK-1 expression were protected against cytokine-induced cell death., Conclusions: MEKK-1 mediates cytokine-induced JNK- and NF-kappaB activation, and this event is necessary for iNOS expression and cell death.

Objective: Protein kinase B/Akt plays a central role in beta-cells, but little is known regarding downstream Akt substrates in these cells. Recently, Rab GTPase-activating protein AS160, a substrate of Akt, was shown to be involved in insulin modulation of GLUT4 trafficking in skeletal muscle and adipose tissue. The aim of this study was to investigate the expression and potential role of AS160 in beta-cells., Research Design and Methods: AS160 mRNA expression was measured in mouse and human islets and fluorescence-activated cell sorted beta-cells and compared in islets from control subjects versus individuals with type 2 diabetes. For knockdown experiments, transformed mouse insulin-secreting MIN6B1 cells were transfected with pSUPER-GFP plasmid encoding a small hairpin RNA against insulin receptor substrate (IRS)-2, AS160, or a negative control. Primary mouse islet cells were transfected with AS160 small interfering RNA., Results: AS160 was expressed in human and mouse pancreatic beta-cells and phosphorylated after glucose stimulation. AS160 mRNA expression was downregulated in pancreatic islets from individuals with type 2 diabetes. In MIN6B1 cells, glucose induced phosphorylation of Akt and AS160, and this was mediated by insulin receptor/IRS-2/phosphatidylinositol 3-kinase independently of changes in cytosolic Ca(2+). Knockdown of AS160 resulted in increased basal insulin secretion, whereas glucose-stimulated insulin release was abolished. Furthermore, beta-cells with decreased AS160 showed increased apoptosis and loss of glucose-induced proliferation., Conclusions: This study shows for the first time that AS160, previously recognized as a key player in insulin signaling in skeletal muscle and adipose tissue, is also a major effector of protein kinase B/Akt signaling in the beta-cell.

The roles played by arachidonic acid and its cyclooxygenase (COX)-generated and lipoxygenase (LOX)-generated metabolites have been studied using rodent islets and insulin-secreting cell lines, but very little is known about COX and LOX isoform expression and the effects of modulation of arachidonic acid generation and metabolism in human islets. We have used RT-PCR to identify mRNAs for cytosolic phospholipase A(2) (cPLA(2)), COX-1, COX-2, 5-LOX, and 12-LOX in isolated human islets. COX-3 and 15-LOX were not expressed by human islets. Perifusion experiments with human islets indicated that PLA(2) inhibition inhibited glucose-stimulated insulin secretion, whereas inhibitors of COX-2 and 12-LOX enzymes enhanced basal insulin secretion and also secretory responses induced by 20 mmol/l glucose or by 50 mumol/l arachidonic acid. Inhibition of COX-1 with 100 mumol/l acetaminophen did not significantly affect glucose-stimulated insulin secretion. These data indicate that the stimulation of insulin secretion from human islets in response to arachidonic acid does not require its metabolism through COX-2 and 5-/12-LOX pathways. The products of COX-2 and LOX activities have been implicated in cytokine-mediated damage of beta-cells, so selective inhibitors of these enzymes would be expected to have a dual protective role in diabetes: they would minimize beta-cell dysfunction while maintaining insulin secretion through enhancing endogenous arachidonic acid levels.

Indoleamine 2,3-dioxygenase (IDO) catalyzes the initial, rate-limiting step of tryptophan (Trp) catabolism along the kynurenine (KYN) pathway, and its induction in cells of the immune system in response to cytokines has been implicated in the regulation of antigen presentation and responses to cell-mediated immune attack. Microarray and quantitative PCR analyses of isolated human islets incubated with interferon (IFN)-gamma for 24 h revealed increased expression of IDO mRNA (>139-fold) and Trp-tRNA synthase (WARS) (>17-fold) along with 975 other transcripts more than threefold, notably the downstream effectors janus kinase (JAK)2, signal transducer and activator of transcription (STAT)1, IFN-gamma regulatory factor-1, and several chemokines (CXCL9/MIG, CXCL10/IP10, CXCL11/1-TAC, CCL2, and CCL5/RANTES) and their receptors. IDO protein expression was upregulated in IFN-gamma-treated islets and accompanied by increased intracellular IDO enzyme activity and the release of KYN into the media. The response to IFN-gamma was countered by interleukin-4 and 1alpha-methyl Trp. Immunohistochemical localization showed IDO to be induced in cells of both endocrine, including pancreatic duodenal homeobox 1-positive beta-cells, and nonendocrine origin. We postulate that in the short term, IDO activation may protect islets from cytotoxic damage, although chronic exposure to various Trp metabolites could equally lead to beta-cell attrition.

The majority of small G-proteins undergo posttranslational modifications (e.g., isoprenylation) at their C-terminal cysteine residues. Such modifications increase their hydrophobicity, culminating in translocation of the modified proteins to their relevant membranous sites for interaction with their respective effectors. Previously, we reported glucose-dependent activation and membrane association of Rac1 in INS 832/13 cells. We also demonstrated modulatory roles for Rac1/GDP dissociation inhibitor in glucose-stimulated insulin secretion (GSIS) in INS 832/13 cells, further affirming roles for Rac1 in GSIS. Herein, we demonstrate that geranylgeranyltransferase inhibitor-2147 (GGTI-2147), an inhibitor of protein prenylation, markedly increased cytosolic accumulation of Rac1 and elicited significant inhibition of GSIS from INS 832/13 cells. In the current study, we also examined the localization of protein prenyltransferases (PPTases) and regulation of GSIS by PPTases in INS 832/13 cells. Western blot analyses indicated that the regulatory alpha-subunit and the structural beta-subunit of PPTase holoenzyme are predominantly cytosolic in their distribution. Overexpression of an inactive mutant of the regulatory alpha-subunit of PPTase markedly attenuated glucose- but not KCl-induced insulin secretion from INS 832/13 cells. Together, our findings provide the first evidence for the regulation of GSIS by PPTase in INS 832/13 cells. Furthermore, they support our original hypothesis that prenylation of specific G-proteins may be necessary for GSIS.

Islet amyloid, formed by aggregation of islet amyloid polypeptide (IAPP; amylin), is a pathological characteristic of the pancreas in type 2 diabetes and may contribute to the progressive loss of beta-cells in this disease. We tested the hypothesis that impaired processing of the IAPP precursor proIAPP contributes to amyloid formation and cell death. GH3 cells lacking the prohormone convertase 1/3 (PC1/3) and IAPP and with very low levels of prohormone convertase 2 (PC2) were transduced with adenovirus (Ad) expressing human or rat (control) proIAPP linked to green fluorescent protein, with or without Ad-PC2 or Ad-PC1/3. Expression of human proIAPP increased the number of transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells 96 h after transduction (+hIAPP 8.7 +/- 0.4% vs. control 3.0 +/- 0.4%; P < 0.05). COOH-terminal processing of human proIAPP by PC1/3 increased (hIAPP+PC1/3 10.4 +/- 0.7%; P < 0.05), whereas NH(2)-terminal processing of proIAPP by addition of PC2 markedly decreased (hIAPP+PC2 5.5 +/- 0.5%; P < 0.05) the number of apoptotic GH3 cells. Islets from mice lacking PC2 and with beta-cell expression of human proIAPP (hIAPP(+/+)/PC2(-/-)) developed amyloid associated with beta-cell death during 2-week culture. Rescue of PC2 expression by ex vivo transduction with Ad-PC2 restored NH(2)-terminal processing to mature IAPP and decreased both the extent of amyloid formation and the number of TUNEL-positive cells (-PC2 26.5 +/- 4.1% vs. +PC2 16.1 +/- 4.3%; P < 0.05). These findings suggest that impaired NH(2)-terminal processing of proIAPP leads to amyloid formation and cell death and that accumulation of the NH(2)-terminally extended human proIAPP intermediate may be a critical initiating step in amyloid formation.

The ubiquitin-proteasome pathway is crucial for protein turnover. Part of the pathway involves deubiquitination, which is carried out by cystein proteases known as ubiquitin COOH-terminal hydrolases. The isoform Uch-L1 was found to be present in large amounts in rat islets by immunostaining, Western blot analysis, and RT-PCR. Culturing islets in high glucose concentrations (16.7 mmol/l) for 24 h led to decreased gene expression. Exposure to chronic hyperglycemia following 90% partial pancreatectomy also led to reduced Uch-L1 expression. Expression of other members of the ubiquitin-proteasome pathway studied after culturing islets at high glucose concentrations revealed little change except for modest declines in parkin, human ubiquitin-conjugating enzyme 5 (UbcH5), and beta-TRCP (transducin repeat-containing protein). With the pancreatectomy model, expression of polyubiquitin-B and c-Cbl were increased and E6-associated protein was reduced. Further insight about the proteasome pathway was obtained with the proteasome inhibitor lactacystin, which in short-term 2-h experiments enhanced glucose-induced insulin secretion. An important role for the ubiquitin-proteasome pathways in beta-cells is suggested by the findings that changes in glucose concentration influence expression of genes in the pathway and that blockade of the proteasome degradation machinery enhances glucose-stimulated insulin secretion.

Hydrogen sulfide (H(2)S) was historically recognized as a toxic gas generated by natural resources. However, its enzymatic production from L-cysteine has recently been demonstrated in mammals. Cystathionine beta-synthase and cystathionine gamma-lyase, both of which can produce H(2)S, were expressed in mouse pancreatic islet cells and the beta-cell line, MIN6. L-cysteine and the H(2)S donor NaHS inhibited glucose-induced insulin release from islets and MIN6 cells. These inhibitory effects were reproduced when insulin release was stimulated by alpha-ketoisocaproate, tolbutamide, or high K+. L-cysteine and NaHS inhibited glucose-potentiated insulin release in the copresence of diazoxide and high K+. Real-time imaging of intracellular Ca2+ concentration ([Ca2+](i)) demonstrated that both L-cysteine and NaHS reversibly suppressed glucose-induced [Ca2+](i) oscillation in a single beta-cell without obvious changes in the mean value. These substances inhibited Ca2+ - or guanosine 5'-0-3-thiotriphosphate-induced insulin release from islets permeabilized with streptolysin-O. L-cysteine and NaHS reduced ATP production and attenuated glucose-induced hyperpolarization of the mitochondrial membrane potential. Finally, L-cysteine increased H(2)S content in MIN6 cells. We suggest here that L-cysteine inhibits insulin release via multiple actions on the insulin secretory process through H(2)S production. Because the activities of H(2)S-producing enzymes and the tissue H(2)S contents are known to increase under diabetic conditions, the inhibition may participate in the deterioration of insulin release in this disease.

Glucose sensing is essential for the ability of pancreatic beta-cells to produce insulin in sufficient quantities to maintain blood glucose within the normal range. Stress causes the release of adrenergic hormones that increase circulating glucose by promoting glucose production and inhibiting insulin release. We have shown that extracellular signal-regulated kinases 1 and 2 (ERK1/2) are responsive to glucose in pancreatic beta-cells and that glucose activates ERK1/2 by mechanisms independent of insulin. Here we show that glucose-induced activation of ERK1/2 is inhibited by epinephrine through the alpha2-adrenergic receptor. Epinephrine and the selective alpha2-adrenergic agonist UK14304 reduced insulin secretion and glucose-stimulated ERK1/2 activation in a pertussis toxin-sensitive manner, implicating the alpha subunit of a Gi family member. Alpha2-adrenergic agonists also reduced stimulation of ERK1/2 by glucagon-like peptide 1 and KCl, but not by phorbol ester or nerve growth factor. Our findings suggest that alpha2-adrenergic agonists act via a Gi family member on early steps in ERK1/2 activation, supporting the idea that ERK1/2 are regulated in a manner that reflects insulin demand.

As the rate-limiting controller of glucose metabolism, glucokinase represents the primary beta-cell "glucose sensor." Inactivation of both glucokinase (GK) alleles results in permanent neonatal diabetes; inactivation of a single allele causes maturity-onset diabetes of the young type 2 (MODY-2). Similarly, mice lacking both alleles (GK(-/-)) exhibit severe neonatal diabetes and die within a week, whereas heterozygous GK(+/-) mice exhibit markedly impaired glucose tolerance and diabetes, resembling MODY-2. Glucose metabolism increases the cytosolic [ATP]-to-[ADP] ratio, which closes ATP-sensitive K(+) channels (K(ATP) channels), leading to membrane depolarization, Ca(2+) entry, and insulin exocytosis. Glucokinase insufficiency causes defective K(ATP) channel regulation, which may underlie the impaired secretion. To test this prediction, we crossed mice lacking neuroendocrine glucokinase (nGK(+/-)) with mice lacking K(ATP) channels (Kir6.2(-/-)). Kir6.2 knockout rescues perinatal lethality of nGK(-/-), although nGK(-/-)Kir6.2(-/-) animals are postnatally diabetic and still die prematurely. nGK(+/-) animals are diabetic on the Kir6.2(+/+) background but only mildly glucose intolerant on the Kir6.2(-/-) background. In the presence of glutamine, isolated nGK(+/-)Kir6.2(-/-) islets show improved insulin secretion compared with nGK(+/-)Kir6.2(+/+). The significant abrogation of nGK(-/-) and nGK(+/-) phenotypes in the absence of K(ATP) demonstrate that a major factor in glucokinase deficiency is indeed altered K(ATP) signaling. The results have implications for understanding and therapy of glucokinase-related diabetes.

A graded relationship has been reported between fasting and postprandial plasma glucose levels and the subsequent risk of cardiovascular morbidity and mortality. We hypothesized that the GCK -30G>A promoter polymorphism is associated with elevated glycemia in the middle-aged general population of whites, as well as with features of the World Health Organization (WHO)-defined metabolic syndrome. The GCK -30G>A polymorphism was genotyped in the population-based Inter99 study cohort (5,965 subjects) and in 332 nondiabetic subjects and 1,063 patients with type 2 diabetes. In the Inter99 cohort, the GCK -30A allele was associated with increased fasting (P < 0.001) and post-oral glucose tolerance test (OGTT) plasma glucose levels (P < 0.001), and in the same cohort, the GCK -30A allele was more frequent among 1,325 subjects with the metabolic syndrome than among 1,679 subjects without any components of the metabolic syndrome (P = 0.002). Moreover, the GCK -30A allele frequency was higher among 2,587 subjects with impaired glucose regulation (IGR) than among 4,773 glucose-tolerant subjects (17.3% [95% CI 16.2-18.3] vs. 15.0% [14.3-15.7], P < 0.001, odds ratio GG vs. GA 1.21 [1.08-1.36], GG vs. AA 1.62 [1.17-2.24]). In conclusion, the GCK -30G>A polymorphism associates with elevated fasting and post-OGTT glycemia in the middle-aged general population of whites, as well as with IGR and other features of the WHO-defined metabolic syndrome.

Reactive oxygen species (ROS) and nitric oxide (NO) are proposed mediators of cytokine-induced beta-cell destruction in type 1 diabetes. We produced transgenic mice with increased beta-cell expression of manganese superoxide dismutase (MnSOD) and catalase. Expression of these antioxidants increased beta-cell ROS scavenging and improved beta-cell survival after treatment with different sources of ROS. MnSOD or catalase conferred protection against streptozotocin (STZ)-induced beta-cell injury. Coexpression of MnSOD and catalase provided synergistic protection against peroxynitrite and STZ. To determine the potential effect of these antioxidants on cytokine-induced toxicity, we exposed isolated islets to a cytokine mixture, including interleukin-1beta and interferon-gamma. Cytokine toxicity was measured as reduced metabolic activity after 6 days and reduced insulin secretion after 1 day. Cytokines increased ROS production, and both antioxidants were effective in reducing cytokine-induced ROS. However, MnSOD and/or catalase provided no protection against cytokine-induced injury. To understand this, the nuclear factor-kappaB (NF-kappaB) signaling cascade was investigated. Antioxidants reduced NF-kappaB activation by ROS, but none of the antioxidants altered activation by cytokines, as measured by inhibitor of kappaB phosphorylation, NF-kappaB translocation, inducible NO synthase activation, and NO production. Our data agree with previous reports that antioxidants benefit beta-cell survival against ROS damage, but they are not consistent with reports that antioxidants reduce cytokine toxicity. ROS appear to have no role in cytokine toxicity in primary beta-cells.

To shed further light on the primary alterations of insulin secretion in type 2 diabetes and the possible mechanisms involved, we studied several functional and molecular properties of islets isolated from the pancreata of 13 type 2 diabetic and 13 matched nondiabetic cadaveric organ donors. Glucose-stimulated insulin secretion from type 2 diabetic islets was significantly lower than from control islets, whereas arginine- and glibenclamide-stimulated insulin release was less markedly affected. The defects were accompanied by reduced mRNA expression of GLUT1 and -2 and glucokinase and by diminished glucose oxidation. In addition, AMP-activated protein kinase activation was reduced. Furthermore, the expression of insulin was decreased, and that of pancreatic duodenal homeobox-1 (PDX-1) and forkhead box O1 (Foxo-1) was increased. Nitrotyrosine and 8-hydroxy-2'-deoxyguanosine concentrations, markers of oxidative stress, were significantly higher in type 2 diabetic than control islets, and they were correlated with the degree of glucose-stimulated insulin release impairment. Accordingly, 24-h exposure to glutathione significantly improved glucose-stimulated insulin release and decreased nitrotyrosine concentration, with partial recovery of insulin mRNA expression. These results provide direct evidence that the defects of insulin secretion in type 2 diabetic islets are associated with multiple islet cell alterations. Most importantly, the current study shows that the functional impairment of type 2 diabetic islets can be, at least in part, reversible. In this regard, it is suggested that reducing islet cell oxidative stress is a potential target of human type 2 diabetes therapy.

Rare mutations in the glucokinase (GCK) gene cause fasting hyperglycemia and considerably influence birth weight when present in a mother or her offspring. The role of common variation of GCK is uncertain. A polymorphism at position -30 of the GCK beta-cell-specific promoter, present in 30% of the population, has been variably associated with type 2 diabetes and diabetes-related quantitative traits. Using 1,763 U.K. Caucasian normoglycemic adult subjects, we demonstrated that the A allele at GCK(-30) is associated with a 0.06-mmol/l increase in fasting plasma glucose (FPG) (P = 0.003). The A allele was also associated with an increase in FPG in 755 women who were 28 weeks pregnant (0.075 mmol/l, P = 0.003). We then went on to analyze the effect of GCK(-30) on birth weight using 2,689 mother/child pairs. The presence of the A allele in the mother was associated with a 64-g (25-102 g) increase in offspring birth weight (P = 0.001). We did not detect a fetal genotype effect. The increase in offspring birth weight in the 30% of mothers carrying an A allele at GCK(-30) is likely to reflect an elevated FPG during pregnancy. This study establishes that common genetic variation, in addition to rare mutations and environmental factors, can affect both FPG and birth weight.

The membrane receptor FAT/CD36 facilitates the major fraction of long-chain fatty acid (FA) uptake by muscle and adipose tissues. In line with the well-known effects of FA metabolism on carbohydrate utilization and insulin responsiveness, altered expression of CD36 has been linked to phenotypic features of the metabolic syndrome including insulin resistance and dyslipidemia. FA metabolism is also known to significantly affect insulin secretion. However, the role of CD36 in this process remains unknown, since its expression levels and function in the pancreas have not been explored. In the present study, freshly isolated human islets and a mouse-derived beta-cell line (MIN6) were shown positive for CD36 expression by RT-PCR, Western blot, and immunofluorescence. The identity of the PCR product was confirmed by microsequencing. The identified transcript was translated and the protein was expressed and subjected to the known posttranslational glycosylation. Fluorescence resonance energy transfer analysis and subcellular protein fractionation indicated that insulin and CD36 are colocalized in the secretory granules of beta-cells. Islet CD36 functioned in FA uptake because this process was blocked by the irreversible CD36 inhibitor sulfosuccinimidyl-oleate. More importantly, sulfosuccinimidyl-oleate reversed enhancing and inhibiting effects, respectively, of acute and long-term palmitate incubations on glucose-dependent insulin secretion. In conclusion, our study demonstrates that human islets express CD36 in the plasma membrane as well as in the insulin secretory granules. CD36 activity appears important for uptake of FA into beta-cells as well as for mediating their modulatory effects on insulin secretion.

Cytokines and free radicals are mediators of beta-cell death in type 1 diabetes. Under in vitro conditions, interleukin-1beta (IL-1beta) + gamma-interferon (IFN-gamma) induce nitric oxide (NO) production and apoptosis in rodent and human pancreatic beta-cells. We have previously shown, by microarray analysis of primary beta-cells, that IL-1beta + IFN-gamma decrease expression of the mRNA encoding for the sarcoendoplasmic reticulum pump Ca(2+) ATPase 2b (SERCA2b) while inducing expression of the endoplasmic reticulum stress-related and proapoptotic gene CHOP (C/EBP [CCAAT/enhancer binding protein] homologous protein). In the present study we show that cytokine-induced apoptosis and necrosis in primary rat beta-cells and INS-1E cells largely depends on NO production. IL-1beta + IFN-gamma, via NO synthesis, markedly decreased SERCA2b protein expression and depleted ER Ca(2+) stores. Of note, beta-cells showed marked sensitivity to apoptosis induced by SERCA blockers, as compared with fibroblasts. Cytokine-induced ER Ca(2+) depletion was paralleled by an NO-dependent induction of CHOP protein and activation of diverse components of the ER stress response, including activation of inositol-requiring ER-to-nucleus signal kinase 1alpha (IRE1alpha) and PRK (RNA-dependent protein kinase)-like ER kinase (PERK)/activating transcription factor 4 (ATF4), but not ATF6. In contrast, the ER stress-inducing agent thapsigargin triggered these four pathways in parallel. In conclusion, our results suggest that the IL-1beta + IFN-gamma-induced decrease in SERCA2b expression, with subsequent depletion of ER Ca(2+) and activation of the ER stress pathway, is a potential contributory mechanism to beta-cell death.

The association of glucokinase with insulin secretory granules has been shown by cell microscopy techniques. We used MIN6 insulin-secretory cells and organelle fractionation to determine the effects of glucose on the subcellular distribution of glucokinase. After permeabilization with digitonin, 50% of total glucokinase remained bound intracellularly, while 30% was associated with the 13,000g particulate fraction. After density gradient fractionation of the organelles, immunoreactive glucokinase was distributed approximately equally between dense insulin granules and low-density organelles that cofractionate with mitochondria. Although MIN6 cells show glucose-responsive insulin secretion, glucokinase association with the granules and low-density organelles was not affected by glucose. Subfractionation of the insulin granule components by hypotonic lysis followed by sucrose gradient centrifugation showed that glucokinase colocalized with the granule membrane marker phogrin and not with insulin. PFK2 (6-phosphofructo-2-kinase-2/fructose-2,6-bisphosphatase)/FDPase-2, a glucokinase-binding protein, and glyceraldehyde phosphate dehydrogenase, which has been implicated in granule fusion, also colocalized with glucokinase after hypotonic lysis or detergent extaction of the granules. The results suggest that glucokinase is an integral component of the granule and does not translocate during glucose stimulation.

Insulin-producing cells are known for their extremely low antioxidant equipment with hydrogen peroxide (H(2)O(2))-inactivating enzymes. Therefore, catalase was stably overexpressed in mitochondria and for comparison in the cytoplasmic compartment of insulin-producing RINm5F cells and analyzed for its protective effect against toxicity of reactive oxygen species (ROS) and proinflammatory cytokines. Only mitochondrial overexpression of catalase provided protection against menadione toxicity, a chemical agent that preferentially generates superoxide radicals intramitochondrially. On the other hand, the cytoplasmic catalase overexpression provided better protection against H(2)O(2) toxicity. Mitochondrial catalase overexpression also preferentially protected against the toxicity of interleukin-1beta (IL-1beta) and a proinflammatory cytokine mixture (IL-1beta, tumor necrosis factor-alpha [TNF-alpha], and gamma-interferon [IFN-gamma]) that is more toxic than IL-1beta alone. Thus, it can be concluded that targeted overexpression of catalase in the mitochondria provides particularly effective protection against cell death in all situations in which ROS are generated intramitochondrially. The observed higher rate of cell death after exposure to a cytokine mixture in comparison with the weaker effect of IL-1beta alone may be due to an additive toxicity of TNF-alpha through ROS formation in mitochondria. The results emphasize the central role of mitochondrially generated ROS in the cytokine-mediated cell destruction of insulin-producing cells.

Cytosolic phospholipase A(2) (cPLA(2)) comprises a widely expressed family of enzymes, some members of which have the properties required of signal transduction elements in electrically excitable cells. Thus, alpha- and beta-isoforms of cPLA(2) are activated by the increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) achieved in depolarized cells. Activation is associated with a redistribution of the enzyme within the cell; activation of cPLA(2) generates arachidonic acid (AA), a biologically active unsaturated fatty acid that can be further metabolized to generate a plethora of biologically active molecules. Studies using relatively nonselective pharmacological inhibitors have implicated cPLA(2) in insulin secretory responses to stimuli that elevate beta-cell [Ca(2+)](i); therefore, we have investigated the role of cPLA(2) in beta-cell function by generating beta-cell lines that under- or overexpress the alpha-isoform of cPLA(2). The functional phenotype of the modified cells was assessed by observation of cellular ultrastructure, by measuring insulin gene expression and insulin protein content, and by measuring the effects of insulin secretagogues on cPLA(2) distribution, on changes in [Ca(2+)](i), and on the rate and pattern of insulin secretion. Our results suggest that cPLA(2) is not required for the initiation of insulin secretion from beta-cells, but that it plays an important role in the maintenance of beta-cell insulin stores. Our data also demonstrate that excessive production of, or exposure to, AA is deleterious to normal beta-cell secretory function through metabolic dysfunction.

Cyclo-oxygenase (COX), the enzyme responsible for conversion of arachidonic acid to prostanoids, exists as two isoforms. In most tissues, COX-1 is a constitutive enzyme involved in prostaglandin-mediated physiological processes, whereas COX-2 is thought to be induced by inflammatory stimuli. However, it has previously been reported that COX-2 is the dominant isoform in islets and an insulin-secreting beta-cell line under basal conditions. We have investigated the relative abundance of COX-1 and COX-2 mRNAs in MIN6 cells, a mouse insulin-secreting cell line, and in primary mouse and human islets. We found that COX-2 was the dominant isoform in MIN6 cells, but that COX-1 mRNA was more abundant than that of COX-2 in freshly isolated mouse islets. Furthermore, COX-2 expression was induced by maintenance of mouse islets in culture, and experiments with human islets indicated that exposure of the islets to hyperglycemic conditions was sufficient to upregulate COX-2 mRNA levels. Given that hyperglycemia has been reported to increase human beta-cell production of interleukin-1beta and that this cytokine can induce COX-2 expression, our observations of glucose-induced induction of COX-2 in human islets suggest that this is one route through which hyperglycemia may contribute to beta-cell dysfunction.

Changes in intracellular Ca(2+) concentration play a key role in the regulation of insulin secretion by glucose and other secretagogues. Here, we explore the importance of the secretory pathway Ca(2+)-ATPase, plasma membrane-related Ca(2+)-ATPase-1 (PMR1; human orthologue ATP2C1) in intracellular Ca(2+) homeostasis in pancreatic islet beta-cells. Endogenous PMR1 mRNA and protein were detected in both isolated rat islets and beta-cell-derived lines (MIN6 and INS1). Subcellular fractionation of the cell lines revealed PMR1 immunoreactivity in both microsomal and dense-core secretory vesicle-enriched fractions. Correspondingly, depletion of cellular PMR1 with small interfering RNAs inhibited Ca(2+) uptake into the endoplasmic reticulum and secretory vesicles by approximately 20%, as assessed using organelle-targeted aequorins in permeabilized INS1 cells. In intact cells, PMR1 depletion markedly enhanced flux though L-type Ca(2+) channels and augmented glucose-stimulated, but not basal, insulin secretion. Whereas average cytosolic [Ca(2+)] increases in response to 30.0 mmol/l glucose were unaffected by PMR1 depletion, [Ca(2+)] oscillation shape, duration, and decay rate in response to glucose plus tetraethylammonium were modified in PMR1-depleted single cells, imaged using fluo-3-acetoxymethylester. PMR1 thus plays an important role, which is at least partially nonoverlapping with that of sarco(endo-)plasmic reticulum Ca(2+)-ATPases, in the control of beta-cell Ca(2+) homeostasis and insulin secretion.

The beta-isoform of group VIA calcium-independent phospholipase A(2) (iPLA(2)beta) does not require calcium for activation, is stimulated by ATP, and is sensitive to inhibition by a bromoenol lactone suicide substrate. Several potential functions have been proposed for iPLA(2)beta. Our studies indicate that iPLA(2)beta is expressed in beta-cells and participates in glucose-stimulated insulin secretion but is not involved in membrane phospholipid remodeling. If iPLA(2)beta plays a signaling role in glucose-stimulated insulin secretion, then conditions that impair iPLA(2)beta functions might contribute to the diminished capacity of beta-cells to secrete insulin in response to glucose, which is a prominent characteristic of type 2 diabetes. Our recent studies suggest that iPLA(2)beta might also participate in beta-cell proliferation and apoptosis and that various phospholipid-derived mediators are involved in these processes. Detailed characterization of the iPLA(2)beta protein level reveals that beta-cells express multiple isoforms of the enzyme, and our studies involve the hypothesis that different isoforms have different functions.

Evidence that group VIA cytosolic calcium-independent phospholipase A(2) (iPLA(2)beta) participates in beta-cell signal transduction includes the observations that inhibition of iPLA(2)beta with the bromoenol lactone suicide substrate suppresses glucose-stimulated insulin secretion and that overexpression of iPLA(2)beta amplifies insulin secretory responses in INS-1 insulinoma cells. Immunofluorescence analyses also reveal that iPLA(2)beta accumulates in the perinuclear region of INS-1 cells stimulated with glucose and forskolin. To characterize this phenomenon further, iPLA(2)beta was expressed as a fusion protein with enhanced green fluorescent protein (EGFP) in INS-1 cells so that movements of iPLA(2)beta are reflected by changes in the subcellular distribution of green fluorescence. Stimulation of INS-1 cells overexpressing iPLA(2)beta-EGFP induced greater insulin secretion and punctate accumulation of iPLA(2)beta-EGFP fluorescence in the perinuclear region. To determine the identity of organelles with which iPLA(2)beta might associate, colocalization of green fluorescence with fluorophores associated with specific trackers targeted to different subcellular organelles was examined. Such analyses reveal association of iPLA(2)beta-EGFP fluorescence with the ER and Golgi compartments. Arachidonate-containing plasmenylethanolamine phospholipid species are abundant in beta-cell endoplasmic reticulum (ER) and are excellent substrates for iPLA(2)beta. Arachidonic acid produced by iPLA(2)beta-catalyzed hydrolysis of their substrates induces release of Ca(2+) from ER stores-an event thought to participate in glucose-stimulated insulin secretion.

The role of protein kinase C (PKC) in glucose-stimulated insulin secretion (GSIS) is controversial. Using recombinant adenoviruses for overexpression of PKC alpha and PKC delta, in both wild-type (WT) and kinase-dead (KD) forms, we here demonstrate that activation of these two PKCs is neither necessary nor sufficient for GSIS from batch-incubated, rat pancreatic islets. In contrast, responses to the pharmacologic activator 12-O-tetradecanoylphorbol-13-acetate (TPA) were reciprocally modulated by overexpression of the PKC alpha WT or PKC alpha KD but not the corresponding PKC delta adenoviruses. The kinetics of the secretory response to glucose (monitored by perifusion) were not altered in either cultured islets overexpressing PKC alpha KD or freshly isolated islets stimulated in the presence of the conventional PKC (cPKC) inhibitor Go6976. However, the latter did inhibit the secretory response to TPA. Using phosphorylation state-specific antisera for consensus PKC phosphorylation sites, we also showed that (compared with TPA) glucose causes only a modest and transient functional activation of PKC (maximal at 2-5 min). However, glucose did promote a prolonged (15 min) phosphorylation of PKC substrates in the presence of the phosphatase inhibitor okadaic acid. Overall, the results demonstrate that glucose does stimulate PKC alpha in pancreatic islets but that this makes little overall contribution to GSIS.

Lipids may serve as coupling factors in K(ATP)-independent glucose sensing in beta-cells. We have previously demonstrated that beta-cells harbor lipase activities, one of which is the hormone-sensitive lipase. Whether beta-cell lipases are critical for glucose-stimulated insulin secretion (GSIS) by providing lipid-derived signals from endogenous lipids is unknown. Therefore, using a lipase inhibitor (orlistat), we examined whether lipase inhibition reduces insulin secretion. Islet lipolysis stimulated by glucose and diglyceride lipase activity was abolished by orlistat. Incubation of rat islets with orlistat dose dependently inhibited GSIS; this inhibition was reversed by 1 mmol/l palmitate, suggesting that orlistat acts via impaired formation of an acylglyceride-derived coupling signal. Orlistat inhibited the potentiating effect of forskolin on GSIS, an effect proposed to be due to activation of a lipase. In perifused islets, orlistat attenuated mainly the second phase of insulin secretion. Because the rise in islet ATP/ADP levels in response to glucose and oxidation of the sugar were unaffected by orlistat whereas the second phase of insulin secretion was reduced, it seems likely that a lipid coupling factor involved in K(ATP)-independent glucose sensing has been perturbed. Thus, beta-cell lipase activity is involved in GSIS, emphasizing the important role of beta-cell lipid metabolism for insulin secretion.

Islet amyloid polypeptide (IAPP) (amylin), the major component of islet amyloid, is produced by cleavage at the COOH- and NH(2)-termini of its precursor, proIAPP, likely by the beta-cell prohormone convertases (PC) 1/3 and PC2. Mice lacking PC2 can process proIAPP at its COOH- but not its NH(2)-terminal cleavage site, suggesting that PC1/3 is capable of initiating proIAPP cleavage at its COOH-terminus. To determine the precise role of PC1/3 in proIAPP processing, Western blot analysis was performed on islets isolated from mice lacking PC1/3 (PC1/3(-/-)). These islets contained not only fully processed IAPP as in PC1/3(+/+) islets, but also elevated levels of a COOH-terminally unprocessed intermediate form, suggesting impaired processing at the COOH-terminus. Next, GH3 cells that do not normally express proIAPP or detectable levels of PC1/3 or PC2 were cotransduced with adenoviruses expressing rat proIAPP and either PC2 or PC1/3. As expected, in GH3 cells transduced to express only proIAPP, no processing was observed. Coexpression of proIAPP and PC2 resulted in production of mature IAPP, whereas in cells that coexpressed proIAPP and PC1/3 only a 6-kDa intermediate was produced. We conclude that PC1/3 is important for processing of proIAPP at the COOH-terminus, but in its absence, PC2 can initiate complete processing of proIAPP to IAPP by cleaving the precursor at either its NH(2)- or COOH-terminal cleavage sites.

Protein tyrosine phosphatase-1b (Ptp1b) inhibits insulin and leptin signaling by dephosphorylating specific tyrosine residues in their activated receptor complexes. Insulin signals are mediated by tyrosine phosphorylation of the insulin receptor and its downstream targets, such as Irs1 and Irs2. Irs2 plays an especially important role in glucose homeostasis because it mediates some peripheral actions of insulin and promotes pancreatic beta-cell function. To determine whether the deletion of Ptp1b compensates for the absence of Irs2, we analyzed mice deficient in both Ptp1b and Irs2. Pancreatic beta-cell area decreased in Ptp1b(-/-) mice, consistent with decreased insulin requirements owing to increased peripheral insulin sensitivity. By contrast, peripheral insulin sensitivity and beta-cell area increased in Irs2(-/-)::Ptp1b(-/-) mice, which improved glucose tolerance in Irs2(-/-)::Ptp1b(-/-) mice and delayed diabetes until 3 months of age. However, beta-cell function eventually failed to compensate for absence of Irs2. Our studies demonstrate a novel role for Ptp1b in regulating beta-cell homeostasis and indicate that Ptp1b deficiency can partially compensate for lack of Irs2.

Parathyroid hormone-related protein (PTHrP) increases the content and mRNA level of insulin in a mouse beta-cell line, MIN6, and primary-cultured mouse islets. We examined the mechanism of PTHrP-induced insulin expression. The PTHrP effect was markedly augmented by SB203580, a mitogen-activated protein (MAP) kinase inhibitor, and SB203580 itself increased insulin expression extensively, even without PTHrP. Because SB203580 inhibits both p38 and c-jun NH(2)-terminal kinases (JNKs), we investigated the JNK-specific inhibitor SP600125. SP600125 also increased insulin content and its mRNA level. PTHrP induced dephosphorylation of JNK1/2, and PTHrP-induced insulin expression was blocked by a dominant-negative type JNK-APF. We suspected that dual specificity MAP kinase phosphatases (MKPs) may be involved in the PTHrP-induced insulin expression by inactivating JNK1/2. MIN6 cells contained at least five MKPs, among which only MKP-1 was inducible by PTHrP. PTHrP-induced insulin expression was blocked by the MKP-1 expression inhibitor Ro-31-8220, indicating that the PTHrP effect is mediated by MKP-1. Indeed, adenoviral MKP-1 expression increased insulin expression by decreasing a phosphorylation form of JNKs and a resulting phosphorylated form of c-jun in MIN6 cells. The phosphorylated form of c-jun is known to repress cAMP-dependent insulin gene promoter activity. Thus, MKP-1 controls the insulin expression by downregulating a JNK/c-jun pathway.

Free radical formation evoked by proinflammatory cytokines has been suggested to be involved in the destruction of beta-cells in the course of type 1 diabetes development. However, there is no direct evidence to support this hypothesis. In this study, we used electron paramagnetic resonance spectroscopy in conjunction with spin-trapping methodology to directly determine whether cytokines give rise to free radical formation in the islets. Our results demonstrate that direct, in vivo administration of tumor necrosis factor-alpha (1,000 units), interleukin-1beta (1,000 units), and interferon-gamma (2,000 units) into the rat pancreas through a bile duct cannula leads to the formation of lipid-derived free radicals in this tissue. These free radicals most likely are generated by the beta-cells because previous depletion of these cells by streptozotocin abolished the cytokine-induced free radical formation. Furthermore, macrophage depletion was found to decrease the production of free radicals. Inhibition of the enzyme inducible cyclooxygenase (COX-2) and the transcription factor nuclear factor-kappaB (NF-kappaB) significantly diminished the free radicals' signal intensity, implicating these factors in the formation of free radicals. We have also demonstrated that cytokine treatment leads to the activation of NF-kappaB in the pancreatic islets of the rats.

Lipid perturbations associated with triglyceride overstorage in beta-cells impair insulin secretion, a process termed lipotoxicity. To assess the role of hormone-sensitive lipase, which is expressed and enzymatically active in beta-cells, in the development of lipotoxicity, we generated transgenic mice overexpressing hormone-sensitive lipase specifically in beta-cells. Transgenic mice developed glucose intolerance and severely blunted glucose-stimulated insulin secretion when challenged with a high-fat diet. As expected, both lipase activity and forskolin-stimulated lipolysis was increased in transgenic compared with wild-type islets. This was reflected in significantly lower triglycerides levels in transgenic compared with wild-type islets in mice receiving the high-fat diet, whereas no difference in islet triglycerides was found between the two genotypes under low-fat diet conditions. Our results highlight the importance of mobilization of the islet triglyceride pool in the development of beta-cell lipotoxicity. We propose that hormone-sensitive lipase is involved in mediating beta-cell lipotoxicity by providing ligands for peroxisome proliferator-activated receptors and other lipid-activated transcription factors, which in turn alter the expression of critical genes. One such gene might be uncoupling protein-2, which was found to be upregulated in transgenic islets, a change that was accompanied by decreased ATP levels.

The presence of a phagocyte-like NAD(P)H oxidase in pancreatic beta-cells was investigated. Three NAD(P)H oxidase components were found in pancreatic islets by RT-PCR: gp91(PHOX), p22(PHOX), and p47(PHOX). The components p67(PHOX) and p47(PHOX) were also demonstrated by Western blotting. Through immunohistochemistry, p47(PHOX) was mainly found in the central area of the islet, confirming the expression of this component by insulin-producing cells. Activation of NAD(P)H oxidase complex in the beta-cells was also examined by immunohistochemistry. The pancreatic islets presented slower kinetics of superoxide production than HIT-T15 cells, neutrophils, and macrophages, but they reached 66% that of the neutrophil nitroblue tetrazolium (NBT) reduction after 2 h of incubation. Glucose (5.6 mmol/l) increased NBT reduction by 75% when compared with control. The involvement of protein kinase C (PKC) in the stimulatory effect of glucose was confirmed by incubation of islets with phorbol myristate acetate (a PKC activator) and bysindoylmaleimide (GF109203X) (a PKC-specific inhibitor). Diphenylene iodonium [an NAD(P)H oxidase inhibitor] abolished the increase of NBT reduction induced by glucose, confirming the NAD(P)H oxidase activity in pancreatic islets. Because reactive oxygen species are involved in intracellular signaling, the phagocyte-like NAD(P)H oxidase activation by glucose may play an important role for beta-cell functioning.

Glucose can activate the mitogen-activated kinases, Erk-1/2, and the ribosomal-S6 kinase, p70(S6K), in beta-cells, contributing to an increase in mitogenesis. However, the signaling mechanism by which glucose induces Erk-1/2 and p70(S6K) phosphorylation activation is undefined. Increased glucose metabolism increases [Ca(2+)](i) and [cAMP], and it was investigated if these secondary signals were linked to glucose-induced Erk-1/2 and p70(S6K) activation in pancreatic beta-cells. Blocking Ca(2+) influx with verapamil, or inhibiting protein kinase A (PKA) with H89, prevented glucose-induced Erk-1/2 phosphorylation. Increasing cAMP levels by GLP-1 potentiated glucose-induced Erk-1/2 phosphorylation via PKA activation. Elevation of [Ca(2+)](i) by glyburide potentiated Erk-1/2 phosphorylation, which was also inhibited by H89, suggesting increased [Ca(2+)](i) preceded PKA for glucose-induced Erk-1/2 activation. Adenoviral-mediated expression of dominant negative Ras in INS-1 cells decreased IGF-1-induced Erk-1/2 phosphorylation but had no effect on that by glucose. Collectively, our study indicates that a glucose-induced rise in [Ca(2+)](i) leads to cAMP-induced activation of PKA that acts downstream of Ras and upstream of the MAP/Erk kinase, MEK, to mediate Erk-1/2 phosphorylation via phosphorylation activation of Raf-1. In contrast, glucose-induced p70(S6K) activation, in the same beta-cells, was mediated by a distinct signaling pathway independent of Ca(2+)/cAMP, most likely via mTOR-kinase acting as an "ATP-sensor."

Insulin resistance as well as pancreatic beta-cell failure can be induced by elevated free fatty acid (FFA) levels. We studied the mechanisms of FFA-induced apoptosis in rat and human beta-cells. Chronic treatment with high physiological levels of saturated fatty acids (palmitate and stearate), but not with monounsaturated (palmitoleate and oleate) or polyunsaturated fatty acids (linoleate), triggers apoptosis in approximately 20% of cultured RIN1046-38 cells. Apoptosis restricted to saturated FFAs was also observed in primary cultured human beta-cells, suggesting that this mechanism is potentially relevant in vivo in humans. To further analyze FFA-induced signaling pathways leading to apoptosis, we used RIN1046-38 cells. Apoptosis was accompanied by a rapid (within 15 min) nuclear translocation of protein kinase C (PKC)-delta and subsequent lamin B1 disassembly. This translocation was impaired by the phospholipase C inhibitor U-73122, which also substantially reduced apoptosis. Furthermore, lamin B1 disassembly and apoptosis were decreased by cell transfection with a dominant-negative mutant form of PKC-delta. These data suggest that nuclear translocation and kinase activity of PKC-delta are both necessary for saturated fatty acid-induced apoptosis.

Glucokinase (GK) gene transcription initiates in the islet (beta-cell), gut, and brain from promoter sequences residing approximately 35 kbp upstream from those used in liver. Expression of betaGK is controlled in beta-cells by cell-enriched (i.e. pancreatic duodenal homeobox 1 [PDX-1]) and ubiquitously (i.e., Pal) distributed factors that bind to and activate from conserved sequence motifs within the upstream promoter region (termed betaGK). Here, we show that a conserved E-box element also contributes to control in the islet and gut. betaGK promoter-driven reporter gene activity was diminished by mutating the specific sequences involved in E-box-mediated basic helix-loop-helix factor activator binding in islet beta-cells and enteroendocrine cells. Gel shift assays demonstrated that the betaGK and insulin gene E-box elements formed the same cell-enriched (BETA2:E47) and generally distributed (upstream stimulatory factor [USF]) protein-DNA complexes. betaGK E-box-driven activity was stimulated in cotransfection assays performed in baby hamster kidney (BHK) cells with BETA2 and E47, but not USF. Chromatin immunoprecipitation assays performed with BETA2 antisera showed that BETA2 occupies the upstream promoter region of the endogenous betaGK gene in beta-cells. We propose that BETA2 (also termed NeuroD1) regulates betaGK promoter activity.

Glucose is the main physiological secretagogue for insulin secretion by pancreatic beta-cells, and the major biochemical mechanisms involved have been elucidated. In particular, an increase in intracellular calcium is important for insulin exocytosis. More recently, it has become apparent that the beta-cell also has many of the elements of the insulin receptor signal transduction pathway, including the insulin receptor and insulin receptor substrate (IRS) proteins 1 and 2. Studies with transgenic models have shown that the beta-cell-selective insulin receptor knockout and the IRS-1 knockout lead to reduced glucose-induced insulin secretion. Overexpression of the insulin receptor and IRS-1 in beta-cells results in increased insulin secretion and increased cytosolic Ca(2+). We have thus postulated the existence of a novel autocrine-positive feedback loop of insulin on its own secretion involving interaction with the insulin receptor signal transduction pathway and regulation of intracellular calcium homeostasis. Our current working hypothesis is that this glucose-dependent interaction occurs at the level of IRS-1 and the sarco(endo)plasmic reticulum calcium ATPase, the calcium pump of the endoplasmic reticulum.

Glucokinase (GK) serves as glucose sensor in pancreatic beta-cells and in other glucose sensor cells in the body. Biochemical genetic studies have characterized many activating and inactivating GK mutants that have been discovered in patients with hyperinsulinemic hypoglycemia or diabetes, all inherited as autosomal dominant traits. Mathematical modeling of the kinetic data of recombinant human wild-type and mutant GK accurately predicts the effects of GK mutations on the threshold of glucose-stimulated insulin release and glucose homeostasis. Structure/function studies of the enzyme suggest the existence of a hitherto unknown allosteric activator site of the enzyme that has significant implications for the physiological chemistry of GK-containing cells, particularly the pancreatic beta-cells. Glucose is the preeminent positive regulator of beta-cell GK expression and involves molecular mechanisms that are still to be elucidated in detail, but seem to have a specific requirement for increased glucose metabolism. Pharmaceutical chemists, motivated by the clear tenets of the GK glucose-sensor paradigm, have searched for and have discovered a novel class of GK activator molecules. The therapeutic application of this basic discovery offers a new principle for drug therapy of diabetes.

Two sarcoendoplasmic reticulum Ca(2+)-ATPases, SERCA3 and SERCA2b, are expressed in pancreatic islets. Immunocytochemistry showed that SERCA3 is restricted to beta-cells in the mouse pancreas. Control and SERCA3-deficient mice were used to evaluate the role of SERCA3 in beta-cell cytosolic-free Ca(2+) concentration ([Ca(2+)](c)) regulation, insulin secretion, and glucose homeostasis. Basal [Ca(2+)](c) was not increased by SERCA3 ablation. Stimulation with glucose induced a transient drop in basal [Ca(2+)](c) that was suppressed by inhibition of all SERCAs with thapsigargin (TG) but unaffected by selective SERCA3 ablation. Ca(2+) mobilization by acetylcholine was normal in SERCA3-deficient beta-cells. In contrast, [Ca(2+)](c) oscillations resulting from intermittent glucose-stimulated Ca(2+) influx and [Ca(2+)](c) transients induced by pulses of high K(+) were similarly affected by SERCA3 ablation or TG pretreatment of control islets; their amplitude was increased and their slow descending phase suppressed. This suggests that, during the decay of each oscillation, the endoplasmic reticulum releases Ca(2+) that was pumped by SERCA3 during the upstroke phase. SERCA3 ablation increased the insulin response of islets to 15 mmol/l glucose. However, basal and postprandial plasma glucose and insulin concentrations in SERCA3-deficient mice were normal. In conclusion, SERCA2b, but not SERCA3, is involved in basal [Ca(2+)](c) regulation in beta-cells. SERCA3 becomes operative when [Ca(2+)](c) rises and is required for normal [Ca(2+)](c) oscillations in response to glucose. However, a lack of SERCA3 is insufficient in itself to alter glucose homeostasis or impair insulin secretion in mice.

IA-2 and phogrin are tyrosine phosphatase-like proteins that may mediate interactions between secretory granules and cytoskeleton in islets and neuroendocrine tissues. We investigated factors that regulate IA-2 and phogrin expression and their relationship to maturation of insulin secretory responses that occur after birth. Islet content of IA-2, but not phogrin, increased during the first 10 days of life in rats, when insulin secretion in response to glucose increased to adult levels. In cultured 5-day-old rat islets, IA-2 protein and mRNA was increased by glucose and agents that potentiate insulin secretion by the cAMP pathway. Addition of insulin increased IA-2 protein levels and insulin biosynthesis without affecting IA-2 mRNA. Blocking insulin secretion with diazoxide or insulin action with insulin receptor antibodies inhibited glucose-induced increases in IA-2 protein, but not those of mRNA. Phogrin expression was unchanged by all agents. Thus, IA-2 is regulated at the mRNA level by glucose and elevated cAMP, whereas locally secreted insulin modulates IA-2 protein levels by stimulating biosynthesis. In contrast, phogrin expression is insensitive to factors that modify beta-cell function. These results demonstrate differential regulation of two closely related secretory granule components and identify IA-2 as a granule membrane protein subject to autocrine regulation by insulin.

Mammalian target of rapamycin (mTOR) is a serine and threonine protein kinase that regulates numerous cellular functions, in particular, the initiation of protein translation. mTOR-mediated phosphorylation of both the translational repressor eukaryotic initiation factor 4E binding protein-1 and p70 S6 kinase are early events that control the translation initiation process. Rapamycin, an inhibitor of mTOR, is a potent immunosuppressant due, in part, to its ability to interfere with T-cell activation at the level of translation, and it has gained a prominent role in preventing the development and progression of rejection in pancreatic islet transplant recipients. The characterization of the insulin signaling cascade that modulates mTOR in insulin-sensitive tissues has been a major focus of investigation. Recently, the ability of nutrients, in particular the branched-chain amino acid leucine, to activate mTOR independent of insulin by a process designated as nutrient signaling has been identified. The beta-cell expresses components of the insulin signaling cascade and utilizes the metabolism of nutrients to affect insulin secretion. These combined transduction processes make the beta-cell an unique cell to study metabolic and autocrine regulation of mTOR signaling. Our studies have described the ability of insulin and IGFs in concert with the nutrients leucine, glutamine, and glucose to modulate protein translation through mTOR in beta-cells. These findings suggest that mitochondria-derived factors, ATP in particular, may be responsible for nutrient signaling. The significance of these findings is that the optimization of mitochondrial function is not only important for insulin secretion but may significantly impact the growth and proliferation of beta-cells through these mTOR signaling pathways.

Ca(2+) may trigger programmed cell death (apoptosis) and regulate death-specific enzymes. Therefore, the development of strategies to control Ca(2+) homeostasis may represent a potential approach to prevent or enhance cell apoptosis. To test this hypothesis, the plasma membrane Na/Ca exchanger (NCX1.7 isoform) was stably overexpressed in insulin-secreting tumoral cells. NCX1.7 overexpression increased apoptosis induced by endoplasmic reticulum (ER) Ca(2+)-ATPase inhibitors, but not by agents increasing intracellular calcium concentration ([Ca(2+)](i)), through the opening of plasma membrane Ca(2+)-channels. NCX1.7 overexpression reduced the rise in [Ca(2+)](i) induced by all agents, depleted ER Ca(2+) stores, sensitized the cells to Ca(2+)-independent proapoptotic signaling pathways, and reduced cell proliferation by approximately 40%. ER Ca(2+) stores depletion was accompanied by the activation of the ER-specific caspase (caspase-12), and the activation was enhanced by ER Ca(2+)-ATPase inhibitors. Hence, Na/Ca exchanger overexpression, by depleting ER Ca(2+) stores, triggers the activation of caspase-12 and increases apoptotic cell death. By increasing apoptosis and decreasing cell proliferation, overexpression of Na/Ca exchanger may represent a new potential approach in cancer gene therapy. On the other hand, our results open the way to the development of new strategies to control cellular Ca(2+) homeostasis that could, on the contrary, prevent the process of apoptosis that mediates, in part, beta-cell autoimmune destruction in type 1 diabetes.

In an effort to better understand the phenomenon of lipotoxicity in human beta-cells, we evaluated the effects of 48-h preculture with 1.0 or 2.0 mmol/l free fatty acid (FFA) (2:1 oleate to palmitate) on the function and survival of isolated human islets and investigated some of the possible mechanisms. Compared with control islets, triglyceride content was significantly increased and insulin content and glucose-stimulated insulin release were significantly reduced in islets precultured with increased FFA concentrations. These changes were accompanied by a significant reduction of glucose utilization and oxidation. By cell death detection techniques, it was observed that exposure to FFAs induced a significant increase of the amount of dead cells. Electron microscopy showed the involvement of beta-cells, with morphological appearance compatible with the presence of apoptotic phenomena. FFA-induced islet cell death was blocked by inhibition of upstream caspases and partially prevented by inhibiton of ceramide synthesis or serine protease activity, whereas inhibition of nitric oxide synthesis had no effect. RT-PCR studies revealed no major change of iNOS and Bax mRNA expression and a marked decrease of Bcl-2 mRNA expression in the islets cultured with FFA. Thus, prolonged exposure to FFAs has cytostatic and pro-apoptotic effects on human pancreatic beta-cells. The cytostatic action is likely to be due to the FFA-induced reduction of intraislet glucose metabolism, and the proapoptotic effects are mostly caspase mediated, partially dependent on ceramide pathway, and possibly Bcl-2 regulated.

Thiazolidinediones, synthetic ligands of peroxisomal proliferator-activated receptor-gamma (PPAR-gamma), improve peripheral insulin sensitivity and glucose-stimulated insulin secretion in pancreatic beta-cells. To explore the role of PPAR-gamma in glucose sensing of beta-cells, we have dissected the beta-cell-specific glucokinase (betaGK) promoter, which constitutes glucose-sensing apparatus in pancreatic beta-cells, and identified a peroxisomal proliferator response element (PPRE) in the promoter. The betaGK-PPRE is located in the region between +47 and +68 bp. PPAR-gamma/retinoid X receptor-alpha heterodimer binds to the element and activates the betaGK promoter. The betaGK promoter lacking or having mutations in PPRE cannot be activated by PPAR-gamma. PPAR-gamma activates the betaGK promoter in beta-cells as well as non-beta-cells. Furthermore, troglitazone increases endogenous GK expression and its enzyme activity in beta-cell lines. These results indicate that PPAR-gamma can regulate GK expression in beta-cells. Taking these results together with our previous work, we conclude that PPAR-gamma regulates gene expression of glucose-sensing apparatus and thereby improves glucose-sensing ability of beta-cells, contributing to the restoration of beta-cell function in type 2 diabetic subjects by troglitazone.

A full biphasic insulin response is the most sensitive index for well-coupled beta-cell signal transduction. While first-phase insulin response is extremely sensitive to potentiating and inhibiting modulations, full expression of second-phase response requires near maximally activated beta-cell fuel metabolism. In the isolated rat pancreas, accelerated calcium entry or activation of protein kinase (PK)-A or PKC result in no insulin response in the absence of fuel metabolism. At submaximal levels of beta-cell fuel secretagogue, arginine (which promotes calcium entry) or glucagon (which activates PKA) produces a small first-phase insulin response but minimal or no second-phase response; carbachol (which activates PKC and promotes calcium entry) generates biphasic insulin response in the presence of minimal fuel (3.3 mmol/l glucose). Glucagon produces full biphasic response in the presence of 10.0 mmol/l glucose, whereas arginine requires near-maximal stimulatory glucose (16.7 mmol) to produce full biphasic insulin response. Thus, PKA and PKC signal pathways potentiate primary signals generated by fuel secretagogues to induce full biphasic insulin response, while calcium recruitment alone is insufficient to potentiate primary signals generated at low levels of fuel secretagogue. We suggest that three families of PKs (calmodulin-dependent PK [CaMK], PKA, and PKC) function as distal amplifiers for stimulus-secretion coupling signals originating from fuel metabolism, as well as from incretins acting through membrane receptors, adenylate cyclase, and phospholipase C. Several isoenzymes of PKA and PKC are present in pancreatic beta-cells, but the specific function of most is still undefined. Each PK isoenzyme is activated and subsequently phosphorylates its specific effector protein by binding to a highly specific anchoring protein. Some diabetes-related beta-cell derangements may be linked to abnormal function of one or more PK isoenzymes. Identification and characterization of the specific function of the individual PK isoenzymes may provide the tool to improve the insulin response of the diabetic patient.

Cytosolic phospholipase A(2) (cPLA(2)) is a Ca(2+)-sensitive enzyme that has been implicated in insulin secretion in response to agents that elevate beta-cell intracellular Ca(2+) ([Ca(2+)](i)). We generated clones of the MIN6 beta-cell line that stably underexpress cPLA(2) by transfection with a vector in which cPLA(2) cDNA had been inserted in the antisense orientation. Reduced expression of cPLA(2) was confirmed by Western blotting. The insulin content of cPLA(2)-deficient MIN6 cells was reduced by approximately 90%, but they showed no decrease in preproinsulin mRNA expression. Measurements of stimulus-dependent changes in [Ca(2+)](i) indicated that reduced expression of cPLA(2) did not affect the capacity of MIN6 cells to show elevations in Ca(2+) in response to depolarizing stimuli. Perifusion experiments indicated that cPLA(2) underexpressing MIN6 pseudoislets responded to glucose, tolbutamide, and KCl with insulin secretory profiles similar to those of cPLA(2) expressing pseudoislets, but that secretion was not maintained with continued stimulus. Analysis of the ultrastructure of cPLA(2)-deficient MIN6 cells by electron microscopy revealed that they contained very few mature insulin secretory granules, but there was an abundance of non-electron-dense vesicles. These data are consistent with a role for cPLA(2) in the maintenance of insulin stores, but they suggest that it is not required for the initiation of insulin secretion from beta-cells.

The role of phosphatidylinositol (PI) 3-kinase in the regulation of pancreatic beta-cell function was investigated. PI 3-kinase activity in p85 alpha regulatory subunit-deficient (p85 alpha(-/-)) islets was decreased to approximately 20% of that in wild-type controls. Insulin content and mass of rough endoplasmic reticula were decreased in beta-cells from p85 alpha(-/-) mice with increased insulin sensitivity. However, p85 alpha(-/-) beta-cells exhibited a marked increase in the insulin secretory response to higher concentrations of glucose. When PI 3-kinase in wild-type islets was suppressed by wortmannin or LY294002, the secretion was also substantially potentiated. Wortmannin's potentiating effect was not due to augmentation in glucose metabolism or cytosolic [Ca(2+)] elevation. Results of p85 alpha(-/-) islets and wortmannin-treated wild-type islets stimulated with diazoxide and KCl showed that inhibition of PI 3-kinase activity exerted its effect on secretion, at least in part, distal to a cytosolic [Ca(2+)] elevation. These results suggest that PI 3-kinase activity normally plays a crucial role in the suppression of glucose-stimulated insulin secretion.

The pyruvate dehydrogenase complex (PDC) has a pivotal role in islet metabolism. The pyruvate dehydrogenase kinases (PDK1-4) regulate glucose oxidation through inhibitory phosphorylation of PDC. Starvation increases islet PDK activity (Am J Physiol Endocrinol Metab 270:E988-E994, 1996). In this study, using antibodies against PDK1, PDK2, and PDK4 (no sufficiently specific antibodies are as yet available for PDK3), we identified the PDK isoform profile of the pancreatic islet and delineated the effects of starvation (48 h) on protein expression of individual PDK isoforms. Rat islets were demonstrated to contain all three PDK isoforms, PDK1, PDK2, and PDK4. Using immunoblot analysis with antibodies raised against the individual recombinant PDK isoforms, we demonstrated increased islet protein expression of PDK4 in response to starvation (2.3-fold; P < 0.01). Protein expression of PDK1 and PDK2 was suppressed in response to starvation (by 27% [P < 0.01] and 10% [NS], respectively). We demonstrated that activation of peroxisome proliferator-activated receptor-alpha (PPAR-alpha) by the selective agonist WY14,643 for 24 h in vivo leads to specific upregulation of islet PDK4 protein expression by 1.8-fold (P < 0.01), in the absence of change in islet PDK1 and PDK2 protein expression but in conjunction with a 2.2-fold increase (P < 0.01) in islet PPAR-alpha protein expression. Thus, although no changes in islet PPAR-alpha expression were observed after the starvation protocol, activation of PPAR-alpha in vivo may be a potential mechanism underlying upregulation of islet PDK4 protein expression in starvation. We evaluated the effects of antecedent changes in PDK profile and/or PPAR-alpha activation induced by starvation or PPAR-alpha activation in vivo on glucose-stimulated insulin secretion (GSIS) in isolated islets. GSIS at 20 mmol/l glucose was modestly impaired on incubation with exogenous triglyceride (1 mmol/l triolein) ( approximately 20% inhibition; P < 0.05) in islets from fed rats. Starvation (48 h) impaired GSIS in the absence of triolein (by 57%; P < 0.001), but GSIS after the further addition of triolein did not differ significantly between islets from fed or starved rats. GSIS by islets prepared from WY14,643-treated fed rats did not differ significantly from that seen with islets from control fed rats, and the response to triolein addition resembled that of islets prepared from fed rather than starved rats. PPAR-alpha activation in vivo led to increased insulin secretion at low glucose concentrations. Our results are discussed in relation to the potential impact of changes in islet PDK profile on the insulin secretory response to lipid and of PPAR-alpha activation in the cause of fasting hyperinsulinemia.