Metformin disrupts insulin secretion, causes proapoptotic and oxidative effects in rat pancreatic beta-cells in vitro.

Metformin is the first-line drug to treat type 2 diabetes mellitus. Its mechanism of action is still debatable, and recent studies report that metformin attenuates oxidative stress. This study evaluated the in vitro antioxidant effects of a broad range of metformin concentrations on insulin-producing cells. The cell cycle, metabolism, glucose-stimulated insulin secretion, and cell death were evaluated to determine the biguanide effects on beta-cell function and survival. Antioxidant potential was based on reactive oxygen species (ROS), reduced glutathione (GSH), oxidative stress biomarker levels, and antioxidant enzyme and transcriptional factor Nrf2 activities. The results demonstrate that metformin disrupted GSIS in a concentration-dependent manner, lowered insulin content, and attenuated beta-cell metabolism. At high concentrations, metformin induced cell death and cell cycle arrest as well as increased ROS generation, consequently reducing GSH content. Although carbonylated protein content was elevated, indicating oxidative stress, the antioxidant enzyme and Nrf2 activities were not altered. In conclusion, our results show that metformin disrupts pancreatic beta-cell functionality but does not exert a putative antioxidant effect. It is important to note that the drug could potentially affect beta-cells, especially at high circulating levels.

Early Cytokine-Induced Transient NOX2 Activity Is ER Stress-Dependent and Impacts ß-Cell Function and Survival.

In type 1 diabetes (T1D) development, proinflammatory cytokines (PIC) released by immune cells lead to increased reactive oxygen species (ROS) production in ß-cells. Nonetheless, the temporality of the events triggered and the role of different ROS sources remain unclear. Isolated islets from C57BL/6J wild-type (WT), NOX1 KO and NOX2 KO mice were exposed to a PIC combination. We show that cytokines increase O2•- production after 2 h in WT and NOX1 KO but not in NOX2 KO islets. Using transgenic mice constitutively expressing a genetically encoded compartment specific H2O2 sensor, we show, for the first time, a transient increase of cytosolic/nuclear H2O2 in islet cells between 4 and 5 h during cytokine exposure. The H2O2 increase coincides with the intracellular NAD(P)H decrease and is absent in NOX2 KO islets. NOX2 KO confers better glucose tolerance and protects against cytokine-induced islet secretory dysfunction and death. However, NOX2 absence does not counteract the cytokine effects in ER Ca2+ depletion, Store-Operated Calcium Entry (SOCE) increase and ER stress. Instead, the activation of ER stress precedes H2O2 production. As early NOX2-driven ROS production impacts ß-cells' function and survival during insulitis, NOX2 might be a potential target for designing therapies against early ß-cell dysfunction in the context of T1D onset.

Effects of lixisenatide treatment on mild cachexia and related metabolic abnormalities in Walker-256 tumour-bearing rats.

Lixisenatide, a glucagon-like peptide-1 (GLP-1) receptor agonist, is used in the treatment of type 2 diabetes mellitus (T2DM). It increases insulin (INS) secretion and can decrease INS resistance, improving metabolic disorders in this disease. However, its effects on metabolic disturbances in cancer-bearing, which also exhibit decreased INS secretion and INS resistance, changes that may contribute to weight loss (cachexia), have not yet been evaluated. The purpose of this study was to investigate the lixisenatide treatment effects on mild cachexia and related metabolic abnormalities in Walker-256 tumour-bearing rats. Lixisenatide (50 µg kg-1 , SC) was administered once daily, for 6 days, after inoculation of Walker-256 tumour cells. Acute lixisenatide treatment did not improve hypoinsulinemia, INS secretion and INS resistance of tumour-bearing rats. It also did not prevent the reduced glucose and increased triacylglycerol and lactate in the blood and nor the loss of retroperitoneal and epididymal fat of these animals. However, acute lixisenatide treatment accentuated the body mass loss of tumour-bearing rats. Therefore, lixisenatide, unlike T2DM, does not improve hypoinsulinemia and INS resistance associated with cancer, evidencing that it does not have the same beneficial effects in these two diseases. In addition, lixisenatide aggravated weight loss of tumour-bearing rats, suggesting that its use for treatment of T2DM patients with cancer should be avoided. SIGNIFICANCE OF THE STUDY: Lixisenatide increases insulin secretion and appears to reduce insulin resistance in T2DM. However, lixisenatide treatment does not improve hypoinsulinemia and insulin resistance associated with cancer, as it does in T2DM, and aggravated weight loss, suggesting that its use for treatment of T2DM patients with cancer should be avoided.

Intermittent Fasting for Twelve Weeks Leads to Increases in Fat Mass and Hyperinsulinemia in Young Female Wistar Rats.

Fasting is known to cause physiological changes in the endocrine pancreas, including decreased insulin secretion and increased reactive oxygen species (ROS) production. However, there is no consensus about the long-term effects of intermittent fasting (IF), which can involve up to 24 hours of fasting interspersed with normal feeding days. In the present study, we analyzed the effects of alternate-day IF for 12 weeks in a developing and healthy organism. Female 30-day-old Wistar rats were randomly divided into two groups: control, with free access to standard rodent chow; and IF, subjected to 24-hour fasts intercalated with 24-hours of free access to the same chow. Alternate-day IF decreased weight gain and food intake. Surprisingly, IF also elevated plasma insulin concentrations, both at baseline and after glucose administration collected during oGTT. After 12 weeks of dietary intervention, pancreatic islets displayed increased ROS production and apoptosis. Despite their lower body weight, IF animals had increased fat reserves and decreased muscle mass. Taken together, these findings suggest that alternate-day IF promote ß -cell dysfunction, especially in developing animals. More long-term research is necessary to define the best IF protocol to reduce side effects.

Short-term high glucose culture potentiates pancreatic beta cell function.

The exposure of pancreatic islets to high glucose is believed to be one of the causal factors of the progressive lowering of insulin secretion in the development of type 2 diabetes. The progression of beta cell failure to type 2 diabetes is preceded by an early positive increase in the insulin secretory response to glucose, which is only later followed by a loss in the secretion capacity of pancreatic islets. Here we have investigated the electrophysiological mechanisms underlying the early glucose-mediated gain of function. Rodent pancreatic islets or dispersed islet cells were cultured in medium containing either 5.6 (control) or 16.7 (high-glucose) mM glucose for 24 h after isolation. Glucose-stimulated insulin secretion was enhanced in a concentration-dependent manner in high glucose-cultured islets. This was associated with a positive effect on beta cell exocytotic capacity, a lower basal KATP conductance and a higher glucose sensitivity to fire action potentials. Despite no changes in voltage-gated Ca2+ currents were observed in voltage-clamp experiments, the [Ca2+]I responses to glucose were drastically increased in high glucose-cultured cells. Of note, voltage-dependent K+ currents were decreased and their activation was shifted to more depolarized potentials by high-glucose culture. This decrease in voltage-dependent K+ channel (Kv) current may be responsible for the elevated [Ca2+]I response to metabolism-dependent and independent stimuli, associated with more depolarized membrane potentials with lower amplitude oscillations in high glucose-cultured beta cells. Overall these results show that beta cells improve their response to acute challenges after short-term culture with high glucose by a mechanism that involves modulation not only of metabolism but also of ion fluxes and exocytosis, in which Kv activity appears as an important regulator.

NADPH oxidase-2 does not contribute to ß-cell glucotoxicity in cultured pancreatic islets from C57BL/6J mice.

High glucose-induced oxidative stress and increased NADPH oxidase-2 (NOX2) activity may contribute to the progressive decline of the functional ß-cell mass in type 2 diabetes. To test that hypothesis, we characterized, in islets from male NOX2 knockout (NOX2-KO) and wild-type (WT) C57BL/6J mice cultured for up to 3 weeks at 10 or 30 mmol/l glucose (G10 or G30), the in vitro effects of glucose on cytosolic oxidative stress using probes sensing glutathione oxidation (GRX1-roGFP2), thiol oxidation (roGFP1) or H2O2 (roGFP2-Orp1), on ß-cell stimulus-secretion coupling events and on ß-cell apoptosis. After 1-2 days of culture in G10, the glucose stimulation of insulin secretion (GSIS) was ∼1.7-fold higher in NOX2-KO vs. WT islets at 20-30 mmol/l glucose despite similar rises in NAD(P)H and intracellular calcium concentration ([Ca2+]i) and no differences in cytosolic GRX1-roGFP2 oxidation. After long-term culture at G10, roGFP1 and roGFP2-Orp1 oxidation and ß-cell apoptosis remained low, and the glucose-induced rises in NAD(P)H, [Ca2+]i and GSIS were similarly preserved in both islet types. After prolonged culture at G30, roGFP1 and roGFP2-Orp1 oxidation increased in parallel with ß-cell apoptosis, the glucose sensitivity of the NADPH, [Ca2+]i and insulin secretion responses increased, the maximal [Ca2+]i response decreased, but maximal GSIS was preserved. These responses were almost identical in both islet types. In conclusion, NOX2 is a negative regulator of maximal GSIS in C57BL/6J mouse islets, but it does not detectably contribute to the in vitro glucotoxic induction of cytosolic oxidative stress and alterations of ß-cell survival and function.

Colonic Fermentation of Unavailable Carbohydrates from Unripe Banana and its Influence over Glycemic Control.

The aim of this study was to evaluate the effect of the colonic fermentation of unavailable carbohydrates from unripe banana (mass - UBM - and starch - UBS) over parameters related to glucose and insulin response in rats. Wistar male rats were fed either a control diet, a UBM diet (5 % resistant starch - RS) or a UBS diet (10 % RS) for 28 days. In vivo (oral glucose tolerance test) and in vitro (cecum fecal fermentation, pancreatic islet insulin secretion) analyses were performed. The consumption of UBM and UBS diets by Wistar rats for 28 days improved insulin/glucose ratio. Also, pancreatic islets isolated from the test groups presented significant lower insulin secretion compared to the control group, when the same in vitro glucose stimulation was done. Total short chain fatty acids produced were higher in both experimental groups in relation to the control group. These findings suggest that UBM and UBS diets promote colonic fermentation and can influence glycemic control, improving insulin sensitivity in rats.

Omega-3 supplementation improves pancreatic islet redox status: in vivo and in vitro studies.

OBJECTIVES: The aim of the study was to evaluate the potential changes induced by fish oil (FO) supplementation on the redox status of pancreatic islets from healthy rats. To test whether these effects were due to eicosapentaenoic acid and docosahexaenoic acid (ω-3), in vitro experiments were performed. METHODS: Rats were supplemented with FO, and pancreatic islets were obtained. Islets were also treated in vitro with palmitate (P) or eicosapentaenoic acid + docosahexaenoic acid (ω-3). Insulin secretion (GSIS), glucose oxidation, protein expression, and superoxide content were analyzed. RESULTS: The FO group showed a reduction in superoxide content. Moreover, FO reduced the expression of NAD(P)H oxidase subunits and increased superoxide dismutase, without altering ß-cell function. Palmitate increased ß-cell reactive oxygen species (ROS) production, apoptosis, and impaired GSIS. Under these conditions, ω-3 triggered a parallel reduction in ROS production and ß-cell apoptosis induced by P and protected against the impairment in GSIS. There was no difference in mitochondrial ROS production. CONCLUSIONS: Our results show that ω-3 protect pancreatic islets from alterations induced by P. In vivo FO supplementation modulates the redox state of pancreatic ß-cell. Considering that in vitro effects do not involve mitochondrial superoxide production, we can speculate that this protection might involve NAD(P)H oxidase activity.

Long-term disruption of maternal glucose homeostasis induced by prenatal glucocorticoid treatment correlates with miR-29 upregulation.

Excess of glucocorticoids (GCs) during pregnancy is strongly associated with the programming of glucose intolerance in the offspring. However, the impact of high GC levels on maternal metabolism is not clearly documented. This study aimed to test the hypothesis that mothers exposed to elevated levels of GCs might also display long-term disturbances in glucose homeostasis. Dexamethasone (DEX) was administered noninvasively to the mothers via drinking water between the 14th and the 19th days of pregnancy. Mothers were subjected to glucose and insulin tolerance tests at 1, 2, 3, 6, and 12 mo postweaning. Pregnant rats not treated with DEX and age-matched virgin rats were used as controls. Pancreatic islets were isolated at the 20th day of pregnancy and 12 mo postweaning in order to evaluate glucose-stimulated insulin secretion. The expression of the miR-29 family was also studied due to its responsiveness to GCs and its well-documented role in the regulation of pancreatic ß-cell function. Rats treated with DEX during pregnancy presented long-term glucose intolerance and impaired insulin secretion. These changes correlated with 1) increased expression of miR-29 and its regulator p53, 2) reduced expression of syntaxin-1a, a direct target of miR-29, and 3) altered expression of genes related to cellular senescence. Our data demonstrate that the use of DEX during pregnancy results in deleterious outcomes to the maternal metabolism, hallmarked by reduced insulin secretion and glucose intolerance. This maternal metabolic programming might be a consequence of time-sustained upregulation of miR-29s in maternal pancreatic islets.

Fish oil supplementation for two generations increases insulin sensitivity in rats.

We investigated the effect of fish oil supplementation for two consecutive generations on insulin sensitivity in rats. After the nursing period (21 days), female rats from the same prole were divided into two groups: (a) control group and (b) fish oil group. Female rats were supplemented with water (control) or fish oil at 1 g/kg body weight as a single bolus for 3 months. After this period, female rats were mated with male Wistar rats fed on a balanced chow diet (not supplemented). Female rats continued to receive supplementation throughout gestation and lactation periods. The same treatment was performed for the next two generations (G1 and G2). At 75 days of age, male offspring from G1 and G2 generations from both groups were used in the experiments. G1 rats did not present any difference with control rats. However, G2 rats presented reduction in glycemia and lipidemia and improvement in in vivo insulin sensitivity (model assessment of insulin resistance, insulin tolerance test) as well as in vitro insulin sensitivity in soleus muscle (glucose uptake and metabolism). This effect was associated with increased insulin-stimulated p38 MAP kinase phosphorylation and lower n-6/n-3 fatty acid ratio, but not with activation of proteins from insulin signaling (IR, IRS-1 and Akt). Global DNA methylation was decreased in liver but not in soleus muscle. These results suggest that long-term fish oil supplementation improves insulin sensitivity in association with increased insulin-stimulated p38 activation and decreased n-6:n-3 ratio in skeletal muscle and decreased global DNA methylation in liver.

Diet-induced obesity impairs AKT signalling in the retina and causes retinal degeneration.

Retinopathy, a common complication of diabetes, is characterized by an unbalanced production of nitric oxide (NO), a process regulated by nitric oxide synthase (NOS). We hypothesized that retinopathy might stem from changes in the insulin receptor substrate (IRS)/PI3K/AKT pathway and/or expression of NOS isoforms. Thus, we analysed the morphology and apoptosis index in retinas of obese rats in whom insulin resistance had been induced by a high-fat diet (HFD). Immunoblotting analysis revealed that the retinal tissue of HFD rats had lower levels of AKT(1) , eNOS and nNOS protein than those of samples taken from control animals. Furthermore, immunohistochemical analyses indicated higher levels of iNOS and 4-hydroxynonenal and a larger number of apoptotic nuclei in HFD rats. Finally, both the inner and outer retinal layers of HFD rats were thinner than those in their control counterparts. When considered alongside previous results, these patterns suggest two major ways in which HFD might impact animals: direct activity of ingested fatty acids and/or via insulin-resistance-induced changes in intracellular pathways. We discuss these possibilities in further detail and advocate the use of this animal model for further understanding relationships between retinopathy, metabolic syndrome and type 2 diabetes.

Expression of NADPH oxidase in human pancreatic islets.

AIMS: NADPH oxidase (NOX) is a known source of superoxide anions in phagocytic and non-phagocytic cells. In this study, the presence of this enzyme in human pancreatic islets and the importance of NADPH oxidase in human ß-cell function were investigated. MAIN METHODS AND KEY FINDINGS: In isolated human pancreatic islets, the expression of NADPH oxidase components was evidenced by real-time PCR (p22(PHOX), p47(PHOX) and p67(PHOX)), Western blotting (p47(PHOX) and p67(PHOX)) and immunohistochemistry (p47(PHOX), p67(PHOX) and gp91(PHOX)). Immunohistochemistry experiments showed co-localization of p47(PHOX), p67(PHOX) and gp91(PHOX) (isoform 2 of NADPH oxidase-NOX2) with insulin secreting cells. Inhibition of NADPH oxidase activity impaired glucose metabolism and glucose-stimulated insulin secretion. SIGNIFICANCE: These findings demonstrate the presence of the main intrinsic components of NADPH oxidase comprising the NOX2 isoform in human pancreatic islets, whose activity also contributes to human ß-cell function.

Metabolic disorders and adipose tissue insulin responsiveness in neonatally STZ-induced diabetic rats are improved by long-term melatonin treatment.

Diabetes mellitus is a product of low insulin sensibility and pancreatic ß-cell insufficiency. Rats with streptozotocin-induced diabetes during the neonatal period by the fifth day of age develop the classic diabetic picture of hyperglycemia, hypoinsulinemia, polyuria, and polydipsia aggravated by insulin resistance in adulthood. In this study, we investigated whether the effect of long-term treatment with melatonin can improve insulin resistance and other metabolic disorders in these animals. At the fourth week of age, diabetic animals started an 8-wk treatment with melatonin (1 mg/kg body weight) in the drinking water at night. Animals were then killing, and the sc, epididymal (EP), and retroperitoneal (RP) fat pads were excised, weighed, and processed for adipocyte isolation for morphometric analysis as well as for measuring glucose uptake, oxidation, and incorporation of glucose into lipids. Blood samples were collected for biochemical assays. Melatonin treatment reduced hyperglycemia, polydipsia, and polyphagia as well as improved insulin resistance as demonstrated by constant glucose disappearance rate and homeostasis model of assessment-insulin resistance. However, melatonin treatment was unable to recover body weight deficiency, fat mass, and adipocyte size of diabetic animals. Adiponectin and fructosamine levels were completely recovered by melatonin, whereas neither plasma insulin level nor insulin secretion capacity was improved in diabetic animals. Furthermore, melatonin caused a marked delay in the sexual development, leaving genital structures smaller than those of nontreated diabetic animals. Melatonin treatment improved the responsiveness of adipocytes to insulin in diabetic animals measured by tests of glucose uptake (sc, EP, and RP), glucose oxidation, and incorporation of glucose into lipids (EP and RP), an effect that seems partially related to an increased expression of insulin receptor substrate 1, acetyl-coenzyme A carboxylase and fatty acid synthase. In conclusion, melatonin treatment was capable of ameliorating the metabolic abnormalities in this particular diabetes model, including insulin resistance and promoting a better long-term glycemic control.

Oleic acid modulates metabolic substrate channeling during glucose-stimulated insulin secretion via NAD(P)H oxidase.

Positive acute effects of fatty acids (FA) on glucose-stimulated insulin secretion (GSIS) and reactive oxygen species (ROS) formation have been reported. However, those studies mainly focused on palmitic acid actions, and reports on oleic acid (OA) are scarce. In this study, the effect of physiological OA levels on ß-cell function and the mechanisms involved were investigated. Analyses of insulin secretion, FA and glucose oxidation, and ROS formation showed that, at high glucose concentration, OA treatment increases GSIS in parallel with increased ROS content. At high glucose, OA oxidation was increased, accompanied by a suppression of glucose oxidation. Using approaches for protein knockdown of FA receptor G protein-coupled receptor 40 (GPR40) and of p47(PHOX), a reduced nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase component, we observed that GPR40 does not mediate OA effects on ROS formation and GSIS. However, in p47(PHOX) knockdown islets, OA-induced ROS formation and the inhibitory effect of OA on glucose metabolism was abolished. Similar results were obtained by pharmacological inhibition of protein kinase C, a known activator of NAD(P)H oxidase. Thus, ROS derived from OA metabolism via NAD(P)H oxidase are an inhibitor of glucose oxidation. Put together, these results indicate that OA acts as a modulator of glucose oxidation via ROS derived from its own metabolism in ß-cells.

Regulation of insulin secretion and reactive oxygen species production by free fatty acids in pancreatic islets.

Free fatty acids regulate insulin secretion through metabolic and intracellular signaling mechanisms such as induction of malonyl-CoA/long-chain CoA pathway, production of lipids, GPRs (G protein-coupled receptors) activation and the modulation of calcium currents. Fatty acids (FA) are also important inducers of ROS (reactive oxygen species) production in ß-cells. Production of ROS for short periods is associated with an increase in GSIS (glucose-stimulated insulin secretion), but excessive or sustained production of ROS is negatively correlated with the insulin secretory process. Several mechanisms for FA modulation of ROS production by pancreatic ß-cells have been proposed, such as the control of mitochondrial complexes and electron transport, induction of uncoupling proteins, NADPH oxidase activation, interaction with the renin-angiotensin system, and modulation of the antioxidant defense system. The major sites of superoxide production within mitochondria derive from complexes I and III. The amphiphilic nature of FA favors their incorporation into mitochondrial membranes, altering the membrane fluidity and facilitating the electron leak. The extra-mitochondrial ROS production induced by FA through the NADPH oxidase complex is also an important source of these species in ß-cells.

NAD(P)H oxidase participates in the palmitate-induced superoxide production and insulin secretion by rat pancreatic islets.

Nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase complex has been shown to be involved in the process of glucose-stimulated insulin secretion (GSIS). In this study, we examined the effect of palmitic acid on superoxide production and insulin secretion by rat pancreatic islets and the mechanism involved. Rat pancreatic islets were incubated during 1 h with 1 mM palmitate, 1% fatty acid free-albumin, 5.6 or 10 mM glucose and in the presence of inhibitors of NAD(P)H oxidase (DPI--diphenyleneiodonium), PKC (calphostin C) and carnitine palmitoyl transferase-I (CPT-I) (etomoxir). Superoxide content was determined by hydroethidine assays. Palmitate increased superoxide production in the presence of 5.6 and 10 mM glucose. This effect was dependent on activation of PKC and NAD(P)H oxidase. Palmitic acid oxidation was demonstrated to contribute for the fatty acid induction of superoxide production in the presence of 5.6 mM glucose. In fact, palmitate caused p47(PHOX) translocation to plasma membrane, as shown by immunohistochemistry. Exposure to palmitate for 1 h up-regulated the protein content of p47(PHOX) and the mRNA levels of p22(PHOX), gp91(PHOX), p47(PHOX), proinsulin and the G protein-coupled receptor 40 (GPR40). Fatty acid stimulation of insulin secretion in the presence of high glucose concentration was reduced by inhibition of NAD(P)H oxidase activity. In conclusion, NAD(P)H oxidase is an important source of superoxide in pancreatic islets and the activity of NAD(P)H oxidase is involved in the control of insulin secretion by palmitate.

Low doses of hydrogen peroxide impair glucose-stimulated insulin secretion via inhibition of glucose metabolism and intracellular calcium oscillations.

The inhibitory effect of hydrogen peroxide (H(2)O(2)) on glucose-stimulated insulin secretion was previously reported. However, the precise mechanism involved was not systematically investigated. In this study, the effects of low concentrations of H(2)O(2) (5-10 micromol/L) on glucose metabolism, intracellular calcium ([Ca(2+)](i)) oscillations, and dynamic insulin secretion in rat pancreatic islets were investigated. Low concentrations of H(2)O(2) impaired insulin secretion in the presence of high glucose levels (16.7 mmol/L). This phenomenon was observed already after 2 minutes of exposure to H(2)O(2). Glucose oxidation and the amplitude of [Ca(2+)](i) oscillations were dose-dependently suppressed by H(2)O(2). These findings indicate that low concentrations of H(2)O(2) reduce insulin secretion in the presence of high glucose levels via inhibition of glucose metabolism and consequent impairment in [Ca(2+)](i) handling.

Short-term modulation of extracellular signal-regulated kinase 1/2 and stress-activated protein kinase/c-Jun NH2-terminal kinase in pancreatic islets by glucose and palmitate: possible involvement of ceramide.

OBJECTIVES: The effect of glucose and palmitate on the phosphorylation of proteins associated with cell growth and survival (extracellular signal-regulated kinase 1/2 [ERK1/2] and stress-activated protein kinase/c-Jun NH2-terminal kinase [SAPK/JNK]) and on the expression of immediate early genes was investigated. METHODS: Groups of freshly isolated rat pancreatic islets were incubated in 10-mmol/L glucose with palmitate, LY294002, or fumonisin B1 for the measurement of the phosphorylation and the content of ERK1/2, JNK/SAPK, and v-akt murine thymoma viral oncongene (AKT) (serine 473) by immunoblotting. The expressions of the immediate early genes, c-fos and c-jun, were evaluated by reverse transcription-polymerase chain reaction. RESULTS: Glucose at 10 mmol/L induced ERK1/2 and AKT phosphorylations and decreased SAPK/JNK phosphorylation. Palmitate (0.1 mmol/L) abolished the glucose effect on ERK1/2, AKT, and SAPK/JNK phosphorylations. LY294002 caused a similar effect. The inhibitory effect of palmitate on glucose-induced ERK1/2 and AKT phosphorylation changes was not observed in the presence of fumonisin B1. Glucose increased c-fos and decreased c-jun expressions. Palmitate and LY294002 abolished these latter glucose effects. The presence of fumonisin B1 abolished the effect induced by palmitate on c-jun expression. CONCLUSIONS: Our results suggest that short-term changes of mitogen-activated protein kinase and AKT signaling pathways and c-fos and c-jun expressions caused by glucose are abolished by palmitate through phosphatidylinositol 3-kinase inhibition via ceramide synthesis.

Palmitate activates insulin signaling pathway in pancreatic rat islets.

OBJECTIVE: To investigate the action of palmitate on insulin receptor (IR) signaling pathway in rat pancreatic islets. The following proteins were studied: IR substrate-1 and -2 (IRS1 and IRS2), phosphatidylinositol 3-kinase, extracellular signal-regulated protein kinase-1 and -2 (ERK1/2), and signal transducer and activator of transcription 3 (STAT3). METHODS: Immunoblotting and immunoprecipitation assays were used to evaluate the phosphorylation states of IRS1 and IRS2 (tyrosine [Tyr]), ERK1/2 (threonine 202 [Thr202]/Tyr204), and STAT3 (serine [Ser727]). RESULTS: The exposure of rat pancreatic islets to 0.1-mmol/L palmitate for up to 30 minutes produced a significant increase of Tyr phosphorylation in IRS2 but not in IRS1. The association of phosphatidylinositol 3-kinase with IRS2 was also upregulated by palmitate. Exposure to 5.6-mmol/L glucose caused a gradual decrease in ERK1/2 (Thr202/Tyr204) and STAT3 (serine [Ser727]) phosphorylations after 30-minute incubation. The addition of palmitate (0.1 mmol/L), associated with 5.6-mmol/L glucose, abolished these latter effects of glucose after 15-minute incubation. CONCLUSIONS: Palmitate at physiological concentration associated with 5.6-mmol/L glucose activates IR signaling pathway in pancreatic beta cells.

Involvement of phosphatidylinositol-3 kinase/AKT/PKCzeta/lambda pathway in the effect of palmitate on glucose-induced insulin secretion.

OBJECTIVES: In the present study, a novel pathway by which palmitate potentiates glucose-induced insulin secretion by pancreatic beta cells was investigated. METHODS: Groups of freshly isolated islets were incubated in 10 mM glucose with palmitate, LY294002, wortmannin, and fumonisin B1 for measurement of insulin secretion by radioimmunoassay (RIA). Also, phosphorylation and content of AKT and PKC proteins were evaluated by immunoblotting. RESULTS: Glucose plus palmitate and glucose plus LY294002 or wortmannin (PI3K inhibitors) increased glucose-induced insulin secretion by isolated pancreatic islets. Glucose at 10 mM induced AKT and PKCzeta/lambda phosphorylation. Palmitate (0.1 mM) abolished glucose stimulation of AKT and PKCzeta/lambda phosphorylation possibly through PI3K inhibition because both LY294002 (50 microM) and wortmannin (100 nM) caused the same effect. The inhibitory effect of palmitate on glucose-induced AKT and PKCzeta/lambda phosphorylation and the stimulatory effect of palmitate on glucose-induced insulin secretion were not observed in the presence of fumonisin B1, an inhibitor of ceramide synthesis. CONCLUSIONS: These findings support the proposition that palmitate increases insulin release in the presence of 10 mM glucose by inhibiting PI3K activity through a mechanism that involves ceramide synthesis.