Prolonged suppression of insulin release by insulin-induced hypoglycemia: demonstration by C-peptide assay.

Pancreatic beta cells secrete the proinsulin connecting peptide (C-peptide) and insulin on an equimolar basis. The C-peptide can thus be used as an indicator of endogenous insulin secretion in the presence of exogenously administered insulin. Using this approach, we have shown suppression of endogenous insulin release in healthy subjects during hypoglycemia induced by intravenous infusion of porcine insulin. Moreover, the suppression persists after the plasma glucose returns to fasting levels, suggesting that the recovery of beta cells from the effects of hypoglycemia is not immediate.     

Protein markers for insulin-producing beta cells with higher glucose sensitivity

Background and Methodology

Pancreatic beta cells show intercellular differences in their metabolic glucose sensitivity and associated activation of insulin production. To identify protein markers for these variations in functional glucose sensitivity, rat beta cell subpopulations were flow-sorted for their level of glucose-induced NAD(P)H and their proteomes were quantified by label-free data independent alternate scanning LC-MS. Beta cell-selective proteins were also identified through comparison with rat brain and liver tissue and with purified islet alpha cells, after geometrical normalization using 6 stably expressed reference proteins.

Principal Findings

All tissues combined, 943 proteins were reliably quantified. In beta cells, 93 out of 467 quantifiable proteins were uniquely detected in this cell type; several other proteins presented a high molar abundance in beta cells. The proteome of the beta cell subpopulation with high metabolic and biosynthetic responsiveness to 7.5 mM glucose was characterized by (i) an on average 50% higher expression of protein biosynthesis regulators such as 40S and 60S ribosomal constituents, NADPH-dependent protein folding factors and translation elongation factors; (ii) 50% higher levels of enzymes involved in glycolysis and in the cytosolic arm of the malate/aspartate-NADH-shuttle. No differences were noticed in mitochondrial enzymes of the Krebs cycle, beta-oxidation or respiratory chain.

Conclusions

Quantification of subtle variations in the proteome using alternate scanning LC-MS shows that beta cell metabolic glucose responsiveness is mostly associated with higher levels of glycolytic but not of mitochondrial enzymes.     

Impaired glucose homeostasis in mice lacking the alpha1b-adrenergic receptor subtype

To assess the role of the alpha1b-adrenergic receptor (AR) in glucose homeostasis, we investigated glucose metabolism in knockout mice deficient of this receptor subtype (alpha1b-AR-/-). Mutant mice had normal blood glucose and insulin levels, but elevated leptin concentrations in the fed state. During the transition to fasting, glucose and insulin blood concentrations remained markedly elevated for at least 6 h and returned to control levels after 24 h whereas leptin levels remained high at all times. Hyperinsulinemia in the post-absorptive phase was normalized by atropine or methylatropine indicating an elevated parasympathetic activity on the pancreatic beta cells, which was associated with increased levels of hypothalamic NPY mRNA. Euglycemic clamps at both low and high insulin infusion rates revealed whole body insulin resistance with reduced muscle glycogen synthesis and impaired suppression of endogenous glucose production at the low insulin infusion rate. The liver glycogen stores were 2-fold higher in the fed state in the alpha1b-AR-/- compared with control mice, but were mobilized at the same rate during the fed to fast transition or following glucagon injections. Finally, high fat feeding for one month increased glucose intolerance and body weight in the alpha1b-AR-/-, but not in control mice. Altogether, our results indicate that in the absence of the alpha1b-AR the expression of hypotalamic NPY and the parasympathetic nervous activity are both increased resulting in hyperinsulinemia and insulin resistance as well as favoring obesity and glucose intolerance development during high fat feeding.     

Glucose-stimulated insulin secretion does not require activation of pyruvate dehydrogenase: impact of adenovirus-mediated overexpression of PDH kinase and PDH phosphate phosphatase in pancreatic islets

Glucose-stimulated increases in mitochondrial metabolism are generally thought to be important for the activation of insulin secretion. Pyruvate dehydrogenase (PDH) is a key regulatory enzyme, believed to govern the rate of pyruvate entry into the citrate cycle. We show here that elevated glucose concentrations (16 or 30 vs 3 mM) cause an increase in PDH activity in both isolated rat islets, and in a clonal beta-cell line (MIN6). However, increases in PDH activity elicited with either dichloroacetate, or by adenoviral expression of the catalytic subunit of pyruvate dehydrogenase phosphatase, were without effect on glucose-induced increases in mitochondrial pyridine nucleotide levels, or cytosolic ATP concentration, in MIN6 cells, and insulin secretion from isolated rat islets. Similarly, the above parameters were unaffected by blockade of the glucose-induced increase in PDH activity by adenovirus-mediated over-expression of PDH kinase (PDK). Thus, activation of the PDH complex plays an unexpectedly minor role in stimulating glucose metabolism and in triggering insulin release.     

Nitric oxide stimulates insulin release in liver cells expressing human insulin

The establishment of surrogate islet beta cells is important for the treatment of diabetes. Hepatocytes have a similar glucose sensing system as beta cells and have the potential to serve as surrogate beta cells. In this report, we demonstrate that infection of Hepa1-6 liver cells with a lentivirus expressing the human insulin cDNA results in expression and secretion of human insulin. Furthermore, we show that l-arginine at low levels of glucose significantly stimulates the release of insulin from these cells, compared to exposure to high concentration of glucose. The arginine-induced insulin release is via the production of nitric oxide, since treatment with N(G)-nitro-l-arginine, an inhibitor of nitric oxide synthase, blocks insulin secretion induced by l-arginine. These results indicate that nitric oxide plays a role in l-arginine-stimulated insulin release in hepatocytes expressing the human insulin gene, and provides a new strategy to induce insulin secretion from engineered non-beta cells.     

Simulated hyperglycemia in rat cardiomyocytes: a proteomics approach for improved analysis of cellular alterations

Diabetic hyperglycemia can lead to stress-related cellular apoptosis of cardiac tissue. However, the mechanism by which hyperglycemia inflicts this damage on the structure and function of the heart is unclear. In this study, we examined the relationship between proteome alterations, mitochondrial function, and major biochemical and electrophysiological changes affecting cardiac performance during simulated short-term hyperglycemia. Two-dimensional comparative proteomics analysis of rat hearts perfused with glucose at high (30 mM) or control (5.5 mM) levels revealed that glucose loading alters cardiomyocyte proteomes. It increased expression levels of initial enzymes of the tricarboxylic acid cycle, and of enzymes of fatty acid beta-oxidation, with consequent up-regulation of enzymes of mitochondrial electron transport. It also markedly decreased expression of enzymes of glycolysis and the final steps of the tricarboxylic acid cycle. Glucose loading increased the rate of Bax-independent apoptosis. High glucose increased the duration of the action potential and elevated level of intracellular cytoplasmic calcium. Surprisingly, glucose loading did not influence levels of nitric oxide or mitochondrial superoxide in isolated cardiomyocytes. In summary, short-term simulated hyperglycemia attenuated expression of many anti-apoptotic proteins. This effect was apparently mediated via alterations in multiple biochemical pathways that collectively increased apoptotic susceptibility.     

Impaired anaplerosis and insulin secretion in insulinoma cells caused by small interfering RNA-mediated suppression of pyruvate carboxylase

Anaplerosis, the synthesis of citric acid cycle intermediates, by pancreatic beta cell mitochondria has been proposed to be as important for insulin secretion as mitochondrial energy production. However, studies designed to lower the rate of anaplerosis in the beta cell have been inconclusive. To test the hypothesis that anaplerosis is important for insulin secretion, we lowered the activity of pyruvate carboxylase (PC), the major enzyme of anaplerosis in the beta cell. Stable transfection of short hairpin RNA was used to generate a number of INS-1 832/13-derived cell lines with various levels of PC enzyme activity that retained normal levels of control enzymes, insulin content, and glucose oxidation. Glucose-induced insulin release was decreased in proportion to the decrease in PC activity. Insulin release in response to pyruvate alone, 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) plus glutamine, or methyl succinate plus beta-hydroxybutyrate was also decreased in the PC knockdown cells. Consistent with a block at PC, the most PC-deficient cells showed a metabolic crossover point at PC with increased basal and/or glucose-stimulated pyruvate plus lactate and decreased malate and citrate. In addition, in BCH plus glutamine-stimulated PC knockdown cells, pyruvate plus lactate was increased, whereas citrate was severely decreased, and malate and aspartate were slightly decreased. The incorporation of 14C into lipid from [U-14C]glucose was decreased in the PC knockdown cells. The results confirm the central importance of PC and anaplerosis to generate metabolites from glucose that support insulin secretion and even suggest PC is important for insulin secretion stimulated by noncarbohydrate insulin secretagogues.     

Pancreatic beta cell heterogeneity in glucose-induced insulin secretion.

Rat pancreatic beta cells differ in their individual sensitivity to glucose-inducible metabolic changes. The present study examines whether beta cells with a higher metabolic threshold require higher glucose levels for stimulation of their secretory activity. Purified beta cells were distributed according to their metabolic redox state at 7.5 mM glucose; the metabolically responsive (high responsive) and unresponsive (low responsive) subpopulations of comparable size and viability were reaggregated in the presence of [3H]tyrosine and then perfused at 2.8 mM glucose with 10-min pulses of increasing glucose concentration. Glucose elicited first-phase insulin release in both high and low responsive subpopulations from, respectively, 4.2 and 8.3 mM on. The amplitude of both secretory responses increased dose dependently, the rates in the high responsive subpopulation being 2-fold higher than in the low responsive one. At all stimulating glucose levels, fractional release of 3H-labeled insulin was 3- to 4-fold higher than that of immunoreactive insulin. Preferential release of newly formed insulin was already maximally stimulated at 4.2 mM glucose in the high responsive subpopulation, whereas it increased dose-dependently in the low responsive one. These results indicate the existence of intercellular differences in the secretory activity of glucose-exposed beta cells, both in terms of glucose sensitivity and of amplitude. This heterogeneity in beta cell secretory responsiveness parallels that which has been previously described for the cellular metabolic and biosynthetic functions. It is concluded that glucose dose-dependently recruits beta cells into both biosynthetic and secretory activities. Co-existence of inactive and activated cells can explain preferential release of newly synthesized over preformed hormone during glucose stimulation.     

Effect of diabetes and insulin-induced hypoglycemia on hexokinase and glucose-6-phosphate dehydrogenase in red blood cells

Activities of hexokinase and glucose-6-phosphate dehydrogenase have been measured in red blood cells from control, diabetic and insulin treated rats. After an initial decrease, the enzyme activities increased, but remained lower than control levels. A reversal of the diabetes effect was seen with insulin administration. Insulin induced hypoglycemia increased both enzymes. An overall control of glucose metabolism by insulin in red blood cells was observed.     

Interleukin-1 beta inhibits glucokinase activity in clonal HIT-T15 beta-cells

Interleukin-1 beta (IL-1 beta) has been implicated in the pathogenesis of insulin-dependent diabetes mellitus. In the present study we have investigated the effects of IL-1 beta on glucose metabolism in clonal HIT-T15 beta cells. In the short-term (1 h), 25 U/ml IL-1 beta significantly increased the rates of insulin release and glucose utilisation, but not glucose oxidation. In contrast, after 48 h, IL-1 beta inhibited insulin release and glucose utilisation and oxidation. By assaying enzymes (hexokinase, glucokinase, pyruvate dehydrogenase, glucose 6-phosphatase) and nucleotides (ATP, ADP) associated with the regulation of glycolysis and glucose oxidation, we conclude that the inhibitory effects of IL-1 beta may be due to impaired glucokinase activity.     

Chronic Reduction of the Cytosolic or Mitochondrial NAD(P)-malic Enzyme Does Not Affect Insulin Secretion in a Rat Insulinoma Cell Line

The cytosolic malic enzyme (ME1) has been suggested to augment insulin secretion via the malate-pyruvate and/or citrate-pyruvate shuttles, through the production of NADPH or other metabolites. We used selectable vectors expressing short hairpin RNA (shRNA) to stably decrease Me1 mRNA levels by 80–86% and ME1 enzyme activity by 78–86% with either of two shRNAs in the INS-1 832/13 insulinoma cell line. Contrary to published short term ME1 knockdown experiments, our long term targeted cells showed normal insulin secretion in response to glucose or to glutamine plus 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid. We found no increase in the mRNAs and enzyme activities of the cytosolic isocitrate dehydrogenase or glucose-6-phosphate dehydrogenase, which also produce cytosolic NADPH. There was no compensatory induction of the mRNAs for the mitochondrial malic enzymes Me2 or Me3. Interferon pathway genes induced in preliminary small interfering RNA experiments were not induced in the long term shRNA experiments. We repeated our study with an improved vector containing Tol2 transposition sequences to produce a higher rate of stable transferents and shortened time to testing, but this did not alter the results. We similarly used stably expressed shRNA to reduce mitochondrial NAD(P)-malic enzyme (Me2) mRNA by up to 95%, with severely decreased ME2 protein and a 90% decrease in enzyme activity. Insulin release to glucose or glutamine plus 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid remained normal. The maintenance of robust insulin secretion after lowering expression of either one of these malic enzymes is consistent with the redundancy of pathways of pyruvate cycling and/or cytosolic NADPH production in insulinoma cells.     

Fatty acid degradation in Caulobacter crescentus.

Fatty acid degradation was investigated in Caulobacter crescentus, a bacterium that exhibits membrane-mediated differentiation events. Two strains of C. crescentus were shown to utilize oleic acid as sole carbon source. Five enzymes of the fatty acid beta-oxidation pathway, acyl-coenzyme A (CoA) synthase, crotonase, thiolase, beta-hydroxyacyl-CoA dehydrogenase, and acyl-CoA dehydrogenase, were identified. The activities of these enzymes were significantly higher in C. crescentus than the fully induced levels observed in Escherichia coli. Growth in glucose or glucose plus oleic acid decreased fatty acid uptake and lowered the specific activity of the enzymes involved in beta-oxidation by 2- to 3-fold, in contrast to the 50-fold glucose repression found in E. coli. The mild glucose repression of the acyl-CoA synthase was reversed by exogenous dibutyryl cyclic AMP. Acyl-CoA synthase activity was shown to be the same in oleic acid-grown cells and in cells grown in the presence of succinate, a carbon source not affected by catabolite repression. Thus, fatty acid degradation by the beta-oxidation pathway is constitutive in C. crescentus and is only mildly affected by growth in the presence of glucose. Tn5 insertion mutants unable to form colonies when oleic acid was the sole carbon source were isolated. However, these mutants efficiently transported fatty acids and had beta-oxidation enzyme levels comparable with that of the wild type. Our inability to obtain fatty acid degradation mutants after a wide search, coupled with the high constitutive levels of the beta-oxidation enzymes, suggest that fatty acid turnover, as has proven to be the case fatty acid biosynthesis, might play an essential role in membrane biogenesis and cell cycle events in C. crescentus.     

Failure of chronic hyperinsulinemia to suppress pancreatic glucagon in vivo in the rat.

To determine the effects of chronic hyperinsulinemia on glucagon release, rats were made hyperinsulinemic for 14 days by supplementation of drinking water with sucrose (10%; sucrose-fed) to increase endogenous release or by implantation of osmotic minipumps (subcutaneous, s.c.; or intraperitoneal, i.p.) to deliver exogenous insulin (6 U/day). Both s.c. and i.p. rats also had sucrose in the drinking water to prevent hypoglycemia. Plasma insulin levels were significantly elevated in sucrose-fed, s.c., and i.p. rats. However, glucose levels were significantly elevated in sucrose-fed rats only. Surprisingly, plasma glucagon concentrations were elevated in i.p. and s.c. rats and were not suppressed in sucrose-fed rats. Inverse relationships were found between the plasma levels of insulin and glucose (n = 65; r = -0.42, p less than 0.0001) and between glucose and glucagon (n = 73; r = -0.46, p less than 0.0001). However, unexpectedly, a positive correlation between insulin and glucagon (n = 65; r = 0.47, p less than 0.0001) was established. As suppression of plasma glucagon levels below basal was not observed in any of the hyperinsulinemic or hyperglycemic rats, we wished to establish further whether pancreatic glucagon release could be suppressed below basal levels in the rat by another means. Thus, high doses of somatostatin (50-100 micrograms.kg-1.min-1) were infused for 45 min into normal rats without or with a concomitant hyperinsulinemic, hyperglycemic glucose clamp. Somatostatin fully suppressed insulin, but although plasma glucagon levels were decreased by somatostatin infusion relative to saline-infused animals, there was still no suppression below basal levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

D-Glyceraldehyde causes production of intracellular peroxide in pancreatic islets, oxidative stress, and defective beta cell function via non-mitochondrial pathways

D-Glyceraldehyde (D-GLYC) is usually considered to be a stimulator of insulin secretion but theoretically can also form reactive oxygen species (ROS), which can inhibit beta cell function. We examined the time- and concentration-dependent effects of D-GLYC on insulin secretion, insulin content, and formation of ROS. We observed that a 2-h exposure to 0.05-2 mM D-GLYC potentiated glucose-stimulated insulin secretion (GSIS) in isolated Wistar rat islets but that higher concentrations inhibited GSIS. A 24-h exposure to 2 mm D-GLYC inhibited GSIS, decreased insulin content, and increased intracellular peroxide levels (2.14 +/- 0.31-fold increase, n = 4, p < 0.05). N-Acetylcysteine (10 mM) prevented the increase in intracellular peroxides and the adverse effects of d-GLYC on GSIS. In the presence of 11.1 but not 3.0 mm glucose, koningic acid (10 microM), a specific glyceraldehyde-3-phosphate dehydrogenase inhibitor, increased intracellular peroxide levels (1.88 +/- 0.30-fold increase, n = 9, p < 0.01) and inhibited GSIS (control GSIS = p < 0.001; koningic acid GSIS, not significant). To determine whether oxidative phosphorylation was the source of ROS formation, we cultured rat islets with mitochondrial inhibitors. Neither rotenone or myxothiazol prevented D-GLYC-induced increases in islet ROS. Adenoviral overexpression of manganese superoxide dismutase also failed to prevent the effect of D-GLYC to increase ROS levels. These observations indicate that exposure to excess D-GLYC increases reactive oxygen species in the islet via non-mitochondrial pathways and suggest the hypothesis that the oxidative stress associated with elevated D-GLYC levels could be a mechanism for glucose toxicity in beta cells exposed chronically to high glucose concentrations.     

Expression of glucose transporter-2, glucokinase and mitochondrial glycerolphosphate dehydrogenase in pancreatic islets during rat ontogenesis.

To gain better insight into the insulin secretory activity of fetal beta cells in response to glucose, the expression of glucose transporter 2 (GLUT-2), glucokinase and mitochondrial glycerol phosphate dehydrogenase (mGDH) were studied. Expression of GLUT-2 mRNA and protein in pancreatic islets and liver was significantly lower in fetal and suckling rats than in adult rats. The glucokinase content of fetal islets was significantly higher than of suckling and adult rats, and in liver the enzyme appeared for the first time on about day 20 of extrauterine life. The highest content of hexokinase I was found in fetal islets, after which it decreased progressively to the adult values. Glucokinase mRNA was abundantly expressed in the islets of all the experimental groups, whereas in liver it was only present in adults and 20-day-old suckling rats. In fetal islets, GLUT-2 and glucokinase protein and their mRNA increased as a function of increasing glucose concentration, whereas reduced mitochondrial citrate synthase, succinate dehydrogenase and cytochrome c oxidase activities and mGDH expression were observed. These findings, together with those reported by others, may help to explain the decreased insulin secretory activity of fetal beta cells in response to glucose.     

High content of mitochondrial glycerol-3-phosphate dehydrogenase in pancreatic islets and its inhibition by diazoxide

Homogenates of isolated pancreatic islets contain 40-70 times as much flavin-linked glycerol-3-phosphate dehydrogenase (EC 1.1.99.5) as homogenates of whole pancreas, liver, heart, or skeletal muscle when the activity is assayed with either iodonitrotetrazolium or with dichloroindophenol as an electron acceptor. Intact mitochondria from islets release 3HOH from [2-3H]glycerol phosphate 7 times faster than do skeletal muscle mitochondria. The activity of the cytosolic, NAD-linked, glycerol phosphate dehydrogenase (EC 1.1.1.8) in pancreatic islets is comparable to that of the mitochondrial dehydrogenase so a glycerol phosphate shuttle is possible in pancreatic islets. Diazoxide, an inhibitor of insulin release in vivo and in vitro, inhibits the islet mitochondrial glycerol phosphate dehydrogenase in all three of the assays mentioned above at concentrations that inhibit insulin release and CO2 formation from glucose by isolated pancreatic islets. Diazoxide does not inhibit the dehydrogenase in mitochondria from skeletal muscle, liver, and heart. A slight inhibition in mitochondria from whole pancreas can be accounted for as inhibition of the islet dehydrogenase because no inhibition is observed in mitochondria from pancreas of rats treated with alloxan, an agent that causes diabetes by destroying pancreatic beta cells. The results of this study are compatible with the hypothesis that the mitochondrial glycerol phosphate dehydrogenase has a key role in stimulus-secretion coupling in the pancreatic beta cell during glucose-induced insulin release.     

Increased oxygen radical formation and mitochondrial dysfunction mediate beta cell apoptosis under conditions of AMP-activated protein kinase stimulation

AMP-activated protein kinase influences cellular metabolism, glucose-regulated gene expression, and insulin secretion of pancreatic beta cells. Its sustained activation by culture at low glucose concentrations or in the presence of 5-aminoimidazole-4-carboxamide riboside (AICAR) was shown to trigger apoptosis in beta cells. This study shows that both low glucose- and AICAR-induced apoptosis are associated with increased formation of mitochondrial superoxide-derived radicals and decreased mitochondrial activity. Mitochondrial dysfunction was reflected by an increased oxidized state of the mitochondrial flavins (FMN/FAD) but not of NAD(P)H. It was accompanied by suppression of glucose oxidation and glucose-induced insulin secretion, while palmitate oxidation appeared unaffected. When the cellular accumulation of superoxide-derived radicals was quenched by the ROS scavengers vitamin E, N-acetylcysteine, or the SOD-mimetic compound MnTBAP, apoptosis was significantly inhibited. Both low glucose and AICAR also elevated the expression of BH3-domain-only Bcl-2 antagonists, and induced caspase-3 activation, causing caspase-dependent truncation of Bcl-2. Overexpression of recombinant human Bcl-2 prevented caspase-3 activation, endogenous Bcl-2 processing, and apoptosis, but did not attenuate oxygen radical formation, AMPK activation, or JNK phosphorylation. We conclude that apoptosis by prolonged AMPK activation in beta cells results from enhanced production of mitochondria-derived oxygen radicals and onset of the intrinsic mitochondrial apoptosis pathway, followed by caspase activation and Bcl-2 cleavage which may amplify the death signal.     

Hepatic overexpression of hormone-sensitive lipase and adipose triglyceride lipase promotes fatty acid oxidation, stimulates direct release of free fatty acids, and ameliorates steatosis

Hepatic steatosis is often associated with insulin resistance and obesity and can lead to steatohepatitis and cirrhosis. In this study, we have demonstrated that hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL), two enzymes critical for lipolysis in adipose tissues, also contribute to lipolysis in the liver and can mobilize hepatic triglycerides in vivo and in vitro. Adenoviral overexpression of HSL and/or ATGL reduced liver triglycerides by 40-60% in both ob/ob mice and mice with high fat diet-induced obesity. However, these enzymes did not affect fasting plasma triglyceride and free fatty acid levels or triglyceride and apolipoprotein B secretion rates. Plasma 3-beta-hydroxybutyrate levels were increased 3-5 days after infection in both HSL- and ATGL-overexpressing male mice, suggesting an increase in beta-oxidation. Expression of genes involved in fatty acid transport and synthesis, lipid storage, and mitochondrial bioenergetics was unchanged. Mechanistic studies in oleate-supplemented McA-RH7777 cells with adenoviral overexpression of HSL or ATGL showed that reduced cellular triglycerides could be attributed to increases in beta-oxidation as well as direct release of free fatty acids into the medium. In summary, hepatic overexpression of HSL or ATGL can promote fatty acid oxidation, stimulate direct release of free fatty acid, and ameliorate hepatic steatosis. This study suggests a direct functional role for both HSL and ATGL in hepatic lipid homeostasis and identifies these enzymes as potential therapeutic targets for ameliorating hepatic steatosis associated with insulin resistance and obesity.     

Metabolic consequence of long-term exposure of pancreatic beta cells to free fatty acid with special reference to glucose insensitivity.

Long-term exposure of the pancreatic beta cells to free fatty acid (FFA) reportedly inhibits glucose-stimulated insulin secretion. We here studied the impact of FFA on glucose and lipid metabolism in pancreatic beta cells with special reference to insulin secretion. Pancreatic beta-cell line MIN6 was exposed to various concentrations of palmitate for 3 days. Glucose-stimulated insulin secretion and insulin content were decreased corresponding to the concentration of the palmitate exposed. Glycolytic flux and ATP synthesis was unchanged, but pyruvate-stimulated change in NAD(P)H concentration was decreased. Pyruvate carboxylase was decreased at the protein level, which was restored by the removal of palmitate or the inhibition of beta-oxidation. Intracellular content of triglyceride and FFA were elevated, beta-oxidation was increased, and de novo lipogenesis from glucose was decreased. NADPH content and citrate output into the medium, which reflected pyruvate malate shuttle flux, were decreased, but malic enzyme activity was unaffected. The malic enzyme inhibitor alone inhibited insulin response to glucose. In conclusion, long-term exposure of FFA to beta cells inhibits glucose-stimulated insulin secretion via the decreased NADPH contents due to the inhibition of pyruvate carboxylase and malate pyruvate shuttle flux.