PTEN deletion in pancreatic α-cells protects against high-fat diet-induced hyperglucagonemia and insulin resistance.

An aberrant increase in circulating catabolic hormone glucagon contributes to type 2 diabetes pathogenesis. However, mechanisms regulating glucagon secretion and α-cell mass are not well understood. In this study, we aimed to demonstrate that phosphatidylinositol 3-kinase (PI3K) signaling is an important regulator of α-cell function. Mice with deletion of PTEN, a negative regulator of this pathway, in α-cells show reduced circulating glucagon levels and attenuated l-arginine-stimulated glucagon secretion both in vivo and in vitro. This hypoglucagonemic state is maintained after high-fat-diet feeding, leading to reduced expression of hepatic glycogenolytic and gluconeogenic genes. These beneficial effects protected high-fat diet-fed mice against hyperglycemia and insulin resistance. The data demonstrate an inhibitory role of PI3K signaling on α-cell function and provide experimental evidence for enhancing α-cell PI3K signaling for diabetes treatment.

The furan fatty acid metabolite CMPF is elevated in diabetes and induces ß cell dysfunction.

Gestational diabetes (GDM) results from failure of the ß cells to adapt to increased metabolic demands; however, the cause of GDM and the extremely high rate of progression to type 2 diabetes (T2D) remains unknown. Using metabolomics, we show that the furan fatty acid metabolite 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF) is elevated in the plasma of humans with GDM, as well as impaired glucose-tolerant and T2D patients. In mice, diabetic levels of plasma CMPF induced glucose intolerance, impaired glucose-stimulated insulin secretion, and decreased glucose utilization. Mechanistically, we show that CMPF acts directly on the ß cell, causing impaired mitochondrial function, decreasing glucose-induced ATP accumulation, and inducing oxidative stress, resulting in dysregulation of key transcription factors and ultimately reduced insulin biosynthesis. Importantly, specifically blocking its transport through OAT3 or antioxidant treatment could prevent CMPF-induced ß cell dysfunction. Thus, CMPF provides a link between ß cell dysfunction and GDM/T2D that could be targeted therapeutically.

SDF2L1 interacts with the ER-associated degradation machinery and retards the degradation of mutant proinsulin in pancreatic ß-cells.

Stromal cell-derived factor 2-like 1 (SDF2L1) is an endoplasmic reticulum (ER)-localized protein whose function is undefined. Here we show that SDF2L1 protein levels are increased in response to ER stress-inducing compounds, but not other cell stressors that we tested in insulinoma cell lines. SDF2L1 protein levels were also induced by expression of misfolded proinsulin in insulinoma cells and in islets from diabetic mice. Immunoprecipitation and binding assays demonstrated that SDF2L1 interacts with the ER chaperone GRP78/BiP, the ER-associated degradation (ERAD) machinery and with misfolded proinsulin. Unexpectedly, knockdown of SDF2L1 in INS-1 (insulin 2 C96Y-GFP) cells increased the degradation kinetics of mutant proinsulin, suggesting that SDF2L1 regulates substrate availability for the ERAD system. We suggest that SDF2L1 increases the time that misfolded proteins have to achieve a correctly folded conformation and thus that SDF2L1 can act as a buffer for substrate availability for ERAD in pancreatic ß-cells.

UCP2 regulates the glucagon response to fasting and starvation.

Glucagon is important for maintaining euglycemia during fasting/starvation, and abnormal glucagon secretion is associated with type 1 and type 2 diabetes; however, the mechanisms of hypoglycemia-induced glucagon secretion are poorly understood. We previously demonstrated that global deletion of mitochondrial uncoupling protein 2 (UCP2(-/-)) in mice impaired glucagon secretion from isolated islets. Therefore, UCP2 may contribute to the regulation of hypoglycemia-induced glucagon secretion, which is supported by our current finding that UCP2 expression is increased in nutrient-deprived murine and human islets. Further to this, we created α-cell-specific UCP2 knockout (UCP2AKO) mice, which we used to demonstrate that blood glucose recovery in response to hypoglycemia is impaired owing to attenuated glucagon secretion. UCP2-deleted α-cells have higher levels of intracellular reactive oxygen species (ROS) due to enhanced mitochondrial coupling, which translated into defective stimulus/secretion coupling. The effects of UCP2 deletion were mimicked by the UCP2 inhibitor genipin on both murine and human islets and also by application of exogenous ROS, confirming that changes in oxidative status and electrical activity directly reduce glucagon secretion. Therefore, α-cell UCP2 deletion perturbs the fasting/hypoglycemic glucagon response and shows that UCP2 is necessary for normal α-cell glucose sensing and the maintenance of euglycemia.

Glutathionylation state of uncoupling protein-2 and the control of glucose-stimulated insulin secretion.

The role of reactive oxygen species (ROS) in glucose-stimulated insulin release remains controversial because ROS have been shown to both amplify and impede insulin release. In regard to preventing insulin release, ROS activates uncoupling protein-2 (UCP2), a mitochondrial inner membrane protein that negatively regulates glucose-stimulated insulin secretion (GSIS) by uncoupling oxidative phosphorylation. With our recent discovery that the UCP2-mediated proton leak is modulated by reversible glutathionylation, a process responsive to small changes in ROS levels, we resolved to determine whether glutathionylation is required for UCP2 regulation of GSIS. Using Min6 cells and pancreatic islets, we demonstrate that induction of glutathionylation not only deactivates UCP2-mediated proton leak but also enhances GSIS. Conversely, an increase in mitochondrial matrix ROS was found to deglutathionylate and activate UCP2 leak and impede GSIS. Glucose metabolism also decreased the total amount of cellular glutathionylated proteins and increased the cellular glutathione redox ratio (GSH/GSSG). Intriguingly, the provision of extracellular ROS (H(2)O(2), 10 µM) amplified GSIS and also activated UCP2. Collectively, our findings indicate that the glutathionylation status of UCP2 contributes to the regulation of GSIS, and different cellular sites and inducers of ROS can have opposing effects on GSIS, perhaps explaining some of the controversy surrounding the role of ROS in GSIS.

Dual role of VAMP8 in regulating insulin exocytosis and islet ß cell growth.

Optimal insulin secretion required to maintain glucose homeostasis is the summation of total pancreatic islet ß cell mass and intrinsic secretory capacity of individual ß cells, which are regulated by distinct mechanisms that could be amplified by glucagon-like-peptide-1 (GLP-1). Because of these actions of GLP-1 on islet ß cells, GLP-1 has been deployed to treat diabetes. We employed SNARE protein VAMP8-null mice to demonstrate that VAMP8 mediates insulin granule recruitment to the plasma membrane, which partly accounts for GLP-1 potentiation of glucose-stimulated insulin secretion. VAMP8-null mice also exhibited increased islet ß cell mass from increased ß cell mitosis, with ß cell proliferative activity greatly amplified by GLP-1. Thus, despite the ß cell exocytotic defect, VAMP8-null mice have an increased total insulin secretory capacity, which improved glucose homeostasis. We conclude that these VAMP8-mediated events partly underlie the therapeutic actions of GLP-1 on insulin secretion and ß cell growth.

A novel high-throughput assay for islet respiration reveals uncoupling of rodent and human islets.

BACKGROUND: The pancreatic beta cell is unique in its response to nutrient by increased fuel oxidation. Recent studies have demonstrated that oxygen consumption rate (OCR) may be a valuable predictor of islet quality and long term nutrient responsiveness. To date, high-throughput and user-friendly assays for islet respiration are lacking. The aim of this study was to develop such an assay and to examine bioenergetic efficiency of rodent and human islets. METHODOLOGY/PRINCIPAL FINDINGS: The XF24 respirometer platform was adapted to islets by the development of a 24-well plate specifically designed to confine islets. The islet plate generated data with low inter-well variability and enabled stable measurement of oxygen consumption for hours. The F1F0 ATP synthase blocker oligomycin was used to assess uncoupling while rotenone together with myxothiazol/antimycin was used to measure the level of non-mitochondrial respiration. The use of oligomycin in islets was validated by reversing its effect in the presence of the uncoupler FCCP. Respiratory leak averaged to 59% and 49% of basal OCR in islets from C57Bl6/J and FVB/N mice, respectively. In comparison, respiratory leak of INS-1 cells and C2C12 myotubes was measured to 38% and 23% respectively. Islets from a cohort of human donors showed a respiratory leak of 38%, significantly lower than mouse islets. CONCLUSIONS/SIGNIFICANCE: The assay for islet respiration presented here provides a novel tool that can be used to study islet mitochondrial function in a relatively high-throughput manner. The data obtained in this study shows that rodent islets are less bioenergetically efficient than human islets as well as INS1 cells.

MK-626, a dipeptidyl peptidase-4 inhibitor, does not improve the hyperglycemia or hyperinsulinemia of nonobese diabetic MKR mice.

Dipeptidyl peptidase-4 (DPP-4) inhibitors increase circulating levels of incretin hormones, which can enhance insulin secretion and ß cell function. The aim of this study was to evaluate the effectiveness of MK-626 (a novel DPP-4 inhibitor) to reduce the hyperglycemia and hyperinsulinemia of nonobese type 2 diabetic MKR mice. Twelve to 14-week-old hyperglycemic MKR mice were gavaged daily with MK-626 (3 mg/kg body weight) or vehicle (0.5% methyl cellulose (MC)) for 2 weeks. MK-626-treated mice displayed no change in body weight or adverse reactions, suggesting good tolerance of the drug. Fed blood glucose was significantly reduced over the 2-week experiment; however, it was also reduced in the MC group, suggesting an effect of gavage alone. Fed plasma insulin and glucagon levels and glucose tolerance of MK-626-treated mice were similar to those of MC mice. Therefore, treatment with MK-626 did not correct the prolonged hyperglycemia and impaired glucose tolerance of MKR mice.

Beta-cell uncoupling protein 2 regulates reactive oxygen species production, which influences both insulin and glucagon secretion.

OBJECTIVE: The role of uncoupling protein 2 (UCP2) in pancreatic ß-cells is highly debated, partly because of the broad tissue distribution of UCP2 and thus limitations of whole-body UCP2 knockout mouse models. To investigate the function of UCP2 in the ß-cell, ß-cell-specific UCP2 knockout mice (UCP2BKO) were generated and characterized. RESEARCH DESIGN AND METHODS: UCP2BKO mice were generated by crossing loxUCP2 mice with mice expressing rat insulin promoter-driven Cre recombinase. Several in vitro and in vivo parameters were measured, including respiration rate, mitochondrial membrane potential, islet ATP content, reactive oxygen species (ROS) levels, glucose-stimulated insulin secretion (GSIS), glucagon secretion, glucose and insulin tolerance, and plasma hormone levels. RESULTS: UCP2BKO ß-cells displayed mildly increased glucose-induced mitochondrial membrane hyperpolarization but unchanged rates of uncoupled respiration and islet ATP content. UCP2BKO islets had elevated intracellular ROS levels that associated with enhanced GSIS. Surprisingly, UCP2BKO mice were glucose-intolerant, showing greater α-cell area, higher islet glucagon content, and aberrant ROS-dependent glucagon secretion under high glucose conditions. CONCLUSIONS: Using a novel ß-cell-specific UCP2KO mouse model, we have shed light on UCP2 function in primary ß-cells. UCP2 does not behave as a classical metabolic uncoupler in the ß-cell, but has a more prominent role in the regulation of intracellular ROS levels that contribute to GSIS amplification. In addition, ß-cell UCP2 contributes to the regulation of intraislet ROS signals that mediate changes in α-cell morphology and glucagon secretion.

Nobiletin attenuates VLDL overproduction, dyslipidemia, and atherosclerosis in mice with diet-induced insulin resistance.

OBJECTIVE: Increased plasma concentrations of apolipoprotein B100 often present in patients with insulin resistance and confer increased risk for the development of atherosclerosis. Naturally occurring polyphenolic compounds including flavonoids have antiatherogenic properties. The aim of the current study was to evaluate the effect of the polymethoxylated flavonoid nobiletin on lipoprotein secretion in cultured human hepatoma cells (HepG2) and in a mouse model of insulin resistance and atherosclerosis. RESEARCH DESIGN AND METHODS: Lipoprotein secretion was determined in HepG2 cells incubated with nobiletin or insulin. mRNA abundance was evaluated by quantitative real-time PCR, and Western blotting was used to demonstrate activation of cell signaling pathways. In LDL receptor-deficient mice (Ldlr(-/-)) fed a Western diet supplemented with nobiletin, metabolic parameters, gene expression, fatty acid oxidation, glucose homeostasis, and energy expenditure were documented. Atherosclerosis was quantitated by histological analysis. RESULTS: In HepG2 cells, activation of mitogen-activated protein kinase-extracellular signal-related kinase signaling by nobiletin or insulin increased LDLR and decreased MTP and DGAT1/2 mRNA, resulting in marked inhibition of apoB100 secretion. Nobiletin, unlike insulin, did not induce phosphorylation of the insulin receptor or insulin receptor substrate-1 and did not stimulate lipogenesis. In fat-fed Ldlr(-/-) mice, nobiletin attenuated dyslipidemia through a reduction in VLDL-triglyceride (TG) secretion. Nobiletin prevented hepatic TG accumulation, increased expression of Pgc1α and Cpt1α, and enhanced fatty acid ß-oxidation. Nobiletin did not activate any peroxisome proliferator-activated receptor (PPAR), indicating that the metabolic effects were PPAR independent. Nobiletin increased hepatic and peripheral insulin sensitivity and glucose tolerance and dramatically attenuated atherosclerosis in the aortic sinus. CONCLUSIONS: Nobiletin provides insight into treatments for dyslipidemia and atherosclerosis associated with insulin-resistant states.

Molecular and metabolic evidence for mitochondrial defects associated with beta-cell dysfunction in a mouse model of type 2 diabetes.

OBJECTIVE The inability of pancreatic beta-cells to appropriately respond to glucose and secrete insulin are primary defects associated with beta-cell failure in type 2 diabetes. Mitochondrial dysfunction has been implicated as a key factor in the development of type 2 diabetes; however, a link between mitochondrial dysfunction and defective insulin secretion is unclear. RESEARCH DESIGN AND METHODS We investigated the changes in islet mitochondrial function and morphology during progression from insulin resistance (3 weeks old), immediately before hyperglycemia (5 weeks old), and after diabetes onset (10 weeks old) in transgenic MKR mice compared with controls. The molecular and protein changes at 10 weeks were determined using microarray and iTRAQ proteomic screens. RESULTS At 3 weeks, MKR mice were hyperinsulinemic but normoglycemic and beta-cells showed negligible mitochondrial or morphological changes. At 5 weeks, MKR islets displayed abrogated hyperpolarization of mitochondrial membrane potential (DeltaPsi(m)), reduced mitochondrial Ca(2+) uptake, slightly enlarged mitochondria, and reduced glucose-stimulated insulin secretion. By 10 weeks, MKR mice were hyperglycemic and hyperinsulinemic and beta-cells contained swollen mitochondria with disordered cristae. beta-Cells displayed impaired stimulus-secretion coupling including reduced hyperpolarization of DeltaPsi(m), impaired Ca(2+)-signaling, and reduced glucose-stimulated ATP/ADP and insulin release. Furthermore, decreased cytochrome c oxidase-dependent oxygen consumption and signs of oxidative stress were observed in diabetic islets. Protein profiling of diabetic islets revealed that 36 mitochondrial proteins were differentially expressed, including inner membrane proteins of the electron transport chain. CONCLUSIONS We provide novel evidence for a critical role of defective mitochondrial oxidative phosphorylation and morphology in the pathology of insulin resistance-induced beta-cell failure.

Naringenin prevents dyslipidemia, apolipoprotein B overproduction, and hyperinsulinemia in LDL receptor-null mice with diet-induced insulin resistance.

OBJECTIVE: The global epidemic of metabolic syndrome and its complications demands rapid evaluation of new and accessible interventions. Insulin resistance is the central biochemical disturbance in the metabolic syndrome. The citrus-derived flavonoid, naringenin, has lipid-lowering properties and inhibits VLDL secretion from cultured hepatocytes in a manner resembling insulin. We evaluated whether naringenin regulates lipoprotein production and insulin sensitivity in the context of insulin resistance in vivo. RESEARCH DESIGN AND METHODS: LDL receptor-null (Ldlr(-/-)) mice fed a high-fat (Western) diet (42% calories from fat and 0.05% cholesterol) become dyslipidemic, insulin and glucose intolerant, and obese. Four groups of mice (standard diet, Western, and Western plus 1% or 3% wt/wt naringenin) were fed ad libitum for 4 weeks. VLDL production and parameters of insulin and glucose tolerance were determined. RESULTS: We report that naringenin treatment of Ldlr(-/-) mice fed a Western diet corrected VLDL overproduction, ameliorated hepatic steatosis, and attenuated dyslipidemia without affecting caloric intake or fat absorption. Naringenin 1) increased hepatic fatty acid oxidation through a peroxisome proliferator-activated receptor (PPAR) gamma coactivator 1alpha/PPARalpha-mediated transcription program; 2) prevented sterol regulatory element-binding protein 1c-mediated lipogenesis in both liver and muscle by reducing fasting hyperinsulinemia; 3) decreased hepatic cholesterol and cholesterol ester synthesis; 4) reduced both VLDL-derived and endogenously synthesized fatty acids, preventing muscle triglyceride accumulation; and 5) improved overall insulin sensitivity and glucose tolerance. CONCLUSIONS: Thus, naringenin, through its correction of many of the metabolic disturbances linked to insulin resistance, represents a promising therapeutic approach for metabolic syndrome.

Metabolic effects of dietary cholesterol in an animal model of insulin resistance and hepatic steatosis.

Although the atherogenic role of dietary cholesterol has been well established, its diabetogenic potential and associated metabolic disturbances have not been reported. Diet-induced hamster models of insulin resistance and dyslipidemia were employed to determine lipogenic and diabetogenic effects of dietary cholesterol. Metabolic studies were conducted in hamsters fed diets rich in fructose (40%), fat (30%), and cholesterol (0.05-0.25%) (FFC) and other test diets. Short-term feeding of the FFC diet induced insulin resistance, glucose intolerance, hypertriglyceridemia, and hypercholesterolemia. Prolonged feeding (6-22 wk) of the FFC diet led to severe hepatic steatosis, glucose intolerance, and mild increases in fasting blood glucose, suggesting progression toward type 2 diabetes, but did not induce beta-cell dysfunction. Metabolic changes induced by the diet, including dyslipidemia and insulin resistance, were cholesterol concentration dependent and were only markedly induced on a high-fructose and high-fat dietary background. There were significant increases in hepatic and plasma triglyceride with FFC feeding, likely due to a 10- to 15-fold induction of hepatic stearoyl-CoA desaturase compared with chow levels (P < 0.03). Hepatic insulin resistance was evident based on reduced tyrosine phosphorylation of the insulin receptor-beta, IRS-1, and IRS-2 as well as increased protein mass of protein tyrosine phosphatase 1B. Interestingly, nuclear liver X receptor (LXR) target genes such as ABCA1 were upregulated on the FFC diet, and dietary supplementation with an LXR agonist (instead of dietary cholesterol) worsened dyslipidemia, glucose intolerance, and upregulation of target mRNA and proteins similar to that of dietary cholesterol. In summary, these data clearly implicate dietary cholesterol, synergistically acting with dietary fat and fructose, as a major determinant of the severity of metabolic disturbances in the hamster model. Dietary cholesterol appears to induce hepatic cholesterol ester and triglyceride accumulation, and diet-induced LXR activation (via cholesterol-derived oxysterols) may possibly be one key underlying mechanism.

UCP2 is highly expressed in pancreatic alpha-cells and influences secretion and survival.

In pancreatic beta-cells, uncoupling protein 2 (UCP2) influences mitochondrial oxidative phosphorylation and insulin secretion. Here, we show that alpha-cells express significantly higher levels of UCP2 than do beta-cells. Greater mitochondrial UCP2-related uncoupling was observed in alpha-cells compared with beta-cells and was accompanied by a lower oxidative phosphorylation efficiency (ATP/O). Conversely, reducing UCP2 activity in alpha-cells was associated with higher mitochondrial membrane potential generated by glucose oxidation and with increased ATP synthesis, indicating more efficient metabolic coupling. In vitro, the suppression of UCP2 activity led to reduced glucagon secretion in response to low glucose; however, in vivo, fasting glucagon levels were normal in UCP2(-/-) mice. In addition to its effects on secretion, UCP2 played a cytoprotective role in islets, with UCP2(-/-) alpha-cells being more sensitive to specific death stimuli. In summary, we demonstrate a direct role for UCP2 in maintaining alpha-cell function at the level of glucose metabolism, glucagon secretion, and cytoprotection.

Inhibition of apoB secretion from HepG2 cells by insulin is amplified by naringenin, independent of the insulin receptor.

Hepatic overproduction of apolipoprotein B (apoB)-containing lipoproteins is characteristic of the dyslipidemia associated with insulin resistance. Recently, we demonstrated that the flavonoid naringenin, like insulin, decreased apoB secretion from HepG2 cells by activation of both the phosphoinositide-3-kinase (PI3-K) pathway and the mitogen-activated protein kinase/extracellular-regulated kinase (MAPK(erk)) pathway. In the present study, we determined whether naringenin-induced signaling required the insulin receptor (IR) and sensitized the cell to the effects of insulin, and whether the kinetics of apoB assembly and secretion in cells exposed to naringenin were similar to those of insulin. Immunoblot analysis revealed that insulin stimulated maximal phosphorylation of IR and IR substrate-1 after 10 min, whereas naringenin did not affect either at any time point up to 60 min. The combination of naringenin and submaximal concentrations of insulin potentiated extracellular-regulated kinase 1/2 activation and enhanced upregulation of the LDL receptor, downregulation of microsomal triglyceride transfer protein expression, and inhibition of apoB-100 secretion. Multicompartmental modeling of apoB pulse-chase studies revealed that attenuation of secreted radiolabeled apoB in naringenin- or insulin-treated cells was similar under lipoprotein-deficient or oleate-stimulated conditions. Naringenin and insulin both stimulated intracellular apoB degradation via a kinetically defined rapid pathway. Therefore, naringenin, like insulin, inhibits apoB secretion through activation of both PI3-K and MAPK(erk) signaling, resulting in similar kinetics of apoB secretion. However, the mechanism for naringenin-induced signaling is independent of the IR. Naringenin represents a possible strategy for reduction of hepatic apoB secretion, particularly in the setting of insulin resistance.

The identification of potential factors associated with the development of type 2 diabetes: a quantitative proteomics approach.

Type 2 diabetes (T2D) arises when pancreatic beta-cells fail to compensate for systemic insulin resistance with appropriate insulin secretion. However, the link between insulin resistance and beta-cell failure in T2D is not fully understood. To explore this association, we studied transgenic MKR mice that initially develop insulin resistance in skeletal muscle but by 8 weeks of age have T2D. In the present study, global islet protein and gene expression changes were characterized in diabetic MKR versus non-diabetic control mice at 10 weeks of age. Using a quantitative proteomics approach (isobaric tags for relative and absolute quantification (iTRAQ)), 159 proteins were differentially expressed in MKR compared with control islets. Marked up-regulation of protein biosynthesis and endoplasmic reticulum stress pathways and parallel down-regulation in insulin processing/secretion, energy utilization, and metabolism were observed. A fraction of the differentially expressed proteins identified (including GLUT2, DNAJC3, VAMP2, RAB3A, and PC1/3) were linked previously to insulin-secretory defects and T2D. However, many proteins for the first time were associated with islet dysfunction, including the unfolded protein response proteins (ERP72, ERP44, ERP29, PPIB, FKBP2, FKBP11, and DNAJB11), endoplasmic reticulum-associated degradation proteins (VCP and UFM1), and multiple proteins associated with mitochondrial energy metabolism (NDUFA9, UQCRH, COX2, COX4I1, COX5A, ATP6V1B2, ATP6V1H, ANT1, ANT2, ETFA, and ETFB). The mRNA expression level corresponding to these proteins was examined by microarray, and then a small subset was validated using quantitative real time PCR and Western blot analyses. Importantly approximately 54% of differentially expressed proteins in MKR islets (including proteins involved in proinsulin processing, protein biosynthesis, and mitochondrial oxidation) showed changes in the proteome but not transcriptome, suggesting post-transcriptional regulation. These results underscore the importance of integrated mRNA and protein expression measurements and validate the use of the iTRAQ method combined with microarray to assess global protein and gene changes involved in the development of T2D.

Free fatty acid-induced reduction in glucose-stimulated insulin secretion: evidence for a role of oxidative stress in vitro and in vivo.

OBJECTIVE: An important mechanism in the pathogenesis of type 2 diabetes in obese individuals is elevation of plasma free fatty acids (FFAs), which induce insulin resistance and chronically decrease beta-cell function and mass. Our objective was to investigate the role of oxidative stress in FFA-induced decrease in beta-cell function. RESEARCH DESIGN AND METHODS: We used an in vivo model of 48-h intravenous oleate infusion in Wistar rats followed by hyperglycemic clamps or islet secretion studies ex vivo and in vitro models of 48-h exposure to oleate in islets and MIN6 cells. RESULTS: Forty-eight-hour infusion of oleate decreased the insulin and C-peptide responses to a hyperglycemic clamp (P < 0.01), an effect prevented by coinfusion of the antioxidants N-acetylcysteine (NAC) and taurine. Similar to the findings in vivo, 48-h infusion of oleate decreased glucose-stimulated insulin secretion ex vivo (P < 0.01) and induced oxidative stress (P < 0.001) in isolated islets, effects prevented by coinfusion of the antioxidants NAC, taurine, or tempol (4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl). Forty-eight-hour infusion of olive oil induced oxidative stress (P < 0.001) and decreased the insulin response of isolated islets similar to oleate (P < 0.01). Islets exposed to oleate or palmitate and MIN6 cells exposed to oleate showed a decreased insulin response to high glucose and increased levels of oxidative stress (both P < 0.001), effects prevented by taurine. Real-time RT-PCR showed increased mRNA levels of antioxidant genes in MIN6 cells after oleate exposure, an effect partially prevented by taurine. CONCLUSIONS: Our data are the first demonstration that oxidative stress plays a role in the decrease in beta-cell secretory function induced by prolonged exposure to FFAs in vitro and in vivo.

Quality assessment of microarray experiments.

OBJECTIVES: We describe a quality assurance procedure to maximize the value of oligonucleotide microarray expression profiles. DESIGN, METHODS, AND RESULTS: Background microarray noise was 82.2+/-54.5 and 51.8+/-12.4 units, respectively, before and after enacting the program (P<0.0001). We also noted improved concordance of microarray expression fold-changes for selected genes with results of RT-PCR validation. CONCLUSIONS: This multi-step procedure, including quantification of RNA sample degradation and detection of outlier data points, has increased data quality from our microarray facility.

Inhibition of microsomal triglyceride transfer protein expression and apolipoprotein B100 secretion by the citrus flavonoid naringenin and by insulin involves activation of the mitogen-activated protein kinase pathway in hepatocytes.

Microsomal triglyceride transfer protein (MTP) is necessary for hepatocyte assembly and secretion of apolipoprotein (apo)B100-containing lipoproteins. The citrus flavonoid naringenin, like insulin, decreased MTP expression in HepG2 cells, resulting in inhibition of apoB100 secretion; however, the mechanism for naringenin is independent of insulin receptor substrate-1/2. Recently, it was reported that insulin decreased MTP expression in HepG2 cells via the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) (MAPK(erk)) pathway. We hypothesized that naringenin acts via a similar mechanism. Inhibition of MAPK kinase (MEK) 1/2 in HepG2 cells significantly attenuated the naringenin- and insulin-induced reduction in MTP expression. Both naringenin and insulin increased ERK1/2 phosphorylation, which was completely inhibited by MEK1/2 inhibition and enhanced by inhibition of MAPK(p38), a negative regulator of MAPK(erk) activity. Inhibition of MEK1/2 significantly attenuated both the naringenin- and insulin-induced decrease in apoB100 secretion demonstrating a direct link between MAPK(erk) activation and apoB100 secretion. Furthermore, both compounds increased MAPK(p38) activation, and therefore inhibition of MAPK(p38) amplified thenaringenin- and insulin-induced decrease in apoB100 secretion. We conclude that MAPK(erk) signaling in hepatocytes is critical for inhibition of apoB100 secretion by naringenin and insulin. Therefore, naringenin may prove useful for activating insulin-signaling pathways important for regulation of hepatocyte lipid homeostasis.