OBJECTIVEThe 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 METHODSLDL 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.RESULTSWe 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) γ coactivator 1α/PPARα-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.CONCLUSIONSThus, naringenin, through its correction of many of the metabolic disturbances linked to insulin resistance, represents a promising therapeutic approach for metabolic syndrome.

Gestational diabetes (GDM) results from failure of the β 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 β 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 β cell dysfunction. Thus, CMPF provides a link between β cell dysfunction and GDM/T2D that could be targeted therapeutically.

OBJECTIVEThe inability of pancreatic β-cells to appropriately respond to glucose and secrete insulin are primary defects associated with β-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 METHODSWe 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.RESULTSAt 3 weeks, MKR mice were hyperinsulinemic but normoglycemic and β-cells showed negligible mitochondrial or morphological changes. At 5 weeks, MKR islets displayed abrogated hyperpolarization of mitochondrial membrane potential (ΔΨm), reduced mitochondrial Ca2+ uptake, slightly enlarged mitochondria, and reduced glucose-stimulated insulin secretion. By 10 weeks, MKR mice were hyperglycemic and hyperinsulinemic and β-cells contained swollen mitochondria with disordered cristae. β-Cells displayed impaired stimulus-secretion coupling including reduced hyperpolarization of ΔΨm, impaired Ca2+-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.CONCLUSIONSWe provide novel evidence for a critical role of defective mitochondrial oxidative phosphorylation and morphology in the pathology of insulin resistance–induced β-cell failure.

Type 2 diabetes (T2D) arises when pancreatic ␤-cells fail to compensate for systemic insulin resistance with appropriate insulin secretion. However, the link between insulin resistance and ␤-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 reticulumassociated 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 ϳ54% 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.

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 ␤-cell function and mass. Our objective was to investigate the role of oxidative stress in FFA-induced decrease in ␤-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 Ͻ 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 Ͻ 0.01) and induced oxidative stress (P Ͻ 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 Ͻ 0.001) and decreased the insulin response of isolated islets similar to oleate (P Ͻ 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 Ͻ 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. T ype 2 diabetes is characterized by both insulin resistance and defective insulin secretion (1). Obesity is the major predisposing factor for type 2 diabetes and is associated with excessive release of fatty acids from the expanded adipose tissue mass, leading to elevated plasma free fatty acids (FFAs), which are known to induce insulin resistance (2,3). Acute FFA exposure stimulates insulin secretion (4), but studies in vitro and in situ have shown that prolonged FFA exposure decreases glucose-stimulated insulin secretion (GSIS) (5). The effect of prolonged FFA elevation on ␤-cell function in vivo has been more controversial, as absolute GSIS was found to be increased (6 -8), unchanged (9,10), or decreased (11-13) by FFA. However, in most of these studies ␤-cell function was inadequate to compensate for FFA-induced insulin resistance (9 -13), at least in predisposed individuals (14). Although the mechanisms behind FFA-induced decrease in ␤-cell function are unclear, one possibility points toward oxidative stress. Pancreatic ␤-cells have low antioxidant defenses (15) and are thus susceptible to reactive oxygen species (ROS)-induced decrease in function and viability (16,17). Oxidative stress has been implicated in the decrease in GSIS induced by prolonged exposure to glucose (18,19), which is in many respects similar to that induced by prolonged exposure to FFA (4,20). However, whether oxidative stress plays a role in FFA-...

OBJECTIVEIncreased 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 METHODSLipoprotein 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.RESULTSIn 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 β-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.CONCLUSIONSNobiletin provides insight into treatments for dyslipidemia and atherosclerosis associated with insulin-resistant states.

BackgroundThe 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 FindingsThe 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/SignificanceThe 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.

INS:GFP(+) cells can be purified from differentiated hESCs, providing a superior source of insulin-producing cells. Genomic analyses revealed that INS:GFP(+) cells collectively resemble immature endocrine cells. However, insulin(+) cells were heterogeneous, a fact that translated into important functional differences within this population. The information gained from this study may now be used to generate new iterations of functioning beta cells that can be purified for transplant.