Model indicating how cytosolic isocitrate dehydrogenase knockdown results in enhanced glucose-induced insulin secretion.

<p>IC, isocitrate; α-KG, α-ketoglutarate; NNT, nicotinamide nucleotide transhydrogenase; Pyr, pyruvate.</p

Knockdown of IDHc expression in dispersed rat islet cells increases glucose-induced hormone release.

<p>ScrAB and siIDH#2 (siIDHc) were co-transfected with human growth hormone (hGH) plasmid in dispersed rat islet cells. Cells were cultured for 48 h prior to the experiment. hGH release was measured in cells incubated at 2 or 16 mM glucose (G) or 2 mM glucose plus 35 mM KCl. hGH release was normalized by hGH cellular content and is expressed as fold increase over the 2 mM glucose condition. Data represent the mean ± SEM of three independent experiments performed in triplicate. * p<0.05 vs ScrAB under the same incubation condition by paired two-tailed Student <i>t</i> test.</p

Knockdown of IDHc expression enhances glucose-induced insulin secretion.

<p>INS 832/13 cells were transfected with ScrAB, siIDHc#1 or siIDHc#2. A, IDHc mRNA expression level normalized with cyclophilin mRNA and presented as percentage vs the ScrAB condition. B, Enzymatic activity of IDHc normalized by protein content. C, Insulin secretion. Insulin release was measured in transfected cells incubated at 1, 5 or 10 mM glucose (G) or 1 mM glucose plus 35 mM KCl. D, Assessment of the amplification pathway of glucose-induced insulin secretion. Insulin secretion was measured in transfected cells incubated at 1, 5 or 10 mM glucose ±150 µM diazoxide plus 35 mM KCl (Dz+KCl). Insulin levels were normalized by protein content. Data represent the mean ± SEM of three to four independent experiments performed in quadruplicate. * <i>p</i><0.05; ** <i>p</i><0.01, vs ScrAB under the same condition, by one-way Anova, Dunnett's post-test.</p

Targeted metabolomics of siRNA-transfected INS 832/13 cells.

<p>INS 832/13 cells were transfected with ScrAB and siIDH#2 (siIDH) and experimental conditions were similar as for insulin secretion. Cells were incubated at 1 (1 G) or 10 mM (10 G) glucose for 45 min. Results are presented by metabolite classes. Data represent the mean ± SEM of four independent experiments performed each with triplicate cell wells. * <i>p</i><0.05; ** <i>p</i><0.01; *** p<0.001 vs 1 G under the same transfection condition; # <i>p</i><0.05; ## <i>p</i><0.02, vs ScrAB under the same incubation condition by unpaired two-tailed Student <i>t</i> test.</p

Effect of the RNA interference delivery method on glucose-induced insulin secretion in INS 832/13 cells.

<p>A, Glucose-induced insulin secretion is not affected by Nucleofactor transfection. INS 832/13 cells were not transfected (No T) or transfected with an empty vector (pBS) or a combination of two siRNA controls (ScrAB) using Nucleofactor electroporation. Cells were cultured for 48 h prior to the experiment. B, Adenoviral infection <i>per se</i> alters glucose-induced insulin secretion. INS 832/13 cells were not infected (No inf) or infected with one control adenovirus containing LacZ or GFP (Ad-LacZ or Ad-GFP) at 10 MOI for 16 h. After the infection period, cells were cultured for 48 h prior to the experiment. Insulin release was measured in cells incubated at 1, 5 or 10 mM glucose (G) or 1 mM glucose plus 35 mM KCl. Insulin levels were normalized by protein content. Data represent the mean ± SEM of two to three independent experiments performed in quadruplicate.</p

Reduction in IDHc expression alters fatty acid metabolism without affecting oxidative glucose metabolism.

<p>Transfected cells were incubated at 1, 5 or 10(G) and results were normalized by protein content. A, Glucose oxidation and B, Glucose incorporation into free fatty acids were monitored using [U-¹⁴C]glucose. C, Fatty acid oxidation was measured using [1-¹⁴C]palmitate. D, Malonyl-CoA levels determined using an enzymatic assay. Data represent means ± SEM of two (A) or three (B, C and D) independent experiments each performed in triplicate cell culture wells. * <i>p</i><0.05; ** <i>p</i><0.01, vs ScrAB under the same incubation condition, by one-way Anova, Dunnett's post-test.</p

Schematic illustrating the metabolite changes induced by the reduction in IDHc expression.

<p>The numbers 1 to 7 refers to points mentioned in the discussion. AlaAT, alanine aminotransferase; AspAT, aspartate aminotransferase; ACC, acetyl-CoA carboxylase; ACL, ATP-citrate lyase; ACOc, cytosolic isoform of aconitase; DHAP, dihydroxyacetone phosphate; FFA, free fatty acids; FUMc, cytosolic isoform of fumarase; GDH, glutamate dehydrogenase; Glnase, glutaminase; Glycerol-3-P, glycerol-3-phosphate; α-KG, alpha-ketoglutarate; IC, isocitrate; IDH1 (or IDHc), cytosolic isoform of NADP⁺-dependent isocitrate dehydrogenase; IDH2, mitochondrial isoform of NADP⁺-dependent isocitrate dehydrogenase; IDH3, mitochondrial isoform of NAD⁺-dependent isocitrate dehydrogenase; MDH1, cytosolic isoform of malate dehydrogenase; MDH2, mitochondrial isoform of malate dehydrogenase; MEc, cytosolic isoform of malic enzyme; NNT, nicotinamide nucleotide transhydrogenase; OAA, oxaloacetate; PC, pyruvate carboxylase; PDH, pyruvate dehydrogenase; Pyr, pyruvate.</p

Pancreatic β-Cell Dysfunction in Diet-Induced Obese Mice: Roles of AMP-Kinase, Protein Kinase Cε, Mitochondrial and Cholesterol Metabolism, and Alterations in Gene Expression

<div><p>Diet induced obese (DIO) mice can be stratified according to their weight gain in response to high fat diet as low responders (LDR) and high responders (HDR). This allows the study of β-cell failure and the transitions to prediabetes (LDR) and early diabetes (HDR). C57BL/6N mice were fed for 8 weeks with a normal chow diet (ND) or a high fat diet and stratified as LDR and HDR. Freshly isolated islets from ND, LDR and HDR mice were studied <i>ex-vivo</i> for mitochondrial metabolism, AMPK activity and signalling, the expression and activity of key enzymes of energy metabolism, cholesterol synthesis, and mRNA profiling. Severely compromised glucose-induced insulin secretion in HDR islets, as compared to ND and LDR islets, was associated with suppressed AMP-kinase activity. HDR islets also showed reduced acetyl-CoA carboxylase activity and enhanced activity of 3-hydroxy-3-methylglutaryl-CoA reductase, which led respectively to elevated fatty acid oxidation and increased cholesterol biosynthesis. HDR islets also displayed mitochondrial membrane hyperpolarization and reduced ATP turnover in the presence of elevated glucose. Expression of protein kinase Cε, which reduces both lipolysis and production of signals for insulin secretion, was elevated in DIO islets. Genes whose expression increased or decreased by more than 1.2-fold were minor between LDR and ND islets (17 differentially expressed), but were prominent between HDR and ND islets (1508 differentially expressed). In HDR islets, particularly affected genes were related to cell cycle and proliferation, AMPK signaling, mitochondrial metabolism and cholesterol metabolism. In conclusion, chronically reduced AMPK activity, mitochondrial dysfunction, elevated cholesterol biosynthesis in islets, and substantial alterations in gene expression accompany β-cell failure in HDR islets. The β-cell compensation process in the prediabetic state (LDR) is largely independent of transcriptional adaptive changes, whereas the transition to early diabetes (HDR) is associated with major alterations in gene expression.</p></div

Defective insulin secretion and mitochondrial dysfunction in DIO islets.

<p>(A) Insulin secretion was measured in freshly isolated islets from normal diet (ND), and obese high fat diet fed low responders (LDR) and high responders (HDR) mice. Groups of 10 islets were incubated 1 h in KRBH at 3, 8, or 16 mM glucose (G) or 3 mM glucose ± 35 mM KCl. Means ± SEM of 10–12 determinations from islets of 6 animals per group in three separate experiments. ***p<0.001 versus ND for the same glucose concentration; ###p<0.001 versus 3 mM glucose; one-way ANOVA, Tukey post-hoc test. (B) Mitochondrial membrane potential (Δψmito) measured by Rhodamine123 fluorescence in dispersed islet cells from ND, LDR and HDR mice. Δψmito was initially measured at 3 mM glucose to set a baseline and then at 16 mM glucose. Data were normalized to baseline fluorescence. Means of 6 (ND) or 5 (LDR and HDR) mice. ***p<0.0001 vs ND; One-way ANOVA, repeated measures, Tukey post-hoc test. (C) Mitochondrial O₂ consumption rate (OCR) measured at 3 mM glucose and then at 16 mM glucose (16G). (D) Baseline respiration at 3 mM glucose, (E) glucose-induced respiration as the difference in OCR between 16 and 3 mM glucose, (F) ATP-turnover at 16G, (G) maximal respiration, (H) uncoupled respiration and (I) non-mitochondrial respiration were determined using mitochondrial inhibitors. Means ± SEM of 5 mice per group, each with quadruplicate observations. *p<0.05 versus ND; One-way ANOVA, Tukey post-hoc test.</p

Individual metabolic parameters of C57BL/6N mice fed with a normal or HFD for 8 weeks used for islet gene expression analysis.

<p>(A) Body weight (BW), (B) glycemia, (C) insulinemia, (D) cholesterolemia, (E) plasma fatty acids and (F) plasma triglycerides. Means ± SEM of 8 animals per group are indicated below the X- axis for each graph. LDR or HDR versus ND: *P<0.05, ***P<0.001; HDR versus LDR: & P<0.05, && P<0.01, &&& P<0.001. One-way ANOVA-Bonferroni’s multiple comparison post hoc test.</p