Insulin secretion in isolated islets from HFD-fed NN or NJ mice.

<p>Insulin secretion at 3 mM glucose plus 35 mM KCl and at 3, 8 and 16 mM glucose in the absence (3G, 8G and 16G) <b>(A)</b> or the presence <b>(B)</b> of palmitate/oleate (OP; 0.15mM each) (3G/OP, 8G/OP and 16G/OP) (n = 4–5 mice, 3–4 replicates/mouse). Insulin release was normalized for the total islet insulin content. Insulin content/10 islets <b>(C)</b>, pancreas weight <b>(D)</b> and beta-cell mass <b>(E)</b> of HFD-fed NN or NJ mice. Results are means ± SEM of 2–3 independent experiments. *p<0.05 and **p<0.01 compared to NN mice (Student’s t-test).</p

Body weight, food intake, glycemia and insulinemia in ND- and HFD-fed NN and NJ mice.

<p>Body weight (A) and food intake (B). Glycemia and insulinemia in overnight fasted (C and E) or fed (D and F) mice. ND, normal diet; HFD, high fat diet. Results are means ± SEM of 7–9 mice in 2–3 independent experiments. *p<0.05 and **p<0.01 compared to NN mice (Student’s t-test).</p

OGTT and ITT in NN and NJ mice.

<p>Glycemia <b>(A)</b> and insulinemia <b>(B)</b> were measured after glucose administration at time 0 in ND or HFD-fed NN and NJ mice and area under the curve (AUC) was calculated for glycemia <b>(D)</b> and insulinemia <b>(E)</b> curves. Glycemia during ITT and area above the curve (AAC) <b>(C and F)</b> in NN or NJ mice fed a HFD. Results are means ± SEM of 7–9 mice in 2–3 independent experiments. Glycemia was also measured after glucose administration in HFD-fed NN and NJ WT <b>(G)</b> or MCre <b>(H)</b> mice. In the same OGTT tests, insulinemia was measured in NN and NJ WT <b>(I)</b> or MCre <b>(J)</b> mice. Insets depict AUC for glycemia and insulinemia curves. Results are means ± SEM of 3 WT and 6 MCre mice/group in 2–3 independent experiments. *p<0.05 and **p<0.01 compared to NN mice under the same diet (two-way ANOVA and Bonferroni post hoc test or Student’s t-test).</p

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

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

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