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

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

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

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

Palmitate β-oxidation and esterification into different lipids in rat islets in the absence or presence of GLP-1.

<p>Islets were processed as described for insulin secretion (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006221#pone.0006221-Prentki1" target="_blank">[Fig. 1]</a>) and after the pre-incubation step they were incubated for 2 h (FA oxidation) or 4 h (FA esterification) in 1 ml KRBH/0.25% defatted BSA containing medium with 1 mM carnitine and 1 µCi/ml [9,10(n)-³H] palmitate (51 Ci/mmol), at 2.8, 8.3 or 16.7 mM glucose in the presence or absence of 20 nM GLP-1. Cold palmitate (pal) was present at 0.1 mM for oxidation and 0.2 mM for esterification experiments. A, Palmitate oxidation; B—H, palmitate incorporation into diacylglycerol, DAG (B), triacylglycerol, TG (C), monoacylglycerol, MAG (D), non-esterified fatty acids, NEFA (E), cholesterol esters, CE (F), phospholipids, PL (G) and total glycerolipids, GL (H). Means±SE of 6–8 separate incubations in 3 independent experiments.</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

Exendin-4 increases [Ca²⁺]_i and cAMP content in mouse β-cells.

<p>A, A single fura-2 loaded and Ad-MtLuc-RFP-infected mouse β-cell was imaged to determine the [Ca²⁺]_I, at 5.6 mM glucose in KRBH. After establishment of a stable baseline [Ca²⁺]_i, 10 nM Ex-4 was applied for 25 sec (indicated by horizontal bars). Note that a repeatable increase of [Ca²⁺]_i was measured. B, Population study conducted at the single cell level in which the action of Ex-4 to increase [Ca²⁺]_i was evaluated in β-cells not infected (open bars) or infected with Ad-MtLuc-RFP (filled bars). For these experiments, the KRB contained 5.6 or 7.5 mM glucose, as indicated. A response to Ex-4 was defined as a >100 nM increase of [Ca²⁺]_i occurring in a single β-cell. C, Ex-4 caused a dose-dependent increase in cAMP content in mouse islet cells in KRB containing 7.5 mM glucose without or with Ad-MtLuc-RFP infection.</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