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

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

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

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

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

Increased total PKCε levels in DIO islets.

<p>Proteins from ND, LDR and HDR islets were probed with an antibody against total PKCε (T-PKCε) and phospho-Ser⁷²⁹ PKCε (P-PKCε). Tubulin or total PKCε were used for normalization. (A) Representative western blot of T-PKCε and P-PKCε, (B) expression level of T-PKCε normalized by tubulin, (C) P-PKCε level normalized by tubulin and (D) by total PKCε. Means ± SEM of 8 (ND), 13 (LDR) and 10 (HDR) mice. *p<0.05, **p<0.01 vs ND, One-way ANOVA, Tukey post-hoc test.</p

mRNA profiling analysis of control and DIO mice.

<p>(A) Top five most significant enriched canonical pathways identified by Ingenuity Pathway Analysis (grey bars) using all the differentially expressed genes in LDR vs ND, HDR vs LDR and HDR vs ND islets comparisons that are listed according to their p-values expressed in—log (Y axis). The ratio of the number of differentially expressed genes over the total number of genes involved in each canonical pathway is reported on top of the bars. (B) AMPK pathways, (C) mitochondrial respiration, (D) mitochondrial metabolism and (E) cholesterol metabolism and trafficking gene expression by microarray analysis (B, C, D and E; +/- 1.2 fold change) and quantitative real time PCR (B; +/- 1.15 fold change) of HDR vs LDR (gray bar) and HDR vs ND (white bar).</p

Increased total cholesterol content in HDR islets.

<p>Total cholesterol content of islets incubated at 3 mM (3G) or 16 mM (16G) glucose for 3h. Means ± SEM from 6 (ND and HDR) and 5 (LDR) mice. *p<0.05 vs ND, &p<0.05, &&p<0.01 for HDR vs LDR, for the same glucose concentration. One-way ANOVA, Tukey post-hoc test.</p

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