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

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

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

Oxygen consumption of rodent islets in the absence or presence of Ex-4.

<p>Single rat (A) and mouse (B) islets were adhered on glass coverslips inside a 35 mm dish using CellTak adhesive. After 30 min equilibration at 4 mM glucose, oxygen consumption was measured in response to 4, 7.5 and 16.6 mM glucose with and without 10 nM Ex-4 (10 nM). Data are means±SE of 3 experiments.</p

Glucose metabolism and lipolysis 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 in 70 µl KRBH/0.25% defatted BSA medium containing 2.8, 8.3 or 16.7 mM glucose plus or minus 20 nM GLP-1 in presence of D-[U-¹⁴C]-glucose (for oxidation) (A) and D-[5-³H]-glucose (for utilization) (B). Incubations were stopped after 90 min as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006221#s2" target="_blank">Methods</a>. Glucose oxidation was measured as ¹⁴CO₂ released, and glucose utilization was determined by measuring the amount of released³H₂O. Results are means±SE of 15 determinations in 3 separate experiments. *p<0.05, **p<0.01, ***p<0.001 when compared to the corresponding 2.8 mM glucose group; #p<0.05 when compared to the corresponding ‘minus GLP-1’ group. For lipolysis determinations (C) overnight-cultured rat islets were washed in KRBH/0.07% BSA medium with 2.8 mM glucose and were transferred into 0.2 mL KRBH/0.07% BSA medium with 2.8, 8.3 or 16.7 mM glucose with or without 20 nM GLP-1. After incubation for 3 h at 37°C, glycerol released into the media and the islet protein content were determined. Means±SE from 4 independent experiments with pentaplicates.</p

Mitochondrial ATP levels of rodent islet cells in the absence or presence of Ex-4.

<p>Dispersed rat (A,B,C) and mouse (D,E,F) islet cells were transduced with Ad-MtLuc-RFP. ATP levels in the presence of 4, 7.5 and 16.6 mM glucose with and without 10 nM Ex-4 were determined as photoemission resulting from luciferase-catalyzed oxidation of luciferin, in populations of approximately 250,000 single islet cells at 5, 15 and 30 min. Data are means±SE of 3 experiments.</p

Acute effects of GLP-1 and Ex-4 on GSIS in rat (A) and mouse (B) islets.

<p>Pancreatic islets were isolated and cultured overnight prior to use as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006221#s2" target="_blank">Methods</a>. Islets were incubated for 1 h (A) or 30 min (B) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006221#s2" target="_blank">Methods</a> for examining insulin secretion at indicated concentrations of glucose and GLP-1 (20 nM) or Ex-4 (20 nM) in A, or 10 nM Ex-4 in B, in the absence or presence of 0.3 mM palmitate. Insulin released into the media and the total islet insulin content were measured. Results shown are mean±SE from 3 independent experiments with quadruplicates (n = 12). For A, *p<0.05, **p<0.01, ***p<0.001 when compared with corresponding 2.8 mM glucose group. #p<0.05, ##p<0.01, ###p<0.001 when compared with corresponding groups without GLP-1 or Ex-4 treatment. For B, †p<0.01 when compared to corresponding minus Ex-4 group. Insulin secretion in mouse islets at basal glucose levels (4 mM) was 1.56±0.3 ng insulin/10 islets/30 min.</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