β-Cell Failure in Diet-Induced Obese Mice Stratified According to Body Weight Gain: Secretory Dysfunction and Altered Islet Lipid Metabolism Without Steatosis or Reduced β-Cell Mass

DIO mice are insulin-resistant, are glucose-intolerant, and have defective first-phase GSIS. A and B: HIEC in 2-h–fasted anesthetized mice. A: Arterial glucose levels at time 0 of the clamp; B: glucose levels during the clamp. The dashed line corresponds to the clamped glucose value at ∼7.2 mmol/l. (C) Glucose infusion rate during the last 30 min of the 2-h HIEC (GIR) and (D) insulin sensitivity index (M/I) calculated as the glucose infusion rate (M) divided by the average insulinemia during the last 30 min of the clamp (I). M/I index is expressed as μmol · kg⁻¹ · min⁻¹ glucose infused per pmol/l insulin. E–I: IVGTT. Plasma glucose (E) and insulin (F) were measured at times 0, 3, 5, 15, 30, 45, and 60 min in response to a glucose challenge (i.v.; 0.5 g/kg) in 16-h overnight fasted anesthetized mice; (G) AUC of the 0–60 min glycemic responses; (H) first-phase insulin response to glucose (Glc) is the AUC of the 0–15 min insulinemic response; (I) second-phase insulin response to glucose is the AUC of the 15–60 min insulinemic response. Means ± SE of 5–8 animals per group. Normal diet (ND) versus LDR or HDR: *P < 0.05, **P < 0.01, ***P < 0.001; LDR versus HDR: §P < 0.05, §§P < 0.01, §§§P < 0.001; one-way (A, C, D, and G–I) or two-way ANOVA (B, E, and F), with Bonferroni post hoc test.

Isolated islets from DIO mice show defective glucose-, KCl-, and arginine-induced insulin release. Insulin secretion was measured in freshly isolated islets from normal diet (ND), LDR, and HDR mice. Group of 10 islets were incubated 1 h in KRBH at 3, 8, or 16 mmol/l glucose (G; A and D) ± 0.25 mmol/l palmitate/0.5% d-BSA (Pal; B and D) or 3 mmol/l glucose ± 35 mmol/l KCl (C) ± Pal (C). Means ± SE are of 14–16 determinations from islets of 21–22 animals per group in five (A, B, and D) and three (C) separate experiments. E–H: Insulin secretion by islets perifused at 3 mmol/l glucose (3G) or 16 mmol/l glucose (16G) with or without 10 mmol/l arginine (Arg). AUC for insulin over the first 15 min (F) and from 15–60 min (G) following glucose concentration increased from 3 to 16 mmol/l. AUC for maximal insulin secretion was determined from 60–80 min after arginine injection (H). Means ± SE are of six normal diet, seven LDR, and seven HDR. Insulin release in the media was determined after the 1-h incubation or at the different time points of the perifusion experiment. *P < 0.05, **P < 0.01, ***P < 0.001 normal diet versus LDR or HDR; #P < 0.05, ##P < 0.01, ###P < 0.001 versus 3 mmol/l glucose (A) plus palmitate (B) or plus KCl (C) or versus the value in absence of palmitate (D) for the same group (normal diet, LDR, HDR); one-way ANOVA–Bonferroni multiple comparison test.

β-Cell mass and proliferation, islet cell cytosolic free calcium, ATP, proinsulin, insulin and protein contents, and glucose metabolism of DIO mice. (A) β-Cell mass and (B) proliferation as indicated by the number of Ki-67 positive β-cells/islet. Means ± SE of six animals for normal diet (ND) and LDR and seven for HDR. Normal diet versus LDR or HDR, *P < 0.05. One-way ANOVA with Dunnett post hoc test. (C) Protein, (D and E) insulin expressed per islet (D) or per islet protein content (E), and (F) total islet proinsulin contents. Means ± SE of 83, 35, and 9 animals per group for (C), (D and E), and (F), respectively. Normal diet versus LDR or HDR, *P < 0.05, **P < 0.01, and ***P < 0.001; LDR versus HDR, §§P < 0.01. One-way ANOVA with Bonferroni post hoc test. Glucose utilization (G) and oxidation (H) were measured in islets incubated in KRBH at 3, 8, and 16 mmol/l glucose (G) with d-[U-¹⁴C]glucose and d-[5-³H]glucose. Means ± SE of 13–15 determinations from islets of 9 normal diet, 8 LDR, and 10 HDR mice in three separate experiments. ###P < 0.001 versus 3 mmol/l glucose for the same group. One-way ANOVA–Bonferroni multiple comparison test. I: Total ATP content was determined in islets incubated for 15 min in KRBH at 3 or 16 mmol/l glucose (G). Means ± SE of 13–15 determinations from islets of 9 normal diet, 9 LDR, and 9 HDR mice in three separate experiments. #P < 0.05 versus 3 mmol/l glucose for the same group. One-way ANOVA–Bonferroni multiple comparison test. J: Cytosolic free calcium was measured by confocal microscopy using Fluo-4 AM dye in dispersed-islet cells of normal diet, LDR, and HDR mice. Cells were perifused for 3 min at 3 mmol/l glucose, then at 16 mmol/l glucose for 10 min, and finally at 3 mmol/l glucose + 35 mmol/l KCl for 2 min. Fluorescence level is expressed in arbitrary units. For clarity purposes, only the mean of six independent experiments from six mice per group is represented. Of note, SE was no more than 15% at all times. One-way ANOVA with repeated measures, Tukey post test. ***P < 0.001 normal diet versus LDR and §§§P < 0.001 LDR versus HDR.

Altered lipid partitioning, lipolysis, and glycerolipid/fatty acid cycling in DIO islets. A: Islet triglyceride content. Means ± SE of 10–12 animals per group. B: Glycerol release, as an index of lipolysis, was determined in islets incubated for 3 h in KRBH at 3 and 16 mmol/l glucose (G). Means ± SE of 19–20 determinations from islets of 17–18 mice per group in four separate experiments. Islets were incubated in KRBH at 3 and 16 mmol/l glucose with [9,10-³H]palmitate to assess (C) FFA oxidation, (D) total intracellular labeled NEFA, and (E) fatty acid (FA) esterification (esterif.) into triglycerides (TG) and (F) cholesterol esters (CE). Means ± SE of 9–12 determinations from islets of 14–18 mice per group in three (C) or four (D–F) separate experiments. (G) Total cholesterol (TC) and free cholesterol (FC) fractions extracted from normal diet (ND), LDR, and HDR islets. Means ± SE of 10–15 animals per group. LDR or HDR versus normal diet: *P < 0.05, **P < 0.01, ***P < 0.001; LDR versus HDR: §P < 0.05; #P < 0.05, ##P < 0.01, ###P < 0.001 versus 3 mmol/l glucose for the same islet group. One-way ANOVA–Bonferroni multiple comparison test.