Distinct Effects of Saturated and Monounsaturated Fatty Acids on β-Cell Turnover and Function
Article Figures & Tables
Figures
- FIG. 1.
Characterization of the effect of palmitic acid on β-cell death by double staining with the TUNEL assay (alkaline phosphatase) and anti-insulin antibody (peroxidase) (A and B) and by double fluorescent staining with Annexin-V—FLUOS (green) (C and D) and propidium iodide (red) (E and F). Islets were plated on extracellular matrix-coated dishes and exposed for 4 days to media containing 11.1 mmol/l glucose alone (A, C, and E) and with 0.5 mmol/l palmitic acid (B, D, and F). The arrows indicate nuclei stained positive for the TUNEL reaction (light and fluorescence microscopy × 400).
- FIG. 3.
Fatty acids and glucose-induced β-cell DNA fragmentation and proliferative activity. Islets were cultured for 4 days in the absence or presence of different fatty acids (0.5 mmol/l) or a mixture of palmitoleic and palmitic acid (0.25 mmol/l [mixture] or 0.5 mmol/l [mixture 0.5] each) in 5.5, 11.1, and 33.3 mmol/l glucose. Results are means ± SE of the relative number of TUNEL+ (A) and Ki-67+ β-cells (B) per islet, normalized to control incubations at 5.5 mmol/l glucose alone (100%) (in absolute value: 0.82 TUNEL+ β-cells per islet and 4.06 Ki-67+ β-cells per islet at 5.5 mmol/l glucose alone). The mean number of islets scored for DNA fragmentation was 40, 68, and 49 and the mean number for proliferative activity by anti-Ki-67 staining was 78, 69, and 63 in media containing 5.5, 11.1, and 33.3 mmol/l glucose, respectively. Islets were isolated from 40 rats. *P < 0.01 between control and palmitic or palmitoleic acid at the same glucose concentration; §P < 0.05 between palmitic acid and fatty acid mixture at the same glucose concentration; **P < 0.01 relative to islets at 5.5 mmol/l glucose.
- FIG. 2.
Double immunostaining for the Ki-67 nuclear antigen (peroxidase) (A and B) and insulin (fluorescein) (C and D). Islets plated on extracellular matrix-coated dishes were exposed for 4 days to media containing 11.1 mmol/l glucose alone (A and C) or including 0.5 mmol/l palmitic acid (B and D). The arrows indicate nuclei stained positive for Ki-67 (light and fluorescence microscopy ×400).
- FIG. 4.
Effects of blockade of ceramide synthesis by fumonisin B1 and of exogenous ceramide on β-cell DNA fragmentation and proliferative activity. Islets were cultured for 4 days in 11.1 mmol/l glucose alone (control) or in the presence of 0.5 mmol/l palmitic acids with or without 15 μmol/l fumonisin B1 (Fumo) or in the presence of 15 μmol/l C2-ceramide. Results are means ± SE of the relative number of TUNEL+ (A) and Ki-67+ β-cells (B) per islet normalized to the solvent-treated control (100%) (in absolute value: 0.61 TUNEL+ β-cells per islet and 7.32 Ki-67+ β-cells per islet at 11.1 mmol/l glucose alone). The mean number of islets scored for DNA fragmentation and for proliferative activity by anti-Ki-67 staining was 85 and 71, respectively. Islets were isolated from 16 rats. *P < 0.01 relative to solvent-treated controls.
- FIG. 5.
Subcellular localization of ANT and cytochrome c in fatty acid-treated islets. Immunoblotting of ANT (molecular weight 30 kDa) and cytochrome c (molecular weight 15 kDa) was performed on mitochondrial (M) and cytosolic (C) fractions of islets cultured at 11.1 mmol/l glucose for 6 or 20 h with 0.5 mmol/l palmitic acid or 0.5 mmol/l palmitoleic acid. Control islets were analyzed after 20 h of incubation. Both antibodies were blotted on the same membrane after stripping. One representative of three experiments is shown.
- FIG. 6.
Confocal images showing cytochrome c diffusely localized to the cytoplasm but not significantly to the mitochondria in palmitic acid-treated islets. Islets were plated on extracellular matrix-coated dishes and were exposed for 4 days to media containing 11.1 mmol/l glucose alone (A) and with 0.5 mmol/l palmitic acid (B) and stained with anti-cytochrome c antibody.
- FIG. 7.
Effect of prolonged exposure of cultured islets to fatty acids on islet insulin content (A), on chronic insulin release into the culture medium (B), and on basal and glucose-stimulated insulin secretion (C). Islets were incubated in 11.1 mmol/l glucose in the absence or presence of 0.5 mmol/l palmitic or palmitoleic acid or a mixture of both (0.5 mmol/l each) for 4 days. Chronic insulin secretion represents the amount secreted into the culture medium during the experiment. Basal and stimulated insulin secretion denotes the amount secreted over 1 h incubation at 3.3 and 16.7 mmol/l glucose, respectively. Each bar represents the mean of 4 separate experiments ± SE. In each experiment, the data were collected from 4 plates per treatment. *P < 0.05 relative to solvent-treated controls; §P < 0.05 for the difference between palmitic acid and palmitoleic acid or fatty acid mixture.
In this Issue
