Fatty acid-induced beta cell apoptosis: a link between obesity and diabetes

Abstract

Like obese humans, Zucker diabetic fatty (ZDF) rats exhibit early beta cell compensation for insulin resistance (4-fold beta cell hyperplasia) followed by decompensation (>50% loss of beta cells). In prediabetic and diabetic ZDF islets, apoptosis measured by DNA laddering is increased 3- and >7-fold, respectively, compared with lean ZDF controls. Ceramide, a fatty acid-containing messenger in cytokine-induced apoptosis, was significantly increased (P < 0.01) in prediabetic and diabetic islets. Free fatty acids (FFAs) in plasma are high (>1 mM) in prediabetic and diabetic ZDF rats; therefore, we cultured prediabetic islets in 1 mM FFA. DNA laddering rose to 19.6% vs. 4.6% in lean control islets, preceded by an 82% increase in ceramide. C2-Ceramide without FFA induced DNA laddering, but fumonisin B1, a ceramide synthetase inhibitor, completely blocked FFA-induced DNA laddering in cultured ZDF islets. [3H]Palmitate incorporation in [3H]ceramide in ZDF islets was twice that of controls, but [3H]palmitate oxidation was 77% less. Triacsin C, an inhibitor of fatty acyl-CoA synthetase, and troglitazone, an enhancer of FFA oxidation in ZDF islets, both blocked DNA laddering. These agents also reduced inducible nitric oxide (NO) synthase mRNA and NO production, which are involved in FFA-induced apoptosis. In ZDF obesity, beta cell apoptosis is induced by increased FFA via de novo ceramide formation and increased NO production.

Figure 1

(A) DNA fragmentation in freshly isolated islets from 5 (preobese)-, 7 (obese and prediabetic)-, and 14 (diabetic)-week-old fa/fa ZDF rats and lean +/+ controls. Ladder (%), fragmented DNA percent; lane 1, 100-bp DNA size marker (Boeringer Mannheim). (B) Ceramide content in pancreatic islets of obese fa/fa ZDF (solid bars) and lean +/+ controls (open bars). Values are the mean ± SEM of three or four experiments. ∗, P < 0.05 vs. +/+; †, P < 0.05 vs. 7 week.

Figure 2

(A) Effect of FFA on DNA fragmentation in pancreatic islets from lean wild-type (+/+) and obese prediabetic homozygous (fa/fa) ZDF rats. Pancreatic islets were isolated from 7-week-old rats and cultured with 1 mM FFA as indicated. Ladder %, fragmented DNA percent. (B) Effect of FFA on ceramide content in pancreatic islets of ZDF rats. Islets from lean wild-type (•) and obese homozygous (□) ZDF rats were cultured with 0 or 1 mM FFA at the indicated times, and ceramide contents were determined. (C) De novo synthesis of [³H]ceramide from [³H]palmitate in islets of wild type (+/+) and fa/fa ZDF rats in the absence and presence of the ceramide synthase inhibitor fumonisin B₁ (FB₁). Rats were 7 weeks of age. Values are the mean ± SEM of triplicate experiments. (D) Comparison of [³H]ceramide and [³H]H₂O to assess relative rates of de novo ceramide synthesis vs. oxidation of [³H]palmitate by isolated islets of +/+ and fa/fa rats. Values are the mean ± SEM of triplicate experiments. ∗, P < 0.05 vs. +/+.

Figure 3

Effect of exogenous ceramide and of blockade of ceramide synthesis on DNA fragmentation in islets from obese fa/fa ZDF rats. Islets isolated from 7-week-old obese prediabetic fa/fa ZDF rats were cultured for 24 hr in medium containing 15 μM C₂-ceramide (C2-Cer) without FFA or 1 mM FFA plus 50 μM fumonisin B₁ (FB₁), an inhibitor of ceramide synthetase. Lane 1, 100-bp DNA size marker (Boeringer Mannheim); Ladder (%), fragmented DNA percent.

Figure 4

(A) Inhibitory effect of triacsin C, troglitazone, and aminoguanidine on FFA-induced DNA fragmentation in islets from obese fa/fa ZDF rats. Islets isolated from 7-week-old fa/fa ZDF rats were cultured for 24 hr at 0 or 1 mM FFA with 10 μM triacsin C, 10 μM troglitazone, or 0.5 mM aminoguanidine. M, 100-bp DNA size marker. (B) Inhibitory effect of triacsin C, troglitazone, and aminoguanidine on FFA-induced iNOS mRNA induction. (C) FFA-induced NO production in islets of obese fa/fa ZDF rats. Effect of triacsin C, troglitazone, and aminoguanidine on islets cultured as described in A except for 48 hr.