Protection against lipoapoptosis of beta cells through leptin-dependent maintenance of Bcl-2 expression

Abstract

Obesity causes its complications through functional and morphologic damage to remotely situated tissues via undetermined mechanisms. In one rodent model of obesity, the Zucker diabetic fatty fa/fa rat, overaccumulation of triglycerides in the pancreatic islets may be responsible for a gradual depletion of beta cells, leading to the most common complication of obesity, non-insulin-dependent diabetes mellitus. At the onset of non-insulin-dependent diabetes mellitus, the islets from fa/fa rats contain up to 100 times the fat content of islets from normal lean rats. Ultimately, about 75% of the beta cells disappear from these fat-laden islets as a consequence of apoptosis induced by long-chain fatty acids (FA). Here we quantify Bcl-2, the anti-apoptosis factor in these islets, and find that Bcl-2 mRNA and protein are, respectively, 85% and 70% below controls. In normal islets cultured in 1 mM FA, Bcl-2 mRNA declined by 68% and completely disappeared in fa/fa islets cultured in FA. In both groups, suppression was completely blocked by the fatty acyl-CoA synthetase inhibitor, triacsin C, evidence of its mediation by fatty acyl-CoA. To determine whether leptin action blocked FA-induced apoptosis, we cultured normal and fa/fa islets in 1 mM FA with or without leptin. Leptin completely blocked FA-induced Bcl-2 suppression in normal islets but had no effect on islets from fa/fa rats, which are unresponsive to leptin because of a mutation in their leptin receptors (OB-R). However, when wild-type OB-R is overexpressed in fa/fa islets, leptin completely prevented FA-induced Bcl-2 suppression and DNA fragmentation.

Figure 1

Bcl-2 and Bax expression in pancreatic islets of lean wild type (+/+) ZDF or obese (fa/fa) ZDF rats. (A) Representative Southern blot of Bcl-2 RT-PCR products. (B) Immunoblot of Bcl-2. (C) Representative Southern blot of Bax RT-PCR products. Bars represent the mean ± SEM of Bcl-2/β-actin (n = 3) or Bax/β-actin ratios (A and C) or arbitrary densitometric unit (B) (n = 3). ∗, P < 0.01 vs. +/+ values.

Figure 2

(A) Effect of FA on Bcl-2 mRNA in pancreatic islets from +/+ and obese homozygous (fa/fa) ZDF rats. (B) The effect of 10 μM triacsin C on the Bcl-2-lowering action of FA in islets of +/+ and fa/fa ZDF rats.

Figure 3

Effect of 1 mM FA alone or in the presence of recombinant leptin on Bcl-2 mRNA in islets from 7-week-old +/+ or fa/fa ZDF rats. Bars represent the mean ± SEM of the Bcl-2/β-actin mRNA ratio. ∗, P < 0.01 vs. 0 mM FA; †, P < 0.01 vs. 1 mM FA.

Figure 4

Effects of overexpressing OB-Rb or, as a control, β-gal in pancreatic islets of 10-week-old obese fa/fa ZDF rats. (A) Expression of OB-Rb mRNA by RT-PCR and protein by immunoblotting. (B) Effects of 20 ng/ml recombinant leptin on Bcl-2 mRNA levels in islets cultured for 24 h with 1 mM FA. Bars represent the mean ± SEM of the Bcl-2/β-actin mRNA ratio. ∗, P < 0.01 vs. 0 mM FFA; †, P < 0.01 vs. 1 mM FA. (C) Effect of 20 ng/ml recombinant leptin on DNA fragmentation in pancreatic islets for 24 h with 1 mM FA. DNA laddering as a percentage of total DNA is indicated by the numbers.