LISOFYLLINE, AN INHIBITOR OF UNSATURATED PHOSPHATIDIC-ACID GENERATION, AMELIORATES INTERLEUKIN-1-BETA-INDUCED DYSFUNCTION IN CULTURED RAT ISLETS

Interleukin-1 beta (IL-1 beta) causes rat islet cell dysfunction throu gh mechanisms that involve inducible nitric oxide synthase (iNOS). How ever, IL-1 beta also activates several lipid pathways, including those generating phosphatidic acid (PA). Lisofylline (LSF), a water-soluble , nontoxic, selective inhibitor of the PA-1 alpha subspecies, which is stimulated by IL-1 beta and tumor necrosis factor-alpha, has been sho wn to prevent cytokine-induced cytotoxicity in in vivo animal models. To evaluate the effect of LSF on acute IL-1 beta-induced islet dysfunc tion, rat islets were exposed to IL-1 beta (0.1 ng/ml) with or without LSF (100 mu M) for 24 h, followed by 25 mM glucose (G) stimulation, m easurement of rat insulin by RIA, and calculation of the insulin secre tion rate. In other experiments, rat islets were precultured for 48 h, then treated for 48 h in 25 mM G with or without IL-1 beta(0.1 ng/ml) and LSF (400 mu M), and aliquots of medium were removed at 0, 24, and 48 h for measurement of rat insulin. In addition, islets were exposed to 25 mM G with or without IL-1 beta and LSF, lipids were then extrac ted, and PA subspecies were identified by TLC and mass spectroscopy, a nd quantitated using normal phase HPLC. Islets were also exposed to IL -1 beta with or without LSF, and Western immunoblots were performed to evaluate the effect of LSF on iNOS protein expression. IL-1 beta caus ed a 44% decrease in islet G-stimulated insulin secretion compared to that in untreated islets (P < 0.0005), which was totally reversed by L SF. In addition, IL-1 beta decreased the G-stimulated medium insulin c ontent by 75% at 24 h (P = 0.0004) and 86% at 48 h compared to that in control islets (P < 0.0001). LSF-treated islets maintained 70% of med ium insulin content at 24 h (P = 0.11) and 50% at 48 h (P < 0.0001) co mpared to control islets. HPLC quantitation of PA-1 alpha extracted fr om islets treated with IL-1 beta alone showed an approximately 15-fold increase over the PA-1 alpha content of islets treated with IL-1 beta and LSF. IL-1 beta-induced expression of MOS was unchanged with the a ddition of LSF. These results suggest that LSF is effective in reducin g IL-1 beta-induced islet dysfunction, thus supporting the role of lip id mediators such as PA in cytokine-induced islet toxicity.