Lisofylline, an inhibitor of unsaturated phosphatidic acid generation, ameliorates interleukin-1β-induced dysfunction in cultured rat islets

Abstract

Interleukin-1β (IL-1β) causes rat islet cell dysfunction through mechanisms that involve inducible nitric oxide synthase (iNOS). However, IL- 1β also activates several lipid pathways, including those generating phosphatidic acid (PA). Lisofylline (LSF), a water-soluble, nontoxic, selective inhibitor of the PA-1α subspecies, which is stimulated by IL-1β and tumor necrosis factor-α, has been shown to prevent cytokine-induced cytotoxicity in in vivo animal models. To evaluate the effect of LSF on acute IL-1β-induced islet dysfunction, rat islets were exposed to IL-1β (0.1 ng/ml) with or without LSF (100 μM) for 24 h, followed by 25 mM glucose (G) stimulation, measurement of rat insulin by RIA, and calculation of the insulin secretion rate. In other experiments, rat islets were precultured for 48 h, then treated for 48 h in 25 mM G with or without IL-1β (0.1 ng/ml) and LSF (400 μM), 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-1β and LSF, lipids were then extracted, and PA subspecies were identified by TLC and mass spectroscopy, and quantitated using normal phase HPLC. Islets were also exposed to IL-1β with or without LSF, and Western immunoblots were performed to evaluate the effect of LSF on iNOS protein expression. IL-1β caused a 44% decrease in islet G-stimulated insulin secretion compared to that in untreated islets (P < 0.0005), which was totally reversed by LSF. In addition, IL-1β decreased the G-stimulated medium insulin content 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 medium insulin content at 24 h (P = 0.11) and 50% at 48 h (P < 0.0001) compared to control islets. HPLC quantitation of PA-1α extracted from islets treated with IL-1β alone showed an approximately 15-fold increase over the PA-1α content of islets treated with IL-1β and LSF. IL-1β-induced expression of iNOS was unchanged with the addition of LSF. These results suggest that LSF is effective in reducing IL-1β-induced islet dysfunction, thus supporting the role of lipid mediators such as PA in cytokine-induced islet toxicity.