TNFalpha impairs glucose stimulated insulin release in human islets by iCOX-independent mechanisms

Abstract

Pro-inflammatory cytokines such as IL-1β, TNFα and IFNγ impair glucose stimulated insulin release (GSIR) and induce apoptosis of human islets in vitro. Previous work has suggested that the inducible cyclooxygenase (iCOX)/prostaglandin E2 (PGE2) pathway mediates inhibition of insulin secretion by IL-1β. We tested the hypothesis that iCOX mediates TNFα-induced injury in human islets. Human islets isolated from cadaveric donors were treated with recombinant human TNFα (1000 U/ml) alone or with a cytokine mixture (CM) containing TNFα, IL-1β (50 U/ml) and IFNγ (750 U/ml) +/− iNOS inhibitor L-NMMA (1 mM), iCOX inhibitor NS-398 (0.01 mM) or caspase inhibitor z-VAD (0.1 mM). At 40 hours, nitrite and PGE2 released to the medium were determined. Islets were subjected to either glucose challenge or RNA extraction. RNase protection assay was used to determine cytokine-regulated gene expression for iCOX and iNOS. At 72 hours, islets were dissociated and flow cytometry used to determine rates of apoptosis. Control islets demonstrated a 15.2 ± 0.5 fold insulin secretion index (ISI, insulin released in high glucose vs. that in low glucose containing solutions). TNFα and CM treated islets demonstrated impaired ISI ( 6.2 ± 0 and 4.1 ± 0.5, respectively). Control islets in culture for 72 hours demonstrated an apoptosis rate of 6.2 ± 0.2%. TNFα induced the rate to 8.0 ± 0.3%, CM treatment 11.1 ± 0.5%. TNFα stimulated iCOX but not iNOS gene expression, while CM stimulated the expression of both genes. PGE2 produced by control islets was 4.4 ± 0.1 pg/ml. TNFα stimulated PGE2 production to 25.2 ± 0.3 pg/ml and CM 100 ± 1.1 pg/ml. Control and TNFα-treated islets had a nitrite production level of 1.6 ± 0.1 μM, CM 3.1 ± 0.2 μM. Co-treatment with NS-398 completely blocked cytokine-induced PGE2 production by the islets. Co-treatment with L-NMMA completely blocked CM-induced nitrite production. z-VAD protected the islets from cytokine-induced apoptosis. NS-398, L-NMMA, and z-VAD all failed, however, to protect the islets from TNFα- or CM-impaired GSIS. Proinflammatory cytokines inhibit islet insulin secretion in response to glucose challenge. iCOX- and iNOS-independent mechanisms are involved in the process.