γ-Tocopherol Partially Protects Insulin-Secreting Cells against Functional Inhibition by Nitric Oxide

Abstract

Preceding the onset of type 1 diabetes mellitus, pancreatic islets are infiltrated by macrophages secreting interleukin-1β (IL-1β) which induces β-cell apoptosis and exerts inhibitory actions on islet β-cell insulin secretion. IL-1β seems to act chiefly through induction of nitric oxide (NO) synthesis. Hence, IL-1β and NO have been implicated as key effector molecules in type 1 diabetes mellitus. In this paper, the influence of endogenously produced and exogenously delivered NO on the regulation of cell proliferation, cell viability and discrete parts of the stimulus-secretion coupling in insulin-secreting RINm5F cells was investigated. Because vitamin E may delay diabetes onset in animal models, we also investigated whether tocopherols may protect β-cells from the suppressive actions of IL-1 and NO in vitro. To this end, the impact of NO on insulin secretory responses to activation of phospholipase C (by carbamylcholine), protein kinase C (by phorbol ester), adenylyl cyclase (by forskolin), and Ca2+ influx through voltage-activated Ca2+ channels (by K+-induced depolarization) was monitored in culture after treatment with IL-1β or by co-incubation with the NO donor spermine-NONOate. It was found that cell proliferation, viability, insulin production and the stimulation of insulin release evoked by carbamylcholine and phorbol ester were impeded by IL-1β or spermine-NONOate, whereas the hormone output by the other secretagogues was not altered by NO. Pretreatment with γ-tocopherol (but not α-tocopherol) afforded a partial protection against the inhibitory effects of NO, whereas specifically inhibiting inducible NO synthase with N-nitro-L-arginine completely reversed the IL-1β effects. In contrast, inhibiting guanylyl cyclase with ODQ (1H-[1,2,4]oxadiazolo[4,3-α]-quinoxaline-1-one) or blocking low voltage-activated Ca2+ channels with NiCl2 failed to influence the actions of NO. In conclusion, our data show that NO inhibits growth and insulin secretion in RINm5F cells, and that γ-tocopherol may partially prevent this. The results suggest that phospholipase C or protein kinase C may be targeted by NO. In contrast, cGMP or low voltage-activated Ca2+ channels appear not to mediate the toxicity of NO in these cells. These adverse effects of NO on the β-cell, and the protection by γ-tocopherol, may be of importance for the development of the impaired insulin secretion characterizing type 1 diabetes mellitus, and offer possibilities for intervention in this process.