Free radical modulation of insulin release in INS-1 cells exposed to alloxan

Generation of free radicals is thought to mediate the cytotoxic action of a lloxan on the pancreatic beta-cell. In this investigation, the early effect s of alloxan on cell function were studied. When INS-1D insulinoma cells we re exposed to alloxan (1 mM) for 45 min followed by a 3-hr recovery period, the drug increased basal insulin release while abolishing the effect of gl ucose in static incubations. This was associated with impaired stimulation of cellular metabolism by glucose and reduced viability, both monitored col orimetrically with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro mide (MTT). These alterations were largely counteracted by the antioxidant butylated hydroxyanisol (BHA). Similar changes occurred when glucose was ad ded directly after 5 min of alloxan treatment, whereas KCl-induced secretio n was only partially inhibited. In perifusion, alloxan caused transient ins ulin secretion to 50% of the rates obtained with glucose 30 min later. Unde r these conditions, epinephrine abolished the stimulation due to both agent s. Membrane potential and cytosolic calcium concentrations ([Ca2+](i)) were recorded to clarify the action of alloxan. Alloxan-induced insulin release correlated with depolarization of INS-ID cells and a rise in [Ca2+](i). Al loxan did not augment [Ca2+](i) in the presence of BHA or the absence of ex tracellular calcium. Nickel chloride blocked the effect of alloxan on [Ca2](1), whereas verapamil was ineffective. This suggests that alloxan promote s Ca2+ influx through channels distinct from L-type channels, perhaps throu gh non-selective cation channels. Thus, alloxan causes changes in INS-ID ce lls prevented by antioxidant treatment, suggesting that free radicals may m odulate the ionic permeability leading to functional activation.