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 alloxan on the pancreatic beta-cell. In this investigation, the early effects of alloxan on cell function were studied. When INS-1D insulinoma cells were 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 glucose in static incubations. This was associated with impaired stimulation of cellular metabolism by glucose and reduced viability, both monitored colorimetrically with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). These alterations were largely counteracted by the antioxidant butylated hydroxyanisol (BHA). Similar changes occurred when glucose was added directly after 5 min of alloxan treatment, whereas KCl-induced secretion was only partially inhibited. In perifusion, alloxan caused transient insulin secretion to 50% of the rates obtained with glucose 30 min later. Under these conditions, epinephrine abolished the stimulation due to both agents. 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-1D cells and a rise in [Ca2+]i. Alloxan did not augment [Ca2+]i in the presence of BHA or the absence of extracellular calcium. Nickel chloride blocked the effect of alloxan on [Ca2+]i, whereas verapamil was ineffective. This suggests that alloxan promotes Ca2+ influx through channels distinct from L-type channels, perhaps through non-selective cation channels. Thus, alloxan causes changes in INS-1D cells prevented by antioxidant treatment, suggesting that free radicals may modulate the ionic permeability leading to functional activation.
Department of Internal Medicine, University Medical Centre, Geneva, Switzerland.
D
D
Janjic
P
P
Maechler
N
N
Sekine
C
C
Bartley
A S
AS
Annen
C B
CB
Wolheim
D000496:Alloxan / Q000494:pharmacology*; D000975:Antioxidants / Q000494:pharmacology; D002083:Butylated Hydroxyanisole / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002470:Cell Survival; D004305:Dose-Response Relationship, Drug; D005609:Free Radicals / Q000378:metabolism*; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007340:Insulinoma; D008564:Membrane Potentials; D014407:Tumor Cells, Cultured
D000975:Antioxidants; D005609:Free Radicals; D007328:Insulin; D002083:Butylated Hydroxyanisole; D000496:Alloxan; D005947:Glucose; D002118:Calcium