EFFECT OF ZN2+ ON THE PROTEOLYTIC INHIBITORY-ACTION OF INSULIN AND BIGUANIDE ANTIHYPERGLYCEMIC DRUGS

The involvement of Zn2+ in the inhibitory action of insulin and phenformin on bulk proteolysis was studied in the Langendorff rat heart with a Zn2+-buffering perfusate (0.1 mM citrate, physiological complete amino acids and 0.2% albumin). Proteins were biosynthetically labeled in vitro for 10 min with [H-3]leucine. Rapidly degraded proteins were eliminated during a 3-h preliminary degradation without insulin or added Zn2+ (2 mM nonradioactive leucine). Insulin (5 nM), the lysosomal inhibitor chloroquine (30-mu-M), and the biguanide antihyperglycemic agent phenformin (2-mu-m) each caused a sustained 35-40% inhibition of [H-3]leucine release beginning within 1-2 min and reaching a maximum at 1-1.5 h. When these agents were combined, their simultaneous proteolytic inhibitory effects were not appreciably greater than the effect of chloroquine alone. Infusion of supraphysiological perfusate Zn2+ (> 15-mu-M) mimicked the inhibitory effect of insulin and chloroquine on lysosomal proteolysis. Infusion of supraphysiological Co2+, Mn2+, Fe2+, and Cr3+ (30-mu-M, 0.5 h) caused no change in proteolysis; however, 30-mu-M Cu2+ caused a slight inhibition. Presumptive chelation of the background (approximately 20 nM) Zn2+ by infusion of 3-mu-M CaNa2 EDTA caused no change in protein degradation over 1-2 h. The infusion of a physiological concentration of 1 or 5-mu-M Zn2+ (as ZnCl2) caused no change in protein degradation over 1-2 h. Biguanides are known to reversibly form a Zn2+ complex with affinity less than that of Zn2+ for EDTA. Prior infusion of 3-mu-M CaNa2 EDTA inactivated the proteolytic inhibitory effect of maximal (2-mu-M) phenformin over at least 1.25 h of concurrent infusion. In identical preparations, the concurrent restoration of physiological 2-mu-M Zn2+ immediately restored the insulin-mimetic proteolytic inhibitory action of phenformin under continued 3-mu-M CaNa2 EDTA (5-mu-M total Zn2+, 2-mu-M nonchelated Zn2+). The time course and potency of submaximal insulin (< 0.1 nM) were decreased or increased by chelation of background perfusate Zn2+ (2-mu-M CaNa2 EDTA) or addition of 1-mu-M Zn2+, respectively. The action of maximal (5 nM) insulin was not changed by 5-mu-M CaNa2 EDTA or addition of 1-mu-M Zn2+. The proteolytic inhibition caused by maximal (5 nM) insulin was not reversed after 2 h of insulin discontinuation. However, 1 mM of the permeant dithiol Zn2+ chelator dimercaptopropane sulfonic acid largely reversed the proteolytic inhibition caused by maximal (5 mM) insulin.