The involvement of Zn2+ in the inhibitory action of insulin and phenformin on bulk proteolysis was studied in the Langendorff rat heart with a Zn(2+)-buffering perfusate (0.1 mM citrate, physiological complete amino acids and 0.2% albumin). Proteins were biosynthetically labeled in vitro for 10 min with [3H]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 μM), and the biguanide antihyperglycemic agent phenformin (2 μm) each caused a sustained 35–40% inhibition of [3H]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 > the effect of chloroquine alone. Infusion of supraphysiological perfusate Zn2+ (>15 μM) mimicked the inhibitory effect of insulin and chloroquine on lysosomal proteolysis. Infusion of supraphysiological Co2+, Mn2+, Fe2+, and Cr3+ (30 μM, 0.5 h) caused no change in proteolysis; however, 30 μM Cu2+ caused a slight inhibition. Presumptive chelation of the background (∼20 nM) Zn2+ by infusion of 3 μM CaNa2 EDTA caused no change in protein degradation over 1–2 h. The infusion of a physiological concentration of 1 or 5 microM Zn2+ (as ZnCl2) caused no change in protein degradation over 1–2 h. Biguanides are known to reversibly form a Zn2+ complex with affinity < that of Zn2+ for EDTA. Prior infusion of 3 μM CaNa2 EDTA inactivated the proteolytic inhibitory effect of maximal (2 μM) phenformin over at least 1.25 h of concurrent infusion. restoration of physiological 2 μM Zn2+ immediately restored the insulin-mimetic proteolytic inhibitory action of phenformin under continued 3 μM CaNa2 EDTA (5 μM total Zn2+, 2 μM nonchelated Zn2+). The time course and potency of submaximal insulin (<0.1 nM) were decreased or increased by chelation of background perfusate Zn2+ (2 μM CaNa2 EDTA) or addition of 1 μM Zn2+, respectively. The action of maximal (5 μM) insulin was not changed by 5 IJLM CaNa2 EDTA or addition of 1 μM 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 nM) insulin.

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