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 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.