The influence of catecholamines and cyclic AMP on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and lipid biosynthesis in isolated rat hepatocytes

Abstract

A modification of existing techniques for isolating rat hepatocytes by perfusion of the liver with collagenase is described in which hepatocyte viability remained greater than 90% and incorporation of [ 3H]leucine into protein remained nearly linear during subsequent cell incubation at 37 °C for 6 h. Both the initial 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity and the rate of sterol synthesis were significantly lower in hepatocytes isolated from rats killed at about the fourth hour of the 12 h light period (L-4) than from those killed at the sixth hour of the dark period (D-6). Incubation of L-4 hepatocytes resulted in increases in both the activity of HMG-CoA reductase and the rate of sterol synthesis from [ 14C]acetate of 200–300% by 3 h, but that from [ 14C]mevalonate did not change. In contrast, D-6 hepatocytes showed no increase in reductase activity or in sterol synthesis. Rat serum elicited a rapid increase in the activity of HMG-CoA reductase and the rate of sterol synthesis in L-4 and D-6 cells. Serum did not stimulate sterol synthesis from [ 14C]mevalonate or fatty acid synthesis from [ 14C]acetate. Epinephrine or norepinephrine resulted in an increase in the hepatocyte reductase activity which was maximal after 3 h of incubation, at an initial hormone concentration of approximately 5.5 × 10 −5 m, with both L-4 and D-6 cells. Epinephrine or norepinephrine also stimulated the rate of incorporation of [ 14C]acetate into nonsaponifiable lipids of L-4 cells but decreased the rate of incorporation into fatty acids. Dibutyryl cyclic AMP at a concentration of 3 × 10 −8 m did not affect the increase in reductase activity observed after a 3 h incubation of L-4 hepatocytes in the presence or absence of rat serum. However, high concentrations (3 × 10 −4 m) partially prevented the increase in enzyme activity. The data indicate that the stimulatory effect of the catecholamines on HMG-CoA reductase activity does not involve elevated concentrations of cyclic AMP as an intermediary.