Insulin regulation of lipoprotein lipase activity and release in 3T3-L1 adipocytes. Separation and dependence of hormonal effects on hexose metabolism and synthesis of RNA and protein.

Insulin regulation of lipoprotein lipase was studied in fully differentiated 3T3-Ll fat cells. Insulin at low concentrations (10e7 to 10-l’ M) elicited a rapid and maximal increase (-lo-fold) in lipoprotein lipase release from the cells to the medium within 30 min and caused a 2to lo-fold increase in total cellular lipoprotein lipase activity over a a-day period. Insulin had no effect on cell lipoprotein lipase activity during the first 4 h of treatment. Prolonged exposure to insulin (% to 4 days) resulted in additional increases in enzyme release rate, and increased the fractional release rate per h approximately lo-fold. Stimulation of cellular lipoprotein lipase activity was dependent on the presence of glucose in the incubation medium. Although half-maximal increases in cell activity required 12 h of insulin treatment in glucose-containing medium, removal of either glucose or insulin resulted in loss of more than 50% of the stimulation in enzyme activity within 90 min. Addition of glucose to cells incubated 20 h with insulin in carbohydrate-free medium elicited a rapid and full response to insulin in 2 h. Fructose, mannose, and glucosamine could substitute for glucose in supporting this rapid response in insulin-pretreated cells, but only glucose, mannose, and fructose would support the effect in 24-h incubations with hormone. Induction of the increase in lipoprotein lipase activity by glucose in insulin-pretreated cells was prevented by cycloheximide, but not by actinomycin D. In contrast, inhibitors of either protein (cycloheximide, puromycin) or RNA (a-amanitin, actinomycin D) synthesis completely blocked the response when added with insulin to cells incubated in medium containing glucose. We propose that insulin regulates lipoprotein lipase in fat cells by three major actions: 1) it elicits a rapid release of enzyme from the cell through mechanisms which are independent of energy metabolism and protein synthesis; 2) it stimulates gene expression and synthesis of RNA independently of effects on hexose and amino acid transport; 3) it stimulates expression of the nuclear effects of the hormone at the translational level by increasing hexose uptake and the levels of regulatory glycolytic metabolites within the cells. Induction of the enzyme and its release thus appear