Lipoprotein Lipase Activity In Cells Research Articles

Treatment of cardiac myocytes with 8-(4-chlorophenylthio)-adenosine 3′,5′-cyclic monophosphate, forskolin or cholera toxin does not stimulate cellular or heparin-releasable lipoprotein lipase activities

Incubation of isolated cardiac myocytes with 500 microM-8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP) or 100 microM-forskolin for 2 1/2 h did not increase the heparin-induced release of lipoprotein lipase (LPL) into the medium. When LPL activity in cardiac myocytes was depleted by treatment of rats with cycloheximide (2 mg/kg; 2.5 h) and inclusion of the protein-synthesis inhibitor in the isolation solutions, incubation with CPT-cAMP or forskolin did not influence the rate of repletion of LPL activity in cells or the recovery of heparin-releasable LPL activity. Although the administration of cholera toxin (0.5 mg/kg; 16-17 h) to rats increased LPL activity in a low-speed supernatant fraction from heparin-perfused hearts, LPL activity was not increased in cardiac myocytes from cholera-toxin-treated rat hearts, and the heparin-induced release of LPL was unchanged. Incubation of cultured ventricular myocytes with 1 microgram of cholera toxin/ml or 500 microM-CPT-cAMP for 24 h did not increase cellular LPL activity or LPL released into the culture medium after a 40 min incubation with heparin. Therefore interventions that stimulate adenylate cyclase activity (forskolin, cholera toxin) or incubation with CPT-cAMP do not increase cellular LPL activity or promote the translocation of LPL to a heparin-releasable fraction in cardiac myocytes.

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

Release of lipoprotein lipase from fat cells in vitro

Release of lipoprotein lipase from rat fat cells incubated at 20°C in medium with albumin, but without glucose proceeded at a constant rate for 30 min. The initial rate of release was increased when serum was present in the medium. Maximal stimulation (100–300%) was produced with 3.8% serum. The maximal increment in release caused by serum was always greater than that produced by heparin and when both were added release was greater than it was with either one alone. The active component(s) of serum, nondialyzable and stable for 30 min at 56°C, was present in sera from humans and rats in the fed or fasted state. Glucose plus insulin (but neither alone) enhanced the rate of lipase release in the presence of serum but not in its absence. The half-life of the lipase in basal medium at 20° C was 90 min. Heparin decreased this to about 50 min and serum markedly prolonged it whether or not heparin was present. Lipoprotein lipase activity in cells and fractions thereof was assayed in extracts of acetone powders. After centrifugation of fat cell homogenates at 600 × g for 15 min, only 50–60% of the activity was recovered in the supernatant. After centrifugation at 100 000 × g for 60 min, the supernatant contained about 10% of the total activity and the sediment 40%. In some experiments, most of the rest was recovered in the floating fat fraction. Total lipoprotein lipase activity of cells plus medium increased steadily during incubation of fat cells for 1 h at 30° C. The major increment occurred in the cells and activity in the medium was always less than 15% of the total. Our observations are consistent with the view that activation may be an important determinant of fat cell lipoprotein lipase activity as well as an integral part of the release process.