The effects of triiodothyronine, hydrocortisone and insulin on lipid synthesis by cultured fibroblasts preincubated in a serum-free medium.

Studies of lipid metabolism in cell cultures are usually carried out after preincubation of cells in media containing lipoprotein-deficient or delipidated serum. The artifacts produced during delipidation prevent the standardization of assays and the study of the role of hormones on lipid metabolism. We studied the effects of triiodothyronine, hydrocortisone, insulin and their combination on cholesterol and fatty acid synthesis in cultured human skin fibroblasts preincubated for 24 h in an artificial medium (medium A) consisting of equal volumes of Dulbecco's modified Eagle's and Ham's F-12 media enriched with transferrin, biotin and calcium pantothenate. In cells preincubated in medium A the incorporation of acetate to cholesterol and the activity of hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase were much lower than in cells preincubated in standard medium containing lipoprotein-deficient serum. Addition of the three hormones caused a marked stimulation of the incorporation of acetate to cholesterol (from 3.1 to 17.7 pmol/min per mg protein), an activity similar to that in cells preincubated in lipoprotein-deficient serum plus hormones. The stimulatory effect of the hormones on HMG-CoA reductase activity was smaller, from 11 to 26 pmol/min per mg protein compared to 83 pmol/min per mg protein in cells preincubated in lipoprotein-deficient serum plus hormones. Most of the stimulatory effect was due to insulin. The lack of coordinate response between these two parameters in cells preincubated in artificial medium could not be explained by (a) stimulation of a post-mevalonate step as measured by the incorporation of mevalonate to cholesterol; (b) the in vitro inactivation of HMG-CoA reductase by phosphorylation: incubation of fibroblast microsomes with Escherichia coli alkaline phosphatase resulted in a decrease in HMG-CoA reductase activity, in contrast to an increase in hepatic microsomes; (c) the presence of inhibitors of HMG-CoA reductase in the microsomal extract. In cells preincubated in medium A the incorporation of acetate to fatty acids and the activities of acetyl-CoA carboxylase and fatty acid synthetase were approximately equal to that of cells preincubated in standard medium containing lipoprotein-deficient serum. Hormones added to medium A caused a stimulation of incorporation of acetate to fatty acids (from 5.1 to 19.8 pmol/min per mg protein), the activity of acetyl-CoA carboxylase (from 494 to 820 pmol/min per mg protein) and of fatty acid synthetase (from 300 to 678 pmol/mg protein). These values were significantly higher than those obtained in cells preincubated with lipoprotein-deficient serum with or without hormones.(ABSTRACT TRUNCATED AT 400 WORDS)