Enhancement of microsomal phosphatidate phosphohydrolase and diacylglycerol acyltransferase activity by insulin during growth of rat adipocyte precursors in culture

Abstract

The availability of a propagating adipocyte precursor culture system has provided the opportunity to study biochemical processes under conditions in which any known interacting influences are controlled. We have studied the activity of various triacylglycerol-biosynthetic enzymes during maturation of rat epididymal adipocyte precursors and any possible effect of insulin on enzyme activity. At certain times in culture, the specific activity of microsomal phosphatidate phosphohydrolase (EC 3.1.3.4) and diacylglycerol acyltransferase (EC 2.3.1.20) is significantly enhanced in cells grown in the presence of added insulin. Under the culture conditions used in this study, the adipocyte precursors acquire several small lipid inclusions and become rounder, but do not assume the signet-ring appearance of mature fat cells during the first 2 weeks of monolayer confluence. Consequently, the effects of the hormone on enzyme activity become evident prior to complete morphological maturation. Phosphatidate phosphohydrolase is believed to be a rate-controlling enzyme in triacylglycerol synthesis in adipose tissue and liver. The fact that the adipocyte precursor microsomal, rather than cytosolic, phosphohydrolase is influenced by insulin suggests that the membrane-bound enzyme is the regulatory phosphohydrolase in intact cells. The enhancement of diacylglycerol acyltransferase activity may be of significance in the reesterification of fatty acids with diacylglycerols, a reaction that by passes the phosphohydrolase step. Thus, in addition to the well-known mechanisms by which insulin promotes triacylglycerol accretion in adipocytes and their precursors, the hormone significantly enhances the specific activity of critical enzymes of triacylglycerol synthesis.