In previous investigations it was demonstrated that low density lipoprotein (LDL) was a principal source of the cholesterol used for steroidogenesis by human fetal adrenal (HFA) tissue. In the present investigation we sought to determine the capacity of HFA to synthesize cholesterol de novo. This was accomplished by measuring the rate of incorporation of [l4C]acetate into cholesterol by HFA tissue maintained in organ culture and determining the specific activity of 3-hydroxy- 3-methylglutaryl coenzyme A (HMG CoA) reductase in microsomes prepared from HFA tissue. Optimal conditions for measuring HMG CoA reductase activity in HFA tissue were established. The Km of HMG CoA reductase for HMG CoA and the Vmax were calculated to be 17 μM and 2.5 nmol mg-1 protein min-1, respectively in freshly prepared microsomes. The specific activities of the enzyme in microsomes prepared from tissue maintained in the presence or absence of ACTH (1 μg ml-1) for 4 days were 0.43 and 0.05 nmol mg-1 protein min-1, respectively. Optimal conditions for measuring the rate of incorporation of [l4C]acetate into cholesterol in HFA tissue were determined. The rate of incorporation of [l4C]acetate into cholesterol was linear with time for 12 h and linear with the concentration of [l4C]acetate up to 592 μM. The effect of duration of exposure of HFA tissue to ACTH on the rate of incorporation of [l4C]acetate nto cholesterol was determined. The rate of incorporation increased after 2 h of exposure to ACTH and continued to increase in a linear fashion until a maximal rate of incorporation was attained after 30 h of ACTH treatment. HFA tissue was maintainedin organ culture for 3 days in the presence and absence of ACTH and LDL (500 μg protein ml-1). Thereafter, the activity of HMG CoA reductase and the rate of incorporation of [MC]- acetate into cholesterol were determined. In the presenc of ACTH, the activity of HMG CoA reductase and the rate of incorporation of [l4C]acetate into cholesterol were markedly stimulated. When LDL was present in the medium there were only minimal reductions in both activities. We conclude from the results of these and previous studies that LDL utilization and de novo cholesterol synthesis are maximally active in ACTH-stimulated HFA tissue, thus ensuring an optimal supply of cholesterol for steroid biosynthesis.
ASJC Scopus subject areas