Changes in sterol biosynthesis accompanying cessation of glial cell growth in serum-free medium

Abstract

C-6 glioma cells, grown in medium supplemented with 5% delipidated foetal calf serum, were induced to enter a quiescent state by removing serum from the medium. Within 24h there was a 75-80% decline in the rate of incorporation of [(14)C]acetate or (3)H(2)O into digitonin-precipitable sterols. Experiments with [(3)H]mevalonolactone as a labelled sterol precursor suggested that the decline in sterol synthesis was regulated primarily at a point in the pathway before the formation of mevalonate. The specific activities of 3-hydroxy-3-methylglutaryl-CoA synthase and 3-hydroxy-3-methylglutaryl-CoA reductase decreased sharply in conjunction with the decline in sterol synthesis in the serum-free cultures; however, the activity of acetoacetyl-CoA thiolase was altered only slightly. The magnitude of the initial decline in reductase activity was not affected when 50-mm-NaF was included in the preincubation and assay buffers to prevent activation of physiologically inactive enzyme. However, after 6h of serum deprivation the decline in 3-hydroxy-3-methylglutaryl-CoA reductase activity was due to a decrease in the amount of latent activity. The sterol concentration in C-6 cells was unchanged after 24h in serum-free medium, although a 20% decrease in the sterol/fatty acid molar ratio occurred as a result of a small increase in the fatty-acid concentration. Incorporation of (3)H(2)O into fatty acids was inhibited in the serum-deprived glial cells; however, this inhibition developed more slowly and was not as pronounced as the diminution in sterol synthesis. The results suggest that in C-6 glia, which resemble the glial stem cells of the developing brain, the decreased demand for membrane sterols in the quiescent state results in a decline in sterol synthesis, mediated primarily through co-ordinate changes in the activities of 3-hydroxy-3-methylglutaryl-CoA synthase and 3-hydroxy-3-methylglutaryl-CoA reductase.

Relation of Cholesterol to Oligodendroglial Differentiation in C‐6 Glial Cells

“…When the sterol-depleted cells were placed in serum-free medium without compactin for 48 h, i.e., conditions previously shown to be necessary for induction of CNP (Maltese and Volpe, 1979), the decreased cholesterol/phospholipid ratios did not change (Table 1, 48 h). This was not unexpected, because under these culture conditions cell growth ceases (Maltese et al, 1980). Thus, by prior treatment of C-6 cells for 24 h in 10% lipoprotein-poor serum with added compactin, graded sterol depletion could be produced, and the degree of sterol depletion could be maintained upon change of medium to serum-free conditions.…”

Inhibition of hydroxymethylglutaryl-coenzyme A synthase by L-659,699.

“…The activity of this enzyme is affected by agents that alter cholesterol synthesis in the livers of chickens (8) and rats (8)(9)(10). Studies in Chinese hamster ovary cells (11,12), HeLa (13), C-6 glia cells (14,15), and L-M cells (16) have also demonstrated that cholesterol biosynthesis was sensitive to changes in the HMG-CoA synthase activity.…”

Studies on the stimulation of dolichol-mediated glycosylation by dexamethasone in HeLa cells

“…In the past few years compelling evidence has accumulated in the literature indicating that cell proliferation is closely linked to the synthesis of cholesterol (Chen et al, 1978: Maltese et al, 1980. As the cells reach confluency the synthesis of cholesterol diminished with concomitant decrease in the activity of HMG-CoA reductase (EC 1.1.1.34), the enzyme that under most circumstances is considered to be rate-limiting (Chen, 1981).…”