A Simple Model for Studies on the Regulation of Cholesterol Synthesis Using Freshly Isolated Hepatocytes

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Abstract
Rat hepatocytes isolated by the procedure described here showed 3-hydroxy-3-methylglutaryl-CoA [HMG-CoA] reductase activity in the range of that reported for rat liver at the maximum of the circadian cycle, even if they were taken from rats at the time of the minimum. The enzyme was present in cells as both its active dephosphorylated (20 .+-. 8%) and the inactive phosphorylated forms. The enzyme activity and the ratio between the 2 forms were unaltered during 3 h of cell incubation. 25-Hydroxycholesterol (50 .mu.M) induced .apprx. 50% inhibition of HMG-CoA reductase activity during 1 h incubation but the relative amount of the 2 forms was not modified by the sterol. Cells isolated by the described procedure may therefore be a useful tool in studies on the regulation of cholesterol neogenesis, both through the synthesis of the enzyme, which can be shown by measuring the activity after complete dephosphorylation of the enzyme, and via the rapid reversible shift of the inactive to the active form, resulting from the ratio between the 2 enzyme forms. The latter mechanism for the modulation of cholesterol synthesis cannot be tested in cell cultures because full activation of the enzyme occurs during hepatocyte plating.