The 3-hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.34) activity of rat liver microsomal fraction varies diurnally: in rats allowed to feed at will and subjected to alternate 12h periods of light and darkness the activity is maximal during the dark period and minimal during the light period (Hamprecht et al., 1969;Shapiro & Rodwell, 1971;Higgins et al., 1971). The present communication describes daily changes in this enzyme activity in rats trained to feed during the daytime; such a study seemed relevant in view of the known tendency of rats to feed during the dark (Suttie, 1968).
ExperimentalFemale Wistar rats were purchased from the Manston Research Centre, Margate, Kent, U.K., and weighed 90g. They were kept in a room artificially illuminated between 09:00h and 21:00h, and in darkness between 21 :00h and 09:00h. Control animals were allowed continuous access to food, but test rats were fed from 09:00h to 12:00h each day. (All times quoted here refer to British Summer Time, which is 1 h ahead of Greenwich Mean Time.) Water was accessible at all times. Animals were maintained for 10 days before the experiment, and, beginning at 24:00h of day 10, measurements of enzyme activity were made; at 09:00h some of the test rats were fed as usual but others were given no food.Liver microsomal fraction was prepared as described by Tata (1969). Enzyme activity was measured by incubating microsomal preparations with 3-hydroxy-3-methyl[3-14C]glutaryl-CoA (specific radioactivity 0.60,uCi/p,mol) and a system for
Isolated luteal cells, prepared from superovulated rat ovaries by digestion with collagenase, were subjected to density-gradient centrifugation on Percoll to give a more highly purified preparation of luteal cells than has been reported previously. The cells formed progesterone when incubated in vitro; lutropin stimulated this steroidogenesis. Progesterone formation was linear for at least 2 h; a minimal lutropin concentration of 1.0 ng/ml was needed for stimulation and concentrations of 3.0 and 100 ng/ml gave half-maximal and maximal responses respectively. The cells were unresponsive towards hormones other than lutropin. Exposure to lutropin raised the cellular cyclic AMP concentration, and dibutyryl cyclic AMP, but not dibutyryl cyclic GMP, was as effective in stimulating steroidogenesis as was lutropin. Aminoglutethimide, an inhibitor of cholesterol side-chain cleavage, completely blocked progesterone formation by the cells, showing cholesterol side-chain cleavage to be an obligatory step in steroidogenesis by these cells. Neither the activity of 3-hydroxy-3-methylglutaryl-CoA reductase nor the incorporation of radioactively labelled acetate or mevalonate into cholesterol by cells incubated in vitro were detectable unless the rats had been treated previously with 4-aminopyrazolo[3,4-d]pyrimidine. In cells from rats so treated, compactin was found to block almost completely the incorporation of radioactively labelled acetate, but not of mevalonate, into cholesterol, indicating that this inhibitor acts in corpus luteum in the same way as it does in other tissues. In cells from rats not treated with 4-aminopyrazolo[3,4-d]pyrimidine compactin had no effect on progesterone formation in vitro, showing cholesterol biosynthesis to be unnecessary for the rapid steroidogenic response by luteal cells to lutropin.
Rat brain contains 3-hydroxy-3-methylglutaryl-CoA reductase activity, but this enzyme is far more active in 7-day-old brain than in adult brain. This difference may partly explain why cholesterol biosynthesis is more rapid in growing than in adult rat brain.
A new method suitable for measuring rat liver 3-hydroxy-3-methylglutaryl-CoA reductase activity is described and its advantages over methods previously available are discussed. An accurate time course was measured for the inhibition of liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase activity by dietary cholesterol; this enzyme was affected 1 1/4 h after the rats began to consume a cholesterol-rich diet. In this experiment there was no correlation between concentrations of microsomal cholesterol ester and the activity of 3-hydroxy-3-methylglutary-CoA reductase.
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