Diurnal changes in the fraction of 3-hydroxy-3-methylglutaryl-CoA reductase in the active form in rat liver microsomal fractions

Abstract

'Initial' and 'total' activities of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) were measured in cold-clamped samples of liver from rats at 2h intervals throughout the 24h light/dark cycle. Initial activities were obtained in microsomes (microsomal fractions) isolated and assayed in the presence of 100mM-KF, whereas 'total' activities were measured in microsomes prepared from the same homogenates but washed free of KF and incubated with exogenous partially purified rat liver protein phosphatase. The initial/total-activity ratio for HMG-CoA reductase underwent a diurnal cycle, which had a nadir 4h into the light phase (when initial activity was 28% of total activity) and a peak 12h later, i.e. 4h into the dark phase (when initial activity was 80% of total activity). These low and high points of the cycle were separated by gradual steady changes in the ratio. The characteristics of this diurnal cycle were different from those of the cycle observed for total activity, which had a plateau of high activity between 2 and 10h into the dark cycle preceded and succeeded by a very rapid increase and decrease, respectively, in the total activity of HMG-CoA reductase. The combination of the two cycles resulted in the dampening of the resultant cycle for the initial or effective activity of HMG-CoA reductase, such that the changes in initial activity around the beginning and and end of the dark phase were more gradual than would otherwise have been the case if the initial/total-activity ratio for HMG-CoA reductase were constant throughout the diurnal cycle. The physiological implications of the observed diurnal variation in the fraction of hepatic HMG-CoA reductase in the active form are discussed.

A common bicyclic protein kinase cascade inactivates the regulatory enzymes of fatty acid and cholesterol biosynthesis

“…700-lOOO-fold to a final specific activity of 30-50 units/mg: details of the purification will be published elsewhere. Rat liver microsomes were prepared as described in [17]. HMG-CoA reductase-overproducing hamster cells (UT-l cells) were grown in roller bottles in 40 PM compactin [18].…”

Acute effects of starvation and treatment of rats with anti-insulin serum, glucagon and catecholamines on the state of phosphorylation of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase in vivo

“…Rats were anaesthetized (60 mg of pentobarbitone/kg body wt.) 20 min before liver sampling; stress was lessened by handling of the animals for 5-7 days before the experiments (see Easom & Zammit, 1984b). No systematic study of the effects of anaesthesia on reductase activity was undertaken.…”

“…However, in these latter studies the E/T activity ratios observed for saline-treated (control) animals were very low (< 15%) and outside the physiological range (Easom & Zammit, 1984b. Consequently, a re-evaluation of the effects of glucagon on hepatic HMG-CoA reductase activity in vivo is required.…”

“…The source and maintenance of the male Wistar rats used (140-160 g) were as described previously (Easom & Zammit, 1984b Sampling of liver and preparation of microsomes (microsomal fractions) These procedures were performed as described previously (Easom & Zammit, 1984a). Rats were anaesthetized (60 mg of pentobarbitone/kg body wt.)…”

“…A physiological role for changes in the phosphorylation state of hepatic HMG-CoA reductase (EC 1.1.1.34) in vivo has been established through the demonstration this parameter varies markedly in response to normal metabolic stimuli, such as those generated during the diurnal feeding cycle of the rat (Easom & Zammit, 1984b). In addition, it has been shown that the fraction of HMG-CoA reductase in the active, dephosphorylated, state can be altered chronically in response to streptozotocin-induced insulin deficiency (Easom & Zammit, 1985).…”