beta-hydroxy-beta-methylglutaryl coenzyme A hydrolase of rat liver.

Beta-Hydroxy-beta-methylglutaryl coenzyme A hydrolase, or deacylase, (EC 3.1.2.5) is important, at least potentially, in the regulation of mammalian cholesterol synthesis. This is so for two reasons, both related to the enzyme generally regarded as rate-limiting for cholesterogenesis, namely beta-hydroxy-beta-methylglutaryl CoA reductase: (i) the hydrolase competes for the same substrate as the reductase and (ii) its end product, hydroxymethylglutamic acid, is a known inhibitor of the reductase. Consequently we have examined some of the properties of the hydrolase, as found in rat liver, after first developing a simple isotopic technique for its assay. Beta-Hydroxy-beta-methylglutaryl CoA hydrolase is both soluble and microsomal. The microsomal enzyme is inactivated by pre-incubation at 37 degree C, but not a 4 degree C, has an apparent pH optimum of approximately 7.6, and has Km and V values of 270 (microM) and 33.3 (nmol HMG/10 per mg protein), respectively, at 37 degree C. For the cytosolic enzyme the corresponding Km and V values are 830 and 111.1. From our observations it seems unlikely that beta-hydroxy-beta-methylglutaryl CoA hydrolase plays a significant role in the regulation of hepatic cholesterol synthesis since, in contrast to microsomal beta-hydroxy-beta-methylglutaryl coenzyme A reductase, we could find for the microsomal hydrolase no evidence of a diurnal rhythm of activity, no inhibition of activity by short-term cholesterol feeding and no evidence from Arrhenius-plot data for any membrane-mediated control of enzyme activity. Thus, the significance of the enzyme in mammalian systems remains unknown.

The lack of effect of fasting and alloxan-diabetes on the rate of fatty acid synthesis by some Morris hepatomas.

We have measured fatty acid synthesis (estimated by incorporation of [1-14C] acetate by tissue slices) by 3 transplantable Morris hepatomas of different growth rates--9618A, 7794A and 5123C--in response to fasting and alloxan-diabetes. Fasting did not alter fatty acid synthesis by the 3 hepatomas, bringing to 5 the number of tumors not tested and found similarly insensitive. Diabetes caused reduced fatty acid synthesis in 7794A and 5123C but not by 9618A. Host liver fatty acid synthesis was influenced by the presence of the tumor.

Exposure to an environment containing the aromatic red cedar, Juniperus virginiana: procarcinogenic, enzyme-inducing and insecticidal effects.

(1) Shavings from the Eastern Red Cedar (Juniperus virginiana) were examined for three diverse biological properties, i.e. enzyme induction, procarcinogenicity and insecticidal activity. (2) The ability of a cedar environment to stimulate liver drug-metabolizing enzymes in mice was confirmed by lowered values for barbiturate sleeping time. (3) In susceptible strains of mice (C3H-Avy, C3H-AvyfB and CBA/J) the use of cedar shavings as bedding increased significantly the incidence of spontaneous tumors of the liver and mammary gland, and also reduced the average time at which tumors appeared. (4) Cedar and some of its derivatives (Oil of Cedarwood, cedrene, cedrol) disrupted the reproductive and developmental cycle of a number of insects, including the Peanut Trash Bug (Elasmolomus sordidus), the Indian Meal Moth (Plodia interpunctella) and the Forage Mite (Tyrophagus putrescentiae).

Membrane homeostasis: is there an optimum level of membrane cholesterol?

The hypothesis is proposed that the relationship between the level of cholesterol in a cellular or sub-cellular membrane and the activity of any membrane function affected by cholesterol is ogival in nature, i.e, there is an optimum level of membrane cholesterol at which functional activity is maximal, and above or below this level activity declines. Data already published from other laboratories, when analysed in this manner, show clearly that this hypothesis appears to hold true for at least several membrane-bound systems, in particular the microsomal enzyme cerebroside sulfotransferase and transmembrane ATP/ADP exchange and glucose transport.

Homoeostatic control of membrane cholesterol and fatty acid metabolism in the rat liver.

Experiments were designed to assess the effect of cholesterol feeding, with or without high levels of either saturated (coconut oil) or unsaturated (sunflower-seed oil) fat on the fatty acid composition of hepatic microsomal membrane lipids, as well as on the activities of several membrane-bound enzymes of cholesterol synthesis and metabolism. Administration of 2% (w/w) cholesterol in the rat diet inhibited hydroxymethylglutaryl-CoA reductase activity, and this inhibition was much more pronounced when cholesterol was fed in combination with unsaturated rather than with saturated fat. Cholesterol 7 alpha-hydroxylase activity was increased by all the high-cholesterol diets and inhibited by both the high-fat diets. Cholesterol esterification, as assessed by acyl-CoA:cholesterol acyltransferase (ACAT) activity, was enhanced after unsaturated-fat feeding. Cholesterol supplement, without any added fat, failed to elicit any significant increase in ACAT activity, whereas consumption of cholesterol in combination with unsaturated fat led to the greatest increase in ACAT activity. After cholesterol feeding, C18:1 and C18:2 fatty acids in the microsomal phospholipids were increased, with concomitant decreases in C18:0, C20:4 and C22:6 fatty acids, leading to an overall decrease in membrane unsaturation, irrespective of the particular fat supplement. It can be concluded that the inhibition of cholesterol biosynthesis and the enhancement of cholesterol utilization, either by increased bile formation or by increased cholesterol esterification, after cholesterol feeding, may not be enough to prevent cholesterol accumulation in the microsomal membranes. Then, to compensate for the altered fluidity resulting from cholesterol enrichment, the unsaturation of membrane phospholipids is decreased, which would in turn have an effect on membrane lipid fluidity opposite to that of increased cholesterol.

Membrane-mediated control of hepatic beta-hydroxy-beta-methylglutaryl-coenzyme A reductase.

Previously we [Sabine & James (1976) Life Sci. 18, 1185--1192] proposed that 'the activity of hepatic beta-hydroxy-beta-methylglutaryl-coenzyme A reductase is critically regulated by the fluidity of its supporting microsomal membrane'. In the present work we examined further this concept of membrane-mediated control, with respect to the specific hypothesis that such control might function as a common mechanism both for the co-ordinated regulation of other enzymes affected by cholesterol feeding and also for the subcellular integration of the several physiological factors known to influence this enzyme's activity. Contrary to earlier expectations, this hypothesis now appears not to hold. We report here that, under those conditions of short-term cholesterol feeding that affected the reductase, a variety of other microsomal enzymes did not display membrane-function interactions, i.e. neither enzymes involved in cholesterol metabolism and also affected by cholesterol feeding (cholesterol 7 alpha-hydroxylase), nor those involved in cholesterol metabolism and not affected by cholesterol feeding (hydroxymethylglutaryl-CoA hydrolase, acyl-CoA:cholesterol acyltransferase), nor those not directly involved in cholesterol metabolism at all (glucose 6-phosphatase). Furthermore, we observed no evidence for the operation of membrane-mediated control of the reductase in other situations known to influence its activity, i.e. starvation, diurnal rhythm, the very early stages of cholesterol feeding and various manipulations in vitro.

Membrane homeostasis: thermotropic behaviour of microsomal membrane lipids isolated from livers of rats fed cholesterol-supplemented diets.

Differential scanning colorimetry (DSC) has been applied to study the phase transition properties of isolated lipids from liver microsomal membranes of rats fed high cholesterol diets with or without high levels of either saturated (coconut oil) or unsaturated (sunflower seed oil) fat. DSC of aqueous buffer dispersions of liver microsomal lipids exhibited two independent, reversible phase transitions. The dietary cholesterol treatments had their major effect on the temperature at which the lower phase transition (T1) occurred. This transition occurred at a lower temperature when cholesterol was added to the diet, irrespective of the nature of the fatty acid supplement. However the magnitude of decrease was more when cholesterol was fed with sunflower seed oil. Inclusion of cholesterol into the rat diets also lowered the enthalpy values for the lower phase transition (T1). No appreciable effect on the temperature of the higher phase transition (T2) was observed, however the enthalpy values were slightly decreased by cholesterol feeding. These results suggest that certain domains of microsomal lipids, probably containing some relatively higher melting-point lipids, independently undergo solidus or gel formation and this transition (T2) is not greatly affected by dietary cholesterol. On the other hand, domains representing the bulk of the microsomal lipids undergo a phase change (T1) at temperatures which are dependent on cholesterol content and fatty acid profiles of the membrane, which are in turn, modified by dietary cholesterol intake.