Increased sensitivity to dietary cholesterol in diabetic and hypothyroid rats associated with low levels of hepatic HMG-CoA reductase expression

We recently postulated that hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase functions as a cholesterol buffer to protect against the serum and tissue cholesterol raising action of dietary cholesterol. This postulate predicts that diminished basal expression of hepatic HMG-CoA reductase results in increased sensitivity to dietary cholesterol. Because diabetic and hypothyroid animals are known to have markedly reduced hepatic HMG-CoA reductase, these animals were selected as models to test our postulate. When rats were rendered diabetic with streptozotocin, their hepatic HMG-CoA reductase activity decreased from 314 to 22 pmol. min(-1). mg(-1), and their serum cholesterol levels increased slightly. When the diabetic animals were challenged with a diet containing 1% cholesterol, their serum cholesterol levels doubled, and their hepatic reductase activity decreased further to 0.9 pmol. min(-1). mg(-1). Hepatic low-density lipoprotein (LDL) receptor immunoreactive protein levels were unaffected in the diabetic rats whether fed cholesterol-supplemented diets or not. In rats rendered hypothyroid by thyroparathyroidectomy, serum cholesterol levels rose from 100 to 386 mg/dl in response to the 1% cholesterol challenge, whereas HMG-CoA reductase activity dropped from 33.8 to 3.4 pmol. min(-1). mg(-1). Hepatic LDL receptor immunoreactive protein levels decreased only slightly in the hypothyroid rats fed cholesterol-supplemented diets. Taken together, these results show that rats deficient in either insulin or thyroid hormone are extremely sensitive to dietary cholesterol largely due to low basal expression of hepatic HMG-CoA reductase.     

Hepatic HMG-CoA reductase expression and resistance to dietary cholesterol

The premise that the intrinsic level of expression of hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase determines the relative sensitivity to the serum cholesterol raising action of dietary cholesterol was examined in 9 strains of rat. For further comparison purposes, hamsters were also examined. The basal expression of hepatic HMG-CoA reductase, extent of feedback regulation by cholesterol, and changes in serum cholesterol levels and the hepatic low-density lipoprotein (LDL) receptor in response to cholesterol challenge were determined in these animals. The Sprague-Dawley, Wistar-Furth, Spontaneously Hypertensive, Lewis, and Wistar-Kyoto rats were all very resistant to dietary cholesterol and exhibited hepatic HMG-CoA reductase activities above 150 pmol / min(-1) / mg(-1). The Buffalo, Brown Norway, and Copenhagen 2331 rats had hepatic HMG-CoA reductase activities below 90 pmol / min(-1) / mg(-1) and had increases in serum cholesterol levels ranging from 12 to 33 mg/dl when given a 4-day, 1% cholesterol challenge. The extent of feedback regulation was reduced to only 3-fold in the Fisher 344 and Brown Norway rats that exhibited significant increases in serum cholesterol levels when given a cholesterol challenge. The Golden Syrian hamsters exhibited the largest increase (197 mg/dl) in serum cholesterol levels in response to dietary cholesterol and the lowest basal expression of hepatic HMG-CoA reductase (3.3 pmol / min(-1) / mg(-1)). Hepatic LDL receptor levels were not significantly decreased by dietary cholesterol in any of the animals. The data from these inbred rats and the hamsters strongly support the conclusion that the animals expressing the highest levels of hepatic HMG-CoA reductase are the most resistant to the serum cholesterol raising action of dietary cholesterol.     

Acyl-CoA: cholesterol acyltransferase and 3-hydroxy-3-methylglutaryl-CoA reductase in carp-liver microsomes: effect of cold acclimation on enzyme activities and on hepatic and plasma lipid composition.

Hepatic microsomal activities of acyl-CoA:cholesterol acyltransferase (ACAT) and 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, rate-limiting enzymes in cholesterol esterification and cholesterol synthesis, and the concentration sand compartmentalization of esterified and unesterified cholesterol, were studied in carp acclimated to 10 and 30 degrees C. Irrespective of acclimation temperature, carp-liver ACAT is characterized by an apparent Km-value for oleoyl-CoA of 11-15 microM and displays an optimum activity at pH 7.4. The enzyme activity is reduced approx. 2-fold upon preincubation of microsomes with alkaline phosphatase. Arrhenius plots of ACAT-activity are curvilinear, with curvatures considerably affected by the acclimation temperature of the fish. Carp HMG-CoA reductase has been characterized previously by Teichert and Wodtke ((1987) Biochim. Biophys. Acta 920, 161-170). When measured at 30 degrees C, ACAT activities from 30 degrees C- and 10 degrees C-acclimated carp are identical (approx. 6 pmol/min per mg protein), whilst 'expressed' HMG-CoA reductase activity (18.1 +/- 12.2 pmol/min per mg protein for 30 degrees C-acclimated carp vs. 159.8 +/- 106.6 pmol/min per mg protein for 10 degrees C-acclimated carp) is enhanced 9-fold in the cold environment. This disparity indicates that cold-acclimation results in a massive increase in the capacity for hepatic cholesterol synthesis relative to hepatic cholesterol esterification. At the same time, hepatic compositional analysis reveals identical contents of unesterified cholesterol in either groups of carp but significantly decreased (3-fold) amounts in cholesterol ester (and also in triacylglycerol, 4-fold) in cold-acclimated carp. Moreover, microsomal fractions display lower cholesterol to phospholipid ratios in the cold. In contrast, concentrations of either cholesterol fractions (and of triacylglycerols) in plasma--the mobile compartment for lipoprotein transport--do not differ in cold- and warm-acclimated carp. Based on current concepts of cholesterol metabolism, it is concluded that the cold-enhanced expression of hepatic HMG-CoA reductase activity is a homeostatic response directed against and compensating for a cold-induced but not yet characterized deficiency in hepatic cholesterol availability.     

Regulation of hepatic cholesterol metabolism in humans: stimulatory effects of cholestyramine on HMG-CoA reductase activity and low density lipoprotein receptor expression in gallstone patients

To characterize the metabolic regulatory response to interruption of the enterohepatic circulation of bile acids, we examined the effects of cholestyramine treatment on the rate-limiting steps in cholesterol biosynthesis (HMG-CoA reductase) and bile acid production (cholesterol 7 alpha-hydroxylase) as well as on the heparin-sensitive binding of low density lipoproteins (LDL) (reflecting LDL receptor expression) in human liver. Altogether, 18 normolipidemic patients with uncomplicated cholesterol gallstone disease were treated with cholestyramine (8 g b.i.d.) for 2-3 weeks prior to cholecystectomy, and another 34 cholesterol gallstone patients served as untreated controls. Cholestyramine treatment stimulated cholesterol 7 alpha-hydroxylase more than sixfold, and increased both HMG-CoA reductase activity (552 +/- 60 pmol/min per mg protein vs 103 +/- 9 pmol/min per mg protein) and LDL receptor expression (6.1 +/- 0.8 ng/mg protein; n = 6 vs 2.2 +/- 0.3 ng/mg protein; n = 7). Moreover, there was a good correlation between HMG-CoA reductase activity and LDL receptor binding (rs = +0.71; n = 13), suggesting a simultaneous stimulatory effect to compensate for the increased hepatic cholesterol catabolism due to bile acid depletion caused by cholestyramine. Further evidence for this assumption was the finding of a significant relationship between cholesterol 7 alpha-hydroxylase activity and both LDL receptor expression (rs = +0.77; n = 13) and HMG-CoA reductase activity (rs = +0.76; n = 46). We conclude that in human liver a parallel stimulation of cholesterol synthesis and LDL receptor expression occurs in response to stimulation of bile acid synthesis.     

Indirect regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase by oestradiol in the rabbit corpus luteum

Rabbits were given 50 i.u. hCG, i.v., to initiate ovulation and pseudopregnancy (Day 0) and were treated, s.c., with or without a 1-cm Silastic oestradiol implant. Serum progesterone concentrations were measured at 4-day intervals and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity was estimated by the conversion of HMG to mevalonate in microsomes from corpora lutea removed on Days 4, 8, 12, 16 and 20 of pseudopregnancy (4 rabbits/day). Total HMG-CoA reductase activity was significantly (P less than 0.05) higher in control rabbits on Days 8 and 12 (5.29 +/- 0.63 and 5.5 +/- 0.28 nmol/min/mg protein, respectively) compared to oestradiol-treated rabbits (2.57 +/- 0.25 and 4.03 +/- 0.23 nmol/min/mg protein, respectively). On Days 16 and 20, total HMG-CoA reductase activity was not different in control and oestradiol-treated animals. There was no difference in the levels of the active fraction of HMG-CoA reductase, which represented less than 20% of the total enzyme activity, in control and oestradiol-treated rabbits (less than 780 pmol/min/mg protein, Day 12). These results indicate that oestradiol does not alter the active form, but can reduce the total activity of HMG-CoA reductase in the rabbit corpus luteum without a decline in serum progesterone. Therefore, neither total nor active forms of HMG-CoA reductase are directly related to progesterone secretion. This suggests that other sources of cholesterol may contribute to progesterone production in the rabbit.     

Increased 3-hydroxy-3-methyl-glutaryl coenzyme A reductase activity in a virilizing adrenal carcinoma

HMG-CoA reductase activity was determined on microsomal preparations of an adrenal carcinoma and on a control adrenal obtained from palliative surgery for breast carcinoma. In both tissues we also measured [14C]pyruvate incorporation to study the formation of sterols. The endogenous adrenal content of cholesterol and its esters was quantitated. The content of various steroids was also determined in tissues and media before and after incubations in Krebs-Ringer. The carcinoma had a HMG-CoA reductase activity of 972.0 pmol/mg protein/min vs 13.8 for the control adrenal. The tumor incorporated 4.6 pmol of [14C]pyruvate per mg protein per 90 min into digitonin precipitable sterols compared to 0.5 pmol found for the control gland. Free cholesterol and cholesterol esters in tumoral tissue were 0.09/100 mg and 0.02/100 mg tissue respectively, compared to 0.18 and 2.56 in control tissue. The output of corticosteroids and androgens was very high when calculated for the whole tumor. These results suggest that the carcinoma had acquired a high capacity for de novo synthesis of cholesterol which could have served as substrate for the observed high plasma androgen level.     

Lipid-lowering efficacy of 3,4-di(OH)-phenylpropionic L-leucine in high-cholesterol fed rats

A preliminary study revealed that 3,4-di(OH)-hydrocinnamate (HC), a polyphenolic compound, lowered the plasma lipids in high-cholesterol fed rats. Accordingly, this study was designed to test the lipid-lowering efficacy of a synthetic derivative, 3,4-di(OH)-phenylpropionic (L-leucine) amide (PPLA), in rats fed a high-cholesterol (1%, wt/wt) diet. As such, HC or PPLA was given as supplement to a high-cholesterol diet for 6 weeks at a dose of 0.137 mmol/100 g diet. The supplementation of HC and PPLA significantly lowered the plasma and hepatic cholesterol and triglyceride levels compared to the control group. The activities of hepatic HMG-CoA reductase (164 +/- 9.12 and 124.74 +/- 17.09 pmol/min/mg protein vs. 245.41 +/- 13.01 pmol/min/mg protein, p < 0.05) and ACAT (411.49 +/- 11.48 and 334.35 +/- 17.68 pmol/min/mg protein vs. 490.41 +/- 16.69 pmol/min/mg protein, p < 0.05) were significantly lower in the HC- and PPLA-supplemented groups than in the control group. However, PPLA was more effective in inhibiting the enzyme activities than HC. The excretion of neutral sterol was significantly higher in HC- and PPLA-supplemented groups than in the control group. Therefore, these results indicate that PPLA, a leucine-attached version of HC, exhibited a similar significant hypocholesterolemic effect to HC in rats fed a high-cholesterol diet.     

Hydroxymethylglutaryl CoA reductase and the modulation of microsomal cholesterol content by the nonspecific lipid transfer protein

The influence of membrane cholesterol content on 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reductase, EC 1.1.1.34) in rat liver microsomes was investigated. Microsomes were enriched in cholesterol by incubation with egg phosphatidylcholine-cholesterol vesicles and the nonspecific lipid transfer protein from rat liver. By this method, the microsomal cholesterol content was 2.5-fold enhanced up to final concentrations of 140 nmol cholesterol per mg microsomal protein. In another experiment, microsomes isolated from rats fed a cholesterol-rich diet were depleted of cholesterol by incubation with egg phosphatidylcholine vesicles and the transfer protein. Both cholesterol enrichment and depletion had virtually no effect on the microsomal HMG-CoA reductase activity. In another set of experiments, normal rat liver microsomes were incubated with human serum, resulting in a rise of microsomal cholesterol content. This was reflected in an increase of acyl-CoA:cholesterol acyltransferase activity but failed to have an effect on HMG-CoA reductase.     

Properties of purified rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase and regulation of enzyme activity

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase from rat liver microsomes has been purified to apparent homogeneity with recoveries of approximately 50%. The enzyme obtained from rats fed a diet supplemented with cholestyramine had specific activities of approximately 21,500 nmol of NADPH oxidized/min/mg of protein. After amino acid analysis a specific activity of 31,000 nmol of NADPH oxidized/min/mg of amino acyl mass was obtained. The s20,w for HMG-CoA reductase was 6.14 S and the Stokes radius was .39 nm. The molecular weight of the enzyme was 104,000 and the enzyme subunit after sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 52,000. Antibodies prepared against the homogeneous enzyme specifically precipitated HMG-CoA reductase from crude and pure fractions of the enzyme. Incubation of rat hepatocytes for 3 h in the presence of lecithin dispersions, compactin, or rat serum resulted in significant increases in the specific activity of the microsomal bound reductase. Immunotitrations indicated that in all cases these increases were associated with an activated form of the reductase. However activation of the enzyme accounted for only a small percentage of the total increase in enzyme activity; the vast majority of the increase was apparently due to an increase in the number of enzyme molecules. In contrast, when hepatocytes were incubated with mevalonolactone the lower enzyme activity which resulted was primarily due to inactivation of the enzyme with little change in the number of enzyme molecules. Immunotitrations of microsomes obtained from rats killed at the nadir or peak of the diurnal rhythm of 3-hydroxy-3-methylglutaryl-CoA reductase indicated that the rhythm results both from enzyme activation and an increased number of reductase molecules.     

3-Hydroxy-3-methylglutaryl coenzyme A reductase activity in the human gastrointestinal tract

Activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34) was measured in intestinal mucosa of the human gastrointestinal tract. Activity was highest in gastric mucosa (18.2 pmol per mg per min) and there was a constant low level in the small bowel and colon (approximately 10 pmol per mg per min). Phosphorylation/dephosphorylation modulation of intestinal reductase activity was demonstrated in normal mucosa. Expressed jejunal reductase activity was significantly higher in celiac sprue mucosa and mucosa from defunctionalized intestine of jejunoileal bypass patients. Enzyme activity also increased during 24-hr mucosal organ culture in the absence of exogenous cholesterol. Addition to the culture medium of pure cholesterol or 25-hydroxycholesterol dissolved in a small volume of ethanol suppressed the culture-induced increase to 86 +/- 3% and 69 +/- 5% of paired controls, respectively. This evidence suggests that a moderate degree of feedback regulation of intestinal cholesterol synthesis by luminal sterol occurs in man. Mucosal HMG-CoA reductase activity was also measured in patients with hyperlipoproteinemia. Patients with either predominant hypercholesterolemia or predominant hypertriglyceridemia lipid profiles had "normal" expressed reductase activity, but feedback regulation by free cholesterol could not be demonstrated in either group under these conditions.     

Abnormal cholesterol metabolism in renal clear cell carcinoma

The clear cell form of renal cell carcinoma is known to derive its histologic appearance from accumulations of glycogen and lipid. We have found that the most consistently stored lipid form is cholesteryl ester. Clear cell cancer tissue contained 8-fold more total cholesterol and 35-fold more esterified cholesterol than found in normal kidney. Cholesteryl ester appeared to be formed intracellularly since it was not membrane-bound and since oleate was the predominant form, as opposed to linoleate in lipoprotein cholesteryl esters. The cholesterol in clear cell tumors did not appear to be a result of excessive synthesis from acetate since HMG-CoA reductase (EC 1.1.1.34) activity was lower in cancer tissue than in normal kidney (2.9 +/- 0.8 vs. 7.2 +/- 1.2 pmol/mg of protein per min). In contrast, intracellular activity of fatty acyl-coenzyme A:cholesterol acyl transferase (ACAT, EC 2.3.1.26) was higher in tumor tissue than in normal kidney (2405 +/- 546 vs. 1326 +/- 301 pmol/mg of protein per 20 min) while cytosolic cholesteryl ester hydrolase activity appeared normal. Cholesteryl ester storage in clear cell renal cancer may be a result of a primary abnormality in ACAT activity or it may be a result of reduced release of free cholesterol (relative to cell content) with a secondary elevation in ACAT activity.     

Instructions for authors

The aim of the present study was to examine hypothesis that the enhanced cholesterologenesis, found in rats with experimental chronic renal failure (CRF) resulted from the increased gene expression of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase – the rate limiting enzyme in the cholesterologenesis pathway, responsible for mevalonate synthesis. Wistar rats were used and experimental CRF was achieved by 5/6 nephrectomy model. We examined: (a) the changes in the rat liver microsomal HMG-CoA reductase activity, (b) the rat liver HMG-CoA reductase mRNA abundance in various times of day. Obtained data indicates that the increased activity of HMG-CoA reductase in the liver of rats with experimental CRF parallel enhanced mRNA level and suggests that enhanced cholesterol biosynthesis, observed in experimental CRF is at least in part due to the increased HMG-CoA reductase gene expression. The results also indicate that the physiological diurnal rhythm of HMG-CoA reductase activity is preserved in the course of experimental CRF.     

Hepatic cholesterol metabolism in cholesterol gallstone disease

Hepatic cholesterol metabolism was examined in 27 Swedish patients with cholesterol gallstone disease and in 13 patients free of gallstones operated for roentgenographically suspect polyps in the gallbladder. All 40 patients underwent cholecystectomy, and a liver biopsy and gallbladder bile were obtained at surgery. The cholesterol saturation of gallbladder bile was significantly higher in patients with gallstones compared to the gallstone-free controls (131 +/- 13 vs. 75 +/- 5%, P less than 0.001). Microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity, governing cholesterol synthesis, did not differ between gallstone and gallstone-free patients (104 +/- 11 vs. and 109 +/- 22 pmol/min per mg protein, respectively). The activity of cholesterol 7 alpha-hydroxylase, catalyzing the catabolism of cholesterol to bile acids, was not significantly decreased in gallstone patients (6.2 +/- 1.1 vs. 8.0 +/- 2.0 pmol/min per mg protein). The capacity to esterify cholesterol, judged by the activity of acyl coenzyme A:cholesterol acyltransferase (ACAT), was similar in gallstone and gallstone-free patients (5.4 +/- 0.4 vs. 6.7 +/- 1.1 pmol/min per mg protein). In the presence of exogenous cholesterol, ACAT activity increased by more than fourfold in both groups. No correlation was found between the saturation of gallbladder bile and any of the mentioned enzyme activities in gallstone patients. It is concluded that distinct abnormalities in cholesterol metabolizing enzymes are not of major importance for development of gallstones in Swedish patients with cholesterol gallstone disease. The results support the contention that the etiology of cholesterol gallstones is multifactorial.     

Lovastatin treatment inhibits sterol synthesis and induces HMG-CoA reductase activity in mononuclear leukocytes of normal subjects

The mechanism by which competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase decrease serum cholesterol is incompletely understood. The few available data in humans suggest that chronic administration of the competitive inhibitor, lovastatin, decreases serum cholesterol with little or no change in total body sterol synthesis. To further define the effect of lovastatin on cholesterol synthesis in normal subjects, we investigated the effect of a single oral dose of lovastatin and a 4-week treatment period of lovastatin on mononuclear leukocyte (ML) sterol synthesis as a reflection of total body sterol synthesis. In parallel, we measured serum lipid profiles and HMG-CoA reductase activity in ML microsomes that had been washed free of lovastatin. ML sterol synthesis did not significantly decrease (23 +/- 5%, mean +/- SEM) at 3 h after a single 40-mg dose of lovastatin. With a single oral 80-mg dose, ML sterol synthesis decreased by 57 +/- 10% (P less than 0.05) and remained low for the subsequent 6 h. With both doses, total HMG-CoA reductase enzyme activity in microsomes prepared from harvested mononuclear leukocytes was induced 4.8-fold (P less than 0.01) over baseline values. Both the 20-mg bid dose and the 40-mg bid dose of lovastatin administered for a 4-week period decreased serum cholesterol by 25-34%. Lovastatin at 20 mg bid decreased ML sterol synthesis by 23 +/- 6% (P less than 0.02) and increased ML HMG-CoA reductase 3.8 times (P less than 0.001) the baseline values. Twenty four hours after stopping lovastatin, ML sterol synthesis and HMG-CoA reductase enzyme activity had returned to the baseline values. The higher dose of lovastatin (40 mg bid) decreased ML sterol synthesis by 16 +/- 3% (P less than 0.05) and induced HMG-CoA reductase to 53.7 times (P less than 0.01) the baseline value at 4 weeks. Stopping this higher dose effected a rebound in ML sterol synthesis to 140 +/- 11% of baseline (P less than 0.01), while HMG-CoA reductase remained 12.5 times baseline (P less than 0.01) over the next 3 days. No rebound in serum cholesterol was observed. From these data we conclude that in normal subjects lovastatin lowers serum cholesterol with only a modest effect on sterol synthesis. The effect of lovastatin on sterol synthesis in mononuclear leukocytes is tempered by an induction of HMG-CoA reductase enzyme quantity, balancing the enzyme inhibition by lovastatin.(ABSTRACT TRUNCATED AT 400 WORDS)     

3-Hydroxy-3-methylglutaryl-coenzyme A reductase in normal and dystrophic hamsters.

The activity and diurnal variation of 3-hydroxy-3-methyglutaryl-CoA reductase (EC 1.1.1.34; HMG-CoA reductase), the rate-limiting enzyme in the cholesterol-biosynthetic pathway, of normal and dystrophic hamsters was determined. Liver enzyme activity showed a diurnal pattern in the normal male, but not in the dystrophic male. Enzyme values in normal males at the midpoint of the 12 h dark period were 10 times those in dystrophic males. No evidence for diurnal variation in the HMG-CoA reductase of the brain was observed, and similar activities were found for normal and dystrophic animals. The apparent Km for HMG-CoA reductase from the liver of normal or dystrophic hamsters was approx. 9 microM, and the Vmax. was 5.9 and 21.7 pmol/min per mg of protein for dystrophic and normal hamsters respectively.     

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.

The fraction of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase in the dephosphorylated (active) form in rat liver in vivo was measured after various experimental treatments of animals. Intraperitoneal injection of glucose (to raise serum insulin concentrations) into rats 4 h into the light phase (L-4) resulted in a transient (30 min) increase in the expressed (E)/total (T) activity ratio of HMG-CoA reductase without any change in total activity (obtained after complete dephosphorylation of the enzyme). Conversely, intravenous injection of guinea-pig anti-insulin serum into rats 4 h into the dark phase (D-4) significantly depressed the E/T ratio within 20 min. Intravenous injection of glucagon into normal rats at this time point did not affect the degree of phosphorylation of the enzyme, in spite of a 10-fold increase in hepatic cyclic AMP concentration induced by the hormone treatment. A 3-fold increase in the concentration of the cyclic nucleotide induced by adrenaline infusion was similarly ineffective in inducing any change in expressed or total activities of hepatic HMG-CoA reductase. However, when insulin secretion was inhibited, either by the induction of streptozotocin-diabetes or by simultaneous infusion of somatostatin, glucagon treatment was able to depress the expressed activity of HMG-CoA reductase (i.e. it increased the phosphorylation of the enzyme). Therefore insulin appears to have a dominant role in the regulation of the phosphorylation state of hepatic HMG-CoA reductase. In apparent corroboration of this suggestion, short-term 4 h food deprivation of animals before D-4 resulted in a marked decrease in the E/T activity ratio of reductase, which was not affected further by an additional 8 h starvation. By contrast, the total activity of the enzyme was not significantly affected by 4 h starvation, but was markedly diminished after 12 or 24 h starvation. Longer-term starvation also produced a chronic increase in the degree of phosphorylation of the enzyme. These results are discussed in relation to the role of reversible phosphorylation in the control of hepatic HMG-CoA reductase activity in vivo.     

The mechanism of lack of hypocholesterolemic effects of pravastatin sodium,a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor,in rats

In order to clarify the reason why pravastatin, a 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitor, did not show hypocholesterolemic effects in rats, the changes of various parameters affecting the serum cholesterol levels by pravastatin were determined in rats and rabbits, as a comparison. In rabbits, pravastatin administration at 50 mg/kg for 14 days decreased serum and liver cholesterol by 40% and 8%, respectively. The hepatic LDL receptor activity was increased 1.7-fold, and VLDL cholesterol secretion was decreased. Cholesterol 7α-hydroxylase activity was not changed. In contrast, in rats, serum cholesterol was increased by 14% at 50 mg/kg and 27% at 250 mg/kg for 7 days, respectively. At 250 mg/kg, liver cholesterol was significantly increased by 11%. Under these conditions, neither the hepatic LDL receptor activity nor cholesterol 7α-hydroxylase was changed, and VLDL cholesterol secretion was increased. At 250 mg/kg, net cholesterol synthesis in rat liver was increased after 7 days of consecutive administration. These results imply that in rats, stimulated net cholesterol synthesis caused the increase of liver cholesterol followed by the increase of VLDL cholesterol secretion, and resulted in the raise of plasma cholesterol. Although hepatic HMG-CoA reductase was induced almost the same fold in both animals at 50 mg/kg, the induced HMG-CoA reductase activity in rats might overcome the inhibitory capability of pravastatin, resulting in an increase of net cholesterol synthesis, but not in rabbits. This overresponse to pravastatin in rats might cause the lack of hypocholesterolemic effects of this drug.     

Isoprenoid synthesis in Halobacterium halobium. Modulation of 3-hydroxy-3-methylglutaryl coenzyme a concentration in response to mevalonate availability

Halobacterium halobium was evaluated as a potentially simpler biological model to study the regulation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity (content) in response to mevalonate availability. H. halobium's HMG-CoA reductase was soluble and required NADPH as its reduced coenzyme. Maximum HMG-CoA reductase activity (4-10 nmol/min/mg of soluble protein) was obtained in buffers which contained 3.5 M KCl. Mevinolin (a) blocked growth of H. halobium, (b) was a competitive inhibitor of HMG-CoA reductase (Ki = 20 nM), (c) did not cause the paradoxical increase in assayable reductase activity, as reported for eukaryotic cells, and (d) caused a rapid (within 30 min) 8-12-fold accumulation of intracellular HMG-CoA. Mevalonate blocked and reversed mevinolin-mediated HMG-CoA accumulation. Although mevinolin-treated cell's growth was restored by mevalonate, HMG-CoA reductase's activity was not. Thus, H. halobium is a unique biological model which allows one to study the regulation of intracellular HMG-CoA concentration and not HMG-CoA reductase activity (content) in response to mevalonate availability.