Two cases of spotted fever group rickettsiosis contracted in southern parts of Africa

The effect of Ogi-Keishi-Gomotsu-To-Ka-Kojin (OKGK), a traditional Chinese herbal medicine (Kampo medicine), on cholesterol metabolism was studied in male Sprague-Dawley rats. Intake of OKGK at doses of 1.38 g/kg for 4 weeks significantly reduced total cholesterol levels in the serum and liver of hypercholesterolemia rats fed a cholesterol-enriched diet. OKGK suppressed cholesterol absorption through the intestine and stimulated excretion of cholesterol into feces as bile acids. Biochemical study indicated that OKGK treatment enhanced cholesterol 7alpha-hydroxylase activity the rate limiting enzyme of cholic acid synthesis, in the liver without any effect on the rate limiting enzyme of cholesterol synthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Further, cholesterol-enriched diet containing cholic acid suppressed cholesterol 7alpha-hydroxylase activity, whereas OKGK administration reversed the suppression. In conclusion, these results supported the idea that OKGK may be an effective agent for treatment of patients with hypercholesterolemia.     

Control of juvenile hormone biosynthesis. Evidence for phosphorylation of the 3-hydroxy-3-methylglutaryl coenzyme A reductase of insect corpus allatum

The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, a key enzyme of juvenile hormone biosynthesis, have been measured in the supernatants of homogenates (10,000 x g) prepared from the corpora allata of the adult tobacco hornworm moth, Manduca sexta. Enzyme activity was inhibited 80% by 50 mM NaF, a known phosphoprotein phosphatase inhibitor, if present during extirpation of the glands and all subsequent workup of the tissue. Reductase activity was also significantly decreased (20-30%) in homogenates preincubated with 4 mM MgCl2 and 2 mM ATP. These results provide evidence that reductase in the insect undergoes phosphorylation and dephosphorylation similar to that occurring with reductase of mammalian liver. If so, this would provide a rapid method for modulating juvenile hormone synthesis.     

Synthesis and biological activity of new 3-hydroxy-3-methylglutaryl-CoA synthase inhibitors: 2-oxetanones with a meta-substituent on the benzene ring in the side chain

Isosteric side chain analogs of 3a were synthesized and tested for inhibitory activities towards 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase and upon cholesterol production in Hep G2 cells and in mouse liver. It became clear that the lipophilic substituent on the aromatic ring and the terminal hydrophilic group in the side chain were important in the enhancement of activity. 4-[2-(3-n-Hexyloxyphenyl)ethyl]-3-hydroxy-methyl-2-oxetanone (5a) showed equivalent inhibitory activity in vivo to that of 1233A.     

Role of newly synthesized cholesterol or its metabolites on the regulation of bile acid biosynthesis after short-term biliary diversion in the rat

Cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme in the bile acid biosynthetic pathway, is thought to be regulated by hydrophobic bile acids through negative feedback control. The role of cholesterol in the regulation of cholesterol 7 alpha-hydroxylase is more controversial, in part because of incomplete understanding of the relationship between the pathways of cholesterol synthesis and degradation. The main objective of this study was to define the interaction between these two pathways in an experimental model in which the supply of newly synthesized cholesterol was interrupted by sustained infusion of mevinolin (lovastatin), an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) or accelerated by a continuous infusion of mevalonate, a cholesterol precursor. The study was carried out in rats subjected to short-term bile fistula. In one set of experiments, rats were treated postoperatively with mevinolin (5 mg/kg loading dose followed by 2 mg/kg/hr infusion), mevalonate (180 mumol/hr infusion) or both for up to 96 hr. In a separate set of experiments, rats were infused intraduodenally with taurocholate (36 mumol/100 gm/hr for up to 96 hr). We determined cholesterol 7 alpha-hydroxylase- and HMG-CoA reductase specific activities at those time intervals, whereas bile acid synthesis rates were determined throughout the study. Compared with rats not subjected to surgery, rats with short-term biliary diversion had increases in cholesterol 7 alpha-hydroxylase activity of 259% and 827% at 48 and 96 hr, respectively. The increase in bile acid biosynthesis was less pronounced. Continuous infusion of mevinolin completely prevented increases in cholesterol 7 alpha-hydroxylase specific activity and bile acid biosynthesis at both time intervals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thyroid hormone is required for dietary fish oil to induce hypersecretion of biliary cholesterol in the rat

In the rat, both fish oil diet and thyroid hormone replacement are reported to augment bile cholesterol secretion out of proportion to bile flow or secretion of other bile lipids. We sought common mechanisms for these effects and evaluated the role of phospholipid fatty acid composition in the process. Methimazole-treated hypothyroid rats were fed low-fat chow or chow supplemented with 10% corn oil or fish oil, and were studied before and after thyroid hormone treatment. Serum, hepatic, and bile lipids were measured, phospholipid fatty acid composition determined, and hepatic 3-hydroxy-3-methylglutaryl CoA reductase activity assayed. Fish oil diet stimulated cholesterol secretion into bile only after thyroid hormone was given, and this action was synergistic with that of thyroid hormone. Reduced serum cholesterol in fish oil-treated rats was associated with increased biliary cholesterol secretion and diminished hepatic cholesterol content. This suggests that augmented biliary cholesterol secretion may contribute to the fish oil-induced reduction of serum cholesterol. No definite relationship between hepatic or biliary phospholipid fatty acid composition and biliary secretion was apparent, although high bile cholesterol secretion was associated with a low percentage of hepatic and bile phospholipid linoleic acid.     

Hepatic cholesterol and bile acid metabolism in subjects with gallstones: comparative effects of short erm feeding of chenodeoxycholic and ursodeoxycholic acid

The activity of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase and 7alpha-hydroxylase, the enzymes controlling the rate of hepatic synthesis, respectively, of cholesterol and bile acids, and the microsomal cholesterol content were evaluated in 25 patients with cholesterol gallstones and 17 subjects without gallstones. The same quantities were estimated in 16 additional patients with gallstones given chenodeoxycholic (CDCA) or ursodeoxycholic acid (UDCA) at a dose of 15 mg/kg per day in order to investigate the comparative effect of a short term (7 days) administration of the two bile acids on the hepatic sterol metabolism. As compared to the controls, subjects with gallstones exhibited a 36% decrease of 7alpha-hydroxylase (26.8 +/- 6.2 versus 41.7 +/- 4.2 pmol/min per mg protein) and a 24% increase of the microsomal cholesterol (78.7 +/- 15.3 versus 63.1 +/- 18.1 nmol/mg protein). Although higher in the gallstone patients, the activity of HMG-CoA reductase did not differ significantly in the two groups of subjects. Administration of CDCA and UDCA changed the bile acid pool composition so that the fed bile acid predominated in the bile (mean CDCA 73% and mean UDCA 54%). Bile lipid composition did not appreciably change. In the eight subjects treated with CDCA the activity of HMG-CoA reductase was reduced to 45% of the value of untreated subjects (27.9 +/- 14.5 versus 63.5 +/- 25.3 pmol/min per mg protein) whereas in the eight subjects treated with UDCA the same enzyme showed a twofold increase (123.5 +/- 20.9). In the treated groups 7alpha-hydroxylase activity was somewhat decreased but the values did not differ significantly from those of the untreated subjects. Microsomal cholesterol content decreased with CDCA (64.8 +/- 11.6 nmol/mg protein) as well as with UDCA (59.1 +/- 10.1) treatment; however in the latter the difference attained statistical significance (P < 0.05). Altogether the results would suggest that in the liver of patients with gallstones the conversion of cholesterol to bile acids is somewhat reduced, and that changing the bile acid pool composition, by exogenous bile acid feeding, has disparate effects on hepatic cholesterol synthesis. The findings could represent the acute changes produced by bile acid feeding, however they could imply that the effects of two bile acids in dissolving cholesterol gallstones might not be related only to the changes in hepatic sterol metabolism.-Carulli, N., M. Ponz De Leon, F. Zironi, A. Pinetti, A. Smerieri, R. Iori, and P. Loria. Hepatic cholesterol and bile acid metabolism in subjects with gallstones: comparative effects of short term feeding of chenodeoxycholic and ursodeoxycholic acid.     

Biliary cholesterol excretion: a novel mechanism that regulates dietary cholesterol absorption

The regulation of dietary cholesterol absorption was examined in C57BL/6 and transgenic mice with liver overexpression of the scavenger receptor BI (SR-BI Tg). In C57BL/6 animals, feeding 0.02 to 1% (wt/wt) dietary cholesterol resulted in a dose-dependent decrease in the percentage of dietary cholesterol absorbed. A plot of total daily mass of dietary cholesterol absorbed versus the percentage by weight of cholesterol in the diet yielded a curve suggesting a saturable process with a Km of 0.4% (wt/wt) and a Vmax of 0.65 mg cholesterol/g body weight per day. Dietary cholesterol suppressed hepatic 3-hydroxy-3-methylglutaryl CoA reductase activity, stimulated cholesterol 7alpha-hydroxylase activity, and enhanced fecal excretion of bile acids, but none of these changes correlated with the percentage of dietary cholesterol absorption. Dietary cholesterol also caused an increase in biliary cholesterol concentration, and in this case the concentration of biliary cholesterol was strongly and inversely correlated with the percentage dietary cholesterol absorption (r = -0.63, P < 0.0001). Biliary cholesterol concentration was also directly correlated with daily cholesterol intake, dietary cholesterol mass absorption, and liver cholesterol ester content. Transgene-induced overexpression of SR-BI resulted in a stimulation of excretion of cholesterol into the bile and suppressed percentage dietary cholesterol absorption. Furthermore, biliary cholesterol levels in SR-BI Tg mice were strongly and inversely correlated with the percentage of dietary cholesterol absorbed (r = -0.99, P < 0.0008). In summary, these results suggest that the excretion of cholesterol into the bile plays an important role in regulating the percentage absorption of dietary cholesterol.     

Therapy of severe familial heterozygous hypercholesterolemia by low-density lipoprotein apheresis with immunoadsorption: effects of the addition of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors to therapy

To establish whether additional therapy with 3-hydroxy-3-methylglutaryl (HMG) coenzyme A (CoA) reductase inhibitors enhances the low-density lipoprotein (LDL) cholesterol lowering effect of LDL apheresis with immunoadsorption in the treatment of patients with familial heterozygous hypercholesterolemia and coronary artery disease we studied eight patients initially on immunoadsorption therapy alone for 3 years. The adding of HMG CoA reductase inhibitors decreased pretreatment LDL cholesterol from 6.76 +/- 0.98 to 4.97 +/- 0.98 mmol/l and posttreatment LDL cholesterol from 2.33 +/- 0.80 to 1.94 +/- 0.67 mmol/l and increased pre- and posttreatment high-density lipoprotein (HDL) cholesterol by 0.08 and 0.13 mmol/l respectively. The LDL/HDL ratio was reduced from 4.0 to 2.8 (prior to any therapy the ratio was 13.4). The increase in LDL cholesterol between weekly treatments was less steep under the combined therapy. At the same time the treated plasma volume during LDL apheresis could be decreased from 5070 +/- 960 to 4370 +/- 1200 ml. We conclude that in patients with severe familial heterozygous hypercholesterolemia LDL apheresis should be combined with HMG CoA reductase inhibitors.     

Lithogenic diet and gallstone formation in mice: integrated response of activities of regulatory enzymes in hepatic cholesterol metabolism

Supersaturation of bile with cholesterol is a prerequisite of the development of gallstones. With the intention to study the integrated response of enzymes regulating hepatic cholesterol metabolism during gallstone formation we used an established model for the induction of cholesterol gallstone disease in mice. Ten mice were fed on a lithogenic diet containing 10 g cholesterol/kg and 5 g cholic acid/kg for 8 weeks and were compared with ten mice fed on a standard pellet diet. Cholesterol crystals or gallstones developed in 90% of gallbladders in treated mice. The lithogenic diet had an inhibitory effect on the rate-limiting enzyme of cholesterol biosynthesis, hepatic 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase (EC 1.1.1.88) activity, 39.6 (SEM 2.8) v. 171.0 (SEM 47.3) pmol/min per mg protein. Cholesterol 7 alpha-hydroxylase (EC 1.14.13.17) activity, regulating bile acid synthesis, was decreased by 80%, and this was assumed to be due to cholic acid in the diet. The cholesterol-enriched diet also induced a tenfold increase in cholesterol esterification rate in the liver, i.e. acyl-CoA:cholesterol acyl transferase (ACAT; EC 2.3.1.26) activity. The total, as well as esterified, cholesterol contents of liver homogenates were significantly higher in cholesterol- and cholic acid-treated mice and correlated well with the ACAT activity (rs 0.72 (P < 0.005), and rs 0.68 (P < 0.01) respectively). A significantly higher ACAT activity was obtained in mice given cholesterol and cholic acid even when the enzyme was saturated with exogenous cholesterol, thus indicating an increased amount of the enzyme. The formation of gallstones is dependent on a delicate balance between lithogenic factors (increased absorption of cholesterol and reduced secretion of bile acids) and defence mechanisms (decreased synthesis and increased esterification of cholesterol). In the specific animal model studied here the two defence mechanisms cannot compensate for the increased absorption of cholesterol and the reduced synthesis of bile acids.     

Evidence that physiologic levels of circulating estrogens and neonatal sex-imprinting modify postpubertal hepatic microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase activity

Intact, but sham-operated female rats had 2- to 3-fold higher levels of hepatic 3-hydroxy-3-methylglutaryl CoA reductase activity than their male counterparts (15--21.5 vs. 6.7--8.7 nmol mevalonate/mg protein per h). The activity of the hepatic enzyme declined to about the same relative degree (40--60%) in male and female rats that were gonadectomized after puberty (53 days of age) and killed 5 weeks later. Implantation of silastic capsules containing 17 beta-estradiol increased the level of hepatic 3-hydroxy-3-methylglutaryl CoA reductase to levels found in sham-operated controls. In rats that were gonadectomized in infancy (12 h old) and killed 7--8 weeks later, the level of enzyme activity was not altered in females, but it was increased from 60--240% in males. Consequently, following neonatal gonadectomy, male-female differences in enzyme activity were no longer apparent. Implantation of silastic capsules containing estradiol in neonatally gonadectomized rats resulted in a doubling of enzyme activity in both males and females. Ovariectomy reduced plasma estrogen levels, but implantation of estradiol in gonadectomized males and females increased the hormone level to that found in sham-operated females. Thus, the results strongly suggest a role for physiologic levels of estrogen as a positive effector of 3-hydroxy-3-methylglutaryl CoA reductase activity. Neonatal sex imprinting also appears to modulate the enzyme activity since sex-mediated differences are effaced by gonadectomy in infancy, but not by gonadectomy following puberty.     

Effect of pregnancy and lactation on lipoprotein and cholesterol metabolism in the rat

Origins of hyperlipidemia and cholestasis that occur during pregnancy were investigated by examining expression of key elements related to plasma and hepatic cholesterol metabolism during pregnancy, lactation, and post-lactation in the rat model. Among major findings were: during pregnancy, the activities of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase, acyl coenzyme A:cholesterol acyltransferase, acyl coenzyme A:diacylglycerol acyltransferase, cholesterol 7alpha-hydroxylase, cholesterol ester hydrolases, low density lipoprotein receptors, LRP, and mdr2 were significantly lower or similar to non-pregnant controls while SR-B1 was elevated. Once lactation began, reductase, cholesterol acyltransferase, 7alpha-hydroxylase activities, low density lipoprotein receptors, and mdr2 increased while SR-B1 decreased. In later stages of lactation most hepatic elements returned to near control levels. Plasma cholesterol levels were higher than control at birth and during lactation with increase in LDL-size particles. By 24 h post-lactation, plasma triglycerides were 3.7-fold higher while cholesterol remained unchanged. Very large lipoproteins were present while LDL-size particles were now absent. Hepatic cholesterol acyltransferase had decreased to 27% of control while diacylglycerol acyltransferase increased 3-fold and low density lipoprotein receptors doubled. Most elements were normalized 3 weeks after weaning except for LRP and low density lipoprotein receptors which were elevated. These studies provide an integrated picture of expression of key elements of hepatic and plasma cholesterol metabolism during pregnancy and lactation and advance understanding of hyperlipidemia and cholestasis during these states.     

Dietary fat saturation and chain length modulate guinea pig hepatic cholesterol metabolism

The effects of dietary fat saturation and saturated fatty acid composition on plasma lipoprotein concentrations and hepatic cholesterol metabolism were investigated in guinea pigs. Animals were fed semipurified diets containing 15 g fat/100 g diet, as palm kernel, palm oil, beef tallow, lard, olive oil or corn oil. Plasma lipoprotein concentrations were significantly altered by the type of dietary fat. The LDL cholesterol concentration was highest in animals fed the diet with palm kernel and lowest in animals fed the diet with corn oil, whereas HDL cholesterol was lowest in beef tallow-fed guinea pigs (P < 0.01). Hepatic cholesteryl ester concentrations were 100% higher in animals fed diets containing polyunsaturated corn oil and monounsaturated olive oil compared with animals fed any of the saturated fat diets (P < 0.01). Hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity varied in the different dietary fat groups independent of hepatic cholesterol pools or plasma LDL concentrations. In contrast, hepatic acyl-CoA: cholesterol acyltransferase (ACAT) activity was significantly correlated with plasma LDL cholesterol across all dietary groups (r = 0.63, P < 0.001). These data demonstrate that regulation of hepatic HMG-CoA reductase activity is relatively independent of changes in plasma lipoprotein levels, whereas hepatic ACAT activity exhibits a positive correlation with plasma LDL cholesterol concentrations.     

Identification and characterization of a mouse oxysterol 7alpha-hydroxylase cDNA

The synthesis of essential 7alpha-hydroxylated bile acids in the liver is mediated by two pathways that involve distinct 7alpha-hydroxylases. One pathway is initiated in the endoplasmic reticulum by cholesterol 7alpha-hydroxylase, a well studied cytochrome P450 enzyme. A second pathway is initiated by a less well defined oxysterol 7alpha-hydroxylase. Here, we show that a mouse hepatic oxysterol 7alpha-hydroxylase is encoded by Cyp7b1, a cytochrome P450 cDNA originally isolated from the hippocampus. Expression of a Cyp7b1 cDNA in cultured cells produces an enzyme with the same biochemical and pharmacological properties as those of the hepatic oxysterol 7alpha-hydroxylase. Cyp7b1 mRNA and protein are induced in the third week of life commensurate with an increase in hepatic oxysterol 7alpha-hydroxylase activity. In the adult mouse, dietary cholesterol or colestipol induce cholesterol 7alpha-hydroxylase mRNA levels but do not affect oxysterol 7alpha-hydroxylase enzyme activity, mRNA, or protein levels. Cholesterol 7alpha-hydroxylase mRNA is reduced to undetectable levels in response to bile acids, whereas expression of oxysterol 7alpha-hydroxylase is modestly decreased. The liver thus maintains the capacity to synthesize 7alpha-hydroxylated bile acids regardless of dietary composition, underscoring the central role of 7alpha-hydroxylated bile acids in lipid metabolism.     

Regulation of classic and alternative bile acid synthesis in hypercholesterolemic rabbits: effects of cholesterol feeding and bile acid depletion

The effect of cholesterol feeding (3 g/day) on bile acid synthesis was examined in 10 New Zealand white rabbits (NZW), 8 Watanabe heterozygous and 10 homozygous rabbits with partial and complete deficiencies of LDL receptors. After 10 days of cholesterol feeding, bile fistulas were constructed and bile acid pool sizes were measured. Cholesterol feeding increased plasma and hepatic cholesterol levels in all rabbit groups. Baseline bile acid pool sizes were smaller (P < 0.01) in heterozygotes (139 +/- 3 mg) and homozygotes (124 +/- 30 mg) than NZW rabbits (254 +/- 44 mg). After feeding cholesterol, bile acid pool sizes doubled with increased cholic acid synthesis in NZW and, to a lesser extent, in Watanabe heterozygous rabbits but not in homozygotes. Baseline cholesterol 7alpha-hydroxylase activity in NZW and heterozygotes declined 69% and 53% (P < 0.001), respectively, after cholesterol feeding. Sterol 27-hydroxylase activity reflecting alternative bile acid synthesis increased 66% (P < 0.01) in NZW and 37% in Watanabe heterozygotes but not in homozygotes after feeding cholesterol. Bile fistula drainage stimulated cholesterol 7alpha-hydroxylase activity but not sterol 27-hydroxylase activity in all three rabbit groups. These results demonstrated that dietary cholesterol increased hepatic sterol 27-hydroxylase activity and alternative bile acid synthesis to expand the bile acid pool and inhibited cholesterol 7alpha-hydroxylase in NZW and in Watanabe heterozygous rabbits but not in homozygotes with absent hepatic LDL receptor function. Thus, in rabbits, sterol 27-hydroxylase is up-regulated by the increased hepatic cholesterol that enters the liver via LDL receptors whereas cholesterol 7alpha-hydroxylase is controlled by the circulating hepatic bile acid flux.     

Ursodeoxycholic acid treatment in cholesterol gallstone disease: effects on hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, biliary lipid composition, and plasma lipid levels

The present study was undertaken to characterize the effects of ursodeoxycholic acid on biliary lipid metabolism in man. Fifteen gallstone patients were treated with ursodeoxycholic acid at a daily dosage of 15 mg per kg body weight for about 4 weeks before cholecystectomy. At operation a liver biopsy, together with gallbladder and hepatic bile, were obtained. Eighteen untreated gallstone patients undergoing cholecystectomy served as controls. During treatment with ursodeoxycholic acid, hepatic bile became unsaturated with cholesterol in all patients investigated. The total biliary lipid concentration remained unchanged. The hepatic cholesterol concentration decreased by about 20%. No significant change in the microsomal HMG CoA reductase activity was observed (38.5 +/- 6.7 pmol . min-1 . mg protein-1 vs 38.3 +/- 4.7 pmol . min-1 . mg protein-1 in the controls; means +/- SEM). Plasma concentrations of total cholesterol were reduced by about 10%, and those of high density lipoprotein (HDL) and low density lipoprotein (LDL) cholesterol by about 15%. Plasma triglyceride levels remained essentially unchanged during treatment. We conclude that, similar to chenodeoxycholic acid therapy, ursodeoxycholic acid treatment results in unsaturation of fasting hepatic bile. In contrast to the changes seen during chenodeoxycholic acid feeding, however, the unsaturation of hepatic bile during ursodeoxycholic acid treatment is not primarily related to a decreased hepatic HMG CoA reductase activity. Furthermore, while chenodeoxycholic acid tends to increase plasma LDL levels, such changes are not seen during ursodeoxycholic acid treatment.     

Three-dimensional reconstructions of carotid bifurcation from CT images: evaluation of different rendering methods

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, involved in de novo cholesterol synthesis and cell-cycle progression, was identified as a potential mediator of the growth inhibitory effects of retinoic acid on human neuroblastoma. Lovastatin, a nonreversible inhibitor of HMG-CoA reductase, induced extensive cytotoxicity that was restricted to drug-resistant P-glycoprotein-expressing neuroblastoma cell lines. This response was potentiated by dibutyryl cyclic AMP but not retinoic acid. Patients with advanced-stage metastatic neuroblastoma often display an acquired chemoresistant phenotype, which may in part be mediated by P-glycoprotein. Our studies support the application or use of HMG-CoA reductase inhibitors as potential therapeutic agents in the treatment of these patients who are refractory to chemotherapy.     

Overexpression of hormone-sensitive lipase in Chinese hamster ovary cells leads to abnormalities in cholesterol homeostasis

Hormone-sensitive lipase (HSL) is an intracellular enzyme that functions as both a neutral triglyceride and cholesteryl ester hydrolase. In order to explore the effects of HSL on cholesterol homeostasis, Chinese hamster ovary (CHO) cells were transfected with rat HSL and several different stable cell lines that overexpress HSL mRNA, HSL protein, and HSL activity approximately 600-fold were isolated. Cells transfected with HSL contained less cholesteryl esters and unesterified cholesterol than control cells. HSL transfectants expressed 20-60% fewer LDL receptors than control cells when grown in lipid-depleted media or in the presence of mevinolin, as assessed by binding and degradation of LDL and immunoblotting of LDL receptors. In contrast, the rate of cholesterol synthesis and the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase were increased 3- to 14-fold in HSL transfectants grown in sterol replete media. The rate of cholesterol synthesis and the activity of HMG-CoA reductase increased when cells were grown in lipid-depleted media, and remained markedly elevated compared to control cells. These results show that the regulation of LDL receptor expression and cholesterol synthesis can be dissociated through the actions of HSL and suggest multiple control mechanisms for sterol-responsive genes.     

Esterified cholesterol accumulation induced by aggregated LDL uptake in human vascular smooth muscle cells is reduced by HMG-CoA reductase inhibitors

Vascular smooth muscle cell (VSMC) proliferation is a key event in the development of atherosclerotic lesions. VSMCs synthesize extracellular matrix, where low density lipoproteins (LDLs) are trapped and become aggregated (agLDL). The objective of this study was to investigate the cholesterol uptake and accumulation triggered by agLDL in comparison with native LDL (nLDL) on unstimulated and platelet-derived growth factor-stimulated human aortic VSMCs and the role of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors on these processes. Esterified cholesterol (EC) accumulation induced by agLDL in VSMCs was correlated with the degree of aggregation and concentration. The EC content of VSMCs treated with 100 microg/mL of agLDL (80% aggregated) increased approximately 70-fold over that in VSMCs incubated with the same concentration of nLDL. Whereas nLDL-derived EC was increased approximately twofold in platelet-derived growth factor-stimulated VSMCs, there was no effect of platelet-derived growth factor (10(-9) mol/L) on the uptake of agLDL. The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor simvastatin (5 micromol/L) reduced EC accumulation derived from agLDL uptake by 58% and 35% in platelet-derived growth factor-stimulated and unstimulated VSMCs, respectively. This inhibition was overcome by geranylgeraniol (10 micromol/L) and partially by farnesol (10 micromol/L). Fluorescence microscopy of the cellular internalization of agLDL labeled with the fluorochrome 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine demonstrated that simvastatin reduces EC accumulation derived from agLDL by inhibiting its endocytosis and that the effect is completely reversed by geranygeraniol. These results indicate that agLDLs are rapidly internalized by human VSMCs and that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors modulate EC accumulation. These data suggest a possible mechanism by which statins could contribute to the passivation and stabilization of actively growing atherosclerotic lesions.     

Sex differences in hepatic sulfation of taurolithocholate in the rat

Metabolism of the bile salts by formation of sulfate esters is catalyzed by bile salt sulfotransferase, an enzyme isolated from rat liver and kidney. The activity of bile salt sulfotransferase was measured in liver and kidney of male and female rats and in oophorectomized rats with or without estrogen replacement. In vitro sulfotransferase activity was correlated with in vivo sulfation by measuring the percentage of an infused dose (0.03 micron per 100 g per min) of taurolithocholate, which was excreted in bile as the sulfate. The activity of sulfotransferase in liver was higher in females (26.3 +/- 3.0 pmoles per mg of protein per min) than in males (9.6 +/- 3.9) and was lower (12.1 +/- 3.8) after oophorectomy. The decrease in activity was prevented by replacement of estrogen. Renal sulfotransferase activity did not differ between the sexes and was unaffected by oophorectomy. Hepatic sulfotransferase activity measured in vitro correlated with in vivo sulfation of taurolithocholate. This study shows definite sex differences in hepatic bile salt sulfotransferase activity, which in females is affected by the presence of estrogen. The correlation between in vitro sulfotransferase activity and in vivo bile salt sulfation suggests that bile salt sulfotransferase is responsible for bile salt sulfation in vivo.