Ontogeny of microsomal activities of triacylglycerol synthesis in guinea pig liver

Because the onset of triacylglycerol-rich lipoprotein synthesis occurs in guinea pig liver during fetal life, we investigated the microsomal enzyme activities of triacylglycerol synthesis in fetal and postnatal guinea pig liver. Hepatic monoacylglycerol acyltransferase specific and total microsomal activities peaked by the 50th day of gestation and declined rapidly after birth to levels that were virtually unmeasurable in the adult. Peak fetal specific activity was more than 75-fold higher than observed in the adult. The specific activities of fatty acid CoA ligase and lysophosphatidic acid acyltransferase increased 2- to 3-fold before birth; lysophosphatidic acid acyltransferase increased a further 2.6-fold during the first week of life. Specific activities of phosphatidic acid phosphatase, microsomal glycerophosphate acyltransferase, and diacylglycerol acyltransferase varied minimally over the time course investigated. These data demonstrate that selective changes occur in guinea pig hepatic microsomal activities of triacylglycerol synthesis before birth. Because of an approximate 11-fold increase in hepatic microsomal protein between birth and the adult, however, major increases in total microsomal activity of all the triacylglycerol synthetic activities occurred after birth. The pattern of monoacylglycerol acyltransferase specific and total microsomal activities differs from that of the rat in occurring primarily during the last third of gestation instead of during the suckling period. This pattern provides evidence that hepatic monoacylglycerol acyltransferase activity probably does not function to acylate 2-monoacylglycerols derived from partial hydrolysis of diet-derived triacylglycerol.     

Gonadotropin modulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in desensitized luteinized rat ovary

These studies were done to examine the effect of gonadotropin on rat luteal 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase activity (the rate-limiting step in cholesterol biosynthesis) in ovaries of pregnant mare's serum gonadotropin (PMSG)-human chorionic gonadotropin (hCG) primed rats. Administration of hCG stimulated HMG CoA reductase activity in a time- and dose-dependent manner: significant increases were noted within 4 h, with maximum effects (30-40-fold increases) seen 24 h after hCG (25 IU) administration. This effect was specific in that only LH, of several hormones tested, was as effective as hCG in stimulating HMG CoA reductase activity, and no change in the activity of either liver microsomal HMG CoA reductase or luteal microsomal NADPH-cytochrome c reductase was seen after hCG. The gonadotropin-induced increase in HMG CoA reductase activity seemed to be due to a net increase in enzyme activity, not to a change in the phosphorylated/dephosphorylated state of the enzyme. Pretreatment of animals with aminoglutethimide, an inhibitor of the conversion of cholesterol to steroid (pregnenolone), prevented the hCG-induced rise in HMG CoA reductase activity, whereas treatment with 4-aminopyrazolo[3,4-d]pyrimidine (4-APP), which depletes cellular cholesterol content, led to striking increases in enzyme activity. However, the combined effects of 4-APP and hCG were additive, suggesting that the stimulating effect of hCG on HMG CoA reductase activity is not entirely due to a depletion of cellular sterol content of luteinized ovaries. Similarly, cholesteryl ester and cholesterol syntheses as measured by [14C]acetate conversion were also increased by hCG and 4-APP treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulatory effects of sterols and bile acids on hepatic 3-hydroxy-3-methylglutaryl CoA reductase and cholesterol 7alpha-hydroxylase in the rat

Specific activities of the hepatic microsomal enzymes 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase and cholesterol 7alpha-hydroxylase were studied in rats fed sterols and bile acids. The administration of bile acids (taurocholate, taurodeoxycholate, taurochenodeoxycholate) at a level of 1% of the diet for 1 wk reduced the activity of HMG CoA reductase. Taurocholate and taurodeoxycholate, but not taurochenodeoxycholate, inhibited cholesterol 7alpha-hydroxylase. Dietary sitosterol produced increases in the specific activity of HMG CoA reductase (3.6-fold) and cholesterol 7alpha-hydroxylase (1.4-fold), and biliary cholesterol concentrations in this group more than doubled. Compared with controls fed the stock diet, the simultaneous administration of sitosterol and taurochenodeoxycholate resulted in a 60% decrease of HMG CoA reductase activity and no change in cholesterol 7alpha-hydroxylase activity or biliary cholesterol concentration. Rats fed sitosterol plus taurocholate had nearly normal HMG CoA reductase activity, but cholesterol 7alpha-hydroxylase was inhibited and biliary cholesterol remained high. Bile acid secretion rates and biliary bile acid composition were similar in controls and sterol-fed animals. In all groups receiving bile acids, biliary secretion of bile acids was nearly doubled and bile acid composition was shifted in the direction of the administered bile acid. It is concluded that the composition of the bile acid pool influences the hepatic concentrations of the rate-controlling enzymes of bile acid synthesis.     

HMG CoA reductase of intestinal mucosa and liver of the rat

Methods were developed for the determination of HMG CoA (3-hydroxy-3-methylglutaryl CoA) reductase activity in subcellular fractions of intestinal mucosa and liver of Wistar strain rats. In the liver, reductase activity was located exclusively in the microsomal fraction. In the intestinal mucosa, activity was found in both mitochondrial and microsomal fractions of crypt cells but not of villi. The microsomal HMG CoA reductases of liver and intestinal mucosa had similar kinetic characteristics and pH optima. However, the activity of the hepatic enzyme differed with age and sex of the experimental animals while that of the intestinal crypt cells did not. Cholestyramine treatment enhanced the activity of the microsomal HMG CoA reductase in both liver and intestinal mucosa. Reductase activity of the intestinal crypt cells was elevated in both jejunum and ileum. The greatest stimulation, both relatively and absolutely, was observed in the distal half of the jejunum.     

In vivo demonstration of the circadian thythm of cholesterol biosynthesis in the liver and intestine of the rat

The existence of a circadian rhythm in the rate of hepatic cholesterol synthesis in the rat has been demonstrated in vivo by measuring the conversion of both [1-(14)C]acetate and (3)H(2)O to cholesterol. By both methods there was observed a similar increase in the rate of hepatic cholesterol synthesis between the nadir at noon and the peak at midnight. Circadian changes in the rate of hepatic cholesterol synthesis measured in vivo with [1-(14)C]acetate were very similar to changes in the activity of hepatic microsomal HMG CoA reductase. Cholesterol synthesis in the jejunum and in the distal ileum was also shown to exhibit the same circadian rhythm in vivo but with smaller amplitude (1.6- and 1.3-fold, respectively). Rats trained to eat during a 4-hr period (9 am-1 pm) while housed under normal illumination showed changes in the timing of the circadian rhythm of cholesterol synthesis; in the liver the maximum rate of cholesterol synthesis occurred at 6 pm, 9 hr after the presentation of food, while the two sections of the intestine investigated exhibited a maximum synthetic response between noon and 6 pm. Results obtained in this study support the hypothesis that the major portion of the rise in HMG CoA reductase activity and the increase in overall rate of cholesterol synthesis in liver and intestine during the circadian rhythm are due to the ingestion of food. Under the limited feeding schedule (food access 9 am-1 pm), the rates of hepatic and intestinal synthesis of fatty acids from the injected acetate also showed a circadian rhythm with a peak at about 3 hr after presentation of food.     

Hepatic cholesterol metabolism in normo- and hyperlipidemic patients with cholesterol gallstones.

In vivo studies have shown abnormalities in cholesterol and bile acid metabolism in primary hyperlipoproteinemia (HLP). The aim of the present investigation was to determine if the increased production of cholesterol in HLP type IV can be attributed to a correspondingly high level of the hepatic 3-hydroxy-3-methylglutaryl (HMG) CoA reductase activity and if the low cholic acid: chenodeoxycholic acid synthesis ratio in HLP type II is due to some hydroxylase deficiency. Liver biopsies from 26 normolipidemic and 25 hyperlipidemic (10 type IIa, 6 type IIb, and 9 type IV) patients undergoing elective cholecystectomy were assayed for HMG CoA reductase activity, 12 alpha-hydroxylase activity, and 25-hydroxylase activity. The HMG CoA reductase activity was normal in HLP type IIa and type IIb and was increased about twice HLP type IV (P less than 0.001). The 12 alpha- and 25-hydroxylase activities were normal in all groups of patients. The results are compatible with a normal cholesterol synthesis in the liver in HLP type II. A reduced 12 alpha- or 25-hydroxylase activity cannot explain the low production of cholic acid relative to chenodeoxycholic acid in this type of HLP. The elevated HMG CoA reductase activity found in the liver of type IV patients may, however, be part of the explanation for the elevated synthesis of cholesterol often seen in these patients.     

Evidence for a common biliary cholesterol and VLDL cholesterol precursor pool in rat liver.

Hepatic free cholesterol levels are influenced by cholesterol synthesis and ester formation, which, in turn, might regulate cholesterol secretion into bile and plasma. We manipulated the rates of hepatic cholesterol synthesis and esterification and measured biliary and very low density lipoprotein (VLDL) cholesterol secretion, and bile acid synthesis. Mevalonate decreased HMG CoA reductase by 80%, increased acyl coenzyme A: cholesterol acyltransferase (ACAT) by 60% and increased [3H]oleate incorporation into microsomal and VLDL cholesteryl esters by 174% and 122%, respectively. Microsomal and biliary free cholesterol remained constant at the expense of increased microsomal and VLDL cholesteryl ester content. Mevalonate did not change bile acid synthesis. 25-OH cholesterol decreased HMG-CoA reductase by 39%, increased ACAT by 24%, but did not effect 7 alpha-hydroxylase. 25-OH cholesterol increased [3H]oleate in microsomal and VLDL cholesterol esters by 71% and 120%. Biliary cholesterol decreased by 40% and VLDL cholesteryl esters increased by 83%. A small and unsustained decrease in bile acid synthesis (14CO2 release) occurred after 25-OH cholesterol. After orotic acid feeding, HMG-CoA reductase increased 352%, and [3H]oleate in microsomal and VLDL cholesteryl esters decreased by 43% and 89%. Orotic acid decreased all VLDL components including free cholesterol (68%) and cholesteryl esters (55%), and increased biliary cholesterol by 160%. No change in bile acid synthesis occurred. Hepatic cholesterol synthesis and esterification appear to regulate a cholesterol pool available for both biliary and VLDL secretion. Changing cholesterol synthesis and esterification did not alter bile acid synthesis, suggesting that either this common bile/VLDL secretory pool is functionally distinct from the cholesterol pool used for bile salt synthesis, or that free cholesterol availability in this precursor pool is not a major determinant of bile acid synthesis.     

Cyclic AMP-sensitive activation of hepatic sterol synthesis and 3-hydroxy-3-methylglutaryl coenzyme A reductase.

We previously showed that preincubation of a 10,000 g supernatant (S(10)) from rat liver for 20 min at 37 degrees C dramatically increased the subsequent incorporation of [(14)C]acetate into sterols. No activation was seen with [(14)C]mevalonate as substrate. In the present studies we have examined the effect of preincubation on HMG CoA reductase. When microsomes were isolated from S(10) by calcium precipitation, preincubation of S(10) increased the specific activity of HMG CoA reductase threefold. No activation of HMG CoA reductase was observed in microsomes isolated by ultracentrifugation. Activation was cyclic AMP-sensitive. When cyclic AMP (0.001-1.0 mM) and MgATP (1 mM) were present during the preincubation period, there was little or no activation of HMG CoA reductase activity or of sterol synthesis from acetate. MgATP alone did not prevent activation. Neither cyclic AMP nor MgATP was inhibitory when present only during the assay of sterol synthesis. We propose that the in vitro activation represents the reversal of a physiologic cyclic AMP-mediated mechanism for the control of hepatic HMG CoA reductase. That a phosphoprotein phosphatase may catalyze the activation was supported by the observation that sodium fluoride, an inhibitor of phosphoprotein phosphatases, inhibited the activation. These results suggest that hormone-induced changes in the cellular level of cyclic AMP may regulate the activity of HMG CoA reductase and the rate of hepatic cholesterol synthesis.     

Hepatic 3-hydroxy-3-methyl-glutaryl-CoA reductase response to litter size in suckling rats.

Rats were raised in litters of 3 or 14 from the 3rd postnatal day. Already, 2 days later on day 5, those raised in groups of three had elevated blood cholesterol and insulin levels and a very much lower hepatic 3-hydroxy-3-methyl-glutaryl-CoA (HMG) CoA) reductase activity, than the other group. When aged 40 days, hepatic HMG CoA reductase activity was higher in the group raised three rats per litter than in the large litter group.     

Mass-spectral identification and purification of phycoerythrobilin and phycocyanobilin.

The hepatic output of triacylglycerol and cholesterol from very-low-density lipoprotein (VLD lipoprotein), and the activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase were compared in the isolated perfused rat-liver preparation and in the intact rat. The output of triacylglycerol and cholesterol from VLD lipoprotein by the perfused liver was stimulated by oleate concomitant with stimulation of hepatic microsomal hydroxymethylglutaryl-coenzyme A reductase activity. In the intact animal treated with Triton WR-1339, the magnitude of secretion of triacylglycerol and cholesterol from VLD lipoprotein coincided with the diurnal rhythm of hepatic hydroxymethylglutaryl-coenzyme A reductase activity, which was maximal at 24:00 h and minimal at 12:00 h. These observations suggest that the stimulation of the reductase and of the secretion of cholesterol from VLD lipoprotein by non-esterified fatty acids, as observed with the isolated perfused rat liver preparation in vitro, may also be an important physiological mechanism in vivo. Hepatic cholesterogenesis may be stimulated under conditions conductive to the secretion of the VLD lipoprotein, the primary transport form for triacylglycerol in the postabsorptive state.     

Analysis of the coordinate expression of 3-hydroxy-3-methylglutaryl coenzyme A synthase and reductase activities in Chinese hamster ovary fibroblasts

Decreased activities of both 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase and HMG CoA reductase are observed in the presence of sterol in the Chinese hamster ovary (CHO) fibroblast. In three different genotypes of CHO cell mutants resistant to 25-hydroxycholesterol both enzyme activities exhibit a decreased response to 25-hydroxycholesterol compared to wild-type cells. Permanently repressed levels of both HMG CoA synthase and HMG CoA reductase activities are observed in another CHO mutant, phenotypically a mevalonate auxotroph. Mevinolin, a competitive inhibitor of HMG CoA reductase, has no effect on HMG CoA synthase activity measured in vitro. Incubation of CHO cells with sublethal concentrations of mevinolin produces an inhibition of the conversion of [14C]acetate to cholesterol and results in elevated levels of both HMG CoA synthase and HMG CoA reductase activities. Studies of CHO cells in sterol-free medium supplemented with cycloheximide indicate that continuous protein synthesis is not required for the maximal expression of HMG CoA synthase activity and provide an explanation for the lack of temporal similarity between HMG CoA synthase and reductase activities after derepression. These results support the hypothesis of a common mode of regulation for HMG CoA synthase and HMG CoA reductase activities in CHO fibroblasts.     

Microsomal and lysosomal enzymes of triacylglycerol metabolism in rat placenta.

The placenta plays a major role in transporting lipid to the developing foetus. Since previous studies have suggested that placental lipid transport involves intermediate esterification steps, we investigated selected microsomal and lysosomal enzymes of triacylglycerol metabolism in rat placenta. Between gestational days 10 and 14, microsomal phosphatidic acid phosphatase specific activity was 6-fold greater than the activity in adult rat liver. Phosphatidic acid phosphatase activity decreased 50% on day 15. Studies employing several different phosphorylated substrates indicated a high degree of substrate specificity. Lysosomal triacylglycerol lipase and cholesterol esterase activities decreased about 50% between days 15 and 18, then rose late in gestation. No changes were observed in the specific activities of fatty acid: CoA ligase, glycerolphosphate acyltransferase, lysophosphatidate acyltransferase, diacylglycerol acyltransferase or diacylglycerol cholinephosphotransferase during the final 12 days of gestation. Kinetic observations (competitive inhibition by alternative substrates, pH-dependence and thermal inactivation) were consistent with the hypothesis that glycerol phosphate and dihydroxyacetone phosphate can be acylated by a single microsomal enzyme in placenta. Except for fatty acid: CoA ligase, the activities of microsomal and lysosomal enzymes of triacylglycerol metabolism were comparable with those in adult rat liver. These observations are consistent with physiological studies suggesting that triacylglycerol synthetic and degradative pathways are very active in rat placenta.     

Effect of a high cholesterol diet on lipid metabolizing enzymes in spontaneously hypertensive rats

A high cholesterol diet induced a fatty liver and an increase in cholesterol oleate in spontaneously hypertensive rats. The activity of microsomal glycerophosphate acyltransferase in liver increased 2-3-fold to meet the increased supply of oleate, the synthesis of which was stimulated by a 10-fold increase in microsomal delta 9-desaturase activity. Hepatic fatty acid synthetase and diacylglycerol acyltransferase activities were decreased somewhat. These results, together with the fact that the large increases in hepatic cholesterol ester and triacylglycerol were not correspondingly reflected in plasma, indicated that the fatty liver resulted from decreased secretion of lipoprotein rather than increased lipogenesis. Endogenous cholesterol in liver microsomes increased 2-fold and hepatic acyl-CoA:cholesterol acyltransferase activity increased 3-fold, whereas plasma lecithin:cholesterol acyltransferase activity was unchanged. Thus, the increase in cholesterol oleate seen in spontaneously hypertensive rats fed a high cholesterol diet is due mainly to increases in acyl-CoA:cholesterol acyltransferase and delta 9-desaturase activities.     

Effect of lipoprotein on 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase activity in rat liver cell culture: special suppressant effect of a lipoprotein isolated from hypercholesterolemic rat plasma.

A significant advance in cell culture methodology has permitted an analysis of the regulation of liver cell cholesterol metabolism by lipoproteins. The effect of rat lipoproteins on the activity of the rate limiting enzyme in cholesterol biosynthesis, HMG CoA reductase, was studied in monolayer cultures of adult rat hepatic parenchymal cells. Lipoproteins isolated from normocholesterolemic rat plasma, including low density lipoprotein, did not suppress HMG CoA reductase activity. However, enzyme activity was profoundly suppressed by a cholesterol rich d < 1.063 lipoprotein(s) isolated from hyperlipemic rat plasma. This lipoprotein may regulate the suppression of endogenous hepatic cholesterol biosynthesis which occurs after cholesterol feeding.     

Growth hormone reduces plasma cholesterol in LDL receptor-deficient mice.

Growth hormone (GH) has pleiotropic effects on cholesterol and lipoprotein metabolism. Pituitary GH is important for the normal regulation of hepatic LDL receptors (LDLR), for the enzymatic activity of bile acid regulatory cholesterol 7alpha-hydroxylase (C7alphaOH), and for the maintenance of resistance to dietary cholesterol. The present study aimed to determine whether GH has beneficial effects on plasma lipids and hepatic cholesterol metabolism in mice devoid of LDLR. Compared with wild-type controls, LDLR-deficient mice had approximately 250% elevated plasma total cholesterol and approximately 50% increased hepatic cholesterol levels; hepatic HMG CoA reductase activity was reduced by 70%, whereas C7alphaOH activity was increased by 40%. In LDLR mice, GH infusion reduced plasma cholesterol and triglycerides up to 40%, whereas HMG CoA reductase and C7alphaOH activities were stimulated by approximately 50% and 110% respectively. GH also stimulated HMG CoA reductase and C7alphaOH activities in control mice, whereas hepatic LDLR and plasma lipoproteins were unchanged. The effects of cholestyramine and atorvastatin on C7alphaOH in LDLR-deficient mice were potentiated by GH, and this was associated with a further reduction in plasma cholesterol. GH treatment reduces plasma cholesterol and triglycerides and stimulates C7alphaOH activity in mice devoid of LDLR, particularly in combination with resin or statin treatment. The potential of GH therapy in patients with homozygous familial hypercholesterolemia should be evaluated.     

Effects of cortisol or corticotropin administration on hepatic 3-hydroxy-3-methylglutaryl-CoA reductase activity and plasma lipids in the pregnant rat and fetuses

Pregnant rats were given pharmacological doses of cortisol or ACTH or no hormone from gestation day 9 to 19 and maternal and fetal hepatic 3-hydroxy-3-methylglutaryl-CoA reductase activity and plasma cholesterol studied on gestation day 20. Reductase activity was also studied in the maternal and fetal adrenal of the rats given cortisol or no hormone. Cortisol administration increased the maternal and fetal plasma cholesterol but had no effect on the hepatic active (phosphorylated) 3-hydroxy-3-methylglutaryl-CoA reductase activity when compared to untreated rats. Total (active + inactive) 3-hydroxy-3-methylglutaryl-CoA reductase activity, however, was reduced in maternal liver but not altered in the fetal liver by cortisol. The maternal cortisol treatment decreased the fetal, but not maternal, adrenal 3-hydroxy-3-methylglutaryl-CoA reductase total enzyme activity. The data support a hypothesis that utilization of plasma cholesterol for adrenal steroidogenesis may be an important determinant of plasma cholesterol homeostasis in the rat fetus. Maternal ACTH administration increased the foetal but not maternal plasma cholesterol, whilst active 3-hydroxy-3-methylglutaryl-CoA reductase activity was increased in the pregnant rat but not her fetuses. This result may suggest coordination of hepatic active reductase activity with adrenal cholesterol utilization in the pregnant rat. The reason for the fetal hypercholesterolaemia caused by ACTH, which is not known to cross the placenta, is uncertain. The studies, however, indicate that fetal cholesterol homeostasis and the rate limiting enzyme of cholesterol synthesis is influenced by maternal glucocorticoid administration.     

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.     

Localization and regulation of epidermal 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity by barrier requirements

Recent studies have shown that epidermal cholesterol synthesis is regulated by HMG CoA reductase activity and that this activity is modulated by changes in the cutaneous permeability barrier. Here, we quantitated HMG CoA reductase activity after acute and chronic barrier disruption in the upper and lower layers of murine epidermis. In unperturbed epidermis, 13 and 87% of enzyme activity localized to the upper and lower epidermis, respectively, with the majority of activity in the stratum basale. Acute barrier disruption with either acetone or sodium dodecylsulfate provoked an increase in HMG CoA reductase activity (54% and 30%) in the lower layers, but only a small change in the upper layers. However, the activation state of the enzyme was increased 50% in the upper epidermis. Correction of barrier function by occlusion with an impermeable Latex wrap prevented the increase both in enzyme activity and activation state. After chronic barrier disruption; i.e., essential fatty acid deficient (EFAD) diet, HMG CoA reductase activity was increased in the upper epidermis (161%); a change prevented by occlusion. These results show: (1) that HMG CoA reductase activity is present in both the upper and lower cell layers; (2) that acute insults to barrier integrity stimulate enzyme activity in both the upper and lower epidermis; and (3) that chronic insults provoke an increase in enzyme activity in the upper layers. These studies provide further insights into the linkage of the permeability barrier with epidermal cholesterol metabolism.     

Stimulation of hepatic cholesterol biosynthesis by fatty acids. Effects of oleate on cytoplasmic acetoacetyl-CoA thiolase, acetoacetyl-CoA synthetase and hydroxymethylglutaryl-CoA synthase.

The effects of oleic acid on the activities of cytosolic HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) synthase, AcAc-CoA (acetoacetyl-CoA) thiolase and AcAc-CoA synthetase, as well as microsomal HMG-CoA reductase, all enzymes in the pathway of cholesterol biosynthesis, were studied in the isolated perfused rat liver. Oleic acid bound to bovine serum albumin, or albumin alone, was infused for 4 h at a rate sufficient to sustain an average concentration of 0.61 +/- 0.05 mM fatty acid during the perfusion. Hepatic cytosol and microsomal fractions were isolated at the termination of the perfusion. Oleic acid simultaneously increased the activities of the cytosolic cholesterol-biosynthetic enzymes 1.4-2.7-fold in livers from normal fed rats and from animals fasted for 24 h. These effects were accompanied by increased net secretion by the liver of cholesterol and triacylglycerol in the very-low-density lipoprotein (VLDL). We confirmed the observations reported previously from this laboratory of the stimulation by oleic acid of microsomal HMG-CoA reductase. In cytosols from perfused livers, the increase in AcAc-CoA thiolase activity was characterized by an increase in Vmax. without any change in the apparent Km of the enzyme for AcAc-CoA. In contrast, oleic acid decreased the Km of HMG-CoA synthase for Ac-CoA, without alteration of the Vmax. of the enzyme. The Vmax. of AcAc-CoA synthetase was increased by oleic acid, and there was a trend towards a small increase in the Km of the enzyme for acetoacetate. These data allow us to conclude that the enzymes that supply the HMG-CoA required for hepatic cholesterogenesis are stimulated, as is HMG-CoA reductase, by a physiological substrate, fatty acid, that increases rates of hepatic cholesterol synthesis and cholesterol secretion. Furthermore, we suggest that these effects of fatty acid on hepatic cholesterol metabolism result from stimulation of secretion of triacylglycerol in the VLDL by fatty acids, and the absolute requirement of cholesterol as an important structural surface component of the VLDL necessary for transport of triacylglycerol from the liver.