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.     

Stimulatory effect of dietary lipid and cholestyramine on hepatic HMG CoA reductase

The diurnal cycle of hepatic HMG CoA reductase activity was studied under conditions of controlled feeding where the percentage of dietary lipid, alone or in combination with 2% cholestyramine, was varied. Cholestyramine caused an increase in HMG CoA reductase activity that began soon after feeding started and peaked 6 hr later. In contrast, a diet containing 20% corn oil was a much weaker inducer of the enzyme but caused a prolonged elevation that began late in the fasting part of the cycle. These patterns suggest two different mechanisms of action.     

Diurnal variation of HMG CoA reductase activity in rat intestine

HMG CoA reductase activity of rat intestinal mucosa has a diurnal rhythm which coincides with the diurnal variation of the hepatic HMG CoA reductase but has a lower amplitude. The rhythmic variation of the intestinal reductase was present in both jejunal and ileal crypt cell microsomes and was not abolished by cholestyramine administration.     

Influence of dietary bile acids on formation of bile acids in rat

Various taurine-conjugated bile acids were fed to rats at the 1%-level in the diet for 3 or 7 days and the effect on several hydroxylations involved in the biosynthesis and metabolism of bile acids was studied. The hydroxylations studied were all catalyzed by the microsomal fraction of liver homogenate fortified with NADPH. The 7α-hydroxylation of cholesterol was inhibited by feeding taurocholic acid, taurocheno-deoxycholic acid and taurodeoxycholic acid for 3 as well as 7 days. No marked inhibition was obtained with taurohyodeoxycholic acid or taurolithocholic acid. The 12α-hydroxylation of 7α-hydroxy-4-cholesten-3-one was inhibited after 3 as well as 7 days by all bile acids except taurohyodeoxycholic acid. With this acid a marked stimulation of 12α-hydroxylation was observed. The effects of the different bile acids on the 7α-hydroxylation of taurodeoxycholic acid were not very marked. The 6β-hydroxylation of lithocholie acid and taurochenodeoxycholic acid was stimulated by taurocholic acid and taurodeoxycholic acid. The reaction was inhibited by taurochenodeoxycholic acid, at least after 7 days. Taurohyodeoxycholic acid inhibited the 6β-hydroxylation slightly and taurolithocholic acid had no effect. The results were discussed in the light of present knowledge concerning mechanisms of regulation of formation and metabolism of bile acids and it was suggested that the mechanisms may be more complex than previously thought.     

Cholesterol synthesis and HMG CoA reductase activity during hepatocarcinogenesis in rats

Two treatment regimes were used to produce preneoplastic foci (as determined by the presence of gamma-glutamyl transferase) in rat liver. Increased [14C]acetate incorporation into cholesterol and 3-hydroxy-3-methyl glutaryl CoA reductase activity were associated with high levels of gamma-glutamyl transpeptidase and foci formation. Dietary feedback inhibition of both [14C]acetate incorporation and 3-hydroxy-3-methyl glutaryl CoA reductase activity was reduced at a selected time when gamma-glutamyl transpeptidase activity was high. These changes could not be accounted for by a regenerative response in the liver.     

Biosynthesis of sterols and bile acids in rat liver epithelial cell lines

A rat liver epithelial cell line growing in a serum-supplemented medium expressed biosynthetic pathways of bile sterols and of free and conjugated chenodeoxycholic and cholic acids, the main primary bile acids of the liver. They were identified and measured by gas chromatography-mass spectrometry. The bile steroid secretion in the serum-supplemented cell line was established upon incubation in a serum-free medium which was demonstrated to sustain cell growth, allowing elimination of the interference of exogenous bile steroids and effectors. The free bile acid secretion was also expressed in a subline adapted to proliferate in this serum-free medium, i.e., a basal medium supplemented with 4 g/l albumin carrying 7.6 muequiv./l of a mixture of six long-chain free fatty acids but without any addition of hormones and growth factors. In addition, the rat liver epithelial cell line growing in the serum-supplemented medium maintained, with time, a steady-state of bile acid secretion over a lifespan of 500 days. In the two types of liver epithelial cell lines, dexamethasone and chenodeoxycholic acid supplementation exerted, individually, either a stimulating or an inhibiting effect on the bile acid secretion concurrently with the hydroxylation of chenodeoxycholic acid into alpha-muricholic acid.     

Spectrum of effects of dietary long-chain fatty acids on rat intestinal glucose and lipid uptake

Isocaloric modification in the ratio of dietary polyunsaturated-to-saturated fatty acids influences intestinal uptake of actively and passively transported nutrients. This study was undertaken to determine which dietary fatty acid was responsible for these alterations in absorption. Adult female rats were fed isocaloric semisynthetic diets high in palmitic and stearic acids (SFA), oleic acid (OA), linoleic acid (LA), or linolenic acid (LNA). An in vitro technique was used to measure the uptake of varying concentrations of glucose as well as a series of fatty acids and cholesterol. Jejunal uptake of 40 mM glucose was highest in rats fed SFA and lowest in those fed LA; ileal glucose uptake was similar in OA, LA, and LNA, but was lowest in SFA. Jejunal uptake of medium-chain fatty acids (8:0-12:0) was higher in OA than in other diet groups; ileal uptake of medium-chain fatty acids was unaffected by diet. Jejunal and ileal uptake of 18:2 was higher in LNA than in SFA or OA; the uptake of the other long-chain saturated or unsaturated fatty acids was unchanged by diet. The ileal but not the jejunal uptake of cholesterol was increased in LA as compared with SFA or OA, and reduced in LNA as compared with LA. These transport changes were not explained by differences in the animals' food consumption, body weight gain, intestinal mass, or mucosal surface area. We postulate that these diet-induced transport alterations may be mediated via changes in brush border membrane phospholipid fatty acyl composition. Thus, intestinal transport of nutrients may be varied by isocaloric changes in the dietary content of individual fatty acids.     

Formation of C21 bile acids from plant sterols in the rat

Formation of bile acids from sitosterol in bile-fistulated female Wistar rats was studied with use of 4-14C-labeled sitosterol and sitosterol labeled with 3H in specific positions. The major part (about 75%) of the 14C radioactivity recovered as bile acids in bile after intravenous administration of [4-14C]sitosterol was found to be considerably more polar than cholic acid, and only trace amounts of radioactivity had chromatographic properties similar to those of cholic acid and chenodeoxycholic acid. It was shown that polar metabolites were formed by intermediate oxidation of the 3 beta-hydroxyl group (loss of 3H from 3 alpha-3H-labeled sitosterol) and that the most polar fraction did not contain a hydroxyl group at C7 (retention of 3H in 7 alpha,7 beta-3H2-labeled sitosterol). Furthermore, the polar metabolites had lost at least the terminal 6 or 7 carbon atoms of the side chain (loss of 3H from 22,23-3H2- and 24,28-3H2-labeled sitosterol). Experiments with 3H-labeled 7 alpha-hydroxysitosterol and 4-14C-labeled 26-hydroxysitosterol showed that none of these compounds was an efficient precursor to the polar metabolites. By analysis of purified most polar products of [4-14C] sitosterol by radio-gas chromatography and the same products of 7 alpha,7 beta-[2H2]sitosterol by combined gas chromatography-mass spectrometry, two major metabolites could be identified as C21 bile acids. One metabolite had three hydroxyl groups (3 alpha, 15, and unknown), and one had two hydroxyl groups (3 alpha, 15) and one keto group. Considerably less C21 bile acids were formed from [4-14C]sitosterol in male than in female Wistar rats. The C21 bile acids formed in male rats did not contain a 15-hydroxyl group. Conversion of a [4-14C]sitosterol into C21 bile acids did also occur in adrenalectomized and ovariectomized rats, indicating that endocrine tissues are not involved. Experiments with isolated perfused liver gave direct evidence that the overall conversion of sitosterol into C21 bile acids occurs in this organ. Intravenously injected 7 alpha,7 beta-3H-labeled campesterol gave a product pattern identical to that of 4-14C-labeled sitosterol. Possible mechanisms for hepatic conversion of sitosterol and campesterol into C21 bile acids are discussed.     

Feedback regulation of cholesterol synthesis: sterol-accelerated ubiquitination and degradation of HMG CoA reductase

3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase produces mevalonate, an important intermediate in the synthesis of cholesterol and essential nonsterol isoprenoids. The reductase is subject to an exorbitant amount of feedback control through multiple mechanisms that are mediated by sterol and nonsterol end-products of mevalonate metabolism. Here, I will discuss recent advances that shed light on one mechanism for control of reductase, which involves rapid degradation of the enzyme. Accumulation of certain sterols triggers binding of reductase to endoplasmic reticulum (ER) membrane proteins called Insig-1 and Insig-2. Reductase-Insig binding results in recruitment of a membrane-associated ubiquitin ligase called gp78, which initiates ubiquitination of reductase. This ubiquitination is an obligatory reaction for recognition and degradation of reductase from ER membranes by cytosolic 26S proteasomes. Thus, sterol-accelerated degradation of reductase represents an example of how a general cellular process (ER-associated degradation) is used to control an important metabolic pathway (cholesterol synthesis).     

Synthesis and HMG CoA reductase inhibition of 4-thiophenyl quinolines as potential hypocholesterolemic agents

A series of novel 4-thiophenyl quinoline-based mevalonolactone derivatives were synthesized from ethyl 6,7,8-trisubstituted-4-chloro-quinoline-3-carboxylates by several reactions and evaluated for their ability to inhibit the rat HMG CoA reductase in vitro. It was found that substitution with a variety of thiophenyl groups at position 4 in quinoline resulted in retention or enhancement of the inhibition and the preferable groups were 4-isopropyl-thiophenyl and 3-methoxy-thiophenyl. (4R,6S)-6-[(E)-2-(6,7,8-trifluoro-4-isopropylthiophenyl-quinoline-3-yl)-ethenyl]-3,4,5,6-tetrahydro-4-hydroxy-2H-pyran-2-one (A16) and (4R, 6S)-6-[(E)-2-(6-fluoro-4,7-di-(3-methoxy-thiophenyl)-quinoline-3-yl)-ethenyl]-3,4,5,6-tetrahydro-4-hydroxy-2H-pyran-2-one (A23) were approximately three times more potent than rosuvastatin or pitavastatin in inhibiting HMG CoA reductase and selected as the hypocholesterolemic candidates for further evaluation.     

Sphingolipids and cellular cholesterol homeostasis. Effect of ceramide on cholesterol trafficking and HMG CoA reductase activity

We previously showed that degradation of cellular sphingomyelin (SM) by SMase C results in a greater stimulation of cholesterol translocation to endoplasmic reticulum, compared to its degradation by SMase D. Here we investigated the hypothesis that the effect of SMase C is partly due to the generation of ceramide, rather than due to depletion of SM alone. Inhibition of hydroxymethylglutaryl CoA reductase (HMGCR) activity was used as a measure of cholesterol translocation. Treatment of fibroblasts with SMase C resulted in a 90% inhibition of HMGCR, whereas SMase D treatment inhibited it by 29%. Treatment with exogenous ceramides, or increasing the endogenous ceramide levels also inhibited HMGCR by 60-80%. Phosphorylation of HMGCR was stimulated by SMase C or exogenous ceramide. The effects of ceramide and SMase D were additive, indicating the independent effects of SM depletion and ceramide generation. These results show that ceramide regulates sterol trafficking independent of cellular SM levels.     

Membrane-bound domain of HMG CoA reductase is required for sterol-enhanced degradation of the enzyme

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase) is a single polypeptide chain with two contiguous domains: a soluble domain (548 amino acids) that catalyzes the rate-controlling step in cholesterol synthesis and a membrane-bound domain (339 amino acids) that anchors the protein to the endoplasmic reticulum (ER). HMG CoA reductase is degraded at least 10-fold more rapidly than other ER proteins; degradation is accelerated in the presence of cholesterol. To understand this controlled degradation, we transfected reductase-deficient Chinese hamster ovary (CHO) cells with a plasmid expression vector containing a reductase cDNA that lacks the segment encoding the membrane domain. The plasmid produced a truncated reductase (37 kd smaller than normal) that was enzymatically active with normal kinetics; most of the truncated enzyme was found in the cytosol. The truncated enzyme was degraded one-fifth as fast as the holoenzyme; degradation was no longer accelerated by sterols. We conclude that the membrane-bound domain of reductase plays a crucial role in the rapid and regulated degradation of this ER protein.     

Effects of dietary fatty acids on hepatic 3-hydroxy-3-methylglutaryl CoA reductase activity in hamsters on a high-glucose diet

Hepatic 3-hydroxy-3-methylglutaryl CoA reductase activity in hamsters given a fat-free high-glucose diet for 21 days was approximately 20 times higher than that in chow-fed hamsters. The increase in enzyme activity by dietary glucose was affected by saturated or unsaturated fatty acids or cholesterol added to the high-glucose diet. Ethyllinoleate or ethyloleate, added to the diet at a concentration of 5%, suppressed the increase in the enzyme activity. In contrast, addition of ethylpalmitate to the diet further stimulated the increase in the enzyme activity. Addition of 2% cholesterol to the high-glucose diet moderately suppressed, and addition of both cholesterol and ethyllinoleate completely prevented, the increase in the enzyme activity. The enzyme activity closely correlated with the incidence of formation of cholesterol gallstones but not with the liver cholesterol level. Marked increase in the enzyme activity was observed by feeding the high-glucose diet to starved hamsters for even a short period. On the third day after feeding was resumed, the enzyme activity was increased 500-fold compared to that during starvation. This increase in the enzyme activity was also reduced by dietary unsaturated fatty acid esters and stimulated by a dietary saturated fatty acid ester.     

Characterization of HMG CoA synthase activity of rat liver and CHO-K1 cells

This report describes the characterization and partial purification of rat liver 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase activity. A preliminary characterization of Chinese hamster ovary (CHO) cell HMG CoA synthase activity is also presented. Ion-exchange chromatography of ammonium sulfate precipitates of rat liver cytosol indicate the existence of two isoenzymes of HMG CoA synthase. These isoenzymes are physically, catalytically, and immunologically distinct. One of these isoenzymes, peak 1, resembles mitochondrial HMG-CoA synthase activity as evidenced by similarities in elution upon ion-exchange chromatography, inhibition by MgCl2, and cross reactivity with an antibody prepared against the mitochondrial enzyme. As peak 1 activity is unstable, further purification studies were performed on peak 2 activity. Peak 2 can be further resolved into two activities (peaks 2A and 2B) by gel filtration. In contrast, CHO-K1 cells (a permanent fibroblast line) possess only peak 2 type HMG CoA synthase activity.