Accumulation of 7α-hydroxycholesterol in liver tissue of patients with cholesterol gallstones

Patients with cholesterol gallstones have a reduced pool of bile acids. This study was undertaken to clarify the mechanism by which bile acid biosynthesis does not increase to supranormal levels to cause a reexpansion of the pool. We investigated the first two steps of the bile acid biosynthesis pathway by assaying the activities of cholesterol 7α-hydroxylase, the rate-limiting enzyme in this pathway, and 3β-hydroxy-Δ⁵-C₂₇-steroid dehydrogenase/isomerase, and by measuring the concentrations of 7α-hydroxycholesterol and 7α-hydroxy-4-cholesten-3-one in liver specimens from ten patients with cholesterol gallstones and ten gallstone-free controls. In the patients with gallstones, cholesterol 7α-hydroxylase activity, 3β-hydroxy-Δ⁵-C₂₇ dehydrogenase/isomerase activity, and hepatic 7α-hydroxy-4-cholesten-3-one concentration did not significantly different from levels in controls, but hepatic 7α-hydroxycholesterol concentration was more than twofold that of controls (12.9 ± 2.6 vs 5.3 ±1.2 nmol/g liver,P<0.01). The concentration of 7α-hydroxycholesterol positively correlated with the ratio of cholesterol 7α-hydroxylase activity to 3β-hydroxy-Δ⁵-C₂₇ dehydrogenase/isomerase activity (r=0.93;P<0.005) in the gallstone-free controls. In contrast, this correlation disappeared in the patients with gallstones. These results suggest a derangement of the normal 7α-hydroxycholesterol metabolism in the patients with gallstones. The reason for the accumulation of 7α-hydroxycholesterol remains unclear; however, it is possible that, in patients with cholesterol gallstones, the accumulated 7α-hydroxycholesterol causes inappropriate suppression of cholesterol 7α-hydroxylase activity by product inhibition.     

Increased bile acid concentration in liver tissue with cholesterol gallstone disease

Patients with cholesterol gallstone disease have a reduced pool of bile acids. Overly sensitive feedback inhibition of bile acid synthesis has been postulated to explain this size reduction. To test this hypothesis, hepatic bile acid concentration and the activity of cholesterol 7-hydroxylase, the rate-limiting enzyme for bile acid biosynthesis, were determined in ten patients with cholesterol gallstones and ten patients without gallstones. The bile acids present in liver tissue are the sum of those returning to liver and those newly synthesized in liver. If an overly sensitive feedback inhibition truly existed in our gallstone patients, a decreased concentration of hepatic bile acids would have been expected. However, patients with cholesterol gallstones had significantly higher total (143.3 ±25.5 vs 64.5±10.8 nmol/g liver,P<0.01), chenodeoxycholic=" (64.1±9.9=" vs=">P<0.01), deoxycholic=" (22.8±10.9=" vs=">P<0.05), and=" ursodeoxycholic=" acid=" (6.2±1.4=" vs=">P<0.01) concentrations=" than=" patients=" without=" gallstones.=" the=" activity=" of=" cholesterol=">-hydroxylase did not differ significantly between the two groups. Impaired hepatic transport or secretion of bile acids is strongly suspected in cholesterol gallstone patients. The findings of the present study showed no evidence of overly sensitive feedback inhibition of bile acid synthesis in cholesterol gallstone patients. Bile acid pool size may be affected by the inappropriate increase of hepatic bile acids rather than by overly sensitive feedback inhibition.     

Sex differences in serum 7α-hydroxycholesterol levels in the rat reflect hepatic activity of 3β-hydroxy-Δ5-C27-steroid dehydrogenase and cholesterol 7α-hydroxylase

Factors that affect serum levels of 7α-hydroxycholesterol were studied in the rat. Serum levels of 7α-hydroxycholesterol differed in male and female rats fed regular chow (male; 0.2±0.1 nmol/ml (mean ±SD)n=8; female; 0.4±0.1 nmol/ml;n=8). When rats were fed with chow to which 3% cholestyramine had been added, the level increased significantly, particularly in female rats (male: 0.6±0.3 nmol/ml;n=8; female; 2.4±1.5 nmol/ml;n=8). The liver activity of cholesterol 7α-hydroxylase, the rate-limiting enzyme for degradation of cholesterol, did not show any sex differences, irrespective of whether the animals were fed with regular chow (male; 51±15 pmol/min per mg protein;n=8; female; 58±21 pmol/min per mg protein;n=8), or the cholestyramine-supplemented chow (male; 162±33pmol/min per mg protein;n=8; female; 172±33 pmol/min per mg protein;n=8). In contrast, the activity of 3β-hydroxy-Δ⁵-C₂₇-steroid dehydrogenase, which acts after cholesterol 7α-hydroxylase in the catabolism of cholesterol, showed a marked difference between the sexes. In both sexes this enzyme activity was higher in cholestyramine-treated rats (male; 963±78 pmol/min per mg protein;n=8; female; 708±106 pmol/min per mg protein,n=8) compared to that in that rats received regular chow (male; 622±83pmol/min per mg protein;n=8). If the serum level of 7α-hydroxycholesterol depended solely on the enzyme activity of cholesterol 7α-hydroxylase, it would be difficult to explain these sex differences, since there were no sex differences in levels of cholesterol, 7α-hydroxylase. These results clearly indicate that, in the rat, the serum level of 7α-hydroxycholesterol depends not only on cholesterol 7α-hydroxylase activity but also on 3β-hydroxy-Δ⁵-C₂₇-steroid dehydrogenase activity. Presented, in part, at the 32nd Annual Meeting of the Japanese Society of Gastroenterology, November 1990, Nara, Japan     

Increased bile acid pool inhibits cholesterol 7 alpha-hydroxylase in cholesterol-fed rabbits

BACKGROUND & AIMS: Cholesterol feeding unexpectedly inhibits cholesterol 7 alpha-hydroxylase in rabbits. The aim of this study was to explore the mechanism. METHODS: Twenty male New Zealand white rabbits were fed regular chow with and without 2% cholesterol for 10 days followed by 7 days of bile drainage. The activities of hepatic cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase that control bile acid synthesis in classic and alternative pathways were related to the size and composition of bile acid pool. RESULTS: After feeding cholesterol, plasma and hepatic cholesterol concentrations increased, the bile acid pool doubled (from 254 +/- 44 to 533 +/- 51 mg; P < 0.001), cholesterol 7 alpha-hydroxylase activity decreased 68% (P < 0.01), but sterol 27-hydroxylase activity increased 66% (P < 0.05) with increased cholic acid synthesis (P < 0.01). Bile drainage in the cholesterol-fed rabbits depleted the bile acid pool and stimulated down- regulated cholesterol 7 alpha-hydroxylase activity 11.4-fold (P < 0.001), although hepatic cholesterol remained elevated. Hepatic sterol 27-hydroxylase activity was unaffected. CONCLUSIONS: Feeding cholesterol increased hepatic cholesterol and stimulated sterol 27- hydroxylase and alternative bile acid synthesis, which expanded the bile acid pool and inhibited cholesterol 7 alpha-hydroxylase in rabbits. In distinction, hepatic sterol 27-hydroxylase was insensitive to changes in the bile acid pool. (Gastroenterology 1997 Dec;113(6):1958-65)     

Reduced hepatic LDL-receptor, 3-hydroxy-3-methylglutaryl coenzyme A reductase and sterol carrier protein-2 expression is associated with pregnancy loss in the diabetic rat

Hepatic low density lipoprotein receptor (LDLR), 3-hydroxy-3-methylglutaryl coenyzme A reductase (HMGR), cholesterol 7α-hydroxylase, and sterol carrier protein-2 are important proteins associated with the uptake, synthesis, degradation and transport of cellular cholesterol. Since cholesterol is critically important for steroid hormone synthesis and is an essential component in membrane biosynthesis, this study investigated whether these proteins are altered in the normal pregnant and streptozotocin (STZ)-induced diabetic pregnant rat. The goal of these experiments was to determine whether diabetic reproductive dysfunction is associated with a significant change in maternal cholesterol homeostasis. Diabetic animals were grouped based on their ability or inability to maintain pregnancy up to day 15 post-conception. LDLR and HMGR mRNA levels were significantly reduced in animals which did not maintain pregnancy whereas diabetic animals with fetuses had normal LDLR and HMGR mRNA levels. Hepatic LDLR, HMGR, and SCP2 protein levels were examined in normal pregnant and diabetic pregnant animals by Western blot analysis. SCP2 levels were reduced in all diabetic animals, particularly in the diabetic animals which lost their fetuses. The decline in SCP2 was associated with an increase in sterol carrier protein-X (SCPx), a protein related to SCP2. SCPx has been shown to have thiolytic activity and is thought to have a role in β-oxidation of fatty acids. HMGR was also significantly reduced in diabetic animals which lost their fetuses. Cholesterol 7α-hydroxylase mRNA was slightly, but not significantly, reduced in all diabetic animals. Serum very low density lipoprotein (VLDL) +LDL cholesterol increased significantly in the STZ-treated diabetic rats while the HDL cholesterol levels declined in these animals. Reduced hepatic LDLR and HMGR mRNA levels were consistently associated with reduced serum progesterone and an inability to maintain pregnancy. The results of this study suggest that the maintenance of maternal cholesterol metabolism is a critical factor directly associated with successful pregnancy outcome in the diabetic rat.     

Regulation of bile acid synthesis. II. Effect of bile acid feeding on enzymes regulating hepatic cholesterol and bile acid synthesis in the rat

Bile acid synthesis is believed to be regulated by bile salts returning to the liver via the portal vein and suppressing cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme in the bile acid biosynthesis pathway. In order to characterize the relative effectiveness of bile salts in regulating bile acid synthesis, seven different bile acids were administered (1% w/w in chow) to rats over a 14-day period. Biliary bile salt composition was determined from bile samples obtained prior to killing; in all cases, the fed bile acid became the predominant bile salt in bile. The specific activities of microsomal cholesterol 7 alpha-hydroxylase, HMG-CoA reductase and acylconenzyme A:cholesterol acyltransferase were determined after killing. Hydrophilic bile salts (ursocholic, hyocholic, ursodeoxycholic and hyodeoxycholic) did not inhibit HMG-CoA reductase or cholesterol 7 alpha-hydroxylase activities. By contrast, more hydrophobic bile salts (cholic, chenodeoxycholic and deoxycholic) inhibited the activities of these two enzymes in order of increasing hydrophobicity. Neither hydrophobic nor hydrophilic bile salts inhibited acylcoenzyme A:cholesterol acyltransferase activity. No consistent effect of bile acid feeding on total microsomal cholesterol was observed. Based on the results of these studies, we propose that the hydrophilic-hydrophobic balance of the bile acid pool may play an important role in the regulation of bile acid synthesis. We postulate that the activities of cholesterol 7 alpha-hydroxylase and HMG-CoA reductase may be regulated by hydrophobic bile acid-induced changes in the lipid composition and physicochemical properties (fluidity) of the microsomal membranes to which both of these rate-limiting enzymes are attached.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative regulation of hepatic sterol 27-hydroxylase and cholesterol 7α-hydroxylase activities in the rat, guinea pig, and rabbit: Effects of cholesterol and bile acids

The regulation of the classic and alternative bile acid synthetic pathways by key hepatic enzyme activities (microsomal cholesterol 7α-hydroxylase and mitochondrial sterol 27-hydroxylase, respectively) was examined in bile acid depletion and replacement and cholesterol-feeding experiments with rats, guinea pigs, and rabbits. The bile acid pool was depleted by creating a bile fistula (BF) and collecting bile for 2 to 5 days, and it was replaced by intraduodenal infusion of the major biliary bile acids (taurocholic acid [TCA], glycochenodeoxycholic acid [GCDCA], and glycocholic acid [GCA] in the rat, guinea pig, and rabbit, respectively) at rates equivalent to the measured hepatic flux of the bile acids. To study the effects of cholesterol, the animals were fed for 7 days on a basal diet with and without 2% cholesterol. Cholesterol 7α-hydroxylase and sterol 27-hydroxylase activities, measured by isotope incorporation assays, were related to bile acid output and composition and hepatic cholesterol concentrations. Intraduodenal infusion of bile acids increased the output of the tested bile acids, but did not significantly change hepatic cholesterol concentrations and had no effect on sterol 27-hydroxylase activity. Neither bile acid depletion nor replacement affected sterol 27-hydroxylase activity when three different substrates (cholesterol, 5β-cholestane-3α,7α-diol, and 5β-cholestane-3α,7α,12α-triol) were tested. In contrast, feeding 2% cholesterol increased hepatic cholesterol concentrations in rats, guinea pigs, and rabbits threefold, twofold, and eightfold, respectively, and increased hepatic mitochondrial sterol 27-hydroxylase activity (conversion of cholesterol to 27-hydroxycholesterol) in all three animal models. The stimulation and feedback inhibition of cholesterol 7α-hydroxylase activity by bile acid depletion and replacement were observed in all three animal models, whereas the effect of cholesterol feeding was species-dependent (cholesterol 7α-hydroxylase activity increased in the rat, did not change in the guinea pig, and was inhibited in the rabbit). Thus, in contrast to sterol 27-hydroxylase, which was upregulated by cholesterol but not affected by bile acid depletion and replacement in all three animal models, cholesterol 7α-hydroxylase activity was controlled consistently and inversely by the hepatic flux of bile acids, but was species-dependent in its response to a 1-week feeding with 2% cholesterol.     

Hepatic cholesterol metabolism in estrogen-treated men.

Operative liver biopsies were obtained from two male patients who developed gallstone disease during estrogen treatment of metastatic prostatic carcinoma. The heparin-sensitive binding of 125I-low-density lipoprotein (LDL) to liver homogenates (reflecting the expression of the LDL receptor) was determined, together with the activities of the rate-limiting enzymes in cholesterol synthesis [3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase], bile acid production (cholesterol 7 alpha-hydroxylase), and cholesterol esterification (acyl CoA:cholesterol acyl transferase). The results were related to data available in 18 patients (5 male, 13 female) who underwent cholecystectomy because of gallstone disease. The hepatic 125I-LDL-binding activity was increased threefold compared with five controls, and the activity of HMG-CoA reductase was increased twofold. There was no major difference in the activities of cholesterol 7 alpha-hydroxylase or acyl CoA:cholesterol acyl transferase. The concentration of free and total cholesterol in liver microsomes was approximately 30% lower in the estrogen-treated men than in 11 controls. The results indicate that estrogen at pharmacological doses stimulates hepatic LDL-receptor expression and HMG-CoA reductase activity in men. The increased LDL-receptor expression could in part explain the enhanced plasma clearance of injected 125I-LDL and hence the reduction in plasma LDL cholesterol previously shown to occur in estrogen-treated men.     

Cholesterol crystals, cholesterol saturation of bile and biliary lithiasis

It has been repeatedly shown that normal human gallbladder bile is commonly supersaturated wih cholesterol. It has been therefore suggested that the crucial step of the formation of cholesterol gallstones might be the nucleation and growth of cholesterol monohydrate crystals. Consequently this work was aimed at determining: 1) if cholesterol crystal formation is really a typical feature of gallbladder bile with cholesterol gallstones; 2) the influence of the degree of cholesterol saturation of bile on the formation of cholesterol crystals. Gallbladder bile from 89 patients (23 from patients with cholesterol gallstones, 7 from patients with non-cholesterol gallstones and 59 from patients free of gallstones) and hepatic bile from 17 previously cholecystectomized patients were studied. Four of these patients had cholesterol stones of the common bile duct. Results: (a) gallbladder bile: cholesterol crystals were present on immediate examination in 19 of the 23 bile samples with cholesterol stones, in 2 of the 7 bile samples with non-cholesterol stones and in 1 of the 59 bile samples without stones. Only 1 bile sample with cholesterol stone developed crystals. Cholesterol saturation of bile with or without crystals did not differ significantly; (b) hepatic bile: cholesterol crystals were detected on immediate examination in one of the 17 bile samples and subsequently appeared in one of the remaining samples. Cholesterol saturation of hepatic bile (2.10 +/- 0.43) was significantly higher (p less than 0.01) than that of gallbladder bile containing cholesterol stones (1.32 +/- 0.43).(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative regulation of hepatic sterol 27-hydroxylase and cholesterol 7alpha-hydroxylase activities in the rat, guinea pig, and rabbit: effects of cholesterol and bile acids

The regulation of the classic and alternative bile acid synthetic pathways by key hepatic enzyme activities (microsomal cholesterol 7alpha-hydroxylase and mitochondrial sterol 27-hydroxylase, respectively) was examined in bile acid depletion and replacement and cholesterol-feeding experiments with rats, guinea pigs, and rabbits. The bile acid pool was depleted by creating a bile fistula (BF) and collecting bile for 2 to 5 days, and it was replaced by intraduodenal infusion of the major biliary bile acids (taurocholic acid [TCA], glycochenodeoxycholic acid [GCDCA], and glycocholic acid [GCA] in the rat, guinea pig, and rabbit, respectively) at rates equivalent to the measured hepatic flux of the bile acids. To study the effects of cholesterol, the animals were fed for 7 days on a basal diet with and without 2% cholesterol. Cholesterol 7alpha-hydroxylase and sterol 27-hydroxylase activities, measured by isotope incorporation assays, were related to bile acid output and composition and hepatic cholesterol concentrations. Intraduodenal infusion of bile acids increased the output of the tested bile acids, but did not significantly change hepatic cholesterol concentrations and had no effect on sterol 27-hydroxylase activity. Neither bile acid depletion nor replacement affected sterol 27-hydroxylase activity when three different substrates (cholesterol, 5beta-cholestane-3alpha,7alpha-diol, and 5beta-cholestane-3alpha,7alpha,12alpha-triol) were tested. In contrast, feeding 2% cholesterol increased hepatic cholesterol concentrations in rats, guinea pigs, and rabbits threefold, twofold, and eightfold, respectively, and increased hepatic mitochondrial sterol 27-hydroxylase activity (conversion of cholesterol to 27-hydroxycholesterol) in all three animal models. The stimulation and feedback inhibition of cholesterol 7alpha-hydroxylase activity by bile acid depletion and replacement were observed in all three animal models, whereas the effect of cholesterol feeding was species-dependent (cholesterol 7alpha-hydroxylase activity increased in the rat, did not change in the guinea pig, and was inhibited in the rabbit). Thus, in contrast to sterol 27-hydroxylase, which was upregulated by cholesterol but not affected by bile acid depletion and replacement in all three animal models, cholesterol 7alpha-hydroxylase activity was controlled consistently and inversely by the hepatic flux of bile acids, but was species-dependent in its response to a 1-week feeding with 2% cholesterol.     

Hepatic cholesterol and bile acid synthesis, low-density lipoprotein receptor function, and plasma and fecal sterol levels in mice: effects of apolipoprotein E deficiency and probucol or phytosterol treatment

We compared hepatic cholesterol metabolism in apolipoprotein (apo) E-knockout (KO) mice with their wild-type counterparts. We also investigated the effects of treatment with phytosterols or probucol on the activity of hepatic 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase (cholesterol synthesis), cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase (bile acid synthesis), and low-density lipoprotein (LDL) receptor function in this animal model of atherogenesis. These findings were then related to treatment-induced changes in plasma, hepatic, and fecal sterol concentrations. Mouse liver membranes have binding sites similar to LDL receptors; the receptor-mediated binding represents 80% of total binding and is LDL concentration-dependent. These binding sites have higher affinity for apo E-containing particles than apo B only-containing particles. Deletion of apo E gene was associated with several-fold increases in plasma cholesterol levels, 1.5-fold increase in hepatic cholesterol concentrations, 50% decrease in HMG-CoA reductase activity, 30% increase in cholesterol 7 alpha-hydroxylase and 25% decrease in LDL receptor function. Treatment of apo E-KO mice with either probucol or phytosterols significantly reduced plasma cholesterol levels. Phytosterols significantly increased the activity of hepatic HMG-CoA reductase, and probucol significantly increased cholesterol 7 alpha-hydroxylase activity. Neither treatment significantly altered hepatic LDL receptor function. Phytosterols, but not probucol, significantly increased fecal sterol excretion and decreased hepatic cholesterol concentrations. Plasma cholesterol lowering effects of phytosterols and probucol are due to different mechanisms: stimulation of cholesterol catabolism via increased bile acid synthesis by probucol and decreased cholesterol absorption by phytosterols. In the absence of apo E, hepatic LDL receptors could not be upregulated and did not contribute to the cholesterol lowering effects of either agent.     

Hepatic cholesterol metabolism in patients with gallstones.

Relative rates of cholesterol and bile acid synthesis were estimated in patients with cholesterol gallstones and biliary obstruction by determining the hepatic activities of 3-hydroxy-3-methylglutaryl-CoA reductase and cholesterol 7 alpha-hydroxylase, the respective rate-determining enzymes for cholesterol and bile acid synthesis. As compared with eight control studies, 3-hydroxy-3-methylglutaryl-CoA reductase activity was 27% higher in 12 gallstone subjects, but 75% lower in 5 subjects with biliary obstruction. Cholesterol 7 alpha-hydroxylase activity was reduced in the gallstone (47% lower) and biliary obstruction (78% lower) subjects. Liver cholesterol concentrations were 56% higher in the gallstone and 53% higher in the biliary obstruction subjects than the control group. These findings suggest that the pathogenesis of gallstones is related to both increased cholesterol synthesis and decreased bile acid formation, whereas cholesterol accumulates in biliary obstruction because of defective removal since cholesterol production is low.     

Klotho protein activates the PKC pathway in the kidney and testis and suppresses 25-hydroxyvitamin D3 1alpha-hydroxylase gene expression

Homozygous Klotho mutant (kl ^−/−) mice exhibit a variety of phenotypes resembling human aging, including arteriosclerosis, infertility, skin atrophy, osteoporosis, and short life span. Calcium abnormality, one of the phenotypes in kl ^−/− mice, is thought to be due to the elevated gene expression of 25-hydroxyvitamin D₃ 1α-hydroxylase in the kidney. We studied 25-hydroxyvitamin D₃ 1α-hydroxylase gene expression using a Klotho plasmid that we had previously constructed for Klotho protein production. It was found that Klotho protein medium upregulated cAMP and the PKC pathway, and suppressed 25-hydroxyvitamin D₃ 1α-hydroxylase in kidney cells. However, both cAMP and PKC are known to elevate 25-hydroxyvitamin D₃ 1α-hydroxylase gene expression, therefore, another unknown calcium regulation pathway using Klotho protein medium might exist. Furthermore, we found that activation of the PKC pathway by Klotho was observed only in the kidney and testis, where the Klotho gene is expressed, although activation of the cAMP pathway was observed in any kind of cell. These data suggest that calcium regulation through 25-hydroxyvitamin D₃ 1α-hydroxylase by Klotho depends on non-cAMP and a non-PKC pathway and that the Klotho protein may have different signaling pathways, depending on the Klotho gene expression in different cells and organs.     

Time course of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and messenger ribonucleic acid, biliary lipid secretion, and hepatic cholesterol content in methimazole-treated hypothyroid and hypophysectomized rats after triiodothyronine administration: possible linkage of cholesterol synthesis to biliary secretion

In an effort to define the mechanism by which thyroid hormone increases the synthesis of hepatic cholesterol, we have investigated both in hypophysectomized and methimazole-treated hypothyroid rats the time course of T3 effects on plasma cholesterol concentration, total hepatic cholesterol, the rate of biliary secretion of cholesterol, bile acids, and phospholipids, and the activity and mRNA levels of 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase, the rate-limiting enzyme in the hepatic synthesis of cholesterol. A single dose of 200 micrograms T3 was estimated to maintain at least 90% nuclear occupancy for the ensuing 54 h of the experiment. In both preparations the relative rise in biliary secretion of cholesterol exceeded that of other biliary constituents and preceded by 12 h an increase in HMG-CoA reductase enzyme activity and its mRNA. The level of total hepatic cholesterol remained constant throughout the experiment. We interpret these findings to suggest that T3-stimulated cholesterol synthesis is mediated by an antecedent T3-induced rise in biliary cholesterol secretion. We postulate that biliary cholesterol secretion is augmented by an intrahepatic shift of cholesterol and depletion of the hepatic sampling center responsible for the feedback regulation of cholesterol synthesis. The level of HMG CoA reductase mRNA appeared to govern enzyme activity in both preparations, but the ratio of mRNA to hepatic enzyme activity was substantially greater in the methimazole-treated compared with the hyphophysectomized animals.     

α-Melanocyte-Stimulating Hormone Gene Transfer Attenuates Inflammation after Bile Duct Ligation in the Rat

Cholestasis occurs in a wide variety of human liver diseases, and hepatocellular injury is an invariant feature of cholestasis causing liver dysfunction and inflammation, promoting fibrogenesis, and ultimately leading to liver failure. α-Melanocyte-stimulating hormone (α-MSH) is a potent anti-inflammatory agent in many models of inflammation, suggesting that it inhibits a critical step common to different forms of inflammation. The aim of this study was to investigate whether the gene transfer of α-MSH could attenuate hepatic inflammation after bile duct ligation in the rat. Studies were performed in bile duct-ligated (BDL) rats. Hydrodynamic-based gene transfection with α-MSH plasmid via rapid tail vein injection was performed 30 min after ligation of bile duct. The endpoints were studied as markers of inflammation 7 days after bile duct ligation. α-MSH expression in liver via a single administration of naked plasmid was demonstrated. Liver inflammation index, including neutrophil infiltration and serum alanine aminotransferase, were significantly reduced in α-MSH gene transfer rats. Markers for liver inflammation, including expression of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and inducible NO synthase (iNOS) mRNA, as assessed by real-time PCR, were also attenuated by α-MSH gene therapy. Expression of iNOS protein in liver diminished after α-MSH gene transfer. Consistent with these data, hepatic stellate cells (HSC) and Kupffer cells were markedly inhibited in α-MSH gene-treated rats. Our findings show that gene transfer of α-MSH could attenuate hepatic inflammation after bile duct ligation in the rat.     

Studies of cholesterol and bile acid metabolism, and early atherogenesis in hamsters fed GT16-239, a novel bile acid sequestrant (BAS)

The purpose of this study was to compare the efficacy of GT16-239, an alkylated, cross-linked poly(allylamine) bile acid sequestrant with cholestyramine on cholesterol and bile acid metabolism, and early aortic atherosclerosis in hypercholesterolemic male F1B Golden Syrian hamsters. In this controlled study, 42 hamsters were divided into six groups and were fed a chow-based hypercholesterolemic diet supplemented with a 10% oil blend (55% coconut/45% corn), 0.1% cholesterol (w/w) (control) and either 0.9 or 1.2% cholestyramine or 0.2, 0.4 or 0.6% GT16-239 for 13 weeks. Laboratory analyses included evaluating plasma lipoprotein cholesterol and triglyceride concentrations, hepatic HMG-CoA reductase and 7 α-hydroxylase activities, fecal excretion of bile acids and neutral sterols, hepatic cholesterol concentrations, and early atherosclerosis (aortic fatty streak area). Relative to the control diet, the 0.6% GT16-239 versus the 1.2% cholestyramine significantly inhibited the elevation of plasma lipoprotein total cholesterol (TC) (−69% vs −40%), high density lipoprotein-cholesterol (HDL-C) (−49% vs −30%), and non-HDL-C (−81% vs −48%) concentrations; increased the activities of both HMG-CoA reductase (1492% vs 62%) and 7 α-hydroxylase (175% vs 86%); lowered the concentration of hepatic cholesteryl ester (−94% vs −59%); increased fecal cholesterol concentration (+28% vs −10%); and decreased aortic fatty streak area (−100% vs −86%). Unexpected findings of this comparison were increased fecal concentrations of cholic acid (533%) and chenodeoxycholic acid (400%) and the reduction in lithocholic acid (−50%) in the 0.6% GT16-239 compared to the 1.2% cholestyramine group. In summary, GT16-239 had a greater impact on cholesterol metabolism and early atherosclerosis in hypercholesterolemic hamsters than cholestyramine.     

Regulation of cholesterol 7α-hydroxylation by cholesterol synthesis in rat liver

Hepatic cholesterol 7α-hydroxylation and cholesterogenesis were determined in vitro at various time intervals 6 hr after oral or intravenous administration to fasted rats. Glucose administration to fasted rats enhanced cholesterol 7α-hydroxylation as well as fatty acid or cholesterol synthesis in the liver, though significant changes were not demonstrated in the cholesterol content of liver and serum throughout the observed periods. Hepatic cholesterol synthesis increased 1 hr after oral glucose ingestion. On the other hand, cholesterol 7α-hydroxylation was stimulated 2 hr after oral glucose administration, with a lag phase of 1 hr following the induction of cholesterol synthesis. From the results of the experiments on the effect of glucose ingestion, it appeared that cholesterol 7α-hydroxylation was correlated with hepatic cholesterogenesis.Hepatic cholesterogenesis was decreased in triparanol-treated rats and was not stimulated by glucose administration. Triparanol did not affect cholesterol hydroxylation in the fasted state, but did abolish the stimulatory effect of glucose.Short-term feeding of a high-cholesterol diet, which suppressed hepatic cholesterol synthesis, stimulated cholesterol 7α-hydroxylation. Oral administration of glucose to cholesterol-fed rats still further accelerated hydroxylation.In rats with ligated bile ducts, hepatic cholesterogenesis increased 24 hr after bileduct ligation while hydroxylation decreased 24 hr after the operation but was stimulated after 72 hr. Glucose ingestion by these rats stimulated both cholesterol synthesis and hydroxylation.It is concluded that increased hepatic cholesterogenesis enhances cholesterol 7α-hydroxylase activity; this activity does not depend upon increased fatty acid or other lipid synthesis, altered bile flow or gastrointestinal factors. Increased dietary cholesterol intake also accelerates hydroxylase activity. These mechanisms may preserve homeostasis in the serum cholesterol level, and an altered balance between these two enzyme systems — anabolic and catabolic — probably induces a change in the size of the body cholesterol pool.     

High vesicular cholesterol and protein in bile are associated with formation of cholesterol but not pigment gallstones

To examine the differentiating parameters between cholesterol and pigment gallstones, we compared the nucleation times, concentrations of biliary lipid and protein, and the distribution of vesicular cholesterol in gallbladder bile of 16 patients with cholesterol, eight patients with black pigment gallstones, and nine gallstone-free control patients. Cholesterol monohydrate crystals were present in the fresh bile of only the cholesterol gallstone group. The nucleation time was significantly faster in the cholesterol stone group (3.3±3.2 days) than in the other two groups (pigment stone: 15.8±6.6, control: 16.9±5.7). The cholesterol saturation indices and the distribution of vesicular cholesterol were significantly higher in the cholesterol gallstone group than those in the other two groups. The total biliary protein concentration was significantly (P<0.01) higher in the cholesterol gallstone group [2.57±1.91 (sd) mg/ml] than that in the black pigment stone group (1.09±0.59). All parameters in patients with black pigment gallstone were essentially similar to the controls. We conclude that the presence of cholesterol crystals, rapid nucleation time, high vesicular cholesterol distribution, elevated cholesterol saturation index, and high protein concentration are associated with cholesterol gallstones but not with black pigment gallstones.     

Deoxycholate and cholate modulate the source of cholesterol substrate for bile acid synthesis in the rat

In the current study, the role of the supply of preformed and newly synthesized cholesterol for the feedback control of the synthesis of different bile acids and the secretion of biliary cholesterol was investigated. To define these cholesterol fluxes and the possibility of a different modulation by bile acids with different suppressive capacities, a continuous labeling with tritiated water was used in rats with an extracorporeal bile duct receiving intraduodenal infusions of taurocholate or taurocholate plus deoxycholate. After bile acid pool depletion (6 to 9 hours) total muricholate, cholate, and chenodeoxycholate synthesis was variably increased (24% to 93%) during an infusion of 304 μmol taurocholate/kg per hour. The increase in bile acid synthesis and biliary cholesterol output was predominantly due to the utilization of preformed (unlabeled) cholesterol. The addition of 52 μmol/kg per hour of deoxycholate to 258 μmol/kg per hour of taurocholate had a comparable effect. In the late period (30 to 54 hours), the taurocholate infusion had little impact on total muricholate and chenodeoxycholate synthesis but caused by a significant increase of the proportion from preformed cholesterol. Both total cholate production and its synthesis from de novo (labeled) cholesterol was inhibited by 30% (P < .05) and 64% (P < .01), respectively. The secretion rate of total and de novo biliary cholesterol was higher (65% and 72%; P < .01) compared with controls. In comparison, the combined bile acid infusion led to a further increase of total muricholate synthesis (P < .05), which was again due to an enhanced synthesis from preformed cholesterol (P < .001). Similar changes were observed in chenodeoxycholate. The more pronounced suppression of total cholate synthesis by 81% (P < .05) was due to a diminished cholate synthesis from both de novo cholesterol by 72% (P < .001) and preformed cholesterol by 91% (P > .05). We conclude that the modulation of the synthesis of the various primary bile acids in the rat differs and feedback regulation of cholate synthesis by taurocholate and deoxycholate is mediated by different mechanisms of control, including inhibition of cholesterol 7α-hydroxylase, HMG-CoA reductase, and uptake of lipoprotein cholesterol.