Effects of gender on hepatic HMG-CoA reductase, cholesterol 7alpha-hydroxylase, and LDL receptor in hereditary analbuminemia

Hypoalbuminemia is accompanied by hypercholesterolemia in both nephrotic syndrome and hereditary analbuminemia. Hypercholesterolemia is more severe in the female than in the male Nagase analbuminemic rats (NAR). The sex difference in plasma cholesterol diminishes after ovariectomy (OVX) and reappears after estrogen replacement in the NAR. The molecular mechanism responsible for the sex difference in severity of hypercholesterolemia in NAR is not known and was investigated here. To this end, hepatic hydroxylmethylglutaryl (HMG)-CoA reductase, cholesterol 7alpha-hydroxylase, and LDL receptor were determined in male, female, and OVX female NAR and Sprague-Dawley (SD) rats. Plasma cholesterol, triglycerides, and hepatic HMG-CoA reductase activities were greater in both female and male NAR than in SD rats. This was coupled with upregulation of cholesterol 7alpha-hydroxylase in both male and female NAR compared with SD controls. LDL receptor in male NAR was similar to that in male SD rats but was significantly reduced in female NAR. OVX partially, but significantly, reduced plasma cholesterol and triglyceride levels in female NAR. This was coupled with a significant rise in hepatic cholesterol 7alpha-hydroxylase and a modest increase in hepatic LDL receptor. In contrast, OVX resulted in a mild elevation of plasma cholesterol and no significant changes in total hepatic HMG-CoA reductase, cholesterol 7alpha-hydroxylase, or LDL receptor in female SD rats. Thus the greater severity of hypercholesterolemia in the female NAR appears to be due, in part, to a combination of the constrained compensatory upregulation of cholesterol 7alpha-hydroxylase and LDL receptor deficiency.     

Role of cholesterol 7alpha-hydroxylase (CYP7A1) in nutrigenetics and pharmacogenetics of cholesterol lowering

The relationship between dietary composition/cholesterol-lowering therapy and final plasma lipid levels is to some extent genetically determined. It is clear that these responses are under polygenic control, with multiple variants in many genes participating in the total effect (and with each gene contributing a relatively small effect). Using different experimental approaches, several candidate genes have been analyzed to date.Interesting and consistent results have been published recently regarding the A-204C promoter variant in the cholesterol 7alpha-hydroxylase (CYP7A1) gene. CYP7A1 is a rate-limiting enzyme in bile acid synthesis and therefore plays an important role in maintaining cholesterol homeostasis. CYP7A1-204CC homozygotes have the greatest decrease in total cholesterol level in response to dietary changes in different types of dietary intervention studies. In contrast, one study has reported that the effect of statins in lowering low-density lipoprotein (LDL)-cholesterol levels was slightly greater in -204AA homozygotes. The CYP7A1 A-204C variant accounts for a significant proportion of the genetic predisposition of the response of plasma cholesterol levels.     

24-hydroxycholesterol is a substrate for hepatic cholesterol 7alpha-hydroxylase (CYP7A)

(24S)-Hydroxycholesterol is formed from cholesterol in the brain and is important for cholesterol homeostasis in this organ. Elimination of (24S)-hydroxycholesterol has been suggested to occur in the liver but little is known about the metabolism of this oxysterol. In the present investigation, we report formation of 7alpha, 24-dihydroxycholesterol in pig and human liver. 7alpha-hydroxylase activity toward both isomers of 24-hydroxycholesterol [(24S) and (24R)] was found in a partially purified and reconstituted cholesterol 7alpha-hydroxylase (CYP7A) enzyme fraction from pig liver microsomes. In contrast, a purified enzyme fraction of pig liver oxysterol 7alpha-hydroxylase with high activity toward 27-hydroxycholesterol did not show any detectable activity toward 24-hydroxycholesterol. 7alpha-Hydroxylation of 24-hydroxycholesterol was strongly inhibited by 7-oxocholesterol, a known inhibitor of CYP7A. Human CYP7A, recombinantly expressed in Escherichia coli and in simian COS cells, showed 7alpha-hydroxylase activity toward both cholesterol and the two isomers of 24-hydroxycholesterol, with a preference for the (24S)-isomer. Our results show that 24-hydroxycholesterol is metabolized by CYP7A, an enzyme previously considered to be specific for cholesterol and cholestanol and not active toward oxysterols. Because CYP7A is the rate-limiting enzyme in the major pathway of bile acid biosynthesis, the possibility is discussed that at least part of the 24-hydroxycholesterol is converted into 7alpha-hydroxylated bile acids by the enzymes involved in the normal biosynthesis of bile acids.     

Expression of hepatic microsomal cholesterol 7 alpha-hydroxylase activity in lean and obese Zucker rats

The activity of hepatic microsomal cholesterol 7 alpha-hydroxylase was studied in genetically obese and lean Zucker rats. The liver microsomal cholesterol 7 alpha-hydroxylase activity in fatty Zucker rats (fa/fa) is about 50% to 70% lower than that of the lean (Fa/-) rats of the same sex, when animals were sacrificed at the middle of the dark cycle. When rats were sacrificed at the middle of the light cycle, cholesterol 7 alpha-hydroxylase activity was the same as in the dark cycle in obese rats of both sexes, but was 65% lower in lean rats. However, cholesterol 7 alpha-hydroxylase activity was stimulated by the treatment with cholestyramine in both obese and lean rats. Our results suggested that the diurnal regulation of cholesterol 7 alpha-hydroxylase activity is lost in obese rats but was present under cholestyramine treatment in the genetically obese strain of rats.     

Roles of nuclear receptors in the up-regulation of hepatic cholesterol 7alpha-hydroxylase by cholestyramine in rats

Nuclear receptors are involved in regulating the expression of cholesterol 7alpha-hydroxylase (CYP7A1), however, their roles in the up-regulation of CYP7A1 by cholestyramine (CSR) are still unclear. In the present study, male Wistar rats were divided into four groups and fed [high sucrose + 10% lard diet] (H), [H + 3% CSR diet] (H + CSR), [H + 0.5% cholesterol + 0.25% sodium cholate diet] (C), or [C + 3% CSR diet] (C + CSR) for 2 weeks. Cholestyramine decreased serum and liver cholesterol levels significantly in rats fed C-based diets, but had no effect on these parameters in rats fed H-based diets. Cholestyramine raised hepatic levels of CYP7A1 mRNA and activity in both groups. The gene expression of hepatic ATP-binding cassettes A1 and G5, regulated by liver X receptor (LXR), were unchanged and down-regulated by cholestyramine, respectively. The mRNA levels of the hepatic ATP-binding cassette B11 and short heterodimer partner (SHP), regulated by farnesoid X receptor (FXR), were not changed by cholestyramine. C-based diets, which contained cholesterol and cholic acid, increased SHP mRNA levels compared to H-based diets. Consequently, in rats fed the C+CSR diet, hepatic FXR was activated by dietary bile acids, but the hepatic CYP7A1 mRNA level was increased 16-fold compared to that in rats fed an H diet. These results suggest that cholestyramine up-regulates the expression of CYP7A1 independently via LXR- or FXR-mediated pathways in rats.     

Regulation of cholesterol 7 alpha-hydroxylase in the liver. Purification of cholesterol 7 alpha-hydroxylase and the immunochemical evidence for the induction of cholesterol 7 alpha-hydroxylase by cholestyramine and circadian rhythm

Two cholesterol 7 alpha-hydroxylase isozymes were purified from liver microsomes of cholestyramine-treated female rats by using anion exchange high performance liquid chromatography. These two cytochrome P-450 isozymes were similar in electrophoretic mobility, immunocross-reactivity, and Vmax but differed in Km for cholesterol, turnover number, and charges. Antibody against the major isozyme was raised in rabbit. This antibody specifically inhibited microsomal cholesterol 7 alpha-hydroxylase activity. Immunoblot of microsomal polypeptides indicated that microsomal cholesterol 7 alpha-hydroxylase enzyme levels were increased in parallel with cholesterol 7 alpha-hydroxylase activity upon the treatment of rats with diet supplemented with cholestyramine. Both cholesterol 7 alpha-hydroxylase activity and enzyme levels were drastically reduced immediately after the removal of cholestyramine from the diet. Cholesterol 7 alpha-hydroxylase activity was also detected in the microsomes of kidney, heart, and lung in about 7-27% of the level found in the liver. 3-Methylcholanthrene treatment induced cholesterol 7 alpha-hydroxylase activity and enzyme level. In contrast, pregnenolone-16 alpha-carbonitrile or dexamethasone treatment greatly depressed enzyme and activity in rats. Cholesterol 7 alpha-hydroxylase enzyme level was 2-3-fold higher in liver microsomes of rats maintained under the reversed light cycle than under the normal light cycle. In genetically obese Zucker rats, cholesterol 7 alpha-hydroxylase activity and enzyme level did not respond to the change in the light cycle, however, were induced to the same levels as in the lean rats by cholestyramine treatment. This study provided the first direct evidence that the bile acid feedback regulation and circadian rhythm of microsomal cholesterol 7 alpha-hydroxylase activity involved the induction of cholesterol 7 alpha-hydroxylase enzyme level.     

Phytosterols and lecithin do not have an additive effect in lowering plasma and hepatic cholesterol levels in diet-induced hypercholesterolemic rats

Both plant sterols and lecithin are used as dietary supplements for lowering blood cholesterol in Western countries. This study evaluated the possibility of an additive effect of these ingredients on the regulation of lipid concentrations and cholesterol metabolism. Male Sprague-Dawley rats were randomly divided into three groups, and fed one of the following diets for 5 weeks; high cholesterol diet (HCD), phytosterol mixture-supplemented diet (PD, HCD+0.25% phytosterols), or phytosterol mixture and lecithin-supplemented diet (PLD, PD+0.15% lecithin). Feeding the PD for 5 weeks resulted in a 34% and 41% decrease in plasma total- and VLDL+LDL-cholesterol levels, respectively, and a 23% decrease in hepatic cholesterol content compared to those for the HCD rats (p < 0.05). These cholesterol-lowering properties of the phytosterol mixture were also associated with the down-regulation of hepatic acyl CoA:cholesterol acytransferase (ACAT) activity (p < 0.05). Addition of lecithin plus phytosterol mixture to the hypercholesterolemic diet did not significantly affect blood and hepatic lipid concentrations (with the exception of 36% decrease in hepatic triglyceride level, p < 0.05) as well as hepatic ACAT activity compared to feeding the hypercholesterolemic diet supplemented with phytosterol alone. These results indicate that combining lecithin, at a 0.15% level, with a phytosterol mixture-supplemented diet does not exhibit an additive effect in regulating hepatic ACAT activity or lowering blood cholesterol in hypercholesterolemic rats.     

Thyroid hormone receptor beta-deficient mice show complete loss of the normal cholesterol 7alpha-hydroxylase (CYP7A) response to thyroid hormone but display enhanced resistance to dietary cholesterol

Thyroid hormone (T3) influences hepatic cholesterol metabolism, and previous studies have established an important role of this hormone in the regulation of cholesterol 7alpha-hydroxylase (CYP7A), the rate-limiting enzyme in the synthesis of bile acids. To evaluate the respective contribution of thyroid hormone receptors (TR) alpha1 and beta in this regulation, the responses to 2% dietary cholesterol and T3 were studied in TRalpha1 and TRbeta knockout mice under hypo- and hyperthyroid conditions. Our experiments show that the normal stimulation in CYP7A activity and mRNA level by T3 is lost in TRbeta-/- but not in TRalpha1-/-mice, identifying TRbeta as the mediator of T3 action on CYP7A and, consequently, as a major regulator of cholesterol metabolism in vivo. Somewhat unexpectedly, T3-deficient TRbeta-/- mice showed an augmented CYP7A response after challenge with dietary cholesterol, and these animals did not develop hypercholesterolemia to the extent as did wild-type (wt) controls. The latter results lend strong support to the concept that TRs may exert regulatory effects in vivo independent of T3.     

Hypocholesterolemic mechanism of Chlorella: Chlorella and its indigestible fraction enhance hepatic cholesterol catabolism through up-regulation of cholesterol 7alpha-hydroxylase in rats

Chlorella powder (CP) has a hypocholesterolemic effect and high bile acid-binding capacity; however, its effects on hepatic cholesterol metabolism are still unclear. In the present study, male Wistar rats were divided into four groups and fed a high sucrose + 10% lard diet (H), an H + 10% CP diet (H+CP), an H + 0.5% cholesterol + 0.25% sodium cholate diet (C), or a C + 10% CP diet (C+CP) for 2 weeks. CP decreased serum and liver cholesterol levels significantly in rats fed C-based diets, but did not affect these parameters in rats fed H-based diets. CP increased the hepatic mRNA level and activity of cholesterol 7alpha-hydroxylase (CYP7A1). CP increased hepatic HMG-CoA reductase (HMGR) activity in the rats fed H-based diets, but not in rats fed C-based diets. CP did not affect hepatic mRNA levels of sterol 27-hydroxylase, HMGR, low-density lipoprotein (LDL) receptor, scavenger receptor class B1, ATP-binding cassette (ABC) A1, ABCG5, or ABCB11. Furthermore, the effect of a 3.08% Chlorella indigestible fraction (CIF, corresponding to 10% CP) on hepatic cholesterol metabolism was determined using the same animal models. CIF also decreased serum and liver cholesterol levels significantly in rats fed C-based diets. CIF increased hepatic CYP7A1 mRNA levels. These results suggest that the hypocholesterolemic effect of CP involves enhancement of cholesterol catabolism through up-regulation of hepatic CYP7A1 expression and that CIF contributes to the hypocholesterolemic effect.     

Leptin induces the hepatic high density lipoprotein receptor scavenger receptor B type I (SR-BI) but not cholesterol 7alpha-hydroxylase (Cyp7a1) in leptin-deficient (ob/ob) mice

Cholesterol elimination from the body involves reverse cholesterol transport from peripheral tissues in which the elimination of high density lipoprotein (HDL) and low density lipoprotein (LDL) cholesterol by the liver and subsequent biliary excretion as free cholesterol and bile acids are important. In situations of peripheral fat and cholesterol accumulation, such as obesity, these pathways may be overloaded, contributing to increased cholesterol deposition. Leptin has an important role in obesity, suppressing food intake and increasing energy expenditure. This hormone, which is absent in genetically obese ob/ob mice, is also thought to be involved in the coordination of lipid excretion pathways, although available data are somewhat inconsistent. We therefore studied the expression of the hepatic HDL receptor, scavenger receptor class B type I (SR-BI), and the LDL receptor as well as the rate-limiting enzyme in bile acid synthesis, cholesterol 7alpha-hydroxylase (Cyp7a1), in leptin-deficient ob/ob mice and their wild-type controls. In ob/ob mice, protein levels of both LDL receptor and SR-BI were reduced, whereas LDL receptor mRNA levels were increased and those of SR-BI were reduced, regardless of challenge with a 2% cholesterol diet. In ob/ob mice, the enzymatic activity and mRNA for Cyp7a1 were reduced, and the increase in response to dietary cholesterol was blunted. Upon short-term (2 days) treatment with leptin, a dose-dependent increase was seen in the SR-BI protein and mRNA, whereas the Cyp7a1 protein and mRNA were reduced. Our findings indicate that leptin is an important regulator of hepatic SR-BI expression and, thus, HDL cholesterol levels, whereas it does not stimulate Cyp7a1 and bile acid synthesis.     

Feeding unsaponifiable compounds from rice bran oil does not alter hepatic mRNA abundance for cholesterol metabolism-related proteins in hypercholesterolemic rats

The hypocholesterolemic effect of rice bran oil (RBO) is defined in human and animal experiments which indicate the presence of active component(s) in the unsaponifiable fraction, but the detailed mechanism is not known yet. Exogenously hypercholesterolemic (ExHC) rats were fed for 2 weeks on a 0.5% cholesterol diet supplemented with 10% each of RBO, RBO-simulated oil (RBOSO) in its fatty acid composition, or RBOSO plus 0.25% unsaponifiable compounds (UC) from RBO. Rats fed RBO or the UC resulted in lowing serum and liver cholesterol concentration and preventing reduction of high density lipoproteinic-cholesterol. Dietary RBO or the UC led to an elevation of fecal neutral sterol excretion, but no significant change in fecal bile acid excretion or in hepatic abundance of mRNAs for 3-hydroxy-3-methylglutaryl-CoA reductase, cholesterol-7alpha-hydroxylase, and low density lipoprotein receptor. Besides, serum and liver alpha-tocopherol concentrations were lowered in RBO or the UC-fed rats. These results show that the UC in RBO leads to a decreased serum cholesterol concentration by interrupting the absorption of intestinal hydrophobic compounds rather than by modifying cholesterol metabolism in the liver.     

Iron utilization and liver mineral concentrations in rats fed safflower oil, flaxseed oil, olive oil, or beef tallow in combination with different concentrations of dietary iron

Diets with a higher proportion of polyunsaturated fatty acids (i.e., linoleic acid) have decreased iron absorption and utilization compared with diets containing a higher proportion of the saturated fatty acid stearic acid (e.g., beef tallow). However, less is known regarding the influence of other polyunsaturated or monounsaturated fatty acids, along with higher dietary iron, on iron absorption and utilization. The present study was conducted to compare the effects of dietary fat sources known to vary in (n-3), (n-6), and (n-9) fatty acids on iron utilization and liver mineral concentrations. Male weanling rats were fed a diet containing 10, 35, or 100 μg/g iron in combination with saffower oil, flaxseed oil, olive oil, or beef tallow for 8 wk. Indicators of iron status, iron utilization, and liver iron concentrations were unaffected by an interaction between the fat source and iron concentration. Plasma copper was the only variable affected by an interaction between the fat source and dietary iron. Findings of this study demonstrate that flaxseed oil and olive oil may alter tissue minerals and affect iron utilization. Further studies should be conducted to establish the effect of varying (n-3), (n-6), and (n-9) fatty acids on trace mineral status and iron utilization. Data were presented in part at Experimental Biology 2000 as a poster session. A. D. Shotton and E. A. Droke, Dietary fat and iron modify immune function, FASEB J. 14, A239 (2000).     

Mitochondrial and microsomal cholesterol mobilization after oxidative stress induced by adriamycin in rats fed with dietary olive and corn oil

The influence of three different dietary fats (8%) and of endogenous lipid peroxidation with regard to cholesterol concentrations in liver mitochondria and microsomes and in serum has been investigated in the rat. Although the different diet fat used did not produce any effect on serum cholesterol, it was possible to show that each experimental diet differently influenced the microsomal and mitochondrial levels of cholesterol. The highest mitochondrial and microsomal cholesterol content was found in case of diet supplemented with virgin olive oil and the lowest with rectified olive oil. An endogenous oxidative stress induced by adriamycin was able to produce a clear decrease in microsomal and mitochondrial cholesterol level and a sharp increase in serum concentration in all three groups. However, dietary fats and adriamycin had no effect on the microsomal and mitochondrial membrane viscosity as detected by fluorescence polarization. These results are consistent with the hypothesis that mitochondrial and microsomal cholesterol can exchange with exogenous pools when phospholipid peroxidation occurs.     

On the possible use of the serum level of 7 alpha-hydroxycholesterol as a marker for increased activity of the cholesterol 7 alpha-hydroxylase in humans

The possibility was investigated that the serum level of 7 alpha-hydroxycholesterol can be used as a marker for cholesterol 7 alpha-hydroxylase activity. Six patients with gallstone disease were found to have a mean level of 7 alpha-hydroxycholesterol in serum of 30 +/- 4 ng/ml (mean +/- SEM) as measured by isotope dilution-mass spectrometry, using deuterated 7 alpha-hydroxycholesterol as internal standard. After treatment with cholestyramine in a dose of 8 g twice daily for 2-3 weeks preoperatively, the serum level increased to 128 +/- 20 ng/ml (P less than 0.001). Eight other patients with gallstone disease had a mean level of 7 alpha-hydroxycholesterol in serum of 29 +/- 7 ng/ml. Treatment with chenodeoxycholic acid, 15 mg per kg body weight per day for 3-4 weeks before surgery, decreased the mean level to 20 +/- 7 ng/ml (P greater than 0.05). The activity of the cholesterol 7 alpha-hydroxylase in liver biopsies taken during operation was found to be 38 +/- 5 pmol/min per mg of protein in the group of patients treated with cholestyramine and 1.3 +/- 0.5 pmol/min per mg in the group of patients treated with chenodeoxycholic acid. Liver biopsies from a group of untreated patients (n = 13) had a mean cholesterol 7 alpha-hydroxylase activity of 7.6 +/- 1.5 pmol/min per mg.(ABSTRACT TRUNCATED AT 250 WORDS)