Improved assay of hepatic microsomal cholesterol 7 alpha-hydroxylase activity by the use of hydroxypropyl-beta-cyclodextrin and an NADPH-regenerating system

Cholesterol 7 alpha-hydroxylase, the key enzyme in bile acid synthesis, has been implicated in atherosclerosis and gallstone disease. The aim of this study was to check if the use of hydroxypropyl-beta-cyclodextrin (HPBCD), a vehicle for solubilizing cholesterol, augmented the rate of 7 alpha-hydroxycholesterol formation in hamster liver microsomes compared to classical assays in which labeled cholesterol was delivered in Tween 80. We observed that [14C]cholesterol carried by HPBCD enhanced the sensitivity of the assay tenfold. However, linearity of 7 alpha-hydroxycholesterol formation with time was short because of the rapid transformation of 7 alpha-hydroxycholesterol into 7 alpha-hydroxy-cholesten-3-one and 7 alpha,12 alpha-dihydroxy-cholesten-3-one when NADPH alone was present in the incubation medium. In order to avoid the transformation of 7 alpha-hydroxycholesterol into 7 alpha-hydroxy-cholesten-3-one, which is essentially NAD(+)-dependent, but is also NADP(+)-dependent, NADPH (1 mmol/l) plus an NADPH-regenerating system must be present in the medium. In this improved assay, the optimal pH was 7.4 and the apparent Km for control and cholestyramine-fed hamsters had a similar value of 315 mumol/l; linearity in the formation of 7 alpha-hydroxycholesterol was also apparent after a relatively short time period (10 min), but with a markedly greater slope of the curve. With a short incubation time (6 min), microsomes from livers of hamsters (five and nine weeks old) that were fed with a commercial ground diet yielded rates of 7 alpha-hydroxycholesterol formation of 115 +/- 10 and 150 +/- 16 pmol/min.mg protein, respectively, whereas microsomes from hamsters fed with a lithogenic sucrose-rich diet (five weeks old) yielded rates of 7 alpha-hydroxycholesterol formation of 77 +/- 7 pmol/min.mg protein, which were significantly lower (-33%) than those of corresponding control hamsters. This improved cholesterol 7 alpha-hydroxylase assay is very sensitive, simple and rapid, and does not necessitate sophisticated equipment. It can be particularly useful for determining cholesterol 7 alpha-hydroxylase activity in liver biopsies from dyslipidemic or lithiasic patients.     

Linkage between cholesterol 7alpha-hydroxylase and high plasma low-density lipoprotein cholesterol concentrations

Interindividual differences in plasma low-density lipoprotein cholesterol (LDL-C) levels reflect both environmental variation and genetic polymorphism, but the specific genes involved and their relative contributions to the variance in LDL-C are not known. In this study we investigated the relationship between plasma LDL-C concentrations and three genes with pivotal roles in LDL metabolism: the low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB), and cholesterol 7alpha-hydroxylase (CYP7). Analysis of 150 nuclear families indicated statistically significant linkage between plasma LDL-C concentrations and CYP7, but not LDLR or APOB. Further sibling pair analyses using individuals with high plasma LDL-C concentrations as probands indicated that the CYP7 locus was linked to high plasma LDL-C, but not to low plasma LDL-C concentrations. This finding was replicated in an independent sample. DNA sequencing revealed two linked polymorphisms in the 5' flanking region of CYP7. The allele defined by these polymorphisms was associated with increased plasma LDL-C concentrations, both in sibling pairs and in unrelated individuals. Taken together, these findings indicate that polymorphism in CYP7 contributes to heritable variation in plasma LDL-C concentrations. Common polymorphisms in LDLR and APOB account for little of the heritable variation in plasma LDL-C concentrations in the general population.     

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.     

Identification and characterization of a putative bile acid-responsive element in cholesterol 7 alpha-hydroxylase gene promoter

Nucleotide sequences of a 7997-base pair SacI fragment spanning 3643 base pairs of the upstream promoter region to exon 4 of the rat cholesterol 7 alpha-hydroxylase gene (CYP7) have been determined. DNase I footprinting and electrophoretic mobility shift assay of the proximal promoter from nucleotides -346 to +36 revealed two protected regions which specifically shifted proteins in rat liver nuclear extracts. Footprint A (nucleotides -81 to -35) contained a cluster of overlapping sequence motifs of TGT3, steroid/thyroid hormone response elements (7 alpha TRE), hepatocyte nuclear factors 1 and 4, and CAAT/enhancer-binding protein alpha and has been shown to confer bile acid repression of the CYP7 gene promoter activity. Footprint B (nucleotides -148 to -129) contained a sequence motif HNF4. When footprint A (-101 to -49) or 7 alpha TRE (-73 to -55) sequence was linked upstream to a heterologous SV40 promoter/luciferase plasmid and transiently transfected into HepG2 cells, taurodeoxycholate suppressed the SV40 promoter activity. Electrophoretic mobility shift assays revealed that one or two bands shifted by the 7 alpha TRE or by a direct repeat sequence in 7 alpha TRE were absent when liver nuclear extracts of deoxycholic acid-treated rats were used. Similar gel shift patterns were also observed when human 7 alpha TRE or human liver nuclear extracts were used. The rat direct repeat sequence interacted with two polypeptides (M(r) = 57,000 and 116,000) in both rat and human liver nuclear extracts. These results suggest that hydrophobic bile acids may suppress the CYP7 gene expression by binding to a bile acid receptor which interacts with and prevents the binding of liver nuclear protein(s) to a bile acid-responsive element and that the core of bile acid-responsive element is a direct repeat.     

Inhibition of rat mammary tumorigenesis by dietary cholesterol

Mitochondrial trifunctional protein deficiency, a recently identified disorder of fatty-acid oxidation, may show characteristic features such as peripheral neuropathy, pigmentary retinopathy, and acute fatty liver degeneration in pregnant women with an affected fetus. We describe a patient with trifunctional protein deficiency whose clinical picture consisted of severe calcium and phosphate abnormalities caused by hypoparathyroidism.     

Regulation of hepatic glutaminase in the streptozotocin-induced diabetic rat

The liver of diabetic animals removes increased quantities of glutamine. We therefore examined factors that affect hepatic glutaminase activity in hepatocytes and mitochondria. Glutamine use, through glutaminase, was measured in isolated rat hepatocytes by monitoring the production of 14CO2 from [1-(14)C]glutamine. Hepatocytes from streptozotocin-induced diabetic rats use glutamine more rapidly than do hepatocytes from normal or insulin-maintained diabetic rats. Glutamine use in all of these hepatocytes was stimulated by glucagon and epinephrine. Glutaminase activity, assayed in broken mitochondrial membranes, was increased approximately 2.5-fold in diabetic rats. The sensitivity of glutaminase, measured in intact liver mitochondria, to phosphate was markedly left-shifted in mitochondria from diabetic rats compared with those from controls. In fact, glutaminase was increased 10-fold at 2.5 mmol/l phosphate compared with controls. This increased sensitivity of glutaminase to physiological concentrations of phosphate is characteristic of its hormonal activation. Therefore, activation of glutaminase plays a major role in diabetes and is as important as increases in its total enzyme amount in determining the increased glutamine uptake in diabetes.     

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

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

Disruption of cholesterol 7alpha-hydroxylase gene in mice. I. Postnatal lethality reversed by bile acid and vitamin supplementation

Mice deficient in cholesterol 7alpha-hydroxylase, the rate-limiting enzyme of bile acid biosynthesis, were constructed by targeted disruption of the Cyp7 gene. The introduced mutation removed exons 3-5 of the gene and gave rise to a null allele that encoded no immunoreactive or enzymatically active protein. Heterozygous carriers of the disrupted gene (Cyp7+/-) were phenotypically normal. Homozygous animals (Cyp7-/-) appeared normal at birth, but died within the first 18 days of life. Approximately 40% of the animals died between postnatal days 1 and 4 and 45% between days 11 and 18. The addition of vitamins to the water of nursing mothers prevented deaths in the early period, whereas the addition of cholic acid to chow prevented deaths in the later period. Newborn Cyp7-/- mice whose mothers were maintained on unsupplemented chow failed to gain weight at a normal rate and developed oily coats, hyperkeratosis, and apparent vision defects. These symptoms waned at 3 weeks of life, and their disappearance was accompanied by a marked increase in survival. In the accompanying study, the induction of an alternate pathway of bile acid biosynthesis is shown to underlie this unusual time course (Schwarz, M., Lund, E. G., Setchell, K. D. R., Kayden, H. J., Zerwekh, J. E., Bj?rkhem, I., Herz, J., and Russell, D. W. (1996) J. Biol. Chem. 271, 18024-18031). We conclude that cholesterol 7alpha-hydroxylase is an essential enzyme for normal postnatal development.     

Regulation of the expression of follistatin in rat hepatocytes

The effect of lipopolysaccharide (LPS) treatment on noradrenergic responses elicited by electrical field stimulation (EFS) was investigated in the rabbit anococcygeus muscle. In the absence of LPS, EFS-induced contractions were enhanced and nitrergic relaxations were inhibited by NG-nitro-L-arginine (L-NOARG) but not by NG-monomethyl-L-arginine (L-NMMA). Administration of L-NMMA prior to L-NOARG inhibited the enhancement of EFS-induced contractions by L-NOARG and reversed the inhibitory effect of L-NOARG on nitrergic relaxations. Treatment with LPS induced a time-dependent loss of phenylephrine-induced tone which was inhibited by cycloheximide, dexamethasone, L-NMMA, or L-NOARG. Treatment of the anococcygeus muscle with LPS also resulted in a time-dependent loss in the magnitude of EFS-induced contractions and an increase in the delay of onset of contractions. These effects were reversible by pretreatment with cycloheximide or by treatment with L-NMMA. These results suggest that LPS induces a loss of tone and of noradrenergic responses through expression of the inducible NO synthase in the rabbit anococcygeus muscle. L-NMMA blocks these effects but does not affect nitrergic transmission, while L-NOARG is active against both.     

Apolipoprotein E polymorphism is associated with plasma cholesterol response in a 7-day hospitalization study for metabolic and dietary control in NIDDM

An 81-year-old woman in whom liver dysfunction had been pointed out 3 years previously was diagnosed as having liver cirrhosis due to lupoid hepatitis. Considering the poor prognosis of cirrhosis and her age, immunosuppressive therapy was not adopted. Nine months later, a small liver tumor was found by ultrasonography and was diagnosed as hepatocellular carcinoma (HCC). The tumor was treated with transcatheter arterial embolization, but grew continuously. She also developed gingival lymphoma that was successfully treated. Three years after initial diagnosis of lupoid hepatitis, she died of hepatic failure. An autopsy was performed and confirmed the clinical diagnosis, liver cirrhosis with HCC. HCC is regarded as a rare complication of lupoid hepatitis, but cases of HCC complicating lupoid hepatitis may increase with progress in treatment methods and elongation of survival. The present case suggests that any malignancy can be developed in long-term surviving patients with lupoid hepatitis.     

Comparison of the effects of cyclic AMP analogues on cholesterol metabolism in cultured rat and hamster hepatocytes

The effects of two cell-permeable cyclic AMP analogues, 8-chloro cyclic AMP (8-Cl cAMP) and 8-(4-chlorophenylthio) cyclic AMP (8-CPT cAMP), on cholesterol esterification, cholesteryl ester hydrolysis and bile acid synthesis were compared in cultured rat and hamster hepatocytes. Cholesterol esterification, as measured by the incorporation of [3H]oleate into cholesteryl ester, was increased by 58-88% by the analogues in rat hepatocytes and by 33-43% in hamster cells. The response in rat hepatocytes, however, was observed after a relatively short incubation time (28% increase after 1 hr), whereas that in hamster cells required a longer period (36% after 12 hr) to become apparent. The activity of the cytosolic neutral cholesteryl ester hydrolase in rat hepatocytes was also stimulated by both cyclic AMP analogues (31-37%, but the microsomal activity was unaffected. In hamster hepatocytes, however, microsomal cholesteryl ester hydrolase activity was increased (47-80%) in the presence of 8-Cl cAMP or 8-CPT cAMP. Bile acid synthesis was increased by 8-CPT cyclic AMP in rat cells (approximately 25%) but was unchanged by both analogues in hamster hepatocytes. These results indicate significant differences in the way in which cholesterol metabolism responds to cyclic AMP in cultured rat and hamster hepatocytes.     

Hepatic adenine nucleotides and microsomal cholesterol 7 alpha-hydroxylase activity in the obstructed and freely draining lobes of the liver after selective bile duct obstruction

BACKGROUND: The effect of selective bile duct obstruction (SBDO) on hepatic reserve function of the bile duct obstructed (BDO) and nonobstructed freely draining (FD) lobes of the liver is obscure. METHODS: The bile duct branches draining from the left lateral and median lobes of the liver were ligated for 4 and 10 days in rats, and hepatic reserve functions in BDO and FD lobes were assessed by microsomal cholesterol 7 alpha-hydroxylase activities and by hepatic adenine nucleotides and energy charge levels. The values were compared with those in the sham-operated control liver. Cholesterol 7 alpha-hydroxylase activities were determined by gas-liquid chromatography--mass spectrometry, and hepatic adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) levels with high-pressure liquid chromatography. RESULTS: The histological examination of the BDO lobes showed proliferation and formation of new bile ductules and fibrous connective tissues linking portal areas. Microsomal cholesterol 7 alpha-hydroxylase activities, hepatic energy charge and each adenine nucleotide level did not differ between FD and BDO lobes, and the values were similar to those in the sham-operated liver. CONCLUSIONS: Selective bile duct obstruction shows no adverse effects on microsomal and mitochondrial functions in both the BDO and FD lobes of the liver.     

Regulation of free choline in rat brain: dietary and pharmacological manipulations

The present study analyses, comparatively, the kinetics of free choline in the brain of rats during dietary and pharmacological manipulations. Low-choline diet halved the choline plasma level but did not cause significant changes of CSF choline. High-choline diet, hypoxia and treatment with nicotinamide increased brain choline availability through a central site of action and increased the CSF choline concentration. CSF choline concentrations were more effectively elevated by nicotinamide treatment (20-25 microM) than by acute choline administration (13-15 microM). Increases of CSF choline, due to brain choline mobilization, were consistently associated with a net release of choline from the brain as reflected by strongly negative arterio-venous differences (AVD) of brain choline. The balance between release and uptake of brain choline was controlled by the arterial plasma choline level in all treatment groups; however, the normal 'reversal point' of 15 microM--representing the plasma choline level where uptake and release of brain choline are balanced--was shifted to more than 40 microM by high-choline diet and nicotinamide. In conclusion, our data characterize the release of choline into the venous blood as an important component of brain choline homeostasis. Furthermore, we demonstrate that the concentration of brain choline (e.g. as a precursor of acetylcholine) can be enhanced more efficiently by manipulating choline homeostatic mechanisms than by acute choline administration.     

Influence of dietary curcumin and cholesterol on the progression of experimentally induced diabetes in albino rat

Effect of feeding 0.5% curcumin diet or 1% cholesterol diet was examined in albino rats rendered diabetic with streptozotocin injection. Diabetic rats maintained on curcumin diet for 8 weeks excreted comparatively less amounts of albumin, urea, creatinine and inorganic phosphorus. Urinary excretion of the electrolytes sodium and potassium were also significantly lowered under curcumin treatment. Dietary curcumin also partially reversed the abnormalities in plasma albumin, urea, creatine and inorganic phosphorus in diabetic animals. On the other hand, glucose excretion or the fasting sugar level was unaffected by dietary curcumin and so also the body weights were not improved to any significant extent. Diabetic rats fed curcumin diet had a lowered relative liver weight at the end of the study compared to other diabetic groups. Diabetic rats fed a curcumin diet also showed lowered lipid peroxidation in plasma and urine when compared to other diabetic groups. The extent of lipid peroxidation on the other hand, was still higher in cholesterol fed diabetic groups compared to diabetic rats fed with control diet. Thus, the study reveals that curcumin feeding improves the metabolic status in diabetic conditions, despite no effect on hyperglycemic status or the body weights. The mechanism by which curcumin improves this situation is probably by virtue of its hypocholesterolemic influence, antioxidant nature and free radical scavenging property.     

Cholesterol 7 alpha-hydroxylase activity in hypothyroidism and hyperthyroidism in humans

The effects of three root-end filling materials on healing following endodontic surgery were assessed radiologically and correlated with histological findings reported elsewhere. The materials compared were a light-cured glass ionomer cement (Vitrebond), a reinforced zinc oxide-eugenol cement (Kalzinol) and amalgam. The root canals of 27 two-rooted mandibular premolar teeth of six beagle dogs were inoculated with endodontic pathogenic bacteria to induce periradicular lesions. The roots were apicected and root-end cavities filled with the tested filling materials. The teeth and surrounding jaw were removed after 4 weeks (30 roots) or 8 weeks (24 roots). Radiographs were taken of each jaw section and subjected to image analysis. Healing was evaluated based on measurements of the size of the periradicular radiolucent areas. ANOVA disclosed no statistically significant differences in the size of the periradicular areas either between time periods or between materials. These results did not correlate with the tissue responses in the same material as assessed histologically and previously reported. The use of radiographs alone to assess healing after endodontic surgery in the dog mandible is unsatisfactory, and should not be regarded as a substitute for histological examination for the determination of healing.