Circadian rhythm of HMG-CoA reductase and insulin in African green monkeys

HMG-CoA reductase in hepatic microsomes and serum insulin display circadian rhythm in two strains of Cercopethicus aethiops. Grivets develop higher levels of serum cholesterol than vervets fed cholesterol. Males (n = 20/gp) were adapted to a light cycle (7:30 a.m.-8:30 p.m.) for 60 days and fed a non-cholesterol diet at 8:30 a.m. and 2:00 p.m. Vervet acrophase, reductase activity was synchronized to serum insulin units with a specific activity of 1480 pmol/min/mg protein changing by 7.5-fold from nadir to acrophase. The activity profile in grivets was asynchronous to vervet fluctuations with peaks at 12 noon (160 pmol/min/mg) and 6 p.m. (275 pmol/min/mg). Insulin levels also peaked near these times. The 24-h reductase activity was over 4 times greater in vervet than grivet livers. The similar rhythmic patterns of reductase and insulin support the notion that insulin plays an important role in the rhythm of HMG-CoA reductase in primates.     

Rate-limiting, diurnal activity of hepatic microsomal cholesterol-7 alpha-hydroxylase in pigeons with high serum cholesterol

Efficiency of regulating serum cholesterol by cholesterol-7 alpha-hydroxylase was studied in pigeon strains hypo-(SR-39) and hypercholesterolemic (SR-37) with respect to dietary cholesterol. Diurnal hydroxylase activity in SR-37 was 10% of that in strain SR-39 adapted to a light-dark cycle and fed a non-cholesterol diet. Acrophase (6 p.m.) activity was 54-fold greater in SR-39 than in SR-37 pigeons. Dietary cholesterol elevated enzyme activity 2.8-fold in SR-37 pigeons. Dietary cholestyramine plus cholesterol increased hydroxylase activity 21-fold in SR-37 and 3-fold in SR-39 strain; yet, activity remained greater in SR-39. Cholestyramine feeding prevented elevated cholesterol levels in both groups. The circadian rhythms of hydroxylase and serum corticosterone were determined. The diurnal activity in SR-37 was 10% of that in SR-39 and acrophase activity was 34-fold greater in SR-39. Hormone levels were comparable. Programmed acrophase was asynchronous between strains. Hydroxylase activity was positively correlated with corticosterone levels and inversely correlated with serum cholesterol. A defect in the up-regulation of cholesterol-7 alpha-hydroxylase is proposed which limits the catabolism of cholesterol in strain SR-37.     

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.     

The effect of portacaval transposition of hepatic cholesterol-7alpha-hydroxylase activity in the rat.

Portacaval anastomosis in the rat results in an increase in the activity of the liver microsomal cholesterol-7alpha-hydroxylase enzyme system. The increase in the activity of this oxygenase occurs despite a decrease in the total amount of cytochrome p450 in the liver microsomes after portacaval anastomosis. It is possible to increase further the activity of the cholesterol-7alpha-hydroxylase enzyme in these portacaval shunted animals by feeding them on a diet containing a bile salt sequestering agent. This suggests that one of the factors influencing the activity of the liver microsomal cholesterol-7alpha-hydroxylase enzyme may be the concentration of bile salts reaching the liver from the blood plasma. Portacaval anastomosis in the rat tended to achieve a small decrease in the plasma cholesterol concentration.     

Evidence for the presence of non-lipoprotein factors in diabetic serum capable of stimulating rat hepatic cholesterol-7 alpha-hydroxylase in vitro

Experimental diabetes in the rat has been shown to result in marked increases in bile acid pool and synthesis. In this study, mechanisms responsible for the increased bile acid synthesis was examined in rats made diabetic with streptozotocin. Our results indicate that a) in diabetic rats, hepatic cholesterol-7 alpha-hydroxylase activity is increased by 100%, b) this increased activity is not due to a higher stimulating activity of cell supernatant factors, but c) may be due to a non-lipoprotein factor(s) in diabetic serum capable of stimulating (by 100%) cholesterol-7 alpha-hydroxylase activity in control livers to the level noted in diabetic animals.     

Impact of an enriched-cholesterol diet on enzymatic cholesterol metabolism during rabbit gestation

An appropriate cholesterol homeostasis is vital for the maintenance and the optimal fetal development. The cholesterol is essential for the synthesis of progesterone and 17beta-estradiol, hormones that actively participate to sustain gestation. However, the administration of 0.2% enriched cholesterol diet (ECD) during rabbit gestation significantly increased the cholesterol blood profile (total-cholesterol, LDL, HDL, esterified-cholesterol and free-cholesterol) of dams and offspring, and induced a reduction of the offspring weight of 15% as compared to the control group. Enzymes involved in cholesterol metabolism (ACAT, HMG-CoA-reductase and cholesterol-7alpha-hydroxylase) are greatly influenced by cholesterol profile. We hypothesized that the administration of an ECD during rabbit gestation modifies the activity of those enzymes. Female rabbits (pregnant or not) were fed with a standard diet or an ECD. At term, livers (dams and offspring) and placentas were collected and ACAT, HMG-CoA-reductase and cholesterol-7alpha-hydroxylase activities were assayed. Our results demonstrate that gestation induced a reduction of ACAT activity (48.9%) in dam's liver and, an augmentation of HMG-CoA-reductase activity (142.4%) whereas it has no effect on cholesterol-7alpha-hydroxylase activity. The administration of the ECD has no additive effect on ACAT, but significantly reduced the HMG-CoA-reductase activity and cholesterol-7alpha-hydroxylase activity as compared with the pregnant control group. In placentas the ECD supplementation has an influence for HMG-CoA-reductase activity, where a 43% increased in observed. Any ACAT activity was detected in placenta and the ECD has no influence on the cholesterol-7alpha-hydroxylase activity. Whereas their offspring's liver present a reduction of ACAT and HMG-CoA-reductase activity. Gestation associated with ECD reduces significantly the HMG-CoA-reductase activity, decreasing the cholesterol synthesis, but placenta seems to compensate this effect by increasing its HMG-CoA-reductase activity.     

Esterified and total 7 alpha-hydroxycholesterol in human serum as an indicator for hepatic bile acid synthesis

Serum levels of 7 alpha-hydroxycholesterol and activities of hepatic microsomal cholesterol 7 alpha-hydroxylase in surgical patients were analyzed by capillary gas-liquid chromatography-selected ion monitoring technique using a new internal standard, 5 alpha-cholestane-3 beta, 7 beta-diol. We found that concentrations of 7 alpha-hydroxycholesterol obtained after alkaline hydrolysis were higher than those without alkaline hydrolysis, indicating that a considerable amount of 7 alpha-hydroxycholesterol in human serum is present in esterified form. Esterified 7 alpha-hydroxycholesterol could also be quantitatively hydrolyzed with cholesterol esterase, suggesting that fatty acid is bound at the 3 beta-position of the cholestenediol. The serum levels of esterified and free 7 alpha-hydroxycholesterol in patients with cholelithiasis were 198.0 +/- 90.3 and 48.3 +/- 19.8 pmol/ml (mean +/- SD), respectively, and were similar to those in patients without hepatobiliary diseases. After treatment with chenodeoxycholic acid (300 mg per day) for 7 to 10 days, esterified and free 7 alpha-hydroxycholesterol levels decreased to 64.9 +/- 33.6 and 20.5 +/- 11.1 pmol/ml, respectively. Activity of cholesterol 7 alpha-hydroxylase was also inhibited. Treatment with ursodeoxycholic acid (600 mg per day) for 7 to 10 days had no inhibitory effect on serum 7 alpha-hydroxycholesterol levels and the enzyme activity. In all groups, high correlations were found between the activity of cholesterol 7 alpha-hydroxylase and serum levels of 7 alpha-hydroxycholesterol: free (r = 0.71, n = 38, P less than 0.001); esterified (r = 0.87, n = 38, P less than 0.001); total (r = 0.87, n = 38, P less than 0.001). Esterified and total 7 alpha-hydroxycholesterol was more highly correlated with the enzyme activity than the free form. We conclude that a significant amount of 3 beta-acyl esters of 7 alpha-hydroxycholesterol is present in human serum and that serum levels of esterified and/or total 7 alpha-hydroxycholesterol are likely to reflect the activity of hepatic cholesterol 7 alpha-hydroxylase and thus the amount of primary bile acids synthesized in the liver.     

CORRELATION BETWEEN TISSULAR AND DIVISION FUNCTIONS IN THE LIVER OF YOUNG RATS

The division and tissue specific functions are studied in the liver of young rats during the first post-natal weeks. The division function is tested by the mitotic and the ³H-thymidine labelling index, the specific tissular function by the cholesterol-7 alpha-hydroxylase activity. The nycthemeral rhythm is triggered simultaneously for the two functions at the 20th day of life. From this time, spontaneous or induced mitoses occur during the day (rest period) and the cholesterol-7-alpha-hydroxylase during the evening (period of activity). The results are explained by the influence of the hypothalamoadrenal axis, which presents a circadian activity with a maximum in the evening.     

On the structural specificity in the regulation of the hydroxymethylglutaryl-CoA reductase and the cholesterol-7 alpha-hydroxylase in rats. Effects of cholestanol feeding

The effect of feeding 2% cholestanol or cholesterol on cholesterol-7 alpha-hydroxylase activity and hydroxymethylglutaryl (HMG)-CoA reductase activity was studied in rats. The rate of 7 alpha-hydroxylation of a trace amount of labelled cholesterol increased by about 80% after the cholestanol feeding, whereas the 7 alpha-hydroxylation of endogenous microsomal cholesterol increased by about 40%. The latter conversion was measured with an accurate technique based on isotope dilution-mass spectrometry. After cholesterol feeding, the corresponding figures were about 50 and 60%, respectively. The cholestanol feeding had no significant effect on the HMG-CoA reductase activity, whereas the cholesterol feeding decreased the activity by about 80%. From the results obtained, it is concluded that the increased 7 alpha-hydroxylation observed after cholesterol feeding can not be explained only by a simple expansion of the substrate pool. The similar effect of both cholesterol and cholestanol on the cholesterol 7 alpha-hydroxylase activity and the diverging effect on the HMG-CoA reductase activity show that there is no coupling between cholesterol synthesis and degradation under the conditions employed. The lack of effect of cholestanol on the HMG-CoA reductase activity indicates a high structural specificity of the receptor involved in regulation of the enzyme. If a receptor mechanism is involved in the stimulation of the cholesterol-7 alpha-hydroxylase by cholesterol and cholestanol, these receptor(s) must be different from those involved in the regulation of the HMG-CoA reductase.     

Role of glutathione in the regulation of hepatic cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme of bile acid biosynthesis

The effect of in vivo variation of hepatic glutathione (using diethyl maleate and L-cysteine) on in vitro cholesterol 7 alpha-hydroxylase activity was studied in male Sprague-Dawley rats. Cholesterol 7 alpha-hydroxylase activity in glutathione-depleted rats (ca. 10% of control glutathione) was significantly reduced compared to that in vehicle-injected controls. While L-cysteine treatment of glutathione-depleted animals increased glutathione levels somewhat (ca. 20% of control glutathione), they were still significantly less than control levels. Similarly, cholesterol 7 alpha-hydroxylase activity in the partially glutathione replete animals was approximately 50% greater than that in the glutathione-depleted animals, but still significantly less than that in the controls. The rate of 7 alpha-hydroxylation of cholesterol was found to be dependent on liver glutathione content. The calculated maximal rate was 34.4 picomoles/mg/min with a half maximal activity at 1.89 mumoles glutathione/gm liver. These results suggest that hepatic glutathione may be an important modulator of in vivo activity of cholesterol 7 alpha-hydroxylase.     

Cholesterol 7 alpha-hydroxylase in isolated rat liver cells.

1. The activity of cholesterol 7 alpha-hydroxylase found in the 10000 x g supernatant prepared from isolated rat liver cells was comparable to that found with microsomal fractions from whole liver. 2. The activity of cholesterol 7 alpha-hydroxylase from cells prepared from livers of rats fed the bile salt sequestering agent cholestyramine was 2--3 fold higher than the activity of this enzyme found in cells isolated from animals on a control diet. 3. On incubation of hepatocytes in a suitable medium at 37 degrees C, cholesterol 7 alpha-hydroxylase activity declined to about 50% of its original value after three hours despite the fact that the cells retained a high level of viability over 5--6 h as measured by various sensitive criteria. 4. The decrease in cholesterol 7 alpha-hydroxylase activity was observed whether cholestyramine was included in the diet or excluded from the diet of the animals used as sources of the liver cells. 5. The change in cholesterol 7 alpha-hydroxylase activity seen on incubation of the cells was not affected by including in the incubation medium additional nutrients such as amino acids, the glucocorticoid cortisol, phospholipid dispersions, or sodium taurocholate. 6. Changing the incubation medium in which the cells were suspended at regular intervals during the three-hour experiments failed to prevent this decline in the cholesterol 7 alpha-hydroxylase activity during the incubation of these cells. 7. Although isolated liver cells have been shown to lose glutathione on incubation, addition of physiological levels of this compound did not prevent the decline in cholesterol 7 alpha-hydroxylase activity. 8. Cycloheximide addition to the incubation medium accelerated the decrease in cholesterol 7 alpha-hydroxylase activity. This suggests that some protein synthesis associated with cholesterol 7 alpha-hydroxylase activity occurs during the incubation and inhibition of such protein synthesis accelerated the decrease in this enzyme activity. 9. The cytochrome P-450 content of the 10000 x g supernatant prepared from hepatocytes declined slowly to about 65% of its original value after four hours of incubation at 37 degrees C. This decline in the 10000 x g supernatant cytochrome P-450 content may partly explain the observed loss of cholesterol 7 alpha-hydroxylase activity during incubations in vitro. 10. Isolated hepatocytes rapidly take up radioactively labelled sodium cholate. Subsequent excretion of the radioactivity was also very rapid even in the presence of large amounts of this bile salt in the medium.     

Insulin is a dominant suppressor of sterol 12 alpha-hydroxylase P450 (CYP8B) expression in rat liver: possible role of insulin in circadian rhythm of CYP8B

Sterol 12 alpha-hydroxylase (CYP8B) is a key enzyme for regulating the cholic acid/chenodeoxycholic acid ratio in bile acid biosynthesis. The hepatic CYP8B level was elevated in streptozotocin-induced diabetic rats, and the elevated CYP8B was suppressed by insulin administration [Ishida, H. et al. (1999) J. Biochem. 126, 19-25]. The streptozotocin-induced elevation of hepatic CYP8B mRNA concomitantly responded to the decrement of the serum insulin level. The CYP8B mRNA level in the cultivated rat hepatoma H4TG cells was strongly suppressed by insulin, although it was affected by dibutyryl cAMP or thyroxine to lesser extents. These observations demonstrate that CYP8B expression is dominantly regulated by the direct action of insulin on hepatocytes. A marked circadian rhythm (maximum at 13:00-16:00 and minimum at 1:00) was observed both on the mRNA level and the activity of CYP8B. This rhythm was shifted from that of cholesterol 7 alpha-hydroxylase, a rate-limiting enzyme of bile acid biosynthesis, showing a maximum at 22:00 and a minimum at 10:00, and this shift might oscillate the cholic acid/chenodeoxycholic acid ratio, which is increased in the late afternoon and decreased at midnight. The rhythm of CYP8B was the inverse of the circadian variation of serum insulin level and was similar to the circadian rhythm of glucose 6-phosphatase. These facts and the potent suppressive effect of insulin on CYP8B indicate that the oscillation of the serum insulin may be a factor in producing the circadian rhythm of CYP8B.     

Purification of cholesterol 7 alpha-hydroxylase from human and rat liver and production of inhibiting polyclonal antibodies

Cholesterol 7 alpha-hydroxylase, the cytochrome P-450-dependent and rate-controlling enzyme of bile acid synthesis, was purified from rat and human liver microsomes. The purified fractions were assayed in a reconstituted system containing [4-14C]cholesterol, and cholesterol 7 alpha-hydroxylase activities in these fractions increased 500-600-fold relative to whole microsomes. Polyacrylamide gel electrophoresis of rat microsomes followed by immunoblotting with polyclonal rabbit antisera raised against purified cholesterol 7 alpha-hydroxylases revealed two peaks at molecular masses of 47,000 and 49,000 daltons for both rat and human fractions. Increasing amounts of rabbit anti-rat and anti-human antibodies progressively inhibited rat microsomal cholesterol 7 alpha-hydroxylase activity up to 80%. In contrast, monospecific antibodies raised against other purified cytochrome P-450 enzymes (P-450f, P-450g, and P-450j) did not inhibit rat or human cholesterol 7 alpha-hydroxylase activity. Immunoblots of rat microsomes with the rabbit anti-rat cholesterol 7 alpha-hydroxylase antibody demonstrated that the antibody reacted quantitatively with the rat microsomal enzyme. Microsomes from cholesterol-fed rats showed increased cholesterol 7 alpha-hydroxylase mass, whereas treatment with pravastatin, an inhibitor of hydroxy-methylglutaryl-coenzyme A reductase, reduced enzyme mass. Microsomes from starved rats contained slightly less cholesterol 7 alpha-hydroxylase protein than chow-fed control rats. These results indicate a similarity in molecular mass, structure, and antigenicity between rat and human cholesterol 7 alpha-hydroxylases; demonstrate the production of inhibiting anti-cholesterol 7 alpha-hydroxylase antibodies that can be used to measure the change in cholesterol 7 alpha-hydroxylase enzyme mass under various conditions; and emphasize the unique structure of cholesterol 7 alpha-hydroxylase with respect to other cytochrome P-450-dependent hydroxylases.     

Neuro-endocrine correlations of hypothalamic-pituitary-thyroid axis in healthy humans

Neuro-endocrine hormone secretion is characterized by circadian rhythmicity. Melatonin, GRH and GH are secreted during the night, CRH and ACTH secretion peak in the morning, determining the circadian rhythm of cortisol secretion, TRH and TSH show circadian variations with higher levels at night. Thyroxine levels do not change with clear circadian rhythmicity. In this paper we have considered a possible influence of cortisol and melatonin on hypothalamic-pituitary-thyroid axis function in humans. Melatonin, cortisol, TRH, TSH and FT4 serum levels were determined in blood samples obtained every four hours for 24 hours from ten healthy males, aged 36-51 years. We correlated hormone serum levels at each sampling time and evaluated the presence of circadian rhythmicity of hormone secretion. In the activity phase (06:00 h-10:00 h-14:00 h) cortisol correlated negatively with FT4, TSH correlated positively with TRH, TRH correlated positively with FT4 and melatonin correlated positively with TSH. In the resting phase (18:00 h-22:00 h-02:00 h) TRH correlated positively with FT4, melatonin correlated negatively with FT4, TSH correlated negatively with FT4, cortisol correlated positively with FT4 and TSH correlated positively with TRH. A clear circadian rhythm was validated for the time-qualified changes of melatonin and TSH secretion (with acrophase during the night), for cortisol serum levels (with acrophase in the morning), but not for TRH and FT4 serum level changes. In conclusion, the hypothalamic-pituitary-thyroid axis function may be modulated by cortisol and melatonin serum levels and by their circadian rhythmicity of variation.     

Regulation of cholesterol 7 alpha-hydroxylase in the liver. Cloning, sequencing, and regulation of cholesterol 7 alpha-hydroxylase mRNA.

Monospecific antibody against purified rat liver cholesterol 7 alpha-hydroxylase cytochrome P-450 was used to screen a lambda gt11 cDNA library constructed from immuno-enriched polysomal RNA of cholestyramine-treated female rat liver. Two types of cDNA clones differing in the length of the 3'-untranslated region were identified, and DNA sequences were determined. The full length clone contains 3561 base pairs plus a long poly(A) tail. The amino acid sequence deduced from the open reading frame revealed a unique P-450 protein containing 503 amino acid residues which belonged to a new gene family designated family VII or CYP7. Southern blot hybridization experiments indicated that the minimal size of P-450 VII gene was 11 kilobase pairs (kb), and there was probably only one gene in this new family. Northern blot hybridization using specific cDNA probes revealed at least two major mRNA species of about 4.0 kb and 2.1 kb, respectively. These two mRNA species may be derived from the use of different polyadenylation signals and reverse-transcribed to two types of cDNA clones. Cholesterol 7 alpha-hydroxylase mRNAs were induced 2- to 3-fold in rat liver by cholestyramine treatment. The mRNA level was rapidly reduced upon the removal of the inducer. Similarly, cholesterol feeding induced enzyme activity, protein, and mRNA levels in the rat by 2-fold, suggesting that cholesterol is an important regulator of cholesterol 7 alpha-hydroxylase in the liver. On the other hand, dexamethasone and pregnenolone-16 alpha-carbonitrile drastically reduced the activity, protein, and mRNA levels. These experiments suggest that the induction of cholesterol 7 alpha-hydroxylase activity by cholestyramine or cholesterol and inhibition of cholesterol 7 alpha-hydroxylase activity by bile acid feedback are results of the rapid turnover of cholesterol 7 alpha-hydroxylase enzyme and mRNA levels.     

The regulation of ovine placental steroid 17 alpha-hydroxylase and aromatase by glucocorticoid

Parturition in the pregnant sheep is preceded by an abrupt alteration in placental steroid metabolism causing a shift from progesterone to estrogen production. This change is believed to be a consequence of the prepartum rise in cortisol in the fetal circulation and involves increases in activities of the enzymes steroid 17 alpha-hydroxylase (cytochrome P-450(17)alpha), steroid C-17,20-lyase, and possibly aromatase. We have investigated the activity levels of aromatase and 17 alpha-hydroxylase in placental microsomes in late pregnancy and dexamethasone-induced labor. Over the gestational period of 118-140 days basal levels of placental aromatase were relatively constant [mean value (+/- SD) of 5.6 +/- 1.6 pmol min-1 mg microsomal protein-1 (n = 10)]. Steroid 17 alpha-hydroxylase activity was undetectable [less than 0.5 pmol min-1 mg microsomal protein-1 (n = 7)]. In six animals in labor induced with infusion of dexamethasone into the fetus, placental aromatase activity had a mean value of 14.0 +/- 2.5 pmol min-1 mg protein-1; placental steroid 17 alpha-hydroxylase, measured in four of the animals, had a mean (+/- SD) activity of 319 +/- 58 pmol min-1 mg microsomal protein-1. Immunoblotting of placental microsomal preparations with specific antibodies to cytochrome P-450(17)alpha and NADPH-cytochrome P-450-reductase indicated that the glucocorticoid-induced activity of 17 alpha-hydroxylase was associated with increased content of cytochrome P-450(17)alpha. Northern blotting with a cDNA probe for cytochrome P-450(17)alpha showed that glucocorticoid increased the levels of mRNA for the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Renal 25-hydroxyvitamin D-3 1 alpha-hydroxylase in guinea-pig: activity variations during development and pregnancy

The renal 25-hydroxyvitamin D-3-1 alpha-hydroxylase (1 alpha-hydroxylase) activity and circulating levels of 1,25-dihydroxyvitamin D (1,25(OH)2D) were measured in pregnant guinea-pigs and their offspring. Serum levels of 1,25(OH)2D were significantly elevated in pregnant guinea-pigs but the renal enzyme activity was not different from non-pregnant animals. The fetal renal 1 alpha-hydroxylase activity was about 6-fold higher than the maternal level, whereas circulating 1,25(OH)2D was low. Treatment with pharmacological doses of 1,25(OH)2D3 increased circulating 1,25(OH)2D and depressed the renal 1 alpha-hydroxylases both in the mother and the fetus. In newborn guinea-pigs the enzyme activity was up to 10-times that seen in adults. It declined over the first 3 weeks, showing no difference between the sexes. In sexually mature animals the males had a significantly higher 1 alpha-hydroxylase activity than the female. However, this higher enzyme activity was not correlated to serum testosterone. Around the time the animals reached sexual maturity serum 1,25(OH)2D increased in both sexes. In the males this rise was correlated to an increase in serum testosterone. It is concluded that the maternal renal 1 alpha-hydroxylase activity is unchange in late pregnancy, compared to non-pregnant females. The data indicate that the fetus produces 1,25(OH)2D, and may contribute to the maternal circulating 1,25(OH)2D. The sex difference in 1 alpha-hydroxylase activity previously demonstrated is manifest at about the time of puberty.     

Circadian rhythms of sterol 12alpha-hydroxylase, cholesterol 7alpha-hydroxylase and DBP involved in rat cholesterol catabolism

Circadian rhythms of important enzymes involved in the conversion of cholesterol to bile acids [sterol 12alpha-hydroxylase (12alpha-hydroxylase) and cholesterol 7alpha-hydroxylase (7alpha-hydroxylase)] and an albumin site D-binding protein (DBP) were examined in rats. When the animals were fed freely, they usually ate in the dark and the circadian rhythms of activities of 12alpha-hydroxylase and 7alpha-hydroxylase showed the same peaks (at 10 p.m.) and lows (at 2 p.m.). Their mRNA levels were determined at four timepoints: 3 a.m., 10 a.m., 3 p.m. and 10 p.m. A maximum of the rhythm of 12alpha-hydroxylase was observed at 3 p.m. and the minimum at 3 a.m. These results are distinct from those of 7alpha-hydroxylase, whose maximum point was at 10 p.m. and minimum at 3 p.m. When the rats were fed only in the day-time (from 9 a.m. to 5 p.m.), a marked shift of the activity and mRNA rhythms was observed with both enzymes. The circadian rhythms of the activities of both enzymes showed the same peaks (at 3 p.m.), but the mRNA levels of 12alpha-hydroxylase were distinct from those of 7alpha-hydroxylase, whose maximum point was at 3 a.m. and minimum at 10 p.m. Differences between the maximum and the minimum points of each enzyme mRNA level were statistically significant (P < 0.01 for 12alpha-hydroxylase and 0.05 for 7alpha-hydroxylase). Moreover, circadian rhythms of DBP were also markedly shifted with the change of feeding period. The maximum mRNA level was observed at 10 p.m. instead of 10 a.m. and the minimum was at 10 a.m. instead of 10 p.m.     

Regulation of bile acid synthesis. IV. Interrelationship between cholesterol and bile acid biosynthesis pathways

Under most experimental conditions, the activities of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA reductase) and cholesterol 7 alpha-hydroxylase, change together in parallel directions. It has been suggested that newly synthesized cholesterol may be the preferred substrate for cholesterol 7 alpha-hydroxylase, which may account for the observed synchronous behavior of the two enzymes. To test this hypothesis, mevinolinic acid, a potent competitive inhibitor of HMG-CoA reductase, was administered as a single intravenous bolus (10 mg/kg) to rats with a chronic bile fistula. Bile acid synthesis was determined following inhibition of HMG-CoA reductase by mevinolinic acid over a 27-h time course and specific activities of HMG-CoA reductase and cholesterol 7 alpha-hydroxylase were determined in liver microsomes. At 3, 6, and 27 h after a bolus dose of mevinolinic acid, bile acid synthesis was reduced by 54 +/- 5%, 42 +/- 8%, and 23 +/- 13%, respectively, from preinfusion baseline. Within 30 min after administration of mevinolinic acid, HMG-CoA reductase activity was inhibited by at least 87%. At 0.5, 1.5, 3, 6, and 27 h after mevinolinic acid injection, cholesterol 7 alpha-hydroxylase activity was decreased by 6%, 25%, 54%, 41%, and 17%, respectively. By 27 h, the activities of both enzymes had returned to baseline levels. The reduction of bile acid synthesis correlated closely with the observed changes in the activities of cholesterol 7 alpha-hydroxylase. In vitro addition of mevinolinic acid (up to 20 microM) to rat liver microsomes failed to inhibit cholesterol 7 alpha-hydroxylase activity, suggesting no direct effect of mevinolinic acid on enzyme activity. When a bolus dose of mevinolinic acid was coupled with a continuous infusion of mevalonate, the product of the reaction catalyzed by HMG-CoA reductase, the mevinolinic acid-induced decrease in cholesterol 7 alpha-hydroxylase activity and bile acid synthesis was prevented. The results of this study provide evidence that, under the experimental conditions described, there is a linkage between the rates of cholesterol synthesis and the activities of cholesterol 7 alpha-hydroxylase. The data also emphasize the importance of the newly synthesized cholesterol in the regulation of cholesterol 7 alpha-hydroxylase activity.     

Biochemical site of regulation of bile acid biosynthesis in the rat

The production of bile salts by rat liver is regulated by a feedback mechanism, but it is not known which enzyme controls endogenous bile acid synthesis. In order to demonstrate the biochemical site of this control mechanism, bile fistula rats were infused intravenously with (14)C-labeled bile acid precursors, and bile acid biosynthesis was inhibited as required by intraduodenal infusion of sodium taurocholate. The infusion of taurocholate (11-14 mg/100 g of rat per hr) inhibited the incorporation of acetate-1-(14)C, mevalonolactone-2-(14)C, and cholesterol-4-(14)C into bile acids by approximately 90%. In contrast, the incorporation of 7alpha-hydroxycholesterol-4-(14)C into bile acids was reduced by less than 10% during taurocholate infusion. These results indicate that the regulation of bile acid biosynthesis is exerted via cholesterol 7alpha-hydroxylase provided that hepatic cholesterol synthesis is adequate.