Effects of mevinolin in rat liver: evidence for a lack of coupling between synthesis of hydroxymethylglutaryl-CoA reductase and cholesterol 7 alpha-hydroxylase activity

The effect of treatment of rats with the hydroxymethylglutaryl-CoA reductase inhibitor, mevinolin, on 7 alpha-hydroxylation of cholesterol was studied. Treatment with 0.1% mevinolin in diet for 3 days was found to have an inhibitory effect on 7 alpha-hydroxylation of cholesterol (about 35%). Treatment with cholestyramine increased 7 alpha-hydroxylation of both exogenously added and endogenous microsomal cholesterol 3-4-fold. Combined treatment with both cholestyramine and mevinolin decreased this stimulation to 2-2.5-fold. Treatment with 2% cholesterol in diet increased 7 alpha-hydroxylation of exogenous cholesterol about 2-fold and 7 alpha-hydroxylation of endogenous cholesterol about 3.5-fold. The stimulatory effect of cholesterol was reduced or abolished when 0.1% mevinolin was added to the cholesterol-containing diet. With the exception of the experiments with cholesterol in the diet, all experiments including mevinolin gave a marked stimulation (up to 60-fold) of the hydroxymethylglutaryl-CoA reductase activity under the in vitro conditions employed. The concentration of free cholesterol in the liver microsomes was not significantly changed in any of these experiments. It is concluded that there is no coupling between induction of synthesis of hydroxymethylglutaryl-CoA reductase protein and cholesterol 7 alpha-hydroxylase activity. The inhibitory effect of mevinolin on cholesterol 7 alpha-hydroxylase activity under experimental conditions where most of the effect of mevinolin on hydroxymethylglutaryl-CoA reductase was abolished by treatment with cholesterol suggest that the effect of mevinolin on the cholesterol 7 alpha-hydroxylase may be independent of its effect on cholesterol synthesis. The over-all results do not favour the hypothesis that cholesterol synthesis and cholesterol availability are the most important determinants for the regulation of the cholesterol 7 alpha-hydroxylase.     

Sensitivity of the soleus muscle to insulin in resting and exercising rats with experimental hypo- and hyper-thyroidism.

1. The effects of hypothyroidism (caused by surgical thyroidectomy followed by treatment for 1 month with propylthiouracil) and of hyperthyroidism [induced by subcutaneous administration of L-tri-iodothyronine (T3)] on glucose tolerance and skeletal-muscle sensitivity to insulin were examined in rats. Glucose tolerance was estimated during 2 h after subcutaneous glucose injection (1 g/kg body wt.). The sensitivity of the soleus muscle to insulin was studied in vitro in sedentary and acutely exercised animals. 2. Glucose tolerance was impaired in both hypothyroid and hyperthyroid rats in comparison with euthyroid controls. 3. In the soleus muscle, responsiveness of the rate of lactate formation to insulin was abolished in hypothyroid rats, whereas the sensitivity of the rate of glycogen synthesis to insulin was unchanged. In hyperthyroid animals, opposite changes were found, i.e. responsiveness of the rate of glycogen synthesis was inhibited and the sensitivity of the rate of lactate production did not differ from that in control sedentary rats. 4. A single bout of exercise for 30 min potentiated the stimulatory effect of insulin on lactate formation in hyperthyroid rats and on glycogen synthesis in hypothyroid animals. 5. The data suggest that thyroid hormones exert an interactive effect with insulin in skeletal muscle. This is likely to be at the post-receptor level, inhibiting the effect of insulin on glycogen synthesis and stimulating oxidative glucose utilization.     

Poly(ADP-ribose) polymerase is affected early by thyroid state during liver regeneration in rats

Poly(ADP-ribose) polymerase (PARP), a nuclear enzyme involved in DNA synthesis, DNA repair, and cell replication and transformation, also plays a role in the early steps of liver regeneration induced by partial hepatectomy (PH). PARP and DNA topoisomerase I (Topo I) activities and de novo DNA synthesis were studied during liver regeneration in rats with altered thyroid state. Hepatic PARP activity, evaluated as [(32)P]NAD incorporated into isolated liver nuclei, was inhibited in hyperthyroid rats and increased in hypothyroid animals. In both euthyroid and hyperthyroid rats PARP activity was rapidly stimulated, peaking 6 h after PH. In hypothyroid animals, an early decrease in activity was found, at a minimum of 6 h after PH, followed by an early onset of DNA synthesis. An inverse relationship between PARP and Topo I activities was a shared feature among euthyroid, hypothyroid, and hyperthyroid rats. Together these data show that, in replicating hepatocytes, thyroid hormones exert a regulatory role on PARP activity, which reflects the control of a number of nuclear proteins involved in DNA metabolism.     

The role of glucocorticoids in the regulation of the diurnal rhythm of hepatic beta-hydroxy-beta-methylglutaryl-coenzyme A reductase and cholesterol 7 alpha-hydroxylase.

The microsomal activities of the hepatic enzymes hydroxymethylglutaryl-CoA reductase and cholesterol 7 alpha-hydroxylase exhibit a diurnal rhythm with maximum activities observed during the dark period and minimum activities around noon (12:00h). This diurnal rhythm was maintained for both enzymes after adrenalectomy, but the amplitude of variation for the activity of both enzymes was greatly decreased. A single injection of cortisol administered to adrenalectomized rats 3h before the expected maximum in enzyme activity resulted in a twofold increase in the activity of both enzymes 3h later, at values similar to those observed for control rats killed at the same time. This response appeared to require protein synthesis, since it was blocked by actinomycin D. However, the administration of cortisol to adrenalectomized rats 3 h before the expected minimum did not result in significant change in the activity of hydroxymethylglutaryl-CoA reductase and cholesterol 7 alpha-hydroxylase 3 h later. Kinetic studies of cholic acid metabolism in vivo demonstrated that adrenalectomy results in a significant decrease in the rate of synthesis of cholic acid and a considerable decrease in the pool size of cholic acid and its metabolic products. Treatment of adrenalectomized rats with cortisol increased the rate oonsistent with the effects of adrenalectomy and cortisol treatment on the activity of cholesterol 7alpha-hydroxylase.     

Effects of thyroid status and fasting on hepatic metabolism of apolipoprotein A-I

Metabolism of apolipoprotein (apo)A-I was studied in normal and chow-fed hyperthyroid rats, in 24-h fasted untreated male rats, and in rats after thyroparathyroidectomy (TXPTX). Rats were made hyperthyroid by administration of T3 (9.6 micrograms/day) or T4 (30 micrograms/day) with an Alzet osmotic minipump. Hyperthyroidism produced a similar two- to threefold elevation in plasma levels of apoA-I in male or female animals. During treatment with T3, plasma levels of T3 ranged from 200 to 400 ng/dl and did not correlate with plasma apoA-I levels. The net mass secretion and synthesis ([3H]leucine incorporation) of apoA-I by perfused livers from male hyperthyroid rats was elevated, while secretion of albumin was not different than that of euthyroid rats. Furthermore, the incorporation of [3H]leucine into total perfusate and hepatic protein was not altered by hyperthyroidism. The effect of thyroid hormone on apoA-I synthesis, therefore, does not appear to be a general effect on protein synthesis. After longer periods of treatment (28 days) with T3 (9.6 micrograms/day), hepatic apoA-I production decreased from that observed after 7 or 14 days of treatment, yet plasma apoA-I concentrations remained elevated. Plasma T3 decreased from 100 ng/dl to 40 ng/dl, in the hypothyroid rat resulting from TXPTX, but the plasma concentration of apoA-I did not change during the 2-week experimental period. The net secretion of apoA-I by livers from hypothyroid animals was depressed and albumin was uneffected compared to the euthyroid. Overnight fasting of euthyroid rats did not alter hepatic apoA-I secretion or plasma apoA-I levels, although under fasting conditions we had reported that hepatic output of apoB and E of VLDL is depressed. The addition of oleic acid to the perfusion medium, sufficient to stimulate VLDL production, did not affect net hepatic secretion of apoA-I by livers from euthyroid, hyperthyroid, or hypothyroid rats. In summary, hepatic synthesis of apoA-I appears to be controlled independently of other apo-lipoproteins and secretory proteins (albumin). Hepatic apoA-I synthesis is sensitive to thyroid status, increased in the hyperthyroid and decreased in the hypothyroid state. The specific stimulation of hepatic synthesis and secretion of apoA-I in the hyperthyroid state, however, tends to normalize over an extended period, perhaps from compensatory effects of a hormonal nature.     

Influence of thyroid states on stress gastric ulcer formation

Thyroid hormones exert a critical developmental and regulatory role on the morphology and biochemistry of gastrointestinal mucosal cells. However, the relationship between thyroid function and stress gastric lesion formation remains undetermined. This study was designed to test the hypothesis that thyroid states may affect the acute development of gastric lesions induced by cold-restraint stress. Normal (euthyroid), hyperthyroid (200 micrograms of T4 i.p. x 7 days) and hypothyroid (thyroidectomized) rats were used. Gastric lesion incidence and severity was significantly (p less than 0.05) increased in hypothyroid rats, whereas in contrast hyperthyroid rats developed significantly less gastric lesions. As anticipated, plasma levels of thyroxin (T4) were significantly (p less than 0.01) elevated in hyperthyroid rats, and undetectable in hypothyroid rats. Acute pretreatment with i.p. cimetidine (100 mg/Kg), but not T4 (200 micrograms/Kg) 1 h prior to stress completely prevented gastric lesions formation in hypothyroid rats. Finally, binding of 3H-dihydroalprenolol to beta-adrenergic receptors on brain membranes prepared from frontal cortex was reduced by 20% in hypothyroid rats after 3 h of stress. These and other data contained herein suggest that thyroid hormones contribute to modulate the responsiveness of the gastric mucosa to stress. The increased rate of ulcerogenesis observed in hypothyroid rats appears to be mediated by gastric acid secretion. The central mechanism of this response may involve decreased brain nonadrenergic receptor function.     

Serum and tissue coenzyme Q9 in rats with thyroid dysfunctions

Serum and tissue CoQ9 levels were determined in hypothyroid, euthyroid and hyperthyroid rats. A significant negative correlation was demonstrated between serum FT4 or T3 and CoQ9 in rats with various states of thyroid functions. Liver CoQ9 was significantly increased in rats rendered mildly hyperthyroid. There was a significant positive correlation between serum FT4 or T3 and liver CoQ9. While liver CoQ9 did not significantly change in severely hyperthyroid animals, liver mitochondrial CoQ9 showed a significant positive correlation with serum T3. Kidney and heart CoQ9 levels did not significantly change in hyperthyroid rats, but those in hypothyroid rats showed a tendency to increase. It was suggested that the synthesis of CoQ9 was increased in the liver in hyperthyroidism.     

Regulation of bile acid synthesis. V. Inhibition of conversion of 7-dehydrocholesterol to cholesterol is associated with down-regulation of cholesterol 7 alpha-hydroxylase activity and inhibition of bile acid synthesis.

In the chronic bile fistula rat, the administration of a bolus dose of mevinolinic acid, an inhibitor of HMG-CoA reductase, was followed by rapid down-regulation of cholesterol 7 alpha-hydroxylase activity and a decrease in bile acid synthesis. These observations suggested that either newly synthesized cholesterol or some other metabolite of mevalonate may be involved in the regulation of bile acid synthesis. In order to distinguish between these two alternatives, we carried out experiments in which cholesterol synthesis was blocked by AY9944, a compound that inhibits the conversion of 7-dehydrocholesterol to cholesterol, a last step in the cholesterol biosynthesis pathway. Rats underwent biliary diversion for 72 h at which time they were given intravenously either a bolus dose of AY9944 (1 mg/kg) or control vehicle. At 0 (pre-treatment control), 0.5, 1.5, and 3 h post bolus, livers were harvested and specific activities of cholesterol 7 alpha-hydroxylase were determined. At 1.5, 3, and 6 h post bolus, AY9944 inhibited bile acid synthesis by 19 +/- 6%, 40 +/- 4%, and 41 +/- 6%, respectively, as compared to pretreatment baseline. Cholesterol 7 alpha-hydroxylase activity determined at 0.5, 1.5, and 3 h was decreased by 44 +/- 6%, 44 +/- 2%, and 36 +/- 2%, respectively, as compared to the control value. In in vitro experiments using microsomes from livers of control bile fistula rats, the addition of AY9944 (up to 100 microM) failed to inhibit cholesterol 7 alpha-hydroxylase activity. The results of this study demonstrate that, in the chronic bile fistula rat, acute inhibition of cholesterol synthesis at either early or late steps leads to a rapid down-regulation of cholesterol 7 alpha-hydroxylase activity and decrease in bile acid synthesis.     

Effect of thyroid state on cyclic AMP-mediated induction of hepatic phosphoenolpyruvate carboxykinase.

Hepatic phosphoenolpyruvate carboxykinase (PEPCK) is significantly increased in the hyperthyroid starved rat, and moderately decreased in the hypothyroid starved rat. As tri-iodothyronine by itself has only a small and sustained effect on the induction of this enzyme, as was previously shown in the isolated perfused organ, the effect of hypo- and hyper-thyroidism on the increase in cytosolic PEPCK provoked by dibutyryl cyclic AMP (Bt2cAMP) was investigated in vivo and in the isolated perfused liver. Compared with euthyroid fed controls, in hypothyroid fed rats Bt2cAMP provoked in 2 h only a small increase in translatable mRNA coding for PEPCK. In contrast, in hyperthyroid animals PEPCK mRNA as measured by translation in vitro was already increased in the fed state, and further enhanced by Bt2cAMP injection to values as in euthyroid controls. Under all thyroid states a close correlation between PEPCK mRNA activity and PEPCK synthesis was observed. In the isolated perfused liver from the hyperthyroid fed rat, the increase in PEPCK provoked by Bt2cAMP or Bt2cAMP + isobutylmethylxanthine was considerably enhanced compared with those obtained in livers of hypothyroid rats. Also, adrenaline provoked a stimulated induction of PEPCK in hyperthyroid rats compared with hypothyroid rats. To summarize, our data indicate that the primary action of thyroid hormones on the synthesis of hepatic cytosolic PEPCK is to accelerate the cyclic AMP- or adrenaline-induction of the enzyme, acting primarily at a pretranslational level.     

Renal phosphoenolpyruvate carboxykinase turnover in hypo- and hyperthyroid rat in vivo

The effect of hypo- and hyperthyroidism on activity, synthesis and degradation of renal cytosolic phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) was studied in the rat by radioimmunological techniques. In hypo- and euthyroid rats, starvation induced similar alterations in enzyme activities and relative rates of synthesis, whereas in hyperthyroid rats the increase in both was significantly reduced. Substitution of l-thyroxine in hypothyroid rats resulted in a decrease in activity and synthesis within 18 h as observed in hyperthyroid animals. The apparent half-life of the enzyme measured by double-pulse labeling experiments was approx. 13 h in euthyroid animals. The rate of degradation was unaffected by the different thyroid states.     

Angiotensinase activity in hypothalamus and pituitary of hypothyroid, euthyroid and hyperthyroid adult male rats.

A local renin-angiotensin system (RAS) that may be involved in their regulatory functions has been identified in hypothalamus and pituitary. Altered thyroid status induces modifications in the secretory function of hypothalamus and pituitary. However, few studies have analyzed the role of the RAS in hypothalamus and, to our knowledge, there is no data on the pituitary RAS during thyroid dysfunction. In the present study, angiotensinase activities (glutamyl, aspartyl and alanyl aminopeptidase: GluAP, AspAP and AlaAP, respectively) were studied in hypothalamus and in the anterior and posterior lobes of pituitary of euthyroid, hypothyroid and hyperthyroid adult male rats. In the anterior pituitary, compared with euthyroid and hyperthyroid rats, hypothyroid animals showed a highly significant increase of GluAP and AspAP activities; the percentage increase in GluAP was markedly higher than the percentage increase in AspAP. This suggests an increased metabolism of angiotensin (Ang) I and Ang II to des-Asp 1-Ang I and Ang III, respectively. We also observed an increase of Ang III-degrading activity (AlaAP) in the hypothalamus of hyperthyroid rats in soluble fraction. Increased Ang I and Ang II metabolism in the anterior pituitary of hypothyroid rats and increased metabolism of Ang III in the hypothalamus of hyperthyroid animals may be related to alterations in the secretory function of hypothalamus and pituitary in these thyroid dysfunctions.     

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.     

Studies on the formation of C7-oxygenated cholesterol and beta-sitosterol metabolites in cell-free preparations of rat liver

The microsomal fraction and the 18,000 g supernatant fluid obtained from livers from normal rats, cholestyraminetreated rats, or from rats with a bile fistula have been used to compare the 7alpha-hydroxylation of [4-(14)C]cholesterol and beta-[4-(14)C]sitosterol (24alpha-ethyl-cholesterol). It was not possible to increase the specific formation of 7alpha-hydroxy-beta-sitosterol above 0.05% with any of the preparations. This conversion was less than 1% of that found for cholesterol. The inhibitory effect of added 7-oxo- and 7beta-hydroxy-beta-sitosterol on the 7alpha-hydroxylation of cholesterol was found to be much less than that of the corresponding cholesterol compounds. 7alpha-Hydroxy-beta-sitosterol was without effect. It is concluded that the activity of the cholesterol 7alpha-hydroxylase is dependent upon the structure of the steroid side chain.     

Regulation by thyroid hormone of the synthesis of a cytosolic thyroid hormone binding protein during liver regeneration.

To understand the regulation by thyroid hormone, 3,3',5-triiodo-L-thyronine (T3), of the synthesis of a cytosolic thyroid hormone binding protein (p58-M2) during liver regeneration, the synthesis of p58-M2 was evaluated. The synthesis of p58-M2 was measured by metabolic labeling of primary cultures derived from the regenerating liver of euthyroid, hypo- or hyperthyroid rats. During regeneration, the increase in the liver/body weight ratio is approximately 25% higher in hyper- than in hypothyroid rats. However, T3 has no effect on the rate of overall liver regeneration observed in four days. In mature liver, T3 increased the synthesis of p58-M2 by approximately 2.5-fold. During regeneration, however, the change in the synthesis of p58-M2 varied with the thyroid status. In euthyroid rats, the synthesis of p58-M2 continued to increase up to 2-fold during liver regeneration. In hyperthyroid rats, after an initial increase by 1.5-fold on day 1, the synthesis of p58-M2 subsequently declined during regeneration. In hypothyroid rats, the synthesis of p58-M2 remained virtually unchanged during regeneration. These results indicate that T3 regulates the synthesis of p58-M2 in mature and regenerating liver.     

Sterol balance studies in the rat. Effects of dietary cholesterol and beta-sitosterol on sterol balance and rate-limiting enzymes of sterol metabolism.

Sterol balance measurements using isotopic and chromatographic techniques were carried out in rats fed diets containing beta-sitosterol (0.8%) and cholesterol (1.2%). The activities of the rate-limiting enzymes of cholesterol synthesis (beta-hydroxy-beta-methylglutaryl-CoA reductase, EC 1.1.1.34) and bile acid synthesis (cholesterol 7 alpha-hydroxylase) were determined in the same animals. Cholesterol feeding increased cholesterol absorption from 1.2 to 70 mg/day. The increased absorption was compensated for by inhibition of hepatic cholesterol synthesis, enhanced conversion of cholesterol to bile acids (from 13.7 to 27.3 mg/day) and a slight increase in the excretion of endogenous neutral steroids (from 7.7 to 11.2 mg/day). Despite the adaptation there was accumulation of cholesterol in the liver (from 2.2 to 9.2 mg/g). Beta-Sitosterol feeding inhibited cholesterol absorption (calculated absorption was zero). In these rats there was enhanced cholesterol synthesis (from 20.0 to 28.8 mg/day, but no change in the rates of bile acid formation. Measurements of the activities of the rate-limiting enzymes showed fair correlation with cholesterol-bile acid balance. In cholesterol fed animals, beta-hydroxy-beta-methylglutaryl-CoA reductase was inhibited 80% and cholesterol 7 alpha-hydroxylase was enhanced 61%. In beta-sitosterol-fed animals, the reductase was increased 2-fold and cholesterol 7 alpha-hydroxylase was not significantly different from controls.     

Role of testosterone levels and of hypophysis in the HCG-induced modifications of the 7 alpha-hydroxylation and 5 alpha-reduction processes in incubated rat testes

The role of a direct or a hypophysis-mediated influence of increased testosterone levels in the effects of a long-term high-dose HCG administration (10 IU/day) upon the 7 alpha-hydroxylation and 5 alpha-reduction activities of incubated testes of mature rats was investigated. Administration of high doses of HCG to hypophysectomized rats resulted in the same metabolic changes as in normal rats, namely, a large decrease in the 7 alpha-hydroxylation and an increase in the 5 alpha-reduction processes. Administration of testosterone-propionate (0.2 mg and 20 mg/day) for several days to hypophysectomized rats and to normal rats receiving and substitutive dose of 1 IU HCG/day, did not modify the testicular metabolization pattern. These findings indicate that the decrease in testicular 7 alpha-hydroxylation activity induced by long-term administration of high doses of HCG is probably not mediated by the hypophysis nor by the extracellular testosterone levels.     

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.     

Changes in lipid peroxidation and free radical scavengers in kidney of hypothyroid and hyperthyroid rats

Kidney weight was significantly decreased in hypothyroidism (induced by Na131I administration) and increased in hyperthyroidism (induced by thyroxine treatment) as compared to control in female Wistar rats. The tissue lipid peroxidation level remained unchanged in hyperthyroid rats but significantly increased in hypothyroid rats. Superoxide dismutase was decreased in both experimental groups but more so in hyperthyroid rats. Catalase was reduced significantly in hyperthyroid rats but remained unaffected in hypothyroid rats. Tissue glutathione peroxidase (GPx) activity was increased while reduced glutathione levels remained unaltered in both hypothyroid and hyperthyroid rats. Plasma GPx activity was significantly low in both the hypothyroid and hyperthyroid rats. The results suggest alterations in the oxidative stress in hypothyroid and hyperthyroid rat kidneys with concomitant changes of free radical scavengers.