Influence of thyroid hormone status on mevalonate metabolism in rats.

Mevalonate, an essential intermediate in cholesterol synthesis, is metabolized either to cholesterol or, by the shunt pathway, to CO2. Previous investigations have demonstrated that the kidneys are the chief site of circulating mevalonate metabolism and that sex hormones as well as insulin markedly influence circulating mevalonate metabolism. The present study examined in rats the influence of thyroid hormone status on mevalonate metabolism in vivo and in vitro. L-thyroxine administration increased renal conversion of circulating mevalonate to cholesterol, 41% in the females and 22% in the males. Conversely, hypothyroidism induced by 6 N propyl-2-thiouracil reduced renal conversion of circulatng mevalonate to cholesterol by 45% in females and 27% in males; thyroid hormone replacement in these animals returned cholesterogenesis in the kidneys to supranormal levels. Neither L-thyroxine nor hypothyroidism altered circulating mevalonate conversion to cholesterol in the liver or carcass. In vitro studies confirmed the in vivo observations. Changes in thyroid hormone produced only minor changes in the shunt pathway of mevalonate metabolism. This study demonstrates that the major effect of the thyroid hormone on the metabolism of circulating mevalonate is to alter the conversion of mevalonate to cholesterol, an effect localized solely to the kidneys.

Sex difference in human mevalonate metabolism.

Two pathways of mevalonate metabolism have been demonstrated: the major (sterol) pathway leads to cholesterol synthesis, whereas the second shunts mevalonate away from sterol production and ultimately results in its oxidation to CO2. Previous studies have demonstrated that the female rat metabolizes circulating mevalonate by the shunt pathway at twice the rate of the male, whereas the male rat converts significantly more circulating mevalonate to cholesterol than the female. The present study extends these observations to humans. Six men and five premenopausal women with normal renal function were injected with R,S-[5-14C]mevalonate, and 14CO2 expired in the breath of the subjects was monitored continuously with an ionization chamber. On an average, the female subjects expired 16.5% and the males 9.8% of the injected R-[5-14C]mevalonate (P less than 0.001). No differences were observed in the plasma and erythrocyte [14C]cholesterol levels. These data demonstrate, in human beings, a sex difference in mevalonate metabolism. The overall impact of the greater mevalonate shunt activity on cholesterol balance in women is unknown.

The effect of cholesterol feeding and alterations in bile acid homeostasis on de novo sterologenesis in diabetic rats.

Previous studies have demonstrated that de novo cholesterol synthesis is increased two- to threefold in the intestines of diabetic animals. This increase is due to a stimulation of cholesterogenesis in both the small and large intestine but, quantitatively, the small intestine is primarily responsible for the observed increase. The present study examined the effect of cholesterol feeding and alterations of bile acid homeostasis on de novo sterol synthesis in intact normal and diabetic animals. Cholesterol feeding in the control animals did not affect sterol synthesis in the small intestine, but in diabetic animals cholesterol feeding markedly inhibited small intestinal sterologenesis. The threefold stimulation of small intestinal sterol synthesis observed in diabetic animals is completely obliterated by cholesterol ingestion. Moreover, this inhibition of sterol synthesis by cholesterol feeding in the small intestine of diabetic animals occurred very rapidly (within 36 h). In the large intestine, cholesterol feeding did not influence sterol synthesis in either the diabetic or control animals. In the liver, cholesterol feeding markedly inhibited sterol synthesis to similar degrees in the diabetics and controls. Colestipol feeding and biliary drainage, procedures that reduce bile acid pool size, stimulated sterol synthesis in the liver and small intestine of both diabetic and control animals. However, reductions in bile acid pool size increased sterologenesis in the large intestine in control animals but had no effect in the diabetics. Bile acid ingestion did not alter either small or large intestinal sterologenesis in the diabetic or control animals. In conclusion, the present study demonstrates the sterol synthesis is enhanced in the small and large intestine of diabetic animals and, moreover, both the cholesterol- and bile acid-mediated regulation of cholesterol synthesis in the intestines of the diabetic animals is altered from normal.

The effect of diabetes mellitus on sterol synthesis in the intact rat.

Diabetes mellitus in both humans and animals is characterized by elevations in plasma cholesterol concentrations. The cause of this hypercholesterolemia is unknown. The present study employed tritiated water to quantity sterol synthesis in intact diabetic and control animals. Sterologenesis was 60-246% greater in the gut of diabetic animals than in controls. This enhancement of sterol synthesis occurred soon after the onset of diabetes and persisted for at least 3 wk. Moreover, insulin therapy markedly decreased gut sterol synthesis in diabetic animals to levels only slightly greater than in controls. Diabetes increased sterol synthesis primarily in the small intestine, but a small increase was also observed in the large intestine. Subfractionation of the small intestine into epithelial cell and muscularis cell layers revealed an increased sterologenesis in both layers. Quantitatively, though, the epithelial layer accounted for the majority of the enhancement of small intestine sterol synthesis observed in diabetic animals. De novo sterologenesis in tissues other than the intestines (liver, skin, stomach, or remaining carcass) was not significantly altered by diabetes. This study demonstrates that diabetes markedly stimulates sterol synthesis, an effect that is specifically localized to the intestines.

Differences in de novo cholesterol synthesis between the intact male and female rat.

Three different isotopes were used to quantify de novo sterologenesis in intact male and female rats. All three substrates (i.e. [14C]acetate, [14C]octanoate, and [3H]water) were incorporated into nonsaponifiable lipids and cholesterol at significantly greater rates in males than in females. Even with cholesterol feeding, male animals synthesized significantly more cholesterol and nonsaponifiable lipids than females. The primary site of this sex difference in sterologenesis is extrahepatic, extraintestinal tissues (e.g. carcass). In the carcass this sex difference is chiefly due to an enhancement of sterol synthesis in the skin of male rats. Cholesterol synthesis is 73% greater and nonsaponifiable lipid synthesis is 85% greater in the skin of males than in females. Moreover, de novo sterologenesis in skin is hormonally dependent. In castrated females, testosterone treatment results in a 2-fold stimulation of skin sterol synthesis compared to that in animals administered estradiol or oil vehicle alone. In castrated males, estradiol treatment caused a 30% reduction in skin cholesterol and nonsaponifiable lipid synthesis compared to that in animals administered testosterone. The effects of sex steroid hormones on skin are, therefore, probably responsible for mediating the observed sex difference in de novo sterol synthesis. Additionally, this study demonstrates that the administration of estradiol and testosterone in physiological doses to castrated animals has no effect on cholesterol or nonsaponifiable lipid synthesis in liver, intestine, or other nondermal tissues.

De novo sterologenesis in the intact rat.

On the basis mainly of in vitro studies, the liver and, to a lesser degree, the small intestine are widely accepted as the major sites of de novo sterologenesis. Utilizing [3H]water, we have investigated de novo sterologenesis in intact rats. Greater than 80% of labeled nonsaponifiable lipids and more than 70% of the labeled cholesterol were localized to extrahepatic, extraintestinal tissues. Feeding cholesterol markedly suppressed hepatic sterologenesis but had little influence on extrahepatic sites of sterol synthesis. Similarly, partial hepatectomy, which greatly decreased sterol synthesis in the liver, also did not significantly affect the accumulation of labeled sterols in extrahepatic tissues; therefore, the transport of sterols from the liver did not account for a significant portion of labeled sterols in extrahepatic tissues. Cannulation of the thoracic duct demonstrated that transport of newly synthesized intestinal sterols to peripheral tissues also did not account for the large accumulation of labeled sterols in extrahepatic, extraintestinal tissues. The primary extrahepatic, extraintestinal sites of sterologenesis were the skin and remaining carcass. The lung, kidney, spleen, heart, ovary, brain, muscle and adipose tissue made minor contributions to de novo sterol synthesis. Therefore, tissues other than the liver and intestine, especially the skin and remaining carcass, are important sites of de novo sterologenesis in vivo.

The quantitative role of the kidneys in the in vivo metabolism of mevalonate.

The roles of the sterol and nonsterol pathways in the metabolism of circulating mevalonate have been estimated in the intact rat. On an average, the sterol pathway accounts for 74 per cent of the mevalonate metabolized, while the nonsterol, or shunt, pathway is responsible for 26 per cent of the mevalonate metabolized in the whole animal. The contribution of the kidneys to each of these processes was evaluated by two approaches. First, the localization of labeled sterols and sterol precursors derived from [14C]mevalonate was determined in each of the major tissues of the body and, second, the effect of nephrectomy upon mevalonate metabolism by the sterol and shunt mechanisms was examined. The results confirm our earlier conclusion that the kidneys represent the primary tissue site of conversion of circulating mevalonate to sterols and sterol precursors. In the present study, it was shown that by 6 h after administration of [14C]mevalonate, the major end product of mevalonate metabolism in the kidneys is cholesterol and that, moreover, the kidneys are responsible for most of the cholestreol synthesized in the intact animal from injected mevalonate. Following nephrectomy, the extrarenal tissues can readily assume the dominant role normally played by the kidneys in synthesizing cholesterol and other sterols from circulating mevalonate. The major observation of the present study is that the kidneys represent the primary site of mevalonate metabolism by the shunt pathway, in that nephrectomy results in approximately a 60 per cent decrease in the mevalonate metabolized by the shunt pathway. These studies, therefore, reinforce and expand the evidence that the kidneys represent the most important single tissue site for the metabolism of circulating mevalonate.

Isopentenyladenine as a mediator of mevalonate-regulated DNA replication.

The mechanism by which the cholesterol precursor, mevalonate, regulates S-phase DNA replication was examined in synchronized BHK cells. As previously demonstrated by this laboratory, blocking 3-hydroxy-3-methylglutaryl-CoA reductase [mevalonate:NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34] with the competitive inhibitor compactin suppresses DNA synthesis specifically during the S phase of the cell cycle. In the present study, known mevalonate derivatives were examined as possible mediators by which mevalonate controls DNA replication. Of the compounds studied, only isopentenyladenine and its 4'-hydroxylated analogue, zeatin, could substitute for mevalonate in restoring DNA replication in compactin-blocked cells. Moreover, these two derivatives proved to be at least 100 times more active than mevalonate, and both restored DNA relication to normal within 15 min of their being added to the medium. In addition, isopentenyladenine, like mevalonate, stimulated DNA synthesis specifically during the S phase of the cell cycle. Isopentenyladenine also reversed the inhibition of DNA synthesis caused by nalidixic acid, an antibiotic that does not inhibit cholesterol synthesis. These findings indicate that isopentenyladenine or a closely related derivative may mediate the regulatory role of mevalonate in DNA replication and suggest that such isoprenes may act upon DNA replication at a site common to that inhibited by nalidixic acid.

Essential role for mevalonate synthesis in DNA replication.

The relationship between 3-hydroxy-3-methylglutaryl (HMG) CoA reductase activity [mevalonate:NADP(+) oxidoreductase (CoA-acylating), EC 1.1.1.34] and DNA synthesis was studied in synchronized cultures of BHK-21 cells. During a 24-hr period of cell replication, two phases of accelerated thymidine incorporation into DNA corresponding to two S phases of the cell cycle occurred. A marked increase in activity of HMG CoA reductase was consistently observed at or just prior to each of these peaks of DNA synthesis. Moreover, when HMG CoA reductase activity was suppressed by the competitive inhibitor compactin, the normal S-phase burst of DNA synthesis was specifically and totally prevented. Finally, the compactin-induced inhibition of DNA synthesis could be completely reversed within minutes by the addition of mevalonate, the product of the HMG CoA reductase reaction. By contrast, addition of cholesterol-rich lipoproteins had no effect upon DNA synthesis in compactin-treated cells. These data demonstrate that HMG CoA reductase activity, and therefore the production of mevalonate, plays an essential role in the synthesis of DNA specifically during the S phase of the cell cycle. Moreover, the results indicate that this function of mevalonate in regulating DNA replication is independent of its conversion to cholesterol.

Isopentenyl adenine as a mediator of mevalonate-regulated DNA replication.

The model in Figure 6 may serve to explain these findings. Our previous studies have shown that mevalonic acid is essential for DNA replication and that this function is independent of its role as a cholesterol precursor. The present study suggests that the isoprene purine, isopentenyl adenine, or a related isoprene, may mediate this essential role of mevalonic acid in DNA replication. The fact that isopentenyl adenine will also reverse the inhibition of DNA synthesis caused by nalidixic acid (a compound that does not influence cholesterol synthesis and acts directly on DNA replication) suggests that isopentenyl adenine and nalidixic acid may act at a common reaction site in the process of DNA replication. Finally, these findings provide a possible mechanism by which the early steps of cholesterol synthesis may influence the rate of cell replication in normal cells. Coupled with our earlier observation that the feedback inhibition of mevalonate is lost in all malignant tumors, the present results also suggest that a derangement in these early steps of mevalonic acid metabolism may figure in the transformation of normal cells into cancer cells.

Tissue distribution of cholesterol feedback control in the guinea pig.

The level of cholesterol synthesis and the activity of the cholesterol feedback system were studied in tissue slices from a number of organs of the guinea pig. In contrast to the tissue distribution of sterol synthesis in the rat, liver slices of the guinea pig have a low rate of sterologenesis, with ileum and lung being the most active sterologenic tissues. More surprising, all tissues studied in the guinea pig, including lung, ileum, and brain, were shown to possess an active cholesterol feedback system. The basis for the widespread organ distribution of cholesterol feedback control in the guinea pig is probably the ability of the various tissues of the guinea pig to take up and concentrate exogenous cholesterol and is not the result of any inherent differences in the lipoprotein composition in this species.

De novo cholesterogenesis in pregnancy.

Hypercholesterolemia occurs during pregnancy in rats and human beings, beginning in the second trimester and increasing progressively throughout the remainder of pregnancy. The present study quantified de novo cholesterol synthesis in vivo and in vitro in pregnant animals using 3H2O as the substrate for measuring cholesterogenesis. In the third trimester, cholesterol synthesis by pregnant rat gut and carcass (all tissues not specifically studied) was not significantly different from that observed in controls. However, hepatic cholesterol synthesis was markedly stimulated in third trimester pregnant rats. Additionally, cholesterol synthesis in the placenta and fetus occurred at a very substantial rate. The magnitude of placental cholesterol synthesis was similar to that observed in the liver of control animals whereas fetal cholesterogenesis was considerably greater. Cholesterol feeding greatly suppressed hepatic cholesterol synthesis in both control and pregnant animals, so that the difference between control and pregnant animals was obliterated. Cholesterol feeding did not significantly affect the accumulation of newly synthesized cholesterol in either the placenta or fetus. In the Saguines fusciollis monkey, pregnancy similarly stimulated hepatic cholesterol synthesis, and the fetus and placenta were important sites of in vivo de novo cholesterogenesis.

Evidence for cAMP-independent inhibition of S-phase DNA synthesis by prostaglandins.

Two prostaglandins, prostaglandin E1 (PGE1) and prostaglandin B1 (PGB1), block S-phase DNA synthesis in synchronous cultured baby hamster kidney (BHK) cells. The prostaglandin inhibition of DNA synthesis does not appear to require elevated levels of cAMP. In BHK-21 cells that have been "desensitized" to prostaglandin stimulation of adenylate cyclase and, therefore, have control levels of cAMP, PGE1 retains its inhibitory effect on the incorporation of tritiated thymidine into DNA. When BHK cells are exposed to PGB1 (a prostaglandin that does not elicit a cAMP response), DNA synthesis is also blocked. In nonsynchronous cells exposed for 1 h to PGE and then incubated for 1 h with PGE removed, a rebound of DNA synthesis occurs, therefore providing evidence that a transient rise of cAMP in itself is not capable of causing a cascade of reactions that block the synthesis of DNA. In addition, the concentration of PGE required for inhibition of DNA synthesis is significantly less than that required for cAMP generation. Addition of 1 x 10(-8) M PGE to BHK cells can be shown to significantly inhibit DNA synthesis within 30 min, with half-maximal inhibition seen at 3 x 10(-7) M PGE. Cyclic AMP levels for controls were 4.9 +/- 0.2 and 4.6 +/- 0.1 for 1 x 10(-6) M PGE1. These findings suggest that the prostaglandins can act independently of cAMP at physiological concentrations; and, therefore, it is possible that prostaglandins have a physiological role in the control of cell growth during S-phase.

The effect of decreased plasma cholesterol concentration on circulatinga mevalonate metabolism in rats.

Circulating mevalonate is metabolized by two mechanisms: the sterol pathways leading to cholesterol and the shunt pathway resulting in CO2 production. The kidney is the chief site of circulating mevalonate metabolism by both pathways. The present study investigated the effect of plasma cholesterol concentration on circulating mevalonate metabolism. 3-Aminopyrazolo(3,4-d)pyrimidine and Triton WR 1339 were utilized to induce "functional hypocholesterolemia". An enhancement of both renal total nonsaponifiable lipid synthesis (36-43%) and cholesterol synthesis (42%) from circulatinga mevalonate was observed when "functional hypocholesterolemia" was induced by either compound. Hepatic total nonsaponifiable lipid synthesis from circulating mevalonate was not enhanced in the Triton-treated animals, but 4-aminopyrazolo(3,4-d)pyrimidine treatment increased accumulation of total labeled nonsaponifiable lipids and cholesterol. No increase in labeled total nonsaponifiable lipids or cholesterol in the carcass was observed after treatment with wither compound. "Functional hypocholesterolemia" reduced the shunt pathway of circulating mevalonate metabolism by approximately 30%. This reduction occurred in both the renal and extrarenal shunt pathways. These data indicate that plasma cholesterol concentration regulates the in vivo metabolism of circulating mevalonate in that hypocholesterolemia reduces the shunt pathway and stimulates sterologenesis, and effect chiefly localized to athe kidneys.

The effect of diabetes on mevalonate metabolism in the rat.

Previous studies have demonstrated a major difference in mevalonate metabolism by male and female rats by both the shunt and sterol pathways. This important sex difference was shown to be related to the presence of oestrogens and/or progesterone. In the present study we have investigated the effects of streptozotocin-induced diabetes on mevalonate metabolism. Firstly, insulin deficiency decreased the ability of the rats to oxidize mevalonate to carbon dioxide by the shunt pathway both in vivo and in vitro. This decrease was reversed by insulin treatment both in the intact animals as well as in tissue slices. Secondly, diabetes caused a marked increase in hepatic sterologenesis in the intact animal. This is the first demonstration that insulin plays significant regulatory role in both the shunt and sterol pathways of mevalonate metabolism.

Altered activation state of hydroxymethylglutaryl-coenzyme A reductase in liver tumors.

Extensive studies have demonstrated that the normal inhibition of cholesterol synthesis by cholesterol feeding is decreased in all hepatomas studied in vivo. This loss of the normal feedback regulation of cholesterol synthesis has been shown to be due to the failure of cholesterol ingestion to inhibit the activity of hydroxymethylglutaryl (HMG)-CoA reductase. The basis for this absence of feedback control of cholesterogenesis is unknown. Studies to date have not demonstrated structural or kinetic differences between the HMG-CoA reductase of normal liver and hepatoma. The present study, however, demonstrates significant differences in the activation state of HMG-CoA reductase from normal liver and hepatoma. In normal liver only approximately 10-20% of the microsomal HMG-CoA reductase is in the dephosphorylated, active form while 80-90% is in the phosphorylated, inactive state. In contrast, in three different Morris hepatomas in vivo, from 53 to 73% of the HMG-CoA reductase is in the active state. That the increased activation state in hepatomas is a property of tumor tissue and is not solely due to rapid growth is demonstrated by the fact that in both fetal and regenerating liver an enhanced activation state of HMG-CoA reductase is not observed. Additionally, preincubation with magnesium and ATP results in the inhibition of HMG-CoA reductase both in tumor and in liver. Presumably, this decrease in HMG-CoA reductase activity is due to the phosphorylation of the enzyme. Similarly, the preincubation of tumor and liver microsomes with phosphatase results in an increase in HMG-CoA reductase activity presumably by the dephosphorylation of the enzyme to its active form. The relationship between the altered activation state of HMG-CoA reductase in hepatomas and the reduction in the feedback regulation of this enzyme in liver tumors remains to be explored.

The in vitro metabolism of mevalonate by sterol and non-sterol pathways.

The metabolism of mevalonic acid by both sterol and non-sterol pathways has been evaluated in nine tissues of the rat. An in vitro estimation of the non-sterol, or "shunt", pathway of mevalonate metabolism was made possible by determining the conversion of [2-14C]mevalonate or [5-14C]mevalonate to 14CO2 in tissue slices. In confirmation of our previous results, the kidney was found to play a major role in the metabolism of mevalonate to sterols and sterol precursors. The shunt pathway accounted for a significant percentage of the mevalonate metabolized in kidney, ileum, spleen, lung and testes, but was of minor importance or undetectable in liver, brain, skin, and adipose tissue. Kidney, however, proved to be by far the most active tissue site of mevalonate metabolism by the shunt mechanism in that, on an average, renal tissue metabolized (R)-[14C]mevalonate over the non-sterol pathway at a rate that was 21 times that of any other tissue examined. These results indicate that the kidneys are of major importance in the metabolism of mevalonate by each of the known pathways of metabolism of this sterol precursor.

Activation of HMG-CoA reductase by microsomal phosphatase.

HMG-CoA reductase activity can be modulated by a reversible phosphorylation-dephosphorylation with the phosphorylated form of the enzyme being inactive and the dephosphorylated form, active. Phosphatases from diverse sources, including cytosol, have been shown to dephosphorylate and activate HMG-CoA reductase. The present study demonstrates phosphatase activity capable of activating HMG-CoA reductase that is associated with purified microsomes. The incubation of microsomes at 37 degrees C for 40 min results in a twofold stimulation of HMG-CoA reductase activity, and this stimulation is blocked by sodium fluoride or phosphate. The ability of microsomes to increase HMG-CoA reductase activity occurs regardless of whether microsomes are prepared by ultracentrifugation or calcium precipitation. Additionally, phosphatases capable of activating HMG-CoA reductase are present in both the smooth and rough endoplasmic reticulum. Freeze-thawing does not prevent microsomes from activating HMG-CoA reductase but preincubation results in a significant decrease in the ability of microsomes to increase HMG-CoA reductase activity. Thus, the present study demonstrates that purified liver microsomes contain phosphatase activity capable of activating HMG-CoA reductase.