Cholesterol Synthesis in Regenerating Peripheral Nerve Is Not Influenced by Serum Cholesterol Levels

Abstract: Following a nerve crush, cholesterol from degenerating myelin is retained within the nerve and reutilized for new myelin synthesis during nerve regeneration, apparently via a lipoprotein-mediated process. Because at least some serum components have access to the endoneurium of injured nerve, it has been suggested that serum lipoproteins are also significant contributors of cholesterol to Schwann cells during nerve regeneration. To test this hypothesis, serum cholesterol levels were reduced by >90% with 4-aminopyrazolopyrimidine, followed by measurement of the activity of the key regulatory enzyme in cholesterol synthesis, 3-hydroxy-3-methylglutaryl-CoA reductase. Treatment with 4-aminopyrazolopyrimidine caused a sevenfold increase in 3-hydroxy-3-methylglutaryl-CoA reductase activity in kidney but had no effect on the activity of this enzyme in either intact or regenerating sciatic nerve. These data indicate that serum-derived cholesterol is neither necessary for nor contributes significantly to myelin synthesis in regenerating nerve.     

Expression of HMGR and corresponding cholesterol content in tissues of two pig breeds

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) is an essential enzyme in cholesterol biosynthesis. To study the expression of HMGR and corresponding cholesterol content in liver, adipose and muscle, six Chinese local breed (Huai pig) and Landrace pigs were selected. The results indicated that significant differences of cholesterol content in adipose (P < 0.01), liver (P < 0.05) and muscle (P < 0.01) tissues were detected between pigs of differing genetic backgrounds. HMGR mRNA expression were noted for adipose, liver and muscle of the two vastly differing genetics. Moreover cholesterol content differed (P < 0.01) among tissues across breed. Likewise, HMGR mRNA expression was different between adipose and liver tissues, muscle and liver tissues in both breeds; however, no difference was noted between adipose and muscle tissues. Results from this study indicate that cholesterol content and HMGR mRNA expression are higher in Huai pig tissues suggesting this gene is expressed in a breed- and tissue-dependent manner in pigs. Understanding the causes of variation in HMGR gene expression may provide crucial information about cholesterol biosynthesis.     

Cholesterol Synthesis Is Down-Regulated During Regeneration of Peripheral Nerve

Abstract: The discovery of apolipoprotein E synthesis and secretion by injured peripheral nerve led to the hypothesis that endoneurial apolipoprotein E serves to salvage degenerating myelin cholesterol. This salvaged cholesterol could then be reutilized by Schwann cells during remyelination via uptake through low-density lipoprotein receptors. As a test of this hypothesis, we measured the rate of cholesterol synthesis in rat sciatic nerve endoneurium during development and at various times following a crush injury at 50 days of age. In control nerves [¹⁴C]acetate incorporation into cholesterol and 3-hydroxy-3-methylglutaryl-CoA reductase activity were closely linked throughout development, indicating that reductase activity in nerve, as in other tissues, is a good indicator of cholesterol's synthetic rate. In the crushed nerves cholesterol synthesis fell to nearly zero during the first week after the crush. There was a partial recovery during the second to fourth weeks, but unlike that of other lipids, cholesterol synthesis remained well below control nerve values throughout most of the 15-week post-crush period examined. Thus, cholesterol synthesis is at very low levels during the myelination of regenerating axons. These results are consistent with a receptor-mediated down-regulation of cholesterol synthesis by lipoproteins, and would be expected if Schwann cells were utilizing an external source of cholesterol as postulated above.     

Effects of cortisol or corticotropin administration on hepatic 3-hydroxy-3-methylglutaryl-CoA reductase activity and plasma lipids in the pregnant rat and fetuses

Pregnant rats were given pharmacological doses of cortisol or ACTH or no hormone from gestation day 9 to 19 and maternal and fetal hepatic 3-hydroxy-3-methylglutaryl-CoA reductase activity and plasma cholesterol studied on gestation day 20. Reductase activity was also studied in the maternal and fetal adrenal of the rats given cortisol or no hormone. Cortisol administration increased the maternal and fetal plasma cholesterol but had no effect on the hepatic active (phosphorylated) 3-hydroxy-3-methylglutaryl-CoA reductase activity when compared to untreated rats. Total (active + inactive) 3-hydroxy-3-methylglutaryl-CoA reductase activity, however, was reduced in maternal liver but not altered in the fetal liver by cortisol. The maternal cortisol treatment decreased the fetal, but not maternal, adrenal 3-hydroxy-3-methylglutaryl-CoA reductase total enzyme activity. The data support a hypothesis that utilization of plasma cholesterol for adrenal steroidogenesis may be an important determinant of plasma cholesterol homeostasis in the rat fetus. Maternal ACTH administration increased the foetal but not maternal plasma cholesterol, whilst active 3-hydroxy-3-methylglutaryl-CoA reductase activity was increased in the pregnant rat but not her fetuses. This result may suggest coordination of hepatic active reductase activity with adrenal cholesterol utilization in the pregnant rat. The reason for the fetal hypercholesterolaemia caused by ACTH, which is not known to cross the placenta, is uncertain. The studies, however, indicate that fetal cholesterol homeostasis and the rate limiting enzyme of cholesterol synthesis is influenced by maternal glucocorticoid administration.     

Tellurium-Induced Neuropathy: Metabolic Alterations Associated with Demyelination and Remyelination in Rat Sciatic Nerve

Rats fed a diet containing 1.25% elemental tellurium initiated on postnatal day 20 undergo a transient neuropathy characterized by synchronous demyelination of peripheral nerves. In sciatic nerve, the extent of demyelination was maximal after 5 days of tellurium exposure; there was a loss of 25% of the myelin, as assayed by concentration of myelin-specific P0 protein. Tellurium-induced alterations in the metabolic capacity of Schwann cells were examined by measuring the synthesis of myelin lipids in vitro in isolated sciatic nerve segments. Exposure to tellurium resulted in an early marked decrease of approximately 50% in overall incorporation of [14C]acetate into lipids, with a preferential depression in synthesis of cerebrosides, cholesterol, and ethanolamine plasmalogens (components enriched in myelin). Most dramatically, within 1 day of initiation of tellurium exposure, there was a profound increase in [14C]acetate-derived radioactivity in squalene; 23% of incorporated label was in this intermediate of cholesterol biosynthesis, compared to less than 0.5% in controls. In association with the remyelinating phase seen after 5 days of tellurium exposure, synthesis of myelin components gradually returned to normal levels. After 30 days, metabolic and morphologic alterations were no longer apparent. We suggest that the sequence of metabolic events in sciatic nerve following tellurium treatment initially involves inhibition of the conversion of squalene to 2,3-epoxysqualene, and that this block in the cholesterol biosynthesis pathway results, either directly or indirectly, in the inhibition of the synthesis of myelin components and breakdown of myelin.     

Tellurium Blocks Cholesterol Synthesis by Inhibiting Squalene Metabolism: Preferential Vulnerability to This Metabolic Block Leads to Peripheral Nervous System Demyelination

Inclusion of 1.1% elemental tellurium in the diet of postweanling rats produces a peripheral neuropathy due to a highly synchronous primary demyelination of sciatic nerve; this demyelination is followed closely by remyelination. Sciatic nerves from animals fed tellurium for various times were removed and incubated ex vivo for 1 h with [14C]acetate, and radioactivity incorporated into individual lipid classes was determined. In nerves from rats exposed to tellurium, there was a profound and selective block in the conversion of radioactive acetate to cholesterol. Another radioactive precursor, [3H]water, gave similar results. We suggest that tellurium feeding inhibits squalene epoxidase activity and that the consequent lack of cholesterol destabilizes myelin, thereby causing destruction of the larger internodes. Ex vivo incubation experiments were also carried out with liver slices. As with nerve, tellurium feeding caused accumulation in squalene of label from radioactive acetate, whereas labeling of cholesterol was greatly inhibited. Unexpectedly, however, incorporation of label from [3H]water into both squalene and cholesterol was increased. Relevant is the demonstration that liver was the primary site of bulk accumulation of squalene, which accounted for 10% of liver dry weight at 5 days. Thus, accumulation of squalene (and other mechanisms, possibly including up-regulation of cholesterol biosynthetic pathways) drives squalene epoxidase activity at normal levels in liver even in the presence of inhibitors of this enzyme. This is reflected by continuing incorporation of [3H]water into cholesterol; incorporation of this precursor takes place at many of the postsqualene biosynthetic steps for sterol formation. [14C]Acetate entering the sterol pathway before squalene in liver is greatly diluted in specific activity when it reaches the large squalene pool, and thus increased squalene epoxidase activity does not transfer significant 14C label to sterols. In contrast to the situation with liver, synthesis of sterols is markedly depressed in sciatic nerve, and squalene does not accumulate to high levels.     

Hormonal Regulation of the mRNA Encoding the Ketogenic Enzyme Mitochondrial 3-Hydroxy-3-Methylglutaryl-CoA Synthase in Neonatal Primary Cultures of Cortical Astrocytes and Meningeal Fibroblasts

Abstract: We have previously identified cerebellum to contain significantly higher levels, compared with other brain regions, of the mRNA encoding the key ketogenic enzyme mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mHS). In this report, we extend these observations, using primary cultures of cerebellar astrocytes and cerebellar granule neurons, and show that mHS mRNA was not readily detected in these cell types, suggesting that other cerebellar cell types account for mHS mRNA abundances observed in cerebellum. In contrast, we report, for the first time, the ready detection of mHS mRNA together with the mRNAs encoding the remaining enzymes of the 3-hydroxy-3-methylglutaryl-CoA cycle, namely, mitochondrial acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglutaryl-CoA lyase, in primary cultures of neonatal meningeal fibroblasts. Based on observations of the effects of fetal calf serum in the culture medium and the documented effects of various hormones on mHS mRNA levels in liver, we show that the glucocorticoid hydrocortisone effects a selective fourfold increase in mHS mRNA abundances in both neonatal meningeal fibroblasts and neonatal cortical astrocytes cultured in a serum-free/hormone-free medium.     

Specificity of the effect of dietary cholesterol on rat liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme a reductase activity.

Dietary cholesterol lowers the activity of rat liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase without affecting various other liver microsomal enzymes. This is consistent with a specific regulatory mechanism and distinguishes the action of cholesterol on 3-hydroxy-3-methylglutaryl-CoA reductase from that of at least one other stimulus known to affect this enzyme.     

A new method for assaying rat liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity and its application in a study of the effect of dietary cholesterol on this effect of dietary cholesterol on this enzyme.

A new method suitable for measuring rat liver 3-hydroxy-3-methylglutaryl-CoA reductase activity is described and its advantages over methods previously available are discussed. An accurate time course was measured for the inhibition of liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase activity by dietary cholesterol; this enzyme was affected 1 1/4 h after the rats began to consume a cholesterol-rich diet. In this experiment there was no correlation between concentrations of microsomal cholesterol ester and the activity of 3-hydroxy-3-methylglutary-CoA reductase.     

Specificity of the effect of dietary cholesterol on rat liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity (Short Communication)

Dietary cholesterol lowers the activity of rat liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase without affecting various other liver microsomal enzymes. This is consistent with a specific regulatory mechanism and distinguishes the action of cholesterol on 3-hydroxy-3-methylglutaryl-CoA reductase from that of at least one other stimulus known to affect this enzyme.     

Rat Brain 3-Hydroxy-3-Methylglutaryl-CoA Reductase: Subcellular Distribution and Cold Lability

Abstract: Data are provided indicating that the rat brain 3-hydroxy-3-methyl-glutaryl-CoA reductase is similar to the enzyme from other tissues as far as diurnal rythmicity, cold lability and half-life measurements at 0°C are concerned. The enzyme activity in the brain decreased with age of the animals. Subcellular fractionation studies demonstrate that while 77% of the activity was associated with the microsomal fraction, 19% of the enzyme activity was recovered in the mitochondrial fraction. The possible function of such a mitochondrially located 3-hydroxy-3-methylglutaryl-CoA reductase in rat brain is discussed.     

Control of 3-hydroxy-3-methylglutaryl coenzyme A reductase by endogenously synthesized sterols in vitro and in vivo.

Isolated rat hepatocytes converted mevalonolactone into sterol intermediates and fatty acids 6- to 8-fold faster than mevalonate salt at concentrations less than 6 X 10(-4) M. Incubation of hepatocytes for 3 h normally results in induction of 3-hydroxy-3-methylglutaryl-CoA reductase. This increase in enzyme activity was inhibited by mevalonolactone and by mevalonate salt; at each concentration between 6 X 10(-4) M and 6 X 10(-8) M the lactone was a more effective inhibitor than the salt. The increase in enzyme activity was completely prevented by 6 X 10(-4) M lactone, and at this concentration the cells synthesized from the lactone an amount of sterol per hour which approximated that leavingthe cells in the same period. Administration of mevalonolactone to intact rats resulted in a dose-dependent inhibition of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase activity. At the highest dose (400 mg of (RS)-mevalonolactone/200 g of rat) enzyme activities declined 85% within 45 min and were still suppressed below normals after 28 h. Mevalonolactone treatment resulted in increases in liver cholesterol content and in the cholesterol ester concentration of liver microsomes. The results demonstrate that the activity of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase can be controlled by the rate of endogenous sterol synthesis both in vitro and in vivo.     

Activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in brains of adult and 7-day-old rats.

Rat brain contains 3-hydroxy-3-methylglutaryl-CoA reductase activity, but this enzyme is far more active in 7-day-old brain than in adult brain. This difference may partly explain why cholesterol biosynthesis is more rapid in growing than in adult rat brain.     

Defective regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in a somatic cell mutant.

A somatic cell mutant of the CHO-K1 cell selected to be resistant to the killing effects of 25-hydroxycholesterol in the absence of cholesterol is shown to be defective in the inhibition of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity by 25-hydroxycholesterol, cholesterol, and lipoproteins, thus maintaining the enzyme activity found in cells in the absence of exogenous sterol constitutively. The mutants phenotype is shown to be dominant with respect to the wild type. Actinomycin D and cycloheximide prevent the increase of HMG-CoA reductase activity that occurs in the CHO-K1 cell when cholesterol is removed from medium. Degradation of the enzyme, measured during inhibition of protein synthesis by cycloheximide, occurs at the same rate in the mutant as in the wild type. Kinetic studies indicate that the Km for two substrates, the activation energy, and a break in the Arrhenius plot are the same for HMG-CoA reductase determined in wild type and mutant cells. From these studies it is concluded that the mutant is defective in the regulation of synthesis of HMG-CoA reductase. Of the four processes which determine cellular cholesterol levels: biosynthesis, esterification, efflux, and uptake, only biosynthesis is altered, demonstrating that these processes are not co-ordinately controlled as has been suggested previously.     

Activation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase during high fat diet feeding

The liver plays a central role in regulating cholesterol homeostasis. High fat diets have been shown to induce obesity and hyperlipidemia. Despite considerable advances in our understanding of cholesterol metabolism, the regulation of liver cholesterol biosynthesis in response to high fat diet feeding has not been fully addressed. The aim of the present study was to investigate mechanisms by which a high fat diet caused activation of liver 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) leading to increased cholesterol biosynthesis. Mice were fed a high fat diet (60% kcal fat) for 5 weeks. High fat diet feeding induced weight gain and elevated lipid levels (total cholesterol and triglyceride) in both the liver and serum. Despite cholesterol accumulation in the liver, there was a significant increase in hepatic HMG-CoA reductase mRNA and protein expression as well as enzyme activity. The DNA binding activity of sterol regulatory element binding protein (SREBP)-2 and specific protein 1 (Sp1) were also increased in the liver of mice fed a high fat diet. To validate the in vivo findings, HepG2 cells were treated with palmitic acid. Such a treatment activated SREBP-2 as well as increased the mRNA and enzyme activity of HMG-CoA reductase leading to intracellular cholesterol accumulation. Inhibition of Sp1 by siRNA transfection abolished palmitic acid-induced SREBP-2 and HMG-CoA reductase mRNA expression. These results suggest that Sp1-mediated SREBP-2 activation contributes to high fat diet induced HMG-CoA reductase activation and increased cholesterol biosynthesis. This may play a role in liver cholesterol accumulation and hypercholesterolemia.     

Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in rat liver and Morris hepatomas 5123C, 9618A and 5123t.c.

Characteristics of 3-hydroxy-3-methylglutaryl-CoA reductase from normal liver, Morris hepatomas 5123C, 5123t.c. and 9618A, and host liver were studied. Animals were fed on control and 5%-cholesterol diets. Microsomal membranes from all tissues were found to accumulate cholesterol after 3 days on the 5%-cholesterol diet. The enzyme of the tumours showed no feedback inhibition by dietary cholesterol, and that of host liver gave a variable response, whereas that of control liver was constantly inhibited by 90% or more. Arrhenius-plot analysis was conducted on the microsomal enzyme isolated from the various tissues. Control animals showed that the phase transition present at 27 degrees C was removed when animals were fed on 5%-cholesterol diet for 12 h. The hepatomas failed to show this change even after 3 days of 5%-cholesterol diet and a significant increase in microsomal cholesterol. This failure to remove the break in Arrhenius plots also occurred in host liver, even though enzyme inhibition occurred. The reason why hepatomas fail to regulate 3-hydroxy-3-methylglutaryl-CoA reductase activity in response to dietary cholesterol may be a decreased membrane-enzyme interaction.     

Regulation of rat liver cell 3-hydroxy-3-methylglutaryl coenzyme A reductase by methoxypolyoxyethylated cholesterol

A water-soluble derivative of cholesterol, methoxypolyoxyethylated (MPOE) cholesterol, has been synthesized and used to study the regulation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the key regulatory enzyme in cholesterol biosynthesis. MPOE cholesterol causes a specific, rapid and linear decline in HMG-CoA reductase in cultured rat liver cells. MPOE cholesterol is not a direct allosteric inhibitor of HMG-CoA reductase, does not appear to regulate HMG-CoA reductase through changes in membrane environment, and does not change the phosphorylation state and level of activation of rat liver cell HMG-CoA reductase. In order to confirm our data, which were consistent with a model in which MPOE cholesterol regulates the amount of HMG-CoA reductase and not its activity, we made direct measurements of reductase mRNA levels. The decline in HMG-CoA reductase in MPOE cholesterol-treated rat liver cells is preceded by the rapid disappearance of HMG-CoA reductase mRNA. As a water-soluble cholesterol derivative, MPOE cholesterol represents a useful model compound for studies on the regulation of the level of HMG-CoA reductase and its cognate mRNA.     

Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in mouse uterine epithelial cells.

The regulation of 3-hydroxy-3-methylglutaryl-CoA reductase was studied in mouse uterine epithelium. The enzyme was rapidly inactivated during incubation with ATP/Mg2+ in vitro, and could be re-activated by incubation with partially purified rat liver phosphoprotein phosphatase. Enzyme activity was rapidly inhibited by mevalonate injection in vivo to approx. 30% of control. The percentage of total enzyme active in vivo was measured by inclusion of NaF in the isolation buffers. The percentage of enzyme active in vivo 18 h after stimulation by oestrogens remained at approx. 25% after inhibition of activity by mevalonate injection, cholesterol feeding or progesterone pretreatment. However, 9 h after oestrogen stimulation, cholesterol feeding inhibited enzyme activity to 57% of control, 94% of which was in the active form. We conclude that, although all components for a reversible phosphorylative regulation of 3-hydroxy-3-methylglutaryl-CoA reductase activity are present in uterine epithelial cells, a role in the rapid changes in epithelial enzyme activity has not been demonstrated.