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PubMed1994

Effect of simvastatin, an inhibitor of hydroxy-methylglutaryl coenzyme A reductase, on the growth of human Ito cells.

During hepatic fibrogenesis, Ito cells proliferate, acquire a myofibroblastlike phenotype and synthesize increased amounts of extracellular matrix components. In this study, we have assessed the effects of simvastatin, an inhibitor of hydroxy-methylglutaryl-coenzyme A reductase, on the growth of human myofibroblastlike Ito cells. Cells were grown from explants of normal human liver and characterized by a positive staining for desmin and smooth muscle alpha-actin. Simvastatin (0.1 to 10 mumol/L) induced a marked dose-dependent decrease of [3H]thymidine incorporation in human Ito cells, whether stimulated by human serum or by purified growth factors. Simvastatin-induced inhibition of DNA synthesis was confirmed by nuclear autoradiography and was not explained by a cytotoxic effect. The growth inhibitory effect of simvastatin was specifically due to inhibition of hydroxy-methylglutaryl-coenzyme A reductase because it was overcome by addition of mevalonic acid, the product of the enzymatic reaction. The reduction in [3H]thymidine incorporation was not affected by supplementation of culture medium with purified cholesterol-low-density lipoprotein or isopentenyl adenine. It was partially reversed by addition of farnesol. These results show that simvastatin decreases the growth of human Ito cells, independently of its effect on cholesterol synthesis. This decrease may be due in part either to reduced farnesylation of proteins involved in growth factor signaling pathway or to inhibition of N-linked protein glycosylation. Whether this effect exists in vivo and could thus lead to a parallel decrease of fibrosis deposition within the liver requires further study.

Actins↗

PubMed1993

Modulation of insulin secretion from normal rat islets by inhibitors of the post-translational modifications of GTP-binding proteins.

Many GTP-binding proteins (GBPs) are modified by mevalonic acid (MVA)-dependent isoprenylation, carboxyl methylation or palmitoylation. The effects of inhibitors of these processes on insulin release were studied. Intact pancreatic islets were shown to synthesize and metabolize MVA and to prenylate several candidate proteins. Culture with lovastatin (to inhibit synthesis of endogenous MVA) caused the accumulation in the cytosol of low-M(r) GBPs (labelled by the [alpha-32P]GTP overlay technique), suggesting a disturbance of membrane association. Concomitantly, lovastatin pretreatment reduced glucose-induced insulin release by about 50%; co-provision of 100-200 microM MVA totally prevented this effect. Perillic acid, a purported inhibitor of the prenylation of small GBPs, also markedly reduced glucose-induced insulin secretion. Furthermore, both N-acetyl-S-trans,trans-farnesyl-L-cysteine (AFC), which inhibited the base-labile carboxyl methylation of GBPs in islets or in transformed beta-cells, and cerulenic acid, an inhibitor of protein palmitoylation, also reduced nutrient-induced secretion; an inactive analogue of AFC (which did not inhibit carboxyl methylation in islets) had no effect on secretion. In contrast with nutrients, the effects of agonists that induce secretion by directly activating distal components in signal transduction (such as a phorbol ester or mastoparan) were either unaffected or enhanced by lovastatin or AFC. These data are compatible with the hypothesis that post-translational modifications are required for one or more stimulatory GBPs to promote proximal step(s) in fuel-induced insulin secretion, whereas one or more inhibitory GBPs might reduce secretion at a more distal locus.

Acetates↗

PubMed1993

Reversal of ras-induced inhibition of gap-junctional intercellular communication, transformation, and tumorigenesis by lovastatin.

The plasma-membrane association and transforming activity of the ras oncoprotein p21 are dependent upon posttranslational farnesylation. Farnesyl synthesis and p21 ras farnesylation are inhibited by hydroxymethylglutaryl-CoA reductase inhibitors such as lovastatin. In this study, we examined whether lovastatin could reverse the transformed phenotype of a v-Ha-ras-transformed rat liver epithelial cell line (WB-ras cells) and if changes were associated with the enhancement of gap-junctional intercellular communication (GJIC). WB-ras cells grow in soft agar, have reduced GJIC, and are highly tumorigenic. Membrane association of p21 ras in these cells was inhibited after in vitro treatment with lovastatin (0.1-0.5 microM) for 48 h. Concomitantly, the cells displayed a more normal morphology, decreased growth in soft agar, and enhanced GJIC. These changes were prevented by cotreatment with mevalonic acid. The morphology and GJIC of rat liver epithelial cells transformed with other oncogenes (src, neu, and raf/myc) were not affected by lovastatin. Intrahepatic WB-ras tumors were induced in male rats by intraportal-vein injection of WB-ras cells. The size and DNA labeling index of these tumors were decreased approximately 75% by administration of lovastatin (5 mg/kg orally twice daily for 2 wk). These results suggest that lovastatin reversed the transformed phenotype of WB-ras cells by inhibiting p21 ras plasma membrane association. Furthermore, the concomitant enhancement of GJIC in lovastatin-treated cells suggests a role for reduced GJIC in the expression of the transformed phenotype.

Animals↗

PubMed1980

Differences in the characteristics of inhibition of lymphocyte stimulation by 25-hydroxycholesterol and by the immunoregulatory serum lipoprotein LDL-In.

Suppression of peripheral blood lymphocyte phytohemagglutinin-stimulated 3H-thymidine uptake in vitro by serum lipoprotein fractions enriched in the immunoregulatory low density lipoprotein (LDL-In) and by 25-hydroxycholesterol was compared to determine whether the biologically active constituent of LDL-In might be an oxygenated sterol. Dose titrations indicated that 25-hydroxycholesterol was 100 times more potent than LDL-In on the basis of total sterol. Both were similar in that maximal inhibitory activity was evident when they were present in the cultures for at least 24 hr before mitogen stimulation. However, the suppression of 3H-thymidine uptake by 25-hydroxycholesterol required the continuous presence of the sterol, whereas equivalent suppression by LDL-In was irreversible and did not require the presence of the lipoprotein beyond the preincubation period. 25-Hydroxycholesterol-mediated suppression of 3H-thymidine uptake was mitigated by the addition of cholesterol or mevalonic acid to the lymphocyte cultures, consistent with an effect on sterol biosynthesis, whereas, LDL-In mediated suppression was not. Finally, when chloroquine was used to inhibit lymphocyte lysosomal hydrolysis, which is required for the intracellular degradation and release of cholesterol esters from internalized low density lipoprotein, the phenotypic expression of LDL-In mediated suppression was not altered, indicating that this known pathway of regulation of cellular function by low density lipoproteins is not the primary mode of action of LDL-In. These data indicated that the mode of suppression of mitogen-stimulated thymidine uptake by serum lipoprotein fractions enriched in LDL-In differs from that produced by 25-hydroxycholesterol, and that LDL-In suppression does not appear to result from inhibition of intracellular cholesterol biosynthesis.

Cell Division↗

PubMedDate not supplied

[The role of vitamin E in the biosynthesis of the isoprenoid region of the ubiquinone molecule in the rat liver].

A decrease in the CoA and acetyl-CoA amount in the rat liver tissue by 34.8 and 29.4%, respectively, as well as inhibition of the biosynthesis rate of mevalonic acid from [I-14C] acetyl-CoA in the postmitochondrial liver fraction by 17.9% as compared to the control are found against a background of E-hypovitaminosis. The last change is not associated with the inhibition of the 3-oxy-3-methylglutaryl-CoA-reductase activity and may not be one of reasons which cause the biosynthesis disturbances in the isoprenoid part of the ubiquinone molecule in this organ. alpha-Tocopherol activates the ubiquinone biosynthesis from [2-14C] sodium acetate in the liver of rats with E-hypovitaminosis under conditions of 30 min preincubation and is not efficient when added in combination with actinomycin D. Probably, such an effect of alpha-tocopherol is realized at the level of RNA synthesis and is associated with the biosynthesis activation of short-living RNA.

Acetyl Coenzyme A↗

PubMed1995

Toxicity of oxidized low density lipoproteins for vascular smooth muscle cells and partial protection by antioxidants.

Oxidized low density lipoprotein (oxLDL) is known to be toxic to a variety of cell types, but relatively little is known about the toxic effects of oxLDL on vascular smooth muscle cells (SMC). We found that LDL oxidized by incubation with 5 microM cupric ions was toxic to cultured porcine SMC when administered at concentrations of 25 micrograms protein/ml and higher. The toxicity was demonstrated whether cells were proliferating or not, and was more evident in the presence of 0.4% lipoprotein-deficient serum than in 10%. Because of recent evidence that 7-ketocholesterol and 7-hydroxycholesterol are toxic species in copper-oxidized LDL, inhibition of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase was hypothesized as a mechanism of toxicity. However, mevalonic acid, the product of this enzyme, failed to protect against the toxicity of either oxLDL or the pure oxysterols. Alpha-tocopherol, alpha-tocopherol acetate, probucol, butylated hydroxytoluene, and deferoxamine provided partial protection to SMC exposed to oxLDL. These results suggested a toxic role for newly initiated lipid peroxidation, either in cells or in media oxLDL. Cellular lipid peroxidation appeared more likely, since no further oxidation of media oxLDL was demonstrated in the presence or absence of antioxidants. Overall, the results suggest that toxicity of copper-oxidized LDL for SMC is multifactorial and differs from the previously described toxicity of iron-oxidized LDL for fibroblasts.

Animals↗

PubMed1997

Upregulation of cholesterol synthesis after acute reduction of low density lipoprotein by apheresis in normocholesterolaemic subjects: evidence for a threshold effect.

The influence of low density lipoproteins (LDL) in the plasma on the regulation of cholesterol biosynthesis is not clear. We studied the changes in plasma mevalonic acid (MVA) concentration and the lathosterol/cholesterol (L/C) ratio, which are well established indices of whole body cholesterol synthesis, in four normocholesterolaemic subjects after each had undergone LDL apheresis on two occasions. LDL apheresis of 75% of the calculated plasma volume reduced LDL-cholesterol by 44% to 1.5 +/- 0.2 mmol/l without changing plasma MVA levels or L/C ratios. Apheresis of 125% of the calculated plasma volume decreased plasma LDL-cholesterol by 69% to 0.9 +/- 0.2 mmol/l, with significant increases in plasma MVA and L/C ratio on the day after the procedure. These results imply that LDL-cholesterol is an integral part of the sterol regulatory pool and suggest that plasma levels cannot be lowered below 1-1.4 mmol/l in normal subjects without upregulating cholesterol biosynthesis.

Adult↗

PubMed1997

Upregulation of low density lipoprotein receptor by gemfibrozil, a hypolipidemic agent, in human hepatoma cells through stabilization of mRNA transcripts.

Gemfibrozil reduces the plasmal levels of cholesterol and triglyceride in patients with hyperlipidemia by a mechanism that is not well understood. The present study evaluated the effect of gemfibrozil on the LDL receptor in human hepatoma cells compared with that of pravastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Exposure to gemfibrozil, 40 mumol/L, for 3 days increased the binding of 125I-LDL to the surface of three lines of human hepatoma cell, HepG2, HuH7, and HLE by 1.5- to 2.0-fold. Similar findings were observed with pravastatin. Scatchard analysis with 125I-LDL indicated an increased number of LDL receptors on the cell surface of HepG2 cells when treated with gemfibrozil and pravastatin. However, the gemfibrozil-treated cells exhibited no increase in the binding of 125I-epidermal growth factor (EGF). Gemfibrozil increased the levels of LDL receptor mRNA and protein in HepG2 cells. The increase in LDL receptor activity induced by pravastatin was abolished by concomitant administration of mevalonic acid, 770 mumol/L. This effect was not seen with gemfibrozil, suggesting the mechanism differs for the two lipid-lowering drugs. To determine whether this increase in mRNA was due to transcriptional activation, we prepared HepG2 cells transfected with an LDL receptor promoter-reporter construct that contained a sterol regulatory element. The expression of LDL receptor regulated by the sterol regulatory element was increased by pravastatin, but not by gemfibrozil. We evaluated the stability of the mRNA in the presence of actinomycin D to explain the increase in the LDL receptor mRNA. Gemfibrozil prolonged the half-life of the mRNA for LDL receptor but not that for the EGF receptor. Stabilization of the LDL receptor mRNA is suggested to be the novel mode of action of gemfibrozil.

Carcinoma, Hepatocellular↗

PubMed1992

Isoprenoid metabolism is required for stimulation of the respiratory burst oxidase of HL-60 cells.

The formation of oxygen radicals by phagocytic cells occurs through the activation of a multiple-component NADPH oxidase system. An unidentified low molecular weight GTP-binding protein has been proposed to modulate the activity of the NADPH oxidase. The low molecular weight GTP-binding proteins undergo posttranslational processing, including an initial covalent incorporation of an isoprenyl group. To test whether such an isoprenylation reaction might be required for the activity of the oxidase, we utilized compactin and lovastatin as inhibitors of the isoprenylation pathway. Treatment of DMSO-differentiated HL-60 cells with compactin produced a concentration-dependent inhibition of O2- formation in response to FMLP or phorbol myristate acetate. Cell viability was not affected nor was normal differentiation of the HL-60 cells into a neutrophil-like cell. The inhibitory effect of compactin was specifically prevented by addition of exogenous mevalonic acid to the HL-60 cells, indicating that the inhibitory effects of the drug were due to blockade of the pathway leading to isoprenoid synthesis. Addition of cholesterol, ubiquinone, or dolichol, which are also downstream products of the isoprenoid pathway, did not override the inhibitory effects of the drug. Subcellular fractions were prepared from compactin-treated cells, and the location of the compactin-sensitive factor was determined by complementation analysis in a cell-free NADPH oxidase system. The inhibited factor was localized to the HL-60 cytosol. These data suggest that an isoprenoid pathway intermediate is necessary for activation of the phagocyte NADPH oxidase. This is likely to represent the requirement for an isoprenoid moiety in the posttranslational modification of a low molecular weight GTP-binding protein. Our studies provide support for the involvement of such a low molecular weight GTP-binding protein in NADPH oxidase activation.

Butadienes↗

PubMed1989

Coordinate regulation of 3-hydroxy-3-methylglutaryl-coenzyme A synthase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase, and prenyltransferase synthesis but not degradation in HepG2 cells.

Human hepatoma HepG2 cells were used to demonstrate coordinate regulation of three enzymes of cholesterol synthesis under a variety of conditions. Addition of either delipidized serum and mevinolin or low density lipoprotein, 25-hydroxycholesterol, or mevalonic acid to HepG2 cells resulted in rapid changes both in the levels of the mRNAs and in the rates of synthesis of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase, HMG-CoA reductase, and farnesyl pyrophosphate synthetase (prenyltranferase). In all cases, the changes in mRNA levels were paralleled by changes in the rates of specific protein synthesis. Pulse-chase techniques were used to determine the half-lives of all three proteins. Addition of low density lipoprotein to the media during the chase increased the rate of degradation of HMG-CoA reductase 4.6-fold but had no affect on the half-lives of HMG-CoA synthase or prenyltransferase. Therefore, we conclude that the coordinate regulation of these three enzymes under a variety of conditions occurs at the level of enzyme synthesis and not at the level of protein stability.

Cell Line↗

PubMed2003

Immunomodulatory effects of HMG-CoA reductase inhibitors.

3-Hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors, or statins, are competitive inhibitors of the rate-limiting enzyme in cholesterol synthesis. Several clinical trials have shown a marked reduction in cholesterol levels associated with decreased cardiovascular mortality in patients treated with statins. However, more recent observations have suggested that the clinical benefits of statins may be, at least in part, independent of the effect of statins on cholesterol synthesis. These so-called pleiotropic or cholesterol-independent effects of statins could be the result of reduction in the formation of intermediaries in the mevalonate pathway as statins, by inhibiting L-mevalonic acid synthesis, also prevent the production of isoprenoids in the cholesterol biosynthetic pathway. Isoprenoids serve as important lipid attachments for the posttranslational modification of a variety of proteins such as small GTP-binding proteins of the Ras superfamily implicated in intracellular signaling. The list of different pleitropic effects of statins is still growing and includes, among others, direct effects of statins on modulating endothelial function, decreasing oxidative stress and, more recently, anti-inflammatory and immunomodulatory actions of statins. For instance, statins decrease T cell activation, the recruitment of inflammatory cells into atherosclerotic lesions, and inhibit IFN-gamma expression of MHC II on antigen-presenting cells. This review article summarizes the anti-inflammatory and immunomodulatory effects of statins and thus provides a new rationale to use statins as a new class of immunosuppressive agents.

Adjuvants, Immunologic↗

PubMed1997

Role of small GTP-binding proteins in lovastatin-induced cataracts.

PURPOSE: To investigate the biochemical mechanisms involved in the cataract induced by lovastatin, a commonly used cholesterol-lowering agent. METHODS: The effects of lovastatin on lens transparency and on lens epithelial cell proliferation and structure have been investigated using organ-cultured rat lenses and cultured epithelial cells from human and rabbit lenses, respectively. Lens histologic and morphologic changes were recorded microscopically. Small GTP-binding protein profiles were determined by [alpha-32P] GTP overlay assays. RESULTS: Rat lenses organ cultured for 7 days with lovastatin, a 3-hydroxy-3-methylglutaryl CoA reductase inhibitor, developed frank subcapsular opacity. Lens epithelial cells (both human and rabbit) demonstrated extensive morphologic changes and inhibition of proliferation when treated with lovastatin. Histologic sections of lovastatin-treated lenses showed partial to complete degeneration of the central epithelium, distortion of elongating epithelial cells, and extensive vacuole formation in the equatorial regions of the cortex. Supplementation of the medium with DL-mevalonic acid (a precursor of isoprenoids whose synthesis is inhibited by lovastatin) prevented the lovastatin-induced changes in whole lenses or in lens epithelial cell cultures, whereas supplementation with cholesterol had no such effect. GTP-binding proteins accumulated in the soluble fractions of lovastatin-treated lens epithelial cells. This was consistent with a blockade in isoprenylation preventing normal association with membranes. CONCLUSIONS: The findings suggest that impairment of the function of small GTP-binding proteins, due to a lovastatin-induced blockade in their isoprenylation, affects lens cell structure and proliferation in tissue culture and induces lens opacity in organ culture. These findings are consistent with the proposed roles of small GTP-binding proteins as molecular switches that regulate fundamental cellular processes, including growth, differentiation, and maintenance of cell structure.

Animals↗

PubMed2001

Clinical and molecular variability in childhood periodic fever with hyperimmunoglobulinaemia D.

OBJECTIVES: The hyperimmunoglobulinaemia D and periodic fever syndrome (HIDS) was found recently to be caused by a deficiency of mevalonate kinase (MK). The aim of this study was to examine whether a relationship exists between the clinical expression of HIDS and the extent of MK deficiency. METHODS: The medical records of children diagnosed with HIDS were reviewed for clinical features and serum immunoglobulin values. The mevalonic acid excretion in urine and MK enzyme activity in patients' cells were measured and the cDNA of the MVK gene was sequenced. RESULTS: Fifteen patients with recurrent fever and raised serum immunoglobulin (Ig) D were included. Their clinical features varied. Eleven patients had a deficiency of MK, caused by mutations in the MVK gene. One mutation (V377I) was common to all 11 patients. Nine patients were compound heterozygotes for V377I and various other MVK mutations. There was no apparent relationship between the observed mutations and the clinical features. Surprisingly, four boys had normal MK activity and no MVK mutations. CONCLUSIONS: Most HIDS patients have mutations in the MVK gene. The clinical variability observed cannot be explained by genotypic differences. Periodic fever and elevated IgD can result from other, still unknown, causes. Hence, testing for MK deficiency is necessary in patients with unexplained periodic fever.

Child↗

PubMed1969

Biosynthesis of squalene and cholesterol by cell-free extracts of adult rat brain.

Cell-free extracts of adult rat brain incubated with mevalonic acid-2-(14)C synthesize (14)C-labeled nonsaponifiable fractions consisting largely of squalene-(14)C. If the cofactor concentrations of the incubation medium are adjusted, much of the squalene can be induced to undergo turnover, with a resultant increase in (14)C-labeled digitonin-precipitable sterols, which include a small amount of cholesterol. The synthesis of labeled sterols is markedly increased in the presence of Mg(++) and depressed by nicotinamide. ATP, NADH, GSH, and glucose-6-phosphate are required for optimal synthesis of digitonin-precipitable material but, unlike Mg(++), are not essential. The cofactor-adjusted extracts also synthesize a complex ester mixture containing, in addition to cholesterol-(14)C, several compounds less polar than cholesterol. The biosynthesis of cholesterol in the extracts is a slow process; at least 12 hr of incubation is required for maximal sterol biosynthesis. A complex mixture of hydrocarbons accompanies squalene in the incubated extracts.

Adenosine Triphosphate↗

PubMed1998

Cholesterol synthesis is increased in mixed hyperlipidaemia.

We showed previously that hypertriglyceridaemia, but not hypercholesterolaemia, is correlated with increases in cholesterol synthesis and apolipoprotein B secretion in patients with secondary hypertriglyceridaemia. The aim of the present study was to compare the rate of cholesterol synthesis, using fasting plasma mevalonic acid (MVA) as an index, in patients with primary mixed hyperlipidaemia (type IIb phenotype, n=45) and primary hypercholesterolaemia (type IIa phenotype, n=92). LDL cholesterol was significantly higher in types IIa (6.38+/-0.18 mmol/l) and IIb (5.89+/-0.25 mmol/l) compared to 40 normolipidaemic controls (2. 99+/-0.1 mmol/l, P<0.0001), whereas serum triglyceride was higher in type IIb (2.62 (range 2.2-3.0) mmol/l) than type IIa (1.22 (range 0. 85-1.60) mmol/l, P<0.001) and controls (0.90 (range 0.68-1.24) mmol/l, P<0.001). Similarly, MVA was higher in type IIb (7.0+/-0.46 ng/ml) than IIa (5.6+/-0.23 ng/ml, P<0.0) and controls (5.6+/-0.36 ng/ml, P<0.05). Plasma MVA correlated positively with serum triglyceride (r=0.22, P=0.004) and negatively with LDL cholesterol (r=-0.21, P=0.014). These results are in accordance with previous observations that VLDL-apolipoprotein B secretion and cholesterol synthesis are linked and demonstrate that the latter is increased in mixed hyperlipidaemia.

Adult↗

PubMed1993

Effects of growth factors on cell cycle arrest in dolichyl phosphate-depleted cultures.

Previously we showed that CHO cell growth is arrested in the G1 or G0 phase within 24 h after the biosynthesis of mevalonic acid is blocked. The growth-limiting factor under these conditions appeared to be dolichyl phosphate or one of its glycosylated derivatives with consequent decrease in the synthesis of N-linked glycoproteins (Doyle, J.W., and A.A. Kandutsch, 1988, J. Cell Physiol. 137:133-140; Kabakoff, B., J.W. Doyle, and A.A. Kandutsch, 1990, Arch. Biochem. Biophys. 276:382-389). We show herein that cell surface glycoproteins are depleted in the inhibited cultures and that growth arrest is delayed when supraphysiological concentrations of insulin, insulin-like growth factor-1 (IGF-1) and bFGF are added to the culture medium. Apparently an elevated level of a growth factor increases the length of time during which a threshold level of occupied receptor is maintained as the number of glycosylated receptor molecules declines. The results support the idea that cellular levels of dolichyl phosphate and its derivatives may limit cell division by controlling the numbers of functional receptors for growth factors and of other glycoproteins on the cell surface.

Animals↗

PubMed1986

An improved enzymatic synthesis of labeled gibberellin A12-aldehyde and gibberellin A12.

The biochemical synthesis of labeled gibberellin A12-7-aldehyde (GA12ald) and and gibberellin A12 (GA12) from labeled R,S-mevalonic acid (MVA) using cell-free extracts from pumpkin (Cucurbita maxima) endosperm has been improved over the previously reported procedure. Three major improvements were developing a one-step HPLC procedure to isolate GA12ald and GA12 in radiochemically pure form; adjusting the pH of the reaction mix to pH 6.9 which increased the GA12ald/GA12 ratio over that at pH 7.8 by ca. 18-fold while reducing the combined yield of these two compounds by less than 17%; and developing a technique that permitted sampling of the fruits without interrupting growth; this doubled the fraction of extracts with "high activity." Conversion of MVA into GA12ald or GA12 displayed Michaelis-Menten kinetics with half-maximal synthesis at 0.4 mM MVA. Four-hour incubations afforded the highest yields from 0.25 mM MVA. Up to 15 and 7% of the MVA was incorporated into GA12ald and GA12, respectively. About one-quarter of the extracts incorporated at least 10% of the 0.25 mM MVA into GA12ald. One pumpkin fruit can provide sufficient endosperm to synthesize ca. 0.6 mumol of GA12ald.

Cell-Free System↗

PubMed2000

Manganese-bilirubin effect on cholesterol accumulation in rat bile canalicular membranes.

Manganese-bilirubin (Mn-BR)-induced cholestasis in rats is associated with altered lipid composition of various hepatic subcellular fractions. Increased bile canalicular (BCM) cholesterol content in Mn-BR cholestasis and the intracellular source of the accumulating cholesterol were investigated. To label the total hepatic cholesterol pool, male Sprague-Dawley rats were given ip 3H-cholesterol, followed 18 h later by 2-14C-mevalonic acid (a precursor of cholesterol synthesis). To induce cholestasis, manganese (Mn, 4.5 mg/kg) and bilirubin (BR, 25 mg/kg) were injected iv; animals were killed 30 min after BR injection; canalicular and sinusoidal membranes, microsomes, mitochondria, and cytosol were isolated. Total cholesterol content of each fraction was determined by spectrophotometric techniques as well as radiolabeled techniques. In Mn-BR cholestasis, the total cholesterol concentrations of BCM and cytosol were significantly increased. Also, the contribution of 14C-labeled cholesterol (newly synthesized cholesterol) was enhanced in all isolated cellular fractions. The results are consistent with the hypothesis that accumulation of newly synthesized cholesterol in BCM is involved in Mn-BR cholestasis. An enhanced rate of synthesis of cholesterol, however, does not appear to be the causal event, as the activity of HMG-CoA reductase (rate-limiting enzyme in cholesterol synthesis), assessed in vitro, was decreased following Mn-BR treatment. Treatment with the Mn-BR combination may affect other aspects of intracellular cholesterol dynamics.

Animals↗