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Biomedical subjects

A M Fogelman

Publications and source records attributed to A M Fogelman.

At least 109 records · Page 6Linked to original sources
PubMed1986

Inhibition of lysosomal protein degradation inhibits the basal degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

The effect of inhibiting lysosomal protein degradation on the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was determined using a mouse mammary cell line (TS-85) which expresses a temperature-sensitive mutation in the ubiquitin degradative pathway. Incubating cells for 18 hr in medium containing 20 mM NH4Cl did not alter total protein synthesis or cell growth, but it did inhibit the rate of total protein degradation by 19%, which is consistent with the known inhibitory effect of NH4Cl on lysosomal protein degradation. NH4Cl treatment also resulted in an increase (81% +/- 20) in HMG-CoA reductase activity. The increase in reductase activity was not correlated with changes in the phosphorylation state of the enzyme or with alteration in the relative rate of reductase synthesis. However, the basal degradation rate of the reductase was significantly inhibited, and after NH4Cl treatment, the half-life of the enzyme increased from 4.0 +/- 0.4 hr to 8.3 +/- 0.8 hr. The change in the rate of reductase degradation can account completely for the increase in reductase activity observed in NH4Cl-treated cells. The accelerated degradation of HMG-CoA reductase induced by 25-hydroxycholesterol treatment was not affected by either NH4Cl or by inactivation of the ubiquitin degradative pathway. Therefore, two different mechanisms may be responsible for the accelerated degradation and basal degradation of HMG-CoA reductase. The latter can be inhibited by NH4Cl and may imply that under basal conditions the enzyme may be degraded in lysosomes.

Ammonium Chloride↗
PubMed1986

The effect of glucagon on the synthesis and degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

Incubation of rat hepatocytes with glucagon results in a time- and dose-dependent decrease in the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase. We demonstrate, using immunoprecipitation of radiolabeled enzyme, that 10 nM glucagon inhibits the synthesis of the enzyme by approximately 50%, but that the apparent rate of degradation of the enzyme is not affected by the hormone. We also demonstrate that the intact reductase polypeptide contained phosphoserine. We conclude that glucagon inhibits the activity of the reductase by inhibition of enzyme synthesis.

Animals↗
PubMed1985

Transcriptional regulation of the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene in rat liver.

This study addresses whether transcriptional control of the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene in rat liver plays a role in determining the level of reductase mRNA. Isolated rat liver nuclei were allowed to elongate nascent RNA transcripts in the presence of [alpha-32P]CTP, and radiolabeled nuclear reductase RNA was quantitated by filter hybridization. Rats fed a diet supplemented with the drugs cholestyramine and mevinolin and having 20-60-fold induced levels of reductase mRNA exhibited levels of reductase transcription which were 20-fold higher than in rats fed an unsupplemented diet. Over 90% of the transcription of the reductase gene was inhibited by concentrations of alpha-amanitin which selectively inhibit RNA polymerase II. Administration of mevalonolactone (the end product of the reaction catalyzed by reductase) to rats fed cholestyramine and mevinolin caused an 80% decrease in the rate of reductase transcription by approximately 1 h. We conclude that under these conditions changes in reductase transcription are primarily responsible for the regulation of reductase mRNA levels.

Amanitins↗
PubMed1985

Receptor-mediated uptake of remnant lipoproteins by cholesterol-loaded human monocyte-macrophages.

Normal human monocyte-macrophages were cholesterol-loaded, and the rates of uptake and degradation of several lipoproteins were measured and compared to rates in control cells. Receptor activities for 125I-rabbit beta-very low density lipoproteins (beta-VLDL), 125I-human low density lipoprotein, and 125I-human chylomicrons were down-regulated in cholesterol-loaded cells; however, the rate of uptake and degradation of 125I-human chylomicron remnants was unchanged from control cells. Cholesterol-loaded alveolar macrophages from a Watanabe heritable hyperlipidemic rabbit, which lack low density lipoprotein receptors, showed receptor down-regulation for 125I-beta-VLDL but not for 125I-human chylomicron remnants. In addition to chylomicron remnants, apo-E-phospholipid complexes competed for 125I-chylomicron remnant uptake, but apo-A-I-phospholipid complexes did not. Chylomicrons competed for lipoprotein uptake in control cells but were not recognized under conditions of cholesterol loading. Chylomicron remnants and beta-VLDL were equally effective in competing for 125I-beta-VLDL and 125I-chylomicron remnant uptake in cholesterol-loaded macrophages. When normal human monocyte-macrophages were incubated in serum supplemented with chylomicron remnants, the cholesteryl ester content increased 4-fold over cells incubated in serum with low density lipoprotein added. We conclude: 1) specific lipoprotein receptor activity persists in cholesterol-loaded cells; 2) this receptor activity recognizes lipo-proteins (at least in part) by their apo-E content; and 3) cholesteryl ester accumulation can occur in monocyte-macrophages incubated with chylomicron remnants.

Animals↗
PubMed1985

Scavenger receptor-mediated recognition of maleyl bovine plasma albumin and the demaleylated protein in human monocyte macrophages.

Maleyl bovine plasma albumin competed on an equimolar basis with malondialdehyde low density lipoprotein (LDL) in suppressing the lysosomal hydrolysis of 125I-labeled malondialdehyde LDL mediated by the scavenger receptor of human monocyte macrophages. Maleyl bovine plasma albumin, in which 94% of the amino groups were modified, exhibited an anodic mobility in agarose electrophoresis 1.7 times that of the native protein. Incubation of maleyl bovine plasma albumin at pH 3.5 regenerated the free amino groups and restored the protein to the same electrophoretic mobility as native albumin. The demaleylated protein suppressed 75% of the hydrolysis of 125I-labeled malondialdehyde LDL and greater than 80% of 125I-labeled maleyl bovine plasma albumin. The ability of the demaleylated protein to compete was abolished after treatment with guanidine hydrochloride. Although ligands recognized by the scavenger receptor typically are anionic, we propose that addition of new negative charge achieved by maleylation, rather than directly forming the receptor binding site(s), induces conformational changes in albumin as a prerequisite to expression of the recognition domain(s). The altered conformation of the modified protein apparently persists after removal of the maleyl groups. We conclude that the primary sequence of albumin, rather than addition of new negative charge, provides the recognition determinant(s) essential for interaction of maleyl bovine plasma albumin with the scavenger receptor.

Animals↗
PubMed1985

Bacterial endotoxin selectively prevents the expression of scavenger-receptor activity on human monocyte-macrophages.

Concentrations of bacterial lipopolysaccharide (LPS) as low as 1 ng/ml suppressed the activity of the scavenger receptor on cultured human monocyte-macrophages. In contrast, concentrations of LPS as high as 100 ng/ml had no effect on the activity of the low density lipoprotein (LDL) receptor. LPS and purified forms of the lipid A moiety of LPS were effective in suppressing scavenger receptor activity. However, acid hydrolysis of the labile phosphate group of the native diphosphorylated lipid A to form monophosphoryl lipid A rendered the molecule ineffective in suppressing scavenger receptor activity. LPS at a concentration of 100 ng/ml had no effect on the secretion of apolipoprotein E, phagocytic activity, tumoricidal activity, or the protein content of monocyte-macrophages. We conclude that the active component of LPS that mediates suppression of scavenger receptor activity is diphosphoryl lipid A.

Apolipoproteins E↗
PubMed1985

Notes on the breeding of the WHHL rabbit: an animal model of familial hypercholesterolemia.

We describe in this study a reliable method for the breeding of the Watanabe heritable hyperlipidemic (WHHL) rabbit. Placing a male and a female WHHL rabbit in the same cage for the purpose of mating resulted in only two pregnancies out of a total of 227 mating attempts (0.9%). After manually assisting the rabbits, 15% of the matings resulted in pregnancies. When the female rabbits were injected with 40 I.U. of human chorionic gonadotropin within 1 hr of this procedure, 56% of the matings resulting in pregnancies. We feel that the inherent difficulty in breeding the WHHL rabbit, a model for the disease familial hypercholesterolemia, can be significantly overcome by the methods discussed in this report.

Animals↗
PubMed1984

Diurnal rhythm of rat liver mRNAs encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase. Correlation of functional and total mRNA levels with enzyme activity and protein.

Rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase exhibits a diurnal rhythm of activity which coincides with a diurnal rhythm of reductase protein and reductase mRNA levels. This diurnal rhythm of reductase activity, polypeptide mass, and mRNA exists in rats fed a normal diet (unsupplemented rat chow) and in rats fed a diet supplemented with cholestyramine plus or minus mevinolin. Levels of reductase protein were determined by 8 M urea/sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Reductase mRNA was measured by in vitro translation or blot hybridization of liver RNA. Functional reductase mRNA levels in rats fed a normal diet were approximately 10-fold higher during the middle of the dark cycle than during the middle of the light cycle. Maximum induction of functional reductase mRNA was observed in rats fed cholestyramine and mevinolin. This latter level was 157-fold higher than the level measured at the diurnal low point in rats fed a normal diet. Blot hybridization of liver RNA showed two predominant mRNAs of 4.6 and 4.2 kilobase pairs and a minor species at 6.9 kilobase pairs. These mRNAs exhibited a diurnal rhythm for rats on all three diets and reached peak levels during the 12-h dark period. These data indicate that the diurnal rhythm of reductase mass and activity is closely paralleled by the level of its mRNA.

Animals↗
PubMed1984

High density lipoproteins and lecithin dispersions increase the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase by increasing the rate of synthesis and decreasing the rate of degradation of the enzyme.

Incubation of rat hepatocytes, prepared from rats fed a normal diet, with either human high density lipoproteins or lecithin dispersions resulted in a dose-and time-dependent increase in the specific activity of 3-hydroxy-3-methylglutaryl-CoA reductase. Incubation of cells with both high density lipoprotein and lecithin dispersions resulted in an additive stimulation of enzymic activity. We used pulse and pulse-chase experiments, followed by immunoprecipitation of the radiolabeled reductase, to demonstrate that high density lipoproteins and lecithin dispersions resulted in increased rates of synthesis and decreased rates of degradation of the reductase. We also demonstrate, by the use of immunoblots, that the increased enzymic activity was paralleled by an equivalent increase in the mass of the reductase. Addition of human low density lipoprotein to rat hepatocytes resulted in a small decrease in both enzymic activity and enzyme synthesis.

Animals↗
PubMed1984

Effect of monocyte migration on low density lipoprotein transport across aortic endothelial cell monolayers.

Endothelial cell monolayers on polycarbonate filters present a barrier to low density lipoprotein (LDL) and albumin transport. These cells form a relatively tight monolayer as shown by measurements of electrical resistance across the monolayer (15 omega-cm2). Monocytes are able to migrate freely across the monolayers in response to chemotactic stimuli. Monocyte chemotaxis across the monolayer caused a marked increase in LDL and albumin transport across the monolayer in the direction of monocyte migration. However, transport in the opposite direction was not significantly increased. These results suggest that monocyte migration across the endothelium could lead to an increased LDL content of the intima.

Aorta↗
PubMed1983

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in avian myeloblasts. Mode of action of 25-hydroxycholesterol.

25-Hydroxycholesterol inhibits cholesterol biosynthesis by inhibiting the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Addition of 25-hydroxycholesterol to chicken myeloblasts caused a rapid inhibition of HMG-CoA reductase activity, producing approximately an 80% decrease in enzyme activity after 60 min. The mode of action of 25-hydroxycholesterol was determined by immunoprecipitating radiolabeled enzyme from 25-hydroxycholesterol-treated myeloblasts. The decline in enzyme activity due to addition of 25-hydroxycholesterol was not associated with increased levels of [32P]PO4 incorporation into the immunoprecipitated reductase polypeptide (Mr = 94,000). Hence, 25-hydroxycholesterol did not appear to regulate reductase activity by enzyme phosphorylation, as observed for other modulators of HMG-CoA reductase. However, 25-hydroxycholesterol was shown to inhibit reductase activity by causing a 350% increase in the relative rate of reductase degradation and a 72% decrease in the relative rate of reductase synthesis. These alterations in the rates of degradation and synthesis occurred rapidly (within 10-30 min after addition of 25-hydroxycholesterol) and can account completely for the 25-hydroxycholesterol-induced inhibition of enzyme activity. The rapid decline in the rate of synthesis of HMG-CoA reductase in 25-hydroxycholesterol-treated cells was not associated with concomitant changes in the levels of reductase mRNA; therefore, suggesting that 25-hydroxycholesterol must inhibit the rate of reductase synthesis by translational regulation. We also present evidence that mRNA purified from chicken myeloblasts codes for two reductase polypeptides of Mr = 94,000 and 102,000.

Animals↗
PubMed1983

Alterations in the rates of synthesis and degradation of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase produced by cholestyramine and mevinolin.

Hepatocytes were prepared from rats previously fed either a normal diet or diets containing cholestyramine or cholestyramine plus mevinolin. The rates of synthesis and degradation of HMG-CoA reductase were determined by immunoprecipitation of the enzyme from cells radiolabeled with [35S]methionine during standard pulse or pulse-chase experiments. In cells prepared from rats fed either a normal diet or a diet supplemented with cholestyramine or cholestyramine plus mevinolin, the relative rates of reductase synthesis were 0.08, 0.21, and 0.99%, respectively, of the rates of total cellular protein synthesis. The corresponding apparent half-lives of the enzyme were approximately 114 min, 153 min, and greater than 10 h. We propose that the elevated reductase activity observed in rats fed cholestyramine and mevinolin (Tanaka, R. D., Edwards, P. A., Lan, S.-F., Knoppel, E. M., and Fogelman, A. M. (1982) J. Lipid Res. 23, 1026-1031) is a result of both increased enzyme synthesis and stabilization of the enzyme. We demonstrate that enzyme stabilization is not an inherent property of the enzyme but is dependent on the presence of mevinolin. We propose that enzyme stabilization results from the decreased cellular levels of some product produced endogenously from mevalonate.

Animals↗
PubMed1983

Mevalonolactone inhibits the rate of synthesis and enhances the rate of degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in rat hepatocytes.

3-Hydroxy-3-methylglutaryl(HMG)-coenzyme A reductase purified from rat liver in the absence of protease inhibitors is composed of two distinct polypeptides of Mr = 51,000 and 52,500. Antibody raised to enzyme purified from rats fed a diet supplemented with cholestyramine and mevinolin inactivated HMG-CoA reductase. The antibody specifically precipitated a polypeptide of Mr = 94,000 from rat liver cells that had been previously incubated with [35S]methionine. The immunoprecipitation of the 35S-labeled polypeptide of Mr = 94,000 was prevented by addition of unlabeled pure HMG-CoA reductase (Mr = 51,000 and 52,500). Incubation of rat liver cells with mevalonolactone resulted in a decreased activity of HMG-CoA reductase and in a 40% decrease in the rate of incorporation of [35S]methionine into the immunoprecipitable reductase polypeptide of Mr = 94,000. In pulse-chase experiments, mevalonolactone enhanced the rate of degradation of the Mr = 94,000 polypeptide 3-fold. We propose that endogenous microsomal HMG-CoA reductase has a subunit of Mr = 94,000 and that the synthesis and degradation of this polypeptide are regulated by either mevalonolactone or, more likely, a product of mevalonolactone metabolism.

Animals↗
PubMed1983

Regulation of the uptake and degradation of beta-very low density lipoprotein in human monocyte macrophages.

In normal human monocyte macrophages 125I-labeled beta-migrating very low density lipoproteins (125I-beta-VLDL), isolated from the plasma of cholesterol-fed rabbits, and 125I-human low density lipoprotein (LDL) were degraded at similar rates at protein concentrations up to 50 micrograms/ml. The high affinity degradation of 125I-labeled human LDL saturated at approximately 50 micrograms/ml; however, 125I-labeled rabbit beta-VLDL high affinity degradation saturated at 100-120 micrograms/ml. The activity of the beta-VLDL receptor was 3-fold higher than LDL receptor activity on freshly isolated normal monocyte macrophages, but with time-in-culture both receptor activities decreased and were similar after several days. The degradations of both beta-VLDL and LDL were Ca2+ sensitive, were markedly down regulated by sterols, and were up regulated by preincubation of the cells in a lipoprotein-free medium. The beta-VLDL receptor is genetically distinct from the LDL receptor as indicated by its presence on monocyte macrophages from a familial hypercholesterolemic homozygote. Human thoracic duct lymph chylomicrons as well as lipoproteins of Sf 20-5000 from fat-fed normal subjects inhibited the degradation of 125I-labeled rabbit beta-VLDL as effectively as nonradioactive rabbit beta-VLDL. We conclude: 1) the beta-VLDL receptor is genetically distinct from the LDL receptor, and 2) intestinally derived human lipoproteins are recognized by the beta-VLDL receptor on macrophages.

Adult↗
PubMed1983

Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase mRNA levels in rat liver.

Addition of cholestyramine or cholestyramine plus mevinolin to the diet has been reported to increase the activity and mass of rat liver 3-hydroxy-3-methylglutaryl-CoA reductase. The present data show that these same dietary manipulations cause an induction of functional reductase mRNA. RNA was isolated from rat livers and added to an in vitro translation system, and the reductase was immunoprecipitated and analyzed by polyacrylamide gel electrophoresis under denaturing conditions. One protein was specifically immunoprecipitated and was found to have a Mr of 90,000 on 0.5 M urea/sodium dodecyl sulfate/polyacrylamide gels and a Mr of 94,000 on 8 M urea/sodium dodecyl sulfate/polyacrylamide gels. In animals fed rat chow supplemented with 5% cholestyramine and 0.1% mevinolin, reductase mRNA levels were 5.7-fold higher than in animals fed rat chow with 5% cholestyramine and were 16-fold higher than in animals fed rat chow with 5% cholestyramine and given mevalonolactone by stomach intubation. RNA isolated from animals fed a normal diet and killed at the nadir of the diurnal cycle of enzyme activity contained no detectable amounts of reductase mRNA as determined by this assay.

Animals↗
PubMed1983

Lymphokines secreted by an established lymphocyte line modulate receptor-mediated endocytosis in macrophages derived from human monocytes.

As little as 1 microliter of serum-free supernatant from Mo(t), an established lymphocyte line, when added to a 500-microliters incubation of macrophages derived from human monocytes, significantly decreased the receptor-mediated uptake and degradation of three cholesterol-rich molecules: low density lipoprotein (LDL); LDL complexed to dextran sulfate; and LDL modified by malondialdehyde (MDA-LDL). In contrast, the receptor-mediated uptake and degradation of mannosyl bovine serum albumin was increased three-fold. The Mo(t) supernatant did not contain competitive inhibitors of the cholesterol-rich ligands, and it did not alter macrophage receptor-independent endocytosis, protein synthesis, or phagocytosis of heat-killed yeast. The effect of the Mo(t) supernatant was specific for macrophages and was abolished by preincubation of the supernatant with trypsin, which indicates that the active substances are protein in nature. The decrease in the uptake and degradation of MDA-LDL induced by preincubating the macrophages with Mo(t) supernatant appeared to result from a decrease in the number of receptors for this ligand at the cell surface. The isolation of these lymphokines should offer new insights into macrophage receptor-mediated endocytosis, and may yield substances useful in preventing foam cell formation in atherosclerosis.

Cell Differentiation↗
PubMed1982

Lymphocyte-conditioned medium protects human monocyte-macrophages from cholesteryl ester accumulation.

Exposure of human monocyte-macrophages to as little as 50 microliters of cultured medium from lymphocytes stimulated by concanavalin A (Con A) resulted in a dramatic decrease in the activities of the low density lipoprotein (LDL) receptor pathway, the LDL-dextran sulfate pathway, and the scavenger receptor pathway. This effect was not seen when the monocyte-macrophages were exposed to culture medium from lymphocytes cultured without Con A or with Con A together with alpha-methyl mannoside or control medium without lymphocytes. The activity of 3-hydroxy-3-methyglutaryl-coenzyme A reductase also decreased in monocyte-macrophages exposed to culture medium from stimulated lymphocytes. Acyl-CoA:cholesterol O-acyltransferase activity, protein synthesis, protein content, phagocytosis of heat-killed yeast, and non-receptor-mediated endocytosis were not inhibited. Monocyte-macrophages exposed to malondialdehyde altered-LDL in the presence of stimulated lymphocyte culture medium accumulated substantially less cholesteryl esters than did cells in control medium. We propose that substances produced by stimulated lymphocytes may be useful in protecting macrophages from cholesteryl ester accumulation.

Cells, Cultured↗