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Biomedical subjects

A M Fogelman

Publications and source records attributed to A M Fogelman.

At least 127 records · Page 7Linked to original sources
PubMed1982

Specificity of receptor-mediated recognition of malondialdehyde-modified low density lipoproteins.

Blood-borne human monocytes and macrophages derived from human monocytes in vitro express an active low density lipoprotein (LDL) receptor and an active receptor for negatively charged proteins, the scavenger receptor. When less than 15% of the lysine residues of human LDL were modified by malondialdehyde while the lipoprotein was in solution, recognition and uptake of the modified lipoprotein occurred via the LDL receptor. Further modification resulted in threshold recognition and uptake by the scavenger receptor with concomitant loss of recognition by the LDL receptor. The rate of degradation via the LDL receptor pathway was inversely related to the degree of modification whereas that mediated by the scavenger receptor was independent of the extent of incorporation of malondialdehyde once threshold recognition was achieved. In contrast to the interaction of LDL with malondialdehyde in solution, modification of less than 15% of the lysine residues of LDL adsorbed to heparin-Sepharose resulted in recognition and uptake by the scavenger receptor. The scavenger receptor-mediated uptake of malondialdehyde-modified LDL may be dependent on formation of recognition sites involving specific modified lysine residues or changes in the conformation of LDL induced by neutralization of specific lysine residues of the apoB polypeptides or both.

Apolipoproteins↗
PubMed1982

The effect of cholestyramine and Mevinolin on the diurnal cycle of rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase.

Rats were fed powdered rat chow or a rat chow diet containing 5% cholestyramine or 5% cholestyramine with Mevinolin (112 mg/100 g food, 200 mg/kg body weight per day). The specific activity of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase was determined at different times during the diurnal cycle of the enzyme. Animals fed cholestyramine had higher specific activities of HMG-CoA reductase at all time points tested when compared to controls. The specific activity at the peak in the diurnal cycle was approximately 8-fold higher in cholestyramine-treated animals. Rats administered the cholestyramine-Mevinolin diet had higher specific activities of the enzyme than either cholestyramine-treated or control animals. In the cholestyramine-Mevinolin-treated animals the peak in the diurnal cycle was shifted to D-12 (12th hour of the dark cycle) and the specific activity at this point was approximately 133-fold greater than the basal (L-6) activity in control animals. Optimal conditions for immunotitration studies were determined such that valid conclusions could be drawn from these data. Based on immunotitration experiments, the increased hepatic HMG-CoA reductase activity in cholestyramine-treated animals resulted in part from a 3-fold activation of the enzyme, while the increased specific activity in the cholestyramine-Mevinolin-treated animals was due solely to increased enzyme mass. Hence the administration of Mevinolin blocked the activation of the enzyme induced by feeding cholestyramine alone. This was confirmed by purifying the enzyme to apparent homogeneity from cholestyramine-Mevinolin-treated animals; the specific activity of this enzyme was 4,000-7,700 nmol NADPH oxidized/min per mg protein. Cholestyramine-Mevinolin treatment affords a novel system for generating milligram quantities of rat hepatic HMG-CoA reductase.-Tanaka, R. D., P. A. Edwards, S. Lan, E. M. Knöppel, and A. M. Fogelman. The effect of cholestyramine and Mevinolin on the diurnal cycle of rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase.

Animals↗
PubMed1982

Purification of 3-hydroxy-3-methylglutaryl coenzyme A reductase from human liver.

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase from human liver was purified to homogeneity. Electrophoresis of the purified human enzyme on non-denaturing gels revealed a single protein-staining band that co-migrated with reductase activity and could be selectively removed by treatment with cross-reacting antibody prepared to the purified reductase from rat liver. On SDS polyacrylamide gel electrophoresis, the purified reductase showed only one protein-staining band corresponding to a molecular weight of approximately 52,000. Based on the sedimentation rate during sucrose density gradient centrifugation, most of the enzyme from crude solubilized preparations had an apparent molecular weight of approximately 104,000. The human reductase, like the rat liver enzyme, was dimeric and composed of subunits of approximately 50,000 molecular weight.

Animals↗
PubMed1981

The metabolism of native and malondialdehyde-altered low density lipoproteins by human monocyte-macrophages.

We have recently shown that cultured human monocyte-macrophages degraded 125I-labeled low density lipoprotein (125I-nativeee-LDL) by a saturable high-affinity process with maximal velocity at 25-30 microgram protein/ml (Fogelman et al., 1980, Proc. Nat. Acad. Sci. USA. 77:2214-2218). We now describe studies of the binding of 125I-native-LDL at 4 degrees C and the effects of chloroquine, Ca2+ concentration, and reductive methylation on high-affinity 125I-native-LDL degradation that indicate that native-LDL is processed by the monocyte-macrophages via the classic LDL receptor pathway. The high-affinity degradation of 125I-native-LDL increased substantially when monocyte-macrophages were exposed to the lipoprotein deficient-fraction of serum (LPDS) for periods as brief as 4 hours, and was 25-fold greater than that of lymphocytes. Freshly isolated monocytes that had never been exposed to LPDS also demonstrated high-affinity degradation of 125I-nativ-LDL. When these monocytes were cultured for 7 days in a medium containing native-LDL at a concentration (186 microgram protein/ml) greatly in excess of that apparently needed to saturate the high-affinity process, there was more than a 10-fold increase in 125I-native-LDL high-affinity degradation. LDL modified by treatment with malondialdehyde was processed by a second high-affinity cell surface receptor that appears identical to the "scavenger" receptor that processes acetylated LDL (Goldstein, et al., 1979, Proc. Nat. Acad. Sci. USA. 76: 333-337).

Cells, Cultured↗
PubMed1981

Factors regulating the activities of the low density lipoprotein receptor and the scavenger receptor on human monocyte-macrophages.

Improved techniques of cell isolation resulted in 90 to 100 million monocytes from a single donor. Addition of low density lipoprotein (LDL) to to cultures of these cells resulted in the down regulation of LDL receptor activity. Addition of malondialdehyde-altered LDL. which enters the cell through a receptor for negatively charged proteins (the scavenger receptor), produced an even greater down regulation of the LDL receptor, indicating that both receptors are present on the same cell. Within hours of adherence of the cells, there was a dramatic decrease in the activity of both receptors. LDL receptor activity was highest during the first week in culture and then declined, despite the maintenance of a constant LDL concentration in the medium. Scavenger receptor activity surpassed LDL receptor activity by the 6th day and was maximally expressed during the second week. Increasing cell density resulted in a slight increase in the activity of the LDL receptor and a dramatic increase in scavenger receptor activity. Insulin had no significant effect on either receptor. Removing serum from the culture medium for 48 hr resulted in a 3.5-fold increase in LDL receptor activity and a 2-fold decrease in scavenger receptor activity. Twenty-four hr after the cells were re-exposed to serum, the activities of both receptors essentially returned to base line. Heat-inactivation of serum was associated with an increased cholesteryl ester content of the cells and depressed receptor activities. Scavenger receptor activity appears related to the maturation of monocytes into macrophages and is promoted by increasing cell density and serum factors that are heat labile.

Cell Membrane↗
PubMed1980

Smooth microsomes. a trap for cholesteryl ester formed in hepatic microsomes.

Acyl-CoA:cholesterol acyltransferase was found predominantly (85%) in RNA-rich microsomes, the rest being in RNA-poor and smooth microsomes. However, the esterified cholesterol concentration of smooth microsomes was 2-fold greater than that of RNA-rich microsomes, suggesting the possibility of an interaction between RNA-rich and smooth microsomes. The distribution of cholesteryl ester between microsome subfractions was examined after incubation of a mixture of RNA-rich and smooth microsomes with [1-14C]palmitoyl-CoA. Based upon specific acyl-CoA:cholesterol acyltransferase activities of the individual fractions, only 31 +/- 3% of the total cholesteryl ester radioactivity should have been found in the smooth component. However, the smooth microsomes contained 54 +/- 3% (p < 0.01) of the radioactive cholesteryl esters. The entrapment of radioactive cholesteryl ester in the smooth microsomes could not be accounted for by passive transfer of cholesteryl ester from RNA-rich microsomes to smooth microsomes. It is proposed that cholesterol in the smooth microsomal membranes may have been esterified by acyl-CoA:cholesterol acyltrasferase located on the surface of RNA-rich microsomes with the resulting cholesteryl ester retained in the smooth microsomes. This hypothesis was strengthened by the observation that acyl-CoA:cholesterol acyl-transferase was located on the cytoplasmic surface of the RNA-rich microsomal vesicle.

Acyltransferases↗
PubMed1980

Properties of purified rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase and regulation of enzyme activity.

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase from rat liver microsomes has been purified to apparent homogeneity with recoveries of approximately 50%. The enzyme obtained from rats fed a diet supplemented with cholestyramine had specific activities of approximately 21,500 nmol of NADPH oxidized/min/mg of protein. After amino acid analysis a specific activity of 31,000 nmol of NADPH oxidized/min/mg of amino acyl mass was obtained. The s20,w for HMG-CoA reductase was 6.14 S and the Stokes radius was .39 nm. The molecular weight of the enzyme was 104,000 and the enzyme subunit after sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 52,000. Antibodies prepared against the homogeneous enzyme specifically precipitated HMG-CoA reductase from crude and pure fractions of the enzyme. Incubation of rat hepatocytes for 3 h in the presence of lecithin dispersions, compactin, or rat serum resulted in significant increases in the specific activity of the microsomal bound reductase. Immunotitrations indicated that in all cases these increases were associated with an activated form of the reductase. However activation of the enzyme accounted for only a small percentage of the total increase in enzyme activity; the vast majority of the increase was apparently due to an increase in the number of enzyme molecules. In contrast, when hepatocytes were incubated with mevalonolactone the lower enzyme activity which resulted was primarily due to inactivation of the enzyme with little change in the number of enzyme molecules. Immunotitrations of microsomes obtained from rats killed at the nadir or peak of the diurnal rhythm of 3-hydroxy-3-methylglutaryl-CoA reductase indicated that the rhythm results both from enzyme activation and an increased number of reductase molecules.

Amino Acids↗
PubMed1980

Malondialdehyde alteration of low density lipoproteins leads to cholesteryl ester accumulation in human monocyte-macrophages.

Glutaraldehyde treatment of (125)I-labeled low density lipoprotein ((125)I-native-LDL) produced a modified LDL ((125)I-glut-LDL) with a molecular weight of 10 x 10(6) or more. Malondialdehyde treatment of (125)I-native-LDL produced a product ((125)I-MDA-LDL) with a molecular weight not appreciably different from that of the original lipoprotein. However, the electrophoretic mobility of MDA-LDL indicated a more negative charge than native-LDL. (125)I-MDA-LDL was degraded by two processes: a high-affinity saturable process with maximal velocity at 10-15 mug of protein per ml and a slower, nonsaturable process. The degradation of (125)I-MDA-LDL was readily inhibited by increasing concentrations of nonradioactive MDA-LDL but was not inhibited by acetylated LDL or native-LDL even at concentrations as high as 1600 mug of protein per ml. After exposure of native-LDL to blood platelet aggregation and release in vitro, 1.73 +/- 0.19 nmol of malondialdehyde per mg of LDL protein was bound to the platelet-modified-LDL. No detectable malondialdehyde was recovered from native-LDL that had been treated identically except that the platelets were omitted from the reaction mixture. After incubation with glut-LDL, MDA-LDL, or platelet-modified-LDL for 3 days, human monocyte-macrophages showed a dramatic increase in cholesteryl ester content whereas the cholesteryl ester content of cells incubated with the same concentration of native-LDL did not. Based on these experiments we propose that modification of native-LDL may be a prerequisite to the accumulation of cholesteryl esters within the cells of the atherosclerotic reaction. We further hypothesize that one modification of LDL in vivo may result from malondialdehyde which is released from blood platelets or is produced by lipid peroxidation at the site of arterial injury.

Arteriosclerosis↗
PubMed1980

A deficiency of mixed function oxidase activities in the cholesterol biosynthetic pathway of human granulocytes.

Highly purified human granulocytes synthesize [14C]farnesol and [14C]squalene but not [14C]sterols from [14C]mevalonic acid. Dimethylsulfoxide was found to be an excellent vehicle for carrying [3H]squalene-2, 3-oxide into the intact cells. The granulocytes synthesized [3H]lanosterol from this substrate, but were unable to further process the newly synthesized lanosterol along the cholesterol biosynthetic pathway. In contrast, intact lymphocytes and monocytes were able to synthesize radioactive cholesterol from either [14C]mevalonic acid or [3H]squalene-2,3-oxide. These results indicate that normal human granulocytes have retained squalene-2,3-oxide-lanosterol cyclase activity but have lost squalene epoxidase activity and at least one other mixed function oxidase activity that is required to transform lanosterol into cholesterol. These results may provide an explanation for the accumulation of farnesol and squalene that has been previously observed in populations of mixed leukocytes (Fogelman, A. M., Edmond, J., Seager, J., and Popják, G. (1975) J. Biol. Chem. 250: 2045-2055 (1); Burns, C. P., Welshman, I. R., Edmond, J., and Spector A. A. (1979) Biochim. Biophys. Acta 572: 345-351) (12).

Cholesterol↗
PubMed1979

Purification and properties of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase.

3-Hydroxy-3-methylglutaryl coenzyme A reductase has been purified from rat liver microsomes with a recovery of approx. 25%. The enzyme was homogeneous on gel electrophoresis and enzyme activity comigrated with the single protein band. The molecular weight of the reductase determined by gel filtration on Sephadex G-200 was 200,000. SDS-polyacrylamide gel electrophoresis gave a subunit molecular weight of 52,000 +/- 2000, suggesting that the enzyme was a tetramer. The specific activities of the purified enzyme obtained from rats fed diets containing 0% or 5% cholestyramine were 11,303 and 19,584 nmol NADPH oxidized/min per mg protein, respectively. The reductase showed unique binding properties to Cibacron Blue Sepharose; the enzyme was bound to the Cibacron Blue via the binding sites for both substrates, NADPH and (S)-3-hydroxy-3-methylglutaryl coenzyme A. Antibodies prepared against purified reductase inactivated 100% of the soluble and at least 91% of the microsomal enzyme activity. Immunotitrations of solubilized enzyme obtained from normal and cholestyramine-fed rats indicated that cholestyramine feeding both increased the amount of enzyme protein and resulted in enzyme activation. Administration of increasing amounts of mevalonolactone to rats decreased the equivalence point obtained from immunotitration studies with solubilized enzyme. These data indicate that the antibody cross-reacts with the inactive enzyme formed after mevalonolactone treatment.

Animals↗
PubMed1979

The effect of glucagon, norepinephrine, and dibutyryl cyclic AMP on cholesterol efflux and on the activity of 3-hydroxy-3-methylglutaryl CoA reductase in rat hepatocytes.

Incubation of rat hepatocytes for 3 hours in a sterol-free medium containing 1.5% albumin resulted in efflux of cellular sterol into the medium and an increased activity of 3-hydroxy-3-methylglutaryl CoA reductase. The secretion of cholesterol was inhibited when cells were incubated with glucagon, norepinephrine, or dibutyryl cyclic AMP. Glucagon and dibutyryl cyclic AMP also inhibited the induction of HMG-CoA reductase. Norepinephrine treatment resulted in a decrease in the synthesis and secretion of proteins but caused an increase in reductase activity. Insulin treatment had no effect either on reductase activity or on sterol efflux from rat hepatocytes.

Animals↗
PubMed1979

Determination of mevalonate in blood plasma in man and rat. Mevalonate "tolerance" tests in man.

A method is described for the determination of mevalonate in ultrafiltrates of blood plasma. The method depends on the phosphorylation of mevalonate with [gamma-32P]ATP and mevalonate kinase to 5-[32P]phosphomevalonate, and the subsequent isolation of the 5-[32P]phosphomevalonate together with known amounts of added 5-phospho[14C]mevalonate by ion-exchange chromatography. The 32P/14C ratio in the isolated 5-phosphomevalonate is a linear function of the mevalonate content of the samples. The smallest amount that can be determined is 1--2 pmol. The fasting level in human plasma varied between 20 and 75 pmol/ml. Human red blood cells absorb mevalonate from plasma relatively slowly; their maximum storage capacity is about 1.3 pmol/10(6) red cells. An oral and intravenous "mevalonate tolerance test" in man is described that can be carried out with 200 and 30 mumol. respectively, of the unlabeled (RS)-mevalonate in a 70-kg man. Beer and wine contain mevalonate at a concentration of 3--8 microns, too low to provide a significant amount of mevalonate even for heavy drinkers. The mevalonate content of the plasma from the blood of the vena cava inferior of male rats varied between 81 and 502 pmol/ml and is positively related to the levels of liver 3-hydroxy-3-methylgultaryl-CoA reductase, suggesting that the liver is probably the main source of mevalonate circulating in blood. The plasma of renal venous blood contained only 33--85% as much mevalonate as the arterial plasma.

Animals↗
PubMed1979

Improved methods for the solubilization and assay of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase.

A method for solubilizing HMG-CoA reductase is described that reproducibly yielded approximately 190% of the activity assayed in rat liver microsomes. Optimal solubilization occurred when microsomal membranes were frozen at a fixed concentration, thawed, homogenized in a buffer containing 50% glycerol, and incubated at 37 degrees C for 60 minutes. A rapid spectrophotometric assay of the reductase has been developed and the optimal conditions defined. Using this assay, the kinetics were determined for HMG-CoA reductase purified to a specific activity of 17,400 nmol NADPH oxidized per minute per mg protein.

Animals↗
PubMed1979

Separation of and cholesterol synthesis by human lymphocytes and monocytes.

We have devised techniques for the isolation of human monocytes which do not require the adherence of the cells to a surface. In 15 consecutive experiments using density-gradient and counterflow centrifugations, a population of mononuclear cells that was 75 +/- 11% monocytes was obtained within 2 hours of venipuncture. These cells had never been pelleted and represented approximately three-fourths of the monocytes that had been present in the whole blood. In another 22 consecutive experiments using sedimentation in gelatin followed by counterflow and density-gradient centrifugations, a population of lymphocytes that was 99.5 +/- 0.5% pure and a population of monocytes that was 94 +/- 3% pure were obtained within 3 hours of venipuncture. When these freshly isolated cells were incubated in the lipoprotein-deficient fraction of serum (d > 1.21 g/ml) or in solvent-extracted serum, the monocytes incorporated 10-20 times more [2-(14)C]acetate into sterols than did the lymphocytes. Monocytes were seen to constitute between 6 and 46% of the mononuclear cells isolated from normal individuals by the usual density-gradient centrifugation of whole blood on Ficoll-Hypaque. We conclude that future studies of cholesterol metabolism utilizing human mononuclear cells must take into account this large variation in the percentage of monocytes and their disproportionately greater activity during short-term incubations in media that induce sterol synthesis.-Fogelman, A. M., J. Seager, M. Hokom, and P. A. Edwards. Separation of and cholesterol synthesis by human lymphocytes and monocytes.

Adult↗
PubMed1977

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.

Alcohol Oxidoreductases↗
PubMed1977

Mechanism of induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase in human leukocytes.

Incubation of leukocytes in buffer alone devoid of lipoproteins does not lead to the induction of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, but incubation of these cells in lipid-depleted serum, abetaliprproteinemic serum, or lipoprotein-deficient serum (d greater than 1.21) leads to sterol loss from the cells and the activation of sterol synthesis from acetate. The latter was shown previously to be proportional to the HMG-CoA reductase levels in the cells (Fogelman, A. M., Edmond, J., Seager, J., and Popják, G. (1975)J. Biol. Chem. 250, 2045-2055). Sterol loss occurs from normal and heterozygous familial hypercholesterolemic leukocytes within 15 min in lipid-depleted serum. Since induction of HMG-CoA reductase activity is not detectable until after the leukocytes have been incubated in the lipid-depleted serum for at least 3 h (Fogelman et al., see above), sterol loss clearly precedes the induction of the enzyme. In six out of six experiments, the nonisotopic sterol content of leukocytes incubated in lipid-depleted serum was equal to or lower than that of the same leukocytes incubated in full serum. This occurred at a time when the leukocytes in the lipid-depleted serum were incorporating 4 to 5 times more [14C]acetate into sterols than the same leukocytes in full serum. This strongly suggests that the induction of the reductase was a compensatory mechanism for sterol loss. Incubation of leukocytes in buffer, or buffer plus lecithin dispersions, or buffer plus albumin did not lead to sterol loss or induction of the reductase, but incubation in buffer and albumin together with lecithin dispersions caused sterol loss into the medium and the activation of sterol synthesis from acetate. It is concluded that a phospholipid-protein-cell interaction, which produces sterol loss, is necessary to induce the reductase in leukocytes. A close correlation between sterol loss and total sterol synthesis (a function of HMG-CoA reductase activity) was demonstrated in normal and heterozygous leukocytes incubated in a variety of incubation media (r = 0.95; p less than 0.005). Heterozygous leukocytes taken fresh from the blood contained no more cholesterol than the leukocytes of their age and sex-matched controls, despite the marked difference in their serum cholesterol concentrations. It is proposed that the abnormality in familial hypercholesterolemia can be accounted for by an abnormal efflux of cholesterol from heterozygous cells.

Adolescent↗