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Biomedical subjects

D Steinberg

Publications and source records attributed to D Steinberg.

At least 235 records · Page 13Linked to original sources
PubMed1981

Enhanced macrophage degradation of low density lipoprotein previously incubated with cultured endothelial cells: recognition by receptors for acetylated low density lipoproteins.

Human low density lipoprotein (LDL) was incubated with an established line of rabbit aortic endothelial cells. Density gradient fractionation showed a time-, concentration-, and temperature-dependent increase in the average density of the LDL (from about 1.036 to as high as 1.070 g/ml). Incubation without cells or with other types of cultured cells (fibroblasts, hepatocytes, 3T3-L1 cells) caused no significant change in density. 125I-Labeled LDL (125I-LDL) recovered after incubation with endothelial cells (EC-modified LDL) was taken up and degraded 3 to 4 times more rapidly than control LDL by resident mouse peritoneal macrophages and by an established tumor line of mouse macrophages (J774 cells). Macrophage degradation of EC-modified 125I-LDL exhibited saturation kinetics (greater than 85% inhibited by excess unlabeled EC-modified LDL). Degradation was also inhibited by unlabeled acetylated LDL and, conversely, unlabeled EC-modified LDL inhibited degradation of acetylated 125I-LDL. Incubation of LDL with conditioned medium-removed from endothelial cell cultures modified neither its density nor its rate of degradation by macrophages. These studies show that endothelial cells have the potential to metabolically modify the LDL molecule, generating a form that is more rapidly degraded by macrophages and that is recognized by the macrophage receptor for acetylated LDL. This process may play a significant role in the pathogenesis of atherosclerosis.

Animals↗
PubMed1981

Hormone-sensitive lipase in differentiated 3T3-L1 cells and its activation by cyclic AMP-dependent protein kinase.

Differentiation of 3T3-L1 fibroblasts to adipocyte-like cells was accompanied by a 19-fold increase in neutral triglyceride lipase activity, a 12-fold increase in diglyceride lipase activity, a 10-fold increase in monoglyceride lipase activity, and a 280-fold increase in cholesterol esterase activity. In contrast, acid acylhydrolase activities did not increase during differentiation. The rate of glycerol release from unstimulated intact cells increased by more than 1 order of magnitude upon differentiation. Isoproterenol (1 microM) and 1-methyl-3-isobutylxanthine (0.1 mM) further stimulated this rate of glycerol release 3-fold. The neutral triglyceride lipase activity in cell-free preparations of differentiated cells was activated 105% by cyclic AMP-dependent protein kinase. Neutral cholesterol esterase, diglyceride lipase, and monoglyceride lipase were also activated (117%, 10%, and 37+, respectively) by cyclic AMP-dependent protein kinase. In contrast, protein kinase had no effect on any of the four lysosomal acid acylhydrolase activities. Thus, hormone-sensitive lipase, the most characteristic and functionally important enzyme of adipose tissue, has been characterized in differentiated 3T3-L1 cells. The 3T3-L1 cell should be a valuable model system in which to study regulation of hormone-sensitive lipase, particularly its long-term regulation.

Adipose Tissue↗
PubMed1981

Metabolism of homologous and heterologous lipoproteins by cultured rat and human skin fibroblasts.

Rat fibroblasts degraded human low density lipoprotein (LDL) very slowly, one-tenth to one-fortieth the rates observed in human fibroblasts. In rat cells, human LDL caused only very small increases in cell cholesterol content and acylCoA:cholesterol acyltransferase (ACAT) activity and caused only small decreases in beta-hydroxy-beta-methylglutaryl CoA (HMG CoA) reductase activity; in human cells, however, human LDL induced very large changes in all three of these parameters, as expected. The binding of human LDL to rat fibroblasts was not reduced by previous incubation with human LDL or with 25-hydroxycholesterol. Thus, in rat fibroblasts there appear to be few, if any, regulated high-affinity receptors that recognize human LDL. Rat LDL fractions (d 1.02-1.05 g/ml), in contrast, were degraded more rapidly than human LDL by rat fibroblasts, caused a significant increase in cell cholesterol content, an increase in ACAT activity, and a significant decrease in HMG CoA reductase activity. Moreover, the degradation of this rat LDL fraction by rat fibroblasts as a function of concentration was biphasic, i.e., there appeared to be a high-affinity component of degradation. Thus, it appears that rat fibroblasts do have a receptor for homologous lipoproteins. However, because both apoprotein B and apoprotein E are present in these rat lipoprotein fractions, the observed effects may relate to recognition of either or both of these apoproteins. The metabolism and metabolic effects of the conventionally defined high density lipoprotein (HDL) fraction of the rat by rat or human fibroblasts resembled those of human LDL in human fibroblasts. It is suggested that rat HDL may, because of its apo E content and higher concentration in rat plasma relative to that of LDL, play an important role in cholesterol homeostasis in vivo.

Animals↗
PubMed1980

Regulation of cholesterol esterification and biosynthesis in monolayer cultures of normal adult rat hepatocytes.

Adult rat parenchymal liver cells were isolated and cultured in monolayers. Cholesterol esterification in the intact cultured cells was determined by measuring incorporation of tritiated oleic acid into cell cholesterol ester. Addition of 10 microgram/ml of 25-hydroxycholesterol to the medium gave a 3- to 6-fold increase in cholesterol esterification, while the incorporation of oleic acid into phospholipids and triglycerides remained unaltered. Similar stimulation of cholesterol esterification by 25-hydroxycholesterol was also found if [14C]mevalonolactone or [3H]cholesterol (the latter presented to the cells in high density lipoproteins) were used as precursors. The stimulatory effect of 25-hydroxycholesterol was maximal after only 15-min incubation and was independent of protein synthesis. After 4 to 6 h of incubation with 25-hydroxycholesterol, its stimulatory effect was reduced significantly, and after 18 h of incubation no stimulation was observed. Thus, the liver cells in some fashion adapt to the continuing presence of 25-hydroxycholesterol. Isolated microsomes prepared from cells previously incubated with 25-hydroxycholesterol showed acyl-CoA:cholesterol acyltransferase activity 3 times that of microsomes from control cells. Incubation of isolated microsomes with 25-hydroxycholesterol increased acyl-CoA:cholesterol acyltransferase activity 2-fold. The net cellular content of ester cholesterol increased after 2 to 6 h incubation of hepatocytes with 25-hydroxycholesterol; there was a net decrease in cellular free cholesterol. Mevalonolactone (10 mM) also stimulated cholesterol esterification and inreased the cellular content of ester cholesterol (3- to 4-fold). The effectiveness of mevalonolactone did not diminish with longer periods of preincubation. Furthermore, the stimulatory effects of 25-hydroxycholesterol and mevalonolactone added together were at least 50 to 100% greater than the effects of either agent alone, suggesting that the mechanisms by which they increase cellular cholesterol esterification are different. Both 25-hydroxycholesterol and mevalonolactone rapidly inhibited microsomal 3-hydroxy-3-methylglutaryl-CoA reductase activity. Pure cholesterol had no effect on cellular cholesterol esterification or on 3-hydroxy-3-methylglutaryl-CoA reductase activity at concentrations up to 10 microgram/ml.

Animals↗
PubMed1980

Hormone-sensitive lipase of rat adipose tissue: correlation of activity with a protein of molecular weight 84 000.

Hormone-sensitive lipase of rat adipose tissue was partially purified. The enzyme retained its capacity to be activated by cyclic AMP-dependent protein kinase throughout purification. When the partially purified 32P-labeled preparation was subjected to two-dimensional gel electrophoresis, the enzyme activity was found to be associated with a 32P-labeled protein of molecular weight 84 000. The result suggests that this 32P-labeled protein represents hormone-sensitive lipase or the catalytic subunit of the enzyme.

Adipose Tissue↗
PubMed1980

Dissociation of the lipid-enzyme complex of hormone-sensitive lipase using high density lipoprotein or apolipoprotein A-I.

Hormone-sensitive lipase in homogenates of adipose tissue occurs as a large, lipid-rich complex including several acylhydrolase activities that emerge quantitatively in the void volume on gel filtration chromatography (2% agarose). Incubation with intact human plasma high density lipoprotein or with lipid-free apolipoprotein A-I, however, disrupted the lipid-rich complex almost completely and most of the enzyme activity eluted from a 2% agarose column at about Ve = 2.3 x Vo. This use of the detergent-like properties of apolipoprotein A-I may be of value for dissociation of other lipid-associated or membrane-bound enzymes.

Adipose Tissue↗
PubMed1980

Metabolism of native and of lactosylated human low density lipoprotein: evidence for two pathways for catabolism of exogenous proteins in rat hepatocytes.

Human low density lipoprotein (LDL) covalently conjugated with 200-250 residues of lactose per LDL particle (Lac-LDL) was bound and rapidly taken up by the galactose-specific receptor of rat hepatocytes. Uptake of Lac-LDL was associated with inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase and stimulation of cholesterol esterification. Uptake of native human LDL had no significant effects on these enzyme activities even when the rates of LDL uptake equaled those of Lac-LDL. When injected into rats, Lac-LDL was selectively removed by the liver (98% of injected dose). The hepatic subcellular distribution of simultaneously injected native 125I-labeled LDL and 131I-labeled Lac-LDL differed significantly, Lac-LDL was associated with fractions enriched in lysosomal hydrolases whereas native LDL was found predominantly in the supernatant fraction enriched in lactate dehydrogenase. Chloroquine (0.1 mM) markedly suppressed uptake of Lac-LDL by cultured rat hepatocytes (> 80%) but had only a small effect on uptake of native LDL. Leupeptin (0.625 mM) inhibited degradation of Lac-LDL more than it did degradation of native LDL. Colchicine (0.25 microM) dramatically suppressed uptake of Lac-LDL (> 70%) but did not affect native LDL uptake even at concentrations as high as 10 microM. Uptake of human LDL by rat hepatocytes occurs largely by nonspecific mechanisms, including fluid endocytosis, whereas Lac-LDL, as shown here, is taken up by a specific receptor-mediated mechanism. The results show further that native human LDL, representing an example of a protein taken up nonspecifically, is processed intracellularly by a pathway qualitatively distinct from that for Lac-LDL, an example of a protein taken up by a specific mechanism. Lac-LDL may serve as a vehicle for specifically delivering drugs, hormones, or radioactive compounds to hepatocytes for therapeutic or diagnostic purposes.

Animals↗
PubMed1980

Dissociation of apoprotein B and triglyceride production in very-low-density lipoproteins.

As a first step toward understanding the regulation of apoprotein B (apoB) secretion in man, we have simultaneously studied the production of very-low-density lipoprotein (VLDL) apoB and VLDL triglycerides (TG) in six subjects with plasma TG levels of 153-344 mg/dl, during consumption of control and high-carbohydrate diets. 125I-labeled VLDL and [3H]glycerol were injected intravenously, and blood samples obtained over a 48-h period were used to isolated VLDL 125I-labeled apoB and VLDL 3H-labeled TG. The complex radioactivity curves obtained were analyzed by a multicompartmental model of VLDL metabolism. In accord with previous data, plasma VLDL TG levels were increased in five of six subjects (mean increase of 76% for the group), and in four of the six subjects the VLDL apoB concentrations were increased significantly during the high-carbohydrate period. The estimated rate of secretion of VLDL TG was higher in all six subjects, from 9.4 to 23.5 mg/kg-1 X h-1 on the control diet to 14.0-65.5 mg X kg-1 X h-1 on the carbohydrate diet (P < 0.001). In contrast, there was no increase in the rate of secretion of VLDL apoB in any of the subjects compared to their control period with values ranging from 0.60 to 1.59 mg X kg-1 X h-1 during the control period to 0.63-1.47 mg X kg-1 X h-1 after carbohydrate induction. These results indicate that physiological perturbations of VLDL TG production need not be accompanied by an alteration in VLDL apoB production.

Adult↗
PubMed1980

Secretion of very low density lipoproteins enriched in cholesteryl esters by cultured rat hepatocytes during simulation of intracellular cholesterol esterification.

Very low density lipoproteins, newly secreted by cultured rat hepatocytes into a serum-free medium, contain some cholesteryl esters although the percentage of total cholesterol in ester form is less than that in plasma very low density lipoproteins. When acyl CoA:cholesterol acyltransferase activity in hepatocytes was stimulated by the addition of 25-hydroxycholesterol (10 microgram/ml) or mevalonolactone (1 mM), the absolute amount of esterified cholesterol secreted in very low density lipoproteins increased significantly, but the amount of free cholesterol decreased or showed no change. Thus the percentage of very low density lipoprotein cholesterol in ester form increased, in some experiments to as much as 50% of the total. These results provide additional evidence that hepatic acyl CoA:cholesterol acyltransferase plays a role in the generation of some of the cholesteryl ester in newly-secreted lipoproteins. They further suggest that changes in the activity of the enzyme can potentially regulate the fraction of cholesterol secreted in esterified form.

Animals↗