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Biomedical subjects

J R Faust

Publications and source records attributed to J R Faust.

At least 37 records · Page 2Linked to original sources
PubMed1982

Appearance of crystalloid endoplasmic reticulum in compactin-resistant Chinese hamster cells with a 500-fold increase in 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

We have developed a line of Chinese hamster ovary cells with a 500-fold increase in 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the membrane-bound enzyme that controls cholesterol synthesis. This line, designated (UT-1, was obtained by stepwise adaptation of cells to growth in increasing concentrations of compactin, a competitive inhibitor of reductase. Reductase accounts for approximately 2% of total cell protein in UT-1 cells, as calculated from enzyme specific activity and by immunoprecipitation of reductase after growth of cells in [35S]methionine. After solubilization in the presence of the protease inhibitor leupeptin and electrophoresis in NaDodSO4/polyacrylamide gels, reductase subunits from UT-1 cells were visualized by immunoblotting as a single band (Mr = 62,000). To accommodate the increased amounts of reductase, UT-1 cells developed marked proliferation of tubular smooth endoplasmic reticulum (ER) membranes, as revealed by immunofluorescence and electron microscopy. The ER tubules were packed in crystalloid hexagonal arrays. When UT-1 cells were incubated with low density lipoprotein, reductase activity was suppressed by 90% in 12 hr and the crystalloid ER disappeared. UT-1 cells should be useful for studies of the regulation of reductase and also for studies of the synthesis and degradation of smooth ER.

Animals↗
PubMed1980

Synthesis of delta 2-isopentenyl tRNA from mevalonate in cultured human fibroblasts.

Human fibroblasts are shown to incorporate [3H]-mevalonolactone into 3H-labeled delta 2-isopentenyl tRNA. This incorporation was observed in cells that were incubated with compactin (ML-236B), an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme. A reductase that blocks mevalonate production by cells. When incubated with low concentrations of [3H]mevalonolactone in the presence of compactin and in the absence of exogenous cholesterol, the cells incorporated small amounts of [3H]mevalonolactone into delta 2-isopentenyl tRNA and large amounts into cholesterol. In the presence of low density lipoprotein, which serves as a source of cholesterol, the incorporation of [3H]mevalonolactone into cholesterol was reduced by 90% and the incorporation into delta 2-isopentenyl tRNA was stimulated by 10-fold. Thus, cultured mammalian cells are now known to use mevalonate for synthesis of three nonsterol products, ubiquinone, dolichol, and delta 2-isopentenyl tRNA, as well as for synthesis of cholesterol.

Alkenes↗
PubMed1979

Squalene synthetase activity in human fibroblasts: regulation via the low density lipoprotein receptor.

Squalene synthetase (farnesyltransferase; farnesyl diphosphate:farnesyl-diphosphate farnesyltransferase, EC 2.5.1.21), the enzyme in the cholesterol biosynthetic pathway that converts farnesyl pyrophosphate into squalene, is subject to regulation in cultured human fibroblasts. When cholesterol-carrying low density lipoprotein (LDL) was removed from the serum of the culture medium, squalene synthetase activity increased 8-fold over 24 hr. When LDL was added back to the medium, squalene synthetase was slowly suppressed, 50% and 90% reduction occurring in 15 and 48 hr, respectively. Suppression of squalene synthetase required uptake of LDL via the LDL receptor; hence, it did not occur in mutant fibroblasts from a patient with homozygous familial hypercholesterolemia that lack receptors. The addition of a mixture of 25-hydroxycholesterol and cholesterol suppressed squalene synthetase equally well in normal and mutant fibroblasts. Coupled with previous data, the current findings indicate that cholesterol derived from LDL regulates at least two enzymes in the cholesterol synthetic pathway in fibroblasts: (i) its primary action is to rapidly suppress 3-hydroxy-3-methylglutaryl coenzyme A reductase [mevalonate:NADP(+), oxidoreductase (CoA-acylating), EC 1.1.1.34], which reduces mevalonate production by 95% within 8 hr, and (ii) its secondary action is to slowly suppress squalene synthetase. The LDL-mediated suppression of squalene synthetase does not regulate de novo cholesterol synthesis; it occurs after 3-hydroxy-3-methylglutaryl coenzyme A reductase is already suppressed. Rather, we hypothesize that it may function to allow the pool size of farnesyl pyrophosphate to be maintained in the presence of LDL so that low levels of mevalonate can be shunted preferentially into nonsterol products, such as ubiquinone-10 and dolichol. This mechanism may explain the earlier observation that the synthesis of ubiquinone-10 in fibroblasts proceeds at a normal rate in the presence of LDL despite a 95% decrease in mevalonate production.

Cells, Cultured↗
PubMed1979

Low density lipoprotein receptors in bovine adrenal cortex. I. Receptor-mediated uptake of low density lipoprotein and utilization of its cholesterol for steroid synthesis in cultured adrenocortical cells.

Functioning bovine adrenocortical cells in monolayer culture were shown to obtain cholesterol for steroid synthesis from plasma low density lipoprotein (LDL). When grown in medium devoid of lipoproteins, the cells developed a minimal enhancement in steroid secretion in response to ACTH or cholera toxin. However, when LDL was available, steroid secretion was stimulated 4- to 9-fold. To determine the mechanism for this effect, we used LDL in which the protein component was labeled with 125I and the cholesteryl ester component was labeled with [3H]cholesteryl linoleate. These studies demonstrated that the cells derived cholesterol from LDL by binding the lipoprotein at a high affinity receptor site, internalizing it, and hydrolyzing its cholesteryl esters within lysosomes. The resultant free cholesterol was used for steroid synthesis and also acted to suppress the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol synthesis within the cell. LDL receptor activity was enhanced several-fold by treatment of the cells with ACTH or cholera toxin. High density lipoprotein, which did not bind to the LDL receptor, was not degraded with high affinity by the cells and did not support steroid synthesis. The current data suggest that the bovine adrenal cortex can obtain cholesterol for steroid hormone secretion from circulating LDL by means of a high affinity LDL receptor pathway. In a subsequent paper in this series, a similar high affinity LDL-binding site is demonstrated in membranes prepared from fresh bovine adrenocortical tissue.

Adrenal Cortex↗
PubMed1978

Inhibition of cholesteryl ester formation in human fibroblasts by an analogue of 7-ketocholesterol and by progesterone.

The synthesis of cholesteryl esters in cultured human fibroblasts is catalyzed by a microsomal acyl-coenzyme A:cholesterol acyltransferase (EC 2.3.1.26). The acyltransferase activity is enhanced when fibroblasts take up cholesterol contained in plasma low density lipoprotein. In the current studies two steroids, SC-31769 (an analogue of 7-ketocholesterol) and progesterone, were shown to inhibit acyltransferase activity in cell-free extracts of human fibroblasts. When added to intact cells, these steroids inhibited the incorporation of [(14)C]oleate into cellular cholesteryl [(14)C]oleate and reduced the accumulation of cholesteryl esters in fibroblasts exposed to low density lipoprotein. The inhibition of cholesteryl ester formation in intact cells by SC-31769 and progesterone was readily reversible. Neither compound inhibited the incorporation of [(14)C]oleate into [(14)C]triglycerides or [(14)C]phospholipids. When incubated with fibroblast monolayers at a concentration of 1 mug/ml, SC-31769 suppressed the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase [mevalonate:NADP(+) oxidoreductase (CoA-acylating); EC 1.1.1.34], the rate-controlling enzyme in cholesterol synthesis. In contrast, progesterone had no effect on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity at concentrations as high as 25 mug/ml. The availability of two types of steroid compounds that inhibit the acyltransferase activity and cholesteryl ester synthesis in human fibroblasts should prove useful in further studies of the regulatory mechanisms responsible for cholesteryl ester accumulation in human cells under normal and pathologic conditions.

Acyltransferases↗
PubMed1978

Similar content of phospholipids and gangliosides in normal and homozygous familial hypercholesterolemia fibroblasts.

The cellular content of total and individual phospholipids and gangliosides was measured in fibroblasts cultured from four normal subjects, three patients with lysosomal lipid storage diseases, and two subjects with homozygous familial hypercholesterolemia. Measurements were made on cells grown in medium containing fetal calf serum under conditions in which normal cells derive cholesterol for cell growth from low density lipoprotein present in the fetal calf serum, whereas familial hypercholesterolemia homozygote cells, which lack cell surface low density lipoprotein receptors, derive cholesterol from endogenous synthesis. No difference was observed in the cellular content of total or individual phospholipids and gangliosides in the normal and familial hypercholesterolemia homozygote cells. In contrast, cells from a patient with Niemann-Pick disease and a patient with Sandhoff disease showed elevations in the content of sphingomyelin and complex gangliosides, respectively.

Adult↗
PubMed1977

Regulation of cholesterol synthesis by low density lipoprotein in isolated human lymphocytes. Comparison of cells from normal subjects and patients with homozygous familial hypercholesterolemia and abetalipoproteinemia.

The rate of cholesterol synthesis from [14C]acetate was low in circulating blood lymphocytes freshly isolated from 17 normal subjects and 4 subjects with homozygous FH. On the other hand, the rate of cholesterol synthesis was two to fourfold above normal in freshly isolated lymphocytes from two subjects with abetalipoproteinemia. When the lymphocytes from subjects with all three genotypes were incubated for 48-72 h in the absence of lipoproteins, the rate of cholesterol synthesis increased by 5-15-fold. The subsequent addition of plasma LDL, but not HDL, rapidly suppressed cholesterol synthesis in the lymphocytes from normal subjects. In contrast, lymphocytes from the FH homozygotes, which have been shown previously to be deficient in cell surface LDL receptors, were resistant to LDL-mediated suppression of cholesterol synthesis. In addition to its ability to suppress cholesterol synthesis after it had been elevated by incubation of the cells in the absence of lipoproteins, LDL was able to suppress the induction of the enhanced rate of sterol synthesis when added to normal lymphocytes immediately after their isolation from the bloodstream. In contrast to the former action of LDL, the latter action of LDL-i.e., the suppression of induction of sterol synthesis-also occurred to a limited extent in lymphocytes from FH homozygotes. However, the FH lymphocytes, but not the normal cells, could be made resistant to this action of LDL by inclusion in the incubation medium of lipoprotein-deficient serum (30 percent, vol/vol) plus HDL (1 mg protein/ml). Considered together with previous data demonstrating a deficiency of LDL receptors in freshly isolated lymphocytes from FH homozygotes, the current studies provide evidence in support of the hypothesis that the interaction of plasma LDL with its cell surface receptor serves to regulate cholesterol synthesis in human lymphocytes.

Abetalipoproteinemia↗
PubMed1977

Lipoprotein-mediated regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and cholesteryl ester metabolism in the adrenal gland of the rat.

In the adrenal gland of the rat, the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-controlling enzyme of cholesterol synthesis, is shown to be regulated by cholesteerol carried in plasma lipoproteins. When plasma cholesterol levels were lowered 90% by administration of the drug 4-aminopyrazolopyrimidine, the cholesteryl ester content of the adrenal gland declined by more than 90% and this was associated with a 150- to 200-fold increase in the activity of adrenal 3-hydroxy-3-methylglutaryl coenzyme A reductase and a 30-fold increase in cholesterol synthesis from [14C]acetate. The subsequent intravenous infusion of cholesterol contained in either rat or human high density or low density lipoproteins restored the adrenal content of cholesteryl esters and reduced the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase to basal levels. The depletion of adrenal cholesteryl esters and the enhancement in the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase that occurred in the 4-aminopyrazolopyrimidine-treated rat required the action of adrenocorticotropic hormone (ACTH) since neither was observed when ACTH secretion was blocked by administration of dexamethasone. The current data indicate that the low rate of cholesterol synthesis normally observed in the rat adrenal gland is due to a suppression of the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase that is mediated by plasma lipoproteins.

Adenine↗
PubMed1976

The biosynthesis of the thiazole moiety of thiamine in Salmonella Typhimurium.

The mechanism of biosynthesis of 4-methyl-5-beta-hydroxyethyl thiazole, the thiazole moiety of thiamine was studied in Salmonella typhimurium. Using the adenosine derepression technique the incorporation of various 14C-labeled precursors was determined. We found that;e1Me-14C]methionine, [2-14C]methionine, [U-14C]alanine, and [2-14C]glycine were not incorporated whereas [2-14C]tyrosine was incorporated. Degradation of the 4-methyl-5-beta-hydroxyethyl thiazole obtained after [2-14C]tyrosine incorporation revealed that all of the activity was located on carbon-2. These findings are discussed and compared with previous findings concerning 4-methyl-5-beta-hydroxyethyl thiazole biosynthesis.

Alanine↗
PubMed1976

Heterozygous familial hypercholesterolemia: failure of normal allele to compensate for mutant allele at a regulated genetic locus.

In normal human fibroblasts, the synthesis of a cell surface receptor for plasma low density lipoprotein (LDL) is regulated by a sensitive system of feedback suppression. The number of functional LDL receptors declines by more than 20 fold when cellular stores of esterified cholesterol are increased by incubation of cells with an exogenous source of cholesterol. Fibroblasts from patients with the heterozygous form of familial hypercholesterolemia (FH) possess one functional allele and one nonfunctional allele at the LDL receptor locus. In the current studies, we have examined the effect that this deficiency produces upon the pattern of regulation of the single functional allele at the LDL receptor locus. Under growth conditions that induced a maximal rate of LDL receptor synthesis (that is, growth in the absence of an exogenous source of cholesterol), the FH heterozygote cells produced about one half as many functional LDL receptors as did the normal cells. More importantly, when grown in the presence of increasing amounts of exogenous cholesterol, the FH heterozygote and normal cells suppressed their respective LDL receptor activities in parallel. Over a wide range of LDL receptor activities, at each level of cellular esterified cholesterol, the FH heterozygote cells expressed about one half as many receptors as did the normal cells. These data indicate that in the FH heterozygote cells, the receptor regulatory mechanism dictates that the normal allele produce only the amount of gene product that it would normally produce at a given level of cellular esterified cholesterol. The failure of the regulatory mechanism to stimulate the normal allele at the LDL receptor locus to produce twice its normal amount of gene product leaves the FH heterozygote cells with a persistent 50% deficiency in LDL receptors under all conditions of cell growth.

Alleles↗
PubMed1976

Evidence for regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and cholesterol synthesis in nonhepatic tissues of rat.

The adenine analogue 4-aminopyrazolopyrimidine has been reported previously to reduce the hepatic secretion of plasma lipoproteins in rats, thereby lowering the plasma cholesterol level. In the current studies, reduction of the plasma cholesterol level by 90% in rats through the administration of aminopyrazolopyrimidine was found to be associated with a 5- to 30-fold increase in the activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase [mevalonate:NADP+ oxidoreductase (CoA-acylating), EC1.1.1.34] in kidney and lung. In both tissues, the enhanced activity of this microsomal enzyme was associated with a 3-fold elevation in the rate of cholesterol synthesis from either [14C]acetate or [14C]octanoate. Comparable increases were not observed in the activities of several other microsomal enzymes or in the rates of [14C]acetate incorporation into saponifiable lipids or CO2. When administration of 4-aminopyrazolopyrimidine was terminated, plasma cholesterol levels rose and 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity declined in the kidney in a reciprocal manner. These data are consistent with the hypothesis that the low levels of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity and cholesterol synthesis that are normally observed in certain nonhepatic tissues of the rat are due to an active form of feedback regulation mediated by cholesterol carried in plasma lipoproteins.

Acetates↗
PubMed1975

Role of lysosomal acid lipase in the metabolism of plasma low density lipoprotein. Observations in cultured fibroblasts from a patient with cholesteryl ester storage disease.

The hydrolysis of cholesteryl esters contained in plasma low density lipoprotein was reduced in cultured fibroblasts derived from a patient with cholesteryl ester storage disease, an inborn error of metabolism in which lysosomal acid lipase activity is deficient. While these mutant cells showed a normal ability to bind low density lipoprotein at its high affinity cell surface receptor site, to take up the bound lipoprotein through endocytosis, and to hydrolyze the protein component of the lipoprotein in lysosomes, their defective lysosomal hydrolysis of the cholesteryl ester component of the lipoprotein led to the accumulation within the cell of unhydrolyzed cholesteryl esters, the fatty acid distribution of which resembled that of plasma lipoprotein. When the cholesteryl ester storage disease cells were incubated with low density lipoprotein, the reduced rate of liberation of free cholesterol by these mutant cells was associated with a delay in the occurrence of two lipoprotein-mediated regulatory events, suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, and activation of endogenous cholesteryl ester formation. In contrast to their defective hydrolysis of exogenously derived lipoprotein-bound cholesteryl esters, the choleseryl ester storage disease cells showed a normal rate of hydrolysis of cholesteryl esters that had been synthesized within the cell. These data lend support to the concept that in cultured human fibroblasts cholesteryl esters entering the cell bound to low density lipoprotein are hydrolyzed within the lysosome and that one of the functions of this intracellular organelle is to supply the cell with free cholesterol.

Cells, Cultured↗
PubMed1975

Role of the low density lipoprotein receptor in regulating the content of free and esterified cholesterol in human fibroblasts.

The transfer of normal human fibroblasts from medium containing whole serum to medium devoid of lipoproteins produced a 90 percent decrease in the cellular content of cholesteryl esters and a 30 percent decrease in the free cholesterol content. When these lipoprotein-deprived cells were subsequently incubated with human low density lipoprotein (LDL), there was a 7-fold increase in the cellular content of esterified cholesterol and a 1.6-fold increase in the cellular content of free cholesterol. The concentration at which LDL produced its half-maximal effect in elevating cellular sterol content (30 mug/ml of LDL-cholesterol) was similar to the half-maximal concentration previously reported for high affinity binding of LDL to its cell surface receptor. High density lipoprotein (HDL) and whole serum from a patient with abetalipoproteinemia (neither of which contains a component that binds to the LDL receptor) did not produce a significant increase in the content of either cholesterol or cholesteryl esters in normal cells. Furthermore, in fibroblasts from patients with the homozygous form of familial hypercholesterolemia, which lack functional LDL receptors, LDL had no effect in raising the cellular content of either free or esterified cholesterol even when present in the medium at concentrations as high as 450 mug sterol/ml. It is concluded that LDL-receptor interactions constitute an important biochemical mechanism for the regulation of the cholesterol content of normal human fibroblasts. Moreover, when considered in light of current concepts of LDL metabolism in intact mammals, the present data suggest that a major function of plasma LDL may be to transport cholesterol from its site of synthesis in liver and intestine to its site of uptake in peripheral tissues.

Abetalipoproteinemia↗