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Biomedical subjects

S N Mathur

Publications and source records attributed to S N Mathur.

At least 55 records · Page 3Linked to original sources
PubMed1988

Modification of CaCo-2 cell membrane fatty acid composition by eicosapentaenoic acid and palmitic acid: effect on cholesterol metabolism.

Membrane fatty acid composition of CaCo-2 cells was modified by incubating the cells for 8 days in medium containing 100 microM eicosapentaenoic acid or palmitic acid. The effect of membrane fatty acid changes on cholesterol metabolism was then studied. Cells incubated with eicosapentaenoic acid had significant changes in membrane fatty acid composition with an accumulation of 20:5 and 22:5 and a reduction in monoenoic fatty acids compared to cells grown in palmitic acid. Intracellular cholesteryl esters could not be detected in CaCo-2 cells grown in the presence of the n-3 polyunsaturated fatty acid. In contrast, cells incubated with the saturated fatty acid contained 2 micrograms/mg protein of cholesteryl esters. Cells grown in eicosapentaenoic acid, however, accumulated significantly more triglycerides compared to cells modified with palmitic acid. The rate of oleic acid incorporation into triglycerides was significantly increased in cells incubated with eicosapentaenoic acid. CaCo-2 cells modified by eicosapentaenoic acid had lower rates of HMG-CoA reductase and ACAT activities compared to cells modified with palmitic acid. The incorporation of the two fatty acids into cellular lipids also differed. Palmitic acid was predominantly incorporated into cellular triglycerides, whereas eicosapentaenoic acid was preferentially incorporated into phospholipids with 60% of it in the phosphatidylethanolamine fraction. The data indicate that membrane fatty acid composition is significantly altered by growing CaCo-2 cells in eicosapentaenoic acid. These modifications in membrane fatty acid saturation are accompanied by a decrease in the rates of cholesterol synthesis and cholesterol esterification.

Adenocarcinoma↗
PubMed1988

Regulation of triglyceride-rich lipoprotein secretion by fatty acids in CaCo-2 cells.

The effect of fatty acids on secretion of triglyceride-rich lipoprotein (d less than 1.006 g/ml) by CaCo-2 cells was studied. Of the fatty acids studied, oleic acid (18:1) was the most potent stimulator of newly synthesized triglyceride secretion in triglyceride-rich lipoproteins followed in descending order by 18:2, 18:3, and 16:0 = 14:0. All the fatty acids increased intracellular triglyceride synthesis. Fatty acids 14:0, 16:0, 18:2, and 18.3 caused similar increases; however, 18:1 caused the highest rates of triglyceride synthesis. Oleic acid (18:1) was used to further study the secretion of lipoproteins of density less than 1.006 g/ml by CaCo-2 cells. There was a step-wise increase in cellular triglyceride synthesis with increasing oleic acid concentration. Above 250 microM of the fatty acid, however, newly synthesized triglyceride secretion in triglyceride-rich lipoproteins plateaued, suggesting saturation of the secretory pathway. After stimulating triglyceride synthesis by oleic acid, radiolabeled triglyceride secreted in triglyceride-rich lipoproteins was initially delayed resulting in a sigmoid-shaped curve for secretion. This was most pronounced in control cells, which were not incubated with the fatty acid. Over 6 hr, cells incubated with oleic acid secreted more newly synthesized triglyceride in triglyceride-rich lipoproteins compared to control cells. The secretion of lipoproteins of density less than 1.006 g/ml was dependent upon protein synthesis and normal microtubular function in as much as cycloheximide and colchicine significantly decreased triglyceride transport without changing cellular triglyceride synthesis. Triglyceride and unesterified cholesterol mass in lipoproteins of density less than 1.006 g/ml were increased 57 and 244%, respectively, in medium from cells incubated with oleic acid compared to control cells. By 24 hr, 0.17% of lipoproteins of density less than 1.006 g/ml were taken up and degraded. Over the same period, approximately 50% of the lipoprotein triglyceride was hydrolyzed. Under conditions whereby lipoprotein secretion was stimulated fourfold by oleic acid, the activities of HMG-CoA reductase and ACAT were unchanged from activities in control cells. The data suggest that CaCo-2 cells secrete triglyceride-rich lipoproteins of density less than 1.006 g/ml in response to fatty acids in the medium. Triglyceride-rich lipoprotein secretion is a saturable process and dependent on protein synthesis and normal microtubular function. An increase in triglyceride-rich lipoprotein secretion is accompanied by an increase in triglyceride mass in lipoproteins of density less than 1.006 g/ml. By 24 hr, significant postsecretory

Cells, Cultured↗
PubMed1987

Effect of cholesterol enrichment on 12-hydroxyeicosatetraenoic acid metabolism by mouse peritoneal macrophages.

The metabolism of 12-hydroxyeicosatetraenoic acid (12-HETE) was investigated in mouse peritoneal macrophages enriched in cholesterol by incubation with acetylated low density lipoproteins. After incubating with labeled arachidonic acid, cholesterol-rich cells released more 12-HETE into the medium than unmodified macrophages. With time, however, 12-HETE decreased in the medium of both cell preparations suggesting re-uptake of this monohydroxyfatty acid and perhaps further metabolism. When control macrophages were incubated with radiolabeled 12-HETE for 2 hr, almost 70% of the cell-associated 12-HETE label was incorporated into phospholipids. In contrast, in cholesterol-rich cells, only 31% of the 12-HETE label was incorporated into phospholipids. Bee venom phospholipase completely hydrolyzed the label, suggesting that the monohydroxyfatty acid was esterified at the sn-2 position of the phospholipid. In cholesterol-rich cells, 69% of the 12-HETE was diverted into neutral lipids. Two major neutral lipids were identified in cholesterol-rich macrophages. One neutral lipid band which migrated with an Rf value of 0.34 contained the hydroxylated fatty acid esterified to a glyceride. The other neutral lipid band having an Rf value of 0.49 contained cholesterol and by further analysis was found to contain predominantly cholesteryl-12-HETE. The labeled fatty acids in these two neutral lipids were mostly oxidized products of 12-HETE in contrast to the native 12-HETE observed in the phospholipids. Cholesterol-rich macrophages released 25% more products of 12-HETE metabolism than control macrophages. Two major products were observed in the medium which eluted in the area of a standard di-HETE, LTB4, on high performance liquid chromatography (HPLC) analysis. We propose that the reincorporation of 12-HETE into these neutral lipids and the increased capacity for further metabolism of this biologically potent hydroxyfatty acid could be a mechanism by which the cholesterol-rich macrophage maintains its membrane function, and regulates the amount of 12-HETE in the pericellular space.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1987

Effect of dietary n-3 fatty acids on HMG-CoA reductase and ACAT activities in liver and intestine of the rabbit.

The regulation of hepatic and intestinal 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase and acyl-CoA; cholesterol acyltransferase (ACAT) activities by dietary fish oil was examined in the rabbit. Rabbits were fed 10% menhaden oil or menhaden oil plus 1% cholesterol for 14 days. They were compared with animals fed a control diet or one enriched with long-chain saturated fats consisting of 10% cocoa butter oil or cocoa butter oil plus 1% cholesterol. Plasma cholesterol was increased in rabbits fed the fish oil and the two cholesterol-containing diets. In the liver, ACAT activity was increased and HMG-CoA reductase activity was decreased in rabbits ingesting the fish oil. The same was true for animals ingesting both cholesterol-containing diets. In the intestine, ACAT activity was not affected by the ingestion of the fish oil compared to control rabbits; however, it was significantly higher in animals fed the fish oil compared to animals ingesting the cocoa butter. HMG-CoA reductase activity was decreased in the distal two-thirds of the intestine in animals fed the menhaden oil compared to activities observed in controls. In animals ingesting the cholesterol diets, intestinal reductase was significantly decreased, whereas intestinal ACAT activity was increased in rabbits ingesting the cocoa butter and cholesterol diet when compared to their controls. Lipid analysis of hepatic and intestinal microsomes demonstrated an enrichment of n-3 polyunsaturated fatty acids in membranes from rabbits ingesting the menhaden oil.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Regulation of cholesterol esterification by micellar cholesterol in CaCo-2 cells.

The regulation of acylcoenzyme A:cholesterol acyltransferase (ACAT) activity by cholesterol was studied in an established enterocyte cell line. CaCo-2 cells were grown in culture to confluency and dome formation. They were characterized morphologically by light and transmission electron microscopy. During the culture period, ACAT activity remained stable while the activities of the brush border enzymes sucrase and alkaline phosphatase progressively increased with time and plateaued 12 days after plating. As determined by the rate of incorporation of oleic acid into the individual lipid classes, the rate of triglyceride synthesis was twice that of phospholipid and 15 times that of cholesteryl ester synthesis in these cells. Incubating CaCo-2 cells with cholesterol solubilized in taurocholate micelles resulted in a significant increase in ACAT activity (149 +/- 5 pmol/dish per 2 hr vs. 366 +/- 5, (P less than 0.001) without changing the rates of triglyceride or phospholipid synthesis. The stimulation of ACAT activity by micellar cholesterol was rapid, occurring within 5 min and reaching a maximal effect by 2 hr. The regulation of ACAT activity by cholesterol was directly dependent upon the concentration of cholesterol solubilized in the micelle and was independent of protein synthesis. Incubating CaCo-2 cells with micellar cholesterol did not increase the esterification of, nor did the cholesterol enter the pool of, newly synthesized or performed cholesterol within 2 hr. The micellar cholesterol that was taken up by the cells was esterified within 5 min after starting the incubation. Progesterone, a known ACAT inhibitor, significantly decreased the rate of esterification of intracellular micellar cholesterol proving that the cholesterol taken up by CaCo-2 cells was indeed entering the ACAT pool. Despite increasing amounts of unesterified cholesterol entering the cells via micelles, the percent of cholesterol that was esterified at any one time remained constant at 1%. The results suggest that ACAT activity in CaCo-2 cells is stimulated by cholesterol delivered to the cells by way of taurocholate micelles. The rapid entry of this sterol into the ACAT substrate pool suggests that ACAT activity in CaCo-2 cells is regulated by the expansion of the cholesterol substrate pool that is being utilized by an unsaturated ACAT enzyme.

Cells, Cultured↗
PubMed1986

Effect of dietary cholesterol on biliary cholesterol content and bile flow in the hypothyroid rat.

The hypothyroid rat model was used to investigate the effect of dietary-induced hypercholesterolemia on biliary cholesterol content and bile flow. Rats were divided into four dietary groups--diet A: Rat Chow; diet B: Rat Chow plus 0.1% propylthiouracil; diet C: Rat Chow plus 0.1% propylthiouracil, 0.3% taurocholate, 5% lard; diet D: Rat Chow plus 0.1% propylthiouracil, 0.3% taurocholate, 5% lard, and 1% cholesterol. After 6 wk, bile was collected and livers were excised for the preparation of membranes. In cholesterol-fed animals, biliary cholesterol content was increased. However, because of a significant decrease in the rate of bile flow that occurred in these animals, biliary cholesterol output was unchanged from the cholesterol output observed in control animals. Dietary cholesterol also caused a threefold increase in liver membrane cholesterol content and a 64% decrease in the activity of sodium-potassium-stimulated adenosine triphosphatase (Na+,K+-ATPase). In a separate group of animals, microsomes prepared from livers of control rats were incubated with phosphatidylserine liposomes, liposomes containing cholesterol, or buffer. The activity of Na+,K+-ATPase was increased in microsomes incubated with phosphatidylserine liposomes. However, when the cholesterol content of the microsomes was increased twofold by incubating the membranes with liposomes containing cholesterol, the stimulation of Na+,K+-ATPase activity was significantly decreased. The data suggest that in the cholesterol-fed hypothyroid rat, biliary cholesterol content is significantly increased; however, because of a decrease in the rate of bile flow, biliary cholesterol output is not changed. The decrease in bile flow is associated with an accumulation of cholesterol and a decrease in the activity of Na+,K+-ATPase in hepatic membranes.

Animals↗
PubMed1986

The effect of hypothyroidism and thyroxine replacement on hepatic and intestinal HMG-CoA reductase and ACAT activities and biliary lipids in the rat.

Hepatic and intestinal cholesterol metabolism were investigated in the hypothyroid and thyroxine-treated hypothyroid rat. Plasma cholesterol levels were significantly increased in hypothyroid animals. After thyroxine administration, plasma cholesterol levels were reduced to levels observed in euthyroid controls. Hypothyroidism caused a significant decrease in biliary cholesterol output, which was reversed with thyroxine treatment. In contrast, biliary bile acid output was unchanged by the thyroid status. Cholesterol synthesis, as estimated by HMG-CoA reductase activity, was decreased in the liver of hypothyroid animals. Thyroxine administration, however, significantly increased reductase activity returning it to control levels. Hypothyroidism did not affect HMG-CoA reductase activity in the intestine, but thyroxine administration markedly stimulated the activity of this enzyme in this organ. Cholesterol esterification, as estimated by ACAT activity, was decreased in the liver of hypothyroid rats, while intestinal ACAT activity was greatly increased. Thyroxine treatment reversed these effects of hypothyroidism on ACAT activity in both organs. An increase in microsomal cholesterol content in the intestine of hypothyroid rats was associated with the observed increase in intestinal ACAT activity. The percent of cholesterol that was absorbed in the intestine was not changed by the thyroid status of the animal. The data suggest that the changes observed in cholesterol metabolism in hypothyroid rats or hypothyroid rats treated with thyroxine for 1 week cannot account for the increase in plasma cholesterol levels observed in the hypothyroid rat. This implies that other factors that were not studied, such as changes in lipoprotein catabolism, are likely to contribute to the hypercholesterolemia of hypothyroidism.

Animals↗
PubMed1986

Lipid metabolism in xanthomatous skin of hypercholesterolemic rabbits.

The authors studied xanthomatous skin in cholesterol-fed rabbits for changes in lipid content and in activities of enzymes regulating intracellular lipid content. After 80 days of hypercholesterolemic diet, xanthomas were widespread and changes in lipid metabolism were marked. In both tissue homogenates and cell membrane pellets, unesterified cholesterol and phospholipids increased 2-fold to 6-fold, and cholesteryl esters increased about 30-fold. Tissue triglycerides, however, decreased to half the levels found in control skin. Cholesterol esterification rates, measured by activity of acyl coenzyme A: cholesterol acyltransferase, increased moderately to markedly; hydrolase activity against 4-methylumbelliferyl oleate also increased at both acid and neutral pH, but hydrolase activity against cholesterol oleate increased only at acid pH. Thus, hypercholesterolemia caused striking increases in intracellular cholesterol esterification rates, increases in lipase activity at both neutral and acid pH, and increases in cholesteryl ester hydrolase activity at acid pH. Increases in cholesterol-esterifying activity uniformly exceeded increases in cholesteryl ester hydrolytic activity in congruence with net accumulation of cholesteryl ester. Skin xanthoma grade, however, had no consistent relation to the cholesterol esterification rates. Instead, the enzyme data suggested that marked abnormalities of lipid metabolism are diffusely distributed through dermal tissue as a precondition for the focal emergence of xanthomas.

Animals↗
PubMed1985

Increased production of lipoxygenase products by cholesterol-rich mouse macrophages.

The metabolism of arachidonic acid by cholesterol-enriched resident mouse peritoneal macrophages was investigated. The amounts of monohydroxyeicosatetraenoic acid (mono-HETE) produced by the cholesterol-rich macrophages were 2.5-fold greater when compared to control macrophages. The major lipoxygenase product, identified by high-performance liquid chromatography in both macrophages was 12-HETE. Since macrophages are important participants in the formation of atheromatous lesions, the increased metabolism of arachidonic acid to HETE products by cholesterol-rich macrophages could contribute to the initiation and progression of the atherosclerotic process.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1985

A defect in mobilization of cholesteryl esters in rabbit macrophages.

Macrophages provide an important way for cholesteryl esters to accumulate in tissues in pathologic amounts. We studied cholesteryl ester metabolism in thioglycollate-induced peritoneal macrophages obtained from normocholesterolemic and hypercholesterolemic rabbits. The macrophage preparations from normocholesterolemic rabbit (MN cells) had 26 nmol esterified cholesterol/mg cellular protein, incorporated 1 nmol of labeled oleate into cholesteryloleate/2 h per mg cellular protein and had an acyl-coenzyme A:cholesterol acyltransferase activity of 22 pmol cholesterylpalmitate formed/min per mg protein in isolated membranes. The macrophage preparations from hypercholesterolemic rabbits (MHC cells) contained a 12-fold greater mass of cholesteryl ester, had an 8-times higher rate of formation of cholesteryloleate, and had 3-times more acyl-coenzyme A:cholesterol acyltransferase activity in the isolated membranes. When a cholesterol acceptor (10% fetal bovine serum or 10 mg of lipid-free fetal bovine serum protein) was added to the culture medium of rabbit MHC cells, the MHC cells retained more than 70% of their cholesteryl esters after 48 h of incubation. In contrast, when a cholesterol acceptor (10% fetal bovine serum) was added to the medium of thioglycollate-induced, cholesterol-enriched macrophages from mice, the mice macrophages retained only 19% of their cholesteryl esters after 48 h of incubation. The limited capacity of rabbit macrophages to release unesterified cholesterol from stored cytoplasmic cholesteryl esters to an exogenous acceptor may be related to the propensity of rabbits to develop atherosclerotic lesions.

Animals↗
PubMed1985

Cholesterol metabolism in regenerating liver of the rat.

The regenerating rat liver was used as a model to investigate the necessity for new cholesterol synthesis prior to the onset of cell division. Plasma cholesterol levels in partially hepatectomized rats were significantly decreased 24 and 48 h after surgery compared with levels in sham-operated animals. Hepatic cholesteryl ester content was also significantly increased in livers from partially hepatectomized animals, but the hepatic content of unesterified cholesterol was not affected. Hepatic triglyceride content was significantly increased within 6 h after surgery in the regenerating liver. The triglyceride levels reached a peak at 24 h, and by 72 h they had decreased back to levels that were no different from control. In the regenerating liver, microsomal 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase activity was increased 12 h after surgery. The activity of this enzyme remained significantly elevated throughout the 72-h period after surgery. In contrast, 12 h after partial hepatectomy the rate of hepatic cholesterol synthesis was significantly lower than that observed in livers from sham-operated rats. An increase in the rate of cholesterol synthesis was not observed until 48 h after partial hepatectomy, some 32 h after the start of DNA synthesis. Microsomal acyl-CoA:cholesterol acyltransferase activity was unchanged except for a 28% decrease at 72 h after partial hepatectomy. The data suggest that new cholesterol synthesis is not a requirement prior to the initiation of DNA synthesis in the regenerating rat liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Acyl Coenzyme A↗
PubMed1984

In vitro regulation of 3-hydroxy-3-methylglutarylcoenzyme A reductase and acylcoenzyme A: cholesterol acyltransferase activities by phosphorylation-dephosphorylation in rabbit intestine.

The regulation of 3-hydroxy-3-methylglutarylcoenzyme A reductase and acylcoenzyme A:cholesterol acyltransferase activities by phosphorylation-dephosphorylation in rabbit intestine was studied in vitro. Preparing intestinal microsomes in the presence of 50 mM NaF caused a 64% decrease in the reductase activity. It had no effect on acyl-CoA:cholesterol acyltransferase activity. Microsomes that were prepared in NaF were incubated with intestinal cytosol, a partially purified phosphatase from cytosol, and Escherichia coli alkaline phosphatase. All three preparations increased 3-hydroxy-3-methylglutaryl-CoA reductase by two- or three-fold suggesting dephosphorylation and 'reactivation' of enzyme activity. Cytosol caused a 78% increase in acyl-CoA:cholesterol acyltransferase activity, but neither the partially purified phosphatase nor the E. coli alkaline phosphatase affected the acyltransferase activity. Microsomes incubated with increasing concentrations of MgCl2 and ATP decreased both the activities of 3-hydroxy-3-methylglutaryl-CoA reductase and acylcoenzyme A:cholesterol acyltransferase in a step-wise fashion. Whereas this inhibitory effect was specific for reductase, the effect on acyl-CoA:cholesterol acyltransferase activity was secondary to the presence of ATP in the assay mixture. The 8500 X g supernatant of intestinal whole homogenate from isolated intestinal cells or scraped mucosa was incubated with MgCl2, ATP and NaF. In microsomes prepared from this supernatant, the activity of 3-hydroxy-3-methylglutaryl-CoA reductase was significantly decreased. Again, no change was observed in the acyltransferase activity. The rate of cholesterol esterification in isolated intestinal cells was not affected by 0.1 mM cAMP or 50 mM NaF. We conclude that under conditions which regulate 3-hydroxy-3-methylglutaryl-CoA reductase activity in rabbit intestine by phosphorylation-dephosphorylation, no regulation of acyl-CoA:cholesterol acyltransferase activity is observed.

Adenosine Triphosphate↗
PubMed1984

Electron-spin resonance studies of lipid-modified microsomes from Friend erythroleukemia cells.

The fatty acid composition of cultured Friend erythroleukemia cells was modified by supplementation of the medium with oleic or linoleic acid. There was a 30% reduction in saturated and a 35% reduction in polyunsaturated fatty acids in microsomal phospholipids when the cells were grown in media supplemented with oleic acid, and a 3-fold increase in polyunsaturated fatty acids when the cells were grown in linoleic acid-supplemented media. Electron-spin resonance studies with the 5- nitroxystearate probe demonstrated that there was no appreciable change in microsomal lipid mobility as measured by the order parameters. In contrast, changes in lipid mobility were detected with the spin-label probe when microsomes were first isolated from Friend erythroleukemia cells and subsequently modified by incubation with liposomes composed of either dioleoyl- or dilinoleoylphosphatidylcholine plus bovine liver phospholipid-exchange protein. The fatty acid compositional changes produced in these microsomes were similar to those obtained when the intact cells were grown in media containing supplemental fatty acids. These findings indicate that the lipid mobility of Friend cell microsomes can be altered by phospholipid replacements in vitro, but that this does not occur when similar microsomal fatty acid modifications are produced during culture of the intact cell.

Animals↗
PubMed1984

Partial purification and properties of thermostable intracellular amylases from a thermophilic Bacillus sp. AK-2.

Intracellular thermostable amylases from a thermophilic Baccilus sp. AK-2 have been isolated and purified. The crude enzyme, having pH optimum at 6.5. and temperature optimum at 68 degrees C was purified by DEAE-cellulose column chromatography. Three separable enzyme fractions having starch hydrolyzing property were eluted by lowering the pH from 8.5 to 7.0. Electrophoretic mobility of these fractions showed a single band. Calcium ion up to a concentration of 20 mM had an activating effect on the three fractions. The optimum temperature for the three fractions (FI, FII and FIII) was 65 degrees C and the pH optimum for each was 6.0, 6.5 and 6.0, respectively. The -SH group in the amylase molecule was essential for enzyme activity. Except for Ca2+, Mg2+, Sr2+ and Mn2+ all other metal ions studied inhibited both alpha and beta-amylase activities. EDTA showed dose dependent non-competitive inhibition. Product formation studies proved FI and FIII to be of the alpha-amylase type and FII of the beta-amylase type. The Km for the substrate (starch) in the presence or absence of EDTA was 0.8 X 10(-3) and 1.13 X 10(-3) g/ml for alpha-amylase and beta-amylase, respectively.

Amylases↗