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PubMed2006

Solvent-free lipase-catalyzed preparation of long-chain alkyl phenylpropanoates and phenylpropyl alkanoates.

An enzymatic method was developed for the preparation of medium- or long-chain alkyl 3-phenylpropenoates (alkyl cinnamates), particularly alkyl hydroxy- and methoxy-substituted cinnamates such as oleyl p-coumarate and oleyl ferulate. The various alkyl cinnamates were formed in high to moderate yield by lipase-catalyzed esterification of cinnamic acid and its analogues with fatty alcohols in vacuo at moderate temperatures in the absence of drying agents and solvents. Immobilized Candida antarctica lipase B was the most effective biocatalyst for the various esterification reactions. The relative esterification activities were of the following order: dihydrocinnamic > cinnamic > 3-methoxycinnamic > dihydrocaffeic approximately 3-hydroxycinnamic > 4-methoxycinnamic > 2-methoxycinnamic > 4-hydroxycinnamic > ferulic approximately 3,4-dimethoxycinnamic > 2-hydroxycinnamic acid. With respect to the position of the substituents at the phenyl moiety, the esterification activity increased in the order meta > para > ortho. Rhizomucor miehei lipase demonstrated moderate esterification activity. Compounds with inverse chemical structure, that is, 3-phenylpropyl alkanoates such as 3-(4-hydroxyphenyl)propyl oleate, were also obtained in high yield by esterification of fatty acids with the corresponding 3-phenylpropan-1-ols.

Alkylation↗

PubMed2002

Characterization of two phases of chlorophyll formation during greening of etiolated barley leaves.

The esterification kinetics of chlorophyllide, obtained by a single flash of light, were investigated in etiolated barley ( Hordeum vulgare L.) and oat ( Avena sativa L.) leaves. A rapid phase, leading to esterification of 15% of total chlorophyllide within 15-30 s, was followed by a lag-phase of nearly 2 min and a subsequent main phase, leading to esterification of 85% of total chlorophyllide within 30-60 min. The presence of additional protochlorophyllide, produced in the leaves by incubation with 5-aminolevulinate, did not change the esterification kinetics. The rapid phase was identical after partial (11-15%) and full (>80%) photoconversion of protochlorophyllide; the ability for a second rapid esterification phase was restored in a dark period of at least 10 min. Cooling the leaves to 0 degrees C abolished the esterification of the main phase while the rapid phase remained unchanged. The prolamellar bodies were already in part transformed into prothylakoid-like structures within 2-5 min after a full flash but not after a weak flash (11% photoconversion); in the latter case, the corresponding transformation required a dark period of about 45 min. The existence of subcomplexes of prolamellar bodies containing NADPH:protochlorophyllide oxidoreductase and chlorophyll synthase in the ratio 7:1 is discussed.

Aminolevulinic Acid↗

PubMed1991

Platelet secreted lipoprotein-like particle is taken up by the macrophage scavenger receptor and enhances cellular cholesterol accumulation.

Enhanced macrophage cholesterol accumulation is associated with foam cell formation in the atherosclerotic lesion. Since platelet activation plays an important role in atherogenesis, we questioned whether products released from activated platelets could affect macrophage cholesterol metabolism. The addition of platelet-conditioned medium (PCM, obtained from collagen activated platelets) to a J-774 macrophage cell line, enhanced cellular cholesteryl ester content by 32%. The cholesterol esterification rate was also increased by 29%. Pre-loading the macrophages with cholesterol by incubation with acetyl-LDL, resulted in a further elevation of 48% in PCM-mediated cholesterol esterification. Possible mechanisms for the enhanced cholesterol esterification by J-774 macrophages following incubation with PCM include increased cholesterol influx and/or decreased cholesterol efflux (These cells were recently shown not to synthesize cholesterol). However, both increased uptake of PCM cholesterol by the macrophages as well as increased cellular cholesterol efflux (by 22%) were noted. The enhancement of cholesterol esterification by PCM was competitively inhibited by fucoidin and polyinosinic acid, implicating PCM binding to the scavenger receptor. This was further evidenced by the observations that apolipoprotein E which reduces cellular uptake via the scavenger receptor but not via the LDL receptor, also inhibited the effect of PCM, whereas IgG C-7, the LDL receptor antibody, did not alter the effect of PCM. Lysosomal involvement in the cellular processing of PCM was observed since PCM activity was inhibited by the lysosomal inhibitor, chloroquine. Partial purification of PCM by gel filtration revealed that the cholesterol component was associated with both phospholipids and proteins in a lipoprotein-like particle. Delipidation of PCM resulted in its inactivation but both heat treatment and tryptic digestion of PCM, revealed that the protein (and not only the cholesterol) component was also essential for the effect of PCM on cellular cholesterol esterification. Furthermore, PCM prepared from platelets of a patient with Gray Platelet Syndrome that lack platelet alfa granules (which contain platelet specific proteins), failed to enhance cholesterol esterification. These results demonstrate that lipoprotein-like particles released during platelet activation can interact with the macrophage scavenger receptor thus leading to enhanced cellular cholesterol accumulation.

Animals↗

PubMed1992

Differential interaction of lecithin-retinol acyltransferase with cellular retinol binding proteins.

Esterification of retinol (vitamin A alcohol) with long-chain fatty acids by lecithin-retinol acyltransferase (LRAT) is an important step in both the absorption and storage of vitamin A. Retinol in cells is bound by either cellular retinol binding protein (CRBP), present in most tissues including liver, or cellular retinol binding protein type II [CRBP(II)], present in the absorptive cell of the small intestine. Here we investigated whether retinol must dissociate from these carrier proteins in order to serve as a substrate for LRAT by comparing Michaelis constants for esterification of retinol presented either free or bound. Esterification of free retinol by both liver and intestinal LRAT resulted in Km values (0.63 and 0.44 microM, respectively) similar to those obtained for esterification of retinol-CRBP (0.20 and 0.78 microM, respectively) and esterification of retinol-CRBP(II) (0.24 and 0.32 microM, respectively). Because Kd values for retinol-CRBP and retinol-CRBP(II) are 10(-8)-10-(-10) M, these similar Km values indicated prior dissociation is not required and that direct binding protein-enzyme interaction must occur. Evidence for such interaction was obtained when apo-CRBP proved to be a potent competitive inhibitor of LRAT, with a KI (0.21 microM) lower than the Km for CRBP-retinol (0.78 microM). Apo-CRBP(II), in contrast, was a poor competitor for esterification of retinol bound to CRBP(II). Apo-CRBP reacted with 4 mM p-(chloromercuri)benzenesulfonic acid lost retinol binding ability but retained the ability to inhibit LRAT, confirming that the inhibition could not be explained by a reduction in the concentration of free retinol.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Chloromercuribenzenesulfonate↗

PubMed1976

The glycerol phosphate, dihydroxyacetone phosphate and monoacylglycerol pathways of glycerolipid synthesis in rat adipose-tissue homogenates.

1. Fat-free homogenates from the epididymal fat-pads of rats were used to measure the rate of palmitate esterification with different substrates. The effectiveness of the acyl acceptors decreased in the order glycerol phosphate, dihydroxyacetone phosphate, 2-octadecenyl-glycerol and 2-hexadecylglycerol. 2. Glycerol phosphate and dihydroxyacetone phosphate inhibited their rates of esterification in a mutually competitive manner. 3. The esterification of glycerol phosphate was also inhibited in a partially competitive manner by 2-octadecenylglycerol and to a lesser extent by 2-hexadecylglycerol. However, glycerol phosphate did not inhibit the esterification of 2-octadecenylglycerol. 4. The esterification of dihydroxyacetone phosphate and 2-hexadecylglycerol was more sensitive to inhibition by clofenapate than was that of glycerol phosphate. Norfenfluramine was more effective in inhibiting the esterification of 2-hexadecylglycerol than that of glycerol phosphate or dihydroxyacetone phosphate. 5 It is concluded that rat adipose tissue can synthesize glycerolipids by three independent routes.

Acyltransferases↗

PubMed1986

Inhibition of fatty acid incorporation into adipose tissue triglycerides in Ehrlich ascites tumor-bearing mice.

Using a recently developed technique of direct tracer injection into selective adipose tissue sites (Baker et al., Mech. Ageing Dev., 27: 295-313, 1984), we have studied the esterification of free fatty acids (FFA) to triglyceride fatty acids in the epididymal fat pads of normal and Ehrlich ascites carcinoma-bearing mice. We have tested the hypothesis that, during Ehrlich ascites carcinoma growth, a defect develops, resulting in the inhibition of the esterification and incorporation of FFA into adipose tissue diglyceride and triglyceride fatty acids. Our technique allowed the measurement of the disappearance of [1-14C]palmitic acid as FFA and its incorporation into di- and triglyceride fatty acids over 1 h. Multicompartmental analysis was used to compute the fractional rates of esterification and turnover. Using measured FFA pool sizes and assuming near-steady-state conditions, we estimated the transport rates (mass/time) of fatty acid esterification and turnover. Our results indicate that, compared to controls (normal mice), the epididymal fat pads of mice bearing early (5-day) and advanced (9-day) Ehrlich ascites carcinoma, respectively, show: 65% and near complete (congruent to 99%) decreases in the fractional rates of FFA esterification; about 2- and 24-fold increases in the FFA pool sizes; and 40% and 70% decreases in the transport rates of esterification.

Adipose Tissue↗

PubMed1976

Factors affecting fatty acid oxidation in fat cells isolated from rat white adipose tissue.

Fat cells isolated from rat epididymal adipose tissue were incubated with albumin-bound [14C]palmitate. Incorporation of 14C into 14CO2 and glycerides was measured. Some evidence is presented to suggest that the exogenous palmitate pool is in isotopic equilibrium with intracellular precursors for these metabolic processes. Precautions were taken to minimize dilution of the exogenous palmitate pool by fatty acids released from the cells. 14CO2 production from [1-14C]palmitate was 3 times that from [16-14C]palmitate. Octanoate increased this differential oxidation of palmitate carbons and also inhibited palmitate oxidation without similarly affecting esterification. Glucose increases palmitate esterification in cells from fed or starved rats. Insulin potentiated this effect of glucose. Glucose influenced palmitate oxidation in a more complex manner, dependent upon the glucose concentration. Both the observation that esterification constitutes 99% of the metabolic flux of fatty acid and the manner in which glucose, insulin, or starvation influence palmitate esterification and oxidation suggested that factors controlling esterification may alter oxidation as a secondary effect, but not vice versa. It is suggested that oxidation and esterification compete for a single intracellular precursor, possibly extramitochondrial long chain fatty acyl CoA.

Adipose Tissue↗

PubMed1992

Use of lipases in the resolution of racemic ibuprofen.

Resolution of (R,S)-ibuprofen enantiomers by esterification in different organic solvents was studied using Candida cylindracea lipase. This enzyme preparation had high enantiospecificity for S(+)-ibuprofen in the esterification reaction of a racemic ibuprofen with primary alcohols. The esterification yields of secondary alcohols were much lower than those of primary alcohols. Esterification with tertiary alcohols was not observed. The synthesis of esters was profoundly affected by the amount of water in the reaction mixture. C. cylindracea lipase was active only in very hydrophobic solvents. The esterification activity of the lipase was reduced significantly by addition of water. The R- and S-enantiomers of ibuprofen were determined without derivatization by HPLC using a chiral column.

Alcohols↗

PubMed1989

Preferential redistribution of lipoprotein-unassociated apoA-IV to an HDL subpopulation with a high degree of LCAT modification.

The relationship between LCAT mediated HDL modification and the redistribution of lipoprotein-unassociated apoA-IV to HDL was investigated in vitro. Immunoaffinity-isolated rat lipoprotein-unassociated apoA-IV was added to apoB-, apoE-, apoA-IV depleted, [3H]cholesterol labelled rat plasma and incubated at 37 degrees C. The addition of lipoprotein-unassociated apoA-IV resulted in a modest (10%) but significant reduction in the rate of cholesterol esterification. Incubations conducted in the presence of active LCAT led to a time-dependent increase in the amount of the 3H label retained by an anti-apoA-IV immunoaffinity column. Lipoproteins retained by the anti-apoA-IV immunoaffinity column had experienced a greater conversion of [3H]cholesterol to [3H]cholesteryl esters (48% esterification at 30 min) than the unretained lipoproteins (19% esterification at 30 min). These data suggest that during the course of LCAT-induced cholesterol esterification, lipoprotein-unassociated apoA-IV transfers to a subpopulation of HDL which has been modified by LCAT to a greater extent than the remaining HDL. Further analysis of the data demonstrates that 48% cholesterol esterification is sufficient to allow apoA-IV to be accommodated on the surface of an HDL particle.

Animals↗

PubMed1996

Liver fatty acid-binding protein expression in transfected fibroblasts stimulates fatty acid uptake and metabolism.

The role of cytosolic liver fatty acid binding protein (L-FABP) in fatty acid uptake and metabolism was examined using cultured L-cell fibroblasts transfected with the cDNA encoding for L-FABP. [3H]Oleic acid was used to determine the effects of intracellular esterification on fatty acid uptake and to determine esterified fatty acid localization to specific lipid classes. cis-Parinaric acid, a poorly esterified fatty acid, was used to determine uptake in the absence of any appreciable esterification. High-expression L-cells had a 80% and 50% greater initial uptake rate for both [3H]oleic acid and cis-parinaric acid, respectively compared to low-expression L-cells. Maximal uptake of [3H]oleic acid did not plateau because of intracellular esterification. In high-expressing cells, maximal cis-parinaric acid uptake rapidly plateaued at a level 34% higher than in low-expression cells. After 1 min of incubation, the majority of cellular [3H]oleic acid was unesterified, with the bulk of the esterified portion preferentially localized to phospholipids. After 5 and 30 min, cells expressing L-FABP esterified a significantly greater amount of [3H]oleic acid into both the neutral lipid and phospholipid fractions than did low-expression cells. L-FABP expression also selectively stimulated [3H]oleic acid incorporation into choline glycerophospholipids. Thus, L-FABP expression not only stimulated fatty acid uptake at all time points, but also stimulated intracellular esterification into specific lipid pools. These results show in detail for the first time using an intact cell culture system that L-FABP expression not only stimulated fatty acid uptake, but also increased intracellular esterification of exogenously supplied fatty acids.

Animals↗

PubMed1994

Lipoprotein phospholipid composition and LCAT activity in nephrotic and analbuminemic rats.

Albumin is an acceptor of lysophosphatidylcholine (LPC), product of the lecithin:cholesterol acyl transferase (LCAT) reaction, and it has been suggested that low LCAT activity and reduced cholesterol esterification rate in patients with the nephrotic syndrome may be linked to depletion of albumin. Effects of low plasma albumin levels on LCAT activity, cholesterol esterification rates and LPC-binding were therefore studied in hyperlipidemic nephrotic (NS) and analbuminemic (NAR) rats. LPC-binding was also measured in normoalbuminemic rats with dietary hypercholesterolemia. Remarkably, LCAT activity, measured with excess exogenous substrate, was not decreased but increased in both NAR and NS rats. Molar esterification rates with endogenous substrate were increased in NAR but normal in NS rats. In normoalbuminemic rats, with or without hypercholesterolemia, LPC was primarily found in the lipoprotein-deficient plasma and the HDL3 fraction. In NAR and NS rats LPC levels were increased in lipoproteins (notably in LDL and HDL2), but, in marked contrast to normoalbuminemic rats, decreased in lipoprotein-deficient plasma. Phosphatidylcholine, quantitatively the major phospholipid, was distributed proportionally over the lipoproteins in NS, NAR and control rats. Therefore, in hypoalbuminemia and analbuminemia LPC is mainly bound to lipoproteins, which is in contrast to the paucity of LPC in these particles in normoalbuminemic rats. Cholesterol esterification in nephrotic plasma is thus not impaired by lack of an acceptor for LPC-binding. The absence of an increase in molar cholesterol esterification in conjunction with increased LCAT activity points to a possible defect of the substrate for this reaction in nephrotic plasma. Increased LPC levels in LDL, a characteristic of oxidized LDL, may be a hitherto unrecognized atherosclerotic risk factor in the nephrotic syndrome.

Animals↗

PubMed1982

Role of glycerol 3-phosphate and glycerophosphate acyltransferase in the nutritional control of hepatic triacylglycerol synthesis.

1. Glycerol 3-phosphate content of isolated hepatocytes from starved rats and of glycogen-depleted hepatocytes from fed rats was low and severely limited triacylglycerol synthesis. 2. Raising the glycerol 3-phosphate content by addition of precursors to the cells resulted in a hyperbolic-like relationship between triacylglycerol synthesis and cellular glycerol 3-phosphate content. Statistical analysis of the curves showed no significant differences between the nutritional states either at saturating or at subsaturating glycerol 3-phosphate content. 3. V(max.) of glycerophosphate acyltransferase measured in homogenized hepatocytes was decreased by 30-40% in starvation. There was no change in apparent K(m) for glycerol 3-phosphate. Since at saturating glycerol 3-phosphate content esterification rates in hepatocytes of both nutritional states were identical, the enzyme is not limiting esterification under this condition. 4. At subsaturating glycerol 3-phosphate content the flux through glycerophosphate acyltransferase necessarily limits esterification. Therefore one would expect a decrease in esterification in starvation under this condition. This was the case when triacylglycerol synthesis was plotted against intracellular glycerol 3-phosphate concentration, calculated from the cellular glycerol 3-phosphate content and the intracellular water space, which was smaller in hepatocytes from starved rats. 5. The data obtained in hepatocytes were extrapolated to the intact liver by using the number of parenchymal cells per g of liver as determined from marker-enzyme analysis and the liver weight per 100g body weight. The extrapolation suggested that glycerol 3-phosphate is limiting esterification in vivo for contents below 0.3-0.4 and 0.5-0.65mumol/g for livers from fed and starved animals respectively. Also for a given fatty acid load and a glycerol 3-phosphate content below 0.3mumol/g the liver may esterify less in the starved state. However, at the glycerol 3-phosphate contents measured in freeze-clamped livers (0.30 and 0.44mumol/g for the fed and starved state respectively), livers in both nutritional states seemed capable of esterifying similar amounts of fatty acids.

Acyltransferases↗

PubMed2000

Cholesterol efflux from normal and Tangier disease fibroblasts into normal, high-density lipoprotein-deficient, and apolipoprotein E-deficient plasmas.

Tangier disease (TD) fibroblasts have defective cholesterol release in the presence of lipid-free apolipoproteins. We compared normolipidemic probands and patients with apolipoprotein A-I (apoA-I) deficiency, apoE deficiency, or TD in terms of the plasma capacity to induce the efflux of [3H]-cholesterol from normal and TD fibroblasts and to esterify this cell-derived cholesterol. Compared with normal fibroblasts, TD fibroblasts released a significantly smaller fraction of [3H]-cholesterol into normal, high-density lipoprotein (HDL)-deficient, and apoE-deficient plasmas. Supplementation of apoE-deficient plasma with exogenous apoE normalized the cholesterol efflux from normal cells but did not fully restore the reduced cholesterol efflux from TD fibroblasts. Compared with control plasma, HDL- and apoE-deficient plasmas had a significantly reduced activity to esterify cell-derived cholesterol. Cholesterol derived from TD fibroblasts was less available for esterification in either patient or normal plasmas than cholesterol derived from normal cells. The esterification defect of TD cell-derived cholesterol was more pronounced in patient plasmas than in control plasma. We conclude that (1) apoA-I and, to a lesser degree, apoE are important determinants of the cholesterol efflux and esterification capacity of plasma, (2) TD fibroblasts have a reduced capacity to release cholesterol into the plasma, and (3) TD cell-derived cholesterol is less available for esterification in plasma than cholesterol from normal fibroblasts. The absence of distinct apoA-I- or apoE-containing subclasses aggravates the defective efflux and esterification of cholesterol derived from TD cells.

Adult↗

PubMed1975

On the reactivity of carboxyl groups of ribonuclease-A in anhydrous methanol.

The esterification of Ribonuclease-A in methanol/0.1 M hydrochloric acid has been studied by measuring the decrease in the number of titratable groups of the protein and estimating the amount of methanol incorporated. Esterification of nearly five of the 11 free carboxyl groups of the protein resulted in almost complete inactivation of the enzyme. The initial products of esterification have been chromatographed on Amberlite columns, and five partially active methyl ester derivatives of Ribonuclease-A have been isolated. The dimethyl ester, the initial product of esterification with reduced catalytic activity, has the carboxyl groups of Glu-49 and Asp-53 modified. Even in the non-aqueous solvent, as in the native structure of the protein in aqueous solution, these carboxyl groups are the fast reacting ones. Subsquently, the esterification reaction appears to proceed preferentially at the C-terminal region of the molecule. Comparison of the reactivities of carboxyl groups of Ribonuclease-A in acidic methanol to that known in aqueous solutions (with carbodiimides) suggests that the structure of Ribonuclease-A in the non-aqueous solvent resembles, at least in part, the structure in aqueous environment.

Carboxylic Acids↗

PubMed2002

Chylomicron and palmitate metabolism by perfused hearts from diabetic mice.

Hydrolysis of triacylglycerols (TG) in circulating chylomicrons by endothelium-bound lipoprotein lipase (LPL) provides a source of fatty acids (FA) for cardiac metabolism. The effect of diabetes on the metabolism of chylomicrons by perfused mouse hearts was investigated with db/db (type 2) and streptozotocin (STZ)-treated (type 1) diabetic mice. Endothelium-bound heparin-releasable LPL activity was unchanged in both type 1 and type 2 diabetic hearts. The metabolism of LPL-derived FA was examined by perfusing hearts with chylomicrons containing radiolabeled TG and by measuring (3)H(2)O accumulation in the perfusate (oxidation) and incorporation of radioactivity into tissue TG (esterification). Rates of LPL-derived FA oxidation and esterification were increased 2.3-fold and 1.7-fold in db/db hearts. Similarly, LPL-derived FA oxidation and esterification were increased 3.4-fold and 2.5-fold, respectively, in perfused hearts from STZ-treated mice. The oxidation and esterification of [(3)H]palmitate complexed to albumin were also increased in type 1 and type 2 diabetic hearts. Therefore, diabetes may not influence the supply of LPL-derived FA, but total FA utilization (oxidation and esterification) was enhanced.

Albumins↗

PubMed2006

Basal lipolysis in epididymal fat cells from streptozotocin-induced diabetic rats.

The level of free fatty acid (FFA) in plasma is increased by diabetes. The increase in plasma FFA levels accompanied the stimulation of basal lipolysis (i.e. lipolysis in the absence of lipolytic agents) in fat cells. Injection of streptozotocin with rats resulted in a significant increase in basal FFA production (5.5 fold) in fat cells. However, basal glycerol production in fat cells was increased only 1.5 fold by streptozotocin-induced diabetes, implying that FFA re-esterification in fat cells was decreased by streptozotocin-induced diabetes. The FFA re-esterification in fat cells was also decreased by 1 d of fasting. Although basal lipolysis was increased by streptozotocin-induced diabetes or 1-d fasting, neutral triacylglycerol lipase activity and the immunoreactive HSL protein content in fat cells from streptozotocin-induced diabetic rats or 1-d fasting rats were not significantly changed. Although beta-blockers inhibited lipolysis induced by norepinephrine at a concentration of 10(-4) M, it failed to inhibit the basal lipolysis and FFA re-esterification in fat cells from streptozotocin-induced diabetic rats. Nor did insulin or H-89, another antilipolytic agent, affect basal lipolysis or FFA re-esterification in fat cells from streptozotocin-induced diabetic rats. These results indicate that basal FFA production may be induced by a decrease of re-esterification of FFA in diabetic rats and is not affected by antilipolytic agents such as insulin, beta-blockers or H-89.

Adenosine↗

PubMed1992

Glycerolipid synthesis in isolated adipocytes: substrate dependence and influence of norepinephrine.

Studies of fatty acid (FA) esterification by adipocytes have led to conflicting views with respect to how the process is regulated by norepinephrine (NE). It remains unclear whether NE directly modulates the pathway or whether its effects are indirect and reflect its well-known action to activate lipolysis. Changes in lipolysis can complicate estimation of esterification rates by altering both medium FA and the hydrolysis of newly formed FA esters. In this report, we describe an experimental approach that determined the effect of NE on FA esterification, amidst the complications introduced by activation of lipolysis. Esterification rates were estimated from the simultaneous incorporations (0.1-60 min) of [14C]glucose and [3H]oleate into diglyceride (DG), phospholipid (PL), and triglyceride (TG). Saturation kinetics of incorporation rates, with respect to FA, and more specifically to unbound or albumin-free FA (ubFA), were determined in both basal and NE-treated cells. To obtain true estimates of ester synthesis, incorporation rates were adjusted for label loss from breakdown of labeled esters. Our findings were: 1) In basal versus NE-treated cells, [3H]oleate, on its pathway to esterification, was diluted, respectively, by 2 and 50% of measured cell FA, and the diluting FA appeared derived from lipolysis. 2) Syntheses of PL, DG, and TG, estimated from incorporation of [14C]glucose, saturated at low ubFA. The Km for TG synthesis (0.06 microM) was within the physiological range of ubFA which meant that changes in plasma FA will modulate TG synthesis. PL synthesis, on the other hand (Km less than 0.01 microM), would be largely saturated under physiological conditions. 3) NE treatment increased the molar ratio of FA to albumin in the medium an average 8-fold and ubFA about 87-fold. In addition, NE accelerated hydrolysis of labeled PL and DG. Adjusting incorporation rates for these changes indicated that NE does not directly regulate glyceride synthesis. The assays described should allow estimation of glycerolipid synthesis under various metabolic or disease states and will distinguish direct effects from those reflecting changes in FA concentration or in hydrolysis of labeled FA esters.

Adipose Tissue↗

PubMed1983

Prevention of the hyperlipidemic serum or LDL-induced cellular cholesterol ester accumulation by 22-hydroxycholesterol and its analogue.

Incubation of monkey arterial smooth muscle cells with hyperlipidemic serum or low-density lipoproteins (LDL) produces intracellular cholesterol ester accumulation. Increased esterification of free cholesterol within the cell may account for this effect. To examine such a possibility, oxygenated sterols were used to block cholesterol esterification. The increased esterification of free cholesterol by smooth muscle cells and human skin fibroblasts, through their exposure to hyperlipidemic lipoproteins, was inhibited by 22-hydroxycholesterol and its analogue, SC-32561 (22-hydroxy-25-fluorocholesterol). The hyperlipidemic LDL-stimulated elevation in the cholesterol ester content of the smooth muscle cells was also prevented by these sterols. This reduction in cellular cholesterol ester did not coincide with an increase in free cholesterol. 22-Hydroxycholesterol also blocked the stimulation of the esterification of cholesterol due to 25-hydroxycholesterol. In the absence of lipoproteins, 22-hydroxycholesterol and SC-32561 had a minor effect on the incorporation of [14C]oleate into cholesterol esters, and efficiently reduced sterol synthesis in fibroblasts and smooth muscle cells. 22-Hydroxycholesterol and SC-32561 had the additional effect of lowering the number of cell-surface LDL receptors to a greater extent than did hyperlipidemic LDL. The presence of 22-hydroxycholesterol did not alter the interaction of normal LDL with the receptor. Oxygenated sterols recovered in the cell represented 4-21% of the total sterol content. The level of intracellular oxygenated sterols was significantly reduced by the presence of lipoproteins in the culture media. Due to the multiple effects of the oxysterols, they were not effective as tools in determining the contribution of acyl-coenzyme A:cholesterol acyltransferase enzyme activity to the intracellular pool of cholesterol esters. These results indicated that 22-hydroxycholesterol and SC-32561 effectively blocked the hyperlipidemic LDL-stimulated increase in smooth muscle cell cholesterol ester content by lowering cholesterol esterification, reducing cholesterol synthesis and down-regulating LDL cell-surface receptors.

Animals↗