Cholesteryl ester metabolism in tissue culture cells: II. Source of accumulated esterified cholesterol in Fu5AH rat hepatoma cells

Previous investigations had demonstrated that Fu5AH rat hepatoma cells accumulated large quantities of esterified cholesterol when grown in hyperlipemic rabbit serum. The present investigation has determined the sources of the cellular esterified cholesterol when the cells were grown in hyperlipemic serum. Cellular esterification of endogenous and exogenous free cholesterol contributed 10% and 30%, respectively. The remaining 60% of the accumulated cellular esterified cholesterol was derived from exogenous (serum) cholesteryl esters. Evidence for the hydrolysis of a portion of the incorporated esterified cholesterol is presented. A stimulation of free cholesterol incorporation and cellular esterification is elicited by hyperlipemic serum and serum lipoproteins when compared to normolipemic serum present at equivalent exogenous cholesterol concentrations. The effect of hyperlipemic serum is reduced by Tween-80 and Triton WR-1339. Comparative data on esterified cholesterol accumulation, free cholesterol incorporation, and cellular cholesterol esterification in Fu5-5 rat hepatoma cells, L-cells, and rabbit aortic medial cells are presented. This work was done during the tenure as Established Investigator of the American Heart Association.     

Cholesteryl ester metabolism in tissue culture cells. I. Accumulation in Fu5AH rat hepatoma cells

Exposure of Fu5AH rat hepatoma tissue culture cells to hyperlipemic rabbit serum results in the accumulation of cellular cholesteryl esters. Accumulation is not a characteristic of all cells in culture, as evidenced by the lack of response of mouse and human fibroblasts. Fu5AH cells grown for 24 hr on 5% hyperlipemic rabbit serum have an 8- to 12-fold increase of cellular cholesteryl esters, small increases in free cholesterol and triglycerides, and no change in phospholipids, when compared to cells grown in normal rabbit serum. Rapid accumulation of cholesteryl esters occurs during the first 8–12 hr of incubation, and maximum cellular concentration is achieved within 24 hr. The maximum level of cellular cholesteryl esters obtained with individual samples of hyperlipemic rabbit serum is correlated with the cholesterol content of the original sera, even when the incubation medium is adjusted to a constant concentration of cholesterol. Heating hyperlipemic rabbit serum (60 C/30 min) does not destroy activity; however, no cholesteryl ester accumulation occurs in heated cells. The stimulatory activity of hyperlipemic rabbit serum primarily is associated with lipoproteins having densities <1.006. High levels of cellular cholesteryl ester are associated with the appearance of cytoplasmic vacuoles containing cholesteryl esters. The increase in cellular cholesteryl esters is accompanied by a decrease of the cholesteryl esters in the growth medium. Cellular cholesteryl esters are not rapidly hydrolyzed or released upon removal of hyperlipemic rabbit serum.     

Inhibition of cholesterol esterification in macrophages and vascular smooth muscle foam cells: Evaluation of E5324, an Acyl-CoA cholesterol acyltransferase inhibitor

Cholesteryl esters (CE) comprise the principal lipid class that accumulates within macrophages and smooth muscle cells of the atherosclerotic lesion. Acyl-CoA cholesterol acyl-transferase (ACAT) is the major enzyme responsible for esterification of intracellular cholesterol. We evaluated the ability of E5324 (n-butyl-N″-[-2-[3-(5-ethyl-4-phenyl-1H-imidazol-1-yl)propoxyl]-6-methyl-phenyllurea), a novel, orally absorbable ACAT inhibitor, to inhibit esterification of fatty acids to cholesterol and CE accumulation in macrophages and in smooth muscle cells. E5324 significantly inhibited cholesterol esterification in rat aortic smooth muscle cells and in macrophages. In addition, E5324 reduced the cellular mass of CE, the significant measure of the efficacy of drugs designed to modulate cholesterol metabolism. E5324 treatment of macrophages exposed to acetylated low-density lipoprotein reduced CE mass by 97%, and treatment of lipid-loaded smooth muscle cells reduced CE mass by 29%. Although free cholesterol increased approximately twofold, this free cholesterol would presumably be accessible to the membrane for effluxin vivo (reverse cholesterol transport). These results demonstrate that E5324 can inhibit cholesterol esterification and CE mass in atherosclerotic foam cells, derived from either macrophages or arterial smooth muscle cells.     

Lipids of cultured hepatoma cells: IV. Effect of serum and lipid upon cellular and media neutral lipids

Minimal deviation hepatoma 7288C cells were cultured in a modified Swim's medium supplemented with decreasing levels of serum, lipid-free serum, lipid-free serum plus fatty acids, and other additives. Cellular and media neutral lipid classes were quantitated, the fatty acids of triglycerides and sterol esters analyzed, and the carbon number distribution of triglycerides determined. Cellular triglyceride biosynthesis virtually was inhibited when the medium was supplemented with bovine serum alone. This inhibition was not observed when the medium was supplemented with fetal calf serum alone or mixtures of fetal calf serum and bovine serum. Cells cultivated on medium supplemented with lipid-free serum plus palmitic or linoleic acids had much lower levels of free and esterified cholesterol. The fatty acid composition of cellular triglycerides and cholesterol esters differed dramatically from the corresponding media lipid classes. Except when linoleic acid was added to the medium, changes in the media serum and lipid levels had only marginal effects upon the fatty acid composition of cellular triglycerides and cholesterol esters. These data, in conjunction with earlier data that showed the media neutral lipid levels did not decrease during cell growth, indicate that these hepatoma cells utilize little or no serum triglycerides and cholesterol esters. Linoleic acid added to the medium dramatically reduced the level of 18∶1 acids in cellular triglycerides and cholesterol esters. Palmitic acid added to the medium did not change the fatty acid compositions significantly. Comparison of experimentally determined and calculated triglyceride carbon number percentages indicated a random distribution of fatty acids in this glyceride. The fatty acid composition of cellular triglycerides was similar to the composition of the cholesterol esters. The lack of characteristic and distinguishable compositions of these two classes that occur in most normal tissues suggests a loss of specificity in the lipid metabolism of this neoplasm at the class level.     

Cholesterol metabolism in human monocyte-derived macrophages: Stimulation of cholesteryl ester formation and cholesterol excretion by serum lipoproteins

The role of lipoproteins and serum in the formation and accumulation of cholesteryl esters in human monocyte-derived macrophages (HMD macrophages) was investigated; studies were also carried out with IC21 cells (a cell line derived from mouse peritoneal macrophages). Following preincubation of HMD macrophages with lipoprotein-depleted serum (LPDS), both native and acetylated low density lipoprotein (LDL and AcLDL, respectively) stimulated the formation of cholesteryl esters with a resultant increase in cellular cholesteryl ester content. Cholesteryl ester formation and accumulation was also stimulated in macrophages exposed continuously to 25-hydroxycholesterol. However, the stimulation of cholesterol esterification by either lipoproteins or 25-hydroxycholesterol was not inhibited by progesterone in HMD macrophages, but was in the IC21 cells. Cholesterol efflux and the hydrolysis of cellular cholesterol ester, promoted by serum components, were studied in HMD macrophages preloaded with cholesteryl ester by incubation with 25-hydroxy cholesterol. Replacement of the medium with one devoid of 25-hydroxycholesterol resulted within 24 hr in at least a 30% decrease in the cholesteryl ester content of the HMD macrophages; replacement with a medium high in cholesterol acceptor content (LPDS or high density lipoprotein) and incubation for three days led to the most marked decreases in cellular cholesterol content. Thus, hydrolysis of the cholesteryl esters by HMD macrophages was not dependent on the presence of cholesterol acceptors in the medium, but cellular cholesterol content was.     

A model for studying LCAT reaction: In vitro cholesterol esterification in pig ovarian follicular fluid

Phosphatidylcholine acyltransferase (lecithin:cholesterol acyltransferase or LCAT; EC 2.3.1.43) activity was found to be present in pig ovarian follicular fluid (POFF), in addition to pig serum (PS). The cholesterol esterification rate in both POFF and PS is linear with incubation time up to 2 hr. The mean absolute rate of POFF-cholesterol esterification was 8.1±0.4 nmoles per ml per hr approximately one-fourth of that in PS. However, the fractional rate (percent of labeled cholesterol esterified per hr) of POFF-cholesterol esterification was similar to that observed in PS. There was little variation of absolute rate of cholesterol esterification in the fluid obtained from different sizes of follicles. Fatty acid or triacylglycerol did not participate in the reaction of cholesterol esterification in POFF. No appreciable change in enzymatic activity was found from storing POFF at 4 C for periods of time up to 24 hr or at −70 C up to 2 months, but activity was lost thereafter. On the other hand, PS showed a much longer period of stability (5 days at 4 C and 9 months at −70 C). A discrepancy between the fatty acid composition of cholesteryl esters formed by the LCAT reaction and the fatty acid composition at the C-2 position of phosphatidylcholine led us to propose a two-step mechanism for the LCAT reaction. It is concluded that the LCAT of POFF, as well as that of plasma, is specific for individual fatty acids rather than for the fatty acid composition of phosphatidylcholine. The fatty acid concentration of lysophosphatidylcholine decreased during prolonged incubation times (6 to 21 hr) suggesting that the increased lysophosphatidylcholine formed as a product of the LCAT reaction may be reused as substrate for the LCAT reaction or for hydrolysis by lysophosphatidylcholine hydrolase. Presented at the AOCS Meeting, New York, May 1977.     

Quantitative and qualitative lipid correlation in experimental endogenous hyperlipemia

The reversible endogenous hyperlipemia in dogs, elicited by the detergent Triton which was given intravenously, was used to study the interrelations of serum lipids. In the cholesterol ester fraction an increase occurs in both monounsaturated and in saturated fatty acids, excepting myristic; while a decrease occurs in polyunsaturated fatty acids. The fatty acids of cholesterol esters of normal dogs contain 22% oleic acid, and only 24% when serum lipids are increased to almost double their normal value (TC=400–500 mg/100 ml). However there is a critical level above which a rapid rise in oleic acid occurs and, in severe hyperlipemia (TC=1500 ±430 mg/100 ml), this acid constitutes almost half of the esterified fatty acid component. Since there is no evidence that Triton directly regulates fatty acid synthesis, the lipid fraction-fatty acid interrelationship may be secondary to lipid mobilization from endogenous sources. This concept is supported by the fact that the increased serum fatty acids are only those which can be synthesized by animals. It is suggested, on the basis of a marked increased of endogenously produced fatty acids, that, at critical lipid levels, shortage of polyunsaturated fatty acids from exogenous sources occurs. This might be of sufficient degree to accelerate fatty acid synthesis to meet the need for fatty acids for energy requirements. There may also be need of fatty acid for esterification of chiefly the accumulated free cholesterol split from lipoprotein by Triton. Triton-induced changes in cholesterol ester fatty acids result in patterns which closely resemble those in the adipose tissue of dog and man and in the serum of human endogenous hyperlipemia.     

Synthesis of cholesterol esters in the plasma and liver of sheep

A study was made with sheep on the formation in vitro of long chain fatty acid esters of cholesterol by the lecithin-cholesterol-acyltransferase system present in the plasma and the acyl CoA-cholesterol-acyltransferase system present in the liver. The rate of cholesterol esterification in the plasma was 0.024 μmoles/ml/hr. The relative pattern of fatty acids esterified during incubation of the plasma remained constant over the 8 hr period of incubation and was similar to the fatty acids in the plasma cholesteryl esters before incubation began and to the fatty acids in the 2-position of the plasma lecithin. The predominant cholesteryl esters synthesized contained monoenoic and dienoic fatty acids. Unlike the bovine, there was no apparent discrimination in favor of the 18∶2 containing species of plasma lecithin as donors of fatty acids. This difference could be accounted for by the similarity in the 18∶2 content of the phospholipids present in the high density (density >1.062 and < 1.21) and the low density (density > 1.006 and <1.063) lipoprotein fractions of the sheep plasma. The possibility of some discrimination against 20∶4 during cholesterol ester synthesis in the plasma of the sheep cannot be excluded. In the liver, the predominant cholesteryl esters synthesized contained saturated and monoenoic fatty acids; cholesteryl linoleate was synthesized to a very much less extent. There was considerable similarity between the composition of the unesterified fatty acid fraction of the liver before incubation began and the fatty acid composition of the cholesteryl esters synthesized during incubation. Addition of sonicated suspensions of free fatty acids altered markedly the fatty acid pattern of the cholesteryl esters synthesized by the liver slices. From the evidence presented it is concluded that the cholesteryl esters in sheep plasma are syntheized mainly by the plasma lecithin-cholesterol-acyltransferase system. The results are discussed in relation to cholesterol esterification systems demonstrated in the plasma and liver of monogastric animals.     

On the rate of cholesterol esterification in cord blood serum

Cholesterol esterification was studied in adult and cord serum by measureing the initial rate of lecithin-cholesterol acyl transferase (LCAT) activity. Cord serum had about one-third as much free and esterified cholesterol and about one-half as much LCAT as adult serum. When the adult LCAT activities are plotted against the individual's serum free cholesterol levels a straight line relationship results (0.101±.005% cholesterol esterified per min). Cord serum LCAT activities (.135±.0407% cholesterol esterified per min) in the main fall above the adult line. Our results show that cord serum can esterify cholesterol at a rate equal to or higher than adult serum when the LCAT activity is related to the amount of serum free cholesterol present.     

The immunosuppressive substance 2-chloro-2-deoxyadenosine modulates lipoprotein metabolism in a murine macrophage cell line (P388 cells)

A recently developed immunosuppressive substance, 2-chloro-2-deoxyadenosine (2-CdA), was reported to inhibit monocyte functions at low concentration. Because macrophages play a key role in the formation of atherosclerotic plaques, it was of interest to study the effect of 2-CdA on cellular lipid metabolism. For this purpose we have used a macrophage cell line (P388) to perform incubation studies in the presence of acetylated low density lipoprotein (Ac-LDL) and 2-CdA. The addition of 2-CdA, in concentrations ranging from 5–20 nM, induced a dose-dependent decrease in cellular cholesterol content and in the amount of extracellular [¹⁴C]oleic acid (OA) incorporated into the cholesteryl ester (CE) fraction. The effect was maximized at 20 nM 2-CdA with an 86% reduction in cholesterol esterification compared to controls (P<0.008). To evaluate the mechanism of interaction of 2-CdA with cellular lipid metabolism, deoxycytidine (dCyt) and 3-methoxybenzamide (3-MOB), substances known to antagonize the effect of 2-CdA in different ways, were co-administered with 2-CdA. dCyt, a competitive inhibitor of dCyt kinase, which catalyzes phosphorylation to the active metabolite, antagonized the effects of 20 nM 2-CdA, producing significantly greater incorporation of extracellular [¹⁴C]OA into the CE fraction than in the presence of 2-CdA alone (P<0.0086). Co-incubation with 2-CdA and the poly-ADP-ribose synthetase inhibitor 3-MOB, which is known to render cells resistant to 2-CdA toxicity by preventing cellular nicotinamide adenine dinucleotide (NAD)- and adenosine triphosphase-depletion, also reversed the effect of 2-CdA on lipid accumulation. However, incubation of P388 cells with 20 nM 2-CdA did not result in a decrease in cellular NAD content. As 20 nM 2-CdA showed no effect on intracellular cholesterol synthesis based on measurement by 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, the decrease in cellular cholesterol content and in [¹⁴C]OA incorporation seems to be primarily due to an interference with Ac-LDL metabolism.     

Lipids of cultured hepatoma cells: I. Effect of serum lipid levels on cell and media lipids

Minimal deviation hepatoma cells were cultured as monolayers to confluency in roller flasks containing modified Swim's medium, supplemented with decreasing amounts of serum, lipid-free serum, and lipid-free serum containing added fatty acids. Good cell growth was observed until serum levels fell below 5% of the medium. Media containing lipid-free serum or lipid-free serum plus linoleic or palmitic acids did not support good growth. Lipids were extracted from cells; media, obtained during the first and last half of the incubation period, resolved into neutral and phospholipid fractions; fatty acid composition of each fraction analyzed by gas liquid chromatography; and lipid class distributions compared by thin layer chromatography. The data showed that the media contained more neutral lipids and phospholipids after incubation than initially, indicating that minimal deviation hepatoma cells excreted lipids into the media. The class composition of the excreted lipids resembled that of the serum. A comparison of media, cells, and serum fatty acid compositions indicated that the lipids secreted into the media were of cellular origin. Although some differences were noted, in general, cells grown on the nine different media had the same ca. neutral lipid and phospholipid class and fatty acid compositions. In contrast, dramatic differences were observed in the class and fatty acid compositions of the serums from that of the cells and media. These results indicate that exogenous serum lipids had little influence on cellular class and fatty acid compositions of the minimal deviation hepatoma cells. This neoplasm did not contain detectable levels of glyceryl ether diesters, indicating that this compound is not characteristic of all tumors. Lipid class profiles and fatty acid compositions of cells grown on various media suggest that the minimal deviation hepatoma cells can synthesize most, if not all, neutral lipid and phosphoglyceride classes found in liver. Presented at the AOCS Meeting, New Orleans, April 1973.     

Fatty acid and sterol specificity of cholesterol esterifying enzyme in developing rat brain

The properties and fatty acid and sterol specificity of cholesterol-esterifying enzyme (EC 3.1.1.13) in rat brain were studied. The enzyme utilized free fatty acid for esterification, and activity was maximal at pH 5.6. Exogenous ATP and CoA did not stimulate the incorporation of free fatty acids into sterol esters. Substrates dispersed in Tween 20 or Triton X-100 were just as effective as the substrates dissolved in acetone solution, while dispersion in propylene glycol or sodium taurocholate was not as effective. Snake venom phospholipase A₂ (EC 3.1.1.4) increased the esterification of cholesterol in the absence of added fatty acid. The fatty acid specificity data indicated that oleic and palmitic acids were the preferred fatty acids. Little or no esterification occurred in the presence of long chain fatty acids (C₂₀–C₂₄). Esterification of cholesterol with palmitate or stearate was not affected by the presence of oleic acid in the mixture. Thus, the nonrequirement of the brain-esterifying enzyme for a bile acid or for an amphiphile such as an unsaturated fatty acid suggests that micellar solubilization of the substrate is not essential for activity. Although the brain enzyme catalyzed the esterification of desmosterol, cholesterol was the preferred substrate. Neither lanosterol (C₂₉ sterols) nor Δ7-dehydrocholesterol was esterified to any significant extent. The presence of low concentrations of desmosterol increased cholesterol esterification slightly, while there was a concentration-dependent inhibition of demosterol esterification by cholesterol. These data on fatty acid and sterol specificity of the esterifying enzyme correlate well with the composition of sterol esters present in developing rat brain.     

Control of lipid metabolism in cultured cells

Several studies are presented which indicate that composition of cell lipid is regulated by interaction between intracellular metabolism and lipid transport processes. When the fatty acid composition of cells cultured in essential fatty acid deficient conditions was studied, activation of synthesis of unusual polyun-saturated fatty acids was observed for a number of cell lines. In addition cells contained persistent residual amounts of linoleic acid, presumably owing to efficient scavenging mechanisms. The source of cell lipids was studied in both chemically defined and serum-supplemented media. In the absence of exogenous lipid, cells synthesize lipids from simple precursors, a process which is inhibited by adding serum. When serum lipid is present, cells preferentially utilize fatty acids as a source of nonsterol lipid. These are subsequently esterified intracellularly to make glycerides and phospholipids. When triglyceride is utilized as a source of cell lipid, it is first hydrolyzed before being taken up. By use of a nonhydrolyzable cholesterol ester analog, it is confirmed that both free and ester cholesterol are taken up and excreted by cells. Intracellular cholesterol content is thus regulated by rates of uptake, hydrolysis and excretion as well as by biosynthesis. One of 13 papers presented at the symposium “Lipid Metabolism in Cells in Culture,” AOCS Meeting, Houston, May 1971.     

Perfluorodecanoic acid and lipid metabolism in the rat

Alterations in lipid metabolism were axamined in adult male Sprague-Dawley rats seven days after a single intraperitoneal injection of perfluorodecanoic acid (PFDA; 20, 40 or 80 mg/kg). Because PFDA treatment caused a dose-related reduction in feed intake, the response of vehicle-treated rats pair-fed to those receiving PFDA was monitored to distinguish direct effects of the perfluorinated fatty acid from those secondary to hypophagia. Carcass content of lipid phosphorus and free cholesterol decreased in dose-dependent fashion in both PFDA-treated and pair-fed rats. Carcass triacylglycerols diminished in a similar manner, yet PFDA-treated rats at each dose had a higher concentration of neutral acylglycerols than their vehicle-treated, pair-fed counterparts. In vehicle-treated, pair-fed rats at the 80 mg/kg dose level, lipid phosphorus and free cholesterol as a proportion of carcass fat increased, whereas the share of the triacyl-glycerols declined. Because of the higher concentration of triacylglycerols in the carcass of rats treated with 80 mg/kg PFDA, enrichment of lipid phosphorus and free cholesterol in carcass fat was less than in their pair-fed partners. The amount of lipid phosphorus and free cholesterol per hepatocyte was similar in both PFDA-treated rats and their pair-fed partners. Liver triacyl-glycerols were markedly increased in PFDA-treated rats. A similar but less extensive augmentary effect of PFDA on hepatic esterified cholesterol was found. Concentration of triacylglycerols in plasma was not elevated in PFDA-treated rats, in spite of hepatic accumulation of esterified compounds. Also, the plasma level of free fatty acids and 3-hydroxybutyrate was similar in all treatment groups, including those receiving PFDA. Thus, the administration of PFDA appears to divert fatty acids from oxidation toward esterification in the liver.     

Effects of Cigarette Smoke on Cell Viability,Linoleic Acid Metabolism and Cholesterol Synthesis,in THP−1 Cells

Cigarette smoke (CS) contains thousands of substances, mainly free radicals that have as a target the polyunsaturated fatty acids (PUFA). Long chain PUFA are produced through elongation and desaturation reactions from their precursors; the desaturation reactions are catalyzed by different enzymes: the conversion of 18:2n-6 (linoleic acid, LA) to 18:3n-6 by Delta6 desaturase, while that of 20:3n-6 to 20:4n-6 by Delta5 desaturase. The aim of this work is to evaluate the effect of serum exposed to cigarette smoke (SE-FBS) on (1) cell viability and proliferation, (2) [1-(14)C] LA conversion and desaturase activities in THP-1 cells, a monocytic cell line. In THP-1, CS inhibits cell proliferation dose-dependently, by producing a modification in the cell cycle with a reduced number of cells in synthesis and mitosis phases at higher concentrations. CS also decreases [1-(14)C] LA conversion to its derivatives in a concentration-dependent manner, inhibiting the activities of Delta6 and mainly Delta5 desaturase. In addition, CS does not modify the incorporation of LA into various lipid classes but it reduces cholesterol synthesis from radiolabelled acetate, and increases free fatty acid, TG and CE levels. In conclusion, CS affects lipid metabolism, inhibiting LA conversion and desaturase activities. CS also shifts the "de novo" lipid synthesis from free cholesterol to TG and CE, where LA is preferentially esterified.     

Free fatty acids in cultured cells

A method for isolation and quantitation of cellular free fatty acid has been developed. When this method was used to quantitate the free fatty acid content of various cells and tissues, their levels of free fatty acids were found to vary over a wide range. In comparing tissue culture cells having different levels of free fatty acid, it was demonstrated that the conditions of culture and the type of serum in the medium are not responsible for the difference in levels. Isotopic studies have shown that the cellular free fatty acid is not biosynthesized, but is derived from the free fatty acid of the medium. Preliminary studies on the fate of the intracellular free fatty acid and a discussion of possible factors controlling the level of this compound in cells are presented.     

Cholesterol-cerebroside interaction: The role of α-hydroxy fatty acids

The esterification of cholesterol by the plasma phosphatidyl choline-cholesterol acyltransferase reaction was studied by two methods, radioisotopic and colorimetric, in the presence of cerebroside, ceramide, or methyl esters of lignoceric or α-hydroxy lignoceric acid. The radioisotopic method measures esterification of exogenous labeled cholesterol which must be taken up into the lipoprotein-bound pool prior to its utilization as a substrate. The colorimetric method measures esterification of endogenous lipoprotein-bound free cholesterol since the exogenous labeled cholesterol is negligible in concentration. Cerebroside and ceramide containing α-hydroxy fatty acids reduced the utilization of exogenous labeled cholesterol as substrate, but had no effect on lipoprotein-bound exogenous cholesterol esterification. Cerebroside and ceramide containing no α-hydroxy fatty acid had no effect on exogenous labeled cholesterol esterification. The methyl esters of lignoceric acid and α-hydroxy lignoceric acid had no effect on the esterification of exogenous cholesterol in plasma. There is a decrease in esterification of exogenous labeled cholesterol with increasing concentration of α-hydroxy fatty acid ceramide. Increasing the concentration of exogenous cholesterol tends to counteract the effect of the ceramide on cholesterol esterification. There was little effect on exogenous cholesterol esterification when the α-hydroxy fatty acid ceramide was exposed to plasma before adding the labeled cholesterol. The findings demonstrate an interaction between free cholesterol and cerebroside or ceramide containing α-hydroxy fatty acids, but the nature of the interaction is not elucidated.     

Utilization of extracellular lipids by HT29/219 cancer cells in culture

Uptake and incorporation of long-chain fatty acids were studied in a human colorectal cancer cell line (HT29/219) grown in culture medium supplemented with either fetal calf serum (FSC) or horse serum (HS). The cells were grown for 120 h with no change of medium; the two major cellular lipid classes, the phospholipids and the triacylglycerols, were analyzed at regular time-points. We observed significant changes in the concentration of most fatty acids throughout culture, and differences in their composition when different sera were used to supplement the medium. Minimal levels of free fatty acids were found in the cells, indicating a very small “free fatty acid pool”. A major difference between the cells grown in media supplemented with different sera was the changes observed in concentrations of cellular polyunsaturated fatty acids during growth. In cells grown with FCS (in which 20∶4n−6 is present), the levels of this acid in the phsopholipid and triacylglycerol fractions declined rapidly during cell growth, suggesting further metabolism. In cells grown in medium supplemented with HS, 18∶2n−6 was the major polyunsaturated acid present. There was clear evidence that this acid accumulated in the cellular triacylglycerol and phospholipid fractions. Furthermore, its concentration did not decline during growth in culture, suggesting minimal conversion to other polyunsaturated n−6 acids. Our results suggest that fatty acids from additional sources in the medium, for example triacylglycerols and phospholipids associated with the lipoproteins, are taken up by the cells. There is also indication of cellular fatty acid synthesis, particularly of monounsaturated and saturated acids during the culture period. HT29/219 cells were shown to take up and incorporate radioactivity when trace amounts of [1-¹⁴C]-labeled arachidonic, linoleic or oleic acids were added to the culture medium. Most (80%) of the label was detected in cellular phospholipids and triacylglycerols, although the specific activities of these various fatty acids were different in the two lipid fractions.     

Metabolism of erucic acid in adipocytes isolated from rat epididymal fat

The metabolism of [14-¹⁴C]erucic acid and [U-¹⁴C]palmitic acid has been investigated in adipocytes isolated from rat epididymal fat. The rate of acylation of [¹⁴C]erucic acid in cellular lipids and oxidation to CO₂ and acid-soluble activity was ca. 1/3 of the rate with [¹⁴C]palmitic acid as substrate. A maximal incorporation of fatty acids in triacylglycerol was found at a fatty acid concentration of 0.8 mM in the medium, both with [¹⁴C]erucic acid and [¹⁴C]palmitic acid as substrate. Glucose added to the medium increased the esterification and decreased the oxidation of both fatty acids. No significant chain-shortening of [¹⁴C]erucic acid to shorter monoenes was identified in the fat cells. Increasing concentrations of unlabeled palmitic acid in the incubation medium markedly inhibited the esterification of [¹⁴C]erucic acid, whereas unlabeled erucic acid had little effect on the rate of esterification of [¹⁴C]palmitic acid.     

Lipid and fatty acid composition and energy partitioning during embryo development in the shrimp Macrobrachium borellii

Energy partitioning, composition of lipids and fatty acids, and their utilization by embryos were determined in the lecithotrophic shrimp Macrobrachium borellii during seven development stages. The biochemical composition at stage I is represented by lipids, proteins, and carbohydrates, with 29.3, 28.7, and 0.2% dry weight, respectively. The former two were identified as the major energy-providing components, contributing 131 and 60 cal/100 mg egg, dry weight, respectively. The overall conversion efficiency (CE) was 45.0% (calculated as percentage of vitelline energy transformed into embryonic tissues). Lipids were the most important energy reserve (CE 39.3%), followed by proteins (CE 57.1%), both being simultaneously utilized during development while carbohydrates were synthesized de novo (CE 587.5%). Variation in the lipid class composition of embryos and vitellus showed an accumulation of triacylglycerols (TAG) and phospholipids (PL) up to stage IV, a more active accumulation and selective utilization phase (stages V and VI), and a consumption and de novo synthesis period until hatching. Structural lipids (PL and cholesterol) and pigment astaxanthin were selectively conserved in embryos, but TAG, hydrocarbons, and esterified sterols were preferentially depleted. Monounsaturated fatty acids (FA) were the major group in TAG, whereas polyunsaturated FA (PUFA) were the major group in PL after organogenesis. Certain PUFA such as 22∶6n−3 and 20∶5n−3 were selectively accumulated in PL.