Interaction of a human plasma lipid transfer protein complex with lipid monolayers

The interaction of a purified human plasma lipid transfer complex with cholesteryl ester, triacylglycerol and phosphatidylcholine in binary and ternary lipid monolayers was investigated. The lipid transfer complex, designated LTC, catalyzes the removal of cholesteryl oleate and triacylglycerol from phosphatidylcholine monolayers. Preincubation of LTC with p-chloromercuriphenyl sulfonate inhibits LTC-catalyzed removal of triacylglycerol; cholesteryl ester removal is not affected. The rate of LTC-facilitated removal of cholesteryl oleate from a phosphatidylcholine monolayer depends on the amount of LTC added to the subphase up to 100 μg protein. In addition, the rate of the LTC-catalyzed transfer of cholesteryl oleate to the subphase increases linearly as the amount of cholesteryl oleate in the monolayer increases to 6 mol%. LTC also removes cholesterol from phosphatidylcholine-cholesterol monolayers, albeit at a rate which is 15% of that for removal of cholesteryl oleate. The ability of LTC to facilitate triacylglycerol and cholesteryl ester removal depends on the composition of the monolayer. Phosphatidylcholine supports cholesteryl ester transfer whereas sphingomyelin-cholesteryl ester monolayers are almost refractory to LTC. In contrast, LTC removes triacylglycerol from either a phosphatidylcholine or a sphingomyelin monolayer. The results suggest the existence of at least two lipid transfer proteins, one of which catalyzes the removal of cholesteryl ester and the other triacylglycerol. The role of these proteins as they relate to lipoprotein metabolism is discussed.     

Alpha-adrenergic suppression of very-low-density-lipoprotein triacylglycerol secretion by isolated rat hepatocytes.

The effect of adrenaline on triacylglycerol synthesis and secretion was examined in isolated rat hepatocytes. Cells were incubated with 0.5 mM-[1-14C]oleate, and the accumulation of triacylglycerol and [14C]triacylglycerol was measured in the incubation medium. Triacylglycerol appearing in the medium was present in a form with properties similar to very-low-density lipoproteins. Triacylglycerol, [14C]triacylglycerol and [14C]phospholipid contents of hepatocytes were also determined. Addition of 10 microM-(-)adrenaline decreased accumulation of glycerolipid in the incubation medium and also decreased cellular [14C]phospholipid content. Prazosin abolished these effects, whereas propranolol did not. The hormone did not affect cellular triacylglycerol content or rates of incorporation of [1-14C]oleate into cell triacylglycerol. The effect of adrenaline on the removal of newly secreted triacylglycerol and the secretion of synthesized glycerolipid was also examined. The catecholamine did not affect rates of removal of newly secreted triacylglycerol. Adrenaline did inhibit the secretion of pre-synthesized lipid by the cells, as assessed by the appearance of radiolabelled triacylglycerol from hepatocytes that had been preincubated with [1,2,3-3H]-glycerol. Adrenaline did not affect rates of fatty acid uptake by hepatocytes, but did stimulate oxidation of [1-14C]oleate, principally to 14CO2.     

Overexpression of rat long chain acyl-coa synthetase 1 alters fatty acid metabolism in rat primary hepatocytes

Long chain acyl-CoA synthetases (ACSL) activate fatty acids (FA) and provide substrates for both anabolic and catabolic pathways. We have hypothesized that each of the five ACSL isoforms partitions FA toward specific downstream pathways. Acsl1 mRNA is increased in cells under both lipogenic and oxidative conditions. To elucidate the role of ACSL1 in hepatic lipid metabolism, we overexpressed an Acsl1 adenovirus construct (Ad-Acsl1) in rat primary hepatocytes. Ad-ACSL1, located on the endoplasmic reticulum but not on mitochondria or plasma membrane, increased ACS specific activity 3.7-fold. With 100 or 750 mum [1-(14)C]oleate, Ad-Acsl1 increased oleate incorporation into diacylglycerol and phospholipids, particularly phosphatidylethanolamine and phosphatidylinositol, and decreased incorporation into cholesterol esters and secreted triacylglycerol. Ad-Acsl1 did not alter oleate incorporation into triacylglycerol, beta-oxidation products, or total amount of FA metabolized. In pulse-chase experiments to examine the effects of Ad-Acsl1 on lipid turnover, more labeled triacylglycerol and phospholipid, but less labeled diacylglycerol, remained in Ad-Acsl1 cells, suggesting that ACSL1 increased reacylation of hydrolyzed oleate derived from triacylglycerol and diacylglycerol. In addition, less hydrolyzed oleate was used for cholesterol ester synthesis and beta-oxidation. The increase in [1,2,3-(3)H]glycerol incorporation into diacylglycerol and phospholipid was similar to the increase with [(14)C]oleate labeling suggesting that ACSL1 increased de novo synthesis. Labeling Ad-Acsl1 cells with [(14)C]acetate increased triacylglycerol synthesis but did not channel endogenous FA away from cholesterol ester synthesis. Thus, consistent with the hypothesis that individual ACSLs partition FA, Ad-Acsl1 increased FA reacylation and channeled FA toward diacylglycerol and phospholipid synthesis and away from cholesterol ester synthesis.     

Triacylglycerol metabolism in isolated rat kidney cortex tubules

Triacylglycerol metabolism has been studied in kidney cortex tubules from starved rats, prepared by collagenase treatment. Triacylglycerol was determined by a newly developed fully enzymic method. Incubation of tubules in the absence of fatty acids led to a decrease of endogenous triacylglycerol by about 50% in 1h. Addition of albuminbound oleate or palmitate resulted in a steady increase of tissue triacylglycerol over 2h. The rate of triacylglycerol synthesis was linearly dependent on oleate concentration up to 0.8mm, reaching a saturation at higher concentrations. Triacylglycerol formation from palmitate was less than that from oleate. This difference was qualitatively the same when net synthesis was compared with incorporation of labelled fatty acids. Quantitatively, however, the difference was less with the incorporation technique. Gluconeogenic substrates, which by themselves had no effect on triacylglycerol concentrations, stimulated neutral lipid formation from fatty acids. Glucose and lysine did not have such a stimulatory effect. Inhibition of gluconeogenesis from lactate by mercaptopicolinic acid likewise inhibited triacylglycerol formation. This inhibitory effect was seen with oleate as well as with oleate plus lactate. When [2-¹⁴C]lactate was used the incorporation of label into triacylglycerol was found in the glycerol moiety exclusively. Addition of dl-β-hydroxybutyrate (5mm) to the incubation medium in the presence of oleate or oleate plus lactate led to a significant increase in triacylglycerol formation. In contrast with the gluconeogenic substrates, dl-β-hydroxybutyrate had no stimulatory effect on fatty acid uptake. The results suggest that renal triacylglycerol formation is a quantitatively important metabolic process. The finding that gluconeogenic substrates, but not glucose, increase lipid formation, indicates that the glycerol moiety is formed by glyceroneogenesis in the proximal tubules. The effect of ketone bodies seems to be caused by the sparing action of these substrates on fatty acid oxidation. The decrease of triacylglycerol in the absence of exogenous substrates confirms previous conclusions that endogenous lipids provide fatty acids for renal energy metabolism.     

Upregulation of cellular triacylglycerol - free fatty acid cycling by oleate is associated with long-term serum-free survival of human breast cancer cells.

We previously showed that exogenous oleate protects human breast cancer cells against palmitate-induced apoptosis in part by increasing esterification of this free fatty acid (FFA) into triacylglycerol (TG). Here, we studied the mechanism whereby oleate protects these cells against apoptosis induced by serum withdrawal. The metabolism of FFA, TG, and glucose, in parallel with long-term cell survival in the absence of serum, was investigated in a panel of human breast cancer cell lines and in nontransformed MCF-10A cells after treatment with exogenous oleate. Short-term (3-24 h) exposure of MDA-MB-231 human breast cancer cells to exogenous oleate resulted in a dose-dependent long-term (10 day) serum-free survival that correlated with the accumulation of TG in lipid droplets and with upregulation of lipolysis. Both effects persisted for several days after oleate removal. Rapid TG lipolysis and FFA re-esterification, supported by high rates of glycolysis that provide the glycerol backbone for TG synthesis, are consistent with the presence of very active TG-FFA cycling in human breast cancer cells. Only the cancer cell lines capable of accumulating TG showed long-term serum-free survival after oleate treatment. The results suggest that upregulation of TG-FFA cycling induced by oleate may be involved in maintenance of human breast cancer cell survival.     

Oxidation affects the regulation of hepatic lipid synthesis by chylomicron remnants

The effects of native and oxidized chylomicron remnants on lipid synthesis in normal and oxidatively stressed liver cells were investigated using MET murine hepatocytes (MMH cells), a nontransformed mouse hepatocyte cell line that maintains a highly differentiated hepatic phenotype in culture. Lipid synthesis was determined by measuring the incorporation of [(3)H]oleate into cholesteryl ester, triacylglycerol, and phospholipid by the cells. The formation of cholesteryl ester and phospholipid was decreased by chylomicron remnants in a dose-dependent manner, while triacylglycerol synthesis was increased. Exposure of MMH cells to mild oxidative stress by incubation with CuSO(4) (2.5 microM) for 24 h led to significantly increased incorporation of [(3)H]oleate into triacylglycerol and phospholipid, but not cholesteryl ester, in the absence of chylomicron remnants. In the presence of the lipoproteins, however, similar effects to those found in untreated cells were observed. Oxidatively modified chylomicron remnants prepared by incubation with CuSO(4) (10 microM, 18 h, 37 degrees C) did not influence cholesteryl ester or phospholipid synthesis in MMH cells, but had a similar effect to that found with native remnants on triacylglycerol synthesis. These findings show that hepatic lipid metabolism is altered by exposure to mild oxidative stress and by lipids from the diet delivered to the liver in chylomicron remnants, and these effects may play a role in the development of atherosclerosis.     

Key role for ceramides in mediating insulin resistance in human muscle cells

Elevated non-esterified fatty acids, triglyceride, diacylglycerol, and ceramide have all been associated with insulin resistance in muscle. We set out to investigate the role of intramyocellular lipid metabolites in the induction of insulin resistance in human primary myoblast cultures. Muscle cells were subjected to adenovirus-mediated expression of perilipin or incubated with fatty acids for 18 h, prior to insulin stimulation and measurement of lipid metabolites and rates of glycogen synthesis. Adenovirus-driven perilipin expression lead to significant accumulation of triacylglycerol in myoblasts, without any detectable effect on insulin sensitivity, as judged by the ability of insulin to stimulate glycogen synthesis. Similarly, incubation of cells with the monounsaturated fatty acid oleate resulted in triacylglycerol accumulation without inhibiting insulin action. By contrast, the saturated fatty acid palmitate induced insulin resistance. Palmitate treatment caused less accumulation of triacylglycerol than did oleate but also induced significant accumulation of both diacylglycerol and ceramide. Insulin resistance was also caused by cell-permeable analogues of ceramide, and palmitate-induced resistance was blocked in the presence of inhibitors of de novo ceramide synthesis. Oleate co-incubation completely prevented the insulin resistance induced by palmitate. Our data are consistent with ceramide being the agent responsible for insulin resistance caused by palmitate exposure. Furthermore, the triacylglycerol derived from oleate was able to exert a protective role in sequestering palmitate, thus preventing its conversion to ceramide.     

S3-12, Adipophilin, and TIP47 package lipid in adipocytes

Animals have evolved mechanisms to maintain circulating nutrient levels when energy demands exceed feeding opportunities. Mammals store most of their energy as triacylglycerol in the perilipin-coated lipid droplets of adipocytes. How newly synthesized triacylglycerol is delivered to perilipin-coated lipid droplets is poorly understood. Perilipin is a member of the evolutionarily related family of PAT proteins (Perilipin, Adipophilin, TIP47), which is defined by sequence similarity and association with lipid droplets. We previously showed that S3-12, which is also a member of this family, associates with a separate pool of lipid droplets that emerge when triacylglycerol storage is driven by adding oleate to the culture medium of adipocytes. Our current data extend these findings to demonstrate that nascent lipid droplets emerge with a coat composed of S3-12, TIP47, and adipophilin. After 100 min of oleate treatment, the nascent lipid droplets are more heterogeneous: S3-12 and TIP47 coat smaller, peripheral droplets and adipophilin coats a more medial population of droplets. Fractionation of untreated and oleate-treated adipocytes shows oleate-dependent redistribution of TIP47 and adipophilin from cytosolic fractions to the lipid droplet fraction. Inhibition of protein synthesis with cycloheximide does not block the oleate-induced formation of the nascent lipid droplets, nor does it prevent TAG accumulation. We suggest that the non-lipid droplet pools of S3-12, adipophilin, and TIP47 constitute a ready reservoir of coat proteins to permit rapid packaging of newly synthesized triacylglycerol and to maximize energy storage during nutrient excess.     

Interruption of triacylglycerol synthesis in the endoplasmic reticulum is the initiating event for saturated fatty acid-induced lipotoxicity in liver cells

The aim of the present study was to investigate in detail the molecular mechanisms by which free fatty acids induce liver toxicity in liver cells. HepG2 and Huh7 human liver cell lines were exposed to varying concentrations of stearate (18:0), oleate (18:1), or mixtures of the two fatty acids, and the effects on cell proliferation, lipid droplet accumulation and induction of endoplasmic reticulum stress and apoptosis were evaluated. It was observed that: (a) stearate, but not oleate, inhibited cell proliferation and induced cell death; (b) stearate-induced cell death had the characteristics of endoplasmic reticulum stress-mediated and mitochondrial-mediated apoptosis; (c) the activation of stearate in the form of stearoyl-CoA was a necessary step for the lipotoxic effect; (d) the capacity of cells to produce and accumulate triacylglycerols in the form of lipid droplets was interrupted following exposure to stearate, whereas it proceeded normally in oleate-treated cells; and (e) the presence of relatively low amounts of oleate protected cells from stearate-induced toxicity and restored the ability of the cells to accumulate triacylglycerols. Our data suggest that interruption of triacylglycerol synthesis in the endoplasmic reticulum, apparently because of the formation of a pool of oversaturated intermediates, represents the key initiating event in the mechanism of saturated fatty acid-induced lipotoxicity.     

Effects of dexamethasone and insulin on the synthesis of triacylglycerols and phosphatidylcholine and the secretion of very-low-density lipoproteins and lysophosphatidylcholine by monolayer cultures of rat hepatocytes.

Rat hepatocytes in monolayer culture were preincubated for 19 h with 1 microM-dexamethasone, and the incubation was continued for a further 23 h with [14C]oleate, [3H]glycerol and 1 microM-dexamethasone. Dexamethasone increased the secretion of triacylglycerol into the medium in particles that had the properties of very-low-density lipoproteins. The increased secretion was matched by a decrease in the triacylglycerol and phosphatidylcholine that remained in the hepatocytes. Preincubating the hepatocytes for the total 42 h period with 36 nM-insulin decreased the amount of triacylglycerol in the medium and in the cells after the final incubation for 23 h with radioactive substrates. However, insulin had no significant effect on the triacylglycerol content of the cell and medium when it was present only in the final 23 h incubation. Insulin antagonized the effects of dexamethasone in stimulating the secretion of triacylglycerol from the hepatocytes, especially when it was present throughout the total 42 h period. The labelling of lysophosphatidylcholine in the medium when hepatocytes were incubated with [14C]oleate and [3H]glycerol was greater than that of phosphatidylcholine. The appearance of this lipid in the medium, unlike that of triacylglycerol and phosphatidylcholine, was not stimulated by dexamethasone, or inhibited by colchicine. However, the presence of lysophosphatidylcholine in the medium was decreased when the hepatocytes were incubated with both dexamethasone and insulin. These findings are discussed in relation to the control of the synthesis of glycerolipids and the secretion of very-low-density lipoproteins and lysophosphatidylcholine by the liver, particularly in relation to the interactions of glucocorticoids and insulin.     

Labelling of glycerolipids in the cotyledons of developing oilseeds by [1-14C]acetate and [2-3H]glycerol

1. 3-sn-Phosphatidylcholine was identified as the major lipid in cotyledons from the developing seeds of soya bean, linseed and safflower when tissue was steamed before lipid extraction. The proportion of oleate in this lipid decreased markedly and that of the polyunsaturated C₁₈ fatty acids increased when detached developing cotyledons were incubated for up to 3h. Similar but less pronounced changes occurred in diacylglycerol, which had a fatty acid composition resembling that of the 3-sn-phosphatidylcholine from cotyledons of the same species. 2. [1-¹⁴C]Acetate supplied to detached cotyledons was incorporated into the acyl moieties of mainly 3-sn-phosphatidylcholine, 1,2-diacylglycerol and triacylglycerol. Initially label was predominantly in oleate, but subsequently entered at accelerating rates the linoleoyl moieties of the above lipids in soya-bean and safflower cotyledons and the linoleoyl and linolenyl moieties of these lipids in linseed cotyledons. In pulse–chase experiments label was rapidly lost from the oleate of 3-sn-phosphatidylcholine and accumulated in the linoleoyl and linolenoyl moieties of this phospholipid and of the di- and tri-acylglycerols. 3. [2-³H]Glycerol was incorporated into the glycerol moieties of mainly 3-sn-phosphatidylcholine and di- and tri-acylglycerols of developing linseed and soya-bean cotyledons. The label entered the phospholipid and diacylglycerol at rates essentially linear with time from the moment the substrate was supplied, and entered the triacylglycerol at an accelerating rate. With linseed cotyledons the labelled glycerol was incorporated initially mainly into species of 3-sn-phosphatidylcholine and diacylglycerol that contained oleate, but accumulated with time in more highly unsaturated species. In pulse–chase experiments with linseed cotyledons, label was lost from both 3-sn-phosphatidylcholine and diacylglycerol, preferentially from the dioleoyl species, and accumulated in triacylglycerol, mainly in species containing two molecules of linolenate. 4. The results suggest a rapid turnover of 3-sn-phosphatidylcholine during triacylglycerol accumulation in developing oilseeds, and are consistent with the operation of a biosynthetic route whereby oleate initially esterified to the phospholipid is first desaturated, then polyunsaturated fatty acids transferred to triacylglycerol, via diacylglycerol. The possible role of oleoyl phosphatidylcholine as a substrate for oleate desaturation is discussed.     

Mitochondrial glycerol phosphate acyltransferase directs the incorporation of exogenous fatty acids into triacylglycerol

The mitochondrial isoform of glycerol-3-phosphate acyltransferase (GPAT), the first step in glycerolipid synthesis, is up-regulated by insulin and by high carbohydrate feeding via SREBP-1c, suggesting that it plays a role in triacylglycerol synthesis. To test this hypothesis, we overexpressed mitochondrial GPAT in Chinese hamster ovary (CHO) cells. When GPAT was overexpressed 3.8-fold, triacylglycerol mass was 2.7-fold higher than in control cells. After incubation with trace [(14)C]oleate ( approximately 3 microm), control cells incorporated 4.7-fold more label into phospholipid than triacylglycerol, but GPAT-overexpressing cells incorporated equal amounts of label into phospholipid and triacylglycerol. In GPAT-overexpressing cells, the incorporation of label into phospholipid, particularly phosphatidylcholine, decreased 30%, despite normal growth rate and phospholipid content, suggesting that exogenous oleate was directed primarily toward triacylglycerol synthesis. Transiently transfected HEK293 cells that expressed a 4.4-fold increase in GPAT activity incorporated 9.7-fold more [(14)C]oleate into triacylglycerol compared with control cells, showing that the effect of GPAT overexpression was similar in two different cell types that had been transfected by different methods. When the stable, GPAT-overexpressing CHO cells were incubated with 100 microm oleate to stimulate triacylglycerol synthesis, they incorporated 1.9-fold more fatty acid into triacylglycerol than did the control cells. Confocal microscopy of CHO and HEK293 cells transfected with the GPAT-FLAG construct showed that GPAT was located correctly in mitochondria and was not present elsewhere in the cell. These studies indicate that overexpressed mitochondrial GPAT directs incorporation of exogenous fatty acid into triacylglycerol rather than phospholipid and imply that (a) mitochondrial GPAT and lysophosphatidic acid acyltransferase produce a separate pool of lysophosphatidic acid and phosphatidic acid that must be transported to the endoplasmic reticulum where the terminal enzymes of triacylglycerol synthesis are located, and (b) this pool remains relatively separate from the pool of lysophosphatidic acid and phosphatidic acid that contributes to the synthesis of the major phospholipid species.     

Effect of long chain fatty acids on triacylglycerol accumulation,fatty acid composition and related gene expression in primary cultured bovine satellite cells

Abstract

This study was conducted to determine the effects of long chain fatty acids (LCFAs) on triacylglycerol (TAG) content, as well as on genes associated with lipid synthesis and fatty acid composition in bovine satellite cells. Both saturated (palmitic and stearic) and unsaturated (oleic and linoleic) fatty acids stimulated the TAG accumulation at a concentration of 100?µM and oleate increased it significantly more than stearate and palmitate. The results revealed that the lipid droplet formation was markedly stimulated by linoleate and oleate at 100?µM. Compared to control, the expressions of adipose triglyceride lipase, carnitine acyltransferase 1 and the fatty acid translocase 36 were upregulated by LCFAs. All the fatty acids also significantly increased diacylglycerol acyltransferase 2 than the untreated control (p?<?0.05). The monounsaturated fatty acids significantly increased (p?<?0.05) in response to oleate and linoleate compared to the control as did the polyunsaturated fatty acids (p?<?0.05), in addition to stearate, linoleate and oleate. In contrast, saturated fatty acids were significantly decreased in the oleate and linoleate-treated groups. The study results contribute to our enhanced understanding of LCFAs’ regulatory roles on the bovine cell lipid metabolism.     

Lipolysis of serum-activated triacylglycerol at the surface of J774.1 macrophages

Summary Cultured mouse (J774.1) macrophages accumulated triacylglycerol, but no cholesteryl ester or cholesterol, when incubated in albumin-poor medium with serum-activated lipid particles containing 84 mol% trioleoylglycerol and 9 mol% cholesteryl oleate. Accumulation of triacylglycerol by cells was associated with hydrolysis of particulate triacylglycerol to fatty acid and glycerol. Both acyl and glyceryl moieties of particulate triacylglycerol were recovered in cellular triacylglycerol with a molar ratio of 3.6. The cells also accumulated fatty acid and monoacylglycerol. Whether acylglycerol was taken up as a single molecular species, such as monoacylglycerol, or as several species can not be determined by the present findings. Macrophages incubated with lipid particles for 24 h had many lipid particles attached to cell surfaces and numerous intracellular lipid droplets. The surface film of attached particles was continuous with the outer leaflet of plasma membrane of the cells. Particles partially depleted of core triacylglycerol and collapsed surface films were found attached to surfaces of macrophages. There was no morphological evidence that lipid particles were taken up intact by cells, through endocytosis or phagocytosis. Macrophages incubated with lipid particles also contained intracellular lamellar structures. They varied in size and shape, and were located in the periphery of cells, sometimes near lipid droplets and endoplasmic reticulum. Only 3% of the lamellar structures were associated with lysosomes, indicating they probably were not of lysosomal origin. Lipid particles attached to cells decreased in size and number, and lamellar structures developed at the surface of particles, or replaced the particles, when glutaraldehyde-fixed specimens were incubated at 25° C, demonstrating lipolytic activity at the surface of macrophages. Our findings suggest that particulate triacylglycerol was hydrolyzed by lipoprotein lipase at the surface of macrophages, and that fatty acid and monoacylglycerol formed by lipolysis were transported directly into the cells to be reesterified. When lipolytic products were taken up faster than they could be utilized, they accumulated as lamellar structures in the cells.Abbreviations MEM Eagle's alpha modification of minimum essential medium     

Storage, mobilization and secretion of cytosolic triacylglycerol in hepatocyte cultures. The role of insulin.

Cytosolic triacylglycerol labelled from [3H]oleate accounted for almost 50% (57 +/- 22 nmol/mg of protein) of the total cellular triacylglycerol which was newly synthesized by cultured hepatocytes during a 24 h incubation. Insulin decreased the export of triacylglycerol as very-low-density lipoprotein (VLDL) during this period. This resulted in a sequestration of newly synthesized triacylglycerol in the cytosol, rather than in the particulate fraction of the cell. Longer periods of incubation with [3H]oleate resulted in increased concentrations of newly synthesized triacylglycerol within the cell, most of which (78 +/- 3% after 48 h; 80 +/- 3% after 72 h) was located within the cytosolic fraction. The quantity of newly synthesized triacylglycerol in the cell cytosol was further increased by insulin. During these periods there were decreases in the amounts of triacylglycerol associated with the particulate fraction of the cell, irrespective of the presence or absence of insulin. In no case was a decrease in VLDL triacylglycerol secretion in response to insulin accompanied by an increased triacylglycerol content in the particulate fraction of the cell. In some experiments, the fate of the cytosolic triacylglycerol was studied by pulse labelling with [3H]oleate. In these cases, when insulin was removed from the medium of cells to which they had previously been exposed, more newly synthesized triacylglycerol was secreted compared with cells which had not been exposed to insulin. This extra triacylglycerol was mobilized from the cytosolic rather than from the particulate fraction of the cell. Subsequent addition of insulin to the medium prevented the mobilization of cytosolic triacylglycerol. These results suggest that insulin enhances the storage of hepatocellular triacylglycerol in a cytosolic pool. Deficiency of insulin in the medium stimulates the mobilization of this pool which is channelled into the secretory pathway, entering the extracellular medium as VLDL.     

Perinatal hepatocyte/hepatoma hybrids: construction of clones that express the developmentally regulated monoacyglycerol acyltransferase activity

Microsomal monoacyglycerol acyltransferase is a developmentally expressed enzyme that catalyzes the synthesis of sn-1,2-diacylglycerol from sn-2-monoacylglycerol and palmitoyl-CoA. The activity is present in liver from fetal and suckling rats but is absent in the adult. In order to obtain a stable permanent cell line that expresses this activity, Fao rat hepatoma cells and hepatocytes from 8-day-old baby rats were hybridized and clones were selected. Two hybrids (HA1 and HA7) expressed monoacylglycerol acyltransferase activity. Like fetal hepatocytes, but unlike hepatocytes from postnatal rats, the HA cells had high rates of [14C]acetate incorporation into glycerolipids, cholesterol, and cholesteryl esters, and they secreted triacylglycerol into the media. Monoacylglycerol acyltransferase specific activity increased 2.5-fold as the cells divided in culture, suggesting growth-dependent regulation. The specific activities of glycerol-P acyltransferase, the committed step of the microsomal pathway of glycerolipid synthesis, and diacylglycerol acyltransferase, the activity unique to triacylglycerol biosynthesis, were comparable to the levels of the corresponding activities in fetal hepatocytes. Addition of insulin or dexamethasone to the media increased the incorporation of [14C]oleate into triacyglycerol about 1.7-fold within 2 h, but had little effect on [14C]oleate incorporation into phospholipid. These hormonally responsive rat-hepatoma/hepatocyte hybrids reflect the fetal stage of hepatocyte development in five major aspects of lipid metabolism: sterol, fatty acid, and triacylglycerol biosynthesis, glycerolipid secretion, and the presence of the developmentally expressed monoacylglycerol pathway.     

Purification and characterization of acylation stimulating protein

We have purified to homogeneity and analyzed the amino acid composition of a small (Mr 14,000), basic (pI 9.0) protein from human plasma. This has been named acylation stimulating protein (ASP) because it markedly stimulates triacylglycerol synthesis in human adipocytes. As well, it stimulates triacylglycerol synthesis in human skin fibroblasts cultured from normal individuals. Characteristic saturation curves for the cell metabolic responses to ASP were observed in both cell types with higher stimulation of oleate incorporation into triacylglycerol being observed in adipocytes. The stimulation of triacylglycerol synthesis was much greater with ASP than with insulin. Neither fatty acid binding protein nor albumin was able to mimic the ASP effect.     

Chylomicron remnant induction of lipid accumulation in J774 macrophages is associated with up-regulation of triacylglycerol synthesis which is not dependent on oxidation of the particles

The influence of chylomicron remnants on lipid accumulation and synthesis and the activity and/or expression of mRNA for some of the key enzymes involved was investigated in the murine macrophage cell line J774. The effects of varying the polyunsaturated fatty acid (PUFA) composition and oxidation state of the remnants were also examined. Chylomicron remnants derived from corn oil (rich in n-6 PUFA) or fish oil (rich in n-3 PUFA) were prepared in vivo and oxidised by incubation with CuSO(4). The native and oxidised remnants caused a marked rise in intracellular triacylglycerol levels, but the rise induced by corn oil remnants (four- to sixfold) was greater than that observed with fish oil remnants (<2-fold). Triacylglycerol synthesis, as measured by the incorporation of [3H]oleate and [3H]glycerol into cellular triacylglycerol, was increased by all four remnant types tested, and corn oil remnants had a significantly greater effect than fish oil remnants. Oxidation of the remnants did not affect the results obtained. Although the incorporation of [3H]oleate into cholesteryl ester by the cells was not significantly changed by any of the four types of remnants tested, the activity and expression of mRNA for acyl Co-enzyme A: cholesterol acyltransferase (ACAT) was increased by corn oil, but not by fish or oxidised corn, remnants. Neutral cholesteryl ester hydrolase (nCEH) activity, however, was also raised by corn oil remnants. These studies indicate that chylomicron remnants induce the accumulation of triacylglycerol in J774 macrophages, and that increased synthesis of triacylglycerol plays a major role in this process. Furthermore, they demonstrate that these effects are enhanced when the remnants are enriched in n-6 PUFA as compared with n-3 PUFA, but not after oxidation of the particles, suggesting that the fatty acid composition of chylomicron remnants may be more important than their oxidation state in their ability to induce foam cell formation.     

The utilization and desaturation of oleate and linoleate during glycerolipid biosynthesis in olive (Olea europaea L.) callus cultures

Callus cultures from olive (Olea europaea L.) were used to study characteristics of desaturation in this oil-rich tissue. The incorporation of [1-(14)C]oleate and [1-(14)C]linoleate into complex lipids and their further desaturation was followed in incubations of up to 48 h. Both radiolabelled fatty acids were rapidly incorporated into lipids, especially phosphatidylcholine and triacylglycerol. Radiolabelling of these two lipids peaked after 1-4 h, after which it fell. In contrast, other phosphoglycerides and the galactosylglycerides were labelled in a more sustained manner. [1-(14)C]Linoleate was almost exclusively found in the galactolipids. With [1-(14)C]linoleate as a precursor, the only significant desaturation to linolenate was in the galactolipids. Monogalactosyldiacylglycerol was the first lipid in which [1-(14)C]linoleate and [1-(14)C]linolenate appeared after incubation of the calli with [1-(14)C]oleate and [1-(14)C]linoleate, respectively. The presence of radioactivity in the plastidial lipids shows that both [1-(14)C]oleate and [1-(14)C]linoleate can freely enter the chloroplast. Two important environmental effects were also examined. Raised incubation temperatures (30-35 degrees C) reduced oleate desaturation and this was also reflected in the endogenous fatty acid composition. Low light also caused less oleate desaturation. The data indicate that lysophosphatidylcholine acyltransferase is important for the entry of oleate and linoleate into olive callus lipid metabolism and phospholipid:diacylglycerol acyltransferase may be involved in triacylglycerol biosynthesis. In addition, it is shown that plastid desaturases are mainly responsible for the production of polyunsaturated fatty acids. Individual fatty acid desaturases were differently susceptible to environmental stresses with FAD2 being reduced by both high temperature and low light, whereas FAD7 was only affected by high temperature.