Lipid and lipoprotein metabolism in Hep G2 cells

Lipid composition, lipid synthesis and lipoprotein secretion by the Hep G2 cell line have been studied with substrate and insulin supplied under different conditions. The lipid composition of Hep G2 cells was close to that of normal human liver, except for a higher content in sphingomyelin (P less than 0.005) and a lower phosphatidylcholine/sphingomyelin ratio. Most of the [14C]triacylglycerols secreted into the medium were recovered by ultracentrifugation at densities of 1.006 to 1.020 g/ml. The main apolipoproteins secreted were apo B-100 and apo A-I. Hep G2 mRNA synthesized in vitro the pro-apolipoproteins A-I and E. Triacylglycerol secretion was 7.38 +/- 1.04 micrograms/mg cell protein per 20 h with 5.5 mM glucose in the medium and increased linearly with glucose concentration. Oleic acid (1 mM) increased the incorporation of [3H]glycerol into the medium and cell triacylglycerols by 251 and 899%, with a concomitant increment in cell triacylglycerols and cholesterol ester. Insulin (1 mU or 7 pmol/ml) inhibited triacylglycerol secretion and [35S]methionine incorporation into secreted protein by 47 and 28%, respectively, with a corresponding increase in the cells. Preincubation of cells with 2.5-10 mM mevalonolactone decreased the incorporation of [14C]acetate into cholesterol 6.2-fold, indicating an inhibitory effect on HMG-CoA reductase. It is concluded that in spite of some differences between Hep G2 and normal human hepatocytes, this line offers an alternative and reliable model for studies on liver lipid metabolism.     

Effect of ethanol on cell growth and cholesterol metabolism in cultured Hep G2 cells.

The Hep G2 human hepatoma cell line has been recognized as an excellent in vitro human model system. For this reason, this line was used to study the effect of ethanol on HMG-CoA reductase activity concerning cell growth and cholesterol metabolism. Cells were incubated in ethanol-containing medium (0-400 mmol/L) for up to 102 h. Ethanol caused an inhibition in the growth rate and in HMG-CoA reductase activity that could be reverted by the removal of ethanol from the culture medium, indicating no cellular damage. These changes cannot be ascribed to the regulatory effect of cholesterol levels, since its content was not modified either in the cells or in the medium. The addition of mevalonate to the culture medium could not revert the growth rate inhibition evoked by ethanol. Moreover, ethanol produced an increment in the cholesterol efflux in [3H]cholesterol-prelabeled cells. We conclude that the decrease in HMG-CoA reductase activity evoked by ethanol treatment on Hep G2 cells would not be the cause but the consequence of the impairment in cellular growth, since this impairment could not be reverted by the addition of mevalonate to the culture medium.     

Dietary trans fatty acids modulate erythrocyte membrane fatty acyl composition and insulin binding in monkeys

The substitution of trans- for half of the cis-monounsaturated fatty acids in the diet of Macaca fasicularis monkeys resulted in alterations in erythrocyte fatty acid composition and insulin receptor properties but not in membrane fluidity. Both cis and trans diets contained 10% fat and similar fatty acid compositions, except that approximately 50% of the cis-octadecenoate (c-18:1) in the cis diet was replaced with trans-octadecenoate isomers (t-18:1) in the trans diet. Compared with the cis diet, the trans diet resulted in the incorporation of approximately 11% t-18:1, an approximately 50% decrease in c-18:1, an approximately 16% decrease in total saturated fatty acids, and an approximately 20% increase in 18:2(n-6) in erythrocyte membrane lipids. The increase in 18:2(n-6) may reflect on homeostatic mechanisms designed to maintain overall membrane fluidity, as no diet-related changes in fluidity were observed with diphenylhexatriene steady state fluorescence polarization. Values observed for insulin binding and insulin receptor number were higher and binding affinity was lower in monkeys fed the cis diet. In the absence of an effect on overall membrane fluidity, altered receptor activity suggests that insulin receptor activity is dynamic, requiring specific fluid membrane subdomains or highly specific fatty acid-protein interactions.     

Alterations in erythrocyte membrane lipids in abetalipoproteinemia: phospholipid and fatty acyl composition

Scanning electron microscopic observation revealed that there were wide variations including typical acanthocytes in morphology of erythrocytes from a patient with abetalipoproteinemia. The erythrocyte membrane phospholipids and cholesterol contents from a patient was higher by 25% compared to an age-matched control subject. Analysis of phospholipid composition of red blood cells showed an increase of sphingomyelin (25.1----30.1%) with a concomitant decrease of lecithin (27.5----21.0%). Thus, the sphingomyelin/lecithin ratio was increased dramatically (0.91----1.43). As for fatty acyl chain composition of main phospholipids, an increased percentage of palmitic acid and docosahexaenoic acid and a decreased proportion of arachidonic acid and lignoceric acid were observed for sphingomyelin. There was an increment of palmitic acid which was accompanied with a decrease of linoleic acid in lecithin. On the other hand, no significant difference was shown in the fatty acid composition of phosphatidylethanolamine and phosphatidylserine plus phosphatidylinositol between a patient and control.     

Fatty acid uptake and metabolism in Hep G2 human-hepatoma cells

The aim of this work was to study the fatty acid metabolism of the human-hepatoma cell line Hep G2. The cultured cells were incubated with either a saturated (palmitic, stearic) or a polyunsaturated (linoleic, -linolenic, eicosatrienoic n-6) radioactive fatty acid. The fatty acids were incorporated into all the basic lipid classes as well as into the main phospholipid subclasses in the cellular membranes. All the fatty acids tested provided a source of carbon for lower members of the saturated fatty-acid family or for cholesterol through -oxidation and a new cycle ofde novo synthesis. Moreover, all radioactive fatty-acid precursors, whether saturated or unsaturated, were anabolized to higher derivatives within their own family. In the case of saturated fatty acids, palmitic and stearic, they were readily monodesaturated to their corresponding products, thus demonstrating the presence of a 9 desaturase. Linoleate and -linolenate were both desaturated and elongated to all the subsequent members of their respective n-6 and n-3 families. These latter observations provide evidence for the incidence of desaturation at the 6 and 5 positions along with the existence of an elongating capacity for fatty acids of all families and chain lengths. In addition, the cellular steady-state fatty-acid profile was seen to be significantly different from the spectrum of exogenous fatty acids available in the growth medium. We conclude that the Hep G2 human-hepatoma line represents an appropriate and relevant experimental model system for investigating the fatty-acid metabolism of adult human liverin vivo.     

Effect of geraniol on fatty-acid and mevalonate metabolism in the human hepatoma cell line Hep G2.

Monoterpenes have multiple pharmacological effects on the metabolism of mevalonate. Geraniol, a dietary monoterpene, has in vitro and in vivo anti-tumor activity against several cell lines. We have studied the effects of geraniol on growth, fatty-acid metabolism, and mevalonate metabolism in the human hepatocarcinoma cell line Hep G2. Up to 100 micromol geraniol/L inhibited the growth rate and 3-hydroxymethylglutaryl coenzyme A reductase (HMG-CoA) reductase activity of these cells. At the same concentrations, it increased the incorporation of cholesterol from the medium in a dose-dependent manner. Geraniol-treated cells incorporated less 14C-acetate into nonsaponifiable lipids, inhibiting its incorporation into cholesterol but not into squalene and lanosterol. This is indicative of an inhibition in cholesterol synthesis at a step between lanosterol and cholesterol, a fact confirmed when cells were incubated with 3H-mevalonate. The incorporation of 3H-mevalonate into protein was also inhibited, whereas its incorporation into fatty acid increased. An inhibition of delta5 desaturase activity was demonstrated by the inhibition of the conversion of 14C-dihomo-gamma-linolenic acid into arachidonic acid. Geraniol has multiple effects on mevalonate and lipid metabolism in Hep G2 cells, affecting cell proliferation. Although mevalonate depletion is not responsible for cellular growth, it affects cholesterogenesis, protein prenylation, and fatty-acid metabolism.     

Drug metabolism by the human hepatoma cell, Hep G2

The human liver-derived cell line, Hep G2, has aryl hydrocarbon hydroxylase and 7-ethoxycoumarin o-de-ethylase activities. Partial purification of cytochrome P-450 from Hep G2 cells provided spectral evidence of this hemeprotein in the purified fraction. These results suggest that Hep G2 cells will be useful for the study of cytochrome P-450 and the regulation of mixed function oxidase activities in liver cells of human origin.     

Effect of 25-hydroxycholesterol and bile acids on the regulation of cholesterol metabolism in Hep G2 cells.

The effect of 25-hydroxycholesterol (25-OH-cholesterol) and chenodeoxycholic (CDC) acid on apoprotein secretion, low-density lipoprotein receptor activity, and [3H]triacylglycerol secretion in Hep G2 cells was studied. Both 25-OH-cholesterol and CDC acid increased the secretion of apolipoprotein (apo) E by Hep G2 cells. The secretion of apo A-I was slightly lowered (less than 10% disease). The maximal increase in apo E secretion was observed in culture medium containing 2 micrograms of 25-OH-cholesterol/ml or 10 micrograms of CDC acid/ml plus 10% fetal calf serum. Cholesterol, 7-OH-cholesterol and other bile acids were ineffective in inducing increases in apo E secretion. Another cholesterol synthesis inhibitor, mevinolin, was also ineffective in generating an increase in apoprotein secretion. The data indicated a specific interaction between 25-OH-cholesterol or CDC acid and apo E secretion in Hep G2 cells. Cholesterol synthesis, as measured by the incorporation of [14C]acetic acid into sterols, was repressed in Hep G2 cells in the presence of 25-OH-cholesterol (17% of control value). CDC acid, on the other hand, increased [14C]acetic acid incorporation (156% of control value). The number of LDL receptors in Hep G2 cells was decreased after incubation with 25-OH-cholesterol (62% of control value), but increased significantly after incubation with CDC acid (149% of control value). The secretion of [3H]triacylglycerol by Hep G2 cells incubated with 25-OH-cholesterol was greatly increased (248% of control value). On the contrary, CDC acid did not cause any increase in [3H]triacylglycerol secretion. The above results suggest that 25-OH-cholesterol and CDC acid have different effects on lipid metabolism in Hep G2 cells. The mRNA levels of apo E increased in cells preincubated with 25-OH-cholesterol and CDC acid, which suggested that the increase in apo E secretion is at least partly due to an increase in synthesis.     

Apolipoprotein B synthesized by Hep G2 cells undergoes fatty acid acylation

Apolipoprotein B is the principal protein associated with cholesterol transport in the blood and has been proposed to play a central role in human atherogenesis. The unique hydrophobic nature of this large (512 kDa), glycosylated apolipoprotein differs from that of the other apolipoproteins. Since another apolipoprotein, apolipoprotein A-I, has been recently shown to have covalently bound fatty acids, potential fatty acid acylation of apolipoprotein B was investigated. The human hepatoma cell line, Hep G2, synthesizes apoB-100 and secretes the apolipoprotein into the culture medium. After a 24-hr incubation with [14C]palmitate and [14C]stearate, the label was incorporated into apoB-100 when assessed by a sodium dodecyl sulfate polyacrylamide gel electrophoresis, autoradiography, immunoblot analysis, and immunoprecipitation. Hydroxylamine treatment, which hydrolyzes ester and thioester bonds, removed the radiolabel. ApoB-100 isolated from Hep G2 cells by ultracentrifugation and preparative sodium dodecyl sulfate gel electrophoresis was hydrolyzed and analyzed by gas-liquid chromatography-mass spectrometry. In contrast to circulating apoB in low density lipoproteins, both palmitate and stearate were present in newly synthesized apoB-100. These results establish that newly synthesized apoB-100 undergoes covalent acylation with palmitate and stearate. The acylation of apoB may play an important role in lipoprotein particle secretion. In addition, derangements in apoB fatty acid acylation may lead to dyslipoproteinemia.     

Effect of osmolarity on LDL binding and internalization in hepatocytes

The present study has been performed to elucidate a possiblerole of cell volume in low-density lipoprotein (LDL) binding andinternalization (LDL_b+i). Asshown previously, increase of extracellular osmolarity (OSMe) andK⁺ depletion, both known to shrinkcells, interfere with the formation of clathrin-coated pits and thuswith LDL_b+i. On the other hand,alterations of cell volume have been shown to modify lysosomal pH,which is a determinant of LDL_b+i.LDL_b+i have been estimated fromheparin-releasable (binding) or heparin-insensitive (internalization)uptake of ¹²⁵I-labeled LDL. OSMewas modified by alterations of extracellular concentrations of ions,glucose, urea, or raffinose. When OSMe was altered by varying NaClconcentrations, LDL_b+i decreased (by 0.5 ± 0.1%/mM) with increasing OSMe andLDL_b+i increased (by 1.2 ± 0.1%/mM) with decreasing OSMe, an effect mainly due to alteredaffinity; the estimated dissociation constant amounted to 20.6, 48.6, and 131.6 µg/ml at 219, 293, and 435 mosM, respectively. A 25%increase of OSMe increased cytosolic (by 0.46 ± 0.03) and decreasedlysosomal (by 0.14 ± 0.02) pH. Conversely, a 25% decrease of OSMedecreased cytosolic (by 0.28 ± 0.02) and increased lysosomal (by0.17 ± 0.02) pH. Partial replacement of extracellularNa⁺ withK⁺ had little effect onLDL_b+i, although it swelledhepatocytes and increased lysosomal and cytosolic pH. Hypertonicglucose, urea, or raffinose did not exert similar effects despite ashrinking effect of hypertonic raffinose. Monensin, which completelydissipates lysosomal acidity, virtually abolishedLDL_b+i. In conclusion, theobservations reveal a significant effect of ionic strength onLDL_b+i. The effect is, however,not likely to be mediated by alterations of cell volume or alterationsof lysosomal pH.

     

Effect of 3-substituted Delta8(14)-15-ketosterols on cholesterol metabolism in hepatoma Hep G2 cells

The effects of 3-substituted Delta8(14)-15-ketosterols--3beta-(2-hydroxyethoxy)-, 3beta-(2-propenyloxy)-, 3beta-[2(R,S),2,3-oxidopropyloxy]-, 3beta-[2(R,S),2,3-dihydroxypropyloxy]-, 3beta-(2-oxoethoxy)-, 3beta-[2(R,S),2-acetoxy-3-acetamidopropyloxy]-, and 3beta-[2(R,S), 2-hydroxy-3-acetamidopropyloxy]-5alpha-cholest-8(14)-en-15-o nes--on cholesterol metabolism were studied in human hepatoma Hep G2 cells. 3beta-(2-Propenyloxy)-, 3beta-(2-oxoethoxy)-, and 3beta-[2(R,S),2, 3-oxidopropyloxy]-5alpha-cholest-8(14)-en-15-ones inhibited cholesterol biosynthesis without any effect on triglyceride biosynthesis, while 3beta-[2(R,S),2-acetoxy-3-acetamidopropyloxy]- and 3beta-[2(R,S), 2-hydroxy-3-acetamidopropyloxy]-5alpha-cholest-8(14)-en-15-o nes inhibited both cholesterol biosynthesis and triglyceride biosynthesis at concentrations exceeding 10 microM. 3beta-[2(R,S),2, 3-Dihydroxypropyloxy]-5alpha-cholest-8(14)-en-15-one, effectively inhibiting cholesterol biosynthesis, was found also to be toxic in Hep G2 cells at micromolar concentrations. 3beta-[2(R,S),2, 3-Oxidopropyloxy]-5alpha-cholest-8(14)-en-15-one effectively inhibited cholesterol acylation. All the tested compounds decreased the HMG-CoA reductase mRNA level at concentrations exceeding 10 microM; however, they did not affect the LDL receptor mRNA level. Among the compounds tested, only 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one decreased the uptake and internalization of LDL-associated cholesteryl esters, being as effective as 25-hydroxycholesterol.     

Regulation of LDL receptor, apoB, and apoE protein and mRNA in Hep G2 cells.

The regulation of low-density lipoprotein (LDL) receptor activity, protein synthesis, and cellular mRNA content was evaluated in the human hepatoma cell line Hep G2. Incubation of the cells with LDL led to a complete downregulation of LDL receptor mRNA and LDL receptor protein synthesis. This LDL regulation of the LDL receptor and its mRNA was both time- and concentration-dependent. In contrast to protein synthesis and cellular mRNA concentrations of the LDL receptor, which were reduced to undetectable levels by prolonged incubation in the presence of LDL, LDL receptor activity was reduced to only 44% of preincubation levels. These findings support the presence of a second metabolic pathway for LDL uptake in human hepatocytic cells. The effect of LDL on cellular LDL receptor expression was specific for LDL because incubation in the presence of HDL did not affect any of these study end points. The potential coordinate regulation of the expression of the LDL receptor with its principal ligands, apolipoproteins (apo) B and E, was also investigated. In contrast to the LDL receptor mRNA downregulation with LDL incubation, cellular apoB and apoE mRNA concentrations were not affected by either LDL or HDL. Secretion of apoB, however, was significantly increased by incubating Hep G2 cells with LDL. These findings indicate that, in contrast to LDL receptor which is regulated at the mRNA level, the ligands for the LDL receptor are regulated either co- or post-translationally.     

Modification of membrane phospholipid fatty acyl composition in a leukemic T cell line: effects on receptor mediated intracellular Ca2+ increase

The effect of modifying fatty acyl composition of cellular membrane phospholipids on receptor-mediated intracellular free Ca²⁺ concentration ([Ca²⁺]_i) increase was investigated in a leukemic T cell line (JURKAT). After growing for 72 h in medium supplemented with unsaturated fatty acids (UFAs) and α-tocopherol, the fatty acyl composition of membrane phospholipids in JURKAT cells was extensively modified. Each respective fatty acid supplemented in the culture medium was readily incorporated into phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine in the JURKAT cells. The total n ? 6 fatty acyl content was markedly reduced in phosphatidylinositol and phosphatidylcholine of cells grown in the presence of n ? 3 fatty acids (α-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid). Conversely, in the presence of n ? 6 fatty acids (linoleic acid and arachidonic acid), the total n ? 3 fatty acyl content was reduced in all the phospholipids examined. In n ? 3 and n ? 6 polyunsaturated fatty acid (PUFA) modified JURKAT cells, the total n ? 9 monounsaturated fatty acyl content in the phospholipids were markedly reduced. Changing the fatty acyl composition of membrane phospholipids in the JURKAT cells appear to have no affect on the presentation of the T cell receptor/CD3 complex or the binding of anti-CD3 antibodies (OKT3) to the CD3 complex. However, the peak increase in [Ca²⁺]_i and the prolonged sustained phase elicited by OKT3 activation were suppressed in n ? 3 and n ? 6 PUFA but not in n ? 9 monounsaturated fatty acid modified cells. In Ca²⁺ free medium, OKT3-induced transient increase in [Ca²⁺]_i, representing Ca²⁺ release from the inositol 1,4,5-triphosphate-sensitive Ca²⁺ stores, were similar in control and UFA modified cells. Using Mn²⁺ entry as an index of plasma membrane Ca²⁺ permeability, the rate of fura-2 fluorescence quenching as a result of Mn²⁺ influx stimulated by OKT3 in n ? 9 monounsaturated fatty acid modified cells was similar to control cells, but the rates in n ? 3 and n ? 6 PUFA modified cells were significantly lower. These results suggest that receptor-mediated Ca²⁺ influx in JURKAT cells is sensitive to changes in the fatty acyl composition of membrane phospholipids and n ? 9 monounsaturated fatty acids appears to be important for the maintenance of a functional Ca²⁺ influx mechanism.     

Stimulation of the LDL receptor activity in the human hepatoma cell line Hep G2 by high-density serum fractions

The regulation of the LDL receptor activity in the human hepatoma cell line Hep G2 was studied. In Hep G2 cells, in contrast with fibroblasts, the LDL receptor activity was increased 2.5-fold upon increasing the concentration of normal whole serum in the culture medium from 20 to 100% by volume. Incubation of the Hep G2 cells with physiological concentrations of LDL (up to 700 micrograms/ml) instead of incubation under serum-free conditions resulted in a maximum 2-fold decrease in LDL receptor activity (10-fold decrease in fibroblasts). Incubation with physiological concentrations of HDL with a density of between 1.16 and 1.20 g/ml (heavy HDL) resulted in an approximately 7-fold increase in LDL receptor activity (1.5-fold increase in fibroblasts). This increased LDL receptor activity is due to an increase in the number of LDL receptors. Furthermore, simultaneous incubation of Hep G2 cells with LDL and heavy HDL (both 200 micrograms/ml) resulted in a 3-fold stimulation of the LDL receptor activity as compared with incubation in serum-free medium. 3-Hydroxy-3-methylglutaryl-CoA reductase activity was also stimulated after incubation of Hep G2 with heavy HDL (up to 3-fold). The increased LDL receptor activity in Hep G2 cells after incubation with heavy HDL was independent of the action of lecithin:cholesterol acyltransferase during that incubation. However, previous modification of heavy HDL by lecithin:cholesterol acyltransferase resulted in an enhanced ability of heavy HDL to stimulate the LDL receptor activity. Our results indicate that in Hep G2 cells the heavy HDL-mediated stimulation of the LDL receptor activity overrules the LDL-mediated down-regulation and raises the suggestion that in man the presence of heavy HDL and the action of lecithin:cholesterol acyltransferase in plasma may be of importance in receptor-mediated catabolism of LDL by the liver.     

Modification of membrane phospholipid fatty acyl composition in a leukemic T cell line: effects on receptor mediated intracellular Ca2+ increase.

The effect of modifying fatty acyl composition of cellular membrane phospholipids on receptor-mediated intracellular free Ca2+ concentration ([Ca2+]i) increase was investigated in a leukemic T cell line (JURKAT). After growing for 72 h in medium supplemented with unsaturated fatty acids (UFAs) and alpha-tocopherol, the fatty acyl composition of membrane phospholipids in JURKAT cells was extensively modified. Each respective fatty acid supplemented in the culture medium was readily incorporated into phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine in the JURKAT cells. The total n-6 fatty acyl content was markedly reduced in phosphatidylinositol and phosphatidylcholine of cells grown in the presence of n-3 fatty acids (alpha-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid). Conversely, in the presence of n-6 fatty acids (linoleic acid and arachidonic acid), the total n-3 fatty acyl content was reduced in all the phospholipids examined. In n-3 and n-6 polyunsaturated fatty acid (PUFA) modified JURKAT cells, the total n-9 monounsaturated fatty acyl content in the phospholipids were markedly reduced. Changing the fatty acyl composition of membrane phospholipids in the JURKAT cells appears to have no affect on the presentation of the T cell receptor/CD3 complex or the binding of anti-CD3 antibodies (OKT3) to the CD3 complex. However, the peak increase in [Ca2+]i and the prolonged sustained phase elicited by OKT3 activation were suppressed in n-3 and n-6 PUFA but not in n-9 monounsaturated fatty acid modified cells. In Ca2+ free medium, OKT3-induced transient increase in [Ca2+]i representing Ca2+ release from the inositol 1,4,5-trisphosphate-sensitive Ca2+ stores, were similar in control and UFA modified cells. Using Mn2+ entry as an index of plasma membrane Ca2+ permeability, the rate of fura-2 fluorescence quenching as a result of Mn2+ influx stimulated by OKT3 in n-9 monounsaturated fatty acid modified cells was similar to control cells, but the rates in n-3 and n-6 PUFA modified cells were significantly lower. These results suggest that receptor-mediated Ca2+ influx in JURKAT cells is sensitive to changes in the fatty acyl composition of membrane phospholipids and monounsaturated fatty acids appears to be important for the maintenance of a functional Ca2+ influx mechanism.     

Sterol carrier protein-2 expression alters phospholipid content and fatty acyl composition in L-cell fibroblasts

The effects sterol carrier protein-2 (SCP-2) expression on L-cell phospholipid levels and fatty acyl composition was assessed using L-cells transfected with the murine cDNA encoding for either the 15 kDa proSCP-2 or 13.2 kDa SCP-2. Expression of these proteins reduced total phospholipid mass (nmol/mg protein) by 24% and reduced the cholesterol to phospholipid ratio 60 and 28%, respectively. In 15 kDa proSCP-2 expressing cells, individual phospholipid class masses, excluding sphingomyelin (CerPCho), were reduced as follows: phosphatidylinositol (PtdIns) and phosphatidylserine (PtdSer) > ethanolamine glycerophospholipid (EtnGpl) > choline glycerophospholipid (ChoGpl). Furthermore, ethanolamine plasmalogen mass was decreased 25%, while choline plasmalogen mass was elevated 30% in 15 kDa proSCP-2 expressing cells. In 13.2 kDa SCP-2 expressing cells, phospholipid class mass was decreased as follows: PtdIns and PtdSer > ChoGpl. These changes in phospholipid mass resulted in altered cellular phospholipid composition. Expression of either protein differentially altered the type of fatty acid esterified onto the phospholipids. These effects included a greater proportion of polyunsaturated fatty acids and a reduction in saturated fatty acids, although 15 kDa proSCP-2 expression had a more robust effect on these parameters than did 13.2 kDa SCP-2 expression. In summary, expression of SCP-2 reduced individual phospholipid class mass, except for CerPCho, and altered the fatty acid composition of each phospholipid class examined.These results clearly demonstrate that SCP-2 expression altered basal phospholipid levels, suggesting that SCP-2 can alter the function of endoplasmic reticulum phospholipid synthetic enzymes.