Fatty acid synthesis in Escherichia coli

1. Fatty acid formation by cells of a strain of Escherichia coli has been studied in the exponential, post-exponential and stationary phases of growth. 2. During the exponential phase of growth, the metabolic quotient (mmumoles of fatty acid synthesized/mg. dry wt. of cells/hr.) for each fatty acid in the extractable lipid was constant. 3. The newly synthesized fatty acid mixtures produced during this phase contained hexadecanoic acid (41%), hexadecenoic acid (31%), octadecenoic acid (21%) and the C(17)-cyclopropane acid, methylenehexadecanoic acid (4%). 4. As the proportion of newly synthesized material increased, changes in the fatty acid composition of the cells during this period were towards this constant composition. 5. Abrupt changes in fatty acid synthesis occurred when exponential growth ceased. 6. In media in which glycerol, or SO(4) (2-) or Mg(2+), was growth-limiting there was a small accumulation of C(17)-cyclopropane acid in cells growing in the post-exponential phase of growth. 7. Where either NH(4) (+) or PO(4) (3-) was growth-limiting and there were adequate supplies of glycerol, Mg(2+) and SO(4) (2-), there was a marked accumulation of C(17)-cyclopropane acid and C(19)-cyclopropane acid appeared. 8. Under appropriate conditions the metabolic quotient for C(17)-cyclopropane acid increased up to sevenfold at the end of exponential growth. Simultaneously the metabolic quotients of the other acids fell. 9. A mixture of glycerol, Mg(2+) and SO(4) (2-) stimulated cyclopropane acid formation in resting cells.     

The peroxisomal Acyl-CoA thioesterase Pte1p from Saccharomyces cerevisiae is required for efficient degradation of short straight chain and branched chain fatty acids

The role of the Saccharomyces cerevisae peroxisomal acyl-coenzyme A (acyl-CoA) thioesterase (Pte1p) in fatty acid beta-oxidation was studied by analyzing the in vitro kinetic activity of the purified protein as well as by measuring the carbon flux through the beta-oxidation cycle in vivo using the synthesis of peroxisomal polyhydroxyalkanoate (PHA) from the polymerization of the 3-hydroxyacyl-CoAs as a marker. The amount of PHA synthesized from the degradation of 10-cis-heptadecenoic, tridecanoic, undecanoic, or nonanoic acids was equivalent or slightly reduced in the pte1Delta strain compared with wild type. In contrast, a strong reduction in PHA synthesized from heptanoic acid and 8-methyl-nonanoic acid was observed for the pte1Delta strain compared with wild type. The poor catabolism of 8-methyl-nonanoic acid via beta-oxidation in pte1Delta negatively impacted the degradation of 10-cis-heptadecenoic acid and reduced the ability of the cells to efficiently grow in medium containing such fatty acids. An increase in the proportion of the short chain 3-hydroxyacid monomers was observed in PHA synthesized in pte1Delta cells grown on a variety of fatty acids, indicating a reduction in the metabolism of short chain acyl-CoAs in these cells. A purified histidine-tagged Pte1p showed high activity toward short and medium chain length acyl-CoAs, including butyryl-CoA, decanoyl-CoA and 8-methyl-nonanoyl-CoA. The kinetic parameters measured for the purified Pte1p fit well with the implication of this enzyme in the efficient metabolism of short straight and branched chain fatty acyl-CoAs by the beta-oxidation cycle.     

Fatty acid synthase plays a role in cancer metabolism beyond providing fatty acids for phospholipid synthesis or sustaining elevations in glycolytic activity

Fatty acid synthase is over-expressed in many cancers and its activity is required for cancer cell survival, but the role of endogenously synthesized fatty acids in cancer is unknown. It has been suggested that endogenous fatty acid synthesis is either needed to support the growth of rapidly dividing cells, or to maintain elevated glycolysis (the Warburg effect) that is characteristic of cancer cells. Here, we investigate both hypotheses. First, we compared utilization of fatty acids synthesized endogenously from ¹⁴C-labeled acetate to those supplied exogenously as ¹⁴C-labeled palmitate in the culture medium in human breast cancer (MCF-7 and MDA-MB-231) and untransformed breast epithelial cells (MCF-10A). We found that cancer cells do not produce fatty acids that are different from those derived from exogenous palmitate, that these fatty acids are esterified to the same lipid and phospholipid classes in the same proportions, and that their distribution within neutral lipids is not different from untransformed cells. These results suggest that endogenously synthesized fatty acids do not fulfill a specific function in cancer cells. Furthermore, we observed that cancer cells excrete endogenously synthesized fatty acids, suggesting that they are produced in excess of requirements. We next investigated whether lipogenic activity is involved in the maintenance of high glycolytic activity by culturing both cancer and non-transformed cells under anoxic conditions. Although anoxia increased glycolysis 2–3 fold, we observed no concomitant increase in lipogenesis. Our results indicate that breast cancer cells do not have a specific qualitative or quantitative requirement for endogenously synthesized fatty acids and that increased de novo lipogenesis is not required to sustain elevations in glycolytic activity induced by anoxia in these cells.     

Docosahexaenoic acid enrichment can reduce L929 cell necrosis induced by tumor necrosis factor

We previously reported that docosahexaenoic acid (DHA) attenuated tumor necrosis factor (TNF)-induced apoptosis in human monocytic U937 cells (J. Nutr. 130: 1095-1101, 2000). In the present study, we examined the effects of DHA and other polyunsaturated fatty acids (PUFA) on TNF-induced necrosis, another mode of cell death, using L929 murine fibrosarcoma cells. After preincubation with PUFA conjugated with BSA for 24 h, cells were treated with TNF or TNF+actinomycin D (Act D). Preincubation of cells with DHA enriched this polyunsaturated acid in the phospholipids and attenuated cell death induced by either TNF or TNF+Act D. When cells were treated with TNF alone, DNA laddering was not detected, and cells were coincidently stained with both annexin V-FITC and propidium iodide, indicating that the death mode was necrotic. TNF+Act D predominantly induced necrosis, although concurrent apoptotic cell death was also observed in this case. Preincubation with oleic acid, linoleic acid or 20:3(n-3) did not affect TNF-induced necrosis. Conversely, supplementation with n-3 docosapentaenoic acid (DPAn-3) or eicosapentaenoic acid (EPA) reduced necrotic cell death, but to a lesser extent in comparison with DHA. Unlike the case of U937 cell apoptosis, arachidonic acid (AA) significantly attenuated L929 cell necrosis, and 20:3(n-6) or 22:4(n-6) showed similar or less activity, respectively. Statistical evaluation indicated that the order of effective PUFA activity was DHA>DPAn-3> or =EPA>AA approximately 20:3(n-6)> or =22:4(n-6). One step desaturation, C2 elongation or C2 cleavage within the n-6 or n-3 fatty acid group was probably very active in L929 cells, because AA, synthesized from 20:3(n-6) or 22:4(n-6), and C22 fatty acids, synthesized from AA or EPA, were preferentially retained in cellular phospholipids. These observations suggested that attenuation of TNF-induced necrosis by the supplementation of various C20 or C22 polyunsaturated fatty acids is mainly attributable to the enrichment of three kinds of polyunsaturated fatty acids, i.e., DHA, DPAn-3 or AA, in phospholipids. Among these fatty acids, DHA was the most effective in the reduction of L929 necrosis as observed in the case of U937 apoptosis. This suggests that DHA-enriched membranes can protect cell against TNF irrespective of death modes and that membranous DHA may abrogate the death signaling common to necrosis and apoptosis.     

Effects of clofibrate feeding on essential fatty acid desaturation and oxidation in isolated rat liver cells.

The effects of clofibrate feeding on the metabolism of polyunsaturated fatty acids were studied in isolated rat hepatocytes. Administration of clofibrate stimulated the oxidation and particularly the peroxisomal beta-oxidation of all the fatty acids used. The increase in oxidation products was markedly higher when n-3 fatty acids were used as substrate, indicating that peroxisomes contribute more to the oxidation of n-3 than n-6 fatty acids. The whole increase in oxidation could be accounted for by a corresponding decrease in acylation in triacylglycerol while the esterification in phospholipids remained unchanged. A marked stimulation of the amounts of newly synthesized C16 and C18 fatty acids recovered, was observed when 18:2(n-6), 20:3(n-6), 18:3 (n-3) and 20:5(n-3), but not when 20:4(n-6) and 22:4(n-6) were used as substrate. This agrees with the view that extra-mitochondrial acetyl-CoA produced from peroxisomal beta-oxidation is more easily used for fatty acid new synthesis than acetyl-CoA from mitochondrial beta-oxidation. The delta 6 and delta 5 desaturase activities were distinctly higher in cells from clofibrate fed rats indicating a stimulating effect.     

Interrelationships between fatty acid biosynthesis and acyl-lipid synthesis in Chlorella vulgaris

1. Fatty acid synthesis from [2-(14)C]acetate by Chlorella vulgaris cells grown and incubated in the dark is limited almost entirely to the production of saturated and monoenoic acids. 2. In light-incubated cells, both saturated and polyunsaturated fatty acids are rapidly synthesized. 3. Two groups of lipids can be distinguished in both dark- and light-incubated cells. The first group, consisting of phosphatidyl-glycerol, monogalactosyl diglyceride, lecithin and neutral glyceride, has a very high turnover rate for certain fatty acids. The second group, consisting of digalactosyl diglyceride, sulpholipid, phosphatidylethanolamine and phosphatidylinositol, has a slow turnover of fatty acids. 4. The lipids with rapid fatty acid turnover may be involved in the sequences of saturated and unsaturated fatty acid synthesis. A classification of lipids is made on the basis of their suggested functions.     

Possible involvement of acetyl coenzyme A carboxylase as well as fatty acid synthetase in the temperature-controlled synthesis of fatty acids in Saccharomyces cerevisiae

Fatty acid synthetase (FAS) preparations from Saccharomyces cerevisiae cells grown at either 35 or 10 degrees C produced the same products at different temperatures and showed quite similar temperature-dependencies in Arrhenius plots, with break points at 25 degrees C. This break point does not appear to reflect a phase transition of phospholipids present in the purified FAS preparations but rather is associated with protein conformational changes. S. cerevisiae cells grown at 35 degrees C and then shifted to 10 degrees C produced fatty acids with a shorter average chain length than those fatty acids synthesized at 10 degrees C by cells already adapted to 10 degrees C (hyper response). Acetyl-CoA carboxylase activity was relatively higher in the cells grown at 35 degrees C than in the cells grown at 10 degrees C; moreover, fatty acids with longer average chain lengths were synthesized in vitro at higher malonyl-CoA concentrations, which was consistent with the difference in the average chain lengths of newly synthesized fatty acids in cells grown at 35 and 10 degrees C. However, the activity levels of acetyl-CoA carboxylase and fatty acid synthetase alone did not account for the hyper response phenomena.     

Long-chain fatty acid esters of 5-androstene-3 beta, 17 beta-diol: composition and turnover in human mammary cancer cells in culture

Long-chain fatty acid esters of the adrenal-derived estrogen 5-androstene-3 beta, 17 beta-diol (ADIOL) were found to accumulate in four human mammary cancer cell lines (MCF-7, ZR-75-1, MDA-MB-231 and MDA-MB-330) when explosed to 10-30 nM ADIOL for variable time periods. At each time point examined, the monoester fraction, which represented the major component of the total lipoidal fraction, contained fatty acids linked to either the 3 beta- or 17 beta-positions. However, there was considerable variation in the ratio of 3 beta- to 17 beta-monoesters in the four cell lines. By means of reverse phase HPLC and referral to authentic synthesized compounds, each monoester fraction was found to contain a number of long-chain fatty acid components whose composition resembled that previously determined for the fatty acid esters formed from 17 beta-estradiol. A specific and measurable turnover of a subfraction of ADIOL-17 beta-monoesters composed of essential fatty acids (22:6, 20:4, 18:3) occurred in MCF-7 cells, and to a lesser extent in ZR-75-1 cells. No changes were observed with time in any of the components of the 3 beta- or 17 beta-monoester fractions in MDA-MB-231 and MDA-MB-330 cells. These results, coupled with other studies, now suggest that a very rapid turnover of some components of these lipoidal derivatives may be occurring. If so, it is possible that the system of acylation-deacylation may be involved in a transport mechanism for estrogens and perhaps other steroid hormones.     

Effect of long-chain fatty acids in the culture medium on fatty acid composition of WEHI-3 and J774A.1 cells

As a first step in determining the mechanism of action of specific fatty acids on immunological function of macrophages, a comparative study of the effect of long-chain polyunsaturated fatty acids (PUFA) in the medium was conducted in two macrophage cell lines, J774A.1 and WEHI-3. The baseline fatty-acid profiles of the two cell lines differed in the % distribution of saturated (SFA) and unsaturated fatty acids (UFA). J774A.1 cells had a higher % of SFA (primarily palmitic acid) than WEHI-3 cells. Conversely, WEHI-3 cells had a higher % of UFA (primarily oleic acid) than J774A.1 cells. Neither cell line had detectable amounts of alpha-linolenic acid (ALA) or eicosapentaenoic acid (EPA). The most abundant polyunsaturated fatty acid in both cells lines was arachidonic acid (AA). The efficiency of transport of fatty acids from the medium to the macrophages by two delivery vehicles (BSA complexes and ethanolic suspensions) was compared. Overall, fatty acids were transported satisfactorily by both delivery systems. Alpha-linolenic acid and doscosahexenoic acid (DHA) were transported more efficiently by the ethanolic suspension system. Linoleic acid (LA) was taken up more completely than ALA, and DHA was taken up more completely than EPA by both cell cultures and delivery systems. A dose-response effect was demonstrated for LA, ALA, EPA and DHA in both J774A.1 and WEHI-3 cells. Addition of polyunsaturated fatty acids (PUFA) to the cell cultures modified the total lipid fatty acid composition of the cells. The presence of ALA in the culture medium resulted in a significant decrease in AA in both cell lines. The omega-3/omega-6 fatty acid ratio (omega-3/omega-6), polyunsaturated/saturated fatty acid ratio (P/S), and unsaturation index (UI) increased directly with the amount of PUFA and omega-3 fatty acid provided in the medium. The results indicate that the macrophage cell lines have similar, but not identical, fatty acid profiles that may be the result of differences in fatty acid metabolism. These distinctions could in turn produce differences in immunological function. The ethanol fatty-acid delivery system, when compared with the fatty acid-BSA complex system, is preferable for measurement of dose-response effects, because the cellular fatty acid content increased in proportion to the amount of fatty acid provided in the medium. Similar dose-response results were observed in a previous in vivo study using flaxseed, rich in ALA, as a source of PUFA.     

Abortive infection of the virulent phage 9NA in a fatty acid auxotroph of Salmonella typhimurium: effect of fatty acid supplementation

A conditional (temperature sensitive) fatty acid biosynthetic mutant (fabB2) of Salmonella typhimurium does not support the development of the virulent bacteriophage 9NA even at permissive temperature (30 degrees C). A limited amount of phage DNA synthesis takes place at this temperature. When the fatty acid composition of the host membrane is altered by growing the cells at 37 degrees C in the presence of exogenous unsaturated fatty acid, differential expression of phage genes was observed. Phage specific lysozyme is induced when the cultures are supplemented with elaidic, palmitelaidic, linoleic and linolelaidic acids but not with oleic and plamitoleic acids. However, in no case were infective particles produced. Under conditions where no lysozyme is synthesized the infected cells increase in length and become filamentous.     

Fatty acid synthetase as a thermoreceptor in Candida utilis

A partially purified fatty acid synthetase from Candida utilis synthesized fatty acids with varying chain lengths that depended on the assay temperature; the stearate/palmitate ratio decreased with decreasing temperature. This temperature-dependency was also observed in vivo for the newly synthesized fatty acids in cells incubated at various temperatures, although to a lesser extent than that observed in vitro. The difference in the temperature-dependencies observed in vivo and in vitro appeared to be due to the difference in the acceptors used in in vitro assays; a temperature-dependency comparable to that observed in vivo was reproduced in vitro on using microsomes rather than bovine serum albumin as the acceptor of the fatty acid synthetase products. Thus, the fatty acid synthetase was identified as a thermoreceptor in Candida cells known to possess a temperature-dependent, inducible desaturase system.     

Synthesis of fatty acids in the perfused mouse liver

1. Fatty acid synthesis de novo was measured in the perfused liver of fed mice. 2. The total rate, measured by the incorporation into fatty acid of (3)H from (3)H(2)O (1-7mumol of fatty acid/h per g of fresh liver), resembled the rate found in the liver of intact mice. 3. Perfusions with l-[U-(14)C]lactic acid and [U-(14)C]glucose showed that circulating glucose at concentrations less than about 17mm was not a major carbon source for newly synthesized fatty acid, whereas lactate (10mm) markedly stimulated fatty acid synthesis, and contributed extensive carbon to lipogenesis. 4. The identification of 50% of the carbon converted into newly synthesized fatty acid lends further credibility to the use of (3)H(2)O to measure hepatic fatty acid synthesis. 5. The total rate of fatty acid synthesis, and the contribution of glucose carbon to lipogenesis, were directly proportional to the initial hepatic glycogen concentration. 6. The proportion of total newly synthesized lipid that was released into the perfusion medium was 12-16%. 7. The major products of lipogenesis were saturated fatty acids in triglyceride and phospholipid. 8. The rate of cholesterol synthesis, also measured with (3)H(2)O, expressed as acetyl residues consumed, was about one-fourth of the basal rate of fatty acid synthesis. 9. These results are discussed in terms of the carbon sources of hepatic newly synthesized fatty acids, and the effect of glucose, glycogen and lactate in stimulating lipogenesis, independently of their role as precursors.     

Polyhydroxyalknoate synthesis in plants as a tool for biotechnology and basic studies of lipid metabolism.

Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyacids naturally synthesized in bacteria as a carbon reserve. PHAs have properties of biodegradable thermoplastics and elastomers and their synthesis in crop plants is seen as an attractive system for the sustained production of large amounts of polymers at low cost. A variety of PHAs having different physical properties have now been synthesized in a number of transgenic plants, including Arabidopsis thaliana, rape and corn. This has been accomplished through the creation of novel metabolic pathways either in the cytoplasm, plastid or peroxisome of plant cells. Beyond its impact in biotechnology, PHA production in plants can also be used to study some fundamental aspects of plant metabolism. Synthesis of PHA can be used both as an indicator and a modulator of the carbon flux to pathways competing for common substrates, such as acetyl-coenzyme A in fatty acid biosynthesis or 3-hydroxyacyl-coenzyme A in fatty acid degradation. Synthesis of PHAs in plant peroxisome has been used to demonstrate changes in the flux of fatty acids to the beta-oxidation cycle in transgenic plants and mutants affected in lipid biosynthesis, as well as to study the pathway of degradation of unusual fatty acids.     

Effect of culture in vitro with eicosatetraenoic (20:4(n-6) ) and eicosapentaenoic (20:5(n-3) ) acids on fatty acid composition, prostaglandin synthesis and chemiluminescence of rat peritoneal macrophages

Rat peritoneal macrophages were cultured in either eicosatetraenoic acid (20:4(n-6) ) or eicosapentaenoic acid (20:5(n-3) ) and the effects on phospholipid fatty acids, prostaglandin synthesizing capacity and the ability of the macrophages to show chemiluminescence were examined. Chemiluminescence is an activity resulting from the synthesis of reactive oxygen species. It has been reported that prostaglandins inhibit this activity. The fatty acid profile of the four major phospholipids reflected the fatty acid component of the medium. Macrophages cultured in 20:4(n-6) synthesized twice the prostaglandin produced by controls and those cultured in 20:5(n-3) synthesized 10% that of controls and 5% that of 20:4(n-6)-cultured cells. Macrophages cultured with 20:4(n-6) for 12 h showed half the chemiluminescence of those cultured with 20:5(n-3), while those cultured with 20:4(n-6) for 24 h showed 10% the chemiluminescence of 20:5(n-3)-cultured cells. Addition of the prostaglandin synthase inhibitor, indomethacin, had no effect on chemiluminescence.     

Comparison of Rapid Methods for Analysis of Bacterial Fatty Acids

When rapid gas-liquid chromatography methods for determination of bacterial fatty acids were compared, results showed that saponification was required for total fatty acid analysis. Transesterification with boron-trihalide reagents (BF(3)-CH(3)OH, BCl(3)-CH(3)OH) caused extensive degradation of cyclopropane acids and was less effective than saponification in releasing cellular hydroxy fatty acids. Digestion of cells with tetramethylammonium hydroxide was unsatisfactory because of extraneous gas-liquid chromatography peaks and because of lower recovery of branched-chain and hydroxy fatty acids. A simple, rapid saponification procedure which can be used for total cellular fatty acid analysis of freshly grown cells is described.     

Absence of monoacylglycerol pathway for triacylglycerol synthesis in goat mammary gland.

Both subcellular fractions of lactating goat mammary gland and dispersed lactating goat mammary-gland cells were able to incorporate [U-3H]2-O-hexadecylglycerol into monoalkyl-monoacylglycerol. However, the incorporation of [U-3H]2-O-hexadecylglycerol into monoalkyl-monoacylglycerol by dispersed cells was not accompanied by incorporation of fatty acid synthesized de novo or added radiolabelled fatty acid. The result therefore shows that an active monoacylglycerol pathway does not exist in goat mammary gland.     

Contributions of fatty acid and sterol synthesis to triglyceride and cholesterol secretion by the perfused rat liver in genetic hyperlipemia and obesity

The relative importance of fatty acid synthesis in triglyceride secretion by perfused livers from lean (normal control) and obese Zucker rats was investigated. Livers from fed animals were perfused in a recirculating system with tritiated water and a constant infusion of oleic acid. Triglyceride secretion was 5 times greater and cholesterol secretion was 35% greater in the obese rat livers. The very-low-density lipoprotein hypersecreted by perfused livers from obese rats contained more apolipoprotein B and exhibited an increased B-48/B-100 ratio. Apo-B was also elevated in the hypertriglyceridemic plasma of obese rats in both fed and fasting states. The very-low-density lipoprotein isolated therefrom was likewise characterized by an increased B-48/B-100 ratio. Ketogenesis was depressed 40% in the obese rat livers and increased hepatic malonyl-CoA was implicated in this alteration. The de novo synthesis and secretion of newly synthesized cholesterol was moderately increased in the perfused livers from obese rats. Tritium incorporation into fatty acids was 15 times greater in the obese genotype. Most of the synthesized fatty acids remained in the liver and were recovered after perfusion in triglyceride and phospholipids. Newly synthesized fatty acids accounted for only 3 and 15% of the triglyceride secreted by the lean and obese rat livers, respectively. A large portion of the secreted triglyceride fatty acids was derived from endogenous liver lipids. When the turnover of newly synthesized fatty acids in these pools was considered, the contribution of de novo fatty acid synthesis to triglyceride secretion was estimated to be 9% in the lean and 44% in the obese rat livers. Therefore, the altered partition of free fatty acids (Fukuda, N., Azain, M. J., and Ontko, J. A. (1982) J. Biol. Chem. 257, 14066-14072) and increased fatty acid synthesis are both major determinants of the hypersecretion of triglyceride-rich lipoproteins by the liver in the genetically obese Zucker rat.     

Effect of eicosapentaenoic acid on triacylglycerol transport in CaCo-2 cells

The human intestinal cell line, CaCo-2, was used to study the effect of the n-3 fatty acid, eicosapentaenoic acid, on triacylglycerol secretion. In cells incubated with 250 microM eicosapentaenoic acid, the incorporation of [3H]glycerol into triacylglycerols secreted into the medium was decreased by 58% compared to cells incubated with 250 microM oleic acid. The incorporation of [3H]glycerol into cellular triacylglycerols was decreased 32% in cells incubated with eicosapentaenoic acid. In cells preincubated with [3H]glycerol to label existing triacylglycerols, the rates of secretion of preformed triacylglycerols were similar in response to the addition of either fatty acid. Initial uptake rates of the n-3 fatty acid were higher than for oleic acid. Both eicosapentaenoic acid and oleic acid were minimally oxidized to CO2. Oleic acid was predominantly incorporated into cellular triacylglycerols (62% vs. 47%), whereas more eicosapentaenoic acid was incorporated into cellular phospholipids (46% vs. 30%). Phospholipids of microsomes prepared from cells incubated with eicosapentaenoic acid were enriched in this fatty acid. The rate of synthesis of triacylglycerol and diacylglycerol acyltransferase activities were significantly less in microsomes prepared from cells incubated with eicosapentaenoic acid. Triacylglycerol mass secreted by CaCo-2 cells incubated with either fatty acid was similar. In CaCo-2 cells, eicosapentaenoic acid decreases the synthesis and secretion of newly synthesized triacylglycerol without decreasing the secretion of triacylglycerol mass. Modification of microsomal membrane phospholipid fatty acid composition is associated with a decrease in microsomal triacylglycerol synthesis and diacylglycerol acyltransferase activities.     

Fish oil fatty acids impair VLDL assembly and/or secretion by cultured rat hepatocytes

We determined the effect of the two major fish oil fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on VLDL assembly and secretion by cultured rat hepatocytes. The incorporation of [3H]glycerol into total triglyceride (cell plus media) was stimulated eight-fold when hepatocytes were incubated for 2 h with 1 mM EPA, DHA, or oleic acid (OA), suggesting that fish oil fatty acids stimulate hepatic triglyceride synthesis to an extent similar to OA. In contrast, mass quantitation of secreted triglyceride showed impaired triglyceride secretion with EPA and DHA compared to OA. During a 42-h time course, cells stimulated with EPA and DHA progressively accumulated triglyceride compared to cells stimulated with OA. To determine whether fish oil fatty acids impair very low density lipoprotein (VLDL) secretion, cells were labeled with [35S]methionine and the secretion of de novo synthesized apoB was measured. Compared to OA, EPA and DHA significantly impaired the secretion of both molecular weight forms of apoB. The cellular content of apoB was not altered by any of the fatty acids. The concordant decrease in the secretion of both triglyceride and apoB suggests that fish oil fatty acids impair VLDL assembly and/or secretion.     

Conversion to adipocytes of a clonal bone marrow preadipocyte line (H-1/A) and fatty acid composition of the resultant adipocytes

Conversion to adipocytes and fatty acid composition were investigated in a clonal bone marrow preadipocyte line (H-1/A). The growing cells exhibited a fibroblastic appearance. After the cessation of growth, triacylglyceride (TG) synthesis in the cells increased as they incorporated precursor from the growth medium and became adipocytes. Hydrocortisone and insulin accelerated the TG synthesis in H-1/A cells in a dose-dependent manner when they were cultured in the growth medium containing 10% horse serum. The rate of conversion to adipocytes was reduced as the concentration of horse serum was decreased, and this reduction was not influenced by the addition of insulin and/or hydrocortisone. These results suggest that conversion to adipocytes of H-1/A cells is primarily dependent on some component(s) of the serum. Conversion to adipocytes of the cells may involve a process of differentiation since the conversion was completely inhibited when the cells were cultured in the presence of bromodeoxyuridine. Fatty acid composition was significantly different between adipose H-1/A cells and adipocytes derived from other marrow preadipocyte line MC3T3-G2/PA6 cells. Unsaturated fatty acids accounted for 76% of the fatty acid composition of adipose H-1/A cells; in contrast, saturated fatty acids constituted 65% of the fatty acid composition of the adipose MC3T3-G2/PA6 cells. These results suggest that there is a heterogeneity of preadipocytes in bone marrow. These two preadipocyte lines thus provide a useful tool for the study of marrow adipocytes and can also be used to analyze the hematopoietic microenvironment through studies of the effect of these cells on hematopoietic cell proliferation.