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1.
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.  相似文献   

2.
We assessed the ability of endothelial lipase (EL) to hydrolyze the sn-1 and sn-2 fatty acids (FAs) from HDL phosphatidylcholine. For this purpose, reconstituted discoidal HDLs (rHDLs) that contained free cholesterol, apolipoprotein A-I, and either 1-palmitoyl-2-oleoylphosphatidylcholine, 1-palmitoyl-2-linoleoylphosphatidylcholine, or 1-palmitoyl-2-arachidonylphosphatidylcholine were incubated with EL- and control (LacZ)-conditioned media. Gas chromatography analysis of the reaction mixtures revealed that both the sn-1 (16:0) and sn-2 (18:1, 18:2, and 20:4) FAs were liberated by EL. The higher rate of sn-1 FA cleavage compared with sn-2 FA release generated corresponding sn-2 acyl lyso-species as determined by MS analysis. EL failed to release sn-2 FA from rHDLs containing 1-O-1'-hexadecenyl-2-arachidonoylphosphatidylcholine, whose sn-1 position contained a nonhydrolyzable alkyl ether linkage. The lack of phospholipase A(2) activity of EL and its ability to liberate [(14)C]FA from [(14)C]lysophosphatidylcholine (lyso-PC) led us to conclude that EL-mediated deacylation of phosphatidylcholine (PC) is initiated at the sn-1 position, followed by the release of the remaining FA from the lyso-PC intermediate. Thin-layer chromatography analysis of cellular lipids obtained from EL-overexpressing cells revealed a pronounced accumulation of [(14)C]phospholipid and [(14)C]triglyceride upon incubation with 1-palmitoyl-2-[1-(14)C]linoleoyl-PC-labeled HDL(3), indicating the ability of EL to supply cells with unsaturated FAs.  相似文献   

3.
Research on fatty acid metabolism in cultured human larynx tumor cells Hep2 was carried out.The cells were incubated with either a saturated (palmitic) or a polyunsaturated (linoleic, alpha-linolenic and eicosatrienoic (n-6)) radioactive fatty acid (0.66 pM, 24 h). The best incorporation capacity was observed in the linoleic acid followed by alpha-linolenic, palmitic and eicosatrienoic acids. All fatty acids tested were anabolized to higher derivatives within their own family. Palmitic acid was primarily monodesaturated rather than elongated, proving to have a very active A9 desaturase activity.With respect to polyunsaturated acid metabolism, the conversion of alpha-linolenic acid to higher homologs, although better than linoleic acid, occurred far less efficiently than that observed in other non-highly undifferentiated human tumor cells. This impairment in higher polyunsaturated fatty acid biosynthesis, reflected in the low levels of arachidonic acid in the fatty acid composition, would not reside in the A5 desaturation step since Hep2 cells can readily convert eicosatrienoic acid into arachidonic acid. Considering the potential regulatory role of specific polyunsaturated fatty acids in the cell proliferative control, the knowledge of the metabolism of fatty acids in this human tumor cell would be important for designing future experiments in order to clarify the mechanism involved in balance, proliferation and cell death.  相似文献   

4.
The biosynthesis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in monocyte-like leukemia U937 cells was monitored by adding [3H]choline, [14C]ethanolamine or [14C]glycerol to the culture media; incorporation into phospholipid (PL) increased with time. The effect of unsaturated fatty acids (UFA) on PC and PE synthesis was investigated by pretreating U937 cells for 72h with 10 μM 18:1 (n –9), 18:2 (n –6), 18:3 (n –3), 20:4 (n –6) and 20:5 (n –3). The UFA caused no alteration in cell growth, as evidenced by light microscopy and the incorporation of [3H]thymidine and [3H]leucine. Total cellular uptake of radioactive precursors remained unaffected by all the treatments. Pretreatment with 20:5 resulted in approximately 25 per cent reduction in the incorporation of [3H]choline into PL, while no significant effect was detected with the other UFAs. 18:3, 20:4 and 20:5 depressed the incorporation of [14C]ethanolamine into PL by 34 per cent, 28 per cent and 49 per cent respectively. However, there was no redistribution of label with any of the treatments. 18:3, 20:4 and 20:5 also antagonized the stimulatory effect of endotoxin (LPS) on PC and PE synthesis. In addition, the incorporation from [14C]glycerol into PC and PE was reduced by 18:3, 20:4 and 20:5. Although the PL composition of the cells remained essentially unaffected, our study shows that chronic treatment of U937 cells with n –3 PUFA (20:5) depressed PC and PE synthesis, and 18:3 and 20:4 also caused inhibition of PE synthesis.  相似文献   

5.
There is controversy about the effect of saturated and polyunsaturated fats on 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, the main regulatory enzyme of cholesterogenic pathway. Results from dietary studies are difficult to interpret because diets normally contain a mixture of fatty acids. Therefore, we have used Reuber H35 hepatoma cells whose phospholipids were enriched in different individual fatty acids and have studied their effects on the cellular reductase activity. Lauric, myristic, eicosapentaenoic (EPA), and docosahexaenoic (DHA) acids were supplemented to the culture medium coupled to bovine serum albumin. The four fatty acids were incorporated into phospholipids from cells grown in media containing whole serum or lipoprotein-poor serum (LPPS). Reductase activity of cells cultivated in a medium with LPPS was three to four times higher than those cultivated in medium with whole serum. Saturated fatty acids increased reductase activity of cells grown in medium with whole serum, whereas n-3 polyunsaturated fatty acids (PUFA) decreased it. However, both saturated and polyunsaturated fatty acids increased reductase activity when serum lipoproteins were removed. In conclusion, this is one of the first reports demonstrating that saturated and n-3 PUFA only show differential effects on HMG-CoA reductase activity in the presence of lipoproteins.  相似文献   

6.
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.  相似文献   

7.
It is believed that free fatty acids contribute to the pathogenesis of type 2 diabetes in humans. We have recently shown that lipoapoptosis of human beta-cells is specifically induced by saturated fatty acids while unsaturated had no effect. In the present study we tested the effect of co-incubation of different saturated and unsaturated free fatty acids on lipoapoptosis in beta-cells. RIN1046-38 cells and isolated human beta-cells were incubated with combinations of saturated fatty acids (palmitate, stearate) and mono- or polyunsaturated fatty acids (palmitoleate, oleate, and linoleate). Cells were incubated for 24-72 h with 1mM fatty acids. All unsaturated fatty acids tested completely prevented palmitate- or stearate-induced apoptosis of rat and human beta-cells as assessed by flow cytometric cell cycle analysis and TUNEL assay. This might suggest that apoptosis in vivo is predominantly determined by the content of unsaturated fatty acids in a mixed fatty acid pool.  相似文献   

8.
We examined the direct effects of unsaturated fatty acids, oleic (18:1 n-9), linoleic (18:2 n-6), eicosapentaenoic (20:5 n-3) and docosahexaenoic (22:6 n-3) on tissue factor (TF) activity in the human leukemia monocytic U937 cell line. After exposing cells to fatty acids for 16 h, there were no significant effects on either TF activity or its activation induced by bacterial endotoxin (LPS). When the cells were primed with fatty acids for 24 h, 48 h or 72 h, the TF activity remained essentially unchanged. However, the extent of TF-activation induced by LPS depended on the length of priming, and the dose and the degree of unsaturation of the fatty acids to which cells were exposed. After a 72-h priming, 18:1 produced 40-60 per cent elevation in LPS-challenge. In contrast, approximately 20-50 per cent reduction in LPS-challenge was achieved by 18:2, 20:5 and 22:6 at high concentrations. The results suggest that chronic exposure of U937 cells to unsaturated fatty acids leads to modulation of the TF-activation in response to LPS.  相似文献   

9.
Hepatomas thrive in a hypoxic environment resulting in the induction of a cluster of hypoxia related genes. The protein phenotypic expression include hypoxia inducible factor-alpha, prolyl-4-hydroxylase, vascular endothelear growth factor and erythropoietin. The present study was undertaken to determine if human hepatoma cells when cultured for 72 h in the presence of serum under normoxia would maintain their cancerous phenotypic expression of certain hypoxia inducible genes. Our positive results affords an in vitro model system to test hypoxia inhibitors on the expression and the intracellular compartmentalization or the secretion of these hypoxia-inducible proteins.  相似文献   

10.
11.
12.
The phosphorylation of insulin-like growth factor binding protein-I (IGFBP-1) alters its binding affinity for insulin-like growth factor I (IGF-I) and thus regulates the bioavailability of IGF-I for binding to the IGF-I receptor. The kinase(s) responsible for the phosphorylation of IGFBP-1 has not been identified. This study was designed to characterize the IGFBP-1 kinase activity in HepG2 human hepatoma cells, a cell line that secretes IGFBP-1 primarily as phosphorylated isoforms. IGFBP-1 kinase activity was partially purified from detergent extracts of the cells by phosphocellulose chromatography and gel filtration. Two kinases of approximate Mr 150,000 (peak I kinase) and Mr 50,000 (peak II kinase) were identified. Each kinase phosphorylated IGFBP-1 at serine residues that were phosphorylated by intact HepG2 cells. The kinases were distinct based on their differential sensitivity to inhibition by heparin (IC50 = 2.5 and 16.5 μg/ml, peak I and II kinase, respectively) and inhibition by the isoquinoline sulfonamide CKI-7 (IC50 = 50 μM and 100 μM, peak I and II kinase, respectively). In addition, a tenfold molar excess of nonradioactive GTP relative to [gamma-32P]ATP lowered the incorporation of 32P into IGFBP-1 by 80% when the reaction was catalyzed by the peak I kinase, whereas GTP had no effect on the reaction catalyzed by the peak II kinase. In the presence of polylysine, IGFBP-1 was radiolabeled by the partially purified kinase activity when [gamma-32P]GTP served as the phosphate donor indicating the presence of casein kinase II activity. Furthermore, IGFBP-1 was phosphorylated by purified casein kinase I and casein kinase II at sites phosphorylated by the peak I and peak II kinases. Our data suggest that at least two kinases could be responsible for the phosphorylation of IGFBP-1 in intact HepG2 cells and that the kinases are related to the casein kinase family of protein kinases. © 1996 Wiley-Liss, Inc.  相似文献   

13.
Summary Long-term cultures of hepatocytes were established from livers of human fetuses obtained by abortion at 18 to 23 wk of gestation. Cells obtained by collagenase dissociation of liver were maintained in defined serum-free medium on a substratum of positively charged plastic. Under these conditions, the cells divide and form a confluent monolayer. After multiple passages over a period of 3 mo., the cells retained an epithelioid morphology and continued to synthesize and secrete albumin. Research was supported, in part, by National Institutes of Health grants AM-17609, AM-17702, and CA-34818.  相似文献   

14.
GPR40 has recently been identified as a G protein-coupled cell-surface receptor for long-chain fatty acids (LCFAs). The mRNA of the bovine ortholog of GPR40 (bGPR40) was detected by RT-PCR in cloned bovine mammary epithelial cells (bMEC) and in the bovine mammary gland at various stages of lactation. Oleate and linoleate caused an increase in intracellular Ca2+ concentrations in these cells, and significantly reduced forskolin-induced cAMP concentrations. Phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and Akt kinase, which regulates cell proliferation and survival, was rapidly increased by oleate. Incubation with oleate and linoleate for 24 h significantly promoted cell proliferation. Moreover, in serum-free medium, oleate significantly stimulated cell proliferation during a 7-day culture. These results suggest that bGPR40 mediates LCFA signaling in mammary epithelial cells and thereby plays an important role in cell proliferation and survival.  相似文献   

15.
The regulation of the human liver-specific cytochrome P450 4F3B (CYP4F3B) isoform, a splice variant of the CYP4F3 gene with strong substrate specificity for long chain fatty acids, is yet an unsolved question. This report provides the first evidence that CYP4F3B is uniquely induced by prostaglandin A(1) (PGA(1)) in human hepatocyte-like HepaRG cells and leads to the synthesis of 20-hydroxy-eicosatetraenoic acids (HETEs). Real time PCR, immunoblot analysis with a specific antipeptide antibody, and determination of fatty acid omega-hydroxylase activity demonstrate that PGA(1) treatment strongly increases expression of CYP4F3B. This induction drives the production of 20-HETE (19-fold increase). SiRNA-mediated-silencing of CYP4F3 suppresses both 20-HETE synthesis and PGA(1) induced 20-HETE production. Taken together, these results provide evidence that CYP4F3B is the key enzyme to produce 20-HETE by omega-hydroxylation of arachidonic acid in liver cells. Since 20-HETE is a potent activator of PPARalpha and an important inflammatory mediator, CYP4F3B may exert important functions in lipid homeostasis and in inflammatory diseases.  相似文献   

16.
N-6 fatty acid metabolism was compared in NIH-3T3 cells and DT cells, which differ only in the presence of the v-Ki-ras oncogene. Non-dividing cells were incubated with [1-14C]-labelled fatty acids (18:2n-6, 18:3n-6, 20:3n-6 and 20:4n-6) at different time intervals (2–24 h) and concentration (0–120 M). In both cells lines, the uptake of different fatty acids from the medium was similar and reached a maximum at 6–8 h. All fatty acids reached the same maximum level in DT cells, whereas, the relative uptake of added fatty acids by NIH-3T3 cells was different: 20:4n-6>20:2n-6>18:2n-6=18:3n-6. Throughout the incubation (2–24 h), desaturation and elongation of n-6 fatty acids was more active in DT cells than in NIH-3T3 cells. However, in both cell lines, incubated with different n-6 fatty acid precursors, the levels of radiolabelled 20:4n-6 were relatively constant. In DT cells, phosphatidylcholine was found to be the major fraction labelled with n-6 fatty acids precursors and those of endogenous synthesis, whereas, in NIH-3T3 cells the neutral lipid fraction, particularly triglycerides, was also strongly labelled. In concentration dependent studies, phospholipid labelling by fatty acids was saturable. At lower concentrations, especially in DT cells, phospholipids were labelled predominantly. As the concentration increased there was an overflow into the triglyceride fraction. Since the differences in fatty acid metabolism between the two cell lines cannot be related to the growth rate, it is suggested that they were a consequence of the expression of the v-Ki-ras oncogene.Abbreviations BSA bovine serum albumin - CE cholesterol ester - DG diglyceride - DMEM Dulbecco's modification of Eagle's medium - EL ether lipids (glyceryl ether diesters) - FAME fatty acid methyl ester - FCS fetal calf serum - FFA free fatty acids - HEPES N-2-(hydroxyethyl)piperazine-N-2-ethanesulphonic acid - MG monoglyceride - NL neutral lipid - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PL phospholipid - s.a specific activity - TG triglyceride - TLC thin layer chromatography  相似文献   

17.
Our hypothesis that the trans fatty acids in hydrogenated fat inhibited the synthesis of polyunsaturated fatty acids in the phospholipid of arterial cells was tested with five groups each with six pregnant porcine fed from d 35 of gestation and during lactation. The basal diet contained 2% corn oil (control). The other four diets included the control + 10% butter or 10% hydrogenated fat plus two levels of Mg. Plasma, milk and aortic phospholipid fatty acids, phospholipid composition and calcium content of the aorta from the piglets were determined. At 48 +/- 2 d of age, the aorta phospholipid of piglets from porcine fed hydrogenated fat contained a significantly higher concentration of linoleic acid, less arachidonic acid, and less long chain polyunsaturated fatty acid (PUFA) than did piglets from porcine fed either butterfat or the control diet. Mg had no effect. These changes in composition in piglets from porcine fed hydrogenated fat indicate that trans fat inhibits the metabolic conversion of linoleic acid to arachidonic acid and to other n-6 PUFA. The aortic calcium content data showed a significant interaction of calcium concentration with age. We concluded: 1) that dietary trans fat perturbed essential fatty acid (EFA) metabolism which led to changes in the phospholipid fatty acid composition in the aorta, the target tissue of atherogenesis, 2) this inhibition of EFA to PUFA by the isomeric fatty acids in hydrogenated fat is a risk factor in the development of coronary heart disease.  相似文献   

18.
The neutral fraction of nonstarch lipids in developing brown rice (Oryza sativa L., cv IR42) was accumulated up to 16 days after flowering (DAF), but phospholipids and glycolipids increased only up to 8 DAF. Fatty acids accumulated in nonstarch lipids until 12 DAF. However, the proportion of linolenic acid in the lipid fraction decreased and that of oleic acid increased during this period. Accumulation of fat-by-hydrolysis in the brown rice occurred until 20 DAF and followed closely that of starch. The proportion of linolenic acid decreased and that of linoleic acid increased until 16 DAF. The fatty acid composition of fat-by-hydrolysis and starch lipids were identical and fat-by-hydrolysis accounted for 48% by weight of starch lipids. Nonstarch lipids were mainly composed of triglycerides and were located in the bran and embryo of mature brown rice. Starch lipids were mainly composed of lysophosphatidyl choline, free fatty acids and lysophosphatidyl ethanolamine, and were located in the endosperm.  相似文献   

19.
Lipids from cultured cells, leaves and seeds of two varieties each of soybean (Glycine max) and oil seed rape (Brassica napus) were separated into neutral lipids, glycolipids and phospholipids and their fatty acids were analysed. Usually, the fatty acid composition differed between corresponding fractions from cultured cells, leaves and seeds. Differences were least marked in (i) the phospholipids from cultured cells and leaves of soybean and (ii) the neutral lipids from cultured cells and seeds of rape. In the cultured cells, the fatty acid composition of the phospholipids differed from that of the glycolipids and neutral lipids, and fatty acids of chain length greater than C18 comprised a large proportion of the fatty acids of the glycolipids.  相似文献   

20.
We have recently identified a neuroprotective role for omega-3 polyunsaturated fatty acids (n-3 PUFAs) in a toxin-induced mouse model of Parkinson's disease (PD). Combined with epidemiological data, these observations suggest that low n-3 PUFA intake is a modifiable environmental risk factor for PD. In order to strengthen these preclinical findings as prerequisite to clinical trials, we further investigated the neuroprotective role of n-3 PUFAs in Fat-1 mice, a transgenic model expressing an n-3 fatty acid desaturase converting n-6 PUFAs into n-3 PUFAs. Here, we report that the expression of the fat-1 transgene increased cortical n-3:n-6 PUFA ratio (+28%), but to a lesser extent than dietary supplementation (92%). Such a limited endogenous production of n-3 PUFAs in the Fat-1 mouse was insufficient to confer neuroprotection against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxicity as assessed by dopamine levels, tyrosine hydroxylase (TH)-positive neurons and fibers, as well as nigral Nurr1 and dopamine transporter (DAT) mRNA expression. Nevertheless, higher cortical docosahexaenoic acid (DHA) concentrations were positively correlated with markers of nigral dopaminergic neurons such as the number of TH-positive cells, in addition to Nurr1 and DAT mRNA levels. These associations are consistent with the protective role of DHA in a mouse model of PD. Taken together, these data suggest that dietary intake of a preformed DHA supplement is more effective in reaching the brain and achieving neuroprotection in an animal model of PD.  相似文献   

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