Trans geometric isomers of EPA decrease LXRα-induced cellular triacylglycerol via suppression of SREBP-1c and PGC-1β

Dietary mono- or di-trans fatty acids with chain lengths of 18–22 increase the risk of cardiovascular diseases because they increase LDL cholesterol and decrease HDL cholesterol in the plasma. However, the effects of trans isomers of PUFAs on lipid metabolism remain unknown. Dietary PUFAs, especially eicosapentaenoic acid (EPA) in marine oils, improve serum lipid profiles by suppressing liver X receptor α (LXRα) activity in the liver. In this study, we compared the effects of trans geometric isomers of eicosapentaenoic acid (TEPA) on triacylglycerol synthesis induced by a synthetic LXRα agonist (T0901317) with the effects of EPA in HepG2 cells. TEPA significantly decreased the amount of cellular triacylglycerol and the expression of mRNAs encoding fatty acid synthase, stearoyl-CoA desaturase-1, and glycerol-3-phosphate acyltransferase induced by T0901317 compared with EPA. However, there was no significant difference between the suppressive effect of TEPA or EPA on the expression of sterol-regulatory element binding protein-1c (SREBP-1c) induced by T0901317. We found that TEPA, but not EPA, decreased the mRNA expression of peroxisome proliferator-activated receptor γ coactivator 1β (PGC-1β), which is a coactivator of both LXRα and SREBP-1. These results suggest that the hypolipidemic effect of TEPA can be attributed to a decrease not only in SREBP-1 but also in PGC-1β expression. Dietary mono- or di-trans fatty acids with chain lengths of 18–22 increase the risk of cardiovascular diseases because they increase LDL cholesterol and decrease HDL cholesterol in the plasma. However, the effects of trans isomers of PUFAs on lipid metabolism remain unknown. Dietary PUFAs, especially eicosapentaenoic acid (EPA) in marine oils, improve serum lipid profiles by suppressing liver X receptor α (LXRα) activity in the liver. In this study, we compared the effects of trans geometric isomers of eicosapentaenoic acid (TEPA) on triacylglycerol synthesis induced by a synthetic LXRα agonist (T0901317) with the effects of EPA in HepG2 cells. TEPA significantly decreased the amount of cellular triacylglycerol and the expression of mRNAs encoding fatty acid synthase, stearoyl-CoA desaturase-1, and glycerol-3-phosphate acyltransferase induced by T0901317 compared with EPA. However, there was no significant difference between the suppressive effect of TEPA or EPA on the expression of sterol-regulatory element binding protein-1c (SREBP-1c) induced by T0901317. We found that TEPA, but not EPA, decreased the mRNA expression of peroxisome proliferator-activated receptor γ coactivator 1β (PGC-1β), which is a coactivator of both LXRα and SREBP-1. These results suggest that the hypolipidemic effect of TEPA can be attributed to a decrease not only in SREBP-1 but also in PGC-1β expression. Most naturally occurring unsaturated fatty acids have only cis double bonds. However, trans fatty acids are found in several foods, including infant formulas, shortenings, vegetable oils and fish oils (1Rantnayake W.M.N. Chardigny J.M. Wolff R.L. Bayard C.C. Sébédio J.L. Martine L. Fatty acids and their trans geometrical isomers in powdered and liquid infant formulas sold in Canada.J. Pediatr. Gastroenterol. Nutr. 1997; 25: 400-407Crossref PubMed Scopus (35) Google Scholar, 2Sébédio J.L. Grandgirard A. Prevost J. Linoleic acid isomers in heat treated sunflower oils.J. Am. Oil Chem. Soc. 1988; 65: 362-366Crossref Scopus (70) Google Scholar, 3Ackman R.G. Composition of fish oils.in: Barlow S.M. Stansby M.E. Nutritional Evaluation of Long-Chain Fatty Acids in Fish Oil. Academic Press, London1982: 25-88Google Scholar), because partial hydrogenation of edible oils converts some cis double bonds to trans double bonds without changing their location. Dietary trans fatty acids increase the risk of cardiovascular diseases. The intake of mono- or di-trans fatty acids with chain lengths of 18–22, from partially hydrogenated edible oils and fats, significantly increases LDL cholesterol and reduces HDL cholesterol in the plasma (4Zock P.L. Katan M.B. Trans fatty acids, lipoproteins, and coronary risk.Can. J. Physiol. Pharmacol. 1995; 75: 211-216Crossref Google Scholar, 5Almendingen K. Jordal O. Kierulf P. Sandstad B. Pedersen J.I. Effects of partially hydrogenated fish oil, partially hydrogenated soybean oil, and butter on serum lipoproteins and Lp[a] in men.J. Lipid Res. 1995; 36: 1370-1384Abstract Full Text PDF PubMed Google Scholar). The trans fatty acid of C18:1 can also stimulate the expression of lipogenic genes in the livers of mice (6Lin J. Yang R. Tarr P.T. Wu P. Handschin C. Li S. Yang W. Pei L. Uldry M. Tontonoz P. et al.Hyperlipidemic effects of dietary saturated fats mediated through PGC-1β coactivation of SREBP.Cell. 2005; 120: 261-273Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar). In contrast, there is little information about the effects of trans geometric isomers, without changing the double bond location, of PUFAs on lipid metabolism. Dietary PUFAs, especially eicosapentaenoic acid (EPA) in marine oils, improve serum lipid profiles (7Nestel P.J. Connor W.E. Reardon M.F. Connor S. Wong S. Boston R. Suppression by diets rich in fish oil of very low density lipoprotein production in man.J. Clin. Invest. 1984; 74: 82-89Crossref PubMed Scopus (459) Google Scholar). The hypolipidemic effect of PUFAs is attributable both to a decrease in lipogenesis and an increase in fatty acid β-oxidation. It has been well established that liver X receptor α (LXRα) and sterol-regulatory element binding protein-1c (SREBP-1c) play crucial roles in the transcriptional regulation of lipogenic genes in the liver (8Edwards P.A. Kast H.R. Anisfeld A.M. BAREing it all: the adoption of LXR and FXR and their roles in lipid homeostasis.J. Lipid Res. 2002; 43: 2-12Abstract Full Text Full Text PDF PubMed Google Scholar, 9Shimano H. Sterol regulatory element-binding proteins (SREBPs): transcriptional regulators of lipid synthetic genes.Prog. Lipid Res. 2001; 40: 439-452Crossref PubMed Scopus (579) Google Scholar). Oxysterols, such as 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 24(S),25-epoxycholesterol, are endogenous LXRα ligands that serve as modulators of LXRα activities. PUFAs compete with LXRα ligands in the activation of the ligand binding domain (LBD) (10Janowski B.A. Willy P.J. Devi T.R. Falck J.R. Mangelsdorf D.J. An oxysterol signaling pathway mediated by the nuclear receptor LXRα.Nature. 1996; 383: 728-731Crossref PubMed Scopus (1468) Google Scholar, 11Yoshikawa T. Shimano H. Yahagi N. Ide T. Amemiya-Kudo M. Matsuzaka T. Nakakuki M. Tomita S. Okazaki H. Tamura Y. et al.Polyunsaturated fatty acids suppress sterol regulatory element-binding protein 1c promoter activity by inhibition of liver X receptor (LXR) binding to LXR response elements.J. Biol. Chem. 2002; 277: 1705-1711Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar). LXRα regulates the expression of many lipogenic genes, including SREBP-1c (8Edwards P.A. Kast H.R. Anisfeld A.M. BAREing it all: the adoption of LXR and FXR and their roles in lipid homeostasis.J. Lipid Res. 2002; 43: 2-12Abstract Full Text Full Text PDF PubMed Google Scholar), which is synthesized as a 125 kDa precursor protein attached to the endoplasmic reticulum (12Brown M.S. Goldstein J.L. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcriptional factor.Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (3004) Google Scholar). In response to insulin stimulation (13Hegarty B.D. Bobard A. Hainault I. Ferré P. Bossard P. Foufelle F. Distinct roles of insulin and liver X receptor in the induction and cleavage of sterol regulatory element-binding protein-1c.Proc. Natl. Acad. Sci. USA. 2005; 102: 791-796Crossref PubMed Scopus (161) Google Scholar), the membrane-bound precursor is cleaved to a 68 kDa N-terminal fragment that translocates to the nucleus and activates the expression of lipogenic genes such as FAS, stearoyl-coenzyme A desaturase-1 (SCD-1), and glycerol-3-phosphate acyltransferase (GPA). PUFAs suppress SREBP-1c expression by inhibiting LXRα binding to liver X receptor response elements (LXREs), which leads to the decrease in expression of lipogenic genes. A recent study reported that peroxisome proliferator-activated receptor γ coactivator 1β (PGC-1β) coactivates the LXRα and SREBP families and increases circulating triacylglycerol and cholesterol in VLDL particles (6Lin J. Yang R. Tarr P.T. Wu P. Handschin C. Li S. Yang W. Pei L. Uldry M. Tontonoz P. et al.Hyperlipidemic effects of dietary saturated fats mediated through PGC-1β coactivation of SREBP.Cell. 2005; 120: 261-273Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar). PGC-1β may be a key cofactor linking the dietary intake of trans fatty acids with hyperlipidemia. Trans geometric isomers of eicosapentaenoic acid (TEPA) have been found as a minor component in vivo (14Chardigny J.M. Sébédio J.L. Juanéda P. Vatèle J.M. Grandgirard A. Occurrence of n-3 trans polyunsaturated fatty acids in human platelets.Nutr. Res. 1993; 13: 1105-1111Crossref Scopus (35) Google Scholar). Trans isomers of α-linolenic acid are elongated and are desaturated to TEPA in rats fed a diet enriched with those isomers (15Grandgirard A. Piconneaux A. Sébédio J.L. Quére L. Occurrence of geometrical isomers of eicosapentaenoic and docosahexaenoic acids in liver lipids of rats fed heated linseed oil.Lipids. 1989; 24: 799-804Crossref PubMed Scopus (64) Google Scholar). In addition, trans isomerization of EPA may occur in vivo, because trans isomers of arachidonic acid are generated by NO2-mediated isomerization (16Jiang H. Kruger N. Lahiri D.R. Wang D. Vatèle J.M. Balazy M. Nitrogen dioxide induces cis-trans-isomerization of arachidonic acid within cellular phospholipids.J. Biol. Chem. 1999; 274: 16235-16241Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) and are found in human blood plasma (17Zghibeh C.M. Gopal V.R. Poff C.D. Falck J.R. Balazy M. Determination of trans-arachidonic isomers in human blood plasma.Anal. Biochem. 2004; 332: 137-144Crossref PubMed Scopus (41) Google Scholar). TEPA has different physiological effects on platelet aggregation (18Loï C. Chardigny J.M. Berdeaux O. Vatèle J.M. Poullain D. Noël J.P. Sébédio J.L. Effects of three trans isomers of eicosapentaenoic acid on rat platelet aggregation and arachidonic acid metabolism.Thromb. Haemost. 1998; 80: 656-661PubMed Google Scholar), oxidative stability, and anti-inflammation (19Zaima N. Sugawara T. Arai H. Nakamura K. Takasugi M. Fukunaga K. Noguchi R. Hirata T. Characterization of trans eicosapentaenoic acid isomers: oxidative stability and anti-inflammatory activity.J. Oleo Sci. 2005; 54: 505-512Crossref Scopus (10) Google Scholar) compared with EPA. However, the effect of TEPA on lipid metabolism remains unknown. The aim of this study was to clarify the effects of TEPA on lipid metabolism. We compared the effect of TEPA on triacylglycerol synthesis induced by a synthetic LXRα agonist (T0901317) with that of EPA in HepG2 cells. We used a mixture of TEPA prepared from p-toluenesulfinic acid, because there are many possible structures of TEPA. The mixture of TEPA was prepared using a chemical catalyst (p-toluenesulfinic acid) and was analyzed by HPLC and GC-MS according to a previously described method (19Zaima N. Sugawara T. Arai H. Nakamura K. Takasugi M. Fukunaga K. Noguchi R. Hirata T. Characterization of trans eicosapentaenoic acid isomers: oxidative stability and anti-inflammatory activity.J. Oleo Sci. 2005; 54: 505-512Crossref Scopus (10) Google Scholar). The TEPA used in this study consisted of 71% trans isomers, without changing the double bond location, and 29% cis isomers (EPA). The sample contained 0.25% conjugated dienes, with no detectable conjugated trienes, conjugated tetraenes, or conjugated pentaenes. HepG2 cells (JCRB 1054; Health Science Research Resources Bank, Osaka, Japan) were plated on six-well plates at 2.0 × 105 cells/ml for 24 h in DMEM supplemented with 10% fetal calf serum and antibiotics (penicillin and streptomycin). The cells were then treated with EPA, TEPA, and/or T0901317 (50 nM) in serum-supplemented medium. Fatty acids and T0901317 were dissolved in ethanol. After incubation for 72 h, lipids were extracted from cells with chloroform-methanol (2:1, v/v). Reference control cells were extracted for cellular lipids before incubation (zero time control). Collected supernatants were evaporated gently under an N2 stream, and triacylglycerol was quantified using a triglyceride E-test kit (Wako Pure Chemical Industries, Osaka, Japan). HepG2 cells were plated on 12-well plates at 2.0 × 105 cells/ml in DMEM supplemented with 10% fetal calf serum and antibiotics as detailed above. After 24 h of incubation, each fatty acid was added to HepG2 cells with T0901317 (50 nM) in serum-free medium containing 0.1% BSA. After 24 h of incubation, total RNA was extracted from the cells using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. RNAs were treated with RNase-free DNase (Invitrogen) to remove contaminating genomic DNA. After inactivating DNase by adding 20 mM EGTA and heating at 65°C for 10 min, each RNA was transcribed to cDNA using SuperScript RNase H− reverse transcriptase (Invitrogen) with random hexamers at 25°C for 10 min and then at 42°C for 50 min. The reactions were stopped by incubation at 70°C for 15 min, then 0.08 μl of the mixture was added to 4 μl of iQ SYBR Green supermix (Bio-Rad Laboratories, Hercules, CA) and 1.6 μl of gene-specific primers in a final volume of 8 μl. Primers used for the quantification of each gene are listed in Table 1 . Primer pairs were selected to yield gene-specific single amplicons based on analyses by melting curves and by agarose gel electrophoresis. Real-time quantitative PCR was performed using a DNA Engine Opticon system (Bio-Rad Laboratories). The thermal cycler parameters were as follows: 3 min at 95°C for one cycle, followed by amplification of the cDNA for 40 cycles with melting for 15 s at 95°C and with annealing and extension for 30 s at 60°C. Values were normalized using 18s rRNA as an endogenous internal standard.TABLE 1Real-time RT-PCR primers for the quantification of human mRNAmRNASequenceReference or Accession NumberSREBP-1c5′-GGAGGGGTAGGGCCAACGGCCT-3′20Field F.J. Born E. Murthy S. Mathur S.N. Polyunsaturated fatty acids decrease the expression of sterol regulatory element-binding protein-1 in CaCo-2 cells: effect on fatty acid synthesis and triacylglycerol transport.Biochem. J. 2002; 368: 855-864Crossref PubMed Scopus (62) Google Scholar5′-CATGTCTTCGAAAGTGCAATCC-3′SREBP-la5′-TCAGCGAGGCGGCTTTGGAGCAG-3′20Field F.J. Born E. Murthy S. Mathur S.N. Polyunsaturated fatty acids decrease the expression of sterol regulatory element-binding protein-1 in CaCo-2 cells: effect on fatty acid synthesis and triacylglycerol transport.Biochem. J. 2002; 368: 855-864Crossref PubMed Scopus (62) Google Scholar5′-CATGTCTTCGATGTCGGTCAG-3′FAS5′-ACAGGGACAACCTGGAGTTCT-3′20Field F.J. Born E. Murthy S. Mathur S.N. Polyunsaturated fatty acids decrease the expression of sterol regulatory element-binding protein-1 in CaCo-2 cells: effect on fatty acid synthesis and triacylglycerol transport.Biochem. J. 2002; 368: 855-864Crossref PubMed Scopus (62) Google Scholar5′-CTGTGGTCCCACTTGATGAGT-3′Stearoyl-coenzyme A desaturase-l5′-TGGTTTCACTTGGAGCTGTG-3′NM_0050635′-GGCCTTGGAGACTTTC'TTCC-3′Glycerol-3-phosphate acyltransferase5′-TTGGGTTTGCGGAATGTTAT-3′NM_0209185′-GGCAGAACCATCAGGGTTTA-3′Apolipoprotein A-II5′-GAGCTTTGGTTCGGAGACAG-3′NM_0016435′-TGTGTTCCAAGTTCCACGAA-3′Peroxisome proliferator-activated receptor γ coactivator 1β5′-ATGACTCCGAGCTCTTCCAG-3′NM_13326318s5′-CGAAGCTGAGGTGCATGATA-3′20Field F.J. Born E. Murthy S. Mathur S.N. Polyunsaturated fatty acids decrease the expression of sterol regulatory element-binding protein-1 in CaCo-2 cells: effect on fatty acid synthesis and triacylglycerol transport.Biochem. J. 2002; 368: 855-864Crossref PubMed Scopus (62) Google Scholar5′-TAAGTCCCTGCCCTTTGTACACA-3′Liver X receptor α5′-GATCCGAGGGCCTCACTAAAC-3′NM_0056935′-GGAGGTACAACCCTGGGAGT-3′ sterol-regulatory element binding in a sterol-regulatory element binding The in the human SREBP-1c promoter was the according to the method of et H. E. F. W. of protein-1c gene expression in HepG2 cells by J. 2004; PubMed Scopus Google Scholar). The contained in the SREBP-1c promoter The cDNA for LXRα was generated from HepG2 cells by RT-PCR amplification of the LXRα A cDNA fragment was using to as the and to as the reverse and was the and were the as in was by HepG2 cells and cells (JCRB Health Science Research Resources were to on including and an internal control were using reagent in serum-free medium. After cells were treated with EPA, TEPA, and/or T0901317 for 24 h in serum-free medium containing 0.1% BSA. After incubation, were with a using the system according to the manufacturer's HepG2 cells were plated on six-well plates at 2.0 × 105 cells/ml for 24 h in DMEM supplemented with 10% fetal calf serum and antibiotics as detailed above. The cells were then treated with EPA, TEPA, and/or T0901317 (50 nM) in serum-free medium containing 0.1% BSA. After incubation for 24 h, and nuclear from cells were prepared by the method of et J. A. Goldstein J.L. M.S. fatty acids SREBP and 1c by in Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). were according to the method A and method for the of of protein the of Biochem. PubMed Scopus Google Scholar). of and nuclear were by and 10% and were then to Japan). The were with were with The of each was quantified using the as a at are reported as The results were analyzed by with significant difference using We in HepG2 cells to the effect of TEPA on lipogenesis 1 The synthetic LXRα significantly cellular 72 h of TEPA at 10 significantly the increase of triacylglycerol EPA. the by which TEPA decreased cellular triacylglycerol FAS, and mRNA were EPA and TEPA decreased the expression of and mRNAs in a TEPA significantly decreased the expression of FAS, and mRNAs EPA. difference in was with TEPA or EPA EPA TEPA effect on mRNA expression as a control gene We generated a containing the that in the human SREBP-1c promoter and performed gene in both HepG2 cells and cells 3 because LXRα and SREBP-1c are key regulators of FAS, and mRNA expression. In the of activity in HepG2 cells and in cells was by and compared with the control EPA and TEPA the T0901317 induction of activity TEPA in HepG2 cells and in cells at 30 and 20 were no significant in the suppressive effects of TEPA and EPA. The expression of SREBP-1c mRNA in cells treated with T0901317 was that in control cells TEPA significantly the T0901317 induction of SREBP-1c mRNA in a and the suppressive effect of TEPA was to that of EPA. the effect of TEPA on SREBP-1 protein the precursor in the and the cleaved in the nuclear were by the used between the SREBP-1c and we the SREBP-1 to to the are in T0901317 the of both the precursor and the of SREBP-1. TEPA as well as EPA decreased the T0901317 induction of both the precursor and of SREBP-1. The decrease of SREBP-1 can be attributed to the of SREBP-1c because TEPA EPA the expression of mRNA not the by which TEPA significantly decreased the expression of lipogenic gene mRNAs EPA, we the effect of TEPA on the mRNA expression of the coactivator of LXRα and We found a significant difference between the effects of TEPA and EPA. TEPA the of PGC-1β mRNA at 40 by compared with the In contrast, EPA significantly the of PGC-1β T0901317 no effect on PGC-1β mRNA expression. study that TEPA triacylglycerol synthesis in HepG2 cells and the expression of lipogenic genes as FAS, and EPA TEPA also activity by T0901317 and SREBP-1c expression which that TEPA, EPA, SREBP-1c expression by with T0901317 in the activation of TEPA PGC-1β mRNA EPA It has been that the in the of LXRα with the of LXRα ligands M. J. J. of LXRα PubMed Scopus Google Scholar, S. T. M. C. K. D. K. D. L. of the of LXRα and in a J. PubMed Scopus Google Scholar). et S. T. M. C. K. D. K. D. L. of the of LXRα and in a J. PubMed Scopus Google Scholar) reported that is the of the in fatty The between and the of fatty acids may be for et T. Shimano H. Yahagi N. Ide T. Amemiya-Kudo M. Matsuzaka T. Nakakuki M. Tomita S. Okazaki H. Tamura Y. et al.Polyunsaturated fatty acids suppress sterol regulatory element-binding protein 1c promoter activity by inhibition of liver X receptor (LXR) binding to LXR response elements.J. Biol. Chem. 2002; 277: 1705-1711Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar) that the of of fatty acids is a for the effect of SREBP-1c but they are n-3 or is The of inhibition of each on LXRα using an expression of the of LXRα to the DNA binding is as follows: arachidonic acid docosahexaenoic acid EPA acid acid acid T. Shimano H. Yahagi N. Ide T. Amemiya-Kudo M. Matsuzaka T. Nakakuki M. Tomita S. Okazaki H. Tamura Y. et al.Polyunsaturated fatty acids suppress sterol regulatory element-binding protein 1c promoter activity by inhibition of liver X receptor (LXR) binding to LXR response elements.J. Biol. Chem. 2002; 277: 1705-1711Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar). In this study, the suppressive effect of TEPA on activity was to that of EPA. An for to LXRα activation to be the of double bonds and the chain but not the double bond PUFAs a and three double bonds be of TEPA significantly decreased FAS, and mRNA EPA, there was no significant difference between the suppressive effects of TEPA or EPA on SREBP-1c expression or on activity These results be attributable in to the of PGC-1β expression by TEPA because PGC-1β lipid synthesis by SREBP-1 and LXRα (6Lin J. Yang R. Tarr P.T. Wu P. Handschin C. Li S. Yang W. Pei L. Uldry M. Tontonoz P. et al.Hyperlipidemic effects of dietary saturated fats mediated through PGC-1β coactivation of SREBP.Cell. 2005; 120: 261-273Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar). suggest that PGC-1β is for the induction of lipogenic genes by SREBP-1 and PGC-1β a for the the EPA increase of PGC-1β EPA decreased the expression of lipogenic genes in this is by the of which is an nuclear for of lipogenic genes. The by which EPA increases PGC-1β mRNA is but the induction of PGC-1β by EPA is with a (6Lin J. Yang R. Tarr P.T. Wu P. Handschin C. Li S. Yang W. Pei L. Uldry M. Tontonoz P. et al.Hyperlipidemic effects of dietary saturated fats mediated through PGC-1β coactivation of SREBP.Cell. 2005; 120: 261-273Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar). trans isomerization of EPA increases the effect of EPA on the expression of lipogenic genes, which leads to that TEPA serum lipid EPA in The activity of TEPA on lipid metabolism may be from that of trans fatty acids a double bonds with chain lengths of The effect of trans fatty acids on serum lipid profiles may on their of and double bonds. the TEPA used in this study was a mixture of trans isomers, it be to clarify the a of trans isomers in In addition, insulin is an of SREBP-1c in It be to the effect of TEPA on lipogenesis both in vivo and in study was in by the Research and also by a for Research from the for the of

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