Polyunsaturated Fatty Acyl Coenzyme A Suppress the Glucose-6-phosphatase Promoter Activity by Modulating the DNA Binding of Hepatocyte Nuclear Factor 4α

Abstract

Glucose-6-phosphatase confers on gluconeogenic tissues the capacity to release endogenous glucose in blood. The expression of its gene is modulated by nutritional mechanisms dependent on dietary fatty acids, with specific inhibitory effects of polyunsaturated fatty acids (PUFA). The presence of consensus binding sites of hepatocyte nuclear factor 4 (HNF4) in the −1640/+60 bp region of the rat glucose-6-phosphatase gene has led us to consider the hypothesis that HNF4α could be involved in the regulation of glucose-6-phosphatase gene transcription by long chain fatty acid (LCFA). Our results have shown that the glucose-6-phosphatase promoter activity is specifically inhibited in the presence of PUFA in HepG2 hepatoma cells, whereas saturated LCFA have no effect. In HeLa cells, the glucose-6-phosphatase promoter activity is induced by the co-expression of HNF4α or HNF1α. PUFA repress the promoter activity only in HNF4α-cotransfected HeLa cells, whereas they have no effects on the promoter activity in HNF1α-cotransfected HeLa cells. From gel shift mobility assays, deletion, and mutagenesis experiments, two specific binding sequences have been identified that appear able to account for both transactivation by HNF4α and regulation by LCFA in cells. The binding of HNF4α to its cognate sites is specifically inhibited by polyunsaturated fatty acyl coenzyme A in vitro. These data strongly suggest that the mechanism by which PUFA suppress the glucose-6-phosphatase gene transcription involves an inhibition of the binding of HNF4α to its cognate sites in the presence of polyunsaturated fatty acyl-CoA thioesters. Glucose-6-phosphatase confers on gluconeogenic tissues the capacity to release endogenous glucose in blood. The expression of its gene is modulated by nutritional mechanisms dependent on dietary fatty acids, with specific inhibitory effects of polyunsaturated fatty acids (PUFA). The presence of consensus binding sites of hepatocyte nuclear factor 4 (HNF4) in the −1640/+60 bp region of the rat glucose-6-phosphatase gene has led us to consider the hypothesis that HNF4α could be involved in the regulation of glucose-6-phosphatase gene transcription by long chain fatty acid (LCFA). Our results have shown that the glucose-6-phosphatase promoter activity is specifically inhibited in the presence of PUFA in HepG2 hepatoma cells, whereas saturated LCFA have no effect. In HeLa cells, the glucose-6-phosphatase promoter activity is induced by the co-expression of HNF4α or HNF1α. PUFA repress the promoter activity only in HNF4α-cotransfected HeLa cells, whereas they have no effects on the promoter activity in HNF1α-cotransfected HeLa cells. From gel shift mobility assays, deletion, and mutagenesis experiments, two specific binding sequences have been identified that appear able to account for both transactivation by HNF4α and regulation by LCFA in cells. The binding of HNF4α to its cognate sites is specifically inhibited by polyunsaturated fatty acyl coenzyme A in vitro. These data strongly suggest that the mechanism by which PUFA suppress the glucose-6-phosphatase gene transcription involves an inhibition of the binding of HNF4α to its cognate sites in the presence of polyunsaturated fatty acyl-CoA thioesters. Polyunsaturated fatty acyl coenzyme A suppress the glucose-6-phosphatase promoter activity by modulating the DNA binding of hepatocyte nuclear factor 4α.Journal of Biological ChemistryVol. 278Issue 7PreviewThere is one error repeated several times in the text and in Fig. 7 on pages 15740–15742. In Fig. 7 in columns A and B, 3a should be 3b, 3b should be 3a, 1a+3a should be 1a+3b, and 1a+3b should be 1a+3a. There is no error in the inset of Fig. 7. In the text on pages 15740–15742, 3b should be substituted for 3a, and 3a should be changed to 3b. Hence, it appears that it is site 3b, such as it has been defined in Fig. 6A, which is more crucial than site 3a for the binding of HNF4 and the sensitivity to PUFA (whereas the contrary is published). Full-Text PDF Open Access glucose-6-phosphatase chloramphenicol acetyltransferase coenzyme A hepatocyte nuclear factor long chain fatty acid(s) luciferase nordihydroguaiaretic acid phosphoenolpyruvate carboxykinase peroxisome proliferator-activated receptor polyunsaturated fatty acid(s) sterol regulatory element-binding protein N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid Glucose-6-phosphatase (Glc6Pase1; EC 3.1.3.9) confers on gluconeogenic tissues, i.e. the liver, the kidney, and the small intestine, the capacity to release endogenous glucose in blood (1Mithieux G. Eur. J. Endocrinol. 1997; 136: 137-145Crossref PubMed Scopus (102) Google Scholar, 2Croset M. Rajas F. Zitoun C. Hurot J.M. Montano S. Mithieux G. Diabetes. 2001; 50: 740-746Crossref PubMed Scopus (158) Google Scholar). The expression of its gene is increased during diabetes and fasting and normalized upon insulin treatment and refeeding, respectively, in all three gluconeogenic tissues (3Mithieux G. Vidal H. Zitoun C. Bruni N. Daniele N. Minassian C. Diabetes. 1996; 45: 891-896Crossref PubMed Scopus (129) Google Scholar, 4Rajas F. Bruni N. Montano S. Zitoun C. Mithieux G. Gastroenterology. 1999; 117: 132-139Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). An increase in the Glc6Pase flux (5Efendic S. Wajngot A. Vranic M. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 2965-2969Crossref PubMed Scopus (64) Google Scholar, 6Efendic S. Karlander S. Vranic M. J. Clin. Invest. 1988; 81: 1953-1961Crossref PubMed Scopus (72) Google Scholar) and maximal velocity (7Clore J.N. Stillman J. Sugerman H. Diabetes. 2000; 49: 969-974Crossref PubMed Scopus (109) Google Scholar) has also been strongly suggested to account for increased glucose production and hepatic insulin resistance in type 2 diabetes mellitus. The Glc6Pase gene expression is also modulated by nutritional mechanisms dependent on dietary fatty acids. In the liver of rats, Glc6Pase mRNA and protein contents are increased upon high fat feeding (8Garg M.L. Sabine J.R. Snoswell A.M. Biochem. Int. 1985; 10: 585-595PubMed Google Scholar) and upon elevation in plasma fatty acid levels (9Massillon D. Barzilai N. Hawkins M. Prus-Wertheimer D. Rossetti L. Diabetes. 1997; 46: 153-157Crossref PubMed Google Scholar). Under these nutritional conditions, the suppression of hepatic glucose production by insulin is impaired (10Mithieux G. Guignot L. Bordet J.C. Wiernsperger N. Diabetes. 2002; 51: 139-143Crossref PubMed Scopus (87) Google Scholar). This suggests that a high plasma fatty acid level may contribute increased production of glucose via increased expression of Glc6Pase, resulting in the development of liver insulin resistance (9Massillon D. Barzilai N. Hawkins M. Prus-Wertheimer D. Rossetti L. Diabetes. 1997; 46: 153-157Crossref PubMed Google Scholar, 11Kraegen E.W. Clark P.W. Jenkins A.B. Daley E.A. Chisholm D.J. Storlien L.H. Diabetes. 1991; 40: 1397-1403Crossref PubMed Scopus (0) Google Scholar, 12Oakes N.D. Cooney G.J. Camilleri S. Chisholm D.J. Kraegen E.W. Diabetes. 1997; 46: 1768-1774Crossref PubMed Google Scholar). In vitro, the treatment of fetal hepatocytes with a high concentration (500 μm) of long chain fatty acids (LCFA), such as oleic and linoleic acids, increases the Glc6Pase mRNA content (13Chatelain F. Pegorier J.P. Minassian C. Bruni N. Tarpin S. Girard J. Mithieux G. Diabetes. 1998; 47: 882-889Crossref PubMed Scopus (79) Google Scholar). We have shown that the likely mechanism involves a stabilizing effect on Glc6Pase mRNA (13Chatelain F. Pegorier J.P. Minassian C. Bruni N. Tarpin S. Girard J. Mithieux G. Diabetes. 1998; 47: 882-889Crossref PubMed Scopus (79) Google Scholar). On the other hand, in vivo, the presence in high fat diets of substantial amounts of polyunsaturated fatty acids (PUFA), such as that in soybean oil (rich in ω:6 fatty acids) or fish oil (rich in ω:3 fatty acids), does not result in an increase in Glc6Pase activity in rats (8Garg M.L. Sabine J.R. Snoswell A.M. Biochem. Int. 1985; 10: 585-595PubMed Google Scholar, 14Venkatraman J.T. Pehowich D. Singh B. Rajotte R.V. Thomson A.B. Clandinin M.T. Lipids. 1991; 26: 441-444Crossref PubMed Scopus (14) Google Scholar). This suggests that among LCFA, PUFA may have specific inhibitory effects on Glc6Pase gene expression. Noteworthy, it is also well known that diets rich in PUFA have only weak deleterious effects on insulin sensitivity, as compared with diets enriched in saturated fat (15Storlien L.H. Kraegen E.W. Chisholm D.J. Ford G.L. Bruce D.G. Pascoe W.S. Science. 1987; 237: 885-888Crossref PubMed Scopus (566) Google Scholar, 16Storlien L.H. Jenkins A.B. Chisholm D.J. Pascoe W.S. Khouri S. Kraegen E.W. Diabetes. 1991; 40: 280-289Crossref PubMed Scopus (953) Google Scholar). It is interesting to note that PUFA may also have opposing effects in regard to hepatic insulin resistance at the level of Glc6Pase activity. Indeed, we have previously shown that PUFA, either added to endoplasmic reticulum membranes or naturally associated to glycogen granules, specifically inhibit the Glc6Pase activity (17Mithieux G. Bordet J.C. Minassian C. Ajzannay A. Mercier I. Riou J.P. Eur. J. Biochem. 1993; 213: 461-466Crossref PubMed Scopus (30) Google Scholar, 18Daniele N. Bordet J.C. Mithieux G. J. Nutr. 1997; 127: 2289-2292Crossref PubMed Scopus (16) Google Scholar). To date, no molecular mechanism involved in the specific inhibitory effect of PUFA on the Glc6Pase gene expression has been described. In the liver, although peroxisome proliferator-activated receptors (PPARs) have emerged as an important factor in the fatty acid regulation at the transcription level, recent evidence indicates that the DNA binding activity and/or the abundance of other factors, such as sterol regulatory element-binding protein 1c (SREBP1c) or hepatocyte nuclear factor 4 (HNF4), may be affected by fatty acids or their metabolites (for reviews, see Refs. 19Jump D.B. Clarke S.D. Annu. Rev. Nutr. 1999; 19: 63-90Crossref PubMed Scopus (546) Google Scholar, J. M.T. Clarke S.D. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, S.D. J. 2001; PubMed Google Scholar). J. I. J. 1998; PubMed Scopus Google Scholar) have that the activity of HNF4α an nuclear could be modulated by LCFA, on their level, the binding of their acyl-CoA to the of saturated LCFA such as the activity of HNF4α in assays, and its the binding of HNF4α to its site in in On the PUFA, such as or inhibit the activity of and their acyl-CoA the binding of HNF4α to its The data a promoter three of the of the gene regulatory region J. I. J. 1998; PubMed Scopus Google Scholar). Noteworthy, as consensus sequences for the binding of HNF4α are in the region of the rat Glc6Pase gene D. S. Diabetes. 1996; 45: PubMed Google has a the to the that a regulation of Glc6Pase gene expression by saturated and polyunsaturated LCFA could at a level and that HNF4α be involved in the We the effects of LCFA on the Glc6Pase promoter activity in HepG2 hepatoma and HeLa cells. Our results strongly suggest that the Glc6Pase promoter activity is by PUFA the modulating effect of their on HNF4α activity. The region of the rat Glc6Pase gene to to the transcription the of a luciferase gene and the which also the The region of the Glc6Pase gene by rat DNA the and D. S. Diabetes. 1996; 45: PubMed Google Scholar) and the to the The by of the by of the A of Glc6Pase with of the Glc6Pase promoter region by either or as a in Fig. mutagenesis of the HNF4α sequences the mutagenesis the resistance in the and specific in Fig. 7 and by the to the of by the of the HNF4 binding sites and the by HNF4α and the PUFA inhibition of the Glc6Pase promoter activity. HeLa with Glc6Pase of the sites and expression HNF4α and are on the and HNF4α binding sites for are in the and compared with the type activity and normalized to the level of activity. The by HNF4α of is to the by HNF4α of the In the B, activity treatment by acid μm) and to the activity in the in the of fatty acids with The results are as of inhibition induced by In both the results are the of at three in and and 3b. B, and not HepG2 hepatoma and HeLa in with (for HepG2 or (for HeLa fetal and in a at the in in The to by the with of the of to for and of a HNF4α expression M. and B. B. A. M. 1997; PubMed Scopus Google or of a expression M. M. I. J. S. M. PubMed Scopus Google as The of DNA by the of of HepG2 cells, in and for HeLa cells, in in and Scholar). The by the of for and the for in for by fatty acid in in the presence of in either or with or or acid In experiments, treatment for in the presence of with or or acid for fatty The three times with and with a and at for at 4 to activity with a the the at for to endogenous and activity as by J.R. 1987; Scholar). The levels of luciferase normalized by of the for in Fig. HNF4α protein in M. and B. and as by B. A. M. 1997; PubMed Scopus Google Scholar). for at 4 in the binding 4 2 in the presence of of HNF4α protein and of B. A. M. 1997; PubMed Scopus Google Scholar). DNA and on a gel in In experiments, the DNA in the to the of the The for gel G. F. M. F. M. J. 1996; PubMed Google Scholar) and/or acyl-CoA with at 4 for the of the and the of the DNA binding a and of protein HeLa cells, HeLa by for HepG2 cells, or rat liver nuclear by gel and to as by B. B. A. M. 1997; PubMed Scopus Google Scholar) G. F. M. F. M. J. 1996; PubMed Google Scholar) at a Glc6Pase a promoter activity in HepG2 in HeLa In HeLa cells, the of an not to promoter activity. Indeed, only a weak activity with of the Glc6Pase promoter the to Fig. This strongly suggested that hepatic specific for Glc6Pase promoter activity. A promoter to bp promoter the to activity in HepG2 A maximal activity with a the bp region of the Glc6Pase In the and the a which to that of the promoter This suggested that the bp region and that the bp regulatory able to the of the regulatory the to bp both the and the a substantial promoter activity than that of the HepG2 with Glc6Pase either a long or a promoter and with for in the presence of and and no effect on promoter activity In all PUFA and the activity of both by with other Glc6Pase promoter of results not These data strongly suggested that the effects of saturated and polyunsaturated LCFA on the Glc6Pase gene expression at a The suppression of the Glc6Pase promoter activity by PUFA not affected in the presence of or μm) In a the PUFA inhibition effect at a in the presence of the μm) in the as that for a This us to the that the effects could be dependent either on the or or on the deleterious of M. F. Biochem. J. 1999; PubMed Scopus (30) Google Scholar). The of in the Glc6Pase gene transcription has been previously Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar, S. J. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, B. DNA 1998; PubMed Scopus Google Scholar). In the of HNF4α has not been described. To the of the HeLa with Glc6Pase and expression of either HNF4α or as a In HeLa cells, the Glc6Pase promoter activity induced by times the the presence of at one site in This in with the presence of a site in region of the rat Glc6Pase promoter D. S. Diabetes. 1996; 45: PubMed Google Scholar). In HeLa cells, the Glc6Pase promoter activity induced by times for the the to the −1640/+60 bp In not the Glc6Pase promoter to bp These results suggested the presence of a site the bp of the Glc6Pase This in with the presence of identified and bp on the and rat Glc6Pase Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google S. J. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). The effects of LCFA on the Glc6Pase promoter activity in HeLa either or In HeLa cells, all PUFA and the Glc6Pase promoter activity by In saturated LCFA and no inhibitory effect The LCFA a weak inhibitory on the Glc6Pase promoter activity In HeLa cells, among the or polyunsaturated LCFA effect on the Glc6Pase promoter activity with Glc6Pase promoter of which results not see also Fig. in HepG2 cells, the inhibition effect in a in the presence of or and of These results strongly suggested that PUFA suppress the Glc6Pase promoter activity via a specific on the The of HNF4α in the by Noteworthy, HNF4α in substantial in HepG2 In HNF4α not in HeLa cells, whereas the in HeLa with times that in HepG2 It could be that the HNF4α level in HeLa in the as that in a rat liver in the data in and in with a of HNF4α in the effects on the Glc6Pase promoter activity in both HepG2 and HeLa cells. sites could be in the rat Glc6Pase gene regulatory region the at and D. S. Diabetes. 1996; 45: PubMed Google see also Fig. The these sites able to HNF4α in gel shift mobility assays, and a in the presence of The binding of by the and by an the specific site of the promoter In an a specific site not able to for the binding of to HNF4α the sites of the Glc6Pase three of and able to for the binding of the binding site to HNF4α The of the binding at of Glc6Pase to the effect as that by of These data suggested that HNF4α could to at three specific binding sites and on the Glc6Pase gene The activity of these HNF4 binding sites in the Glc6Pase gene transcription via or mutagenesis as in The the by HNF4α as that of the that the presence of the HNF4α site 4 not for the transactivation of the Glc6Pase The of the on the the by HNF4α by and the on the promoter in a in the transactivation effect. In the of the no effect These results strongly suggested that the site is crucial for the HNF4α transactivation and for the promoter activity. The of the site 3a on the the HNF4α transactivation by and induced a inhibition of the activity of the on site both of site and site 3a on the in a in the HNF4α transactivation by In of site 3b no substantial effect on the or inhibition effect on the on site These data strongly suggested that the 3a is crucial for the of the Glc6Pase We to the HNF4α binding sites for the PUFA suppression of the Glc6Pase promoter activity. A of inhibition by of the promoter activity for and and all a site The activity of the an site and a site also by by In the of both sites and in a of the suppression of promoter activity by These results strongly suggested that of the two sites and able to that both to account suppression of transcription of the Glc6Pase promoter by We the effects of fatty acyl-CoA on the binding of HNF4α to site the binding site the in by gel shift mobility The binding inhibited in a in the presence of and A maximal inhibitory effect of a concentration of inhibition at 2 for for and 7 for In and no inhibitory effects on the HNF4α binding the of whereas a weak effect at These data strongly suggested that the mechanism by which PUFA suppress the Glc6Pase promoter activity involves an inhibition of the binding of HNF4α to its cognate sites in the presence of polyunsaturated fatty acyl-CoA thioesters. The results the that LCFA are able to the expression of the Glc6Pase gene at a We that PUFA, and to a fatty acid may specific effects on the Glc6Pase promoter activity with regard to saturated fatty acids. In we the likely molecular mechanism of such an strongly that it may be via a of the transactivation effect induced by a transactivation factor we have shown that HNF4 a crucial in the Glc6Pase promoter the binding to two specific DNA cognate and that LCFA, via their metabolites fatty acyl-CoA are able to the of HNF4 by of a of its DNA binding activity. In regard to the of the of HNF4α in the regulation of the Glc6Pase it be that 2 of 4 sites with the consensus G. L. PubMed Scopus Google Scholar) to have a in the HNF4α transactivation of the Glc6Pase Indeed, an site 2 to for the binding of HNF4α to its specific binding of the promoter in gel shift and the of site 4 the no effect on the transactivation induced by HNF4α of Fig. and This is in with the that site 2 site 4 is an of a of the type Fig. In both sites and are an of a with with the consensus in the in In with a crucial of these sites in the of the Glc6Pase the by of or both results in in the transcription of the and Glc6Pase promoter in both the sites and of with the consensus appear to be the crucial in the HNF4α It be that HNF4α the sites and This suggest the presence of HNF4α binding site promoter HNF4α also the be an effect of HNF4α by on the Glc6Pase A binding site has been in the bp region of the rat Glc6Pase promoter Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar, S. J. 1997; Full Text Full Text PDF PubMed Scopus Google of the consensus the consensus in sequences is in the the consensus that are at three times in the The in in the Open in a In the consensus the consensus in sequences is in the the consensus that are at three times in the The in in the to the regulation of Glc6Pase transcription by LCFA, it is of note that both sites and is able to the suppression of the transactivation by PUFA and that the of both sites results in a of the effect. an has been that site has a compared with site in experiments, with regard to both the transactivation of transactivation effect of the for site for site and to PUFA inhibition for site for site see Fig. In site a in HNF4α binding in gel shift and for inhibition of the binding in the presence of We have no for the It likely that the of J.C. G. J. J. G. J. 2001; PubMed Scopus Google Scholar) a in cells, whereas they are in in the results are in with the molecular mechanism previously by J. I. J. 1998; PubMed Scopus Google Scholar) that the LCFA regulatory effects are via a of the binding of HNF4α to its DNA cognate sites induced by their results are with the of J. I. J. 1998; PubMed Scopus Google Scholar) in regard to two we have that PUFA inhibit the transactivation of the Glc6Pase promoter in cells, and we have that polyunsaturated fatty acyl-CoA inhibit the binding of HNF4α to its cognate sites in gel shift mobility Our results in in regard to other important we have not of transactivation by in HeLa or of the binding of HNF4α to its DNA binding sites in the presence of in the gel shift mobility we have not that and inhibitory effects to of PUFA and and we have that and at conditions, effects to of PUFA and a We have no for these the have been with a gene promoter a promoter could at of the of the effect of on the HNF4α binding in gel shift Fig. which has been suggested to be to a effect on HNF4α G. L. J. 2000; PubMed Scopus Google we have effects induced by we with the of and G. L. J. 2000; PubMed Scopus Google Scholar) that be as a for There is no that HNF4α specific binding sites for J. I. J. 1998; PubMed Scopus Google Scholar). the effects induced by PUFA in and in binding assays, respectively, led us to that may be as a of transactivation of the inhibitory PUFA are of it is of note that the in the presence of of the and/or us to the hypothesis that the of PUFA via these could be involved in the effects It should also be that the expression of liver the has been to be inhibited by PUFA at the level of transcription M. Clarke S. D.B. Endocrinol. Google Scholar). The molecular mechanism not been in the it is interesting to that the promoter region the PUFA a M. Clarke S. D.B. Endocrinol. Google Scholar). J. I. J. 1998; PubMed Scopus Google Scholar) in their that dietary and PUFA have been effects on the one hand, and to of dietary on the other hand, in such as blood or the level of blood Our that and and their may be the with regard to the effects induced on the HNF4α transactivation in more in with the in their in to their It that a the level of of LCFA and their and the effects induced on either the transactivation of the Glc6Pase promoter by HNF4α in or the binding of HNF4α to its DNA cognate sites in vitro. LCFA and not suppress and their not inhibit the The LCFA a weak effect on the HNF4α transactivation in HeLa cells, and a inhibitory effect on the binding only at the in gel shift mobility PUFA the effect on the Glc6Pase and their inhibited the binding of HNF4α to its cognate DNA binding sites the This suggests that the inhibitory of could be at in dependent on either the of in the chain or on the by these We have also in the that either or could be involved in the PUFA of Glc6Pase gene in of μm) the Glc6Pase promoter activity not In no in the abundance of the Glc6Pase mRNA in the liver in C. J. A. J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) as compared with the with or not not These data strongly suggested that not be involved in the PUFA of the Glc6Pase gene results have shown that the of in HepG2 or HeLa not the activity of the Glc6Pase promoter not This us to the hypothesis that suppression of the Glc6Pase promoter activity be dependent on a in the abundance of as has been for the effects on the transcription of involved in fatty acid D.B. Clarke S.D. Annu. Rev. Nutr. 1999; 19: 63-90Crossref PubMed Scopus (546) Google Scholar, J. M.T. Clarke S.D. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). In we that PUFA are able to the Glc6Pase gene transcription by of a specific inhibition of the binding of HNF4α to specific sites on the Glc6Pase These results the of J. I. J. 1998; PubMed Scopus Google Scholar). of the crucial of the Glc6Pase gene on the one and of the nutritional on the other in the of hepatic insulin resistance and type 2 the data may likely at in the effects of PUFA on insulin resistance at the level of endogenous glucose We and for expression of HNF4α and HNF4α and for the of expression of Bruce for the expression of and for the of membranes with and liver