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• W2034299473 abstract "Stearoyl-CoA desaturase (SCD) synthesizes oleate necessary for the biosynthesis of triglycerides and other lipids. Mice with a targeted disruption of the SCD1 gene are deficient in tissue oleate and have reduced expression of the sterol regulatory element-binding protein (SREBP) and its target genes. The SREBP-1c isoform is a known mediator of insulin action on hepatic gene expression, but its transcriptional effects due to glucose or fructose are still unclear. We found that fructose compared with glucose is a stronger inducer of SREBP-1c and lipogenic gene expression, causing a dramatic increase in hepatic triglyceride levels. However, when fed to the SCD1–/– mice, fructose failed to induce SREBP-1 or lipogenic genes and the triglyceride levels were not increased. Instead fructose feeding caused a decrease in hepatic glycogen and plasma glucose levels. The induction of SREBP-1 and lipogenic gene expression as well as the levels of liver triglycerides, glycogen, and plasma glucose was partially restored when the fructose diet was supplemented with very high levels of oleate (20% by weight) but not with palmitate, stearate, or linoleate. Fructose in a long term feeding induced the expression of SCD1 and that of other lipogenic genes in the liver of SREBP-1c–/– mice, and a further increase in expression of these genes occurred when the fructose diet was supplemented with oleate. Our observations demonstrated that oleate produced by SCD is necessary for fructose-mediated induction of lipogenic gene expression through SREBP-1c-dependent and -independent mechanisms and suggested that SCD1 gene expression is important in lipid and carbohydrate homeostasis. Stearoyl-CoA desaturase (SCD) synthesizes oleate necessary for the biosynthesis of triglycerides and other lipids. Mice with a targeted disruption of the SCD1 gene are deficient in tissue oleate and have reduced expression of the sterol regulatory element-binding protein (SREBP) and its target genes. The SREBP-1c isoform is a known mediator of insulin action on hepatic gene expression, but its transcriptional effects due to glucose or fructose are still unclear. We found that fructose compared with glucose is a stronger inducer of SREBP-1c and lipogenic gene expression, causing a dramatic increase in hepatic triglyceride levels. However, when fed to the SCD1–/– mice, fructose failed to induce SREBP-1 or lipogenic genes and the triglyceride levels were not increased. Instead fructose feeding caused a decrease in hepatic glycogen and plasma glucose levels. The induction of SREBP-1 and lipogenic gene expression as well as the levels of liver triglycerides, glycogen, and plasma glucose was partially restored when the fructose diet was supplemented with very high levels of oleate (20% by weight) but not with palmitate, stearate, or linoleate. Fructose in a long term feeding induced the expression of SCD1 and that of other lipogenic genes in the liver of SREBP-1c–/– mice, and a further increase in expression of these genes occurred when the fructose diet was supplemented with oleate. Our observations demonstrated that oleate produced by SCD is necessary for fructose-mediated induction of lipogenic gene expression through SREBP-1c-dependent and -independent mechanisms and suggested that SCD1 gene expression is important in lipid and carbohydrate homeostasis. Stearoyl-CoA desaturase (SCD) 1The abbreviations used are: SCD, stearoyl-CoA desaturase; SREBP, sterol regulatory element-binding protein; FAS, fatty-acid synthase; LFAE, long chain fatty acid elongase; ACC, acetyl-CoA carboxylase; ANOVA, analysis of variance; RT, reverse transcriptase. is a microsomal rate-limiting enzyme in the biosynthesis of mono-unsaturated fatty acids, mainly oleate. Oleate is necessary for the biosynthesis of triglycerides, cholesterol esters, and phospholipids (1Ntambi J.M. Prog. Lipid. Res. 1995; 34: 139-150Crossref PubMed Scopus (290) Google Scholar, 2Ntambi J.M. J. Lipid. Res. 1999; 40: 1549-1558Abstract Full Text Full Text PDF PubMed Google Scholar, 3Ntambi J.M. Miyazaki M. Curr. Opin. Lipidol. 2003; 14: 255-261Crossref PubMed Scopus (216) Google Scholar, 4Ntambi J.M. Miyazaki M. Prog. Lipid Res. 2004; 43: 91-104Crossref PubMed Scopus (552) Google Scholar, 5Miyazaki M. Ntambi J.M. Prostaglandins Leukot. Essent. Fatty Acids. 2003; 68: 113-121Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). Oleate also serves as a mediator of signal transduction and cellular differentiation (6Garbay B. Boiron-Sargueil F. Shy M. Chbihi T. Jiang H. Kamholz J. Cassagne C. J. Neurochem. 1998; 71: 1719-1726Crossref PubMed Scopus (51) Google Scholar, 7Medina J.M. Tabernero A. J. Physiol. (Paris). 2002; 96: 265-271Crossref PubMed Scopus (59) Google Scholar, 8Kaestner K.H. Ntambi J.M. Kelly Jr., T.J. Lane M.D. J. Biol. Chem. 1989; 264: 14755-14761Abstract Full Text PDF PubMed Google Scholar, 9Ntambi J.M. Buhrow S.A. Kaestner K.H. Christy R.J. Sibley E. Kelly Jr., T.J. Lane M.D. J. Biol. Chem. 1988; 263: 17291-17300Abstract Full Text PDF PubMed Google Scholar) and has recently been shown to regulate food intake in the brain (10Obici S. Feng Z. Morgan K. Stein D. Karkanias G. Rossetti L. Diabetes. 2002; 51: 271-275Crossref PubMed Scopus (530) Google Scholar). Oleate also influences apoptosis (11Listenberger L.L. Han X. Lewis S.E. Cases S. Farese Jr., R.V. Ory D.S. Schaffer J.E. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3077-3082Crossref PubMed Scopus (1407) Google Scholar, 12Roche E. Buteau J. Aniento I. Reig J.A. Soria B. Prentki M. Diabetes. 1999; 48: 2007-2014Crossref PubMed Scopus (124) Google Scholar, 13Lee Y. Song S.M. Park H.S. Kim S. Koh E.H. Choi M.S. Choi M.U. Immunology. 2002; 107: 435-443Crossref PubMed Scopus (13) Google Scholar) and may have some role in mutagenesis in some tumors (11Listenberger L.L. Han X. Lewis S.E. Cases S. Farese Jr., R.V. Ory D.S. Schaffer J.E. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3077-3082Crossref PubMed Scopus (1407) Google Scholar, 12Roche E. Buteau J. Aniento I. Reig J.A. Soria B. Prentki M. Diabetes. 1999; 48: 2007-2014Crossref PubMed Scopus (124) Google Scholar, 13Lee Y. Song S.M. Park H.S. Kim S. Koh E.H. Choi M.S. Choi M.U. Immunology. 2002; 107: 435-443Crossref PubMed Scopus (13) Google Scholar, 14Hardy S. Langelier Y. Prentki M. Cancer Res. 2000; 60: 6353-6358PubMed Google Scholar, 15Hardy S. El-Assad W. Przybytkowski E. Joly E. Prentki M. Langelier Y. J. Biol. Chem. 2003; 12 (31861-31870): 278Google Scholar). A proper ratio of mono-unsaturated fatty acids to their saturated precursors is necessary to ensure proper membrane fluidity, and changes in this ratio have been implicated in various pathological conditions including diabetes, atherosclerosis, cancer, and obesity (3Ntambi J.M. Miyazaki M. Curr. Opin. Lipidol. 2003; 14: 255-261Crossref PubMed Scopus (216) Google Scholar, 4Ntambi J.M. Miyazaki M. Prog. Lipid Res. 2004; 43: 91-104Crossref PubMed Scopus (552) Google Scholar, 5Miyazaki M. Ntambi J.M. Prostaglandins Leukot. Essent. Fatty Acids. 2003; 68: 113-121Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 16Sun Y. Hao M. Luo Y. Liang C.P. Silver D.L. Cheng C. Maxfield F.R. Tall A.R. J. Biol. Chem. 2003; 278: 5813-5820Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Four SCD genes have been cloned in mice (8Kaestner K.H. Ntambi J.M. Kelly Jr., T.J. Lane M.D. J. Biol. Chem. 1989; 264: 14755-14761Abstract Full Text PDF PubMed Google Scholar, 9Ntambi J.M. Buhrow S.A. Kaestner K.H. Christy R.J. Sibley E. Kelly Jr., T.J. Lane M.D. J. Biol. Chem. 1988; 263: 17291-17300Abstract Full Text PDF PubMed Google Scholar, 17Zheng Y. Prouty S.M. Harmon A. Sundberg J.P. Stenn K.S. Parimoo S. Genomics. 2001; 71: 182-191Crossref PubMed Scopus (148) Google Scholar, 18Miyazaki M. Jacobson M.J. Man W.C. Cohen P. Asilmaz E. Friedman J.M. Ntambi J.M. J. Biol. Chem. 2003; 18: 33904-33911Abstract Full Text Full Text PDF Scopus (163) Google Scholar), and two have been characterized in humans (19Zhang L. Ge L. Parimoo S. Stenn K. Prouty S.M. Biochem. J. 1999; 340: 255-264Crossref PubMed Scopus (204) Google Scholar, 20Beiraghi S. Zhou M. Talmadge C.B. Went-Sumegi N. Davis J.R. Huang D. Saal H. Seemayer T.A. Sumegi J. Gene (Amst.). 2003; 309: 11-21Crossref PubMed Scopus (65) Google Scholar). SCD homologs have also been described in yeast, flies, worms, sheep, and hamster, indicating that the SCD gene has served a vital metabolic function throughout evolution (3Ntambi J.M. Miyazaki M. Curr. Opin. Lipidol. 2003; 14: 255-261Crossref PubMed Scopus (216) Google Scholar, 4Ntambi J.M. Miyazaki M. Prog. Lipid Res. 2004; 43: 91-104Crossref PubMed Scopus (552) Google Scholar). We recently found that mice with a disruption in the SCD1 gene have increased energy expenditure, reduced body adiposity, and increased insulin sensitivity. They are resistant to diet- and leptin deficiency-induced obesity (3Ntambi J.M. Miyazaki M. Curr. Opin. Lipidol. 2003; 14: 255-261Crossref PubMed Scopus (216) Google Scholar, 4Ntambi J.M. Miyazaki M. Prog. Lipid Res. 2004; 43: 91-104Crossref PubMed Scopus (552) Google Scholar, 21Cohen P. Miyazaki M. Socci N.D. Hagge-Greenberg A. Liedtke W. Soukas A.A. Sharma R. Hudgins L.C. Ntambi J.M. Friedman J.M. Science. 2002; 297: 240-243Crossref PubMed Scopus (664) Google Scholar, 22Ntambi J.M. Miyazaki M. Stoehr J.P. Lan H. Kendziorski C.M. Yandell B.S. Song Y. Cohen P. Friedman J.M. Attie A.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11482-11486Crossref PubMed Scopus (880) Google Scholar). Triglyceride synthesis in liver was decreased relative to wild-type mice, which suggests that high SCD1 gene expression is highly correlated with liver steatosis (3Ntambi J.M. Miyazaki M. Curr. Opin. Lipidol. 2003; 14: 255-261Crossref PubMed Scopus (216) Google Scholar, 4Ntambi J.M. Miyazaki M. Prog. Lipid Res. 2004; 43: 91-104Crossref PubMed Scopus (552) Google Scholar, 21Cohen P. Miyazaki M. Socci N.D. Hagge-Greenberg A. Liedtke W. Soukas A.A. Sharma R. Hudgins L.C. Ntambi J.M. Friedman J.M. Science. 2002; 297: 240-243Crossref PubMed Scopus (664) Google Scholar, 22Ntambi J.M. Miyazaki M. Stoehr J.P. Lan H. Kendziorski C.M. Yandell B.S. Song Y. Cohen P. Friedman J.M. Attie A.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11482-11486Crossref PubMed Scopus (880) Google Scholar, 23Asilmaz E. Cohen P. Miyazaki M. Dobrzyn P. Ueki K. Fayzikhodjaeva G. Soukas A.A. Kahn C.R. Ntambi J.M. Socci N.D. Friedman J.M. J. Clin. Investig. 2004; 113: 414-424Crossref PubMed Scopus (170) Google Scholar, 24Miyazaki M. Kim Y.C. Gray-Keller M.P. Attie A.D. Ntambi J.M. J. Biol. Chem. 2000; 275: 30132-30138Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar, 25Miyazaki M. Kim Y.C. Ntambi J.M. J. Lipid Res. 2001; 42: 1018-1024Abstract Full Text Full Text PDF PubMed Google Scholar). The SCD1–/– mice exhibited down-regulation in the expression of genes such as FAS and GPAT that encode enzymes of lipid synthesis and up-regulation in the expression of genes that encode enzymes involved in fatty acid oxidation (22Ntambi J.M. Miyazaki M. Stoehr J.P. Lan H. Kendziorski C.M. Yandell B.S. Song Y. Cohen P. Friedman J.M. Attie A.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11482-11486Crossref PubMed Scopus (880) Google Scholar). The increase in fatty acid oxidation has been shown to be partly due to activation of AMP-activated kinase (26Dobrzyn P. Dobrzyn A. Miyazaki M. Cohen P. Asilmaz E. Hardie D.G. Friedman J.M. Ntambi J.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 6409-6414Crossref PubMed Scopus (329) Google Scholar). However, the mechanism by which SCD1 deficiency leads to a decrease in lipogenesis in liver has not been established. The SCD1 gene is regulated at the transcriptional level by a number of dietary factors including cholesterol, polyunsaturated fatty acids, glucose, and fructose (27Hasty A.H. Shimano H. Yahagi N. Amemiya-Kudo M. Perrey S. Yoshikawa T. Osuga J. Okazaki H. Tamura Y. Iizuka Y. Shionoiri F. Ohashi K. Harada K. Gotoda T. Nagai R. Ishibashi S. Yamada N. J. Biol. Chem. 2000; 275: 31069-31077Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 28Shimizu S. Ugi S. Maegawa H. Egawa K. Nishio Y. Yoshizaki T. Shi K. Nagai Y. Morino K. Nemoto K. Nakamura T. Bryer-Ash M. Kashiwagi A. J. Biol. Chem. 2003; 278: 43095-43101Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 29Nagai Y. Nishio Y. Nakamura T. Maegawa H. Kikkawa R. Kashiwagi A. Am. J. Physiol. 2002; 282: E1180-E1190Crossref PubMed Scopus (172) Google Scholar, 30Repa J.J. Liang G. Ou J. Bashmakov Y. Lobaccaro J.M. Shimomura I. Shan B. Brown M.S. Goldstein J.L. Mangelsdorf D.J. Genes Dev. 2000; 14: 2819-2830Crossref PubMed Scopus (1419) Google Scholar, 31Xu J. Nakamura M.T. Cho H.P. Clarke S.D. J. Biol. Chem. 1999; 274: 23577-23583Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar) in a sterol regulatory element-binding protein-1c (SREBP-1c)-dependent mechanism (32Tabor D.E. Kim J.B. Spiegelman B.M. Edwards P.A. J. Biol. Chem. 1999; 274: 20603-20610Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 33Tabor D.E. Kim J.B. Spiegelman B.M. Edwards P.A. J. Biol. Chem. 1998; 273: 22052-22058Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 34Shimomura I. Shimano H. Korn B.S. Bashmakov Y. Horton J.D. J. Biol. Chem. 1998; 273: 35299-352306Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). Sterol regulatory element-binding proteins are members of the basic helix-loop-helix leucine zipper family of transcription factors and regulate enzymes responsible for the synthesis of cholesterol, fatty acids, and triglycerides (35Horton J.D. Goldstein J.L. Brown M.S. J. Clin. Investig. 2002; 109: 1125-1131Crossref PubMed Scopus (3787) Google Scholar). There are three characterized isoforms of SREBP. SREBP-1a and SREBP-1c are derived from a single gene through the use of alternative promoters that give rise to the diverse first exon. SREBP-1a is the predominant form in cultured cells and a stronger activator of transcription of genes controlling lipogenesis and cholesterol synthesis (36Shimomura I. Shimano H. Horton J.D. Goldstein J.L. Brown M.S. J. Clin. Investig. 1997; 99: 838-845Crossref PubMed Scopus (641) Google Scholar). The roles of SREBP-1c and SREBP-2 are more restricted than that of SREBP-1a. Transgenic animal studies showed that SREBP-1c preferentially stimulates transcription of lipogenic genes in response to insulin and high carbohydrate feeding, whereas SREBP-2 preferentially activates genes involved in cholesterol synthesis (37Shimano H. Horton J.D. Hammer R.E. Shimomura I. Brown M.S. Goldstein J.L. J. Clin. Investig. 1996; 98: 1575-1584Crossref PubMed Scopus (698) Google Scholar, 38Shimano H. Horton J.D. Shimomura I. Hammer R.E. Brown M.S. Goldstein J.L. J. Clin. Investig. 1997; 99: 846-854Crossref PubMed Scopus (684) Google Scholar, 39Shimano H. Shimomura I. Hammer R.E. Herz J. Goldstein J.L. Brown M.S. Horton J.D. J. Clin. Investig. 1997; 100: 2115-2124Crossref PubMed Scopus (353) Google Scholar). The worldwide prevalence of obesity is increasing, and although it is probable that no single factor is responsible, environmental factors interacting with predisposing genetic factors are involved (40Ogden C.L. Flegal K.M. Carroll M.D. Johnson C.L. J. Am. Med. Assoc. 2002; 288: 1728-1732Crossref PubMed Scopus (3275) Google Scholar, 41Flegal K.M. Carroll M.D. Ogden C.L. Johnson C.L. J. Am. Med. Assoc. 2002; 288: 1723-1727Crossref PubMed Scopus (5360) Google Scholar, 42Kuczmarski R.J. Flegal K.M. Campbell S.M. Johnson C.L. J. Am. Med. Assoc. 1994; 272: 205-211Crossref PubMed Scopus (2415) Google Scholar). One of these environmental elements is diet, and currently, changes in the diet are being studied as contributing factors to the development of obesity. Along with an increase in total energy consumption over the past decades, there has been a shift in the types of nutrients consumed worldwide (43Elliott S.S. Keim N.L. Stern J.S. Teff K. Havel P.J. Am. J. Clin. Nutr. 2002; 76: 911-922Crossref PubMed Scopus (823) Google Scholar). The consumption of fructose has increased, largely because of an increase in the consumption of soft drinks and many other beverages that contain high levels of fructose (43Elliott S.S. Keim N.L. Stern J.S. Teff K. Havel P.J. Am. J. Clin. Nutr. 2002; 76: 911-922Crossref PubMed Scopus (823) Google Scholar). The mechanisms by which fructose may contribute to the development of obesity and accompanying abnormalities of insulin resistance have not been well addressed. We show in this study that when mice are fed either glucose or fructose individually, fructose is a stronger inducer of liver SREBP-1c and lipogenic gene expression than glucose. However, fructose failed to induce SREBP-1c and lipogenic gene expression in SCD1–/– mice. Fructose feeding was instead accompanied by a decrease in liver glycogen and plasma glucose levels. SREBP-1c and lipogenic gene expression, as well as triglyceride and glycogen levels, were partially restored in the SCD1–/– mice when the fructose diet was supplemented with high levels of dietary oleate and after feeding for 7 days. Stearate or palmitate supplementation to the fructose diet did not induce SREBP-1c or lipogenic gene expression. Liver triglyceride, liver glycogen, and plasma glucose levels were not rescued. Long term feeding of fructose also induced the expression of SCD1 and other lipogenic genes in SREBP-1c–/– mice. Supplementing the fructose diet with oleate did change SCD1 mRNA levels but caused additional increase in the mRNA levels of FAS and ACC. Taken together, our observations demonstrated that fructose-mediated induction of SREBP-1c or lipogenic genes is highly dependent on SCD1 gene expression and suggested that regulation of SCD1 plays an important role in carbohydrate and lipid homeostasis. Animals and Diets—The generation of SCD1–/– mice has been described previously (44Miyazaki M. Man W.C. Ntambi J.M. J. Nutr. 2001; 13: 2260-2268Crossref Scopus (225) Google Scholar). Pre-bred homozygous (SCD1–/–) and wild-type (SCD1+/+) mice on a pure 129 SV background were used. The SREBP-1c–/– mice were from the Jackson Laboratory and were bred into 129S6/SvEv background at least for five generations. 16–20-week-old mice were used in all of the experiments. Three mice were housed/caged and fed a standard chow diet (number 7001, Harlan Teklad), a 60% fructose diet (catalog number TD02518, Harlan Teklad), or a 60% glucose diet (TD02519) for 2 or 7 days. Triolein, tripalmitin, and tristearin (99% purity, Sigma) were supplemented to the fructose diet at 20 g weight % of diet. Dietary fat absorption was assessed by delivering 2mm bovine serum albumin complex containing 1 μCi of [14C]palmitate and phosphatidylcholine-liposome containing 1 μCi of [3H]sitostanol (American Radiolabeled Chemicals) by oral gavage to the mouse after a 4-h fast. Animals were given ad libitum access to food and water after gavage. Total feces were collected for 3 days, and the ratio of 14C to 3H radioactivity in aliquots of fecal extracts were used to calculate the percent dietary fat absorption. The breeding and care of the animals are in accordance with the protocols approved by the Animal Care Research Committee (ACRC) of the University of Wisconsin-Madison. Materials—Radioactive [32P]dCTP (3000 Ci/mmol) was obtained from PerkinElmer Life Sciences. Thin layer chromatography plates (TLC Silica Gel G60) were from Merck (Darmstadt, Germany). The cDNA probes for SCD1, FAS, and SREBP-1 have been described previously (25Miyazaki M. Kim Y.C. Ntambi J.M. J. Lipid Res. 2001; 42: 1018-1024Abstract Full Text Full Text PDF PubMed Google Scholar). All of the other chemicals were purchased from Sigma. SREBP-1 and SREBP-2 antibodies were from Dr. Jay Horton (University of Texas Southwestern Medical Center). Isolation and Analysis of RNA—Total RNA was isolated from livers of SCD1–/– mice and SREBP-1c–/– mice as well as the appropriate wild-type controls using TRIzol reagent (Invitrogen). The isolated RNA from livers of 4–6 mice in each group were pooled, and 15 μg of total RNA were separated by 1.0% agarose/2.2 m formaldehyde gel electrophoresis and transferred onto a nylon membrane. The membrane was hybridized with 32P-labeled cDNA probes. The cDNA probes for long chain fatty acyl-CoA elongase (LFAE, GenBank™ accession number AY053453), acetyl-CoA carboxylase (ACC, GenBank™ accession numbers J03808 and NM000664), fructokinase (FK, GenBank™ accession number Y09335), and aldolase B (GenBank™ accession number AH011101) were prepared by RT-PCR. The primers used for PCR were as follows. For ACC, the 5′ primer was 5′-GGGACTTCATGAATTTGCTGATTCTCA-3′ and the 3′ primer was 5′-GTCATTACCATCTTCATTTACCTCAATC-3′. For long chain fatty acyl-CoA elongase, the 5′ primer was 5′-CCTGTTTTCTGCGCTGTACGG-3′ and the 3′ primer was 5′-GCATGCACGCCATAGTTCAT-3′. For FK, the 5′ primer was 5′-GAATGCATCGGAACAGGTGA-3′ and the 3′ primer was 5′-GCTTGCCTCTCACACAATGC-3′. For aldolase B, the 5′ primer was 5′-CAATGCTCTGGCTCGCTATG-3′ and the 3′ primer was 5′-CTTCCTGGGTTGCCTTCTTG-3′. SREBP-1c, SREBP-1a, and SREBP-2 isoform mRNAs were analyzed by RT-PCR (18Miyazaki M. Jacobson M.J. Man W.C. Cohen P. Asilmaz E. Friedman J.M. Ntambi J.M. J. Biol. Chem. 2003; 18: 33904-33911Abstract Full Text Full Text PDF Scopus (163) Google Scholar). Specific primers for SREBP isoforms were designed as described previously (45Liang G. Yang J. Horton J.D. Hammer R.E. Goldstein J.L. Brown M.S. J. Biol. Chem. 2002; 277: 9520-9528Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar). Western Blot Analysis—For the immunoblotting of the SREBP proteins, nuclear extracts and membrane fractions of mice livers were prepared according to the methods of Shimomura et al. (46Shimomura I. Bashmakov Y. Shimano H. Horton J.D. Goldstein J.L. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12354-12359Crossref PubMed Scopus (126) Google Scholar). Aliquots of nuclear extracts (20 μg) were mixed with SDS loading buffer, subjected to SDS/PAGE on an 8% gel, transferred, and immobilized on Immobilon-P transfer membranes. After blocking with 3% bovine serum albumin in Tris-buffered saline buffer (pH 8.0) plus Tween 20 at 4 °C overnight, the membranes were washed and incubated with polyclonal anti-SREBP-1 or anti-SREBP-2 as primary antibody and anti-rabbit IgG-HRP conjugate as the secondary antibody. Visualization of the SREBP proteins was performed with Western blotting detection system kit (Pierce). Lipid Analysis—Total lipids were extracted from liver according to the method of Bligh and Dyer (47Bligh E.G. Dyer W.J. Can. J. Med. Sci. 1959; 37: 911-917Google Scholar). The lipid extracts were separated by TLC (hexane/diethyl ether/acetic acid, 80:20:1), and the bands were scraped from the plates, methylated, and analyzed by gas-liquid chromatography as described previously (48Miyazaki M. Kim H.J. Man W.C. Ntambi J.M. J. Biol. Chem. 2001; 276: 39455-39461Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Pentadecanoic acid (Sigma) was added as an internal standard for the quantitation of fatty acids. Glucose and Glycogen Determinations—Plasma glucose was analyzed using colorimetric glucose oxidase method (Sigma), and glycogen content was measured according to the methods of Roehrig and Allred (49Roehrig K.L. Allred J.B. Anal. Biochem. 1974; 58: 414-421Crossref PubMed Scopus (220) Google Scholar). Statistical Analysis—Statistical analysis was performed with either one-way ANOVA or Student's t test with statistical significance set at p < 0.05. Fig. 1 shows a Northern blot of total RNA isolated from the liver of wild-type mice that were fed chow, high glucose, or fructose diets for 7 days measuring the mRNA levels of SREBP-1 and lipogenic genes. The 7-day feeding period was used to ensure maximum induction of mRNA levels. Dietary glucose increased the mRNA levels of SREBP-1, SCD1, LFAE, FAS, and ACC by 1.5-, 4.7-, 2.8-, 2.3-, and 1.9-fold, respectively, whereas fructose increased the levels of these mRNAs by 2.6-, 19.6-, 7.9-, 7.0-, and 9.1-fold, respectively, relative to chow diet. The effect of glucose or fructose on the expression of SREBP isoforms was analyzed by RT-PCR. Fig. 1B shows that glucose induced the mRNA level of SREBP-1c isoform by 1.5-fold, whereas fructose induced it by 2.9-fold relative to chow diet. Glucose or fructose did not induce SREBP-1a and SREBP-2 mRNA expression. Consistent with the Northern blot, fructose induced the mature form of SREBP-1 protein 3.8-fold, whereas glucose did not induce it (Fig. 1C). The levels of the mature form of SREBP-2 protein were not altered by either sugar. These experiments indicated that fructose is a stronger inducer of SREBP-1c and lipogenic gene expression than glucose. Mice with a targeted disruption of the SCD1 isoform have reduced body adiposity and down-regulated expression of several genes encoding enzymes of fatty acid and triglyceride biosynthesis (22Ntambi J.M. Miyazaki M. Stoehr J.P. Lan H. Kendziorski C.M. Yandell B.S. Song Y. Cohen P. Friedman J.M. Attie A.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11482-11486Crossref PubMed Scopus (880) Google Scholar). We sought to explore the mechanism by which SCD1 deficiency causes reduced lipogenesis by feeding the highly lipogenic fructose diet to the SCD1–/– mice and measuring the expression of SREBP-1c and its target genes. Fig. 2 shows that, whereas fructose induced SREBP-1, SCD1, FAS, and ACC mRNA levels by 2.3-, 16.3-, 9.8-, and 13.3-fold, respectively, in the wild-type mice relative to chow diet, fructose did not induce SREBP-1, FAS, and ACC mRNA expression in the SCD1–/– mice. Food intake of the fructose diet was not different between SCD1–/– and wild-type mice (3.1 ± 0.3 g/day SCD1–/– mice versus 2.9 ± 0.4 g/day wild-type mice). To check the nutritional status of the mice, we measured the mRNA expression of fructokinase and aldolase B genes. These genes encode for fructokinase and aldolase B enzymes, which are increased by fructose feeding (50Adelman R.C. Spolter P.D. Weinhouse S. J. Biol. Chem. 1966; 241: 5467-5472Abstract Full Text PDF PubMed Google Scholar). Fig. 2A shows that fructose induced fructokinase and aldolase B mRNA levels by >2-fold in both wild type and SCD1–/– mice, demonstrating that both groups of mice were correctly fed fructose. Consistent with the Northern blot (Fig. 2A), Fig. 2B shows that fructose increased the content of the precursor and mature forms of SREBP-1 protein by 1.8- and 4-fold, respectively, in the wild-type mice, but no such induction was observed in the SCD1–/– mice. The content of the precursor and mature forms of SREBP-2 proteins was not changed in the wild-type and SCD1–/– mice in response to fructose feeding. Oleate is the major product of SCD (48Miyazaki M. Kim H.J. Man W.C. Ntambi J.M. J. Biol. Chem. 2001; 276: 39455-39461Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). To determine whether oleate is required for the induction of SREBP-1c and lipogenic gene expression, SCD1–/– mice were fed a fructose diet supplemented with 5% triolein (18:1) for 2 or 7 days and mRNA expression of SREBP-1, FAS, and ACC were measured. Because there was no increase in the levels of these mRNAs in the liver of SCD1–/– mice (data not shown), the amount of triolein supplemented to the fructose diet was increased to 20% by weight. A two-day feeding of fructose induced SREBP-1 and lipogenic genes in the wild-type mice but not in the SCD1–/– mice (data not shown). Fig. 2A shows that 20% triolein (18:1) supplementation to the fructose diet for 2 days still did not increase the mRNA for SREBP-1, FAS, and ACC in the SCD1–/– mice, but a 4.3-, 3.5-, and 7.0-fold induction, respectively, relative to the fructose-fed mice occurred after a 7-day feeding period. Triolein supplementation caused a further increase in the expression of SREBP-1, FAS, and ACC mRNA levels in the wild-type mice, but the mRNA level of SCD1 was not increased. Consistent with the Northern blot (Fig. 2), 20% triolein feeding increased the content of the mature SREBP-1 protein in the wild-type and SCD1–/– mice 6.7- and 5.3-fold, respectively, relative to a chow diet feeding (Fig. 2B). The content of precursor and mature forms of SREBP-2 proteins were not affected by triolein feeding. Fructose supplemented with tristearin (18:0) did not induce SREBP-1, FAS, and ACC mRNA levels, and the content of the mature form of SREBP-1 protein was not increased in SCD1–/– mice (Fig. 2B). The content of the precursor and mature form of SREBP-2 protein was not changed. In another experiment, 20% tripalmitin (16:0) or trilinolein (18:2n-6) supplementation to the fructose diet did not induce the mRNA levels for SREBP-1, ACC, and FAS in the SCD1–/– mice (Fig. 2C) and the levels of the mature form of SREBP-1 protein were not increased (Fig. 2D). Dietary fat absorption as assessed by the ratio of 14C to 3H radioactivity of [14C]palmitate and of phosphatidylcholine-liposome containing [3H]sitostanol in the feces of SCD1–/– and wild type mice was not different (data not shown). These experiments suggested that fructose-mediated induction of SREBP-1 and lipogenic gene expression requires oleate. To determine whether oleate is required for fructose-mediated changes in triglyceride levels, we measured liver and plasma triglycerides in the SCD1+/+ and SCD1–/– mice after 7 days of feeding. Fig. 3A shows that fructose caused a 2.7-fold increase in liver triglyceride levels in the wild-type mice but that no increase in triglyceride content was observed in the SCD1–/– mice. Triolein (20%) supplementation caused a further 1.5-fold increase in liver triglyceride content relative to fructose feeding in wild-type mice but caused a 4.8-fold increase in triglyceride content in the SCD1–/– mice. Tristearin or tripalmitin supplementation did" @default.
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• W2034299473 date "2004-06-01" @default.
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• W2034299473 title "Stearoyl-CoA Desaturase 1 Gene Expression Is Necessary for Fructose-mediated Induction of Lipogenic Gene Expression by Sterol Regulatory Element-binding Protein-1c-dependent and -independent Mechanisms" @default.
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• W2034299473 doi "https://doi.org/10.1074/jbc.m402781200" @default.
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