Fish Oil Feeding Decreases Mature Sterol Regulatory Element-binding Protein 1 (SREBP-1) by Down-regulation of SREBP-1c mRNA in Mouse Liver
1999; Elsevier BV; Volume: 274; Issue: 36 Linguagem: Inglês
10.1074/jbc.274.36.25892
ISSN1083-351X
AutoresHyoun-Ju Kim, Mayumí Takáhashi, Osamu Ezaki,
Tópico(s)Cancer, Lipids, and Metabolism
ResumoDietary fish oil induces hepatic peroxisomal and microsomal fatty acid oxidation by peroxisome proliferator-activator receptor α activation, whereas it down-regulates lipogenic gene expression by unknown mechanism(s). Because sterol regulatory element-binding proteins (SREBPs) up-regulated lipogenic genes, investigation was made on the effects of fish oil feeding on SREBPs and sterol regulatory element (SRE)-dependent gene expression in C57BL/6J mice. Three forms of SREBPs, SREBP-1a, -1c, and -2, are expressed in liver, and their truncated mature forms activate transcription of sterol-regulated genes. C57BL/6J mice were divided into three groups; the first group was given a high carbohydrate diet, and the other two groups were given a high fat diet (60% of total energy), with the fat in the form of safflower oil or fish oil, for 5 months. Compared with safflower oil feeding, fish oil feeding decreased triglyceride and cholesterol concentrations in liver. There were no differences in amount of SREBP-1 and -2 in both precursor and mature forms between carbohydrate- and safflower oil-fed mice. However, compared with safflower oil feeding, fish oil feeding reduced the amounts of precursor SREBP-1 in membrane fraction by 90% and of mature SREBP-1 in liver nuclei by 57%. Fish oil feeding also reduced precursor SREBP-2 by 65% but did not alter the amount of mature SREBP-2. Compared with safflower oil feeding, fish oil feeding decreased liver SREBP-1c mRNA level by 86% but did not alter SERBP-1a mRNA. Consistent with decrease of mature SREBP-1, compared with safflower oil feeding, fish oil feeding down-regulated the expression of liver SRE-dependent genes, such as low density lipoprotein receptor, 3-hydroxy-3-methylglutaryl-CoA reductase, 3-hydroxy-3-methylglutaryl-CoA synthase, fatty acid synthase, acetyl-CoA carboxylase, and stearoyl-CoA desaturase-1. These data suggested that in liver, fish oil feeding down-regulates the mature form of SREBP-1 by decreasing SREBP-1c mRNA expression, with corresponding decreases of mRNAs of cholesterologenic and lipogenic enzymes. Dietary fish oil induces hepatic peroxisomal and microsomal fatty acid oxidation by peroxisome proliferator-activator receptor α activation, whereas it down-regulates lipogenic gene expression by unknown mechanism(s). Because sterol regulatory element-binding proteins (SREBPs) up-regulated lipogenic genes, investigation was made on the effects of fish oil feeding on SREBPs and sterol regulatory element (SRE)-dependent gene expression in C57BL/6J mice. Three forms of SREBPs, SREBP-1a, -1c, and -2, are expressed in liver, and their truncated mature forms activate transcription of sterol-regulated genes. C57BL/6J mice were divided into three groups; the first group was given a high carbohydrate diet, and the other two groups were given a high fat diet (60% of total energy), with the fat in the form of safflower oil or fish oil, for 5 months. Compared with safflower oil feeding, fish oil feeding decreased triglyceride and cholesterol concentrations in liver. There were no differences in amount of SREBP-1 and -2 in both precursor and mature forms between carbohydrate- and safflower oil-fed mice. However, compared with safflower oil feeding, fish oil feeding reduced the amounts of precursor SREBP-1 in membrane fraction by 90% and of mature SREBP-1 in liver nuclei by 57%. Fish oil feeding also reduced precursor SREBP-2 by 65% but did not alter the amount of mature SREBP-2. Compared with safflower oil feeding, fish oil feeding decreased liver SREBP-1c mRNA level by 86% but did not alter SERBP-1a mRNA. Consistent with decrease of mature SREBP-1, compared with safflower oil feeding, fish oil feeding down-regulated the expression of liver SRE-dependent genes, such as low density lipoprotein receptor, 3-hydroxy-3-methylglutaryl-CoA reductase, 3-hydroxy-3-methylglutaryl-CoA synthase, fatty acid synthase, acetyl-CoA carboxylase, and stearoyl-CoA desaturase-1. These data suggested that in liver, fish oil feeding down-regulates the mature form of SREBP-1 by decreasing SREBP-1c mRNA expression, with corresponding decreases of mRNAs of cholesterologenic and lipogenic enzymes. fatty acid synthase peroxisome proliferator-activated receptor sterol regulatory element sterol regulatory element-binding protein low density lipoprotein 3-hydroxy-3-methylglutaryl-CoA lipoprotein lipase acyl-CoA synthetase acetyl-Co A carboxylase stearoyl-CoA desaturase Dietary fish oil contains n-3 fatty acids, such as eicosapentaenoic acid and docosahexaenoic acid, which decrease blood triglyceride concentrations in hypertriglycemic patients and are considered to have protective effects against cardiovascular diseases (1Nestel P.J. Annu. Rev. Nutr. 1990; 10: 149-167Crossref PubMed Scopus (197) Google Scholar). This effect of n-3 fatty acids mainly results from the combined effects of inhibition of lipogenesis and stimulation of fatty acid oxidation in liver (2Rustan A.C. Nossen J.O. Christiansen E.N. Drevon C.A. J. Lipid Res. 1988; 29: 1417-1426Abstract Full Text PDF PubMed Google Scholar, 3Halminski M.A. Marsh J.B. Harrison E.H. J. Nutr. 1991; 121: 1554-1561Crossref PubMed Scopus (104) Google Scholar). It has been shown that n-3 fatty acids in vivo or in cell culture inhibited the transcription of genes coding for lipogenesis enzymes, such as fatty acid synthase (FAS),1acetyl-CoA carboxylase (ACC), stearoyl-CoA desaturase (SCD), and S14 protein (4Clarke S.D. Jump D.B. Annu. Rev. Nutr. 1994; 14: 83-98Crossref PubMed Scopus (267) Google Scholar, 5Pegorier J.-P. Curr. Opin. Clin. Nutr. Metab. Care. 1998; 1: 329-334Crossref PubMed Scopus (36) Google Scholar). On the other hand, n-3 fatty acids increased the transcription of the regulatory enzymes of fatty acid oxidation, such as lipoprotein lipase (LPL), fatty acid-binding protein, acyl-CoA synthetase (ACS), carnitine palmitoyltransferase 1, acyl-CoA dehydrogenese, and acyl-CoA oxidase (4Clarke S.D. Jump D.B. Annu. Rev. Nutr. 1994; 14: 83-98Crossref PubMed Scopus (267) Google Scholar, 5Pegorier J.-P. Curr. Opin. Clin. Nutr. Metab. Care. 1998; 1: 329-334Crossref PubMed Scopus (36) Google Scholar). The molecular mechanisms by which n-3 fatty acids regulate gene transcription have not yet been clarified, but on the basis of in vitro assays and in comparison with peroxisome proliferators, such as fibrate compounds, it has been suggested that n-3 fatty acids can regulate gene transcription through the activation of a transcription factor, peroxisome proliferator-activated receptor (PPAR) α (6Reddy J.K. Mannaerts G.P. Annu. Rev. 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Interestingly, enzymes for peroxisomal (cytochrome P450 4A2) and microsomal (acyl-CoA oxidase) oxidation are PPARα-dependent and are up-regulated, whereas enzymes for lipid synthesis (FAS and S14) are PPARα-independent and are downregulated (10Ren B. Thelen A.P. Peters J.M. Gonzalez F.J. Jump D.B. J. Biol. Chem. 1997; 272: 26827-26832Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). On the other hand, sterol regulatory element-binding proteins (SREBPs) are other important transcription factors that regulate fatty acid and cholesterol metabolism in liver (11Sakai J. Duncan E.A. Rawson R.B. Hua X. Brown M.S. Goldstein J.L. Cell. 1996; 85: 1037-1046Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar). In sterol depletion, SREBPs are cleaved and become mature forms to bind sterol regulatory elements (SREs) (12Briggs M.R. Yokoyama C. Wang X. Brown M.S. Goldstein J.L. J. Biol. Chem. 1993; 268: 14490-14496Abstract Full Text PDF PubMed Google Scholar, 13Wang X. Briggs M.R. Hua X. Yokoyama C. Goldstein J.L. Brown M.S. J. Biol. Chem. 1993; 268: 14497-14504Abstract Full Text PDF PubMed Google Scholar) and/or E-box sequences (14Kim J.B. Spotts G.D. Halvorsen Y.D. Shih H.M. Ellenberger T. Towle H.C. Spiegelman B.M. Mol. Cell. Biol. 1995; 15: 2582-2588Crossref PubMed Scopus (297) Google Scholar) and then activate the target gene expression. Thus, both expression levels and processing of SREBPs regulate the target gene expression. Furthermore, three forms of SREBPs, SREBP-1a, SREBP-1c, and SREBP-2, are expressed in liver, and they use different promoters for their own expression (15Hua X. Wu J. Goldstein J.L. Brown M.S. Hobbs H.H. Genomics. 1995; 25: 667-673Crossref PubMed Scopus (248) Google Scholar, 16Miserez A.R. Cao G. Probst L.C. Hobbs H.H. Genomics. 1997; 40: 31-40Crossref PubMed Scopus (90) Google Scholar). In addition, studies on transgenic mice that over-expressed SREBP-1a (17Shimano H. Horton J.D. Hammer R.E. Shimomura I. Brown M.S. Goldstein J.L. J. Clin. Invest. 1996; 98: 1575-1584Crossref PubMed Scopus (699) Google Scholar), SERBP-1c (18Shimano H. Horton J.D. Shimomura I. Hammer R.E. Brown M.S. Goldstein J.L. J. Clin. Invest. 1997; 99: 846-854Crossref PubMed Scopus (688) Google Scholar), and SREBP-2 (19Horton J.D. Shimomura I. Brown M.S. Hammer R.E. Goldstein J.L. Shimano H. J. Clin. Invest. 1998; 101: 2331-2339Crossref PubMed Google Scholar) in liver demonstrated that they have different potencies for regulation of target gene expression. The target genes of SREBPs involved in cholesterol metabolism include LDL receptor, 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, HMG-CoA synthase, and SREBP itself (17Shimano H. Horton J.D. Hammer R.E. Shimomura I. Brown M.S. Goldstein J.L. J. Clin. Invest. 1996; 98: 1575-1584Crossref PubMed Scopus (699) Google Scholar, 18Shimano H. Horton J.D. Shimomura I. Hammer R.E. Brown M.S. Goldstein J.L. J. Clin. Invest. 1997; 99: 846-854Crossref PubMed Scopus (688) Google Scholar, 19Horton J.D. Shimomura I. Brown M.S. Hammer R.E. Goldstein J.L. Shimano H. J. Clin. Invest. 1998; 101: 2331-2339Crossref PubMed Google Scholar). Genes involved in fatty acid and triglyceride synthesis that are regulated by SREBPs include ACC, FAS, and SCD-1 (17Shimano H. Horton J.D. Hammer R.E. Shimomura I. Brown M.S. Goldstein J.L. J. Clin. Invest. 1996; 98: 1575-1584Crossref PubMed Scopus (699) Google Scholar, 18Shimano H. Horton J.D. Shimomura I. Hammer R.E. Brown M.S. Goldstein J.L. J. Clin. Invest. 1997; 99: 846-854Crossref PubMed Scopus (688) Google Scholar, 19Horton J.D. Shimomura I. Brown M.S. Hammer R.E. Goldstein J.L. Shimano H. J. Clin. Invest. 1998; 101: 2331-2339Crossref PubMed Google Scholar). We examined SREBP mRNAs and their protein levels in liver from fish oil-fed mice. Our experiment demonstrated that in liver, fish oil feeding decreased SREBP-1c gene expression and resulted in decrease of mature SREBP-1 protein. These data provide a new mechanism for down-regulation of mRNAs of cholesterologenic and lipogenic enzymes observed in fish oil feeding. Female C57BL/6J mice were obtained from Tokyo Laboratory Animals Science Co. (Tokyo, Japan) at 7 weeks of age and fed a normal laboratory diet (CE2, Clea, Tokyo, Japan) for 1 week to stabilize the metabolic conditions. Mice were exposed to a 12-h light/12-h dark cycle and maintained at a constant temperature of 22 °C. Mice were divided into three groups (n = 13–16 in each group). Each group was divided into three cages, with each cage containing 4–6 mice. The first group was given a high carbohydrate diet that, on a calorie basis, contained 63% carbohydrate, 11% fat, and 26% protein. In the high carbohydrate diet, safflower oil was used as source of fat. The second group was given a safflower oil-rich diet containing 14% carbohydrate, 60% safflower oil, and 26% protein. The third group was given a high fish oil diet containing 14% carbohydrate, 60% fish oil (mainly from tuna), and 26% protein. Fatty acid compositions of dietary oils were measured by gas-liquid chromatography. Safflower oil (high oleic type) contained 46% oleic acid (18:1 n-9) and 45% linoleic acid (18:2 n-6) from total fatty acids; fish oil contained 7% eicosapentaenoic acid (20:5 n-3) and 24% docosahexaenoic acid (22:6 n-3). The materials and methods of diet preparation and those of estimation of energy intake were the same as those used in our previous studies (20Ikemoto S. Takahashi M. Tsunoda N. Maruyama K. Itakura H. Ezaki O. Metabolism. 1996; 45: 1539-1546Abstract Full Text PDF PubMed Scopus (226) Google Scholar, 21Tsunoda N. Ikemoto S. Takahashi M. Maruyama K. Itakura H. Watanabe H. Goto N. Ezaki O. Metabolism. 1998; 47: 724-730Abstract Full Text PDF PubMed Scopus (36) Google Scholar). Mice were fed each diet for 5 months. At the end of the experiments, animals were anesthetized at about 10:00 a.m. by intraperitoneal injection of pentobarbital sodium (0.08 mg/g of body weight; Nembutal, Abbot, North Chicago, IL). Liver was isolated immediately, weighed, and homogenized in guanidine-thiocyanate, and RNA was prepared by the method described by Chirgwin et al. (22Chirgwin J.M. Przybyla A.E. MacDonald R.J. Rutter W.J. Biochemistry. 1979; 18: 5294-5299Crossref PubMed Scopus (16652) Google Scholar). A part of liver of each mouse was immediately homogenized to obtain membrane fractions and nuclear extracts (23Sheng Z. Otani H. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 935-938Crossref PubMed Scopus (279) Google Scholar), and the other portion of liver was frozen for measurement of triglyceride and cholesterol as described in previously (24Ikemoto S. Takahashi M. Tsunoda N. Maruyama K. Itakura H. Kawanaka K. Tabata I. Higuchi M. Tange T. Yamamoto T. Ezaki O. Am. J. Physiol. 1997; 273: E37-E45Crossref PubMed Google Scholar). Female C57BL/6J mice (n = 5; 7 weeks of age) were treated for 2 weeks with fenofibrate (Sigma) mixed in high carbohydrate diet that had the same ingredient used in fish oil diet study. Control mice (n= 5) were fed under the same conditions but in the absence of fenofibrate. Because each mouse consumed approximately 1.5–2.0 g of chow/day, doses of 0.5% (w/w) mixed in diet correspond to 410–550 mg/kg of body weight/day. Mice were fed each diet for 2 weeks. Mice were sacrificed in a method similar to that of the fish oil diet experiment. The cDNA fragments for mouse SREBP-1, SREBP-2, HMG-CoA reductase, HMG-CoA synthase, apoE, and LPL were obtained by polymerase chain reaction from first strand cDNA using mouse liver total RNA. Total RNA from mouse liver was isolated by the method of Chirgwin et al. (22Chirgwin J.M. Przybyla A.E. MacDonald R.J. Rutter W.J. Biochemistry. 1979; 18: 5294-5299Crossref PubMed Scopus (16652) Google Scholar). First strand cDNA was prepared using a Superscript II kit (Life Technologies, Inc.) primed with oligo-dT. The polymerase chain reaction primers used were as follows (17Shimano H. Horton J.D. Hammer R.E. Shimomura I. Brown M.S. Goldstein J.L. J. Clin. Invest. 1996; 98: 1575-1584Crossref PubMed Scopus (699) Google Scholar, 31Shimomura I. Shimano H. Horton J.D. Goldstein J.L. Brown M.S. J. Clin. Invest. 1997; 99: 838-845Crossref PubMed Scopus (642) Google Scholar): SREBP-1, 5′ primer, 5′-TCAACAACCAAGACAGTGACTTCCCTGGCC-3′, and 3′ primer, 5′-GTTCTCCTGCTTGAGCTTCTGGTTGCTGTG-3′; SREBP-2, 5′ primer, 5′-CATGGACACCCTCACGGAGCTGGGCGACGA-3′, and 3′ primer, 5′-TGCATCATCCAATAGAGGGCTTCCTGGCTC-3′; HMG-CoA reductase, 5′ primer, 5′-GGGACGGTGACACTTACCATCTGTATGATG-3′, and 3′ primer, 5′-ATCATCTTGGAGAGATAAAACTGCCA-3′; HMG-CoA synthase, 5′ primer, 5′-TATGATGGTGTAGATGCTGGGAAGTATACC-3′, and 3′ primer, 5′-TAAGTTCTTCTGTGCTTTTCATCCAC-3′; apoE, 5′ primer, 5′-TGGGAGCAGGCCCTGAACCGCTTC-3′, and 3′ primer, 5′-GAGTCGGGCCTGTGCCGCCTGCAC-3′; and LPL, 5′ primer, 5′-GTGGCCGCAGCAGACGCAGGAAGA-3′, and 3′ primer, 5′-CATCCAGTTGATGAATCTGGCCAC-3′. Polymerase chain reaction was performed with a Tag DNA polymerase (Takara, Shiga, Japan). Thirty-two cycles of amplification were made by using the following program: 94 °C, 40 s; 68 °C, 1 min; and 72 °C, 2 min. The amplified products were subcloned into pGEM-T Easy vector (Promega, Madison, WI). The cDNA probes for rat LDL receptor and rat ACS were kindly provided by Dr. T. Yamamoto at Tohoku University (25Kim D. Iijima H. Goto K. Sakai J. Ishii H. Kim H. Suzuki H. Kondo H. Saeki S. Yamamoto T. J. Biol. Chem. 1996; 271: 8373-8380Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar, 26Suzuki H. Kawarabayasi Y. Kondo J. Abe T. Nishikawa K. Kimura S. Hashimoto T. Yamamoto T. J. Biol. Chem. 1990; 265: 8681-8685Abstract Full Text PDF PubMed Google Scholar), mouse SCD-1 was provided by Dr. Daniel M. Lane at Johns Hopkins University (27Ntambi J.M. Buhrow S.A. Kaestner K.H. Christry 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 rat ACC and rat FAS were provided by Dr. N. Iritani at Tezukayama Gakuin College (28Katsurada A. Iritani N. Fukuda H. Matsumura Y. Nishimoto N. Noguchi T. Tanaka T. Eur. J. Biochem. 1990; 190: 427-433Crossref PubMed Scopus (139) Google Scholar, 29Katsurada A. Iritani N. Fukuda H. Matsumura Y. Nishimoto N. Noguchi T. Tanaka T. Eur. J. Biochem. 1990; 190: 435-441Crossref PubMed Scopus (136) Google Scholar). These cDNAs were used as probes for Northern blotting. Aliquots of total RNA(10–15 μg) were denatured with glyoxal and dimethyl sulfoxide, subjected to electrophoresis in a 1% agarose gel, and transferred to nylon membranes (NEN Life Science Products). After transfer and UV cross-linking, RNA blots were stained with methylene blue to locate 28 S and 18 S rRNAs and to ascertain the amount of loaded RNAs (30Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 187-206Google Scholar). The membranes were hybridized with each cDNA probe labeled with [α-32P]dCTP (NEN Life Science Products) by a random primer labeling kit (Amersham Pharmacia Biotech). The membranes were hybridized overnight at 42 °C in hybridization buffer, subsequently washed one time with 1× SSC, 0.1% SDS at 22 °C, two times for 30 min at 50 °C, and one time for 30 min at 65 °C. The membranes were exposed to Kodak XAR-5 film at −80 °C with intensifying screens. Quantitative analysis was performed with an image analyzer (BAS 2000, Fuji Film, Tokyo, Japan) and expressed as the intensity of phosphostimulated luminescence. The cDNA fragment specific to either mouse SREBP-1a or SREBP-1c was amplified by polymerase chain reaction from first strand cDNA prepared using mouse liver total RNA. The following primers were used. SREBP-1a: 5′ primer, 5′-TAGTCCGAAGCCGGGTGGGCGCCGGCGCCAT-3′; 3′ primer, 5′-GATGTCGTTCAAAACCGCTGTGTGTCCAGTTC-3′ (Table I from Ref. 31Shimomura I. Shimano H. Horton J.D. Goldstein J.L. Brown M.S. J. Clin. Invest. 1997; 99: 838-845Crossref PubMed Scopus (642) Google Scholar). SREBP-1c: 5′ primer, 5′-ATCGGCGCGGAAGCTGTCGGGGTAGCGTC-3′; 3′ primer, 5′-ACTGTCTTGGTTGTTGATGAGCTGGAGCAT-3′ (31Shimomura I. Shimano H. Horton J.D. Goldstein J.L. Brown M.S. J. Clin. Invest. 1997; 99: 838-845Crossref PubMed Scopus (642) Google Scholar). The amplified products were subcloned into pGEM-T Easy vector (Promega, Madison, WI). After linearization of plasmid, antisense RNA was transcribed with [α-32P]CTP (800 Ci/mmol) using bacteriophage T7 or SP6 RNA polymerase (Promega). Specific activities of the transcribed RNAs were measured in each experiment and were in the range of 0.8–1.2 × 109 cpm/μg. Aliquots of total RNA(10 μg) from mouse liver were subjected to RNase protection assay using a RPA IITM kit (Ambion, Inc., Austin, TX). After digestion with RNase A/T1, protected fragments were separated on 8 murea/4.8% polyacylamide gels. The gels were dried and then subjected to autoradiography. Quantitative analysis was performed with an image analyzer (BAS 2000). Pooled liver membranes and nuclear extracts from 5–6 mice of each group were prepared by the method described by Sheng et al. (23Sheng Z. Otani H. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 935-938Crossref PubMed Scopus (279) Google Scholar). The same amount of protein from each fraction was applied to 7% SDS-polyacrylamide gel electrophoresis and transferred to Hybond-P membranes (Amersham Pharmacia Biotech). Immunoblot analysis was performed by using the ECL Western blotting detection system kit (Amersham Pharmacia Biotech). Membrane sheets were first incubated with antibody against SREBP-1 or SREBP-2 for 1 h at 22 °C and then washed several times and incubated with horseradish peroxidase-conjugated anti-mouse IgG according to the protocol supplied by the manufacturer. The bands were quantified by scanning with Canon IX-4015 (Canon Inc., Tokyo, Japan). Monoclonal antibodies to SREBP-1 (IgG-2A4) and SREBP-2 (IgG-7D4) were purified by protein A-Sepharose (Amersham Pharmacia Biotech) from the supernatant of hybridoma cell lines CRL 2121 and CRL 2198, respectively. These cell lines were purchased from American Tissue Culture Collection (Manassas, VA). Blood samples were obtained by cutting the tail end under feeding conditions. Triglyceride concentrations were measured by enzyme assays, determiner LTG (Kyowa Medics, Tokyo, Japan) and cholesterol by determiner LTC (Kyowa Medics). Comparisons of data from multiple groups were made by one-way analysis of variance. When they were significant, each group was compared with the others by Fisher's protected least significant difference test (Statview 4.0, Abacus Concepts). Comparisons of data from two experimental groups were made by unpaired Student's t test. Statistical significance is defined as p < 0.05. Values are mean ± S.E. The phenotypic comparison of mice fed three different diets for 5 months is summarized in Table I. In agreement with our previous data (20Ikemoto S. Takahashi M. Tsunoda N. Maruyama K. Itakura H. Ezaki O. Metabolism. 1996; 45: 1539-1546Abstract Full Text PDF PubMed Scopus (226) Google Scholar, 21Tsunoda N. Ikemoto S. Takahashi M. Maruyama K. Itakura H. Watanabe H. Goto N. Ezaki O. Metabolism. 1998; 47: 724-730Abstract Full Text PDF PubMed Scopus (36) Google Scholar, 24Ikemoto S. Takahashi M. Tsunoda N. Maruyama K. Itakura H. Kawanaka K. Tabata I. Higuchi M. Tange T. Yamamoto T. Ezaki O. Am. J. Physiol. 1997; 273: E37-E45Crossref PubMed Google Scholar), compared with high carbohydrate-fed mice, safflower oil-fed mice showed a 1.4-fold increase in body weight (p < 0.001), a 3.5-fold increase of parametrial white adipose tissue weight (p< 0.001), and a 1.5-fold increase of triglyceride accumulation in liver (p < 0.001). In contrast, fish oil-fed mice did not develop obesity or triglyceride accumulation in liver. However, the average energy intake among these three groups was not significantly different (7.4 ± 0.5, 7.7 ± 0.9, and 8.0 ± 0.5 kcal/mouse/day in carbohydrate, safflower oil, and fish oil-fed mice, respectively; n = 5). Fish oil feeding also affected lipid metabolism. Liver cholesterol and triglyceride concentrations from fish oil-fed mice were lower by 35% (p < 0.05) and 62% (p < 0.001), respectively, than those from safflower oil-fed mice. Plasma cholesterol and triglycerides concentrations from fish oil-fed mice were also lower by 32% (p < 0.001) and 36% (p = 0.19), respectively, than those from safflower oil-fed mice. Liver weight from fish oil-fed mice was 25% greater than that from safflower oil-fed mice (p < 0.001). This might be due to the well known effects of fish oil on peroxisomal proliferation (32Reddy J.K. Chu R. Ann. N. Y. Acad. Sci. 1996; 804: 176-201Crossref PubMed Scopus (100) Google Scholar).Table IPhenotypic comparison of high carbohydrate-, high safflower oil-, and high fish oil-fed miceCarbohydrateSafflower oilFish oilInitial body weight (g)17.8 ± 0.3 (15)17.6 ± 0.3 (13)17.5 ± 0.3 (16)Final body weight (g)24.2 ± 0.6 (15)33.3 ± 1.2 (13)1-aP < 0.001, safflower oil compared with carbohydrate; fish oil compared with safflower oil by Fisher's protected least significant difference test.22.6 ± 0.4 (16)1-aP < 0.001, safflower oil compared with carbohydrate; fish oil compared with safflower oil by Fisher's protected least significant difference test.WAT weight (g)0.6 ± 0.1 (14)2.1 ± 0.2 (13)1-aP < 0.001, safflower oil compared with carbohydrate; fish oil compared with safflower oil by Fisher's protected least significant difference test.0.4 ± 0.0 (16)1-aP < 0.001, safflower oil compared with carbohydrate; fish oil compared with safflower oil by Fisher's protected least significant difference test.Liver weight (g)1.0 ± 0.0 (15)1.2 ± 0.0 (13)1-bP < 0.01.1.5 ± 0.1 (16)1-aP < 0.001, safflower oil compared with carbohydrate; fish oil compared with safflower oil by Fisher's protected least significant difference test.Liver cholesterol (μmol/g)15.2 ± 1.6 (8)11.5 ± 0.8 (8)1-cP < 0.05.7.5 ± 0.8 (8)1-cP < 0.05.Liver triglyceride (μmol/g)51.4 ± 7.2 (8)76.3 ± 2.5 (8)1-aP < 0.001, safflower oil compared with carbohydrate; fish oil compared with safflower oil by Fisher's protected least significant difference test.29.3 ± 2.2 (8)1-aP < 0.001, safflower oil compared with carbohydrate; fish oil compared with safflower oil by Fisher's protected least significant difference test.Plasma cholesterol (mmol/liter)2.30 ± 0.08 (6)2.54 ± 0.09 (5)1.72 ± 0.09 (7)1-aP < 0.001, safflower oil compared with carbohydrate; fish oil compared with safflower oil by Fisher's protected least significant difference test.Plasma triglycerides (mmol/liter)0.20 ± 0.05 (6)0.28 ± 0.08 (5)0.18 ± 0.03 (7)Mice were killed at 5 months of feeding, and final body weight, wet parametrial white adipose tissue (WAT) and liver weight, and liver cholesterol and triglyceride were measured. Initial body weight is body weight at the beginning of diet experiments. Plasma triglycerides and cholesterol were measured under feeding conditions at 4 months of feeding. Each data point represents mean ± S.E. of 5–16 mice, and number of mice is shown in parentheses.1-a P < 0.001, safflower oil compared with carbohydrate; fish oil compared with safflower oil by Fisher's protected least significant difference test.1-b P < 0.01.1-c P < 0.05. Open table in a new tab Mice were killed at 5 months of feeding, and final body weight, wet parametrial white adipose tissue (WAT) and liver weight, and liver cholesterol and triglyceride were measured. Initial body weight is body weight at the beginning of diet experiments. Plasma triglycerides and cholesterol were measured under feeding conditions at 4 months of feeding. Each data point represents mean ± S.E. of 5–16 mice, and number of mice is shown in parentheses. Fig. 1 shows immunoblots of the precursor and mature SREBP-1 and -2 in liver of mice fed high carbohydrate, high safflower oil, and high fish oil diets for 5 months. Because antibody to SREBP-1 reacted to both SREBP-1a and -1c forms, we could not distinguish these two forms, and we used the noncommittal term SREBP-1. However, in mouse liver, the ratio of SREBP-1c to -1a transcripts is 9:1 (31Shimomura I. Shimano H. Horton J.D. Goldstein J.L. Brown M.S. J. Clin. Invest. 1997; 99: 838-845Crossref PubMed Scopus (642) Google Scholar), and thus the -1c form accounted for most of SREBP-1 observed on the immunoblots. In preliminary experiments, to confirm that 125- and 68-kDa proteins we observed are really the precursor and mature SREBP-1, fasting and refeeding experiments were conducted. Both precursor and mature SREBP-1 were decreased by 48 h fasting and increased above nonfasted levels by refeeding (data not shown). This confirms not only previous finding (33Horton J.D. Bashmakov Y. Shimomura I. Shimano H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5987-5992Crossref PubMed Scopus (540) Google Scholar) but also confirms that the bands at which we aimed were SREBP-1. There were no differences in the amount of SREBP-1 and -2 in both precursor and mature forms between carbohydrate- and safflower oil-fed mice. However, compared with safflower oil feeding, fish oil feeding reduced the amount of precursor SREBP-1 in membrane fraction by 90% and that of mature SREBP-1 in liver nuclei by 57% (Fig. 1 A). Fish oil feeding also reduced the precursor SREBP-2 by 65% but did not alter the amount of mature SREBP-2 (Fig. 1 B). In this experiment and others (23Sheng Z. Otani H. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 935-938Crossref PubMed Scopus (279) Google Scholar,33Horton J.D. Bashmakov Y. Shimomura I. Shimano H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5987-5992Crossref PubMed Scopus (540) Google Scholar), SREBP bands often appeared as closely spaced doublets, but the reason is not clear. To examine whether a marked decrease of SREBP-1 protein and a moderate decrease of SREBP-2 protein level by fish oil feeding reflected their mRNA levels, SREBP-1 and -2 mRNA levels in liver were measured by Northern blotting (Fig. 2). Parallel to their protein levels, compared with safflower oil-fed mice, fish oil-fed mice showed markedly decreased SREBP-1 mRNA level by 80% (p < 0.001). As for SREBP-2 mRNA, parallel to the amount of its precursor form, it also decreased by 47% in fish oil-fed mice (p < 0.001). The SREBP-1 gene uses two different promoters to produce two different transcripts, -1a and -1c, that differ in the first exon (15Hua X. W
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