A low fish oil inhibits SREBP-1 proteolytic cascade, while a high-fish-oil feeding decreases SREBP-1 mRNA in mice liver: relationship to anti-obesity
2003; Elsevier BV; Volume: 44; Issue: 2 Linguagem: Inglês
10.1194/jlr.m200289-jlr200
ISSN1539-7262
AutoresTeruyo Nakatani, Hyoun-Ju Kim, Yasushi Kaburagi, Kazuki Yasuda, Osamu Ezaki,
Tópico(s)Cancer, Lipids, and Metabolism
ResumoRodents fed fish oil showed less obesity with a reduction of triglyceride synthesis in liver, relative to other dietary oils, along with a decrease of mature form of sterol regulatory element binding protein-1 (SREBP-1) and activation of peroxisome proliferator-activated receptor α (PPARα). Decrease of mature SREBP-1 protein by fish oil feeding was due to either inhibition of SREBP-1 proteolytic cascade or to decrease of its mRNA. To clarify its mechanism and relation to antiobesity effect, mice were fed fish oil in a range from 10 to 60 energy percent (en%). Fish oil feeding decreased body weight and fat mass in a dose-dependent manner, in parallel with PPARα activation and a decrease of SREBP-1 mRNA. However, compared with 0 en% fish oil feeding, 10 en% fish oil feeding decreased mature SREBP-1 protein by 50% with concomitant decreases of lipogenic genes, while precursor SREBP-1 protein rather increased by 1.3-fold.These data suggest that physiological doses of fish oil feeding effectively decrease expression of liver lipogenic enzymes by inhibiting SREBP-1 proteolytic cascade, while substantial decrease of SREBP-1 expression is observed in its pharmacological doses, and that activation of PPARα rather than SREBP-1 decrease might be related to the antiobesity effect of fish oil feeding. Rodents fed fish oil showed less obesity with a reduction of triglyceride synthesis in liver, relative to other dietary oils, along with a decrease of mature form of sterol regulatory element binding protein-1 (SREBP-1) and activation of peroxisome proliferator-activated receptor α (PPARα). Decrease of mature SREBP-1 protein by fish oil feeding was due to either inhibition of SREBP-1 proteolytic cascade or to decrease of its mRNA. To clarify its mechanism and relation to antiobesity effect, mice were fed fish oil in a range from 10 to 60 energy percent (en%). Fish oil feeding decreased body weight and fat mass in a dose-dependent manner, in parallel with PPARα activation and a decrease of SREBP-1 mRNA. However, compared with 0 en% fish oil feeding, 10 en% fish oil feeding decreased mature SREBP-1 protein by 50% with concomitant decreases of lipogenic genes, while precursor SREBP-1 protein rather increased by 1.3-fold. These data suggest that physiological doses of fish oil feeding effectively decrease expression of liver lipogenic enzymes by inhibiting SREBP-1 proteolytic cascade, while substantial decrease of SREBP-1 expression is observed in its pharmacological doses, and that activation of PPARα rather than SREBP-1 decrease might be related to the antiobesity effect of fish oil feeding. In rodents, fish oil feeding prevents lipid accumulation in white adipose tissue (WAT) compared with other types of dietary oils (1Cunnane S.C. McAdoo K.R. Horrobin D.F. N-3 essential fatty acids decrease weight gain in genetically obese mice.Br. J. Nutr. 1986; 56: 87-95Google Scholar, 2Belzung F. Raclot T. Groscolas R. Fish oil n-3 fatty acids selectively limit the hypertrophy of abdominal fat depots in growing rats fed high-fat diets.Am. J. Physiol. 1993; 264: R1111-R1118Google Scholar, 3Hill J.O. Peters J.C. Lin D. Yakubu F. Greene H. Swift L. Lipid accumulation and body fat distribution is influenced by type of dietary fat fed to rats.Int. J. Obes. Relat. Metab. Disord. 1993; 17: 223-236Google Scholar, 4Ikemoto S. Takahashi M. Tsunoda N. Maruyama K. Itakura H. Ezaki O. High-fat diet-induced hyperglycemia and obesity in mice. Differential effects of dietary oils.Metabolism. 1996; 45: 1539-1546Google Scholar). It is known that triglyceride stored in fat cells is largely derived from circulation triglyceride, especially high-fat feeding. It is speculated that fish oil feeding limits triglyceride supply to adipose tissues by decreased VLDL synthesis in liver. Thus, increased fatty acid oxidation and inhibition of triglyceride synthesis in liver may play an important role in fish oil-induced body weight (BW) decrease (5Couet C. Delarue J. Ritz P. Antoine J-M. Lamisse F. Effect of dietary fish oil on body fat mass and basal fat oxidation in healthy adults.Int. J. Obes. 1997; 21: 637-643Google Scholar). It has been shown that n-3 fatty acids, which are abundant in fish oil in vivo or in cell culture inhibited the transcription of genes coding for lipogenesis enzymes such as fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), stearoyl-CoA desaturase (SCD), and S14 protein with decrease of mature form of sterol regulatory element binding (SREBP-1) protein (6Worgall T.S. Sturley S.L. Seo T. Osborne T.F. Deckelbaum R.J. Polyunsaturated fatty acids decrease expression of promoters with sterol regulatory elements by decreasing levels of mature sterol regulatory element-binding protein.J. Biol. Chem. 1998; 273: 25537-25540Google Scholar, 7Xu J. Nakamura M.T. Cho H.P. Clarke S.D. Sterol regulatory element binding protein-1 expression is suppressed by dietary polyunsaturated fatty acids. A mechanism for the coordinate suppression of lipogenic genes by polyunsaturated fats.J. Biol. Chem. 1999; 274: 23577-23583Google Scholar, 8Kim H-J. Takahashi M. Ezaki O. Fish oil feeding decreases mature sterol regulatory element-binding protein 1 (SREBP-1) by down-regulation of SREBP-1c mRNA in mouse liver. A possible mechanism for down-regulation of lipogenic enzyme mRNAs.J. Biol. Chem. 1999; 274: 25892-25898Google Scholar, 9Mater M.K. Thelen A.P. Pan D.A. Jump D.B. Sterol response element-binding protein 1c (SREBP1c) is involved in the polyunsaturated fatty acid suppression of hepatic S14 gene transcription.J. Biol. Chem. 1999; 274: 32725-32732Google Scholar, 10Yahagi N. Shimano H. Hasty A.H. Amemiya-Kudo M. Okazaki H. Tamura Y. Iizuka Y. Shionoiri F. Ohashi K. Osuga J. Harada K. Gotoda T. Nagai R. Ishibashi S. Yamada N. A crucial role of sterol regulatory element-binding protein-1 in the regulation of lipogenic gene expression by polyunsaturated fatty acids.J. Biol. Chem. 1999; 274: 35840-35844Google Scholar, 11Hannah V.C. Ou J. Luong A. Goldstein J.L. Brown M.S. Unsaturated fatty acids down-regulate SREBP isoforms 1a and 1c by two mechanisms in HEK-293 cells.J. Biol. Chem. 2001; 276: 4365-4372Google Scholar). On the other hand, n-3 fatty acids increased the transcription of the regulatory enzymes of fatty acid oxidation, such as acyl-CoA oxidase (ACO), medium-chain acyl-CoA dehydrogenase (MCAD), lipoprotein lipase (LPL), fatty acid binding protein, acyl-CoA synthetase (ACS), uncoupling protein-2 (UCP2), and carnitine palmitoyltransferase-1, with activation of peroxisome proliferator-activated receptor (PPAR)α (12Reddy J.K. Mannaerts G.P. Peroxisomal lipid metabolism.Annu. Rev. Nutr. 1994; 14: 343-370Google Scholar, 13Schoonjans K. Staels B. Auwerx J. The peroxisome proliferator activated receptors (PPARS) and their effects on lipid metabolism and adipocyte differentiation.Biochim. Biophys. Acta. 1996; 1302: 93-109Google Scholar, 14Krey G. Braissant O. L'Horset F. Kalkhoven E. Perroud M. Parker M.G. Wahli W. Fatty acids, eicosanoids, and hypolipidemic agents identified as ligands of peroxisome proliferator-activated receptors by coactivator-dependent receptor ligand assay.Mol. Endocrinol. 1997; 11: 779-791Google Scholar, 15Latruffe N. Vamecq J. Peroxisome proliferators and peroxisome proliferator activated receptors (PPARs) as regulators of lipid metabolism.Biochimie. 1997; 79: 81-94Google Scholar, 16Nakatani T. Tsuboyama-Kasaoka N. Takahashi M. Miura S. Ezaki O. Mechanism for peroxisome proliferator-activated receptor-alpha activator-induced up-regulation of UCP2 mRNA in rodent hepatocytes.J. Biol. Chem. 2002; 277: 9562-9569Google Scholar).SREBPs are master transcription factors that regulate fatty acid and cholesterol metabolism in liver (17Horton J.D. Goldstein J.L. Brown M.S. SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver.J. Clin. Invest. 2002; 109: 1125-1131Google Scholar). In sterol depletion, SREBPs are cleaved and become mature forms to bind sterol regulatory elements (SREs) (18Briggs M.R. Yokoyama C. Wang X. Brown M.S. Goldstein J.L. Nuclear protein that binds sterol regulatory element of low density lipoprotein receptor promoter. I. Identification of the protein and delineation of its target nucleotide sequence.J. Biol. Chem. 1993; 268: 14490-14496Google Scholar, 19Wang X. Briggs M.R. Hua X. Yokoyama C. Goldstein J.L. Brown M.S. Nuclear protein that binds sterol regulatory element of low density lipoprotein receptor promoter. Purification and characterization.J. Biol. Chem. 1993; 268: 14497-14504Google Scholar) and/or E-box sequences (20Kim J.B. Spotts G.D. Halvorsen Y.D. Shih H.M. Ellenberger T. Towle H.C. Spiegelman B.M. Dual DNA binding specificity of ADD1/SREBP1 controlled by a single amino acid in the basic helix-loop-helix domain.Mol. Cell. Biol. 1995; 15: 2582-2588Google Scholar) and then activate the target gene expression. SREBP cleavage-activating protein (SCAP) escorts the SREBPs to the Golgi complex, where they are cleaved sequentially by two membrane-bound proteases designated Site-1 protease (S1P) and Site-2 protease (S2P), thereby liberating the NH2-terminal domain so that it can enter the nucleus (21Brown M.S. Goldstein J.L. A proteolytic pathway that controls the cholesterol content of membranes, cells, and blood.Proc. Natl. Acad. Sci. USA. 1999; 96: 11041-11048Google Scholar). 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; SREBP-1c plays a crucial role in the dietary regulation of the most hepatic lipogenic genes, whereas SREBP-2 is actively involved in the transcription of cholesterol biosynthetic genes (22Shimano H. Horton J.D. Hammer R.E. Shimomura I. Brown M.S. Goldstein J.L. Overproduction of cholesterol and fatty acids causes massive liver enlargement in transgenic mice expressing truncated SREBP-1a.J. Clin. Invest. 1996; 98: 1575-1584Google Scholar, 23Shimano H. Horton J.D. Shimomura I. Hammer R.E. Brown M.S. Goldstein J.L. Isoform 1c of sterol regulatory element binding protein is less active than isoform 1a in livers of transgenic mice and in cultured cells.J. Clin. Invest. 1997; 99: 846-854Google Scholar, 24Horton J.D. Shimomura I. Brown M.S. Hammer R.E. Goldstein J.L. Shimano H. Activation of cholesterol synthesis in preference to fatty acid synthesis in liver and adipose tissue of transgenic mice overproducing sterol regulatory element-binding protein-2.J. Clin. Invest. 1998; 101: 2331-2339Google Scholar).Consistent with the notion that SREBP-1c is a dominant regulator for lipogenesis, several reports have been published demonstrating that administration of polyunsaturated fatty acid (PUFA) such as n-6 and n-3 fatty acids suppresses mature SREBP-1 protein and/or its mRNA in cultured cells and rodent liver tissues (6Worgall T.S. Sturley S.L. Seo T. Osborne T.F. Deckelbaum R.J. Polyunsaturated fatty acids decrease expression of promoters with sterol regulatory elements by decreasing levels of mature sterol regulatory element-binding protein.J. Biol. Chem. 1998; 273: 25537-25540Google Scholar, 7Xu J. Nakamura M.T. Cho H.P. Clarke S.D. Sterol regulatory element binding protein-1 expression is suppressed by dietary polyunsaturated fatty acids. A mechanism for the coordinate suppression of lipogenic genes by polyunsaturated fats.J. Biol. Chem. 1999; 274: 23577-23583Google Scholar, 8Kim H-J. Takahashi M. Ezaki O. Fish oil feeding decreases mature sterol regulatory element-binding protein 1 (SREBP-1) by down-regulation of SREBP-1c mRNA in mouse liver. A possible mechanism for down-regulation of lipogenic enzyme mRNAs.J. Biol. Chem. 1999; 274: 25892-25898Google Scholar, 9Mater M.K. Thelen A.P. Pan D.A. Jump D.B. Sterol response element-binding protein 1c (SREBP1c) is involved in the polyunsaturated fatty acid suppression of hepatic S14 gene transcription.J. Biol. Chem. 1999; 274: 32725-32732Google Scholar, 10Yahagi N. Shimano H. Hasty A.H. Amemiya-Kudo M. Okazaki H. Tamura Y. Iizuka Y. Shionoiri F. Ohashi K. Osuga J. Harada K. Gotoda T. Nagai R. Ishibashi S. Yamada N. A crucial role of sterol regulatory element-binding protein-1 in the regulation of lipogenic gene expression by polyunsaturated fatty acids.J. Biol. Chem. 1999; 274: 35840-35844Google Scholar, 11Hannah V.C. Ou J. Luong A. Goldstein J.L. Brown M.S. Unsaturated fatty acids down-regulate SREBP isoforms 1a and 1c by two mechanisms in HEK-293 cells.J. Biol. Chem. 2001; 276: 4365-4372Google Scholar). However, the mechanisms of decrease of mature SREBP-1 protein were diverse; in culture cells, PUFA addition inhibited the SREBP-1 proteolytic cascade in CV-1 and human embryonic kidney (HEK)-293 cells and down-regulated SREBP-1 mRNA in HEK-239 cells (6Worgall T.S. Sturley S.L. Seo T. Osborne T.F. Deckelbaum R.J. Polyunsaturated fatty acids decrease expression of promoters with sterol regulatory elements by decreasing levels of mature sterol regulatory element-binding protein.J. Biol. Chem. 1998; 273: 25537-25540Google Scholar, 11Hannah V.C. Ou J. Luong A. Goldstein J.L. Brown M.S. Unsaturated fatty acids down-regulate SREBP isoforms 1a and 1c by two mechanisms in HEK-293 cells.J. Biol. Chem. 2001; 276: 4365-4372Google Scholar). In primary culture of rat hepatocytes, PUFA suppressed SREBP-1 mRNA by accelerating transcript decay (25Xu J. Teran-Garcia M. Park J.H.Y. Nakamura M.T. Clarke S.D. Polyunsaturated fatty acids suppress hepatic sterol regulatory element-binding protein-1 expression by accelerating transcript decay.J. Biol. Chem. 2001; 276: 9800-9807Google Scholar). In rodent studies, 25 energy percent (en%) menhaden fish oil, 60 en% tuna fish oil, and 25 en% fish oil feeding decreased SREBP-1 mRNA by 60, 86, and 80%, respectively, compared with their appropriate controls (7Xu J. Nakamura M.T. Cho H.P. Clarke S.D. Sterol regulatory element binding protein-1 expression is suppressed by dietary polyunsaturated fatty acids. A mechanism for the coordinate suppression of lipogenic genes by polyunsaturated fats.J. Biol. Chem. 1999; 274: 23577-23583Google Scholar, 8Kim H-J. Takahashi M. Ezaki O. Fish oil feeding decreases mature sterol regulatory element-binding protein 1 (SREBP-1) by down-regulation of SREBP-1c mRNA in mouse liver. A possible mechanism for down-regulation of lipogenic enzyme mRNAs.J. Biol. Chem. 1999; 274: 25892-25898Google Scholar, 9Mater M.K. Thelen A.P. Pan D.A. Jump D.B. Sterol response element-binding protein 1c (SREBP1c) is involved in the polyunsaturated fatty acid suppression of hepatic S14 gene transcription.J. Biol. Chem. 1999; 274: 32725-32732Google Scholar), while 40 en% sardine or tuna fish oil decreased mature SREBP-1 protein but not its mRNA (10Yahagi N. Shimano H. Hasty A.H. Amemiya-Kudo M. Okazaki H. Tamura Y. Iizuka Y. Shionoiri F. Ohashi K. Osuga J. Harada K. Gotoda T. Nagai R. Ishibashi S. Yamada N. A crucial role of sterol regulatory element-binding protein-1 in the regulation of lipogenic gene expression by polyunsaturated fatty acids.J. Biol. Chem. 1999; 274: 35840-35844Google Scholar).In this study, to examine the mechanism of decrease of mature SREBP-1 by fish oil feeding in greater detail, mice were given a different amount of fish oils in place of safflower oil and then premature and mature SREBP-1 protein conten and SREBP-1 mRNA levels in liver were measured. In addition, to examine the correlation between body fat and alteration of activities of SREBP-1 and PPARα, body composition was assessed by dual-energy X-ray absorptiometry (DEXA) and expression levels of target genes of SREBP-1 and PPARα were examined.EXPERIMENTAL PROCEDURESAnimalsFemale 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 12-h light/12-h dark cycle and maintained at a constant temperature of 22°C.DietMice were divided into seven groups (n = 5–6 in each group). All groups of mice were fed a high-fat diet containing 14 en% carbohydrate, 60 en% dietary oil, and 26 en% protein. The dietary fats are a mixture of safflower oil and fish oil. The amount of fish oil increased from 0 to 60 en% with concomitant decrease of safflower oil from 60 to 0 en%, maintaining the total amount of fat constant at 60 en%. Detailed composition of the experimental diets is described in Table 1. Fatty acid compositions of dietary oils were measured by gas-liquid chromatography. Safflower oil (high-oleic type) contained 46% oleic acid (18:1n-9) and 45% linoleic acid (18:2n-6) from total fatty acids; fish oil contained 7% eicosapentaenoic acid (EPA, 20:5n-3) and 24% docosahexaenoic acid (DHA, 22:6n-3). The materials and methods of diet preparation were the same as those used in our previous studies (8Kim H-J. Takahashi M. Ezaki O. Fish oil feeding decreases mature sterol regulatory element-binding protein 1 (SREBP-1) by down-regulation of SREBP-1c mRNA in mouse liver. A possible mechanism for down-regulation of lipogenic enzyme mRNAs.J. Biol. Chem. 1999; 274: 25892-25898Google Scholar, 16Nakatani T. Tsuboyama-Kasaoka N. Takahashi M. Miura S. Ezaki O. Mechanism for peroxisome proliferator-activated receptor-alpha activator-induced up-regulation of UCP2 mRNA in rodent hepatocytes.J. Biol. Chem. 2002; 277: 9562-9569Google Scholar, 26Tsuboyama-Kasaoka N. Takahashi M. Kim H. Ezaki O. Up-regulation of liver uncoupling protein-2 mRNA by either fish oil feeding or fibrate administration in mice.Biochem. Biophys. Res. Commun. 1999; 257: 879-885Google Scholar). Fish oil was provided by NOF (Tokyo, Japan). Food consumption was measured for three consecutive days. The mean food intake per day was estimated by subtracting the food weight of the day from the initial food weight of the previous day and dividing by the number of mice housed in the cage. Thus, standard error for food intake shown in Fig. 1Awas variation of daily intake, but not from the individual mouse. Mice were fed each diet for 1 or 13 weeks and were anesthetized at about 10 AM by intraperitoneal injection of pentobarbital sodium (0.08 mg/g BW, Nembutal, Abbot, North Chicago, IL). Liver, gastrocnemius, and parametrial WAT were isolated immediately, weighed, and homogenized in guanidine-thiocyanate, and RNA was prepared by the method described by Chirgwin et al. (27Chirgwin J.M. Przybyla A.E. MacDonald R.J. Rutter W.J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease.Biochemistry. 1979; 18: 5294-5299Google Scholar). A part of liver of each mouse was immediately homogenized to obtain membrane fractions and nuclear extracts (28Sheng Z. Otani H. Brown M.S. Goldstein J.L. Independent regulation of sterol regulatory element-binding proteins 1 and 2 in hamster liver.Proc. Natl. Acad. Sci. USA. 1995; 92: 935-938Google Scholar).TABLE 1Composition of the experimental dietsExperimental Diet0 en%aPercent of energy from total energy intake. Fish Oil10 en% Fish Oil20 en% Fish Oil30 en% Fish Oil40 en% Fish Oil50 en% Fish Oil60 en% Fish OilFish oil (%bPercent of weight from total food weight.)05.51116.52227.533.5Safflower oil (%)33.52822.51711.560Casein (%)29292929292929Sucrose (%)23.2923.2923.2923.2923.2923.2923.29Vitamin mix (%)1.451.451.451.451.451.451.45Mineral mix (%)5.085.085.085.085.085.085.08Cellulose powder (%)7.257.257.257.257.257.257.25l-Cystein (%)0.440.440.440.440.440.440.44Fat energy (en%)60.560.960.960.760.760.660.6DHA (en%)02.44.87.29.61214.4EPA (en%)00.71.42.12.83.54.2DHA, docosahexaenoic; EPA, eicosapentaenoic acid.a Percent of energy from total energy intake.b Percent of weight from total food weight. Open table in a new tab ImmunoblottingPooled liver membranes and nuclear extracts from five mice of each group were prepared by the method described by Sheng et al. (28Sheng Z. Otani H. Brown M.S. Goldstein J.L. Independent regulation of sterol regulatory element-binding proteins 1 and 2 in hamster liver.Proc. Natl. Acad. Sci. USA. 1995; 92: 935-938Google Scholar). The same amount of protein from each fraction was applied to 8% SDS-PAGE transferred to Hybond-P membranes (Amersham Pharmacia Biotech, Tokyo, Japan). Immunoblot analysis was performed by using Enhanced Chemiluminescence Western Blotting Detection System kit (Amersham Pharmacia Biotech). Membrane sheets were first incubated with antibody against SREBP-1 for 1 h at 22°C, 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) were purified by protein A sepharose (Amersham Pharmacia Biotech) from the supernatant of hybridoma cell lines CRL 2121. These cell lines were purchased from American Tissue Culture Collection (Rockville, MD).Preparation of cDNA probe for Northern blotThe cDNA fragments for mouse SREBP-1, UCP2, ACO, and MCAD were obtained by PCR as described in our previous studies (8Kim H-J. Takahashi M. Ezaki O. Fish oil feeding decreases mature sterol regulatory element-binding protein 1 (SREBP-1) by down-regulation of SREBP-1c mRNA in mouse liver. A possible mechanism for down-regulation of lipogenic enzyme mRNAs.J. Biol. Chem. 1999; 274: 25892-25898Google Scholar, 29Tsuboyama-Kasaoka N. Tsunoda N. Maruyama K. Takahashi M. Kim H-J. Ikemoto S. Ezaki O. Up-regulation of uncoupling protein 3 (UCP3) mRNA by exercise training and down-regulation of UCP3 by denervation in skeletal muscles.Biochem. Biophys. Res. Commun. 1998; 247: 498-503Google Scholar, 30Ikeda S. Miyazaki H. Nakatani T. Kai Y. Kamei Y. Miura S. Tsuboyama-Kasaoka N. Ezaki O. Up-regulation of SREBP-1c and lipogenic genes in skeletal muscles after exercise training.Biochem. Biophys. Res. Commun. 2002; 296: 395-400Google Scholar). A part of cDNAs of mouse SCAP and S1P were obtained by PCR from first strand DNA using mouse liver total RNA. First strand cDNA was prepared using a Ready-To-Go T-Primed First Strand kit (Amersham Pharmacia Biotech). The PCR primers used were as follows: SCAP (XM033294), 5′ primer, 5′-TGTGAAGGATTACTTCGCC-3′ and 3′ primer, 5′-CCAGTCATTCTGCCAGAAGT-3′; S1P (NM019709), 5′ primer, 5′-CAACTGTGGTGGAGTACGAA-3′ and 3′ primer, 5′-CGACCTGGCGAGGAA-3′. PCR was performed with a Taq DNA polymerase (Takara, Shiga, Japan). The cDNA probes for rat ACS was kindly provided by Dr. T. Yamamoto at Tohoku University, mouse SCD-1 by Dr. Daniel M. Lane at Johns Hopkins University, and rat ACC and rat FAS by Dr. N. Iritani at Tezukayama Gakuin College. These cDNA were used as probes for Northern blotting.Body composition analysisAt 13 weeks of feeding, mice were anesthetized by pentobarbital sodium (0.08 mg/g BW, Nembutal) and scanned with a Lunar PIXI mus2 densitometer (31Nagy T.R. Clair A-L. Precision and accuracy of dual-energy x-ray absorptiometry for determining in vivo body composition of mice.Obes. Res. 2000; 8: 392-398Google Scholar) (Lunar Corporation, Madison, WI).Northern blottingAliquots of total RNA (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, Boston, MA). After transfer and UV cross-linking, RNA blots were stained with methylene blue to locate 28S and 18S rRNAs and to ascertain the amount of loaded RNAs (32Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982: 206Google Scholar). The membranes were hybridized with each cDNA probe labeled with [α-32P]dCTP (NEN) by a Megaprime DNA labeling kit (Amersham Pharmacia Biotech). The membranes were hybridized overnight at 42°C in hybridization buffer subsequently washed two times for 20 min at 50°C with 1XSSC, 0.1% SDS and one time for 20 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 (PSL).Statistical analysisComparisons of data from multiple groups were made by one-way ANOVA. When they were significant, each group was compared with the others by Fisher's protected least significant difference (PLSD) test (Statview 4.0, Abacus Concepts). Statistical significance is defined as P < 0.05. Values are mean ± SE.RESULTSBW, parametrial WAT, and liver weight in mice fed 0, 10, 20, 30, 40, 50, and 60 en% fish oil for 1 weekAccording to our previous data (4Ikemoto S. Takahashi M. Tsunoda N. Maruyama K. Itakura H. Ezaki O. High-fat diet-induced hyperglycemia and obesity in mice. Differential effects of dietary oils.Metabolism. 1996; 45: 1539-1546Google Scholar, 8Kim H-J. Takahashi M. Ezaki O. Fish oil feeding decreases mature sterol regulatory element-binding protein 1 (SREBP-1) by down-regulation of SREBP-1c mRNA in mouse liver. A possible mechanism for down-regulation of lipogenic enzyme mRNAs.J. Biol. Chem. 1999; 274: 25892-25898Google Scholar, 26Tsuboyama-Kasaoka N. Takahashi M. Kim H. Ezaki O. Up-regulation of liver uncoupling protein-2 mRNA by either fish oil feeding or fibrate administration in mice.Biochem. Biophys. Res. Commun. 1999; 257: 879-885Google Scholar), compared with high-carbohydrate-fed mice, 60 en% safflower oil-fed mice showed increases of BW and WAT weight, while 60 en% fish oil feeding increased energy expenditure and did not develop obesity. To examine whether the antiobesity effect by 60 en% fish oil feeding is also observed in lower fish oil feeding, mice were fed 0, 10, 20, 30, 40, 50, and 60 en% fish oil for 1 week under 60 en% high-fat diet feeding conditions. Although there were no significant differences in the average energy intake of mice fed each diet (Fig. 1A), even in a relatively short term feeding period there were significant decreases of BW in 40–60 en% fish oil feeding compared with 0 en% fish oil feeding (Fig. 1B). Corresponding with BW decreases, wet weight of parametrial WAT in fish-oil-fed mice decreased in a dose-dependent manner and reached significance in more than 50 en% feeding (Fig. 1C). Liver weight from fish-oil-fed mice also increased in a dose-dependent manner and reached significance in more than 20 en% feeding (Fig. 1D). This might be due to the well-known effects of fish oil on peroxisomal proliferation (33Reddy J.K. Chu R. Peroxisome proliferator-induced pleiotropic responses. Pursuit of a phenomenon.Ann. N. Y. Acad. Sci. 1996; 804: 176-201Google Scholar), and also suggested that a dose-dependent intake of fish oils might have occurred in this experiment. The increase of liver weight was not accompanied by liver damage, since there were no increases of transaminases in 60 en% fish oil feeding (data not shown).SREBP-1 protein in membrane fractions (precursor form) and nuclear extracts (mature form) from livers of mice fed 0, 10, 20, 30, 40, 50, and 60 en% fish oil for 1 weekIn our previous study, 60 en% fish oil feeding decreased mature SREBP-1c with decrease of its mRNA (8Kim H-J. Takahashi M. Ezaki O. Fish oil feeding decreases mature sterol regulatory element-binding protein 1 (SREBP-1) by down-regulation of SREBP-1c mRNA in mouse liver. A possible mechanism for down-regulation of lipogenic enzyme mRNAs.J. Biol. Chem. 1999; 274: 25892-25898Google Scholar). To examine whether a lower dose of fish oil feeding also decreases mature SREBP-1c protein, the precursor and mature SREBP-1 protein in liver of mice fed 0, 10, 20, 30, 40, 50, and 60 en% fish oil for 1 week were measured by immunoblotting (Fig. 2A, B). Since antibody to SREBP-1 reacted to both SREBP-1a and -1c forms, we could not distinguish these two forms and thus used the noncommittal term SREBP-1. In mouse liver, the ratio of SREBP-1c to 1a transcripts is 9 to 1 (34Shimomura I. Shimano H. Horton J.D. Goldstein J.L. Brown M.S. Differential expression of exons 1a and 1c in mRNAs for sterol regulatory element binding protein-1 in human and mouse organs and cultured cells.J. Clin. Invest. 1997; 99: 838-845Google Scholar), and thus 1c form accounted for most of SREBP-1 observed on the immunoblots. In preliminary experiments, to confirm that 125 kDa and 68 kDa proteins we observed are really the precursor and mature SREBP-1, fasting and refeeding experiments were conducted (data not shown).Fig. 2Immunoblot analysis of SREBP-1 in membrane fractions (A) and nuclear extracts (B) and its ratio (C) from livers of mice fed 0, 10, 20, 30, 40, 50, and 60 en% fish oil for 1 week. Mice were fed on a 60 en% high-fat diet with various doses of fish oil in place of safflower oil. Mice were killed at 1 week feeding. For each group, livers from mice (n = 5) were pooled, and aliquots of membrane fractions (90 μg protein) and nuclear extracts (30 μg protein) were subjected to 8% SDS-PAGE and electrophoretically transferred to Hybond-P membranes. The membranes were incubated with 5 μg/ml of mouse monoclonal antibody IgG 2A4 against amino acid 301–407 of human SREBP-1. Immunoblot analysis was carried out by the enhanced chemiluminescence system. Filters were exposed to film for 60 s. The bands were quantified by scanning with Canon IX-4015 (Canon Inc., Tokyo, Japan). A typical autoradiogram of SREBP-1 from membrane fractions (A) and nuclear extracts (B) and their relative levels are shown. P and M denote the precursor and mature forms of SREBP-1. Each value represents mean ± SE of three independent experiments. In panel C, the ratio of the mature SREBP-1 to its precursor is plotted. *P < 0.05, **P < 0.01, ***P < 0.001, various doses of fish oil feeding compared with 0 en% fish oil feeding by Fisher's PLSD test.View Large Image
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