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Single Nucleotide Polymorphism (–468 Gly to Ala) at the Promoter Region of Sterol Regulatory Element-binding Protein-1c Associates with Genetic Defect of Fructose-induced Hepatic Lipogenesis

2004; Elsevier BV; Volume: 279; Issue: 28 Linguagem: Inglês

10.1074/jbc.m309449200

1083-351X

Ryoko Nagata, Yoshihiko Nishio, Osamu Sekine, Yoshio Nagai, Yasuhiro Maeno, Satoshi Ugi, Hiroshi Maegawa, Atsunori Kashiwagi,

Cholesterol and Lipid Metabolism

To evaluate the genetic susceptibility to metabolic disorders induced by high fructose diet, we investigated the metabolic characteristics in 10 strains of inbred mice and found that they were separated into CBA and DBA groups according to the response to high fructose diet. The hepatic mRNA expression of the sterol regulatory element-binding protein-1 (SREBP-1) in CBA/JN was remarkably enhanced by high fructose diet but not in DBA/2N. Similar results were observed in primary hepatocytes after exposure to fructose. The nucleotide sequence at –468 bp from the putative starting point of the SREBP-1c gene was adenine in the DBA group while it was guanine in the CBA group. In hepatocytes from CBA/JN, the activity of CBA-SREBP-1c promoter was significantly increased by 2.4- and 2.2-fold, in response to 30 mm fructose or 10 nm insulin, respectively, whereas the activity of DBA-SREBP-1c promoter responded to insulin but not to fructose. In hepatocytes from DBA/2N, both types of SREBP-1c promoter activities in response to insulin were attenuated. Furthermore, electrophoretic mobility shift assay revealed an unidentified nuclear protein bound to the oligonucleotides made from the region between –453 to –480 bp of the SREBP-1c promoter of CBA/JN but not to the probe from DBA/2N. Thus, in DBA/2N, the reduced mRNA expression of SREBP-1 after fructose refeeding appeared to associate with two independent mechanisms, 1) loss of binding of unidentified proteins to the region between –453 to –480 bp of the SREBP-1c promoter and 2) impaired insulin stimulation of SREBP-1c promoter activity. To evaluate the genetic susceptibility to metabolic disorders induced by high fructose diet, we investigated the metabolic characteristics in 10 strains of inbred mice and found that they were separated into CBA and DBA groups according to the response to high fructose diet. The hepatic mRNA expression of the sterol regulatory element-binding protein-1 (SREBP-1) in CBA/JN was remarkably enhanced by high fructose diet but not in DBA/2N. Similar results were observed in primary hepatocytes after exposure to fructose. The nucleotide sequence at –468 bp from the putative starting point of the SREBP-1c gene was adenine in the DBA group while it was guanine in the CBA group. In hepatocytes from CBA/JN, the activity of CBA-SREBP-1c promoter was significantly increased by 2.4- and 2.2-fold, in response to 30 mm fructose or 10 nm insulin, respectively, whereas the activity of DBA-SREBP-1c promoter responded to insulin but not to fructose. In hepatocytes from DBA/2N, both types of SREBP-1c promoter activities in response to insulin were attenuated. Furthermore, electrophoretic mobility shift assay revealed an unidentified nuclear protein bound to the oligonucleotides made from the region between –453 to –480 bp of the SREBP-1c promoter of CBA/JN but not to the probe from DBA/2N. Thus, in DBA/2N, the reduced mRNA expression of SREBP-1 after fructose refeeding appeared to associate with two independent mechanisms, 1) loss of binding of unidentified proteins to the region between –453 to –480 bp of the SREBP-1c promoter and 2) impaired insulin stimulation of SREBP-1c promoter activity. Single nucleotide polymorphism (-468 Gly to Ala) at the promoter region of sterol regulatory element-binding protein-1c associates with genetic defect of fructose-induced hepatic lipogenesis. Vol. 279 (2004) 29031-29042Journal of Biological ChemistryVol. 279Issue 35PreviewThe title was incorrect. The correct title should be: Full-Text PDF Open Access The prevalence of type 2 diabetes has been rising over the last few decades (1Zimmet P. Alberti K.G.M.M. Shaw J. Nature. 2001; 414: 782-787Crossref PubMed Scopus (4545) Google Scholar). An important correlate of this alarming phenomenon is the recent increase in prevalence among the population of metabolic syndrome, which consists of several metabolic disorders including hyperlipidemia, visceral obesity, impaired glucose tolerance, and hyperinsulinemia (2Reaven G.M. Diabetes. 1988; 37: 1595-1607Crossref PubMed Google Scholar, 3Kaplan N.M. Arch. Intern. Med. 1989; 149: 1514-1520Crossref PubMed Google Scholar). Metabolic syndrome may be induced by such environmental factors as increased consumption of processed calorie-dense foods and/or lack of exercise in certain people who have as yet an unidentified genetic predisposition. A high fructose diet in rats induces metabolic derangements similar to those that accompany with metabolic syndrome (4Shinozaki K. Kashiwagi A. Nishio Y. Okamura T. Yoshida Y. Masada M. Toda N. Kikkawa R. Diabetes. 1999; 48: 2437-2445Crossref PubMed Scopus (310) Google Scholar, 5Shinozaki K. Nishio Y. Okamura T. Yoshida Y. Maegawa H. Kojima H. Masada M. Toda N. Kikkawa R. Kashiwagi A. Circ. Res. 2000; 87: 566-573Crossref PubMed Scopus (216) Google Scholar), and rats fed high fructose diet have been used as animal models for metabolic syndrome (6Sleder J. Chen Y.D. Cully M.D. Reaven G.M. Metabolism. 1980; 29: 303-305Abstract Full Text PDF PubMed Scopus (89) Google Scholar, 7Nagai Y. Nishio Y. Nakamura T. Maegawa H. Kikkawa R. Kashiwagi A. Am. J. Physiol. Endocrinol. Metab. 2002; 282: E1180-E1190Crossref PubMed Scopus (172) Google Scholar). We previously reported that the high fructose diet up-regulated hepatic expression of the sterol regulatory element-binding protein-1 (SREBP-1), 1The abbreviations used are: SREBP, sterol regulatory element (SRE)-binding protein; AP4, activator protein 4; EMSA, electrophoretic mobility sift assay; FAS, fatty acid synthase; PPARα, peroxisome proliferator-activated receptor α; OCT-1, octamer transcription factor 1; NS, not significant.1The abbreviations used are: SREBP, sterol regulatory element (SRE)-binding protein; AP4, activator protein 4; EMSA, electrophoretic mobility sift assay; FAS, fatty acid synthase; PPARα, peroxisome proliferator-activated receptor α; OCT-1, octamer transcription factor 1; NS, not significant. a key transcription factor for hepatic expression of lipogenic enzymes, but down-regulated the expression of peroxisome proliferator-activated receptor α (PPARα), a ligand-activated nuclear receptor for expression of enzymes involved in fatty acid oxidation (7Nagai Y. Nishio Y. Nakamura T. Maegawa H. Kikkawa R. Kashiwagi A. Am. J. Physiol. Endocrinol. Metab. 2002; 282: E1180-E1190Crossref PubMed Scopus (172) Google Scholar). These alterations in the expression of the transcription factors may play a central role in the pathogenesis of metabolic derangements in rats fed a high fructose diet. The molecular mechanisms, however, by which fructose induces the SREBP-1 gene expression and suppresses the PPARα gene expression are not yet fully understood. Likewise, the genetic predisposition to metabolic disorders in response to a high fructose diet has not yet been determined. Therefore, we hypothesized that inducibility of SREBP-1 and PPARα expressions in response to a high fructose diet could be genetically determined. To investigate the genetic heterogeneity in the regulation of hepatic SREBP-1 and PPARα gene expressions after consumption of high fructose diet, we selected two inbred mouse strains, one of which is highly responsive to a high fructose diet. We found that there were marked differences in CBA/JN mice and DBA/2N mice between the hepatic mRNA expressions of SREBP-1 in response to a high fructose diet. We also observed a single nucleotide mutation in DBA/2N mice from guanine to adenine at –468 bp from the putative starting point of the SREBP-1c gene, which caused impaired activation of SREBP-1c gene transcription in response to fructose. These results indicate that the loss of nuclear protein binding to the specific site at the promoter region of SREBP-1c may cause impaired hepatic lipogenesis in mice. Materials—All materials were of reagent grade and were purchased from Nacalai Tesque (Kyoto, Japan) or Sigma unless otherwise indicated. Animals—Five-week-old male mice of CBA/JN, DBA/2N, C57BL/6N, C57BL/6J, C3H/HeJ, and DBA/1JN strains were purchased from CLEA Japan, Inc. (Tokyo, Japan). Mice of C57BL/6 strain were purchased from Charles River Japan (Kanagawa, Japan). Mice of C3H/He, C3H/HeN, and BALB/c strains were purchased from SLC Japan, Inc. (Shizuoka, Japan). The mice were housed in an environmentally controlled room with a 12-h light/dark cycle and provided free access to a laboratory diet and water. The animals were divided into two groups (a control diet group and a high fructose diet group) and pair-fed for 8 weeks. The control diet (Oriental Yeast, Tokyo, Japan) consisted of 58% carbohydrate (no fructose), 12% fat, and 30% protein (energy percent of diet). The high fructose diet (oriental yeast) contained 67% carbohydrate (98% of which was fructose), 13% fat, and 20% protein. The day before the experiment we withdrew the food from all animals at 20:00. The next day half of the animals were fed either the control diet or the high fructose diet in the dark from 6:00 to 8:00 before the experiment, and the other half were kept in the fasting state. At 10:00, after 10 mg/kg intraperitoneal pentobarbital injection and under deep anesthetization, the liver and the epididymal fat were excised, immediately frozen in liquid nitrogen, and stored at –80 °C. Blood samples were also taken for several blood tests such as triglyceride, total cholesterol, blood sugar, and insulin. RNA and DNA were extracted from the frozen samples. Shiga University of Medical Science Animal Care Committees approved all experiments. Mouse Primary Hepatocytes—Mouse primary hepatocytes were isolated by the collagenase method with minor modifications (9Gorski K. Carneiro M. Schibler U. Cell. 1986; 47: 767-776Abstract Full Text PDF PubMed Scopus (973) Google Scholar). Under deep anesthetization the liver of each mouse was perfused in situ via the portal vein with 150 ml of Krebs-Ringer buffer followed by 100 ml of Krebs-Ringer buffer containing collagenase (Sigma-Aldrich). The cells were dispersed in an equal volume of ice-cold William's E medium (Sigma-Aldrich). The cells were precipitated and washed twice at 4 °C with the same medium. Aliquots of 1 × 106 cells in William's E medium supplemented with 5% (v/v) fetal calf serum, 1 nm insulin, 100 nm triiodothyronine, 100 nm dexamethasone, 100 units/ml penicillin, and 100 μg/ml streptomycin were plated onto 6-well rat collagen I-coated dishes (Asahi Techno Glass, Chiba, Japan). After incubation for 2 h at 37 °C in 9% CO2, the cells were incubated with William's E medium supplemented with 10% (v/v) fetal calf serum, 1 nm insulin, 1 nm triiodothyronine, 100 nm dexamethasone, 100 units/ml penicillin, and 100 μg/ml streptomycin. Northern Blot Analysis—Total hepatic RNA was isolated from the livers with TRIzol reagent (Invitrogen) after perfusion of ice-cold phosphate-buffered saline (–) in situ via the portal vein. In cases of primary hepatocytes they were starved in William's E medium supplemented with 0.75% bovine serum albumin, 100 units/ml penicillin, and 100 μg/ml streptomycin followed by 6 h with insulin (100 nm), mannitol (30 mm), or fructose (30 mm). Total RNA was isolated with TRIzol reagent. Then 10–30 μg of RNA samples were run on a 1.2% agarose gel containing formaldehyde and transferred onto a nylon membrane (Nytran N; Schleicher & Schuell). The cDNA probes for Northern blot analyses were generated as previously described (7Nagai Y. Nishio Y. Nakamura T. Maegawa H. Kikkawa R. Kashiwagi A. Am. J. Physiol. Endocrinol. Metab. 2002; 282: E1180-E1190Crossref PubMed Scopus (172) Google Scholar). The probes were labeled with [α-32P]dCTP (Amersham Biosciences) using a labeling kit (Takara, Shiga, Japan), hybridized to ultraviolet cross-linked blots overnight at 68 °C in the hybridization buffer (Perfecthyb; Toyobo, Tokyo, Japan), and then washed at 68 °C over 40 min with 1× saline-sodium citrate and 0.1% SDS. The blots were exposed to Kodak Biomax (Eastman Kodak Co.) film at –80 °C. The signal was quantified with a densitometer, and loading differences were normalized to the signal generated with a probe for 18 S ribosomal RNA (8Tenenhouse H.S. Martel J. Biber J. Murer H. Am. J. Physiol. Renal Physiol. 1995; 268: 1062-1069Crossref PubMed Google Scholar). Sequencing of SREBP-1c Promoter Region—DNA sequences were analyzed by the dideoxynucleotide chain termination method. Construction of Plasmids—The 1.2-kilobase pair sequence from the 5′-flanking regions of SREBP-1c genes of both CBA/JN and DBA/2N mice were amplified using each genomic DNA by PCR with the sense primer 5′-GGATCCAGAACTGGATCATC and the antisense primer 5′-CCTAGGGCGTGCAGACGCTA. The resultant amplicons were gel-purified, inserted into pCR2.1-TOPO vector (Invitrogen), digested with KpnI/BglII, and inserted into KpnI/BglII-linearized pGL3-basic to form a reporter plasmid, pGL3-CBA/JN or pGL3-DBA/2N. Cell Transfection and Luciferase Assays—Primary hepatocytes were prepared as described. All transfections were performed using Superfect (Invitrogen) according to the manufacturer's instructions. Transfections were carried out with 1 μg of pRSV-β-gal expression plasmid and 2 μg of each reporter plasmid. After the transfection for 18 h, the medium was replaced with William's E medium supplemented with 0.75% bovine serum albumin for starving followed by 8 h with either insulin (10 nm), mannitol (30 mm), or fructose (30 mm). The results were quantified with a luminometer and normalized to the β-galactosidase activity measured in the extract of the cells. Preparation of Nuclear Protein Extracts—Nuclear protein of HeLa (HeLaScribe) was purchased from Promega (Madison, WI). Nuclear protein extract from the livers of mice and primary hepatocytes were isolated according to the procedure of Gorski et al. (9Gorski K. Carneiro M. Schibler U. Cell. 1986; 47: 767-776Abstract Full Text PDF PubMed Scopus (973) Google Scholar). The nuclear extract was suspended in 20 mm HEPES (pH 7.9), 330 mm NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 25% glycerol, 0.5 mm dithiothreitol, and 0.2 mm phenylmethylsulfonyl fluoride, and aliquots were frozen in liquid nitrogen and stored at –80 °C. Western Blot Analysis—For Western blot analysis, whole cell lysates (50 μg of protein per lane) were denatured by boiling in Laemmli sample buffer containing 100 mm dithiothreitol and resolved by SDS-PAGE. Gels were transferred to nitrocellulose by electroblotting in Towbin buffer containing 20% methanol. For immunoblotting, membranes were blocked and probed with the specified antibodies. Blots were then incubated with horseradish peroxidase-linked second antibody followed by chemiluminescence detection, according to the manufacturer's (PerkinElmer Life Sciences) instructions. Electrophoretic Mobility Shift Assays (EMSA)—EMSAs were performed using radiolabeling double-strand oligonucleotides corresponding to the following pairs from the promoter regions: DBA/2N mice, 5′-TATCTAAAGGCAACTATTGG and 5′-GGAAGGCCAATAGTTG; CBA/JN mice, 5′-TATCTAAAGGCAGCTATTGG and 5′-GGAAGGCCAATAGCTG; AP4, 5′-AAAGAACCAGCTGTGGAAT and 5′-ATTCCACAGCTGGTTCTTT (10Mermod N. Williams T.J. Tjian R. Nature. 1988; 6164: 557-561Crossref Scopus (224) Google Scholar). For control studies EMSAs were performed using radiolabeling OCT-1 consensus oligonucleotides (Promega). The CBA/JN and DBA/2N probes were labeled with [α-32P]dCTP using DNA polymerase (Takara). OCT-1 and AP4 probes were labeled with [γ-32P]dATP using T4PNK (Takara). For EMSAs, the reaction mixture contained 10 μg of nuclear extract, 1 μg of poly(dI-dC), 10 mm HEPES (pH 7.9), 60 mm KCl, 1 mm EDTA, 7% glycerol, and 50,000 cpm-labeled probes. Samples were incubated for 20 min at room temperature (11Nishio Y. Kashiwagi A. Taki H. Shinozaki K. Maeno Y. Kojima H. Maegawa H. Haneda M. Hidaka H. Yasuda H. Horiike K. Kikkawa R. Diabetes. 1998; 47: 1318-1325Crossref PubMed Scopus (79) Google Scholar) before the addition of antibodies directed against AP4 (Santa Cruz Biotechnology, CA) and incubated for a further 20 min at room temperature. Then the samples were loaded onto 6% polyacrylamide gels in 0.25× Tris borate-EDTA buffer and run at 150 V. The gels were dried and exposed to the film. We also carried out competition assay using non-labeled oligonucleotides. Statistical Analysis—The data are expressed as the means ± S.E. unless otherwise stated. Tukey-Welsh's step-down multiple comparison test was used to determine the significance of any differences among four or more groups. p < 0.05 was considered significant. Characteristics of Experimental Animals—In 10 inbred mouse strains (BALB/c, C3H/He, C3H/HeJ, C3H/HeN, CBA/JN, C57BL/6, C57BL/6N, C57BL/6J, DBA/1JN, and DBA/2N) we investigated the responses of metabolic characteristics to the high fructose diet. The metabolic characteristics of the six representatives are shown in Table I. Compared with the control mice fed a normal laboratory chew, C3H/He showed significantly increased body weights after feeding of the high fructose diet for 8 weeks. The epididymal fat weights for all strains except the DBA/2N and DBA/1JN strains a showed significant increase after the high fructose diet for 8 weeks compared with those of the control animals. The levels of blood glucose did not significantly differ in the control and the high fructose diet groups for any strains.Table ICharacteristics of the experimental animalsMouse strainsDietConditionBody weightEpididymal fat weightBlood glucoseTotal cholesterolTriglycerideInsulinggmmol/litermg/dlmg/dlpmol/literC3H/HeControlFasting26 ± 1.10.4 ± 0.17 ± 1.2102 ± 351 ± 1740 ± 24Postprandial--11 ± 1.8ap < 0.01 versus the control-fasting.108 ± 7168 ± 11ap < 0.01 versus the control-fasting.154 ± 100ap < 0.01 versus the control-fasting.FructoseFasting30 ± 1.6ap < 0.01 versus the control-fasting.0.9 ± 0.3ap < 0.01 versus the control-fasting.8 ± 2.4174 ± 25ap < 0.01 versus the control-fasting.80 ± 2230 ± 19Postprandial--14 ± 0.8bp < 0.01 versus the fructose-fasting.201 ± 22cp < 0.01 versus the control-postprandial.214 ± 39bp < 0.01 versus the fructose-fasting.,cp < 0.01 versus the control-postprandial.415 ± 107bp < 0.01 versus the fructose-fasting.,cp < 0.01 versus the control-postprandial.CBA/JNControlFasting28 ± 1.70.5 ± 0.15 ± 2.765 ± 491 ± 720 ± 12Postprandial--20 ± 4.9ap < 0.01 versus the control-fasting.77 ± 5262 ± 100ap < 0.01 versus the control-fasting.422 ± 186ap < 0.01 versus the control-fasting.FructoseFasting30 ± 1.31.1 ± 0.1ap < 0.01 versus the control-fasting.8 ± 2.9112 ± 9ap < 0.01 versus the control-fasting.150 ± 34ap < 0.01 versus the control-fasting.70 ± 31ap < 0.01 versus the control-fasting.Postprandial--14 ± 0.3bp < 0.01 versus the fructose-fasting.113 ± 19cp < 0.01 versus the control-postprandial.392 ± 12bp < 0.01 versus the fructose-fasting.,cp < 0.01 versus the control-postprandial.751 ± 153bp < 0.01 versus the fructose-fasting.,cp < 0.01 versus the control-postprandial.BALB/cControlFasting29 ± 10.4 ± 0.16 ± 1.786 ± 796 ± 4105 ± 16Postprandial--15 ± 2ap < 0.01 versus the control-fasting.92 ± 11107 ± 12235 ± 120ap < 0.01 versus the control-fasting.FructoseFasting30 ± 10.7 ± 0.1ap < 0.01 versus the control-fasting.8 ± 1143 ± 19ap < 0.01 versus the control-fasting.104 ± 1674 ± 29Postprandial--21 ± 4.3bp < 0.01 versus the fructose-fasting.152 ± 11cp < 0.01 versus the control-postprandial.162 ± 33bp < 0.01 versus the fructose-fasting.,cp < 0.01 versus the control-postprandial.385 ± 41bp < 0.01 versus the fructose-fasting.,cp < 0.01 versus the control-postprandial.C57BL/6NControlFasting24 ± 0.90.2 ± 0.18 ± 1.265 ± 554 ± 1433 ± 26Postprandial--16 ± 2.5ap < 0.01 versus the control-fasting.54 ± 1368 ± 7151 ± 98ap < 0.01 versus the control-fasting.FructoseFasting24 ± 2.60.6 ± 0.1ap < 0.01 versus the control-fasting.6 ± 1.7113 ± 10ap < 0.01 versus the control-fasting.31 ± 447 ± 31Postprandial--15 ± 1.5bp < 0.01 versus the fructose-fasting.110 ± 857 ± 11224 ± 173bp < 0.01 versus the fructose-fasting.DBA/2NControlFasting23 ± 1.70.3 ± 0.28 ± 1.989 ± 899 ± 855 ± 35Postprandial--15 ± 2.7ap < 0.01 versus the control-fasting.92 ± 10106 ± 13168 ± 131ap < 0.01 versus the control-fasting.FructoseFasting25 ± 2.20.5 ± 0.26 ± 1.7120 ± 19ap < 0.01 versus the control-fasting.108 ± 3145 ± 23Postprandial--14 ± 1.5bp < 0.01 versus the fructose-fasting.105 ± 11126 ± 28135 ± 97bp < 0.01 versus the fructose-fasting.DBA/1JNControlFasting24 ± 0.90.3 ± 0.17 ± 2.384 ± 1299 ± 3042 ± 21Postprandial--13 ± 2.0ap < 0.01 versus the control-fasting.60 ± 994 ± 4188 ± 51ap < 0.01 versus the control-fasting.FructoseFasting23 ± 1.80.3 ± 0.16 ± 1.288 ± 468 ± 2140 ± 7Postprandial--15 ± 1.2bp < 0.01 versus the fructose-fasting.75 ± 1492 ± 26159 ± 66bp < 0.01 versus the fructose-fasting.a p < 0.01 versus the control-fasting.b p < 0.01 versus the fructose-fasting.c p < 0.01 versus the control-postprandial. Open table in a new tab As shown in Table I, the levels of serum triglycerides in the postprandial state were significantly elevated after feeding the control diet in the C3H/He and CBA/JN strains and after feeding the high fructose diet in the C3H/He, CBA/JN, and BALB/c strains. In addition, these three strains showed higher postprandial plasma insulin levels after feeding the high fructose diet than those after feeding the control diet. In contrast, in C57BL/6N, DBA/1JN, and DBA/2N, the levels of serum triglycerides in the postprandial state were not significantly elevated after feeding control or high fructose diet. Although the postprandial plasma insulin levels in these strains after feeding of either control or high fructose diet were increased compared with the corresponding fasting level, the postprandial insulin levels did not differ between the control and the high fructose groups, respectively. The mRNA Expression of SREBP-1 in the Liver—To explore the molecular mechanisms of the differences in response to the high fructose diet, we compared the hepatic mRNA expressions of SREBP-1, a key transcription factor regulating fatty acid synthesis in various strains of mice. As shown in Fig. 1, A and B, the hepatic mRNA expression of SREBP-1 in the CBA/JN mice was increased after feeding. In the control-postprandial group, it was stimulated by 3.9-fold (p < 0.001) as compared with that of the control-fasting group. The fructose-fasting group showed stimulation by 1.9-fold (p < 0.05) as compared with the control-fasting group. Furthermore, the fructose-postprandial group showed enhancement by 23-fold (p < 0.001) compared with the control-fasting group. Thus, the fructose diet stimulated the level of SREBP-1 mRNA expression by 11-fold (p < 0.001) compared with fructose-fasting levels and by 6-fold (p < 0.001) compared with the control-postprandial group. Similar changes in the hepatic mRNA expression of SREBP-1 were observed in the C3H/He strain (Fig. 1, C and D). The hepatic mRNA expression of SREBP-1 in the C3H/He control-postprandial group was stimulated by 3.8-fold (p < 0.05) compared with that in the control-fasting group. The level in the fructose postprandial group was enhanced by 5.8-fold (p < 0.001) compared with the control-fasting group. As a result, a fructose diet stimulated the level of SREBP-1 mRNA expression by 3.5-fold (p < 0.001) compared with the fructose-fasting level and by 1.5-fold (p < 0.001) compared with the control-postprandial group. In addition, compared with that in the control-fasting group, the hepatic mRNA expression of SREBP-1 in the BALB/c strain was also significantly increased by 4.2-fold (p < 0.05) or by 6.1-fold (p < 0.001), respectively, after feeding the control or the high fructose diet. On the other hand, the hepatic mRNA expressions of SREBP-1 in the DBA/2N mice (Fig. 1, E and F) and the DBA/1JN mice (Fig. 1, G and H) were not affected by either the control or the high fructose diet. The hepatic mRNA expression of SREBP-1 in the C57BL/6N mice was increased significantly but to a lesser degree, i.e. the control postprandial group showed a 2.1-fold (p < 0.05) increase, and the fructose-post-prandial group showed a 2.5-fold (p < 0.01) increase compared with the control fasting group, respectively. However, hepatic SREBP-1 mRNA expressions in either fasting or postprandial state were not different between the control and the high fructose diet groups. The Hepatic mRNA Expression of Fatty Acid Synthase (FAS)— To investigate the effect of increased hepatic SREBP-1 expression on the downstream activation of the enzyme, we examined the mRNA expression of hepatic FAS, one of the target genes of SREBP-1, by Northern blot analysis. The hepatic mRNA expression of FAS from postprandial CBA/JN mice fed the high fructose diet was increased by 6-fold (p < 0.001) over that of the postprandial mice fed the control diet and by 4-fold (p < 0.001) when compared with that of the fructose-fasting mice (Fig. 2, A and B). In contrast, the hepatic mRNA expression of FAS in the DBA/2N mice was not significantly affected by either control diet or high fructose diet (Fig. 2, C and D). The mRNA Expression of PPARα in the Livers from the CBA/JN and the DBA/2N Mice—As shown in Fig. 3, A and B, the mRNA expression of PPARα, a key transcription factor for fatty acid oxidation, in the livers from the CBA/JN mice was decreased after intake of either the control (p < 0.01) or the high fructose (p < 0.01) diet. Similar significant reductions of the hepatic mRNA expression of PPARα were also found in DBA/2N mice after intake of either diet (Fig. 3, C and D). The mRNA Expression of SREBP-1 in Primary Cultured Hepatocytes—To study the direct effect of fructose on the expression of SREBP-1 in the liver, we examined the mRNA contents of SREBP-1 in primary cultured hepatocytes isolated from the CBA/JN (Fig. 4A) or the DBA/2N (Fig. 4B) mice in the presence or absence of 30 mm fructose. In this experiment we used cells cultured with 30 mm mannitol as a control for fructose-treated or insulin-treated cells, since 30 mm mannitol did not affect the mRNA expression of SREBP-1 in either CBA/JN or DBA/2N primary hepatocytes; the values for hepatocytes cultured with 5 mm glucose and with 30 mm mannitol were 0.9 ± 0.4 and 1.1 ± 0.2, for CBA/JN (arbitrary units, n = 8, NS) and 1.1 ± 0.2 and 1.0 ± 0.2 for DBA/2N (arbitrary units, n = 8, NS). As shown in Fig. 4A, compared with the controls, the primary cultured hepatocytes from the CBA/JN mice showed a 73% (p < 0.01) increase in the mRNA expression of SREBP-1 in the presence of 30 mm fructose in the media and 92% (p < 0.01) increase in the presence of 100 nm insulin. In contrast, as shown in Fig. 4B, the incubation with 30 mm fructose did not induce the mRNA expression of SREBP-1 in the primary hepatocytes isolated from the DBA/2N mice. However, 100 nm insulin significantly increased the expression of SREBP-1 mRNA by 24% (p < 0.01) in these hepatocytes. Thus, the effect of insulin on the SREBP-1 mRNA expression in hepatocytes from DBA/2N mice was significantly blunted compared with that on the hepatocytes from CBA/JN mice (p < 0.01). The mRNA Expression of FAS in Primary Cultured Hepatocytes—The mRNA expressions of FAS in primary cultured hepatocytes from both DBA/2N and CBA/JN mice were studied in the presence or absence of 30 mm fructose. In this experiment we used cells cultured with 30 mm mannitol as the control for fructose-treated or insulin-treated cells, since the 30 mm mannitol did not affect the mRNA expression of FAS in either CBA/JN and DBA/2N primary hepatocytes; in the presence of 5 mm glucose or 30 mm mannitol the expression was 1.0 ± 0.01 and 1.0 ± 0.1 for hepatocyte from CBA/JN (arbitrary units, n = 8, NS) and 1.0 ± 0.1 and 1.0 ± 0.2 for hepatocytes from DBA2N (arbitrary units, n = 8, NS). Comparable with the results from the experiment on the mRNA expression of SREBP-1, both 30 mm fructose and 100 nm insulin significantly increased (p < 0.01) the mRNA expression of FAS in the hepatocytes from CBA/JN mice, by 100 and 96%, respectively (Fig. 5A). On the other hand, the mRNA expression of FAS in primary hepatocytes from DBA/2N mice was not affected by 30 mm fructose (Fig. 5B). However, a 32% (p < 0.01) increase in mRNA expression of FAS was induced by 100 nm insulin; the effect of insulin on the FAS mRNA expression was reduced significantly (p < 0.01) compared with that on the hepatocytes from CBA/JN mice. Single Nucleotide Polymorphism in SREBP-1c Promoter Region—We have cloned a 1.2-kilobase pair fragment of the 5′ upstream region of SREBP-1c gene from each inbred mouse strain and found a single nucleotide polymorphism at –468 bp from the putative starting point of the SREBP-1c gene. The nucleotide at –468 bp in the C3H/HeN, C3H/He, C3H/HeJ, BALB/c, and CBA/JN strains is guanine, whereas it is adenine in the C57BL/6, C57BL/6J, C57BL/6N, DBA/1JN, and DBA/2N strains. Luciferase Activities of SREBP-1c Promoters—To investigate the significance of the single nucleotide mutation from guanine to adenine at –468 bp of the SREBP-1c promoter, we analyzed the promoter activity using the luciferase reporter carrying the 1.2-kilobase pair SREBP-1c promoter region of the CBA/JN or DBA/2N mice with 5 individual experiments in a total of 12 determinations. The activity of SREBP-1c promoter in primary hepatocytes isolated from CBA/JN mice was significantly increased by 2.4-fold (p < 0.01) by exposure of the cells to 30 mm fructose (Fig. 6A), but no increase was observed in primary hepatocytes isolated from the DBA/2N mice (Fig. 6B). The activity of the SREBP-1c promoter from DBA/2N mice was not induced by 30 mm fructose in primary hepatocytes isolated from either the CBA/JN or the DBA/2N mice. We also investigated the effect of insulin with 5 individual experiments in a total of 12 determinations. As shown in Fig. 6C, the activity of SREBP-1c promoter from either the DBA/2N or CBA/JN strain in hepatocytes isolated f