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Endocrinology, doi:10.1210/en.2007-1461
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Endocrinology Vol. 149, No. 5 2293-2305
Copyright © 2008 by The Endocrine Society

Regulation of SREBP1c Gene Expression in Skeletal Muscle: Role of Retinoid X Receptor/Liver X Receptor and Forkhead-O1 Transcription Factor

Yasutomi Kamei, Shinji Miura, Takayoshi Suganami, Fumiko Akaike, Sayaka Kanai, Satoshi Sugita, Aki Katsumata, Hiroyuki Aburatani, Terry G. Unterman, Osamu Ezaki and Yoshihiro Ogawa

Department of Molecular Medicine and Metabolism (Y.K., T.S., F.A., S.K., S.S., Y.O.) and Center of Excellence Program for Frontier Research on Molecular Destruction and Reconstitution of Tooth and Bone (S.S., Y.O.), Medical Research Institute, Tokyo Medical and Dental University, Tokyo 101-0062, Japan; Nutritional Science Program (S.M., A.K., O.E.), National Institute of Health and Nutrition, Tokyo 162-8636, Japan; Research Center for Advanced Science and Technology (H.A.), University of Tokyo, Tokyo 153-8904, Japan; and Departments of Medicine and Physiology and Biophysics (T.G.U.), University of Illinois College of Medicine and Jesse Brown Veterans Affairs Medical Centers, Chicago, Illinois 60612

Address all correspondence and requests for reprints to: Osamu Ezaki M.D., Ph.D., Nutritional Science Program, National Institute of Health and Nutrition, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8636, Japan. E-mail: ezaki{at}nih.go.jp; or Yoshihiro Ogawa M.D, Ph.D., Department of Molecular Medicine and Metabolism, Medical Research Institute, Tokyo Medical and Dental University, 2-3-10 Kanda-surugadai, Chiyoda-ku, Tokyo 101-0062, Japan. E-mail: ogawa.mmm{at}mri.tmd.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Sterol regulatory element binding protein 1c (SREBP1c) is a master regulator of lipogenic gene expression in liver and adipose tissue, where its expression is regulated by a heterodimer of nuclear receptor-type transcription factors retinoid X receptor-{alpha} (RXR{alpha}) and liver X receptor-{alpha} (LXR{alpha}). Despite the potential importance of SREBP1c in skeletal muscle, little is known about the regulation of SREBP1c in that setting. Here we report that gene expression of RXR{gamma} is markedly decreased by fasting and is restored by refeeding in mouse skeletal muscle, in parallel with changes in gene expression of SREBP1c. RXR{gamma} or RXR{alpha}, together with LXR{alpha}, activate the SREBP1c promoter in vitro. Moreover, transgenic mice overexpressing RXR{gamma} specifically in skeletal muscle showed increased gene expression of SREBP1c with increased triglyceride content in their skeletal muscles. In contrast, transgenic mice overexpressing the dominant-negative form of RXR{gamma} showed decreased SREBP1c gene expression. The expression of Forkhead-O1 transcription factor (FOXO1), which can suppress the function of multiple nuclear receptors, is negatively correlated to that of SREBP1c in skeletal muscle during nutritional change. Moreover, transgenic mice overexpressing FOXO1 specifically in skeletal muscle exhibited decreased gene expression of both RXR{gamma} and SREBP1c. In addition, FOXO1 suppressed RXR{alpha}/LXR{alpha}-mediated SREBP1c promoter activity in vitro. These findings provide in vivo and in vitro evidence that RXR/LXR up-regulates SREBP1c gene expression and that FOXO1 antagonizes this effect of RXR/LXR in skeletal muscle.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
STEROL REGULATORY ELEMENT binding protein 1c (SREBP1c) is a master transcription factor which regulates the expression of a number of metabolic genes involved in the partitioning of nutrients into lipid storage (1). It enhances the expression of multiple lipogenic genes such as fatty acid synthase (FAS) (2). So far, the role of SREBP1c in lipogenesis has been investigated primarily in liver and adipose tissue (1, 3). Unlike liver and adipose tissue, skeletal muscle has not been regarded as highly lipogenic. However, recent studies suggest that lipogenesis and triglyceride storage in skeletal muscle are important (4, 5). Elevated skeletal muscle triglyceride storage has been associated with insulin resistance and the development of type 2 diabetes (5, 6). On the other hand, endurance-trained athletes have increased intramuscular triglyceride stores, which may serve as fuel during muscle contraction (5, 7). Given the well-established role of SREBP1c in lipogenesis in liver and adipose tissue and relatively high expression of SREBP1c in skeletal muscle (8, 9), it is conceivable that SREBP1c may play an important role in lipid metabolism in skeletal muscle as well. Indeed, fasting lowers the SREBP1c gene expression and protein levels in skeletal muscle, which are restored by refeeding (8, 10, 11, 12), similar to changes observed in liver and adipose tissue (13, 14).

Liver X receptors (LXRs), nuclear receptor proteins that form heterodimers with retinoid X receptors (RXRs) (15, 16, 17), have been reported to regulate the gene expression of SREBP1c in liver and adipose tissue (18, 19, 20, 21, 22, 23). RXR/LXR is known to play an important role in insulin-mediated activation of SREBP1c transcription; insulin activates the SREBP1c promoter at least in part by increasing the transcriptional activity of RXR/LXR (24). The LXR subfamily consists of LXR{alpha} and LXRβ (15, 25). The RXR subfamily consists of RXR{alpha}, RXRβ, and RXR{gamma} (15, 16, 17). Because RXR{alpha} and LXR{alpha} are abundantly expressed subtypes in liver (15, 16, 17), most studies on regulation of SREBP1c gene expression have been done using RXR{alpha}/LXR{alpha}. On the other hand, RXR{gamma} is preferentially expressed in skeletal muscle (16, 26). However, its role in regulation of SREBP1c gene expression has not been studied.

In addition to RXR/LXR, recent studies have suggested that Forkhead-O1 transcription factor (FOXO1), a member of the Forkhead ‘Other’ group of transcription factors and an important target of insulin and growth factor signaling (27), also may contribute to the regulation of SREBP1c. FOXO1 plays an important role in stimulating hepatic gluconeogenic gene expression in fasting, when insulin levels are low (27), and insulin disrupts this effect of FOXO1 by promoting its phosphorylation by Akt (also known as protein kinase B) and promoting its exclusion from the nucleus. Hepatic expression of constitutively active FOXO1 in transgenic mice was associated with reduced SREBP1c and lipogenic gene expression, and FOXO1 also suppresses SREBP1c gene expression in isolated hepatocytes (28). Genetic studies in mice with targeted deletion of FOXO1 in the liver also support the concept that FOXO1 negatively regulates SREBP1c expression in the liver when insulin signaling is disrupted (29).

We previously demonstrated that the FOXO1 gene expression in skeletal muscle is increased by fasting and decreased by refeeding (12). Because SREBP1c gene expression in skeletal muscle is decreased by fasting and restored by refeeding (8, 10, 11, 12), we considered the possibility that SREBP1c gene expression may be negatively regulated by FOXO1 in skeletal muscle. FOXO1 is known to suppress the function of multiple nuclear receptor proteins such as the estrogen receptor (ER) and peroxisome proliferators-activated receptor-{gamma} (PPAR{gamma}) (30, 31). However, whether FOXO1 suppresses RXR/LXR-mediated SREBP1c gene expression remains unknown.

Despite the potential importance of SREBP1c in skeletal muscle, the regulation of SREBP1c gene expression and the involvement of RXR/LXR in skeletal muscle are largely unknown. In this study, we attempt to elucidate the transcriptional regulation of SREBP1c gene in skeletal muscle. Our in vitro and in vivo data suggest that RXR/LXR can be a positive regulator of SREBP1c gene expression in skeletal muscle, which is negatively regulated by FOXO1.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmids
Mouse RXR{gamma} cDNA was obtained by RT-PCR with mouse skeletal muscle RNA as template. pSG5-mRXR{alpha} was kindly provided by Dr. Chambon (Strasbourg, France). Human LXR{alpha} cDNA was obtained by screening a human liver cDNA library. The coding region of mouse RXR{gamma} cDNA and human LXR{alpha} cDNA was subcloned into a mammalian expression plasmid, pCMX (32). A dominant-negative (DN)-RXR{gamma} was made by substituting glutamic acid at amino acid position 454 by glutamine, using the QuikChange mutagenesis kit (Stratagene, La Jolla, CA), and confirmed by sequencing. DN-RXR{gamma} cDNA was subcloned into pCMX. The full-length cDNA encoding constitutively active human FOXO1, where threonine-24, serine-256, and serine-319 have been replaced with alanine (28), was inserted into expression vector pCAG (33).

Transfection and luciferase assays
HEK293 cells were plated at a density of 1 x 105 cells per 12-well plate in DMEM containing 10% fetal bovine serum. Luciferase gene constructs containing a 550-bp mouse SREBP1c promoter fragment with or without mutation of LXR response elements (LXREs) were prepared (SREBP1c-luc) (20, 22). Luciferase reporter plasmid (0.8 µg), expression plasmids (pCMX-LXR{alpha}, pCMX-RXR{gamma} and empty pCMX; pCAG-FOXO1; or pCMX-DN-RXR{gamma}, total 0.8 µg), and a phRL-TK vector (25 ng; Promega, Madison, WI) as an internal control for transfection efficiency were transfected into cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). After an overnight transfection period, cells were treated with 9-cis retinoic acid (9-cis RA) (1 µM; Sigma-Aldrich, St. Louis, MO) and/or T0901317 (1 µM; Calbiochem, San Diego, CA) for 24 h. After treatment, cells were lysed and assayed for luciferase activity using the dual luciferase assay kit (Promega). The activity was calculated as the ratio of firefly luciferase activity to Renilla luciferase activity (internal control) and represented as the average of triplicate experiments.

Gel mobility shift assay
Gel mobility shift assay was performed as described previously (20). In vitro-translated human LXR{alpha}, mouse RXR{gamma}, mouse DN-RXR{gamma}, and mouse RXR{alpha} were generated from pCMX-LXR{alpha}, pCMX-RXR{gamma}, pCMX-DN-RXR{gamma}, and pSG5-RXR{alpha} plasmids, respectively, using the TNT T7 Quick Coupled Transcription/Translation System (Promega) according to the manufacturer’s manual. Double-stranded oligonucleotide probes used in gel mobility shift assays were prepared by annealing both strands of each LXRE in the SREBP1c promoter (LXREa, 5'-CAGTGACCGCCAGTAACCCCAGC-3'; LXREb, 5'-GGACGCCCGCTAGTAACCCCGGC-3'; LXREa mutant, 5'-CAGAGTCCGCCAGAATCCCCAGC-3'; and LXREb mutant, 5'-GGAAGTCCGCTAGAATCCCCGGC-3') and labeling with [{gamma}-32P]ATP (PerkinElmer Life Sciences, Waltham, MA) using T4 polynucleotide kinase (Roche Applied Science, Indianapolis, IN). The labeled probes (50,000 dpm) were incubated with extracts containing in vitro-translated human LXR{alpha} and mouse RXR{gamma} in a mixture (total volume of 25 µl) containing 10 mM Tris (pH 7.5), 50 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, and 4.4% glycerol with 1 mg poly (dI-dC) for 30 min on ice and then separated by electrophoresis on a 6% polyacrylamide gel in 45 mM Tris (pH 8.0), 45 mM borate, and 1 mM EDTA. After electrophoresis, gels were dried and analyzed with BAS-2500 (Fuji Film, Tokyo, Japan).

Stable cell lines
Phoenix 293 cells (34) were cultured in 90-mm dishes and transfected at 70% confluence using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions, with 2 µg pLNCX-derived expression plasmid (BD Biosciences, San Jose, CA) containing LXR{alpha} cDNA or vector alone. Virus-containing supernatants were harvested 48 h after transfection. The supernatants were added onto dishes (90 mm in diameter) of C2C12 cells at 50% confluence in DMEM containing 10% fetal calf serum and 5 µg/ml polybrene in a final volume of 5 ml. Cells were split 1:10 24 h after infection and replated in DMEM containing 10% fetal calf serum and 0.5 mg/ml G418 to eliminate uninfected cells. After selection with G418, a secondary infection was performed with a pMX-derived expression plasmid (35) containing RXR{gamma} cDNA, and cells were selected using 5 µg/ml puromycin to eliminate uninfected cells. After the second drug selection, virally infected stable cells were cultured to confluence in DMEM containing 10% fetal calf serum. Cells were refed every 2 d. At 3 d after confluence, cells were incubated for 6 h with 1 µM 9-cis retinoic acid and/or 1 µM T0901317 and used for the preparation of RNA.

Chromatin immunoprecipitation (ChIP) analysis
ChIP was carried out using the ChIP assay kit (Upstate, Temecula, CA) according to the manufacturer’s guidelines. Briefly, cell cultures were incubated for 24 h with 1 µM 9-cis retinoic acid and 1 µM T0901317. Proteins were cross-linked to DNA with the addition of formaldehyde (final concentration 1%). Cells were washed and lysed in SDS lysis buffer and sonicated for 10 sec and allowed to recover for 30 sec over ice (this was repeated seven times). Lysates were cleared with protein A-agarose for 30 min, pelleted, and incubated overnight with anti-Flag antibody (M5; Sigma-Aldrich). Before the incubation, input samples were removed from the lysate and stored at 4 C until extraction. After incubation with the antibody, protein-DNA complexes were eluted (1% SDS and 0.1 M NaHCO3), and the cross-links were reversed. DNA was purified by phenol/chloroform extraction. PCR primers were designed to locate LXREs of the SREBP1c promoter: first primer set forward 5'-AGTTCTGGGTGTGTGCGAAC-3' and reverse 5'-TGAGCGCACATGGCCAATCA-3'; and nested primer set forward 5'-GAACCAGCGGTGGGAACACAGAGC-3' and reverse 5'-GACGGCGGCAGCTCGGGTTT CTC-3'.

Transgenic mice
The human skeletal muscle {alpha}-actin promoter (36) was provided by Drs. E. D. Hardeman and K. Guven (Children’s Medical Research Institute, Australia). The transgenes (see Figs. 3AGo and 6BGo) were excised and purified for injection (2 ng/µl). Fertilized eggs were recovered from C57BL/6 females crossed with C57BL/6 males and microinjected at Japan SLC Inc. (Hamamatsu, Japan). The mice were maintained at a constant temperature of 24 C with fixed artificial light (12 h light and 12 h dark). Generation of transgenic mice overexpressing FOXO1 in skeletal muscle was described previously (37). Male transgenic mice (heterozygotes, C57BL6 background) and female wild-type C57BL6 mice were crossed, and the offspring (heterozygote and wild-type littermates) were used for the experiments. All animal experiments were conducted in accordance with the guidelines of National Institute of Health and Nutrition (no. 0706) and Tokyo Medical and Dental University Committee on Animal Research (no. 0060028).


Figure 3
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  		FIG. 3. Generation of RXR{gamma} transgenic mice (RXR{gamma} mice). A, A schematic of the 5-kb RXR{gamma} transgene structure. The transgene was under the control of the human skeletal muscle {alpha}-actin promoter and included exon 1 and the intron of the human skeletal muscle {alpha}-actin gene as well as the bovine growth hormone polyadenylation site (36 ). B, Expression of the RXR{gamma} transgene in mice. Northern blot analysis of RXR{gamma} gene expression in tissues from RXR{gamma} mice (line 5-3) and wild-type mice. Total RNAs extracted from the brain, heart, kidney, liver, skeletal muscle [gastrocnemius (Gastro.) and quadriceps (Quadri.)] and adipose tissue were analyzed. The blots were rehybridized with the SREBP1 and 36B4 probes. Each lane contained 20 µg total RNA. In each lane, 28 S rRNA staining confirmed similar loading (not shown). C, Expression of RXR{gamma} protein in skeletal muscle from RXR{gamma} mice. Protein extracts from RXR{gamma} mice (line 5-3) were subjected to SDS-PAGE (30 µg per lane). The RXR{gamma} protein was detected by immunoblotting. The immunoreactive band is indicated by the arrow. The approximate estimated molecular size was 55 kDa. A similar RXR{gamma} protein level was observed in the other line of RXR{gamma} mice (line 4-3, not shown).

 

Figure 6
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  		FIG. 6. Effect of DN-RXR{gamma} on SREBP1c gene expression. A, DN effect of DN-RXR{gamma} on the RXR{alpha}/LXR{alpha}-induced SREBP1c promoter activity. SREBP1c-luc with RXR{gamma} and LXR{alpha} was transfected into HEK293 cells. Increasing amounts of DN-RXR{gamma} plasmid (0–0.4 µg) were cotransfected. After an overnight transfection period, cells were treated with 1 µM 9-cis RA plus 1 µM T0901317 for 24 h. The relative values compared with those for mock transfection (set as 100) are shown. Each value represents means ± SE (n = 4). For some points, error bars were too small to be shown. B, A schematic of the 5-kb DN-RXR{gamma} transgene structure. Glutamic acid (E) at amino acid position 454 of mouse RXR{gamma} was substituted by glutamine (Q). The {alpha}-actin promoter and polyadenylation signal are as used in RXR{gamma} mice (Fig. 3AGo). C, Gene expression in skeletal muscle from DN-RXR{gamma} mice. Quantitative real-time PCR analysis was performed on total RNA (5 µg per sample) isolated from skeletal muscle (quadriceps) from DN-RXR{gamma} mice (lines 3-6, 6-6, and 8-2, black bars) and respective wild-type controls (white bars). The names of the genes examined are shown on the top of the graph (the control value is set as 100). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Numbers of animals used are shown in Table 2Go.

 
Northern blot analysis
Northern blot analysis was performed as described previously (37). The cDNA fragments for SREBP1 and 36B4 were obtained by PCR with first-strand cDNA using mouse skeletal muscle total RNA. First-strand cDNA was prepared using a T-primed first-strand kit (GE Healthcare UK Ltd., Buckinghamshire, UK). The amplified products were subcloned into pGEM-T Easy Vector (Promega) and confirmed by sequencing.

Immunoblotting
Skeletal muscle protein extracts were prepared by centrifugation of tissue homogenates as described previously (37). Protein extracts (30 µg) separated by SDS-PAGE were electrophoretically transferred onto Immobilon P (Millipore, Bedford, MA). Immunoblotting was performed using rabbit anti-RXR{gamma} IgG (Y-20; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and antirabbit IgG conjugated with horseradish peroxidase as secondary antibody (1:1000). Bands were visualized with the enhanced chemiluminescence system (GE Healthcare UK).

Skeletal muscle triglyceride analysis
Skeletal muscles of mice (quadriceps) were dissected and attached fat pad carefully removed. Lipids in skeletal muscle were extracted quantitatively with ice-cold 2:1 (vol/vol) chloroform/methanol. Triglyceride concentrations in skeletal muscle homogenates were measured by enzymatic colorimetric methods using triglyceride E tests (Wako Pure Chemicals, Osaka, Japan).

Blood analysis
Blood samples were obtained from mice tail tips for hormone and metabolite determination under feeding conditions. Immunoreactive insulin was measured using an insulin assay kit (Morinaga, Kanagawa, Japan), free fatty acid using NEFA C-test (Wako, Osaka, Japan), and glucose by using a TIDEX glucose analyzer (Sankyo, Tokyo, Japan).

Quantitative real-time PCR
Quantitative real-time PCR was performed as described below. Total RNA was prepared using Sepazol (Nakalai Tesque, Kyoto, Japan). cDNA was synthesized from 5 µg total RNA using Superscript II reverse transcriptase (Invitrogen) with random primers. Gene expression levels were measured with an ABI PRISM 7700 using SYBR Green PCR Core Reagents (Applied Biosystems, Tokyo, Japan). The primers used were as follows: RXR{alpha} forward 5'-AACCCCAGCTCACCAAATGACC-3' and reverse 5'-AACAGGACAATGGCTCGCAGG-3'; RXRβ forward 5'-GCCAAGCTGCTGTTACGTCTT-3' and reverse 5'-ACAGGTGCTCCAGA-CACTTGAG-3'; RXR{gamma} forward 5'-CCGCTGCCAGTACTGTCG-3' and reverse 5'-ACCTGGTCCTCCAAGGTGAG-3'; LXR{alpha} forward 5'-GGGAGGAGTGTGTGCTGTCAG-3' and reverse 5'-GAGCGCCTGTTACACTGTTGC-3'; LXRβ forward 5'-GGCCTGGACGATGCAGAGT-3' and reverse 5'-CGATCGGCTGAGAAGATGTTG-3'; FOXO1 forward 5'-AATCCAGCATGAGCCCTTTG-3' and reverse, 5'-TCCAGTTCCTTCATTCTGCA-3'; SREBP1a forward 5'-GGCCGAGATGTGCGAAC-3' and reverse 5'-GTTGATGAGCTGGAGCATGT-3'; SREBP1c forward 5'-AGCTGTCGGGGTAGCGTCTG-3' and reverse 5'-GAGAGTTGGCACCTGGGCTG-3'; FAS forward 5'-GCTGCGGAA-ACTTCAGGAAAT-3' and reverse 5'-AGAGACGTGTCACTCCTGGACTT-3'; ACC1 forward 5'-TAAACCAGCACTCCCGATTC-3' and reverse 5'-CCATCCTGTAAGCCAGAGAT-3'; ACC2 forward 5'-AGACAGCTGATGACCAG-CTT-3' and reverse 5'-TGTTCTCGGCCTCTCTTCAC-3'; SCD1 forward 5'-CCGGAGACCCCTTAGATCGA-3' and reverse 5'-TAGCCTGTAAAAGATTTCTGCAAACC-3'; ABCD2 forward 5'-GGCATGGAGG-AATGGCAGT-3' and reverse 5'-GGCAATAGAACTCGGGACCC-3'; RXR{gamma}1 forward 5'-TTCCCACCGGCTTTGGT-3' and reverse 5'-CAAGGCTACTGAAGGGCTCATG-3'; RXR{gamma}2 forward 5'-CGCTGCTAAA-AGGCTTTGGT-3' and reverse 5'-GAACAATCCCCACCCAAGGT-3'; and 36B4 forward 5'-GGCCCTGCACTCTCGCTTTC-3' and reverse 5'-TGCCAGGACGCGCTTGT-3'.

Statistical analysis
Statistical comparisons of data from experimental groups were made by one-way ANOVA, and groups were compared using Fisher’s protected least significant difference test (Statview 5.0; Abacus Concepts, Inc., Berkeley, CA). When differences were significant, groups were compared using Fisher’s protected least significant difference test. Statistical significance was defined as P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Gene expression of SREBP1s, RXRs, LXRs, and FOXO1 during fasting and refeeding
To obtain insight into the possible role of RXRs, LXRs, and FOXO1 in skeletal muscle expression of SREBP1c, we examined their gene expression in the mouse skeletal muscle during fasting and refeeding. SREBP1c gene expression was decreased by fasting and restored by refeeding, which is consistent with previous findings (8, 10, 11, 12). In this study, gene expression of SREBP1a, an isoform of SREBP1c, was unchanged during fasting. The gene expression of FAS, a target gene of SREBP1c (2), was decreased by fasting and then increased above the basal level by refeeding (Fig. 1Go).


Figure 1
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  		FIG. 1. Gene expression of SREBP1s, RXRs, LXRs, and FOXO1 in skeletal muscle and liver during fasting and refeeding. A, Gene expression levels in skeletal muscle and liver during fasting and refeeding. Mice were divided into three experimental groups. They were either fed ad libitum (fed) or subjected to a 48-h fast (fast). The other mice were subjected to a 32-h fast followed by 16 h of feeding (refed). Quantitative real-time PCR analysis was performed on total RNA isolated from skeletal muscle (quadriceps) or liver. The relative gene expression level (the value of the fed sample is set as 100) is shown. Each value represents mean ± SE (n = 5). ***, P < 0.001; **, P < 0.01; *, P < 0.05. The gene expression levels in the liver (fed) relative to those in skeletal muscle sample (fed) are as follows: SREBP1c, 471; SREBP1a, 190; FAS, 792; RXR{gamma}, 4; RXR{alpha}, 173: RXRβ, 64; LXR{alpha}, 614; LXRβ, 129; and FOXO1, 212 (the value of the fed sample of skeletal muscle is set as 100); the values are shown above each column of the bar graph.

 
Among three RXR subtypes, only the RXR{gamma} gene expression was decreased by fasting, and it was restored by refeeding (Fig. 1Go). In the fed state, RXR{alpha} and RXR{gamma} were highly expressed at comparable levels, whereas RXRβ expression was relatively low compared with RXR{alpha} and RXR{gamma}. The relative gene expression values of RXR{alpha}, RXRβ, and RXR{gamma} are 100, 33, and 72 (supplemental Fig. 1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org), being consistent with a previous report (38). The gene expression of LXR{alpha} and LXRβ was not decreased, but rather increased, by fasting. By microarray analysis, we also examined gene expression of other nuclear receptor family members and found that only RXR{gamma} and neuronal growth factor-induced clone B (NGFI-B), an orphan nuclear receptor (39, 40, 41), are markedly decreased in skeletal muscle by fasting (supplemental Fig. 2). The FOXO1 gene expression was increased by fasting and decreased by refeeding, which is consistent with the data of our previous study (12).

For comparison, we examined the expression of the above genes in the mouse liver obtained simultaneously (Fig. 1Go, right). The relative expression level of each gene in liver of the fed mice compared with that in skeletal muscle of each gene is shown in Fig. 1Go. Consistent with previous reports (8, 11, 12), gene expression of SREBP1c, SREBP1a, and FAS in liver are decreased by fasting and restored by refeeding. It is of particular notice that in the fed mice, the RXR{gamma} gene expression is very low in liver (approximately 4% of that in skeletal muscle), where it is not decreased but rather increased by fasting (Fig. 1Go). The FOXO1 gene expression in liver was slightly increased by fasting and then decreased below the basal level by refeeding. These observations, taken together, indicate that among RXRs and LXRs, only RXR{gamma} gene expression is correlated to SREBP1c gene expression in skeletal muscle during fasting and refeeding. They also demonstrate that FOXO1 gene expression is negatively correlated to SREBP1c gene expression in skeletal muscle.

RXR{gamma}/LXR{alpha} stimulates the SREBP1c promoter activity in vitro
To examine whether RXR{gamma} and LXR{alpha} can activate the SREBP1c promoter, we performed luciferase reporter analysis. The SREBP1c promoter containing 550 bp upstream from the transcription start site of the SREBP1c gene contains two LXREs (20, 21, 23). Because LXR{alpha} and LXRβ have similar transcriptional activities (25, 42, 43), in this study, we used LXR{alpha} as a representative of LXR subtypes. As shown in Fig. 2AGo, in the absence of ligand, either RXR{gamma} or LXR{alpha} significantly increased the reporter Luc activity (P < 0.01). LXR{alpha} and RXR{gamma} synergistically increased the Luc activity (P < 0.001). The addition of 9-cis RA, which is a ligand for RXRs, stimulated the SREBP1c promoter activity even in the absence of RXR{gamma} and LXR{alpha} expression (see lanes 1 and 5), suggesting endogenous expression of RXR and LXR is sufficient to mediate effects in HEK293 cells. The addition of both 9-cis RA and LXR ligands (T0901317) further stimulated SREBP1c promoter activity (lanes 9–12). Mutation of the two consensus LXREs of the SREBP1c promoter markedly diminished ligand/receptor-induced activation of the SREBP1c promoter (lanes 13–16) (Fig. 2AGo). In addition to the RXR{gamma}/LXR{alpha} combination, coexpression of RXR{alpha} with LXR{alpha} also activated the SREBP1c promoter (not shown). We also observed RXR{gamma}/LXR{alpha}-dependent activation of the SREBP1c promoter in C2C12 myoblasts cells, similar to HEK293 cells (data not shown). These observations demonstrate that RXR{gamma}/LXR{alpha} stimulates the SREBP1c promoter activity in vitro.


Figure 2
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  		FIG. 2. In vitro analysis of SREBP1c gene expression by RXR{gamma}/LXR{alpha}. A, Transient transfection-reporter analysis of LXREs in the SREBP1c promoter. The SREBP1c promoter containing LXREs and mutated LXREs was fused to a luciferase reporter plasmid. This construct (SREBP1c-luc) or the mutant construct, with or without RXR{gamma} and/or LXR{alpha} expression vectors, was transfected into HEK293 cells. After an overnight transfection period, cells were treated with 1 µM 9-cis RA or 1 µM 9-cis RA plus 1 µM T0901317 for 24 h. The relative values compared with those obtained for mock transfection (lane 1, set as 100) are shown. Each value represents means ± SE (n = 3). For most points, the error bars are too small to be shown. B, Gel mobility shift assay of binding of RXR{gamma} and LXR{alpha} to LXREs in the SREBP1c promoter. Sequences of LXREa (–237 to –215) and LXREb (–186 to –164) in the mouse SREBP1c promoter and those of the mutated oligonucleotides are shown. Asterisks indicate the mutated nucleotide positions. Double-stranded oligonucleotides, LXREa (lanes 1–4) and LXREb (lanes 6–9) and the mutated oligonucleotides (lanes 5 and 10) were labeled and incubated with the in vitro-synthesized RXR{gamma} and LXR{alpha} recombinant proteins. The DNA-protein complexes were resolved in a 6% polyacrylamide gel. Asterisks in the autoradiogram denote nonspecific binding. The shifted LXRE probes are indicated by arrows (lanes 4 and 9). C, SREBP1c gene expression in cultured myocytes overexpressing RXR{gamma} and LXR{alpha}. RXR{gamma} and LXR{alpha} were overexpressed in C2C12 cells. The cells were treated with ligand (9-cis RA plus T0901317) for 24 h. Total RNA was isolated from the cells and analyzed by real-time PCR with primers for SREBP1c and FAS. Each value represents mean ± SE (n = 3). The relative values are shown (the control is set as 100). **, P < 0.01; ***, P < 0.001.

 
Binding of the RXR{gamma}/LXR{alpha} heterodimer complex to LXREs in the SREBP1c promoter in vitro
We performed gel mobility shift assay to examine the binding of the RXR{gamma}/LXR{alpha} heterodimer complex to the LXREs (LXREa and LXREb) in the SREBP1c promoter (Fig. 2BGo). In vitro-translated RXR{gamma} and LXR{alpha} proteins were tested to examine whether these proteins would bind to labeled LXREa or LXREb (20, 21, 23). As shown in Fig. 2BGo, both RXR{gamma} and LXR{alpha} were required for the binding to LXREa or LXREb. The signal intensity of the gel shift complex of RXR{gamma}/LXR{alpha} was similar to that of the complex of RXR{alpha}/LXR{alpha} (not shown). Mutation of the consensus LXREs abolished the binding of RXR{gamma} and LXR{alpha} (Fig. 2BGo, lanes 5 and 10). These observations suggest that RXR{gamma} and LXR{alpha} can activate the SREBP1c promoter via the LXREs.

Overexpression of RXR{gamma} and LXR{alpha} in C2C12 myoblasts increases SREBP1c gene expression
In this study, RXR{gamma} and LXR{alpha} were overexpressed in C2C12 myoblasts using retroviral vectors, and the endogenous SREBP1c gene expression was examined (Fig. 2CGo). Consistent with the transfection data, overexpression of RXR{gamma} and LXR{alpha} increased SREBP1c gene expression in the presence of ligands (9-cis RA and T0901317). We performed a ChIP analysis using C2C12 cells expressing RXR{gamma}/LXR{alpha} (as used in Fig. 2CGo) and observed that the RXR{gamma}/LXR{alpha} complex was recruited to the SREBP1c promoter (supplemental Fig. 3). In this study, the gene expression level of FAS increased in parallel to that of SREBP1c (Fig. 2CGo), suggesting that RXR{gamma} and LXR{alpha} increase functional SREBP1c protein expression and thereby up-regulate FAS gene expression. Because there is an LXRE in the FAS promoter (4), RXR{gamma}/LXR{alpha} also may affect directly FAS gene expression. These observations suggest that RXR{gamma}/LXR{alpha} increases the endogenous SREBP1c gene expression.

Increased SREBP1c gene expression in transgenic mice overexpressing RXR{gamma} in skeletal muscle
To examine the involvement of RXR{gamma} in the regulation of SREBP1c gene expression in skeletal muscle in vivo, we produced transgenic mice overexpressing RXR{gamma} under the control of the human skeletal muscle {alpha}-actin promoter. Figure 3AGo shows the schematic design of the plasmid construct used. This promoter is active only after birth and avoids any possible developmental effects of the transgene (36). We obtained two independent lines of transgenic mice (RXR{gamma} mice) harboring 10 (line 4-3) and eight (line 5-3) copies of the transgene. The tissue distribution examined by Northern blot analysis revealed the transgene expression only in skeletal muscle (gastrocnemius and quadriceps) but not in other tissues (Fig. 3BGo). SREBP1 gene expression (both SREBP1c and SREBP1a subtypes are hybridized by the probe used) was increased by 4- to 5-fold in skeletal muscle from RXR{gamma} mice but not other tissues, including liver and adipose tissue, indicating that the transgene produces functional RXR{gamma} protein and activates the SREBP1 gene expression in skeletal muscle. In this study, the SREBP1 gene expression level in skeletal muscle was similar to that in liver and adipose tissue of RXR{gamma} mice (Fig. 3BGo). The level of RXR{gamma} protein in skeletal muscle of transgenic mice was also analyzed by Western blotting. The RXR{gamma} protein level was about 10-fold higher in RXR{gamma} mice than in wild-type mice (Fig. 3CGo). Figure 3Go shows the data on line 5-3, and we obtained essentially the same results using line 4-3 (not shown). Table 1Go shows the data on blood parameters and body weights of both lines of RXR{gamma} mice. RXR{gamma} mice appeared to show no adverse effects on systemic glucose and lipid metabolism (Table 1Go).


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  		TABLE 1. Blood parameters in RXR{gamma} mice and their wild-type littermates

 
The gene expression changes in skeletal muscle of both lines of RXR{gamma} mice were examined by real-time PCR (Fig. 4Go). As expected, gene expression of RXR{gamma} was increased due to overexpression of the transgene. Consistent with the data obtained by Northern blot analysis (Fig. 3BGo), the SREBP1c gene expression was markedly increased in both lines of RXR{gamma} mice. Because fasting induced marked down-regulation of RXR{gamma} transgene expression under the control of the human skeletal muscle {alpha}-actin promoter (not shown), we examined SREBP1c gene expression in skeletal muscle from RXR{gamma} mice only when fed ad libitum. FAS gene expression was also increased in RXR{gamma} mice. Gene expression of RXR{alpha} was decreased in RXR{gamma} mice (both lines, P < 0.05 for line 4-3 and P < 0.01 for line 5-3), suggesting that overexpression of RXR{gamma} alters the expression of other RXR subtypes in skeletal muscle. Gene expression of RXRβ (P < 0.05) and LXRβ (P < 0.01) was slightly decreased in RXR{gamma} mice (line 5-3). LXR{alpha} and FOXO1 gene expression levels did not differ significantly between wild-type and RXR{gamma} mice.


Figure 4
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  		FIG. 4. Gene expression in skeletal muscle of RXR{gamma} mice. Quantitative real-time PCR analysis was performed on total RNA (5 µg per sample) isolated from skeletal muscle (quadriceps) from RXR{gamma} mice (line 4-3 and line 5-3, black bars) and respective wild-type controls (white bars). The names of the genes examined are on the top of the graph (the control value is set as 100). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Numbers of animals used are shown in Table 1Go. We used mice at 12 wk of age in this experiment, and we also observed increased gene expression of SREBP1c at 30 wk of age (not shown).

 
Next, we examined whether the increase in SREBP1c and FAS mRNAs led to the accumulation of triglyceride in skeletal muscle of RXR{gamma} mice. The triglyceride content was markedly increased in skeletal muscle of both lines of RXR{gamma} mice compared with control wild-type mice (Fig. 5Go). These observations suggest that RXR{gamma}-induced SREBP1c expression stimulates lipid synthesis and storage in skeletal muscle in vivo.


Figure 5
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  		FIG. 5. Triglyceride content in skeletal muscle of RXR{gamma} mice. Triglyceride content was measured (100 mg tissue weight per sample) isolated from skeletal muscle (quadriceps) from RXR{gamma} mice (line 4-3 and line 5-3, black bars) and respective wild-type controls (white bars). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Animals used are shown in Fig. 4Go and Table 1Go.

 
Decreased SREBP1c gene expression in transgenic mice overexpressing a DN form of RXR{gamma} in skeletal muscle
In addition to overexpression of RXR{gamma} (RXR{gamma} mice), we also attempted to block RXR{gamma} activity in skeletal muscle in vivo. We previously demonstrated that substitution of glutamic acid (E) 454 (located in the AF2 domain, which is conserved among many nuclear receptors) with glutamine (Q) in RXR{gamma} abolishes the interaction with coactivators, such as CBP/p300 (44). In the case of RXR{alpha}, deletion of the AF2 domain (19-amino-acid deletion) results in DN function in vivo in transgenic mice (45). In this study, we examined whether expression of RXR{gamma} (E454Q) can suppress the RXR{gamma}/LXR{alpha}-induced increase in the SREBP1c promoter activity. Initial studies in HEK293 cells showed that increasing amounts of RXR{gamma} (E454Q) clearly suppressed RXR{gamma}/LXR{alpha}-mediated activation of SREBP1c transcription (Fig. 6AGo), thereby establishing the RXR{gamma} mutant (E454Q) as a DN-RXR{gamma} (DN-RXR{gamma}). Gel shift studies confirmed that DN-RXR{gamma}/LXR{alpha} complexes can bind LXREs of SREBP1c promoter as well as RXR{gamma}/LXR{alpha} complexes in gel shift assays (not shown).

Using the human skeletal muscle {alpha}-actin promoter, we produced transgenic mice overexpressing DN-RXR{gamma} in skeletal muscle (DN-RXR{gamma} mice, Fig. 6BGo). As expected, the transgene was expressed specifically in skeletal muscle (not shown).

Table 2Go shows data on blood parameters of DN-RXR{gamma} mice, suggesting that they are healthy (Table 2Go). Interestingly, SREBP1c gene expression was markedly lower than that in wild-type mice in three independent lines of DN-RXR{gamma} mice (Fig. 6CGo). However, the gene expression of FAS was not decreased in DN-RXR{gamma} mice, suggesting that other factors besides SREBP1c contribute to the regulation of FAS in skeletal muscle under these conditions (46). Alternatively, the DN construct used in this study may not suppress all function of RXR-mediated transcriptional activity. We examined expression of other known SREBP1c target genes, including acetyl coenzyme A carboxylase 1 and 2 (ACC1 and ACC2), stearoyl coenzyme A desaturase 1 (SCD1), and the ATP-binding cassette transporter ABCD2, in skeletal muscle of RXR{gamma} mice and DN-RXR{gamma} mice. The results of quantitative real-time PCR analysis are shown in supplemental Tables 1 and 2. Gene expression of ACC1 and ABCD2 was increased in RXR{gamma} mice and decreased in DN-RXR{gamma} mice compared with their respective control mice. In contrast, gene expression of ACC2 and SCD1 did not differ both in RXR{gamma} mice and in DN-RXR{gamma} mice compared with their respective controls. This may reflect tissue-specific regulatory mechanisms involved in mediating effects of SREBP1c on target genes, which remain to be clarified. Together, these observations indicate that DN mutant of RXR{gamma} suppresses SREBP1c gene expression both in vivo and in vitro.


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  		TABLE 2. Blood parameters in DN-RXR{gamma} mice and their wild-type littermates

 
Decreased SREBP1c gene expression in transgenic mice overexpressing FOXO1 in skeletal muscle
Zhang et al. (28) reported that FOXO1 can suppress the expression of SREBP1c gene expression in liver and isolated hepatocytes. Furthermore, fasting increases FOXO1 gene expression in skeletal muscle (Fig. 1Go), and FOXO1 activity is expected to be increased under fasting conditions where insulin levels are low (27). It is, therefore, tempting to speculate that FOXO1 may function as a negative regulator of SREBP1c gene expression in skeletal muscle. In this study, we examined whether SREBP1c gene expression is decreased in transgenic mice overexpressing FOXO1 in skeletal muscle (FOXO1 mice) (37). In skeletal muscle from FOXO1 mice (two independent lines A1 and A2), the expression level of SREBP1c was markedly decreased (Fig. 7AGo). In addition, the gene expression of RXR{gamma}, but not those of RXR{alpha} or -β or LXR{alpha} or -β, also was decreased in both lines of FOXO1 mice (Fig. 7AGo and not shown). Blood glucose and insulin levels showed no difference between wild-type and transgenic mice (37) (and not shown). These observations indicate that increasing FOXO1 function decreases SREBP1c gene expression in skeletal muscle.


Figure 7
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  		FIG. 7. SREBP1c gene expression in skeletal muscle from FOXO1 mice. A, Gene expression of SREBP1c and RXR{gamma} in skeletal muscle from transgenic mice overexpressing FOXO1 (FOXO1 mice). Quantitative real-time PCR analysis in skeletal muscle from FOXO1 mice (lines A1 and A2, black bars) and age- and sex- matched wild-type control mice (open bars). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Numbers of animals used are as follows: line A1, n = 4; line A2, n = 6; and wild-type, n = 5. Results of female mice at 7 months of age are shown. We obtained essentially the same data using male mice (not shown). B, Effect of FOXO1 on the RXR{alpha}/LXR{alpha}-induced SREBP1c promoter activity. SREBP1c-luc with RXR{alpha} and LXR{alpha} was transfected into HEK293 cells. Constitutive nuclear form of FOXO1 expression plasmid was cotransfected. After an overnight transfection period, cells were treated with 1 µM 9-cis RA plus 1 µM T0901317 for 24 h. The relative values compared with those for mock transfection (set as 100) are shown. Each value represents means ± SE (n = 4). For some points, error bars are too small to be shown. ***, P < 0.001.

 
FOXO1 suppresses the RXR/LXR-stimulated SREBP1c promoter activity in vitro
FOXO1 is reported to suppress the function of multiple nuclear receptors such as ER and PPAR{gamma} (30, 31), but the effect of FOXO1 on the RXR/LXR transcriptional activity has not been reported so far. Thus, we examined whether FOXO1 can suppress the transcriptional activity of RXR/LXR on the SREBP1c promoter. Because RXR{gamma} gene expression is decreased during fasting, when FOXO1 gene expression is increased, we tested the effect of FOXO1 on the RXR{alpha}/LXR{alpha}-induced SREBP1c promoter activity. A constitutively nuclear form of FOXO1 (28 ; see Materials and Methods) was coexpressed with RXR{alpha}/LXR{alpha} and the SREBP1c-luc reporter in HEK293 cells. In this study, FOXO1 significantly repressed RXR{alpha}/LXR{alpha}-induced SREBP1c promoter activity (P < 0.001, Fig. 7BGo). Interestingly, in the absence of RXR{alpha}/LXR{alpha} expression vectors, FOXO1 did not suppress the SREBP1c promoter activity (Fig. 7BGo), indicating that this effect of FOXO1 is LXR/RXR-dependent. Similarly, FOXO1 also repressed RXR{gamma}/LXR{alpha}-stimulated SREBP1c promoter activity in 293 cells and in C2C12 cells (data not shown). At the same time, FOXO1 stimulated a control promoter containing two FOXO1 binding sites (47) more than 10-fold (not shown). These observations indicate that FOXO1 can down-regulate the SREBP1c promoter via the RXR/LXR-mediated transcriptional mechanism.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
SREBP1c is a master transcription factor that regulates the expression of a number of lipogenic genes in liver and adipose tissue, where it is largely regulated by a heterodimer of RXR{alpha} and LXR{alpha} (18, 19, 20, 21, 22, 23). Despite the potential importance of SREBP1c in skeletal muscle, the regulation of SREBP1c gene expression in skeletal muscle remains largely unknown. In this study, we demonstrate for the first time that RXR{gamma} gene expression is changed in skeletal muscle by nutritional conditions, in a manner correlated with the SREBP1c gene expression (Fig. 1Go). In vitro experiments showed that RXR{gamma} and RXR{alpha} combined with LXR{alpha} can activate the SREBP1c promoter and increase SREBP1c mRNA levels. Using RXR{gamma} (Figs. 3Go and 4Go) and DN-RXR{gamma} mice (Fig. 6Go), we also observed increased and decreased SREBP1c gene expression in skeletal muscle, respectively. Taken together, these observations suggest that RXR{gamma}/LXR{alpha} is a positive regulator of SREBP1c gene expression in skeletal muscle in vivo and in vitro. Because DN-RXR{gamma} also has a DN effect on RXR{alpha} (unpublished data) (and possibly RXRβ) as well as RXR{gamma} (Fig. 6AGo), the data of this study do not exclude the possible involvement of RXR{alpha} and RXRβ in the regulation of SREBP1c gene expression. Because LXRβ is also expressed in skeletal muscle (19) and LXR{alpha} and LXRβ have similar transcriptional activities (25, 42, 43), LXRβ also may be involved in the up-regulation of SREBP1c gene expression in skeletal muscle. Among nuclear receptors examined, only RXR{gamma} and NGFI-B gene expression are decreased in skeletal muscle by fasting (supplemental Fig. 1). In this regard, we have recently produced transgenic mice overexpressing NGFI-B specifically in skeletal muscle, where we found no appreciable increase in SREBP1c gene expression (Miura, S., and O. Ezaki, unpublished data). It is conceivable that NGFI-B is not important for the regulation of SREBP1c gene expression in skeletal muscle. Meanwhile, RXR heterodimerizes with not only LXR but also other nuclear receptors involved in metabolic regulation in skeletal muscle, such as thyroid hormone receptors (TRs) and PPARs (15, 16). The LXREs of the SREBP1c promoter is reported to be bound by TR (48). However, TR and thyroid hormone do not transactivate the mouse SREBP1c promoter through the LXREs but rather repress through a different element in the promoter (48). Thus, the increased SREBP1c gene expression in RXR{gamma} mice was not likely to have been mediated by TR. To our knowledge, no other nuclear receptor binding elements, including PPAR-responsive elements, have been reported in the promoter of SREBP1c. Given that among the nuclear receptors tested, only RXR{gamma} is functionally correlated to SREBP1c gene expression in skeletal muscle, these observations support the view that SREBP1c gene expression in skeletal muscle is mediated at least in part by the RXR{gamma}/LXR{alpha} heterodimer (model is shown in Fig. 8Go).


Figure 8
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  		FIG. 8. A proposed model for the regulation of SREBP1c gene expression in skeletal muscle during fasting (A) and refeeding (B). A, During the fasting state, gene expression of FOXO1 is increased and that of RXR{gamma} is decreased. Decreased RXR{gamma} level may decrease the activation of SREBP1c promoter. FOXO1, when increased, may act as a corepressor of the RXR{alpha}/β and LXR{alpha}/β heterodimer on the SREBP1c promoter. FOXO1 may also suppress RXR{gamma} promoters. Overall, SREBP1c gene expression is decreased by fasting. B, During refeeding, RXR{gamma} (or possibly RXR{alpha}/β) and LXR{alpha}/β heterodimer binds to LXREs of the SREBP1c promoter and activates its transcription. (For details, see the Discussion.) Overall, SREBP1c gene expression is increased by refeeding.

 
In this study, we also found that FOXO1 suppresses RXR/LXR-induced SREBP1c promoter transcriptional activity in vitro (Fig. 7BGo). We also observed decreased gene expression of SREBP1c in skeletal muscle from FOXO1 mice (Fig. 7AGo). These observations, taken together, suggest that FOXO1 can suppress the RXR/LXR-mediated SREBP1c gene expression in skeletal muscle in vivo. To our knowledge, there are no previous reports of the effect of FOXO1 on RXR/LXR-mediated gene transcription. There is considerable evidence for convergence of various nuclear receptors and FOXO1. For instance, FOXO1 antagonizes the transcriptional activities of ER and progesterone and glucocorticoid receptors, whereas it enhances those of TR and retinoic acid receptors (31). Schuur et al. (49) reported that FOXO1 enhances the transcriptional activity of ER, whereas ER antagonizes that of FOXO1. Moreover, FOXO1 antagonizes the ability of RXR{alpha}/PPAR{gamma} to activate transcription (30). FOXO1 also enhances the transcriptional activity of constitutive activator of retinoic acid response element (CAR) and pregnane X receptor (PXR), orphan nuclear receptors (50). In this context, Wagner et al. (43) previously reported that the RXR{alpha}/LXR{alpha}-mediated SREBP1c gene expression is negatively regulated by the corepressor complex including nuclear receptor corepressor (NCoR). Whether FOXO1 acts as a component of the complex on the SREBP1c promoter or interferes with RXR/LXR activation of the SREBP1c promoter at another level must await further investigation.

We also found that the expression of FOXO1 and RXR{gamma} are regulated in opposite directions in skeletal muscle during fasting (increased FOXO1, decreased RXR{gamma}) and refeeding (decreased FOXO1, increased RXR{gamma}) (Fig. 1Go). Furthermore, the gene expression of RXR{gamma} is reduced in mice overexpressing FOXO1 in skeletal muscle compared with wild-type mice. These observations suggest that FOXO1 also may down-regulate RXR{gamma} gene expression. A close inspection of the human and mouse RXR{gamma} upstream regulation regions revealed the presence of at least two different promoters that produce two different isoforms of RXR{gamma} (RXR{gamma}1 and RXR{gamma}2) in both species (26, 51, 52). RXR{gamma}1 and RXR{gamma}2 differ in their first exon, and RXR{gamma}1 is shorter than RXR{gamma}2 in its N terminus (26). RXR{gamma}1 and RXR{gamma}2 are expressed at similar levels in skeletal muscle (26), and the expression of both isoforms is decreased during fasting (supplemental Fig. 4). Gene expression of both RXR{gamma}1 and RXR{gamma}2 are also decreased in skeletal muscle from FOXO1 mice relative to wild-type mice (supplemental Fig. 4). Thus, the transcription activity of the two different promoters may be similarly regulated. Because there are no consensus core FOXO1 binding sites (5'-TTGTTTAC-3') (47) in the 2 kb upstream from exon 1 of the mouse RXR{gamma}1 and RXR{gamma}2 genes (26), FOXO1 may suppress RXR{gamma}1 and RXR{gamma}2 promoters through an indirect mechanism that remains to be determined.

In this study, to further understand the regulation of SREBP1c gene expression in skeletal muscle in vivo, we generated RXR{gamma} mice and DN-RXR{gamma} mice. We demonstrated a marked increase in SREBP1c gene expression and triglyceride content in skeletal muscle from RXR{gamma} mice, suggesting that RXR{gamma} mice may have increased lipogenesis in skeletal muscle. In this regard, Brown et al. (53) showed that RXR{gamma}-deficient mice (RXR{gamma}-knockout mice) have a higher metabolic rate relative to their wild-type littermates. Because RXR{gamma} is expressed not only in skeletal muscle but also in other tissues such as pituitary and brain, the metabolic phenotypes of RXR{gamma}-knockout mice may be attributable to RXR{gamma} deficiency in nonskeletal muscle tissues. However, given that skeletal muscle plays an important role in the regulation of energy expenditure (54, 55), we speculate that increased metabolic rate reported in RXR{gamma}-knockout mice may be mediated by RXR{gamma} deficiency in skeletal muscle (53). This is consistent with the concept that RXR{gamma} has an anabolic (lipogenic) effect in skeletal muscle through the up-regulation of SREBP1c. It is noteworthy that because RXR{gamma} can heterodimerize with multiple nuclear receptors (15, 16), complex gene expression changes may occur in skeletal muscle from RXR{gamma} mice and DN-RXR{gamma} mice. Additional studies with both RXR{gamma} and DN-RXR{gamma} mice will help to define the functional roles of RXR{gamma} in skeletal muscle. Detailed metabolic phenotypes are being analyzed in our laboratory.

Figure 8Go shows a proposed model for the regulation of SREBP1c gene expression in skeletal muscle by nutritional conditions. During fasting (Fig. 8AGo), RXR{gamma} gene expression is decreased in skeletal muscle, thereby leading to the reduction of the SREBP1c gene expression. Because RXR{alpha}/β gene expression is not decreased during fasting, they might replace RXR{gamma} on the SREBP1c promoter. On the other hand, FOXO1 gene expression is increased in skeletal muscle in fasting. Under these conditions, FOXO1 may act to suppress SREBP1c expression either directly, acting as a corepressor, or indirectly through interaction with other factors, e.g. by sequestering coactivators or stimulating the expression of repressor proteins. In addition, FOXO1 may suppress RXR{gamma} gene expression. Thus, FOXO1 appears to suppress SREBP1c through several mechanisms, both by reducing the level of RXR{gamma} and by suppressing transactivation by both RXR{alpha}/β/LXR and RXR{gamma}/LXR. So, even though RXR{gamma} is not the only form of RXR expressed in skeletal muscle, it is conceivable that FOXO1 suppresses transactivation of SREBP1c by RXR/LXR nucleoprotein complexes. During refeeding (Fig. 8BGo), the expression of RXR{gamma} is increased and RXR{gamma} (and possibly RXR{alpha}/β) can heterodimerize with LXR{alpha} (or LXRβ), binding to LXREs in the SREBP1c promoter and activate its transcription. Conversely, FOXO1 gene expression and function is down-regulated in skeletal muscle. Collectively, our data suggest that the RXR/LXR heterodimer promotes SREBP1c gene expression in skeletal muscle and contributes to negative regulation of SREBP1c by FOXO1.

In conclusion, this study provides new insight into the role of RXR{gamma} and FOXO1 in the regulation of SREBP1c gene expression, and the molecular mechanisms involved in the regulation of lipogenesis and triglyceride storage in skeletal muscle.


   Acknowledgments
 
We thank Dr. Christopher K. Glass (University of California, San Diego) and Dr. Ichizo Nishino (National Center of Neurology and Psychiatry, Tokyo, Japan) for discussion and critical reading of the manuscript.


   Footnotes
 
This work was supported in part by a Grant-in-Aid for scientific research KAKENHI from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (MEXT, Tokyo, Japan); Ground-Based Research Program for Space Utilization promoted by Japan Space forum; research grants from the Japanese Ministry of Health, Labor, and Welfare; a grant for the Promotion of Fundamental Studies in Health Sciences from the Organization for Pharmaceutical Safety and Research (OPSR) and National Institute of Biomedical Innovation (NIBIO); and research grants from Japan Diabetes Foundation, Astellas Foundation for Research on Metabolic Disorders, Ono Medical Research Foundation, Suzuken Memorial Foundation, Chiyoda Mutual Life Foundation, and the U.S. Department of Veterans Affairs Merit Review Program (T.G.U.).

Disclosure statement: The authors have nothing to disclose.

First Published Online January 17, 2008

Abbreviations: ACC, Acetyl coenzyme A carboxylase; ChIP, chromatin immunoprecipitation; DN, dominant-negative; ER, estrogen receptor; FAS, fatty acid synthase; FOXO1, Forkhead-O1 transcription factor; LXR, liver X receptor; LXRE, LXR response element; NGFI-B, neuronal growth factor-induced clone B; PPAR, peroxisome proliferators-activated receptor; RA, retinoic acid; RXR, retinoid X receptor; SCD1, stearoyl CoA desatulase 1; SREBP1c, sterol regulatory element binding protein 1c; TR, thyroid hormone receptor.

Received October 24, 2007.

Accepted for publication January 10, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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