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and Liver X Receptor (LXR) in Nutritional Regulation of Fatty Acid Metabolism. II. LXRs Suppress Lipid Degradation Gene Promoters through Inhibition of PPAR Signaling
Department of Internal Medicine (T.I., H.S., T.M., M.N., S.Y., A.T., H.S., N.S.), Institute of Clinical Medicine, University of Tsukuba, Ibaraki 305-8575, Japan; and Department of Metabolic Diseases (T.Y., N.Y., M.A.-K., Y.I., S.T., K.O., T.G., J.O., S.I.), Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113-8655, Japan
Address all correspondence and requests for reprints to: Hitoshi Shimano, M.D., Ph.D., Department of Internal Medicine, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan. E-mail: shimano-tky{at}umin.ac.jp.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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controls fatty acid degradation, whereas sterol regulatory element-binding protein-1c activated by liver X receptor (LXR) regulates fatty acid synthesis. To explore potential interactions between LXR and PPAR, the effect of LXR activation on PPAR signaling was investigated. In luciferase reporter gene assays, overexpression of LXR or ß suppressed PPAR-induced peroxisome proliferator response element-luciferase activity in a dose-dependent manner. LXR agonists, T0901317 and 22(R)-hydroxycholesterol, dose dependently enhanced the suppressive effects of LXRs. Gel shift assays demonstrated that LXR reduced binding of PPAR/retinoid X receptor (RXR) to peroxisome proliferator response element. Addition of increasing amounts of RXR restored these inhibitory effects in both luciferase and gel shift assays, suggesting the presence of RXR competition. In vitro protein binding assays demonstrated that activation of LXR by an LXR agonist promoted formation of LXR/RXR and, more importantly, LXR/PPAR heterodimers, leading to a reduction of PPAR/RXR formation. Supportively, in vivo administration of the LXR ligand to mice and rat primary hepatocytes substantially decreased hepatic mRNA levels of PPAR-targeted genes in both basal and PPAR agonist-induced conditions. The amount of nuclear PPAR/RXR heterodimers in the mouse livers was induced by treatment with PPAR ligand, and was suppressed by superimposed LXR ligand. Taken together with data from the accompanying paper (Yoshikawa, T., T. Ide, H. Shimano, N. Yahagi, M. Amemiya-Kudo, T. Matsuzaka, S. Yatoh, T. Kitamine, H. Okazaki, Y. Tamura, M. Sekiya, A. Takahashi, A. H. Hasty, R. Sato, H. Sone, J. Osuga, S. Ishibashi, and N. Yamada, Endocrinology 144:1240–1254) describing PPAR suppression of the LXR-sterol regulatory element-binding protein-1c pathway, we propose the presence of an intricate network of nutritional transcription factors with mutual interactions, resulting in efficient reciprocal regulation of lipid degradation and lipogenesis. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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is abundantly expressed in tissues, which have high lipid catabolic activity, such as liver, kidney, heart, skeletal muscle, and brown adipose tissue (4, 5). PPAR is activated by fatty acids, eicosanoids, and fibrates, a known class of hypolipidemic drugs. Like other nuclear receptors such as: TR, VDR, and retinoic acid receptor (RAR) (6, 7, 8, 9), PPAR forms a heterodimer with RXR, which enhances its binding to DNA sequence elements termed peroxisome proliferator response elements (PPRE) (10, 11, 12). PPREs have been recently identified in the 5'-flanking sequences of genes involved in lipid degradation such as the ACO [acyl-coenzyme A (CoA) oxidase] (13), mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mHMG-CoA Syn) (14), L-carnitine palmitoyltransferase (CPTI) (15), and L-fatty acid binding protein genes (16). Studies using PPAR-deficient mice have established that PPAR plays a crucial role in fatty acid degradation (17, 18, 19).
LXRs (LXR/NR1H3 and LXRß/NR1H2) were identified as orphan nuclear receptors and are now thought to regulate the metabolism of several important lipids, including cholesterol and bile acid (20, 21). LXRs regulate intracellular cholesterol levels by induction of the gene expression of cholesterol 7-hydroxylase (22, 23), which is the rate-limiting enzyme of the classic bile acid synthesis pathway, and ATP-binding cassette transporter A1 (ABCA1) (24), which modulates apolipoprotein mediated-efflux of cholesterol. Further evidence supporting an important role of LXR in lipid homeostasis is provided by the loss of capacity to regulate catabolism of dietary cholesterol in LXR deficient mice, an effect for which the isoform, LXRß, could not compensate (25). Differences in the physiological functions between LXR and LXRß including target and tissue specificity have been suggested (26). LXRß is ubiquitously expressed, whereas LXR is restricted to metabolically active tissues, such as liver, kidney, intestines, and adrenal glands (27, 28). Recently, we (29) and others (30) reported that both LXR and ß are dominant activators for SREBP-1c/adipocyte determination and differentiation 1 (ADD1). Previous in vivo studies established that SREBP-1c plays a crucial role in the dietary regulation of most hepatic genes of fatty acid synthetic enzymes (31, 32, 33, 34). Therefore, LXRs could be also important in fatty acid synthesis (35).
As PPAR and LXR-SREBP-1c are reciprocal regulators for fatty acid metabolism, nuclear receptors could interact with each other as was previously observed in a cross-talk between PPAR and TR (36, 37). The current study examined the effects of LXR activation on PPAR signaling. The results demonstrate that LXR ligand activation represses PPAR signaling through reduction of stimulated-PPAR/RXR heterodimerization in the liver. Taken together with the accompanying paper (38) describing PPAR suppression of the LXR-SREBP-1c pathway, we propose a novel aspect of nutritional regulation with these mutual interactions forming a network of transcription factors regulating fatty acid metabolism.
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RESULTS |
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-Mediated Transactivation activation on PPAR signaling, transfection studies with human embryonic kidney (HEK) 293 cells were performed using luciferase (Luc) reporter gene assays containing a PPRE from the ACO gene promoter [pPPRE-Luc (PPRE-Luc plasmid)], a representative PPAR target. As shown in Fig. 1
, the Luc activity of PPRE-Luc was markedly (60-fold) induced by cotransfection of PPAR due to lack of endogenous PPAR expression in HEK293 cells (data not shown). The PPAR-inducible expression of Luc activity was slightly, but dose dependently suppressed by coexpression of LXR (Fig. 1A) (50% inhibition at a DNA dose ratio of 1:10). Expression level of transfected PPAR or LXR gene in HEK293 cells were roughly comparable to that in mouse liver as estimated by Northern blotting, and thus was within a physiological range (data not shown). This observation is consistent with a previous report describing LXR interaction with PPAR signaling (39). More interestingly, addition of LXR ligands such as 22RHC and T0901317 markedly enhanced the inhibitory effects of LXR. LXRß alone, without an LXR ligand, substantially inhibit PPAR activation of PPRE-Luc in a dose-dependent manner (Fig. 1B). A 50% inhibition was observed at a DNA ratio of 1:5. Additional LXR ligands further augmented the inhibitory effect of LXRß. These effects by LXRs and LXR ligands are not due to direct inhibition of Luc activity because Luc activity from constitutive expression of Luc gene by TK promoter-Luc construct was not affected by LXR coexpression, by addition of LXR ligand, or a combination of both (Fig. 1C). These data demonstrate that LXRs and their ligands inhibit PPAR signaling with overexpressing nuclear receptors system.
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/RXR Binding to PPRE. As a positive control, a dose-response curve of LXRE (LXR response element)-Luc activation by T0901317 and 22RHC with coexpression of LXR is shown in the upper panel, which is consistent with previous data (29). With a similar efficiency, these LXR agonists inhibited PPAR activation of PPRE-Luc, making both curves a mirror image of each other. LXRß coexpression gave similar results with a slightly higher efficiency.
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by LXR, gel mobility shift assays were performed. The binding specificity of LXR/RXR to LXRE, and PPAR/RXR to PPRE was confirmed in the experiments shown in Fig. 3, A and B. RXR is required for specific binding of both LXR and PPAR. Neither LXR/RXR nor LXRß/RXR bound to PPRE within the sensitivity of this assay. Coincubation of PPAR and LXR or ß essentially caused no shift using PPRE probe. These data exclude the possibility that LXR/RXR competes with PPAR/RXR binding to a response element. Next, PPAR/RXR binding to PPRE was estimated in the presence of LXR and/or its ligand (Fig. 4). PPAR/RXR binding to PPRE was partially inhibited by addition of LXR protein. LXR ligand enhanced this inhibition. These data are consistent with results from reporter assays, suggesting that suppression of PPAR activity by LXR is mediated at least partly through inhibition of PPAR/RXR binding to PPRE.
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/RXR-Mediated Transactivation and LXRs share RXR as a heterodimer partner for their specific functions, it is conceivable that LXR inhibition of PPAR activation on PPRE-Luc is mediated through competition for RXR when RXR is in limiting amounts in cultured cells. Thus, the effect of supplementation with RXR on the LXR inhibition of PPRE-Luc activity was evaluated (Fig. 5). By cotransfection of an increasing amount of RXR, PPAR-activated PPRE-Luc activity was enhanced, indicating that the amount of RXR is not saturated for PPAR/RXR activation (Fig. 5A). Figure 5B shows effect of each amount of RXR on the percent inhibition of PPAR-induced PPRE-Luc values by LXR activation (LXR coexpression with or without an LXR ligand, T0901317 or 22RHC). The inhibitory efficiency of LXR activation was attenuated by coexpressed RXR in a dose-dependent manner (Fig. 5B). Similar results were obtained in the repression by LXRß (Fig. 5C).
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). TRß strongly suppressed PPRE-Luc activity, which is consistent with a previous report (37). Inhibition by FXR was modest. The ability of inhibitory action on PPAR-mediated transactivation appears to be different among LXR, TRß, and FXR, suggesting their constitutive activity based on levels of intracellular natural ligands may influence cross-talk in heterodimeric receptors with RXR.
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-induced PPRE-Luc activity (Fig. 6B
).
The RXR restoration of LXR-dependent suppression was also evaluated in gel mobility shift assays. Without LXR, the signal of PPRE shifted by PPAR was enhanced with increasing amounts of RXR, suggesting that the amount of RXR is a limiting factor in the range of PPAR/RXR ratio used here (Fig. 7A). With the lowest amount of RXR, addition of LXRs and T0901317 caused a significant decrease in PPAR/RXR binding to PPRE (Fig. 7, A and B). The inhibitory effect of LXR activation was abolished with increasing concentrations of RXR. In the case of LXRß, RXR restoration of the binding was partial even at the highest amount, reflecting stronger LXRß inhibitory effect. These data are consistent with the results from cell reporter assays (Fig. 5), providing additional evidence for the hypothesis that LXR inhibits PPAR activation by competing with it for heterodimerization with RXR.
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and Inhibits Formation of PPAR/RXR Heterodimer). First, LXR protein was labeled with 35S-methionine, incubated with unlabeled RXR protein, and precipitated with anti-RXR antibody. Resolution of immunocomplex on SDS-PAGE detected labeled LXR, indicating formation of an LXR/RXR heterodimer. The complex formation was markedly enhanced by addition of T0901317, suggesting that LXR ligand increases affinity for LXR/RXR heterodimerization (Fig. 8A). We also tested the possibility of formation of PPAR/LXR heterodimers, as was previously described (39). Labeled LXR was incubated with PPAR and immunoprecipitated with anti-PPAR antibody. PPAR/LXR complex was detected with an intensity comparable to LXR/RXR, suggesting that LXR can heterodimerize with PPAR with similar efficiency to RXR (Fig. 8C). T0901317 enhanced this interaction. LXRß gave very similar results (Fig. 8, B and D). The gel shift assay (Fig. 3B) showed no retardation of the PPRE probe in the presence of both PPAR and LXR, indicating that PPAR/LXR heterodimers do not bind to PPRE. Therefore, LXR could show inhibition of PPAR signaling by forming not only LXR/RXR, but also PPAR/LXR in a competition with functional PPAR/RXR formation. To show this, effects of LXRs on PPAR/RXR interaction were estimated in this protein interaction assay. PPAR protein was labeled, incubated with RXR in the presence or absence of LXR, precipitated with anti-RXR antibody, and run on gels. PPAR/RXR heterodimer formation was inhibited by addition of LXR or LXRß with T0901317 (Fig. 8, E and F), suggesting that PPAR/LXR as well as LXR/RXR could prevent formation of PPAR/RXR and participate in repressing PPAR signaling.
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/RXR Binding to PPRE in Hepatic Nuclear Extracts and RXR were abundant on immunoblot analysis, although PPAR was decreased after refeeding (Fig. 9A) as well as at mRNA level. Gel shift mobility assays demonstrated that the PPRE probe was shifted by coincubation with hepatic nuclear extracts. The signal of the complexes was stronger from fasted mice than in a refed state, was enhanced by addition of Wy14,643 and declined after addition of anti-PPAR or anti-RXR antibody, confirming the presence of PPAR/RXR in the hepatic nuclear extracts from fasted mice and its specific binding to PPRE (Fig. 9B). The PPRE complex also contained a small amount of unidentified RXR heterodimer(s). T0901713 suppressed the signal of the PPAR/RXR-PPRE complex induced by Wy14,643 (Fig. 9C). Thus, these data indicate that LXR ligand suppressed PPAR ligand-induced PPAR/RXR-PPRE complexes in a condition with physiological levels and spectrum of various nuclear receptors.
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Target Gene in Primary Hepatocytes and Mouse Livers signaling was estimated using their ligands in rat primary hepatocytes. Hepatocytes were incubated with increasing amounts of T0901317 and expression of related target genes was estimated by Northern blot analysis (Fig. 10). LXR activation was confirmed by a dose-dependent increases in SREBP-1c and ABCA1 mRNA levels (Fig. 10A). LXR ligand dose dependently suppressed a representative PPAR gene, mHMG-CoA syn mRNA level. In the presence of Wy14,643 at a concentration sufficient to induce mHMG-CoA syn, a clear dose-dependent suppression by T0901317 was still observed (Fig. 10B). These data demonstrate that LXR activation by its ligand suppresses PPAR-target genes, in a competitive manner with PPAR activation, supporting LXR interference with PPAR signaling in primary hepatocytes. To further evaluate the in vivo physiological significance of LXR interference to PPAR-mediated transactivation, similar conditions in mouse livers were recreated. Mice were fasted to induce hepatic endogenous PPAR and its downstream genes (17, 31) and were treated with T0901317 to activate LXR. Various PPAR-target genes in the livers such as mHMG CoA Syn, ACO, CPTI, and cytochrome P450A2 were estimated by Northern blot analysis. Wy14,643 was also administered to fasted mice as a positive control for activation of PPAR target genes. As shown in Fig. 11A, hepatic mRNA levels of these PPAR-regulated genes from T0901317 treated animals were considerably decreased, whereas Wy14643 treatment increased expression of these genes. The activation of LXR by T0901317 was confirmed by observational increases in SREBP-1 and ABCA1 mRNA levels, both of which are well-known LXR target genes (Fig. 11B). These data demonstrate that LXR activation by LXR ligand can affect PPAR signaling in mouse livers. In contrast, Wy14,643 alone slightly increased ABCA1 mRNA (Fig. 11B), which is consistent with recently reports of PPAR-LXR-ABCA1 pathway in macrophages (43, 44, 45). Moreover, we studied LXR interference in mice treated with both PPAR and LXR ligands. As estimated by Northern blot analysis of mHMG-CoA syn and ACO mRNA levels, these PPAR-target genes were suppressed by T0901317 in both Wy14,643-induced and untreated conditions (Fig. 11D). Immunoprecipitation assays of liver nuclear extracts from these mice demonstrated that PPAR/RXR heterodimers were increased by Wy14,643 treatment and this induction was suppressed by T0901317. These results suggested that LXR ligand could repress hepatic expression of the PPAR target genes via reduction of PPAR/RXR formation in liver nucleus (Fig. 11E), extending the hypothesis from transfection studies to in vivo conditions.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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signaling. The Luc, gel shift, and protein-protein binding assays show that LXR activation inhibits formation of PPAR/RXR, and thus, their binding to PPRE. RXR supplementation restored this LXR inhibition of PPAR action, indicating that the mechanism could be at least partially explained by RXR competition between PPAR and LXR. Protein-protein interaction experiments established the formation of PPAR/LXR and PPAR/LXRß as well as LXR/RXR heterodimers. Direct binding between PPAR and LXR has been found in a two-hybrid system, and involvement of LXR in PPAR signaling has been implicated (39). We further found that PPAR/LXR formation is enhanced by LXR ligand. PPAR/LXR complex cannot bind to either PPRE or LXRE. Therefore, this heterodimer can interfere with both PPAR and LXR action and can contribute to PPAR inhibition of the LXR-SREBP-1c system as proposed in the accompanying paper (38) as well as LXR inhibition of PPAR signaling as described in the current study. Consistently, the combined treatment with both PPAR and LXR agonists resulted in simultaneous reduction of both PPAR/RXR and LXR/RXR [Fig. 11E in this paper and Fig. 9B in the accompanying paper (38)], implicating an important contribution of PPAR/LXR formation in reciprocal inhibition of LXR and PPAR signaling. This can also explain the restoration by supplementation with RXR, not only by providing sufficient RXR for functional RXR heterodimers, but also by preventing nonfunctional PPAR/LXR formation.
LXR inhibition of PPAR signaling observed from transfection studies with cultured cells is likely to be extendable to the in vivo regulation of the hepatic energy metabolism. It is noteworthy that administration of LXR agonist reduced hepatic nuclear PPAR/RXR and impaired expression of hepatic fatty acid degradation enzyme genes both in rat primary hepatocytes and livers of fasted mice (Figs. 10 and 11). In a fasted state, hepatic PPAR expression is highly induced and fatty acids are recruited as ligands for PPAR activation (17, 18). Nutritional condition does not markedly change the expression of hepatic LXR (29). Therefore, LXR ligand can activate hepatic LXR and cause cross-talk with PPAR/RXR leading to suppression of expression of PPAR target genes involved in lipid degradation. These data demonstrate that LXR interference with PPAR signaling can occur in vivo. In the accompanying paper (38), we observed a mirror image observation, that PPAR activation inhibits ligand-induced LXR signaling in hepatocytes.
The triangle relationship among LXR, PPAR, and RXR could be crucial for mutual regulation of both LXR and PPAR activities, and thus nutritional regulation of their downstream genes. PPAR is involved in fatty acid degradation as an adaptic control of energy depletion. Meanwhile, SREBP-1c, whose expression is dominated by LXR is involved in fatty acid synthesis for storage of excess energy. These opposite nutritional regulators are reciprocally up- and down-regulated depending upon energy states. As summarized in Fig. 12, our findings suggest that the mutual suppression efficiently facilitates these reciprocal actions of PPAR and LXR-SREBP-1c systems. These studies should open up a new paradigm of a novel cross-talk of nutritional transcription factors in energy metabolism where the nuclear concentrations of each receptor and ligand are crucial for nutritional regulation for fatty acid metabolism. It is also important to investigate whether the cross-talk of these receptors involves a potential competition for their coactivators such as CBP/p300 (42, 46, 47), steroid receptor coactivator-1 (48, 49, 50), and PPAR
coactivator-1 (51, 52). Further studies should focus on the precise mechanisms for this network.
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MATERIALS AND METHODS |
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sc-9000, RXR D20 sc-553, and LXR C-19 sc-1201 antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA), Redivue [-32P]deoxy-CTP (6,000 Ci/mmol) and Protein G Sepharose from Amersham Biosciences Inc. (Uppsala, Sweden), in vitro transcription and translation kit (TNT Coupled Reticulocyte Lysate System) from Promega (Madison, WI), and other chemicals from Sigma (St. Louis, MO). T0901317 (N-methyl-N-[4-(2, 2, 2- trifluoro-1-hydroxy-1-trifluoromethylethyl)-phenyl]-benzenesulfonamide) was provided by Kyorin Pharmaceutical Co. Ltd. (Tochigi, Japan).
Plasmids
Cytomegalovirus (CMV) promoter expression plasmid of human RXR and the Luc reporter gene construct, PPRE from ACO gene promoter fused upstream from thymidine kinase (TK) promoter (pPPRE-Luc), and pCMV-RXR were kind gifts from Dr. D. J. Mangelsdorf (University of Texas Southwestern Medical Center, Dallas, TX). pCMV-FXR, pCMX-TRß, and pCMV-CBP were from Dr. H. Fujii (Graduate School of Agricultural and Life Science, The University of Tokyo, Tokyo, Japan), Dr. R. M. Evans (The Salk Institute for Biological Studies, La Jolla, CA), and Dr. T. Nakajima (St. Marianna University School of Medicine, Kawasaki, Japan), respectively. The LXRE of SREBP-1c promoter-Luc construct (pLXRE-Luc), pCMV-mLXR and pCMV-mLXRß were prepared as previously described (29). CMV or T7 promoter expression plasmid of mouse (m) PPAR (1–468, amino acid) (16) was prepared by PCR, and the cDNA was introduced into pCMV7 or pBluescript II SK vector, respectively. T7 promoter expression plasmids of mLXR and mLXRß were prepared using pBluescript II SK vector.
Transfections and Luc Assays
HEK293 were grown at 37 C in an atmosphere of 5% CO2 in DMEM containing 25 mM glucose, 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate supplemented with 10% fetal bovine serum (FBS). Transfection studies were carried out with cells plated on 12-well plates as previously described (29). The indicated amount of each expression plasmid was transfected simultaneously with a Luc reporter plasmid (0.25 µg) and simian virus 40-ß-galactosidase (ß-gal) plasmid (0.2 µg) or (0.05 µg). The total amount of DNA in each transfection was adjusted to 1.5 µg/well with the vector DNA, pCMV7. 22RHC and T0901713 were dissolved in ethanol. Each agent was treated to the cells immediately after transfection in DMEM with 10% FBS, and incubated for 24 h. After incubation, the amount of Luc activity in transfectants was measured and normalized to the amount of ß-gal activity.
Gel Mobility Shift Assays
Gel mobility shift assays were performed as previously described (53). Briefly, mLXR, mLXRß, mPPAR, and human (h) RXR proteins were generated from the expression vectors using a coupled in vitro transcription/translation system (Promega). Double-stranded oligonucleotides used in gel mobility shift assays were the LXREb of the SREBP-1c promoter (29) and PPRE of the ACO promoter (16). These were then labeled with [-32P]deoxy-CTP by klenow enzyme, followed by purification on sephadex G50 columns. The labeled probes (30,000–100,000 cpm) were incubated with lysates (1 to 4 µl), or hepatic nuclear extract (1 µg) in a mixture (20 µl) containing 10 mM HEPES (pH 7.6), 50 mM KCl, 0.05 mM EDTA, 2.5 mM MgCl2, 8.5% glycerol, 1 mM dithiothreitol, 0.4 µg/ml poly(deoxyinosine-deoxycytidine), 0.1% Triton X-100, and 1 mg/ml nonfat milk for 60 min on ice. The DNA-protein complexes were resolved on a 4.5% PAGE at 100 V for 2 h at 4 C. Gels were dried and exposed to the filter of BAS2000 with BAStation software (Fuji Photo Film, Kanagawa, Japan).
Coimmunoprecipitation of Receptors
In vitro translated [35S]methionine-labeled receptors with unlabeled receptors or hepatic nuclear extracts from ligand-treated mice were brought to a final volume of 20 or 200 µl with buffer containing 10 mM HEPES (pH 7.6), 50 mM KCl, 0.05 mM EDTA, 2.5 mM MgCl2, 8.5% glycerol, 1 mM dithiothreitol, 0.1% Triton X-100, and 1 mg/ml nonfat milk for 2 h at 4 C and incubated with 10 µl of rabbit or goat polyclonal antibodies binding to protein G-sepharose for overnight at 4 C. The precipitations were washed with PBS containing 0.2% Tween-20 and 3% BSA. After microcentrifugation, the pellet was washed four times with 1 ml of ice-cold PBS containing 0.2% Tween-20. Twenty microliters of SDS-PAGE sample buffer were added to the final pellet and boiled for 5 min at 95 C. The supernatant was subjected to electrophoresis on 10% SDS-PAGE.
Hepatocyte Isolation and Culture
Primary hepatocytes were isolated from male Sprague- Dawley rats (160–180 g, Japan Clea, Tokyo, Japan) using the collagenase perfusion method as described previously (54). The viability of isolated cells was over 90% as determined by the trypan blue. Cells were resuspended in DMEM containing 100 U/ml penicillin and 100 µg/ml streptomycin sulfate supplemented with 5% FBS, seeded on collagen-coated dishes 100 mm at a final density of 4 x 104 cells/cm2. After an attachment for 4 h, cells were cultured with medium containing the indicated agonists for 24 h.
Animals
Male mice (C57BL/6J) were obtained from Charles River Japan (Yokohama, Japan). All mice were given a standard diet and tap water ad libitum. All institutional guidelines for animal care and use were applied in this study. Vehicle (0.5% carboxymethyl-cellulose) T0901317 (50 mg/kg), Wy14,643 (50 mg/kg), or both their agonists was orally administered to the mice before 18 h fasting. For fasting and refeeding treatment, mice were fasted for 24 h and fed a high sucrose/fat free diet for 12 h as described (55). Hepatic nuclear protein were prepared from the livers as previously described (56), were subjected to immunoblotting with the anti-PPAR, anti-LXR, or anti RXR antibodies.
Northern Blot Analysis
Total RNA was extracted from livers and rat primary hepatocytes using TRIZOL Reagent (Invitrogen Corp., Carlsbad, CA). Equal aliquots of total RNA from mice in each group were pooled (total 10 µg), subjected to formalin-denatured agarose electrophoresis, and transferred to nylon membrane (Hybond N, Amersham Pharmacia Biotech, Uppsala, Sweden). Blot hybridization was performed with the cDNA probes labeled with [-32P]CTP (6000 Ci/mmol) using the Megaprime DNA Labeling System (Amersham Biosciences Inc.). The cDNA probes for SREBP-1, fatty acid synthase, ACO, cytochrome P450A2, PPAR, LXR and ß, ABCA1 and acidic ribosomal phosphoprotein PO (36B4) were prepared as previously described (29, 31). The cDNA probes for L-FABP, CPTI, and mHMG-CoA Syn were provided by Kyorin Pharmaceutical Co. LTD. Each signal was analyzed with BAS2000 and BAStation software (Fuji Photo Film).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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T.I. and H.S. equally contributed to this work.
Abbreviations: ABCA1, ATP-binding cassette transporter A1; ACO, acyl-CoA oxidase; 36B4, acidic ribosomal phosphoprotein PO; CBP, cAMP-response element binding protein-binding protein; CMV, cytomegalovirus; CoA, coenzyme A; CPTI, L-carnitine palmitoyltransferase; FBS, fetal bovine serum; FXR, farnesoid X receptor; ß-gal, ß-galactosidase; HEK, human embryonic kidney; Luc, luciferase; LXR, liver X receptor; LXRE, liver X receptor response element; m, mouse; mHMG-CoA syn, mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase; PGC-1, peroxisome proliferator-activated receptor coactivator-1; PPAR, peroxisome proliferator-activated receptor; PPRE, peroxisome proliferator responsive element; pSV, simian virus 40 promoter plasmid; 22RHC, 22(R)-hydroxycholesterol; RXR, retinoid X receptor; SREBP-1c, sterol regulatory element-binding protein 1c; TK, thymidine kinase; TR, thyroid hormone receptor; VDR, vitamin D3 receptor.
Received for publication May 23, 2002. Accepted for publication April 14, 2003.
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Y.-Y. Liu, R. S. Heymann, F. Moatamed, J. J. Schultz, D. Sobel, and G. A. Brent A Mutant Thyroid Hormone Receptor {alpha} Antagonizes Peroxisome Proliferator-Activated Receptor {alpha} Signaling in Vivo and Impairs Fatty Acid Oxidation Endocrinology, March 1, 2007; 148(3): 1206 - 1217. [Abstract] [Full Text] [PDF]
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 M. C. Rudolph, J. L. McManaman, T. Phang, T. Russell, D. J. Kominsky, N. J. Serkova, T. Stein, S. M. Anderson, and M. C. Neville Metabolic regulation in the lactating mammary gland: a lipid synthesizing machine Physiol Genomics, February 12, 2007; 28(3): 323 - 336. [Abstract] [Full Text] [PDF]
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 R. J Deckelbaum, T. S Worgall, and T. Seo n-3 Fatty acids and gene expression Am. J. Clinical Nutrition, June 1, 2006; 83(6): S1520 - 1525S. [Abstract] [Full Text] [PDF]
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 M. Loffler, M. Bilban, M. Reimers, W. Waldhausl, and T. M. Stulnig Blood Glucose-Lowering Nuclear Receptor Agonists Only Partially Normalize Hepatic Gene Expression in db/db Mice J. Pharmacol. Exp. Ther., February 1, 2006; 316(2): 797 - 804. [Abstract] [Full Text] [PDF]
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 S. K. Cheema, A. Agarwal-Mawal, C. M. Murray, and S. Tucker Lack of stimulation of cholesteryl ester transfer protein by cholesterol in the presence of a high-fat diet J. Lipid Res., November 1, 2005; 46(11): 2356 - 2366. [Abstract] [Full Text] [PDF]
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 C. Le May, M. Cauzac, C. Diradourian, D. Perdereau, J. Girard, A.-F. Burnol, and J.-P. Pegorier Fatty Acids Induce L-CPT I Gene Expression through a PPAR{alpha}-Independent Mechanism in Rat Hepatoma Cells J. Nutr., October 1, 2005; 135(10): 2313 - 2319. [Abstract] [Full Text] [PDF]
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M. L. Fernandez and K. L. West Mechanisms by which Dietary Fatty Acids Modulate Plasma Lipids1 J. Nutr., September 1, 2005; 135(9): 2075 - 2078. [Abstract] [Full Text] [PDF]
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 S. P. Anderson, C. Dunn, A. Laughter, L. Yoon, C. Swanson, T. M. Stulnig, K. R. Steffensen, R. A.S. Chandraratna, J.-A. Gustafsson, and J. C. Corton Overlapping Transcriptional Programs Regulated by the Nuclear Receptors Peroxisome Proliferator-Activated Receptor {alpha}, Retinoid X Receptor, and Liver X Receptor in Mouse Liver Mol. Pharmacol., December 1, 2004; 66(6): 1440 - 1452. [Abstract] [Full Text] [PDF]
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 T. Kino and G. P. Chrousos Combating Atherosclerosis With LXR{alpha} And PPAR{alpha} Agonists: Is Rational Multitargeted Polypharmacy the Future of Therapeutics in Complex Diseases? Mol. Interv., October 1, 2004; 4(5): 254 - 257. [Abstract] [Full Text] [PDF]
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C. G Taylor and P. Zahradka Dietary conjugated linoleic acid and insulin sensitivity and resistance in rodent models Am. J. Clinical Nutrition, June 1, 2004; 79(6): 1164S - 1168S. [Abstract] [Full Text] [PDF]
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 J. M. Paterson, N. M. Morton, C. Fievet, C. J. Kenyon, M. C. Holmes, B. Staels, J. R. Seckl, and J. J. Mullins Metabolic syndrome without obesity: Hepatic overexpression of 11{beta}-hydroxysteroid dehydrogenase type 1 in transgenic mice PNAS, May 4, 2004; 101(18): 7088 - 7093. [Abstract] [Full Text] [PDF]
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 D. S. Ng, C. Xie, G. F. Maguire, X. Zhu, F. Ugwu, E. Lam, and P. W. Connelly Hypertriglyceridemia in Lecithin-cholesterol Acyltransferase-deficient Mice Is Associated with Hepatic Overproduction of Triglycerides, Increased Lipogenesis, and Improved Glucose Tolerance J. Biol. Chem., February 27, 2004; 279(9): 7636 - 7642. [Abstract] [Full Text] [PDF]
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 G. F. Gbaguidi and L. B. Agellon The inhibition of the human cholesterol 7{alpha}-hydroxylase gene (CYP7A1) promoter by fibrates in cultured cells is mediated via the liver x receptor {alpha} and peroxisome proliferator-activated receptor {alpha} heterodimer Nucleic Acids Res., February 11, 2004; 32(3): 1113 - 1121. [Abstract] [Full Text] [PDF]
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