Interrelationship between Liver X Receptor α, Sterol Regulatory Element-binding Protein-1c, Peroxisome Proliferator-activated Receptor γ, and Small Heterodimer Partner in the Transcriptional Regulation of Glucokinase Gene Expression in Liver*

Liver glucokinase (LGK) plays an essential role in controlling blood glucose levels and maintaining cellular metabolic functions. Expression of LGK is induced mainly regulated by insulin through sterol regulatory element-binding protein-1c (SREBP-1c) as a mediator. Since LGK expression is known to be decreased in the liver of liver X receptor (LXR) knockout mice, we have investigated whether LGK might be directly activated by LXRα. Furthermore, we have studied interrelationship between transcription factors that control gene expression of LGK. In the current studies, we demonstrated that LXRα increased LGK expression in primary hepatocytes and that there is a functional LXR response element in the LGK gene promoter as shown by electrophoretic mobility shift and chromatin precipitation assay. In addition, our studies demonstrate that LXRα and insulin activation of the LGK gene promoter occurs through a multifaceted indirect mechanism. LXRα increases SREBP-1c expression and then insulin stimulates the processing of the membrane-bound precursor SREBP-1c protein, and it activates LGK expression through SREBP sites in its promoter. LXRα also activates the LGK promoter by increasing the transcriptional activity and induction of peroxisome proliferator-activated receptor (PPAR)-γ, which also stimulates LGK expression through a peroxisome proliferator-responsive element. This activation is tempered through a negative mechanism, where a small heterodimer partner (SHP) decreases LGK gene expression by inhibiting the transcriptional activity of LXRα and PPARγ by directly interacting with their common heterodimer partner RXRα. From these data, we propose a mechanism for LXRα in controlling the gene expression of LGK that involves activation through SREBP-1c and PPARγ and inhibition through SHP.


Liver glucokinase (LGK) plays an essential role in controlling blood glucose levels and maintaining cellular metabolic functions. Expression of LGK is induced mainly regulated by insulin through sterol regulatory element-binding protein-1c (SREBP-1c) as a mediator. Since LGK expression is known to be decreased in the liver of liver X receptor (LXR) knockout mice, we have investigated whether LGK might be directly activated by LXR␣. Furthermore, we have studied interrelationship between transcription factors that control gene expression of
LGK. In the current studies, we demonstrated that LXR␣ increased LGK expression in primary hepatocytes and that there is a functional LXR response element in the LGK gene promoter as shown by electrophoretic mobility shift and chromatin precipitation assay. In addition, our studies demonstrate that LXR␣ and insulin activation of the LGK gene promoter occurs through a multifaceted indirect mechanism. LXR␣ increases SREBP-1c expression and then insulin stimulates the processing of the membrane-bound precursor SREBP-1c protein, and it activates LGK expression through SREBP sites in its promoter. LXR␣ also activates the LGK promoter by increasing the transcriptional activity and induction of peroxisome proliferator-activated receptor (PPAR)-␥, which also stimulates LGK expression through a peroxisome proliferator-responsive element. This activation is tempered through a negative mechanism, where a small heterodimer partner (SHP) decreases LGK gene expression by inhibiting the transcriptional activity of LXR␣ and PPAR␥ by directly interacting with their common heterodimer partner RXR␣. From these data, we propose a mechanism for LXR␣ in controlling the gene expression of LGK that involves activation through SREBP-1c and PPAR␥ and inhibition through SHP.
Several tissues are involved in maintaining optimal blood glucose levels. Among these, the liver plays a major role by maintaining the balance between the storage and release of glucose (1). Conversion of glucose to glucose 6-phosphate is a ratecontrolling reaction in the hepatic glucose metabolism. If the hepatic glycogen stores are replete, glucose 6-phosphate enters the glycolytic pathway to produce pyruvate for de novo lipogenesis.
Glucose entering the liver is phosphorylated by hepatic glucokinase (liver glucokinase; LGK) 4 (ATP:D-glucose 6-phosphotransferase; EC 2.7.1.1). Since the K m value of LGK is considerably higher than normal blood glucose concentrations and LGK is not subjected to allosteric regulation by the end product, the rate of glucose phosphorylation is directly proportional to the blood glucose level. Thus, LGK is considered to play an essential role for sensing and maintaining proper blood glucose levels (2,3). GK is mainly expressed in the liver, pancreatic ␤ cells, and neuroendocrine cells of the brain. Two alternate promoters regulate the tissue-specific expression of the GK gene in the liver and pancreatic ␤ cells (4). An upstream promoter regulates ␤ cell-specific GK expression, whereas the promoter that regulates LGK gene expression is controlled by a downstream promoter. In the liver, LGK gene expression is regulated in response to fasting and refeeding (5), with insulin and glucagon serving as the mediators of this response. Insulin stimulates LGK gene expression in primary cultured hepatocytes regard-* This work was supported by Korea Science and Engineering Foundation less of glucose concentration, and glucagon inhibits LGK gene expression (6,7). The action of insulin on the up-regulation of LGK transcription is mediated by the SREBP-1c (8,9).
LXRs are nuclear receptors that sense oxysterols and regulate cholesterol and lipid metabolism (10,11). The LXR family consists of the LXR␣ and LXR␤ isoforms. LXR␣ is expressed primarily in liver, adipose tissue, kidney, macrophage and intestine, whereas LXR␤ is present ubiquitously (12). LXRs form heterodimers with RXR␣ and bind to target DNA sequences known as the LXR response element (LXRE) (13). In response to oxysterols, LXRs activate genes involved in reverse cholesterol transport and hepatic cholesterol metabolism (14). LXR␣ also increases the synthesis of fatty acids by either up-regulating SREBP-1c or binding to the promoters of some lipogenic genes directly (15)(16)(17). Other diverse roles of LXR␣ include the inhibition of the expression of gluconeogenic genes, such as phosphoenolpyruvate carboxykinase and glucose-6-phosphatase (18). In addition, LXR␣ stimulates adipocyte differentiation through induction of PPAR␥ expression (19).
SHP is an atypical nuclear receptor that lacks a conventional DNA binding domain (20,21). It is known to interact with other transcription factors and represses transcriptional activity by either competing with other coactivators for binding to an affected transcription factor or by recruiting corepressors directly to the transcriptional repression domain of SHP (22). SHP is expressed in liver and plays an important role in the regulation of cholesterol homeostasis (23,24). Also, it is known that SHP regulates LXR transcriptional activity and augments the transcriptional activity of PPAR␥ (25,26).
In this study, we identified an interactive mechanism for regulation of LGK expression through LXR␣ with critical roles for SREBP-1c, PPAR␥, and SHP. LXR␣ directly activated LGK expression by binding to the LXRE in the LGK promoter, and LXR␣-mediated LGK gene expression was also indirectly upregulated by increasing SREBP-1c gene expression and increasing transcriptional activity of PPAR␥. We also show that SHP inhibited LXR␣-dependent transcriptional activation of LGK by interacting with RXR␣. These results suggest a multicomponent mechanism for regulation of LGK expression by diverse nuclear receptors.

EXPERIMENTAL PROCEDURES
Plasmids and Materials-Construction of the luciferase reporter of the rat LGK promoter, pRGKL-1448, and its mutants m2 and mab were described earlier (9,27). Mutant clones m4, m5, m6, and m7 were constructed by introducing substitution mutations into pRGKL-1448. Expression plasmids for mPPAR␥, mRXR␣, and ␤-galactosidase and their control vectors were previously described (9,28). Human SHP expression vector was a kind gift from Dr. Heung-Sik Choi, and mouse LXR␣ expression vector was a kind gift from Dr. David J. Mangelsdorf (29,30). The expression plasmid for myc-Histagged human LXR␣ was received from Dr. J. B. Kim (Seoul National University, Korea).
LGK-PPREϫ3tkLuc and LGK-LXREϫ3tkLuc were prepared by inserting three copies of Ϫ119/Ϫ98 and Ϫ56/Ϫ32 regions of the LGK promoter, respectively. APRE or GPRE was prepared by inserting three copies of PPRE from the acyl-CoA oxidase promoter (APRE) or ␤ cell-specific GK promoter (GPRE) in front of the thymidine kinase minimal promoter in the luciferase reporter (ptkLuc). To generate Gal4DBD-PPAR␥, Gal4DBD-RXR␣, Gal4DBD-SHP, VP16-PPAR␥, VP16-RXR␣, and VP16-SHP constructs used in the mammalian two-hybrid assay, full-length cDNAs of PPAR␥, RXR␣, and SHP were prepared by PCR amplification, and then these fragments were subcloned into pM and pVP16 vectors, respectively (Clontech). All of the plasmid constructs were confirmed by DNA sequencing. Rosiglitazone was a kind gift from GlaxoSmithKline Korea (Seoul, Korea), and 9-cis-retinoic acid (9-CR), 22-hydroxycholesterol, and insulin were purchased from Sigma. TO-901317 (T1317) was purchased from Cayman (Ann Arbor, MI).
Cell Culture and Transient Transfection Assay-Alexander cells (human hepatoma cell lines; ATCC number CRL8024) and CV-1 cells (Monkey kidney cell lines; ATCC number CCL70) were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% (v/v) fetal bovine serum, 100 units/ml penicillin, and 100 g/ml streptomycin. The culture medium for Alexander cells was supplemented with 50 g/ml tylosine for an anti-pleuropneumonia-like organisms (PPLO) agent. Primary cultured hepatocytes were isolated from Sprague-Dawley rats (ϳ200 g) and cultured as described previously (9). Transient transfection and luciferase assays were performed using Lipofectamine PLUS reagent (Invitrogen) and a luciferase assay kit (Promega, Madison, WI) (27). Luciferase activities were normalized with ␤-galactosidase activities to adjust transfection efficiency. Normalized luciferase activities are shown as the mean Ϯ S.D. of three independent experiments performed in triplicate and are expressed as -fold increases relative to the basal activity of the reporters in the absence of overexpression vectors.
Preparation of Recombinant LXR␣ Protein and Anti-LXR␣ Antibody-To prepare bacterial recombinant fusion proteins, we cloned cDNA of mouse LXR␣ into pET21 bacterial expression plasmid and transformed into Escherichia coli (BL21-DE3). The recombinant LXR␣ expression was induced for 4 h with 0.5 mM isopropyl-␤-D-thiogalactopyranoside. The recombinant protein containing polyhistidine (His) tag was purified by Ni 2ϩ -nitrilotriacetic acid-agarose resin chromatography (Peptron, Taejeon, Korea). One milligram of recombinant LXR␣ protein was suspended in 1 ml of phosphate-buffered saline and emulsified with 1 mM Freund's complete adjuvant (Sigma), which was injected subcutaneously into the dorsal side of a New Zealand White rabbit. The second and third immunizations were performed in the same way to boost production of anti-LXR␣ antibody. One month after the third immunization, we collected blood from immunized rabbit and prepared serum. Specificity of immunized rabbit serum (IgG) was confirmed by Western blot using myc-His-LXR␣-overexpressed HEK293 cell extract and used for the experiment.
Isolation of Total RNA, Reverse Transcription, and Quantitative PCR-Total RNA was extracted from primary cultured hepatocytes using the Illustra RNAspin Mini RNA isolation kit (GE Healthcare) according to the manufacturer's protocol. First strand cDNA was synthesized from 2 g of total RNA in a 20-l volume using random hexamers and ImProm II Reverse transcriptase (Promega, Madison, WI). Real time quantitative PCR was performed using the ABI PRISM 7500 sequence detection system instrumentation and software (Applied Biosystems, Foster City, CA) according to the manufacturer's protocol with minor modifications. Briefly, the appropriate amount of the reverse transcription reaction mixture was amplified with specific primers using SYBR Green PCR master mix (Applied Biosystems, Foster City, CA). Expression levels of target genes were determined by generating a five-point serial standard curve. This curve was used to calculate the amount of target gene mRNA in vehicle-and ligand-treated samples based on real time PCR. RNA samples were normalized by determining ␤-actin mRNA level. Primers used in PCR were as follows: LGK-f, 5Ј-CTCAGGAGTCAGGAACATCT-3Ј; LGK-r, 5Ј-TGACCAGCATCACTCTGAAG-3Ј; SREBP-1c-f, 5Ј-GGAG-CCATGGATTGCACATT-3Ј; SREBP-1c-r, 5Ј-AGGAAGGC-TTCCAGAGAGGA-3Ј; LXR␣-f, 5Ј-GAGAAGCTGGTGGC-TGCCCA-3Ј; LXR␣-r, 5Ј-AGCTGTAGGAAGCCAGGGAG-3Ј; PPAR␥-f, 5Ј-TCCGTGATGGAAGACCACTC-3Ј; PPAR␥-r, Electrophoretic Mobility Shift Assay-In vitro translated proteins of LXR␣-myc-His and RXR␣-FLAG were prepared by using a coupled transcription/translation kit (Promega, Madison, WI). Synthesis of full-length LXR␣-myc-His and RXR␣-FLAG proteins was confirmed by Western blotting. An oligonucleotide covering the Ϫ59/Ϫ29 region of the rat LGK promoter was used as a wild-type probe. The probes were labeled as previously described (31). Briefly, 10 pmol of singlestranded sense oligonucleotide was labeled with [␥-32 P]ATP using T4 polynucleotide kinase (New England Biolabs Inc., Ipswich, MA) and annealed with a 5-fold molar excess of antisense oligonucleotide. 32 P-Labeled double-stranded oligonucleotides were purified with Sephadex G25 spin columns (Amersham Biosciences). Ten thousand cpm (ϳ0.08 pmol) of probe was incubated with the in vitro translated protein for 30 min in 10 mM Hepes (pH 7.9), 1 mM MgCl 2 , 30 mM KCl, 1 mM dithiothreitol, 5% glycerol. One microgram of poly(dI-dC) was added to each reaction to suppress nonspecific binding. For competition assays, excessive unlabeled GLUT4 LXRE oligonucleotides were added to the reaction mixture (32). Two l of anti-myc antibody were added to the reaction for the supershift assay (Cell signaling, Denver, MA). The oligonucleotides used in the electrophoretic mobility shift assay were as follows: wild type, 5Ј-CTGGCCCTGACCTTGTGACACTAGGCAGGG-3Ј; GLUT4 LXRE, 5Ј-CAGCCCCGGGTTACTTTGGGGCA-TTgCTCC-3Ј.
Chromatin Immunoprecipitation Assay-The chromatin immunoprecipitation assay was performed based on a previously described method with minor modification (33). Proteins were cross-linked to DNA by adding formaldehyde directly to the culture medium to a final concentration of 1% and incubating for 10 min at 37°C. The cells were harvested after washing and lysed with SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris, pH 8.1). The livers of adult male rats (ad libitum) were perfused with serum-free Dulbecco's modified Eagle's medium for 5 min through the portal vein and then cross-linked with 3% formaldehyde in serum-free Dulbecco's modified Eagle's medium. Chromatin complexes in the supernatants were immunoprecipitated overnight with 2 g of preimmune serum or antibody specific for LXR␣ and RXR␣ (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 4°C with rotation. The GLUT2 promoter was used as a negative control (34).
Small Interfering RNA Experiments-The siRNAs sequences for rat LXR␣ and scramble are AUUAGCAUCCGUGGGAA-CAUCAGUC and CCUACGCCACCAAUUUCGU-dTdT, respectively. For siRNA transfection, rat primary cultured hepatocytes were incubated with Dulbecco's modified Eagle's medium containing 10% fetal bovine serum in the absence of antibiotics for 12 h and transfected with 200 nM siRNA-scramble or siRNA-LXR␣ using Lipofectamine RNAiMAX following the manufacturer's protocol (Invitrogen). After 5 h, the media containing the respective ligands were changed and incubated for 24 h.
Adenoviral Transduction-The cDNA encoding full-length human SHP into pAdTrack-CMV shuttle vector was received from Dr. I. K. Lee (36). The cDNA encoding SREBP-1c-DN was described (9). The recombinant adenoviruses were amplified in 293A cells and purified using CsCl gradient centrifugation. Isolated primary hepatocytes were infected with 10 plaque-forming units/cell of Ad-SHP, Ad-DN-SREBP-1c, or Ad-null virus and incubated for 3 h.
Western Blot-Cell lysates were prepared using passive lysis buffer (Promega, Madison, WI) containing protease inhibitors. Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane (GE Healthcare). The membranes were blocked and incubated with anti-HA (Covance Inc., CA), anti-␣-tubulin (Calbiochem, Darmstadt, Germany) in fresh phosphate-buffered saline containing 1% nonfat dry milk overnight at 4°C with agitation. Incubated membranes were washed and treated with horseradish peroxidase-conjugated secondary antibody. Then horseradish peroxidase signal was detected by using ECL substrate (GE Healthcare).

LXR␣ Activates
LGK Expression Directly-SREBP-1c is known to mediate the regulation of LGK gene expression by insulin. Both LGK and SREBP-1c gene expressions are known to be increased in the livers of mice after administration of LXR␣ ligand for 3 days, and the effect was lost in LXR null mice (37,38). Oral administration of LXR␣ ligand for 12 h did not increase LGK gene expression in the liver of SREBP-1c null mice, which suggested that SREBP-1c is required for LGK gene expression by LXR␣ ligand (39). However, we observed that LXR␣ activated the rat LGK promoter in Alexander cells, where SREBP-1a is predominantly expressed instead of SREBP-1c. As shown in Fig. 1, LGK promoter activity was not activated by T1317 and/or 9-CR when LXR␣ and/or RXR␣ expression vector were not transfected. Treatment of 9-CR resulted in an increase in the LGK promoter activity in the Alexander cells with the expression of RXR␣. The coexpression of LXR␣ and RXR␣ activated the LGK promoter in the absence of ligands, probably due to a low concentration of endogenous oxysterol LXR ligands. The promoter activity was further increased by T1317/9-CR with the expression of LXR␣ and RXR␣, suggesting that the LGK promoter was directly activated by the LXR␣/ RXR␣ heterodimer.
To identify the region responsible for the activation by LXR␣, serial deletion constructs of LGK promoter-reporter were transfected into Alexander cells with the LXR␣ and RXR␣ expression vectors in the presence or absence of added T1317/ 9-CR (Fig. 1B). Deletion of the promoter down to Ϫ120 resulted in slight loss of LXR␣-dependent LGK promoter activity; however, the LGK promoter was still substantially activated by LXR␣. Further deletion down to Ϫ76 and Ϫ25 resulted in almost complete loss of LXR␣ responsiveness. If LXR␣dependent activation of LGK promoter is entirely mediated by SREBP-1c, deletion down to Ϫ120 is likely to result in complete loss of LXR␣-dependent activation, because two SREs are present between bp Ϫ238 and Ϫ128 of the rat LGK promoter. But this deletion study suggested that the region between Ϫ120 and Ϫ25 was more responsible for the LXR␣-dependent activation of the LGK promoter. Considering that a PPRE is present in the Ϫ120/Ϫ76 bp region of the LGK gene promoter (27) and that PPAR␥ is also categorized as a permissive binding partner of RXR␣, the region between bp Ϫ76 and Ϫ25 is more likely to contain an LXRE. Unfortunately, this region did not contain an easily identifiable DR4, which is the high affinity LXRE (16). In order to find putative LXRE, five scanning mutants were prepared by introducing substitution mutations into the putative nuclear receptor half-site elements contained within the Ϫ120/ Ϫ25 bp region (Fig. 1C), and the mutants were tested for responsiveness to LXR␣ (Fig. 1D). The PPRE mutation (m2) showed a 50% decrease in LXR␣-dependent activation of LGK promoter, but m4 and m5 mutations retained LXR␣ responsiveness. Mutations at the m6 and m7 sites resulted in a 75% decrease in LXR␣-dependent activation, suggesting the presence of a functional LXRE in this region. From these data, it is assumed that the Ϫ52/Ϫ37 bp region may be an LXRE, and LGK could be activated by LXR␣ through this site and the PPRE.
To know whether LXR␣ could bind to the putative LXRE in the Ϫ52/Ϫ37 bp region of the LGK promoter, we performed electrophoretic mobility shift assays using in vitro translated LXR␣-myc-His and RXR␣-FLAG. As shown in Fig. 2A, neither LXR␣ nor RXR␣ formed specific protein-DNA complex alone (lanes 3 and 4). When the probes were incubated with both LXR␣ and RXR␣, a specific shifted band was observed, indicat-ing the binding of an LXR␣/RXR␣ heterodimer to the LGK-LXRE (lane 5). The addition of an excessive amount of unlabeled competitor LXRE (50-fold) from the GLUT4 promoter completely blocked LXR␣/RXR␣ binding to the Ϫ59/Ϫ29 bp region of LGK promoter (lane 6), but unlabeled m7 mutant did not compete the binding of LXR␣/RXR␣ to LXRE (lane 7). The specificity of the shifted complex was confirmed once again by anti-myc antibody (lane 8). The direct binding of LXR␣ to LGK-LXRE was further confirmed by a chromatin immunoprecipitation assay in both primary cultured hepatocytes (Fig. 2C) and liver (Fig. 2D). In addition to identifying the LXRE in LGK promoter through both in vitro and in vivo approaches, we needed to confirm whether the LGK-LXRE itself could respond to LXR␣/RXR␣, because its sequence was different from the conventional LXRE consensus sequence. To this end, three copies of LGK-LXRE were subcloned in front of the thymidine kinase minimal promoter (tk-Luc) and used to test its responsiveness to LXR␣/RXR␣. As shown in Fig. 7, LGK-LXRE was well activated by LXR␣/RXR␣, indicating that LGK-LXRE is functional both in the LGK promoter and the artificial promoter context. These results suggested that LXR␣/RXR␣ could bind to the LGK promoter and directly activate LGK transcription in the liver.
The promoter study led us to explore the precise molecular interplay between LXR␣ and SREBP-1c in the regulation of LGK gene expression. To delineate the relationship between LXR␣ and SREBP-1c upon LGK expression, primary hepatocytes were isolated from rat liver and treated with the LXR␣ ligand (T1317, 1 M) and/or the RXR␣ ligand (9-CR, 1 M) for 24 h. The mRNA level of LGK was increased either by T1317 (3-fold) or 9-CR (18-fold), respectively (Fig. 3A). Furthermore, combined treatment of T1317 and 9-CR resulted in a synergistic increase in LGK mRNA level (43-fold). In contrast, the pattern of SREBP-1c gene expression in response to the LXR␣ ligand did not match with that of LGK gene expression. SREBP-1c mRNA was increased by T1317 (39-fold), but 9-CR did not increase SREBP-1c gene expression. In addition, the combined treatment of T1317 and 9-CR did not result in the synergistic increase in the SREBP-1c mRNA level (Fig. 3B). If LXR␣-dependent transcriptional induction of LGK gene is entirely mediated by SREBP-1c, the transcriptional induction pattern of LGK by the ligands of LXR␣ and/or RXR␣ could be similar to that of SREBP-1c. The different pattern of transcriptional induction by the ligands of LXR␣ and/or RXR␣ for LGK versus SREBP-1c suggested again that LXR␣ could activate LGK expression independently of SREBP-1c. We also confirmed the effect of LXR␣ on LGK expression by knocking down LXR␣ in primary hepatocytes. As shown in Fig. 3C, transfection of siRNA for LXR␣, siLXR␣, resulted in a 90% decrease in the LXR␣ expression. Knockdown of LXR␣ could block the increased gene expression of LGK as well as SREBP-1c by T1317/9-CR, and it also blocked the LXR␣-induced gene expression of FAS and SHP, which are well known targets of LXR␣ (16,40). In addition, we tested endogenous ligands of LXR␣ on LGK expression in primary hepatocytes. 22-Hydroxycholesterol increased the LGK mRNA level by 2-fold, and combined treatment of 22-hydroxycholesterol and 9-CR increased LGK mRNA level synergistically (Fig. 3D). Taken together, we concluded that T1317/9-CR could stimulate LGK expression through direct activation of LXR␣ in hepatocytes.
LXR␣ Activates LGK Expression Indirectly-As shown in Fig.  1C, activation of the LGK promoter by LXR␣ was compromised by a mutation in the previously characterized PPRE (m2). Because LXR␣ is known to increase PPAR␥ expression and increase adiposity in white adipose tissue (19), it is likely that some effects of LXR␣ could be exerted through increasing PPAR␥ expression. In addition, LXR␣ is known to increase the expression of SREBP-1c, which in turn binds to two SREs in the LGK promoter and increases LGK gene expression in response to insulin (41). Together, PPAR␥ and SREBP-1c are likely to be involved in the regulation of the LGK promoter by LXR␣. To explore these interrelationships further, we prepared LGK promoter reporter constructs with various combinations of mutations in the SRE, PPRE, and LXRE (Fig. 4). The mutated LGK promoter constructs were cotransfected into Alexander cells with expression vectors for LXR␣/RXR␣ and treated with their respective ligands. Mutation of the PPRE (m2) or the LXRE (m7) resulted in an approximately 60% decrease in LXR␣-dependent activation, whereas the SRE mutations (mab) did not cause any decrease in the LXR␣-dependent activation. Introduction of SRE mutations into the PPRE mutant (m2ab) or LXRE mutant (m7ab) did not result in a further decrease in LXR␣-dependent activation of the LGK promoter when compared with the PPRE mutant (m2) or LXRE mutant (m7). Double mutations in LXRE and PPRE (m27) and triple mutations (m27ab) caused the most significant decrease in the LXR␣-dependent activation of LGK promoter. These results indicate that LXR␣-dependent activation of the LGK promoter in Alexander cells is partly through the direct activation of the LXRE and partly through indirect activation of PPRE.
To test our hypothesis for indirect activation of the PPRE in the LGK promoter by LXR␣, three copies of each of the LGK- A chromatin immunoprecipitation assay was adopted to confirm the binding of LXR␣/RXR␣ to the LGK promoter in primary cultured hepatocytes (C) and liver (D). Primary cultured hepatocytes isolated from rats were treated with T1317 and 9-CR for 24 h and then were cross-linked using formaldehyde. The liver was fixed with 3% formaldehyde by perfusing the portal vein. Chromatins were incubated with anti-LXR␣ antiserum and anti-RXR␣ antibody. DNA in the presence or in the absence of antibody was immunoprecipitated, and PCR amplification of the DNA fragments was performed using primer pairs specific to the Ϫ148/ϩ50 bp region of the rat LGK gene (B and D).
PPRE and LGK-LXRE were subcloned in front of a thymidine kinase minimal promoter in the luciferase reporter, and the resulting constructs were tested for responsiveness to LXR␣/ RXR␣ or PPAR␥/RXR␣ (Fig. 7). LGK-PPRE was activated both by LXR␣/RXR␣ and PPAR␥/RXR␣, and the LGK-LXRE was activated only by LXR␣/RXR␣, suggesting the indirect activation of PPRE by LXR␣.
In an attempt to confirm the effects of LXR␣ in connection with SREBP-1c, primary hepatocytes were treated with T1317/ 9-CR for 24 h in the presence or absence of insulin and with or without expression of a dominant negative form of SREBP-1c (DN-SREBP-1c). As shown in Fig. 5A, T1317/9-CR increased LGK mRNA (7-fold) without insulin, and insulin increased LGK mRNA level (6-fold). The combination of T1317/9-CR/ insulin increased the LGK mRNA level synergistically (22-fold). Although expression of DN-SREBP-1c inhibited the stimulation of LGK expression by T1317/9-CR in the presence of insulin, it did not inhibit its expression in the absence of insulin. We also observed that treatment of T1317/9-CR and/or insulin and the expression of DN-SREBP-1c had similar effects on the gene expression of FAS, which is known to be activated by both SREBP-1c and LXR␣ (Fig. 5B). These results suggest that LXR␣ directly activates LGK gene expression. In addition, the activation occurs indirectly by increasing SREBP-1c gene expression.
SHP Functions as a Negative Modulator of LGK Gene Transcription-SHP interacts with various transcriptional factors and represses their transcriptional activity either by competing with coactivators or recruiting corepressors (22,42). SHP is also known to interact with LXR␣ and RXR␣ and inhibit the transcriptional activity of these receptors (25,42). In contrast to LXR␣ and RXR␣, SHP was reported to augment PPAR␥ transcriptional activity by competing with the binding of a corepressor (26). These opposite effects of SHP on LXR␣ and PPAR␥ led us to explore the role of SHP in transcriptional regulation of LGK by PPAR␥ and LXR␣.
To examine the effect of SHP on LGK, the LGK promoter reporter construct (pRGKL-1448) was transfected to Alexander cells, and an expression vector for SHP was cotransfected with vectors for LXR␣ or PPAR␥. As shown in Fig. 6A, LXR␣dependent activation of LGK promoter was decreased by SHP in a dose-dependent manner. When equal amounts of SHP and LXR␣ were transfected, most of the LXR␣-dependent transcriptional activation was abolished. SHP also inhibited PPAR␥dependent activation of the LGK promoter (Fig. 6B). To know whether these repressive effects of SHP on LXR␣and PPAR␥dependent activation of the LGK promoter could inhibit the activation of LGK gene expression by LXR␣ and PPAR␥, we transduced adenovirus expressing SHP into primary hepatocytes and treated cells with the ligands of PPAR␥ and LXR␣. As shown in Fig. 6, C and D, adenoviral expression of SHP inhibited the LXR␣and PPAR␥-dependent stimulation of LGK gene expression in primary hepatocytes. The repressive effect of SHP was further confirmed by using LGK-PPRE and LGK-LXRE artificial reporter promoter constructs (Fig. 7A). LGK-PPRE was activated by either PPAR␥ or LXR␣, and both of the activations were inhibited by SHP. In contrast, LGK-LXRE was activated by LXR␣, which was also inhibited by SHP. These results suggested that SHP could inhibit the LXR␣and PPAR␥dependent transactivation of LGK promoter.
In order to know whether the inhibitory action of SHP on PPAR␥ is LGK-specific or not, we repeated the same experiment using reporter constructs that contain three copies of APRE or GPRE in front of the thymidine kinase minimal promoter (Fig. 7, B and C). The APRE reporter construct was activated by PPAR␥/RXR␣ and suppressed by ectopic expression of SHP in both Alexander cells and CV-1 cells. However, activa-  tion of GPRE by PPAR␥/RXR␣ was not affected by SHP in these cells. To explore a precise mechanism of how SHP inhibits the activation of the LGK promoter by LXR␣ and PPAR␥, we per-formed a mammalian two-hybrid assay using a luciferase reporter that has upstream activating sequence in front of the thymidine kinase minimal promoter. We employed a system utilizing a GAL4-PPAR␥ fusion and SHP fused with or without the VP16 activation domain or the reciprocal combination of fusion proteins. As shown in Fig. 8A, expression of the Gal4-PPAR␥ fusion protein increased luciferase activity in the presence of rosiglitazone (1 M). However, neither SHP nor VP16-SHP affected Gal4-PPAR␥ activity. We further confirmed the interaction of PPAR␥ and SHP in the reciprocal combination. Gal4-SHP expression decreased luciferase activity because of the intrinsic repression function of SHP.
Coexpression of Gal4-SHP and VP16-PPAR␥ did not stimulate luciferase activity that was repressed in Gal4-SHP. This result suggested that PPAR␥ did not interact with SHP in hepatocytes. By contrast, SHP did repress the promoter activity that was activated by the Gal4-RXR␣ (Fig. 8B), and VP16-SHP restored the promoter activity that was repressed by SHP, suggesting that the VP16 activation domain compensated for the repressive effect of SHP on RXR␣-dependent activation. When we checked the interaction between RXR␣ and SHP with the reciprocal combination, Gal4-SHP decreased luciferase activity, and coexpression of VP16-RXR␣ with Gal4-SHP stimulated the luciferase activity from the repressed state. The interaction between RXR␣ and SHP was further confirmed by the GST pull-down assay using in vitro translated HA-SHP and GST-RXR␣ fusion protein (Fig. 8C). These data do not agree with earlier reports that SHP increases the transcriptional activity of PPAR␥ by directly interacting with PPAR␥ (26,43). It is not clear whether repression of PPAR␥ by SHP is a genespecific phenomenon or not. Based on our experiments using Alexander cells and CV-1 cells, it is likely that this phenomenon may not be cell-specific. We also tried coimmunoprecipitation to confirm the interaction of PPAR␥ and SHP in a different way, but interaction between PPAR␥ and SHP was not observed (data not shown). Therefore, our results suggested that SHP functioned as a negative modulator of the PPAR␥ and that LXR␣ mediated LGK gene transcription by interacting with RXR␣.

DISCUSSION
Intracellular glucose 6-phosphate is the primary physiologic stimulus for the hepatic glucose metabolism, since it is a sub-strate for both glycolysis and glycogen synthesis (44), and small changes in the expression of the enzyme LGK, which produces glucose 6-phosphate from glucose, can have a profound impact on the blood glucose concentration (2,3,45). Here, we present data indicating that LXR␣ plays an orchestrated role in the regulation of LGK expression. We have identified an LXRE in the LGK promoter and shown that LXR␣ could activate LGK expression directly through binding to its promoter. We have also shown that LXR␣ activates the LGK promoter indirectly through a mechanism requiring PPAR␥ and SREBP-1c. In addition, we showed that SHP functions as a negative modulator of LGK transcription by inhibiting the transcriptional activity of LXR␣ and RXR␣.
LXR␣ has an insulin-like effect on hepatic carbohydrate metabolism by stimulating genes involved in glucose storage and inhibiting gluconeogenesis, which would be consistent with a role in promoting energy storage in a model of dietinduced obesity and insulin resistance (37). Recently, Oosterveer et al. (46) reported defective induction of lipogenic gene expression after fasting/refeeding in LXR␣ KO mice. Thus, LXR␣ has an important role in the coordination of lipid and glucose metabolism, which suggests that LXR␣ ligands may have therapeutic potential through the modulation of glucose homeostasis. LXR␣ is known to activate SREBP-1c expression and thereby increases the activity of genes involved in glycolysis and lipogenesis (15,47). However, there are still some questions of whether the relationship between SREBP-1c and LXR␣ exists, LXR␣ is involved directly in the physiologic effects of insulin, or the role of LXR␣ is solely to activate SREBP-1c, and these questions need to be explored.
Insulin is known to increase LXR␣-dependent SREBP-1c expression by producing endogenous LXR␣ ligands (47). Recently, Hegarty et al. (41) showed that conversion of the membrane-bound SREBP-1c precursor into the mature soluble transcription factor is enhanced after acute exposure to insulin, and LXR␣ increases LGK expression in primary cultured hepatocytes by increasing SREBP-1c gene expression in the presence of insulin. Administration of the LXR␣ ligand GW3965 for 3 days increased LGK expression in mouse liver. In addition, treatment of mice with another synthetic LXR ligand, T1317, for 12 h did not increase LGK gene expression in the SREBP-1c null mouse (38,39). These data suggested that LGK induction by LXR␣ is mediated by SREBP-1c. But there is a caveat to be considered. When Liang et al. (39) tested GK gene expression in the liver of the SREBP-1c null mouse, they treated mice with GW3695 instead of T1317 only for 12 h in a chow diet. This treatment protocol seemed to be less effective than 3 days' gavage feeding of T1317, which potentially makes it more difficult to observe changes in gene expression that is not strongly activated by LXR␣. In addition, we used rat instead of mouse LGK genes and rat primary cultured hepatocytes. We compared the 3-kb upstream region from exon 1 of the mouse LGK gene, but we could not find a similar sequence with LGK-LXRE of rats. Thus, there might be a species difference in the regulation mechanism of LGK expression by LXR␣. We have also observed that LXR␣ activates the LGK promoter through a PPRE. This is likely to be indirect, because it has been reported that LXR␣ could increase the production of endogenous PPAR␥ agonists through the up-regulation of SREBP-1c gene as well as the expression of PPAR␥ (19,48). In these results, a role for ligand activation of RXR␣, a permissive partner of LXR␣, cannot be ruled out. However, overexpression of RXR␣ alone did not activate LGK-PPRE in the artificial promoter context (data not shown). Based on all of our observations, we propose a novel mechanism of activation for LGK by LXR␣, which involves both SREBP-1c and PPAR␥.
Finally, we also showed that SHP inhibited the transcriptional activation of the LGK promoter by LXR␣ and PPAR␥. We also showed that adenoviral expression of SHP significantly decreased LGK expression in the primary hepatocytes. Our results suggest that the inhibitory effects of SHP on LXR␣or PPAR␥-mediated LGK gene expression are supposed to occur through mutual interaction with RXR␣, resulting in the decrease in LGK promoter activity. Considering that SHP is also known to work as a negative regulator for the transcriptional activators involved in the gluconeogenic genes as well as the LGK gene (49), we speculate that SHP plays a fine tuning role in the hepatic glucose metabolism by modulating transcription factors. We provide a model summarizing the orchestrated role of PPAR␥, LXR␣, SREBP-1c, and the negative modulator SHP in the regulation of LGK gene transcription (Fig. 9). In this model, LXR␣ up-regulates the gene expression of LGK and SREBP-1c by direct binding to a cis-element in their promoters. Insulin activates the SREBP-1c gene as well as its maturation, resulting in the synergistic increase of LXR␣-mediated LGK gene expression. In addition, we propose that LXR␣ increases LGK gene expression by increasing the transcriptional activity of PPAR␥, possibly either by increasing the production of endogenous PPAR␥ ligand or by increasing PPAR␥ expression in hepatocytes (19,48). LXR␣ and PPAR␥ increase the transcription of the SHP gene (40,50), which in turn represses the transcription of the LXR␣or PPAR␥-mediated LGK gene by interacting with RXR␣.