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Molecular Endocrinology 21 (11): 2687-2697
Copyright © 2007 by The Endocrine Society

The Nuclear Receptor Cofactor, Receptor-Interacting Protein 140, Is Required for the Regulation of Hepatic Lipid and Glucose Metabolism by Liver X Receptor

Birger Herzog, Magnus Hallberg, Asha Seth, Angela Woods, Roger White and Malcolm G. Parker

Institute of Reproductive and Developmental Biology (B.H., M.H., A.S., R.W., M.G.P.), and Medical Research Council Cellular Stress Group, Clinical Sciences Centre (A.W.), Imperial College London, Faculty of Medicine, London W12 0NN, United Kingdom

Address all correspondence and requests for reprints to: Malcolm G. Parker, Institute of Reproductive and Developmental Biology, Imperial College London, Faculty of Medicine, Du Cane Road, London W12 0NN, United Kingdom. E-mail: m.parker{at}imperial.ac.uk.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The liver X receptors (LXRs) are nuclear receptors that play important roles in the regulation of lipid metabolism. In this study, we demonstrate that receptor-interacting protein 140 (RIP140) is a cofactor for LXR in liver. Analysis of RIP140 null mice and hepatocytes depleted of RIP140 indicate that the cofactor is essential for the ability of LXR to activate the expression of a set of genes required for lipogenesis. Furthermore we demonstrate that RIP140 is required for the ability of LXR to repress the expression of the phosphoenolpyruvate carboxykinase gene in Fao cells and mice. Thus, we conclude that the function of RIP140 as a cofactor for LXR in liver varies according to the target genes and metabolic process, serving as a coactivator in lipogenesis but as a corepressor in gluconeogenesis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
LIVER X RECEPTORS (LXRs) are members of the nuclear receptor superfamily of transcription factors that regulate the expression of a number of genes involved in lipid, cholesterol, and glucose metabolism in hepatocytes and other cell types (1, 2, 3, 4, 5). LXRs serve as intracellular sensors of cholesterol, and oxidized derivatives of cholesterol (oxysterols) have been identified as endogenous ligands (6, 7). This signaling pathway is important for the control of three important processes in hepatocytes, namely the synthesis of steroid hormones, bile acids, and cholesterol (6).

In liver, excess cholesterol is converted into bile acids and exported from the cell while, at the same time, cholesterol biosynthesis and uptake of lipoprotein cholesterol is reduced. Functional response elements for LXR{alpha} and ß have been identified in the promoters of several genes that encode rate-limiting enzymes, transporters, and regulators of these processes (7, 8, 9, 10, 11). Insights into the function of LXR in cholesterol metabolism were provided by the generation of mice devoid of LXR{alpha} (LXR{alpha}–/–) (12). For example, transcription of the gene encoding cholesterol 7{alpha}-hydroxylase, the rate-limiting enzyme in bile acid synthesis, is impaired in LXR{alpha}–/– mice leading to cholesterol accumulation and ultimately defective liver function.

LXR also plays a key role in the regulation of hepatic lipid metabolism by activating lipogenesis. This is achieved by increasing the expression of sterol regulatory element-binding protein (SREBP)-1c that controls the expression of fatty acid synthase (FAS) and other key genes involved in fatty acid biosynthesis (13, 14, 15). Basal LXR activity is essential for the expression of SREBP1c in hepatocytes, underlining the importance of LXR for hepatic lipogenesis (16). In addition, LXR regulates the expression of the FAS and other lipogenic genes directly (17) whereas fatty acids were identified as positive regulators of LXR{alpha} gene expression in cultured hepatocytes (18). These observations suggest an important cross talk between fatty acid- and cholesterol-mediated regulation of lipid metabolism.

LXRs are also regulators of glucose metabolism. LXR agonists have been shown to improve glucose tolerance and insulin sensitivity in diabetic animals by increasing glucose transporter 4 expression and glucose uptake in adipocytes and by suppressing gluconeogenesis, in particular the genes encoding rate-determining enzymes, such as phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) (19, 20, 21).

A number of transcriptional cofactors have been found to play a crucial role in the integration of metabolic processes, including peroxisomal proliferator-activated receptor coactivator (PGC)-1{alpha}, PGC-1ß, steroid receptor coactivator 1, and transcriptional intermediary factor 2 (22, 23). In addition, corepressors can regulate networks of metabolic genes. For example, receptor-interacting protein 140 (RIP140) promotes the storage of lipids in adipose tissue by inhibiting the expression of genes involved in mitochondrial biogenesis, fatty acid oxidation, and oxidative phosphorylation (24, 25, 26). RIP140 interacts with a number of nuclear receptors, including peroxisomal proliferator-activated receptors, estrogen-related receptors, and LXR that regulate metabolic pathways (27, 28, 29). In this study we address the role of RIP140 in LXR-regulated hepatic lipid and glucose metabolism. Experiments in WT and RIP140 null mice fed different diets and cultured cells depleted of RIP140 reveal that RIP140 is required for LXR function in two different ways: the induction of lipogenesis and the repression of the PEPCK gene. Therefore we conclude that RIP140 is involved in both positive and negative effects of LXR in transcriptional regulation in hepatocytes.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Regulation of Hepatic Lipid Metabolism in RIP140 Null Mice
To study whether RIP140 plays a regulatory role in hepatic lipid metabolism, we monitored the levels of triglycerides in wild-type (WT) and RIP140 null mice fed chow or western diet for 4 wk. Hepatic and plasma triglyceride levels were similar in both WT and RIP140 null mice fed a chow diet; however, the expected increase in hepatic triglycerides after challenge with a western diet was less marked in mice devoid of RIP140 (Fig. 1AGo). Plasma triglyceride levels are also slightly less in RIP140 null mice after feeding with western diet (Fig. 1AGo). Given the importance of LXR in hepatic lipogenesis (13), we investigated the possibility that RIP140 might function as a cofactor for LXR in the regulation of this process. Mice were fed a synthetic LXR agonist (T0901317) for 3 d. Analysis of hepatic lipogenic gene expression indicated that whereas LXR mRNA levels were essentially unchanged after T0901317 administration (data not shown), there was a significant increase in SREBP1c, steroyl-coenzyme A desaturase (SCD-1), FAS, and acetyl-coenzyme A carboxylase 1 expression in WT but not RIP140 null mice (Fig. 1BGo). Next we measured the amount of hepatic triglycerides after LXR activation. As expected, the treatment with T0901317 resulted in lipid accumulation in the liver of WT mice. In contrast, mice devoid of RIP140 have much less hepatic triglycerides after the same treatment (Fig. 1CGo). These results suggest that the failure of RIP140 null mice to increase lipogenesis in response to LXR activation contributes to the reduced accumulation of triglycerides in the liver of these animals.


Figure 1
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  		Fig. 1. Hepatic Lipid Metabolism in WT and RIP140 Null Mice

A, Triglyceride amounts in liver and plasma of WT and RIP140 null mice maintained on chow or western diet for 4 wk. Results are expressed as the mean ± SEM of five to 10 animals in each group. ***, P < 0.001 vs. WT fed with western diet. B, WT and RIP140 null mice were maintained on a standard chow diet and fed a synthetic LXR agonist (T0901317) or vehicle alone for 3 d (50 mg/kg/d). Gene expression was determined by real-time PCR analysis. Shown are relative mRNA levels normalized to cyclophilin as mean ± SEM of four to five animals in each group. C, Hepatic triglyceride content in WT and RIP140 null mice maintained on a standard chow diet and fed T0901317 or vehicle alone. *, P < 0.05; **, P < 0.01 vs. WT treated with T0901317. KO, Knockout; rel., relative.

 
RIP140 Function in Isolated Hepatocytes
We isolated hepatocytes from WT and RIP140 null mice to test whether the observed effects of RIP140 are autonomous to hepatocytes. First we measured the total lipid content in both cell types after stimulation with the LXR agonist. Treatment of WT hepatocytes with T0901317 resulted in an increased lipid accumulation; however, hepatocytes derived from RIP140 null mice lacked this increase (Fig. 2AGo). Next we measured the expression of the lipogenic genes FAS, SCD-1, and SREBP1c in cells treated with either T0901317 or vehicle (dimethylsulfoxide). As expected, this treatment resulted in an increased expression of these genes in WT cells. In contrast, the expression of these genes remains unchanged in the cells derived from RIP140 null mice (Fig. 2BGo). These findings suggest that the effect of RIP140 on LXR is autonomous for hepatocytes.


Figure 2
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  		Fig. 2. Function of RIP140 in Primary Hepatocytes

A, Total lipid content was measured in primary hepatocytes isolated from WT or RIP140 null mice (RIP KO). Cells were stimulated for 24 h with T0901317 or vehicle alone. Data are expressed as fold change compared with WT control samples. B, Cells were treated as described above and gene expression was determined by real-time PCR. Shown are relative mRNA levels normalized to cyclophilin as mean ± SEM; *, P < 0.05 vs. WT treated with T0901317. KO, Knockout; rel., relative.

 
RIP140 Function in LXR-Mediated Lipogenesis in HuH7 Cells
To investigate the function of RIP140 in lipogenesis in more detail, we used human HuH7 hepatoma cells. Cells were infected with an adenovirus expression small interfering RNA (siRNA) for RIP140 or a control virus, respectively. Treatment with the small interfering RIP140 (siRIP140) virus resulted in an effective knockdown of RIP140 protein as shown by Western blotting (Fig. 3AGo). The expression of LXR, SREBP1c, SCD-1, and FAS was markedly increased in response to treatment with T0901317 in control samples; however, this was completely abrogated when RIP140 was depleted by siRNA (Fig. 3BGo). Thus, it appears that RIP140 functions as a positive cofactor for LXR and is required to stimulate expression of the lipogenic program.


Figure 3
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  		Fig. 3. RIP140 Function in LXR-Mediated Lipogenesis

A, HuH7 cells were treated with siRNA for RIP140 (siRIP140) or control (pAd-GFP) as indicated. After 48 h, RIP140 and CtBP protein was detected by Western blotting. B, Gene expression of lipogenic genes was determined after treatment with 1 µM T0901317 or vehicle alone for 12–16 h in HuH7 cells depleted of RIP140 (siRIP140) or controls (pAd-GFP). mRNA levels were measured by real-time PCR and normalized to cyclophilin. Data are expressed as fold change relative to control samples and represent the average of three experiments performed in triplicate ± SEM. *, P < 0.05; **, P < 0.01 vs. control treated with T0901317. rel., Relative.

 
RIP140 Binding to LXR Target Genes in HuH7 Cells
Nuclear extracts were prepared from HuH7 cells, which had been treated with 1 µM T0901317 or vehicle alone for 2 h. Coimmunoprecipitation experiments were performed using conjugated RIP140 antibody or a control antibody. RIP140 and LXR are expressed in HuH7 cells, and LXR protein was detected by Western blot in the precipitates with RIP140 antibody but not the control antibody. Consistent with previous in vitro observations, the LXR agonist had very little effect on the interaction of RIP140 and LXR (28)(Fig. 4AGo). Next we performed chromatin immunoprecipitation assays to test whether RIP140 binds to known target genes of LXR involved in lipogenesis in these cells. Chromatin fragments for FAS, SREBP1c, and LXR{alpha} gene promoters were amplified by PCR using primers for reported functional LXR response elements (LXREs) in these genes (11, 15, 17). Our experiments indicate that both LXR and RIP140 bind directly to the FAS, SREBP1c, and LXR{alpha} gene promoter in the vicinity of the LXRE but not to a distal region of the FAS gene, which does not contain a LXRE and was used as a control for specific binding in this assay (Fig. 4BGo). Consistent with the LXR-RIP140 interaction studies, there is little effect of the LXR agonist on the promoter occupation in these cells. These results suggest that RIP140 is directly involved in the transcriptional regulation of these genes in HuH7 cells.


Figure 4
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  		Fig. 4. Interaction of LXR and RIP140 in HuH7 Cells

A, Coimmunopreciptiation assays were performed with nuclear extracts from HuH7 cells treated with 1 µM T0901317 or vehicle alone for 2 h. B, Chromatin immunopreciptiation assay for LXR and RIP140 in HuH7 cells treated with T0901317 or vehicle alone for 2 h. Purified DNA from precipitated chromatin fragments was amplified by PCR using primers encompassing the LXREs in the promoter region of the indicated genes or a distal region of the FAS gene. The distal region of the FAS gene does not harbor a LXRE and was used as a control. C, Cotransfection of WT or mutant FAS reporter genes, LXR{alpha}, and increasing amounts of RIP140 expression plasmids. Cells were treated with T0901317 as indicated. Data represent the mean luciferase activity of three to four experiments ± SEM. IP, Immunoprecipitation; luc., luciferase; mut., mutant; rel., relative.

 
Finally, we investigated the ability of RIP140 to stimulate transcription from the FAS promoter in transiently transfected HuH7 cells (Fig. 4CGo). The activity of a FAS luciferase reporter gene was increased approximately 3-fold in the presence of T0901317 and was further increased up to 10-fold with increasing expression of RIP140. It should be noted that the ability of RIP140 to potentiate FAS promoter activity is dependent on a functional LXRE in the promoter (Fig. 4CGo). Thus we conclude that RIP140 is required for the induction of FAS expression by LXR.

Function of RIP140 in the Repression of Gluconeogenesis
A role for LXR in the regulation of hepatic glucose metabolism has recently emerged (19, 20, 21). We therefore investigated the possibility that RIP140 is also important for this aspect of LXR function in liver. In particular LXR has been shown to inhibit the expression of two key gluconeogenic enzymes, PEPCK and G6Pase (20). We therefore determined the effect of T0901317 administration on the expression of these genes in RIP140 null mice and WT controls. The expression of PEPCK and G6Pase was found to be down-regulated in WT animals after administration of the LXR agonist, as expected (Fig. 5AGo). However, the repression of PEPCK, but not G6Pase, by T0901317 was abrogated in mice devoid of RIP140 (Fig. 5AGo). Next we measured the effect of LXR activation in hepatocytes isolated from WT or RIP140 null mice. Glucose production (Fig. 5BGo) and PEPCK gene expression (Fig. 5CGo) were reduced in WT but not in RIP140 null cells. We next investigated the expression of PEPCK and G6Pase in Fao cells, which were previously used to study their transcriptional regulation (30). We were able to confirm that the expression of both genes was suppressed by LXR ligand and that the depletion of RIP140 by means of siRNA results in a loss of PEPCK but not G6Pase repression by T0901317 (Fig. 5DGo). These results suggest that the repression of PEPCK gene transcription by LXR is dependent on RIP140.


Figure 5
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  		Fig. 5. RIP140 Function in the Regulation of Gluconeogenesis

A, PEPCK and G6Pase gene expression in WT and RIP140 null mice treated with T0901317 (50 mg/kg/d) or vehicle alone for 3 d. Shown are the mRNA amounts relative to the control animals as the means of four to five animals in each group ± SEM. B, Primary hepatocytes were isolated from WT and RIP140 null mice. Cells were treated with T0901317 or vehicle and glucose production was measured. C, PEPCK gene expression in primary hepatocytes form WT and RIP KO mice was determined by real-time PCR. D, Fao cells were depleted of RIP140 by siRNA (siRIP140). After 48 h, control and siRIP140 cells were treated with T0901317 (T) or vehicle (V) alone for 12–16 h. PEPCK and G6Pase mRNA relative to control samples were measured by real-time PCR and represent the mean of three experiments ± SEM. *, P < 0.05; **, P < 0.01 vs. animals or samples treated with vehicle alone. KO, Knockout; rel., relative.

 
LXR and RIP140 Binding to the PEPCK Gene Promoter
We next investigated the ability of RIP140 and LXR{alpha} to interact in Fao cells by coimmunoprecititation experiments. As shown in Fig. 6AGo, RIP140 and LXR{alpha} interact but the LXR agonist had very little effect on the interaction. Next we performed chromatin immunoprecipitation assays to determine whether RIP140 is recruited to the PEPCK gene in Fao cells. We found that LXR and RIP140 bind in the vicinity of the putative LXRE in the PEPCK gene promoter but not to a distal region of the PEPCK gene, which does not contain a LXRE and was used as a control for specific binding in this assay (Fig. 6BGo). In contrast, we could not detect LXR or RIP140 binding to the proximal promoter region of the G6Pase gene, which has been shown to bind nuclear receptors such as hepatocyte nuclear factor 4 (HNF4) (31). HNF4 binds to both genes as shown earlier and was used as positive control for the chromatin immunoprecipitation assay (Fig. 6BGo). These findings demonstrate that RIP140 is directly involved in the repression of PEPCK by LXR and suggest that different mechanisms are involved in the repression of G6Pase and PEPCK expression by LXR in these cells.


Figure 6
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  		Fig. 6. RIP140 Binding to the PEPCK Gene Promoter

A, Coimmunopreciptiation assays were performed with nuclear extracts from Fao cells treated with 1 µM T0901317 or vehicle alone for 2 h. B, Fao cells were treated with T0901317 or vehicle alone. Chromatin fragments were precipitated with the indicated antibodies, and purified DNA was amplified by PCR using primers encompassing the LXREs in the promoter region of the PEPCK and G6Pase genes or a distal region of the PEPCK gene. The distal region of the PEPCK gene does not harbor a LXRE and was used as a control. IP, Immunoprecipitation.

 
An inspection of the PEPCK gene promoter sequence revealed a putative LXRE, which is very similar to known response elements for LXR (3). This sequence is relatively well conserved between species (Fig. 7AGo). To test whether LXR can bind to this element in the PEPCK promoter, we incubated biotin-labeled PEPCK probe with retinoic X receptor (RXR){alpha} and LXR{alpha} protein. Gel mobility shift analysis showed that this PEPCK sequence indeed binds LXR/RXR heterodimers (Fig. 7BGo). Competition assays using unlabeled oligonucleotides confirmed that the LXR/RXR binding was specific for the LXRE sequence in the PEPCK gene promoter (Fig. 7BGo).


Figure 7
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  		Fig. 7. Identification of a LXRE in the PEPCK Gene Promoter

A, Sequence alignment of putative LXREs in the rat, human, and mouse (mus) PEPCK promoter and human FAS LXRE. B, Biotin-labeled PEPCK probe was incubated with LXR{alpha} and RXR{alpha} protein in the presence or absence of 10- to 50-fold molar excess of competitor oligonucleotides, as indicated. The resulting DNA-protein complexes were separated from the free probe in a polyacrylamide gel and detected using strepavidin conjugated to horseradish peroxidase. ApoE, Apolipoprotein E; hum, human; mt, mutant; PEP, phosphoenol pyruvate.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Previous studies indicate that RIP140 plays a crucial role in fat accumulation (24) by suppressing the expression of genes involved in oxidative metabolism, uncoupling, and mitochondrial biogenesis in adipose tissue (25, 26). The role of RIP140 in other tissues is much less clear although the broad expression pattern suggests additional functions in transcriptional regulation (24). Gene expression profiling and the analysis of biochemical parameters in WT and RIP140 null mice led us to identify genes involved in hepatic lipogenesis as targets of RIP140. In this paper, we report that RIP140 is required for the ability of LXR to stimulate the expression of genes involved in lipogenesis. These observations prompted us to investigate whether RIP140 is also involved in other functions of LXR in liver. As a result, we have identified the PEPCK gene, a rate-controlling gene in the process of gluconeogenesis, as a direct target for LXR and RIP140. In contrast to the stimulation of lipogenesis, RIP140 acts as a corepressor for the inhibition of PEPCK by LXR (20). Thus, we propose that the function of RIP140 as a cofactor for LXR in hepatocytes varies according to the target genes and metabolic process.

Impaired accumulation of triglycerides in RIP140 null mice is accompanied by a failure of LXR to stimulate expression of the lipogenic genes SREBP1c, acetyl-coenzyme A carboxylase 1, SCD-1, and FAS. That RIP140 functions as a coactivator for LXR is supported by the failure of the LXR agonist T0901317 to stimulate the expression of these genes in cultured HuH7 cells devoid of the cofactor. Further evidence is provided by our observation that RIP140 potentiates the ability of LXR to stimulate transcription from the FAS promoter in transfected cells and by the binding of LXR and RIP140 to the LXRE in chromatin immunoprecipitation assays. The reduction of hepatic lipogenesis might contribute to the lean phenotype of the RIP140 null mice similar to the LXR–/– mice (24, 32). LXRs are also important for the regulation of lipid metabolism in muscle and white adipose tissue (32, 33). Future work will address a possible role of RIP140 in the function of LXR in these tissues. This will involve the treatment of isolated adipocytes with LXR agonists and the generation of tissue-specific knock-out mice for RIP140.

The agonist for LXR is required for lipogenic gene expression, both in vivo and in vitro; however, its effect on the binding of LXR and RIP140 to the FAS promoter is minimal. This is consistent with our observation that endogenous LXR and RIP140 interact in a ligand-independent manner and with previous findings characterizing the in vitro interaction (28). In fact, RIP140 seems to bind to LXR in an atypical way involving regions of the RIP140 protein other than the nuclear receptor boxes (34). We presume that ligand binding to LXR serves another function, possibly the recruitment of other cofactors. Recent studies identified PGC-1ß as a coactivator for SREBP in hepatocytes (35). It is thus conceivable that RIP140 and PGC-1ß costimulate the expression of key genes for hepatic lipogenesis.

Although RIP140 does not seem to be involved in glucose homeostasis during fasting (data not shown), it is important for the repression of PEPCK expression by dietary cholesterol. Indeed, the repression of PEPCK by LXR depends on RIP140, both in vivo and in cultured cells. The function of RIP140 as a corepressor for LXR is also supported by chromatin immunoprecipitation assays showing the binding of both LXR and RIP140 to the PEPCK promoter. It should be noted that the down-regulation of another key gluconeogenic enzyme, G6Pase, is independent of RIP140. Thus, we conclude that the ability of LXR to suppress the expression of PEPCK and G6Pase is achieved by distinct mechanisms. Indeed the indirect regulation of G6Pase gene expression by LXR has been observed (36). These findings may have implications for the development of antidiabetic drugs. Partial agonists of LXR (37), which bypass the requirement for RIP140, should still repress the expression of G6Pase and hepatic glucose output but would not stimulate lipogenesis (38, 39). A role for LXR in the regulation of glucose metabolism is suggested by a recent report that LXR acts as a glucose sensor (40).

The function of RIP140 as a corepressor has been shown to involve the recruitment of repressive enzyme complexes such as histone deacetylases and C-terminal binding protein (CtBP) (41, 42). At present we can only speculate about the underlying molecular mechanisms for the activation of transcription. Recently, however, Cavailles and co-workers (43) have demonstrated that RIP140 is also capable of activating transcription from estrogen-related receptor target genes, possibly by sequestration of repressive enzymes. On the other hand, it is conceivable that RIP140 is capable of binding either repressive or activating enzyme complexes, such as PGC-1 (44). Thus, RIP140 bound to LXR in the liver may activate transcription from lipogenic genes and repress PEPCK gene transcription to regulate metabolic pathways and lipid homeostasis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Animal Studies
Generation of RIP140 knockout mice (RIP140 null) has been described elsewhere (45). Mice (age matched and 3–4 months old a time of analysis) were housed under standard conditions and fed ad libitum with chow diet (Special Diet Services, Witham, UK) or a western diet (D12079B; Research Diets, New Brunswick, NJ) for 4 wk. The LXR agonist T0901317 was administered by gavage feeding of 50 mg/kg·d for 3 d. Mice were killed 24 h after the last treatment and tissue samples were collected. All procedures were performed in accordance with the guidelines for animal care and use of the United Kingdom Home Office.

Biochemical Analysis
Triglyceride content in saponified, neutralized liver extracts, plasma, and cell extracts was measured using the Triglyceride (GPO Trinder) Reagent (Sigma Chemical Co., St. Louis, MO). Glucose production assay was performed as described previously (46).

Plasmids and Adenoviral Constructs
FAS luciferase reporter constructs and RIP140 expression plasmid have been described elsewhere (17, 25). LXR{alpha} expression plasmid was a gift from M. Needham (Astra Zeneca, Alderley Edge, UK). The adenoviral vector expressing siRNA for RIP140 was generated as described previously (47) and targets the sequence 5'-AGAAGATCAAGATACCTCA-3' of human, rat, and mouse RIP140.

Cell Culture
Hepatocytes were isolated by collagenase perfusion of liver from WT and RIP140 null mice as described elsewhere (48). HuH7 and Fao cells were grown in DMEM containing 10% fetal calf serum. Cells were transfected using the FuGENE 6 reagent (Roche Clinical Laboratories, Indianapolis, IN). Luciferase reporter activities were determined approximately 18 h after transfections using the Dual-Glo Luciferase Assay System (Promega Corp., Madison, WI). For adenovirus infection, cells were cultured in six-well format and infected with the siRIP140 or a control virus [expressing green fluorescent protein (GFP)] at an estimated multiplicity of infection of 20 (HuH7 cells) or 50 (Fao cells) for 36 h and then treated with 1 µM T0901317 or vehicle alone for approximately 12 h and harvested for RNA isolation.

EMSA
RXR{alpha} and LXR{alpha} protein (ProteinOne, Bethesda, MD) and biotin-labeled rat PEPCK probe (5'-CAAGAGGCGTCCCGGCCA-GCCCTGTCCTTGACCCCCACCTGA-3') were incubated at room temperature for 20 min. The competition reactions were performed by adding 10- to 50-fold excess unlabeled double-stranded apoE ME enhancer (5'-GATCGCTGCCAGGGTC-ACTGGCGGTCAAAGGCAG-3'), mouse PEPCK (5'-CAA-GAGGCGTCTCGGCTAGGCCTGCCCTTGACCCCCACCTGA-3'), rat PEPCK or mutated LXRE probe 5'-CAAGAGGCGCGGAGGACTGTCCTCCGACCAACCCCCACCTGA-3') oligonucleotide to the reaction mixture. The reactions were electrophoresed on a 6% precasted Tris-borate-EDTA gel at 100 V for 1 h in a 100 mM Tris-borate-EDTA buffer and transferred to a nylon membrane (Invitrogen, Carlsbad, CA). The biotin-labeled DNA was detected with LightShift chemiluminescent EMSA kit (Pierce Chemical Co., Rockford, IL).

Antibodies and Western Blot
Antibodies against RIP140 used in this study were 6D7 (mouse monoclonal against AA301–478 of human RIP140) and a rabbit polyclonal antiserum provided by Dr. H. Chen, UCDCC, Sacramento, CA); Western blot analysis was performed using antibodies against RIP140 (6D7), LXR (sc-1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and CtBP (sc-5963).

Coimmunoprecipitation and Chromatin Immunoprecipitation Assays
For coimmunoprecipitations, 5 mg nuclear extracts were precleared with 50 µl A/G plus agarose (Santa Cruz Biotechnology). Precleared extracts were incubated with 20 µl anti-RIP140-conjugated agarose beads (Pierce) for 4 h with rotation and washed three times with wash buffer [50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10% glycerol 0.1% Nonidet P-40]. Bound proteins were eluted with elution buffer (0.1 M glycine, pH 3.2) for 10 min at room temperature. Chromatin immunoprecipitations were performed with antibodies against RIP140 (Chen), LXR{alpha}/ß (sc-1000), HNF4{alpha} (sc-8987) or IgG (sc-2027, Santa Cruz Biotechnology), as described elsewhere (49, 50) using gene-specific primers (supplemental Table S1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org).

Quantitative PCR
Total RNA was isolated using the TRIzol reagent (Invitrogen). RNA transcripts were quantified by real-time PCR using the Opticon 2 (MJ Research, Inc., Watertown, MA) and the SYBR Green JumpStart Taq ReadyMix system (Sigma) using gene-specific primers (supplemental Table S1).

Statistical Analysis
Data are expressed as mean ± SEM. The statistical significance of differences between samples was determined by a two-tailed Student’s t test.


   ACKNOWLEDGMENTS
 
We thank Drs. T. Osborne, P. Tontonoz, and D. Granner for sharing their reagents and A. Soutar for a critical review of the manuscript.


   FOOTNOTES
 
This work was supported by the Wellcome Trust (Grant 061930 to B.H. and A.S.) and the Biotechnology and Biological Sciences Research Council (Grant BB/C5O4327/1 to M.H.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online August 7, 2007

Abbreviations: CtBP, C-terminal binding protein; FAS, fatty acid synthase; GFP, green fluorescent protein; G6Pase, glucose-6-phosphatase; HNF4, hepatocyte nuclear factor 4; LXR, liver X receptor; LXRE, LXR response element; PEPCK, phosphoenolpyruvate carboxykinase; PGC, PPAR coactivator; RIP140, receptor-interacting protein 140; RXR, retinoic X receptor; SCD, steroyl-coenzyme A desaturase; siRIP140, small interfering RIP140; siRNA, small interfering RNA; SREBP1c, sterol regulatory element-binding protein-1c; WT, wild type.

Received for publication April 25, 2007. Accepted for publication July 31, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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NURSA Molecule Pages Link:

Nuclear Receptors:   LXRβ  |  LXRα
Coregulators:   RIP140
Ligands:   T0901317



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