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Institute of Reproductive and Developmental Biology, Imperial College London, Faculty of Medicine, Du Cane Road, London W12 0NN, United Kingdom,1 Arexis AB, Arvid Wallgrens Backe, 20 SE-413 46 Göteborg, Sweden2
Received 4 March 2005/ Returned for modification 8 April 2005/ Accepted 5 August 2005
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ABSTRACT |
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INTRODUCTION |
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One of the key genes in BAT (Ucp1) encodes uncoupling protein 1, a member of the mitochondrial carrier family of proteins. Upon Ucp1 activation, respiration is uncoupled from ATP synthesis, resulting in an increased metabolic rate and the release of chemical energy from the brown fat in the form of heat (8). Functionally defective BAT has been associated with obesity (9, 12), and transgenic mice with reduced BAT are obese, with symptoms of hyperglycemia and insulin resistance (21). In addition, ectopic expression of Ucp1 in WAT in transgenic mice results in resistance to diet-induced obesity and diabetes (15). Thus, the amount of BAT and the expression of Ucp1 correlate with protection from obesity.
Adipose tissue is controlled by humoral factors, by para- and intracrine factors, and by neural regulation. The differentiation of direct effects from systemic effects following genetic manipulation requires the analysis of in vitro and in vivo systems. Cell culture systems provide the opportunity to study the cell-autonomous action of genes. Many transcriptional events that mediate adipogenesis have been elucidated in cell lines, particularly in 3T3-L1 cells (22, 24). The primary regulators include the CCAAT/enhancer binding proteins (C/EBPß, C/EBP
, and C/EBP
) and peroxisome proliferator-activated receptor 
2 (PPAR2), a member of the nuclear receptor (NR) family of transcription factors (22). These factors seem to have similar roles in both white and brown adipocyte differentiation. Little is known about the potential interconversion of white and brown adipose tissue in terms of whether specific white or brown preadipocytes give rise to each type of adipose tissue or whether a single preadipocyte may be able to differentiate into either form of adipose cell. However, a number of transcription factors, coactivators, and cell cycle regulators, including NRs and NR cofactors, have been implicated in the control of white and brown adipocyte differentiation (30). One key regulator of many metabolic processes is the PPAR coactivator 1 (PGC1), which is required for adaptation to metabolic and physiologic stimuli (18, 20, 27). PGC1 coordinates a number of signaling pathways in adipocytes, including those involving additional coactivators for NRs, such as the p160 family members SRC-1 and TIF2. The relative expression levels of these coactivators regulate the development and functions of WAT and BAT (26).
The role of NR corepressors in adipogenesis and adipocyte function is less well understood. The corepressor RIP140 binds to the ligand binding domain of NRs in the presence of agonists, and lack of RIP140 results in reduced fat accumulation in vivo (17). The aim of this study was to determine the intrinsic role of RIP140 in adipose biology by using differentiated wild-type and RIP140-null primary adipogenic cultures, a RIP140-null cell line that retains its ability to undergo adipogenesis, and cells into which RIP140 has been reintroduced. Comparison of gene expression profiles in undifferentiated cells and adipocytes lacking and expressing RIP140 identifies derepressed and repressed genes that contribute to adipocyte function.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Morphological and immunohistochemical analysis. Tissue sections prepared as described previously (17) were incubated with polyclonal goat anti-mouse primary antibody to Cidea (sc-8732; Santa Cruz Biotechnology), diluted to a ratio of 1:200 in phosphate-buffered saline (PBS), and detected using a Vectastain Elite ABC kit (Vector Laboratories). Sections were counterstained with hematoxylin.
Cell culturing. Primary WAT cultures were prepared from inguinal fat depots. The tissue was finely minced and digested with collagenase (500 µg/ml) and DNase (100 µg/ml), followed by centrifugation at 1,000 rpm for 5 min. The cells were passed through a 70-µm filter and cultured in Dulbecco's modified Eagle's medium (DMEM)-F12 medium supplemented with 10% fetal bovine serum (Invitrogen). Mouse embryonic fibroblasts (MEFs) from embryos at embryonic day 11.5 were isolated and cultured in DMEM-F12 medium supplemented with 10% fetal bovine serum (Invitrogen). The RIPKO-1 cell line was generated by continuous culturing of MEFs. Differentiation of the cells was performed as previously described (35) in the absence or presence of 2.5 µM rosiglitazone (Ro) as indicated below. Differentiated cells were visualized with oil red O staining.
Lentiviral transduction of RIPKO-1 cells. The PCR-amplified coding sequence for human RIP140 was cloned into pLenti6/V5-D-TOPO (Invitrogen). This vector was cotransfected into 293FT cells with ViraPower packaging mix to generate the lentivirus. RIPKO-1 cells were transduced with the lentivirus and stable cell lines (RIPKO-L) generated by selecting with blasticidin.
Transient transfection.
Ucp1 promoter reporter constructs were generated by cloning a 4-kb fragment or 220-bp enhancer element (bp –2530 to –2310 relative to the transcriptional start site) of the 5'-flanking region of the murine Ucp1 gene into pGL3/basic vector (Promega). A 4-kb promoter construct lacking the enhancer region (
Enh; deletion of bp –2615 to –2228) was generated by PCR. RIPKO-1 cells were transfected in 24-well plates by using Fugene6 (Roche) with 1 µg of reporter gene, 250 ng of pRL-CMV, and/or 500 ng of pCI-RIP. Cells were harvested for luciferase assay 1, 3, and 5 days following the addition of the differentiation cocktail. Renilla luciferase activity was used to correct for differences in transfection efficiencies.
Expression analysis. RNA extraction from cell lines and tissue and cDNA preparation were performed as described previously (17). RIP140, L19, and UCP1 gene expression levels were determined using specific primers and TaqMan probes. Expression levels for all other genes were determined with SYBR green reagent by using specific primers. Expression levels for all genes were correlated to that for the L19 ribosomal coding gene. Primer sequences may be obtained on request.
Quantitation of mitochondrial DNA. DNA was extracted from untreated and differentiated primary MEFs using a DNeasy kit (QIAGEN). Real-time PCR with SYBR green reagent and specific primers was used to monitor levels of the mitochondrial cytochrome c oxidase subunit II gene and normalized to levels of the nucleus-encoded Ucp1 promoter.
Western blots. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and immunodetection were performed as described previously (6) using rabbit polyclonal anti-mouse UCP1 (AB1426; Chemicon International) at a ratio of 1/1,000 and mouse monoclonal anti-ß-actin (ab6276; Abcam Ltd., Cambridge, United Kingdom) at 1/5,000. Bands were visualized using secondary peroxidase-conjugated antibody and enhanced chemiluminescence.
Immunocytochemical staining. RIPKO-1 cells cultured on chamber slides were fixed in methanol and permeabilized in 0.2% Triton. UCP1 was detected using a rabbit polyclonal anti-mouse UCP1 antibody (AB3038; Chemicon International) diluted 1/100, a procedure followed by incubation with fluorescein isothiocyanate-conjugated swine anti-rabbit immunoglobulin G (IgG) (DAKO) diluted 1/50. Cell numbers were assessed by counting nuclei stained in the presence of a DAPI (4',6'diamidino-2-phenylindole)-containing Vectashield mountant (Vector Labs).
Mitochondrial stain. Cells were incubated for 30 min in MitoTracker Green (Molecular Probes) at 100 nM, washed in PBS, and mounted in Vectashield medium (Vector Labs).
[3H]palmitate oxidation assay. Following differentiation, primary MEFs and RIPKO-1, RIPKO-L, and 3T3-L1 cells were assayed for 3H2O production from excess [3H]palmitic acid as described previously (38).
Chromatin immunoprecipitation (ChIP) assay. Cells were incubated in the protein-protein cross-linking reagent dimethyl adipimidate · 2HCl (Pierce) at 10 mM in PBS for 30 min, followed by incubation in 1% formaldehyde in DMEM for 5 min at 37°C. The cross-linked cells were lysed, sonicated, and immunoprecipitated with protein A/G PLUS-agarose (Santa Cruz) according to the manufacturer's instructions using mouse anti-V5 antibody (Invitrogen) or control normal mouse IgG (Santa Cruz). DNA fragments were purified with a QIAquick PCR purification kit (QIAGEN) and used for templates in PCRs.
Affymetrix array hybridization and data analysis.
Total RNA was isolated from three samples each of RIPKO-1 and RIPKO-L cells, both undifferentiated and after adipocyte differentiation (10 days) in the presence of the PPAR ligand GW1929 (2.5 µm) (Sigma). Two RIP140-lentivirus-transduced cell lines were used for profiling in order to avoid variation due to differences in the sites of incorporation of the virus. Affymetrix array hybridization and scanning were performed by the CSC/IC Microarray Centre, Imperial College London, Hammersmith Campus, using murine 430 2.0 chips. Array data were analyzed with d-CHIP software (19). The microarray data are available at http://www.ebi.ac.uk/arrayexpress/ under accession number E-MIMR-42.
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RESULTS |
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agonist rosiglitazone. In these primary cell systems, both wild-type and RIP140-null cells underwent adipogenesis, as judged morphologically by the accumulation of cytosolic fat droplets (data not shown). In addition to these changes, the induction of the adipocyte marker aP2 gene and the adipocyte transcription factor PPAR2 was detected in all cell types following differentiation (Fig. 1A and B).
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We generated a MEF line lacking the RIP140 gene (termed RIPKO-1) by continuous culturing of the primary MEFs. This cell line retained the potential to differentiate into adipocytes, as judged morphologically by cytosolic fat droplet accumulation (data not shown). Consistent with these changes, aP2 and PPAR were induced in the cells, with maximal expression being reached by day 6 of treatment (Fig. 1C, upper panel). Ucp1 and Cpt1b displayed progressive increases in expression in the RIPKO-1 cells (Fig. 1C, lower panel). The expression of Ucp1 in BAT is normally associated with the transcription factor PGC1. However, expression levels of PGC1 or of the related gene PGC1ß were similar in wild-type and RIP140-null primary WAT adipocytes (data not shown).
Identification of RIP140 target genes following reexpression of RIP140 in RIPKO-1 cells.
To verify an essential role for RIP140 in gene regulation, it was reexpressed in the RIPKO-1 cells using a lentiviral vector. Adipocyte differentiation of stably expressing cell lines (termed RIPKO-L cells) was unaltered compared to that of the parental null cells, as determined by oil red O staining (Fig. 2A). Furthermore, both aP2 and PPAR2 gene expression levels were progressively increased during differentiation, as observed in both RIPKO-1 and 3T3-L1 cells (data not shown). The expression levels of RIP140 in the RIPKO-L and 3T3-L1 cells were similar at days 5 and 14 following differentiation (Fig. 2B). However, the patterns of expression differed between the cell lines; expression increased in 3T3-L1 cells following differentiation, whereas in RIPKO-L cells, expression was reduced. This difference is due to the reintroduced RIP140 being under the control of the cytomegalovirus promoter rather than that of its endogenous regulatory mechanisms.
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, PPAR, and SREBP1, which are essential for normal adipocyte differentiation and function, were induced following differentiation in both RIPKO-1 and RIPKO-L cells (Fig. 2C, upper panel). Genes that are associated with adipocyte function, such as those for SCD1, aP2, and adipsin, were also induced.
The microarray data were analyzed to identify genes that are induced in mature adipocytes and may be targeted for repression by RIP140. We selected genes that are upregulated by at least twofold in RIPKO-1 cells upon differentiation yet are repressed fivefold upon RIP140 reexpression (Fig. 2C, middle panel). The Ucp1 gene was identified within this gene cluster, showing that the reexpression of RIP140 in RIPKO-1 cells ablates the adipocyte-dependent expression of this gene. This analysis identified a number of additional genes repressed by the reexpression of RIP140 (Fig. 2C, middle panel). These include genes implicated in adipocyte function, namely, the Cidea (cell-death-inducing DFF45-like effector A) (41), Aquaporin 7 (AQPap) (13), ß3-adrenergic receptor (2), carboxylesterase 3 (34), acetyl coenzyme A synthetase 2 (33), and membrane metalloendopeptidase (31) genes. In addition, a number of other genes are similarly repressed by RIP140 expression (Fig. 2C, middle panel). In contrast, the expression levels of key regulators and markers of BAT function (37) are unaltered or only moderately affected (Fig. 2C, lower panel). For example, PGC1 and FOXC2 were expressed at lower levels in RIP140-null cells.
Cidea expression is altered in RIP140–/– cells in vitro and in vivo. We next investigated the regulation of the Cidea gene, identified by the microarray study as highly expressed only in differentiated RIPKO-1 cells, which has been implicated in thermogenesis through the direct modulation of Ucp1 activity (41). Cidea mRNA expression was undetectable in RIPKO-1, RIPKO-L, and 3T3-L1 adipocytes prior to differentiation and, following adipogenesis, increased only in RIP140-null cells (Fig. 3A, left panel); the expression profile closely resembled that of Ucp1 (Fig. 4A).
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RIP140 targets the Ucp1 promoter to repress transcription. To investigate further the mechanism of action of RIP140, we analyzed its ability to repress transcription from the Ucp1 promoter. Real-time PCR showed that Ucp1 expression was reduced in RIPKO-1 cells upon exogenous RIP140 expression to generate the RIPKO-L cells (Fig. 4A). Western blot analysis showed an induction of a 32-kDa immunoreactive band corresponding to UCP1 in fully differentiated RIP140-null cells (Fig. 4B). Immunocytochemical staining of RIPKO-1 cells showed a progressive increase in UCP1 as differentiation proceeded (Fig. 4B). This staining verified that UCP1 was confined to the lipid-containing differentiated cells and localized to the cytoplasm in accordance with its role as a protein confined to the mitochondrial compartment.
In mouse brown adipocytes, the transcription from the Ucp1 gene promoter is under the control of a region up to 4 kb upstream of the transcriptional start site (4, 16). Subsequent studies with transgenic mice have identified a 220-bp enhancer from kb –2.5 to –2.3 that controls tissue-specific expression (32). RIPKO-1 cells were transiently transfected with a luciferase reporter gene under the control of either the wild-type 4-kb Ucp1 promoter or a construct with the 220-bp enhancer region deleted (4kbEnh). Reporter activity was reduced by 50% with the deletion of the enhancer element (Fig. 4C). Exogenous expression of RIP140 inhibited luciferase activity from both the wild-type and 4kbEnh constructs. Following the demonstration that the enhancer region was necessary for the full induction of Ucp1 promoter activity, RIPKO-1 cells were transiently transfected with a luciferase reporter gene under the control of either the 4-kb Ucp1 promoter or the 220-bp enhancer element linked to the thymidine kinase promoter from –105 to +50. There was a progressive increase in promoter activity as the cells differentiated. Coexpression of RIP140 inhibited the Ucp1 promoter activity from both the full-length promoter and the 220-bp enhancer element, thus identifying this specific region as a target for RIP140 suppression of Ucp1 gene transcription (Fig. 4D).
Direct association of RIP140 with the Ucp1 enhancer element was determined using ChIP assays. The expression of RIP140-V5 in the RIPKO-L cells does not exceed that of the endogenous RIP140 in differentiated 3T3-L1 adipocytes (Fig. 2B). An antibody specific for V5 precipitated the Ucp1 enhancer element in differentiated RIPKO-L cells but not a control region 15 kb upstream of the mouse Ucp1 gene (Fig. 4E). Enrichment of the Ucp1 enhancer element was not observed with the V5 antibody in RIPKO-1 cells devoid of RIP140. Thus, RIP140 is recruited to the Ucp1 enhancer element in differentiated RIPKO-L cells.
Increased ß-oxidation in RIP140-null adipocytes. Following the identification of the Ucp1 gene as a target for RIP140, we investigated the functional consequences of increased Ucp1 expression. The induction of factors that facilitate fatty acid oxidation and uncouple respiration leads to an increased capacity for ß-oxidation. Measurement of the rate of 3H2O production derived from a [3H]palmitic acid substrate showed that RIP140-null adipocytes derived from primary MEFs exhibited a total fatty acid oxidation 1.5-fold higher than that of wild-type adipocytes (Fig. 5A). The cells had differentiated to similar extents, as judged by the expression of aP2; however, Ucp1 levels were elevated in the RIP140-null adipocytes (Fig. 5A). Measurement of the ratio of mitochondrial DNA to nuclear DNA in primary MEFs showed that mitochondrial biogenesis occurred in both wild-type and RIP140-null cells following differentiation (Fig. 5A). However, the absence of RIP140 did not significantly affect the level of mitochondrial DNA in the differentiated cells. The specific uptake of a MitoTracker mitochondrion-selective probe revealed that both RIPKO-1 and RIPKO-L differentiated cells contained more mitochondria than undifferentiated cells did (Fig. 5B).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, C/EBP, SCD1, aP2, and adipsin, are induced. Thus, the adipogenic program is not perturbed either by the absence of or by the exogenous expression of RIP140. Affymetrix microarray analysis was used to identify genes that are targeted for repression by RIP140 in mature adipocytes. These studies confirm the in vivo observations that Ucp1, a key factor in energy expenditure in BAT which is upregulated in the WAT in RIP140-null mice, is also derepressed following the loss of RIP140 in isolated adipocytes. The Cpt1b gene, which was also shown to be elevated in expression in vivo, is similarly increased in null cells, but to a lesser extent than Ucp1 is. In addition to Ucp1, we identified a number of genes regulated with similar expression profiles. These include the gene encoding Cidea, a protein reported to interact directly with Ucp1 to modulate the process of thermogenesis (41), which is downregulated by obesity in human WAT (23). We hypothesize therefore that RIP140 may be directly involved in the prevention of the expression of genes such as those encoding UCP1, CPT1b, and Cidea. In particular, the demonstration that novel RIP140 targets such as Cidea are derepressed in primary cells, mouse embryo fibroblasts, and in vivo WAT provides evidence to validate the expression profile differences determined by the microarray studies.
The increased Ucp1, Cpt1b, and Cidea expression in the absence of RIP140 occurs progressively during differentiation, suggesting that it is dependent on factors generated during adipogenesis. This progressive increase was also observed when transcription from a transfected Ucp1 gene promoter was analyzed for RIPKO-1 cells. The upregulation seems to be mediated predominantly by a previously characterized 220-bp enhancer element in the promoter containing binding sites for PPARs and TR/RXR (1, 29, 32). Importantly, we found that this element was a target for repression by RIP140 in transfected RIPKO-1 cells. This was further confirmed using ChIP experiments, demonstrating that RIP140 is recruited directly to this regulatory element in differentiated RIPKO-L cells where Ucp1 expression is abrogated. In brown fat cells, PPARs, together with PGC-1, have been shown to stimulate transcription from the Ucp1 promoter (1, 28, 32). Ucp1 expression is also regulated by neural signals under the control of the sympathetic nervous system that result in the activation of transcription factors such as ATF2 (2, 3). The levels of PGC-1 and PGC-1ß did not differ between wild-type and RIP140-null adipocytes, suggesting that altered expression of these coactivators is not essential for Ucp1 expression in the absence of RIP140.
In addition to the Ucp1 gene, a number of other genes are regulated with similar expression profiles, many of which are expressed in adipocytes and may also be nuclear receptor targets, including the AQPap gene, encoding a protein forming adipose-specific glycerol channels (14). Whether such genes are also direct targets of RIP140 or are regulated indirectly by additional factors is still to be determined. In contrast, other genes implicated in BAT function, such as the FOXC2 gene, were not increased in cells lacking RIP140. It is apparent that the absence of RIP140 in adipose cells results in increased levels of a subset of genes normally restricted to BAT, some of which regulate metabolic uncoupling processes and hence energy expenditure.
It is noteworthy that the abilities of primary MEFs and 3T3-L1, RIPKO-1, and RIPKO-L cells to metabolize palmitate correlated with the expression levels of Ucp1, Cpt1b, and Cidea, with the highest rates observed in RIP140-null cells. Mitochondrial biogenesis is a fundamental aspect of white adipose cell differentiation (40); however, the degrees of mitochondrial biogenesis did not differ significantly between wild-type adipocytes and those null for the RIP140 gene. The increased number of mitochondria in differentiated cells, along with the elevation of Ucp1, provides increased capacity for the enhanced ß-oxidation in cells lacking RIP140. These data demonstrating altered gene expression and mitochondrial respiration in RIP140-null adipocytes are in agreement with both expression-profiling studies and the increased O2 consumption in vivo found in RIP140-null mice (17).
Our studies demonstrate that processes that control metabolism and energy homeostasis in the adipocyte involve the direct action of a ligand-dependent NR corepressor and are independent of systemic effects. In summary, it has been demonstrated by a number of studies that NRs and their coactivators, by integrating different hormonal signals to regulate gene expression, play a fundamental role in the regulation of both the differentiation and the metabolic function of adipocytes. Here we describe a major role in adipocytes for a ligand-dependent NR corepressor in the prevention of the expression of genes that are associated with energy dissipation. Therefore, the prevention of RIP140 recruitment to receptors or a reduction in the levels of RIP140 itself may provide novel mechanisms for the control of energy use in adipose cells and assist in the treatment of obesity-related disorders.
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ACKNOWLEDGMENTS |
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This work was supported by the Wellcome Trust (M.C., E.K., and G.L.) grant no. 061930 and for a PhD studentship (D.D.) grant no. 069361.
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FOOTNOTES |
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