1 Graduate Center for Nutritional Sciences, University of Kentucky, Lexington, Kentucky
2 Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado
3 Department of Microbiology, University of Colorado Health Sciences Center, Denver, Colorado
4 Department of Pathology, University of Colorado Health Sciences Center, Denver, Colorado
5 Nestle Research Center, Lausanne, Switzerland
6 Department of Food Science & Human Nutrition, Colorado State University, Fort Collins, Colorado
7 Department of Pediatrics, University of Colorado Health Sciences Center, Denver, Colorado
8 Department of Biochemistry & Molecular Genetics, University of Colorado Health Sciences Center, Denver, Colorado
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ABSTRACT |
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Adiponectin is exclusively expressed in adipocytes (14) and has diverse roles that include regulation of insulin sensitivity and energy homeostasis, immunological responses, and the development of vascular disease (58). Expression and serum levels of adiponectin are diminished in humans and animals with insulin resistance, obesity, and type 2 diabetes (912). Although there has been rapid progress in understanding of the physiologic functions of adiponectin and the signaling pathways mediating adiponectin action (1315), little information is available regarding the transcriptional regulation of adiponectin expression.
The human adiponectin gene spans 16 kb and is composed of three DNA sequences encoding exons from 184,277 bp in size with consensus splice sites (16,17). The two introns span 10 and 0.9 kb. Both human and mouse adiponectin promoters are TATA-less but contain a classical CCAAT box (1618). Transient transfection studies indicate that a 1.3-kb fragment located upstream of the transcriptional start site of the human adiponectin gene, or 1.13 kb counterpart of mouse, confers basal transcriptional activity (17,18). However, promoter activity was increased only twofold in 3T3-L1 adipocytes compared with the activity in fibroblasts (17), suggesting that only modest adipocyte-specific regulatory elements are present in the proximal promoter region of the adiponectin gene. Transcriptional activity of basal promoters is often regulated by enhancer elements found at some distance from the start site of transcription, and the large DNA encoding the first intron often contains cis-acting regulatory elements (19). However, Das et al. (17) demonstrated that upstream sequences in the 5' flanking region of the mouse adiponectin gene do not increase adiponectin promoter activity in adipocytes. Therefore, the current study was designed to explore whether sequences within the first intron functioned to enhance adiponectin transcription in adipocytes.
Peroxisome proliferatoractivated receptors (PPAR)s and CCAAT/enhancer-binding proteins (C/EBPs) are the two major transcription factor families that transcriptionally transactivate adipose-specific genes, including adipocyte-specific fatty acidbinding protein (aP2), fatty acid synthase, leptin, and resistin (20). PPAR may directly bind to the human adiponectin promoter by forming heterodimers with retinoid X receptor (RXR) (21,22). Moreover, PPAR
2 stimulates the adipocyte differentiation program in C/EBP
/ fibroblasts and induces adiponectin expression at a modest level, but restoration of C/EBP
caused a remarkable increase in the expression of adiponectin (23,24). Therefore, full activation of adiponectin gene transcription requires both PPAR
and C/EBP
. Although sequence analysis revealed two putative C/EBPß sites and a C/EBP consensus region in the adiponectin proximal promoter region, most available data suggest that no nuclear protein appears to bind to these sites, and ectopic expression of C/EBP
had no effect on adiponectin promoter activity (17,21,22,25,26). However, during the preparation of this manuscript, Park et al. (23) reported that both C/EBP
and C/EBPß bind at the proximal promoter of mouse adiponectin gene. Therefore, whether C/EBPs regulate adiponectin gene transcription directly through the promoter or another region is controversial and warrants further investigation.
In this study, we show that the DNA encoding the first intron of the human adiponectin gene confers significant activity on the adiponectin promoter. We have identified adipocyte-specific regulatory elements in the DNA encoding the first intron of the human adiponectin gene. These elements are highly sensitive to the adipogenic transcription factor C/EBP but not to PPAR
. Furthermore, in both human Chub-S7 or 3T3-L1 adipocytes, adiponectin mRNA levels were increased or decreased, respectively, by overexpressing or knocking down C/EBP
expression. In total, the results suggest that the DNA encoding the first intron of the human adiponectin gene contains an intronic enhancer and regulates adiponectin gene transcription in an adipose tissuespecific manner.
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RESEARCH DESIGN AND METHODS |
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Experimental animals.
Male C57BLKS/J-db/db mice weighing 3941 g and age-matched (810 weeks of age) wild-type littermates were purchased from The Jackson Laboratory (Bar Harbor, ME). Homozygous C/EBPß gene knockout mice were obtained by cross-breeding female mice heterozygous for a null mutation of the C/EBPß gene with homozygous male mice (30). After overnight fasting, the mice were killed and epididymal fat tissues collected and stored at 80°C for mRNA and nuclear protein isolation.
Plasmid constructs.
A reporter plasmid encoding luciferase under the control of the human adiponectin promoter (Pro-Luc) was generated by excising the 1.3-kb promoter fragment and 18-bp DNA encoding exon 1 from human genomic DNA and insertion into the KpnI and NheI sites of the pGL3 basic luciferase expression vector (Promega). To generate a gene reporter construct with the adiponectin promoter and DNA encoding exon 1 and intron 1, intron 1 encoding DNA was cloned by PCR from human genomic DNA, and the NheI-XhoI PCR fragment was inserted into the Pro-Luc plasmid (Pro-Int1-Luc). Serial deletion of the human promoter intron 1 luciferase plasmids was achieved by PCR. Site-directed mutations of the adiponectin Pro-Int1-Luc plasmids were constructed using a Quickchange mutagenesis kit following the instructions provided by the supplier (Stratagene). The mutations of the C/EBP consensus binding sites in intron 1 were confirmed by DNA sequencing. Heterologus gene reporter constructs were created using the human adiponectin promoter and the DNA encoding the intron from the adenovirus type 5 E1A 13S mRNA, intron 1 of the mouse phosphoinositide 3-kinase regulatory subunit p85 gene, or intron 1 of the mouse adiponectin gene. The diagrams of the constructs are illustrated in Fig. 1B.
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Generation of the siRNA expression cassette.
The siRNA expression cassette was created by ligating the human U6 pol III promoter with sense and antisense oligonucleotides of self-complementary hairpin sequence. The human U6 pol III promoter was amplified by PCR using human genomic DNA as template. The PCR fragments were digested with KpnI and HindIII and cloned into pBluescriptII SK+ (Stragagene, La Jolla, CA) to create pBluescriptII+U6. C/EBP target sequence (human, rat, and mouse) for siRNA was selected according to the Ambion web-based criteria and further analyzed by BLAST search to avoid significant similarity with other genes. The sequences of the oligonucleotides are 5'-AGCTTGCGACGAGTTCCTGGCCGACTTCAAGAGAGTCGGCCAGGAACTCGTCGTTTTTTGGAAT-3'; and 5'-CTAGATTCCAAAAAACGACGAGTTCCTGGCCGACTCTCTTGAAGTCGGCCAGGAACTCGTCGCA-3'. The oligonucleotides contain the termination signal of five thymidines and cohesive ends for HindIII and XbaI at the 5'- and 3'- ends. The annealed oligonucleotides were inserted into the HindIII and XbaI sites of pBluescriptII+U6. The C/EBP
siRNA-expression cassette was digested with the enzymes KpnI and XbaI and then cloned into the same sites in the adenoviral shuttle vector pAdTrack. Recombinant adenoviruses containing the cDNAs encoding mouse C/EBPß, the human C/EBP
and PPAR
2 or siRNA against C/EBP
were generated using the AdEasy system (31) as previously modified (32).
Luciferase assay.
Reporter constructs were introduced into cells by electroporation or chemical reagent. Differentiated (day 10) 3T3-L1 adipocytes were transfected by a modification of the electroporation method (33). For comparison, confluent 3T3-L1 fibroblasts or HEK293 cells were also transfected by electroporation. In some studies, the adiponectin promoter-luciferase gene reporter construct was cotransfected with pcDNA-hC/EBP, pcDNA-hPPAR
2, and/or CMX-hRXR
into HEK293 cells using FuGENE 6 transfection reagent (Roche, Indianapolis, IN). A pCMVß-galactosidase plasmid was cotransfected to control for transfection efficiency (Clontech Laboratories, Palo Alto, CA). Twenty-four hours after transfection, cells were lysed and luciferase activity measured. Luciferase data were normalized relative to ß-galactosidase luminescence and expressed as the relative luciferase activity.
Electrophoretic mobility shift assay.
Electrophoretic mobility shift assay (EMSA) was performed using the LightShift Chemiluminescent EMSA kit following the protocol provided by manufacture (Pierce Biotechnology, Rockford, IL). Double-stranded DNA fragments corresponding to the C/EBP consensus sequences or mutant sequences and oligonucleotides from the DNA encoding the first intron of the human adiponectin gene were 5' end-labeled with biotin (IDT, Coralville, IA). C/EBP protein was synthesized in HEK293 cells transduced with the adenovirus encoding human C/EBP
. Double-stranded oligonucleotides composed of the following sequences were used for binding and competition assays: C/EBP consensus, 5'-TGCAGATTGCGCAATCTGCA-3'; C/EBP mutant, 5'-TGCAGAGACTAGTCTCTGCA-3'; C/EBP
binding sites in the DNA encoding the first intron of human adiponectin gene at +3062/+3082 (+1 nt refer to the transcription start site), 5'-GAAGATTAAGTAAAATAAATT-3'; at +3076/+3096, 5'-ATAAATTTTGAAAATGCCTTA-3'; AT +3089/+3109, 5'-ATGCCTTATGAAAATTACACT-3'; at +5913/+5931, 5'-ACTTATTTTACCTAATCTT-3'; at +6570/+6588, 5'-AGTTTTATTGCCAAAGCCA-3'; at +7872/+7890, 5'-TAATTTCTTACAGAATATA-3'; at +7922/+7940, 5'-AGTTTGATGAAATATAAAT-3'; at +9935/+9953, 5'-TGAATGGAGCAATCTGTGT-3'.
Nuclear protein extraction and Western blot analysis.
Nuclear protein was extracted as described previously (34). Protein levels of C/EBPß, C/EBP, or PPAR
were measured by Western blot using antibody raised against the COOH-terminus of human C/EBPß, C/EBP
, or PPAR
(Santa Cruz Biotechnology, Santa Cruz, CA) (30).
RNA extraction and Northern blot analysis.
Total RNA was prepared with Trizol reagent (Invitrogen, Carlsbad, PA). RNA was denatured, separated by electrophoresis on 1% agarose gels, and transferred to positively charged nylon membranes (Roche). The cDNA fragment of mouse or human adiponectin, aP2, and GAPDH were labeled with [-32P]dCTP using the Prime-It II Random Primer Labeling kit (Stratagene, Cedar Creek, TX). Membranes were blotted with labeled probes, and radioactive signals were quantified and analyzed with PhosphorImager and Imagequant software (Molecular Dynamics, Sunnyvale, CA).
Data analysis.
Data are expressed as mean ± SE. Statistical analyses were performed using the Students t test or ANOVA analyses followed by contrast test with Tukey or Dunnett error protection. Differences were considered significant at P < 0.05.
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RESULTS |
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One recent study reported that the heterologous Hbb intron enhances human adiponectin promoter-driven luciferase activity in adipocytes and has been used for tissue-specific gene expression (35). The human and rabbit ß-globin (also named Hbb) genes have two introns. Both introns are essential for the accumulation of stable cytoplasmic mRNA and Hbb gene expression (36,37). However, there are no enhancer elements in the DNA encoding these introns, and regulation is exerted posttranscriptionally through increased efficiency of 3'-end formation (36,37). The finding of increased efficiency of adiponectin promoter-driven gene expression dependent on the Hbb intron raises the question of whether the enhancement of adiponectin promoter activity by intron 1 of the human adiponectin gene is gene-specific and whether there are enhancer elements in the DNA encoding the intron. To examine the mechanism(s), heterologous promoter constructs were created using the herpes simplex virus thymidine kinase (TK) promoter and the first intron of the human adiponectin gene. Transient transfection studies indicated that the presence of the DNA encoding intron 1 of the adiponectin gene increased TK promoter activity in response to C/EBP (data not shown). Most importantly, the enhancement was not dependent on the location of the DNA encoding intron 1. However, upregulation of luciferase expression by the Hbb intron is position dependent (35). Generally, transcriptional enhancer elements exhibit a position-independent effect on gene expression. Therefore, these studies suggest that there are enhancer elements in the DNA encoding the first intron of the human adiponectin gene.
To test whether the adiponectin intron 1dependent increased promoter activity occurs at the level of transcription or at a posttranscriptional step, two heterologous gene reporter constructs were created using the mouse p85 intron 1 and the 13S mRNA intron of adenovirus type 5 E1A. The first intron of the mouse p85
gene is 9.934 kb in length, similar to the size of intron 1 of the human adiponectin gene, and p85
is expressed in most tissues. To date, there are no reports indicating that the p85
intron 1 regulates gene expression. The DNA encoding the E1A 13S mRNA intron, which is only 116 bp in length, has no known function other than in splicing (35). As shown in Fig. 1D, the presence of the DNA encoding the E1A intron led to a slight increase, and the presence of the DNA encoding the p85
intron led to a slight decrease in adiponectin promoter-driven luciferase expression in adipocytes. However, neither intron had a statistically significant effect on the level of luciferase (P > 0.05). We also tested the functional similarity of the first intron of human and mouse adiponectin genes. Consistent with a previous study (17), the presence of mouse intron 1 did not increase the level of adiponectin promoter-directed luciferase activity in adipocytes (Fig. 1D). These data suggest that the enhancement of the human adiponectin promoter activity by the DNA encoding the first intron of the human adiponectin gene is dependent directly on the DNA sequence rather than on the presence of an intron in the primary transcript.
C/EBP increases adiponectin promoter transactivation activity through the DNA encoding the first intron.
C/EBP and PPAR
are major adipogenic transcription factors that transactivate many adipocyte-specific genes. We tested the ability of these transcription factors to regulate adiponectin gene expression using transient transfection assays in HEK293 cells, which are a well established transient transfection cellular model for studying transcriptional regulation of adipocyte gene expression (17,21,22). As shown in Fig. 2A, endogenous C/EBP
and PPAR
in HEK293 cells are barely detectable. Ectopic expression significantly increased C/EBP
, PPAR
2, or RXR
protein levels (Fig. 2A). Coexpression of PPAR
2 and RXR
increased adiponectin promoter activity twofold (P < 0.05, Fig. 2B). Although the luciferase activity directed by the Pro-Int1-Luc reporter construct was increased significantly in the presence of high levels of PPAR
and RXR
, the magnitude of the stimulation was less than that of the construct with the adiponectin promoter sequence alone (Fig. 2B). The reduced response from the Pro-Int1-Luc construct may be caused by the presence of the DNA encoding the first intron. These sequences may contain repressive elements (see Elements responsible for the C/EBP
-mediated transcriptional regulation of the adiponectin gene) that suppress adiponectin gene expression, especially in nonadipocytes. Nevertheless, the data suggest that PPAR
upregulation of human adiponectin gene transcription is mediated by the proximal basal promoter. In contrast, overexpression of C/EBP
did not increase luciferase activity directed by the Pro-Luc construct (after excluding the increment driven by the elements in the pGl3 vector, Fig. 2C). However, C/EBP
led to a robust increase in the activity of the adiponectin promoter when the DNA encoding the first intron was present in the construct (Fig. 2C).
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C/EBP regulates adiponectin gene expression in adipocytes.
To further assess the role of C/EBP, C/EBPß, and PPAR
in adiponectin gene expression in adipocytes, we overexpressed C/EBP
, C/EBPß, or PPAR
in mature Chub-S7 (day 17 after induction of differentiation) or 3T3-L1CAR
1 adipocytes (day 10 after induction of differentiation) or knocked down endogenous C/EBP
expression. As shown in Fig. 5A, 24h after transduction with adenovirus vectors, C/EBP
, C/EBPß, or PPAR
protein levels were significantly increased. Studies have suggested that during the adipocyte differentiation process, expression of these transcription factors can induce or regulate one another (20). Interestingly, in this study, over expression of C/EBP
, C/EBPß, or PPAR
did not affect levels of the other proteins, neither did thiazolidinedione (TZD) treatment (Fig. 5A). The differences in results relative to the earlier studies may be explained by the relatively long period of treatment in this study and the fact that that the cells used here are differentiated mature human adipocytes. Surprisingly, in adipocytes transduced with adenovirus encoding C/EBPß, C/EBP
expression was significantly increased (P < 0.001, Fig. 5A). Expression of siRNA directed against C/EBP
knocked down endogenous C/EBP
protein levels by 78% in Chub-S7 adipocytes (P < 0.001, Fig. 5B), without any significant morphological change or change of lipid droplets (data not shown). The C/EBP
siRNA did not alter PPAR
2 protein level but slightly increased C/EBPß and C/EBP
protein levels without statistical significance (Fig. 5B).
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Similar to the results from Chub-S7 adipocytes, overexpression or knocking down of C/EBP in 3T3-L1CAR
1 adipocytes resulted in a significant increase or decrease of adiponectin mRNA levels, respectively (Fig. 5D). Interestingly, consistent with a previous observation (38), overexpression of PPAR
2 and TZD treatment only slightly increased adiponectin mRNA levels (P > 0.05) in the 3T3-L1CAR
1 adipocytes but markedly elevated aP2 mRNA (P < 0.05, Fig. 5D). The mechanism for the differences in the response to PPAR
2 and TZD treatment between Chub-S7 and 3T3-L1CAR
1 adipocytes is currently unknown.
C/EBPß gene deletion does not alter adiponectin mRNA and serum protein level in mice.
Four members of the C/EBP family are involved in adipocyte differentiation (20). To determine the role of C/EBPß in regulating adiponectin expression, we measured adiponectin mRNA levels and serum protein from C/EBPß/, db/db (leptin receptor gene mutation) diabetic and wild-type control mice. Consistent with former studies, adiponectin mRNA and serum protein levels were significantly reduced in db/db mice (Fig. 6). Surprisingly, although there is a significantly low fat mass in C/EBPß/ mice (data not shown), there were no significant differences in adiponectin gene expression and serum protein levels compared with wild-type control (Fig. 6).
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DISCUSSION |
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Available data indicate that the coding sequences and adiponectin protein structure are well conserved between human and mouse. However, alignment of the first intron (10,304 bp) of the human adiponectin gene with its mouse counterpart (8,584 bp) shows no significant homology. Consistent with a previous study (17), our study also showed that the presence of the DNA encoding the first intron of the mouse adiponectin gene did not increase reporter gene expression even in adipocytes (Fig. 1D). Thus, the regulatory elements in the first intron of the human adiponectin gene may not exist in mouse. However, the current study demonstrates that ectopic expression of C/EBP indeed increases the adiponectin mRNA levels in both Chub-S7 and 3T3-L1 adipocytes, which are originally from mouse, suggesting that C/EBP
upregulates adiponectin gene expression in both human and mouse. It raises the possibility that C/EBP
upregulates adiponectin gene expression perhaps through a mechanism that is conserved between human and mouse. Support for this hypothesis comes from a recent study demonstrating that both C/EBP
and C/EBPß can bind to the mouse adiponectin proximal promoter and increase the promoter activity (39). This study also revealed significant sequence similarity for the C/EBP binding sites at the proximal promoter region between mouse and human (39). Our study demonstrated that cotransfection of C/EBP
significantly increased (12- to 16-fold) the human adiponectin promoter-driven luciferase expression. The vector we used for the luciferase gene reporter construct exhibits regulation by C/EBP
. Most likely, the C/EBP
-stimulated increase in luciferase activity (approximately twofold) (39) was obscured by the background vector response in our study. The discrepancy between our results and those of the previous study may also result from the use of different cell lines. Therefore, further studies are warranted to investigate whether C/EBP
binds on the proximal promoter region and regulates adiponectin gene transcription in humans. We also hypothesize that there might be an indirect pathway that mediates the regulatory effect of C/EBP
. The mechanistic difference between C/EBP
regulation of adiponectin gene expression in mice and humans is currently under investigation.
Because adiponectin is exclusively expressed in adipocytes, there must be a mechanism by which adipose tissuespecific transcription factors activate the gene in adipocytes. Although a PPAR response element has been identified in the promoter of the human adiponectin gene and TZD treatment can increase the expression and secretion of adiponectin in both human subjects and db/db diabetic mice (21,38), the mechanism by which PPAR
activates adiponectin gene expression is not completely clear. Furthermore, the role by which PPAR
regulates adiponectin expression in an adipocyte-specific manner was not examined. The results from this study show that the presence of DNA encoding the first intron significantly increased the adiponectin promoter activity only in adipocytes (Fig. 1C), and the presence of this DNA fragment does not further increase the promoter activity in response to PPAR
2 (Fig. 2B). Thus, PPAR
is required but not totally responsible for adipocyte-specific activation of adiponectin gene transcription. It appears that there are elements in the DNA encoding the first intron of the human adiponectin gene responsible for adipocyte-specific expression (Fig. 1C). C/EBP
dramatically increased adiponectin promoter activity but in a manner dependent on the presence of the DNA encoding the first intron (Fig. 2C). Thus, C/EBP
, acting through the intronic enhancer, contributes to the tissue-specific expression of adiponectin. Although ectopic expression of either C/EBP
or PPAR
alone in preadipocytes can stimulate the adipogenic program, there are numerous lines of evidence to indicate that these two transcription factors act synergistically with one another (20,40,41). However, there is a subset of adipocyte-specific genes, including leptin and resistin, that rely on C/EBP
for expression (42,43). Furthermore, both leptin and resistin expression are not only dependent on C/EBP
, but are downregulated by liganded PPAR
(42,43).
C/EBPs are a family of leucine zipper transcription factors. During adipocyte differentiation, C/EBPß and C/EBP increase transiently at the onset of adipocyte differentiation and decrease during terminal differentiation, but C/EBP
increases later and remains at a high level in mature adipocytes (20,44). Adiponectin expression parallels and occurs immediately after the rise of C/EBP
and is maintained at a relatively high level in mature adipocytes (2). However, C/EBPß and C/EBP
expression transiently increase at the beginning of adipocyte differentiation and drop rapidly after day 2 of differentiation (45). In contrast, C/EBP
and adiponectin expression are then activated, and protein levels are increased and maintained at a high level in the late stages of adipocyte differentiation and in differentiated mature adipocytes (2,45). Thus, during adipocyte differentiation, C/EBPß and C/EBP
protein levels correlate poorly with adiponectin expression. It is logical to speculate that C/EBPß and C/EBP
are not key regulators of adiponectin gene expression in differentiated mature adipocytes. Further support comes from the finding that there is a significant reduction of fat tissue mass in the C/EBPß/ mice relative to the wild-type mice (30), but there is no difference in serum adiponectin protein or adiponectin mRNA levels from the white adipose tissues (Fig. 6). However, in this study, the effects of ectopic expression of C/EBPß and C/EBP
were examined in differentiated mature adipocytes. Therefore, the current study cannot rule out roles for C/EBPß or C/EBP
in regulating adiponectin gene expression during differentiation. A recent study has reported that phosphorylation of C/EBPß at a consensus ERK/GSK3 site is required for both C/EBP
and adiponectin gene expression during the differentiation of mouse fibroblasts into adipocytes (23).
In summary, this is the first report showing the DNA encoding the first intron of the human adiponectin gene acts as an enhancer in regulating adiponectin gene expression. The enhancer specifically responds to C/EBP.
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ACKNOWLEDGMENTS |
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We gratefully acknowledge Dr. Harvey F. Lodishand and Dr. David Mangelsdorf for providing PPRE-Luc and CMX-hRXR plasmids. We thank Dr. T. Jackson and Dr. A. Bradford for technical assistance with luciferase assays.
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FOOTNOTES |
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Address correspondence and reprint requests to Jianhua Shao, MD, PhD, Graduate Center for Nutritional Sciences, University of Kentucky, 900 S. Limestone, Lexington, KY 40536. E-mail: jianhuashao{at}uky.edu
Received for publication October 11, 2004 and accepted in revised form March 16, 2005
aP2, adipocyte-specific fatty acidbinding protein; C/EBP, CCAAT/enhancer-binding protein; EMSA, electrophoretic mobility shift assay; HEK, human embryonic kidney; PPAR, peroxisome proliferatoractivated receptor; RXR, retinoid X receptor
; TK, thymidine kinase; TZD, thiazolidinedione
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REFERENCES |
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