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Molecular Endocrinology, doi:10.1210/me.2003-0028
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Molecular Endocrinology 17 (8): 1522-1533
Copyright © 2003 by The Endocrine Society

Functional Characterization of the Human Resistin Promoter with Adipocyte Determination- and Differentiation-Dependent Factor 1/Sterol Regulatory Element Binding Protein 1c and CCAAT Enhancer Binding Protein-{alpha}

Jong Bae Seo, Mun Ju Noh, Eung Jae Yoo, So Yun Park, Jiyoung Park, In Kyu Lee, Sang Dai Park and Jae Bum Kim

School of Biological Sciences (J.B.S., M.J.N., E.J.Y., S.Y.P., J.P., S.D.P., J.B.K.), Seoul National University, Seoul 151-742, Korea; and Department of Internal Medicine (I.K.L.), School of Medicine, Keimyung University, Dongsan-Dong, Jungu, Taegu 700-712, Korea

Address all correspondence and requests for reprints to:Jae Bum Kim, School of Biological Sciences, Building 20, Room 109, Seoul National University, NS-70, San 56-1, Sillim-Dong, Kwanak-Gu, Seoul 151-742, Korea. E-mail: jaebkim{at}snu.ac.kr.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Recent studies with murine models propose that resistin would be a possible mediator to link between obesity and insulin resistance. Although it has been reported that resistin is highly expressed and secreted by adipocytes, transcription factors that are involved in resistin gene expression have not been well characterized. To investigate the molecular mechanisms of resistin gene expression, we cloned and characterized the human resistin promoter. Sequence analysis of the resistin promoter revealed several putative binding sites for adipogenic transcription factors including adipocyte determination- and differentiation-dependent factor 1 (ADD1)/sterol regulatory element binding protein 1c (SREBP1c) and CCAAT enhancer binding protein-{alpha} (C/EBP{alpha}). EMSA and chromatin immunoprecipitation assays demonstrated that ADD1/SREBP1c binds to the human resistin promoter in vitro and in vivo. Expression of ADD1/SREBP1c transactivated the luciferase reporter gene activity, the promoter region of which contains a human resistin promoter in a sterol regulatory element (SRE)-dependent manner. Furthermore, ectopic expression of ADD1/SREBP1c by adenovirus significantly increased the expression of resistin mRNA in adipocytes. Human resistin promoter was also activated by C/EBP{alpha} expression, although ectopic expression of both transcription factors did not show any synergistic effects on the activation of resistin promoter. Together, these data suggest that ADD1/SREBP1c and C/EBP{alpha} may play discrete roles in the regulation of the resistin gene expression.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ADIPOSE TISSUE HAS been considered previously as a passive organ for energy homeostasis since it is known to synthesize and store triglycerides as a major energy source and release them into other peripheral tissues when necessary. However, it is now recognized that adipose tissue actively executes whole-body energy metabolism as an endocrine organ by producing and secreting signaling molecules. These biologically active molecules, so called "adipocytokines," include TNF{alpha}, leptin, adiponectin (also known as AdipoQ or Acrp30), and resistin (also known as adipose tissue-specific secretory factor or Fizz3) (1). These factors are abundantly expressed in adipose tissue and are involved in energy metabolisms linked with obesity and diabetes. Many recent studies demonstrated that most adipocytokines play crucial roles in the regulation of lipid metabolism, feeding behavior, energy balance, and insulin sensitivity. For example, TNF{alpha} is overproduced from the adipose tissue of obese animals and contributes to the development of insulin resistance by decreasing insulin receptor kinase activity (2, 3, 4). Adipocyte-produced leptin acts on the hypothalamus to regulate food intake and energy balance. It is also involved in body weight control, fatty acid oxidation, and insulin sensitivity in peripheral tissues (5, 6). Adiponectin is a novel adipose-specific protein that belongs to the complement-related protein family (7, 8, 9). Expression of adiponectin is reduced in obese or diabetic animals, implying that it would function as an "adipostat" in regulating energy balance and that its deficiency might contribute to the obesity-dependent development of diabetes (10, 11).

Resistin is a newly identified adipocytokine (12). Like other adipocytokines, resistin expression was detected in adipose tissue and was markedly increased during differentiation of 3T3-L1 adipocytes (12, 13). The resistin gene encodes 114 amino acids and produces a secretory form containing 94 amino acids with 11 cysteine residues (14). A recent study showed that cysteine 26 is required for dimerization of the secreted form of resistin in serum (15). Resistin is a member of the resistin-like molecules family, which was the homolog to resistin (14). In murine models, resistin was also identified as a cysteine-rich adipose tissue-specific secretory factor, the overexpression of which inhibits adipocyte differentiation (13, 15). A human homolog of resistin, identified as hFizz3 (16), contains 54% amino acid homology to the mouse resistin protein (14). Due to its diverse family members, it is possible that mouse resistin and its human homologs among its family members might have different functional roles with different tissue specificity.

The serum levels of resistin protein were significantly elevated in obese mice, and administration of resistin impaired insulin action in vivo and in cultured cells (12). Interestingly, its expression was decreased by administration of thiazolidinedines (TZDs), which are antidiabetic drugs and synthetic ligands for peroxisomal proliferator-activated receptor-{gamma} (PPAR{gamma}) (12, 17, 18). Because TZDs increase insulin sensitivity, it has been proposed that TZD may reverse insulin resistance by repressing the expression of some genes that could cause insulin resistance (19, 20, 21). Therefore, Steppan et al. (12) proposed that resistin would represent an adipocyte-derived mediator that links obesity and insulin resistance.

In contrast to this model, several recent studies have suggested that resistin might not be tightly associated with insulin resistance. Way et al. (22) showed that resistin mRNA expression was significantly decreased in white adipose tissue of several obese and diabetic mouse models while TZDs increased resistin expression in both mice and rats. Moreover, administration of TZDs stimulated resistin mRNA levels in the white fat of obese mice, implying that resistin may not be the cause of insulin resistance (22, 23, 24). Furthermore, it has been reported that resistin mRNA expression was quite low in human adipose tissue and that it has a little correlation with body mass index of humans (17, 23). Thus, functional roles of resistin, especially regarding obesity and insulin resistance, remain to be elucidated.

In this study to investigate the molecular mechanisms of human resistin gene expression, the human resistin promoter was cloned by PCR. Sequence analysis of human resistin promoter reveals putative binding sites for adipocyte determination- and differentiation-dependent factor 1 (ADD1)/sterol regulatory binding protein 1c (SREBP1c) and CCAAT enhancer binding protein-{alpha} (C/EBP{alpha}). We show here that ADD1/SREBP1c binds directly to the human resistin promoter and transactivates it via a sterol regulatory element (SRE) motif. C/EBP{alpha} also activates the human resistin promoter. However, this transactivation is independent of ADD1/SREBP1c. These observations suggest a possible role of ADD1/SREBP1c and C/EBP{alpha} in the control of resistin expression.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Binding of ADD1/SREBP1c to the Human Resistin Promoter
To investigate the molecular mechanism of resistin gene expression, we cloned a human resistin promoter from genomic DNA by using PCR. Computer-assisted analysis (the TFSEARCH program based upon the TRANSFAC database) of human resistin promoter revealed that there are several putative cis-elements that might have potential roles in the transcriptional regulation of resistin gene expression (Fig. 1AGo). Notably, several SREs were identified in the region from -760 to -600 bp, and a C/EBP{alpha}-binding site was found at -231 bp upstream of the ATG site (Fig. 1AGo). In addition, human resistin promoter contains several E-box motifs and Sp-1 binding sites as marked in Fig. 1AGo. When the proximal promoter region of mouse resistin gene was compared with that of human resistin gene, we found that there is 49.8% sequence identity between two species (Fig. 1BGo). Several SREs and the C/EBP{alpha}-binding site are relatively well conserved in both human and mouse resistin promoters (Fig. 1BGo). These results suggest the possible involvement of ADD1/SREBPc and/or C/EBP{alpha} in the regulation of resistin gene expression.



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  		Fig. 1. Putative Binding Sites of Several Transcription Factors at the Human Resistin Promoter (-816 bp)

A, The sequence of the human resistin promoter is shown and numbered relative to the first ATG codon. This DNA fragment was used as the probe for EMSA (Fig. 2Go) and as the luciferase reporter construct designated pRstn820-Luc (Figs. 4Go and 5Go). Three SRE motifs, four putative E boxes, and a putative C/EBP site are marked with arrows. B, Schematic representation of the human and mouse resistin promoters. The putative SREs (black ellipses) and C/EBP{alpha}-binding site (black square) are indicated.

 
Since it appears that sequence of the human resistin promoter might contain at least three putative SRE motifs from -760 and -600 bp, we decided to examine whether ADD1/SREBP1c is able to bind to this region of the human resistin promoter. Radiolabeled DNA probe containing the region from -816 to +89 bp of the human resistin promoter was incubated with recombinant ADD1/SREBP1c proteins to perform EMSAs. As shown in Fig. 2AGo, ADD1/SREBP1c binds to the human resistin promoter by forming stable DNA-protein complexes (lanes 2 and 3). These DNA-protein complexes disappeared in the presence of an unlabeled competitor, SRE oligonucleotides corresponding to the preferential binding site of ADD1/SREBP1c (Fig. 2AGo, lane 4). In contrast, a nonspecific competitor, PPAR{gamma}/retinoid X receptor-{alpha} binding element (ARE7), did not compete with ADD1/SREBP1c for the binding to the resistin promoter (Fig. 2AGo, lane 3). These results indicate that ADD1/SREBP1c stably associates with the human resistin promoter in a sequence-specific manner.



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  		Fig. 2. Binding of ADD1/SREBP1c to the Resistin Promoter

A, ADD1/SREBP1c binds to the human resistin promoter. Double-stranded DNA fragments corresponding to the MluI-BglII fragment of the human resistin promoter (from -816 to +89) were radiolabeled with [{gamma}-32P] ATP and incubated in the reaction mixture with (lanes 2–4) or without (lane 1) recombinant ADD1/SREBP1c proteins. For competition assays, a 100-fold molar excess of unlabeled ARE7 (lane 3) or SRE (lane 4) oligonucleotides were used. DNA-ADD1/SREBP1c protein complexes and free probe are indicated by the arrow and arrowhead, respectively. The putative SREs (black ellipses) are indicated. B, ADD1/SREBP1c binds to SRE-A (-617 to -598) in the human resistin promoter. Radiolabeled DNA probe was incubated in the reaction mixture with (lanes 2–6) or without (lane 1) recombinant ADD1/SREBP1c proteins. In competition assays (lanes 3–7), a 100-fold molar excess of an unlabeled ARE7 (lane 3), SRE (core SRE lane 4), A (putative SRE-A, lane 5), B (putative SRE-B, lane 6), or C (putative SRE-C, lane 7) were added into the reaction mixture. C, Sequence comparison of putative SREs in the human and mouse resistin promoters with conserved SRE motifs.

 
Because there are three SREs in the proximal region (-760 to -600 bp) of the human resistin promoter, we investigated which SRE motif(s) would be the preferential binding site(s) of ADD1/SREBP1c. To address this issue, competition analyses were performed with three independent SRE oligonucleotides (Fig. 2CGo). When the control SRE found in low-density lipoprotein receptor promoter was added into binding reactions, formation of a DNA-protein complex was completely abolished (Fig. 2Go, A and B). Interestingly, stable DNA-protein complexes were also effectively competed by the addition of unlabeled SRE-A oligonucleotides, but not by ARE7, SRE-B, or SRE-C (Fig. 2BGo). Furthermore, we observed from in vitro assays that ADD1/SREBP1c protein can directly bind to the SRE-A site (data not shown). As in the human resistin promoter, three SREs were also identified within the region from -685 to -596 bp in the mouse resistin promoter (Fig. 2CGo). Therefore, it is likely that the ADD1/SREBP1c would preferentially and specifically bind to the SRE-A (-617 to -598) motif in the human resistin promoter.

Next, we performed chromatin immunoprecipitation (ChIP) assays to address the question whether ADD1/SREBP1c directly binds the chromatin-containing human resistin promoter in vivo. ADD1/SREBP1c proteins were ectopically expressed in h293 cells and immunoprecipitated from nuclear lysates after formaldehyde cross-linking. Direct association of ADD1/SREBP1c onto the endogenous human resistin promoter was detected by PCR amplification from ADD1/SREBP1c-immunoprecipitated DNA pellets. As shown in Fig. 3Go, ADD1/SREBP1c clearly bound to the endogenous chromatin DNA containing a human resistin promoter region. We also examined the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene fragment from the same immunoprecipitated DNA pellets to normalize the quantities of each PCR-amplified DNA as background control. This observation strongly implicates that ADD1/SREBP1c protein physically binds to the endogenous human resistin promoter at the chromatin level. Taken together with in vitro EMSA (Fig. 2Go), these results suggest that ADD1/SREBP1c would be able to bind to the resistin promoter for the regulation of resistin gene expression.



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  		Fig. 3. ChIP Assay of the Human Resistin Promoter

A, Direct binding of ADD1/SREBP1c to the human resistin promoter in vivo. h293 Cells were transfected with ADD1 expression vector. Transfected cells were cross-linked and immunoprecipitated by either rabbit polyclonal anti-ADD1 antibodies (lane 3) or rabbit preimmune serum as a negative control (lane 2). The immunoprecipitated DNA fragments were amplified by PCR (see Materials and Methods). Lane 1 shows the amplified resistin promoter and GAPDH from 1% of input DNA. GAPDH fragments were also amplified for the normalization of input DNA. B, Quantitation of PCR-amplified products of human resistin promoter normalized with the amplified GAPDH fragments.

 
Transactivation of the Human Resistin Promoter by ADD1/SREBP1c via the SRE Motif
We tested the ability of ADD1/SREBP1c to transactivate the resistin promoter since ADD1/SREBP1c can bind to the human resistin promoter both in vitro and in vivo. A luciferase reporter plasmid was constructed by inserting the 5'-flanking region of the human resistin promoter into a pGL3 basic vector (pRstn820-Luc). As shown in Fig. 4AGo, ectopic expression of ADD1/SREBP1c stimulated the transcriptional activity of the human resistin promoter.



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  		Fig. 4. Transactivation of the Resistin Promoter by ADD1/SREBP1c

A, The luciferase reporter pRstn820-Luc containing the -816 to +89 bp fragment of the human resistin promoter was cotransfected with ADD1/SREBP1c expression vector (ADD1) and pCMV-ßgal into Rat1-IR cells. The amounts of pSV-ADD1 expression vector cotransfected were 0, 50, 200, 400, and 1000 ng in lanes 1–5, respectively. B, Transactivation of FAS promoter by ADD1/SREBP1c is mediated via E box rather than SRE. Promoter activity of the luciferase reporter, pFAS-Luc, containing the -220 to +25 bp fragment of the fatty acid synthase gene was increased by either ADD1 (lane 2) or ADD1 (Y->R) (lane 3). C, The luciferase activity of the pRstn820-Luc reporter vector (wild type) was induced by ADD1, but not by ADD1 (Y->R). D, The luciferase activity of the pRstn820mSRE-A-Luc (see Materials and Methods) reporter (mutant: mSRE-A) was not induced by ADD1 or ADD1 (Y->R). The luciferase activities were normalized by ß-galactosidase activities, and the values are expressed as fold of the activity relative to that of control (lane 1). The putative SREs (black ellipses) and E boxes (white triangles) are indicated.

 
Unlike other transcription factors, ADD1/SREBP1c has a unique dual DNA binding specificity, allowing it to bind to both E-box (CANNTG) and non-E-box (SRE) motifs (25). Therefore, ADD1/SREBP1c has a potential to transactivate its target genes via either an E-box or SRE motif depending upon the promoter context of target genes (26). To determine whether ADD1/SREBP1c transactivates the human resistin promoter via either an E-box or SRE motif, we used a wild-type and point mutant form of ADD1/SREBP1c. The former encodes the amino-terminal fragment of the wild-type protein that has a maximal transcription activity (ADD1), and the latter contains a tyrosine-to-arginine mutation at the position of amino acid 320 [termed ADD1 (Y->R)]. Previously, this mutation has been shown to disrupt the dual DNA binding specificity of ADD1/SREBP1c, allowing its binding to an E-box but abolishing its binding to SRE motifs (27, 28). Fatty acid synthase (FAS) is one of the best characterized target genes of ADD1/SREBP1c (29). Consistent with the previous works, expression of the wild-type ADD1/SREBP1c transactivated the FAS promoter (Fig. 4BGo and Refs. 29, 30, 31). ADD1 (Y->R) showed a magnitude of transactivation of the FAS promoter similar to that of to wild-type ADD1 because ADD1/SREBP1c transactivates the FAS promoter via an E-box motif, but not via the SRE-motif (Fig. 4BGo and Ref. 29). In contrast to the FAS promoter, the ADD1 (Y->R) mutant was unable to transactivate the human resistin promoter, indicating that transactivation of this promoter by ADD1/SREBP1c is mediated by the SRE rather than the E-box motif (Fig. 4CGo). In addition, a mutation of the SRE-A (-617 to -598) motif (Fig. 4DGo) rendered this promoter completely resistant to activation by ADD1/SREBP1c or ADD1 (Y->R). These results suggest that ADD1/SREBP1c is capable of inducing the expression of human resistin gene via a specific SRE motif (-617 to -589) in its promoter.

Transactivation of the Human Resistin Promoter by C/EBP{alpha}
C/EBP{alpha} and PPAR{gamma} are key adipogenic transcriptional factors that modulate the expression of many adipocyte-specific genes. Expression of C/EBP{alpha} mRNA is induced at the late stage of adipocyte differentiation, which triggers the expression of many subsequent adipogenic genes at the terminal phase of differentiation (32). When the sequence of human resistin promoter was analyzed, a potential C/EBP{alpha} binding site was found (Figs. 1Go and 5Go). To examine the binding ability of C/EBP{alpha} to the human resistin promoter, nuclear extracts of h293 cells overexpressing C/EBP{alpha} were used for EMSA. As shown in Fig. 5BGo, C/EBP{alpha} binding to the resistin promoter was detected (lane 3), and its binding was left intact upon the addition of a nonspecific competitor (lane 4). However, addition of C/EBP consensus oligonucleotides specifically abolished the binding activity of C/EBP{alpha} to the resistin promoter without affecting nonspecific bindings (Fig. 5BGo, lane 5).



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  		Fig. 5. Transactivation of Resistin Promoter by C/EBP{alpha}

A, Sequences comparison of C/EBP{alpha} sites in the human and mouse resistin promoters. B, C/EBP{alpha} binds to the human resistin promoter. A double-stranded DNA fragment with the human resistin promoter (from -816 to +89) was radiolabeled with [{gamma}-32P]ATP and incubated in the reaction mixture with each nuclear extract (lane 2, mock-transfected nuclear extract; lanes 3–5, C/EBP{alpha}-transfected nuclear extract). For competition assays, a 100-fold molar excess of an unlabeled ARE7 (lane 4) or C/EBP{alpha} (lane 5) oligonucleotides were added. DNA-C/EBP{alpha} protein complexes and free probe are indicated by the arrow and black arrowhead, respectively. Nonspecific DNA-protein complex bands are indicated by white arrowheads. The putative C/EBP{alpha} site (black square) is indicated. C, The pRstn820-Luc was cotransfected with C/EBP{alpha} expression vector and pCMV-ßgal into Rat1-IR cells. The amounts of C/EBP{alpha} expression vector cotransfected were 0, 4, and 8 µg in lanes 1, 2, and 3, respectively. D, ADD1/SREBP1c and C/EBP{alpha} independently transactivate the resistin promoter. The pRstn820-Luc reporter vector was cotransfected with ADD1 (lane 2), C/EBP{alpha} (lane 3), or both (lane 4). The luciferase activities were normalized by ß-galactosidase activities, and the values are expressed as fold changes relative to that of control (lane1). The putative SREs (black ellipses), E boxes (white triangles), and C/EBP{alpha} site (black square) are indicated.

 
To study the involvement of C/EBP{alpha} in resistin gene expression, a C/EBP{alpha} expression vector was cotransfected with the pRstn820-luc reporter containing human resistin promoter. Overexpression of C/EBP{alpha} transactivated the human resistin promoter although the degree of transactivation by C/EBP{alpha} appeared to be lower than that by ADD1/SREBP1c (Fig. 5Go, C and D). These results imply that resistin gene expression is possibly regulated by C/EBP{alpha} as well as by ADD1/SREBP1c (Figs. 4Go and 5Go). Additionally, to investigate whether ADD1/SREBP1c functionally interacts with C/EBP{alpha} in modulating the resistin promoter, we examined the luciferase activities in the presence of both ADD1/SREBP1c and C/EBP{alpha} (Fig. 5DGo). The promoter activity of the resistin gene was enhanced solely by either ADD1/SREBP1c or C/EBP{alpha} alone, but coexpression of the two transcription factors did not show any synergistic effects. These results suggest that ADD1/SREBP1c and C/EBP{alpha} would independently activate the resistin promoter.

Induction of Resistin mRNA Expression by ADD1/SREBP1c in Adipocytes
To directly address the question whether ADD1/SREBP1c is involved in the regulation of resistin gene expression, we overexpressed ADD1/SREBP1c into differentiated 3T3-L1 adipocytes via adenovirus infection. To validate the effects of ADD1/SREBP1c on resistin gene expression, resistin mRNA levels were carefully examined by the use of quantitative RT-PCR. As shown in Fig. 6Go, ADD1/SREBP1c-infected adipocytes stimulated the expression of resistin mRNA about 2.63-fold more than control null-infected adipocytes. Previously, it was demonstrated that ADD1/SREBP1c transactivated its own promoter as an autoregulatory mechanism (33, 34). Adenovirally overexpressed ADD1/SREBP1c also enhanced the expression of ADD1/SREBP1c mRNA in adipocytes (Fig. 6Go). Furthermore, adenoviral expression of ADD1/SREBP1 significantly promoted the expression of FAS mRNA, which is a well known target gene of ADD1/SEBP1c. These results evidently indicate that ADD1/SREBP1c controls resistin gene expression in adipocytes.



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  		Fig. 6. Induction of Resistin mRNA by ADD1/SREBP1c in Adipocytes

A, Adenoviral overexpression of ADD1/SREBP1c stimulates resistin mRNA expression in adipocytes. Adenovirus containing ADD1 (ADD1/SREBP1c) or null-infected 3T3-L1 adipocytes were harvested, and total RNAs were isolated and analyzed with quantitative RT-PCR. B, Quantitation of induced mRNA levels of ADD1/SREBP1c, FAS, and resistin by infection of adenovirus containing null or ADD1. All the expression levels were normalized to GAPDH RT-PCR products. The RT-PCR products were analyzed by 0.7% agarose gel electrophoresis, and band intensities were compared by imaging of ethidium bromide staining.

 
Expressional Control of Resistin and ADD1/SREBP1c upon Nutritional Status
There are several reports that ADD1/SREBP1c mRNA expression is induced at the early stage of adipocyte differentiation (26, 35, 36). To elucidate the expression profiles of SREBP1, C/EBP{alpha}, and resistin during adipogenesis, we performed Northern blot analysis with differentiating 3T3-L1 cells (Fig. 7AGo). As shown in Fig. 7AGo, induction of resistin mRNA expression was observed somewhat later than that of SREBP1 during adipogenesis (Fig. 7AGo), supporting the hypothesis that ADD1/SREBP1c and C/EBP{alpha} might regulate the expression of resistin gene during adipocyte differentiation.



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  		Fig. 7. mRNA Expression Profile of Resistin, SREBP1, and C/EBP{alpha} in Adipocytes

A, Resistin mRNA is induced later than that of SREBP1 and C/EBP{alpha} during adipogenesis (lanes 2 and 3). Each mRNA was detected by Northern blot analysis during 3T3-L1 differentiation. B, Expression of ADD1/SREBP1c and resistin are closely regulated by nutritional status. mRNA expression levels of each adipogenic gene including SREBP1 and resistin were analyzed by Northern blotting from preadipocyte, adipocyte, and WATs from feeding or fasting animals. A feeding control was allowed free access to food. The fasting group was restricted free access to food for 12 h. Equivalent amounts (20 µg) of loaded RNA were run in each lane.

 
It has been demonstrated that expression of ADD1/SREBP1c is tightly regulated by the nutritional status of animals (29, 37, 38). Fasting dramatically reduces the expression of SREBP1 mRNA while feeding or refeeding induces it in both liver and adipose tissue (Fig. 7BGo and Refs. 29 ,37 , and 38). To determine whether the expression of resistin gene is regulated by dietary treatments, we examined the mRNA levels of resistin upon feeding and fasting. Compared with the normal feeding conditions, resistin mRNA expression was detected in low levels when mice were fasted (Fig. 7BGo). It appeared that the expression of resistin gene also was also regulated by the nutritional status in white adipose tissue (WAT), which was closely correlated with the nutritional regulation of ADD1/SREBP1c in WAT. However, the expression of C/EBP{alpha} did not dramatically change by feeding or fasting conditions (data not shown and Ref. 29), implying that ADD1/SREBP1c might be more closely involved in the nutritional regulation of resistin gene in adipose tissue. In parallel, we compared the expression level of several other adipogenic genes in preadipocytes to those in adipocytes of 3T3-L1.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Obesity is a common and chronic disorder associated with a variety of diseases including type 2 diabetes mellitus, hypertension, stroke, and coronary heart disease. Obesity results from a disorder of energy balance, which occurs when the energy intake chronically exceeds the expenditure. One of the major concerns in obesity is insulin resistance, which is closely associated with type 2 diabetes. Although it has not been yet completely understood how insulin resistance is associated with increased adiposity found in obesity, it has been reported that high levels of free fatty acids and TNF{alpha}, which are overproduced from adipose tissue of obese people, would induce insulin resistance in muscle, liver, and fat tissues (4, 39, 40). A recently identified gene, resistin, was also proposed to be a mediator to link obesity and insulin resistance (12), although this idea has been challenged by conflicting data on resistin expression as described above (17, 22, 24). Recently, Steppan and Lazar (41) proposed several explanations to reconcile the discrepancies for resistin expression in obese animals. One of the possible models is that the reduction of resistin mRNA per obese fat cell might reflect an inhibitory feedback mechanism since the secretion of resistin protein from adipocytes in obese mice might overcome the reduced amounts of resistin per cell. The other model is that the correlation between the levels of adipocyte resistin mRNA and resistin protein might vary in obesity.

Recent studies revealed that several insulin sensitivity-modulating molecules are involved in the regulation of resistin gene expression. TZDs, in particular, which are the ligands for PPAR{gamma} and function as antidiabetic drugs by increasing insulin sensitivity have been studied intensively (12, 17, 18). TZDs stimulate adipocyte differentiation and increase free fatty acid uptake into fat cells and thereby reduce the free fatty acid level in serum and increase insulin signaling to confer insulin-dependent glucose uptake in peripheral tissues (42). As described above, although TZDs can modulate resistin gene expression, further studies are necessary to clarify the expression regulation for the resistin gene because TZD treatment into obese animal models showed opposite effects on resistin expression by different groups (12, 18, 22). In addition to TZD, insulin has also been shown to increase resistin gene expression (13, 22). This observation is somewhat consistent with the nutritional regulation of resistin expression because feeding status, which induces insulin secretion, stimulates resistin expression (Fig. 7BGo and Refs. 13 and 22). However, a very high concentration of insulin treatment (>1 µM) showed inhibitory effects on resistin expression in 3T3-L1 cells (43). On the contrary, TNF{alpha}, free fatty acids, or ß-adrenergic agonist were all shown to repress resistin expression, although it is not clear by which mechanisms these molecules regulate resistin gene expression (44, 45, 46).

In this report, we aimed to elucidate the molecular mechanisms of resistin expression with several transcription factors. To address this issue, we cloned a human resistin promoter and characterized it with in vitro and in vivo analyses. Our data revealed that ADD1/SREBP1c and C/EBP{alpha} are involved in the regulation of resistin gene expression. In vivo ChIP assay and in vitro EMSA clearly demonstrated that ADD1/SREBP1c binds to the human resistin promoter through a specific SRE motif. These results were further confirmed by luciferase reporter assays showing that ADD1/SREBP1c expression transactivates the human resistin promoter in a SRE-dependent manner. Along the same line, ectopic expression of ADD1/SREBP1c by adenoviral infection remarkably stimulated the expression of resistin mRNA in adipocytes. Furthermore, sequence analysis of mouse resistin promoter reveals that there are three SREs, implying that mouse resistin gene expression may also be regulated by ADD1/SREBP1c during adipocyte differentiation. Therefore, it is likely that ADD1/SREBP1c is involved in the regulation of resistin gene expression by transactivation of the resistin promoter, which is independent of C/EBP{alpha}, although expression of some adipocyte-specific genes appears to be cooperatively modulated by several transcription factors. Recently, Hartman et al. (47) reported that C/EBP{alpha} is involved in the activation of mouse resistin promoter. They also confirmed that TZD reduced resistin mRNA expression in 3T3-L1 cells although functional PPAR response element was not found within 6.2 kb upstream of the mouse resistin promoter (12, 17, 18, 47). Similarly, within 3 kb upstream of the human resistin promoter, PPAR{gamma} did not transactivate the promoter or bind to the proximal region of the promoter (data not shown). Of course, we cannot rule out the possibility that PPAR{gamma}-dependent resistin gene expression is regulated at the far distal enhancer region of the resistin gene.

ADD1/SREBP1c mRNA is highly expressed in adipose tissues and liver, and its mRNA expression is induced at the early stage of adipocyte differentiation (26, 35, 36). This notion indicates that ADD1/SREBP1c is involved in the induction of many adipogenic genes to coordinate adipocyte differentiation and energy homeostasis. In fact, it has been demonstrated that ADD1/SREBP1c stimulates expression of PPAR{gamma}, FAS, LPL, leptin, and acetyl coenzyme A carboxylase (26, 29, 36, 48, 49). Interestingly, both ADD1/SREBP1c and resistin mRNAs are increased by insulin stimuli in vivo and in vitro (12, 13, 29, 37, 50). In fact, expression of lipogenic enzymes such as FAS and acetyl coenzyme A carboxylase, which are target genes of ADD1/SREBP1c, are regulated by the nutritional status of animals (38, 50, 51). Similar to these genes, feeding increases the expression of ADD1/SREBP1c and resistin while fasting decreases the expression of both genes in adipose tissue (12, 13, 26). In this respect, it appears that increased expression of lipogenic genes and resistin by feeding might be stimulated through the induction of ADD1/SREBP1c. Although the functional role(s) of resistin remains to be elucidated, the present results support the notion that ADD1/SREBP1c regulates expression of resistin gene upon nutritional regulation and adipogenesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Animal Treatment
Male C57BL/6 mice (10–12 wk, 18–22 g) were housed (five mice per cage) and water was given ad libitum, with a 12-h light, 12-h dark cycle beginning at 0700 h. In experiments, food was withdrawn during the daylight hours (12 h) before onset of the dark cycle. Four mice were used in each feeding and fasting group. In the refeeding experiment, food was reintroduced after 12 h of fasting. Animals were scarified for the isolation of epididymal fat.

Cell Culture
3T3-L1 cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS, HyClone, Logan, UT) at 5% CO2 and 37 C. Differentiation of preadipocytes to adipocytes was achieved by allowing the cells to reach confluence before the addition of DMEM supplemented with 10% FBS, 0.5 mM 3-isobutyl-1-methylxanthine, 1 µM dexamethasone, and 5 µg/ml insulin at 5% CO2 and 37 C. After 2 d, and every 2 d thereafter, fresh medium (DMEM plus 10% FBS and 5 µg/ml insulin) was added to the cells. Rat1-IR and h293 cells were maintained in DMEM supplemented with 10% (vol/vol) bovine calf serum (JBI, Daegu, Korea), 100 U of antibiotic-antimycotic and cultured at 37 C in a 10% CO2 incubator.

Northern Blot Analysis
Total RNA was isolated from the epididymal adipose tissue of mice and cultured cells by a guanidine isothiocyanate extraction protocol described previously (26, 43). RNA (20 µg) was denatured in formamide and formaldehyde and subsequently separated by electrophoresis in formaldehyde-containing agarose gels. RNA was transferred to nytran membrane, and membranes were cross-linked, hybridized, and washed as described by the manufacturer (Schleicher & Schuell, Dassel, Germany). Probes were labeled by random priming using the Klenow fragment of DNA polymerase I (Promega Corp., Madison, WI) and [{alpha}-32P]dCTP (Amersham Pharmacia Biotech, Arlington Heights, IL). cDNAs used as probes are as follows: ADD1/SREBP1c, resistin, FAS, and adipsin.

Cloning of Human Resistin Promoter and Construction of Luciferase Reporter
Human genomic DNA was isolated from HeLa cells using lysis buffer [50 mM Tris (pH 7.5), 50 mM EDTA, 100 mM NaCl, 2% sodium dodecyl sulfate (SDS)]. Conditions for PCR were as follows: 2 µM each primer, 0.6 mM each deoxynucleotide triphosphate, 1x PCR buffer, 5 U long amplification Taq polymerase (TaKaRa, Kyoto, Japan), in 50 µl reaction volume. The PCR cycle was once 30 sec at 95 C, followed by 30 cycles of 12 sec at 95 C, 30 sec at 60 C, and 1 min at 72 C, and then once 5 min at 72 C. The primers used for PCR are as follows: forward, 5'-TAC ACG CGT GAG CCA CCG GCC ATA AAC CAT GAT TTT ATT TT-3'; reverse, 5'-GAG GAG GAG ACA GAG ATC TTT CAT CCT GCA GGC GCT GAA A-3'. The primers included the sequences for the MluI (5'-primer) and BglII (3'-primer) restriction sites. The PCR products were digested with MluI and BglII and subcloned into the pGEM easy vector (Promega Corp.) and pGL3-basic vector (Promega Corp.). Site-directed mutagenesis of pRstn820-Luc plasmids was performed with the QuikChange kit (Stratagene, La Jolla, CA) using the following mutagenic primer (mutated sites are underlined): 5'-GGC TCA AGC ATT CTC AAA CGT CAG CCT CCT TAG TAG CTG-3' for mutation of the putative SRE-A at -611bp (pRstn820mSRE-A-Luc). DNA sequences of each construct were examined by DNA sequencing analysis.

Transient Transfection and Luciferase Assay
Rat1-IR cells were transfected with several DNA constructs 1 d before confluence by the calcium phosphate method as described previously (25, 29, 30, 52). The mammalian expression vectors for ADD1 contain amino-terminal ADD1/SREBP1c from 1–403 amino acids, ADD1 (Y->R), and CCAAT-enhancer-binding protein (C/EBP){alpha} was derived from pSV-SPORT1 (Life Technologies, Inc., Gaithersburg, MD) as described (25, 29). After incubation for 24 h, cell extracts were prepared with lysis buffer [25 mM Tris-phosphate (pH 7.8), 2 mM dithiothreitol, 2 mM 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid, 10% glycerol, 1% Triton X-100], and the activities of ß-galactosidase and luciferase were determined according to the manufacturer’s instructions (Promega Corp.). The luciferase activity in relative light units was normalized to ß-galactosidase activity of each sample.

Preparation of Nuclear Extracts
Nuclear extracts from h293 cells were prepared as described previously (53). Protein concentration was determined by Bradford assay (54). The extracts were aliquoted and stored at -70 C.

EMSA Conditions
EMSAs were performed as described previously (22). Briefly, binding reactions were performed in a 20 µl volume containing purified bacterial protein (20 ng) in the reaction buffer [10 mM Tris (pH 7.5), 50 mM KCl, 2.5 mM MgCl2, 0.05 mM EDTA, 0.1% (vol/vol) Triton X-100, 8.5% (vol/vol) glycerol, 1 mg of poly (dI-dC), 1 mM dithiothreitol, and 0.1% (wt/vol) nonfat dry milk]. Radiolabeled probe (0.1 pmol) was added into the reaction mixture and incubated on ice for 30 min. Electrophoresis was performed on a 4% polyacrylamide gel with 0.25x Tris-borate-EDTA buffer and processed for autoradiography. For binding competition analysis, unlabeled oligonucleotides (100-fold molar excess) were added into the reaction mixture just before the addition of the radiolabeled probe. The DNA sequence of the double-stranded oligonucleotides are as follows (only one strand is shown): ARE7, 5'-GAT CTG TGA ACT CTG ATC CAG TAA G-3'; SRE, 5'-GAT CCT GAT CAC CCC ACT GAG GAG-3'; putative SRE-A: 5'-ATT CTC TCA CGT CAG CCT CC-3'; putative SRE-B: 5'-GTG CAG TGC TGT GAT CAT AA-3'; putative SRE-C: 5'-CAT TCT CAC CCA GAG ACA TA-3'; C/EBP{alpha}, 5'-GAT CCG CGT TGC GCC ACG ATG-3'.

ChIP Assay
Subconfluent h293 cells were transiently transfected with pSV-ADD1–403 and incubated at 37 C for 48 h. Transfected cells were cross-linked in 1% formaldehyde at 37 C for 10 min and resuspended in 200 µl of Nonidet P-40 (NP40)-containing buffer: 5 mM piperazine-1,4-bis[2-ethanesulfonic acid] (pH 8.0), 85 mM KCl, 0.5% NP40. The crude nuclei were precipitated and lysed in 200 µl of lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1). Nuclear lysates were sonicated and diluted 10-fold with an IP buffer [16.7 mM Tris-HCl (pH 8.1), 167 mM NaCl, 1.2 mM EDTA, 0.01% SDS, 1.1% Triton X-100]. Lysates were incubated with Protein A-Sepharose CL-4B (Amersham Pharmacia Biotech) and either anti-ADD1 antibodies (26) or preimmune sera for 2 h at 4 C. The immunoprecipitates were successively washed for 5 min each with 1 ml of TSE 150 [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 150 mM NaCl], 1 ml of TSE 500 [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 500 mM NaCl], buffer III [0.25 M LiCl, 1% NP40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl (pH 8.1)], and 1 ml of TE 10 [mM Tris-HCl (pH 8.0), 1 mM EDTA]. Immune complexes were eluted with 2 volumes of 250 µl elution buffer (1% SDS, 0.1 M NaHCO3), and 20 µl of 5 M NaCl were added to reverse formaldehyde cross-linking. DNA was extracted with phenol/chloroform and precipitated with isopropyl alcohol and 80 µg of glycogen. Precipitated DNA was amplified by PCR. Conditions for PCRs are as follows: 0.25 µM each primer, 0.1 mM each deoxynucleoside triphosphate, 1x PCR buffer, 1 U Ex Taq polymerase (TaKaRa), 0.06 mCi/ml [{alpha}-32P]dCTP in 20 µl reaction volume. PCR products were resolved in 8% polyacrylamide/1x Tris-borate-EDTA gels. Primers used in this study are as follows: -816 Resistin-f, 5'-AGC CAC CGG CCA TAA ACC AT-3'; -409 Resistin-r, 5'-ACA GGG CCT CCG TCT TCA TG-3'; GAPDH-f, 5'-GTG TTC CTA CCC CCA ATG TG-3'; GAPDH-r, 5'-CTT GCT CAG TGT CCT TGC TG-3'.

Adenovirus Infection and Semiquantitative RT-PCR
Differentiated 3T3-L1 adipocytes were infected with 1 ml of adenovirus-containing DMEM at a titer of 10 pfu/cell for 12 h at 37 C. Then, culture medium was adjusted to 2 ml with DMEM supplemented with 10% fetal calf serum. After viral infection for 48 h, infected cells were harvested for RNA isolation. Total cellular RNA was isolated using TRIzol reagent (Invitrogen, San Diego, CA) according to the manufacturer’s protocol. Concentration of RNA was spectrophotometrically measured at OD260. RT-PCRs were performed using the SuperScript First-Strand Synthesis System for RT-PCR kit (Invitrogen) using 250 ng of total RNA. ADD1/SREBP1c and resistin cDNA were amplified for 30 and 27 cycles, respectively, which are not saturating stages. RT-PCR products were analyzed by 0.7% agarose gel electrophoresis, and band intensities were compared by imaging of ethidium bromide staining (Scion Image, Scion Corp., Frederick, MD). Primers used in this study are as follows: Resistin-up, 5'-GAC AGA AGC TTA TAC CCA GAA CTG AG-3'; Resistin-down, 5'-CTG GAA ACC ACG CTG AAT TCC CCG AC-3'; FAS-up, 5'-TGC TCC CAG CTG CAG GC-3'; FAS-down, 5'-GCC CGG TAG CTC TGG GTG TA-3'; GAPDH-up, 5'-TGC ACC ACC AAC TGC TTA G-3'; GAPDH-down, 5'-GGA TGC AGG GAT GAT GTT C-3'.


   ACKNOWLEDGMENTS
 
We are grateful to Pascal Ferre (Institut National de la Santé et de la Recherche Médicale, France) for providing ADD1/SREBP1c adenovirus. We are also grateful to Cecilia Roh and Heekyung Chung for critically reading the manuscript.


   FOOTNOTES
 
This work was supported by grant Molecular Medicine Research Group Program (Grant M1-0106-02-0003) from the Ministry of Science and Technology. J.B.S., S.Y.P., J.P., S.D.P., and J.B.K. are supported by the BK21 Research Fellowship from the Ministry of Education and Human Resources Development.

Abbreviations: ADD1, Adipocyte determination- and differentiation-dependent factor 1; ARE7, PPAR{gamma}/retinoid X receptor-{alpha} binding element; ChIP, chromatin immunoprecipitation; C/EBP{alpha}, CCAAT enhancer binding protein-{alpha}; FAS, fatty acid synthase; FBS, fetal bovine serum, GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NP40, Nonidet P-40; PPAR, peroxisomal proliferator-activated receptor; SDS, sodium dodecyl sulfate; SRE, sterol regulatory element; SREBP1c, sterol regulatory element binding protein 1c; TZD, thiazolidinedine; WAT, white adipose tissue.

Received for publication January 24, 2003. Accepted for publication April 22, 2003.


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