Published online before print July 31, 2008, doi: 10.1161/CIRCRESAHA.108.175893
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© 2008 American Heart Association, Inc.
Molecular Medicine |
The Helix–Loop–Helix Factors Id3 and E47 Are Novel Regulators of Adiponectin
From the Cardiovascular Research Center/Cardiovascular Division (A.C.D., N.M., A.C., H.D., R.P.S., S.N.O., C.A.M.), Division of Endocrinology and Metabolism (S.R.K.), Department of Medicine, University of Virginia, Charlottesville; and School of Biological Sciences (J.B.K.), Seoul National University, Korea.
Correspondence to Coleen A. McNamara, PO Box 801394, University of Virginia, Charlottesville, VA 22908. E-mail cam8c{at}virginia.edu
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Abstract |
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Adiponectin is an adipocyte-derived cytokine with beneficial effects on insulin sensitivity and the development of atherosclerosis. Id3 is a helix–loop–helix factor that binds to E-proteins such as E47 and inhibits their binding to DNA. Although the helix–loop–helix factor sterol regulatory element binding protein (SREBP)-1c is a known activator of adiponectin transcription, this study provides the first evidence of a role for Id3 and E47 in adiponectin expression. Decreased Id3 in differentiating adipocytes correlates with increased adiponectin expression and forced expression of Id3 inhibits adiponectin expression. Moreover, Id3-null mice have increased adiponectin expression in visceral fat tissue and in serum. We demonstrate that E47 potentiates SREBP-1c–mediated adiponectin promoter activation and that Id3 can dose-dependently inhibit this action via interaction with E47. Mutation of a consensus E47 binding site results in nearly complete loss of promoter activation. Furthermore, we demonstrate E47 binding to the endogenous adiponectin promoter both in vitro and in vivo by chromatin immunoprecipitation analysis. Binding is not detected in undifferentiated cells which express Id3 but peaks during differentiation in parallel with Id3 decline. This promoter binding can be completely abolished by the overexpression of Id3 and is enhanced in adipose tissue null for Id3. These data establish Id3 and E47 as novel regulators of SREBP-1c–mediated adiponectin expression in differentiating adipocytes and provide evidence that Id3 regulates adiponectin expression in vivo.
Key Words: basic helix–loop–helix proteins • differentiation • gene regulation • adiponectin • adipocytes
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Introduction |
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Adiponectin is produced by adipocytes and is secreted into the circulation.1,2 Adiponectin levels are reduced in obese, insulin-resistant, and diabetic rodents,3 monkeys,4 and humans.5 Administration of adiponectin in rodent models increases insulin sensitivity and lowers plasma glucose.6,7 Low adiponectin levels are associated with the development of coronary artery disease8,9 and ApoE–/– mice overexpressing adiponectin demonstrate significantly less atherosclerosis in response to high-fat feeding.10,11 Although these studies implicate adiponectin as an important modulator of insulin sensitivity and the development of atherosclerosis, the transcriptional mechanisms that regulate adiponectin expression are incompletely understood.
Both the human and mouse adiponectin promoters contain binding sites for transcription factors including sterol regulatory elements (SREs), peroxisome proliferator-activated receptor (PPAR)-response elements, C/EBP sites, and E-boxes.12 Accordingly, many factors influence adiponectin expression either directly or indirectly, including sterol regulatory element binding protein (SREBP)-1c,12 PPAR
,12,13 and C/EBP.12,14 To date, no study has addressed the role of the 3 putative E-boxes present in the adiponectin promoter.12
SREBP-1c is a member of the basic helix–loop–helix (bHLH)–leucine zipper (bHLH-LZ) family of proteins that binds to SREs.15 Originally implicated in cholesterol-regulated gene expression,16 SREBP-1c is also a major regulator of adipogenic genes, including fatty acid synthase and adiponectin.12,17 Also known as adipocyte determination– and differentiation–dependent factor-1 (ADD1), SREBP-1c is expressed preferentially in adipose tissue and is upregulated as adipocyte differentiation progresses.12,15
The inhibitor of differentiation (Id) family of proteins contains 4 members, Id1 to -4, which have both redundant and unique functions.18 Ids are HLH proteins that function as dominant-negative transcription factors that are incapable of binding to DNA. Instead, the Ids bind to a subset of bHLH factors known as E-proteins, including E12, E47, ITF2, and HEB, thereby preventing their dimerization and DNA binding. Although the Ids bind preferentially to E-proteins, 1 report has suggested that Id3 binds to and inhibits SREBP-1c directly.19
Previous studies have proposed a role for Id proteins in adipocytes. The mRNAs for Ids 1 to 3 are expressed in 3T3-L1 preadipocytes and decrease to undetectable levels as they differentiate into adipocytes.19–21 Id3 has been shown to negatively regulate fatty acid synthase in vitro19; however, no other adipocyte target genes have been identified.
In the present study, we examine the role of Id3 and E47 in the control of adiponectin transcription. We demonstrate that Id3 inhibits adiponectin expression in vitro and in vivo. In addition, we show that E47 and SREBP-1c bind the endogenous adiponectin promoter in vitro and in vivo and synergistically activate the adiponectin promoter. We refute previous suggestions that Id3 directly binds to SREBP-1c and propose instead that Id3 regulates SREBP-1c activity indirectly by interacting with E47, thereby preventing E47 binding to the adiponectin promoter and inhibiting adiponectin expression.
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Materials and Methods |
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An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
Transfections, adenoviral transductions, and promoter–reporter assays, Western blotting, and 2-hybrid analysis were performed as described previously.22,23 OP9 and 3T3-L1 cell culture and differentiation were carried out as described by Wolins et al.24 Chromatin immunoprecipitation (ChIP) methods were based on Francis et al and are described in the online data supplement.25
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Results |
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Increased Levels of Adiponectin Protein and mRNA in Id3 Knockout Mice
To address the potential in vivo regulation of adiponectin by Id3 in an atherogenic model, total adiponectin levels in ApoE–/– and Id3–/–ApoE–/– mice were determined. Sera were collected from 8-week-old, weight-matched ApoE–/– and Id3–/–ApoE–/– mice, and adiponectin levels were measured by radioimmunoassay. Loss of Id3 resulted in a significant increase in serum adiponectin levels (Figure 1A). To determine whether this was attributable to an effect on adiponectin production, we analyzed visceral adipose tissue, the major site of adiponectin production in rodents.1,2 Epididymal and mesenteric fat depots from ApoE–/– and Id3–/–ApoE–/– mice were harvested from 8-week-old, weight-matched animals. Relative amounts of adiponectin were determined by Western blotting. When compared to ApoE–/– mice, Id3–/–ApoE–/– mice had increased adiponectin in both the epididymal (3.7-fold) and mesenteric (2.8-fold) fat pads (Figure 1B and 1C). To determine whether Id3 affects adiponectin expression at the mRNA level, total RNA was isolated from fat pads, and adiponectin transcripts were quantified by real-time PCR. As compared to ApoE–/– mice, Id3–/–ApoE–/– animals expressed 2.7- and 3.1-fold more adiponectin mRNA in the epididymal and mesenteric fat pads, respectively (Figure 1D).
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Forced Expression of Id3 Inhibits Adiponectin Expression
Throughout our study, we have confirmed many of our results in 2 adipocyte cell lines: 3T3-L1, a widely used adipocyte line; and OP9, a new alternative model.24 Wolins et al have demonstrated that OP9 cells express the same adipocyte lineage markers as 3T3-L1 cells but differentiate more rapidly (3 to 7 days versus 2 weeks26 for complete differentiation after plating).24 We have confirmed that OP9 cells can be transiently transfected with high efficiency and are efficiently transduced with an adenovirus (95% transduction [data not shown] versus 50% or less with 3T3-L1 cells27), enabling us to assay changes in expression of endogenous proteins in the total cell population. In addition, the shorter differentiation time allows OP9 cells to maintain expression of transfected genes over the course of differentiation.
We evaluated the expression of Id3 and adiponectin in both 3T3-L1 and OP9 cells. Undifferentiated (preadipocytes) or fully differentiated (adipocytes) cells were analyzed by Western blotting, revealing that Id3 is detected in undifferentiated but not in differentiated 3T3-L1 or OP9 cells. Conversely, adiponectin is present in differentiated but not undifferentiated cells (Figure 2A).
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To determine whether Id3 modulates adiponectin expression, undifferentiated OP9 or 3T3-L1 cells were transduced with an adenovirus expressing either Id3 (Ad-Id3) or green fluorescent protein (Ad-GFP) and then differentiated for 3 days (OP9) or 5 days (3T3-L1). Expression of adiponectin, Id3, or GLUT4 (a marker of differentiated adipocytes28) was analyzed by Western blotting. Exogenous Id3 expression significantly decreased adiponectin protein levels (Figure 2B) by approximately 3-fold in OP9 cells and 2-fold in 3T3-L1 cells (Figure 2C). GLUT4 expression in the Ad-Id3 and Ad-GFP groups was similar, indicating that the effect of Id3 on adiponectin expression is not attributable to inhibition of differentiation. To ascertain whether Id3 inhibits adiponectin expression independent of differentiation state, differentiated OP9 cells were transduced with Ad-GFP or Ad-Id3 and analyzed 72 hours later. Differentiated OP9 adipocytes transduced with Id3 showed a 2-fold decrease in adiponectin expression as compared to vehicle or Ad-GFP-treated cells (Figure I in the online data supplement).
To assess whether forced Id3 expression inhibits adiponectin at the transcriptional level, we also compared adiponectin mRNA levels in cells transduced with increasing amounts of Ad-GFP or Ad-Id3. Dose-dependent increases in Id3 mRNA expression in Ad-Id3 treated cells were confirmed by real-time PCR. Whereas increasing doses of GFP did not alter adiponectin expression, expression of Id3 in either OP9 or 3T3-L1 cells reduced adiponectin mRNA levels in a dose-dependent manner (Figure 2D).
Id3 Inhibits Adiponectin Promoter Activation
Because the level of Id3 expression affected adiponectin mRNA levels, regulation of the adiponectin promoter by Id3 was tested next. NIH3T3 fibroblasts were transfected with a 0.41-kb adiponectin promoter/luciferase reporter construct in combination with SREBP-1c (a potent activator of the adiponectin promoter12) and/or Id3 (Figure 3). NIH3T3 cells were used to eliminate the potentially confounding effect of adipocyte differentiation state and to avoid background activity from the endogenous expression of SREBP-1c. Id3 alone had no effect on basal luciferase activity, whereas SREBP-1c expression resulted in a 12-fold activation of the promoter. When SREBP-1c and Id3 were cotransfected, a dose-dependent reduction in SREBP-1c–mediated activation of the adiponectin promoter was observed. As previously shown, basal and SREBP-1c–induced adiponectin promoter activity were similar with either the proximal 0.41- or 0.98-kb promoter fragments (data not shown).12
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SREBP-1c Partners With E47 but Not Id3 in Adipocytes
The established paradigm of Id function involves dimerization with E-proteins such as E47 and not a direct interaction with bHLH-LZ proteins such as SREBP-1c.21,29 A previous study demonstrated an Id3:SREBP-1c interaction using in vitro translated proteins outside of a cellular context.19 To determine whether SREBP-1c interacts with either Id3 or E47 in adipocytes, mammalian 2-hybrid analysis in differentiated OP9 cells was used (see methods). Large T antigen and p53, which are known to interact, were used as a positive control, and partners producing equal or greater signal than these were considered positive (Figure 4A). SREBP-1c bound to E47 but not to Id3, which is in contrast with the aforementioned study, but consistent with previous results demonstrating E47:SREBP-1c interaction in other tissues.30 To extend the 2-hybrid findings, coimmunoprecipitation of Id3 with E47 or SREBP-1c was tested. COS7 cells were transfected with HA-Id3 and either FLAG–SREBP-1c or FLAG-E47 before immunoprecipitation with anti-FLAG beads. HA-Id3 coimmunoprecipitated with FLAG-E47 but not with FLAG–SREBP-1c (Figure 4B), confirming the results of the 2-hybrid experiment. These findings provide evidence that, in adipocytes, SREBP-1c binds to E47 and not to Id3.
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E47 Potentiates SREBP-1c–Mediated Adiponectin Promoter Activation
To determine whether E47:SREBP-1c interaction influences adiponectin promoter–reporter activation, NIH3T3 cells were transfected with the 0.41-kb adiponectin promoter–reporter construct together with SREBP1-c and varying amounts of E47. E47 alone did not activate the promoter, but coexpression of E47 with SREBP-1c augmented adiponectin promoter activation 6-fold more than the same dose of SREBP-1c alone (Figure 5).
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To identify possible sites at which E47 acts, the 2 E-boxes within the proximal 400 bp of the adiponectin promoter were mutated. The most distal E-box was not tested because, as mentioned above, the 0.41-kb promoter–reporter construct, in which this E-box is deleted, responds to E47 and Id3 in the same manner as the 0.98-kb construct. Approximate positions of the E-boxes are shown in the schematic (Figure 6A). Mutations within E-box no. 1 but not E-box no. 2 inhibited SREBP-1c–mediated adiponectin promoter activity (Figure 6B). Moreover, the mutation of E-box no. 1, but not E-box no. 2, led to the loss of promoter activation by SREBP-1c and E47 (Figure 6C).
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E47 Binds to the Adiponectin Promoter During Adipocyte Differentiation
To determine whether E47 binds the endogenous adiponectin promoter, ChIP was performed. OP9 and 3T3-L1 cells were harvested during differentiation, at days 3 and 5, respectively, and precipitated with anti-E47 antibody. Because SREBP-1c is known to bind the adiponectin promoter, it was used as a positive control. Adiponectin promoter recovery was determined by real-time PCR using primers spanning E-box no. 1 (Figure 7A). In OP9 cells, precipitation with E47 antibody led to a 4-fold enrichment of the adiponectin promoter compared to isotype control and in 3T3-L1 cells, 5-fold enrichment was obtained (Figure 7B). Precipitation with an antibody for E12, an alternate splice product of the E2A gene that also produces E47, did not lead to any detectable recovery of the adiponectin promoter in either cell line. Similar results were obtained using a second set of adiponectin promoter primers, whereas a negative set of primers gave no specific signal (supplemental Figure IIA and IIB). To investigate whether Id3 expression can interrupt binding of E47 to the promoter, OP9 cells were transduced with Ad-Id3 during differentiation (day 3), when endogenous Id3 had dropped to undetectable levels. OP9 cells transduced with Ad-GFP and precipitated with E47 antibody showed a 4.8-fold increase in adiponectin promoter recovery in the E47 immunoprecipitate compared to isotype control whereas cells transduced with Ad-Id3 had no detectable promoter binding of E47 (Figure 7C). Immunoblotting of lysates transduced with either GFP or Id3 revealed that levels of E47 and SREBP-1c were similar between the 2 groups (Figure 7C), confirming that the observed decrease in binding was not attributable to a reduction in E47 or SREBP-1c expression. Transduction with Ad-Id3 also inhibited SREBP-1c binding to the adiponectin promoter. Results using a second set of primers were similar and no specific signal was obtained using negative control primers (supplemental Figure IIC).
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To determine the temporal pattern of Id3, E47, SREBP-1c, and adiponectin expression in differentiating adipocytes, OP9 cells were harvested at day 0 (undifferentiated), day 3 (differentiating), and day 6 (fully differentiated). Immunoblotting revealed that E47 is expressed in undifferentiated preadipocytes and during differentiation, whereas SREBP-1c is expressed in differentiating and fully differentiated cells (Figure 7D). To test whether endogenous E47 and SREBP-1c binding to the adiponectin promoter is consistent with the temporal pattern of protein expression during differentiation, we performed ChIP analysis of samples harvested in parallel to those used for Western blotting (Figure 7E). Consistent with our data that Id3 levels are high in undifferentiated OP9 and 3T3-L1 cells, E47 did not bind the adiponectin promoter. At that stage, SREBP-1c is not expressed in undifferentiated cells and no detectable adiponectin promoter binding was observed. In OP9 cells undergoing differentiation, Id3 levels are declining, E47 levels remain high, and SREBP-1c is expressed. During this phase, both SREBP-1c and E47 bound the promoter with a 1.8- and 5.0-fold increase in binding compared to isotype, respectively. In mature adipocytes, Id3 is undetectable, E47 is reduced, and SREBP-1c is abundantly expressed. At this stage, both E47 and SREBP-1c bound the promoter (1.5- and 5.9-fold increased binding, respectively). Consistent with Western blot data in Figure 7D, E47 binding peaks during differentiation when E47 levels are high, and SREBP-1c binding peaks later in differentiation when SREPB-1c levels are maximal (Figure 7E). To confirm that E47 and SREBP-1c bind the adiponectin promoter in vivo and that Id3 regulates this, in vivo ChIP was performed. Epididymal adipose tissue from ApoE–/– and Id3–/–ApoE–/– mice was harvested from 8-week-old animals and ChIP analysis was performed as above. Precipitation with an E47 antibody led to a 3.4-fold enrichment of the adiponectin promoter in ApoE–/– mice, whereas in Id3–/–ApoE–/– mice, the enrichment was 6.0-fold compared to isotype control. Precipitation with an SREBP-1c antibody resulted in an 8.8-fold enrichment of adiponectin promoter from ApoE–/– adipose tissue and 10.1-fold enrichment from Id3–/–ApoE–/– adipose tissue. Taken together, these data provide evidence that E47 promotes and Id3 inhibits the expression of adiponectin in adipocytes in vitro and in vivo.
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Discussion |
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A strong link has been established between adiponectin levels and clinical atherosclerosis and type 2 diabetes; however, the molecular regulation of adiponectin is incompletely understood. The present study provides the first evidence that the HLH proteins Id3 and E47 regulate adiponectin transcription during adipocyte differentiation.
Here, we have shown that Id3 can negatively regulate adiponectin expression. Id proteins are unable to bind DNA; therefore, they regulate transcription by dimerizing with other HLH proteins, known as E-proteins, to prevent the DNA binding of these factors. We sought to identify an E-protein that may play a role in the regulation of adiponectin. A previous study demonstrated expression of E-proteins in 3T3-L1 cells at the mRNA level.31 The present study is the first to attribute a function to any E-protein in adipocyte biology. We show that E47 is expressed in preadipocytes and differentiating adipocytes and binds the adiponectin promoter. Furthermore, our data suggest that E47 interacts with SREBP-1c, a known positive regulator of adiponectin, to enhance SREBP-1c–mediated promoter activation. Consistent with our findings in adipocytes, a recent study reported that E47 partners with SREBP-1c in pancreatic islet cells to activate the insulin promoter.30
Like Id3 and E47, SREBP-1c is a member of the HLH family of transcription factors. In contrast to the Ids and the E-proteins, SREBP-1c also possesses a leucine zipper (HLH-LZ).15 Based on structural and empirical data, the HLH-LZ factors are not capable of partnering with Id proteins.29,32 Contrary to this, 1 previous report suggested that Id3 and SREBP-1c bind directly to each other, proposing this as a possible mechanism for Id3 antagonism of SREBP-1c activation of the fatty acid synthase promoter. Using in vitro translated versions of Id3 and SREBP-1c, the authors showed that Id3 altered SREBP-1c binding to the promoter in a gel-shift mobility assay and that Id3 and SREBP-1c coimmunoprecipitated in a cell-free system.19 We were unable to reproduce these results in a cellular context using either mammalian 2-hybrid or immunoprecipitation techniques. Instead, both Id3:E47 and E47:SREBP-1c were shown to interact. This suggests that the action of Id3 on SREBP-1c is via inhibition of the SREBP-1c partner, E47. Accordingly, E47 binding to the promoter was dramatically reduced by the overexpression of Id3 in vitro and was increased in Id3–/–ApoE–/– adipose tissue. Although we demonstrate that Id3 inhibits SREBP-1c activity in NIH3T3 cells (Figure 3), we believe that this effect is not direct but, instead, attributable to the interaction of Id3 with endogenous E47 present in NIH3T3 cells.33
Id3 has been proposed to regulate the differentiation of adipocytes because constitutive expression of Id3 in 3T3-F442A cells inhibits the expression of adipsin and adipocyte lipid binding protein mRNAs.20 Our data show that acute overexpression of Id3 inhibits adiponectin but does not change the levels of GLUT4, another marker of differentiation. This is true whether Id3 is expressed before or after the cells undergo differentiation. This suggests that the effect of Id3 on adiponectin is specific rather than via a global blockade of differentiation.
The present study shows that E47 is highly expressed in undifferentiated cells and during differentiation, although expression is significantly decreased in differentiated cells. Correspondingly, E47 binding to the adiponectin promoter occurs in a differentiation state-specific manner (Figure 8). No binding is observed in undifferentiated cells when Id3 expression is high. Maximal binding occurs during differentiation when Id3 levels have fallen and a small amount of binding persists in fully differentiated cells that have reduced E47 levels. SREBP-1c expression and promoter binding increase throughout differentiation and are maximal in fully differentiated adipocytes. The expression of E47 and SREBP-1c overlap during differentiation when Id3 is undetectable, which may allow E47:SREBP-1c interaction and activation of the adiponectin promoter.
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SREBP-1c is known to act at 2 distinct SREs within the mouse adiponectin promoter.12 This same study reported the existence of 3 putative E-boxes within the promoter, which we investigated here. These E-boxes are relatively conserved in the human adiponectin promoter, which shares 56% similarity to the mouse promoter.12 This similarity includes several E-boxes in the same region as mouse E-box no. 1, raising the possibility that at least 1 of these elements could function similarly in the human promoter. In our study, the elimination of E-box no. 3 (the 0.41-kb promoter construct) and the mutation of E-box no. 2 (Figure 5A) did not significantly change promoter activation by SREBP-1c and E47. Although these sequences can be identified as E-boxes based on their similarity to the "CANNTG" DNA sequence, neither of them represent the type of E-box that is considered to be the classic binding site (CAGCTG or CACCTG) for E-proteins such as E47.34 In addition, E-box no. 2 overlaps with a functional SRE site, making it less likely to be the location of E-protein binding.12 E-box no. 1 represents a classic E-box for E-protein binding and mutational analysis shows that this E-box is necessary for the activation observed with E47 and SREBP-1c (Figure 5). Interestingly, mutation of E-box no. 1 also decreases the effect seen with SREBP-1c alone. Although it is plausible that SREBP-1c binds to E-box no. 1 itself as SREBP-1c proteins can bind to both SRE and E-box elements,35 this latter scenario is less likely, given that SREBP-1c binding sites within the adiponectin promoter have been mapped by DNase footprinting and that SREBP-1c binding does not protect the region of the promoter containing E-box no. 1.12 Id3 overexpression during differentiation inhibits not only E47 but also SREBP-1c binding to the adiponectin promoter (Figure 7C). Id3 does not appear to bind to SREBP-1c (Figure 6), indicating that SREBP-1c binding at this stage may depend on E47 binding.
Other protein partners for E47 and SREBP-1c are likely to exist, particularly in the case of SREBP-1c, which continues to be expressed and to bind the adiponectin promoter later in differentiation even after E47 expression has been downregulated. Taken together, these data suggest that during differentiation, efficient adiponectin promoter activation by SREBP-1c is dependent on interaction with E47 and E47 binding to E-box no. 1. Little E47 and prominent SREBP-1c binding to the adiponectin promoter in differentiated cells suggest the existence of E47-independent mechanisms to activate adiponectin expression by SREBP-1c in mature adipocytes. Given the role for Id3 in regulating adiponectin expression both in vitro and in vivo, it will be important to determine what controls Id3 expression in adipocytes. Id3 is known to be an early response gene whose expression is stimulated in response to mitogenic stimuli21 and decreased when cells are growth arrested or induced to differentiate.36 To date, no specific factor has been demonstrated to directly control Id3 expression in adipocytes. Further studies to identify such factors in these cells seem warranted given the significant role Id3 plays in the control of adiponectin expression.
In summary, we have shown that Id3 regulates the expression of adiponectin in differentiating adipocytes in culture and in adipose tissue in vivo. During differentiation, E47 is capable of partnering with SREBP-1c, a previously identified positive regulator of adiponectin expression, to enhance SREBP-1c–mediated adiponectin transcription. It will be important to identify other HLH factors that may regulate adiponectin as well as other adipocyte markers to more fully understand the role of HLH factors in adipocyte biology.
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Acknowledgments |
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We thank Toni Barbera and the Diabetes Center Animal Characterization Core at the University of Virginia (NIH DK063609) for assistance.
Sources of Funding
This work was supported by NIH grant P01 HL55798 (to C.A.M.), NIH Training Grant 5-T32 HL007355-29 (to A.C. and H.D.), and an American Heart Association Predoctoral Fellowship (A.C.D.).
Disclosures
None.
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Footnotes |
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Original received March 17, 2008; revision received July 22, 2008; accepted July 23, 2008.
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