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Proc Natl Acad Sci U S A. Jun 8, 2010; 107(23): 10508–10513.
Published online May 24, 2010. doi:  10.1073/pnas.1000788107
PMCID: PMC2890788
Cell Biology

A circadian-regulated gene, Nocturnin, promotes adipogenesis by stimulating PPAR-γ nuclear translocation

Abstract

Nocturnin (NOC) is a circadian-regulated protein related to the yeast family of transcription factors involved in the cellular response to nutrient status. In mammals, NOC functions as a deadenylase but lacks a transcriptional activation domain. It is highly expressed in bone-marrow stromal cells (BMSCs), hepatocytes, and adipocytes. In BMSCs exposed to the PPAR-γ (peroxisome proliferator-activated receptor-γ) agonist rosiglitazone, Noc expression was enhanced 30-fold. Previously, we reported that Noc−/− mice had low body temperature, were protected from diet-induced obesity, and most importantly exhibited absence of Pparg circadian rhythmicity on a high-fat diet. Consistent with its role in influencing BMSCs allocation, Noc−/− mice have reduced bone marrow adiposity and high bone mass. In that same vein, NOC overexpression enhances adipogenesis in 3T3-L1 cells but negatively regulates osteogenesis in MC3T3-E1 cells. NOC and a mutated form, which lacks deadenylase activity, bind to PPAR-γ and markedly enhance PPAR-γ transcriptional activity. Both WT and mutant NOC facilitate nuclear translocation of PPAR-γ. Importantly, NOC-mediated nuclear translocation of PPAR-γ is blocked by a short peptide fragment of NOC that inhibits its physical interaction with PPAR-γ. The inhibitory effect of this NOC-peptide was partially reversed by rosiglitazone, suggesting that effect of NOC on PPAR-γ nuclear translocation may be independent of ligand-mediated PPAR-γ activation. In sum, Noc plays a unique role in the regulation of mesenchymal stem-cell lineage allocation by modulating PPAR-γ activity through nuclear translocation. These data illustrate a unique mechanism whereby a nutrient-responsive gene influences BMSCs differentiation, adipogenesis, and ultimately body composition.

Several lines of evidence demonstrate a relationship between circadian networks and adipogenesis. For example, alterations in time-keeping among individuals on psychotropic drugs, nighttime shift workers, or those with hormonal imbalances are often associated with fat redistribution (13). The nuclear receptor and transcription factor PPAR (peroxisome proliferator-activated receptor)-γ2 and PGC-1α (PPAR-γ coactivator 1-α) both exhibit cell-autonomous circadian patterns of gene expression (4). PPAR-γ2 is also critical in mesenchymal cell specification (5), a process under the influence of circadian networks. Activation of PPAR-γ2 with aging or by the PPAR-γ agonist rosiglitazone is negatively correlated with osteogenesis and positively with bone marrow adipogenesis, although Pparg haploinsufficiency in mice results in high bone mass, leanness, and little marrow fat (5, 6).

Mammalian time-keeping is regulated by hypothalamic and peripheral clock genes (711). Nocturnin (Noc; Ccrn4l) is one of several cell-autonomous circadian genes (12) whose expression is high in liver, hypothalamus, and adipocytes, and whose production peaks in early night (13). Noc encodes a deadenylase that removes poly-A tails from the 3′ ends of mRNAs (14, 15). Previously, we reported that Noc gene expression was up-regulated nearly 30-fold in bone-marrow stromal cells (BMSCs) transfected with Pparg2 and exposed to rosiglitazone (16). Similarly, in a congenic mouse with low bone mass and increased Pparg activity, Noc transcripts were markedly enhanced in liver, fat, and bone marrow (17). In hepatic and skeletal tissues from aging rodents, there is increased Noc expression coincident with greater PPAR-γ2 activity, low bone mass, and greater bone-marrow adiposity (18). In addition, we have also shown that Noc−/− mice are protected from diet-induced obesity and exhibited absence of circadian Pparg expression, even on a high-fat diet (19). These data led us to hypothesize there is an important interaction between NOC and PPAR-γ that facilitates adipogenesis. Here, we present the molecular mechanism underlying this interaction in which NOC stimulates PPAR-γ function by facilitating its nuclear localization. These observations suggest that Noc modulates BMSCs fate by shifting stem cells into the adipogenic lineage and away from osteoblast differentiation. More importantly, these lines of evidence reinforce the importance of circadian networks in the regulation of body composition.

Results

Noc Stimulates Adipogenesis and Suppresses Osteoblastogenesis.

First, to understand the role of Noc during mesenchymal stromal cell differentiation, the critical first step in osteoblast and adipocyte differentiation, we examined the temporal profile of Noc expression. We found Noc was up-regulated during adipogenesis in both 3T3-L1 and OP9 cells (Fig. 1 A and B and Fig S1). Surprisingly, Noc expression was induced coincident with PPAR-γ1 in early adipogenesis and before PPAR-γ2 (Fig. 1B). Overexpression of Noc in 3T3-L1 cells markedly stimulated adipogenesis and was accompanied by a significant increase in aP2 (fatty acid-binding protein 4) and Pparg2 expression (Fig. 1 C and D and Fig S2). Consistently, knockdown of Noc in 3T3-L1 cells suppressed adipogenesis and was associated with reduced expression of adipogenic markers including Pparg2, aP2, and Lpl (lipoprotein lipase) (Fig. 1 E and F and Fig S2). In contrast, Noc expression was down-regulated during osteoblastogenesis of primary calvarial osteoblasts (COBs) (Fig. 1 G and H), and COBs derived from Noc−/− mice had lower Pparg and higher Runx2 expression compared with controls (Fig S3A). Overexpression of Noc in MC3T3-E1 cells suppressed osteoblastogenesis with reduced expression of alkaline phosphatase (ALP) and Osteocalcin (OC) (Fig. 1 I and J and Fig S2), whereas knockdown of Noc in MC3T3-E1 cells stimulated osteoblastogenesis with increased expression of ALP and OC (Fig. 1 K and L and Fig S2). Consistent with this, Noc −/− COBs, when exposed to osteogenic media, also showed enhanced osteogenesis compared with control cells (Fig. 1 M and N). The findings that NOC enhances Pparg2 expression and decreases Runx2 expression led us to hypothesize that NOC affects the transcription of these genes. To test this tenet, we performed reporter assays using luciferase vectors fused with either the Runx2 promoter (0.6 kb) or the Pparg2 promoter (0.9 kb). To investigate whether the deadenylase activity of NOC is involved in this regulation, we also used a magnesium-binding motif mutant of NOC (E193A-Noc), which lacked deadenylase activity. However, neither WT nor mutant NOC affected the luciferase activity of these constructs (Fig S3B). The increase in Runx2 expression in Noc−/− COBs was not accompanied by a craniofacial phenotype, which was assessed at postnatal day 7 by inspection with microCT (Fig S3C). Notwithstanding, because the promoter size inserted in these luciferase vectors is small, we cannot exclude the possibility that the regulatory region may exist outside of tested promoter fragments. In sum, Noc modulates BMSCs’ fate by shifting cells into the adipogenic pathway and away from the osteoblast lineage.

Fig. 1.
NOC favors adipogenesis over osteoblastogenesis. (A and B) 3T3-L1 cells were treated with control media (cont or C) or adipogenic mixture (Adipo or A) for 9 d. Expression of aP2 and Noc was analyzed by real-time PCR (n = 3) (A), and protein levels of ...

NOC Is Physically and Functionally Associated with PPAR-γ Independent of Its Deadenylase Activity.

Based on the temporal profile of Noc expression in adipocytes, we next sought to determine whether Noc influenced adipogenesis by regulating PPAR-γ. As described previously, NOC had no effect on the activity of the luciferase vector containing Pparg2 promoter, but interestingly, we found that NOC coimmunoprecipitated with both PPAR-γ1 and PPAR-γ2, demonstrating a physical interaction between these proteins (Fig. 2A and Fig S4A). Based on the exclusive expression and the critical role of Pparg2 in adipocytes, we analyzed the interaction between Pparg2 and Noc in this study. In a reporter assay using a luciferase vector containing a Pparg response element, WT Noc did not affect the basal Pparg2 transcriptional activity, but Pparg2 transcriptional activation by rosiglitazone was significantly increased in the presence of WT Noc in murine marrow-derived UAMS-33 cells stably expressing Pparg2 expression vector (U-33/γ2 cells) (Fig. 2B). Consistently, Pparg2 transcriptional activation by rosiglitazone was impaired in Noc-deficient mouse embryonic fibroblasts (MEFs) compared with WT MEFs, although basal transcriptional activity was comparable between WT and Noc−/− MEFs (Fig. 3A). To examine whether physical binding of NOC to PPAR-γ2 caused any alteration in the stability of PPAR-γ2, C3H10T1/2 cells over-expressing either GFP or Noc were transfected with a Pparg2 expression vector and treated with cyclohexamide to inhibit protein synthesis. Western blot analysis for PPAR-γ2 revealed that, although PPAR-γ2 levels before cyclohexamide treatment were increased in cells overexpressing Noc, Noc did not influence the PPAR-γ2 degradation rate (Fig. 2C).

Fig. 2.
NOC is physically and functionally associated with PPAR-γ2 in a deadenylase-independent manner. (A) HEK293 cells were tranfected with WT-Noc-Flag or E193A-Noc-Flag, or a Pparg2-expression vector. Immunoprecipitation with Flag was followed by Western ...
Fig. 3.
Lack of Noc in MEFs decreases PPAR-γ transcriptional activation by rosiglitazone and PPAR-γ nuclear expression. (A) Mouse MEFs from WT or Noc−/− mice were transfected with Pparg2 expression vector and PPRE containing luciferase ...

Next, we investigated whether the physical association between NOC and PPAR-γ2 was part of the deadenylation process. For this purpose, we used a mutant of NOC (E193A-Noc) that lacked deadenylase activity. Coimmunoprecipitation analysis revealed that E193A-Noc bound to PPAR-γ2 in a manner identical to the WT Noc (Fig. 2A). U-33/γ2 cells expressing E193A-Noc also showed a significant increase in PPAR-γ2 transcriptional activation by rosiglitazone (Fig. 2B). To test whether NOC functions as a transcriptional coactivator for PPAR-γ2, we analyzed the intracellular localization of WT NOC and E193A-NOC. Immunofluorescence and Western blotting analyses confirmed that NOC was present exclusively in the cytoplasm even in the presence of PPAR-γ2 activation (Fig. 2 D and E), thereby ruling out the possibility that NOC acts as a transcriptional coactivator of PPAR-γ. Taken together, these data demonstrate that NOC binds to PPAR-γ2 and activates PPAR-γ2 transcriptional activity in a deadenylase-independent manner.

NOC Stimulates PPAR-γ Nuclear Translocation.

To clarify the role of NOC in PPAR-γ2 activation, we performed immunofluorescence and found that NOC exhibited perinuclear localization (Fig. 4A). This finding led us to hypothesize that NOC assisted PPAR-γ2 translocation into the nucleus in part by retaining it at the nuclear membrane. To answer this question, we examined intracellular localization of PPAR-γ2 using HEK293 cells transfected with Pparg2 and Noc. In HEK293 cells PPAR-γ2 mainly resided in the cytoplasm. However, with overexpression of Noc, PPAR-γ2 translocated into the nucleus even in the absence of a PPAR-γ2 agonist (Fig. 4 B and C). We also analyzed the nuclear translocation of PPAR-γ1, and found the same effect of NOC on PPAR-γ1 nuclear translocation (Fig S4B). Consistent with these data, the percentage of cells exhibiting nuclear PPAR-γ expression was reduced in Noc−/− MEFs compared with WT cells (Fig. 3 B and C). E193A-Noc, the mutated NOC without deadenylase activity, also facilitated PPAR-γ2 nuclear translocation (Fig. 4 B and C), demonstrating that NOC facilitates PPAR-γ2 nuclear translocation in a deadenylase-independent manner. In addition, cells expressing N-terminal truncation of NOC (S64-F429 NOC), which lacks the first 63 amino acids, also showed enhancement of PPAR-γ2 nuclear transport (Fig S4C). Finally, to determine the binding site of NOC to PPAR-γ, we studied the full-length NOC protein and identified a region (amino acids 341–351) with sequence similarity to an Src homology 2 domain binding motif. We then generated a short peptide that corresponded to amino acids 341 to 351 in NOC and fused it to a TAT protein (20) in its N terminus (NOC-peptide) to test whether this sequence could prevent the NOC/PPAR-γ interaction. Addition of NOC-peptide (1 μM) inhibited the binding of NOC to PPAR-γ2 (Fig. 5A) and significantly blocked NOC-stimulated endogenous PPAR-γ nuclear translocation in 3T3-L1 cells expressing WT-NOC (Fig. 5 B and C). In addition, NOC-peptide inhibited endogenous PPAR-γ transcriptional activity in 3T3-L1 cells (Fig. 5D).

Fig. 4.
NOC shows perinuclear localization, and enhances nuclear transport of PPAR-γ2 in a deadenylase-independent manner. (A) HEK293 cells were transfected with WT-Noc-Flag expression vector. Noc expression was visualized by immunofluoresence using anti-Flag ...
Fig. 5.
Amino acid 341–351 in the NOC protein is responsible for the binding between NOC and PPAR-γ2. (A) HEK293 cells were tranfected with a Pparg2 or WT-Noc-Flag expression vector in the presence of 5 μg/mL NOC-peptide or control peptide, ...

Next, we investigated whether nuclear translocation of PPAR-γ by NOC is affected by ligand activation with rosiglitazone and if rosiglitazone could antagonize the effect of NOC-peptide. Rosiglitazone induced nuclear translocation of PPAR-γ in 3T3-L1 cells expressing GFP (3T3-L1-GFP cells) treated with control peptide, and treatment with rosiglitazone in 3T3-L1 cells expressing Noc (3T3-L1-Noc cells) further increased nuclear translocation of PPAR-γ compared with 3T3-L1-Noc cells, although this did not reach statistical significance (Fig. 5C). In addition, rosiglitazone partially but significantly blocked the inhibitory effect of NOC-peptide on PPAR-γ nuclear translocation. These lines of evidence suggest that the effect of NOC on PPAR-γ nuclear translocation is in part mediated by the mechanism independent of rosiglitazone-induced PPAR-γ nuclear translocation (Fig. 5C). In sum, NOC mediates nuclear activation of PPAR-γ by facilitating nuclear transport through binding to the region corresponding to amino acid 341 to 351 in the NOC protein.

Body Composition Phenotype of Noc−/− Mice.

Previously, we reported that Noc−/− mice were protected from high-fat diet (HFD)-induced obesity (19). Generally, Pparg exhibits a circadian rhythm in liver that is amplified by a HFD (4, 19). However, Pparg circadian gene expression was absent on a HFD in Noc−/− mice, suggesting Pparg function could be altered in Noc−/− mice (19). Thus, we further characterized the adipogenic and skeletal phenotype of Noc−/− mice to clarify the in vivo consequence of the lack of Noc. We found that total body weight did not differ from controls across several time points (Fig. 6A), although percent body fat was less than controls at 4 wk of age (Fig. 6B). At 16 wk, Noc−/− mice accumulated more ovarian fat accompanied by hypertrophic adipocytes (Fig. 6 CF). In addition, Noc−/− mice developed moderate glucose intolerance (Fig. 6G). In the bone marrow of Noc−/− mice there were fewer adipocytes compared with controls (Fig S5). The size of brown adipose tissue (BAT) was not different among the three genotypes of Noc, but BAT from Noc−/− mice showed an increase in accumulation of lipid droplets compared with Noc+/+ or Noc+/− mice (Fig S6 A and B). There was no difference in brown adipogenesis of the stromal vascular fraction from BAT among the three genotypes (Fig S6C). Furthermore, BAT-related gene expression was not different between Noc+/+ and Noc+/− brown adipocytes (Fig S6D), but expression of Pparg2, aP2, and LRP130 (leucine-rich PPR motif-containing protein; LRPPRC), which is downstream of PGC-1α and modulates brown adipocyte function (21), were significantly decreased in Noc−/− brown adipocytes (Fig. 6H). Basal expression of UCP-1 (uncoupling protein 1) and PGC1α did not differ by genotype (Fig. 6H and Fig S6D), but induction of UCP-1 and PGC1α by forskolin was significantly impaired in the Noc−/− cells vs. control cells (Fig. 6I). In sum, Noc−/− mice have impaired ex vivo BAT function, which would be consistent with the lower basal body temperature reported previously (19).

Fig. 6.
Body composition and fat characteristics of Noc−/− mice. (A and B) Body weight (A) and percent body fat (B) was measured in Noc+/+ (n = 13–16) and Noc−/− (n = 10) mice at the ages indicated. (C and D) The gross ...

In respect to skeletal composition, Noc−/− mice exhibited higher areal whole-body and femoral bone mineral density (aBMD) than controls (Table S1). Femoral areal bone mineral content (aBMC) was significantly higher in Noc−/− mice vs. controls (Table S1). This phenotype was primarily because of a larger cortical compartment and longer femorae (Fig. 6J and Tables S2 and S3). Bone volume fraction in the femur as measured by microCT in 16-wk Noc−/− mice was significantly greater than controls principally becuase of enhanced trabecular thickness (Fig. 6J and Table S3). Thus, NOC plays an important role in regulating body composition by affecting both hard and soft tissues.

Discussion

The process of adipogenesis includes the differentiation of BMSCs under the influence of adipocyte-specific transcription factors (22, 23). PPAR-γ activation is essential for this process and here we show that NOC enhances PPAR-γ nuclear translocation. Of importance is that although NOC facilitates PPAR-γ nuclear translocation, a PPAR-γ ligand (endogenous or exogenous) is required to activate PPAR-γ transcriptional activity. NOC is a member of a family of proteins that include transcription factors, deadenylases and phosphatases (24). It shares significant sequence similarity to the multifunctional yeast protein Ccr4p, a transcription factor that is involved in regulation of global gene expression in response to nutrient status, in addition to its role as a deadenylase (12, 24, 25). Our initial findings raised the possibility that NOC was functioning at the level of transcription to regulate cell differentiation. However, mammalian NOC lacks the N-terminus activating domain, which Ccr4p possesses (12). In addition, NOC was not detected in the nucleus even with PPAR-γ2 activation, ruling out the possibility that it was a coactivator of PPAR-γ.

Previously PPAR-γ was considered solely in the context of its role as a nuclear receptor and transcription factor, even though cytosolic localization of PPAR-γ has been reported frequently (2629). However, a nuclear localization signal has never been identified for PPAR-γ, although there is evidence that it is exported from the nucleus in a MEK-dependent manner (30). Thus, the precise mechanism underlying PPAR-γ nuclear translocation remains elusive. Nuclear transport must involve either molecular size-based diffusion or a transport factor acting as a chaperone (31, 32). Our findings raise the intriguing possibility that NOC is involved in the translocation of PPAR-γ, although its absence in the nucleus implies that NOC must function in collaboration with other transport mechanisms to complete the nuclear translocation of PPAR-γ.

In support of that tenet, we found that Noc is induced transiently in early adipogenesis, and then is robustly up-regulated during the latter phases of differentiation in 3T3-L1 cells. The early up-regulation of Noc can be induced by FCS or insulin (15) and may be a critical step in activating PPAR-γ. Indeed, we found that silencing Noc resulted in impaired induction of Pparg transcripts during early adipogenesis in 3T3-L1 cells. Previously, we reported that in U-33 mesenchymal stromal cells transfected with a Pparg2 expression construct and treated with rosiglitazone, Noc expression was enhanced nearly 30-fold, implying that Noc was a down-stream target of Pparg (16). In addition, a Pparg “gain-of-function” 6T congenic mouse, which exhibits low bone mass and high marrow adiposity, showed increased Noc expression in fat, liver, and bone marrow (17, 33). Thus, the robust up-regulation of Noc expression in late adipogenesis may be explained in part by increased Pparg expression. In sum, Noc and Pparg may activate each other during adipogenesis, although we cannot rule out the possibility that Noc may also function to deadenylate one of the 3′UTR forms of Pparg, thereby acting as negative feedback on nuclear receptor activation.

Further evidence that NOC is important in PPAR-γ regulation comes from our in vivo studies of Noc−/− mice. These mice have high bone mass and reduced marrow adiposity, a phenotype remarkably similar to that reported in Pparg+/− mice (6). Moreover, the absence of Pparg circadian rhythmicity in the Noc−/− mice, even on a HFD, suggests that NOC modulates PPAR-γ activity in a forward direction.

It is well established that PPAR-γ is an important regulator of mesenchymal stem-cell fate, and its activation favors adipogenesis over osteoblastogenesis partly by suppressing osteogenic signaling pathways, such as Wnt, TGFβ/BMP, IGF-I, and transcription factors, including Runx2 and OSX (osterix) (16, 3436). Because Pparg expression is lower and Runx2 expression is higher in Noc−/− COBs compared with control cells, alterations in cell specification of BMSCs may be responsible for the skeletal phenotype of Noc−/− mice. Similarly, mice with a conditional deletion of circadian genes in osteoblasts, such as period and cryptochrome, also exhibit a high bone-mass phenotype (11). Moreover, type I collagen and osteocalcin exhibit significant periodicity during a 24-h cycle, suggesting that bone remodeling is under the control of the circadian clock (37). Because Noc may also be regulated by other clock genes (38, 39) that are important in bone turnover, it is conceivable that Noc modulates skeletal acquisition by functioning as a downstream modulator of osteoblast differentiation.

Adjusting cell behavior to energy availability is a central function of cell physiology (1). Recently, Lamia et al. reported that AMPK, a peripheral sensor of nutrient availability, phosphorylates cryptochrome 1, thereby promoting its ubiquitination and subsequent degradation (40). Previously, we have shown that Noc is rapidly induced in NIH 3T3 cells by FCS (15). In this study, we found that Noc was up-regulated by FCS and insulin in 3T3-L1 preadipocytes (Fig S7). Noc was also up-regulated by FCS in BMSCs, but the induction was not as great as that in 3T3-L1 cells (Fig S7). Insulin did not have any effect on Noc induction in BMSCs. These findings suggest that Noc expression is regulated by external stimuli, although the effect on Noc induction is likely to be cell-specific. The possibility that NOC regulates PPAR-γ nuclear transport provides a unique mechanism whereby fine tuning of adipogenesis can occur in response to entrainment from nutrient excess. The evolution of Noc in the mammalian organism as a nuclear activator for PPAR-γ raises a series of provocative questions about the role energy sufficiency plays in skeletal and adipogenic homeostasis and how environmentally induced alterations could ultimately affect body composition.

Methods

Mice.

Generation of Ccrn4ltmlBjc, which we refer to nocturnin knockout mice (Noc−/− mice) has been described previously (19). The original mice were backcrossed onto C57BL/6J background for at least seven generations. All experimental studies were performed using female mice. All of the animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of Maine Medical Center Research Institute.

Cell Culture.

See SI Methods for details.

Constructs.

The pCMV-WT-Noc-Flag, and pCMV-E193A-Noc-Flag vector, which harbors a point mutation in the Mg2+ binding motif leading to loss of deadenylase activity, have been previously described (15). Details for other constructs are described in SI Methods.

Body Composition and Skeletal Phenotyping of Noc−/− Mice.

Whole-body aBMD and aBMC, femoral aBMD and aBMC, and body composition were analyzed using dual-energy x-ray absorptiometry. Peripheral quantitative CT was used to analyze femur length and cortical thickness. Microarchitecture of distal trabecular bone and midshaft cortical bone of femur were evaluated by MicroCT. See SI Methods for details.

Analysis for the Role of NOC in Adipogenesis and Osteoblastogenesis.

Noc was overexpressed using retrovirus in 3T3-L1 cells and MC3T3-E1 cells to evaluate the role of NOC in adipogenesis and osteoblastogenesis. Noc expression was suppressed using small interfering RNA or short-hairpin RNA in 3T3-L1 cells and MC3T3-E1 cells. The expression of marker genes and specific staining were used to evaluate adipogenesis and osteoblastogenesis as described in SI Methods.

Intracellular Localization of NOC and PPAR-γ2.

Intracellular localization of NOC was analyzed by immunofluorescence and Western blot analyses as described in SI Methods. A lipophilic tracer [either DiIC18(5) (DiD) or DOPE-rhodamine] was used to visualize nuclear membrane. For nuclear staining, cells were stained with either TOPRO3 or SYTOX (Invitrogen).

Analysis of Interaction Between NOC and PPAR-γ2.

To analyze the physical interaction between NOC and PPAR-γ2, HEK293 cells were transfected with Pparg2 expression vector and either WT-Noc-Flag or E193A-Noc-Flag expression vector, and immunoprecipitated with anti-Flag antibody. Then, Western blot analysis for PPAR-γ was performed as described in SI Methods. Reporter assay using PPRE-Luc vector was performed in HEK293 cells to analyze the role of NOC in Pparg transcriptional activity as described in SI Methods.

Statistical Analysis.

All data are expressed as the mean ± SEM. Results were analyzed for statistically significant differences using Student's t test or ANOVA followed by Bonferroni multiple comparison post hoc test. The glucose tolerance test was analyzed using repeat measures ANOVA. Statistical significance was set at P < 0.05.

Supplementary Material

Supporting Information:

Acknowledgments

We thank A. Breggia and S. Bornstein for discussion and mouse genotyping (Maine Medical Center Research Institute). This work is supported by National Institutes of Health Grants AR45433-12, AR54604-03, and AR54604-S2 (to C.J.R.), AG030910 (to C.A.-B.), and GM076626 (to C.B.G.).

Footnotes

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1000788107/-/DCSupplemental.

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