Retinoic Acid-Related Orphan Receptor γ (RORγ): A Novel Participant in the Diurnal Regulation of Hepatic Gluconeogenesis and Insulin Sensitivity

The hepatic circadian clock plays a key role in the daily regulation of glucose metabolism, but the precise molecular mechanisms that coordinate these two biological processes are not fully understood. In this study, we identify a novel connection between the regulation of RORγ by the clock machinery and the diurnal regulation of glucose metabolic networks. We demonstrate that particularly at daytime, mice deficient in RORγ exhibit improved insulin sensitivity and glucose tolerance due to reduced hepatic gluconeogenesis. This is associated with a reduced peak expression of several glucose metabolic genes critical in the control of gluconeogenesis and glycolysis. Genome-wide cistromic profiling, promoter and mutation analysis support the concept that RORγ regulates the transcription of several glucose metabolic genes directly by binding ROREs in their promoter regulatory region. Similar observations were made in liver-specific RORγ-deficient mice suggesting that the changes in glucose homeostasis were directly related to the loss of hepatic RORγ expression. Altogether, our study shows that RORγ regulates several glucose metabolic genes downstream of the hepatic clock and identifies a novel metabolic function for RORγ in the diurnal regulation of hepatic gluconeogenesis and insulin sensitivity. The inhibition of the activation of several metabolic gene promoters by an RORγ antagonist suggests that antagonists may provide a novel strategy in the management of metabolic diseases, including type 2 diabetes.


Introduction
RORc constitutes with RORa and RORb, the retinoic acidrelated orphan receptor (ROR; NR1F1-3) subfamily of the nuclear receptors, which regulate transcription by binding as monomers to ROR-responsive elements (ROREs) in the regulatory region of target genes [1,2]. Through alternative promoter usage, the RORc gene generates 2 isoforms, RORc1 and RORc2 (RORct), that regulate different physiological functions. RORct is restricted to several distinct immune cells and is essential for thymopoiesis, lymph node development, and Th17 cell differentiation [1,[3][4][5]. RORc antagonists inhibit Th17 cell differentiation and may provide a novel therapeutic strategy in the management of several autoimmune diseases [4,6].
In contrast to RORct, relatively little is known about the physiological functions of RORc1. The expression of RORc1 is highly restricted to tissues that have major functions in metabolism and energy homeostasis, including liver and adipose tissue, and in contrast to RORa and RORb, RORc is not expressed in the central nervous system, including the hypothalamus and suprachiasmatic nucleus [1,[6][7][8][9][10][11][12][13]. In several peripheral tissues RORc1 exhibits a robust rhythmic pattern of expression with a peak at zeitgeber time (ZT) 16-20 that is directly regulated by the clock proteins, brain and muscle ARNTlike (Bmal1) and circadian locomotor output cycles kaput (Clock), and the Rev-Erb nuclear receptors [1,[8][9][10][11][12]14,15]. Although RORc is recruited to ROREs in the regulatory regions of several clock genes, including Bmal1, Clock, Rev-Erba, and cryptochrome 1 (Cry1); the loss of RORc has little influence on the expression of Bmal1 and Clock, and only modestly reduces the expression of Rev-Erba and Cry1 [10,12]; The robust oscillatory regulation of RORc1 expression by the clock machinery raised the possibility that RORc might regulate the expression of certain target genes in a ZT-dependent manner. Because the clock machinery plays a critical role in the circadian regulation of many metabolic pathways, including glucose metabolism [13,[16][17][18][19], RORc may function as an intermediary between the clock machinery and the regulation of metabolic genes. Since recent studies indicated an association between the level of RORc expression and obesityassociated insulin resistance in mice and humans [20,21], these observations led us to propose that RORc1 might be an important participant in the diurnal regulation of glucose metabolic pathways [10,16,18,22].
To study this hypothesis further, we examined the effect of the loss of RORc on the diurnal regulation of glucose metabolism in ubiquitous and the hepatocyte-specific RORc knockout mice. This analysis showed that loss of RORc enhances glucose tolerance and insulin sensitivity particularly during early daytime (ZT4-6) and reduces the peak expression of several glucose metabolic genes. RORc cistrome and promoter analysis indicated that several of these metabolic genes were regulated directly by RORc and involved ZT-dependent recruitment of RORc to ROREs in their regulatory region. Together, our observations are consistent with the concept that RORc directly regulates the diurnal expression of a number of glucose metabolic genes in the liver downstream of the hepatic clock machinery, thereby enhancing gluconeogenesis and decreasing insulin sensitivity and glucose tolerance. The inhibition of the activation of several glucose metabolic gene promoters by an RORc antagonist suggests that such antagonists might provide a novel therapeutic strategy in the management of insulin resistance and type 2 diabetes.

Loss of RORc improves insulin sensitivity and glucose tolerance in a ZT-dependent manner
Glucose tolerance and insulin sensitivity, as RORc1 expression, have been reported to be under endogenous circadian control [23,24]. Recently, we proposed that RORc1 might be an important participant in the diurnal regulation of several glucose metabolic pathways downstream of the circadian clock [10,22]. To study the potential role of RORc in glucose homeostasis, we examined the effect of the loss of RORc on insulin sensitivity, glucose tolerance and the rhythmic expression pattern of glucose metabolic genes in ubiquitous and hepatocyte-specific RORc knockout mice. Our data revealed that the loss of RORc expression had a significant effect on insulin tolerance (ITT) and glucose tolerance (GTT) in mice fed with a high-fat diet (HFD). Comparison of the insulin responsiveness at two different time periods, ZT4-6 (daytime) and ZT18-20 (nighttime) showed that in wild type mice fed a HFD (WT(HFD)) insulin was more effective in controlling glucose levels at ZT18-20 than at ZT4-6 indicating that insulin sensitivity was ZT dependent [23,24] ( Figure 1A). Interestingly, this ZT-dependent difference in insulin responsiveness was greatly diminished in RORc 2/2 (HFD) mice. ITT analysis showed that at ZT4-6 blood glucose levels remained significantly lower in RORc 2/2 (HFD) mice after insulin injection than in WT(HFD) mice particularly after reaching a trough at 60 min ( Figure 1A and Table S1). ITT performed at CT4-6 under constant darkness similarly showed improved insulin sensitivity in RORc 2/2 (HFD) mice ( Figure S1A), suggesting that RORc significantly affects insulin sensitivity also under a Zeitgeber-free condition. At ZT18-20 the difference in ITT response between WT(HFD) and RORc 2/2 (HFD) mice was significantly smaller than at ZT4-6. Consistent with the improved insulin sensitivity, GTT analysis showed that RORc 2/2 (HFD) mice were more glucose tolerant than WT(HFD) particularly at ZT4-6 ( Figure 1C). Although the difference was smaller than in mice fed with a HFD, RORc 2/2 (ND) mice fed with a normal diet (ND) were also significantly more insulin sensitive and glucose tolerant at ZT4-6 than WT(ND) mice ( Figure S1C and S1D). Because of the larger difference in mice fed a HFD, we focused much of our further analysis particularly on these mice. Altogether our observations indicate that the loss of RORc enhanced glucose tolerance and insulin sensitivity particularly at ZT4-6 and CT4-6. Analysis of the areas under the curves (AUC) for ITT and GTT was consistent with this conclusion ( Figure 1B and 1D).
To obtain further insights into the improved insulin sensitivity in RORc 2/2 mice, we compared the level of insulin-induced activation of Akt phosphorylation (P-Akt), one of the most sensitive phosphorylation targets in the insulin signaling pathway, in liver and several other metabolic tissues ( Figure 1E). No significant difference in P-Akt was observed at ZT4-6 in liver, brown and white adipose tissue (BAT, WAT), skeletal muscle between WT(HFD) and RORc 2/2 (HFD) mice after insulin stimulation. Moreover, no significant difference in P-Akt was observed between insulin-treated WT and RORc 2/2 primary hepatocytes ( Figure 1F). These results suggest that loss of RORc does not alter insulindependent phosphorylation of Akt in several metabolic tissues.

RORc participates in the diurnal regulation of hepatic gluconeogenesis
Next, we examined insulin sensitivity and glucose fluxes at daytime by the hyperinsulinemic-euglycemic clamp test. Consistent with the results of ITT, the glucose infusion rate (GIR) required to maintain blood glucose level under constant insulin infusion was significantly higher in RORc 2/2 (HFD) mice than in WT(HFD) mice at daytime (ZT2-9), while their glucose absorption rate estimated by whole-body glucose disappearance (Rd) was almost equal during the clamp (Figure 2A, S2A, S2B). Importantly, basal hepatic glucose production (HGP) and clamp HGP were significantly lowered in RORc 2/2 mice. Insulin equally suppressed the HGP about 70% in both WT and RORc 2/2 mice ( Figure 2B), indicating that the insulin responsiveness was not changed in RORc 2/2 mice, consistent with the observation in Figures 1E and  1F. Glucose turnover estimated from the steady-state infusion of 3 H-glucose (Basal HGP and Rd) [25] was lower in RORc 2/2 mice, indicating that the glucose absorption rate might also be reduced. These results suggest that the increased GIR required to maintain

Author Summary
The circadian clock plays a critical role in the regulation of many physiological processes, including metabolism and energy homeostasis. The retinoic acid-related orphan receptor c (RORc) functions as a ligand-dependent transcription factor that regulates transcription by binding as a monomer to ROR-responsive elements. In liver, RORc exhibits a robust circadian pattern of expression that is under direct control of the hepatic circadian clock. However, the connection between the circadian regulation of RORc and its control of downstream metabolic processes is not well understood. In this study, by using ubiquitous and liver-specific RORc-deficient mice as models, we demonstrate that hepatic RORc modulates daily insulin sensitivity and glucose tolerance by regulating hepatic gluconeogenesis. Genome-wide cistromic profiling, gene expression, and promoter analysis revealed that RORc is targeting and regulating a number of novel metabolic genes critical in the control of glycolysis and gluconeogenesis pathways. We provide evidence for a model in which RORc regulates the circadian expression of glucose metabolic genes in the liver downstream of the hepatic circadian clock, thereby enhancing gluconeogenesis and decreasing insulin sensitivity and glucose tolerance. This study suggests that attenuating RORc activity by antagonists might be beneficial for the management of glucose metabolic diseases including type 2 diabetes. blood glucose level in RORc 2/2 mice was due to reduced hepatic glucose production and not due to improved insulin responsiveness.
The clamp test suggested that the output of hepatic glucose produced by gluconeogenesis and glycogenolysis was reduced in RORc 2/2 mice. Because hepatic gluconeogenesis is under close control of the circadian clock [18,23,26], we analyzed gluconeogenesis efficiency at 2 different ZTs in WT and RORc 2/2 mice fed with either a ND or HFD. The pyruvate tolerance test (PTT) indicated that gluconeogenesis was significantly higher at ZT4-6 than at ZT18-20 in both WT mice RORc 2/2 mice with fed either a HFD or ND ( Figure S1E). However, gluconeogenesis was greatly reduced at ZT4-6 in RORc 2/2 mice compared to WT mice independent of whether the mice were fed a ND or HFD, while little difference in pyruvate tolerance was observed at ZT18-20 between the two genotypes ( Figure 2C, S1E). Analysis of the AUC for PTT supported this conclusion ( Figure 2D, S1E).
RORc 2/2 (HFD) mice also showed a reduced gluconeogenesis at CT4-6, a subjective daytime, under constant darkness ( Figure  S1B). Together, these observations indicate that loss of RORc affects pyruvate tolerance particularly at ZT4-6 and support a regulatory role for RORc in the circadian control of hepatic gluconeogenesis.
To obtain additional evidence that RORc enhances hepatic gluconeogenesis, we analyzed PTT in RORc 2/2 mice in which RORc was over-expressed in liver by adenovirus administration. As shown in Figure 2E, gluconeogenesis was significantly increased in mice injected with RORc-expressing adenovirus compared to mice injected with empty adenovirus. Further support for a role of RORc in gluconeogenesis was provided by data showing that over-expression of RORc in RORc 2/2 primary hepatocytes increased glucose production ( Figure S2C). Together these results suggested that RORc modulates insulin resistance and glucose tolerance by regulating hepatic gluconeogenesis. and ZT18-20 in WT and RORc 2/2 mice fed a HFD for 6 weeks (n = 7-12). Data represent mean 6SEM, * P,0.05, ** P,0.01, *** P,0.001 by ANOVA. (B, D) Comparison of AUC for ITT and GTT by one way ANOVA. AUC was also calculated by 2-way ANOVA; for ITT: Time period P = 0.080 and Genotype P = 0.0002; for GTT: Time period P = 0.073 and Genotype P = 0.013 (not shown). (E) Loss of RORc did not affect Akt activation. Total and phosphorylated of Akt were examined by Western blot analysis in liver, BAT, WAT, and skeletal muscle (SM) isolated from WT(HFD) and RORc 2/2 (HFD) mice 30 min after intraperitoneal injection of either 0.75 U/kg insulin or PBS. (F) Representative Western blot analysis (n = 2) of total and phosphorylated Akt in primary mouse hepatocytes isolated from WT and RORc 2/2 mice. Cells were treated with 20 nM insulin or PBS for 10 min before proteins were isolated. doi:10.1371/journal.pgen.1004331.g001 Blood insulin and hepatic glycogen levels are reduced in RORc 2/2 mice Food intake during daytime and nighttime was not significantly changed in RORc 2/2 (HFD) mice ( Figure 3A) and although glucose levels tended to be somewhat lower during daytime, a period in which gluconeogenesis was reduced, serum glucose levels were largely maintained in RORc 2/2 (HFD) mice ( Figure 3B). Serum insulin levels in WT mice exhibited a circadian pattern reaching peak levels at ZT16, while insulin levels were significantly lower in both RORc 2/2 (HFD) and RORc 2/2 (ND) mice particularly during ZT12-20 ( Figure 3B, S3A). Glucose-stimulated insulin secretion (GSIS) experiments indicated no difference in insulin secretion between WT and RORc 2/2 mice fed with either a ND or HFD ( Figure 3C). In addition, little difference was observed in the level of pancreatic insulin at ZT16, the time at which the difference in serum insulin levels was the greatest ( Figure 3D). These results suggested that lower serum insulin levels in RORc 2/2 mice were not due to impaired insulin secretion or reduced pancreatic b-cell mass. Moreover, the amount of insulin secretion in response to the same quantity of glucose injected was not changed, suggesting that the reduced insulin level in RORc 2/2 mice is likely due to reduced glucose production.
Glyconeogenesis and glycogenolysis play an important part in glucose homeostasis; 10-20% of hepatic glucose production in mice fasting for 4 h depends on glycogenolysis [27]. Hepatic glycogen reached its highest level at ZT0 and its lowest between ZT8-12 in both WT(HFD) and RORc 2/2 (HFD) mice; however, its peak level was significantly lower in RORc 2/2 (HFD) mice ( Figure 3E). After 16 h fasting, the level of hepatic glycogen was dramatically reduced in both WT(HFD) and RORc 2/2 (HFD) mice, but levels remained significantly lower in RORc 2/2 (HFD) mice ( Figure 3F). The level of hepatic glycogen was also reduced in RORc 2/2 mice fed with a ND ( Figure S3B). Glycogen accumulation was increased in RORc 2/2 (HFD) mice injected with RORcexpressing adenovirus ( Figure 3G), indicating that RORc positively contributes to hepatic glycogen accumulation. Altogether, these results indicate that RORc 2/2 mice are able to maintain blood glucose levels at lower insulin levels due to reduced hepatic glucose production and possibly reduced glucose uptake by the liver. The latter is consistent with the reduced glycogen accumulation and clamp test data showing that basal HGP/Rd was reduced in RORc 2/2 mice ( Figure 2A).

Loss of RORc affects energy homeostasis in a diurnal manner
We next examined the behavior activity and energy homeostasis in WT(ND) and RORc 2/2 (ND) mice in relationship to the effect of RORc on circadian rhythm and hepatic glucose metabolism. No significant difference in total body weight was observed between WT and RORc 2/2 mice fed a ND ( Figure S3C). The wheel running test showed that the circadian phase of behavioral activity was not changed in RORc 2/2 (ND) mice consistent with a previous report [12], but peak activity was lower than in WT mice ( Figure  S3D). Indirect calorimetry showed that oxygen consumption (VO 2 ), CO 2 production (VCO 2 ), respiratory exchange ratio (RER), and heat production were significantly lower in RORc 2/2 (ND) mice compared to WT(ND) mice particularly at nighttime ( Figure 3H and Figure S3E). Lower RER particularly at nighttime might indicate a preference for fatty acid consumption over glucose for energy production. Plotting of these parameters as a ratio between RORc 2/2 (ND) and WT(ND) mice showed that the largest difference between WT and RORc 2/2 mice occurred around ZT20 ( Figure 3I), which corresponds closely to the peak expression of RORc [10]. These results indicate that the change in glucose metabolism in RORc 2/2 mice is associated with reduced energy expenditure.

RORc cistrome is enriched for genes involved in lipid and glucose metabolism
To obtain further insights into the mechanism underlying the regulation of hepatic glucose metabolism by RORc, we performed ChIP-Seq analysis to determine the genome-wide map of cisacting targets (cistrome) of RORc in murine liver at ZT22, a few hours after the peak expression of RORc ( Figure S4A) [10]. This analysis identified 3,061 RORc binding sites (P,0.001) that were localized within intergenic regions (40.5%), introns (34.5%), within a 5 kb region upstream of the transcription start site (TSS)(11.5%), and the 59UTR (10.8%) ( Figure 4A, 4B). Notably, RORc-binding sites were enriched near the transcription start sites ( Figure 4C). De novo motif analysis using MEME program identified a classic RORE motif, AGGTCA preceded by an AT-rich region ( Figure 4D and 4E) as well as direct repeat 1 (DR1)-like nuclear receptor binding motif and a RORE variant motif. Interestingly, a similar DR1 and variant RORE motifs were recently found within , CO 2 production (VCO 2 ), respiratory exchange ratio (RER), and heat production were measured during 3 successive days using metabolic cages and the average in each ZT was plotted as a ratio between RORc 2/2 (ND) and WT(ND) mice (n = 8). Data represent mean 6SEM, * P,0.05, ** P,0.01, *** P,0.001 by ANOVA. doi:10.1371/journal.pgen.1004331.g003 the binding sites of Rev-Erbs [14,28]. Gene ontology analysis of 1,443 RORc candidate target genes, defined as those that have one or more detected RORc binding site within 5 Kb upstream of the TSS and/or within the gene body, indicated that the RORc cistrome was enriched for genes involved in fatty acid, amino acid, and carbohydrate metabolism (Table 1 and Table S2). Comparison of the ChIP-Seq data with those obtained from our previous microarray analysis [29] indicated that about 23% of the RORc candidate target genes were differentially expressed between WT and RORc 2/2 liver. CircaDB (http://bioinf.itmat.upenn.edu/ circa/) database analysis indicated that about 25% of the RORc target genes exhibited a rhythmic expression pattern.
Because RORa and RORc bind similar DNA response elements, we examined the degree of functional redundancy between RORc and RORa in regulating hepatic gene expression by comparing the RORa and RORc binding sites identified by ChIP-Seq analyses. The specificity of each anti-ROR antibody was confirmed by WB and ChIP assays using chromatin of RORdeficient mice as a negative control ( Figure S4B and S4C). ChIP-Seq analysis identified 1,319 RORa binding sites (P,0.001) and 957 candidate target genes ( Figure 4F). Comparison of the RORa and RORc cistromes revealed that 288 sites, including the ROREs within several clock genes reported previously [10], recruited both RORa and RORc ( Figure 4G and Table S3). Thus, the relatively small overlap indicates that in liver RORa and RORc exhibit a limited functional redundancy.

RORc regulates the circadian expression of glucose metabolic genes
Our ChIP-Seq analysis indicated that RORc is recruited to regulatory regions of several genes implicated in hepatic glucose metabolism, including G6pase, Pepck, Glut2, Pklr, Gck, Gckr, Gys2, Ppard, Pcx and Klf15 ( Figure 4G, S5). Loss of RORc resulted in a ZT-dependent decrease in the hepatic expression of most of these genes ( Figure 5A-5D) and are consistent with our ChIP-Seq data indicating that their transcription is directly regulated by RORc. The expression of G6pase was repressed in RORc 2/2 liver during most of the circadian cycle, while Pepck expression was reduced during ZT4-12; both genes play a key role in gluconeogenesis ( Figure 5A). Peak expression of Gys2, encoding a rate-limiting enzyme for glycogenesis, and Ppard, which regulates several genes involved in glucose and lipid metabolism [30], was decreased between ZT4-16 and ZT16-4, respectively. The expression of several other gluconeogenic genes, including Pcx and Klf15, the glucose transporter Glut2, and several genes important in the glycolysis pathway, including Plkr, Gck, and Gckr, was also diminished in RORc 2/2 liver ( Figure 5A-5D). Decreased expression of these genes was also observed in liver of RORc 2/2 mice fed with a HFD ( Figure 5C). Importantly, the loss of RORc had very little effect on the expression of Bmal1 and Clock, and a limited influence on the expression of Cry1 and Rev-Erba [10], which all play a critical role in the circadian regulation of lipid/glucose metabolic genes ( Figure S6) [10,12]. These results are consistent with the conclusion that the changes in the circadian pattern of expression of glucose metabolic genes are directly related the loss of RORc rather than changes in the regulation of clock genes by RORc.
We further showed that exogenous expression of RORc in RORc 2/2 liver tissue by adenovirus significantly increased the expression of G6pase, Pepck, Gck, Gckr, Ppard, Pcx, and Klf15 as well as the RORc-target gene, Avpr1a ( Figure 5E) [10]. Similarly, exogenous expression of RORc in RORc 2/2 primary hepatocytes significantly activated the expression of several of these genes ( Figure 5F). These data are consistent with the conclusion that these genes are positively regulated by RORc.
To examine whether any of these changes in gene expression translated into alterations in corresponding protein, we analyzed the expression of Pklr, which plays a key role in catalyzing the formation of pyruvate from phosphoenolpyruvate. As shown in Figure 5A and 5B, the level of Pklr protein in WT and RORc 2/2 liver correlated rather well with the level of RNA expression. The levels of Pklr protein and RNA were higher at ZT16 than at ZT4 and clearly repressed in RORc 2/2 liver.

RORc activates the target genes through novel ROREs
Our ChIP-Seq analysis indicated that in liver both RORa and RORc are recruited to the proximal promoter of G6pase and to intron 2 of Ppard ( Figure 4G and Figure S5A). ChIP-QPCR analysis showed higher association of RORc with these regulatory regions at ZT22 compared to ZT10, whereas relatively little recruitment was observed in RORc 2/2 liver at either ZT10 or ZT22 ( Figure S5D, S5E). Analysis of the G6pase proximal promoter (2500/+58) identified, in addition to a classical RORE (RORE1) [31], a RORE variant motif (RORE2), and a PPAR responsive-element (PPRE) ( Figure 6A), which has been reported to mediate the transactivation of G6pase by PPARa [32]. Reporter gene analysis showed that both RORc and RORa were able to highly activate the G6pase promoter ( Figure 6A), while the RORc-selective antagonist ''A'' [10] inhibited the activation by RORc at concentrations as low as 100 nM ( Figure 6B). Mutation of either the RORE1 or RORE2 greatly reduced the activation by RORs. Interestingly, these RORE mutations also inhibited the transcriptional activation of the G6pase promoter by PPARa. Inversely, a PPRE mutation significantly reduced the activation by RORs as well as by PPARa, while mutation of both ROREs and PPRE almost totally abolished G6pase transactivation ( Figure 6A). These observations suggested that RORs and PPARa collectively regulate G6pase expression.
The ROR binding region in intron 2 of Ppard contains three putative ROREs, including a variant sequence ( Figure 6C). Reporter analysis showed that RORc and RORa activated the Luc reporter gene driven by this regulatory region about 45-and 140-fold, respectively. Mutation of any of these 3 ROREs strongly reduced the activation of the reporter by RORc, while the triple mutation almost totally abolished activation. The RORc antagonist inhibited this activation in a dose-responsive manner ( Figure 6D). These results support the conclusion that Ppard transcription is directly regulated by RORc through these response elements and suggest that the circadian regulation of certain metabolic outputs by RORc may be in part due to its regulation of Ppard expression.
Although RORa was recruited to the RORE-containing regions of G6pase and Ppard ( Figure S5D, S5E) and activated the G6pase and the Ppard regulatory region in reporter assays, loss of RORa had little effect on the circadian expression of G6pase and Ppard ( Figure 6E). The expression of these genes in double knockout RORa sg/sg RORc 2/2 liver was reduced to a similar degree as in RORc 2/2 liver ( Figure 6F). These results suggest that under the conditions tested RORc rather than RORa, plays a significant role in the hepatic regulation of G6pase and Ppard in vivo.
In addition to G6pase and Ppard, RORc was recruited to several other genes important in glucose homeostasis, including intron 1 of Gck, the proximal promoter (2685/+42) of Gckr ( Figure 6G and 6H, Figure S5B), intron 2 of Glut2, the promoter of Gys2, and the promoter of Dlat ( Figure S7A). RORc was able to activate the Luc reporter gene driven by these regulatory regions. Mutation or deletion of the RORE(s) in the Gck and Gckr regulatory region as well as addition of the RORc antagonist significantly reduced the activation by RORc ( Figure 6G, 6H, S7B). ChIP-Seq analysis showed that RORa was not associated with these genes, and except for Gys2, RORa-deficiency had little effect on the expression of these genes in vivo ( Figure S7C, S7D). Together, these results support the conclusion that RORc directly regulates the transcription of a series of genes important in glucose metabolism and homeostasis.
Liver-specific RORc 2/2 mice exhibit reduced gluconeogenesis and improved insulin sensitivity To determine whether the effects on hepatic glucose metabolism were based on the hepatocyte-specific loss of RORc function rather than loss of RORc in other metabolic tissues or immune cells, we analyzed liver-specific RORcdeficient (RORc fx/fx Alb-Cre + ) mice. Our data confirmed that RORc expression was completely lost in the liver of RORc fx/ fx Alb-Cre + mice and was not changed in the kidney ( Figure 7A). ITT, GTT, and PTT analysis showed that, as demonstrated for the RORc ubiquitous knockout mice, RORc fx/fx Alb-Cre + (HFD) mice exhibited a greater glucose tolerance, were more responsive to insulin, and showed reduced gluconeogenesis, respectively ( Figure 7B-7D). Moreover, as in RORc 2/ 2 mice, the blood insulin concentration at ZT16 was significantly reduced in RORc fx/fx Alb-Cre + (HFD) mice and so was the peak accumulation of hepatic glycogen at ZT0 ( Figure 7E). Moreover, gene expression analysis showed that the hepatic expression of a series of RORc target genes important in glucose metabolism, including G6pase and Ppard, were also decreased in RORc fx/fx Alb-Cre + mice as seen in RORc 2/2 mice ( Figure 7F). Together, these observations suggest that the changes in hepatic glucose metabolism are related directly to the loss of RORc function in the liver and support the conclusion that RORc directly contributes to the regulation of hepatic gluconeogenesis and glucose metabolism.

Discussion
In this study, we identify a novel function for RORc in the daily regulation of hepatic glucose metabolism and insulin sensitivity. Our results demonstrate that at ZT4-6 RORc 2/2 mice are significantly more insulin sensitive and glucose tolerant than WT mice, while there was a smaller difference between the two strains at ZT18-20. The euglycemic clamp test revealed that hepatic glucose production was considerably reduced in RORc 2/2 mice (Figure 2A). This was supported by PTT data showing that the conversion of exogenously administered pyruvate to glucose was significantly lower in RORc 2/2 mice particularly at ZT4-6 ( Figure 2C). Inversely, ectopic RORc expression in RORc 2/2 liver tissue or primary hepatocytes increased glucose production ( Figure 2E, S2C). Our ITT and PTT data indicate that the regulation of glucose metabolism by RORc is also functional at subjective daytime, CT4-6, under constant darkness ( Figure S1A, S1B). Together, these observations demonstrate that gluconeo-  genesis is less efficient in RORc 2/2 liver and support the conclusion that RORc is an important positive regulator of hepatic gluconeogenesis and insulin sensitivity particularly during early daytime. The regulation of glucose metabolism is complex and not only depends on hepatic metabolism, but also involves control of metabolic pathways in other tissues in which RORc is expressed, such as adipose and skeletal muscle. It also involves certain regions of the brain, including the SCN and the hypothalamus, which are implicated in the regulation of the central circadian clock and appetite, respectively [16][17][18]. However, in contrast to RORa and RORb, RORc is not or very poorly expressed in the SCN,  hypothalamus or other parts of the brain [11,33]. Therefore, it appears unlikely that the brain plays a major role in the phenotypic changes observed in RORc 2/2 mice. In addition, many of the changes in RORc 2/2 mice, including the reduction in glucose metabolic gene expression, were also observed in liverspecific RORc-deficient mice, indicating that these effects are directly related to the loss of RORc in hepatocytes and separate from the loss of RORc in other metabolic tissues ( Figure 7F).
Since RORc functions as a transcription factor, the reduced gluconeogenesis in RORc-deficient mice must involve alterations in the transcription of RORc target genes. De novo motif analysis of the RORc cistrome identified, in addition to the classic RORE, two variant RORE-like motifs. The variant ROREs appear to allow a greater diversity in ROR binding than expected from the in vitro binding assays [34,35]. A greater promiscuity in in vivo DNA binding has also been observed for other nuclear receptors, and might be due to promoter context, chromatin structure, and histone modifications. Gene ontology analysis showed that many of the potential RORc-target genes are linked to metabolic pathways (Table 1 and Table S2), including glucose homeostasis (e.g., G6pase, Pepck, Pklr, Ppard, Gck, Gckr, Glut2, Gys2, Dlat, Pcx, and Klf15). Analysis of their rhythmic pattern of expression demonstrated that RORc deletion reduced peak expression of most of these genes, without affecting their phase. Regulation of these Figure 7. Liver-specific RORc deficient mice exhibit improved insulin sensitivity and reduced gluconeogenesis. (A) RORc expression in liver and kidney collected from RORc fx/fx Alb-Cre + and -Alb-Cre 2 mice at ZT8 and ZT20 (n = 4-5). GTT (B), ITT (C), and PTT (D) were examined during ZT4-6 in RORc fx/fx Alb-Cre + and -Alb-Cre 2 mice fed with a HFD (n = 7-11). (E) Serum insulin levels were measured at ZT4 (n = 8) and ZT16 (n = 15-16) in RORc fx/fx Alb-Cre + and -Alb-Cre 2 mice on a HFD. Hepatic glycogen was measured at ZT0. Data represent mean 6SEM, * P,0.05, ** P,0.01 by ANOVA. (F) The expression of a series of glucose metabolic genes was analyzed in the liver collected at ZT8 or ZT20 (n = 4-5). Data represent mean 6SD, * P, 0.05, ** P,0.01, *** P,0.001 by ANOVA. (G) RORc coordinates the regulation of circadian rhythm, hepatic glucose metabolism, and insulin sensitivity. Genome-wide cistromic profiling and promoter analysis revealed that RORc is targeting and regulating a number of metabolic genes critical in the control of glycolysis, gluconeogenesis and glycogenesis pathways. The loss of RORc in hepatocytes reduces the expression of these genes and hepatic gluconeogenesis in a diurnal time-dependent manner that results in improved insulin sensitivity. Due to reduced hepatic glucose production, RORc 2/2 mice may require less insulin than WT mice to maintain blood glucose levels. A decrease in glucose uptake due to lower insulin levels as well as reduced Gys2 expression may in part be responsible for the reduced accumulation of liver glycogen. Our study supports the model that the circadian regulation of several glucose metabolic genes by RORc in liver is linked to its circadian control of gluconeogenesis, insulin sensitivity, and glucose tolerance and is consistent with the idea that RORc functions as a positive regulator of gluconeogenesis and is positively linked to increased risk for type 2 diabetes. doi:10.1371/journal.pgen.1004331.g007 genes by RORc was supported by data showing that exogenous expression of RORc in RORc 2/2 liver and primary hepatocytes significantly enhanced their level of expression ( Figure 5E, 5F). Promoter and mutation analysis demonstrated that RORc was able to activate several of the RORE-containing promoters, while mutation of either the classic or variant ROREs significantly reduced this activation by RORc indicating that these motifs are functional. The RORc-mediated promoter activation was further supported by data showing that treatment with a RORc-selective antagonist considerably inhibited this activation ( Figure 6B, 6D,  S7B). Our RORc cistrome data together with the mRNA expression and promoter analysis support the model that in murine liver, RORc positively regulates the expression of a series of glucose metabolic genes directly through RORE binding. The reduced peak expression of several key metabolic genes, including G6pase and Pepck, which are critical for gluconeogenesis, the glucose transporter Glut2, and several genes important in the glycolysis pathway, including Plkr, Gck, and Gckr, likely contribute to the reduced glucose uptake, the less efficient gluconeogenesis and the lower glycogen accumulation observed in RORc deficient liver.
In addition to RORc, glucose metabolism is under the control of a number of other transcription factors. Although loss of RORc reduced peak expression of several glucose metabolic genes, most of these genes still exhibited a substantial rhythmic pattern of expression, indicating that additional factors are involved. For example, analysis of the G6pase promoter showed that in addition to the classic and variant RORE proximal promoter, it contained a PPRE ( Figure 6A), which has been reported to mediate the transactivation of G6pase by PPARa [32]. Mutation of either the ROREs or PPRE caused a significant reduction in the activation of this promoter suggesting that RORc and PPARa cooperatively regulate G6pase. Although comparison of the RORa and RORc cistromes indicated that RORa and RORc have largely distinct functions, there was a 10% overlap in target genes that included several glucose metabolic genes, such as G6pase and Ppard ( Figure S5). However, in contrast to RORc 2/2 mice, loss of RORa did not affect the expression of G6pase or Ppard ( Figure 6E, 6F) suggesting that under the conditions tested these genes are regulated by RORc rather than RORa.
Although several studies have demonstrated a role for Bmal1 and Clock in the regulation of several metabolic genes and shown that RORc is recruited to ROREs in Clock and Bmal1, the loss of RORc had little effect on the hepatic expression of Bmal1 and Clock ( Figure S6) [8,10]. These observations suggest that changes in glucose metabolic genes in RORc 2/2 liver are not due to changes in Clock or Bmal1 expression and are consistent with the hypothesis that RORc regulates these genes downstream of the clock machinery. However, cistrome analysis has shown that Bmal1 can also be recruited to certain glucose metabolic genes, such as G6pase, suggesting that Bmal1 in conjunction with RORc positively regulates the expression of these genes. In addition, RORc might cause changes in chromatin structure and as such influences the recruitment of Bmal1 or Clock to common target genes. The Rev-Erb nuclear receptors also play a critical regulatory role in the robust oscillation of circadian expression of a number genes [14]. RORs and Rev-Erb receptors can interfere with each other's activity by competing for RORE binding [10]. Despite the modest reduction in peak expression of Rev-Erba in RORc 2/2 liver ( Figure S6), which should result in increased target gene expression, the loss of RORc may reduce the competition with Rev-Erba for RORE binding and as a consequence increase the repression of gene transcription by Rev-Erba. A more comprehensive comparison between the cistrome of RORs, clock proteins, and Rev-Erbs is needed to provide further insights into the crosstalk between these transcription factors.
Although insulin levels were significantly lower in RORc 2/2 mice, blood glucose levels were largely maintained ( Figure 3B). At daytime, hepatic glucose production is less efficient in the knockout mice and consistent with this, blood insulin level was significantly reduced at ZT4. We hypothesize that insulin sensitivity in RORc 2/2 mice is also improved during nighttime due to reduced hepatic glucose production, which as a consequence would require less insulin to maintain blood glucose level and explain the lower level of blood insulin in RORc 2/2 mice. This is supported by AUC analysis for ITT, which indicates that also at nighttime insulin sensitivity was significantly better in RORc 2/2 mice ( Figure 1B). When mice eat during nighttime, more insulin is required to maintain blood glucose levels and this may account for the greater difference in blood insulin level compared to the difference at daytime. The observation that the PTT indicated little changed in gluconeogenesis efficiency at nighttime may be related to the fact that the PPT determines the efficiency of the gluconeogenesis pathway by measuring the formation of glucose from pyruvate after exogenous pyruvate injection, which is not a total reflection of all the pathways involved in the regulation of hepatic gluconeogenesis in vivo because pyruvate for gluconeogenesis can be supplied by other metabolic pathways.
A lower RER is considered to indicate that fat is increasingly preferred as a fuel source, whereas a higher RER is indicative for an increased use of carbohydrates. Thus, the lower RER observed at daytime in both WT and knockout mice indicates a greater preference for fatty acid consumption over glucose compared to nighttime ( Figure 3H), while the lower nighttime RER levels in RORc 2/2 mice compared to WT mice indicate a greater preference for fatty acid consumption over glucose. The latter is likely related to reduced glucose production and reduced glucose uptake in RORc knockout liver. Our data show that hepatic glycogen accumulation was reduced in RORc knockout mice during ZT16-0 clearly indicating that loss of RORc also affects glucose homeostasis at nighttime. This reduction in glycogen is likely due a reduced glucose uptake, which correlate with the lower levels of blood insulin in RORc knockout mice ( Figure 3B and 3E). Further analyses will be needed to precisely understand the precise interrelationships between various transcription factors, their diurnal regulation of various metabolic pathways and glucose and energy homeostasis.
In summary, our study identifies a novel function for RORc in the regulation of gluconeogenesis and insulin resistance. Our data are consistent with the model in which RORc directly regulates the expression of glucose metabolic genes in the liver downstream of the hepatic circadian clock, thereby enhancing gluconeogenesis, and decreasing insulin sensitivity and glucose tolerance ( Figure 7G). The temporal organization of tissue metabolism is coordinated by reciprocal crosstalk between the core clock machinery and key metabolic enzymes and transcription factors. Our study indicates that RORc is a novel important participant in this crosstalk. The improved insulin sensitivity and glucose tolerance observed in RORc-deficient mice suggest that the loss of RORc might be beneficial in controlling glucose homeostasis and in the management of metabolic diseases. This is supported by recent studies showing that in human patients the level of RORc expression positively correlates with insulin resistance [20,21]. The inhibition of the activation of several glucose metabolic gene promoters by an RORc-selective antagonist, thereby mimicking the effects in RORc 2/2 liver, suggests that such antagonists might provide a novel therapeutic strategy in the management of insulin resistance and type 2 diabetes.

Experimental animals
Heterozygous C57BL/6 staggerer (RORa +/sg ) were obtained from the Jackson Laboratories (Bar Harbor, ME). RORc 2/2 and RORa sg/sg RORc 2/2 double knockout (DKO) mice were described previously [10,36]. Liver-specific RORc knockout mice, referred to as RORc fx/fx Alb-Cre + , were generated by crossing B6(Cg)-Rorc tm3Litt /J (RORc fx/fx ) with B6.Cg-Tg(Alb-cre)21Mgn/J transgenic mice (Jackson Laboratories). Mice were supplied ad libitum with NIH-A31 formula (normal diet, ND) and water, and maintained at 23uC on a constant 12 h light:12 h dark cycle. Two month-old male mice were fed with a high fat diet (40% kcal fat) (HFD: D12079B Research Diets Inc., New Brunswick, NJ) for 6 weeks. Littermate wild type (WT) mice were used as controls. All animal protocols followed the guidelines outlined by the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at the NIEHS.

Glucose tolerance test (GTT), insulin tolerance test (ITT), and pyruvate tolerance test (PTT)
After 16 h fasting, WT and RORc 2/2 mice (n = 8-10) fed a ND or HFD for 6 weeks were injected intraperitoneally with glucose (2 g/kg), insulin (0.75 U/kg) (Eli Lilly, Indianapolis, IN) or sodium pyruvate (2 g/kg) (Sigma-Aldrich) at ZT4 or ZT18. The blood glucose was measured every 20 min for up to 140 min with glucose test strips (Nova Biomedical, Waltham, MA). These tests were performed in the same way using RORc fx/fx Alb-Cre + and RORc fx/fx Alb-Cre 2 mice (n = 11) fed a HFD. ITT and PTT were also performed under red light at CT4 after WT(HFD) and RORc 2/2 (HFD) mice (n = 12) were kept for 1 day under constant darkness. Total AUC (Area under the curve) was calculated by the trapezoid rule. Two-way ANOVA was performed using Graph-Pad PRISM software.

Western blot analysis
To evaluate insulin signaling, liver, BAT, WAT, and skeletal muscle were isolated from fasting WT(HFD) and mice RORc 2/2 (HFD) mice 30 min after injection with either 0.75 U/kg insulin or PBS. Protein from these tissues was extracted with lysis buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS). In a separate experiment, primary hepatocytes isolated from WT and RORc 2/2 mice were treated with 20 nM insulin in serum-free 199 medium (Sigma-Aldrich) for 10 min. Phosphorylated Akt (Ser473) and whole Akt proteins were detected by Western blot analysis with antibodies 7408 and 7102 from Cell Signaling Technology. Pklr and Gapdh were detected in liver lysates from WT and RORc 2/2 mice (n = 3) at ZT4 and ZT16 by Western blot analysis with anti-Pklr (22456-1-AP, Proteintech Group Inc., Chicago, IL, USA) and anti-Gapdh (Cell Signaling Technology) antibodies.

Hyperinsulinemic-euglycemic clamp test
WT and RORc 2/2 mice (n = 5) fed a HFD for 6 weeks underwent surgery under anesthesia to attach catheters to the jugular vein and carotid artery. Mice were left at least 2 days to recover. After a 3.5 h fasting, the basal rates of glucose turnover were measured by continuous infusion of HPLC-purified D-[3-3 H] glucose (0.05 mCi/min) (Perkin Elmer, Boston, MA) for 90 minutes following a bolus of 1 mCi. Blood samples (about 40 ml) were taken from the carotid artery catheter at 75 and 85 min after the infusion to determine the plasma [3-3 H] glucose concentration. Subsequently the hyperinsulinemic euglycemic clamp test was performed for 120 min in conscious, restrained mice. Human insulin (HumulinR, Eli Lilly) was infused at a constant rate (30 mU/kg/min) through the end of the experiment following a bolus of 90 mU/kg/min for 3 min. Glucose was measured every 10 min in blood from tail vein with glucose test strips. The glucose concentration was maintained at 110-130 mg/dl by a variable rate of 20% glucose infusion under a continuous infusion of [3-3 H] glucose (0.1 mCi/min). Blood samples (about 40 ml) were taken from the carotid artery catheter every 10 min during the last 40 min. [ 3 H]-glucose was used to trace hepatic glucose production and glucose turnover. The experiment was performed during daytime at ZT2-9.
For the determination of the plasma 3 H-glucose concentration, plasma samples were deproteinized with 0.3 N Ba(OH) 2 and ZnSO 4 and dried to remove 3 H 2 O before the radioactivity was measured in a liquid scintillation counter. Basal hepatic glucose production (Basal HGP) was calculated as the ratio of the preclamp [ 3 H]-glucose infusion rate (GIR) (dpm/min) to the specific activity of plasma glucose. Clamp whole-body glucose disappearance (Rd) was calculated as the ratio of the clamp [3-3 H] GIR (dpm/min) to the specific activity of plasma glucose. Clamp glucose production (Clamp HGP) was determined by subtracting the average GIR in the last 40 min from the Rd.

Preparation and injection of recombinant adenovirus
Recombinant adenoviruses were generated using the AdEasy adenoviral system (Agilent Technologies, Palo Alto, CA). Fulllength RORc1 cDNA was inserted to pShuttle-IRES-hrGFP-1 vector, and co-transformed with pAdEasy-1 in BJ5183-AD-1 bacteria by electroporation. The recombinant adenovirus plasmid was then transfected in AD-293 cells. The amplified adenoviruses were purified and concentrated by cesium chloride density gradient centrifugation. The empty control and RORc expressing adenoviruses were injected into the retro-orbital sinus of RORc 2/2 (HFD) mice (n = 6-7). Pyruvate tolerance test was performed 4 days later and after an additional four days, liver was collected at ZT8 to analyze glycogen accumulation and gene expression.

Primary hepatocyte isolation and glucose production assay
Hepatocytes from 2 month-old WT and RORc 2/2 mouse were isolated with a Hepatocyte Isolation System (Worthington Biochemical Corporation, New Jersey, USA) according to the manufacturer's instructions. Primary hepatocytes were cultured in collagen-coated dishes with Medium 199 supplemented with 100 nM dexamethasone, 1 nM insulin, 10 nM triiodothyronine, 5% fetal bovine serum, and penicillin/streptomycin. After 8-12 h, cells were infected with empty lentivirus pLVX-mCherry-N1 or RORc1-expressing lentivirus. 24 h later cells were washed twice in PBS and then incubated in serum-free medium 199 in the presence or absence of 100 nM insulin or 100 nM glucagon (Sigma-Aldrich) for 6 h before RNA was isolated. Glucose production was measured with a glucose production buffer (glucose/phenol red-free DMEM (Sigma-Aldrich), 1 mM lactose, 2 mM sodium pyruvate) in RORc 2/2 hepatocyte infected with lentivirus for each empty and RORc expression (n = 3). Glucose in the medium was measured with a Glucose assay kit (Sigma-Aldrich).
Insulin, liver glycogen, pyruvate measurement Serum and liver samples were collected from WT and RORc 2/2 mice on a HFD (n$5) every 4 h over a period of 24 h. Serum insulin was measured by a sandwich ELISA with a Rat/Mouse Insulin ELISA kit (EZRMI-13K, Millipore). Glucose stimulated insulin secretion (GSIS) was measured at ZT4 in WT and RORc 2/2 mice on a HFD (n = 5-6) or ND (n = 2-3). Serum was collected at 2.5, 5, 15, and 30 min after intraperitoneal injection of glucose (2 g/ kg). Pancreatic insulin was determined by rapidly removing the pancreas from WT and RORc 2/2 mice (n = 10-14) on a HFD. Pancreas was then homogenized and extracted overnight with acidethanol at 220uC. Insulin in the extracts was measured with the insulin ELISA kit. Insulin was normalized by total pancreatic protein. Glycogen extracted from liver with 30% KOH at 100uC for 2 h followed by precipitation by ethanol, was measured with a Glycogen Assay Kit (BioVison Inc., Mountain View, CA).

LabMaster metabolic analysis
To analyze metabolic parameters including oxygen consumption, CO 2 production, respiratory exchange ratio, heat production, and food/water consumptions were measured in WT and RORc 2/2 mice (n = 8) with a LabMaster system (TSE systems Inc., Chesterfield, MO) during 4 successive days.

Chromatin immunoprecipitation (ChIP) assay
The ChIP assay was performed using a ChIP assay kit from Millipore (Billerica, MA) according to the manufacturer's protocol with minor modifications as described previously [10]. Briefly, livers collected from WT, RORa sg/sg , and RORc 2/2 mice at ZT10 and ZT22 were homogenized with a polytron PT 3000 (Brinkmann Instruments) and crosslinked by 1% formaldehyde for 10 min at room temperature. After a wash in PBS, an aliquot of the crosslinked chromatin was sonicated and incubated overnight with an anti-RORa or anti-RORc antibody [10] generated against amino acids 129-231 and 121-213 in mouse RORc1 and RORa4, respectively. After incubation with protein G agarose beads for 2 h, DNA-protein complexes were eluted. The crosslinks were reversed by overnight incubation at 65uC in the presence of 25 mM NaCl, digested with RNase A and proteinase K, and then the ChIPed-DNA was purified. The amount of ChIPed-DNA relative to each input DNA was determined by QPCR. All QPCR reactions were carried out in triplicate. Sequences of primers for ChIP-QPCR are listed in Table S4.

ChIP-Seq data analysis
ChIPed-DNA and input DNA as a control were prepared using RORc-and RORa-specific antibodies as described previously [10]. ChIP-Seq analysis was performed by the NIH Intramural Sequencing Center and data were analyzed as reported previously [37]. The sequencing reads were obtained from base-calling of Illumina Genome Analyzer. The wiggle-formatted alignment results were visualized on UCSC Genome Browser using mouse mm9 reference genome. SISSRs (Site Identification from Short Sequence Reads) were used for identification of significant RORc and RORa binding sites (P,0.001) that have enriched reads in each ChIPed-DNA versus input control across the whole genome [38]. The distance from each ROR peak to the nearest transcriptional start sites was determined using custom scripts. De novo consensus motif search within ROR binding sites was performed using MEME. ChIP-Seq data was compared with gene expression data using Kolmogorov-Smirnov (KS) plot. Gene ontology analysis was performed using the NIH Database for Annotation, Visualization, and Integrated Discovery (DAVID) online web-server, and based on PANTHER Biological process definitions.

QRT-PCR analysis
To quantify gene expression during circadian time, liver tissues were collected from WT, RORc 2/2 , and RORa sg/sg mice every 4 h over a period of 24 h, processed overnight in RNAlater solution (Ambion, Austin, TX) at 4uC, and then stored at 280uC until use. Tissues were then homogenized with a Polytron PT-3000 (Brinkmann Instruments, Westbury, NY). Liver tissues were also collected from RORa sg/sg RORc 2/2 DKO mice and littermate control WT mice, and RORc fx/fx Alb-Cre + and RORc fx/fx Alb-Cre 2 mice at zeitgeber time (ZT) 8 and ZT20. RNA was then extracted using a QIAshredder column and RNeasy Mini kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. The RNA was reverse-transcribed using a High-Capacity cDNA Archive Kit (Applied Biosystems). QPCR analysis was performed using SYBR Green I (Applied Biosystems, Foster City, CA). The reactions were carried out in triplicate using 20 ng of cDNA and the following conditions: 10 min at 95uC, followed by 40 cycles of 15 sec at 95uC and 60 sec at 60uC. The results were normalized by the amount of Gapdh mRNA. Primer sequences are listed in Table S4.