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CRTC2 (TORC2) Contributes to the Transcriptional Response to Fasting in the Liver but Is Not Required for the Maintenance of Glucose Homeostasis

John Le Lay,1,3 Geetu Tuteja,1,3 Peter White,1,3 Ravindra Dhir,1,2 Rexford Ahima,1,2 and Klaus H. Kaestner1,3,* 1Department of Genetics 2Department of Medicine 3Institute for Diabetes, Obesity, and Metabolism University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA *Correspondence: [email protected] DOI 10.1016/j.cmet.2009.06.006

SUMMARY The gluconeogenic program is largely regulated at the level of transcription, a process thought to be coordinated by the cAMP The liver contributes to glucose homeostasis by response element-binding (CREB) and its ability to acti- promoting either storage or production of glucose, vate PPARg -1a (PGC-1a)(Herzig et al., 2001). Two depending on the physiological state. The cAMP mechanisms have been proposed to explain how CREB response element-binding protein (CREB) is a prin- responds to glucagon and/or epinephrine stimulation in order cipal regulator of involved in coordinating the to elicit the appropriate changes in hepatic expression. hepatic response to fasting, but its mechanism of Originally, phosphorylation of CREB at serine 133 by the cAMP-responsive protein kinase A (PKA) and the subsequent gene activation remains controversial. We derived recruitment of CREB-binding protein (CBP) or its paralog p300 CRTC2 (CREB-regulated transcription coactivator 2, were suggested to be the pivotal regulatory events (Chrivia previously TORC2)-deficient mice to assess the et al., 1993; Gonzalez and Montminy, 1989; Kwok et al., 1994; contribution of this cofactor to hepatic glucose metab- Mayr and Montminy, 2001). CBP and p300 are cofactors that olism in vivo. CRTC2 mutant hepatocytes showed promote gene transcription through their intrinsic histone deace- reduced glucose production in response to glucagon, tylase activity and through direct interactions with the basal tran- which correlated with decreased CREB binding to scription machinery (Bannister and Kouzarides, 1996; Goodman several gluconeogenic genes. However, despite and Smolik, 2000; Shiama, 1997). More recently, the simplicity of attenuated expression of CREB target genes, in- this model has been challenged with the discovery of the CREB- cluding PEPCK, G6Pase,andPGC-1a,nohypogly- regulated transcription coactivator (CRTC) family of cofactors cemia was observed in mutant mice. Collectively, (Conkright et al., 2003; Iourgenko et al., 2003). CRTC2, also called transducer of regulated CREB activity 2 (TORC2), is the these results provide genetic evidence supporting most abundant CRTC protein in the liver and has been implicated a role for CRTC2 in the transcriptional response to as the foremost driving CREB target fasting, but indicate only a limited contribution of this (Conkright et al., 2003; Koo et al., 2005). Upon glucagon stimula- cofactor to the maintenance of glucose homeostasis. tion, cytoplasmic CRTC2 is dephosphorylated and translocates to the nucleus, where it engages CREB in a phosphorylation- INTRODUCTION independent manner to direct the gluconeogenic program (Bittinger et al., 2004; Screaton et al., 2004). In attempts to unify Maintenance of blood glucose levels within a relatively tight the two models, it has been suggested that CRTC2 is required in range is of critical importance, as evidenced by the complica- addition to CREB phosphorylation for the proper recruitment tions arising from diabetes or hyperinsulinism. The liver, a key of CBP and p300 to regulatory complexes at CREB target target of both insulin and glucagon signaling, contributes to the sequences (Ravnskjaer et al., 2007; Xu et al., 2007). However, control of glucose metabolism by facilitating glucose uptake it has also been proposed that the transcriptional response to and output, depending on the physiologic condition (Pilkis and fasting is only transiently dependent on CRTC2, with the demon- Granner, 1992). Insulin is released from pancreatic b cells stration that it is deacetylated and degraded after long-term food when nutrient availability is high and instructs peripheral tissues, deprivation, at which point FOXO1 takes over as the predomi- such as liver and muscle, to remove glucose from the blood- nant transcriptional regulator (Liu et al., 2008). stream and store it for later use, effectively preventing prolonged In order to begin to address this complex issue using genetic hyperglycemia. In times of food deprivation, however, the liver is means, we assessed the hepatic response to fasting in mice central in the metabolic response to glucagon, epinephrine, and harboring a null mutation in the Crtc2 gene. Surprisingly, glucocorticoids, which act to stimulate hepatic glucose produc- CRTC2 null (CRTC2/) mice did not display fasting hypogly- tion (HGP) via glycogenolysis and gluconeogenesis in order to cemia despite impairments in the transcription of several meta- prevent extended periods of hypoglycemia. bolic genes. These transcriptional defects were observed only

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following prolonged fasting, indicating that CRTC2 continues to regulate gene expression beyond the short term. We also show that CREB association with target sequences, although unchanged during the initial stages of fasting, is reduced in the livers of 24 -fasted CRTC2/ mice, possibly accounting for the observed transcriptional deficiencies. Collectively, our results indicate a supporting role for CRTC2 in the regulation of CREB target genes in response to fasting but challenge its status as the key mediator of the gluconeogenic program.

RESULTS AND DISCUSSION

Derivation of the CRTC2/ Allele To assess CRTC2 function in vivo, we derived a CRTC2/ allele via homologous recombination in mouse embryonic stem cells. The targeting strategy used to construct this mutant allele is shown in Figure 1A. loxP sites were positioned upstream of exon 3 and downstream of exon 11 to force deletion of the sequence spanning exon 4 to exon 11 of the Crtc2 gene upon exposure to Cre recombinase. Crosses to the ubiquitous EIIa- Cre line resulted in recombination between loxP sites in the germline, yielding a global null allele. Western blotting confirmed the effective deletion of the full-length Crtc2 gene product (Figure 1B). CRTC2/ animals were born at the expected Mendelian frequency, gained weight appropriately with age, and were generally indistinguishable from littermate controls (data not shown). The livers of adult CRTC2/ mice also ap- peared histologically normal (data not shown). In addition, there were no appreciable differences in most plasma metabolic parameters of fasted CRTC2 mutant mice compared to controls, except for a small but significant decrease in corticosterone levels (Table S1).

CRTC2 Contributes to the Regulation of Genes Involved in the Hepatic Response to Fasting CRTC2 has been described as a critical component of the early transcriptional fasting response in the liver (Koo et al., 2005; Liu et al., 2008). When circulating glucose and insulin levels are low, glucagon and/or epinephrine signaling in hepatocytes results in the dephosphorylation of CRTC2 and its translocation to the nucleus, where it drives the transcription of several genes impor- tant in gluconeogenesis. Following long-term stimulation, however, it has been proposed that CRTC2 is deacetylated and targeted for degradation in the proteasome (Liu et al., 2008). CRTC2 activity is further controlled by insulin signaling, which results in CRTC2 phosphorylation and exclusion from the nucleus, where it can no longer impact gene expression (Dentin et al., 2007). Thus, in the absence of CRTC2, hypogly- Figure 1. Derivation of the CRTC2/ Mouse cemia due to impaired HGP would occur during the initial stages (A) loxP sites were positioned to flank exon 4 and exon 11 of the Crtc2 gene as well as a Neomycin (Neo) selection cassette. Mice harboring the CRTC2 loxP of fasting. To test this hypothesis, blood glucose measurements / allele were crossed to EIIa-Cre transgenic mice, resulting in a null allele lacking were taken from CRTC2 mice along with littermate controls at the loxP-flanked exons and the Neomycin selection cassette. regular intervals during a 24 hr fasting time course. As expected, (B) Western blot analysis of whole-cell liver lysates taken from mice homozy- no difference in blood glucose levels was observed in mice fed gous for the null allele (/) or littermate controls (+/+). Livers were dissected ad libitum (Figures 2A and 2B). Strikingly, we also found no differ- from mice fed ad libitum or fasted for the amount of time indicated. ences between groups at any time point measured during the (C) Quantitative RT-PCR to measure relative CRTC1 mRNA levels in the liver of / 24 hr fast (Figures 2A and 2B). Furthermore, glucose production control (+/+) and CRTC2 (/) mice fasted for the indicated amount of time or allowed to feed ad libitum (fed). Transcript levels in control mice were following injection of a pyruvate bolus was not affected in mutant assigned an arbitrary value of 1.0 for comparison. n = 5 for each group. Values mice (Figure 2C). Finally, hepatic glucose production as deter- are presented as average ± SD. mined by radioisotopic tracer techniques before or during

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Figure 2. CRTC2 Knockout Mice Maintain Normal Fed and Fasting Glycemia (A and B) Blood glucose levels of control (+/+) and CRTC2/ (/) mice during the course of a 24 hr fast. n = 8–12 for each group. (C) Blood glucose levels of control (+/+) and CRTC2/ (/) mice after overnight fasting (0 min) and following injection of a pyruvate bolus at the times indicated. n = 6 for each group. (D) Hepatic glucose production (HGP) was measured before (Basal HGP) and during a hyperinsulinemic-hypoglycemic clamp. Glucose infusion rate (GIR) and glucose disposal (Rd) were also measured during the clamp. n = 4 for each group. Values are presented as average ± SD. hyperinsulinemic-hypoglycemic clamps was not statistically that, despite its involvement in the regulation of G6Pase, PEPCK, different between CRTC2 mutants and littermate controls (Fig- and PGC-1a, CRTC2 is not required for an adequate metabolic ure 2D). Collectively, these results indicate that CRTC2 is not response to hypoglycemia. required to maintain the appropriate physiological response to fasting. Hepatocyte Function Is Impaired by CRTC2 Deficiency Next, we examined the hepatic mRNA expression levels of Many mechanisms in multiple tissues are in place to help G6Pase, PEPCK, and PGC-1a, three cAMP-inducible genes respond to metabolic challenges. During a fast, blood glucose involved in the response to fasting (Yoon et al., 2001). As ex- levels are maintained through the coordinated efforts of glyco- pected, gene expression levels were indistinguishable in animals genolysis and gluconeogenesis and, ultimately, the shift from that were refed following an overnight fast or allowed to feed ad glucose utilization to fatty acids and ketones as substrates for libitum (Figure 3A). Despite the apparently normal glucose energy production (Wang et al., 2006). Despite having compro- homeostasis of CRTC2/ animals, the expression of G6Pase, mised expression of several genes involved in the hepatic PEPCK, and PGC-1a was moderately but significantly reduced response to fasting, no overt glycemic phenotype was observed in CRTC2 mutants, but only after a prolonged 24 hr fast (Fig- in CRTC2/ mice. Indeed, similar outcomes have been ob- ure 3A). In light of the recent report implicating a role for served upon deletion of other metabolic genes, including PEPCK CRTC2 primarily in the early phases of fasting (Liu et al., 2008), and PGC-1a, which also fail to elicit dramatic metabolic pheno- it is worth noting that the timing of these transcriptional defi- types (Leone et al., 2005; She et al., 2000). Based on our expres- ciencies suggests the continued contribution of CRTC2 to the sion analysis indicating that several key genes involved in gluco- control of gene expression in the long term. In accordance with neogenesis are impacted by CRTC2 deletion, we suspected that this finding, total CRTC2 protein remained abundant in the liver hepatocytes in mutant animals might have functional impair- even following the 24 hr fast when we expected it to be absent ments that are masked by compensation in other tissues when due to degradation (Figure 1B). Protein levels of FOXO1, we analyzed glucose homeostasis in vivo. To address this point, CREB, serine 133-phosphorylated CREB, STAT3, HNF4a,C/ hepatocytes from control and mutant mice were isolated and as- EBPa, and C/EBPb were similar in the livers of control and sayed in culture. As expected, the induction of G6Pase, PEPCK, mutant mice, indicating that these factors are not likely to and PGC-1a mRNA levels in response to glucagon stimulation, compensate for the lack of CRTC2 in the short term (Figure 1B). as a surrogate for fasting, was blunted in CRTC2 mutant hepato- Furthermore, CRTC1 message levels were unaltered by the loss cytes (Figure 3B). Notably, the induced expression of these of CRTC2 activity (Figure 1C). These experiments demonstrate genes was impaired to a greater extent in culture than we had

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Figure 3. The Transcriptional Response to Fasting Is Blunted in CRTC2/ Hepatocytes (A) Quantitative RT-PCR to measure relative G6Pase, PEPCK, and PGC-1a mRNA levels in the liver of control (+/+) and CRTC2/ (/) mice fasted for the indicated amount of time, refed following an overnight fast (refed), or allowed to feed ad libitum (fed). (B) Quantitative RT-PCR to measure relative G6Pase, PEPCK, and PGC-1a mRNA levels in primary hepatocytes isolated from control (+/+) and CRTC2/ (/) mice 2 hr after treatment with 50 nM glucagon. Transcript levels in control cells were assigned an arbitrary value of 1.0 for comparison. (C) Glucose levels were measured in the culture medium of primary hepatocytes isolated from control (+/+) and CRTC2/ (/) mice 5 hr after treatment with or without 50 nM glucagon. n = 6–10 for each group. (D and E) Glycogen levels were measured in liver and muscle taken from mice allowed to feed ad libitum. n = 5 for each group. All values are presented as average ± SD. *p < 0.05. observed in the liver (Figure 2B), again implicating additional their decreased expression. To test this hypothesis, we per- mechanisms, such as activation by glucocorticoids, that may formed chromatin immunoprecipitation (ChIP) assays on chro- be at play in the whole animal to maintain sufficient hepatic ex- matin isolated from the livers of fasted CRTC2/ and control pression of these relevant transcripts. Furthermore, CRTC2/ mice. Strikingly, we found no significant differences in the ability hepatocytes failed to secrete as much glucose as controls of CBP or p300 to interact with regulatory complexes at any of the following glucagon stimulation (Figure 3C), indicating a func- promoters tested (Figures 4A and 4B). Furthermore, despite tional impairment that is presumably overcome by additional having previously been described as direct targets for CREB acti- processes unaffected by CRTC2 deletion in vivo. Indeed, we vation (Herzig et al., 2001; Schmoll et al., 1999; Waltner-Law observed that liver and muscle glycogen levels were significantly et al., 2003; Zhang et al., 2005), CREB binding to these promoter reduced in fed CRTC2 mutants (Figures 3D and 3E). This obser- regions was relatively weak and, in the cases of the PGC-1a and vation could be indicative of an unrecognized role for CRTC2 in G6Pase promoters, nearly undetectable (Figure 4C). regulating glycogen metabolism, but it may also represent Based on these results, we hypothesized that previously un- a mechanism by which hepatocytes and other tissues are able appreciated control sequences that confer CREB responsive- to offset any insufficiencies provoked by the absence of CRTC2. ness to these metabolic genes may exist. To localize these elements, we performed ChIP-Seq experiments on chromatin CREB Recruitment to Target Sequences Is Reduced prepared from the livers of fasted mice as an unbiased approach in the Absence of CRTC2 toward the genome-wide identification of CREB target sites. It has been suggested that CRTC2 imparts regulation on target With a focus on the PEPCK, PGC-1a, and G6Pase loci, we genes by specifically interacting with CREB and facilitating were able to identify cAMP response element (CRE)-containing recruitment of the coactivators CBP and p300 to CREB target sequences strongly bound by CREB in vivo approximately sequences (Liu et al., 2008; Ravnskjaer et al., 2007; Xu et al., 5.8 kb upstream of the PEPCK transcription start site (Figure 4D) 2007). We therefore expected that the association of CBP or and in an intronic region approximately 8 kb downstream of p300 with PEPCK, PGC-1a, and G6Pase control regions would the PGC-1a promoter (Figure 4E). No new regions bound by be attenuated in the livers of CRTC2/ mice, thus resulting in CREB were discovered upon examination of the G6Pase locus.

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Figure 4. Binding of CREB to Select Target Sequences Is Reduced in CRTC2 Knockout Hepatocytes (A–C) ChIP assays were performed on chromatin isolated from the livers of CRTC2/ (/) or control (+/+) mice fasted for 6 or 24 hr using antibodies against CBP (A), p300 (B), or CREB (C). Quantitative PCR was performed to determine enrichment of sequences associated with the PEPCK, G6Pase, and PGC-1a genes. n = 5–9 for each group. (D–E) Schematics of the PEPCK (D) and PGC-1a (E) genes, indicating the location of the unique CREB-binding regions as determined by ChIP-Seq analysis. The distal PEPCK element is located at 5870 bp relative to its transcription start site, while the PGC-1a region is located at +8020 bp within intron 2. The CRE sequences are underlined. Notice that peaks near the respective promoters, where presumptive CRE sequences have been described (arrowheads), are not as pronounced, indicating enhanced CREB occupancy at the sites. All values are presented as average ± SEM. *p < 0.05.

Robust CREB occupancy at these target sites in the PEPCK and hepatocytes of CRTC2/ mice fasted for 24 hr (Figure 4C). In PGC-1a genes was confirmed by qPCR in chromatin isolated accordance with the gene expression pattern, CREB binding from livers of fasted control mice (Figure 4C). While CBP and was not affected in the livers of mice fasted for 6 hr, thus p300 binding at these sites was similarly unaffected by the revealing a correlation between CREB binding and the transcrip- absence of CRTC2 (Figure 4B), we found that CREB occupancy tional activity of these genes. These data indicate a direct role for at the PEPCK and PGC-1a target sequences was reduced in CRTC2 in facilitating CREB binding to select target elements but

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also raise questions with regard to the nature of CRTC2-medi- visualized using the ECL Plus Western Blotting Detection System (GE Health- ated G6Pase regulation. Neither CBP nor p300 recruitment to care Bio-Sciences Corp). the G6Pase promoter region was impacted by the CRTC2 dele- Determination of Blood Glucose Levels and Pyruvate Challenge tion, while CREB binding to its presumptive CRE was tenuous, Blood was sampled from the tail vein of mice allowed to feed ad libitum (fed) or leaving open the possibility of indirect or CREB-independent fasted for 6, 9, 12, or 24 hr. Glucose levels were measured with a OneTouch mechanisms of control. Overall, these results support a role for Ultra Glucometer (LifeScan; Milpitas, CA). For gene expression analyses of CRTC2 in regulating the expression of gluconeogenic genes in refed animals, mice were fasted overnight and then allowed to feed for 2 hr the liver, but they also indicate that the overall contribution of before sacrifice. For pyruvate challenge experiments, animals were fasted CRTC2 to glucose homeostasis in vivo is limited. overnight, and a blood glucose measurement was taken and considered time 0. Mice were then injected intraperitoneally with 2 g of pyruvate (Sigma-Aldrich) per kg of body weight. Glucose levels were then measured EXPERIMENTAL PROCEDURES at 15, 30, 60, 90, and 120 min postinjection.

/ Derivation of CRTC2 Mice Hyperinsulinemic-Hypoglycemic Clamp An 18.7 kb DNA fragment containing all CRTC2 coding sequences was Glucose homeostasis was measured in 12- to 15-week-old male control and retrieved from C57BL/6J mouse bacterial artificial clone RP23- CRTC2/ mice using insulin clamp and radioisotopic tracer techniques (Alen- 237D16 via bacterial recombination (Copeland et al., 2001) into plasmid ghat et al., 2008; Varela et al., 2008). An indwelling catheter was inserted in the PL253 that contains an Mc1-driven thymidine kinase cassette for negative right internal jugular vein under sodium pentobarbital anesthesia and extended selection. One loxP site was inserted upstream of exon 3, while a pair of to the right atrium. Four days after recovery, the mice were fasted overnight for loxP sites flanking a neomycin selection cassette were placed downstream 16 hr. They were placed in restrainers and administered a bolus injection of of exon 11, also by bacterial recombination. The linearized targeting vector 5 mCi of [3-3H]glucose, followed by continuous intravenous infusion at was electroporated into B6 embryonic stem cells (ESCs) (Chemicon; Billerica, 0.05 mCi/min. Baseline glucose kinetics were measured for 60 min, followed by MA), and clones that survived G418 and ganciclovir selection were screened hyperinsulinemic-hypoglycemic clamp for 120 min. A priming dose of regular for homologous recombination by Southern blot analysis. Targeted clones insulin (16 mU/kg [Humulin, Eli Lilly; Indianapolis, IN]) was given intravenously, were injected into C57BL/6J-derived blastocysts that were then transferred followed by continuous infusion at 2.5 mU/kg1/min1. Blood glucose was to pseudopregnant females. Male offspring were mated to C57BL/6J females, maintained at 60–70 mg/dl via a variable infusion rate of 20% glucose. The and ESC-derived offspring were identified by PCR-based genotyping. Mice mice were euthanized, and liver samples were excised, frozen immediately harboring the targeted insertion of the three loxP sites in the Crtc2 gene locus in liquid nitrogen, and stored at 80C. The rates of basal glucose turnover were then crossed to the EIIa-Cre line to achieve germline deletion of loxP- and whole-body glucose uptake are measured as the ratio of the [3H]glucose flanked sequences (Holzenberger et al., 2000). Mice heterozygous for the infusion rate (GIR) (dpm) to the specific activity of plasma glucose. HGP during +/ CRTC2 deletion (CRTC2 ) were then mated to C57BL/6J to segregate clamp was measured by subtracting the GIR from the whole-body glucose +/ away the EIIa-Cre transgene. CRTC2 mice were then intercrossed to obtain uptake (Rd). CRTC2/ animals. Genotyping of all offspring was performed by PCR using 50 0 0 0 0 and 3 primers for CRTC2 (5 -GCCTGATTTTTCCCCCTATT-3 and 5 -CATCT Glycogen Assay 0 CAATGAAGGCCACCT-3 ), which yield a 431 bp product from the wild-type Tissue samples from mice fed ad libitum were homogenized in 6% PCA / allele and a 608 bp product from the CRTC2 allele. All PCR was performed (500 ml/100 mg tissue). Precipitates were pelleted, and the supernatant was for 35 cycles under the following conditions: 95C, 30 s; 60C, 30 s; 72C, 60 s. collected. One volume of H2O was added, and the solution was adjusted to pH 6.5 with 10 N KOH. A fraction of each sample was then incubated with Plasma Metabolic Profile Analysis five volumes of amyloglucosidase (1 mg/ml in 0.2 M [pH 4.8] acetate buffer) / Whole venous blood collected from the inferior vena cava of CRTC2 and at 40C for 2 hr. Glucose concentrations were then determined using the control mice was applied to plasma separator tubes with lithium heparin anti- Amplex Red Glucose/Glucose Oxidase Assay Kit (Invitrogen). Samples incu- coagulant (BD Biosciences) and centrifuged for 2 min at 14,0003 g, and the bated in the absence of amyloglucosidase were used as baseline controls. plasma was collected. Metabolic parameters were determined by Ani Lytics, Inc. (Gaithersburg, MD). RNA Isolation and Quantitative RT-PCR Analysis Total cellular RNA was extracted from liver fragments or cultured primary Whole-Cell Lysate Preparation and Western Blot Analysis hepatocytes using RNeasy Kit (QIAGEN), then assayed for quantity and quality Liver fragments from CRTC2/ and control mice were homogenized by hand with the Agilent 2100 Bioanalyzer (Agilent Technologies; Santa Clara, CA). in 200 ml of lysis buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, 1 mM Approximately 1 mg of total RNA was reverse transcribed using oligo (dT) EDTA, 1% Triton X-100, 0.5% deoxycholic acid, and 1% SDS; supplemented and Superscript II Reverse Transcriptase (Invitrogen). The resulting cDNA with protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktails 1 samples were the template for qPCR experiments performed with SYBR and 2 (Sigma); then sonicated using a Bioruptor (Diagenode; Sparta, NJ). GreenER qPCR Supermix (Invitrogen) and the SYBR Green (with dissociation Whole-cell lysates were then centrifuged at maximum speed for 15 min to curve) program on the Mx3000 Multiplex Quantitative PCR System (Strata- pellet cellular debris, and the supernatant was collected. Protein concentra- gene). Reactions were performed in triplicate and normalized relative to the tions were determined by Bradford assay using Protein Assay Reagent (Bio- ROX reference dye. Median cycle threshold values were determined and Rad). Approximately 50 mg of whole-cell lysates were separated by SDS- used for analyses. Expression levels were normalized to those of hypoxan- PAGE and transferred to Immobilon-P membranes (Millipore). Membranes thine-guanine phosphoribosyltransferase (HPRT) as the internal control. were blocked in buffer consisting of 5% milk and 0.1% Tween 20 in 13 PBS Primer information is available at http://www.med.upenn.edu/kaestnerlab/ (PBST milk) for 4 hr and then incubated overnight at 4C with anti-CRTC2 index.shtml. (ST1099, Calbiochem) diluted 1:5000, anti-FOXO1 (C29H4, Cell Signaling) diluted 1:1000, anti-CREB (48H2, Cell Signaling) diluted 1:1000, anti-phos- Primary Hepatocyte Isolation and Culture pho-CREB (87G3, Cell Signaling) diluted 1:1000, anti-HNF4a (H6939, R&D Hepatocytes were isolated from 3-month-old mice. The vena cava was cannu- Systems) diluted 1:1000, anti-STAT3 (9132, Cell Signaling) diluted 1:1000, lated and the liver was perfused with 37C Liver Perfusion Buffer (GIBCO) for anti-C/EBPa (sc-61, Santa Cruz) diluted 1:500, anti-C/EBPb (sc-150, Santa 2 min, then 50 ml of 37C Liver Digestion Buffer (GIBCO) supplemented with Cruz) diluted 1:500, or anti-b-actin (13E5, Cell Signaling) diluted 1:2000 in 30 mg of collagenase D (Roche). The liver was then excised, dispersed in PBST milk. After washing, membranes were incubated with HRP-conjugated DMEM containing 10% FBS and penicillin/streptomycin, filtered through goat-anti-rabbit IgG (GE Healthcare Bio-Sciences Corp.; Piscataway, NJ) a 100 mm mesh, pelleted, and subjected to a Percoll gradient (45% Percoll diluted 1:12,500 in PBST milk for 45 min at RT. After washing, were in DMEM) to isolate viable cells. After two washes in DMEM containing 10%

60 Cell Metabolism 10, 55–62, July 8, 2009 ª2009 Elsevier Inc. Cell Metabolism Limited Role for CRTC2 in Glucose Metabolism

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