Targeted Disruption of the CREB Coactivator Crtc2 Increases Insulin Sensitivity

Targeted disruption of the CREB coactivator Crtc2 increases insulin sensitivity

Yiguo Wang, Hiroshi Inoue, Kim Ravnskjaer, Kristin Viste, Nina Miller, Yi Liu, Susan Hedrick, Liliana Vera, and Marc Montminy1

Salk Institute for Biological Studies, Peptide Biology Laboratory, La Jolla, CA 92037

Contributed by Marc R. Montminy, December 24, 2009 (sent for review December 22, 2009) Under fasting conditions, increases in circulating concentrations of Results pancreatic glucagon maintain glucose homeostasis through induc- Exposure to cAMP agonist promotes CRTC2 dephosphorylation tion of gluconeogenic genes by the CREB coactivator CRTC2. and nuclear entry (Fig. 1A). Following its translocation, CRTC2 is Hepatic CRTC2 activity is elevated in obesity, although the extent recruited to CREB binding sites on the PEPCK promoter as to which this cofactor contributes to attendant increases in insulin measured by chromatin immunoprecipitation (ChIP) assay (Fig. resistance is unclear. Here we show that mice with a knockout of 1B). Although CREB binding is readily detected over this pro- the CRTC2 gene have decreased circulating glucose concentrations moter under basal conditions, exposure to FSK further enhances during fasting, due to attenuation of the gluconeogenic program. CREB occupancy. CRTC2 was found to stimulate hepatic gene expression in part CRTCs associate with CREB through a conserved, 50–amino through an N-terminal CREB binding domain that enhanced CREB acid N-terminal CREB binding domain (CBD), which specifi- occupancy over relevant promoters in response to glucagon. cally recognizes the bZIP region of CREB (11). The importance Deletion of sequences encoding the CREB binding domain in −/− of the CREB bZIP domain for DNA binding led us to test CRTC2 mice lowered circulating blood glucose concentrations whether its association with the CBD increases CREB binding to and improved insulin sensitivity in the context of diet-induced relevant promoters. In gel mobility shift assays, a GST (GST)– obesity. Our results suggest that small molecules that attenuate CRTC2 (aa 1–120) polypeptide containing the CBD exhibited no – fi the CREB CRTC2 pathway may provide therapeutic bene t to indi- DNA binding activity, but it enhanced binding of CREB to a viduals with type 2 diabetes. double-stranded CRE oligonucleotide probe 4-fold (Fig. 1C and Fig. S1). In keeping with these effects, overexpression of wild- gluconeogenesis | obesity | CREB | CRTC2 | insulin type CRTC2 enhanced the activity of a cAMP responsive (EVX- luciferase [luc]) reporter in HEK293T cells exposed to FSK, but uring fasting, increases in hepatic gluconeogenesis ensure a mutant CRTC2 lacking the CBD did not (Fig. 1D). By contrast, Denergy balance for glucose-dependent tissues such as brain a CRTC2 mutant lacking only the central regulatory region, but and the red blood cell compartment. Hepatic glucose pro- containing the CBD and C-terminal transactivation domains, duction is elevated in type 2 diabetes, reflecting decreases in exhibited near wild-type activity. MEDICAL SCIENCES insulin signaling that otherwise inhibit the gluconeogenic pro- We deleted sequences encoding the N-terminal CREB bind- gram (1–3). ing domain (CBD), having shown that this domain increases Increases in circulating glucagon are thought to trigger gluco- CREB occupancy over relevant binding sites in the PEPCK neogenic gene expression in part through the cAMP-dependent promoter (Fig. 2A). Full-length CRTC2 protein was detected in tissues from wild-type but not CRTC2 KO mice (Fig. 2B). phosphorylation of the transcription factor CREB and through the − − Truncated CRTC2 polypeptides were also absent from CRTC2 / dephosphorylation of its cognate coactivator CRTC2 (4–6). In hepatic extracts by immunoblot assay with a C-terminally direc- − − parallel, decreases in circulating insulin concentrations during ted CRTC2 antiserum (aa 454–607; Fig. S2). CRTC2 / animals fasting also stimulate gluconeogenic genes by the dephosphor- were born at the expected Mendelian frequency; they were ylation of the forkhead activator FOXO1 (7). indistinguishable from control littermates at birth (Fig. S2). Localized in the cytoplasm under basal conditions through a Previous studies showing that RNAi-mediated knockdown of phosphorylation-dependent association with 14-3-3 proteins, CRTC2 in liver promotes fasting hypoglycemia (4, 6) prompted CRTC2 shuttles to the nucleus following its dephosphorylation us to examine effects of CRTC2 gene disruption on glucose −/− at Ser171, where it mediates induction of cellular genes by homeostasis. Relative to wild-type controls, CRTC2 mice binding to the bZIP domain of CREB over relevant pro- maintained lower fasting blood glucose concentrations on a moters (8, 9). The importance of CRTC2 for gluconeogenic normal chow diet (Fig. 2C, Upper Left). Gluconeogenic capacity, gene expression is supported by RNAi-mediated knockdown measured by pyruvate tolerance testing (PTT), was also reduced in CRTC2 mutant males (Fig. 2C, Lower Left). Correspondingly, studies, where acute depletion of CRTC2 lowered hepatic glu- hepatic mRNA amounts for G6Pase and PEPCK1 were down- − − cose production in fasted mice and by overexpression studies in regulated in fasted CRTC2 / mice compared with controls (Fig. which phosphorylation-defective, active CRTC2 increases cir- 2C, Upper Right). Consistent with a transcriptional effect, hep- culating glucose levels under both fasting and feeding conditions atic glucose-6-phosphatase (G6Pase)–luc reporter activity was − − (4, 6, 10). also reduced in fasted CRTC2 / mice compared with control To determine the importance of the CRTC2–CREB association for induction of the gluconeogenic program, we charac- terized mice lacking the conserved N-terminal CREB binding Author contributions: Y.W. and M.M. designed research; Y.W., H.I., K.R., K.V., N.M., Y.L., S.H., and L.V. performed research; Y.W., H.I., K.R., K.V., Y.L., and M.M. analyzed data; and domain of CRTC2. We found that gluconeogenic gene expres- Y.W. and M.M. wrote the paper. sion and hepatic glucose production were reduced in CRTC2- The authors declare no conflict of interest. fi de cient mice during fasting and in the setting of insulin resist- 1To whom correspondence should be addressed. E-mail: [email protected]. ance. Our results support an important role for CRTC2 in This article contains supporting information online at www.pnas.org/cgi/content/full/ mediating effects of fasting signals on hepatic gluconeogenesis. 0914897107/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.0914897107 PNAS | February 16, 2010 | vol. 107 | no. 7 | 3087–3092 Downloaded by guest on September 26, 2021 Fig. 1. CRTC2 increases CREB promoter occupancy in response to fasting signals. (A) Immunoblot (Upper) and immunocytochemical (Lower) analysis showing CRTC2 dephosphorylation and nuclear translocation in primary hepatocytes exposed to forskolin (FSK). Scale bar, 5 um. (B) Chromatin immunoprecipitation (ChIP) assay of CREB (Upper) and CRTC2 (Lower) occupancy over the human PEPCK promoter in HEK293T cells exposed to FSK (black bars) or vehicle (white bars) (n =3,P < 0.05; SEM). Location of CREB binding sites indicated. (C) Gel mobility shift assay of CREB and GST-CRTC2 proteins using a 32P-labeled double- stranded CRE oligonucleotide. Protein–DNA complexes and free probe indicated. (D)(Upper Left) Schematic diagram showing organiziation of wild-type or mutant CRTC2 vectors lacking either the N-terminal CREB binding domain (aa 1–50) or central regulatory region (aa 51–539) indicated. (Upper Right) Immunoblot with anti-ﬂag epitope antiserum showing relative expression of each CRTC2 polypeptide in transfected cells. (Lower) Transient assay of HEK293T cells transfected with cAMP responsive EVX-luc reporter vector and exposed to FSK as indicated (n =3,P < 0.05; SEM).

− − littermates (Fig. 2C, Lower Right). The effects of CRTC2 on increased in CRTC2 / mice by glucose and insulin tolerance − − hepatic glucose output in CRTC2 / mice appear to be cell testing (Fig. 3B). Although they experienced similar weight − − autonomous because glucose output and G6Pase reporter gain, CRTC2 / mice remained relatively insulin sensitive under activity were also down-regulated in primary cultures of high-fat diet (HFD) conditions, when insulin resistance is typi- − − CRTC2 / hepatocytes compared with wild-type cells exposed to cally increased (Fig. 3 C and D and Fig. S3). Taken together, glucagon (Fig. 2D). these results suggest that CRTC2 contributes to the devel- Chronic increases in hepatic glucose production are thought to opment of insulin resistance in part through its effects on contribute to the development of type 2 diabetes because they hepatic gluconeogenesis. stimulate compensatory increases in insulin secretion that ulti- We examined whether CRTC2 re-expression is sufﬁcient to − − mately lead to islet cell failure (3). In line with their reduced rescue gluconeogenic gene expression in CRTC2 / hepatocytes. hepatic glucose production, CRTC2 mutant mice had lower ad Exposure to glucagon increased G6Pase and PEPCK mRNA − − libitum circulating concentrations of insulin; circulating trigly- amounts in wild-type but not CRTC2 / hepatocytes; adenoviral cerides and cholesterol were also down-regulated (Fig. 3A and delivery of CRTC2 restored PEPCK and G6Pase mRNA − − Fig. S3). Correspondingly, whole body insulin sensitivity was induction by glucagon in CRTC2 / cells (Fig. 4A). In keeping

3088 | www.pnas.org/cgi/doi/10.1073/pnas.0914897107 Wang et al. Downloaded by guest on September 26, 2021 MEDICAL SCIENCES

Fig. 2. Fasting hypoglycemia in CRTC2−/− mice. (A) Schematic diagram of wild-type and mutant CRTC2 alleles following homologous recombination with targeting vector lacking exon 1 sequences. (B)(Left) PCR analysis showing CRTC2 fragments generated from wild-type, heterozygous, or homozygous CRTC2 mutant mice. (Right) Immunoblot of CRTC2 protein amounts in hepatic extracts from from wild-type and CRTC2 mutant mice. *Nonspeciﬁc band. CRTC2 antiserum was developed against aa 454–607 of mouse CRTC2. (C)(Top) Circulating glucose concentrations (Left) and hepatic mRNA amounts for gluco- − − neogenic genes (Right; G6Pase, PEPCK) in wild-type and CRTC2 / mice (n = 10, P < 0.05; SEM). (Lower) pyruvate tolerance testing (n =5,P < 0.05; SEM) (Left) and G6Pase-luc reporter activity (Right) in fasted wild-type and CRTC2 mutant mice. (D) G6Pase reporter activity (Top) and glucose output (Bottom) from primary hepatocytes (wild-type, CRTC2+/−, CRTC2−/−) under basal conditions and following exposure to glucagon (n =3,P < 0.05; SEM).

with these effects, exposure to glucagon also increased CREB in their study, lacking exons 4–11, is capable of generating an in- occupancy over G6Pase and PEPCK promoters in wild-type cells frame polypeptide containing the N-terminal CREB binding − − but not in CRTC2 / hepatocytes (Fig. 4C). In addition, ade- domain fused to the C-terminal transactivation domain. noviral CRTC2 expression also rescued effects of glucagon on Although the expression levels of this CRTC2 product are CREB occupancy over G6Pase and PEPCK promoters in unknown, we found that a similar truncated CRTC2 polypeptide − − CRTC2 / cells (Fig. 4 B and C), confirming the ability for lacking the central regulatory domain exhibits near wild- CRTC2 to enhance CREB binding to target promoters. type activity on a CRE-luc reporter (Fig. 1D), potentially ex- plaining why hepatic glucose production is relatively unaffected Discussion in that study. During fasting, increases in circulating pancreatic glucagon To avoid residual regulatory contributions from a mutant promote glucose homeostasis by stimulating the gluconeogenic CRTC2 allele, we deleted sequences encoding the N-terminal program in liver. Hepatic glucose output is up-regulated in CREB binding domain (CBD), which we show here increases insulin resistance, when it contributes to chronic increases in CREB occupancy over relevant binding sites in the PEPCK circulating glucose levels and ultimately to islet cell failure and promoter. Although CREB has been shown to modulate cellular type 2 diabetes. Our results suggest that CRTC2 contributes gene expression in response to a variety of stimuli, the extent to significantly to the development of insulin resistance through its which these increases reflect changes in promoter occupancy has effects on hepatic CREB activity in this setting. been unclear (13). In a recent study, calcium and cAMP agonists Although hepatic glucose production was reduced in our were found to enhance CREB occupancy independently of their − − CRTC2 / mice, Kaestner et al. observed minimal effects on effects on CREB phosphorylation (14). Our results suggest that − − circulating glucose concentrations in their CRTC2 / animals such increases in CREB binding are likely transmitted through under normal chow conditions (12). In addition to possible dif- recruitment of CRTC2 to relevant sites. Because it interacts ferences in mouse strains, we note that the mutant CRTC2 gene directly with residues in the bZIP domain, the CREB binding

Wang et al. PNAS | February 16, 2010 | vol. 107 | no. 7 | 3089 Downloaded by guest on September 26, 2021 − − Fig. 3. CRTC2 / mice have enhanced insulin sensitivity under high-fat diet conditions. (A) Circulating triglyceride, cholesterol, and insulin concentrations in wild-type and CRTC2 mutant mice on a normal chow (NC) diet (n =15,P < 0.05; SEM). (B) Whole-body insulin sensitivity of NC-fed wild-type and CRTC2 mutant mice by IP glucose and insulin tolerance testing (GTT, ITT) (n =5,P < 0.05; SEM). (C) Effect of high-fat diet (HFD) feeding on weight gain, circulating insulin concentrations, and whole-body insulin sensitivity (GTT, ITT) of wild-type and CRTC2 mutant mice (n =5,P < 0.05; SEM). (D) Quantitative PCR analysis of gluconeogenic gene expression in wild-type and CRTC2 mutant mice under NC or HFD conditions (n =5,P < 0.05; SEM).

domain of CRTC2 may increase CREB occupancy by stabilizing generate chimeric mice. Heterozygous mice were backcrossed with C57BL/ its secondary structure. Future studies should reveal whether 6J for 2 generations and then intercrossed (het × het) to obtain homo- − − CRTC2 mediates these effects on specific target gene subsets, zygous CRTC2 / mice confirmed by PCR-based genotyping. Experiments − − depending on the nucleotide sequence or location of CREB were performed with CRTC2 / mice and their CRTC2 +/+ littermates. binding sites on cAMP responsive promoters. Genotyping of the wild-type CRTC2 allele was performed using primer pair: 5′-gctgggtatattgggacagg-3′ and 5′-gccagaggccacttgtgtag-3′,which Materials and Methods generates a 391-bp product. Primers for detection of the disrupted CRTC2 Mice. Mice were housed in a temperature-controlled environment under allele were 5′-ctgggaagcaagaaaccaag-3′ and 5′-gccagaggccacttgtgtag-3′, 12-h light/dark cycle conditions with free access to water and standard which gave rise to a 222-bp product. chow diet (Lab diet 5001). For high-fat diet feeding experiments, standard chow was replaced with chow containing 60% calories from fat. To obtain Live Imaging. Mice were imaged as described (15). Mice were injected − − / − CRTC2 mice, the CRTC2 targeting vector was constructed by replacing intraperitoneally with glucagon (100 mg·kg 1; Sigma) or vehicle. Before − sequences in exon 1 of the CRTC2 gene with a phosphoglycerol kinase imaging, mice were injected intraperitoneally with 50 mg·kg 1 Nembutal (pgk) neomycin cassette for positive selection and a pgk-diphtheria toxin- − (Abbott Laboratories) and 100 mg·kg 1 firefly D-luciferin (Biosynth AG). A cassette for negative selection. The CRTC2 targeting vector was line- Mice were imaged on the IVIS 100 Imaging System (Boston Scientific) and arized and electroporated into R1 embryonic stem cells, and G418-resist- ant clones were screened for homologous recombination by Southern blot analyzed with Living Image software (Xenogen) 1 h after glucagon analysis. Targeted clones were injected into 129-derived blastocysts to injection.

3090 | www.pnas.org/cgi/doi/10.1073/pnas.0914897107 Wang et al. Downloaded by guest on September 26, 2021 PAGE analysis. Blood glucose values were determined using a LifeScan automatic glucometer. Glucose tolerance tests (GTT) were performed by − glucose i.p.administration (1 g·kg 1) after overnight fasting. Insulin tolerance − tests were performed by i.p. injection of human regular insulin (1 U·kg 1) after 5 h fasting. For pyruvate challenge experiments, mice were fasted overnight − and injected i.p. with pyruvate (2 g·kg 1). Serum triglycerides and ketones were evaluated by cardio-check analyzer. Serum cholesterol (Biovison), insulin (Mercodia), and leptin (Millipore) levels were measured according to the manufacturer’s instructions.

Immunoblot, Immunostaining, and Gel Shift Analysis. Immunoblot and immnuostaining assays were performed as previously described (15). Recombinant CREB and GST-CRTC2 (aa 1–120) were isolated as described previously (15, 16). CRTC2 antiserum was developed against residues 454– 607 of mouse CRTC2 (8). For gel shift assays, binding reactions were in buffer containing 12 mM Hepes, pH 7.9, 12% glycerol, 1 mM EDTA, 75 mM −1 −1 KCl, 5 mM MgCl2, with 0.5 mg·ml BSA, 2 mM TCEP, 25 μg·ml poly dI:dC, and protease inhibitors. Proteins were incubated with 32P end-labeled somatostatin promoter duplex oligo for 30 min at room temperature before electrophoresis on nondenaturing 4% polyacrylamide gels with 2.5% glycerol in TBE buffer.

Cell Culture, Luciferase Activity, and Glucose Output. HEK293T (ATCC) cells were cultured in DMEM containing 10% FBS (HyClone), 100 mg·ml−1 pen- icillin–streptomycin. Mouse primary hepatocytes were isolated and cultured as previously described (15). Transient assays were performed as previously described using adenoviral (Ad-G6Pase-luc) reporters for primary hepatocytes and plasmid based reporters (EVX-luc) for HEK293T cells (4, 6, 10). Cells were exposed to glucagon (100 nm) or FSK (10 μM) for 4 h, and luciferase activities were normalized to β-galactosidase activity from adenoviral or plasmid-encoded RSV β-gal. Glucose output from primary hepatocytes was determined enzymatically, after 1-h collection in glucose-free M199 media, supplemented with 10 mM lactate and 1 mM pyruvate (15).

Chromatin Immunoprecipitation and Quantitative PCR. HEK293T cells or mouse primary hepatocytes were treated with forskolin (10 μM) or glucagon (100 nM). Cells were cross-linked on the plates with 0.75% form-

aldehyde and chromatin prepared essentially as described (8). For CREB and MEDICAL SCIENCES CRTC2 IPs, rabbit polyclonal antibody raised against respective antigens (11) was used along with rabbit IgG for negative controls. After removing crosslinks, DNA was extracted using phenol-chloroform, and CREB-target promoters were quantified using SYBR green real-time PCR. All signals were normalized to input chromatin signals. For quantitative PCR to test Fig. 4. CRTC2 increases CREB occupancy over gluconeogenic genes. (A)Q-PCR gene expression, total cellular RNAs from whole liver or primary hep- − − analysis of gluconeogenic gene expression in wild-type or CRTC2 / primary atocytes were extracted using the RNeasy kit (Qiagen). mRNA levels were hepatocytes under basal conditions and following exposure to glucagon (n =3,P measured as previously described (15). < 0.05; SEM). Effect of adenoviral CRTC2 or green fluorescent protein (GFP) expression shown. (B and C) ChIP assays of CRTC2 (B) and CREB (C) occupancy Statistical Analyses. All studies were performed on at least three independent −/− over G6Pase and PEPCK promoters in wild-type or CRTC2 hepatocytes (n =3,P experiments. Results are reported as mean and SEM. The comparison of < 0.05; SEM). Exposure to glucagon indicated. Effect of adenoviral CRTC2 different groups was carried out using a two-tailed unpaired Student’s t test. expression relative to control (Ad-GFP) shown. (D) Model for activation of the Differences were considered statistically significant at P < 0.05. gluconeogenic program by CREB and CRTC2 during fasting. Increases in circulating glucagon trigger CRTC2 dephosphorylation and nuclear entry. Binding of ACKNOWLEDGMENTS. We thank members of the Montminy laboratory for nuclear CRTC2 to CREB increases CREB binding to relevant promoters. helpful discussions. This work was supported by National Institutes of Health Grants R01-DK083834 and R01-DK049777, by the Clayton Foundation Laboratories for Medical Research, and by the Helmsley Foundation. M.M. is supported by the Keickhefer foundation; K.V. is supported by the In Vivo Analysis. Mouse tissues were sonicated and centrifuged, and super- Department of Biomedicine, University of Bergen; and Y.W. is supported natants were reserved for β-gal activity, protein determinations, and SDS– by a mentor-based fellowship from the American Diabetes Association.

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