Derivation of CRTC2-/- Mice
An 18.7kb DNA fragment containing all CRTC2 coding sequences was retrieved from C57BL/6J mouse BAC clone RP23-237D16 via bacterial recombination (Copeland et al., 2001
) into plasmid PL253 that contains an Mc1-driven Thymidine Kinase (TK) cassette for negative selection. One loxP site was inserted upstream of exon 3 while a pair of loxP sites flanking a neomycin selection cassette were placed downstream of exon 11, also by bacterial recombination. The linearized targeting vector was electroporated into B6 ES cells (Chemicon) and clones that survived G418 and gancyclovir selection were screened for homologous recombination by Southern blot analysis. Targeted clones were injected into C57BL/6J-derived blastocysts that were then transferred to pseudopregnant females. Male offspring were mated to C57BL/6J females and ES cell-derived offspring were identified by PCR-based genotyping. Mice harboring the targeted insertion of the three-loxP sites in the CRTC2 gene locus were then crossed to the EIIa-Cre line to achieve germline deletion of loxP-flanked sequences (Holzenberger et al., 2000
). Mice heterozygous for the CRTC2 deletion (CRTC2+/-
) were then mated to C57BL/6J to segregate away the EIIa-Cre transgene. CRTC2+/-
mice were then inter-crossed to obtain CRTC2-/-
animals. Genotyping of all offspring was performed by PCR using 5′ and 3′ primers for CRTC2 (5′-GCCTGATTTTTCCCCCTATT-3′ and 5′-CATCTCAATGAAGGCCACCT-3′), which yield a 431bp product from the wild-type allele and a 608bp product from the CRTC2 null allele. All PCR was performed for 35 cycles under the following conditions (95°C, 30sec; 60°C, 30sec; 72°C, 60sec).
Plasma Metabolic Profile Analysis
Whole venous blood collected from the inferior vena cava of CRTC2-/- and control mice was applied to plasma separator tubes with lithium heparin anticoagulant (BD Biosciences), centrifuged for 2 min at 14,000 × g, and the plasma was collected. Metabolic parameters were determined by Ani Lytics, Inc. (Gaithersburg, Md.).
Whole Cell Lysate Preparation and Western Blot Analysis
Liver fragments from CRTC2-/- and control mice were homogenized by hand in 200μL of lysis buffer containing 50mM Tris (pH8.0), 150mM NaCl, 1mM EDTA, 1% Triton-X100, 0.5% deoxycholic acid, and 1% SDS, supplemented with protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktails 1 and 2 (Sigma), then sonicated using a Bioruptor (Diagenode). Whole-cell lysates were then centrifuged at maximum speed for 15 minutes to pellet cellular debris, and the supernatant was collected. Protein concentrations were determined by Bradford assay using Protein Assay Reagent (Bio-Rad). Approximately 50μg of whole-cell lysates were separated by SDS-PAGE and transferred to Immobilon-P membranes (Millipore). Membranes were blocked in buffer consisting of 5% milk and 0.1% Tween-20 in 1X PBS (PBST-milk) for 4 hrs, then incubated overnight at 4°C with anti-CRTC2 (Calbiochem ST1099) diluted 1:5000, anti-FOXO1 (Cell Signaling C29H4) diluted 1:1000, anti-CREB (Cell Signaling 48H2) diluted 1:1000, anti-phoshpo-CREB (Cell Signaling 87G3) diluted 1:1000, anti-HNF4α (R&D Systems H6939) diluted 1:1000, anti-STAT3 (Cell Signaling 9132) diluted 1:1000, anti-C/EBPα (Santa Cruz sc-61) diluted 1:500, anti-C/EBPβ (Santa Cruz sc-150) diluted 1:500, or anti-βActin (Cell Signaling 13E5) diluted 1:2000 in PBST-milk. After washing, membranes were incubated with HRP-conjugated goat-anti-rabbit IgG (Amersham) diluted 1:12,500 in PBST-milk for 45 minutes at room temperature. After washing, proteins were visualized using the ECL-plus Western Blotting Detection System (Amersham).
Determination of Blood Glucose Levels and Pyruvate Challenge
Blood was sampled from the tail vein of mice allowed to feed ad libitum (fed) or fasted for 6, 9, 12, or 24 hours. Glucose levels were measured with a OneTouch Ultra Glucometer (Lifescan). For gene expression analyses of re-fed animals, mice were fasted overnight and then allowed to feed for two hours before sacrifice. For pyruvate challenge experiments, animals were fasted overnight, and a blood glucose measurement was taken and considered time zero. Mice were then injected intraperitoneally with 2g of pyruvate (Sigma-Aldrich) per kg of body weight. Glucose levels were then measured at 15, 30, 60, 90, and 120 min postinjection.
Glucose homeostasis was measured in 12-15 week old male control and CRTC2-/-
mice using insulin clamp and radioisotopic tracer techniques (Alenghat et al., 2008
; Varela et al., 2008
). An indwelling catheter was inserted in the right internal jugular vein under sodium pentobarbital anesthesia and extended to the right atrium. Four days after recovery, the mice were fasted overnight for 16 hours. They were placed in restrainers and administered a bolus injection of 5 μCi of [3-3
H] glucose, followed by continuous intravenous infusion at 0.05 μCi/min. Baseline glucose kinetics were measured for 60 min, followed by hyperinsulinemic-hypoglycemic clamp for 120 min. A priming dose of regular insulin (16 mU/kg, Humulin; Eli Lilly, Indianapolis, IN) was given intravenously, followed by continuous infusion at 2.5 mU/kg-1
. Blood glucose was maintained at 60-70 mg/dL via a variable infusion rate of 20% glucose. The mice were euthanized and liver samples were excised, frozen immediately in liquid nitrogen and stored at −80°C. The rates of basal glucose turnover and whole body glucose uptake are measured as the ratio of the [3
H] glucose infusion rate (dpm) to the specific activity of plasma glucose. Hepatic glucose production (HGP) during clamp was measured by subtracting the glucose infusion rate (GIR) from the whole body glucose uptake (Rd).
Tissue samples from mice fed ad libitum were homogenized in 6% PCA (500μL/100μg tissue). Precipitates were pelleted and the supernatant was collected. One volume of H2O was added and the solution was adjusted to pH 6.5 with 10N KOH. A fraction of each sample was then incubated with five volumes of amyloglucosidase (1mg/mL in 0.2M, pH 4.8 acetate buffer) at 40°C for 2 hours. Glucose concentrations were then determined using the Amplex Red glucose/glucose oxidase assay (Invitrogen). Samples incubated in the absence of amyloglucosidase were used as baseline controls.
RNA Isolation and Quantitative RT-PCR Analysis
Total cellular RNA was extracted from liver fragments or cultured primary hepatocytes using RNeasy Kit (Qiagen), then assayed for quantity and quality with the Agilent 2100 Bioanalyzer (Agilent Technologies). Approximately 1μg of total RNA was reverse transcribed using oligo (dT) and Superscript II reverse transcriptase (Invitrogen). The resulting cDNA samples were the template for qPCR experiments performed with SYBR GreenER qPCR Supermix (Invitrogen) and the SYBR Green (with dissociation curve) program on the Mx3000 Multiplex Quantitative PCR System (Stratagene). Reactions were performed in triplicate and normalized relative to the ROX reference dye. Median cycle threshold values were determined and used for analyses. Expression levels were normalized to those of hypoxanthine-guanine phosphoribosyltransferase (HPRT) as the internal control. Primer information is available at http://www.med.upenn.edu/kaestnerlab/index.shtml
Primary Hepatocyte Isolation and Culture
Hepatocytes were isolated from 3-month-old mice. The vena cava was cannulated and the liver was perfused with 37°C Liver Perfusion Buffer (Gibco) for 2 min, then 50mL of 37°C Liver Digestion Buffer (Gibco) supplemented with 30mg of collagenase D (Roche). The liver was then excised, dispersed in DMEM containing 10% FBS and penicillin/streptomycin, filtered through a 100 micron mesh, pelleted, and subjected to a Percoll gradient (45% Percoll in DMEM) to isolate viable cells. After two washes in DMEM containing 10% FBS and penicillin/streptomycin, cells were plated in 12-well, collagen I-coated plates (BD BioCoat) at a density of 150k cells/well in DMEM containing 10% FBS and penicillin/streptomycin. For gene expression analysis, cells were cultured for 48 hours, then stimulated with 50nM glucagon for 2 hours before harvesting for RNA isolation. For glucose production measurements, cells were cultured for 24 hours, then switched to glucose-free medium containing 20mM sodium lactate and 2mM sodium pyruvate before stimulating for 5 hrs with 50nM glucagon. The culture medium was collected for glucose concentration determination via Amplex Red glucose/glucose oxidase assay (Invitrogen).
Chromatin Immunoprecipitation (ChIP)-Seq and Quantitative RT-PCR Analysis
Chromatin from control and CRTC2-/-
livers was prepared as previously described (Tuteja et al., 2008
). Immunoprecipitations were also performed as previously described (Tuteja et al., 2008
), except that the herring sperm DNA was excluded from the agarose bead blocking step. Sheared chromatin (10μg) was incubated overnight at 4°C with 2μg of anti-CREB (Santa Cruz sc-186X), anti-CBP (Santa Cruz sc-369X and sc-583X), or anti p300 (Santa Cruz sc-584X and sc-585X), and immune complexes were recovered with protein-G conjugated agarose beads for 45min at 4°C. Enrichment of target DNA fragments was determined via qPCR using SYBR GreenER qPCR Supermix (Invitrogen) and the SYBR Green (with dissociation curve) program on the Mx3000 Multiplex Quantitative PCR System (Stratagene). qPCR experiments were performed in triplicate on both input (sheared genomic DNA) and ChIP sample DNA, and median cycle threshold values were used for analysis. Enrichment of bound regions was calculated using the myelin basic protein (MBP) promoter as a reference for nonspecific binding, and comparing to amplification values obtained from input DNA.
For ChIP-Seq experiments, the remainder of the immunoprecipitated DNA was modified for sequencing following the manufacturer’s protocol (Illumina). Briefly, DNA samples were blunted with a combination of T4 DNA polymerase, Klenow polymerase, and T4 PNK, then a single 3′-end “A” base was added using Klenow exo (3′ to 5′ exo minus). Adapters provided by Illumina were then ligated to the ends of the modified DNA before size selection of ~200bp fragments via PAGE extraction. The isolated DNA was then amplified by 18 cycles of PCR as described. PCR products were column purified with the QIAquick PCR Purification Kit (Qiagen) and assayed for quantity and quality with the Agilent 2100 Bioanalyzer (Agilent Technologies), then diluted to a concentration of 10nM. The library was sequenced on an Illumina 1G Genome Analyzer, at a concentration of 3-4pM. The sequencing output was analyzed using the Genome Analyzer pipeline provided by Illumina. Sequence tags that aligned uniquely to the mouse genome build MM8 with zero, one, or two mismatches, according to the ELAND alignment algorithm, were used for further analysis.