Generation of knock-in mice
We amplified the Foxo1
locus around exon 2 from 129/SvEv DNA using Phusion polymerase (Finnzymes), and replaced the EcoR
III fragment with sequences encoding either FoxO1-KR or FoxO1-KQ (Kitamura et al., 2005
). We selected homologous recombinants using a floxed pGK-neomycin cassette inserted 1 kb upstream of exon 2, and a diphtheria toxin-A cassette and detection by Southern blotting of ES cell DNA digested with Kpn
I with a probe external to the targeting vector. ES cell manipulations were as described (Accili et al., 1996
), and were followed by intercrosses with ROSA26
-cre transgenics to delete the neomycin cassette prior to breeding to homozygosity. PCR genotyping primers are: 5′-GCACCTTCAGTCGCCGTCAA and 5′-CCACAGGAGAATACAAGAGGAAGGC. The wild-type allele is 394-bp and the knock-in allele is 448 bp. Animals were backcrossed onto C57BL/6Jdb/+
, JAX stock 000697] for ten generations prior to diet studies and indirect calorimetry.
Adenovirus Generation and Cell Culture
We generated adenoviruses encoding WT-, KR- or KQ-FoxO1-GFP using AdEasy (Stratagene) and obtained primary hepatocytes as described (Banks et al., 2008
). We carried out adenovirus transduction 4 hr after isolation.
Mice and Embryo collection
We collected embryos from timed matings between Foxo1KQ/+ mice, considering 12 PM on the day of appearance of a vaginal plug embryonic day (E) 0.5. We assessed litters at E9.5, E10.5, E11.5, and postnatal day (P) 12, and carried out embryo dissections in cold PBS, using a dissecting microscope (Leica MZ8). We fixed embryos in 4% paraformaldehyde (PFA), washed them three times in PBS, 1:1 PBS/MeOH, and 100% MeOH, and performed immunostaining. We genotyped embryos using yolk sac specimens.
We carried out wholemount immunohistochemistry with rat anti-mouse CD31 and platelet endothelial cell adhesion molecule-1 (PECAM-1) antibodies (BD Biosciences). Briefly, after bleaching with MeOH:DMSO:H2O2 (4:1:1) for 5-hr, we rehydrated embryos with 50% MeOH in PBT (PBS, 0.2% BSA, 0.5% Triton-X), followed by one wash in PBT and five washes in PBSMT (PBS, 2% non-fat milk powder, 0.5% Triton-X). We incubated embryos with primary antibody in PBSMT, washed and incubated them with horseradish peroxidase-conjugated antibody (sc-2005, Santa Cruz). We performed DAB staining in PBT containing 0.08% NaCl, 0.25 mg/ml DAB, and 0.03% H2O2 for 20–60 min, followed by fixation in 4% PFA, or fluorescent staining with Cy3-donkey anti-rat secondary antibody (Jackson Immunoresearch). We imaged embryos using a Nikon SMZ1500 microscope or a Zeiss LSM5 Exciter confocal microscope.
For H&E histological analysis, we embedded embryos in OCT, after fixation with 4% PFA and cryopreservation in 30% sucrose. We obtained transverse frozen sections (8 μm) using a cryostat (Microm HM525, Thermo Scientific), and imaged them using a Leica CTR6500 microscope.
Metabolic characterization and energy balance studies
We measured blood or plasma glucose using a glucose monitor (One Touch Ultra, Lifescan); insulin, leptin, resistin, and adiponectin (Linco/Millipore) and TNF–α (BD Biosciences) by ELISA, plasma triglyceride (Infinity), cholesterol and non-esterified fatty acids by colorimetric assays (Cholesterol E and NEFA C, Wako Pure Chemicals). Glucose, insulin, and pyruvate tolerance tests have been described (Matsumoto et al., 2007
; Nakae et al., 2002
). We collected blood between 10:00 AM and 12:00 PM to reduce variability. For lipoprotein separation, we pooled serum samples of six mice per genotype and applied them to FPLC gel filtration on two Superose 6 columns in series (Amersham Bioscience). We collected eluates in 0.5ml fractions at a flow rate of 0.7 ml/min. We assayed TG and cholesterol from column eluates and from liver extracts as described (Haeusler et al., 2010
). To measure hepatic glycogen content, we homogenized frozen liver in 6% perchloric acid, adjusted to pH 6–7 with KOH followed by incubation with 1 mg/ml amyloglucosidase (Sigma) in 0.2 M acetate (pH 4.8) and quantification of glucose released (glycogen breakdown value – PCA value). We carried out hyperinsulinemic-euglycemic clamp studies as described (Lin et al., 2010
). We performed indirect calorimetry using the Oxymax Comprehensive Lab Animal Monitoring System (Columbus Instruments) and measured body composition with either Piximus DEXA scanner (GE Healthcare) or NMR (Bruker Optics) (Banks et al., 2008
RNA and protein analysis
We used standard RNA extraction and real-time RT-PCR and western blotting procedures (Matsumoto et al., 2006
). Primer sequences are available on request. Anti-acetylated-FoxO1 antibody was from Santa Cruz Biotechnology (#sc-49437).
All results are presented as mean ± SEM. We calculate P values by unpaired Student’s t – tests or 2-way ANOVA.