FCoR inhibits Foxo1 transcriptional activity. FCoR inhibits CN Foxo1-induced IGFBP1 (A) and G6PC (B) promoter activity. After transient transfection with IGFBP1/luciferase (p925GL3) (A) or a G6Pase/luciferase reporter vector (PicaGene/human G6Pase promoter-luciferase) (B), SV40-transformed hepatocytes were infected with the indicated adenovirus. phRL-SV40 was used as an internal control for transfection efficiency. After overnight serum deprivation and induction with dexamethasone/8-Br-cAMP/IBMX, cells were harvested and luciferase activity was measured. Single and double asterisks indicate statistically significant difference between luciferase activity in the absence and the presence of FCoR (*P<0.005 and **P<0.05, respectively, by one-way ANOVA). Data represent the mean±s.e.m. from three independent experiments. (C) FCoR inhibits Foxo1-induced 5XGAL4-luciferase activity. After transient transfection, HEK293 cells were stimulated with forskolin (20 μM) for 6 h, harvested, and luciferase activity was measured. An asterisk indicates a statistically significant difference between luciferase activity in the absence and presence of FCoR (*P<0.02 by one-way ANOVA). Data represent the mean±s.e.m. from three independent experiments. (D) Effect of overexpression of FCoR on endogenous Igfbp1 gene expression. SV40-transformed hepatocytes were transduced with adenovirus encoding FLAG-CN Foxo1. After 2 h, cells were transduced again with adenovirus encoding cMyc-FCoR. After 36 h, cells were incubated with dexamethasone/cAMP/IBMX for 8 h and harvested. Total RNA was isolated from cells and subjected to real-time PCR to analyse the Igfbp1 and β-actin levels. Data were corrected using the β-actin expression level and then represented as relative mRNA abundance. Data represent the mean values±s.e.m. from three independent experiments. An asterisk indicates a statistically significant difference between endogenous Igfbp1 expression induced by FLAG-CN Foxo1 in the absence and presence of cMyc-FCoR (*P<0.001 by one-way ANOVA). (E) Interaction between Foxo1, Foxo3a or FOXO4 and FCoR. HEK293 cells were co-transfected with pCMV5-cMyc-Foxo1, pCMV5-cMyc-Foxo3a or pCMV5/cMyc-FOXO4 and pFLAG-CMV2-WT FCoR and immunoprecipitated with anti-FLAG, anti-cMyc or normal mouse IgG and blotted with anti-cMyc or anti-FLAG antibody. (F) FCoR inhibits Foxo1-induced and Foxo3a-induced, but not FOXO4-induced 5XGAL4-luciferase activity. After transient transfection with FCoR, HEK293 cells were stimulated with forskolin (20 μM) for 6 h, harvested, and luciferase activity was measured. The grey bar indicates mock-transfected cells and the blue bar indicates pCMV5-cMyc-WT FCoR-transfected cells. An asterisk indicates a statistically significant difference between luciferase activity in the absence and presence of FCoR (*P<0.01 by one-way ANOVA). Data represent the mean±s.e.m. from three independent experiments. Figure source data can be found with the Supplementary data.

FCoR is phosphorylated and translocated into the nucleus in a PKA-dependent manner. (A) Subcellular localization of exogenous FCoR in HEK293 cells. At 36 h after transfection with pCMV5-cMyc-WT FCoR, HEK293 cells were stimulated with forskolin (20 μM) for 30 min, fixed, and stained with anti-cMyc mouse monoclonal antibody. (B) Western blotting of cytosolic and nuclear extracts from HEK293 cells transfected with cMyc-FCoR. At 36 h after transfection with pCMV5-cMyc-WT FCoR, HEK293 cells were stimulated with forskolin (20 μM) at the indicated time and harvested. Lysates were fractionated into cytosolic and nuclear extracts and subjected to western blotting with the indicated antibodies. (C) Immunofluorescence of endogenous FCoR in 3T3-F442A cells. 3T3-F442A cells were cultured and induced differentiation into mature adipocytes as described in Materials and Methods except supplementation with 500 nM of IBMX for 48 h. Thereafter, cells were cultured in DMEM containing 10% fetal calf serum and 1.7 μM of insulin. Cells were fixed at the indicated days and stained with anti-FCoR rabbit polyclonal antibody. (D) Phosphorylation of FCoR by forskolin. After transfection with pCMV5-cMyc-WT FCoR, HEK293 cells were stimulated with forskolin (20 μM) at the indicated time and harvested. Cell lysates were immunoprecipitated with anti-cMyc mouse monoclonal antibody, subjected to western blotting, and phosphorylated FCoR was detected. (E) Inhibition of phosphorylation of FCoR by H89. At 48 h after transient transfection with pCMV5-cMyc-WT FCoR, HEK293 cells were incubated with or without H89 (1 μM) for 3 h and stimulated with forskolin (20 μM) for 15 min and harvested. Phosphorylated FCoR was detected as described above. (F) Phosphorylation of mutant FCoR. After transfection with pCMV5-cMyc-WT (lanes 1 and 2), S92A (lanes 3 and 4), T93A (lanes 5 and 6), or S92A/T93A (lanes 7 and 8) FCoR, HEK293 cells were stimulated with forskolin (20 μM) for 15 min. Phosphorylated FCoR was detected as described above. (G) Detection of phosphorylated Threonine 93 by anti-phospho-T93-specific (anti-pT93) antibody. At 48 h after transfection with pCMV5-cMyc-WT FCoR, HEK293 cells were incubated with or without H89 (1 μM) for 3 h and stimulated with or without forskolin (20 μM) for 15 min and harvested. Lysates were subjected to western blotting with anti-pT93. (H) In-vitro phosphorylation assay of FCoR. GST, GST-WT, GST-T93A, GST-T93D FCoR, and GST-RRAS, which is a short sequence of salt-inducible kinase (SIK) 2, was phosphorylated in vitro by incubating with a catalytic subunit from cAMP-dependent protein kinase as described in ‘Materials and methods'. Reaction products were subjected to western blotting, phosphorylated products were detected using Phos-tag^R BTL-104 (top panel) and then blotted with anti-GST antibody (bottom panel). (I) Subcellular localization of exogenous T93A (the top panel) or T93D FCoR (the bottom panel) in HEK293 cells. At 36 h after transfection with pCMV5-cMyc-T93A or T93D FCoR, HEK293 cells were fixed as described in Materials and methods and stained with anti-cMyc mouse monoclonal antibody. (J) Western blotting of cytosolic and nuclear extracts from HEK293 cells transfected with cMyc-WT (lanes 1 and 2), T93D (lanes 3 and 4), or T93A (lanes 5 and 6) FCoR. At 36 h after transfection, HEK293 cells were harvested. Lysates were fractionated into cytosolic and nuclear extracts and subjected to western blotting with the indicated antibodies. (K) 5XGAL4-luciferase assay of PM-Foxo1 with pCMV5/cMyc-WT, T93A, and T93D FCoR. At 36 h after transfection with pTAL-5XGAL4, phRL-SV40, and the indicated PM-Foxo1 with or without the FCoR expression vector, HEK293 cells were incubated with forskolin (20 μM) for 6 h and harvested. Luciferase activity was measured in the lysates. Data represent the mean values±s.e.m. from three independent experiments. Asterisks indicate statistically significant difference (*P<0.001, **P<0.005, and ***P<0.05 by one-way ANOVA). (L) The T93D FCoR mutant acetylates Foxo1 the most. After transfection with pFLAG-CMV2-WT Foxo1 with or without pCMV5-cMyc-WT (lane 4), T93A (lane 5), or T93D (lane 6) FCoR, HEK293 cells were incubated with (lanes 3–6) or without H₂O₂ (lanes 1 and 2) (500 μM), nicotinamide (NAM) (50 mM), and trichostatin A (TSA) (2 μM) for 3 h and harvested. Lysates were immunoprecipitated with anti-FLAG mouse monoclonal antibody (M2) and subjected to western blotting with the indicated antibodies. (M) Interaction between Foxo1 and FCoR. HEK293 cells were co-transfected with pCMV5-cMyc-Foxo1 and pFLAG-CMV2-WTFCoR and culture at the indicated condition. Cell lysates were immunoprecipitated with anti-FLAG, anti-cMyc or normal mouse IgG and blotted with anti-cMyc or anti-FLAG antibody. Figure source data can be found with the Supplementary data.

Generation of transgenic mice expressing wild-type FCoR driven by the aP2 promoter. (A) Diagram of the transgenic construct. In addition to 5.4 kb of the aP2 promoter, the construct contains the cMyc-WT Fcor cDNA and the human GH polyadenylation sequences. (B) Tissue survey of transgene expression in WAT (lanes 1 and 6), BAT (lanes 2 and 7), liver (lanes 3 and 8), skeletal muscle (lanes 4 and 9), and hypothalamus (lanes 5 and 10) in line N5 (lanes 1–5) and line 1 (lanes 6–10). Total RNA was isolated from the tissues indicated at the bottom of the autoradiogram and analysed by northern blotting using Fcor or β-actin cDNA probe. (C) Real-time PCR of transgene expression in WAT and BAT from transgenic lines. Data represent the mean values±s.e.m. of RNA samples from each of five transgenic mice for each tissue. The grey, sky blue, and red bars indicate wild-type, aP2-FCoR-1 (WFCoR), and aP2-FCoR-N5 (BFCoR), respectively. (D) Western blotting of transgenic cMyc-FCoR expression in WAT (lanes 1–3) and BAT (lanes 4–6). Lysates from the tissue of the indicated types of mice were subjected to western blotting with the indicated antibodies. (E) Body weight of wild-type and transgenic mice (WFCoR) fed a normal chow diet. Data represent the mean values±s.e.m. of 15–20 male mice for each genotype. The open and closed circles indicate wild-type and WFCoR mice, respectively. An asterisk indicates a statistically significant difference between wild-type and WFCoR (*P<0.01 by one-way ANOVA). (F) Adiposity of 5-month-old WFCoR transgenic mice fed a normal chow diet. Skeletal muscle mass, adipose tissue mass (sum of visceral and subcutaneous fat mass), visceral fat mass, and subcutaneous fat mass were calculated. The data are reported as percentage of body weight and represent the mean±s.e.m. of five mice of each genotype. Asterisks indicate statistically significant differences between wild-type and WFCoR mice (*P<0.02 and **P<0.05 by one-way ANOVA). (G) Average of size of adipocytes from epididymal fat of 5-month-old wild-type (grey bar) and WFCoR (sky blue bar) mice fed a normal chow diet. An asterisk indicates a statistically significant difference between wild-type and WFCoR mice (*P<0.001 by one-way ANOVA). (H) Food intake, oxygen consumption, and respiratory quotient of 5-month-old wild-type (grey bar) and WFCoR (sky blue bar) mice fed a normal chow diet. An asterisk indicates a statistically significant difference between wild-type and WFCoR mice (*P<0.001 by one-way ANOVA). (I) Intraperitoneal glucose tolerance test in wild-type (n=10) and WFCoR transgenic mice (n=10) fed a normal chow diet. The open and closed circles indicate wild-type and WFCoR mice, respectively. Data represent the mean values±s.e.m. Asterisks indicate statistically significant differences between wild-type and WFCoR mice (*P<0.005 and **P<0.02 by one-way ANOVA). (J) Intraperitoneal insulin tolerance test in wild-type (n=8) and WFCoR (n=10) mice fed a normal chow diet. Data represent the mean values±s.e.m. Asterisks indicate statistically significant differences between wild-type and WFCoR mice (*P<0.005 and **P<0.05 by one-way ANOVA). (K) Real-time PCR of WAT-specific genes using WAT from wild-type (grey bar) and WFCoR transgenic mice (sky blue bar) fed a normal chow diet. Data represent the mean values±s.e.m. of RNA samples from five mice of each genotype. An asterisk indicates a statistically significant difference between wild-type and WFCoR transgenic mice (*P<0.05 by one-way ANOVA). (L) Real-time PCR of Ccl2, Emr1, and Ccr2 genes using WAT from wild-type (grey bar) and WFCoR transgenic mice (sky blue bar) fed a normal chow diet. Data represent the mean values±s.e.m. of RNA samples from five mice of each genotype. Asterisks indicate statistically significant differences between wild-type and WFCoR transgenic mice (*P<0.001 and **P<0.005 by one-way ANOVA). (M) Real-time PCR of Foxo1-target genes using WAT from wild-type (grey bar) and WFCoR transgenic mice (sky blue bar). Data represent the mean values±s.e.m. of RNA samples from five mice of each genotype. Asterisks indicate statistically significant differences between wild-type and WFCoR transgenic mice (*P<0.001 and **P<0.005 by one-way ANOVA). (N) Acetylation of endogenous Foxo1 in WAT from 5-month-old wild-type (lane 1) and WFCoR transgenic mice (lane 2). Tissue lysates containing nicotinamide (NAM) (50 mM) and trichostatin A (TSA) (2 μM) were immunoprecipitated with anti-FOXO1 antibody and blotted with the indicated antibodies.

Deletion of Fcor induces insulin resistance. (A) Body weight of wild-type and FcorKO mice fed a normal chow diet. Data represent the mean values±s.e.m. of 20–30 male mice for each genotype. The open and yellow closed circles indicate wild-type and FcorKO mice, respectively. An asterisk indicates statistically significant difference between wild-type and FcorKO (*P<0.01 by one-way ANOVA). (B) Intraperitoneal glucose tolerance test in wild-type (n=10) and FcorKO mice (n=10) under normal chow diet. The open and yellow closed circles indicate wild-type and FcorKO mice, respectively. Data represent mean values±s.e.m. Asterisks indicate statistically significant differences between wild-type and FcorKO mice (*P<0.001 and **P<0.02 by one-way ANOVA). (C) Intraperitoneal insulin tolerance test in wild-type (n=10) and FcorKO (n=10) fed a normal chow diet. Data represent the mean values±s.e.m. Asterisks indicate statistically significant differences between wild-type and FcorKO mice (*P<0.005 and **P<0.05 by one-way ANOVA). (D) Distribution of adipocyte size. The sizes of single adipocytes from 5-month-old wild-type (grey bar) or FcorKO (yellow bar) mice fed a normal chow diet were measured as described in ‘Supplementary Experimental Procedures'. The data represent the mean values±s.e.m. of the percentage of all adipocytes with the indicated size. Asterisks indicate statistically significant differences between wild-type and FcorKO mice (*P<0.05 by one-way ANOVA). (E) The average of size of adipocytes from (D). An asterisk indicates a statistically significant difference between wild-type and FcorKO mice (*P<0.005 by one-way ANOVA). (F, G) Real-time PCR of Foxo1-target genes using WAT (F) and BAT (G) from wild-type (grey bar) and FcorKO (yellow bar) mice. Data represent the mean±s.e.m. of RNA samples from 10 mice of each genotype. Asterisks indicate statistically significant differences between wild-type and WFCoR transgenic mice (*P<0.05 by one-way ANOVA). (H) Real-time PCR of WAT-specific genes using WAT from wild-type (grey bar) and FcorKO (yellow bar) mice fed a normal chow diet. Data represent the mean values±s.e.m. of RNA samples from five mice of each genotype. (I) Real-time PCR of Ccl2, Emr1, and Ccr2 genes using WAT from wild-type (grey bar) and FcorKO (yellow bar) mice. Data represent the mean values±s.e.m. of RNA samples from five mice of each genotype. Asterisks indicate statistically significant differences between wild-type and FcorKO mice (*P<0.05 by one-way ANOVA). (J) Food intake of 5-month-old wild-type (grey bar) and FcorKO (yellow bar) mice. Data represent the mean values±s.e.m. of food intake for 4 days. (K, L) The effects of deletion of Fcor on energy expenditure. Average oxygen consumption (K) and respiratory quotient (L) of 5-month-old wild-type (grey bar) and FcorKO (yellow bar) mice fed a normal diet. Data represent the mean values±s.e.m. of six male mice for each genotype. Measurements of oxygen consumption were performed for 72 h after allowing the mice to acclimate to the cage environment. An asterisk indicates a statistically significant difference between wild-type and FcorKO mice (*P<0.05 by one-way ANOVA). (M) Western blotting of PGC-1α (top panel) and tubulin (bottom panel) in BAT from wild-type (lanes 1–3) and FcorKO (lanes 4–7) mice. (N) Real-time PCR of Ppargc1a and Ucp1 using BAT from wild-type (grey bar) and FcorKO (yellow bar) mice. Data represent the mean values±s.e.m. of RNA samples from 10 mice of each genotype. Asterisks indicate statistically significant differences between wild-type and WFCoR transgenic mice (*P<0.05 by one-way ANOVA). Figure source data can be found with the Supplementary data.

Interaction between FCoR and Foxos and expression profiling of FCoR. (A) In-vitro translation of FCoR. Lysates from liver (lane 1), WAT (lane 2), and BAT (lane 3) from wild-type mice, along with in vitro-translated FCoR were analysed by western blotting using anti-mouse FCoR antiserum. (B) Interaction between exogenous FCoR and Foxo1. HEK293 cells were co-transfected with pFLAG-CMV2-WT Foxo1 or pFLAG-CMV2-Δ256 Foxo1 plus pCMV5-cMyc-WT FCoR and cultured in the presence of serum. At 48 h after transfection, cells were harvested and lysates were immunoprecipitated with anti-cMyc (lanes 1 and 5), anti-FLAG antibody (lanes 3 and 7), or normal mouse IgG (lanes 2, 4, 6, and 8) and blotted with anti-FLAG (lanes 1, 2, 5, and 6) or anti-cMyc antibody (lanes 3, 4, 7, and 8). (C) Interaction between endogenous Foxo1 and FCoR in WAT and BAT. Lysates from WAT (lanes 1, 2, 5, and 6) and BAT (lanes 3, 4, 7, and 8) were immunoprecipitated with anti-FOXO1 (lanes 1 and 3), anti-FCoR (lanes 5 and 6), or normal rabbit IgG (lanes 2, 4, 6, and 8) and blotted with anti-FCoR (lanes 1–4) or anti-FOXO1 (lanes 5–8), respectively. (D) Direct interaction of FCoR with Foxo1. GST-FCoR was subjected to a pull-down assay. Aliquots of in vitro-translated WT Foxo1 were incubated with glutathione-Sepharose beads coated with bacterially expressed GST-P13 (lane 2) or GST alone (lane 3) for 6 h at 4°C. The in vitro-translated Foxo1 proteins retained on the column were eluted and separated by SDS–PAGE followed by western blotting with anti-FOXO1 antibody. The bottom panel shows GST or GST-GST-P13 (10% of input, lane 3) blotted with anti-GST antibody. (E) Expression profiling of Fcor in various tissues. Total RNA isolated from WAT (lane 1), BAT (lane 2), liver (lane 3), skeletal muscle (lane 4), and whole brain (lane 5) of wild-type mice was subjected to northern blotting with Fcor (top panel) or β-actin (bottom panel). (F) Real-time PCR of Fcor using the adipocyte or stromal vascular fractions of fractionated WAT. (G) Northern blotting of 3T3-F442A cells during differentiation. Total RNA isolated from 3T3-F442A cells on the indicated day after induction of differentiation was subjected to Northern blotting with Fcor (top panel) or β-actin (bottom panel). (H) Western blotting of FCoR protein from 3T3-F442A cells during differentiation. Lysates from 3T3-F442A cells on the indicated day after induction of differentiation were subjected to western blotting using anti-FCoR polyclonal antiserum (top panel) or anti-tubulin monoclonal antibody (bottom panel). (I) Effects of the feeding state on Fcor gene expression. Total RNA from liver (lanes 1–3), WAT (lanes 4–6), and BAT (lanes 7–9) from C57Bl6J mice in the fed, fasting, or refed states was subjected to northern blotting with Fcor (top panel) or β-actin (bottom panel). (J) Western blotting of the FCoR protein from WAT (lanes 1 and 2) and BAT (lanes 3 and 4) from C57Bl6J mice in fed (lanes 1 and 3) or fasting state (lanes 2 and 4). Tissue lysates were subjected to western blotting using anti-FCoR polyclonal antiserum (top panel) or anti-tubulin monoclonal antibody (bottom panel). (K) Fcor gene expression in WAT and BAT from Lepr^db/db mice. Total RNA isolated from WAT (lanes1 and 2) and BAT (lanes 3 and 4) of C57Bl6J (lanes 1 and 3) or Lepr^db/db mice (lanes 2 and 4) was subjected to northern blotting with Fcor (top panel) or β-actin (bottom panel). (L) Effect of cold exposure on Fcor gene expression in BAT. Total RNA isolated from the BAT of C57Bl6J mice exposed to the cold (4°C for 6 h) was subjected to northern blotting with Fcor (top panel) or β-actin (bottom panel). (M) Immunohistochemistry of WAT (left panel) and BAT (right panel) from C57Bl6J mice using anti-FCoR anti-sera. Scale bars indicate 20 μm.

FCoR enhances Foxo1 acetylation. (A) Effect of FCoR on Foxo1 acetylation. After transfection with pFLAG-CMV2-WT Foxo1 with or without pCMV5-cMyc-WT FCoR, HEK293 cells were incubated with H₂O₂ (500 μM), nicotinamide (NAM) (50 mM), and trichostatin A (TSA) (2 μM) for 3 h and harvested. Lysates were immunoprecipitated with anti-FLAG mouse monoclonal antibody (M2) and subjected to western blotting with the indicated antibodies. The bottom panel shows western blotting of the lysates with and anti-cMyc mouse monoclonal antibody. (B) FCoR disrupts the interaction between Foxo1 and Sirt1. After transfection with pCMV5-cMyc-WT Foxo1 and pTOPO-Sirt1 with or without pCMV5-cMyc-WT FCoR, HEK293 cells were incubated with H₂O₂ for 3 h and harvested. Cell lysates were immunoprecipitated with an anti-cMyc mouse monoclonal antibody and subjected to western blotting with the indicated antibodies. (C) 5XGAL4-luciferase assay of PM-WT, -6KQ, and -6KR Foxo1. At 36 h after transfection with pTAL-5XGAL4, phRL-SV40, and the indicated PM-Foxo1 with or without the FCoR expression vector, HEK293 cells were incubated with forskolin (20 μM) for 6 h and harvested. Luciferase activity was measured in the cell lysates. The grey bar indicates mock-transfected cells and the blue bar indicates pCMV5-cMyc-WT FCoR-transfected cells. Data represent the mean values±s.e.m. from three independent experiments. Asterisk indicates statistically significant difference (*P<0.005 by one-way ANOVA). (D) Sequence alignment of FCoR with various acetyltransferases. Residues that are identical or chemically similar to those in mouse FCoR are shown with a red background. FCoR has sequence similarity to the acetyl-CoA binding motifs of the MYST family of HATs (yeast Esa1, yeast Sas3, Drosophila MOF, human TIP60, and human P/CAF). (E) In-vitro acetylation assay of Foxo1. The GST-Foxo1-C1 (aa251–409) protein was subjected to in-vitro acetylation assays with GST (lanes 1, 4, and 5) or GST-FCoR (lanes 2 and 3) as described in ‘Materials and methods'. Reaction products were analysed by Coomassie brilliant blue staining and autoradiography (¹⁴C). (F) In-vitro acetylation assay of Foxo1. The GST-Foxo1-C1 (aa251–409) protein was subjected to in-vitro acetylation assays with GST-FCoR (lane 1), GST (lane 2), or recombinant p300 (lane 3) as described in ‘Materials and methods'. Reaction products were analysed by Coomassie brilliant blue staining and autoradiography (¹⁴C). (G) Quantification of acetylation activity of FCoR. The intensity of each acetylated band and Coomassie brilliant blue staining band was measured by NIH Image1.62. The relative intensity was corrected by the weight of GST-FCoR or recombinant p300. Data represent the mean values±s.e.m. from two independent experiments. An asterisk indicates statistically significant differences (*P<0.001 by one-way ANOVA). (H) 5XGAL4-luciferase assay of PM-Foxo1 with pCMV5/cMyc-WT, I78A, T80A, L81A, L85A, and L87A FCoR. At 36 h after transfection with pTAL-5XGAL4, phRL-SV40, and the indicated PM-Foxo1 with or without the FCoR expression vector, HEK293 cells were incubated with forskolin (20 μM) for 6 h and harvested. Luciferase activity was measured in the cell lysates. Data represent the mean values±s.e.m. from three independent experiments. Asterisks indicate statistically significant differences (*P<0.001 and **P<0.05 by one-way ANOVA). Figure source data can be found with the Supplementary data.

The effects of knockdown of FCoR on adipocyte differentiation. (A) Oil-red O staining of 3T3-F442A cells. At day 14 after induction of differentiation and transduction with adenoviruses encoding scrambled (the top panel), shRNA1 (the middle panel), and cMyc FCoR (the bottom panel), cells were stained with Oil-red O. (B) Knockdown of endogenous Fcor mRNA in 3T3-F442A cells during differentiation. 3T3-F442 cells were induced to differentiation into mature adipocytes as described in ‘Supplementary Experimental Procedures' and transduced with adenovirus encoding shRNA1 (blue bar) or scrambled shRNA (grey bar). Total RNA was isolated from cells on the indicated day and subjected to real-time PCR of Fcor and β-actin. The data were corrected using the β-actin expression level and represent the mean values±s.e.m. from two independent experiments. Asterisks indicate statistically significant differences between Fcor expression levels in scrambles- and shRNA1-transduced cells (*P<0.001 by one-way ANOVA). (C) Western blotting of endogenous FCoR protein. On day 10 after transduction with adenovirus encoding scrambled or shRNA1, 3T3-F442A cells were harvested and the lysates were subjected to western blotting with the indicated antibodies. (D) Effects of knockdown of FCoR on the expression of adipocyte-specific genes during differentiation of 3T3-F442A cells. After induction of differentiation and transduction, total RNA was isolated on the indicated days and subjected to real-time PCR of Pparg (top panel), Slc2a4 (middle panel), and Adipoq (bottom panel). The grey bar indicates scrambled-transduced cells and the blue bar indicates shRNA1-transduced cells. The data were corrected using the β-actin expression level and represent the mean values±s.e.m. from two independent experiments. Asterisks indicate statistically significant difference between scrambled- and shRNA1-transduced cells (*P<0.001, **P<0.005, and ***P<0.01 by one-way ANOVA). (E) Effects of knockdown of FCoR on the expression levels of Foxo1-target genes during differentiation of 3T3-F442A cells. Total RNA from scrambled- (grey bar) or shRNA1-transduced cells (blue bar) on the indicated days was subjected to real-time PCR of the indicated Foxo1-target genes. The data were corrected using the β-actin expression level and represent the mean values±s.e.m. from two independent experiments. Asterisks indicate statistically significant difference between scrambled- and shRNA1-transduced cells (*P<0.001 and **P<0.005 by one-way ANOVA). (F) Chromatin immuunoprecipitation (Chip) assay of the Cdkn1a promoter. The Chip assay was performed using 3T3-F442A cells during (lane 1) or at the end of clonal expansion (lane 2) with the indicated antibodies. The lower panel shows western blotting of 3T3-F442A cells lysates at the indicated period with the indicated antibodies. (G) Acetylation of endogenous Foxo1 in 3T3-F442A cells transduced with adenoviruses encoding LacZ (lanes 1–4) or cMyc-FCoR (lanes 5–8) during differentiation. After induction of differentiation and transduction, cells were harvested on the indicated day, immunoprecipitated with anti-FOXO1 antibody, and blotted with the indicated antibodies.

FCoR in BAT affects thermoregulation through suppression of Ppargc1a expression. (A) Changes in the rectal temperature of 6-month-old wild-type (open circle) and BFCoR (closed circle) mice after cold exposure. Data represent the mean values±s.e.m. (n=6 for each genotype). Asterisks indicate statistically significant differences between wild-type and BFCoR transgenic mice (*P<0.05 by one-way ANOVA). (B) Real-time PCR of Ppargc1a and Ucp1 in BAT from wild-type (grey bar) and BFCoR transgenic mice (red bar). The data were corrected using the β-actin expression level and represent the mean values±s.e.m. of three independent experiments (n=6 for each genotype). Asterisks indicate statistically significant differences between wild type and BFCoR transgenic mice (*P<0.05 by one-way ANOVA). (C) Western blotting of PGC-1α and UCP-1 protein in BAT from wild-type (lane 1) and BFCoR transgenic mice (lane 2). Lysates (200 μg) of BAT from wild-type and BFCoR transgenic mice were subjected to western blotting with the indicated antibodies. (D) Real-time PCR of mitochondrial component genes using BAT from wild-type (grey bar) and BFCoR transgenic mice (red bar). Data represent the mean values±s.e.m. of RNA samples from five mice of each genotype. Asterisks indicate statistically significant differences between wild-type and BFCoR transgenic mice (*P<0.001 and **P<0.005 by one-way ANOVA). (E) The effects of overexpression of cMyc-FCoR on endogenous Ppargc1a gene expression in T37i cells. T37i cells were transduced with adenovirus encoding LacZ or cMyc-FCoR. Cells were harvested 48 h after transduction. Total RNA was isolated and subjected to real-time PCR of Ppargc1a. Data were corrected using the β-actin expression level, shown as ‘fold change′ compared with LacZ-transduced cells, and represent the mean values±s.e.m. from three independent experiments. An asterisk indicates a statistically significant difference between LacZ- and cMyc-FCoR-transduced cells (*P<0.001 by one-way ANOVA). (F) PGC-1α protein expression was decreased in a dose-dependent manner by overexpression of FCoR in T37i cells. (G) Effect of FCoR on Ppargc1a promoter activity. Data were obtained in five experiments and are represented as the mean values±s.e.m. of fold change compared with mock vector-transfected activity in T37i cells transfected with truncated Ppargc1a (S3). *P<0.001 (one-factor ANOVA of cells transfected with pCMV5/cMyc and with pCVM5/cMyc-FCoR vector). (H) Chip assays of T37i cells transduced with an adenovirus encoding cMyc-FCoR and harvested 36 h after transduction. The PCR primers used to amplify the mouse Ppargc1a promoter sequence as indicated as S3 and S4. PCRss with total input chromatin are shown as controls. Figure source data can be found with the Supplementary data.

A model for the roles of FCoR in fine-tuning of Foxo1 activity. (A) When mice are in the fed state, Foxo1 is phosphorylated and FCoR expression is increased. Both proteins remain in the cytosol. FCoR acetylates Foxo1 directly and keeps it in the acetylated state. (B) When mice are in the fasting state, insulin level decreases and Foxo1 remains in the nucleus and is activated. At the same time, glucagon from pancreatic α-cells is increased and activates cAMP-dependent protein kinase (PKA). Furthermore, upon exposure to the cold, adrenergic stimuli also activate PKA. These events lead to the nuclear localization of FCoR, which increases acetylation of Foxo1 by interrupting the association between Foxo1 and Sirt1. FCoR thus regulates energy storage, energy expenditure, and insulin sensitivity by fine-tuning Foxo1 function.