(A) Whole-body energy expenditure measured by indirect calorimetry (22 weeks of age). 6,406±339 and 5,291±331 kJ/day/kg BW0.75), KRAP−/− (KO) and wild-type (WT) mice, respectively. P<0.05; F (1, 13) = 4.8; n = 7–8 per group. (B) Spontaneous ambulatory activity of mice evaluated for 24 h. Total ambulatory activity per day was 12,076±1,351 and 7,716±1,100 counts/day, KO and WT, respectively. P<0.05; F (1, 10) = 6.3; n = 6. (C) Rectal temperature in the fed or overnight-fasted conditions (n = 7–8). All the data are presented as mean±S.E.M.; *P<0.05; **P<0.01 compared with wild-type controls.

Northern blot analysis in liver (A), skeletal muscle (B), white adipose tissue (WAT; C) and brown adipose tissue (BAT; D) isolated from KRAP−/− (KO) and wild-type (WT) mice. (E) Western blot analysis of total-, phospho-ACC (P-ACC) and FAS of liver isolated from KO and WT mice (left). Densitometric quantification of ACC-1, ACC-2 and FAS normalized by actin content (right; n = 6; *P<0.001). Data are presented as mean±S.E.M. (F) Western blot analysis of ACC and FAS expressions in HepG2 cells 72 h after the transfection of siRNAs. Scramble-siRNAs were used as controls.

(A) Glucose uptake into differentiated adipocytes, from KRAP−/− (KO)- and wild-type (WT)-BAT, measured with or without the pretreatment of insulin. One representative data of three independent experiments performed in quadruplicate. Data are presented as mean±S.E.M.; NS, not significant. (B) Accumulation of lipid droplets detected by Oil Red-O staining in the differentiated adipocytes. Scale bar, 100 μm. (C) Western blot analysis of ACC and PGC-1 in the differentiated adipocytes. (D) Western blot analysis of total- and phospho-Akt (P-Akt) in the differentiated adipocytes treated with 100 nM of insulin for the indicated period (upper) or with the indicated concentrations of insulin for 10 min (lower).

(A) Targeted disruption of the KRAP gene. A region of the murine KRAP gene containing exons 11–16 was replaced with a neomycin resistance cassette. DT-A, Diphtheria-toxin A fragment; E, exon; neo, pGKneo. (B) Southern blotting of EcoRV digested tail DNA using the 5′ external probe A. WT, wild-type; HT, heterozygous; KO, KRAP−/−. (C) Western blotting with the KRAP antibody of liver lysates. (D) Body weight (BW) for KO and WT mice (n = 5–14 per group). P<0.01; F (1, 8) = 160.3 for male; P<0.01; F (1, 11) = 146.4 for female. (E) Food intake for KO and WT mice normalized by BW0.75. 30–36 weeks of age; n = 6. P<0.01; F (1, 10) = 12.6. (F) Percent body fat mass of epididymal white adipose tissue (WAT) and interscapular brown adipose tissue (BAT) (20 weeks of age; n = 9). (G) Tissue triglyceride content of livers and skeletal muscles, n = 9. (H) Daily ash mass of adult mice normalized to their food intake, n = 10. All the data are presented as mean±S.E.M.; *P<0.05; **P<0.01 compared with wild-type controls.

(A) BW-gain under high-fat diet (HFD) for KRAP−/− (KO) and wild-type (WT) mice. n = 6–7; P<0.02; F (1, 11) = 9.5. (B) Food intake under HFD for KO and WT mice normalized by BW0.75. n = 6–7; P<0.05; F (1, 11) = 5.0. (C) Whole-body energy expenditure measured by indirect calorimetry after 4 weeks of an HFD. 4,694±598 and 3,389±496 kJ/day/kg BW0.75, KO and WT, respectively. n = 5–6; P<0.05; F (1, 9) = 5.9. (D) Percent body fat mass of epididymal white adipose (WAT) after 4 weeks of an HFD (left panel) or brown adipose tissue (BAT) after 18 weeks of an HFD (right panel). n = 6–7. (E) Gross appearance of liver from KO and WT mice after 14 weeks of an HFD. (F) Histological analysis of the livers represented in (E) performed by Oil Red-O and hematoxylin staining. Scale bar, 100 μm. (G) Blood glucose concentrations before and 120 min after the single glucose i.p. injection at a dose of 2 g glucose/kg BW. Glucose tolerance test performed after 8 weeks of an HFD (n = 7–8 per group). (H) Insulin sensitivity test performed after 8 weeks of an HFD. n = 7–8; P<0.01; F (1, 13) = 9.9. All the data are presented as mean±S.E.M.; *P<0.05; **P<0.01; #P<0.001 compared with wild-type controls.

(A) Intraperitoneal glucose tolerance test in age-matched pair (15–17 weeks of age) of male mice on a standard diet. n = 10; P<0.01; F (1, 18) = 12.1. (B) Serum insulin concentrations before and 10 min after the single glucose i.p. injection at a dose of 2 g glucose/kg BW. Age-matched pair (9–11 weeks of age) of male mice. n = 8–10; P<0.01; F (1, 15) = 10.8. (C) Insulin sensitivity test in age-matched pair (21–27 weeks of age) of female mice on a standard diet. n = 14–15; P = 0.07; F (1, 27) = 3.5. (D) PET image showing the enhanced accumulation of 18F-FDG in the brown adipose tissue (BAT) of KRAP−/− (KO) mice. Arrow indicates a location of the BAT. A separate experiment in the same procedure was performed on five pairs of mice and the similar results were obtained. Gradation bar indicates signal intensity. (E) Time-activity curves of 18F-FDG in BAT (red circle), brain (dark blue circle), liver (green circle), kidney (pale blue circle) and muscle (brown circle) were obtained from the mean pixel radioactivity in ROI of the images shown in (D) (n = 5). (F) Quantification of the 18F-FDG uptakes in blood, brain, heart, muscle (Musc), liver, kidney (Kid), pancreas (Panc), BAT, epididymal white adipose tissue (WAT) and bladder (Blad) after the PET imaging (n = 5). All the data are presented as mean±S.E.M.; *P<0.05; **P<0.01 compared with wild-type controls.