Fujimoto et al. 10.1073/pnas.0707328104. |
Fig. 6. Histology of epididymal adipose tissues. The paraffin sections were stained with H&E. (Scale bar, 20 mm.)
Fig. 7. Phenotype of Dmbx1-/-;Lepob/ob mice. (A) Growth curves of Dmbx1+/+ (white circles, Dmbx1-/- (filled circles), Lepob/ob (white triangles), and Dmbx1-/-;Lepob/ob (filled triangles) male mice. (B) Changes in blood glucose levels of Dmbx1+/+ (white circles), Dmbx1-/- (filled circles), Lepob/ob (white triangles), and Dmbx1-/-;Lepob/ob (filled triangles) male mice. (C) Serum insulin levels in mice aged 16 weeks. The data are from male mice for each genotype.
Fig. 8. STAT3 phosphorylation by leptin in the arcuate nucleus. (Upper) Phosphorylated STAT3 protein was detected by anti-pSTAT3 (Y705) antibody. (Lower) The same membrane was reprobed with anti-STAT3 antibody.
Fig. 9. Histology of liver of Dmbx1+/+, Dmbx1-/-, Ay/a, and Dmbx1-/-;Ay/a mice. The paraffin sections were stained with H&E. (Scale bar, 100 mm.)
Fig. 10. Immunostaining for AgRP and a-MSH and Nissl staining of the dorsal tegment of the pons. (Top and Middle) Serial sections of the dorsal tegment of the pons were immunostained for AgRP (Top) or a-MSH (Middle). (Bottom) The same sections for a-MSH were used for Nissl staining. (Scale bar, 100 mm.) MPB, medial parabrachial nucleus; Me5, mesencephalic 5 nucleus; LC, locus coeruleus; Bar, Barrington' nucleus.
Fig. 11. Immunostaining for tyrosine hydroxylase in the hypothalamus (Upper) and the ventral tegmental area (Lower). (Scale bar, 500 mm.) DM, dorsomedial hypothalamic nucleus; VMH, ventromedial hypothalamic nucleus; VTA, ventral tegmental area; MM, medial mammillary nucleus.
Fig. 12. Histology of the pons. The paraffin sections were stained with H&E. (Scale bar, 20 mm.) Vsc, ventral spinocerebellar tract; scp, superior cerebellar peduncle; PB, parabrachial nucleus; 4V, fourth ventricle; 7n, root of facial nerve; RET, reticular nucleus.
Fig. 13. Targeted disruption of Dmbx1. (A) Illustration of mouse Dmbx1 genomic organization, wild-type allele, targeting vector, and mutant allele. (B) Southern blot analysis of mouse tail genomic DNA. Genomic DNA (5 mg) digested with BamHI was prepared from mouse tails. The probe used for Southern blotting was a genomic DNA fragment as shown in A. (C) Northern blot analysis of whole body of E10.5 embryos. Total RNA (10 mg) was prepared from whole body of E10.5 fetus. The probe for Northern blotting was the cDNA fragment of mouse Dmbx1 (corresponding to nucleotide + 238 ~ +1082 of GenBank accession no. NM_130865). (B and C) Hybridization was performed under highly stringent conditions with digoxigenin-labeled probes.
SI Materials and Methods
Animals.
Dmbx1-deficient (Dmbx1-/-) mice were generated by replacing a part of exon 3 and exon 4 of the Dmbx1 gene with the b-galactosidase gene LacZ (SI Fig. 13). We analyzed the Dmbx1-/- mice in the ICR background (Japan SLC, Hamamatsu, Japan) because Dmbx1-/- mice are neonatal lethal in the pure C57BL6 background. All animal studies were approved by the Animal Care and Use Committees of Kobe University and by the National Institute for Physiological Sciences in Japan. Dmbx1-/- mice were genotyped by postnatal day 7, and nonknockout (Dmbx1+/+ and Dmbx1+/-) littermates were removed from the cage by day 7. Dmbx1-/-;Lepob/ob and Dmbx1-/-;Ay/a mice (Japan SLC) were also generated in ICR background, and genotype of Ay/a was determined by the coat color. Wild-type (Dmbx1+/+) mice of the same genetic background were used as controls.
Feeding Behavior and Activity.
For monitoring feeding after separation, female mice that had been housed in groups (four to six mice per cage) until 20-24 wk of age were separated into individual cages (at 1000 hours), and food intake and body weight were monitored every morning (at 1000 hours) for 10 d. Except for the separation study (Fig. 2A), food intake was measured in mice acclimatized to single-housing for at least 2 wk. For acclimatization, the mouse was separated from its cohabitants, and its body weight was monitored. The mice were provided regular pellet food (CE-2; Japan Clea, Tokyo, Japan) placed on the floor. Locomotor activities were monitored in individually caged male mice using a Chronobiology Kit (Stanford Software Systems, Santa Cruz, CA) after acclimatization (>2 wk). Activity was evaluated by an infraredbeam-splitting paradigm.
STAT3 Phosphorylation by Leptin.
Murine leptin (Calbiochem, La Jolla, CA) was administered i.p. (2 mg per gram of body weight) to mice fasted for 16 h. For control groups, the vehicle (PBS) alone was administered. Mice were killed 45 min later, and the arcuate nucleus was rapidly dissected and homogenized in radioimmunoprecipitation assay buffer containing protease inhibitor cocktails from Sigma (P8340 and P5726; Sigma-Aldrich, St. Louis, MO). Lysate (20 mg) from each mouse was subjected to SDS/PAGE and immunoblotting by using anti-pStat3 (Y705) and anti-Stat3 antibodies (Cell Signaling Technology, Danvers, MA). The membranes were probed with anti-pStat3 antibody (´1,000) first and then reprobed with anti-Stat3 antibody (´1,000).
Real-Time PCR Analysis.
Dmbx1+/+mice with reduced body weight were prepared by food restriction for 8-10 w. Female mice at 20 wk of age were introduced to the food restriction in which they had free access to food only for 1 h, twice a day. The mice demonstrating body weight loss of >20% from the initial body weight but devoid of any further body weight loss were sampled at 28-30 w. The probe for Agrp was reported previously (1).
1. Minokoshi Y, Alquier T, Furukawa N, Kim YB, Lee A, Xue B, Mu J, Foufelle F, Ferré P, Birnbaum MJ, et al. (2004) Nature 428:569-574.