Ryu et al. 10.1073/pnas.0800096105. |
Fig. 6. Adult-onset hyperleptinemia in Ub-/- mice. (A) Blood glucose levels of 5- to 6-month-old mice [Ubb+/+ (+/+), n = 11; Ubb-/- (-/-), n = 11] after fasting for 18 h. (B) Serum insulin levels of fed and 48-h fasted 5- to 6-month-old mice (+/+, n = 4; -/-, n = 3). (C) Serum leptin levels of young (1- to 2-month-old) and adult (5- to 6-month-old) male and female mice fed ad libitum. Each symbol represents a single mouse. Horizontal bar indicates mean. (D) Serum leptin levels before and after 48-h fasting of 6-month-old mice (+/+, n = 3; -/-, n = 3). All data are expressed as means ± SEM from the indicated number of mice. In C: *, P < 0.05; **, P < 0.01. In D: **, P < 0.01 vs. Ubb+/+ before fasting; #, P < 0.05 vs. Ubb+/+ before fasting.
Fig. 7. Preferential expression of Ubb in different brain regions. Representative confocal images for direct visualization of GFP-puror fluorescence from 4-month-old Ubb+/+ (n = 3), Ubc+/- (n = 4), and Ubb+/- (n = 3) mouse hypothalamus (A) and cerebral cortex (B). GFP-puror is a direct reporter of Ubb or Ubc transcriptional activity in Ubb+/- or Ubc+/- mice, respectively. Ubb+/+ lack this reporter and constitute a background control. 3V, third ventricle; ARC, arcuate nucleus; CTX, cerebral cortex; MOp, primary motor area; SSp, primary somatosensory area (indicated by arrowhead); VMH, ventromedial hypothalamus. (Scale bar, 100 mm.)
SI Materials and Methods
Hormone Assays.
Serum samples were collected in the morning to minimize the diurnal variation of hormone levels. Blood samples were allowed to clot at room temperature for 90 min and centrifuged at 2,000 ´ g for 15 min. Supernatants were collected and serum leptin and insulin levels were measured using the ELISA kit (ALPCO Diagnostics). Blood glucose levels were measured from tail bleed using conventional glucometer (One Touch Ultra).Quantitative Real-Time RT-PCR.
Brains from ad libitum-fed or fasted mice were removed after CO2 euthanasia and placed in ice-cold RNase-free PBS for 30 sec. Hypothalamus was isolated as a 1.5-mm thick cubic tissue block along the midline with anterior margin at optic chiasm and posterior margin at mammilary bodies, and stabilized with RNAlater (Qiagen) immediately. Total RNA was isolated using RNeasy kit (Qiagen) with DNaseI treatment. RNA concentration was determined and 10 ng of total RNA was used as a template. Primers used for NPY, AgRP, POMC real-time RT-PCR are as follows: NPY-F (5'-GGA CTG ACC CTC GCT CTA TC-3'); NPY-R (5'-AGT GTC GCA GAG CGG AGT AG-3'); AgRP-F (5'-GAG TCC TGC TTG GGA CAG C-3'); AgRP-R (5'-CTT GCG GCA GTA GCA AAA GG-3'); POMC-F (5'-TGG AAG ATG CCG AGA TTC TGC-3'); POMC-R (5'-CAA GCC AGC AGG TTG CTC TC-3').In Situ
Hybridization. Ad libitum-fed mice were euthanized, brains were removed and snap frozen in isopentane/dry ice bath at -35°C, and stored at -80°C. Brain sections (20 mm thick) were cut on a Leica cryostat through the hypothalamus, thaw-mounted onto polylysine-coated slides and stored at -80°C until processing for in situ hybridization. Antisense cRNA probes directed against mouse AgRP or NSE mRNA were generated and labeled with digoxigenin-11-UTP (Roche Diagnostics). Brain sections were fixed in 4% paraformaldehyde for 1 h and rinsed twice in 2´ SSC (300 mM NaCl, 30 mM sodium citrate, pH 7.2). Sections were then acetylated in 0.1 M triethanolamine, pH 8.0, with 0.25% acetic anhydride for 10 min, dehydrated through a graded series of ethanol (50-100%, 30 sec each) and subsequently air-dried. Brain sections were hybridized with cRNA probes in 50% hybridization buffer (50% formamide, 10% dextran sulfate, 3x SSC, 50 mM sodium phosphate buffer, pH 7.4, 1´ Denhardt's solution, 0.1 mg/ml yeast tRNA, and 30 mM DTT). Diluted probes were placed on each slide, and the sections were cover slipped. Brain slides were placed in plastic trays moistened with 50% formamide and incubated at 55°C for 18 h. The following day, brain sections were washed with SSC, treated with RNase A (200 mg/ml) for 1 h at 37°C. Following the final wash in 0.1´ SSC at 68°C for 1 h, sections were then processed for immunohistochemistry to visualize the digoxigenin-labeled mRNA. Briefly, brain sections were treated with a blocking solution (0.1 M phosphate buffer containing 0.5% Triton X-100 and 0.25% carrageenan, pH 7.5) for 4 h, and then incubated overnight with Fab fragments from an anti-digoxigenin antibody from sheep, conjugated with horse-radish peroxidase (1:15,000; Roche Diagnostics). After rinsing in both 0.1 M phosphate buffer and 0.1 M Tris buffer (30 min each), sections were incubated with a color reaction buffer (0.45% nitroblue tetrazolium chloride, 0.35% 5-bromo-4-chloro-3-indoylphosphate 4-toluidine salt, 5% polyvinyl alcohol, and 0.24% levamizole). The color reaction was completed overnight. Sections were then rinsed in water and incubated with 0.1 M glycine buffer, pH 2.2, containing 0.5% Triton X-100 for 10 min. Finally, brain sections were fixed in 25% glutaraldehyde for 2 h. After rinsing in water and dehydrating in a graded series of ethanol (50-100%), the brain slides were cover slipped in a xylene-based mounting medium (Permount; Fisher Scientific). Digoxigenin-labeled AgRP and NSE mRNAs were visualized under a bright field as a blue-purple precipitate. Cell counts were determined at ´100 magnification bilaterally within the entire arcuate nucleus anatomically matched between animals.Indirect Competitive ELISA.
Brains and isolated hypothalamus were homogenized in hypotonic buffer (10 mM sodium phosphate, pH 7.4, with protease inhibitor mixture, Roche) with 0.1% digitonin and incubated on ice for 20-30 min. Tissue lysates were centrifuged at 13,000 rpm for 10 min at 4°C and the supernatant was removed to measure protein concentration by BCA protein assay (Pierce). To prepare samples for ELISA, tissue lysates (24 mg) were treated with 2.4 mg Usp2-cc in the presence of 1.4 mM b-mercaptoethanol and 140 mM NaCl in 20-m l reaction volume for 30 min at 37°C. Usp2-cc treated tissue lysates were further diluted with 1% BSA/PBS containing 0.1% digitonin and total Ub levels were measured by ELISA.