Hoffmann-La Roche, Basel,

Hoffmann-La Roche, Basel, http://www.selleckchem.com/products/Cisplatin.html Switzerland) according to the manufacturer’s instructions. Overnight fasting blood glucose was measured using the Ascensia Contour glucometer (Bayer HealthCare, IN). FFAs were measured in mouse serum using the Half Micro Test (Roche Diagnostics, Mannheim, Germany). Hormone measurements were also taken at 32 weeks of age. Fasting serum insulin was measured using the ultrasensitive mouse insulin ELISA kit from Mercodia (Uppsala, Sweden) according to the manufacturer’s instructions. Fasting serum leptin and adiponectin/Acrp30 were measured by ELISA using commercial assay kits (MRP300 mouse Adiponectin/Acrp30; MOB00 mouse leptin, R and D Systems, Minneapolis, MN). Fasting serum Retinol Binding Protein 4 (RBP4) was measured using the Dual mouse/rat RBP4 ELISA kit (RB0642EK; AdipoGen, Seoul, Korea).

Histology and Oil-Red-O staining of murine liver and adipose tissue Liver and visceral adipose tissues were removed and weighed. Formalin-fixed, paraffin-embedded liver and adipose tissue from eight of the 32-week-old male mice were processed, and 4-��m-thick serial sections were cut and stained with Oil-Red-O or hematoxylin and eosin (HE) for lipid analysis according to standard pathology laboratory procedures. After mounting with glycerol gelatin, images were captured using Axio Vision Rel4.5 software (Carl Zeiss). For measurement of adipocyte cell areas, images were converted to TIFF files for analysis using the Discovery SeriesTM Quantity One? 1-D analysis software (Bio-Rad Laboratories).

Hepatic TG quantitation Levels of mouse liver TG were quantified using the Triglyceride Determination Kit TRO100 with appropriate TG standards (Sigma-Aldrich, St. Louis, MO). Frozen liver samples from 32-week-old mice were first powdered under liquid nitrogen and 120 mg of the frozen liver powder was weighed into 2 ml of chloroform:methanol mix (2:1, v/v) and incubated for 2 h at room temperature with occasional shaking. Following the addition of 0.2 volumes of water, vortexing, and centrifuging at 2,500 g, the lower phase containing the lipids was collected and dried under vacuum in a rotary evaporator for 5�C6 h. The dried pellets were resuspended in the reaction buffer provided in the kit. Results were expressed as mean TG (mg/g tissue) �� SEM, n = 6 per diet group.

RNA isolation To detect early changes in gene expression, total RNA was prepared from the liver and visceral adipose tissue taken from 16-week-old mice Brefeldin_A (n = 4) in the four different diet groups using the Qiagen RNeasy kit according to the manufacturer’s instructions and stored at ?80oC. RNA quality was verified using a 2100 Bioanalyzer instrument and an RNA 6000 Nano LabChip assay (Agilent Technologies, Santa Clara, CA). RNA concentrations were determined by absorption at 260-nm wavelength with an ND-1000 spectrometer (Nanodrop Technologies, Wilmington, DE).

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