conjugated donkey polyclonal anti-goat IgG, IRDyeH 680CW conjugated donkey anti- mouse IgG (Licor Biosceinecs, Lincoln, NE); 50% Glutaraldehyde solution-EM grade (Electron Micros- copy Sciences, Hatfield, PA); oligonucleotide primers for gene expression analysis (Eurofins MWG Operon, Huntsville AL); rabbit polyclonal CD36 (H-300), rabbit polyclonal b- tubulin antibody (Santa Cruz Biotechnology, Santa Cruz, CA); TFAM MaxPab rabbit polyclonal antibody D01 (Abnova, Taipei, Taiwan); mouse monoclonal complex IV subunit I (COXI) antibody (MitoSciences, Eugene, OR); rabbit polyclonal phos- pho-AMPKaThr172 antibody (pAMPK), rabbit polyclonal AMPKa antibody, rabbit polyclonal phospho-HSLSer660 antibody, and rabbit polyclonal phospho-HSLSer563 antibody (Cell Signaling Technology, Danvers, MA); mouse monoclonal lipoprotein lipase (LPL) antibody, and rabbit polyclonal HSL antibody (Abcam Inc, Cambridge, MA). All other common reagents were purchased from Fisher Scientific (Pittsburg, PA) or VWR (Westchester, PA). Animals The generation of the mGHRKO mice has been described previously [8]. Male mice on the C57BL/6 background were used for all experiments, and were housed in the pathogen-free AAALAC accredited animal facilities of the Mount Sinai School of Medicine. All experimental procedures were in accordance with the Institutional Animal Care and Use Committee of the Mount Sinai School of Medicine (IACUC # LA12-00052). The mice were kept on a 12 h light/dark cycle, and had ad libitum access to water and either standard mouse chow (LabDiet, Brentwood, MO), or, where indicated, high-fat diet (60% fat calories) (starting from 7 weeks of age) (BioServ, New Brunswick, NJ). In all experiments the mGHRKO (GHRfloxed/floxed/muscle creatine kinase-Cre+) mice were compared to control (GHRfloxed/floxed) mice. For the 24 h fasting experiments, 14wk-HFD fed mice were separated into 4 groups- control-fed, control-fasted, mGHRKO- fed, mGHRKO-fasted. Mice were anesthesized with Avertin (1:40 dilution of a 100% solution of 2,2,2 tribromothanol in tert-amyl alcohol), and the tissues were rapidly extracted and snap-frozen in liquid nitrogen. However, prior to freezing the epididymal adipose tissue, a small piece of tissue was taken for the ex vivo lipolysis study (described below). Blood Chemistry Blood glucose levels were measured from the tail using an automated glucometer (Elite; Bayer, Mishawaka, IN). Serum or plasma collected from the tail vein or retro-orbital sinus was used for measurement of circulating insulin, GH and FFA levels as per manufacturers’ instructions. Fasting/Re-feeding At 6:00 p.m of the first day, basal blood glucose levels of the mice were measured from the tail using an automated glucometer (Elite; Bayer, Mishawaka, IN), and the mice were bled from the tail vein for serum and plasma. Subsequently, food was removed from the cage, but the mice had ad libitum access to water. The mice were sampled for blood glucose, serum and plasma at 6:00 p.m the next day, after which a known amount of food (either RC or HFD) was added to the cage. The mice were sampled for blood glucose, serum and plasma again at 9:00 pm; at which time the amount of food remaining in the cage was weighed. Food intake during the re-feeding period was calculated by the formula (gfoodinitial- gfoodfinal)/number of mice per cage/(body weight)0.75/h. The mice were closely monitored for the duration of the study to ensure that they were not adversely affected as a result of the fast. Serum FFA levels and plasma insulin levels were Muscle GH Receptor Signaling and Lipid Oxidation measured from the collected samples as per manufactures’ instructions. Ex-vivo Lipolysis Epididymal adipose tissue was extracted from fed and 24 h fasted mice, rinsed in ice-cold PBS, and minced. 50–70 mg of minced tissue was placed into two wells of a 24-well plate with 1 ml of Krebs-Ringer buffer (calcium chloride 1 mM, magnesium sulfate 1.2 mM, dihydrogen potassium phosphate 1.2 mM, potassium chloride 1.4 mM, sodium chloride 130 mM, pyruvic acid 2 mM, HEPES 20 mM, and bovine serum albumin 2.5%). 1 mM isoproteronol hydrochloride was added into one of the two wells. The plate was incubated at 37uC with mild agitation. 50 ml of buffer was sampled at times 0, 1, 2, and 3 h after isoproteronol addition. FFA levels were measured using 10 ml of sample as per manufacturers’ instructions. FFA secretion rate was determined as the difference in FFA levels in the medium at 0 h and 3 h divided by the duration of the experiment (3 h). Indirect Calorimetry For locomotor acitivity, 14wk-HFD-fed mice were singly housed in metabolic chambers maintained at thermo-neutrality on a 12 h light/dark cycle. Locomotor activity was determined as beam breaks in the horizontal and vertical directions using a CLAMS (Columbus Instruments) open circuit indirect calorimetry system. For the treadmill challenge, 14wk- HFD- fed control and mGHRKO mice underwent two training sessions on the treadmill and on the day of the challenge, the mice were made to run on treadmill, connected to an indirect calorimetry instrument, and the belt speed was incrementally increased every 3 min and the metabolic measurements were made. Transmission Electron Microscopy 16wk-old or 14wk-HFD-fed control and mGHRKO mice were anesthetized with Avertin, and were perfused with ice-cold PBS, followed by ice-cold 3% glutaraldehyde. The quadriceps was immediately extracted, minced into 262 pieces, fixed in 3% glutaraldehyde, and processed by increased concentrations of ethanol through propylene oxide and embedded in Epon. Thick one micron section were cut and stained with methylene blue and azure II and observed by light microscopy. Representative areas were ultra-thinned and stained with 4% uranyl acetate and Reynold’s lead citrate by the Electron Microscopy Core Lab at the Mount Sinai School of Medicine. Electron micrographs were taken at 5000x magnification using a Hitachi H7650 instrument linked to SIA (Scientific Instruments and Applications) digital camera controlled by Maxim CCD software. 7–10 images from non-overlapping fields were obtained for each animal. ImageJ software (NIH Bethesda, MD) was used to determine the area of individual mitochondria and the mitochondrial density was determined as the ratio of total mitochondrial area to total area of the field. The mitochondrial density for each animal was determined by averaging the mitochondrial density of the different fields. Histology and Cytochrome Oxidase Staining 14wk-HFD fed mice were sacrificed and the quadriceps and gastrocnemius were extracted and fixed in 10% PBS-buffered formalin or frozen in liquid-nitrogen cooled isopentane respec- tively. The quadriceps was subsequently embedded in paraffin, sections were cut, stained with H&E and pictures were obtained using the Olympus AX70 camera (Olympus, Center Valley, PA). NIH-ImageJ software (NIH, Bethesda, MD) was used to quantify PLOS ONE | www.plosone.org 9 September 2012 | Volume 7 | Issue 9 | e44777