the cross-sectional area of at least 170 muscle fibers. Cytochrome oxidase activity in cryosections of the gastrocnemius was assessed by incubating the sections with a buffer containing cytochrome C and catalase. Fibers which were positive for cytochrome oxidase acitivity stained brown due to use of the substrate 3, 3 diaminobenzidine tetrahydrochloride (DAB). Western Blot Analysis The quadriceps and epididymal fat pad were homogenized in protein extraction buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1.25% CHAPS, 1 mM sodium orthovanadate, 2 mM sodium flourdie, 10 mM sodium pyrophosphate, 8 mM b- glycerophosphate and CompleteH Protease Inhibitor Cocktail, pH 7.4), proteins were extracted, and protein concentration was determined using the BCA protein assay using bovine serum albumin as a protein standard. 25–30 mg of proteins were re- suspended in 3x blue loading buffer supplemented with DTT, denatured by boiling at 96uC, and subjected to SDS-PAGE (8– 16% acrylamide gradient gels), and transferred to a nitrocellulose membrane. The membranes were blocked in 5% Milk/TBS for an hour at room temperature, incubated with primary antibody diluted in 0.5% Milk/TBS/0.1% Tween-20 overnight at 4uC, and followed by fluorescent secondary antibodies diluted in 0.5%Milk/ TBS/0.1% Tween-20 for an hour at room temperature. The signal was developed using the Odyssey Infrared Imaging System (Licor Biosciences, Lincoln, NE). The blots were quantified using the Odyssey Infrared Imaging System Application Software Version 3.0 (Licor Biosciences, Lincoln, NE). RNA Analysis Total RNA from quadriceps and epididymal fat pad was extracted with phenol/chloroform using the RNeasy Lipid Tissue Mini kit the with on-column DNase treatment, and the concentration of RNA was determined using the NanoDrop ND-1000 Spectrophotometer (Thermo Scientific, Wilmington, DE). 1000 ng of RNA was subjected to reverse transcription according to the manufacturer’s instructions (Invitrogen Corp., Carlsbad, CA). The resulting cDNA was diluted to about 200 ul with DNase/RNase- free water, and 3 ml of sample was used per well of a 384-well real-time PCR plate. Real time-PCR was performed using the QuantiTectTM SYBRH green PCR kit (Qiagen, Valencia, CA) in ABI PRISM 7900HT sequence detection systems (Applied Biosystems, Foster City, CA). For each gene, a single sample was assayed 3 times and gene expression was normalized to gapdh or b-actin. The expression of the MHC isoforms was determined by reverse transcriptase-PCR (RT-PCR) using the following reaction: 95uC –2 min, (94uC –30 sec, 60uC – 45 sec, 72uC –1 min)X 25 cycles. gapdh was used as an internal control. The band intensity was quantified using ImageJ and the expression of the MHC isoform was normalized to that of gapdh and was expressed as a fold change compared to that of the control mice. The primer sequences used were : pgc-1a forward: ATGTGTCGCCTTGCTCT and reverse: ATC- TACTGCCTGCCCACCTT; pparb/d forward: AGGCCCGG- GAAGAGGAGAAAGAGG and reverse: CGCGTGGACCCCGTA-GTGGA; ppara forward: ATGCCAG- TACTGCCGTTTTC and reverse: GGCCTT-GACCTTGTT- CATGT; erra forward: GGAAGTGCTGGTGCTGGGTGT and reverse: GAATTGGCAAGGGCCAGAGCT pdk4 forward: AA- GATGCTCTGCGACCAGTAT and reverse: GAAGGTGT- GAAGGAACGTACA nrf1 forward: GGAGCACTTACTG- GAGTCC and reverse: CTGTCCGATATCCTGGTGGT; tfam forward: CCTGAGGAAAA-GCAGGCATA and reverse: ATGTCTCCGGATCGTTTCAC; sirt1 forward: GTAATGT- Muscle GH Receptor Signaling and Lipid Oxidation GAGGAGTCAGCAC and reverse: TTGGACATTAC- CACGTCTGC; sirt3 forward: ACTACAGGCCCAATGTCACT and reverse: TTCAACCAGCTTTGAGGCAG; ghr forward: GCCTGGGGACAAGTTCTTCTG and reverse: GCAGCTTGTCGTTGGC-TTTCC; mhcI forward: CTCCCAAGGAGAGACGACTG and reverse: TTAAG- CAGGTCGGCTGAGTT; mhcIIa forward: GAACCCTCC- CAAGTACGACA and reverse: TAAGGGTTGACGGTGA- CACA; mhcIIx forward: CTTCAACCACCACAT-GTTCG and reverse: GAGCTTCAACCTTGCCTTTG; mhcIIb forward: CCAGAGTCACCTTCCAGCTC and reverse: CTTCCCTTTGCTTTTGCTTG; gapdh forward: TGAAGGTCGGTGTCAACGGATTTGGC and reverse: CATGTAGGCCAT-GAGGTCCACCAC; b-actin forward: ATATCGCTGCGCTGGTCGTC and reverse: AG- GATGGCGTGAGGGAGAGC. Tissue TG Total TG were extracted from the liver and gastrocnemius using the chloroform-methanol method [44]. Briefly, approxi- mately 100 mg of gastrocnemius tissue was crushed on dry ice using a mortar and pestle and transferred to a glass tube. The tissues were incubated in 3 mL of 2:1 chloroform: methanol mixture for 4 h with agitation, after which 1.5 mL of 0.1 M NaCl was added to the tubes, mixed by vortexing and centrifuged at 1000 rpm for 10 min at room temperature to separate the organic and aqueous phases. The lower organic phase containing the TG was transferred to a new glass tube using a glass Pasteur pipette and the liquid was evaporated under a steady stream of nitrogen gas. The remaining TG pellet was re-suspended in 200 uL of a 3 M potassium hydroxide in 65% ethanol solution and incubated at 70uC for 60 min in a water bath, and stored at 220uC. The samples were diluted 1:10 in water and TG content was quantified using the TG reagent. Statistical Analysis All data are represented as mean 6 S.E.M. One-way (for comparison of two groups) or two-way (for comparison of three or more groups) ANOVA with Holm-Sidak post-hoc test was performed using SigmaStat for Windows (version 3.5; Systat Software, Inc., Chicago, IL). Supporting Information Figure S1 Analysis of fiber type in the mGHRKO mice. (A,C). Average muscle cross sectional area in about 175 fibers was measured using ImageJ software in RC-fed (A) and HFD-fed (C) mice (n = 6–7/genotype/diet). (B,D) mRNA expression of myosin heavy chain (MHC) isoforms as measured by RT-PCR in quadriceps muscles of control and mGHRKO mice under RC- fed (B) and HFD-fed (D) conditions. Band intensity was quantified using ImageJ software and represented as a fold change compared to control mice (n = 6–14/genotype/diet). E. Muscle cytochrome oxidase acitivity in gastrocnemius muscle of HFD-fed control and mGHRKO mice. Scale bar represent 500 mm (n = 4/ genotype, representative images are shown). All values are represented as mean 6 S.E.M. *2 p#0.05 control versus mGHRKO. (TIF) Figure S2 Basal expression of sirtuins. mRNA expression of sirt1 and sirt3 as measured by realtime PCR analysis in quadriceps muscles of control and mGHRKO mice under HFD- fed conditions (n = 7–8/genotype). All values are represented as mean 6 S.E.M. PLOS ONE | www.plosone.org 10 September 2012 | Volume 7 | Issue 9 | e44777