[25]. Further, PGC1a knockout mice display lower expression of genes involved in mitochondrial biogenesis in the heart and skeletal muscle, but this is not associated with changes in mitochondrial content or the content of oxidative fibers (which are rich in mitochondria) in the heart and skeletal muscle respectively [26]. Moreover, studies in whole-body and muscle- specific PGC1a knockout mice suggest that PGC1a-independent increase in mitochondrial function is possible. The muscle-specific PGC1a knockout mice display compromised mitochondrial structural integrity, which is normalized by voluntary activity [27,28]. In light of these studies, the higher locomotor activity in the HFD-fed mGHRKO mice may result in activation of alternative pathways of mitochondrial biogenesis and may explain why they do not display alterations in mitochondrial content despite lower expression of nrf1 and tfam. When challenged with fasting, a condition that activates lipid oxidation, the HFD-fed mGHRKO mice demonstrate increased expression of lipid oxidation genes such as pgc1a, ppara, pparb/d, and pdk4 to the similar extent as the fasted controls. We next tested our hypothesis by subjecting the mice to a treadmill running challenge, another paradigm that activates lipid oxidation. The HFD-fed mGHRKO mice demonstrated lower RER during the treadmill challenge. We have previously shown that the HFD-fed mGHRKO mice have higher RER in the light phase under standard housing conditions [8]. Thus, taken together our data suggest that while demonstrating reduced lipid oxidation under normal conditions, the HFD-fed mGHRKO are able to adapt to increased energy demand by activating lipid oxidation pathways, a regulation that is essentially lost in obesity and insulin resistance. The HFD-fed mGHRKO demonstrate up-regulation of sirt1 expression upon fasting. SIRT1 is a NAD+-dependent histone deacetylase that can deacetylate PGC1a and increase its activity [12]. Further, activation of SIRT1 using a synthetic molecule protects mice from diet-induced obesity and increases lipid oxidation in the skeletal muscle by mediating the deacetylation of PGC1a and FOXO1 [29]. Thus, higher sirt1 expression in the mGHRKO mice upon fasting could result in increased activation of PGC1a in the mGHRKO mice. PGC1a can also be activated by phosphorylation which is mediated by AMPK. However, we did not detect a significant difference in AMPK phosphorylation in the muscles of the control and mGHRKO mice upon fasting. While AMPK is believed to be activated in conditions of low cellular energy, recent studies have in fact reported that AMPK activity is not elevated after a 24 h fast in the skeletal muscle and liver [30]. SIRT3 has been show to regulate global mitochondrial deacetylation; and its expression in the skeletal muscle is down- regulated on HFD feeding, while it is increased in response to exercise and fasting [31,32]. Further SIRT3 has also been shown to be induced by PGC1a in the ERRa-dependent manner [33]. Fasting induced the expression of sirt3 only in the HFD- fed mGHRKO mice, but not the control mice. This could either be reflective of their improved insulin sensitivity or greater PGC1a activity (mediated by its deacetylation by SIRT1). Transgenic over-expression of PGC1a, either in the whole body or specifically in the skeletal muscle, results in increased expression of genes involved in FA uptake in the skeletal muscle [10,34]. Additionally, we observe a tendency towards increased FAT/ CD36 protein content in the mGHRKO, but not control, mice on fasting suggesting greater muscle FA uptake in the fasted mGHRKO mice. Moreover, adipose tissue explants of the fasted mGHRKO mice are significantly more lipolytic than that of the fasted controls. Further, it has been shown that adipose tissue lipolysis and increased delivery of FA to the muscle during exercise Muscle GH Receptor Signaling and Lipid Oxidation occurs before hydrolysis of muscle TG [35,36]. We also did not observe a significant up-regulation of lipolytic pathways in the muscles of the fasted mGHRKO mice. Thus, we propose that under states of energy demand such as fasting, the adipose tissue of the mGHRKO mice exhibits increased lipolysis and FFA secretion in order to provide substrates for the muscle. Interestingly, we found higher fed-state circulating GH levels as well as significantly greater adipose tissue GHR expression in the HFD-fed mGHRKO mice. GH mediates adipose tissue lipolysis [37,38,39,40], and this could potentially mediate the increased lipolysis in the fasted mGHRKO mice. There are several factors that could possibly mediate these effects. The higher GH levels in the HFD- fed mGHRKO could be due to their lower insulin levels [8], as insulin has been reported to decrease GH secretion from the anterior pituitary gland [41,42]. GH has been hypothesized to induce the expression of its receptor, and this may explain the increased expression of GHR in the adipose tissue of the HFD-fed mGHRKO mice (reviewed in [43]). Alternatively, we have also previously reported altered gene expression of myokines, such as myostatin and interleukin-15, in the skeletal muscle of the mGHRKO mice [8], which could potentially also mediate secondary changes in other tissues and may account for the increased GHR expression in the adipose tissue. However, while these are exciting possibilities, further investigation is required to explain these adaptive changes. In summary, increased skeletal muscle lipid oxidation can have beneficial metabolic effects as seen under exercise-stimulated conditions, or results in metabolic deterioration when activated in the context of obesity and insulin resistance. The predominant difference between the two states is the loss of metabolic flexibility, or the ability of the muscle to shift substrate utilization based on energy demand, under obese, insulin resistant conditions. Our study implicates muscle GHR signaling in regulating basal skeletal muscle lipid oxidation, but not in response to states of increased energy demand such as fasting and exercise. Materials and Methods Materials Chemicals and materials were obtained from the following sources: 0.2 micron nitrocellulose membranes, SpectraTM multi- color broad range protein ladder (Thermo Scientific, Rockford, IL); NovexH Tris-Glycine gels, BenchmarkTM, NovexH Tris- glycine SDS running buffer (10X), NovexH Tris-glycine transfer buffer (25X), SuperscriptTM II reverse transcriptase, random primers (Invitrogen, Carlsbad, CA); BCA protein assay, bovine albumin (Pierce, Indianapolis, IN); CompleteH protease inhibitor cocktail, free fatty acids, half micro test kit (Roche, Indianapolis, IN); sodium flourdie, sodium orthovanadate, tetrasodium pyro- phosphate, TritonX-100, pyruvic acid, isoproterenol hydrochlo- ride, anti-growth hormone receptor developed in goat, mouse monoclonal b-actin antibody (clone AC-15), tert-amyl alcohol, 2,2,2 tribromoethanol, cytochrome C from horse heart, catalase from bovine liver, 3,39-diaminobenzidine tetrahydrochloride (Sigma-Aldrich, Saint Louis, MO); b-glycerophosphate (MP Biochemicals, Solon OH); NaCl, HEPES buffer (1M) (Mediatech, Manassas, VA); CHAPS (Affymetrix, Santa Clara, CA ); fat-free milk powder (Lab Scientific, Livingston, NJ); rat/mouse growth hormone 96-well plate assay, Sensitive rat insulin RIA kit (Millipore, Billerica, MA); dNTP mix (10 mM) (Promega, Madison, WI); RNase- free DNase set, RNeasyH Lipid Tissue Mini Kit, QuantiTectH SYBRH Green PCR kit (Qiagen, Valencia, CA); TG reagent (Pointe Scientific, Canton, MI); IRDyeH 800CW conjugated goat polyclonal anti-rabbit IgG, IRDyeH 800CW PLOS ONE | www.plosone.org 8 September 2012 | Volume 7 | Issue 9 | e44777