higher in the HFD-fed mGHRKO mice in the light phase, indicative of enhanced carbohydrate utilization [8]. Thus, using the mGHRKO mice we sought to analyze the effect of muscle GHR signaling on lipid oxidation, and the ability of the muscle to switch between fuel sources in the fed-to-fasted transition. PGC1a (peroxisome proliferator-activated receptor c coactiva- tor 1-a) is a master regulator of lipid metabolism and mitochon- drial function in the skeletal muscle, and is induced in response to cues of increased energy demand such as fasting, and exercise (reviewed in [9]). The importance of PGC1a in mediating the metabolic benefits of exercise is evident from the skeletal muscle- specific PGC1a knockout mice that demonstrate decreased locomotor activity as well as a lower threshold for endurance exercise, while the opposite is observed in PGC1a transgenic mice [10,11]. PGC1a is subject to transcriptional as well as post- translational regulation. One important post-translational modifi- cation of PGC1a that increases its activity is deacetylation, and the NAD+-dependent histone deacetylase sirtuin1 (SIRT1) has been implicated in this process [12]. In the muscle, PGC1a is involved in lipid uptake and oxidation, mitochondrial biogenesis and oxidative phosphorylation, glucose uptake, and fiber-type specifi- cation. These diverse effects are mediated by the association of PGC1a with various transcription factors such as the peroxisome proliferator activated receptors (PPARs), estrogen- related recep- tors (ERRs), nuclear respiratory factor (NRF), and myocyte- specific enhancer factor 2C (MEF2C). In addition to increasing their transcriptional activity, PGC1a has also been shown to induce the expression of some of these transcription factors (reviewed in [13]). Thus, the gene expression signature of transcription factors associated with PGC1a is a good indicator of the state of activation of lipid oxidation pathways, and we applied this to our present study. Our data ascribe a role for skeletal muscle GHR signaling in regulating basal lipid oxidation, but not in response to fasting. The mGHRKO mice demonstrate down-regulation of genes involved in PGC1a-dependent lipid oxidation in the muscle under both regular chow (RC)- and HFD- fed conditions, suggesting a direct role of GHR signaling in mediating muscle lipid oxidation. However, under states of increased energy demand such as fasting and exercise the HFD-fed mGHRKO mice are able to effectively activate a program of lipid oxidation similar to the HFD-fed control mice. Furthermore, we also show that the adipose tissue of the HFD-fed mGHRKO mice demonstrates increased lipolysis and free fatty acid (FFA) secretion under fasted conditions, suggesting a metabolic adaptation that increases FA supply to the muscle. Results mGHRKO Mice Demonstrate Down-regulation of Components of the PGC1a-dependent Lipid Oxidation Pathway in the Muscle Since PGC1a is implicated in activation of lipid oxidation pathways, we analyzed the expression of components of this pathway in the mGHRKO mice under RC- and HFD- fed conditions. While no difference in the expression of pgc1a and pparb/d (peroxisome proliferator- activated receptor b/d) was observed in the muscles of 16wk-old RC-fed mGHRKO mice when compared to controls, the expression of erra (estrogen-related receptor a) and pdk4 (pyruvate dehydrogenase kinase 4) were significantly reduced in the mGHRKO mice relative to the control mice [Figure 1A]. However, under HFD-fed conditions, the expression of pparb/d, erra, and pdk4 were significantly down-regulated in the mGHRKO mice compared to the control mice, despite similar expression of Muscle GH Receptor Signaling and Lipid Oxidation Figure 1. The muscles of mGHRKO mice display down- regulation of lipid oxidation genes. mRNA expression of indicated genes was determined by real-time PCR in quadriceps muscles of A. 16 wk-old regular chow (RC) -fed (n = 6 mice/genotype), and B. 14wk- high-fat diet (HFD) -fed (n = 14–16/group) control (open bars) and mGHRKO mice (closed bars). Each sample was probed three times and gene expression was normalized to b-actin (A) or gapdh (B) and represented as a fold change when compared to respective control (either RC- or HFD- fed). All values are represented as mean 6 S.E.M. *2p#0.05, One-way ANOVA. doi:10.1371/journal.pone.0044777.g001 pgc1a [Figure 1B]. These data suggest that the lipid oxidation is suppressed in the skeletal muscle of the mGHRKO mice under RC- as well as HFD- fed conditions. Muscle Mitochondrial Content in the mGHRKO Mice is Similar to the Controls Muscle fibers can be categorized as oxidative and glycolytic fibers based on their fuel source, that is, triglycerides and glycogen respectively. PGC1a has been shown to enhance muscle oxidative capacity by promoting mitochondrial biogenesis via activation of the transcription factor NRF1 (nuclear respiratory factor 1) and its downstream target, mitochondrial transcription factor A (TFAM). The expression of nrf1 and tfam was not altered in the quadriceps of 16 week old RC-fed mGHRKO mice [Figure 2A], but was significantly decreased in that of the HFD-fed mGHRKO mice relative to the controls [Figure 2B]. This was associated with 40% reduction or unchanged TFAM protein content in the RC- and HFD- fed mGHRKO mice respectively [Figure 2C]. Muscle fiber cross-sectional area was significantly lower in the mGHRKO mice under RC-fed conditions, but there was no difference between the genotypes under HFD-fed conditions [Figure S1A, S1C]. The expression of the different myosin heavy chain (MHC) isoforms is indicative of fiber type with PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e44777