Muscle GH Receptor Signaling and Lipid Oxidation Figure 2. Mitochondrial content is not altered in the muscle of the HFD-fed mGHRKO mice. mRNA expression of nrf1 and tfam were determined by real-time PCR in quadriceps muscles of A. 16 wk-old RC-fed (n = 6 mice/genotype), and B. 14wk HFD-fed (n = 14–16/group) control and mGHRKO mice. 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). C. Protein content of TFAM and COXI were determined in protein lysates isolated from quadriceps muscle of RC- and HFD- fed control and mGHRKO mice (n = 3–4/group). b- tubulin was used to determine equal loading (top). The protein content was normalized to that of b- tubulin, and represented as a fold change compared to respective control (either RC- or HFD- fed) (bottom). Arrow indicates appropriate TFAM band. D. Electron micrographs of quadriceps muscle of 16wk-old or 14wk-HFD-fed mice (n = 3–5/ group; representative images are shown). The mitochondria in the section are encircled. Scale bar represents 2 mm (left). The area of the mitochondria was quantified using ImageJ software and mitochondrial density was determined as a ratio of the total area of the mitochondria to the total area of the field (right). All values are represented as mean 6 S.E.M. *- p#0.05, One-way ANOVA. doi:10.1371/journal.pone.0044777.g002 progression from oxidative to glycolytic fibers entailing a switch in expression of the isoforms from MHCI.MCHIIa.MH- CIIx.MHCIIb respectively. The expression of mhcI and mhcIIa, which are expressed in oxidative fibers, in the quadriceps was similar between the control and mGHRKO mice under both RC- and HFD- fed conditions [Figure S1B, S1D]. The protein expression of mitochondrial-encoded cytochrome oxidase subunit I (COXI) was either not altered or significantly increased in the quadriceps of the mGHRKO mice relative to controls under RC- and HFD- fed conditions respectively [Figure 2C]. Further, mitochondrial density was also not different between the control and mGHRKO mice under both RC- and HFD- fed conditions [Figure 2D]. Also, staining of gastrocnemius muscles for cytochrome oxidase activity also did not reveal any significant differences between the genotypes under HFD-fed conditions [Figure S1E]. However, the HFD-fed mGHRKO mice displayed significantly higher expression of mhcIIb which is suggestive of greater glycolytic capacity [Figure S1D]. These data suggest that the loss of GHR signaling in the skeletal muscle did not affect mitochondrial capacity of the muscle under RC- and HFD- fed conditions and also indicate the existence of PGC1a-independent pathways regulating mitochondrial DNA (mtDNA) replication and transcription, as well as, muscle fiber type as will be discussed later. HFD-fed mGHRKO Mice Display Induction of Lipid Oxidation Genes in Response to Fasting Nutrient availability determines substrate preference; fasting promotes lipid utilization and activation of lipid oxidation pathways in a PGC1a-dependent manner. Thus, we subjected the HFD-fed mice to a 24 h fasting, and analyzed the induction of genes involved in the PGC1a-dependent lipid oxidation pathway. Fasting increased the expression of pgc1a, pparb/d, and ppara in both control and mGHRKO mice when compared to the fed mice of each genotype [Figure 3A, 3B, 3C]. The expression erra tended to be higher in the fasted mGHRKO mice relative to the fed mGHRKO mice, while no alteration in erra expression was PLOS ONE | www.plosone.org 3 September 2012 | Volume 7 | Issue 9 | e44777