seen in the fasted control mice [Figure 3D]. pdk4 expression was significantly increased in both the fasted controls and mGHRKO mice [Figure 3E]. Fasting significantly increased the expression of sirt3 in the muscles of the HFD-fed mGHRKO mice, and this was not evident in the fasted control mice [Figure 3F]. Interestingly, there was no difference in basal sirt3 expression in the control and mGHRKO muscles in the fed state [Figure S2]. PGC1a is activated by deacetylation and phosphorylation which are mediated by SIRT1 and AMP-activated protein kinase (AMPK). While there was no difference in the basal expression of sirt1 in the fed state [Figure S2], fasting was associated with a significant induction of sirt1 only in the mGHRKO mice, but not the control mice [Figure 3G]. Further, there was no significant difference in AMPK phosphorylation upon fasting in both the genotypes [Figure 3H]. Thus, despite lower basal expression, the HFD-fed mGHRKO mice were able to up-regulate the expression of lipid oxidation genes in response to fasting. Muscle GH Receptor Signaling and Lipid Oxidation HFD-fed mGHRKO Mice Exhibit Increased Lipid Utilization during a Treadmill Running Challenge Having observed activation of lipid oxidation genes in the muscles of the HFD-fed mGHRKO mice in response to fasting, we subjected the HFD-fed mice to a treadmill running challenge after two training sessions to stimulate lipid utilization. During the study, the HFD-fed mGHRKO mice displayed a significant increase in oxygen consumption (VO2) and energy expenditure, normalized to body weight, indicating that they have greater aerobic capacity than the control mice [Figure 4A, 4B]. Additionally, the mGHRKO mice demonstrated lower RER indicating their reliance on lipids as the predominant fuel source [Figure 4C]. We also measured 24 h locomotor activity and found that the HFD-fed mGHRKO had significantly greater dark phase locomotor activity when compared to the controls [Figure 4D]. Thus, the HFD- fed mGHRKO mice are able to activate lipid oxidation in response to exercise, perhaps to a greater extent than HFD- fed control mice. Figure 3. Activation of lipid utilizing pathways in muscles of HFD-fed control and mGHRKO mice in response to 24 h fasting challenge. 14-wk HFD-fed control and mGHRKO mice were subjected to a 24 h fasting challenge. (A–G). mRNA expression of indicated genes in quadriceps muscles of fed, and 24 h fasted mice (n = 6–8/group). Each sample was probed three times and gene expression was normalized to b- actin and represented as a fold change when compared to the fed mice for each genotype. H. Phosphorylation of AMPKa at Thr172 was determined in protein lysates isolated from quadriceps muscle of fed and fasted control and mGHRKO mice. Levels of phosphorylated AMPKa was normalized to total protein content of AMPKa and represented as a fold changed compared to fed mice of each genotype. b- tubulin was used to determine equal loading (n = 6–8/group; representative blots are provided). All values are represented as mean 6 S.E.M. *2p#0.05 versus respective fed group, #2p#0.05 fasted control versus fasted mGHRKO, Two-way ANOVA. doi:10.1371/journal.pone.0044777.g003 PLOS ONE | www.plosone.org 4 September 2012 | Volume 7 | Issue 9 | e44777