Muscle GH Receptor Signaling and Lipid Oxidation Figure 5. Lipid handling the control and mGHRKO mice in response to 24 h fasting. 14-wk HFD-fed control and mGHRKO mice were subjected to a 24 h fasting challenge. A. Muscle TG content in the gastrocnemius was determined in the fed and fasted mice (n = 6–8/group). (B–F). Western blots were performed to determine total protein content of ATGL and HSL, and the phosphorylated levels of HSL at residues Ser660 and Ser563 in the quadriceps muscle. b-tubulin was used to determine equal loading. Levels of phosphorylated HSL at Ser660 and Ser563 were normalized to total HSL content, while total protein content of ATGL and HSL were normalized to b-tubulin. The data are represented as a fold change compared to the fed state levels for each genotype (n = 6–8/group; representative blots are provided). All values are represented as mean 6 S.E.M. *2 p#0.05 versus respective fed group, One-way ANOVA. doi:10.1371/journal.pone.0044777.g005 adipose tissue of the fasted mGHRKO mice has a higher lipolytic rate than those of their fed counterparts. GH is known to mediate lipolytic effects in the adipose tissue, thus we looked at the expression of GHR in the epididymal adipose tissue, which displays a high rate of lipolysis. Surprisingly, we found a 5- and 1.5- fold increase in mRNA expression and protein content of GHR in adipose tissue lysates of HFD-fed mGHRKO mice, respectively, as compared to the control mice [Figure 6D, 6E]. Further, GH measurements made in the mice in the fed state suggested that the HFD-fed mGHRKO mice may have a trend towards higher GH levels [Figure 6F]. Together these data suggest that GH activity is higher in the HFD-fed mGHRKO mice and this could possibly account for the higher rate of lipolysis in the fasted mGHRKO mice. Discussion We have previously shown that loss of GHR signaling in the skeletal muscle in the mGHRKO mice is associated with a marked protection from the development of HFD-induced insulin resistance, as assessed by several parameters. In addition, the HFD-fed mGHRKO mice had a higher RER in the light phase under conditions of ad libitum feeding, indicative of greater whole- body carbohydrate utilization [8]. This indicated that lipid oxidation is suppressed in the mGHRKO mice, and we explored this hypothesis in this study. Our data suggests that basal lipid oxidation is reduced in the skeletal muscle of the mGHRKO mice. However, the induction of lipid oxidation in states of increased energy demand such as fasting and exercise was similar in both the HFD- fed control and mGHRKO mice, suggesting that muscle GHR signaling is not involved in this process. Our results are in line with other studies in GHR knockout (GHRKO) and GH transgenic (GH-Tg) mice, which show an up-regulation of lipid oxidation pathways in response to 30% calorie restriction [14,15]. PGC1a is a master regulator of lipid oxidation in the skeletal muscle and exerts its effects by associating with several transcrip- tion factors. One such transcription factor is ERRa which is an orphan nuclear receptor that is highly expressed in tissues with high energy demand, in a manner similar to PGC1a. Its expression is induced by PGC1a, and it also interacts with PGC1a to activate lipid oxidation [16]. PGC1a also interacts with the PPAR family of transcription factors to regulate lipid metabolism in a tissue-specific manner. In the skeletal muscle, PGC1a/PPAR interaction results in increased FA uptake and activation of lipid oxidation [17,18]. PPARb/d has also been shown to induce PGC1a expression in the muscle [19]. Both ERRa and PPARb/d have been shown to induce the expression of PDK4 in a forkhead transcription factor (FoxO1)-dependent manner. PDK4 phosphorylates and inactivates pyruvate dehydro- genase complex resulting in a suppression of glucose oxidation [18,20]. While we did not find changes in pgc1a expression, the expression of erra, pparb/d, and pdk4 expression were significantly PLOS ONE | www.plosone.org 6 September 2012 | Volume 7 | Issue 9 | e44777