Muscle GH Receptor Signaling and Lipid Oxidation Figure 6. Adipose tissue of the fasted mGHRKO mice is more lipolytic than that of control mice. A. FFA levels were determined in the serum of 10–12-wk HFD-fed mice at baseline (0), after 24 h fasting (24), and after 3 h of re-feeding (+3). B. The suppression of lipolysis was determined as the percent change in FFA levels at the start and end of the re-feeding phase. C. FFA secretion rate was determined in adipose tissue explants of fed and 24 h fasted 14wk HFD-fed mice either in the absence (2Iso) or presence (+Iso) of 1 mM isoproterenol (n = 2–4/group). D. mRNA expression of ghr was determined by real-time PCR in the epididymal adipose tissue of 14wk-HFD-fed mice (n = 7/group). Each sample was probed three times and gene expression was normalized to gapdh and represented as a fold change when compared to control. E. Protein content of GHR was determined in protein lysates isolated from epididymal adipose tissue 14wk-HFD-fed control and mGHRKO mice. Liver lysate was used as a positive control (+) (n = 11–15/group, representative blots are shown). b-actin was used to determine equal loading (top). The protein content of GHR was normalized to that of b-actin, and represented as a fold change compared to controls (bottom). F. Serum GH levels in the fed state were measured in 14-wk HFD-fed control and mGHRKO mice. Each point on the graph represents an individual mouse (n = 6–8/group). All values are represented as mean 6 S.E.M. *2 p#0.05 HFD-fed control versus HFD-fed mGHRKO, #2 p#0.05 mGHRKO/fed versus mGHRKO/fasted, @2 p#0.05 control/fed versus mGHRKO/fed, One-way ANOVA. doi:10.1371/journal.pone.0044777.g006 reduced in muscles of the HFD-fed mGHRKO mice, suggesting a suppression of lipid oxidation in the muscles of the HFD-fed mGHRKO mice. Additionally, we observed higher expression of the mhcIIb isoform in the quadriceps muscles of the HFD-fed mGHRKO mice, suggesting an increased glycolytic capacity which is also in agreement with the higher RER in the HFD-fed mGHRKO [8]. Any changes in gene expression in the HFD-fed mGHRKO mice could be attributed to their improved insulin sensitivity. We have previously shown that the mGHRKO mice have only a mild improvement in insulin sensitivity under RC-fed conditions [8]. Thus, in our initial analysis we included a cohort of RC-fed mice to determine if intrinsic changes exist in the mGHRKO muscles. Indeed, we found a reduction in the expression of erra and pdk4 in the muscles of the RC-fed mGHRKO mice, suggesting that these effects were mediated by the direct loss of GHR signaling in the muscle. It is also likely that the changes in gene expression in the RC-fed mGHRKO mice do not manifest in an overt metabolic phenotype, but in the setting of HFD-induced obesity, lower expression of PGC1a-dependent lipid oxidation genes may in fact be metabolically beneficial. This hypothesis is in line with a report of the lack of an effect of GH treatment on muscle lipid and carbohydrate oxidation in normal, healthy rats [21]. PGC1a has also been shown to regulate mitochondrial biogenesis by the activating the transcription factor NRF1 via direct interaction or indirectly via ERRa [22,23]. NRF1 activates mitochondrial transcription factors such as TFAM which promote mtDNA replication and stabilization [24]. Despite lower expres- sion of nrf1 and tfam in the HFD-fed mGHRKO mice relative to the controls, we did not detect any changes in mitochondrial content or cytochrome oxidase activity. This observation supports the existence of alternative pathways that could regulate mtDNA replication and maintenance independent of PGC1a. Heterozy- gous loss of TFAM in mice did not result in a significant reduction in mtDNA copy number in the skeletal muscle, despite lower mtDNA copy number in other tissues like the heart and kidney PLOS ONE | www.plosone.org 7 September 2012 | Volume 7 | Issue 9 | e44777