Muscle GH Receptor Signaling and Lipid Oxidation Figure 4. Increased lipid utilization during a treadmill running challenge and greater locomotor activity in the HFD-fed mGHRKO mice. 14wk-HFD-fed control and mGHRKO mice were subjected to a treadmill running study. A. Oxygen consumption (VO2), B. Energy expenditure after normalization to BW, and C. Respiratory exchange ratio were measured during the treadmill running study (n = 8/group). D. 24 h locomotor activity (left) was measured as beam breaks in the horizontal and vertical axes in 14wk-HFD-fed control and mGHRKO mice, and summarized as light phase, dark phase and total locomotor activity (right). Bar represents dark phase. All values are represented as mean 6 S.E.M. *2p#0.05, One-way (D) or Two-way (A–C) ANOVA. doi:10.1371/journal.pone.0044777.g004 Adipose Tissue of HFD-fed mGHRKO Mice Demonstrates Increased Lipolysis under Fasted Conditions 24 h fasting resulted in slightly lower intramuscular TG content in both the control and mGHRKO mice [Figure 5A]. This was further associated with a significant increase in the protein content of the triglyceride (TG) hydrolase, adipose triglyceride lipase (ATGL) in the muscles of the HFD-fed control mice but not the HFD-fed mGHRKO mice [Figure 5B, 5C]. We also looked at the activation status of the other important TG hydrolase, hormone sensitive lipase (HSL). The control, but not mGHRKO, mice demonstrated significantly higher phosphorylation of HSL at residue Ser660, but not Ser563 [Figure 5B, 5D, 5E]. Interestingly, fasting significantly decreased the total protein content of HSL in the control mice, but this was not observed in the mGHRKO mice [Figure 5B, 5F]. Further we detected a trend towards higher protein content of the FA transporter (FAT/CD36) in the skeletal muscle of fasted mGHRKO, but not control, mice relative to their fed counterparts (data not shown). These data suggest that fasting did not significantly induce lipolysis of intramuscular TG in the mGHRKO mice, and perhaps there was a greater influx of FA from the circulation. Interestingly, the HFD- fed mGHRKO mice had significantly higher serum FFA levels after a 24 h fast [Figure 6A]. We also observed a similar rise in FFA levels in the RC-fed mGHRKO relative to the control mice, suggesting that this adaptation was a consequence of loss of muscle GHR signaling and independent of diet [Figure S3A]. Re-feeding for 3 h was associated with significantly greater suppression of lipolysis in the HFD-fed mGHRKO mice, which is in line with their improved insulin sensitivity [Figure 6B]. The lack of an effect of re-feeding on the FFA levels in the HFD-fed control mice was not due to a reduction in food intake or low insulin levels but mainly due to their insulin resistance [Figure S3B, S3C, S3D]. The elevation in FFA levels in the mGHRKO mice at the end of the 24 h fasting period suggested higher FFA flux from the adipose tissue. Thus, we analyzed FFA secretion from adipose tissue explants of fed and fasted HFD-fed control and mGHRKO mice over a period of 3 hours. Adipose tissue explants of 24 h fasted mGHRKO mice demonstrated significantly greater rate of FFA secretion when compared to those from the fed mGHRKO mice; while there was no difference in FFA secretion between the fed and fasted control explants. Interestingly, the FFA secretion rate from the explants of fed mGHRKO mice was significantly lower than that from the fed control mice. However, when lipolysis was stimulated with a b-adrenergic receptor agonist (isoproterenol) all groups, except the fed controls, displayed a significant increase in the rate of FFA secretion, indicating that the mice were responsive to parasympathetic cues [Figure 6C]. Thus, the PLOS ONE | www.plosone.org 5 September 2012 | Volume 7 | Issue 9 | e44777