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Western blots were performed as previously described (Buettner et?al., 2008). Detailed description and antibodies used can be found in Supplemental Experimental Procedures. All data are represented as mean �� SEM. Comparisons among groups were made using one-way ANOVA followed by unpaired two-tailed Student's t tests if not otherwise indicated. Differences were considered statistically significant BYL719 supplier at p?SKI-606 order making available the NIRKO mice; Case Western University MMPC, which is supported by U24 DK76169, for the mass spectrometry analyses; and the Yale Center of Clinical Investigations, which is supported by CTSA Grant UL1 RR024139 from the National Center for Research Resources, for the NE measurements. We further thank Nir Barzilai and Radhika Muzumdar for WAT from calorically restricted, aged rats and Gary Schwartz for his advice and helpful discussions. This work was supported by NIH grants DK074873, DK083568, and DK082724 to C.B. and DK073683 to S.J.F. and by a European Foundation for the Study of Diabetes grant to T.S. C.B. is the recipient of a Junior Faculty Award from the American Diabetes Association. ""Increased energy intake and decreased energy expenditure contribute to the development of obesity and its comorbidities, such as type 2 diabetes, making this problem one of the most topical health issues facing Western society. Alteration in energy balance is the Pramipexole main determinant of body composition and body weight, and is coordinated by central nervous system (CNS) pathways, including the central melanocortin system (Cone, 2005, Cone, 2006?and?Morton et?al., 2006). An important component of this system is the melanocortin-4 receptor (MC4R), which is expressed by a number of neurons in the CNS (Mountjoy et?al., 1994, Kishi et?al., 2003?and?Liu et?al., 2003). The role of MC4Rs in regulation of energy balance is demonstrated by severe obesity in MC4R knockout mice, in which obesity is caused by the combined effects of increased food intake and decreased energy expenditure (Huszar et?al., 1997, Chen et?al., 2000?and?Ste Marie et?al., 2000). Importantly, this obesity syndrome also occurs in humans with MC4R mutations (Vaisse et?al., 1998?and?Yeo et?al., 1998). The role of melanocortins in regulating energy balance has also been demonstrated through pharmacologic experiments.