The long-range goal of this revised project is to understand major molecular mechanisms that regulate insulin-sensitivity and metabolic flux in adipocytes. Disruptions in adipocyte glucose metabolism in mice can cause muscle insulin resistance and diabetes, while augmenting glucose utilization in adipocytes enhances whole body glucose tolerance. Thus, the identification and detailed characterization of novel genes and regulators of adipocyte metabolism are fundamental objectives in this field. Exciting preliminary data we obtained using high-throughput RNAi-based screens have revealed two proteins as novel global regulators of glucose and fatty acid oxidation in adipocytes - the corepressor RIP140 and the protein kinase MAP4K4. Our data suggest the hypothesis that the actions of both RIP140 and MAP4K4 converge at PPAR-?, which is negatively regulated by these proteins, and these actions may be highly integrated. Silencing either RIP140 or MAP4K4 in cultured adipocytes: 1.) enhances expression of enzymes in carbohydrate and fatty acid oxidation, 2.) enhances insulin-sensitive glucose uptake, and 3.) enhances expression of enzymes that control mitochondrial biogenesis and oxygen consumption. Markedly increased oxidative metabolism is observed in adipocytes lacking RIP140. Strikingly, RIP140 KO mice are resistant both to obesity and to glucose intolerance when fed a high fat diet. We propose here to identify the set of genes that is common to RIP140 and MAP4K4 repression and TZD regulation to test the hypothesis that PPAR? is a target of their regulation. Two mechanisms of potential PPAR? repression will be evaluated: 1.) possible direct interactions between PPAR? and RIP140 or MAP4K4, and, 2.) direct phosphorylation of PPAR? or regulators of PPAR? such as RIP140 itself by MAP4K4. ChIP analysis of promoter regions of PPAR?-sensitive and insensitive genes will be performed to determine whether RIP140 or MAP4K4 are present in complexes with PPAR? at these promoters. Experiments to probe molecular mechanisms of RIP140 and MAP4K4 actions on PPAR? are now proposed, using a confirmed TZD-sensitive luciferase-based reporter of PPAR? promoter activity in adipocytes. Mass spectrometry will identify possible phosphorylation sites on RIP140, PPAR?, and other potential substrates of MAP4K4, followed by their functional analysis in transcriptional regulation. We shall also test the molecular basis of TZD action on PPAR? as it relates to MAP4K4 and RIP140 functions. Finally, newly proposed studies address how the metabolic actions of RIP140 are linked to glucose tolerance in mice. We shall test the hypothesis that greatly enhanced fatty acid oxidation in fat and perhaps muscle in RIP140 KO mice releases the inhibition by fatty acid derivatives on insulin signaling. These studies will reveal underlying mechanisms of action of these novel global regulators of adipocyte metabolism and function.