The overall goal of this research is to elucidate mechanisms involved in the control of metabolism by insulin in muscle and fat cells. A defect in the ability of these cells to respond to insulin is a primary cause of Type 2 diabetes mellitus, which in turn is a leading cause of vision disorders, neuropathy, kidney disease, peripheral vascular disease, and heart disease. This proposal is to investigate lipin and mTORC2, two new targets of insulin action. Lipin is the protein product of the gene that is mutated in Lpn1fld/fld mice, and mTORC2 is a newly discovered rapamycin-insensitive signaling complex that controls both the actin cytoskeleton and phosphorylation of Akt. Lpn1fld/fld mice exhibit fatty liver, defective adipogenesis, glucose intolerance and insulin resistance. It is clear from these abnormalities that lipin is essential for normal insulin action;however, the biochemical function of lipin is unknown. Defining this function will be a major objective. Lipin is phosphorylated in response to insulin.
In AIM 1 a plan involving peptide mapping, site directed mutagenesis, and mass spectrometry is presented to determine the sites of phosphorylation. Experiments to identify the kinases that phosphorylate lipin are also described.
AIM 2 is to determine the mechanism of action of lipin. Preliminary results indicate that lipin interacts with NFAT3, a transcription factor that has been implicated in the control of PPAR?2 expression and adipogenesis. Other findings, including results with the S. cerevesiae lipin, Smp2, provide a strong reason to test the hypothesis that lipin represses ChREBP, a transcription factor that promotes expression of multiple genes encoding enzymes involved in lipogenesis. The role of lipin phosphorylation on lipin interactions with ChREBP and NFAT3 will be investigated. ChIP analyses are proposed to determine whether lipin associates with the promoter regions of genes controlled by NFAT3 or ChREBP, and reporter assays are described to determine whether lipin enhances or represses the activity of these transcription factors. Since other proteins that interact with lipin may hold the key to lipin function, we will search for new interacting proteins by using multiple approaches including identification of proteins that co purify with lipin or that bind to a lipin-agarose resin.
AIM 3 is to test the hypothesis that mTORC2 is a mediator of the metabolic effects of insulin. Glucose transport and oxidation, lipid and glycogen synthesis, GLUT4 translocation, and several parameters of insulin signaling will be measured after increasing or decreasing levels of rictor, the defining subunit of mTORC2. Lentivirus will be used to overexpress rictor or to express shRNA to knockdown rictor in 3T3-L1 adipocytes. To investigate mTORC2 function in vivo, we propose to knockout rictor in adipocytes and skeletal muscle of mice.
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