Diabetes results from deficiencies in insulin production and/or insulin signaling. Insulin signals adipocytes to alter the expression of genes for enzymes and hormones that regulate energy balance. Transcription factors that control the expression of these genes include peroxisome proliferator activated receptor gamma (PPARy) and CCAAT enhancer binding protein alpha (C/EBPa). Insulin signaling alters the phosphorylation status of C/EBPa. PPARy is the receptor for thiazolidenediones (TZDs). PPARy heterodimerizes with RXR, the receptor for 9-cis retinoic acid, which enhances the insulin-sensitizing actions of TZDs. The interactions of these factors with themselves, their ligands and co-activators in response to insulin are poorly defined. Understanding these interactions in the cellular milieu will lead to improved insulin-sensitizing therapies.We uniquely have developed powerful fluorescence microscopy techniques that measure the amounts, structure and interactions of proteins at tens of thousands of locations within cells. Transcription factors and co-factors involved in insulin regulation will be tagged with spectrally distinct derivatives of green fluorescent protein (GFP), and expressed pairwise in 3T3-L1 pre-adipocyte/adipocyte model cells. The relative locations of each factor will be determined microscopically within the living cell by comparing the locations of fluorescence emitted from each fluorophore-factor fusion. Factor location will be compared to gene location by in situ hybridizations in fixed cells. Direct interactions between, and conformations within, the factors will be measured at each subcellular location as the degree to which fluorescence energy excited in the fluorophore attached to one factor (or factor domain) is transferred to a fluorophore attached to the second factor (or domain). Thus, regulation of these cooperative factors will be determined, in 3T3-L1 cells, by measuring the separate and combined effects of insulin, the TZD rosiglitazone and 9-cis retinoic acid on the:1. location, dimerization and interactions of C/EBPa, PPARg, RXRa and their co-activators PGC-1, PGC-2, SRC-la, CBP and TRAP2202. conformation of C/EBPa, PPARg, RXRa and the same co-activators, and3. conformations, dimers and interactions of the above factors specifically in the neighborhood of the genes that they regulate.
