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Insulin increases cellular glucose uptake and metabolism in the postprandial state by acutely stimulating the translocation of the Glut4 glucose transporter from intracellular membrane compartments to the cell surface in muscle and fat cells. The intracellular targeting of Glut4 is dictated by specific structural motifs within cytoplasmic domains of the transporter. We demonstrate that two leucine residues at the extreme C-terminus of Glut4 are critical components of a motif (IRM, insulin responsive motif) involved in the sorting of the transporter to insulin responsive vesicles in 3T3L1 adipocytes. Light microscopy, immunogold electron microscopy, subcellular fractionation, and sedimentation analysis indicate that mutationsmore » in the IRM cause the aberrant targeting of Glut4 to large dispersed membrane vesicles that are not insulin responsive. Proteomic characterization of rapidly and slowly sedimenting membrane vesicles (RSVs and SSVs) that were highly enriched by immunoadsorption for either wild-type Glut4 or an IRM mutant revealed that the major vesicle fraction containing the mutant transporter (IRM-RSVs) possessed a relatively small and highly distinct protein population that was enriched for proteins associated with stress granules. We suggest that the IRM is critical for an early step in the sorting of Glut4 to insulin responsive subcellular membrane compartments and that IRM mutants are miss-targeted to relatively large, amorphous membrane vesicles that may be involved in a degradation pathway for miss-targeted or miss-folded proteins or represent a transitional membrane compartment that Glut4 traverses en route to insulin responsive storage compartments.« less

Prior research demonstrated that environmental enrichment creates individual differences in behavior leading to a protective addiction phenotype in rats. Understanding the mechanisms underlying this phenotype will guide selection of targets for much-needed novel pharmacotherapeutics. The current study investigates differences in proteome expression in the nucleus accumbens of enriched and isolated rats and the proteomic response to cocaine self-administration using a liquid chromatography mass spectrometry (LCMS) technique to quantify 1917 proteins. Results of complementary Ingenuity Pathways Analyses (IPA) and gene set enrichment analyses (GSEA), both performed using protein quantitative data, demonstrate that cocaine increases vesicular transporters for dopamine and glutamate asmore » well as increasing proteins in the RhoA pathway. Further, cocaine regulates proteins related to ERK, CREB and AKT signaling. Environmental enrichment altered expression of a large number of proteins implicated in a diverse number of neuronal functions (e.g., energy production, mRNA splicing, and ubiquitination), molecular cascades (e.g., protein kinases), psychiatric disorders (e.g., mood disorders), and neurodegenerative diseases (e.g., Huntington’s and Alzheimer’s diseases). Upregulation of energy metabolism components in EC rats was verified using RNA sequencing. Most of the biological functions and pathways listed above were also identified in the Cocaine X Enrichment interaction analysis, providing clear evidence that enriched and isolated rats respond quite differently to cocaine exposure. The overall impression of the current results is that enriched saline-administering rats have a unique proteomic complement compared to enriched cocaine-administering rats as well as saline and cocaine-taking isolated rats. These results identify possible mechanisms of the protective phenotype and provide fertile soil for developing novel pharmacotherapeutics. Proteomics data are available via ProteomeXchange with identifier PXD000990.« less