Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. Over 130 million Americans suffer from the devastating consequences of type 2 diabetes (T2D) or prediabetes. Skeletal muscle accounts for up to 85% of insulin-induced blood glucose clearance, and insulin resistance for muscle glucose uptake is an essential, and perhaps primary defect for T2D. One exercise bout can enhance subsequent insulin-stimulated glucose uptake (ISGU) by muscle, but the mechanisms have remained elusive. The long-range goal is to fully understand the molecular, cellular, and physiological events responsible for this significant health benefit. Recent research using a unique Akt substrate of 160 kDa-knockout (AS160-KO) rat model revealed that expression of AS160 (a key regulator of GLUT4 glucose transporter localization) is essential for the elevated postexercise ISGU. Specific Aim 1 will identify specific Rab proteins that collaborate with AS160 to regulate postexercise insulin-stimulated glucose uptake by skeletal muscle. Research using L6 myocytes identified the key Rab proteins that regulate insulin-stimulated GLUT4 translocation, but the specific Rab proteins required for AS160-dependent regulation of ISGU in authentic skeletal muscle are unknown. An adeno-associated virus (AAV) vector-approach will be used to identify the roles of these Rab proteins on ISGU in muscles from sedentary and exercised rats. Specific Aim 2 will elucidate AS160’s regulation of subcellular GLUT4 localization in muscle postexercise. Knowledge of AS160’s role in GLUT4 distribution is limited to insulin’s ability to elevate GLUT4 exocytosis to cell surface membranes in unexercised muscle. A powerful new microscopy-based approach (STERM, Sample Thinning Enhanced Resolution Microscopy) will examine GLUT4 distribution in 7 different myocellular compartments. Coupling STERM with the AS160-KO model will be used to test if AS160 is crucial for postexercise regulation of GLUT4 distribution in both cell surface and intracellular membrane compartments. We will also determine insulin and exercise effects on AS160’s distribution in intracellular compartments. Specific Aim 3 will elucidate estrogen receptor dependent mechanisms contributing to greater postexercise insulin-stimulated glucose uptake by skeletal muscle. Although AS160 expression is essential for elevated postexercise ISGU in both sexes, AS160 phosphorylation of key sites is required only in male rats. Estrogen receptor (ER) mediated processes are major regulators of muscle ISGU under resting conditions, but the ER’s role for enhanced PEX-ISGU is unknown. We will use an AAV-approach to alter skeletal muscle expression of ER and probe pAS160’s sexually dimorphic role on PEX-ISGU. We will then identify ER-linked differences in skeletal muscle protein expression. We will also determine the extent to which ER-linked effects on muscle expression of specific proteins are r...