Sodium-glucose cotransporter-2 inhibitors (SGLT2i) are type-2 diabetes (T2D) medications designed to promote urinary glucose excretion that are increasingly associated with unanticipated health benefits. Growing evidence indicates SGLT2i have off-target effects that improve insulin action, including studies in humans and mouse models of obesity. The effect has been attributed to enhanced skeletal muscle insulin action, yet remains untested. The mechanistic explanation as to how SGLT2i may enhance skeletal muscle insulin action also remains unknown. These gaps in knowledge indicate unrealized therapeutic potential of SGLT2i. Taken together, there is a critical need to determine the impact and underlying mechanisms of SGLT2i treatment on regulation of skeletal muscle metabolism. Our long-term goal is to identify strategies to improve skeletal muscle metabolic health and lower disease risk. Our objective in this application is to determine mechanisms by which SGLT2i modulate skeletal muscle metabolism as part of an expanded model of SGLT2i treatment. Our overall hypothesis is that SGLT2i have direct actions on skeletal muscle to increase glucose uptake as a function of inhibiting mitochondrial complex I and activation of AMP-activated protein kinase (AMPK). The hypothesis is supported by strong preliminary data. We provide new evidence that SGLT2i treatment improves glucose uptake in skeletal muscle as a function of enhanced insulin signaling. We demonstrate these effects are likely attributable to direct actions on skeletal muscle, whereby all 4 FDA-approved SGLT2i cause dose- dependent inhibition of mitochondrial complex I and activation of AMPK. Importantly, our new findings of off- target mechanisms of action of SGLT2i on muscle identify clinical relevance to determine overlap with metformin (a common T2D co-therapy) on complex I metabolism. The following aims test specific hypotheses of our overall model. Aim 1: Determine the role of complex I inhibition for SGLT2i regulation of skeletal muscle metabolism. We will determine the effects of SGLT2i treatments on skeletal muscle mitochondrial bioenergetics and insulin action in western diet fed mice using both acute and chronic treatment strategies. Aim 2: Identify the requirement of AMPK for SGLT2i regulation of skeletal muscle metabolism. We will use an inducible, skeletal muscle-specific AMPK knockout model then determine glucose uptake in isolated tissues and mitochondrial bioenergetics. Aim 3: Identify combination of canagliflozin and metformin treatment on complex I metabolism. We will treat mice with metformin then determine combination with either canagliflozin or empagliflozin on complex I respiratory function in skeletal muscle and liver. The proposed studies can be expected to provide new information regarding how SGLT2i regulate skeletal muscle metabolism and contribute to improved metabolic health. The work will establish a new proposed mechanism of action for a widely used class of me...