PROJECT SUMMARY / ABSTRACT In the US, approximately 0.7-1.8% or ~ 275,000 youth identify as transgender, a number that is likely to increase with greater recognition of this condition. Gender affirming hormone therapy (GAHT), which attempts to more align the physical appearance with the identified gender, is the primary medical intervention for transgender people and is recognized as medically necessary. GAHT has been associated with increased cardiometabolic risk (increased blood pressure, dyslipidemia, insulin resistance, and endothelial dysfunction) in transgender adults. While treatment standards have evolved to initiate GAHT in adolescence, little is known about the cardiometabolic changes that occur with GAHT in adolescence, a critical gap in the optimal care of trans youth. For the first time using state-of-the-art techniques, this proposal aims to characterize the metabolic and cardiovascular changes that occur in trans youth with the initiation of GAHT. We hypothesize that the altered hormonal milieu is the major driver of increased cardiometabolic risk in transgender youth. The initiation of testosterone or estrogen will have characteristic effects on markers of insulin resistance, lipid profiles, vascular health, and endothelial function. Transgender subjects will be examined twice: prior to the initiation of GAHT and again six months later when hormone replacement strategies typically result in hormonal levels comparable to cis- gender adults. Using the hyperinsulinemic-euglycemic clamp technique with stable isotope tracers, precise peripheral and hepatic insulin sensitivity measures will be quantified and analyzed to isolate the effects of exogenous hormones (testosterone and estrogen). Additional metabolic metrics will be determined from oral glucose tolerance tests (whole-body insulin sensitivity, insulinogenic index) and Bod Pod (body composition) performed at baseline and six months. The conduit-level endothelial function will be assessed by flow-mediated vasodilation of the brachial artery, and microvascular endothelial function (skin blood flow) will be determined by local forearm skin heating with laser Doppler flowmetry, at baseline and after six months of GAHT. Over time, we will compare the cardiometabolic changes in transgender subjects to those of cisgender male and female controls. These novel and innovative studies will illuminate the early metabolic and vascular changes accompanying GAHT, enabling a more informed understanding of the cardiometabolic risks in trans youth undergoing treatment. This is a critical gap in our knowledge; once these early changes can be characterized, subsequent studies can track these changes over longer treatment trajectories, and effective interventions to reduce adverse metabolic and cardiovascular outcomes can be designed and evaluated.