PROJECT SUMMARY/ABSTRACT Insulin resistance is a key driver of type 2 diabetes (T2D). Interestingly, people living with HIV (PLWH) are prone to developing insulin resistance and T2D, even with viral suppression. In determining what drives this insulin resistance, we have focused on the fact that whereas obesity is a common trait in humans, T2D risk is increased by “metabolically unhealthy obesity” (MUO). Emerging observations point to two factors promoting MUO in the general population: 1) reduced mitochondrial content and fewer mitochondria-enriched “beige” adipocytes in the subcutaneous fat (SCAT) and 2) Development of SCAT fibrosis. Moreover, reducing beige fat biogenesis in mice promotes SCAT fibrosis and insulin resistance, whereas increasing it remarkably blocks fibrosis and improves insulin sensitivity. With this in mind, we and others have observd that PLWH have markedly increased SCAT fibrosis, a response replicated in SIV-infected macaques. This prompts us to wonder whether PLWH also have suppressed beige adipocyte biogenesis, and what factors dictate this. To this end, we recently identified a subset of adipogenic precursor cells (APCs) expressing signature markers (Lin–: PDGFRa+: CD81+) that control a reciprocal balance between beige fat biogenesis and SCAT fibrosis. Specifically, CD81+ APCs give rise to mitochondria-rich beige adipocytes in the SCAT, whereas CD81 deficiency causes both SCAT fibrosis and insulin resistance in mice. These data highlight the intriguing possibility that CD81+ APCs in the SCAT may influence development of MUO in PLWH. We also reported that beige/brown fat is a metabolic sink for branched- chain amino acids (BCAAs), the circulating levels of which are linked to human insulin resistance. Activating brown/beige fat biogenesis clears circulating BCAAs in humans; indeed, BCAA clearance is a noninvasive indicator of brown/beige fat activity. However, the impact of BCAA usage in fat has not been assessed in PLWH. We hypothesize that a reduced capacity of CD81+ APCs to maintain normal SCAT function, reflected by SCAT fibrosis and impaired BCAA clearance, drives insulin resistance in PLWH, and that reversing this may improve metabolic health. Leveraging an innovative cohort of PLWH and uninfected controls, including those with MUO, we propose to test this hypothesis by pursuing two aims, each integrating a comprehensive human study with mechanistic studies in innovative mouse models. In Aim 1, we examine the extent to which loss of CD81+ APC abundance, proliferative capacity, or differentiation potential predicts SCAT fibrosis and insulin resistance in PLWH, and whether manipulating these cells can modulate SCAT fibrosis in mice. In Aim 2, we probe adipose clearance of BCAAs as a determinant of glucose homeostasis in PLWH, and determine the impact of disrupting beige/brown fat BCAA usage on SAT fibrosis and metabolic health in mice. By validating a mechanistic index of SCAT health (CD81+ APCs, fibrosis, BCAA clear...