Isolation and Proteomics of Human Skeletal Muscle Lipid Droplets Identify Anchored Mitochondria and Associated TPD52L2

Abstract Skeletal muscle is the primary site for postprandial glucose disposal, and insulin resistance in this tissue plays a central role in the pathogenesis of type 2 diabetes mellitus (T2DM). Lipid droplets (LDs) are critical for lipid storage and metabolic regulation in skeletal muscle, yet their molecular composition in human skeletal muscle remains unexplored, hindering mechanistic understanding of their role in insulin resistance. Here, we established the first comprehensive proteomic database of human skeletal muscle LDs, using an athlete cohort as a physiologically advantageous model. Our analysis identified PLIN4, rather than PLIN2 or PLIN5, as the predominant PLIN family protein on human skeletal muscle LDs. Comparative proteomics of LD-anchored mitochondria (LDAM) and whole-tissue mitochondria revealed selective enrichment of complex IV proteins within LDAM, with COX6B1 exhibiting the most pronounced increase, pointing to COX6B1 as a defining feature of skeletal muscle LDAM. Additionally, we discovered TPD52L2 as a previously uncharacterized LD-associated protein that dynamically localizes to LDs upon oleic acid treatment. Collectively, this work provides the first molecular atlas of human skeletal muscle LDs, establishing a mechanistic framework for the athlete's paradox and a critical foundation for T2DM therapeutic development.