Lipid Droplet Proteome Reveals that Associated ATGL and Anchored Mitochondria Lead to Higher Skeletal Muscle Insulin Sensitivity in Endurance Athletes than Type 2 Diabetes Mellitus

As the key organelle governed lipid homeostasis, lipid droplets (LDs) in skeletal muscle plays a critical role in regulating systemic insulin sensitivity. In type 2 diabetes mellitus (T2DM) patients intramyocellular lipid (IMCL) content negatively associates with insulin sensitivity, while endurance athletes exhibit IMCL levels were positively correlated to insulin sensitivity, which is known as the athletes paradox. To solve this paradox, LDs were isolated from skeletal muscle samples of seven athletes and nine T2DM patients, and the first proteome dataset of human skeletal muscle LD was established. Comparative proteomic analysis revealed 733 upregulated proteins in athlete LDs, primarily enriched in mitochondria, and 755 downregulated proteins, mainly associated with cytoskeletal components, relative to T2DM patients. Integrated analysis with tissue proteomics further indicated enhanced energy metabolism activity of LD-anchored mitochondria (LDAM) in athletes. Notably, adipose triglyceride lipase (ATGL) was specifically upregulated by 2.97-fold on LDs in athletes, while its overall tissue expression remained unchanged. Moreover, deletion of ATGL in myoblasts significantly reduced insulin-stimulated AKT phosphorylation. Additionally, skeletal muscle LDs from db/db and ob/ob mice exhibited reduced levels of mitochondrial proteins and ATGL. Together, our findings reveal a LD-based solution for the athletes paradox, and identify ATGL as a key regulator of skeletal muscle insulin sensitivity. The results also suggest that the "bad LDs" in the skeletal muscle of T2DM patients are characterized by higher levels of cytoskeletal proteins, reduced LDAM, and lower ATGL compared to the "good LDs" observed in athletes.