Insulin regulates lymphatic endothelial integrity via palmitoylation.

Lipid metabolism plays a critical role in lymphatic endothelial cell (LEC) development and vessel maintenance. Altered lipid metabolism is associated with loss of lymphatic vessel integrity, which compromises organ function, protective immunity, and metabolic health. Thus, understanding how lipid metabolism affects LECs is critical for uncovering the mechanisms underlying lymphatic dysfunction. Protein palmitoylation, a lipid-based post-translational modification, has emerged as a critical regulator of protein function, stability, and interaction networks. Insulin, a master regulator of systemic lipid metabolism, also regulates protein palmitoylation. However, the role of insulin-driven palmitoylation in LEC biology remains unexplored. To examine the role of palmitoylation in LEC function, we generated the first palmitoylation proteomics profile in human LECs, validated insulin-regulated targets, and determined the role of palmitoylation in LEC barrier function. In unstimulated conditions, palmitoylation occurred primarily on proteins involved in vesicular and membrane trafficking, and in translation initiation. Insulin treatment, instead, enriched palmitoylation of proteins involved in LEC integrity, namely junctional proteins such as claudin 5, along with small GTPases and ubiquitination enzymes. We also investigated the role of the long-chain fatty acid transporter CD36, a major mediator of palmitate uptake into cells, in regulating optimal lymphatic protein palmitoylation. CD36 silencing in LECs increased by 2-fold palmitoylation of proteins involved in inflammation and immune cell activation. Overall, our findings provide novel insights into the intricate relationship between lipid modification and LEC function, suggesting that insulin and palmitoylation play a critical role in lymphatic endothelial function.