PROJECT SUMMARY Clinical hypertriglyceridemia is common and is only partially responsive to available drugs. Conversely, excess triglyceride delivery to individual tissues can lead to skeletal muscle insulin resistance, cardiac lipotoxicity, or severe inflammatory responses. Given the importance of proper triglyceride delivery, it is imperative that we understand the mechanisms that regulate this process. Two proteins normally required for triglyceride clearance are lipoprotein lipase (LPL) and GPIHBP1. LPL is required for the actual hydrolysis of plasma triglyceides, liberating fatty acids for tissue uptake. The endothelial cell protein GPIHBP1 is required to properly localize LPL to the vascular lumen where lipolysis takes place. The long term goal of the proposed research is to understand and ultimately modulate the delivery of triglyceride-derived fatty acids to specific tissues. The objectives in this application are to advance our understanding of how the angiopoietin-like (ANGPTL) proteins, ANGPTL3 and ANGPTL4, modulate triglyceride clearance. ANGPTL3 and ANGPTL4 can both inhibit LPL in vitro, and ANGPTL3 or ANGPTL4 deficiency leads to lower plasma triglyceride levels. However, if and how ANGPTL3 and ANGPTL4 directly modulate LPL/GPIHBP1-mediated triglyceride clearance is not clear. Moreover, the plasma triglyceride levels in mice deficient in both GPIHBP1 and ANGPTL4 indicate that ANGPTL4 may regulate an uncharacterized, GPIHBP1-independent triglyceride clearance pathway. These issues will be addressed by pursuing two specific aims: 1) Define the mechanisms by which ANGPTL3 and ANGPTL4 modulate GPIHBP1-dependent triglyceride clearance; and 2) Identify novel mechanisms of GPIHBP1- independent triglyceride clearance. The studies in aim 1 employ cell culture–based binding and lipase activity assays, as well as in vivo injection of recombinant proteins and tissue-specific knockout mice to investigate how ANGPTL proteins interact with GPIHBP1/LPL complexes on the surface of endothelial cells. The second aim will address the remarkably lower triglycerides in Angptl4–/–Gpihbp1–/– mice. Triglyceride clearance and fatty acid uptake assays will be used to identify the tissues to which triglycerides get cleared. Expression, protein, and lipase activity analysis will be used to identify the proteins responsible for this clearance. The approaches in the proposed research are innovative because they combine standard methods with novel mouse models to address previously untested questions about how triglyceride clearance is regulated. The proposed studies are significant because they will elucidate how and where ANGPTL3 and ANGPTL4 modulate levels of functional LPL to influence triglyceride delivery and will uncover novel mechanisms of triglyceride clearance that do not require vascular LPL, ultimately permitting new strategies designed to prevent or treat dyslipidemia.