The PIs’ laboratories focus on the pathways leading to lipid uptake by tissues and their metabolic implications. We showed that the FA transporter, CD36, mediates uptake of VLDL- but not chylomicron (CM)-derived FAs and established in vivo that there are at least two pathways for FA uptake into tissues. We recently floxed the CD36 gene and created knockouts in multiple tissues. Most germane to this proposal, we showed that endothelial cell (EC) specific CD36 deletion reproduces many of the in vivo effects of global CD36 deletion including reduced long-chain FA uptake into muscle and adipose tissues. These findings documented importance of the endothelium in regulating tissue lipid uptake and together with novel preliminary data form the basis for the current application, which is focused on understanding how ECs function in uptake of non-esterified FAs and of FAs derived from CMs and VLDL. Our preliminary data show that exogenous FAs in complex with EC-CD36 and in conjunction with Caveolin 1 (Cav1) are internalized into vesicles and are released from ECs within exosome- like small extracellular vesicles or sEVs. In Aim 1 we will dissect the cellular events associated with the FA- CD36-Cav1 interaction and FA transcytosis by ECs and define the processes required for sEV secretion. In addition, we propose that at high FA levels, e.g. during CM lipolysis, a non-CD36 paracellular pathway of FA transfer is activated and we will determine the mechanisms that mediate FA-induced changes in EC junctions and barrier function. Other preliminary data show that ECs internalize and metabolize nascent CMs via a non- lipoprotein lipase and non-CD36 requiring process that appears to involve scavenger receptor-B1. In Aim 2 we will examine how EC delivery of FAs from CMs and VLDL differs. We will determine the molecular requirements for CM interaction with the EC surface, the cellular processes for internalization and whether CM lipids are transcytosed across ECs and released in EVs. The CM uptake pathway will be compared to that for VLDL where we will define the role of CD36. We will also examine how internalization of FAs, CMs, VLDL and EV products changes EC and macrophage biology. These studies will illustrate the pathways by which the body distributes calories between organs and will define basic processes that underlie the physiologic and pathological uptake and storage of lipids in tissues. Such information will suggest novel ways to disrupt the pathological consequences of excess calorie intake that occur with metabolic syndrome and type 2 diabetes.