Project Summary Postprandial plasma triacylglyceride levels are independently associated with risk of cardiovascular disease. The intestine plays a key role in the regulation of dietary triacylglyceride absorption by packaging and secreting lipids in chylomicron particles. The intracellular steps of chylomicron biogenesis and transport have been fairly well described, however, there is a significant gap in our understanding of the mechanism for the final steps of chylomicron secretion by enterocytes, specifically post-Golgi transport and secretion. We recently identified a protein that plays a critical role in this pathway, DENND5B. Our newly generated Dennd5b-/- mouse demonstrates an essential role for this gene in Golgi to plasma membrane transport of chylomicron secretory vesicles. Dennd5b-deficient mice are resistant to diet-induced: obesity, changes in plasma lipids, and atherosclerosis. In humans, exome sequencing studies reveal that a common DENND5B variant is correlated with body mass index. These studies establish an important role for DENND5B in post-Golgi chylomicron secretion and a subsequent influence on body composition and peripheral lipoprotein metabolism. The overall objective of this proposal is to gain fundamental knowledge about the process of dietary lipid absorption by the intestine and to examine the impact of this process on systemic metabolic health. This objective aligns well with the NIH mission to seek fundamental knowledge about living systems and to apply this knowledge to enhance health. The Specific Aims of this proposal use our novel mouse model and in vitro experiments to examine the mechanistic role of DENND5B in the intestine and its systemic metabolic effects. Aim 1 will quantify the impact of Dennd5b on dietary lipid absorption and generate a comprehensive spatial single-cell RNAsequencing data set for examination of gene networks that participate in regulation of dietary lipid absorption. Aim 2 will examine the mechanism underlying Dennd5b action in CM transport. Furthermore, this aim will use mass spectrometry and interactomics analysis to expand our understanding of this process by identifying other interacting proteins which may form a complex to facilitate chylomicron secretion by the intestine. Aim 3 will determine the impact of Dennd5b on enterocyte metabolic function and peripheral lipid metabolism. Successful completion of this proposal will fill a significant knowledge gap in the process of dietary lipid absorption. This work will advance the field by providing mechanistic details for a key step in chylomicron secretion that can affect systemic lipid homeostasis and metabolic health.