Examining the Role of Fat-Related Proteins in Endothelial Cell Fatty Acid Uptake

Fat plays a variety of important roles in the human body, providing insulation and temperature regulation, protecting essential areas like organs, and storing energy for long-term use. However, fat can become harmful in excess and when its uptake and use is poorly regulated, contributing to issues like obesity, diabetes, and cardiovascular diseases. Endothelial cells are a critical factor in fat uptake, as they line the inside of blood vessels to form a layer that regulates the transport of substances between the blood and surrounding tissues, helping maintain homeostasis by providing cells with nutrients (including lipids) and removing waste. A wide variety of proteins are involved in the cellular uptake of fat, such as fatty acid binding protein (FABP), fatty acid transport protein (FATP), long-chain acyl-CoA synthetase (ACSL), and Caveolin (Cav), all of which exist in multiple isoforms. How these proteins regulate the uptake and intracellular metabolism of fat specifically in endothelial cells is not completely known. This project aims to identify the fat-related proteins that are most abundantly expressed in endothelial cells, as I hypothesize that such proteins will likely play significant roles in fat uptake. I conducted a literature search to compile RNA-seq data from published experiments, and normalized the data to compare expression values across different types of endothelial cells. This showed that genes for FABP4 and 5, FATP1, 3, 4, Cav1, Cav2, and ACSL1, 3, and 4 are consistently the most highly expressed. We performed siRNA treatments for siACSL1, siFABP4, and siFABP5 in endothelial cells, followed by a fatty acid uptake assay, to determine whether genetically knocking down these proteins affects cellular fatty acid uptake. We confirmed siRNA effectiveness through qPCR. With this preliminary experiment, we found that knockdown of each of these proteins resulted in a modest but significant reduction in fat uptake. Future work will entail repeating this experiment under varying conditions, as well as continuing to test further proteins, possibly in combination with each other. Determining which proteins play a significant role in endothelial fat transport will broaden our understanding of how fat from the blood crosses into metabolic tissue, and will contribute valuable information for the treatment of fat-related diseases at a cellular level.