Testing Depletion of RNF213 in Endothelial Cells To Reduce Lipotoxicity

A key component of a person's health can be driven by their diet. If an individual consumes excess lipids, especially saturated fatty acids (FAs), this can result in a systemic condition known as lipotoxicity. This occurs when excess lipids accumulate in cell types other than adipocytes (fat cells), which leads to negative metabolic effects in these tissues that are not equipped to handle such a high lipid load. Prior to the lipids accumulating, the saturated FAs have to move into the tissues from the bloodstream, and the blood vessel walls that the FAs must cross are composed of endothelial cells (ECs). With excess bloodborne saturated FAs, the ECs suffer from cellular lipotoxicity, leading to diseases like cardiovascular dysfunction. It was recently found that a protein, RNF213, once depleted, can protect against cellular lipotoxicity, but the specific mechanisms are largely unclear. We hypothesize that genetically depleting RNF213 will ameliorate lipotoxicity in ECs, and thus help us further grasp cellular and metabolic pathways that regulate lipid uptake and handle cell stress responses. To test this hypothesis, we will use two orthogonal assays to quantify endothelial lipotoxicity: SYTOX & MTT. When the ECs are damaged from excess FA accumulation, it harms cell metabolism and leads to cell death. The MTT assay measures metabolic activity in live cells, and the SYTOX assay separately measures permeabilized (dying) cells. To induce lipotoxicity in the ECs, we will use palmitate, the most commonly consumed saturated fat, at a dose and duration previously determined to be effective in preliminary experiments. We will genetically deplete RNF213 in ECs by treating them with siRNA, a transfection technique to reduce the expression of selected genes. Finally, we will use the two aforementioned assays to quantify whether the RNF213-depleted ECs exhibit decreased lipotoxicity. We have so far established protocols for the MTT and SYTOX assays, and have begun combining experiments to answer this main question. Success here will allow us to move forward with follow-up experiments, such as determining the mechanism for RNF213-mediated lipotoxicity and finding other proteins involved in endothelial lipotoxicity. Being able to test for and reduce endothelial lipotoxicity could create potential new therapeutic methods for treating various metabolic diseases.