Comparative proteomic analysis of SLC13A5 knockdown reveals elevated ketogenesis and enhanced cellular toxic response to chemotherapeutic agents in HepG2 cells.

Solute carrier family 13 member 5 (SLC13A5) is an uptake transporter mainly expressed in the liver and transports citrate from blood circulation into hepatocytes. Accumulating evidence suggests that SLC13A5 is involved in hepatic lipogenesis, cell proliferation, epilepsy, and bone development in mammals. However, the molecular mechanisms behind SLC13A5-mediated physiological/pathophysiological changes are largely unknown. In this regard, we conducted a differential proteome analysis in HepG2 and SLC13A5-knockdown (KD) HepG2 cells. A total of 3826 proteins were quantified and 330 proteins showed significant alterations (fold change ≥1.5; p < .05) in the knockdown cells. Gene ontology enrichment analysis reveals that 38 biological processes were significantly changed, with ketone body biosynthetic process showing the most significant upregulation following SLC13A5-KD. Catalytic activity and binding activity were the top two molecular functions associated with differentially expressed proteins, while HMG-CoA lyase activity showed the highest fold enrichment. Further ingenuity pathway analysis predicted 40 canonical pathways and 28 upstream regulators (p < .01), of which most were associated with metabolism, cell proliferation, and stress response. In line with these findings, functional validation demonstrated increased levels of two key ketone bodies, acetoacetate and β-hydroxybutyrate, in the SLC13A5-KD cells. Additional experiments showed that SLC13A5-KD sensitizes HepG2 cells to cellular stress caused by a number of chemotherapeutic agents. Together, our findings demonstrate that knockdown of SLC13A5 promotes hepatic ketogenesis and enhances cellular stress response in HepG2 cells, suggesting a potential role of this transporter in metabolic disorders and liver cancer.