Abstract 13357: Dhtkd1 Knock-out Alters Cellular Respiration by Preventing Electron Transport Chain Related Proteins From Entering Into Mitochondria

Abstract

The metabolite α-aminoadipic acid (2-AAA) has been identified as a predictor of diabetes and atherosclerosis in large human prospective studies. However, the mechanisms linking this metabolite to disease pathophysiology remain unknown. DHTKD1 is a central gene in the 2-AAA pathway, and has been linked to variation in 2-AAA levels in humans and animals. However, little is known about the role of DHTKD1 in cellular metabolism. We hypothesized that DHTKD1 is an important regulator of mitochondrial energy metabolism, with potential involvement in cardiometablic disease. Methods and Results: We investigated the consequences of loss of DHTKD1 function in a human HAP-1 cell line. Treatment with 2-AAA increased mitochondrial respiration in wild-type (WT) cells, but had no effect in DHTKD1 knock-out (KO) cells. DHTKD1 KO cells had significantly lower mitochondrial respiration (Mito Stress Test, Seahorse Analyzer), with no differences in glycolytic function, supporting an important role for DHTKD1 in mitochondrial metabolism. Membrane potential and mitochondrial content were up-regulated in KO compared to WT cells, suggesting a potential compensatory mechanism. We investigated the mechanisms underlying impaired mitochondrial function, and found TOM40 and TIM23, the pivotal proteins required for the movement of proteins into mitochondria, were decreased in DHTKD1 KO cells. Further, DHTKD1 KO resulted in reduced expression of electron transport chain related proteins (NDUF88, SDHB, MTCO1, UQCRC2, ATP5A) in mitochondria, but increased expression in the cytosol, suggesting impaired ability to cross the mitochondrial membrane. Conclusions: Our data suggest that the absence of DHTKD1 leads to less TOM40 and TIM23 expression, preventing key proteins in the electron transport chain from entering into mitochondria, resulting in impaired mitochondrial respiration. These findings highlight the vital role of DHTKD1 in cellular metabolism, and establish DHTKD1-mediated mitochondrial dysfunction as a potential novel pathway in cardiometabolic disease. Elevated 2-AAA observed in individuals prior to onset of diabetes and atherosclerosis may be an early biomarker of dysfunction in this pathway.