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  • br Materials and methods br Results To determine the


    Materials and methods
    Results To determine the effect of NAT1 deletion on mitochondrial function, OCR and ECAR were measured using a Seahorse XFe96 Flux Analyzer. In MDA-MB-231 cells, basal oxygen consumption, ATP-coupled oxygen consumption and reserve respiratory capacity decreased in the NAT1 knockout cells compared to parental cells (Fig. 1A & B, left panels). These results are consistent with a decrease in glucose flux through the mitochondria following NAT1 deletion. In the HT-29 cells, OCR was also decreased with the largest, and most significant, change seen in the reserve respiratory capacity (Fig. 1A & B, right panels). In addition to oxidative phosphorylation, ATP requirements can be met by aerobic glycolysis where glucose is diverted to lactic Cholesterol instead of entering the TCA cycle. This is common in cells with mitochondrial dysfunction. Glycolysis was measured in each cell line by quantification of ECAR following the addition of glucose. In both MDA-MB-231 and HT-29 cells, glycolysis significantly decreased following NAT1 deletion (Fig. 2A & B). However, no change was seen with the glycolytic reserve. To compare glucose metabolism via oxidative phosphorylation to that via glycolysis, a bioenergetics plot was constructed (Fig. 3A). In most cells, a decrease in one bioenergetics pathway is compensated by an increase in the other. However, following NAT1 deletion, there was a decrease in both oxidative phosphorylation and glycolysis indicating a shifted to a lower overall bioenergetic state. These results suggest that NAT1 knockout cells do not utilize glucose for glycolysis or oxidative phosphorylation to the same extent as the parental cells. To determine whether this difference was due to a decrease in glucose uptake, the accumulation of the glucose transporter probe 2-NBDG was measured. However, there was no significant difference in glucose transport in either cell line following NAT1 deletion (Fig. 3B). Taken together, these results suggest that glucose flux in the knockout cells is diverted away from the glycolysis/oxidative phosphorylation pathway. The reserve respiratory capacity is essential for cell survival during mitochondrial stress. It is partly dependent on activity of the mitochondrial pyruvate dehydrogenase complex and a loss in activity can reduce or eliminate reserve respiratory capacity (Pfleger et al., 2015; Prabhu et al., 2015). To determine whether deletion of NAT1 altered pyruvate dehydrogenase complex function, enzyme activity was measured in both parental and knockout MDA-MB-231 and HT-29 cells (Fig. 4A & B). For both cell lines, there was a significant decrease in activity following NAT1 deletion. Pyruvate dehydrogenase-E1α (PDH-E1α) is an essential component of the pyruvate dehydrogenase complex and is regulated by reversible phosphorylation catalysed by pyruvate dehydrogenase kinase (PDHK). Phosphorylation of PDH-E1α results in a decrease in activity of the pyruvate dehydrogenase complex. When MDA-MB-231 and HT-29 knockout cells were treated with the PDHK inhibitor dichloroacetate (DCA), the changes seen in OCR were completely rescued (Fig. 4C & D) suggesting that NAT1 deletion may induce PDH-E1α phosphorylation. To test this, both total and phosphorylated PDH-E1α were quantified in parental and NAT1 knockout cells (Fig. 5A). For the MDA-MD-231 cells, there was a 3-fold increase in phosphorylated PDH-E1α following NAT1 knockout. DCA treatment of the NAT1 deleted cells reversed this increase to levels seen in the parental cells (Fig. 5B). By contrast, total PDH-E1α decreased in the HT-29 NAT1 knockout cells. When these cells were treated with DCA, PDH-E1α increased to levels similar to the parental cells (Fig. 5C) showing that loss of PDH-E1α following NAT1 knockout was reversed by DCA treatment.