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  • br Conflicts of interest br Introduction Arylamine N

    2020-03-27


    Conflicts of interest
    Introduction Arylamine N-acetyltransferase 1 (NAT1, EC 2.3.1.5) is a cytosolic enzyme that catalyses the acetylation of small molecule arylamines, hydrazines and sulphonamides (Hein, 2002). Found in most erk inhibitors in the body, NAT1 expression is regulated at the transcriptional and post-transcriptional levels (Butcher et al., 2007, 2008). A detailed evolutionary study of mammalian arylamine N-acetyltransferase s supports the notion that NAT1 has evolved through strong negative selection to conserve functionality over time (Sabbagh et al., 2013). This observation, along with its wide distribution in the body, suggests NAT1 has a role in addition to xenobiotic metabolism. The only known endogenous substrate for NAT1 is the folate catabolite p-aminobenzoylglutamate, but the physiological importance of p-aminobenzoylglutamate acetylation in vivo has been questioned (Witham et al., 2013). There is emerging evidence that NAT1 expression is associated with changes in cell growth and survival, cell morphology, and various intracellular metabolic pathways (Carlisle et al., 2016; Stepp et al., 2018; Tiang et al., 2011, 2010, 2015; Witham et al., 2013, 2017). In addition to NAT1, humans express a second arylamine N-acetyltransferase (NAT2), the result of a gene duplication on chromosome 8. NAT2 levels are highest in the liver and gastrointestinal tract where it is important in the metabolism of drugs and other xenobiotics (Hein, 2002). Recently, single nucleotide polymorphisms in the NAT2 gene, which result in a slow acetylator phenotype, were shown to be associated with insulin resistance (Knowles et al., 2015). Moreover, deletion of the murine homolog of human NAT2 (Nat1) recapitulated the insulin resistance phenotype (Camporez et al., 2017), increased mitochondrial dysfunction and the production of reactive oxygen species (ROS) (Chennamsetty et al., 2016). Nat1 knockdown in 3T3-L1 cells with sh-RNA decreased both basal respiration as well as reserve respiratory capacity. A similar finding was reported for hepatocytes isolated from Nat1 null mice (Camporez et al., 2017), suggesting that human NAT2 (or mouse Nat1) is important for mitochondrial function. Previously, we reported that deletion of NAT1 in various human cells including MDA-MB-231 cells, using CRISPR/Cas9 technology decreased oxidative phosphorylation (Lichter et al., 2017). Moreover, in human HT-29 cells, glucose utilisation decreased and ROS production increased following NAT1 deletion (Wang et al., 2018). These studies suggested that NAT1 affected mitochondrial function in a similar manner to that seen following Nat1 knockdown in mice. However, a recent study using human breast MDA-MB-231 cells reported that deletion of NAT1 increased reserve respiratory capacity and glycolytic reserve (Carlisle et al., 2018). To better understand the effects of NAT1 on cell metabolism, glucose-mediated oxygen consumption and glycolysis were compared in 2 independent cell models following deletion of the NAT1 gene. The results show mitochondrial changes that may explain the link between arylamine N-acetyltransferase N-acetyltransferase expression and mitochondrial function.
    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 acid 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.