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  • br Discussion When NAT was deleted from MDA

    2020-07-27


    Discussion When NAT1 was deleted from MDA-MB-231 and HT-29 cells, there was a marked decrease in oxidative phosphorylation, which was associated with a decrease in PDH-E1α activity. Inhibition of the pyruvate dehydrogenase complex limits pyruvate entry into the TCA cycle and lowers ATP generation. A common response in cancer purinergic receptor to a change in oxidative phosphorylation is metabolically switching to aerobic glycolysis, which can maintain ATP production. However, this was not the case following NAT1 deletion in either cell line as glycolysis was also diminished. Since glucose uptake was not altered, these results suggest glucose was shunted away from the glycolytic/oxidative phosphorylation pathways towards other pathways such as glycogenesis, the pentose phosphate pathway or the hexosamine synthesis pathway (Hay, 2016). The mechanism for this remains to be determined. However, a recent metabolomics study using NAT1 deleted MDA-MB-231 cells identified changes in numerous polar metabolites, although most remain to be definitively identified (Carlisle et al., 2016). Nevertheless, that study demonstrated marked changes in metabolism following NAT1 deletion. The changes in mitochondrial function in the MDA-MB-231 and HT-29 cells are similar to those reported in murine cells following Nat1 knockout (Camporez et al., 2017; Chennamsetty et al., 2016). Nat1 is the murine homolog of human NAT2, not NAT1, so the similarity in responses following gene deletion was unexpected. These observations suggest that NAT1 and NAT2 may have common or redundant biological roles in regulating mitochondrial function. Alternatively, since NAT1 and NAT2 are differentially expressed in vivo, they may have similar roles but in different tissues in the body. Interestingly, when we quantified NAT2 expression in the MDA-MD-231 and HT-29 cells by qPCR following NAT1 knockout, there was a 3.5 ± 0.6 and 2.0 ± 0.5 fold purinergic receptor increase in mRNA compared to the parental cells, respectively (p < 0.01). This suggests that expression of NAT1 and NAT2 are not completely independent, an observation supported by a positive association between the expression of the 2 genes in human breast cancer cells (Carlisle and Hein, 2018). The effect of NAT1 deletion on mitochondrial function in MDA-MB-231 cells has been reported elsewhere (Carlisle et al., 2018). However, unlike the data presented here, increases in reserve capacity and glycolytic reserve were seen. The reasons for this variance between our study and that of Carlisle et al studies is not immediately obvious. There were differences in the gene deletion protocols that may have contributed to varying phenotypes. There may also be important differences associated with how the gene-deleted cells were selected and cultured. It will be important in the future to identify the molecular mechanisms for the difference in mitochondrial function reported here and by Carlisle et al (Carlisle et al., 2018). In HT-29 cells, mitochondrial function also decreased following NAT1 deletion due to an attenuated PDH activity. However, the mechanism involved a decrease in total PDH protein, not an increase in phosphorylation as seen in the MDA-MB-231 cells. While there is much known about the regulation of PDH by PDHKs, very little is known about its stability. PDH is down-regulated in rat liver following treatment with β-hydroxybutyrate (Sharma et al., 2005) and during the development of dilated cardiomyopathy (Missihoun et al., 2009). Importantly, the protein is stabilized by DCA, both in vitro and in vivo. This was also seen in the present study (Fig. 5C) and explains why DCA was able to rescue the changes in mitochondrial function following NAT1 deletion.