To compare glucose metabolism via oxidative
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 DL-TBOA mg 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.
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 cells 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 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).