The effect of NAT deletion on mitochondrial function in MDA

The effect of NAT1 deletion on mitochondrial function in MDA-MB-231 Chelerythrine Chloride receptor 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. Regardless of whether PDH activity was down-regulated by phosphorylation or by loss of total PDH protein, the metabolic outcomes were similar suggesting that the effect of NAT1 deletion was to decrease metabolic capacity. Many functional changes observed following NAT1 inhibition are cell-type dependent. For example, in HT-29 cells (Tiang et al., 2011) and MDA-MB-231 cells (Stepp et al., 2018), low NAT1 activity was associated with slower growth and inhibition of colony formation in soft agar. This is consistent with a lower metabolic capacity. By contrast, no changes were observed in HeLa cells (Witham et al., 2017). In MDA-MB-231 and MDA-MB-436 cells, knockdown of NAT1 with shRNA induced morphological changes and decreased invasiveness. However, this was not seen in BT-549 cells (Tiang et al., 2015). Deletion of the NAT1 gene increased reactive oxygen (ROS) production and apoptosis, especially under nutrient stress, in HT-29 cells but not in HeLa cells (Wang et al., 2018). The current study has identified diminished PDH activity as central to the changes in mitochondrial function following NAT1 deletion, a least in MDA-MB-231 and HT-29 cells. These results may help explain some of the cellular changes that have been reported recently in cell and animal models of NAT1 and NAT2 deficiency. For NAT2, there is a strong insulin resistance phenotype reported in humans carrying the slow acetylator allele (rs1208 “A”) (Knowles et al., 2015). It will be important to determine whether these individuals have altered PDH activity in peripheral tissues. If so, genotyping diabetic patients for NAT1 /or NAT2 polymorphisms may identify a patient sub-population that could benefit from treatment with PDH activators, such as DCA
Author contributions
Conflict of interest
Acknowledgement This work was supported by the National Health and Medical Research Council of Australia (Grant # 1024769)
Introduction Methoxychlor (MXC), an organochlorine pesticide that has a similar structure to dichlorodiphenyl trichloroethane, has been widely used as a preferred alternative to dichlorodiphenyl trichloroethane due to its lower levels of persistence in the environment [1,2]. People expose to MXC primarily via contaminated air, soil, and water. After entering into the human body, MXC is predominantly metabolized into mono- and bis-demethylated metabolites, including 2-(4-hydroxyphenyl)-2- (4-methoxyphenyl)-1,1,1-trichloroethane and 2,2-bis(4- hydroxyphenyl)- 1,1,1-trichloroethane (hydroxychlor, HPTE) [2]. Previous observations indicate that both MXC and HPTE are endocrine disruptors [3,4]. MXC and HPTE could potently reduce steroid biosynthesis in the testis and ovary, possibly via directly suppressing cytochrome P450 side chain cleavage enzyme, cytochrome P450 17α-hydroxylase/20-lyase, 3β-hydroxysteroid dehydrogenase, and 17β-hydroxysteroid dehydrogenase 3 activities [[4], [5], [6]].