Arylamine N-acetyltransferase 2 (NAT2) is a phase II metabolic enzyme commonly known for its role in biotransformation of aromatic amines and hydrazines as reviewed previously (McDonagh et al., 2014). NAT2 expresses well-defined genetic polymorphisms, and previous studies have shown that single nucleotide polymorphisms (SNPs) in the NAT2 coding region often lead to changes in protein stability or altered substrate affinity of NAT2 (Hein, 2002). Combination of variant NAT2 alleles manifest as three different acetylator phenotypes in the general population based on the relative activity level they produce: slow, intermediate, and rapid acetylators (Hein, 2002; Sim et al., 2014). The acetylator phenotype has a profound impact on the rate of certain drug (e.g., isoniazid and hydralazine) and carcinogen (e.g., 4-aminobiphenyl) metabolism in individuals (Hein and Millner, 2021).

In recent years, studies have suggested that NAT2 plays unexpected roles in human pathophysiology. In a genome wide association study (GWAS), NAT2 coding SNPs, rs1208 and rs1801280, has been associated with insulin resistance (assessed by the euglycemic clamp method), independent of body-mass index (Knowles et al., 2015). Follow up studies reported that Nat1 (the functional ortholog of human NAT2) knockout mice exhibit multiple signs of metabolic dysfunction, including elevated fasting blood glucose, insulin resistance, mitochondrial dysfunction, decreased fat utilization, and marked increases in tissue fat content (Chennamsetty et al., 2016; Camporez et al., 2017), supporting the role of murine NAT1 in insulin sensitivity and energy utilization in vivo. Although the mechanism remains obscure, these studies indicate that variable levels (or activities) of NAT2 may influence the state of energy storage and utilization.

Recent reports from our group implicated NAT2 in regulation of lipid/cholesterol homeostasis in the liver. NAT2 transcript is upregulated by glucose and insulin in liver cancer cell lines (Hong et al., 2022), suggesting that its expression is regulated by the nutrient status. Moreover, according to our in silico analysis, human NAT2 is co-expressed with the genes involved in lipid and cholesterol synthesis and transport, such as APOB, ABCG8, ANGPTL3, FABP1, MOGAT2, and PLA2G12B (Hong et al., 2022). Numerous GWAS reports also implicate NAT2 in regulation of plasma lipid and cholesterol levels (Hong et al., 2023), as multiple NAT2 genetic variants have been associated with differential plasma lipid and cholesterol levels. Interestingly, the risk alleles for dyslipidemia of NAT2 gene (e.g., rs1495741-A) are associated with the rapid acetylator phenotype in humans (Hong et al., 2023), which suggests that individuals with increased levels of NAT2 activity are at a higher risk of developing dyslipidemia. The link between NAT2 acetylator phenotype and plasma lipid levels is also supported by our previous study on rats congenic for rapid or slow Nat2 acetylator genotypes. Regardless of the diet, rapid acetylator rats were more prone to develop dyslipidemia (i.e., higher triglyceride; higher LDL; and lower HDL), compared to slow acetylator rats (Hong et al., 2020).

Taken together, it appears that the level of NAT2 is not only regulated by the nutrient status (e.g., glucose and insulin) but also may play an important role in regulating lipid/cholesterol homeostasis, presumably in liver and intestines which are two tissues with relatively high expression. Although many studies have examined the functional outcomes of genetic variants of NAT2 in the context of xenobiotic metabolism, so far, few studies have explored its transcriptional regulation (Zou et al., 2020; Zhu et al., 2021; Hong et al., 2022). In our previous study, we reported that the genes that are co-expressed with NAT2 include those encoding hepatic nuclear receptors, e.g., farnesoid X receptor (FXR; NR1H4) and pregnane X receptor (PXR; NR1I2). Nuclear receptors function as ligand-activated transcription factors (Frigo et al., 2021). For example, PXR is commonly known to transactivate genes that encode proteins involved in xenobiotic metabolism, but also plays a role in glucose homeostasis and insulin sensitivity (Kliewer et al., 2002; Spruiell et al., 2014). FXR, liver X receptor alpha and beta (LXRα/β), and peroxisome proliferator activated receptor alpha (PPARα) respond to changes in cellular levels of endogenous lipid ligands by regulating the expression of genes involved in lipid metabolism (Calkin and Tontonoz, 2012; Hong and Tontonoz, 2014). This prompted us to investigate the mechanisms of transcriptional regulation of NAT2 by nuclear receptors expressed in the liver.

In the present study, we tested if FXR, PXR, LXR, or PPARα regulate NAT2 expression. We treated cryopreserved human hepatocytes with specific agonists for the nuclear receptors and compared the transcript levels of NAT2 as well as known target genes of the receptors.

The activation of nuclear receptors, FXR, PXR, and LXR, by corresponding agonists did not result in significant alterations in the NAT2 mRNA level in cryopreserved human hepatocytes, which suggests that they are not major transcriptional regulators of NAT2 under the test conditions. However, it is worth noting that activation of PPARα by GW-7647 resulted in a marginal, yet significant, reduction in the NAT2 transcript level. However, it is not clear if such a minute decrease has any biological impact.

PPARα is activated by endogenous fatty acids and their derivatives (Chakravarthy et al., 2009; Pawlak et al., 2015). PPARα activation promotes fatty acid transport and metabolism (β-oxidation) as well as lipogenesis via transcriptional activation of the genes involved in these processes, including sterol regulatory element binding protein 1c (SREBP-1c; SREBF1), fatty acid synthase (FASN), and acetyl-CoA carboxylase 1 (ACC1; ACACA) (Patel et al., 2001; Fernández-Alvarez et al., 2011). While ACOX1 and FABP1 represented the target genes upregulated by PPARα (Rakhshandehroo et al., 2010), we chose to examine CYP7A1 and GLUT2 (SLC2A2) as downregulated target genes (see Figure 4). CYP7A1 encodes cholesterol 7-α-monooxygenase, the first and rate-limiting enzyme in bile acid synthesis from cholesterol (Noshiro and Okuda, 1990). It has been shown to be suppressed by Wy14643, a PPARα agonist, in HepG2 and primary hepatocytes (Marrapodi and Chiang, 2000; Rakhshandehroo et al., 2009). Rakhshandehroo and colleagues reported that the treatment with Wy14643 (another PPARα agonist) results in a significant downregulation of genes involved in glucose absorption, such as glucose transporter 2 (GLUT2; SLC2A2) and villin-1 (VIL1) in both mouse and human primary hepatocytes (Rakhshandehroo et al., 2009). In our study, GW-7647 failed to suppress CYP7A1 expression, whereas it significantly downregulated GLUT2 (SLC2A2) expression in cryopreserved human hepatocytes. It is not clear why CYP7A1 was not downregulated following PPARα activation in the present study. Marrapodi and Chiang reported that downregulation of CYP7A1 by Wy14643 is unlikely due to direct regulation by PPARα, but rather indirectly via downregulation of HNF4α (Marrapodi and Chiang, 2000). Notably, their experiments were carried out using HepG2 cells. In the study by Rakhshandehroo et al. (2009). The authors used a different agonist (Wy14643), which may account for the difference. Additionally, differences in culture condition and batch-to-batch variability between cryopreserved human hepatocytes might have contributed to this discrepancy. Differences between previous and our current results may attribute to the level of HNF4α in hepatocytes used and/or the use of different PPARα agonists.

The amino acid catabolism is one of the processes suppressed by PPARα activation. By promoting the proteasomal degradation of hepatocyte nuclear factor 4 alpha (HNF4α), PPARα indirectly downregulates genes encoding amino acid-degrading enzymes, including histidine ammonia-lyase (HAL) and serine dehydratase (SDS) (Rakhshandehroo et al., 2009; Alemán et al., 2013; Tobón-Cornejo et al., 2021). In support of this data, a global transcriptomic analysis of genes differentially regulated by Wy14643 in both mouse and human primary hepatocytes revealed that one of the biological processes that are significantly downregulated include “alpha-amino acid catabolic process” (GO:1901606) and “cellular amino acid catabolic process” (GO:0009063) (Rakhshandehroo et al., 2009). Thus, it is plausible that downregulation of the NAT2 transcript level by GW-7647 is not a direct result of PPARα activation but of subsequent downregulation of HNF4α. It would be of interest to test if NAT2 is transcriptionally regulated by HNF4α.

Our previous study showed that NAT2 mRNA levels in HepG2 cells vary according to the glucose concentration in culture media (Hong et al., 2022). Thus, downregulation of GLUT2/SLC2A2 expression by PPARα activation could potentially lead to a reduction in glucose uptake and indirectly downregulate NAT2 expression. However, despite the statistical significance, the reduction in GLUT2 expression was minute, and we question the functional significance of the change.

Although variable NAT2 activity has been linked to physiologically and clinically relevant outcomes (e.g., drug toxicity and dyslipidemia), few studies have investigated its transcriptional regulation. One recent study reported that NAT2 expression is positively regulated by the vitamin D receptor (VDR) (Zhu et al., 2021). The study showed that treatment with the biologically active form of vitamin D (1,25(OH)₂D₃) increases NAT2 expression and that VDR binds to the NAT2 promoter in colorectal cancer cells (Zhu et al., 2021). Whether or not NAT2 is regulated similarly by VDR in the liver remains to be tested.

In summary, we did not observe marked alterations in the NAT2 transcript level following treatment of synthetic agonists for FXR, PXR, LXR, and PPARα in cryopreserved human hepatocytes. Additional potential transcriptional regulators of hepatic NAT2 expression, among nuclear receptors, include HNF4α and VDR, which remains to be tested.