Nicotinamide- N -methyltransferase is essential for SAM and 1- 1 methylnicotinamide homeostasis in the AML12 hepatocyte cell line 2

30 Nicotinamide- N -methyltransferase (NNMT) is an enzyme that consumes S -adenosyl- 31 methionine (SAM) and nicotinamide (NAM) to produce S -adenosyl-homocysteine (SAH) and 32 1-methylnicotinamide (MNAM). How much NNMT contributes to the quantity regulation of 33 these four metabolites depends on whether NNMT is a major consumer or producer of these 34 metabolites, which varies among various cellular contexts. Yet, whether NNMT critically 35 regulates these metabolites in the AML12 hepatocyte cell line has been unexplored. To address 36 this, we knock down Nnmt in AML12 cells and investigate the effects of Nnmt RNAi on 37 metabolism and gene expression. We find that Nnmt RNAi accumulates SAM and SAH, 38 whereas it reduces MNAM with NAM being unaltered. These results indicate that NNMT is a 39 significant consumer of SAM and critical for MNAM production in this cell line. Moreover, 40 transcriptome analyses reveal that altered SAM and MNAM homeostasis is accompanied by 41 various detrimental molecular phenotypes, as exemplified by the down-regulations of lipogenic 42 genes such as Srebf1 . Consistent with this, oil-red O-staining experiments demonstrate the 43 decrease of total lipids upon Nnmt RNAi. These results suggest that NNMT maintains proper 44 SAM and MNAM homeostasis, providing an additional example where NNMT plays a critical 45 role in regulating SAM and MNAM metabolism. 46 Collectively, our data showed that NNMT is critical for maintaining global gene expression and lipogenesis in this hepatocyte cell line. These data deepened our understanding 173 of how NNMT regulates metabolism and gene expression in various cellular contexts.


Introduction 48
Nicotinamide-N-methyltransferase (NNMT) transfers a methyl group from S-adenosyl-49 methionine (SAM) to nicotinamide (NAM) and produces S-adenosyl-homocysteine (SAH) and 50 1-methylnicotinamide (MNAM) (1). SAM is a methyl donor that contributes to various 51 methylation events in the cytoplasm and nucleus (2-4). In addition, recent studies demonstrate 52 that MNAM retains biological activities such as anti-inflammation (5-10). Via regulating the 53 NNMT-related metabolites, NNMT plays a critical role in a series of phenomena such as energy 54 metabolism (1,5,7,8,(11)(12)(13)(14)(15)(16)(17)(18). 55 How much NMNT contributes to the quantity of SAM, NAM, MNAM, and SAH is 56 context-dependent: it depends on how many other enzymes are involved in the metabolism of 57 these four metabolites in a certain cellular context. For example, suppression of NNMT results 58 in the loss of MNAM in all cell types reported so far (1, 5, 7, 10-13). This simplicity is attributed 59 to the fact that only NNMT produces MNAM in worms, mice, and humans. On the other hand, 60 the homeostasis of SAM and NAM is more complex, as many other enzymes consume and 61 produce these two metabolites. In the case of murine livers, deletion of NNMT does not 62 accumulate SAM and NAM, suggesting that NNMT is not a significant consumer for them in 63 the liver. Intriguingly, when GNMT, the major consumer for SAM in the liver, is reduced, 64 suppression of NNMT leads to the accumulation of SAM (i.e., the contribution of NNMT to 65 SAM homeostasis is increased upon GNMT suppression) (7, 13). These studies exemplify that 66 NNMT context-dependently contributes to SAM homeostasis. As such, the effects of altered 67 NNMT metabolism on cellular homeostasis should differ among contexts. In this regard, 68 investigation of the roles of NNMT in various cellular contexts is essential to deepen our 69 understanding of the significance of NNMT-dependent metabolism in living cells. 70 In the current study, we examine the roles of NNMT in metabolism and gene expression 71 in the AML12 hepatocyte cell line. AML12 is one of the commonly used murine hepatocyte 72 cell lines established from mice overexpressing human transforming growth factor-alpha (TGF-73  Fig.  118 3a, Nnmt RNAi resulted in down-regulation of 747 genes and up-regulation of 65 genes (> 2-119 fold change and q value < 0.05; Table S2), suggesting that the NNMT-dependent metabolism 120 is important in maintaining proper gene expression in AML12 cells. Such crucial roles of 121 NNMT were exemplified by the reduction of Albumin (Alb), the marker gene for AML12 122 hepatocytes (Fig. 3b). Gene ontology analyses demonstrated that the down-regulated genes 123 represent dampened "lipid metabolic process" in this cell line (Fig. 3c). Genes including 124 diacylglycerol acyltransferase 2 (Dgat2) (21) and sterol regulatory element binding 125 transcription factor 1 (Srebf1) (22) were severely reduced upon Nnmt RNAi (Fig. 3d). It has 126 been shown that these genes are involved in lipogenesis, implicating a role of NNMT in 127 lipogenesis in AML12 cells. To further test this hypothesis, we performed oil-red O-staining 128 against AML12 cells treated with either siLuc or siNnmt. We quantified total lipid levels using 129 a spectrophotometer (OD = 540 nm), finding that total lipids were reduced upon Nnmt RNAi 130 In the current study, we examined the roles of NNMT in metabolism and gene expression in 137 the AML12 hepatocytes cell line. We found that NNMT is critical to maintaining SAM and 138 MNAM homeostasis and contributes to lipogenesis in this cell line. 139 As discussed earlier, the degree of contribution of NNMT to SAM homeostasis differs 140 depending on cell types. We previously demonstrated that, in the murine livers, deletion 141 of Nnmt does not alter the steady-state amount of SAM in healthy conditions (7) (Fig. 4). We 142 reasoned that this was because GNMT is the major consumer of SAM in the liver in vivo and 143 because the trigonelline pathway might have received excess methyl groups from SAM (Fig.  144   4). Trigonelline is a methylated form of nicotinic acids (23). Interestingly, the murine livers do 145 not have trigonelline-producing activity, suggesting that trigonelline is synthesized with the 146 help of, for example, microbiomes (7, 23). Consistent with this assumption, we found that 147 trigonelline was undetectable in AML12 cells that must have been free from microbiomes 148 (Table S1). Hence, in contrast to living mice, AML12 cells seemingly lack the trigonelline 149 pathway that could receive methyl groups from SAM. In addition, the expression of Gnmt is 150 low in this cell line (Fig. 2e). We suggest that these biological contexts establish NNMT as a 151 crucial regulator of SAM homeostasis in AML12 cells. In summary, this study provides 152 additional evidence that the methyl-donor balance is maintained in context-dependent manners. 153 In line with the critical roles in SAM and MNAM homeostasis (Fig. 2), we found that 154 NNMT significantly contributes to global gene expression in AML12 cells (Fig. 3)

Liquid chromatography coupled with tandem mass spectrometry
Metabolites from AML12 cells were extracted using the Blight and Dyer's method (25) with some modifications. Briefly, each sample was mixed with 1 ml of cold methanol containing 10camphorsulfonic acid (1.0 nmol or 1.5 nmol) as internal standard (IS) for mass spectrometrybased metabolomic analysis. The samples were vigorously mixed by vortexing for 1 min followed by 5 min of sonication. The extracts were then centrifuged at 16,000 × g for 5 min at 4°C, and the resultant supernatant was collected. After mixing supernatant with chloroform and water

Transcriptome analysis
Total RNAs were extracted from AML12 cells as described above with RNase-Free DNase Set in Supplementary Data 2. The volcano plot was depicted using ggplot2 to visualize differentially expresses genes (https://ggplot2.tidyverse.org/index.html). Differentially expressed genes were further subjected to gene ontology analyses using g:Profiler (32).

Oil-red O staining
To measure total lipids, we stained AML12 cells using the Lipid Assay Kit (COSMO BIO, Tokyo, Japan) according to the manufacturer's instructions. Briefly, AML12 cells were treated with either siLuc or siNnmt for 48 hours. The cells were then washed with PBS and fixed with 10% formalin at room temperature overnight. The fixed AML12 cells were washed three times with distilled water and stained with oil-red O at room temperature for 15 min. Following staining, the cells were washed three times with distilled water and dried at room temperature overnight. The dye extractions from these cells were quantified by measuring 540 nm by a spectrophotometer Multiskan GO (Thermo Fisher Scientific).

Statistics and Data visualization
GraphPad Prism Software was used to analyze data. Data were displayed as mean ± SEM. Student's t test was performed to analyze the statistical significance between groups, and p value < 0.05 was considered statistically significant. In RNA-seq analyses, q value was calculated using the Storey's method (https://www.bioconductor.org/packages/release/bioc/html/qvalue.html).

Data availability
RNA-seq data obtained in this study are available from DNA Databank of Japan under the accession number of DRA014854.        The contribution of NNMT in the maintenance of SAM is context-dependent. In the liver, SAM homeostasis is maintained even in the absence of NNMT. This is likely owing to the strong contribution of GNMT in SAM homeostasis. In addition, nicotinic acid methyltransferase (NAMT), which is derived most likely from the microbiome (7, 23), seemingly contributes to SAM homeostasis in the liver. Compared to the liver, the expression of Gnmt in AML12 cells is relatively low. In addition, AML12 cells lack the NAMT-trigonelline pathway. In these conditions, NNMT plays a critical role in SAM homeostasis.