Department of Integrative Bioanalytics, Institute of Development, Aging and Cancer (IDAC), Tohoku University, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan.
Inter-Organ Communication Research Team, Institute for Life and Medical Sciences, Kyoto University, 53 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan.
J Biochem. 2023 Jun 30;174(1):89-98. doi: 10.1093/jb/mvad028.
Nicotinamide-N-methyltransferase (NNMT) is an enzyme that consumes S-adenosyl-methionine (SAM) and nicotinamide (NAM) to produce S-adenosyl-homocysteine (SAH) and 1-methylnicotinamide (MNAM). How much NNMT contributes to the quantity regulation of these four metabolites depends on whether NNMT is a major consumer or producer of these metabolites, which varies among various cellular contexts. Yet, whether NNMT critically regulates these metabolites in the AML12 hepatocyte cell line has been unexplored. To address this, we knockdown Nnmt in AML12 cells and investigate the effects of Nnmt RNAi on metabolism and gene expression. We find that Nnmt RNAi accumulates SAM and SAH, whereas it reduces MNAM with NAM being unaltered. These results indicate that NNMT is a significant consumer of SAM and critical for MNAM production in this cell line. Moreover, transcriptome analyses reveal that altered SAM and MNAM homeostasis is accompanied by various detrimental molecular phenotypes, as exemplified by the down-regulations of lipogenic genes, such as Srebf1. Consistent with this, oil-red O-staining experiments demonstrate the decrease of total neutral lipids upon Nnmt RNAi. Treating Nnmt RNAi AML12 cells with cycloleucine, an inhibitor of SAM biogenesis suppresses SAM accumulation and rescues the decrease of neutral lipids. MNAM also shows activity to elevate neutral lipids. These results suggest that NNMT contributes to lipid metabolism by maintaining proper SAM and MNAM homeostasis. This study provides an additional example where NNMT plays a critical role in regulating SAM and MNAM metabolism.
烟酰胺-N-甲基转移酶（NNMT）是一种消耗 S-腺苷甲硫氨酸（SAM）和烟酰胺（NAM）产生 S-腺苷同型半胱氨酸（SAH）和 1-甲基烟酰胺（MNAM）的酶。NNMT 对这四种代谢物数量的调节作用有多大，取决于 NNMT 是这些代谢物的主要消耗者还是生产者，这在不同的细胞环境中是不同的。然而，NNMT 是否在 AML12 肝细胞系中对这些代谢物进行关键调节尚未得到探索。为了解决这个问题，我们在 AML12 细胞中敲低 Nnmt，并研究 Nnmt RNAi 对代谢和基因表达的影响。我们发现 Nnmt RNAi 积累了 SAM 和 SAH，而 MNAM 减少，NAM 则保持不变。这些结果表明，NNMT 是 SAM 的重要消耗者，对该细胞系中 MNAM 的产生至关重要。此外，转录组分析显示，SAM 和 MNAM 动态平衡的改变伴随着各种有害的分子表型，例如脂肪生成基因 Srebf1 的下调。与此一致，油红 O 染色实验表明，Nnmt RNAi 后总中性脂质减少。用 SAM 生物合成抑制剂环亮氨酸处理 Nnmt RNAi AML12 细胞可抑制 SAM 积累并挽救中性脂质的减少。MNAM 也显示出增加中性脂质的活性。这些结果表明，NNMT 通过维持适当的 SAM 和 MNAM 动态平衡来促进脂质代谢。本研究提供了一个额外的例子，说明 NNMT 在调节 SAM 和 MNAM 代谢方面起着关键作用。
