Hepatocyte-specific depletion of Nnmt protects mice from non-alcoholic steatohepatitis

ER stress-induced upregulation of NNMT contributes to alcohol-related fatty liver developmentJournal of HepatologyVol. 73Issue 4PreviewN-nicotinamide methyltransferase (NNMT) is emerging as an important enzyme in the regulation of metabolism. NNMT is highly expressed in the liver. However, the exact regulatory mechanism(s) underlying NNMT expression remains unclear and its potential involvement in alcohol-related liver disease (ALD) is completely unknown. Full-Text PDF NNMT is an enzyme that mediates the conversion of nicotinamide (NAM) to 1-methylnicotinamide (MNA), while consuming a methyl group from S-Adenosyl methionine (SAM). The important role of NNMT in metabolic homeostasis has been investigated in various context.1Kraus D. Yang Q. Kong D. Banks A.S. Zhang L. Rodgers J.T. et al.Nicotinamide N-methyltransferase knockdown protects against diet-induced obesity.Nature. 2014; 508: 258-262Crossref PubMed Scopus (307) Google Scholar, 2Hong S. Moreno-Navarrete J.M. Wei X. Kikukawa Y. Tzameli I. Prasad D. et al.Nicotinamide N-methyltransferase regulates hepatic nutrient metabolism through Sirt1 protein stabilization.Nat Med. 2015; 21: 887-894Crossref PubMed Scopus (138) Google Scholar, 3Hong S. Zhai B. Pissios P. Nicotinamide N-methyltransferase interacts with enzymes of the methionine cycle and regulates methyl donor metabolism.Biochemistry. 2018; 57: 5775-5779Crossref PubMed Scopus (23) Google Scholar With great interest, we read the research article by Song et al. The authors demonstrated that the expression of Nnmt can be regulated by unfolded protein response (UPR)-related PERK-ATF4 signaling in the context of alcohol-induced liver damage.[4]Song Q. Chen Y. Wang J. Hao L. Huang C. Griffiths A. et al.ER stress-induced upregulation of NNMT contributes to alcohol-related fatty liver development.J Hepatol. 2020; 73: 783-793Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar Notably, similar phenotypes have also been identified in the context of palmitate-induced hepatocyte damage in cell culture.[5]Griffiths A. Wang J. Song Q. Iyamu I.D. Liu L. Park J. et al.Nicotinamide N-methyltransferase upregulation via the mTORC1-ATF4 pathway activation contributes to palmitate-induced lipotoxicity in hepatocytes.Am J Physiol Cell Physiol. 2021; 321: C585-C595Crossref PubMed Scopus (3) Google Scholar But the specific role of NNMT in the development of non-alcoholic steatohepatitis (NASH) has not been systemically investigated. Previously, many investigators including Song et al. and ourselves have tried to study the specific effects of NNMT in livers using adenovirus- or adeno-associated virus (AAV)-based transient overexpression or knockdown. The fact that the viruses will be eliminated in a short period of time has prevented us from performing long-term experiments. Additionally, virus-induced immune responses may also confound such experiments. A non-virus-based tool to study the long-term physiological function of NNMT in a tissue-specific manner is still missing. To address these issues, a conditional knockout (KO) mouse model of Nnmt was established by knocking in the LoxP cassettes around the second exon (Nnmt flox/flox). By crossing the Nnmt flox/flox mice to Alb-cre mice, we created the hepatocyte-specific Nnmt KO mouse model Alb-cre::Nnmt flox/flox (referred to as Nnmt KO mice hereafter) (Fig. 1A). The KO efficiency was validated by reverse-transcription quantitative PCR and western blot (Fig. 1B,C and Fig. S1A,B). The Nnmt KO mice have similar body weight as control mice (Nnmt flox/flox) when fed a normal chow diet (Fig. S1C). The mice were then challenged with carbon tetrachloride (intraperitoneally), fructose and glucose (via drinking water) and western diet to establish a NASH model. No bodyweight differences were observed between Nnmt KO mice and the control mice (Fig. S1D). Lower concentrations of total cholesterol, LDL-c, ALT and AST were observed in the serum of Nnmt KO mice (Fig. 1D-E). A lower proportion of liver was also identified in the Nnmt KO mice (Fig. 1F). Notably, the serum concentrations of TC, LDL-c, ALT and the proportion of liver in the Nnmt KO mice were comparable to the control mice on chow diet (Fig. S1E-G). The changes in serum lipid profile as well as the liver proportion were not described in the original articles from Song et al. but are consistent with their major conclusions. The expression of genes related to fibrosis and inflammation in the liver was downregulated in Nnmt KO mice (Fig. 1G-H). We then applied RNA-seq analysis to understand the genome-wide transcriptional changes in the liver. Principle component analysis revealed dramatic differences in gene expression profiles (Fig. S1H). We identified 1,752 genes with at least 2-fold changes and p <0.05 using t tests (Fig. S1I). The top differentially expressed genes were presented in a heatmap (Fig. S1J). Gene set enrichment analysis showed downregulation of genes enriched in gene sets related to fibrosis and inflammation (Fig. 1I-J). The results of Picrosirius red, α-SMA and CD68 staining assays indicated less fibrosis and inflammation in the livers of Nnmt KO mice (Fig. 1K). In contrast, no differences in fibrosis or inflammation were observed in the livers of Nnmt KO mice fed a chow diet (Fig. S1K). These phenotypic changes suggested Nnmt KO protected the mice from NASH. Similarly, the protective effects of Nnmt knockdown or pharmacological inhibition against alcohol-induced liver damage were also observed by Song et al. As to the mechanism, Song et al. proposed that Nnmt knockdown protected against liver damage by suppressing the de novo lipogenesis induced by alcohol or endoplasmic reticulum stress. We thus investigated the effects of Nnmt depletion on lipid metabolism in mouse primary hepatocytes. Few lipid contents were detected in the Nnmt KO hepatocytes with the oil red staining assay, when the cells were treated with palmitic acid and oleic acid (Fig. 1L-M). In addition, genes related to lipogenesis were also downregulated by Nnmt KO (Fig. 1N). The inhibitory effects of Nnmt depletion on fatty acid-induced de novo lipogenesis are consistent with the phenotypes Song et al. observed in alcohol-induced de novo lipogenesis. It has been reported that whole body depletion of Nnmt leads to profound metabolic changes in mice,[6]Brachs S. Polack J. Brachs M. Jahn-Hofmann K. Elvert R. Pfenninger A. et al.Genetic nicotinamide N-methyltransferase (Nnmt) deficiency in male mice improves insulin sensitivity in diet-induced obesity but does not affect glucose tolerance.Diabetes. 2019; 68: 527-542Crossref PubMed Scopus (24) Google Scholar but the functions of NNMT in different tissues are variable.[7]Trammell S.A. Brenner C. NNMT: a bad actor in fat makes good in liver.Cell Metab. 2015; 22: 200-201Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar The Nnmt flox/flox mice we generated are a useful tool for the community to study the physiological function of NNMT in a tissue-specific manner. We identified the protective effects of Nnmt depletion on NASH, which is consistent with its protective effects on alcohol-induced liver damage. We therefore propose that NNMT is a general and key mediator of liver damage. UPR-NNMT signaling has the potential to be developed as a therapeutic target in liver disease. This work was supported by MOST ( 2021YFA0804801 ), NSFC ( 91957117 and 31971082 ) and IDH1322092/010 to S.H., MOST 2020YFA0803600 , 2018YFA0801300 , NSFC 32071138 and SKLGE - 2118 to J.L., and MOST 2019YFA0801900 and 2020YF1402600 to H.H. Conceptualization, D.L., C.Y., H.H., S.H. and J.L.; Investigation, D.L., C.Y., H.H., S.H. and J.L.; Analysis, D.L., C.Y., H.H., S.H. and J.L.; Writing, D.L., C.Y., H.H., S.H. and J.L.; Data Visualization, D.L., C.Y., H.H., S.H. and J.L.; Funding Acquisition, H.H., S.H. and J.L.; Supervision, S.H., J.L.. The authors claim no conflict of interests. Please refer to the accompanying ICMJE disclosure forms for further details. The following are the supplementary data to this article: Download .pdf (1.43 MB) Help with pdf files Multimedia component 1 Download .pdf (.83 MB) Help with pdf files Multimedia component 2