Investigating RNA expression profiles altered by nicotinamide mononucleotide therapy in a chronic model of alcoholic liver disease

Abstract

Background: Chronic alcohol consumption is a significant cause of liver disease worldwide. Several biochemical mechanisms have been linked to the initiation and progression of alcoholic liver disease (ALD) such as oxidative stress, inflammation, and metabolic dysregulation, including the disruption of NAD⁺/NADH. Indeed, an ethanol-mediated reduction in hepatic NAD⁺ levels is thought to be one factor underlying ethanol-induced steatosis, oxidative stress, steatohepatitis, insulin resistance, and inhibition of gluconeogenesis. Therefore, we applied a NAD⁺ boosting supplement to investigate alterations in the pathogenesis of early-stage ALD.

Methods: To examine the impact of NAD⁺ therapy on the early stages of ALD, we utilized nicotinamide mononucleotide (NMN) at 500 mg/kg intraperitoneal injection every other day, for the duration of a Lieber-DeCarli 6-week chronic ethanol model in mice. Numerous strategies were employed to characterize the effect of NMN therapy, including the integration of RNA-seq, immunoblotting, and metabolomics analysis.

Results: Our findings reveal that NMN therapy increased hepatic NAD⁺ levels, prevented an ethanol-induced increase in plasma ALT and AST, and changed the expression of 25% of the genes that were modulated by ethanol metabolism. These genes were associated with a number of pathways including the MAPK pathway. Interestingly, our analysis revealed that NMN treatment normalized Erk1/2 signaling and prevented an induction of Atf3 overexpression.

Conclusions: These findings reveal previously unreported mechanisms by which NMN supplementation alters hepatic gene expression and protein pathways to impact ethanol hepatotoxicity in an early-stage murine model of ALD. Overall, our data suggest further research is needed to fully characterize treatment paradigms and biochemical implications of NAD⁺-based interventions.

Keywords: ATF3; Alcoholic liver disease; ERK1/2; Liver; NMN; RNA-seq; Sirtuin.

Fig. 1

Differential ethanol effects on alanine aminotransferase (ALT) and aspartate aminotransferase (AST). a Treatment with NMN prevents an ethanol-induced increase in plasma ALT. b NMN treatment prevents an ethanol-mediated increase in AST plasma levels (mean +/− SEM) (n = 4 to 5 per treatment combination) (*p < 0.05)

Fig. 2

Metabolomics profile of hepatic tissue assessing the impact of NMN therapy in a model of ethanol toxicity. a NMN treatment significantly alters metabolites of nicotinamide metabolism. b TCA cycle metabolites are moderately altered due to NMN supplementation (mean +/− SEM) (n ≥ 4) (*p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 3

Ethanol effects on RNA expression levels via RNA sequencing analysis. a Pipeline for identification of NMN-dependent and NMN-independent ethanol effects on RNA expression levels. b RNA expression differences across the treatment (NMN and ethanol) combinations for genes with NMN-dependent ethanol effects. Each row represents a gene with a significant ethanol effect (FDR < 0.05) that was classified as NMN-dependent (differential ethanol p < 0.05; 437 genes). Expression levels are shown as the ratio of average expression level in each treatment combination (columns) to the average expression in the Control Saline group. Expression levels were log base 2 transformed prior to analyses and graphing. Values in the key have be back transformed to increase interpretability

Fig. 4

Pathway analysis and enrichment (p < 0.05) of a NMN-dependent ethanol genes and b NMN-independent ethanol genes. The top three (via p value) pathways enriched for genes with NMN-dependent ethanol effects: c amino sugar and nucleotide sugar metabolism, d steroid hormone biosynthesis, and e MAPK signaling pathway. In these heatmaps, each row represents a gene with a significant ethanol effect (FDR < 0.05) that was classified as NMN-dependent (differential ethanol p < 0.05). Expression levels are shown as the ratio of average expression level in each treatment combination (columns) to the average expression in the Control Saline group. Expression levels were log base 2 transformed prior to analyses and graphing. Values in the key have be back transformed to increase interpretability

Fig. 5

Effect of NMN treatment on Erk, P38, and Atf3. a Western blot image of phosho-Erk1/2, Erk1/2, phospho-P38, P38, and Atf3. b Densitometric analysis of phospho-Erk1/2 immunoblot normalized to total Erk1/2. c Densitometric analysis of phosphorylated P38 normalized to total P38. d Densitometric analysis of Atf3 western blot (mean +/− SEM) (***p < 0.001)