Rewired NAD+ metabolism promotes NF-κB-mediated oxidative stress and disrupts lipid homeostasis in liver fibrosis progression

ABSTRACT Chronic liver fibrosis significantly increases the risk of hepatocellular carcinoma (HCC), a leading cause of cancer-related deaths. However, the molecular mechanisms linking fibrosis to inflammation-associated HCC development remain unclear, complicating early diagnosis and intervention. In this study, we employ multi-omics analyses, including untargeted and targeted metabolomics, lipidomics, and transcriptomics, in a mouse model of chemically induced liver fibrosis and HCC, integrating publicly available transcriptome data from LX-2 human hepatic stellate cell (HSC) line. Our results reveal a profound rewiring of NAD+ metabolism as a central driver of metabolic disturbance. Analysis of bulk liver tissue shows increased activity of the kynurenine pathway of tryptophan metabolism, enhancing NAD+ precursor production. Hepatic nicotinamide (NAM) levels decrease due to elevated expression of NAM N-methyltransferase (Nnmt) in HSCs. Despite reduced hepatic NAM, serum NAD+ level rise and is compartmentalized, triggering a disruption in NAD+ homeostasis and activating NF-κB-mediated oxidative stress pathways. Moreover, lipid dysregulation occurs, with NF-κB dominating the regulation of SIRT1/SREBP-controlled lipogenic and cholesterogenic genes, leading to imbalances in hepatic and serum lipids. These insights elucidate connections between NAD+ metabolism, inflammation, and lipid dysregulation, potentially aiding in developing diagnostic biomarkers and therapeutic targets for non-viral HCC. Key Points The integration of untargeted and targeted metabolomics identifies the metabolic pathways that are associated with the development of hepatocellular carcinoma (HCC) and the potential role of disturbances in NAD+ metabolism as a central driver of metabolic dysregulation in hepatic fibrosis, validated across transcriptomics. Fibrosis-associated HCC is linked to the depletion of nicotinamide and a cascade of metabolic disturbances that promote inflammation and oxidative stress in hepatic stellate cells. Targeted analysis of the metabolites in the discriminative pathways along a continuum of fibrosis-cirrhosis-HCC discovers several potential tissue and serum biomarkers for monitoring disease progression. Multi-omics analysis (i.e., metabolomics, lipidomics, and transcriptomics) reveals complex interplay between NAD+ metabolism, NF-κB-mediated oxidative stress, and lipid homeostasis in fibrosis-associated HCC development. Competing Interest Statement The authors have declared no competing interest. Footnotes Format changed. Key Points: 1.The integration of untargeted and targeted metabolomics identifies the metabolic pathways that are associated with the development of hepatocellular carcinoma (HCC) and the potential role of disturbances in NAD+ metabolism as a central driver of metabolic dysregulation in hepatic fibrosis, validated across transcriptomics. 2.Fibrosis-associated HCC is linked to the depletion of nicotinamide and a cascade of metabolic disturbances that promote inflammation and oxidative stress in hepatic stellate cells. 3.Targeted analysis of the metabolites in the discriminative pathways along a continuum of fibrosis-cirrhosis-HCC discovers several potential tissue and serum biomarkers for monitoring disease progression. 4.Multi-omics analysis (i.e., metabolomics, lipidomics, and transcriptomics) reveals complex interplay between NAD+ metabolism, NF-κB-mediated oxidative stress, and lipid homeostasis in fibrosis-associated HCC development. Abbreviations - 3-HAA - 3-Hydroxyanthranilate - AAG - Alanine/aspartate/glutamate - Acat2 - Acyl-CoA:cholesterol acyltransferase 2 - Acly - ATP citrate lyase - AMC - Age-matched control - Aox1 - Aldehyde oxidase 1 - BA - Bile acid - CCl4 - Carbon tetrachloride - Cpt1a - Carnitine palmitoyltransferase 1A - DEG - Differentially expressed gene - DEN - Diethylnitrosamine - DG - Diglyceride - DNL - De novo lipogenesis - ESI - Electrospray ionization - FA - Fatty acid - FAO - Fatty acid oxidation - Fasn - Fatty acid synthase - Haao - 3-Hydroxyanthranilate 3,4-dioxygenase - HBV - Hepatitis B virus - HCC - Hepatocellular carcinoma - Hmgcr - 3-Hydroxy-3-methylglutaryl-CoA reductase - Hmgcs1 - 3-Hydroxy-3-methylglutaryl-CoA synthase 1 - HSC - Hepatic stellate cell - IFN-γ - Interferon gamma - m/z - Mass-to-charge ratio - MS - Mass spectrometry - NA - Nicotinic acid - NAD+ - Nicotinamide adenine dinucleotide - NAFLD - Non-alcoholic fatty liver disease - NAM - Nicotinamide - Nampt - Nicotinamide phosphoribosyltransferase - NASH - Non-alcoholic steatohepatitis - Nfkb1 - Nuclear factor kappa B subunit 1 - Nmnat1 - Nicotinamide mononucleotide adenylyltransferase 1 - Nnmt - Nicotinamide N-methyltransferase - Prkag2 - 5’-AMP-activated protein kinase subunit gamma-2 - QC - Quality control - Qprt - Quinolinate phosphoribosyl transferase - RNA-Seq - RNA sequencing - RPLC - Reverse-phase liquid chromatography - Sirt - Sirtuin - Srebf1 - Sterol regulatory element-binding protein 1 - Srebf2 - Sterol regulatory element-binding protein 2 - SREBP - Sterol regulatory element-binding protein - Tdo2 - Tryptophan 2,3-dioxygenase - TG - Triglyceride - TGF-β1 - Transforming growth factor beta 1