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Background & Aims: Expression of P21, encoded by the CDKN1A gene, has been associated with fibrosis progression in steatotic liver disease (SLD); however, the underlying mechanisms remain unknown. In the present study, we investigated the function of CDKN1A in SLD. Methods: CDKN1A expression levels were evaluated in different patient cohorts with SLD, fibrosis, and advanced chronic liver disease (ACLD). Cdkn1a-/- and Cdkn1a+/+ mice were fed with either a Western diet (WD), a Lieber-DeCarli (LdC) diet plus multiple EtOH (ethanol) binges, or a DuAL diet (metabolic dysfunction-associated fatty liver disease and alcohol-related liver). Primary hepatocytes were isolated and functional assays performed. Results: A significant increase in CDKN1A expression was observed in patients with steatohepatitis and fibrosis (with a positive correlation with both NAFLD Activity Score and fibrosis staging scores), cirrhosis and ACLD. Cdkn1a+/+ mice, fed a DuAL diet exhibited liver injury and cell death increased reactive oxygen species (ROS), and markers of senescence (γH2AX, β-GAL, Cdkn1a/p53) contributing to steatosis and inflammation. In contrast, Cdkn1a-/- mutant mice showed a significant decrease in senescence-associated markers as well as in markers of liver injury, hepatic steatosis and an increase in fatty acid oxidation and reduction in free fatty acid uptake as well as de novo lipogenesis. Mechanistically, activation of the AMPK-SIRT3 was observed in Cdkn1a-deleted animals. Conclusions: Cdkn1a deletion protected against preclinical SLD by promoting fatty acid oxidation and preventing free fatty acid uptake and de novo lipogenesis via the AMPK-SIRT3 axis. CDKN1A expression was found to be directly correlated with increased severity of NAFLD Activity Score and fibrosis in patients with SLD. CDKN1A could be a potential theragnostic target for the treatment of metabolic dysregulation in patients with SLD, with and without alcohol consumption. Impact and implications:: Expression of p21, encoded by the CDKN1A gene, has been associated with fibrosis progression in steatotic liver disease (SLD), but the molecular mechanisms remain elusive. Interestingly, in this study we found that Cdkn1a deletion protected against preclinical SLD by promoting fatty acid oxidation and preventing free fatty acid uptake and de novo lipogenesis, via the AMPK-SIRT3 axis. Translationally, Cdkn1a expression was found to be directly correlated with increased severity of NAFLD Activity Score (NAS) and fibrosis in SLD patients, and therefore, CDKN1A might be used potential theragnostic target for the treatment of metabolically induced SLD, with and without alcohol consumption.

Objectives: Lately, many countries have restricted or even banned transfat, and palm oil has become a preferred replacement for food manufacturers. Whether palm oil is potentially an unhealthy food mainly due to its high content of saturated Palmitic Acid (PA) is a matter of debate. The aim of this study was to test whether qualitative aspects of diet such as levels of PA and the fat source are risk factors for Metabolic Syndrome (MS) and Metabolic Associated Fatty Liver Disease (MAFLD). Methods: C57BL/6 male mice were fed for 14 weeks with three types of Western diet (WD): 1. LP-WD—low concentration of PA (main fat source—corn and soybean oils); 2. HP-WD—high concentration of PA (main fat source—palm oil); 3. HP-Trans-WD—high concentration of PA (mainly transfat). Results: All types of WD caused weight gain, adipocyte enlargement, hepatomegaly, lipid metabolism alterations, and steatohepatitis. Feeding with HP diets led to more prominent obesity, hypercholesterolemia, stronger hepatic injury, and fibrosis. Only the feeding with HP-Trans-WD resulted in glucose intolerance and elevation of serum transaminases. Brief withdrawal of WDs reversed MS and signs of MAFLD. However, mild hepatic inflammation was still detectable in HP groups. Conclusions: HP and HP-Trans-WD play a crucial role in the genesis of MS and MAFLD.

We aimed to identify a microRNA (miRNA)‐E3 ubiquitin ligase regulatory network for protein substrates enriched in cell death pathways and investigate the underlying molecular mechanisms in alcohol‐associated hepatitis (AH). An miRNA‐E3 ubiquitin ligase regulatory network for protein substrates enriched in cell death pathways was constructed using integrated bioinformatics analysis. Differentially expressed hub miRNAs (GSE59492) and their validated miRNA target genes (GSE28619) were identified in the liver of patients with AH compared with healthy controls. Liver samples from patients with AH and healthy individuals and mice exposed to Gao‐binge (acute on chronic) ethanol were used for experimental validation. Using hub miRNAs identified by weighted correlation network analysis, a miRNA‐E3 ubiquitin ligase regulatory network was established based on 17 miRNAs and 7 E3 ligase genes targeted by these miRNAs that were down‐regulated in AH. Among the miRNAs in this regulatory network, miR‐150‐5p was the only miRNA regulating the E3 ligase cytokine‐inducible SH2 containing protein (CISH), the E3 ligase that regulates the largest number of substrates among all E3 ligase family members. Therefore, the CISH regulatory pathway for ubiquitinated substrates was selected for subsequent experimental validation. Consistent with the bioinformatics analysis results, expression of miR‐150‐5p was markedly increased, while CISH was decreased, in the livers of patients with AH and mice exposed to Gao‐binge ethanol. Moreover, ubiquitination of Fas‐associated protein with death domain, a predicted CISH substrate involved in the regulation of programmed cell death, was reduced in livers from mice after Gao‐binge ethanol. Conclusion: Identification of the miRNA‐E3 ubiquitin ligase regulatory network for protein substrates enriched in the cell death pathways provides insights into the molecular mechanisms contributing to hepatocyte death in AH.

Interferon regulatory factor 3 (IRF3) has both transcriptional and nontranscriptional functions. Transcriptional activity is dependent on serine phosphorylation of IRF3, while transcription‐independent IRF3‐mediated apoptosis requires ubiquitination. IRF3 also binds to inhibitor of nuclear factor kappa B kinase (IKKβ) in the cytosol, restricting nuclear translocation of p65. IRF3‐deficient mice are highly sensitive to high‐fat diet (HFD)‐induced liver injury; however, it is not known if transcriptional and/or nontranscriptional activity of IRF3 confers protection. Using a mouse model only expressing nontranscriptional functions of IRF3 (Irf3S1/S1), we tested the hypothesis that nontranscriptional activity of IRF3 protects mice from HFD‐induced liver injury. C57BL/6, Irf3−/−, and Irf3S1/S1 mice were fed an HFD for 12 weeks. In C57BL/6 mice, the HFD increased expression of interferon (IFN)‐dependent genes, despite a decrease in IRF3 protein in the liver. The HFD had no impact on IFN‐dependent gene expression Irf3−/− or Irf3S1/S1 mice, both lacking IRF3 transcriptional activity. Liver injury, apoptosis, and fibrosis were exacerbated in Irf3−/− compared to C57BL/6 mice following the HFD; this increase was ameliorated in Irf3S1/S1 mice. Similarly, expression of inflammatory cytokines as well as numbers of neutrophils and infiltrating monocytes was increased in Irf3−/− mice compared to C57BL/6 and Irf3S1/S1 mice. While the HFD increased the ubiquitination of IRF3, a response associated with IRF3‐mediated apoptosis, in Irf3S1/S1 mice, protection from liver injury was not due to differences in apoptosis of hepatocytes or immune cells. Instead, protection from HFD‐induced liver injury in Irf3S1/S1 mice was primarily associated with retardation of nuclear translocation of p65 and decreased expression of nuclear factor kappa B (NFκB)‐dependent inflammatory cytokines. Conclusion: Taken together, these data identify important contributions of the nontranscriptional function of IRF3, likely by reducing NFκB signaling, in dampening the hepatic inflammatory environment in response to an HFD.

Hepatocellular death contributes to progression of alcohol–associated (ALD-associated) and non–alcohol-associated (NAFL/NASH) liver diseases. However, receptor-interaction protein kinase 3 (RIP3), an intermediate in necroptotic cell death, contributes to injury in murine models of ALD but not NAFL/NASH. We show here that a differential role for mixed-lineage kinase domain–like protein (MLKL), the downstream effector of RIP3, in murine models of ALD versus NAFL/NASH and that RIP1-RIP3-MLKL can be used as biomarkers to distinguish alcohol-associated hepatitis (AH) from NASH. Phospho-MLKL was higher in livers of patients with NASH compared with AH or healthy controls (HCs). MLKL expression, phosphorylation, oligomerization, and translocation to plasma membrane were induced in WT mice fed diets high in fat, fructose, and cholesterol but not in response to Gao-binge (acute on chronic) ethanol exposure. Mlkl–/– mice were not protected from ethanol-induced hepatocellular injury, which was associated with increased expression of chemokines and neutrophil recruitment. Circulating concentrations of RIP1 and RIP3, but not MLKL, distinguished patients with AH from HCs or patients with NASH. Taken together, these data indicate that MLKL is differentially activated in ALD/AH compared with NAFL/NASH in both murine models and patients. Furthermore, plasma RIP1 and RIP3 may be promising biomarkers for distinguishing AH and NASH.