Background and Aims: Overdose of acetaminophen (APAP) is the major cause of acute liver failure in the western world. We report a novel signaling interaction between hepatocyte nuclear factor 4 alpha (HNF4α) cMyc and nuclear factor erythroid 2–related factor 2 (Nrf2) during liver injury and regeneration after APAP overdose. Approach and Results: APAP-induced liver injury and regeneration were studied in male C57BL/6J (WT) mice, hepatocyte-specific HNF4α knockout mice (HNF4α-KO), and HNF4α-cMyc double knockout mice (DKO). C57BL/6J mice treated with 300 mg/kg maintained nuclear HNF4α expression and exhibited liver regeneration, resulting in recovery. However, treatment with 600-mg/kg APAP, where liver regeneration was inhibited and recovery was delayed, showed a rapid decline in HNF4α expression. HNF4α-KO mice developed significantly higher liver injury due to delayed glutathione recovery after APAP overdose. HNF4α-KO mice also exhibited significant induction of cMyc, and the deletion of cMyc in HNF4α-KO mice (DKO mice) reduced the APAP-induced liver injury. The DKO mice had significantly faster glutathione replenishment due to rapid induction in Gclc and Gclm genes. Coimmunoprecipitation and ChIP analyses revealed that HNF4α interacts with Nrf2 and affects its DNA binding. Furthermore, DKO mice showed significantly faster initiation of cell proliferation resulting in rapid liver regeneration and recovery. Conclusions: These data show that HNF4α interacts with Nrf2 and promotes glutathione replenishment aiding in recovery from APAP-induced liver injury, a process inhibited by cMyc. These studies indicate that maintaining the HNF4α function is critical for regeneration and recovery after APAP overdose.

Overdose of acetaminophen (APAP) is the major cause of acute liver failure (ALF) in the Western world with very limited treatment options. Previous studies from our groups and others have shown that timely activation of liver regeneration is a critical determinant of transplant-free survival of APAP-induced ALF patients. Here, we report that hepatocyte-specific deletion of Yes-associated protein (Yap), the downstream mediator of the Hippo Kinase signaling pathway results in faster recovery from APAP-induced acute liver injury. Initial studies performed with male C57BL/6J mice showed a rapid activation of Yap and its target genes within first 24 h after APAP administration. Treatment of hepatocyte-specific Yap knockout (Yap-KO) mice with 300 mg/kg APAP resulted in equal initial liver injury but a significantly accelerated recovery in Yap-KO mice. The recovery was accompanied by significantly rapid hepatocyte proliferation supported by faster activation of Wnt/β-catenin pathway. Furthermore, Yap-KO mice had significantly earlier and higher pro-regenerative inflammatory response following APAP overdose. Global gene expression analysis indicated that Yap-KO mice had a robust activation of transcription factors involved in response to endoplasmic reticulum stress (XBP1) and maintaining hepatocyte differentiation (HNF4α). In conclusion, these data indicate that inhibition of Yap in hepatocytes results in rapid recovery from APAP overdose due to an earlier activation of liver regeneration.

Background: O-GlcNAcylation is a post-translational modification catalyzed by the enzyme O-GlcNAc transferase, which transfers a single N-acetylglucosamine sugar from UDP-GlcNAc to the protein on serine and threonine residues on proteins. Another enzyme, O-GlcNAcase (OGA), removes this modification. O-GlcNAcylation plays an important role in pathophysiology. Here, we report that O-GlcNAcylation is essential for hepatocyte differentiation, and chronic loss results in fibrosis and HCC. Methods: Single-cell RNA-sequencing (RNA-seq) was used to investigate hepatocyte differentiation in hepatocyte-specific O-GlcNAc transferase-knockout (OGT-KO) mice with decreased hepatic O-GlcNAcylation and in O-GlcNAcase-KO mice with increased O-GlcNAcylation in hepatocytes. Patients HCC samples and the diethylnitrosamine-induced HCC model were used to investigate the effect of modulation of O-GlcNAcylation on the development of liver cancer. Results: Loss of hepatic O-GlcNAcylation resulted in disruption of liver zonation. Periportal hepatocytes were the most affected by loss of differentiation, characterized by dysregulation of glycogen storage and glucose production. O-GlcNAc transferase-KO mice exacerbated diethylnitrosamine-induced HCC development with increased inflammation, fibrosis, and YAP signaling. Consistently, O-GlcNAcase -KO mice with increased hepatic O-GlcNAcylation inhibited diethylnitrosamine-induced HCC. A progressive loss of O-GlcNAcylation was observed in patients with HCC. Conclusions: Our study shows that O-GlcNAcylation is a critical regulator of hepatic differentiation, and loss of O-GlcNAcylation promotes hepatocarcinogenesis. These data highlight increasing O-GlcNAcylation as a potential therapy in chronic liver diseases, including HCC. Export

Nuclear receptors such as Constitutive Androstane Receptor (CAR), Pregnane X Receptor (PXR), and Peroxisome Proliferator-Activated Receptor-alpha (PPARα), and transcription factors with nuclear receptor type activity such as Aryl hydrocarbon Receptor (AhR) function as xenobiotic sensors. Hepatocyte nuclear factor 4alpha (HNF4α) is a highly conserved orphan nuclear receptor essential for liver function. We tested the hypothesis that HNF4α is essential for the function of these four major xenosensors. Wild-type (WT) and hepatocyte-specific Hnf4a null (HNF4α-KO) mice were treated with the mouse-specific activators of AhR (TCDD, 30 µg/kg), CAR (TCPOBOP, 2.5 µg/g), PXR, (PCN, 100 µg/g), and PPARα (WY-14643, 1 mg/kg). Blood and liver tissue samples were collected to study receptor activation. TCDD (AhR agonist) treatment did not affect the liver-to-body weight ratio (LW/BW) in either WT or HNF4α-KO mice. Further, TCDD activated AhR in both WT and HNF4α-KO mice, confirmed by increase in expression of AhR target genes. TCPOBOP (CAR agonist) significantly increased the LW/BW ratio and CAR target gene expression in WT mice, but not in HNF4α-KO mice. PCN (a mouse PXR agonist) significantly increased LW/BW ratio in both WT and HNF4α-KO mice however, it failed to induce PXR target genes in HNF4α-KO mice. The treatment of WY-14643 (PPARα agonist) increased LW/BW ratio and PPARα target gene expression in WT mice but not in HNF4α-KO mice. Together, these data indicate that the function of CAR, PXR, and PPARα but not of AhR was disrupted in HNF4α-KO mice. These results demonstrate that HNF4α function is critical for the activation of hepatic xenosensors, which are critical for toxicological responses.

Non‐alcoholic fatty liver disease (NAFLD), newly renamed metabolic dysfunction‐associated liver disease (MASLD), is a leading cause of liver disease in children and adults. There is a paucity of data surrounding potential biomarkers and therapeutic targets, especially in pediatric NAFLD. Leukocyte cell‐derived chemotaxin 2 (LECT2) is a chemokine associated with both liver disease and skeletal muscle insulin resistance. Our aim was to determine associations between LECT2 and common clinical findings of NAFLD in pediatric patients. Enzyme‐linked immunosorbent assay (ELISA) was used to measure serum LECT2 concentrations in children (aged 2–17 years) with and without NAFLD. LECT2 concentrations were then correlated to clinical parameters in NAFLD. Mean LECT2 was significantly elevated in children with NAFLD versus healthy controls (n = 63 vs. 42, 5.83 ± 1.98 vs. 4.02 ± 2.02 ng/mL, p < 0.005). Additionally, LECT2 had strong correlations with body mass index (BMI) (Pearson r = 0.301, p = 0.002). A LECT2 concentration of 3.76 mg/mL predicts NAFLD with a sensitivity of 90.5% and specificity of 54.8%. Principal component analysis and logistic regression models further confirmed associations between LECT2 and NAFLD status. This study demonstrates increased serum LECT2 concentrations in pediatric NAFLD, which correlates with BMI and shows strong predictive value within these patients. Our data indicate that LECT2 is a potential diagnostic biomarker of disease and should be further investigated in pediatric as well as adult NAFLD.

Hepatocyte nuclear factor 4 alpha (HNF4α) is a nuclear factor essential for liver function that regulates the expression of cMyc and plays an important role in proliferation and differentiation during liver regeneration. This study investigated the role of HNF4α-cMyc interaction in regulating liver injury and regeneration using the choline-deficient and ethionine-supplemented (CDE) diet model. WT, hepatocyte-specific HNF4α-KO, cMyc-KO, and HNF4α-cMyc double knockout (DKO) mice were fed a CDE diet for one week to induce subacute liver injury. To study regeneration, CDE diet was followed by normal chow diet for one week. WT mice showed significant liver injury and decreased HNF4α mRNA and protein expression after one week of a CDE diet. HNF4α deletion resulted in significantly higher injury with increased inflammation, fibrosis, proliferation, and HPC activation compared to WT mice after CDE diet feeding but similar recovery. Deletion of cMyc substantially lowered liver injury with activation of inflammatory genes activation compared to WT and HNF4α-KO mice after CDE diet feeding. DKO mice resulted in phenotype comparable to the HNF4α-KO mice after CDE diet feeding and led to complete recovery. DKO mice showed a significant increase in HPC markers both following CDE-induced injury and after one week of recovery. Taken together, these data show that HNF4α protects against inflammatory and fibrotic change following CDE diet-induced injury, which is driven by cMyc.