Udayan Apte

Liver regeneration is critical determinant of survival following acetaminophen (APAP) overdose, driven primarily by proliferation of surviving hepatocytes. Hepatic progenitor cells (HPCs) are bipotential cells activated when hepatocyte proliferation is impaired. The contribution of HPCs to liver regeneration after APAP overdose remains unclear. This study investigated the contribution of HPCs to hepatocyte repopulation when regeneration is impaired after severe APAP overdose using a lineage-tracing mouse model. Two-month-old male C57BL/6J mice were fasted overnight and then administered a 600 mg/kg overdose of APAP (600APAP). For lineage tracing, male ROSA^mT-mG mice were injected with AAV8-TBG-Cre to label hepatocytes. Two weeks after AAV administration, mice were fasted overnight and then administered a 600 mg/kg overdose of APAP. Liver and blood samples were collected multiple times. C57BL/6J mice treated with severe acetaminophen overdose (600APAP) showed substantial liver injury, with peak serum ALT and hepatic necrosis at 72 h, followed by progressive recovery. HPCs activation was evidenced by increased expression of EpCAM, A6. CK19, cholangiocyte marker, increase during the recovery phase. APAP administration to ROSA^mT-mG mice induced liver injury and subsequent regeneration and recovery comparable to wild type mice. Detection of HNF4α/tdTomato double-positive cells, hepatocytes originating from a non-parenchymal source, were detected after injury but represented only 0.3–0.6% of total hepatocytes. Taken together, this study indicates that despite an increase of HPC/cholangiocyte markers, HPC/cholangiocyte transdifferentiation contributes minimally to hepatocyte replenishment during an impaired regeneration after severe APAP-induced liver injury.

Liver tumors are the most common carcinogenic outcome observed in two-year bioassays used for regulatory risk assessment of chemicals and pharmaceuticals. Because these traditional long-term studies are resource-intensive and not feasible for most chemicals, there is a critical need to develop alternative approaches that can identify potential rodent liver carcinogens in short-term studies (e.g., ≤ 1 month). Biomarkers associated with molecular initiating events (MIEs) of early rodent liver tumorigenesis may enable the prediction of two-year outcomes from such studies. Here, we identified transcriptional changes associated with six key initiators of rat liver tumorigenesis: five transcription factors (TFs) associated with non-genotoxic modes of action (AhR, PXR, CAR, PPARα, and ERα), and tumor suppressor protein p53, which is activated by diverse cellular stressors including genotoxicity. Three known chemical activators for each TF were administered orally once daily for five days at doses associated with tumorigenic responses in previous rat cancer bioassays. RNA-seq identified differentially expressed genes (DEGs) that were consistently regulated by at least two activators in wild-type but not in corresponding TF knockout animals. Gene set enrichment analysis of ranked differential expression results demonstrated enrichment of pathways reflecting established TF biology. When comparing the consensus DEGs for each TF to a library of chemically induced, liver-specific gene expression profiles, we found that in most cases the chemicals with the most similar profiles to the consensus lists were known to activate the corresponding TF. Collectively, these findings identified candidate gene sets that may have utility for monitoring the induction of carcinogenesis-associated MIEs in short-term studies.

Liver regeneration is critical determinant of survival following acetaminophen (APAP) overdose, is driven primarily by proliferation of surviving hepatocytes. Hepatic progenitor cells (HPCs) are bipotential cells activated when hepatocyte proliferation is impaired. Contribution of HPCs to liver regeneration after APAP overdose remains unclear. This study investigated the contribution of HPCs to hepatocyte repopulation after severe APAP overdose using lineage tracing mouse models. Two-month-old male C57BL/6J mice were fasted overnight and then administered a 600 mg/kg overdose of APAP (600APAP). For lineage tracing, male ROSA^mT-mG mice were injected with AAV8-TBG-Cre to label hepatocytes. Two weeks after AAV administration, mice were fasted overnight and then administered a 600 mg/kg overdose of APAP. Liver and blood samples were collected multiple times. C57BL/6J mice treated with severe acetaminophen overdose (600APAP) show substantial liver injury, with peak serum ALT and hepatic necrosis at 72 h, followed by progressive recovery. HPC activation was evidenced by increased expression of EpCAM, A6, and CK19 during recovery phase. APAP administration to ROSA^mT-mG mice induced liver injury and subsequent regeneration and recovery comparable to wild type mice. Detection of HNF4α/tdTomato double-positive cells, hepatocytes originating from HPCs, were detected after injury but represented only 0.3–0.6% of total hepatocytes. Taken together, this study indicates that despite an increase of HPCs markers, lineage tracing model demonstrate that HPCs transdifferentiation contribute minimally to hepatocyte replenishment following severe APAP-induced liver injury.

Metabolic dysfunction‐associated liver disease (MASLD) is a leading cause of liver disease in children. There is a paucity of data on potential biomarkers and therapeutic targets, especially in pediatric MASLD. We used mass spectrometry (MS)‐mediated proteomics followed by enzyme‐linked immunosorbent assay (ELISA) to identify potential biomarkers and therapeutic targets in pediatric MASLD. Serum samples were collected from pediatric subjects without (n = 56) and with MASLD (n = 72). Initial screen using MS‐based proteomics identified 6 upregulated (adenosine deaminase 2, sex hormone‐binding globulin (SHBG), inter‐alpha‐trypsin inhibitor heavy chain H1 (ITIH1), fructose‐bisphosphate aldolase A, type II cytoskeletal 2 epidermal keratine, N‐acetylmuramoyl‐L‐alanine amidase) and 3 downregulated (alcohol dehydrogenase 4 (ADH4), fructose‐bisphosphate aldolase B (ALDOB), serum albumin) proteins in the MASLD group. Confirmatory studies using ELISA were performed for the 2 strongest upregulated proteins (SHBG and ITIH1) and two top downregulated proteins (ADH4 and ALDOB). Correlation of ELISA results with clinical data revealed that SHBG had strong associations with BMI, ALT, and HgbA1c (p < 0.05). ADH4 had strong associations with BMI and HgbA1c (p < 0.05). ITIH1 and ALDOB had no strong correlations with common clinical parameters of MASLD. Area under ROC Curve revealed statistically significant ability of SHBG (494 nmol/L, sensitivity = 98%, specificity 80%) and ADH4 (2.14 ng/mL, sensitivity = 65%, specificity = 66%) to diagnosis MASLD (p < 0.05). MS with confirmation ELISA identified SHBG and ADH4 as potential biomarkers of pediatric MASLD.

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Background: Hepatocyte nuclear factor 4 alpha (HNF4α) is the master regulator of hepatic differentiation. Recent studies have also revealed the role of HNF4α in hepatocyte proliferation via negatively regulating the expression of proto-mitogenic genes, including cMyc. Here, we aimed to study the interaction between HNF4α-cMyc during liver regeneration after partial hepatectomy (PHX).Methods: Wild-type (WT), hepatocyte-specific knockout of HNF4α (HNF4α-KO), cMyc (cMyc-KO), and HNF4α-cMyc double knockout (DKO) mice were subjected to PHX to induce liver regeneration. Blood and liver tissue samples were collected at 0h, 24h, 48h, 7D, and 14D after PHX for further analysis.Results: WT, HNF4α-KO, cMyc-KO and DKO mice regained liver weight by 14 days after PHX. The deletion of cMyc did not affect liver regeneration, which was similar to the WT mice. WT and cMyc-KO mice started regaining liver weight as early as 24 hours after PHX, with a peak proliferation response at 48 hours after PHX. HNF4α- KO and DKO showed a delayed response with liver weight increase by day 7 after PHX. The overall hepatocyte proliferation response by DKO mice following PHX was lower than that of other genotypes. Interestingly, the surviving HNF4α-KO and DKO mice showed re-expression of HNF4α at mRNA and protein levels on day 14 after PHX. This was accompanied by a significant increase in the expression of Krt19 and Epcam, hepatic progenitor cell markers, in the DKO mice on day 14 after PHX.Conclusion: These data indicate that, in the absence of HNF4α, cMyc contributes to hepatocyte-driven proliferation to compensate for the lost tissue mass. Furthermore, in the absence of both HNF4α and cMyc, HPC-driven proliferation occurs to support liver regeneration.

Liver cancer is the sixth most common cancer and the fourth leading cause of cancer-related deaths worldwide. Hepatocellular carcinoma (HCC) is the most prevalent primary liver cancer and the incidence of HCC is on the rise. Liver cancers in general and HCC in particular do not respond to chemotherapy. Radiological ablation, surgical resection, and liver transplantation are the only medical therapies currently available. Hepatocyte nuclear factor 4 α (HNF4α) is an orphan nuclear receptor expressed only in hepatocytes in the liver. HNF4α is considered the master regulator of hepatic differentiation because it regulates a significant number of genes involved in various liver-specific functions. In addition to maintaining hepatic differentiation, HNF4α also acts as a tumor suppressor by inhibiting hepatocyte proliferation by suppressing the expression of promitogenic genes and inhibiting epithelial to mesenchymal transition in hepatocytes. Loss of HNF4α expression and function is associated with rapid progression of chronic liver diseases that ultimately lead to liver cirrhosis and HCC, including metabolism-associated steatohepatitis, alcohol-associated liver disease, and hepatitis virus infection. This review summarizes the role of HNF4α in liver cancer pathogenesis and highlights its potential as a potential therapeutic target for HCC.

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.

Per- and polyfluoroalkyl substances (PFAS) such as perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) and perfluoro-2-methyl-3-oxahexanoic acid (GenX), the new replacement PFAS, are major environmental contaminants. In rodents, these PFAS induce several adverse effects on the liver, including increased proliferation, hepatomegaly, steatosis, hypercholesterolemia, nonalcoholic fatty liver disease and liver cancers. Activation of peroxisome proliferator receptor alpha by PFAS is considered the primary mechanism of action in rodent hepatocyte-induced proliferation. However, the human relevance of this mechanism is uncertain. We investigated human-relevant mechanisms of PFAS-induced adverse hepatic effects using FRG liver-chimeric humanized mice with livers repopulated with functional human hepatocytes. Male FRG humanized mice were treated with 0.067 mg/L of PFOA, 0.145 mg/L of PFOS, or 1 mg/L of GenX in drinking water for 28 days. PFOS caused a significant decrease in total serum cholesterol and LDL/VLDL, whereas GenX caused a significant elevation in LDL/VLDL with no change in total cholesterol and HDL. All three PFAS induced significant hepatocyte proliferation. RNA-sequencing with alignment to the human genome showed a total of 240, 162, and 619 differentially expressed genes after PFOA, PFOS, and GenX exposure, respectively. Upstream regulator analysis revealed that all three PFAS induced activation of p53 and inhibition of androgen receptor and NR1D1, a transcriptional repressor important in circadian rhythm. Further biochemical studies confirmed NR1D1 inhibition and in silico modeling indicated potential interaction of all three PFAS with the DNA-binding domain of NR1D1. In conclusion, our studies using FRG humanized mice have revealed new human-relevant molecular mechanisms of PFAS including their previously unknown effect on circadian rhythm.