One Sentence Summary:Inhibiting injury-induced senescence mediated by TGFβ signaling in regenerative epithelium improves liver regeneration. Accessible Summary:The liver is a paradigm of organ regeneration, however regeneration may fail in a previously normal liver following acute severe injury such as acetaminophen poisoning. We show that, a process with prevents proliferation termed senescence, which is classically associated with aging and carcinogenesis, stops the liver's regenerative cells. This senescence can be spread from cell to cell by the signaling molecule TGFβ. When TGFβ signaling is inhibited during acetaminophen poisoning in mice, senescence is impeded, regeneration accelerates, and survival is improved. Therefore targeting senescence induced by acute tissue injury is an attractive therapeutic approach to improve regeneration. Abstract:Liver injury results in rapid regeneration through hepatocyte proliferation and hypertrophy.However, after acute severe injury, such as acetaminophen poisoning, effective regeneration may fail. We investigated how senescence may underlie this regenerative failure. In human acute liver disease, and murine models, p21-dependent hepatocellular senescence was proportionate to disease severity and was associated with impaired regeneration. In an acetaminophen injury mouse model, a transcriptional signature associated with the induction 4 of paracrine senescence was observed within twenty four hours, and was followed by one of impaired proliferation. In mouse genetic models of hepatocyte injury and senescence we observed transmission of senescence to local uninjured hepatocytes. Spread of senescence depended upon macrophage-derived TGFβ1 ligand. In acetaminophen poisoning, inhibition of TGFβ receptor 1 (TGFβR1) improved mouse survival. TGFβR1 inhibition reduced senescence and enhanced liver regeneration even when delivered after the current therapeutic window for acetaminophen poisoning. This mechanism, in which injury-induced senescence impairs liver regeneration, is an attractive therapeutic target for developing treatments for acute liver failure.

Dysfunctional mitochondria and generation of reactive oxygen species (ROS) promote chronic diseases, which have spurred interest in the molecular mechanisms underlying these conditions. Previously, we have demonstrated that disruption of post-translational modification of proteins with β-linked -acetylglucosamine (-GlcNAcylation) via overexpression of the GlcNAc-regulating enzymesGlcNAc transferase (OGT) or GlcNAcase (OGA) impairs mitochondrial function. Here, we report that sustained alterations inGlcNAcylation either by pharmacological or genetic manipulation also alter metabolic function. Sustained GlcNAc elevation in SH-SY5Y neuroblastoma cells increased OGA expression and reduced cellular respiration and ROS generation. Cells with elevatedGlcNAc levels had elongated mitochondria and increased mitochondrial membrane potential, and RNA-sequencing analysis indicated transcriptome reprogramming and down-regulation of the NRF2-mediated antioxidant response. Sustained GlcNAcylation in mouse brain and liver validated the metabolic phenotypes observed in the cells, and OGT knockdown in the liver elevated ROS levels, impaired respiration, and increased the NRF2 antioxidant response. Moreover, elevatedGlcNAc levels promoted weight loss and lowered respiration in mice and skewed the mice toward carbohydrate-dependent metabolism as determined by indirect calorimetry. In summary, sustained elevation in GlcNAcylation coupled with increased OGA expression reprograms energy metabolism, a finding that has potential implications for the etiology, development, and management of metabolic diseases.

Perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS), chemicals present in a multitude of consumer products, are persistent organic pollutants. Both compounds induce hepatotoxic effects in rodents, including steatosis, hepatomegaly and liver cancer. The mechanisms of PFOA- and PFOS-induced hepatic dysfunction are not completely understood. We present evidence that PFOA and PFOS induce their hepatic effects via targeting hepatocyte nuclear factor 4-alpha (HNF4α). Human hepatocytes treated with PFOA and PFOS at a concentration relevant to occupational exposure caused a decrease in HNF4α protein without affecting HNF4α mRNA or causing cell death. RNA sequencing analysis combined with Ingenuity Pathway Analysis of global gene expression changes in human hepatocytes treated with PFOA or PFOS indicated alterations in the expression of genes involved in lipid metabolism and tumorigenesis, several of which are regulated by HNF4α. Further investigation of specific HNF4α target gene expression revealed that PFOA and PFOS could promote cellular dedifferentiation and increase cell proliferation by down regulating positive targets (differentiation genes such as CYP7A1) and inducing negative targets of HNF4α (pro-mitogenic genes such as CCND1). Furthermore, in silico docking simulations indicated that PFOA and PFOS could directly interact with HNF4α in a similar manner to endogenous fatty acids. Collectively, these results highlight HNF4α degradation as novel mechanism of PFOA and PFOS-mediated steatosis and tumorigenesis in human livers.

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Macroautophagy is a major cellular degradation pathway for long-lived proteins and cellular organelles to maintain cellular homeostasis. Reduced autophagy has been implicated in neurodegenerative diseases, metabolic syndrome, and tumorigenesis. In contrast, increased autophagy has been shown to protect against tissue injury and aging. Here we employed a cell-based quantitative high-throughput image screening (qHTS) for autophagy modulators using mouse embryonic fibroblasts (MEFs) that are stably expressing GFP-LC3. The library of pharmacologically active compounds (LOPAC) was used to screen for the autophagy modulators in compounds alone or in combination with the lysosome inhibitor chloroquine (CQ). The GFP-LC3 puncta were then quantified to measure autophagic flux. The primary screening revealed 173 compounds with efficacy more than 40%. These compounds were cherry-picked and re-tested at multiple different concentrations using the same assay. A number of novel autophagy inducers, inhibitors, and modulators with dual-effects on autophagy were identified from the cherry-pick screening. Interestingly, we found a group of compounds that induce autophagy are related to dopamine receptors and are commonly used as clinical psychiatric drugs. Among them, indatraline hydrochloride (IND), a dopamine inhibitor, and chlorpromazine hydrochloride (CPZ) and fluphenazine dihydrochloride (FPZ), two dopamine receptor antagonists, were further evaluated. We found that FPZ-induced autophagy through mTOR inhibition but IND and CPZ induced autophagy in an mTOR-independent manner. Our data suggest that image-based autophagic flux qHTS can efficiently identify autophagy inducers and inhibitors.