Activins are a subgroup of the TGFβ superfamily of growth and differentiation factors, dimeric in nature and consisting of two inhibin beta subunits linked via a disulfide bridge. Canonical activin signaling occurs through Smad2/3, with negative feedback initiated by Smad6/7 following signal transduction, which binds activin type I receptor preventing phosphorylation of Smad2/3 and activation of downstream signaling. In addition to Smad6/7, other inhibitors of activin signaling have been identified as well, including inhibins (dimers of an inhibin alpha and beta subunit), BAMBI, Cripto, follistatin, and follistatin-like 3 (fstl3). To date, activins A, B, AB, C, and E have been identified and isolated in mammals, with activin A and B having the most characterization of biological activity. Activin A has been implicated as a regulator of several important functions of liver biology, including hepatocyte proliferation and apoptosis, ECM production, and liver regeneration; the role of other subunits of activin in liver physiology are less understood. There is mounting data to suggest a link between dysregulation of activins contributing to various hepatic diseases such as inflammation, fibrosis, and hepatocellular carcinoma, and emerging studies demonstrating the protective and regenerative effects of inhibiting activins in mouse models of liver disease. Due to their importance in liver biology, activins demonstrate utility as a therapeutic target for the treatment of hepatic diseases such as cirrhosis, NASH, NAFLD, and HCC; further research regarding activins may provide diagnostic or therapeutic opportunity for those suffering from various liver diseases.

Patients with end-stage liver disease exhibit progressive skeletal muscle atrophy, highlighting a negative crosstalk between the injured liver and muscle. Our study was to determine whether TGFβ ligands function as the mediators. Acute or chronic liver injury was induced by a single or repeated administration of carbon tetrachloride. Skeletal muscle injury and repair was induced by intramuscular injection of cardiotoxin. Activin type IIB receptor (ActRIIB) ligands and growth differentiation factor 8 (Gdf8) were neutralized with ActRIIB-Fc fusion protein and a Gdf8-specific antibody, respectively. We found that acute hepatic injury induced rapid and adverse responses in muscle, which was blunted by neutralizing ActRIIB ligands. Chronic liver injury caused muscle atrophy and repair defects, which were prevented or reversed by inactivating ActRIIB ligands. Furthermore, we found that pericentral hepatocytes produce excessive Gdf8 in injured mouse liver and cirrhotic human liver. Specific inactivation of Gdf8 prevented liver injury-induced muscle atrophy, similar to neutralization of ActRIIB ligands. Inhibition of Gdf8 also reversed muscle atrophy in a treatment paradigm following chronic liver injury. Direct injection of exogenous Gdf8 protein into muscle along with acute focal muscle injury recapitulated similar dysregulated muscle regeneration as that observed with liver injury. The results indicate that injured liver negatively communicate with the muscle largely via Gdf8. Unexpectedly, inactivation of Gdf8 simultaneously ameliorated liver fibrosis in mice following chronic liver injury. In vitro, Gdf8 induced human hepatic stellate (LX-2) cells to form a septa-like structure and stimulated expression of profibrotic factors. Our findings identified Gdf8 as a novel hepatomyokine contributing to injured liver–muscle negative crosstalk along with liver injury progression.

We found that maternal hepatocytes exhibit developmental phenotypes in a temporal and spatial manner, similarly to fetal hepatocytes. They acquire this new property partially via yes-associated protein 1.

The transcription factor Nrf2 modulates the initiation and progression of a number of diseases including liver disorders. We evaluated whether Nrf2 mediates hepatic adaptive responses to cholestasis. Wild-type and Nrf2-null mice were subjected to bile duct ligation (BDL) or a sham operation. As cholestasis progressed to day 15 post-BDL, hepatocytes in the wild-type mice exhibited a tendency to dedifferentiate, indicated by the very weak expression of hepatic progenitor markers: CD133 and tumor necrosis factor-like weak induced apoptosis receptor (Fn14). During the same period, Nrf2 deficiency augmented this tendency, manifested by higher CD133 expression, earlier, stronger, and continuous induction of Fn14 expression, and markedly reduced albumin production. Remarkably, as cholestasis advanced to the late stage (40 days after BDL), hepatocytes in the wild-type mice exhibited a Fn14+ phenotype and strikingly upregulated the expression of deleted in malignant brain tumor 1 (DMBT1), a protein essential for epithelial differentiation during development. In contrast, at this stage, hepatocytes in the Nrf2-null mice entirely inhibited the upregulation of DMBT1 expression, displayed a strong CD133+/Fn14+ phenotype indicative of severe dedifferentiation, and persistently reduced albumin production. We revealed that Nrf2 maintains hepatocytes in the differentiated state potentially via the increased activity of the Nrf2/DMBT1 pathway during cholestasis.

BACKGROUND & AIMS:Maternal liver shows robust adaptations to pregnancy to accommodate the metabolic needs of the developing and growing placenta and fetus by largely unknown mechanisms. We found that Ascl1, a gene encoding a basic helix-loop-helix transcription factor essential for neuronal development, is highly activated in maternal hepatocytes during the second half of gestation in mice.METHODS:To investigate whether and how Ascl1 plays a pregnancy-dependent role, we deleted the Ascl1 gene specifically in maternal hepatocytes from midgestation until term.RESULTS:As a result, we identified multiple Ascl1-dependent phenotypes. Maternal livers lacking Ascl1 showed aberrant hepatocyte structure, increased hepatocyte proliferation, enlarged hepatocyte size, reduced albumin production, and increased release of liver enzymes, indicating maternal liver dysfunction. Simultaneously, maternal pancreas and spleen and the placenta showed marked overgrowth; and the maternal ceca microbiome showed alterations in relative abundance of several bacterial subpopulations. Moreover, litters born from maternal hepatic Ascl1-deficient dams experienced abnormal postnatal growth after weaning, implying an adverse pregnancy outcome. Mechanistically, we found that maternal hepatocytes deficient for Ascl1 showed robust activation of insulin-like growth factor 2 expression, which may contribute to the Ascl1-dependent phenotypes widespread in maternal and uteroplacental compartments.CONCLUSIONS:In summary, we show that maternal liver, via activating Ascl1 expression, modulates the adaptations of maternal organs and the growth of the placenta to maintain a healthy pregnancy. Our studies show that Ascl1 is a novel and critical regulator of the physiology of pregnancy.

Abstract The role of activin B, a transforming growth factor β (TGFβ) superfamily cytokine, in liver health and disease is largely unknown. We aimed to investigate whether activin B modulates liver fibrogenesis. Liver and serum activin B, along with its analog activin A, were analyzed in patients with liver fibrosis from different etiologies and in mouse acute and chronic liver injury models. Activin B, activin A, or both was immunologically neutralized in mice with progressive or established carbon tetrachloride (CCl4)–induced liver fibrosis. Hepatic and circulating activin B was increased in human patients with liver fibrosis caused by several liver diseases. In mice, hepatic and circulating activin B exhibited persistent elevation following the onset of several types of liver injury, whereas activin A displayed transient increases. The results revealed a close correlation of activin B with liver injury regardless of etiology and species. Injured hepatocytes produced excessive activin B. Neutralizing activin B largely prevented, as well as improved, CCl4‐induced liver fibrosis, which was augmented by co‐neutralizing activin A. Mechanistically, activin B mediated the activation of c‐Jun‐N‐terminal kinase (JNK), the induction of inducible nitric oxide synthase (iNOS) expression, and the maintenance of poly (ADP‐ribose) polymerase 1 (PARP1) expression in injured livers. Moreover, activin B directly induced a profibrotic expression profile in hepatic stellate cells (HSCs) and stimulated these cells to form a septa structure. Conclusions: We demonstrate that activin B, cooperating with activin A, mediates the activation or expression of JNK, iNOS, and PARP1 and the activation of HSCs, driving the initiation and progression of liver fibrosis.

Following partial hepatectomy (PH), the majority of remnant hepatocytes synchronously enter and rhythmically progress through the cell cycle for three major rounds to regain lost liver mass. Whether and how the circadian clock core component Bmal1 modulates this process remains elusive. We performed PH on Bmal1+/+ and hepatocyte-specific Bmal1 knockout (Bmal1hep-/-) mice and compared the initiation and progression of the hepatocyte cell cycle. After PH, Bmal1+/+ hepatocytes exhibited three major waves of nuclear DNA synthesis. In contrast, in Bmal1hep-/-hepatocytes, the first wave of nuclear DNA synthesis was delayed by 12 h, and the third such wave was lost. Following PH, Bmal1+/+ hepatocytes underwent three major waves of mitosis, whereas Bmal1hep-/-hepatocytes fully abolished mitotic oscillation. These Bmal1-dependent disruptions in the rhythmicity of hepatocyte cell cycle after PH were accompanied by suppressed expression peaks of a group of cell cycle components and regulators, and dysregulated activation patterns of mitogenic signaling molecules c-Met and EGFR. Moreover, Bmal1+/+ hepatocytes rhythmically accumulated fat as they expanded following PH, whereas this phenomenon was largely inhibited in Bmal1hep-/-hepatocytes. In addition, during late stages of liver regrowth, Bmal1 absence in hepatocytes caused the activation of redox sensor Nrf2, suggesting an oxidative stress state in regenerated liver tissue. Collectively, we demonstrated that during liver regeneration, Bmal1 partially modulates the oscillation of S-phase progression, fully controls the rhythmicity of M-phase advancement, and largely governs fluctuations in fat metabolism in replicating hepatocytes, and eventually determines the redox state of regenerated livers. New and Noteworthy We demonstrated that Bmal1 centrally controls the synchronicity and rhythmicity of the cell cycle and lipid accumulation in replicating hepatocytes during liver regeneration. Bmal1 plays these roles, at least in part, by ensuring formation of the expression peaks of cell cycle components and regulators, as well as the timing and levels of activation of mitogenic signaling molecules. Graphical Abstract

ABSTRACTBackgroundAn emerging clinical phenomenon in patients with end stage liver disease is progressive skeletal muscle atrophy. This loss in lean mass predicts poor survival outcomes for liver disease patients and highlights an underappreciated crosstalk between injured liver and muscle that lacks defined mediators. The purpose of our study was to identify potential liver-muscle mediator(s) in pre-clinicalin vivomodels of liver injury which may contribute to the muscle loss observed in liver disease.MethodsUtilizing a mouse model of carbon tetrachloride CCl4-induced liver injury in the presence or absence of cardiotoxin-induced muscle injury, we evaluated whether neutralizing Activin type IIB receptor (ActRIIB) ligands, or specifically growth differentiation factor 8 (Gdf8), could preserve or reverse muscle atrophy associated with liver disease.ResultsWe found that hepatic injury via CCl4or bile duct ligation (BDL) similarly caused significant muscle atrophy along with decreased gene expression in key myogenesis markers. This adverse effect of injured liver on muscle were completely prevented and reversed by the intervention of Activin type IIB receptor (ActRIIB)-Fc fusion protein, which neutralizes the ActRIIB ligands, including Activins and growth differentiation factor 8 (Gdf8 or myostatin). The results indicate that ActRIIB ligands promoted muscle atrophy which was manifested in response to hepatic injury/disease and conferred the negative communication of injured liver with muscle. Indeed, direct injection of exogenous Gdf8 protein into muscle along with acute focal muscle injury recapitulated similar dysregulated muscle regeneration as observed with liver injury. Furthermore, we found that hepatocytes produced Gdf8 in response to liver injury in rodents and in patients with end stage liver disease. A neutralizing antibody to Gdf8 attenuated muscle atrophy and unexpectedly ameliorated liver fibrosis in both CCl4and BDL models. Following this observation, we demonstrated Gdf8’s ability to induce fibrogenesis in stellate cells, potentially identifying a novel hepatic role for this protein. Moreover, hepatic Gdf8 promoted muscle wasting in response to liver damage and hindered skeletal muscle regeneration.ConclusionOur findings identified Gdf8 as a novel hepatomyokine contributing to injured liver-muscle negative crosstalk and liver injury progression. Moreover, we demonstrated a promising therapeutic strategy for muscle atrophy accompanying liver diseases.

Liver resection induces robust liver regrowth or regeneration to compensate for the lost tissue mass. In a clinical setting, pregnant women may need liver resection without terminating pregnancy in some cases. However, how pregnancy affects maternal liver regeneration remains elusive. We performed 70% partial hepatectomy (PH) in nonpregnant mice and gestation day 14 mice, and histologically and molecularly compared their liver regrowth during the next 4 days. We found that compared with the nonpregnant state, pregnancy altered the molecular programs driving hepatocyte replication, indicated by enhanced activities of epidermal growth factor receptor and STAT5A, reduced activities of cMet and p70S6K, decreased production of IL-6, TNFα, and hepatocyte growth factor, suppressed cyclin D1 expression, increased cyclin A1 expression, and early activated cyclin A2 expression. As a result, pregnancy allowed the remnant hepatocytes to enter the cell cycle at least 12 h earlier, increased hepatic fat accumulation, and enhanced hepatocyte mitosis. Consequently, pregnancy ameliorated maternal liver regeneration following PH. In addition, a report showed that maternal liver regrowth after PH is driven mainly by hepatocyte hypertrophy rather than hyperplasia during the second half of gestation in young adult mice. In contrast, we demonstrate that maternal liver relies mainly on hepatocyte hyperplasia instead of hypertrophy to restore the lost mass after PH. Overall, we demonstrate that pregnancy facilitates maternal liver regeneration likely via triggering an early onset of hepatocyte replication, accumulating excessive liver fat, and promoting hepatocyte mitosis. The results from our current studies enable us to gain more insights into how maternal liver regeneration progresses during gestation.NEW & NOTEWORTHY We demonstrate that pregnancy may generate positive effects on maternal liver regeneration following partial hepatectomy, which are manifested by early entry of the cell cycle of remnant hepatocytes, increased hepatic fat accumulation, enhanced hepatocyte mitosis, and overall accelerated liver regrowth.

Prolactin (PRL) signaling has been implicated in the regulation of glucose homeostatic adaptations to pregnancy. In this report, the PRL receptor (Prlr) gene was conditionally disrupted in the pancreas, creating an animal model which proved useful for investigating the biology and pathology of gestational diabetes including its impacts on fetal and placental development. In mice, pancreatic PRLR signaling was demonstrated to be required for pregnancy-associated changes in maternal β cell mass and function. Disruption of thePrlrgene in the pancreas resulted in fewer insulin-producing cells, which failed to expand appropriately during pregnancy resulting in reduced blood insulin levels and maternal glucose intolerance. This inability to sustain normal blood glucose balance during pregnancy worsened with age and a successive pregnancy. The etiology of the insulin insufficiency was attributed to deficits in regulatory pathways controlling β cell differentiation. Additionally, the disturbance in maternal blood glucose homeostasis was associated with fetal overgrowth and dysregulation of inflammation and PRL-associated transcripts in the placenta. Overall, these results indicate that the PRLR, acting within the pancreas, mediates maternal pancreatic adaptations to pregnancy. PRLR dysfunction is associated with glucose intolerance during pregnancy and pathological features consistent with gestational diabetes.

Autophagy is important for liver homeostasis, and the deficiency leads to injury, inflammation, ductular reaction (DR), fibrosis, and tumorigenesis. It is not clear how these events are mechanistically linked to autophagy deficiency. Here, we reveal the role of high-mobility group box 1 (HMGB1) in two of these processes. First, HMGB1 was required for DR, which represents the expansion of hepatic progenitor cells (HPCs) implicated in liver repair and regeneration. DR caused by hepatotoxic diets (3,5-diethoxycarbonyl-1,4-dihydrocollidine [DDC] or choline-deficient, ethionine-supplemented [CDE]) also depended on HMGB1, indicating that HMGB1 may be generally required for DR in various injury scenarios. Second, HMGB1 promoted tumor progression in autophagy-deficient livers. Receptor for advanced glycation end product (RAGE), a receptor for HMGB1, was required in the same two processes and could mediate the proliferative effects of HMBG1 in isolated HPCs. HMGB1 was released from autophagy-deficient hepatocytes independently of cellular injury but depended on NRF2 and the inflammasome, which was activated by NRF2. Pharmacological or genetic activation of NRF2 alone, without disabling autophagy or causing injury, was sufficient to cause inflammasome-dependent HMGB1 release. In conclusion, HMGB1 release is a critical mechanism in hepatic pathogenesis under autophagy-deficient conditions and leads to HPC expansion as well as tumor progression.

Autophagy is important for liver homeostasis, and the deficiency leads to injury, inflammation, ductular reaction (DR), fibrosis, and tumorigenesis. It is not clear how these events are mechanistically linked to autophagy deficiency. Here, we reveal the role of high-mobility group box 1 (HMGB1) in two of these processes. First, HMGB1 was required for DR, which represents the expansion of hepatic progenitor cells (HPCs) implicated in liver repair and regeneration. DR caused by hepatotoxic diets (3,5-diethoxycarbonyl-1,4-dihydrocollidine [DDC] or choline-deficient, ethionine-supplemented [ CDE]) also depended on HMGB1, indicating that HMGB1 may be generally required for DR in various injury scenarios. Second, HMGB1 promoted tumor progression in autophagy-deficient livers. Receptor for advanced glycation end product (RAGE), a receptor for HMGB1, was required in the same two processes and could mediate the proliferative effects of HMBG1 in isolated HPCs. HMGB1 was released from autophagy-deficient hepatocytes independently of cellular injury but depended on NRF2 and the inflammasome, which was activated by NRF2. Pharmacological or genetic activation of NRF2 alone, without disabling autophagy or causing injury, was sufficient to cause inflammasome-dependent HMGB1 release. In conclusion, HMGB1 release is a critical mechanism in hepatic pathogenesis under autophagy-deficient conditions and leads to HPC expansion as well as tumor progression.

1Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN, USA; 2MRC Center for Inflammation Research, The Queen’s Medical Research Institute, The University of Edinburgh, United Kingdom; 3Department of Biology, Purdue University School of Science, Indianapolis, IN, USA; 4Department of Biology, Institute of Molecular Health Sciences, Swiss Federal Institute of Technology (ETH) Zurich, 8093 Zurich, Switzerland; 5Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, USA; 6Department of Anatomy and Histology, Fukushima Medical University, School of Medicine, Fukushima, Japan; 7Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; 8Department of Nephrology, the Second Xiangya Hospital, Central South University, Changsha, Hunan, China; 9Department of Cellular Biology and Anatomy, Medical College of Georgia at Augusta University and Charlie Norwood VA Medical Center, Augusta, GA, USA

Epalrestat (EPS), an aldose reductase inhibitor, is widely prescribed to manage diabetic neuropathy. It is generally believed that EPS is beneficial to diabetic patients because it can protect endothelial cells, Schwann cells, or other neural cells from oxidative stress. However, several clinical studies revealed that EPS therapy led to liver dysfunction, which limited its clinical applications. Currently, the underlying mechanism by which EPS causes liver dysfunction is unknown. This study aimed to investigate the mechanism responsible for EPS-induced liver injury. In mouse liver, EPS 1) increased oxidative stress, indicated by increased expression of manganese superoxide dismutase, Ho-1, and Nqo1, 2) induced inflammation, indicated by infiltration of inflammatory cells, and induced expression of tumor necrosis factor-alpha, CD11b, and CD11c, as well as 3) predisposed to induce fibrosis, evidenced by increased mRNA and protein expression of early profibrotic biomarker genes procollagen I and alpha-smooth muscle actin, and by increased collagen deposition. In cultured mouse and human hepatoma cells, EPS treatment induced oxidative stress, decreased cell viability, and triggered apoptosis evidenced by increased Caspase-3 cleavage/activation. In addition, EPS increased mRNA and protein expression of cytoglobin in mouse liver, indicating that EPS activated hepatic stellate cells (HSCs). Furthermore, EPS treatment in cultured human HSCs increased cell viability. In summary, EPS administration induced oxidative stress and inflammation in mouse liver, and stimulated liver fibrogenesis. Therefore, cautions should be exercised during EPS therapy.

Transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) regulates multiple biologic processes, including hepatic lipid metabolism. Estrogen exerts actions affecting energy homeostasis, including a liver fat-lowering effect. Increasing evidence indicates the crosstalk between these two molecules. The aim of this study was to evaluate whether Nrf2 modulates estrogen signaling in hepatic lipid metabolism. Nonalcoholic fatty liver disease (NAFLD) was induced in wild-type and Nrf2-null mice fed a high-fat diet and the liver fat-lowering effect of exogenous estrogen was subsequently assessed. We found that exogenous estrogen eliminated 49% and 90% of hepatic triglycerides in wild-type and Nrf2-null mice with NAFLD, respectively. This observation demonstrates that Nrf2 signaling is antagonistic to estrogen signaling in hepatic fat metabolism; thus, Nrf2 absence results in striking amplification of the liver fat-lowering effect of estrogen. In addition, we found the association of trefoil factor 3 and fatty acid binding protein 5 with the liver fat-lowering effect of estrogen. In summary, we identified Nrf2 as a novel and potent inhibitor of estrogen signaling in hepatic lipid metabolism. Our finding may provide a potential strategy to treat NAFLD by dually targeting Nrf2 and estrogen signaling.