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.

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.

Iron (Fe) deficiency is a global health problem, especially in underdeveloped countries. Biofortification with genetic engineering methods has been used to improve Fe nutrition in a number of crops. Various steps, e.g., uptake, distribution, and storage, involved in Fe homeostasis have been manipulated to increase the Fe concentration in the edible portions of plants. Nicotianamine (NA) is an important metal ion chelator in plants. It promotes the mobility of Fe and decreases cellular Fe toxicity. Increasing the Fe content in crops by promoting NA synthesis could help decrease human diseases associated with Fe deficiency. In the present study, Arabidopsis thaliana nicotianamine synthase 1 (AtNAS1) was overexpressed in potato (Solanum tuberosum, St) under the control of the cauliflower mosaic virus 35S promoter. Transgenic plants had a significantly increased amount of Fe in tubers (52.7 µg/g dry weight, 2.4-fold the amount in wild-type tubers), while no differences in plant phenotype or yield were detected between transgenic and wild-type plants. The expression of genes involved in root mineral uptake and homeostasis, such as StYSL1, StIRT1, StFRO1, and StNAS, was also altered in the roots and leaves of the transgenic plants. Our results demonstrate that the manipulation of Fe chelation is a useful strategy for Fe nutrition improvement, and the increased Fe accumulation in tubers of transgenic potato plants is most likely caused by the increased movement of Fe from the leaf to the tuber.

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.

Activin A, a member of the transforming growth factor-beta superfamily, provides pleiotropic regulation of fibrosis and inflammation. We aimed at determining whether selective inhibition of activin A would provide a regenerative benefit. The introduction of activin A into normal muscle increased the expression of inflammatory and muscle atrophy genes Tnf, Tnfrsf12a, Trim63, and Fbxo32 by 3.5-, 10-, 2-, and 4-fold, respectively. The data indicate a sensitive response of muscle to activin A. Two hours after cardiotoxin-induced muscle damage, Local activin A protein expression increased by threefold to ninefold. Neutralization of activin A with a specific monoclonal antibody in this muscle injury model decreased the muscle protein Levels of Lymphotoxin alpha and Il17a by 32% and 42%, respectively. Muscle histopathological features showed that activin A antibody treated mice displayed an increase in muscle degradation, with the concomitant 9.2-fold elevation in F4/80-positive cells 3 days after injury. At the same time, the number of Pax7/Myod1-positive cells also increased, indicative of potentiated muscle precursor activation. Ultimately, activin A inhibition resulted in rapid recovery of muscle contractile properties indicated by a restoration of maximum and specific force. In summary, selective inhibition of activin A with a monoclonal antibody in muscle injury leads to the early onset of tissue degradation and subsequent enhanced myogenesis, thereby accelerating muscle repair and functional recovery.

Follistatin (FST) is a member of the tissue growth factor β family and is a secreted glycoprotein that antagonizes many members of the family, including activin A, growth differentiation factor 11, and myostatin. The objective of this study was to explore the use of an engineered follistatin therapeutic created by fusing FST315 lacking heparin binding activity to the N terminus of a murine IgG1 Fc (FST315-ΔHBS-Fc) as a systemic therapeutic agent in models of muscle injury. Systemic administration of this molecule was found to increase body weight and lean muscle mass after weekly administration in normal mice. Subsequently, we tested this agent in several models of muscle injury, which were chosen based on their severity of damage and their ability to reflect clinical settings. FST315-ΔHBS-Fc treatment proved to be a potent inducer of muscle remodeling and regeneration. FST315-ΔHBS-Fc induced improvements in muscle repair after injury/atrophy by modulating the early inflammatory phase allowing for increased macrophage density, and Pax7-positive cells leading to an accelerated restoration of myofibers and muscle function. Collectively, these data demonstrate the benefits of a therapeutically viable form of FST that can be leveraged as an alternate means of ameliorating muscle regeneration.