ABSTRACT
Hepatic steatosis plays a detrimental role in the onset and progression of alcohol-associated liver disease (ALD). Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an evolutionarily conserved protein related to the unfolded protein response. Recent studies have demonstrated that MANF plays an important role in liver diseases. In this study, we investigated the role of MANF in ethanol-induced steatosis and the underlying mechanisms. We showed that the hepatic MANF expression was markedly upregulated in mouse model of ALD by chronic-plus-single-binge ethanol feeding. Moreover, after chronic-plus-binge ethanol feeding, hepatocyte-specific MANF knockout (HKO) mice displayed more severe hepatic steatosis and liver injury than wild-type (WT) control mice. Immunoprecipitation-coupled MS proteomic analysis revealed that arginosuccinate synthase 1 (ASS1), a rate-limiting enzyme in the urea cycle, resided in the same immunoprecipitated complex with MANF. Hepatocyte-specific MANF knockout led to decreased ASS1 activity, whereas overexpression of MANF contributed to enhanced ASS1 activity in vitro. In addition, HKO mice displayed unique urea cycle metabolite patterns in the liver with elevated ammonia accumulation after ethanol feeding. ASS1 is known to activate AMPK by generating an intracellular pool of AMP from the urea cycle. We also found that MANF supplementation significantly ameliorated ethanol-induced steatosis in vivo and in vitro by activating the AMPK signaling pathway, which was partly ASS1 dependent. This study demonstrates a new mechanism in which MANF acts as a key molecule in maintaining hepatic lipid homeostasis by enhancing ASS1 activity and uncovers an interesting link between lipid metabolism and the hepatic urea cycle under excessive alcohol exposure.
Subject(s)
Fatty Liver , Liver Diseases, Alcoholic , Animals , Mice , AMP-Activated Protein Kinases/metabolism , Astrocytes/metabolism , Ethanol/toxicity , Fatty Liver/chemically induced , Hepatocytes/metabolism , Liver/metabolism , Mice, Knockout , Nerve Growth Factors/metabolism , Proteomics , Urea/metabolismABSTRACT
Acute lung injury (ALI) is an urgent respiratory disease without effective treatment. Mesencephalic astrocyte-derived neurotrophic factor (MANF)has been demonstrated to play a suppressive role in some inflammatory conditions. However, the effect of MANF on ALI has not yet been reported. In this study, we collected bronchoalveolar lavage fluid (BALF) from the patients with or without pulmonary inflammation, and used lipopolysaccharide (LPS) to induce mice ALI model. Mono-macrophage-specific MANF knockout (MKO) mice were constructed and recombinant human MANF protein was used to ALI mice. We found that the endogenous MANF protein in both human BALF and mice lung tissues was increased in inflammatory conditions. MANF level in the macrophages of inflammatory lung was higher than that in normal controls in both human and mice. MANF deficiency in macrophages induced lung inflammation and aggravated LPS-induced lung injury. MANF lowered LPS-induced lung injury, inhibited macrophage polarization to M1 functional type. Meanwhile, MANF inhibited-LPS induced activation of NF-κB signal pathway by down regulating phosphorylated p65in lung tissue and macrophages. These results indicate that MANF acts as a suppressor in ALI via negatively regulating NF-κB activation and macrophages polarization, which may be a novel potential target and shed light on ALI therapy.
Subject(s)
Acute Lung Injury , Lipopolysaccharides , Macrophages , Nerve Growth Factors , Acute Lung Injury/genetics , Acute Lung Injury/immunology , Acute Lung Injury/metabolism , Acute Lung Injury/pathology , Animals , Astrocytes/metabolism , Astrocytes/pathology , Disease Models, Animal , Humans , Lipopolysaccharides/pharmacology , Lung , Macrophage Activation , Macrophages/immunology , Macrophages/pathology , Mice , Mice, Knockout , NF-kappa B/metabolism , Nerve Growth Factors/genetics , Nerve Growth Factors/immunology , Nerve Growth Factors/metabolismABSTRACT
Succinyl-CoA:3-ketoacid CoA transferase (SCOT) plays a crucial role in ketone-body metabolism. SCOT from Drosophila melanogaster (DmSCOT) was purified and crystallized. The crystal structure of DmSCOT was determined at 2.64â Å resolution and belonged to space group P212121, with unit-cell parameters a=76.638, b=101.921, c=122.457â Å, α=ß=γ=90°. Sequence alignment and structural analysis identified DmSCOT as a class I CoA transferase. Compared with Acetobacter aceti succinyl-CoA:acetate CoA transferase, DmSCOT has a different substrate-binding pocket, which may explain the difference in their substrate specificities.