Deletion of GPR81 activates CREB/Smad7 pathway and alleviates liver fibrosis in mice.

BACKGROUND: Enhanced glycolysis is a crucial metabolic event that drives the development of liver fibrosis, but the molecular mechanisms have not been fully understood. Lactate is the endproduct of glycolysis, which has recently been identified as a bioactive metabolite binding to G-protein-coupled receptor 81 (GPR81). We then questioned whether GPR81 is implicated in the development of liver fibrosis. METHODS: The level of GPR81 was determined in mice with carbon tetrachloride (CCl4)-induced liver fibrosis and in transforming growth factor beta 1 (TGF-ß1)-activated hepatic stellate cells (HSCs) LX-2. To investigate the significance of GPR81 in liver fibrosis, wild-type (WT) and GPR81 knockout (KO) mice were exposed to CCl4, and then the degree of liver fibrosis was determined. In addition, the GPR81 agonist 3,5-dihydroxybenzoic acid (DHBA) was supplemented in CCl4-challenged mice and TGF-ß1-activated LX-2 cells to further investigate the pathological roles of GPR81 on HSCs activation. RESULTS: CCl4 exposure or TGF-ß1 stimulation significantly upregulated the expression of GPR81, while deletion of GPR81 alleviated CCl4-induced elevation of aminotransferase, production of pro-inflammatory cytokines, and deposition of collagen. Consistently, the production of TGF-ß1, the expression of alpha-smooth muscle actin (α-SMA) and collagen I (COL1A1), as well as the elevation of hydroxyproline were suppressed in GPR81 deficient mice. Supplementation with DHBA enhanced CCl4-induced liver fibrogenesis in WT mice but not in GPR81 KO mice. DHBA also promoted TGF-ß1-induced LX-2 activation. Mechanistically, GPR81 suppressed cAMP/CREB and then inhibited the expression of Smad7, a negative regulator of Smad3, which resulted in increased phosphorylation of Smad3 and enhanced activation of HSCs. CONCLUSION: GPR81 might be a detrimental factor that promotes the development of liver fibrosis by regulating CREB/Smad7 pathway.

Negative regulation of pro-apoptotic AMPK/JNK pathway by itaconate in mice with fulminant liver injury.

Accumulating evidence indicates that metabolic responses are deeply integrated into signal transduction, which provides novel opportunities for the metabolic control of various disorders. Recent studies suggest that itaconate, a highly concerned bioactive metabolite catalyzed by immune responsive gene 1 (IRG1), is profoundly involved in the regulation of apoptosis, but the underlying mechanisms have not been fully understood. In the present study, the molecular mechanisms responsible for the apoptosis-modulatory activities of IRG1/itaconate have been investigated in mice with lipopolysaccharide (LPS)/D-galactosamine (D-Gal)-induced apoptotic liver injury. The results indicated that LPS/D-Gal exposure upregulated the level of IRG1 and itaconate. Deletion of IRG1 resulted in exacerbated hepatocytes apoptosis and liver injury. The phospho-antibody microarray analysis and immunoblot analysis indicated that IRG1 deletion enhanced the activation of AMP-activated protein kinase (AMPK)/c-jun-N-terminal kinase (JNK) pathway in LPS/D-Gal exposed mice. Mechanistically, IRG1 deficiency impaired the anti-oxidative nuclear factor erythroid-2 related factor 2 (Nrf2) signaling and then enhanced the activation of the redox-sensitive AMPK/JNK pathway that promotes hepatocytes apoptosis. Importantly, post-insult supplementation with 4-octyl itaconate (4-OI), a cell-permeable derivate of itaconate, resulted in beneficial outcomes in fulminant liver injury. Therefore, IRG1/itaconate might function as a negative regulator that controls AMPK-induced hepatocyte apoptosis in LPS/D-Gal-induced fulminant liver injury.

Sirtuin 1 activator alleviated lethal inflammatory injury via promotion of autophagic degradation of pyruvate kinase M2.

Upregulation of pyruvate kinase M2 (PKM2) is critical for the orchestration of metabolism and inflammation in critical illness, while autophagic degradation is a recently revealed mechanism that counter-regulates PKM2. Accumulating evidence suggests that sirtuin 1 (SIRT1) function as a crucial regulator in autophagy. The present study investigated whether SIRT1 activator would downregulate PKM2 in lethal endotoxemia via promotion of its autophagic degradation. The results indicated that lethal dose of lipopolysaccharide (LPS) exposure decreased the level of SIRT1. Treatment with SRT2104, a SIRT1 activator, reversed LPS-induced downregulation of LC3B-II and upregulation of p62, which was associated with reduced level of PKM2. Activation of autophagy by rapamycin also resulted in reduction of PKM2. The decline of PKM2 in SRT2104-treated mice was accompanied with compromised inflammatory response, alleviated lung injury, suppressed elevation of blood urea nitrogen (BUN) and brain natriuretic peptide (BNP), and improved survival of the experimental animals. In addition, co-administration of 3-methyladenine, an autophagy inhibitor, or Bafilomycin A1, a lysosome inhibitor, abolished the suppressive effects of SRT2104 on PKM2 abundance, inflammatory response and multiple organ injury. Therefore, promotion of autophagic degradation of PKM2 might be a novel mechanism underlying the anti-inflammatory benefits of SIRT1 activator.

Immune response gene 1 deficiency aggravates high fat diet-induced nonalcoholic fatty liver disease via promotion of redox-sensitive AKT suppression.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disorder worldwide. Immune response gene 1 (IRG1) catalyzes the production of bio-active itaconate, which is actively involved in the regulation of signal transduction. A recent study has found that the expression of IRG1 was significantly down-regulated in obesity-associated fatty liver, but the potential roles of IRG1 in the development NAFLD remain unclear. The present study found that genetic deletion of IRG1 aggravated high fat diet (HFD)-induced metabolic disturbance, including obesity, dyslipidemia and insulin resistance. In addition, HFD induced more severe liver steatosis and higher serum ALT and AST level in IRG1 KO mice, which were accompanied with altered expression of genes involved in lipid uptake, synthesis and catabolism. RNA-seq and immunoblot analysis indicated that deficiency of IRG1 is associated with suppressed activation of AKT, a master metabolic regulator. Mechanistically, IRG1/itaconate enhanced the antioxidative NRF2 pathway and prevented redox-sensitive suppression of AKT. Interestingly, supplementation with 4-octyl itaconate (4-OI), a cell-permeable derivate of itaconate, alleviated HFD-induced oxidative stress, AKT suppression and liver steatosis. Therefore, IRG1 probably functions as a protective regulator in the development of NAFLD and the cell-permeable 4-OI might have potential value for the pharmacological intervention of NAFLD.

Protective effects of glutamine on lipopolysaccharide/D-galactosamine-induced fulminant hepatitis in mice.

Fulminant hepatitis remains a critical health problem owing to its high mortality rate and the lack of effective therapies. An increasing number of studies have shown that glutamine supplementation provides protective benefits in inflammation-related disorders, but the pharmacological significance of glutamine in lipopolysaccharide (LPS)/D-galactosamine (D-Gal)-induced fulminant hepatitis remains unclear. In the present study, the potential effects of glutamine on LPS/D-Gal-induced fulminant hepatitis were investigated. Pretreatment with glutamine decreased plasma activities of alanine and aspartate aminotransferases, and ameliorated hepatic morphological abnormalities in LPS/D-Gal-exposed mice. Glutamine pretreatment also inhibited LPS/D-Gal-induced tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) production. In addition, glutamine pretreatment decreased the level of cleaved cysteinyl aspartate-specific proteinase 3 (caspase-3), suppressed the activities of caspase-3, caspase-8, and caspase-9, and reduced the number of cells positive for TdT-mediated dUTP nick-end labeling in LPS/D-Gal-challenged mice. Interestingly, post-treatment with glutamine also provided protective benefits against LPS/D-Gal-induced acute liver injury, although these effects were less robust than those of glutamine pre-treatment. Thus, glutamine may have potential value as a pharmacological intervention in fulminant hepatitis.

Deficiency of IRG1/ itaconate aggravates endotoxemia-induced acute lung injury by inhibiting autophagy in mice.

Itaconate, produced by aconitate decarboxylase 1 (ACOD1), which is encoded by immune-responsive gene 1 (Irg1), is one of the metabolites derived from the tricarboxylic acid cycle. It has been reported that exogenous itaconate plays an anti-inflammatory role in the progression of multiple diseases and pathological processes, including activated macrophage, ischemia-reperfusion injury, and acute lung injury. However, the role and specific mechanism of endogenous itaconate in endotoxemia-induced acute lung injury (ALI) remain unclear. The animal model of ALI in wild-type and Irg1-/- mice was constructed by LPS intraperitoneal injection. Ultrahigh-performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS) analysis was performed to measure the quantity of endogenous itaconate. The protective effect of itaconate was investigated by the behavioral assessment and the levels of inflammatory cytokines. Acute lung injury was assessed by hematoxylin and eosin staining, total protein in BALF, and Evans blue leakage. Western blotting was used to detect the IRG1 expression and autophagic protein in the lung. We demonstrated that IRG1 was highly expressed in ALI and that endogenous itaconate was produced simultaneously and was 100 times higher. Using Irg1-/- mice, we found that endogenous itaconate was likely to exert an anti-inflammatory effect by activating NRF2 and promoting autophagy. Furthermore, autophagy was restrained by LPS but enhanced by 4-octyl itaconate (4-OI) pretreatment. Our study illustrated that a deficiency of IRG1/Itaconate aggravates ALI and that the IRG1/itaconate pathway protects against ALI. The protective mechanisms could be related to the facilitation of autophagy. Such findings may provide a theoretical foundation for the treatment of endotoxemia-induced ALI.

Activation of GPR81 Aggravates Remote Organ Injury During Hepatic Ischemia-Reperfusion Injury.

Hepatic ischemia-reperfusion injury (HIRI) is a serious situation with high morbidity and mortality, which is usually accompanied with hyperlactatemia due to impaired lactate clearance in liver. G-protein-coupled receptor 81 (GPR81) has recently been identified as the bioactive receptor of lactate. GPR81 is profoundly involved in the modulation of metabolism and inflammation, but its significance in HIRI remains unclear. The present study investigated the potential roles of GPR81 in HIRI by using the GPR81 agonist 3-chloro-5-hydroxybenzoic acid (CHBA). The results indicated that treatment with CHBA had no obvious effects on HIRI-induced histologic abnormalities and elevation of serum aspartate aminotransferase, alanine aminotransferase. However, CHBA significantly upregulated the serum level of tumor necrosis factor alpha and interleukin-6 in mice with HIRI. Administration of CHBA also exacerbated HIRI-induced histologic lesions in lung, increased the level of myeloperoxidase in lung tissue and the protein concentration in bronchoalveolar lavage fluid. In addition, the serum levels of brain natriuretic peptide and creatinine also increased in CHBA-treated mice. The results indicate that activation of GPR81 might aggravate HIRI-induced remote organ injury and result in serious outcomes.

Immune response gene 1 deficiency impairs Nrf2 activation and aggravates liver fibrosis in mice.

A growing body of evidence suggests that metabolic events play essential roles in the development of liver fibrosis. Immune response gene 1 (IRG1) catalyzes the generation of itaconate, which function as a metabolic checkpoint under several pathological circumstances. In the present study, the hepatic level of IRG1 was determined in mice with carbon tetrachloride (CCl4)-induced liver fibrosis. And then the pathological significance of IRG1 and the pharmacological potential of 4-octyl itaconate (4-OI), a cell-permeable derivate of itaconate, in liver fibrosis were investigated in mice. The results indicated that the hepatic level of IRG1 was upregulated in mice with liver fibrosis. CCl4-induced formation of fibrotic septa and deposition of collagen was aggravated in IRG1 KO mice. IRG1 deletion also resulted in increased expression of transforming growth factor beta 1 (TGF-ß1), enhanced phosphorylation of Smad3, elevated level of alpha smooth muscle actin (α-SMA) and hydroxyproline, which were associated with compromised activation of nuclear erythroid 2-related factor 2 (Nrf2)-mediated antioxidant system and exacerbated oxidative stress. Interestingly, supplementation with 4-OI activated Nrf2 pathway, suppressed TGF-ß1 signaling and attenuated fibrogenesis. Our data indicated that upregulation of IRG1 might function as a protective response during the development of liver fibrosis, and 4-OI might have potential value for the pharmacological intervention of liver fibrosis.

Epidemiology and evolution of novel deltacoronaviruses in birds in central China.

The variety and widespread of coronavirus in natural reservoir animals is likely to cause epidemics via interspecific transmission, which has attracted much attention due to frequent coronavirus epidemics in recent decades. Birds are natural reservoir of various viruses, but the existence of coronaviruses in wild birds in central China has been barely studied. Some bird coronaviruses belong to the genus of Deltacoronavirus. To explore the diversity of bird deltacoronaviruses in central China, we tested faecal samples from 415 wild birds in Hunan Province, China. By RT-PCR detection, we identified eight samples positive for deltacoronaviruses which were all from common magpies, and in four of them, we successfully amplified complete deltacoronavirus genomes distinct from currently known deltacoronavirus, indicating four novel deltacoronavirus stains (HNU1-1, HNU1-2, HNU2 and HNU3). Comparative analysis on the four genomic sequences showed that these novel magpie deltacoronaviruses shared three different S genes among which the S genes of HNU1-1 and HNU1-2 showed 93.8% amino acid (aa) identity to that of thrush coronavirus HKU12, HNU2 S showed 71.9% aa identity to that of White-eye coronavirus HKU16, and HNU3 S showed 72.4% aa identity to that of sparrow coronavirus HKU17. Recombination analysis showed that frequent recombination events of the S genes occurred among these deltacoronavirus strains. Two novel putative cleavage sites separating the non-structural proteins in the HNU coronaviruses were found. Bayesian phylogeographic analysis showed that the south coast of China might be a potential origin of bird deltacoronaviruses existing in inland China. In summary, these results suggest that common magpie in China carries diverse deltacoronaviruses with novel genomic features, indicating an important source of environmental coronaviruses closed to human communities, which may provide key information for prevention and control of future coronavirus epidemics.

REV-ERBα Agonist GSK4112 attenuates Fas-induced Acute Hepatic Damage in Mice.

Fas-induced apoptosis is a central mechanism of hepatocyte damage during acute and chronic hepatic disorders. Increasing evidence suggests that circadian clock plays critical roles in the regulation of cell fates. In the present study, the potential significance of REV-ERBα, a core ingredient of circadian clock, in Fas-induced acute liver injury has been investigated. The anti-Fas antibody Jo2 was injected intraperitoneally in mice to induce acute liver injury and the REV-ERBα agonist GSK4112 was administered. The results indicated that treatment of GSK4112 decreased the level of plasma ALT and AST, attenuated the liver histological changes, and promoted the survival rate in Jo2-insulted mice. Treatment with GSK4112 also downregulated the activities of caspase-3 and caspase-8, suppressed hepatocyte apoptosis. In addition, treatment with GSK4112 decreased the level of Fas and enhanced the phosphorylation of Akt. In conclusion, treatment with GSK4112 alleviated Fas-induced apoptotic liver damage in mice, suggesting that REV-ERBα agonist might have potential value in pharmacological intervention of Fas-associated liver injury.

Nuclear accumulation of pyruvate kinase M2 promotes liver regeneration via activation of signal transducer and activator of transcription 3.

AIMS: Pyruvate kinase M2 (PKM2), a unique isoform of the pyruvate kinases, not only acts as a crucial metabolic enzyme when it locates in the cytoplasm, but also plays important roles in tumor formation and growth when it accumulates in the nuclei. Our aim was to investigate the potential role of PKM2 in liver regeneration in mice insulted with carbon tetrachloride (CCl4). MATERIAL AND METHODS: The liver regeneration model was established by intraperitoneal injection of CCl4 for 48 h in male BALB/c mice. The expression of PKM2, phospho-STAT3, STAT3, proliferating cell nuclear antigen (PCNA) and Cyclin D1 were evaluated by western blot. The distribution of PKM2 was verified by immunofluorescence staining. The degree of injured region was assessed by hematoxylin and eosin (HE) staining. The proliferation of liver cells was tested by Immunohistochemistry. KEY FINDINGS: The nuclear accumulation of PKM2 increased in the liver treated with CCl4, but treatment with ML-265 significantly suppressed CCl4-induced nuclear accumulation of PKM2. In addition, treatment with ML-265 suppressed the level of cyclin D1 and proliferating cell nuclear antigen (PCNA), reduced the count of Ki67-positive hepatocytes, and expanded the damaged region in histological examination. Meanwhile, treatment with ML-265 suppressed the phosphorylation of nuclear signal transducer and activator of transcription 3 (STAT3). Inhibition of STAT3 by stattic made the same effects as ML-265. SIGNIFICANCE: These data uncovered the role of nuclear PKM2 in liver regeneration and the pro-proliferation effects of nuclear PKM2 may be through targeting its downstream transcription factor STAT3.

Lipopolysaccharide-Induced Dephosphorylation of AMPK-Activated Protein Kinase Potentiates Inflammatory Injury via Repression of ULK1-Dependent Autophagy.

AMP-activated protein kinase (AMPK) is a crucial metabolic regulator with profound modulatory activities on inflammation. Although the anti-inflammatory benefits of AMPK activators were well documented in experimental studies, the pathological significance of endogenous AMPK in inflammatory disorders largely remains unknown. This study investigated the phosphorylation status of endogenous AMPK and the potential roles of AMPK in mice with lipopolysaccharide (LPS)-induced lethal inflammation. The results indicated that LPS dose-dependently decreased the phosphorylation level of AMPK and its target protein acetyl-CoA carboxylase (ACC). Reactivation of AMPK with the AMPK activator A-769662 suppressed LPS-induced elevation of interleukin 6, alleviated histological abnormalities in lung and improved the survival of LPS-challenged mice. Treatment with A-769662 restored LPS-induced suppression of autophagy, inhibition of autophagy by 3-MA reversed the beneficial effects of A-769662. Treatment with A-769662 suppressed LPS-induced activation of mammalian target of rapamycin (mTOR), co-administration of mTOR activator abolished the beneficial effects of A-769662, and the suppressive effects of A-769662 on uncoordinated-51-like kinase 1 (ULK1) phosphorylation. Inhibition of ULK1 removed the beneficial effects of A-769662. These data indicated that LPS-induced dephosphorylation of AMPK could result in weakened inhibition of mTOR and repression of ULK1-dependent autophagy, which might potentiate the development of LPS-induced inflammatory injury. These data suggest that pharmacological restoration of AMPK activation might be a beneficial approach for the intervention of inflammatory disorders.

Caloric Restriction Mimetic 2-Deoxyglucose Alleviated Inflammatory Lung Injury via Suppressing Nuclear Pyruvate Kinase M2-Signal Transducer and Activator of Transcription 3 Pathway.

Inflammation is an energy-intensive process, and caloric restriction (CR) could provide anti-inflammatory benefits. CR mimetics (CRM), such as the glycolytic inhibitor 2-deoxyglucose (2-DG), mimic the beneficial effects of CR without inducing CR-related physiologic disturbance. This study investigated the potential anti-inflammatory benefits of 2-DG and the underlying mechanisms in mice with lipopolysaccharide (LPS)-induced lethal endotoxemia. The results indicated that pretreatment with 2-DG suppressed LPS-induced elevation of tumor necrosis factor alpha and interleukin 6. It also suppressed the upregulation of myeloperoxidase, attenuated Evans blue leakage, alleviated histological abnormalities in the lung, and improved the survival of LPS-challenged mice. Treatment with 2-DG had no obvious effects on the total level of pyruvate kinase M2 (PKM2), but it significantly suppressed LPS-induced elevation of PKM2 in the nuclei. Prevention of PKM2 nuclear accumulation by ML265 mimicked the anti-inflammatory benefits of 2-DG. In addition, treatment with 2-DG or ML265 suppressed the phosphorylation of nuclear signal transducer and activator of transcription 3 (STAT3). Inhibition of STAT3 by stattic suppressed LPS-induced inflammatory injury. Interestingly, posttreatment with 2-DG at the early stage post-LPS challenge also improved the survival of the experimental animals. This study found that treatment with 2-DG, a representative CRM, provided anti-inflammatory benefits in lethal inflammation. The underlying mechanisms included suppressed nuclear PKM2-STAT3 pathway. These data suggest that 2-DG might have potential value in the early intervention of lethal inflammation.

Metformin mitigates carbon tetrachloride-induced TGF-ß1/Smad3 signaling and liver fibrosis in mice.

Increasing evidence suggests that the widely used antidiabetic drug metformin have an extensive therapeutic range beyond hypoglycemic therapy. We previously found that metformin significantly attenuated acute hepatitis. The present study investigated the potential effects of metformin on carbon tetrachloride (CCl4)-induced liver fibrosis in mice. The results indicated that treatment with metformin suppressed CCl4-induced elevation of liver index, formation of fibrotic septa, accumulation of connective tissue and upregulation of collagen I. These effects were not associated with increase of the antioxidative transcription factor nuclear erythroid 2-related factor 2 (NRF2) or its downstream targets, heme oxygenase 1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO1). However, treatment with metformin suppressed CCl4-induced expression of transforming growth factor beta 1 (TGF-ß1) and phosphorylation of Smad3, which was accompanied with decreased level of alpha smooth muscle actin (α-SMA), a marker for the activated hepatic stellate cells (HSCs). These data indicates that metformin could mitigate CCl4-induced liver fibrosis and these beneficial effects might result from suppressed TGF-ß1/Smad3 signaling.