Inhibition of glycogen synthase kinase 3β promotes autophagy to protect mice from acute liver failure mediated by peroxisome proliferator-activated receptor α

Our previous studies have demonstrated that inhibition of glycogen synthase kinase 3β (GSK3β) activity protects mice from acute liver failure (ALF), whereas its protective and regulatory mechanism remains elusive. Autophagy is a recently recognized rudimentary cellular response to inflammation and injury. The aim of the present study was to test the hypothesis that inhibition of GSK3β mediates autophagy to inhibit liver inflammation and protect against ALF. In ALF mice model induced by d-galactosamine (d-GalN) and lipopolysaccharide (LPS), autophagy was repressed compared with normal control, and d-GalN/LPS can directly induce autophagic flux in the progression of ALF mice. Autophagy activation by rapamycin protected against liver injury and its inhibition by 3-methyladenine (3-MA) or autophagy gene 7 (Atg7) small interfering RNA (siRNA) exacerbated liver injury. The protective effect of GSK3β inhibition on ALF mice model depending on the induction of autophagy, because that inhibition of GSK3β promoted autophagy in vitro and in vivo, and inhibition of autophagy reversed liver protection and inflammation of GSK3β inhibition. Furthermore, inhibition of GSK3β increased the expression of peroxisome proliferator-activated receptor α (PPARα), and the downregulated PPARα by siRNA decreased autophagy induced by GSK3β inhibition. More importantly, the expressions of autophagy-related gene and PPARα are significantly downregulated and the activity of GSK3β is significantly upregulated in liver of ALF patients with hepatitis B virus. Thus, we have demonstrated the new pathological mechanism of ALF that the increased GSK3β activity suppresses autophagy to promote the occurrence and development of ALF by inhibiting PPARα pathway.Acute liver failure (ALF), an inflammation-mediated hepatocellular injury process, is a clinical syndrome that results from hepatocellular apoptosis and hemorrhagic necrosis.¹ ALF frequently results from viral hepatitis, ingestion of drugs or toxic substances, or hepatic ischemia-reperfusion injury, among others. The prognosis for ALF is extremely poor, and there is currently no effective therapy for the end stage of the disease other than liver transplantation.² Although the nature of ALF has been extensively studied, the mechanisms by which organ damage occurs are not completely understood.Glycogen synthase kinases 3 are a group of ubiquitously expressed serine/threonine kinases that are initially found to regulate glycogen synthesis. There are two highly homologous isoforms, designated as glycogen synthase kinase 3 (GSK3)α and GSK3β, respectively. Constitutively active in resting cells, GSK3β has a broad range of substrates, and regulates cell activation, differentiation and survival.^{3, 4} Among the diverse functions that are regulated by GSK3β, inflammation has recently emerged as one of the major interesting focuses. Studies showed that GSK3β is an important positive regulator in inflammatory process.^{5, 6, 7, 8} GSK3β deletion results in embryonic lethality caused by severe liver degeneration during development.⁹ Particularly, GSK3β-deficient cells become more sensitive to tumor necrosis factor α (TNF-α)-induced apoptosis.¹⁰ Our studies have shown that the activity of GSK3β is promoted in the progression of ALF and inhibition of GSK3β mitigates liver inflammation to ameliorate ALF model of mice,^{11, 12} but its protective mechanisms are not well defined.Macroautophagy (referred to hereafter as autophagy) is a highly evolutionarily conserved process found in virtually all types of eukaryotic cells. Autophagy involves the sequestration of regions of cytosol within double-membrane-bound compartments followed by lysosome-based degradation of the contents. Previous studies have suggested that autophagy represents an adaptive strategy by which cells can remove damaged organelles and enhance survival following bioenergetics-induced stress, and have multiple roles of autophagy in the regulation of cell death, differentiation and the anti-microbial response in mammals.^{13, 14, 15} In recent years, emerging evidence has indicated that the autophagy process may have an essential role for the host during bacterial clearance and may also interact with inflammatory processes, which consequently may impact the outcomes of disease progression.^{16, 17} There is a complex reciprocal relationship between autophagy pathway/proteins and inflammation.^{18, 19} Recent observations have revealed a relationship between autophagy and inflammasome-associated pro-inflammatory cytokine maturation in macrophages.^{20, 21}Given the above information, we speculated that autophagy activation may serve a protective function to restrain liver inflammation in cases of ALF. The study has also showed that inhibition of hepatocyte autophagy increases TNFα-dependent liver injury by promoting caspase-8 activation.²² So, we hypothesized that inhibition of GSK3β may promote autophagy to protect mice from ALF. To test these hypotheses, we used the ALF model induced by the co-injection of d-galactosamine (d-GalN) and lipopolysaccharide (LPS), which has been widely used to examine the underlying mechanisms of ALF,^{23, 24} to explore the protective mechanisms of GSK3β inhibition and its regulatory pathway in the context of ALF, and further measure the expression of autophagic gene in human liver samples from patients with ALF. Our findings demonstrate that inhibition of GSK3β increase autophagy to alleviate liver inflammation and protect mice from ALF mediated by peroxisome proliferator-activated receptor α (PPARα).     

Hypoxia potentiates LPS-induced inflammatory response and increases cell death by promoting NLRP3 inflammasome activation in pancreatic β cells

Hypoxia and islet inflammation are involved in β-cell failure in type 2 diabetes (T2D). Elevated plasma LPS levels have been verified in patients with T2D, and hypoxia occurs in islets of diabetic mice. Activation of inflammasomes in ischemic or hypoxic conditions was identified in various tissues. Here, we investigated whether hypoxia activates the inflammasome in β cells and the possible mechanisms involved. In mouse insulinoma cell line 6 (MIN6), hypoxia (1% O₂) primes the NLRP3 inflammasome along with NF-κB signaling activation. Our results demonstrate that hypoxia can activate the NLRP3 inflammasome in LPS-primed MIN6 to result in initiating the β cell inflammatory response and cell death in vitro. Reactive oxygen species (ROS) and the thioredoxin-interacting protein (TXNIP) are up-regulated in response to hypoxia. Finally, the role of the ROS-TXNIP axis in mediating the activation of the NLRP3 inflammasome and cell death was characterized by pretreating with the ROS scavenger N-acetylcysteine (NAC) and performing TXNIP knockdown experiments in MIN6. Our data indicate for the first time that the inflammasome is involved in the inflammatory response and cell death in hypoxia-induced β cells through the ROS-TXNIP-NLRP3 axis in vitro. This provides new insight into the relationship between hypoxia and inflammation in T2D.     

The atypical interaction of peroxisome proliferator-activated receptor α with liver X receptor α antagonizes the stimulatory effect of their respective ligands on the murine cholesterol 7α-hydroxylase gene promoter

Cholesterol 7α-hydroxylase (cyp7a) mediates cholesterol elimination in the liver by catalyzing the first and rate-limiting step in the conversion of cholesterol into bile acids. Peroxisome proliferator-activated receptor α (PPARα; NR1C1) and liver X receptor α (LXRα; NR1H3) are two nuclear receptors that stimulate the murine Cyp7a1 gene. Here we report that co-expression of PPARα and LXRα in hepatoma cells abolishes the stimulation of Cyp7a1 gene promoter in response to their respective agonists. PPARα and LXRα form an atypical heterodimer that binds to two directly adjacent hexameric sequences localized within overlapping PPARα and LXRα response elements (termed Site I), antagonizing the interaction of PPARα:retinoid X receptor α (RXRα) or RXRα:LXRα with the Cyp7a1 gene promoter. Mutations within either hexameric sequences that specifically abolished LXRα:PPARα heterodimer binding to the murine Cyp7a1 Site I also relieved promoter inhibition. The LXRα:PPARα heterodimer may be important in coordinating the expression of genes that encode proteins involved in metabolism of fats and cholesterol.     

Selenium bioisosteric replacement of adenosine derivatives promoting adiponectin secretion increases the binding affinity to peroxisome proliferator-activated receptor δ

N⁶-(3-Iodobenzyl)adenosine-5′-N-methyluronamide (1a, IB-MECA) exhibited polypharmacological characteristics targeting A₃ adenosine receptor (AR), peroxisome proliferator-activated receptor (PPAR) γ, and PPARδ, simultaneously. The bioisosteric replacement of oxygen in 4′-oxoadenosines with selenium significantly increased the PPARδ-binding activity. 2-Chloro-N⁶-(3-iodobenzyl)-4′-selenoadenosine-5′-N-methyluronamide (3e) and related 4′-selenoadenosine derivatives significantly enhanced adiponectin biosynthesis during adipogenesis in human bone marrow mesenchymal stem cells (hBM-MSCs). The PPARδ-binding affinity, but not the A₃ AR binding affinity, of 4′-selenoadenosine derivatives correlated with their adiponectin secretion stimulation. Compared with the sugar ring of 4′-oxoadenosine, that of 4′-selenoadenosine was more favorable in forming the South sugar conformation. In the molecular docking simulation, the South sugar conformation of compound 3e formed additional hydrogen bonds inside the PPARδ ligand-binding pocket compared with the North conformation. Therefore, the sugar conformation of 4′-selenoadenosine PPAR modulators affects the ligand binding affinity against PPARδ.     

Rosuvastatin alleviated the liver ischemia reperfusion injury by activating the expression of peroxisome proliferator-activated receptor gamma (PPARγ)

Journal of Bioenergetics and Biomembranes - Liver ischemia and reperfusion could cause serious damage to liver tissues. Abnormal liver function could induce serious damage and threaten human...     

Mas-related G protein-coupled receptor D participates in inflammatory pain by promoting NF-κB activation through interaction with TAK1 and IKK complex

Mas-related G protein-coupled receptor D (MrgprD) is mainly expressed in small-diameter sensory neurons of the dorsal root ganglion (DRG). Results from previous studies suggest that MrgprD participates in mechanical hyperalgesia and nerve injury-induced neuropathic pain. However, it remains elusive whether and how MrgprD is involved in inflammatory pain. Here, we used a mouse model of chronic inflammatory pain established by intraperitoneal administration of lipopolysaccharide (LPS). The LPS injection induced an evident peripheral neuroinflammation and mechanical hyperalgesia in the mice and increased MrgprD expression in the DRG. The LPS administration also augmented the proportion of MrgprD-expressing neurons in the lumbar 4 DRG. Behaviorally, the LPS-induced hypersensitivities to mechanical and cold stimuli, but not to a heat stimulus, were substantially attenuated in Mrgprd-knockout mice compared with wildtype littermates. Mrgprd deletion in DRGs suppressed the LPS-triggered activation of the NF-κB signaling pathway and attenuated LPS-induced up-regulation of pro-inflammatory factors. Moreover, ectopic overexpression of MrgprD in HEK293 cells stably expressing mouse toll-like receptor 4 (TLR4) markedly promoted the LPS-induced NF-κB activation and enhanced NF-κB's DNA-binding activity. Furthermore, MrgprD physically interacted with TGF-β-activated kinase 1 (TAK1) and I-kappa-B-kinase (IKK) complexes, but not with mitogen-activated protein kinases (MAPKs) in mouse DRGs. In macrophage-like RAW 264.7 cells, MrgprD also interacted with TAK1 and IKK complex, and the treatment of MrgprD agonist elicited the activation of NF-κB signaling, but not of mitogen-activated protein kinases (MAPKs) signaling pathway. Our findings indicate that MrgprD facilitates the development of LPS-triggered persistent inflammatory hyperalgesia by promoting canonical NF-κB activation, highlighting the important roles of MrgprD in NF-κB-mediated inflammation and chronic pain.     

Agonist-induced activation releases peroxisome proliferator-activated receptor β/δ from its inhibition by palmitate-induced nuclear factor-κB in skeletal muscle cells

The mechanisms by which elevated levels of free fatty acids cause insulin resistance are not well understood, but there is a strong correlation between insulin resistance and intramyocellular lipid accumulation in skeletal muscle. In addition, accumulating evidence suggests a link between inflammation and type 2 diabetes. The aim of this work was to study whether the exposure of skeletal muscle cells to palmitate affected peroxisome proliferator-activated receptor (PPAR) β/δ activity. Here, we report that exposure of C2C12 skeletal muscle cells to 0.75 mM palmitate reduced (74%, P<0.01) the mRNA levels of the PPARβ/δ-target gene pyruvatedehydrogenase kinase 4 (PDK-4), which is involved in fatty acid utilization. This reduction was not observed in the presence of the PPARβ/δ agonist L-165041. This drug prevented palmitate-induced nuclear factor (NF)-κB activation. Increased NF-κB activity after palmitate exposure was associated with enhanced protein–protein interaction between PPARβ/δ and p65. Interestingly, treatment with the PPARβ/δ agonist L-165041 completely abolished this interaction. These results indicate that palmitate may reduce fatty acid utilization in skeletal muscle cells by reducing PPARβ/δ signaling through increased NF-κB activity.