Retention of bile acids (BAs) in the liver during cholestasis plays an important role in the development of cholestatic liver injury. Several studies have reported that high concentrations of certain ...BAs induce cell death and inflammatory response in the liver, and BAs may promote liver tumorigenesis. Macroautophagy (hereafter referred to as autophagy) is a lysosomal degradation process that regulates organelle and protein homeostasis and serves as a cell survival mechanism under a variety of stress conditions. However, it is not known if BAs modulate autophagy in hepatocytes. In the present study, we determined autophagic flux in livers of farnesoid X receptor (FXR) knockout (KO) mice that have increased concentrations of hepatic BAs and in primary cultured mouse hepatocytes treated with BAs. The results showed that autophagic flux was impaired in livers of FXR KO mice and in BA-treated primary mouse hepatocytes. Mechanistically, BAs did not affect the activities of cathepsin or the proteasome, but impaired autophagosomal-lysosomal fusion likely due to reduction of Rab7 protein expression and targeting to autophagosomes. In conclusion, BAs suppress autophagic flux in hepatocytes by impairing autophagosomal-lysosomal fusion, which may be implicated in bile acid-induced liver tumor promotion observed in FXR KO mice.
Alcohol-associated liver disease (ALD) is a major chronic liver disease around the world without successful treatment. Acute alcoholic hepatitis is one of the most severe forms of ALD with high ...mortality, which is often associated with binge drinking. Alcohol drinking dysregulates lipid metabolism, increases adipose tissue lipolysis, and induces liver steatosis and adipose tissue atrophy. Increasing evidence implicates that crosstalk of liver and adipose tissue in the pathogenesis of ALD. Mechanistic target of rapamycin (mTOR) is a phosphatidylinositol 3-kinase (PI3K)-like serine/threonine protein kinase that regulates lipid metabolism, cell proliferation and autophagy. However, the role of mTOR in regulating adipose-liver crosstalk in binge drinking-induced organ damage remains unclear.
We generated liver-specific and adipocyte-specific regulatory-associated protein of mTOR (Rptor) knockout (RptorLKO and RptorAKO) as well as Mtor knockout (MtorLKO and MtorAKO) mice, by crossing Rptorflox and Mtorflox mice with albumin Cre or adiponectin Cre mice, respectively. In addition, we generated liver and adipocyte double deletion of Rptor or Mtor (MtorLAKO and RptorLAKO) mice. The knockout mice with their matched wild-type littermates (RptorWT and MtorWT) were subjected to acute gavage of 7 g/kg ethanol.
Mice with adipocyte deletion of Rptor or Mtor developed hepatomegaly and adipose tissue atrophy. Alcohol gavage increased liver injury, hepatic steatosis and inflammation in mouse livers as demonstrated by elevated serum alanine aminotransferase activities, increased hepatic levels of triglyceride and increased hepatic numbers of CD68 positive macrophages in mouse livers after alcohol gavage. Liver injury was further exacerbated by deletion of adipocyte Rptor or Mtor. Serum adipokine array analysis revealed that increased levels of pro-inflammatory cytokines IL-6 and TNFα as well as chemokine MCP-1 following acute alcohol gavage in wild-type mice, which were further increased in adipocyte-specific Mtor or Rptor knockout mice. Conversely, levels of anti-inflammatory cytokine IL-10 decreased in adipocyte-specific Mtor or Rptor knockout mice. The levels of circulating fibroblast growth factor 21 (FGF21) increased whereas levels of circulating adiponectin and fetuin A decreased in wild-type mice after alcohol gavage. Intriguingly, adipocyte-specific Mtor or Rptor knockout mice already had decreased basal level of FGF21 which increased by alcohol gavage. Moreover, adipocyte-specific Mtor or Rptor knockout mice already had increased basal level of adiponectin and decreased fetuin A which were not further changed by alcohol gavage.
Adipocyte but not hepatocyte ablation of Mtor pathway contributes to acute alcohol-induced liver injury with increased inflammation. Our results demonstrate the critical role of adipocyte mTOR in regulating the adipose-liver crosstalk in ALD.
•Novel and unique genetic modified animal models are used in the study.•mTOR or Raptor regulates liver – adipose tissue crosstalk in ALD.•Adipocyte deletion but not hepatic deletion of mTOR or Raptor contributes to ALD.
Despite the growing global burden of alcoholic liver diseases, therapeutic options are limited, and novel targets are urgently needed. Accumulating evidence suggests that mitochondria adapt in ...response to ethanol and formation of megamitochondria in the livers of patients is recognized as a hallmark of alcoholic liver diseases. The processes involved in ethanol-induced hepatic mitochondrial changes, the impact on mitochondria-shaping proteins, and the significance of megamitochondria formation remain unknown. In this study, we investigated the mitochondrial and cellular response to alcohol in hepatoma cell line VL-17A. The mitochondrial architecture rapidly changed after 3 or 14 days of ethanol exposure with double-pronged presentation of hyperfragmentation and megamitochondria, and cell growth was inhibited. Dynamin-1–like protein (Drp1) was identified as the main mediator driving these mitochondrial alterations, and its genetic inactivation was determined to foster megamitochondria development, preserving the capacity of the cells to grow despite alcohol toxicity. The role of Drp1 in mediating megamitochondria formation in mice with liver-specific inactivation of Drp1 was further confirmed. Finally, when these mice were fed with ethanol, the presentation of hepatic megamitochondria was exacerbated compared with wild type fed with the same diet. Ethanol-induced toxicity was also reduced. Our study demonstrates that megamitochondria formation is mediated by Drp1, and this phenomenon is a beneficial adaptive response during alcohol-induced hepatotoxicity.
Motor neurons in amyotrophic lateral sclerosis (ALS) patients and animal models show degeneration from the nerve terminal, known as dying-back neuropathy. To investigate the mechanism underlying this ...neuropathy, we analyzed the neuromuscular junctions (NMJs) and motor neuron cell bodies in SOD1
mice using electron microscopy. NMJs of SOD1
mice exhibited significantly higher numbers of autophagosomes and degenerated mitochondria compared to wild-type controls. Mitophagosomes were identified in the NMJ presynaptic terminals of wild-type mice and SOD1
mice. However, the number of mitophagosomes did not increase significantly in SOD1
NMJs indicating a defect in mitophagy, the autophagic process to degrade mitochondria. Consistent with this, proteins essential for mitophagy, p62/SQSTM1, Bnip3, Pink1, and Parkin were down-regulated in motor neurons in SOD1
mice. Importantly, SQSTM1 is one of the genes mutated in familial ALS patients. We evaluated the effect of impaired mitophagy on motor neurons by analyzing the double knockout mice of Pink1 and Parkin, two genes responsible for sensing depolarized mitochondria and delivering degenerated mitochondria to mitophagosomes. The double knockout mice exhibited NMJ degeneration, including axon swelling and NMJ fragmentation at 4 months of age. These phenotypes were rarely observed in wild-type control mice of the same age. The protein level of ATP synthase β subunit increased in the NMJ presynaptic terminals, suggesting the accumulation of mitochondria at NMJs of the double knockout mice. Importantly, NMJ denervation was observed in the double knockout mice. These data suggest that the reduced mitophagy function in motor neurons of SOD1
mice is one of the mechanisms causing degeneration of ALS NMJs.
Anthocyanins are a group of naturally occurring phenolic compounds widely available in fruits and vegetables in human diets. They have broad biological activities including anti-mutagenesis and ...anticarcinogenesis, which are generally attributed to their antioxidant activities. We studied the effects and the mechanisms of the most common type of anthocyanins, cyanidin-3-rutinoside, in several leukemia and lymphoma cell lines. We found that cyanidin-3-rutinoside extracted and purified from the black raspberry cultivar Jewel induced apoptosis in HL-60 cells in a dose- and time-dependent manner. Paradoxically, this compound induced the accumulation of peroxides, which are involved in the induction of apoptosis in HL-60 cells. In addition, cyanidin-3-rutinoside treatment resulted in reactive oxygen species (ROS)-dependent activation of p38 MAPK and JNK, which contributed to cell death by activating the mitochondrial pathway mediated by Bim. Down-regulation of Bim or overexpression of Bcl-2 or Bcl-xL considerably blocked apoptosis. Notably, cyanidin-3-rutinoside treatment did not lead to increased ROS accumulation in normal human peripheral blood mononuclear cells and had no cytotoxic effects on these cells. These results indicate that cyanidin-3-rutinoside has the potential to be used in leukemia therapy with the advantages of being widely available and selective against tumors.
Acetaminophen (APAP) overdose is the most frequent cause of acute liver failure in the US and many western countries. It is well known that APAP induces mitochondrial damage to trigger centrilobular ...necrosis. Emerging evidence suggests that autophagic removal of damaged mitochondria may protect against APAP-induced liver injury. Electron and confocal microscopy analysis of liver tissues revealed that APAP overdose triggers unique biochemical and pathological zonated changes in the mouse liver, which includes necrosis (zone 1), mitochondrial spheroid formation (zone 2), autophagy (zone 3) and mitochondrial biogenesis (zone 4). In this graphic review, we discuss the role of autophagy/mitophagy in limiting the expansion of necrosis and promoting mitochondrial biogenesis and liver regeneration for the recovery of APAP-induced liver injury. We also discuss possible mechanisms that could be involved in regulating APAP-induced autophagy/mitophagy and the formation of mitochondrial spheroids.
Arsenite is a known carcinogen and its exposure has been implicated in a variety of noncarcinogenic health concerns. Increased oxidative stress is thought to be the primary cause of arsenite toxicity ...and the toxic effect is thought to be linear with detrimental effects reported at all concentrations of arsenite. But the paradigm of linear dose response in arsenite toxicity is shifting. In the present study we demonstrate that arsenite effects on mitochondrial respiration in primary hepatocytes follow a nonlinear dose response. In vitro exposure of primary hepatocytes to an environmentally relevant, moderate level of arsenite results in increased oxidant production that appears to arise from changes in the expression and activity of respiratory Complex I of the mitochondrial proton circuit. In primary hepatocytes the excess oxidant production appears to elicit adaptive responses that promote resistance to oxidative stress and a propensity to increased proliferation. Taken together, these results suggest a nonlinear dose-response characteristic of arsenite with low-dose arsenite promoting adaptive responses in a process known as mitohormesis, with transient increase in ROS levels acting as transducers of arsenite-induced mitohormesis.
Cystic echinococcosis is highly prevalent in northwest China. A cost-effective, easy to operate diagnostic tool with high sensitivity and specificity would greatly facilitate the monitoring of ...Echinococcus infections in canine definitive hosts.
The primers used in the LAMP assay were based on the mitochondrial nad5 gene of E. granulosus sensu stricto (E. granulosus s.s., or E.g.s.s.) and were designed using Primer Explorer V4 software. The developed LAMP assay was compared with a conventional PCR method, copro-ELISA and microscopy, using the faeces of dogs experimentally infected with E.g.s.s., and field-collected faeces of domestic dogs including 190 from Qinghai province highly endemic for E.g.s.s. and 30 controls from an area in Gansu, where a domestic dog de-worming program was in operation.
The positivity rates obtained for the field-collected faecal samples were 12.6%, 1.6% and 2.1% by the LAMP, PCR and copro-ELISA assays, respectively. All samples obtained from the control dogs were negative. Compared with the conventional PCR, the LAMP assay provided 88.8% specificity and 100% sensitivity. The higher sensitivity of the LAMP method was also shown by the fact that it could detect the presence of laboratory challenge dog infections of E. granulsous s.s. four days earlier than the PCR method. Three copro-samples shown positive by the commercial copro-ELISA were all negative by LAMP, PCR and microscopy, which suggests these samples may have originated from another infection rather than E. granulsous s.s., possibly E. shiquicus or E. Canadensis, which is also present in China.
We have developed a potentially useful surveillance tool for determining the prevalence of canine E. granulosus s.s. infections in the field. The LAMP assay may lead to a more cost-effective and practicable way of tracking Echinococcus infections in canids, especially when combined with the copro-ELISA.
Chronic alcohol causes liver hypoxia and steatosis, which eventually develops into alcoholic liver disease (ALD). While it has been known that alcohol consumption activates hepatic hypoxia inducing ...factor-1α (HIF-1α), conflicting results regarding the role of HIF-1α in alcohol-induced liver injury and steatosis in mice have been reported. In the present study, we aimed to use hepatocyte-specific HIF-1β knockout mice to eliminate the possible compensatory effects of the single knockout of the 1α subunit of HIF to study the role of HIFs in ALD. C57BL/6 wild type mice were treated with acute ethanol to mimic human binge drinking. Matched wild-type and hepatocyte specific HIF-1β knockout mice were also subjected to a recently established Gao-binge alcohol model to mimic chronic plus binge conditions, which is quite common in human alcoholics. We found that acute alcohol treatment increased BNIP3 and BNIP3L/NIX expression in primary cultured hepatocytes and in mouse livers, suggesting that HIF may be activated in these models. We further found that hepatocyte-specific HIF-1β knockout mice developed less steatosis and liver injury following the Gao-binge model or acute ethanol treatment compared with their matched wild type mice. Mechanistically, protection against Gao-binge treatment-induced steatosis and liver injury was likely associated with increased FoxO3a activation and subsequent induction of autophagy in hepatocyte-specific HIF-1β knockout mice.
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