-
Hepatology (Baltimore, Md.) Jan 2023Increased megamitochondria formation and impaired mitophagy in hepatocytes have been linked to the pathogenesis of alcohol-associated liver disease (ALD). This study...
BACKGROUND AND AIMS
Increased megamitochondria formation and impaired mitophagy in hepatocytes have been linked to the pathogenesis of alcohol-associated liver disease (ALD). This study aims to determine the mechanisms by which alcohol consumption increases megamitochondria formation in the pathogenesis of ALD.
APPROACH AND RESULTS
Human alcoholic hepatitis (AH) liver samples were used for electron microscopy, histology, and biochemical analysis. Liver-specific dynamin-related protein 1 (DRP1; gene name DNM1L, an essential gene regulating mitochondria fission ) knockout (L-DRP1 KO) mice and wild-type mice were subjected to chronic plus binge alcohol feeding. Both human AH and alcohol-fed mice had decreased hepatic DRP1 with increased accumulation of hepatic megamitochondria. Mechanistic studies revealed that alcohol feeding decreased DRP1 by impairing transcription factor EB-mediated induction of DNM1L . L-DRP1 KO mice had increased megamitochondria and decreased mitophagy with increased liver injury and inflammation, which were further exacerbated by alcohol feeding. Seahorse flux and unbiased metabolomics analysis showed alcohol intake increased mitochondria oxygen consumption and hepatic nicotinamide adenine dinucleotide (NAD + ), acylcarnitine, and ketone levels, which were attenuated in L-DRP1 KO mice, suggesting that loss of hepatic DRP1 leads to maladaptation to alcohol-induced metabolic stress. RNA-sequencing and real-time quantitative PCR analysis revealed increased gene expression of the cGAS-stimulator of interferon genes (STING)-interferon pathway in L-DRP1 KO mice regardless of alcohol feeding. Alcohol-fed L-DRP1 KO mice had increased cytosolic mtDNA and mitochondrial dysfunction leading to increased activation of cGAS-STING-interferon signaling pathways and liver injury.
CONCLUSION
Alcohol consumption decreases hepatic DRP1 resulting in increased megamitochondria and mitochondrial maladaptation that promotes AH by mitochondria-mediated inflammation and cell injury.
Topics: Mice; Humans; Animals; Hepatitis, Alcoholic; Mitochondrial Swelling; Liver Diseases, Alcoholic; Mitochondria; Ethanol; Nucleotidyltransferases; Inflammation; Interferons; Mitochondrial Dynamics
PubMed: 35698731
DOI: 10.1002/hep.32604 -
Cell Reports Nov 2021Tumor necrosis factor (TNF) is a key driver of several inflammatory diseases, such as rheumatoid arthritis, inflammatory bowel disease, and psoriasis, in which affected...
Tumor necrosis factor (TNF) is a key driver of several inflammatory diseases, such as rheumatoid arthritis, inflammatory bowel disease, and psoriasis, in which affected tissues show an interferon-stimulated gene signature. Here, we demonstrate that TNF triggers a type-I interferon response that is dependent on the cyclic guanosine monophosphate-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway. We show that TNF inhibits PINK1-mediated mitophagy and leads to altered mitochondrial function and to an increase in cytosolic mtDNA levels. Using cGAS-chromatin immunoprecipitation (ChIP), we demonstrate that cytosolic mtDNA binds to cGAS after TNF treatment. Furthermore, TNF induces a cGAS-STING-dependent transcriptional response that mimics that of macrophages from rheumatoid arthritis patients. Finally, in an inflammatory arthritis mouse model, cGAS deficiency blocked interferon responses and reduced inflammatory cell infiltration and joint swelling. These findings elucidate a molecular mechanism linking TNF to type-I interferon signaling and suggest a potential benefit for therapeutic targeting of cGAS/STING in TNF-driven diseases.
Topics: Animals; Arthritis, Experimental; DNA, Mitochondrial; Female; Immunity, Innate; Inflammation; Interferon Regulatory Factor-3; Interferon Type I; Macrophages; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitophagy; Nucleotidyltransferases; Tumor Necrosis Factor-alpha
PubMed: 34758308
DOI: 10.1016/j.celrep.2021.109977 -
Journal of Hepatology Jan 2022Patients with acute-on-chronic liver failure (ACLF) present a systemic hyperinflammatory response associated with increased circulating levels of small-molecule...
BACKGROUND & AIMS
Patients with acute-on-chronic liver failure (ACLF) present a systemic hyperinflammatory response associated with increased circulating levels of small-molecule metabolites. To investigate whether these alterations reflect inadequate cell energy output, we assessed mitochondrial morphology and central metabolic pathways with emphasis on the tricarboxylic acid (TCA) cycle in peripheral leukocytes from patients with acutely decompensated (AD) cirrhosis, with and without ACLF.
METHODS
The study included samples from patients with AD cirrhosis (108 without and 128 with ACLF) and 41 healthy individuals. Leukocyte mitochondrial ultrastructure was visualized by transmission electron microscopy and cytosolic and mitochondrial metabolic fluxes were determined by assessing NADH/FADH production from various substrates. Plasma GDF15 and FGF21 were determined by Luminex and acylcarnitines by LC-MS/MS. Gene expression was analyzed by RNA-sequencing and PCR-based glucose metabolism profiler array.
RESULTS
Mitochondrial ultrastructure in patients with advanced cirrhosis was distinguished by cristae rarefication and swelling. The number of mitochondria per leukocyte was higher in patients, accompanied by a reduction in their size. Increased FGF21 and C6:0- and C8:0-carnitine predicted mortality whereas GDF15 strongly correlated with a gene set signature related to leukocyte activation. Metabolic flux analyses revealed increased energy production in mononuclear leukocytes from patients with preferential involvement of extra-mitochondrial pathways, supported by upregulated expression of genes encoding enzymes of the glycolytic and pentose phosphate pathways. In patients with ACLF, mitochondrial function analysis uncovered break-points in the TCA cycle at the isocitrate dehydrogenase and succinate dehydrogenase level, which were bridged by anaplerotic reactions involving glutaminolysis and nucleoside metabolism.
CONCLUSIONS
Our findings provide evidence at the cellular, organelle and biochemical levels that severe mitochondrial dysfunction governs immunometabolism in leukocytes from patients with AD cirrhosis and ACLF.
LAY SUMMARY
Patients at advanced stages of liver disease have dismal prognosis due to vital organ failures and the lack of treatment options. In this study, we report that the functioning of mitochondria, which are known as the cell powerhouse, is severely impaired in leukocytes of these patients, probably as a consequence of intense inflammation. Mitochondrial dysfunction is therefore a hallmark of advanced liver disease.
Topics: Acute-On-Chronic Liver Failure; Humans; Immunologic Factors; Leukocytes; Leukocytes, Mononuclear; Mitochondrial Diseases; Tandem Mass Spectrometry
PubMed: 34450236
DOI: 10.1016/j.jhep.2021.08.009 -
Cell Cycle (Georgetown, Tex.) May 2020Pyroptosis is a form of programmed cell death initiated by inflammasomes and is critical for immunity. SIRT1, a NAD+-dependent deacetylase, plays multiple roles in...
Pyroptosis is a form of programmed cell death initiated by inflammasomes and is critical for immunity. SIRT1, a NAD+-dependent deacetylase, plays multiple roles in inflammatory response and immunity. Metformin can activate SIRT1 to participate in different biological processes and exert its anticancer effects. However, the mechanism by which metformin activates SIRT1 to drive cancer cell pyroptosis has not been reported. In this study, we treated cancer cells with metformin for diverse periods of time (0-24 h) and found that cell viability was decreased obviously. Interestingly, pyroptosis occurred when cancer cells were treated with metformin for the indicated time (4, 8 and 12 h), which was elucidated by the cell swelling and bubbles blowing in the membrane. Metformin also increased the release of lactate dehydrogenase (LDH, an indication of pyroptotic cell cytotoxicity) remarkably. The underlying mechanisms were that metformin enhanced AMPK/SIRT1 pathway and further increased NF-κB p65 expression to stimulate Bax activation and cytochrome c release, triggering caspase3 cleavage of GSDME, which is a characteristic pyroptotic marker. Depletion of SIRT1 inhibited metformin-induced these protein expression, revealing that metformin promotes AMPK/SIRT1/NF-κB signaling to drive cancer cell pyroptosis. Meantime, metformin induced mitochondrial dysfunction to trigger activation of caspase3 and generation of GSDME-N. Moreover, mitochondrial dysfunction activated AMPK/SIRT1 pathway to cause pyroptotic death upon metformin treatment. This research firstly reveals that metformin as a sensitizer amplifies AMPK/SIRT1/NF-κB signaling to induce caspase3/GSDME-mediated cancer cell pyroptosis. Induction of cellular pyroptosis by metformin is considered as a novel therapeutic option against various cancers.
Topics: AMP-Activated Protein Kinases; Antineoplastic Agents; Caspase 3; Cell Proliferation; Cell Survival; Gene Knockdown Techniques; HT29 Cells; Hep G2 Cells; Humans; MCF-7 Cells; Metformin; Mitochondria; NF-kappa B; Neoplasms; Pyroptosis; Receptors, Estrogen; Signal Transduction; Sirtuin 1; Transfection
PubMed: 32286137
DOI: 10.1080/15384101.2020.1743911 -
Oxalate-induced apoptosis through ERS-ROS-NF-κB signalling pathway in renal tubular epithelial cell.Molecular Medicine (Cambridge, Mass.) Aug 2022Kidney stones are composed of approximately 70-80% calcium oxalate. However, the exact mechanism of formation of calcium oxalate kidney stones remains unclear. In this...
BACKGROUND
Kidney stones are composed of approximately 70-80% calcium oxalate. However, the exact mechanism of formation of calcium oxalate kidney stones remains unclear. In this study, we investigated the roles of endoplasmic reticulum stress (ERS), reactive oxygen species (ROS), and the NF-κB signalling pathway in the pathogenesis of oxalate-induced renal tubular epithelial cell injury and its possible molecular mechanisms.
METHODS
We established a model to evaluate the formation of kidney stones by intraperitoneal injection of glyoxylic acid solution into mice and assessed cell morphology, apoptosis, and the expression levels of ERS, ROS, and NF-κB signalling pathway-related proteins in mouse renal tissues. Next, we treated HK-2 cells with potassium oxalate to construct a renal tubular epithelial cell injury model. We detected the changes in autophagy, apoptosis, and mitochondrial membrane potential and investigated the ultrastructure of the cells by transmission electron microscopy. Western blotting revealed the expression levels of apoptosis and autophagy proteins; mitochondrial structural and functional proteins; and ERS, ROS, and NF-κB (p65) proteins. Lastly, we studied the downregulation of NF-κB activity in HK-2 cells by lentivirus interference and confirmed the interaction between the NF-κB signalling and ERS/ROS pathways.
RESULTS
We observed swelling of renal tissues, increased apoptosis of renal tubular epithelial cells, and activation of the ERS, ROS, and NF-κB signalling pathways in the oxalate group. We found that oxalate induced autophagy, apoptosis, and mitochondrial damage in HK-2 cells and activated the ERS/ROS/NF-κB pathways. Interestingly, when the NF-κB signalling pathway was inhibited, the ERS/ROS pathway was also inhibited.
CONCLUSION
Oxalate induces HK-2 cell injury through the interaction between the NF-κB signalling and ERS/ROS pathways.
Topics: Animals; Apoptosis; Calcium Oxalate; Endoplasmic Reticulum Stress; Epithelial Cells; Kidney Calculi; Mice; NF-kappa B; Oxalates; Reactive Oxygen Species
PubMed: 35922749
DOI: 10.1186/s10020-022-00494-5 -
Cell Death and Differentiation Aug 2023The mitochondrial permeability transition (mPT) describes a Ca-dependent and cyclophilin D (CypD)-facilitated increase of inner mitochondrial membrane permeability that... (Review)
Review
The mitochondrial permeability transition (mPT) describes a Ca-dependent and cyclophilin D (CypD)-facilitated increase of inner mitochondrial membrane permeability that allows diffusion of molecules up to 1.5 kDa in size. It is mediated by a non-selective channel, the mitochondrial permeability transition pore (mPTP). Sustained mPTP opening causes mitochondrial swelling, which ruptures the outer mitochondrial membrane leading to subsequent apoptotic and necrotic cell death, and is implicated in a range of pathologies. However, transient mPTP opening at various sub-conductance states may contribute several physiological roles such as alterations in mitochondrial bioenergetics and rapid Ca efflux. Since its discovery decades ago, intensive efforts have been made to identify the exact pore-forming structure of the mPT. Both the adenine nucleotide translocase (ANT) and, more recently, the mitochondrial FF (F)-ATP synthase dimers, monomers or c-subunit ring alone have been implicated. Here we share the insights of several key investigators with different perspectives who have pioneered mPT research. We critically assess proposed models for the molecular identity of the mPTP and the mechanisms underlying its opposing roles in the life and death of cells. We provide in-depth insights into current controversies, seeking to achieve a degree of consensus that will stimulate future innovative research into the nature and role of the mPTP.
Topics: Mitochondrial Permeability Transition Pore; Mitochondrial Membrane Transport Proteins; Consensus; Mitochondria; Mitochondrial Membranes
PubMed: 37460667
DOI: 10.1038/s41418-023-01187-0 -
Cardiovascular Diabetology Jan 2021Sodium-glucose linked transporter type 2 (SGLT-2) inhibition has been shown to reduce cardiovascular mortality in heart failure independently of glycemic control and...
BACKGROUND
Sodium-glucose linked transporter type 2 (SGLT-2) inhibition has been shown to reduce cardiovascular mortality in heart failure independently of glycemic control and prevents the onset of atrial arrhythmias, a common co-morbidity in heart failure with preserved ejection fraction (HFpEF). The mechanism behind these effects is not fully understood, and it remains unclear if they could be further enhanced by additional SGLT-1 inhibition. We investigated the effects of chronic treatment with the dual SGLT-1&2 inhibitor sotagliflozin on left atrial (LA) remodeling and cellular arrhythmogenesis (i.e. atrial cardiomyopathy) in a metabolic syndrome-related rat model of HFpEF.
METHODS
17 week-old ZSF-1 obese rats, a metabolic syndrome-related model of HFpEF, and wild type rats (Wistar Kyoto), were fed 30 mg/kg/d sotagliflozin for 6 weeks. At 23 weeks, LA were imaged in-vivo by echocardiography. In-vitro, Ca transients (CaT; electrically stimulated, caffeine-induced) and spontaneous Ca release were recorded by ratiometric microscopy using Ca-sensitive fluorescent dyes (Fura-2) during various experimental protocols. Mitochondrial structure (dye: Mitotracker), Ca buffer capacity (dye: Rhod-2), mitochondrial depolarization (dye: TMRE) and production of reactive oxygen species (dye: H2DCF) were visualized by confocal microscopy. Statistical analysis was performed with 2-way analysis of variance followed by post-hoc Bonferroni and student's t-test, as applicable.
RESULTS
Sotagliflozin ameliorated LA enlargement in HFpEF in-vivo. In-vitro, LA cardiomyocytes in HFpEF showed an increased incidence and amplitude of arrhythmic spontaneous Ca release events (SCaEs). Sotagliflozin significantly reduced the magnitude of SCaEs, while their frequency was unaffected. Sotagliflozin lowered diastolic [Ca] of CaT at baseline and in response to glucose influx, possibly related to a ~ 50% increase of sodium sodium-calcium exchanger (NCX) forward-mode activity. Sotagliflozin prevented mitochondrial swelling and enhanced mitochondrial Ca buffer capacity in HFpEF. Sotagliflozin improved mitochondrial fission and reactive oxygen species (ROS) production during glucose starvation and averted Ca accumulation upon glycolytic inhibition.
CONCLUSION
The SGLT-1&2 inhibitor sotagliflozin ameliorated LA remodeling in metabolic HFpEF. It also improved distinct features of Ca-mediated cellular arrhythmogenesis in-vitro (i.e. magnitude of SCaEs, mitochondrial Ca buffer capacity, diastolic Ca accumulation, NCX activity). The safety and efficacy of combined SGLT-1&2 inhibition for the treatment and/or prevention of atrial cardiomyopathy associated arrhythmias should be further evaluated in clinical trials.
Topics: Animals; Arrhythmias, Cardiac; Atrial Function, Left; Atrial Remodeling; Calcium Signaling; Disease Models, Animal; Glycosides; Heart Atria; Heart Failure; Metabolic Syndrome; Mitochondria, Heart; Mitochondrial Dynamics; Mitochondrial Swelling; Rats, Inbred WKY; Rats, Zucker; Reactive Oxygen Species; Sodium-Calcium Exchanger; Sodium-Glucose Transporter 1; Sodium-Glucose Transporter 2; Sodium-Glucose Transporter 2 Inhibitors; Rats
PubMed: 33413413
DOI: 10.1186/s12933-020-01208-z -
Pharmacological Research Aug 2021Pyroptosis, a type of programmed cell death (PCD), is characterized by cell swelling with bubbles, and the release of inflammatory cell cytokines. Cucurbitacin B (CuB),...
Pyroptosis, a type of programmed cell death (PCD), is characterized by cell swelling with bubbles, and the release of inflammatory cell cytokines. Cucurbitacin B (CuB), extracted from muskmelon pedicel, is a natural bioactive product that could effectively exert anti-tumor activities in lung cancer. However, the exact molecular mechanisms and the direct targets of CuB in non-small cell lung cancer (NSCLC) remain to be discovered. Here, we firstly found that CuB exerted an anti-tumor effect via pyroptosis in NSCLC cells and NSCLC mice models. Next, based on the molecular docking and cellular thermal shift assay (CETSA), we identified that CuB directly bound to Toll-like receptor 4 (TLR4) to activate the NLRP3 inflammasome, which further caused the separation of N- and C-terminals of Gasdermin D (GSDMD) to execute pyroptosis. Moreover, CuB enhanced the mitochondrial reactive oxygen species (ROS), mitochondrial membrane protein Tom20 accumulation, and cytosolic calcium (Ca) release, leading to pyroptosis in NSCLC cells. Silencing of TLR4 inhibited CuB-induced pyroptosis and decreased the level of ROS and Ca in A549 cells. In vivo study showed that CuB treatment suppressed lung tumor growth in mice via pyroptosis without dose-dependent manner, and CuB at 0.75 mg/kg had a better anti-tumor effect compared to the Gefitinib group. Taken together, our findings revealed the mechanisms and targets of CuB triggering pyroptosis in NSCLC, thus supporting the notion of developing CuB as a promising therapeutic agent for NSCLC.
Topics: A549 Cells; Animals; Antineoplastic Agents, Phytogenic; Carcinoma, Non-Small-Cell Lung; Gene Expression Regulation, Neoplastic; Humans; Inflammasomes; Lung Neoplasms; Male; Mice, Inbred C57BL; Mice, Nude; NLR Family, Pyrin Domain-Containing 3 Protein; Phosphate-Binding Proteins; Pore Forming Cytotoxic Proteins; Pyroptosis; Signal Transduction; Toll-Like Receptor 4; Triterpenes; Tumor Burden; Xenograft Model Antitumor Assays; Mice
PubMed: 34217831
DOI: 10.1016/j.phrs.2021.105748 -
Redox Biology Oct 2022Bile acids are steroid synthesized in liver, which are essential for fat emulsification, cholesterol excretion and gut microbial homeostasis. However, the role of bile...
PURPOSE
Bile acids are steroid synthesized in liver, which are essential for fat emulsification, cholesterol excretion and gut microbial homeostasis. However, the role of bile acids in leukemia progression remains unclear. We aim at exploring the effects and mechanisms of chenodeoxycholic acid (CDCA), a type of bile acids, on acute myeloid leukemia (AML) progression.
RESULTS
Here, we found that CDCA was decreased in feces and plasma of AML patients, positively correlated with the diversity of gut microbiota, and negatively associated with AML prognosis. We further demonstrated that CDCA suppressed AML progression both in vivo and in vitro. Mechanistically, CDCA bound to mitochondria to cause mitochondrial morphology damage containing swelling and reduction of cristae, decreased mitochondrial membrane potential and elevated mitochondrial calcium level, which resulted in the production of excessive reactive oxygen species (ROS). Elevated ROS further activated p38 MAPK signaling pathway, which collaboratively promoted the accumulation of lipid droplets (LDs) through upregulating the expression of the diacylglycerol O-acyltransferase 1 (DGAT1). As the consequence of the abundance of ROS and LDs, lipid peroxidation was enhanced in AML cells. Moreover, we uncovered that CDCA inhibited M2 macrophage polarization and suppressed the proliferation-promoting effects of M2 macrophages on AML cells in co-cultured experiments.
CONCLUSION
Our findings demonstrate that CDCA suppresses AML progression through synergistically promoting LDs accumulation and lipid peroxidation via ROS/p38 MAPK/DGAT1 pathway caused by mitochondrial dysfunction in leukemia cells and inhibiting M2 macrophage polarization.
Topics: Bile Acids and Salts; Calcium; Chenodeoxycholic Acid; Cholesterol; Diacylglycerol O-Acyltransferase; Humans; Leukemia, Myeloid, Acute; Lipid Peroxidation; Macrophages; Reactive Oxygen Species; p38 Mitogen-Activated Protein Kinases
PubMed: 36084349
DOI: 10.1016/j.redox.2022.102452 -
CGG repeat expansion in causes mitochondrial dysfunction and progressive neurodegeneration in model.Proceedings of the National Academy of... Oct 2022Neuronal intranuclear inclusion disease (NIID) is a neuromuscular/neurodegenerative disease caused by the expansion of CGG repeats in the 5' untranslated region (UTR) of...
Neuronal intranuclear inclusion disease (NIID) is a neuromuscular/neurodegenerative disease caused by the expansion of CGG repeats in the 5' untranslated region (UTR) of the gene. These repeats can be translated into a polyglycine-containing protein, uN2CpolyG, which forms protein inclusions and is toxic in cell models, albeit through an unknown mechanism. Here, we established a transgenic model expressing uN2CpolyG in multiple systems, which resulted in progressive neuronal cell loss, locomotor deficiency, and shortened lifespan. Interestingly, electron microscopy revealed mitochondrial swelling both in transgenic flies and in muscle biopsies of individuals with NIID. Immunofluorescence and immunoelectron microscopy showed colocalization of uN2CpolyG with mitochondria in cell and patient samples, while biochemical analysis revealed that uN2CpolyG interacted with a mitochondrial RNA binding protein, LRPPRC (leucine-rich pentatricopeptide repeat motif-containing protein). Furthermore, RNA sequencing (RNA-seq) analysis and functional assays showed down-regulated mitochondrial oxidative phosphorylation in uN2CpolyG-expressing flies and NIID muscle biopsies. Finally, idebenone treatment restored mitochondrial function and alleviated neurodegenerative phenotypes in transgenic flies. Overall, these results indicate that transgenic flies expressing uN2CpolyG recapitulate key features of NIID and that reversing mitochondrial dysfunction might provide a potential therapeutic approach for this disorder.
Topics: 5' Untranslated Regions; Animals; Animals, Genetically Modified; Drosophila; Intranuclear Inclusion Bodies; Leucine; Mitochondria; Neurodegenerative Diseases; RNA-Binding Proteins; Trinucleotide Repeat Expansion
PubMed: 36191230
DOI: 10.1073/pnas.2208649119