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1.
Cell ; 171(3): 628-641.e26, 2017 Oct 19.
Article in English | MEDLINE | ID: mdl-29053969

ABSTRACT

Ferroptosis is a form of programmed cell death that is pathogenic to several acute and chronic diseases and executed via oxygenation of polyunsaturated phosphatidylethanolamines (PE) by 15-lipoxygenases (15-LO) that normally use free polyunsaturated fatty acids as substrates. Mechanisms of the altered 15-LO substrate specificity are enigmatic. We sought a common ferroptosis regulator for 15LO. We discovered that PEBP1, a scaffold protein inhibitor of protein kinase cascades, complexes with two 15LO isoforms, 15LO1 and 15LO2, and changes their substrate competence to generate hydroperoxy-PE. Inadequate reduction of hydroperoxy-PE due to insufficiency or dysfunction of a selenoperoxidase, GPX4, leads to ferroptosis. We demonstrated the importance of PEBP1-dependent regulatory mechanisms of ferroptotic death in airway epithelial cells in asthma, kidney epithelial cells in renal failure, and cortical and hippocampal neurons in brain trauma. As master regulators of ferroptotic cell death with profound implications for human disease, PEBP1/15LO complexes represent a new target for drug discovery.


Subject(s)
Acute Kidney Injury/pathology , Asthma/pathology , Brain Injuries, Traumatic/pathology , Cell Death , Phosphatidylethanolamine Binding Protein/metabolism , Acute Kidney Injury/metabolism , Animals , Apoptosis , Asthma/metabolism , Brain Injuries, Traumatic/metabolism , Cell Death/drug effects , Cell Line , Humans , Isoenzymes/metabolism , Lipoxygenase/chemistry , Lipoxygenase/metabolism , Mice , Models, Molecular , Oxazolidinones/pharmacology , Oxidation-Reduction , Phosphatidylethanolamine Binding Protein/chemistry
2.
J Am Chem Soc ; 145(20): 11311-11322, 2023 05 24.
Article in English | MEDLINE | ID: mdl-37103240

ABSTRACT

Reliable probing of cardiolipin (CL) content in dynamic cellular milieux presents significant challenges and great opportunities for understanding mitochondria-related diseases, including cancer, neurodegeneration, and diabetes mellitus. In intact respiring cells, selectivity and sensitivity for CL detection are technically demanding due to structural similarities among phospholipids and compartmental secludedness of the inner mitochondrial membrane. Here, we report a novel "turn-on" fluorescent probe HKCL-1M for detecting CL in situ. HKCL-1M displays outstanding sensitivity and selectivity toward CL through specific noncovalent interactions. In live-cell imaging, its hydrolyzed product HKCL-1 efficiently retained itself in intact cells independent of mitochondrial membrane potential (Δψm). The probe robustly co-localizes with mitochondria and outperforms 10-N-nonyl acridine orange (NAO) and Δψm-dependent dyes with superior photostability and negligible phototoxicity. Our work thus opens up new opportunities for studying mitochondrial biology through efficient and reliable visualization of CL in situ.


Subject(s)
Cardiolipins , Fluorescent Dyes , Fluorescent Dyes/chemistry , Cardiolipins/chemistry , Mitochondria/chemistry , Phospholipids/analysis , Mitochondrial Membranes
3.
Nat Chem Biol ; 17(4): 465-476, 2021 04.
Article in English | MEDLINE | ID: mdl-33542532

ABSTRACT

Ferroptosis, triggered by discoordination of iron, thiols and lipids, leads to the accumulation of 15-hydroperoxy (Hp)-arachidonoyl-phosphatidylethanolamine (15-HpETE-PE), generated by complexes of 15-lipoxygenase (15-LOX) and a scaffold protein, phosphatidylethanolamine (PE)-binding protein (PEBP)1. As the Ca2+-independent phospholipase A2ß (iPLA2ß, PLA2G6 or PNPLA9 gene) can preferentially hydrolyze peroxidized phospholipids, it may eliminate the ferroptotic 15-HpETE-PE death signal. Here, we demonstrate that by hydrolyzing 15-HpETE-PE, iPLA2ß averts ferroptosis, whereas its genetic or pharmacological inactivation sensitizes cells to ferroptosis. Given that PLA2G6 mutations relate to neurodegeneration, we examined fibroblasts from a patient with a Parkinson's disease (PD)-associated mutation (fPDR747W) and found selectively decreased 15-HpETE-PE-hydrolyzing activity, 15-HpETE-PE accumulation and elevated sensitivity to ferroptosis. CRISPR-Cas9-engineered Pnpla9R748W/R748W mice exhibited progressive parkinsonian motor deficits and 15-HpETE-PE accumulation. Elevated 15-HpETE-PE levels were also detected in midbrains of rotenone-infused parkinsonian rats and α-synuclein-mutant SncaA53T mice, with decreased iPLA2ß expression and a PD-relevant phenotype. Thus, iPLA2ß is a new ferroptosis regulator, and its mutations may be implicated in PD pathogenesis.


Subject(s)
Ferroptosis/physiology , Group VI Phospholipases A2/metabolism , Animals , Arachidonate 15-Lipoxygenase/metabolism , Disease Models, Animal , Female , Group VI Phospholipases A2/physiology , Humans , Iron/metabolism , Leukotrienes/metabolism , Lipid Metabolism/physiology , Lipid Peroxides/metabolism , Lipids/physiology , Male , Mice , Mice, Inbred C57BL , Oxidation-Reduction , Parkinson Disease/metabolism , Phosphatidylethanolamine Binding Protein/metabolism , Phospholipases/metabolism , Phospholipids/metabolism , Rats , Rats, Inbred Lew
4.
J Cardiovasc Pharmacol ; 80(1): 158-170, 2022 07 01.
Article in English | MEDLINE | ID: mdl-35500215

ABSTRACT

ABSTRACT: Limited treatments are available for alleviating heart remodeling in postmenopausal hypertension. The cardioprotective effect of naoxintong (NXT) has been widely accepted. This study aimed to explore the effects of NXT on pathological heart remodeling in a postmenopausal hypertension mouse model in vivo and H9c2 cardiomyocytes in vitro. In vivo, ovariectomy combined with chronic angiotensin II infusion was used to establish the postmenopausal hypertension animal model. NXT significantly ameliorated cardiac remodeling as indicated by a reduced ratio of heart weight/body weight and left ventricle weight/body weight, left ventricular wall thickness, diameter of cardiomyocytes, and collagen deposition in the heart. NXT also significantly increased the expression of estrogen receptors (ERs) and downregulated the expression of nicotinamide adenine dinucleotide phosphate oxidase 2 (Nox2). In vitro, NXT treatment greatly suppressed angiotensin II-induced cardiac hypertrophy, cardiac fibrosis, and excessive oxidative stress as proven by reducing the diameter of H9c2 cardiomyocytes, expression of hypertrophy and fibrosis markers, intracellular reactive oxygen species, and oxidative enzymes. Mechanistically, NXT significantly upregulated the expression of ERs, which activated the Nrf2/HO-1 signaling pathway and inhibited the phosphorylation of the p38α pathway. Collectively, the results indicated that NXT administration might attenuate cardiac remodeling through upregulating the expression of ERs, which activated the Nrf2/HO-1 signaling pathway, inhibited the phosphorylation of the p38α signaling pathway, and reduced oxidative stress.


Subject(s)
Hypertension , NF-E2-Related Factor 2 , Angiotensin II/metabolism , Animals , Body Weight , Drugs, Chinese Herbal , Female , Fibrosis , Heme Oxygenase-1/metabolism , Hypertension/chemically induced , Hypertension/drug therapy , Hypertension/metabolism , Membrane Proteins/metabolism , Mice , Myocytes, Cardiac/metabolism , NF-E2-Related Factor 2/metabolism , Oxidative Stress , Postmenopause , Receptors, Estrogen/metabolism , Signal Transduction , Ventricular Remodeling
5.
Nat Chem Biol ; 13(1): 91-98, 2017 Jan.
Article in English | MEDLINE | ID: mdl-27842070

ABSTRACT

Ferroptosis is a form of regulated necrotic cell death controlled by glutathione peroxidase 4 (GPX4). At present, mechanisms that could predict sensitivity and/or resistance and that may be exploited to modulate ferroptosis are needed. We applied two independent approaches-a genome-wide CRISPR-based genetic screen and microarray analysis of ferroptosis-resistant cell lines-to uncover acyl-CoA synthetase long-chain family member 4 (ACSL4) as an essential component for ferroptosis execution. Specifically, Gpx4-Acsl4 double-knockout cells showed marked resistance to ferroptosis. Mechanistically, ACSL4 enriched cellular membranes with long polyunsaturated ω6 fatty acids. Moreover, ACSL4 was preferentially expressed in a panel of basal-like breast cancer cell lines and predicted their sensitivity to ferroptosis. Pharmacological targeting of ACSL4 with thiazolidinediones, a class of antidiabetic compound, ameliorated tissue demise in a mouse model of ferroptosis, suggesting that ACSL4 inhibition is a viable therapeutic approach to preventing ferroptosis-related diseases.


Subject(s)
Apoptosis , Coenzyme A Ligases/metabolism , Glutathione Peroxidase/metabolism , Mammary Neoplasms, Experimental/metabolism , Animals , Cell Death/drug effects , Cell Line , Cell Survival/drug effects , Coenzyme A Ligases/antagonists & inhibitors , Coenzyme A Ligases/deficiency , Female , Glutathione Peroxidase/deficiency , Humans , Hypoglycemic Agents/pharmacology , Mammary Neoplasms, Experimental/drug therapy , Mammary Neoplasms, Experimental/pathology , Mice , Mice, Knockout , Necrosis , Phospholipid Hydroperoxide Glutathione Peroxidase , Thiazolidinediones/pharmacology
6.
Nat Chem Biol ; 13(1): 81-90, 2017 Jan.
Article in English | MEDLINE | ID: mdl-27842066

ABSTRACT

Enigmatic lipid peroxidation products have been claimed as the proximate executioners of ferroptosis-a specialized death program triggered by insufficiency of glutathione peroxidase 4 (GPX4). Using quantitative redox lipidomics, reverse genetics, bioinformatics and systems biology, we discovered that ferroptosis involves a highly organized oxygenation center, wherein oxidation in endoplasmic-reticulum-associated compartments occurs on only one class of phospholipids (phosphatidylethanolamines (PEs)) and is specific toward two fatty acyls-arachidonoyl (AA) and adrenoyl (AdA). Suppression of AA or AdA esterification into PE by genetic or pharmacological inhibition of acyl-CoA synthase 4 (ACSL4) acts as a specific antiferroptotic rescue pathway. Lipoxygenase (LOX) generates doubly and triply-oxygenated (15-hydroperoxy)-diacylated PE species, which act as death signals, and tocopherols and tocotrienols (vitamin E) suppress LOX and protect against ferroptosis, suggesting a homeostatic physiological role for vitamin E. This oxidative PE death pathway may also represent a target for drug discovery.


Subject(s)
Arachidonic Acid/metabolism , Fatty Acids, Unsaturated/metabolism , Phospholipids/metabolism , Animals , Arachidonic Acid/antagonists & inhibitors , Cell Death/drug effects , Cell Line , Coenzyme A Ligases/antagonists & inhibitors , Coenzyme A Ligases/deficiency , Coenzyme A Ligases/metabolism , Fatty Acids, Unsaturated/antagonists & inhibitors , Female , Male , Mice , Mice, Inbred C57BL , Mice, Knockout
7.
J Am Chem Soc ; 140(51): 17835-17839, 2018 12 26.
Article in English | MEDLINE | ID: mdl-30525572

ABSTRACT

sn2-15-Hydroperoxy-eicasotetraenoyl-phosphatidylethanolamines ( sn2-15-HpETE-PE) generated by mammalian 15-lipoxygenase/phosphatidylethanolamine binding protein-1 (15-LO/PEBP1) complex is a death signal in a recently identified type of programmed cell demise, ferroptosis. How the enzymatic complex selects sn2-ETE-PE as the substrate among 1 of ∼100 total oxidizable membrane PUFA phospholipids is a central, yet unresolved question. To unearth the highly selective and specific mechanisms of catalytic competence, we used a combination of redox lipidomics, mutational and computational structural analysis to show they stem from (i) reactivity toward readily accessible hexagonally organized membrane sn2-ETE-PEs, (ii) relative preponderance of sn2-ETE-PE species vs other sn2-ETE-PLs, and (iii) allosteric modification of the enzyme in the complex with PEBP1. This emphasizes the role of enzymatic vs random stochastic free radical reactions in ferroptotic death signaling.


Subject(s)
Arachidonate 15-Lipoxygenase/metabolism , Cell Death/physiology , Phosphatidylethanolamines/metabolism , Animals , Arachidonate 15-Lipoxygenase/chemistry , Catalysis , Cell Line , Mice , Mutation , Oxidation-Reduction , Phosphatidylethanolamine Binding Protein/genetics , Phosphatidylethanolamine Binding Protein/metabolism , Phosphatidylethanolamines/chemistry , Substrate Specificity
8.
Mol Biol Rep ; 43(1): 31-9, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26671656

ABSTRACT

The rebuilding of the connective tissue during wound healing requires the recruitment of fibroblasts to the wound area as well as reentry of quiescent fibroblasts to the proliferative cycle. Whether this process can be modulated by a small molecular weight thiol antioxidant N-acetyl-L-cysteine (NAC) was tested in normal human skin fibroblasts (NHFs) using a uni-directional wound healing assay. NAC treated cells demonstrated a decreased migration rate but increased number of proliferating cells recruited into the wound area post wounding. Fifteen day quiescent control and NAC treated NHFs were re-plated at a lower density and cell numbers counted at different days post-plating. Interestingly, NAC treated cells exhibited increased cellular proliferation indicated by both decreased cell population doubling time and increased S phase cells. NAC treated cells demonstrated decreased steady state levels of reactive oxygen species as well as increased protein and activity levels of manganese superoxide dismutase (MnSOD). NAC treatment failed to induce proliferation in quiescent cells lacking MnSOD expression. These results demonstrate that NAC enhanced the recruitment of quiescent NHFs into proliferation cycle during wound healing. Our results also suggest that the wound healing properties of NAC might be due to its ability to induce and enhance MnSOD expression and activity. Altogether, these findings suggest NAC might be potentially developed as a dietary intervention to improve tissue injury in animals and humans.


Subject(s)
Acetylcysteine/pharmacology , Fibroblasts/drug effects , Superoxide Dismutase/metabolism , Wound Healing/drug effects , Animals , Antioxidants/pharmacology , Cell Cycle/drug effects , Cell Proliferation/drug effects , Fibroblasts/cytology , Fibroblasts/metabolism , Humans , Mice , Mice, Knockout , Oxidation-Reduction , Reactive Oxygen Species/metabolism
9.
J Biol Chem ; 289(47): 32488-98, 2014 Nov 21.
Article in English | MEDLINE | ID: mdl-25278024

ABSTRACT

Cytochrome c is a multifunctional hemoprotein in the mitochondrial intermembrane space whereby its participation in electron shuttling between respiratory complexes III and IV is alternative to its role in apoptosis as a peroxidase activated by interaction with cardiolipin (CL), and resulting in selective CL peroxidation. The switch from electron transfer to peroxidase function requires partial unfolding of the protein upon binding of CL, whose specific features combine negative charges of the two phosphate groups with four hydrophobic fatty acid residues. Assuming that other endogenous small molecule ligands with a hydrophobic chain and a negatively charged functionality may activate cytochrome c into a peroxidase, we investigated two hydrophobic anionic analogues of vitamin E, α-tocopherol succinate (α-TOS) and α-tocopherol phosphate (α-TOP), as potential inducers of peroxidase activity of cytochrome c. NMR studies and computational modeling indicate that they interact with cytochrome c at similar sites previously proposed for CL. Absorption spectroscopy showed that both analogues effectively disrupt the Fe-S(Met(80)) bond associated with unfolding of cytochrome c. We found that α-TOS and α-TOP stimulate peroxidase activity of cytochrome c. Enhanced peroxidase activity was also observed in isolated rat liver mitochondria incubated with α-TOS and tBOOH. A mitochondria-targeted derivative of TOS, triphenylphosphonium-TOS (mito-VES), was more efficient in inducing H2O2-dependent apoptosis in mouse embryonic cytochrome c(+/+) cells than in cytochrome c(-/-) cells. Essential for execution of the apoptotic program peroxidase activation of cytochrome c by α-TOS may contribute to its known anti-cancer pharmacological activity.


Subject(s)
Cytochromes c/chemistry , Peroxidase/chemistry , alpha-Tocopherol/analogs & derivatives , alpha-Tocopherol/chemistry , Animals , Apoptosis/drug effects , Apoptosis/genetics , Binding Sites/genetics , Cell Line , Cytochromes c/genetics , Cytochromes c/metabolism , Enzyme Activation/drug effects , Horses , Hydrophobic and Hydrophilic Interactions , Magnetic Resonance Spectroscopy , Male , Mice, Knockout , Models, Molecular , Molecular Structure , Peroxidase/metabolism , Protein Binding , Protein Structure, Tertiary , Spectrophotometry , Vitamins/chemistry , Vitamins/metabolism , Vitamins/pharmacology , alpha-Tocopherol/pharmacology
10.
J Cardiovasc Transl Res ; 15(5): 971-984, 2022 10.
Article in English | MEDLINE | ID: mdl-35478454

ABSTRACT

Abnormal phenotype switch in vascular smooth muscle cells (VSMCs) plays an important role in the initiation and progression of vascular proliferative diseases. Annexin A2 (ANXA2), related to the pro-inflammatory response, contributes to the proliferation and migration of VSMCs. This study explored the mechanisms involved in the regulation of VSMC phenotype modulation via ANXA2. The results revealed that ANXA2 promotes the phosphorylation of p65 and co-translocates with p65 into the nucleus, resulting in VSMC proliferation, migration, and dedifferentiation. Based on bioinformatics predictions and RNA immunoprecipitation assays, LINC00281 was confirmed to be an upstream regulator of ANXA2. Taken together, ANXA2, which is negatively regulated by the long noncoding RNA (lncRNA) LINC00281, has significant importance in the regulation of VSMC proliferation, migration, and phenotype switching via the nuclear factor-kappa B (NF-кB) p65 signaling pathway. This indicates that the lncRNA LINC00281/ANXA2/NF-кB p65 signaling pathway might be a new therapeutic target for vascular proliferative diseases.


Subject(s)
Annexin A2 , RNA, Long Noncoding , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Muscle, Smooth, Vascular/metabolism , NF-kappa B/genetics , NF-kappa B/metabolism , Annexin A2/genetics , Annexin A2/metabolism , Cell Proliferation , Cell Movement , Myocytes, Smooth Muscle/metabolism , Signal Transduction , Phenotype , Cells, Cultured
11.
J Hypertens ; 40(4): 819-834, 2022 04 01.
Article in English | MEDLINE | ID: mdl-35142739

ABSTRACT

BACKGROUND: CACNA1D gene, which encodes the α1 subunit of the Cav1.3 L-type calcium channel effectively regulates intracellular Ca2+ stability. In recent years, clinical studies have shown that the CACNA1D polymorphisms were associated with hypertension. OBJECTIVE: The purpose of this study was to evaluate the effects of CACNA1D exon mutation on blood pressure (BP) in Sprague-Dawley rats. METHODS: The rats with CACNA1D p.D307G, CACNA1D p.V936I or CACNA1D p.R1516Q were constructed using CRISPR-Cas9 technology. SBP measurements of rats were taken for 32 weeks. Tissue morphology of rats and vasoactive substances in serum was tested. Furthermore, the effects of L-type calcium channel blocker isradipine and endothelin-1 (ET-1) inhibitor BQ-123 on BP of double mutation rats (CACNA1D p.D307G/p.R1516Q) were tested. Then we examined the effects of CACNA1D gene mutation on gene expression in human umbilical vein endothelial cells (HUVECs) and vascular smooth muscle cells (VSMCs). RESULTS: Elevated SBP and increased circulating ET-1 was observed in CACNA1D p.D307G mutant rats. Morphological assessments showed that the vascular, cardiac and renal remodeling could also be observed in rats with p.D307G mutant. Cav1.3 protein expression and calcineurin phosphatase activity in VSMCs of rats with CACNA1D p.D307G were increased in vitro, and the vascular ring tension test of mesenteric grade 3 arteries in CACNA1D p.D307G rats were increased in vivo. Furthermore, ET-1 expression were increased in isolated primary aortic endothelial cells in p.D307G mutant rats and transfected p.D307G mutant HUVECs. Finally, double heterozygosity rats with CACNA1D p.D307G/p.R1516Q or CACNA1D p.D307G/p.V936I further accelerated the rise of SBP compared with p.D307G mutation rats, and isradipine and BQ-123 reduced BP to the same extent in CACNA1D p.D307G/p.R1516Q rats. CONCLUSION: CACNA1D gene is key players in the regulation of blood pressure. CACNA1D mutation rat may be a new hypertension animal model.


Subject(s)
Calcium Channels, L-Type , Endothelial Cells , Animals , Blood Pressure/genetics , Calcium Channels, L-Type/genetics , Calcium Channels, L-Type/metabolism , Endothelial Cells/metabolism , Exons/genetics , Humans , Mutation , Rats , Rats, Sprague-Dawley
12.
Redox Biol ; 50: 102232, 2022 04.
Article in English | MEDLINE | ID: mdl-35101798

ABSTRACT

Ferroptosis and necroptosis are two pro-inflammatory cell death programs contributing to major pathologies and their inhibition has gained attention to treat a wide range of disease states. Necroptosis relies on activation of RIP1 and RIP3 kinases. Ferroptosis is triggered by oxidation of polyunsaturated phosphatidylethanolamines (PUFA-PE) by complexes of 15-Lipoxygenase (15LOX) with phosphatidylethanolamine-binding protein 1 (PEBP1). The latter, also known as RAF kinase inhibitory protein, displays promiscuity towards multiple proteins. In this study we show that RIP3 K51A kinase inactive mice have increased ferroptotic burden and worse outcome after irradiation and brain trauma rescued by anti-ferroptotic compounds Liproxstatin-1 and Ferrostatin 16-86. Given structural homology between RAF and RIP3, we hypothesized that PEBP1 acts as a necroptosis-to-ferroptosis switch interacting with either RIP3 or 15LOX. Using genetic, biochemical, redox lipidomics and computational approaches, we uncovered that PEBP1 complexes with RIP3 and inhibits necroptosis. Elevated expression combined with higher affinity enables 15LOX to pilfer PEBP1 from RIP3, thereby promoting PUFA-PE oxidation and ferroptosis which sensitizes Rip3K51A/K51A kinase-deficient mice to total body irradiation and brain trauma. This newly unearthed PEBP1/15LOX-driven mechanism, along with previously established switch between necroptosis and apoptosis, can serve multiple and diverse cell death regulatory functions across various human disease states.


Subject(s)
Apoptosis , Ferroptosis , Animals , Cell Death , Mice , Necrosis , Oxidation-Reduction , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism
13.
Br J Nutr ; 106(1): 87-95, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21324214

ABSTRACT

The objective of the present study was to determine whether a mitochondria-targeted vitamin E derivative (MitoVit E) would affect certain mitochondrial parameters, as well as systemic oxidative stress. A total of sixty-four mice were fed a high-fat (HF) diet for 5 weeks. They were then switched to either a low-fat (LF) or a medium-fat (MF) diet, and administered orally with MitoVit E (40 mg MitoVit E/kg body weight) or drug vehicle (10 % (v/v) ethanol in 0·9 % (w/v) NaCl solution), every other day for 5 weeks. Mitochondrial ATP and H(2)O(2) production rates in both the liver and the gastrocnemius were not affected by MitoVit E administration in either LF or MF diet-fed mice. However, the number and average size of the subsarcolemmal mitochondria, but not the intermyofibrillar mitochondria, from the soleus muscle were significantly higher in the MF group receiving MitoVit E (MF-E) than in the MF group receiving vehicle only (MF-C). After the mice were switched from the HF diet to the four dietary treatments (LF-C, LF-E, MF-C and MF-E), the decrease in urinary isoprostane concentration was significantly greater in the LF-E group than in the other three groups during the whole study (weeks 6-10). In addition, MitoVit E significantly increased plasma superoxide dismutase (SOD) activity in the MF diet-fed group without affecting plasma glutathione peroxidase activity or H(2)O(2) levels. Overall, these data suggest that MitoVit E affects subsarcolemmal mitochondrial density and systemic oxidative stress parameters such as plasma SOD activity and urinary isoprostane concentration.


Subject(s)
Drug Delivery Systems , Mitochondria, Liver/drug effects , Mitochondria, Muscle/drug effects , Organophosphorus Compounds/pharmacology , Oxidative Stress/drug effects , Adenosine Triphosphate/metabolism , Animals , Body Weight/drug effects , Dietary Supplements , Hydrogen Peroxide/metabolism , Isoprostanes/urine , Liver/metabolism , Male , Mice , Mice, Inbred C57BL , Mitochondria, Liver/metabolism , Mitochondria, Muscle/metabolism , Oxidation-Reduction , Ubiquinone/pharmacology
14.
J Nutr ; 140(8): 1425-31, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20554905

ABSTRACT

Our objective in this study was to determine whether a mitochondria-targeted vitamin E derivative (MitoVit E) would decrease oxidative stress and associated obesity by preventing a previously proposed aconitase inhibition cascade. Sixty-four mice were fed a high-fat (HF) diet for 5 wk. They were then switched to either a low-fat (LF) or a medium-fat (MF) diet and gavaged with MitoVit E (40 mg MitoVit E x kg body weight(-1)) or drug vehicle (10% ethanol in 0.9% NaCl solution) every other day for 5 wk. Epididymal fat weight, as well as liver lipid and remaining carcass lipid, were significantly lower in the MF group receiving MitoVit E (MF-E) than in the MF group receiving vehicle only (MF-C). Liver mitochondrial H(2)O(2) production and the protein carbonyl level were also significantly lower in MF-E than in MF-C mice. In contrast, none of the biochemical variables (aconitase activity, ATP and H(2)O(2) production, and protein carbonyl level) in the muscle mitochondria were modified by MitoVit E in either MF or LF groups. Expression of acetyl-CoA carboxylase and fatty acid synthase in both liver and adipose tissue of MF groups was not affected by MitoVit E. However, expression of carnitine palmitoyltransferase 1a in the liver and uncoupling protein 2 in adipose tissue were significantly enhanced by MitoVit E in both LF and MF groups. In conclusion, MitoVit E attenuates hepatic oxidative stress and inhibits fat deposition in mice but not through alleviation of the aconitase inhibition cascade.


Subject(s)
Adipose Tissue/metabolism , Liver/metabolism , Organophosphorus Compounds/pharmacology , Oxidative Stress/drug effects , Aconitate Hydratase/antagonists & inhibitors , Aconitate Hydratase/genetics , Adenosine Triphosphate/metabolism , Adipose Tissue/anatomy & histology , Animals , Body Composition , Body Weight , Diet , Dietary Fats/administration & dosage , Eating , Fatty Acid Synthases/genetics , Gene Expression/drug effects , Hydrogen Peroxide/metabolism , Lipids/analysis , Liver/chemistry , Liver/drug effects , Male , Mice , Mice, Inbred C57BL , Mitochondria, Liver/enzymology , Mitochondria, Liver/metabolism , Mitochondria, Muscle/enzymology , Mitochondria, Muscle/metabolism , Muscle, Skeletal/ultrastructure , Obesity/prevention & control , Organ Size/drug effects , Organophosphorus Compounds/administration & dosage , RNA, Messenger/analysis , Ubiquinone/administration & dosage , Ubiquinone/pharmacology
15.
Antioxid Redox Signal ; 29(13): 1333-1358, 2018 11 01.
Article in English | MEDLINE | ID: mdl-28835115

ABSTRACT

SIGNIFICANCE: Oxygenated polyunsaturated lipids are known to play multi-functional roles as essential signals coordinating metabolism and physiology. Among them are well-studied eicosanoids and docosanoids that are generated via phospholipase A2 hydrolysis of membrane phospholipids and subsequent oxygenation of free polyunsaturated fatty acids (PUFA) by cyclooxygenases and lipoxygenases. Recent Advances: There is an emerging understanding that oxygenated PUFA-phospholipids also represent a rich signaling language with yet-to-be-deciphered details of the execution machinery-oxygenating enzymes, regulators, and receptors. Both free and esterified oxygenated PUFA signals are generated in cells, and their cross-talk and inter-conversion through the de-acylation/re-acylation reactions is not sufficiently explored. CRITICAL ISSUES: Here, we review recent data related to oxygenated phospholipids as important damage signals that trigger programmed cell death pathways to eliminate irreparably injured cells and preserve the health of multicellular environments. We discuss the mechanisms underlying the trans-membrane redistribution and generation of oxygenated cardiolipins in mitochondria by cytochrome c as pro-apoptotic signals. We also consider the role of oxygenated phosphatidylethanolamines as proximate pro-ferroptotic signals. FUTURE DIRECTIONS: We highlight the importance of sequential processes of phospholipid oxygenation and signaling in disease contexts as opportunities to use their regulatory mechanisms for the identification of new therapeutic targets.


Subject(s)
Oxygen/metabolism , Phospholipids/metabolism , Signal Transduction , Animals , Cell Differentiation , Humans , Oxidation-Reduction
16.
J Clin Invest ; 128(10): 4639-4653, 2018 10 01.
Article in English | MEDLINE | ID: mdl-30198910

ABSTRACT

Ferroptosis is a death program executed via selective oxidation of arachidonic acid-phosphatidylethanolamines (AA-PE) by 15-lipoxygenases. In mammalian cells and tissues, ferroptosis has been pathogenically associated with brain, kidney, and liver injury/diseases. We discovered that a prokaryotic bacterium, Pseudomonas aeruginosa, that does not contain AA-PE can express lipoxygenase (pLoxA), oxidize host AA-PE to 15-hydroperoxy-AA-PE (15-HOO-AA-PE), and trigger ferroptosis in human bronchial epithelial cells. Induction of ferroptosis by clinical P. aeruginosa isolates from patients with persistent lower respiratory tract infections was dependent on the level and enzymatic activity of pLoxA. Redox phospholipidomics revealed elevated levels of oxidized AA-PE in airway tissues from patients with cystic fibrosis (CF) but not with emphysema or CF without P. aeruginosa. We believe that the evolutionarily conserved mechanism of pLoxA-driven ferroptosis may represent a potential therapeutic target against P. aeruginosa-associated diseases such as CF and persistent lower respiratory tract infections.


Subject(s)
Apoptosis , Cystic Fibrosis/metabolism , Phosphatidylethanolamines/metabolism , Pseudomonas Infections/metabolism , Pseudomonas aeruginosa/metabolism , Respiratory Mucosa/metabolism , Respiratory Tract Infections/metabolism , Cell Line , Cystic Fibrosis/microbiology , Cystic Fibrosis/pathology , Epithelial Cells/metabolism , Epithelial Cells/microbiology , Epithelial Cells/pathology , Humans , Pseudomonas Infections/pathology , Pseudomonas aeruginosa/pathogenicity , Respiratory Mucosa/microbiology , Respiratory Mucosa/physiology , Respiratory Tract Infections/microbiology , Respiratory Tract Infections/pathology
17.
ACS Chem Biol ; 11(2): 530-40, 2016 Feb 19.
Article in English | MEDLINE | ID: mdl-26697918

ABSTRACT

While opto-genetics has proven to have tremendous value in revealing the functions of the macromolecular machinery in cells, it is not amenable to exploration of small molecules such as phospholipids (PLs). Here, we describe a redox opto-lipidomics approach based on a combination of high affinity light-sensitive ligands to specific PLs in mitochondria with LC-MS based redox lipidomics/bioinformatics analysis for the characterization of pro-apoptotic lipid signals. We identified the formation of mono-oxygenated derivatives of C18:2-containing cardiolipins (CLs) in mitochondria after the exposure of 10-nonylacridine orange bromide (NAO)-loaded cells to light. We ascertained that these signals emerge as an immediate opto-lipidomics response, but they decay long before the commencement of apoptotic cell death. We found that a protonophoric uncoupler caused depolarization of mitochondria and prevented the mitochondrial accumulation of NAO, inhibited the formation of C18:2-CL oxidation product,s and protected cells from death. Redox opto-lipidomics extends the power of opto-biologic protocols to studies of small PL molecules resilient to opto-genetic manipulations.


Subject(s)
Apoptosis , Cardiolipins/metabolism , Mitochondria/metabolism , Oxygen/metabolism , Acridine Orange/analogs & derivatives , Acridine Orange/metabolism , Apoptosis/radiation effects , Cardiolipins/chemistry , Coloring Agents/metabolism , Computational Biology , HeLa Cells , Humans , Light , Mitochondria/chemistry , Mitochondria/radiation effects , Oxidation-Reduction , Oxygen/chemistry
18.
Food Funct ; 6(8): 2578-87, 2015 Aug.
Article in English | MEDLINE | ID: mdl-26114447

ABSTRACT

The beneficial effect of caffeine-containing food on non-alcoholic fatty liver disease (NAFLD) has been widely reported. The aim of this study was to explore the effect of caffeine on hepatic steatosis. C57BL/6 mice were randomly assigned to a normal diet or a high energy diet (HED). Caffeine was given to HED mice by oral gavage. Body weights, lipids in the liver and liver damage were measured. Meanwhile, cAMP, SIRT3 or AMPK inhibitors were treated respectively before incubation with caffeine in oleate-treated HepG2 cells. SIRT3 was further silenced by siRNA to confirm the results. Caffeine significantly decreased the mass of fat tissues, lipids, ALT and AST levels in the liver of HED-treated mice. Caffeine increased the transformation of ADP to ATP and activated the cAMP/CREB/SIRT3/AMPK/ACC pathway in the liver. Nile red staining demonstrated that suppression of cAMP, SIRT3 or AMPK in oleate-treated HepG2 cells counteracted the effect of caffeine. Moreover, knocking down SIRT3 could down-regulate AMPK and ACC phosphorylation by caffeine. These results demonstrate that caffeine could improve HED-induced hepatic steatosis by promoting lipid metabolism via the cAMP/CREB/SIRT3/AMPK/ACC pathway. SIRT3 functioned as a molecular bridge connecting caffeine and lipid metabolism.


Subject(s)
Caffeine/administration & dosage , Lipid Metabolism , Non-alcoholic Fatty Liver Disease/drug therapy , Sirtuin 3/metabolism , AMP-Activated Protein Kinases/genetics , AMP-Activated Protein Kinases/metabolism , Acetyl-CoA Carboxylase/genetics , Acetyl-CoA Carboxylase/metabolism , Animals , CREB-Binding Protein/genetics , CREB-Binding Protein/metabolism , Diet, High-Fat/adverse effects , Humans , Liver/drug effects , Liver/enzymology , Liver/metabolism , Male , Mice , Mice, Inbred C57BL , Non-alcoholic Fatty Liver Disease/enzymology , Non-alcoholic Fatty Liver Disease/genetics , Non-alcoholic Fatty Liver Disease/metabolism , Sirtuin 3/genetics
19.
Antioxid Redox Signal ; 20(9): 1423-35, 2014 Mar 20.
Article in English | MEDLINE | ID: mdl-23919724

ABSTRACT

AIMS: This study determined whether acute radiation-induced liver injury seen in Sirtuin3(-/-) mice after exposure to Cs-137 γ-rays was mediated by superoxide anion (O2(•-)). RESULTS: Male wild-type (WT) and SIRT3(-/-) mice were given 2×2 Gy whole-body radiation doses separated by 24 h and livers were harvested 20 h after the second dose. Ex vivo measurements in fresh frozen liver sections demonstrated 50% increases in dihydroethidium oxidation from SIRT3(-/-) animals, relative to WT animals, before irradiation, but this increase was not detected 20 h after radiation exposure. In addition, irradiated livers from SIRT3(-/-) animals showed significant hydropic degeneration, loss of MitoTracker Green FM staining, increased immunohistochemical staining for 3-nitrotyrosine, loss of Ki67 staining, and increased mitochondrial localization of p53. These parameters of radiation-induced injury were significantly attenuated by an intraperitoneal injection of 2 mg/kg of the highly specific superoxide dismutase mimic, GC4401, 30 min before each fraction. INNOVATION: Sirtuin 3 (SIRT3) is believed to regulate mitochondrial oxidative metabolism and antioxidant defenses in response to acute radiation-induced liver injury. This work provides strong evidence for the causal role of O2(•-) in the liver injury process initiated by whole-body irradiation in SIRT3(-/-) mice. CONCLUSION: These results support the hypothesis that O2(•-) mediates acute liver injury in SIRT3(-/-) animals exposed to whole-body γ-radiation and suggest that GC4401 could be used as a radio-protective compound in vivo.


Subject(s)
Chemical and Drug Induced Liver Injury/genetics , Chemical and Drug Induced Liver Injury/metabolism , Sirtuin 3/deficiency , Superoxides/metabolism , Animals , Cell Proliferation/drug effects , Cell Proliferation/radiation effects , Chemical and Drug Induced Liver Injury/pathology , Electron Transport/radiation effects , Enzyme Activation , Ethidium/analogs & derivatives , Ethidium/metabolism , Liver/metabolism , Liver/pathology , Male , Mice , Mice, Knockout , Mitochondria/drug effects , Mitochondria/metabolism , Mitochondria/radiation effects , Oxidation-Reduction , Protein Transport , Superoxide Dismutase/metabolism , Tumor Suppressor Protein p53/metabolism , Whole-Body Irradiation
20.
Redox Biol ; 2: 847-54, 2014.
Article in English | MEDLINE | ID: mdl-25009786

ABSTRACT

Retroviral transformation has been associated with pro-proliferative oncogenic signaling in human cells. The current study demonstrates that transduction of human breast carcinoma cells (MDA-MB231) with LXSN and QCXIP retroviral vectors causes significant increases in growth rate, clonogenic fraction, and aldehyde dehydrogenase-1 positive cells (ALDH1+), which is associated with increased steady-state levels of cancer stem cell populations. Furthermore, this retroviral-induced enhancement of cancer cell growth in vitro was also accompanied by a significant increase in xenograft tumor growth rate in vivo. The retroviral induced increases in cancer cell growth rate were partially inhibited by treatment with 100 U/ml polyethylene glycol-conjugated-(PEG)-superoxide dismutase and/or PEG-catalase. These results show that retroviral infection of MDA-MB231 human breast cancer cells is capable of enhancing cell proliferation and cancer stem cell populations as well as suggesting that modulation of reactive oxygen species-induced pro-survival signaling pathways may be involved in these effects.


Subject(s)
Breast Neoplasms/pathology , Isoenzymes/metabolism , Neoplastic Stem Cells/enzymology , Retinal Dehydrogenase/metabolism , Retroviridae/physiology , Aldehyde Dehydrogenase 1 Family , Animals , Breast Neoplasms/enzymology , Breast Neoplasms/virology , Catalase/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Female , Humans , Mice , Mice, Nude , Neoplastic Stem Cells/cytology , Neoplastic Stem Cells/virology , Polyethylene Glycols/pharmacology , Superoxide Dismutase/pharmacology , Transplantation, Heterologous
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