-
Journal of Controlled Release :... Feb 2023Ischemic stroke causes brain endothelial cell (BEC) death and damages tight junction integrity of the blood-brain barrier (BBB). We harnessed the innate mitochondrial...
Ischemic stroke causes brain endothelial cell (BEC) death and damages tight junction integrity of the blood-brain barrier (BBB). We harnessed the innate mitochondrial load of BEC-derived extracellular vesicles (EVs) and utilized mixtures of EV/exogenous 27 kDa heat shock protein (HSP27) as a one-two punch strategy to increase BEC survival (via EV mitochondria) and preserve their tight junction integrity (via HSP27 effects). We demonstrated that the medium-to-large (m/lEV) but not small EVs (sEV) transferred their mitochondrial load, that subsequently colocalized with the mitochondrial network of the recipient primary human BECs. Recipient BECs treated with m/lEVs showed increased relative ATP levels and mitochondrial function. To determine if the m/lEV-meditated increase in recipient BEC ATP levels was associated with m/lEV mitochondria, we isolated m/lEVs from donor BECs pre-treated with oligomycin A (OGM, mitochondria electron transport complex V inhibitor), referred to as OGM-m/lEVs. BECs treated with naïve m/lEVs showed a significant increase in ATP levels compared to untreated OGD cells, OGM-m/lEVs treated BECs showed a loss of ATP levels suggesting that the m/lEV-mediated increase in ATP levels is likely a function of their innate mitochondrial load. In contrast, sEV-mediated ATP increases were not affected by inhibition of mitochondrial function in the donor BECs. Intravenously administered m/lEVs showed a reduction in brain infarct sizes compared to vehicle-injected mice in a mouse middle cerebral artery occlusion model of ischemic stroke. We formulated binary mixtures of human recombinant HSP27 protein with EVs: EV/HSP27 and ternary mixtures of HSP27 and EVs with a cationic polymer, poly (ethylene glycol)-b-poly (diethyltriamine): (PEG-DET/HSP27)/EV. (PEG-DET/HSP27)/EV and EV/HSP27 mixtures decreased the paracellular permeability of small and large molecular mass fluorescent tracers in oxygen glucose-deprived primary human BECs. This one-two punch approach to increase BEC metabolic function and tight junction integrity may be a promising strategy for BBB protection and prevention of long-term neurological dysfunction post-ischemic stroke.
Topics: Mice; Humans; Animals; HSP27 Heat-Shock Proteins; Brain; Blood-Brain Barrier; Stroke; Infarction, Middle Cerebral Artery; Heat-Shock Proteins; Ischemic Stroke; Mitochondria; Extracellular Vesicles; Adenosine Triphosphate
PubMed: 36642252
DOI: 10.1016/j.jconrel.2023.01.025 -
Cancer Science Apr 2023To meet cellular bioenergetic and biosynthetic demands, cancer cells remodel their metabolism to increase glycolytic flux, a phenomenon known as the Warburg effect and...
To meet cellular bioenergetic and biosynthetic demands, cancer cells remodel their metabolism to increase glycolytic flux, a phenomenon known as the Warburg effect and believed to contribute to cancer malignancy. Among glycolytic enzymes, phosphofructokinase-1 (PFK1) has been shown to act as a rate-limiting enzyme and to facilitate the Warburg effect in cancer cells. In this study, however, we found that decreased PFK1 activity did not affect cell survival or proliferation in cancer cells. This raised a question regarding the importance of PFK1 in malignancy. To gain insights into the role of PFK1 in cancer metabolism and the possibility of adopting it as a novel anticancer therapeutic target, we screened for genes that caused lethality when they were knocked down in the presence of tryptolinamide (TLAM), a PFK1 inhibitor. The screen revealed a synthetic chemical-genetic interaction between genes encoding subunits of ATP synthase (complex V) and TLAM. Indeed, after TLAM treatment, the sensitivity of HeLa cells to oligomycin A (OMA), an ATP synthase inhibitor, was 13,000 times higher than that of untreated cells. Furthermore, this sensitivity potentiation by TLAM treatment was recapitulated by genetic mutations of PFK1. By contrast, TLAM did not potentiate the sensitivity of normal fibroblast cell lines to OMA, possibly due to their reduced energy demands compared to cancer cells. We also showed that the PFK1-mediated glycolytic pathway can act as an energy reservoir. Selective potentiation of the efficacy of ATP synthase inhibitors by PFK1 inhibition may serve as a foundation for novel anticancer therapeutic strategies.
Topics: Humans; Early Detection of Cancer; Glycolysis; HeLa Cells; Neoplasms; Phosphofructokinase-1; RNA Interference; Adenosine Triphosphatases
PubMed: 36601784
DOI: 10.1111/cas.15713 -
Cells Dec 2022Mitochondrial dysfunction is central to breaking the barrier integrity of retinal endothelial cells (RECs) in various blinding eye diseases such as diabetic retinopathy...
Relative Importance of Different Elements of Mitochondrial Oxidative Phosphorylation in Maintaining the Barrier Integrity of Retinal Endothelial Cells: Implications for Vascular-Associated Retinal Diseases.
PURPOSE
Mitochondrial dysfunction is central to breaking the barrier integrity of retinal endothelial cells (RECs) in various blinding eye diseases such as diabetic retinopathy and retinopathy of prematurity. Therefore, we aimed to investigate the role of different mitochondrial constituents, specifically those of oxidative phosphorylation (OxPhos), in maintaining the barrier function of RECs.
METHODS
Electric cell-substrate impedance sensing (ECIS) technology was used to assess in real time the role of different mitochondrial components in the total impedance (Z) of human RECs (HRECs) and its components: capacitance (C) and the total resistance (R). HRECs were treated with specific mitochondrial inhibitors that target different steps in OxPhos: rotenone for complex I, oligomycin for complex V (ATP synthase), and FCCP for uncoupling OxPhos. Furthermore, data were modeled to investigate the effects of these inhibitors on the three parameters that govern the total resistance of cells: Cell-cell interactions (R), cell-matrix interactions (α), and cell membrane permeability (Cm).
RESULTS
Rotenone (1 µM) produced the greatest reduction in Z, followed by FCCP (1 µM), whereas no reduction in Z was observed after oligomycin (1 µM) treatment. We then further deconvoluted the effects of these inhibitors on the R, α, and C parameters. Rotenone (1 µM) completely abolished the resistance contribution of R, as the R became zero immediately after the treatment. Secondly, FCCP (1 µM) eliminated the resistance contribution of R only after 2.5 h and increased C without a significant effect on α. Lastly, of all the inhibitors used, oligomycin had the lowest impact on R, as evidenced by the fact that this value became similar to that of the control group at the end of the experiment without noticeable effects on C or α.
CONCLUSION
Our study demonstrates the differential roles of complex I, complex V, and OxPhos coupling in maintaining the barrier functionality of HRECs. We specifically showed that complex I is the most important component in regulating HREC barrier integrity. These observed differences are significant since they could serve as the basis for future pharmacological and gene expression studies aiming to improve the activity of complex I and thereby provide avenues for therapeutic modalities in endothelial-associated retinal diseases.
Topics: Infant, Newborn; Humans; Oxidative Phosphorylation; Rotenone; Endothelial Cells; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Mitochondria; Diabetic Retinopathy; Oligomycins
PubMed: 36552890
DOI: 10.3390/cells11244128 -
Toxics Dec 2022Industrial and consumer products, such as pesticides, lubricants, and cosmetics, can contain perfluorinated compounds (PFCs). Although many short-chain PFCs have been...
Industrial and consumer products, such as pesticides, lubricants, and cosmetics, can contain perfluorinated compounds (PFCs). Although many short-chain PFCs have been linked to physiological and behavioral changes in fish, there are limited data on longer-chain PFCs. The objective of this study was to determine the potential impact of perfluorotetradecanoic acid (PFTeDA) exposure on zebrafish () during early developmental stages. We measured several endpoints including gene expression, mitochondrial bioenergetics, and locomotor activity in zebrafish. Survival, timing of hatching, and deformity frequency were unaffected by PFTeDA at the concentrations tested (0.01, 0.1, 1, and 10 µM) over a 7-day exposure period. The expression levels of mitochondrial-related genes ( and ) and oxidative stress-related genes ( and ) were increased in larval fish with exposure to 10 µM PFTeDA; however, there was no change in oxidative respiration of embryos (i.e., basal respiration and oligomycin-induced ATP-linked respiration). Reactive oxygen species were reduced in larvae treated with 10 µM PFTeDA, coinciding with the increased transcription of antioxidant defense genes. Both the visual motor response test and light-dark preference test were conducted on 7 dpf larvae and yielded no significant findings. This study improves current knowledge regarding toxicity mechanisms for longer-chain PFCs such as PFTeDA.
PubMed: 36548609
DOI: 10.3390/toxics10120776 -
Journal of Biosciences 2022Eukaryotic cells contain multiple copies of mitochondrial DNA (mtDNA) in discrete organelles or as tubular networks throughout the cytoplasm. The mtDNA copy number is...
Eukaryotic cells contain multiple copies of mitochondrial DNA (mtDNA) in discrete organelles or as tubular networks throughout the cytoplasm. The mtDNA copy number is dynamically regulated by mitochondrial biogenesis and mitophagy processes. However, the conditions regulating mtDNA replication, an essential component of biogenesis, are unknown. We observed that short-term (2 h) treatment of rat myoblasts with oligomycin, a specific inhibitor of the mitochondrial F1F0 ATP synthase, resulted in stimulation of mtDNA synthesis from the O replication origin. This effect was abrogated by Compound C, an antagonist of the AMP-dependent protein kinase (AMPK), a universal intracellular energy sensor, and in AMPK-knockdown cells, indicating that mtDNA replication is regulated by AMPK under oxidative phosphorylation (OXPHOS)- deficient conditions. Using antibody decoration, enzymatically active AMPK, phosphorylated at T172 of the α1 subunit, was found to be located on the mitochondrial surface. Furthermore, oligomycin induced the compartmentalization of several mRNAs encoding OXPHOS components and mtDNA replication factors to mitochondria. Compartmentalization of mRNAs was inhibited by Compound C. We infer that AMPK is locally activated by inhibition of the F1F0 ATP synthase to stimulate association of mtDNA replication factor mRNAs, leading to stimulation of mtDNA synthesis. The findings have implications for the clonal expansion of OXPHOS-deficient mtDNA mutant mitochondria in human patients, with clinical consequences.
Topics: Humans; Rats; Animals; AMP-Activated Protein Kinases; RNA, Messenger; DNA, Mitochondrial; Oligomycins; Adenosine Triphosphate
PubMed: 36408541
DOI: No ID Found -
Experimental & Molecular Medicine Nov 2022Transarterial chemoembolization (TACE) is the first-line treatment for unresectable intermediate-stage hepatocellular carcinoma (HCC). It is of high clinical...
Transarterial chemoembolization (TACE) is the first-line treatment for unresectable intermediate-stage hepatocellular carcinoma (HCC). It is of high clinical significance to explore the synergistic effect of TACE with antiangiogenic inhibitors and the molecular mechanisms involved. This study determined that glucose, but not other analyzed nutrients, offered significant protection against cell death induced by sorafenib, as indicated by glucose deprivation sensitizing cells to sorafenib-induced cell death. Next, this synergistic effect was found to be specific to sorafenib, not to lenvatinib or the chemotherapeutic drugs cisplatin and doxorubicin. Mechanistically, sorafenib-induced mitophagy, as indicated by PINK1 accumulation, increased the phospho-poly-ubiquitination modification, accelerated mitochondrial membrane protein and mitochondrial DNA degradation, and increased the amount of mitochondrion-localized mKeima-Red engulfed by lysosomes. Among several E3 ubiquitin ligases tested, SIAH1 was found to be essential for inducing mitophagy; that is, SIAH1 silencing markedly repressed mitophagy and sensitized cells to sorafenib-induced death. Notably, the combined treatment of glucose restriction and sorafenib abolished ATP generation and mitophagy, which led to a high cell death rate. Oligomycin and antimycin, inhibitors of electron transport chain complexes, mimicked the synergistic effect of sorafenib with glucose restriction to promote cell death mediated via mitophagy inhibition. Finally, inhibition of the glucose transporter by canagliflozin (a clinically available drug used for type-II diabetes) effectively synergized with sorafenib to induce HCC cell death in vitro and to inhibit xenograft tumor growth in vivo. This study demonstrates that simultaneous treatment with sorafenib and glucose restriction is an effective approach to treat HCC, suggesting a promising combination strategy such as transarterial sorafenib-embolization (TASE) for the treatment of unresectable HCC.
Topics: Humans; Carcinoma, Hepatocellular; Sorafenib; Chemoembolization, Therapeutic; Liver Neoplasms; Mitophagy; Glucose; Niacinamide; Antineoplastic Agents
PubMed: 36385558
DOI: 10.1038/s12276-022-00878-x -
International Journal of Molecular... Oct 2022Trained immune responses, based on metabolic and epigenetic changes in innate immune cells, are de facto innate immune memory and, therefore, are of great interest in...
Trained immune responses, based on metabolic and epigenetic changes in innate immune cells, are de facto innate immune memory and, therefore, are of great interest in vaccine development. In previous studies, the recombinant fusion protein rFlaA:Betv1, combining the adjuvant and toll-like receptor (TLR)5-ligand flagellin (FlaA) and the major birch pollen allergen Bet v 1 into a single molecule, significantly suppressed allergic sensitization in vivo while also changing the metabolism of myeloid dendritic cells (mDCs). Within this study, the immune-metabolic effects of rFlaA:Betv1 during mDC activation were elucidated. In line with results for other well-characterized TLR-ligands, rFlaA:Betv1 increased glycolysis while suppressing oxidative phosphorylation to different extents, making rFlaA:Betv1 a suitable model to study the immune-metabolic effects of TLR-adjuvanted vaccines. In vitro pretreatment of mDCs with cerulenin (inhibitor of fatty acid biosynthesis) led to a decrease in both rFlaA:Betv1-induced anti-inflammatory cytokine Interleukin (IL) 10 and T helper cell type (TH) 1-related cytokine IL-12p70, while the pro-inflammatory cytokine IL 1β was unaffected. Interestingly, pretreatment with the glutaminase inhibitor BPTES resulted in an increase in IL-1β, but decreased IL-12p70 secretion while leaving IL-10 unchanged. Inhibition of the glycolytic enzyme hexokinase-2 by 2-deoxyglucose led to a decrease in all investigated cytokines (IL-10, IL-12p70, and IL-1β). Inhibitors of mitochondrial respiration had no effect on rFlaA:Betv1-induced IL-10 level, but either enhanced the secretion of IL-1β (oligomycin) or decreased IL-12p70 (antimycin A). In extracellular flux measurements, mDCs showed a strongly enhanced glycolysis after rFlaA:Betv1 stimulation, which was slightly increased after respiratory shutdown using antimycin A. rFlaA:Betv1-stimulated mDCs secreted directly antimicrobial substances in a mTOR- and fatty acid metabolism-dependent manner. In co-cultures of rFlaA:Betv1-stimulated mDCs with CD4+ T cells, the suppression of Bet v 1-specific TH2 responses was shown to depend on fatty acid synthesis. The effector function of rFlaA:Betv1-activated mDCs mainly relies on glycolysis, with fatty acid synthesis also significantly contributing to rFlaA:Betv1-mediated cytokine secretion, the production of antimicrobial molecules, and the modulation of T cell responses.
Topics: Toll-Like Receptor 5; Allergens; Interleukin-10; Flagellin; Hexokinase; Glutaminase; Ligands; Antimycin A; Cerulenin; Dendritic Cells; Recombinant Proteins; Cytokines; Adjuvants, Immunologic; Vaccines; Recombinant Fusion Proteins; Glycolysis; TOR Serine-Threonine Kinases; Deoxyglucose; Oligomycins; Fatty Acids
PubMed: 36293550
DOI: 10.3390/ijms232012695 -
MicroLife 2022is a facultative intracellular pathogen that can replicate and disseminate in mammalian macrophages. In this study, we analyzed fungal proteins identified in murine...
is a facultative intracellular pathogen that can replicate and disseminate in mammalian macrophages. In this study, we analyzed fungal proteins identified in murine macrophage-like cells after infection with . To accomplish this, we developed a protocol to identify proteins released from cryptococcal cells inside macrophage-like cells; we identified 127 proteins of fungal origin in infected macrophage-like cells. Among the proteins identified was urease, a known virulence factor, and others such as transaldolase and phospholipase D, which have catalytic activities that could contribute to virulence. This method provides a straightforward methodology to study host-pathogen interactions. We chose to study further Yeast Oligomycin Resistance (Yor1), a relatively uncharacterized protein belonging to the large family of ATP binding cassette transporter (ABC transporters). These transporters belong to a large and ancient protein family found in all extant phyla. While ABC transporters have an enormous diversity of functions across varied species, in pathogenic fungi they are better studied as drug efflux pumps. Analysis of strains revealed defects in nonlytic exocytosis, capsule size, and dimensions of extracellular vesicles, when compared to wild-type strains. We detected no difference in growth rates and cell body size. Our results indicate that releases a large suite of proteins during macrophage infection, some of which can modulate fungal virulence and are likely to affect the fungal-macrophage interaction.
PubMed: 36247839
DOI: 10.1093/femsml/uqac015 -
International Journal of Molecular... Sep 2022Platelets produce inorganic polyphosphate (polyP) upon activation to stimulate blood coagulation. Some researchers have linked polyP metabolism to ATP production,...
Platelets produce inorganic polyphosphate (polyP) upon activation to stimulate blood coagulation. Some researchers have linked polyP metabolism to ATP production, although the metabolic linkage is yet to be elucidated. We found evidence for this possibility in our previous study on professional athletes (versus non-athletes), and proposed that the regulatory mechanism might be different for these two groups. To explore this aspect further, we investigated the effects of modulated ATP production on polyP levels. Blood samples were obtained from Japanese healthy, non-athletes in the presence of acid-citrate-dextrose. The platelets in the plasma were treated with oligomycin, rotenone, and GlutaMAX to modulate ATP production. PolyP level was quantified fluorometrically and visualized using 4',6-diamidino-2-phenylindole. Correlations between polyP and ATP or NADH were then calculated. Contrary to the hypothesis, inhibitors of ATP production increased polyP levels, whereas amino acid supplementation produced the opposite effect. In general, however, polyP levels were positively correlated with ATP levels and negatively correlated with NADH levels. Since platelets are metabolically active, they exhibit high levels of ATP turnover rate. Therefore, these findings suggest that ATP may be involved in polyP production in the resting platelets of non-athletes.
Topics: Adenosine Triphosphate; Amino Acids; Citrates; Glucose; Humans; NAD; Oligomycins; Polyphosphates; Rotenone
PubMed: 36232597
DOI: 10.3390/ijms231911293 -
Autophagy May 2023Macroautophagy/autophagy or mitophagy plays crucial roles in the maintenance of pancreatic β-cell function. PPP3/calcineurin can modulate the activity of TFEB, a master...
Macroautophagy/autophagy or mitophagy plays crucial roles in the maintenance of pancreatic β-cell function. PPP3/calcineurin can modulate the activity of TFEB, a master regulator of lysosomal biogenesis and autophagy gene expression, through dephosphorylation. We studied whether PPP3/calcineurin inhibitors can affect the mitophagy of pancreatic β-cells and pancreatic β-cell function employing FK506, an immunosuppressive drug against graft rejection. FK506 suppressed rotenone- or oligomycin+antimycin-A-induced mitophagy measured by Mito-Keima localization in acidic lysosomes or RFP-LC3 puncta colocalized with TOMM20 in INS-1 insulinoma cells. FK506 diminished nuclear translocation of TFEB after treatment with rotenone or oligomycin+antimycin A. Forced TFEB nuclear translocation by a constitutively active TFEB mutant transfection restored impaired mitophagy by FK506, suggesting the role of decreased TFEB nuclear translocation in FK506-mediated mitophagy impairment. Probably due to reduced mitophagy, recovery of mitochondrial potential or quenching of mitochondrial ROS after removal of rotenone or oligomycin+antimycin A was delayed by FK506. Mitochondrial oxygen consumption was reduced by FK506, indicating reduced mitochondrial function by FK506. Likely due to mitochondrial dysfunction, insulin release from INS-1 cells was reduced by FK506 in vitro. FK506 treatment also reduced insulin release and impaired glucose tolerance in vivo, which was associated with decreased mitophagy and mitochondrial COX activity in pancreatic islets. FK506-induced mitochondrial dysfunction and glucose intolerance were ameliorated by an autophagy enhancer activating TFEB. These results suggest that diminished mitophagy and consequent mitochondrial dysfunction of pancreatic β-cells contribute to FK506-induced β-cell dysfunction or glucose intolerance, and autophagy enhancement could be a therapeutic modality against post-transplantation diabetes mellitus caused by PPP3/calcineurin inhibitors.
Topics: Humans; Mitophagy; Autophagy; Calcineurin Inhibitors; Tacrolimus; Antimycin A; Glucose Intolerance; Rotenone; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Lysosomes; Oligomycins; Insulins
PubMed: 36217215
DOI: 10.1080/15548627.2022.2132686