Most cited article - PubMed ID 26887443
Hypoxic HepG2 cell adaptation decreases ATP synthase dimers and ATP production in inflated cristae by mitofilin down-regulation concomitant to MICOS clustering
Mitochondria (mt) represent the vital hub of the molecular physiology of the cell, being decision-makers in cell life/death and information signaling, including major redox regulations and redox signaling. Now we review recent advances in understanding mitochondrial redox homeostasis, including superoxide sources and H2O2 consumers, i.e., antioxidant mechanisms, as well as exemplar situations of physiological redox signaling, including the intramitochondrial one and mt-to-cytosol redox signals, which may be classified as acute and long-term signals. This review exemplifies the acute redox signals in hypoxic cell adaptation and upon insulin secretion in pancreatic beta-cells. We also show how metabolic changes under these circumstances are linked to mitochondrial cristae narrowing at higher intensity of ATP synthesis. Also, we will discuss major redox buffers, namely the peroxiredoxin system, which may also promote redox signaling. We will point out that pathological thresholds exist, specific for each cell type, above which the superoxide sources exceed regular antioxidant capacity and the concomitant harmful processes of oxidative stress subsequently initiate etiology of numerous diseases. The redox signaling may be impaired when sunk in such excessive pro-oxidative state.
- MeSH
- Antioxidants metabolism MeSH
- Insulin-Secreting Cells metabolism MeSH
- Humans MeSH
- Mitochondria * metabolism MeSH
- Oxidation-Reduction * MeSH
- Oxidative Stress physiology MeSH
- Signal Transduction physiology MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
- Names of Substances
- Antioxidants MeSH
Significance: Mitochondrial (mt) reticulum network in the cell possesses amazing ultramorphology of parallel lamellar cristae, formed by the invaginated inner mitochondrial membrane. Its non-invaginated part, the inner boundary membrane (IBM) forms a cylindrical sandwich with the outer mitochondrial membrane (OMM). Crista membranes (CMs) meet IBM at crista junctions (CJs) of mt cristae organizing system (MICOS) complexes connected to OMM sorting and assembly machinery (SAM). Cristae dimensions, shape, and CJs have characteristic patterns for different metabolic regimes, physiological and pathological situations. Recent Advances: Cristae-shaping proteins were characterized, namely rows of ATP-synthase dimers forming the crista lamella edges, MICOS subunits, optic atrophy 1 (OPA1) isoforms and mitochondrial genome maintenance 1 (MGM1) filaments, prohibitins, and others. Detailed cristae ultramorphology changes were imaged by focused-ion beam/scanning electron microscopy. Dynamics of crista lamellae and mobile CJs were demonstrated by nanoscopy in living cells. With tBID-induced apoptosis a single entirely fused cristae reticulum was observed in a mitochondrial spheroid. Critical Issues: The mobility and composition of MICOS, OPA1, and ATP-synthase dimeric rows regulated by post-translational modifications might be exclusively responsible for cristae morphology changes, but ion fluxes across CM and resulting osmotic forces might be also involved. Inevitably, cristae ultramorphology should reflect also mitochondrial redox homeostasis, but details are unknown. Disordered cristae typically reflect higher superoxide formation. Future Directions: To link redox homeostasis to cristae ultramorphology and define markers, recent progress will help in uncovering mechanisms involved in proton-coupled electron transfer via the respiratory chain and in regulation of cristae architecture, leading to structural determination of superoxide formation sites and cristae ultramorphology changes in diseases. Antioxid. Redox Signal. 39, 635-683.
- Keywords
- ATP-synthase dimeric rows, MICOS, OPA1, mitochondrial cristae, mitochondrial superoxide formation, respiratory chain supercomplexes,
- MeSH
- Adenosine Triphosphate metabolism MeSH
- Homeostasis MeSH
- Mitochondrial Membranes * metabolism MeSH
- Mitochondrial Proteins metabolism MeSH
- Oxidation-Reduction MeSH
- Superoxides * metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
- Names of Substances
- Adenosine Triphosphate MeSH
- Mitochondrial Proteins MeSH
- Superoxides * MeSH
Redox signaling from mitochondria (mt) to the cytosol and plasma membrane (PM) has been scarcely reported, such as in the case of hypoxic cell adaptation or (2-oxo-) 2-keto-isocaproate (KIC) β-like-oxidation stimulating insulin secretion in pancreatic β-cells. Mutual redox state influence between mitochondrial major compartments, the matrix and the intracristal space, and the cytosol is therefore derived theoretically in this article to predict possible conditions, when mt-to-cytosol and mt-to-PM signals may occur, as well as conditions in which the cytosolic redox signaling is not overwhelmed by the mitochondrial antioxidant capacity. Possible peroxiredoxin 3 participation in mt-to-cytosol redox signaling is discussed, as well as another specific case, whereby mitochondrial superoxide release is diminished, whereas the matrix MnSOD is activated. As a result, the enhanced conversion to H2O2 allows H2O2 diffusion into the cytosol, where it could be a predominant component of the H2O2 release. In both of these ways, mt-to-cytosol and mt-to-PM signals may be realized. Finally, the use of redox-sensitive probes is discussed, which disturb redox equilibria, and hence add a surplus redox-buffering to the compartment, where they are localized. Specifically, when attempts to quantify net H2O2 fluxes are to be made, this should be taken into account.
Transcript levels for selected ATP synthase membrane FO-subunits-including DAPIT-in INS-1E cells were found to be sensitive to lowering glucose down from 11 mM, in which these cells are routinely cultured. Depending on conditions, the diminished mRNA levels recovered when glucose was restored to 11 mM; or were elevated during further 120 min incubations with 20-mM glucose. Asking whether DAPIT expression may be elevated by hyperglycemia in vivo, we studied mice with hyaluronic acid implants delivering glucose for up to 14 days. Such continuous two-week glucose stimulations in mice increased DAPIT mRNA by >5-fold in isolated pancreatic islets (ATP synthase F1α mRNA by 1.5-fold). In INS-1E cells, the glucose-induced ATP increment vanished with DAPIT silencing (6% of ATP rise), likewise a portion of the mtDNA-copy number increment. With 20 and 11-mM glucose the phosphorylating/non-phosphorylating respiration rate ratio diminished to ~70% and 96%, respectively, upon DAPIT silencing, whereas net GSIS rates accounted for 80% and 90% in USMG5/DAPIT-deficient cells. Consequently, the sufficient DAPIT expression and complete ATP synthase assembly is required for maximum ATP synthesis and mitochondrial biogenesis, but not for insulin secretion as such. Elevated DAPIT expression at high glucose further increases the ATP synthesis efficiency.
- Keywords
- ATP synthase oligomers mitochondrial cristae morphology, USMG5/DAPIT, glucose-induced expression, glucose-stimulated insulin secretion, membrane subunits of ATP synthase, mitochondria,
- MeSH
- Adenosine Triphosphate metabolism MeSH
- Insulin-Secreting Cells cytology drug effects metabolism MeSH
- Cell Culture Techniques MeSH
- Cell Line MeSH
- Glucose administration & dosage pharmacology MeSH
- Protein Conformation MeSH
- Rats MeSH
- Hyaluronic Acid chemistry MeSH
- Membrane Proteins chemistry genetics metabolism MeSH
- DNA, Mitochondrial drug effects genetics MeSH
- Mitochondria drug effects genetics metabolism MeSH
- Models, Molecular MeSH
- Mice MeSH
- Up-Regulation * MeSH
- DNA Copy Number Variations drug effects MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Adenosine Triphosphate MeSH
- Atp5md protein, rat MeSH Browser
- Glucose MeSH
- Hyaluronic Acid MeSH
- Membrane Proteins MeSH
- DNA, Mitochondrial MeSH
Pancreatic β-cells are vulnerable to oxidative stress due to their low content of redox buffers, such as glutathione, but possess a rich content of thioredoxin, peroxiredoxin, and other proteins capable of redox relay, transferring redox signaling. Consequently, it may be predicted that cytosolic antioxidants could interfere with the cytosolic redox signaling and should not be recommended for any potential therapy. In contrast, mitochondrial matrix-targeted antioxidants could prevent the primary oxidative stress arising from the primary superoxide sources within the mitochondrial matrix, such as at the flavin (IF) and ubiquinone (IQ) sites of superoxide formation within respiratory chain complex I and the outer ubiquinone site (IIIQ) of complex III. Therefore, using time-resolved confocal fluorescence monitoring with MitoSOX Red, we investigated various effects of mitochondria-targeted antioxidants in model pancreatic β-cells (insulinoma INS-1E cells) and pancreatic islets. Both SkQ1 (a mitochondria-targeted plastoquinone) and a suppressor of complex III site Q electron leak (S3QEL) prevented superoxide production released to the mitochondrial matrix in INS-1E cells with stimulatory glucose, where SkQ1 also exhibited an antioxidant role for UCP2-silenced cells. SkQ1 acted similarly at nonstimulatory glucose but not in UCP2-silenced cells. Thus, UCP2 can facilitate the antioxidant mechanism based on SkQ1+ fatty acid anion- pairing. The elevated superoxide formation induced by antimycin A was largely prevented by S3QEL, and that induced by rotenone was decreased by SkQ1 and S3QEL and slightly by S1QEL, acting at complex I site Q. Similar results were obtained with the MitoB probe, for the LC-MS-based assessment of the 4 hr accumulation of reactive oxygen species within the mitochondrial matrix but for isolated pancreatic islets. For 2 hr INS-1E incubations, some samples were influenced by the cell death during the experiment. Due to the frequent dependency of antioxidant effects on metabolic modes, we suggest a potential use of mitochondria-targeted antioxidants for the treatment of prediabetic states after cautious nutrition-controlled tests. Their targeted delivery might eventually attenuate the vicious spiral leading to type 2 diabetes.
- MeSH
- Antioxidants pharmacology MeSH
- Insulin-Secreting Cells drug effects metabolism pathology MeSH
- Phenanthridines MeSH
- Cells, Cultured MeSH
- Mitochondrial Membranes drug effects metabolism pathology MeSH
- Mitochondria drug effects metabolism pathology MeSH
- Mice, Inbred C57BL MeSH
- Mice MeSH
- Organophosphorus Compounds MeSH
- Oxidation-Reduction MeSH
- Oxidative Stress drug effects MeSH
- Reactive Oxygen Species metabolism MeSH
- Uncoupling Protein 2 metabolism MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Antioxidants MeSH
- Phenanthridines MeSH
- MitoSox Red MeSH Browser
- Organophosphorus Compounds MeSH
- Reactive Oxygen Species MeSH
- Uncoupling Protein 2 MeSH
Human hepatocellular carcinoma HepG2 cells are forced to oxidative phosphorylation (OXPHOS), when cultured in aglycemic conditions at galactose and glutamine. These Oxphos cells represent a prototype of cancer cell bioenergetics with mixed aerobic glycolysis and OXPHOS. We aimed to determine fractions of (i) glutaminolytic pathway involving aminotransferase reaction supplying 2-oxoglutarate (2OG) to the Krebs cycle vs. (ii) active segment of the Krebs cycle with aconitase and isocitrate dehydrogenase-3 (ACO-IDH3), which is typically inactive in cancer cells due to the citrate export from mitochondria. At normoxia, Oxphos cell respiration was decreased down to ~15 and ~10% by the aminotransferase inhibitor aminooxyacetate (AOA) or with AOA plus the glutamate-dehydrogenase inhibitor bithionol, respectively. Phosphorylating to non-phosphorylating respiration ratios dropped from >6.5 to 1.9 with AOA and to zero with AOA plus bithionol. Thus, normoxic Oxphos HepG2 cells rely predominantly on glutaminolysis. Addition of membrane-permeant dimethyl-2-oxoglutarate (dm2OG) to inhibited cells instantly partially restored respiration, evidencing the lack of 2OG-dehydrogenase substrate upon aminotransferase inhibition. Surprisingly, after 72 hr of 5% O2 hypoxia, the AOA (bithionol) inhibition ceased and respiration was completely restored. Thus in aglycemic HepG2 cells, the hypoxia-induced factor (HIF) upregulation of glycolytic enzymes enabled acceleration of glycolysis pathway, preceded by galactolysis (Leloir pathway), redirecting pyruvate via still incompletely blocked pyruvate dehydrogenase toward the ACO-IDH3. Glycolytic flux upregulation at hypoxia was evidently matched by a higher activity of the Leloir pathway in Oxphos cells. Hypoxic Oxphos cells increased 2-fold the NADPH oxidase activity, whereas hypoxic glycolytic cells decreased it. Oxphos cells and glycolytic cells at 5 mM glucose decreased their reduced glutathione fraction. In contrast to aglycemic cells, glycolytic HepG2 cells decreased their respiration at hypoxia despite the dm2OG presence, i.e., even at unlimited respiratory substrate availability for 72 hr at 5% O2, exhibiting the canonical HIF-mediated adaptation. Nevertheless, their ATP content was much higher with dm2OG as compared to its absence during hypoxic adaptation. Thus, the metabolic plasticity of cancer cells is illustrated under conditions frequently established for solid tumors in vivo, such as aglycemia plus hypoxia. Consequently, a wide acceptance of the irreversible and exclusive Warburg phenotype in cancer cells is incorrect.
- Keywords
- HepG2 cells, Warburg phenotype, aminotransferase inhibiton, cancer mitochondria, glutaminolysis, hypoxia,
- Publication type
- Journal Article MeSH
