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Significance: The architecture of the mitochondrial network and cristae critically impact cell differentiation and identity. Cells undergoing metabolic reprogramming to aerobic glycolysis (Warburg effect), such as immune cells, stem cells, and cancer cells, go through controlled modifications in mitochondrial architecture, which is critical for achieving the resulting cellular phenotype. Recent Advances: Recent studies in immunometabolism have shown that the manipulation of mitochondrial network dynamics and cristae shape directly affects T cell phenotype and macrophage polarization through altering energy metabolism. Similar manipulations also alter the specific metabolic phenotypes that accompany somatic reprogramming, stem cell differentiation, and cancer cells. The modulation of oxidative phosphorylation activity, accompanied by changes in metabolite signaling, reactive oxygen species generation, and adenosine triphosphate levels, is the shared underlying mechanism. Critical Issues: The plasticity of mitochondrial architecture is particularly vital for metabolic reprogramming. Consequently, failure to adapt the appropriate mitochondrial morphology often compromises the differentiation and identity of the cell. Immune, stem, and tumor cells exhibit striking similarities in their coordination of mitochondrial morphology with metabolic pathways. However, although many general unifying principles can be observed, their validity is not absolute, and the mechanistic links thus need to be further explored. Future Directions: Better knowledge of the molecular mechanisms involved and their relationships to both mitochondrial network and cristae morphology will not only further deepen our understanding of energy metabolism but may also contribute to improved therapeutic manipulation of cell viability, differentiation, proliferation, and identity in many different cell types. Antioxid. Redox Signal. 39, 684-707.
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Mitocondrias , Dinámicas Mitocondriales , Mitocondrias/metabolismo , Metabolismo Energético , Fosforilación Oxidativa , Redes y Vías Metabólicas , Glucólisis , Reprogramación CelularRESUMEN
Previously, a number of ~ 1.4 of mitochondrial DNA (mtDNA) molecules in a single nucleoid was reported, which would reflect a minimum nucleoid division. We applied 3D-double-color direct stochastic optical reconstruction microscopy (dSTORM), i.e. nanoscopy with ~ 25-40 nm x,y-resolution, together with our novel method of Delaunay segmentation of 3D data to identify unbiased 3D-overlaps. Noncoding D-loops were recognized in HeLa cells by mtDNA fluorescence in situ hybridization (mtFISH) 7S-DNA 250-bp probe, containing biotin, visualized by anti-biotin/Cy3B-conjugated antibodies. Other mtFISH probes with biotin or Alexa Fluor 647 (A647) against ATP6-COX3 gene overlaps (1,100 bp) were also used. Nucleoids were imaged by anti-DNA/(A647-)-Cy3B-conjugated antibodies. Resulting histograms counting mtFISH-loci/nucleoid overlaps demonstrated that 45% to 70% of visualized nucleoids contained two or more D-loops or ATP6-COX3-loci, indicating two or more mtDNA molecules per nucleoid. With increasing number of mtDNA per nucleoid, diameters were larger and their distribution histograms peaked at ~ 300 nm. A wide nucleoid diameter distribution was obtained also using 2D-STED for their imaging by anti-DNA/A647. At unchanged mtDNA copy number in osteosarcoma 143B cells, TFAM expression increased nucleoid spatial density 1.67-fold, indicating expansion of existing mtDNA and its redistribution into more nucleoids upon the higher TFAM/mtDNA stoichiometry. Validation of nucleoid imaging was also done with two TFAM mutants unable to bend or dimerize, respectively, which reduced both copy number and nucleoid spatial density by 80%. We conclude that frequently more than one mtDNA molecule exists within a single nucleoid in HeLa cells and that mitochondrial nucleoids do exist in a non-uniform size range.
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ADN Mitocondrial , Proteínas de Unión al ADN , Humanos , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células HeLa , Hibridación Fluorescente in Situ , Proteínas Mitocondriales/metabolismoRESUMEN
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.
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Membranas Mitocondriales , Superóxidos , Membranas Mitocondriales/metabolismo , Superóxidos/metabolismo , Homeostasis , Oxidación-Reducción , Adenosina Trifosfato/metabolismo , Proteínas Mitocondriales/metabolismoRESUMEN
Significance: Mitochondria determine glucose-stimulated insulin secretion (GSIS) in pancreatic ß-cells by elevating ATP synthesis. As the metabolic and redox hub, mitochondria provide numerous links to the plasma membrane channels, insulin granule vesicles (IGVs), cell redox, NADH, NADPH, and Ca2+ homeostasis, all affecting insulin secretion. Recent Advances: Mitochondrial redox signaling was implicated in several modes of insulin secretion (branched-chain ketoacid [BCKA]-, fatty acid [FA]-stimulated). Mitochondrial Ca2+ influx was found to enhance GSIS, reflecting cytosolic Ca2+ oscillations induced by action potential spikes (intermittent opening of voltage-dependent Ca2+ and K+ channels) or the superimposed Ca2+ release from the endoplasmic reticulum (ER). The ATPase inhibitory factor 1 (IF1) was reported to tune the glucose sensitivity range for GSIS. Mitochondrial protein kinase A was implicated in preventing the IF1-mediated inhibition of the ATP synthase. Critical Issues: It is unknown how the redox signal spreads up to the plasma membrane and what its targets are, what the differences in metabolic, redox, NADH/NADPH, and Ca2+ signaling, and homeostasis are between the first and second GSIS phase, and whether mitochondria can replace ER in the amplification of IGV exocytosis. Future Directions: Metabolomics studies performed to distinguish between the mitochondrial matrix and cytosolic metabolites will elucidate further details. Identifying the targets of cell signaling into mitochondria and of mitochondrial retrograde metabolic and redox signals to the cell will uncover further molecular mechanisms for insulin secretion stimulated by glucose, BCKAs, and FAs, and the amplification of secretion by glucagon-like peptide (GLP-1) and metabotropic receptors. They will identify the distinction between the hub ß-cells and their followers in intact and diabetic states. Antioxid. Redox Signal. 36, 920-952.
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Células Secretoras de Insulina , Islotes Pancreáticos , Adenosina Trifosfato/metabolismo , Calcio/metabolismo , Glucosa/metabolismo , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Mitocondrias/metabolismo , NAD/metabolismo , NADP/metabolismo , Secretagogos/metabolismoRESUMEN
We compared the activity of complex 1, complex 2, and the expression of the complex 1 subunit, NDUFA9, in isolated brown adipose tissue mitochondria from wild type and mitochondrial uncoupling protein 1 (UCP1) knockout mice. Direct spectrophotometric measurement revealed that complex 2 activity was similar, but complex 1 activity was greater (~2.7 fold) in isolated mitochondria from wild-type mice compared to UCP1 knockout mice, an observation endorsed by greater complex 1 subunit expression (NDUFA9) in mitochondria of wild-type mice. We also measured reactive oxygen species (ROS) production by isolated brown adipose mitochondria respiring on succinate, without rotenone, thus facilitating reverse electron flow through complex 1. We observed that reverse electron flow in isolated mitochondria from wild-type mice, with UCP1 inhibited, produced significantly greater (~1.6 fold) ROS when compared with isolated brown adipose mitochondria from UCP1 knockout mice. In summary, we demonstrate that ROS production by succinate-driven reverse electron flow can occur in brown adipose tissue mitochondria and is a good index of complex 1 activity.
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Adipocitos Marrones/efectos de los fármacos , Tejido Adiposo Pardo/efectos de los fármacos , Complejo I de Transporte de Electrón/metabolismo , Mitocondrias/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Ácido Succínico/farmacología , Adipocitos Marrones/enzimología , Tejido Adiposo Pardo/enzimología , Animales , Biomarcadores/metabolismo , Western Blotting , Fraccionamiento Celular , Complejo I de Transporte de Electrón/genética , Electroforesis en Gel de Poliacrilamida , Fluorometría , Ratones Noqueados , Mitocondrias/enzimología , Mitocondrias/genética , Ratas , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismoRESUMEN
Pancreatic ß-cell insulin secretion, which responds to various secretagogues and hormonal regulations, is reviewed here, emphasizing the fundamental redox signaling by NADPH oxidase 4- (NOX4-) mediated H2O2 production for glucose-stimulated insulin secretion (GSIS). There is a logical summation that integrates both metabolic plus redox homeostasis because the ATP-sensitive K+ channel (KATP) can only be closed when both ATP and H2O2 are elevated. Otherwise ATP would block KATP, while H2O2 would activate any of the redox-sensitive nonspecific calcium channels (NSCCs), such as TRPM2. Notably, a 100%-closed KATP ensemble is insufficient to reach the -50 mV threshold plasma membrane depolarization required for the activation of voltage-dependent Ca2+ channels. Open synergic NSCCs or Cl- channels have to act simultaneously to reach this threshold. The resulting intermittent cytosolic Ca2+-increases lead to the pulsatile exocytosis of insulin granule vesicles (IGVs). The incretin (e.g., GLP-1) amplification of GSIS stems from receptor signaling leading to activating the phosphorylation of TRPM channels and effects on other channels to intensify integral Ca2+-influx (fortified by endoplasmic reticulum Ca2+). ATP plus H2O2 are also required for branched-chain ketoacids (BCKAs); and partly for fatty acids (FAs) to secrete insulin, while BCKA or FA ß-oxidation provide redox signaling from mitochondria, which proceeds by H2O2 diffusion or hypothetical SH relay via peroxiredoxin "redox kiss" to target proteins.
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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.
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Glucosa/administración & dosificación , Células Secretoras de Insulina/citología , Proteínas de la Membrana/genética , Regulación hacia Arriba , Adenosina Trifosfato/metabolismo , Animales , Técnicas de Cultivo de Célula , Línea Celular , Variaciones en el Número de Copia de ADN/efectos de los fármacos , ADN Mitocondrial/efectos de los fármacos , ADN Mitocondrial/genética , Glucosa/farmacología , Ácido Hialurónico/química , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/genética , Mitocondrias/metabolismo , Modelos Moleculares , Conformación Proteica , RatasRESUMEN
Aims: Glucose-stimulated insulin secretion (GSIS) in pancreatic ß cells was expected to enhance mitochondrial superoxide formation. Hence, we elucidated relevant redox equilibria. Results: Unexpectedly, INS-1E cells at transitions from 3 (11 mM; pancreatic islets from 5 mM) to 25 mM glucose decreased matrix superoxide release rates (MitoSOX Red monitoring validated by MitoB) and H2O2 (mitoHyPer, subtracting mitoSypHer emission). Novel double-channel fluorescence lifetime imaging, approximating free mitochondrial matrix NADHF, indicated its â¼20% decrease. Matrix NAD+F increased on GSIS, indicated by the FAD-emission lifetime decrease, reflecting higher quenching of FAD by NAD+F. The participation of pyruvate/malate and pyruvate/citrate redox shuttles, elevating cytosolic NADPHF (iNAP1 fluorescence monitoring) at the expense of matrix NADHF, was indicated, using citrate (2-oxoglutarate) carrier inhibitors and cytosolic malic enzyme silencing: All changes vanished on these manipulations. 13C-incorporation from 13C-L-glutamine into 13C-citrate reflected the pyruvate/isocitrate shuttle. Matrix NADPHF (iNAP3 monitored) decreased. With decreasing glucose, the suppressor of Complex III site Q electron leak (S3QEL) suppressor caused a higher Complex I IF site contribution, but a lower superoxide fraction ascribed to the Complex III site IIIQo. Thus, the diminished matrix NADHF/NAD+F decreased Complex I flavin site IF superoxide formation on GSIS. Innovation: Mutually validated methods showed decreasing superoxide release into the mitochondrial matrix in pancreatic ß cells on GSIS, due to the decreasing matrix NADHF/NAD+F (NADPHF/NADP+F) at increasing cytosolic NADPHF levels. The developed innovative methods enable real-time NADH/NAD+ and NADPH/NADP+ monitoring in any distinct cell compartment. Conclusion: The export of reducing equivalents from mitochondria adjusts lower mitochondrial superoxide production on GSIS, but it does not prevent oxidative stress in pancreatic ß cells.
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Glucosa/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Mitocondrias/metabolismo , NAD/metabolismo , Superóxidos/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Respiración de la Célula , Cromatografía Liquida , Ácido Cítrico/metabolismo , Metabolismo Energético , Flavina-Adenina Dinucleótido/metabolismo , Peróxido de Hidrógeno/metabolismo , Espectrometría de Masas , Potencial de la Membrana Mitocondrial , Redes y Vías Metabólicas , Metabolómica/métodos , Ratas , Transducción de SeñalRESUMEN
We have previously reported that transient knock-down of ATPase inhibitory factor 1 (IF1) by siRNA upregulates ATP levels and subsequently augments insulin secretion in model pancreatic ß-cells INS-1E. Here we investigated how long-term IF1-overexpression impacts pancreatic ß-cell bioenergetics and insulin secretion. We generated INS-1E cell line stably overexpressing native IF1. We revealed that IF1 overexpression leads to a substantial decrease in ATP levels and reduced glucose-stimulated insulin secretion. A decrease in total cellular ATP content was also reflected in decreased free ATP cytosolic and mitochondrial levels, as monitored with ATeam biosensor. Consistently, cellular respiration of IF1-overexpressing cells was decreased. 3D structured illumination microscopy (SIM) revealed a higher amount of insulin granules with higher volume in IF1-overexpressing cells. Similar effects occurred when cells were incubated at low glucose concentrations. Noteworthy, activation of PKA by dibutyryl cAMP entirely abolished the inhibitory effect of IF1 overexpression on ATP production and insulin secretion. Mitochondrial network morphology and cristae ultrastructure in INS-1E overexpressing IF1 remained mostly unchanged. Finally, we show that INS-1E cells decrease their IF1 protein levels relative to ATP synthase α-subunit in response to increased glucose. In conclusion, IF1 actively downregulates INS-1E cellular metabolism and reduces their ability to secrete insulin.
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Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Proteínas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , CMP Cíclico/análogos & derivados , CMP Cíclico/metabolismo , Regulación hacia Abajo , Glucosa/metabolismo , Proteínas/genética , ARN Interferente Pequeño/genética , Ratas , Transducción de Señal , Regulación hacia Arriba , Proteína Inhibidora ATPasaRESUMEN
Type 2 diabetes progression stems from dysfunction of ß-cells, besides the peripheral insulin resistance. Mitochondria as glucose sensor and regulation center are impaired at various stages of this progression. Their biogenesis and functional impairment is reflected by altered morphology of the mitochondrial network and ultramorphology of cristae and mitochondrial DNA loci, termed nucleoids. Aspects of all above changes are reviewed here together with a brief introduction to proteins involved in mitochondrial network dynamics, cristae shaping, and mtDNA nucleoid structure and maintenance. Most frequently, pathology is reflected by the fragmentation of network, cristae inflation or absence and declining number of nucleoids.
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Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Mitocondrias/metabolismo , Dinámicas Mitocondriales , Membranas Mitocondriales/metabolismo , Animales , Diabetes Mellitus Tipo 2/patología , Humanos , Células Secretoras de Insulina/ultraestructura , Mitocondrias/ultraestructura , Membranas Mitocondriales/ultraestructuraRESUMEN
Hypoxia causes mitochondrial cristae widening, enabled by the ~20% degradation of Mic60/mitofilin, with concomitant clustering of the MICOS complex, reflecting the widening of crista junctions (outlets) (Plecitá-Hlavatá et al. FASEB J., 2016 30:1941-1957). Attempting to accelerate metabolism by the addition of membrane-permeant dimethyl-2-oxoglutarate (dm2OG) to HepG2 cells pre-adapted to hypoxia, we found cristae narrowing by transmission electron microscopy. Glycolytic HepG2 cells, which downregulate hypoxic respiration, instantly increased respiration with dm2OG. Changes in intracristal space (ICS) morphology were also revealed by 3D super-resolution microscopy using Eos-conjugated ICS-located lactamase-ß. Cristae topology was resolved in detail by focused-ion beam/scanning electron microscopy (FIB/SEM). The spatial relocations of key cristae-shaping proteins were indicated by immunocytochemical stochastic 3D super-resolution microscopy (dSTORM), while analyzing inter-antibody-distance histograms: i) ATP-synthase dimers exhibited a higher fraction of shorter inter-distances between bound F1-α primary Alexa-Fluor-647-conjugated antibodies, indicating cristae narrowing. ii) Mic60/mitofilin clusters (established upon hypoxia) decayed, restoring isotropic random Mic60/mitofilin distribution (a signature of normoxia). iii) outer membrane SAMM50 formed more focused clusters. Less abundant fractions of higher ATP-synthase oligomers of hypoxic samples on blue-native electrophoresis became more abundant fractions at the high dm2OG load and at normoxia. This indicates more labile ATP-synthase dimeric rows established at crista rims upon hypoxia, strengthened at normoxia or dm2OG-substrate load. Hypothetically, the increased Krebs substrate load stimulates the cross-linking/strengthening of rows of ATP-synthase dimers at the crista rims, making them sharper. Crista narrowing ensures a more efficient coupling of proton pumping to ATP synthesis. We demonstrated that cristae morphology changes even within minutes.
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Ácidos Cetoglutáricos/farmacología , Mitocondrias/ultraestructura , Membranas Mitocondriales/ultraestructura , Respiración de la Célula , Dimerización , Células Hep G2 , Humanos , Hipoxia , Microscopía Electrónica de Transmisión , Membranas Mitocondriales/efectos de los fármacos , Proteínas Mitocondriales/metabolismo , ATPasas de Translocación de Protón Mitocondriales/metabolismoRESUMEN
3D super-resolution microscopy based on the direct stochastic optical reconstruction microscopy (dSTORM) with primary Alexa-Fluor-647-conjugated antibodies is a powerful method for accessing changes of objects that could be normally resolved only by electron microscopy. Despite the fact that mitochondrial cristae yet to become resolved, we have indicated changes in cristae width and/or morphology by dSTORM of ATP-synthase F1 subunit α (F1α). Obtained 3D images were analyzed with the help of Ripley's K-function modeling spatial patterns or transferring them into distance distribution function. Resulting histograms of distances frequency distribution provide most frequent distances (MFD) between the localized single antibody molecules. In fasting state of model pancreatic ß-cells, INS-1E, MFD between F1α were ~80â¯nm at 0 and 3â¯mM glucose, whereas decreased to 61â¯nm and 57â¯nm upon glucose-stimulated insulin secretion (GSIS) at 11â¯mM and 20â¯mM glucose, respectively. Shorter F1α interdistances reflected cristae width decrease upon GSIS, since such repositioning of F1α correlated to average 20â¯nm and 15â¯nm cristae width at 0 and 3â¯mM glucose, and 9â¯nm or 8â¯nm after higher glucose simulating GSIS (11, 20â¯mM glucose, respectively). Also, submitochondrial entities such as nucleoids of mtDNA were resolved e.g. after bromo-deoxyuridine (BrDU) pretreatment using anti-BrDU dSTORM. MFD in distances distribution histograms reflected an average nucleoid diameter (<100â¯nm) and average distances between nucleoids (~1000â¯nm). Double channel PALM/dSTORM with Eos-lactamase-ß plus anti-TFAM dSTORM confirmed the latter average inter-nucleoid distance. In conclusion, 3D single molecule (dSTORM) microscopy is a reasonable tool for studying mitochondrion.
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ADN Mitocondrial/química , ADN Mitocondrial/metabolismo , Proteínas de Unión al ADN/metabolismo , Imagenología Tridimensional/métodos , Microscopía Fluorescente/instrumentación , Membranas Mitocondriales/metabolismo , Animales , Células Cultivadas , Células Hep G2 , Humanos , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Proteínas Mitocondriales/metabolismo , Ratas , Ratas WistarRESUMEN
ATPase Inhibitory factor 1 (IF1) is an endogenous regulator of mitochondrial ATP synthase, which is involved in cellular metabolism. Although great progress has been made, biological roles of IF1 and molecular mechanisms of its action are still to be elucidated. Here, we show that IF1 is present in pancreatic ß-cells, bound to the ATP synthase also under normal physiological conditions. IF1 silencing in model pancreatic ß-cells (INS-1E) increases insulin secretion over a range of glucose concentrations. The left-shifted dose-response curve reveals excessive insulin secretion even under low glucose, corresponding to fasting conditions. A parallel increase in cellular respiration and ATP levels is observed. To conclude, our results indicate that IF1 is a negative regulator of insulin secretion involved in pancreatic ß-cell glucose sensing.
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Glucosa/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Consumo de Oxígeno/fisiología , Proteínas/metabolismo , Animales , Línea Celular Tumoral , Células Secretoras de Insulina/citología , Ratas , Ratas Wistar , Proteína Inhibidora ATPasaRESUMEN
Hypertrophic pancreatic islets (PI) of Goto Kakizaki (GK) diabetic rats contain a lower number of ß-cells vs. non-diabetic Wistar rat PI. Remaining ß-cells contain reduced mitochondrial (mt) DNA per nucleus (copy number), probably due to declining mtDNA replication machinery, decreased mt biogenesis or enhanced mitophagy. We confirmed mtDNA copy number decrease down to <30% in PI of one-year-old GK rats. Studying relations to mt nucleoids sizes, we employed 3D superresolution fluorescent photoactivable localization microscopy (FPALM) with lentivirally transduced Eos conjugate of mt single-stranded-DNA-binding protein (mtSSB) or transcription factor TFAM; or by 3D immunocytochemistry. mtSSB (binding transcription or replication nucleoids) contoured "nucleoids" which were smaller by 25% (less diameters >150 nm) in GK ß-cells. Eos-TFAM-visualized nucleoids, composed of 72% localized TFAM, were smaller by 10% (immunochemically by 3%). A theoretical ~70% decrease in cell nucleoid number (spatial density) was not observed, rejecting model of single mtDNA per nucleoid. The ß-cell maintenance factor Nkx6.1 mRNA and protein were declining with age (>12-fold, 10 months) and decreasing with fasting hyperglycemia in GK rats, probably predetermining the impaired mtDNA replication (copy number decrease), while spatial expansion of mtDNA kept nucleoids with only smaller sizes than those containing much higher mtDNA in non-diabetic ß-cells.
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Diabetes Mellitus Experimental/genética , Proteínas de Homeodominio/genética , Células Secretoras de Insulina/patología , Factores de Transcripción/genética , Animales , Variaciones en el Número de Copia de ADN/genética , Replicación del ADN/genética , ADN Mitocondrial/genética , Proteínas de Unión al ADN/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Humanos , Células Secretoras de Insulina/metabolismo , Masculino , Mitocondrias/genética , Mitocondrias/patología , Mitofagia/genética , Páncreas Exocrino/metabolismo , Ratas , Ratas WistarRESUMEN
The relationship of the inner mitochondrial membrane (IMM) cristae structure and intracristal space (ICS) to oxidative phosphorylation (oxphos) is not well understood. Mitofilin (subunit Mic60) of the mitochondrial contact site and cristae organizing system (MICOS) IMM complex is attached to the outer membrane (OMM) via the sorting and assembly machinery/topogenesis of mitochondrial outer membrane ß-barrel proteins (SAM/TOB) complex and controls the shape of the cristae. ATP synthase dimers determine sharp cristae edges, whereas trimeric OPA1 tightens ICS outlets. Metabolism is altered during hypoxia, and we therefore studied cristae morphology in HepG2 cells adapted to 5% oxygen for 72 h. Three dimensional (3D), super-resolution biplane fluorescence photoactivation localization microscopy with Eos-conjugated, ICS-located lactamase-ß indicated hypoxic ICS expansion with an unchanged OMM (visualized by Eos-mitochondrial fission protein-1). 3D direct stochastic optical reconstruction microscopy immunocytochemistry revealed foci of clustered mitofilin (but not MICOS subunit Mic19) in contrast to its even normoxic distribution. Mitofilin mRNA and protein decreased by â¼20%. ATP synthase dimers vs monomers and state-3/state-4 respiration ratios were lower during hypoxia. Electron microscopy confirmed ICS expansion (maximum in glycolytic cells), which was absent in reduced or OMM-detached cristae of OPA1- and mitofilin-silenced cells, respectively. Hypoxic adaptation is reported as rounding sharp cristae edges and expanding cristae width (ICS) by partial mitofilin/Mic60 down-regulation. Mitofilin-depleted MICOS detaches from SAM while remaining MICOS with mitofilin redistributes toward higher interdistances. This phenomenon causes partial oxphos dormancy in glycolytic cells via disruption of ATP synthase dimers.-Plecitá-Hlavatá, L., Engstová, H., Alán, L., Spacek, T., Dlasková, A., Smolková, K., Spacková, J., Tauber, J., Strádalová, V., Malínský, J., Lessard, M., Bewersdorf, J., Jezek, P. Hypoxic HepG2 cell adaptation decreases ATP synthase dimers and ATP production in inflated cristae by mitofilin down-regulation concomitant to MICOS clustering.
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Complejos de ATP Sintetasa/metabolismo , Adaptación Fisiológica/fisiología , Adenosina Trifosfato/biosíntesis , Mitocondrias/fisiología , Proteínas Mitocondriales/metabolismo , Oxígeno , Regulación hacia Abajo , Regulación de la Expresión Génica/fisiología , Células Hep G2 , Humanos , Dinámicas Mitocondriales/fisiología , Proteínas Mitocondriales/genética , Complejos Multiproteicos/fisiología , Dominios y Motivos de Interacción de Proteínas , Subunidades de ProteínaRESUMEN
Oligomer aggregation of green-to-red photoconvertible fluorescent protein Eos (EosFP) is a natural feature of the wildtype variant. The aim of the present study was to follow up mitochondrial nucleoid behavior under natural conditions of living cells transfected with mitochondrial singlestrand DNAbinding protein (mtSSB) conjugated with EosFP. HEPG2 and SHSY5Y cells were subjected to lentiviral transfection and subsequently immunostained with antiDNA, antitranscription factor A, mitochondrial (TFAM) or antitranslocase of the inner membrane 23 antibodies. Fluorescent microscopy, conventional confocal microscopy, superresolution biplane fluorescence photo-activation localization microscopy and direct stochastic optical reconstruction microscopy were used for imaging. In the two cell types, apparent couples of equallysized mtSSBEosFPvisualized dots were observed. During the time course of the ongoing transfection procedure, however, a small limited number of large aggregates of mtSSBEosFPtagged protein started to form in the cells, which exhibited a great colocalization with the noted coupled positions. Antibody staining and 3D immunocytochemistry confirmed that nucleoid components such as TFAM and DNA were colocalized with these aggregates. Furthermore, the observed reduction of the mtDNA copy number in mtSSBEosFPtransfected cells suggested a possible impairment of nucleoid function. In conclusion, the present study demonstrated that coupled nucleoids are synchronized by mtSSBEosFP overexpression and visualized through their equal binding capacity to mtSSBEosFPtagged protein. This observation suggested parallel replication and transcription activity of nucleoid couples native from a parental one. Preserved coupling in late stages of artificial EosFPmediated aggregation of tagged proteins suggested a rational manner of mitochondrial branching that may be cell-type specifically dependent on hierarchical nucleoid replication.
Asunto(s)
ADN Mitocondrial/metabolismo , Proteínas de Unión al ADN/metabolismo , Mitocondrias/metabolismo , Multimerización de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ADN/química , Dosificación de Gen , Humanos , Inmunohistoquímica , Microscopía Confocal , Proteínas Mitocondriales/metabolismo , Unión Proteica , Transporte de Proteínas , Proteínas Recombinantes de Fusión/química , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
AIMS: Pancreatic ß-cell chronic lipotoxicity evolves from acute free fatty acid (FA)-mediated oxidative stress, unprotected by antioxidant mechanisms. Since mitochondrial uncoupling protein-2 (UCP2) plays antioxidant and insulin-regulating roles in pancreatic ß-cells, we tested our hypothesis, that UCP2-mediated uncoupling attenuating mitochondrial superoxide production is initiated by FA release due to a direct H2O2-induced activation of mitochondrial phospholipase iPLA2γ. RESULTS: Pro-oxidant tert-butylhydroperoxide increased respiration, decreased membrane potential and mitochondrial matrix superoxide release rates of control but not UCP2- or iPLA2γ-silenced INS-1E cells. iPLA2γ/UCP2-mediated uncoupling was alternatively activated by an H2O2 burst, resulting from palmitic acid (PA) ß-oxidation, and it was prevented by antioxidants or catalase overexpression. Exclusively, nascent FAs that cleaved off phospholipids by iPLA2γ were capable of activating UCP2, indicating that the previously reported direct redox UCP2 activation is actually indirect. Glucose-stimulated insulin release was not affected by UCP2 or iPLA2γ silencing, unless pro-oxidant activation had taken place. PA augmented insulin secretion via G-protein-coupled receptor 40 (GPR40), stimulated by iPLA2γ-cleaved FAs (absent after GPR40 silencing). INNOVATION AND CONCLUSION: The iPLA2γ/UCP2 synergy provides a feedback antioxidant mechanism preventing oxidative stress by physiological FA intake in pancreatic ß-cells, regulating glucose-, FA-, and redox-stimulated insulin secretion. iPLA2γ is regulated by exogenous FA via ß-oxidation causing H2O2 signaling, while FAs are cleaved off phospholipids, subsequently acting as amplifying messengers for GPR40. Hence, iPLA2γ acts in eminent physiological redox signaling, the impairment of which results in the lack of antilipotoxic defense and contributes to chronic lipotoxicity.
Asunto(s)
Fosfolipasas A2 Grupo II/metabolismo , Insulina/metabolismo , Canales Iónicos/metabolismo , Mitocondrias/efectos de los fármacos , Proteínas Mitocondriales/metabolismo , Estrés Oxidativo/efectos de los fármacos , Receptores Acoplados a Proteínas G/metabolismo , Animales , Antioxidantes/farmacología , Línea Celular Tumoral , Peróxido de Hidrógeno/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/efectos de los fármacos , Lípidos/toxicidad , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratas , Transducción de Señal/efectos de los fármacos , Superóxidos/metabolismo , Proteína Desacopladora 2 , terc-Butilhidroperóxido/farmacologíaRESUMEN
Mitochondrial nucleoids are confined sites of mitochondrial DNA existing in complex clusters with the DNA-compacting mitochondrial (mt) transcription factor A (TFAM) and other accessory proteins and gene expression machinery proteins, such as a mt single-stranded-DNA-binding protein (mtSSB). To visualize nucleoid distribution within the mt reticular network, we have employed three-dimensional (3D) double-color 4Pi microscopy. The mt network was visualized in hepatocellular carcinoma HepG2 cells via mt-matrix-addressed GFP, while 3D immunocytochemistry of mtSSB was performed. Optimization of iso-surface computation threshold for nucleoid 4Pi images to 30 led to an average nucleoid diameter of 219 ± 110 and 224 ± 100 nm in glucose- and galactose-cultivated HepG2 cells (the latter with obligatory oxidative phosphorylation). We have positioned mtDNA nucleoids within the mt reticulum network and refined our model for nucleoid redistribution within the fragmented network--clustering of up to ten nucleoids in 2 µm diameter mitochondrial spheroids of a fragmented mt network, arising from an original 10 µm mt tubule of a 400 nm diameter. However, the theoretically fragmented bulk parts were observed most frequently as being reintegrated into the continuous mt network in 4Pi images. Since the predicted nucleoid counts within the bulk parts corresponded to the model, we conclude that fragmentation/reintegration cycles are not accompanied by mtDNA degradation or that mtDNA degradation is equally balanced by mtDNA replication.
Asunto(s)
ADN Mitocondrial/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Mitocondriales/metabolismo , Modelos Moleculares , Factores de Transcripción/metabolismo , Técnicas de Cultivo de Célula , ADN Mitocondrial/genética , Proteínas de Unión al ADN/genética , Proteínas Fluorescentes Verdes/metabolismo , Células Hep G2 , Humanos , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Proteínas Mitocondriales/genética , Conformación de Ácido Nucleico , Factores de Transcripción/genéticaRESUMEN
Mitochondrial DNA (mtDNA) is organized in nucleoids in complex with accessory proteins, proteins of mtDNA replication and gene expression machinery. A robust mtDNA genome is represented by hundreds to thousands of nucleoids in cell mitochondrion. Detailed information is lacking about the dynamics of nucleoid distribution within the mitochondrial network upon physiological and pathological events. Therefore, we used confocal microscopy to study mitochondrial nucleoid redistribution upon mitochondrial fission and following reintegration of the mitochondrial network. Fission was induced by oxidative stress at respiration inhibition by rotenone or upon elimination of the protonmotive force by uncoupling or upon canceling its electrical component, ΔΨ(m), by valinomycin; and by silencing of mitofusin MFN2. Agent withdrawal resulted in concomitant mitochondrial network reintegration. We found two major principal morphological states: (i) a tubular state of the mitochondrial network with equidistant nucleoid spacing, 1.10±0.2 nucleoids per µm, and (ii) a fragmented state of solitary spheroid objects in which several nucleoids were clustered. We rarely observed singular mitochondrial fragments with a single nucleoid inside and very seldom we observed empty fragments. Reintegration of fragments into the mitochondrial network re-established the tubular state with equidistant nucleoid spacing. The two major morphological states coexisted at intermediate stages. These observations suggest that both mitochondrial network fission and reconnection of the disintegrated network are nucleoid-centric, i.e., fission and new mitochondrial tubule formation are initiated around nucleoids. Analyses of combinations of these morphological icons thus provide a basis for a future mitochondrial morphology diagnostics.
Asunto(s)
Replicación del ADN/genética , ADN Mitocondrial/ultraestructura , Mitocondrias/ultraestructura , Dinámicas Mitocondriales/genética , ADN Mitocondrial/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células Hep G2 , Humanos , Microscopía Confocal , Dinámicas Mitocondriales/fisiología , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/ultraestructuraRESUMEN
We reviewed mechanisms that determine reactive oxygen species (redox) homeostasis, redox information signaling and metabolic/regulatory function of autocrine insulin signaling in pancreatic ß cells, and consequences of oxidative stress and dysregulation of redox/information signaling for their dysfunction. We emphasize the role of mitochondrion in ß cell molecular physiology and pathology, including the antioxidant role of mitochondrial uncoupling protein UCP2. Since in pancreatic ß cells pyruvate cannot be easily diverted towards lactate dehydrogenase for lactate formation, the respiration and oxidative phosphorylation intensity are governed by the availability of glucose, leading to a certain ATP/ADP ratio, whereas in other cell types, cell demand dictates respiration/metabolism rates. Moreover, we examine the possibility that type 2 diabetes mellitus might be considered as an inevitable result of progressive self-accelerating oxidative stress and concomitantly dysregulated information signaling in peripheral tissues as well as in pancreatic ß cells. It is because the redox signaling is inherent to the insulin receptor signaling mechanism and its impairment leads to the oxidative and nitrosative stress. Also emerging concepts, admiting participation of redox signaling even in glucose sensing and insulin release in pancreatic ß cells, fit in this view. For example, NADPH has been firmly established to be a modulator of glucose-stimulated insulin release.