RESUMEN
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.
Asunto(s)
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
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.
Asunto(s)
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
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
The mitochondrion owns an autonomous genome. Double-stranded circular mitochondrial DNA (mtDNA) is organized in complexes with a packing/stabilizing transcription factor TFAM, having multiple roles, and proteins of gene expression machinery in structures called nucleoids. From hundreds to thousands nucleoids exist distributed in the matrix of mitochondrial reticulum network. A single mtDNA molecule contained within the single nucleoid is a currently preferred but questioned model. Nevertheless, mtDNA replication should lead transiently to its doubling within a nucleoid. However, nucleoid division has not yet been documented in detail. A 3D superresolution microscopy is required to resolve nucleoid biology occurring in â¼100 nm space, having an advantage over electron microscopy tomography in resolving the particular protein components. We discuss stochastic vs. stimulated emission depletion microscopy yielding wide vs. narrow nucleoid size distribution, respectively. Nucleoid clustering into spheroids fragmented from the continuous mitochondrial network, likewise possible nucleoid attachment to the inner membrane is reviewed.
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ADN Mitocondrial/metabolismo , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Humanos , Microscopía Confocal/métodos , Microscopía Fluorescente/métodosRESUMEN
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
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.
Asunto(s)
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
AIMS: Expression of the HER2 oncogene in breast cancer is associated with resistance to treatment, and Her2 may regulate bioenergetics. Therefore, we investigated whether disruption of the electron transport chain (ETC) is a viable strategy to eliminate Her2high disease. RESULTS: We demonstrate that Her2high cells and tumors have increased assembly of respiratory supercomplexes (SCs) and increased complex I-driven respiration in vitro and in vivo. They are also highly sensitive to MitoTam, a novel mitochondrial-targeted derivative of tamoxifen. Unlike tamoxifen, MitoTam efficiently suppresses experimental Her2high tumors without systemic toxicity. Mechanistically, MitoTam inhibits complex I-driven respiration and disrupts respiratory SCs in Her2high background in vitro and in vivo, leading to elevated reactive oxygen species production and cell death. Intriguingly, higher sensitivity of Her2high cells to MitoTam is dependent on the mitochondrial fraction of Her2. INNOVATION: Oncogenes such as HER2 can restructure ETC, creating a previously unrecognized therapeutic vulnerability exploitable by SC-disrupting agents such as MitoTam. CONCLUSION: We propose that the ETC is a suitable therapeutic target in Her2high disease. Antioxid. Redox Signal. 26, 84-103.
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Antineoplásicos/farmacología , Neoplasias de la Mama/metabolismo , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Receptor ErbB-2/metabolismo , Antineoplásicos/química , Biomarcadores , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Respiración de la Célula/efectos de los fármacos , Proteínas del Complejo de Cadena de Transporte de Electrón/antagonistas & inhibidores , Proteínas del Complejo de Cadena de Transporte de Electrón/química , Complejo I de Transporte de Electrón/antagonistas & inhibidores , Complejo I de Transporte de Electrón/química , Complejo I de Transporte de Electrón/metabolismo , Femenino , Humanos , Concentración 50 Inhibidora , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Modelos Moleculares , Conformación Molecular , Terapia Molecular Dirigida , Unión Proteica , Especies Reactivas de Oxígeno/metabolismo , Receptor ErbB-2/antagonistas & inhibidores , Tamoxifeno/farmacologíaRESUMEN
Mitochondrial DNA (mtDNA) is compacted in ribonucleoprotein complexes called nucleoids, which can divide or move within the mitochondrial network. Mitochondrial nucleoids are able to aggregate into clusters upon reaction with intercalators such as the mtDNA depletion agent Ethidium Bromide (EB) or anticancer drug Doxorobicin (DXR). However, the exact mechanism of nucleoid clusters formation remains unknown. Resolving these processes may help to elucidate the mechanisms of DXR-induced cardiotoxicity. Therefore, we addressed the role of two key nucleoid proteins; mitochondrial transcription factor A (TFAM) and mitochondrial single-stranded binding protein (mtSSB); in the formation of mitochondrial nucleoid clusters during the action of intercalators. We found that both intercalators cause numerous aberrations due to perturbing their native status. By blocking mtDNA replication, both agents also prevented mtDNA association with TFAM, consequently causing nucleoid aggregation into large nucleoid clusters enriched with TFAM, co-existing with the normal nucleoid population. In the later stages of intercalation (>48h), TFAM levels were reduced to 25%. In contrast, mtSSB was released from mtDNA and freely distributed within the mitochondrial network. Nucleoid clusters mostly contained nucleoids with newly replicated mtDNA, however the nucleoid population which was not in replication mode remained outside the clusters. Moreover, the nucleoid clusters were enriched with p53, an anti-oncogenic gatekeeper. We suggest that mitochondrial nucleoid clustering is a mechanism for protecting nucleoids with newly replicated DNA against intercalators mediating genotoxic stress. These results provide new insight into the common mitochondrial response to mtDNA stress and can be implied also on DXR-induced mitochondrial cytotoxicity.
Asunto(s)
ADN Mitocondrial/metabolismo , Proteínas de Unión al ADN/metabolismo , Mitocondrias Hepáticas/metabolismo , Proteínas Mitocondriales/metabolismo , Factores de Transcripción/metabolismo , Daño del ADN , Doxorrubicina , Dinaminas , Etidio , GTP Fosfohidrolasas/metabolismo , Células Hep G2 , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Data segmentation and object rendering is required for localization super-resolution microscopy, fluorescent photoactivation localization microscopy (FPALM), and direct stochastic optical reconstruction microscopy (dSTORM). We developed and validated methods for segmenting objects based on Delaunay triangulation in 3D space, followed by facet culling. We applied them to visualize mitochondrial nucleoids, which confine DNA in complexes with mitochondrial (mt) transcription factor A (TFAM) and gene expression machinery proteins, such as mt single-stranded-DNA-binding protein (mtSSB). Eos2-conjugated TFAM visualized nucleoids in HepG2 cells, which was compared with dSTORM 3D-immunocytochemistry of TFAM, mtSSB, or DNA. The localized fluorophores of FPALM/dSTORM data were segmented using Delaunay triangulation into polyhedron models and by principal component analysis (PCA) into general PCA ellipsoids. The PCA ellipsoids were normalized to the smoothed volume of polyhedrons or by the net unsmoothed Delaunay volume and remodeled into rotational ellipsoids to obtain models, termed DVRE. The most frequent size of ellipsoid nucleoid model imaged via TFAM was 35 × 45 × 95 nm; or 35 × 45 × 75 nm for mtDNA cores; and 25 × 45 × 100 nm for nucleoids imaged via mtSSB. Nucleoids encompassed different point density and wide size ranges, speculatively due to different activity stemming from different TFAM/mtDNA stoichiometry/density. Considering twofold lower axial vs. lateral resolution, only bulky DVRE models with an aspect ratio >3 and tilted toward the xy-plane were considered as two proximal nucleoids, suspicious occurring after division following mtDNA replication. The existence of proximal nucleoids in mtDNA-dSTORM 3D images of mtDNA "doubling"-supported possible direct observations of mt nucleoid division after mtDNA replication.
Asunto(s)
Algoritmos , ADN Mitocondrial/metabolismo , Imagenología Tridimensional , Microscopía Fluorescente , Análisis de Componente Principal , ADN Mitocondrial/química , Proteínas de Unión al ADN/metabolismo , Células Hep G2 , Humanos , Proteínas Mitocondriales/metabolismo , Modelos Moleculares , Conformación de Ácido NucleicoRESUMEN
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
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
Insulin production in pancreatic beta-cells is critically linked to mitochondrial oxidative phosphorylation. Increased ATP production triggered by blood glucose represents the beta-cells' glucose sensor. Type-2 diabetes mellitus results from insulin resistance in peripheral tissues and impaired insulin secretion. Pathology of diabetic beta-cells might be reflected by the altered morphology of mitochondrial network. Its characterization is however hampered by the complexity and density of the three-dimensional (3D) mitochondrial tubular networks in these cell types. Conventional confocal microscopy does not provide sufficient axial resolution to reveal the required details; electron tomography reconstruction of these dense networks is still difficult and time consuming. However, mitochondrial network morphology in fixed cells can also be studied by 4Pi microscopy, a laser scanning microscopy technique which provides an approximately 7-fold improved axial resolution (approximately 100 nm) over conventional confocal microscopy. Here we present a quantitative study of these networks in insulinoma INS-1E cells and primary beta-cells in Langerhans islets. The former were a stably-transfected cell line while the latter were transfected with lentivirus, both expressing mitochondrial matrix targeted redox-sensitive GFP. The mitochondrial networks and their partial disintegration and fragmentation are revealed by carefully created iso-surface plots and their quantitative analysis. We demonstrate that beta-cells within the Langerhans islets from diabetic Goto Kakizaki rats exhibited a more disintegrated mitochondrial network compared to those from control Wistar rats and model insulinoma INS-1E cells. Standardization of these patterns may lead to development of morphological diagnostics for Langerhans islets, for the assessment of beta-cell condition, before their transplantations.
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Diabetes Mellitus Tipo 2/patología , Células Secretoras de Insulina/patología , Microscopía Confocal/métodos , Mitocondrias/patología , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Proteínas Fluorescentes Verdes/genética , Imagenología Tridimensional , Técnicas In Vitro , Células Secretoras de Insulina/ultraestructura , Insulinoma/patología , Mitocondrias/ultraestructura , Neoplasias Pancreáticas/patología , Ratas , Ratas Wistar , Proteínas Recombinantes de Fusión/genética , TransfecciónRESUMEN
Increased ATP/ADP ratio resulting from enhanced glycolysis and oxidative phosphorylation represents a plausible mechanism controlling the glucose-stimulated insulin secretion (GSIS) in pancreatic beta-cells. Although specific bioenergetics might be involved, parallel studies of cell respiration and mitochondrial membrane potential (DeltaPsi(m)) during GSIS are lacking. Using high resolution respirometry and parallel DeltaPsi(m) monitoring by two distinct fluorescence probes we have quantified bioenergetics in rat insulinoma INS-1E cells representing a suitable model to study in vitro insulin secretion. Upon glucose addition to glucose-depleted cells we demonstrated a simultaneous increase in respiration and DeltaPsi(m) during GSIS and showed that the endogenous state 3/state 4 respiratory ratio hyperbolically increased with glucose, approaching the maximum oxidative phosphorylation rate at maximum GSIS. Attempting to assess the basis of the "toxic" effect of fatty acids on insulin secretion, GSIS was studied after linoleic acid addition, which diminished respiration increase, DeltaPsi(m) jump, and magnitude of insulin release, and reduced state 3/state 4 dependencies on glucose. Its effects were due to protonophoric function, i.e. uncoupling, since without glucose, linoleic acid accelerated both state 3 and state 4 respiration by similar extent. In turn, state 3 respiration increased marginally with linoleic acid at 10-20mM glucose. We conclude that upon glucose addition in physiological range, the INS-1E cells are able to regulate the oxidative phosphorylation rate from nearly zero to maximum and that the impairment of GSIS by linoleic acid is caused by mitochondrial uncoupling. These findings may be relevant to the pathogenesis of type 2 diabetes.
Asunto(s)
Glucosa/farmacología , Insulinoma/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Neoplasias Pancreáticas/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Ácido Linoleico/farmacología , Microscopía Electrónica de Transmisión , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Ratas , Ratas Wistar , Células Tumorales CultivadasRESUMEN
Thermogenic uncoupling has been proven only for UCP1 in brown adipose tissue. All other isoforms of UCPs are potentially acting in suppression of mitochondrial reactive oxygen species (ROS) production. In this contribution we show that BAT mitochondria can be uncoupled by lauric acid in the range of approximately 100 nM when endogenous fatty acids are combusted by carnitine cycle and beta-oxidation is properly separated from the uncoupling effect. Respiration increased up to 3 times when related to the lowest fatty acid content (BSA present plus carnitine cycle). We also illustrated that any effect leading to more coupled states leads to enhanced H2O2 generation and any effect resulting in uncoupling gives reduced H2O2 generation in BAT mitochondria. Finally, we report doubling of plant UCP transcript in cells as well as amount of protein detected by 3H-GTP-binding sites in mitochondria of shoots and roots of maize seedlings subjected to the salt stress.
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Tejido Adiposo Pardo/metabolismo , Proteínas Portadoras/fisiología , Ácidos Grasos/metabolismo , Proteínas de la Membrana/fisiología , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Sitios de Unión , Carnitina/metabolismo , Respiración de la Célula , Guanosina Trifosfato/metabolismo , Peróxido de Hidrógeno/metabolismo , Técnicas In Vitro , Canales Iónicos , Ácidos Láuricos/metabolismo , Ácidos Láuricos/farmacología , Mamíferos , Proteínas Mitocondriales , Estrés Oxidativo , Proteínas de Plantas/fisiología , Raíces de Plantas/metabolismo , Brotes de la Planta/metabolismo , Desacopladores/metabolismo , Desacopladores/farmacología , Proteína Desacopladora 1 , Zea mays/metabolismoRESUMEN
Undecanesulfonate is transported by uncoupling protein-1. Its inability to induce H+ uniport with reconstituted uncoupling protein-1 supports fatty acid cycling hypothesis. Rial et al. [Rial, E., Aguirregoitia, E., Jimenez-Jimenez, J., & Ledesma, A. (2004). Alkylsulfonates activate the uncoupling protein UCP1: Implications for the transport mechanism. Biochimica et Biophysica Acta, 1608, 122-130], have challenged the fatty acid cycling by observing uncoupling of brown adipose tissue mitochondria due to undecanesulfonate, interpreted as allosteric activation of uncoupling protein-1. We have estimated undecanesulfonate effects after elimination of endogenous fatty acids by carnitine cycle in the presence or absence of bovine serum albumin. We show that the undecanesulfonate effect is partly due to fatty acid release from albumin when undecanesulfonate releases bound fatty acid and partly represents a non-specific uncoupling protein-independent acceleration of respiration, since it proceeds also in rat heart mitochondria lacking uncoupling protein-1 and membrane potential is not decreased upon addition of undecanesulfonate without albumin. When the net fatty acid-induced uncoupling was assayed, the addition of undecanesulfonate even slightly inhibited the uncoupled respiration. We conclude that undecanesulfonate does not allosterically activate uncoupling protein-1 and that fatty acid cycling cannot be excluded on a basis of its non-specific effects.
Asunto(s)
Tejido Adiposo Pardo/metabolismo , Alcanosulfonatos/farmacología , Proteínas Portadoras/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Tejido Adiposo Pardo/efectos de los fármacos , Alcanosulfonatos/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Cricetinae , Ácidos Grasos/metabolismo , Canales Iónicos , Transporte Iónico/efectos de los fármacos , Masculino , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Cardíacas/metabolismo , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/fisiología , Proteínas Mitocondriales , Modelos Biológicos , Consumo de Oxígeno/efectos de los fármacos , Protones , Ratas , Albúmina Sérica Bovina/farmacología , Proteína Desacopladora 1RESUMEN
Rat liver mitochondria contain a negligible amount of mitochondrial uncoupling protein UCP2 as indicated by 3H-GTP binding. UCP2 recruitment in hepatocytes during infection may serve to decrease mitochondrial production of reactive oxygen species (ROS), and this, in turn, would counterbalance the increased oxidative stress. To characterize in detail UCP2 recruitment in hepatocytes, we studied rats pretreated with lipopolysaccharide (LPS) or hepatocytes isolated from them, as an in vitro model for the systemic response to bacterial infection. LPS injection resulted in 3.3- or 3-fold increase of UCP2 mRNA in rat liver and hepatocytes, respectively, as detected by real-time RT-PCR on a LightCycler. A concomitant increase in UCP2 protein content was indicated either by Western blots or was quantified by up to three-fold increase in the number of 3H-GTP binding sites in mitochondria of LPS-stimulated rats. Moreover, H2O2 production was increased by GDP only in mitochondria of LPS-stimulated rats with or without fatty acids and carboxyatractyloside. When monitored by JC1 fluorescent probe in situ mitochondria of hepatocytes from LPS-stimulated rats exhibited lower membrane potential than mitochondria of unstimulated rats. We have demonstrated that the lower membrane potential does not result from apoptosis initiation. However, due to a small extent of potential decrease upon UCP2 recruitment, justified also by theoretical calculations, we conclude that the recruited UCP2 causes only a weak uncoupling which is able to decrease mitochondrial ROS production but not produce enough heat for thermogenesis participating in a febrile response.