Mitochondrial Metabolic State Research Articles

Mitochondria: An Emerging Unavoidable Link in the Pathogenesis of Periodontitis Caused by Porphyromonas gingivalis.

Porphyromonas gingivalis (P. gingivalis) is a key pathogen of periodontitis. Increasing evidence shows that P. gingivalis signals to mitochondria in periodontal cells, including gingival epithelial cells, gingival fibroblast cells, immune cells, etc. Mitochondrial dysfunction affects the cellular state and participates in periodontal inflammatory response through the aberrant release of mitochondrial contents. In the current review, it was summarized that P. gingivalis induced mitochondrial dysfunction by altering the mitochondrial metabolic state, unbalancing mitochondrial quality control, prompting mitochondrial reactive oxygen species (ROS) production, and regulating mitochondria-mediated apoptosis. This review outlines the impacts of P. gingivalis and its virulence factors on the mitochondrial function of periodontal cells and their role in periodontitis.

Kisspeptin Modulates Mitochondrial Metabolic State and Potentiates Glucose Stimulated Insulin Secretion in INS 832/3 Cells and Pseudo-Islets

Glucagon Like Peptide-1 (GLP-1) and Kisspeptin (KP) receptors are co-expressed on pancreatic β-cells, and activation of both receptors potentiates glucose-stimulated insulin secretion (GSIS). Due to their roles in enhancing GSIS, GLP-1 and KP represent targets for controlling blood glucose in patients with diabetes. Unlike GLP-1, KP has a low affinity for receptor activation (EC50 GLP-1 ~20 nM vs KP ~1 μM) making it difficult to use as a therapeutic agent. GLP-1 has been shown to potentiate insulin secretion via cAMP dependent pathways. Conversely, previous studies in isolated rat islets of Langerhans indicated that KP potentiation of GSIS was not dependent on cAMP and may be related to changes in beta cell metabolism (Schwetz TA, et al., Differential Stimulation of Insulin Secretion by GLP-1 and Kisspeptin-10. PLoS ONE 9: e113020, 2014). In the current studies, we evaluated KP signaling in comparison to GLP-1, in order to determine if there are unique components to target in the KP signaling pathway. The effects of GLP-1 and a 10 amino acid analog of KP (KP10) on insulin secretion and cell signaling were compared in insulinoma INS 832/3 cells. As previously described, KP had no effect on insulin secretion in the absence of glucose. In the presence of stimulatory glucose (15.5 mM), KP10 (10 μM) doubled insulin secretion (GSIS without KP10= 471.3 +/- 83.8, GSIS with KP10 = 865.0 +/- 23.8 pgm/min; +/- SEM) with an EC50 of ~5-10 μM. At saturating doses, KP10 (100 μm) and GLP-1 (100 nM) had similar effects on GSIS. Importantly, in combination, they had an additive effect (GLP-1 = 612.5 +/- 25.0, GLP-1 + KP-10 = 739.8 +/- 70.0 pgm/min) consistent with activation of independent downstream signaling pathways. GLP-1 increased cAMP production (165.0 +/- 17.4% over control), whereas KP-10 had no effect. Neither GLP-1 nor KP-10 altered intracellular Ca2+ levels at basal or activating glucose concentrations. Conversely, the addition of KP-10 enhanced glucose-stimulated oxygen consumption (% change over baseline O_2 consumption= 221.2 +/- 44.2%) from INS 832/3 pseudo-islets (Hart et al. Insulinoma-derived pseudo-islets for diabetes research. Am. J. Physio. 321, C247-C256, 2021) which was correlated with a sustained increase in mitochondrial NADH fluorescence. Our findings indicate that KP modulation of mitochondrial metabolic state is related to its ability to potentiate GSIS. Moreover, the KP induced potentiation is synergistic with that promoted by GLP-1 indicating the potential for targeting the KP activated metabolic pathway to further enhance GLP-1 therapeutic effectiveness. Funded by the Juvenile Diabetes Research Foundation (2-SRA-2018-685-S-B), and Procyon Technologies LLC This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Metabolic profiling of transferred mitochondria using FLIM intensity based image segmentation (FIBIS).

Bulk metabolic assays on cell populations have shown that mitochondria transfer trigger breast cancer cells turn towards oxidative phosphorylation (OXPHOS) in favor of proliferation, migration, and cell growth. Yet, the current methods lack single cell resolution of interaction between endogenous and exogenous mitochondria to help understand the consequence metabolic alterations and other cell fate decisions. The phasor approach to fluorescence lifetime imaging microscopy (FLIM) has been widely used to measure the free to bound fraction of reduced form of NADH to quantify metabolic changes in live cells but not at the single mitochondrial scale. Here, we developed the FIBIS algorithm as a robust approach to recognize mitochondria from NADH intensity and further analyze mitochondrial metabolic states to investigate mitochondria transfer. We demonstrate that the NADH autofluorescence perfectly localizes with Mito7-mRuby labeled mitochondria and is free from artifactual emission spectral overlaps. Our data indicates that there was a 40% and a 20% increased fraction of bound NADH after mitochondria transfer in MCF7 and MB231 cells, respectively. This suggests that there is an enhancement of OXPHOS for both breast cancer cells. This was in correlation with the results from the Seahorse XF analyzer where the oxygen consumption rate was significantly higher after uptake of isolated mitochondria from breast epithelial cells. We also used Mitometer to show that there were increasing numbers of mitochondria and branching which imply that the fusion of exogenous and endogenous mitochondria are highly adaptable and increase cellular respiration. In summary, the FIBIS enables single mitochondrial metabolic profiling and aids in understanding the effects of transferred mitochondria to recipient cells. Our results also indicate that transferred mitochondria in cancer cells enhances OXPHOS, increased branching, and mitochondrial numbers, thereby decreasing cell viability and increasing sensitivity to chemotherapy agents.

Total and reduced/oxidized forms of coenzyme Q10 in fibroblasts of patients with mitochondrial disease

Coenzyme Q10 (CoQ10) is involved in ATP production through electron transfer in the mitochondrial respiratory chain complex. CoQ10 receives electrons from respiratory chain complex I and II to become the reduced form, and then transfers electrons at complex III to become the oxidized form. The redox state of CoQ10 has been reported to be a marker of the mitochondrial metabolic state, but to our knowledge, no reports have focused on the individual quantification of reduced and oxidized CoQ10 or the ratio of reduced to total CoQ10 (reduced/total CoQ10) in patients with mitochondrial diseases.We measured reduced and oxidized CoQ10 in skin fibroblasts from 24 mitochondrial disease patients, including 5 primary CoQ10 deficiency patients and 10 respiratory chain complex deficiency patients, and determined the reduced/total CoQ10 ratio.In primary CoQ10 deficiency patients, total CoQ10 levels were significantly decreased, however, the reduced/total CoQ10 ratio was not changed. On the other hand, in mitochondrial disease patients other than primary CoQ10 deficiency patients, total CoQ10 levels did not decrease. However, the reduced/total CoQ10 ratio in patients with respiratory chain complex IV and V deficiency was higher in comparison to those with respiratory chain complex I deficiency.Measurement of CoQ10 in fibroblasts proved useful for the diagnosis of primary CoQ10 deficiency. In addition, the reduced/total CoQ10 ratio may reflect the metabolic status of mitochondrial disease.

Therapeutic implications of mitochondrial stress-induced proteasome inhibitor resistance in multiple myeloma.

The connections between metabolic state and therapy resistance in multiple myeloma (MM) are poorly understood. We previously reported that electron transport chain (ETC) suppression promotes sensitivity to the BCL-2 antagonist venetoclax. Here, we show that ETC suppression promotes resistance to proteasome inhibitors (PIs). Interrogation of ETC-suppressed MM reveals integrated stress response–dependent suppression of protein translation and ubiquitination, leading to PI resistance. ETC and protein translation gene expression signatures from the CoMMpass trial are down-regulated in patients with poor outcome and relapse, corroborating our in vitro findings. ETC-suppressed MM exhibits up-regulation of the cystine-glutamate antiporter SLC7A11, and analysis of patient single-cell RNA-seq shows that clusters with low ETC gene expression correlate with higher SLC7A11 expression. Furthermore, erastin or venetoclax treatment diminishes mitochondrial stress–induced PI resistance. In sum, our work demonstrates that mitochondrial stress promotes PI resistance and underscores the need for implementing combinatorial regimens in MM cognizant of mitochondrial metabolic state.

Energy Metabolism on Mitochondrial Maturation and Its Effects on Cardiomyocyte Cell Fate.

Alterations in energy metabolism play a major role in the lineage of cardiomyocytes, such as the dramatic changes that occur in the transition from neonate to newborn. As cardiomyocytes mature, they shift from a primarily glycolytic state to a mitochondrial oxidative metabolic state. Metabolic intermediates and metabolites may have epigenetic and transcriptional roles in controlling cell fate by increasing mitochondrial biogenesis. In the maturing cardiomyocyte, such as in the postnatal heart, fatty acid oxidation increases in conjunction with increased mitochondrial biogenesis driven by the transcriptional coregulator PGC1-α. PGC1-α is necessary for mitochondrial biogenesis in the heart at birth, with deficiencies leading to postnatal cardiomyopathy. While stem cell therapy as a treatment for heart failure requires further investigation, studies suggest that adult stem cells may secrete cardioprotective factors which may regulate cardiomyocyte differentiation and survival. This review will discuss how metabolism influences mitochondrial biogenesis and how mitochondrial biogenesis influences cell fate, particularly in the context of the developing cardiomyocyte. The implications of energy metabolism on stem cell differentiation into cardiomyocytes and how this may be utilized as a therapy against heart failure and cardiovascular disease will also be discussed.

Thermoplasmonic Regulation of the Mitochondrial Metabolic State for Promoting Directed Differentiation of Dental Pulp Stem Cells.

Regulating stem cell differentiation in a controllable way is significant for regeneration of tissues. Herein, we report a simple and highly efficient method for accelerating the stem cell differentiation of dental pulp stem cells (DPSCs) based on the synergy of the electromagnetic field and the photothermal (thermoplasmonic) effect of plasmonic nanoparticles. By simple laser irradiation at 50 mW/cm2 (10 min per day, totally for 5 days), the thermoplasmonic effect of Au nanoparticles (AuNPs) can effectively regulate mitochondrial metabolism to induce the increase of mitochondrial membrane potential and further drive energy increase during the DPSC differentiation process. The proposed method can specifically regulate DPSCs' cell differentiation toward odontoblasts, with the differentiation time reduced to only 5 days. Simultaneously, the molecular profiling change of mitochondria within DPSCs during the cell differentiation process is revealed by in situ surface-enhanced Raman spectroscopy. It clearly demonstrates that the expression of hydroxyproline and glutamate gradually increases with prolonging of the differentiation days. The developed method is simple, robust, and rapid for stem cell differentiation of DPSCs, which would be beneficial to tissue engineering and regenerative medicine.

A novel HIF-2α targeted inhibitor suppresses hypoxia-induced breast cancer stemness via SOD2-mtROS-PDI/GPR78-UPRER axis

Hypoxic tumor microenvironment (TME) plays critical roles in induction of cancer stem cell-like phenotype in breast cancer and contribute to chemoresistance. However, the mechanism underlying stemness reprogramming of breast cancer cells (BCs) by hypoxic TME remains largely unknown. In the present study, we illustrated that HIF-2α, but not HIF-1α, induces stemness in BCs under hypoxia through SOD2-mtROS-PDI/GRP78-UPRER pathway, linking mitochondrial metabolic state to endoplasmic reticulum (ER) response via mitochondrial reactive oxygen species (mtROS) level. HIF-2α activates endoplasmic reticulum unfolded protein response (UPRER) in drug-sensitive MCF7 and T47D cells to induce drug-resistant stem-like phenotype. Genetic depletion or pharmacological inhibition (YQ-0629) of HIF-2α abolished hypoxia-induced stem-like phenotype in vitro and in vivo. Mechanistically, HIF-2α activates transcription of superoxide dismutase 2 (SOD2) under hypoxia and thereby decreases mtROS level. With less mtROS transported to endoplasmic reticulum, the expression and activity of protein disulfide isomerase (PDI) is suppressed, allowing glucose-regulated protein 78 (GRP78) to dissociate from receptor proteins of UPRER and bind misfolded protein to activate UPRER, which eventually confer chemoresistance and stem-like properties to BCs. Moreover, the increase in mtROS and PDI levels caused by HIF-2α knockdown and the subsequent UPRER inhibition could be substantially rescued by mitoTEMPOL (a mtROS scavenger), 16F16 (a PDI inhibitor), or GRP78 overexpression. Overall, we reported the critical roles of HIF-2α-SOD2-mtROS-PDI/GRP78-UPRER axis in mediating hypoxia-induced stemness in BCs, highlighting the interaction between organelles and providing evidence for further development of targeted HIF-2α inhibitor as a promising therapeutic strategy for chemoresistant breast cancer.

Twist1 as a pivotal regulator of hematopoietic stem cell fate.

Twist1 as a pivotal regulator of hematopoietic stem cell fate.

Enhanced PRL-1 expression in placenta-derived mesenchymal stem cells accelerates hepatic function via mitochondrial dynamics in a cirrhotic rat model

BackgroundPlacenta-derived mesenchymal stem cells (PD-MSCs) have been highlighted as an alternative cell therapy agent that has become a next-generation stem cell treatment. Phosphatase of regenerating liver-1 (PRL-1), an immediate early gene, plays a critical role during liver regeneration. Here, we generated enhanced PRL-1 in PD-MSCs (PD-MSCsPRL-1, PRL-1+) using lentiviral and nonviral gene delivery systems and investigated mitochondrial functions by PD-MSCPRL-1 transplantation for hepatic functions in a rat bile duct ligation (BDL) model.MethodsPD-MSCsPRL-1 were generated by lentiviral and nonviral AMAXA gene delivery systems and analyzed for their characteristics and mitochondrial metabolic functions. Liver cirrhosis was induced in Sprague-Dawley (SD) rats using common BDL for 10 days. PKH67+ naïve and PD-MSCsPRL-1 using a nonviral sysyem (2 × 106 cells/animal) were intravenously administered into cirrhotic rats. The animals were sacrificed at 1, 2, 3, and 5 weeks after transplantation and engraftment of stem cells, and histopathological analysis and hepatic mitochondrial functions were performed.ResultsPD-MSCsPRL-1 were successfully generated using lentiviral and nonviral AMAXA systems and maintained characteristics similar to those of naïve cells. Compared with naïve cells, PD-MSCsPRL-1 improved respirational metabolic states of mitochondria. In particular, mitochondria in PD-MSCsPRL-1 generated by the nonviral AMAXA system showed a significant increase in the respirational metabolic state, including ATP production and mitochondrial biogenesis (*p < 0.05). Furthermore, transplantation of PD-MSCsPRL-1 using a nonviral AMAXA system promoted engraftment into injured target liver tissues of a rat BDL cirrhotic model and enhanced the metabolism of mitochondria via increased mtDNA and ATP production, thereby improving therapeutic efficacy.ConclusionsOur findings will further our understanding of the therapeutic mechanism of enhanced MSCs and provide useful data for the development of next-generation MSC-based cell therapy and therapeutic strategies for regenerative medicine in liver disease.

Photobiomodulation preserves mitochondrial redox state and is retinoprotective in a rodent model of retinitis pigmentosa

Photobiomodulation (PBM) by far-red (FR) to near-infrared (NIR) light has been demonstrated to restore the function of damaged mitochondria, increase the production of cytoprotective factors and prevent cell death. Our laboratory has shown that FR PBM improves functional and structural outcomes in animal models of retinal injury and retinal degenerative disease. The current study tested the hypothesis that a brief course of NIR (830 nm) PBM would preserve mitochondrial metabolic state and attenuate photoreceptor loss in a model of retinitis pigmentosa, the P23H transgenic rat. P23H rat pups were treated with 830 nm light (180 s; 25 mW/cm2; 4.5 J/cm2) using a light-emitting diode array (Quantum Devices, Barneveld, WI) from postnatal day (p) 10 to p25. Sham-treated rats were restrained, but not treated with 830 nm light. Retinal metabolic state, function and morphology were assessed at p30 by measurement of mitochondrial redox (NADH/FAD) state by 3D optical cryo-imaging, electroretinography (ERG), spectral-domain optical coherence tomography (SD-OCT), and histomorphometry. PBM preserved retinal metabolic state, retinal function, and retinal morphology in PBM-treated animals compared to the sham-treated group. PBM protected against the disruption of the oxidation state of the mitochondrial respiratory chain observed in sham-treated animals. Scotopic ERG responses over a range of flash intensities were significantly greater in PBM-treated rats compared to sham controls. SD-OCT studies and histological assessment showed that PBM preserved the structural integrity of the retina. These findings demonstrate for the first time a direct effect of NIR PBM on retinal mitochondrial redox status in a well-established model of retinal disease. They show that chronic proteotoxic stress disrupts retinal bioenergetics resulting in mitochondrial dysfunction, and retinal degeneration and that therapies normalizing mitochondrial metabolism have considerable potential for the treatment of retinal degenerative disease.

The effect of hyperbaric oxygen on mitochondrial and glycolytic energy metabolism: the caloristasis concept.

We present new data on the effects of HBOT on human kidney (HK-2) cell metabolism using a SeaHorse XF Analyzer to evaluate separately the state of mitochondrial and glycolytic energy metabolism. The data are discussed in the context of the concept of cellular caloristasis networks. The information on the changes in cellular energy metabolism stimulated by HBOT presented here provides new insights into the cellular energy state and mitochondrial environment in which sHSPs function. These data will be useful in forming testable hypotheses about the functions of translocated sHSPs in human mitochondria responding to stressors.

Metabolic support of the respiratory burst in blood and sputum neutrophils of patients with community-acquired pneumonia

The aim of the study was to investigate relationships between the respiratory burst in blood and sputum neutrophils and intracellular enzyme activity in patients with pneumonia. Methods. The study involved 82 patients with moderate to severe community-acquired pneumonia (CAP). The chemiluminescent assay was used to investigate synthesis of primary and secondary reactive oxygen species (ROS). The bioluminescent method was used to examine NAD(P)-dependent dehydrogenase activity in neutrophils. Results. Low-intensity respiratory burst in blood neutrophils and its predominant relations to the state of mitochondrial metabolism and malic enzyme activity were observed in healthy subjects. Blood neutrophils in patients with CAP were activated; the anaerobic respiration and mitochondrial metabolism increased in the cells. Additionally, intensity of the respiratory burst was related to terminal glycolysis reactions and the key reaction of the pentose phosphate cycle. Sputum neutrophils in patients with CAP were also activated. Conclusion. Abnormalities of metabolic support of the respiratory burst, such as decreased production of primary and secondary ROS, could appear under unfavorable conditions. Peroxidation increased in the cells, the intensity of terminal glycolysis reactions decreased, and substrates transferred from the citric acid cycle to amino acid exchange reactions. Under these conditions, malic enzyme remained the only substrate that stimulated the respiratory burst in sputum neutrophils, while substrate transfer from the tricarboxylic acid cycle inhibited synthesis of the secondary ROS.

Exposure to Whole Body Vibration Impairs the Functional Activity of the Energy Producing System in Rabbit Myocardium

Indices of the patterns of energy metabolism in the rabbit myocardium were studied after vertical whole body vibration with an amplitude of 0.5 mm at frequencies of 8 and 44 Hz generated by an industrial unit. The energy dependent reactions of native mitochondria were investigated by a polarographic technique using a Clark-type closed membrane electrode. The metabolic states of mitochondria were modeled in vitro by varying exogenous energy substrates (succinic acid, a mixture of glutamic and malic acids) before and after the effect of the 2,4-dinitrophenol uncoupler of oxidative phosphorylation. Amital or malonate-inhibitory analysis was used to estimate the contributions of nicotinamide dinucleotide and flavinadenin dinucleotide-dependent substrates to the endogenous respiratory activity of mitochondria. The results show that changes in the functional activity of myocardial mitochondria in response to vibration depend on the frequency and duration of exposure and are seen as inhibition of the NAD-dependent link of the respiratory chain and activation of the oxidation system of succinic acid, the ligand of metabotropic purinergic G-protein-conjugated receptor GPR91 from the P2Y-family. Due to a systematic deregulated effect vibration can be used as a factor to model bio-energetic cellular hypoxia, to study vibration phenomena at the level of energy producing systems of tissues and organs and for assessment of the vibroprotective properties of drugs.

Cytotoxic Activity of the Histone Deacetylase 3-Selective Inhibitor Pojamide on MDA-MB-231 Triple-Negative Breast Cancer Cells.

We examined the effects of the ferrocene-based histone deacetylase-3 inhibitor Pojamide (N1-(2-aminophenyl)-N8-ferrocenyloctanediamide) and its two derivatives N1-(2-aminophenyl)-N6-ferrocenyladipamide and N1-(2-aminophenyl)-N8-ferroceniumoctanediamide tetrafluoroborate on triple-negative MDA-MB-231 breast cancer cells. Viability/growth assays indicated that only the first two compounds at 70 μM concentration caused an approximate halving of cell number after 24 h of exposure, whereas the tetrafluoroborate derivative exerted no effect on cell survival nor proliferation. Flow cytometric and protein blot analyses were performed on cells exposed to both Pojamide and the ferrocenyladipamide derivative to evaluate cell cycle distribution, apoptosis/autophagy modulation, and mitochondrial metabolic state in order to assess the cellular basis of the cytotoxic effect. The data obtained show that the cytotoxic effect of the two deacetylase inhibitors may be ascribed to the onset of non-apoptotic cell death conceivably linked to a down-regulation of autophagic processes and an impairment of mitochondrial function with an increase in intracellular reactive oxygen species. Our work expands the list of autophagy-regulating drugs and also provides a further example of the role played by the inhibition of autophagy in breast cancer cell death. Moreover, the compounds studied may represent attractive and promising targets for subsequent molecular modeling for anti-neoplastic agents in malignant breast cancer.

Spectrally and spatially resolved laser-induced photobleaching of endogenous flavin fluorescence in cardiac myocytes.

Naturally occurring endogenous fluorescence of flavins, arising in response to excitation by visible light, offers broad opportunity to investigate mitochondrial metabolic state directly in living cells and tissues, including in clinical settings. However, photobleaching, the loss of the autofluorescence intensity following prolonged exposure to light is an inherent phenomenon occurring during the fluorescence acquisition, which can have a negative impact on the recorded data, particularly in the context of measurement of metabolic modulations in pathophysiological conditions. In the presented study, we present a detailed analysis of endogenous flavins fluorescence photobleaching arising in living cardiac cells during spectrally‐resolved confocal imaging. We demonstrate significant nonuniform photobleaching related to different bleaching rates of individual flavin components, resolved by linear spectral unmixing of the recorded signals. Induced photodamage was without effect on the cell morphology, but lead to significant modifications of the cell responsiveness to metabolic modulators and its contractility, suggesting functional metabolic alterations in the recorded cells. These findings point to the necessity of inducing limited photobleaching during metabolic screening in all studies involving visible light excitation and fluorescence acquisition in living cells. © 2018 The Authors Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry

Mitochondrial Membrane Potential (ΔΨ) Fluctuations Associated with the Metabolic States of Mitochondria.

The proton electrochemical gradient generated by the respiratory chain activity accounts for over 90% of the available respiratory energy, and, as such, its evaluation and accurate measurement regarding total values and fluctuations are an invaluable component of the understanding of mitochondrial function. Consequently, alterations in electric potential across the inner mitochondrial membrane generated by differential protonic accumulation and transport is known as the mitochondrial membrane potential, or ΔΨ, and is reflective of the functional metabolic status of mitochondria. There are several experimental approaches to measure ΔΨ, ranging from fluorometric evaluations to electrochemical probes. Here, we will expose a particular method for ΔΨ evaluation, which is dependent on the movement of a particular ion, tetraphenylphosphonium (TPP+) with a selective electrode. The evaluation of the accumulation and movements of TPP+ across the inner mitochondrial membrane is a sensitive, immediate, accurate, and simple method of evaluation of ΔΨ in isolated, respiring mitochondria.

VDAC electronics: 4. Novel electrical mechanism and thermodynamic estimations of glucose repression of yeast respiration

Inhibition of cell respiration by high concentrations of glucose (glucose repression), known as “Crabtree effect”, has been demonstrated for various cancerous strains, highly proliferating cells and yeast lines. Although significant progress in understanding metabolic events associated with the glucose repression of cell respiration has been achieved, it is not yet clear whether the Crabtree effect is the result of a limited activity of the respiratory chain, or of some glucose-mediated regulation of mitochondrial metabolic state. In this work we propose an electrical mechanism of glucose repression of the yeast S. cerevisiae, resulting from generation of the mitochondrial outer membrane potential (OMP) coupled to the direct oxidation of cytosolic NADH in mitochondria. This yeast-type mechanism of OMP generation is different from the earlier proposed VDAC-hexokinase-mediated voltage generation of cancer-type, associated with the mitochondrial outer membrane. The model was developed assuming that VDAC is more permeable to NADH than to NAD+. Thermodynamic estimations of OMP, generated as a result of NADH(2-)/NAD+(1-) turnover through the outer membrane, demonstrated that the values of calculated negative OMP match the known range of VDAC voltage sensitivity, thus suggesting a possibility of OMP-dependent VDAC-mediated regulation of cell energy metabolism. According to the proposed mechanism, we suggest that the yeast-type Crabtree effect is the result of a fast VDAC-mediated electrical repression of mitochondria due to a decrease in the outer membrane permeability to charged metabolites and owing their redistribution between the mitochondrial intermembrane space and the cytosol, both controlled by metabolically-derived OMP.

Mitochondrial permeability transition pore: sensitivity to opening and mechanistic dependence on substrate availability

Mitochondrial Ca2+ uptake has a key role in cellular Ca2+ homeostasis. Excessive matrix Ca2+ concentrations, especially when coincident with oxidative stress, precipitate opening of an inner mitochondrial membrane, high-conductance channel: the mitochondrial permeability transition pore (mPTP). mPTP opening has been implicated as a final cell death pathway in numerous diseases and therefore understanding conditions dictating mPTP opening is crucial for developing targeted therapies. Here, we have investigated the impact of mitochondrial metabolic state on the probability and consequences of mPTP opening. Isolated mitochondria were energised using NADH- or FADH2-linked substrates. The functional consequences of Ca2+-induced mPTP opening were assessed by Ca2+ retention capacity, using fluorescence-based analysis, and simultaneous measurements of mitochondrial Ca2+ handling, membrane potential, respiratory rate and production of reactive oxygen species (ROS). Succinate-induced, membrane potential-dependent reverse electron transfer sensitised mitochondria to mPTP opening. mPTP-induced depolarisation under succinate subsequently inhibited reverse electron transfer. Complex I-driven respiration was reduced after mPTP opening but sustained in the presence of complex II-linked substrates, consistent with inhibition of complex I-supported respiration by leakage of matrix NADH. Additionally, ROS generated at complex III did not sensitise mitochondria to mPTP opening. Thus, cellular metabolic fluxes and metabolic environment dictate mitochondrial functional response to Ca2+ overload.

ROS regulation of axonal mitochondrial transport is mediated by Ca2+ and JNK in Drosophila.

Mitochondria perform critical functions including aerobic ATP production and calcium (Ca2+) homeostasis, but are also a major source of reactive oxygen species (ROS) production. To maintain cellular function and survival in neurons, mitochondria are transported along axons, and accumulate in regions with high demand for their functions. Oxidative stress and abnormal mitochondrial axonal transport are associated with neurodegenerative disorders. However, we know little about the connection between these two. Using the Drosophila third instar larval nervous system as the in vivo model, we found that ROS inhibited mitochondrial axonal transport more specifically, primarily due to reduced flux and velocity, but did not affect transport of other organelles. To understand the mechanisms underlying these effects, we examined Ca2+ levels and the JNK (c-Jun N-terminal Kinase) pathway, which have been shown to regulate mitochondrial transport and general fast axonal transport, respectively. We found that elevated ROS increased Ca2+ levels, and that experimental reduction of Ca2+ to physiological levels rescued ROS-induced defects in mitochondrial transport in primary neuron cell cultures. In addition, in vivo activation of the JNK pathway reduced mitochondrial flux and velocities, while JNK knockdown partially rescued ROS-induced defects in the anterograde direction. We conclude that ROS have the capacity to regulate mitochondrial traffic, and that Ca2+ and JNK signaling play roles in mediating these effects. In addition to transport defects, ROS produces imbalances in mitochondrial fission-fusion and metabolic state, indicating that mitochondrial transport, fission-fusion steady state, and metabolic state are closely interrelated in the response to ROS.