Oxidative Phosphorylation Flux Research Articles

13 C-MFA helps to identify metabolic bottlenecks for improving malic acid production in Myceliophthora thermophila

BackgroundMyceliophthora thermophila has been engineered as a significant cell factory for malic acid production, yet strategies to further enhance production remain unclear and lack rational guidance. 13C-MFA (13C metabolic flux analysis) offers a means to analyze cellular metabolic mechanisms and pinpoint critical nodes for improving product synthesis. Here, we employed 13C-MFA to investigate the metabolic flux distribution of a high-malic acid-producing strain of M. thermophila and attempted to decipher the crucial bottlenecks in the metabolic pathways.ResultsCompared with the wild-type strain, the high-Malic acid-producing strain M. thermophila JG207 exhibited greater glucose uptake and carbon dioxide evolution rates but lower oxygen uptake rates and biomass yields. Consistent with these phenotypes, the 13C-MFA results showed that JG207 displayed elevated flux through the EMP pathway and downstream TCA cycle, along with reduced oxidative phosphorylation flux, thereby providing more precursors and NADH for malic acid synthesis. Furthermore, based on the 13C-MFA results, we conducted oxygen-limited culture and nicotinamide nucleotide transhydrogenase (NNT) gene knockout experiments to increase the cytoplasmic NADH level, both of which were shown to be beneficial for malic acid accumulation.ConclusionsThis work elucidates and validates the key node for achieving high malic acid production in M. thermophila. We propose effective fermentation strategies and genetic modifications for enhancing malic acid production. These findings offer valuable guidance for the rational design of future cell factories aimed at improving malic acid yields.

Pan-tissue mitochondrial phenotyping reveals lower OXPHOS expression and function across cancer types

Targeting mitochondrial oxidative phosphorylation (OXPHOS) to treat cancer has been hampered due to serious side-effects potentially arising from the inability to discriminate between non-cancerous and cancerous mitochondria. Herein, comprehensive mitochondrial phenotyping was leveraged to define both the composition and function of OXPHOS across various murine cancers and compared to both matched normal tissues and other organs. When compared to both matched normal tissues, as well as high OXPHOS reliant organs like heart, intrinsic expression of the OXPHOS complexes, as well as OXPHOS flux were discovered to be consistently lower across distinct cancer types. Assuming intrinsic OXPHOS expression/function predicts OXPHOS reliance in vivo, these data suggest that pharmacologic blockade of mitochondrial OXPHOS likely compromises bioenergetic homeostasis in healthy oxidative organs prior to impacting tumor mitochondrial flux in a clinically meaningful way. Although these data caution against the use of indiscriminate mitochondrial inhibitors for cancer treatment, considerable heterogeneity was observed across cancer types with respect to both mitochondrial proteome composition and substrate-specific flux, highlighting the possibility for targeting discrete mitochondrial proteins or pathways unique to a given cancer type.

Neuroinflammation, Energy and Sphingolipid Metabolism Biomarkers Are Revealed by Metabolic Modeling of Autistic Brains

Autism spectrum disorders (ASD) are a heterogeneous group of neurodevelopmental disorders generally characterized by repetitive behaviors and difficulties in communication and social behavior. Despite its heterogeneous nature, several metabolic dysregulations are prevalent in individuals with ASD. This work aims to understand ASD brain metabolism by constructing an ASD-specific prefrontal cortex genome-scale metabolic model (GEM) using transcriptomics data to decipher novel neuroinflammatory biomarkers. The healthy and ASD-specific models are compared via uniform sampling to identify ASD-exclusive metabolic features. Noticeably, the results of our simulations and those found in the literature are comparable, supporting the accuracy of our reconstructed ASD model. We identified that several oxidative stress, mitochondrial dysfunction, and inflammatory markers are elevated in ASD. While oxidative phosphorylation fluxes were similar for healthy and ASD-specific models, and the fluxes through the pathway were nearly undisturbed, the tricarboxylic acid (TCA) fluxes indicated disruptions in the pathway. Similarly, the secretions of mitochondrial dysfunction markers such as pyruvate are found to be higher, as well as the activities of oxidative stress marker enzymes like alanine and aspartate aminotransferases (ALT and AST) and glutathione-disulfide reductase (GSR). We also detected abnormalities in the sphingolipid metabolism, which has been implicated in many inflammatory and immune processes, but its relationship with ASD has not been thoroughly explored in the existing literature. We suggest that important sphingolipid metabolites, such as sphingosine-1-phosphate (S1P), ceramide, and glucosylceramide, may be promising biomarkers for the diagnosis of ASD and provide an opportunity for the adoption of early intervention for young children.

P-cadherin induces anoikis-resistance of matrix-detached breast cancer cells by promoting pentose phosphate pathway and decreasing oxidative stress

Successful metastatic spreading relies on cancer cells with stem-like properties, glycolytic metabolism and increased antioxidant protection, allowing them to escape anoikis and to survive in circulation. The expression of P-cadherin, a poor prognostic factor in breast cancer, is associated with hypoxic, glycolytic and acidosis biomarkers. In agreement, P-cadherin-enriched breast cancer cell populations presents a glycolytic and an acid-resistance phenotype.Our aim was to evaluate whether P-cadherin expression controls the glycolytic and oxidative phosphorylation fluxes of matrix-detached breast cancer cells, acting as an antioxidant and enhancing their survival in anchorage-independent conditions. By using matrix-detached breast cancer cells, we concluded that P-cadherin increases glucose-6-phosphate dehydrogenase expression, up-regulating the carbon flux through the pentose phosphate pathway, while inhibiting pyruvate oxidation to acetyl-coA via pyruvate dehydrogenase kinase-4 (PDK-4) activation. Accordingly, P-cadherin expression conferred increased sensitivity to dichloroacetate (DCA), a PDK inhibitor. P-cadherin expression also regulates oxidative stress in matrix-detached breast cancer cells, through the control of antioxidant systems, such as catalase and superoxide dismutases (SOD)1 and 2, providing these cells with an increased resistance to doxorubicin-induced anoikis. Importantly, this association was validated in primary invasive breast carcinomas, where an enrichment of SOD2 was found in P-cadherin-overexpressing breast carcinomas.In conclusion, we propose that P-cadherin up-regulates carbon flux through the pentose phosphate pathway and decreases oxidative stress in matrix-detached breast cancer cells. These metabolic remodeling and antioxidant roles of P-cadherin can promote the survival of breast cancer cells in circulation and in metastatic sites, being a possible player in breast cancer therapeutic resistance to pro-oxidant-based interventions.

Temporal fluxomics reveals oscillations in TCA cycle flux throughout the mammalian cell cycle

Cellular metabolic demands change throughout the cell cycle. Nevertheless, a characterization of how metabolic fluxes adapt to the changing demands throughout the cell cycle is lacking. Here, we developed a temporal‐fluxomics approach to derive a comprehensive and quantitative view of alterations in metabolic fluxes throughout the mammalian cell cycle. This is achieved by combining pulse‐chase LC‐MS‐based isotope tracing in synchronized cell populations with computational deconvolution and metabolic flux modeling. We find that TCA cycle fluxes are rewired as cells progress through the cell cycle with complementary oscillations of glucose versus glutamine‐derived fluxes: Oxidation of glucose‐derived flux peaks in late G1 phase, while oxidative and reductive glutamine metabolism dominates S phase. These complementary flux oscillations maintain a constant production rate of reducing equivalents and oxidative phosphorylation flux throughout the cell cycle. The shift from glucose to glutamine oxidation in S phase plays an important role in cell cycle progression and cell proliferation.

Intermittent hypoxia leads to functional reorganization of mitochondria and affects cellular bioenergetics in marine molluscs.

Fluctuations in oxygen (O2) concentrations represent a major challenge to aerobic organisms and can be extremely damaging to their mitochondria. Marine intertidal molluscs are well-adapted to frequent O2 fluctuations, yet it remains unknown how their mitochondrial functions are regulated to sustain energy metabolism and prevent cellular damage during hypoxia and reoxygenation (H/R). We used metabolic control analysis to investigate the mechanisms of mitochondrial responses to H/R stress (18 h at <0.1% O2 followed by 1 h of reoxygenation) using hypoxia-tolerant intertidal clams Mercenaria mercenaria and hypoxia-sensitive subtidal scallops Argopecten irradians as models. We also assessed H/R-induced changes in cellular energy balance, oxidative damage and unfolded protein response to determine the potential links between mitochondrial dysfunction and cellular injury. Mitochondrial responses to H/R in scallops strongly resembled those in other hypoxia-sensitive organisms. Exposure to hypoxia followed by reoxygenation led to a strong decrease in the substrate oxidation (SOX) and phosphorylation (PHOS) capacities as well as partial depolarization of mitochondria of scallops. Elevated mRNA expression of a reactive oxygen species-sensitive enzyme aconitase and Lon protease (responsible for degradation of oxidized mitochondrial proteins) during H/R stress was consistent with elevated levels of oxidative stress in mitochondria of scallops. In hypoxia-tolerant clams, mitochondrial SOX capacity was enhanced during hypoxia and continued rising during the first hour of reoxygenation. In both species, the mitochondrial PHOS capacity was suppressed during hypoxia, likely to prevent ATP wastage by the reverse action of FO,F1-ATPase. The PHOS capacity recovered after 1 h of reoxygenation in clams but not in scallops. Compared with scallops, clams showed a greater suppression of energy-consuming processes (such as protein turnover and ion transport) during hypoxia, indicated by inactivation of the translation initiation factor EIF-2α, suppression of 26S proteasome activity and a dramatic decrease in the activity of Na(+)/K(+)-ATPase. The steady-state levels of adenylates were preserved during H/R exposure and AMP-dependent protein kinase was not activated in either species, indicating that the H/R exposure did not lead to severe energy deficiency. Taken together, our findings suggest that mitochondrial reorganizations sustaining high oxidative phosphorylation flux during recovery, combined with the ability to suppress ATP-demanding cellular functions during hypoxia, may contribute to high resilience of clams to H/R stress and help maintain energy homeostasis during frequent H/R cycles in the intertidal zone.

Changes in mitochondrial function and mitochondria associated protein expression in response to 2-weeks of high intensity interval training.

Purpose: High-intensity short-duration interval training (HIT) stimulates functional and metabolic adaptation in skeletal muscle, but the influence of HIT on mitochondrial function remains poorly studied in humans. Mitochondrial metabolism as well as mitochondrial-associated protein expression were tested in untrained participants performing HIT over a 2-week period.Methods: Eight males performed a single-leg cycling protocol (12 × 1 min intervals at 120% peak power output, 90 s recovery, 4 days/week). Muscle biopsies (vastus lateralis) were taken pre- and post-HIT. Mitochondrial respiration in permeabilized fibers, citrate synthase (CS) activity and protein expression of peroxisome proliferator-activated receptor gamma coactivator (PGC-1α) and respiratory complex components were measured.Results: HIT training improved peak power and time to fatigue. Increases in absolute oxidative phosphorylation (OXPHOS) capacities and CS activity were observed, but not in the ratio of CCO to the electron transport system (CCO/ETS), the respiratory control ratios (RCR-1 and RCR-2) or mitochondrial-associated protein expression. Specific increases in OXPHOS flux were not apparent after normalization to CS, indicating that gross changes mainly resulted from increased mitochondrial mass.Conclusion: Over only 2 weeks HIT significantly increased mitochondrial function in skeletal muscle independently of detectable changes in mitochondrial-associated and mitogenic protein expression.

The shock of vacuolar PrA on glycolytic flux, oxidative phosphorylation, and cell morphology by industrial Saccharomyces cerevisiae WZ65

Proteinase A (PrA) is one of the most significant vacuolar proteinase in S. cerevisiae, and it plays an important role in S. cerevisiae physiology and metabolism, especially under unfavorable environment. In this study, the differences in pyruvate kinase (PYK) level under fructose-1,6-diphosphate (FDP) induction and ATP synthesis block among SC1 (the wild-type yeast that was industrial Saccharomyces cerevisiae WZ65), SC2 (PEP4 partial deletion) and SC3 (PEP4 complete deletion) were examined. Results showed that the induction caused by FDP clearly increased PYK expression no matter for which strain, but the increasing effect is more significant for SC2 (P < 0.05). The comparative results of intracellular ATP accumulation showed that the induction by FDP may be affected at the presence of PrA. The block experiment of ATP synthesis showed that PYK activities in PEP4-modified strains are lower than that of the wild type, but the intracellular ATP levels in the wild-type one are generally higher than the PEP4-modified strains after rotenone treatment (P < 0.01). This implies that the effect of PrA deficiency on intracellular ATP accumulation was much more pronounced than the effect of rotenone on oxidative phosphorylation. The cell morphology of three strains was comparatively examined by means of transmission electron microscopy (TEM). The PEP4-modified strains possessed more vacuoles, and cell structure were more integrated than the wild-type strain. Current data preliminarily indicated that the deletion of PEP4 gene in industrial S. cerevisiae WZ65 may not only affected PYK expression but also modulated the oxidative phosphorylation flux.

A radical approach to beating hypoxia: depressed free radical release from heart fibres of the hypoxia-tolerant epaulette shark (Hemiscyllum ocellatum)

Hypoxia and warm ischemia are primary concerns in ischemic heart disease and transplant and trauma. Hypoxia impacts tissue ATP supply and can induce mitochondrial dysfunction that elevates reactive species release. The epaulette shark, Hemiscyllum ocellatum, is remarkably tolerant of severe hypoxia at temperatures up to 34 °C, and therefore provides a valuable model to study warm hypoxia tolerance. Mitochondrial function was tested in saponin permeabilised ventricle fibres using high-resolution respirometry coupled with purpose-built fluorospectrometers. Ventricular mitochondrial function, stability and reactive species production of the epaulette shark was compared with that of the hypoxia-sensitive shovelnose ray, Aptychotrema rostrata. Fibres were prepared from each species acclimated to normoxic water conditions, or following a 2 h, acute hypoxic exposure at levels representing 40% of each species' critical oxygen tension. Although mitochondrial respiratory fluxes for normoxia-acclimated animals were similar for both species, reactive species production in the epaulette shark was approximately half that of the shovelnose ray under normoxic conditions, even when normalised to tissue oxidative phosphorylation flux. The hypoxia-sensitive shovelnose ray halved oxidative phosphorylation flux and cytochrome c oxidase flux was depressed by 34% following hypoxic stress. In contrast, oxidative phosphorylation flux of the epaulette shark ventricular fibres isolated from acute hypoxia exposed the animals remained similar to those from normoxia-acclimated animals. However, uncoupling of respiration revealed depressed electron transport systems in both species following hypoxia exposure. Overall, the epaulette shark ventricular mitochondria showed greater oxidative phosphorylation stability and lower reactive species outputs with hypoxic exposure, and this may protect cardiac bioenergetic function in hypoxic tropical waters.

Bayesian flux balance analysis applied to a skeletal muscle metabolic model

In this article, the steady state condition for the multi-compartment models for cellular metabolism is considered. The problem is to estimate the reaction and transport fluxes, as well as the concentrations in venous blood when the stoichiometry and bound constraints for the fluxes and the concentrations are given. The problem has been addressed previously by a number of authors, and optimization-based approaches as well as extreme pathway analysis have been proposed. These approaches are briefly discussed here. The main emphasis of this work is a Bayesian statistical approach to the flux balance analysis (FBA). We show how the bound constraints and optimality conditions such as maximizing the oxidative phosphorylation flux can be incorporated into the model in the Bayesian framework by proper construction of the prior densities. We propose an effective Markov chain Monte Carlo (MCMC) scheme to explore the posterior densities, and compare the results with those obtained via the previously studied linear programming (LP) approach. The proposed methodology, which is applied here to a two-compartment model for skeletal muscle metabolism, can be extended to more complex models.

Modular Kinetic Analysis of the Adenine Nucleotide Translocator–Mediated Effects of Palmitoyl-CoA on the Oxidative Phosphorylation in Isolated Rat Liver Mitochondria

To test whether long-chain fatty acyl-CoA esters link obesity with type 2 diabetes through inhibition of the mitochondrial adenine nucleotide translocator, we applied a system-biology approach, dual modular kinetic analysis, with mitochondrial membrane potential (Deltapsi) and the fraction of matrix ATP as intermediates. We found that 5 mumol/l palmitoyl-CoA inhibited adenine nucleotide translocator, without direct effect on other components of oxidative phosphorylation. Indirect effects depended on how oxidative phosphorylation was regulated. When the electron donor and phosphate acceptor were in excess, and the mitochondrial "work" flux was allowed to vary, palmitoyl-CoA decreased phosphorylation flux by 38% and the fraction of ATP in the medium by 39%. Deltapsi increased by 15 mV, and the fraction of matrix ATP increased by 46%. Palmitoyl-CoA had a stronger effect when the flux through the mitochondrial electron transfer chain was maintained constant: Deltapsi increased by 27 mV, and the fraction of matrix ATP increased 2.6 times. When oxidative phosphorylation flux was kept constant by adjusting the rate using hexokinase, Deltapsi and the fraction of ATP were not affected. Palmitoyl-CoA increased the extramitochondrial AMP concentration significantly. The effects of palmitoyl-CoA in our model system support the proposed mechanism linking obesity and type 2 diabetes through an effect on adenine nucleotide translocator.

Energetics of isolated hepatocyte swelling induced by sodium co-transported amino acids.

This study was designed to investigate the energetics of isolated rat hepatocyte swelling due to sodium-cotransported amino acid accumulation in a medium containing either glucose or octanoate as basal substrate. We show that the size of the increase in cytosolic volume is directly correlated with the total amino acid accumulation, which depends on the difference of electrical potential across the plasma membrane. Such a change in cell volume, with either glucose or octanoate, does not modify the mitochondrial volume. Addition of sodium-cotransported amino acids for which the metabolism was avoided showed that the rise in cell volume, per se, did not change the respiratory rate, deltap, or phosphate potential in either mitochondrial or cytosolic compartments. Conversely, the large increase in oxidative phosphorylation flux was due to the metabolism of amino acids as a consequence of a rise in electron supply for the respiratory chain rather than an increase in cellular ATP demand, as indicated by the increase in cytosolic phosphate potential. Moreover, although we confirm that octanoate addition largely increases the respiratory rate by a process different from uncoupling, we observed that the same overall thermodynamic driving force through the respiratory chain and the same mitochondrial or cytosolic phosphate potential were maintained for much higher oxygen consumption when octanoate was present. We propose that these octanoate effects are due to a decrease in the actual protons/2 electrons stoichiometry as a consequence of a shift in electron supply toward a two-coupling site instead of a three-coupling site. The change in the FADH2/NADH formation flux ratio in either fatty acid or carbohydrate oxidation explains such results.