Cytosolic ATP Research Articles

Aggregation-prone Tau impairs mitochondrial import, which affects organelle morphology and neuronal complexity.

Mitochondrial protein import is essential for organellar biogenesis, and thereby for the sufficient supply of cytosolic ATP - which is particularly important for cells with high energy demands like neurons. This study explores the prospect of import machinery perturbation as a cause of neurodegeneration instigated by the accumulation of aggregating proteins linked to disease. We found that the aggregation-prone Tau variant (TauP301L) reduces the levels of components of the import machinery of the outer (TOM20, encoded by TOMM20) and inner membrane (TIM23, encoded by TIMM23) while associating with TOM40 (TOMM40). Intriguingly, this interaction affects mitochondrial morphology, but not protein import or respiratory function; raising the prospect of an intrinsic rescue mechanism. Indeed, TauP301L induced the formation of tunnelling nanotubes (TNTs), potentially for the recruitment of healthy mitochondria from neighbouring cells and/or the disposal of mitochondria incapacitated by aggregated Tau. Consistent with this, inhibition of TNT formation (and rescue) reveals Tau-induced import impairment. In primary neuronal cultures, TauP301L induced morphological changes characteristic of neurodegeneration. Interestingly, these effects were mirrored in cells where the import sites were blocked artificially. Our results reveal a link between aggregation-prone Tau and defective mitochondrial import relevant to disease.

Pulsed Radiofrequency Decreases pERK and Affects Intracellular Ca2+ Influx, Cytosolic ATP Level, and Mitochondrial Membrane Potential in the Sensitized Dorsal Root Ganglion Neuron Induced by N-Methyl D-Aspartate.

The molecular mechanism of pulsed radiofrequency (PRF) in chronic pain management is not fully understood. Chronic pain involves the activation of specific N-Methyl D-Aspartate receptors (NMDAR) to induce central sensitization. This study aims to determine the effect of PRF on central sensitization biomarker phosphorylated extracellular signal-regulated kinase (pERK), Ca2+ influx, cytosolic ATP level, and mitochondrial membrane potential (Δψm) of the sensitized dorsal root ganglion (DRG) neuron following NMDAR activation. This study is a true experimental in-vitro study on a sensitized DRG neuron induced with 80 µM NMDA. There are six treatment groups including control, NMDA 80 µM, Ketamine 100 µM, PRF 2Hz, NMDA 80 µM + PRF 2 Hz, and NMDA 80 µM + PRF 2 Hz + ketamine 100 µM. We use PRF 2 Hz 20 ms for 360 seconds. Statistical analysis was performed using the One-Way ANOVA and the Pearson correlation test with α=5%. In the sensitized DRG neuron, there is a significant elevation of pERK. There is a strong correlation between Ca2+, cytosolic ATP level, and Δψm with pERK intensity (p<0.05). PRF treatment decreases pERK intensity from 108.48 ± 16.95 AU to 38.57 ± 5.20 AU (p<0.05). PRF exposure to sensitized neurons also exhibits a Ca2+ influx, but still lower than in the unexposed neuron. PRF exposure in sensitized neurons has a higher cytosolic ATP level (0.0458 ± 0.0010 mM) than in the unexposed sensitized neuron (0.0198 ± 0.0004 mM) (p<0.05). PRF also decreases Δψm in the sensitized neuron from 109.24 ± 6.43 AU to 33.21 ± 1.769 AU (p<0.05). PRF mechanisms related to DRG neuron sensitization are by decreasing pERK, altering Ca2+ influx, increasing cytosolic ATP level, and decreasing Δψm which is associated with neuron sensitization following NMDAR activation.

Stem bark methanolic extract of Bauhinia thonningii (Schum) demonstrates gastroprotective properties against in vivo ethanol-induced injury

Background: Stomach ulcer is one of the diseases that many patients suffer from all over the world, and it causes damage to the intestinal wall. Some research has shown that Bauhinia thonningii extract, which has been traditionally used to treat a variety of ailments, including stomach ulcers, has powerful antioxidant and mucus-increasing properties. Objective:This research is aimed at studying the mechanism(s) through which methanolic (MBST) and chloroform (CBST) extracts of B. thonningii stem bark protects against ethanol-induced stomach damage in rats. Material and Methods: MBST and CBST were evaluated in vivo for their cytoprotective qualities employing stomach acidity, mucus content, macroscopic and microscopic methods, lipid peroxidation, nitric oxide, mass spectroscopy, 2D gel electrophoresis, and biochemical indicators. These extracts were tested in vitro on lipopolysaccharide/interferon-stimulated rat cells. Additionally, in-vivo, and in-vitro toxicity investigations were conducted. FRAP, DPPH, and ORAC tests were used to determine the antioxidant activity of MBST and CBST. Chemical and spectroscopic approaches were used to analyze the phytochemistry of MBST and CBST.Results: The gross and histological characteristics of B. thonningii proved its anti-ulcerogenic effects. MBST has gastroprotective effects by modulating lipid peroxidation and proteomics indicators [heat shock protein (Hsp60), disulfide isomerase (ERp60), protein disulfide-isomerase, creatine kinase B, cytosolic malate dehydrogenase, peroxiredoxin-6, and ATP synthase]. MBST and CBST have substantial impacts on nitric oxide both in-vitro and in-vivo. In comparison to CBST, methanolic extract (MBST) demonstrated greater gastroprotective properties, polyphenolic content, and antioxidant activity. The plant extracts did not show any toxicity in-vitro or in-vivo. MBST shows anti-ulcer action, which may be ascribed to its ability to reduce ethanol-triggered oxidative injury, to intervene in proteomic mechanisms, and to modulate nitric oxide.Conclusion:The present study found that stem bark extracts of Bauhinia thonningii Schum have cytoprotective activities against in vivo ethanol-induced damage due to its powerful antioxidant and mucus-increasing capabilities. Bauhinia thonningii warrants more attention since it may provide an exciting novel medication for the treatment of acute erosive gastropathy.Keywords: Bauhinia thonningii Schum;polar and non-polar extraction; peptic ulcer; nitric oxide; proteomics

Neurons require glucose uptake and glycolysis in vivo

Neurons require large amounts of energy, but whether they can perform glycolysis or require glycolysis to maintain energy remains unclear. Using metabolomics, we show that human neurons do metabolize glucose through glycolysis and can rely on glycolysis to supply tricarboxylic acid (TCA) cycle metabolites. To investigate the requirement for glycolysis, we generated mice with postnatal deletion of either the dominant neuronal glucose transporter (GLUT3cKO) or the neuronal-enriched pyruvate kinase isoform (PKM1cKO) in CA1 and other hippocampal neurons. GLUT3cKO and PKM1cKO mice show age-dependent learning and memory deficits. Hyperpolarized magnetic resonance spectroscopic (MRS) imaging shows that female PKM1cKO mice have increased pyruvate-to-lactate conversion, whereas female GLUT3cKO mice have decreased conversion, body weight, and brain volume. GLUT3KO neurons also have decreased cytosolic glucose and ATP at nerve terminals, with spatial genomics and metabolomics revealing compensatory changes in mitochondrial bioenergetics and galactose metabolism. Therefore, neurons metabolize glucose through glycolysis invivo and require glycolysis for normal function.

The SGLT2 inhibitor empagliflozin improves cardiac energy status via mitochondrial ATP production in diabetic mice

Empagliflozin, a sodium-glucose co-transporter 2 inhibitor developed, has been shown to reduce cardiovascular events in patients with type 2 diabetes and established cardiovascular disease. Several studies have suggested that empagliflozin improves the cardiac energy state which is a partial cause of its potency. However, the detailed mechanism remains unclear. To address this issue, we used a mouse model that enabled direct measurement of cytosolic and mitochondrial ATP levels. Empagliflozin treatment significantly increased cytosolic and mitochondrial ATP levels in the hearts of db/db mice. Empagliflozin also enhanced cardiac robustness by maintaining intracellular ATP levels and the recovery capacity in the infarcted area during ischemic-reperfusion. Our findings suggest that empagliflozin enters cardiac mitochondria and directly causes these effects by increasing mitochondrial ATP via inhibition of NHE1 and Nav1.5 or their common downstream sites. These cardioprotective effects may be involved in the beneficial effects on heart failure seen in clinical trials.

Mitochondrial alternative NADH dehydrogenases NDA1 and NDA2 promote survival of reoxygenation stress in Arabidopsis by safeguarding photosynthesis and limiting ROS generation.

Plant submergence stress is a growing problem for global agriculture. During desubmergence, rising O2 concentrations meet a highly reduced mitochondrial electron transport chain (mETC) in the cells. This combination favors the generation of reactive oxygen species (ROS) by the mitochondria, which at excess can cause damage. The cellular mechanisms underpinning the management of reoxygenation stress are not fully understood. We investigated the role of alternative NADH dehydrogenases (NDs), as components of the alternative mETC in Arabidopsis, in anoxia-reoxygenation stress management. Simultaneous loss of the matrix-facing NDs, NDA1 and NDA2, decreased seedling survival after reoxygenation, while overexpression increased survival. The absence of NDAs led to reduced maximum potential quantum efficiency of photosystem II linking the alternative mETC to photosynthetic function in the chloroplast. NDA1 and NDA2 were induced upon reoxygenation, and transcriptional activation of NDA1 was controlled by the transcription factors ANAC016 and ANAC017 that bind to the mitochondrial dysfunction motif (MDM) in the NDA1 promoter. The absence of NDA1 and NDA2 did not alter recovery of cytosolic ATP levels and NADH : NAD+ ratio at reoxygenation. Rather, the absence of NDAs led to elevated ROS production, while their overexpression limited ROS. Our observations indicate that the control of ROS formation by the alternative mETC is important for photosynthetic recovery and for seedling survival of anoxia-reoxygenation stress.

Phosphate-deprivation and damage signalling by extracellular ATP.

Phosphate deprivation compromises plant productivity and modulates immunity. DAMP signalling by extracellular ATP (eATP) could be compromised under phosphate deprivation by the lowered production of cytosolic ATP and the need to salvage eATP as a nutritional phosphate source. Phosphate-starved roots of Arabidopsis can still sense eATP, indicating robustness in receptor function. However, the resultant cytosolic free Ca2+ signature is impaired, indicating modulation of downstream components. This perspective on DAMP signalling by extracellular ATP (eATP) addresses the salvage of eATP under phosphate deprivation and its promotion of immunity, how Ca2+ signals are generated and how the Ca2+ signalling pathway could be overcome to allow beneficial fungal root colonization to fulfill phosphate demands. Safe passage for an endophytic fungus allowing root colonization could be achieved by its down-regulation of the Ca2+ channels that act downstream of the eATP receptors and by also preventing ROS accumulation, thus further impairing DAMP signalling.

Mitochondrial H2S donor AP39 induces stomatal closure by modulating guard cell mitochondrial activity.

Hydrogen sulfide (H2S) is a gaseous signaling molecule involved in numerous physiological processes in plants, including gas exchange with the environment through the regulation of stomatal pore width. Guard cells (GCs) are pairs of specialized epidermal cells that delimit stomatal pores and have a higher mitochondrial density and metabolic activity than their neighboring cells. However, there is no clear evidence on the role of mitochondrial activity in stomatal closure induction. In this work, we showed that the mitochondrial-targeted H2S donor AP39 induces stomatal closure in a dose-dependent manner. Experiments using inhibitors of the mitochondrial electron transport chain (mETC) or insertional mutants in cytochrome c (CYTc) indicated that the activity of mitochondrial CYTc and/or complex IV are required for AP39-dependent stomatal closure. By using fluorescent probes and genetically encoded biosensors we reported that AP39 hyperpolarized the mitochondrial inner potential (Δψm) and increased cytosolic ATP, cytosolic hydrogen peroxide levels, and oxidation of the glutathione pool in GCs. These findings showed that mitochondrial-targeted H2S donors induce stomatal closure, modulate guard cell mETC activity, the cytosolic energetic and oxidative status, pointing to an interplay between mitochondrial H2S, mitochondrial activity, and stomatal closure.

Contraction of the rigor actomyosin complex drives bulk hemoglobin expulsion from hemolyzing erythrocytes

Erythrocyte ghost formation via hemolysis is a key event in the physiological clearance of senescent red blood cells (RBCs) in the spleen. The turnover rate of millions of RBCs per second necessitates a rapid efflux of hemoglobin (Hb) from RBCs by a not yet identified mechanism. Using high-speed video-microscopy of isolated RBCs, we show that electroporation-induced efflux of cytosolic ATP and other small solutes leads to transient cell shrinkage and echinocytosis, followed by osmotic swelling to the critical hemolytic volume. The onset of hemolysis coincided with a sudden self-propelled cell motion, accompanied by cell contraction and Hb-jet ejection. Our biomechanical model, which relates the Hb-jet-driven cell motion to the cytosolic pressure generation via elastic contraction of the RBC membrane, showed that the contributions of the bilayer and the bilayer-anchored spectrin cytoskeleton to the hemolytic cell motion are negligible. Consistent with the biomechanical analysis, our biochemical experiments, involving extracellular ATP and the myosin inhibitor blebbistatin, identify the low abundant non-muscle myosin 2A (NM2A) as the key contributor to the Hb-jet emission and fast hemolytic cell motion. Thus, our data reveal a rapid myosin-based mechanism of hemolysis, as opposed to a much slower diffusive Hb efflux.

Suicidal Death of Human Erythrocytes Following Exposure to Pentostatin

Background/Aims: Pentostatin (2'-deoxycoformycin), a purine analog, is used for the treatment of diverse B and T-cell malignancies as well as for immunosuppression. Pentostatin is at least in part effective by triggering apoptosis. Pentostatin sensitive mechanisms leading to apoptosis include accumulation of DNA strand breaks, altered transcription and mitochondrial depolarization. Erythrocytes lack nuclei and mitochondria but nevertheless may enter eryptosis, an apoptosis-like suicidal cell death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i), ceramide formation and energy depletion. The present study tested, whether and how pentostatin induces eryptosis. Methods: The phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, hemolysis from hemoglobin release, [Ca2+]i from Fluo3-fluorescence, ceramide abundance utilizing specific antibodies, and cytosolic ATP concentration utilizing a luciferin–luciferase assay kit. Results: A 48 hours exposure of human erythrocytes to pentostatin (≥5 µg/ml) significantly increased the percentage of annexin-V-binding cells and significantly decreased forward scatter. Pentostatin significantly increased [Ca2+]i, and significantly decreased cytosolic ATP, but did not significantly modify ceramide abundance. The effect of pentostatin on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+. Conclusion: Pentostatin triggers erythrocyte shrinkage and phospholipid scrambling of the erythrocyte cell membrane, effects paralleled by and at least partially due to entry of extracellular Ca2+ and cellular energy depletion.

Effect of TP53 deficiency and KRAS signaling on the bioenergetics of colon cancer cells in response to different substrates: A single cell study.

Metabolic reprogramming is a hallmark of cancer. Somatic mutations in genes involved in oncogenic signaling pathways, including KRAS and TP53, rewire the metabolic machinery in cancer cells. We here set out to determine, at the single cell level, metabolic signatures in human colon cancer cells engineered to express combinations of activating KRAS gene mutations and TP53 gene deletions. Specifically, we explored how somatic mutations in these genes and substrate availability (lactate, glucose, substrate deprivation) from the extracellular microenvironment affect bioenergetic parameters, including cellular ATP, NADH and mitochondrial membrane potential dynamics. Employing cytosolic and mitochondrial FRET-based ATP probes, fluorescent NADH sensors, and the membrane-permeant cationic fluorescent probe TMRM in HCT-116 cells as a model system, we observed that TP53 deletion and KRAS mutations drive a shift in metabolic signatures enabling lactate to become an efficient metabolite to replenish both ATP and NADH following nutrient deprivation. Intriguingly, cytosolic, mitochondrial and overall cellular ATP measurements revealed that, in WT KRAS cells, TP53 deficiency leads to an enhanced ATP production in the presence of extracellular lactate and glucose, and to the greatest increase in ATP following a starvation period. On the other hand, oncogenic KRAS in TP53-deficient cells reversed the alterations in cellular ATP levels. Moreover, cell population measurements of mitochondrial and glycolytic metabolism using a Seahorse analyzer demonstrated that WT KRAS TP53-silenced cells display an increase of the basal respiration and tightly-coupled mitochondria, in the presence of glucose as substrate, compared to TP53 competent cells. Furthermore, cells possessing oncogenic KRAS, independently of TP53 status, showed less pronounced mitochondrial membrane potential changes in response to metabolic nutrients. Furthermore, analysis of cytosolic and mitochondrial NADH levels revealed that the simultaneous presence of TP53 deletion and oncogenic KRAS showed the most pronounced alteration in cytosolic and mitochondrial NADH during metabolic stress. In conclusion, our findings demonstrate how activating KRAS mutation and loss of TP53 remodel cancer metabolism and lead to alterations in bioenergetics under metabolic stress conditions by modulating cellular ATP production, NADH oxidation, mitochondrial respiration and function.

Adenine nucleotides transport across the inner mitochondrial membrane in cancer cells: role of ANTs and SCaMCs

Introduction: The regulation of adenine nucleotide levels in the mitochondria and the cytosol is essential for cell metabolism. To sustain mitochondrial respiration, ATP generated by ATP synthase must be exported to the cytosol, whereas mitochondria must be refilled with ADP and Pi. Adenine Nucleotide Translocases (ANTs), which catalyze the electrogenic ATP4-/ADP3- exchange, have been proposed to work together with ATP synthase and the mitochondrial phosphate carrier to support mitochondrial oxidative phosphorylation by forming the so-called ATP synthasome. Nevertheless, disruption of the main ANT isoform or pharmacological inhibition of ANTs in cancer cells does not decrease oxidative phosphorylation. Furthermore, the import of cytosolic ATP to the mitochondria when oxidative phosphorylation is inhibited (required, for example, for the maintenance of the ΔΨm) is neither mediated by ANTs in cancer cells. Here, we study the possibility that calcium-regulated mitochondrial ATP-Mg2+/Pi or ADP/Pi carriers, (also called SCaMCs), might participate in the mitochondrial transport of adenine nucleotides in cancer cells. Material and methods: To study the function of SCaMC-1, the main isoform in cancer cells, we have generated SCaMC-1 KO HeLa cells using CRISPR-Cas9 genome editing. These cells have been characterized in terms of OCR (with Seahorse XF24), mitochondrial membrane potential (with TMRM) and mitochondrial ATP levels (with mitoGoATeam genetic probe) in basal conditions and in the presence of ETC inhibitors. Results and conclusions: We show that SCaMC-1, the isoform that is abundant in tumor cells, mediates ATP import to the mitochondria after histamine stimulation of HeLa cells, probably to buffer calcium entry to the mitochondria. However, SCaMC-1 does not participate in the transport of cytosolic ATP for the maintenance of ΔΨm after OXPHOS inhibition, neither decreases mitochondrial respiration. Future studies based on the deletion of the other SCaMCs isoforms (SCaMC-2, SCaMC-3, SCaMC-3 like) together with simultaneous deletion of ANTs and SCaMCs will be required to shed light on the control of adenine nucleotides transport between the mitochondria and the cytosol in cancer cells.

Arabidopsis guard cell chloroplasts import cytosolic ATP for starch turnover and stomatal opening

Stomatal opening requires the provision of energy in the form of ATP for proton pumping across the guard cell (GC) plasma membrane and for associated metabolic rearrangements. The source of ATP for GCs is a matter of ongoing debate that is mainly fuelled by controversies around the ability of GC chloroplasts (GCCs) to perform photosynthesis. By imaging compartment-specific fluorescent ATP and NADPH sensor proteins in Arabidopsis, we show that GC photosynthesis is limited and mitochondria are the main source of ATP. Unlike mature mesophyll cell (MC) chloroplasts, which are impermeable to cytosolic ATP, GCCs import cytosolic ATP through NUCLEOTIDE TRANSPORTER (NTT) proteins. GCs from ntt mutants exhibit impaired abilities for starch biosynthesis and stomatal opening. Our work shows that GCs obtain ATP and carbohydrates via different routes from MCs, likely to compensate for the lower chlorophyll contents and limited photosynthesis of GCCs.

The broad range di- and tri-nucleotide exchanger SLC35B1 displays asymmetrical affinities for ATP transport across the ER membrane

In eukaryotic cells, uptake of cytosolic ATP into the endoplasmic reticulum (ER) lumen is critical for the proper functioning of chaperone proteins. The human transport protein SLC35B1 was recently postulated to mediate ATP/ADP exchange in the ER; however, the underlying molecular mechanisms mediating ATP uptake are not completely understood. Here, we extensively characterized the transport kinetics of human SLC35B1 expressed in yeast that was purified and reconstituted into liposomes. Using [α32P]ATP uptake assays, we tested the nucleotide concentration dependence of ATP/ADP exchange activity on both sides of the membrane. We found that the apparent affinities of SLC35B1 for ATP/ADP on the internal face were approximately 13 times higher than those on the external side. Because SLC35B1-containing liposomes were preferentially inside-out oriented, these results suggest a low-affinity external site and a high-affinity internal site in the ER. Three different experimental approaches indicated that ATP/ADP exchange by SLC35B1 was not strict, and that other di- and tri-nucleotides could act as suitable counter-substrates for ATP, although mononucleotides and nucleotide sugars were not transported. Finally, bioinformatic analysis and site-directed mutagenesis identified that conserved residues K117 and K120 from transmembrane helix 4 and K277 from transmembrane helix 9 play critical roles in transport. The fact that SLC35B1 can promote ATP transport in exchange for ADP or UDP suggest a more direct coupling between ATP import requirements and the need for eliminating ADP and UDP, which are generated as side products of reactions taking place in the ER-lumen.

Compartment-specific 13C metabolic flux analysis reveals boosted NADPH availability coinciding with increased cell-specific productivity for IgG1 producing CHO cells after MTA treatment.

Increasing cell‐specific productivities (CSPs) for the production of heterologous proteins in Chinese hamster ovary (CHO) cells is an omnipresent need in the biopharmaceutical industry. The novel additive 5′‐deoxy‐5′‐(methylthio)adenosine (MTA), a chemical degradation product of S‐(5′‐adenosyl)‐ʟ‐methionine (SAM) and intermediate of polyamine biosynthesis, boosts the CSP of IgG1‐producing CHO cells by 50%. Compartment‐specific 13C flux analysis revealed a fundamental reprogramming of the central metabolism after MTA addition accompanied by cell‐cycle arrest and increased cell volumes. Carbon fluxes into the pentose‐phosphate pathway increased 22 fold in MTA‐treated cells compared to that in non‐MTA‐treated reference cells. Most likely, cytosolic ATP inhibition of phosphofructokinase mediated the carbon detour. Mitochondrial shuttle activity of the α‐ketoglurarate/malate antiporter (OGC) reversed, reducing cytosolic malate transport. In summary, NADPH supply in MTA‐treated cells improved three fold compared to that in non‐MTA‐treated cells, which can be regarded as a major factor for explaining the boosted CSPs.

Mitochondrial Contributions in the Genesis of Delayed Afterdepolarizations in Ventricular Myocytes.

Mitochondria fulfill the cell’s energy demand and affect the intracellular calcium (Ca2+) dynamics via direct Ca2+ exchange, the redox effect of reactive oxygen species (ROS) on Ca2+ handling proteins, and other signaling pathways. Recent experimental evidence indicates that mitochondrial depolarization promotes arrhythmogenic delayed afterdepolarizations (DADs) in cardiac myocytes. However, the nonlinear interactions among the Ca2+ signaling pathways, ROS, and oxidized Ca2+/calmodulin-dependent protein kinase II (CaMKII) pathways make it difficult to reveal the mechanisms. Here, we use a recently developed spatiotemporal ventricular myocyte computer model, which consists of a 3-dimensional network of Ca2+ release units (CRUs) intertwined with mitochondria and integrates mitochondrial Ca2+ signaling and other complex signaling pathways, to study the mitochondrial regulation of DADs. With a systematic investigation of the synergistic or competing factors that affect the occurrence of Ca2+ waves and DADs during mitochondrial depolarization, we find that the direct redox effect of ROS on ryanodine receptors (RyRs) plays a critical role in promoting Ca2+ waves and DADs under the acute effect of mitochondrial depolarization. Furthermore, the upregulation of mitochondrial Ca2+ uniporter can promote DADs through Ca2+-dependent opening of mitochondrial permeability transition pores (mPTPs). Also, due to much slower dynamics than Ca2+ cycling and ROS, oxidized CaMKII activation and the cytosolic ATP do not appear to significantly impact the genesis of DADs during the acute phase of mitochondrial depolarization. However, under chronic conditions, ATP depletion suppresses and enhanced CaMKII activation promotes Ca2+ waves and DADs.

Creatine supplementation and muscles: From metabolism to medical practice

Creatine has become the most popular dietary supplement in sport and exercise physiology. In humans creatine is synthesized by the kidneys, pancreas and liver and transported mainly into brain, skeletal and cardiac muscle. Phosphocreatine is a high-energy content molecule, essential for the ADP to ATP conversion during intensive physical activity. Creatine and phosphocreatine are crucial in the energy shuttle system of high-energy phosphates between the mitochondrial ATP production and the cytosolic ATP consumption. Creatine supplementation increases lean body mass acting on myogenic regulatory factors. During muscular recovery, creatine supplementation regulates the regeneration process by reduction of muscle damage-induced inflammation and oxidative stress, activation and proliferation of satellite cells and regulation of calcium transport in muscle. The effects of creatine supplementation on muscle physiology are beneficial in anaerobic/aerobic exercises. In several muscle disorders (muscular dystrophies, in idiopathic inflammatory myopathies) creatine improved functional performance, but apparently not in metabolic myopathies; in McArdle diseases it may even have paradoxical effects. More research is warranted to better understand the short and long-term effects and safety of creatine supplementation among adolescents or elderly, as well as in different types of muscle diseases; for the two enzymatic genetic defects of creatine biosynthesis – arginine: glycine amidinotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT), respectively – normal neurodevelopment has been achieved in early initiation of creatine therapy.

The Complementary Roles of Chloroplast Cyclic Electron Transport and Mitochondrial Alternative Oxidase to Ensure Photosynthetic Performance.

Chloroplasts use light energy and a linear electron transport (LET) pathway for the coupled generation of NADPH and ATP. It is widely accepted that the production ratio of ATP to NADPH is usually less than required to fulfill the energetic needs of the chloroplast. Left uncorrected, this would quickly result in an over-reduction of the stromal pyridine nucleotide pool (i.e., high NADPH/NADP+ ratio) and under-energization of the stromal adenine nucleotide pool (i.e., low ATP/ADP ratio). These imbalances could cause metabolic bottlenecks, as well as increased generation of damaging reactive oxygen species. Chloroplast cyclic electron transport (CET) and the chloroplast malate valve could each act to prevent stromal over-reduction, albeit in distinct ways. CET avoids the NADPH production associated with LET, while the malate valve consumes the NADPH associated with LET. CET could operate by one of two different pathways, depending upon the chloroplast ATP demand. The NADH dehydrogenase-like pathway yields a higher ATP return per electron flux than the pathway involving PROTON GRADIENT REGULATION5 (PGR5) and PGR5-LIKE PHOTOSYNTHETIC PHENOTYPE1 (PGRL1). Similarly, the malate valve could couple with one of two different mitochondrial electron transport pathways, depending upon the cytosolic ATP demand. The cytochrome pathway yields a higher ATP return per electron flux than the alternative oxidase (AOX) pathway. In both Arabidopsis thaliana and Chlamydomonas reinhardtii, PGR5/PGRL1 pathway mutants have increased amounts of AOX, suggesting complementary roles for these two lesser-ATP yielding mechanisms of preventing stromal over-reduction. These two pathways may become most relevant under environmental stress conditions that lower the ATP demands for carbon fixation and carbohydrate export.

O-039 Mitochondrial ATP generation in mammalian eggs and Ca2+ signaling

Abstract text In metaphase II arrested mammalian oocytes (eggs) and cleavage stage embryos the mitochondria are responsible for nearly all ATP production because glycolysis is inactivated. Luciferase assays show that ATP levels in eggs are strictly dependent upon pyruvate and fatty acid oxidation. The level of ATP in eggs appears to be maximal in conventional medium because the addition of extra mitochondrial substrates to eggs does not increase cytosolic ATP. The only clear elevation of ATP is seen at fertilization and is associated with sperm induced Ca2+ oscillations. Our recent findings suggest that the level of ATP modulates events at fertilization. At fertilization, the egg is activated by sperm derived PLCzeta which triggers a series of Ca2+ oscillations, with each Ca2+ release event causes by inositol trisphosphate (InsP3). Previous studies have shown that mouse eggs are more sensitive to PLCzeta, and generate higher frequency Ca2+ oscillations, than human eggs. Mouse eggs also generate Ca2+ oscillations and activate in response to Sr2+ that directly stimulates InsP3 receptors. In contrast, human eggs that contain the same type of InsP3 receptors do not generate Ca2+ oscillations in response to Sr2+. The difference in sensitivity of Ca2+ release between species can be explained by the fact that mouse eggs are about ten times more sensitive to InsP3 than human eggs. The reason for this difference appears to be due to ATP. The ATP level in unfertilized mouse eggs is about twice that in human eggs. Furthermore, the ability of mouse eggs to Sr2+ medium can be abolished by removing the mitochondrial substrate pyruvate, which reduces the ATP level. Adding back pyruvate to such eggs restores ATP levels promotes Sr2+ induced Ca2+ levels in mouse eggs. These data suggest that the level of ATP, possibly as ATP4-, modulates the sensitivity of the InsP3 receptor and the ability of eggs to generate Ca2+ oscillations. The level of cytosolic ATP may represent a significant ‘egg factor’ in determining the success of fertilization in humans. Enhancing mitochondrial ATP production could be useful in improving activation and embryo development after fertilization, or after artificial egg activation.

Abstract 2332: Modifying bioavailability of ATP and measuring metabolic response in HeLa cells

Abstract Introduction: Since studies by Otto Warburg demonstrated that cancer cells had markedly reduced oxidative phosphorylation (OXPHOS) under conditions of high oxygen tension (Warburg Effect/aerobic glycolysis), metabolic reprogramming has become a fundamental hallmark of cancer. While numerous discoveries have revealed highly specific metabolic differences between cancer cells, utilization of aerobic glycolysis as the primary means of ATP generation remains one of the most common metabolic alterations observed across a range of cancers. There is growing interest in understanding the multifaceted role ATP plays in cancer development, progression and chemotherapeutic resistance. Using the man-made protein DX, which chelates ATP with high specificity and affinity, we have begun to untangle these roles by significantly reducing cytosolic ATP and investigating cellular response. Hypothesis: Delivery of DX into HeLa cancer cells will reduce intracellular bioavailable ATP and induce an adaptive metabolic response. Methods: A cationic lipid mixture was complexed with active, purified DX protein to generate a DX/lipid complex capable of delivering the protein to the cytoplasm of HeLa cells. To confirm the successful delivery of DX, immunofluorescent microscopy was performed (DYKDDDDK tag monoclonal antibody, Alexa Fluor 647) at various time points following DX transfection. The impact of DX on cell viability was determined using a tetrazolium-based colorimetric cell viability assay and a caspase 3/7 assay. To correlate phenotypic/viability change with DX activity, bioavailable ATP levels were measured at specific time points following DX delivery. Additionally, the relative contribution of glycolysis and OXPHOS to the total ATP production rate was measured using the label-free XF Real Time ATP Rate Assay (XFe96 Seahorse, Agilent Technologies) over time post DX transfection. Results: In a time- and dose- dependent manner, DX negatively impacted cell growth and induced cell death via apoptosis, at a time concomitant with a decrease in bioavailable ATP. In response to DX over time, the total ATP production rates in HeLa cells significantly decreased. Importantly, this reduction in the rate of ATP production was associated with a metabolic program that was primarily glycolytic (70%) with a smaller contribution from OXPHOS (30%) 24 h post DX transfection. Conclusion: Advances in protein engineering have made it possible to create artificial proteins with specific functions. In addition to direct clinical applications, synthetic proteins can be developed into powerful experimental tools to resolve biological enigmas. Direct delivery of DX potentiated energy flux in HeLa cells in an unbiased manner, independent of metabolic pathway interference. This work establishes DX as a useful tool for examination of cancer cell metabolism in response to ATP stress. Citation Format: Taha Muhammad, Ashley Brown, Selina Martinez, John C. Chaput, Jeffrey Norris, Shaleen Korch. Modifying bioavailability of ATP and measuring metabolic response in HeLa cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2332.