Rate Of ATP Consumption Research Articles

A genomic database furnishes minimal functional glycyl-tRNA synthetases homologous to other, designed class II urzymes.

The hypothesis that conserved core catalytic sites could represent ancestral aminoacyl-tRNA synthetases (AARS) drove the design of functional TrpRS, LeuRS, and HisRS 'urzymes'. We describe here new urzymes detected in the genomic record of the arctic fox, Vulpes lagopus. They are homologous to the α-subunit of bacterial heterotetrameric Class II glycyl-tRNA synthetase (GlyRS-B) enzymes. AlphaFold2 predicted that the N-terminal 81 amino acids would adopt a 3D structure nearly identical to our designed HisRS urzyme (HisCA1). We expressed and purified that N-terminal segment and the spliced open reading frame GlyCA1-2. Both exhibit robust single-turnover burst sizes and ATP consumption rates higher than those previously published for HisCA urzymes and comparable to those for LeuAC and TrpAC. GlyCA is more than twice as active in glycine activation by adenosine triphosphate as the full-length GlyRS-B α2 dimer. Michaelis-Menten rate constants for all three substrates reveal significant coupling between Exon2 and both substrates. GlyCA activation favors Class II amino acids that complement those favored by HisCA and LeuAC. Structural features help explain these results. These minimalist GlyRS catalysts are thus homologous to previously described urzymes. Their properties reinforce the notion that urzymes may have the requisite catalytic activities to implement a reduced, ancestral genetic coding alphabet.

Targeting SIRT5 By Succinylating Hadha Synergizes with Venetoclax in Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) is genetically diverse, driven by multiple oncogenic factors. Metabolic dysregulation is a critical vulnerability in AML cells for targeted therapy. BCL2 has been previously reported to maintain mitochondrial oxidative phosphorylation (OXPHOS) in AML cells. One of the mechanisms of action of the combination of venetoclax and azacitidine is to inhibit amino acid metabolism, leading to cell death. Although venetoclax-based treatment regimens have shown promising clinical outcomes in AML patients, treatment resistance has become a newly emerged challenge. Leukemia stem cells isolated from venetoclax-resistant patients exhibit distinctive metabolic features, including elevated nicotinamide metabolism. SIRT5 is an NAD+-dependent deacylase enzyme, and its knockout leads to elevated succinylation of various enzymes involved in OXPHOS. Hence, we hypothesize that inhibiting SIRT5 may augment venetoclax's cytotoxic effect by dampening metabolism. To further elucidate this mechanism, we employed proteome analyses of succinylation to identify specific target molecules regulated by SIRT5. Methods: To determine the effects of combination therapy on cell proliferation, apoptosis, and colony formation, AML cell lines were transduced with lentiviruses carrying either shNC or shSIRT5, or treated with NRD167 (a specific inhibitor of SIRT5), followed by venetoclax treatment. To evaluate the clearance of leukemia cells in vivo, NSG mice were transplanted with MOLM-13 cells (carrying shNC or shSIRT5) and then subjected to vehicle or venetoclax treatment (80 mg/kg/day) via oral gavage for 14 days. Weekly live animal in vivo imaging was conducted to monitor tumor engraftment and growth. The synergistic effect on the metabolic level of AML was assessed by measuring ATP, glutathione, mitochondrial superoxide, and oxygen consumption rates (OCR). Electron microscopy was employed to detect changes in cellular mitochondrial structure after SIRT5 knockdown and venetoclax utilization. Furthermore, lysine succinylation proteome analyses were performed to identify proteins and sites with altered succinylation on lysine before and after SIRT5 knockdown in MOLM-13 cells. Immunoprecipitation assays and Western blot were conducted to verify the interaction and altered succinylation levels of the relevant proteins. Results: The combination of SIRT5 inhibition with venetoclax treatment resulted in further suppression of cell proliferation, reduced colony formation, and increased apoptosis in AML cells compared to venetoclax treatment alone.The combination led to alterations in the metabolic profile of leukemia cells, increased the generation of mitochondrial reactive oxygen species (ROS), reduced intracellular glutathione levels, inhibited ATP production, and suppressed OCR. Notably, combination therapy resulted in a significant reduction in the engraftment levels of human leukemia cells in NSG mice. Mechanistically, electron microscopy revealed that combination treatment with SIRT5 knockdown increased mitochondrial damage. Lysine succinylation proteome analyses demonstrated upregulation of succinylation modification on mitochondrial trifunctional enzyme subunit α (HADHA), a protein involved in the fatty acid oxidation (FAO) pathway. Co-immunoprecipitation of SIRT5 confirmed its interaction with HADHA, and the relative succinylation level of precipitated HADHA was upregulated after SIRT5 knockdown. Moreover, the combination of trimetazidine (a specific inhibitor of HADHA) with venetoclax demonstrated a preferential reduction in cell proliferation and induction of apoptosis in AML cells. Conclusion: Targeting of SIRT5 in combination with venetoclax treatment results in synergistic cytotoxic effects in AML cells. We hypothesize that SIRT5 desuccinylase activity promotes FAO and reduces intracellular mitochondrial ROS levels by desuccinylating HADHA. Targeting SIRT5 inhibits fatty acid metabolism in AML cells, thereby enhancing the therapeutic effect of venetoclax. We will further investigate the site(s) of SIRT5 desuccinylation modification of HADHA through lysine site mutation and assess changes in HADHA enzyme activity to elucidate its regulatory mechanism.

Depressed myocardial cross-bridge cycling kinetics in a female guinea pig model of diastolic heart failure.

Cardiac hypertrophy is associated with diastolic heart failure (DHF), a syndrome in which systolic function is preserved but cardiac filling dynamics are depressed. The molecular mechanisms underlying DHF and the potential role of altered cross-bridge cycling are poorly understood. Accordingly, chronic pressure overload was induced by surgically banding the thoracic ascending aorta (AOB) in ∼400 g female Dunkin Hartley guinea pigs (AOB); Sham-operated age-matched animals served as controls. Guinea pigs were chosen to avoid the confounding impacts of altered myosin heavy chain (MHC) isoform expression seen in other small rodent models. In vivo cardiac function was assessed by echocardiography; cardiac hypertrophy was confirmed by morphometric analysis. AOB resulted in left ventricle (LV) hypertrophy and compromised diastolic function with normal systolic function. Biochemical analysis revealed exclusive expression of β-MHC isoform in both sham control and AOB LVs. Myofilament function was assessed in skinned multicellular preparations, skinned single myocyte fragments, and single myofibrils prepared from frozen (liquid N2) LVs. The rates of force-dependent ATP consumption (tension-cost) and force redevelopment (Ktr), as well as myofibril relaxation time (Timelin) were significantly blunted in AOB, indicating reduced cross-bridge cycling kinetics. Maximum Ca2+ activated force development was significantly reduced in AOB myocytes, while no change in myofilament Ca2+ sensitivity was observed. Our results indicate blunted cross-bridge cycle in a β-MHC small animal DHF model. Reduced cross-bridge cycling kinetics may contribute, at least in part, to the development of DHF in larger mammals, including humans.

A streamlined process for discovery and characterization of inhibitors against phenylalanyl-tRNA synthetase of Mycobacterium tuberculosis.

Aminoacyl-tRNA synthetases (aaRSs) catalyze aminoacylation of tRNAs to produce aminoacyl-tRNAs for protein synthesis. Bacterial aaRSs have distinctive features, play an essential role in channeling amino acids into biomolecular assembly, and are vulnerable to inhibition by small molecules. The aaRSs continue to be targets for potential antibacterial drug development. The first step of aaRS reaction is the activation of amino acid by hydrolyzing ATP to form an acyladenylate intermediate with the concomitant release of pyrophosphate. None-radioactive assays usually measure the rate of ATP consumption or phosphate generation, offering advantages in high-throughput drug screening. These simple aaRS enzyme assays can be adapted to study the mode of inhibition of natural or synthetic aaRS inhibitors. Taking phenylalanyl-tRNA synthetase (PheRS) of Mycobacterium tuberculosis (Mtb) as an example, we describe a process for identification and characterization of Mtb PheRS inhibitor.

Effect of acute hypoxia on the brain energy metabolism of the scorpionfish Scorpaena porcus Linnaeus, 1758: the pattern of oxidoreductase activity and adenylate system.

The activity of oxidoreductases, malate dehydrogenase and lactate dehydrogenase (MDH, 1.1.1.37; LDH, 1.1.1.27), as well as parameters of adenylate system-[ATP], [ADP], [AMP], total adenylate pool (AP), and adenylate energy charge (AEC) in medulla oblongata (MB) and forebrain, midbrain, and diencephalon (FDMB)-were studied in the scorpionfish under acute hypoxia (0.9-1.2mgO2·L-1, 90min). A higher MDH activity level was observed in MB and FDMB, as compared to LDH (p < 0.05). At the same time, MB showed a higher adenylate content and increased AP (p < 0.05). AEC did not exceed ~ 0.7 (vs. the maximum of this index ~ 0.9-1.0) in the brain of the scorpionfish indicating adaptation of the tissue energy status to hypoxia. A rapid decrease in MDH activity (p < 0.05) was observed in MB under acute hypoxia. These changes were accompanied by insignificant LDH activation. A pronounced LDH activation (p < 0.05), a decrease in MDH activity, and the highest AP raise (p < 0.05) were observed in FDMB, suggesting activation of glycolysis and simultaneous decrease in the rate of ATP consumption. MB and FDMB demonstrated the ability to a relative retention of AEC during hypoxia. The unidirectional metabolic adaptation was based on the intensification of glycolysis, a decrease of ATP consumption, and a subsequent increase in adenylate concentration that allowed the scorpionfish brain structures to maintain the energy status under acute hypoxia.

Transgenerational effects and phenotypic plasticity in sperm and larvae of the sea urchin Paracentrotus lividus under ocean acidification.

In marine organisms, differing degree of sensitivity to ocean acidification (OA) is expected for each life stage, and disturbance at one stage can carry over into the following stage or following generation. In this study we investigated phenotypic changes of sperm and larvae of the sea urchin Paracentrotus lividus in response to different pH conditions (8.0, 7.7, 7.4) experienced by the parents during gametogenesis. In sperm from two-months exposed males, sperm motility, velocity, ATP content, ATP consumption and respiration rate were evaluated at three pH values of the activating medium (8.0, 7.7 and 7.4). Moreover, larvae from each parental group were reared at pH 8.0 and 7.7 for 20 days and larval mortality and growth were then assessed. Sperm motility and respiration rate were not affected either by exposure of males to low pH or by the post-activation pH. Sperm velocity did not differ among post-activation pH values in all sperm groups, but it decreased slower in sperm developed under acidified conditions, suggesting the presence of positive carryover effect on sperm longevity. This positive carryover effect of exposure of males to low pH values was highlighted also for the sperm ATP content, which was higher in these groups of sperm. ATP consumption rate was affected by post-activation pH with higher values at pH 8.0 in sperm from males maintained at control condition and pH 7.7 while the energy consumption appeared to be differently modulated at different experimental conditions. A negative carry over effect of OA was observed on survival of larvae from parents acclimated at pH 7.4 and additive negative effects of both parental and larval exposure to low pH can be suggested. In all groups of larvae, decreased somatic growth was observed at low rearing pH, thus larvae from parents maintained at low pH did not show an increased capability to cope with OA.

Age-related changes in the energy of human mesenchymal stem cells.

Aging is a physiological process that leads to a higher risk for the most devastating diseases. There are a number of theories of human aging proposed, and many of them are directly or indirectly linked to mitochondria. Here, we used mesenchymal stem cells (MSCs) from young and older donors to study age-related changes in mitochondrial metabolism. We have found that aging in MSCs is associated with a decrease in mitochondrial membrane potential and lower NADH levels in mitochondria. Mitochondrial DNA content is higher in aged MSCs, but the overall mitochondrial mass is decreased due to increased rates of mitophagy. Despite the higher level of ATP in aged cells, a higher rate of ATP consumption renders them more vulnerable to energy deprivation compared to younger cells. Changes in mitochondrial metabolism in aged MSCs activate the overproduction of reactive oxygen species in mitochondria which is compensated by a higher level of the endogenous antioxidant glutathione. Thus, energy metabolism and redox state are the drivers for the aging of MSCs/mesenchymal stromal cells.

Simulating Metabolic Flexibility in Low Energy Expenditure Conditions Using Genome-Scale Metabolic Models.

Metabolic flexibility is the ability of an organism to adapt its energy source based on nutrient availability and energy requirements. In humans, this ability has been linked to cardio-metabolic health and healthy aging. Genome-scale metabolic models have been employed to simulate metabolic flexibility by computing the Respiratory Quotient (RQ), which is defined as the ratio of carbon dioxide produced to oxygen consumed, and varies between values of 0.7 for pure fat metabolism and 1.0 for pure carbohydrate metabolism. While the nutritional determinants of metabolic flexibility are known, the role of low energy expenditure and sedentary behavior in the development of metabolic inflexibility is less studied. In this study, we present a new description of metabolic flexibility in genome-scale metabolic models which accounts for energy expenditure, and we study the interactions between physical activity and nutrition in a set of patient-derived models of skeletal muscle metabolism in older adults. The simulations show that fuel choice is sensitive to ATP consumption rate in all models tested. The ability to adapt fuel utilization to energy demands is an intrinsic property of the metabolic network.

Extracellular ATP hydrolysis in Caco-2 human intestinal cell line

Extracellular nucleotides and nucleosides activate signaling pathways that play major roles in the physiology and pathophysiology of the gastrointestinal tract. Ectonucleotidases hydrolyze extracellular nucleotides and thus regulate ligand exposure to purinergic receptors. In this study, we investigated the expression, localization and activities of ectonucleotidases using Caco-2 cells, a model of human intestinal epithelial cells. In addition, by studying ATP release and the rates of extracellular ATP (eATP) hydrolysis, we analyzed the contribution of these processes to the regulation of eATP in these cells. Results show that Caco-2 cells regulate the metabolism of eATP and by-products by ecto-nucleoside triphosphate diphosphohydrolase-1 and -2, a neutral ecto-phosphatase and ecto-5′-nucleotidase. All these ectoenzymes were kinetically characterized using intact cells, and their presence confirmed by denatured and native gels, western blot and cytoimmunofluorescence techniques.In addition, regulation of eATP was studied by monitoring the dynamic balance between intracellular ATP release and ectoATPase activity. Following mechanical and hypotonic stimuli, Caco-2 cells triggered a strong but transient release of intracellular ATP, with almost no energy cost, leading to a steep increase of eATP concentration, which was later reduced by ectoATPase activity. A data-driven algorithm allowed quantifying and predicting the rates of ATP release and ATP consumption contributing to the dynamic accumulation of ATP at the cell surface.

Alteration of the F1Fo ATP Synthase Causes Metabolic Remodeling in Breast Cancer Cells

Abstract Objectives The F1Fo ATP synthase is a multienzyme complex that produces mitochondrial ATP. Aberrant expression or assembly of F1Fo ATP synthase subunits leads to alterations in energy metabolism. We recently found that breast cancer cells exposed to fluid shear stress (FSS) have significantly enhanced metastatic behavior including chemoresistance and cell proliferation. Chemoresistance depends upon active transport systems, and cell division and growth require ATP. Therefore, we hypothesized that circulating breast cancer cells undergo altered energy metabolism via FSS-induced changes in F1Fo ATP synthase subunits and subsequent mitochondrial remodeling. Methods Non-metastatic MCF7 and metastatic MDA-MB-231 human breast cancer cells were treated with or without FSS and cultured. Cellular proliferation was assayed by measuring cell number and gap distance. Metabolic profile including intracellular ATP and oxygen consumption rate were analyzed. We also quantified abundance of F1Fo ATP synthase subunits using immunoblotting. Results Treatment with FSS significantly increased proliferation of both MCF7 and MDA-MB-231 human breast cancer cells. FSS significantly increased intracellular ATP in MDA-MB-231 breast cancer cells while ATP levels in MCF7 were not significantly changed. MDA-MB-231 cells retained increased ATP after treatment with the uncoupler FCCP, indicating remodeling and decreased reliance on mitochondrial energy metabolism. Interestingly, oxygen consumption rate was significantly increased in both MCF7 and MDA-MB-231 by FSS. We further quantified the abundance of F1Fo ATP synthase subunits in both cell lines. The β- and c-subunits of the F1Fo ATP synthase were significantly depleted in both lines of FSS-treated breast cancer cells. Conclusions Our data show that FSS alters abundance of the F1Fo ATP synthase subunits leading to metabolic remodeling. We suggest that FSS may influence non-metastatic (MCF7) and metastatic cancer cells (MDA-MB-231) differently. Underlying changes in mitochondrial and cytoplasmic ATP production in these cells is still under investigation. However, it is possible that reactive oxygen species generated during FSS may signal a switch to cytoplasmic intracellular energy metabolism. Funding Sources Alabama Life Research Institute Pilot Project (University of Alabama)

Metabolic Homeostasis in Life as We Know It: Its Origin and Thermodynamic Basis.

Living organisms require continuous input of energy for their existence. As a result, life as we know it is based on metabolic processes that extract energy from the environment and make it available to support life (energy metabolism). This metabolism is based on, and regulated by, the underlying thermodynamics. This is important because thermodynamic parameters are stable whereas kinetic parameters are highly variable. Thermodynamic control of metabolism is exerted through near equilibrium reactions that determine. (1) the concentrations of metabolic substrates for enzymes that catalyze irreversible steps and (2) the concentrations of small molecules (AMP, ADP, etc.) that regulate the activity of irreversible reactions in metabolic pathways. The result is a robust homeostatic set point (−ΔGATP) with long term (virtually unlimited) stability. The rest of metabolism and its regulation is constrained to maintain this set point. Thermodynamic control is illustrated using the ATP producing part of glycolysis, glyceraldehyde-3-phosphate oxidation to pyruvate. Flux through the irreversible reaction, pyruvate kinase (PK), is primarily determined by the rate of ATP consumption. Change in the rate of ATP consumption causes mismatch between use and production of ATP. The resulting change in [ATP]/[ADP][Pi], through near equilibrium of the reactions preceding PK, alters the concentrations of ADP and phosphoenolpyruvate (PEP), the substrates for PK. The changes in ADP and PEP alter flux through PK appropriately for restoring equality of ATP production and consumption. These reactions appeared in the very earliest lifeforms and are hypothesized to have established the set point for energy metabolism. As evolution included more metabolic functions, additional layers of control were needed to integrate new functions into existing metabolism without changing the homeostatic set point. Addition of gluconeogenesis, for example, resulted in added regulation to PK activity to prevent futile cycling; PK needs to be turned off during gluconeogenesis because flux through the enzyme would waste energy (ATP), subtracting from net glucose synthesis and decreasing overall efficiency.

Cellular and molecular pathways controlling muscle size in response to exercise.

From the discovery of ATP and motor proteins to synaptic neurotransmitters and growth factor control of cell differentiation, skeletal muscle has provided an extreme model system in which to understand aspects of tissue function. Muscle is one of the few tissues that can undergo both increase and decrease in size during everyday life. Muscle size depends on its contractile activity, but the precise cellular and molecular pathway(s) by which the activity stimulus influences muscle size and strength remain unclear. Four correlates of muscle contraction could, in theory, regulate muscle growth: nerve-derived signals, cytoplasmic calcium dynamics, the rate of ATP consumption and physical force. Here, we summarise the evidence for and against each stimulus and what is known or remains unclear concerning their molecular signal transduction pathways and cellular effects. Skeletal muscle can grow in three ways, by generation of new syncytial fibres, addition of nuclei from muscle stem cells to existing fibres or increase in cytoplasmic volume/nucleus. Evidence suggests the latter two processes contribute to exercise-induced growth. Fibre growth requires increase in sarcolemmal surface area and cytoplasmic volume at different rates. It has long been known that high-force exercise is a particularly effective growth stimulus, but how this stimulus is sensed and drives coordinated growth that is appropriately scaled across organelles remains a mystery.

Monitoring and modeling of lymphocytic leukemia cell bioenergetics reveals decreased ATP synthesis during cell division

The energetic demands of a cell are believed to increase during mitosis, but the rates of ATP synthesis and consumption during mitosis have not been quantified. Here, we monitor mitochondrial membrane potential of single lymphocytic leukemia cells and demonstrate that mitochondria hyperpolarize from the G2/M transition until the metaphase-anaphase transition. This hyperpolarization was dependent on cyclin-dependent kinase 1 (CDK1) activity. By using an electrical circuit model of mitochondria, we quantify mitochondrial ATP synthesis rates in mitosis from the single-cell time-dynamics of mitochondrial membrane potential. We find that mitochondrial ATP synthesis decreases by approximately 50% during early mitosis and increases back to G2 levels during cytokinesis. Consistently, ATP levels and ATP synthesis are lower in mitosis than in G2 in synchronized cell populations. Overall, our results provide insights into mitotic bioenergetics and suggest that cell division is not a highly energy demanding process.

Regulation of mitochondrial dynamics and energetics in the diabetic renal proximal tubule by the β2-adrenergic receptor agonist formoterol.

Diabetes is a prevalent metabolic disease that contributes to ∼50% of all end-stage renal disease and has limited treatment options. We previously demonstrated that the β2-adrenergic receptor agonist formoterol induced mitochondrial biogenesis and promoted recovery from acute kidney injury. Here, we assessed the effects of formoterol on mitochondrial dysfunction and dynamics in renal proximal tubule cells (RPTCs) treated with high glucose and in a mouse model of type 2 diabetes. RPTCs exposed to 17 mM glucose exhibited increased electron transport chain (ETC) complex I, II, III, and V protein levels and reduced ATP levels and uncoupled oxygen consumption rate compared with RPTCs cultured in the absence of glucose or osmotic controls after 96 h. ETC proteins, ATP, and oxygen consumption rate were restored in RPTCs treated with formoterol. RPTCs exposed to high glucose had increased phospho-dynamin-related protein 1 (Drp1), a mitochondrial fission protein, and decreased mitofusin 1 (Mfn1), a mitochondrial fusion protein. Formoterol treatment restored phospho-Drp1 and Mfn1 to control levels. Db/db and nondiabetic (db/m) mice (10 wk old) were treated with formoterol or vehicle for 3 wk and euthanized. Db/db mice showed increased renal cortical ETC protein levels in complexes I, III, and V and decreased ATP; these changes were prevented by formoterol. Phospho-Drp1 was increased and Mfn1 was decreased in db/db mice, and formoterol restored both to control levels. Together, these findings demonstrate that hyperglycemic conditions in vivo and exposure of RPTCs to high glucose similarly alter mitochondrial bioenergetic and dynamics profiles and that treatment with formoterol can reverse these effects. Formoterol may be a promising strategy for treating early stages of diabetic kidney disease.

Impaired Myocardial Energetics Causes Mechanical Dysfunction in Decompensated Failing Hearts.

Cardiac mechanical function is supported by ATP hydrolysis, which provides the chemical-free energy to drive the molecular processes underlying cardiac pumping. Physiological rates of myocardial ATP consumption require the heart to resynthesize its entire ATP pool several times per minute. In the failing heart, cardiomyocyte metabolic dysfunction leads to a reduction in the capacity for ATP synthesis and associated free energy to drive cellular processes. Yet it remains unclear if and how metabolic/energetic dysfunction that occurs during heart failure affects mechanical function of the heart. We hypothesize that changes in phosphate metabolite concentrations (ATP, ADP, inorganic phosphate) that are associated with decompensation and failure have direct roles in impeding contractile function of the myocardium in heart failure, contributing to the whole-body phenotype. To test this hypothesis, a transverse aortic constriction (TAC) rat model of pressure overload, hypertrophy, and decompensation was used to assess relationships between metrics of whole-organ pump function and myocardial energetic state. A multiscale computational model of cardiac mechanoenergetic coupling was used to identify and quantify the contribution of metabolic dysfunction to observed mechanical dysfunction. Results show an overall reduction in capacity for oxidative ATP synthesis fueled by either fatty acid or carbohydrate substrates as well as a reduction in total levels of adenine nucleotides and creatine in myocardium from TAC animals compared to sham-operated controls. Changes in phosphate metabolite levels in the TAC rats are correlated with impaired mechanical function, consistent with the overall hypothesis. Furthermore, computational analysis of myocardial metabolism and contractile dynamics predicts that increased levels of inorganic phosphate in TAC compared to control animals kinetically impair the myosin ATPase crossbridge cycle in decompensated hypertrophy/heart failure.

Red-Light Irradiation of Horse Spermatozoa Increases Mitochondrial Activity and Motility through Changes in the Motile Sperm Subpopulation Structure.

Previous studies in other mammalian species have shown that stimulation of semen with red-light increases sperm motility, mitochondrial activity, and fertilizing capacity. This study sought to determine whether red-light stimulation using a light emitting diode (LED) at 620–630 nm affects sperm motility and structure of motile subpopulations, sperm viability, mitochondrial activity, intracellular ATP levels, rate of O2 consumption and DNA integrity of horse spermatozoa. For this purpose, nine ejaculates were collected from nine different adult stallions. Upon collection, semen was diluted in Kenney extender, analyzed, its concentration was adjusted, and finally it was stimulated with red-light. In all cases, semen was packaged in 0.5-mL transparent straws, which were randomly divided into controls and 19 light-stimulation treatments; 6 consisted of a single exposure to red-light, and the other 13 involved irradiation with intervals of irradiation and darkness (light-dark-light). After irradiation, sperm motility was assessed using a Computerized Semen Analysis System (CASA). Flow cytometry was used to evaluate sperm viability, mitochondrial membrane potential and DNA fragmentation. Intracellular levels of ATP and O2 consumption rate were also determined. Specific red-light patterns were found to modify kinetics parameters (patterns: 4, 2-2-2, 3-3-3, 4-4-4, 5-1-5, and 5-5-5 min), the structure of motile sperm subpopulations (patterns: 2, 2-2-2, 3-3-3, and 4-1-4 min), mitochondrial membrane potential (patterns: 4, 3-3-3, 4-4-4, 5-1-5, 5-5-5, 15-5-15, and 15-15-15 min), intracellular ATP levels and the rate of O2 consumption (pattern: 4 min), without affecting sperm viability or DNA integrity. Since the increase in some kinematic parameters was concomitant with that of mitochondrial activity, intracellular ATP levels and O2 consumption rate, we suggest that the positive effect of light-irradiation on sperm motility is related to its impact upon mitochondrial activity. In conclusion, this study shows that red LED light stimulates motility and mitochondrial activity of horse sperm. Additional research is needed to address the impact of red-light irradiation on fertilizing ability and the mechanisms through which light exerts its effects.

Impact of Isoorientin on Metabolic Activity and Lipid Accumulation in Differentiated Adipocytes.

The current study explored the effect of isoorientin on the metabolic activity and lipid accumulation in fully differentiated 3T3-L1 adipocytes. To achieve this, the 3T3-L1 pre-adipocytes were differentiated for eight days and treated with various concentrations of isoorientin (0.1–100 μM) for four hours. Subsequently, the metabolic activity, lipid accumulation, and mitochondrial respiration were assessed. Furthermore, to unravel the molecular mechanisms that might elucidate the bioactivity of isoorientin, protein expression of the genes involved in insulin signaling and energy expenditure, such as AKT and AMPK, were investigated. The results showed that isoorientin, at different doses, could block lipid storage and enhance glycerol release, with a concomitant improvement of the metabolic activity and mitochondrial function. Although the observed beneficial effects of isoorientin on these cultured 3T3-L1 adipocytes were not consistent at all concentrations, it was clear that doses between 1 and 10 μM were most effective compared to the untreated control. Moreover, the activity of isoorientin was comparable to tested positive controls of CL-316,2431, isoproterenol, insulin, and metformin. Mechanistically, protein expression of AKT and AMPK, was enhanced with isoorientin exposure, suggesting their partial role in modulating lipid metabolism and mitochondrial biogenesis. Indeed, our results showed that isoorientin has the ability to enhance mitochondrial respiration, as we observed an increase in the ATP and oxygen consumption rate. Therefore, we concluded that isoorientin has a potential to impact mitochondrial activity, lipid metabolism and energy expenditure using an in vitro experimental model of obesity.

Total Cellular ATP Production Varies With Primary Substrate in Breast Cancer Cells

Cancer cell growth is predicted to require substantial increases in the rates of substrate catabolism and ATP turnover to drive biosynthesis and cell growth. Strategic limitation of substrate supply is therefore a potentially effective way to slow tumor growth. However, the effects of substrate limitation on the rates of ATP production and consumption, especially over the acute timescales relevant to most experimental design, are not straightforward. First, bioenergetic homeostasis will oppose substrate limitation by increasing catabolism of substrates that remain accessible, in order to meet existing ATP demand. Second, ATP demand itself may be different under different substrate conditions. Empirical evaluation of total cellular ATP production is therefore required. Cultured cells remain an important model system for understanding these mechanisms of bioenergetic control.Using extracellular flux analysis to estimate total cellular ATP production rate, we demonstrate that the MCF7 breast cancer model supports rates of basal ATP production over an approximately 2‐fold range, in the presence of the single substrates glucose, glutamine, and pyruvate, the main substrates commonly present in standard culture media. In comparison, a nontransformed cell culture model shows maintenance of a single ATP production rate across the same single substrates. These novel findings demonstrate that MCF7 cells are highly flexible with respect to total cellular ATP production, over a timeframe that is independent of the slower cellular remodeling caused by altered transcriptional regulation.Support or Funding InformationNIH 1R15ES025917‐01A1; Touro University California College of Pharmacy

ELUCIDATION OF UNIQUE ATP PRODUCTION MECHANISM BY HEMATOPOIETIC STEM CELLS BY COMPREHENSIVE AND QUANTITATIVE ANALYSIS OF ATP CONCENTRATION / AMOUNT IN HEMATOPOIETIC CELLS

Metabolic status governs the fate of hematopoietic stem/progenitor cells (HSPCs). ATP dynamics reflects the intracellular metabolic status; therefore, quantification of the amount and concentration of ATP at high temporal resolution in living cells will increase understanding of metabolism in HSPCs. Here, we developed a method to directly measure the concentration and amount of ATP in HSPCs using an ATP-biosensor-knock-in mouse model and determined the ATP source in each HSPC fraction. The ATP concentration was highest in hematopoietic stem cells (HSCs) among HSPCs; however, the ATP amount was relatively low in HSCs. To elucidate the relationship between ATP metabolism and the nutritional environment, we examined ATP dynamics in HSPC fractions under various glucose concentrations. In the absence of glucose, ATP was produced by oxidative phosphorylation (OXPHOS) in all HSPC fractions. On the other hand, in the presence of a sufficient amount of glucose, HSCs persistently produced ATP only by glycolysis, whereas hematopoietic progenitor cells (HPCs) produced ATP by both glycolysis and OXPHOS. The dependency of HSCs on glycolysis increased with the extracellular glucose concentration, suggesting that metabolic plasticity of HSCs is dependent on their environmental nutrients. By contrast, HPCs were persistently dependent on both OXPHOS and glycolysis for ATP production. Among HPCs, while granulocyte/macrophage progenitors consumed ATP at a high rate, megakaryocyte/erythroid progenitors and common lymphoid progenitors consumed ATP at a low rate similarly to HSCs. Therefore, ATP production is not simply determined by the rate of ATP consumption but by the hematopoietic differentiation stage. Overall, HSCs uniquely exhibit high plasticity in ATP metabolism. Our results also overwrite the conventional view that HSCs always preferentially perform glycolysis rather than OXPHOS.

Effect of subzero temperature treatment at −2 °C before thawing on prevention of thaw rigor, biochemical changes and rate of ATP consumption in frozen chub mackerel (Scomber japonicus)

Prevention of thaw rigor in frozen fish with high ATP content is crucial for the quality of the fish supplied for sashimi. In this study, the effect of subzero temperature treatment at −2 °C on the biochemical and quality characters of thawed meat was investigated with an aim to develop a rapid thawing method to prevent thaw rigor in chub mackerel meat frozen in pre-rigor stage. The meat, which was stored at −2 °C in a water bath for 3 h prior to thawing, did not undergo thaw rigor, and both drip loss and hardness decreased. The average ATP consumption rate in the meat with occurrence and non-occurrence of thaw rigor during thawing was 284.0 μmol/(g·h) and 51.6 μmol/(g·h), respectively. The ATP consumption rate during pretreatment at −2 °C was 75.2 μmol/(g·h). Thaw rigor did not occur in chub mackerel when the rate of ATP consumption was below 50–75 μmol/(g·h). These results suggest that this rapid thawing method could serve useful to prevent thaw rigor in pre-rigor-frozen chub mackerel meat.