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Research Question: Female age is the single greatest factor influencing reproductive performance and granulosa cells are considered as potential biomarkers of oocyte quality. Is there an age effect on the energy metabolism of human mural granulosa cells?, Design: Observational prospective cohort and experimental study including 127 women who had undergone IVF cycles. Women were allocated to two groups: a group of infertile patients aged over 38 years and a control group comprising oocyte donors aged less than 35 years. Individuals with pathologies that could impair fertility were excluded from both groups. Following oocyte retrieval, cumulus and granulosa cells were isolated and their bioenergetic properties (oxidative phosphorylation parameters, rate of aerobic glycolysis and adenine nucleotide concentrations) were analysed and compared., Results: Human mural luteinized granulosa and cumulus cells present high rates of aerobic glycolysis that cannot be increased further when mitochondrial ATP synthesis is inhibited. Addition of follicular fluid to the experimental media is necessary to reach the full respiratory capacity of the cells. Granulosa cells from aged women present lower mitochondrial respiration (12.8 ± 1.6 versus 11.2 ± 1.6 pmol O 2 /min/mg; P = 0.046), although mitochondrial mass is not decreased, and lower aerobic glycolysis, than those from young donors (12.9 ± 1.3 versus 10.9 ± 0.5 mpH/min/mg; P = 0.009). The concurrent decrease in the two energy supply pathways leads to a decrease in the cellular energy charge (0.87 ± 0.01 versus 0.83 ± 0.02; P < 0.001)., Conclusions: Human mural luteinized granulosa cells exhibit a reduction in their energy metabolism as women age that is likely to influence female reproductive potential., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

ATP/ADP depicts the bioenergetic state of Mycobacterium tuberculosis (Mtb). However, the metabolic state of Mtb during infection remains poorly defined due to the absence of appropriate tools. Perceval HR (PHR) was recently developed to measure intracellular ATP/ADP levels, but it cannot be employed in mycobacterial cells due to mycobacterial autofluorescence. Here, we reengineered the ATP/ADP sensor Perceval HR into PHR-mCherry to analyze ATP/ADP in fast- and slow-growing mycobacteria. ATP/ADP reporter strains were generated through the expression of PHR-mCherry. Using the Mtb reporter strain, we analyzed the changes in ATP/ADP levels in response to antimycobacterial agents. As expected, bedaquiline induced a decrease in ATP/ADP. Interestingly, the transcriptional inhibitor rifampicin led to the depletion of ATP/ADP levels, while the cell wall synthesis inhibitor isoniazid did not affect the ATP/ADP levels in Mtb. The usage of this probe revealed that Mtb faces depletion of ATP/ADP levels upon phagocytosis. Furthermore, we observed that the activation of macrophages with interferon gamma and lipopolysaccharides leads to metabolic stress in intracellular Mtb. Examination of the bioenergetics of mycobacteria residing in subvacuolar compartments of macrophages revealed that the bacilli residing in phagolysosomes and autophagosomes have significantly less ATP/ADP than the bacilli residing in phagosomes. These observations indicate that phagosomes represent a niche for metabolically active Mtb, while autophagosomes and phagolysosomes harbor metabolically quiescent bacilli. Interestingly, even in activated macrophages, Mtb residing in phagosomes remains metabolically active. We further observed that macrophage activation affects the metabolic state of intracellular Mtb through the trafficking of Mtb from phagosomes to autophagosomes and phagolysosomes. IMPORTANCE ATP/ADP levels guide bacterial cells, whether to replicate or to enter nonreplicating persistence. However, tools for measuring ATP/ADP levels with spatiotemporal resolution are lacking. Here, we describe a method for tracking ATP/ADP levels at the single-cell and population levels. Using this tool, we have demonstrated that the transcription inhibitor rifampicin induces metabolic stress. In contrast, the cell wall synthesis inhibitor isoniazid does not alter the metabolic state of the bacilli, suggesting that transcription is tightly intertwined with metabolism, while cell wall synthesis is not. Furthermore, we analyzed the metabolic state of mycobacteria residing in different compartments of macrophages. We observed that Mtb cells residing inside phagosomes have healthy ATP/ADP levels. In contrast, the bacteria residing inside phagolysosomes and autophagosomes face depletion of ATP. Interestingly, the activation of macrophages facilitates the trafficking of mycobacterial cells from metabolism-conducive phagosomes to metabolism-averse phagolysosomes and autophagosomes. We believe that this tool holds the key to the identification of inhibitors of mycobacterial metabolism.

1. The purpose of this study was to investigate ATP levels and the activities of important enzymes involved in glycolysis and TCA cycle in livers of embryonated chicken eggs infected by infectious bursal disease virus (IBDV).2. Embryonated chicken eggs (9 days) were randomly divided into two groups (50 eggs per group). The first group was inoculated with a very virulent IBDV (vvIBDV) isolate into the chorioallantoic membrane. The second group was maintained as uninfected control eggs and inoculated with physiological saline. Embryo survival was assessed daily, and six embryos were sacrificed at 24, 48, 72, 96, and 120 hpi for examining livers. Viral loads in the livers were evaluated by qRT-PCR. A comparative analysis of markers associated with the regulation of energy metabolism across several functional classes (ATP, pyruvic and lactic acids, mitochondrial protein, NAD+/NADH ratios, and enolase, lactic acid dehydrogenase and the respiratory chain complex I activities) were examined in the context of IBDV infection.3. The results indicated that increases in the enzymatic activities associated with glycolytic metabolism in turn affected the synthesis and cytoplasmic concentrations of ATP at early timepoints in infected chicken embryos. Subsequently, energy metabolism was inhibited through the pathological perturbations of metabolic enzymes and mitochondrial damage, as inferred from reduced ATP generation.4. These results suggested impaired bioenergetics, which may lead to liver dysfunction consequent to IBDV infection, contributing to the disease pathogenesis.

To avoid chilling injury (CI) of nectarines during storage, the impact of near-freezing temperature (NFT) (-1.4 ± 0.1 °C), 0 ± 0.1 °C and 5 ± 0.1 °C on CI incidence, ion leakage, levels of soluble sugars and enzymatic activities related to soluble sugars and energy metabolism, were investigated over five weeks. NFT-stored fruit showed no CI symptoms and significantly (P < 0.05) lower increase of ion leakage than those kept at 0 and 5 °C. NFT significantly (P < 0.05) diminished the activities of sucrose metabolism-associated enzymes leading to a higher level of sucrose in fruit, and maintained higher activities of hexokinase and fructokinase. Additionally, NFT-stored fruit exhibited significantly (P < 0.05) higher activities of energy metabolism-associated enzymes than fruit stored at 0 and 5 °C, leading to high levels of adenosine triphosphate and energy in fruit. These results indicated that NFT storage can effectively enhance chilling tolerance of nectarine fruit by inducing the metabolism of soluble carbohydrates and energy., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The effects of postharvest treatment with sodium silicate (Si) (100 mM) on mitochondrial ROS production and energy metabolism of the muskmelon fruits (cv. Yujinxiang) on development of defense responses to Trichothecium roseum were studied. Si treatment decreased decay severity of inoculated muskmelons, enhanced the activities of energy metabolism of key enzymes and kept the intracellular ATP at a higher level; meanwhile, Si also induced the mtROS accumulation such as H 2 O 2 and superoxide anion. TMT-based quantitative proteomics analysis revealed that a total of 24 proteins with significant differences in abundance involved in energy metabolism, defense and stress responses, glycolytic and TCA cycle, and oxidation-reduction process. It is suggested by our study that melon fruit mitochondria, when induced by Si treatments, play a key role in priming of host resistance against T. roseum infection through the regulation of energy metabolism and ROS production in the pathogen infected muskmelon fruits., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Transcranial direct current stimulation (tDCS) is a neuromodulatory method that has been tested experimentally and has already been used as an adjuvant therapeutic option to treat a number of neurological disorders and neuropsychiatric diseases. Beyond its well known local effects within the brain, tDCS also transiently promotes systemic glucose uptake and reduces the activity of the neurohormonal stress axes. We aimed to test whether the effects of a single tDCS application could be replicated upon double stimulation to persistently improve systemic glucose tolerance and stress axes activity in humans. In a single-blinded cross-over study, we examined 15 healthy male volunteers. Anodal tDCS vs sham was applied twice in series. Systemic glucose tolerance was investigated by the standard hyperinsulinaemic-euglycaemic glucose clamp procedure, and parameters of neurohormonal stress axes activity were measured. Because tDCS-induced brain energy consumption has been shown to be part of the mechanism underlying the assumed effects, we monitored the cerebral high-energy phosphates ATP and phosphocreatine by 31 phosphorus magnetic resonance spectroscopy. As hypothesised, analyses revealed that double anodal tDCS persistently increases glucose tolerance compared to sham. Moreover, we observed a significant rise in cerebral high-energy phosphate content upon double tDCS. Accordingly, the activity of the neurohormonal stress axes was reduced upon tDCS compared to sham. Our data demonstrate that double tDCS promotes systemic glucose uptake and reduces stress axes activity in healthy humans. These effects suggest that repetitive tDCS may be a future non-pharmacological option for combating glucose intolerance in type 2 diabetes patients., (© 2019 British Society for Neuroendocrinology.)

Type 2 diabetic women have a high risk of mortality via myocardial infarction even with anti-diabetic treatments. Resveratrol (RSV) is a natural polyphenol, well-known for its antioxidant property, which has also shown interesting positive effects on mitochondrial function. Therefore, we aim to investigate the potential protective effect of 1 mg/kg/day of RSV on high energy compounds, during myocardial ischemia-reperfusion in type 2 diabetic female Goto-Kakizaki (GK) rats. For this purpose, we used 31 P magnetic resonance spectroscopy in isolated perfused heart experiments, with a simultaneous measurement of myocardial function and coronary flow. RSV enhanced adenosine triphosphate (ATP) and phosphocreatine (PCr) contents in type 2 diabetic hearts during reperfusion, in combination with better functional recovery. Complementary biochemical analyses showed that RSV increased creatine, total adenine nucleotide heart contents and citrate synthase activity, which could be involved in better mitochondrial functioning. Moreover, improved coronary flow during reperfusion by RSV was associated with increased eNOS, SIRT1, and P-Akt protein expression in GK rat hearts. In conclusion, RSV induced cardioprotection against ischemia-reperfusion injury in type 2 diabetic female rats via increased high energy compound contents and expression of protein involved in NO pathway. Thus, RSV presents high potential to protect the heart of type 2 diabetic women from myocardial infarction.

Insufficient or dysregulated energy metabolism may underlie diverse inherited and degenerative diseases, cancer, and even aging itself. ATP is the central energy carrier in cells, but critical pathways for regulating ATP levels are not systematically understood. We combined a pooled clustered regularly interspaced short palindromic repeats interference (CRISPRi) library enriched for mitochondrial genes, a fluorescent biosensor, and fluorescence-activated cell sorting (FACS) in a high-throughput genetic screen to assay ATP concentrations in live human cells. We identified genes not known to be involved in energy metabolism. Most mitochondrial ribosomal proteins are essential in maintaining ATP levels under respiratory conditions, and impaired respiration predicts poor growth. We also identified genes for which coenzyme Q10 (CoQ10) supplementation rescued ATP deficits caused by knockdown. These included CoQ10 biosynthetic genes associated with human disease and a subset of genes not linked to CoQ10 biosynthesis, indicating that increasing CoQ10 can preserve ATP in specific genetic contexts. This screening paradigm reveals mechanisms of metabolic control and genetic defects responsive to energy-based therapies., Competing Interests: The authors have declared that no competing interests exist.

Study Objectives: Obstructive sleep apnea (OSA) is a well-established cause of impaired daytime functioning. However, there is a complex inter-individual variability in neurobehavioral performance in OSA patients. We previously reported compromised brain bioenergetics during apneic sleep in severe OSA. In this study, we investigate whether brain bioenergetics during resting wakefulness are related to neurobehavioral performance., Methods: Patients attended the sleep laboratory in the evening and were kept awake over-night. Repeated testing on the 10-minute psychomotor vigilance task (PVT, at 9 pm, 11 pm, 1 am, 3 am, 5 am) and 30-minute AusEd driving simulator task (9 pm and 5 am) was performed. Brain bioenergetics (inorganic phosphate/adenosine triphosphate ratio, Pi/ATP) were measured in the temporal lobe during resting wakefulness at 7 am in a 1.5T MRI scanner using phosphorus magnetic resonance spectroscopy (31P MRS)., Results: Fifteen males with severe OSA (age 47.7 ± 10.4 years, body mass index [BMI] 34 ± 6.6 kg/m2, apnea hypopnea index [AHI] 79.7 ± 21.8/hour) were investigated. A higher Pi/ATP ratio in the brain (lower phosphorylation potential) was correlated with worse PVT and driving simulator performance across the testing period (PVT lapses: r = 0.632, r2 = 0.399, p = 0.012; and AusEd braking reaction time: r = 0.609, p = 0.016). In contrast, the conventional AHI measure of disease severity was not significantly correlated with performance (PVT lapses: r = -0.084, p = 0.8; and AusEd braking reaction time: r = -0.326, p = 0.2)., Conclusions: Lower phosphorylation potential was associated with worse performance. Compromised brain bioenergetics may in part underlie the neurobehavioral deficits in untreated OSA. We speculate that better brain bioenergetics may explain why some OSA patients are relatively asymptomatic compared with others.

Background/aims: Diabetic cardiomyopathy (DCM) is a serious complication of diabetes. It is therefore crucial to elucidate the characteristic metabolic changes that occur during the development of diabetes to gain an understanding the pathogenesis of this disease and identify potential drug targets involved., Methods: 1H nuclear magnetic resonance-based metabonomics combined with HPLC measurements were used to determine the metabolic changes in isolated cardiac tissues after 5 weeks, 9 weeks, and 15 weeks in rats treated with streptozotocin., Results: Pattern recognition analysis clearly discriminated the diabetic rats from time-matched control rats, suggesting that the metabolic profile of the diabetic group was markedly different from that of the controls. Quantitative analysis showed that the levels of energy metabolites, such as the high-energy phosphate pool (ATP and creatine), significantly decreased in a time-dependent manner. Correlation analysis revealed the inhibition of glycolysis and the tricarboxylic acid (TCA) cycle, enhanced lipid metabolism, and changes in some amino acids, which may have led to the decline in energy production in the DCM rats., Conclusions: The results indicated that the administration of energy substances or the manipulation of myocardial energy synthesis induced by increased glucose oxidation may contribute to the amelioration of cardiac dysfunction in diabetes., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

Fish generally have much higher requirements for dietary protein than mammals, and this long-standing puzzle remains unsolved. The present study was conducted with zebrafish (omnivores) and hybrid striped bass (HSB, carnivores) to test the hypothesis that AAs are oxidized at a higher rate than carbohydrates (e.g., glucose) and fatty acids (e.g., palmitate) to provide ATP for their tissues. Liver, proximal intestine, kidney, and skeletal muscle isolated from zebrafish and HSB were incubated at 28.5 °C (zebrafish) or 26 °C (HSB) for 2 h in oxygenated Krebs-Henseleit bicarbonate buffer (pH 7.4, with 5 mM D-glucose) containing 2 mM L-[U- 14 C]glutamine, L-[U- 14 C]glutamate, L-[U- 14 C]leucine, or L-[U- 14 C]palmitate, or a trace amount of D-[U- 14 C]glucose. In parallel experiments, tissues were incubated with a tracer and  a mixture of unlabeled substrates [glutamine, glutamate, leucine, and palmitate (2 mM each) plus 5 mM D-glucose]. 14 CO 2 was collected to calculate the rates of substrate oxidation. In the presence of glucose or a mixture of substrates, the rates of oxidation of glutamate and ATP production from this AA by the proximal intestine, liver, and kidney of HSB   were much higher than those for glucose and palmitate. This was also true for glutamate in the skeletal muscle and glutamine in the liver of both species, glutamine in the HSB kidney, and leucine in the zebrafish muscle, in the presence of a mixture of substrates. We conclude that glutamate plus glutamine plus leucine contribute to ~80% of ATP production in the liver, proximal intestine, kidney, and skeletal muscle of zebrafish and HSB. Our findings provide the first direct evidence that the major tissues of fish use AAs (mainly glutamate and glutamine) as primary energy sources instead of carbohydrates or lipids.

Growth and development of plants is ultimately driven by light energy captured through photosynthesis. ATP acts as universal cellular energy cofactor fuelling all life processes, including gene expression, metabolism, and transport. Despite a mechanistic understanding of ATP biochemistry, ATP dynamics in the living plant have been largely elusive. Here, we establish MgATP 2- measurement in living plants using the fluorescent protein biosensor ATeam1.03-nD/nA. We generate Arabidopsis sensor lines and investigate the sensor in vitro under conditions appropriate for the plant cytosol. We establish an assay for ATP fluxes in isolated mitochondria, and demonstrate that the sensor responds rapidly and reliably to MgATP 2- changes in planta. A MgATP 2- map of the Arabidopsis seedling highlights different MgATP 2- concentrations between tissues and within individual cell types, such as root hairs. Progression of hypoxia reveals substantial plasticity of ATP homeostasis in seedlings, demonstrating that ATP dynamics can be monitored in the living plant.

Energy metabolism of "Fuyan" longan fruit treated with hydrogen peroxide (H 2 O 2 ), the most stable of the reactive oxygen, and its relationship to pericarp browning were investigated in this work. The results displayed that H 2 O 2 significantly decreased contents of adenosine triphosphate (ATP) and adenosine diphosphate (ADP). It also inhibited activities of H + -ATPase, Ca 2+ -ATPase and Mg 2+ -ATPase in membranes of plasma, vacuole and mitochondria during the early-storage and mid-storage (except for mitochondrial membrane Mg 2+ -ATPase). These results gave convincing evidence that the treatment of H 2 O 2 accelerating pericarp browning in harvested longans was due to a decrease of ATPase activity and available ATP content. This might break the ion homeostasis and the integrity of mitochondria, which might reduce energy charge and destroy the function and compartmentalization of cell membrane. These together aggravated browning incidence in pericarp of harvested longan fruit., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Objective: To observe the changes of Prohibitin1(PHB1) contents in rat brain, heart, skeletal muscle tissue and mitochondria with acute exhaustive exercise and the effects of acute exhaustive exercise on mitochondrial function in rats, to explore the relationship among PHB1 and mitochondrial function and energy metabolism., Methods: Acute exhaustive exercise model:The rats carried acute exhaustive exercise after 8 weeks of feeding. The heart, brain and skeletal muscle samples were collected and the mitochondria were collected to detect the changes of respiratory function and reactive oxygen species(ROS). The expression of PHB1 protein in tissues and mitochondria was detected by Western blot. The ATP content in the organs and the activity of complexes (ATP synthase activity) in mitochondria were measured by spectrophotometer., Results: ① The ATP contents of brain, myocardium and skeletal muscle were decreased significantly after acute exhaustive exercise. ②The activities of complex V, respiratory control rates (RCR) and ROS in mitochondria of brain, myocardium and skeletal muscle were decreased significantly after acute exhaustive exercise, respiration rate state 4(ST4) was increased significantly, at the same time, respiration rate state 3(ST3) had no significant difference. ③ The expression of PHB1 in mitochondria of skeletal muscle was decreased significantly after acute exhaustive exercise, while there was no significant change in PHB1 in myocardial tissue and mitochondria. ④ The correlation analysis showed that the ATP contents in the brain, myocardium and skeletal muscle were positively correlated with the activity of complex V and the expression of PHB1 after acute exhaustive exercise., Conclusions: After acute exhaustive exercise, the mitochondrial oxidative phosphorylation was reduced, the ROS production was increased, the expression of PHB1 was decreased, the ATP content and the activity of complex V were decreased in the brain and skeletal muscle of rats. Acute exhaustive exercise reduced the expression of PHB1 in mitochondria, decreased mitochondrial function, and reduced energy metabolism.

Background: Sulfonylureas, such as glibenclamide, are antidiabetic drugs that stimulate beta-cell insulin secretion by binding to the sulfonylureas receptors (SURs) of adenosine triphosphate-sensitive potassium channels (KATP). Glibenclamide may be also cardiotoxic, this effect being ascribed to interference with the protective function of cardiac KATP channels for which glibenclamide has high affinity. Prompted by recent evidence that glibenclamide impairs energy metabolism of renal cells, we investigated whether this drug also affects the metabolism of cardiac cells., Methods: The cardiomyoblast cell line H9c2 was treated for 24 h with glibenclamide or metformin, a known inhibitor of the mitochondrial respiratory chain. Cell viability was evaluated by sulforodhamine B assay. ATP and AMP were measured according to the enzyme coupling method and oxygen consumption by using an amperometric electrode, while Fo-F1 ATP synthase activity assay was evaluated by chemiluminescent method. Protein expression was measured by western blot., Results: Glibenclamide deregulated energy balance of H9c2 cardiomyoblasts in a way similar to that of metformin. It inhibited mitochondrial complexes I, II and III with ensuing impairment of oxygen consumption and ATP synthase activity, ATP depletion and increased AMPK phosphorylation. Furthermore, glibenclamide disrupted mitochondrial subcellular organization. The perturbation of mitochondrial energy balance was associated with enhanced anaerobic glycolysis, with increased activity of phosphofructo kinase, pyruvate kinase and lactic dehydrogenase. Interestingly, some additive effects of glibenclamide and metformin were observed., Conclusions: Glibenclamide deeply alters cell metabolism in cardiac cells by impairing mitochondrial organization and function. This may further explain the risk of cardiovascular events associated with the use of this drug, alone or in combination with metformin., (© 2017 The Author(s). Published by S. Karger AG, Basel.)

We developed an effective and rapid assay to detect both bio-energetic and envelope permeability (BEEP) alterations of Pseudomonas aeruginosa. The assay is based on quantification of extracellular ATP in bacterial cultures using luciferase as a reporter. To demonstrate the validity of our assay we conducted a biased screen of a transposon insertion library in P. aeruginosa strain PAO1 in order to expedite the isolation of mutants with defects in bioenergetic pathways. We successfully isolated insertion mutants that were reduced for extracellular ATP accumulation and identified the corresponding mutations that caused the phenotype. Most of the genes identified from this analysis were associated with energy metabolism and several appeared to be potentially novel bioenergetic targets. In addition, we show that treatment of P. aeruginosa strain PAO1 with antibiotics that disrupt the bacterial cell envelope leads to greater extracellular ATP accumulation. In summary, increases in extracellular ATP accumulation above wild type levels indicated a perturbation of membrane permeability while decreases in extracellular ATP accumulation indicated defects in bioenergetics., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Mitochondrial dysfunction has recently been implicated as an underlying factor to several common neurodegenerative diseases, including Parkinson's disease, Alzheimer's and amyotrophic lateral sclerosis (ALS). Valosin containing protein (VCP)-associated multisystem proteinopathy is a new hereditary disorder associated with inclusion body myopathy, Paget disease of bone (PDB), frontotemporal dementia (FTD) and ALS. VCP has been implicated in several transduction pathways including autophagy, apoptosis and the PINK1/Parkin cascade of mitophagy. In this report, we characterized VCP patient and mouse fibroblasts/myoblasts to examine their mitochondrial dynamics and bioenergetics. Using the Seahorse XF-24 technology, we discovered decreased spare respiratory capacity (measurement of extra ATP that can be produced by oxidative phosphorylation in stressful conditions) and increased ECAR levels (measurement of glycolysis), and proton leak in VCP human fibroblasts compared with age- and sex-matched unaffected first degree relatives. We found decreased levels of ATP and membrane potential, but higher mitochondrial enzyme complexes II+III and complex IV activities in the patient VCP myoblasts when compared to the values of the control cell lines. These results suggest that mutations in VCP affect the mitochondria's ability to produce ATP, thereby resulting in a compensatory increase in the cells' mitochondrial complex activity levels. Thus, this novel in vitro model may be useful in understanding the pathophysiology and discovering new drug targets of mitochondrial dynamics and physiology to modify the clinical phenotype in VCP and related multisystem proteinopathies (MSP)., (Copyright © 2015 © Elsevier B.V. and Mitochondria Research Society. Published by Elsevier B.V. All rights reserved.)

Unlabelled: ATP is a key molecule of cell physiology, but despite its importance, there are currently no methods for monitoring single-cell ATP fluctuations in live bacteria. This is a major obstacle in studies of bacterial energy metabolism, because there is a growing awareness that bacteria respond to stressors such as antibiotics in a highly individualistic manner. Here, we present a method for long-term single-cell tracking of ATP levels in Mycobacterium smegmatis based on a combination of microfluidics, time-lapse microscopy, and Förster resonance energy transfer (FRET)-based ATP biosensors. Upon treating cells with antibiotics, we observed that individual cells undergo an abrupt and irreversible switch from high to low intracellular ATP levels. The kinetics and extent of ATP switching clearly discriminate between an inhibitor of ATP synthesis and other classes of antibiotics. Cells that resume growth after 24 h of antibiotic treatment maintain high ATP levels throughout the exposure period. In contrast, antibiotic-treated cells that switch from ATP-high to ATP-low states never resume growth after antibiotic washout. Surprisingly, only a subset of these nongrowing ATP-low cells stains with propidium iodide (PI), a widely used live/dead cell marker. These experiments also reveal a cryptic subset of cells that do not resume growth after antibiotic washout despite remaining ATP high and PI negative. We conclude that ATP tracking is a more dynamic, sensitive, reliable, and discriminating marker of cell viability than staining with PI. This method could be used in studies to evaluate antimicrobial effectiveness and mechanism of action, as well as for high-throughput screening., Importance: New antimicrobials are urgently needed to stem the rising tide of antibiotic-resistant bacteria. All antibiotics are expected to affect bacterial energy metabolism, directly or indirectly, yet tools to assess the impact of antibiotics on the ATP content of individual bacterial cells are lacking. The method described here for single-cell tracking of intracellular ATP in live bacteria has many advantages compared to conventional ensemble-averaged assays. It provides a continuous real-time readout of bacterial ATP content, cell vitality, and antimicrobial mechanism of action with high temporal resolution at the single-cell level. In combination with high-throughput microfluidic devices and automated microscopy, this method also has the potential to serve as a novel screening tool in antimicrobial drug discovery., (Copyright © 2015 Maglica et al.)

A literal mountain of documentation generated in the past five decades showing unmistakable health hazards associated with extremely low-frequency electromagnetic fields (ELF-EMFs) exposure. However, the relation between energy mechanism and ELF-EMF exposure is poorly understood. In this study, Caenorhabditis elegans was exposed to 50 Hz ELF-EMF at intensities of 0.5, 1, 2, and 3 mT, respectively. Their metabolite variations were analyzed by GC-TOF/MS-based metabolomics. Although minimal metabolic variations and no regular pattern were observed, the contents of energy metabolism-related metabolites such as pyruvic acid, fumaric acid, and L-malic acid were elevated in all the treatments. The expressions of nineteen related genes that encode glycolytic enzymes were analyzed by using quantitative real-time PCR. Only genes encoding GAPDH were significantly upregulated (P < 0.01), and this result was further confirmed by western blot analysis. The enzyme activity of GAPDH was increased (P < 0.01), whereas the total intracellular ATP level was decreased. While no significant difference in lifespan, hatching rate and reproduction, worms exposed to ELF-EMF exhibited less food consumption compared with that of the control (P < 0.01). In conclusion, C. elegans exposed to ELF-EMF have enhanced energy metabolism and restricted dietary, which might contribute to the resistance against exogenous ELF-EMF stress.

Background: Postoperative fatigue syndrome (POFS) is a common clinical complication followed by almost every major abdominal surgery. Ginsenoside Rb1 (GRb1), a principle ginsenoside in ginseng, could exert a potent anti-fatigue effect on POFS. However, the mechanism is still unknown. Previous studies revealed that alterations in the energy metabolism in the skeletal muscle may play a vital role in the development and progression of fatigue. In the present study, we investigate the effect of GRb1 on energy metabolism in the skeletal muscle of a rat model of POFS induced by major small intestinal resection., Methods: GRb1 (10 mg/kg) was intraperitoneally administrated once daily for 1, 3, 7, and 10 d from the operation day, respectively. The locomotor activity was recorded every day, and total food intake was calculated starting from 24 h after surgery. After GRb1 treatment was completed, blood and skeletal muscle were sampled. The level of blood glucose was determined by an automatic biochemical analyzer. The content of adenosine triphosphate (ATP) in skeletal muscle was determined by high-performance liquid chromatography. The activity of energy metabolic enzymes Na(+)-K(+)-ATPase, pyruvate kinase, and succinate dehydrogenase (SDH) was assessed by commercially available kits., Results: The results revealed that GRb1 could increase locomotor activity of POFS rats and significantly increase their total food intake postoperatively (P < 0.05). Furthermore, GRb1 also significantly increased ATP content in the skeletal muscle of POFS rats (P < 0.05). Meanwhile, the activity of Na(+)-K(+)-ATPase and SDH in the skeletal muscle of POFS rats was enhanced by GRb1 (P < 0.05). However, no significant differences in blood glucose and pyruvate kinase were found between the POFS and GRb1 treatment rats (P > 0.05)., Conclusions: These results suggest that GRb1 may improve skeletal muscle energy metabolism in POFS, and the underlying mechanism may be associated with an increase in the content of ATP and an enhancement in the activity of energy metabolic enzymes such as Na(+)-K(+)-ATPase ATPase and SDH in the skeletal muscle., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Importance: Abnormalities in neural activity and cerebral bioenergetics have been observed in schizophrenia (SZ). Further defining energy metabolism anomalies would provide crucial information about molecular mechanisms underlying SZ and may be valuable for developing novel treatment strategies., Objective: To investigate cerebral bioenergetics in SZ via measurement of creatine kinase activity using in vivo 31P magnetization transfer spectroscopy., Design, Setting, and Participants: Cross-sectional case-control study in the setting of clinical services and a brain imaging center of an academic psychiatric hospital. Twenty-six participants with chronic SZ (including a subgroup diagnosed as having schizoaffective disorder) and 26 age-matched and sex-matched healthy control subjects (25 usable magnetic resonance spectroscopy data sets from the latter)., Intervention: 31P magnetization transfer spectroscopy., Main Outcomes and Measures: The primary outcome measure was the forward rate constant (k(f)) of the creatine kinase enzyme in the frontal lobe. We also collected independent measures of brain intracellular pH and steady-state metabolite ratios of high-energy phosphate-containing compounds (phosphocreatine and adenosine triphosphate [ATP]), inorganic phosphate, and the 2 membrane phospholipids phosphodiester and phosphomonoester., Results: A substantial (22%) and statistically significant (P = .003) reduction in creatine kinase kf was observed in SZ. In addition, intracellular pH was significantly reduced (7.00 in the SZ group vs 7.03 in the control group, P = .007) in this condition. The phosphocreatine to ATP ratio, inorganic phosphate to ATP ratio, and phosphomonoester to ATP ratio were not substantially altered in SZ, but a significant (P = .02) reduction was found in the phosphodiester to ATP ratio. The abnormalities were similar between SZ and schizoaffective disorder., Conclusions and Relevance: Using a novel 31P magnetization transfer magnetic resonance spectroscopy approach, we provide direct and compelling evidence for a specific bioenergetic abnormality in SZ. Reduced kf of the creatine kinase enzyme is consistent with an abnormality in storage and use of brain energy. The intracellular pH reduction suggests a relative increase in the contribution of glycolysis to ATP synthesis, providing convergent evidence for bioenergetic abnormalities in SZ. The similar phosphocreatine to ATP ratios in SZ and healthy controls suggest that the underlying bioenergetics abnormality is not associated with change in this metabolite ratio.

Intervertebral disc (IVD) degeneration is closely associated with low back pain (LBP), which is a major health concern in the U.S. Cellular biosynthesis of extracellular matrix (ECM), which is important for maintaining tissue integrity and preventing tissue degeneration, is an energy demanding process. Due to impaired nutrient support in avascular IVD, adenosine triphosphate (ATP) supply could be a limiting factor for maintaining normal ECM synthesis. Therefore, the objective of this study was to investigate the energy metabolism in the annulus fibrosus (AF) and nucleus pulposus (NP) of porcine IVD under static and dynamic compressions. Under compression, pH decreased and the contents of lactate and ATP increased significantly in both AF and NP regions, suggesting that compression can promote ATP production via glycolysis and reduce pH by increasing lactate accumulation. A high level of extracellular ATP content was detected in the NP region and regulated by compressive loading. Since ATP can serve not only as an intra-cellular energy currency, but also as a regulator of a variety of cellular activities extracellularly through the purinergic signaling pathway, our findings suggest that compression-mediated ATP metabolism could be a novel mechanobiological pathway for regulating IVD metabolism., (© 2013 Orthopaedic Research Society.)

A plethora of data is accumulating from high throughput methods on metabolites, coenzymes, proteins, and nucleic acids and their interactions as well as the signalling and regulatory functions and pathways of the cellular network. The frozen moment viewed in a single discrete time sample requires frequent repetition and updating before any appreciation of the dynamics of component interaction becomes possible. Even then in a sample derived from a cell population, time-averaging of processes and events that occur in out-of-phase individuals blur the detailed complexity of single cell organization. Continuously-grown cultures of yeast can become spontaneously self-synchronized, thereby enabling resolution of far more detailed temporal structure. Continuous on-line monitoring by rapidly responding sensors (O2 electrode and membrane-inlet mass spectrometry for O2, CO2 and H2S; direct fluorimetry for NAD(P)H and flavins) gives dynamic information from time-scales of minutes to hours. Supplemented with capillary electophoresis and gas chromatography mass spectrometry and transcriptomics the predominantly oscillatory behaviour of network components becomes evident, with a 40 min cycle between a phase of increased respiration (oxidative phase) and decreased respiration (reductive phase). Highly pervasive, this ultradian clock provides a coordinating function that links mitochondrial energetics and redox balance to transcriptional regulation, mitochondrial structure and organelle remodelling, DNA duplication and cell division events. Ultimately, this leads to a global partitioning of anabolism and catabolism and the enzymes involved, mediated by a relatively simple ATP feedback loop on chromatin architecture.

The aim of this study was to examine the effect of the archetypal pro-inflammatory cytokine, interleukin-1beta (IL-1β), on high-energy phosphate levels within an ex vivo rat organotypic hippocampal-slice culture (OHSC) preparation using phosphorus ((31)P) magnetic resonance spectroscopy (MRS). Intrastriatal microinjection of IL-1β induces a chronic reduction in the apparent diffusion coefficient (ADC) of tissue water, which may be indicative of metabolic failure as established by in vivo models of acute cerebral ischaemia. The OHSC preparation enables examination of the effects of IL-1β on brain parenchyma per se, independent of the potentially confounding effects encountered in vivo such as perfusion changes, blood-brain barrier (BBB) breakdown and leukocyte recruitment. (31)P MRS is a technique that can detect multiple high-energy phosphate metabolites within a sample non-invasively. Here, for the first time, we characterise the energy metabolism of OHSCs using (31)P MRS and demonstrate that IL-1β does not compromise high-energy phosphate metabolism. Thus, the chronic reduction in ADC observed in vivo is unlikely to be a consequence of metabolic failure., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Neuronal signaling consumes much of the brain energy, mainly through the restoration of the membrane potential (MP) by ATP-consuming ionic pumps. We have reported that, compared with waking, ATP levels increase during the initial hours of natural slow-wave sleep, a time with prominent electroencephalogram (EEG) delta oscillations (0.5-4.5 Hz). We have hypothesized that there is a delta oscillation-ATP increase coupling, since, during delta waves, neurons exhibit a prolonged hyperpolarizing phase followed by a very brief phase of action potentials. However, direct proof of this hypothesis is lacking, and rapid changes in EEG/neuronal activity preclude measurement in the naturally sleeping brain. Thus, to induce a uniform state with pure delta oscillations and one previously shown to be accompanied by a similar pattern of neuronal activity during delta waves as natural sleep, we used ketamine-xylazine treatment in rats. We here report that, with this treatment, the high-energy molecules ATP and ADP increased in frontal and cingulate cortices, basal forebrain, and hippocampus compared with spontaneous waking. Moreover, the degree of ATP increase positively and significantly correlated with the degree of EEG delta activity. Supporting the hypothesis of decreased ATP consumption during delta activity, the ATP-consuming Na+-K+-ATPase mRNA levels were significantly decreased, whereas the mRNAs for the ATP-producing cytochrome c oxidase (COX) subunits COX III and COX IVa were unchanged. Taken together, these data support the hypothesis of a cortical delta oscillation-dependent reduction in ATP consumption, thus providing the brain with increased ATP availability, and likely occurring because of reduced Na+-K+-ATPase-related energy consumption., (Published by Elsevier Ltd.)

Background: Differences in brain energy metabolism have been found between migraine patients and controls in previous phosphorus magnetic resonance spectroscopy ((31)P-MRS) studies, most of them emphasizing migraine with aura (MwA). The aim of this study was to verify potential changes in resting-state brain energy metabolism in patients with migraine without aura (MwoA) compared to control subjects by (31)P-MRS at 3 tesla., Methods: Quantification was performed using the phantom replacement technique. MRS measurements were performed interictally and in the medial occipital lobe of 19 MwoA patients and 26 age-matched controls., Results: A significantly decreased phosphocreatine concentration ([PCr]) was found as in previous studies. While adenosine triphosphate concentration ([ATP]) was considered to be constant in previously published work, this study found a significant decrease in the measured [ATP] in MwoA patients. The inorganic phosphate ([P(i)]) and magnesium ([Mg(2+)]) concentrations were not significantly different between MwoA patients and controls., Conclusions: The altered metabolic concentrations indicate that the energy metabolism in MwoA patients is impaired, certainly in a subgroup of patients. The actual decrease in [ATP] adds further strength to the theory of the presence of a mitochondrial component in the pathophysiology of migraine.

Objective: Increased pro-inflammatory cytokines and reactive oxygen and nitrogen species (RONS) occur in osteoarthritis (OA). Oxygen tension can alter the levels of RONS induced by interleukin-1 (IL-1). RONS such as nitric oxide (NO) can alter energy metabolism. The aim of this study was to determine if oxygen tension alters energy metabolism, in articular cartilage, in response to IL-1 or NO and to determine if cell death occurred., Design: Porcine articular chondrocytes were incubated with IL-1 or the NO donor NOC-18 for 48 h in either 1, 5 or 20% O(2). Adenosine triphosphate (ATP) levels were measured and immunoblots for adenosine monophosphate-activated protein kinase (AMPK) were done. Protein translation was measured by S6 activation. Senescence and autophagy were determined by increased caveolin or conversion of LC3-I to LC3-II respectively., Results: One percent O(2) significantly reduced ATP levels compared with 20% O(2). Five percent O(2) significantly increased ATP levels compared with 20% O(2). One percent O(2) significantly increased phospho-AMPK (pAMPK) protein expression compared with 5 or 20% O(2). Oxygen tension had no effects on pS6, caveolin or LC3-II levels. IL-1-induced NO production was significantly reduced with decreased oxygen tension, and significantly reduced ATP levels at all oxygen tensions, but pAMPK was only significantly increased at 5% O(2). IL-1 significantly reduced pS6 at all oxygen tensions. IL-1 had no effects on caveolin and significantly increased LC3-II at 20% O(2) only. NOC-18 significantly reduced ATP levels at all oxygen tensions, and significantly increased pAMPK at 5% O(2) only, and significantly decreased pAMPK at 1% O(2). NOC-18 significantly reduced pS6 at 1% O(2) and significantly increased caveolin at 5% O(2), and LC3-II at 1% O(2)., Conclusion: Our data suggest 5% O(2) is optimal for energy metabolism and protective to some effects of IL-1 and NO. NO has the greatest effects on ATP levels and the induction of autophagy at 1% O(2)., (Copyright 2010 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Impaired energy metabolism is considered a possible cause of fatigue. The thiamine derivative, thiamine tetrahydrofurfuryl disulfide (TTFD), is prescribed and is also an over-the-counter drug for the attenuation of fatigue. It is readily absorbed from the intestinal tract and converted into thiamine pyrophosphate (TPP), which plays an important role as a cofactor for enzymes of metabolic pathways involved in the production of adenosine triphosphate (ATP). We postulated that TTFD has an anti-fatigue effect by improving energy metabolism during physical-fatigue loading. Here, we initially used the forced swimming test to determine whether daily TTFD or thiamine for 5 days has anti-fatigue effects on weight-loaded rats. The swimming duration of TTFD-, but not of thiamine-treated rats, was significantly longer than that of control rats (P < .05). Based on these findings, we examined changes in the levels of thiamine and its phosphate esters in various organs and the effect of TTFD on ATP levels in skeletal muscle after forced swimming, to determine the cellular mechanisms of the anti-fatigue effect of TTFD. Daily TTFD resulted in a characteristic distribution of thiamine and its phosphate esters in rat skeletal muscle, liver, kidney, heart, brain, and plasma. Furthermore, daily TTFD attenuated the decrease in ATP content in the skeletal muscle caused by forced swimming with a weight load for a defined period (150 s). These results indicate that TTFD exerts anti-fatigue effects by improving energy metabolism during physical fatigue.

Mitochondrial dysfunction hypothetically contributes to neuronal degeneration in patients with Parkinson's disease. While several in vitro data exist, the measurement of cerebral mitochondrial dysfunction in living patients with Parkinson's disease is challenging. Anatomical magnetic resonance imaging combined with phosphorus and proton magnetic resonance spectroscopic imaging provides information about the functional integrity of mitochondria in specific brain areas. We measured partial volume corrected concentrations of low-energy metabolites and high-energy phosphates with sufficient resolution to focus on pathology related target areas in Parkinson's disease. Combined phosphorus and proton magnetic resonance spectroscopic imaging in the mesostriatal region was performed in 16 early and 13 advanced patients with Parkinson's disease and compared to 19 age-matched controls at 3 Tesla. In the putamen and midbrain of both Parkinson's disease groups, we found a bilateral reduction of high-energy phosphates such as adenosine triphophosphate and phosphocreatine as final acceptors of energy from mitochondrial oxidative phosphorylation. In contrast, low-energy metabolites such as adenosine diphophosphate and inorganic phosphate were within normal ranges. These results provide strong in vivo evidence that mitochondrial dysfunction of mesostriatal neurons is a central and persistent phenomenon in the pathogenesis cascade of Parkinson's disease which occurs early in the course of the disease.

Adult male Wistar rats were intoxicated with 1% lead acetate (PbAc) administered in drinking water for nine months, which amounts to a period five times longer than the duration of one spermatogenesis. There were mitochondrial ultrastructure disorders of epididymal epithelial cells observed in PbAc-treated rats; also a significant lead-induced decrease in ATP concentration in epididymal epithelial cells (by 32%, P < 0.05), Adenylate Energy Charge value (AEC) (by 8%, P < 0.05) and an increase in ADP (28.5%, P < 0.05), AMP (27%, P < 0.05) and adenosine (by 56%, P < 0.05). The results were measured using high performance liquid chromatography (HPLC) and detected even at low lead concentrations in whole blood (M:7.03 μg/dL; Q1-Q3: 2.99-7.65). The function of mitochondria in cultured epididymal epithelial cells of control and PbAc-treated animals were evaluated using fluorophores: Mitotracker Green FM and JC-1. After incubation with Mitotracker Green FM, we observed active mitochondria producing bright green fluorescence in the cytoplasm of cultured epididymal epithelial cells, both in the control group and the Pb-treated animals. Incubation of cultured epididymal epithelial cells of animals from both groups produced red-orange fluorescence with the mitochondrial JC-1 probe indicating mitochondria with high membrane potential (ΔΨm > 80-100 mV) and green fluorescence in the mitochondria with low membrane potential (ΔΨm < 80 mV). The results showed that a chronic low-level exposure to lead, even without severe clinical symptoms of contamination, disrupted the ultrastructure and energy metabolism of mitochondria in epididymal epithelial cells.

The hepatic energy state, defined by adenine nucleotide levels, couples metabolic pathways with energy requirements. This coupling is fundamental in the adaptive response to many conditions and is impaired in metabolic disease. We have found that the hepatic energy state is substantially reduced following exercise, fasting, and exposure to other metabolic stressors in C57BL/6 mice. Glucagon receptor signaling was hypothesized to mediate this reduction because increased plasma levels of glucagon are characteristic of metabolic stress and because this hormone stimulates energy consumption linked to increased gluconeogenic flux through cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) and associated pathways. We developed what we believe to be a novel hyperglucagonemic-euglycemic clamp to isolate an increment in glucagon levels while maintaining fasting glucose and insulin. Metabolic stress and a physiological rise in glucagon lowered the hepatic energy state and amplified AMP-activated protein kinase signaling in control mice, but these changes were abolished in glucagon receptor- null mice and mice with liver-specific PEPCK-C deletion. 129X1/Sv mice, which do not mount a glucagon response to hypoglycemia, displayed an increased hepatic energy state compared with C57BL/6 mice in which glucagon was elevated. Taken together, these data demonstrate in vivo that the hepatic energy state is sensitive to glucagon receptor activation and requires PEPCK-C, thus providing new insights into liver metabolism.

Phenylpyrazoles are relatively new insecticides designed to manage problematic insect resistance and public health hazards encountered with older pesticide families. In vitro cytotoxicity induced by the phenylpyrazole insecticides, Ethiprol and Fipronil, and Fipronil metabolites, sulfone and sulfide, was studied in Caco-2 cells. This cellular model was chosen because it made possible to mimic the primary site of oral exposure to xenobiotics, the intestinal epithelium. Assessment of the barrier function of Caco-2 epithelium was assessed by TEER measurement and showed a major loss of barrier integrity after exposure to Fipronil and its metabolites, but not to Ethiprol. The disruption of the epithelial barrier was attributed to severe ATP depletion independent of cell viability, as revealed by LDH release. The origin of energetic metabolism failure was investigated and revealed a transient enhancement of tetrazolium salt reduction and an increase in lactate production by Caco-2 cells, suggesting an increase in glucose metabolism by pesticides. Cellular symptoms observed in these experiments lead us to hypothesize that phenylpyrazole insecticides interacted with mitochondria.

Indirect evidence from laboratory studies suggests that mitochondrial energy metabolism is impaired in progressive supranuclear palsy (PSP), but brain energy metabolism has not yet been studied directly in vivo in a comprehensive manner in patients. We have used combined phosphorus and proton magnetic resonance spectroscopy to measure adenosine-triphosphate (ATP), adenosine-diphosphate (ADP), phosphorylated creatine, unphosphorylated creatine, inorganic phosphate and lactate in the basal ganglia and the frontal and occipital lobes of clinically probable patients (N=21; PSP stages II to III) and healthy controls (N=9). In the basal ganglia, which are severely affected creatine in PSP patients, the concentrations of high-energy phosphates (=ATP+phosphorylated creatine) and inorganic phosphate, but not low-energy phosphates (=ADP+unphosphorylated creatine), were decreased. The decrease probably does not reflect neuronal death, as the neuronal marker N-acetylaspartate was not yet significantly reduced in the early-stage patients examined. The frontal lobe, also prone to neurodegeneration in PSP, showed similar alterations, whereas the occipital lobe, typically unaffected, showed less pronounced alterations. The levels of lactate, a product of anaerobic glycolysis, were elevated in 35% of the patients. The observed changes in the levels of cerebral energy metabolites in PSP are consistent with a functionally relevant impairment of oxidative phosphorylation.

Negative pressure wound therapy (NPWT) using reticulated open cell foam dressing (ROCF) is effective for treatment of recalcitrant wounds; however, the effects of this therapy on cellular metabolism remain to be elucidated. The effect of two different subatmospheric pressure applications on the cell energetics of human fibroblasts grown in a 3D fibrin matrix was studied using two different pressure-manifolding materials, an ROCF or gauze under suction (GUS). It was found that levels of cytochrome c oxidase, energy charge, and adenosine triphosphate/adenosine diphosphate were significantly increased following the application of NPWT using ROCF vs. GUS (p<0.05). Increases in these parameters likely reflect an improved energetic status. In addition, levels of transforming growth factor-beta and platelet-derived growth factor (alpha and beta isoforms) were significantly increased (80 and 53%, respectively; p<0.05) over static control cultures following treatment with NPWT using ROCF but not following GUS. These growth factors are known to be important during wound healing. Clearly, both the material used as the dressing to manifold the subatmospheric pressure and the pressure used have a dramatic effect on cellular response.

Adenosine triphosphate (ATP) is well known to be released from injured or inflamed tissues, and to excite/sensitize nociceptors in response to heat and mechanical stimulation. To determine whether muscle releases ATP when it is compressed, we measured ATP release from the extensor digitorum longus muscle (EDL). In addition, we investigated whether there is any difference in ATP release from the EDL of rats 2 days after lengthening contraction (LC), since the condition of the muscle is different, i.e., mechanically hyperalgesic and swollen. The EDL was put in a small chamber and superfused with Krebs-Henseleit solution equilibrated with a gas mixture of 95% oxygen and 5% carbon dioxide. The muscle was quantitatively stimulated with a servo-controlled mechanical stimulator. Reproducibility of ATP release was examined with stimulation using a 20 g force. Stimulus intensity-dependency of ATP release was also examined with 5 time compression with intensities of 5, 10, 20 and 40 g force. Bioluminescent determination by the luciferin-luciferase method was used to quantify ATP in the sample. The ATP release was decreased by repetitive mechanical stimulation of the EDL with 30 min intervals, and it was stimulus intensity (5-40 g force)-dependent. The amount of ATP released from the muscle preparations was not different between the non-treated control and the LC group. These results provide clear evidence that ATP is released from rat skeletal muscle by compression.

Background/aims: Juvenile visceral steatosis (jvs-/-) mice lack the activity of the carnitine transporter OCTN2 and are dependent on carnitine substitution. The effects of carnitine deprivation on carnitine homeostasis and energy metabolism are not known in jvs-/- mice., Methods: jvs-/- mice were studied 3, 6 and 10 days after carnitine deprivation, and compared to jvs-/- mice substituted with carnitine, wild-type (jvs+/+) and jvs+/- mice. Carnitine concentrations were assessed radioenzymatically., Results: Compared to wild-type mice, carnitine-treated jvs-/- mice had decreased plasma beta-hydroxybutyrate levels and showed hepatic fat accumulation. The carnitine levels in plasma, liver and skeletal muscle were decreased by 58, 16 and 17%, respectively. After ten days of carnitine deprivation, the plasma carnitine concentration had fallen by 87% (to 2.3 mumol/l) and the tissue carnitine levels by approximately 50% compared to carnitine-treated jvs-/- mice. Carnitine deprivation was associated with a further drop in plasma beta-hydroxybutyrate and increased hepatic fat. Skeletal muscle glycogen stores decreased and lactate levels increased with carnitine deprivation, whereas tissue ATP levels were maintained., Conclusions: In jvs-/- mice, tissue carnitine stores are more resistant than carnitine plasma concentrations to carnitine deprivation. Metabolic changes (liver steatosis and loss of muscle glycogen stores) appear also early after carnitine deprivation., (2008 S. Karger AG, Basel.)

Gastrointestinal nematodes require energy for active establishment in the gut against intestinal flow and peristaltic motion. In this study we employed CellTiter-Glo Luminescent Cell Viability Assay to measure the ATP value of individual adult Nippostrongylus brasiliensis during the course of immune-mediated expulsion from the small intestine in rats. The ATP values of adult worms taken from the lumen of the distal small intestine were lower than worms collected from the lumen of the proximal small intestine. Moreover, values from worms in the lumen of the proximal small intestine were lower than those from worms in the mucosa, the preferred site of adult N. brasiliensis. The reduction of ATP values in worms from each region was observed not only at expulsion phase, but also at established phases of the infection suggesting that energy metabolism of the parasites is independent of host immune response. When adult worms with low ATP values on day 12 post-infection were implanted surgically into the small intestine of naïve rats, the worms re-established in recipients and completely restored the ATP values. Short in vitro culture of adult worms under low oxygen tension resulted in low ATP value in the worms. These results suggested that adult worms were dislodged from their preferred site by intact energy metabolism activity.

Maintenance of ATP levels is a critical feature of all cells. Mitochondria are responsible for most ATP synthesis in eukaryotes. We show here that mammalian cells respond to a partial chemical uncoupling of mitochondrial oxidative phosphorylation with a decrease in ATP levels, which recovers over several hours to control levels. This recovery occurs through an increased expression of the transcriptional coactivator peroxisome proliferator-activated receptor-coactivator 1alpha (PGC-1alpha) and mitochondrial genes. Cells and animals lacking PGC-1alpha lose this compensatory mechanism and cannot defend their ATP levels or increase mitochondrial gene expression in response to reduced oxidative phosphorylation. The induction of PGC-1alpha and its mitochondrial target genes is triggered by a burst of intracellular calcium, which causes an increase in cAMP-response-element-binding protein and transducer of regulated cAMP-response-element-binding proteins actions on the PGC-1alpha promoter. These data illustrate a fundamental transcriptional cycle that provides homeostatic control of cellular ATP. In light of this compensatory system that limits the toxicity of mild uncoupling, the use of chemical uncoupling of mitochondria as a means of treating obesity should be re-evaluated.

The present study examined the effect of elevated temperature on muscle energy turnover during dynamic exercise. Nine male subjects performed 10 min of dynamic knee-extensor exercise at an intensity of 43 W (SD 10) and a frequency of 60 contractions per minute. Exercise was performed under normal (C) and elevated muscle temperature (HT) through passive heating. Thigh oxygen uptake (V(O2)) was determined from measurements of thigh blood flow and femoral arterial-venous differences for oxygen content. Anaerobic energy turnover was estimated from measurements of lactate release as well as muscle lactate accumulation and phosphocreatine utilization based on analysis of muscle biopsies obtained before and after each exercise. At the start of exercise, muscle temperature was 34.5 degrees C (SD 1.7) in C compared with 37.2 degrees C (SD 0.5) during HT (P < 0.05). Thigh V(O2) after 3 min was 0.52 l/min (SD 0.11) in C and 0.63 l/min (SD 0.13) in HT, and at the end of exercise it was 0.60 l/min (SD 0.14) and 0.61 l/min (SD 0.10) in C and HT, respectively (not significant). Total lactate release was the same between the two temperature conditions, as was muscle lactate accumulation and PCr utilization. Total ATP production (aerobic + anaerobic) was the same between each temperature condition [505.0 mmol/kg (SD 107.2) vs. 527.1 mmol/kg (SD 117.6); C and HT, respectively]. In conclusion, within the range of temperatures studied, passively increasing muscle temperature before exercise has no effect on muscle energy turnover during dynamic exercise.

Four indirect estimations of energy expenditure were examined, (i) O(2) debt, (ii) O(2) deficit, (iii) blood lactate concentration, and (iv) excess CO(2) production during and after 6 exercise durations (2, 4, 10, 15, 30, and 75 s) performed at 3 different intensities (50%, 100%, and 200% of VO(2) max). Analysis of variance (ANOVA) was used to determine if significant differences existed among these 4 estimations of anaerobic energy expenditure and among 4 estimations of total energy expenditure (that included exercise O(2) uptake and excess post-exercise oxygen consumption, or EPOC, measurements). The data indicate that estimations of anaerobic energy expenditure often differed for brief (2, 4, and 10 s) bouts of exercise, but this did not extend to total energy expenditure. At the higher exercise intensities with the longest durations O(2) deficit, blood lactate concentration, and excess CO(2) estimates of anaerobic and total energy expenditure revealed high variability; however, they were not statistically different. Moreover, they all differed significantly from the O(2) debt interpretation (p < 0.05). It is concluded that as the contribution of rapid substrate-level ATP turnover with lactate production becomes larger, the greatest error in quantifying total energy expenditure is suggested to occur not with the method of estimation, but with the omission of a reasonable estimate of anaerobic energy expenditure.

Changes in metabolism of Japanese medaka (Oryzias latipes) embryos exposed to dinoseb (2-sec-butyl-4,6-dinitrophenol), a substituted dinitrophenol herbicide, were determined by in vivo (31)P NMR, high-pressure liquid chromatography (HPLC)-UV, and (1)H NMR metabolomics. ATP and phosphocreatine (PCr) metabolism were characterized within intact embryos by in vivo (31)P NMR; concentrations of ATP, GTP, ADP, GDP, AMP and PCr were determined by HPLC-UV; and changes in numerous polar metabolites were characterized by (1)H NMR-based metabolomics. Rangefinding exposures determined two sublethal doses of dinoseb, 50 and 75 ppb, in which embryos survived from 1-day post fertilization (DPF) through the duration of embryogenesis. In vivo (31)P NMR data were acquired from 900 embryos in 0, 50, and 75 ppb dinoseb at 14, 62, and 110 h (n = 6 groups) after initiation of exposure. After 110 h, embryos were observed for normal development and hatching success, then either preserved in 10% formalin for growth analysis or flash frozen and extracted for HPLC-UV and (1)H NMR analysis. Dinoseb exposure at both concentrations resulted in significant declines in [ATP] and [PCr] at 110 h as measured by in vivo (31)P NMR (p < 0.01), HPLC-UV (p < 0.001) and NMR-based metabolomics. Reduced eye growth and diminished heart rate occurred in a concentration-dependent fashion. Metabolic effects measured by in vivo (31)P NMR showed a significant increase in orthophosphate levels (P(i); p < 0.05), and significant decreases in [ATP], [PCr] and the PCr/P(i) ratio (p < 0.05). Metabolomics revealed a dose-response relationship between dinoseb and endogenous metabolite changes, with both dinoseb concentrations producing significantly different metabolic profiles from controls (p < 0.05). Metabolic changes included decreased concentrations of ATP, PCr, alanine and tyrosine, and increased concentrations of lactate with medaka embryotoxicity. This study demonstrated that medaka embryos respond to dinoseb with significant changes in metabolism, reduced growth and heart rates, and increased abnormal development and post-exposure mortality. All three analytical methods confirmed similar trends, and utilization of PCr to compensate for ATP loss was found to be a consistent indicator of sublethal stress-one that could be used to quantify stress associated with medaka embryotoxicity.

Background: Recent studies have shown a relationship between alterations in tissue oxygen metabolism and cellular changes following ischemia and reperfusion, such as energy store depletion and intracellular acidosis. The aim of this study was to evaluate the relationship between tissue energy metabolism and intramuscular tissue oxygen tension in the mobilized latissimus dorsi muscle., Material and Methods: The latissimus dorsi muscle was raised in New Zealand white rabbits (n = 10, 2.5 +/- 0.5 kg). During 4 h of ischemia and 2 h of reperfusion, the intramuscular tissue oxygen tension (Licox PO2-microcatheter probe) and the status of phosphorylated muscle energy metabolites were measured using a high-field 31P-NMR spectrometer. Linear correlation was performed between 31P-NMR data and tissue oxygen tension readings., Results: The tissue oxygen tension (PO2) values correlated significantly with phosphocreatine (PCr) (r = 0.96, P < 0.001), beta-adenosin triphosphate (beta-ATP) (r = 0.64, P <0.01), and intracellular pH (r = 0.82, P <0.001)., Conclusions: On the basis of these findings, we conclude that the data provided by tissue oxygen tension measurement offer a real time minimally invasive estimate of muscle oxidative metabolism during ischemia and reperfusion.

This study investigated the energy metabolism of masseter muscles by 31P-Magnetic Resonance Spectroscopy (MRS) during increased blood flow induced by hot pack application to clarify the influence of changes in blood flow on muscle fatigue. Twelve healthy subjects with no history of muscle pain in the masticatory system participated in this study. The 31P-MRS measurements were performed before and after hot pack application and the ratio of phosphocreatine (PCr) acting as the energy source to reproduce ATP to beta-ATP, the PCr/beta-ATP ratio, was analyzed. Results showed that PCr/beta-ATP ratios increased significantly by an average of 22.4% after the hot pack application. The results suggest that changes in blood flow volume influence the energy metabolism in masseter muscles and that blood flow increases due to the hot pack cause higher energy levels in masseter muscles and offer an advantageous condition for preventing and relieving muscle fatigue.

Children and adolescents aged 4-16 years with the diagnosis of acute respiratory viral infection with long-lasting fever, manifestations of intoxication syndrome, and catarrhal symptoms were examined. In children and adolescents suffering from frequent diseases and presented with acute respiratory viral infection we found disorders in the immune status (depression of the cellular component, helper/suppressor imbalance, suppressed production of IgA and hyperproduction of IgM, decreased concentration of secretory IgA in the saliva) in comparison with children rarely falling ill. The redox potential and lymphocyte cytochrome C content were decreased in adolescents often falling ill, while the content of cytochrome oxidase did not change. A negative multiple correlation (R=6.8, p<0.005) was detected between the decrease in cytochrome C content and NADP/NADPH redox potential and increase in the immunoregulatory index. ATP content in lymphocyte from adolescents frequently falling ill remained 21% decreased during the first 2 weeks after acute respiratory viral infection, while the ATP/ADP ratio was shifted towards dinucleotide, which also indicated disorders in ATP synthesis in lymphocytes.

Hypothesis: Recent evidence suggests that sepsis may induce an uncoupling of oxidative phosphorylation. The purpose of this study was to quantify temporal changes in hepatic oxygen consumption and cellular energy state with increasing severity of sepsis and thus assess the interrelationship of these parameters as either primary defect or compensatory response., Main Outcome Measures: Pseudomonas aeruginosa was infused intravenously in eight instrumented anesthetized swine inducing a progressive severity of sepsis to shock. Eight other animals served as instrumented controls. Hepatic blood flow, oxygen use, and concentrations of ATP, ADP, AMP, NAD(+), and NADH were measured at baseline and then sequentially during the study., Results: Except for an increase in heart rate, there were no temporal changes in measured values for the control animals. For swine receiving P. aeruginosa, hepatic oxygen delivery and consumption increased with early sepsis whereas there were no alterations in the concentrations of adenine nucleotides or NAD(+)/NADH within liver. Septic shock was notable for a decrease in oxygen delivery yet oxygen consumption remained elevated because of an increase in percent oxygen extraction. The hepatic concentrations of ATP and NADH decreased during septic shock., Conclusions: These findings suggest that any sepsis-induced limitation in phosphorylation may be initially compensated by an increase in oxygen use. This study also suggests that decreases in NADH availability may be a principal factor in the decompensation of sepsis to shock.

We examined the effects of exhaustive exercise and post-exercise recovery on white muscle substrate depletion and metabolite distribution between white muscle and blood plasma in the Pacific spiny dogfish, both in vivo and in an electrically stimulated perfused tail-trunk preparation. Measurements of arterial-venous lactate, total ammonia, beta-hydroxybutyrate, glucose, and L-alanine concentrations in the perfused tail-trunk assessed white muscle metabolite fluxes. Exhaustive exercise was fuelled primarily by creatine phosphate hydrolysis and glycolysis as indicated by 62, 71, and 85% decreases in ATP, creatine phosphate, and glycogen, respectively. White muscle lactate production during exercise caused a sustained increase (approximately 12 h post-exercise) in plasma lactate load and a short-lived increase (approximately 4 h post-exercise) in plasma metabolic acid load during recovery. Exhaustive exercise and recovery did not affect arterial PO2, PCO2, or PNH3 but the metabolic acidosis caused a decrease in arterial HCO3- immediately after exercise and during the first 8 h recovery. During recovery, lactate was retained in the white muscle at higher concentrations than in the plasma despite increased lactate efflux from the muscle. Pyruvate dehydrogenase activity was very low in dogfish white muscle at rest and during recovery (0.53 +/- 0.15 nmol g wet tissue(-1) min(-1); n=40) indicating that lactate oxidation is not the major fate of lactate during post-exercise recovery. The lack of change in white muscle free-carnitine and variable changes in short-chain fatty acyl-carnitine suggest that dogfish white muscle does not rely on lipid oxidation to fuel exhaustive exercise or recovery. These findings support the notion that extrahepatic tissues cannot utilize fatty acids as an oxidative fuel. Furthermore, our data strongly suggest that ketone body oxidation is important in fuelling recovery metabolism in dogfish white muscle and at least 20% of the ATP required for recovery could be supplied by uptake and oxidation of beta-hydroxybutyrate from the plasma.

beta cell dysfunction is an important component of type 2 diabetes, but the molecular basis for this defect is poorly understood. The transcriptional coactivator PGC-1alpha mRNA and protein levels are significantly elevated in islets from multiple animal models of diabetes; adenovirus-mediated expression of PGC-1alpha to levels similar to those present in diabetic rodents produces a marked inhibition of glucose-stimulated insulin secretion from islets in culture and in live mice. This inhibition coincides with changes in metabolic gene expression associated with impaired beta cell function, including the induction of glucose-6-phosphatase and suppression of GLUT2, glucokinase, and glycerol-3-phosphate dehydrogenase. These changes result in blunting of the glucose-induced rise in cellular ATP levels and membrane electrical activity responsible for Ca(2+) influx and insulin exocytosis. These results strongly suggest that PGC-1alpha plays a key functional role in the beta cell and is involved in the pathogenesis of the diabetic phenotype.

Aims and Methods: Recently it was suggested that ischaemic preconditioning (IP) protects the myocardium in a graded pattern as assessed by myocardial infarct size estimation. Using tissue biopsies we investigated the impact of the proposed graded pattern of protection on myocardial energy state in an open-chest porcine model of IP with either one (1xIP) or four (4xIP) episodes of preconditioning. Furthermore, we evaluated the relationship between interstitial energy-related metabolite levels obtained by the microdialysis technique and the degree of subsequent ischaemic insult., Results: During the long ischaemia the difference between pre-ischaemic and post-ischaemic total adenylate pools and the sum of adenylate breakdown products (adenosine, inosine and hypoxanthine) as well as tissue lactate levels appeared as follows: non-IP > 1xIP > 4xIP (P < 0.05). Moreover interstitial peak levels of lactate, hypoxanthine and taurine displayed a graded pattern analogous to the development of ischaemic damage, where non-IP > 1xIP > 4xIP., Conclusions: We present for the first time concordant energy metabolic and morphometric data in support of IP being a stepwise phenomenon for protection of the ischaemic myocardium. Furthermore, IP resulted in proportionally higher levels of hypoxanthine (relative to inosine) in the ischaemic myocardium, suggesting a different handling of adenine nucleotide breakdown products in the IP myocardium.