ADP Levels Research Articles

Melatonin treatment maintains the quality properties and storability of carambola fruit by modulating energy metabolism

The influences of 150 μmol/L melatonin treatment on the quality properties, storability, and energy metabolism in carambola fruit were explored. The results showed that, compared to the control, melatonin treatment significantly retained higher rate of commercially acceptable fruit, and retarded the development of fruit browning and yellowing. Additionally, melatonin treatment displayed higher levels of chromaticity L⁎ and h° values, titratable acid, total soluble solids, total soluble sugars, sucrose, and vitamin C, but lower respiration rate, chromaticity a⁎ and b⁎ values, and reducing sugar content. Moreover, melatonin treatment presented higher levels of ATP, ADP, and energy charge, as well as higher activities of H+-ATPase, Mg2+-ATPase, and Ca2+-ATPase in the membranes of vacuole, mitochondria, and plasma, thereby reducing the damage to cell membranes. These results will provide a scientific basis and practical guidance for melatonin to maintain the quality properties and storability, and to prolong the shelf life of postharvest carambola fruit.

Utility of melatonin on brain injury, synaptic transmission, and energy metabolism in rats with sepsis.

Melatonin is a powerful endogenous antioxidant hormone. Its healing effects on energy balance and neuronal damage associated with oxidative metabolism disorders have been reported in pathologic conditions. We aimed to determinate the utility of melatonin on neuronal damage, synaptic transmission, and energy balance in the brain tissue of rats with sepsis induced with LPS. Rats was divided into four groups such as control, LPS (20mg/kg i.p.), melatonin (10mg/kg i.p. × 3), and LPS + Melatonin (LPS + Mel). After 6h from the first injection, rats were decapitated, and also tissue and serum samples were taken. Lipid peroxidation and neuron-specific enolase (NSE) levels were determined from the serum in all group. High energy compounds, creatine, and creatine phosphate are measured by HPLC methods from the homogenized tissue. Counts of living neurons are marked with NeuN (neuronal nuclei), degenerated neurons are marked with S100-ß and synaptic vesicles transmission is analyzed with synaptophysin antibodies immunoreactivities. One-way ANOVA and post hoc Tukey tests were used to statistical analysis. In LPS group, AMP, ATP, creatine, and creatine phosphate levels were significantly decreased (p < 0.05), and also ADP levels were significantly increased compared with the other groups (p < 0.01). Living neurons counts were significantly decreased in LPS (p < 0.01), melatonin, and LPS + Melatonin (p < 0.05) groups compared with control. Degenerated neurons counts were increased in LPS group compared with control (p < 0.01) and also decreased in both of melatonin and LPS + Melatonin groups (p < 0.01). Synaptophysin immunoreactivity was decreased in LPS group compared with the other groups (p < 0.05). We observed that melatonin administration prevents neuronal damage, regulates energy metabolism, and protects synaptic vesicle proteins from sepsis-induced reduction.

Energy Budget in Terms of ATP, ADP, AMP, and Energy Charge In The Osmoregulatory Tissues of Indian Major Carp Catla catla (Hamilton) Under Thermal-Stress And Thermal-Adaptation

The heat-adapted (32°C) Catla catla registered as increased level of ATP and energy charge with concomitant lower levels of ADP, and AMP when compared to cold-adapted (22°C) fishes. All the osmoregulatory tissues like the gill, kidney, and intestine indicate greater ATP turnover adaptation to higher temperatures. In order to differentiate thermal stress from thermal adaptation, the 22°C adapted were readapted to a temperature change of 22°C 32°C at the rate of 1°C/hour as in the case of stress and at the rate of l°C/ 60 hrs as in the case of adaptation During the time course of experiments there is a gradual stepping-up in the levels of ATP and energy charge and a gradual stepping-down in ADP and AMP in all tissues. Further, there is a complete filling-up process in ATP and values reached gradually the original level of these parameters of the control of 32°C adapted fishes. The heat-adapted fishes exhibited a fairly good amount of recovery ranging from 83% to 88% in these adenylate nucleotides. On the other hand, in the temperature-stressed fishes the adenylate nucleotides did not reach the control values and the filling processes with ATP could not be completed. Further, the % recovery in these parameters is far less 47 % to 57%, when compared to temperature adapted fishes. The continuous thermal-stress (1°C/hour) action upon this fish Catla catla resulted in stress-adaptation (adaptation resulted due to stress).

Differences in Brain Region Mitochondrial Bioenergetics Detected Using CK Clamp

The brain is a non-uniform organ comprised of discrete structures of varying function and cell composition. The differing function of brain regions ostensibly would necessitate disparate energetic demands, thereby suggesting brain bioenergetics would be different among brain regions. This study aimed to address whether brain regions indeed have different bioenergetics by evaluating substrate preference, fuel-specific mitochondrial function at physiologically relevant energy demands as well as associated membrane potential and electron conductance using high-resolution respirometry (Oroboros O2k) in the frontal cortex, hypothalamus, and hippocampus. We hypothesize there will be different bioenergetics among brain regions. Substrate preference was established using a substrate-uncoupler-inhibitor-titration (SUIT) with additions, in order: ADP, Pyruvate/Malate, UK5099, Octanoylcarnitine, Rotenone, Succinate, and Antimycin A. There was no statistically significant interaction between region and substrate (p=0.15) nor a main effect of region (p=0.91), however, there was a main effect of substrate in which pyruvate/malate (+275%) and succinate/rotenone (+170%) had significantly greater respiration than octanoylcarnitine, independent of region (p&lt;0.001). Given the results of the SUIT test, pyruvate/malate was used for subsequent tests. The SUIT method relies upon saturating ADP levels and non-physiological ATP:ADP concentration ratios, the extent to which this test obscures the interpretation of brain region respiration is unclear. Instead, an alternative approach that leverages the enzymatic reaction of creatine kinase and phosphocreatine to assess mitochondrial respiration and membrane potential at clamped physiological ATP:ADP ratios, “CK Clamp,” was used. Function of mitochondria with a pyruvate fuel was then evaluated in all three brain regions in which energetic demand was clamped at ΔGATP = -12.87 (high demand), -14.08, -14.5, and -14.85 (low demand). There was a statistically significant interaction between brain region and clamped ΔGATP values (p = 0.02). Frontal cortex displayed higher respiration than hippocampus at -12.87 ΔGATP (33%). There was no statistically significant interaction between region and clamp for mitochondrial membrane potential assessed during the CK clamp (p=0.16), however there was a significant main effect of clamped ΔGATP (p&lt;0.001) in which membrane potential increased (i.e., hyperpolarized) as energetic demand decreased. This agrees with the bioenergetic model that a decrease in energetic demand increases the proton motive force (aka, hyperpolarization), thereby applying a backpressure on the electron transport chain, slowing respiration. Electron conductance was then assessed by calculating the slope of respiration versus clamped ΔGATP. Electron conductance was greater in the frontal cortex than hypothalamus (+70%, p = 0.015). The conclusion of this study is there are differences in mitochondrial bioenergetics among brain regions and the CK clamp was more sensitive at detecting these differences than the SUIT method. None. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

YÜKSEK FRUKTOZLA BESLENEN SIÇANLARDA QUERCETİN’İN PERİRENAL YAĞ DOKUSUNDAKİ ADİPONEKTİN VE REZİSTİN DÜZEYLERİNE ETKİSİ

Obesity is defined as the accumulation of adipose tissue to excess and to an extent that impairs both physical and psychosocial health and well-being. The increase in adipose tissue observed in obesity causes proinflammatory and anti-inflammatory cytokine imbalance. Accumulating evidence on quercetin, one of the antioxidants frequently used in obesity treatment, suggests that quercetin has significant anti-obesity and lipid-lowering effects simultaneously. We hypothesized that quercetin supplementation could improve obesity parameters and restore adipokine balance in obese mice. Sprague Dawley rats, 8-10 weeks of age, were divided into 4 groups, including a control group (C), high fructose (HF) group, quercetine (Q) group, and high fructose+quercetin (HF+Q) group. Fructose was administered to HF groups as a 20% solution in drinking water for 6 weeks. The rats in the Q groups were given 50 mg quercetin per kg BW by gavage for the next 4 weeks. The body weight, lee index, TG, HDL, fasting insülin, fasting glucose, and HOMA-IR were determined in rats. ADP and resistin levels were determined by ELISA assay from perirenal adipose tissue homogenates. We showed that quercetin acts to improve TG, fasting glucose and insulin resistance in high fructose-fed rats. In this study, we found no effect of quercetin on perirenal adipose tissue ADP and resistin levels. These results showed that high fructose could induce obesity in rats, while quercetin could favorably affect these parameters.

Activities of antioxidant enzymes and Hsp levels in response to elevated temperature in land snail species with varied latitudinal distribution.

Land snails occupy a variety of habitats, with differing temperature and humidity regimes and exhibit a wide span of adaptations, to withstand abiotic condition changes. The present work's aim was to examine the correlation of habitat's thermal adversity in different Mediterranean type habitats with the land snail's antioxidant and heat shock responses. For this purpose, snails of different species from populations along a north-south axis from the islands and mainland of Greece were exposed to elevated temperature and antioxidant enzyme activities, and Hsp70 and Hsp90 levels were determined in their tissues. The ATP, ADP, and AMP levels and the adenylate energy charge (AEC) were also determined. The comparison of protein levels and enzymatic activities across time intervals revealed significant differences for all factors examined. While the gradation pattern over time for a given factor was similar in all populations the absolute values over time differed. Catalase activity and the Hsp90 protein levels had the higher contribution in separating the different species and populations, followed by the activity of glutathione reductase and Hsp70 protein levels which contributed to a lesser degree. In general, populations from the southern part of their distribution in Greece tend to display a faster increase than northern populations in induction levels of all factors examined. Our data seem to be in line with the concept of preparation for oxidative stress (POS) while the changes in the AEC indicate an early preparation to cover the energy demand for the induction and synthesis of antioxidant enzymes and Hsps.

Erythrocyte Intracellular Adenosine Regulates Oxygen Release Combating Tissue Hypoxia, Inflammation and Fibrosis

Hypoxia is considered a common cause of myriad diseases and drives tissue damage and progression. Erythrocytes are vital under hypoxia since they can sense and act rapidly to hypoxia by promoting metabolomic reprogramming and oxygen (O 2) release. Adenosine monophosphate deaminase 3 (AMPD3) is highly enriched in erythrocytes and plays an important role in maintaining erythrocyte intracellular purine homeostasis. However, how it senses and regulates oxygen release remains poorly understood. Here we report that genetic ablation of AMPD3 led to more O 2 release from erythrocytes, resulting in less tissue hypoxia, damage, inflammation and fibrosis in two independent pathological hypoxia models including Angiotensin II infusion and unilateral ureteric obstruction (UUO) models. Metabolically, untargeted high-throughput metabolomic profiling in erythrocytes revealed that nucleotides including AMP, ADP and ATP levels were significantly increased in WT mice and further elevated in Ampd3 -/-mice. Isotopically adenosine flux experiment demonstrated that adenosine was rapidly and largely converted to AMP. At molecular level, we further demonstrated that hypoxia-induced AMPD3 activity as a compensatory mechanism led to accumulation of AMP, subsequently inducing AMP kinase (AMPK)-dependent metabolic reprogramming by inducing bisphosphoglycerate mutase (BPGM), which shifts glycolysis toward erythroid unique Rapoport-Leubering Shunt (RSL) to promote 2,3-BPG production, O 2 delivery and anti-ROS capacity to mitigate tissue hypoxia, dysfunction, inflammation and fibrosis. At cellular level, we revealed that hypoxia directly inhibited AMPD3, leading to activation of AMPK by lowering ROS in both cultured primary human and murine erythrocytes. Finally, we conducted human translational studies demonstrating that the production of 2,3-BPG, BPGM activity, p-AMPK level, and P50 were increased, while AMPD3 activity was decreased in patients with chronic kidney disease (a common pathological condition) compared with normal controls. Altogether, eAMPD3is a previously unrecognized master intracellular purinergic componentsensing hypoxia and promoting adaptative metabolic reprogramming tomitigate tissue hypoxia, insufficient energy, tissue damage and fibrosis by enhancing O 2 delivery and antioxidative stress capacity in a positive feedforward manner.

KATP channel activity and slow oscillations in pancreatic beta cells are regulated by mitochondrial ATP production.

Pancreatic beta cells secrete insulin in response to plasma glucose. The ATP-sensitive potassium channel (KATP ) links glucose metabolism to islet electrical activity in these cells by responding to increased cytosolic [ATP]/[ADP]. It was recently proposed that pyruvate kinase (PK) in close proximity to beta cell KATP locally produces the ATP that inhibits KATP activity. This proposal was largely based on the observation that applying phosphoenolpyruvate (PEP) and ADP to the cytoplasmic side of excised inside-out patches inhibited KATP . To test the relative contributions of local vs. mitochondrial ATP production, we recorded KATP activity using mouse beta cells and INS-1 832/13 cells. In contrast to prior reports, we could not replicate inhibition of KATP activity by PEP+ADP. However, when the pH of the PEP solutions was not corrected for the addition of PEP, strong channel inhibition was observed as a result of the well-known action of protons to inhibit KATP . In cell-attached recordings, perifusing either a PK activator or an inhibitor had little or no effect on KATP channel closure by glucose, further suggesting that PK is not an important regulator of KATP . In contrast, addition of mitochondrial inhibitors robustly increased KATP activity. Finally, by measuring the [ATP]/[ADP] responses to imposed calcium oscillations in mouse beta cells, we found that oxidative phosphorylation could raise [ATP]/[ADP] even when ADP was at its nadir during the burst silent phase, in agreement with our mathematical model. These results indicate that ATP produced by mitochondrial oxidative phosphorylation is the primary controller of KATP in pancreatic beta cells. KEY POINTS: Phosphoenolpyruvate (PEP) plus adenosine diphosphate does not inhibit KATP activity in excised patches. PEP solutions only inhibit KATP activity if the pH is unbalanced. Modulating pyruvate kinase has minimal effects on KATP activity. Mitochondrial inhibition, in contrast, robustly potentiates KATP activity in cell-attached patches. Although the ADP level falls during the silent phase of calcium oscillations, mitochondria can still produce enough ATP via oxidative phosphorylation to close KATP . Mitochondrial oxidative phosphorylation is therefore the main source of the ATP that inhibits the KATP activity of pancreatic beta cells.

Hippocampal mitochondrial Ca++ in experimentally induced Alzheimer's disease, link to calpains and impact of vitamin D3 supplementation

ObjectiveVitamin D impact on hippocampal mitochondrial Ca++ and calpains was not previously investigated in Alzheimer's disease (AD). The current work aimed to assess the alteration in hippocampal mitochondrial Ca++, ATP & ADP and hippocampal calpains' level in (AlCl3)-induced AD model, and the effect of 2 regimens of vitamin D supplementation on these alterations. Methods: Forty male Wistar rats were randomized into 4 groups; control, AD (AlCl3100 mg/kg, p.o. daily for 42 days), AD and vitamin D co-treated group (AlCl3 as in AD group with vitamin D3 400 IU/kg/day, p.o. for 42 days) and AD, followed by vitamin D3 group (AlCl3 was given as in AD group for 42 days, then vitamin D3 for two weeks). AD was assessed by hippocampal levels of Aβ42, p-tau and spatial memory assessment in Morris water maze. Hippocampal mitochondrial Ca++, ATP and ADP levels besides to calpain-1 & 2 and cytochrome C were assessed in addition to CA1 histological examination. Results: AD animals showed impaired mitochondrial function as denoted by high Ca++ and decreased ATP and ADP and elevated calpain-1 & 2 and cytochrome C. Hippocampal CA1 region showed increased degenerated neurons and reduced thickness of its pyramidal layer. Vitamin D administration minimized the hippocampal mitochondrial impairement induced by AD and mitigated histological alterations even when supplemented post AD establishment. Conclusion: Vitamin D administration to AD rats breaks the deleterious loop in the hippocampus that involves increased Ca++, calpain activation, mitochondrial failure, neuronal degeneration and AD disease progression.

Serum level of certain cytokines associated with psoriasis progression in Iraqi patients

Introduction and Aim: Psoriasis is a skin ailment that typically affects the scalp, lower back, knees, elbows, and results in flaky skin patches. The purpose of this study was to compare cytokines, leptin, and adiponectin levels, as well as several physiological characteristics, in psoriasis patients with and without treatment to levels in healthy people in Iraq. Materials and Methods: The study included 310 subjects, males and females aged (25-65) years. The patients were attending a dermatology consultant at Al-Yarmouk Teaching Hospital in Baghdad. Blood samples were collected by venipuncture from all individuals. The Sandwich ELISA was used to estimate serum levels of IL-2, IL-8, IL-22, TNF-alpha, IFN-gamma, ADP and Leptin. Results: Patients with psoriasis had considerably decreased lymphocyte and RBC counts while having significantly higher neutrophil, platelet, NLR, and PLR counts. WBC mean values in females and males among control groups were 7.16 ± 3.09x 103/µl and 8.6 ±3.62 x103/µl respectively. In psoriasis patients the WBC counts varied from 3.69 x103/µl to 12.8 x103/µl with a mean of 7.27 x 103/µl in female patients and from 6.47 x 103/µl to 13.62 x103/µl in males. Serum levels of cytokines, leptin and adiponectin were significantly higher in Group I patients, while in Group II it was almost like levels in the control group. Conclusion: Smoking is a risk factor for psoriasis. Lymphocytes, erythrocytes, and hemoglobin levels are decreased in patients with psoriasis, while platelets and cytokines are elevated, signifying a relationship between these immunological parameters and development and severity of psoriasis disease.

Regulation of AMPK activation by extracellular matrix stiffness in pancreatic cancer

The adenosine monophosphate (AMP)-activated protein kinase (AMPK) sits at a central node in the regulation of energy metabolism and tumor progression. AMPK is best known to sense high cellular ADP or AMP levels, which indicate the depletion of energy stores. Previous studies have shown that the low expression of phosphorylated AMPK is associated with a poor prognosis of pancreatic cancer. In this study, we report that AMPK is also highly sensitive to extracellular matrix (ECM) stiffness. We found that AMPK is activated in cells when cultured under low ECM stiffness conditions and is functionally required for the metabolic switch induced by ECM stiffness. This regulation of AMPK requires the Hippo kinases but not LKB1/CaMKKβ. Hippo kinases directly phosphorylate AMPKα at Thr172 to activate AMPK at low ECM stiffness. Furthermore, we found AMPK activity is inhibited in patients with pancreatic ductal adenocarcinoma (PDAC) with high ECM stiffness and is associated with a poor survival outcome. The activation of Hippo kinases by ROCK inhibitor Y-27632 in combination with the mitochondrial inhibitor metformin synergistically activates AMPK and dramatically inhibits PDAC growth. Together, these findings establish a novel model for AMPK regulation by the mechanical properties of ECMs and provide a rationale for simultaneously targeting the ECM stiffness–Hippo kinases–AMPK signaling and low glucose–LKB1–AMPK signaling pathways as an effective therapeutic strategy against PDAC.

BIO14: Platelet energy metabolism in an in-vitro ECMO model

Background: Extracorporeal membrane oxygenation (ECMO) exposes blood to mechanical shear stress and artificial surfaces, leading to platelet activation and thrombosis. We hypothesize that ECMO-induced platelet activation leads to changes in platelet energy metabolism. Methods: Based on an in vitro ECMO model (Cardiohelp platform; HLS Advanced 5.0 Oxygenator), human whole blood was circulated at low (1 L/min) or nominal flow (4L/min) for 6 hours. A static control sample was drawn after priming and held in the same tubing at 37 °C. Experiments were terminated if clot formation caused device failure. Samples were drawn at baseline as well as hourly throughout the experiment (T0 – T6) and assessed for cell counts and intracellular levels of bioenergetic intermediates, which were normalized to platelet count. Results: Preliminary data documents that 83% of ECMO runs completed 6 hours of perfusion. One nominal ECMO run was terminated due to clot formation after 5 hours. We found a statistically significant difference in platelet ATP levels after 6 hours between the control and the nominal group (181 nM ± 13 vs 107 nM ± 11, p = 0.02). Furthermore, we saw a statistically significant difference in ADP levels at T6 between the control group and the nominal group (517 nM ± 45 vs 255 nM ± 16, p = 0.001) as well as between the control group and the low-flow-group (330 nM ± 9, p = 0.001). We observed a trend in difference between low and nominal flow groups for ATP and ADP. Conclusion: This preliminary analysis indicates that ECMO pump flow rate effects on ATP and ADP levels in platelets. These insights into energy metabolism in platelets exposed to ECMO might lead to novel therapeutical approaches to prevent clotting events during ECMO.

Dependence of cardiomyocyte force, velocity, and power generation on ATP concentration

Theoretical analysis and computer modeling of myocardial energy metabolism under physiological and heart failure conditions predicts that cytoplasmic ATP and ADP levels both fall roughly in proportion to reductions in total adenine nucleotide pool observed in heart failure. Assessing sub-maximal force and velocity in loaded myocardial muscle at MgATP levels in the range of 2 to 8 mM, Beard et al. ( Biophys. J. 2022. 121: 3213–3223) observed effects of MgATP on myocardial power generation that suggest that although maximal force and velocity are not substantially sensitive to differences in [MgATP] that occur in the normal versus failing myocardium, maximal power generation, which occurs at submaximal force and velocity, is sensitive to [MgATP]. Previously developed computational models are not able to reproduce the observed dependence on MgATP. The goals of the present study are to construct and analyze a computational model of muscle dynamics to analyze data on the dependence of maximal force, velocity, and power generation on [MgATP] in order to determine a potential mechanism to explain the MgATP-dependent phenomena. Model development and identification was guided by dynamic measurements on permeabilized mouse trabecular fibers. The data include force-velocity relation and time constant of tension development. The mathematical model used to simulate cardiomyocyte crossbridge mechanics was based on the previous crossbridge and calcium models from Tewari et al. (J Mol Cell Cardiol, 2016. 96: 11-25) and Campbell et al. (Biophys J, 2018. 115: 543-553). The developed model includes strain- and metabolite-dependent transition between attachment states, and ATP-dependent destabilization of the super-relaxed (SRX) myosin state. Model-based analysis of force and velocity dyanmics is consistent with the hypothesis that ATP assocition with the myosin protein influence the kinetics and thermodynamics of transitions between the SRX and disorded (DRX) states. Funding: Department of Veterans Affairs Merit Review Award I01BX000740 (A.J.B.) and National Heart, Lung, and Blood Institute grant R01 HL154624 (A.J.B., D.A.B.) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Low production of mitochondrial reactive oxygen species after anoxia and reoxygenation in turtle hearts.

Extremely anoxia-tolerant animals, such as freshwater turtles, survive anoxia and reoxygenation without sustaining tissue damage to their hearts. In contrast, for mammals, the ischemia-reperfusion (IR) injury that leads to tissue damage during a heart attack is initiated by a burst of superoxide (O2·-) production from the mitochondrial respiratory chain upon reperfusion of ischemic tissue. Whether turtles avoid oxidative tissue damage because of an absence of mitochondrial superoxide production upon reoxygenation, or because the turtle heart is particularly protected against this damage, is unclear. Here, we investigated whether there was an increase in mitochondrial O2·- production upon the reoxygenation of anoxic red-eared slider turtle hearts in vivo and in vitro. This was done by measuring the production of H2O2, the dismutation product of O2·-, using the mitochondria-targeted mass-spectrometric probe in vivo MitoB, while in parallel assessing changes in the metabolites driving mitochondrial O2·- production, succinate, ATP and ADP levels during anoxia, and H2O2 consumption and production rates of isolated heart mitochondria. We found that there was no excess production of in vivo H2O2 during 1 h of reoxygenation in turtles after 3 h anoxia at room temperature, suggesting that turtle hearts most likely do not suffer oxidative injury after anoxia because their mitochondria produce no excess O2·- upon reoxygenation. Instead, our data support the conclusion that both the low levels of succinate accumulation and the maintenance of ADP levels in the anoxic turtle heart are key factors in preventing the surge of O2·- production upon reoxygenation.

Turtle hearts can cope with having low oxygen

Living trapped under the ice in a pond all winter doesn't sound like much fun, especially for an animal that normally breathes air. However, several turtle species in North America do just that. While these animals may have drawn the short straw when it comes to the habitat they reside in, they have also developed some remarkable adaptations which allow them to survive in such a difficult environment. One such adaptation is their ability to tolerate having very low or no oxygen and then suddenly having normal levels of oxygen again. In humans, this type of event happens in the short term when someone suffers a heart attack or stroke, generally resulting in the mitochondria of cells producing reactive oxygen species, chemicals such as hydrogen peroxide and superoxide, which damage or even kill cells. However, the turtles don't seem to incur any damage from this process, which led Amanda Bundgaard and Angela Fago of Aarhus University, Denmark, to delve into the mechanisms by which red-eared sliders (Trachemys scripta elegans) solve the problem of dealing with these harmful chemicals.Working with researchers from the University of Cambridge, UK, and the University of Manchester, UK, Bundgaard and Fago placed the shelled reptiles in water that had no oxygen dissolved in it and measured the amount of reactive oxygen species after 3 h underwater followed by 1 h on land. Surprisingly, the turtles’ hearts didn't have higher levels of these damaging chemicals after being resupplied with normal amounts of oxygen than they did when they were starved of oxygen under the water. Even more surprisingly, the sliders had even lower levels of reactive oxygen species than turtles that never experienced the absence of oxygen. Bundgaard and the team then set about discovering how these remarkable reptiles were able to keep their production of reactive oxygen species to a minimum.From previous research in mammals, the researchers knew that the build-up of succinate (a chemical involved in the Krebs cycle) and depleting energy sources (ATP and ADP) are important factors in generating reactive oxygen species such as superoxide. The amount of succinate in the heart cells nearly doubled in the turtles that experienced extremely low oxygen levels. However, mammals that experience heart attack- or stroke-like events have increases in succinate levels that are 10 times higher than normal, suggesting that the turtles are doing something to keep their succinate levels lower. During this time, the turtles were also able to maintain relatively normal levels of ADP while experiencing low oxygen levels, suggesting that they are able to make energy efficiently without oxygen. Keeping succinate levels low is not the only way that an animal can deal with these chemicals, they can also produce large quantities of antioxidants. However, when the team measured how much superoxide and hydrogen peroxide turtles’ hearts could remove, the levels were comparable to those of mice and iguanas, neither of which experience prolonged periods without oxygen.Although the team is quick to point out that surviving without oxygen is very different from having a stroke, as blood is still flowing while the turtles are underwater, these turtles’ remarkable ability to keep their generation of reactive oxygen species in check seems to be what is driving their ability to not accumulate damage to their tissues after their winter-long hibernation under the ice. While the sliders may have drawn the short straw in terms of their habitat, they have certainly come up with some remarkable adaptations to make the best of their situation.

The bioenergetic "CK Clamp" technique detects substrate-specific changes in mitochondrial respiration and membrane potential during early VML injury pathology.

Volumetric muscle loss (VML) injuries are characterized by non-recoverable loss of tissue resulting in contractile and metabolic dysfunction. The characterization of metabolic dysfunction in volumetric muscle loss-injured muscle has been interpreted from permeabilized myofiber respiration experiments involving saturating ADP levels and non-physiologic ATP:ADP concentration ratios. The extent to which this testing condition obscures the analysis of mitochondrial (dys) function after volumetric muscle loss injury is unclear. An alternative approach is described that leverages the enzymatic reaction of creatine kinase and phosphocreatine to assess mitochondrial respiration and membrane potential at clamped physiologic ATP:ADP ratios, "CK Clamp." The objective of this study was to validate the CK Clamp in volumetric muscle loss-injured muscle and to detect differences that may exist between volumetric muscle loss-injured and uninjured muscles at 1, 3, 5, 7, 10, and 14days post-injury. Volumetric muscle loss-injured muscle maintains bioenergetic features of the CK Clamp approach, i.e., mitochondrial respiration rate (JO2) titters down and mitochondrial membrane potential is more polarized with increasing ATP:ADP ratios. Pyruvate/malate/succinate-supported JO2 was significantly less in volumetric muscle loss-injured muscle at all timepoints compared to uninjured controls (-26% to -84%, p < 0.001) and electron conductance was less at day 1 (-60%), 5 (-52%), 7 (-35%), 10 (-59%), and 14 (-41%) (p < 0.001). Palmitoyl-carnitine/malate-supported JO2 and electron conductance were less affected following volumetric muscle loss injury. volumetric muscle loss-injury also corresponded with a more polarized mitochondrial membrane potential across the clamped ATP:ADP ratios at day 1 and 10 (pyruvate and palmitoyl-carnitine, respectively) (+5%, p < 0.001). This study supports previous characterizations of metabolic dysfunction and validates the CK Clamp as a tool to investigate bioenergetics in traumatically-injured muscle.

ATPergic signaling disruption in human sepsis as a potential source of biomarkers for clinical use.

Sepsis is a life-threatening organ dysfunction caused by a dysregulated inflammatory response to infection. To date, there is no specific treatment established for sepsis. In the extracellular compartment, purines such as adenosine triphosphate (ATP) and adenosine play essential roles in the immune/inflammatory responses during sepsis and septic shock. The balance of extracellular levels among ATP and adenosine is intimately involved in the signals related to immune stimulation/immunosuppression balance. Specialized enzymes, including CD39, CD73, and adenosine deaminase (ADA), are responsible to metabolize ATP to adenosine which will further sensitize the P2 and P1 purinoceptors, respectively. Disruption of the purinergic pathway had been described in the sepsis pathophysiology. Although purinergic signaling has been suggested as a potential target for sepsis treatment, the majority of data available were obtained using pre-clinical approaches. We hypothesized that, as a reflection of deregulation on purinergic signaling, septic patients exhibit differential measurements of serum, neutrophils and monocytes purinergic pathway markers when compared to two types of controls (healthyand ward). It was observed that ATP and ADP serum levels were increased in septic patients, as well as the A2a mRNA expression in neutrophils and monocytes. Both ATPase/ADPase activities were increased during sepsis. Serum ATP and ADP levels, and both ATPase and ADPase activities were associated with the diagnosis of sepsis, representing potential biomarkers candidates. In conclusion, our results advance the translation of purinergic signaling from pre-clinical models into the clinical setting opening opportunities for so much needed new strategies for sepsis and septic shock diagnostics and treatment.

Effects of vitamin D on proliferation, invasion and energy metabolism of MCF-7 breast cancer cell line

Background and Aims: 1,25(OH)2D3 (vitamin D) is a pleiotropic hormone with anti-proliferative, pro-apoptotic, and prodifferentiation effects on various cell types, which suggest anti-cancer activity in addition to its classical regulatory action on calcium and phosphate metabolism. Methods: We aimed to put forward the effects of vitamin D in various concentrations and time intervals on cell proliferation and invasion of human estrogen receptor-positive breast cancer (MCF-7) cells by real-time cell electronic sensing system (xCELLigence). A determined dose of the IC50 was applied on samples taken from cell lysates and analyzed the levels of the energy. We also aimed to clarify how vitamin D effects the activity of the protease uPA and their relations with each other. Results: Vitamin D showed a cytotoxic effect on MCF-7 cells in a time and dose dependent manner, with dose of IC50 found to be 140 nM. ATP, ADP, and AMP levels, as well as uPA activities were respectively increased in vitamin D treatment group compared to the control group for the first 24 hours while decreasing at 48, 72, and 96 hours. We determined that 70 and 140 nM vitamin D were decreased in invasion of MCF-7 cells compared to control cells. Conclusion: We observed that proliferation and invasion of breast cancer cells were inhibited by vitamin D treatment on a dose and time dependent manner, and also vitamin D supplementation decreased uPA activity and energy levels. Further studies on the mechanisms of vitamin D and the formulation of none-hypercalcemic analogues in featured are needed.

Dysregulated Autophagy in Platelets from JAK2V617F-Positive Polycythemia Vera and Essential Thrombocythemia Underlies Bioenergetic Failure As a Possible Mechanism for Thrombohemorrhagic Complications

Background: JAK2V617F-positive polycythemia vera (PV) and essential thrombocythemia (ET) are the most common Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). ET/PV patients have high levels of inflammation and are at increased risk of thrombosis. Platelets from patients with ET/PV are characterized by hyperreactivity (low activation threshold), contributing to the increased incidence of thrombosis in the MPNs. We previously described that platelets from PV patients accumulate dysmorphic mitochondria. Autophagy is a housekeeping and an inducible cellular process that eliminates damaged proteins and organelles (such as mitochondria and endoplasmic reticulum), providing biosynthetic and bioenergetic substrates for the cell. Dysregulated autophagy has been described in cancer, aging, and neurodegenerative, cardiac, and muscular diseases. We hypothesized that impairment of the platelet autophagic flux in ET/PV Jak2V617F alters platelet metabolism and function at the early and late stages of platelet activation. Methods: Platelets from sex and age-matched healthy subjects and patients with PV were analyzed. Platelets were incubated for two hours with chloroquine (CQ) [50 and 100 µM] to inhibit autophagy. CQ blocks the late stage of the autophagy process; therefore, functional autophagy was assessed by quantifying the accumulation of autophagic-related proteins by western blot. Oxidative stress (4-hydroxynonenal protein adducts) was measured by western blot. Platelet bioenergetics was studied by the Seahorse extracellular flux analyzer. Semi-quantitative metabolomics was performed using the Vanquish UHPLC system. Clot retraction was assessed by measuring the weight of serum extruded from the clot. Results: Platelets from healthy patients showed basal and inducible autophagy characterized by the accumulation of autophagic markers such as LC3II (p<0.0024), GABARAP, the autophagic cargo receptor p62 and the mitochondrial marker TOM20 (p<0.021). However, ET/PV platelets showed dysfunctional basal and inducible autophagy. Moreover, PV platelets and platelets treated with CQ had higher levels of ROS-damaged proteins (p<0.0001) compared to healthy platelets. Platelets from PV patients exhibited hyperreactivity by flow cytometry (PAC-1 binding, p<0.05) and under flow conditions (p<0.03). Moreover, platelets from ET/PV patients showed marked bioenergetic failure characterized by increased mitochondrial depolarization, higher intracellular calcium, depressed mitochondrial respiration, and lower levels of ADP and ATP. Pharmacological inhibition of autophagy of platelets from healthy individuals with CQ recapitulated the bioenergetic and functional profiles of platelets from patients with ET/PV. Bioenergetically, autophagy inhibition resulted in depressed basal (p<0.03) and ATP-linked respiration (p<0.01). Functionally, pharmacological inhibition of the autophagic flux led to significantly decreased clot retraction, as observed with the platelets from patients with ET/PV. Conclusion: Our results suggest that impairment of autophagy in platelets from ET/PV patients might exert a negative bioenergetic effect on platelet function, promoting platelet hyperreactivity and dysfunctional clot retraction, which would likely favor the formation of unstable clots. Our findings may explain why patients with Jak2V617F ET and PV are at increased risk for both thrombosis and hemorrhage and suggest that targeting autophagy might lower this risk. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Acidic electrolyzed water treatment suppresses Phomopsis longanae Chi-induced the decreased storability and quality properties of fresh longans through modulating energy metabolism

Longan fruit loses its market value rapidly due to postharvest pathogenic infestation and quality deterioration. Here, we hypothesized that acidic electrolyzed water (AEW) could maintain higher quality of P. longanae-inoculated longans via regulating energy metabolism. Results indicated that AEW reduced fruit disease index and decay incidence. Significantly, AEW treatment retained higher levels of ATP, ADP, and energy charge, and higher activities of Ca2+-ATPase, Mg2+-ATPase, and H+-ATPase in the membranes of plasma, vacuole, and mitochondria, which maintained the structural and functional integrity of cell membrane. Furthermore, indirectly sustaining cell membrane function via AEW treatment could maintain the storability and quality properties of longans, including keeping higher values of color chromaticity (L*, a*, and b*), higher amounts of vitamin C, total soluble solids, sucrose, and total soluble sugars, lower titratable acid and reducing sugar contents. This work elucidated the potential regulation of AEW on the balance of energy metabolism and fruit quality.