High-energy Phosphate Research Articles

Intracellular Energy-Transfer Networks and High-Resolution Respirometry: A Convenient Approach for Studying Their Function.

Compartmentalization of high-energy phosphate carriers between intracellular micro-compartments is a phenomenon that ensures efficient energy use. To connect these sites, creatine kinase (CK) and adenylate kinase (AK) energy-transfer networks, which are functionally coupled to oxidative phosphorylation (OXPHOS), could serve as important regulators of cellular energy fluxes. Here, we introduce how selective permeabilization of cellular outer membrane and high-resolution respirometry can be used to study functional coupling between CK or AK pathways and OXPHOS in different cells and tissues. Using the protocols presented here the ability of creatine or adenosine monophosphate to stimulate OXPHOS through CK and AK reactions, respectively, is easily observable and quantifiable. Additionally, functional coupling between hexokinase and mitochondria can be investigated by monitoring the effect of glucose on respiration. Taken together, high-resolution respirometry in combination with permeabilization is a convenient approach for investigating energy-transfer networks in small quantities of cells and tissues in health and in pathology.

The creatine kinase system as a therapeutic target for myocardial ischaemia–reperfusion injury

Restoring blood flow following an acute myocardial infarction saves lives, but results in tissue damage due to ischaemia–reperfusion injury (I/R). Ameliorating this damage is a major research goal to improve recovery and reduce subsequent morbidity due to heart failure. Both the ischaemic and reperfusion phases represent crises of cellular energy provision in which the mitochondria play a central role. This mini-review will explore the rationale and therapeutic potential of augmenting the creatine kinase (CK) energy shuttle, which constitutes the primary short-term energy buffer and transport system in the cardiomyocyte. Proof-of-principle data from several transgenic mouse models have demonstrated robust cardioprotection by either raising myocardial creatine levels or by overexpressing specific CK isoforms. The effect on cardiac function, high-energy phosphates and myocardial injury will be discussed and possible directions for future research highlighted. We conclude that the CK system represents a viable target for therapeutic intervention in I/R injury; however, much needed translational studies will require the development of new pharmacological tools.

Protective Effects of a Novel Agonist of Galanin Receptors Against Doxorubicin-Induced Cardiotoxicity in Rats.

The clinical use of antineoplastic agent doxorubicin (DOX) is limited due to its cardiotoxic action. [βAla14, His15]-galanine (2-15) (G) is a novel synthetic agonist of galanin receptors GalR1-3 having cardioprotective properties in animal models in vivo. The aim of the present study was to explore effects of G on DOX-induced cardiotoxicity. Wistar rats were divided into four groups and treated with DOX (D group), DOX and G (D + G group), G (G group), and saline (control). Before treatment and at the end of the study, concentration of thiobarbituric acid reactive substances (TBARS) and activity of creatine kinase-MB (CK-MB) were determined in blood plasma, the animals were weighed, and cardiac function was evaluated by echocardiography. At the end of experiments, the hearts were used to determine energy metabolites and mitochondrial respiration in permeabilized fibers. After an 8-week study, D group exhibited a pronounced cardiac failure, the absence of weight gain, an increased plasma TBARS concentration, and CK-MB activity. These disorders were accompanied by a reduced myocardial content of high-energy phosphates and mitochondrial respiratory parameters. Co-administration of G with DOX significantly decreased plasma TBARS level and prevented an increase in plasma CK-MB activity. In D + G group, myocardial contents of ATP, PCr, total adenine nucleotides, and total creatine as well as myocardial PCr/ATP ratio and the respiratory control index were higher than in D group at the end of the experiments. Peptide G significantly improved parameters of left ventricular (LV) function and caused weight gain in animals of D + G group. These results suggest that peptide G may be a potential pharmacological agent that attenuates the cardiotoxic effects of DOX.

Skeletal Muscle Abnormalities and Iron Deficiency in Chronic Heart FailureAn Exercise 31P Magnetic Resonance Spectroscopy Study of Calf Muscle.

Heart failure (HF) is often associated with iron deficiency (ID). Skeletal muscle abnormalities are common in HF, but the potential role of ID in this phenomenon is unclear. In addition to hemopoiesis, iron is essential for muscle bioenergetics. We examined whether energetic abnormalities in skeletal muscle in HF are affected by ID and if they are responsive to intravenous iron. Forty-four chronic HF subjects and 25 similar healthy volunteers underwent 31P magnetic resonance spectroscopy of calf muscle at rest and during exercise (plantar flexions). Results were compared between HF subjects with or without ID. In 13 ID-HF subjects, examinations were repeated 1 month after intravenous ferric carboxymaltose administration (1000 mg). As compared with controls, HF subjects displayed lower resting high-energy phosphate content, lower exercise pH, and slower postexercise PCr recovery. Compared with non-ID HF, ID-HF subjects had lower muscle strength, larger PCr depletion, and more profound intracellular acidosis with exercise, consistent with an earlier metabolic shift to anaerobic glycolysis. The exercise-induced PCr drop strongly correlated with pH change in HF group ( r=-0.71, P<0.001) but not in controls ( r=0.13, P=0.61, interaction: P<0.0001). Short-term iron administration corrected the iron deficit but had no effect on muscle bioenergetics assessed 1 month later. HF patients display skeletal muscle myopathy that is more severe in those with iron deficiency. The presence of ID is associated with greater acidosis with exercise, which may explain early muscle fatigue. Further study is warranted to identify the strategy to restore iron content in skeletal muscle.

Myocardial fatty acid uptake through CD36 is indispensable for sufficient bioenergetic metabolism to prevent progression of pressure overload-induced heart failure

The energy metabolism of the failing heart is characterized by reduced fatty acid (FA) oxidation and an increase in glucose utilization. However, little is known about how energy metabolism-function relationship is relevant to pathophysiology of heart failure. Recent study showed that the genetic deletion of CD36 (CD36KO), which causes reduction in FA use with an increased reliance on glucose, accelerates the progression from compensated hypertrophy to heart failure. Here, we show the mechanisms by which CD36 deletion accelerates heart failure in response to pressure overload. CD36KO mice exhibited contractile dysfunction and death from heart failure with enhanced cardiac hypertrophy and interstitial fibrosis when they were subjected to transverse aortic constriction (TAC). The pool size in the TCA cycle and levels of high-energy phosphate were significantly reduced in CD36KO-TAC hearts despite an increase in glycolytic flux. De novo synthesis of non-essential amino acids was facilitated in CD36KO-TAC hearts, which could cause a further decline of the pool size. The ingestion of a diet enriched in medium-chain FA improved cardiac dysfunction in CD36KO-TAC hearts. These findings suggest that myocardial FA uptake through CD36 is indispensable for sufficient ATP production and for preventing an increased glycolytic flux-mediated structural remodeling during pressure overload-induced hypertrophy.

The effect of succinate on brain NADH/NAD+ redox state and high energy phosphate metabolism in acute traumatic brain injury

A key pathophysiological process and therapeutic target in the critical early post-injury period of traumatic brain injury (TBI) is cell mitochondrial dysfunction; characterised by elevation of brain lactate/pyruvate (L/P) ratio in the absence of hypoxia. We previously showed that succinate can improve brain extracellular chemistry in acute TBI, but it was not clear if this translates to a change in downstream energy metabolism. We studied the effect of microdialysis-delivered succinate on brain energy state (phosphocreatine/ATP ratio (PCr/ATP)) with 31P MRS at 3T, and tissue NADH/NAD+ redox state using microdialysis (L/P ratio) in eight patients with acute major TBI (mean 7 days). Succinate perfusion was associated with increased extracellular pyruvate (+26%, p < 0.0001) and decreased L/P ratio (−13%, p < 0.0001) in patients overall (baseline-vs-supplementation over time), but no clear-cut change in 31P MRS PCr/ATP existed in our cohort (p > 0.4, supplemented-voxel-vs-contralateral voxel). However, the percentage decrease in L/P ratio for each patient following succinate perfusion correlated significantly with their percentage increase in PCr/ATP ratio (Spearman's rank correlation, r = −0.86, p = 0.024). Our findings support the interpretation that L/P ratio is linked to brain energy state, and that succinate may support brain energy metabolism in select TBI patients suffering from mitochondrial dysfunction.

Sex-Mediated Response to the Beta-Blocker Landiolol in Sepsis: An Experimental, Randomized Study.

To investigate any gender effect of the beta-1 adrenergic blocker, landiolol, on cardiac performance and energy metabolism in septic rats, and to explore the expression of genes and proteins involved in this process. Randomized animal study. University research laboratory. Male and female Wistar rats. One hour after cecal ligation and puncture, male and female rats were randomly allocated to the following groups: sham male, cecal ligation and puncture male, cecal ligation and puncture + landiolol male, sham female, cecal ligation and puncture female, and cecal ligation and puncture + landiolol female. Cardiac MRI was carried out 18 hours after cecal ligation and puncture to assess in vivo cardiac function. Ex vivo cardiac function measurement and P magnetic resonance spectroscopy were subsequently performed using an isovolumic isolated heart preparation. Finally, we assessed cardiac gene and protein expression. In males, landiolol increased indexed stroke volume by reversing the indexed end-diastolic volume reduction without affecting left ventricle ejection fraction. In females, landiolol did not increase indexed stroke volume and indexed end-diastolic volume but decreased left ventricle ejection fraction. Landiolol had no effect on ex vivo cardiac function and on high-energy phosphate compounds. The effect of landiolol on the gene expression of natriuretic peptide receptor 3 and on protein expression of phosphorylated-AKT:AKT ratio and endothelial nitric oxide synthase was different in males and females. Landiolol improved the in vivo cardiac performance of septic male rats while deleterious effects were reported in females. Expression of natriuretic peptide receptor 3, phosphorylated-AKT:AKT, and endothelial nitric oxide synthase are signaling pathways to investigate to better understand the sex differences in sepsis.

The effects of ginsenoside Rb1 on fatty acid β-oxidation, mediated by AMPK, in the failing heart.

Objective(s):This study intended to investigate the effects of Ginsenoside-Rbl (Gs-Rbl) on fatty acid β-oxidation (FAO) in rat failing heart and to identify potential mechanisms of Gs-Rbl improving heart failure (HF) by FAO pathway dependent on AMP-activated protein kinase (AMPK). Materials and Methods:Rats with chronic HF, induced by adriamycin (Adr), were randomly grouped into 7 groups. Gs-Rb1, adenine 9-β-D-arabinofuranoside (Ara A, specific AMPK inhibitor), and 5'-aminoimidazole-4-carboxamide riboside (Aicar, specific AMPK activator) were administered to rats with HF, singly and/or combinedly. Myocardial high-energy phosphate (such as phosphocreatine, ADP, and ATP), free L-Carnitine, malonyl-CoA, and the activity of FAO-related enzymes in left ventricle from different groups were measured by using the corresponding molecular biological techniques. Results: Gs-Rb1 improved HF significantly, accompanied by a significant increase in phosphocreatine (PCr), ADP, ATP, PCr/ATP ratio, free carnitine, malonyl-CoA, mRNA, activity of carnitine palmitoyltransferase (Cpt), medium-chain Acyl-CoA Dehydrogenase (MCAD) and long-chain acyl-CoA Synthetase (ACSL) and a significant decrease of the ADP/ATP ratio in the left ventricular myocardium. However, all those effects were almost abolished by Ara A and were not further improved by Aicar. Conclusion:Taken together, it suggests that Gs-Rb1 may modulate cardiac metabolic remodeling by improving myocardial fatty acid β-oxidation in failing heart. In addition, the effects of Gs-Rb1 may be mediated via activating AMPK.

Unraveling the cause of white striping in broilers using metabolomics.

ABSTRACTWhite striping (WS) is a major problem affecting the broiler industry. Fillets affected by this myopathy present pathologies that compromise the quality of the meat, and most importantly, make the fillets more prone to rejection by the consumer. The exact etiology is still unknown, which is why a metabolomics analysis was performed on breast samples of broilers. The overall objective was to identify biological pathways involved in the pathogenesis of WS. The analysis was performed on a total of 51 muscle samples and distinction was made between normal (n = 19), moderately affected (n = 24) and severely affected (n = 8) breast fillets. Samples were analyzed using gas chromatographic mass spectral analysis and liquid chromatography quadrupole time-of-flight mass spectrometry. Data were subsequently standardized, normalized and analyzed using various multivariate statistical procedures. Metabolomics allowed for the identification of several pathways that were altered in white striped breast fillets. The tricarboxylic acid cycle exhibited opposing directionalities. This is described in literature as the backflux and enables the TCA cycle to produce high-energy phosphates through matrix-level phosphorylation and, therefore, produce energy under conditions of hypoxia. Mitochondrial fatty acid oxidation was limited due to disturbances in especially cis-5–14:1 carnitine (log2 FC of 2, P < 0.01). Because of this, accumulation of harmful fatty acids took place, especially long-chain ones, which damages cell structures. Conversion of arginine to citrulline increased presumably to produce nitric oxide, which enhances blood flow under conditions of hypoxia. Nitric oxide however also increases oxidative damage. Increases in taurine (log2 FC of 1.2, P < 0.05) suggests stabilization of the sarcolemma under hypoxic conditions. Lastly, organic osmolytes (sorbitol, taurine, and alanine) increased (P < 0.05) in severely affected birds; likely this disrupts cell volume maintenance. Based on the results of this study, hypoxia was the most likely cause/initiator of WS in broilers. We speculate that birds suffering from WS have a vascular support system in muscle that is borderline adequate to support growth, but triggers like activity results in local hypoxia that damages tissue.

OXPHOS Defects Due to mtDNA Mutations: Glutamine to the Rescue!

Mutations in mtDNA associated with OXPHOS defects preclude energy harnessing by OXPHOS. The work of Chen etal. (2018) is previewed, reporting flux pathways of glutamine catabolism in mtDNA mutant cells yielding high-energy phosphates through substrate-level phosphorylation and the influence exerted by the severity of OXPHOS impairment.

Metabolic remodeling of substrate utilization during heart failure progression.

Heart failure (HF) is a clinical syndrome caused by a decline in cardiac systolic or diastolic function, which leaves the heart unable to pump enough blood to meet the normal physiological requirements of the human body. It is a serious disease burden worldwide affecting nearly 23 million patients. The concept that heart failure is "an engine out of fuel" has been generally accepted and metabolic remodeling has been recognized as an important aspect of this condition; it is characterized by defects in energy production and changes in metabolic pathways involved in the regulation of essential cellular functions such as the process of substrate utilization, the tricarboxylic acid cycle, oxidative phosphorylation, and high-energy phosphate metabolism. Advances in second-generation sequencing, proteomics, and metabolomics have made it possible to perform comprehensive tests on genes and metabolites that are crucial in the process of HF, thereby providing a clearer and comprehensive understanding of metabolic remodeling during HF. In recent years, new metabolic changes such as ketone bodies and branched-chain amino acids were demonstrated as alternative substrates in end-stage HF. This systematic review focuses on changes in metabolic substrate utilization during the progression of HF and the underlying regulatory mechanisms. Accordingly, the conventional concepts of metabolic remodeling characteristics are reviewed, and the latest developments, particularly multi-omics studies, are compiled.

Reduction of 2-methoxy-1,4-naphtoquinone by mitochondrially-localized Nqo1 yielding NAD+ supports substrate-level phosphorylation during respiratory inhibition

Provision of NAD+ for oxidative decarboxylation of alpha-ketoglutarate to succinyl-CoA by the ketoglutarate dehydrogenase complex (KGDHC) is critical for maintained operation of succinyl-CoA ligase yielding high-energy phosphates, a process known as mitochondrial substrate-level phosphorylation (mSLP). We have shown previously that when NADH oxidation by complex I is inhibited by rotenone or anoxia, mitochondrial diaphorases yield NAD+, provided that suitable quinones are present (Kiss G et al., FASEB J 2014, 28:1682). This allows for KGDHC reaction to proceed and as an extension of this, mSLP. NAD(P)H quinone oxidoreductase 1 (NQO1) is an enzyme exhibiting diaphorase activity. Here, by using Nqo1−/− and WT littermate mice we show that in rotenone-treated, isolated liver mitochondria 2-methoxy-1,4-naphtoquinone (MNQ) is preferentially reduced by matrix Nqo1 yielding NAD+ to KGDHC, supporting mSLP. This process was sensitive to inhibition by specific diaphorase inhibitors. Reduction of idebenone and its analogues MRQ-20 and MRQ-56, menadione, mitoquinone and duroquinone were unaffected by genetic disruption of the Nqo1 gene. The results allow for the conclusions that i) MNQ is a Nqo1-preferred substrate, and ii) in the presence of suitable quinones, mitochondrially-localized diaphorases other than Nqo1 support NADH oxidation when complex I is inhibited. Our work confirms that complex I bypass can occur by quinones reduced by intramitochondrial diaphorases oxidizing NADH, ultimately supporting mSLP. Finally, it may help to elucidate structure-activity relationships of redox-active quinones with diaphorase enzymes.

NF-κB-mediated metabolic remodelling in the inflamed heart in acute viral myocarditis

Acute viral myocarditis (VM), characterised by leukocyte infiltration and dysfunction of the heart, is an important cause of sudden cardiac death in young adults. Unfortunately, to date, the pathological mechanisms underlying cardiac failure in VM remain incompletely understood. In the current study, we investigated if acute VM leads to cardiac metabolic rewiring and if this process is driven by local inflammation. Transcriptomic analysis of cardiac biopsies from myocarditis patients and a mouse model of VM revealed prominent reductions in the expression of a multitude of genes involved in mitochondrial oxidative energy metabolism. In mice, this coincided with reductions in high-energy phosphate and NAD levels, as determined by Imaging Mass Spectrometry, as well as marked decreases in the activity, protein abundance and mRNA levels of various enzymes and key regulators of cardiac oxidative metabolism. Indicative of fulminant cardiac inflammation, NF-κB signalling and inflammatory cytokine expression were potently induced in the heart during human and mouse VM. In cultured cardiomyocytes, cytokine-mediated NF-κB activation impaired cardiomyocyte oxidative gene expression, likely by interfering with the PGC-1 (peroxisome proliferator-activated receptor (PPAR)-γ co-activator) signalling network, the key regulatory pathway controlling cardiomyocyte oxidative metabolism. In conclusion, we provide evidence that acute VM is associated with extensive cardiac metabolic remodelling and our data support a mechanism whereby cytokines secreted primarily from infiltrating leukocytes activate NF-κB signalling in cardiomyocytes thereby inhibiting the transcriptional activity of the PGC-1 network and consequently modulating myocardial energy metabolism.

Influence of Mesenchymal Stem Cells on Qualitative and Quantitative Characteristics of Energy Transformation in Muscle Tissue of Lower Extremity in Ischemia.

Energy transformation required for the performance of multiple energy dependent functions of the body, i.e. mechanical, chemical, and electrical, is important part of cell activity. Energy dependent functions become slower at intracellular level due to decreased ATP by 10–15% and stops completely with 25–30% decrease in ATP in the cell. Adenylic nucleotide concentration measures the mismatch between the energy demand and energy production in the cell's mitochondrial oxidative phosphorylation.A study was done with pubescent Wistar rats with body mass of 160–200 grams. The ischemia of the lower extremities was achieved by ligation of femoral artery. Bone marrow was taken from femoral, humoral and tibial bones. Cell culture was grown in Eagle medium IGLA MEM, enriched with L‐glutamine, 10% calf embryo serum with antibiotics added and 5 million of cell were injected intramuscularly in the experimental animals. The concentration of ATP, ADP and AMP in the gastrocnemius muscle measured by means of thin‐layer filtration on silica gel plates on 3rd, 5th, 7th, 14th, 21st and 28th day after injection of mesenchymal stem cells (MSC).The concentration of ATP decreased significantly in the control group after ischemia in consecutive study on specified days. It has been proved that MSC ejection redistributes the pool of adenylic nucleotides towards the increased ATP biosynthesis, so that such parameters of energy metabolism as energy charge, energy potential, thermodynamic control of respiration are increased and the correlation between the components of adenylate kinase reaction is decreased.This study showed that the ability of MSC provide protective action on energy homeostasis by means of the synthesis of large amount of cytokines and growth factors which stimulate the synthesis of glycolytic and Krebs cycle enzymes, increase glucose utilisation and ATP synthesis. Thus, activation of ATP synthesis restores the level of ATP and high energy phosphate bonds.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Role of the Creatine Kinase Energy System in Intestinal Inflammation

Inflammatory bowel disease (IBD) is a chronic inflammation of the intestine affecting more than 1 million people in the United States. Intestinal epithelia facilitate nutrient transport as well as protect against luminal bacteria and disruption of the intestinal epithelial barrier is a hallmark of IBD. The regulation of intestinal epithelial barrier is made possible by junctional complexes including tight junctions and adherens junctions that are supported by the actin cytoskeleton. Maintenance of the actin cytoskeleton is an energy demanding process and intestinal epithelial cells shuttle energy to the locations of the cellular junctions through the creatine energy circuit. Within the cell, creatine and its associated enzyme creatine kinase (CK) facilitate the shuttling of high energy phosphates in the form of phosphocreatine between sites of ATP generation. Previous work in our lab has shown that CK contributes to apical junction assembly in vitro and that supplementation of creatine in murine colitis models has a protective effect. Further studies have shown that mice lacking the brain and mitochondrial isoforms of CK have worse outcomes in a DSS model of colitis. In addition to impaired barrier function, this may be due in part to exacerbated intestinal bleeding observed in these mice. To that end, we measured platelet counts and aggregation and found that platelet counts are lower in CK double knockout mice compared to wild‐type mice. The platelets from the knockout mice also showed a hyporesponsive phenotype in a collagen adhesion assay. Together, these results provide evidence that the CK energy circuit is essential in maintaining intestinal epithelial barrier function as well as supporting proper platelet function.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Is visual activation associated with changes in cerebral high-energy phosphate levels?

Phosphorus magnetic resonance spectroscopy (31P MRS) has been employed before to assess phosphocreatine (PCr) and other high-energy phosphates in the visual cortex during visual stimulation with inconsistent results. We performed functional 31P MRS imaging in the visual cortex and control regions during a visual stimulation paradigm at an unprecedented sensitivity, exploiting a dedicated RF coil design at a 7 T MR system. Visual stimulation in a 3 min 24 s on–off paradigm in eight young healthy adults generated a clear BOLD effect with traditional 1H functional MRI in the visual cortex (average z score 9.9 ± 0.2). However, no significant event-related changes in any of the 31P metabolite concentrations, linewidths (7.9 ± 1.8 vs 7.8 ± 1.9 Hz) or tissue pH (7.07 ± 0.13 vs 7.06 ± 0.07) were detectable. Overall, our study of 31P MRSI in 15 cm3 voxels had a detection threshold for changes in PCr, Pi and γ-ATP between stimulation and rest of 5, 17 and 10%, respectively. In individual subjects, the mean coefficients of variance for PCr and Pi levels of control voxels were 6 ± 3 and 19 ± 8% (three time point average of 3 min 24 s). Altogether this indicates that energy supply for neuronal activation at this temporal resolution does not drain global PCr resources.

Neuroprotective effect of phosphocreatine on oxidative stress and mitochondrial dysfunction induced apoptosis in vitro and in vivo: Involvement of dual PI3K/Akt and Nrf2/HO-1 pathways.

Methylglyoxal (MGO), an active metabolite of glucose, is observed in high levels in the tissues and blood of diabetic patients. Phosphocreatine (PCr), a high-energy phosphate compound, exhibits a range of pharmacological actions but little is well known of its neuroprotective action. The aim of the present study was to investigate the neuroprotective effects and the possible mechanisms of PCr. Diabetes is closely associated with neurodegenerative diseases, leading not only to the peripheral nervous system (PNS) and but also to central nervous system (CNS) damage. Therefore, we established two rat models of diabetes in vivo induced by MGO and streptozocin (STZ) respectively, while utilized differentiated PC-12 cells in vitro. Treatment of PC-12 cells with PCr markedly attenuated MGO-induced change of viability, apoptosis, accompanied by decreased levels of caspase-3, casapse-9 and Bcl-2/Bax protein ratio. Determination of cellular respiratory function was performed with intact PC-12 cells and homogenized hippocampal neuron tissue of rat. Reactive oxygen species (ROS) generation was assessed by membrane permeable fluorescent probe DCFH-DA. The expressions of Akt, Nrf2 and HO-1 were examined by Western blot. PCr pretreatment significantly reduced oxidative stress-induced high LDH, MDA level, and ROS production of PC-12 cells. PCr pretreatment also significantly decreased mitochondrial dysfunction in vitro and in vivo. In addition, PCr pretreatment increased the expression of p-Akt, Nrf2 and HO-1, and reduced the apoptosis. Moreover, the expression of Cleaved caspase3 was partially increased and the p-Akt, Nrf2 and HO-1 was partially reduced by a PI3K inhibitor (LY294002). While, compared with LY294002 groups, pre-treatment with PCr at the concentrations of 20 mM significantly reduced the expression of Cleaved caspase3 and increased the expression of p-Akt, Nrf2 and HO-1. Molecular docking assay showed that PCr possessed powerful affinity towards to Akt with lower binding energy. In conclusion, the neuroprotective effects of PCr in vitro and in vivo rely on normalizing mitochondrial function and reducing oxidative stress via Akt mediated Nrf2/HO-1 pathway, suggesting that PCr may be a novel therapeutic candidate for the treatment of diabetes-associated neurodegenerative diseases.

Impaired brain energy gain upon a glucose load in obesity

BackgroundThere is evidence that the brain's energy status is lowered in obesity despite of chronic hypercaloric nutrition. The underlying mechanisms are unknown. We hypothesized that the brain of obese people does not appropriately generate energy in response to a hypercaloric supply. MethodsGlucose was intravenously infused in 17 normal weights and 13 obese participants until blood glucose concentrations reached the postprandial levels of 7 mmol/L and 10 mmol/L. Changes in cerebral adenosine triphosphate (ATP) and phosphocreatine (PCr) content were measured by 31phosphorus magnetic resonance spectroscopy and stress hormonal measures regulating glucose homeostasis were monitored. Because vitamin C is crucial for a proper neuronal energy synthesis we determined circulating concentrations during the experimental testing. ResultsCerebral high-energy phosphates were increased at blood glucose levels of 7 mmol/L in normal weights, which was completely missing in the obese. Brain energy content moderately raised only at blood glucose levels of 10 mmol/L in obese participants. Vitamin C concentrations generally correlated with the brain energy content at blood glucose concentrations of 7 mmol/L. ConclusionsOur data demonstrate an inefficient cerebral energy gain upon a glucose load in obese men, which may result from a dysfunctional glucose transport across the blood-brain barrier or a downregulated energy synthesis in mitochondrial oxidation processes. Our finding offers an explanation for the chronic neuroenergetic deficiency and respectively missing satiety perception in obesity.

EXPLOSIVE MUSCLE POWER ASSESSMENT IN ELITE ATHLETES USING WINGATE ANAEROBIC TEST

ABSTRACT Introduction: Maximal effort physiological tests provide information about the current functional capacity of athletes. Objective: The aim of this study was to evaluate anaerobic performance parameters in elite athletes and to compare them in terms of the specific demands of each sport. We also created and applied the new software which enables us to quantify a new parameter -explosive muscle power (EP), a major component in sports requiring explosive bursts of movement lasting from a few seconds to 1 or 2 minutes. This new parameter reflects the velocity of energy transformation from intramuscular ATP and high-energy phosphates into mechanical power. Methods: All Wingate test parameters (standard parameters) - anaerobic power (AP), anaerobic capacity (AC), and explosive power (EP) as the new parameter were recorded in 104 subjects: 30 non-athletes and 74 athletes divided into different groups depending on their sport specialty (20 rowers, 28 wrestlers and 26 soccer players). Results: Anaerobic power (AP), anaerobic capacity (AC) and explosive power (EP) were significantly higher in the group of athletes compared to non-athletes. Among athletes, significant differences were observed in some parameters according to the type of activities they are involved in. The highest values were recorded in the group of wrestlers (AP=836W; AC=16.6kJ; EP=139W/s). The values of AP (absolute values) and EP (absolute and relative values) were significantly higher in wrestlers than in soccer players and rowers, but there was no significant difference in AC among these groups. The EP variable had a distribution similar to AP. Conclusions: Alongside anaerobic power and anaerobic capacity, the assessment of explosive power may complement the anaerobic profile of athletes. Experts in the field of sports medicine and exercise physiology could find these results useful in improving test variables, which are more important for specific sports, and for evaluating and monitoring training progress. Level of Evidence I; Diagnostic studies - Investigating a diagnostic test.

Interaction of Calmodulin with the cSH2 Domain of the p85 Regulatory Subunit.

Calmodulin (CaM) is a calcium sensor protein that directly interacts with the dual-specificity (lipid and protein) kinase PI3Kα through the SH2 domains of the p85 regulatory subunit. In adenocarcinomas, the CaM interaction removes the autoinhibition of the p110 catalytic subunit of PI3Kα, leading to activation of PI3Kα and promoting cell proliferation, survival, and migration. Here we demonstrate that the cSH2 domain of p85α engages its two CaM-binding motifs in the interaction with the N- and C-lobes of CaM as well as the flexible central linker, and our nuclear magnetic resonance experiments provide structural details. We show that in response to binding CaM, cSH2 exposes its tryptophan residue at the N-terminal region to the solvent. Because of the flexible nature of both CaM and cSH2, multiple binding modes of the interactions are possible. Binding of CaM to the cSH2 domain can help release the inhibition imposed on the p110 subunit, similar to the binding of the phosphorylated motif of RTK, or phosphorylated CaM (pCaM), to the SH2 domains. Amino acid sequence analysis shows that CaM-binding motifs are common in SH2 domains of non-RTKs. We speculate that CaM can also activate these kinases through similar mechanisms.