Activation Of Anaerobic Glycolysis Research Articles

ДЫХАНИЕ МИТОХОНДРИЙ И НЕКОТОРЫЕ ПОКАЗАТЕЛИ ГЛИКОЛИЗА ГОЛОВНОГО МОЗГА В УСЛОВИЯХ ЕГО ЧАСТИЧНОЙ ИШЕМИИ

Background. Estimation of changes in neuronal bioenergetics in the brain its partial ischemia (BPI) is crucial for finding new ways of correcting brain ischemic lesions. The aim of the study was to investigate the changes both in mitochondrial respiration (MR) and processes of anaerobic glycolisis in the rats` homogenate of the brain in terms of partial ischemia. Material and methods. In an experiment on 19 white female rats we studied the dynamics of changes MR of the brain after 1 hour and 3 days of BPI by means of the polarographic method using succinate or L-malate/L-glutamate as well as ADP. The activity of anaerobic glycolysis was assessed with respect to the changes in the concentration of lactate and pyruvate in plasma and homogenate of the brain. Results. We identified inhibition of the mitochondrial respiration in rat brain homogenate as well as increased level of lactate in cerebral homogenate, which was more pronounced in plasma after 1 hour of BPI compared to 3 days of BPI which was more pronounced in cerebral homogenate. The level of pyruvate was more pronounced in plasma after 1 hour of BPI compared to 3 days of BPI only. Conclusion. Partial cerebral ischemia of the rat due to the ligation of the right common carotid artery leads to a remarkable reduction in the respiratory activity of the brain's mitochondria and to the dissociation of oxidation and phosphorylation in the mitochondria, which in turn aggravates the neuronal energy deficit induced by the disturbance of the supply of oxygen by blood. The search for effective methods for correcting energy processes in the mitochondria of the brain is one of the key directions in the pathogenetic therapy of cerebral ischemia. Partial cerebral ischemia can be of interest to researchers for use as a model for screening the means of pharmacological correction of mitochondrial respiration disorders in brain cells during its ischemia.

178 TRANSCRIPTIONAL DIFFERENCES IN GENES RELATED TO GLUCOSE METABOLISM BETWEEN COMPETENT AND INCOMPETENT BOVINE IMMATURE CUMULUS-OOCYTE COMPLEXES SELECTED BY BCB

In vitro maturation is a key step of in vitro embryo production, being the main factor responsible for the low blastocyst yield. In vitro maturation requires the selection of competent immature cumulus-oocytes complexes (COC), which is usually accomplished based on morphological criteria and follicle size. Competent immature COC have finished their growth phase and show decreased G6PDH activity. Brilliant cresol blue (BCB) is a dye that is degraded by G6PDH and, therefore, can be used to distinguish COC that have finished their growth phase (BCB+) from those that are still growing and are less competent (BCB-). The objective of this study was to determine the metabolic differences between BCB- and BCB+ COC by performing a transcriptional analysis of genes related to glucose metabolism. The COC obtained from slaughterhouse ovaries were selected based on BCB staining. The BCB+ and BCB- COC were fertilized and cultured in vitro to determine the differences in developmental ability. For gene expression analysis, BCB+ and BCB- COC were denuded by vortexing and 5 groups of 10 oocytes; their corresponding cumulus cells per group were snap-frozen until analysis. Messenger RNA was extracted by Dynabeads (Dynal Biotech, Lake Success, NY, USA) and relative mRNA abundance was analysed by quantitative PCR using PPIA1 as housekeeping. Statistical differences were determined based on ANOVA (P < 0.05). The genes analysed were G6PDH, its positive regulator SIRT2, 2 glucose transporters SLC2A1 and SLC2A5, 2 genes involved in anaerobic glycolysis GAPDH and LDHA, 2 genes related with Krebs cycle CS and ATP5A1, and 1 gene related to glutathione metabolism GPX1. As expected, the BCB+ group showed a higher cleavage rate (85.6 ± 1.8 v. 74.2 ± 1.3%, BCB+ v. BCB-; P < 0.05) and blastocyst yield (Day 9: 33.3 ± 3.8 v. 16.1 ± 1.4%, BCB+ v. BCB-; P < 0.05) compared with BCB-. Genes SIRT2, GAPDH, and LDHA were significantly up-regulated in BCB- cumulus cells (SIRT2: 1 ± 0.04 v. 1.45 ± 0.21; GAPDH: 1 ± 0.17 v. 1.46 ± 0.15; LDHA: 1 ± 0.22 v. 1.65 ± 0.12; BCB+ v. BCB-; P < 0.05), whereas no significant differences were found in the other genes and in oocytes. In conclusion, the differences in G6PDH activity between BCB+ and BCB- COC are not controlled by G6PDH transcript abundance, but seem to be mediated by SIRT2 regulation of G6PDH activity. The BCB- cumulus cells showed an up-regulation of GAPDH and LDHA, suggesting a higher activity of anaerobic glycolysis in BCB- COC compared with BCB+.

Synthetic Strategy for Controllable Electrochemical Regulation of Metabolism in Living Cells Using Biocompatible Amphiphilic Redox-Active Polymers

In general, metabolism in living cells is closely related to the intracellular redox state. Focusing on the fact, a lot of studies examined the electrochemical regulation of cellular metabolism using lipophilic electron mediators with transmembrane ability. In these systems, electron mediators transport electrons between the intracellular redox active species and the extracellular redox active species or electrodes, across cell membranes. This process is called extracellular electron transfer (EET). Although conventional lipophilic mediators show cytotoxicity and are not suitable for long-term cultivation of living cells, we have recently developed a redox-active polymer as a biocompatible electron mediator (hereafter, the mediator is called PMF)[1]. PMF is an amphiphilic, transmembrane random copolymer polymerized by a free radical polymerization of hydrophilic 2-methacryloyloxyethyl phosphorylcholine (MPC) and hydrophobic vinyl ferrocene (VFc). The MPC and the VFc unit plays a role as a biocompatible and a redox active unit, respectively. Using thus synthesized PMF, we have successfully achieved electrochemical regulations of microbial metabolism without any genetic manipulations[2]. In the present work, with the aim of expanding the potential of this method, we attempted to regulate EET efficiency by tuning the molecular structure of the PMF polymers. It is known that the transmembrane ability is one of the rate determining factors in EET with conventional lipophilic electron mediators. Therefore, we assumed that cell membrane permeability could also influence the efficiency of the PMF-mediated EET. To verify this hypothesis, we added ethylene diamine tetra acetic acid (EDTA), which is known as a reagent to increase the cell-membrane permeability of gram-negative bacteria, during the measurement of the EET current from E. coli. Consequently, the EET current was increased by the addition of EDTA, and thus, the importance of the cell-membrane permeability was confirmed also for the case of the PMF-mediated EET. On the basis of the results, we decided to control the transmembrane ability of PMF for improving the EET efficiency. Theoretical calculations on amphiphilic copolymers have indicated that there is an suitable ratio between hydrophilic and hydrophobic units (R) for efficient cell-membrane permeability[3]. In addition, in the case of random copolymers including PMFs, cell-membrane permeability is expected to increase with decreasing molecular weight (Mw ). Based on these assumption, three kinds of PMF polymers with different Mw and R values (in this case, the ratio of VFc in the PMF polymer) were synthesized in this study. To evaluate the effects of three PMF polymers on EET, EET current from E. coli and S. cerevisiae was measured. Here, E. coli and S. cerevisiae were used as model living cells as prokaryotic cells and eukaryotic cells, respectively. From both experiments, the tendency of the relationship between EET efficiency and properties of PMF was revealed to be nearly the same for these two species. Experimental results suggested that R has a more significant influence on EET efficiency. Furthermore, it was also suggested that the EET efficiency could be increased with decreasing Mw at similar R values, as expected. Next, we tried to investigate the relationship between EET efficiency and the metabolisms by evaluating the activity of anaerobic glycolysis of S. cerevisiae as a model system. It is reported that lipophilic electron mediators with the redox potential lower than redox active species in electron transport chain (ETC) change the metabolic pathway of S. cerevisiae from the anaerobic glycolysis to anaerobic respiration by intercepting intracellular electrons from the ETC[4]. To evaluate the change of the metabolic pathway, we measured ethanol concentrations and the biomass in cell culture cultivated in the presence or the absence of the oxidized form of PMF. It was anticipated that the ethanol production becomes lower and the biomass becomes higher. In fact, the metabolic pathway was revealed to change in the presence of the oxidized form of PMF. Reasonably, the degree of the metabolism change was higher when the PMF with higher EET efficiency was used. These results indicated that the properties of the PMFs affects the efficiency of electrochemical regulation of cellular metabolism. Reference [1]Nishio et al, Chemphyschem, 2013, 14, 2159–2163. [2]Nishio et al, Environ. Sci. Technol. Lett., 2014, 1, 40–43. [3]Werner et al, Biomacromolecules, 2015, 16, 125–135. [4]Zhao et al, Anal. Chim. Acta, 2007, 597, 67–74. Figure 1

Hypoventilation Training at Supramaximal Intensity Improves Swimming Performance.

This study aimed to determine whether hypoventilation training at supramaximal intensity could improve swimming performance more than the same training conducted under normal breathing conditions. For a 5-wk period, 16 triathletes (12 men and 4 women) were asked to include one supramaximal set of 12 to 20 × 25-m front crawl swimming twice a week into their usual swimming session, performed either with hypoventilation at low lung volume (VHL group) or with normal breathing (CONT group). Before (Pre) and after (Post) training, all triathletes performed all-out front crawl trials for 100, 200, and 400 m. Time performance was significantly improved in VHL in all trials (100 m: -3.7 ± 3.7 s [-4.4% ± 4.0%]; 200 m: -6.9 ± 5.0 s [-3.6% ± 2.3%]; 400 m: -13.6 ± 6.1 s [-3.5% ± 1.5%]) but did not change in CONT. In VHL, the maximal lactate concentration (+2.35 ± 1.3 mmol·L on average) and the rate of lactate accumulation in blood (+41.7% ± 39.4%) were higher at Post than at Pre in the three trials, whereas they remained unchanged in CONT. Arterial oxygen saturation, heart rate, breathing frequency, and stroke length were not altered in both groups at the end of the training period. On the other hand, stroke rate was higher at Post compared with Pre in VHL but not different in CONT. The measurements of gas exchange for the 400-m trial revealed no change in peak oxygen consumption as well as in any pulmonary variable in both groups. This study demonstrated that VHL training, when performed at supramaximal intensity, represents an effective method for improving swimming performance, partly through an increase in the anaerobic glycolysis activity.

The molecular and ultrastructural aspects of the formation of mitochondrial dysfunction in the modeling of chronic cerebral ischemia: The mitoprotective effects of Angiolin

Modeling of cerebral circulation disorder by bilateral occlusion of carotids was accompanied by formation of severe neurological symptoms and their preservation for up to 18 days of the experiment. We found a significant decrease in the level of the HSP70 heat shock protein and the formation of persistent mitochondrial dysfunction. Administration of Angiolin to experimental animals resulted in activation of the expression of the HSP70 gene in neurons and ultrastructural improvement of mitochondria and neurons of CA1 hippocampal zone. The substantial mitoprotective activity of Angiolin is based on its antioxidant activity and the ability to positively influence the activity of anaerobic glycolysis, thus stabilizing the cellular energy metabolism under conditions of ischemic injury.

HIF-1-driven skeletal muscle adaptations to chronic hypoxia: molecular insights into muscle physiology.

Skeletal muscle is a metabolically active tissue and the major body protein reservoir. Drop in ambient oxygen pressure likely results in a decrease in muscle cells oxygenation, reactive oxygen species (ROS) overproduction and stabilization of the oxygen-sensitive hypoxia-inducible factor (HIF)-1α. However, skeletal muscle seems to be quite resistant to hypoxia compared to other organs, probably because it is accustomed to hypoxic episodes during physical exercise. Few studies have observed HIF-1α accumulation in skeletal muscle during ambient hypoxia probably because of its transient stabilization. Nevertheless, skeletal muscle presents adaptations to hypoxia that fit with HIF-1 activation, although the exact contribution of HIF-2, I kappa B kinase and activating transcription factors, all potentially activated by hypoxia, needs to be determined. Metabolic alterations result in the inhibition of fatty acid oxidation, while activation of anaerobic glycolysis is less evident. Hypoxia causes mitochondrial remodeling and enhanced mitophagy that ultimately lead to a decrease in ROS production, and this acclimatization in turn contributes to HIF-1α destabilization. Likewise, hypoxia has structural consequences with muscle fiber atrophy due to mTOR-dependent inhibition of protein synthesis and transient activation of proteolysis. The decrease in muscle fiber area improves oxygen diffusion into muscle cells, while inhibition of protein synthesis, an ATP-consuming process, and reduction in muscle mass decreases energy demand. Amino acids released from muscle cells may also have protective and metabolic effects. Collectively, these results demonstrate that skeletal muscle copes with the energetic challenge imposed by O2 rarefaction via metabolic optimization.

LMP1 Increases Expression of NADPH Oxidase (NOX) and Its Regulatory Subunit p22 in NP69 Nasopharyngeal Cells and Makes Them Sensitive to a Treatment by a NOX Inhibitor.

Oxidative stress is thought to contribute to cancer development. Epstein–Barr virus (EBV) and its encoded oncoprotein, latent membrane protein 1 (LMP1), are closely associated with the transformation of nasopharyngeal carcinoma (NPC) and Burkitt’s lymphoma (BL). In this study, we used LMP1-transformed NP cells and EBV-related malignant cell lines to assess the effects of LMP1 on reactive oxygen species (ROS) accumulation and glycolytic activity. Using NPC tissue samples and a tissue array to address clinical implications, we report that LMP1 activates NAD(P)H oxidases to generate excessive amount of ROS in EBV-related malignant diseases. By evaluating NAD(P)H oxidase (NOX) subunit expression, we found that the expression of the NAD(P)H oxidase regulatory subunit p22phox was significantly upregulated upon LMP1-induced transformation. Furthermore, this upregulation was mediated by the c-Jun N-terminal kinase (JNK) pathway. In addition, LMP1 markedly stimulated anaerobic glycolytic activity through the PI3K/Akt pathway. Additionally, in both NPC cells and tissue samples, p22phox expression correlated with LMP1 expression. The NAD(P)H oxidase inhibitor diphenyleneiodonium (DPI) also exerted a marked cytotoxic effect in LMP1-transformed and malignant cells, providing a novel strategy for anticancer therapy.

Differential proteomic profiling unveils new molecular mechanisms associated with mitochondrial complex III deficiency

Despite considerable knowledge about their genetic origins, the pathophysiological mechanisms that contribute to the clinical manifestations of mitochondrial disorders remain poorly understood. We have investigated the molecular pathways and metabolic adaptations that take place in primary skin fibroblasts from patients with mutations in the BCS1L gene, a primary cause of mitochondrial complex III enzyme deficiency. Two-dimensional DIGE together with MALDI-TOF/TOF mass spectrometry and physiological validation analyses revealed a significant metabolic and genetic reprogramming as an adaptive response to mitochondrial respiratory chain dysfunction. Our data provide information about specific protein targets that regulate the transmitochondrial functional responses to complex III deficiency, thereby opening new doors for future research.

Gasto energético aeróbico y anaeróbico en un circuito con cargas a seis intensidades diferentes. (Aerobic and anaerobic energy expenditure during at circuit weight training through six different intensities).

Resistance training is an intense anaerobic glycolytic activity and has been shown that estimates of energy expenditure in this activity turn out into an error that varies between 13 and 30%. The main aim of this paper is to describe the anaerobic energy contribution in circuit weight training. Twelve men (20-26 years) and seventeen women (18-29 years) students in Science of Physical Activity and Sport performed a circuit training at six different intensities (between 30% and 80% of 15RM). During all the circuits aerobic energy expenditure was registered by indirect calorimetry, heart rate with Polar® monitors and lactate concentration in capillary blood to measure the anaerobic contribution. The increased due to anaerobic energy was between 5,1%, and a maximum of 13,5% which clearly means that to measure or not the anaerobic contribution in circuit training can lead to an average error of 9,65%. There are significant differences (P <0,05) between aerobic energy expenditure and total (aerobic+anaerobic) at all the intensities, in a circuit weight training with progressive loads.

Fipronil is a powerful uncoupler of oxidative phosphorylation that triggers apoptosis in human neuronal cell line SHSY5Y

Fipronil is a phenylpyrazole insecticide known to elicit neurotoxicity via an interaction with ionotropic receptors, namely GABA and glutamate receptors. Recently, we showed that fipronil and other phenylpyrazole compounds trigger cell death in Caco-2 cells. In this study, we investigated the mode of action and the type of cell death induced by fipronil in SH-SY5Y human neuroblastoma cells. Flow cytometric and western blot analyses demonstrated that fipronil induces cellular events belonging to the apoptosis process, such as mitochondrial potential collapse, cytochrome c release, caspase-3 activation, nuclear condensation and phosphatidylserine externalization. In addition, fipronil induces a rapid ATP depletion with concomitant activation of anaerobic glycolysis. This cellular response is characteristic of mitochondrial injury associated with a defect of the respiration process. Therefore, we also investigated the effect of fipronil on the oxygen consumption in isolated mitochondria. Interestingly, we show for the first time that fipronil is a strong uncoupler of oxidative phosphorylation at relative low concentrations. Thus in this study, we report a new mode of action by which the insecticide fipronil could triggers apoptosis.

Muscle energetics changes throughout maturation: a quantitative31P-MRS analysis

We quantified energy production in 7 prepubescent boys (11.7 ± 0.6 yr) and 10 men (35.6 ± 7.8 yr) using (31)P-magnetic resonance spectroscopy to investigate whether development affects muscle energetics, given that resistance to fatigue has been reported to be larger before puberty. Each subject performed a finger flexions exercise at 0.7 Hz against a weight adjusted to 15% of their maximal voluntary strength for 3 min, followed by a 15-min recovery period. The total energy cost was similar in both groups throughout the exercise bout, whereas the interplay of the different metabolic pathways was different. At the onset of exercise, children exhibited a higher oxidative contribution (50 ± 15% in boys and 25 ± 8% in men, P < 0.05) to ATP production, whereas the phosphocreatine breakdown contribution was reduced (40 ± 10% in boys and 53 ± 12% in men, P < 0.05), likely as a compensatory mechanism. The anaerobic glycolysis activity was unaffected by maturation. The recovery phase also disclosed differences regarding the rates of proton efflux (6.2 ± 2.5 vs. 3.8 ± 1.9 mM · pH unit(-1) · min(-1), in boys and men, respectively, P < 0.05), and phosphocreatine recovery, which was significantly faster in boys than in men (rate constant of phosphocreatine recovery: 1.3 ± 0.5 vs. 0.7 ± 0.4 min(-1); V(max): 37.5 ± 14.5 vs. 21.1 ± 12.2 mM/min, in boys and men, respectively, P < 0.05). Our results obtained in vivo clearly showed that maturation affects muscle energetics. Children relied more on oxidative metabolism and less on creatine kinase reaction to meet energy demand during exercise. This phenomenon can be explained by a greater oxidative capacity, probably linked to a higher relative content in slow-twitch fibers before puberty.

Tumour Reactions to Hypoxia

Fast growing solid tumors generally lack an inner organisation, which causes the problem of a sufficient nutrient of each part of the tumor that then happens only by diffusion. The low oxygen supply leads to the activation of hypoxia-inducible factors, which regulate a plethora of genes. The reaction of tumor cells to hypoxia can be divided into two parts: On the one hand, there are signal substances, predominantly growth factors and cytokines, which provoke the vascularisation (angiogenesis), lymph vessel development (lymphangiogenesis), and the innervation (neoneurogenesis) of tumors and thus connect the tumor to structures of the environment. On the other hand, genes for intracellular proteins and receptors are regulated, which lead to changes of the tumor cell functions. Best characterized is the metabolic shift, a high anaerobic glycolytic activity and simultaneously a reduction of respiration. Furthermore, proliferation, dedifferentiation, resistance to apoptosis, and the metastatic potential are affected. With regard to the latter, we herein show that the migratory activity and velocity of PC-3 human prostate carcinoma cells significantly increases under oxygen-deprivation, which might be an explanation for the increasing number of experimental and clinical hints, that an anti-angiogenic therapy can promote the metastasis formation.

Glycolysis activity in flight muscles of birds according to their physiological function. An experimental model in vitro to study aerobic and anaerobic glycolysis activity separately

An experimental system in vitro is presented to assess the activity of the entire glycolysis in tissue extracts, which allows determining aerobic and anaerobic glycolysis activities separately. Glycolysis activity has been measured in pectoral and supracoracoideus muscles of the homing pigeon and the domestic fowl. These muscles support different aspects of flight in the two birds and are representative models of the two kinds of basic movements, endurance and sprint. The results obtained showed that in type I red fibers (pigeon pectoral), glucose produced a high glycolytic activity, while it was a poor substrate for type IIb white fibers (fowl pectoral and the two supracoracoideus). White fibers, however, attained its maximum glycolytic activity with phosphorylated glucose as substrate. These results demonstrated the validity of the experimental system as a method for assaying the two kinds of glycolytic activity in tissues, and supply new information about the biochemical and physiological features of these types of fibers.

Deoxygenated Hemoglobin/Myoglobin Kinetics of Forearm Muscles from Rest to Exercise in Patients with Chronic Obstructive Pulmonary Disease

Exercise capacity is frequently decreased in patients with chronic obstructive pulmonary disease (COPD), and muscle dysfunction is one factor in this reduction. Studies using (31)-phosphorus magnetic resonance spectroscopy ((31)P-MRS) have shown that phosphocreatine (PCr) and muscle pH (pHi) are significantly decreased in patients with COPD during mild exercise, suggesting the early activation of anaerobic glycolysis in their muscles. Thus, muscle oxygenation states during exercise might differ between patients with COPD and healthy individuals. We simultaneously measured oxygenation state and pHi in the muscles of patients with COPD during the transition from rest to exercise (on-transition) using near infrared spectroscopy (NIRS) and (31)P-MRS. Sixteen patients with COPD (aged 68.6 +/- 7.5 years) and 7 healthy males (controls; aged 63.3 +/- 7.5 years) performed dynamic handgrip exercise (lifting a weight by gripping at a rate of 20 grips per min for 3 min). Patients were classified based on pHi data at the completion of exercise as having a normal (>or= 6.9; n = 8) or a low (< 6.9; n = 8) pHi. The deoxygenated hemoglobin/myoglobin (deoxy-Hb/Mb) in NIRS recordings remained constant or slightly decreased initially (time delay), then increased to reach a plateau. We calculated the time delay and the time constant of deoxy-Hb/Mb kinetics during the on-transition. The time delay was shorter in the group with a low pHi than in the controls. These findings might reflect a slower increase in O(2) delivery in patients with a low pHi, which might partly account for altered muscle energy metabolism.

Anaerobic glyoxylate cycle activity during simultaneous utilization of glycogen and acetate in uncultured Accumulibacter enriched in enhanced biological phosphorus removal communities

Enhanced biological phosphorus removal (EBPR) communities protect waterways from nutrient pollution and enrich microorganisms capable of assimilating acetate as polyhydroxyalkanoate (PHA) under anaerobic conditions. Accumulibacter, an important uncultured polyphosphate-accumulating organism (PAO) enriched in EBPR, was investigated to determine the central metabolic pathways responsible for producing PHA. Acetate uptake and assimilation to PHA in Accumulibacter was confirmed using fluorescence in situ hybridization (FISH)-microautoradiography and post-FISH chemical staining. Assays performed with enrichments of Accumulibacter using an inhibitor of glyceraldehyde-3-phosphate dehydrogenase inferred anaerobic glycolysis activity. Significant decrease in anaerobic acetate uptake and PHA production rates were observed using inhibitors targeting enzymes within the glyoxylate cycle. Bioinformatic analysis confirmed the presence of genes unique to the glyoxylate cycle (isocitrate lyase and malate synthase) and gene expression analysis of isocitrate lyase demonstrated that the glyoxylate cycle is likely involved in PHA production. Reduced anaerobic acetate uptake and PHA production was observed after inhibition of succinate dehydrogenase and upregulation of a succinate dehydrogenase gene suggested anaerobic activity. Cytochrome b/b(6) activity inferred that succinate dehydrogenase activity in the absence of external electron acceptors may be facilitated by a novel cytochrome b/b(6) fusion protein complex that pushes electrons uphill to more electronegative electron carriers. Identification of phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase genes in Accumulibacter demonstrated the potential for interconversion of C(3) intermediates of glycolysis and C(4) intermediates of the glyoxylate cycle. Our findings along with previous hypotheses from analysis of microbiome data and metabolic models for PAOs were used to develop a model for anaerobic carbon metabolism in Accumulibacter.

Ca 2+-dependent glycolysis activation mediates apoptotic ATP elevation in HeLa cells

It was previously shown that cells die with increased cytosolic ATP after stimulation with apoptotic inducers including staurosporine (STS). To identify the source of apoptotic ATP elevation, we monitored, in real time, the cytosolic ATP level in luciferase-expressing HeLa cells. A mitochondrial uncoupler or a respiration chain inhibitor was found to decrease cytosolic ATP by about 50%. However, even when mitochondrial ATP synthesis was suppressed, STS induced a profound elevation of intracellular ATP. In contrast, the STS-induced ATP increase was prevented by any of three inhibitors of the glycolytic pathway: 2-deoxyglucose, iodoacetamide, and NaF. The STS effect strongly depended on intracellular calcium and was mimicked by a calcium ionophore. We conclude that Ca 2+-dependent activation of anaerobic glycolysis, but not aerobic mitochondrial oxidative phosphorylation, is responsible for the STS-induced elevation of ATP in apoptotic HeLa cells.

Diversities in hepatic HIF-1, IGF-I/IGFBP-1, LDH/ICD, and their mRNA expressions induced by CoCl2 in Qinghai-Tibetan plateau mammals and sea level mice

Ochotona curzoniae and Microtus oeconomus are the native mammals living on the Qinghai-Tibetan-Plateau of China. The molecular mechanisms of their acclimatization to the Plateau-hypoxia remain unclear. Expressions of hepatic hypoxia-inducible factor (HIF)-1alpha, insulin-like growth factor-I (IGF-I)/IGF binding protein (BP)-1(IGFBP-1; including genes), and key metabolic enzymatic genes [lactate dehydrogenase (LDH)-A/isocitrate dehydrogenase (ICD)] are compared in Qinghai-Tibetan-Plateau mammals and sea-level mice after injection of CoCl(2) (20, 40, or 60 mg/kg) and normobaric hypoxia (16.0% O(2), 10.8% O(2), and 8.0% O(2)) for 6 h, tested by histochemistry, Western blot analysis, ELISA, and RT-PCR. Major results are CoCl(2) markedly increased 1) HIF-1alpha only in mice, 2) hepatic and circulatory IGF-I in M. oeconomus, 3) hepatic IGFBP-1 in mice and O. curzoniae, and 4) LDH-A but reduced ICD mRNA in mice (CoCl(2) 20 mg/kg) but were unchanged in the Tibetan mammals. Normobaric hypoxia markedly 1) increased HIF-1alpha and LDH-A mRNA in mice and M. oeconomus (8.0% O(2)) not in O. curzoniae, and 2) reduced ICD mRNA in mice and M. oeconomus (8.0% O(2)) not in O. curzoniae. Results suggest that 1) HIF-1alpha responsiveness to hypoxia is distinct in lowland mice and plateau mammals, reflecting a diverse tolerance of the three species to hypoxia; 2) CoCl(2) induces diversities in HIF-1, IGF-I/IGFBP-1 protein or genes in mice, M. oeconomus, and O. curzoniae. In contrast, HIF-1 mediates IGFBP-1 transcription only in mice and in M. oeconomus (subjected to severe hypoxia); 3) differences in IGF-I/IGFBP-1 expressions induced by CoCl(2) reflect significant diversities in hormone regulation and cell protection from damage; and 4) activation of anaerobic glycolysis and reduction of Krebs cycle represents strategies of lowland-animals vs. the stable metabolic homeostasis of plateau-acclimatized mammals.

Free fatty acid metabolism in the air-breathing African catfish ( Clarias gariepinus) during asphyxia

In several waterbreathing fish species, hypoxia induces a decrease in plasma free fatty acid (FFA) levels as opposed to an increase in air-breathing mammals. We hypothesised that this change is coupled to the mode of breathing. Therefore, we followed the metabolic response of cannulated air-breathing African catfish to an 8-h asphyxia period. The hematocrit and hemoglobin increased significantly upon asphyxia. However, no change was observed in the mean cellular hemoglobin concentration, indicating that more erythrocytes were brought into circulation. A continuous increase in plasma lactate concentration during asphyxia showed permanent activation of anaerobic glycolysis, pointing to a persistent oxygen shortage. Plasma glucose levels did not change, but FFA levels decreased significantly upon asphyxia with a concomitant increase in plasma noradrenaline levels. Thus, these results suggest that in the air-breathing African catfish noradrenaline mediated a decrease in plasma FFA levels similar to that in waterbreathing fish species.

Skeletal muscle metabolism in Duchenne muscular dystrophy (DMD): an in-vitro proton NMR spectroscopy study

The metabolic differences in the skeletal muscle of patients with Duchenne muscular dystrophy (DMD) and normal subjects (controls) were investigated using in-vitro high-resolution proton NMR spectroscopy. In all, 56 metabolites were unambiguously identified in the perchloric acid extract of muscle tissue using one- and two-dimensional NMR. The concentrations of glycolytic substrate glucose (Glc; p < 0.05), gluconeogenic amino acids such as glutamine (Gln; p < 0.05) and alanine (Ala; p < 0.05) and the glycolytic product lactate (Lac; p < 0.05) were statistically significantly lower in DMD patients as compared to controls. A significant reduction in the concentrations of total creatine (TCr; p < 0.05), glycerophosphoryl choline + phosphoryl choline + carnitine (GPC/PC/Car; p < 0.05), choline (Cho; p < 0.05) and acetate (Ace; p < 0.05) was also observed in these patients. Decrease in the level of glucose may be attributed to the reduction in the concentrations of gluconeogenic substrates or membrane abnormalities in degenerated muscle of DMD patients. Lower levels of choline containing compounds indicate membrane abnormalities. Decrease in the concentration of lactate in the muscle of DMD patients may be due to the reduction in anaerobic glycolytic activity or lower substrate concentration. The decrease in the concentration of acetate may reflect reduced transport of fatty acids into mitochondria due to decreased concentration of carnitine in DMD patients. Kreb’s cycle intermediate α-ketoglutarate was observed only in the diseased muscle, which is suggestive of predominant oxidative metabolism for energy generation.

Oxygen Regulation of Energy Metabolism in Isolated Pig Hearts: A Near-IR Spectroscopy Study

Intracellular pO2in cardiac muscle is determined by the balance between its diffusion from vascular (VS) to intracellular space (IS) and uptake by mitochondria. Cessation of mechanical work decreases O2demand and should reduce the O2gradient between VS and IS. In this work, the effects of arterial pO2(pO2a) on myoglobin (Mb) oxygenation, O2uptake and lactate formation rates [V(O2) and V(Lac),μmol/min/g wet weight, respectively] in beating and KCl-arrested pig hearts were compared. pO2awas decreased stepwise (five steps, 10 min/step) from 528±24 to 54±19 mmHg by changing the ratio of O2/CO2(95:5%) to N2/CO2(95:5%) in the gas mixture from 100/0 through 50/50, 25/75, 10/90 to 0/100. While recording mechanical function, visible/near-infrared (near-IR) spectra (400–2500 nm) were acquired from the left ventricular wall every 45 s (32 scans) using a fiber-optic light guide connected to an NIRSystems spectrometer. Relative MbO2in the epicardium + midmural layer and the epicardium was estimated from absorbance changes (second derivative) at 764 and 584 nm, respectively. In beating hearts, a decrease in pO2aresulted in a decrease in MbO2(similar in both layers) and V(O2) (from 1.26±0.15 to 0.22±0.10) and an increase in V(Lac) from 0 to 2.43±0.76. In arrested hearts an increase in V(Lac) (0.55±0.24) was observed only at the lowest pO2aand the Mb oxygenation curve was shifted towards lower vascular pO2[(pO2a+pO2v)/2] by 160 mmHg. Oxygen consumption (oxygen flux) correlated linearly with calculated values of the oxygen gradients between the vascular and cytoplasmic spaces in both beating and arrested hearts. However, the diffusion rate constant was 3.5 times lower in arrested hearts. Conclusions: (1) hypoxia in KCl-arrested hearts results in only moderate activation of anaerobic glycolysis; (2) oxygenation of the epicardial and midmural LV layers is similar; and (3) a large pO2gradient exists between VS and IS in beating and arrested crystalloid-perfused hearts.