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ATP Levels (atp + level)
Kinds of ATP Levels Selected AbstractsCellular oxygen sensing, signalling and how to survive translational arrest in hypoxiaACTA PHYSIOLOGICA, Issue 2 2009M. Fähling Abstract Hypoxia is a consequence of inadequate oxygen availability. At the cellular level, lowered oxygen concentration activates signal cascades including numerous receptors, ion channels, second messengers, as well as several protein kinases and phosphatases. This, in turn, activates trans -factors like transcription factors, RNA-binding proteins and miRNAs, mediating an alteration in gene expression control. Each cell type has its unique constellation of oxygen sensors, couplers and effectors that determine the activation and predominance of several independent hypoxia-sensitive pathways. Hence, altered gene expression patterns in hypoxia result from a complex regulatory network with multiple divergences and convergences. Although hundreds of genes are activated by transcriptional control in hypoxia, metabolic rate depression, as a consequence of reduced ATP level, causes inhibition of mRNA translation. In a multi-phase response to hypoxia, global protein synthesis is suppressed, mainly by phosphorylation of eIF2-alpha by PERK and inhibition of mTOR, causing suppression of 5,-cap-dependent mRNA translation. Growing evidence suggests that mRNAs undergo sorting at stress granules, which determines the fate of mRNA as to whether being translated, stored, or degraded. Data indicate that translation is suppressed only at ,free' polysomes, but is active at subsets of membrane-bound ribosomes. The recruitment of specific mRNAs into subcellular compartments seems to be crucial for local mRNA translation in prolonged hypoxia. Furthermore, ribosomes themselves may play a significant role in targeting mRNAs for translation. This review summarizes the multiple facets of the cellular adaptation to hypoxia observed in mammals. [source] Acute action of rotenone on nigral dopaminergic neurons , involvement of reactive oxygen species and disruption of Ca2+ homeostasisEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 10 2009Peter S. Freestone Abstract Rotenone is a toxin used to generate animal models of Parkinson's disease; however, the mechanisms of toxicity in substantia nigra pars compacta (SNc) neurons have not been well characterized. We have investigated rotenone (0.05,1 ,m) effects on SNc neurons in acute rat midbrain slices, using whole-cell patch-clamp recording combined with microfluorometry. Rotenone evoked a tolbutamide-sensitive outward current (94 ± 15 pA) associated with increases in intracellular [Ca2+] ([Ca2+]i) (73.8 ± 7.7 nm) and intracellular [Na+] (3.1 ± 0.6 mm) (all with 1 ,m). The outward current was not affected by a high ATP level (10 mm) in the patch pipette but was decreased by Trolox. The [Ca2+]i rise was abolished by removing extracellular Ca2+, and attenuated by Trolox and a transient receptor potential M2 (TRPM2) channel blocker, N -(p -amylcinnamoyl) anthranilic acid. Other effects included mitochondrial depolarization (rhodamine-123) and increased mitochondrial reactive oxygen species (ROS) production (MitoSox), which was also abolished by Trolox. A low concentration of rotenone (5 nm) that, by itself, did not evoke a [Ca2+]i rise resulted in a large (46.6 ± 25.3 nm) Ca2+ response when baseline [Ca2+]i was increased by a ,priming' protocol that activated voltage-gated Ca2+ channels. There was also a positive correlation between ,naturally' occurring variations in baseline [Ca2+]i and the rotenone-induced [Ca2+]i rise. This correlation was not seen in non-dopaminergic neurons of the substantia nigra pars reticulata (SNr). Our results show that mitochondrial ROS production is a key element in the effect of rotenone on ATP-gated K+ channels and TRPM2-like channels in SNc neurons, and demonstrate, in these neurons (but not in the SNr), a large potentiation of rotenone-induced [Ca2+]i rise by a small increase in baseline [Ca2+]i. [source] Increased glucose metabolism and ATP level in brain tissue of Huntington's disease transgenic miceFEBS JOURNAL, Issue 19 2008Judit Oláh Huntington's disease (HD) is a progressive neurodegenerative disorder characterized by multifarious dysfunctional alterations including mitochondrial impairment. In the present study, the formation of inclusions caused by the mutation of huntingtin protein and its relationship with changes in energy metabolism and with pathological alterations were investigated both in transgenic and 3-nitropropionic acid-treated mouse models for HD. The HD and normal mice were characterized clinically; the affected brain regions were identified by immunohistochemistry and used for biochemical analysis of the ATP-producing systems in the cytosolic and the mitochondrial compartments. In both HD models, the activities of some glycolytic enzymes were somewhat higher. By contrast, the activity of glyceraldehyde-3-phosphate dehydrogenase was much lower in the affected region of the brain compared to that of the control. Paradoxically, at the system level, glucose conversion into lactate was enhanced in cytosolic extracts from the HD brain tissue, and the level of ATP was higher in the tissue itself. The paradox could be resolved by taking all the observed changes in glycolytic enzymes into account, ensuing an experiment-based detailed mathematical model of the glycolytic pathway. The mathematical modelling using the experimentally determined kinetic parameters of the individual enzymes and the well-established rate equations predicted the measured flux and concentrations in the case of the control. The same mathematical model with the experimentally determined altered Vmax values of the enzymes did account for an increase of glycolytic flux in the HD sample, although the extent of the increase was not predicted quantitatively. This suggested a somewhat altered regulation of this major metabolic pathway in HD tissue. We then used the mathematical model to develop a hypothesis for a new regulatory interaction that might account for the observed changes; in HD, glyceraldehyde-3-phosphate dehydrogenase may be in closer proximity (perhaps because of the binding of glyceraldehyde-3-phosphate dehydrogenase to huntingtin) with aldolase and engage in channelling for glyceraldehyde-3-phosphate. By contrast to most of the speculation in the literature, our results suggest that the neuronal damage in HD tissue may be associated with increased energy metabolism at the tissue level leading to modified levels of various intermediary metabolites with pathological consequences. [source] hnRNP K interacts with RNA binding motif protein 42 and functions in the maintenance of cellular ATP level during stress conditionsGENES TO CELLS, Issue 2 2009Toshiyuki Fukuda Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a conserved RNA-binding protein that is involved in multiple processes of gene expression, including chromatin remodeling, transcription, RNA splicing, mRNA stability and translation, together with diverse groups of molecular partners. Here we identified a previously uncharacterized protein RNA binding motif protein 42 (RBM42) as hnRNP K-binding protein. RBM42 directly bound to hnRNP K in vivo and in vitro. RBM42 also directly bound to the 3, untranslated region of p21 mRNA, one of the target mRNAs for hnRNP K. RBM42 predominantly localized within the nucleus and co-localized with hnRNP K there. When cells were treated with agents, puromycin, sorbitol or arsenite, which induced the formation of stress granules (SGs), cytoplasmic aggregates of stalled translational pre-initiation complexes, both hnRNP K and RBM42 localized at SGs. Depletion of hnRNP K by RNA interference decreased cellular ATP level following release from stress conditions. Simultaneous depletion of RBM42 with hnRNP K enhanced the effect of the hnRNP K depletion. Our results indicate that hnRNP K and RBM42 are components of SGs and suggest that hnRNP K and RBM42 have a role in the maintenance of cellular ATP level in the stress conditions possibly through protecting their target mRNAs. [source] Gene expression profile analysis of regenerating liver after portal vein ligation in rats by a cDNA microarray systemLIVER INTERNATIONAL, Issue 3 2004Y Nagano Abstract: Aims: We assessed changes in gene expression of hypertrophied liver after portal vein ligation (PL) in a test group of rats compared to a control group, which had the same size liver but no PL. Methods: The portal veins of the left and median lobes in the test group were ligated in an initial operation. Four days after the PL, the liver volume of the posterior caudate lobe (5%) increased two-fold and comprised 10% of the liver. A 90% hepatectomy was then performed, leaving only the hypertrophied posterior caudate lobe, and leaving the normal anterior and posterior caudate lobes (10%) in the control (sham) group. A comparison of the expression profiles between two groups was performed using cDNA microarrays and the hepatic ATP level was measured. Results: The survival rate for the PL group was significantly higher than for the sham group at 4 days after the hepatectomy (56.3% and 26.7%, P<0.05). Gene expression of cyclin D1, proliferating cell nuclear antigen, cyclin A and B was upregulated, and the cyclin-dependent kinase inhibitor was downregulated. Increases were observed in: (i) pyruvate dehydrogenase, the tricarboxylic acid cycle cycle regulator, (ii) acyl-CoA dehydrogenase, the oxidation regulator, and (iii) cytochrome oxidases, the oxidative phosphorylation regulator. Hepatic ATP concentration after hepatectomy was better maintained in the PL group than in the sham group (0.48±0.01 ,mol/ml vs. 0.33±0.01 ,mol/ml, P<0.05). Conclusion: The regenerating liver increased tolerance for extended hepatectomy compared to normal liver. It is believed that this is because the induced rapid regeneration of the remaining liver after hepatectomy increases ATP metabolism. [source] Chemoprotective effect of plant phenolics against anthracycline-induced toxicity on rat cardiomyocytes.PHYTOTHERAPY RESEARCH, Issue 2 2004Part I. Silymarin, its flavonolignans Abstract Silymarin, an extract of ,avonolignans from the dried fruits of milk thistle (Silybum marianum L. Gaertneri) and its constituents silibinin, dehydrosilibinin, silychristin and silydianin were tested for protective effects on rat cardiomyocytes exposed to doxorubicin. Silymarin and individual ,avonolignans did not exert cytotoxicity in the range 25,100 µm (incubation 9 h). Dehydrosilibinin was tested only at 25 µm concentration due to its low solubility. All substances increased the cell ATP level. Silymarin and ,avonolignans displayed a dose-dependent cytoprotection against doxorubicin (100 µm, incubation 8 h). The protective effects of silymarin, silibinin, dehydrosilibinin and silychristin were comparable to that of dexrasoxane, while silydianin exerted the best protective effect. The ability of silymarin complex and its components to protect cardiomyocytes against doxorubicin-induced oxidative stress is due mainly to their cell membrane stabilization effect, radical scavenging and iron chelating potency. Copyright © 2004 John Wiley & Sons, Ltd. [source] Enhanced IFN, production in adenosine-treated CHOCells: A mechanistic studyBIOTECHNOLOGY PROGRESS, Issue 3 2009William P. K. Chong Abstract Adenosine causes growth arrest in recombinant mammalian cell cultures, which results in enhanced productivity of the recombinant protein. Adenosine is also known to increase intracellular ATP level when added to mammalian cells. As a cell's energy level affects its protein expression capacity, we investigated the factors that contribute to the increase in recombinant protein productivity. Chinese hamster ovary (CHO) cells expressing human interferon-gamma (IFN,) were treated with 1 mM adenosine on Day 2 of culture. The growth arrest resulted in 60% reduction in integral viable cell density when compared with control. However, IFN, titer improved 1.4-fold alongside a 2.5-fold increase in average specific productivity. The adenosine-treated cells also experienced a two-fold increase in ATP level that sustained for 3 days. Western blot studies revealed a relatively short-lived but strong activation of the energy sensor AMP-activated protein kinase (AMPK) in adenosine-treated cells. Activation of AMPK was probably due to adenosine being temporarily converted to AMP. Activated AMPK should have down-regulated protein translation by preventing mammalian target of rapamycin (mTOR) from phosphorylating and inactivating 4E-binding protein 1 (4E-BP1), a key repressor of protein translation initiation. However, Western blots showed increased phosphorylation of 4E-BP1 on Day 2 that lasted 3 days. This implied that a high concentration of ATP could keep 4E-BP1 inhibited, probably by directly modulating mTOR. This corroborated with an earlier in vitro observation (Dennis et al., Science. 2001;294:1102-1105). Inhibition of translation initiation repression is thus likely to contribute in part to the improvement in IFN,-specific productivity and titer. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] AMP-activated protein kinase: role in metabolism and therapeutic implicationsDIABETES OBESITY & METABOLISM, Issue 6 2006Greg Schimmack AMP-activated protein kinase (AMPK) is an enzyme that works as a fuel gauge which becomes activated in situations of energy consumption. AMPK functions to restore cellular ATP levels by modifying diverse metabolic and cellular pathways. In the skeletal muscle, AMPK is activated during exercise and is involved in contraction-stimulated glucose transport and fatty acid oxidation. In the heart, AMPK activity increases during ischaemia and functions to sustain ATP, cardiac function and myocardial viability. In the liver, AMPK inhibits the production of glucose, cholesterol and triglycerides and stimulates fatty acid oxidation. Recent studies have shown that AMPK is involved in the mechanism of action of metformin and thiazolidinediones, and the adipocytokines leptin and adiponectin. These data, along with evidence that pharmacological activation of AMPK in vivo improves blood glucose homeostasis, cholesterol concentrations and blood pressure in insulin-resistant rodents, make this enzyme an attractive pharmacological target for the treatment of type 2 diabetes, ischaemic heart disease and other metabolic diseases. [source] Augmentation of cellular adenosine triphosphate levels in PC12 cells by extracellular adenosineDRUG DEVELOPMENT RESEARCH, Issue 1 2003Hiroyuki Fujimori Abstract The effects of extracellular adenosine (Ado) on cellular levels of adenosine triphosphate (ATP) in PC12 cells were studied. Ado and inosine but not adenine nucleotides, guanosine, cytosine, uridine, thymidine, and various P1 receptor agonists of Ado, significantly enhanced cellular ATP levels in PC12 cells by about 2.5-fold. The ATP-enhancing effect of Ado was potentiated by dipyridamole, an inhibitor of Ado uptake, and was also observed when PC12 cells were incubated in glucose-free medium. These results suggest that augmentation of cellular ATP levels in PC12 cells by extracellular Ado might be acceleration of ATP synthesis through the Ado salvage system utilizing hypoxanthine-guanine phosphoribosyltransferase rather than Ado kinase, since 5,-iodotubercidin, an Ado kinase inhibitor, had no effect on the enhancement induced by Ado. Drug Dev. Res. 59:8,13, 2003. © 2003 Wiley-Liss, Inc. [source] Structural myocardial changes after coronary artery surgeryEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 11 2000F. Eberhardt Background Postoperative contractile dysfunction or ,myocardial stunning' has been described after coronary artery bypass grafting (CABG). In the present study we sought to determine if and to what extent clinical, structural and histochemical evidence of myocardial changes associated with stunning could be found in patients after CABG and cold crystalloid cardioplegia. Materials and methods Left ventricular (LV) biopsies were obtained from CABG patients (n = 10) prior to and at the end of cardiopulmonary bypass (CPB). These biopsies were immunostained for the inducible heat-shock protein 70 (HSP-70i), intercellular adhesion molecule-1 (ICAM-1) and actin. ATP was measured by bioluminescence. Results Biopsies pre-CPB showed no evidence of myocardial damage as HSP-70i was absent and a regular actin cross-striation pattern and only constitutive ICAM-1-expression were present. After CPB we found significantly increased HSP-70i and ICAM-1 levels as well as a deranged actin cross-striation pattern with a widening of actin bands. ATP levels declined from 10 mmol L,1 pre-CPB to 4.9 mmol L,1 after CPB. Correspondingly, coronary sinus effluent showed a significant lactate production. Although, cardiac function determined by transoesophageal echocardiography did not deteriorate, significant inotropic support was necessary to maintain cardiac output. Conclusions Our results present clinical and structural evidence of ,myocardial stunning' after CABG and cold crystalloid cardioplegia. Increased HSP-70i and ICAM-1 expression, as well as a deranged actin cross-striation pattern, might be structural markers to determine ,myocardial stunning' in clinical settings. [source] Serum or target deprivation-induced neuronal death causes oxidative neuronal accumulation of Zn2+ and loss of NAD+EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 6 2010Christian T. Sheline Abstract Trophic deprivation-mediated neuronal death is important during development, after acute brain or nerve trauma, and in neurodegeneration. Serum deprivation (SD) approximates trophic deprivation in vitro, and an in vivo model is provided by neuronal death in the mouse dorsal lateral geniculate nucleus (LGNd) after ablation of the visual cortex (VCA). Oxidant-induced intracellular Zn2+ release ([Zn2+]i) from metallothionein-3 (MT-III), mitochondria or ,protein Zn2+', was implicated in trophic deprivation neurotoxicity. We have previously shown that neurotoxicity of extracellular Zn2+ required entry, increased [Zn2+]i, and reduction of NAD+ and ATP levels causing inhibition of glycolysis and cellular metabolism. Exogenous NAD+ and sirtuin inhibition attenuated Zn2+ neurotoxicity. Here we show that: (1) Zn2+ is released intracellularly after oxidant and SD injuries, and that sensitivity to these injuries is proportional to neuronal Zn2+ content; (2) NAD+ loss is involved , restoration of NAD+ using exogenous NAD+, pyruvate or nicotinamide attenuated these injuries, and potentiation of NAD+ loss potentiated injury; (3) neurons from genetically modified mouse strains which reduce intracellular Zn2+ content (MT-III knockout), reduce NAD+ catabolism (PARP-1 knockout) or increase expression of an NAD+ synthetic enzyme (Wlds) each had attenuated SD and oxidant neurotoxicities; (4) sirtuin inhibitors attenuated and sirtuin activators potentiated these neurotoxicities; (5) visual cortex ablation (VCA) induces Zn2+ staining and death only in ipsilateral LGNd neurons, and a 1 mg/kg Zn2+ diet attenuated injury; and finally (6) NAD+ synthesis and levels are involved given that LGNd neuronal death after VCA was dramatically reduced in Wlds animals, and by intraperitoneal pyr vate or nicotinamide. Zn2+ toxicity is involved in serum and trophic deprivation-induced neuronal death. [source] Metabolic gene switching in the murine female heart parallels enhanced mitochondrial respiratory function in response to oxidative stressFEBS JOURNAL, Issue 20 2007M. Faadiel Essop The mechanisms underlying increased cardioprotection in younger female mice are unclear. We hypothesized that serine-threonine protein kinase (protein kinase B; Akt) triggers a metabolic gene switch (decreased fatty acids, increased glucose) in female hearts to enhance mitochondrial bioenergetic capacity, conferring protection against oxidative stress. Here, we employed male and female control (db/+) and obese (db/db) mice. We found diminished transcript levels of peroxisome proliferator-activated receptor-alpha, muscle-type carnitine palmitoyltransferase 1 and pyruvate dehydrogenase kinase 4 in female control hearts versus male hearts. Moreover, females displayed improved recovery of cardiac mitochondrial respiratory function and higher ATP levels versus males in response to acute oxygen deprivation. All these changes were reversed in female db/db hearts. However, we found no significant gender-based differences in levels of Akt, suggesting that Akt-independent signaling mechanisms are responsible for the resilient mitochondrial phenotype observed in female mouse hearts. As glucose is a more energetically efficient fuel substrate when oxygen is limiting, this gene program may be a crucial component that enhances tolerance to oxygen deprivation in female hearts. [source] Calcium-independent cytoskeleton disassembly induced by BAPTAFEBS JOURNAL, Issue 15 2004Yasmina Saoudi In living organisms, Ca2+ signalling is central to cell physiology. The Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane- N,N,N,,N,-tetraacetic acid (BAPTA) has been widely used as a probe to test the role of calcium in a large variety of cell functions. Here we show that in most cell types BAPTA has a potent actin and microtubule depolymerizing activity and that this activity is completely independent of Ca2+ chelation. Thus, the depolymerizing effect of BAPTA is shared by a derivative (D-BAPTA) showing a dramatically reduced calcium chelating activity. Because the extraordinary depolymerizing activity of BAPTA could be due to a general depletion of cell fuel molecules such as ATP, we tested the effects of BAPTA on cellular ATP levels and on mitochondrial function. We find that BAPTA depletes ATP pools and affects mitochondrial respiration in vitro as well as mitochondrial shape and distribution in cells. However, these effects are unrelated to the Ca2+ chelating properties of BAPTA and do not account for the depolymerizing effect of BAPTA on the cell cytoskeleton. We propose that D-BAPTA should be systematically introduced in calcium signalling experiments, as controls for the known and unknown calcium independent effects of BAPTA. Additionally, the concomitant depolymerizing effect of BAPTA on both tubulin and actin assemblies is intriguing and may lead to the identification of a new control mechanism for cytoskeleton assembly. [source] Intracellular pH homeostasis in the filamentous fungus Aspergillus nigerFEBS JOURNAL, Issue 14 2002Stephan J. A. Hesse Intracellular pH homeostasis in the filamentous fungus Aspergillus niger was measured in real time by 31P NMR during perfusion in the NMR tube of fungal biomass immobilized in Ca2+ -alginate beads. The fungus maintained constant cytoplasmic pH (pHcyt) and vacuolar pH (pHvac) values of 7.6 and 6.2, respectively, when the extracellular pH (pHex) was varied between 1.5 and 7.0 in the presence of citrate. Intracellular metabolism did not collapse until a ,pH over the cytoplasmic membrane of 6.6,6.7 was reached (pHex 0.7,0.8). Maintenance of these large pH differences was possible without increased respiration compared to pHex 5.8. Perfusion in the presence of various hexoses and pentoses (pHex 5.8) revealed that the magnitude of ,pH values over the cytoplasmic and vacuolar membrane could be linked to the carbon catabolite repressing properties of the carbon source. Also, larger ,pH values coincided with a higher degree of respiration and increased accumulation of polyphosphate. Addition of protonophore (carbonyl cyanide m -chlorophenylhydrazone, CCCP) to the perfusion buffer led to decreased ATP levels, increased respiration and a partial (1 µm CCCP), transient (2 µm CCCP) or permanent (10 µm CCCP) collapse of the vacuolar membrane ,pH. Nonlethal levels of the metabolic inhibitor azide (N3,, 0.1 mm) caused a transient decrease in pHcyt that was closely paralleled by a transient vacuolar acidification. Vacuolar H+ influx in response to cytoplasmic acidification, also observed during extreme medium acidification, indicates a role in pH homeostasis for this organelle. Finally, 31P NMR spectra of citric acid producing A. niger mycelium showed that despite a combination of low pHex (1.8) and a high acid-secreting capacity, pHcyt and pHvac values were still well maintained (pH 7.5 and 6.4, respectively). [source] Cytochrome c oxidase isoform IV-2 is involved in 3-nitropropionic acid-induced toxicity in striatal astrocytesGLIA, Issue 14 2009Shilpee Singh Abstract Astrocyte mitochondria play an important role for energy supply and neuronal survival in the brain. Toxic and degenerative processes are largely associated with mitochondrial dysfunction. We, therefore, investigated the effect of 3-nitropropionic acid (NPA), a mitochondrial toxin and in vitro model of Huntington's disease (HD), on mitochondrial function and viability of primary striatal astrocytes. Although NPA is known as an irreversible inhibitor of succinate dehydrogenase, we observed an increase of astrocyte ATP levels after NPA treatment. This effect could be explained by NPA-mediated alterations of cytochrome c oxidase subunit IV isoform (COX IV) expression. The up-regulation of COX isoform IV-2 caused an increased enzyme activity at the expense of elevated mitochondrial peroxide production causing increased cell death. The application of a small interfering RNA against COX IV-2 revealed the causal implication of COX isoform IV-2 in NPA-mediated elevation of oxidative stress and necrotic cell death. Thus, we propose a novel, additional mechanism of NPA-induced cell stress and death which is based on structural and functional changes of astrocyte COX and which could indirectly impair neuronal survival. © 2009 Wiley-Liss, Inc. [source] Nitric oxide suppresses transforming growth factor-,1,induced epithelial-to-mesenchymal transition and apoptosis in mouse hepatocytes,HEPATOLOGY, Issue 5 2009Xinchao Pan Nitric oxide (NO) is a multifunctional regulator that is implicated in various physiological and pathological processes. Here we report that administration of NO donor S-nitroso-N-acetylpenicillamine (SNAP) inhibited transforming growth factor-,1 (TGF-,1)-induced epithelial-to-mesenchymal transition (EMT) and apoptosis in mouse hepatocytes. Overexpression of inducible NO synthase (iNOS) by transfection of the iNOS-expressing vector, which increased NO production, also inhibited the TGF-,1-induced EMT and apoptosis in these cells. Treatment of cells with proinflammatory mediators, including tumor necrosis factor (TNF)-,, interleukin (IL)-1,, and interferon (IFN)-,, which increased the endogenous NO production, produced the same inhibitory effect. Furthermore, exogenous NO donor SNAP treatment caused a decrease in the intracellular adenosine triphosphate (ATP) levels. Consistently, depletion of intracellular ATP by mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) inhibited the TGF-,1-induced EMT and apoptosis, suggesting that an NO-induced decrease of ATP involved in the NO-mediated inhibition of TGF-,1-induced EMT and apoptosis. NO and FCCP also inhibited TGF-,1-induced STAT3 activation, suggesting that signal transducer and activator of transcription 3 inactivation is involved in the NO-induced effects on TGF-,1-induced EMT and apoptosis. Conclusion: Our study indicates that NO plays an important role in the inhibition of TGF-,1-induced EMT and apoptosis in mouse hepatocytes through the downregulation of intracellular ATP levels. The data provide an insight into the in vivo mechanisms on the function of NO during the processes of both EMT and apoptosis. (HEPATOLOGY 2009.) [source] Improving cellular function through modulation of energy metabolismINTERNATIONAL JOURNAL OF COSMETIC SCIENCE, Issue 5 2004D. Maes The ambivalent consequences of mitochondrial stimulation on cellular activity have been well established. Mitochondria supply the cell with energy through a process of oxidative phosphorylation but thereby generate free radicals, resulting in the accumulation of hydrogen peroxide in the cytoplasm. We have investigated the impact of cellular senescence as well as UV irradiation, on the balance between these two activities. The adenosine triphosphate (ATP) level, DNA and protein synthesis in fibroblasts obtained from donors between 30 and 90 years of age appeared to be significantly influenced by the aging process. Both DNA and protein synthesis could be stimulated by increasing intracellular ATP levels. In-vitro senescent fibroblasts showed a reduction in the level of ATP as well as a shift in mitochondrial membrane potential. At the same time, there was an increase in intracellular hydrogen peroxide with increasing population doubling, indicating a clear dysfunction of the metabolic machinery in the mitochondria of senescent cells. To counteract this degradation of the energy pool, we treated cells with creatine, which is known to restore the pool of phosphocreatine in the mitochondria. Creatine treatment significantly increased cell survival after UV exposure, stimulated the repair of UVB-induced DNA damage in keratinocytes and caused a significant reduction in the number of sunburn cells in a UVB-exposed reconstituted skin model. These results clearly indicate that restoration of the energy pool in mitochondria increased cellular self-defense mechanism. These data show the important role played by the mitochondrial energy metabolism on the aging process, and indicate a possible therapy that can be used to counteract this negative effect. Treatment with creatine seems to provide the necessary boost to the cellular metabolism, which leads to an induction of a significant amount of protection and repair to human skin cells. [source] The role of PAS kinase in regulating energy metabolismIUBMB LIFE, Issue 4 2008Huai-Xiang Hao Abstract Metabolic disorders, such as diabetes and obesity, are fundamentally caused by cellular energy imbalance and dysregulation. Therefore, understanding the regulation of cellular fuel and energy metabolism is of great importance to develop effective therapies for metabolic disease. The cellular nutrient and energy sensors, AMPK and TOR, play a key role in maintaining cellular energy homeostasis. Like AMPK and TOR, PAS kinase (PASK) is also a nutrient responsive protein kinase. In yeast, PAS kinase phosphorylates the enzyme Ugp1 and thereby shifts glucose partitioning toward cell wall glucan synthesis at the expense of glycogen synthesis. Consistent with this function, yeast PAS kinase is activated by both cell integrity stress and growth in non-fermentative carbon sources. PASK is also important for proper regulation of glucose metabolism in mammals at both the hormonal and cellular level. In cultured pancreatic ,-cells, PASK is activated by elevated glucose concentrations and is required for glucose-stimulated transcription of the insulin gene. PASK knockdown in cultured myoblasts causes increased glucose oxidation and elevated cellular ATP levels. Mice lacking PASK exhibit increased metabolic rate and resistance to diet-induced obesity. Interestingly, PGC-1 expression and AMPK and TOR activity were not affected in PASK deficient mice, suggesting PASK may exert its metabolic effects through a new mechanism. We propose that PASK plays a significant role in nutrient sensing, metabolic regulation, and energy homeostasis, and is a potential therapeutic target for metabolic disease. © 2008 IUBMB IUBMB Life, 60(4): 204,209, 2008 [source] Attenuation of TCDD-induced oxidative stress by 670 nm photobiomodulation in developmental chicken kidneyJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 4 2008Jinhwan Lim 2,3,7,8-Tetrachlorodibenzo- p -dioxin (TCDD), a potent developmental teratogen inducing oxidative stress and sublethal changes in multiple organs, provokes developmental renal injuries. In this study, we investigated TCDD-induced biochemical changes and the therapeutic efficacy of photobiomodulation (670 nm; 4 J/cm2) on oxidative stress in chicken kidneys during development. Eggs were injected once prior to incubation with TCDD (2 pg/g or 200 pg/g) or sunflower oil vehicle control. Half of the eggs in each dose group were then treated with red light once per day through embryonic day 20 (E20). Upon hatching at E21, the kidneys were collected and assayed for glutathione peroxidase, glutathione reductase, catalase, superoxide dimutase, and glutathione- S -transferase activities, as well as reduced glutathione and ATP levels, and lipid peroxidation. TCDD exposure alone suppressed the activity of the antioxidant enzymes, increased lipid peroxidation, and depleted available ATP. The biochemical indicators of oxidative and energy stress in the kidney were reversed by daily phototherapy, restoring ATP and glutathione contents and increasing antioxidant enzyme activities to control levels. Photobiomodulation also normalized the level of lipid peroxidation increased by TCDD exposure. The results of this study suggest that 670 nm photobiomodulation may be useful as a noninvasive treatment for renal injury resulting from chemically induced cellular oxidative and energy stress. © 2008 Wiley Periodicals, Inc. J Biochem Mol Toxicol 22:230,239, 2008; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20233 [source] Prostate carcinoma cells selected by long-term exposure to reduced oxygen tension show remarkable biochemical plasticity via modulation of superoxide, HIF-1, levels, and energy metabolismJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2007Jeanne Bourdeau-Heller Cancer cells are able to tolerate levels of O2 that are damaging or lethal to normal cells; we hypothesize that this tolerance is the result of biochemical plasticity which maintains cellular homeostasis of both energy levels and oxidation state. In order to examine this hypothesis, we used different O2 levels as a selective agent during long-term culture of DU145 prostate cancer cells to develop three isogenic cell lines that grow in normoxic (4%), hyperoxic (21%), or hypoxic (1%) O2 conditions. Growth characteristics and O2 consumption differed significantly between these cell lines without changes in ATP levels or altered sensitivity to 2-deoxy- D -glucose, an inhibitor of glycolysis. O2 consumption was significantly higher in the hyperoxic line as was the level of endogenous superoxide. The hypoxic cell line regulated the chemical gradient of the proton motive force (PMF) independent of the electrical component without O2 -dependent changes in Hif-1, levels. In contrast, the normoxic line regulated Hif-1, without tight regulation of the chemical component of the PMF noted in the hypoxic cell line. From these studies, we conclude that selection of prostate cancer cells by long-term exposure to low ambient levels of O2 resulted in cells with unique biochemical properties in which energy metabolism, reactive oxygen species (ROS), and HIF-1, levels are modulated to allow cell survival and growth. Thus, cancer cells exhibit remarkable biochemical plasticity in response to various O2 levels. J. Cell. Physiol. 212:744,752, 2007. © 2007 Wiley-Liss, Inc. [source] Regulation of protein phosphatase 1, activity in hypoxia through increased interaction with NIPP1: Implications for cellular metabolismJOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2006Kathrina M. Comerford Eukaryotic cells sense decreased oxygen levels and respond by altering their metabolic strategy to sustain non-respiratory ATP production through glycolysis, and thus promote cell survival in a hypoxic environment. Protein phosphatase 1 (PP1) has been recently implicated in the governance of the rational use of energy when metabolic substrates are abundant and contributes to cellular recovery following metabolic stress. Under conditions of hypoxia, the expression of the gamma isoform of PP1 (PP1,), is diminished, an event we have hypothesized to be involved in the adaptive cellular response to hypoxia. Decreased PP1, activity in hypoxia has a profound impact on the activity of the cAMP response element binding protein (CREB), a major transcriptional regulator of metabolic genes and processes. Here, we demonstrate a further mechanism leading to inhibition of PP1 activity in hypoxia which occurs at least in part through increased association with the nuclear inhibitor of PP1 (NIPP1), an event dependent upon decreased basal cAMP/PKA-dependent signaling. Using a dominant negative NIPP1 construct, we provide evidence that NIPP1 plays a major role in the regulation of both CREB protein expression and CREB-dependent transcription in hypoxia. Furthermore, we demonstrate functional sequellae of such events including altered gene expression and recovery of cellular ATP levels. In summary, we demonstrate that interaction with NIPP1 mediates decreased PP1, activity in hypoxia, an event which may constitute an inherent part of the cellular oxygen-sensing machinery and may play a role in physiologic adaptation to hypoxia. J. Cell. Physiol. 209: 211,218, 2006. © 2006 Wiley-Liss, Inc. [source] Two modes of mitochondrial dysfunction lead independently to lifespan extension in Caenorhabditis elegansAGING CELL, Issue 3 2010Wen Yang Summary In Caenorhabditis elegans, longevity is increased by a partial loss-of-function mutation in the mitochondrial complex III subunit gene isp-1. Longevity is also increased by RNAi against the expression of a variety of mitochondrial respiratory chain genes, including isp-1, but it is unknown whether the isp-1(qm150) mutation and the RNAi treatments trigger the same underlying mechanisms of longevity. We have identified nuo-6(qm200), a mutation in a conserved subunit of mitochondrial complex I (NUDFB4). The mutation reduces the function of complex I and, like isp-1(qm150), results in low oxygen consumption, slow growth, slow behavior, and increased lifespan. We have compared the phenotypes of nuo-6(qm200) to those of nuo-6(RNAi) and found them to be distinct in crucial ways, including patterns of growth and fertility, behavioral rates, oxygen consumption, ATP levels, autophagy, and resistance to paraquat, as well as expression of superoxide dismutases, mitochondrial heat-shock proteins, and other gene expression markers. RNAi treatments appear to generate a stress and autophagy response, while the genomic mutation alters electron transport and reactive oxygen species metabolism. For many phenotypes, we also compared isp-1(qm150) to isp-1(RNAi) and found the same pattern of differences. Most importantly, we found that, while the lifespan of nuo-6, isp-1 double mutants is not greater than that of the single mutants, the lifespan increase induced by nuo-6(RNAi) is fully additive to that induced by isp-1(qm150), and the increase induced by isp-1(RNAi) is fully additive to that induced by nuo-6(qm200). Our results demonstrate that distinct and separable aspects of mitochondrial biology affect lifespan independently. [source] Neuronal expression of a single-subunit yeast NADH,ubiquinone oxidoreductase (Ndi1) extends Drosophila lifespanAGING CELL, Issue 2 2010Sepehr Bahadorani Summary The ,rate of living' theory predicts that longevity should be inversely correlated with the rate of mitochondrial respiration. However, recent studies in a number of model organisms, including mice, have reported that interventions that retard the aging process are, in fact, associated with an increase in mitochondrial activity. To better understand the relationship between energy metabolism and longevity, we supplemented the endogenous respiratory chain machinery of the fruit fly Drosophila melanogaster with the alternative single-subunit NADH,ubiquinone oxidoreductase (Ndi1) of the baker's yeast Saccharomyces cerevisiae. Here, we report that expression of Ndi1 in fly mitochondria leads to an increase in NADH,ubiquinone oxidoreductase activity, oxygen consumption, and ATP levels. In addition, exogenous Ndi1 expression results in increased CO2 production in living flies. Using an inducible gene-expression system, we expressed Ndi1 in different cells and tissues and examined the impact on longevity. In doing so, we discovered that targeted expression of Ndi1 in fly neurons significantly increases lifespan without compromising fertility or physical activity. These findings are consistent with the idea that enhanced respiratory chain activity in neuronal tissue can prolong fly lifespan. [source] 2-Deoxyglucose and NMDA inhibit protein synthesis in neurons and regulate phosphorylation of elongation factor-2 by distinct mechanismsJOURNAL OF NEUROCHEMISTRY, Issue 3 2006M. Maus Abstract Cerebral ischaemia is associated with brain damage and inhibition of neuronal protein synthesis. A deficit in neuronal metabolism and altered excitatory amino acid release may both contribute to those phenomena. In the present study, we demonstrate that both NMDA and metabolic impairment by 2-deoxyglucose or inhibitors of mitochondrial respiration inhibit protein synthesis in cortical neurons through the phosphorylation of eukaryotic elongation factor (eEF-2), without any change in phosphorylation of initiation factor eIF-2,. eEF-2 kinase may be activated both by Ca2+ -independent AMP kinase or by an increase in cytosolic Ca2+. Although NMDA decreases ATP levels in neurons, only the effects of 2-deoxyglucose on protein synthesis and phosphorylation of elongation factor eEF-2 were reversed by Na+ pyruvate. Protein synthesis inhibition by 2-deoxyglucose was not as a result of a secondary release of glutamate from cortical neurons as it was not prevented by the NMDA receptor antagonist 5-methyl-10,11-dihydro-5H-dibenzo-(a,d)-cyclohepten-5,10-imine hydrogen maleate (MK 801), nor to an increase in cytosolic-free Ca2+. Conversely, 2-deoxyglucose likely activates eEF-2 kinase through a process involving phosphorylation by AMP kinase. In conclusion, we provide evidence that protein synthesis can be inhibited by NMDA and metabolic deprivation by two distinct mechanisms involving, respectively, Ca2+ -dependent and Ca2+ -independent eEF-2 phosphorylation. [source] Uncoupling protein 2 influences dopamine secretion in PC12h cellsJOURNAL OF NEUROCHEMISTRY, Issue 2 2003Shigeru Yamada Abstract Uncoupling protein 2 (UCP2) belongs to the UCP family, and is distributed in many organs including the brain. Although UCP2 is known to be related to many functions such as the regulation of insulin secretion or the scavenging of the radicals, the role of UCP2 in the central nervous system remains unclear. In this report, rat UCP2 (rUCP2) and its mutants were overexpressed in the PC12h cells to determine the physiological roles played by UCP2 in neural cells and to elucidate the mechanisms that regulate these functions. It was found that rUCP2 was activated by the stimulation of the cAMP-protein kinase A (PKA) cascade. Moreover, the activation of rUCP2 suppressed intracellular ATP levels and inhibited the cAMP-dependent increase of dopamine secretion. Thus, UCP2 appears to be regulated by the excitatory stimulus via the cAMP-PKA cascade and serves to negatively control the synaptic output by reducing intracellular ATP levels. [source] Diazepam Promotes ATP Recovery and Prevents Cytochrome c Release in Hippocampal Slices After In Vitro IschemiaJOURNAL OF NEUROCHEMISTRY, Issue 3 2000Francesca Galeffi Abstract: Benzodiazepines protect hippocampal neurons when administered within the first few hours after transient cerebral ischemia. Here, we examined the ability of diazepam to prevent early signals of cell injury (before cell death) after in vitro ischemia. Ischemia in vitro or in vivo causes a rapid depletion of ATP and the generation of cell death signals, such as the release of cytochrome c from mitochondria. Hippocampal slices from adult rats were subjected to 7 min of oxygen-glucose deprivation (OGD) and assessed histologically 3 h after reoxygenation. At this time, area CA1 neurons appeared viable, although slight abnormalities in structure were evident. Immediately following OGD, ATP levels in hippocampus were decreased by 70%, and they recovered partially over the next 3 h of reoxygenation. When diazepam was included in the reoxygenation buffer, ATP levels recovered completely by 3 h after OGD. The effects of diazepam were blocked by picrotoxin, indicating that the protection was mediated by an influx of Cl - through the GABAA receptor. It is interesting that the benzodiazepine antagonist flumazenil did not prevent the action of diazepam, as has been shown in other studies using the hippocampus. Two hours after OGD, the partial recovery of ATP levels occurred simultaneously with an increase of cytochrome c (,400%) in the cytosol. When diazepam was included in the reoxygenation buffer, it completely prevented the increase in cytosolic cytochrome c. Thus, complete recovery of ATP and prevention of cytochrome c release from mitochondria can be achieved when diazepam is given after the loss of ATP induced by OGD. [source] Mitochondrial dysfunction in a neural cell model of spinal muscular atrophyJOURNAL OF NEUROSCIENCE RESEARCH, Issue 12 2009Gyula Acsadi Abstract Mutations of the survival motor neuron (SMN) gene in spinal muscular atrophy (SMA) lead to anterior horn cell death. The cause is unknown, but motor neurons depend substantially on mitochondrial oxidative phosphorylation (OxPhos) for normal function. Therefore, mitochondrial parameters were analyzed in an SMA cell culture model using small interfering RNA (siRNA) transfection that decreased Smn expression in NSC-34 cells to disease levels. Smn siRNA knock-down resulted in 35% and 66% reduced Smn protein levels 48 and 72 hr posttransfection, respectively. ATP levels were reduced by 14% and 26% at 48 and 72 hr posttransfection, respectively, suggesting decreased ATP production or increased energy demand in neural cells. Smn knock-down resulted in increased mitochondrial membrane potential and increased free radical production. Changes in activity of cytochrome c oxidase (CcO), a key OxPhos component, were observed at 72 hr with a 26% increase in oxygen consumption. This suggests a compensatory activation of the aerobic pathway, resulting in increased mitochondrial membrane potentials, a condition known to lead to the observed increase in free radical production. Further testing suggested that changes in ATP at 24 hr precede observable indices of cell injury at 48 hr. We propose that energy paucity and increased mitochondrial free radical production lead to accumulated cell damage and eventual cell death in Smn-depleted neural cells. Mitochondrial dysfunction may therefore be important in SMA pathology and may represent a new therapeutic target. © 2009 Wiley-Liss, Inc. [source] Mercury compounds disrupt neuronal glutamate transport in cultured mouse cerebellar granule cellsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 4 2005Elena Fonfría Abstract Cerebellar granule cells are targeted selectively by mercury compounds in vivo. Despite the affinity of mercury for thiol groups present in all cells, the molecular determinant(s) of selective cerebellar degeneration remain to be elucidated fully. We studied the effect of mercury compounds on neuronal glutamate transport in primary cultures of mouse cerebellar granule cells. Immunoblots probed with an antibody against the excitatory amino acid transporter (EAAT) neuronal glutamate transporter, EAAT3, revealed the presence of a specific band in control and mercury-treated cultures. Micromolar concentrations of both methylmercury and mercuric chloride increased the release of endogenous glutamate, inhibited glutamate uptake, reduced mitochondrial activity, and decreased ATP levels. All these effects were completely prevented by the nonpermeant reducing agent Tris-(2-carboxyethyl)phosphine (TCEP). Reduction of mitochondrial activity by mercuric chloride, but not by methylmercury, was inhibited significantly by 4,4,-diisothiocyanato-stilbene-2,2,-disulfonic acid (DIDS) and by reduced extracellular Cl, ion concentration. In addition, DIDS and low extracellular Cl, completely inhibited the release of glutamate induced by mercuric chloride, and produced a partial although significant reduction of that induced by methylmercury. We suggest that a direct inhibition of glutamate uptake triggers an imbalance in cell homeostasis, leading to neuronal failure and Cl, -regulated cellular glutamate efflux. Our results demonstrate that neuronal glutamate transport is a novel target to be taken into account when assessing mercury-induced neurotoxicity. © 2005 Wiley-Liss, Inc. [source] Exogenous melatonin enhances bile flow and ATP levels after cold storage and reperfusion in rat liver: implications for liver transplantationJOURNAL OF PINEAL RESEARCH, Issue 4 2005Mariapia Vairetti Abstract:, ,Although the use of melatonin in the transplantation field has been suggested, it has not been previously tested in a liver cold-storage model. We used a rat liver model to study (a) the dose-dependent effect of melatonin on bile production, and (b) the potential of melatonin to improve liver function after cold-storage. Male Wistar rats were perfused with Krebs,Henseleit bicarbonate buffer (KHB) at 37°C without or with 25, 50, 100 and 200 ,m melatonin. Each dose of melatonin stimulated bile production. For cold-storage studies, livers were flushed with either University of Wisconsin (UW) or Celsior solution and stored for 20 hr at 4°C. Reperfusion (120 min) was performed with KHB at 37°C. In subsequent studies, 100 ,m melatonin were added to the perfusate during the reperfusion period. ATP and melatonin levels in the tissue were measured. Bile analysis was performed by measuring melatonin, bilirubin and gamma-glutamyl transpeptidase (, -GT) levels in the fluid. A dose-dependent increase in bile secretion, associated with an enhanced melatonin and bilirubin levels in the bile were observed. Also, tissue levels of melatonin increased in a dose-dependent manner. When melatonin was added during the reperfusion period, bile production and bile bilirubin levels increased both with UW and Celsior solutions. The analysis of , -GT in the bile showed an increase in the Celsior-preserved liver and the addition of melatonin to the perfusate reduced this effect. Tissue ATP levels were higher when melatonin was added to the perfusion medium. Higher levels of melatonin in bile than in tissue were found. In conclusion, we demonstrate that melatonin improves significantly the restoration of liver function after cold-storage and reperfusion. [source] S-Adenosyl-L-Methionine Co-administration Prevents the Ethanol-Elicited Dissociation of Hepatic Mitochondrial Ribosomes in Male RatsALCOHOLISM, Issue 1 2009Peter Sykora Background:, Chronic ethanol feeding to male rats has been shown to result in decreased mitochondrial translation, depressed respiratory complex levels and mitochondrial respiration rates. In addition, ethanol consumption has been shown to result in an increased dissociation of mitoribosomes. S-adenosyl-L-methionine (SAM) is required for the assembly and subsequent stability of mitoribosomes and is depleted during chronic ethanol feeding. The ability of dietary SAM co-administration to prevent these ethanol-elicited lesions was investigated. Methods:, Male Sprague-Dawley rats were fed a nutritionally adequate liquid diet with ethanol comprising 36% of the calories according to a pair-fed design for 28 days. For some animals, SAM was supplemented in the diet at 200 mg/l. Liver mitochondria were prepared and mitoribosomes isolated. Respiration rates, ATP levels, respiratory complex levels, and the extent of mitoribosome dissociation were determined. Results:, Twenty-eight days of ethanol feeding were found to result in decreased SAM content, depressed respiration, and increased mitoribosome dissociation. No changes in mitochondrial protein content; levels of respiratory complexes I, III, and V; complex I activities; and ATP levels were detected. Co-administration of SAM in the diet was found to prevent ethanol-induced SAM depletion, respiration decreases and mitoribosome dissociation. Conclusions:, Taken together, these findings suggest (1) that mitoribosome dissociation precedes respiratory complex depressions in alcoholic animals and (2) that dietary supplementation of SAM prevents some of the early mitochondrial lesions associated with chronic ethanol consumption. [source] | |||||||||||||||||||||||||||||||