Adenosine Triphosphate Levels In Cells Research Articles

Mitochondria fission accentuates oxidative stress in hyperglycemia-induced H9c2 cardiomyoblasts in vitro by regulating fatty acid oxidation.

Oxidative stress plays a pivotal role in the development of diabetic cardiomyopathy (DCM). Previous studies have revealed that inhibition of mitochondrial fission suppressed oxidative stress and alleviated mitochondrial dysfunction and cardiac dysfunction in diabetic mice. However, no research has confirmed whether mitochondria fission accentuates hyperglycemia-induced cardiomyoblast oxidative stress through regulating fatty acid oxidation (FAO). We used H9c2 cardiomyoblasts exposed to high glucose (HG) 33 mM to simulate DCM in vitro. Excessive mitochondrial fission, poor cell viability, and lipid accumulation were observed in hyperglycemia-induced H9c2 cardiomyoblasts. Also, the cells were led to oxidative stress injury, lower adenosine triphosphate (ATP) levels, and apoptosis. Dynamin-related protein 1 (Drp1) short interfering RNA(siRNA) decreased targeted marker expression, inhibited mitochondrial fragmentation and lipid accumulation, suppressed oxidative stress, reduced cardiomyoblast apoptosis, and improved cell viability and ATP levels in HG-exposed H9c2 cardiomyoblasts, but not in carnitine palmitoyltransferase 1 (CPT1) inhibitor etomoxir treatment cells. We also found subcellular localization of CPT1 on the mitochondrial membrane, FAO, and levels of nicotinamide adenine dinucleotide phosphate(NADPH) were suppressed after exposure to HG treatment, whereas Drp1 siRNA normalized mitochondrial CPT1, FAO, and NADPH. However, the blockade of FAO with etomoxir abolished the above effects of Drp1 siRNA in hyperglycemia-induced H9c2 cardiomyoblasts. The preservation of mitochondrial function through the Drp1/CPT1/FAO pathway is the potential mechanism of inhibited mitochondria fission in attenuating oxidative stress injury of hyperglycemia-induced H9c2 cardiomyoblasts.

Biocompatible aggregation-induced emission active polyphosphate-manganese nanosheets with glutamine synthetase-like activity in excitotoxic nerve cells

Glutamine synthetase (GS) is vital in maintaining ammonia and glutamate (Glu) homeostasis in living organisms. However, the natural enzyme relies on adenosine triphosphate (ATP) to activate Glu, resulting in impaired GS function during ATP-deficient neurotoxic events. To date, no reports demonstrate using artificial nanostructures to mimic GS function. In this study, we synthesize aggregation-induced emission active polyP-Mn nanosheets (STPE-PMNSs) based on end-labeled polyphosphate (polyP), exhibiting remarkable GS-like activity independent of ATP presence. Further investigation reveals polyP in STPE-PMNSs serves as phosphate source to activate Glu at low ATP levels. This self-feeding mechanism offers a significant advantage in regulating Glu homeostasis at reduced ATP levels in nerve cells during excitotoxic conditions. STPE-PMNSs can effectively promote the conversion of Glu to glutamine (Gln) in excitatory neurotoxic human neuroblastoma cells (SH-SY5Y) and alleviate Glu-induced neurotoxicity. Additionally, the fluorescence signal of nanosheets enables precise monitoring of the subcellular distribution of STPE-PMNSs. More importantly, the intracellular fluorescence signal is enhanced in a conversion-responsive manner, allowing real-time tracking of reaction progression. This study presents a self-sustaining strategy to address GS functional impairment caused by ATP deficiency in nerve cells during neurotoxic events. Furthermore, it offers a fresh perspective on the potential biological applications of polyP-based nanostructures.

Co-delivery of Paclitaxel/Atovaquone/Quercetin to regulate energy metabolism to reverse multidrug resistance in ovarian cancer by PLGA-PEG nanoparticles

Ovarian cancer is a malignant tumor that seriously endangers the lives of women, with chemotherapy being the primary clinical treatment. However, chemotherapy encounters the problem of generating multidrug resistance (MDR), mainly due to drug efflux induced by P-glycoprotein (P-gp), which decreases intracellular accumulation of chemotherapeutic drugs. The drugs efflux mediated by P-gp requires adenosine triphosphate (ATP) hydrolysis to provide energy. Therefore, modulating energy metabolism pathways and inhibiting ATP production may be a potential strategy to reverse MDR. Herein, we developed a PTX-ATO-QUE nanoparticle (PAQNPs) based on a PLGA-PEG nanoplatform capable of loading the mitochondrial oxidative phosphorylation (OXPHOS) inhibitor atovaquone (ATO), the glycolysis inhibitor quercetin (QUE), and the chemotherapeutic drug paclitaxel (PTX) to reverse MDR by inhibiting energy metabolism through multiple pathways. Mechanistically, PAQNPs could effectively inhibit the OXPHOS and glycolytic pathways of A2780/Taxol cells by suppressing the activities of mitochondrial complex III and hexokinase II (HK II), respectively, ultimately decreasing intracellular ATP levels in tumor cells. Energy depletion can effectively inhibit cell proliferation and reduce P-gp activity, increasing the chemotherapeutic drug PTX accumulation in the cells. Moreover, intracellular reactive oxygen species (ROS) is increased with PTX accumulation and leads to chemotherapy-resistant cell apoptosis. Furthermore, PAQNPs significantly inhibited tumor growth in the A2780/Taxol tumor-bearing NCG mice model. Immunohistochemical (IHC) analysis of tumor tissues revealed that P-gp expression was suppressed, demonstrating that PAQNPs are effective in reversing MDR in tumors by inducing energy depletion. In addition, the safety study results, including blood biochemical indices, major organ weights, and H&E staining images, showed that PAQNPs have a favorable in vivo safety profile. In summary, the results suggest that the combined inhibition of the two energy pathways, OXPHOS and glycolysis, can enhance chemotherapy efficacy and reverse MDR in ovarian cancer.

Mitochondrial protein isoleucyl-tRNA synthetase 2 in tumor cells as a potential therapeutic target for cervical cancer.

Isoleucyl-tRNA synthetase 2 (IARS2) is crucial for mitochondrial activity and function in cancer cells. Cervical cancer is a highly prevalent malignancy affecting the female reproductive system on a global scale. This research investigates the expression and potential roles of IARS2 in cervical cancer cells. Initially, we examined the IARS2 expression profile in cervical cancer cells using Western blot technique and quantitative reverse transcription polymerase chain reaction methodologies. Subsequently, cervical cancer cell models with IARS2 silencing and overexpression were constructed using Short Hairpin RNA (ShRNA) (IARS2) and pcMV-FLAG-IARS2, respectively. The impact of IARS2 silencing or overexpression on Hela cell mitochondrial membrane potential, mitochondrial complex I, adenosine triphosphate (ATP) levels, reactive oxygen species activity, viability, proliferation, migration, apoptosis-related proteins, and apoptosis levels was examined through fluorescence staining, enzyme-linked immunosorbent assay, cell counting kit-8 assay, Transwell experiments, Western blot technique, and Terminal deoxynucleotidyl transferase dUTP nick end labeling assay techniques. The expression of IARS2 is upregulated in cervical cancer cells. Silencing IARS2 with ShRNA (IARS2) disrupts mitochondrial function in cervical cancer cells, resulting in mitochondrial depolarization, heightened oxidative stress, suppression of mitochondrial complex I, and a decrease in ATP levels. Moreover, the depletion of IARS2 significantly impedes the viability, proliferation, and migration of cervical cancer cells, inducing apoptotic processes. In contrast, the overexpression of IARS2 augments the proliferation, migration, and ATP levels in cervical cancer cells. IARS2 plays a pivotal role as a mitochondrial protein in fostering the growth of cervical cancer cells, presenting itself as an innovative target for tumor diagnosis and treatment.

Heme oxygenase‑1 inhibits renal tubular epithelial cell pyroptosis by regulating mitochondrial function through PINK1.

Endotoxin-induced acute kidney injury (AKI) is commonly observed in clinical practice. Renal tubular epithelial cell (RTEC) pyroptosis is one of the main factors leading to the development of endotoxin-induced AKI. Mitochondrial dysfunction can lead to pyroptosis. However, the biological pathways involved in the potential lipopolysaccharide (LPS)-induced pyroptosis of RTECs, notably those associated with mitochondrial dysfunction, are poorly understood. Previous studies have demonstrated that heme oxygenase (HO)-1 confers cell protection via the induction of PTEN-induced putative kinase 1 (PINK1) expression through PTEN to regulate mitochondrial fusion/fission during endotoxin-induced AKI in vivo. Therefore, the present study investigated the role of HO-1/PINK1 in maintaining mitochondrial function and inhibiting the pyroptosis of RTECs exposed to LPS. Primary cultures of RTECs were obtained from wild-type (WT) and PINK1-knockout (PINK1KO) rats. An in vitro model of endotoxin-associated RTEC injury was established following treatment of the cells with LPS. The WT RTECs were divided into the control, LPS, Znpp + LPS and Hemin + LPS groups, and the PINK1KO RTECs were divided into the control, LPS and Hemin + LPS groups. RTECs were exposed to LPS for 6 h to assess cell viability, inflammation, pyroptosis and mitochondrial function. In the LPS-treated RTECs, the mRNA and protein expression levels of HO-1 and PINK1 were upregulated. Cell viability, adenosine triphosphate (ATP) levels and the mitochondrial oxygen consumption rate were decreased, whereas the inflammatory response, pyroptosis and mitochondrial reactive oxygen species (ROS) levels were increased. The cell inflammatory response and the induction of pyroptosis were inhibited, whereas the levels of mitochondrial ROS were decreased. In addition, the cell viability and ATP levels were increased in the WT RTECs following the upregulation of HO-1 expression. These effects were reversed by the downregulation of HO-1 expression. However, no statistically significant differences were noted between the LPS and the Hemin + LPS groups in the PINK1KO RTECs. Collectively, the findings of the present study indicate that HO-1 inhibits inflammation and regulates mitochondrial function by inhibiting the pyroptosis of LPS-exposed RTECs via PINK1.

KIAA0101 knockdown inhibits glioma progression and glycolysis by inactivating the PI3K/AKT/mTOR pathway.

KIAA0101, a proliferating cell nuclear antigen (PCNA)-associated factor, is reported to be overexpressed and identified as an oncogene in several human malignancies. The purpose of this study is to determine the function and possible mechanism of KIAA0101 in glioma progression. KIAA0101 expression in glioma patients was analyzed by GSE50161 and GEPIA datasets. Kaplan-Meier survival analysis was used to evaluate the survival distributions. KIAA0101 expression in glioma cells were detected by qRT-PCR and western blot analyses. The function of KIAA0101 was investigated using MTT, flow cytometry, caspase-3 activity, and Transwell assays. Additionally, glycolytic flux was determined by measuring extracellular acidification rate (ECAR), glucose consumption, lactate production, and adenosine triphosphate (ATP) level. The changes of phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway were detected by western blot analysis. Results showed that KIAA0101 was upregulated in glioma tissues and cells. High KIAA0101 expression predicted a poor prognosis in glioma patients. KIAA0101 depletion impeded cell proliferation, migration, and invasion and triggered apoptosis in glioma cells. KIAA0101 silencing reduced the ECAR, glucose consumption, lactate production, and ATP level in glioma cells, suggesting that KIAA0101 knockdown inhibited glycolysis in glioma cells. Mechanistically, KIAA0101 knockdown inhibited the PI3K/AKT/mTOR pathway. In conclusion, KIAA0101 silencing inhibited glioma progression and glycolysis by inactivating the PI3K/AKT/mTOR pathway.

Exosomal microRNA-503-3p derived from macrophages represses glycolysis and promotes mitochondrial oxidative phosphorylation in breast cancer cells by elevating DACT2

MicroRNAs (miRNAs) are emerging drivers in tumor progression, while the role of miR-503-3p in breast cancer (BC) remains largely unknown. We aimed to explore the impact of macrophage-derived exosomal miR-503-3p in the development of BC by regulating disheveled-associated binding antagonist of beta-catenin 2 (DACT2). miR-503-3p and DACT2 expression in BC tissues and cells was assessed, and the expression of Wnt/β-catenin signaling pathway-related proteins in BC cells was also evaluated. Macrophages were induced and exosomes were extracted. The screened BC cell lines were, respectively, treated with exosomes, miR-503-3p inhibitor/mimic or upregulated/inhibited DACT2, and then the phenotypes, glucose intake, oxygen consumption rate, and adenosine-triphosphate (ATP) level of BC cells were determined. Cell growth in vivo was also observed. MiR-503-3p was elevated, DACT2 was reduced, and Wnt/β-catenin signaling pathway was activated in BC cells. Macrophage-derived exosomes, upregulated miR-503-3p or inhibited DACT2 promoted malignant behaviors of BC cells, glucose intake, and activity of the Wnt/β-catenin signaling pathway, while repressed oxygen consumption rate and ATP level in BC cells. Reversely, reduced miR-503-3p or upregulated DACT2 exerted opposite effects. This study revealed that reduction of macrophage-derived exosomal miR-503-3p repressed glycolysis and promoted mitochondrial oxidative phosphorylation in BC by elevating DACT2 and inactivating Wnt/β-catenin signaling pathway. Our research may provide novel targets for BC treatment.

Sensory neurons derived from diabetic rats exhibit deficits in functional glycolysis and ATP that are ameliorated by IGF-1

ObjectiveThe distal dying-back of the longest nerve fibres is a hallmark of diabetic neuropathy, and impaired provision of energy in the form of adenosine triphosphate (ATP) may contribute to this neurodegenerative process. We hypothesised that energy supplementation via glycolysis and/or mitochondrial oxidative phosphorylation is compromised in cultured dorsal root ganglion (DRG) sensory neurons from diabetic rodents, thus contributing to axonal degeneration. Functional analysis of glycolysis and mitochondrial respiration and real-time measurement of ATP levels in live cells were our specific means to test this hypothesis. MethodsDRG neuron cultures from age-matched control or streptozotocin (STZ)-induced type 1 diabetic rats were used for in vitro studies. Three plasmids containing ATP biosensors of varying affinities were transfected into neurons to study endogenous ATP levels in real time. The Seahorse XF analyser was used for glycolysis and mitochondrial respiration measurements. ResultsFluorescence resonance energy transfer (FRET) efficiency (YFP/CFP ratio) of the ATP biosensors AT1.03 (low affinity) and AT1.03YEMK (medium affinity) were significantly higher than that measured using the ATP-insensitive construct AT1.03R122/6K in both cell bodies and neurites of DRG neurons (p < 0.0001). The ATP level was homogenous along the axons but higher in cell bodies in cultured DRG neurons from both control and diabetic rats. Treatment with oligomycin (an ATP synthase inhibitor in mitochondria) decreased the ATP levels in cultured DRG neurons. Likewise, blockade of glycolysis using 2-deoxy-d-glucose (2-DG: a glucose analogue) reduced ATP levels (p < 0.001). Cultured DRG neurons derived from diabetic rats showed a diminishment of ATP levels (p < 0.01), glycolytic capacity, glycolytic reserve and non-glycolytic acidification. Application of insulin-like growth factor-1 (IGF-1) significantly elevated all the above parameters in DRG neurons from diabetic rats. Oligomycin pre-treatment of DRG neurons, to block oxidative phosphorylation, depleted the glycolytic reserve and lowered basal respiration in sensory neurons derived from control and diabetic rats. Depletion was much higher in sensory neurons from diabetic rats compared to control rats. In addition, an acute increase in glucose concentration, in the presence or absence of oligomycin, elevated parameters of glycolysis by 1.5- to 2-fold while having no impact on mitochondrial respiration. ConclusionWe provide the first functional evidence for decreased glycolytic capacity in DRG neurons derived from type 1 diabetic rats. IGF-1 protected against the loss of ATP supplies in DRG cell bodies and axons in neurons derived from diabetic rats by augmenting various parameters of glycolysis and mitochondrial respiration.

Mitochonic acid 5 regulates mitofusin 2 to protect microglia

Microglial apoptosis is associated with neuroinflammation and no effective strategies are currently available to protect microglia against inflammation-induced apoptosis. Mouse microglial BV-2 cells (5 × 106) were incubated with 10 μg/mL lipopolysaccharides for 12 hours to mimic an inflammatory environment. Then the cells were co-cultured with mitochonic acid 5 (MA-5) for another 12 hours. MA-5 improved the survival of lipopolysaccharide-exposed cells. MA-5 decreased the activity of caspase-3, which is associated with apoptosis. MA-5 reduced the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells, and increased adenosine triphosphate levels in cells. MA-5 decreased the open state of the mitochondrial permeability transition pore and reduced calcium overload and diffusion of second mitochondria-derived activator of caspase (Smac). MA-5 decreased the expression of apoptosis-related proteins (mitochondrial Smac, cytoplasmic Smac, pro-caspase-3, cleaved-caspase-3, and caspase-9), and increased the levels of anti-apoptotic proteins (Bcl2 and X-linked inhibitor of apoptosis protein), mitochondria-related proteins (mitochondrial fusion protein 2, mitochondrial microtubule-associated proteins 1A/1B light chain 3B II), and autophagy-related proteins (Beclin1, p62 and autophagy related 5). However, MA-5 did not promote mitochondrial homeostasis or decrease microglial apoptosis when Mitofusin 2 expression was silenced. This shows that MA-5 increased Mitofusin 2-related mitophagy, reversed cellular energy production and maintained energy metabolism in BV-2 cells in response to lipopolysaccharide-induced inflammation. These findings indicate that MA-5 may promote the survival of microglial cells via Mitofusin 2-related mitophagy in response to lipopolysaccharide-induced inflammation.

Phase 1 Multiple Ascending Dose Study of Safety, Tolerability, and Pharmacokinetics/Pharmacodynamics of Mitapivat (AG-348) in Subjects with Sickle Cell Disease

Phase 1 Multiple Ascending Dose Study of Safety, Tolerability, and Pharmacokinetics/Pharmacodynamics of Mitapivat (AG-348) in Subjects with Sickle Cell Disease

In vitro neuroprotective potential of Clivia miniata and Nerine humilis (Amaryllidaceae) in MPP+-induced neuronal toxicity in SH-SY5Y neuroblastoma cells

Parkinson's disease (PD) is one of the most common neurodegenerative diseases (NDD) and mainly affects the ageing population. A significant feature of PD pathogenesis is the progressive loss of dopaminergic neurons in the substantia nigra pars compacta part of the midbrain in affected persons. This neuronal loss occurs partly due to the increased generation of reactive oxygen species (ROS) in the mitochondrial and the depletion of adenosine triphosphate (ATP) in affected neurons. In this study, the methanolic extracts of Clivia miniata (CME) and Nerine humilis (NHE) belonging to the plant family Amaryllidaceae, were investigated for their neuroprotective potential in MPP+-induced neurotoxicity in SH-SY5Y neuroblastoma cells. Cell viability was determined using the 3-(4,5-dimethyithiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and cell morphology was analysed using light microscopy. Furthermore, the effects of the extracts on apoptosis and ATP production were investigated using caspase 3/7 apoptosis kit and the Promega Mitochondrial ToxGlo ATP assay kit respectively. Additionally, the antioxidant contents of extracts were evaluated using routine laboratory procedures. The results show that pre-treatment of cells with the extracts at 2 and 4 μg/mL concentrations improved cell viability as well as cell morphology by inhibiting the toxicity induced by MPP+. The extracts also improved ATP levels in cells and attenuated the apoptosis induced by MPP+. Furthermore, antioxidant assays showed that both extracts had low antioxidant activity. Findings from this study indicate that CME and NHE may be promising as neuroprotective agents for PD and warrant further investigation to determine the bioactive components of the plants that may be responsible for the observed effects.

KUS121, a valosin-containing protein modulator, attenuates ischemic stroke via preventing ATP depletion

Reduced adenosine triphosphate (ATP) levels in ischemic stroke constitute an upstream contributor to neuronal cell death. We have recently created a small chemical, named Kyoto University Substance 121 (KUS121), which can reduce cellular ATP consumption. In this study, we examined whether KUS121 has neuroprotective effects in rodent cerebral ischemia models. We evaluated cell viability and ATP levels in vitro after oxygen glucose deprivation (OGD) in rat cortical primary neuronal cultures incubated with or without KUS121. We found that KUS121 protected neurons from cell death under OGD by preventing ATP depletion. We also used in vivo ischemic stroke models of transient distal middle cerebral artery occlusion in C57BL/6 and B-17 mice. Administration of KUS121 in these models improved functional deficits and reduced brain infarction volume after transient focal cerebral ischemia in both C57BL/6 and B-17 mice. These results indicate that KUS121 could be a novel type of neuroprotective drug for ischemic stroke.

The Effect of Adenosine on Extracellular Matrix Production in Porcine Intervertebral Disc Cells

Compressive loading promotes adenosine triphosphate (ATP) production and release by intervertebral disc (IVD) cells. Extracellular ATP can be rapidly hydrolyzed by ectonucleotidases. Adenosine, one of the adenine derivatives of ATP hydrolysis, can modulate diverse cellular actions via adenosine receptors. The objectives of this study were to investigate the effects of exogenous adenosine on the production of extracellular matrix (ECM; i.e., collagen type II and aggrecan) and ATP of IVD cells and explore the underlying mechanism of action. It was found that adenosine treatment significantly upregulated aggrecan and type II collagen gene expression and the ATP level in IVD cells. Dipyridamole, an adenosine transport blocker, completely suppressed the effects of adenosine on the ATP production and ECM gene expression of the IVD cells, whereas antagonists of adenosine receptors did not significantly affect adenosine-treated IVD cells. The findings suggested that elevated intracellular ATP and upregulation of ECM gene expression by adenosine treatment are mainly due to adenosine uptake rather than receptor activation. Since ECM biosynthesis is a high ATP demanding process, supplementing adenosine could be beneficial as IVD cells are able to utilize it to replenish intracellular ATP and sequentially promote ECM production, which is constantly suppressed by limited nutrition supply due to the avascular nature of the IVD.

GRP75 overexpression rescues frataxin deficiency and mitochondrial phenotypes in Friedreich ataxia cellular models.

Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disease caused by the deficiency of frataxin, a mitochondrial protein crucial for iron-sulfur cluster biogenesis and adenosine triphosphate (ATP) production. Currently, there is no therapy to slow down the progression of FRDA. Recent evidence indicates that posttranslational regulation of residual frataxin levels can rescue some of the functional deficit of FRDA, raising the possibility of enhancing levels of residual frataxin as a treatment for FRDA. Here, we present evidence that mitochondrial molecular chaperone GRP75, also known as mortalin/mthsp70/PBP74, directly interacts with frataxin both in vivo in mouse cortex and in vitro in cortical neurons. Overexpressing GRP75 increases the levels of both wild-type frataxin and clinically relevant missense frataxin variants in human embryonic kidney 293 cells, while clinical GRP75 variants such as R126W, A476T and P509S impair the binding of GRP75 with frataxin and the effect of GRP75 on frataxin levels. In addition, GRP75 overexpression rescues frataxin deficiency and abnormal cellular phenotypes such as the abnormal mitochondrial network and decreased ATP levels in FRDA patient-derived cells. The effect of GRP75 on frataxin might be in part mediated by the physical interaction between GRP75 and mitochondrial processing peptidase (MPP), which makes frataxin more accessible to MPP. As GRP75 levels are decreased in multiple cell types of FRDA patients, restoring GRP75 might be effective in treating both typical FRDA patients with two guanine-adenine-adenine repeat expansions and compound heterozygous patients with point mutations.

Avian reovirus σA-modulated suppression of lactate dehydrogenase and upregulation of glutaminolysis and the mTOC1/eIF4E/HIF-1α pathway to enhance glycolysis and the TCA cycle for virus replication.

Adenosine triphosphate (ATP) is an energy source for many types of viruses for facilitating virus replication. This is the first report to demonstrate that the structural protein σA of avian reovirus (ARV) functions as an activator of cellular energy. Three cellular factors, isocitrate dehydrogenase 3 subunit beta (IDH3B), lactate dehydrogenase A (LDHA), and vacuolar-type H+-ATPase (vATPase) co-immunoprecipitated with ARV σA and were identified by 2D-LC/MS/MS. ARV enhances glycolytic flux through upregulation of glycolytic enzymes. Increased ATP levels in both ARV-infected and σA-transfected cells were observed by a fluorescence resonance energy transfer-based genetically encoded indicator, Ateams. Furthermore, σA upregulates IDH3B and glutamate dehydrogenase (GDH) to promote glutaminolysis, activating HIF-1α. Both HIF-1α level and viral yield in IDH3B-depleted and glutamine-deprived cells, and inhibition of glutaminolysis was significantly reduced. The σAR155/273A mutant loses its ability to enter the nucleolus, impairing its ability to regulate glycolysis. In addition, we have identified the conserved untranslated regions (UTR) of the 5'- and 3'-termini of the ARV genome segments that are required for viral protein synthesis in an ATP-dependent manner. Deletion of either the 5'- or 3'-UTR impaired viral protein synthesis. Knockdown of σA reduced the ATP level and significantly decreased virus yield, suggesting that σA enhances ATP formation to promote virus replication. Collectively, this study provides novel insights into σA-modulated suppression of LDHA and activation of IDH3B and GDH to activate the mTORC1/eIF4E/HIF-1α pathways to upregulate glycolysis and the TCA cycle for virus replication.

Relationship between post-thaw adenosine triphosphate content, motility and viability in cryopreserved bovine semen applying two different preservation methods.

Motility and energy level in sperm cells are tightly linked, but not totally understood. The aim of this study was to examine whether adenosine triphosphate (ATP) content as a sperm quality parameter for bull semen could give additional information together with viability and motility. The objective was therefore to examine the relationships between alterations in sperm ATP content, motility and viability in bovine semen samples immediately after thawing and following post-thaw incubation at physiological temperature. Two different cryopreservation methods were compared. Ejaculates from ten young bulls were split into two and cryopreserved using conventional procedure with Biladyl® (B) extender and with SpermVital® (SV) immobilization technology. From each sample, simultaneous analysis of ATP content, motility and viability was performed post-thaw (T0) and after incubation at physiological temperature for three hours (T3). Multivariate correlation analysis showed high correlation at T0 between ATP content and viability (p<0.05), ATP and total motility (p<0.05), as well as progressive motility and viability (p<0.05). However, there was no significant correlation between progressive motility and ATP content at T3, neither for B nor SV semen. We conclude that both preservation method and post-thaw incubation at physiological temperature affect ATP level in bull sperm cells partly independent of motility and viability. The ATP level of bovine spermatozoa post-thaw is therefore implicated to give supplementary information of sperm quality.

Mild therapeutic hypothermia protects against cerebral ischemia/reperfusion injury by inhibiting miR-15b expression in rats.

OBJECTIVE:Mild hypothermia has a protective effect on ischemic stroke, but the mechanisms remain elusive. Here, we investigated microRNA (miRNA) profiles and the specific role of miRNAs in ischemic stroke treated with mild hypothermia.MATERIALS AND METHODS:Male adult Sprague Dawley rats were subjected to focal transient cerebral ischemia. Mild hypothermia was induced by applying ice packs around the neck and head of the animals. miRNAs expression profiles were detected in ischemic stroke treated with mild therapeutic hypothermia through miRNA chips. Reverse transcription-polymerase chain reaction (RT-PCR) was used to verify the change of miRNA array. Western blot and adenosine triphosphate (ATP) assay kits were used to detect the changes of protein expression and ATP levels, respectively. miR-15b mimic and its control were injected into the right lateral ventricle 60 min before the induction of ischemia.RESULTS:The results showed that mild hypothermia affected miRNAs profiles expression. We verified the expression of miR-15b and miR-598-3p by miRNA RT-PCR. miR-15b mimic inhibited the expression of its target, ADP ribosylation factor-like 2 (Arl2) protein, and decreased ATP levels in PC12 cells. Compared with the control, miR-15b mimic increased the infarct volume and aggravated the neurological function under normothermia or hypothermia treatment. Furthermore, the expression of Arl2 was decreased in the miR-15b mimic group under normothermia or hypothermia treatment.CONCLUSIONS:Mild therapeutic hypothermia affected miRNA profiles and protected against cerebral ischemia/reperfusion by inhibiting miR-15b expression in rats. miR-15b may be a potential target for therapeutic intervention in stroke.

Hypoxia-induced tumor cell resistance is overcome by synergistic GAPDH-siRNA and chemotherapy co-delivered by long-circulating and cationic-interior liposomes.

Chemotherapeutic drug resistance of tumor cells under hypoxic conditions is caused by the inhibition of apoptosis by autophagy and drug efflux via adenosine triphosphate (ATP)-dependent transporter activation, among other factors. Here, we demonstrate that disrupting glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression can reduce the autophagy and ATP levels in tumor cells. To test whether GAPDH knockdown is sufficient to overcome drug resistance, a nanocarrier (asymmetry-membrane liposome) was designed to encapsulate GAPDH-siRNA with a low dose of paclitaxel (PTX). Liposomes were prepared using novel cryogenic inner-outer dual reverse phase emulsion liposome manufacturing technology to obtain a high loading of siRNA. The results of dynamic light scattering (DLS) indicated that the liposomes had an average hydrodynamic diameter of 250.5 nm and polydispersity index (PDI) of 0.210, which was confirmed by (Transmission Electron Microscope) TEM images. In in vitro tests, the siRNA liposomes presented a high specificity in the suppression of GAPDH expression and significant synergy in cytotoxicity with co-delivery of PTX against tumor cells (HeLa and MCF-7) under hypoxic conditions. Moreover, in vivo studies (a HeLa tumor xenograft model using female BALB/c nude mice) demonstrate that the liposomes could not only increase the concentration of drugs in tumors over time but also successfully boosted the chemotherapeutic efficacy of PTX (synergistic therapy with GAPDH-siRNA). Tumor cells appeared to lose their resistance against PTX therapy, becoming more sensitive to PTX when GAPDH-siRNA was simultaneously administered in long-circulating liposomes. Consequently, the novel delivery of GAPDH-siRNA using nanotargeted liposomes provides a useful and potential tool to overcome multidrug resistant (MDR) tumors and presents a bright prospect compared with the traditional chemotherapeutic strategies in clinic cancer therapy.

Safety, Tolerability, Pharmacokinetics and Pharmacodynamics of Multiple Doses of AG-519, an Allosteric Activator of Pyruvate Kinase-R, in Healthy Subjects

BACKGROUNDPyruvate kinase (PK) deficiency is a congenital hemolytic anemia caused by deficiency of the glycolytic enzyme red cell PK (PK-R) due to mutations in the PKLR gene. PK catalyzes the last enzymatic step in the glycolytic pathway and is the main source of adenosine triphosphate (ATP) production in red blood cells. PKLR mutations lead to defective proteins that are hypothesized to reduce ATP levels in red cells, leading to hemolysis. Small molecule allosteric activation of PK-R resulting in increases in ATP and decreases in 2,3-diphosphoglycerate (2,3-DPG) in healthy volunteers has been observed with an earlier molecule, AG-348, the first small molecule PK-R activator to enter clinical trials (Yang et al. EHA 2015, S138). AG-519 is the second small molecule PK-R activator to enter clinical trials. AG-519 is a potent, highly selective and orally bioavailable PK-R activator devoid of the aromatase inhibitory effects that were observed with AG-348.AIMSAG-519 is currently being tested in a randomized, double-blind, phase 1 study in healthy volunteers (NCT02630927), with the objective of identifying a safe and pharmacodynamically active dose and schedule to support potential ongoing development in patients with PK deficiency. Here we report the first 4 cohorts of the multiple ascending dose (MAD) phase of this study. The single ascending dose (SAD) phase of the study and the first two cohorts of the MAD phase of the study have been reported previously (Barbier et al. EHA 2016, P752).METHODSHealthymen and women (non-childbearing potential) aged 18-60 years who provided informed consent were eligible. The MAD phase of the study consisted of 5 dose cohorts. The dose levels administered were determined during interim data reviews of each completed MAD cohort, as well as data from completed SAD cohorts. At each dose level, 8 subjects were enrolled and randomized to receive AG-519 (n=6) or placebo (n=2) twice daily (BID; approximately every 12 hours) for 14 days. Safety assessments included adverse events (AEs), vital signs, electrocardiogram and clinical laboratory parameters. Serial blood samples were drawn to measure plasma concentrations of AG-519 and whole blood concentrations of 2,3-DPG and ATP for pharmacokinetic and pharmacodynamic (PD) assessments.RESULTSData are available for 32 subjects enrolled across 4 dose cohorts in the MAD phase of the study: 8 subjects each in cohort 1 (125 mg BID), cohort 2 (375 mg BID), cohort 3 (25 mg BID), and cohort 4 (300 mg BID). Blinded safety reviews indicated that multiple doses up to 375 mg have been well tolerated with no serious AEs or dose-limiting toxicities reported to date. One case of probable drug-induced Grade 2 thrombocytopenia was previously reported in 1 subject in the 375 mg cohort; the event was rapidly reversible with no clinical sequelae. The protocol was amended to require daily monitoring of platelets in subsequent cohorts and no other subjects have developed thrombocytopenia during treatment. The preliminary analysis of free testosterone and estradiol confirmed the absence of aromatase inhibitory activity.AG-519 steady-state was reached the third day after the first dose based on trough concentration values. The clearance of AG-519 after multiple doses was similar to that observed after single doses in the SAD cohorts. Dose-dependent increases in ATP in blood (Figure 1) and decreases in 2,3-DPG in blood correlated with dose-dependent increases in exposure of AG-519, with a peak effect at or below 375 mg BID. ATP response at 25 mg appears to be greater than 50% of maximal response. Results from the fifth MAD cohort, which evaluated the PD results with 10 mg BID, will be presented.ATP = adenosine triphosphate; BID = twice dailyCONCLUSIONAG-519 is well tolerated in healthy subjects at doses ranging from 25 mg to 375 mg BID for 14 days. The robust dose-dependent changes in ATP and 2,3-DPG concentrations in blood from healthy volunteers are consistent with increased activity of PK-R, the expected PD effect of AG-519. These data support the hypothesis that AG-519 may be able to enhance glycolytic activity in red cells of patients with PK deficiency to address the underlying cause of the disease. [Display omitted] DisclosuresBarbier:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Bodie:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Connor:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Merica:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Kung:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Le:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Yang:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Kosinski:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Silverman:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Yuan:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Bowden:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Cohen:Agios Pharmaceuticals, Inc.: Consultancy.

2-deoxy-D-glucose enhances anesthetic effects in mice.

The mechanisms of general anesthesia by volatile drugs remain largely unknown. Mitochondrial dysfunction and reduction in energy levels have been suggested to be associated with general anesthesia status. 2-Deoxy-D-glucose (2-DG), an analog of glucose, inhibits hexokinase and reduces cellular levels of adenosine triphosphate (ATP). 3-Nitropropionic acid is another compound which can deplete ATP levels. In contrast, idebenone and L-carnitine could rescue deficits of energy. We therefore sought to determine whether 2-DG and/or 3-nitropropionic acid can enhance the anesthetic effects of isoflurane, and whether idebenone and L-carnitine can reverse the actions of 2-DG. C57BL/6J mice (8 months old) received different concentrations of isoflurane with and without the treatments of 2-DG, 3-nitropropionic acid, idebenone, and L-carnitine. Isoflurane-induced loss of righting reflex (LORR) was determined in the mice. ATP levels in H4 human neuroglioma cells were assessed after these treatments. Finally, 31P-magnetic resonance spectroscopy was used to determine the effects of isoflurane on brain ATP levels in the mice. 2-DG enhanced isoflurane-induced LORR (P = 0.002, N = 15). 3-Nitropropionic acid also enhanced the anesthetic effects of isoflurane (P = 0.005, N = 15). Idebenone (idebenone + saline versus idebenone + 2-DG: P = 0.165, N = 15), but not L-carnitine (L-carnitine + saline versus L-carnitine + 2-DG: P < 0.0001, N = 15), inhibited the effects of 2-DG on enhancing isoflurane-induced LORR in the mice, as evidenced by 2-DG not enhancing isoflurane-induced LORR in the mice pretreated with idebenone. Idebenone (idebenone + saline versus idebenone + 2-DG: P = 0.177, N = 6), but not L-carnitine (L-carnitine + saline versus L-carnitine + 2-DG: P = 0.029, N = 6), also mitigated the effects of 2-DG on reducing ATP levels in cells, as evidenced by 2-DG not decreasing ATP levels in the cells pretreated with idebenone. Finally, isoflurane decreased ATP levels in both cultured cells and mouse brains (β-ATP: P = 0.003, N = 10; β-ATP/phosphocreatine: P = 0.006, N = 10; β-ATP/inorganic phosphate: P = 0.001, N = 10). These results from our pilot studies have established a system and generated a hypothesis that 2-DG enhances anesthetic effects via reducing energy levels. These findings should promote further studies to investigate anesthesia mechanisms.