scholarly journals Increased demand for NAD+ relative to ATP drives aerobic glycolysis

2020 ◽  
Author(s):  
Alba Luengo ◽  
Zhaoqi Li ◽  
Dan Y. Gui ◽  
Lucas B. Sullivan ◽  
Maria Zagorulya ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Mitochondrial Respiration ◽  
Nicotinamide Adenine Dinucleotide ◽  
Atp Hydrolysis ◽  
Aerobic Glycolysis ◽  
Pyruvate Dehydrogenase Complex ◽  
Atp Synthesis ◽  
Oxidation Reactions ◽  
Nadh Ratio ◽  
Metabolic Consequence

AbstractAerobic glycolysis, or preferential fermentation of glucose-derived pyruvate to lactate despite available oxygen, is a hallmark of proliferative metabolism that is observed across many organisms and conditions. To better understand why aerobic glycolysis is associated with cell proliferation, we examined the metabolic consequence of activating the pyruvate dehydrogenase complex (PDH) to increase mitochondrial pyruvate oxidation at the expense of fermentation. We find that increasing PDH activity impairs cell proliferation by reducing the nicotinamide adenine dinucleotide cofactor ratio (NAD+/NADH). This change in NAD+/NADH ratio is caused by an increase in mitochondrial membrane potential that impairs mitochondrial electron transport and NAD+ regeneration. Uncoupling mitochondrial respiration from ATP synthesis or increasing ATP hydrolysis restores NAD+/NADH homeostasis and proliferation even when glucose oxidation is increased. These data suggest that when the demand for NAD+ to support oxidation reactions exceeds the demand for ATP consumption in cells, NAD+ regeneration by mitochondrial respiration becomes constrained, promoting fermentation despite available oxygen. This argues that cells engage in aerobic glycolysis when the cellular demand for NAD+ is in excess of the cellular demand for ATP.

scholarly journals Cesium Treatment Depresses Glycolysis Pathway in HeLa Cell

2021 ◽  
Vol 55 (4) ◽  
pp. 477-488
Keyword(s):  
Hela Cell ◽  
Nicotinamide Adenine Dinucleotide ◽  
Aerobic Glycolysis ◽  
Biological Effects ◽  
Glycolytic Enzyme ◽  
Nicotinamide Adenine ◽  
Reduced Form ◽  
Dependent Manner ◽  
Nadh Ratio ◽  
Glycolysis Pathway

Background/Aims: Cesium (Cs) is an alkali metal element that is of no essential use for humans; it has no known beneficial function that is verified by clinical research. When used as an alternative cancer therapy, it even causes toxicity in high doses. Thus, before using Cs as treatment in clinical settings, it is important to clearly determine its biological effects on cells. However, Cs was found to suppress the proliferation of human cervical cancer cells in a dose-dependent manner, and it was assumed that Cs inhibits the glycolysis pathway. In this study, we clearly determined the step of the glycolysis pathway that is affected by Cs. Methods: The glycolytic enzyme expressions, activities, and metabolite concentrations in HeLa cells were measured by PCR, western blotting, and enzymatic methods, after treating the cells with Cs for 3 days. Results: Cs treatment decreased transcriptional and expression levels of hexokinase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase (PK), and lactate dehydrogenase and the activity of PK. Analysis of glycolysis pathway metabolites revealed that Cs treatment reduces lactate level and increases the level of nicotinamide adenine dinucleotide (oxidized form, NAD+); however, it did not affect the levels of pyruvate and nicotinamide adenine dinucleotide (reduced form, NADH). Increase of the [NAD+]/[NADH] ratio and decrease of the [lactate]/[pyruvate] ratio indicate that Cs treatment inhibits the aerobic glycolysis pathway. Conclusion: Cs treatment inhibits PK activity and increases the [NAD+]/[NADH] ratio. Hence, Cs has been determined to inhibit glycolysis, especially the aerobic glycolysis pathway. These results suggest that suppression of HeLa cell proliferation following Cs treatment was caused by inhibition of aerobic glycolysis by Cs.

scholarly journals Anti-Warburg Effect of Melatonin: A Proposed Mechanism to Explain its Inhibition of Multiple Diseases

2021 ◽  
Vol 22 (2) ◽  
pp. 764
Author(s):  
Russel J. Reiter ◽  
Ramaswamy Sharma ◽  
Sergio Rosales-Corral
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Warburg Effect ◽  
Coenzyme A ◽  
Aerobic Glycolysis ◽  
Pyruvate Dehydrogenase Complex ◽  
Pyruvate Dehydrogenase Kinase ◽  
Metabolic Phenotype ◽  
Common Denominator ◽  
Acetyl Coenzyme A ◽  
Acetyl Coenzyme

Glucose is an essential nutrient for every cell but its metabolic fate depends on cellular phenotype. Normally, the product of cytosolic glycolysis, pyruvate, is transported into mitochondria and irreversibly converted to acetyl coenzyme A by pyruvate dehydrogenase complex (PDC). In some pathological cells, however, pyruvate transport into the mitochondria is blocked due to the inhibition of PDC by pyruvate dehydrogenase kinase. This altered metabolism is referred to as aerobic glycolysis (Warburg effect) and is common in solid tumors and in other pathological cells. Switching from mitochondrial oxidative phosphorylation to aerobic glycolysis provides diseased cells with advantages because of the rapid production of ATP and the activation of pentose phosphate pathway (PPP) which provides nucleotides required for elevated cellular metabolism. Molecules, called glycolytics, inhibit aerobic glycolysis and convert cells to a healthier phenotype. Glycolytics often function by inhibiting hypoxia-inducible factor-1α leading to PDC disinhibition allowing for intramitochondrial conversion of pyruvate into acetyl coenzyme A. Melatonin is a glycolytic which converts diseased cells to the healthier phenotype. Herein we propose that melatonin’s function as a glycolytic explains its actions in inhibiting a variety of diseases. Thus, the common denominator is melatonin’s action in switching the metabolic phenotype of cells.

scholarly journals Mitochondrial Respiration in KRAS and BRAF Mutated Colorectal Tumors and Polyps

Cancers ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 815 ◽  
Author(s):  
Egle Rebane-Klemm ◽  
Laura Truu ◽  
Leenu Reinsalu ◽  
Marju Puurand ◽  
Igor Shevchuk ◽  
...  
Keyword(s):  
Mitochondrial Respiration ◽  
Atp Synthesis ◽  
Colorectal Polyps ◽  
Metabolic Phenotype ◽  
Mitochondrial Outer Membrane ◽  
Control Group ◽  
Colon Polyps ◽  
Wild Type ◽  
Tissue Samples ◽  
Potential Component

This study aimed to characterize the ATP-synthesis by oxidative phosphorylation in colorectal cancer (CRC) and premalignant colon polyps in relation to molecular biomarkers KRAS and BRAF. This prospective study included 48 patients. Resected colorectal polyps and postoperative CRC tissue with adjacent normal tissue (control) were collected. Patients with polyps and CRC were divided into three molecular groups: KRAS mutated, BRAF mutated and KRAS/BRAF wild-type. Mitochondrial respiration in permeabilized tissue samples was observed using high resolution respirometry. ADP-activated respiration rate (Vmax) and an apparent affinity of mitochondria to ADP, which is related to mitochondrial outer membrane (MOM) permeability, were determined. Clear differences were present between molecular groups. KRAS mutated CRC group had lower Vmax values compared to wild-type; however, the Vmax value was higher than in the control group, while MOM permeability did not change. This suggests that KRAS mutation status might be involved in acquiring oxidative phenotype. KRAS mutated polyps had higher Vmax values and elevated MOM permeability as compared to the control. BRAF mutated CRC and polyps had reduced respiration and altered MOM permeability, indicating a glycolytic phenotype. To conclude, prognostic biomarkers KRAS and BRAF are likely related to the metabolic phenotype in CRC and polyps. Assessment of the tumor mitochondrial ATP synthesis could be a potential component of patient risk stratification.

Mitochondrial Inhibition Prior to Oxygen-Withdrawal Facilitates the Occurrence of Hypoxia-Induced Spreading Depression in Rat Hippocampal Slices

2006 ◽  
Vol 96 (1) ◽  
pp. 492-504 ◽  
Author(s):  
Florian J. Gerich ◽  
Sebastian Hepp ◽  
Irmelin Probst ◽  
Michael Müller
Keyword(s):  
Mitochondrial Respiration ◽  
Spreading Depression ◽  
Hippocampal Slices ◽  
Atp Synthesis ◽  
Atp Depletion ◽  
Mitochondrial Uncoupling ◽  
Ca1 Neurons ◽  
Optical Signals ◽  
Nitropropionic Acid ◽  
Carbonyl Cyanide

Oxygen withdrawal blocks mitochondrial respiration. In rat hippocampal slices, this triggers a massive depolarization of CA1 neurons and a negative shift of the extracellular DC potential, the characteristic sign of hypoxia-induced spreading depression (HSD). To unveil the contribution of mitochondria to the sensing of hypoxia and the ignition of HSD, we modified mitochondrial function. Mitochondrial uncoupling by carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, 1 μM) prior to hypoxia hastened the onset and shortened the duration of HSD. Blocking mitochondrial ATP synthesis by oligomycin (10 μg/ml) was without effect. Inhibition of mitochondrial respiration by rotenone (20 μM), diphenyleneiodonium (25 μM), or antimycin A (20 μM) also hastened HSD onset and shortened HSD duration. 3-nitropropionic acid (1 mM) increased HSD duration. Cyanide (100 μM) hastened HSD onset and increased HSD duration. At higher concentrations, cyanide (1 mM), azide (2 mM), and FCCP (10 μM) triggered SD episodes on their own. Compared with control HSD, the spatial extent of the intrinsic optical signals of cyanide- and azide-induced SDs was more pronounced. Monitoring NADH (nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide) autofluorescence and mitochondrial membrane potential verified the mitochondrial targeting by the drugs used. Except 1 mM cyanide, no treatment reduced cellular ATP levels severely and no correlation was found between ATP, NADH, or FAD levels and the time to HSD onset. Therefore ATP depletion or a cytosolic reducing shift due to NADH/FADH2 accumulation cannot serve as a general explanation for the hastening of HSD onset on mitochondrial inhibition. Additional redox couples (glutathione) or events downstream of the mitochondrial depolarization need to be considered.

82 Methyl Donor Supplementation Alters Cytosine Methylation and Biological Processes of Cells Cultured in Divergent Glucose Media Reflecting Improvements in Mitochondrial Respiration and Cell Growth Rate

2021 ◽  
Vol 99 (Supplement_1) ◽  
pp. 109-109
Author(s):  
Matthew S Crouse ◽  
Wellison Jarles Da Silva Diniz ◽  
Joel Caton ◽  
Carl R Dahlen ◽  
Lawrence P Reynolds ◽  
...  
Keyword(s):  
Growth Rate ◽  
Cell Proliferation ◽  
Cell Growth ◽  
Vitamin B12 ◽  
Mitochondrial Respiration ◽  
Cytosine Methylation ◽  
Biological Processes ◽  
Cell Growth Rate ◽  
Metabolic Processes ◽  
Positive Regulation

Abstract We hypothesized that supplementation of one-carbon metabolites (OCM: methionine, folate, choline, and vitamin B12) to bovine embryonic tracheal fibroblasts in divergent glucose media would alter cytosine methylation, and alterations in cytosine methylation will reflect biological processes matching previously improved mitochondrial respiration, cell proliferation, and cell growth rate data. Cells were cultured with 1g/L glucose (Low) or 4.5g/L glucose (High). Control medium (CON) contained basal concentrations of folate (0.001g/L), choline (0.001g/L), vitamin B12 (4µg/L), and methionine (0.015g/L). The OCM were supplemented at 2.5 and 5 times (2.5X and 5X, respectively) the CON media, except methionine was limited to 2X across all supplemented treatments. Cells were passaged three times in their treatment media before DNA extraction. Reduced representation bisulfite sequencing was adopted to analyze and compare the genomic methylation patterns within and across treatments using edgeR. Biological processes (BP) were retrieved based on the nearest genes of differentially methylated cytosines (P < 0.01) for each comparison between treatments. In both Low and High treatments, greater OCM increased the proportion of hypomethylated vs. hypermethylated cytosines. Functional analyses pointed out positive regulation of BP related to energy metabolism, except for the contrasts within the High group. Among the BP, we can highlight positive regulation of: GTPase activity, catalytic activity, molecular function, protein modification processes, phosphorylation, protein phosphorylation, cellular protein metabolic processes, MAPK cascade, and metabolic processes. These data support previously reported results from this experiment that showed increased mitochondrial respiration, cell proliferation, and growth rates with increasing OCM levels. We interpret these data to imply that when energy and OCM requirements are met for growth and basal methylation levels, DNA methylation levels decrease which may allow for greater transcription. Thus, OCM can be utilized for other functions such as polyamine synthesis, nucleotide synthesis, energetic metabolites, and phosphatidylcholine synthesis. USDA is an equal opportunity provider and employer.

Abstract 90: Bendavia (MTP-131), a Novel Mitochondria-Targeting Peptide, Improves ADP-Stimulated Mitochondrial Respiration in Cardiomyocytes Isolated From Dogs with Chronic Heart failure

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Hani Sabbah ◽  
Ramesh C Gupta ◽  
Sharad Rastogi ◽  
Paula Mohyi ◽  
Kristina Szekely
Keyword(s):  
Heart Failure ◽  
Mitochondrial Respiration ◽  
Systolic Function ◽  
Atp Synthesis ◽  
Vehicle Control ◽  
Electrode System ◽  
Lv Function ◽  
Targeting Peptide ◽  
Lv Systolic Function ◽  
Mitochondria Targeting

Background: Mitochondria (MITO) of failed human hearts and hearts of dogs with experimental heart failure (HF) manifest structural and functional abnormalities characterized by hyperplasia and reduced organelle size and reduced respiration. These abnormalities lead to reduced ATP synthesis that adversely impacts LV function. We previously showed that chronic therapy (3 months) with Bendavia (MTP-131), a novel mitochondria-targeting peptide, improves LV systolic function in dogs with heart failure (HF), reverses MITO abnormalities and normalizes mitochondria ATP synthesis in myocardium from Bendavia-treated HF dogs. In the present study we examined the direct effects of Bendavia on mitochondria ADP-stimulated state 3 respiration in freshly isolated cardiomyocytes from dogs with advanced chronic HF. Methods: Cardiomyocytes were isolated from LV free wall of 3 untreated dogs with HF produced by intracoronary microembolizations (LV ejection fraction <30%). A standard collagenase-based enzymatic process was used for isolation that yielded ~70% viable rod-shaped cardiomyocytes that excluded trypan blue. Equal aliquotes of cardiomyocytes were incubated in 0, 0.01, 0.10, 1.0 and 10 μM concentration of Bendavia for one hour at 37°C. At the end of incubation, ADP-stimulated state-3 respiration was measured using a Clark electrode system and quatified in nAtom Oxygen/min/mg protein. Results: State-3 respiration in the absence of Bendavia (Vehicle-Control) was 248±9 nAtom Oxygen/min/mg protein. Compared to vehicle-control, incubation of failing cardiomyocytes with Bendavia significantly increased state-3 respiration to 303±33 at 0.01 μM, p<0.05; 405±39 at 0.10 μM, p<0.05; 371±28 at 1.0 μM, p<0.05; and 346±29 at 10.0 μM, p<0.05. Conclusions: Results of this study indicate that the effects of Bendavia on mitochondrial respiration in cardiomyocytes is direct and not a consequence of improved global LV structure or function. Furthermore, the results indicate that the improvement in mitochondrial respiration after treatment with Bendavia can occur early after initiation of therapy (within one hour) and is dose-dependent up to concentrations of 0.10 μM.

scholarly journals Aerobic Growth of Escherichia coli Is Reduced, and ATP Synthesis Is Selectively Inhibited when Five C-terminal Residues Are Deleted from the ϵ Subunit of ATP Synthase

2015 ◽  
Vol 290 (34) ◽  
pp. 21032-21041 ◽  
Author(s):  
Naman B. Shah ◽  
Thomas M. Duncan
Keyword(s):  
Escherichia Coli ◽  
Atp Synthase ◽  
Atp Hydrolysis ◽  
Atp Synthesis ◽  
Intrinsic Rate ◽  
Synthesis Rate ◽  
Final Segment ◽  
Rotary Catalysis

F-type ATP synthases are rotary nanomotor enzymes involved in cellular energy metabolism in eukaryotes and eubacteria. The ATP synthase from Gram-positive and -negative model bacteria can be autoinhibited by the C-terminal domain of its ϵ subunit (ϵCTD), but the importance of ϵ inhibition in vivo is unclear. Functional rotation is thought to be blocked by insertion of the latter half of the ϵCTD into the central cavity of the catalytic complex (F1). In the inhibited state of the Escherichia coli enzyme, the final segment of ϵCTD is deeply buried but has few specific interactions with other subunits. This region of the ϵCTD is variable or absent in other bacteria that exhibit strong ϵ-inhibition in vitro. Here, genetically deleting the last five residues of the ϵCTD (ϵΔ5) caused a greater defect in respiratory growth than did the complete absence of the ϵCTD. Isolated membranes with ϵΔ5 generated proton-motive force by respiration as effectively as with wild-type ϵ but showed a nearly 3-fold decrease in ATP synthesis rate. In contrast, the ϵΔ5 truncation did not change the intrinsic rate of ATP hydrolysis with membranes. Further, the ϵΔ5 subunit retained high affinity for isolated F1 but reduced the maximal inhibition of F1-ATPase by ϵ from >90% to ∼20%. The results suggest that the ϵCTD has distinct regulatory interactions with F1 when rotary catalysis operates in opposite directions for the hydrolysis or synthesis of ATP.

scholarly journals Purification and Biochemical Characterization of the F1Fo-ATP Synthase from Thermoalkaliphilic Bacillus sp. Strain TA2.A1

2003 ◽  
Vol 185 (15) ◽  
pp. 4442-4449 ◽  
Author(s):  
Gregory M. Cook ◽  
Stefanie Keis ◽  
Hugh W. Morgan ◽  
Christoph von Ballmoos ◽  
Ulrich Matthey ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Biochemical Characterization ◽  
Atp Hydrolysis ◽  
Sodium Dodecyl ◽  
Electrical Potential ◽  
Atp Synthesis ◽  
Intrinsic Property ◽  
Protein Sequencing ◽  
Hydrolysis Activity

ABSTRACT We describe here purification and biochemical characterization of the F1Fo-ATP synthase from the thermoalkaliphilic organism Bacillus sp. strain TA2.A1. The purified enzyme produced the typical subunit pattern of an F1Fo-ATP synthase on a sodium dodecyl sulfate-polyacrylamide gel, with F1 subunits α, β, γ, δ, and ε and Fo subunits a, b, and c. The subunits were identified by N-terminal protein sequencing and mass spectroscopy. A notable feature of the ATP synthase from strain TA2.A1 was its specific blockage in ATP hydrolysis activity. ATPase activity was unmasked by using the detergent lauryldimethylamine oxide (LDAO), which activated ATP hydrolysis >15-fold. This activation was the same for either the F1Fo holoenzyme or the isolated F1 moiety, and therefore latent ATP hydrolysis activity is an intrinsic property of F1. After reconstitution into proteoliposomes, the enzyme catalyzed ATP synthesis driven by an artificially induced transmembrane electrical potential (Δψ). A transmembrane proton gradient or sodium ion gradient in the absence of Δψ was not sufficient to drive ATP synthesis. ATP synthesis was eliminated by the electrogenic protonophore carbonyl cyanide m-chlorophenylhydrazone, while the electroneutral Na+/H+ antiporter monensin had no effect. Neither ATP synthesis nor ATP hydrolysis was stimulated by Na+ ions, suggesting that protons are the coupling ions of the ATP synthase from strain TA2.A1, as documented previously for mesophilic alkaliphilic Bacillus species. The ATP synthase was specifically modified at its c subunits by N,N′-dicyclohexylcarbodiimide, and this modification inhibited ATP synthesis.

scholarly journals Structure of a bacterial ATP synthase

2018 ◽  
Author(s):  
Hui Guo ◽  
Toshiharu Suzuki ◽  
John L. Rubinstein
Keyword(s):  
Atp Synthase ◽  
Biochemical Analysis ◽  
Atp Hydrolysis ◽  
Genetic Manipulation ◽  
Proton Translocation ◽  
Atp Synthesis ◽  
Mitochondrial Complex ◽  
Rotational States ◽  
Membrane Region ◽  
Atp Synthases

AbstractATP synthases produce ATP from ADP and inorganic phosphate with energy from a transmembrane proton motive force. Bacterial ATP synthases have been studied extensively because they are the simplest form of the enzyme and because of the relative ease of genetic manipulation of these complexes. We expressed theBacillusPS3 ATP synthase inEschericia coli, purified it, and imaged it by cryo-EM, allowing us to build atomic models of the complex in three rotational states. The position of subunitεshows how it is able to inhibit ATP hydrolysis while allowing ATP synthesis. The architecture of the membrane region shows how the simple bacterial ATP synthase is able to perform the same core functions as the equivalent, but more complicated, mitochondrial complex. The structures reveal the path of transmembrane proton translocation and provide a model for understanding decades of biochemical analysis interrogating the roles of specific residues in the enzyme.

scholarly journals Circ-BANP Contributes to Cell Carcinogenesis and Aerobic Glycolysis by Regulating MAPK1 Through miR-874-3p in Colorectal Cancer

2021 ◽  
Author(s):  
Yunxin Zhang ◽  
Kexin Shen ◽  
Hanyi Zha ◽  
Wentao Zhang ◽  
Haishan Zhang
Keyword(s):  
Colorectal Cancer ◽  
Cell Proliferation ◽  
Aerobic Glycolysis ◽  
Lactate Production ◽  
Xenograft Model ◽  
Mitogen Activated Protein Kinase ◽  
Cell Counting ◽  
Nuclear Antigen ◽  
Luciferase Reporter

Abstract BackgroundCircular RNA-BTG3 associated nuclear protein (circ-BANP) was identifified to involve in cell proliferation of colorectal cancer (CRC). The aerobic glycolysis is a key metabolism mediating cancer progression. However, the role of circ-BANP on aerobic glycolysis in CRC remains unknown. MethodsThe expression of circ-BANP, microRNA (miR)-874-3p, and mitogen-activated protein kinase 1 (MAPK1) mNRA was detected using quantitative real-time polymerase chain reaction. Cell viability and invasion were measured by cell counting kit-8 assay or transwell assay. Glucose consumption and lactate production were assessed by a glucose and lactate assay kit. XF Extracellular Flux Analyzer was used to determine extracellular acidifification rate (ECAR). Western blot was used to analyze the levels of hexokinase-2 (HK2), pyruvate kinase M2 (PKM2), MAPK1, proliferating cell nuclear antigen (PCNA), Cyclin D1, N-cadherin, E-cadherin, hypoxia inducible factor-1α (HIF-1α), glucose transport protein 1(GLUT1), and c-Myc. The interaction between miR-874-3p and circ-BANP or MAPK1 was confifirmed by dual luciferase reporter assay. In vivo experiments were conducted through the murine xenograft model. ResultsCirc-BANP was up-regulated in CRC tissues and cell lines. Circ-BANP knockdown suppressed CRC cell proliferation, invasion and aerobic glycolysis in vitro as well as inhibited tumor growth in vivo. Circ-BANP was a sponge of miR-874-3p and performed anti-tumor effffects by binding to miR-874-3p in CRC cells. Subsequently, we confifirmed MAPK1 was a target of miR-874-3p and circ-BANP indirectly regulated MAPK1 expression by sponging miR-874-3p. After that, we found MAPK1 overexpression partially reversed circ-BANP deletion-mediated inhibition on cell carcinogenesis and aerobic glycolysis in CRC. ConclusionCirc-BANP accelerated cell carcinogenesis and aerobic glycolysis by regulating MAPK1 through miR- 874-3p in CRC, suggesting a promising therapeutic strategy for CRC treatment.

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