Bioenergetic characterization of mouse podocytes

Mitochondrial dysfunction contributes to podocyte injury, but normal podocyte bioenergetics have not been characterized. We measured oxygen consumption rates (OCR) and extracellular acidification rates (ECAR), using a transformed mouse podocyte cell line and the Seahorse Bioscience XF24 Extracellular Flux Analyzer. Basal OCR and ECAR were 55.2 ± 9.9 pmol/min and 3.1 ± 1.9 milli-pH units/min, respectively. The complex V inhibitor oligomycin reduced OCR to ∼45% of baseline rates, indicating that ∼55% of cellular oxygen consumption was coupled to ATP synthesis. Rotenone, a complex I inhibitor, reduced OCR to ∼25% of the baseline rates, suggesting that mitochondrial respiration accounted for ∼75% of the total cellular respiration. Thus ∼75% of mitochondrial respiration was coupled to ATP synthesis and ∼25% was accounted for by proton leak. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), which uncouples electron transport from ATP generation, increased OCR and ECAR to ∼360% and 840% of control levels. FCCP plus rotenone reduced ATP content by 60%, the glycolysis inhibitor 2-deoxyglucose reduced ATP by 35%, and 2-deoxyglucose in combination with FCCP or rotenone reduced ATP by >85%. The lactate dehydrogenase inhibitor oxamate and 2-deoxyglucose did not reduce ECAR, and 2-deoxyglucose had no effect on OCR, although 2-deoxyglucose reduced ATP content by 25%. Mitochondrial uncoupling induced by FCCP was associated with increased OCR with certain substrates, including lactate, glucose, pyruvate, and palmitate. Replication of these experiments in primary mouse podocytes yielded similar data. We conclude that mitochondria play the primary role in maintaining podocyte energy homeostasis, while glycolysis makes a lesser contribution.

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

Journal of Neurophysiology ◽

10.1152/jn.01015.2005 ◽

2006 ◽

Vol 96 (1) ◽

pp. 492-504 ◽

Cited By ~ 38

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.

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Nitric oxide alters metabolism in isolated alveolar type II cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1996.271.1.l23 ◽

1996 ◽

Vol 271 (1) ◽

pp. L23-L30 ◽

Cited By ~ 3

Author(s):

P. R. Miles ◽

L. Bowman ◽

L. Huffman

Keyword(s):

Nitric Oxide ◽

Oxygen Consumption ◽

Atp Synthesis ◽

Alveolar Type ◽

Type Ii Cells ◽

Type Ii ◽

Atp Content ◽

Alveolar Type Ii Cells ◽

Type Ii Cell ◽

Cellular Oxygen

Alveolar type II cells may be exposed to nitric oxide (.NO) from external sources, and these cells can also generate .NO. Therefore we studied the effects of altering .NO levels on various type II cell metabolic processes. Incubation of cells with the .NO generator, S-nitroso-N-acetylpenicillamine (SNAP; 1 mM), leads to reductions of 60-70% in the synthesis of disaturated phosphatidylcholines (DSPC) and cell ATP levels. Cellular oxygen consumption, an indirect measure of cell ATP synthesis, is also reduced by SNAP. There is no direct effect of SNAP on lung mitochondrial ATP synthesis, suggesting that .NO does not directly inhibit this process. On the other hand, incubation of cells with NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase (NOS), the enzyme responsible for .NO synthesis, results in increases in DSPC synthesis, cell ATP content, and cellular oxygen consumption. The L-NAME effects are reversed by addition of L-arginine, the substrate for NOS. Production of .NO by type II cells is inhibited by L-NAME, a better inhibitor of constitutive NOS (cNOS) than inducible NOS (iNOS), and is reduced in the absence of external calcium. Aminoguanidine, a specific inhibitor of iNOS, has no effect on cell ATP content or on .NO production. These results indicate that alveolar type II cell lipid and energy metabolism can be affected by .NO and suggest that there may be cNOS activity in these cells.

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Relative sensitivity of soluble guanylate cyclase and mitochondrial respiration to endogenous nitric oxide at physiological oxygen concentration

Biochemical Journal ◽

10.1042/bj20070033 ◽

2007 ◽

Vol 405 (2) ◽

pp. 223-231 ◽

Cited By ~ 26

Author(s):

Félix Rodríguez-Juárez ◽

Enara Aguirre ◽

Susana Cadenas

Keyword(s):

Nitric Oxide ◽

Oxygen Consumption ◽

Oxygen Concentration ◽

Guanylate Cyclase ◽

Mitochondrial Respiration ◽

Soluble Guanylate Cyclase ◽

Atmospheric Oxygen ◽

Cellular Respiration ◽

Cell Physiology ◽

Experimental Conditions

Nitric oxide (NO) is a widespread biological messenger that has many physiological and pathophysiological roles. Most of the physiological actions of NO are mediated through the activation of sGC (soluble guanylate cyclase) and the subsequent production of cGMP. NO also binds to the binuclear centre of COX (cytochrome c oxidase) and inhibits mitochondrial respiration in competition with oxygen and in a reversible manner. Although sGC is more sensitive to endogenous NO than COX at atmospheric oxygen tension, the more relevant question is which enzyme is more sensitive at physiological oxygen concentration. Using a system in which NO is generated inside the cells in a finely controlled manner, we determined cGMP accumulation by immunoassay and mitochondrial oxygen consumption by high-resolution respirometry at 30 μM oxygen. In the present paper, we report that the NO EC50 of sGC was approx. 2.9 nM, whereas that required to achieve IC50 of respiration was 141 nM (the basal oxygen consumption in the absence of NO was 14±0.8 pmol of O2/s per 106 cells). In accordance with this, the NO–cGMP signalling transduction pathway was activated at lower NO concentrations than the AMPKs (AMP-activated protein kinase) pathway. We conclude that sGC is approx. 50-fold more sensitive than cellular respiration to endogenous NO under our experimental conditions. The implications of these results for cell physiology are discussed.

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Evaluation of the mitochondrial respiration of cardiac myocytes in rats submitted to mechanical bile duct obstruction

Acta Cirurgica Brasileira ◽

10.1590/s0102-86502008000700012 ◽

2008 ◽

Vol 23 (suppl 1) ◽

pp. 66-71 ◽

Cited By ~ 4

Author(s):

Rafael Kemp ◽

Orlando de Castro-e-Silva ◽

José Sebastião dos Santos ◽

Ajith Kumar Sankarankutty ◽

Rodrigo Borges Correa ◽

...

Keyword(s):

Oxygen Consumption ◽

Statistical Analysis ◽

Bile Duct ◽

Mitochondrial Respiration ◽

Respiratory Control ◽

Cellular Respiration ◽

Sham Operation ◽

Significant Drop ◽

Stage 4 ◽

Male Wistar Rats

PURPOSE: The objective of the present study was to evaluate the capacity of the myocardium for energy production by the analysis of mitochondrial respiration in rats with jaundice submitted to bile duct ligature. METHODS: Sixteen male Wistar rats were divided into 2 Groups: Group SO submitted to nontherapeutic laparotomy (sham operation) and Group IC (icteric group) submitted to bile duct ligature. After 7 days, laparotomy was again performed in all animals for cardiac muscle extraction and analysis. Mitochondrial oxygen consumption was determined by stage 3 velocity and stage 4 velocity. The respiratory control ratio (RCR) was obtained by the ratio of stage 3 to stage 4 velocity. Statistical analysis was performed by the Mann-Whitney test, with the level of significance set at 5% (p<0.05). RESULTS: Statistical analysis revealed a significant drop in oxygen consumption during stage 3 mitochondrial respiration in group IC compared with SO, whereas the values obtained during stage 4 were basically identical for the two groups. Likewise, RCR values exhibited a significant reduction. CONCLUSION: The cellular respiration of the "jaundiced heart" is depressed. This was demonstrated by the reduced capacity of cardiac mitochondria to consume oxygen and synthesize ATP, supporting the idea of a latent cardiac impairment responsible for the hemodynamic decompensation occurring during cholestasis.

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ATP-consuming futile cycles as energy dissipating mechanisms to counteract obesity

Reviews in Endocrine and Metabolic Disorders ◽

10.1007/s11154-021-09690-w ◽

2021 ◽

Author(s):

Alexandra J. Brownstein ◽

Michaela Veliova ◽

Rebeca Acin-Perez ◽

Marc Liesa ◽

Orian S. Shirihai

Keyword(s):

Energy Dissipation ◽

Energy Homeostasis ◽

Atp Synthesis ◽

Heat Energy ◽

Mitochondrial Uncoupling ◽

Energy Consuming ◽

Futile Cycles ◽

Import And Export ◽

Metabolic Reactions ◽

Oxidative Function

AbstractObesity results from an imbalance in energy homeostasis, whereby excessive energy intake exceeds caloric expenditure. Energy can be dissipated out of an organism by producing heat (thermogenesis), explaining the long-standing interest in exploiting thermogenic processes to counteract obesity. Mitochondrial uncoupling is a process that expends energy by oxidizing nutrients to produce heat, instead of ATP synthesis. Energy can also be dissipated through mechanisms that do not involve mitochondrial uncoupling. Such mechanisms include futile cycles described as metabolic reactions that consume ATP to produce a product from a substrate but then converting the product back into the original substrate, releasing the energy as heat. Energy dissipation driven by cellular ATP demand can be regulated by adjusting the speed and number of futile cycles. Energy consuming futile cycles that are reviewed here are lipolysis/fatty acid re-esterification cycle, creatine/phosphocreatine cycle, and the SERCA-mediated calcium import and export cycle. Their reliance on ATP emphasizes that mitochondrial oxidative function coupled to ATP synthesis, and not just uncoupling, can play a role in thermogenic energy dissipation. Here, we review ATP consuming futile cycles, the evidence for their function in humans, and their potential employment as a strategy to dissipate energy and counteract obesity.

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BAX supports the mitochondrial network, promoting bioenergetics in nonapoptotic cells

AJP Cell Physiology ◽

10.1152/ajpcell.00325.2010 ◽

2011 ◽

Vol 300 (6) ◽

pp. C1466-C1478 ◽

Cited By ~ 29

Author(s):

Rebecca J. Boohaker ◽

Ge Zhang ◽

Adina Loosley Carlson ◽

Kathleen N. Nemec ◽

Annette R. Khaled

Keyword(s):

Oxygen Consumption ◽

Citrate Synthase ◽

Atp Synthesis ◽

Cellular Respiration ◽

Small Interfering Rnas ◽

Glycolytic Activity ◽

Mitochondrial Dna Content ◽

Hct 116 ◽

Subsequent Loss ◽

Hct 116 Cells

The dual functionality of the tumor suppressor BAX is implied by the nonapoptotic functions of other members of the BCL-2 family. To explore this, mitochondrial metabolism was examined in BAX-deficient HCT-116 cells as well as primary hepatocytes from BAX-deficient mice. Although mitochondrial density and mitochondrial DNA content were the same in BAX-containing and BAX-deficient cells, MitoTracker staining patterns differed, suggesting the existence of BAX-dependent functional differences in mitochondrial physiology. Oxygen consumption and cellular ATP levels were reduced in BAX-deficient cells, while glycolysis was increased. These results suggested that cells lacking BAX have a deficiency in the ability to generate ATP through cellular respiration. This conclusion was supported by detection of reduced citrate synthase activity in BAX-deficient cells. In nonapoptotic cells, a portion of BAX associated with mitochondria and a sequestered, protease-resistant form was detected. Inhibition of BAX with small interfering RNAs reduced intracellular ATP content in BAX-containing cells. Expression of either full-length or COOH-terminal-truncated BAX in BAX-deficient cells rescued ATP synthesis and oxygen consumption and reduced glycolytic activity, suggesting that this metabolic function of BAX was not dependent upon its COOH-terminal helix. Expression of BCL-2 in BAX-containing cells resulted in a subsequent loss of ATP measured, implying that, even under nonapoptotic conditions, an antagonistic interaction exists between the two proteins. These findings infer that a basal amount of BAX is necessary to maintain energy production via aerobic respiration.

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Cell-Permeable Succinate Rescues Mitochondrial Respiration in Cellular Models of Statin Toxicity

International Journal of Molecular Sciences ◽

10.3390/ijms22010424 ◽

2021 ◽

Vol 22 (1) ◽

pp. 424

Author(s):

Vlad F. Avram ◽

Imen Chamkha ◽

Eleonor Åsander-Frostner ◽

Johannes K. Ehinger ◽

Romulus Z. Timar ◽

...

Keyword(s):

Oxygen Consumption ◽

Mitochondrial Dysfunction ◽

Mitochondrial Respiration ◽

Complex I ◽

Ex Vivo ◽

Coupling Efficiency ◽

Human Platelets ◽

Lipid Lowering ◽

Lipid Lowering Therapy ◽

Cellular Models

Statins are the cornerstone of lipid-lowering therapy. Although generally well tolerated, statin-associated muscle symptoms (SAMS) represent the main reason for treatment discontinuation. Mitochondrial dysfunction of complex I has been implicated in the pathophysiology of SAMS. The present study proposed to assess the concentration-dependent ex vivo effects of three statins on mitochondrial respiration in viable human platelets and to investigate whether a cell-permeable prodrug of succinate (complex II substrate) can compensate for statin-induced mitochondrial dysfunction. Mitochondrial respiration was assessed by high-resolution respirometry in human platelets, acutely exposed to statins in the presence/absence of the prodrug NV118. Statins concentration-dependently inhibited mitochondrial respiration in both intact and permeabilized cells. Further, statins caused an increase in non-ATP generating oxygen consumption (uncoupling), severely limiting the OXPHOS coupling efficiency, a measure of the ATP generating capacity. Cerivastatin (commercially withdrawn due to muscle toxicity) displayed a similar inhibitory capacity compared with the widely prescribed and tolerable atorvastatin, but did not elicit direct complex I inhibition. NV118 increased succinate-supported mitochondrial oxygen consumption in atorvastatin/cerivastatin-exposed platelets leading to normalization of coupled (ATP generating) respiration. The results acquired in isolated human platelets were validated in a limited set of experiments using atorvastatin in HepG2 cells, reinforcing the generalizability of the findings.

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Circadian Regulation of Mitochondrial Uncoupling and Lifespan

Innovation in Aging ◽

10.1093/geroni/igaa057.2648 ◽

2020 ◽

Vol 4 (Supplement_1) ◽

pp. 740-741

Author(s):

Matthew Ulgherait

Keyword(s):

Stem Cells ◽

Circadian Rhythm ◽

Uncoupling Protein ◽

Intestinal Stem Cells ◽

Intestinal Stem Cell ◽

Cellular Respiration ◽

Circadian Regulation ◽

Lifespan Extension ◽

Diet Restriction ◽

Mitochondrial Uncoupling

Abstract Because old age is associated with defects in circadian rhythm, loss of circadian regulation is thought to be pathogenic and contribute to mortality. We show instead that loss of specific circadian clock components Period (Per) and Timeless (Tim) in male Drosophila significantly extends lifespan. This lifespan extension is not mediated by canonical diet-restriction longevity pathways, but is due to altered cellular respiration via increased mitochondrial uncoupling. Lifespan extension of per mutants depends on mitochondrial uncoupling in the intestine. Moreover, up-regulated uncoupling protein UCP4C in intestinal stem cells and enteroblasts is sufficient to extend lifespan and preserve proliferative homeostasis in the gut with age. Consistent with inducing a metabolic state that prevents over-proliferation, mitochondrial uncoupling drugs also extend lifespan and inhibit intestinal stem cell overproliferation due to aging or even tumorigenesis. These results demonstrate that circadian-regulated intestinal mitochondrial uncoupling controls longevity in Drosophila and suggest a new potential anti-aging therapeutic target.

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Mitochondrial Respiration in KRAS and BRAF Mutated Colorectal Tumors and Polyps

Cancers ◽

10.3390/cancers12040815 ◽

2020 ◽

Vol 12 (4) ◽

pp. 815 ◽

Cited By ~ 1

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.

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