REDUCED CITRATE SYNTHASE ACTIVITY EFFECT ON OXYGEN CONSUMPTION RATES IN ISOLATED MITOCHONDRIA FROM MICE LIVER AND MUSCLES

Background. Liver and skeletal muscles play the major role in metabolism. Mitochondria are of particular importance in functioning of these organs. We tested the hypothesis that reduced citrate synthase (CS) activity could induce improved fatty substrate and carbohydrate oxidation in mitochondria extracted from liver and hind limb muscles of mice. Methods. Eight mice each of 12-week-old control C57B6/J (B6) and congenic B6.A-(rs3676616-D10Utsw1)/ Kjn (B6.A) mice were studied. The mitochondria were isolated by differential centrifugation method followed by assessment of mitochondrial respiration and citrate synthase (CS) activity. Mitochondrial respiration was measured as oxygen consumption with Clark-type oxygen electrode by using polarography system. CS enzyme activity was measured spectrophotometrically. Results. The activity of CS was by ~32% lower for mitochondria for B6.A compared to B6 mice (603.9 ± 135.6 U/g and 894.2 ± 193.2 U/g, respectively). Mitochondrial respiration did not differ significantly between the strains. Conclusions. 30% reduction in citrate synthase activity does not impair mitochondrial respiration.

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Chronic Hypoxia Enhances β-Oxidation-Dependent Electron Transport via Electron Transferring Flavoproteins

Cells ◽

10.3390/cells8020172 ◽

2019 ◽

Vol 8 (2) ◽

pp. 172 ◽

Cited By ~ 2

Author(s):

Dominik C. Fuhrmann ◽

Catherine Olesch ◽

Nina Kurrle ◽

Frank Schnütgen ◽

Sven Zukunft ◽

...

Keyword(s):

Oxygen Consumption ◽

Fatty Acid ◽

Electron Transport ◽

Mitochondrial Respiration ◽

Chronic Hypoxia ◽

Acute Hypoxia ◽

Acid Synthesis ◽

Acid Oxidation ◽

Fatty Acid Production ◽

Consumption Rates

Hypoxia poses a stress to cells and decreases mitochondrial respiration, in part by electron transport chain (ETC) complex reorganization. While metabolism under acute hypoxia is well characterized, alterations under chronic hypoxia largely remain unexplored. We followed oxygen consumption rates in THP-1 monocytes during acute (16 h) and chronic (72 h) hypoxia, compared to normoxia, to analyze the electron flows associated with glycolysis, glutamine, and fatty acid oxidation. Oxygen consumption under acute hypoxia predominantly demanded pyruvate, while under chronic hypoxia, fatty acid- and glutamine-oxidation dominated. Chronic hypoxia also elevated electron-transferring flavoproteins (ETF), and the knockdown of ETF–ubiquinone oxidoreductase lowered mitochondrial respiration under chronic hypoxia. Metabolomics revealed an increase in citrate under chronic hypoxia, which implied glutamine processing to α-ketoglutarate and citrate. Expression regulation of enzymes involved in this metabolic shunting corroborated this assumption. Moreover, the expression of acetyl-CoA carboxylase 1 increased, thus pointing to fatty acid synthesis under chronic hypoxia. Cells lacking complex I, which experienced a markedly impaired respiration under normoxia, also shifted their metabolism to fatty acid-dependent synthesis and usage. Taken together, we provide evidence that chronic hypoxia fuels the ETC via ETFs, increasing fatty acid production and consumption via the glutamine-citrate-fatty acid axis.

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An optimized protocol for coupling oxygen consumption rates with β-oxidation in isolated mitochondria from mouse soleus

STAR Protocols ◽

10.1016/j.xpro.2021.100735 ◽

2021 ◽

Vol 2 (3) ◽

pp. 100735

Author(s):

Cristina Sánchez-González ◽

Laura Formentini

Keyword(s):

Oxygen Consumption ◽

Isolated Mitochondria ◽

Optimized Protocol ◽

Consumption Rates

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Repeated Exposure to Hyperbaric Hyperoxia Affects Mitochondrial Functions of the Lung Fibroblasts

Physiological Research ◽

10.33549/physiolres.934046 ◽

2018 ◽

pp. S633-S643 ◽

Cited By ~ 2

Author(s):

J. DEJMEK ◽

M. KOHOUTOVÁ ◽

M. KRIPNEROVÁ ◽

M. ČEDÍKOVÁ ◽

Z. TŮMA ◽

...

Keyword(s):

Oxygen Consumption ◽

Mitochondrial Respiration ◽

Citrate Synthase ◽

Fluorescent Microscopy ◽

Repeated Exposure ◽

Cellular Growth ◽

Lung Fibroblasts ◽

Pure Oxygen ◽

Complex Ii ◽

Mitochondrial Functions

Hyperbaric oxygen (HBO) therapy, i.e. breathing pure oxygen under increased environmental pressures serves as a treatment for diverse medical conditions. However, elevated oxygen concentration can be detrimental to central nervous system or lungs. Our study aimed to evaluate the effects of repeated exposure to HBO on mitochondrial respiration assessed by high-resolution respirometry (HRR), cell viability estimated by PrestoBlue® reaction, morphology analyzed by routine phase contrast and fluorescent microscopy, and superoxide dismutase (SOD) and citrate synthase (CS) activities using human lung fibroblasts. The cells were exposed to HBO for 2 h per day for 5 consecutive days. One day after the last exposure, HBO cells displayed significantly smaller area and perimeter, compromised viability and elevated SOD activity. No changes were detected in CS activity or quality of mitochondrial network. HRR revealed impaired mitochondrial oxygen consumption manifested by increased leak respiration, decreased activity of complex II and compromised ATP-related oxygen consumption when fatty acids were oxidized. Our findings document that in conditions mimicking chronic intermittent exposure to HBO, lung fibroblasts suffer from compromised mitochondrial respiration linked to complex II and impaired cellular growth in spite of increased antioxidant defense. Underlying mechanism of this HBO-induced mitochondrial dysfunction should be further explored.

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Aquatic Oxygen Analysis

Measuring Metabolic Rates ◽

10.1093/oso/9780198830399.003.0005 ◽

2018 ◽

pp. 43-48

Author(s):

John R. B. Lighton

Keyword(s):

Oxygen Consumption ◽

Aquatic Organisms ◽

Practical Method ◽

Oxygen Electrode ◽

Metabolic Rates ◽

Consumption Rates ◽

Common Electrode ◽

Flow Through ◽

Common Problems ◽

Electrode Membrane

Measuring oxygen consumption rates in aquatic media is the only practical method for determining the metabolic rates of cell cultures and aquatic organisms. This chapter describes the three principal variations of aquatic respirometry—closed and open system (or flow-through) respirometry, and headspace respirometry—together with procedures for calibrating aquatic respirometry systems, acquiring data from them, and analyzing the resulting data. Appendix 2 describes the operation of the widely used Clark dissolved oxygen electrode, the characteristics of common electrode membrane materials, and necessary routine maintenance. Common problems are discussed and trouble-shooting guides are included.

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Mitochondrial Respiration Correlates with Prognostic Markers in Chronic Lymphocytic Leukemia and Is Normalized by Ibrutinib Treatment

Cancers ◽

10.3390/cancers12030650 ◽

2020 ◽

Vol 12 (3) ◽

pp. 650 ◽

Cited By ~ 2

Author(s):

Subir Roy Chowdhury ◽

Eric D. J. Bouchard ◽

Ryan Saleh ◽

Zoann Nugent ◽

Cheryl Peltier ◽

...

Keyword(s):

Oxygen Consumption ◽

Chronic Lymphocytic Leukemia ◽

B Lymphocytes ◽

Mitochondrial Respiration ◽

Prognostic Markers ◽

Response To Therapy ◽

Lymphocytic Leukemia ◽

Mitochondrial Bioenergetics ◽

Respiration Rates ◽

Consumption Rates

Mitochondrial bioenergetics profiling, a measure of oxygen consumption rates, correlates with prognostic markers and can be used to assess response to therapy in chronic lymphocytic leukemia (CLL) cells. In this study, we measured mitochondrial respiration rates in primary CLL cells using respirometry to evaluate mitochondrial function. We found significant increases in mitochondrial respiration rates in CLL versus control B lymphocytes. We also observed amongst CLL patients that advanced age, female sex, zeta-chain-associated protein of 70 kD (ZAP-70+), cluster of differentiation 38 (CD38+), and elevated β2-microglobulin (β2-M) predicted increased maximal respiration rates. ZAP-70+ CLL cells exhibited significantly higher bioenergetics than B lymphocytes or ZAP-70− CLL cells and were more sensitive to the uncoupler, carbonyl cyanide-p-trifluoro-methoxyphenylhydrazone (FCCP). Univariable and multivariable linear regression analysis demonstrated that ZAP-70+ predicted increased maximal respiration. ZAP-70+ is a surrogate for B cell receptor (BCR) activation and can be targeted by ibrutinib, which is a clinically approved Bruton’s tyrosine kinase (BTK) inhibitor. Therefore, we evaluated the oxygen consumption rates (OCR) of CLL cells and plasma chemokine (C-C motif) ligands 3 and 4 (CCL3/CCL4) levels from ibrutinib-treated patients and demonstrated decreased OCR similar to control B lymphocytes, suggesting that ibrutinib treatment resets the mitochondrial bioenergetics, while diminished CCL3/CCL4 levels indicate the down regulation of the BCR signaling pathway in CLL. Our data support evaluation of mitochondrial respiration as a preclinical tool for the response assessment of CLL cells.

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Turbidity and Temperature Effects on Oxygen Consumption in the Zebra Mussel (Dreissena polymorpha)

Canadian Journal of Fisheries and Aquatic Sciences ◽

10.1139/f94-020 ◽

1994 ◽

Vol 51 (1) ◽

pp. 179-184 ◽

Cited By ~ 34

Author(s):

James E. Alexander Jr. ◽

James H. Thorp ◽

Ronald D. Fell

Keyword(s):

Oxygen Consumption ◽

Zebra Mussel ◽

Dreissena Polymorpha ◽

Bentonite Clay ◽

Oxygen Electrode ◽

Acclimation Temperature ◽

Ohio River ◽

Maintenance Costs ◽

Normal Ranges ◽

Consumption Rates

The effects of acute exposure to suspended inorganic sediment (bentonite clay) on oxygen consumption rates of the zebra mussel (Dreissena polymorpha) were examined to estimate their maintenance costs for colonizing the Ohio River. Adult D. polymorpha were acclimated to 10, 18, or 26 °C for 2 wk (10 replicate groups for each acclimation temperature) and respiration was measured in a closed recirculating system using oxygen electrode probes. Each replicate group of mussels was tested at their acclimation temperature under five turbidity levels: 0 (clear water), 5, 20, 80, and 160 NTU (nephelometric turbidity units). Respiration rates were significantly affected by acclimation temperature (direct relationship) and turbidity (inverse relationship, which leveled off with increasing turbidity). Warmer-acclimated animals showed a more dramatic drop in oxygen consumption with increasing turbidity, and all size-classes were equally affected by turbidity. Our results suggest that the normal ranges of temperatures and turbidity levels in the Ohio River may depress growth rates of adult D. polymorpha by increasing maintenance costs but probably will not by themselves prevent establishment of viable populations in this river.

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Seabream Larval Physiology under Ocean Warming and Acidification

Fishes ◽

10.3390/fishes5010001 ◽

2019 ◽

Vol 5 (1) ◽

pp. 1 ◽

Cited By ~ 1

Author(s):

Marta S. Pimentel ◽

Filipa Faleiro ◽

Jorge Machado ◽

Pedro Pousão-Ferreira ◽

Rui Rosa

Keyword(s):

Oxygen Consumption ◽

Citrate Synthase ◽

Sparus Aurata ◽

Future Research ◽

Activity Levels ◽

Important Species ◽

Consumption Rates ◽

Commercially Important ◽

Ocean Conditions ◽

Enzymatic Machinery

The vulnerability of early fish stages represents a critical bottleneck for fish recruitment; therefore, it is essential to understand how climate change affects their physiology for more sustainable management of fisheries. Here, we investigated the effects of warming (OW; +4 °C) and acidification (OA; ΔpH = 0.5) on the heart and oxygen consumption rates, metabolic enzymatic machinery—namely citrate synthase (CS), lactate dehydrogenase (LDH), and ß-hydroxyacyl CoA dehydrogenase (HOAD), of seabream (Sparus aurata) larvae (fifteen days after hatch). Oxygen consumption and heart rates showed a significant increase with rising temperature, but decreased with pCO2. Results revealed a significant increase of LDH activity with OW and a significant decrease of the aerobic potential (CS and HOAD activity) of larvae with OA. In contrast, under OA, the activity levels of the enzyme LDH and the LDH:CS ratio indicated an enhancement of anaerobic pathways. Although such a short-term metabolic strategy may eventually sustain the basic costs of maintenance, it might not be adequate under the future chronic ocean conditions. Given that the potential for adaptation to new forthcoming conditions is yet experimentally unaccounted for this species, future research is essential to accurately predict the physiological performance of this commercially important species under future ocean conditions.

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The Role of Dietary Saturated Fat in Associations between Exposure to Ambient Fine Particulate Matter and Mitochondrial Respiratory Functions in Circulating Platelets

Current Developments in Nutrition ◽

10.1093/cdn/nzab041_038 ◽

2021 ◽

Vol 5 (Supplement_2) ◽

pp. 523-523

Author(s):

Wan Shen ◽

Hao Chen ◽

James Samet ◽

Haiyan Tong

Keyword(s):

Oxygen Consumption ◽

Mitochondrial Respiration ◽

Saturated Fatty Acids ◽

Fine Particulate Matter ◽

Saturated Fat ◽

Basal Respiration ◽

Mitochondrial Oxygen Consumption ◽

Atp Production ◽

Consumption Rates ◽

Mitochondrial Respiratory

Abstract Objectives Exposure to ambient fine particulate matter (PM2.5) is associated with platelet activation and increased mitochondrial respiration. The impact of dietary saturated fat on the circulating platelets is not understood. This project aimed to determine whether dietary saturated fatty acids moderate mitochondrial respiratory function in circulating platelets after short-term exposure to PM2.5. Methods Platelets were isolated from 22 healthy male volunteers (mean age ± SD, 37 ± 8.2) in a panel study and measured for mitochondrial oxygen consumption rates using an extracellular flux analyzer. Intakes of saturated fat were determined from 24 hr dietary recalls the day before the assay. Daily ambient PM2.5 concentrations during the study period were obtained from ambient air quality monitoring stations. Data were fitted with a moderation model, where the level of ambient PM2.5 was the independent variable, saturated fat intake was the moderator, and mitochondrial respiratory functions in circulating platelets were the dependent variables. Results After controlling for age, dietary consumption of saturated fat moderated the mitochondrial oxygen consumption rates of non-mitochondrial respiration, basal respiration, maximum respiration, ATP production, and spare respiratory capacity after exposure to ambient PM2.5 with 2 days lag. Specifically, the negative associations between the above mentioned mitochondrial respiratory measurements and PM2.5 levels reached statistical significance (95% Confident Intervals did not include 0) in subjects with a high intake of total saturated fat. Further, results for individual saturated fatty acid showed similar patterns, specifically that negative association between mitochondrial oxygen consumption rates of non-mitochondrial respiration, basal respiration and ATP production and levels of exposed PM2.5 was moderated by intakes of short-chain (C4:0), medium-chain (C6:0, C8:0, C10:0, C12:0), long-chain (C14:0, C16:0) saturated fatty acids. Conclusions Taken together, these preliminary findings suggest that consumption of saturated fat moderates platelet mitochondrial respiration after exposure to PM2.5. THIS ABSTRACT OF A PROPOSED PRESENTATION DOES NOT NECESSARILY REFLECT EPA POLICY. Funding Sources This project was supported by the U.S. EPA Intramural Research Program.

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Effects of 5-bromo-2′-deoxyuridine on Friend erythroleukaemia cells. II. Oxidative metabolism and enzyme content of whole cells and isolated mitochondria

Journal of Cell Science ◽

10.1242/jcs.52.1.37 ◽

1981 ◽

Vol 52 (1) ◽

pp. 37-54

Author(s):

R.J. Walter

Keyword(s):

Oxygen Consumption ◽

Respiratory Control ◽

High Energy ◽

Inhibitory Effect ◽

Mitochondrial Enzymes ◽

Morphological Alterations ◽

Isolated Mitochondria ◽

Outer Compartment ◽

Consumption Rates ◽

Electrode Chamber

Untreated or bromodeoxyuridine (BrdUrd)-treated Friend erythroleukaemia (EL) cells from 15- and 72-h-cultures were harvested and the mitochondria were isolated by homogenization and differential centrifugation. Aliquots of the original cell suspensions or the final mitochondrial suspensions were either fixed for electron microscopy, assayed for enzyme activities, or introduced into a 1-ml Clarke oxygen electrode chamber. Whole BrdUrd-treated cells exhibited notable morphological alterations (see accompanying paper) but no effect was observed on whole-cell respiration. Morphologically, isolated mitochondria exhibited a highly condensed matrix and a greatly expanded outer compartment. Functionally, these mitochondria oxidized a variety of substrates at high state-3 (ADP-stimulated) rates (60-210 ng atoms O/min per mg protein) and displayed adequate respiratory control and ADP/O ratios. In the mitochondria isolated from BrdUrd-treated EL cells (both 15- and 72-h cultures), the state-3 oxygen consumption rates, respiratory control ratios, and ADP/O ratios generally decreased compared to their matched controls. These functional deficiencies coincided with in situ increases in the volume of the mitochondrial compartment and conspicuous (30-100%) increases in the specific and total activities of several mitochondrial enzymes. In addition, the mitochondria isolated from the 72-h group (treated and untreated) displayed immediate alterations when BrdUrd was added to the electrode chamber. Oxygen consumption rates dropped, respiratory control ratios changed moderately, and ADP/O ratios increased. BrdUrd may act both as an inhibitor of oxidative phosphorylation and perhaps as a sink for some of the high energy phosphate that is being generated. Thus, BrdUrd may exert its inhibitory effect on cell differentiation by interfering with mitochondrial function.

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Chronic DDE Exposure Modifies Mitochondrial Respiration during Differentiation of Human Adipose-Derived Mesenchymal Stem Cells into Mature Adipocytes

Biomolecules ◽

10.3390/biom11081068 ◽

2021 ◽

Vol 11 (8) ◽

pp. 1068

Author(s):

Iva Kladnicka ◽

Miroslava Cedikova ◽

Jan Jedlicka ◽

Michaela Kohoutova ◽

Ludek Muller ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Oxygen Consumption ◽

Mitochondrial Respiration ◽

Molecular Mechanisms ◽

Citrate Synthase ◽

Environmental Pollutants ◽

Intact Cells

The contribution of environmental pollutants to the obesity pandemic is still not yet fully recognized. Elucidating possible cellular and molecular mechanisms of their effects is of high importance. Our study aimed to evaluate the effect of chronic, 21-day-long, 2,2-bis (4-chlorophenyl)-1,1-dichlorethylenedichlorodiphenyldichloroethylene (p,p′-DDE) exposure of human adipose-derived mesenchymal stem cells committed to adipogenesis on mitochondrial oxygen consumption on days 4, 10, and 21. In addition, the mitochondrial membrane potential (MMP), the quality of the mitochondrial network, and lipid accumulation in maturing cells were evaluated. Compared to control differentiating adipocytes, exposure to p,p′-DDE at 1 μM concentration significantly increased basal (routine) mitochondrial respiration, ATP-linked oxygen consumption and MMP of intact cells on day 21 of adipogenesis. In contrast, higher pollutant concentration seemed to slow down the gradual increase in ATP-linked oxygen consumption typical for normal adipogenesis. Organochlorine p,p′-DDE did not alter citrate synthase activity. In conclusion, in vitro 1 μM p,p′-DDE corresponding to human exposure is able to increase the mitochondrial respiration per individual mitochondrion at the end of adipocyte maturation. Our data reveal that long-lasting exposure to p,p′-DDE could interfere with the metabolic programming of mature adipocytes.

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