scholarly journals Measurement of Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) in Culture Cells for Assessment of the Energy Metabolism

BIO-PROTOCOL ◽  
2018 ◽  
Vol 8 (10) ◽  
Author(s):  
Birte Plitzko ◽  
Sandra Loesgen
Hypertension ◽  
2020 ◽  
Vol 76 (1) ◽  
pp. 121-132
Author(s):  
Xiao Chun Li ◽  
Xinchun Zhou ◽  
Jia Long Zhuo

The present study tested the hypotheses that overexpression of an intracellular Ang II (angiotensin II) fusion protein, mito-ECFP/Ang II, selectively in the mitochondria of mouse proximal tubule cells induces mitochondrial oxidative and glycolytic responses and elevates blood pressure via the Ang II/AT 1a receptor/superoxide/NHE3 (the Na + /H + exchanger 3)-dependent mechanisms. A PT-selective, mitochondria-targeting adenoviral construct encoding Ad-sglt2-mito-ECFP/Ang II was used to test the hypotheses. The expression of mito-ECFP/Ang II was colocalized primarily with Mito-Tracker Red FM in mouse PT cells or with TMRM in kidney PTs. Mito-ECFP/Ang II markedly increased oxygen consumption rate as an index of mitochondrial oxidative response (69.5%; P <0.01) and extracellular acidification rate as an index of mitochondrial glycolytic response (34%; P <0.01). The mito-ECFP/Ang II–induced oxygen consumption rate and extracellular acidification rate responses were blocked by AT 1 blocker losartan ( P <0.01) and a mitochondria-targeting superoxide scavenger mito-TEMPO ( P <0.01). By contrast, the nonselective NO inhibitor L-NAME alone increased, whereas the mitochondria-targeting expression of AT 2 receptors (mito-AT 2 /GFP) attenuated the effects of mito-ECFP/Ang II ( P <0.01). In the kidney, overexpression of mito-ECFP/Ang II in the mitochondria of the PTs increased systolic blood pressure 12±3 mm Hg ( P <0.01), and the response was attenuated in PT-specific PT- Agtr1a −/− and PT- Nhe3 −/− mice ( P <0.01). Conversely, overexpression of AT 2 receptors selectively in the mitochondria of the PTs induced natriuretic responses in PT- Agtr1a −/− and PT- Nhe3 −/− mice ( P <0.01). Taken together, these results provide new evidence for a physiological role of PT mitochondrial Ang II/AT 1a /superoxide/NHE3 and Ang II/AT 2 /NO/NHE3 signaling pathways in maintaining blood pressure homeostasis.


2022 ◽  
Vol 20 (1) ◽  
Author(s):  
Mengling Wang ◽  
Feng Zeng ◽  
Fengling Ning ◽  
Yinhang Wang ◽  
Shilin Zhou ◽  
...  

Abstract Background and aims Renal fibrosis is the common outcome in all progressive forms of chronic kidney disease. Unfortunately, the pathogenesis of renal fibrosis remains largely unexplored, among which metabolic reprogramming plays an extremely crucial role in the evolution of renal fibrosis. Ceria nanoparticles (CeNP-PEG) with strong ROS scavenging and anti-inflammatory activities have been applied for mitochondrial oxidative stress and inflammatory diseases. The present study aims to determine whether CeNP-PEG has therapeutic value for renal fibrosis. Methods The unilateral ureteral obstructive fibrosis model was used to assess the therapeutic effects in vivo. Transforming growth factor beta1-induced epithelial-to-mesenchymal transition in HK-2 cells was used as the in vitro cell model. The seahorse bioscience X96 extracellular flux analyzer was used to measure the oxygen consumption rate and extracellular acidification rate. Results In the present study, CeNP-PEG treatment significantly ameliorated renal fibrosis by increased E-cadherin protein expression, and decreased α-SMA, Vimentin and Fibronectin expression both in vitro and in vivo. Additionally, CeNP-PEG significantly reduced the ROS formation and improved the levels of mitochondrial ATP. The seahorse analyzer assay demonstrated that the extracellular acidification rate markedly decreased, whereas the oxygen consumption rate markedly increased, in the presence of CeNP-PEG. Furthermore, the mitochondrial membrane potential markedly enhanced, hexokinase 1 and hexokinase 2 expression significantly decreased after treatment with CeNP-PEG. Conclusions CeNP-PEG can block the dysregulated metabolic status and exert protective function on renal fibrosis. This may provide another therapeutic option for renal fibrosis. Graphical Abstract


2019 ◽  
Vol 30 (5) ◽  
pp. 542-553 ◽  
Author(s):  
Dong Jun Kim ◽  
Mai-Tram Vo ◽  
Seong Hee Choi ◽  
Ji-Heon Lee ◽  
So Yeon Jeong ◽  
...  

Hexokinase 2 (HK2) catalyzes the first step of glycolysis and is up-regulated in cancer cells. The mechanism has not been fully elucidated. Tristetraprolin (TTP) is an AU-rich element (ARE)-binding protein that inhibits the expression of ARE-containing genes by enhancing mRNA degradation. TTP expression is down-regulated in cancer cells. We demonstrated that TTP is critical for down-regulation of HK2 expression in cancer cells. HK2 mRNA contains an ARE within its 3′-UTR. TTP binds to HK2 3′-UTR and enhances degradation of HK2 mRNA. TTP overexpression decreased HK2 expression and suppressed the glycolytic capacity of cancer cells, measured as glucose uptake and production of glucose-6-phosphate, pyruvate, and lactate. TTP overexpression reduced both the extracellular acidification rate (ECAR) and the oxygen consumption rate (OCR) of cancer cells. Ectopic expression of HK2 in cancer cells attenuated the reduction in glycolytic capacity, ECAR, and OCR from TTP. Taken together, these findings suggest that TTP acts as a negative regulator of HK2 expression and glucose metabolism in cancer cells.


Nematology ◽  
2000 ◽  
Vol 2 (5) ◽  
pp. 551-559 ◽  
Author(s):  
Lihong Qiu ◽  
Robin Bedding

AbstractEnergy metabolism of the infective juveniles (IJ) of Steinernema carpocapsae under aerobic conditions and its relation to survival and infectivity of the IJ was studied by monitoring the changes in mean dry weight, levels of key energy reserve compounds, oxygen consumption rate, respiratory quotient, survival and infectivity of freshly harvested IJ incubated in tap water on a shaker at 28°C over time. The survival rate of the IJ exceeded 90% for the first 6 weeks and then dropped sharply to about 50% at week 8. The infectivity of the IJ did not change markedly within the first 3 weeks, dropped slightly during weeks 4 and 5 and then decreased sharply to week 6. The mean dry weight of the IJ dropped at a roughly constant rate from an initial level of 74 ng/IJ to 41 ng/IJ at week 6. Thus, during the period of 6 weeks at 28°C, each IJ consumed nearly half of its initial dry weight. Consumption of the lipids, proteins, glycogen and trehalose accounted for 91, 41, 56 and 78% of the initial levels of these compounds and 47, 41, 9 and 3% of the total dry weight of materials consumed, respectively. Most lipids were consumed in the first 3 weeks while most proteins, glycogen and trehalose were consumed in the last 2 or 3 weeks. Oxygen consumption rate decreased from 3.0 to 0.5 ml/million IJ/day from day 1 to week 6 while the respiratory quotient remained constant at about 0.7 for the first 4 weeks then dropped sharply to 0.3 at week 6. Both survival time and the rate of reduction in mean dry weight of IJ were severely affected by temperature and buffering. The functions of the individual energy reserve materials, characteristics of the energy metabolism and the relationship between energy metabolism and the survival and infectivity of the IJ are discussed. Der Energiestoffwechsel und seine Beziehung zum Überleben und zur Infektivität der Infektionsjuvenilen von Steinernema carpocapsae unter aeroben Bedingungen - Der Energiestoffwechsel der Infektionsjuvenilen (IJ) von Steinernema carpocapsae unter aeroben Bedingungen und seine Beziehungen zum Überleben und zur Infektivität der IJ wurde untersucht durch die Erfassung der Änderungen im mittleren Trockengewicht, des Niveaus der wichtigen Energiereservestoffe, des Sauerstoffverbrauchs, des Respirationsquotienten sowie der Überlebensrate und der Infektivität frisch gewonnener IJ, die in Leitungswasser bei 28°C auf einem Schüttler gehalten wurden. Die Überlebensrate überschritt während der ersten sechs Wochen 90% und fiel dann in der achten Woche schnell auf etwa 50% ab. Die Infektivität änderte sich innerhalb der ersten 3 Wochen wenig, fiel dann im Laufe der Wochen 4 und 5 etwas ab, verminderte sich dann aber schnell in Woche 6. Das mittlere Trockengewicht der IJ fiel ungefähr gleichmäßig von einem Ausgangsniveau von 74 ng/IJ auf 41 ng/IJ in Woche 6 ab. Jedes IJ verbrauchte also bei 28°C innerhalb von 6 Wochen fast die Hälfte seines ursprünglichen Trockengewichts. Vom ursprünglichen Gehalt an Lipiden wurden 91% verbraucht, von Proteinen 41%, von Glykogen 56% und von Trehalose 78%. Vom Gesamtverbrauch entfielen 47% auf Lipide, 41% auf Proteine, 9% auf Glykogen und 3% auf Trehalose. Die meisten Lipide wurden in den ersten drei Wochen verbraucht, während die meisten Proteine, Glykogen und Trehalose in den letzten 2 oder 3 Wochen verbraucht wurden. Der Sauerstoffverbrauch fiel von 3,0 auf 0,5 ml/million IJ/Tag von Tag 1 bis Woche 6, während der Respirationsquotient in den ersten 4 Wochen konstant bei 0,7 und dann schnell auf 0,3 in Woche 6 abnahm. Überlebensdauer und Abnahme des mittleren Trockengewichts wurden durch Temperatur und Pufferung stark beeinflusst. Die Funktionen der einzelnen Energiereservestoffe, die Kennzeichen des Energiestoffwechsels und die Beziehungen zwischen Energiestoffwechsel, Überleben und Infektivität der IJ werden diskutiert.


Author(s):  
Grażyna Mazurkiewicz-Boroń ◽  
Teresa Bednarz ◽  
Elżbieta Wilk-Woźniak

Microbial efficiency in a meromictic reservoirIndices of microbial efficiency (expressed as oxygen consumption and carbon dioxide release) were determined in the water column of the meromictic Piaseczno Reservoir (in an opencast sulphur mine), which is rich in sulphur compounds. Phytoplankton abundances were low in both the mixolimnion (up to 15 m depth) and monimolimnion (below 15 m depth). In summer and winter, carbon dioxide release was 3-fold and 5-fold higher, respectively, in the monimolimnion than in the mixolimnion. Laboratory enrichments of the sulphur substrate of the water resulted in a decrease in oxygen consumption rate of by about 42% in mixolimnion samples, and in the carbon dioxide release rate by about 69% in monimolimnion samples. Water temperature, pH and bivalent ion contents were of major importance in shaping the microbial metabolic efficiency in the mixolimnion, whilst in the monimolimnion these relationships were not evident.


2021 ◽  
Vol 22 (9) ◽  
pp. 4366
Author(s):  
Rebecca L. Paszkiewicz ◽  
Richard N. Bergman ◽  
Roberta S. Santos ◽  
Aaron P. Frank ◽  
Orison O. Woolcott ◽  
...  

The authors wish to make the following corrections to this paper [...]


2021 ◽  
Vol 22 (16) ◽  
pp. 8367
Author(s):  
Hien Lau ◽  
Shiri Li ◽  
Nicole Corrales ◽  
Samuel Rodriguez ◽  
Mohammadreza Mohammadi ◽  
...  

Pre-weaned porcine islets (PPIs) represent an unlimited source for islet transplantation but are functionally immature. We previously showed that necrostatin-1 (Nec-1) immediately after islet isolation enhanced the in vitro development of PPIs. Here, we examined the impact of Nec-1 on the in vivo function of PPIs after transplantation in diabetic mice. PPIs were isolated from pancreata of 8–15-day-old, pre-weaned pigs and cultured in media alone, or supplemented with Nec-1 (100 µM) on day 0 or on day 3 of culture (n = 5 for each group). On day 7, islet recovery, viability, oxygen consumption rate, insulin content, cellular composition, insulin secretion capacity, and transplant outcomes were evaluated. While islet viability and oxygen consumption rate remained high throughout 7-day tissue culture, Nec-1 supplementation on day 3 significantly improved islet recovery, insulin content, endocrine composition, GLUT2 expression, differentiation potential, proliferation capacity of endocrine cells, and insulin secretion. Adding Nec-1 on day 3 of tissue culture enhanced the islet recovery, proportion of delta cells, beta-cell differentiation and proliferation, and stimulation index. In vivo, this leads to shorter times to normoglycemia, better glycemic control, and higher circulating insulin. Our findings identify the novel time-dependent effects of Nec-1 supplementation on porcine islet quantity and quality prior to transplantation.


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