scholarly journals TCA Cycle and Mitochondrial Membrane Potential Are Necessary for Diverse Biological Functions

2016 ◽  
Vol 61 (2) ◽  
pp. 199-209 ◽  
Author(s):  
Inmaculada Martínez-Reyes ◽  
Lauren P. Diebold ◽  
Hyewon Kong ◽  
Michael Schieber ◽  
He Huang ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Tca Cycle ◽  
Biological Functions

scholarly journals Effects of inflammatory and anti-inflammatory environments on the macrophage mitochondrial function

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Dong Ji ◽  
Jian-yun Yin ◽  
Dan-feng Li ◽  
Chang-tai Zhu ◽  
Jian-ping Ye ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Citrate Synthase ◽  
Tca Cycle ◽  
Raw 264.7 Cells ◽  
Metabolic Adaptation ◽  
Isocitrate Dehydrogenase 2 ◽  
Anti Inflammatory

AbstractMitochondrial response to inflammation is crucial in the metabolic adaptation to infection. This study aimed to explore the mitochondrial response under inflammatory and anti-inflammatory environments, with a focus on the tricarboxylic acid (TCA) cycle. Expression levels of key TCA cycle enzymes and the autophagy-related protein light chain 3b (LC3b) were determined in raw 264.7 cells treated with lipopolysaccharide (LPS) and metformin (Met). Additionally, reactive oxygen species (ROS) levels and mitochondrial membrane potential were assessed using flow cytometry. Moreover, 8-week-old C57BL/6J mice were intraperitoneally injected with LPS and Met to assess the mitochondrial response in vivo. Upon LPS stimulation, the expression of key TCA enzymes, including citrate synthase, α-ketoglutarate dehydrogenase, and isocitrate dehydrogenase 2, and the mitochondrial membrane potential decreased, whereas the levels of LC3b and ROS increased. However, treatment with Met inhibited the reduction of LPS-induced enzyme levels as well as the elevation of LC3b and ROS levels. In conclusion, the mitochondrial TCA cycle is affected by the inflammatory environment, and the LPS-induced effects can be reversed by Met treatment.

30 OXYGEN DEPRIVATION DOES NOT FURTHER AUGMENT MITOCHONDRIAL MEMBRANE POTENTIAL IN PHARMACOLOGICALLY TREATED FIBROBLASTS FOR USE IN SOMATIC CELL NUCLEAR TRANSFER

2017 ◽  
Vol 29 (1) ◽  
pp. 122
Author(s):  
B. R. Mordhorst ◽  
S. N. Bogue ◽  
K. D. Wells ◽  
J. A. Green ◽  
R. S. Prather
Keyword(s):  
Membrane Potential ◽  
Somatic Cell ◽  
Mitochondrial Membrane Potential ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Mitochondrial Membrane ◽  
Somatic Cells ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Donor Cells

Somatic cells commonly used in nuclear transfer primarily utilise the tricarboxylic acid cycle and cellular respiration for energy production. Comparatively, the metabolism of somatic cells contrasts that of cells within early embryos, which predominantly use glycolysis and exhibit Warburg Effect (WE)-like characteristics. We hypothesised that fibroblast cells can become more blastomere-like if driven either pharmacologically or by oxygen constraint and could result in improved in vitro embryonic development after somatic cell nuclear transfer (SCNT). The pharmaceuticals used (PS48 and CPI-613) should decrease mitochondrial use of the tricarboxylic acid (TCA) cycle and promote the PI3K pathway, respectively. Furthermore, we hypothesised that oxygen constraint (1.3%) would hinder TCA cycle activity and promote glycolysis. The goal was to achieve a WE-like effect in donor cells before nuclear transfer (NT) by treating Day 35 porcine fetal fibroblasts with CPI-613 (100 µM), PS48 (10 µM), both drugs combined (MIX), or as controls (CON, 0 µM) for 7 days under stepwise oxygen constraint (OC; 1.3%) or under normal conditions (ON; 5%). Three biological replicates were collected and data were analysed for main effect of treatment via GLM procedure of SAS 9.4 (SAS Institute Inc., Cary, NC, USA). To determine if our treatments affected mitochondria respiratory capacity (thereby TCA cycle capability) within embryos, we measured mitochondrial membrane potential (Δψm) using JC-10, a biphasic cationic dye. Mitotracker green (MTG) was used to estimate mitochondrial quantity. The percentage of cells with low Δψm was increased (P = 0.02) with any CPI or MIX treatment (treatments ≥ 95%) compared with OC-PS48 and both control (ON and OC) treatments (treatments ≥ 77.4%), whereas ON-PS48 had an intermediate level (90.4%; error = 4.9%). Contrary to our prediction, MTG intensity was lower across all ON treatments compared with OC treatments (NO treatments ≤ 736 AU v. OC treatments ≥ 872 AU; error = 23 AU; P < 0.01). Regardless of oxygen level, controls and PS48 treatments yielded the highest percentages of viable cells (treatments ≥ 94%) and OC-CPI and NO-MIX the lowest (treatments ≤ 86%) with NO-CPI and OC-MIX being intermediate (treatments ≥ 90%; error = 3%; P < 0.01). Oxygen constraint did not promote a reduction in mitochondrial membrane potential in pharmacologically treated fibroblasts. Additionally, intensity of MTG was increased in fibroblasts cultured under oxygen constraint compared with those cultured in 5% oxygen. Our results warrant further investigation of the mitochondrial changes occurring with oxygen deprivation in donor-cells. Experiments are underway to determine if gene expression in cells treated pharmacologically and with oxygen constraint are augmented, and whether these treatments will result in better development after SCNT. This study was funded by Food for the 21 st Century and NIH R01HD080636.

scholarly journals Monitoring of relative mitochondrial membrane potential in living cells by fluorescence microscopy.

1981 ◽  
Vol 88 (3) ◽  
pp. 526-535 ◽  
Author(s):  
L V Johnson ◽  
M L Walsh ◽  
B J Bockus ◽  
L B Chen
Keyword(s):  
Membrane Potential ◽  
Fluorescence Microscopy ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Specific Interaction ◽  
Living Cells ◽  
Cellular Level ◽  
Energy Requirements ◽  
Active Movement ◽  
Biological Functions

Permeant cationic fluorescent probes are shown to be selectively accumulated by the mitochondria of living cells. Mitochondria-specific interaction of such molecules is apparently dependent on the high trans-membrane potential (inside negative) maintained by functional mitochondria. Dissipation of the mitochondrial trans-membrane and potential by ionophores or inhibitors of electron transport eliminates the selective mitochondrial association of these compounds. The application of such potential-dependent probes in conjunction with fluorescence microscopy allows the monitoring of mitochondrial membrane potential in individual living cells. Marked elevations in mitochondria-associated probe fluorescence have been observed in cells engaged in active movement. This approach to the analysis of mitochondrial membrane potential should be of value in future investigations of the control of energy metabolism and energy requirements of specific biological functions at the cellular level.

Platelets apoptosis in rats after global brain ischemia

2018 ◽  
pp. 27-35
Author(s):  
А.А. Соколовская ◽  
Э.Д. Вирюс ◽  
В.В. Александрин ◽  
А.С. Роткина ◽  
К.А. Никифорова ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Ischemic Stroke ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Brain Ischemia ◽  
Mitochondrial Membrane ◽  
Male Rats ◽  
Global Brain Ischemia ◽  
Platelet Apoptosis ◽  
Global Brain

Цель исследования. Ишемические повреждения головного мозга, являются одной из наиболее частой причин инвалидности и смертности во всем мире. Недавно была установлена роль апоптоза тромбоцитов в патофизиологии инсульта, однако его механизмы до сих пор остаются невыясненными. Несмотря на различные экспериментальные модели, направленные на мониторинг апоптоза тромбоцитов, результаты, относительно изучения и выявления апоптоза тромбоцитов при ишемии головного мозга у крыс, весьма немногочисленны. Цель исследования - анализ апоптоза тромбоцитов с помощью метода проточной цитофлуориметрии на модели глобальной ишемии мозга у крыс. Методика. В экспериментах использовано 6 крыс-самцов Вистар в возрасте от 5 до 6 мес., разделенных на 2 группы: интактный контроль (К) и глобальная ишемия головного мозга. Модель глобальной ишемии головного мозга у крыс воспроизводилась путём билатеральной окклюзии общих сонных артерий на фоне гипотензии. Уровень системного артериального давления снижали посредством кровопотери до 40-45 мм рт. ст. Суспензию тромбоцитов крыс получали методом гельфильтрации с использованием сефарозы 2B. Для анализа экстернализации фосфатидилсерина (ФС) тромбоциты крыс инкубировали с Аннексином V-PE в связывающем буфере. Для оценки митохондриального мембранного потенциала (ММП) тромбоциты инкубировали с катионным красителем JC-1. После инкубации образцы немедленно анализировали на проточном цитофлуориметре FACSCalibur (Becton Dickinson, США). Результаты. Согласно полученным данным, экстернализация ФС на тромбоцитах крыс, перенесших инсульт, была значительно выше (53,45 ± 4,21%), чем в контрольной группе крыс (5,27 ± 2,40%). Данный эффект подтверждается выраженной деполяризацией митохондриальных мембран (DYm). После экспериментальной ишемии мозга почти 40% тромбоцитов было деполяризовано. Заключение. Использованный в работе подбор методов и маркеров обеспечивает понимание механизмов апоптоза тромбоцитов как в экспериментальных, так и в клинических условиях. Полученные данные позволяют сделать заключение, что апоптоз тромбоцитов является одним из факторов развития глобальной ишемии головного мозга у крыс. Результаты могут быть использованы для понимания механизмов, участвующих в развитии ишемического повреждения, что, в свою очередь, может быть использовано при разработке новых терапевтических стратегий. Aim. Stroke is one of the most common causes of disability and mortality worldwide. Multiple experimental models of stroke have focused on monitoring of platelet apoptosis. However, studies on and detection of platelet apoptosis in rats with ischemic stroke are very scarce. We investigated platelet apoptosis in rats with global brain ischemia using flow cytometry. Methods. Experiments were carried out on healthy, adult Wistar male rats weighing 300-350 g. The rats were divided into the following 2 groups: intact rats and rats with global brain ischemia. Global brain ischemia was induced by two-vessel (2-VO) carotid occlusion in combination with hypotension. Systemic blood pressure was reduced by 40-45 mm Hg by inducing haemorrhage. Platelets were isolated by gel filtration on Sepharose 2B. For evaluation of phosphatidylserine (PS) externalization, platelets were incubated with Annexin V-PE and analyzed on FACSCalibur (BD Biosciences). Mitochondrial membrane potential (DY) was measured during platelets apoptosis using JC-1, a mitochondrial membrane potential indicator. Platelets were analyzed by flow cytometry immediately after the incubation. Results. PS externalization on platelets was significantly greater after global brain ischemia (53.45 ± 4.21%) than in the control group (5.27 ± 2.40%). Pronounced depolarization of mitochondrial membrane potential (DYm) confirmed this finding. In the rat group with experimental brain ischemia, almost 40% (35.24 ± 5.21%) of platelets were depolarized. Conclusion. Our results provide insight into mechanisms involved in platelet apoptosis during ischemic stroke and can be used in further development of new therapeutic strategies.

Reserpine Induces Apoptosis and Cell Cycle Arrest in Hormone Independent Prostate Cancer Cells through Mitochondrial Membrane Potential Failure

2019 ◽  
Vol 18 (9) ◽  
pp. 1313-1322 ◽  
Author(s):  
Manjula Devi Ramamoorthy ◽  
Ashok Kumar ◽  
Mahesh Ayyavu ◽  
Kannan Narayanan Dhiraviam
Keyword(s):  
Prostate Cancer ◽  
Reactive Oxygen Species ◽  
Cell Cycle ◽  
Membrane Potential ◽  
Cancer Cells ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Reactive Oxygen ◽  
Oxygen Species ◽  
Prostate Cancer Cells

Background: Reserpine, an indole alkaloid commonly used for hypertension, is found in the roots of Rauwolfia serpentina. Although the root extract has been used for the treatment of cancer, the molecular mechanism of its anti-cancer activity on hormonal independent prostate cancer remains elusive. Methods: we evaluated the cytotoxicity of reserpine and other indole alkaloids, yohimbine and ajmaline on Prostate Cancer cells (PC3) using MTT assay. We investigated the mechanism of apoptosis using a combination of techniques including acridine orange/ethidium bromide staining, high content imaging of Annexin V-FITC staining, flow cytometric quantification of the mitochondrial membrane potential and Reactive Oxygen Species (ROS) and cell cycle analysis. Results: Our results indicate that reserpine inhibits DNA synthesis by arresting the cells at the G2 phase and showed all standard sequential features of apoptosis including, destabilization of mitochondrial membrane potential, reduced production of reactive oxygen species and DNA ladder formation. Our in silico analysis further confirmed that indeed reserpine docks to the catalytic cleft of anti-apoptotic proteins substantiating our results. Conclusion: Collectively, our findings suggest that reserpine can be a novel therapeutic agent for the treatment of androgen-independent prostate cancer.

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