proton leak
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Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 957-957
Author(s):  
Orlando Esparza ◽  
Giovanny Hernandez ◽  
Rachelle Nuss ◽  
David Irwin ◽  
Marguerite Kelher ◽  
...  

Abstract BACKGROUND: Sickle cell disease (SCD) is a group of inherited hemoglobinopathies that continues to be highly morbid and lethal. SCD-associated platelet hyperreactivity is a well-recognized contributor to the pathophysiology of the disease via complex interactions with the immune system and endothelium. Aberrant platelet bioenergetics have been implicated as a biological mechanism for SCD-associated platelet hyperreactivity, however, little is known about the impact current medical interventions (e.g., hydroxyurea [HU] and red blood cell [RBC] exchange transfusions) have on the platelet functional-bioenergetic profile. In this study we investigate the effects of hydroxyurea and RBC exchange transfusions on reprograming the platelet functional-bioenergetic profile and provide insight into biological pathways that may be amenable to intervention. METHODS: Platelets from sex-, race-, and aged-matched adult healthy control subjects and adult patients with homozygous SCD (HbSS), actively being treated with hydroxyurea (HU group) or RBC exchange transfusions (RBC exchange transfusion group), were isolated and washed following standard protocols. Platelet activation by flow cytometry was determined at baseline and following activation with thrombin (0.075U/ml) and ADP (1.25uM). Platelet-activated fibrinogen binding site (αIIbβIII), P-selectin, and phosphatidylserine (PS) surface marker expression (as measured by mean fluorescence intensity [MFI]) was determined with PAC-1, P-selectin, and lactadherin antibodies, respectively. The bioenergetic profile of washed platelets was determined by the 24-well format Seahorse extracellular flux analyzer. Statistical analyses were performed using the one-way ANOVA. Correlations were performed by 2-tailed nonparametric Spearman correlations and linear regression analysis with 95% confidence interval (GraphPad Software v9.1.2). Data expressed as mean plus or minus standard error of the mean (SEM). Differences were considered significant at p < 0.05. RESULTS: Platelets from patients in the HU group exhibited increased surface marker expression of αIIbβIII (p = 0.004), P-selectin (p = 0.003), and PS (p = 0.003) at resting conditions when compared to the RBC exchange transfusion group and healthy controls. Additionally, an increase in PS expression was seen in the HU group upon activation with ADP (p = 0.0003). No significant differences were seen in the platelet functional profile after activation with thrombin. The platelet bioenergetic profile in the HU group demonstrated an elevated proton leak (p = 0.03) when compared to the RBC exchange transfusion group. Elevated proton leak in SCD was found to have positive correlation with P-selectin and PS expression (Figure 1). CONCLUSION: While therapeutic interventions have improved overall outcomes in patients with SCD, adverse events continue to be a deterrent to many patients prompting the need for safer, more tolerable, and cost-effective alternatives. We have identified that while HU has little impact on the hyperreactive and procoagulant platelet phenotype in SCD, RBC exchange transfusions appear to mitigate the phenotype and reprogram the bioenergetic profile. Amongst treatment groups, a strong correlation was found between platelet activation markers (i.e., P-selectin and PS) and proton leak, suggesting an interplay between alterations in platelet bioenergetics and SCD-associated platelet hyperreactivity. Further studies are needed to elucidate the metabolic pathways that are responsible for the aberrant platelet functional-bioenergetic profile seen in SCD. These observations are important as targeting the platelet bioenergetic profile via less invasive and toxic therapeutic modalities may be equally efficacious as current interventions. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.


Author(s):  
Jennifer B. M. Steffen ◽  
Fouzia Haider ◽  
Eugene P. Sokolov ◽  
Christian Bock ◽  
Inna M. Sokolova

Oxygen fluctuations are common in marine waters, and hypoxia/reoxygenation (H/R) stress can negatively affect mitochondrial metabolism. The long-lived ocean quahog, Arctica islandica, is known for its hypoxia tolerance associated with metabolic rate depression, yet the mechanisms that sustain mitochondrial function during oxygen fluctuations are not well understood. We used top-down metabolic control analysis (MCA) to determine aerobic capacity and control over oxygen flux in the mitochondria of quahogs exposed to short-term hypoxia (24 h <0.01% O­2) and subsequent reoxygenation (1.5 h 21% O­2) compared to normoxic control animals (21% O­2). We demonstrated that flux capacities of the substrate oxidation and proton leak subsystems were not affected by hypoxia, while the capacity of the phosphorylation subsystem was enhanced during hypoxia associated with a depolarization of the mitochondrial membrane. Reoxygenation decreased oxygen flux capacities of all three mitochondrial subsystems. Control over oxidative phosphorylation (OXPHOS) respiration was mostly exerted by substrate oxidation regardless of H/R stress, whereas the control of the proton leak subsystem over LEAK respiration increased during hypoxia and returned to normoxic level during reoxygenation. During hypoxia, reactive oxygen species (ROS) efflux was elevated in the LEAK state, while suppressed in the OXPHOS state. Mitochondrial ROS efflux returned to normoxic control levels during reoxygenation. Thus, mitochondria of A. islandica appear robust to hypoxia by maintaining stable substrate oxidation and upregulating phosphorylation capacity, but remain sensitive to reoxygenation. This mitochondrial phenotype might reflect adaptation of A. islandica to environments with unpredictable oxygen fluctuations and its behavioural preference for low oxygen levels.


2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 105-106
Author(s):  
Mia Y Kawaida ◽  
Oh-Sung Kwon ◽  
Ahram Anh ◽  
Amanda S Reiter ◽  
Nicole M Tillquist ◽  
...  

Abstract Mitochondria are critical for oxidative phosphorylation in skeletal muscle, especially in athletic species such as the horse. Mitochondrial respiration increases with physical exercise, but the relationship between mitochondrial respiration and cardiovascular functions are not well described in the horse. The objective of this study was to determine if there is a relationship between heart rate (HR) during and after submaximal exercise tests (SETs) and skeletal muscle mitochondrial respiration in polo ponies. We hypothesized that horses with greater maximum HR and average HR during the exercise tests would have greater mitochondrial respiration in skeletal muscle. Twelve fit polo ponies (14.8 ± 1.7 years old, 10 mares and 2 geldings) were equipped with Polar equine heart rate monitors (Polar Electro Inc., Lake Success, NY) and underwent 26-minute SETs designed to mimic a polo chukker followed by a 30-minute recovery period. Muscle biopsy samples from the semitendinosus muscle were taken 2 weeks prior to the SET to determine mitochondrial oxygen consumption using the Oroboros O2k high-resolution respirometer (Oroboros Instruments, Innsbruck, Austria). Data were analyzed using the PROC CORR procedure (SAS Inst. Inc., Cary, NC). Correlations were considered strong at r > 0.6 and significant at P < 0.05. Maximum HR during SET and state IV respiration were positively correlated (P = 0.02, r = 0.68). Average HR during SET and state IV respiration were also positively correlated (P = 0.01, r = 0.72). However, correlations between maximum and average HR and state III respiration were not as strong (P ≥ 0.05, r < 0.6). These data suggest that state IV mitochondrial respiration (proton leak) in equine skeletal muscle may impact cardiac responses to high-intensity exercise. Horses with higher HR during exercise may have less efficient oxidative phosphorylation, resulting in earlier fatigue and/or greater formation of reactive oxygen species resulting from proton leak.


2021 ◽  
Author(s):  
Wen-Wei Tseng ◽  
Ching-Hsiang Chu ◽  
Chen Chang ◽  
Yi-Ju Lee ◽  
Shirui Zhao ◽  
...  

Cellular bioenergetics and mitochondrial dynamics are crucial for the secretion of insulin by pancreatic beta cells in response to elevated blood glucose concentrations. To obtain better insights into the interactions between energy production and mitochondrial fission/fusion dynamics, we combine live-cell mitochondria imaging with biophysical-based modeling and network analysis to elucidate the principle regulating mitochondrial morphology to match metabolic demand in pancreatic beta cells. A minimalistic differential equation-based model for beta cells was constructed to include glycolysis, oxidative phosphorylation, simple calcium dynamics, and graph-based fission/fusion dynamics controlled by ATP synthase flux and proton leak flux. The model revealed that mitochondrial fission occurs in response to hyperglycemia, starvation, ATP synthase inhibition, uncoupling, and diabetic condition, in which the rate of proton leak exceeds the rate of mitochondrial ATP synthesis. Under these metabolic challenges, the propensities of tip-to-tip fusion events simulated from the microscopic images of the mitochondrial networks were lower than those in the control group and prevented mitochondrial network formation. The modeling and network analysis could serve as the basis for further detailed research on the mechanisms of bioenergetics and mitochondrial dynamics coupling.


Author(s):  
Danielle E. Levitt ◽  
Tekeda F Ferguson ◽  
Stefany DePrato Primeaux ◽  
Jeanette A Zavala ◽  
Jameel Ahmed ◽  
...  

At-risk alcohol use is prevalent and increases dysglycemia among people living with human immunodeficiency virus (PLWH). Skeletal muscle (SKM) bioenergetic dysregulation is implicated in dysglycemia and type 2 diabetes. The objective of this study was to determine the relationship between at-risk alcohol, glucose tolerance, and SKM bioenergetic function in PLWH. Thirty-five PLWH (11 females, 24 males, age: 53±9 yrs, body mass index: 29.0±6.6 kg/m2) with elevated fasting glucose enrolled in the ALIVE-Ex study provided medical history and alcohol use information (Alcohol Use Disorders Identification Test, AUDIT), then underwent an oral glucose tolerance test (OGTT) and SKM biopsy. Bioenergetic health and function and mitochondrial volume were measured in isolated myoblasts. Mitochondrial gene expression was measured in SKM. Linear regression adjusting for age, sex, and smoking was performed to examine the relationship between glucose tolerance (2-h glucose post-OGTT), AUDIT, and their interaction with each outcome measure. Negative indicators of bioenergetic health were significantly (p<0.05) greater with higher 2-h glucose (proton leak) and AUDIT (proton leak, non-mitochondrial oxygen consumption, and bioenergetic health index). Mitochondrial volume was increased with the interaction of higher 2-h glucose and AUDIT. Mitochondrial gene expression decreased with higher 2-h glucose (TFAM, PGC1B, PPARG, MFN1), AUDIT (MFN1, DRP1, MFF), and their interaction (PPARG, PPARD, MFF). Decreased expression of mitochondrial genes were coupled with increased mitochondrial volume and decreased bioenergetic health in SKM of PLWH with higher AUDIT and 2-h glucose. We hypothesize these mechanisms reflect poorer mitochondrial health and may precede overt SKM bioenergetic dysregulation observed in type 2 diabetes.


2021 ◽  
Vol 12 ◽  
Author(s):  
Erik Rollwitz ◽  
Martin Jastroch

Oxygen consumption allows measuring the metabolic activity of organisms. Here, we adopted the multi-well plate-based respirometry of the extracellular flux analyzer (Seahorse XF96) to investigate the effect of temperature on the bioenergetics of zebrafish embryos (Danio rerio) in situ. We show that the removal of the embryonic chorion is beneficial for oxygen consumption rates (OCR) and penetration of various mitochondrial inhibitors, and confirm that sedation reduces the variability of OCR. At 48h post-fertilization, embryos (maintained at a routine temperature of 28°C) were exposed to different medium temperatures ranging from 18°C to 37°C for 20h prior OCR measurement. Measurement temperatures from 18°C to 45°C in the XF96 were achieved by lowering the room temperature and active in-built heating. At 18°C assay temperature, basal OCR was low due to decreased ATP-linked respiration, which was not limited by mitochondrial power, as seen in substantial spare respiratory capacity. Basal OCR of the embryos increased with assay temperature and were stable up to 37°C assay temperature, with pre-exposure of 37°C resulting in more thermo-resistant basal OCR measured at 41°C. Adverse effects of the mitochondrial inhibitor oligomycin were seen at 37°C and chemical uncouplers disrupted substrate oxidation gradually with increasing assay temperature. Proton leak respiration increased at assay temperatures above 28°C and compromised the efficiency of ATP production, calculated as coupling efficiency. Thus, temperature impacts mitochondrial respiration by reduced cellular ATP turnover at lower temperatures and by increased proton leak at higher temperatures. This conclusion is coherent with the assessment of heart rate, an independent indicator of systemic metabolic rate, which increased with exposure temperature, peaking at 28°C, and decreased at higher temperatures. Collectively, plate-based respirometry allows assessing distinct parts of mitochondrial energy transduction in zebrafish embryos and investigating the effect of temperature and temperature acclimation on mitochondrial bioenergetics in situ.


2021 ◽  
Vol 2021 ◽  
pp. 1-7
Author(s):  
Gisela Chelimsky ◽  
Pippa Simpson ◽  
Liyun Zhang ◽  
Doug Bierer ◽  
Steve Komas ◽  
...  

Background. Fatigue is often the primary complaint of children with functional gastrointestinal disorders (FGDI) and other chronic overlapping pain disorders (COPC). The basis for this symptom remains unknown. We evaluated mitochondrial function in the white blood cells of these patients. Methods. This prospective Children’s Wisconsin IRB approved study recruited subjects aging 10–18 years from pediatric neurogastroenterology clinics and healthy comparison subjects (HC). Environmental and oxidative stressors can damage the mitochondrial respiratory chain. The known low-grade inflammation in COPC could, therefore, impact the respiratory chain and theoretically account for the disabling fatigue so often voiced by patients. Mitochondrial energy generation can be easily measured in peripheral mononuclear cells (PMC) as a general marker by the Seahorse XF96 Extracellular Flux Analyzer. We measured 5 parameters of oxygen consumption using this methodology: basal respiration (BR), ATP linked oxygen consumption (ATP-LC), maximal oxygen consumption rate (max R), spare respiratory capacity (SRC), and extracellular acidification rate (ECAR), which reflect non-electron chain energy generation through glycolysis. In health, we expect high ATP linked respiration, high reserve capacity, low proton leak, and low non-mitochondrial respiration. In disease, the proton leak typically increases, ATP demand increases, and there is decreased reserve capacity with increased non-mitochondrial respiration. Findings and clinical data were compared to healthy control subjects using a Mann–Whitney test for skewed variables, Fisher’s exact test for dichotomous variables, and regression tree for association with functional outcome (functional disability inventory, FDI). Results. 19 HC and 31 COPC showed no statistically significant difference in age. FGID, orthostatic intolerance, migraine, sleep disturbance, and chronic fatigue were present in the majority of COPC subjects. BR, ECAR, and ATP-LC rates were lower in the COPC group. The low BR and ATP-LC suggest that mitochondria are stressed with decreased ability to produce ATP. Tree analysis selected SRC as the best predictor of functional disability: patients with SRC >150 had a greater FDI (more disability) compared to patients with SRC <=150, p -value = 0.021. Conclusion. Subjects with COPC have reduced mitochondrial capacity to produce ATP. Predisposing genetic factors or reversible acquired changes may be responsible. A higher SRC best predicts disability. Since a higher SRC is typically associated with more mitochondrial reserve, the SRC may indicate an underutilized available energy supply related to inactivity, or a “brake” on mitochondrial function. Prospective longitudinal studies can likely discern whether these findings represent deconditioning, true mitochondrial dysfunction, or both.


Life Sciences ◽  
2021 ◽  
pp. 119614
Author(s):  
Yanhong Xu ◽  
Shiqiao Peng ◽  
Xinyu Cao ◽  
Shengnan Qian ◽  
Shuang Shen ◽  
...  

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 569
Author(s):  
Vadim S. Ten ◽  
Anna A. Stepanova ◽  
Veniamin Ratner ◽  
Maria Neginskaya ◽  
Zoya Niatsetskaya ◽  
...  

This review discusses the potential mechanistic role of abnormally elevated mitochondrial proton leak and mitochondrial bioenergetic dysfunction in the pathogenesis of neonatal brain and lung injuries associated with premature birth. Providing supporting evidence, we hypothesized that mitochondrial dysfunction contributes to postnatal alveolar developmental arrest in bronchopulmonary dysplasia (BPD) and cerebral myelination failure in diffuse white matter injury (WMI). This review also analyzes data on mitochondrial dysfunction triggered by activation of mitochondrial permeability transition pore(s) (mPTP) during the evolution of perinatal hypoxic-ischemic encephalopathy. While the still cryptic molecular identity of mPTP continues to be a subject for extensive basic science research efforts, the translational significance of mitochondrial proton leak received less scientific attention, especially in diseases of the developing organs. This review is focused on the potential mechanistic relevance of mPTP and mitochondrial dysfunction to neonatal diseases driven by developmental failure of organ maturation or by acute ischemia-reperfusion insult during development.


2021 ◽  
Author(s):  
Elham Khosrowabadi ◽  
Antti Rivinoja ◽  
Maija Risteli ◽  
Anne Tuomisto ◽  
Tuula Salo ◽  
...  

AbstractProper functioning of each secretory and endocytic compartment relies on its unique pH micro-environment that is known to be dictated by the rates of V-ATPase-mediated H+ pumping and its leakage back to the cytoplasm via an elusive “H+ leak” pathway. Here, we show that this proton leak across Golgi membranes involves AE2a (SLC4A2a)-mediated bicarbonate-chloride exchange, as it is strictly dependent on both bicarbonate import (in exchange of chloride export) and the AE2a expression level in the cells. Imported bicarbonate anions and luminal protons then facilitate a common buffering reaction that yields carbon dioxide and water before their egress back to the cytoplasm via diffusion or water channels. The high surface-volume ratio of flattened Golgi cisternae helps this process, as their shape is optimal for water and gas exchange. Interestingly, this pathway is often upregulated in cancers and established cancer cell lines, and responsible for their markedly elevated Golgi resting pH and attenuated glycosylation potential. Accordingly, AE2 knockdown in SW-48 colorectal cancer cells was able to restore these two phenomena, and at the same time, to reverse cells’ invasive and anchorage-independent growth phenotype. These findings suggest that a malignant cell can be returned to a benign state by normalizing its Golgi resting pH.


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