Role of band 3 in regulating metabolic flux of red blood cells.
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ABSTRACT:
SUBMITTER: Lewis IA
PROVIDER: S-EPMC2773988 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Under static conditions, mammalian red blood cells (RBCs) require a continuous supply of energy, typically via glucose, to maintain their biconcave disc shape. Mechanical distortion, in a complementary way, should lead to increased energy demand that is manifest in accelerated glycolysis. The experimental challenge in observing this phenomenon was met by reversibly and reproducibly distorting the cells and noninvasively measuring glycolytic flux. This was done with a gel-distorting device that was coupled with 13C nuclear magnetic resonance (NMR) spectroscopy. We measured [3-13C]l-lactate production from [1,6-13C]d-glucose in the RBCs suspended in gelatin gels, and up to 90% rate enhancements were recorded. Thus, for the first time, we present experiments that demonstrate the linkage of mechanical distortion to metabolic changes in whole mammalian cells. In seeking a mechanism for the linkage between shape and energy supply, we measured transmembrane cation flux with Cs+ (as a K+ congener) using 133Cs NMR spectroscopy, and the cation flux was increased up to fivefold. The postulated mechanism for these notable (in terms of whole-body energy consumption) responses is stimulation of Ca-adenosine triphosphatase by increased transmembrane flux of Ca2+ via the channel protein Piezo1 and increased glycolysis because its flux is adenosine triphosphate demand-regulated.
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Project description:BACKGROUND:Red blood cell exchange (RCE) transfusions are a mainstay in the treatment of sickle cell anemia (SCA), and allow a temporary restoration of physiological parameters with respect to erythrocyte oxygen carrying capacity and systems metabolism. Recently, we noted that 1) RCE significantly impacts recipients' metabolism in SCA; 2) fresh and end-of-storage red blood cell (RBC) units differently impact systems of metabolism in healthy autologous recipients; and 3) phosphate/inosine/pyruvate/adenine (PIPA) solution reverses the metabolic age of stored RBCs. Therefore, we hypothesized that RCE with PIPA-treated RBC units could further increase the metabolic benefits of RCE in SCA patients. STUDY DESIGN AND METHODS:Circulating plasma and erythrocytes were collected from patients with SCA before and after RCE, with either conventional or PIPA-treated RBC units, prior to metabolomics analyses. RESULTS:Consistent with prior work, RCE significantly decreased circulating levels of markers of systemic hypoxemia (lactate, succinate) and decreased plasma levels of acyl-carnitines and amino acids. However, PIPA-treated exchanges were superior to untreated RCEs, with a higher energy state and an increased capacity to activate the pentose phosphate pathway and glutamine metabolism. In addition, RBCs and plasma from recipients of PIPA-treated RBC units resulted in significantly decreased levels of post-transfusion plasticizers, though at the expense of higher circulating levels of oxidized purines (hypoxanthine, xanthine, and the antioxidant urate). CONCLUSION:Transfusion of PIPA-treated RBCs further increases the metabolic benefits of RCE to patients with SCA, significantly reducing the levels of post-transfusion plasticizers.
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Project description:Cellular metabolic fluxes are determined by enzyme activities and metabolite abundances. Biochemical approaches reveal the impact of specific substrates or regulators on enzyme kinetics but do not capture the extent to which metabolite and enzyme concentrations vary across physiological states and, therefore, how cellular reactions are regulated. We measured enzyme and metabolite concentrations and metabolic fluxes across 25 steady-state yeast cultures. We then assessed the extent to which flux can be explained by a Michaelis-Menten relationship between enzyme, substrate, product, and potential regulator concentrations. This revealed three previously unrecognized instances of cross-pathway regulation, which we biochemically verified. One of these involved inhibition of pyruvate kinase by citrate, which accumulated and thereby curtailed glycolytic outflow in nitrogen-limited yeast. Overall, substrate concentrations were the strongest driver of the net rates of cellular metabolic reactions, with metabolite concentrations collectively having more than double the physiological impact of enzymes.
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Project description:Metabolic rate is a key ecological variable that quantifies the energy expenditure needed to fuel almost all biological processes in an organism. Metabolic rates are typically measured at the whole-organism level (woMR) with protocols that can elicit stress responses due to handling and confinement, potentially biasing resulting data. Improved, non-stressful methodology would be especially valuable for measures of field metabolic rate, which quantifies the energy expenditure of free-living individuals. Recently, techniques to measure cellular metabolic rate (cMR) in mitochondria of blood cells have become available, suggesting that blood-based cMR can be a proxy of organismal aerobic performance. Aerobic metabolism actually takes place in the mitochondria. Quantifying cMR from blood samples offers several advantages such as direct estimates of metabolism and minimized disturbance of individuals. To our knowledge, the hypothesis that blood-based cMR correlates with woMR has not yet been directly tested. We measured cMR in red blood cells of captive great tits (Parus major), first during their morning activity period and second after subjecting them to a 2.5 h day-time respirometry protocol to quantify woMR. We predicted cMR to decrease as individuals transitioned from an active to a resting state. In the two blood samples we also assessed circulating corticosterone concentrations to determine the perceived disturbance of individuals. From respirometry traces we extracted initial and final woMR measures to test for a predicted positive correlation with cMR measures, while accounting for corticosterone concentrations. Indeed, cMR declined from the first to the second measurement. Furthermore, woMR and cMR were positively related in individuals that had relatively low corticosterone concentrations and displayed little locomotor activity throughout respirometry. By contrast, woMR and cMR covaried negatively in birds that increased corticosterone concentrations and activity levels substantially. Our results show that red blood cell cMR represents a proxy for woMR when birds do not display signs of stress, i.e., either before increases in hormonal or behavioral parameters have occurred or after they have abated. This method represents a valuable tool for obtaining metabolic data repeatedly and in free-living individuals. Our findings also highlight the importance of accounting for individual stress responses when measuring metabolic rate at any level.
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Project description:Anaerobic red blood cell (RBC) storage reduces oxidative damage, maintains adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (DPG) levels, and has superior 24-hour recovery at 6 weeks compared to standard storage. This study will determine if removal of CO2 during O2 depletion by gas exchange may affect RBCs during anaerobic storage.This is a matched three-arm study (n?=?14): control, O2 and CO2 depleted with Ar (AN), and O2 depleted with 95%Ar/5%CO2 (AN[CO2 ]). RBCs in additives AS-3 or OFAS-3 were evenly divided into three bags, and anaerobic conditions were established by gas exchange. Bags were stored at 1 to 6°C in closed chambers under anaerobic conditions or ambient air, sampled weekly for up to 9 weeks for a panel of in vitro tests. A full metabolomics screening was conducted for the first 4 weeks of storage.Purging with Ar (AN) results in alkalization of the RBC and increased glucose consumption. The addition of 5% CO2 to the purging gas prevented CO2 loss with an equivalent starting and final pH and lactate to control bags (p?>?0.5, Days 0-21). ATP levels are higher in AN[CO2 ] (p?<?0.0001). DPG was maintained beyond 2 weeks in the AN arm (p?<?0.0001). Surprisingly, DPG was lost at the same rate in both control and AN[CO2 ] arms (p?=?0.6).Maintenance of ATP in the AN[CO2 ] arm demonstrates that ATP production is not solely a function of the pH effect on glycolysis. CO2 in anaerobic storage prevented the maintenance of DPG, and DPG production appears to be pH dependent. CO2 as well as O2 depletion provides metabolic advantage for stored RBCs.
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Project description:We present the light scattering properties of individual human red blood cells (RBCs). We show that both the RBC static and dynamic scattering signals are altered by adenosine 5'-triphosphate (ATP)-driven membrane metabolic remodeling. To measure the light scattering signal from individual RBCs, we use diffraction phase microscopy together with a Fourier transform light scattering technique. RBC cytosolic ATPs are both chemically and metabolically depleted, and the corresponding scattering signals are compared with the light scattering signal of normal RBCs having physiologic levels of ATP.
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Project description:1. Whole blood was incubated at 37 degrees , while being dialysed against a large volume of iso-osmotic bicarbonate buffer, pH7.4. The buffer contained glucose and the essential inorganic components of blood plasma in proportion. 2. After 3hr. of incubation in vitro there is a loss of red-cell 2,3-diphosphoglycerate. 3. Isotope experiments show that this is due to an accelerated rate of destruction of this compound. 4. Simultaneously, there is an increase in the median of red-cell osmotic fragility. 5. After extended periods of incubation there is a decrease in the metabolic rate and a decrease in the ratio of the rates of lactate production to glucose consumption. 6. There is a continuous loss of total adenine nucleotide and, after the first 12hr. of incubation, a tendency for the intracellular Na(+) and K(+) to equilibrate with the plasma. 7. The standard deviation of red-cell osmotic fragility expressed among the red-cell population increases exponentially with the time of incubation.
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Project description:Primitive nucleated erythroid cells in the bloodstream have long been suggested to be more similar to nucleated red cells of fish, amphibians, and birds than the red cells of fetal and adult mammals. Rainbow trout Ficoll-purified red blood cells (RBCs) cultured in vitro undergo morphological changes, especially when exposed to stress, and enter a new cell stage that we have coined shape-shifted RBCs (shRBCs). We have characterized these shRBCs using transmission electron microscopy (TEM) micrographs, Wright⁻Giemsa staining, cell marker immunostaining, and transcriptomic and proteomic evaluation. shRBCs showed reduced density of the cytoplasm, hemoglobin loss, decondensed chromatin in the nucleus, and striking expression of the B lymphocyte molecular marker IgM. In addition, shRBCs shared some features of mammalian primitive pyrenocytes (extruded nucleus surrounded by a thin rim of cytoplasm and phosphatidylserine (PS) exposure on cell surface). These shRBCs were transiently observed in heat-stressed rainbow trout bloodstream for three days. Functional network analysis of combined transcriptomic and proteomic studies resulted in the identification of proteins involved in pathways related to the regulation of cell morphogenesis involved in differentiation, cellular response to stress, and immune system process. In addition, shRBCs increased interleukin 8 (IL8), interleukin 1 β (IL1β), interferon ɣ (IFNɣ), and natural killer enhancing factor (NKEF) protein production in response to viral hemorrhagic septicemia virus (VHSV). In conclusion, shRBCs may represent a novel cell stage that participates in roles related to immune response mediation, homeostasis, and the differentiation and development of blood cells.
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Project description:The YPEL family genes are highly conserved across a diverse range of eukaryotic organisms and thus potentially involved in essential cellular processes. Ypel4, one of five YPEL family gene orthologs in mouse and human, is highly and specifically expressed in late terminal erythroid differentiation. In this study, we investigated the role of Ypel4 in murine erythropoiesis, providing for the first time an in-depth description of a Ypel4-null phenotype in vivo. We demonstrated that the Ypel4-null mice displayed a secondary polycythemia with macro- and reticulocytosis. While lack of Ypel4 did not affect steady-state erythropoiesis in the bone marrow or spleen, the anemia-recovering capacity of Ypel4-null cells was diminished. Furthermore, Ypel4-null red blood cells (RBC) were cleared from the circulation at an increased rate, demonstrating an intrinsic defect of RBCs. Scanning electron micrographs revealed an ovalocytic morphology of Ypel4-null RBCs and functional testing confirmed reduced deformability. Even though Band 3 protein levels were shown to be reduced in Ypel4-null RBC membranes, we could not find support for a physical interaction between YPEL4 and the Band 3 protein. In conclusion, our findings provide crucial insights into the role of Ypel4 in preserving normal red cell membrane functionality.
2021-08-11 | GSE171959 | GEO
Project description:Human red blood cells (RBC) are highly differentiated cells that have lost all organelles and most intracellular machineries during their maturation process. RBC are fundamental for the nearly all basic physiologic dynamics and they are key cells in the body's respiratory system by being responsible for the oxygen transport to all cells and tissues, and delivery of carbon dioxide to the lungs. With their flexible structure RBC are capable to deform in order to travel through all blood vessels including very small capillaries. Throughout their in average 120 days lifespan, human RBC travel in the bloodstream and come in contact with a broad range of different cell types. In fact, RBC are able to interact and communicate with endothelial cells (ECs), platelets, macrophages, and bacteria. Additionally, they are involved in the maintenance of thrombosis and hemostasis and play an important role in the immune response against pathogens. To clarify the mechanisms of interaction of RBC and these other cells both in health and disease as well as to highlight the role of important key players, we focused our interest on RBC membrane components such as ion channels, proteins, and phospholipids.
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