Kaempferol as a phytochemical increases ATP content in C2C12 myotubes under hypoxic conditions

Brain edema that forms during the early stages of stroke involves increased transport of Na+ and Cl− across an intact blood-brain barrier (BBB). Our previous studies have shown that a luminal BBB Na+-K+-Cl− cotransporter is stimulated by conditions present during ischemia and that inhibition of the cotransporter by intravenous bumetanide greatly reduces edema formation in the rat middle cerebral artery occlusion model of stroke. The present study focused on investigating the effects of hypoxia, which develops rapidly in the brain during ischemia, on the activity and expression of the BBB Na+-K+-Cl− cotransporter, as well as on Na+-K+-ATPase activity, cell ATP content, and intracellular volume. Cerebral microvascular endothelial cells (CMECs) were assessed for Na+-K+-Cl− cotransporter and Na+-K+-ATPase activities as bumetanide-sensitive and ouabain-sensitive 86Rb influxes, respectively. ATP content was assessed by luciferase assay and intracellular volume by [3H]-3-O-methyl-d-glucose and [14C]-sucrose equilibration. We found that 30-min exposure of CMECs to hypoxia ranging from 7.5% to 0.5% O2 (vs. 19% normoxic O2) significantly increased cotransporter activity as did 7.5% or 2% O2 for up to 2 h. This was not associated with reduction in Na+-K+-ATPase activity or ATP content. CMEC intracellular volume increased only after 4 to 5 h of hypoxia. Furthermore, glucose and pyruvate deprivation increased cotransporter activity under both normoxic and hypoxic conditions. Finally, we found that hypoxia increased phosphorylation but not abundance of the cotransporter protein. These findings support the hypothesis that hypoxia stimulation of the BBB Na+-K+-Cl− cotransporter contributes to ischemia-induced brain edema formation.

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Deoxygenation Affects Composition of Membrane-Bound Proteins in Human Erythrocytes

Cellular Physiology and Biochemistry ◽

10.1159/000445607 ◽

2016 ◽

Vol 39 (1) ◽

pp. 81-88 ◽

Cited By ~ 6

Author(s):

Oksana G. Luneva ◽

Svetlana V. Sidorenko ◽

Olga O. Ponomarchuk ◽

Artem M. Tverskoy ◽

Aleksander A. Cherkashin ◽

...

Keyword(s):

Molecular Mechanisms ◽

Atp Release ◽

Human Erythrocytes ◽

Atp Content ◽

Hypoxic Conditions ◽

Sds Page ◽

Membrane Bound ◽

Molecular Origin ◽

Free Environment ◽

Page Electrophoresis

Background/Aims: ATP release from erythrocyte plays a key role in hypoxia-induced elevation of blood flow in systematic circulation. We have previously shown that hemolysis contributes to erythrocyte ATP release triggered by several stimuli, including hypoxia, but the molecular mechanisms of hypoxia-increased membrane fragility remain unknown. Methods: In this study, we compared the action of hypoxia on hemolysis, ATP release and the composition of membrane-bound proteins in human erythrocytes. Results: Twenty minutes incubation of human erythrocytes in the oxygen-free environment increased the content of extracellular hemoglobin by ∼1.5 fold. Paired measurements of hemoglobin and ATP content in the same samples, showed a positive correlation between hemolysis and ATP release. Comparative analysis of SDS-PAGE electrophoresis of erythrocyte ghosts obtained under control and deoxygenated conditions revealed a ∼2-fold elevation of the content of membrane-bound protein with Mr of ∼60 kDa. Conclusion: Deoxygenation of human erythrocytes affects composition of membrane-bound proteins. Additional experiments should be performed to identify the molecular origin of 60 kDa protein and its role in the attenuation of erythrocyte integrity and ATP release in hypoxic conditions.

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H2S-stimulated bioenergetics in chicken erythrocytes and the underlying mechanism

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00348.2019 ◽

2020 ◽

Vol 319 (1) ◽

pp. R69-R78

Author(s):

Zhuping Jin ◽

Quanxi Zhang ◽

Eden Wondimu ◽

Richa Verma ◽

Ming Fu ◽

...

Keyword(s):

Electron Transport ◽

Mammalian Cells ◽

Electron Transport Chain ◽

Structural Integrity ◽

Underlying Mechanism ◽

Atp Content ◽

Hypoxic Conditions ◽

Transport Chain ◽

Chicken Erythrocytes

The production of H2S and its effect on bioenergetics in mammalian cells may be evolutionarily preserved. Erythrocytes of birds, but not those of mammals, have a nucleus and mitochondria. In the present study, we report the endogenous production of H2S in chicken erythrocytes, which was mainly catalyzed by 3-mercaptopyruvate sulfur transferase (MST). ATP content of erythrocytes was increased by MST-generated endogenous H2S under normoxic, but not hypoxic, conditions. NaHS, a H2S salt, increased ATP content under normoxic, but not hypoxic, conditions. ATP contents in the absence or presence of NaHS were eliminated by different inhibitors for mitochondrial electron transport chain in chicken erythrocytes. Succinate and glutamine, but not glucose, increased ATP content. NaHS treatment similarly increased ATP content in the presence of glucose, glutamine, or succinate, respectively. Furthermore, the expression and activity of sulfide:quinone oxidoreductase were enhanced by NaHS. The structural integrity of chicken erythrocytes was largely maintained during 2-wk NaHS treatment in vitro, whereas most of the erythrocytes without NaHS treatment were lysed. In conclusion, H2S may regulate cellular bioenergetics as well as cell survival of chicken erythrocytes, in which the functionality of the electron transport chain is involved. H2S may have different regulatory roles and mechanisms in bioenergetics of mammalian and bird cells.

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Naringenin Attenuates Non-Alcoholic Fatty Liver Disease by Enhancing Energy Expenditure and Regulating Autophagy via AMPK

Frontiers in Pharmacology ◽

10.3389/fphar.2021.687095 ◽

2021 ◽

Vol 12 ◽

Author(s):

Ying Yang ◽

Yue Wu ◽

Jie Zou ◽

Yu-Hao Wang ◽

Meng-Xia Xu ◽

...

Keyword(s):

Western Blotting ◽

Lipid Accumulation ◽

C2c12 Myotubes ◽

Autophagic Flux ◽

Atp Content ◽

Alcoholic Fatty Liver ◽

Compound C ◽

Oil Red O Staining ◽

Alcoholic Fatty Liver Disease ◽

Oil Red O

Background: The prevalence of non-alcoholic fatty liver disease (NAFLD) keeps growing recently.Purpose: To investigate the effects and mechanisms of naringenin (NAR) on NAFLD.Methods: High-fat diet (HFD)-induced NAFLD rats were orally administered with NAR at 10, 30, and 90 mg/kg for 2 weeks. The serum level of triglyceride (TG), total cholesterol (TC), glutamic-oxaloacetic transaminase (AST), and glutamic-pyruvic transaminase (ALT) was measured. The hepatic histology was detected by H&E and oil red O staining. L02 and Huh-7 cells were induced by sodium oleate to establish a NAFLD cell model. The effects of NAR on lipid accumulation were detected by oil red O staining. The glucose uptake and ATP content of 3T3-L1 adipocytes and C2C12 myotubes were measured. The expression of proteins of the AMPK signaling pathway in 3T3-L1 adipocytes and C2C12 myotubes was assessed by Western blotting. The mitochondrial biogenesis of 3T3-L1 adipocytes and C2C12 myotubes was measured by mitotracker orange staining and Western blotting. The biomarkers of autophagy were detected by Western blotting and immunofluorescence. The binding of NAR to AMPKγ1 was analyzed by molecular docking. Chloroquine and compound C were employed to block autophagic flux and AMPK, respectively.Results: NAR alleviated HFD-induced NAFLD in rats at 10, 30, and 90 mg/kg. NAR attenuated lipid accumulation in L02 and Huh-7 cells at 0.7, 2.2, 6.7, and 20 μM. NAR increased glucose uptake, decreased the ATP content, activated the CaMKKβ/AMPK/ACC pathway, and enhanced the mitochondrial biogenesis in 3T3-L1 adipocytes and C2C12 myotubes. NAR increased autophagy and promoted the initiation of autophagic flux in 3T3-L1 preadipocytes and C2C12 myoblasts, while it inhibited autophagy in NAFLD rats, 3T3-L1 adipocytes, and C2C12 myotubes. Molecular docking showed that NAR binds to AMPKγ1. Compound C blocked effects of NAR on lipid accumulation and autophagy in L02 cells.Conclusion: NAR alleviates NAFLD by increasing energy expenditure and regulating autophagy via activating AMPK directly and indirectly. The direct binding of NAR and AMPKγ1 needs further validation.

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Regulation of ATP synthase subunit e gene expression by hypoxia: cell differentiation stage-specific control

AJP Cell Physiology ◽

10.1152/ajpcell.1997.272.2.c457 ◽

1997 ◽

Vol 272 (2) ◽

pp. C457-C465 ◽

Cited By ~ 27

Author(s):

F. H. Levy ◽

D. P. Kelly

Keyword(s):

Cell Differentiation ◽

Atp Synthase ◽

C2c12 Cells ◽

C2c12 Myotubes ◽

Hypoxic Exposure ◽

Mrna Levels ◽

Atp Production ◽

Hypoxic Conditions ◽

Subunit E ◽

Differentiation Stage

Using the technique of differential display, we identified genes that are expressed differentially under normoxic and hypoxic conditions. One regulated gene encoded subunit e of mitochondrial F1F0-ATP synthase (subunit e). The hypoxia-mediated regulation of subunit e expression in C2C12 cells was influenced by the stage of cellular differentiation. Under normoxic conditions, subunit e expression was markedly upregulated during the transition from myoblast to myotube. After exposure to hypoxia for 24 h, subunit e mRNA expression markedly decreased (>70%) in C2C12 myotubes. In contrast, subunit e mRNA levels increased slightly in response to hypoxia in C2C12 myoblasts. Studies performed with primary rat cardiocytes demonstrated that expression of subunit e mRNA and a cardiac-enriched related transcript was downregulated after a hypoxic exposure. We conclude that expression of subunit e is regulated, at the pretranslational level, by oxygen availability via cell differentiation stage-specific mechanisms consistent with the proposed regulatory role of this protein in cellular ATP production.

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