Qingyou Du
University of Dundee
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Publication
Featured researches published by Qingyou Du.
Journal of Cellular Physiology | 2007
Andrey Sukhodub; Sofija Jovanović; Qingyou Du; Grant R. Budas; Allyson K. Clelland; Mei Shen; Kei Sakamoto; Rong Tian; Aleksandar Jovanović
Brief periods of ischemia and reperfusion that precede sustained ischemia lead to a reduction in myocardial infarct size. This phenomenon, known as ischemic preconditioning, is mediated by signaling pathway(s) that is complex and yet to be fully defined. AMP‐activated kinase (AMPK) is activated in cells under conditions associated with ATP depletion and increased AMP/ATP ratio. In the present study, we have taken advantage of a cardiac phenotype overexpressing a dominant negative form of the α2 subunit of AMPK to analyze the role, if any, that AMPK plays in preconditioning the heart. We have found that myocardial preconditioning activates AMPK in wild type, but not transgenic mice. Cardiac cells from transgenic mice could not be preconditioned, as opposed to cells from the wild type. The cytoprotective effect of AMPK was not related to the effect that preconditioning has on mitochondrial membrane potential as revealed by JC‐1, a mitochondrial membrane potential‐sensitive dye, and laser confocal microscopy. In contrast, experiments with di‐8‐ANEPPS, a sarcolemmal‐potential sensitive dye, has demonstrated that intact AMPK activity is required for preconditioning‐induced shortening of the action membrane potential. The preconditioning‐induced activation of sarcolemmal KATP channels was observed in wild type, but not in transgenic mice. HMR 1098, a selective inhibitor of sarcolemmal KATP channels opening, inhibited preconditioning‐induced shortening of action membrane potential as well as cardioprotection afforded by AMPK. Immunoprecipitation followed by Western blotting has shown that AMPK is essential for preconditioning‐induced recruitment of sarcolemmal KATP channels. Based on the obtained results, we conclude that AMPK mediates preconditioning in cardiac cells by regulating the activity and recruitment of sarcolemmal KATP channels without being a part of signaling pathway that regulates mitochondrial membrane potential. J. Cell. Physiol. 210: 224–236, 2007.
The FASEB Journal | 2006
Qingyou Du; Sofija Jovanović; Allyson K. Clelland; Andrey Sukhodub; Grant R. Budas; Karen Phelan; Victoria Murray-Tait; Lorraine Malone; Aleksandar Jovanović
ATP‐sensitive K+ (KATP) channels are present in the sarcolemma of cardiac myocytes where they link membrane excitability with the cellular bioen‐ergetic state. These channels are in vivo composed of Kir6.2, a pore‐forming subunit, SUR2A, a regulatory subunit, and at least four accessory proteins. In the present study, real‐time RT‐PCR has demonstrated that of all six sarcolemmal KATP channel‐forming proteins, SUR2A was probably the least expressed protein. We have generated mice where the SUR2A was under the control of a cytomegalovirus promoter, a promoter that is more efficient than the native promoter. These mice had an increase in SUR2A mRNA/protein levels in the heart whereas levels of mRNAs of other channel‐forming proteins were not affected at all. Imunopre‐cipitation/Western blot and patch clamp electrophysi‐ology has shown an increase in KATP channel numbers in the sarcolemma of transgenic mice. Cardiomyocytes from transgenic mice responded to hypoxia with shortening of action membrane potential and were significantly more resistant to this insult than cardiomyocytes from the wild‐type. The size of myocardial infarction in response to ischemia‐reperfusion was much smaller in hearts from transgenic mice compared to those in wild‐type. We conclude that overexpression of SUR2A generates cardiac phenotype resistant to hypoxia/isch‐emia/reperfusion injury due at least in part to increase in levels of sarcolemmal KATP channels.—Du, Q., Jovanovic, S., Clelland, A., Sukhodub, A., Budas, G., Phelan, K., Murray‐Tait, V., Malone, L., Jovanovic, A. Overexpression of sur2a generates cardiac phenotype resistant to ischemia. FASEBJ. 20, 1131–1141 (2006)
EMBO Reports | 2005
Sofija Jovanović; Qingyou Du; Russell M. Crawford; Grant R. Budas; Igor Stagljar; Aleksandar Jovanović
Cardiac sarcolemmal ATP‐sensitive K+ (KATP) channels, composed of Kir6.2 and SUR2A subunits, are regulated by intracellular ATP and they couple the metabolic status of the cell with the membrane excitability. On the basis of previous studies, we have suggested that glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) may be a part of the sarcolemmal KATP‐channel protein complex. A polypeptide of ∼42 kDa was immunoprecipitated with an anti‐SUR2A antibody from guinea‐pig cardiac membrane fraction and identified as GAPDH. Immunoprecipitation/western blotting analysis with anti‐Kir6.2, anti‐SUR2A and anti‐GAPDH antibodies showed that GAPDH is a part of the sarcolemmal KATP‐channel protein complex in vivo. Further studies with immunoprecipitation/western blotting and the membrane yeast two‐hybrid system showed that GAPDH associates physically with the Kir6.2 but not the SUR2A subunit. Patch‐clamp electrophysiology showed that GAPDH regulates KATP‐channel activity irrespective of high intracellular ATP, by producing 1,3‐bisphosphoglycerate, a KATP‐channel opener. These results suggest that GAPDH is an integral part of the sarcolemmal KATP‐channel protein complex, where it couples glycolysis with the KATP‐channel activity.
Diabetologia | 2012
Craig Beall; David Lee Hamilton; Jennifer Gallagher; Lisa Logie; Karen A Wright; Marc P.M. Soutar; Selma Dadak; Fiona B Ashford; Elizabeth Haythorne; Qingyou Du; Aleksandar Jovanović; Rory J. McCrimmon; Michael L.J. Ashford
Aims/hypothesisHypothalamic glucose-excited (GE) neurons contribute to whole-body glucose homeostasis and participate in the detection of hypoglycaemia. This system appears defective in type 1 diabetes, in which hypoglycaemia commonly occurs. Unfortunately, it is at present unclear which molecular components required for glucose sensing are produced in individual neurons and how these are functionally linked. We used the GT1-7 mouse hypothalamic cell line to address these issues.MethodsElectrophysiological recordings, coupled with measurements of gene expression and protein levels and activity, were made from unmodified GT1-7 cells and cells in which AMP-activated protein kinase (AMPK) catalytic subunit gene expression and activity were reduced.ResultsHypothalamic GT1-7 neurons express the genes encoding glucokinase and ATP-sensitive K+ channel (KATP) subunits Kir6.2 and Sur1 and exhibit GE-type glucose-sensing behaviour. Lowered extracellular glucose concentration hyperpolarised the cells in a concentration-dependent manner, an outcome that was reversed by tolbutamide. Inhibition of glucose uptake or metabolism hyperpolarised cells, showing that energy metabolism is required to maintain their resting membrane potential. Short hairpin (sh)RNA directed to Ampkα2 (also known as Prkaa2) reduced GT1-7 cell AMPKα2, but not AMPKα1, activity and lowered the threshold for hypoglycaemia-induced hyperpolarisation. shAmpkα1 (also known as Prkaa1) had no effect on glucose-sensing or AMPKα2 activity. Decreased uncoupling protein 2 (Ucp2) mRNA was detected in AMPKα2-reduced cells, suggesting that AMPKα2 regulates UCP2 levels.Conclusions/interpretationWe have demonstrated that GT1-7 cells closely mimic GE neuron glucose-sensing behaviour, and reducing AMPKα2 blunts their responsiveness to hypoglycaemic challenge, possibly by altering UCP2 activity. These results show that suppression of AMPKα2 activity inhibits normal glucose-sensing behaviour and may contribute to defective detection of hypoglycaemia.
Journal of Molecular Biology | 2015
Qingyou Du; Yoshinori Kawabe; Christina Schilde; Zhi-Hui Chen; Pauline Schaap
Aggregative multicellularity, resulting in formation of a spore-bearing fruiting body, evolved at least six times independently amongst both eukaryotes and prokaryotes. Amongst eukaryotes, this form of multicellularity is mainly studied in the social amoeba Dictyostelium discoideum. In this review, we summarise trends in the evolution of cell-type specialisation and behavioural complexity in the four major groups of Dictyostelia. We describe the cell–cell communication systems that control the developmental programme of D. discoideum, highlighting the central role of cAMP in the regulation of cell movement and cell differentiation. Comparative genomic studies showed that the proteins involved in cAMP signalling are deeply conserved across Dictyostelia and their unicellular amoebozoan ancestors. Comparative functional analysis revealed that cAMP signalling in D. discoideum originated from a second messenger role in amoebozoan encystation. We highlight some molecular changes in cAMP signalling genes that were responsible for the novel roles of cAMP in multicellular development.
The International Journal of Biochemistry & Cell Biology | 2009
Sofija Jovanović; Qingyou Du; Andriy Sukhodub; Aleksandar Jovanović
Muscle form of lactate dehydrogenase (M-LDH) physically associate with KATP channel subunits, Kir6.2 and SUR2A, and is an integral part of the ATP-sensitive K+ (KATP) channel protein complex in the heart. Here, we have shown that concomitant introduction of viral constructs containing truncated and mutated forms of M-LDH (ΔM-LDH) and 193gly-M-LDH respectively, generate a phenotype of rat heart embryonic H9C2 cells that do not contain functional M-LDH as a part of the KATP channel protein complex. The K+ current was increased in wild type cells, but not in cells expressing ΔM-LDH/193gly-M-LDH, when they were exposed to chemical hypoxia induced by 2,4 dinitrophenol (DNP; 10 mM). At the same time, the outcome of chemical hypoxia was much worse in ΔM-LDH/193gly-M-LDH phenotype than in the control one, and that was associated with increased loss of intracellular ATP in cells infected with ΔM-LDH/193gly-M-LDH. On the other hand, cells expressing Kir6.2AFA, a Kir6.2 mutant that abolishes KATP channel conductance without affecting intracellular ATP levels, survived chemical hypoxia much better than cells expressing ΔM-LDH/193gly-M-LDH. Based on the obtained results, we conclude that M-LDH physically associated with Kir6.2/SUR2A regulates the activity of sarcolemmal KATP channels as well as an intracellular ATP production during metabolic stress, both of which are important for cell survival.
Pharmacological Research | 2010
Andriy Sukhodub; Qingyou Du; Sofija Jovanović; Aleksandar Jovanović
Graphical abstract
Biochimica et Biophysica Acta | 2010
Qingyou Du; Sofija Jovanović; Andriy Sukhodub; Aleksandar Jovanović
Transgenic mice overexpressing SUR2A, a subunit of ATP-sensitive K+ (KATP) channels, acquire resistance to myocardial ischaemia. However, the mechanism of SUR2A-mediated cytoprotection is yet to be fully understood. Adenoviral SUR2A construct (AV-SUR2A) increased SUR2A expression, number of KATP channels and subsarcolemmal ATP in glycolysis-sensitive manner in H9C2 cells. It also increased K+ current in response to chemical hypoxia, partially preserved subsarcolemmal ATP and increased cell survival. Kir6.2AFA, a mutant form of Kir6.2 with largely decreased K+ conductance, abolished the effect of SUR2A on K+ current, did not affect SUR2A-induced increase in subsarcolemmal ATP and partially inhibited SUR2A-mediated cytoprotection. Infection with 193gly-M-LDH, an inactive mutant of muscle lactate dehydrogenase, abolished the effect of SUR2A on K+ current, subsarcolemmal ATP and cell survival; the effect of 193gly-M-LDH on cell survival was significantly more pronounced than those of Kir6.2AFA. We conclude that AV-SUR2A increases resistance to metabolic stress in H9C2 cells by increasing the number of sarcolemmal KATP channels and subsarcolemmal ATP.
Clinical and Translational Science | 2008
Sofija Jovanović; Qingyou Du; Somnath Mukhopadhyay; Robert Swingler; Richard Buckley; Jane McEachen; Aleksandar Jovanović
Hypokalemic periodic paralysis (HOPP) is a rare disease associated with attacks of muscle weakness and hypokalemia. In the present study, immunoprecipitation/Western blotting has shown that a HOPP patient was deficient in sarcolemmal KATP channels. Real‐time RT‐PCR has revealed that HOPP has decreased mRNA levels of Kir6.2, a pore‐forming KATP channel subunit, without affecting the expression of other KATP channel‐forming proteins. Based on these findings, we conclude that HOPP could be associated with impaired expression of Kir6.2 which leads to deficiency in skeletal muscle KATP channels, which may explain the symptoms and clinical signs of this disease.
Biochimica et Biophysica Acta | 2009
Sofija Jovanović; Qingyou Du; Andriy Sukhodub; Aleksandar Jovanović
Muscle form of lactate dehydrogenase (M-LDH), a minor LDH form in cardiomyocytes, physically interacts with ATP-sensitive K+ (KATP) channel-forming subunits. Here, we have shown that expression of 193gly-M-LDH, an inactive mutant of M-LDH, inhibit regulation of the KATP channels activity by LDH substrates in embryonic rat heart H9C2 cells. In cells expressing 193gly-M-LDH chemical hypoxia has failed to activate KATP channels. The similar results were obtained in H9C2 cells expressing Kir6.2AFA, a mutant form of Kir6.2 with largely decreased K+ conductance. Kir6.2AFA has slightly, but significantly, reduced cellular survival under chemical hypoxia while the deleterious effect of 193gly-M-LDH was significantly more pronounced. The levels of total and subsarcolemmal ATP in H9C2 cells were not affected by Kir6.2AFA, but the expression of 193gly-M-LDH led to lower levels of subsarcolemmal ATP during chemical hypoxia. We conclude that M-LDH regulates both the channel activity and the levels of subsarcolemmal ATP and that both mechanism contribute to the M-LDH-mediated cytoprotection.