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Glutamine Reduces the Apoptosis of H9C2 Cells Treated with High-Glucose and Reperfusion through an Oxidation-Related Mechanism.

Li K, Cui YC, Zhang H, Liu XP, Zhang D, Wu AL, Li JJ, Tang Y - PLoS ONE (2015)

Bottom Line: Data indicated that high glucose and hypoxia-reoxygenation were associated with a dramatic decline of intercellular glutamine and increase in apoptosis.Glutamine supplementation was also associated with less S-glutathionylation and increased the activity of complex I, leading to less mitochondrial ROS formation.We conclude that apoptosis induced by high glucose and hypoxia-reoxygenation was reduced by glutamine supplementation, via decreased oxidative stress and inactivation of the intrinsic apoptotic pathway.

View Article: PubMed Central - PubMed

Affiliation: Animal Experimental Center, Beijing Key Laboratory of Preclinical Research and Evaluation for Cardiovascular Implant Materials, State key Laboratory of Cardiovascular Disease, Fu Wai Hospital, National Center for Cardiovascular Disease, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.

ABSTRACT
Mitochondrial overproduction of reactive oxygen species (ROS) in diabetic hearts during ischemia/reperfusion injury and the anti-oxidative role of glutamine have been demonstrated. However, in diabetes mellitus the role of glutamine in cardiomyocytes during ischemia/reperfusion injury has not been explored. To examine the effects of glutamine and potential mechanisms, in the present study, rat cardiomyoblast H9C2 cells were exposed to high glucose (33 mM) and hypoxia-reoxygenation. Cell viability, apoptosis, intracellular glutamine, and mitochondrial and intracellular glutathione were determined. Moreover, ROS formation, complex I activity, membrane potential and adenosine triphosphate (ATP) content were also investigated. The levels of S-glutathionylated complex I and mitochondrial apoptosis-related proteins, including cytochrome c and caspase-3, were analyzed by western blot. Data indicated that high glucose and hypoxia-reoxygenation were associated with a dramatic decline of intercellular glutamine and increase in apoptosis. Glutamine supplementation correlated with a reduction in apoptosis and increase of glutathione and glutathione reduced/oxidized ratio in both cytoplasm and mitochondria, but a reduction of intracellular ROS. Glutamine supplementation was also associated with less S-glutathionylation and increased the activity of complex I, leading to less mitochondrial ROS formation. Furthermore, glutamine supplementation prevented from mitochondrial dysfunction presented as mitochondrial membrane potential and ATP levels and attenuated cytochrome c release into the cytosol and caspase-3 activation. We conclude that apoptosis induced by high glucose and hypoxia-reoxygenation was reduced by glutamine supplementation, via decreased oxidative stress and inactivation of the intrinsic apoptotic pathway.

No MeSH data available.


Related in: MedlinePlus

Effects of Gln on mitochondrial membrane potential stability and ATP level.H9C2 cells were treated as described in Fig 3. (A). Representative images of JC-1 confocal images of each group; the red image detected in the left panel represents JC-1 aggregates, and the green image represents JC-1 monomer; the blue image represents DAPI, right panel shows the merged image. (B) Mean ± S.D. of the changes in radiometric JC-1 fluorescence for each group as indicated. (C) Cellular ATP levels of each group were detected. * P < 0.05 compare with the control group; # P < 0.05 compare with the 1 mM Gln and 4 mM Gln groups. Data are expressed as the mean ±S.D. of 3 independent experiments. a.u. = arbitrary units.
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pone.0132402.g006: Effects of Gln on mitochondrial membrane potential stability and ATP level.H9C2 cells were treated as described in Fig 3. (A). Representative images of JC-1 confocal images of each group; the red image detected in the left panel represents JC-1 aggregates, and the green image represents JC-1 monomer; the blue image represents DAPI, right panel shows the merged image. (B) Mean ± S.D. of the changes in radiometric JC-1 fluorescence for each group as indicated. (C) Cellular ATP levels of each group were detected. * P < 0.05 compare with the control group; # P < 0.05 compare with the 1 mM Gln and 4 mM Gln groups. Data are expressed as the mean ±S.D. of 3 independent experiments. a.u. = arbitrary units.

Mentions: Mitochondrial transmembrane potential (ΔΨm) is a marker of mitochondrial function, and dissipation of ΔΨm always indicates mitochondrial dysfunction [28]. In the present study, ΔΨm of the control group was normal, evident from the significant amount of JC-1 aggregates (red fluorescence; Fig 6). On the other hand, HG + H/R treatment depolarized ΔΨm (decreasing the red/green fluorescence ratio) relative to the control group (P < 0.05). The ratio of red-to-green fluorescence was significantly higher in the 16 mM Gln group than in the 1 mM and 4 mM groups (P < 0.05).


Glutamine Reduces the Apoptosis of H9C2 Cells Treated with High-Glucose and Reperfusion through an Oxidation-Related Mechanism.

Li K, Cui YC, Zhang H, Liu XP, Zhang D, Wu AL, Li JJ, Tang Y - PLoS ONE (2015)

Effects of Gln on mitochondrial membrane potential stability and ATP level.H9C2 cells were treated as described in Fig 3. (A). Representative images of JC-1 confocal images of each group; the red image detected in the left panel represents JC-1 aggregates, and the green image represents JC-1 monomer; the blue image represents DAPI, right panel shows the merged image. (B) Mean ± S.D. of the changes in radiometric JC-1 fluorescence for each group as indicated. (C) Cellular ATP levels of each group were detected. * P < 0.05 compare with the control group; # P < 0.05 compare with the 1 mM Gln and 4 mM Gln groups. Data are expressed as the mean ±S.D. of 3 independent experiments. a.u. = arbitrary units.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493145&req=5

pone.0132402.g006: Effects of Gln on mitochondrial membrane potential stability and ATP level.H9C2 cells were treated as described in Fig 3. (A). Representative images of JC-1 confocal images of each group; the red image detected in the left panel represents JC-1 aggregates, and the green image represents JC-1 monomer; the blue image represents DAPI, right panel shows the merged image. (B) Mean ± S.D. of the changes in radiometric JC-1 fluorescence for each group as indicated. (C) Cellular ATP levels of each group were detected. * P < 0.05 compare with the control group; # P < 0.05 compare with the 1 mM Gln and 4 mM Gln groups. Data are expressed as the mean ±S.D. of 3 independent experiments. a.u. = arbitrary units.
Mentions: Mitochondrial transmembrane potential (ΔΨm) is a marker of mitochondrial function, and dissipation of ΔΨm always indicates mitochondrial dysfunction [28]. In the present study, ΔΨm of the control group was normal, evident from the significant amount of JC-1 aggregates (red fluorescence; Fig 6). On the other hand, HG + H/R treatment depolarized ΔΨm (decreasing the red/green fluorescence ratio) relative to the control group (P < 0.05). The ratio of red-to-green fluorescence was significantly higher in the 16 mM Gln group than in the 1 mM and 4 mM groups (P < 0.05).

Bottom Line: Data indicated that high glucose and hypoxia-reoxygenation were associated with a dramatic decline of intercellular glutamine and increase in apoptosis.Glutamine supplementation was also associated with less S-glutathionylation and increased the activity of complex I, leading to less mitochondrial ROS formation.We conclude that apoptosis induced by high glucose and hypoxia-reoxygenation was reduced by glutamine supplementation, via decreased oxidative stress and inactivation of the intrinsic apoptotic pathway.

View Article: PubMed Central - PubMed

Affiliation: Animal Experimental Center, Beijing Key Laboratory of Preclinical Research and Evaluation for Cardiovascular Implant Materials, State key Laboratory of Cardiovascular Disease, Fu Wai Hospital, National Center for Cardiovascular Disease, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.

ABSTRACT
Mitochondrial overproduction of reactive oxygen species (ROS) in diabetic hearts during ischemia/reperfusion injury and the anti-oxidative role of glutamine have been demonstrated. However, in diabetes mellitus the role of glutamine in cardiomyocytes during ischemia/reperfusion injury has not been explored. To examine the effects of glutamine and potential mechanisms, in the present study, rat cardiomyoblast H9C2 cells were exposed to high glucose (33 mM) and hypoxia-reoxygenation. Cell viability, apoptosis, intracellular glutamine, and mitochondrial and intracellular glutathione were determined. Moreover, ROS formation, complex I activity, membrane potential and adenosine triphosphate (ATP) content were also investigated. The levels of S-glutathionylated complex I and mitochondrial apoptosis-related proteins, including cytochrome c and caspase-3, were analyzed by western blot. Data indicated that high glucose and hypoxia-reoxygenation were associated with a dramatic decline of intercellular glutamine and increase in apoptosis. Glutamine supplementation correlated with a reduction in apoptosis and increase of glutathione and glutathione reduced/oxidized ratio in both cytoplasm and mitochondria, but a reduction of intracellular ROS. Glutamine supplementation was also associated with less S-glutathionylation and increased the activity of complex I, leading to less mitochondrial ROS formation. Furthermore, glutamine supplementation prevented from mitochondrial dysfunction presented as mitochondrial membrane potential and ATP levels and attenuated cytochrome c release into the cytosol and caspase-3 activation. We conclude that apoptosis induced by high glucose and hypoxia-reoxygenation was reduced by glutamine supplementation, via decreased oxidative stress and inactivation of the intrinsic apoptotic pathway.

No MeSH data available.


Related in: MedlinePlus