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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

Effect of Gln on cytosolic GSH, GSSG and intracellular ROS.H9C2 cells were exposed to HG+H/R with different concentrations of Gln (1, 4, or 16 mM). (A) Cytosolic GSH, (B) GSSG, and (C) the GSH/GSSG ratio in each group were detected. (D) Representative images of DCFH flow cytometry of each group. (E) Mean DCFH fluorescence intensities are presented as percentage relative to the control value. (F) Representative images of DHE flow cytometry of each group. (G) Mean DHE fluorescence intensities are presented as percentage relative to the control value. These data are expressed as the mean ± S.D. of 3 independent experiments. * P < 0.05 compare with the control group, # P < 0.05 compare with the 1 mM Gln and 4 mM Gln groups, a.u. = arbitrary units.
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pone.0132402.g003: Effect of Gln on cytosolic GSH, GSSG and intracellular ROS.H9C2 cells were exposed to HG+H/R with different concentrations of Gln (1, 4, or 16 mM). (A) Cytosolic GSH, (B) GSSG, and (C) the GSH/GSSG ratio in each group were detected. (D) Representative images of DCFH flow cytometry of each group. (E) Mean DCFH fluorescence intensities are presented as percentage relative to the control value. (F) Representative images of DHE flow cytometry of each group. (G) Mean DHE fluorescence intensities are presented as percentage relative to the control value. These data are expressed as the mean ± S.D. of 3 independent experiments. * P < 0.05 compare with the control group, # P < 0.05 compare with the 1 mM Gln and 4 mM Gln groups, a.u. = arbitrary units.

Mentions: Gln is a precursor amino acid of glutathione, which can improve cellular tolerance to oxidative stress. Furthermore, de novo GSH synthesis occurs exclusively in the cytosolic compartment [26]. To detect whether apparent anti-apoptotic effects of Gln were further associated with GSH-GSSG balance, cytoplasmic GSH, GSSG, and the GSH/GSSG ratio were measured (Fig 3A, 3B and 3C). Cytoplasmic GSH, GSSG, and GSH/GSSG levels were markedly lower in HG+H/R-treated H9C2 cells than in the control groups (P < 0.05). This effect was reversed by 16 mM Gln although the lower doses of Gln had no such effect.


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)

Effect of Gln on cytosolic GSH, GSSG and intracellular ROS.H9C2 cells were exposed to HG+H/R with different concentrations of Gln (1, 4, or 16 mM). (A) Cytosolic GSH, (B) GSSG, and (C) the GSH/GSSG ratio in each group were detected. (D) Representative images of DCFH flow cytometry of each group. (E) Mean DCFH fluorescence intensities are presented as percentage relative to the control value. (F) Representative images of DHE flow cytometry of each group. (G) Mean DHE fluorescence intensities are presented as percentage relative to the control value. These data are expressed as the mean ± S.D. of 3 independent experiments. * P < 0.05 compare with the control group, # P < 0.05 compare with the 1 mM Gln and 4 mM Gln groups, a.u. = arbitrary units.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493145&req=5

pone.0132402.g003: Effect of Gln on cytosolic GSH, GSSG and intracellular ROS.H9C2 cells were exposed to HG+H/R with different concentrations of Gln (1, 4, or 16 mM). (A) Cytosolic GSH, (B) GSSG, and (C) the GSH/GSSG ratio in each group were detected. (D) Representative images of DCFH flow cytometry of each group. (E) Mean DCFH fluorescence intensities are presented as percentage relative to the control value. (F) Representative images of DHE flow cytometry of each group. (G) Mean DHE fluorescence intensities are presented as percentage relative to the control value. These data are expressed as the mean ± S.D. of 3 independent experiments. * P < 0.05 compare with the control group, # P < 0.05 compare with the 1 mM Gln and 4 mM Gln groups, a.u. = arbitrary units.
Mentions: Gln is a precursor amino acid of glutathione, which can improve cellular tolerance to oxidative stress. Furthermore, de novo GSH synthesis occurs exclusively in the cytosolic compartment [26]. To detect whether apparent anti-apoptotic effects of Gln were further associated with GSH-GSSG balance, cytoplasmic GSH, GSSG, and the GSH/GSSG ratio were measured (Fig 3A, 3B and 3C). Cytoplasmic GSH, GSSG, and GSH/GSSG levels were markedly lower in HG+H/R-treated H9C2 cells than in the control groups (P < 0.05). This effect was reversed by 16 mM Gln although the lower doses of Gln had no such effect.

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