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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 mitochondrial GSH, GSSG, S-glutathionylation, and complex I activity.H9C2 cells were treated as described in Fig 3. (A) Mitochondrial GSH, (B) GSSG, and (C) the GSH/GSSG ratio in each group were analyzed. (D and E) S-glutathionylated Complex I protein expression (51—and 75—kDa subunits) from each group were detected by immunoprecipitation and western blot analysis. The up panel shows the anti-complex I 75 and 51 kDa subunits positive-IP sample followed by identification by an anti-GSH antibody. As a control, mitochondrial protein (20 μg) was also immunoblotted with complex I 75 kDa and 51 kDa subunit antibody. Bar graphs summarize the protein band intensities of S- glutathionylated Complex I. (F) Complex I activity (200 μg mitochondrial protein) of each group are detected. * P < 0.05 compare with the control group, # P < 0.05 compare with the 1 mM Gln and 4 mM Gln groups. The data are expressed as the mean ±S.D. of 3 independent experiments, a.u. = arbitrary units.
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pone.0132402.g004: Effect of Gln on mitochondrial GSH, GSSG, S-glutathionylation, and complex I activity.H9C2 cells were treated as described in Fig 3. (A) Mitochondrial GSH, (B) GSSG, and (C) the GSH/GSSG ratio in each group were analyzed. (D and E) S-glutathionylated Complex I protein expression (51—and 75—kDa subunits) from each group were detected by immunoprecipitation and western blot analysis. The up panel shows the anti-complex I 75 and 51 kDa subunits positive-IP sample followed by identification by an anti-GSH antibody. As a control, mitochondrial protein (20 μg) was also immunoblotted with complex I 75 kDa and 51 kDa subunit antibody. Bar graphs summarize the protein band intensities of S- glutathionylated Complex I. (F) Complex I activity (200 μg mitochondrial protein) of each group are detected. * P < 0.05 compare with the control group, # P < 0.05 compare with the 1 mM Gln and 4 mM Gln groups. The data are expressed as the mean ±S.D. of 3 independent experiments, a.u. = arbitrary units.

Mentions: As the primary antioxidant in mitochondria, GSH also has an important role in mitochondrial oxidative stress [27]. Here, we investigated the effect of Gln on mtGSH levels in cells exposed to high glucose and H/R (Fig 4A, 4B and 4C). The levels of mtGSH and mtGSH/GSSG in the HG+H/R groups treated with low-dose Gln (1 mM or 4 mM) were significantly lower than that of the control group (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)

Effect of Gln on mitochondrial GSH, GSSG, S-glutathionylation, and complex I activity.H9C2 cells were treated as described in Fig 3. (A) Mitochondrial GSH, (B) GSSG, and (C) the GSH/GSSG ratio in each group were analyzed. (D and E) S-glutathionylated Complex I protein expression (51—and 75—kDa subunits) from each group were detected by immunoprecipitation and western blot analysis. The up panel shows the anti-complex I 75 and 51 kDa subunits positive-IP sample followed by identification by an anti-GSH antibody. As a control, mitochondrial protein (20 μg) was also immunoblotted with complex I 75 kDa and 51 kDa subunit antibody. Bar graphs summarize the protein band intensities of S- glutathionylated Complex I. (F) Complex I activity (200 μg mitochondrial protein) of each group are detected. * P < 0.05 compare with the control group, # P < 0.05 compare with the 1 mM Gln and 4 mM Gln groups. The data are expressed as the mean ±S.D. of 3 independent experiments, a.u. = arbitrary units.
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Related In: Results  -  Collection

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pone.0132402.g004: Effect of Gln on mitochondrial GSH, GSSG, S-glutathionylation, and complex I activity.H9C2 cells were treated as described in Fig 3. (A) Mitochondrial GSH, (B) GSSG, and (C) the GSH/GSSG ratio in each group were analyzed. (D and E) S-glutathionylated Complex I protein expression (51—and 75—kDa subunits) from each group were detected by immunoprecipitation and western blot analysis. The up panel shows the anti-complex I 75 and 51 kDa subunits positive-IP sample followed by identification by an anti-GSH antibody. As a control, mitochondrial protein (20 μg) was also immunoblotted with complex I 75 kDa and 51 kDa subunit antibody. Bar graphs summarize the protein band intensities of S- glutathionylated Complex I. (F) Complex I activity (200 μg mitochondrial protein) of each group are detected. * P < 0.05 compare with the control group, # P < 0.05 compare with the 1 mM Gln and 4 mM Gln groups. The data are expressed as the mean ±S.D. of 3 independent experiments, a.u. = arbitrary units.
Mentions: As the primary antioxidant in mitochondria, GSH also has an important role in mitochondrial oxidative stress [27]. Here, we investigated the effect of Gln on mtGSH levels in cells exposed to high glucose and H/R (Fig 4A, 4B and 4C). The levels of mtGSH and mtGSH/GSSG in the HG+H/R groups treated with low-dose Gln (1 mM or 4 mM) were significantly lower than that of the control group (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