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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

Influence of Gln on mitochondrial apoptosis-related protein.H9C2 cells were treated as described in Fig 3. The expression of cytosolic (A) and mitochondrial (B) cytochrome c (cyto-c) as well as cleaved caspase-3 and pro caspase-3 (C) in each group were measured by western blot. Beta-actin and VADC1 were used as internal protein loading controls. Bar graphs summarize the protein band intensities of cyto-c and cleaved caspase 3. * P < 0.05 compare with the control group; # P <0.05 compare with the 1 mM and 4 mM Gln groups.
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pone.0132402.g007: Influence of Gln on mitochondrial apoptosis-related protein.H9C2 cells were treated as described in Fig 3. The expression of cytosolic (A) and mitochondrial (B) cytochrome c (cyto-c) as well as cleaved caspase-3 and pro caspase-3 (C) in each group were measured by western blot. Beta-actin and VADC1 were used as internal protein loading controls. Bar graphs summarize the protein band intensities of cyto-c and cleaved caspase 3. * P < 0.05 compare with the control group; # P <0.05 compare with the 1 mM and 4 mM Gln groups.

Mentions: Western blot analysis revealed that in the 1 mM and 4 mM Gln groups exposed to high glucose and H/R, the levels of cytochrome c in mitochondrial fractions was higher, and the levels of cytochrome c in cytosolic fractions was lower, compared with the control (Fig 7A and 7B). No significant difference in these fractions was found in HG+H/R cells exposed to 16 mM Gln. Furthermore, in the 1 mM and 4 mM Gln groups, the levels of cleaved caspase 3 at 17-and 19-kDa were significantly higher than that of the control cells, but these higher levels were not observed in the cells treated with 16 mM Gln (Fig 7C).


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)

Influence of Gln on mitochondrial apoptosis-related protein.H9C2 cells were treated as described in Fig 3. The expression of cytosolic (A) and mitochondrial (B) cytochrome c (cyto-c) as well as cleaved caspase-3 and pro caspase-3 (C) in each group were measured by western blot. Beta-actin and VADC1 were used as internal protein loading controls. Bar graphs summarize the protein band intensities of cyto-c and cleaved caspase 3. * P < 0.05 compare with the control group; # P <0.05 compare with the 1 mM and 4 mM Gln groups.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493145&req=5

pone.0132402.g007: Influence of Gln on mitochondrial apoptosis-related protein.H9C2 cells were treated as described in Fig 3. The expression of cytosolic (A) and mitochondrial (B) cytochrome c (cyto-c) as well as cleaved caspase-3 and pro caspase-3 (C) in each group were measured by western blot. Beta-actin and VADC1 were used as internal protein loading controls. Bar graphs summarize the protein band intensities of cyto-c and cleaved caspase 3. * P < 0.05 compare with the control group; # P <0.05 compare with the 1 mM and 4 mM Gln groups.
Mentions: Western blot analysis revealed that in the 1 mM and 4 mM Gln groups exposed to high glucose and H/R, the levels of cytochrome c in mitochondrial fractions was higher, and the levels of cytochrome c in cytosolic fractions was lower, compared with the control (Fig 7A and 7B). No significant difference in these fractions was found in HG+H/R cells exposed to 16 mM Gln. Furthermore, in the 1 mM and 4 mM Gln groups, the levels of cleaved caspase 3 at 17-and 19-kDa were significantly higher than that of the control cells, but these higher levels were not observed in the cells treated with 16 mM Gln (Fig 7C).

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