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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 cell viability and apoptosis.H9C2 cells were exposed to HG + H/R with different concentrations of Gln (0.5, 1, 2, 4, 8, 16, and 32 mM). (A) Cell viability of each group was determined by MTT assay. (B) Results of the TUNEL assay and DAPI staining of each group. Green spots represented apoptotic bodies and blue spots represent cell nuclei. (C) Bars represent the percentage of TUNEL-positive cells based on the total number of cells stained by DAPI. * P < 0.05 compare with the control group, # P < 0.05 compare with the 1 mM and 4 mM Gln groups. Data are expressed as mean ± S.D. of 3 independent experiments; a.u. = arbitrary units.
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pone.0132402.g002: Effect of Gln on cell viability and apoptosis.H9C2 cells were exposed to HG + H/R with different concentrations of Gln (0.5, 1, 2, 4, 8, 16, and 32 mM). (A) Cell viability of each group was determined by MTT assay. (B) Results of the TUNEL assay and DAPI staining of each group. Green spots represented apoptotic bodies and blue spots represent cell nuclei. (C) Bars represent the percentage of TUNEL-positive cells based on the total number of cells stained by DAPI. * P < 0.05 compare with the control group, # P < 0.05 compare with the 1 mM and 4 mM Gln groups. Data are expressed as mean ± S.D. of 3 independent experiments; a.u. = arbitrary units.

Mentions: To observe the effect of Gln on cell viability or apoptosis, H9C2 cells were exposed to high glucose and H/R, with various concentrations of Gln (0.5–32 mM). The MTT assay showed a concentration-dependent association between Gln treatment and cell viability (Fig 2A). In accord with the MTT results, the TUNEL assay also revealed a Gln concentration-dependent decrease in the number of TUNEL-positive nuclei in the high glucose and H/R treated cells, indicating that Gln could inhibit cell apoptosis (Fig 2B and 2C). The least number of apoptotic cells was found in the group treated with 16 mM Gln. More apoptotic cells were found in the group treated with 32 mM Gln than in those treated with Gln concentrations of 8–16 mM. The results suggested that apoptosis induced by high glucose and H/R could be reduced, at least partly, by Gln supplementation. Based on these results, in subsequent studies, 4 experimental treatments were used to elucidate the potential protective mechanism of Gln: HG+HR with 1, 4, or 16 mM Gln, and the control.


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 cell viability and apoptosis.H9C2 cells were exposed to HG + H/R with different concentrations of Gln (0.5, 1, 2, 4, 8, 16, and 32 mM). (A) Cell viability of each group was determined by MTT assay. (B) Results of the TUNEL assay and DAPI staining of each group. Green spots represented apoptotic bodies and blue spots represent cell nuclei. (C) Bars represent the percentage of TUNEL-positive cells based on the total number of cells stained by DAPI. * P < 0.05 compare with the control group, # P < 0.05 compare with the 1 mM and 4 mM Gln groups. Data are expressed as 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.g002: Effect of Gln on cell viability and apoptosis.H9C2 cells were exposed to HG + H/R with different concentrations of Gln (0.5, 1, 2, 4, 8, 16, and 32 mM). (A) Cell viability of each group was determined by MTT assay. (B) Results of the TUNEL assay and DAPI staining of each group. Green spots represented apoptotic bodies and blue spots represent cell nuclei. (C) Bars represent the percentage of TUNEL-positive cells based on the total number of cells stained by DAPI. * P < 0.05 compare with the control group, # P < 0.05 compare with the 1 mM and 4 mM Gln groups. Data are expressed as mean ± S.D. of 3 independent experiments; a.u. = arbitrary units.
Mentions: To observe the effect of Gln on cell viability or apoptosis, H9C2 cells were exposed to high glucose and H/R, with various concentrations of Gln (0.5–32 mM). The MTT assay showed a concentration-dependent association between Gln treatment and cell viability (Fig 2A). In accord with the MTT results, the TUNEL assay also revealed a Gln concentration-dependent decrease in the number of TUNEL-positive nuclei in the high glucose and H/R treated cells, indicating that Gln could inhibit cell apoptosis (Fig 2B and 2C). The least number of apoptotic cells was found in the group treated with 16 mM Gln. More apoptotic cells were found in the group treated with 32 mM Gln than in those treated with Gln concentrations of 8–16 mM. The results suggested that apoptosis induced by high glucose and H/R could be reduced, at least partly, by Gln supplementation. Based on these results, in subsequent studies, 4 experimental treatments were used to elucidate the potential protective mechanism of Gln: HG+HR with 1, 4, or 16 mM Gln, and the control.

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