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C-peptide prevents hyperglycemia-induced endothelial apoptosis through inhibition of reactive oxygen species-mediated transglutaminase 2 activation.

Bhatt MP, Lim YC, Hwang J, Na S, Kim YM, Ha KS - Diabetes (2012)

Bottom Line: High glucose (33 mmol/L) induced apoptotic cell death in endothelial cells via sequential elevation of intracellular Ca(2+) and reactive oxygen species (ROS) as well as subsequent activation of transglutaminase 2 (TG2).In addition, C-peptide prevented hyperglycemia-induced activation of transamidation activity and apoptosis in the heart and renal cortex of streptozotocin diabetic mice.Furthermore, TG2 may be a promising avenue of therapeutic investigation to treat diabetic vasculopathies.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biochemistry, Kangwon National University School of Medicine, Chuncheon, Kangwon-do, Korea.

ABSTRACT
C-peptide is a bioactive peptide with a potentially protective role in diabetes complications; however, its molecular mechanism of protection against cardiovascular damage caused by hyperglycemia-induced apoptosis remains unclear. We investigated the protective mechanism of C-peptide against hyperglycemia-induced apoptosis using human umbilical vein endothelial cells and streptozotocin diabetic mice. High glucose (33 mmol/L) induced apoptotic cell death in endothelial cells via sequential elevation of intracellular Ca(2+) and reactive oxygen species (ROS) as well as subsequent activation of transglutaminase 2 (TG2). C-peptide (1 nmol/L) prevented endothelial cell death by inhibiting protein kinase C- and NADPH oxidase-dependent intracellular ROS generation and by abolishing high glucose-induced TG2 activation, without affecting intracellular Ca(2+) levels. Consistently, in the aorta of streptozotocin diabetic mice, hyperglycemia stimulated transamidating activity and endothelial cell apoptosis that was inhibited by C-peptide replacement therapy (35 pmol/min/kg) using osmotic pumps (control and diabetes, n = 8; diabetes + C-peptide, n = 7). In addition, C-peptide prevented hyperglycemia-induced activation of transamidation activity and apoptosis in the heart and renal cortex of streptozotocin diabetic mice. Thus, C-peptide protects endothelial cells from hyperglycemia-induced apoptotic cell death by inhibiting intracellular ROS-mediated activation of TG2. Furthermore, TG2 may be a promising avenue of therapeutic investigation to treat diabetic vasculopathies.

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High glucose (HG)–induced generation of intracellular ROS is prevented by various inhibitors. Cells were incubated with HG (33 mmol/L d-glucose) in the presence of the indicated inhibitors for 48 h, and the levels of intracellular ROS were determined as described in RESEARCH DESIGN AND METHODS. A: Time course changes in intracellular ROS by control, l-glucose, and d-glucose. Dose-dependent inhibition of HG-induced ROS generation by Trolox and BAPTA-AM (B), PKC inhibitors (C), NADPH oxidase inhibitors DPI (diphenylene iodonium) and apocynin (D), and carbonyl cyanide m-chlorophenyl hydrazone (CCCP), dinitrophenol (DNP), and rotenone (E). Results are expressed as mean ± SD from three independent experiments. ***P < 0.001.
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Figure 3: High glucose (HG)–induced generation of intracellular ROS is prevented by various inhibitors. Cells were incubated with HG (33 mmol/L d-glucose) in the presence of the indicated inhibitors for 48 h, and the levels of intracellular ROS were determined as described in RESEARCH DESIGN AND METHODS. A: Time course changes in intracellular ROS by control, l-glucose, and d-glucose. Dose-dependent inhibition of HG-induced ROS generation by Trolox and BAPTA-AM (B), PKC inhibitors (C), NADPH oxidase inhibitors DPI (diphenylene iodonium) and apocynin (D), and carbonyl cyanide m-chlorophenyl hydrazone (CCCP), dinitrophenol (DNP), and rotenone (E). Results are expressed as mean ± SD from three independent experiments. ***P < 0.001.

Mentions: High d-glucose increased intracellular ROS in a time-dependent manner, with significant stimulation at 48 h (P < 0.001), whereas l-glucose did not (Fig. 3A). As expected, Trolox inhibited ROS generation in a dose-dependent manner (Fig. 3B). In addition, BAPTA-AM inhibited ROS generation in a dose-dependent manner (Fig. 3B), indicating the importance of intracellular Ca2+ in high glucose–induced ROS generation.


C-peptide prevents hyperglycemia-induced endothelial apoptosis through inhibition of reactive oxygen species-mediated transglutaminase 2 activation.

Bhatt MP, Lim YC, Hwang J, Na S, Kim YM, Ha KS - Diabetes (2012)

High glucose (HG)–induced generation of intracellular ROS is prevented by various inhibitors. Cells were incubated with HG (33 mmol/L d-glucose) in the presence of the indicated inhibitors for 48 h, and the levels of intracellular ROS were determined as described in RESEARCH DESIGN AND METHODS. A: Time course changes in intracellular ROS by control, l-glucose, and d-glucose. Dose-dependent inhibition of HG-induced ROS generation by Trolox and BAPTA-AM (B), PKC inhibitors (C), NADPH oxidase inhibitors DPI (diphenylene iodonium) and apocynin (D), and carbonyl cyanide m-chlorophenyl hydrazone (CCCP), dinitrophenol (DNP), and rotenone (E). Results are expressed as mean ± SD from three independent experiments. ***P < 0.001.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3526059&req=5

Figure 3: High glucose (HG)–induced generation of intracellular ROS is prevented by various inhibitors. Cells were incubated with HG (33 mmol/L d-glucose) in the presence of the indicated inhibitors for 48 h, and the levels of intracellular ROS were determined as described in RESEARCH DESIGN AND METHODS. A: Time course changes in intracellular ROS by control, l-glucose, and d-glucose. Dose-dependent inhibition of HG-induced ROS generation by Trolox and BAPTA-AM (B), PKC inhibitors (C), NADPH oxidase inhibitors DPI (diphenylene iodonium) and apocynin (D), and carbonyl cyanide m-chlorophenyl hydrazone (CCCP), dinitrophenol (DNP), and rotenone (E). Results are expressed as mean ± SD from three independent experiments. ***P < 0.001.
Mentions: High d-glucose increased intracellular ROS in a time-dependent manner, with significant stimulation at 48 h (P < 0.001), whereas l-glucose did not (Fig. 3A). As expected, Trolox inhibited ROS generation in a dose-dependent manner (Fig. 3B). In addition, BAPTA-AM inhibited ROS generation in a dose-dependent manner (Fig. 3B), indicating the importance of intracellular Ca2+ in high glucose–induced ROS generation.

Bottom Line: High glucose (33 mmol/L) induced apoptotic cell death in endothelial cells via sequential elevation of intracellular Ca(2+) and reactive oxygen species (ROS) as well as subsequent activation of transglutaminase 2 (TG2).In addition, C-peptide prevented hyperglycemia-induced activation of transamidation activity and apoptosis in the heart and renal cortex of streptozotocin diabetic mice.Furthermore, TG2 may be a promising avenue of therapeutic investigation to treat diabetic vasculopathies.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biochemistry, Kangwon National University School of Medicine, Chuncheon, Kangwon-do, Korea.

ABSTRACT
C-peptide is a bioactive peptide with a potentially protective role in diabetes complications; however, its molecular mechanism of protection against cardiovascular damage caused by hyperglycemia-induced apoptosis remains unclear. We investigated the protective mechanism of C-peptide against hyperglycemia-induced apoptosis using human umbilical vein endothelial cells and streptozotocin diabetic mice. High glucose (33 mmol/L) induced apoptotic cell death in endothelial cells via sequential elevation of intracellular Ca(2+) and reactive oxygen species (ROS) as well as subsequent activation of transglutaminase 2 (TG2). C-peptide (1 nmol/L) prevented endothelial cell death by inhibiting protein kinase C- and NADPH oxidase-dependent intracellular ROS generation and by abolishing high glucose-induced TG2 activation, without affecting intracellular Ca(2+) levels. Consistently, in the aorta of streptozotocin diabetic mice, hyperglycemia stimulated transamidating activity and endothelial cell apoptosis that was inhibited by C-peptide replacement therapy (35 pmol/min/kg) using osmotic pumps (control and diabetes, n = 8; diabetes + C-peptide, n = 7). In addition, C-peptide prevented hyperglycemia-induced activation of transamidation activity and apoptosis in the heart and renal cortex of streptozotocin diabetic mice. Thus, C-peptide protects endothelial cells from hyperglycemia-induced apoptotic cell death by inhibiting intracellular ROS-mediated activation of TG2. Furthermore, TG2 may be a promising avenue of therapeutic investigation to treat diabetic vasculopathies.

Show MeSH
Related in: MedlinePlus