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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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C-peptide and inhibitors of intracellular ROS, Ca2+, and TG2 inhibit high glucose (HG)–induced apoptotic cell death in HUVECs. Confluent cells were preincubated with 1 nmol/L C-peptide, 5 μmol/L Ac-DEVD-cho, 1 μmol/L Z-VAD-fmk, 0.5 mmol/L Trolox, 5 μmol/L BAPTA-AM, 50 μmol/L cystamine, or 10 μmol/L monodansylcadaverine (MDC) for 30 min, followed by incubation with HG (33 mmol/L d-glucose) for 72 h. A: Cell viability was determined using an MTT reduction assay. B: HG-induced apoptotic cell death was measured by DiOC6/PI double-staining. Early and late apoptotic cell death rates were determined as described in RESEARCH DESIGN AND METHODS. Results are expressed as mean ± SD from three independent experiments. **P < 0.01.
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Figure 1: C-peptide and inhibitors of intracellular ROS, Ca2+, and TG2 inhibit high glucose (HG)–induced apoptotic cell death in HUVECs. Confluent cells were preincubated with 1 nmol/L C-peptide, 5 μmol/L Ac-DEVD-cho, 1 μmol/L Z-VAD-fmk, 0.5 mmol/L Trolox, 5 μmol/L BAPTA-AM, 50 μmol/L cystamine, or 10 μmol/L monodansylcadaverine (MDC) for 30 min, followed by incubation with HG (33 mmol/L d-glucose) for 72 h. A: Cell viability was determined using an MTT reduction assay. B: HG-induced apoptotic cell death was measured by DiOC6/PI double-staining. Early and late apoptotic cell death rates were determined as described in RESEARCH DESIGN AND METHODS. Results are expressed as mean ± SD from three independent experiments. **P < 0.01.

Mentions: High glucose decreased the viability of HUVECs dramatically, and this effect was overcome by 1 nmol/L C-peptide (Fig. 1A). The high glucose–induced decrease in cell viability was also inhibited by a caspase-3 inhibitor, Ac-DEVD-cho, and a broad-spectrum caspase inhibitor, Z-VAD-fmk (P < 0.01). Glucose treatment increased the number of early and late apoptotic cells, as evaluated by DiOC6/PI staining, and cell death was prevented by C-peptide, Ac-DEVD-cho, or Z-VAD-fmk (Fig. 1B).


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)

C-peptide and inhibitors of intracellular ROS, Ca2+, and TG2 inhibit high glucose (HG)–induced apoptotic cell death in HUVECs. Confluent cells were preincubated with 1 nmol/L C-peptide, 5 μmol/L Ac-DEVD-cho, 1 μmol/L Z-VAD-fmk, 0.5 mmol/L Trolox, 5 μmol/L BAPTA-AM, 50 μmol/L cystamine, or 10 μmol/L monodansylcadaverine (MDC) for 30 min, followed by incubation with HG (33 mmol/L d-glucose) for 72 h. A: Cell viability was determined using an MTT reduction assay. B: HG-induced apoptotic cell death was measured by DiOC6/PI double-staining. Early and late apoptotic cell death rates were determined as described in RESEARCH DESIGN AND METHODS. Results are expressed as mean ± SD from three independent experiments. **P < 0.01.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: C-peptide and inhibitors of intracellular ROS, Ca2+, and TG2 inhibit high glucose (HG)–induced apoptotic cell death in HUVECs. Confluent cells were preincubated with 1 nmol/L C-peptide, 5 μmol/L Ac-DEVD-cho, 1 μmol/L Z-VAD-fmk, 0.5 mmol/L Trolox, 5 μmol/L BAPTA-AM, 50 μmol/L cystamine, or 10 μmol/L monodansylcadaverine (MDC) for 30 min, followed by incubation with HG (33 mmol/L d-glucose) for 72 h. A: Cell viability was determined using an MTT reduction assay. B: HG-induced apoptotic cell death was measured by DiOC6/PI double-staining. Early and late apoptotic cell death rates were determined as described in RESEARCH DESIGN AND METHODS. Results are expressed as mean ± SD from three independent experiments. **P < 0.01.
Mentions: High glucose decreased the viability of HUVECs dramatically, and this effect was overcome by 1 nmol/L C-peptide (Fig. 1A). The high glucose–induced decrease in cell viability was also inhibited by a caspase-3 inhibitor, Ac-DEVD-cho, and a broad-spectrum caspase inhibitor, Z-VAD-fmk (P < 0.01). Glucose treatment increased the number of early and late apoptotic cells, as evaluated by DiOC6/PI staining, and cell death was prevented by C-peptide, Ac-DEVD-cho, or Z-VAD-fmk (Fig. 1B).

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