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Diabetologia. 2011 Oct;54(10):2702-12. doi: 10.1007/s00125-011-2251-0. Epub 2011 Jul 20.

C-peptide reduces high-glucose-induced apoptosis of endothelial cells and decreases NAD(P)H-oxidase reactive oxygen species generation in human aortic endothelial cells.

Author information

1
Division of Immunogenetics, Department of Pediatrics, Rangos Research Center, Children's Hospital of Pittsburgh, 530 45th Street, Pittsburgh, PA 15201, USA.

Abstract

AIMS/HYPOTHESIS:

Reactive oxygen species (ROS) generated during hyperglycaemia are implicated in the development of diabetic vascular complications. High glucose increases oxidative stress in endothelial cells and induces apoptosis. A major source of ROS in endothelial cells exposed to glucose is the NAD(P)H oxidase enzyme. Several studies demonstrated that C-peptide, the product of proinsulin cleavage within the pancreatic beta cells, displays anti-inflammatory effects in certain models of vascular dysfunction. However, the molecular mechanism underlying this effect is unclear. We hypothesised that C-peptide reduces glucose-induced ROS generation by decreasing NAD(P)H oxidase activation and prevents apoptosis

METHODS:

Human aortic endothelial cells (HAEC) were exposed to 25 mmol/l glucose in the presence or absence of C-peptide and tested for protein quantity and activity of caspase-3 and other apoptosis markers by ELISA, TUNEL and immunoblotting. Intracellular ROS were measured by flow cytometry using the ROS sensitive dye chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H(2)-DCDFA). NAD(P)H oxidase activation was assayed by lucigenin. Membrane and cytoplasmic levels of the NAD(P)H subunit ras-related C3 botulinum toxin substrate 1 (rho family, small GTP binding protein Rac1) (RAC-1) and its GTPase activity were studied by immunoblotting and ELISA. RAC-1 (also known as RAC1) gene expression was investigated by quantitative real-time PCR.

RESULTS:

C-peptide significantly decreased caspase-3 levels and activity and upregulated production of the anti-apoptotic factor B cell CLL/lymphoma 2 (BCL-2). Glucose-induced ROS production was quenched by C-peptide and this was associated with a decreased NAD(P)H oxidase activity and reduced RAC-1 membrane production and GTPase activity.

CONCLUSIONS/INTERPRETATION:

In glucose-exposed endothelial cells, C-peptide acts as an endogenous antioxidant molecule by reducing RAC-1 translocation to membrane and NAD(P)H oxidase activation. By preventing oxidative stress, C-peptide protects endothelial cells from glucose-induced apoptosis.

PMID:
21773684
DOI:
10.1007/s00125-011-2251-0
[Indexed for MEDLINE]

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