title: 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.
abstract: OBJECTIVE
  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
  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.
authors.affiliation: Division of Immunogenetics, Department of Pediatrics, Rangos Research Center, Children's Hospital of Pittsburgh, 530 45th Street, Pittsburgh, PA 15201, USA.
authors.forename: V
authors.initials: V
authors.lastname: Cifarelli
authors.forename: X
authors.initials: X
authors.lastname: Geng
authors.forename: A
authors.initials: A
authors.lastname: Styche
authors.forename: R
authors.initials: R
authors.lastname: Lakomy
authors.forename: M
authors.initials: M
authors.lastname: Trucco
authors.forename: P
authors.initials: P
authors.lastname: Luppi
mesh_terms: D001011:Aorta / Q000166:cytology; D017209:Apoptosis / Q000187:drug effects; D002096:C-Peptide / Q000494:pharmacology*; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D005947:Glucose / Q000494:pharmacology*; D006801:Humans; D015151:Immunoblotting; D020287:In Situ Nick-End Labeling; D019255:NADPH Oxidases / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D020830:rac1 GTP-Binding Protein / Q000235:genetics
chemical_list: D002096:C-Peptide; D017382:Reactive Oxygen Species; D019255:NADPH Oxidases; D020830:rac1 GTP-Binding Protein; D005947:Glucose