Vasculopathy is a major complication of diabetes; however, molecular mechanisms mediating the development of vasculopathy and potential strategies for prevention have not been identified. We have previously reported that C-peptide prevents diabetic vasculopathy by inhibiting reactive oxygen species (ROS)-mediated endothelial apoptosis. To gain further insight into ROS-dependent mechanism of diabetic vasculopathy and its prevention, we studied high glucose–induced cytosolic and mitochondrial ROS production and its effect on altered mitochondrial dynamics and apoptosis. For the therapeutic strategy, we investigated the vasoprotective mechanism of C-peptide against hyperglycemia-induced endothelial damage through the AMP-activated protein kinase α (AMPKα) pathway using human umbilical vein endothelial cells and aorta of diabetic mice. High glucose (33 mmol/L) increased intracellular ROS through a mechanism involving interregulation between cytosolic and mitochondrial ROS generation. C-peptide (1 nmol/L) activation of AMPKα inhibited high glucose–induced ROS generation, mitochondrial fission, mitochondrial membrane potential collapse, and endothelial cell apoptosis. Additionally, the AMPK activator 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside and the antihyperglycemic drug metformin mimicked protective effects of C-peptide. C-peptide replacement therapy normalized hyperglycemia-induced AMPKα dephosphorylation, ROS generation, and mitochondrial disorganization in aorta of diabetic mice. These findings highlight a novel mechanism by which C-peptide activates AMPKα and protects against hyperglycemia-induced vasculopathy.

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 Ca²⁺ 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 Ca²⁺ 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.

Lack of C-peptide, along with insulin, is the main feature of Type 1 diabetes mellitus (DM) and is also observed in progressive β-cell loss in later stage of Type 2 DM. Therapeutic approaches to hyperglycaemic control have been ineffective in preventing diabetic vasculopathy, and alternative therapeutic strategies are necessary to target both hyperglycaemia and diabetic complications. End-stage organ failure in DM seems to develop primarily due to vascular dysfunction and damage, leading to two types of organ-specific diseases, such as micro- and macrovascular complications. Numerous studies in diabetic patients and animals demonstrate that C-peptide treatment alone or in combination with insulin has physiological functions and might be beneficial in preventing diabetic complications. Current evidence suggests that C-peptide replacement therapy might prevent and ameliorate diabetic vasculopathy and organ-specific complications through conservation of vascular function, as well as prevention of endothelial cell death, microvascular permeability, vascular inflammation, and neointima formation. In this review, we describe recent advances on the beneficial role of C-peptide replacement therapy for preventing diabetic complications, such as retinopathy, nephropathy, neuropathy, impaired wound healing, and inflammation, and further discuss potential beneficial effects of combined C-peptide and insulin supplement therapy to control hyperglycaemia and to prevent organ-specific complications.

Human C-peptide has a beneficial effect on the prevention of diabetic neuropathy, nephropathy, and vascular complications; however, its role in protection against increased vascular permeability in diabetes remains unclear. Our purpose was to explore the potential protective role of C-peptide against microvascular permeability mediated by vascular endothelial growth factor (VEGF)-induced reactive oxygen species (ROS) generation in diabetes. Generation of intracellular ROS, real-time changes in intracellular Ca²⁺, ROS-dependent stress fibre formation, and the disassembly of the adherens junctions were studied by a confocal microscopy in human umbilical vein endothelial cells (HUVECs). VEGF-induced vascular leakage was investigated in the skin of diabetic mice using a Miles vascular permeability assay. Microvascular leakage in the retina of streptozotocin diabetic mice was investigated using a confocal microscopy after left ventricle injection of fluorescein isothiocyanate (FITC)-dextran. C-peptide inhibited the VEGF-induced ROS generation, stress fibre formation, disassembly of vascular endothelial cadherin, and endothelial permeability in HUVECs. Intradermal injection of C-peptide prevented VEGF-induced vascular leakage. Consistent with this, intravitreal injection of C-peptide prevented the extravasation of FITC-dextran in the retinas of diabetic mice, which was also prevented by anti-VEGF antibody and ROS scavengers in diabetic mice. C-peptide prevents VEGF-induced microvascular permeability by inhibiting ROS-mediated intracellular events in diabetic mice, suggesting that C-peptide replacement is a promising therapeutic strategy to prevent diabetic retinopathy.

Proteomics is one of the most important issues in the post-genomic area, because it can greatly contribute to identifying protein biomarkers for disease diagnosis and drug screening. Protein array is a key technology for proteome researches and has been analyzed by various methods including fluorescence, mass spectrometry, atomic force microscopy and surface plasmon resonance (SPR). SPR biosensor is a promising technology in proteomics, since it has various advantages including real-time measurement of biomolecular interactions without labeling and the simple optical system for the device. SPR biosensors have a strong potential for analyzing proteomes by SPR imaging and SPR spectroscopic imaging, even though the challenge is to produce proteins on a proteomic scale.