C‐peptide and Insulin Work in Concert to Mitigate Hyperglycemia‐induced Oxidative Stress and Facilitate Cellular Recovery

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The role of C‐peptide in the prevention and resolution of diabetes‐induced vascular injuries is not fully understood. C‐peptide is a 31 amino acid peptide that is co‐produced and co‐secreted with insulin by pancreatic beta islet cells. In insulin‐dependent diabetics C‐peptide levels correlate with the delayed onset of comorbidities that arise as a consequence of vascular compromise. These comorbidities include increased incidence of cardiovascular disease, retinopathies, renal failure, and compromised wound healing. In 2013, G‐protein‐coupled‐receptor 146 was identified by our group as the receptor for C‐peptide. GPR146 is highly expressed on endothelial cells, fibroblasts, red blood cells, and lymphocytes. We hypothesized that C‐peptide and insulin act in concert to regulate and reduce hyperglycemia‐induced oxidative stress and normalize cellular function in hyperglycemia. In this study, primary human dermal microvascular endothelial cells (HDMEC), primary normal human dermal fibroblasts (NHDF), and human microvascular endothelial cells (HMEC), were used to investigate cellular response to incubation in normal glucose(NG) or high glucose (25 mM)(HG) alone or with the addition of C‐peptide (1.5 nM), insulin (0.5 nM), or both C‐peptide and insulin. All cell lines were pre‐screened for the presence of the insulin receptor and GPR 146 by qPCR and the responses to C‐peptide and insulin by relative expression of cFOS mRNA. In cells incubated with 25 mM glucose for 3 days, the ratio of phosphorylated ERK1–2/ERK 1–2 was not significantly increased by C‐peptide or insulin alone but was significantly increased by incubation with both. Cells incubated in HG for 3 days demonstrated phosphorylated AKT/AKT levels which were not altered by C‐peptide alone, but were increased by insulin and markedly enhanced by the presence of both. In wound healing assays cells in HG demonstrated impaired migration relative to those in NG and the greatest improvement in migration was in the presence of paired C‐peptide and insulin. Endothelial cell nitric oxide (NO) production was significantly elevated in HG, and while there was no significant inhibition by C‐peptide or insulin alone, incubation with both resulted in complete restoration to normal levels. NO generated by ECs in response to HG is produced in conditions of mitochondrial oxidative stress and production of radical oxygen species (ROS), in this environment NO complexes with ROS to form peroxynitrites. Peroxynitrites are potent mediators of cellular damage, forming complexes with proteins, enzymes, and lipids. ROS and superoxide (SO) levels were increased in cells exposed to high glucose conditions, and incubation with both C‐peptide and insulin resulted in the reduction of ROS and SO levels. This study demonstrated the cytoprotective potential of C‐peptide when paired with insulin. These results support the hypothesis that co‐administration of C‐peptide with insulin to insulin‐dependent diabetics may reduce the onset of comorbidities.Support or Funding InformationHJ121456This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.