Diabetes mellitus, a global health concern, is a disorder of chronic hyperglycemia associated with deficiency or resistance to insulin. Although manageable with treatments aimed at increasing the body's utilization of insulin, often, complications associated with poor microvascular blood flow persist. Erythrocytes play an important role in regulation of microvascular blood flow by their ability to release ATP, a known stimulus of NO. Although insulin produced by the pancreatic Îø-cells acts on GLUT4, erythrocytes and most other bloodstream cells possess GLUT1, suggesting that a different molecular secretion may stimulate GLUT1 trafficking to the cell membrane. Fewer microvascular complications are observed for diabetic patients who possess residual pancreatic Îø-cell activity, suggesting that other pancreatic secretions are important.C-peptide, the 31 amino acid peptide that connects the alpha and beta chains of insulin during its synthesis, is released into the bloodstream in equimolar quantities to insulin after its cleavage from proinsulin. The Spence group, and others, have shown that C-peptide increases erythrocyte-derived ATP release and downstream NO production. However, unlike all other groups, the Spence group has only observed cellular activity of C-peptide in the presence a carrier protein and transition metal. In vivo, the candidate metal is likely Zn2+, due to its high concentrations in the pancreatic Îø-cells. In 2015, it was verified by ITC that HSA can carry C-peptide, with a Ka of 1.75 ℗ł 0.64 x 10-5 M-1 and binding stoichiometry of two C-peptide to a single HSA.Due to the long half-life of HSA (~12-21 days) it is subject to the slow process of non-enzymatic glycation, impacting its ability to act as a carrier molecule, which is particularly problematic in conditions of persistent hyperglycemia. For this reason, Spence group hypothesized that other molecules be able to carry C-peptide and Zn2+ to the erythrocyte. Leptin, a recently discovered adipocyte-derived hormone with a well-known role in metabolism, and a half-life of ~25 minutes, has been implicated in a variety of physiological processes, including glucose regulation, nitric oxide release and blood flow. It was confirmed that leptin, like albumin, can carry C-peptide and Zn2+, increase erythrocyte-derived ATP release and stimulate GLUT1 trafficking.This dissertation investigates of leptin's binding interactions with erythrocytes and C-peptide and how they are impacted by Zn2+, albumin, and hyperglycemia. The results reported herein demonstrate that leptin has a saturable and specific binding site on the erythrocyte with Kd = (1.79 ℗ł 0.46) x 10-7 M and a binding affinity in the presence of 20 nM C-peptide with Kd = (2.05 ℗ł 0.20) x 10-7 M. Specific binding between C-peptide and leptin has been confirmed by SPR, with a Kd of 2.40 x 10-6 M. The data presented herein provide important information toward understanding the mechanism by which C-peptide and leptin are involved in erythrocyte energetics and will aid in creation of more targeted therapeutics to address the microvascular complications of diabetes.
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