Erythrocytes (ERYs) deliver oxygen to organs. However, another important role of ERYs is to regulate blood flow by responding to various stimuli. In blood vessels, flowing ERYs deform and release adenosine triphosphate (ATP). ATP is able to stimulate the production of a well-known vasodilator, nitric oxide (NO) in the endothelium, thereby serving as an indirect determinant of blood flow. NO relaxes vascular smooth muscle cells enabling the dilation of blood vessels to maintain normal blood flow. In addition to deformation, ERYs also respond to other stimuli in the bloodstream such as C-peptide and zinc to release ATP.C-peptide is a 31 amino acid peptide co-secreted with insulin from pancreatic β-cells. Insulin is known for controlling blood glucose levels, and insulin replacement is approved therapy for type 1 diabetic patients, whose pancreas fails to produce insulin and C-peptide. In the 1990s, beneficial effects of C-peptide in reducing diabetes complications were discovered, and were thought to be related to improved blood flow. However, the mechanism of C-peptide remains elusive, which became the major obstacle for C-peptide administration to be used along with insulin.Previously, it has been shown that C-peptide, when combined with zinc, can enhance ATP release from ERYs by 50%. In turn, an increase in NO production from the endothelium was also measured using an in vitro platform. However, the interaction of C-peptide and zinc with ERYs is unclear. Enzyme-linked immunosorbent assay (ELISA) and scintillation counting results showed that both C-peptide and zinc could bind to the ERY, although no binding between C-peptide and zinc could be detected. The results in this dissertation provide evidence that albumin is indispensable for uptake of both species, and that C-peptide uptake by the ERY is requisite for zinc uptake. Isotheral titration calorimetry experiments revealed specific binding of C-peptide and zinc to albumin, suggesting that albumin transports C-peptide and zinc to ERYs for biological effects. Uptake of C-peptide and zinc led to an improvement in ERY membrane deformability, which could explain the subsequent increase of ATP release when subjected to flow-induced shear stress.To demonstrate the broader impact of these findings, the response of stored ERYs to C-peptide and zinc is found compromised in the current FDA approved AS-1 solution. Decreased response was reversed up to 12-15 days of storage. Excess glucose in the AS-1 solution was found responsible for the loss of response. C-peptide and zinc are components of pancreatic β-cell secretion, therefore suggesting that transfusion of ERYs stored in AS-1 for longer than 15 days can cause more health issues than post transfusion complications by not properly responding to healthy organs such as the pancreas, whereas a normoglycemic storage conditions may be a solution to the issues.The ability of C-peptide and zinc to have combined effects with molecules secreted from other tissues was also investigated. Specifically, leptin, a hormone secreted from adipose cells, acts on receptors in the arcuate nucleus of the hypothalamus to control appetite, and achieve energy balance. Results of ATP release experiments showed that, in the presence of C-peptide and zinc, leptin further enhanced ATP release from ERYs by an additional 30%. This may suggest another mechanism of leptin for glucose clearance that involves a site of action on the ERY also governed by the efficacy of C-peptide and zinc.
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