ABSTRACT MECHANISTIC AND FUNCTIONAL STUDIES OF ZINC (II) ACTIVATION OF C-PEPTIDE AND ITS EFFECT ON RED BLOOD CELL METABOLISM By Wathsala Medawala C-peptide, a 31 amino acid peptide co-secreted with insulin from β-cells in the pancreas, has long been considered a by-product of insulin synthesis. Later it was discovered that C-peptide could ameliorate diabetic complications such as neuropathy, nephropathy, retinopathy and microvascular disease. However, the molecular mechanism behind the effects of C-peptide is not yet completely understood. The effect of C-peptide on the microcirculation is important, because problems in the circulation have been linked to other diabetic complications. C-peptide improves the microcirculation by increasing endothelial-derived nitric oxide (NO), red blood cell (RBC) deformability and Na+/K+-ATPase activity. C-peptide increases glucose utilization and ATP release from RBCs and endothelial cells. ATP is a stimulus for the production of endothelial-derived NO, which is a known vasodilator. Therefore, the C-peptide mediated increase in RBC-derived ATP release can lead to the improvement of blood flow.C-peptide alone did not increase RBC-derived ATP release. The presence of a metal ion such as Zn2+ was needed to elicit this response. However, the role of Zn2+ in C-peptide activity is not fully understood. A fluorescence based study was used to investigate the binding of Zn2+ to C-peptide. Results indicate that C-peptide binds one Zn2+ ion and has a binding constant of 1.02 x 107 M-1 with Zn2+ at pH 5.5, which is the pH inside a mature β-cell granule. At physiological pH of 7.4, C-peptide binds with two Zn2+ ions and has a binding constant of 7.99 x 106 M-1. This indicates that C-peptide may bind with Zn2+ inside the β-cell granule and release Zn2+ upon entering the blood stream. Circular dichroism studies suggest that a 1:1 Zn2+ to C-peptide ratio elicits a decrease in the randomness of the peptide chain, which is lost at higher Zn2+ to C-peptide ratios.Five single amino acid peptide mutants of C-peptide were used to study the effect of the acidic amino acid residues on Zn2+ binding. Substitution of glutamate residues at positions 27, 11 and 3 decreased the Zn2+ binding to C-peptide by ~50%, agreeing with the activity of these mutants in other bio-assays. Several studies have indicated the necessity of insulin for C-peptide activity. The Zn2+ that available with insulin hexamers may be responsible for activation of C-peptide. Using a RBC-derived ATP assay, it was shown that Zn2+ added in the form of Zn-insulin activates C-peptide, as observed by the increase in ATP release. The C-peptide interaction with RBCs was studied using an ELISA. Zn2+-activated C-peptide bound to RBCs in a dose dependent manner, 2 pmol of Zn2+-activated C-peptide binding to 1 mL of 7% red blood cell sample at saturation (~1500 molecules/ cell). The interaction did not change significantly even in the absence of Zn2+. However, Zn2+ uptake in RBCs was observed only in the presence of C-peptide, indicating a possible role for C-peptide as a Zn2+ carrier. One argument against C-peptide being a potential medication is that patients with type 2 diabetes develop the complications of the disease despite having circulating C-peptide. Red blood cells incubated in high glucose (>10 mM), showed lower interaction with Zn2+-activated C-peptide and lower ATP release, showing that patients with uncontrolled blood glucose levels might show resistance to the action of the peptide. Also, the exposure of Zn2+-activated C-peptide to serum albumin and mM levels of other cations and anions decreases its activity. Taken together, the work presented in this dissertation explains the role of Zn2+ in C-peptide action and how reproducible results may be obtained in C-peptide clinical trials.
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