Zinc-activated C-peptide resistance to the type 2 diabetic erythrocyte is associated with hyperglycemia-induced phosphatidylserine externalization and reversed by metformin

Abstract

Insulin resistance can broadly be defined as the diminished ability of cells to respond to the action of insulin in transporting glucose from the bloodstream into cells and tissues. Here, we report that erythrocytes (ERYs) obtained from type 2 diabetic rats display an apparent resistance to Zn(2+)-activated C-peptide. Thus, the aims of this study were to demonstrate that Zn(2+)-activated C-peptide exerts potentially beneficial effects on healthy ERYs and that these same effects on type 2 diabetic ERYs are enhanced in the presence of metformin. Incubation of ERYs (obtained from type 2 diabetic BBZDR/Wor-rats) with Zn(2+)-activated C-peptide followed by chemiluminescence measurements of ATP resulted in a 31.2 +/- 4.0% increase in ATP release from these ERYs compared to a 78.4 +/- 4.9% increase from control ERYs. Glucose accumulation in diabetic ERYs, measured by scintillation counting of (14)C-labeled glucose, increased by 35.8 +/- 1.3% in the presence of the Zn(2+)-activated C-peptide, a value significantly lower than results obtained from control ERYs (64.3 +/- 5.1%). When Zn(2+)-activated C-peptide was exogenously added to diabetic ERYs, immunoassays revealed a 32.5 +/- 8.2% increase in C-peptide absorbance compared to a 64.4 +/- 10.3% increase in control ERYs. Phosphatidylserine (PS) externalization and metformin sensitization of Zn(2+)-activated C-peptide were examined spectrofluorometrically by measuring the binding of FITC-labeled annexin to PS. The incubation of diabetic ERYs with metformin prior to the addition of Zn(2+)-activated C-peptide resulted in values that were statistically equivalent to those of controls. Summarily, data obtained here demonstrate an apparent resistance to Zn(2+)-activated C-peptide by the ERY that is corrected by metformin.

Molecular Effects of C-Peptide in Microvascular Blood Flow Regulation

“…Figure 3 presents the effect of the different Cpeptide fragments on erythrocyte deformability, and the inhibition with oubain or EDTA at a shear stress rate of 1.2 Pa. In a type 2 diabetes rat model, Zn 2+ -activated C-peptide was shown to exert less ATP release from erythrocytes, as compared with controls [46]. Pretreatment of the type 2 diabetic erythrocytes with metformin restored the C-peptide-stimulated release of ATP from the erythrocyte.…”

A Molecular Level Understanding of Zinc Activation of C-peptide and its Effects on Cellular Communication in the Bloodstream

“…Previous studies involving C-peptide interactions with RBCs clearly demonstrate that Cpeptide is able to stimulate glucose uptake into the RBC, resulting in the release of ATP into the extracellular matrix [18,52]. This glucose uptake results in an increase of ATP production by the RBC.…”

“…A small scale study involving RBCs obtained from a rat model of type 1 diabetes revealed that the overall percentage increase in ATP release was not statistically different to the release measured from RBCs obtained from control rats [18]. In contrast, in a rat model of type 2 diabetes, ATP release from these cells were significantly less (approximately 50%) than that obtained from control RBCs [18]. Subsequent studies suggested that exogenously added C-peptide was interacting with the RBCs obtained from the type 2 rat models to a much lesser extent than the control RBCs (again, approximately a 50% reduction).…”

“…The knowledge of Cpeptide's role in this breakdown remains incomplete, although studies have shown that C-peptide does participate in the breakdown process. One possible mechanism is that the low pH of the granules keeps glutamic acid residues (structure shown inside the beta-cell) protonated until secretion where it changes to glutamate (structure shown above the beta-cell), a substance capable of binding Zn overcome the reduced interactions between C-peptide and the type 2 RBC [18].…”

“…Insulin-independent effects of Cpeptide and metal activation C-peptide has been shown by our group and other groups to interact with RBCs [15][16][17][18]. In the studies involving deformability [15][16], the RBC samples were incubated with C-peptide alone and, after an incubation period, improvements in deformability of the RBCs obtained from diabetic patients were measured.…”