Enhancement of homocysteine toxicity to insulin-secreting BRIN-BD11 cells in combination with alloxan

Abstract

Previous studies have shown that homocysteine (HC) has a detrimental impact on insulin secretion and pancreatic beta cell function. The aim of the present study was to determine the role of reactive oxygen species (ROS) in the in vitro toxic effects of HC on insulin secretion and function of BRIN-BD11 insulin-secreting cells. In this study, insulin secretion from BRIN-BD11 cells was determined radioimmunologically, cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and glucokinase activity by a glucose phosphorylation assay following culture with HC plus alloxan (Alx). Treatment with HC resulted in concentrationdependent inhibition of insulin secretion induced by glucose and other insulinotropic agents. HC in combination with Alx resulted in a more pronounced decline in insulin secretion, including that induced by 20 mM alanine, by 43% (P!0 . 001) and 30 mM KCl by 60% (P!0 . 001), compared with control culture. The glucokinase phosphorylating capacity in cells cultured with HC plus Alx was significantly lower, compared with control cells. The cells also displayed a significant 84% (P!0 . 001) decline in cell viability. Prolonged, 72-h culture of insulin-secreting cells with HC followed by 18-h culture without HC did not result in full restoration of beta cell responses to insulinotropic agents. In vitro oxygen consumption was enhanced by a combination of Alx with HC. The study arrived at the conclusion that HC generates ROS in a redox-cycling reaction with Alx that explains the decline in viability of insulin-secreting cells, leading to reduced glucokinase phosphorylating ability, diminished insulin secretory responsiveness and cell death.

Improved antioxidative defence protects insulin-producing cells against homocysteine toxicity

“…This confirms that these thiol containing amino acids can reduce alloxan to dialuric acid, alloxan's redox cycling partner. These results are in line with our earlier observation in BRIN-BD11 cells that alloxan can be rendered cytotoxic to insulin-producing cells by interaction with HC [19]. While concentrations of HC increase, the cell viability declines in parallel to the generation of ROS.…”

“…Moreover, our data confirm the previous observation that HC redox cycles with alloxan much more vigorously than cysteine [19]. Thus, cumulating evidence strongly suggests that the more pronounced cytotoxic effect of HC compared with cysteine depends on a higher rate of ROS formation in the cytoplasm.…”

“…Consequently, INS1E were susceptible to alloxan toxicity, and this toxicity was further potentiated by HC. The strongest inhibition of GSIS was observed when both alloxan and HC were present, as has been reported also for BRIN-BD11 cells [19].…”

“…Thus acute and chronic exposure of insulin-secreting cells to HC results in defective insulin secretion and beta cell demise [16e19]. We have previously shown that HC generates reactive oxygen species in a redox cycling reaction with alloxan [19] explaining the decline in viability of insulin-secreting cells, leading to reduced glucokinase phosphorylating ability, diminished insulin secretory responsiveness and cell death [19].…”

“…Alloxan and its redox cycling products target SH groups, forming disulfide bonds thereby causing beta-cell dysfunction and death [21,22]. Sulphur-containing HC serves also as a redox cycling partner for alloxan thereby potentiating alloxan toxicity [19].…”