Mechanism of action of diabetogenic zinc-chelating agents. Model system studies

Using model systems, we have studied the properties of a number of zinc-chelating agents which are known to cause diabetes in laboratory animals. The abilities to permeate membranes and to complex zinc inside liposomes with the release of protons are suggested as chemical properties that can enhance diabetogenicity. When such complexing agents are added to lipid vesicles at pH 6 containing entrapped zinc ions, they acidify the contents of these vesicles. We have demonstrated this effect by measuring intravesicular pH both with a fluorine-containing F NMR probe as well as with the fluorescent probe, quinine. For example, using quinine, we observed that 0.1 mM 8-hydroxyquinoline reduced the intravesicular pH of sonicated phospholipid vesicles containing entrapped Zn2+ (as sulfate) from pH 6.0 to 2.8. These diabetogenic chelating agents also solubilized zinc-insulin precipitates from unbuffered suspensions at pH 6.0. The solubilization results from the acidification of these suspensions. Dithizone and 8-hydroxyquinoline at 4 mM solubilized 97 and 42%, respectively, of the suspended insulin. We suggest that if such proton release occurs within the zinc-containing insulin storage granules of pancreatic beta-cells, solubilization of insulin would be induced. Such an event would lead to osmotic stress and eventually to rupture of the granule. The effects of diethyldithiocarbamate (DDC), an agent that has been found to protect rabbits against the induction of diabetes by some other zinc-chelating agents, were also studied. DDC caused a decrease of 3.5 units in the intravesicular pH of zinc-containing vesicles by a mechanism not involving the release of protons upon chelation of zinc. We have demonstrated several properties of DDC which may contribute to its ability to protect against the induction of diabetes. These include its ability to store zinc as a hydrophobic complex in membranes, its consumption of protons upon spontaneous decomposition, and the ability of one of its decomposition products, diethylamine, to accelerate the dissipation of pH gradients across lipid bilayers. Diethylamine is particularly effective in stimulating a rapid dissipation of such pH gradients, even at micromolar concentrations. We have attempted to estimate quantitatively the extent of proton liberation by various zinc-chelating agents. This analysis demonstrated that partitioning of the ligand between organic and aqueous phases, ligand acidity, and zinc complex stability determine the extent of proton release.