Investigation of transport mechanisms and regulation of intracellular Zn2+ in pancreatic α-cells

During insulin secretion, pancreatic alpha-cells are exposed to Zn2+ released from insulin-containing secretory granules. Although maintenance of Zn2+ homeostasis is critical for cell survival and glucagon secretion, very little is known about Zn2+-transporting pathways and the regulation of Zn2+ in alpha-cells. To examine the effect of Zn2+ on glucagon secretion and possible mechanisms controlling the intracellular Zn2+ level ([Zn2+](i)), we employed a glucagon-producing cell line (alpha-TC6) and mouse islets where non beta-cells were identified using islets expressing green fluorescent protein exclusively in beta-cells. In this study, we first confirmed that Zn2+ treatment resulted in the inhibition of glucagon secretion in alpha-TC6 cells and mouse islets in vitro. The inhibition of secretion was not likely via activation of KATP channels by Zn2+. We then determined that Zn2+ was transported into alpha-cells and was able to accumulate under both low and high glucose conditions, as well as upon depolarization of cells with KCl. The nonselective Ca2+ channel blocker Gd3+ partially inhibited Zn2+ influx in alpha-TC cells, whereas the L-type voltage-gated Ca2+ channel inhibitor nitren-dipine failed to block Zn2+ accumulation. To investigate Zn2+ transport further, we profiled alpha-cells for Zn2+ transporter transcripts from the two families that work in opposite directions, SLC39 (ZIP, Zrt/Irt-like protein) and SLC30 (ZnT, Zn2+ transporter). We observed that Zip1, Zip10, and Zip14 were the most abundantly expressed Zips and ZnT4, ZnT5, and ZnT8 the dominant ZnTs. Because the redox state of cells is also a major regulator of [Zn2+](i), we examined the effects of oxidizing agents on Zn2+ mobilization within alpha-cells. 2,2'-Dithiodipyridine (-SH group oxidant), menadione (superoxide generator), and SIN-1 (3-morpholinosydnonimine) (peroxynitrite generator) all increased [Zn2+](i) in alpha-cells. Together these results demonstrate that Zn2+ inhibits glucagon secretion, and it is transported into alpha-cells in part through Ca2+ channels. Zn2+ transporters and the redox state also modulate [Zn2+](i).