In vitro and in vivo characterization of insulin vesicles by electron microscopy

Insulin is the main hypoglycemic hormone, promoting the absorption and storage of glucose and inhibiting its production. It is a hexamer composed of six insulin macromolecules and a Zn²⁺ and clustered in insulin vesicles of pancreatic β cell. Most current research has focused on the in vivo imaging of whole cells while there are few detailed studies on structure of insulin vesicles. The precise content of Zn²⁺ in vesicles is not clear, and the aggregation state and location of insulin in insulin vesicles is not fully characterized, which hinders a thorough understanding of insulin secretion process and diseases caused by blood sugar regulation. Here, we performed electron microscopy (EM) studies on both whole cells (in vivo) and extracted isolated insulin vesicles by supercentrifugation (in vitro) to explore the location and distribution of insulin vesicles in pancreatic β cells. Meanwhile, we analyzed the content of Zn²⁺ and Ca²⁺ through EM imaging and energy dispersive spectroscopy (EDS) mapping, and the content of Zn²⁺ was found to be proportional to the size of insulin vesicles. In addition, by taking advantage of TEM tomography, the three-dimensional structure of insulin vesicle was obtained by acquisition projections in different angles of insulin vesicle. This study provides a promising way to quantitative analysis of intracellular insulin, which may be of great significance to the study of diabetes and other blood sugar diseases.