Imaging the Glucagon-Secreting Alpha Cells

Pancreatic islets secrete insulin and glucagon, two critical hormones for the blood glucose homeostasis. Islets are composed mainly of insulin-secreting beta-cells (∼80%) and glucagon-secreting alpha-cells (10-15%). Typically, insulin is secreted after a meal to reduce blood glucose levels, whereas glucagon is released in times of starvation. In order for insulin and glucagon to exert their opposite effects, glucose must also have an inverse effect on the secretory function of the two cell types. While the mechanisms of glucose-stimulated insulin secretion by beta-cells are fairly well understood, the study of the mechanisms underlying glucagon secretion has been hindered by the lack of reliable methods to distinguish between alpha- and beta-cells. Thus, it is not clear how glucose suppresses glucagon secretion. There is still little agreement as to whether this inhibition is a direct effect on alpha-cells or mediated by a paracrine inhibition from beta-cells via secretion of molecules such as insulin, Zn2+, GABA.Recently, a fragment of a rat glucagon promoter has been successfully used to specifically drive the expression of fluorescent proteins in mouse alpha-cells. Thus, it is now possible to easily identify glucagon-secreting cells within isolated islets or to flow sort a purified population of alpha-cells. Here, we report the use of dynamic fluorescence imaging techniques on these transgenic islets and cells. In particular, we investigated how glucose affects 1) alpha-cell metabolism via measurement of NAD(P)H autofluorescence by two-photon excitation microscopy; 2) alpha-cell intracellular calcium concentrations, via the loading of alpha-cells expressing red fluorescent proteins (tdRFP) with the calcium indicator dye Fluo4-AM; and 3) alpha-cell membrane potential, via application of the fluorescent probe DiSBAC2(3). The results of these biophysical measurements will also be compared to parallel glucagon secretion in response to glucose from both intact islets and sorted alpha-cells.