Identification of alpha- and beta-cells in intact isolated islets of Langerhans by their characteristic cytoplasmic Ca2+ concentration dynamics and immunocytochemical staining.

Abstract

Ratiometric images of cytoplasmic Ca2+ concentration ([Ca2+]c) in individual cells were recorded simultaneously with a confocal ultraviolet-laser microscope in the Indo-1-loaded islets isolated from mice. After changes in [Ca2+]c in response to glucose or amino acids were recorded, the islet was fixed, permeabilized, and stained by the indirect immunofluorescence method against insulin or glucagon in situ; the individual cells were then identified in the focal plain identical to that used for the [Ca2+]c imaging. Almost all cells identified as insulin-positive (beta-cells) by their distinct immunofluorescence responded to the increase in glucose concentration from 3 to 11 mmol/l with an increase in [Ca2+]c. Major populations of cells (approximately 65%) identified as glucagon-positive (alpha-cells) responded to the addition of arginine (5-10 mmol/l) to 3 mmol/l glucose solution with an increase in [Ca2+]c. About half of the alpha-cells (47.6%) responded to the addition of alanine (5-10 mmol/l) to 3 mmol/l glucose solution with an increase in [Ca2+]c. In contrast, <13% of beta-cells responded to the addition of alanine (5-10 mmol/l) or arginine (5-10 mmol/l) to 3 mol/l glucose with an increase in [Ca2+]c. More than one-fourth of alpha-cells responded with an increase in [Ca2+]c when glucose concentration in perifusion solution was reduced from 11 to 0 mmol/l. These results indicate that [Ca2+]c changes in islet cells stimulated by glucose or amino acid were characteristic of the cell species, at least in the alpha- and beta-cell. This technique provides a useful tool to investigate not only the intracellular signal transduction but also the intercellular signal transmission in the intact islet.

Glucose Suppression of Glucagon Secretion

“…Several models of glucose-mediated suppression of glucagon secretion rely on this assumption; however, there is poor consensus regarding glucose-mediated calcium dynamics in the ␣-cell. Some studies report a decrease in cytoplasmic calcium levels or a slowing down of oscillation frequencies in response to glucose (59 -61), and some report a weak or negligible effect (25,79), although some others describe glucose-mediated increases in [Ca 2ϩ ] i (24,58). Our calcium measurements on intact islets (FuraRed and Fluo4 imaging) indicate that ␣-cells respond to glucose by elevating their averaged [Ca 2ϩ ] i (Figs.…”

Glucose Induces Opposite Intracellular Ca2+ Concentration Oscillatory Patterns in Identified α- and β-Cells Within Intact Human Islets of Langerhans

“…Immunocytochemistry. After Ca 2ϩ signals were recorded in an optical section, intact islets were washed with PBS for 10 min, fixed using 4% (wt/vol) paraformaldehyde for 10 min, and permeabilized with 0.5% Triton X-100 for 10 min, as previously described (12,13,40). To reduce nonspecific antibody binding, cells were first preincubated with a blocking buffer (2% goat serum in PBS) for 15 min at room temperature before primary antibodies were applied in the same buffer.…”

Glucose-induced [Ca2+]i abnormalities in human pancreatic islets: important role of overstimulation.

“…Regarding synchronous oscillations in response to glucose, studies using confocal microscopy in mouse islets indicate that only ␤-cells oscillate (42,43). Arginine-induced [Ca 2+ ] i effects could, on the other hand, be influenced by ␣-cell participation because this amino acid is a potent stimulator of glucagon secretion (41) and induces an increase in [Ca 2+ ] i in mouse islets (37,42). Note, however, that [Ca 2+ ] i responses to arginine tended to be higher rather than lower after high-glucose culture.…”