Glucose suppression of glucagon secretion: metabolic and calcium responses from alpha-cells in intact mouse pancreatic islets

Abstract

Glucagon is released from alpha-cells present in intact pancreatic islets at glucose concentrations below 4 mm, whereas higher glucose levels inhibit its secretion. The mechanisms underlying the suppression of alpha-cell secretory activity are poorly understood, but two general types of models have been proposed as follows: direct inhibition by glucose or paracrine inhibition from non-alpha-cells within the islet of Langerhans. To identify alpha-cells for analysis, we utilized transgenic mice expressing fluorescent proteins targeted specifically to these cells. Measurements of glucagon secretion from pure populations of flow-sorted alpha-cells show that contrary to its effect on intact islets, glucose does stimulate glucagon secretion from isolated alpha-cells. This observation argues against a direct inhibition of glucagon secretion by glucose and supports the paracrine inhibition model. Imaging of cellular metabolism by two-photon excitation of NAD(P)H autofluorescence indicates that glucose is metabolized in alpha-cells and that glucokinase is the likely rate-limiting step in this process. Imaging calcium dynamics of alpha-cells in intact islets reveals that inhibiting concentrations of glucose increase the intracellular calcium concentration and the frequency of alpha-cell calcium oscillations. Application of candidate paracrine inhibitors leads to reduced glucagon secretion but did not decrease the alpha-cell calcium activity. Taken together, the data suggest that suppression occurs downstream from alpha-cell calcium signaling, presumably at the level of vesicle trafficking or exocytotic machinery.

FIGURE 1.

Fluorescence-based visualization of α-cells within an islet. A, overlay of differential interference contrast and anti-GFP antibody (green) images from a fixed permeabilized islet harvested from an EYFP-labeled α-cell animal. B, same islet stained with anti-glucagon antibodies (red). C, overlay of anti-GFP (green) and anti-glucagon (red) antibodies, co-localization presented in yellow. D, NAD(P)H autofluorescence signal of an islet perifused at 12 mm glucose. E, EYFP signal (yellow) emitted by α-cells within the islet presented in D. F, overlay of NAD(P)H and EYFP signals. Scale bars, 20 μm. Bar in A is valid for A–C. Bar in D is valid for D and E.

FIGURE 2.

Hormone secretion from intact islets and sorted α-cells. A, percent of islet glucagon (solid squares) and insulin (solid circles) content secreted per h in response to glucose. When glucose levels are greater than or equal to 7 mm, both insulin and glucagon secretion are statistically different from base-line release at 1 mm (n = 7 islets, p < 0.01, ANOVA-independent samples). B, percent of glucagon content secreted from sorted α-cells (open squares) and intact islets (solid squares). Secretion from sorted cells is significantly greater at 12 and 20 mm than at 1 mm (p < 0.05, ANOVA). Between 14 and 30 assays were performed at any given glucose concentration. C, glucagon secretion responses from intact islets. D, glucagon secretion responses from flow-sorted α-cells. Glc, glucose; Ins, insulin; SST, somatostatin; Arg, arginine. Error bars represent the mean ± S.E. *, p < 0.05, **, p < 0.01, ANOVA.

FIGURE 3.

Glucose-dependent NAD(P)H responses from intact islets and isolated cells. Black traces indicate β-cells; gray traces indicate α-cells, both with S.E. error bars. A, representative time course of the NAD(P)H response from an islet exposed to a step increase in glucose from 1 to 20 mm; NAD(P)H emission was collected every 18 s. Inset, NAD(P)H islet image denotes α-cell locations by gray circles based on EYFP signal, and β-cell region of interest is shown by the black rectangle. B, normalized percent change in NAD(P)H response relative to base line (1 mm glucose). Application of 12 mm glucose increases cellular redox state in both α- and β-cells (gray and black columns, respectively). Addition of 10 mm d-mannoheptulose significantly inhibits NAD(P)H responses from both cell types (p < 0.01, n = 12 for α-cells and n = 7 for β-cells). C, normalized glucose dose-response curve of NAD(P)H from islet α-cells (solid squares, n = 250) and β-cells (solid circles, core regions averaged from 67 islets). D, % change in NAD(P)H autofluorescence to step increases in glucose from isolated α-cells compared with values at 1 mm glucose (open squares, n = 146) and isolated β-cells (open circles, n = 269). All NAD(P)H responses in C and D were significantly different from base line at 1 mm glucose (p < 0.01, ANOVA, independent samples).

FIGURE 4.

Glucose-dependent changes in [Ca²⁺]_i by FuraRed imaging from intact islets and isolated cells. Black lines and gray lines with S.E. error bars represent β- and α-cells, respectively. A, glucose-dependent [Ca²⁺]_i increases from intact islets. At glucose levels greater than or equal to 7 mm, changes were statistically significant to base line at 1 mm glucose (n = 22 α-cells from 7 islets, p < 0.01, ANOVA). B, glucose-dependent [Ca²⁺]_i changes from sorted cells. Two distinct responses were apparent from sorted α-cells. The dashed gray line represents α-cells responding to glucose by an increase in [Ca²⁺]_i (11 of 20 cells). The dotted gray plot indicates α-cells that decrease their [Ca²⁺]_i (7 of 20 cells). At glucose levels greater than or equal to 7 mm, changes were statistically significant from base line (n = 9 β-cells, n = 20 α-cells, p < 0.01, ANOVA, correlated samples).

FIGURE 5.

Calcium oscillations from α-cells within intact islets as measured by Fluo4. A, B, and D are time series of the Fluo4 signal relative to the minimal intensity recorded during the experiment. A, single calcium pulse from one islet α-cell perifused with 1 mm glucose. The inset images show the cell before and at the maximum of the calcium transient. B, two adjacent α-cells within an islet exposed to 1 mm glucose show no synchronization between their [Ca²⁺]_i responses. C, representative time course of the Fluo4 response from an islet exposed to a step increase in glucose from 1 to 7 mm. The black line indicates a region of β-cells, and the gray plots show the responses of two individual α-cells. Fluo4 signal was recorded every 2 s. D, representative time course of Fluo4 signals from an islet exposed to 12 mm glucose. The black line indicates β-cells, and the gray line represents an α-cell. Fluo4 signal was recorded every 2.5 s, and the confocal pinhole diameter was 1 airy unit.