A Role for Glutamate Transporters in the Regulation of Insulin Secretion
Figure 5
The glutamate analogue D-aspartate is neither taken up through the plasma membrane in intact β-cells nor into SGs in permeabilized β-cells.
A–C, Acutely prepared slices of islet tissue were incubated with exogenous D-aspartate (100 µM) before aldehyde fixation and labelling with antibodies that selectively recognize D-aspartate. (A–B) Immunoperoxidase labelling shows that the tissue not exposed to D-aspartate (Control) is unlabelled, while in islets exposed to D-aspartate (D-Asp) labelling is observed only in the peripheral α-cell area, not in the central β-cell area of the islet. (C) Immunofluorescence shows that the central insulin positive β-cells are negative for exogenous D-aspartate and that the peripheral non-insulin α-cells are labelled. (D–F) Streptolysin-O permeabilized INS-1E cells were exposed to different concentrations of exogenous D-aspartate (0–3 mM) before fixation and labelling with the D-aspartate antibodies. (D) In cells not exposed to D-aspartate (Control) there was no labelling for D-aspartate, only for insulin (red). (E) In cells exposed to 1 mM D-aspartate staining with the D-aspartate antibodies (green) is observed. There was some weak co-localization (yellow) with insulin (red) that is attributable to extra granular fixation of D-aspartate (see F). (F) Electron micrograph of permeabilized INS cells exposed to 1 mM D-aspartate shows no significant D-aspartate labelling inside the secretory garnules (indicated in transparent yellow). Note some labelling along the limiting membrane of secretory granules and in the cytosol, reflecting fixation of exogenous D-aspartate to extragranular proteins.