Abstract
In the brain, glutamate is an extracellular transmitter that mediates cell-to-cell communication. Prior to synaptic release it is pumped into vesicles by vesicular glutamate transporters (VGLUTs). To inactivate glutamate receptor responses after release, glutamate is taken up into glial cells or neurons by excitatory amino acid transporters (EAATs). In the pancreatic islets of Langerhans, glutamate is proposed to act as an intracellular messenger, regulating insulin secretion from β-cells, but the mechanisms involved are unknown. By immunogold cytochemistry we show that insulin containing secretory granules express VGLUT3. Despite the fact that they have a VGLUT, the levels of glutamate in these granules are low, indicating the presence of a protein that can transport glutamate out of the granules. Surprisingly, in β-cells the glutamate transporter EAAT2 is located, not in the plasma membrane as it is in brain cells, but exclusively in insulin-containing secretory granules, together with VGLUT3. In EAAT2 knock out mice, the content of glutamate in secretory granules is higher than in wild type mice. These data imply a glutamate cycle in which glutamate is carried into the granules by VGLUT3 and carried out by EAAT2. Perturbing this cycle by knocking down EAAT2 expression with a small interfering RNA, or by over-expressing EAAT2 or a VGLUT in insulin granules, significantly reduced the rate of granule exocytosis. Simulations of granule energetics suggest that VGLUT3 and EAAT2 may regulate the pH and membrane potential of the granules and thereby regulate insulin secretion. These data suggest that insulin secretion from β-cells is modulated by the flux of glutamate through the secretory granules.
Highlights
Glutamate is the transmitter used at most excitatory synapses in the brain [1]
Immunoblots revealed that VGLUT3 is present in islet tissue (Figure 1G), and immunofluorescent microscopy demonstrated that VGLUT3 is present in both b-cells and a-cells (Figure 1A), Glutamate Transporters and Insulin Secretion whereas VGLUT2 is present in a-cells only (Figure 1B)
VGLUT3 was co-localized with insulin containing granules (Figure 1A, C), and there was a significant co-localization with the synaptic-like microvesicle (SLMV) marker synaptophysin (Figure 1D)
Summary
Glutamate is the transmitter used at most excitatory synapses in the brain [1]. Glutamate is carried from the cytosol into synaptic vesicles by vesicular glutamate transporters (VGLUTs) in a process dependent on a vacuolar ATP-driven H+ pump. Electron immunogold microscopy using antibodies to glutamate showed that glutamate is located in secretory granules in b-cells, it is present at lower levels than in the cytosol and much lower than in SLMVs (Figure 2A–B, E).
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