Human Insulin Vesicle Dynamics During Pulsatile Secretion

Abstract

In healthy individuals, plasma insulin levels oscillate in both fasting and fed states. Numerous studies of isolated pancreata and pancreatic islets support the hypothesis that insulin oscillations arise because the underlying rate of insulin secretion also oscillates; yet, insulin secretion has never been observed to oscillate in individual pancreatic ␤-cells. Using expressed fluorescent vesicle cargo proteins and total internal reflection fluorescence (TIRF) microscopy, we demonstrate that glucose stimulates human pancreatic ␤-cells to secrete insulin vesicles in short, coordinated bursts of ϳ70 vesicles each. Randomization tests and spectral analysis confirmed that the temporal patterns of secretion were not random, instead exhibiting alternating periods of secretion and rest, recurring with statistically significant periods of 15-45 s. Although fluorescent vesicles arrived at the plasma membrane before, during, and after stimulation, their rate of arrival was significantly slower than their rate of secretion, so that their density near the plasma membrane dropped significantly during the cell's response. To study in greater detail the vesicle dynamics during cyclical bursts of secretion, we applied trains of depolarizations once a minute and performed simultaneous membrane capacitance measurements and TIRF imaging. Surprisingly, young fluorescent insulin vesicles contributed at least half of the vesicles secreted in response to a first train, even though young vesicles were vastly outnumbered by older, nonfluorescent vesicles. For subsequent trains, young insulin vesicles contributed progressively less to total secretion, whereas capacitance measurements revealed that total stimulated secretion did not decrease. These results suggest that in human pancreatic ␤-cells, young vesicles are secreted first, and only then are older vesicles recruited for secretion.

Contact-induced clustering of syntaxin and munc18 docks secretory granules at the exocytosis site

“…In chromaffin cells, a few granules (15%) are in a dead-end docked state and remain resistant to repeated stimulation 4 , and we would not be surprised to find this also in Ins1 cells. In contrast, immediate exocytosis of newly arriving insulin granules (crash fusion) has been observed by some authors 12,57,58 , but not others 2,4,6 , particularly during 2nd phase secretion. The finding suggests that some granules may be able to prime before docking.…”

“…Exocytosis of neurotransmitter-and hormone-containing secretory vesicles is triggered within milliseconds by a rise in cytosolic Ca 2 þ . To achieve this temporal precision, a pool of release-ready vesicles is morphologically docked at the plasma membrane with the exocytosis machinery in place [1][2][3][4][5][6] . Sustained secretion therefore requires tethering and docking of new vesicles at the plasma membrane followed by a set of slower priming reactions that render docked vesicles fusion-competent 1,3 .…”

Myrip Couples the Capture of Secretory Granules by the Actin-Rich Cell Cortex and Their Attachment to the Plasma Membrane

“…SG mobility is high in BON cells compared to primary neuroendocrine cells such as adrenal chromaffin cells and pancreatic ␤ cells (Degtyar et al, 2007;Michael et al, 2007 A compartment model to analyze Myrip-dependent SG trafficking Our data indicate that Myrip has profound effects on SG distribution, mobility, and membrane attachment. To evaluate the different parameters controlling SG dynamics at the cell cortex, we designed a simple compartment model to describe SG delivery to the PM (Fig.…”

The isolated pancreatic islet as a micro-organ and its transplantation to cure diabetes

“…Careful application of these approaches may yield evidence as to whether granules from distinct granule pools differentially release soluble vs. condensed contents, and how rapidly granule membrane is retrieved and resorted. 30,31 In addition, stimulus-secretion coupling by a-cells has also received intensive study using single cell techniques presented above. Outlines have emerged as to how tonic glucagon secretion results at very low levels of glucose and insulin, how glucagon secretion is curtailed in response to a post-prandial rise in glucose and how glucagon secretion reappears at high levels of glucose or in the presence of high concentrations of adrenalin.…”

“…Specific tags for exocytosis include red or green fluorescent protein labeled peptides, including C-peptide of derived from pro-insulin, intrinsic granule membrane proteins (syncollin, or phogrin), granule membrane attached proteins (tissue plasminogen activator), and even insulin bound Zn 2+ released into the ECS with decondensation of crystalline insulin granule core and then detected by a bath applied fluorescent indicator. [27][28][29][30][31] Endocytosis can be tracked by the filling of granule exocytotic figures with otherwise excluded fluorescent dextran, applied as a fluid phase tracer or with quenching of pH-sensitive fluorescence on granule re-acidification after their recapture. With simultaneous capacitance and amperometry recordings from the same cell it is becoming possible to ask what fraction of attempted exocytoses lead to exit of the crystalline insulin (with its associated Zn 2+ ) into the extracellular space, as opposed to more transient and restricted fusion pore formation with diffusion of more soluble contents, including peptides such as C-peptide or small molecular weight molecules such ATP and GABA.…”