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Human Insulin Vesicle Dynamics During Pulsatile Secretion

¹Department of Physiology and Biophysics, Keck School of Medicine, Zilkha Neurogenetic Institute, University of Southern California, Los Angeles, CA 90089
²Department of Cell Biology and Physiology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15261

Correspondence: rchow{at}usc.edu

Key Words: pancreatic beta cells • insulin • exocytosis • secretory vesicles • fluorescence microscopy

In healthy individuals, plasma insulin levels oscillate in both fasting and fed states. Numerous studies of isolated pancreas 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 beta cells. Using expressed fluorescent vesicle cargo proteins and total internal reflection fluorescence microscopy, we demonstrate that glucose stimulates human pancreatic beta cells to secrete insulin vesicles in short, coordinated bursts of about 70 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 to 45 seconds. 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. Simultaneous monitoring of secretion by membrane capacitance measurements and TIRF imaging revealed that young fluorescent insulin vesicles contributed at least half of the vesicles secreted in response to a first train of depolarizations, even though young vesicles are vastly outnumbered by older, non-fluorescent vesicles. For subsequent trains, the contribution of young insulin vesicles to secretion decreased significantly, whereas capacitance measurements revealed that total stimulated secretion did not decrease. These results suggest that in human pancreatic beta cells, young vesicles are secreted first, and only then are older vesicles recruited for secretion.

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