Visualization of Sequential Exocytosis in Rat Pancreatic Islet β Cells

Leung, Yuk‐Man; Sheu, Laura; Kwan, Eugene E.; Wang, Guotang; Tsushima, Robert G.; Gaisano, Herbert Y.

doi:10.1006/bbrc.2002.6712

Cited by 34 publications

(38 citation statements)

References 24 publications

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“…Immunohistochemical labeling of live cells. This protocol was based on previously published protocols (17)(18)(19). The cells were neither fixed nor permeabilized.…”

Section: Methodsmentioning

confidence: 99%

“…Second, the fraction of ␤-cells with insulin on their surface increases as a function of time (17), in particular when cells are exposed to factors known to enhance the regulated secretion of insulintheophylline and elevated extracellular concentrations of calcium and glucose (17). Third, cell surface insulin is not distributed uniformly around the cell's perimeter; instead, it is found in discrete spots (17)(18)(19). Finally, images from ultrastructural techniques such as electron microscopy and autometallography have shown what appear to be insulin-dense cores outside of pancreatic ␤-cells (18,20).…”

mentioning

confidence: 99%

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Pancreatic β-Cells Secrete Insulin in Fast- and Slow-Release Forms

et al. 2006

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Insulin vesicles contain a chemically rich mixture of cargo that includes ions, small molecules, and proteins. At present, it is unclear if all components of this cargo escape from the vesicle at the same rate or to the same extent during exocytosis. Here, we demonstrate through real-time imaging that individual rat and human pancreatic ␤-cells secrete insulin in heterogeneous forms that disperse either rapidly or slowly. In healthy pancreatic ␤-cells maintained in culture, most vesicles discharge insulin in its fastrelease form, a form that leaves individual vesicles in a few hundred milliseconds. The fast-release form of insulin leaves vesicles as rapidly as C-peptide leaves vesicles. Healthy ␤-cells also secrete a slow-release form of insulin that leaves vesicles more slowly than C-peptide, over times ranging from seconds to minutes. Individual ␤-cells make vesicles with both forms of insulin, though not all vesicles contain both forms of insulin. In addition, we confirm that insulin vesicles store their cargo in two functionally distinct compartments: an acidic solution, or halo, and a condensed core. Thus, our results suggest two important features of the condensed core: 1) It exists in different states among the vesicles undergoing exocytosis and 2) its dissolution determines the availability of insulin during exocytosis. Diabetes 55:600 -607, 2006 I nsufficient insulin secretion in the face of insulin resistance leads to the disease type 2 diabetes (1). The mechanism responsible for insufficient insulin secretion remains unclear. Before it is possible to understand why insulin secretion fails to compensate for insulin resistance during the progression of type 2 diabetes (2), it is essential to understand insulin secretion at all levels of biological complexity, ranging from the whole pancreas within a living animal to the pancreatic ␤-cell and its fundamental unit of secretion, the insulin secretory vesicle.Insulin vesicles contain a chemically complex mixture of ions, small molecules, and proteins (3,4). Among the ions, there are large amounts of two divalent cations: zinc and calcium (5). Both of these ions are believed to play important roles in the regulated secretion of insulin (6). Among the vesicle proteins, insulin-related peptides comprise ϳ75% of the mass (7). The remaining 25% of the protein mass includes more than a hundred different membrane and cargo proteins. Although our understanding of regulated secretion of insulin has progressed greatly in recent years, the mechanisms that control the trafficking, processing, and secretion of insulin vesicle cargo are still matters of debate and extensive research (8).Previously, we (9) and others (10 -13) have studied secretion from single insulin vesicles by imaging fluorescently tagged proteins consisting of a vesicle cargo protein linked to a fluorescent protein. The fluorescently tagged vesicle cargo proteins, hereafter referred to as fluorescent cargo proteins, have provided a valuable and powerful tool for studies of single vesicle exocytosis. H...

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“…Immunohistochemical labeling of live cells. This protocol was based on previously published protocols (17)(18)(19). The cells were neither fixed nor permeabilized.…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Pancreatic β-Cells Secrete Insulin in Fast- and Slow-Release Forms

et al. 2006

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“…Calcium Imaging-Imaging was performed using equipment described previously (22). Cells on glass coverslips were loaded with 4 M Fura-2-AM (Molecular Probes, Eugene, OR) for 45 min at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Inhibition of Kv2.1 Voltage-dependent K+Channels in Pancreatic β-Cells Enhances Glucose-dependent Insulin Secretion

MacDonald¹,

Sewing²,

Wang³

et al. 2002

Journal of Biological Chemistry

167

171

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Voltage-dependent (Kv) outward K ؉ currents repolarize ␤-cell action potentials during a glucose stimulus to limit Ca 2؉ entry and insulin secretion. Dominant-negative "knockout" of Kv2 family channels enhances glucose-stimulated insulin secretion. Here we show that a putative Kv2.1 antagonist (C-1) stimulates insulin secretion from MIN6 insulinoma cells in a glucose-and dosedependent manner while blocking voltage-dependent outward K ؉ currents. C-1-blocked recombinant Kv2.1-mediated currents more specifically than currents mediated by Kv1, -3, and -4 family channels (Kv1.4, 3.1, 4.2). Additionally, C-1 had little effect on currents recorded from MIN6 cells expressing a dominant-negative Kv2.1 ␣-subunit. The insulinotropic effect of acute Kv2.1 inhibition resulted from enhanced membrane depolarization and augmented intracellular Ca 2؉ responses to glucose. Immunohistochemical staining of mouse pancreas sections showed that expression of Kv2.1 correlated highly with insulin-containing ␤-cells, consistent with the ability of C-1 to block voltage-dependent outward K ؉ currents in isolated mouse ␤-cells. Antagonism of Kv2.1 in an ex vivo perfused mouse pancreas model enhanced first-and second-phase insulin secretion, whereas glucagon secretion was unaffected. The present study demonstrates that Kv2.1 is an important component of ␤-cell stimulus-secretion coupling, and a compound that enhances, but does not initiate, ␤-cell electrical activity by acting on Kv2

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“…Optical methods, such as TIRF, confocal, and multiphoton microscopies (13,(23)(24)(25)(26)(27)(28)(29)(30)(31)(32), are well suited for studies of vesicle trafficking to the readily releasable pool. According to one previous study in the MIN-6 cell line (27), the readily releasable pool is refilled by vesicles that move from stores deep inside the cell to the plasma membrane.…”

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confidence: 99%

Human Insulin Vesicle Dynamics During Pulsatile Secretion

Michael

Xiong²,

Geng³

et al. 2007

Diabetes

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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.

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Visualization of Sequential Exocytosis in Rat Pancreatic Islet β Cells

Cited by 34 publications

References 24 publications

Pancreatic β-Cells Secrete Insulin in Fast- and Slow-Release Forms

Pancreatic β-Cells Secrete Insulin in Fast- and Slow-Release Forms

Inhibition of Kv2.1 Voltage-dependent K+Channels in Pancreatic β-Cells Enhances Glucose-dependent Insulin Secretion

Human Insulin Vesicle Dynamics During Pulsatile Secretion

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