Detection of Secretion from Single Pancreatic β-Cells Using Extracellular Fluorogenic Reactions and Confocal Fluorescence Microscopy

Qian, Weijun; Aspinwall, Craig A.; Battiste, Merle A.; Kennedy, Robert T.

doi:10.1021/ac991085t

Cited by 102 publications

(89 citation statements)

References 40 publications

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“…Although we limited our analysis to averaged fluorescence changes over the cell membrane, we did note that different regions of the cell membrane increased in fluorescence intensity preferentially over others. These areas may correspond to "secretory addresses" along the cell membrane where various molecules and channels involved in exocytosis are co-localized, as has been reported by others in the literature (30,35,39,40).…”

Section: Discussionmentioning

confidence: 52%

Exogenous Nitric Oxide and Endogenous Glucose-Stimulated β-Cell Nitric Oxide Augment Insulin Release

et al. 2002

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The role nitric oxide (NO) plays in physiological insulin secretion has been controversial. Here we present evidence that exogenous NO stimulates insulin secretion, and that endogenous NO production occurs and is involved in the regulation of insulin release. Radioimmunoassay measurement of insulin release and a dynamic assay of exocytosis using the dye FM1-43 demonstrated that three different NO donors-hydroxylamine (HA), sodium nitroprusside, and 3-morpholinosydnonimine (SIN-1)-each stimulated a marked increase in insulin secretion from INS-1 cells. Pharmacological manipulation of the guanylate cyclase/guanosine 3,5-cyclic monophosphate pathway indicated that this pathway was involved in mediating the effect of the intracellular NO donor, HA, which was used to simulate endogenous NO production. This effect was further characterized as involving membrane depolarization and intracellular T he endogenous synthesis of nitric oxide (NO) from L-arginine (L-arg) has been shown to play a critical role in a wide variety of physiological functions including neurotransmission, vascular tone, platelet aggregation, immunological reactions, penile erection, and endocrine and exocrine function (1). Cellular NO is synthesized by a family of NO synthase (NOS) enzymes, comprised of constitutively expressed, Ca 2ϩ / calmodulin-dependent neuronal NOS (nNOS) and endothelial NOS (eNOS), and a Ca 2ϩ /calmodulin-independent inducibly expressed NOS (iNOS) (2).Pancreatic ␤-cells are able to express the iNOS enzyme in response to inflammatory stimuli, leading to high cytotoxic levels of NO production, which appear to be involved in ␤-cell damage, dysfunction, and death and the pathogenesis of type 1 diabetes (3,4). The presence of a constitutive NOS (cNOS) enzyme in ␤-cells is substantiated by considerable evidence, including biochemical, histochemical, immunohistochemical, immunofluorescence, RT-PCR, and protein immunoblot analyses (5-11). Given that the amino acid NO precursor L-arg has long been known to stimulate insulin release (12), it has been postulated that a low level of NO produced from the ␤-cell cNOS isoform functions in the regulation of insulin release. However, several reports in which cNOS activity was manipulated or exogenous NO was applied have yielded seemingly conflicting results. NOS inhibition has been reported to produce an inhibitory effect (5,13-15), a stimulatory effect (6,9,16 -18), and no effect at all (19,20) on insulin release. Similarly, exogenously applied NO has been reported to exert a stimulatory (5,13,14,21,22) and an inhibitory effect (6,18,23-27) on insulin release. These discrepant data may be the result of variations in the specifics of the experimental conditions, including differences in the agents used (e.g., NOS substrate, NOS inhibitors, NO donors), the concentration of these agents, whether other stimulatory pathways were activated concurrently (e.g., with glucose), the experimental model used (e.g., ␤-cell line, islets, pancreas), and the species. These critical differences may result in d...

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Section: Discussionmentioning

confidence: 52%

Exogenous Nitric Oxide and Endogenous Glucose-Stimulated β-Cell Nitric Oxide Augment Insulin Release

et al. 2002

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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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“…We make the comparison here, for the first time. To monitor insulin vesicle exocytosis, we used two independent markers that report exocytosis: a fluorescent cargo protein and a fluorescent zinc indicator (21). Examined separately, neither of these probes was able to faithfully track the fates of all native cargo.…”

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confidence: 99%

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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Detection of Secretion from Single Pancreatic β-Cells Using Extracellular Fluorogenic Reactions and Confocal Fluorescence Microscopy

Cited by 102 publications

References 40 publications

Exogenous Nitric Oxide and Endogenous Glucose-Stimulated β-Cell Nitric Oxide Augment Insulin Release

Exogenous Nitric Oxide and Endogenous Glucose-Stimulated β-Cell Nitric Oxide Augment Insulin Release

Human Insulin Vesicle Dynamics During Pulsatile Secretion

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

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