Detection of exocytosis at individual pancreatic beta cells by amperometry at a chemically modified microelectrode.

Huang, Lan; Shen, Hong; Atkinson, Mark A.; Kennedy, Robert T.

doi:10.1073/pnas.92.21.9608

Cited by 112 publications

(111 citation statements)

References 26 publications

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“…The number of granules undergoing exocytosis per beta cell and minute was estimated from the release data of [77] assuming that every secretory granule contains 1.6 amol [37] of insulin and that a typical mouse islet consists of 1000 beta cells tonin [33,34,35,36]. The carbon fibre technique has subsequently been improved to detect insulin itself [37]. Analysis of the amperometric currents suggests that a single secretory granule in a human beta cell contains 1.7 amol of insulin, which equates to an intragranular insulin concentration of 118 mmol/l.…”

Section: Novel Methods To Study Releasementioning

confidence: 99%

Insulin granule dynamics in pancreatic beta cells

2003

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Section: Novel Methods To Study Releasementioning

confidence: 99%

Insulin granule dynamics in pancreatic beta cells

2003

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“…Amperometric Detection of Exocytosis-Microelectrodes were constructed of carbon fibers sealed in glass micropipettes and were polished to a 45°angle and cleaned prior to use (23,24). Amperometric measurements were made by positioning microelectrodes ϳ1 m from a cell and applying stimulant from a micropipette ϳ 30 m from the cell as described elsewhere (23,24 Amperometry was performed using a battery to apply potential to a sodium-saturated calomel reference electrode as described previously (24).…”

Section: Methodsmentioning

confidence: 99%

“…Amperometric measurements were made by positioning microelectrodes ϳ1 m from a cell and applying stimulant from a micropipette ϳ 30 m from the cell as described elsewhere (23,24 Amperometry was performed using a battery to apply potential to a sodium-saturated calomel reference electrode as described previously (24). For measurements of 5-hydrotryptamine (5-HT) secretion, dispersed ␤-cells were incubated in tissue culture medium containing 0.5 ] i increases in the interior region (red line) and the edge of the cell (blue line).…”

Section: Methodsmentioning

confidence: 99%

Roles of Insulin Receptor Substrate-1, Phosphatidylinositol 3-Kinase, and Release of Intracellular Ca2+ Stores in Insulin-stimulated Insulin Secretion in β-Cells

Aspinwall¹,

Qian²,

Roper³

et al. 2000

Journal of Biological Chemistry

158

124

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The signaling pathway by which insulin stimulates insulin secretion and increases in intracellular freeInsulin secreted by pancreatic ␤-cells is the primary regulator of serum glucose concentrations in mammals. Although substantial progress has been made in elucidating the mechanisms responsible for normal regulation of insulin secretion from the ␤-cell, many aspects of this process remain unclear. In particular, chemical and physiological interactions between cells within the islet exert an important level of control in the physiological regulation of insulin secretion that is not entirely understood. Both hormonal and neuronal influences within islets may modulate ␤-cell activity and insulin secretion in vitro and in vivo (1-3). Although such influences have been demonstrated, the existence of significant autocrine effects of insulin on ␤-cells remained controversial for many years because a variety of studies yielded conflicting evidence on the modulation of insulin secretion by insulin in whole islets or in vivo. Recently, however, a variety of new methods have been utilized that demonstrate potent and possibly clinically important autocrine actions of insulin.Several recent studies have indicated that ␤-cells express components of insulin signaling systems including insulin receptors (4 -6), insulin receptor substrates (IRS-1 and IRS-2) 1 (7-9), phosphatidylinositol 3-kinase (PI3-K) (10, 11), and protein kinase B (12). Evidence has also been obtained indicating that insulin released by glucose can activate these components in addition to other proteins in the cells. Insulin binds to receptors on the surface of ␤-cells (4, 13) and activates tyrosine phosphorylation of insulin receptors (6), insulin receptor substrates (8), and PHAS-I (an inhibitor of mRNA cap-binding protein) (14). Furthermore, maximal glucose-stimulated production of phosphatidylinositol 3,4,5-triphosphate (PIP 3 ), a major product of PI3-K activity, coincides with the early peak phase insulin secretion in islets and clonal ␤-cells (10). Thus, autocrine activation of the ␤-cell insulin receptors and several downstream proteins has been demonstrated.Some of the physiological consequences of insulin receptor activation at ␤-cells have recently been revealed. Activation of the insulin signaling pathway in ␤-cells leads to initiation of insulin synthesis at both transcriptional and translational levels, increasing the cellular content of releasable hormone in primary and clonal ␤-cell cultures (14 -16). In ␤TC6-F7 cells transfected to overexpress the insulin receptor, basal and glucose-stimulated insulin secretion was enhanced compared with kinase negative controls (15). In another report, clonal cells lacking the IRS-1 protein showed both decreased insulin content and glucose-stimulated secretion (17). These latter studies suggest that insulin can exert positive control over synthesis and/or secretion. Direct evidence for the effects of insulin on insulin secretion has been obtained by application of exogenous insulin to isolated ␤-cells and d...

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“…More recently, studies on individual beta cells were made possible with the development of electrophysiological approaches including amperometry [5] and measurements of membrane capacitance [6,7]. Though the latter techniques provide remarkable temporal resolution, they are, however, limited with respect to the spatial aspects of insulin release.…”

Section: Introductionmentioning

confidence: 99%

Visualising insulin secretion. The Minkowski Lecture 2004

Rutter

2004

Diabetologia

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Insulin secretion from pancreatic islet beta cells is a tightly regulated process, under the close control of blood glucose concentrations, neural inputs and circulating hormones. Defects in glucose-triggered insulin secretion, possibly exacerbated by a decrease in beta cell mass, are ultimately responsible for the development of type 2 diabetes. A full understanding of the mechanisms by which glucose and other nutrients trigger insulin secretion will probably be essential to allow for the development of new therapies of type 2 diabetes and for the derivation of "artificial" beta cells from embryonic stem cells as a treatment for type 1 diabetes. I focus here on recent developments in our understanding of beta cell glucose sensing, achieved in part through the development of recombinant targeted probes (luciferase, green fluorescent protein) that allow islet beta cell metabolism and Ca 2+ handling to be imaged in situ in the intact islet with single cell resolution. Combined with classical biochemistry, these techniques show that the beta cell is uniquely poised, thanks to the expression of low levels of lactate dehydrogenase and plasma membrane lactate/monocarboxylate transporters, to channel glucose carbons towards oxidative metabolism, ATP synthesis and inhibition of AMP-activated protein kinase, a newly defined regulator of insulin release. I also discuss the molecular basis of the recruitment of secretory vesicles to the cell surface, analysed by the use of new imaging techniques including total internal reflection of fluorescence, as well as the "nanomechanics" of the exocytotic event itself.

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Detection of exocytosis at individual pancreatic beta cells by amperometry at a chemically modified microelectrode.

Cited by 112 publications

References 26 publications

Insulin granule dynamics in pancreatic beta cells

Insulin granule dynamics in pancreatic beta cells

Roles of Insulin Receptor Substrate-1, Phosphatidylinositol 3-Kinase, and Release of Intracellular Ca2+ Stores in Insulin-stimulated Insulin Secretion in β-Cells

Visualising insulin secretion. The Minkowski Lecture 2004

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