Insulin targeting to the regulated secretory pathway after fusion with green fluorescent protein and firefly luciferase

Pouli, Aristea E.; Kennedy, Helen J; Schofield, Jill; Rutter, Guy A.

doi:10.1042/bj3310669

Cited by 80 publications

(60 citation statements)

References 34 publications

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“…This was followed by blocking in 3% (w/v) BSA in PBS for 15 min. Cells were then incubated with the primary antibodies overnight and with the secondary antibodies for 1.5 h at room temperature in 3% (w/v) BSA in PBS, before mounting and confocal imaging as described previously [38].…”

Section: Methodsmentioning

confidence: 99%

Intracellular ATP-sensitive K+ channels in mouse pancreatic beta cells: against a role in organelle cation homeostasis

et al. 2006

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Aims/hypothesis: ATP-sensitive K + (K ATP ) channels located on the beta cell plasma membrane play a critical role in regulating insulin secretion and are targets for the sulfonylurea class of antihyperglycaemic drugs. Recent reports suggest that these channels may also reside on insulin-containing dense-core vesicles and mitochondria. The aim of this study was to explore these possibilities and to test the hypothesis that vesicle-resident channels play a role in the control of organellar Ca 2+ concentration or pH. Methods: To quantify the subcellular distribution of the pore-forming subunit Kir6.2 and the sulfonylurea binding subunit SUR1 in isolated mouse islets and clonal pancreatic MIN6 beta cells, we used four complementary techniques: immunoelectron microscopy, density gradient fractionation, vesicle immunopurification and fluorescence-activated vesicle isolation. Intravesicular and mitochondrial concentrations of free Ca 2+ were measured in intact or digitonin-permeabilised MIN6 cells using recombinant, targeted aequorins, and intravesicular pH was measured with the recombinant fluorescent probe pHluorin. Results: SUR1 and Kir6.2 immunoreactivity were concentrated on dense-core vesicles and on vesicles plus the endoplasmic reticulum/Golgi network, respectively, in both islets and MIN6 cells. Reactivity to neither subunit was detected on mitochondria. Glibenclamide, tolbutamide and diazoxide all failed to affect Ca 2+ uptake into mitochondria, and K ATP channel regulators had no significant effect on intravesicular free Ca 2+ concentrations or vesicular pH. Conclusions/Interpretation: A significant proportion of Kir6.2 and SUR1 subunits reside on insulin-secretory vesicles and the distal secretory pathway in mouse beta cells but do not influence intravesicular ion homeostasis. We propose that densecore vesicles may serve instead as sorting stations for the delivery of channels to the plasma membrane.

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Section: Methodsmentioning

confidence: 99%

Intracellular ATP-sensitive K+ channels in mouse pancreatic beta cells: against a role in organelle cation homeostasis

et al. 2006

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“…Fusion of EGFP to the C-terminus of phogrin also allows the photoprotein to face the cell cytosol, rather than the lumen, thus avoiding fluorescence quenching at low intraluminal pH. In contrast, the fusion of insulin with EGFP [70] is more problematic [77], unless steps are taken to reduce expression levels (T. Tsuboi, G.A. Rutter, unpublished results).…”

Section: Glucose-stimulated Insulin Secretion Is Biphasic: Visualisatmentioning

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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“…After transfection, MIN6 or INS-1 (832/13) cells were fixed and incubated with primary and secondary antibodies as described (35,36). Foxa2 was probed with the same antibody used for Western blotting (Santa Cruz Biotechnology, Inc.; 1:2500) and revealed with Alexa Fluor 568 donkey anti-goat IgG (Molecular Probes, Eugene, OR; 1:35,000).…”

Section: Methodsmentioning

confidence: 99%

MicroRNA-124a Regulates Foxa2 Expression and Intracellular Signaling in Pancreatic β-Cell Lines

Baroukh

Ravier

Loder

et al. 2007

Journal of Biological Chemistry

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323

309

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MicroRNAs (miRNAs) are short non-coding RNAs that have been implicated in fine-tuning gene regulation, although the precise roles of many are still unknown. Pancreatic development is characterized by the complex sequential expression of a gamut of transcription factors. We have performed miRNA expression profiling at two key stages of mouse embryonic pancreas development, e14.5 and e18.5. miR-124a2 expression was strikingly increased at e18.5 compared with e14.5, suggesting a possible role in differentiated ␤-cells. Among the potential miR124a gene targets identified by biocomputation, Foxa2 is known to play a role in ␤-cell differentiation. To evaluate the impact of miR-124a2 on gene expression, we overexpressed or down-regulated miR-124a2 in MIN6 ␤-cells. As predicted, miR-124a2 regulated Foxa2 gene expression, and that of its downstream target, pancreatic duodenum homeobox-1 (Pdx-1). Foxa2 has been described as a master regulator of pancreatic development and also of genes involved in glucose metabolism and insulin secretion, including the ATP-sensitive K ؉ (K ATP ) channel subunits, Kir6.2 and Sur-1. Correspondingly, miR-124a2 overexpression decreased, and anti-miR-124a2 increased Kir6.2 and Sur-1 mRNA levels. Moreover, miR-124a2 modified basal and glucose-or KCl-stimulated intracellular free Ca 2؉ concentrations in single MIN6 and INS-1 (832/13) ␤-cells, without affecting the secretion of insulin or co-transfected human growth hormone, consistent with an altered sensitivity of the ␤-cell exocytotic machinery to Ca 2؉ . In conclusion, whereas the precise role of microRNA-124a2 in pancreatic development remains to be deciphered, we identify it as a regulator of a key transcriptional protein network in ␤-cells responsible for modulating intracellular signaling.Studies implicating small regulatory RNAs in the control of gene expression have demonstrated that transcriptional regulation is controlled not only by protein factors, but also by small endogenous RNA molecules of ϳ19 -23 nucleotides in length called microRNAs (miRNAs or miRs) 3 (1, 2). The first miRNAs discovered were lin-4 and let-7, which are crucial for regulating the developmental timing in the nematode, Caenorhabditis elegans (1, 3). Since this initial report, several hundred miRNAs have been identified in plants and animals that regulate diverse biological processes ranging from cell metabolism to cell differentiation and growth, apoptosis, and immune responses (4 -8). Moreover, it has been shown that miRNAs are characterized by differential spatial and temporal expression patterns supporting their role in such processes (3, 9). miRNAs serve as regulators of gene expression by binding to complementary sites on their target transcripts and, by an ill-defined mechanism, significantly induce the cleavage of mRNA or the repression of translation, depending on the partial or complete sequence homology, respectively (2, 10 -12). It has been estimated that miRNA genes represent ϳ1% of the genome of complex organisms. It appears that they share a certain...

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Insulin targeting to the regulated secretory pathway after fusion with green fluorescent protein and firefly luciferase

Cited by 80 publications

References 34 publications

Intracellular ATP-sensitive K+ channels in mouse pancreatic beta cells: against a role in organelle cation homeostasis

Intracellular ATP-sensitive K+ channels in mouse pancreatic beta cells: against a role in organelle cation homeostasis

Visualising insulin secretion. The Minkowski Lecture 2004

MicroRNA-124a Regulates Foxa2 Expression and Intracellular Signaling in Pancreatic β-Cell Lines

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