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Fluorescent proteins have been extensively used as protein "tags" to study the subcellular localization of proteins and/or their translocation upon stimulation or as markers for transfection in transient and stable expression systems. However, they have not been frequently used as reporter genes to monitor stimulus-induced gene expression in mammalian cells. Here we demonstrate the use of fluorescent proteins to study stimulus-induced gene transcription. The general applicability of the approach is exemplified by doxycyclin-(Tet-On) and phorbol 12-myristate 13-acetate-induced (c-fos) promoter activation, with green fluorescent protein (GFP) and red fluorescent protein (DsRed) as semiquantitative and immediate reporters, of transcription activation. Under the control of beta-cell-specific promoters, such as the rat insulin 1 promoter or the rat upstream glucokinase promoter, this approach allowed us to monitor online glucose-induced gene transcription in primary beta-cells at the single-cell level as well as in the context of the islet of Langerhans. Applying discretely detectable fluorescent proteins, for example GFP and DsRed, enabled us to simultaneously monitor stimulus-induced transcription by two different promoters in the same cell. [ABSTRACT FROM AUTHOR]

OBJECTIVE--Nephrin, an immunoglobulin-like protein essential for the function of the glomerular podocyte and regulated in diabetic nephropathy, is also expressed in pancreatic β-cells, where its function remains unknown. The aim of this study was to investigate whether diabetes modulates nephrin expression in human pancreatic islets and to explore the role of nephrin in β-cell function. RESEARCH DESIGN AND METHODS--Nephrin expression in human pancreas and in MIN6 insulinoma cells was studied by Western blot, PCR, confocal microscopy, subcellular fractionation, and immunogold labeling. Islets from diabetic (n = 5) and nondiabetic (n = 7) patients were compared. Stable transfection and siRNA knockdown in MIN-6 cells/human islets were used to study nephrin function in vitro and in vivo after transplantation in diabetic immunodeficient mice. Live imaging of green fluorescent protein (GFP)-nephrin--transfected cells was used to study nephrin endocytosis. RESULTS--Nephrin was found at the plasma membrane and on insulin vesicles. Nephrin expression was decreased in islets from diabetic patients when compared with nondiabetic control subjects. Nephrin transfection in MIN-6 cells/pseudoislets resulted in higher glucose-stimulated insulin release in vitro and in vivo after transplantation into immunodeficient diabetic mice. Nephrin gene silencing abolished stimulated insulin release. Confocal imaging of GFP-nephrin-transfected cells revealed nephrin endocytosis upon glucose stimulation. Actin stabilization prevented nephrin trafficking as well as nephrin-positive effect on insulin release. CONCLUSIONS--Our data suggest that nephrin is an active component of insulin vesicle machinery that may affect vesicle-actin interaction and mobilization to the plasma membrane. Development of drugs targeting nephrin may represent a novel approach to treat diabetes. Diabetes 59:190-199, 2010 [ABSTRACT FROM AUTHOR]

Tomosyn, a syntaxin-binding protein, is capable of dissociating mammalian homolog of the Caenorhabditis elegans unc-18 gene from syntaxin and is involved in the regulation of exocytosis. We have investigated the expression, cellular localization, and functional role of tomosyn in pancreatic β-cells. Western blotting revealed a 130-kDa protein corresponding to tomosyn in insulin-secreting β-cell lines. RT-PCR amplification showed that b-, m-, and s-tomosyn isoform mRNAs are expressed in β-cell lines and rat pancreatic islets. Immunohistochemistry revealed punctate tomosyn immunoreactivity in the cytoplasm of insulin-, glucagon-, pancreatic polypeptide-, and somatostatin-containing islet cells. Syntaxin 1 coimmunoprecipitated with tomosyn in extracts of insulin-secreting cells. Overexpression of m-tomosyn in mouse β-cells significantly decreased exocytosis, whereas inhibition of tomosyn expression by small interfering RNA increased exocytosis. Hence, in the pancreatic beta;-cell, tomosyn negatively regulates insulin exocytosis. [ABSTRACT FROM AUTHOR]

Pancreatic islet transplantation has emerged as a therapy for type 1 diabetes and is today performed using both freshly isolated and cultured islets. Islet blood vessels are disrupted during islet isolation; therefore, proper revascularization of the transplanted islets is of great importance for islet graft function and survival. We have studied intraislet endothelial cells after islet isolation, during islet culture, and following islet transplantation. By isolating islets from the transgenic Tie2-GFP (green fluorescent protein) mouse, characterized by an endothelial cell--specific expression of GFP, living endothelial cells could be studied in intact islets utilizing two-photon laser-scanning microscopy (TPLSM). Intraislet endothelial cells were found to survive islet transplantation but to rapidly disappear during islet culture. By transplanting freshly isolated Tie2-GFP islets and applying a novel ex vivo model for simultaneous perfusion and TPLSM imaging of the graft-bearing kidneys, GFP fluorescent endothelial cells were found to extensively contribute to vessels within the islet graft vasculature. Real-time imaging of the flow through the islet graft vasculature confirmed that the donor-derived vessels were functionally integrated. Hence, intraislet endothelial cells have the capability of participating in revascularization of pancreatic islets subsequent to transplantation. Therefore, preservation of intraislet endothelial cell mass may improve long-term graft function. Diabetes 54:2287-2293, 2005 [ABSTRACT FROM AUTHOR]

Because sulfonylureas directly activate the exocytotic machinery, we were interested in the extent to which these compounds penetrate the β-cell plasma membrane and the underlying molecular mechanism(s). We now provide evidence that sulfonylureas cross phospholipid bilayer membranes rapidly and effectively by a free-diffusion mechanism. Two sulfonylurea compounds investigated by ¹H nuclear magnetic resonance spectroscopy, glibenclamide and tolbutamide, were found to incorporate into phospholipid bilayers, with the ionizable sulfonamide exposed to the aqueous interface and its apparent dissociation constant (pK[sub a]) increased to ∼7.0. Diffusion of weak amphiphilic acids across membranes is associated with a measurable change in pH. Thus, by using a fluorescencebased pH assay, we could investigate the diffusion of sulfonylurea compounds across phospholipid bilayer membranes. A fluorescent pH indicator (pyranin or [2',7'-bis (2-carboxyethyl)-5(6)-carboxyfluorescein][BCECF]) was trapped in egg phosphatidylcholine vesicles. Addition of glibenclamide decreased internal pH (pH[sub in]), and addition of albumin reversed this drop by 50%. With the same amount of tolbutamide, the decrease in pH[sub in] was much smaller, primarily because of the lower partitioning of tolbutamide into phospholipid bilayers. Using similar protocols, we also demonstrated diffusion by the same mechanism across the β-cell plasma membrane. Thus, we now provide a molecular mechanism by which sulfonylureas can penetrate the plasma membrane and reach intracellular sites regulating exocytosis. [ABSTRACT FROM AUTHOR]

The insulinotropic activity of the novel imidazoline compound BL11282 was investigated. Intravenous administration of BL11282 (0.3 mg x kg(-1) x min(-1)) to anesthetized rats did not change blood glucose and insulin levels under basal conditions, but produced a higher increase in blood insulin levels and a faster glucose removal from the blood after glucose infusion. Similarly, in isolated Wistar rat pancreatic islets, 0.1-100 micromol/l BL11282 potently stimulated glucose-induced insulin secretion but did not modulate basal insulin secretion. Unlike previously described imidazolines, BL11282 did not block ATP-dependent K+ channels. Furthermore, the compound stimulated insulin secretion in islets depolarized with high concentrations of KCl or permeabilized with electric shock. Insulinotropic activity of BL11282 was dependent on activity of protein kinases A and C. In pancreatic islets from spontaneously diabetic GK rats, the imidazoline compound restored the impaired insulin response to glucose. In conclusion, the imidazoline BL11282 constitutes a new class of insulinotropic compounds that exerts an exclusive glucose-dependent insulinotropic activity in pancreatic islets by stimulating insulin exocytosis. [ABSTRACT FROM AUTHOR]

Discusses the cloning, identification and sequencing of cDNA encoding a T-type calcium channel isoform in insulin-secreting cells. Composition of the cDNA; Expression of the splice variants in rats islets.

The effects of RX871024, a compound with an imidazoline structure, on cytoplasmic-free Ca2+ concentration ([Ca2+]i) in mouse pancreatic beta-cells were studied. RX871024 modulates [Ca2+]i by at least two mechanisms. One mechanism involves closure of ATP-regulated K+ channels, resulting in membrane depolarization, opening of voltage-gated L-type Ca2+ channels, and a subsequent increase in [Ca2+]i. Another mechanism, reported here for the first time, deals with RX871024-induced mobilization of Ca2+ from nonmitochondrial thapsigargin-sensitive intracellular stores. Reduced glutathione, inhibitors of cytochrome P-450, and monoaminooxidases A and B blocked this Ca2+ mobilization. It is concluded that the mechanism of RX871024-induced Ca2+ mobilization from intracellular stores involves changes in the oxidation/reduction state of the pancreatic beta-cell and may be controlled by cytochrome P-450. [ABSTRACT FROM AUTHOR]