Online monitoring of stimulus-induced gene expression in pancreatic beta-cells.

Abstract

T he use of fluorescent proteins as a "protein-tag" in cell biology sheds new light on the mechanisms that determine key processes in cell function, such as subcellular localization of proteins, colocalization of proteins, physical interaction of proteins, and protein translocation. Used in a rational experimental set-up and in combination with techniques that allow high resolution and/or high dynamics of the obtained images, fluorescent proteins provide a tool that makes the phrase "seeing is believing" a reality in cell physiology. The generation of different colored members of the green fluorescent protein (GFP) family (1), i.e., blue (BFP, EBFP), cyan (ECFP), green (GFP, EGFP), and yellow (EYFP), as well as the cloning of new fluorescent proteins, such as the red fluorescent protein DsRed from Discosoma sp.(2), permitted fluorescent probes exhibiting distinct spectra of light/energy excitation and emission. This allowed on the one hand the use of a combination of different colors to monitor distinct proteins at the same time; on the other hand, it made it possible to develop tailored molecular probes to monitor protein interactions, substrate concentrations, enzyme activities, etc., at the subcellular level, partially based on fluorescence resonance energy transfer. A timely review on the potential application of fluorescent proteins based on the GFP family was published by Tsien (1).The use of fluorescent proteins as reporter genes, although proposed as genetic markers from the beginning by Chalfie et al. (3), has been scarce, except as a marker in transfection experiments, despite their potential for use as dynamic "reporters" compared with classical reporter genes.Here we show that fluorescent proteins, i.e., GFP S65T and DsRed, can serve as semiquantitative and dynamic reporters for stimulus-induced gene expression in general and in the context of stimulus-induced cell type-specific gene expression, exemplified in the insulin-producing pancreatic ␤-cell. Moreover, we show that fluorescent proteins do not only allow monitoring of stimulus-induced gene expression at the single-cell level but also at the level of a micro-organ-here, the islet of Langerhans. Finally, use of fluorescent proteins with discrete excitation/emission properties, i.e., GFP S65T and DsRed, allows the simultaneous monitoring of two genes in the same cell, here exemplified by the stimulus-induced rat insulin 1 (rIns1) promoter-driven DsRed expression and the rat ␤-cell-active glucokinase (r␤GK) gene promoter-driven GFP expression. RESEARCH DESIGN AND METHODSExpression vectors. The construction of prIns1.GFP and pCMV.GFP was described elsewhere (4). Adenovirus (Ad)-rIns1.GFP (5) was provided by Dr. Lina Moitoso de Vargas (New England Medical School, Boston, MA). Plasmid prIns1.DsRed was generated by exchanging the GFP S65T -bGHpA cassette with the DsRed-SV40pA cassette from pDsRed1-1 (Clontech, Palo Alto, CA). Plasmid pr␤GK.GFP was constructed by exchanging the CAT-SV40pA cassette in pr␤GK-278.CAT (6) with the GFP S65T -bGHpA cassette...

Glucagon regulates its own synthesis by autocrine signaling

“…This allowed the analysis of stimulus-induced glucagon promoter activity in living α cells at the single-cell level. Similar to the dynamics described for insulin promoter-driven or c-fos promoter-driven DsRed2 expression/fluorescence in insulin-producing cells (12,13), DsRed2 fluorescence in glucagon-producing αTC1-9 cells began to increase by 60 min after the start of glucagon stimulation for 15 min and continued to increase up to 240 min (Fig. 1D).…”

Diet‐induced β‐cell insulin resistance results in reversible loss of functional β‐cell mass

“…We developed a biosensor that allowed us to monitor β-cell function in response to glucose stimulation by changes in fluorescence intensity. As we have previously shown (27–29, 41, 42), the rat insulin gene-1 promoter (RIP1) and the rat β-cell–active glucokinase gene promoter contain metabolic response elements that allow an increase in promoter activity upon stimulation with glucose or insulin in vitro . To generate βFLUOMETRI for in vivo monitoring, we combined 3 expression cassettes in a single adenoviral vector ( Fig.…”

“…Image analysis for in vitro experiments was performed as described in (27–30) using ImageJ. Average fluorescence intensities for each cell was determined for t = 60 min (start) and t = 240 min.…”

A Novel Dual-Color Reporter for Identifying Insulin-Producing Beta- Cells and Classifying Heterogeneity of Insulinoma Cell Lines

“…The “direct" dual-color reporter systems that have been described use the ubiquitous CMV or a cell-/tissue-specific promoter to drive one color and another cell-/tissue-specific reporter for the second color [30], [31], [32], [33], [34], [35]. Thus, the cells in which the cell-/tissue-specific promoter is activated should express two colors, while all other cells should express only one color.…”