Probing cell type–specific functions of Gi in vivo identifies GPCR regulators of insulin secretion
Jean B. Regard, … , Matthias Hebrok, Shaun R. Coughlin
Jean B. Regard, … , Matthias Hebrok, Shaun R. Coughlin
Published November 8, 2007
Citation Information: J Clin Invest. 2007;117(12):4034-4043. https://doi.org/10.1172/JCI32994.
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Research Article Technical Advance Cardiology

Probing cell type–specific functions of Gi in vivo identifies GPCR regulators of insulin secretion

Abstract

The in vivo roles of the hundreds of mammalian G protein–coupled receptors (GPCRs) are incompletely understood. To explore these roles, we generated mice expressing the S1 subunit of pertussis toxin, a known inhibitor of Gi/o signaling, under the control of the ROSA26 locus in a Cre recombinase–dependent manner (ROSA26PTX). Crossing ROSA26PTX mice to mice expressing Cre in pancreatic β cells produced offspring with constitutive hyperinsulinemia, increased insulin secretion in response to glucose, and resistance to diet-induced hyperglycemia. This phenotype underscored the known importance of Gi/o and hence of GPCRs for regulating insulin secretion. Accordingly, we quantified mRNA for each of the approximately 373 nonodorant GPCRs in mouse to identify receptors highly expressed in islets and examined the role of several. We report that 3-iodothyronamine, a thyroid hormone metabolite, could negatively and positively regulate insulin secretion via the Gi-coupled α2A-adrenergic receptor and the Gs-coupled receptor Taar1, respectively, and protease-activated receptor–2 could negatively regulate insulin secretion and may contribute to physiological regulation of glucose metabolism. The ROSA26PTX system used in this study represents a new genetic tool to achieve tissue-specific signaling pathway modulation in vivo that can be applied to investigate the role of Gi/o-coupled GPCRs in multiple cell types and processes.

Authors

Jean B. Regard, Hiroshi Kataoka, David A. Cano, Eric Camerer, Liya Yin, Yao-Wu Zheng, Thomas S. Scanlan, Matthias Hebrok, Shaun R. Coughlin

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Figure 1

Generation and characterization of the conditional ROSA26PTX allele.

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Generation and characterization of the conditional ROSA26PTX allele. ...
(A) Diagram of the wild-type ROSA26 locus, the ROSA26PTX targeting vector, the ROSA26PTX allele, and Cre-recombined locus. Black arrowheads represent LoxP sites. (B) Cre-dependent expression of PTX S1 mRNA. RNA from endothelial cells that were isolated from ROSA26PTX mice and then infected with Cre- or GFP-expressing (control) adenovirus was analyzed by Northern blot. Blots were hybridized with probe for PTX S1 and for GAPDH mRNA to assess loading. (C) Cre-dependent ADP-ribosylation of Gαi/o. Fibroblasts from ROSA26PTX mouse embryos carrying a CMV-IE-ER-Cre transgene were incubated with or without 1 μM 4-hydroxy-tamoxifen for 4 days to induce Cre expression, ROSA26PTX recombination, and expression of PTX S1. Membranes from these cells were incubated with the ADP-ribose donor [32P]NAD and exogenous pertussis toxin, then analyzed by SDS-PAGE and autoradiography (upper panel) or stained for protein loading (lower panel). Note the absence of Gαi/o available for in vitro labeling in membranes from tamoxifen-induced cells, indicating that ADP-ribosylation of Gαi/o had already occurred in vivo. (D) Cell-autonomous function of ROSA26PTX. Mouse thymocytes were cocultured with Cre-expressing ROSAPTX mouse embryonic fibroblasts (MEFs; as in Figure 1C). Where indicated (+pre-treat), thymocytes were treated with pertussis holotoxin (50 ng/ml). After 8 hours of coculture, thymocytes were isolated, and their membrane fractions were assayed for Gαi/o available for in vitro ADP-ribosylation as in Figure 1C. Note that coculture of thymocytes with PTX S1–expressing MEFs did not cause detectable loss of ADP-ribosylatable Gαi/o, but addition of exogenous holotoxin did.