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PNAS 104 (38): 15069-15074

Copyright © 2007 by the National Academy of Sciences.

Gut-expressed gustducin and taste receptors regulate secretion of glucagon-like peptide-1

Hyeung-Jin Jang*, Zaza Kokrashvili{dagger}, Michael J. Theodorakis*, Olga D. Carlson*, Byung-Joon Kim*, Jie Zhou*, Hyeon Ho Kim*, Xiangru Xu*, Sic L. Chan*, Magdalena Juhaszova*, Michel Bernier*, Bedrich Mosinger{dagger}, Robert F. Margolskee{dagger},{ddagger}, and Josephine M. Egan*

*National Institute on Aging/National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224; and {dagger}Department of Neuroscience, Mount Sinai School of Medicine, 1425 Madison Avenue, Box 1065, New York, NY 10029


Figure 1
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Fig. 1.. Presence of taste signaling elements in L cells of human duodenum. (A) Indirect immunofluorescent imaging showing coexpression of taste signaling elements (Left) with GLP-1 (Center). Nuclei in the merged images (Right) are stained blue. (Scale bars, 15 µm.) (B) (Top) Cells showing {alpha}-gustducin cytosolic expression and dense apical immunostaining (arrows) projecting into the gut lumen. (Scale bars, 5 µm.) (Middle) Low-magnification fields showing immunostaining of {alpha}-gustducin, GLP-1, and GIP. (Bottom) Solitary gustducin-expressing, L (GLP-1), and K (GIP) cells amongst the more numerous enterocytes are shown: nuclei are stained red. (Scale bars, 50 µm.) (C) Coexpression of T1R2 sweet taste receptor subunit with {alpha}-gustducin ({alpha}-gust), GLP-1, and T1R3 in duodenal enteroendocrine cells. (Scale bars, 15 µm.) (D) Triple staining, showing expression of both GLP-1 and GIP in an {alpha}-gustducin-expressing cell (Upper, arrow). The same image, taken at a different depth, shows a cell that expresses GLP-1 and {alpha}-gustducin but not GIP (Lower, arrowhead). (Scale bars, 15 µm.) (E) Quantitation of cells expressing {alpha}-gustducin, GLP-1, or GIP. Statistically significant results determined by Student's t test; values are means ± SEM. (F) RT-PCR amplification of {alpha}-gustducin mRNA in the indicated subpopulations of laser-captured cells.

 

Figure 2
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Fig. 2.. Altered secretion of GLP-1, GIP, and insulin in response to gavage-administered glucose in {alpha}-gustducin null ({alpha}-gust–/–) vs. wild-type ({alpha}-gust+/+) mice. (A) Plasma GLP-1 (Top), GIP (Middle) and insulin (Lower) levels after glucose gavage (5 g/kg body weight). (B) Plasma glucose after glucose gavage (2 g/kg body weight). (C) Plasma glucose after postfasting feeding on chow. (D) Plasma GLP-1 responses from surgically isolated duodenum in vivo: the duodenum was ligated away from the stomach and rest of the intestines, and circulatory contact maintained. Ten percent glucose was infused directly into the isolated duodenum. (E) GLP-1 secretory responses to 10% glucose from minced proximal duodenum. For in vivo experiments, n = 6–12 animals per genotype; in vitro experiments were carried out in triplicate and replicated at least twice. Statistical significance determined by ANOVA, values are means ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 

Figure 3
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Fig. 3.. Secretion of GLP-1 in response to glucose, sucrose, and sucralose in NCI-H716 cells. (A and B) Glucose-, sucrose-, and sucralose-mediated GLP-1 secretion from NCI-H716 cells. The sweet receptor inhibitor lactisole inhibited sucralose-mediated GLP-1 secretion. (C) siRNA-mediated diminution of both {alpha}-gustducin protein levels (by immunoblotting) and glucose-induced [but not basal (BSL)] GLP-1 secretion from NCI-H716 cells. (D) Immunoblotting of ERK and pERK phosphorylated from NCI-H716 cells in response to increasing concentrations of glucose and sucralose. The inhibitor of Erk phosphorylation, PD98059, inhibited sucralose-mediated Erk phosphorylation. The sweet receptor inhibitor lactisole diminished Erk phosphorylation. BSL, basal. Experiments were carried out in triplicate and replicated at least twice. Statistical significance determined by ANOVA; values are means ± SEM; *, P < 0.05; ***, P < 0.001.

 

Figure 4
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Fig. 4.. Coupling of taste receptors to G protein {alpha}-subunits in NCI-H716 cells. Membranes from NCI-H716 cells were preincubated with the indicated concentrations of glucose and sucrose for 10min at 25°C in the presence of 5–10µCi [32P] GTP-azidoanilide, then irradiated to cross link the GTP analog to G proteins. G protein-specific immune complexes (anti-G{alpha}-gustducin, anti-G{alpha}i1,2, anti-G{alpha}s, and control IgG) were separated by SDS/PAGE, transferred to membranes, and autoradiographically imaged. A positive control for G protein activation is shown in SI Fig. 12.

 


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