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

Abstract

Glucagon-like peptide-1 (GLP-1), released from gut endocrine L cells in response to glucose, regulates appetite, insulin secretion, and gut motility. How glucose given orally, but not systemically, induces GLP-1 secretion is unknown. We show that human duodenal L cells express sweet taste receptors, the taste G protein gustducin, and several other taste transduction elements. Mouse intestinal L cells also express alpha-gustducin. Ingestion of glucose by alpha-gustducin null mice revealed deficiencies in secretion of GLP-1 and the regulation of plasma insulin and glucose. Isolated small bowel and intestinal villi from alpha-gustducin null mice showed markedly defective GLP-1 secretion in response to glucose. The human L cell line NCI-H716 expresses alpha-gustducin, taste receptors, and several other taste signaling elements. GLP-1 release from NCI-H716 cells was promoted by sugars and the noncaloric sweetener sucralose, and blocked by the sweet receptor antagonist lactisole or siRNA for alpha-gustducin. We conclude that L cells of the gut "taste" glucose through the same mechanisms used by taste cells of the tongue. Modulating GLP-1 secretion in gut "taste cells" may provide an important treatment for obesity, diabetes and abnormal gut motility.

Conflict of interest statement

Conflict of interest statement: Dr. Margolskee has a personal financial interest in the form of stock ownership in the Redpoint Bio company, receives consulting fees from the Redpoint Bio company, and is an inventor on patents and patent applications which have been licensed to the Redpoint Bio company.

Figures

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 α-gustducin cytosolic expression and dense apical immunostaining (arrows) projecting into the gut lumen. (Scale bars, 5 μm.) (Middle) Low-magnification fields showing immunostaining of α-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 α-gustducin (α-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 α-gustducin-expressing cell (Upper, arrow). The same image, taken at a different depth, shows a cell that expresses GLP-1 and α-gustducin but not GIP (Lower, arrowhead). (Scale bars, 15 μm.) (E) Quantitation of cells expressing α-gustducin, GLP-1, or GIP. Statistically significant results determined by Student's t test; values are means ± SEM. (F) RT-PCR amplification of α-gustducin mRNA in the indicated subpopulations of laser-captured cells.

Fig. 2.

Altered secretion of GLP-1, GIP, and insulin in response to gavage-administered glucose in α-gustducin null (α-gust−/−) vs. wild-type (α-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.

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 α-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.

Fig. 4.

Coupling of taste receptors to G protein α-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-Gα-gustducin, anti-Gαi1,2, anti-Gαs, 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.