Probes for GLP-1R

Therapies based on glucagon-like peptide-1 receptor (GLP-1R) agonism are highly effective in treating type 2 diabetes and obesity, but the localization of GLP-1Rs mediating the antidiabetic and other possible actions of GLP-1 is still debated. The purpose with this study was to identify sites of GLP-1R mRNA and protein expression in the mouse gastrointestinal system by means of GLP-1R antibody immunohistochemistry, Glp1r mRNA fluorescence in situ hybridization, and 125I-exendin (9-39) autoradiography. As expected, GLP-1R staining was observed in almost all β-cells in the pancreatic islets, but more rarely in α- and δ-cells. In the stomach, GLP-1R staining was found exclusively in the gastric corpus mucous neck cells, known to protect the stomach mucosa. The Brunner glands were strongly stained for GLP-1R, and pretreatment with GLP-1 agonist exendin-4 caused internalization of the receptor and mucin secretion, while pretreatment with phosphate-buffered saline or antagonist exendin (9-39) did not. In the intestinal mucosa, GLP-1R staining was observed in intraepithelial lymphocytes, lamina propria lymphocytes, and enteroendocrine cells containing secretin, peptide YY, and somatostatin, but not cholecystokinin. GLP-1R staining was seen in nerve fibers within the choline acetyl transferase- and nitric oxide-positive myenteric plexuses from the gastric corpus to the distal large intestine being strongest in the mid- and hindgut area. Finally, intraperitoneal administration of radiolabeled exendin (9-39) strongly labeled myenteric fibers. In conclusion, this study expands our knowledge of GLP-1R localization and suggests that GLP-1 may serve an important role in modulating gastrointestinal health and mucosal protection.

Abstract Objective Analogues of GDF15 (Growth Differentiation Factor 15) are promising new anti-obesity therapies as pharmacological treatment with GDF15 results in dramatic reductions of food intake and body weight. GDF15 exerts its central anorexic effects by binding to the GFRAL receptor exclusively expressed in the Area Postrema (AP) and the Nucleus of the Solitary Tract (NTS) of the hindbrain. We sought to determine if GDF15 is an indispensable factor for other interventions that cause weight loss and which are also known to act via these hindbrain regions. Methods To explore the role of GDF15 on food choice we performed macronutrient intake studies in mice treated pharmacologically with GDF15 and in mice having either GDF15 or GFRAL deleted. Next we performed vertical sleeve gastrectomy (VSG) surgeries in a cohort of diet-induced obese Gdf15-null and control mice. To explore the anatomical co-localization of neurons in the hindbrain responding to GLP-1 and/or GDF15 we used GLP-1R reporter mice treated with GDF15, as well as naïve mouse brain and human brain stained by ISH and IHC, respectively, for GLP-1R and GFRAL. Lastly we performed a series of food intake experiments where we treated mice with targeted genetic disruption of either Gdf15 or Gfral with liraglutide; Glp1r-null mice with GDF15; or combined liraglutide and GDF15 treatment in wild-type mice. Results We found that GDF15 treatment significantly lowered the preference for fat intake in mice, whereas no changes in fat intake were observed after genetic deletion of Gdf15 or Gfral. In addition, deletion of Gdf15 did not alter the food intake or bodyweight after sleeve gastrectomy. Lack of GDF15 or GFRAL signaling did not alter the ability of the GLP-1R agonist liraglutide to reduce food intake. Similarly lack of GLP-1R signaling did not reduce GDF15’s anorexic effect. Interestingly, there was a significant synergistic effect on weight loss when treating wild-type mice with both GDF15 and liraglutide. Conclusion These data suggest that while GDF15 does not play a role in the potent effects of VSG in mice there seems to be a potential therapeutic benefit of activating GFRAL and GLP-1R systems simultaneously.

Enterochromaffin (EC) cell-derived 5-hydroxytryptamine (5-HT) is a mediator of toxin-induced reflexes, initiating emesis via vagal and central 5-HT3 receptors. The amine is also involved in gastrointestinal (GI) reflexes that are prosecretory and promotile, and recently 5-HT's roles in chemosensation in the distal bowel have been described. We set out to establish the efficacy of 5-HT signaling, local 5-HT levels and pharmacology in discrete regions of the mouse small and large intestine. We also investigated the inter-relationships between incretin hormones, glucagon-like peptide-1 (GLP-1) and gastric inhibitory polypeptide (GIP) and endogenous 5-HT in mucosal and motility assays.Adult mouse GI mucosae were mounted in Ussing chambers and area-specific studies were performed to establish the 5-HT3 and 5-HT4 pharmacology, the sidedness of responses, and the inter-relationships between incretins and endogenous 5-HT. Natural fecal pellet transit in vitro and full-length GI transit in vivo were also measured.We observed the greatest level of tonic and exogenous 5-HT-induced ion transport and highest levels of 5-HT in ascending colon mucosa. Here both 5-HT3 and 5-HT4 receptors were involved but elsewhere in the GI tract epithelial basolateral 5-HT4 receptors mediate 5-HT's prosecretory effect. Exendin-4 and GIP induced 5-HT release in the ascending colon, while L cell-derived PYY also contributed to GIP mucosal effects in the descending colon. Both peptides slowed colonic transit.We provide functional evidence for paracrine interplay between 5-HT, GLP-1 and GIP, particularly in the colonic mucosal region. Basolateral epithelial 5-HT4 receptors mediated both 5-HT and incretin mucosal responses in healthy colon.