Probes for INS

Cleft palate is one of the most common craniofacial congenital defects in humans. It is associated with multiple genetic and environmental risk factors, including mutations in the genes encoding signaling molecules in the sonic hedgehog (Shh) pathway, which are risk factors for cleft palate in both humans and mice. However, the function of Shh signaling in the palatal epithelium during palatal fusion remains largely unknown. Although components of the Shh pathway are localized in the palatal epithelium, specific inhibition of Shh signaling in palatal epithelium does not affect palatogenesis. We therefore utilized a hedgehog (Hh) signaling gain-of-function mouse model, K14-Cre;R26SmoM2, to uncover the role of Shh signaling in the palatal epithelium during palatal fusion. In this study, we discovered that constitutive activation of Hh signaling in the palatal epithelium results in submucous cleft palate and persistence of the medial edge epithelium (MEE). Further investigation revealed that precise downregulation of Shh signaling is required at a specific time point in the MEE during palatal fusion. Upregulation of Hh signaling in the palatal epithelium maintains the proliferation of MEE cells. This may be due to a dysfunctional p63/Irf6 regulatory loop. The resistance of MEE cells to apoptosis is likely conferred by enhancement of a cell adhesion network through the maintenance of p63 expression. Collectively, our data illustrate that persistent Hh signaling in the palatal epithelium contributes to the etiology and pathogenesis of submucous cleft palate through its interaction with a p63/Irf6-dependent biological regulatory loop and through a p63-induced cell adhesion network.

Among children with the most severe presentation of Marfan syndrome (MFS), an inherited disorder of connective tissue caused by a deficiency of extracellular fibrillin-1, heart failure is the leading cause of death. Here, we show that, while MFS mice (Fbn1C1039G/+ mice) typically have normal cardiac function, pressure overload (PO) induces an acute and severe dilated cardiomyopathy in association with fibrosis and myocyte enlargement. Failing MFS hearts show high expression of TGF-β ligands, with increased TGF-β signaling in both nonmyocytes and myocytes; pathologic ERK activation is restricted to the nonmyocyte compartment. Informatively, TGF-β, angiotensin II type 1 receptor (AT1R), or ERK antagonism (with neutralizing antibody, losartan, or MEK inhibitor, respectively) prevents load-induced cardiac decompensation in MFS mice, despite persistent PO. In situ analyses revealed an unanticipated axis of activation in nonmyocytes, with AT1R-dependent ERK activation driving TGF-β ligand expression that culminates in both autocrine and paracrine overdrive of TGF-β signaling. The full compensation seen in wild-type mice exposed to mild PO correlates with enhanced deposition of extracellular fibrillin-1. Taken together, these data suggest that fibrillin-1 contributes to cardiac reserve in the face of hemodynamic stress, critically implicate nonmyocytes in disease pathogenesis, and validate ERK as a therapeutic target in MFS-related cardiac decompensation.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone increasing postprandial insulin release. GLP-1 also induces diuresis and natriuresis in humans and rodents. The GLP-1 receptor is extensively expressed in the renal vascular tree in normotensive rats where acute GLP-1 treatment leads to increased mean arterial pressure (MAP) and increased renal blood flow (RBF). In hypertensive animal models, GLP-1 has been reported both to increase and decrease MAP. The aim of this study was to examine expression of renal GLP-1 receptors in spontaneously hypertensive rats (SHR) and to assess the effect of acute intrarenal infusion of GLP-1. We hypothesized that GLP-1 would increase diuresis and natriuresis and reduce MAP in SHR. Immunohistochemical staining and in situ hybridization for the GLP-1 receptor were used to localize GLP-1 receptors in the kidney. Sevoflurane-anesthetized normotensive Sprague-Dawley rats and SHR received a 20 min intrarenal infusion of GLP-1 and changes in MAP, RBF, heart rate, dieresis, and natriuresis were measured. The vasodilatory effect of GLP-1 was assessed in isolated interlobar arteries from normo- and hypertensive rats. We found no expression of GLP-1 receptors in the kidney from SHR. However, acute intrarenal infusion of GLP-1 increased MAP, RBF, dieresis, and natriuresis without affecting heart rate in both rat strains. These results suggest that the acute renal effects of GLP-1 in SHR are caused either by extrarenal GLP-1 receptors activating other mechanisms (e.g., insulin) to induce the renal changes observed or possibly by an alternative renal GLP-1 receptor.

Abstract

Objectives

G protein-coupled receptors (GPCRs) act as transmembrane molecular sensors of neurotransmitters, hormones, nutrients, and metabolites. Because unmyelinated vagalafferents richly innervate the gastrointestinal mucosa, gut-derived molecules may directly modulate the activity of vagal afferents through GPCRs. However, the types of GPCRs expressed in vagal afferents are largely unknown. Here, we determined the expression profile of all GPCRs expressed in vagal afferents of the mouse, with a special emphasis on those innervating the gastrointestinal tract.

Methods

Using a combination of high-throughput quantitative PCR, RNA sequencing, and in situhybridization, we systematically quantified GPCRs expressed in vagal unmyelinated Nav1.8-expressing afferents.

Results

GPCRs for gut hormones that were the most enriched in Nav1.8-expressing vagal unmyelinated afferents included NTSR1, NPY2R, CCK1R, and to a lesser extent, GLP1R, but not GHSR and GIPR. Interestingly, both GLP1R and NPY2R were coexpressed with CCK1R. In contrast, NTSR1 was coexpressed with GPR65, a marker preferentially enriched in intestinal mucosal afferents. Only few microbiome-derived metabolite sensors such as GPR35 and, to a lesser extent, GPR119 and CaSR were identified in the Nav1.8-expressing vagal afferents. GPCRs involved in lipid sensing and inflammation (e.g. CB1R, CYSLTR2, PTGER4), and neurotransmitters signaling (CHRM4, DRD2, CRHR2) were also highly enriched in Nav1.8-expressing neurons. Finally, we identified 21 orphan GPCRs with unknown functions in vagal afferents.

Conclusion

Overall, this study provides a comprehensive description of GPCR-dependent sensing mechanisms in vagal afferents, including novel coexpression patterns, and conceivably coaction of key receptors for gut-derived molecules involved in gut-brain communication.