Probes for INS

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.

Objective

The supramammillary nucleus (SuM) is nestled between the lateral hypothalamus (LH) and the ventral tegmental area (VTA). This neuroanatomical position is consistent with a potential role of this nucleus to regulate ingestive and motivated behavior. Here neuroanatomical, molecular, and behavior approaches are utilized to determine whether SuM contributes to ingestive and food-motivated behavior control.

Methods

Through the application of anterograde and retrograde neural tract tracing with novel designer viral vectors, the current findings show that SuM neurons densely innervate the LH in a sex dimorphic fashion. Glucagon-like peptide-1 (GLP-1) is a clinically targeted neuro-intestinal hormone with a well-established role in regulating energy balance and reward behaviors. Here we determine that GLP-1 receptors (GLP-1R) are expressed throughout the SuM of both sexes, and also directly on SuM LH-projecting neurons and investigate the role of SuM GLP-1R in the regulation of ingestive and motivated behavior in male and female rats.

Results

SuM microinjections of the GLP-1 analogue, exendin-4, reduced ad libitum intake of chow, fat, or sugar solution in both male and female rats, while food-motivated behaviors, measured using the sucrose motivated operant conditioning test, was only reduced in male rats. These data contrasted with the results obtained from a neighboring structure well known for its role in motivation and reward, the VTA, where females displayed a more potent response to GLP-1R activation by exendin-4. In order to determine the physiological role of SuM GLP-1R signaling regulation of energy balance, we utilized an adeno-associated viral vector to site-specifically deliver shRNA for the GLP-1R to the SuM. Surprisingly, and in contrast to previous results for the two SuM neighboring sites, LH and VTA, SuM GLP-1R knockdown increased food seeking and adiposity in obese male rats without altering food intake, body weight or food motivation in lean or obese, female or male rats.

Conclusion

Taken together, these results indicate that SuM potently contributes to ingestive and motivated behavior control; an effect contingent on sex, diet/homeostatic energy balance state and behavior of interest. These data also extend the map of brain sites directly responsive to GLP-1 agonists, and highlight key differences in the role that GLP-1R play in interconnected and neighboring nuclei.

Kisspeptin (Kiss1) neurons in the hypothalamic arcuate nucleus (ARC) are key components of the hypothalamic-pituitary-gonadal axis, as they regulate the basal pulsatile release of gonadotropin releasing hormone (GnRH). ARC Kiss1 action is dependent on energy status and unmasking metabolic factors responsible for modulating ARC Kiss1 neurons is of great importance. One possible factor is glucagon-like peptide-1 (GLP-1), an anorexigenic neuropeptide produced by brainstem preproglucagon neurons. As GLP fiber projections and the GLP-1 receptor (GLP-1R) are abundant in the ARC, we hypothesized that GLP-1R signaling could modulate ARC Kiss1 action. Using ovariectomized (OVX) mice, we found that GLP-producing fibers come in close apposition with ARC Kiss1 neurons; these neurons also contain Glp1r mRNA. Electrophysiological recordings revealed that liraglutide (a long-acting GLP-1R agonist) increased action potential firing and caused a direct membrane depolarization of ARC Kiss1 cells in brain slices. We determined that brainstem preproglucagon mRNA is decreased following a 48 h fast in mice, a negative energy state in which ARC Kiss1 expression and downstream GnRH/luteinizing hormone (LH) release are potently suppressed. However, activation of GLP-1R signaling in fasted mice with liraglutide was not sufficient to prevent LH inhibition. Furthermore, chronic central infusions of the GLP-1R antagonist, exendin (9-39) in ad libitum fed mice did not alter ARC Kiss1 mRNA or plasma LH. As a whole, these data identify a novel interaction of the GLP-1 system with ARC Kiss1 neurons but indicate that CNS GLP-1R signaling alone is not critical for the maintenance of LH during fasting or normal feeding.

Significance Statement Reproductive dysfunction is associated with metabolic imbalance, and identifying the underlying molecular mechanisms linking metabolic status with reproductive function is of great importance. Kisspeptin neurons (Kiss1) located in the arcuate nucleus of the hypothalamus (ARC) are essential for fertility and are potently inhibited during negative energy balance; this inhibition occurs in the presence or absence of ovarian steroids. Preproglucagon-expressing neurons located in the brainstem send abundant fiber projections to the ARC where they release the anorexigenic neuropeptide, glucagon-like peptide-1 (GLP-1). The aim of these studies was to determine the interaction of the CNS GLP-1 system with ARC Kiss1 activity to potentially provide a link between systems that control energy balance with those that control reproductive neuroendocrine output.