Probes for GLP-1R

Abstract

Glucagon-like peptide-1 (GLP-1) is a physiological regulator of appetite and long-acting GLP-1 receptor agonists (GLP-1RA) lower food intake and bodyweight in both human and animal studies. The effects are mediated through brain GLP-1Rs, and several brain nuclei expressing the GLP-1R may be involved. To date, mapping the complete location of GLP-1R protein in the brain has been challenged by lack of good antibodies and the discrepancy between mRNA and protein especially relevant in neuronal axonal processes. Here, we present a novel and specific monoclonal GLP-1R antibody for immunohistochemistry with murine tissue and show detailed distribution of GLP-1R expression as well as mapping of GLP-1R mRNA by non-radioactive in situ hybridization. Semi-automated image analysis was performed to map the GLP-1R distribution to atlas plates from the Allen Institute of Brain Science (AIBS). The GLP-1R was abundantly expressed in numerous regions including the septal nucleus, the hypothalamus and the brain stem. GLP-1R protein expression was also observed on neuronal projections in brain regions devoid of any mRNA which has not been observed in earlier reports. Taken together, these findings provide new knowledge on GLP-1R expression in neuronal cell bodies and neuronal projections.

Semaglutide is a human glucagon-like peptide-1 (GLP-1) analogue that is in development for the treatment of type 2 diabetes. In the pre-approval cardiovascular outcomes trial SUSTAIN 6, semaglutide was associated with a significant increase in the risk of diabetic retinopathy (DR) complications vs placebo. GLP-1 receptor (GLP-1R) expression has previously been demonstrated in the retina in animals and humans; however, antibodies used to detect expression have been documented to be non-specific and fail to detect the GLP-1R using immunohistochemistry (IHC), a problem common for many G-protein coupled receptors. Using a validated GLP-1R antibody for IHC and in situ hybridization for GLP-1R mRNA in normal human eyes, GLP-1Rs were detected in a small fraction of neurons in the ganglion cell layer. In advanced stages of DR, GLP-1R expression was not detected at the protein or mRNA level. Specifically, no GLP-1R expression was found in the eyes of people with long-standing proliferative DR (PDR). In conclusion, GLP-1R expression is low in normal human eyes and was not detected in eyes exhibiting advanced stages of PDR.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone which stimulates insulin release and inhibits glucagon secretion from the pancreas in a glucose-dependent manner. Incretin-based therapies, consisting of GLP-1 receptor (GLP-1R) agonists and dipeptidyl peptidase-4 (DPP-4) inhibitors, are used for the treatment of T2D. Immunohistochemical studies for GLP-1R expression have previously been hampered by the use of unspecific polyclonal antibodies. This study used a new monoclonal antibody to assess GLP-1R expression in pancreatic tissue from 23 patients with T2D, including 7 with a DPP-4 inhibitor and 1 with a GLP-1R agonist treatment history. A software-based automated image analysis algorithm was used for quantitating intensities and area fractions of GLP-1R positive compartments. The highest intensity GLP-1R immunostaining was seen in beta-cells in islets (average signal intensity 76,1 (± 8, 1)). GLP-1R/insulin double-labelled single cells or small clusters of cells were also frequently located within or in close vicinity of ductal epithelium in all samples and with the same GLP-1R immunostaining intensity as found in beta-cells in islets. In the exocrine pancreas a large proportion of acinar cells expressed GLP-1R with a 3-fold lower intensity of immunoreactivity as compared to beta-cells (average signal intensity 25,5 (± 3,3)). Our studies did not unequivocally demonstrate GLP-1R immunoreactivity on normal-appearing ductal epithelium. Pancreatic intraepithelial neoplasia (PanINs; a form of non-invasive pancreatic ductular neoplasia) were seen in most samples, and a minority of these expressed low levels of GLP-1R. These data confirm the ubiquity of early stage PanIN lesions in patients with T2D and do not support the hypothesis that incretin-based therapies are associated with progression towards the more advanced stage PanIN lesions.

Abstract

Objective

Glucagon-like peptide-1 (GLP-1) neurons in the hindbrain densely innervate the dorsomedial hypothalamus (DMH), a nucleus strongly implicated in body weight regulation and the sympathetic control of brown adipose tissue (BAT) thermogenesis. Therefore, DMH GLP-1 receptors (GLP-1R) are well placed to regulate energy balance by controlling sympathetic outflow and BAT function.

Methods

We investigate this possibility in adult male rats by using direct administration of GLP-1 (0.5 ug) into the DMH, knocking down DMH GLP-1R mRNA with viral-mediated RNA interference, and by examining the neurochemical phenotype of GLP-1R expressing cells in the DMH using in situ hybridization.

Results

GLP-1 administered into the DMH increased BAT thermogenesis and hepatic triglyceride (TG) mobilization. On the other hand, Glp1r knockdown (KD) in the DMH increased body weight gain and adiposity, with a concomitant reduction in energy expenditure (EE), BAT temperature, and uncoupling protein 1 (UCP1) expression. Moreover, DMH Glp1r KD induced hepatic steatosis, increased plasma TG, and elevated liver specific de-novo lipogenesis, effects that collectively contributed to insulin resistance. Interestingly, DMH Glp1r KD increased neuropeptide Y (NPY) mRNA expression in the DMH. GLP-1R mRNA in the DMH, however, was found in GABAergic not NPY neurons, consistent with a GLP-1R-dependent inhibition of NPY neurons that is mediated by local GABAergic neurons. Finally, DMH Glp1r KD attenuated the anorexigenic effects of the GLP-1R agonist exendin-4, highlighting an important role of DMH GLP-1R signaling in GLP-1-based therapies.

Conclusions

Collectively, our data show that DMH GLP-1R signaling plays a key role for BAT thermogenesis and adiposity.

Glucagon-like peptide-1 receptor (GLP-1R) agonists, used to treat type 2 diabetes and obesity, reduce rates of myocardial infarction and cardiovascular death. The GLP-1R has been localized to the human sinoatrial node; however, its expression in ventricular tissue remains uncertain. Here we studied GLP-1R expression in the human heart using GLP-1R-directed antisera, quantitative PCR, reverse transcription PCR to detect full length mRNA transcripts, and in situ hybridization. GLP1R mRNA transcripts, encompassing the entire open reading frame, were detected in all four cardiac chambers from 15 hearts at levels approximating those detected in human pancreas. In contrast, cardiac GLP2R expression was relatively lower, whereas cardiac GCGR expression was sporadic and not detected in the left ventricle. GLP1R mRNA transcripts were not detected in RNA from human cardiac fibroblasts, coronary artery endothelial, or vascular smooth muscle cells. Human Brunner's glands and pancreatic islets exhibited GLP-1R-immunopositivity and abundant expression of GLP1R mRNA transcripts by in situ hybridization. GLP1R transcripts were also detected by in situ hybridization in human cardiac sinoatrial node tissue. However definitive cellular localization of GLP1R mRNA transcripts or immunoreactive GLP-1R protein within human cardiomyocytes (CMs) or cardiac blood vessels remained elusive. Moreover, validated GLP-1R antisera lacked sufficient sensitivity to detect expression of the endogenous islet or cardiac GLP-1R by Western blotting. Hence, although human cardiac ventricles express the GLP1R, the identity of one or more ventricular cell type(s) that express a translated GLP1R protein requires further clarification with highly sensitive methods of detection.

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.

Glutamatergic neurons that express pre-proglucagon (PPG) and are immunopositive (+) for glucagon-like peptide-1 (i.e., GLP-1+ neurons) are located within the caudal nucleus of the solitary tract (cNTS) and medullary reticular formation in rats and mice. GLP-1 neurons give rise to an extensive central network in which GLP-1 receptor (R) signaling suppresses food intake, attenuates rewarding, increases avoidance, and stimulates stress responses, partly via . GLP-1R signaling within the cNTS. In mice, noradrenergic (A2) cNTS neurons express GLP-1R, whereas PPG neurons do not. In the present study, confocal microscopy in rats confirmed that prolactin-releasing peptide (PrRP)+ A2 neurons are closely apposed by GLP-1+ axonal varicosities. Surprisingly, GLP-1+ appositions were also observed on dendrites of PPG/GLP-1+ neurons in both species, and electron microscopy in rats revealed that GLP-1+ boutons form asymmetric synaptic contacts with GLP-1+ dendrites. However, RNAscope confirmed that rat GLP-1 neurons do not express GLP-1R mRNA. Similarly, Ca2+ imaging of somatic and dendritic responses in mouse ex vivo slices confirmed that PPG neurons do not respond directly to GLP-1, and a mouse cross-breeding strategy revealed that fewer than 1% of PPG neurons co-express GLP-1R. Collectively, these data suggest that GLP-1R signaling pathways modulate the activity of PrRP+ A2 neurons, and also reveal a local "feed-forward" synaptic network among GLP-1 neurons that apparently does not utilize GLP-1R signaling. This local GLP-1 network may instead use glutamatergic signaling to facilitate dynamic and potentially selective recruitment of GLP-1 neural populations that shape behavioral and physiological responses to internal and external challenges.