Activation of arcuate nucleus glucagon-like peptide-1 receptor-expressing neurons suppresses food intake

Abstract

Background: Central nervous system (CNS) control of metabolism plays a pivotal role in maintaining energy balance. In the brain, Glucagon-like peptide 1 (GLP-1), encoded by the proglucagon 'Gcg' gene, produced in a distinct population of neurons in the nucleus tractus solitarius (NTS), has been shown to regulate feeding behavior leading to the suppression of appetite. However, neuronal networks that mediate endogenous GLP-1 action in the CNS on feeding and energy balance are not well understood.

Results: We analyzed the distribution of GLP-1R-expressing neurons and axonal projections of NTS GLP-1-producing neurons in the mouse brain. GLP-1R neurons were found to be broadly distributed in the brain and specific forebrain regions, particularly the hypothalamus, including the arcuate nucleus of the hypothalamus (ARC), a brain region known to regulate energy homeostasis and feeding behavior, that receives dense NTS^Gcg neuronal projections. The impact of GLP-1 signaling in the ARC GLP-1R-expressing neurons and the impact of activation of ARC GLP-1R on food intake was examined. Application of GLP-1R specific agonist Exendin-4 (Exn-4) enhanced a proportion of the ARC GLP-1R-expressing neurons and pro-opiomelanocortin (POMC) neuronal action potential firing rates. Chemogenetic activation of the ARC GLP-1R neurons by using Cre-dependent hM3Dq AAV in the GLP-1R-ires-Cre mice, established that acute activation of the ARC GLP-1R neurons significantly suppressed food intake but did not have a strong impact on glucose homeostasis.

Conclusions: These results highlight the importance of central GLP-1 signaling in the ARC that express GLP-1R that upon activation, regulate feeding behavior.

Keywords: Chemogenetics; Exendin-4; Feeding; Glucagon-like peptide-1; Glucose tolerance; Hypothalamus; Pro-opiomelanocortin.

Fig. 1

Quantification of GLP-1 receptor distribution in the whole brain. A Representative images of GLP-1R-ires-cre mouse crossed with Ai14 tdTomato (LSL-TdTomato) reporter mice in Suprachiasmatic nucleus (Sch), Paraventricular nucleus of the hypothalamus (PVN), Basomedial amygdaloid nucleus (BMA), anterior cortical amygdaloid area (ACo), medial amygdaloid nucleus (MeP). B Whole brain slice preparation of GLP-1R-ires-Cre::Ai14 tdTomato. C Percentage (%) of GLP-1R neuronal density in the brain. D Quantification of the total number of GLP-1R neurons area (density) in the brain. Data are presented as mean ± standard error of the mean (SEM), [n = 3]

Fig. 2

Axonal projection patterns of NTS Gcg endogenous GLP-1 neurons. A Gcg-Cre mouse injected with AAV-DIO-Chr2-EYFP in the NTS. B Representative image of the injection site in the Gcg-Cre mouse injected with AAV-DIO-Chr2-EYFP in the NTS. C Representative images of the axon terminals in the brain regions of Gcg-Cre mouse injected with AAV-DIO-ChR2-EYFP. D Identified brain regions with axon terminals from NTS^Gcg (GLP-1) neurons. A map representing the projection patterns of the GLP-1 neurons in the brain (n = 3)

Fig. 3

Expression of GLP-1R in the ARC and the effects of Exn4 on ARC neurons. A GLP-1 neuronal axonal projections in the ARC region. AAV-DIO-Chr2-EYFP was injected into the NTS region of the Gcg-Cre mouse. B GLP-1R-expressing neurons in the ARC region revealed by tdTomato fluorescence in GLP-1R-ires-Cre::Ai14 (LSL-TdTomato) mice. C Expression of GLP-1R in the ARC using a conditional viral-mediated approach. AAV-DIO-eGFP was injected into the ARC region of the GLP-1R-ires-Cre mouse. D Experimental paradigm for recordings from GLP-1R-expressing neurons labeled with tdTomato in GLP-1R-ires-cre::Ai14 tdTomato (Lox-Stop-Lox-TdTomato) reporter mice. E and F The resting membrane potential (RMP) of ARC GLP-1R-expressing neurons in the absence or presence of Exn4. Paired t-test, *p < 0.05, ***p < 0.001. G Pie chart of the proportion of ARC GLP-1R-expressing neurons showing RMP changes in the presence of Exn-4. H Represent traces of membrane potentials with action potentials (APs) in the ARC GLP-1R neurons with or without Exn4. I Pooled data. Recordings were conducted in 4 animals. Paired t-test, *p < 0.05. J and K Experimental paradigm for recordings in POMC-neurons. POMC-Cre mice were injected with AAV-DIO-eYFP in the ARC. L Representative traces of APs in POMC neurons in the absence or presence of Exn4. M and N Pooled data. Data are presented as mean + S.E.M., n = 5/2 cells/animals. Paired t-test, *p < 0.05

Fig. 4

Acute activation response of ARC GLP-1R neurons on blood glucose. A Experimental paradigm for virus delivery and chemogenetic stimulation of the GLP-1R neurons in ARC. B Representative image of hM3Dq (red) and c-Fos (white) staining in the GLP-1R-ires-Cre ARC region. C Quantification of c-Fos and GLP-1R co-localized neurons in the ARC after chemogenetic activation (CNO) control n = 4, hM3Dq n = 5. D Average bodyweight of the control and hM3Dq groups after overnight fasting (12 h) control n = 7, hM3Dq n = 8. E Fasting glucose levels control n = 11, hM3Dq n = 12. F IP GTT glucose levels upon ARC GLP-1R neuronal activation, p-value = 0.0633 control n = 11, hM3Dq n = 12. G Insulin levels after 1 h ARC GLP-1R neuron activation via CNO delivery. Data are presented as mean ± standard error of the mean (SEM). Student’s t-test (Bar-graph); *p < 0.05; **p < 0.01, ***p < 0.001. Two-way Anova, with Geisser-Greenhouse correction

Fig. 5

Acute activation response of ARC GLP-1R neuron on feeding behavior. A Experimental paradigm for virus delivery and chemogenetic stimulation of the GLP-1R neurons in ARC. B Food intake consumption upon activation of the GLP-1R neurons in ARC (0.5 g food fed overnight), p value = 0.0261*, control n = 7, hM3Dq n = 8. C Food intake consumption after 24 h CNO injection. D In an exploratory open field test, the dependent locomotor activity of control and hM3Dq mice after CNO delivery, measuring various parameters. E Active count. F Total active time (min). G Total traveled distance (cm). H Time spent in the cage center (min). Data are presented as mean ± standard error of the mean (SEM). Student’s t-test; *p < 0.05; **p < 0.01, ***p < 0.001. Two-way Anova, with Geisser-Greenhouse correction