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Original Article
Diabetes, obesity and metabolism Exendin-4(1-32)K-Capric Acid, a Glucagon-Like Peptide-1 Receptor Agonist, Suppresses Food Intake via Arcuate Pro-Opiomelanocortin Neurons
Keypoint
-Anorexia induced by Ex-4c depends on the presence of POMC neurons.
-Ex-4c activates arcuate POMC neurons largely via the PKA-dependent closure of ATP-sensitive K+ channels.
-Activation of POMC neurons by Ex-4 and GLP-1 involves different ion channel mechanisms compared to Ex-4c.
Sujin Yoo1orcid, Eun-Seon Yoo1, Jae Il Kim2, Jong-Woo Sohn1orcid
Endocrinology and Metabolism 2025;40(3):434-447.
DOI: https://doi.org/10.3803/EnM.2024.2185
Published online: April 14, 2025

1Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Korea

2School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju, Korea

Corresponding author: Jong-Woo Sohn. Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Korea Tel: +82-42-350-2631, Fax: +82-42-350-2610, E-mail: jwsohn@kaist.ac.kr
• Received: September 23, 2024   • Revised: November 18, 2024   • Accepted: December 18, 2024

Copyright © 2025 Korean Endocrine Society

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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  • Background
    Glucagon-like peptide-1 (GLP-1) is an incretin known for its anti-obesity effects, and several effective drugs targeting GLP-1 receptors (GLP-1Rs) have recently been developed to treat obesity. Although GLP-1Rs are expressed by various populations of central neurons, it is still unclear which specific populations mediate the anti-obesity effects of GLP-1R agonists.
  • Methods
    In this study, we utilized the previously reported GLP-1R agonist, exendin-4(1-32)K-capric acid (Ex-4c), and conducted whole-cell patch-clamp recordings, immunohistochemistry experiments, and in vivo food intake measurements.
  • Results
    Our findings indicate that the appetite-suppressing effects of Ex-4c depend on pro-opiomelanocortin (POMC) neurons. Fos immunochemistry experiments and whole-cell patch-clamp recordings showed that Ex-4c activated POMC neurons in the arcuate nucleus of the hypothalamus. Additionally, we observed that Ex-4c stimulated GLP-1Rs and activated the protein kinase A (PKA)-dependent signaling pathway, which in turn closed putative adenosine triphosphate-sensitive K+ (KATP) channels, leading to the depolarization of POMC neurons.
  • Conclusion
    Our results demonstrate that the appetite-suppressing effects of Ex-4c are mediated through the activation of arcuate POMC neurons. Furthermore, the PKA-dependent closure of putative KATP conductance is identified as the cellular mechanism responsible for the activation of POMC neurons.
  • Keywords: Incretins; Hypothalamus; Electrophysiology; Immunohistochemistry; Appetite; Obesity
Glucagon-like peptide-1 (GLP-1) belongs to the incretin family, renowned for its glucose-dependent insulinotropic and appetite-suppressing functions [1]. Recently, GLP-1’s role in appetite regulation has garnered significant attention, leading to the market release of several GLP-1 receptor (GLP-1R) agonists, such as liraglutide and semaglutide, as effective anti-obesity drugs. Although these drugs have proven to be quite successful, the exact mechanisms behind their appetite-suppressing effects remain only incompletely understood.
Multiple brain regions reportedly express functional GLP-1Rs [1], and previous studies have shown that GLP-1R agonists influence the activity of neurons that regulate appetite. For example, exendin-4 (Ex-4), a GLP-1R agonist, increases excitatory synaptic inputs to the anorexigenic corticotropin-releasing hormone (CRH) neurons in the paraventricular nucleus of the hypothalamus (PVH) [2,3]. Ex-4 also activates GLP-1R-expressing neurons in the dorsomedial nucleus of the hypothalamus (DMH) and the dorsolateral septum (dLS) [4,5]. Additionally, a series of studies have shown that liraglutide activates the anorexigenic pro-opiomelanocortin (POMC) neurons while inhibiting the orexigenic neuropeptide Y (NPY)/agouti-related peptide (AgRP) neurons in the arcuate nucleus of the hypothalamus (ARH) [6,7]. A more recent study found that liraglutide activates GLP-1R-expressing neurons in the DMH, which then inhibit NPY/AgRP neurons in the ARH, thereby suppressing food intake [8]. Furthermore, the involvement of GLP-1R-expressing neurons in the dorsal vagal complex (DVC) in the anorexigenic effects of semaglutide has been demonstrated [9]. Therefore, it appears that GLP-1R agonists induce anorexia by acting on multiple brain areas.
Previously, Lee et al. [10] developed a modified version of Ex-4, incorporating various fatty acids into Ex-4(1-33), where Ser33 was replaced by Lys to facilitate acylation. They discovered that exendin-4(1-32)K-capric acid (Ex-4c) demonstrated enhanced effects in reducing blood glucose and body weight. However, the mechanisms by which Ex-4c reduces food intake to lower body weight remain unclear. In this study, we explored the central mechanisms through which Ex-4c suppresses food intake. We found that arcuate POMC neurons are essential for the anorexigenic effects of Ex-4c and that Ex-4c activates these neurons via a protein kinase A (PKA)-dependent inhibition of putative adenosine triphosphate-sensitive K+ (KATP) channels. Our findings offer insights into how GLP-1R agonism may reduce food intake through hypothalamic pathways.
Animals
All experimental procedures were approved by the Institutional Animal Care and Use Committee of the Korea Advanced Institute of Science and Technology (KAIST; Protocol No. KA2021-126). The experiments involved 6- to 12-week-old male mice with a C57BL/6J genetic background. These mice were housed in a temperature-controlled, specific pathogen-free facility at the KAIST Laboratory Animal Resource Center. The housing conditions included a 12-hour light-dark cycle (lights on at 7:00 AM) and ad libitum access to water and standard mouse chow (2018S, Envigo, Indianapolis, IN, USA). For electrophysiology and immunohistochemistry experiments, Pomc-humanized Renilla reniformis green fluorescent protein (hrGFP) mice (#006421, Jackson Laboratory, Bar Harbor, ME, USA) were utilized. In fasting-refeeding experiments, Pomc-Cre mice (#005965, Jackson Laboratory) were crossbred with inducible diphtheria toxin receptor (iDTR) mice (#007900, Jackson Laboratory) to selectively ablate POMC neurons.
Cannulation and intracerebroventricular infusion
Male iDTR and Pomc-Cre::iDTR mice, aged 6 to 12 weeks, were anesthetized using 1%–4% isoflurane inhalation and secured in a stereotaxic frame. Prior to making a small incision, 0.5% lidocaine was applied topically as an anesthetic to the skin. The skull was then carefully drilled, and a guide cannula (C315G/SPC, 26 gauge, Plastics One, Roanoke, VA, USA) was accurately positioned in the right lateral ventricle (coordinates: anterior-posterior –0.6 mm; medial-lateral 1.1 mm; dorsal-ventral –2.0 mm from the bregma). The cannula was securely attached to the skull using jeweler’s screws. To prevent any blockage, a dummy cannula extending 0.5 mm beyond the guide cannula’s tip was installed. The mice were then allowed a recovery period of 7 days. After recovery, the dummy cannula was removed, and an internal cannula (C315I/SPC, 33 gauge, Plastics One) was inserted. This internal cannula was connected to a Hamilton syringe through a polyethylene tube. To verify the accurate placement of the cannula, angiotensin II (ATII; 50 μg/mL, 2 μL) was administered, which triggered water consumption within 10 minutes. After confirming the placement with the ATII test, the internal cannula was removed and replaced with the dummy cannula. The mice underwent a week of acclimation through handling before any measurements were taken.
POMC neuron ablation was performed on 5-week-old iDTR::Pomc-Cre mice by intracerebroventricular (i.c.v.) infusion of 2.5 ng of diphtheria toxin (DT) dissolved in 2.0 µL of saline, followed by a 2-week period to ensure complete cell-lineage ablation [11-13]. DT-injected iDTR mice were used as controls. To evaluate the acute effects of Ex-4c on food intake, Ex-4c was administered i.c.v. at doses of 0.1, 0.3, or 1.0 μg, each dissolved in 2.0 μL of saline. The injections were administered at a rate of 2.0 μL/min. A group receiving only saline (2.0 µL) served as the vehicle control.
Fasting-refeeding
Wild-type, iDTR, or iDTR::Pomc-Cre mice were subjected to an overnight fast (16 hours) before receiving an i.c.v. injection of either saline or Ex-4c at 10:30 AM. Thirty minutes later, the mice were allowed to feed, and both food intake and body weight were recorded at 1, 2, 4, 6, and 24 hours post-refeeding. Before conducting these fasting-refeeding experiments, all mice were housed individually for at least 1 week. The median effective dose (ED50) of Ex-4c for the suppression of food intake was obtained by plotting normalized food intake at 6 hours since refeeding on the Y-axis versus the common logarithm of Ex-4c dose (μg) on the X-axis, and performing curve fitting using the equation shown below:
Y=Min+Max−Min1+10(log⁡ED50−x)× Hill
where Hill=Hill slope, Max=maximum asymptote, and Min=minimum asymptote.
Tissue processing and immunohistochemistry
Isoflurane-anesthetized mice were perfused with phosphate-buffered saline (PBS), followed by cold 4% paraformaldehyde (PFA, 158127, Merck, Rahway, NJ, USA) in PBS. The extracted brain was stored in PFA overnight at 4°C and then dehydrated by incubating in a 30% sucrose solution for 48 hours. Subsequently, the brain was embedded in the Tissue-Tek® O.C.T. (Sakura Finetek USA, Torrance, CA, USA) and cryo-sectioned into 30 µm coronal slices using a sliding microtome (CM 1860, Leica, Wetzlar, Germany). Sections containing ARH were washed with PBS three times for 5 minutes each and treated with a blocking solution (3.0% goat serum, 0.3% triton-X, and filtered PBS) for 1 hour at room temperature. The sections were then incubated at room temperature for 24 hours with a rabbit anti-beta endorphin antibody (H-022-33, Phoenix Pharmaceuticals, Burlingame, CA, USA) diluted to 1:1,000 in the blocking solution. For Fos immunostaining, anti-Fos antibody (ab190289, Abcam, Cambridge, UK) was diluted to 1:1,000 in the blocking solution. After washing the slices with PBS three times, we incubated them with Alexa Fluor 647 goat anti-rabbit secondary antibody (A-21245, Thermo Fisher Scientific, Waltham, MA, USA), diluted 1:500 in the blocking solution, for 2 hours at room temperature. The sections were washed three times for 10 minutes each and mounted on slides using Vectashield antifade mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) (FV-93952-24, Vector Laboratories, Newark, CA, USA). Images of arcuate POMC neurons were acquired with a confocal microscope (LSM 780, Zeiss, Oberkochen, Germany), and analyzed using ZEN and ImageJ software (National Institutes of Health, Bethesda, MD, USA). The area of interest (AOI) was defined based on the location of ARH in the brain atlas (bregma –1.46 to –2.18 mm) and the number of cells in the AOI was counted manually.
Electrophysiology
Mice were deeply anesthetized using isoflurane inhalation and perfused with a modified artificial cerebrospinal fluid (ACSF), in which NaCl was replaced by equiosmolar concentrations of sucrose. Following decapitation, the brain was removed, and the block containing the ARH was submerged in ice-cold ACSF. It was then sectioned into 250 μm coronal slices using a vibrating microtome (VT1200S, Leica). The slices were incubated for at least 1 hour at 34°C in ACSF composed of 126 mM NaCl, 2.8 mM KCl, 26 mM NaHCO3, 1.25 mM NaH2PO4, 1.2 mM MgSO4, 2.5 mM CaCl2, and 5 mM glucose (pH 7.3), while being continuously bubbled with 95% O2 and 5% CO2. Subsequently, the slices were transferred to a recording chamber where they were maintained in a continuous flow (approximately 2 mL/min) of oxygenated ACSF at temperatures ranging from 32°C to 34°C. Epifluorescence was initially used to locate fluorescent cells; thereafter, the light source was switched to infrared differential interference contrast imaging for whole-cell patch-clamp recordings (Nikon Eclipse FN1 equipped with a fixed stage and an optiMOS scientific CMOS camera, Nikon, Tokyo, Japan). Electrophysiological signals were captured using an Axopatch 700B amplifier (Molecular Devices, San Jose, CA, USA) and subsequently analyzed offline on a PC using pCLAMP software (Clampfit 10.4, Molecular Devices).
The recording electrodes had a resistance of 3 to 5 MΩ when filled with pipette solutions containing 120 mM K-gluconate, 10 mM KCl, 10 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), 1 mM CaCl2, 1 mM MgCl2, 5 mM ethylene glycol-bis(2-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), 2 mM Mg-ATP, and 0.03 mM Alexa Fluor 488 hydrazide dye. The baseline membrane potential was determined as a stable voltage maintained for at least 3 minutes. To assess input resistance, we measured the voltage deflection amplitudes at the end of responses to small hyperpolarizing rectangular current pulse steps, lasting 500 ms and ranging from 0 to –50 pA. We considered the slope of the linear fit in the voltage (V)-current (I) relationship as the input resistance. For voltage-clamp experiments, we replaced K+ with equimolar concentrations of Cs+. We recorded excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) at holding potentials of –60 and –10 mV, respectively. We only analyzed traces when the series resistance was consistently maintained below 20 MΩ throughout the recording session. A neuron was classified as depolarized or hyperpolarized if its membrane potential shifted by at least 2 mV in amplitude. It is important to note that membrane potential values were not compensated for the junction potential of –8 mV.
Drugs
H-89 (2910), KT5702 (1288), forskolin (1099), 8-Br-cyclic adenosine monophosphate (cAMP; 1140), XE991 (2000), and linopirdine (1999) were obtained from Tocris Bioscience (Bristol, UK). ATII (A9525), kynurenic acid (K3375), picrotoxin (P1675), and tolbutamide (T0891) were obtained from Sigma Aldrich (St. Louis, MO, USA). Tetrodotoxin (TTX; T-550) and tertiapin-Q (STT-170) were sourced from Alomone Labs (Jerusalem, Israel). Ex(9-39)amide (Ex(9-39); AGP-8494), Ex-4 (AGP-8090), and GLP-1 (AGP-8047) were procured from Anygen (Gwangju, Korea). All stock solutions were prepared by dissolving them in de-ionized water, except for tolbutamide, which was dissolved in ethanol.
Statistical analysis
All statistical analyses were performed using GraphPad Prism version 10 (GraphPad Software Inc., San Diego, CA, USA). All data were presented as mean±standard error of the mean, with n representing the number of animals or cells included for analyses. The statistical significance of differences between groups was evaluated using the unpaired t test, paired t test, or two-way analysis of variance with the Tukey honestly significant difference (HSD) test. Statistical significance was defined at P<0.05, P<0.01, P<0.001, or P<0.0001.
POMC neurons are required for the anorexigenic effects of Ex-4c
Given the body weight-lowering effects of Ex-4c treatments [10], we tested whether Ex-4c could suppress food intake. We fasted wild-type mice for 18 hours and administered i.c.v. injections of either saline or three different doses (0.1, 0.3, and 1 μg) of Ex-4c (n=8) (Fig. 1A, B). We measured food intake for 6 hours after refeeding and found that Ex-4c significantly reduced food intake at 0.3 and 1 μg (Fig. 1A), which was also accompanied by decreased body weight gain after refeeding (Fig. 1B). The median ED50 of Ex-4c for suppression of food intake was 0.32 μg at 6 hours after refeeding.
Since arcuate POMC neurons have been shown to be activated by GLP-1R agonists [6,7,14], we used the iDTR mouse model to selectively ablate POMC neurons [11-13]. We performed i.c.v. injections of DT (2.5 ng) into iDTR::Pomc-Cre mice, while using DT-injected iDTR mice as controls. Two weeks after DT injections, the body weights of iDTR mice and iDTR::Pomc-Cre mice were 24.2±2.2 g (n=11) and 25.6±1.4 g (n=8), respectively. We counted the number of POMC neurons as determined by immunohistochemistry for β-endorphin (β-end) in the ARH (Fig. 1C). The average number of β-end (+) cells from eight coronal sections (between bregma –1.22 and –2.42 mm, 30 μm thickness) per mouse was 699±35 (n=4) in iDTR mice (Fig. 1D). In contrast, the average number of β-end (+) cells was significantly lower, at 242±10 (n=3), in iDTR::Pomc-Cre mice (Fig. 1E), confirming the successful ablation of POMC neurons. Indeed, we found that i.c.v. injections of Ex-4c (1 μg) significantly suppressed the food intake of iDTR mice (Fig. 1E), which is consistent with the results shown in Fig. 1A, B. As expected, the anorexigenic effects of Ex-4c were not observed in iDTR:: Pomc-Cre mice (Fig. 1F). Taken together, these results suggest that the anorexigenic effects of Ex-4c require POMC neurons.
Ex-4c stimulates GLP-1Rs to directly activate arcuate POMC neurons
While POMC neurons are present in both the nucleus tractus solitarius (NTS) and the ARH, previous studies have shown that NTS POMC neurons do not express GLP-1Rs [15]. Consequently, our research focused on the effects of Ex-4c on arcuate POMC neurons. We administered i.c.v. injections of either saline or Ex-4c (1 μg) to Pomc-hrGFP mice and sacrificed them after 2 hours to assess Fos expression, a marker of increased neuronal activity. We observed that, compared to the saline-injected group, mice injected with Ex-4c exhibited a significant increase in Fos expression by POMC neurons within the ARH (Fig. 2A). We counted the number of ARH neurons from seven hypothalamic sections in the rostrocaudal axis between bregma –1.46 and –2.18 mm and found that the number of hrGFP (+) neurons was comparable between the two groups (1,163.7±16.2, n=3, for saline injections; and 1,246.7±47.8, n=3, for Ex-4c injections, P=0.1755, unpaired t test; Fig. 2B). These results suggest that the number of POMC neurons is not affected by Ex-4c treatments. We noted a tendency toward an increased number of Fos (+) neurons in the ARH (283.0±67.8, n=3, for saline injections; and 510.0±76.1, n=3, for Ex-4c injections; Fig. 2C); however, this difference was not statistically significant (P=0.090, unpaired t test). Importantly, the number of Fosexpressing POMC neurons was 22.3±7.8 (n=3) for saline injections and 161.0±33.7 (n=3) for Ex-4c injections (P=0.016, unpaired t test; Fig. 2D). Taken together, these results suggest that arcuate POMC neurons constitute the major neuronal population activated by Ex-4c, although other ARH neuron populations may also be activated.
Subsequently, we used Pomc-hrGFP mice to perform whole-cell patch-clamp recordings of fluorescently labeled POMC neurons from acute hypothalamic slices that contained areas of the ARH (Fig. 3A). We applied Ex-4c (100 nM) to bath solutions and found that 17 of 44 cells (38.6%) are depolarized by 4.6±0.6 mV (Fig. 3B, G). One cell (2.3%) was hyperpolarized by –3.5 mV, while the remaining 26 cells (59.1%) did not respond (0.3±0.2 mV) to Ex-4c application (Table 1, Fig. 3G). We noted a significant increase in the action potential (AP) firing frequency in response to Ex-4c (from 2.8±0.3 to 3.6±0.4 Hz, n=44, P=0.002, paired t test), which further supported the excitatory effects of Ex-4c on arcuate POMC neurons (Supplemental Fig. S1A). We also confirmed that acute effects of Ex-4c were not observed (0.2±0.3 mV, n=8) by the pretreatment of slices with 100 nM Ex(9-39), a GLP-1R blocker (Fig. 3C, G). Subsequently, we performed the same series of experiments in the presence of TTX (0.5 μM) and a blend of fast synaptic blockers (1 mM kynurenic acid and 50 μM picrotoxin). In this condition, Ex-4c still depolarized eight of 15 cells (53.3%) by 3.7±0.5 mV (n=8) (Fig. 3D, G), suggesting a postsynaptic location of Ex-4c action. We also tested the effects of Ex-4c on spontaneous EPSC (sEPSC) and spontaneous IPSC (sIPSC) and found that the frequency and mean amplitude of sEPSC and sIPSC are not affected by Ex-4 (Supplemental Fig. S2). Together, these results suggest that Ex-4c directly activates arcuate POMC neurons via the stimulation of postsynaptic GLP-1Rs.
To examine the ion channel mechanisms for the depolarizing responses, we applied small hyperpolarizing current steps during the recording (arrows in Fig. 3D, from –50 to 0 pA, 500 ms) (Fig. 3E). We found that all depolarizing responses were accompanied by increased input resistance (17.6%±4.4%, from 1.13±0.18 to 1.30±0.18 GΩ, n=8, green dots in Fig. 3G), and the reversal potential (Erev) was –89.0±8.2 mV (n=8) (Fig. 3F). Since this value was close to the calculated equilibrium potential of K+ (EK=–99.7 mV), these results suggest that a reduction of putative potassium conductance is responsible for the depolarizing responses. We also performed the same series of experiments in the absence of TTX and synaptic blockers. In this condition, 12 of 17 (70.6%) depolarizing responses were accompanied by increased input resistance (26.5%±9.4%, from 1.17±0.10 to 1.46±0.14 GΩ, n=12, green dots in Fig. 3G) and Erev was –89.0±5.4 mV (n=12). These results are consistent with the observations in the presence of TTX and synaptic blockers (Fig. 3E, F). However, we also noted decreases in input resistance (–21.7%±1.8%, from 1.48±0.21 to 1.17±0.17 GΩ, n=5, blue dots in Fig. 3G) in five of 17 cells (29.4%) that were depolarized by Ex-4c, where Erev was calculated to be –23.7±3.0 mV (n=5) (Table 1). These results suggest that putative non-selective cation (NSC) conductance may also underlie the depolarizing responses to Ex-4c application in a minor population of POMC neurons.
PKA-dependent signaling mechanisms underlie acute effects of Ex-4c
GLP-1Rs typically signal via the cAMP-PKA pathway [16]. To examine whether activation of this signaling pathway may mimic the acute effects of Ex-4c, we applied 10 μM forskolin (an adenylate cyclase activator) or 500 μM 8-Br-cAMP (a PKA activator), both of which activate the PKA signaling pathway, to the bath solutions. We found that forskolin depolarized four of eight cells (50%) by 4.9±0.8 mV (n=4) (Fig. 4A), and input resistance was increased by forskolin (15.1%±8.2%, from 1.27±0.22 to 1.45±0.22 GΩ, Erev=–105.9±19.2 mV) in three cells (green dots in Fig. 4E) and decreased (–17.4%, from 1.03 to 0.85 GΩ, Erev=–14.5 mV) in one cell (blue dots in Fig. 4E). 8-Br-cAMP depolarized three of eight cells (37.5%) by 6.1±1.4 mV (n=3) (Fig. 4B) and input resistance was increased by 8-Br-cAMP (13.5%±5.8%, from 1.32±0.04 to 1.50±0.10 GΩ, Erev=–101.1±19.3 mV) in all three cells (green dots in Fig. 4E). Therefore, activating the PKA signaling pathway depolarizes the membrane potential either by the closure of putative K+ conductance (increased input resistance) or by the opening of putative NSC conductance (decreased input resistance), mirroring the acute effects of Ex-4c.
Subsequently, we pretreated the slices with PKA inhibitors (1 μM H-89 or 100 nM KT5720) for 5 minutes prior to the applications of Ex-4c. We found that in the presence of H-89 membrane potential of POMC neurons was not changed in response to Ex-4c (0.3±0.3 mV, n=11) (Fig. 4C, E). In addition, only one of 15 cells (6.7%) was depolarized by 2.7 mV in response to Ex-4c (green dots in Fig. 4E) while the remaining 14 cells (93.3%) remained non-responsive to Ex-4c (0.6±0.2 mV, n=14) (Fig. 4D, E). Together, these results suggest that the acute effects of Ex-4c are mediated by the PKA-dependent signaling pathway.
Acute effects of Ex-4c are largely mediated by the closure of KATP channels
It has previously been shown that the M-type K+ channels and the G protein-gated inwardly rectifying K+ (GIRK) channels contribute to maintaining resting membrane potential (RMP) of POMC neurons [17,18]. Therefore, we tested the possibility that Ex-4c inhibits these K+ channels to activate POMC neurons. We treated the slices with M-type K+ channel blockers (10 μM linopirdine+10 μM XE991) prior to the application of Ex-4c, and we found that treatments by linopirdine and XE991 depolarized membrane potential in three of six cells (50%) by 3.9±1.7 mV (from –43.9±5.3 to –40.0±7.0 mV, n=3) (Fig. 5A). These results suggest that M-type K+ channels maintain the RMP of POMC neurons. Importantly, M-type K+ channel blockers increased input resistance by 21.1%±2.1% (from 1.12±0.10 to 1.37±0.14 GΩ, n=3) with a calculated Erev of –74.0±3.7 mV (n=3), which suggested a closure of putative K+ conductance. Subsequent applications of Ex-4c further depolarized the membrane potential in three of six cells (50%) by 5.1±0.8 mV (from –43.6±3.5 to –38.5±4.2 mV, n=3) (Fig. 5B, E), and input resistance increased in two cells (from 1.59 to 1.70 GΩ, Erev=–87.4 mV; and from 1.10 to 1.22 GΩ, Erev=–100.5 mV, green dots in Fig. 5E) and decreased in one cell (from 1.25 to 1.03 GΩ, Erev=–23.5 mV, blue dots in Fig. 5E). When we treated the slices with 100 nM tertiapin-Q, a GIRK channel blocker, membrane potential was depolarized by 2.9±0.3 mV (from –52.4±2.1 to –49.5±1.9 mV, n=4) in four of seven POMC neurons (50%) (Fig. 5A). Again, input resistance increased by 32.7%±4.4% (n=4) from 0.99±0.19 to 1.32±0.28 GΩ (n=4) and the calculated Erev was –72.1±2.8 mV (n=4), which suggested a contribution of GIRK channels to RMP of POMC neurons. Subsequent applications of Ex-4c resulted in depolarization of membrane potential by 4.3±0.7 mV (from –50.1±1.7 to –45.8±1.5 mV, n=4) in four of seven cells (57.1%) regardless of their responses to tertiapin-Q (Fig. 5C, E). Input resistance increased by 24.0%±12.2% in three cells (from 1.05±0.18 to 1.26±0.19 GΩ, Erev=–84.8±4.4 mV, green dots in Fig. 5E) and decreased in one cell (from 2.07 to 1.72 GΩ, Erev=–32.4 mV, blue dot in Fig. 5E). Taken together, the depolarizing responses by Ex-4c are not likely to involve the closure of M-type K+ channels or GIRK channels.
KATP channels reportedly maintain the RMP of arcuate POMC neurons [19]. To test the possibility that Ex-4c may inhibit KATP channels to activate POMC neurons, we pretreated the slices with 200 μM tolbutamide, a KATP channel blocker, and found that five of 12 cells (41.7%) were depolarized by 3.9±0.5 mV (from –48.0±1.7 to –44.1±1.4 mV, n=5) (Fig. 5A). The input resistance increased by 26.7%±11.4% (n=5) from 0.94±0.04 GΩ (n=5) to 1.20±0.14 GΩ (n=5), and the calculated Erev was –73.3±4.6 mV (n=5). These results confirmed the contribution of KATP channels to the RMP of POMC neurons. Interestingly, subsequent applications of Ex-4c did not affect the membrane potential of POMC neurons (0.4±0.3 mV, from –46.1±1.9 to –45.6±1.9 mV, n=12) (Fig. 5D, E). These results support the hypothesis that the closure of KATP channels underlies the activation of arcuate POMC neurons by Ex-4c.
Acute effects of Ex-4 and GLP-1
Since Ex-4c is derived from Ex-4 and GLP-1 is an endogenous peptide agonist, we wanted to compare the effects of these peptides to those of Ex-4c. Bath applications of 10 μM Ex-4 resulted in depolarization of the membrane potential by 5.0±0.7 mV (from –48.0±2.2 to –43.0±1.8 mV, n=6) in six of 21 cells tested (28.5%) (Table 1, Fig. 6A, C). Input resistance increased in two cells by Ex-4 (from 3.73 to 4.12 GΩ, Erev=–82.8 mV; and from 0.76 to 0.84 GΩ, Erev=–106.8 mV, green dots in Fig. 6C) (Table 1). The input resistance of the other four cells decreased from 1.72±0.44 GΩ (n=4) to 1.17±0.31 GΩ (n=4), Erev=–25.7±6.6 mV (n=4) (blue dots in Fig. 6C). These results show that Ex-4 activates arcuate POMC neurons preferentially by opening putative NSC conductance. One cell (4.8%) was hyperpolarized by –17.0 mV (from –49.0 to –66.0 mV, red dot in Fig. 6C). This hyperpolarizing effect was associated with a decreased input resistance (from 2.75 to 1.01 GΩ) and an Erev of –75.1 mV, suggesting the opening of putative K+ conductance. Indeed, subsequent treatment of this cell with 200 μM tolbutamide completely reversed the membrane potential and input resistance to baseline values (–48.0 mV and 2.66 GΩ, respectively). The remaining 14 cells (66.7%) did not respond to Ex-4 (0.1±0.3 mV, n=14). The average AP firing frequency was increased by Ex-4 (from 3.7±0.8 to 4.2±0.8 Hz, n=21), but this increase was not statistically significant (P=0.282, paired t test) (Supplemental Fig. S1B).
Subsequently, we applied 10 μM GLP-1 to the bath solutions and found that 18 of 32 cells (56.2%) were depolarized by 4.5±0.5 mV (from –47.0±0.8 to –42.5±0.8 mV, n=18) (Table 1, Fig. 6B, C). Interestingly, input resistance increased in nine cells (from 1.29±0.08 to 1.46±0.10 GΩ with an Erev of –103.0±8.8 mV, green dots in Fig. 6C) and decreased in the other nine cells (from 1.59±0.15 to 1.23±0.10 GΩ with an Erev of –23.3±1.5 mV, blue dots in Fig. 6C) (Table 1). These results suggest that GLP-1 either closes putative K+ conductance or opens putative NSC conductance with equal probability to activate arcuate POMC neurons. The remaining 14 cells (43.8%) did not respond to GLP-1 (0.1±0.3 mV, n=14). Finally, we noted a significant increase in the AP firing frequency in response to GLP-1 (from 3.0±0.5 to 4.9±0.5 Hz, n=32, P=0.0004, paired t test) (Supplemental Fig. S1C).
In this study, we investigated the ability of Ex-4c, a modified version of Ex-4, to suppress food intake in mice. We provide evidence that arcuate POMC neurons are essential for the anorexigenic effects of Ex-4c (Fig. 1). Our experiments demonstrated that Ex-4c directly activated arcuate POMC neurons via the GLP-1R/PKA pathway and that the closure of KATP channels largely mediated the acute effects of Ex-4c (Figs. 2-5). Finally, we compared the ion channel mechanisms by which different GLP-1R agonists activated arcuate POMC neurons (Fig. 6). Our study provides insights into how GLP-1R agonists suppress food intake. However, we did not directly test the effects of Ex-4c on the activity of POMC neurons that reside in the NTS. Therefore, it is possible that Ex-4c may also work via NTS POMC neurons, which needs to be tested in future studies.
GLP-1R agonists such as liraglutide (Saxenda, Novo Nordisk, Plainsboro, NJ, USA) and semaglutide (Wegovy, Novo Nordisk) are undoubtedly among the most successful appetite suppressants. Importantly, previous studies have shown that both drugs activate arcuate POMC neurons to suppress food intake [6,7,14]. It has been demonstrated that the activation of arcuate POMC neurons by liraglutide involves the opening of the transient receptor potential classical 5 (TRPC5) channel, a key member of the NSC channel family [6]. Additionally, liraglutide indirectly activates POMC neurons by enhancing excitatory synaptic inputs to these neurons [6]. However, the opening of putative NSC conductance only explains the acute effects of Ex-4c in a minor population of arcuate POMC neurons, while the closure of KATP channels largely accounts for the activation of arcuate POMC neurons by Ex-4c (Fig. 5). This discrepancy may stem from the use of different GLP-1R agonists (liraglutide vs. Ex-4c) in this and previous studies. Supporting this idea, we observed different ion conductance values involved in the acute effects of Ex-4 and GLP-1 (Fig. 6). Thus, it appears that different GLP-1R agonists activate arcuate POMC neurons through distinct ion channels, as previously discussed in the context of biased agonism among different melanocortin-4 receptor agonists [20]. In this study, we also demonstrated that the cAMP/PKA-dependent signaling pathway mediates the acute effects of Ex-4c on arcuate POMC neurons (Figs. 3, 4), suggesting the involvement of Gs proteins. Since GLP-1Rs may also signal via Gq or Gi/o, as well as through G protein-independent mechanisms [21-26], further research is needed to determine which signaling molecules other GLP-1R agonists might recruit to activate POMC neurons due to biased agonism.
In this study, we demonstrated the critical role of POMC neurons in mediating the anorexigenic effects of Ex-4c (Fig. 1). Recent studies have highlighted the significance of GLP-1R-expressing neurons within the DMH and DVC in the anorexigenic effects of liraglutide [8,9]. Interestingly, inhibiting GLP-1R-expressing neurons within the ARH did not alter the suppression of food intake induced by liraglutide [9]. These findings indicate that different GLP-1R agonists may induce anorexia through distinct central mechanisms. One possible explanation for these differences is that GLP-1Rs expressed in neurons across various brain areas may have varying affinities for specific GLP-1R agonists. Thus, while liraglutide might activate arcuate POMC neurons in ex vivo preparations, it could preferentially bind to DVC rather than ARH neurons in vivo. Since POMC neurons in the NTS do not express GLP-1Rs [15], Ex-4c administered in vivo might bind and activate arcuate POMC neurons, leading to anorexia. As previously noted, Ex-4 is known to activate CRH neurons in the PVH and GLP-1R neurons in the DMH and dLS [2-5]. It remains unclear which neurons express the GLP-1Rs necessary for the anorexigenic effects of Ex-4 in vivo; however, the responsible brain areas may differ from those implicated in the effects of Ex-4c and liraglutide. Thus, we hypothesize that different GLP-1R agonists may interact with GLP-1Rs expressed by distinct neuronal populations, thereby inducing anorexia. The involvement of unique neuronal circuitry in the anorexigenic effects of each GLP-1R agonist could partly explain the variations in drug efficacy.
Pharmaceutical companies are currently developing numerous appetite suppressants that act through GLP-1R agonism. For instance, tirzepatide (Zepbound, Eli Lilly, Indianapolis, IN, USA) is the first injectable dual agonist targeting both the GLP-1R and the glucose-dependent insulinotropic peptide receptor (GIPR). This drug has been shown to cause significant body weight loss and is already in clinical use [27]. Another promising weight loss medication, retatrutide, acts as a triple agonist on GLP-1R, GIPR, and the glucagon receptor, and is poised for market release soon [28]. Additionally, a recent animal study demonstrated that combining the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 with GLP-1 effectively suppresses food intake and reduces body weight [29]. However, the exact mechanism of action of these complex drugs remains to be identified. As more drugs targeting GLP-1Rs are being used and developed, it is increasingly important to understand the neurobiology of anorexia by GLP-1R agonists.

Supplemental Fig. S1.

Acute effects of glucagon-like peptide-1 receptor (GLP-1R) agonists on the action potential (AP) firing frequency of arcuate pro-opiomelanocortin (POMC) neurons. (A-C) Dots and lines summarize the changes of AP firing frequency in response to (A) exendin-4(1-32)K-capric acid (Ex-4c), (B) exendin-4 (Ex-4), and (C) GLP-1. ACSF, artificial cerebrospinal fluid; NS, not significant, aP<0.01, bP<0.001, unpaired t test.
enm-2024-2185-Supplemental-Fig-S1.pdf

Supplemental Fig. S2.

Acute effects of exendin-4(1-32)K-capric acid (Ex-4c) on spontaneous excitatory postsynaptic current (sEPSC) and spontaneous inhibitory postsynaptic current (sIPSC) recorded in arcuate pro-opiomelanocortin (POMC) neurons. (A) Traces demonstrate voltage-clamp recordings of sEPSC in the absence (upper) and the presence (lower) of Ex-4c at a holding potential (HP) of –60 mV. (B) Dots and lines summarize the changes of sEPSC frequency (left) and mean amplitude (right) in response to Ex-4c. (C) Traces demonstrate voltage-clamp recordings of sIPSC in the absence (upper) and the presence (lower) of Ex-4c at a HP of –10 mV. (D) Dots and lines summarize the changes of sIPSC frequency (left) and mean amplitude (right) in response to Ex-4c. Paired t test. ACSF, artificial cerebrospinal fluid; NS, not significant.
enm-2024-2185-Supplemental-Fig-S2.pdf

CONFLICTS OF INTEREST

No potential conflict of interest relevant to this article was reported.

ACKNOWLEDGMENTS

This work was supported by the National Research Foundation of Korea (RS-2020-NR046282 and RS-2022-NR070560 to Jong-Woo Sohn) funded by the Korean Ministry of Science and ICT.

AUTHOR CONTRIBUTIONS

Conception or design: S.Y., J.W.S. Acquisition, analysis, or interpretation of data: S.Y., E.S.Y., J.I.K., J.W.S. Drafting the work or revising: S.Y., J.W.S. Final approval of the manuscript: S.Y., E.S.Y., J.I.K., J.W.S.

Fig. 1.
Exendin-4(1-32)K-capric acid (Ex-4c) suppresses food intake via pro-opiomelanocortin (POMC) neurons. (A, B) Effects of intracerebroventricular Ex-4c (0.1, 0.3, and 1.0 μg) vs. saline on food intake (A) and body weight (B) of wild-type mice (n=8). Data are presented as mean±standard error of the mean. Two-way analysis of variance (ANOVA) with Tukey honestly significant difference (HSD) test. (C) Immunofluorescence of β-endorphin (β-end, green) and 4′,6-diamidino-2-phenylindole (DAPI; blue) in the arcuate nucleus of the hypothalamus of diphtheria toxin (DT)-injected inducible diphtheria toxin receptor (iDTR) mice (left) and DT-injected iDTR::Pomc-Cre mice (right). Scale bar=200 μm. (D) Bar graphs and dots summarize the number of β-end (+) cells from DT-injected iDTR mice (black, n=4) and DT-injected iDTR::Pomc-Cre mice (red, n=3). Unpaired t test. (E) Normalized food intake of DT-injected iDTR mice (n=11) after saline (empty circle) and Ex-4c (1.0 μg, filled circle) injections. Two-way ANOVA with Tukey’s HSD test. (F) Normalized food intake of DT-injected iDTR::Pomc-Cre (n=8) after saline (empty triangle) and Ex-4c (1.0 μg, filled triangle) injections. aP<0.05, bP<0.01, and cP<0.0001 (saline vs. 1.0 μg Ex-4c); dP<0.01 (saline vs. 0.3 μg Ex-4c); eP<0.0001; fP<0.01; gP<0.001.
enm-2024-2185f1.jpg
Fig. 2.
Exendin-4(1-32)K-capric acid (Ex-4c) induces Fos expression in arcuate pro-opiomelanocortin (POMC) neurons. (A) Confocal images demonstrate humanized Renilla reniformis green fluorescent protein (hrGFP; green), Fos immunoreactivity (red), and 4′,6-diamidino2-phenylindole (DAPI; blue) in arcuate nucleus of the hypothalamus sections obtained from Pomc-hrGFP mice that received intracerebroventricular (i.c.v.) injections of either saline (top row) or 1.0 μg Ex-4c (bottom row). Scale bar=100 μm. (B-D) Bar graphs summarize number of cells that are positive for (B) hrGFP, (C) Fos, and (D) both hrGFP and Fos. NS, not significant. aP<0.05, unpaired t test.
enm-2024-2185f2.jpg
Fig. 3.
Exendin-4(1-32)K-capric acid (Ex-4c) directly activates pro-opiomelanocortin (POMC) neurons via the stimulation of glucagon-like peptide-1 receptors (GLP-1Rs). (A) Bright-field, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), and merged images (from left to right) of a targeted neuron. Scale bar=10 μm. (B) Membrane potential is depolarized in response to Ex-4c (100 nM) treatment. The dashed line indicates baseline membrane potential. Scale bar=1 minute (horizontal), 20 mV (vertical). (C) Ex-4c had no effect on membrane potential when the cell was pretreated with 100 nM Ex(9-39). The dashed line indicates baseline membrane potential. Scale bar=1 minute (horizontal), 20 mV (vertical). (D) Membrane potential was depolarized in response to Ex-4c (100 nM) treatment in the presence of tetrodotoxin (TTX; 500 nM) and synaptic blocker (SB; 1 mM kynurenic acid+50 μM picrotoxin). The dashed line indicates baseline membrane potential. Scale bar=1 minute (horizontal), 20 mV (vertical). (E) Voltage responses to hyperpolarizing current steps from –50 to 0 pA, applied as indicated by arrows in (D). Scale bar=0.5 second (horizontal), 20 mV (vertical). (F) The voltage-current relationship demonstrates increases in input resistance in response to Ex-4c. (G) Bar graphs and dots summarize the acute effects of Ex-4c. Green dots represent depolarized cells with increased input resistance, while blue dots denote depolarized cells with decreased input resistance. Red dots correspond to hyperpolarized cells. Empty dots indicate cells with no response.
enm-2024-2185f3.jpg
Fig. 4.
Exendin-4(1-32)K-capric acid (Ex-4c) activates pro-opiomelanocortin (POMC) neurons via a protein kinase A-dependent signaling pathway. (A, B) The membrane potential was depolarized in response to (A) 10 μM forskolin or (B) 500 μM 8-Br-cyclic adenosine monophosphate (cAMP) treatment. The dashed line indicates baseline membrane potential. Scale bar=1 minute (horizontal), 20 mV (vertical). (C, D) Ex-4c had no effect on membrane potential when the cell was pretreated with (C) 1 μM H-89 or (D) 100 nM KT5720. The dashed line indicates baseline membrane potential. Scale bar=1 minute (horizontal), 20 mV (vertical). (E) Bar graphs and dots summarize the acute effects of forskolin, 8-Br-cAMP, and Ex-4c. Green dots represent depolarized cells with increased input resistance, while blue dots denote depolarized cells with decreased input resistance. Red dots correspond to hyperpolarized cells. Empty dots indicate cells with no response.
enm-2024-2185f4.jpg
Fig. 5.
Exendin-4(1-32)K-capric acid (Ex-4c) activates pro-opiomelanocortin (POMC) neurons via the closure of adenosine triphosphatesensitive K+ (KATP) channels. (A) Bar graphs and dots summarize the acute effects of K+ channel blockers. Green dots indicate depolarized cells with increased input resistance. Empty dots indicate cells with no response. (B-D) Traces demonstrate acute effects of Ex-4c on membrane potential when the cell was pretreated with (B) 10 μM linopirdine+10 μM XE991, (C) 100 nM tertiapin-Q, or (D) 200 μM tolbutamide. The dashed line indicates baseline membrane potential. Scale bar=1 minute (horizontal), 20 mV (vertical). (E) Bar graphs and dots summarize the acute effects of Ex-4c. Green dots represent depolarized cells with increased input resistance, while blue dots denote depolarized cells with decreased input resistance. Empty dots indicate cells with no response.
enm-2024-2185f5.jpg
Fig. 6.
Acute effects of glucagon-like peptide-1 receptor (GLP-1R) agonists on arcuate pro-opiomelanocortin (POMC) neurons. (A, B) Traces demonstrate the acute effects of (A) exendin-4 (Ex-4) or (B) GLP-1 on membrane potential. The dashed line indicates the baseline membrane potential. Scale bar=1 minute (horizontal), 20 mV (vertical). (C) Bar graphs and dots summarize the acute effects of exendin-4(1-32)K-capric acid (Ex-4c), Ex-4, and GLP-1. Green dots represent depolarized cells with increased input resistance, while blue dots denote depolarized cells with decreased input resistance. Empty dots indicate cells with no response.
enm-2024-2185f6.jpg
enm-2024-2185f7.jpg
Table 1.
Acute Effects of GLP-1R Agonists on POMC Neuron Activity
Acute responses to agonists Ex-4c Ex-4 GLP-1
Depolarized
 ↑ Input resistance 12 (27.3) 2 (9.5) 9 (28.1)
Erev =–89.0±5.4 mV Erev =–82.8 mV & –106.8 mV Erev =–103.0±8.8 mV
 ↓ Input resistance 5 (11.3) 4 (19.0) 9 (28.1)
Erev =–23.7±3.0 mV Erev =–25.7±6.6 mV Erev =–23.3±1.5 mV
Hyperpolarized 1 (2.3) 1 (4.8) 0
Erev =–111.6 mV Erev =–75.1 mV
No response 26 (59.1) 14 (66.7) 14 (43.8)
Total 44 (100) 21 (100) 32 (100)

Values are expressed as number (%) or mean±standard error of the mean.

GLP-1R, glucagon-like peptide-1 receptor; POMC, pro-opiomelanocortin; Ex-4c, exendin-4(1-32)K-capric acid; Ex-4, exendin-4; Erev, reversal potential.

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    Exendin-4(1-32)K-Capric Acid, a Glucagon-Like Peptide-1 Receptor Agonist, Suppresses Food Intake via Arcuate Pro-Opiomelanocortin Neurons
    Image Image Image Image Image Image Image
    Fig. 1. Exendin-4(1-32)K-capric acid (Ex-4c) suppresses food intake via pro-opiomelanocortin (POMC) neurons. (A, B) Effects of intracerebroventricular Ex-4c (0.1, 0.3, and 1.0 μg) vs. saline on food intake (A) and body weight (B) of wild-type mice (n=8). Data are presented as mean±standard error of the mean. Two-way analysis of variance (ANOVA) with Tukey honestly significant difference (HSD) test. (C) Immunofluorescence of β-endorphin (β-end, green) and 4′,6-diamidino-2-phenylindole (DAPI; blue) in the arcuate nucleus of the hypothalamus of diphtheria toxin (DT)-injected inducible diphtheria toxin receptor (iDTR) mice (left) and DT-injected iDTR::Pomc-Cre mice (right). Scale bar=200 μm. (D) Bar graphs and dots summarize the number of β-end (+) cells from DT-injected iDTR mice (black, n=4) and DT-injected iDTR::Pomc-Cre mice (red, n=3). Unpaired t test. (E) Normalized food intake of DT-injected iDTR mice (n=11) after saline (empty circle) and Ex-4c (1.0 μg, filled circle) injections. Two-way ANOVA with Tukey’s HSD test. (F) Normalized food intake of DT-injected iDTR::Pomc-Cre (n=8) after saline (empty triangle) and Ex-4c (1.0 μg, filled triangle) injections. aP<0.05, bP<0.01, and cP<0.0001 (saline vs. 1.0 μg Ex-4c); dP<0.01 (saline vs. 0.3 μg Ex-4c); eP<0.0001; fP<0.01; gP<0.001.
    Fig. 2. Exendin-4(1-32)K-capric acid (Ex-4c) induces Fos expression in arcuate pro-opiomelanocortin (POMC) neurons. (A) Confocal images demonstrate humanized Renilla reniformis green fluorescent protein (hrGFP; green), Fos immunoreactivity (red), and 4′,6-diamidino2-phenylindole (DAPI; blue) in arcuate nucleus of the hypothalamus sections obtained from Pomc-hrGFP mice that received intracerebroventricular (i.c.v.) injections of either saline (top row) or 1.0 μg Ex-4c (bottom row). Scale bar=100 μm. (B-D) Bar graphs summarize number of cells that are positive for (B) hrGFP, (C) Fos, and (D) both hrGFP and Fos. NS, not significant. aP<0.05, unpaired t test.
    Fig. 3. Exendin-4(1-32)K-capric acid (Ex-4c) directly activates pro-opiomelanocortin (POMC) neurons via the stimulation of glucagon-like peptide-1 receptors (GLP-1Rs). (A) Bright-field, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), and merged images (from left to right) of a targeted neuron. Scale bar=10 μm. (B) Membrane potential is depolarized in response to Ex-4c (100 nM) treatment. The dashed line indicates baseline membrane potential. Scale bar=1 minute (horizontal), 20 mV (vertical). (C) Ex-4c had no effect on membrane potential when the cell was pretreated with 100 nM Ex(9-39). The dashed line indicates baseline membrane potential. Scale bar=1 minute (horizontal), 20 mV (vertical). (D) Membrane potential was depolarized in response to Ex-4c (100 nM) treatment in the presence of tetrodotoxin (TTX; 500 nM) and synaptic blocker (SB; 1 mM kynurenic acid+50 μM picrotoxin). The dashed line indicates baseline membrane potential. Scale bar=1 minute (horizontal), 20 mV (vertical). (E) Voltage responses to hyperpolarizing current steps from –50 to 0 pA, applied as indicated by arrows in (D). Scale bar=0.5 second (horizontal), 20 mV (vertical). (F) The voltage-current relationship demonstrates increases in input resistance in response to Ex-4c. (G) Bar graphs and dots summarize the acute effects of Ex-4c. Green dots represent depolarized cells with increased input resistance, while blue dots denote depolarized cells with decreased input resistance. Red dots correspond to hyperpolarized cells. Empty dots indicate cells with no response.
    Fig. 4. Exendin-4(1-32)K-capric acid (Ex-4c) activates pro-opiomelanocortin (POMC) neurons via a protein kinase A-dependent signaling pathway. (A, B) The membrane potential was depolarized in response to (A) 10 μM forskolin or (B) 500 μM 8-Br-cyclic adenosine monophosphate (cAMP) treatment. The dashed line indicates baseline membrane potential. Scale bar=1 minute (horizontal), 20 mV (vertical). (C, D) Ex-4c had no effect on membrane potential when the cell was pretreated with (C) 1 μM H-89 or (D) 100 nM KT5720. The dashed line indicates baseline membrane potential. Scale bar=1 minute (horizontal), 20 mV (vertical). (E) Bar graphs and dots summarize the acute effects of forskolin, 8-Br-cAMP, and Ex-4c. Green dots represent depolarized cells with increased input resistance, while blue dots denote depolarized cells with decreased input resistance. Red dots correspond to hyperpolarized cells. Empty dots indicate cells with no response.
    Fig. 5. Exendin-4(1-32)K-capric acid (Ex-4c) activates pro-opiomelanocortin (POMC) neurons via the closure of adenosine triphosphatesensitive K+ (KATP) channels. (A) Bar graphs and dots summarize the acute effects of K+ channel blockers. Green dots indicate depolarized cells with increased input resistance. Empty dots indicate cells with no response. (B-D) Traces demonstrate acute effects of Ex-4c on membrane potential when the cell was pretreated with (B) 10 μM linopirdine+10 μM XE991, (C) 100 nM tertiapin-Q, or (D) 200 μM tolbutamide. The dashed line indicates baseline membrane potential. Scale bar=1 minute (horizontal), 20 mV (vertical). (E) Bar graphs and dots summarize the acute effects of Ex-4c. Green dots represent depolarized cells with increased input resistance, while blue dots denote depolarized cells with decreased input resistance. Empty dots indicate cells with no response.
    Fig. 6. Acute effects of glucagon-like peptide-1 receptor (GLP-1R) agonists on arcuate pro-opiomelanocortin (POMC) neurons. (A, B) Traces demonstrate the acute effects of (A) exendin-4 (Ex-4) or (B) GLP-1 on membrane potential. The dashed line indicates the baseline membrane potential. Scale bar=1 minute (horizontal), 20 mV (vertical). (C) Bar graphs and dots summarize the acute effects of exendin-4(1-32)K-capric acid (Ex-4c), Ex-4, and GLP-1. Green dots represent depolarized cells with increased input resistance, while blue dots denote depolarized cells with decreased input resistance. Empty dots indicate cells with no response.
    Graphical abstract

    Fig. 1.

    Fig. 2.

    Fig. 3.

    Fig. 4.

    Fig. 5.

    Fig. 6.

    Graphical abstract

    Exendin-4(1-32)K-Capric Acid, a Glucagon-Like Peptide-1 Receptor Agonist, Suppresses Food Intake via Arcuate Pro-Opiomelanocortin Neurons
    Acute responses to agonists Ex-4c Ex-4 GLP-1
    Depolarized
     ↑ Input resistance 12 (27.3) 2 (9.5) 9 (28.1)
    Erev =–89.0±5.4 mV Erev =–82.8 mV & –106.8 mV Erev =–103.0±8.8 mV
     ↓ Input resistance 5 (11.3) 4 (19.0) 9 (28.1)
    Erev =–23.7±3.0 mV Erev =–25.7±6.6 mV Erev =–23.3±1.5 mV
    Hyperpolarized 1 (2.3) 1 (4.8) 0
    Erev =–111.6 mV Erev =–75.1 mV
    No response 26 (59.1) 14 (66.7) 14 (43.8)
    Total 44 (100) 21 (100) 32 (100)
    Table 1. Acute Effects of GLP-1R Agonists on POMC Neuron Activity

    Values are expressed as number (%) or mean±standard error of the mean.

    GLP-1R, glucagon-like peptide-1 receptor; POMC, pro-opiomelanocortin; Ex-4c, exendin-4(1-32)K-capric acid; Ex-4, exendin-4; Erev, reversal potential.

    Table 1.

    Endocrinol Metab : Endocrinology and Metabolism
    © Korean Endocrine Society.
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