GLP-1 and Its Derived Peptides Mediate Pain Relief Through Direct TRPV1 Inhibition Without Affecting Thermoregulation

Hormonal regulation during food ingestion and its association with pain prompted the investigation of the impact of glucagon-like peptide-1 (GLP-1) on the transient receptor potential vanilloid 1 (TRPV1). Both endogenous and synthetic GLP-1 and an antagonist of GLP-1, exendin 9–39, reduced heat sensitivity in naïve mice. GLP-1-derived peptides (liraglutide, exendin-4, and exendin 9–39) effectively inhibited capsaicin (CAP)-induced currents and calcium responses in cultured sensory neurons and TRPV1-expressing cell lines. Notably, the exendin 9–39 alleviated CAP-induced acute pain, as well as chronic pain induced by complete Freund’s adjuvant (CFA) and spared nerve injury (SNI) in mice, without causing hyperthermia associated with other TRPV1 inhibitors. Electrophysiological analyses revealed that exendin 9–39 binds to the extracellular side of TRPV1, functioning as a noncompetitive inhibitor of CAP. Exendin 9–39 did not affect proton-induced TRPV1 activation, suggesting its selective antagonism. Among exendin 9–39 fragments, exendin 20–29 specifically binds to TRPV1, alleviating pain in both acute and chronic pain models without interfering with GLP-1R function. Our study revealed a novel role for GLP-1 and its derivatives in pain relief, proposing exendin 20–29 as a promising therapeutic candidate.


INTRODUCTION
Individuals experiencing pain often report overeating calorie-dense, high-sugar, and high-fat foods 1 as a coping mechanism 2 .This phenomenon, termed "ingestion analgesia," has been also observed in animals [3][4][5][6] .Noxious heat-evoked withdrawal behaviors in rats are suppressed during self-initiated chocolate eating and ingestion of sucrose 7 .While much research has focused on mechanisms within the central nervous system to explain this pain suppression 8 , we hypothesize additional mechanisms occurring in the peripheral nervous system based on hormonal regulation during digestion.
Upon food ingestion and its subsequent entry into the small intestine, intestinal L-cells undergo posttranslational processing of the proglucagon gene, leading to the production of glucagon-like peptide-1 (GLP-1), a potent incretin peptide hormone 9 .GLP-1 plays an important role in glucose homeostasis and is secreted into the hepatic portal system in response to elevated glucose levels from food intake 10 , stimulating insulin synthesis and release from the pancreas 11 .The short half-life of native GLP-1, typically only 1-2 min due to rapid degradation, has facilitated the development of structurally modi ed GLP-1 analogs with longer half-lifes, such as liraglutide, exendin-4, and dulaglutide, which exhibit 97%, 53%, and 90% sequence homology, respectively 12 .
Regarding pain modulation, various studies have reported that GLP-1 analogs exert antinociceptive effects on the central nervous system.This modulation occurs by regulating the spinal dorsal horn microglial pathway through GLP-1R activation, leading to alterations in spinal excitatory synaptic transmission under neuropathic pain conditions [13][14][15][16][17][18] .However, the effects of GLP-1 on the peripheral nervous system, and its relationship with pain modulation, remain largely unexplored.
The transient receptor potential vanilloid 1 (TRPV1) channel plays a crucial role in heat pain perception 19 .TRPV1, known for its thermosensitive and polymodal nociceptor properties, is expressed in sensory neurons and is activated by stimuli such as capsaicin (CAP) and protons 20 .In chronic pain states, TRPV1 channels are upregulated in nociceptive neurons, which lowers stimulation thresholds and increases pain perception, as reported in hyperalgesia or allodynia 21 .Inhibiting TRPV1 has proven effective in mitigating pain in diverse neuropathic pain models, thereby garnering interest from numerous pharmaceutical companies 22,23 .However, the challenge with current TRPV1 antagonists lies in their associated adverse effects, such as hyperthermia [24][25][26][27][28][29][30][31] and hypothermia [32][33][34] , caused by failures in thermoregulation.This issue has been attributed to the mode of action of these antagonists: those blocking CAP-, proton-, and heat-induced TRPV1 activation result in hyperthermia, while those sparing proton-induced activation do not lead to hyperthermia 33,35 .Conversely, potentiation of proton-induced TRPV1 activation leads to hypothermia 32 .
In light of these challenges, in this study, we assess the impact of GLP-1 and its derivatives on pain behavior and their in uence on the peripheral nervous system.Preliminary ndings indicate that GLP-1 harbors a key sequence that directly binds to and inhibits the activation of TRPV1 channels in sensory neurons.Importantly, we also demonstrate that GLP-1 and its derivatives directly anatgonize TRPV1 channels in a mode-selective manner that can offer pain relief without the adverse thermoregulatory side effects commonly associated with current TRPV1 inhibitors.

Animals
Adult wild-type male C57BL/6N mice were purchased from Orient Bio (Sungnam, South Korea).Mice were group-housed at constant temperature and humidity under a 12-h/12-h light-dark cycle with free access to standard food and water for at least 1 week prior to the beginning of the experimental procedures.All animal experiments were approved by the Institutional Animal Care and Use Committee of the College of Medicine at Gachon University (approval number: LCDI-2020-0135).

Patch-clamp recordings
Whole-cell voltage-clamp and cell-attached patch-clamp recordings were conducted at room temperature to measure capsaicin (CAP)-or proton-induced currents in dissociated dorsal root ganglion (DRG) neurons and hTRPV1-expressing CHO K1 cells, respectively.An EPC10 ampli er (HEKA, Stuttgart, Germany) was utilized for these recordings.Patch pipettes, prepared with a micropipette puller (Narishige, Tokyo, Japan), had resistances of 4-5 MΩ for whole-cell recordings and 6-8 MΩ for cellattached recordings.The recording chamber, with a volume of 500 µL, was continuously superfused at a rate of 1-2 mL/min.CAP-or low pH-induced currents were recorded at a holding potential of − 60 mV.
Inside-out recordings from hTRPV1-expressing CHO K1 cells were conducted using a pipette resistance of 8-9 MΩ.The internal solution for inside-out recordings matched that used for whole-cell and cellattached methods, whereas the external solution (intracellular side) differed slightly, containing 2 mM CaCl 2 instead of EGTA.The open probability and average single-channel opening and closing times for inside-out recordings were analyzed using a 50% threshold criterion, with all events double-checked before analysis.Ca 2+ imaging in dissociated DRG neurons and hTRPV1-CHO K1 cells At room temperature, Ca 2+ imaging was conducted in mouse DRG, HEK293T, and hTRPV1-expressing CHO K1 cells.Cells on poly-D-lysine-coated coverslips were loaded with 2 µM Fura-2 AM (Thermo Fisher Scienti c, Waltham, MA) at 37°C for 40 min in DMEM.The cells were then rinsed three times with the medium and incubated for 30 min, following which they were placed on the stage of an inverted microscope (BX51W1; Olympus, Tokyo, Japan) and continuously superfused at a ow speed of 1 mL/min with a bath solution containing 140 mM NaCl, 5 mM KCl, 1 mM CaCl 2 , 2 mM MgCl 2 , 10 mM HEPES, and 10 mM glucose, adjusted to pH 7.4 with NaOH.Using illumination with a 175-W xenon arc lamp, excitation wavelengths (340/380 nm) were selected using a Lambda DG-4 monochromator wavelength changer (Shutter Instrument, Novato, CA).The uorescence 340/380 ratio was measured using digital video micro uorometry with an intensi ed camera (OptiMOS; QImaging, Surrey, Canada) coupled to the microscope.Data were analyzed using SlideBook 6 (Intelligent Imaging Innovations, Denver, CO).At the end of the experiment, cells were identi ed based on their response to high concentrations of KCl.

Pull-down assay and immunoblotting
Interactions between exendin 9-39 and TRPV1 were examined via His-mediated pull-down assays using a modi ed protocol from the Pierce Pull-down PolyHis Protein:Protein Interaction Kit (Thermo Fisher Scienti c).His-tagged exendin 9-39 was bound to HisPur Cobalt Resin as the bait protein and incubated with lysates of hTRPV1-CHO K1 or native CHO K1 cells overnight at 4°C.After washing ve times with lysis buffer, the complexes were mixed with lysis buffer containing 290 mM imidazole, and the bound proteins were eluted by boiling in 5× sodium dodecyl sulfate (SDS) loading buffer for 5 min.
The products were then separated using SDS-polyacrylamide gel electrophoresis, transferred onto nitrocellulose membranes, and blotted with an anti-TRPV1 antibody (#ACC-030; Alomone Labs, Jerusalem, Israel).The membrane was washed with TBST and incubated for 1 h with the secondary antibody, anti-rabbit Ig-HRP (1:10,000, 9910; Cell Signaling Technologies, Danvers, MA).After washing with TBST, the immune complexes were detected by chemiluminescence (Beyotime, Shanghai, China) using a Pierce western blotting kit (Thermo Fisher Scienti c).Quantitative densitometric analysis was performed using a UVP BioSpectrum multispectral imaging system (Image Quant LAS 4000; GE Healthcare, Chicago, IL).

Confocal uorescence imaging
Approximately 1 × 10 5 mL − 1 of hTRPV1-expressing CHO K1 cells or naïve CHO K1 cells were seeded into confocal dishes and cultured overnight for cell adherence.The cells were washed with phosphatebuffered saline (PBS) and xed with 2% paraformaldehyde for 10 min at 25°C.After washing with PBS, the cells were permeabilized with 0.1% Triton X-100 for 5 min at room temperature and blocked with 3% bovine serum albumin with glycine for 30 min.Cells were incubated with primary antibody against TRPV1 (1:250; #ACC-030, Alomone Labs) for 1 h at room temperature.After washing with PBS three times, Alexa Fluor 594-conjugated secondary antibody (1:400; Invitrogen) and Hoechst 33342 (Thermo Fisher Scienti c) were added for another hour of incubation at 4°C.The cells were washed with PBS and incubated with FITC-tagged exendin 9-39 dissolved in cold PBS at a concentration of 10 µM for 30 min at 4°C.Finally, after washing with cold PBS, images were acquired using a confocal laser-scanning microscope with a 100× oil-immersion objective (LSM 700; Carl Zeiss, Oberkochen, Germany).

Behavioral tests in mice
Baseline heat sensitivity was measured to determine the systemic effect of glucose or GLP-1(7-36) administration using a hot plate (Ugo Basile, Italy).The PWL to heat was measured after intraperitoneal administration of glucose (2.0 g/kg body weight) in a volume of 200 µL or after administration of the same volume of vehicle (0.9% saline) (n = 5 each).The local effect of GLP-1(7-36) and exendin 9-39 in heat sensitivity was tested by measuring PWL to heat using the Hargreaves radiant heat apparatus after intraplantar administration of 10 µg GLP-1 (7-36) or vehicle (0.9% saline) (n = 5 each) in a volume of 10 µL.
To evaluate nociceptive behavior, the time spent licking the paw, the paw withdrawal threshold (PWT) to mechanical stimuli, and the PWL to heat were measured as described previously 36 .Either vehicle, exendin 9-39, exendin 20-29, or BCTC was rst injected via the intraplantar route, and 30 min later, an additional exendin 9-39, exendin 20-29, or BCTC in combination with CAP was injected.
Licking time was recorded for 5 min after CAP injection in each group.Mechanical allodynia and thermal hyperalgesia were evaluated in separate experiments in a time-dependent manner.Mechanical allodynia was assessed using von Frey laments (NC12775-99; North Coast Medical, CA).The 50% PWT was calculated using the up-down method.Thermal hyperalgesia was assessed by recording PWL using the Hargreaves radiant heat apparatus (IITC Life Sciences, Woodland Hills, CA).A cutoff value of 20 s was used to prevent tissue damage.For rectal temperature recording, the rectal temperature was measured with a digital thermometer (Therma-1; ETI, West Sussex, UK) by inserting a corn oil-soaked exible bead probe into the rectum after intraperitoneal administration of 200 µL of vehicle, exendin 9-39 (50 µg/kg), or BCTC 5 mg/kg in wild-type mice (n = 6).The acute IPGTT was performed in mice aged 8 weeks.Baseline blood glucose levels were measured and mice were intraperitoneally injected with 200 µL of vehicle, exendin 20-29 (10 µg/kg), and exendin-4 (10 µg/kg) in wild-type mice (n = 6).After 15 min, the mice were challenged with glucose (2.0 g/kg body weight).Blood glucose levels were measured using an Accu-Chek Performa glucometer (Roche, Mannheim, Germany).

CFA-induced pain
Under temporal anesthesia with 3% iso urane, the CFA-induced in ammatory pain model was established by intraplantar injection of 20 µL of CFA.Mice were then treated with either vehicle, exendin 9-39, or exendin 20-29 in 20 µL, administered on the same paw where CFA was injected.Heat hyperalgesia and mechanical allodynia induced by CFA were assessed using the Hargreaves and von Frey tests, respectively.Paw thickness was measured in millimeters using a digimatic caliper CD-15APX (Mitutoyo Corporation, Kawasaki, Japan).

SNI-induced pain
Under continuous anesthesia with iso urane, mice underwent surgical manipulation to expose the left sciatic nerve by separating the muscle tissue.Upon visualization of the sciatic nerve, the peroneal and tibial nerves were ligated and transected at the lower end of the ligature using silk thread, while the sural nerve remained intact.The surgical site was then sutured, and iodine was applied for debridement.After a recovery period of 14 days post-surgery, mice received either intraplantar or intraperitoneal administration (20 or 200 µL, respectively) of vehicle, exendin 9-39, or exendin 20-29.Subsequently, the heat sensitivity of the neuropathic pain model mice was assessed using the Hargreaves test.

Statistical analysis
Statistical analyses were conducted using GraphPad Prism 8 (GraphPad Software, San Diego, CA).All data are presented as the mean ± standard error of the mean (S.E.M.).Differences between groups were compared using a two-tailed unpaired t-test for two groups, one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test for multiple groups, or two-way repeated measures ANOVA followed by the Bonferroni multiple comparison test for multiple groups and time courses.The statistical signi cance thresholds were * p < 0.05, ** p < 0.01, *** p < .001,and **** p < .0001(likewise indicated with # and †).

Release of GLP-1 by glucose application alleviates heat sensitivity
Given the essential role that GLP-1 plays in regulating glucose levels in the body, we primarily utilized intraperitoneal glucose administration and assessed changes in heat sensitivity in mice using hot plate tests.The role of endogenous GLP-1 in relation to heat sensitivity was demonstrated by the observation that the intraperitoneal glucose-treated group exhibited a decrease in heat sensitivity as compared with the vehicle-treated group (Fig. 1a), as the administration of glucose signi cantly increased blood glucose levels from 1 to 2 h (Supplementary Fig. 1).To con rm whether the reduction in heat sensitivity after glucose application was due to systemic release of GLP-1, we intraperitoneally administered 10 µg/kg GLP-1(7-36), one of the two primary biologically active forms of secreted GLP-1.Similar to the results observed with glucose administration, the hot plate test revealed a reduction in heat sensitivity from 1 h (Fig. 1b, e).
Upon discovering the impact of systemic GLP-1 on heat sensitivity, we evaluated its in uence on the peripheral nervous system through local intraplantar injection of 10 µg GLP-1 (7-36).The results of the Hargreaves test demonstrated a reduction in pain sensitivity (Fig. 1c).As changes in heat sensitivity are dependent on TRPV1 activity, we sought to determine whether intraplantar injection of 10 µg GLP-1(7-36) modulates TRPV1 agonist CAP-induced spontaneous pain behavior in mice.We found that intraplantar injection of 10 µg GLP-1(7-36) for 10 min signi cantly reduced pain-like (licking) behavior induced by intraplantar injection of 1.6 µg CAP (Fig. 1d).Therefore, we hypothesized that GLP-1 and its metabolites affect peripheral pain regulation through the TRPV1 channel (Fig. 1f).
We initially sought to examine whether exendin 9-39 could reverse the decrease in pain sensitivity through local intraplantar injection.However, the results of the Hargreaves test indicated that the administration of 10 µg exendin 9-39 decreased heat sensitivity upon intraplantar injection (Fig. 3a).
To determine the direct effect of exendin 9-39 on the time mice spent licking their hind paw in the CAPinduced spontaneous pain model, intraplantar administration of exendin 9-39 (doses of 5 and 10 µg) was followed by intraplantar administration of CAP (1.6 µg).Exendin 9-39 demonstrated a dosedependent reduction in paw-licking time, similar to the effect observed with BCTC (0.5 µg) as the control (Fig. 3d).In addition, the paw withdrawal latency (PWL) in the Hargreaves test was signi cantly lower in the vehicle + CAP than vehicle groups from 30 to 120 min (Fig. 3f).Similarly, intraplantar CAP injection signi cantly reduced the PWT in the von Frey test from 30 to 120 min compared with that in the vehicle group (Fig. 3f), whereas mechanical allodynia was dose-dependently alleviated in the groups treated with exendin 9-39 (5 or 10 µg) or BCTC (0.5 µg), a widely used TRPV1 inhibitor 39 .These results suggest that the analgesic effects of exendin 9-39 via TRPV1 were similar to those of BCTC.
To determine whether exendin 9-39 treatment causes hyperthermia, which is a typical adverse effect of TRPV1 inhibitors 40 , body temperature was measured following the administration of exendin 9-39.
Previous animal studies have shown that intraperitoneal administration of various TRPV1 antagonists results in hyperthermia 41 ; therefore, in this study, exendin 9-39 was injected intraperitoneally.As exendin 9-39 showed analgesic effects on the nociceptive behavior of mice at a 10 times higher mass compared with that of BCTC, the same factor (50 mg/kg exendin 9-39 vs. 5 mg/kg BCTC) was used in this experiment.
The rectal temperature in the group treated with exendin 9-39 was similar to that in the vehicle-treated group.By contrast, the rectal temperature in the BCTC-treated group rapidly increased by 0.7-1.5°Cand returned to baseline values at 12 min post-administration (Fig. 3e).

Exendin 9-39 alleviates in ammatory and neuropathic chronic pain behaviors
The analgesic potential of exendin 9-39 was con rmed using an acute pain mice model, followed by an assessment of its effectiveness in the chronic pain model.Considering the documented changes in sensitization and upregulation of TRPV1 in the complete Freund's adjuvant (CFA)-induced in ammatory pain model, CFA was utilized to induce chronic in ammatory pain in mice 42 .The effects of exendin 9-39 on heat hyperalgesia and mechanical allodynia were assessed using the Hargreaves and von Frey tests, respectively (Fig. 4a).In the Hargreaves test, mice with CFA-induced in ammation displayed delayed recovery from heat hyperalgesia following intraplantar vehicle administration.However, a single intraplantar administration of 5 or 10 µg exendin 9-39 effectively relieved heat hyperalgesia and accelerated the recovery process (Fig. 4b).Similarly, in the von Frey test, mice injected with intraplantar vehicle post-CFA induction displayed prolonged mechanical allodynia, which was signi cantly reversed by single intraplantar administration of 5 or 10 µg exendin 9-39 (Fig. 4c).Additionally, exendin 9-39 administration mitigated paw swelling resulting from CFA induction (Supplementary Fig. 2).Furthermore, peripheral nerve injury leads to neuropathic pain, and the spared nerve injury (SNI) model in mice induces persistent heat hyperalgesia 43 .After 14 days of SNI surgery, mice showed heat hyperalgesia compared with those in the sham group with intraplantar vehicle administration (Fig. 4d).
However, SNI-challenged mice when administered intraplantar injections of 10 µg exendin 9-39 showed alleviated heat hyperalgesia starting from 1 h post-administration, peaking at 3 h, compared with those in the vehicle-administered group (Fig. 4e).Similarly, when the same dose of exendin 9-39 was administered intraperitoneally to test systemic analgesic effects, it alleviated heat hyperalgesia starting from 1 h post-administration, peaking at 3 h, compared with the intraperitoneal administration of the vehicle (Fig. 4f).These ndings suggest that the analgesic effects of exendin 9-39 extend not only to chronic in ammatory pain but also to neuropathic pain.
Exendin 9-39 directly binds to the TRPV1 channel To assess the direct interaction between exendin 9-39 and the TRPV1 channel, we conducted a protein binding assay using His-tagged exendin 9-39, which was able to pull down the TRPV1 channel (Fig. 5i) in the CHO K1 cell line stably expressing human TRPV1.In contrast, the naïve CHO K1 cell line showed no TRPV1 channel binding.We tested the binding of exendin 9-39 to TRPV1 by incubating the two cell lines with uorescein isothiocyanate (FITC)-labeled exendin 9-39.Immuno uorescence imaging revealed that FITC-labeled exendin 9-39 was bound to TRPV1 on the cell surface in CHO K1 cells expressing human TRPV1 but not in naïve CHO K1 cells (Fig. 5j).Although exendin 9-39 was tagged with FITC, it still blocked the CAP-induced inward currents of TRPV1 channels (Supplementary Fig. 5a, b).Exendin 9-39 targets the extracellular side of the TRPV1 channel but does not share the CAP binding site Next, we examined the molecular target of exendin 9-39 on the TRPV1 channel using a patch-clamp competition assay in CHO K1 cells expressing human TRPV1.BCTC, a highly potent TRPV1 antagonist that competitively interacts with CAP to bind to TRPV1 channels 39 , was used for this assay.BCTC (10 nM) was applied in the presence of 10 nM CAP until saturation, and twice the BCTC concentration (20 nM) was sequentially applied.While 10 nM BCTC blocked the CAP-induced TRPV1 currents, 20 nM BCTC did not signi cantly potentiate this TRPV1-inhibiting effect (Fig. 6a).In contrast, 100 nM exendin 9-39 application following 10 nM BCTC elicited a signi cant further inhibition of CAP-evoked TRPV1 inward currents (Fig. 6b).These results indicate that exendin 9-39 does not share the binding site with CAP and inhibits TRPV1 activation by interacting noncompetitively with CAP.
To validate the TRPV1 binding site, we performed single-channel recordings from cells expressing human TRPV1 channels.Cell-attached patch recordings showed that the external bath application of 10 nM CAP and 100 nM exendin 9-39 did not reduce the frequency of single-channel open events.In contrast, when the pipette solution contained 100 nM exendin 9-39, which allowed the interaction of exendin 9-39 with the extracellular surface of TRPV1, single-channel opening events were signi cantly blocked (Fig. 6c-e).We further conducted inside-out patch-clamp recordings to determine whether bath application of CAP and exendin 9-39 to the intracellular surface of TRPV1 changed single-channel opening events (Fig. 6f-h).The results showed that only if the pipette solution contained 100 nM exendin 9-39, facilitating the exposure of exendin 9-39 to the extracellular surface of TRPV1, were the single-channel openings blocked while CAP was applied to the intracellular side of the channel.In contrast, single-channel opening events were not detected when CAP and exendin 9-39 were administered via the external bath, allowing exposure of CAP and exendin 9-39 to the intracellular surface of TRPV1.Thus, exendin 9-39 binds to the extracellular side of TRPV1 but cannot penetrate the membrane.
Exendin 9-39 does not affect TRPV1 activation by protons suggesting its mode selectivity As the inhibitory effect of exendin 9-39 on CAP-induced activation of TRPV1 was con rmed, its effect on proton-induced activation of TRPV1 was also tested using patch-clamp recordings and calcium imaging.BCTC is also a potent antagonist of TRPV1 activated by protons 44 ; therefore, it was used as a control antagonist.Unlike exendin 9-39 during CAP-induced TRPV1 activation, 1 µM exendin 9-39 did neither reduce nor potentiate low pH-induced inward currents (Fig. 7a, b) and calcium in ux (Fig. 7c, d) in hTRPV1-expressing CHO K1 cells, whereas 1 µM BCTC completely blocked them.The exendin 9-39 concentration used in this experiment was approximately 30 times higher than its IC 50 value in CAPinduced TRPV1 activation, indicating the mode-selective characteristics of exendin 9-39.This suggests that exendin 9-39 may be less likely to cause adverse effects associated with thermoregulation, such as hyperthermia or hypothermia, as found with previous TRPV1 antagonists.

Exendin 20-29 alleviates in ammatory and neuropathic chronic pain behaviors
Exendin 20-29 was further assessed for its e cacy in alleviating in ammatory and neuropathic chronic pain behaviors.In CFA-induced chronic in ammatory pain model, mice with CFA-induced in ammation showed prolonged heat hyperalgesia following intraplantar vehicle administration, while a single intraplantar administration of 20 or 50 µg exendin 20-29 effectively alleviated heat, with complete recovery observed by day 8 (Supplementary Fig. 8a).Similarly, in the von Frey test, mice injected with intraplantar vehicle post-CFA induction displayed slow recovery of mechanical allodynia.Nevertheless, a single intraplantar administration of 20 or 50 µg exendin 20-29 alleviated mechanical allodynia (Supplementary Fig. 8b).Additionally, administration of exendin 20-29 resulted in a reduction in paw swelling induced by CFA (Supplementary Fig. 8c).Moreover, in the SNI-induced chronic neuropathic pain model, mice challenged with SNI and administered intraperitoneal injections of 50 µg exendin 20-29 exhibited alleviated heat hyperalgesia, with effects observed as early as 1 hour post-administration and peaking at 2 hours, compared to the vehicle-administered group (Supplementary Fig. 8d).These ndings highlight the therapeutic potential of exendin 20-29 as an analgesic agent for chronic pain management.

DISCUSSION
Our study has identi ed a novel function for GLP-1-derived peptides in providing pain relief.We speci cally found that exendin 20-29 inhibits TRPV1 activity in sensory neurons in a direct and modespeci c manner, reducing pain behaviors without noticeable adverse effects.This discovery is particularly relevant in the context of the ingestion analgesia phenomenon, which relates to the suppression of noxious heat-induced withdrawal behaviors in rats during the consumption of chocolate or sweet liquids, contrasting with salt ingestion [3][4][5][6][7] .Our primary aim was to dissect the role of the incretin peptide hormone, GLP-1, in this context by systemically administering GLP-1 and glucose via intraperitoneal injection.Our ndings demonstrated that both natural and synthetic GLP-1 administrations altered heat sensitivity and that local administration of GLP-1 notably reduced heat sensitivity and CAP-induced spontaneous pain behavior.This suggests that GLP-1 and its metabolites might modulate TRPV1 activity, which plays a pivotal role in heat pain perception 19,46 .Moreover, our study highlighted that GLP-1 analogs, including GLP-1, liraglutide, and exendin-4, along with the GLP-1 antagonist exendin 9-39, which exhibits 53% sequence homology with native GLP-1 38 , effectively inhibited CAP-induced TRPV1 activation in mouse DRG neurons.Unlike BCTC, exendin 9-39 also mitigated both thermal and mechanical nociceptive behaviors in mice without inducing hyperthermia, marking it as a viable analgesic without the common adverse effects associated with existing pain management strategies, such as opioids and traditional TRPV1 antagonists 32,41,[47][48][49] .Thereby, our study indicated that GLP-1-derived peptides offer effective pain relief without these signi cant adverse effects, suggesting their potential as a safer alternative for chronic pain management.
Previous studies have shown that GLP-1 analogs can prevent or improve diabetic neuropathy in animal models [50][51][52][53] .In addition to their neuroprotective role, GLP-1 analogs have also been studied for their in uence on spinal excitatory synaptic transmission under pathophysiological conditions such as neuropathic pain.However, previous studies have not investigated GLP-1 analogs and exendin 9-39 for pain transmission at peripheral sites, especially in small-sized nociceptive DRG neurons, where the TRPV1 channel is crucial in chronic pain-inducing mechanical allodynia and thermal hyperalgesia [54][55][56][57] .
To identify the mechanism behind the analgesic effects of GLP-1 analogs and exendin 9-39, we utilized a cell line lacking GLP-1R to transfect the rat TRPV1 plasmid.Both GLP-1 analogs and exendin 9-39 were found to reduce CAP-induced inward currents and calcium in ux through the TRPV1 channel.Determination of the IC50 values for these compounds in CHO K1 cells expressing human TRPV1 revealed that exendin 9-39 exhibited the lowest IC 50 value for inhibiting CAP-induced TRPV1 activation.
These ndings suggest exendin 9-39 directly interacts with TRPV1, independently of GLP-1R, to inhibit its activation.This interaction was further corroborated by pull-down assays and confocal imaging, indicating a direct binding between exendin 9-39 and TRPV1 channels.Our electrophysiological analyses revealed that exendin 9-39 likely binds to the extracellular side of TRPV1, offering selective and potent antagonism for pain relief.This nding is particularly signi cant as CAP is known to bind to the transmembrane domains of TRPV1 channels 56 , suggesting exendin 9-39 as a selective and potent antagonist for pain relief without competitively displacing CAP at its binding site.
Although the intraperitoneal administration of exendin 9-39 did not cause the adverse effect of hyperthermia, a critical aspect of developing TRPV1 antagonists is minimizing interference with TRPV1 activation by protons, and hence, identifying mode-selective antagonists 32 .Unlike its in uence on CAPinduced TRPV1 activation, exendin 9-39 did not block or potentiate proton-induced channel activity, even at a concentration approximately 30 times higher than its IC 50 value inhibiting CAP-induced TRPV1 activity, indicating mode-speci c inhibition of exendin 9-39.
Critical to our analysis was the examination of the half-maximal effective concentration values for GLP-1 analogs to activate GLP-1R, which are known to be at picomolar levels 58 .To exclude any potential effects of exendin 9-39 on insulin secretion via GLP-1R interaction, we analyzed key protein sequences involved in TRPV1 inhibition.Three distinct fragments of exendin 9-39 were found to reduce CAPinduced inward currents and calcium in ux through TRPV1.Their common sequence, exendin 20-29, speci cally inhibited CAP-induced TRPV1 activation without affecting proton-induced activation, and similarly alleviated CAP-induced nociceptive behaviors.To ensure that exendin 20-29 does not interact with GLP-1R, we utilized an acute IPGTT.Unlike exendin-4, exendin 20-29 did not affect blood glucose levels, supporting its lack of GLP-1R interaction.This was further validated by the absence of blood glucose changes in wild-type mice following administration of exendin 20-29 at effective doses, contrasting with the hypoglycemic effect observed with exendin-4.

Figure 1 Reduced
Figure 1