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GLP-1RAs attenuated obesity and reversed leptin resistance partly via activating the microbiome-derived inosine/A2A pathway
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Chunyan Dong, Bailing Zhou, Binyan Zhao, Ke Lin, Yaomei Tian, Rui Zhang, Daoyuan Xie, Siwen Wu, Li Yang*
Acta Pharmaceutica Sinica B | 2025, 15(2) : 1023 - 1038
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Acta Pharmaceutica Sinica B | 2025, 15(2): 1023-1038
• ORIGINAL ARTICLE •
GLP-1RAs attenuated obesity and reversed leptin resistance partly via activating the microbiome-derived inosine/A2A pathway
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Chunyan Dong, Bailing Zhou, Binyan Zhao, Ke Lin, Yaomei Tian, Rui Zhang, Daoyuan Xie, Siwen Wu, Li Yang*
Affiliations
  • State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center of Biotherapy, Chengdu 610041, China
About Author:

E-mail address: (Li Yang).

Author contributions

Chunyan Dong: Writing – original draft. Bailing Zhou: Methodology, Formal analysis. Binyan Zhao: Methodology. Ke Lin: Investigation. Yaomei Tian: Investigation. Rui Zhang: Resources. Daoyuan Xie: Resources. Siwen Wu: Resources, Methodology. Li Yang: Writing – review & editing, Project administration, Data curation, Conceptualization.

doi: 10.1016/j.apsb.2024.12.006
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Extensive evidence has demonstrated that glucagon-like peptide-1 receptor agonists (GLP-1RAs) can ameliorate obesity. Our previous studies revealed that (Ex-4)2-Fc, a long-acting GLP-1RA we developed, depends on the leptin pathway to treat obesity. However, the mechanisms linking (Ex-4)2-Fc and leptin resistance remain largely unclear. To address this question, we explored the mechanism of GLP-1RAs from the perspective of the gut microbiota, as increasing evidence indicates an important link between the gut microbiota and obesity. This study aimed to explore the potential role of the gut microbiota in the treatment of GLP-1RAs. We found that (Ex-4)2-Fc treatment reshaped obesity-induced gut microbiota disturbances and substantially increased the abundance of Akkermansia muciniphila (Am). In addition, (Ex-4)2-Fc did not respond well in antibiotic-treated (ATB) Obese mice. Subsequent studies have shown that this defect can be overcome by gavage with Am. In addition, we found that Am enhanced (Ex-4)2-Fc therapy by producing the metabolite inosine. Inosine regulates the macrophage adenosine A2A receptor (A2A) pathway to indirectly reduce leptin levels in adipocytes Thus, elucidating the role of metabolites in regulating the leptin pathway will provide new insights into GLP-1RAs therapy and may lead to more effective strategies for guiding the clinical use of antidiabetic agents.

Obesity  /  GLP-1RAs  /  Gut microbiota  /  Metabolite  /  Obesity-related inflammation  /  Leptin resistance  /  Macrophage-adipocyte  /  A2A pathway
Chunyan Dong, Bailing Zhou, Binyan Zhao, Ke Lin, Yaomei Tian, Rui Zhang, Daoyuan Xie, Siwen Wu, Li Yang. GLP-1RAs attenuated obesity and reversed leptin resistance partly via activating the microbiome-derived inosine/A2A pathway[J]. Acta Pharmaceutica Sinica B, 2025 , 15 (2) : 1023 -1038 . DOI: 10.1016/j.apsb.2024.12.006
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1.　Introduction
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Obesity has become a public health problem of great concern in the past few decades. According to the latest updated records from the World Health Organization (WHO), approximately 40% of people worldwide are overweight, whereas 13% are obese1. Improving one's lifestyle, adjusting one's eating habits, and increasing one's physical exercise help to treat obesity. However, these approaches do not completely solve the problem of obesity, so some research has focused on treating obesity with drugs2-4. Glucagon-like peptide-1 (GLP-1) is a 30-amino acid incretin hormone secreted mainly by L-cells in the distal portion5. GLP-1 can control body weight and enhance glucose sensitivity, but endogenous GLP-1 is easily degraded by dipeptidyl peptidase IV (DPP-IV), which has a half-life of 1–2 min, thus limiting the clinical therapeutic potential of natural GLP-16,7. Exendin-4 (Ex-4) is a 39-amino acid peptide isolated from the salivary glands of the Gila monster lizard (Heloderma suspectum) that shares 53% sequence homology with GLP-1. Unlike GLP-1, Ex-4 is a stable peptide because it is not easily degraded by DPP-IV8. Although Ex-4 has a longer half-life than natural GLP-1, it still needs to be injected once or twice a day9. In our previous study, we designed a long-acting GLP-1RA, Exendin-4-IgG4 Fc fusion protein ((Ex-4)2-Fc), which is essentially two tandem copies of the Ex-4 molecule fused onto a mutated human IgG4 constant heavy chain10,11. The half-life of (Ex-4)2-Fc was significantly extended to 122 h in male Sprague–Dawley (SD) rats10. For decades, GLP-1 analogs and their long-acting formulations have been continuously developed and are as effective as GLP-1. For example, from exenatide twice daily (the first approved GLP-1RA) to liraglutide once a day, to albiglutide, dulaglutide, and semeglutide once a week, and even the oral GLP-1RA (semeglutide) approved in 2019. In addition to diabetes, Novo Nordisk's semeglutide injection, Wegovy, has shown promising results in the clinic and was approved by the FDA for the treatment of obesity in 202112-16, and extensive evidence has demonstrated that GLP-1RAs can effectively treat obesity17-20. Similarly, we found that (Ex-4)2-Fc can also achieve good weight loss, and this is partly achieved by reducing the leptin levels10, and Zhao et al.21 also demonstrated that partial leptin reduction can effectively ameliorate obesity. However, the underlying mechanism by which (Ex-4)2-Fc reduces leptin to improve obesity remains unclear.
In recent years, the gut microbiota is associated with several diseases, such as obesity, type 2 diabetes (T2D), and alcoholic liver injury22-25. It has been observed in clinical practice that not all T2D patients reach therapeutic targets in response to GLP-1-based therapies because their HbA1c is lower than 6.5%, indicating a state of GLP-1 resistance26,27. Grasset et al.28 reported that a specific set of bacteria impaired the GLP-1-activated gut–brain axis to control insulin secretion and gastric emptying and that gut microbiota dysbiosis induced GLP-1 resistance through the gut–brain axis mechanism in animal models. Some studies have shown that obesity induces greater abundances of Firmicutes, Fusobacteria, Proteobacteria, and Actinobacteria and a lower abundance of Bacteroidete29-31. Remarkably, there is also evidence demonstrating the interactions between antidiabetic drugs and the gut microbiota, and drugs could affect host function by altering the gut microbiome and its metabolites (drug–microbial–metabolic axis)32. Several studies have highlighted the key role of the gut microbiota in drug therapy, such as the immune checkpoint inhibitors CTLA-4, PD-1, and PD-L131,33-37. Therefore, regulating the gut microbiota composition by drug therapy may be a potential way to treat obesity. Currently, although studies have reported changes in the gut microbiota composition after GLP-1RA treatment, whether the gut microbiota influences its therapeutic efficacy and the related molecular mechanisms remain unclear. The development of GLP-1RAs and next-generation GLP-1 therapies requires a more precise understanding of the mechanisms of action and greater insight into the control of GLP-1R signaling. Our previous research revealed that (Ex-4)2-Fc can treat obesity by partially lowering leptin10, and Zhao et al.21 also demonstrated that partial leptin reduction can effectively ameliorate obesity. However, the underlying mechanism by which (Ex-4)2-Fc reduces leptin to improve obesity remains unclear.
In our study, we first found that (Ex-4)2-Fc treatment altered the microbiota and its driven metabolite inosine. In addition, inosine reduced adipocyte leptin levels by regulating adipocyte macrophage A2A signaling, ultimately reversing obesity-induced hypothalamic leptin resistance and improving obesity. Finally, we demonstrated that (Ex-4)2-Fc plays a therapeutic role through A2A signaling. Taken together, our findings revealed that (Ex-4)2-Fc reversed obesity-induced hypothalamic leptin antagonism via microbiome-derived inosine.
2.　Materials and methods
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2.1.　 Chemicals and reagents
High fat diet (HFD) was purchased from Research Diets, Inc. (New Brunswick, USA). Ampicillin, RIPA lysis buffer, protease inhibitors, phosphatase inhibitors, and loading buffer were purchased from Beijing Solarbio Biotechnology Co., Ltd. (Beijing, China). Streptomycin and colistin were obtained from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China). Tryptic Soy Agar (TSA) blood plates and brain heart infusion broth were purchased from Elite Media, Inc. (London, UK). Inosine, isobutylmethylxanthine (IBMX), dexamethasone (DEX), insulin (INS), and ZM241385 were purchased from MedChemexpress (MCE) Biotechnology Co., Ltd. (Monmouth Junction, NJ, USA). Stool DNA isolation kit and RNA isolation kit were purchased from Chengdu Foregene Biotechnology Co., Ltd. (Chengdu, China). The AxyPrep DNA Gel Extraction Kit was purchased from Corning Inc. (Silicon Valley, CA, USA). ChamQ Universal SYBR qPCR Master Mix kit Nanjing Vazyme Biotechnology Co., Ltd. (Nanjing, China). Taq DNA Polymerase Kit was purchased from Takara Holdings Inc. (Dalian, China). The A2A, F4/80, P65, and p-P65 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The JNK, p-JNK, P38, p-P38, ERK, p-ERK, and GAPDH antibodies were purchased from Hangzhou HuaAn Biotechnology (Hangzhou, China). The p-STAT3 antibody was purchased from Cell Signaling Technology (CST), Inc. (Boston, MA, USA). Leptin enzyme-linked immunosorbent assay (ELISA) kit was purchased from Bio-Techne (Minneapolis, MN, USA). The insulin ELISA kit was purchased from Merck Millipore (Darmstadt, Germany). Recombinant murine macrophage colony stimulating factor (M-CSF) was purchased from Beijing PrimeGene Science and Technology Co., Ltd. (Beijing, China). Lipopolysaccharide (LPS) was purchased from Beyotime Biotech Inc. (Shanghai, China). Type I collagenase was purchased from Absin Bioscience Inc. (Shanghai, China).
2.2.　 Animals
Male C57BL/6J mice aged 6–8 weeks were purchased from Beijing Wei Tong Li Hua Biological Technology Co., Ltd. (Beijing, China). The db/db and ob/ob mice (male, 8 weeks old) were purchased from Hua Fu Kang Biological Technology Co., Ltd. (Sichuan, China). A2A−/− mice were purchased from the Model Animal Research Center (MARC) of Nanjing University (Nanjing, China). A2A−/− mice bred on the C57BL/6J genetic background and their wild-type mice were maintained at the School of Sichuan University (Chengdu, China). All animal experiments were conducted following the guidelines approved by the Ethics Committee of Sichuan University (20190923028).
2.3.　 Animal studies
2.3.1.　 (Ex-4)2-Fc treatment
Male C57BL/6J mice were fed a high-fat diet containing 60% caloric fat (Research Diets, USA) to induce DIO model mice (HFD group), while the mice in the normal group (NC) were fed normal chow. DIO model mice were divided into HFD and (Ex-4)2-Fc groups. The (Ex-4)2-Fc group mice were injected with (Ex-4)2-Fc (1.8 mg/kg, once a week) by subcutaneous injection for 14 days, and the mice were sacrificed after fecal and serum samples were collected. Body weight and food intake were measured during the experiments. WT and A2A−/− mice were fed a high-fat diet to induce obesity. WT mice were divided into the WT and WT+(Ex-4)2-Fc groups, and A2A−/− mice were divided into the A2A−/− and A2A−/−+(Ex-4)2-Fc groups. The WT+(Ex-4)2-Fc and A2A−/−+(Ex-4)2-Fc group mice were injected with (Ex-4)2-Fc (1.8 mg/kg, once a week) by subcutaneous injection for 14 days, and the body weight and food intake of the mice were monitored daily.
2.3.2.　 Antibiotic treatment experiment
DIO model mice were divided into an antibiotic-treated group (ATB) and an untreated group (HFD). The ATB group mice were supplemented with mixed antibiotics (ampicillin 1 mg/mL; streptomycin 5 mg/mL; colistin 1 mg/mL) in sterile drinking water, which was changed twice a week33. The untreated group was divided into HFD and (Ex-4)2-Fc groups and the treated group was divided into ATB and ATB+(Ex-4)2-Fc groups. Mice in the (Ex-4)2-Fc and ATB+(Ex-4)2-Fc groups were subcutaneously injected with (Ex-4)2-Fc (1.8 mg/kg, once a week) for 14 days. The body weight and food intake of the mice were monitored daily.
2.3.3.　 Supplemental Am cotherapy
ATB mice were divided into ATB, ATB+(Ex-4)2-Fc and ATB+(Ex-4)2-Fc + Am groups, and HFD-fed mice were divided into HFD and (Ex-4)2-Fc groups. In the ATB+(Ex-4)2-Fc + Am group, Am was administered by oral gavage (108 CFU/200 μL, once daily) starting two weeks before (Ex-4)2-Fc treatment. Am (ATCC BAA-835) was purchased from the American Type Culture Collection (ATCC) and cultured at 37 ℃ in an anaerobic incubator containing 100% nitrogen. Am was evenly coated on TSA blood plates (ELITE-MEDIA, UK) and cultured for 24 h. On the second day, Am was collected and diluted to the desired concentration using brain heart infusion broth (ELITE-MEDIA, UK). Then, the diluted Am suspension was treated by gavage, and part of the suspension was evenly coated on a TSA blood plate for further culture. The body weight and food intake of the mice were monitored daily after the (Ex-4)2-Fc treatment began.
2.3.4.　 Supplemental inosine co-therapy
ATB mice were divided into ATB, ATB+(Ex-4)2-Fc and ATB+(Ex-4)2-Fc + Inosine groups, and HFD-fed mice were divided into HFD and (Ex-4)2-Fc groups. The ATB+(Ex-4)2-Fc + Inosine group mice were given inosine (MCE, USA) by oral gavage (300 mg/kg, once daily) for 14 days from the first day of (Ex-4)2-Fc treatment33. The body weight and food intake of the mice were monitored daily.
2.3.5.　 Inosine treatment
DIO model mice were divided into HFD and inosine groups. The inosine group was given inosine (300 mg/kg, once daily) for 14 days. The db/db mice were divided into db/db NS and db/db NS + Inosine groups. The ob/ob mice were divided into the ob/ob NS and ob/ob NS + Inosine groups. The db/db NS + Inosine group and ob/ob NS + Inosine group mice were given inosine (300 mg/kg, once daily) for 14 days. The body weight and food intake of the mice were monitored daily after inosine treatment. Similarly, inosine treatment was administered to WT and A2A obese mice, and the body weight and food intake of the mice were monitored daily.
2.4.　 Glucose tolerance test
For the glucose tolerance tests (IPGTTs), mice were fasted for 15 h before receiving an intraperitoneal injection of glucose at a dose of 2 g/kg. Blood glucose at different time points (0, 30, 60, and 120 min) was measured with a portable glucometer.
2.5.　 Fecal microbiome 16S rRNA detection and analysis
Fecal samples were collected after 14 days of (Ex-4)2-Fc treatment. DNA in the fecal samples was extracted using a Stool DNA isolation kit (Foregene, China) according to the manufacturer's instructions. The variable region 4 (V4) region of the microbial 16S rRNA gene was amplified. PCR products were then extracted from agarose gels and purified using the AxyPrep DNA Gel Extraction Kit (Axygen, USA). Purified amplicons were sequenced using an Illumina HiSeq sequencer and QIIME or R to analyze the gut microbiota diversity at Beijing Novogene Co., Ltd. (Beijing, China).
2.6.　 Untargeted metabonomics
Serum samples were processed and injected into the LC–MS/MS system according to the methods of the Beijing Novogene Co., Ltd. (Beijing, China). UHPLC‒MS/MS analyses were performed using a Vanquish UHPLC system (ThermoFisher, Germany) coupled with an Orbitrap Q Exactive HF mass spectrometer (ThermoFisher, Germany) by Beijing Novogene Co., Ltd. (Beijing, China). Statistical analyses were performed using the statistical software R (R version R-3.4.3), Python (Python 2.7.6 version), and CentOS (CentOS release 6.6).
2.7.　 High-performance liquid chromatography (HPLC)
The serum sample inosine concentration was determined by HPLC (Agilent, Germany). The mobile phase was methanol and formic acid in water. The stationary phase was a C18 column (250 mm × 4.6 mm, Agilent, Germany), and the column temperature was 25 ℃. The flow rate was 1 mL/min.
2.8.　 PCR analysis
Real-time PCR (RT-PCR) analysis. RNA isolation was performed according to the manufacturer's instructions (Foregene, China). RT-PCR was conducted with a ChamQ Universal SYBR qPCR Master Mix kit (Vazyme, China). The relative expression of genes was normalized to that of Gapdh. All of the sequences of primers used for RT-PCR are listed in Supporting Information Table S1.
Genomic DNA isolation and PCR. The tails of the partially propagated mice were subjected to PCR to determine the effect of the gene knockout. The tails were added to a tube filled with 75 μL of liquid A and 1 mL of 1 mol/L NaOH +80 μL of 0.1 mol/L EDTA. After cooling at 4 ℃ to room temperature, 75 μL of Tris-HCl was added, the mixture was vortexed, oscillated for 5 s, and centrifuged at 12,000 rpm for 5 min. Genomic DNA was extracted from the supernatant for PCR identification using a Taq DNA Polymerase Kit (Takara, China). Based on the design results, a 623 bp target band was obtained from the WT mice by PCR amplification with the P3 and P4 primers, while a 948 bp target band was obtained from the A2A−/− mice by PCR amplification with the P1 and P2 primers.
2.9.　 Protein extraction and Western blot analysis
Lysates were prepared using RIPA (Solarbio, China) buffer containing protease inhibitors (Solarbio, China) and phosphatase inhibitors (Solarbio, China) on ice and centrifuged at 12,000 × g for 10 min at 4 ℃, after which the protein in the supernatant was collected. The extracts were added to the loading buffer (Solarbio, China) and denatured by boiling at 100 ℃ for 10 min. The protein samples were electrophoresed on 10% SDS-PAGE gels and transferred to a PVDF membrane. The membranes were blocked with 5% nonfat milk and then incubated with antibodies against A2A (Santa Cruz, USA), P65 (Santa Cruz, USA), p-P65 (Santa Cruz, USA), JNK (Huabio, China), p-JNK (Huabio, China), P38 (Huabio, China), p-P38 (Huabio, China), ERK (Huabio, China), p-ERK (Huabio, China), and GAPDH (Huabio, China). After incubation with the corresponding secondary antibodies and washing with TBST, images were collected using a chemiluminescence imaging system (Clinx, Shanghai, China).
2.10.　 Histological analysis
Hematoxylin and eosin (HE) staining of eWAT was performed by Lilai (Chengdu, China). Briefly, the sections were deparaffinized and rehydrated, and the nuclei were stained with hematoxylin for 15 min. The sections were then rinsed in running tap water, stained with eosin for 3 min, dehydrated, and mounted. Images were obtained with BA210Digital (Motic). The size of adipocytes was analyzed in HE-stained sections with ImageJ software. The size of the adipocytes was analyzed by ImageJ software, and the average adipocyte area of each group was analyzed statistically by GraphPad software.
Immunofluorescence (IF) staining of eWAT was performed by Hubei Biossci Co., Ltd. (Wuhan, China). Briefly, the sections were blocked for 30 min at room temperature, incubated with primary antibodies against F4/80 (Santa Cruz, USA) and A2A (Santa Cruz, USA) at 4 ℃ overnight, and then incubated with secondary Alexa Fluor 488 donkey anti-mouse antibody and Alexa Fluor 594 donkey anti-rabbit antibody at 37 ℃ for 45 min. Images were obtained with a BX51 microscope (Olympus). For the hypothalamus tissues, the sections were blocked for 1 h at room temperature, incubated with primary antibodies against p-STAT3 (CST, USA) overnight at 4 ℃, and then incubated with a secondary goat anti-rabbit IgG H&L (Alexa Fluor 555) antibody for 1 h at room temperature. Images were captured using a Zeiss Axioplan 2 imaging system (Zeiss). The positive cells were statistically analyzed by ImageJ software.
2.11.　 ELISA analysis
Serum leptin concentrations were determined using mouse ELISA kits (R&D, USA). Serum insulin concentrations were determined using a mouse insulin ELISA kit (Millipore, Germany).
2.12.　 Cell culture
The preadipocyte cell line 3T3-L1 (ATCC, CL-173) was purchased from ATCC. The cells were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM) with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. 3T3-L1 cells were induced to differentiate into mature adipocytes. Briefly, 2 days after confluence (Day 0), the cells were placed in differentiation medium consisting of DMEM, 10% FBS, 0.5 mmol/L IBMX (MCE, USA), 1 μmol/L DEX (MCE, USA), and 10 μg/mL INS (MCE, USA) and then switched to maintenance medium supplemented with DMEM, 10% FBS and 10 μg/mL INS. The medium was replenished every other day.
BMDMs were isolated from C57BL/6J mice. BMDMs were cultured in 1640 medium with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 20 ng/mL M-CSF (prime gene, China) for one week. For cytokine detection, BMDMs were treated with 100 ng/mL LPS (Beyotime, China), 20 mmol/L inosine (MCE, China) or 1 μmol/L ZM241385 (MCE, China) for 6 h38. In vitro, BMDMs and 3T3-L1 cells were co-cultured. BMDMs were induced with 100 ng/mL LPS (Beyotime, China), 20 mmol/L inosine (MCE, China) or 1 μmol/L ZM241385 (MCE, China) for 24 h before co-culture with mature 3T3-L1 cells39. Leptin expression was detected in the cells after 24 h co-culture.
SVFs were isolated from eWAT. Briefly, eWAT was cut into small pieces and digested with type I collagenase (Absin, China) for 30 min at 37 ℃. The digestion was terminated, and red cell lysate was cleaved to dissolve the red cells. The SVFs were cultured in DMEM supplemented with 10% FBS overnight. The next day, the cells were incubated with inosine (20 mmol/L) or ZM241385 (1 μmol/L) at 37 ℃ for 24 h, after which the expression level of leptin was measured.
2.13.　 Statistical analysis
Unless otherwise specified, statistical analyses were performed with Student's t-test (two groups) and two-way ANOVA (more than two groups), followed by Tukey's multiple comparisons test, where appropriate, using GraphPad Prism software. The data are presented as the mean ± standard error of mean (SEM), and a P value < 0.05 was considered to indicate statistical significance.
3.　Results
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3.1.　 (Ex-4)2-Fc reshaped obesity-induced gut microbiota dysbiosis
Growing evidence indicates that obesity induces gut microbiota dysbiosis. We analyzed the changes in the gut microbiota after (Ex-4)2-Fc treatment by 16S rRNA sequencing of fecal samples. The results revealed alterations in alpha (Fig. 1A) and beta diversities (Fig. 1B) after (Ex-4)2-Fc treatment. At the phylum level, the abundances of Bacteroidetes and Verrucomicrobia increased, while those of Firmicutes, Actinobacteria, and Proteobacteria decreased significantly after (Ex-4)2-Fc treatment (Fig. 1C). At the species level, obesity-induced some changes in bacterial abundance, while (Ex-4)2-Fc treatment reversed these bacteria from returning to normal and increased the abundances of some specific bacteria, especially Am (Fig. 1D and E). These findings showed that the gut microbiota composition was reshaped after (Ex-4)2-Fc treatment.
3.2.　 ATB mice showed a diminished response to GLP-1RAs, but gavage of Am overcame this deficiency
Next, we determined whether the efficacy of (Ex-4)2-Fc therapy was related to the gut microbiota (Fig. 2A). We determined that antibiotics in the drinking water eliminated bacteria in HFD-fed mice by detecting the fecal genome content (Supporting Information Fig. S1A) and culturing bacteria (Fig. S1B). Compared to the ATB+(Ex-4)2-Fc group, the (Ex-4)2-Fc group showed more significant weight loss and less body weight gain (Fig. 2B). Relatively lower food intake was observed in the (Ex-4)2-Fc group (Fig. S1C). Lower insulin levels were observed in the (Ex-4)2-Fc group than in the other groups, which led to the alleviation of obesity-induced insulin resistance (Fig. 2C). The (Ex-4)2-Fc group had lower glucose levels and areas under the curve (AUCs) than the other groups according to the IPGTTs (Fig. 2D). HE staining revealed that the adipocyte area in the (Ex-4)2-Fc group was smaller than that in the ATB+(Ex-4)2-Fc group (Fig. 2F), and the epididymal adipose tissue (eWAT) weight in the (Ex-4)2-Fc group was lower (Fig. 2E). The above results indicate that the gut microbiota was closely related to (Ex-4)2-Fc therapy.
We performed an Am and (Ex-4)2-Fc co-therapy test in ATB mice to validate whether supplementation with Am restored (Ex-4)2-Fc efficacy (Fig. 2G). Interestingly, the ATB+(Ex-4)2-Fc + Am group and (Ex-4)2-Fc group had similar therapeutic effects, with more significant weight loss and lower body weight gain than the ATB+(Ex-4)2-Fc group (Fig. 2H) but no significant change in food intake (Supporting Information Fig. S2A). The insulin levels and glucose tolerance of the ATB+(Ex-4)2-Fc + Am and (Ex-4)2-Fc groups were better than those of the ATB+(Ex-4)2-Fc group (Fig. 2I and J). Additionally, HE staining revealed that the adipocyte areas in the ATB+(Ex-4)2-Fc + Am and (Ex-4)2-Fc groups were smaller than those in the ATB+(Ex-4)2-Fc group, and the eWAT weights in the ATB+(Ex-4)2-Fc group were significantly greater than those in the ATB+(Ex-4)2-Fc + Am and (Ex-4)2-Fc groups (Fig. 2K). These results suggested that supplementation with Am restored the side effects of (Ex-4)2-Fc therapy on the gut microbiota.
3.3.　 Microbiota-driven inosine enhanced the efficacy of (Ex-4)2-Fc in ATB mice
To understand the mechanism by which the gut microbiota affects (Ex-4)2-Fc efficacy, we analyzed serum metabolism by untargeted metabolomics (Fig. 3A). The PCA ordination plot revealed that the overall distribution of metabolites significantly differed after (Ex-4)2-Fc therapy (Fig. 3B). There were 154 different metabolites after (Ex-4)2-Fc treatment and the top 3 metabolites were xanthine, hypoxanthine, and inosine (Fig. 3C and D). These three metabolites are products of purine metabolism, and inosine metabolism results in the production of xanthine and hypoxanthine28. In addition, the use of HPLC technology also demonstrated that the serum creatinine concentration increased after treatment (Supporting Information Fig. S3). To determine the relationship between the gut microbiota and metabolites, we performed an association analysis between the gut microbiota and metabolites. The results showed that Am was positively correlated with inosine (Fig. 3E). Mager et al. proved that inosine was the metabolite produced by Am33. We also confirmed that the serum inosine concentration increased after Am administration (Fig. 3F). The above results indicated that changes in the gut microbiota affected purine metabolism after (Ex-4)2-Fc treatment. Therefore, we speculated that the gut microbiota might impact (Ex-4)2-Fc therapy via the metabolite inosine.
We performed the inosine and (Ex-4)2-Fc co-therapy test in ATB mice (Fig. 3G). Excitingly, inosine and (Ex-4)2-Fc co-therapy also enhanced the effect of (Ex-4)2-Fc in ATB mice. The ATB+(Ex-4)2-Fc + Inosine and (Ex-4)2-Fc groups had similar therapeutic effects, with more significant weight loss and lower body weight gain than the ATB+(Ex-4)2-Fc group (Fig. 3H), but no significant change in food intake (Fig. S2B). Insulin levels, glucose tolerance, and fat-related indicators in the ATB+(Ex-4)2-Fc + Inosine and (Ex-4)2-Fc groups were greater than those in the ATB+(Ex-4)2-Fc group (Fig. 3I–K). These results suggest that supplementation with inosine enhanced (Ex-4)2-Fc efficacy in ATB mice.
3.4.　 Inosine reversed obesity-induced hypothalamic leptin resistance and improved obesity
Based on the above studies, we hypothesized that (Ex-4)2-Fc reversed obesity-induced hypothalamic leptin resistance through the metabolite inosine. We therefore designed experiments to verify whether inosine can treat obesity and reverse obesity-induced leptin resistance (Fig. 4A). Lower body weights and body weight gain were observed in the inosine-treated group (Fig. 4B). Inosine treatment effectively reduced food intake (Fig. S2C). Insulin (Fig. 4C) and glucose levels (Fig. 4D) were lower in the NC and inosine groups than in the HFD group. HE staining revealed that the adipocyte area and eWAT weight in the NC and inosine groups were smaller than those in the HFD group (Fig. 4E).
Further studies revealed lower circulating leptin and eWAT leptin expression levels in the inosine group (Fig. 4F). Leptin acts on the hypothalamus to decrease food intake and increase energy expenditure, requiring signal transducer and activator of transcription 3 (STAT3) signaling23. We also found that inosine-treated mice had increased phosphorylated STAT3 (p-STAT3) protein expression and p-STAT3+ cell numbers in the hypothalamus (Fig. 4G and H), and supplementation with the STAT3 antagonist S3I-201 partially blocked the therapeutic effects of inosine (Supporting Information Fig. S4). These results support the hypothesis that inosine could improve obesity and reverse obesity-induced leptin resistance (Fig. 4I).
3.5.　 Inosine therapy for obesity via the leptin pathway
Next, we tested whether inosine could cure obesity via the leptin pathway using leptin-deficient (ob/ob) and leptin receptor-deficient (db/db) mice (Fig. 5A). Body weight, weight gain, and food intake did not differ between db/db and ob/ob mice after inosine treatment (Fig. 5B, Fig. S2D). In addition, inosine treatment did not significantly improve the insulin sensitivity, glucose tolerance, or adipose tissue of db/db or ob/ob mice (Fig. 5C–F). These results suggest that inosine action depended on the leptin pathway.
3.6.　 Inosine inhibited obesity-related inflammation by regulating the macrophage A2A pathway
We next explored the mechanism by which inosine reduces leptin production in adipocytes. Adenosine catabolism produces inosine, which also plays an anti-inflammatory role through adenosine receptor signaling24. Therefore, we examined whether inosine regulated adipocyte leptin secretion by targeting adenosine receptors (Supporting Information Fig. S5A). Transcriptome sequencing analysis of four adenosine receptors, namely, Adaor1 receptor (A1), A2A, Adaor2b receptor (A2B), and Adaor3 receptor (A3), in eWAT, after (Ex-4)2-Fc treatment revealed that only A2A expression increased (Fig. S5B). Thus, we speculated that inosine may regulate adipocyte leptin secretion by activating A2A. Western blot and RT-PCR analyses also revealed the inosine-activated A2A expression in eWAT (Fig. 6A). Immunofluorescence analysis revealed that A2A was co-expressed with macrophage marker F4/80, but not with adipocytes (Fig. 6B). The number of F4/80++A2A+ cells in the inosine group was greater than that in the HFD group (Fig. 6B and C). This result indicated that inosine mainly activated A2A in eWAT macrophages. Using in vitro simulated cell models, we also demonstrated that inosine activated A2A expression in bone marrow-derived macrophages (BMDMs) but not in 3T3-L1 cells (Fig. S5C).
Obesity induces adipose tissue-related inflammation, such as increased IL-6 and TNF-α expression levels40. Previous studies have shown that A2A signaling attenuates LPS-induced macrophage inflammation39,40. Our results demonstrate that inosine regulated A2A signaling-related downstream protein expression and the expression of proinflammatory cytokines in eWAT (Fig. 6D, Fig. S5D). Inosine also inhibited proinflammatory cytokine expression in BMDMs, and this effect was partially reversed by treatment with ZM241385 (an A2A antagonist) (Fig. 6E). Moreover, flow cytometry was used to analyze the proportions of M1-type macrophages with pro-inflammatory effects and M2-type macrophages with anti-inflammatory effects in eWAT. The results showed that inosine therapy increased the number of M2-type cells and decreased the number of M1-type cells (Fig. S5E). These results indicate that inosine inhibited obesity-related inflammation by regulating macrophage A2A signaling.
3.7.　 Inosine indirectly reduced adipocyte leptin levels by regulating macrophages
To better mimic the environment of inosine in eWAT, we isolated and cultured adipose tissue stromal vascular cells (SVFs) from normal and obese mice. The results showed that inosine reduced the expression of leptin, and the ability of inosine to inhibit leptin expression was partially abrogated after treatment with ZM241385 (Fig. 7A). In addition, inosine did not directly inhibit leptin secretion in LPS-stimulated 3T3-L1 cells but increased leptin expression (Fig. 7B). Therefore, we hypothesized that inosine may indirectly reduce adipocyte leptin expression by inhibiting macrophage inflammation. We tested our hypothesis by performing validation experiments, coculturing inosine-treated BMDMs with 3T3-L1 cells, and detecting leptin expression (Fig. 7C). The results showed that inosine inhibited leptin expression, and the inosine concentration had a certain positive correlation with leptin levels (Fig. 7C). In addition, the ability of inosine to reduce leptin expression levels in 3T3-L1 and BMDM cocultured cells treated with ZM241385 (Fig. 7C) or isolated from A2A−/− mice (Fig. 7D) was partially disrupted. In summary, inosine indirectly influenced adipocyte leptin secretion by regulating macrophage A2A signaling (Fig. 7E).
3.8.　 (Ex-4)2-Fc partially reversed obesity-induced hypothalamic leptin resistance and improved obesity via A2A signaling
Finally, we used A2A−/− mice to verify whether (Ex-4)2-Fc reversed obesity-induced hypothalamic leptin resistance via A2A signaling (Fig. 8A). We identified wild-type (WT) and A2A−/− mice by PCR (Supporting Information Fig. S6A). WT and A2A−/− mice were fed a high-fat diet to induce obesity (Fig. S6B–S6D). Compared to A2A−/− mice, WT mice showed more significant weight loss and less body weight gain after (Ex-4)2-Fc treatment (Fig. 8B), while food intake was greater (Fig. S2E). In addition, the improvement effect of (Ex-4)2-Fc on insulin, glucose tolerance, and eWAT was also partially disrupted by the loss of A2A (Fig. 8C–E). The WT+(Ex-4)2-Fc group showed lower leptin levels in serum and eWAT (Fig. 8F) and higher p-STAT3 expression in the hypothalamus (Fig. 8G and H). Similarly, the levels of relevant inflammatory markers in the WT (Ex-4)2-Fc group were lower than those in the A2A−/− +(Ex-4)2-Fc group (Fig. 8I and J, Supporting Information Fig. S7). Overall, these results show that (Ex-4)2-Fc partially reversed obesity-induced hypothalamic leptin resistance and improved obesity via A2A signaling.
4.　Discussion
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The gut microbiota was shown to make a crucial contribution to the development of obesity and metabolic disease22-24. Some studies have shown that GLP-1RAs, such as liraglutide, modulate the gut microbiota41. For example, liraglutide treatment in patients with T2D significantly increased the diversity and richness of the gut microbiota, particularly Bacteroides, Proteobacteria, and Bacillus42. In addition, it was also suggested that the reason why the use of GLP-1RAs had to be terminated in the trial due to GLP-1 resistance in clinical application may be related to gut microbiota imbalance43,44. In 2017, how gut microbiota dysbiosis induces GLP-1 resistance was well exhibited in mice28. In one human study, different groups of gut microbes were found to respond differently to GLP-1RAs45. Many studies have shown that GLP-1 is closely related to the functional gut microbiota28,46,47. Therefore, the characteristics of the gut microbiota can predict the efficacy of GLP-1RAs. However, a randomized controlled trial showed that liraglutide and sitagliptin did not change the alpha or beta diversity of the gut microbiota in adults with T2D48. In addition, a fixed combination of liraglutide and degludec for 6 months did not change the microbiome biodiversity or community among a group of very old T2D patients49. The possible reason was that the combination of drugs masked the effect47. Our research aimed to explore the mechanism of (Ex-4)2-Fc from the perspective of the gut microbiota. We found that (Ex-4)2-Fc induced a decrease in Firmicutes and Proteobacteria and an increase in Bacteroidetes and Verrucomicrobia. In particular, Am in Verrucobacteria increased significantly after (Ex-4)2-Fc treatment. Similarly, some previous studies have also shown significant increases in intestinal Am abundance in wild-type obese mice and db/db mice treated with liraglutide40,41,50,51. However, the relationships between the gut microbiota, GLP-1, and the host are still unclear. At present, many studies on the correlation between GLP-1 and the gut microbiota are limited. In the future, the development of technologies will help explain and validate the relationship between GLP-1 and the gut microbiota. The clinical use of GLP-1RAs should consider the impact of the gut microbiota and should provide a personalized regimen.
To better elucidate the relationship between (Ex-4)2-Fc treatment and the gut microbiota, previous studies have shown that the gut microbiota can interact with hosts through metabolites41,52-55 and that drugs can also affect metabolism by changing the intestinal microflora and its metabolites32. Therefore, we explored the mechanism of (Ex-4)2-Fc therapy through the gut microbiota or metabolites. The results confirmed that the therapeutic effect of (Ex-4)2-Fc was partly related to the gut microbiota, and its therapeutic effect was related to Am and its metabolite inosine. In our previous studies, we found that (Ex-4)2-Fc improved obesity and inhibited obesity-related inflammation by reducing leptin levels10. Leptin is a hormone secreted from adipocytes that acts on the hypothalamus to decrease food intake and increase energy expenditure, effects that require STAT3 signaling32. Studies have indicated that leptin signaling in the hypothalamus of obese mice not only remains functional but is also permanently activated56. This persistent activation was due to endogenous high circulating leptin concentrations, and there was a saturation of leptin signaling with a failure to further respond to even higher concentrations of exogenous leptin. Some recent reports indicated that partial leptin reduction in obese mice improved glucose tolerance and insulin sensitivity and reduced body weight21,57, which is consistent with our current partial leptin reduction strategy. However, the potential mechanism of action of GLP-1RAs in the treatment of obesity through the leptin pathway has not been understood until now. Based on the finding that (Ex-4)2-Fc treatment can increase inosine and reverse obesity-induced hypothalamic leptin antagonism, we hypothesized that it may regulate body leptin levels through the metabolite inosine. Indeed, we found that inosine therapy reversed obesity-induced hypothalamic leptin antagonism and ameliorated obesity through the leptin pathway in db/db and ob/ob mice. Moreover, studies have demonstrated that inosine mediates proinflammatory cytokine production, which is partially reversed by the A2A antagonist DMPX38. Similarly, our study revealed that the reduction in proinflammatory cytokine production induced by inosine was partially reversed by ZM241385. Moreover, inosine decreased adipocyte leptin levels indirectly by regulating macrophage A2A. In addition, inosine-mediated improvements in obesity were partly dependent on the A2A pathway (Supporting Information Fig. S8). We further verified that (Ex-4)2-Fc reversed leptin antagonism through A2A signaling in A2A−/− mice.
5.　Conclusions
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Our study revealed that GLP-1RAs partially reversed obesity-induced hypothalamic leptin antagonism through microbiota-driven inosine and provided new insights into guiding the clinical use of antidiabetic agents.
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Appendix
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Year 2025 volume 15 Issue 2
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doi: 10.1016/j.apsb.2024.12.006
  • Receive Date：2024-04-04
  • Online Date：2026-09-17
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  • Received：2024-04-04
  • Revised：2024-06-26
  • Accepted：2024-07-24
Affiliations
    State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center of Biotherapy, Chengdu 610041, China

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表12种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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