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IL1R1 is required for celastrol’s leptin-sensitization and antiobesity effects

Nature Medicine volume 25, pages 575–582 (2019)Cite this article

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Abstract

Celastrol, a pentacyclic triterpene, is the most potent antiobesity agent that has been reported thus far1. The mechanism of celastrol’s leptin-sensitizing and antiobesity effects has not yet been elucidated. In this study, we identified interleukin-1 receptor 1 (IL1R1) as a mediator of celastrol’s action by using temporally resolved analysis of the hypothalamic transcriptome in celastrol-treated DIO, lean, and db/db mice. We demonstrate that IL1R1-deficient mice are completely resistant to the effects of celastrol in leptin sensitization and treatment of obesity, diabetes, and nonalcoholic steatohepatitis. Thus, we conclude that IL1R1 is a gatekeeper for celastrol’s metabolic actions.

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Fig. 1: Identification of IL1R1 as a candidate mediator of celastrol’s action.
Fig. 2: IL1R1 is required for celastrol’s antiobesity effects.
Fig. 3: IL1R1 is required for celastrol’s ability to enhance the actions of exogenous leptin.
Fig. 4: Administration of anakinra attenuates celastrol action.

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Data availability

Raw data from the mouse hypothalamic microarray analysis are available from the Gene Expression Omnibus (GEO) under accession numbers GSE124353 (DIO mice), GSE124355 (db/db mice), and GSE124356 (lean mice). The source images for p-STAT3 staining can be found at https://figshare.com/s/0741ab007eefdf3f6605. All other data that support the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

This work was mainly supported by the funds provided to U.O. from the Department of Medicine, the Smith President’s Innovation Award from Boston Children’s Hospital, and grants R01DK098496 and R56DK098496 provided to U.O. by the National Institutes of Health, as well as by support from the Fidelity Biosciences Research Initiative.

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Author notes

  1. These authors contributed equally: Xudong Feng, Dongxian Guan.

Authors and Affiliations

  1. Division of Endocrinology, Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA

    Xudong Feng, Dongxian Guan, Thomas Auen, Jae Won Choi, Mario Andrés Salazar Hernández, Jaemin Lee, Farhana Faruk, Esther Kaplun, Kyle D. Copps & Umut Ozcan

  2. Department of Mathematics and Statistics, Boston University, Boston, MA, USA

    Hyonho Chun

  3. Molecular Biology Core Facilities, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA

    Zachary Herbert

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Contributions

U.O. posited that IL1R1 might have a role in celastrol’s leptin-sensitizing effects and directed initial and subsequent analyses. X.F., D.G., T.A., J.L., J.W.C., M.A.S.H., F.F., and E.K. performed experiments under the direction of U.O. Data were analyzed by U.O., X.F., D.G., and J.L. H.C. performed limma F-test analysis, and Z.H. provided help for principal-component analysis and sample–sample correlation analysis. K.D.C. provided scientific criticism and help during revision of the manuscript. The finalized manuscript was prepared by X.F., K.D.C., and U.O.

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Correspondence to Umut Ozcan.

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U.O. is a scientific founder, shareholder, and member of the scientific advisory board and board of directors of ERX Pharmaceuticals.

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Extended data

Extended Data Fig. 1 Microarray analysis of the hypothalamic transcriptome.

a, Flow chart for microarray analysis. b, Heat map depicting the average log2 FC (Cel/Veh) for the 38 upregulated genes and 15 downregulated genes in pattern 10 and pattern 17 from Fig. 1h. c, Heat map showing clustering of 74 upregulated genes and 29 downregulated genes from limma F-test analysis. d, GO pathways significantly enriched in 74 upregulated genes from limma F-test analysis. e, log2-transformed fold change (log2 FC) in the nine genes present in the GO pathway regulation of inflammatory response in DIO mice after 6 h, 1 d, and 4 d of vehicle or celastrol administration. n = 4 mice (6 h, 1 d) and n = 3 mice (4 d). Values indicate means ± s.e.m. f, Heat map showing log2-transformed expression of the nine identified genes present in the GO pathway regulation of inflammatory response in DIO, lean, and db/db mice at 6 h, 1 d, and 4 d. g, Geometric mean of the fold change values at 1 d and 4 d for the identified genes in DIO, lean, and db/db mice. h,i, Hypothalamic mRNA levels for the identified genes in DIO mice treated with vehicle or celastrol for 1 d (Il1r1, P = 0.0002; Ada, P = 0.0001; Nfkbia, P = 0.01; Ptgs2, P = 0.003; Tgm2, P = 0.0002; Zfp36, P = 0.01; n = 6 mice for each group) (h) and 4 d (Il1r1, P = 0.01; Ada, P = 0.03; Nfkbia, P = 0.01; Ptgs2, P = 0.009; Tgm2, P = 0.007; Zfp36, P = 0.02; n = 4 mice for each group) (i). Values indicate averages ± s.e.m. P values were determined by two-tailed Student’s t test in h and i. *P < 0.05, **P < 0.01, ***P < 0.001.

Source Data

Extended Data Fig. 2 IL1R1 deficiency leads to significantly higher levels of obesity.

Il1r1–/– mice and their wild-type (Il1r1+/+) littermates were fed a HFD for 16 weeks to induce obesity. The experiments were repeated two times with similar results. a, PCR analysis of the Il1r1 locus in genomic DNA from Il1r1+/+ and Il1r1–/– mice. b,c, qPCR analysis of Il1r1 mRNA levels in the hypothalamus (P < 0.0001) (b), and liver (P = 0.007) (c). n = 6 for the Il1r1+/+ group and n = 4 for the Il1r1–/– group. d, Body weights of Il1r1+/+ and Il1r1–/– mice during HFD feeding (n = 10 mice for the Il1r1+/+ group and n = 9 mice for the Il1r1–/– group; P < 0.0001). e, Average 24-h food intake per mouse after 4, 8, 12, or 16 weeks of HFD feeding (n = 3 cages for each group). f–h, DEXA analysis of body composition after 16 weeks of HFD (n = 10 mice for the Il1r1+/+ group and n = 9 mice for the Il1r1–/– group), including lean mass (P = 0.163) (f), fat mass (P = 0.003) (g), and fat percentage (P = 0.001) (h). i, Plasma leptin levels of Il1r1+/+ and Il1r1–/– mice at 4, 8, 12, and 16 weeks of HFD feeding (n = 5 mice for each group; 4 weeks, P = 0.001; 8 weeks, P = 0.02). j,k, Blood glucose levels of Il1r1+/+ and Il1r1–/– mice during GTT performed after 10 weeks on the HFD (j) and AUC analysis of GTT results (n = 10 mice for the Il1r1+/+ group and n = 9 mice for the Il1r1–/– group; P = 0.03) (k). l,m, Blood glucose levels of Il1r1+/+ and Il1r1–/– mice during ITT performed after 12 weeks on HFD feeding (l) and AUC analysis of ITT results (n = 10 mice for the Il1r1+/+ group and n = 9 mice for the Il1r1–/– group; P = 0.007) (m). n, Fifteen-hour fasting blood glucose levels of Il1r1+/+ and Il1r1–/– mice after 10 weeks of HFD feeding (n = 10 mice for the Il1r1+/+ group and n = 9 mice for the Il1r1–/– group; P = 0.03). o, Six-hour fasting blood glucose levels of Il1r1+/+ and Il1r1–/– mice after 12 weeks on HFD feeding (n = 10 mice for the Il1r1+/+ group and n = 9 mice for the Il1r1–/– group; P = 0.003). p, Plasma insulin levels in Il1r1+/+ and Il1r1–/– mice after 4, 8, 12, or 16 weeks on HFD feeding (n = 5 mice for the Il1r1+/+ group and n = 4 mice for the Il1r1–/– group; 4 weeks, P = 0.007; 8 weeks, P = 0.184). Values indicate averages ± s.e.m. P values were determined by two-way ANOVA with Bonferroni’s multiple-comparisons test (d,j,l) or two-tailed Student’s t test (b,c,e–i,k,m–p). *P < 0.05, **P < 0.01, ***P < 0.001; n.s., not significant (P > 0.05).

Source Data

Extended Data Fig. 3 Celastrol does not change the body temperature of Il1r1+/+ or Il1r1–/– mice.

a–c, Il1r1+/+ and Il1r1–/– mice were fed a HFD for 20 weeks and treated with vehicle or celastrol (100 μg/kg body weight, i.p., once a day) for 3 weeks. Graphs show core body temperature (°C) for Il1r1+/+ and Il1r1–/– mice after 1 week (a), 2 weeks (b), or 3 weeks (c) of vehicle or celastrol treatment. Values indicate averages ± s.e.m. n = 9 for both the vehicle- and celastrol-treated groups for Il1r1+/+ mice; n = 12 for the vehicle-treated and n = 13 for the celastrol-treated groups for Il1r1–/– mice. P values were determined by two-way ANOVA with Bonferroni’s multiple-comparisons test; P > 0.99 between each group at 1 week, 2 weeks, and 3 weeks of vehicle or celastrol treatment.

Source Data

Extended Data Fig. 4 Celastrol does not affect home-cage, anxiety, or social behavior in DIO mice.

a–o, DIO mice were treated with vehicle or celastrol (100 μg/kg body weight, i.p., once a day) for 3 d. On the fourth day, home-cage behavior (grooming) (d,e), open-field tests (f–l), and social behavior (m–o) were assessed under the ad libitum–fed condition. a, Average 24-h food intake per mouse during the treatment (n = 3 cages for each group; P = 0.0001). b,c, Body weight (day 0 vs. day 4, P > 0.99 for the vehicle-treated group and P = 0.002 for the celastrol-treated group) (b) and percent body weight reduction before (day 0) and after 4 d (day 4) of vehicle or celastrol treatment (P < 0.0001) (c). d, Frequency (numbers) of grooming bouts (P = 0.572). e, Total time the mice spent grooming during the home-cage behavior test (P = 0.255). n = 10 mice for both the vehicle- and celastrol-treated groups. f, Total distance traveled by the mice (P = 0.956). g, Average velocity of movement during the open-field test (P = 0.956). h, Duration (percent of time in the assay) the mice spent moving (P = 0.999). i, Frequency (number of times during assay) that mice traveled to the central portion of the field (P = 0.331). j, Total time spent in the central portion of the field (P = 0.591). k, Latency (in seconds) until initial entry into the central portion of the open field (P = 0.913). l, Representative movement-tracking plots of vehicle- and celastrol-treated mice in the open-field assay. n = 8 mice for each group. m, Total time the mice spent in the object-paired side chamber (object) versus the novel mouse 1–paired side chamber (novel mouse 1). Object chamber vs. novel mouse chamber, P = 0.007 in the vehicle-treated group and P < 0.0001 in the celastrol-treated group. n, Total time the mice spent with the object, which was in the left chamber, and with novel mouse 1, in the right chamber during the sociability test. Object vs. novel mouse, P = 0.0003 in the vehicle-treated group and P < 0.0001 in the celastrol-treated group. o, Total time the mice spent in the novel mouse 1–paired (novel mouse 1) or novel mouse 2–paired (novel mouse 2) side chamber during the social novelty test (novel mouse 1 vs. novel mouse 2, P = 0.01 in the vehicle-treated group and P = 0.02 in the celastrol-treated group). n = 8 mice for both groups during the sociability test and n = 7 for both groups during the social novelty test. Values indicate averages ± s.e.m. P values were determined by two-tailed Student’s t test (a,d–k) or two-way ANOVA with Bonferroni’s multiple-comparisons test (b,c,m–o). * P < 0.05, **P < 0.01, ***P < 0.001; n.s., not significant (P > 0.05).

Source Data

Extended Data Fig. 5 Celastrol treatment decreases motivation for food seeking in only fasted mice.

a–f, Conditioned place preference (CPP) assay under the ad libitum feeding condition: DIO mice were treated with celastrol (100 μg/kg body weight, i.p., once a day) for 3 d. On the fourth day, 1 h after the beginning of the light cycle, the mice were administered vehicle or celastrol (200 μg/kg body weight, i.p.) and the CPP assay was performed 6 h later under the ad libitum feeding condition (n = 8 mice for both the vehicle- and celastrol-treated groups). a, Total distance that the mice traveled during the test (P = 0.941). b, Average velocity of movement during the CPP assay (P = 0.934). c, Frequency (numbers during assay) with which mice traveled to the food-paired side chamber (P = 0.688). d, Total time the mice spent in the food-paired side chamber (P = 0.641). e, Percentage of total assay time the mice spent in the food-paired side chamber (P = 0.641). f, Frequency (times during assay) that mice traveled to the food-containing zone within the food-paired chamber (P = 0.557). g–l, CPP assay under a 20-h fasting condition: DIO mice were treated with celastrol (100 μg/kg body weight, i.p.) once a day and fasted for 15 h after the third injection. On the fourth day, 1 h after the beginning of the light cycle, the mice were administered vehicle or celastrol (200 μg/kg body weight, i.p.) and the CPP assay was performed 5 h after this injection under the fasting condition (n = 7 mice for both the vehicle- and celastrol-treated groups). g, Total distance traveled by the mice during the CPP assay (P = 0.02). h, Average velocity of movement (P = 0.02). i, Frequency with which the mice traveled to the food-paired side chamber (P = 0.104). j, Total time the mice spent in the food-paired side chamber during the test (P = 0.02). k, Percent of total assay time the mice spent in the food-paired side chamber (P = 0.02). l, Frequency with which the mice traveled to the food-containing zone within the food-paired side chamber (P = 0.01). m,n, Representative traces showing the movements of individual vehicle- and celastrol-treated mice in the CPP assays conducted under the ad libitum feeding (m) or 20-h fasting (n) condition. Values indicate averages ± s.e.m. P values were determined by two-tailed Student’s t test. *P < 0.05; n.s., not significant (P > 0.05).

Source Data

Extended Data Fig. 6 Celastrol fails to increase STAT3 phosphorylation and gene expression in the hypothalamus of Il1r1–/– mice.

Il1r1+/+ and Il1r1–/– male mice were fed a HFD for 20 weeks and then administered either vehicle (Veh) or celastrol (Cel; 100 μg/kg body weight, i.p.) daily for 3 d. Each group of mice subsequently received a single dose of vehicle or celastrol (200 μg/kg body weight, i.p.) on the morning of the fourth day and was then fasted for 6 h prior to extraction of the hypothalamus. Phosphorylation of STAT3 (p-STAT3Tyr705) in the medial basal hypothalamus (MBH) was analyzed by immunofluorescence staining using a phospho-specific antibody. a,c,e, Representative images of p-STAT3Tyr705 immunostaining in the arcuate nucleus (ARC) (a), ventromedial hypothalamus (VMH) (c), and dorsomedial hypothalamus (DMH) (e) of Il1r1+/+ and Il1r1–/– mice after 4 d of vehicle or celastrol treatment. b,d,f, Quantification of total p-STAT3Tyr705-positive cell numbers and total fluorescence intensity for p-STAT3Tyr705 in the ARC (Il1r1+/+–Veh vs. Il1r1+/+–Cel, P = 0.0007, and Il1r1+/+–Cel vs. Il1r1–/––Cel, P = 0.006 for cell number; Il1r1+/+–Veh vs. Il1r1+/+–Cel, P = 0.001, and Il1r1+/+–Cel vs. Il1r1–/––Cel, P = 0.008 for fluorescence intensity) (b), VMH (Il1r1+/+–Veh vs. Il1r1+/+–Cel, P = 0.0003, and Il1r1+/+–Cel vs. Il1r1–/––Cel, P = 0.0006 for cell number; Il1r1+/+–Veh vs. Il1r1+/+–Cel, P = 0.0004, and Il1r1+/+–Cel vs. Il1r1–/––Cel, P = 0.0007 for fluorescence intensity) (d), and DMH (Il1r1+/+–Veh vs. Il1r1+/+–Cel, P = 0.002, and Il1r1+/+–Cel vs. Il1r1–/––Cel, P = 0.02 for cell number; Il1r1+/+–Veh vs. Il1r1+/+–Cel, P = 0.004, and Il1r1+/+–Cel vs. Il1r1–/––Cel, P = 0.01 for fluorescence intensity) (f). The experiments in a–f were repeated in two independent cohorts with similar outcomes, and the results in b, d, and f represent the combination of two independent experiments (total n = 7 mice for both vehicle- and celastrol-treated mice in the Il1r1+/+ group; n = 8 for both vehicle- and celastrol-treated mice in the Il1r1–/– group). Scale bars, 100 μm. 3V, third ventricle. g–j, Expression levels of genes in the hypothalamus of Il1r1+/+ and Il1r1–/– mice treated with vehicle or celastrol (100 μg/kg body weight, i.p., once a day) for 4 d, including for Agrp (Il1r1+/+–Veh vs. Il1r1+/+–Cel, P = 0.005; Il1r1–/––Veh vs. Il1r1–/––Cel, P > 0.99) (g), Npy (Il1r1+/+–Veh vs. Il1r1+/+–Cel, P = 0.362; Il1r1–/––Veh vs. Il1r1–/––Cel, P = 0.871) (h), Pomc (Il1r1+/+–Veh vs. Il1r1+/+–Cel, P = 0.757; Il1r1–/––Veh vs. Il1r1–/––Cel, P > 0.99) (i), and Socs3 (Il1r1+/+–Veh vs. Il1r1+/+–Cel, P = 0.03; Il1r1–/––Veh vs. Il1r1–/––Cel, P > 0.99) (j) (n = 6 vehicle-treated and n = 7 celastrol-treated mice in the Il1r1+/+ group; n = 4 vehicle- and celastrol-treated mice in the Il1r1–/– group). Values indicate averages ± s.e.m. P values were determined by two-way ANOVA with Bonferroni’s multiple-comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001; n.s., not significant (P > 0.05).

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Extended Data Fig. 7 IL1R1 deficiency or IL1R1 antagonist treatment reduces MAP kinase phosphorylation.

a–d, Ad libitum–fed Il1r1+/+ mice and food-restricted (FR) Il1r1–/– mice (~0.5 g of food per day) were treated with vehicle (Veh) or celastrol (Cel; 100 μg/kg body weight, i.p., daily) for 7 d and the phosphorylation status of hypothalamic or hepatic MAP kinases (p38, Erk1/2) was analyzed by western blot. a, Representative immunoblots for phoshorylated p38 MAP kinase (p-p38) and total p38 MAP kinase (p38) in the hypothalamus of Il1r1+/+ and FR Il1r1–/– mice. b, Ratio of quantified p-p38 density to p38 density. n = 3 mice for each group. Il1r1+/+–Veh vs. Il1r1–/––Veh, P < 0.0001; Il1r1+/+–Cel vs. Il1r1–/––Cel, P < 0.0001; Il1r1–/––Veh vs. Il1r1–/––Cel, P > 0.99. c, Representative immunoblots for phosphorylated MAP kinase (p-Erk1/2) and Erk1/2 MAP kinase in the liver of Il1r1+/+ and FR Il1r1–/– mice. d, Ratio of quantified p-Erk1/2 density to Erk1/2 density. n = 3 mice for each group. Il1r1+/+–Veh vs. Il1r1+/+–Cel, P = 0.06; Il1r1+/+–Cel vs. Il1r1–/––Cel, P = 0.01; Il1r1–/––Veh vs. Il1r1–/––Cel, P > 0.99. e,f, DIO mice were administered the IL1R1 antagonist anakinra (Ank; 5 μg/mouse per day) through intracerebroventricular injection into the lateral ventricle in combination with celastrol (100 μg/kg body weight, i.p., daily) for 7 d. e, Representative immunoblots for p-p38 and total p38 in the hypothalamus. f, Ratio of quantified p-p38 density to p38 density. n = 5 mice for each group. P = 0.03. g,h, DIO mice were treated with IL1R1 antagonist (Ank; 30 mg/kg body weight, i.p., twice a day) in combination with celastrol (100 μg/kg body weight, i.p., daily) for 7 d. g, Representative immunoblots for p-Erk1/2 and total Erk1/2 in the liver. h, Ratio of quantified p-Erk1/2 density to Erk1/2 density. n = 6 mice for each group. Sal + Veh vs. Sal + Cel, P = 0.0004; Ank + Veh vs. Ank + Cel, P > 0.99; Sal + Cel vs. Ank + Cel, P = 0.0004. Values indicate averages ± s.e.m. P values were determined by two-way ANOVA with Bonferroni’s multiple-comparisons test (b,d,h) or two-tailed Student’s t test (f). *P < 0.05, ***P < 0.001; n.s., not significant (P > 0.05).

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Extended Data Fig. 8 Effect of celastrol on mice with heterozygous deletion of Il1r1.

a, Hypothalamic Il1r1 mRNA levels in Il1r1+/+ and Il1r1+/– mice determined by qPCR. n = 7 mice for each group. Values indicate averages ± s.e.m.; P = 0.0009, determined by two-tailed Student’s t test. b–g, Il1r1+/+ and Il1r1+/– or Il1r1–/– mice (with average body weight of 37–40 g) were fed on a chow diet and treated with vehicle or celastrol (100 μg/kg body weight, i.p., once a day) for 7 d. n = 7 mice for each group. b, Body weight. Il1r1+/+–Veh vs. Il1r1+/+–Cel, P = 0.0005; Il1r1+/––Veh vs. Il1r1+/––Cel, P < 0.0001; Il1r1–/––Veh vs. Il1r1–/––Cel, P = 0.753. c, Percent body weight change during treatment. Il1r1+/+–Veh vs. Il1r1+/+–Cel and Il1r1+/––Veh vs. Il1r1+/––Cel, P < 0.0001; Il1r1–/––Veh vs. Il1r1–/––Cel, P = 0.576. d, Average daily food intake during treatment. Il1r1+/+–Veh vs. Il1r1+/+–Cel, P = 0.001; Il1r1+/––Veh vs. Il1r1+/––Cel, P = 0.002; Il1r1–/––Veh vs. Il1r1–/––Cel, P > 0.99; Il1r1+/+–Cel vs. Il1r1+/––Cel, P > 0.99; Il1r1+/––Cel vs. Il1r1–/––Cel, P = 0.02. e, Plasma leptin levels after 7 d of treatment. Il1r1+/+–Veh vs. Il1r1+/+–Cel, P = 0.03; Il1r1+/––Veh vs. Il1r1+/––Cel, P = 0.04; Il1r1–/––Veh vs. Il1r1–/––Cel, P > 0.99; Il1r1+/+–Cel vs. Il1r1+/––Cel, P > 0.99; Il1r1+/––Cel vs. Il1r1–/––Cel, P = 0.06. f, Six-hour fasting blood glucose levels after 7 d of treatment. Il1r1+/+–Veh vs. Il1r1+/+–Cel and Il1r1+/––Veh vs. Il1r1+/––Cel, P < 0.0001; Il1r1–/––Veh vs. Il1r1–/––Cel and Il1r1+/+–Cel vs. Il1r1+/––Cel, P > 0.99; Il1r1+/––Cel vs. Il1r1–/––Cel, P = 0.0002. g, Plasma IL-1β levels after 7 d of treatment (P > 0.99, between each group). Values indicate averages ± s.e.m. P values were determined by two-way ANOVA with Bonferroni’s multiple-comparisons test. *P < 0.05, ***P < 0.001; n.s., not significant (P > 0.05).

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Extended Data Fig. 9 IL1R1 interacts with LepRb.

HEK 293 cells were transfected with plasmids expressing LacZ, human IL1R1 (hIL1R1), Flag-tagged LepRb, or hIL1R1 and Flag-tagged LepRb together. The experiments were repeated two times with similar outcomes. Representative immunoblots depict the immunoblotting results for IL1R1 and Flag in the Flag immunoprecipitates (top). The expression levels of IL1R1 and HSP90 in the input total-cell lysates are shown in the lower panels.

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Feng, X., Guan, D., Auen, T. et al. IL1R1 is required for celastrol’s leptin-sensitization and antiobesity effects. Nat Med 25, 575–582 (2019). https://doi.org/10.1038/s41591-019-0358-x

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