Il1r1 is required for celastrol’s leptin-sensitization and antiobesity effects nature medicine gas vs electric oven

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a, Flow chart for microarray analysis. b, Heat map depicting the average log 2 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 i gas shares and 29 downregulated genes from limma F-test analysis. d, GO pathways significantly enriched in 74 upregulated genes from limma F-test analysis. e, log 2-transformed fold change (log 2 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 log 2-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.

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 gas mask tattoo 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 electricity and magnetism ppt). 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).

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-STAT3 Tyr705) in the medial basal hypothalamus (MBH) was analyzed by immunofluorescence staining using a phospho-specific antibody. a, c, e, Representative images of p-STAT3 Tyr705 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-STAT3 Tyr705-positive cell numbers and total fluorescence intensity gas after eating fruit for p-STAT3 Tyr705 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 electricity and circuits 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).

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  0.99. c, Representative immunoblots for phosphorylated MAP kinase (p-Erk1/2) and Erk1/2 MAP kinase in the liver of Il1r1 +/+ and FR Il1r1 b games unblocked –/– 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 mp electricity bill payment online indore, 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).

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  0.99; 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  0.99; 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).