Interleukin-13 drives metabolic conditioning of muscle to endurance exercise

Abstract

Repeated bouts of exercise condition muscle mitochondria to meet increased energy demand-an adaptive response associated with improved metabolic fitness. We found that the type 2 cytokine interleukin-13 (IL-13) is induced in exercising muscle, where it orchestrates metabolic reprogramming that preserves glycogen in favor of fatty acid oxidation and mitochondrial respiration. Exercise training-mediated mitochondrial biogenesis, running endurance, and beneficial glycemic effects were lost in Il13^-/- mice. By contrast, enhanced muscle IL-13 signaling was sufficient to increase running distance, glucose tolerance, and mitochondrial activity similar to the effects of exercise training. In muscle, IL-13 acts through both its receptor IL-13Rα1 and the transcription factor Stat3. The genetic ablation of either of these downstream effectors reduced running capacity in mice. Thus, coordinated immunological and physiological responses mediate exercise-elicited metabolic adaptations that maximize muscle fuel economy.

Fig. 1.. IL-13 is an exercise-inducible factor regulating endurance capacity

(A) Resting plasma concentrations of IL-13 and IL-6 in sedentary (n=36 obese; n=23 normal weight) and exercise-trained (n=25) women. Linear regressions were used to examine associations adjusting for age and obesity status. Detailed data are in table S1. (B) Resting plasma concentrations of IL-13 and IL-6 in normal-weight sedentary controls (n=48) and exercise-trained men (n=25 cross-country; n=24 football). Linear regressions were used to examine associations adjusting for age and training type. Detailed data are in table S2. (C) mRNA expression of Il13 and Il6 in primary myotubes (differentiated from primary myoblasts or satellite cells) or muscle immune/stromal cells (Percoll gradient-isolated) measured by qPCR. n=4–6/group, experiment performed twice, statistical analysis performed using unpaired Student’s t-test. (D) mRNA expression of Il13 and Il6 in muscle immune/stromal cells from untrained or 4-week endurance–trained mice measured by qPCR. n=6/group, experiment performed twice, statistical analysis performed using unpaired Student’s t-test. (E) Quantification of % ILC2 (Gata3⁺), ILC3 (Rorγt⁺), and T cells (CD3⁺) among CD45⁺ cells in skeletal muscle of untrained or 4-week endurance–trained mice. n=6/group, 10-week-old male mice, experiment performed twice, statistical analysis performed using unpaired Student’s t-test. (F) Quantification of % IL-13⁺ ILC2 (Gata3⁺) and T cells (CD3⁺) among CD45⁺ cells in skeletal muscle of untrained or 4-week endurance–trained mice. n=6/group, 10-week old male mice, experiment performed twice, statistical analysis performed using unpaired Student’s t-test. (G) Endurance capacity test performed by treadmill running in WT and Il13^−/− mice. n=9/group, 16-week-old male mice, experiment performed four times, statistical analysis performed using unpaired Student’s t-test. Additional mouse cohort data included in table S3. Error bars indicate SEM. *p<0.05, **p<0.01, ***p<0.001.

Fig. 2.. IL-13 regulates metabolic substrate utilization in exercising muscle.

(A) Illustration of key metabolic genes in muscle metabolism regulated by endurance training in gastrocnemius of WT and Il13^−/− mice identified by RNA-seq. Data presented as heatmap (log₂-fold change, trained versus untrained of the same genotype) with WT animals on the left and Il13^−/− on the right. Red indicates higher expression in exercised muscle, whereas blue indicates lower expression. n=4/group, 20-week-old male mice. (B) Fatty acid uptake and oxidation and (C) glucose uptake in C2C12 myotubes treated with rIL-13 (10 ng/mL) overnight. n=5–6 biological replicates/group/experiment, experiment performed four times, statistical analysis performed using unpaired Student’s t-test. (D) Triglyceride (TG) and glycogen levels in the quadriceps of non-exercised (control) and single-session exercised, WT and Il13^−/− mice. n=4–5/group, 24-week-old male mice, experiment performed twice, statistical analysis (non-exercise vs single-session of the same genotype) performed using unpaired Student’s t-test. (E) Serum concentrations of free fatty acids (FFA), triglyceride (TG) and glycerol in control and single-session exercised, WT and Il13^−/− mice. n=4–5/group, 24-week-old male mice, experiment performed twice, statistical analysis (non-exercise vs single-session of the same genotype) performed using unpaired Student’s t-test. (F) Respiration of exercising WT and Il13^−/− mice at increasing running speeds. Maximum oxygen uptake (VO₂ max) under the experimental setting was assessed and shown in the right panel. n=4/group, 24-week-old female mice, experiment performed twice, statistical analysis performed using two-way ANOVA with Bonferroni post-hoc test with animal group and time points modeled as variables (respiration) and unpaired Student’s t-test (VO₂ max). Error bars indicate SEM. *p<0.05, **p<0.01, ***p<0.001.

Fig. 3.. Mitochondrial biogenesis in endurance-trained muscle requires IL-13 signaling.

(A) Representative mitochondrial genes differentially regulated in the gastrocnemius of endurance-trained WT and Il13^−/− mice identified by RNA-seq. Data presented as a heatmap (log₂-fold change, trained versus untrained of the same genotype) with WT animals on the left and Il13^−/− on the right. Red indicates higher expression in exercised muscle, whereas blue indicates lower expression. n=4/group, 20-week-old male mice. (B) Left panel: mitochondrial respiration of C2C12 myotubes treated with rIL-13 (10 ng/mL) overnight. Oligomycin (1) was added to block ATP-coupled respiration, FCCP (2) to induce maximal respiration, and antimycin A/rotenone (3) to block mitochondrial electron transport. n=5 biological replicates/group/experiment, experiments performed six times, statistical analysis performed using two-way ANOVA with Bonferroni post-hoc test with treatment and time points modeled as variables. Right panel: mtDNA content of C2C12 myotubes treated with rIL-13 (10 ng/mL) overnight. Relative mitochondrial DNA (mtDNA) content was measured by qPCR normalized to nuclear DNA (nDNA). n=3 biological replicates/group/experiment, experiments performed six times, statistical analysis performed using unpaired Student’s t-test. (C) Immunoblot analyses of gastrocnemius mitochondrial protein content from untrained and endurance-trained WT and Il13^−/− mice. n=4/group, experiment performed twice, 20-week-old male mice. (D) mRNA expression of oxidative and glycolytic muscle fiber type markers measured by qPCR in gastrocnemius muscle from untrained and endurance-trained WT and Il13^−/− mice. n=6–7/group, 20-week-old male mice, experiment performed three times, statistical analysis (untrained vs trained of the same genotype) performed using unpaired Students t-test. (E) Quantification of succinate dehydrogenase positive (SDH⁺) muscle fibers in cross-sections of gastrocnemius from untrained and endurance-trained WT and Il13^−/− mice. n=8/group, 20-week-old male mice, experiment performed twice, statistical analysis (untrained vs trained of the same genotype) performed using unpaired Student’s t-test. Error bars indicate SEM. *p<0.05, **p<0.01, ***p<0.001.

Figure 4.. Endurance training increases running capacity and glucose tolerance via IL-13 signaling.

(A) Electron flow assay using Seahorse bioanalyzer with mitochondria isolated from gastrocnemius of untrained and endurance-trained WT and Il13^−/− mice. Complex I (C-I) respiration was measured using pyruvate and malate as substrates and blocked with rotenone. Complex II (C-II) was measured using succinate as substrate and blocked with antimycin A. Complex IV (C-IV) respiration was measured by injecting tetramethyl-p-phenylenediamine/ascorbate. n=15–25 replicates/group, data combined from three experiments, statistical analysis (untrained vs trained of the same genotype) performed using unpaired Student’s t-test. (B) Complex IV activity assay performed with gastrocnemius mitochondria from untrained and endurance-trained WT and Il13^−/− mice. The activity was assessed by the rate of cytochrome c oxidation measured by the decline in reduced cytochrome c (absorbance at 550 nm). n=3–5/group, 20-week-old female mice, experiment performed twice, statistical analysis performed using two-way ANOVA with Bonferroni post-hoc test with animal group and time points modeled as variables. (C) Endurance capacity test performed by treadmill running in untrained and endurance-trained WT and Il13^−/− mice. n=6–7/group, 16-week-old male mice, experiment performed twice, statistical analysis (untrained vs trained of the same genotype) performed using unpaired Student’s t-test. (D) Left panel: glucose tolerance test (GTT) of untrained and endurance-trained WT and Il13^−/− mice. Middle panel: area under the curve (AUC) of the GTT. Right panel: fasting serum insulin levels of untrained and endurance-trained WT and Il13^−/− mice. n=5/group for GTT/AUC and n=4/group for serum insulin, 20-week-old male mice, experiment performed three times, statistical analysis (untrained vs trained of the same genotype) performed using two-way ANOVA with Bonferroni post-hoc test with animal group and time points modeled as variables (GTT) and unpaired Student’s t-test (AUC and serum insulin). Error bars indicate SEM. *p<0.05, **p<0.01, ***p<0.001.

Fig. 5.. IL-13Rα1 and Stat3 are downstream effectors of IL-13 in muscle.

(A) Endurance capacity test of WT and Il13ra1^−/− mice performed by treadmill running. n=6/group, 20-week-old male mice, experiment performed twice, statistical analysis performed using unpaired Student’s t-test. (B) Endurance capacity test performed by treadmill running and ex vivo fatty acid oxidation of isolated soleus muscle from Il13ra1^f/f (control) and skmIl13ra1^−/− mice. n=6–8/group (endurance capacity) and n=5–8/group (fatty acid oxidation), 24-week-old male mice, experiment performed twice, statistical analysis performed using unpaired Student’s t-test. (C) Immunoblot analyses and quantification of Stat3 phosphorylation (Y705) in quadriceps muscle of untrained and endurance-trained WT and Il13^−/− mice. n=6/group, 16-week-old female mice, experiment performed three times, statistical analysis (untrained vs trained of the same genotype) performed using unpaired Student’s t-test. p-Stat3: phospho-Stat3; t-Stat3: total-Stat3. (D) Endurance capacity test performed by treadmill running and ex vivo fatty acid oxidation of isolated soleus muscle from Stat3^f/f (control) and skmStat3^−/− mice. n=12/group for endurance capacity and n=5/group for fatty acid oxidation, 24-week-old male mice, experiment performed twice, statistical analysis performed using unpaired Student’s t-test. Error bars indicate SEM. *p<0.05, **p<0.01.

Figure 6.. Increased IL-13 signaling in muscle drives a Stat3-dependent, exercise-like effect.

(A) Endurance capacity test performed by treadmill running of Stat3^f/f and skmStat3^−/− mice injected with adGFP or adIL-13 into gastrocnemius muscle. n=6–8/group, 24-week-old male mice, experiment performed twice, statistical analysis (adGFP vs adIL-13 of the same genotype) performed using unpaired Student’s t-test. Right panel: Ex vivo fatty acid oxidation of isolated soleus muscle from Stat3^f/f and skmStat3^−/− mice injected with adGFP or adIL-13 into gastrocnemius muscle. n=5–6/group, 24-week-old male mice, experiment performed once, statistical analysis (adGFP vs adIL-13 of the same genotype) performed using unpaired Student’s t-test. (B) Glucose tolerance test (GTT) of mice injected with adGFP or adIL-13 into gastrocnemius muscle. n=6–8/group, 24-week-old male mice, experiment performed twice, statistical analysis performed using two-way ANOVA with Bonferroni post-hoc test with animal group and time points modeled as variables. (C) mRNA expression of Esrra and Esrrg in gastrocnemius muscle of untrained and endurance-trained WT and Il13^−/− mice measured by qPCR. n=6–7/group, 20-week-old male mice, experiment performed twice, statistical analysis (untrained vs trained of the same genotype) performed using unpaired Student’s t-test. (D) mRNA expression of Esrra and Esrrg in quadriceps muscle of Stat3^f/f and skmStat3^−/− mice after gastrocnemius injection of adGFP or adIL-13 measured by qPCR. n=6–8/group, 24-week-old males, experiment performed once, statistical analysis (adGFP vs adIL-13 of the same genotype) performed using unpaired Student’s t-test. (E) Stat3 regulation of Esrrg gene promoter in reporter transient transfection assays. Relative luciferase activity of reporters driven by Esrrg promoters in AD293 cells co-transfected with increasing amounts of Stat3 expression vector. “1-kb”, “2-kb WT”, and “2-kb mutant” refer to reporter constructs containing 1-kb, 2-kb, and 2-kb with the mutated Stat3 binding site of Esrrg promoter, respectively. n=6 biological replicates/group/experiment, experiment performed four times, statistical analysis performed using unpaired Student’s t-test. (F) Relative luciferase activity of 2-kb Esrrg promoter in WT and Stat3^−/− C2C12 myoblasts. Stat3^−/− cells were co-transfected with a control or Stat3 expression vector ± rIL-13 overnight. n=12 biological replicates/group/experiment, experiment performed three times, statistical analysis performed using unpaired Student’s t-test. Error bars indicate SEM. *p<0.05, **p<0.01, ***p<0.001.