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. 2020 Jun 1;130(6):2888-2902.
doi: 10.1172/JCI133572.

Muscle-derived interleukin 6 increases exercise capacity by signaling in osteoblasts

Subrata Chowdhury  1 , Logan Schulz  1 , Biagio Palmisano  1 , Parminder Singh  2 , Julian M Berger  1 , Vijay K Yadav  1   2 , Paula Mera  1   3   4   5 , Helga Ellingsgaard  6   7 , Juan Hidalgo  8 , Jens Brüning  9 , Gerard Karsenty  1
Affiliations

Muscle-derived interleukin 6 increases exercise capacity by signaling in osteoblasts

Subrata Chowdhury et al. J Clin Invest. .

Abstract

Given the numerous health benefits of exercise, understanding how exercise capacity is regulated is a question of paramount importance. Circulating interleukin 6 (IL-6) levels surge during exercise and IL-6 favors exercise capacity. However, neither the cellular origin of circulating IL-6 during exercise nor the means by which this cytokine enhances exercise capacity has been formally established yet. Here we show through genetic means that the majority of circulating IL-6 detectable during exercise originates from muscle and that to increase exercise capacity, IL-6 must signal in osteoblasts to favor osteoclast differentiation and the release of bioactive osteocalcin in the general circulation. This explains why mice lacking the IL-6 receptor only in osteoblasts exhibit a deficit in exercise capacity of similar severity to the one seen in mice lacking muscle-derived IL-6 (mIL-6), and why this deficit is correctable by osteocalcin but not by IL-6. Furthermore, in agreement with the notion that IL-6 acts through osteocalcin, we demonstrate that mIL-6 promotes nutrient uptake and catabolism into myofibers during exercise in an osteocalcin-dependent manner. Finally, we show that the crosstalk between osteocalcin and IL-6 is conserved between rodents and humans. This study provides evidence that a muscle-bone-muscle endocrine axis is necessary to increase muscle function during exercise in rodents and humans.

Keywords: Bone Biology; Osteoclast/osteoblast biology; Skeletal muscle.

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Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. IL-6 is necessary for the increase in circulating osteocalcin levels observed in response to a training intervention in humans.
(A–C) Circulating IL-6 levels in (A) rhesus monkeys (14 ± 0.8 years old), (B) rats (4 months old), and (C) mice (3 months old) treated with vehicle or osteocalcin (Ocn) (13.5 ng/g for monkeys, 30 ng/g for rats, and 30 ng/g for mice). n = 12 per treatment. Statistical analyses were conducted using 1-way ANOVA followed by Tukey’s post hoc test (A) or 2-tailed unpaired t test (B and C). (D) Circulating uncarboxylated and bioactive Ocn levels in obese, nondiabetic subjects treated with either placebo or a neutralizing antibody against the IL-6 receptor in combination with or without intensive endurance exercise for 45 minutes, 3 times a week, for 12 consecutive weeks. n = 11 per group except no exercise with the tocilizumab group, n = 12. Statistical analyses were conducted using 1-way ANOVA followed by the Holm-Sidak post hoc test. All results presented as the mean ± SEM. *P < 0.05; **P < 0.01.
Figure 2
Figure 2. Muscle-derived IL-6 is needed for maximal exercise capacity.
(A) The promoter of the human α-skeletal actin (HSA) gene drives expression of the MerCreMer (MCM) gene, which harbors a mutated estrogen receptor (Mer) ligand-binding domain on each end of Cre recombinase. After crossing with Il6fl/fl mice and treatment with tamoxifen, a Cre-mediated recombination event results in the deletion of the Il6 gene. (B) Detection of Il6 deletion by PCR on genomic DNA isolated from various tissues of Il6Hsa–/– mice. (C) Detection of Il6 deletion by PCR on genomic DNA isolated from various skeletal muscles of Il6Hsa–/– mice. (D) Circulating IL-6 levels in 3-month-old male Il6fl/fl and Il6Hsa–/– mice at rest and after exercise. n = 8 per group. (E) Performance during an endurance run of 3- and 6-month-old Il6fl/fl and Il6Hsa–/– female mice. (F) Performance during an endurance test (running on a treadmill at 30 cm/s until exhaustion) of 8-month-old mice treated with vehicle or osteocalcin (Ocn, 500 ng/g) and an antibody against IL-6 or a control IgG, n = 7 per group. Results presented as the mean ± SEM. Data were analyzed with 2-tailed unpaired t test (D and E) or 1-way ANOVA followed by Tukey’s post hoc test (F) *P < 0.05; **P < 0.01.
Figure 3
Figure 3. mIL-6 favors exercise capacity in part through osteocalcin.
(A) Circulating bioactive osteocalcin (Ocn) levels in 3-month-old Il6fl/fl and Il6Hsa–/– mice at rest and after exercise, n = 12. (B) Performance during an endurance run of 3-month-old Ocn+/– Il6Hsa+/– and control (Il6Hsa+/–, Ocn+/–, and WT) mice, n = 8–12. (C) Circulating Ocn levels in 3-month-old control (Il6fl/fl, Ocn+/–, and WT mice) and Ocn+/– Il6Hsa+/– mice at rest and after exercise, n = 8. (D) Performance during an endurance run of 3-month-old Il6fl/fl and Il6Hsa–/– mice after an i.p. injection of IL-6 (3 ng/g), n = 8. (E) Circulating Ocn levels during an endurance run in 3-month-old Il6fl/fl and Il6Hsa–/– mice after an i.p. injection of IL-6 (3 ng/g), n = 8. (F) Circulating IL-6 levels during an endurance run in 3-month-old Il6fl/fl and Il6Hsa–/– mice after an i.p. injection of IL-6 (3 ng/g), n = 8. (G) Performance during an endurance run of 3-month-old control (Il6fl/fl, Ocn+/–, and WT mice) and Ocn+/– Il6Hsa+/– mice after an i.p. injection of IL-6 (3 ng/g), n = 8. (H) Performance during an endurance run of 3-month-old Il6fl/fl and Il6Hsa–/– mice after an i.p. Ocn injection (120 ng/g), n = 7. (I) Circulating IL-6 levels during an endurance run in 3-month-old Il6fl/fl and Il6Hsa–/– mice after an i.p. injection of Ocn (120 ng/g), n = 8. These results are representative of 3 independent experiments. Data were analyzed by 1-way ANOVA followed by Tukey’s post hoc test. Results presented as the mean ± SEM. *P < 0.05; **P < 0.01.
Figure 4
Figure 4. mIL-6 contributes to the maintenance of muscle mass in an osteocalcin-independent manner.
(A) Weight of hind limb muscles of 3-month-old female Il6Hsa–/– and control littermates, n = 6–11. (B) Weight of hind limb muscles in 3-month-old Ocn+/– Il6Hsa+/– compound mutant mice and control littermates (control group includes WT, Ocn+/–, and Il6Hsa+/– mice), n = 5–7. (C) Representative H&E staining of soleus muscle fibers in 3-month-old female Il6Hsa–/– and control littermates. Scale bars: 100 μm. (D and E) Representative histology with (D) N0Q7 (MHC I slow twitch fibers) and (E) MY32 (MHC II fast twitch fibers) staining of soleus muscle of 3-month-old female Il6Hsa–/– and control littermates. Scale bars: 100 μm. (F) Distribution of type 1 and type II myofibers in 3-month-old female Il6Hsa–/– and control littermates. Scale bars: 100 μm. (G) Measurement of cross-sectional area (CSA) of muscle fibers in 3-month-old female Il6Hsa–/– and control littermates. (H) Expression of myosin heavy chain genes in gastrocnemius muscle measured by qRT-PCR. (I) Representative histology with N0Q7 and MY32 staining of soleus muscle after endurance exercise in 3-month-old female Il6Hsa–/– and control littermates. Scale bars: 100 μm. These results are representative of 3 independent experiments. Data shown in A, F, and G were analyzed by 2-tailed unpaired t test and data in B and H by 1-way ANOVA followed by Tukey’s post hoc test. Data are presented as the mean ± SEM. *P < 0.05.
Figure 5
Figure 5. IL-6 favors osteoclastogenesis by signaling in osteoblasts.
(A and B) Coculture of Il6rfl/fl osteoblasts with Il6rfl/fl osteoclast precursor cells (OPCs), Il6rosb–/– osteoblasts with Il6fl/fl OPCs, and Il6rfl/fl osteoblasts with Il6rosb–/– OPCs in the presence of IL-6 and sIL-6r. (A) Quantification of the number of osteoclasts (OCs), TRAP activity, and the number of nuclei per TRAP+ osteoclast. (B) Expression of resorbing-activity markers Acp5, cathepsin K (Ctsk), Atp6v0d2, Dc-stamp, and Clcn7 in mouse osteoclasts (WT OCs). (C) Coculture in the presence of IL-6 and sIL-6r of (i) Il6rfl/fl osteoblasts with Il6rfl/fl OPCs, (ii) Il6rosb–/– osteoblasts with Il6fl/fl OPCs, or (iii) Il6r–/– osteoblasts with Il6rosb–/– OPCs. TRAP activity and quantification of the number of osteoclasts. (D and E) IL6r–/– osteoblasts were generated by infecting Il6rfl/fl osteoblasts with adenovirus expressing Cre recombinase and coculturing with Il6rfl/fl osteoblasts in the absence or presence of IL-6 in the culture medium. Similarly, IL6r–/– OPCs were generated by infecting Il6rfl/fl OPCs with adenovirus expressing Cre recombinase and coculturing with Il6rfl/fl osteoblasts in the absence or presence of IL-6. (D) Quantification of TRAP activity, of the number of nuclei per TRAP+ osteoclast, and gene expression of resorption markers (Acp5, Dc-stamp, and Clcn7). (E) Cocultures in the presence of IL-6 in the culture medium, quantification of TRAP activity, the number of nuclei per TRAP+ osteoclast, and expression of resorption markers (Acp5, Atp6v0d2, Dc-stamp, and Clcn7). These results are representative of 3 independent experiments with triplicate samples. Data in A–E were analyzed by 2-way ANOVA followed by Tukey’s post hoc test; the relative TRAP activity data in A and C–E were analyzed by 2-tailed unpaired t test. Error bars represent SEM. *P < 0.05; **P < 0.01.
Figure 6
Figure 6. IL-6 signaling in osteoblasts is needed to enhance exercise capacity during endurance exercise.
(A) Crossing of Il6rfl/fl mice with Ocn-Cre mice to delete Il6r in differentiated osteoblasts after birth and generate Il6rOsb–/– mice. (B) Detection of Il6r deletion by PCR on genomic DNA isolated from various tissues of Il6rOsb–/– mice. (C) Circulating IL-6 levels in 3-month-old Il6rfl/fl and Il6rOsb–/– mice before and after exercise, n = 8. (D) Performance during an endurance run of 3- and 6-month-old Il6rfl/fl and Il6rOsb–/– mice, n = 12–18. (E) Performance during an endurance run of 3-month-old Il6rfl/fl and Il6rHsa–/– mice, n = 7–9. (F) Circulating Ocn levels at rest and after exercise in bones of 3-month-old Il6rfl/fl and Il6rOsb–/– mice, n = 9–10. (G) Performance during an endurance run of 3-month-old control (Il6rOsb+/–, Ocn+/–, and WT) and Ocn+/– Il6rOsb+/– mice, n = 7–13. (H) Circulating Ocn levels in 3-month-old controls (WT, Il6rOsb+/–, and Ocn+/–) and Ocn+/– Il6rOsb+/– mice at rest and after exercise, n = 10 each. These results are representative of 4 independent experiments. Data were analyzed by 1-way ANOVA followed by Tukey’s post hoc test. Data presented as the mean ± SEM. *P < 0.05; **P < 0.01.
Figure 7
Figure 7. IL-6 signaling in osteoblasts is needed to enhance exercise capacity during endurance exercise.
(A) Performance during an endurance run of 3-month-old Il6rfl/fl and Il6rOsb–/– mice after an i.p. injection of IL-6 (3 ng/g), n = 6. (B) Performance during an endurance run of 3-month-old Il6rOsb+/– Ocn+/– and control (Il6rOsb+/–, Ocn+/–, and WT) mice after an i.p. injection of IL-6 (3 ng/g), n = 5. (C) Circulating IL-6 levels during an endurance exercise in 3-month-old Il6rfl/fl and Il6rOsb–/– mice after i.p. injection of IL-6 (3 ng/g), n = 6. (D) Circulating IL-6 levels in 3-month-old Il6rfl/fl and Il6rOsb–/– mice after i.p. injection of Ocn (120 ng/g), n = 8. (E) Performance during an endurance exercise of 3-month-old Il6rfl/fl and Il6rosb–/– mice treated with osteocalcin (Ocn, 120 ng/g), n = 5. (F) Performance during an endurance exercise of 3-month-old Il6rOsb+/– Ocn+/–, Il6rOsb+/–, Ocn+/–, and WT mice after an i.p. injection of Ocn (120 ng/g), n = 8. (G) Circulating IL-6 levels in 3-month-old controls (WT, Il6rOsb+/–, and Ocn+/–) and Ocn+/– Il6rOsb+/– mice treated with Ocn (120 ng/g), n = 6. These results are representative of 4 independent experiments. Data were analyzed by 1-way ANOVA followed by Tukey’s post hoc test. Results presented as the mean ± SEM. *P < 0.05; **P < 0.01.
Figure 8
Figure 8. mIL-6 favors glucose uptake and catabolism in myofibers during exercise through osteocalcin.
(A and B) Uptake of 3H-2-DG in glycolytic (Gly, white quadriceps) and oxidative (Ox, red quadriceps) muscle in 3-month-old mice. (A) Il6fl/fl versus Il6Hsa–/– and (B) Il6rfl/fl versus Il6rOsb–/– mice after exercise, n = 6. (C) Pgma1 expression at rest and after exercise in gastrocnemius muscle of 3-month-old Il6fl/fl versus Il6Hsa–/– mice, n = 5 (3 replicates for each mouse). (D and E) Uptake of 3H-2-DG in oxidative (Ox, red quadriceps) muscle in 3-month-old (D) controls (Il6Hsa+/– and Ocn+/–) versus Ocn+/– Il6Hsa+/–, and (E) controls (Il6rOsb+/– and Ocn+/–) versus Ocn+/– Il6rOsb+/– mice, n = 6. (F–I) Uptake of 3H-2-DG in oxidative (Ox, red quadriceps) muscle after exercise in 3-month-old (F) Il6fl/fl versus Il6Hsa–/–, (G) Il6rfl/fl versus Il6rOsb–/–, (H) controls (WT, Il6Hsa+/–, and Ocn+/–) versus Ocn+/– Il6Hsa+/–, and (I) controls (WT, Il6rOsb+/–, and Ocn+/–) versus Ocn+/– Il6rOsb+/– mice treated with osteocalcin (Ocn, 120 ng/g) i.p., n = 6. (J and K) Uptake of 3H-2-DG in oxidative (Ox, red quadriceps) muscle after exercise in 3-month-old (J) Il6fl/fl versus Il6Hsa–/– and (K) Il6rfl/fl versus Il6rosb–/– mice treated with IL-6 (3 ng/g) i.p., n = 6. These results are representative of 3 independent experiments. Data were analyzed by 1-way ANOVA followed by Tukey’s post hoc test. Results presented as the mean ± SEM. *P < 0.05; **P < 0.01.
Figure 9
Figure 9. mIL-6 favors fatty acid uptake and catabolism in myofibers during exercise through osteocalcin.
(A and B) Circulating NEFA levels at rest and after exercise in 3-month-old Il6Hsa–/– and Il6rOsb–/– mice and their respective controls. (C–F) Expression of Fatp1 and Cpt1b at rest and after exercise in gastrocnemius of 3-month-old (C) Il6fl/fl and Il6Hsa–/– mice, (D) Il6rfl/fl versus Il6rOsb–/– mice, (E) controls (WT, Il6Hsa+/–, and Ocn+/–) versus Ocn+/– Il6Hsa+/– mice, and (F) controls (WT, Il6rOsb+/–, and Ocn+/–) versus Ocn+/– Il6rOsb+/– mice. (G–J) Western blot analysis of HSL phosphorylation (Ser563) in tibialis muscles of 3-month-old (G) Il6fl/fl versus Il6Hsa–/–, (H) Il6rfl/fl versus Il6rOsb–/–, (I) controls (WT, Il6Hsa+/–, and Ocn+/–) versus Ocn+/– Il6Hsa+/–, and (J) controls (WT, Il6rOsb+/–, and Ocn+/–) versus Ocn+/– Il6rOsb+/– mice after exercise. These results are representative of 3 independent experiments. Data in A–E were analyzed by 1-way ANOVA followed by Tukey’s post hoc test. Results presented as the mean ± SEM. *P < 0.05.
Figure 10
Figure 10. Schematic representation of how muscle-derived IL-6 increases exercise capacity in an osteocalcin-dependent manner.
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References

    1. Neufer PD, et al. Understanding the cellular and molecular mechanisms of physical activity-induced health benefits. Cell Metab. 2015;22(1):4–11. - PubMed
    1. Zierath JR, Wallberg-Henriksson H. Looking ahead perspective: where will the future of exercise biology take us? Cell Metab. 2015;22(1):25–30. doi: 10.1016/j.cmet.2015.06.015. - DOI - PubMed
    1. Pedersen BK, Febbraio MA. Muscles, exercise and obesity: skeletal muscle as a secretory organ. Nat Rev Endocrinol. 2012;8(8):457–65. doi: 10.1038/nrendo.2012.49. - DOI - PubMed
    1. Ostrowski K, Rohde T, Zacho M, Asp S, Pedersen BK. Evidence that interleukin-6 is produced in human skeletal muscle during prolonged running. J Physiol (Lond) 1998;508(pt 3):949–953. - PMC - PubMed
    1. Steensberg A, van Hall G, Osada T, Sacchetti M, Saltin B, Klarlund Pedersen B. Production of interleukin-6 in contracting human skeletal muscles can account for the exercise-induced increase in plasma interleukin-6. J Physiol (Lond) 2000;529(pt 1):237–242. - PMC - PubMed

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