Osteocalcin Signaling in Myofibers Is Necessary and Sufficient for Optimum Adaptation to Exercise

Abstract

Circulating levels of undercarboxylated and bioactive osteocalcin double during aerobic exercise at the time levels of insulin decrease. In contrast, circulating levels of osteocalcin plummet early during adulthood in mice, monkeys, and humans of both genders. Exploring these observations revealed that osteocalcin signaling in myofibers is necessary for adaptation to exercise by favoring uptake and catabolism of glucose and fatty acids, the main nutrients of myofibers. Osteocalcin signaling in myofibers also accounts for most of the exercise-induced release of interleukin-6, a myokine that promotes adaptation to exercise in part by driving the generation of bioactive osteocalcin. We further show that exogenous osteocalcin is sufficient to enhance the exercise capacity of young mice and to restore to 15-month-old mice the exercise capacity of 3-month-old mice. This study uncovers a bone-to-muscle feedforward endocrine axis that favors adaptation to exercise and can reverse the age-induced decline in exercise capacity.

Figure 1. Regulation of circulating osteocalcin levels by exercise and age

A. Serum total osteocalcin (Ocn) and insulin (Ins) levels in 3 month-old mice at rest or after exercise. B. Serum undercarboxylated Ocn (uncarb Ocn) levels in 3 month-old mice at rest (−1), 0, 1, 2 and 4 hours after exercise. C. Serum CTX levels in 3 month-old mice at rest and after exercise. D. Serum OCN levels in women at rest and after exercise. E–F. Serum total and uncarb Ocn levels in female and male mice of various ages. G. Serum PINP in mice of various ages. H. Osteocalcin (Ocn) expression in femur in mice of various ages. I. Performance during an endurance test (running on a treadmill at 30cm/s until exhausted) of 3, 6 and 12 month-old female WT mice. J. Serum uncarb Ocn levels in 3, 6 and 12 month-old female mice at rest and after exercise. K. Serum total Ocn levels in 2 to 33 year-old female and male rhesus macaque monkeys. L. Serum total OCN levels in women and men 11 to 78 year-old. M. Performance during an endurance test (running on a treadmill at 30cm/s until exhausted) of 3 month-old and N. 12 and 15 month-old WT mice treated with Ocn and O. 10 month-old WT mice receiving Ocn for 28 days. (Exercise refers to 40 min running at 30cm/s on a treadmill).

Figure 2. Osteocalcin signaling in myofibers is necessary for adaptation to exercise

A. Performance during an endurance test (running on a treadmill at 30cm/s until exhausted) of 3 month-old Ocn−/− and WT mice, B. Ocnf/f and Ocn_Osb−/− mice and C. Gprc6a−/− and WT mice. D. Gprc6a expression in various tissues and E. in EDL and soleus muscles. F. In situ hybridization analysis of Gprc6a expression in soleus muscle. G. Gprc6a expression in WT and Ocn−/− gastrocnemius muscles. H. cAMP accumulation in WT and Gprc6a−/− myotubes treated with vehicle or osteocalcin (Ocn). I. Performance during an endurance test (running on a treadmill at 30cm/s until exhausted) of 3 month-old Gprc6af/f and Gprc6a_Mck−/− mice and J. Ocn+/−; Gprc6a_Mck+/− and control mice. K. Performance during an endurance test (running on a treadmill at 30cm/s until exhausted) of 6 month-old Ocn−/− or L. 12 month-old Gprc6a_Mck−/− mice treated with Ocn.

Figure 3. Osteocalcin signaling in myofibers promotes uptake and utilization of glucose during exercise

A. VO₂, B. VO₂ max and C. RER in 3 month-old Gprc6af/f and Gprc6a_Mck−/− mice running on a treadmill at increasing speed until exhausted. D. Glycogen content and breakdown in 3 month-old Gprc6af/f and Gprc6a_Mck−/− tibialis muscles at rest and after exercise. E. Uptake of ³H-2-deoxyglucose (³H-2-DG) in WT and Gprc6a−/− myotubes and F. WT EDL and soleus muscles treated with vehicle or osteocalcin (Ocn). G. Uptake of ³H-2-DG in glycolytic (Gly, white quadriceps) and oxidative (Ox, red quadriceps) muscles after exercise in 3 month-old Gprc6af/f and Gprc6a_Mck−/− mice and H. 15 month-old WT mice treated with Ocn. I. Glycolysis, determined by the extracellular acidification of the media (ECAR), in WT and Gprc6a−/− myofibers treated with vehicle or Ocn. J. GLUT4 translocation in C2C12 myoblasts treated with Ocn determined by optic microscopy. K. Western blot analyses of Akt phosphorylation (Ser473) in tibialis muscles of 3 month-old Gprc6af/f, Gprc6a_Mck−/−, WT and Ocn−/− mice after exercise. L. Aspartate and M–N. TCA cycle metabolites accumulation in quadriceps of 3 month-old Gprc6af/f and Gprc6a_Mck−/− mice at rest and after exercise. O. ¹³C-labeled TCA metabolites and lactate in quadriceps muscles of 3 month-old Gprc6af/f and Gprc6a_Mck−/− mice receiving a bolus of ¹³C-glucose prior to exercise. P. Oxygen consumption rate (OCR) in myofibers cultured in Krebs-Ringer HEPES buffer with 25mM glucose. Q. Blood glucose levels in 3 month-old Gprc6af/f and Gprc6a_Mck−/− mice at rest and after running on a treadmill for 40 min or until exhaustion. (Exercise refers to 40 min running at 30cm/s on a treadmill).

Figure 4. Osteocalcin signaling in myofibers favors FAs utilization during exercise

A. Acylcarnitine levels in quadriceps muscles and B. plasma of 3 month-old Gprc6af/f and Gprc6a_Mck−/− mice at rest and after exercise. C. ¹⁴C-oleate oxidation in WT and Gprc6a−/− myotubes treated with osteocalcin (Ocn). D. Oxygen consumption rate (OCR) in myofibers cultured in Krebs-Ringer HEPES buffer with 3 mM oleic acid. E. Plasma NEFAs levels in 3 month-old Gprc6af/f and Gprc6a_Mck−/− mice at rest and after exercise. F. Western blot analysis after exercise of AMPK phosphorylation (Thr172) in tibialis muscles of 3 month-old Gprc6af/f and Gprc6a_Mck−/− mice or G. of 15 month-old WT mice injected with Ocn. H. ¹⁴C-oleate oxidation in WT and Ampkα2−/− myotubes treated with Ocn. I. Uptake of ³H-2-deoxyglucose (³H-2-DG) in WT and Ampkα2−/− myotubes treated with vehicle or Ocn. J. Western blot analysis after exercise of HSL phosphorylation (Ser563) in tibialis muscles of 3 month-old Gprc6af/f, Gprc6a_Mck−/−, WT and Ocn−/− mice and K. 15 month-old WT mice injected with Ocn. (Exercise refers to 40 min running at 30cm/s on a treadmill)

Figure 5. Osteocalcin signaling in myofibers favors expression of FAs transporters during exercise

A. Cd36, Fatp1 and Cpt1b expression at rest and after exercise in gastrocnemius muscles of 3 month-old WT and Ocn−/− mice. B. Cd36, Fatp1 and Cpt1b expression at rest and after exercise in gastrocnemius muscles of 3 month-old Gprc6af/f, Gprc6a_Mck−/− and Ocn+/−;Gprc6a_Mck+/− mice and C. in gastrocnemius muscles of 15 month-old WT mice injected with vehicle or osteocalcin (Ocn). D. Western blot analysis after exercise of CREB phosphorylation (Ser133) in tibialis muscles of 3 month-old Gprc6af/f and Gprc6a_Mck−/− mice and E. in WT myotubes treated with vehicle or Ocn. F. Performance during an endurance test (running on a treadmill at 30cm/s until exhausted) of 3 month-old Gprc6a_Mck+/−;Creb_Mck+/−, Creb_Mck+/− and control mice. G. Uptake of ³H-2-deoxyglucose (³H-2-DG) in Crebf/f and Creb−/− myotubes treated with vehicle or Ocn. H. Glycolysis determined by the extracellular acidification of the media (ECAR), in Crebf/f and Creb_Mck−/− myofibers treated with vehicle or Ocn. I. ¹⁴C-oleate oxidation in Crebf/f and Creb−/− myotubes treated with Ocn. J. ATP accumulation in quadriceps muscles of 3 month-old Gprc6af/f and Gprc6a_Mck−/− mice after exercise. (Exercise refers to 40 min running at 30cm/s on a treadmill)

Figure 6. Osteocalcin is necessary for the increase in Interleukin-6 expression in muscle during exercise

A. RNASeq analyses in gastrocnemius muscles of 3 month-old Gprc6af/f and Gprc6a_Mck−/− mice after exercise. B. Expression after exercise of Il6 and Il6rα in gastrocnemius muscles of 3 month-old Gprc6af/f and Gprc6a_Mck−/− and C. WT and Ocn−/−, mice. D. Circulating IL-6 at rest and after exercise in 3 month-old Gprc6af/f and Gprc6a_Mck−/− mice, E. WT and Ocn−/− mice and F. in women (45 min run on a treadmill (6.5km/h)). G. Circulating IL-6 at rest and after exercise in 3 and 15 month-old mice and 15 month-old mice treated with vehicle or osteocalcin (Ocn). H. Performance during an endurance test (running on a treadmill at 30cm/s until exhausted) of 15 month-old mice treated with vehicle or Ocn and an antibody against IL-6 or a control IgG (control group include mice treated with vehicle alone, vehicle and IL-6 antibody, vehicle and IgG). I. Performance during an endurance test (running on a treadmill at 30cm/s until exhausted) of 6 month-old WT and Ocn−/− mice treated with IL-6. J. Uptake of ³H-2-deoxyglucose (³H-2-DG), K. Glycolysis, ad determined by the extracellular acidification of the media (ECAR) and L. ¹⁴C-Oleate oxidation in WT and Il6−/− myotubes treated with vehicle or osteocalcin. (Exercise refers to 40 min running at 30cm/s on a treadmill, nd = non-detected)

Figure 7. IL-6 favors the production of active osteocalcin during exercise

A. Ocn, Rankl and Opg expression in osteoblasts treated with IL-6 and soluble IL-6Rα. B. Serum CTX levels in 2 month-old WT and Il6−/− mice after exercise. C. Serum undercarboxylated osteocalcin (uncarb Ocn) levels in 2 month-old WT and Il6−/− mice at rest and after exercise. D. Schematic representation of how osteocalcin signaling in myofibers and muscle-derived IL-6 cooperate to favor adaptation to exercise. (Exercise refers to 40 min running at 30cm/s on a treadmill).