title: The effects of muscle contraction and recombinant osteocalcin on insulin sensitivity ex vivo.
abstract: We tested whether GPRC6A, the putative receptor of undercarboxylated osteocalcin (ucOC), is present in mouse muscle and whether ucOC increases insulin sensitivity following ex vivo muscle contraction. GPPRC6A is expressed in mouse muscle and in the mouse myotubes from a cell line. ucOC potentiated the effect of ex vivo contraction on insulin sensitivity.
  
  
  BACKGROUND
  Acute exercise increases skeletal muscle insulin sensitivity. In humans, exercise increases circulating ucOC, a hormone that increases insulin sensitivity in rodents. We tested whether GPRC6A, the putative receptor of ucOC, is present in mouse muscle and whether recombinant ucOC increases insulin sensitivity in both C2C12 myotubes and whole mouse muscle following ex vivo muscle contraction.
  
  
  METHODS
  Glucose uptake was examined in C2C12 myotubes that express GPRC6A following treatment with insulin alone or with insulin and increasing ucOC concentrations (0.3, 3, 10 and 30 ng/ml). In addition, glucose uptake, phosphorylated (p-)AKT and p-AS160 were examined ex vivo in extensor digitorum longus (EDL) dissected from C57BL/6J wild-type mice, at rest, following insulin alone, after muscle contraction followed by insulin and after muscle contraction followed by recombinant ucOC then insulin exposure.
  
  
  RESULTS
  We observed protein expression of the likely receptor for ucOC, GPRC6A, in whole muscle sections and differentiated mouse myotubes. We observed reduced GPRC6A expression following siRNA transfection. ucOC significantly increased insulin-stimulated glucose uptake dose-dependently up to 10 ng/ml, in differentiated mouse C2C12 myotubes. Insulin increased EDL glucose uptake (∼30 %, p < 0.05) and p-AKT and p-AKT/AKT compared with rest (all p < 0.05). Contraction prior to insulin increased muscle glucose uptake (∼25 %, p < 0.05), p-AKT, p-AKT/AKT, p-AS160 and p-AS160/AS160 compared with contraction alone (all p < 0.05). ucOC after contraction increased insulin-stimulated muscle glucose uptake (∼12 % p < 0.05) and p-AS160 (<0.05) more than contraction plus insulin alone but without effect on p-AKT. In the absence of insulin and/or of contraction, ucOC had no significant effect on muscle glucose uptake.
  
  
  CONCLUSIONS
  GPRC6A, the likely receptor of osteocalcin (OC), is expressed in mouse muscle. ucOC treatment augments insulin-stimulated skeletal muscle glucose uptake in C2C12 myotubes and following ex vivo muscle contraction. ucOC may partly account for the insulin sensitizing effect of exercise.
authors.affiliation: Clinical Exercise Science Research Program, Institute of Sport, Exercise and Active Living (ISEAL) College of Sport and Exercise Science, Victoria University, PO Box 14428, Melbourne, VIC, 8001, Australia. itamar.levinger@vu.edu.au.
authors.forename: I
authors.initials: I
authors.lastname: Levinger
authors.affiliation: Clinical Exercise Science Research Program, Institute of Sport, Exercise and Active Living (ISEAL) College of Sport and Exercise Science, Victoria University, PO Box 14428, Melbourne, VIC, 8001, Australia.
authors.forename: X
authors.initials: X
authors.lastname: Lin
authors.affiliation: Clinical Exercise Science Research Program, Institute of Sport, Exercise and Active Living (ISEAL) College of Sport and Exercise Science, Victoria University, PO Box 14428, Melbourne, VIC, 8001, Australia.
authors.forename: X
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Department of Physiology and Bosch Institute for Medical Research, University of Sydney, Sydney, Australia.
authors.forename: T C
authors.initials: TC
authors.lastname: Brennan-Speranza
authors.affiliation: Department of Physiology and Bosch Institute for Medical Research, University of Sydney, Sydney, Australia.
authors.forename: B
authors.initials: B
authors.lastname: Volpato
authors.affiliation: Clinical Exercise Science Research Program, Institute of Sport, Exercise and Active Living (ISEAL) College of Sport and Exercise Science, Victoria University, PO Box 14428, Melbourne, VIC, 8001, Australia.|Centre for Chronic Disease, College of Health and Biomedicine, Victoria University, Melbourne, Australia.
authors.forename: A
authors.initials: A
authors.lastname: Hayes
authors.affiliation: Department of Endocrinology, Austin Health, University of Melbourne, Melbourne, Australia.
authors.forename: G
authors.initials: G
authors.lastname: Jerums
authors.affiliation: Department of Endocrinology, Austin Health, University of Melbourne, Melbourne, Australia.
authors.forename: E
authors.initials: E
authors.lastname: Seeman
authors.affiliation: Clinical Exercise Science Research Program, Institute of Sport, Exercise and Active Living (ISEAL) College of Sport and Exercise Science, Victoria University, PO Box 14428, Melbourne, VIC, 8001, Australia.|Centre for Chronic Disease, College of Health and Biomedicine, Victoria University, Melbourne, Australia.
authors.forename: G
authors.initials: G
authors.lastname: McConell
mesh_terms: D000818:Animals; D004305:Dose-Response Relationship, Drug; D055785:Gene Knockdown Techniques / Q000379:methods; D005947:Glucose / Q000378:metabolism; D007333:Insulin Resistance / Q000502:physiology*; D008297:Male; D008810:Mice, Inbred C57BL; D009119:Muscle Contraction / Q000502:physiology*; D018485:Muscle Fibers, Skeletal / Q000187:drug effects / Q000378:metabolism; D009124:Muscle Proteins / Q000378:metabolism; D018482:Muscle, Skeletal / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D015675:Osteocalcin / Q000008:administration & dosage / Q000494:pharmacology*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000494:pharmacology; D046509:Tissue Culture Techniques
chemical_list: C515750:GPRC6A protein, mouse; D009124:Muscle Proteins; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; D015675:Osteocalcin; D005947:Glucose
keywords: Bone-muscle interactions; Glucose uptake; Insulin sensitivity; Muscle contraction; Osteocalcin