Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1999 Apr 15;339(Pt 2):443–451.

Insulin-like growth factor (IGF-I) induces myotube hypertrophy associated with an increase in anaerobic glycolysis in a clonal skeletal-muscle cell model.

C Semsarian 1, P Sutrave 1, D R Richmond 1, R M Graham 1
PMCID: PMC1220176  PMID: 10191278

Abstract

Insulin-like growth factor-I (IGF-I) is an important autocrine/paracrine mediator of skeletal-muscle growth and development. To develop a definitive cultured cell model of skeletal-muscle hypertrophy, C2C12 cells were stably transfected with IGF-I and clonal lines developed and evaluated. Quantitative morphometric analysis showed that IGF-I-transfected myotubes had a larger area (2381+/-60 micrometer2 versus 1429+/-39 micrometer2; P<0.0001) and a greater maximum width (21.4+/-0.6 micrometer versus 13.9+/-0.3 micrometer; P<0.0001) than control C2C12 myotubes, independent of the number of cell nuclei per myotube. IGF-I-transfected myotubes had higher levels of protein synthesis but no difference in DNA synthesis when compared with control myotubes, indicating the development of hypertrophy rather than hyperplasia. Both lactate dehydrogenase and alanine aminotransferase activities were increased (3- and 5-fold respectively), and total lactate levels were higher (2.3-fold) in IGF-I-transfected compared with control myotubes, indicating an increase in anaerobic glycolysis in the hypertrophied myotubes. However, expression of genes involved in skeletal-muscle growth or hypertrophy in vivo, e.g. myocyte nuclear factor and myostatin, was not altered in the IGF-I myotubes. Finally, myotube hypertrophy could also be induced by treatment of C2C12 cells with recombinant IGF-I or by growing C2C12 cells in conditioned media from IGF-I-transfected cells. This quantitative model should be uniquely useful for elucidating the molecular mechanisms of skeletal-muscle hypertrophy.

Full Text

The Full Text of this article is available as a PDF (218.9 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Andrés V., Walsh K. Myogenin expression, cell cycle withdrawal, and phenotypic differentiation are temporally separable events that precede cell fusion upon myogenesis. J Cell Biol. 1996 Feb;132(4):657–666. doi: 10.1083/jcb.132.4.657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baker J., Liu J. P., Robertson E. J., Efstratiadis A. Role of insulin-like growth factors in embryonic and postnatal growth. Cell. 1993 Oct 8;75(1):73–82. [PubMed] [Google Scholar]
  3. Barnes D. M., Calvert C. C., Klasing K. C. Source of amino acids for tRNA acylation. Implications for measurement of protein synthesis. Biochem J. 1992 Apr 15;283(Pt 2):583–589. doi: 10.1042/bj2830583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brunetti A., Maddux B. A., Wong K. Y., Goldfine I. D. Muscle cell differentiation is associated with increased insulin receptor biosynthesis and messenger RNA levels. J Clin Invest. 1989 Jan;83(1):192–198. doi: 10.1172/JCI113858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  6. Coleman M. E., DeMayo F., Yin K. C., Lee H. M., Geske R., Montgomery C., Schwartz R. J. Myogenic vector expression of insulin-like growth factor I stimulates muscle cell differentiation and myofiber hypertrophy in transgenic mice. J Biol Chem. 1995 May 19;270(20):12109–12116. doi: 10.1074/jbc.270.20.12109. [DOI] [PubMed] [Google Scholar]
  7. Coolican S. A., Samuel D. S., Ewton D. Z., McWade F. J., Florini J. R. The mitogenic and myogenic actions of insulin-like growth factors utilize distinct signaling pathways. J Biol Chem. 1997 Mar 7;272(10):6653–6662. doi: 10.1074/jbc.272.10.6653. [DOI] [PubMed] [Google Scholar]
  8. Duerr R. L., Huang S., Miraliakbar H. R., Clark R., Chien K. R., Ross J., Jr Insulin-like growth factor-1 enhances ventricular hypertrophy and function during the onset of experimental cardiac failure. J Clin Invest. 1995 Feb;95(2):619–627. doi: 10.1172/JCI117706. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Engert J. C., Berglund E. B., Rosenthal N. Proliferation precedes differentiation in IGF-I-stimulated myogenesis. J Cell Biol. 1996 Oct;135(2):431–440. doi: 10.1083/jcb.135.2.431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ewton D. Z., Florini J. R. Effects of the somatomedins and insulin on myoblast differentiation in vitro. Dev Biol. 1981 Aug;86(1):31–39. doi: 10.1016/0012-1606(81)90312-2. [DOI] [PubMed] [Google Scholar]
  11. Ewton D. Z., Florini J. R. Effects of the somatomedins and insulin on myoblast differentiation in vitro. Dev Biol. 1981 Aug;86(1):31–39. doi: 10.1016/0012-1606(81)90312-2. [DOI] [PubMed] [Google Scholar]
  12. Florini J. R., Ewton D. Z., Coolican S. A. Growth hormone and the insulin-like growth factor system in myogenesis. Endocr Rev. 1996 Oct;17(5):481–517. doi: 10.1210/edrv-17-5-481. [DOI] [PubMed] [Google Scholar]
  13. Florini J. R., Ewton D. Z., Evinger-Hodges M. J., Falen S. L., Lau R. L., Regan J. F., Vertel B. M. Stimulation and inhibition of myoblast differentiation by hormones. In Vitro. 1984 Dec;20(12):942–958. doi: 10.1007/BF02619668. [DOI] [PubMed] [Google Scholar]
  14. Florini J. R., Ewton D. Z., Magri K. A. Hormones, growth factors, and myogenic differentiation. Annu Rev Physiol. 1991;53:201–216. doi: 10.1146/annurev.ph.53.030191.001221. [DOI] [PubMed] [Google Scholar]
  15. Florini J. R., Ewton D. Z., Roof S. L. Insulin-like growth factor-I stimulates terminal myogenic differentiation by induction of myogenin gene expression. Mol Endocrinol. 1991 May;5(5):718–724. doi: 10.1210/mend-5-5-718. [DOI] [PubMed] [Google Scholar]
  16. Florini J. R. Hormonal control of muscle growth. Muscle Nerve. 1987 Sep;10(7):577–598. doi: 10.1002/mus.880100702. [DOI] [PubMed] [Google Scholar]
  17. Florini J. R., Magri K. A. Effects of growth factors on myogenic differentiation. Am J Physiol. 1989 Apr;256(4 Pt 1):C701–C711. doi: 10.1152/ajpcell.1989.256.4.C701. [DOI] [PubMed] [Google Scholar]
  18. Garry D. J., Yang Q., Bassel-Duby R., Williams R. S. Persistent expression of MNF identifies myogenic stem cells in postnatal muscles. Dev Biol. 1997 Aug 15;188(2):280–294. doi: 10.1006/dbio.1997.8657. [DOI] [PubMed] [Google Scholar]
  19. Grobet L., Martin L. J., Poncelet D., Pirottin D., Brouwers B., Riquet J., Schoeberlein A., Dunner S., Ménissier F., Massabanda J. A deletion in the bovine myostatin gene causes the double-muscled phenotype in cattle. Nat Genet. 1997 Sep;17(1):71–74. doi: 10.1038/ng0997-71. [DOI] [PubMed] [Google Scholar]
  20. Han V. K., D'Ercole A. J., Lund P. K. Cellular localization of somatomedin (insulin-like growth factor) messenger RNA in the human fetus. Science. 1987 Apr 10;236(4798):193–197. doi: 10.1126/science.3563497. [DOI] [PubMed] [Google Scholar]
  21. Kaliman P., Viñals F., Testar X., Palacín M., Zorzano A. Phosphatidylinositol 3-kinase inhibitors block differentiation of skeletal muscle cells. J Biol Chem. 1996 Aug 9;271(32):19146–19151. doi: 10.1074/jbc.271.32.19146. [DOI] [PubMed] [Google Scholar]
  22. Lassar A. B., Skapek S. X., Novitch B. Regulatory mechanisms that coordinate skeletal muscle differentiation and cell cycle withdrawal. Curr Opin Cell Biol. 1994 Dec;6(6):788–794. doi: 10.1016/0955-0674(94)90046-9. [DOI] [PubMed] [Google Scholar]
  23. Le Roith D. Seminars in medicine of the Beth Israel Deaconess Medical Center. Insulin-like growth factors. N Engl J Med. 1997 Feb 27;336(9):633–640. doi: 10.1056/NEJM199702273360907. [DOI] [PubMed] [Google Scholar]
  24. Leferovich J. M., Lana D. P., Sutrave P., Hughes S. H., Kelly A. M. Regulation of c-ski transgene expression in developing and mature mice. J Neurosci. 1995 Jan;15(1 Pt 2):596–603. doi: 10.1523/JNEUROSCI.15-01-00596.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Levinovitz A., Jennische E., Oldfors A., Edwall D., Norstedt G. Activation of insulin-like growth factor II expression during skeletal muscle regeneration in the rat: correlation with myotube formation. Mol Endocrinol. 1992 Aug;6(8):1227–1234. doi: 10.1210/mend.6.8.1406701. [DOI] [PubMed] [Google Scholar]
  26. Liu J. P., Baker J., Perkins A. S., Robertson E. J., Efstratiadis A. Mice carrying null mutations of the genes encoding insulin-like growth factor I (Igf-1) and type 1 IGF receptor (Igf1r). Cell. 1993 Oct 8;75(1):59–72. [PubMed] [Google Scholar]
  27. Mathews L. S., Hammer R. E., Behringer R. R., D'Ercole A. J., Bell G. I., Brinster R. L., Palmiter R. D. Growth enhancement of transgenic mice expressing human insulin-like growth factor I. Endocrinology. 1988 Dec;123(6):2827–2833. doi: 10.1210/endo-123-6-2827. [DOI] [PubMed] [Google Scholar]
  28. McPherron A. C., Lawler A. M., Lee S. J. Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member. Nature. 1997 May 1;387(6628):83–90. doi: 10.1038/387083a0. [DOI] [PubMed] [Google Scholar]
  29. Morrison K. S., Mackie S. C., Palmer R. M., Thompson M. G. Stimulation of protein and DNA synthesis in mouse C2C12 satellite cells: evidence for phospholipase D-dependent and -independent pathways. J Cell Physiol. 1995 Nov;165(2):273–283. doi: 10.1002/jcp.1041650208. [DOI] [PubMed] [Google Scholar]
  30. Nagulesparen M., Trickey R., Davies M. J., Jenkins J. S. Muscle changes in acromegaly. Br Med J. 1976 Oct 16;2(6041):914–915. doi: 10.1136/bmj.2.6041.914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Olwin B. B., Hauschka S. D. Cell surface fibroblast growth factor and epidermal growth factor receptors are permanently lost during skeletal muscle terminal differentiation in culture. J Cell Biol. 1988 Aug;107(2):761–769. doi: 10.1083/jcb.107.2.761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Palmer R. M., Thompson M. G., Knott R. M., Campbell G. P., Thom A., Morrison K. S. Insulin and insulin-like growth factor-I responsiveness and signalling mechanisms in C2C12 satellite cells: effect of differentiation and fusion. Biochim Biophys Acta. 1997 Feb 4;1355(2):167–176. doi: 10.1016/s0167-4889(96)00127-9. [DOI] [PubMed] [Google Scholar]
  33. Pardee A. B. G1 events and regulation of cell proliferation. Science. 1989 Nov 3;246(4930):603–608. doi: 10.1126/science.2683075. [DOI] [PubMed] [Google Scholar]
  34. Powell-Braxton L., Hollingshead P., Warburton C., Dowd M., Pitts-Meek S., Dalton D., Gillett N., Stewart T. A. IGF-I is required for normal embryonic growth in mice. Genes Dev. 1993 Dec;7(12B):2609–2617. doi: 10.1101/gad.7.12b.2609. [DOI] [PubMed] [Google Scholar]
  35. Prysor-Jones R. A., Jenkins J. S. Effect of excessive secretion of growth hormone on tissues of the rat, with particular reference to the heart and skeletal muscle. J Endocrinol. 1980 Apr;85(1):75–82. doi: 10.1677/joe.0.0850075. [DOI] [PubMed] [Google Scholar]
  36. Quinn L. S., Haugk K. L., Grabstein K. H. Interleukin-15: a novel anabolic cytokine for skeletal muscle. Endocrinology. 1995 Aug;136(8):3669–3672. doi: 10.1210/endo.136.8.7628408. [DOI] [PubMed] [Google Scholar]
  37. Rosenthal S. M., Cheng Z. Q. Opposing early and late effects of insulin-like growth factor I on differentiation and the cell cycle regulatory retinoblastoma protein in skeletal myoblasts. Proc Natl Acad Sci U S A. 1995 Oct 24;92(22):10307–10311. doi: 10.1073/pnas.92.22.10307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Rotwein P. Structure, evolution, expression and regulation of insulin-like growth factors I and II. Growth Factors. 1991;5(1):3–18. doi: 10.3109/08977199109000267. [DOI] [PubMed] [Google Scholar]
  39. Sherr C. J. G1 phase progression: cycling on cue. Cell. 1994 Nov 18;79(4):551–555. doi: 10.1016/0092-8674(94)90540-1. [DOI] [PubMed] [Google Scholar]
  40. Tollefsen S. E., Lajara R., McCusker R. H., Clemmons D. R., Rotwein P. Insulin-like growth factors (IGF) in muscle development. Expression of IGF-I, the IGF-I receptor, and an IGF binding protein during myoblast differentiation. J Biol Chem. 1989 Aug 15;264(23):13810–13817. [PubMed] [Google Scholar]
  41. Ullrich A., Bell J. R., Chen E. Y., Herrera R., Petruzzelli L. M., Dull T. J., Gray A., Coussens L., Liao Y. C., Tsubokawa M. Human insulin receptor and its relationship to the tyrosine kinase family of oncogenes. 1985 Feb 28-Mar 6Nature. 313(6005):756–761. doi: 10.1038/313756a0. [DOI] [PubMed] [Google Scholar]
  42. Wang J., Walsh K. Resistance to apoptosis conferred by Cdk inhibitors during myocyte differentiation. Science. 1996 Jul 19;273(5273):359–361. doi: 10.1126/science.273.5273.359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Yaffe D., Saxel O. Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle. Nature. 1977 Dec 22;270(5639):725–727. doi: 10.1038/270725a0. [DOI] [PubMed] [Google Scholar]
  44. Yang Q., Bassel-Duby R., Williams R. S. Transient expression of a winged-helix protein, MNF-beta, during myogenesis. Mol Cell Biol. 1997 Sep;17(9):5236–5243. doi: 10.1128/mcb.17.9.5236. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES