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Vol. 15, Issue 10, 4544-4555, October 2004

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Insulin and Wnt1 Pathways Cooperate to Induce Reserve Cell Activation in Differentiation and Myotube Hypertrophy

Anne Rochat ^* , Anne Fernandez ^* , Marie Vandromme ^* , Jeàn-Pierre Molès ^* ||, Triston Bouschet ^* ¶, Gilles Carnac ^* # @, and Ned J. C. Lamb ^* @

^* Institut de Génétique Humaine, Centre National de la Recherche Scientifique, UPR-1142, 34396 Montpellier Cedex 5, France; ^|| Laboratoire de Dermatologie Moléculaire, Institut Universitaire de Recherche Clinique, 34396 Montpellier Cedex 5, France; and ^¶ Laboratoire de Génomique Fonctionnelle, Centre National de la Recherche Scientifique, Institute National de la Santé et de la Recherche Médicale de Pharmacologie/Endocrinologie, UPR-2580, 34094 Montpellier Cedex 5, France

Submitted November 14, 2003; Revised July 5, 2004; Accepted July 12, 2004
Monitoring Editor: Tony Hunter

During ex vivo myoblast differentiation, a pool of quiescent mononucleated myoblasts, reserve cells, arise alongside myotubes. Insulin/insulin-like growth factor (IGF) and PKB/Akt-dependent phosphorylation activates skeletal muscle differentiation and hypertrophy. We have investigated the role of glycogen synthase kinase 3 (GSK-3) inhibition by protein kinase B (PKB)/Akt and Wnt/-catenin pathways in reserve cell activation during myoblast differentiation and myotube hypertrophy. Inhibition of GSK-3 by LiCl or SB216763, restored insulin-dependent differentiation of C2ind myoblasts in low serum, and cooperated with insulin in serum-free medium to induce MyoD and myogenin expression in C2ind myoblasts, quiescent C2 or primary human reserve cells. We show that LiCl treatment induced nuclear accumulation of -catenin in C2 myoblasts, thus mimicking activation of canonical Wnt signaling. Similarly to the effect of GSK-3 inhibitors with insulin, coculturing C2 reserve cells with Wnt1-expressing fibroblasts enhanced insulin-stimulated induction of MyoD and myogenin in reserve cells. A similar cooperative effect of LiCl or Wnt1 with insulin was observed during late ex vivo differentiation and promoted increased size and fusion of myotubes. We show that this synergistic effect on myotube hypertrophy involved an increased fusion of reserve cells into preexisting myotubes. These data reveal insulin and Wnt/-catenin pathways cooperate in muscle cell differentiation through activation and recruitment of satellite cell-like reserve myoblasts.

Abbreviations used: C2ind, C2inducible cell line; DM, differentiation medium; GSK-3, glycogen synthase kinase-3; GM. growth medium; IGF, insulin-like growth factor; PI3K, phosphatidyl inositol 3 kinase; PKB/Akt, protein kinase B.

Present address: Hubrecht Laboratory, NIOB, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands

Present address: Centre de Biologie du Developpement, 118 route de Narbonne, 31062 Toulouse Cedex 01, France

^# Present address: CRBM, CNRS FRE 2593, 1919 Route de Mende, 34293 Montpellier Cedex, France.

^@ These authors contributed equally to this study.

Corresponding author. E-mail address: anne.fernandez{at}acrux.igh.cnrs.fr.

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