Effect of Wnt signaling on glucose transport in contracting myotubes.

<p>(<b>A</b>) Myotubes cultured in 5 mM glucose (G5) or in 25 mM glucose (G25) for 48 hours were transfected with a mouse Wnt10b cDNA or treated with 1 µM BIO. 2-deoxyglucose (2-DG) uptake was then measured in the presence or absence of 10 nM insulin for 30 minutes as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008509#s4" target="_blank">Material and Methods</a>. Data are expressed as mean±SE from 5 independent experiments performed in triplicate. Significant difference from G5, (***) p<0.0001; (**) p<0.02; Significant difference from G25, (###) p<0.0001; (##) p<0.001. (<b>B</b>) BIO induced GLUT4 translocation to the plasma membrane. Myotubes were cultured in 5 mM (G5) or 25 mM glucose (G25) for 48 hours in the presence or absence of 1 µM BIO. Myotubes were treated or not with 10 nM insulin for 30 minutes, then plasma membranes were isolated. Western blot analysis showed that insulin and BIO induced GLUT4 translocation to the plasma membrane, whereas GLUT1 was unaffected. (<b>C</b>) Quantification of GLUT4 and GLUT1 translocation. Data are expressed as mean±SE from 4 independent experiments. Significant difference from G5, (***) p<0.0001; (**) p<0.01; (*) p<0.05.</p

Activation of Wnt signaling in contracting myotubes.

<p>(<b>A</b>) Over-expression of Wnt10b cDNA down-regulated SREBP-1c protein. Myotubes were transfected with a plasmid encoding the mouse Wnt10b cDNA, then treated or not with 10 nM insulin for 24 hours. Western blot analysis of cytoplasmic (left panel) and nuclear (right panel) protein extracts showing the down-regulation of precursor and mature forms of SREBP-1c following the activation of the Wnt/β-catenin pathway. Wnt10b over-expression induced the nuclear accumulation of active β-catenin and MyoD. Blots were normalized using antibodies raised against the cytoplasmic protein GAPDH or the nuclear protein Lamin A/C. (<b>B</b>) Myotubes were submitted to a 48 hour-treatment with 1 µM BIO, then 10 nM insulin was added for 24 hours. BIO-mediated activation of the Wnt/β-catenin pathway induced SREBP-1c down-regulation, even in the presence of insulin.</p

Comparison between insulin and BIO effects on intracellular signaling.

<p>Myotubes cultured in 5 mM glucose (G5) or 25 mM glucose (G25) concentration were treated with 10 nM insulin for 30 minutes, or with 1 µM BIO for 30 and 60 minutes. (<b>A</b>) Insulin increased GSK-3β^S9 phosphorylation in myotubes cultured in G5 or G25, whereas BIO had no effect. In contrast, BIO decreased GSK-3β^Y216 phosphorylation, whereas insulin had no effect. (<b>B</b>) Insulin increased Akt2^S473 phosphorylation in myotubes cultured in G5, whereas myotubes cultured in G25 were resistant to insulin. BIO increased Akt1^T308 phosphorylation in myotubes cultured in G5, and had a biphasic effect in myotubes cultured in G25. (<b>C</b>) BIO stimulated AMPK-α1^S485 but not AMPK-α2^T172 phosphorylation whatever the glucose concentration, whereas insulin had no effect on AMPK phosphorylation. (<b>D</b>) In contrast to BIO, insulin failed to increase AS160^S588 phosphorylation. BIO showed a biphasic effect in myotubes cultured in G25. (<b>E</b>) Insulin increased Erk1/2 phosphorylation only in myotubes cultured in G5, whereas BIO diminished Erk1/2 phosphorylation in myotubes whatever the glucose concentration. Lower panels show quantifications of 3 independent experiments. Data are expressed as mean±SE. Significant difference between BIO and insulin, (###) p<0.001; (##) p<0.01. Significant difference between control and insulin (***) p<0.001; (**) p<0.01. NS: non significant.</p

Effect of BIO on intramyocellular lipid accumulation.

<p>Oil red O staining of intramyocellular lipids in myotubes cultured in 5 mM glucose (<b>A</b>) 25 mM glucose (<b>B</b>) or 25 mM glucose in the presence of 1 µM BIO for 5 days (<b>C</b>). Phase-contrast microphotographs of the same myotubes cultured in 5 mM glucose (<b>D</b>), or 25 mM glucose in the absence (<b>E</b>) or presence (<b>F</b>) of BIO. BIO totally abolished intramyocellular lipid deposition. Scale bar 20 µm.</p

Wnt10b and SREBP-1c are also inversely expressed in cultured satellite cells.

<p>(<b>A</b>) Western blot analysis showing an inverse expression pattern between Wnt10b and SREBP-1c proteins according to the differentiation stage. In contrast, Wnt3 remained almost unchanged throughout differentiation. SREBP-1c induced the up-regulation of the lipogenic enzyme FAS in myotubes. (<b>B</b>) Wnt10b knockdown was sufficient to up-regulate SREBP-1c and PPARγ mRNAs, whereas Wnt10b over-expression down-regulated their expression. RT-PCR was performed on myoblasts transfected with a scrambled siRNA (lane 1), a pool of 3 Wnt10b siRNAs (lane 2), or a plasmid encoding the mouse Wnt10 cDNA (lane 3) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008509#s4" target="_blank">Material and Methods</a>. (<b>C</b>) SREBP-1 knockdown stimulated Wnt signaling in contracting myotubes. Myotubes were transfected with SREBP-1 siRNAs or a scrambled siRNA, then treated or not with 10 nM insulin for 24 hours. SREBP-1 knockdown was sufficient to induce Wnt10b protein expression in myotubes, particularly in the presence of insulin, and to activate the Wnt/β-catenin pathway, as shown by GSK-3β and β-catenin activities. (<b>D</b>) Wnt10b knockdown induced SREBP-1c protein expression in myoblasts. Myoblasts were transfected with 30 pmoles or 60 pmoles of a pool of 3 Wnt10b siRNAs, or with a scrambled siRNA as a control. Silencing Wnt10b was sufficient to induce SREBP-1c protein expression in myoblasts through the inhibition of Wnt/β-catenin signaling. The blots are representative of 3 independent experiments.</p

Differential expression of SREBP-1c and Wnt proteins during skeletal muscle ontogenesis and regeneration.

<p>(<b>A</b>) Western blot analysis showing inverse expression patterns between Wnt10b and SREBP-1c during ontogenesis. The developmental stages are underlined using antibodies against developmental (MyHC-Dev) and fast (MyHC-2) myosin heavy chains. (<b>B</b>) Western blot analysis of Wnt10b and SREBP-1c protein levels in regenerating (R) adult EDL muscles at 2, 8, and 30 days after crush injury as compared to contralateral control (C) EDL. The down-regulation of SREBP-1c was concomitant with the up-regulation of Wnt10b throughout regeneration. The blots are representative of 3 independent experiments.</p