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, 4 (12), e8509

Activation of Wnt/beta-catenin Signaling Increases Insulin Sensitivity Through a Reciprocal Regulation of Wnt10b and SREBP-1c in Skeletal Muscle Cells

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Activation of Wnt/beta-catenin Signaling Increases Insulin Sensitivity Through a Reciprocal Regulation of Wnt10b and SREBP-1c in Skeletal Muscle Cells

Mounira Abiola et al. PLoS One.

Abstract

Background: Intramyocellular lipid accumulation is strongly related to insulin resistance in humans, and we have shown that high glucose concentration induced de novo lipogenesis and insulin resistance in murin muscle cells. Alterations in Wnt signaling impact the balance between myogenic and adipogenic programs in myoblasts, partly due to the decrease of Wnt10b protein. As recent studies point towards a role for Wnt signaling in the pathogenesis of type 2 diabetes, we hypothesized that activation of Wnt signaling could play a crucial role in muscle insulin sensitivity.

Methodology/principal findings: Here we demonstrate that SREBP-1c and Wnt10b display inverse expression patterns during muscle ontogenesis and regeneration, as well as during satellite cells differentiation. The Wnt/beta-catenin pathway was reactivated in contracting myotubes using siRNA mediated SREBP-1 knockdown, Wnt10b over-expression or inhibition of GSK-3beta, whereas Wnt signaling was inhibited in myoblasts through silencing of Wnt10b. SREBP-1 knockdown was sufficient to induce Wnt10b protein expression in contracting myotubes and to activate the Wnt/beta-catenin pathway. Conversely, silencing Wnt10b in myoblasts induced SREBP-1c protein expression, suggesting a reciprocal regulation. Stimulation of the Wnt/beta-catenin pathway i) drastically decreased SREBP-1c protein and intramyocellular lipid deposition in myotubes; ii) increased basal glucose transport in both insulin-sensitive and insulin-resistant myotubes through a differential activation of Akt and AMPK pathways; iii) restored insulin sensitivity in insulin-resistant myotubes.

Conclusions/significance: We conclude that activation of Wnt/beta-catenin signaling in skeletal muscle cells improved insulin sensitivity by i) decreasing intramyocellular lipid deposition through downregulation of SREBP-1c; ii) increasing insulin effects through a differential activation of the Akt/PKB and AMPK pathways; iii) inhibiting the MAPK pathway. A crosstalk between these pathways and Wnt/beta-catenin signaling in skeletal muscle opens the exciting possibility that organ-selective modulation of Wnt signaling might become an attractive therapeutic target in regenerative medicine and to treat obese and diabetic populations.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Differential expression of SREBP-1c and Wnt proteins during skeletal muscle ontogenesis and regeneration.
(A) 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) 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.
Figure 2
Figure 2. Wnt10b and SREBP-1c are also inversely expressed in cultured satellite cells.
(A) 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) 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 Material and Methods. (C) 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. (D) 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.
Figure 3
Figure 3. Activation of Wnt signaling in contracting myotubes.
(A) 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) 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.
Figure 4
Figure 4. Effect of BIO on intramyocellular lipid accumulation.
Oil red O staining of intramyocellular lipids in myotubes cultured in 5 mM glucose (A) 25 mM glucose (B) or 25 mM glucose in the presence of 1 µM BIO for 5 days (C). Phase-contrast microphotographs of the same myotubes cultured in 5 mM glucose (D), or 25 mM glucose in the absence (E) or presence (F) of BIO. BIO totally abolished intramyocellular lipid deposition. Scale bar 20 µm.
Figure 5
Figure 5. Effect of Wnt signaling on glucose transport in contracting myotubes.
(A) 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 Material and Methods. 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) 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. (C) 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.
Figure 6
Figure 6. Time-course of BIO effects on GSK-3β activity, Akt, AMPK and MAPK signaling in contracting myotubes.
Myotubes were cultured in 5 mM (G5) or 25 mM (G25) glucose concentration for 48 hours, then 1 µM BIO was added. Upper panels show western blots whereas lower panels show quantifications of 3 to 5 independent experiments. (A) BIO decreased within 30 minutes GSK-3βY216 phosphorylation in myotubes cultured either in G5 or G25, but had no effect on GSK-3βS9 phosphorylation. (B) BIO induced PDK1S241, Akt1T308 and AS160S588 phosphorylations, but had no effect on Akt2S473 phosphorylation in myotubes cultured either in G5 or G25. (C) BIO increased AMPK-α1S485 phosphorylation, but had no effect on AMPK-αT172 phosphorylation in myotubes cultured in G5 (left panels). In contrast, BIO had a biphasic effect in myotubes cultured in G25: AMPK-α1S485 phosphorylation was decreased to a basal level within 30 minutes, then increased with a time-course similar to that observed in myotubes cultured in G5 (right panels). (D) BIO decreased Erk1/2 phosphorylation with a similar time-course in myotubes cultured either in G5 or G25. Data are expressed as mean±SE from 3 independent experiments.
Figure 7
Figure 7. Comparison between insulin and BIO effects on intracellular signaling.
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. (A) Insulin increased GSK-3βS9 phosphorylation in myotubes cultured in G5 or G25, whereas BIO had no effect. In contrast, BIO decreased GSK-3βY216 phosphorylation, whereas insulin had no effect. (B) Insulin increased Akt2S473 phosphorylation in myotubes cultured in G5, whereas myotubes cultured in G25 were resistant to insulin. BIO increased Akt1T308 phosphorylation in myotubes cultured in G5, and had a biphasic effect in myotubes cultured in G25. (C) BIO stimulated AMPK-α1S485 but not AMPK-α2T172 phosphorylation whatever the glucose concentration, whereas insulin had no effect on AMPK phosphorylation. (D) In contrast to BIO, insulin failed to increase AS160S588 phosphorylation. BIO showed a biphasic effect in myotubes cultured in G25. (E) 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.
Figure 8
Figure 8. Hypothesis for an interplay between insulin and BIO signaling in contracting myotubes.
Wnt10b (a) and BIO (b) activate the Wnt/β-catenin pathway through the inactivation of GSK-3βY216 phosphorylation, which results in the nuclear translocation of active β-catenin, stimulation of myogenic genes transcription such as myoD, and inhibition of Srebp-1c transcription (f). Insulin-induced Srebp-1c transcription is mediated by the MAPK pathway in muscle cells. BIO inhibits the MAPK pathway, which could explain the down-regulation of Srebp-1c gene expression (c). In parallel, inactivation of GSK-3βY216 is followed by autophosphorylation of PDK1S241 which phosphorylates Akt1T308 (but not Akt2S473), then the subsequent phosphorylation of AS160S588 induces GLUT4 translocation (e). In contrast, insulin stimulates GLUT4 translocation through the PI3K/Akt2S473/AS160 pathway (d). BIO activates the AMP kinase pathway by phosphorylating AMPK-α1S485, which also induces GLUT4 translocation (e). These results strongly suggest that Wnt signaling, in contrast to insulin signaling, increases glucose transport in both insulin-sensitive and insulin-resistant myotubes through the activation of AMPK-α1 and Akt2/AS160 pathways.

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