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, 589 (Pt 7), 1831-46

Early Activation of mTORC1 Signalling in Response to Mechanical Overload Is Independent of Phosphoinositide 3-kinase/Akt Signalling

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Early Activation of mTORC1 Signalling in Response to Mechanical Overload Is Independent of Phosphoinositide 3-kinase/Akt Signalling

Mitsunori Miyazaki et al. J Physiol.

Abstract

The mammalian target of rapamycin complex 1 (mTORC1) functions as a central integrator of a wide range of signals that modulate protein metabolism and cell growth. However, the contributions of individual pathways regulating mTORC1 activity in skeletal muscle are poorly defined. The purpose of this study was to determine the regulatory mechanisms that contribute to mTORC1 activation during mechanical overload-induced skeletal muscle hypertrophy. Consistent with previous studies, mechanical overload induced progressive hypertrophy of the plantaris muscle which was associated with significant increases in total RNA content and protein metabolism. mTORC1 was activated after a single day of overload as indicated by a significant increase in S6K1 phosphorylation at T389 and T421/S424. In contrast, Akt activity, as assessed by Akt phosphorylation status (T308 and S473), phosphorylation of direct downstream targets (glycogen synthase kinase 3 β, proline-rich Akt substrate 40 kDa and tuberous sclerosis 2 (TSC2)) and a kinase assay, was not significantly increased until 2–3 days of overload. Inhibition of phosphoinositide 3-kinase (PI3K) activity by wortmannin was sufficient to block insulin-dependent signalling but did not prevent the early activation of mTORC1 in response to overload. We identified that the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)-dependent pathway was activated at day 1 after overload. In addition, a target of MEK/ERK signalling, phosphorylation of TSC2 at S664, was also increased at this early time point. These observations demonstrate that in vivo, mTORC1 activation at the early phase of mechanical overload in skeletal muscle occurs independently of PI3K/Akt signalling and provide evidence that the MEK/ERK pathway may contribute to mTORC1 activation through phosphorylation of TSC2.

Figures

Figure 1
Figure 1. Increased muscle weight, RNA content and protein metabolism in response to mechanical overload of the plantaris muscle
A, plantaris muscle weight (mg) was significantly increased in response to mechanical overload (OV) after 1 day (OV-1) and continued to progressively increase to OV-10 (n = 4/time point). B, total RNA content significantly increased in response to OV after 5 days (OV-5) and remained elevated for the remainder of the time course (n = 4/time point). C, in vitro protein synthesis rate showed a significant 14.2-fold increase in the OV-7 group compared to the control group (n = 8/group). D, in vitro protein degradation rate was significantly increased 1.7-fold in the OV-7 group compared to the control group (n = 8/group). All results are expressed as the mean ± SEM with a significant difference (P < 0.05) from the control group (OV-0) designated by an asterisk.
Figure 2
Figure 2. Early activation of mTORC1 signalling in response to mechanical overload
A, representative images of S6K1 showing phosphorylation status of T389 and T421/S424 and total S6K1 protein. B, relative (normalized to OV-0) phosphorylation levels of T389 and T421/S424 sites and total protein level of S6K1 at each time point were quantified (n = 4–5/group). Significant differences: #quantification of S6K1-T389 phosphorylation compared to control (OV-0) group, P < 0.05; *quantification of S6K1-T421/S424 phosphorylation compared to control (OV-0) group, P < 0.05. All results are expressed as the mean ± SEM. C, phosphorylation state of rpS6 was determined by immunofluorescence analysis. Upper panels were from sham-operated control muscle and lower panels were from the plantaris muscle with a single day of mechanical overload. rpS6 phosphorylation at S235/236 was detected using Texas Red-conjugated goat anti-rabbit antibody (red), and nuclei were visualized by DAPI (blue). Images with low (×10, left panels) and high (×40, right panels) magnification were indicated. Each image was merged using SPOT Advanced Software (SPOT Imaging Solutions, Sterling Heights, MI, USA). Scale bars represent 400 μm in low magnification images and 100 μm in high magnification images.
Figure 3
Figure 3. Delayed phosphorylation of Akt and direct Akt targets following mechanical overload of the plantaris muscle
Mechanical overload-induced alteration in the phosphorylation state of Akt and downstream targets of Akt including PRAS40 (at T246 site) and GSK3-β (at S9 site) were determined. A, representative images showing Akt phosphorylation (S473 and T308 sites) states and total Akt protein. B, relative phosphorylation (S473 and T308 sites) and total protein levels of Akt at each time point were quantified (n = 4–5/group). Significant differences: #quantification of Akt-S473 phosphorylation, compared to the control (OV-0) group, P < 0.05; *quantification of Akt-T308 phosphorylation, compared to the control (OV-0) group, P < 0.05; †quantification of Akt-total, compared to the control (OV-0) group, P < 0.05. C, representative images of PRAS40 phosphorylation (T246 site) state and total protein expression. D, the relative (normalized to OV-0) phosphorylation (T246 site) and total protein levels of PRAS40 in each group were quantified. Significant differences: #quantification of PRAS40-T246 phosphorylation compared to the control (OV-0) group, P < 0.05. E, representative images of GSK3-β phosphorylation (S9 site) state and total protein expression. F, relative phosphorylation (S9 site) and total protein levels of GSK3-β in each group were quantified. Significant differences: #quantification of GSK3-β-S9 phosphorylation compared to the control (OV-0) group, P < 0.05. GAPDH was used as a loading control among the experimental conditions. All results are expressed as the mean ± SEM (n = 4–5).
Figure 4
Figure 4. Increased expression of TSC1 and TSC2 following mechanical overload
Mechanical overload-induced phosphorylation of TSC2 (S939 and T1462 sites) and complex formation between TSC1 and TSC2 were determined in the plantaris muscle. A, representative images of phosphorylation (TSC2-S939 and TSC2-T1462) state and total protein expression of TSC1 and TSC2. Total lysates from the plantaris muscle were immunoprecipitated with either anti-TSC1 or anti-TSC2 antibody. GAPDH was used as a loading control among the experimental conditions. B and C, the relative expression (normalized to OV-0) levels of total TSC1 (B) and total TSC2 (C) in each group were quantified. All results are expressed as the mean ± SEM (n = 3/group). Significant differences: #compared to the control (OV-0) group, *compared to 1 day of mechanical overload (OV-1) group, P < 0.05. The phosphorylation level of TSC2 at S939 site was increased in the plantaris muscle following 7 days of mechanical overload with concomitant increase in the total TSC2 protein. Co-immunoprecipitation assay showed that mechanical overload did not affect the physical association between TSC1 and TSC2 in the plantaris muscle. No changes in the phosphorylation state of TSC2 at S939 or T1462 site was observed at day 1 of overload.
Figure 5
Figure 5. Inhibition of PI3K signalling did not prevent early activation of mTORC1 in response to mechanical overload
A, effect of wortmannin or rapamycin treatment on insulin-induced phosphorylation of Akt (S473 and T308 sites) and S6K1 (T389 and T421/S424 sites) in the plantaris muscle. Insulin (0.75 U kg−1), wortmannin (3 mg kg−1) and rapamycin (1.5 mg kg−1) were administered by intraperitoneal injection. DMSO was used as a solvent for wortmannin and rapamycin. Wortmannin was administered twice (12 h and 24 h prior to the insulin stimulation), and rapamycin was injected once (24 h prior to the insulin stimulation). Twenty-four hours after the first shot of solvent (DMSO) or inhibitors, insulin was administered by intraperitoneal injection and muscle samples collected 20 min after insulin treatment. As expected, wortmannin blocked insulin-induced phosphorylation of Akt and S6K1 whereas rapamycin only prevented S6K1 phosphorylation. B, effect of wortmannin or rapamycin treatment on mechanical overload-induced phosphorylation of Akt and S6K1 and in vitro Akt kinase activity in the plantaris muscle. Wortmannin was administered twice (immediately and 12 h after the synergist ablation surgery) and rapamycin was injected once (immediately after the surgery). Sham-operated (sham) served as a control. Muscle samples were collected 24 h after adminstration of DMSO or inhibitor. There was no change in Akt phosphorylation at this early time point (OV-1). However, phosphorylation of S6K1 at T389 was blocked by rapamycin but not by wortmannin. The in vitro Akt kinase assay revealed no change in Akt activity confirming the Akt phosphorylation data. Each set of experiments were repeated at least 3 times using different animals.
Figure 6
Figure 6. Early activation of MEK1/2 and ERK1/2 signalling in plantaris muscle following mechanical overload
A, representative images of phosphorylation state (S217/221 sites) and total protein expression of MEK1/2. B, relative (normalized to OV-0) phosphorylation (S217/221 sites) and total protein levels of MEK1/2 in each group were quantified. All results are expressed as the mean ± SEM (n = 4–5). Significant differences: #compared to the control (OV-0) group, P < 0.05. C, representative images of phosphorylation states (T202/204) and total protein expression of ERK1/2. D, relative (normalized to OV-0) phosphorylation (T202/204) and total protein levels of ERK1/2 in each group were quantified. All results are expressed as the mean ± SEM (n = 4–5). Significant differences: #quantification of ERK1/2-T202/204 phosphorylation compared to the control (OV-0) group, P < 0.05; †quantification of ERK1/2-total compared to the control (OV-0) group, P < 0.05. For both MEK1/2 and ERK1/2 there was a significant increase in phosphorylation after 1 day of overload (OV-1) that was maintained until OV-7.
Figure 7
Figure 7. Increased TSC2 phosphorylation following 1 day of mechanical overload
Phosphorylation state of TSC2 at S664 site was examined by Western blotting and chromogenic immunohistochemical analysis. A, representative images of TSC2 phosphorylation at S664 site and total protein expression of TSC2. Total lysates from the plantaris muscle were immunoprecipitated with anti-TSC2 antibody. For the Western blotting analysis, phospho-specific antibody for TSC2 at the S664 site was kindly provided by Dr Pier Paolo Pandolfi, Beth Israel Deaconess Medical Center, Harvard Medical School. B–D, the plantaris muscles for immunohistochemical analysis were collected from sham-operated control (Sham) and 1 day of mechanical overload. For immunohistochemistry, the phospho-specific antibody for TSC2 at S664 site was obtained from Biolegend. The absence of staining in the no antibody control (B) demonstrated no background staining whereas the sham control (C) showed very low TSC2 phosphorylation under resting conditions. Following a single day of overload (D) muscle fibres showed enhanced chromogenic staining indicating increased TSC2 phosphorylation at site S664. Scale bars represent 100 μm in all images.
Figure 8
Figure 8. mTORC1 inhibitor rapamycin did not prevent MEK/ERK/RSK signalling in response to mechanical overload
The effect of rapamycin treatment on mechanical overload-induced phosphorylation of S6K1 (T389), rpS6 (S235/236 and S240/244), MEK1/2 (S217/221), ERK1/2 (T202/204) and RSK (T359/S363 and S380) was examined. Rapamycin (1.5 mg kg−1) was administered immediately after surgery by intraperitoneal injection. DMSO was used as a solvent for rapamycin. Sham-operated (sham) served as a control. Muscle samples were collected 24 h after adminstration of DMSO or rapamycin. The mTORC1 inhibitor rapamycin completely blocked overload-induced phosphorylation of S6K1; however, it was not sufficient for complete inhibition of rpS6 phosphorylation at both S235/236 and S240/244 sites in response to mechanical overload. No inhibitory effects of rapamycin treatment on overload-induced phosphorylation of MEK1/2, ERK1/2 and RSK were observed. Each set of experiments was repeated at least 3 times using different animals.
Figure 9
Figure 9. Schematic representation of the molecular mechanisms in the regulation of mTORC1 signalling pathway during early phase of mechanical overload-induced skeletal muscle hypertrophy
Activation of mTORC1 signalling at the early phase in response to mechanical overload occurs independently of PI3K/Akt signalling in skeletal muscle. MEK/ERK pathway through TSC2 phosphorylation at S664 may contribute to mTORC1 activation during skeletal muscle hypertrophy.

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