Mol Cell Biochem DOI 10.1007/s11010-015-2446-7

Mechanical stretch activates mammalian target of rapamycin and AMP-activated protein kinase pathways in skeletal muscle cells Naoya Nakai1,2 • Fuminori Kawano1 • Ken Nakata1

Received: 6 January 2015 / Accepted: 7 May 2015 Ó Springer Science+Business Media New York 2015

Abstract Cellular protein synthesis is believed to be antagonistically regulated by mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) signaling pathways. In the present study, we examined the relationship between mTOR/p70 S6 kinase (p70S6K) and AMPK in response to mechanical stretch. C2C12 myoblasts were grown on a silicone elastomer chamber to confluence and further cultured in differentiation medium for 4 days to form multinucleated myotubes. Cells were subjected to 15 % cyclic uniaxial stretch for 4 h at a frequency of 1 Hz. Phosphorylation of p70S6K at threonine 389 and AMPK at threonine 172 of the catalytic a subunit were concomitantly increased by mechanical stretch. Stimulation of the mTOR pathway by adding leucine and insulin increased the phosphorylation of p70S6K without inactivation of AMPK. In contrast, addition of compound C, a pharmacological inhibitor of AMPK, increased the phosphorylation of p70S6K in stretched cells. Activation of AMPK by the addition of 5-amino-4-imidazolecarboxamide ribonucleoside reduced the phosphorylation of p70S6K in response to mechanical stretch. In conclusion, crosstalk between mTOR and AMPK signaling was not tightly regulated in response to physiological stimuli, such as mechanical stress and/or

& Naoya Nakai [email protected]; [email protected] 1

Department of Health and Sports Sciences, Graduate School of Medicine, Osaka University, 1-17 Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan

2

Present Address: Department of Nutrition, School of Human Cultures, University of Shiga Prefecture, 2500 Hassaka-cho, Hikone, Shiga 522-8533, Japan

nutrients. However, pharmacological modulation of AMPK influenced the mTOR/p70S6K signaling pathway. Keywords Mechanical stimulus  Mammalian target of rapamycin  AMP-activated protein kinase  Protein synthesis

Introduction The most widely recognized stimuli for increasing skeletal muscle mass are appropriate nutritional intake and resistance exercise. Signaling through the mammalian target of rapamycin (mTOR) plays an important role in the control of muscle protein synthesis [1, 2]. In eukaryotes, mTOR is found in two independently regulated, functionally distinct complexes, namely, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) [3]. mTORC1 is thought to be the nutrient- and exercise-responsive mediator of skeletal muscle protein synthesis [4]. Resistance exercise and/or nutrients activate the mTOR pathway in a process mediated in part via its downstream targets, e.g., p70 S6 kinase (p70S6K) [5, 6]. Phosphorylation of p70S6K at threonine 389 (T389) results in maximal kinase activity and is associated with increased activation of protein translation initiation [7–9]. Mechanical stimuli have been recognized as an essential factor for the maintenance of skeletal muscle mass in response to resistance exercise. Mechanical activation of the mTORC1 pathway has been demonstrated by Hornberger et al. [10–12]. We also previously reported that mechanical stretch increases the phosphorylation of p70S6K in C2C12 myoblasts [13]. With regard to the nutrient-induced activation of mTORC1, the branched-chain amino acid (BCAA) leucine has been reported as the primary nutrient

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signal that regulates the initiation phase of protein synthesis [14]. Numerous studies have shown that leucine induces activation of the mTOR pathway in human [15, 16] and rodent [17, 18] skeletal muscle and in cultured myoblasts [19, 20]. Thus, the mTOR pathway is intimately involved in determining skeletal muscle mass in response to nutrients and exercise. In contrast to the mTOR/p70S6K signaling pathway, AMP-activated protein kinase (AMPK) is activated under conditions of low energy status and negatively regulates mTOR signaling [21, 22]. The regulation of skeletal muscle mass in response to nutrition and exercise is mediated by a complex interplay between mTOR and AMPK signaling [23]. Phosphorylation of AMPK at threonine 172 (T172) of the catalytic a subunit activates its kinase activity [24]. AMPK has been reported to be activated in muscles during treadmill running [25] and electrically stimulated contraction [26]. These studies suggested that contraction-activated AMPK may act as a negative regulator of mTOR/p70S6K signaling immediately after contraction or exercise in rats [27, 28]. In fact, mTOR/ p70S6K signaling has been shown to be decreased during contraction with concomitant activation of AMPK [29]. However, this assumption has been challenged in several recent studies in which AMPK and mTOR phosphorylation have been shown to increase concomitantly in response to acute endurance exercise [30, 31]. The aim of the present study was to elucidate the relationship between mTOR/p70S6K and AMPK signaling pathways in response to mechanical stimuli and nutrient in vitro. Our results showed that the crosstalk between mTOR and AMPK signaling was not tightly regulated in response to physiological stimuli. However, pharmacological modulation of AMPK affected the activation of the mTOR/p70S6K signaling pathway in response to mechanical stimuli.

Materials and methods Materials Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine albumin (FBS), and antibiotics (penicillin–streptomycin) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The AMPK inhibitor compound C (CC) and the AMPK activator 5-amino-4-imidazolecarboxamide ribonucleoside (AICAR) were purchased from Calbiochem (San Diego, CA, USA) and Sigma-Aldrich, respectively, and dissolved in dimethyl sulfoxide (DMSO). Antibodies against phospho-T389-p70S6K, phospho-T172-AMPKa, total AMPKa, phospho-S2448-mTOR, and phospho-S473Akt were obtained from Cell Signaling Technology

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(Beverly, MA, USA). Antibodies against total p70S6K were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). C2C12 culture C2C12 cells (a mouse myoblast cell line) were plated in fibronectin-coated silicone elastomer chambers (32 mm long, 32 mm wide, and 10 mm deep) in DMEM, supplemented with 10 % FBS, 100 units/mL penicillin, and 100 mg/mL streptomycin sulfate in a 5 % CO2-humidified incubator at 37 °C. Cells were grown to approximately 90–100 % confluence, and culture medium was then changed to differentiation medium. Differentiation medium consisted of DMEM supplemented with 2 % calf serum, 100 units/mL penicillin, and 100 mg/mL streptomycin sulfate. Cells were cultured in a differentiation medium for 4 days to form multinucleated myotubes. Differentiation medium was changed every 24 h. Mechanical stretch Differentiated myotubes were subjected to uniaxial mechanical cyclic stretch using a stretch apparatus (ST-150; Strex, Osaka, Japan) [13]. At 1.5 h before subjection to mechanical stretch, differentiation medium was replaced with serum-free DMEM supplemented with 100 units/mL penicillin and 100 mg/mL streptomycin sulfate. The chamber was uniaxially stretched at 15 % of the initial length for 60 stretch-relaxation cycles/min. In experiments using the pharmacological inhibitor or activator of AMPK, 20 lM compound C or 2 mM AICAR was added to the cells 1 h prior to stretching. The cells were then subjected to mechanical stretch in the presence of the indicated compounds. The same amount of DMSO without compounds was added to the control cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting Cells were rinsed with cold phosphate-buffered saline (PBS) and lysed in RIPA lysis buffer (Santa Cruz Biotechnology). Equal amounts of protein were subjected to SDS-PAGE and immunoblotting. Separated proteins were transferred to polyvinylidenedifluoride (PVDF) membranes. Nonspecific binding to the membrane was blocked with Tris-buffered saline (pH 7.4), containing 0.05 % (v/v) Tween-20 and 5 % bovine serum albumin (BSA) or 5 % skim milk. Following an overnight incubation with the indicated primary antibody, the membranes were washed and then incubated with horseradish peroxidase-conjugated anti-rabbit IgG. Immunoreactive protein bands were visualized by ECL-prime (GE Healthcare

Mol Cell Biochem

UK Ltd., Buckinghamshire, UK) using an ECL mini camera (GE Healthcare UK Ltd.) or myECL Imager (Thermo Fisher Scientific Inc., MA, USA). The band intensity was quantified by a computer analysis package (NIH ImageJ). Statistics Data are presented as means ± standard deviations (SDs). Statistical analysis for multiple comparisons was performed using one-way or two-way analysis of variance (ANOVA) followed by the Scheffe’s post-hoc test. Differences with P values of less than 0.05 were considered statistically significant.

Results Effects of mechanical stretch on p70S6K and AMPKa Phosphorylation of p70S6K at threonine 389 (T389) results in the maximal kinase activity [7]. Therefore, we first examined the effects of mechanical stretch on the phosphorylation of p70S6K by Western blot analysis (Fig. 1A). Phosphorylation of p70S6K at T389 was increased by 0.5

Fig. 1 Effects of mechanical stretch on the phosphorylation of p70 S6 kinase (p70S6K) (A) and AMP-activated protein kinase a (AMPKa) (B). Cells were subjected to 0.5 or 4 h of uniaxial mechanical stretch at 15 % of the initial length. Photographs of representative Western blots for total (T)-p70S6K and phospho (P)T389-p70S6K are shown at the top (A). Photographs of representative

and 4 h of mechanical stretch, with a significant increase observed after 4 h of stretch compared with the control group. No changes in total p70S6K expression were observed among the three groups (Fig. 1A). Activation of AMPK requires phosphorylation at threonine 172 (T172) of the catalytic a subunit [24]. At 0.5 and 4 h, mechanical stretch significantly increased the phosphorylation of T172 in AMPKa (Fig. 1B), while the expression of total AMPKa was not different among groups (Fig. 1B). Effects of leucine and insulin on the phosphorylation of p70S6K The BCAA leucine [32] and the hormone insulin [33] are well-known activators of the mTOR/p70S6K pathway. Therefore, we next examined the phosphorylation of p70S6K in response to leucine and insulin in control and stretched cells. The addition of leucine tended to increase the phosphorylation of p70S6K both in the control and stretched groups (Fig. 2). Moreover, the addition of insulin significantly increased the phosphorylation of p70S6K both in control and stretched cells (Fig. 2), and insulin-induced phosphorylation of p70S6K was significantly higher in stretched cells than in control cells (Fig. 2).

Western blots of total (T)-AMPKa and phospho (P)-AMPKa are shown at the top (B). Data for P-T389-p70S6K (A) and P-T172AMPKa (B) are expressed as the percentage relative to the nonstretched control cells (Cont) (100 %). *Significantly different from Cont (P \ 0.05)

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Fig. 2 Effects of leucine and insulin on the phosphorylation of p70 S6 kinase (p70S6K) in mechanically stretched cells. Cells were subjected to 4 h of uniaxial mechanical stretch at 15 % of the initial length. Immediately after the mechanical stretch, PBS, 2 mM leucine, or 1 lM insulin was added to the medium, and cells were incubated for 45 min. Photographs of representative Western blots of total (T)p70S6K and phospho (P)-T389-p70S6K are shown at the top. Data for P-T389-p70S6K are expressed as the percentage relative to the control-PBS group (100 %). Values with different letters are significantly different (P \ 0.05). *Significantly different from control cells in the same treatment group (P \ 0.05)

Effects of leucine and insulin on the phosphorylation of AMPKa The activity and phosphorylation of AMPK at T172 of the catalytic a subunit have been reported to be suppressed by insulin [34, 35]. Thus, we next examined the effects of leucine and insulin on the phosphorylation of AMPKa in control and stretched cells. The expression of total AMPKa was not affected by the addition of leucine or insulin (Fig. 3). Additionally, the phosphorylation of AMPKa did not change in control and stretched cells treated with PBS, leucine, or insulin (Fig. 3). Effects of leucine and insulin on the phosphorylation of mTOR and Akt The phosphorylation statuses of mTOR at S2448 and Akt at S473 have been reported to regulate intracellular signaling for protein synthesis in response to nutrients and exercise [4]. Phosphorylation of mTOR tended to increase following stretching and leucine treatment; however, there were no statistically significant differences among groups (Fig. 4B). Phosphorylation of Akt was markedly increased by the addition of insulin in both control and stretch groups (Fig. 4C).

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Fig. 3 Effects of leucine and insulin on the phosphorylation of AMPactivated protein kinase a (AMPKa) in mechanically stretched cells. Cells were subjected to 4 h of uniaxial mechanical stretch at 15 % of the initial length. Immediately after the mechanical stretch, PBS, 2 mM leucine, or 1 lM insulin was added to the medium, and cells were incubated for 45 min. Photographs of representative Western blots of total (T)-AMPKa and phospho (P)-AMPKa are shown at the top. Data for P-T172-AMPKa are expressed as the percentage relative to the control-PBS group (100 %). *Significantly different from control cells in the same treatment group (P \ 0.05)

Effects of compound C on the phosphorylation of p70S6K and AMPK To determine the effects of AMPK inhibition on the mTOR/p70S6K pathway, we utilized the AMPK inhibitor compound C. Treatment of the cells with 20 lM compound C further increased the phosphorylation of p70S6K in the stretch group (Fig. 5A). The expression of total p70S6K did not differ among the groups (Fig. 5A). Treatment of the cells with compound C tended to decrease the phosphorylation of AMPK both in control and stretch groups, but this difference was not significant (Fig. 5B). Effects of AICAR on the phosphorylation of p70S6K and AMPK AICAR is widely used as an AMPK activator. Treatment of the cells with 2 mM AICAR slightly decreased the phosphorylation of p70S6K in control cells. In stretched cells, pretreatment of the cells with AICAR blocked the stretch-induced phosphorylation of p70S6K (Fig. 6A). The expression of total p70S6K was not significantly different among the groups (Fig. 6A). Treatment of the cells with AICAR significantly increased the phosphorylation of AMPK both in control and stretched groups (Fig. 6B).

Mol Cell Biochem

Fig. 4 Effects of leucine and insulin on the phosphorylation of mTOR and Akt in mechanically stretched cells. Cells were subjected to 4 h of uniaxial mechanical stretch at 15 % of the initial length. Immediately after the mechanical stretch, PBS, 2 mM leucine, or 1 lM insulin was added to the medium, and cells were incubated for 45 min. Photographs of representative Western blots of phospho (P)mTOR, P-Akt, and b-actin are shown at the top (A). Data for P-S2448-mTOR (B) and P-S473-Akt (C) are expressed as the percentage relative to the control-PBS group (100 %). *Values with different letters are significantly different (P \ 0.05)

Discussion In this study, we examined the relationship between the mTOR/p70S6K and AMPK signaling pathways in response to mechanical stretch in C2C12 myotubes. We showed that mechanical stretch activated both p70S6K and AMPK simultaneously. Moreover, the addition of leucine and insulin increased the phosphorylation of p70S6K without dephosphorylation of AMPKa. These results suggested that the crosstalk between mTOR and AMPK signaling was not tightly regulated in response to physiological stimuli, such as mechanical stress and/or nutrients. However, pharmacological modulation of AMPK influenced activation of the

mTOR/p70S6K signaling pathway in response to mechanical stretch. Thus, our data provided important insights into the signaling pathways mediating mTOR and AMPK activity in response to physiological stimuli in muscle cells. Mechanical stimuli are major regulators of protein synthesis in skeletal muscle [36]. Mechanical activation of the mTOR/p70S6K signaling pathway has also been reported in muscle cells in vitro [13, 37]. In the present study, 4-h exposure to 15 % cyclic uniaxial stretch at the frequency of 1 Hz significantly increased the phosphorylation of p70S6K in myotubes, suggesting that mechanical stretch activates the initiation of protein translation. In contrast, mechanical stimulus-induced activation of AMPK has also been demonstrated in skeletal muscle cell in vivo [38], ex vivo [39, 40], and in vitro [41]. In the present study, phosphorylation of AMPK at T172 of the catalytic a subunit was significantly higher in stretched cells, suggesting that mechanical stretch activated AMPK. A number of studies have proposed that AMPK acts antagonistically against the mTOR signaling pathway and protein synthesis [29, 42, 43]. However, this assumption has been challenged by several recent studies. For example, phosphorylation levels of AMPKa and mTOR have been shown to increase in response to acute endurance exercise [30, 31]. The results of the present study confirmed the concomitant activation of AMPK and mTOR/ p70S6K pathways. Phosphorylation of p70S6K tended to be increased by leucine and was significantly increased by insulin in control and stretched cells. Insulin-induced p70S6K phosphorylation was significantly higher in stretched cells than in control cells, suggesting that insulin and mechanical stretch had an additive effect on the activation of p70S6K. Moreover, insulin-induced activation of p70S6K was achieved without dephosphorylation of T172 of the AMPKa subunit in stretched cells. These results suggested that activation of AMPK did not inhibit the phosphorylation of p70S6K in response to leucine and insulin. A recent report by Valentine et al. [44] showed that phosphorylation of S485/491 of AMPKa was involved in insulin-induced deactivation of AMPK. Therefore, future studies are required to determine the phosphorylation levels at this site and the actual activity of AMPK because of this phosphorylation. We further examined the phosphorylation of mTOR and Akt, which is involved in the regulation of muscle protein synthesis [1, 36, 45]. Phosphorylation of mTOR at S2448 tended to increase following leucine treatment and mechanical stretch and decreased following insulin treatment; however, these differences were not significant. In contrast, the phosphorylation of Akt at S473 was markedly increased by the addition of insulin in both control and stretched

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Fig. 5 Effects of compound C on the stretch-induced phosphorylation of p70 S6 kinase (p70S6K) and AMP-activated protein kinase a (AMPKa). One hour prior to mechanical stretch, DMSO or 20 lM compound C (CC) was added to the cells. The cells were then subjected to 4 h of uniaxial mechanical stretch at 15 % of the initial length. Western blots for total (T)-p70S6K and phospho (P)-T389p70S6K are shown at the top (A). Western blots for total (T)-AMPK,

phospho (P)-T172-AMPKa, and b-actin are shown at the top (B). Data for P-T389-p70S6K (A) are expressed as the percentage relative to the control-DMSO group (100 %). Data for P-T172-AMPKa (B) are expressed as the percentage relative to the control-DMSO group (100 %). #Significantly different from control cells in the same treatment group (P \ 0.05). *Significantly different from DMSO groups (P \ 0.05)

cells. Barazzoni et al. [46] reported that sustained insulin infusion without amino acid replacement causes sustained stimulation of Akt phosphorylation, without downstream stimulatory effects on mTOR phosphorylation. These results suggest that Akt-induced upregulation of mTOR phosphorylation at S2448 may not be essential for p70S6K activation by mechanical stretch and nutrient supplementation in C2C12 cells. In the present study, addition of the AMPK inhibitor compound C increased the phosphorylation of p70S6K in stretched cells. In contrast, AICAR-induced activation of AMPK inhibited the phosphorylation of p70S6K in response to mechanical stretch. Reciprocal regulation of nitric oxide-induced cardiomyogenesis of embryonic stem cells by AMPK and mTOR has been clearly demonstrated by AICAR and compound C [47]. The results of the present study and former report both support that pharmacological

modulation of AMPK activity regulates the mTOR/ p70S6K signaling pathway. The current data indicated that mechanical stimuli increased both mTOR/p70S6K and AMPK signaling pathways and showed that insulin-induced activation of p70S6K was achieved without inactivation of AMPK. These results suggested that the antagonistic relationship between mTOR/p70S6K and AMPK was not observed in response to mechanical stretch and subsequent stimulation with insulin in C2C12 myotubes. In contrast, pharmacological modulation of AMPK activity affected mechanical stretch-induced phosphorylation of p70S6K. Thus, mTOR/ p70S6K and AMPK may be highly coordinated, rather than antagonistic, in the regulation of cellular responses to various stimuli. Future studies are needed to clarify the mechanisms mediating cellular responses to mechanical stimuli.

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Fig. 6 Effects of AICAR on the stretch-induced phosphorylation of p70 S6 kinase (p70S6K) and AMP-activated protein kinase a (AMPKa). One hour prior to mechanical stretch, DMSO or 2 mM AICAR was added to the cells. The cells were then subjected to 4 h of uniaxial mechanical stretch at 15 % of the initial length. Western blots for total (T)-p70S6K and phospho (P)-T389-p70S6K are shown at the top (A). Western blots for total (T)-AMPK, phospho (P)-T172-

AMPKa, and b-actin are shown at the top (B). Data for P-T389p70S6K (A) are expressed as the percentage relative to the controlDMSO group (100 %). Data for P-T172-AMPKa (B) are expressed as the percentage relative to the control-DMSO group (100 %). # Significantly different from control cells in the same treatment group (P \ 0.05). *Significantly different from DMSO groups (P \ 0.05)

Funding This work was supported by a Grant-in-Aid for Scientific Research C (25350813 to N.N.) from the Japan Society for the Promotion of Science (JSPS), Japan.

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Conflict of interest of interest.

6.

The authors declare that there are no conflicts 7.

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