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ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
Department of Animal Science, University of Wyoming, Laramie 82071
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Abstract |
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subunit at Thr172 (28.6 ± 4.9% reduction, P < 0.05) and inhibited AMPK activity (43.6 ± 3.5% reduction, P < 0.05). In addition, leucine increased the phosphorylation of mTOR at Ser2448 by 63.5 ± 10.0% (P < 0.05) and protein synthesis by 30.6 ± 6.1% (P < 0.05). Applying 5-aminoimidazole-4-carbox-amide 1-beta-d-ribonucleoside, an activator of AMPK, abolished the stimulation of mTOR signaling by leucine, showing that AMPK negatively controls mTOR signaling. To further show the role of AMPK in mTOR signaling, myoblasts expressing a dominant negative AMPK subunit were employed. Negative myoblasts had very low AMPK activity. The activation of mTOR induced by leucine in these cells was abated, showing that AMPK contributed to mTOR activation. In conclusion, leucine stimulates mTOR signaling in part through AMPK inhibition. This study implicates AMPK as an important target for nutritional management to enhance mTOR signaling and protein synthesis in muscle cells, thereby increasing muscle growth.
Key Words: adenosine monophosphate-activated protein kinase • leucine • myoblast cell • mammalian target of rapamycin • protein synthesis
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INTRODUCTION |
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TopAbstractINTRODUCTIONRESULTSDISCUSSIONLITERATURE CITED |
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; Bolster et al., 2003; Sakamoto et al., 2003). Activation of mTOR upregulates protein production through phosphorylating eukaryotic initiation factor 4E binding protein 1 (4E-BP1) and ribosomal protein S6 kinase (S6K; Schmelzle and Hall, 2000; Hara et al., 2002; Shen et al., 2005).
Adenosine monophosphate (AMP)-activated protein kinase (AMPK), a heterotrimeric enzyme with , ß, and 
subunits, is mainly recognized as a critical regulator of energy metabolism (Kim et al., 2004). The AMPK is activated when the cellular energy level is low. Activation of AMPK phosphorylates tuberous sclerosis-2 (TSC2; Figure 1), which negatively controls mTOR signaling, linking energy availability to mTOR signaling.
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; Lynch et al., 2003; Lang and Frost, 2005). This stimulation of protein synthesis involves upregulation of mTOR signaling (Lynch et al., 2003; Tokunaga et al., 2004). The mechanisms associated with enhanced mTOR signaling due to leucine, however, are poorly understood (Martin and Hall, 2005). We hypothesized that leucine activates mTOR in part through AMPK inhibition. Leucine and other branched-chain amino acids (BCAA) are not degraded in the liver due to the absence of branched-chain amino acid aminotransferase (BCAT), whereas other amino acids are catabolized mainly in the liver. However, BCAT is present in skeletal muscle (Sweatt et al., 2004), and degradation of BCAA in skeletal muscle is hypothesized to provide energy that inhibits AMPK activity, leading to mTOR activation (Figure 1
). The objective of this study was to examine whether leucine stimulates mTOR signaling in part through inhibition of AMPK activity.
All animal procedures were approved by the University of Wyoming Animal Care and Use Committee.
Antibodies and Chemicals for Cell Culture
Antibodies against phospho-mTOR at Ser2448, phospho-AMPK at Thr172, and phospho-S6K at Thr389 were purchased from Cell Signalling Technology Inc. (Beverly, MA). Antibody against actin was obtained from the Developmental Studies Hybridoma Bank (Iowa City, IA). Cell culture medium [Dulbecco’s Modified Eagle’s Medium (DMEM)] and fetal bovine serum (FBS) were obtained from Sigma (St. Louis, MO). The C2C12 myoblast cells were purchased from the American Type Culture Collection (Manassas, VA). The L-[2,3,4,5,6-3H]phenylalanine was bought from Amersham Biosciences (Buckinghamshire, UK). L-Leucine and other chemicals were purchased from Sigma.
C2C12 Myoblast Cells
The C2C12 myoblast cells were grown to 60% confluence in DMEM supplemented with 10% heat-inactivated FBS plus 50 U/mL of penicillin and 50 µg/mL of streptomycin in an incubator under a humidified atmosphere of 5% CO2 and 95% air at 37°C. Then, the cells were starved in low-glucose DMEM supplemented with 0.2% FBS for 4 h. The cells were subjected to various treatments in the same starvation media by adding 2 mM L-leucine, 2 mM AICAr, 2 mM D-leucine, 2 mM glucose, or 2 mM sodium pyruvate, and collected for the analyses 30 min after application of the treatments, unless specifically indicated.
Primary skeletal muscle cells were obtained from the skeletal muscle of wild-type mice and mice that had muscle-specific expression of dominant negative AMPK; the AMPK activity in skeletal muscle of such mice was very low (Mu et al., 2001). Mice that specifically expressed dominant negative AMPK were obtained from M. J. Birnbaum (Department of Medicine and Howard Hughes Medical Institute, University of Pennsylvania). The skeletal muscle was minced in DMEM after trimming off all visible fat and connective tissue, and then incubated in 1 mg/mL of protease (P5985, Sigma) at 37°C for 1 h. The tissue slurry was centrifuged at 1,500 x g for 10 min, and the pellet was suspended in DMEM and centrifuged at 400 x g for 5 min. The supernatant was transferred to another tube and centrifuged at 1,500 x g for 10 min. The resulting pellet containing myoblast cells was used for cell culture. Before treatments, primary myoblast cells were induced to fuse into myotubes by providing them with 2% horse serum (Sigma).
Preparation of Cell Lysates
Cells were lysed in a buffer containing 50 mM HEPES, pH 7.4; 137 mM NaCl; 1% NP-40; 10% glycerol; 2 mM Na3VO4; 100 mM NaF; 1 mM MgCl2; 1 mM CaCl2; 2.5 mM EDTA; and 1% protease inhibitor cocktail (Sigma). The cell lysate was centrifuged at 12,000 x g for 15 min at 4°C, and the protein concentration of the supernatant was determined by the bicinchoninic acid method (Biorad, Hercules, CA; Zhu et al., 2006).
Electrophoresis and Immunoblotting
Cell lysates containing equivalent amounts of protein were boiled in Laemmli sample buffer with 5% mercaptoethanol at 95°C for 5 min. A 5 to 20% gradient gel was used for SDS-PAGE separation of the proteins. A Hoefer mini-gel system (Hoefer Inc., San Francisco, CA) was used for casting gels and running electrophoresis. After electrophoresis, the proteins on the gel were transferred to a nitrocellulose membrane (BioRad, Hercules, CA) in a transfer buffer containing 20 mM Trisbase, 192 mM glycine, 0.1% SDS, and 20% methanol.
The nitrocellulose membranes were incubated in a blocking solution consisting of 5% nonfat dry milk in TBS/T (0.1% Tween-20; 50 mM Tris-HCl, pH 7.6; and 150 mM NaCl) for 1 h followed by an overnight incubation at 4°C with primary antibodies appropriately diluted with TBS/T containing 1% nonfat dry milk. At the end of the incubation with primary antibody, the membranes were washed 3 times for 5 min each with 20 mL of TBS/T. After that, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies at an appropriate dilution for 1 h in TBS/T with gentle agitation. After three 10-min washes, the secondary antibody was visualized using the ECL Western blotting reagents (Amersham Bioscience, Piscataway, NJ) and exposure to film (MR, Kodak, Rochester, NY). The density of the bands was quantified by using an Imager Scanner II and ImageQuant TL software (Amersham Bioscience; Du et al., 2004). To reduce the variation between blots, cell lysates from all treatments were run on each gel. The band density among different blots was normalized according to the density of the reference band, which was loaded on all gels. The band density was also normalized to the actin content (Yang et al., 2005).
For reprobing, the membranes were incubated in a stripping solution (100 mM 2-mercaptoethanol; 2% SDS; 62.5 mM Tris-Cl, pH 6.7) at 50°C for 30 min with shaking. Then, the membranes were thoroughly washed with TBS/T and blocked with 5% nonfat dry milk in TBS/T. The membranes were used for actin content measurement by immunoblotting as described above.
Measurement of Protein Synthesis
Protein synthesis was measured by the rate of incorporation of [3H]phenylalanine into total mixed proteins. Two microcurie of L-[2,3,4,5,6-3H]phenylalanine (120 Ci/mmol) was added to each flask of C2C12 myoblast cells or myotubes and incubated for 30 min. Then, the cells were lysed as described above, and the cell lysates were mixed with an equal volume of 20% trichloroacetic acid (Sigma). The resultant precipitate was solubilized by treatment with 200 µL of 1 M NaOH for 30 min and mixed with 5 mL of ScintiVerse (Fisher Scientific, Hanover Park, IL). The incorporation of the radioisotopes was measured by liquid scintillation counting (Shen et al., 2005).
Measurement of AMPK Activity
The AMPK activity was measured as previously reported (Zhu et al., 2004), with modifications. Briefly, the cell lysates were centrifuged for 5 min at 13,000 x g at 4°C. Then, AMPK in the cell lysates was immunoprecipitated by using an antibody against AMPK and sepharose protein-A beads (Rockland Immunochemicals, Gilbertsville, PA; Hao et al., 2004). The beads were incubated for 10 min at 37°C in 40 mM HEPES, 0.2 mM SAMS peptide (His-Met-Arg-Ser-Ala-Met-Ser-Gly-Leu-His-Leu-Val-Lys-Arg-Arg; Invitrogen), 0.2 mM AMP, 80 mM NaCl, 8% (wt/vol) glycerol, 0.8 mM EDTA, 0.8 mM DTT, 5 mM MgCl2, and 0.2 mM ATP + 2 µCi [32P]ATP (Amersham Biosciences), pH 7.0, in a final volume of 50 µL. An aliquot (20 µL) was removed and spotted onto a 2 x 2–cm piece of Whatman P81 filter paper. The remaining [32P]ATP was removed with 3 washes of 1% phosphoric acid (Sigma) and once with 100 mL of acetone (Sigma) to remove the water. The filter paper was air-dried, and radioactivity was quantified after immersing the filter paper in 3 mL of ScintiV-erse (Fisher Scientific).
Measurement of AMP and ATP Content
Adenosine triphosphate and AMP content were determined by high performance liquid chromatography (HPLC, Beckman Instruments Inc., Fullerton, CA), as previously described (Shen et al., 2006). Briefly, cultured cells were collected and homogenized in 3 volumes of ice-cold 0.9 N perchloric acid. After extraction for 30 min on ice, the supernatant was obtained by centrifuging at 13,000 x g, 4°C for 10 min, and neutralized with 2 M KOH and centrifuged again under the same condition to remove KClO4. The neutralized supernatant was passed through a 0.2-µm filter (Pall Corp., East Hills, NY). Ten-microliter aliquots of the final muscle extract were injected into the chromatography column (Phenomenex C18-MC1, 250 x 4.60 mm, 5 µm). Mobile phase A was phosphate buffer (0.04 M potassium dihydrogen orthophosphate and 0.06 M dipotassium hydrogen orthophosphate, pH 7.0). Mobile phase B was acetonitrile. The UV detection was carried out at a wavelength of 254 nm. The peaks were identified and quantified by comparison for retention time and peak area with known external standards (Sigma).
Statistics
The data were analyzed as a complete randomized design using the GLM procedure (SAS Inst. Inc., Cary, NC). Three separate experiments were conducted, and each experiment was regarded as a replicate. The differences (P < 0.05) between the means were evaluated with Tukey’s multiple comparison test, and the means and SEM were reported.
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RESULTS |
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TopAbstractINTRODUCTIONRESULTSDISCUSSIONLITERATURE CITED |
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shows the time-course of mTOR phosphorylation at Ser2448 following leucine treatment. The phosphorylation of mTOR reached maximal at 30 min following leucine treatment (Figure 2).
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). No difference in actin content was detected, showing that the differences observed for the phosphorylated mTOR and S6K were not due to sample loading differences (Figure 3a). This upregulation of mTOR signaling due to leucine treatment might be related to the inhibition of AMPK activity. Leucine treatment reduced the activity of AMPK by 43.6 ± 3.5% (P < 0.05, Figure 3c). The phosphorylation of AMPK at Thr172, which is correlated with the AMPK activity, was also reduced for 28.6 ± 4.9% (P < 0.05, Figure 3b).
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). When applying AICAr, leucine treatment did not change phosphorylation or activity of AMPK (Figure 3b,c). No significant increase in the phosphorylation of mTOR and S6K due to leucine treatment was observed in cells treated with AICAr.
The ATP content in cells was significantly increased following leucine treatment. The AMP/ATP ratio was decreased from 0.22 ± 0.04 to 0.14 ± 0.02 (P < 0.05). Because AMPK is activated when the AMP/ATP ratio in cell increases, the reduction in AMPK activity is likely due to the decrease in AMP/ATP ratio after leucine treatment. To further identify whether the increase of AMP/ATP ratio was due to leucine specific effect or due to a general increase in energy sources, other substrates, including 2 mM glucose, 2 mM sodium pyruvate, and 2 mM D-leucine, were added separately into medium. Phosphorylatoin of mTOR and AMP/ATP ratio were analyzed after 30-min incubation (Figure 4). L-Leucine and D-leucine were equally effective in enhancing mTOR signaling, whereas glucose and pyruvate slightly increased mTOR signaling. Addition of L-Leucine, D-Leucine, and pyruvate decreased the AMP/ATP ratio in myoblast cells (P < 0.05, Figure 4).
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subunit (negative) were employed. Such myoblasts have a very low AMPK activity (Mu et al., 2001). In these myoblast cells, leucine stimulated the phosphorylation of mTOR by 26.1 ± 10.2% (Figure 5a,b). This mTOR phosphorylation was weaker compared with the phosphorylation of mTOR (59.2 ± 5.7%) in wild-type myoblasts following leucine treatment (Figure 5c,d). Because the major difference between negative and wild-type myoblasts was AMPK activity, these data show that AMPK is involved in the mTOR activation induced by leucine.
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) in C2C12 myoblast cells. This result is in agreement with the activation of mTOR signaling following leucine treatment and inhibition of mTOR signaling by AICAr (Figures 3 and 6). In contrast, no difference in protein synthesis was observed for negative cells due to leucine or AICAr treatment, though a similar numerical change was observed as in C2C12 cells (Figure 6). This numerical change could be due to the residual AMPK activity in negative cells.
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DISCUSSION |
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TopAbstractINTRODUCTIONRESULTSDISCUSSIONLITERATURE CITED |
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). Phosphorylation of 4E-BP1 by mTOR releases eIF4E, which instead binds to eIF4G and initiates translation. The mTOR directly phosphorylates S6K and S6K phosphorylates ribosomal protein S6 (Jefferies et al., 1997; Bodine et al., 2001). Phosphorylation of ribosomal protein S6 drives translation of a small family of abundant transcripts that encode primarily ribosomal proteins and components of the translational apparatus (Jefferies et al., 1997; Schmelzle and Hall, 2000). Thus, activation of mTOR upregulates the translational machinery and promotes protein translation (Schmelzle and Hall, 2000; Shen et al., 2005). Mammalian target of rapamycin is a large protein with a molecular weight around 290 kDa. Growth factors, such as insulin and IGF-I, activate PI3K/Akt pathway, leading to mTOR phosphorylation, cell growth, and proliferation (Anthony et al., 2001). The nature of the upstream kinases involved in the activation of mTOR remains controversial (McManus and Alessi, 2002; Inoki et al., 2003; Ma et al., 2005).
Adenosine monophosphate activated-protein kinase is mainly recognized as a critical regulator of energy metabolism, which is a heterotrimeric enzyme with , ß, and subunits (Hardie, 2005). Each subunit exists as isoforms (1, 2, ß1, ß2, 1, 2, 3; Hardie, 2004). The subunit is the catalytic unit, the subunit has a regulatory function, and the ß unit provides anchorage sites for and (Sambandam and Lopaschuk, 2003). Adenosine monophosphate activated-protein kinase acts as a monitor of cellular AMP/ATP ratio, and is switched on by an increase in the AMP/ATP ratio, an indicator of low energy level in cells (Hardie, 2004).
Numerous studies show that leucine supplementation stimulates protein synthesis in skeletal muscle (Anthony et al., 2001; Lynch et al., 2003; Lang and Frost, 2005). This stimulation of protein synthesis involves the upregulation of mTOR signaling (Lynch et al., 2003; Tokunaga et al., 2004). Amino acids, leucine in particular, independently stimulate protein synthesis through pathways separated from insulin signaling (O’Connor et al., 2003). Mechanisms of amino acid in the control of mTOR signaling are poorly understood.
Leucine and other BCAA have a distinct metabolic pathway compared with other amino acids. Other amino acids are mainly degraded in liver. However, due to the absence of BCAT in liver, BCAA are degraded primarily in skeletal muscle (Sweatt et al., 2004). Leucine is oxidized to isovaleryl-CoA and NADH in skeletal muscle, resulting in increased ATP synthesis (Lynch et al., 2003; Tokunaga et al., 2004). In spite of studies regarding to the oxidation of leucine for energy, no study was conducted to assess the contribution of AMPK inhibition to the activation of mTOR signaling induced by leucine. Here, we assessed the contribution of AMPK to the activation of mTOR signaling induced by leucine in a model system.
Adenosine monophosphate-activated protein kinase may control the activation of mTOR and protein synthesis through several possible mechanisms. First, AMPK phosphorylates TSC2 directly, thereby enhancing the activity of TSC1/TSC2 complex (Inoki et al., 2003). Activated TSC2 acts through Rheb to inhibit mTOR function (Gao et al., 2002). Second, AMPK may also inhibit the activity of mTOR directly by phosphorylation at Thr2446. The phosphorylation at Thr2446 and Ser2448 is mutually exclusive and might act as a switch to integrate energy status with protein synthesis (Cheng et al., 2004). Third, AMPK phosphorylates and inhibits the activity of eukaryotic elongation factor 2 (eEF2). The activation of AMPK by AICAr activates eEF2 kinase, which phosphorylates and inactivates eEF2 (Horman et al., 2002; Browne et al., 2004), leading to the inhibition of protein synthesis (Bolster et al., 2002; Browne et al., 2004).
Leucine treatment increased ATP content in muscle cells and reduced the AMP/ATP ratio, confirming that leucine was used to generate energy in muscle cells. To test whether this is due to a L-leucine specific effect, D-leucine, glucose, and pyruvate were used to treat cells. As did L-leucine, D-leucine also reduced AMP/ATP ratio in cells. The reason for this reduction in AMP/ATP ratio could be due to the presence of D-amino acid oxidase in skeletal muscle (Wang and Zhu, 2003). D-Leucine is converted to -ketoisocaproic acid by D-amino acid oxidase and used for oxidation (Hasegawa et al., 2002). Similar to L-leucine and D-leucine, glucose and pyruvate also reduced AMP/ATP ratio in muscle cells. Alteration in AMP/ATP ratio inhibits AMPK activity (Hardie, 2004). Indeed, the AMPK activity was reduced due to leucine treatment. To show that AMPK indeed contributes to the upregulation of mTOR stimulated by leucine, we used myoblasts expressing dominant negative AMPK subunit. In these cells, AMPK activity was very low. Without functional AMPK, the mTOR signaling induced by leucine was muted, showing that AMPK is involved in the leucine induced mTOR activation. Activation of AMPK by AICAr dramatically inhibited mTOR signaling, eliciting that AMPK is an important negative regulator of mTOR signaling. Thus, inhibition of AMPK activity due to leucine treatment contributes significantly to the activation of mTOR signaling and is a potential strategy to enhance protein synthesis and skeletal muscle growth. In a previous report in adipose tissue in rats, the major effect of leucine on mTOR signaling was demonstrated to be mainly through the direct stimulation on mTOR itself, rather than the metabolites of leucine (Lynch et al., 2003). This result is partially in agreement with our results because we showed that leucine stimulated mTOR signaling despite abolishing the function of AMPK. In addition, despite similar effects of leucine, glucose, and pyruvate in reducing AMP/ATP ratio in cells, leucine was more effective in enhancing mTOR signaling compared with glucose and pyruvate, showing that leucine stimulated mTOR signaling through an energy-independent mechanism. Furthermore, leucine supplementation provides energy to muscle cells, which inhibits AMPK and promotes mTOR signaling. Due to the unique catabolism pathway of BCAA compared with other amino acids, leucine is effectively oxidized by skeletal muscle in vivo (Sweatt et al., 2004), which provides energy and contributes to mTOR activation through AMPK inhibition.
This study shows that AMPK is an important negative regulator of mTOR signaling in muscle. Data suggested that mTOR signaling and protein synthesis in muscle can be enhanced by proper nutritional management to inhibit AMPK activity, resulting in enhanced muscle growth. The important role of AMPK in muscle growth is elegantly shown in Hampshire pigs carrying Rendement Napole (RN–) gene. A mutation in the AMPK 3 subunit was identified as the reason for the RN– genotype whereby lean growth is enhanced (Enfalt et al., 1997; Milan et al., 2000). It is quite possible that AMPK mutation in RN– pigs causes enhanced mTOR signaling which leads to enhanced muscle growth, and further investigation is needed to confirm this hypothesis.
In summary, we demonstrate that AMPK activation downregulates mTOR signaling and protein synthesis in myoblasts. Leucine stimulates mTOR signaling and protein synthesis in muscle cells in part through inhibition of AMPK. These data implicate that nutritional management to inhibit AMPK is a potential strategy to promote mTOR signaling and protein synthesis in muscle cells, enhancing lean growth in livestock.
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Footnotes |
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2 Corresponding author: mindu{at}uwyo.edu
Received for publication May 26, 2006. Accepted for publication December 11, 2006.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONRESULTSDISCUSSIONLITERATURE CITED |
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AMPK pathway. Curr. Opin. Cell Biol. 17:167–173.[CrossRef][Medline]This article has been cited by other articles:
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  T. G. Anthony and J. C. Anthony AMPing Down Leucine Action in Skeletal Muscle J. Nutr., December 1, 2008; 138(12): 2307 - 2308. [Full Text] [PDF]
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A. M. Pruznak, A. A. Kazi, R. A. Frost, T. C. Vary, and C. H. Lang Activation of AMP-Activated Protein Kinase by 5-Aminoimidazole-4-Carboxamide-1-{beta}-D-Ribonucleoside Prevents Leucine-Stimulated Protein Synthesis in Rat Skeletal Muscle J. Nutr., October 1, 2008; 138(10): 1887 - 1894. [Abstract] [Full Text] [PDF]
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