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Lysine suppresses myofibrillar protein degradation by regulating the autophagic-lysosomal system through phosphorylation of Akt in C2C12 cells.

Sato T, Ito Y, Nagasawa T - Springerplus (2014)

Bottom Line: Lys suppressed the phosphorylation of AMPK, but this effect was also abolished by Akti.These results indicate that regulation of AMPK activity is not essential for the regulation of autophagy by Lys.Taken together, our results show that Lys suppresses myofibrillar protein degradation by the autophagic-lysosomal system through the phosphorylation of Akt in C2C12 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioresources Science, The United Graduate School of Agricultural Sciences, Iwate University, Morioka, Iwate 020-8550 Japan.

ABSTRACT
The prevention of muscle wasting is important for maintaining quality of life, since loss of muscle mass can lead to a bedridden state and decreased resistance to diseases. The prevention of muscle wasting requires an increase in protein synthesis and a decrease in protein degradation in skeletal muscle. We previously showed that lysine (Lys) markedly suppressed myofibrillar protein degradation by inhibiting the autophagic-lysosomal system via the mammalian target of rapamycin (mTOR) and other signal molecules in C2C12 cells. In this study, we investigated the involvement of Akt and adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK), two regulators of autophagy, on the suppressive effects of Lys on myofibrillar protein degradation in C2C12 cells. Lys induced the phosphorylation of Akt, but the suppressive effects of Lys on myofibrillar protein degradation and autophagy were completely abolished in the presence of Akt1/2 kinase inhibitor (Akti). Lys suppressed the phosphorylation of AMPK, but this effect was also abolished by Akti. On the other hand, AMPK activation by 5-aminoimidazole-4-carboxamide-1-β-D-ribonucleoside (AICAR) did not affect either Akt activity or the autophagic-lysosomal system in C2C12 cells treated with Lys. These results indicate that regulation of AMPK activity is not essential for the regulation of autophagy by Lys. Taken together, our results show that Lys suppresses myofibrillar protein degradation by the autophagic-lysosomal system through the phosphorylation of Akt in C2C12 cells.

No MeSH data available.


Related in: MedlinePlus

Lys regulates the mTOR and AMPK pathways by activating Akt in C2C12 myotubes. Cells were treated for 30 min with DMSO (C), 10 μM Akti (Ai), 10 mM Leu (L), 10 mM Leu and 10 μM Akti (AiL), 10 mM Lys (K), or 10 mM Lys and 10 μM Akti (AiK). The phosphorylation level of 4E-BP1 (a) and AMPK (b) in the lysates was analyzed by Western blotting. The level of 4E-BP1 phosphorylation was estimated from the ratio of the γ-form to that of total 4E-BP1. Results are expressed as the level relative to the level in the control group. Representative immunoblots are shown. Values are means with SE (n = 3–4). Different letters indicate significant differences among the groups (p < 0.05).
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Fig4: Lys regulates the mTOR and AMPK pathways by activating Akt in C2C12 myotubes. Cells were treated for 30 min with DMSO (C), 10 μM Akti (Ai), 10 mM Leu (L), 10 mM Leu and 10 μM Akti (AiL), 10 mM Lys (K), or 10 mM Lys and 10 μM Akti (AiK). The phosphorylation level of 4E-BP1 (a) and AMPK (b) in the lysates was analyzed by Western blotting. The level of 4E-BP1 phosphorylation was estimated from the ratio of the γ-form to that of total 4E-BP1. Results are expressed as the level relative to the level in the control group. Representative immunoblots are shown. Values are means with SE (n = 3–4). Different letters indicate significant differences among the groups (p < 0.05).

Mentions: Next, we investigated the effect of Akt activation by Leu and Lys on the mTOR and AMPK pathways. One downstream target of mTOR, eIF4E-binding protein 1 (4E-BP1), was significantly phosphorylated by treatment with Leu (L) or Lys (K), and the up-regulation of 4E-BP1 phosphorylation was abolished by Akti (AiK) (Figure 4a). The level of AMPK phosphorylation was suppressed by 40% when the medium was supplemented with Leu or Lys, and the suppressive effect of Lys on AMPK phosphorylation was abolished by Akti (Figure 4b). These results indicated that Lys regulates the mTOR and AMPK pathways through Akt activation.Figure 4


Lysine suppresses myofibrillar protein degradation by regulating the autophagic-lysosomal system through phosphorylation of Akt in C2C12 cells.

Sato T, Ito Y, Nagasawa T - Springerplus (2014)

Lys regulates the mTOR and AMPK pathways by activating Akt in C2C12 myotubes. Cells were treated for 30 min with DMSO (C), 10 μM Akti (Ai), 10 mM Leu (L), 10 mM Leu and 10 μM Akti (AiL), 10 mM Lys (K), or 10 mM Lys and 10 μM Akti (AiK). The phosphorylation level of 4E-BP1 (a) and AMPK (b) in the lysates was analyzed by Western blotting. The level of 4E-BP1 phosphorylation was estimated from the ratio of the γ-form to that of total 4E-BP1. Results are expressed as the level relative to the level in the control group. Representative immunoblots are shown. Values are means with SE (n = 3–4). Different letters indicate significant differences among the groups (p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4197202&req=5

Fig4: Lys regulates the mTOR and AMPK pathways by activating Akt in C2C12 myotubes. Cells were treated for 30 min with DMSO (C), 10 μM Akti (Ai), 10 mM Leu (L), 10 mM Leu and 10 μM Akti (AiL), 10 mM Lys (K), or 10 mM Lys and 10 μM Akti (AiK). The phosphorylation level of 4E-BP1 (a) and AMPK (b) in the lysates was analyzed by Western blotting. The level of 4E-BP1 phosphorylation was estimated from the ratio of the γ-form to that of total 4E-BP1. Results are expressed as the level relative to the level in the control group. Representative immunoblots are shown. Values are means with SE (n = 3–4). Different letters indicate significant differences among the groups (p < 0.05).
Mentions: Next, we investigated the effect of Akt activation by Leu and Lys on the mTOR and AMPK pathways. One downstream target of mTOR, eIF4E-binding protein 1 (4E-BP1), was significantly phosphorylated by treatment with Leu (L) or Lys (K), and the up-regulation of 4E-BP1 phosphorylation was abolished by Akti (AiK) (Figure 4a). The level of AMPK phosphorylation was suppressed by 40% when the medium was supplemented with Leu or Lys, and the suppressive effect of Lys on AMPK phosphorylation was abolished by Akti (Figure 4b). These results indicated that Lys regulates the mTOR and AMPK pathways through Akt activation.Figure 4

Bottom Line: Lys suppressed the phosphorylation of AMPK, but this effect was also abolished by Akti.These results indicate that regulation of AMPK activity is not essential for the regulation of autophagy by Lys.Taken together, our results show that Lys suppresses myofibrillar protein degradation by the autophagic-lysosomal system through the phosphorylation of Akt in C2C12 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioresources Science, The United Graduate School of Agricultural Sciences, Iwate University, Morioka, Iwate 020-8550 Japan.

ABSTRACT
The prevention of muscle wasting is important for maintaining quality of life, since loss of muscle mass can lead to a bedridden state and decreased resistance to diseases. The prevention of muscle wasting requires an increase in protein synthesis and a decrease in protein degradation in skeletal muscle. We previously showed that lysine (Lys) markedly suppressed myofibrillar protein degradation by inhibiting the autophagic-lysosomal system via the mammalian target of rapamycin (mTOR) and other signal molecules in C2C12 cells. In this study, we investigated the involvement of Akt and adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK), two regulators of autophagy, on the suppressive effects of Lys on myofibrillar protein degradation in C2C12 cells. Lys induced the phosphorylation of Akt, but the suppressive effects of Lys on myofibrillar protein degradation and autophagy were completely abolished in the presence of Akt1/2 kinase inhibitor (Akti). Lys suppressed the phosphorylation of AMPK, but this effect was also abolished by Akti. On the other hand, AMPK activation by 5-aminoimidazole-4-carboxamide-1-β-D-ribonucleoside (AICAR) did not affect either Akt activity or the autophagic-lysosomal system in C2C12 cells treated with Lys. These results indicate that regulation of AMPK activity is not essential for the regulation of autophagy by Lys. Taken together, our results show that Lys suppresses myofibrillar protein degradation by the autophagic-lysosomal system through the phosphorylation of Akt in C2C12 cells.

No MeSH data available.


Related in: MedlinePlus