Using an in vitro assay we assessed whether the acute exposure of soleus muscle of adult and aged rats to essential amino acid enriched mixture (EAAem) activates mTOR signaling pathway (mTOR and p70S6K) even after prolonged supplementation with the same mixture. A total of 20 adult (9 months of age at the end of treatment) and 20 aged (18 months of age at the end of treatment) male Wistar rats were used. Ten of each group were treated with EAAem (1.5 gr/kg/day in tap water) for 6 months. At the end of treatment the rats were grouped (n = 5 each group) as follows: adult (AD) and aged (AG) untreated controls; adult (AD_EAAem) and aged (AG_EAAem) chronically supplemented with EAAem; adult (AD+EAAem) and aged (AG+EAAem) acutely incubated with EAAem (soleus in 1 percent EAAem for 30 min); AD_EAAem+ and AG_EAAem+ acutely incubated with EAAem. Following treatment the activation level of mTOR and p70S6K was measured by Western blot. The basal level of mTOR and p70S6K activation appeared to be higher in AD compared with AG. In AG+EAAem a significant change in the level of p70S6K activation, unlike mTOR, was observed whereas no change was observed in AD+EAAem. In AD_EAAem muscles the basal level of p70S6K activation, unlike mTOR, was significantly lower than in AD and the acute exposure to EAAem produced a significant reduction of mTOR activation. Contrarily to AG, in AG_EAAem+ the acute exposure to EAAem produced a significant activation of mTOR, unlike p70S6K. Results in the adults indicated a higher basal level of activation and a lower responsiveness of the pathway to acute and chronic exposure to EAA-enriched mixture. On the contrary, in the aged, a lower basal level of activation was associated with a higher responsiveness to EAAem. In particular, although with a different timing, acute exposure to EAAem activated mTOR signaling even following prolonged supplementation.
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