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Impaired anabolic response of muscle protein synthesis is associated with S6K1 dysregulation in elderly humans

Unité du Métabolisme Protéino-Energétique, UMR Université d’Auvergne/INRA, CRNH, Centre Hospitalier Universitaire, Clermont-Ferrand, France; and
^* Unité de Nutrition et du Métabolisme Protéique, INRA-Theix, CRNH, Clermont-Ferrand, France

¹ Correspondence: Unité du Métabolisme protéino-énergétique, Laboratoire de Nutrition Humaine, BP 321, 58 rue Montalembert, 63009 Clermont-Ferrand cedex 1, France. E-mail: boirie{at}clermont.inra.fr

SPECIFIC AIMS

We hypothesized that the age-related defect of nutrients action on muscle protein synthesis may occur at some selective steps of the signaling pathway. Therefore, the combined effect of insulin and amino acid was investigated simultaneously on muscle protein synthesis and on the expression of factors implicated in protein translation initiation, in young and healthy elderly subjects, by using euglycemic hyperinsulinemic clamp techniques.

PRINCIPAL FINDINGS

1. The response of muscle protein synthesis to insulin and amino acid is impaired in elderly subjects
The fractional synthesis rate of mixed muscle protein was measured from the incorporation of L-[1-¹³C]leucine in muscle protein from vastus lateralis biopsies taken before (basal state) and during a 4 h hyperinsulinemic, euglycemic, hyperaminoacidemic clamp. In the basal state, mixed muscle protein fractional synthesis rate (Fig. 1 ) was lower in elderly subjects (0.061±0.004%.h^–1) compared with young subjects (0.082±0.010%.h^–1, P<0.05, elderly vs. young). Synthesis of muscle protein was stimulated after insulin and amino acid infusion in young (0.119±0.006%.h^–1, P<0.01, clamp vs. basal) and in elderly subjects (0.084±0.005%.h^–1, P<0.05, clamp vs. basal) but the increase was significantly lower in elderly in comparison with young subjects (+0.023±0.004%.h^–1 vs. +0.041±0.008%.h^–1, P<0.05, elderly vs. young).

View larger version (8K): [in this window] [in a new window]   Figure 1. Fractional synthesis rate (FSR) of muscle proteins in young and elderly subjects in basal (open columns) and during hyperinsulinemic, euglycemic, hyperaminoacidemic clamp (filled columns). *Significant differences between young and elderly subjects, P < 0.05. Significant differences between basal and insulin and amino acid infusion, P < 0.05.

2. This alteration is associated with a defect in S6K1 activation
Using Western blot analysis, we investigated the phosphorylation state of some factors implicated in the insulin signaling transduction pathway in the basal state and during insulin and amino acid infusion: protein kinase B (PKB), mammalian target of rapamycin protein (mTOR), eukaryotic initiation factor 4E binding protein-1 (4E-BP1), 70 kDa ribosomal protein S6 kinase (S6K1).

In the basal state, phosphorylation of factors implicated in the insulin signaling transduction pathway was not different between young and elderly subjects. Phosphorylation of S6K1 (Fig. 2 D) tended to be greater in elderly subjects than in young subjects. Hyperinsulinemia and hyperaminoacidemia increased significantly the phosphorylation of PKB (Fig. 2A ), mTOR (Fig. 2B ), and 4E-BP1 (Fig. 2C ) in both young and elderly subjects (P<0.05, clamp vs. basal). The infusion of insulin and amino acids did not stimulated S6-kinase phosphorylation in elderly subjects, in contrast to young subjects (P<0.05, clamp vs. basal).

View larger version (25K): [in this window] [in a new window]   Figure 2. Quantification of phosphorylation and Western blot (above histograms) of protein kinase B (A), mTOR (B), 4E-BP1 (C), and S6K1 (D) in the basal state (open columns) and during insulin and amino acid infusion (filled columns) in young and elderly subjects. Significant differences between basal and insulin and amino acid infusion, P < 0.05.

CONCLUSIONS
The present study demonstrates for the first time in elderly humans the association of a reduced response of muscle protein synthesis to anabolic factors, together with a defect in translation factors activation. Insulin and amino acid, the most potent anabolic factors, are known to increase synergistically muscle protein synthesis in human adults. Nevertheless, amino acid availability seemed to be the most important factor in increasing muscle protein synthesis in elderly humans. Indeed, infusion or ingestion of high amounts of amino acid can stimulate muscle protein synthesis in elderly subjects to a similar extent as in young subjects. By contrast, muscle protein anabolism in response to the intake of an amino acid/glucose mixture, increasing plasma insulin, and amino acid concentrations has been reported to be blunted in healthy elderly subjects, suggesting that the action of insulin and amino acid on muscle protein synthesis may be reduced in this population. This observation is in agreement with previous animal studies showing that, in old rats, mixed muscle protein synthesis is less responsive to meal intake. Moreover, a resistance to insulin action of muscle protein synthesis had been reported in adult and in old rats compared with young animals. Taken together, these observations and our results suggested that, with age, the effect of insulin on protein metabolism was impaired and this alteration may prevent the stimulating effect of amino acid on muscle protein synthesis.

Insulin and amino acid regulate protein synthesis through activation of factors implicated in the initiation of translation. In the present study, the phosphorylation of S6K1 was not increased after insulin and amino acid infusion in elderly subjects, contrary to young subjects. However, the phosphorylation of PKB, mTOR, and 4E-BP1 was normally activated by insulin and amino acid. To our knowledge, possible alterations in insulin transduction signal during aging have not been investigated. In old rat muscle, phosphorylation of S6K1 appeared not to be altered in the basal state, but it was less sensitive to the stimulating action of a specific amino acid such as leucine compared with young and adult rats. This modification was also related to a lower response of muscle protein synthesis to leucine in old rats. Our results in humans agreed with these data, since we observed reduced stimulation of both muscle protein synthesis and phosphorylation of S6K1 by insulin and amino acid in elderly subjects, whereas the other translation factors normally were stimulated.

In conclusion, the response of muscle protein synthesis to insulin and amino acid was impaired in elderly subjects. The most important finding of this study was that the lower stimulation of protein synthesis is associated with a defect in S6K1 activation. This specific defect provides a mechanistic basis for an understanding of the loss in muscle protein mass occurring during aging. Future studies now should focus on the regulation of all the factors implicated in the insulin signaling transduction pathway in order to find a therapeutic strategy against the alterations of muscle protein synthesis with age.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1341fje;

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