Amino acids are necessary for the insulin-induced activation of mTOR/S6K1 signaling and protein synthesis in healthy and insulin resistant human skeletal muscle

Abstract

Background: Amino acids (AA) activate the mammalian target of rapamycin (mTOR) signaling pathway but overactivation has a negative feedback effect on insulin signaling which may lead to insulin resistance and type 2 diabetes (T2DM).

Purpose: To determine the effect of reduced AA concentrations on mTOR and insulin signaling during increased nutrient and insulin availability.

Methods: Six control and six T2DM subjects were studied at baseline and following a 5h AA lowering high energy and insulin clamp. Stable isotopic techniques in combination with femoral catheterizations were used to measure AA kinetics across the leg while muscle biopsies were used to measure mTOR and insulin signaling proteins using immunoblotting techniques.

Results: AA concentrations decreased by approximately 30-60% in both groups (p<0.05). Phospho-mTOR, S6K1, eEF2, and eIF2alpha were unchanged in both groups following the clamp (p>0.05). In T2DM subjects, IRS-1 serine phosphorylation was unchanged while phospho-AMPKalpha decreased and phospho-Akt, phospho-AS160 and glucose uptake increased following the clamp (p<0.05). In comparison, AA concentrations were maintained in a separate group during an insulin infusion. In this group, phospho-Akt, mTOR and S6K1 (n=4) increased.

Conclusion: Amino acids are necessary for insulin-induced activation of mTOR signaling and protein synthesis in both healthy and insulin resistant skeletal muscle.

Figures

Figure 1

Histograms represent arterial AA concentrations on left Y-axis and AA delivered to leg on right Y-axis (n=6) for A) Leucine (n=6), B) Isoleucine (n=6), and C) Valine (n=6) at Basal and following Reduced AA clamp in Control and T2DM subjects. All values are expressed as mean ± SE. *Significantly different than Basal (P#Significantly different from Control (P<0.05).

Figure 2

Phosphorylation status of A) mTOR at Ser2448 (n=6) and B) S6K1 at Thr389 (n=5) at Basal and following a Reduced AA clamp in Control and T2DM subjects. Phosphorylation values are relative to total protein. All values are expressed as mean ± SE.

Figure 3

Phosphorylation status of A) eEF2 at Thr56 (n=5) and B) eIF2α at Ser52 (n=6) at Basal and following a Reduced AA clamp in Control and T2DM subjects. Representative immunoblot images are shown above each figure. Phosphorylation values are relative to total protein in Fig. 3A and relative to standard in Fig. 3B. All values are expressed as mean ± SE. #Significantly different from Control (P<0.05).

Figure 4

Phosphorylation status of IRS-1 at Basal and following a Reduced AA clamp in Control and T2DM subjects. A) IRS-1 at Ser312 (n=5) and B) Ser636/639 (n=6). Representative immunoblot images are shown above each figure. Phosphorylation values are relative to total protein. All values are expressed as mean ± SE.

Figure 5

Phosphorylation status of A) Akt at Ser473 (n=5) and B) AS160 (n=6) at Basal and following a Reduced AA clamp in Control and T2DM subjects. Representative immunoblot images are shown above each figure. Phosphorylation values are relative to total protein. All values are expressed as mean ± SE. *Significantly different than Basal (P

Figure 6

Histograms represent the phosphorylation status of A) AMPKα (n=5) and B) ACCβ at Ser79 (n=5) at Basal and following a Reduced AA clamp in Control and T2DM subjects. Representative immunoblot images are shown above each figure. Phosphorylation values are relative to total protein in Fig. 6A and relative to standard in Fig. 6B. All values are expressed as mean ± SE. *Significantly different than Basal (P

Figure 7

Histograms represent the phosphorylation status of A) Akt at Ser473 (n=4), B) mTOR at Ser2448 (n=4), and C) S6K1 at Thr389 at Basal and following treatment with Maintained AA in young, healthy subjects. Representative immunoblot images are shown above each figure. Phosphorylation values are relative to total protein. All values are expressed as mean ± SE. *Significantly different than Basal (P