Insulin stimulates human skeletal muscle protein synthesis via an indirect mechanism involving endothelial-dependent vasodilation and mammalian target of rapamycin complex 1 signaling

Abstract

Objective: Our objective was to determine whether endothelial-dependent vasodilation is an essential mechanism by which insulin stimulates human skeletal muscle protein synthesis and anabolism.

Subjects: Subjects were healthy young adults (n=14) aged 31+/-2 yr.

Design: Subjects were studied at baseline and during local leg infusion of insulin alone (control, n=7) or insulin plus the nitric oxide synthase inhibitor NG-monomethyl-L-arginine (L-NMMA, n=7) to prevent insulin-induced vasodilation.

Methods: We measured skeletal muscle protein metabolism with stable isotope tracers, blood flow with indocyanine green, capillary recruitment with contrast enhanced ultrasound, glucose metabolism with stable isotope tracers, and phosphorylation of proteins associated with insulin (Akt) and amino acid-induced mammalian target of rapamycin (mTOR) complex 1 (mTORC1) signaling (mTOR, S6 kinase 1, and eukaryotic initiation factor 4E-binding protein 1) with Western blot analysis.

Results: No basal differences between groups were detected. During insulin infusion, blood flow and capillary recruitment increased in the control (P<0.05) group only; Akt phosphorylation and glucose uptake increased in both groups (P<0.05), with no group differences; and mTORC1 signaling increased more in control (P<0.05) than in L-NMMA. Phenylalanine net balance increased (P<0.05) in both groups, but with opposite mechanisms: increased protein synthesis (basal, 0.051+/-0.006 %/h; insulin, 0.077+/-0.008 %/h; P<0.05) with no change in proteolysis in control and decreased proteolysis (P<0.05) with no change in synthesis (basal, 0.061+/-0.004 %/h; insulin, 0.050+/-0.006 %/h; P value not significant) in L-NMMA.

Conclusions: Endothelial-dependent vasodilation and the consequent increase in nutritive flow and mTORC1 signaling, rather than Akt signaling, are fundamental mechanisms by which insulin stimulates muscle protein synthesis in humans. Additionally, these data underscore that insulin modulates skeletal muscle proteolysis according to its effects on nutritive flow.

Figure 1

Study design. Blood and muscle sampling is indicated by arrows. A detailed description of the study design is provided in the text.

Figure 2

Leg blood flow by dye dilution (ICG) (A), muscle microvascular flow by contrast enhanced ultrasound (CEU) (B), and endothelin-1 concentrations in the femoral vein (C) in two groups of healthy young subjects at baseline and during local insulin infusion in one leg with (l-NMMA) or without (control) concomitant infusion of the eNOS inhibitor l-NMMA. Data are the mean ± se. *, P < 0.05 vs. baseline. AU, Arbitrary units.

Figure 3

Phosphorylation of Akt^Ser473 (A), Akt^Thr308 (B), TSC2^Thr1462 (C), mTOR^Ser2448 (D), S6K1^Thr389 (E), and 4E-BP1^Thr37/46 (F) in the skeletal muscle of two groups of healthy young subjects at baseline (biopsy 1 at 120 min, Bx1), and during local insulin infusion (biopsy 3 at 330 min, Bx3; and biopsy 4 at 420 min, Bx4) in one leg with (l-NMMA, n = 7) or without (control, n = 7) concomitant infusion of the eNOS inhibitor l-NMMA. Blots for phosphorylated and total proteins are from a single representative control and l-NMMA subject, respectively. Data are the mean ± se. *, P < 0.05 vs. baseline; #, P < 0.05 vs. control. AU, Arbitrary units.

Figure 4

Skeletal muscle mixed protein FSR in two groups of healthy young subjects at baseline and during local insulin infusion in one leg with (l-NMMA) or without (control) concomitant infusion of the eNOS inhibitor l-NMMA. Data are the mean ± se. *, P < 0.05 vs. baseline.