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. 2008 Sep;295(3):E595-604.
doi: 10.1152/ajpendo.90411.2008. Epub 2008 Jun 24.

Disassociation between the effects of amino acids and insulin on signaling, ubiquitin ligases, and protein turnover in human muscle

P L Greenhaff  1 , L G Karagounis, N Peirce, E J Simpson, M Hazell, R Layfield, H Wackerhage, K Smith, P Atherton, A Selby, M J Rennie
Affiliations

Disassociation between the effects of amino acids and insulin on signaling, ubiquitin ligases, and protein turnover in human muscle

P L Greenhaff et al. Am J Physiol Endocrinol Metab. 2008 Sep.

Abstract

We determined the effects of intravenous infusion of amino acids (AA) at serum insulin of 5, 30, 72, and 167 mU/l on anabolic signaling, expression of ubiquitin-proteasome components, and protein turnover in muscles of healthy young men. Tripling AA availability at 5 mU/l insulin doubled incorporation of [1-(13)C]leucine [i.e., muscle protein synthesis (MPS), P < 0.01] without affecting the rate of leg protein breakdown (LPB; appearance of d(5)-phenylalanine). While keeping AA availability constant, increasing insulin to 30 mU/l halved LPB (P < 0.05) without further inhibition at higher doses, whereas rates of MPS were identical to that at 5 mU/l insulin. The phosphorylation of PKB Ser(473) and p70(S6k) Thr(389) increased concomitantly with insulin, but whereas raising insulin to 30 mU/l increased the phosphorylation of mTOR Ser(2448), 4E-BP1 Thr(37/46), or GSK3beta Ser(9) and decreased that of eEF2 Thr(56), higher insulin doses to 72 and 167 mU/l did not augment these latter responses. MAFbx and proteasome C2 subunit proteins declined as insulin increased, with MuRF-1 expression largely unchanged. Thus increasing AA and insulin availability causes changes in anabolic signaling and amounts of enzymes of the ubiquitin-proteasome pathway, which cannot be easily reconciled with observed effects on MPS or LPB.

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Figures

Fig. 1.
Fig. 1.
Study protocol.
Fig. 2.
Fig. 2.
Arterialized venous serum insulin concentrations before and during 3 h of continuous insulin infusion aimed at achieving steady-state insulin concentrations of ∼5, 30, 70, and 170 mU/l. Values are means ± SE (some error bars lie within the symbols).
Fig. 3.
Fig. 3.
Blood phenylalanine concentrations (means ± SE) in postabsorptive state and 3 h after infusion of mixed amino acids (AA; Glamin) at 18 g/h. Different letters denote significance of difference, at least P < 0.01.
Fig. 4.
Fig. 4.
Leg blood flow (means ± SE) in the postabsorptive (post-abs) state and after insulin infusion to serum concentrations shown. The values during insulin infusion are collectively different from the post-abs value, i.e., no added AA (ANOVA, P < 0.007), and the values at 5 mU/l are significantly different from the post-abs value ( P < 0.01).
Fig. 5.
Fig. 5.
A: PKB Ser473, p70 S6 kinase (p70S6k) Thr389, and eukaryotic elongation factor-2 (eEF2) Thr56 phosphorylation after a 3-h insulin clamp at 5, 30, 72, and 167 mU/l. Significantly different from 5 mU/l: *P < 0.05; ***P < 0.001. Significantly different from 30 mU/l−1: ††P < 0.01; †††P < 0.001. Significantly different from 72 mU/l: ++P < 0.01. Values are means ± SE (arbitrary units) corrected for actin. Representative Western blots are shown above the bars. B: mammalian target of rapamycin (mTOR) Ser2448, GSK-3β Ser9, and 4E-binding protein-1 (4E-BP1) Thr37/46 phosphorylation after a 3-h insulin clamp at 5, 30, 72, and 167 mU/l. Significantly different from 5 mU/l: *P < 0.05; **P < 0.01; ***P < 0.001. Values are means ± SE (arbitrary units) corrected for actin. Representative Western blots are shown above the bars.
Fig. 6.
Fig. 6.
Proteasome C2 subunit, muscle atrophy F-box (MAFbx), and muscle-specific RING finger-1 (MuRF-1) total protein concentrations after a 3-h insulin clamp at 5, 30, 72, and 167 mU/l. Significantly different from 5 mU/l: *P < 0.05; **P < 0.01; ***P < 0.001. Significantly different from 30 mU/l: ††P < 0.01. Significantly different from 72 mU/l: +P < 0.05; ++P < 0.01. Values are means ± SE (arbitrary units) corrected for actin. Representative Western blots are shown above the bars.
Fig. 7.
Fig. 7.
Leg protein turnover as leg phenylalanine kinetics. LPB, leg protein breakdown as phenylalanine appearance; LPS, leg protein synthesis as phenylalanine disappearance; Bal, leg phenylalanine balance. Significantly different from postabsorptive: *P < 0.05; **P < 0.01. †P < 0.05, significantly different from value at 5 mU/l. The values of LPS, LPB, and Bal were not significantly different from each other at 30, 72, and 167 mU/ml of insulin. Values are means ± SE.
Fig. 8.
Fig. 8.
Muscle protein synthesis as incorporation of [1-13C]leucine. Values are not different from one another but about twice the expected value in post-abs human muscle, i.e., 0.044%/h (see text). FSR, fractional synthesis rate.
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