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. 2013 Jul 19;288(29):21074-21081.
doi: 10.1074/jbc.M113.456228. Epub 2013 Jun 6.

Differential contribution of insulin and amino acids to the mTORC1-autophagy pathway in the liver and muscle

Takako Naito  1 , Akiko Kuma  2 , Noboru Mizushima  3
Affiliations

Differential contribution of insulin and amino acids to the mTORC1-autophagy pathway in the liver and muscle

Takako Naito et al. J Biol Chem. .

Abstract

Autophagy is a highly inducible intracellular degradation process. It is generally induced by nutrient starvation and suppressed by food intake. Mammalian (or mechanistic) target of rapamycin complex 1 (mTORC1) is considered to be the major regulator of autophagy, but the precise mechanism of in vivo regulation remains to be fully characterized. Here, we examined the autophagy-suppressive effect of glucose, insulin, and amino acids in the liver and muscle in mice starved for 1 day. Refeeding after starvation with a standard mouse chow rapidly suppressed autophagy in both tissues, and this suppression was inhibited by rapamycin administration almost completely in the liver and partially in muscle, confirming that mTORC1 is indeed a crucial regulator in vivo. As glucose administration showed no major suppressive effect on autophagy, we examined the role of insulin and amino acids using hyperinsulinemic-euglycemic clamp and intravenous amino acid infusion techniques. Insulin administration showed a clear effect on the mTORC1-autophagy pathway in muscle, but had only a very weak effect in the liver. By contrast, amino acids were able to regulate the mTORC1-autophagy pathway in the liver, but less effectively in muscle. These results suggest that autophagy is differentially regulated by insulin and amino acids in a tissue-dependent manner.

Keywords: Amino Acid; Autophagy; Insulin; Protein Degradation; mTOR Complex (mTORC).

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Figures

FIGURE 1.
FIGURE 1.
Autophagy is suppressed by refeeding but not by glucose administration in muscle and liver. A, mice were starved for 24 h, and then fed for 1 h (refed) or injected with glucose or saline (starved). Glucose was injected (2.5 g/kg intraperitoneal) at 1 h before sampling of blood and tissues. Plasma insulin and blood glucose concentrations were measured after feeding and glucose administration. Blood samples were collected from the tail vein. B, gastrocnemius muscle and liver samples were obtained from the 3 groups of mice described in A and subjected to immunoblot analysis of insulin-mTORC1 signaling and LC3 conversion. α-tubulin was used as a loading control. C, immunoblot analysis showing the effect of feeding and glucose administration on phosphorylation levels of AMPKα and its substrate acetyl-CoA carboxylase (ACC). Data from two independent mice are shown for each experimental group (A–C).
FIGURE 2.
FIGURE 2.
mTORC1 contributes to autophagy suppression after feeding. A, mice were starved for 24 h (starved) and then refed for 1 h (refed). Rapamycin (0.4 mg/kg), a specific inhibitor of mTORC1, or saline with 5% Tween 20 and 10% DMSO (vehicle) was intraperitoneally injected at 2 h and 16 h before tissue sampling. Muscle and liver samples were subjected to immunoblot analysis using the indicated antibodies. B, quantitative analysis of the immunoblot data in skeletal muscle and liver samples using densitometry scanning. The LC3-II/total LC3 (LC3-I+II) ratio were calculated. The anti-LC3 antibody used in this study reacts to both LC3-I and LC3-II with similar affinities (supplemental Fig. S2). Data are mean ± S.E. of 4 mice (two are shown in A and the other two are not shown). *, p < 0.05; **, p < 0.01; n.s., not significant.
FIGURE 3.
FIGURE 3.
The effect of insulin on the mTORC1-autophagy pathway is greater in muscle than in liver. A, mice were starved for 24 h, and then fed for 1 h (refed) or insulin (10 or 40 mU/kg/min) was administered for 1 h using the euglycemic glucose clamp technique. Plasma insulin and blood glucose concentrations were measured during feeding and insulin administration. Data are mean ± S.E. of 4 mice for the starved and refed groups, and of 5 mice for the insulin-treated group. B, representative immunoblot analysis showing the effect of feeding and insulin administration (10 and 40 mU/kg/min) on phosphorylation levels of AKT, PRAS40, S6K1, S6, and 4E-BP1 and protein levels of LC3. C and D, quantitative analysis of the immunoblot data (starved, refed, and 10 mU/kg/min insulin) of skeletal muscle (C) and liver (D) samples using densitometry scanning. The phosphorylation levels of AKT Ser-473 and S6 Ser-235/236 were normalized relative to total protein content. Data are mean ± S.E. of 4 mice for the starved and refed groups, and of 5 mice for the insulin-treated group (two are shown in B and the others are not shown).*, p < 0.05; **, p < 0.01; n.s., not significant.
FIGURE 4.
FIGURE 4.
Amino acids induce mTORC1 activation greater in the liver than in muscle. A, mice were starved for 24 h, and then refed for 1 h or an amino acid mixture (18.2 mg/kg/min with somatostatin) was administered for 1 h. Free amino acid concentrations of a total of 20 amino acids in the liver and muscle were measured and expressed as mmol per mg wet weight. B, plasma insulin and blood glucose concentrations were measured during feeding and amino acid administration in the mice described in A. C, immunoblot analysis showing the effect of amino acid administration on phosphorylation levels of AKT, PRAS40, S6K1, S6, and 4E-BP1 and protein levels of LC3 in the gastrocnemius muscle and liver in the mice described in A. D and E, quantitative analysis of the immunoblot data of skeletal muscles (D) and liver (E) obtained from starved, refed, or amino acid-treated (18.2 mg/kg/min) mice. The phosphorylation levels of S6 Ser-235/236 were normalized relative to total protein contents. Data are mean ± S.E. of 3 mice for the starved group, and of 4 mice for the refed and amino acid-treated groups. *, p < 0.05; **, p < 0.01; n.s., not significant.
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