Amino Acid- and Insulin-Induced Activation of mTORC1 in Neonatal Piglet Skeletal Muscle Involves Sestin2-GATOR2, Rag A/C-mTOR, and RHEB-mTOR Complex Formation

Abstract

Background: Feeding stimulates protein synthesis in skeletal muscle of neonates and this response is regulated through activation of mechanistic target of rapamycin complex 1 (mTORC1). The identity of signaling components that regulate mTORC1 activation in neonatal muscle has not been fully elucidated.

Objective: We investigated the independent effects of the rise in amino acids (AAs) and insulin after a meal on the abundance and activation of potential regulators of mTORC1 in muscle and whether the responses are modified by development.

Methods: Overnight-fasted 6- and 26-d-old pigs were infused for 2 h with saline (control group) or with a balanced AA mixture (AA group) or insulin (INS group) to achieve fed levels while insulin or AAs, respectively, and glucose were maintained at fasting levels. Muscles were analyzed for potential mTORC1 regulatory mechanisms and results were analyzed by 2-factor ANOVA followed by Tukey's post hoc test.

Results: The abundances of DEP domain-containing mTOR-interacting protein (DEPTOR), growth factor receptor bound protein 10 (GRB10), and regulated in development and DNA damage response 2 (REDD2) were lower (65%, 73%, and 53%, respectively; P < 0.05) and late endosomal/lysosomal adaptor, MAPK and mTOR activator 1/2 (LAMTOR1/2), vacuolar H+-ATPase (V-ATPase), and Sestrin2 were higher (94%, 141%, 145%, and 127%, respectively; P < 0.05) in 6- than in 26-d-old pigs. Both AA and INS groups increased phosphorylation of GRB10 (P < 0.05) compared with control in 26- but not in 6-d-old pigs. Formation of Ras-related GTP-binding protein A (RagA)-mTOR, RagC-mTOR, and Ras homolog enriched in brain (RHEB)-mTOR complexes was increased (P < 0.05) and Sestrin2-GTPase activating protein activity towards Rags 2 (GATOR2) complex was decreased (P < 0.05) by both AA and INS groups and these responses were greater (P < 0.05) in 6- than in 26-d-old pigs.

Conclusion: The results suggest that formation of RagA-mTOR, RagC-mTOR, RHEB-mTOR, and Sestrin2-GATOR2 complexes may be involved in the AA- and INS-induced activation of mTORC1 in skeletal muscle of neonates after a meal and that enhanced activation of the mTORC1 signaling pathway in neonatal muscle is in part due to regulation by DEPTOR, GRB10, REDD2, LAMTOR1/2, V-ATPase, and Sestrin2.

FIGURE 1

Current concepts of the regulation of mTORC1 activation by AAs and insulin. AA, amino acids; ACC, acetyl CoA carboxylase; AMPK, AMP-activated protein kinase; DEPTOR, DEP domain-containing mechanistic target of rapamycin-interacting protein; eIF4E, eukaryotic translation initiation factor 4E; eIF4G, eukaryotic translation initiation factor 4G; GATOR1/2, GAP activity toward Rags 1/2; GβL, G protein beta protein subunit-like; GRB10, growth factor receptor bound protein 10; IRS-1, insulin receptor substrate 1; LAMTOR1/2, late endosomal/lysosomal adaptor, MAPK and mechanistic target of rapamycin activator 1/2; LRS, leucyl-tRNA synthetase; mTOR, mechanistic target of rapamycin; mTORC1, mechanistic target of rapamycin complex 1; PI3-K, phosphatidylinositol-3-kinase; PKB, protein kinase B; Rag A/B C/D, RAS-related GTP-binding protein A/B C/D; RAPTOR, regulatory associated protein of mechanistic target of rapamycin complex 1; REDD1/2, regulated in development and DNA damage response 1/2; RHEB, Ras homolog enriched in brain; rpS6, ribosomal protein S6; Sestrin1/2, stress response protein 1/2; SLC38A9, Solute Carrier Family 38 Member 9; S6K1, p70 ribosomal protein S6 kinase 1; V-ATPase, vacuolar H⁺-ATPase; 4EBP1, eukaryotic translation initiation factor 4E binding protein 1.

FIGURE 2

The abundance of IRS-1 (A), phosphorylation of IRS-1 at Ser636/639 (B), abundance of GRB10 (C), phosphorylation of GRB10 at Ser501/503 (D), and abundance of DEPTOR (E), REDD1 (F), and REDD2 (G) in longissimus dorsi muscle of 6- and 26-d-old pigs after 2-h infusion of saline (control), amino acids, or insulin. The values for protein abundance were normalized by β-actin abundance. The phosphorylation values were corrected by their abundance in the samples. White lines between bands indicate where images from the same blots were spliced to adjust sample order on the membrane for presentation. Values are least square means ± SEM; n = 4–6. Means with uncommon letters are significantly different: a > b. P < 0.05 was considered significant. AA, amino acid group; AU, arbitrary unit; DEPTOR, DEP domain-containing mechanistic target of rapamycin-interacting protein; GRB10, growth factor receptor bound protein 10; INS, insulin group; IRS-1, insulin receptor substrate 1; p, phosphorylated; REDD, regulated in development and DNA damage response; Trt, treatment.

FIGURE 3

The abundance of ACC (A), phosphorylation of ACC at Ser79 (B), and the abundance of V-ATPase (C), SLC38A9 (D), LAMTOR1 (E), and LAMTOR2 (F) in longissimus dorsi muscle of 6- and 26-d-old pigs after 2-h infusion of saline (control), amino acids, or insulin. The values for protein abundance were normalized by β-actin abundance. The phosphorylation values of ACC were corrected by the ACC abundance in the samples. White lines between bands indicate where images from the same blots were spliced to adjust sample order on the membrane for presentation. Values are least square means ± SEM; n = 4–6. Means with uncommon letters are significantly different: a > b. P < 0.05 was considered significant. AA, amino acid group; ACC, acetyl CoA carboxylase; AU, arbitrary unit; INS, insulin group; LAMTOR, late endosomal/lysosomal adaptor, MAPK and mechanistic target of rapamycin actvator; p, phosphorylated; SLC38A9, Solute Carrier Family 38 Member 9; Trt, treatment; V-ATPase, vacuolar H⁺-ATPase.

FIGURE 4

The abundance of Sestrin1 (A), Sestrin2 (B), GATOR1 (C), GATOR2 (D), RagA-mTOR complex (E), and RagC-mTOR complex (F) in longissimus dorsi muscle of 6- and 26-d-old pigs after 2-h infusion of saline (control), amino acids, or insulin. The values were normalized by β-actin abundance. White lines between bands indicate where images from the same blots were spliced to adjust sample order on the membrane for presentation. Values are least square means ± SEM; n = 4–6. Means with uncommon letters are significantly different: a > b > c. P < 0.05 was considered significant. AA, amino acid group; AU, arbitrary unit; GATOR, GAP activity toward Rags; INS, insulin group; mTOR, mechanistic target of rapamycin; Rag, RAS-related GTP-binding protein; Sestrin, stress response protein; Trt, treatment.

FIGURE 5

The abundance of RHEB-mTOR complex (A), GATOR2-Sestrin2 complex (B), Folliculin (C), and LRS (D) in longissimus dorsi muscle of 6- and 26-d-old pigs after 2-h infusion of saline (control), amino acids, or insulin. The values were normalized by β-actin abundance. White lines between bands indicate where images from the same blots were spliced to adjust sample order on the membrane for presentation. Values are least square means ± SEM; n = 4–6. Means with uncommon letters are significantly different: a > b > c. P < 0.05 was considered significant. AA, amino acid group; AU, arbitrary unit; GATOR2, GAP activity toward Rags 2; INS, insulin group; LRS, leucyl-tRNA synthetase; mTOR, mechanistic target of rapamycin; RHEB, Ras homolog enriched in brain; Sestrin2, stress response protein 2; Trt, treatment.