The abundance and activation of mTORC1 regulators in skeletal muscle of neonatal pigs are modulated by insulin, amino acids, and age

Abstract

Mammalian target of rapamycin complex 1 (mTORC1) signaling is crucial for the regulation of protein synthesis. Most of known mTORC1 regulators have been isolated and characterized using cell culture systems, and the physiological roles of these regulators have not been fully tested in vivo. Previously we demonstrated that the insulin (INS) and amino acid (AA)-induced activation of mTORC1 is developmentally regulated in skeletal muscle (Suryawan A et al. Am J Physiol Endocrinol Metab 293: E1597-E1605, 2007). The present study aimed to characterize in more detail the effects of the postprandial rise in INS and AA on the activation and abundance of mTORC1 regulators in muscle and how this is modified by development. Overnight fasted 6- and 26-day-old pigs were studied during 1) euinsulinemic-euglycemic-euaminoacidemic conditions (control), 2) euinsulinemic-euglycemic-hyperaminoacidemic clamps (AA), and 3) hyperinsulinemic-euglycemic-euaminoacidemic clamps (INS). INS, but not AA, enhanced the PRAS40 phosphorylation, and this effect was greater in 6- than in 26-day old pigs. Phospholipase D1 (PLD1) abundance and phosphorylation, and the association of PLD1 with Ras homolog enriched in brain (Rheb), were greater in the younger pigs. Neither INS, AA, nor age altered the abundance of Rheb, vacuolar protein sorting 34 (Vps34), or FK506-binding protein 38 (FKBP38). Although INS and AA had no effect, the abundance of ras-related GTP binding B (RagB) and the association of RagB with Raptor were greater in 6- than in 26-day-old pigs. Neither INS, AA, nor age altered AMPK-induced phosphorylation of Raptor. Our results suggest that the enhanced activation of mTORC1 in muscle of neonatal pigs is in part due to regulation by PRAS40, PLD1, and the Rag GTPases.

Fig. 1.

Recent concepts of the insulin and amino acid signaling pathways leading to the activation of mammalian target of rapamycin complex 1 (mTORC1)-induced protein synthesis. IRS1, insulin receptor substrate 1; PI3K, phosphoinositide-3 kinase; PKB, protein kinase B; TSC1/2, tuberous sclerosis complex 1/2; Rheb, Ras homolog enriched in brain; PLD1, phospholipase D1; FKBP38, FK506-binding protein 38; RagA–D, ras-related GTP binding A–D; mVps34, mammalian vacuolar protein sorting 34; 4EBP1, eukaryotic initiation factor 4E binding protein 1; S6K1, S6 kinase 1.

Fig. 2.

Phosphorylation (p) of PRAS40 at Thr-246 (A) and phosphorylation of Raptor at Ser-792 (B) in longissimus dorsi muscle of 6- and 26-day-old pigs in response to euinsulinemic-euglycemic-euaminoacidemic condition (control; C), euinsulinemic-euglycemic-hyperaminoacidemic clamps (AA), and hyperinsulinemic-euglycemic-euaminoacidemic clamps (INS). Values of the phosphoproteins were normalized for protein abundance in samples. Values are means ± SE; n = 4–6/treatment. T, total. Means with different letters differ at P < 0.05.

Fig. 3.

PLD1 abundance (A), phosphorylation of PLD1 at Thr-147 (B), phosphorylation of PLD1 at Ser-561 (C), and PLD1-Rheb association (D) in longissimus dorsi muscle of 6- and 26-day-old pigs in response to C, AA, and INS. Values of the phospho-PLD1 were normalized for PLD1 protein abundance in samples. Values for protein complexes were normalized for Rheb content in the immunoprecipitant. Values are means ± SE; n = 4–6/treatment. Means with different letters differ at P < 0.05.

Fig. 4.

FKBP38 abundance (A) and Vps34 abundance (B) in longissimus dorsi muscle of 6- and 26-day-old pigs in response to C, AA, and INS. Values are means ± SE; n = 4–6/treatment.

Fig. 5.

RagB abundance (A) and RagB-Raptor association (B) in longissimus dorsi muscle of 6- and 26-day-old pigs in response to C, AA, and INS. Values for protein complexes were normalized for Raptor content in the immunoprecipitant. Values are means ± SE; n = 4–6/treatment. Means with different letters differ at P < 0.05.