Preterm birth alters the feeding-induced activation of Akt signaling in the muscle of neonatal piglets

Abstract

Background: Postnatal lean mass accretion is commonly reduced in preterm infants. This study investigated mechanisms involved in the blunted feeding-induced activation of Akt in the skeletal muscle of preterm pigs that contributes to lower protein synthesis rates.

Methods: On day 3 following cesarean section, preterm and term piglets were fasted or fed an enteral meal. Activation of Akt signaling pathways in skeletal muscle was determined.

Results: Akt1 and Akt2, but not Akt3, phosphorylation were lower in the skeletal muscle of preterm than in term pigs (P < 0.05). Activation of Akt-positive regulators, PDK1 and mTORC2, but not FAK, were lower in preterm than in term (P < 0.05). The formation of Akt complexes with GAPDH and Hsp90 and the abundance of Ubl4A were lower in preterm than in term (P < 0.05). The abundance of Akt inhibitors, PHLPP and SHIP2, but not PTEN and IP6K1, were higher in preterm than in term pigs (P < 0.05). PP2A activation was inhibited by feeding in term but not in preterm pigs (P < 0.05).

Conclusions: Our results suggest that preterm birth impairs regulatory components involved in Akt activation, thereby limiting the anabolic response to feeding. This anabolic resistance likely contributes to the reduced lean accretion following preterm birth.

Impact: The Akt-mTORC1 pathway plays an important role in the regulation of skeletal muscle protein synthesis in neonates. This is the first evidence to demonstrate that, following preterm birth, the postprandial activation of positive regulators of Akt in the skeletal muscle is reduced, whereas the activation of negative regulators of Akt is enhanced. This anabolic resistance of Akt signaling in response to feeding likely contributes to the reduced accretion of lean mass in premature infants. These results may provide potential novel molecular targets for intervention to enhance lean growth in preterm neonates.

Fig. 1.. Current models of Akt activation.

The activation of Akt is highly complex and is tightly controlled via a multistep process that depends on physiological conditions, the cell type, and types of stimuli. 4EBP1, eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1; Akt1/2/3 Akt/PKB protein kinase B 1/2/3; eEF2, elongation factor 2; eIF2α, eukaryotic translation initiation factor 2α; eIF4E, eukaryotic translation initiation factor 4E; eIF4G, eukaryotic translation initiation factor 4G; FAK, focal adhesion kinase; IP6K1, inositol hexakisphosphate kinase 1; IP7, 5-diphosphoinositolpentakisphosphate, IR, insulin receptor; IRS1, insulin receptor substrate 1; mTORC1/2, mechanistic target of rapamycin complex 1/2; PDK1, 3-phosphoinositide-dependent protein kinase-1; PI3K, phosphatidylinositol-3-kinase; PHLPP, PH domain leucine-rich repeat protein phosphatase; PP2A, protein phosphatase 2A; PTEN, phosphatase and tensin homolog deleted on chromosome ten; rpS6, ribosomal protein S6; S6K1, p70 ribosomal protein S6 kinase 1; SHIP2, SH2 domain-containing inositol phosphatase; TSC1/2, Ulb4A, ubiquitin-like protein 4A.

Fig. 2.. Preterm attenuates the phosphorylation of Akt1 and Akt2, but not Akt3.

The phosphorylation of individual Akt isoform was analyzed by immunoprecipitation followed by Western blot. a-c phosphorylated Akt1 at Ser472, Akt2 at Ser473, and Akt3 at Ser474. Phosphorylation values were corrected by their protein abundance in the immunoprecipitates. For Akt1, there was a significant interaction (P < 0.01) between GAB and STATE. Representative blots are shown. Data are expressed as mean ± SEM; n = 21–25/group (*P < 0.05 and **P < 0.01). AU, arbitrary units.

Fig. 3.. Preterm blunts positive regulators of Akt activation.

a Western analysis of phosphorylated PDK1 at Ser241. The phosphorylation of mTORC2, Akt-GAPDH complex abundance, and Akt-Hsp90 complex abundance were analyzed by immunoprecipitation followed by Western blot. b-f Western blot analysis of phosphorylated mTORC2 at Ser2481, phosphorylated FAK at Tyr397, Ubl4A abundance, Akt-GAPDH complex abundance, and Akt-Hsp90 complex abundance. Values were corrected by their protein abundance in the immunoprecipitates or tissue lysate samples. Ubl4A abundance values were normalized by GAPDH in the samples. For PDK1, there was a significant interaction (P < 0.001) between GAB and STATE. Representative blots are shown. Data are expressed as mean ± SEM; n = 21–25/group (*P < 0.05 and **P < 0.01). AU, arbitrary units.

Fig. 4.. Preterm upregulates Akt endogenous inhibitors.

a-e Western analysis of phosphorylated PTEN at Ser380, phosphorylated PP2A at Tyr307, SHIP2, PHLPP, and IP6K1. Phosphorylation values were corrected by their protein abundance in the samples. Total protein abundance values were normalized either by GAPDH or vinculin abundances in the samples. For PP2A, there was a significant interaction (P < 0.001) between GAB and STATE. Representative blots are shown. Data are expressed as mean ± SEM; n = 21–25/group (*P < 0.05 and **P < 0.01). AU, arbitrary units.