Many babies are born preterm and despite improvements in their care, their extrauterine growth frequently falters (¹–³). Although premature infants may be initially provided parenteral nutrition, the goal is to initiate and advance enteral feeding (⁴). However, enteral feedings is often limited by concern for the preterm infant’s ability to metabolize nutrients and clinical complications related to feeding intolerance and necrotizing enterocolitis (⁵–⁷). Thus, enteral feeding may not achieve the level of protein required to sustain optimal growth.

To identify strategies to optimize the nutrition of neonates, we have used the neonatal pig as a model of the human infant. We found that feeding stimulates PS in skeletal muscle of neonatal pigs (⁸), and this response is independently mediated by the rise in insulin and amino acids (AA) (⁹–¹⁰). Leucine is the most effective single AA to trigger the activation of translation initiation factors that regulate muscle PS (¹¹–¹⁶), but the effects of leucine in visceral tissues are not well understood. We have shown that parenteral AA infusion in neonates stimulates the intracellular signaling proteins that regulate PS (¹⁷), however, less is known about the effects of enteral AA delivery on the activation of this pathway.

AA, especially leucine, serve as substrates for PS and also as nutrient signals to stimulate translation initiation (¹¹–¹⁷). Unlike insulin, AA do not activate protein kinase B (PKB) but activate the mammalian target of rapamycin (mTOR) (¹⁶, ¹⁸), which functions in two distinct protein complexes (mTORC1 and mTORC2) (¹⁹). mTORC1, a major player for mRNA translation, consists of several components, including regulatory associated protein of mammalian target of rapamycin (raptor, an activator) and proline-rich Akt substrate of 40 kDa (PRAS40, an inhibitor) (²⁰), whose association is crucial for mTORC1 activation. Activated mTORC1 promotes eukaryotic initiation factor (eIF) 4E-binding protein 1 (4EBP1) and ribosomal protein S6 kinase-1 (S6K1) phosphorylation. Phosphorylated 4EBP1 allows active eIF4E·eIF4G complex formation and activated S6K1 phosphorylates ribosomal protein S6. Both processes are crucial for translation initiation (²¹).

Other crucial steps for mRNA translation are the eukaryotic initiation factor (eIF2) pathway (²²) and the peptide elongation process. Phosphorylation of eIF2α (an inhibitor) is a limiting step in the eIF2 pathway while elongation factor 2 (eEF2) phosphorylation regulates elongation (²³). Insulin and AA modulate eIF2α and eEF2, resulting in PS progression.

Recently, we showed that enteral leucine supplementation of a low protein meal acutely stimulates PS (²⁴). However, it is unclear whether the effects could be sustained by prolonged enteral leucine supplementation. Although prolonged intravenous leucine infusion alone can reduce circulating levels of essential AA, which can limit PS (¹⁷), whether this occurs with prolonged enteral leucine supplementation is unknown as a decline in other AA was not observed in our previous acute enteral leucine supplementation study (²⁴). Therefore, the aims of this study were to determine whether feeding of a low protein diet supplemented with leucine for 24 h can sustain enhanced rates of muscle and visceral tissue PS in neonatal pigs to rates similar to those achieved with a high protein diet and to examine the mechanisms involved.

Postnatal nutrition has a critical impact on the long-term outcome of preterm infants (³²). Short-term decreases in dietary intake are inevitable consequences in the complex care of preterm infants (³³). Preterm infants frequently have poor tolerance to enteral feeding and thus consume a lower volume of milk formula than that necessary to meet their protein requirements for adequate growth. Recently, we showed that feeding a meal of a low protein milk formula supplemented with leucine compared to a low protein formula meal alone acutely increases PS in most tissues to rates similar to that achieved with a high protein meal (²⁴). In the current study, we wished to determine whether the anabolic response to leucine supplementation could be sustained for a more prolonged period. The results of the current study indicate that prolonged leucine supplementation of a milk formula with a low protein content can enhance PS compared to the low protein diet alone. Although the leucine-induced increase in PS was lower than that with a high protein milk formula, our findings suggest a potential beneficial effect of including leucine supplementation in the nutritional management plan for sick premature infants. However, limitations of the study include the lack of a normal suckling pig control and the use of term rather than preterm piglets.

Previously, we showed that parenteral leucine infusion for 24 h reduced circulating levels of other AA and limited the leucine-induced increase in PS, likely because other AA were needed as substrates for PS (¹⁷). However, when the fall in other AA was prevented by performing an AA clamp, the leucine-induced increase in PS was sustained. Recently, we found that acute enteral leucine supplementation of a LP meal did not elicit a fall in other AA and increased PS similar to that of a HP meal (²⁴). In the current study, consumption of the LP+L compared to LP diet for 24 h reduced circulating isoleucine and valine concentrations, likely due to activation of enzymes that metabolize BCAA, namely branched-chain aminotransferase and branched-chain α-ketoacid dehydrogenase (³⁴). Unlike in our previous acute enteral leucine supplementation study (²⁴), the reduction in isoleucine and valine in the current study may be responsible for the failure to achieve maximum PS rates obtained with the HP diet. On the other hand, circulating insulin levels in the current study were higher in the HP than LP and LP+L groups, unlike our previous acute leucine supplementation study, and may have contributed to the higher PS rate, at least in muscle, but only in the HP group.

The positive effect of leucine administration on muscle PS in humans and animal models has been recognized (¹¹–¹³, ³⁵). In this study, we determined effects of leucine supplementation on muscles of different fiber types including longissimus dorsi (mainly glycolytic fibers), gastrocnemius (mixed glycolytic and oxidative fibers), masseter (oxidative fibers) and cardiac muscles (oxidative fibers). Consistent with our 24 h parenteral leucine infusion study (¹⁷), in the current study enteral leucine supplementation stimulated PS in muscles of different fiber types, although the leucine effect was less potent than the HP diet, which was used as a positive control to elicit maximum PS rates. In our previous study, acute leucine supplementation of a LP meal enhanced PS in these muscles comparable to the HP meal (²⁴), likely due to maintenance of isoleucine and valine levels.

To better understand the overall anabolic effect of leucine supplementation in neonates, we determined PS rates in visceral tissues. We showed that leucine supplementation enhanced PS in liver, pancreas, and jejunum, but not kidney. In our previous 24 h parenteral leucine infusion study, the leucine-induced increase in PS was detected in liver and pancreas, although not jejunum or kidney, and only when AA availability was maintained (¹⁷). Differences between these two studies in the jejunum response could be due to the different route of leucine administration (i.e., parenteral versus enteral). The lack of effect in the kidney may be due to rerouting excess AA to gluconeogenesis (³⁶).

To evaluate molecular mechanisms by which leucine stimulates PS in vivo, we determined the leucine-induced activation of signaling components leading to mRNA translation. More detailed study of signaling component activation was conducted in longissimus dorsi muscle. PKB activation is a hallmark for insulin-induced PS (³⁷). Consistent with other studies (¹⁶, ¹⁸), we found that leucine supplementation had no effect on PKB activation in muscle. The recent consensus is that AA, particularly leucine, stimulate PS in an mTORC1-dependent pathway downstream of PKB (³⁸) although the exact molecular mechanism is not completely understood. Phosphorylation of two major targets of mTORC1, S6K1 and 4EBP1, has been used as a read-out for mTORC1 activation (³⁸). Likewise, S6K1 and 4EBP1 association with mTORC1 through raptor is a bona fide indicator of mTORC1 activation (³⁹, ⁴⁰). Cell culture studies suggest that mTOR-raptor association positively and PRAS40-raptor association negatively regulates mTORC1 activation (⁴¹). In our study, leucine supplementation enhanced S6K1 and 4EBP1 association with raptor, which elicits increased phosphorylation of these two mTOR substrates. These results are consistent with cell culture studies that showed that, for mTORC1 to phosphorylate S6K1 and 4EBP1, raptor has to bind to them through their TOR signaling motif (³⁹, ⁴⁰). We did not find a leucine effect on raptor association with mTOR or PRAS40, which could be due to a transient nature of their interaction in vivo. A leucine-induced increase in S6K1 and 4EBP1 phosphorylation was also observed in muscles with different fiber types and visceral tissues, except kidney. Increased 4EBP1 phosphorylation leads to decreased association with eIF4E, allowing active eIF4G·eIF4E complex formation (³⁸). In this study we found that leucine supplementation reduced 4EBP1·eIF4E complex formation and enhanced eIF4G·eIF4E complex formation.

In the current study, plasma glutamine levels were elevated in the LP+L compared to LP group suggesting de novo synthesis from exogenous leucine. Cell culture studies suggest that glutamine is needed for leucine-induced activation of mTORC1 (⁴²), and thus, together with leucine, glutamine plays a crucial role in the regulation of PS.

Another potential signaling pathway mediating the leucine-induced stimulation of PS is the eIF2 pathway (²²). In this study, we examined phosphorylation of eIF2α, an inhibitor and key player in this pathway. We found that no effect of leucine on eIF2α phosphorylation in all tissues studied. This result is consistent with the notion that leucine does not directly affect eIF2α phosphorylation but eIF2α phosphorylation status is affected by essential AA deficiency (⁴³).

The elongation process in mRNA translation is another crucial step in the PS process (²³). In cell culture studies, AA, especially leucine, activate eEF2, a marker for peptide chain elongation, by dephophorylation (⁴⁴). Thus, we determined whether leucine has similar effects in vivo. As in our previous study (¹⁶), leucine had no effect on eEF2 phosphorylation in all tissues studied. This suggests that regulation of elongation by AA in vivo is complex and may differ from responses found in vitro.

In summary, 24 h enteral leucine supplementation of a LP formula diet enhanced PS in most tissues of neonatal pigs in an mTORC1-dependent fashion. Although the leucine-induced increase in PS was less than that achieved with a HP diet, the results of our study suggest that leucine supplementation may have a beneficial effect on neonatal growth. Further studies are required to better understand the role of leucine and BCAA in the regulation of neonatal growth.

Statement of financial support: This study was supported by Ajinomoto Amino Acid Research Program, NIH R01 AR44474, NIH KO8 AR051563, and USDA/ARS 6250-510000-055.

We thank Rosemarie Almonaci for technical assistance, Jerome Stubblefield and Rickey Bryant for care of animals, E. O’Brian Smith for statistical assistance, Adam Gillum for graphics, and Linda Kemper for secretarial assistance.