Skeletal muscle in the neonate grows at a rapid rate due in part to an enhanced sensitivity to the postprandial rise in amino acids, particularly leucine. To elucidate the molecular mechanism by which leucine stimulates protein synthesis in neonatal muscle, overnight-fasted 7-day-old piglets were treated with rapamycin [an inhibitor of mammalian target of rapamycin (mTOR) complex (mTORC)1] for 1 h and then infused with leucine for 1 h. Fractional rates of protein synthesis and activation of signaling components that lead to mRNA translation were determined in skeletal muscle. Rapamycin completely blocked leucine-induced muscle protein synthesis. Rapamycin markedly reduced raptor-mTOR association, an indicator of mTORC1 activation. Rapamycin blocked the leucine-induced phosphorylation of mTOR, S6 kinase 1 (S6K1), and eukaryotic initiation factor (eIF)4E-binding protein-1 (4E-BP1) and formation of the eIF4E.eIF4G complex and increased eIF4E.4E-BP1 complex abundance. Rapamycin had no effect on the association of mTOR with rictor, a crucial component for mTORC2 activation, or G protein beta-subunit-like protein (GbetaL), a component of mTORC1 and mTORC2. Neither leucine nor rapamycin affected the phosphorylation of AMP-activated protein kinase (AMPK), PKB, or tuberous sclerosis complex (TSC)2, signaling components that reside upstream of mTOR. Eukaryotic elongation factor (eEF)2 phosphorylation was not affected by leucine or rapamycin, although current dogma indicates that eEF2 phosphorylation is mTOR dependent. Together, these in vivo data suggest that leucine stimulates muscle protein synthesis in neonates by enhancing mTORC1 activation and its downstream effectors.

Insulin and amino acids act independently to stimulate protein synthesis in skeletal muscle of neonatal pigs, and the responses decrease with development. The purpose of this study was to compare the separate effects of fed levels of INS and AA on the activation of signaling components leading to translation initiation and how these responses change with development. Overnight-fasted 6- ( n = 4/group) and 26-day-old ( n = 6/ group) pigs were studied during 1) euinsulinemic-euglycemiceuaminoacidemic conditions (controls), 2) euinsulinemic-euglycemichyperaminoacidemic clamps (AA), and 3) hyperinsulinemic-euglycemic-euaminoacidemic clamps (INS). INS, but not AA, increased the phosphorylation of protein kinase B (PKB) and tuberous sclerosis 2 (TSC2). Both INS and AA increased protein synthesis and the phosphorylation of mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase-1, and eukaryotic initiation factor (eIF)4E-binding protein 1 (4E-BP1), and these responses were higher in 6-day-old compared with 26-day-old pigs. Both INS and AA decreased the binding of 4E-BP1 to eIF4E and increased eIF4E binding to eIF4G; these effects were greater in 6-day-old than in 26-day-old pigs. Neither INS nor AA altered the composition of mTORC1 (raptor, mTOR, and GβL) or mTORC2 (rictor, mTOR, and GβL) complexes. Furthermore, neither INS, AA, nor age had any effect on the abundance of Rheb and the phosphorylation of AMP-activated protein kinase and eukaryotic elongation factor 2. Our results suggest that the activation by insulin and amino acids of signaling components leading to translation initiation is developmentally regulated and parallels the developmental decline in protein synthesis in skeletal muscle of neonatal pigs.

Skeletal muscle protein synthesis is elevated in neonates in part due to an enhanced response to the rise in insulin and amino acids after eating. In vitro studies suggest that glucose plays a role in protein synthesis regulation. To determine whether glucose, independently of insulin and amino acids, is involved in the postprandial rise in skeletal muscle protein synthesis, pancreatic-substrate clamps were performed in neonatal pigs. Insulin secretion was inhibited with somatostatin and insulin was infused to reproduce fasting or fed levels, while glucose and amino acids were clamped at fasting or fed levels. Fractional protein synthesis rates and translational control mechanisms were examined. Raising glucose alone increased protein synthesis in fast-twitch glycolytic muscles but not in other tissues. The response in muscle was associated with increased phosphorylation of protein kinase B (PKB) and enhanced formation of the active eIF4E·eIF4G complex but no change in phosphorylation of AMP-activated protein kinase (AMPK), tuberous sclerosis complex 2 (TSC2), mammalian target of rapamycin (mTOR), 4E-binding protein-1 (4E-BP1), ribosomal protein S6 kinase (S6K1), or eukaryotic elongation factor 2 (eEF2). Raising glucose, insulin, and amino acids increased protein synthesis in most tissues. The response in muscle was associated with phosphorylation of PKB, mTOR, S6K1, and 4E-BP1 and enhanced eIF4E·eIF4G formation. The results suggest that the postprandial rise in glucose, independently of insulin and amino acids, stimulates protein synthesis in neonates, and this response is specific to fast-twitch glycolytic muscle and occurs by AMPK- and mTOR-independent pathways.

In skeletal muscle of adults, sepsis reduces protein synthesis by depressing translation initiation and induces resistance to branched-chain amino acid stimulation. Normal neonates maintain a high basal muscle protein synthesis rate that is sensitive to amino acid stimulation. In the present study, we determined the effect of amino acids on protein synthesis in skeletal muscle and other tissues in septic neonates. Overnight-fasted neonatal pigs were infused with endotoxin (LPS, 0 and 10 g ⅐ kg Ϫ1 ⅐ h Ϫ1 ), whereas glucose and insulin were maintained at fasting levels; amino acids were clamped at fasting or fed levels. In the presence of fasting insulin and amino acids, LPS reduced protein synthesis in longissimus dorsi (LD) and gastrocnemius muscles and increased protein synthesis in the diaphragm, but had no effect in masseter and heart muscles. Increasing amino acids to fed levels accelerated muscle protein synthesis in LD, gastrocnemius, masseter, and diaphragm. LPS stimulated protein synthesis in liver, lung, spleen, pancreas, and kidney in fasted animals. Raising amino acids to fed levels increased protein synthesis in liver of controls, but not LPS-treated animals. The increase in muscle protein synthesis in response to amino acids was associated with increased mTOR, 4E-BP1, and S6K1 phosphorylation and eIF4G-eIF4E association in control and LPS-infused animals. These findings suggest that amino acids stimulate skeletal muscle protein synthesis during acute endotoxemia via mTOR-dependent ribosomal assembly despite reduced basal protein synthesis rates in neonatal pigs. However, provision of amino acids does not further enhance the LPS-induced increase in liver protein synthesis. growth; sepsis; mammalian target of rapamycin; eukaryotic initiation factor 4G; ribosomal protein S6 kinase THE SYSTEMIC INFLAMMATORY RESPONSE and sepsis syndromes elicited in the host in response to a bacterial pathogen or its products (35) are associated with hypermetabolism, increased oxygen consumption and energy expenditure, activation of peripheral protein catabolism, and alteration in the flux of metabolic substrates between skeletal muscle and liver (4, 39). Derangements in skeletal muscle glucose and amino acid metabolism evoked by sepsis result from the body's attempt to maintain the balance between peripheral substrate stores and the increased metabolic demands required to activate the immune system and the stress response (4, 37). These adaptative responses are different from those in the normal fasting person in that the immune response is the drive for the metabolic enhancement (11,59). In an attempt to establish homeostasis, amino acids are released from peripheral tissues and shifted to the liver for promotion of hepatic protein synthesis, gluconeogenesis, and urea synthesis (24, 25). Plasma amino acid concentrations in patients with sepsis or disease are lower than in persons without sepsis (17,30,49), and attempts have been made to use the extent of this difference as a marker of the severity and prognosis of the ...

OBJECTIVES: To determine the association between preintubation respiratory support and outcomes in patients with acute respiratory failure and to determine the impact of immunocompromised (IC) diagnoses on outcomes after adjustment for illness severity. DESIGN: Retrospective multicenter cohort study. SETTING: Eighty-two centers in the Virtual Pediatric Systems database. PATIENTS: Children 1 month to 17 years old intubated in the PICU who received invasive mechanical ventilation (IMV) for greater than or equal to 24 hours. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: High-flow nasal cannula (HFNC) or noninvasive positive-pressure ventilation (NIPPV) or both were used prior to intubation in 1,825 (34%) of 5,348 PICU intubations across 82 centers. When stratified by IC status, 50% of patients had no IC diagnosis, whereas 41% were IC without prior hematopoietic cell transplant (HCT) and 9% had prior HCT. Compared with patients intubated without prior support, preintubation exposure to HFNC (adjusted odds ratio [aOR], 1.33; 95% CI, 1.10–1.62) or NIPPV (aOR, 1.44; 95% CI, 1.20–1.74) was associated with increased odds of PICU mortality. Within subgroups of IC status, preintubation respiratory support was associated with increased odds of PICU mortality in IC patients (HFNC: aOR, 1.50; 95% CI, 1.11–2.03; NIPPV: aOR, 1.76; 95% CI, 1.31–2.35) and HCT patients (HFNC: aOR, 1.75; 95% CI, 1.07–2.86; NIPPV: aOR, 1.85; 95% CI, 1.12–3.02) compared with IC/HCT patients intubated without prior respiratory support. Preintubation exposure to HFNC/NIPPV was not associated with mortality in patients without an IC diagnosis. Duration of HFNC/NIPPV greater than 6 hours was associated with increased mortality in IC HCT patients (HFNC: aOR, 2.41; 95% CI, 1.05–5.55; NIPPV: aOR, 2.53; 95% CI, 1.04–6.15) and patients compared HCT patients with less than 6-hour HFNC/NIPPV exposure. After adjustment for patient and center characteristics, both preintubation HFNC/NIPPV use (median, 15%; range, 0–63%) and PICU mortality varied by center. CONCLUSIONS: In IC pediatric patients, preintubation exposure to HFNC and/or NIPPV is associated with increased odds of PICU mortality, independent of illness severity. Longer duration of exposure to HFNC/NIPPV prior to IMV is associated with increased mortality in HCT patients.