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.

The postprandial rise in amino acids, particularly leucine, stimulates muscle protein synthesis in neonates. Previously, we showed that a 1-h infusion of leucine increased protein synthesis, but this response was not sustained for 2 h unless the leucine-induced decrease in amino acids was prevented. To determine whether a parenteral leucine infusion can stimulate protein synthesis for a more prolonged, clinically relevant period if baseline amino acid concentrations are maintained, overnight food-deprived neonatal pigs were infused for 24 h with saline, leucine (400 mumol.kg(-1). h(-1)), or leucine with replacement amino acids. Amino acid replacement prevented the leucine-induced decrease in amino acids. Muscle protein synthesis was increased by leucine but only when other amino acids were supplied to maintain euaminoacidemia. Leucine did not affect activators of mammalian target of rapamycin (mTOR), i.e. protein kinase B, AMP-activated protein kinase, tuberous sclerosis complex 2, or eukaryotic elongation factor 2. There was no effect of treatment on the association of mTOR with regulatory associated protein of mammalian target of rapamycin (raptor), G-protein beta subunit-like protein, or rictor or the phosphorylation of raptor or proline-rich Akt substrate of 40 kDa. Phosphorylation of mTOR and its downstream targets, eukaryotic initiation factor (eIF) 4E binding protein and ribosomal protein S6 kinase, and the eIF4E . eIF4G association were increased and eIF2alpha phosphorylation was reduced by leucine and was not further altered by correcting for the leucine-induced hypoaminoacidemia. Thus, prolonged parenteral infusion of leucine activates mTOR and its downstream targets in neonatal skeletal muscle, but the stimulation of protein synthesis also is dependent upon amino acid availability.

Orogastric tube feeding, using either continuous or intermittent bolus delivery, is common in infants for whom normal feeding is contraindicated. To compare the impact of different feeding strategies on muscle protein synthesis, after withholding food overnight, neonatal pigs received a complete formula orally as a bolus feed every 4 h or were continuously fed. Protein synthesis rate and translational mechanisms in skeletal muscle were examined after 0, 24, and 25.5 h. Plasma amino acid and insulin concentrations increased minimally and remained constant in continuously fed compared to feed-deprived pigs; however, the pulsatile meal feeding pattern was mimicked in bolus-fed pigs. Muscle protein synthesis was stimulated by feeding and the greatest response occurred after a bolus meal. Bolus but not continuous feeds increased polysome aggregation, the phosphorylation of protein kinase B, tuberous sclerosis complex 2, proline-rich Akt substrate of 40 kDa, eukaryotic initiation factor (eIF) 4E binding protein (4EBP1), and rp S6 kinase and enhanced dissociation of the 4EBP1 ·eIF4E complex and formation of the eIF4E ·eIF4G complex compared to feed deprivation (P < 0.05). Activation of insulin receptor substrate-1, regulatory associated protein of mammalian target of rapamycin, AMP-activated protein kinase, eukaryotic elongation factor 2, and eIF2α phosphorylation were unaffected by either feeding modality. These results suggest that in neonates, intermittent bolus feeding enhances muscle protein synthesis to a greater extent than continuous feeding by eliciting a pulsatile pattern of amino acid- and insulin-induced translation initiation.

Flavonoids, dietary components in vegetables, fruits and beverages, may protect against coronary heart disease, stroke and cancer. However, the bioavailability of these compounds is questionable. A previous study in ileostomy patients of the most abundant flavonoid, quercetin, suggested a 52% absorption of its major dietary forms, monoglucoside (QMG) and diglucoside (QDG), from an onion meal. However, this was based on indirect measurements after acid hydrolysis. Because human intestinal Caco-2 cell monolayers showed minimal absorption of the glucosides, we repeated the study in ileostomy patients, using molecularly specific analytical methodology for the intact glucosides and quercetin. The onion meal had high concentrations of both QMG and QDG with only trace amounts of quercetin. The intake of QMG and QDG in four patients ranged from 10.9 to 51.6 mg. No QMG or QDG was detected in the ileostomy fluid. In contrast, the amounts of the aglycone quercetin were substantial, 2.9-11.3 mg. This corresponded to 19.5-35.2% of total quercetin glucosides ingested, implying absorption of 64.5-80.7%. These findings suggest a different interpretation than that from the previous study, i.e., that both QMG and QDG are efficiently hydrolyzed in the small intestine by beta-glucosidases to quercetin, most of which is then absorbed.

A cDNA clone encoding the major intestinal cytosolic 14-kDa bile acid-binding protein (14-kDa I-BABP) was isolated from a rat ileal lambda gt22A library following immunoscreening using a monospecific antiserum raised against a 14-kDa polypeptide found in the rat ileal cytosol. One clone of 516 bp encoded a 128-amino acid protein with a predicted molecular mass of 14,544 Da. The deduced amino acid sequence of 14-kDa I-BABP showed 100% homology to rat intestinal 15-kDa protein (I-15P) and 72% homology to porcine 15-kDa gastrotropin, whereas comparison of I-BABP to rat 14-kDa fatty acid-binding proteins of liver, intestine, and heart revealed homologies of 44%, 25%, and 28%, respectively. Northern blot analysis revealed a single transcript of approximately 0.5 kb in ileum and ovary; however, the abundance of I-BABP mRNA was much greater in ileum than in ovary. No transcript was seen in RNA extracted from stomach, jejunum, colon, liver, adrenal, brain, heart, kidney, or testis. Transfection of the I-BABP cDNA into COS-7 cells resulted in the expression of a 14-kDa protein that was identical to the ileal cytosolic I-BABP as determined by immunoblotting. Photoaffinity labeling of expressed 14-kDa protein was saturable with respect to increasing concentrations of 7,7-azo[3H]taurocholate (Km, 83.3 microM; Vmax, 6.7 pmol/mg per 5 min). Taurocholate inhibited 7,7-azotaurocholate labeling by > 96% with lesser inhibition by taurochenodeoxycholate (83.1%), chenodeoxycholate (74.6%), cholate (50.5%), and progesterone (38.5%), whereas oleic acid and estradiol did not inhibit binding.