Supplementing monosodium glutamate to partial enteral nutrition slows gastric emptying in preterm pigs(1-3).

Emerging evidence suggests that free glutamate may play a functional role in modulating gastroduodenal motor function. We hypothesized that supplementing monosodium glutamate (MSG) to partial enteral nutrition stimulates gastric emptying in preterm pigs. Ten-day-old preterm, parenterally fed pigs received partial enteral nutrition (25%) as milk-based formula supplemented with MSG at 0, 1.7, 3.0, and 4.3 times the basal protein-bound glutamate intake (468 mg·kg(-1)·d(-1)) from d 4 to 8 of life (n = 5-8). Whole-body respiratory calorimetry and (13)C-octanoic acid breath tests were performed on d 4, 6, and 8. Body weight gain, stomach and intestinal weights, and arterial plasma glutamate and glutamine concentrations were not different among the MSG groups. Arterial plasma glutamate concentrations were significantly higher at birth than after 8 d of partial enteral nutrition. Also at d 8, the significant portal-arterial concentration difference in plasma glutamate was substantial (∼500 µmol/L) among all treatment groups, suggesting that there was substantial net intestinal glutamate absorption in preterm pigs. MSG supplementation dose-dependently increased gastric emptying time and decreased breath (13)CO2 enrichments, (13)CO2 production, percentage of (13)CO2 recovery/h, and cumulative percentage recovery of (13)C-octanoic acid. Circulating glucagon-like peptide-2 (GLP-2) concentration was significantly increased by MSG but was not associated with an increase in intestinal mucosal growth. In contrast to our hypothesis, our results suggest that adding MSG to partial enteral nutrition slows the gastric emptying rate, which may be associated with an inhibitory effect of increased circulating GLP-2.

Arginine-induced stimulation of protein synthesis and survival in IPEC-J2 cells is mediated by mTOR but not nitric oxide.

Arginine is an indispensable amino acid in neonates and is required for growth. Neonatal intestinal epithelial cells (IEC) are capable of arginine transport, catabolism, and synthesis and express nitric oxide (NO) synthase to produce NO from arginine. Our aim was to determine whether arginine directly stimulates IEC growth and protein synthesis and whether this effect is mediated via mammalian target of rapamycin (mTOR) and is NO-dependent. We studied neonatal porcine IEC (IPEC-J2) cultured in serum- and arginine-free medium with increasing arginine concentrations for 4 or 48 h. Our results show that arginine enhances IPEC-J2 cell survival and protein synthesis, with a maximal response at a physiological concentration (0.1-0.5 mM). Addition of arginine increased the activation of mTOR, p70 ribosomal protein S6 (p70 S6) kinase, and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) in a time- and dose-dependent manner. The arginine-induced protein synthesis response was not inhibited by the NO inhibitors nitro-l-arginine methyl ester (l-NAME) and aminoguanidine, despite inducible NO synthase expression in IPEC-J2 cells. Moreover, protein synthesis was not increased or decreased in some cases by addition of an NO donor (S-nitroso-N-acetylpenicillamine), arginine precursors (proline and citrulline) in the absence of arginine, or insulin; S-nitroso-N-acetylpenicillamine suppressed phosphorylation of mTOR, p70 S6 kinase, and 4E-BP1. We found a markedly higher arginase activity in IPEC-J2 cells than in primary pig IEC. Furthermore, mTOR inhibition by rapamycin partially (42%) reduced the arginine-induced protein synthesis response and phosphorylation of mTOR and 4E-BP1. We conclude that arginine-dependent cell survival and protein synthesis signaling in IPEC-J2 cells are mediated by mTOR, but not by NO.

Intestinal metabolism of sulfur amino acids.

The gastrointestinal tract (GIT) is a metabolically significant site of sulfur amino acid (SAA) metabolism in the body and metabolises about 20 % of the dietary methionine intake which is mainly transmethylated to homocysteine and trans-sulfurated to cysteine. The GIT accounts for about 25 % of the whole-body transmethylation and trans-sulfuration. In addition, in vivo studies in young pigs indicate that the GIT is a site of net homocysteine release and thus may contribute to the homocysteinaemia. The gut also utilises 25 % of the dietary cysteine intake and the cysteine uptake by the gut represents about 65 % of the splanchnic first-pass uptake. Moreover, we recently showed that SAA deficiency significantly suppresses intestinal mucosal growth and reduces intestinal epithelial cell proliferation, and increases intestinal oxidant stress in piglets. These recent findings indicate that intestinal metabolism of dietary methionine and cysteine is nutritionally important for intestinal mucosal growth. Besides their role in protein synthesis, methionine and cysteine are precursors of important molecules. S-adenosylmethionine, a metabolite of methionine, is the principal biological methyl donor in mammalian cells and a precursor for polyamine synthesis. Cysteine is the rate-limiting amino acid for glutathione synthesis, the major cellular antioxidant in mammals. Further studies are warranted to establish how SAA metabolism regulates gut growth and intestinal function, and contributes to the development of gastrointestinal diseases. The present review discusses the evidence of SAA metabolism in the GIT and its functional and nutritional importance in gut function and diseases.

Delayed initiation but not gradual advancement of enteral formula feeding reduces the incidence of necrotizing enterocolitis (NEC) in preterm pigs.

Enteral formula feeding is a risk factor for necrotizing enterocolitis (NEC) in premature infants, yet studies are conflicting regarding the safest timing for introduction and advancement of feeds. Our aim was to test the effects of early vs. late initiation and abrupt vs. gradual advancement of enteral feeding of an intact vs. hydrolyzed protein formula on NEC incidence and severity in preterm pigs. In Experiment 1, preterm pigs received total parenteral nutrition (TPN) at birth with abrupt initiation of enteral formula feeds (50% full intake) on d of life (DOL) 2 (EA) or 5 (LA) while PN continued. Pigs were also fed formula containing either intact or hydrolyzed protein. In Experiment 2, preterm pigs received TPN at birth with enteral, hydrolyzed-protein formula feeds introduced on DOL 2 either abruptly (EA; 50% full feeds) or gradually (EG; 10-50% full feeds over 5 d) while PN continued. NEC incidence and severity were assessed based on macroscopic and histological scoring. In Experiment 1, NEC incidence (41% vs. 70%, P<0.05) and severity were reduced in LA vs. EA groups and LA was associated with a higher survival rate, daily weight gain and jejunum villus height. Piglets fed hydrolyzed vs. intact protein formula had lower stomach content weights and similar NEC incidence. In Experiment 2, NEC incidence and severity were not different between pigs the EG vs. EA group. Proinflammatory gene expression (IL-1ß, IL-6 and S100A9) in the ileum was lower in both LA and EG vs. EA groups. In conclusion, delayed initiation but not gradual advancement of enteral feeding is protective against NEC in preterm pigs. Feeding hydrolyzed vs. intact protein formula improved gastric transit without affecting the NEC incidence.

Continuous parenteral and enteral nutrition induces metabolic dysfunction in neonatal pigs.

BACKGROUND: We previously showed that parenteral nutrition (PN) compared with formula feeding results in hepatic insulin resistance and steatosis in neonatal pigs. The current aim was to test whether the route of feeding (intravenous [IV] vs enteral) rather than other feeding modalities (diet, pattern) had contributed to the outcome. METHODS: Neonatal pigs were fed enterally or parenterally for 14 days with 1 of 4 feeding modalities as follows: (1) enteral polymeric formula intermittently (FORM), (2) enteral elemental diet (ED) intermittently (IEN), (3) enteral ED continuously (CEN), and (4) parenteral ED continuously (PN). Subgroups of pigs underwent IV glucose tolerance tests (IVGTT) and hyperinsulinemic-euglycemic clamps (CLAMP). Following CLAMP, pigs were euthanized and tissues collected for further analysis. RESULTS: Insulin secretion during IVGTT was significantly higher and glucose infusion rates during CLAMP were lower in CEN and PN than in FORM and IEN. Endogenous glucose production rate was suppressed to zero in all groups during CLAMP. In the fed state, plasma glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide (GLP)-1, and GLP-2 were different between feeding modalities. Insulin receptor phosphorylation in liver and muscle was decreased in IEN, CEN, and PN compared with FORM. Liver weight was highest in PN. Steatosis and myeloperoxidase (MPO) activity tended to be highest in PN and CEN. Enterally fed groups had higher plasma GLP-2 and jejunum weight compared with PN. CONCLUSIONS: PN and enteral nutrition (EN) when given continuously as an elemental diet reduces insulin sensitivity and the secretion of key gut incretins. The intermittent vs continuous pattern of EN produced the optimal effect on metabolic function.

Sulfur amino acid deficiency upregulates intestinal methionine cycle activity and suppresses epithelial growth in neonatal pigs.

We recently showed that the developing gut is a significant site of methionine transmethylation to homocysteine and transsulfuration to cysteine. We hypothesized that sulfur amino acid (SAA) deficiency would preferentially reduce mucosal growth and antioxidant function in neonatal pigs. Neonatal pigs were enterally fed a control or an SAA-free diet for 7 days, and then whole body methionine and cysteine kinetics were measured using an intravenous infusion of [1-(13)C;methyl-(2)H(3)]methionine and [(15)N]cysteine. Body weight gain and plasma methionine, cysteine, homocysteine, and taurine and total erythrocyte glutathione concentrations were markedly decreased (-46% to -85%) in SAA-free compared with control pigs. Whole body methionine and cysteine fluxes were reduced, yet methionine utilization for protein synthesis and methionine remethylation were relatively preserved at the expense of methionine transsulfuration, in response to SAA deficiency. Intestinal tissue concentrations of methionine and cysteine were markedly reduced and hepatic levels were maintained in SAA-free compared with control pigs. SAA deficiency increased the activity of methionine metabolic enzymes, i.e., methionine adenosyltransferase, methionine synthase, and cystathionine beta-synthase, and S-adenosylmethionine concentration in the jejunum, whereas methionine synthase activity increased and S-adenosylmethionine level decreased in the liver. Small intestine weight and protein and DNA mass were lower, whereas liver weight and DNA mass were unchanged, in SAA-free compared with control pigs. Dietary SAA deficiency induced small intestinal villus atrophy, lower goblet cell numbers, and Ki-67-positive proliferative crypt cells in association with lower tissue glutathione, especially in the jejunum. We conclude that SAA deficiency upregulates intestinal methionine cycle activity and suppresses epithelial growth in neonatal pigs.