2013 Annual Report
1a.Objectives (from AD-416):
Objective 1: Establish a model using caesarean-delivered animal model to investigate the impact of prematurity on the gastrointestinal and metabolic response to perinatal nutrition.
Subobjective 1A: Quantify the effect of chronic parenteral nutrition in the first 2 weeks after birth on the short-term (3 months) and long-term (12 months) development of body composition and glucose homeostasis in models delivered preterm and at term.
Subobjective 1B: Quantify whether manipulation of dietary macronutrients and supplementation with bioactive food ingredients fed to premature animal models prevents the onset of mucosal inflammation and gastrointestinal disease, specifically necrotizing enterocolitis.
Objective 2: Compare the impact of continuous versus intermittent bolus delivery of nutrients provided enterally or parenterally on protein synthesis and accretion in neonatal animal models and identify the intracellular signaling mechanism involved.
Subobjective 2A: Compare the short-term effects of enteral or parenteral amino acids provided in a continuous vs. intermittent bolus delivery pattern.
Subobjective 2B: Compare the short-term effects of an enteral or parenteral complete diet provided in a continuous vs. intermittent bolus delivery pattern.
Subobjective 2C: Compare the long-term effects of an enteral or parenteral complete diet provided in a continuous vs. intermittent bolus delivery pattern.
1b.Approach (from AD-416):
The goal of our research is to determine how key nutrients, bioactive ingredients, and the pattern of nutrient delivery affects nutrient metabolism, body composition and incidence of disease in early postnatal life. Children's Nutrition Research Center researchers will use multiple experimental approaches involving innovative neonatal animal models to specifically examine the effects of prematurity, parenteral nutrition, and intermittent bolus feeding versus continuous feeding on glucose and protein metabolism. Main endpoints of metabolism will include measurements of glucose tolerance and insulin sensitivity using hyperinsulinemic, euglycemic clamps. The metabolic fate of amino acids and glucose will be measured using oxidation and incorporation of stable isotopic tracers into end-products such as CO2 and protein. Our research team will also examine whether specific carbohydrates and treatment with the bioactive gut peptide, glucagon-like peptide-2, can prevent the onset of necrotizing enterocolitis using a novel premature animal model. We will quantify endpoints of gastrointestinal function, such as blood flow and nutrient absorption, as well as disease based on measures of proinflammatory cytokine expression and gut microbiota communities. This research will provide novel information that will be directly useful to optimize the nutritional management of low birth weight infants and reduce the risk of early postnatal diseases.
In Objective 1A, we have continued progress on the study designed to examine the long-term effects of enteral (EN) and parenteral nutrition (PN) in neonatal, term piglets. We previously reported that neonatal PN increased body fat content and this persisted for up to 8 weeks even after the pigs in both groups were fed the same diet. Further analysis shows that there were no differences in glucose tolerance or liver function, which suggest that they were not developing symptoms similar to diabetes. We are finalizing analysis of these results and plan to submit a manuscript for publication later this year. We have continued work on a study designed to examine the impact of new generation parenteral lipid emulsions on metabolic function and the prevention of liver disease in preterm PN-fed piglets. This project generated highly clinically relevant results showing that fish-oil containing lipid emulsions prevented liver disease. We finalized results summarizing the impact of lipid emulsions on liver disease and submitted a manuscript to the Journal of Lipid Research. Additional findings from this study were disseminated in an oral presentation and a published abstract at a national meeting this year. A second manuscript from this study describing the metabolomic and transcriptomic findings is in preparation. Additionally we extended the studies examining lipid emulsions to explore the relative impact of phytosterols and vitamin E contained in new lipid emulsions in the prevention of liver disease in preterm total parenteral nutrition-fed piglets.
Phytosterols are plant forms of cholesterol that are contained in soybean lipid emulsions. We completed a study in 2013 showing that vitamin E added to soybean oil-based lipid emulsion protects against liver disease. We also found that adding phytosterols to the fish oil-based emulsion (Omegaven) does not induce cholestasis or steatosis. These results were presented at a national meeting, and a manuscript reporting the findings is being prepared.
In Objective 1B, we completed the study designed to test the effect of a gut hormone called glucagon-like peptide 2 (GLP-2) pretreatment on the incidence of necrotizing enterocolitis (NEC), a devastating inflammatory disease that occurs in premature babies. Our results showed that GLP-2 pretreatment delays the onset, but does not prevent the incidence of NEC in our preterm piglet model. This finding was disappointing and may be related to the short duration of GLP-2 treatment prior to enteral formula feeding. Given these results, we have elected not to continue on Obj.1B with studies designed to test whether GLP-2 pretreatment improves digestive function in preterm TPN-fed piglets. In collaborative studies related to GLP-2 function, we completed a study that showed that feeding a tiny amount of bile acid prevented liver disease and gut atrophy that occurs during PN and liver disease. This work was recently published, showing that treatment of PN-fed piglets with enteral bile acid prevents the development of hepatic cholestasis and steatosis and reversed the mucosal atrophy. We published a review article based on this work this year.
In Objective 2A, the results of our studies, which have identified intracellular mechanisms by which amino acids regulate protein synthesis in neonates, are being utilized to develop new strategies to improve the nutritional management of low birth weight infants. Our studies, using the neonatal pig as an animal model, have shown that the branched-chain amino acid leucine acts as a nutrient signal to stimulate protein synthesis in neonatal muscle and that supplementation with leucine, parenterally or enterally, can enhance protein synthesis. Because an increase in mass requires that more proteins be synthesized than degraded, a nutritional supplement that promotes protein synthesis can lead to an increase in growth. We recently demonstrated that supplementation of milk formula with leucine enhances protein synthesis in skeletal muscle and visceral tissues, such as the liver and intestine, of neonatal pigs. These results suggest that leucine supplementation has the potential to improve the growth of low birth weight infants. Current studies are testing whether supplementation with metabolites of leucine may also promote protein synthesis in skeletal muscle of the neonate.
Studies to address Objective 2B have examined if feeding modality can impact the synthesis, degradation, and deposition of protein in the neonate. Our studies have shown that the intermittent bolus pattern of feeding has a greater stimulatory effect on muscle protein synthesis than continuous feeding of formula in neonates. Studies we recently reported in the journal Pediatric Research have shown that the enhanced rate of protein synthesis occurs in muscles of different fiber types as well as in visceral tissues. This greater increase in protein synthesis with intermittent bolus feeding is due to the rapid rise of amino acids and insulin in the blood after the meal. Both amino acids and insulin stimulate the intracellular signaling pathways that regulate protein synthesis, resulting in an increase in the amount of protein deposited in the body. Because some infants must be fed continuously due to feeding intolerance, we investigated if leucine infusion can be used to enhance protein synthesis during continuous feeding. In studies recently published in the American Journal of Physiology Endocrinology and Metabolism and presented at Experimental Biology meeting, we showed that leucine pulses delivered every 4 hours by parenteral infusion increased muscle protein synthesis in piglets that were continuously fed formula by orogastric tube. However, the rate of protein synthesis in most muscles was not as great as that obtained by intermittent bolus feeding. Nonetheless, the results suggest that leucine pulses in infants and children who need continuous tube feeding may improve protein deposition in muscle.
Studies in Objective 2C are determining whether feeding modality affects growth and lean tissue accretion and are identifying the mechanisms for the response. The results suggest that intermittent feeding enhances lean tissue accretion as compared to continuous feeding, and that this difference is apparent by 9 days and persists for the duration of feeding. The increased rate of lean tissue accretion in pigs fed intermittently occurs in response to an increased activation of translation initiation. In addition, intermittent feeding enhances lean tissue accretion by increasing amino acid transport and protein turnover. These findings have been disseminated in two oral presentations and published abstracts at national meetings.
GLP-2 treatment does not prevent NEC in preterm neonates. Premature infants are at an increased risk for an intestinal disease called necrotizing enterocolitis (NEC) that has devastating effects on the health and long-term development of the infant. Based on previous findings, researchers at the Children's Nutrition Research Center in Houston, Texas, tested whether infusing GLP-2 intravenously immediately after birth could augment intestinal growth and prevent NEC in premature piglets fed infant formula. We found that GLP-2 treatment delayed the onset of NEC but did not prevent the ultimate incidence of the disease, such that approximately 70% of pigs developed NEC. We found that GLP-2 treatment increased intestinal growth as expected, but did not reduce the inflammation that occurs in association with NEC. Our findings suggest that GLP-2 treatment was not effective in the prevention of NEC.
Leucine supplementation enhances protein synthesis in neonates. The growth rate of low birth weights infants is frequently suboptimal, thus better strategies are needed to optimize the nutritional management of these infants whose growth has been compromised. On the basis of studies in cells and rodents demonstrating positive effects of the branched-chain amino acid leucine, scientists at the Children's Nutrition Research Center in Houston, Texas, examined whether leucine may stimulate the synthesis of muscle proteins in the neonate. Because an increase in muscle mass requires that more proteins be synthesized than degraded, a nutritional supplement that promotes protein synthesis can lead to an increase in muscle mass. We found that leucine rapidly stimulates protein synthesis in the muscle of neonatal piglets and that this stimulation can be sustained for at least 24 hours, provided that the supply of other amino acids is not limiting. The response to leucine occurs whether the leucine is provided parenterally by infusion or enterally in the formula. These results have implications for the pediatric nutrition community as they suggest that leucine supplementation has the potential to enhance lean growth in early life.