2008 Annual Report 1a.Objectives (from AD-416) Establish the role of critical nutritional, endocrine and genetic factors on growth, development and function of mammalian cells, tissues and organs. Determine metabolic fate and regulatory role of essential amino acids, especially methionine and cysteine, in intestinal epithelial cells in neonates. Determine the regulatory role of branched-chain amino acids, especially leucine, and glucose on activation of cellular protein synthesis signaling mechanisms in neonates. Determine the molecular function of vitamin A in vascular development and embryonic hematopoiesis and the key signaling molecules involved. Determine how maternal gestational malnutrition affects glucocorticoid status, growth factor expression and satellite cell proliferation in skeletal muscle and whether these factors contribute to postnatal growth impairment of offspring. Determine the impact of glutamine, simple sugars, soluble fiber, and probiotics on intestinal sensory, motor and immune function in infants and children. Understand the role of obestatin and GPR39 gene receptor which will increase our knowledge about critical development of obesity and type II diabetes in human. 1b.Approach (from AD-416) These objectives will be accomplished by quantifying the metabolism of isotopic labeled sulfur amino acids when given enterally and parenterally to neonatal piglets in vivo and in cultured intestinal epithelial cells in vitro. The in vivo rates and cellular localization of sulfur amino acid metabolism via transmethylation into homocysteine, transsulfuration into cysteine and incorporation into glutathione will be quantified. The fractional rates of tissue protein synthesis and the activation and/or protein-protein interaction of nutrient signaling proteins will be determined in tissues from neonatal piglets infused with amino acids and glucose to achieve levels within the fasting to fed range. Modulators of cellular nutrient signaling (rapamycin, LY294002 and AICAR) will be infused to distinguish the specificity of key signaling pathways. Endodermal differentiation and subsequent induction of endothelial cell growth, maturation, and vessel assembly will be characterized in retinoic acid deficient embryos cultured in the presence and absence of endodermally derived signals. The production of hematopoietic cells from mesodermal progenitors and the expression of specific target genes will be measured in normal and mutant cultured embryos in response to retinoic acid sufficiency and deficiency. Protein synthesis, growth factor expression, satellite cell cycle activity, rDNA transcription, and rRNA abundance will be measured in skeletal muscle of offspring from dams subjected to manipulation of nutrition and glucocorticoid status during gestation. Bowel motor and sensory patterns, stool transit time, permeability, and fecal calprotectin will be measured in children randomized and stratified by age to receive in a double blind fashion either fiber psyllium, probiotic, or glucose for four weeks. Children with bowel pain that do not respond to treatment will be placed on a lactose, sorbitol, fructose restricted diet and reassessed for intestinal functional endpoints. Infection rate and duration of hospitalization will be measured in preterm infants fed glutamine-supplemented or placebo formulas. Implement a series of experiments utilizing the mouse model and analysis of the hypothalamus in order to understand GPR39 gene function. 3.Progress Report Researchers completed studies examining the impact of sulfur amino acid deficiency on intestinal growth in neonatal piglets. This work showed that sulfur amino acid deficiency significantly reduces intestinal growth & cell proliferation within one week of treatment. We examined whether dysregulation of gut methionine & homocysteine metabolism contributes to pathologies of gastrointestinal disease, specifically inflammatory bowel disease. Our studies showed that disruption of methionine metabolism using B-vitamin deficient diets protected mice from colitis. We speculate that these protective effects are due to accumulation of the methionine metabolite, methylthioadenosine. Further studies have shown that dietary supplementation with methylthioadenosine can reduced the severity of colitis (Project 1). CNRC researchers examined the individual effect of amino acids & insulin on skeletal muscle protein synthesis & the signaling components leading to mRNA translation in 6- & 26-day-old animal models. Our results show that the stimulation of muscle protein synthesis by insulin & amino acids is due to the activation of the amino acid as well as growth factor signaling pathways & these responses decrease with development (Project 2). We determined the signaling hierarchy that mediates endodermal induction of mesoderm differentiation toward vascular endothelium. We also identified candidate molecules that mediate retinoic acid regulation of embryonic hematopoiesis, & are investigating the function of these in vitro & in vivo (Project 3). We analyzed additional time points following the nutritional rehabilitation of mice pups that had been undernourished from birth to weaning. In addition to evaluating the muscle growth after 2 wks of rehabilitation, measurements were performed after 2, 7, & 140 days of refeeding (Project 4). We spent the past year working with the FDA to obtain an investigational new drug approval. Because of the FDA requirement first to test the probiotic in adults, we are working with them to design that study (Project 5). We created a glp2r-Flox/Flox germ-line mouse. This conditional knockout mouse model will provide a powerful tool to define physiological role & signaling network of GLP-2 receptor in tissue- or cell–specific manner. In addition, we established 13C-octanoic acid breath test to assess gastric emptying in the conscious mouse, & found that central GLP-2 does not alter gastric emptying though it stimulates intestinal cell proliferation. Finally, we established a primary neuron culture model to test cellular action and molecular signaling of GLP-2 receptor, & found that GLP-2R activation stimulates protein synthesis by enhanced activation of the PI3K-Akt-dependent mTOR signaling pathway in primary neurons (Project 6). [NP107, Components 2 & 4] 4.Accomplishments 1. Understanding Key Ingredients for Normal Intestinal Growth: A lack of knowledge exists of how sulfur amino acid deficiency specifically affects the growth of the neonatal intestine. Children's Nutrition Research Center researchers showed that diets deficient in both methionine and cysteine, two important amino acids, resulted in significantly reduced intestinal growth, cell proliferation, and antioxidant function. Researchers accomplished this by feeding newborn animal models a sulfur amino acid-deficient diet for 1 week and quantified the impact on small intestinal morphology and cell proliferation. Our results provide a cellular basis to explain why methionine is essential for normal intestinal mucosal growth. This work will provide the basis for further studies to determine what other normal cell functions require methionine metabolism. [NP107, Component 4] (CNRC Project 1) 2. Improving Nutrition of Low Birth Weight Infants: Our research has shown that a rise in insulin and amino acids after a meal stimulates protein synthesis (the process in which cells build proteins) in skeletal muscle of newborns, however this response decreases with development and it is unclear why this occurs. Children's Nutrition Research Center researchers examined the separate effects of insulin and amino acids on the activation of intracellular signaling components that regulate protein synthesis and how these responses to insulin and amino acids change with development over time. Our results suggest that insulin stimulates skeletal muscle protein synthesis by activating three specific parts of the signaling pathway (protein kinase B, tuberous sclerosis protein 2, and mammalian target of rapamycin); however amino acids stimulate only one of these (mammalian target of rapamycin) parts of the signaling pathway. Both stimulate the activation of the translation initiation factors, the first stage of protein synthesis and we conclude that insulin and amino acid signaling pathways converge at the mammalian target of rapamycin, and that the effect of these signaling components decrease with development. The developmental changes in the activation of the insulin and amino acid signaling pathways in response to the rise in insulin and amino acids after a meal contribute to the high rate of protein synthesis and rapid gain in skeletal muscle mass in newborns. These findings provide valuable information to improve strategies for the nutritional management of low birth weight infants. [NP 107, Component 2 Bioavailability of Nutrients and Food Components] (CNRC Project 2) 3. Regulation of Endothelial Development and Specialization: Children's Nutrition Research Center researchers investigated the signals that regulate endothelial cell development and specialization to blood-forming endothelium, the thin layer of cells that line the interior surface of blood vessels. Through these studies, our lab has determined the signaling hierarchy that mediates endoderm induction of vascular endothelial cell differentiation. We also determined that retinoic acid is needed for the specialization of a subset of endothelial cells to blood forming, or hemogenic, endothelium. These results are significant since insight gained from our developmental studies further our understanding of mammalian blood and blood vessel formation, and will be applicable to discerning underlying causes of human blood and vessel diseases. [NP 107, Component 2] (CNRC Project 3) 4. Generating the glp2r Gene Tissue-Specific Knockout Mouse: Glucagon–like peptide-2 (GLP-2) is a nutrient-responsive gut hormone that plays an important role in the regulation of intestinal growth, function, and microcirculation. To act as a molecular signal, GLP-2 binds to a specific protein, namely GLP-2 receptor (GLP-2R) that is mainly localized to the intestine and brain. Children's Nutrition Research Center scientists wanted to investigate how GLP-2R functions in the body. In order to document its physiological roles at specific tissues, we wanted to generate a genetically modified mouse model. Researchers screened out embryonic stem cells that contained a glp2r-floxed DNA construct, and then microinjected them into blastocytes, and after obtaining three males of 100% chimeras, our lab successfully generated a glp2r-floxed agouti mouse. Tissuespecific glp2r deficient mice (knockouts) will be generated by crossing the glp2rfloxed agouti mouse with tissue-specific Cre transgenic lines. Therefore, this glp2r-floxed/floxed mouse model will provide a powerful tool to define physiological role and signaling network of GLP-2 receptor in a tissue–specific manner. [NP107, Component 4 Nutrient Requirements] (CNRC project 6) 5. Central GLP-2 Regulates Gut Growth and Function: Since the receptor of the Glucagon–like peptide-2(GLP-2), a hormone that regulates intestinal growth, function, and microcirculation, is highly expressed in the hypothalamus, Children's Nutrition Research Center researchers wanted to test if central infusion of GLP-2 will affect gut growth and function. Weaning mice were administrated with either artificial cerebrospinal fluid or human GLP-2. Central infusion of GLP-2 in mice suppressed food intake and increased glucose tolerance. Interestingly, central GLP-2 increased intestinal crypt depth with enhanced cell proliferation without significant change in villous height or enterocyte (intestinal absorptive cell) death. However, central GLP-2 did not affect gastric emptying in the conscious mouse. These findings are important because GLP-2 may be essential for controlling intestinal balance, improving absorption efficiency, and maintaining immune defense; and thus effective in treatment of total parenteral nutrition-induced gut atrophy and morbidity, short bowel syndrome, and inflammatory bowel diseases. [NP107, Component 4 Nutrient Requirements] (CNRC project 6) 6. Cellular Action and Signaling Network of GLP-2 Receptor In Vitro: Little is known about the GLP-2 receptor activation-mediated cellular action and signaling network. To investigate them, Children's Nutrition Research Center researchers have established two systems: [1] GLP-2R-overexpressed transiently in HEK 293 cells, and [2] primary cultured hippocampal neurons. Our lab has found that GLP-2 dose-dependently stimulated protein synthesis through the phosphatidylinositol-3- kinase (PI3K)-dependent Akt-mTOR signaling pathway in HEK 293 cells. Both Akt(protein kinase B) and mTOR (the mammalian target of rapamycin) are key serine/threonine-specific protein kinases that regulate cell proliferation, survival and protein synthesis. In addition, mTOR functions as an intracellular sensor of nutrient and energy availability and redox status. Interestingly, we have found that primary cultured neurons expressed the GLP-2R protein responded to GLP-2 stimulation and displayed a typical saturation curve of specific ligand binding with high binding affinity (Kd= 0.08 nM). Moreover, GLP-2 receptor binds to p85alpha, a regulatory subunit of PI3K in primary neurons. Finally, GLP-2 stimulates protein synthesis via the mTOR signaling pathway in primary hippocampal neurons. This primary neuron culture model will be further employed to establish cellular action and signaling network of GLP-2 receptor in vitro. [NP107, Component 4 Nutrient Requirements] (CNRC project 6) 7. Determining Optimal GI Integrity Measuring: The gastrointestinal (GI) tract provides a critical interface between the body and the environment; and it must allow for the digestion and absorption of dietary constituents and at the same time act as a barrier against potential noxious dietary components. Recent data has begun to suggest that dietary components previously thought to be benign may, in fact, be responsible for GI disturbances in a large population of adults and children, thus it is critical that we learn more about the interaction of diet and the GI tract. One of the issues in evaluating the effect of diet on the gastrointestinal (GI) tract is how best to measure GI integrity (permeability). Children's Nutrition Research Center researchers carried out a study to determine the optimal method to do so and to accomplish this research, scientists evaluated GI permeability in healthy adults and children over a 24 hour period. Our lab found that the test should be analyzed in two parts: an overnight period when the subjects are fasting and then a second period that continues up to 24 hr after the permeability test has started. These findings are important because our method will be used by other investigators world wide and has revealed new variables in testing that must be accounted for. [NP107, Component 6 Prevention of Obesity and Disease: Relationship between Diet, Genetics, and Lifestyle] (CNRC Project 5) 8. Understanding Origins of Pediatric Abdominal Pain: Children often have abdominal pain that may be associated with GI normal function and/or eating, and greater knowledge is need to determine what factors contribute to this problem. Children's Nutrition Research Center researchers showed that children with pain often have evidence of abnormal GI integrity and inflammation. Our lab evaluated GI permeability and inflammation in healthy children with frequent or infrequent abdominal pain. Our observations point to there being a fundamental change in the GI tract of certain children that may predispose them to having more abdominal complaints. Such findings are important for the pediatric community. [NP107,Component 6] (CNRC Project 5) 9. Effects of Undernutrition on Skeletal Muscle Mass Development: Researchers have identified that if animals are undernourished during development, skeletal muscle mass appears to be permanently compromised, which is unexpected because skeletal muscle has a huge regenerative capacity. Children's Nutrition Research Center researchers aimed to identify the cause of the loss in muscle growth potential. When pups were suckled on undernourished mothers they developed a deficit in muscle mass, myonuclear abundance, and satellite cells. Upon refeeding, there was some initial compensatory growth, but it was not sufficient to restore muscle size; thus our researchers concluded that the animals' capacity to increase myonuclear number upon nutritional rehabilitation may be limited by the reduction in the number of satellite cells and an apparent inability to replenish their population with nutritional rehabilitation. This data clearly establishes that alterations in the nutritional environment of the growing organism are a determinant of adult skeletal mass, and this effect appears to be mediated by satellite cells, a population of adult stem cells responsible for the remodeling and regeneration of adult skeletal muscle. These data are consistent with the possibility that the nutritional environment of a growing organism promotes epigenetic changes in muscle stem cells, which then has lifelong consequences for the organism. [NP107, Component 2] (CNRC Project 4) 6.Technology Transfer None Review Publications Janeczko, M.J., Stoll, B., Chang, X., Guan, X., Burrin, D.G. 2007. Extensive gut metabolism limits the intestinal absorption of excessive supplemental dietary glutamate loads in infant pigs. Journal of Nutrition. 137:2384-2390. Takai, K., Bier, D.M., Cynober, L., Morris, S.M., Shimomura, Y. 2007. Aromatic amino acids and related substances: Chemistry, biology, medicine, and application. Journal of Nutrition (Supplement). 137(6S-1):1501S-1598S. Enciso, J.M., Hirschi, K.K. 2007. Nutrient regulation of tumor and vascular endothelial cell proliferation. Current Cancer Drug Targets. 7(5):432-437. Suryawan, A., Orellana, R.A., Nguyen, H.V., Jeyapalan, A.S., Fleming, J.R., Davis, T.A. 2007. Activation by insulin and amino acids of signaling components leading to translation initiation in skeletal muscle of neonatal pigs is developmentally regulated. American Journal of Physiology - Endocrinology and Metabolism. 293(6):E1597-E1605. Wu, G., Bazer, F.W., Davis, T.A., Jaeger, L.A., Johnson, G.A., Kim, S.W., Knabe, D.A., Meininger, C.J., Spencer, T.E., Yin, Y-L. 2007. Important roles for the arginine family of amino acids in swine nutrition and production. Livestock Science. 112(1-2):8-22. Escobar, J., Frank, J.W., Suryawan, A., Nguyen, H.V., Davis, T.A. 2007. Amino acid availability and age affect the leucine stimulation of protein synthesis and eIF4F formation in muscle. American Journal of Physiology-Endocrinology and Metabolism. 293(6):E1615-E1621. Orellana, R.A., Jeyapalan, A., Escobar, J., Frank, J.W., Nguyen, H.V., Suryawan, A., Davis, T.A. 2007. Amino acids augment muscle protein synthesis in neonatal pigs during acute endotoxemia by stimulating mTOR-dependent translation initiation. American Journal of Physiology-Endocrinology and Metabolism. 293(5):E1416-E1425. Shulman, R.J., Phillips, S. 2007. Parenteral nutrition indications, administration, and monitoring. In: Baker, S.S., Baker, R.D., Davis, A. M., editors. Pediatric Nutrition Support. Boston, MA, Jones and Barlett Publishers. p. 273-285. Davis, T.A., Suryawan, A., Orellana, R.A., Nguyen, H.V., Fiorotto, M.L. 2008. Postnatal ontogeny of skeletal muscle protein synthesis in pigs. Journal of Animal Science. 86(14 Suppl):E13-E18. Bergeron, K., Julien, P., Davis, T.A., Myre, A., Thivierge, M.C. 2007. Long-chain n-3 fatty acids enhance neonatal insulin-regulated protein metabolism in piglets by differentially altering muscle lipid composition. Journal of Lipid Research. 48:2396-2410. Wilson, F.A., Orellana, R.A., Suryawan, A., Nguyen, H.V., Jeyapalan, A.S., Frank, J., Davis, T.A. 2008. Stimulation of muscle protein synthesis by somatotropin in pigs is independent of the somatotropin-induced increase in circulating insulin. American Journal of Physiology - Endocrinology and Metabolism. 295(1):E187-E194. McOmber, M.E., Shulman, R.J. 2007. Recurrent abdominal pain and irritable bowel syndrome in children. Current Opinion in Pediatrics. 19(5):581-585. Amaizu, N., Shulman, R.J., Schanler, R.J., Lau, C. 2008. Maturation of oral feeding skills in preterm infants. Acta Paediatrica. 97(1):61-67. Janeczko, M., Burrin, D.G. 2008. Trophic factors and regulation of gastrointestinal tract and liver development. In: Neu, J. editor. Gastroenterology and Nutrition, Neonatology Questions and Controversies. Philadelphia, PA: Elsevier. p. 121-134. Burrin, D.G., Janeczko, M.J., Stoll, B. 2008. Emerging aspects of dietary glutamate metabolism in the developing gut. Asia Pacific Journal of Clinical Nutrition. 17(Suppl. 1):368-371.