2007 Annual Report 1a.Objectives (from AD-416) The objective of this cooperative research is to establish the role of critical nutritional, endocrine and genetic factors on growth, development and function of mammalian cells, tissues, and organs. 1) Intestinal Amino Acid Requirements in Neonates - Determine metabolic fate and regulatory role of essential amino acids, especially methionine and cysteine, in intestinal epithelial cells in neonates. 2) Nutritional Regulation of Tissue Anabolism 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. 3) Nutrient Regulation of Blood and Blood Vessel Formation - Determine the molecular function of vitamin A in vascular development and embryonic hematopoiesis and the key signaling molecules involved. 4) Consequences of Perinatal Undernutrition for Satellite Cell Function and Skeletal Muscle Growth - 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. 5) Nutritional Influence on Gastrointestinal Function - Determine the impact of glutamine, simple sugars, soluble fiber, and probiotics on intestinal sensory, motor and immune function in infants and children. 6) Physiological Role of Obestatin Receptor in Control of Energy Homeostasis and Obesity Development - 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) 1) Intestinal Amino Acid Requirements in Neonates - Quantify 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. 2) Nutritional Regulation of Tissue Anabolism in Neonates - 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. 3) Nutrient Regulation of Blood and Blood Vessel Formation - 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. 4) Consequences of Perinatal Undernutrition for Satellite Cell Function and Skeletal Muscle Growth - 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. 5) Nutritional Influence on Gastrointestinal Function - 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. 6) Physiological Role of Obestatin Receptor in Control of Energy Homeostasis and Obesity Development - Implement a series of experiments utilizing the mouse model and analysis of the hypothalamus in order to understand GPR39 gene function. 3.Progress Report The ADODR monitors activities for the project through communication with the Center Director, consulation with the On-Site Representative, review of cooperator reports, conference calls, and site visits. 4.Accomplishments The Amino Acid Homocysteine is produced in the Infant Gut: Homocysteine is considered to be a significant risk factor for cardiovascular disease and stroke. Children's Nutrition Research Center researchers demonstrated that the infant gut is an important site of homocysteine production and remethylation. Using highly sensitive isotopic tracers of 13C-methionine in an in vivo animal model of the human infant, our lab showed that the gut is a key site of methionine metabolism to homocysteine. CNRC researchers showed that the gut releases homocysteine into the blood and thus may contribute to the development of homocysteinemia, a disorder due to elevated homocysteine levels. Our lab will continue to examine whether dysregulation of gut methionine and homocysteine metabolism contributes to the development of gastrointestinal disease, specifically inflammatory bowel disease. [NP107, Component 4 Nutrient Requirements] (CNRC Project 1) Understanding the Role of Retinoic Acid: Greater insight is needed on our understanding of human blood and vascular development, and the role of specific nutrients such as Vitamin A in the regulation of these processes. Children's Nutrition Research Center researchers determined that retinoic acid is needed to specify a subset of endothelial cells to become blood-forming, or hemogenic, endothelium. These findings were learned from cellular and molecular studies in the mouse embryonic model system. This is important since we now know more about the role of Vitamin A during the development of blood vessels and blood. [NP 107, Component 2 Bioavailability of Nutrients and Food Components] (CNRC Project 3) Glucose Stimulates Protein Synthesis in Skeletal Muscle through an AMPK and mTOR Independent Process: Children's Nutrition Research Center researchers have demonstrated that skeletal muscle protein synthesis is increased in newborns, partially due to an increased response to insulin and amino acids upon eating. Our lab examined the role of glucose in the rise in skeletal muscle protein synthesis that occurs after eating meals (postprandial). The results showed that the postprandial rise in glucose, independent of insulin and amino acids, stimulates protein synthesis in newborns and this response occurs by AMPK (activated protein kinase) and mTOR (mammalian target of rapamycin) independent pathways. These results demonstrate that multiple nutrient factors impact protein synthesis, and thus, growth in young animals. This research emphasizes the importance of a balanced carbohydrate and protein containing diet in the growth of skeletal muscle in the newborn child, and provides valuable information to enhance strategies for the nutritional management of low birth weight infants. [NP 107, Component 2 Bioavailability of Nutrients and Food Components] (CNRC Project 2) Inadequate Satellite Cell Replication Compromises Muscle Regrowth Following Postnatal Nutrient Restriction: When muscle growth is reduced by nutritional deficiencies during the suckling period, resuming feeding of that animal accelerates the rate of muscle growth but not sufficiently to restore the animal's muscle mass. This impediment to muscle growth is likely due to a limited increase in myonuclear number as a result of the limited capacity of the existing satellite cells to multiply. At the Children's Nutrition Research Center researcher’s in Houston, TX, mouse dams were fed a low-protein diet during lactation, resulting in pups with muscles weighing 80% of the control's muscle weight. When the low-protein diet fed mice were fed a normal diet, muscle mass gain accelerated to age appropriate levels, however remained proportionately smaller than the control's muscles. This research is important since it addresses the potential mechanism that in mammals, birth weight and early newborn growth rates are significant determinants of adult muscle mass. [NP107, Component 2 Bioavailability of Nutrients and Food Components] (CNRC Project 4) Researchers Identify Leads to Fatty Liver: Children's Nutrition Research Center researchers showed that chronic (parenteral) intravenous nutrition leads to hepatic steatosis or fatty liver, thus this finding suggests that fatty liver may contribute to liver dysfunction and metabolic problems associated with intravenous feedings in premature infants. Utilizing the newborn piglet as a model, animals were fed exclusively intravenously through a vascular catheter for 7 days and compared to those fed orally with a milk-based formula. Our lab discovered that intravenous nutrition resulted in a higher content of fat in liver tissue and that this was associated with increased death of liver cells. These studies show that a relatively short period of intravenous nutrition can lead to fatty liver and perhaps dysfunction, and further studies will be pursued to specifically assess how fatty liver might contribute to poor liver function in this population of infants. We hope to use these research findings to design nutritional approaches to reduce fatty liver and maintain healthy liver function. [NP107, Component 4 Nutrient Requirements] (CNRC Project 1) Generating GLP-2 Receptor Conditional Knockout Mouse Model: Children's Nutrition Research Center researchers have completed the full construct for generating glucagon-like peptide 2 (GLP-2) receptor conditional knockout mice. This construct was generated through utilization of the Bacterial Artificial Chromosomes (BAC) recombineering approach and electroporated into the mouse embryonic cells. After screening, the embryonic stem cells were microinjected into blastocytes to obtain the glp2r conditional knockout mice. These knockout mice will be used by researchers to establish physiological significance and signaling network of GLP-2 receptor in controlling gut development, growth, and function. [NP107, Component 4 Nutrient Requirements] (CNRC Project 6) Establishment of Primary Neuron Culture Model: Children's Nutrition Research Center researchers have developed a primary culture of central and peripheral neurons that play a significant role in the control of intestinal function. Our lab has found that the GLP-2R protein was localized to the spinal ganglion neurons and hippocampal (brain) neurons, and the primary cultured neurons respond to the exogenous GLP-2. Development of this primary culture model is important since it can be used to address at cellular and molecular levels how the GLP-2 receptor functions, thus furthering science. [NP107, Component 4 Nutrient Requirements] (CNRC Project 6)