2009 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.
Researchers completed studies examining the impact of sulfur amino acid deficiency on intestinal growth in neonatal piglets, and the results showed that sulfur amino acid deficiency significantly reduces intestinal growth and cell proliferation within 1 wk of treatment. Other studies are examining the methionine cycle activity in human colonic epithelial cell line (HT-29). Preliminary results show that treatment of HT-29 cells with endotoxin or lipopolysaccharide (LPS) induces inflammation as measured by interleukin-8 (IL-8) secretion and that combined treatment with LPS+MTA partially blocks IL-8 secretion. This suggests that MTA suppressed inflammation. Other studies taking place are to examine whether dysregulation of gut methionine metabolism contributes to pathologies of gastrointestinal disease, specifically inflammatory bowel disease (IBD). (Project.
1)Protein synthesis in neonate skeletal muscle increases markedly after a meal due to an enhanced sensitivity to the post-prandial rise in amino acids and insulin. Researchers demonstrated that the activation of a number of components in the amino acid and insulin signaling pathways is enhanced in neonatal skeletal muscle. Our team also demonstrated that leucine, acting through kinase, the mammalian target of rapamycin, is critical to the regulation of protein synthesis and growth, in skeletal muscle of neonates. (Project.
2) Our team sought to define the consequences for skeletal muscle development of fetal exposure to maternal glucocorticoid during the later two-thirds of gestation. A substantial part of the project has been completed, and has revealed some novel insights into the factors that regulate muscle growth during early life. (Project.
3) CNRC researchers have determined the signaling hierarchy that mediates endodermal induction of mesoderm differentiation toward vascular endothelium, and migration of endothelial cells during vascular remodeling. We have also identified candidate molecules that mediate RA regulation of embryonic hematopoiesis, and are investigating the function of these in vitro and in vivo. (Project.
4)The design of the study was changed so that a comparison could be made between fiber and placebo. We continue to work with the FDA to seek approval to use the probiotic. (Project.
5)Researchers have created central POMC neuron-specific glp2r knockout mice. We have found that GLP-2 receptor is essential for maintaining glucose (and energy) homeostasis. This conditional knockout mouse model will provide a powerful tool to define physiological role and signaling network of central GLP-2 receptor in regulation of glucose (and energy homeostasis). In addition, we have used the whole-cell patch clamp technique to explore GLP-2-mediated action and signaling in the hypothalamus, and found that GLP-2 specifically excites POMC neurons by increasing firing rate and membrane depolarization. Finally, we have used a primary neuron culture model to explore cellular action and molecular signaling of GLP-2 receptor, and found that GLP-2 receptor activation potentiates L-type Ca2+ channel activity with stimulated glucose uptake in Hippocampal neurons. (Project 6)
Regulation of Vascular Endothelial Cell Development: Children's Nutrition Research Center scientists are investigating the signals that regulate the development of vascular endothelium, which is a critical cellular component that lines blood vessels. They are attempting to understand how some of these endothelial cells become specialized to make blood cells. CNRC researchers found that the biologically active form of Vitamin A, retinoic acid, is needed for the specialization of a subset of endothelial cells to become blood forming. Although these processes are being studied in mouse models, the researchers are trying to determine whether these findings would apply to the human endothelial cells, in order to be translated into the development of human clinical therapies. Insights gained from these developmental studies not only further our understanding of mammalian blood and blood vessel formation, but can also help to determine underlying causes of human blood and vessel diseases. (CNRC Project 4: Nutrient regulation of blood and blood vessel formation)
Insulin Contributes to Protein Synthesis and Skeletal Muscle Gain: The stimulation of protein synthesis in skeletal muscle after a meal is modulated by the rise in amino acids and insulin, and this response decreases with development. To identify the mechanism by which this occurs, Children's Nutrition Research Center researchers examined the separate effects of consumed levels of insulin and amino acids in young animal models on the activation of components in the amino acid and insulin signaling pathways and how these changed with development. We found that the rise in insulin after a meal stimulates protein synthesis through activation of insulin signaling components that regulate mRNA translation initiation but not elongation. Regulation by amino acids involves the kinase, mammalian target of rapamycin, and eukaryotic initiation factors 4E and 4G, but not tuberous sclerosis 2 or AMP-activated protein kinase. These results demonstrate that the ontogenic changes in the activation of these signaling components contribute to the high rate of protein synthesis and rapid gain in skeletal muscle mass in newborns. (CNRC Project 2: Nutritional regulation of tissue anabolism in neonate)
Beneficial Aspects of the Amino Acid - Leucine: Leucine appears to be unique among the amino acids in its ability to rapidly stimulate the synthesis of skeletal muscle protein. Children's Nutrition Research Center scientists examined the molecular mechanism by which this occurs. Newborn animal models were treated with an inhibitor of the intracellular nutrient signaling pathway, and we examined the amino acid signaling components that regulate protein synthesis. We discovered the molecular target that is used by leucine to stimulate protein synthesis in skeletal muscle of newborn. The importance of the amino acid leucine in the stimulation muscle protein synthesis in newborns was clearly demonstrated, and future studies can now be conducted to gain a further insight. (CNRC Project 2: Nutritional regulation of tissue anabolism in neonate)
Prolonged Leucine Exposure Aids Skeletal Muscle Development: Previous Children's Nutrition Research Center studies showed that the amino acid leucine rapidly increases protein synthesis in skeletal muscle but the response to leucine is not maintained for a long period. We observed that other amino acids in the blood fell as we continued to provide leucine alone. To determine whether leucine could continue to promote protein synthesis when the leucine-induced fall in other amino acids is prevented, CNRC scientists infused newborn animal models for 24 hours with leucine, with and without replacement amino acids. Our team found that prolonged infusion of leucine activates the intracellular nutrient signaling pathway that regulates protein synthesis. However, in order for protein to be synthesized for a prolonged period in newborn muscle, other amino acids must be provided. The results suggest that leucine supplementation may be a potentially useful additive to the nutritional management of infants. (CNRC Project 2: Nutritional regulation of tissue anabolism in neonate)
Understanding the Effects of Stress Hromones on Skeletal Muscle Growth: When a pregnant woman is undernourished or stressed, her baby is exposed to high levels of stress hormones called glucococorticoids which impair the growth of the baby's muscles and overall growth in utero, and ultimately these babies often grow into adults with smaller muscles. Children's Nutrition Research Center researchers conducted a study to determine if the effects of stress hormones and maternal undernutrition on the baby's muscle are different. The study compared how well rat fetuses could make muscle proteins and grow their muscles when their mothers either were allowed to eat only 85% of the food they would normally have eaten, or were given glucocorticoids in addition to eating only 85% of their normal food intake. The muscles of the fetuses from food-restricted mothers were significantly smaller than normal, and those from mothers that also received the stress hormone were twice as small. The measurements suggest that this additive response occurred because an inadequate food intake and stress hormones impair different aspects of muscle growth. This study highlights the detrimental consequences of maternal stress and undernutrition on the offspring and justifies further investigation of its long-term consequences and the evaluation of interventions to remedy the problem. (CNRC Project 4: Consequences of perinatal undernutrition for satellite cell function and skeletal muscle growth)
Factors that Inflame the Intestine: Certain foods cause abdominal pain and inflammation in children and adults. Whether this is related to genetic or environmental factors is unclear. Children's Nutrition Research Center researchers demonstrated that there are both genetic and environmental factors at play. The gastrointestinal integrity of siblings and parents of children with abdominal pain were compared to those of siblings and parents of children without pain. Our results show the importance of studying genes that regulate inflammation in the intestine to understand how they affect the response to food, as well as the need to study the role of intestinal bacteria and how they may affect the response to food. (CNRC Project 5: Nutritional influences on gastrointestinal function in health and disease)
Studying Sugars That Cause Abdominal Pain in Children: Individuals vary as to their ability to digest and absorb various types of sugars. Inefficiency at digestion can lead to significant gastrointestinal discomfort. Children's Nutrition Research Center scientists demonstrated that dietary fructose can be a significant source of abdominal discomfort in children. We evaluated the role of fructose breath testing in the management of children with abdominal pain. Our results stress the need to evaluate the role of other dietary carbohydrates, including simple sugars, in producing adverse gastrointestinal symptoms in individuals and determining why individuals vary in their tolerance to carbohydrates that are not completely digested. (CNRC Project 5: Nutritional influences on gastrointestinal function in health and disease)
Understanding the Foundation of Tropical Enteropathy: Tropical enteropathy is responsible for stunted growth in a large proportion of the world's children. Children's Nutrition Research Center researchers demonstrated that tropical enteropathy is not related to small intestinal bacterial overgrowth. A randomized, double-blind, placebo-controlled trial of rifaximin, a non-absorbable antibiotic, was carried out to evaluate its efficacy in the treatment of tropical enteropathy. The failure of antibiotic treatment to improve tropical enteropathy suggests that nutritional etiologies also must be considered in the fundamental development of this disorder. (CNRC Project 5: Nutritional influences on gastrointestinal function in health and disease)
Monitoring Feeding Skills of Infants: Safe and successful oral feeding in infants requires proper maturation for sucking, swallowing, and respiration; however, how age affects these factors is unclear. Children's Nutrition Research Center scientists demonstrated that the differences in functional stability confirm that maturation levels depend on infants' gestational rather than postmenstrual age. Specific feeding skills were monitored in preterm infants as indirect markers for the maturational process of oral feeding musculatures. Our research results demonstrate that interventions to improve feeding skills in preterm infants must take into account that components of sucking, swallowing, respiration, and their coordinated activity mature at different times and rates based on gestational age. (CNRC Project 5: Nutritional influences on gastrointestinal function in health and disease)
Cellular Action and Signaling Network of GLP-2 Receptor In Vivo: Since GLP-2 receptor is highly expressed in the hypothalamus, Children's Nutrition Research Center reseachers wanted to test if central infusion of GLP-2 will affect energy homeostasis. We found that it suppresses food intake and increases glucose tolerance by enhancing the pro-opiomelanocortin (POMC) neurons and expression of POMC expression at the arcuate nucleus. Moreover, the POMC neuron-specific glp2r knockout mice show decreased basal levels of blood glucose, increased food intake, and impaired energy expenditure. Thus, central GLP-2R activation is essential for maintenance of energy homeostasis. (CNRC Project 6: Physiological role of obestatin receptor in control of energy homeostasis and obesity development)
|Number of Invention Disclosures Submitted||1|
Bertolo, R.F., Burrin, D.G. 2008. Comparative aspects of tissue glutamine and proline metabolism. Journal of Nutrition. 138:2032S-2039S.
Bjornvad, C.R., Thymann, T., Deutz, N.E., Burrin, D.G., Jensen, S.K., Jensen, B.B., Molbak, L., Boye, M., Larsson, L., Schmidt, M., Sangild, P.T. 2008. Enteral feeding induces diet-dependent mucosal dysfunction, bacterial overgrowth and necrotizing enterocolitis in preterm parenterally-fed pigs. American Journal of Physiology Gastrointestinal Liver Physiology. 295:1092-1103.
Davis, T.A., Fiorotto, M.L. 2009. Regulation of muscle growth in neonates. Current Opinion in Clinical Nutrition and Metabolic Care. 12(1):78-85.
Thivierge, M.C., Bush, J.A., Suryawan, A., Nguyen, H.V., Orellana, R.A., Burrin, D.G., Jahoor, F., Davis, T.A. 2008. Positive net movements of amino acids in the hindlimb after overnight food deprivation contribute to sustaining the elevated anabolism of neonatal pigs. Journal of Applied Physiology. 105(6):1959-1966.
Wilson, F.A., Suryawan, A., Orellana, R.A., Nguyen, H.V., Jeyapalan, A.S., Gazzaneo, M.C., Davis, T.A. 2008. Fed levels of amino acids are required for the somatotropin-induced increase in muscle protein synthesis. American Journal of Physiology: Endocrinology and Metabolism. 295(4):E876-E883.
Suryawan, A., Jeyapalan, A.S., Orellana, R.A., Wilson, F.A., Nguyen, H.V., Davis, T.A. 2008. Leucine stimulates protein synthesis in skeletal muscle of neonatal pigs by enhancing mTORC1 activation. American Journal of Physiology: Endocrinology and Metabolism. 295(4):E868-E875.
Davis, T.A. 2008. Insulin and amino acids are critical regulators of neonatal muscle growth. Nutrition Today. 43(4):143-149.
Kellermayer, R., Tatevian, N., Klish, W., Shulman, R.J. 2008. Steroid responsive eosinophilic gastric outlet obstruction in a child. World Journal of Gastroenterology. 14(14):2270-2271.