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United States Department of Agriculture

Agricultural Research Service

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Location: Children's Nutrition Research Center

2010 Annual Report

1a. Objectives (from AD-416)
It has now been convincingly demonstrated by human epidemiological investigations that the origins of many of the major chronic diseases that are manifest in adult life have their origins during development. Additionally, molecular, cellular and animal studies have now shown conclusively that interference with, or alteration of, developmental pathways during critical windows of development can provide the pathophysiological basis for the events that take place later in adult life. Our research attempts to answer the following: 1) differentiate the effects of fetal versus postnatal maternal dietary protein restriction on satellite cell accretion and skeletal muscle mass; 2) determine if impaired catch-up growth upon nutritional rehabilitation is due to aberrant epigenetic mechanisms intrinsic to the satellite cell and/or an absence of the extracellular cues necessary to sufficiently accelerate satellite cell division; 3) develop novel techniques to study amino acid metabolism in conscious mouse models, with special emphasis on hepatic and enteral metabolism; 4)determine the role of urea cycle intermediates in maintaining nitric oxide and ureagenesis during different physiological and pathophysiological conditions; 5) determine gene expression in human lactating mammary epithelium; 6) characterize inbred mouse strains for lactation performance, gene expression, and weight gain among offspring in lean and obese animals, making use of a cross-fostering design where appropriate; 7) study the effect of nutrients on mammary gland development and function in mouse models; define the critical window for effects on mammary gland development and function; 8) elucidate the role of vitamin A in vascular development and hematopoiesis using mouse embryos, in vitro assays, and complementary techniques; and 9) identify target genes downstream of retinoic acid signaling that are required for blood and blood vessel development.

1b. Approach (from AD-416)
These research studies will use various techniques to accomplish the research to be undertaken. Establishing the critical window of development during which an inadequate nutrient supply permanently compromises the growth of the skeletal muscle and to understand the responsible mechanisms is of great importance. Research studies will focus on the satellite cell in animal models in order to understand how the nutrition of the fetus and infant has lifelong consequences for the health of the individual. Studies will take place on the role of amino acids in the urea cycle (crucial for nitric oxide synthesis), which is in demand for physiological (growth, pregnancy) and pathophysiological (trauma, sepsis) conditions. Lactation research will be conducted in mouse models to obtain an improved understanding of the genomic factors regulating mammary gland function that is central to providing therapeutic interventions that can aid women to establish and maintain a productive lactation. Since the abnormal formation of blood and blood vessels in adults is central to the progression of prevalent pathologies, including atherosclerosis, tumor angiogenesis, and anemia, researchers will develop an understanding of the cellular and molecular regulation of blood and blood vessel formation. Scientists will study retinoids in their role in the formation and maturation of blood vessels, as well as hematopoiesis, during embryonic development. Additionally, CNRC researchers will investigate, on a cellular and molecular level, the role of retinoid signaling in the regulation of these processes.

3. Progress Report
Project 1. In animal experiments, we undernourished pregnant mothers or their pups after birth, and then re-fed the offspring and examined the ability of their muscles to recover from nutritionally induced growth retardation. The analyses performed on the muscles we have collected so far include muscle mass determinations, histology, satellite cell number, total myonuclei, and newly added myonuclei. To complete these analyses we captured 7680 images. We conducted infusion studies in knockout mice to determine the origin of citrulline. Dietary and plasma precursors for synthesis of this non-protein amino acid were established. Our multitracer approach in conscious mice show that dietary arginine is the main precursor for citrulline synthesis and when arginine is absent from the diet, plasma arginine and ornithine are the main precursors. Lack of enteral arginine in Arginase II knockout mice reduced utilization of dietary arginine but increased contribution of dietary glutamine and proline. Oral ornithine supplementation was mostly channeled to citrulline synthesis. Project 2. We recruited 7 normal women and completed milk collection in 6. We completed studies in 2 obese women. There is a large amount of RNA in human colostrum, but isolation of high quality RNA has been challenging. With modifications in our protocol, we substantially improved quality of the RNA. Milk-derived RNA has been isolated, quantitated, and the cDNA and cRNA produced. We measured traits associated with milk production in 32 inbred strains of mice and collected milk, blood, urine, and mammary tissue for future use. We compared maternal behavior, food intake, and body composition and measured maternal ability to mobilize body fat during early lactation. For animal breeding for experiments it was necessary to breed together 3 separate mouse lines into one mouse line to obtain certain transgenes. We have started collecting phenotypic data and samples for further analysis. The current approach is limited by unforeseen deficiencies in the used model. We found that gene expression of the inactivating gene is not developmental-stage-specific, is not exclusively expressed in the mammary gland, is also expressed in some other tissues, and is expressed regardless of administration of inducing substance. We will have to adjust strategy. We plan to use a transplant model. Based on recent published this will better address anticipated effects of deletion of the gene of interest on mammary gland development. Project 3. We determined the role of retinoic acid in regulating endothelial cell migration during blood vessel formation and remodeling, and determined the signaling pathways downstream of retinoic acid that regulate this process. We also isolated and characterized specialized hemogenic endothelial cells from the embryo and are now trying to determine how retinoic acid regulates its formation and function during embryonic development.

4. Accomplishments
1. Impact of malnutrition on muscle growth. Malnutrition early in life can result in a permanent deficit in muscle size; however, we don't know if the muscle response differs depending upon the age when the malnutrition occurs. Children's Nutrition Research Center scientists in Houston, Texas, showed that if malnourished mouse models were re-fed when their muscles were still immature, they were able to return to their normal size, but not if the refeeding was delayed until their muscles were more mature. These results indicate that if the growth of an individual is impaired during early life (primarily during the suckling period) because they are not getting sufficient nutrients, the consequences on their muscles will persist throughout life unless nutritional interventions to reverse the effects are implemented before the muscles become completely mature. The results of these studies have implication for both human health and animal production.

2. Functional genomics of lactation. Researchers at the Children's Nutrition Research Center in Houston, Texas, have established a unique method to isolate and measure mRNA from human milk. mRNA is the material that takes the information from the DNA to different parts of cells to cause specific proteins to be made. We have completed the collection of mRNA from the time of delivery through the first 6 weeks of life of the infant in 6 normal and 2 obese women. With these data we will have established for the first time the post-partum pattern of gene expression in normal lactating women and compared this to women who traditionally struggle with breastfeeding (teenage mothers, mothers with premature infants, and obese mothers).

3. The effect of nutrients on mammary gland development and function. The effect of undernutrition on mammary gland development is not known, and this is important as it impacts the biological processes of the mother and offspring. As part of their research, scientists at the Children's Nutrition Research Center in Houston, Texas, have established that a mouse model system intended to result in mammary-gland-specific inactivation of genes of interests, is not functioning as intended. These findings are important for researchers, as this data implies that this particular mouse model is not suitable for these particular studies. As a result, alternative research methods will need to be identified and used.

4. Impact of Vitamin A on blood vessel development. Researchers are interested in understanding how blood vessels form, and what role specific nutrients play in this process. In these studies, we sought to investigate the signals that regulate the development of the inner layer of blood vessels, called endothelial cells. Researchers at the Children's Nutrition Research Center, Houston, Texas, found that Vitamin A (retinoic acid) not only regulates the growth of endothelial cells, but also regulates their ability to move (migrate) and organize into blood vessel structures. We found that Vitamin A is critically important for blood vessels to form and grow, and we also defined the biological pathway that is regulated by Vitamin A to control endothelial cell behavior. From this work, we gained a better understanding of the cellular role of Vitamin A in the process of blood vessel formation, which was previously unknown. This advances the field of vascular development and provides more insight into human defects resulting from Vitamin A deficiency.

Review Publications
Khan, A.S., Bodles-Brakhop, A.M., Fiorotto, M.L. Draghia-Akli, R. 2010. Effects of maternal plasmid GHRH treatment on offspring growth. Vaccine. 28(8):1905-1910.

Khan, A.S., Draghia-Akli-R., Shypailo, R.J., Ellis, K.I., Mersmann, H., Fiorotto, M.L. 2010. A comparison of the growth responses following intramuscular GHRH plasmid administration versus daily growth hormone injections in young pigs. Molecular Therapy. 18(2):327-333.

Trehan, I., Shulman, R.J., Ou, C., Maleta, K., Manary, M.J. 2009. A randomized, double-blind, placebo-controlled trial of Rifaximin, a nonabsorbable antibiotic, in the treatment of tropical enteropathy. American Journal of Gastroenterology. 104(9):2326-2333.

Thakkar, K., Chen, L., Tatevian, N., Shulman, R.J., McDuffie, A., Tsou, M., Gilger, M.A., El-Serag, H.B. 2009. Diagnostic yield of oesophagogastroduodenoscopy in children with abdominal pain. Alimentary Pharmacology & Therapeutics. 30(6)662-669.

Kress, C., Ballester, M., Devinoy, E., Rijnkels, M. 2010. Epigenetic modifications in 3D: Nuclear organization of the differentiating mammary epithelial cell. Journal of Mammary Gland Biology and Neoplasia. 15:73-83.

Kabotyanski, E.B., Rijnkels, M., Freeman-Zadrowski, C., Buser, A.C., Edwards, D.P., Rosen, J.M. 2009. Lactogenic hormonal induction of long distance interactions between beta-casein gene regulatory elements. Journal of Biological Chemistry. 281:22815-22824.

Lemay, D.G., Lynn, D.J., Martin, W.F., Neville, M.C., Casey, T.M., Rincon, G., Kriventseva, E.V., Barris, W.C., Hinrichs, A.S., Molenaar, A.J., Pollard, K.S., Maqbool, N.J., Singh, K., Murney, R., Zdobnov, E.M., Tellam, R.L., Medrano, J.F., German, J.B., Rijnkels, M. 2009. The bovine lactation genome: Insights into the evolution of mammalian milk. Genome Biology. 10(4):R43.1-R43.18.

Sonabend, R.Y., McKay, S.V., Okcu, M.F., Yan, J., Haymond, M.W., Margolin, J.F. 2008. Hyperglycemia during induction therapy is associated with increased infectious complications in childhood acute lymphocytic leukemia. Pediatric Blood and Cancer. 51(3):387-392

Boles, M.K., Wilkinson, B.M., Maxwell, A., Lai, L., Mills, A.A., Nishijima, I., Salinger, A.P., Moskowitz, I., Hirschi, K.K., Liu, B., Bradley, A., Justice, M.J. 2009. A mouse chromosome 4 balancer ENU-mutagenesis screen isolates eleven lethal lines. Biomed Central (BMC) Genetics. 10:12.

Goldie, L.C., Nix, M.K., Hirschi, K.K. 2008. Embryonic vasculogenesis and hematopoietic specification. Organogenesis. 4(4):257-263.

Hirschi, K.K., Ingram, D.A., Yoder, M.C. 2008. Assessing identity, phenotype, and fate of endothelial progenitor cells. Arteriosclerosis Thrombosis and Vascular Biology. 28(9):1584-1595.

Chao, H., Hirschi, K.K. 2010. Hemato-vascular origins of endothelial progenitor cell? Microvascular Research. 79(3):169-173.

Hirschi, K.K. 2010. Vascular precursors: origin, regulation and function. Arteriosclerosis Thrombosis and Vascular Biology. 30(6):1078-1079.

Iacobas, I., Vats, A., Hirschi, K.K. 2010. Vascular potential of human pluripotent stem cells. Arteriosclerosis Thrombosis and Vascular Biology. 30(6):1110-1117.

Heptulla, R.A., Rodriguez, L.M., Mason, K.J., Haymond, M.W. 2008. Gastric emptying and postprandial glucose excursions in adolescents with type 1 diabetes. Pediatric Diabetes. 9(6):561-566.

Sonabend, R.Y., Mckay, S.V., Okcu, M.F., Yan, J., Haymond, M.W., Margolin, J. 2009. Hyperglycemia during induction therapy is associated with poorer survival in children with acute lymphocytic leukemia. Journal of Pediatrics. 155(1):73-78.

Hadsell, D.L., George, J., Abraham, P.A., Collier, R.J., Lambert, B.D. 2009. Technical note: Assessing the functional capacity of mitochondria isolated from lactating mammary tissue: Choose your chelating agent wisely. Journal of Dairy Science. 92:2038-2045.

Last Modified: 06/25/2017
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