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United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR, CELLULAR, AND REGULATORY ASPECTS OF NUTRITIONAL METABOLISM DURING CHILDHOOD DEVELOPMENT
2011 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
Proj. 1 The effects of fetal versus postnatal undernutrition on adult muscle mass were compared, and we showed that when pregnant mouse dams were protein-restricted, the muscle masses and body composition of their offspring at weaning and as adults were the same as for offspring of well-nourished pregnant dams. We established that when mice malnourished during the suckling period were allowed to recover by eating a complete diet ad libitum from weaning, their muscles were still smaller 4 and 18 mo later. We have refined surgical procedures for feeding adult mice intravenously for 3 days, with minimum weight loss and have begun to collect data on citrulline production in intravenously fed mice. We conducted studies to determine arginine utilization by the gut and liver in control and arginase II knockout mice. Proj. 2 We completed the collection/analysis of milk samples obtained from normal mothers from delivery through 6 weeks post partum and have carried out the initial determination of the messenger RNA in the milk but have only begun to do the more complex job of interpreting massive amounts of data. We analyzed milk and mammary tissue samples collected from 32 inbred mice strains during the first 10 days of lactation. This data is being used to map the locations of genes that could cause variations in milk production. We used an alternative cell-culture-based method to inactivate genes of interest. Proj. 3 We determined the characteristics of endothelial cells within the aorta that can make blood during embryonic development. We demonstrated their blood-forming ability in vitro and within embryos using real time imaging of transgenic mice. To establish the hedgehog (Hh) pathway as an important regulator of adipose tissue formation, we examined the gene expression levels and cellular localization of the Hh pathway components in several previously published preadipocyte cell lines. We found that the Hh pathway plays an inhibitory role in adipocyte maturation.


4.Accomplishments
1. Identifying genes to improve breastfeeding rates for premature infants. It is well established that breastfeeding an infant has significant health benefits for both the infant and the mother and are related to the duration of breastfeeding. However women who deliver premature infants, and teenage or obese mothers have a high rate of lactation failure, and it is important to understand the molecular events that potentially lead to these failures. Researchers at the Children's Nutrition Research Center completed the analysis of the induction of gene expression from delivery through the first 6 weeks post partum in normal women. We were able to determine that a number of the genes normally expressed over the first 96 hour after birth in normal healthy women are not normally expressed in women with premature infants, or obese and/or teenage mothers, and we now have a genetic target that would lead us to attempt to determine the molecular and cellular mechanisms that might be suppressing or factors that might turn on these genes. These results may lead to an increase in the success of breastfeeding in all women, thus increasing the health and nutrition of their infants and decreasing the health risks of women who might otherwise have stopped breastfeeding their infants.

2. Effects of inadequate maternal protein intake on skeletal muscle growth of offspring. An inadequate intake of nutrients during early development can have long-term effects on the health of the individual. These effects depend on the individual tissue/organ and on the stage of development when the nutrients are limiting; for instance, skeletal muscle is especially vulnerable to an inadequate supply of amino acids. Scientists at the Children's Nutrition Research Center in Houston, TX, conducted a study that showed if maternal protein intake is insufficient during pregnancy, the offspring can develop a normal skeletal muscle mass if they are provided with sufficient nutrients at birth. However, if the offspring are malnourished from birth until they are weaned, and are then provided with a complete diet containing ample amounts of all nutrients, the muscle growth rate attained is insufficient to support complete recovery, and their muscle mass is permanently compromised. These studies emphasize the importance of providing appropriate and adequate amino acids as protein to newborns for skeletal muscle development, and especially in those cases where the mother has been malnourished during pregnancy.

3. Mapping genomic regions associated with variation in lactation performance in the mouse. Genetic variation underlying milk production for lactation is not well defined. Children's Nutrition Research Center scientists identified 25 regions within the mouse genome that influence various levels of lactation performance. The regions contained 44 genes that could play an important role in determining milk production. Such findings are important as researchers discover the genetic background for what influences milk production levels.

4. Characterization of hemogenic endothelial cells within the embryonic aorta (heart). Children's Nutrition Research Center researchers previously knew that blood was derived from aortic endothelial cells (a thin layer of cells that lines the interior surface of blood vessels) during development, but did not know the specific characteristics of these cells. These scientists, based in Houston, TX, discovered the cells' physical characteristics, and demonstrated their function in vitro and in vivo on a single cell level. These studies will allow researchers to prospectively isolate such cells and/or derive them from human stem cells so that we can use them to generate blood cells in vitro for hematopoietic (blood making) cell therapy. These findings provide possible technologies to address health concerns in the future.


Review Publications
Marini, J.C., Didelija, I.C., Castillo, L., Lee, B. 2010. Plasma arginine and ornithine are the main citrulline precursors in mice infused with arginine-free diets. Journal of Nutrition. 140(8):1432-1437.

Rijnkels, M., Kabotyanski, E., Montazer-Torbati, M.B., Beauvis, C.H., Vassetzky, Y., Rosen, J.M., Devinoy, E. 2010. The epigenetic landscape of mammary gland development and functional differentiation. Journal of Mammary Gland Biology and Neoplasia. 15:85-100.

Devinoy, E., Rijnkels, M. 2010. Epigenetics in mammary gland biology and cancer. Journal of Mammary Gland Biology and Neoplasia. 15:1-4.

Marini, J.C., Didelija, I.C., Castillo, L., Lee, B. 2010. Glutamine: Precursor or nitrogen donor for citrulline synthesis?. American Journal of Physiology - Endocrinology and Metabolism. 299(1):69-79.

The TODAY Study Group, Haymond, M., Cuttler, L., Geffner, M., Katz, L. 2010. Design of a family-based lifestyle intervention for youth with type 2 diabetes: the TODAY study. International Journal of Obesity. 34:217-226.

Boles, M.K., Wilkinson, B.M., Wilming, L.G., Liu, B., Probst, F.J., Harrow, J., Grafham, D., Hentges, K.E., Woodward, L.P., Maxwell, A., Mitchell, K., Risley, M.D., Johnson, R., Hirschi, K., Lupski, J.R., Funato, Y., Miki, H., Marin-Garcia, P., Matthews, L., Coffey, A.J., Parker, A., Hubbard, T.J., Rogers, J., Bradley, A., Adams, D.J., Justice, M.J. 2009. Discovery of candidate disease genes in ENU-induced mouse mutants by large-scale sequencing, including a splice-site mutation in nucleoredoxin. PLoS Genetics. 5(12):e1000759.

Mohammad, M.A., Sunehag, A.L., Haymond, M.W. 2009. Effect of dietary macronutrient composition under moderate hypocaloric intake on maternal adaptation during lactation. American Journal of Clinical Nutrition. 89(6):1821-1827.

Lemay, D.G., Rijnkels, M., German, J.B. 2009. Lessons from the bovine genome: Implications for human nutrition and research. Journal of Nutrition. 139:1271-1271.

Heptulla, R.A., Rodriguez, L.M., Mason, K.J., Haymond, M.W. 2009. Twenty-four-hour simultaneous subcutaneous basal-bolus administration of insulin and amylin in adolescents with type 1 diabetes decreases postprandial hyperglycemia. Journal of Clinical Endocrinology and Metabolism. 94(5):1608-1611.

Thame, M.M., Fletcher, H.M., Baker, T.M., Marini, J.C., Kao, C.C., Jahoor, F. 2010. Arginine flux, but not nitric oxide synthesis, decreases in adolescent girls compared with adult women during pregnancy. Journal of Nutrition. 141:71-74.

Ma, X., Lin, L., Qin, G., Lu, X., Fiorotto, M.L., Dixit, V.D., Sun, Y. 2011. Ablations of ghrelin and ghrelin receptor exhibit differential metabolic phenotypes and thermogenic capacity during aging. PLoS One. 6(1),e16391:1-10.

Hadsell, D.L., Olea, W., Wei, J., Fiorotto, M.L., Matsunami, R.K., Engler, D.A., Collier, R.J. 2011. Developmental regulation of mitochondrial biogenesis and function in the mouse mammary gland during a prolonged lactation cycle. Physiological Genomics. 43(6):271-285.

Levy-Marchal, C., Arslanian, S., Cutfield, W., Sinaiko, A., Druet, C., et al. 2010. Insulin resistance in children: consensus, perspective, and future directions. Journal of Clinical Endocrinology and Metabolism. 95:5189-5198.

Marini, J.C. 2011. Quantitative analysis of 15N labeled positional isomers of glutamine and citrulline via electrospray ionization tandem mass spectrometry of their dansyl derivatives. Rapid Communications in Mass Spectrometry. 25(9):1291-1296.

Sen, P., Choudhury, T., Smith, E., Langston, C. 2010. Expression of angiogenic and vasculogenic proteins in the lung in alveolar capillary dysplasia/misalignment of pulmonary veins: an immunohistochemical study. Pediatric and Developmental Pathology. 13:354-361.

Ying, A.K., Lairson, D.R., Giardino, A.P., Bondy, M.L., Zaheer, I., Haymond, M.W., Heptulla, R.A. 2011. Predictors of direct cost of diabetes care in pediatric patients with type 1 diabetes. Pediatric Diabetes. 12:177-182.

Marini, J.C., Lanpher, B.C., Scaglia, F., O'Brien, W.E., Sun, Q., Garlick, P.J., Jahoor, F., Lee, B. 2011. Phenylbutyrate improves nitrogen disposal via alternative pathway without eliciting an increase in protein breakdown and catabolism in control and ornithine transcarbamylace-deficient patients patients. American Journal of Clinical Nutrition. 93(6):1248-1254.

Marini, J.C., Keller, B., Didelija, I.C., Castillo, L., Lee, B. 2011. Enteral arginase II provides ornithine for citrulline synthesis. American Journal of Physiology - Endocrinology and Metabolism. 300:E188-E194.

Ross, J., Czernichow, P., Biller, B.M., Colao, A., Reiter, E., Kiess, W., and the participants in the advisory panel meeting on the effects of growth hormone. 2010. Growth hormone: health considerations beyond height gain. Pediatrics. 125:e906-e919.

The Pediatric Diabetes Consortium 2010. The Pediatric Diabetes Consortium: Improving care of children with Type 1 diabetes through collaborative research. Diabetes Technology & Theraputics. 12(9):685-688.

Maningat, P.D., Sen, P., Rijnkels, M., Hadsell, D.L., Bray, M.S., Haymond, M.W. 2011. Short-term administration of rhGH increases markers of cellular proliferation, but not milk protein gene expression in normal lactating women. Physiological Genomics. 43:381-391.

Murgas-Torrazza, R., Suryawan, A., Gazzaneo, M.C., Orellana, R.A., Frank, J.W., Nguyen, H.V., Fiorotto, M.L., El-Kadi, S.W., Davis, T.A. 2010. Leucine supplementation of a low-protein meal increases skeletal muscle and visceral tissue protein synthesis in neonatal pigs by stimulating mTOR-dependent translation initiation. Journal of Nutrition. 140(12):2145-2152.

Suryawan, A., Orellana, R.A., Fiorotto, M.L., Davis, T.A. 2010. Triennial growth symposium: Leucine acts as a nutrient signal to stimulate protein synthesis in neonatal pigs. Journal of Animal Science. 89(7):2004-2016.

Kelly, M.A., Hirschi, K.K. 2009. Signaling hierarchy regulating human endothelial cell development. Arteriosclerosis Thrombosis and Vascular Biology. 29(5):718-724.

Last Modified: 10/20/2014
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