2007 Annual Report 1a.Objectives (from AD-416) 1) Develop a greater understanding for the enhanced development and host protection that is observed in the breast-fed versus formulae-fed infants. . 2)Gain an understanding of the mechanisms of insulin/IGF-I actions in the lactating mammary gland.. 3)Determine if there is a finite period during which the human neonate maximally utilizes protein intake for growth, to define this period and to determine if a higher protein intake during this period improves early growth without unacceptable metabolic consequences and reduces subsequent growth and neurodevelopmental deficits.. 4)Provide a better understanding of how maternal obesity and undernutrition alter the metabolic/physiologic adaptations necessary for a successful pregnancy and thereby will test nutritional therapies aimed at reducing or correcting their adverse outcomes.. 5)Poor complementary feeding practices and zinc deficiency are two of the most important nutritional problems for young children worldwide. The consequences are retarded physical and intellectual development, as well in increased child morbidity and mortality. The objective is to develop and test novel strategies that have the potential to solve these problems in a population of high-risk children.. 6)Understand the regulation of the casein genes and the genomic domain in which these genes reside, since Caseins (CSN) constitute the major nutritional proteins in milk and supply basic amino acids, calcium, phosphates and bioactive peptides (e.g. anti-microbial and opioid).. 7)Understand amino acid metabolism and requirements for nutritional and functional balance under conditions of health and disease in children. 1b.Approach (from AD-416) 1) Use generated LF knockout mice (LFKO) and two novel genetic mouse models that direct overexpression of LF to the small or large intestine, respectively, using intestinal-specific promoters. These unique gain-of-function transgenic mice will be used to investigate the effects of LF on iron homeostasis and host protection in the intestine of post weaning and adult mice thus eliminating any confounding factors in milk that may mask the functional properties(s) of LF during the suckling period.. 2)Use mice that carry targeted inactivating germline mutations in the genes for IRS-1 or IRS-2 in combination with mammary tissue transplantation and primary cell culture approaches to determine the importance of activation of these signaling proteins in mammary cells to milk synthesis and/or mammary cell survival. . 3)By testing if there is a finite period during early life when the infant maximally utilizes protein for growth and that failure to provide sufficient protein during this period results not only in short-and long-term growth deficits but also suboptimal neurodevelopment and by defining the period of maximum protein utilization for growth and the impact of size for gestational age as well as formula-feeding vs. breast-feeding on this period will be defined.. 4)Perform a series of experiments of obese pregnant women and underweight teenagers in Houston and in underweight and normal weight adult women in India to test a series of hypotheses.. 5)Develop a micronutrient-rich, energy-dense ready-to-use food and compare it to a fish-fortified porridge as a complementary food in 6-18 month old children. The quantity and quality of consumed breast milk will be measured and children will be followed longitudinally. The role of asymptomatic intestinal malabsorption in zinc homeostasis will be investigated in 3-5 year old children, using site specific gastrointestinal sugar absorption tests and zinc stable isotope techniques. . 6)Analyze the chromatin structure and by analysis of transgenic mice harboring large BAC based transgenes with deletions or mutations of evolutionary conserved regions.. 7)Methionine requirements that maintain nutritional balance and glutathione synthesis rates in health adolescent children will be studied through using intravenous indicator amino acid oxidation and balance technique. 4.Accomplishments Nitric Oxide Production: Greater in Underweight versus Normal Weight Pregnant Women: An adaptation necessary for a successful pregnancy is the expansion of the vascular compartment; a failure to appropriately expand blood volume results in hypertension, with underweight women having a low BMI (body mass index) being more likely to have a greater incidence of hypertension during pregnancy. Our previous work showed that pregnant women produced more nitric oxide (NO), which we believe is involved in the blood volume expansion of pregnancy. In this study Children's Nutrition Research Center researchers tested the hypothesis that underweight women, because of their lower body protein mass, may not be able to produce sufficient quantities of the amino acid arginine (the precursor from which NO is synthesized), causing lower NO production. Our team discovered that in the first trimester underweight women produced more arginine than normal weight women, and this was associated with a higher NO production in both the first and second trimesters. These findings suggest that compared to normal weight women, underweight women need to elicit a greater increase in arginine and NO production in order initiate and maintain blood volume expansion during pregnancy. [NP 107, Component 2 Bioavailability of Nutrients and Food Components] (CNRC Project 4) Association between Maternal Gluconeogenesis and Infant Birthweight: Normal growth of the human fetus to result in a normal weight infant at birth is dependent upon, among other things, an adequate glucose supply by the pregnant mother. During pregnancy, increased glucose production is maintained by increased gluconeogenesis. Researchers at the Children's Nutrition Research Center, Houston, TX, tested the hypothesis that underweight women with low BMI (body mass index) have a higher incidence of low birthweight (LBW) babies because of an inability to increase gluconeogenesis. Our team discovered that glucose production and oxidation were significantly higher in the low BMI group during the first trimester, but there were no significant differences in gluconeogenesis; when maternal weight was used as a covariate, these differences disappeared. When the results were analyzed by grouping mothers into those who gave birth to LBW infants and normal birthweight (NBW) infants, LBW mothers had a significantly slower rate of gluconeogenesis. These findings suggest that fetal growth is dependent upon maternal capacity to synthesize glucose. [NP 107, Component 4 Nutrient Requirements] (CNRC Project 4) Improved Complementary Feeding in the Developing World: Children's Nutrition Research Center scientists compared the effect of using a peanut/soy-based fortified spread (FS) and a corn porridge fortified with fish powder (FP) as complementary foods on growth in rural Malawian children. Our team enrolled 240 children at the age of 6 months and randomized them to receive FS or FP. Children were followed monthly for physical measurements and every two weeks for symptoms of fever, cough, or diarrhea until they were 18 months old; and zinc and selenium status were assessed at 6 and 12 months of age. We discovered that children who received FS gained 110 g more from 6 to 12 months of age than children receiving FP; there was no significant difference in weight gain between children receiving FS and FP between 12 and 18 months of age, nor in statural growth from 6 to 12 months or 12 to 18 months. Twenty-three percent of all children were zinc deficient at 6 months of age, and this increased to 37% at 12 months; neither FS nor FP was associated with improved zinc status. Ultimately we learned that the FS was associated with better weight gain from 6 to 12 mo of age. This may be useful in conjunction with additional interventions to improve infant growth in the developing world. [NP 107, Component 5 Health Promoting Properties of Plant and Animal Foods] (CNRC Project 5) Nutritional Methionine Requirements in Healthy Adolescents: Children's Nutrition Research Center researchers defined the amino acid methionine requirement for nutritional balance in healthy adolescents using the indicator amino acid oxidation and balance technique. Methionine requirements for nutritional balance had not been determined in the healthy adolescent population. Our lab determined that methionine balance is obtained at intakes of 23 mg.kg-1.d-1. Safe level of intake is found at 25 mg.kg-1.d-1. This finding is important since the factorial method, which has been considered the standard for dietary methionine recommendations, underestimates methionine requirements in healthy adolescents. [NP 107, Component 4 Nutrient Requirements] (CNRC Project 7) Gene Mammary Expression during the Lactation Cycle: The need exists to identify genes that play a role in regulating the capacity of the mammary gland to produce milk during lactation. Researchers at the Children's Nutrition Research Center successfully identified 1,056 genes expressed within the lactating mammary gland that play a potential role in determining the capacity to produce milk during lactation. Our lab collected mammary tissue samples from lactating mice and conducted gene expression microarray analysis on RNA (Ribonucleic acid) prepared from these samples. This finding is important since genes identified by this study may serve as targets in future strategies to enhance milk production during lactation. [NP 107, Component 6 Prevention of Obesity and Disease: Relationship between Diet, Genetics, and Lifestyle] (CNRC Project 2) Progressive Changes in Packaging of DNA During Mammary Gland Development: Although the way DNA is packaged in the cell (chromatin) plays an important role in gene regulation, there is limited knowledge of its role in the regulation of casein genes. Children's Nutrition Research Center researchers have demonstrated a progressive change in the chromatin in mammary cells during development that results in an open chromatin structure at the proximal promoters and distal regulatory elements when the casein genes are fully active. Our lab analyzed several markers for the chromatin conformation during post-natal mammary gland development, and our findings fit with a model in which the initial changes in the chromatin enable access of signaling molecules to fully activate the casein genes. These studies have enabled us to identify potentially new regulatory elements in the casein gene cluster, and they provide insight into the mechanisms regulating milk composition. Furthermore, they improve our understanding of normal lactation and aid in improving the composition of synthetic formulas, milk production in livestock, and the production of biologically important proteins in the milk of transgenic animals. [NP 107, Component 5 Health Promoting Properties of Plant and Animal Foods] (CNRC Project 6) Evaluation of a Non-invasive Method of Studying Protein Turnover: To minimize the invasiveness of protein turnover (the balance between the creation of proteins and protein degradation) studies, particularly in small infants, Children's Nutrition Research Center researchers investigated other means of administration. A less invasive method in which enteral versus intravenous administration of the tracer amino acid and measurement of urinary versus plasma enrichment of the tracer amino acid was conducted. To do so, our lab administered a leucine tracer orally as well as intravenously and measured the enrichment of the tracer in both plasma and urine. Our team has concluded that it is possible to use the less invasive method of determining protein turnover, provided a tracer free of the D-enantiomer is used or the urinary L-enantiomer is specifically measured. This is significant since it may permit more extensive studies of protein turnover in vulnerable populations such as low-birth-weight infants. [NP 107, Component 6 Prevention of Obesity and Disease: Relationship between Diet, Genetics, and Lifestyle] (CNRC Project 3) Role of Lactoferrin in Host Defense: Children's Nutrition Research Center researchers have carried out a very thorough, comprehensive study of the effects of lactoferrin ablation on white blood cell function. Lactoferrin is important, since it is present at high levels in human milk and in the milk of other mammals, and serves as the first line of defense in protection against pathogens. Using lactoferrin knock-out mice has allowed our lab, for the first time, to establish the essential, non-redundant function of lactoferrin in the white blood cell. This data has revealed the specificity of lactoferrin function in cell biology. More importantly we were able to demonstrate an unexpected, essential role for lactoferrin in the optimal oxidative burst response of the white blood cell in response to stimulation. [NP 107, Component 6 Prevention of Obesity and Disease: Relationship between Diet, Genetics, and Lifestyle] (CNRC Project 1) 6.Technology Transfer Number of non-peer reviewed presentations and proceedings 10 Review Publications Hadsell, D., George, J., Torres, D. 2007. The declining phase of lactation: Peripheral or central, programmed or pathological? Journal of Mammary Gland Biology and Neoplasia. 12:59-70. Dearth, R.K., Cui, X., Kim, H., Kuiatse, I., Lawrence, N.A., Zhang, X., Divisova, J., Britton, O.L., Mohsin, S., Allred, D.C., Hadsell, D.L., Lee, A.V. 2006. Mammary tumorigenesis and metastasis caused by overexpression of insulin receptor substrate 1 (IRS-1) or IRS-2. Molecular and Cellular Biology. 26:9302-9314. Hadsell, D.L., Olea, W., Lawrence, N., George, J., Torres, D., Kadowaki, T., Lee, A.V. 2007. Decreased lactation capacity and altered milk composition in insulin receptor substrate null mice is associated with decreased maternal body mass and reduced insulin-dependent phosphorylation of mammary Akt. Journal of Endocrinology. 194:327-336. Dearth, R.K., Cui, X., Kim, H., Hadsell, D.L., Lee, A.V. 2007. Oncogenic transformation by the signaling adaptor proteins insulin receptor substrate (IRS)-1 and IRS-2. Cell Cycle. 6(6):705-713. Heird, W.C., Kashyap, S. 2006. Protein and amino acid metabolism and requirements. In: Polin, R.A., Fox, W.W., Abman, S.H., editors. Fetal and Neonatal Physiology. 3rd edition. Philadelphia, PA: W.B. Sanders. p. 527-539. Heird, W.C. 2006. Intravenous feeding. In: Thureen, P., Hay, W., editors. Neonatal Nutrition and Metabolism. 2nd Edition. NY: Cambridge University Press. p. 312-331. Heird, W.C. 2006. The role of essential fatty acids in development. In: Thureen, P., Hay, W., editors. Neonatal Nutrition and Metabolism. 2nd edition. New York, NY: Cambridge University Press. p. 147-160. Heird, W.C. 2006. Nutrient requirements of term and preterm infants. In: Burg, F.D., Ingelfinger, J.R., Polin, R.A., Gershon, A.A., editors. Current Pediatric Therapy. 18th edition. Philadelphia, PA: Saunders Elsevier. p. 66-71. Heird, W.C., Cooper, A. 2006. Infancy and childhood. In: Shils, M.E., Shike, M., Ross, A.C., Caballero, B., Cousins, R.J., editors. Modern Nutrition in Health and Desease. 10th edition. Baltimore, MD: Lippincott Williams & Wilkins. p. 797-817. Cooper, A., Heird, W.C. 2006. Nutritional management of infants and children with specific diseases or other conditions. In: Shils, M.E., Shike, M., Ross, A.C., Caballero, B., Cousins, R.J., editors. Modern Nutrition in Health and Disease. 10th edition. Baltimore, MD: Lippincott Williams & Wilkins. p. 991-1003. Heird, W.C., Lapillonne, A. 2005. The role of essential fatty acids in development. Annual Review of Nutrition. 25:549-571. Ciliberto, M.A., Manary, M.J., Ndekha, M.J., Briend, A., Ashorn, P. 2006. Home-based therapy for oedematous malnutrition with ready-to-use therapeutic food. Acta Paediatrica. 95(8):1012-1015. Ciliberto, M.A., Sandige, H., Ndekha, M.J., Ashorn, P., Briend, A., Ciliberto, H.M., Manary, M.J. 2005. Comparison of home-based therapy with ready-to-use therapeutic food with standard therapy in the treatment of malnourished Malawian children: a controlled, clinical effectiveness trial. American Journal of Clinical Nutrition. 81(4):864-870. Sullivan, J., Ndekha, M., Maker, D., Hotz, C., Manary, M.J. 2006. The quality of the diet in Malawian children with kwashiorkor and marasmus. Maternal and Child Nutrition. 2(2):114-122. Patel, M.P., Sandige, H.L., Ndekha, M.J., Briend, A., Ashorn, P., Manary, M.J. 2005. Supplemental feeding with ready-to-use therapeutic food in Malawian children at risk of malnutrition. Journal of Health, Population, and Nutrition. 23(4):351-357. Reid, M., Badaloo, A., Forrester, T., Jahoor, F. 2006. In vivo rates of erythrocyte glutathione synthesis in adults with sickle cell disease. American Journal of Physiology Endocrinology and Metabolism. 291:E73-E79. Badaloo, A.V., Forrester, T., Reid, M., Jahoor, F. 2006. Lipid kinetic differences between children with kwashiorkor and those with marasmus 1-3. The American Journal of Clinical Nutrition. 83:1283-1288. Jahoor, F., Badaloo, A., Reid, M., Forrester, T. 2006. Glycine production in severe childhood undernutrition. American Journal of Clinical Nutrition. 84:143-149. Balasubramanyam, A., Mersmann, H., Jahoor, F., Phillips, T.M., Sekhar, R.V., Schubert, U., Brar, B., Iyer, D., Smith, E.O., Takahashi, H., Lu, H., Anderson, P., Kino, T., Henklein, P., Kopp, J.B. 2007. Effects of transgenic expression of HIV-1 Vpr on lipid and energy metabolism in mice. American Journal of Physiology - Endocrinology and Metabolism. 292(1):E40-E48. Jahoor, F., Badaloo, A., Reid, M., Forrester, T. 2005. Protein kinetic differences between children with edematous and nonedematous severe childhood undernutrition in the fed and postabsorptive states. The American Journal of Clinical Nutrition. 82:792-800. Balasubramanyam, A., Sekhar, R.V., Jahoor, F., Pownall, H.J., Lewis, D. 2006. Pathophysiology of adipocyte defects and dyslipidemia in HIV lipodystrophy: New evidence from metabolic and molecular studies. American Journal of Infectious Diseases. 2(3):167-172. Ginger, M.R., Shore, A.N., Contreras, A., Rijnkels, M., Miller, J., Gonzalez-Rimbau, M.F., Rosen, J.M. 2006. A noncoding RNA is a potential marker of cell fate during mammary gland development. Proceedings of the National Academy of Sciences. 103(15):5781-5786. Divisova, J., Kuiatse, I., Lazard, Z., Weiss, H., Vreeland, F., Hadsell, D.L., Schiff, R., Osborne, C.K., Lee, A.V. 2006. The growth hormone receptor antagonist pegvisomant blocks both mammary gland development and MCF-7 breast cancer xenograft growth. Breast Cancer Research and Treatment. 98(3):315-327. Ward, P.P., Paz, E., Conneely, O.M. 2005. Multifunctional roles of lactoferrin: a critical overview. Cellular and Molecular Life Sciences. 62(22):2540-2548. Galpin, L., Thakwalakwa, C., Phuka, J., Ashorn, P., Maleta, K., Wong, W.W., Manary, M.J. 2007. Breast milk intake is not reduced more by the introduction of energy dense complementary food than by typical infant porridge. Journal of Nutrition. 137(7):1828-1833. Jahoor, F., Badaloo, A., Reid, M., Forrester, T. 2006. Sulfur amino acid metabolism in children with severe childhood undernutrition: Methionine kinetics. American Journal of Clinical Nutrition. 84(6):1400-1405. Jahoor, F., Badaloo, A., Reid, M., Forrester, T. 2006. Sulfur amino acid metabolism in children with severe childhood undernutrition: Cysteine kinetics. American Journal of Clinical Nutrition. 84(6):1393-1399. Villalpando, S., Gopal, J., Balasubramanyam, A., Bandi, V.P., Guntupalli, K., Jahoor, F. 2006. In vivo arginine production and intravascular nitric oxide synthesis in hypotensive sepsis. American Journal of Clinical Nutrition. 84(1):197-203. Jahoor, F., Badaloo, A., Reid, M., Forrester, T. 2006. Unique metabolic characteristics of the major syndromes of severe childhood malnutrition. In: Picou, D., Forrester, T., Walker, S.P., editors. The Tropical Metabolism Research Unit, the University of the West Indies Jamaica 1956-2006. The House that John Built. Kingston: Ian Randle Publishers. p. 25-60. Kabotyanski, E.B., Huetter, M., Xian, W., Rijnkels, M., Rosen, J. M. 2006. Integration of prolactin and glucocorticoid signaling at the beta-casein promoter and enhancer by ordered recruitment of specific transcription factors and chromatin modifiers. Molecular Endocrinology. 20:2355-2368. Schmidt, R.M., Wiemann, C.M., Rickert, V.I., Smith, E.O. 2006. Moderate to severe depressive symptoms among adolescent mothers followed four years postpartum. Journal of Adolescent Health. 38(6):712-718. Badaloo, A., Reid, M., Soares, D., Forrester, T., Jahoor, F. 2005. Relationship between liver fat content and the rate of VLDL apolipoprptein B-100 synthesis in children with protein-energy malnutrition. American Journal for Clinical Nutrition. 81:1126-1132. Kaplan, W., Haymond, M., McKay, S., Karaviti, L.P. 2006. Osteopenic effects of MgSO4 in multiple pregnancies. Journal of Pediatric Endocrinology & Metabolism. 19(10):1225-1230. Le Bacquer, O., Mauras, N., Welch, S., Haymond, M., Darmaun, D. 2007. Acute depletion of plasma glutamine increases leucine oxidation in prednisone-treated humans. Clinical Nutrition. 26:231-238.