2009 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. 3.Progress Report We developed an enhanced technique for isolating & enriching functional mitochondria from different cell types within the lactating mammary gland. Our goal was to facilitate the analysis of mitochondrial function & of the mitochondrial proteome within the epithelial cell fraction of the mammary gland. We used 2-dimensional differential in gel electrophoresis to compare the mammary mitochondrial proteome in mitochondrial fractions isolate from whole tissue in comparison to mitochondrial fractions prepared from an epithelial cell–enriched fraction. We also determined the impact of decreasing milk removal frequency in the lactating mouse on mammary gland development, gene expression & mitochondrial function during prolonged lactation. (Proj. 2)To date, 204 infants have been enrolled & 179 have completed the first phase of this study (enrollment to hospital discharge). The mothers of 91 infants were still breastfeeding at discharge. Mothers of 60 infants were discharged on one of the assigned formulas. As expected, enrollment was complete in March 2009. (Proj. 3)We have completed studies of glucose, arginine & nitric oxide metabolism in pregnant teenagers & adult women in the first & beginning of the 3rd trimester of pregnancy. We completed studies of leucine kinetics & the production rates of the two major nitrogen transporting amino acids, glutamine & alanine, & two amino acid precursors of glutathione synthesis, glycine & cysteine, in pregnant teenagers & in adults. In addition, glutathione kinetics has also been measured. We are currently recruiting pregnant underweight women into a protocol to determine the effect of a balanced dietary supplement of energy & protein on whole body protein kinetics, rate of NO synthesis, gluconeogenesis & glucose production, maternal weight gain & neonatal birth weight, length & head circumference. (Proj. 4)26 children at risk for zinc deficiency & tropical enteroapthy from Masika village southern Malawi were studied, with complete stool & urine collections on a prescribed diet. Samples were transported to the USDA CNRC & analyzed for isotopic enrichment & absorption of non-metaboliozed sugars. These data were analyzed & summarized in a manuscript. (Proj. 5)We investigated the association of genetic variants in potential regulatory elements in the casein gene region with milk production traits in cattle, expression of casein & other milk protein genes, & binding of protein factors to these DNA elements. We established with collaborators, the expression in milk & lactating mammary gland of 2 non-casein genes located in the casein genomic region, Statherin (STATH) & Histatherin (HSTN). HSTN has a different promoter for mammary gland & salivary gland specific expression & the gene product has antimicrobial properties. As part of the bovine genome sequencing & analysis consortium we analyzed the organization & localization of milk protein genes, including the casein gene region, in the bovine genome, variation in the bovine genome structure affecting milk protein genes, & the evolutionary relationship of milk protein genes of different mammals. (Proj 6) 4.Accomplishments 1. The Bovine Lactation Genome: Insights Into the Evolution of Mammalian Milk: As part of the Bovine Lactation Genome Consortium, Children's Nutrition Research Center researchers investigated cow milk and lactation genetic data in relationship to other mammals. Over 6,000 genes that are present in the mammary gland of the cow were identified, and we found that about one-fourth of these genes are located together in the genome. Additionally we found that milk and mammary genes are more likely to be present in all mammals and they are more highly conserved across all mammals. Proteins associated with nutritional and immunological components of milk differ most between mammals, whereas proteins associated with secretion are highly conserved. Our findings support that milk is essential for the survival of mammalian newborns and that mechanisms for milk secretion were established more than 160 million years ago. (Project 6: Functional genomics of the casein gene cluster region) 2. The Bovine Lactation Genome: Insights in Bovine Milk Composition: As part of the annotation and analysis of the bovine genome, genes encoding cow milk proteins and proteins expressed in the mammary gland were investigated in relation to the cow genome. Children's Nutrition Research Center researchers together with research collaborators showed that structural changes in the genome contribute to differences in milk composition and that many of the genes affected by these changes have immune related functions. At least three new milk protein genes were identified, all having immune related functions. This is important since the presence of these genes in bovine milk may enhance milk's ability to fight harmful pathogens in both the mammary gland and in the newborn gastro-intestinal track. (Project 6: Functional genomics of the casein gene cluster region) 3. Regulation of Mammary Mitochondria During Lactation: Lactation is of great importance for offspring development and overall health, and a greater understanding of the biological activities that regulate milk production is needed. Children's Nutrition Research Center researchers wanted to identify mitochondrial proteins that might regulate milk production within the mammary gland during lactation. Using a technique known as mass spectrometry, our research team successfully identified 206 mammary mitochondrial proteins that change during lactation. Changes in these proteins could regulate the ability of the lactating female to produce milk. (Project 2: Insulin and IGF signaling in lactogenesis and maintenance of lactation) 4. Regulation of Mammary Cell Energy For Milk Production: Additional knowledge is necessary to understand how energy is made available to mammary cells for the production of milk during lactation. Children's Nutrition Research Center researchers developed a procedure to isolate and analyze mitochondria, the energy producing structures, from mammary cells. The research indicated that the ability of these mitochondria to produce energy changes during lactation and further suggests that these mitochondria are important to the amount of milk that a female produces during lactation. This observation is important and provides a scientific basis for further studies on the regulation of mammary cell mitochondrial function during lactation. (Project 2: Insulin and IGF signaling in lactogenesis and maintenance of lactation) 5. Regulation of Milk Protein Expression Through the Hormonal Control: Beta-casein is an important protein found in cow's milk, and it makes up about a third of the total protein content in milk. The hormones associated with lactation regulate beta-casein but how these hormones exert their effects is not known. Children's Nutrition Research Center scientists, in collaboration with researchers at Baylor College of Medicine, investigated the effect of different hormones on their ability to regulate beta-casein. We show that the hormones of lactation stimulate the physical interaction of two regions of the genome that regulate beta-casein and that this interaction is present in mammary gland tissue of lactating animals, but not in tissue from non-lactating virgin animals. A hormone known to repress lactation inhibits the expression of beta-casein and the interaction of its regulatory regions. These results indicate that hormones play a role in the regulation of milk protein expression in part through their effect on the interaction of regulatory elements. Project 6: Functional genomics of the casein gene cluster region) 6. Variation in the DNA of the Cow is Associated with Milk Production: Children's Nutrition Research Center researchers, in collaboration with researchers at the University of California-Davis, previously identified variation (single-nucleotide polymorphism or SNP) in parts of the genetic code (DNA) of the cow that regulates the production of milk protein genes (casein). The association of these SNP with milk production traits was investigated. We identified 3 SNP associated with production traits. These results highlight the importance of SNP variation in DNA sequences that regulate milk protein expression in relation to milk production and management traits in dairy cattle as well as the performance of the mammary gland in general. (Project 6: Functional genomics of the casein gene cluster region) 7. Association of Tropical Enteropathy to Zinc Homeostasis in Malawian children: Tropical enteropathy and zinc deficiency are major public health problems worldwide, with tropical enteropathy being characterized by an increased urinary lactulose-mannitol ratio and zinc homeostasis measured with a test of zinc absorption and excretion. Children's Nutrition Research Center researchers tested the hypothesis that zinc homeostasis would be correlated with tropical enteropathy. A dual sugar absorption test and dual stable isotope test was performed on 25 healthy Malawian children aged 3-5 years at high risk for enteropathy and zinc deficiency. From this study we were able to conclude that zinc deficiency is associated with tropical enteropathy in children. (Project 5: Nutritional pathogenesis, treatment and prevention of protein-energy malnutrition in young children) 8. Growth of Premature Infants Fed an Increased Protein Intake: Identifying the optimal protein intake for increased growth in preterm babies is of importance for pediatricians and nutritionist alike. Children's Nutrition Research Center researchers have been conducting studies over the past four years concerning the effects of a protein intake of 4 g/kg/d on growth, and this past year we successfully completed enrollment for this project. With all data available, preliminary results indicate that the higher protein intake was well tolerated and resulted in a greater rate of growth in preterm infants. A formula similar to the one studied is now on the market and is expected to replace formulas with less protein. These findings have the potential to improve the growth of preterm infants. However due to the development portion of this study is still being underway, we do not know if the higher protein diet results in greater cognitive development in these infants. (Project 3: Utilization of dietary protein during early infancy) 9. Nutrient Metabolism in Pregnant Teenagers Compared with Adult Women: When teenage girls become pregnant they give birth to smaller babies, and it is believed this happens because teenagers are still growing and their individual nutrient needs are in competition with that of their growing baby's nutritional needs. Scientists at the Children's Nutrition Research Center decided to study two particular nutrients that are used by the growing baby, glucose and amino acids, to determine how well-nourished teenagers were making and using these nutrients in the early part of pregnancy and late in their pregnancy, when compared to pregnant adult women. We found that the pregnant teenagers were unable to make as much glucose as the adult women and their bodies were using more energy than the adult women. However this did not have a bad effect on their baby's weight, as there was no difference in the weight of their babies and the babies of the adult women. Nevertheless, because the teenager cannot make as much glucose as the adult woman, we think that if she does not have adequate food intake at all times she will be at high risk of having a smaller baby. (Project 4: Relationship between maternal nutritional status and pregnancy outcome) Review Publications Balazs, A.E., Athanassaki, I., Gunn, S.K., Tatevian, N., Huang, S.A., Haymond, M.W., Karaviti, L.P. 2007. Rapid resolution of consumptive hypothyroidism in a child with hepatic hemangioendothelioma following liver transplantation. Annals of Clinical and Laboratory Science. 37(3):280-284. Heird, W.C. 2007. The feeding of infants and children. In: Kliegman, R.M., Jenson, H.B., Behrman, R.E., Stanton, B.F., editors. Nelson Textbook of Pediatrics. 18th edition. Saunders Elsevier, Philadelphia, PA. p. 214-225. Elsik, C.G., Gibbs, R., Skow, L., Tellam, R., Weinstock, G., Worley, K., Kappes, S.M., Green, R.D., Alexander, L.J., Bennett, G.L., Carroll, J.A., Chitko Mckown, C.G., Hamernik, D.L., Harhay, G.P., Keele, J.W., Liu, G., Macneil, M.D., Matukumalli, L.K., Rijnkels, M., Roberts, A.J., Smith, T.P., Snelling, W.M., Stone, R.T., Waterman, R.C., White, S.N. 2009. The Genome Sequence of Taurine Cattle: A Window to Ruminant Biology and Evolution. Science. 324:522-528. Phuka, J.C., Maleta, K., Thakwalakwa, C., Cheung, Y.B., Briend, A., Manary, M.J., Ashorn, P. 2008. Complementary feeding with fortified spread and incidence of severe stunting in 6- to 18-month-old rural Malawians. Archives of Pediatrics and Adolescent Medicine. 162(7):619-626. Lin, C.A., Manary, M.J., Maleta, K., Briend, A., Ashorn, P. 2008. An energy-dense complementary food is associated with a modest increase in weight gain when compared with a fortified porridge in Malawian children aged 6-18 months. Journal of Nutrition. 138(3):593-598. Maningat, P.D., Sen, P., Sunehag, A.L., Hadsell, D.L., Haymond, M.W. 2007. Regulation of gene expression in human mammary epithelium: Effect of breast pumping. Journal of Endocrinology. 195(3):503-511. Heckman, B.M., Chakravarty, G., Vargo-Gogola, T., Gonzales-Rimbau, M., Hadsell, D.L., Lee, A.V., Settleman, J., Rosen, J.M. 2007. Crosstalk between the p190-B RhoGAP and IGF signaling pathways is required for embryonic mammary bud development. Developmental Biology. 309:137-149. Kaplan-Lefko, P.J., Sutherland, B.W., Evagelou, A.I., Hadsell, D.L., Barrios, R.J., Foster, B.A., Demayo, F., Greenberg, N.M. 2008. Enforced epithelial expression of IGF-1 causes hyperplastic prostate growth while negative selection is requisite for spontaneous metastogenesis. Oncogene. 27(20):2868-2876. Hadsell, D.L., Parlow, A.F., Torres, D., George, J., Olea, W. 2008. Enhancement of maternal lactation performance during prolonged lactation in the mouse by mouse GH and long-R3-IGF-I is linked to changes in mammary signaling and gene expression. Journal of Endocrinology. 198:61-70. Heikens, G.T., Amadi, B.C., Manary, M., Rollins, N., Tomkins, A. 2008. Nutrition interventions need improved operational capacity. Lancet. 371(9608):181-182. Heikens, G.T., Bunn, J., Amadi, B., Manary, M., Chhagan, M., Berkley, J.A., Rollins, N., Kelly, P., Adamczick, C., Maitland, K., Tomkins, A. 2008. Case management of HIV-infected severely malnourished children: Challenges in the area of highest prevalence. Lancet. 371(9620):1305-1307. Flax, V.L., Ashorn, U., Phuka, J., Maleta, K., Manary, M.J., Ashorn, P. 2008. Feeding patterns of underweight children in rural Malawi given supplementary fortified spread at home. Maternal and Child Nutrition. 4(1):65-73. Maningat, P.D., Sen, P., Rijnkels, M., Sunehag, A.L., Hadsell, D.L., Bray, M., Haymond, M.W. 2009. Gene expression in the human mammary epithelium during lactation: The milk fat globule transcriptome. Physiological Genomics. 37(1):12-22.