2008 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.
We evaluated the penalty of loss of lactoferrin (LF) function on the susceptibility of mice to opportunistic infection, as well as responses to acute bacterial challenge with two bacterial strains. We completed analysis of impaired neutrophil functions in the absence of LF (Proj 1). We developed a technique for isolating & enriching functional mitochondria from the lactating mammary gland. We cataloged the changes that occured within the mammary mitochondrial proteome during a single prolonged lactation cycle. We validated chemical & gene expression markers for mammary gland oxidative metabolism by comparing inbred strains of mice with low or high milk production capacity & low or high mitochondrial ATP synthesis activity (Proj 2). 180 infants have been enrolled & finished the 1st phase. 120 mothers elected to provide human milk for feeding their infant & 67 of these infants were breastfeeding at discharge. 34 infants were switched to the assigned formula before discharge. Mothers of 60 infants elected to formulafeed their infant & 58 were still being fed the assigned formula at discharge (Proj 3). We completed studies of glucose & protein metabolism in pregnant women with low & normal BMI in the 1st & 2nd trimester of pregnancy & completed studies of nitric oxide kinetics & the production rates of glutamine & alanine, & glycine & cysteine. These studies were performed in pregnant teenagers & in adults, & glutathione kinetics were also been measured (Proj 4). Data was analyzed for randomized, double-blinded trial of rifiamixin treatment in 3- to 5-year-old rural Malawian children on the effect of gut function. Treatment had no effect on gut function, & an experiment was designed to look for a correlation between gut function & zinc homeostasis (Proj 5). Genome sequences were analyzed computationally to identify transcription factor binding sites in regulatory regions in the casein gene cluster, & mutation in the DNA sequence of these regions was identified in different dairy cattle breeds. Analyses of the packaging of DNA (chromatin) were performed on mammary gland tissue at different developmental stages & mouse mammary epithelial cell lines. The physical interaction of important regulatory elements under influence of lactogenic hormones & changes in chromatin during the lactation cycle was established. Genomic analyses were performed on the new bovine genome sequences assembly to further investigate the casein gene cluster organization in the cow. New insights were gathered about possible regulation of casein genes & non-casein genes in the region (Proj 6). We initiated studies on the effect of arginine supplementation on insulin resistance in obese and overweight adolescents. These studies are completed & the analytical phase is underway. We are conducting studies of methionine splanchnic metabolism in healthy adolescents to better define the utilization of methionine into its various substrates (Proj 7).[NP107 Comp. 4]
Nutrient Needs of Pregnant Low BMI Women:
Evidence that underweight women with low body mass index (BMI) have a higher risk of having a low birth weight (LBW) baby suggests that the underweight mother is not capable of providing adequate nutrients, especially energy and protein, for growth of the reproductive tissues and the fetus. The metabolic mechanisms linking maternal weight and fetal growth are not fully understood but there is evidence that in women with normal BMI the extra amino acids needed during pregnancy are met through increased protein breakdown and reduced amino acid oxidation. Yet it is not known, however, whether the underweight pregnant woman with a low BMI can make these adaptations successfully. Children's Nutrition Research Center researchers measured protein and amino acid metabolism in groups of 10 low and 10 normal BMI women in the first and second trimesters of pregnancy and found that there were no significant differences in gestational weight gain, or in baby birth weight and that the extra amino acids needed for increased protein synthesis in pregnancy are obtained through a common mechanism of increased protein breakdown and decreased oxidation in both normal and underweight women. The former was more marked in low BMI women, who, possibly as an adaptive response, also had a lower efficiency of utilization of dietary protein; however, they compensated for this decreased efficiency by consuming 50% more protein starting the first trimester. This latter finding has important implications for feeding pregnant low BMI women extra protein and energy very early in pregnancy to prevent LBW babies. [NP 107, Component 4] (CNRC Project 4)
Impact of Rifamixin on Gut Function in Rural Malawian Children:
Tropical enteropathy (diseases developing in the intestine) is ubiquitous in rural African children, and its role in nutritional compromise is not well understood. Children's Nutrition Research Center researchers tested the gut function in a population of 147 rural 3- to 5-year-old children before and 21 days after completion of a 7-day treatment with either rifamixin, a non-absorbed antibiotic, or a placebo. The children receiving the antibiotic showed no improvement over those receiving placebo. This questions the role of bacterial contamination of the small bowel as the cause of tropical enteropathy. [NP 107, Component 5] (CNRC Project 5)
Bicarbonate Kinetics and Predicted energy Expenditure in Critically Ill Children:
The objective of these studies was to find out the amount of calories that children in the pediatric intensive care unit need when they are feed intravenously or fed by a nasogastric tube. Children's Nutrition Research Center researchers used carbon oxidation research techniques and observed that the amount of calories that critically ill children need are less than what has been recommended. Sick children seldom receive the calories that they need, and are over or under fed, regardless of the route of nutrient administration. [NP 107, Component 4 Nutrient Requirements] (CNRC Project 7)
Adaptation to an Arginine and Precursor Free Diet:
Arginine is an important amino acid because it serves as a precursor for important compounds such as nitric oxide, a gaseous molecule that intervenes in multiple functions, and creatine, which intervenes in energy metabolism. To determine how this important building block of protein (arginine) is used by the human body, Children's Nutrition Research Center researchers conducted a study in healthy subjects receiving a diet devoid of arginine for 4 weeks, provided as an amino acid formula. We observed that during an arginine-free intake, there is a decrease in the rates of arginine oxidation; therefore there is a conservation of arginine. However, the synthesis of arginine remains unchanged and from a nutritional standpoint, arginine is not an essential amino acid. This situation may change under conditions of disease. [NP 107, Component 4 Nutrient Requirements] (CNRC Project 7)
A New Cow Milk Protein with Anti-microbial Properties:
The region of the cattle genome that harbors the major milk protein genes (caseins) also contains several other genes and this is important because the proteins encoded by these genes might be expressed in milk, and could have important functions in the health and wellbeing of the mammary gland and suckling newborn. Children's Nutrition Research Center researchers previously established the presence in the cattle genome of a gene now named Histatherin, (HSTN) which is located close to one of the casein genes, beta-casein. Upon further analysis of the new cattle genome sequence, we showed that this gene is adjacent to DNA that are important for the expression of the beta-casein gene. Collaborators in New Zealand determined that HSTN is expressed in the mammary gland, thereby is present in cow's milk and that this synthesized peptide has anti-microbial effects. These results indicate that the location of Histatherin in the cattle genome has led to its lactation related expression in the mammary gland and that its presence in cow's milk could help fight pathogenic insults in both the mammary gland and the newborn gastrointestinal track. [NP 107, Component 5 Health Promoting Properties of Plant and Animal Foods] (CNRC Project 6)
Binding Sites for DNA-Binding Proteins and Genetic Variation at Distal Regulatory Elements are Identified:
The way DNA is packaged in the cell, specifically chromatin, plays an important role in gene regulation. Children's Nutrition Research Center researchers have shown that chromatin changes during the development of the mammary gland at distal regions in the genome thought to be important for casein gene regulation, yet there is limited knowledge of the role that DNA-binding proteins and genetic changes in these regions play. Our laboratory analyzed distal regions of the genome for the presence of potential binding sites for DNA-binding proteins and found that a number of sites for factors with inhibitory or activating properties. Collaborators from UC Davis analyzed these regions for genetic variation (single nucleotide polymorphism or SNP) in three different dairy cattle breeds and successfully identified five SNP that show different milk production traits with most of the SNP being close to conserved
binding sites which can potentially interfere with the function of these regions. These findings indicate that genetic variation at regulatory elements of lactation related genes could play a role in lactation performance. Moreover, changes in chromatin, induced through external influences such as infection or nutrition, could impact genetic variation. [NP 107, Component 5 Health Promoting Properties of Plant and Animal Foods] (CNRC Project 6)
Hormones of Lactation Induce Interactions Between Regulatory Elements of Milk Protein Genes:
The way DNA is packaged in the cell, specifically chromatin, plays an important role in gene regulation yet there is limited knowledge of its role in the regulation of the major milk proteins (casein). Scientists at the Children's Nutrition Research Center have previously demonstrated progressive changes in the chromatin and binding of trans-acting factors at DNA in mammary cells during development. This resulted in an open chromatin structure at the proximal and distal regulatory elements of fully active casein genes. We analyzed the physical interaction between a proximal and distal regulatory element of one of the casein genes and were able to show that the hormones of lactation induce the physical interaction of these regulatory elements. These studies have provided insight into the mechanisms regulating milk composition.
Furthermore, it improves 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)
Role of Lactoferrin (LF) in Neutrophil Mediated Host Defense:
Children's Nutrition Research Center researchers have addressed the functions of LF in mediating neutrophil-dependent immune defense responses by comparative analysis of the functional activities of neutrophils derived from wild type versus LF deficient neutrophils obtained from mice that were genetically modified to inhibit LF production. Using this system, our lab has demonstrated that loss of LF results in increased sensitivity to opportunistic infections, impaired neutrophil functions, and abnormal numbers of white blood cells. Our studies provide strong evidence for an essential role of LF in regulation of immune defense responses. Our studies also suggest that supplementation with lactoferrin, a natural antimicrobial protein, may be beneficial in protecting against opportunistic infections in immune-compromised patient groups. [NP 107, Component 6] (CNRC Project.
Microarray Analysis of Mammary Gene Expression:
Scientific researchers need additional knowledge in order to understand what are the genetic pathways that regulate changes in mammary cell function during a prolonged lactation cycle. Children's Nutrition Research Center researchers identified 1,056 different genes whose expression in the mammary gland changed over the course of a single lactation cycle in the mouse. Our lab analyzed mammary tissue RNA by microarrays to identify genes with changes in expression over the lactation cycle. The genes identified in these studies provide CNRC researchers new targets that could impact milk production in lactating females. [NP 107, Component 4](CNRC Project 2)
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Lin, C.A., Boslaugh, S., Ciliberto, H.M., Maleta, K., Ashorn, P., Briend, A., Manary, M.J. 2007. A prospective assessment of food and nutrient intake in a population of Malawian children at risk for kwashiorkor. Journal of Pediatric Gastroenterology and Nutrition. 44(4):487-493.
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Verbruggen, S.C., Joosten, K.F., Castillo, L., Van Goudoever, J.B. 2007. Insulin therapy in the pediatric intensive care unit. Clinical Nutrition. 26(6):677-690.
Sy, J., Gourishankar, A., Gordon, W.E., Griffin, D., Zurakowski, D., Roth, R.M., Coss-Bu, J., Jefferson, L., Heird, W., Castillo, L. 2008. Bicarbonate kinetics and predicted energy expenditure in critically ill children. American Journal of Clinical Nutrition. 88(2):340-347.
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Chacko, S.K., Sunehag, A.L., Sharma, S., Sauer, P.J.J., Haymond, M.W. 2008. Measurement of gluconeogenesis using glucose fragments and mass spectrometry after ingestion of deuterium oxide. Journal of Applied Physiology. 104:944-951.
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Llorente, A.M., Voigt, R., Jensen, C.L., Fraley, J.K., Heird, W.C., Rennie, K.M. 2008. The test of variables of attention (TOVA): Internal consistency (Q1 vs. Q2 and Q3 vs. Q4) in children with Attention Deficit/Hyperactivity Disorder (ADHD). Child Neuropsychology. 14(4):314-322.
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