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Research Project: Impact of Early Dietary Factors on Child Development and Health

Location: Arkansas Children's Nutrition Center

2015 Annual Report

Overall objective is to gather evidence about important environmental factors that have long-term consequences on child development/health, and their health as children become adults. 1: Continue Beginnings study, longitudinal study of growth/development/body composition in infants breast-fed or fed soy/cow milk formula. 2: Determine if soy feeding affects estrogen receptor beta using chromatin immunoprecipitation analysis; gene deletion studies in appropriate animal models to ascertain if soy protein affects reproductive development/metabolism/body composition via activation of ERbeta-mediated signaling. 3: Determine body composition/metabolism of prepubertal pigs fed breast milk/milk formula/soy formula as neonates, then weaned onto either low or high fat diet. Compare data with body composition/metabolism in children age 5 from Beginnings Study. 4: Determine effects of early diet in pig model on gut-associated lymphoid tissue development/function; ... 5: Characterize effects of neonatal diet on composition of intestinal microbiota, Collect urine/fecal samples from Beginnings Study infants/children at 3/6/9/12 months and yearly thereafter. 6: Determine if feeding blueberries (BB) and other fruits/vegetables containing phenolic acid (PA) chlorogenic acid (CA) or treatment with CA metabolite hippuric acid stimulate bone growth in rodent model; examine role of G-coupled receptor protein GPR109A as potential mediator of PA effects on bone. 7: Perform peripheral quantitative computerized tomography to study bone morphology; measure urine/serum bone turnover markers in lean/obese prepubertal children with/without additional symptoms of insulin resistance. 8: Determine ability of diets containing BB to block obesity-induced impairment in bone quality/strength in weaning rats; determine method of activiation in underlying mechanisms by which this occurs. Determine if bone turnover markers in urine/serum of obese children are improved after short-term intervention with BB-containing diets. 9: Determine if toll-like receptor/early response protein-1 signaling is mediating embryonic/placental inflammation and epigenetic dysregulation in offspring due to maternal obesity in rodent models/human subjects. 10: Characterize effect of maternal obesity on programming offspring's metabolism and risk of obesity later in life in the Glowing study. 11: Determine if interventions prior to/during pregnancy that alter maternal hyperinsulinemia, gastrointestinal microbiome and/or inflammation prevent obesity-induced metabolic programming in animal models. 12: Determine if reprogramming of offspring energy metabolism/mitochondrial function can occur by promoting neonatal physical activity or dietary factors in rodent models. 13: Determine effects of early diet on neurocognitive development by completing neurophysiological/behavioral aspects of Beginnings study. 14: Determine effects of diet composition, meal pattern/frequency on brain development/function, and behavioral dynamics important for learning in well-characterized lean/obese school children. 15: Characterize how neurocognitive functions associated with obesity can be improved by physical activity.

Studies will focus on the various dietary factors found in foods commonly consumed by children, such as breast milk, infant formulas, fruits, grain, milk, and soy to determine their long-term health effects in infants and children. We will analyze how the early exposure to protein sources and fruits normally consumed by infants and children prevents the initiation of and protects against development of chronic diseases by altering tissue differentiation, inflammation, and/or oxidative status. We will use animal models to mechanistically address the molecular and cellular pathways regulated by intake of various dietary factors (such as in soy foods, berries, grains and milk) in mammary tissue, liver, adipose tissue, pancreas, gastrointestinal tract, bone, skeletal muscle and the immune system; identify tissue and serum biomarkers of healthy status associated with these diets; and provide new molecular targets and processes underlying chronic diseases that may be influenced by proper nutrition. Additional work will be undertaken in an observational study of infants, The Beginnings Study, which is a longitudinal study of breast-fed, milk formula-fed, and soy formula-fed children from birth through puberty, will evaluate growth, development, body composition, metabolism, bone development, and immune system development and function. Animal models such as the neonatal pig will be utilized to explore molecular mechanisms underlying the effects of early dietary exposures. Rodent models will be used to understand the parental epigenetic transmission of the effects of maternal obesity and high fat feeding to future generations and underlying molecular, biochemical, and endocrine mechanisms, in the offspring. These studies will be translated in an ongoing longitudinal clinical study of infant body composition in children of lean and overweight women (The Glowing Study). Work will be accomplished by evaluating critical periods of development and vulnerable stages of life (i.e. the nutritional status of women at the moment of conception; nutritional and developmental issues during pregnancy and lactation). Children (infants, toddlers, and school-aged youths) will be studied to evaluate the effects of infant diet (specifically, breast-milk and a variety of infant formulas) on morphological, neurophysiological, behavioral, and cognitive development in infants and children. Nutritional status assessments, anthropometric measurements, body composition, energy metabolism, physical activity fitness, urine and blood analysis, and measures of brain structure (MRI imaging), psychological and behavioral status (standardized testing), neuropsychological, and cognitive measures will be obtained and analyzed. The effects of diet composition, meal patterns, meal frequency, physical activity, and body composition on brain function, behavioral dynamics, learning, and school performance will be assessed in normal weight and overweight/obese school children using validated survey instruments and state-ofthe-art research equipment.

Progress Report
Significant research progress was accomplished during the year. To review the progress, please refer to project 6026-51000-010-01S and 6026-51000-010-05S.

1. Maternal obesity affects boys' body fat during the first 6 years of life. Maternal obesity can influence neonatal body composition (fat and lean mass) and has been linked to higher risk of childhood obesity; yet, very few studies have been able to investigate this relationship longitudinally during childhood. Investigators at the Arkansas Children's Nutrition Center in Little Rock, AR have prospectively examined body composition trajectories during the first 6 years of life of children born to either normal weight, overweight or obese mothers. The findings demonstrate that boys born to obese mothers had higher body fat from ages 2 to 6 years when compared to boys born to normal weight and overweight mothers. These findings are interesting especially when compared to the findings of girls, where their body composition was not different across the maternal groups. These results demonstrate for the first time a sexual dimorphism in response to maternal obesity in children, a phenomenon that had been previously predicted from studies in animal models.

2. Childhood obesity associated with lower brain grey matter. There is increasing evidence that brain grey matter is reduced in obese adults, but there is little information about brain development in obese, but otherwise healthy, children. Grey matter is important because it contains networks of neurons responsible for information processing in the brain. Scientists at the Arkansas Children's Nutrition Center in Little Rock, AR used advanced neuroimaging methods to evaluate grey matter volume in healthy 8-10 year old children who were either normal weight or obese. The amount of grey matter was significantly lower in obese compared to normal weight children in widespread brain regions, including those involved in the regulation of attention, motor control and working memory. These findings increase understanding of brain structural features in obese children that may be associated with reported differences between obese and normal weight children in cognitive function, and suggest further that obesity-linked body composition changes include the brain as well as other tissues.

3. Mother's obesity alters epigenetic marks at neurodevelopmental genes in umbilical cord tissue. The mother's body weight and body fat during pregnancy have significant influence on her baby's development in the womb. One avenue via which such changes may occur is through epigenetic alterations, which involve changes in the "decorative marks" on DNA, such as methylation of specific residues. Investigators at the Arkansas Children's Nutrition Center in Little Rock, AR utilized umbilical cord tissue at birth from infants born to either lean or obese mothers to study DNA methylation, and found that offspring born to obese mothers had changes in methylation marks around genes involved in neuronal development. In light of infant brain scans that show lesser "white matter" in children born to obese mothers, the findings strongly suggest that exposure to maternal obesity in the womb may detrimentally alter brain development; nonetheless, the precise functional impact, if any, of these changes is under investigation.

4. Maternal exercise during pregnancy impacts offspring metabolism. Exercise in general has many health benefits, but the impact of maternal exercise during pregnancy on both the mother and developing baby is much less studied. Researchers at the Arkansas Children's Nutrition Center in Little Rock, AR investigated how maternal physical activity impacts the fetus and placenta by providing running wheels to pregnant mice, and then following the expression of numerous genes important for metabolism. Maternal exercise led to marked changes in select genes in the placenta, which were dependent on the gender of the offspring; fat metabolism pathways were predominantly affected in males and cellular differentiation were more strongly influenced in females. Overall, these studies led to the discovery that maternal exercise influences offspring physiology in a sexually-dimorphic fashion, which may have important implications for body function and metabolism into adulthood.

5. Infant diet and frontal brain activity predict child temperament differences at 3 years. Early temperament is an important factor in personality, affect and social development, yet it is unknown how early infant diet and associated brain neurodevelopment relate to temperament development. Scientists at the Arkansas Children's Nutrition Center in Little Rock, AR studied these relationships by comparing standardized measures of temperament in 3 yr old children that had been exclusively breast-fed, fed milk formula, or fed soy formula as infants. Temperament ratings for all children were in the normal range, but soy fed children differed on several temperament scales associated with emotion reactivity and regulation, as well as on relationships of their brain activity at 9 months with 3 year temperament measures. These results provide the first evidence that early infant diets contribute to the differential development of temperament and that these differences are related to measures of diet-associated brain activity during infancy. The findings suggest that the development of some temperament-sensitive behaviors, such as socio-emotional relations and executive functions, will differ in soy formula-fed compared with breastfed and milk formula-fed children.

6. Infants fed soy formula had normal reproductive organ size. Reports suggest that some chemicals found in soybeans may impair reproductive function in animals, raising the potential concern that consuming soy infant formula could alter hormone-sensitive organ development in children. Investigators at the Arkansas Children's Nutrition Center in Little Rock, AR used ultrasonography to examine the effects of feeding infants breast-milk, cow's milk-based formula or soy-based formula on the volumes and morphological characteristics of breast buds, uterus, ovaries, prostate, and testicles at age 5 years. The findings demonstrated that there were no differences between the groups. These results are the first to investigate prospectively the effect of soy-based infant formula on hormone sensitive organs in boys and girls in early childhood. The results suggest that feeding soy-based formula to infants do not change volumes or the morphology of hormone-sensitive organs at age 5 years.

7. Trace mineral status differs in breast-fed and formula-fed neonates. Trace metals such as zinc and iron are critical nutrients that impact many of the body's systems, yet there is a lack of studies testing if the trace metal composition of infant formula influences nutrient status or function. Scientists at the Arkansas Children's Nutrition Center in Little Rock, AR examined this question in a newborn piglet model (shares many physiological attributes of human newborns) fed soy infant formula (soy), cow's milk formula (milk), or sow-fed from postnatal day 2 to 21. Serum zinc values were higher in milk formula-fed animals compared to breastfed or soy groups, and the small intestine expression of some zinc-associated transporter proteins were altered by diet. Differences in serum zinc after cow's milk formula compared to soy formula consumption suggests compensatory changes in expression of zinc-regulated genes. The results support the idea that there are factors inherent to breastmilk that could lead to different availability of some trace metals, and this is relevant to future ingredient designs of infant formulas.

8. Gastrointestinal (GI) tract development and gut-associated immunity markers are altered by formula feeding. The effects of early diet on intestinal development and function are only partially characterized; this is a major issue, considering the gut's central role in nutrient uptake, protection against pathogens, and hence normal growth and development of the newborn. Scientists at the Arkansas Children's Nutrition Center in Little Rock, AR have characterized GI tract immune and anatomical/functional development in piglets (as a model of neonatal human gut development), fed a formula diet vs. breastfed on sow's milk. Animals fed a formula diet displayed a more pronounced inflammatory signature in the GI tract, and less robust development of the GI tract. These results highlight that infant breastfeeding leads to positive effects in the GI tract when compared to formula; studies are underway to determine the specific components of breast milk that lead to these effects, which will be relevant to new formula ingredient design to better mimic the health benefits of breastfeeding.

9. Soy protein isolate consumption promotes bone development. Adult bone health is influenced by diet, exercise and obesity status during childhood. To understand how dietary factors early in development impact bone integrity and growth, Arkansas Children's Nutrition Center scientists in Little Rock, AR have studied the effects of dietary soy protein isolate (SPI) and oxidative stress on bone outcomes in rodent models. By using young and old animals, it was demonstrated that dietary SPI protects against osteoblast (bone-forming cell) senescence ("aging"), i.e. that brought about by high fat, obesity-promoting diet. These findings raise the possibility that consumption of a soy-rich diet during early life may affect growth and development of bones and the risk of fractures resulting from degenerative bone diseases in later adult life.

10. Maternal obesity disrupts mitochondrial health in offspring. More and more women are either overweight or obese when entering pregnancy, and evidence indicates that the mother's weight during pregnancy is an important factor in her baby's health. Researchers at the Arkansas Children's Nutrition Center (ACNC) in Little Rock, AR examined whether an imbalance in the mitochondria (the powerhouses of our cells) may be responsible for changes in offspring metabolism. ACNC scientists found in animal models that specific factors associated with healthy mitochondria and mitochondrial fat burning were reduced in the placenta and important tissues of offspring born to obese mothers. Reductions of some of these factors were also evident in human placenta from overweight mothers, highlighting for the first time that a mother's obesity can detrimentally alter the energy-producing cellular powerhouses of her offspring, in turn contributing to altered energy metabolism.

11. Thyroid hormone signaling in offspring is a target of maternal obesity. An unhealthy diet and lack of exercise contributes to obesity, but increasing evidence suggests that exposure to factors in the womb of obese women may itself increase obesity and alter metabolism offspring. Researchers at the Arkansas Children's Nutrition Center in Little Rock, AR utilized a cutting-edge methodology to monitor the expression of genes (called RNAseq). Examination of placenta of lean and obese pregnant rats (whose offspring display higher propensity toward obesity) showed that high maternal adiposity (body fat abundance) persistently decreased genes involved in thyroid hormone signaling. Since thyroid hormones impact metabolism, the findings provide evidence regarding a potential pathway by which maternal obesity alters the offspring metabolism and contributes to increased risk of obesity.

12. Phonetic processing is better in preadolescents who were breast-fed as infants. The critical neurodevelopment taking place during infancy is fueled by nutrients provided by diets (breast-milk or formula) that differ significantly in composition, but little is known about how these variations in infant diet relate to later development of brain function and behavior. Scientists at the Arkansas Children's Nutrition Center in Little Rock, AR used functional magnetic resonance imaging (fMRI) methods to study brain activation in 8 year old children (breast-fed or fed milk based-formula as infants) as they evaluated if pairs of words rhymed, a decision involving phonetic processes fundamental to normal language development. Children in the breast-fed group showed significantly greater activation of brain regions specific to phonetic processing as well as better behavioral performance (number of correct responses). These findings are the first to demonstrate a relationship between infant diet and preadolescent brain function and behavior related to phonetic processing and show advantages in these relationships for children who were breast-fed.

Review Publications
Mercer, K.E., Hennings, L., Ronis, M.J. 2014. Alcohol consumption, Wnt/ß-catenin cignaling, and hepatocarcinogenesis. Advances in Experimental Medicine and Biology. 815:185-195.
Ronis, M.J., Miousse, I.R., Mason, A.Z., Sharma, N., Blackburn, M.L., Badger, T.M. 2015. Trace element status and zinc homeostasis differ in breast and formula-fed piglets. Experimental Biology and Medicine. 240(1):58-66.
Andres, A., Moore, M.B., Linam, L., Casey, P.H., Cleves, M., Badger, T.M. 2015. Compared with Feeding Infants Breast Milk or Cow-milk formula, soy formula feeding does not affect subsequent reproductive organ size at 5 years of age. Journal of Nutrition. 145(5):871-875.
Yang, C.S., Mercer, K.E., Alund, A.W., Suva, L.J., Badger, T.M., Ronis, M.J. 2014. Genistein supplementation increases bone turnover but does not prevent alcohol-induced bone loss in male mice. Experimental Biology and Medicine. 239(10):1380-1389.
Ou, X., Thakali, K.M., Shankar, K., Andres, A., Badger, T.M. 2015. Maternal adiposity negatively influences infant brain white matter development. Obesity. 23(5):1047-1054.
Chen, J., Lazarenko, O.P., Blackburn, M.L., Mercer, K.E., Badger, T.M., Ronis, M.J. 2015. p47phox-Nox2-dependent ROS signaling inhibits early bone development in mice but protects against skeletal aging. Journal of Biological Chemistry. 290(23):14692-14704.
Andres, A., Hull, H., Cleves, M., Casey, P., Badger, T., Shankar, K. 2015. Longitudinal body composition of children born to normal weight, overweight and obese mothers. Obesity. 23(6):1252-1258.
Fang, N., Yu, S., Ronis, M.J., Badger, T.M. 2015. Matrix effects break the LC behavior rule for analytes in LC-MS/MS analysis of biological samples.Experimental Biology and Medicine. 240(4):488-497.
Saben, J., Kang, P., Zhong, Y., Thakali, K., Gomez-Acevedo, H., Borengasser, S.J., Andres, A., Badger, T.M., Shankar, K. 2014. RNA-seq analysis of the rat placentation site reveals maternal obesity-associated changes in placental and offspring thyroid hormone signaling. Placenta. 35(12):1013-1020.
Chen, J., Lazarendo, O.P., Blackburn, M.L., Badger, T.M., Ronis, M.J. 2015. Soy protein isolate inhibits high-Ffat diet-induced senescence pathways in osteoblasts to maintain bone acquisition in male rats. Endocrinology. 156(2):475-487.
Ou, X., Andres, A., Cleves, M.A., Pivik, R.T., Snow, J.H., Ding, Z., Badger, T.M. 2014. Sex-specific association between infant diet and white matter integrity in 8-y-old children. Pediatric Research. 76(6):535-543.
Piccolo, B.D., Keim, N.L., Fiehn, O., Adams, S.H., Van Loan, M.D., Newman, J.W. 2014. Habitual physical activity and plasma metabolomic patterns distinguish individuals with low vs. high weight loss during controlled energy restriction. Journal of Nutrition. 145(4):681-690.
Bedinger, D.H., Goldfine, I.D., Corbin, J.A., Roell, M.K., Adams, S.H. 2015. Differential pathway coupling efficiency of the activated insulin receptor drives signaling selectivity by xmeta, an allosteric partial agonist antibody. Journal of Pharmacology and Experimental Therapeutics. 353(1):35-43.
Meissen, J.K., Hirahatake, K.M., Adams, S.H., Fiehn, O. 2015. Temporal metabolomic responses of cultured HepG2 liver cells to high fructose and high glucose exposures. Metabolomics. 11(3):707-721.
Vaziri, N.D., Oiu, S., Lau, W., Nazertehrani, S., Farzaneh, S.H., Kieffer, D.A., Adams, S.H., Martin, R.J. 2014. High amylose resistant starch diet ameliorates oxidative stress, inflammation, and progression of chronic kidney disease. PLoS One. 9(12) doi:
Piccolo, B.D., Comerford, K.B., Karakas, S.E., Knotts, T.A., Fiehn, O., Adams, S.H. 2015. Whey protein supplementation does not alter plasma branched-chained amino acid profiles but results in unique metabolomics patterns in obese women enrolled in an 8-week weight loss trial. Journal of Nutrition. 145(4):691-700, R1-R134.
Diaz, E.C., Herndon, D.N., Lee, J., Porter, C., Cotter, M., Suman, O.E., Sidossis, L.S., Borsheim, E. 2015. Predictors of muscle protein synthesis after severe pediatric burns. Journal of Trauma and Acute Care Surgery. 78(4):816-822.
Borengasser, S.J., Faske, J., Kang, P., Blackburn, M.L., Badger, T.M., Shankar, K. 2014. In utero exposure to prepregnancy maternal obesity and postweaning high-fat diet impair regulators of mitochondrial dynamics in rat placenta and offspring. Physiological Genomics. 46(23):841-850.
Pivik, R.T., Andres, A., Tennal, K.B., Gu, Y., Cleves, M.A., Badger, T.M. 2015. Infant diet, gender and the development of vagal tone stability during the first two years of life. International Journal of Psychophysiology. 96(2):104-114.
Ronis, M.J., Mercer, K.E., Gannon, B.M., Engi, B., Zimniak, P., Shearn, C., Orlicky, D., Albano, E., Badger, T.M., Petersen, D.R. 2015. Increased 4-hydroxynonenal protein adducts in male GSTA4–4/PPAR-alpha double knockout mice enhance injury during early stages of alcoholic liver disease. American Journal of Physiology - Gastrointestinal and Liver Physiology. 308(5):G403-G415.
Mccoin, C.S., Knotts, T.A., Adams, S.H. 2015. Acylcarnitines: Old actors auditioning for new roles in metabolic physiology. Nature Reviews Endocrinology. doi:10.1038/2015.129.
Lynch, C.J., Adams, S.H. 2014. Branched-chain amino acids in metabolic signaling and insulin resistance. Nature Reviews Endocrinology. 10(12):723-736. doi: 10.1038/nrendo.2014.171.