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United States Department of Agriculture

Agricultural Research Service

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Location: Arkansas Children's Nutrition Center

2012 Annual Report

1a. Objectives (from AD-416):
Dietary factors may significantly impact long-term human health during adult life as a result of the influences on early developmental events. Certain common dietary factors appear to be capable of affecting growth and development; transiently and permanently altering metabolism; influencing body composition; and preventing some diseases. For example, fruits, vegetables, grains, and milk contain natural compounds (phytochemicals, peptides, and proteins) that can alter development, physiology, and metabolism, which can ultimately lead to disease prevention and phenotypic changes. Additionally isoflavones are particularly concentrated in soybeans and can have many of the same actions as the major female hormones in women, the estrogens. Countries with regular consumption of large amounts of soy foods report lower incidence of cancer, cardiovascular disease, and obesity; and factors in soy (isoflavones and peptides) are postulated as being partially responsible. The objectives of this research include: 1) determine the effects of diet and physical activity in humans and animal models on development and organ function; 2) determine how early exposure to soy proteins and fruits confers resistance to chronic diseases such as mammary cancer, cardiovascular disease, and type 2 diabetes in later adult life; 3) evaluate multiple molecular mechanisms and identify bioactive components for chronic disease prevention by diets using appropriate models as measured by tumor suppressors and oncogenes; oxidative modification; inflammation; immunomodulation; and insulin sensitivity; 4) examine consequences of early intake of combinations of foods (soy, fruits) on chronic disease prevention, organ development and signaling pathways, relative to dietary intake of a single food; 5) investigate the mechanisms of maternal obesity-induced fetal programming; 6) examine the impact of type and amount of dietary macronutrient components on development of obesity and associated metabolic sequence in an animal model of pediatric total enteral nutrition and in clinical studies; 7) identify the potential of dietary factors for mitigating risk of obesity via nutritional programming; and 8) determine the effects of genetic and epigenetic interactions with diet, nutritional status, weight gain, and behavior during gestation on placental and offspring development, health and susceptibility to chronic diseases, including obesity. It is essential to ascertain the long-term health consequences, both positive and negative, of early consumption of these phytochemicals since it may impact a major segment of our American population.

1b. Approach (from AD-416):
Studies will focus on the various dietary factors found in foods commonly consumed by children, such as infant formula, fruits, rice, 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 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, aorta, liver, adipose tissue, pancreas, and skeletal muscle; 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 from birth to age 6 years, The Beginnings Study. Breast-fed, milk formula-fed, and soy formula-fed children are studied for growth, development, body composition, and metabolism. In addition, bone development, and immune system development and function will be studied in children, and animal models will be utilized to explore molecular mechanisms underlying the effects of early dietary exposures. The rat model will be used to understand the parental genetic transmission of the susceptibility to high fat feeding to future generations and underlying molecular, biochemical, and endocrine mechanisms, in the offspring. 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) and the development of eating behaviors during childhood, adolescence, and later stages of life.

3. Progress Report:
In FY12, the Arkansas Children's Nutrition Center (ACNC) devoted significant effort towards understanding the potential differences in development and chronic health consequences associated with breast feeding or formula feeding. Breast milk differs from formula in many components, including: proteins, carbohydrates, fats, vitamins, and minerals, and while the American Academy of Pediatrics considers "breast to be best", the majority of U.S. infants are fed infant formula for a substantial period within the first year of life. Thus, understanding the differences is considered important to health of both children and adults. In the case of soy-based formula, there is a long running controversy over potential benefits or hazards for child development and long-term health consequences, such as growth and development; body composition; brain function and behavior; reproductive development; cancers of the reproductive organs; and bone quality. These issues have been raised mainly because of the presence of so called "phytoestrogens" bound to soy proteins, that act like estrogens under some conditions. During FY12, the ACNC has conducted studies in breast-fed and formula-fed infants to determine their growth and development differences. Because it is neither ethical nor practical to carefully and completely study most tissues of healthy infants to learn about organ development and function, we also study neonatal piglets that were either breast-fed or formula-fed. These animals are excellent models of human babies, and they allow for in-depth tissue analyses not possible in children. We have also studied the soy or milk proteins used in infant formulas in rats and mice to uncover mechanistic actions of these proteins and the plant- and animal-derived chemicals associated with them. The ACNC has been conducting a longitudinal study of infants fed breast milk, milk formula, or soy formula (called the Beginnings Study) from ages 3 months through 6 years. We have recruited more than 600 infants and nearly completed the data collection on children through age one year. Our studies in children demonstrated that breast-fed infants have some advantages over formula-fed infants in standardized behavioral testing of cognitive, motor, and language development during the first year of life. These effects were very small, but nonetheless, breast-fed infants scored better than formula-fed infants, suggesting that brain development can be impacted by early diet (nutritional content) and breast milk may be superior to formula. We also developed and standardized body composition procedures using a new quantitative nuclear magnetic resonance instrument we had built specifically for children. We compared this technique to other methods in the study of body composition in infants and found it to be excellent. We observed major differences in the body composition (fat and lean mass) amount of infants fed breast milk and formulas. Soy-fed infants had less fat and were leaner, and they grew at a faster rate over the first year of life. It is as yet unclear as to what advantages or disadvantages there are to each of these unique body composition and growth patterns. This year we compared the metabolic differences produced by cow's milk formula and soy formula in piglets, and found almost no evidence for estrogenic effects of soy formulas in the neonatal liver. We also compared bone in young rats fed the same protein source used in soy formulas with rats treated with estrogen. We found that soy and estrogen have different effects on bone prior to puberty. This occurs because while estrogen and soy turn-on and turn-off some of the same genes, soy and estrogens activate unique sets of genes, resulting in differences in liver metabolism and bone growth. The long-term health consequences of these effects will be investigated over the following years; however, for bone it appears that consumption of the soy protein used in soy formula improves bone development in rats and pigs. Our ongoing studies include analyses of different organs in the neonatal animals to examine these effects at the biochemical and molecular level. In piglet studies, we also found major differences in genes involved in iron storage and cholesterol metabolism of liver between breast- and formula-fed piglets. Similar to humans, these effects reflected differences in iron and cholesterol content of breast milk and formulas. "Maternal programming of fetal metabolism" is a term that refers to a process by which pregnant women can alter the metabolism of their fetus. This process can permanently alter the metabolism of the offspring. It is hypothesized that a significant proportion of children of obese women were programmed by their mothers in utero, resulting in childhood obesity. Because treatment of childhood obesity is difficult and more often than not is unsuccessful, the ACNC considers prevention to be the best long-term approach to ending childhood obesity. Obesity can be caused by several mechanisms, one of which is thought to be "metabolic programming". Therefore, women who are overweight when they become pregnant constitute an important research target population of the ACNC. We are studying maternal programming of fetal metabolism in two ongoing clinical studies (the largest is the Glowing Study), as well as animal studies (translational research). In fact, our clinical studies are designed based upon results from the animal studies. Thus, while we are studying lean and obese women during their pregnancy and their children until age 2 years, we are continuing our animal studies to learn about mechanisms to inform our future clinical studies. For example, this year we reported that the uterus of obese rats is inflamed and that an inflammatory response in the developing embryo could be responsible for the metabolic programming leading to reduced metabolic rate and increased fat accumulation in their offspring. In addition, we have been studying the placenta, which is the organ linking the mother’s body to the fetus and through which nutrients and oxygen pass. We studied placentas from obese rats that have different profiles of genes being turned on and off. These animal studies led to similar studies in human tissues. During the FY12 period, there were 212 women screened, resulting in 75 women being admitted to the Glowing study, with 66 pregnancies and 17 babies being born. The final goal is to enroll sufficient participants to study 320 infants from lean or obese women. In addition to obese women, we are studying overweight women. We have known that childhood obesity is higher in families where other family members are obese. This supports the hypothesis that obese parents could "transmit" obesity to their offspring, and we have focused on obese women in the Glowing study described above. However, this year our data demonstrated that even overweight women may transfer the risk of becoming obese to their children. This is potentially an extremely important finding, because it demonstrates that so many more children are exposed to conditions that may increase their risk for obesity. Another aspect to these data is the realization that even breast-fed infants can be metabolically programmed, suggesting that breast feeding does not necessarily protect against obesity. We have previously shown that diets containing soy or blueberries consumed during early development may significantly delay or prevent diseases later in life, such as atherosclerosis, breast cancer, and osteoporosis. In FY12, we have further characterized the mechanisms underlying long-lasting bone-preserving effects associated with blueberry consumption and identified compounds known as phenolic acids to be the bioactive compounds found in blood following consumption of blueberries to be responsible. These studies uncovered a novel mechanism by which these compounds extend the functional life of bone cells. These results suggest that blueberries may form the basis of new bone-enhancing dietary supplements. In similar studies, breast cancer prevention is being studied. Breast cancer may be initiated by a population of specific cancer-causing mammary stem/progenitor cells. Preliminary studies of the soy isoflavone genistein and blueberry polyphenols raise the possibility of breast cancer protective effects, whereby there is a reduction in the numbers of these mammary cancer-causing cells. Our studies demonstrated that genistein and polyphenols repressed the expression of inflammatory genes and proteins in the mammary tumor-initiating cells, thus linking reduced inflammation by diet in the prevention of breast cancer. Interestingly, genistein also inhibited formation of mammary fat cells that may contribute to initiating mammary cancer, especially in obese women where the risk is greater. We also continued our research into dietary prevention of atherosclerosis and found that blueberries prevent the formation of foam cells that form the basis of the fatty streaks that cause the lesions in aortic walls and which are the hallmarks of atherosclerosis. The above studies are a small part of a concerted effort to learn how early childhood diet may prevent heart disease, cancer, and bone disorders later in life.

4. Accomplishments
1. Breast-fed infants score higher than formula-fed infants on standardized tests. Questions remain as to whether infant formula supports brain development to the same extent as breast feeding. Researchers at the Arkansas Children's Nutrition Center in Little Rock, Arkansas, conducted a longitudinal study of 391 infants at ages 3, 6, 9, and 12 months to determine if the mental, motor, and language development differed between breast-fed, milk formula-fed, and soy formula-fed infants using the Bayley Scales of Infant Development. They found that all infants, regardless of feeding methods, scored within normal ranges, but breast-fed infants scored slightly higher on all tests than formula-fed infants, demonstrating that breast-feeding may provide an advantage to cognitive development. Importantly, there were no differences between milk formula and soy formula. Although the advantage for breast feeding was very small, these results support the recommendations of the American Academy of Pediatrics to breast feed during the first year of life.

2. Infants born to overweight women have more body fat. While there is evidence that maternal pre-pregnancy obesity results in greater infant body fat and elevated risk of obesity later in life, there is no evidence that women who are simply overweight deliver infants with higher body fat. Researchers at the Arkansas Children's Nutrition Center in Little Rock, Arkansas, studied the body composition of infants born to lean or overweight women. They found that total body fat mass was higher in infants born to overweight mothers compared to infants born to lean mothers, at ages 2 weeks and 3 months. Thus, infants of overweight mothers have a greater body fat mass accretion in utero compared to infants of lean mothers, suggesting that maternal overweight may predispose the fetal metabolism to favor fat storage. The long-term health consequences (such as type 2 diabetes or obesity in later life) of this greater fat storage very early in life is as yet unknown and the topic of ongoing research. These data are extremely important because there are more overweight than obese women of child-bearing age, and knowing if these women may be contributing to childhood obesity could mean that future development of prevention strategies that should target both obese and overweight women.

3. A new state-of-the-art method to assess body composition in children. Measurement of the amount of fat, muscle, bone, and water in children (i.e., body composition) is extremely important for understanding child development and the potential for developing obesity. While there are several methods to measure body composition in infants and children, they all have major deficiencies. A technical advance studied by researchers at the Arkansas Children's Nutrition Center in Little Rock, Arkansas, known as Quantitative Nuclear Magnetic Resonance (QMR), was previously tested in pigs and found to be very promising for children. Using a Pediatric QMR constructed specifically for infants and children, researchers assessed body composition in 113 infants and children and compared the results to three of the most widely used methods. The researchers found that after developing a new mathematical adjustment for analyzing the data, the Pediatric QMR provides a fast and precise method for longitudinally assessing body composition in infants and in children weighing up to 50 kg (110 pounds). Although an expensive technology, these results suggest that QMR may be the future method of choice for assessing fat mass in children.

4. Blueberries may protect the heart. Cardiovascular disease is the leading cause of death in the US, and diet is a risk factor that can contribute to or help prevent cardiovascular disease. One food recently reported to reduce atherosclerosis in experimental models is blueberries. Researchers at the Arkansas Children's Nutrition Center in Little Rock, Arkansas, have been conducting studies to learn how blueberries prevent atherosclerosis and what factors in the fruit are responsible. Atherosclerosis is caused by fatty deposits within the aorta, formed by cells containing low density lipoproteins. Results from four separate studies demonstrated that blueberries: 1) block cholesterol transport into cells (macrophages); and 2) prevent oxidation of cholesterol in these cells, thereby reducing inflammation that impairs macrophage function. These researchers further determined that the phenolic acids in the serum following consumption of blueberries appear to be responsible for many of these protective effects. In summary, the present study identified mechanisms by which blueberries may be cardio-protective and further identified phenolic acids as the causative factors.

5. Distinct gene expression signatures in the rat placentation site. The placenta is the earliest maternal/fetal organ to develop in all pregnant eutherian mammals (mammals that have a placenta) and is essential for fetal development. The placenta is entrusted with a number of functions, including the transport of nutrients and gases to support fetal growth, and providing protection against maternal immune response during pregnancy. To achieve these diverse functions, the rat placenta is composed of three diverse layers containing distinctive cell types. Scientists at the Arkansas Children's Nutrition Center, in Little Rock, Arkansas, utilized sequencing techniques to study the gene expression of these layers. Sequencing-based analysis allowed discovery of small proteins at very low levels and allowed researchers to elucidate unique gene networks that orchestrate functions within these placenta compartments. Most importantly, this work provides a novel resource to understand gene expression and function in different placenta compartments.

6. Maternal obesity promotes a pro-inflammatory signature in rat uterus and blastocyst. Understanding how maternal obesity influences the developing offspring is critical in mitigating the rise in obesity. Using a model of maternal overweight at conception, researchers at the Arkansas Children's Nutrition Center in Little Rock, Arkansas, studied regulation of genes in the uterus of obese pregnant rats shortly after conception. Using a technique to assay thousands of genes at one time (microarrays), these investigators discovered that pregnancy of obese rats led to accumulation of fat in the implantation site. This was accompanied with an increase in pro-inflammatory genes and proteins which led to biochemical changes in the developing embryo (blastocyst). These studies highlight the possibility that maternal body composition may alter the development of the offspring by influencing the milieu in which early development occurs.

7. Blueberries consumed in early life protect against adult bone loss. The effects of early diet on bone turnover (a lifelong process where mature bone tissue is removed and new bone tissue is formed) may influence how much peak bone mass is attained in individuals and the risk of osteoporosis in later life. Investigators at the Arkansas Children's Nutrition Center in Little Rock, Arkansas, have determined that blueberries fed during pre-pubertal development can enhance the formation of new bone cells (osteoblasts) and prevent them from losing activity as they age (senescence). The effect of blueberries appears to be through increases in cytoskeletal proteins (such as myosin) which control the shape of cells, and movement of other protein factors (such as Runx2) from the cytosol to the nucleus. Therefore, early blueberry consumption can influence osteoporosis risk in old age by increasing the life of bone cells, resulting in higher peak bone mass as adults.

8. Maternal obesity impairs fetal bone development. It has been suggested that the environment the fetus develops in during pregnancy may permanently alter later organ development. Investigators at the Arkansas Children's Nutrition Center in Little Rock, Arkansas, have shown that maternal obesity reduced the ability of fetal bone cell precursors to develop into mature bone cells (osteoblasts) and increased their likelihood of becoming fat cells instead. This was found to be associated with a specific modification (known as methylation) of an important gene in skeletal development (the HoxA10 gene). The effect of obesity was mimicked in cell culture by a certain type of free fatty acids (non-esterified) that increases as the level of fat tissue increases during development of obesity. These studies suggest that maternal obesity may result in permanent reductions in skeletal mass in offspring and increase fracture risk.

9. Targeting distinct breast cancer subtypes with diets. Breast cancer is a complex disease characterized by more than two dozen types of cells capable of becoming cancerous, making it extremely difficult to treat. The major challenge faced by clinicians is how to provide chemotherapy in sufficient doses that specifically target and kill mammary tumor-initiating sub-populations without eliciting side effects that dramatically impair quality of life. Using human mammary cancer cells, scientists at the Arkansas Children's Nutrition Center in Little Rock, Arkansas, found that the soy isoflavone genistein can inhibit the expansion of mammary stem/progenitor cells from well-differentiated estrogen receptor-expressing breast tumors (as well as from more invasive, non-estrogen receptor-expressing breast tumors). They further found that polyphenolic acids found in serum following consumption of blueberries, specifically hippuric acid, only targeted mammary stem/progenitor cells from the more deadly triple-negative breast tumors. These findings provide support to the role of genistein (and perhaps soy foods) and hippuric acid (and perhaps blueberries) in mammary tumor protection and raise the novel possibility of using these compounds or diet as an adjunct to standard breast cancer treatments for increased drug efficacy.

10. Formula feeding changes gene expression profiles and iron storage in liver. Although breast feeding is supported by the American Academy of Pediatrics, reports of iron deficiency in breast-fed infants are not uncommon. Investigators at the Arkansas Children's Nutrition Center in Little Rock, Arkansas, have used a neonatal pig model and a technique studying thousands of liver genes at once (called microarray technology) to show that formula feeding produces a liver gene expression signature and metabolic profile substantially different from that of breast feeding. Formula-fed piglets had lower liver iron stores, altered expression of iron-regulated genes, and lower cholesterol than breast-fed piglets. Cow's milk formula and soy formula-fed piglets differed significantly in which genes in the liver were turned on or turned off. Furthermore, genes typically regulated by estrogens were not affected by either formula. These studies suggest that metabolic responses of neonates to breast milk and formula differ significantly and that there is no evidence to suggest that soy formula has estrogenic actions in the neonatal liver.

Last Modified: 06/22/2017
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