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United States Department of Agriculture

Agricultural Research Service

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Location: Arkansas Children's Nutrition Center

2013 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:
The ACNC translational research program focused on two major areas: the effects of early infant feeding on growth and development (the Beginnings study); and the effects of maternal body composition childhood obesity (the Glowing study). The Beginnings study looks at the effects of infant diets on growth, body composition, and brain development of 600 children from birth through age 6 y who were fed one of three major infant diets used in the US (breast milk, milk-based and soy-based formula). Two hundred children/group were recruited, and data collection is nearly completed through age 2 y. This year, 239 study visits were completed. During these visits we studied various aspects of brain function using standardized psychological testing, and are correlating these effects to electrical brain activity outlined in our other ACNC project. Data from these studies are providing answers to questions regarding how and to what extent early nutritional status and diet influence child development during the formative period from birth through early childhood. Additionally, Beginnings is providing significant data on healthy infant and childhood body composition (fat, bone, muscle, and water content). In view of international concerns about the use of soy formula, documentation of child development in soy-fed infants is of particular importance to parents and pediatricians. To date, our results have shown that brain development and function are within the normal ranges for all three diet groups, although there are diet-related differences within the normal range that may have implications for future development. Importantly, the results to date have not revealed any adverse effects of soy formula on study measures. We continued animal studies to determine more in-depth mechanistic effects of early infant diet in development that cannot be conducted in children because of their invasive nature. Although population studies and some clinical studies have linked consumption of soy foods with health benefits (e.g., reduced blood cholesterol and certain cancers), questions have been raised concerning estrogen-like actions of isoflavones in the soybean. We compared the effects of the major estrogen (estradiol) on mammary gland development in young rats before puberty to the effects of a soy-containing diet in which 100% of the protein is soy. In young female animals, estrogens made the mammary gland more highly developed; whereas in young males, estrogens turned on and turned off many genes and stimulated cell division pathways that have been linked to breast cancer. In contrast to estrogens, the soy diet had little or no effect on the structure of the mammary gland and expression of only a very few genes were changed, suggesting that soy has negligible estrogenicity. These results thus far provide no evidence for concern of soy formula use. Our previous study results demonstrated that young pigs and rats fed soy-containing diets had better bone development than animals fed non-soy diets. It is also known that obesity impairs bone health and results in insulin resistance in bone. We recently observed that feeding a soy-containing diet improved insulin sensitivity in bone cells, and this appears to be a mechanism by which soy diets protect against obesity-associated bone loss. These effects were found to be at least in part associated with phytochemicals found in soy called isoflavones. We extended our studies on the bone-preserving effects of blueberries and uncovered a new mechanism whereby blueberries improve bone quality. The above studies are part of our research program designed to learn how early diet can optimize development and prevent adult diseases. Fatty liver disease is currently the number one cause of pediatric liver transplants, and diet composition is an important factor in the development of this disease. This year, we demonstrated that the type of dietary fat plays a key role in determining how much fat accumulates in the liver and whether it progresses to more serious inflammation and fibrosis. We demonstrated that dietary fats made with medium chain saturated fatty acids, such as MCT oil, are metabolized differently than longer chain fatty acids and that they stimulate the burning of fat, resulting in reduced weight gain and prevention of fatty liver when substituted for polyunsaturated fats, such as corn oil. Even when monounsaturated fats, such as olive oil, were overfed to rats to cause fatty liver, it prevented the progression of liver injury. These results suggest that even in high fat diets, such as the American diet, substitution of medium chain or monounsaturated fats for polyunsaturated fats can protect the liver, and suggest that a diet high in olive oil is health beneficial. In FY13, we also used a genetically modified mouse model to examine the role of oxidative stress in the development of obesity and fatty liver. We showed that mice lacking a key protein (P47phox) do not become fat or develop fatty liver when fed a high-fat diet during early development. These studies uncovered potential new targets for the prevention of obesity and fatty liver disease. In our Glowing Study, we have been studying a process known as "maternal programming of offspring metabolism" in animal models. We demonstrated that male rat offspring born to obese mothers are hyper-responsive to high-fat diets consumed later in life and become obese at a greater rate and to a greater extent than offspring from lean mothers. Based on these findings, we initiated a clinical study of infants of lean and obese women (the Glowing study), and we continue to conduct mechanistic animal studies to address questions not possible in humans. A central premise of these studies is that obesity during pregnancy produces a fetal environment that permanently changes the offspring's metabolism which leads to obesity in the offspring. The Glowing study is designed to test the hypothesis that fetal exposure to factors from obese women predisposes the offspring to early onset of overweight and risk of obesity later in life. Body composition (fat, muscle, bone contents) and measures of body growth (e.g., weight-for-length Z-scores) during the first two years of life will serve as the most important outcome variables of interests. Participants are recruited to enter the study between conception and the tenth week of their pregnancy. Future mothers are carefully characterized during pregnancy (metabolism, dietary intake, energy expenditure) and their children are studied for the first two years of life. Inclusion criteria include: pre-pregnancy body mass index between 18.5 and 35; pregnant with the second child; and singleton pregnancy. Exclusion criteria include: fertility treatments; preexisting medical conditions or medical complications during pregnancy; and smoking or alcohol use. Following a pilot study in FY11 (n=20), enrollment in Glowing started in June 2011. Since then, we have enrolled 172 women (as of 8/1/2013). A total of 96 women have completed pregnancy with 74 successful births. Five children have completed their 18-mo visit, 22 completed the 12-mo visit, and 47 are within their first year of assessments in the study. The targeted enrollment for Glowing is 300 women to yield 130 children at age 2 y (65/group). In FY13, we: enrolled 88 women (7/mo); completed 1044 study visits, including 595 mothers, 418 infants, and 32 father visits; collected 44 placenta, umbilical cord and cord blood out of 59 births; completed 416 blood collections; 606 urine collections; 73 subcutaneous fat biopsies; 270 breast milk collections; 339 indirect calorimetry assessments; and 594 food records. Our preliminary data demonstrate that women with higher fat mass had higher serum levels of insulin, leptin, C-reactive protein, IL-6 during pregnancy than women with lower fat mass (P<0.05) and gave birth to infants with higher fat mass at 2 wk and at 3 mo of age despite gestational weight gain within the Institute of Medicine recommendations. The following projects were worked on in FY13 and a peer-reviewed paper was published and 4 papers on human placenta or umbilical cord are in final stages of preparation: 1) Maternal obesity-associated placental dysfunction; 2) Umbilical cord gene expression in "appropriate for gestational age" infants born to lean or obese mothers; 3) A comprehensive analysis of human placenta transcriptome; 4) Changes in human umbilical cord stem cells leading to formation and maturation of fat cells; and 5) Obesity induces inflammation in human placenta. This latter paper was recently published (Am J Physiol Endo Metabol) and addressed the potential link between placental inflammation and maternal programming of fetal metabolism. It was determined that placental inflammation is orchestrated differently by key proteins in placentas from obese women than in lean women, and this may play a role in maternal programming of fetal metabolism. Data from mechanistic studies in animals are expected to be published in 6 peer-reviewed papers that are either in press, in review, or in final preparation: 1) maternal obesity disrupts circadian rhythms of offspring and leads to metabolic programming in liver; 2) maternal obesity influences mitrochondrial dynamics in the placenta and offspring; 3) maternal obesity produces transgenerational effects on metabolism; 4) maternal obesity disrupts thyroid signaling in placenta and offspring; 5) maternal obesity enhances DNA methylation in white adipose tissue; and 6) Obesity increases inflammation, oxidative stress, and insulin resistance in the ovary. We are also conducting a pilot study to test the hypothesis that oocyte gene expression profiles differ significantly between lean and obese women, leading to metabolic programming of their offspring. Thirty-nine women (17 lean, 8 overweight, 13 obese) have been recruited and oocytes collected from 34. Sample and data analyses should be completed next year.

4. Accomplishments
1. Body fat and bone accrual profiles differ for breast-fed and formula-fed infants. Breast feeding has been reported to have several developmental effects that are beneficial to child health and perhaps adult health. However, few studies have documented the differential effects of breast and formula feeding (or between different formula types) using modern technology for analysis of body composition. Researchers at the Arkansas Children's Nutrition Center in Little Rock, Arkansas, have studied fat mass and bone mineral content in 207 infants during the first year of life. They found unique total body fat (% body fat mass) profiles whereby breast-fed infants were significantly fatter than formula-fed infants until about age 9 months. Furthermore, infants fed soy formula were leaner and accumulated bone and body length faster than breast-fed or milk formula-fed infants. The long-term consequences of the rather dramatic and unique profiles early in life are unknown, but because neonatal programming of metabolism is thought to be of great importance to childhood and adult health, these results provide a basis upon which to conduct further studies.

2. The effects of body fat mass on psychological behavior measures. Psychomotor development is highly correlated with brain development and function. Although mental development has been reported to be impaired in obese children, less is known about psychomotor development in obesity. Researchers at the Arkansas Children's Nutrition Center in Little Rock used the Bayley Scales of Infant Development (psychomotor developmental index) and dual X-ray absorptiometry to study the relationship of body fat to psychomotor development. They found that greater fat mass and overweight status were associated with lower motor development in early childhood, although the overall mean scores remained within normal limits. These results provide significant evidence that increasing body fat in children can lead to impaired gross motor skills, and since this is also correlated to cognitive skills, future interventions to reduce pediatric obesity should be considered to maximize child development.

3. New biomarkers for monitoring the effects of environmental estrogens. Although there are many potentially estrogenic compounds in our environment (ranging from pesticides and pollutants to phytoestrogens in some plant foods) that might have reproductive toxicity or increase the risk of some cancers, there are few good biomarkers available to measure the full range of estrogenic actions in whole animal studies. Researchers at the Arkansas Children's Nutrition Center in Little Rock, Arkansas, have studied the dose-dependent effects of estrogen treatment on structure and gene expression in the immature breast tissue of male and female rats. They found that the immature male mammary gland is exquisitely sensitive to estrogen treatment which produced structural changes and a pattern of gene expression associated with increased cell division at very low doses. These changes represent new sensitive biomarkers of in vivo estrogenic actions in a reproductive tissue and can be utilized for the toxicological screening of new compounds, environmental samples and foodstuffs. This is an important discovery that should greatly improve the ability to identify relevant environmental estrogens.

4. Soy protein has minimal estrogenic actions on the adult mammary gland. There is considerable controversy over the potential for phytochemicals associated with soy-based food products (particularly isoflavones, such as genistein) to have estrogenic effects that might result in reproductive toxicity or increased cancer risk (e.g., breast cancer). Yet, few studies have actually compared the effects of estrogens and soy foods directly in target tissues, such as the mammary gland. Researchers at the Arkansas Children's Nutrition Center in Little Rock, Arkansas, addressed the question "is Soy Protein Isolate (SPI) estrogenic" by directly comparing the effects of the major natural estrogen 17-beta estradiol (E2) and SPI, the sole protein source in soy infant formulas, on mammary gland structure and gene expression in adult female rats. Whereas E2 significantly changed mammary structure and increased cell division pathways linked to breast cancer, SPI had no effect on mammary structure and only minimal effects on gene expression. Moreover, the combination of SPI and E2 resulted in a blunting of E2 effects. These data suggest that SPI has minimal estrogenic properties and actually acts as an anti-estrogen in the adult mammary tissue. These observations are consistent with epidemiological studies suggesting that eating soy foods reduces rather than increases the risk of breast cancer or its reoccurrence.

5. Diets rich in olive oil do not promote the progression of fatty liver disease. Nonalcoholic fatty liver disease (NAFLD) is currently the number one cause of liver transplants in children and is associated with overconsumption of high fat diets and obesity. Few studies have compared the effects of different dietary fat types on progression of simple fatty liver to NAFLD to more serious pathology, including development of inflammation and fibrosis with reduced liver function (known as NASH). Researchers at the Arkansas Children's Nutrition Center in Little Rock, Arkansas, have demonstrated that overfeeding rats diets rich in olive oil resulted in fatty liver that did not progress to liver injury, whereas diets made with corn oil did progress to severe liver injury. These studies suggest that even when high fat diets are eaten in excess sufficient to produce obesity, monounsaturated fats can protect the liver. This provides additional evidence that a Mediterranean diet, high in olive oil, is health beneficial.

6. Blueberries prevent bone cell death and bone resorption. Recent studies have shown that blueberries have powerful effects to increase bone thickness and quality when consumed during early development and can increase the formation of new bone cells. Researchers at the Arkansas Children's Nutrition Center in Little Rock, Arkansas, have uncovered new mechanisms whereby blueberries improve bone quality. They found that blueberries and phytochemicals from blueberries inhibit the development of osteoclasts (cells that eat away bone) and prevent cell death of osteoblasts (cells that make bone). These studies are part of a research program designed to learn how early diet can optimize development and prevent adult diseases and have revealed new targets for novel diet-derived neutraceuticals that can be used to prevent degenerative bone diseases such as osteoporosis.

7. Molecular pathways regulate fat accumulation and development of fatty liver disease. It is known that sex steroids such as the primary female hormone (estradiol) play a role in the regulation of fat accumulation and that removal of sex steroids by menopause or castration increases fat mass. In addition, it has recently been shown that development of obesity is accompanied by appearance of oxidative stress and low grade inflammation in the fat tissue. Researchers at the Arkansas Children's Nutrition Center in Little Rock, Arkansas, have demonstrated that young developing female mice lacking a key protein (P47phox) required for the activity of a member of the NADPH-oxidase (NOX) family of enzymes to generate oxidative stress do not become obese, and neither males or female develop fatty liver when fed a high fat diets. These studies provide new fundamental insights into the control of fat metabolism and storage, and have uncovered potential new targets for developing a prevention strategy for obesity and fatty liver disease.

8. Soy protects bone from the effects of obesity. Recent studies have suggested that diets containing soy foods can improve glucose tolerance by increasing insulin sensitization in animals fed high fat diets, and this may prevent the development of metabolic syndrome. Researchers at the Arkansas Children's Nutrition Center in Little Rock, Arkansas, have now shown that the previously observed insulin sensitizing effects of soy: 1) extend to bone cells and other non-liver tissues; and 2) reversal of insulin resistance in bone contributes to the protection soy diets provide toward preventing obesity-associated bone loss. Furthermore, the researchers were able to show that the effects of soy diets on bone cells were at least in part associated with phytochemicals found in soy called isoflavones. These data suggest that soy foods and dietary isoflavone supplements may improve bone quality in addition to reducing blood sugar in obese individuals.

9. The effects of soy protein and the soy isoflavone genistein on mammary gland fat cells. Fat cells (adipocytes) are thought to play an important role in breast cancer development and progression. Consumption of soy foods is associated with lower breast cancer rates and lower body fat, but the relationship between soy consumption, mammary gland fat content, and breast cancer has not been established. Researchers at the Arkansas Children's Nutrition Center in Little Rock, Arkansas, found that diets containing soy protein or the soy phytochemical genistein blocked fat cell formation, reduced mammary gland fat content, increased molecules known as tumor suppressors, and blocked cancer-stem cells from progressing toward a cancerous condition in cell culture. These results suggest a potential mechanism by which soy can help prevent breast cancer and may result in the development of a strategy to help prevent the initiation of breast cancer.

10. Soy beans may act to prevent breast cancer. Breast cancer is one of the major chronic diseases of women, and prevention of this disease is much preferred to treatment. Soy food consumption is associated with lower breast cancer rates, but the identity of the active components within soy is still unclear. Two potentially important factors in soybeans are a 43 amino acid peptide (lunasin) and a soy isoflavone (genistein), each of which has been reported to be potential cancer preventive agents. Researchers at the Arkansas Children's Nutrition Center in Little Rock, Arkansas, have studied these compounds to determine if they act through the same mechanisms. Results from this study suggest different mechanisms of action, but the combination of both factors provided increased death (apoptosis) to malignant and non-malignant mammary epithelial cell, actions that are thought to be important in prevention of breast cancer. These results provide potentially important information to help support the use of soy foods in the development of a healthy lifestyle regimen for women at high risk of developing breast cancer. These data also demonstrate that the health effects of food and diets are the result of additive or synergistic actions of many separate food components (phytochemicals, peptides, etc.), and may not be replicated by supplements made from an individual or even several bioactive components.

11. Maternal blueberry consumption may program mammary gland development and risk of breast cancer. Breast cancer is one of the major chronic diseases of women, and environmental exposure (e.g., to dietary factors) is an area of intense research to find either causes or preventions to this disease. Researchers at the Arkansas Children's Nutrition Center in Little Rock, Arkansas, have been studying blueberries because they have been shown to affect metabolism of several organs, including the mammary gland, and these actions could lead to a strategy to prevent breast cancer. They found that maternal consumption of a blueberry-containing diet during pregnancy and lactation reduced tumor size in the mammary gland of offspring genetically predisposed to mammary gland cancer in association with lower serum concentrations of insulin which may also act as a tumor growth factor. Results from this study add to previous results showing how diet can be an important factor in disease prevention. There is an expectation that this can help lead to a dietary strategy that helps reduce breast cancer risks in the future.

12. Saturated fatty acids can lead to inflammation of the placenta. Obesity is associated with low-grade chronic inflammation, which contributes to cellular dysfunction promoting metabolic disease in the offspring. Using an established cell culture model of placental cells, obesity researchers at the Arkansas Children's Nutrition Center in Little Rock, Arkansas, studied the mechanisms by which saturated fatty acids induce inflammation in placenta. These investigators found that saturated fatty acid treatment predominantly altered expression of genes that led to increased inflammation and immediate-early response. These results in cell culture shed new light on how saturated fatty acids and high fat diets may contribute to the development of placental inflammation and dysfunction, and provide an underlying basis for more in-depth studies of human placentas to increase our understanding of how mothers may influence long-term health of their offspring.

13. Maternal obesity is associated with increased inflammation of placentas. Obesity during pregnancy leads to a placental inflammation; however, the underlying causes for obesity-induced placental inflammation in women remain unclear. Recent work in animals by scientists at the Arkansas Children's Nutrition Center in Little Rock, Arkansas, suggests that obese women can program metabolism of their offspring and this may be liked to inflammation. To determine the extent to which inflammatory processes of obese women differed from that of lean women from the Glowing study, metabolic pathways known to produce inflammation were studied in placentas from term pregnancies. Researchers were able to determine that placental inflammation is orchestrated differently by key proteins in placentas from obese women than in lean women, and this may play a role in maternal programming of fetal metabolism.

14. Excess caloric intake recognized as a major source of toxicity. Now that the American Medical Association has declared that obesity is a disease, significantly more attention is being paid to the mechanisms underlying obesity development. There is a need to educate trainees and professionals in both industry and government in the current science underlying obesity. Scientists at the Arkansas Children's Nutrition Center in Little Rock, Arkansas, are at the forefront of new research into molecular mechanisms underlying increased fat accumulation and the health consequences of obesity and were asked to write a new chapter in the sixth edition of the textbook "Casarett & Doull's Toxicology" on the toxic effects of excess calories. This book is the major textbook used by graduate programs in toxicology in the U.S. and is the text used for Board Certification in Toxicology by professionals in industry and government worldwide. Addition of a new chapter on obesity represents recognition that this is a new field of research for the toxicology community and recognition that the ACNC is a leader in this area.

Last Modified: 08/15/2017
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