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Research Project: Effects of Diet and Physical Activity on Maternal/Child/Adolescent Health and Development

Location: Arkansas Children's Nutrition Center Microbiome and Metabolism Research Unit

2022 Annual Report

The mission of Cooperator's Arkansas Children's Nutrition Center (ACNC) is to determine the effects of diet, physical activity and nutritional status in childhood and in mothers during the pre- and post-conception periods on growth, development and health by conducting research on: 1) central nervous system and cognitive development and function of infants and children; 2) roles of maternal and child body composition, metabolism and genes (including responses to food macronutrients, phytochemicals and microbe-derived factors) on infant and child growth, development and the long-term health consequences of these factors; and 3) impact of parental and child physical activity on these parameters, singly or in combination with diet, and the biological basis for exercise-associated behaviors and health outcomes. To accomplish this mission, the ACNC has the following objectives and as appropriate, others as mutually agreed upon: A. Determine how maternal/parental diet, exercise, and metabolic health associate with psychological/behavioral, neurologic and physiological function and development in children. B. Understand how postnatal diet, childhood physical activity, and nutrition associate with psychological/behavioral, neurologic and physiological function and development in children. C. Characterize the interaction between the microbiome, host health and nutrition to modify physiology and promote health and development (skeleton, gastrointestinal and hepatic tissues, immune system, cardiovascular, and muscle, e.g.).

To accomplish these objectives, a research plan encompassing the specific research described below is authorized. Research is to be accomplished over a multi-year period as resources are available and as readiness for research and management capabilities develops. The plan shall be divided into the following areas: A. Determine how dietary components, dietary patterns, and metabolic health influence psychological-behavioral outcomes, neurodevelopment, and physiological function in children. Children will be studied longitudinally and cross-sectionally to evaluate the effects of child/parental diet, metabolic health, and body composition on psychological-behavioral, neurologic and physiological function. B. Determine how physical activity and fitness influence psychological-behavioral outcomes, neurodevelopment, and physiological function in children. Children will be studied longitudinally and cross-sectionally to evaluate the effects of child/parental physical activity patterns, fitness, metabolic health, and body composition on psychological-behavioral, neurologic and physiological function. C. Characterize the impact of host-microbe interactions on health and development. Children will be studied longitudinally and cross-sectionally to evaluate the factors linking child/parental microbiome with metabolic health, body composition, physical activity outcomes, psychological/behavioral, neurologic and physiological functions. D. Center Development. Cooperator has the following general areas of research in the Arkansas Children’s Nutrition Center (ACNC), which address the mission area of parental/child health: Brain Development and Central Nervous System Function; Dietary Factors and Physical Activity Effects on Health, Development and Function of Organ Systems (skeleton, gastrointestinal and hepatic tissues, immune system, cardiovascular, and muscle, e.g.); and Obesity and its Effects on Cellular and Physiologic Function in mothers, infants and children.

Progress Report
For Project 1, Subobjective 1A is examining the impact of maternal high fat diet (HFD) on energy homeostasis, adipose tissue development and metabolism. Cooperating researchers had issues breeding high fat diet-fed dams resulting in limiting numbers of offspring. Metabolic phenotyping of postnatal diet studies and postnatal cold exposure studies are ongoing. Subobjective 1B is determining the role of Ezh2 signaling in maternal HFD programming of postnatal skeletal development. LsyM-Cre Ezh2flox/flox mice have been developed that lack Ezh2 in pre-osteoclastic macrophage. Deletion of Ezh2 increased gene expression of osteoclast suppressors IRF8, MafB and Arg1, and decreased expression of NFATc1 and Cathepsin k. Maternal HFD also alters histone trimethylation and acetylation of genes controlling osteoblastogenesis. Subobjective 1C explores how maternal HFD programs offspring vascular cells and cardiovascular outcomes. We completed arterial contractility studies examining function of perivascular adipose tissue, smooth muscle cells, and endothelial cells in offspring at 20 weeks of age. Investigation of heat shock protein expression and analysis of vascular inflammation markers has begun. In Subobjective 2A research continues on the role of oocyte MyD88/TLR signaling in maternal HFD programming of offspring. Collaborating scientists are generating flox control and oocyte specific MyD88 knockout mice for generation of offspring for postnatal studies. Subobjective 2B studies placental MyD88/TLR signaling in maternal HFD programming of offspring. A collaborating researcher generated flox control and trophoblast specific MyD88 knockout mice. Male flox control offspring from HFD-dams fed HFD had greater weight gain relative to controls. Effects of maternal HFD were not present in placental MyD88 knockout offspring from HFD-dams fed HFD, revealing a pivotal role for placental TLR signaling in programming of obesity. A manuscript is in preparation. For Project 2, Subobjective 1B explores postnatal feeding practices and impacts to long-term systems development. To date, collaborating scientist in Little Rock, AR have enrolled 14 participants (14 years). Another 9 participants will turn 14 years between now and June 30. In total, 121 participants have been enrolled. Preliminary analyses indicate no significant differences between children fed human milk, soy-protein or dairy based infant formulas on growth, body composition, serum markers, age of menarche and bone mass accretion. Objective 2 explores if early life exposure to metabolites generated from gut microbial metabolism of blueberry phenolics can suppress bone resorption activities of osteoclasts and limit osteoblast senescence. A collaborating researcher made tissue specific gene knockout mice to determine the role of GPR109A in childhood bone modeling finding thatphenolic metabolites promoted bone growth and development through direct interaction with GPR109A. Subobjective 3A and 3B continue to explore if breastfeeding results in stronger immune function relative to formula feeding, how this relates to gut microbiota and how microbial metabolites including novel cyclopropane fatty acids (CpFAs) affect immune cell response. Subobjective 3C studies changes in host metabolic health as influence by composition and function of gut microbiota. Diabetes progression was found to change ileal transcriptome and ileal-colonic morphology in a rat model of spontaneous diabetes. A linear increase in ileal villi length, ileal crypt depth, and colonic crypt depth was associated with diabetes progression. Diabetes progression had little effect on the colonic transcriptome, suggesting the upper bowel is more susceptible to diabetes. For Project 3, Subobjective 1A investigates if greater maternal weight during gestation increases infant weight and risk of childhood obesity at age 5y and 8y. This year, a collaborating researcher has completed 27 study visits and collection of flow mediated dilatation data. Analyses have been completed on diet, body composition, gut microbial ecology, human milk branched-chained amino acids, neonatal brain cortical thickness and dietary fatty acid oxidation. Subobjective 1B continues to study the impact of maternal BMI on umbilical cord stem cell differentiation into adipose tissue. Adipogenic differentiation, gene expression and protein characterization of key targets, lipid staining, and supernatant triglyceride estimation have been completed. Subobjective 2A, 2B and 2C continues to explore if maternal exercise during gestation can decrease a child’s susceptibility to obesity by age 2y, alters composition of maternal gut microbiota, or decreases placental pro-inflammatory signatures. This year, 117 study visits were completed, 37 prenatal and 49 postnatal stool samples were acquired, and 5 placenta/umbilical cord samples were collected. Collection of placenta was impacted due to COVID restrictions at delivery hospitals. Subobjective 2D continues to examine the feasibility and demonstrate fidelity of a community-adapted exercise intervention during pregnancy. A collaborating researcher completed in FY22 adaptation of the Community Expecting study for virtual delivery and a pre-test of the virtual protocol with enrollment of 8 women. Results were presented at a national conference. Community sites and trainers have been onboarded for a hybrid pre-test, but recruitment was delayed by COVID-19. Research in Subobjective 3A continues to investigate effects of exercise and a healthy diet on reproductive health. All study procedures have been piloted and tested successfully and a collaborating researcher has enrolled 5 participants in FY22, but recruitment/enrollment were significantly impacted by COVID restrictions in this population. For Project 4, Subobjective 1A, 1B, and 1C studies impacts of physical activity, physical fitness, and obesity on mitochondrial function, substrate usage, and protein requirement in children. A total of 114 children have been enrolled in 1A, and 36 enrolled in 1B to test substrate usage at submaximal exercise intensities. A methodological study on platelet mitochondrial function was completed. For Subobjective 1C the protocol is being refined and a pilot study is ongoing to test feasibility of experimental diet preparation and palatability and protocol flow. Subobjective 2A explores maternal weight status (overweight vs. normal) before/early in pregnancy and affects offspring physical activity behaviors. Data collection from 2 and 5 yr-old children is completed, and analyses from 2-yr-old completed. Testing of 8-yr-olds progresses. Subobjective 2B is testing feasibility of a physical activity intervention in pre-school age children born to mothers with obesity. The population-level needs assessment including 267 surveys and 30 interviews are complete and have influenced adaptations to serve Arkansas families. Data collection protocols and intervention were tested with 3 mother-child dyads. A focus group was then conducted to inform future intervention designs. Subobjective 2C explores if early-life physical activity will result in persistent effects on energy and substrate metabolism. A collaborating researcher found that rat pups born to parents with low cardiorespiratory fitness weigh more, have greater adiposity, altered glucose homeostasis, and exhibit altered liver and muscle bioenergetics compared to pups born to parents with high cardiorespiratory fitness. Studies are examining if early life physical activity can rescue metabolic phenotype of rats born to parents with low cardiorespiratory fitness. Subobjective 3 continues to determine the role of muscle myoglobin in lipid trafficking. Mouse phenotyping studies are complete. In silico binding studies of oxy- and deoxy-Mb to mitochondrial membrane have confirmed that Mb releases O2 at a faster rate upon interaction with lactate in acidic conditions compared to physiological conditions. For Project 5, Objective 1 continues to study effects of early diet on neurocognitive development in healthy children and adolescents. This year, the impact of diet on development of infant brain including language-specific perception by the infant has been completed and presented at the Organization for Human Brain Mapping. A manuscript is in preparation with submission planned next year. In Objective 2 Data collection is underway to evaluate the impact of maternal obesity on brain development and function of children. Children undertake two tasks testing their cognitive development, and the study team continues to process data with approximately half of datasets now preprocessed. Objective 3A continues to study effects of diet composition, meal pattern and exercise on brain function and behavioral dynamics in normal weight and obese children. Recruitment and enrollment is largely complete and preprocessed. Initial findings suggest obesity in preadolescents affects attention and inhibitory resources. Obese preadolescents had less ability to modulate conflict-monitoring and attention allocation suggesting differences in development between normal weight and obese preadolescent children. Preliminary results were submitted for presentation at the Society for Neuroscience. Preparations for Subobjective 3B continue. For this study, a new cognitive task was designed, and pilot tested on study team members. Pilot testing in pre-adolescents and recruitment will follow. For Project 6, research begins on Subobjective 1A which will determine the role of maternal obesity on the immune system during pregnancy. Sample have been submitted for single cell sequencing. Research continues on Subobjective 2A to determine the role of infant microbiota on gut and immune system. An animal breeding colony has been set-up and studies are ongoing.

1. Maternal weight status and breastfeeding duration impacts how children oxidize dietary fatty acids. To better understand how excessive maternal weight impact children’s metabolism, cooperative researchers in Little Rock, Arkansas, have investigated the dietary fatty acid (palmitate) oxidation in children age 2-year-old who were born to mothers with normal weight, overweight or obesity. Results demonstrated that breastfeeding duration modifies the association between maternal weight status early in pregnancy and dietary fatty acid oxidation of their 2-year-old children. The study also identified that maternal physical activity early in pregnancy is a factor that independently associates with dietary fatty acid oxidation in 2-year-old children. These results suggest that exercise in early pregnancy may favorably modify physiological responses in the offspring directly associated with fat deposition and utilization.

2. Maternal obesity impacts infant gut microbiome in the first year of life. To better understand how excessive maternal weight changes infant microbiome, cooperative researchers in Little Rock, Arkansas, have investigated the difference in gut content of infant born to mothers with normal weight compared to mothers with overweight or obesity. Results demonstrated that maternal excessive weight was associated with relative depletion of butyrate producing microbes and fecal butyrate in the early infant microbiome. Findings also identified that microbial richness may aid in prediction of elevated adiposity in later infancy.

3. Maternal exercise in sedentary women with obesity does not impact long-chain fatty acid availability or oxidation. To better understand how exercise during pregnancy impacts metabolism, cooperative researchers in Little Rock, Arkansas, have investigated the longitudinal changes in long-chain acylcarnitine concentrations during the first and third trimesters of gestation in sedentary women with obesity who were assigned to an exercise intervention or standard of care. Results demonstrated that a moderate-intensity exercise intervention during pregnancy, consisting of both aerobic and resistance training, in women with obesity did not negatively impact normal alterations in substrate supply and demand for the mother and the offspring throughout gestation.

4. Maternal overweight impacts human milk branched-chain amino acids which influence infant growth. To better understand how excessive maternal weight changes human milk composition, cooperative researchers in Little Rock, Arkansas, have investigated the difference in specific amino acids (branched-chain amino acids) content of human milk from mothers with normal weight compared to mothers with overweight or obesity. Not only was the human milk from mothers with overweight and obesity different in amino acid content, but also, infant intakes of these specific amino acids were associated with growth and body composition.

5. Maternal excessive weight impacts neonatal brain cortical development. To better understand how excessive maternal weight changes neurodevelopmental outcomes in children, cooperative researchers in Little Rock, Arkansas, have investigated the relationships between maternal obesity status during pregnancy and neonatal brain cortical development. Results demonstrated that maternal obesity status during pregnancy is associated with lower neonatal brain cortical thickness in several frontal lobe regions of the brain important for language and executive functions.

6. Maternal diet and body composition are associated with gut microbial ecology in pregnancy. To better understand how offspring obesity risk is linked to maternal body composition, gestational weight gain and dietary intake, cooperative researchers in Little Rock, Arkansas, have investigated the gut microbiome at each trimester of gestation to evaluate microbial ecology. Results indicated that there are pregnancy- and maternal obesity-dependent interactions between dietary factors and the maternal gut microbiome.

7. Protocol to investigate the effect of a dietary and exercise intervention in women with overweight and obesity on oocyte gene expression and follicular fluid content. To better understand how a peri-conception nutrition and exercise intervention mitigates obesity-associated changes in samples collected during in vitro fertilization treatments, cooperative researchers in Little Rock, Arkansas, have designed and published a study protocol to collect rigorous and reproducible data to evaluate both the dietary and exercise impact on oocyte gene expression and follicular fluid metabolites.

8. Digitally-based interventions for supporting physical activity for children are more effective when they include child-directed components. To better understand the effects of digitally based interventions to stimulate physical activity (PA) in young children, cooperative researchers in Little Rock, Arkansas, conducted a systematic review of the literature to determine the quality of studies using digital PA intervention strategies with preschool-aged children. The review also assessed the efficacy of digital interventions and approaches designed to improve PA in preschool-aged children and examined theoretical application and implementation. The published review identified 8 prior studies in this area, 2 of which showed promising effects. Interventions that targeted parents as an indirect means to increase child activity were not effective; whereas interventions that included child-directed activities showed positive effects.

9. Sex Differences in Skeletal Muscle. The foundations and characteristics of inherent sex differences in skeletal muscle functions remain to be elaborated.. To better understand these differences, cooperative researchers in Little Rock, Arkansas, evaluated muscle fibers, blood capillaries, and large-scale gene expression in female and male mice. The results highlight thousands of innate transcript differences. Data analyses and literature review revealed a specific set of genes that appear to trigger and then maintain sexual dimorphism in muscle of mice and humans. These discovered pathways provide a foundation to design evidence-based, sex-specific interventions to optimize muscle function and metabolism.

10. Cardiorespiratory fitness determines health outcomes in children. High blood pressure (HBP) is a classic cardiovascular disease risk factor known to damage the heart, blood vessels, and kidneys. Children who suffer HBP are likely to also have HBP as adults. In adults, strong evidence supports that physical fitness is a major determinant of HBP and health, whereas less is known in children. To better understand the associations between fitness and risk of HBP in children, cooperative researchers in Little Rock, Arkansas, measured peak aerobic capacity and health outcomes in children ages 7-10 years. Results showed that both components of blood pressure (systolic and diastolic) improve with increasing fitness in all children, regardless of weight status, and the probability of HBP in children with excessive weight decreased when fitness increased. Liver function, sugar metabolism and kidney function were also improved in children that were more fit. In conclusion, all children benefit from improving fitness levels as evidenced by healthier blood pressure values, improved kidney function, sugar metabolism and liver health.

11. Circulating microRNAs in children with increased liver fat and insulin resistance. To better understand if regulation of gene expression is impacted in children with obesity and metabolic complications, cooperative researchers in Little Rock, Arkansas, studied the circulating microRNAs, sugar metabolism, and insulin, as well as liver fat content in adolescent children with obesity. The microRNAs are small molecules that regulate the type and amount of proteins a cell is making. The investigators found that several miRNAs were differentially expressed in children with sugar metabolism impairment and elevated liver fat content. Measurements of circulating microRNAs may be a useful tool in studies aiming to prevent future diabetes and fatty liver in children and adolescents with obesity.

12. Obesity increases inflammation in perivascular adipose tissue. How maternal obesity alters offspring perivascular adipose tissue remains to be fully understood. To better understand how maternal obesity leads to increased cardiovascular disease risk in offspring, collaborating researchers in Little Rock, Arkansas, performed a thorough review of the published literature to identify what is known about the molecular mechanisms by which maternal obesity induces inflammation in offspring perivascular adipose tissue. Findings point to further investigations of the interaction between perivascular adipose tissue and leukocytes, specific type of immune cell important in mediating tissue inflammation.

13. Human milk feeding shapes beneficial microbial composition in the large intestine. Exclusive breastfeeding is recommended to newborns during the first 6 months of life by the World Health Organization and American Academy of Pediatrics. Using a piglet model, our group has previously reported differences in the gut bacterial composition of piglets that were human milk fed compared to milk-formula. However, less is known about the fungal composition and how that associates to bacterial composition in the gut. To address this knowledge gap, an ARS researcher in Little Rock, Arkansas, utilized piglet model of human milk feeding. Data demonstrated the colonization and growth of beneficial bacterial abundance (i.e Lactobacillus sps, Bacteroides) in human milk fed group while gram negative bacteria (i.e Proteobacteria and Burkholderiales bacterium) that were associated with inflammation and asthma outcomes were higher in formula fed group. For fungal taxa, Aspergillus spp which was associated adiposity and weight gain and increased incidence of rhinitis was higher in formula-fed relative to HM group. It is possible that inter-kingdom communication occurs between bacterial and fungal species. Overall, our findings indicate that human milk diet associated microbial changes are beneficial to the infant’s health outcomes.

14. Plant Based Formula was safe and likely improves liver function. Human milk is the gold standard for infant nutrition and health during the first year of life, while infant formula is recommended as an alternative nutrition source to human milk. In the U.S., approximately 25% of the infant formulas available in the market are soy protein based. Studies demonstrated that soy-fed infants have a similar growth and development compared to breastfed infants. However, some concerns including the soy-based formula diet composition and the risk of lower bone mineralization among others risks have raised. Therefore, an ARS researcher in Little Rock, Arkansas, evaluated the safety and the effects on growth and blood biomarkers of a plant-based infant formula containing almonds, and buckwheat as the main ingredients using piglet model. No diarrhea was observed after PND 8 and all the piglets completed the trial. Body growth, kcal intake, and intestinal organs development was similar between the two groups. Blood glucose was higher in the dairy-based fed piglets relative to the plant-based at 2 weeks of age. Liver function biomarkers levels were greater in the blood of the plant-based compared to the dairy-based fed group. In addition, calcium levels were higher in the plant-based fed piglets at 1 week of age. Hematological parameters were within the reference range in both groups. Thus, the plant-based formula tested in this study was well-tolerated by the piglets and supported similar growth to dairy based milk formula. Additionally, the positive clinical outcomes observed in the blood of the plant-based fed group suggest an improvement in the liver function during the neonatal period in comparison to dairy-based formula feeding.

15. Human milk oligosaccharides impact cellular and inflammatory gene expression and immune response. Human milk contains several bioactives that support infant growth and development. Human milk oligosaccharides (HMOs) are the third most abundant bioactive component present in human milk. HMOs have been shown to change intestinal microbiota (i.e., gut milieu) composition, and prevent pathogenic bacteria binding to the gut by serving as decoy receptors. However, the direct effect of HMOs in the absence of the gut microbiota on intestinal function and immunity remains to be elucidated. To address this knowledge gap, ARS researchers in Little Rock, Arkansas, used a germ-free mouse model, measured gut and immune system outcomes. Gut epithelial layer length (i.e., villi and crypt) was reduced in HMOs treated group in comparison to controls. In addition, HMOs treated mice showed gene changes involved in the mucus layer, immune system, transport and absorption of nutrients and changes in immune cell composition were noted. These data suggest that HMOs have a direct effect on the gastrointestinal tract metabolism and immune system even in the absence of host microbiota.

16. Diabetes progression has a larger effect on total gene translation in the upper bowel. Gut bacteria contribute to health and disease. More recently, gut bacteria have been implicated as a contributing factor for several disease that effect a large amount of children and adults, such as obesity and diabetes. ARS researchers in Little Rock, Arkansas, have previously shown that the profile of bacteria in the gut can be altered by changes in blood sugar levels using a rat model that spontaneously develops diabetes without changes in diet. To date, the way that diabetes changes the profile of bacteria within the gut has not been clearly identified. Researchers at the Arkansas Children’s Nutrition Center, in collaboration with scientists at the University of California Davis, measured which genes are actively transcribed in the upper and lower bowel during the progression diabetes in the spontaneous rat model of diabetes and found that the upper bowel is more strongly affected than the lower bowel. As the vast majority of bacteria reside in the lower gut, this suggests that diabetes may not directly impact the gut environment associated with gut bacteria. However, diabetes may indirectly influence lower bowel bacteria via changes in upstream physiology.

17. Oxygen regulation in intense exercise. Myoglobin (Mb) is a central protein for storage and transport of oxygen (O2) in the muscle tissues, but it is not fully understood how cellular molecules regulate Mb-O2 binding. By using advanced machines to measure binding of molecules, cooperative researchers in Little Rock, Arkansas, evaluated how various pH levels caused by varying concentrations of lactate affect the Mb-O2 binding. Through this, the scientists could mimic conditions of rest and exercise. Researchers found that lactate interaction with Mb led to rapid release of O2 from the Mb. This supports findings that molecules released during exercise regulate O2 release from the Mb. The O2 is then delivered to the mitochondria in the cells where it is used in the process of creating ATP which again enable further muscle work in periods of shortage of O2 delivery.

Review Publications
Piccolo, B.D., Graham, J.L., Kang, P., Randolph, C.E., Shankar, K., Yeruva, V., Fox, R., Robeson, M.S., Moody, B., LeRoith, T., Stanhope, K.L., Adams, S.H., Havel, P.J. 2021. Progression of diabetes is associated with changes in the ileal transcriptome and ileal-colon morphology in the UC Davis Type 2 diabetes mellitus rat. Physiological Reports. 9(22):e15102.