Project : USDA ARS

ARS Home » Plains Area » Houston, Texas » Children's Nutrition Research Center » Research » Research Project #444343

Research Project: Regulatory Aspects of Nutritional Metabolism

Location: Children's Nutrition Research Center

2025 Annual Report

Objectives
Researchers will 1) determine the immune protective function of human milk and bovine colostrum-derived immunoglobulin A and protection against necrotizing enterocolitis (NEC) in a formula-fed, preterm pig model; 2) determine the impact of citrulline and arginine supplementation on metabolism and prevention of NEC incidence in formula-fed preterm pigs; 3) determine the nutritional regulation of muscle growth following preterm birth and develop targeted amino acid supplementation to promote lean growth and optimal development; 4) determine the optimum bioavailability of different chemical forms of choline that maximize the plasma and tissue deposition and function of long chain fatty acids in preterm pigs; 5) determine the impact of a maternal high-fat diet on the development of thermogenic adipocytes in the offspring; 6) define how early-life transient overexpression of gap junction protein Connexin43 slows fat accretion when mice are fed a high-fat diet in adulthood; 7) define the critical window when gut microbial exposure shapes the epigenetic regulation of intestinal stem cell function; and 8) determine the impact of perinatal nutrition and exercise on cardiovascular development and health.

Approach
This research will be accomplished using a variety of models and scientific studies. Researchers will use neonatal piglet models to determine the extent to which the anabolic resistance to nutrition contributes to reduced muscle growth in the preterm and whether targeted amino acid supplementation will promote lean growth. We will determine the immune protective function of human milk and bovine colostrum-derived immunoglobulin A (IgA) and protection against necrotizing enterocolitis (NEC) in the preterm. We will determine the optimum bioavailability of different chemical forms of choline that maximize the plasma and tissue deposition and function of long chain fatty acids in the preterm. We will use mouse models to study the direct contribution of maternal high-fat diet to the development of thermogenic adipocytes in offspring and identify the underlying transcriptional regulators. We will investigate how a gap junction protein Connexin43 affects the gene expression by epigenetic mechanisms and whether an early-life intervention on adipocytes can reprogram fat deposition, energy balance, and glucose and lipid metabolism in adulthood. We will utilize germ-free mice and gut microbiota transplant experiments to delineate epigenetic cross-talks between gut microbiome and intestinal stem cells at distinct developmental stages. We will also use mouse models to understand the relationship between diet in early life and the risk for developing cardiovascular dysfunction, and how these differ between sexes.

Progress Report
In Project 1 Role of Perinatal Nutrition on Overall Health and the Prevention of Disease, Objective 1, we will examine the benefits of feeding human milk to preterm infants. Human milk is vital for the health and development of preterm infants. Preterm infants are at risk for developing necrotizing enterocolitis (NEC) which is the most common gastrointestinal emergency in preterm infants. Development of NEC depends on nutrition, poor intestinal blood flow, and inflammation. We are utilizing an approach involving enteroids which are obtained from a biopsy from preterm infant intestine and grown in vitro. We tested the optimum conditions to culture preterm infant enteroids, measure their growth, and how human milk protects against gut bacteria. We have grown different isolates of Clostridium perfringens strains in the presence of donor human milk and infant formula. We are interested in C. perfringens because the amount of this bacteria is increased in the intestine of preterm piglets fed formula and infants that contract NEC. We obtained C. perfringens isolates from preterm piglets that develop NEC when fed infant formula. We tested C. perfringens strains derived from preterm infant stool samples. We show that C. perfringens grows slower when cultured with donor human milk than in infant formula. We also showed that C. perfringens grows slower when cultured with specific carbohydrates present in human milk (lactose) than those in infant formula (maltodextrin). We have optimized conditions for culturing preterm infant enteroids to examine their functional capacity to form a barrier to invasion from gut bacteria. We show that the culture of preterm infant enteroids with donor human milk and infant formula does not affect the barrier function. Next year we will test how exposure of preterm infant enteroids to C. perfringens impacts the barrier function and immune response when mixed with and without human milk. In Objective 2, we explored the potential benefit of supplementing arginine and citrulline to preterm piglets to prevent NEC in preterm infants. Arginine is a conditionally essential amino acid and can be deficient in preterm infants. Citrulline is a non-protein amino acid that can be converted into arginine by enzymes in the kidney and liver. It is also important to note that arginine is an important precursor to nitric oxide (NO) which can facilitate vasodilation and blood flow when it is produced by the cells that line blood vessels. However, when NO is produced by activated white blood cells, it can act as a pro-inflammatory cytokine that can perpetuate and worsen inflammation. Strikingly, we found that the supplementation of both arginine and citrulline were ineffective at preventing NEC in preterm pigs. When both arginine and citrulline were supplemented to infant formula, it also did not increase the amount of arginine present in the blood. We believe this reflects the rapid metabolism of both arginine and citrulline by the liver, resulting in higher levels of plasma and liver ornithine in the arginine and citrulline-supplemented pigs. These results suggest that supplementing arginine and citrulline is not an effective strategy to prevent NEC in preterm infants. In Objective 3, we used the infant pig as a model of the human infant to identify the mechanisms by which nutrition regulates muscle growth following preterm birth. We showed that the growth promoting effects of nutrition are reduced following preterm birth and this is due to defects in the response of intracellular signaling pathways to the rise in amino acids and insulin after each meal. In studies to develop targeted nutrition therapies to promote lean growth following preterm birth, we showed that supplementation with the amino acid, leucine, enhances the activation of the intracellular amino acid signaling pathway that promotes translation initiation and stimulates protein synthesis in muscle. However, a higher amount of leucine is required in preterm pigs than in those born at term. Studies conducted this year showed that this increase in protein synthesis with leucine can be sustained long-term and promote muscle growth. We further showed that supplementation with the amino acid, citrulline, is ineffective in acutely stimulating muscle protein synthesis in the short-term but long-term trials are ongoing. Together our studies suggest that targeted nutritional therapies applied to preterm infants could support muscle growth and overcome their deficit in lean mass in early childhood. The goal of Objective 4 is to conduct a pilot study to detect choline metabolites in premature piglets following the infusion of D9-choline. The pilot is scheduled later this year to accommodate the lead time required from sow breeding to the delivery of piglets. We have established analytical procedures for the measurement of how D9-choline is metabolized in the body. Project 2 aims to understand how diet, gut microbes, and physical activity during key stages of early development can cause permanent changes in tissue structure, function, and epigenetic regulation throughout life. Our findings could guide the development of early-life strategies to promote long-term health and prevent adult chronic disease. A high-fat diet during pregnancy can have lasting effects on maternal health and the development of fat tissue in offspring. In Objective 1, we studied how a maternal high-fat diet influences the development of brown fat in offspring, using mice as our animal model. We generated mice with brown fat marked by the gene Paired Related Homeobox 1 (Prx1) and established procedures to identify Prx1-marked brown fat cells throughout the offspring’s lifespan. We have measured the body and brown fat tissue weights of the female mice and their offspring that were fed a regular diet. Additionally, we analyzed tissue sections containing the Prx1-marked brown fat cells to determine their distribution and shape. We have also begun feeding female mice a high-fat diet during pregnancy to produce offspring exposed to maternal high-fat intake. We will compare the body weights, brown fat tissue weights, and Prx1-marked cells of these mice to those of the mice fed with a regular diet. Research under Objective 2 focuses on the role of a gap junction protein Connexin43 in adipose tissues. We have amended our protocol to include body composition analysis, metabolic cage experiments to assess substrate utilization, and several metabolic phenotyping tests—including glucose tolerance, insulin tolerance, triglyceride tolerance, and hyperinsulinemic-euglycemic clamp experiments—for the study of doxycycline-inducible Connexin43 overexpressing mice. In addition, we have generated transgenic mice for the proposed experiments in the upcoming year. The goal of Objective 3 is to understand whether the expansion of gut microbes after weaning (when the infant transitions from milk to solid food) affects how genes are regulated in intestinal stem cells. These stem cells are important because they control how the gut grows, repairs itself, and defends against harmful microbes. We developed the necessary mouse models and used advanced genomics techniques to study how early-life exposure to microbes influences gene activity in intestinal stem cells. In addition, we created new experimental systems, including precise DNA-editing tools to test how changes in epigenetic gene regulation affect stem cell function. We also developed organoid models that allow us to study stem cell behavior in a controlled environment. Research under Objective 4 is designed to test if the relatively small heart found in individuals who were malnourished during early life can be permanently restored to a normal size by endurance exercise training during the period of adolescence, but not during adulthood. The animal protocol for the studies was approved and the breeding colony was established. We have started dietary interventions. All of the milestones proposed could not be undertaken due to critical vacancies and alternative logistics had to be developed for in vivo cardiovascular measurements because the DeBakey Heart Center Animal Murine Non-Invasive Cardiovascular Core Laboratory closed. As a result, we procured the necessary instrumentation to perform the measurements at the Children’s Nutrition Research Center. We anticipate that we can continue the proposed objective as described.

Accomplishments

U.S. DEPARTMENT OF AGRICULTURE

Children's Nutrition Research Center: Houston, TX