Location: Children's Nutrition Research Center
2020 Annual Report
Objectives
Objective 1: Determine the effect of enteral nutrition on FGF19 secretion and the activation of FGF19 receptors and downstream signaling pathways and metabolism in various tissues in neonatal pigs.
Objective 2: Determine whether increased FGF19 availability using parenteral administration of porcine FGF19 and oral FXR agonist treatment controls the rate of growth, tissue protein synthesis and intestinal development in neonatal pigs.
Objective 3: Determine whether being born prematurely blunts the protein and glucose metabolic responses to the feeding-induced rise in amino acids and insulin and identify the mechanisms involved.
Objective 4: Identify the mechanisms by which amino acids, particularly leucine and its metabolites, regulate protein synthesis, degradation, and accretion and how these responses change with development.
Objective 5: Identify the mechanisms (molecular and metabolic) that limit citrulline production in premature, neonatal, and young pigs of both sexes; determine the basis for the greater citrulline production observed in females and determine the utilization of citrulline for endogenous arginine synthesis in vivo at different developmental stages.
SubObjective 5A: Identify the molecular and metabolic mechanisms that limit citrulline production in premature, neonatal, and young pigs of both sexes; and to determine the basis for the greater citrulline production observed in females.
SubObjective 5B: To determine the utilization of citrulline for endogenous arginine synthesis in vivo at different developmental stages.
Objective 6: Establish the molecular mechanisms and functional significance of differences in gene expression identified in satellite cell-derived myoblasts isolated from the offspring of dams fed a low-protein versus an adequate protein diet over critical windows of postnatal development.
Objective 7: Determine the impact of maternal dietary protein level during lactation on biomarkers of one-carbon metabolism in their offspring and establish if the observed effects translate into differences for DNA methylation and/or histone post-translational modifications in satellite cell-derived myoblasts isolated from the skeletal muscles of these offspring.
Approach
Despite improvements in their nutritional management, most premature and low birth weight infants have experienced growth faltering by discharge. Many remain small to adulthood and are at an increased risk for developing metabolic diseases such as obesity and type 2 diabetes. The goal of this project is to identify the mechanisms that regulate the diminished growth and altered metabolic responses to nutrition in premature and low birth weight infants and to develop new nutritional strategies to optimize their growth and development. Our approach will be to use neonatal piglet and rodent models to fill these knowledge gaps. We will determine whether being born prematurely blunts the anabolic response to feeding and identify mechanisms by which amino acids, particularly leucine, regulate lean growth. We will determine the role of the enterokine, FGF19, in the anabolic response to enteral feeding in the preterm and whether augmentation of its secretion will enhance growth and metabolic function. We will identify mechanisms that limit citrulline production and the impact of gender and age. We will establish the mechanisms by which undernutrition during critical windows of postnatal development impacts proliferation of skeletal muscle stem cells and the mature muscle nuclear number. Further we will test whether methyl group deficiency induced by inadequate amino acid supply results in permanent epigenetic modifications that impact muscle growth. This project is expected to have a positive impact by providing novel information that will be directly useful in optimizing the nutritional management of premature and low birth weight infants and improving their long-term metabolic health and growth.
Progress Report
As part of Objective 1, we completed a study to test how the stage of pregnancy and feeding influence the secretion of a novel gut hormone called fibroblast growth factor 19 (FGF19). We used neonatal piglets that were delivered via cesarean at either 10 days before birth or at near the normal term birth date. In each gestation group, either premature or term, some piglets were studied immediately after birth prior to being fed and the remaining piglets were fed for three days after birth. We collected samples of tissue, blood, and bile from all piglets and completed some preliminary analysis. The results confirm our report published last year that premature pigs have significantly lower blood levels of FGF19 than those born at term. We also found that after three days, the blood level of FGF19 does not increase significantly in the four hours after a feeding. Moreover, the blood level of FGF19 after three days of feeding remains lower in premature pigs when comparted to term. We also found a lower content of bile in the gut after feeding in premature compared to term piglets. These results suggest that bile acid production by the liver and secretion into the gut may be underdeveloped in premature compared to term piglets. Underdeveloped bile acid production in premature piglets could explain why premature infants have poor fat digestion and absorption. Objective 3 studies were conducted in infant piglets and showed that being born prematurely reduces the synthesis of proteins in the muscle in response to formula feeding. The studies suggest that in premature piglets there is a reduced capacity for insulin and amino acids to activate tissue signaling pathways involved in muscle growth. To determine whether this blunted response to feeding is due to a reduced sensitivity to insulin, amino acids, or both, the individual responses to insulin and amino acids were examined in additional studies. We showed that the individual responses of the intracellular signaling pathways that are regulated independently by amino acids and insulin are blunted in the preterm compared to the term pigs. This diminished capacity of muscle to respond adequately to insulin or amino acids alone or concurrently suggests that skeletal muscle in preterm neonates is resistant to the rise in both amino acids and insulin after a meal. This resistance to amino acids and growth factors like insulin likely contribute to the reduced growth and lean body mass of premature infants. These reductions in muscle mass may have profound effects on their risk for developing metabolic and cardiovascular diseases. We are currently undertaking studies to identify strategies to promote lean growth in these infants in order to mitigate poor health outcomes. In studies completed as part of Objective 5, we measured the production of citrulline and its molecular basis in piglets from different stages of development (from -10 d preterm to 35 d old). Citrulline is an amino acid not found in most foods, but is produced in the gut and is a critical precursor for synthesis of arginine, an essential amino acid in neonates. These studies were done by infusing isotopic tracers of citrulline and measuring the metabolism of these tracers in blood samples collected after infusion of the tracers. We found that the production of citrulline is increased with the stage of development. In addition, we collected intestinal stem cells from the pigs studied in Sub-objective 5B and used these biopsy samples to produce enteroids. These enteroids are primary cells derived from the pig that can be studied in a dish to examine how the stage of development affects the production of citrulline in intestinal cells (Sub-objective 5A). Enteroid cultures are underway and the production of citrulline and metabolic pathways are being characterized. In support of Objective 6 a subordinate study was performed to assess the validity of the underlying hypothesis. Newborn mice were undernourished either between birth and postnatal day 11 (PN11) or day 22 (PN22). After PN11 or PN22, respectively, the pups were provided ample nutrition to support rehabilitation and promote rapid recuperation of growth. We quantified satellite cell (SC; adult muscle stem cells) numbers in muscle after 2, 7 and 21 days of rehabilitation in both groups. Our data showed that undernutrition, regardless of age, reduced SC numbers, their rate of division, and the total number of nuclei in the muscle. The capacity to recover the deficit when refed a normal diet was dependent on the age of the mice. The younger mice (PN11) dramatically increased the rate of SC division when refed and they completely recuperated the deficit in muscle nuclei and muscle mass. In contrast, in the older mice (PN22) the SC were unable to divide sufficiently to recuperate the deficit in muscle nuclei; their muscles remained smaller. These data support our hypothesis that early vs later nutritional rehabilitation is more effective at restoring a normal growth trajectory in infants; this is consistent with our overall hypothesis in Objective 6.
Accomplishments
1. Prematurity blunts the synthesis of muscle proteins after a meal. Worldwide, approximately 15 million infants are born preterm and these infants typically have a lower lean mass than those born full term. The lower lean mass in premature infants may contribute to their poorer short- and long-term health outcomes. Using the neonatal piglet as a model for the human infant, researchers in Houston, Texas, conducted a study to determine if preterm birth alters the intracellular mechanisms that regulate muscle growth in response to feeding. We showed that the synthesis of proteins in muscle after a meal was lower in preterms than those born at term, leading to a reduction in growth. This reduced response in the preterms can be attributed to a blunting of the activation of the intracellular signaling pathways in muscle that are regulated by amino acids and insulin in the blood. These animal studies provide the mechanisms that underlie the reduced lean growth in premature births and may provide relevance for the bedside care of preterm infants.
2. New generation lipid emulsions prevent liver disease. In the U.S., the main lipid emulsion approved for use in total parenteral nutrition (TPN) for preterm infants is based on soybean oil, called Intralipid. Studies in preterm babies and our studies in preterm piglets have shown that long-term infusion of soybean emulsions leads to a life-threatening condition known as parenteral nutrition-associated liver disease (PNALD). Researchers in Houston, Texas, tested two new lipid emulsions in premature, newborn piglets. One group received the new emulsion containing four different fats, called SMOF, and a second group (EXP) got a similar emulsion, but with enriched levels of key omega-fatty acids that are important for immune function. Our results showed that the SMOF and EXP emulsions prevented PNALD when compared to a control group given Intralipid. We also found that these new emulsions maintained the normal flow of bile into the gut and this led to marked changes in the gut bacterial population. These findings confirm previous work and show that new generation lipid emulsions prevent liver disease and that the underlying explanation may be related to changes in the bacteria in the gut.
