Location: Children's Nutrition Research Center
2021 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 all animal and most laboratory analysis of 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). FGF19 is mainly secreted from epithelial cells lining the intestinal tissue. We used neonatal piglets that were delivered via cesarean section at either 10 days before birth (preterm) or at near the normal term birth date (term). 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. Our results showed that premature pigs have significantly lower blood levels of FGF19 than those born at term. Moreover, after three days, the blood level of FGF19 does not increase significantly after piglets are given a formula feeding. We also found a lower content of bile in the gut after feeding in premature compared to term piglets. We developed a new in vitro model using small sections of intestinal tissue, called explants, to examine how the pig intestinal FGF19 production responds to bile acids. We found that intestinal explants from term pigs produced significantly more FGF19 in response to bile acid treatment than explants from preterm pigs. These results suggest that bile acid production by the liver and secretion into the gut is underdeveloped in premature compared to term piglets. In addition, the preterm intestinal tissue FGF19 production is less responsive to bile acids. Our results imply that underdeveloped bile acid production in premature piglets could explain why premature infants have poor fat digestion and absorption. Future studies are now exploring the metabolic and nutritional implications of lower FGF19 secretion in preterm pigs. Previous studies in our laboratory, using the infant pig as a model for the human infant, have demonstrated that leucine supplementation of a milk replacement formula can promote lean growth. Our previous work in Objective 3 showed that pigs born prematurely have a lower rate of growth and that this is linked to reduced growth of skeletal muscle in response to feeding. The key underlying defect in preterm pigs is the reduced ability of the skeletal muscle to synthesize protein in response to the rise in circulating insulin and amino acids after each meal. In the past year, we further identified specific defects in the intracellular insulin and amino acids signaling pathways that regulate protein synthesis in the skeletal muscle of the preterm pig.
Previous studies in our laboratory have demonstrated that the branched-chain amino acid, leucine, which is abundant in milk protein, can act like a signaling molecule to stimulate the synthesis of protein in the skeletal muscle of the neonate. Supplementation with leucine in neonatal pigs continuously fed a milk replacement formula can promote skeletal muscle growth. In studies over the past year to address Objective 4, we combined plasma metabolomic analysis with transcriptome expression and protein catabolic pathways in skeletal muscle to investigate the mechanisms involved in the growth promoting action of leucine. The results suggest that leucine supplementation enhances the gain in muscle mass by increasing the activity of the intracellular signaling pathways that regulate protein synthesis. Leucine supplementation also reduces the activity of intracellular signaling pathways that regulate the degradation of protein in muscle. Further studies to address Objective 4 were conducted in neonatal pigs to investigate the mechanisms by which leucine, as well as its metabolite, beta-hydroxy-beta-methylbutyrate (HMB), regulate the synthesis of protein in skeletal muscle. The study demonstrated that both leucine and HMB stimulate protein synthesis in muscle of the neonate by activating the master protein kinase, mechanistic target of rapamycin (mTOR) that regulates translation initiation. However, leucine and HMB differed in how they affect several key intracellular signal transduction processes upstream of mTOR, as well the processes that regulate myonuclear accretion.
In studies completed as part of Objective 5, we measured the production of citrulline and its molecular basis in piglets from different stages (from -10 days preterm to 35 days old) to study the effect on development. Citrulline is an amino acid not found in most foods but is produced mainly in the intestine, and is a critical precursor for the synthesis of arginine, an essential amino acid in neonates. Arginine is not only needed for protein synthesis and growth, but it is also converted into many other molecules involved in energy and nitrogen metabolism. Arginine is also the precursor for nitric oxide, a signaling molecule involved in blood pressure regulation and in the immune response. These studies were done by infusing isotopic tracers of citrulline and measuring the metabolism of these tracers in blood samples collected after the infusion of the tracers. We found that the production of citrulline increases with the stage of development. To understand the cellular basis for this finding, we collected intestinal stem cells from the pigs in this study and used these biopsy samples to produce enteroids. Enteroids are intestinal cell structures derived from the pig tissue that grow into a mini-gut. These enteroids can be studied in a cell culture dish to examine how the stage of development affects the production of citrulline in intestinal cells. Enteroids derived from pigs of different ages failed to replicate the developmental changes in enzyme activity observed in the live animals. It is possible that other cues are needed in the media to recapitulate the live phenotype.
The studies in Objective 6 are designed to establish the mechanisms whereby undernutrition during critical windows of postnatal development alters the ability of skeletal muscle stem (satellite) cells to divide and support the growth and maintenance of skeletal muscles. Studies conducted in mice in vivo showed that when pups were briefly undernourished (by suckling dams fed a low protein diet) just before weaning, their muscle growth was reduced, and the deficit could not be entirely recovered, even after a prolonged period of good nutrition. However, when the same treatment occurred immediately after birth, it could be recovered following nutritional rehabilitation and there were no long-term repercussions. We have completed a histological analysis of muscles from a subset of the treatments proposed, and established that the disparate responses were associated with differences in the ability of satellite cells to divide at the different ages. In vitro studies on isolated satellite cells to identify the mechanisms responsible for the age-dependent differences in response to undernutrition were limited due to COVID-19.
The goal of Objective 7 is to identify if the maternal low protein diet provided to lactating dams alters the availability of important nutrient products (metabolites) that are utilized by the offspring to regulate epigenetic mechanisms that alter the activity of DNA in their satellite cells. In preparation for this work, we have begun to develop the methodologies required for identifying key epigenetic parameters (histone profiles) in satellite cells, and for measuring relevant muscle metabolite concentrations.
Accomplishments
1. Prematurity dampens the anabolic response to nutrition. The lean growth of preterm infants is typically lower than infants born at term which likely contributes to undesirable short-term and long-term health outcomes. Using the neonatal pig as a model for the human infant, researchers in Houston, Texas, conducted studies to determine the mechanisms by which preterm birth alters the response to nutritional growth. Our work showed that preterm birth impairs the ability of skeletal muscle to increase the synthesis of protein after a meal. These studies provide vital information on the mechanisms underpinning the reduced growth of lean muscle mass after birth in preterm infants, and will aid in the development of nutritional therapies to improve lean growth.
