Location: Children's Nutrition Research Center
2022 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: removed due to investigator departure
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 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 laboratory analysis of a study to test how the stage of pregnancy and mode of birth (vaginal vs cesarean delivery) influence the secretion of a novel gut hormone called fibroblast growth factor 19 (FGF19). FGF19 is a novel gut hormone that is important in controlling the amount of bile made in the liver. Hepatic bile production is critical for fat digestion in newborn infants. Fat digestion is not well developed in preterm infants and may explain why they experience poor growth. FGF19 is secreted mainly from epithelial cells that line 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). We also completed studies with neonatal piglets that were born naturally via vaginal delivery at term. Our team found that piglets born vaginally at term have a significantly higher blood level of FGF19 at birth compared to those that are born via cesarean delivery at preterm or term gestational age. We also found that the intestinal tissue explants from the vaginal term piglets produce more FGF19 when incubated with bile acids in tissue culture plates. We measured several hormones that change markedly during the process of labor during parturition and found that cortisol, a key stress-related glucocorticoid hormone, was most highly correlated with the plasma FGF19 levels at birth among all three groups. Moreover, we found that intestinal tissue explants from piglets produce FGF19 when treated with dexamethasone (synthetic glucocorticoid). We also found that dexamethasone increased FGF19 production in human intestinal enteroids, or miniguts, made from human intestine tissue collected by biopsies during hospital visits. These new enteroids are an exciting new in vitro model to examine how human intestines function. Our results suggest that the surge in cortisol that occurs in late pregnancy and during labor triggers an increase in the expression and secretion of FGF19 in neonatal piglets. These results suggest that treating preterm pigs with glucocorticoid hormones prior to birth may correct the deficiency levels of FGF19 and restore normal production of FGF19 in preterm pigs. This is clinically important because expectant mothers of preterm infants and newborn preterm infants are often treated with glucocorticoids to promote maturation of lung function in the infant.
In Objective 2 we conducted studies to see whether treatment of preterm piglets with an infusion of FGF19 for one week increases growth rate and protein synthesis. Preterm infants frequently experience growth faltering after birth and have a lower lean body mass later in life than infants born at full term.
In studies conducted as part of Objective 3, using the infant pig as a model for the human infant, we sought to identify the intracellular mechanisms that contribute to the reduced growth of lean tissue in the premature infant. We showed that the stimulation of protein synthesis in skeletal muscle in response to feeding is reduced in the preterm. We further identified specific defects in both the insulin and the amino acid signaling pathways that regulate translation initiation and protein synthesis in skeletal muscle of the preterm pig. This blunted response in muscle due to feeding likely contributes to the reduced growth of lean tissue and extrauterine growth restriction of premature infants. Because our previous studies in the neonatal pig born at term showed that intermittent bolus feeding (similar to meal feeding) enhances lean growth more than continuous feeding, further studies in our lab were completed to determine whether intermittent feeding could be effective in promoting lean growth in the premature infant. We showed that intermittent bolus feeding did not enhance muscle growth more than continuous feeding in pigs born preterm, likely because premature birth attenuates the capacity of muscle to respond to the cyclical surges in insulin and amino acids that occur with intermittent bolus feeding.
Previous studies in our lab have demonstrated that the branched-chain amino acid leucine, which is abundant in whey protein, promotes protein synthesis by stimulating the intracellular amino acid signaling pathway that regulates protein synthesis in the neonate. As part of Objective 4, we investigated whether supplementation with leucine during continuous parenteral nutrition could be used as an intervention to promote protein synthesis in skeletal muscle of the preterm. Our results showed no apparent benefit of leucine supplementation on protein synthesis in skeletal muscle in pigs born preterm that received continuous parenteral nutrition. Our findings are in stark contrast to pigs born at term which benefit from intermittent pulses of leucine during continuous enteral feeding. However, these data support our previous findings in the preterm piglet model which showed that preterm pigs exhibit a blunted anabolic response to the rise in insulin and amino acids after a meal. These results are directly relevant to the clinical care of premature infants because current nutritional support is often unable to match the growth rate and body composition that approximates a normal fetus of the same gestational age.
The proliferation of muscle stem cells is critical for postnatal muscle growth effects on muscle mass. For Objective 6, we seek to identify how these cells are affected by brief periods of insufficient food intake during early life, and why the lifelong effects depend on the age of the organism when the deficit in food intake occurs. In Objective 7 we planned to determine how a deficiency in dietary nutrients, particularly protein, could lead to the response we observe in Objective 6. The milestones for Objectives 6 and 7 have not been met due to COVID-19-related staffing and supply shortages. Staff with the necessary expertise to carry out the cell culture studies was not available. Thus, only preliminary studies to establish the methodology were completed. Additionally, we were not permitted to maintain the animal numbers required for completing the study. It is our plan to utilize some of the resources that are needed and have become available recently to begin undertaking Objective 7 in the coming year. Availability of results from these studies would then be used to stream-line the experiments in Objective 6 and answer the central question relating to the developmental programming of muscle stem cells by the postnatal diet. Despite the setbacks, we were able to make progress on other elements of the project. This year, time was also spent on setting up the instrumentation to build an indirect calorimetry system for Children's Nutrition Research Center research that uses newborn pigs. This included the construction of the system, the development of software and operation procedures, data analysis, and validation of the new system. Because of these efforts, these new calorimeters are now operational and are being used for Objective 4C of this project.
Accomplishments
