Location: Children's Nutrition Research Center2010 Annual Report
1a. Objectives (from AD-416)
It has now been convincingly demonstrated by human epidemiological investigations that the origins of many of the major chronic diseases that are manifest in adult life have their origins during development. Additionally, molecular, cellular and animal studies have now shown conclusively that interference with, or alteration of, developmental pathways during critical windows of development can provide the pathophysiological basis for the events that take place later in adult life. Our research attempts to answer the following: 1) differentiate the effects of fetal versus postnatal maternal dietary protein restriction on satellite cell accretion and skeletal muscle mass; 2) determine if impaired catch-up growth upon nutritional rehabilitation is due to aberrant epigenetic mechanisms intrinsic to the satellite cell and/or an absence of the extracellular cues necessary to sufficiently accelerate satellite cell division; 3) develop novel techniques to study amino acid metabolism in conscious mouse models, with special emphasis on hepatic and enteral metabolism; 4)determine the role of urea cycle intermediates in maintaining nitric oxide and ureagenesis during different physiological and pathophysiological conditions; 5) determine gene expression in human lactating mammary epithelium; 6) characterize inbred mouse strains for lactation performance, gene expression, and weight gain among offspring in lean and obese animals, making use of a cross-fostering design where appropriate; 7) study the effect of nutrients on mammary gland development and function in mouse models; define the critical window for effects on mammary gland development and function; 8) elucidate the role of vitamin A in vascular development and hematopoiesis using mouse embryos, in vitro assays, and complementary techniques; and 9) identify target genes downstream of retinoic acid signaling that are required for blood and blood vessel development.
1b. Approach (from AD-416)
These research studies will use various techniques to accomplish the research to be undertaken. Establishing the critical window of development during which an inadequate nutrient supply permanently compromises the growth of the skeletal muscle and to understand the responsible mechanisms is of great importance. Research studies will focus on the satellite cell in animal models in order to understand how the nutrition of the fetus and infant has lifelong consequences for the health of the individual. Studies will take place on the role of amino acids in the urea cycle (crucial for nitric oxide synthesis), which is in demand for physiological (growth, pregnancy) and pathophysiological (trauma, sepsis) conditions. Lactation research will be conducted in mouse models to obtain an improved understanding of the genomic factors regulating mammary gland function that is central to providing therapeutic interventions that can aid women to establish and maintain a productive lactation. Since the abnormal formation of blood and blood vessels in adults is central to the progression of prevalent pathologies, including atherosclerosis, tumor angiogenesis, and anemia, researchers will develop an understanding of the cellular and molecular regulation of blood and blood vessel formation. Scientists will study retinoids in their role in the formation and maturation of blood vessels, as well as hematopoiesis, during embryonic development. Additionally, CNRC researchers will investigate, on a cellular and molecular level, the role of retinoid signaling in the regulation of these processes.
3. Progress Report
Project 1. In animal experiments, we undernourished pregnant mothers or their pups after birth, and then re-fed the offspring and examined the ability of their muscles to recover from nutritionally induced growth retardation. The analyses performed on the muscles we have collected so far include muscle mass determinations, histology, satellite cell number, total myonuclei, and newly added myonuclei. To complete these analyses we captured 7680 images. We conducted infusion studies in knockout mice to determine the origin of citrulline. Dietary and plasma precursors for synthesis of this non-protein amino acid were established. Our multitracer approach in conscious mice show that dietary arginine is the main precursor for citrulline synthesis and when arginine is absent from the diet, plasma arginine and ornithine are the main precursors. Lack of enteral arginine in Arginase II knockout mice reduced utilization of dietary arginine but increased contribution of dietary glutamine and proline. Oral ornithine supplementation was mostly channeled to citrulline synthesis. Project 2. We recruited 7 normal women and completed milk collection in 6. We completed studies in 2 obese women. There is a large amount of RNA in human colostrum, but isolation of high quality RNA has been challenging. With modifications in our protocol, we substantially improved quality of the RNA. Milk-derived RNA has been isolated, quantitated, and the cDNA and cRNA produced. We measured traits associated with milk production in 32 inbred strains of mice and collected milk, blood, urine, and mammary tissue for future use. We compared maternal behavior, food intake, and body composition and measured maternal ability to mobilize body fat during early lactation. For animal breeding for experiments it was necessary to breed together 3 separate mouse lines into one mouse line to obtain certain transgenes. We have started collecting phenotypic data and samples for further analysis. The current approach is limited by unforeseen deficiencies in the used model. We found that gene expression of the inactivating gene is not developmental-stage-specific, is not exclusively expressed in the mammary gland, is also expressed in some other tissues, and is expressed regardless of administration of inducing substance. We will have to adjust strategy. We plan to use a transplant model. Based on recent published this will better address anticipated effects of deletion of the gene of interest on mammary gland development. Project 3. We determined the role of retinoic acid in regulating endothelial cell migration during blood vessel formation and remodeling, and determined the signaling pathways downstream of retinoic acid that regulate this process. We also isolated and characterized specialized hemogenic endothelial cells from the embryo and are now trying to determine how retinoic acid regulates its formation and function during embryonic development.