2010 Annual Report
1a.Objectives (from AD-416)
Objective 1: Differentiate the effects of fetal versus postnatal maternal dietary protein restriction on satellite cell accretion and skeletal muscle mass.
Sub-objective 1.A. Determine in vivo the number of skeletal muscle satellite cells undergoing division, apoptosis, and differentiation in term fetuses of mouse dams that are fed a protein-restricted (PR) or a control (C) diet ad libitum during gestation.
Sub-objective 1.B. Determine in vivo the number of skeletal muscle satellite cells undergoing division, differentiation, and apoptosis in 21-d-old mouse pups that are suckled by dams fed either a PR or a C diet ad libitum from birth.
Sub-objective 1.C. Determine satellite cell and myonuclear numbers, myofiber cross-sectional area, and muscle mass in the 15-wk-old and 18-mo-old offspring of dams fed a PR diet either during gestation or during the suckling period, and then refed from birth (suckled on C dams) or after weaning (C diet, ad libitum), respectively.
Objective 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.
Sub-objective 2.A.1. Quantify and compare the in vitro replicative, differentiation, and fusion capacities of satellite cells isolated from muscles of 21-d-old offspring that were suckled on C or PR dams when they are cultured in vitro under identical conditions.
Sub-objective 2.A.2. Quantify and compare the in vitro replicative, differentiation, and fusion capacities of satellite cells isolated from muscles of 21-d-old offspring of dams fed the PR or C diet during pregnancy and then suckled on C dams when they are cultured in vitro under identical conditions.
Sub-objective 2.B. Determine the regenerative capacity of whole skeletal muscles transplanted from 21-d-old PR and C pups into 10-d-old C pups.
Objective 3: Develop novel techniques to study amino acid metabolism in conscious mouse models, with special emphasis on hepatic and enteral metabolism.
Sub-Objective 3.A. Determine the effect of a loss of small intestinal function on arginine availability.
Sub-Objective 3.B. Verify the function of arginase II in first pass metabolism of arginine by the small intestine.
Objective 4: Determine the role of urea cycle intermediates in maintaining nitric oxide and ureagenesis during different physiological and pathophysiological conditions.
Sub-objective 4A: Determine the role of arginine availability in sustaining nitric oxide production during conditions of increased arginine demand.
Sub-objective 4B: Determine the liver requirements of urea cycle intermediates for ureagenesis in urea cycle transgenic mice.
1b.Approach (from AD-416)
Children's Nutrition Research Center researchers will study the offspring of mouse dams that have been protein malnourished during pregnancy and/or lactation, and then nutritionally rehabilitated by suckling on well-nourished dams or by feeding on a control diet after weaning. In vivo satellite cell responses and skeletal muscle growth will be assessed primarily by immunohistofluorescence imaging with morphometry to assess cell division, apoptosis, differentiation, and muscle mass. To assess the role of epigenetic mechanisms intrinsic to the satellite cell, cells will be harvested from mice with different nutritional histories and their activity studied in vitro. Additionally, we will use different transgenic mouse models, including conditional knockout models, and stable isotope tracer infusions to explore various pathways. Mice will also have surgical implantation of intravenous or intragastric catheters for the delivery of nutrients and tracers. These infusions will be performed to further explore the transorgan metabolism of arginine and related molecules.
In FY2010 we completed most of the animal experiments. This means that 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 the nutritionally induced growth retardation. We have collected the muscles from mice that had been undernourished and then re-fed either before they were biologically and functionally mature, i.e., before they were weaned or after they were weaned at 22 d of age once their muscles are fully mature. We also examined the muscles after they had been allowed to recover for 18 months. The long-term studies of the offspring whose mothers were undernourished only during pregnancy are still in progress. The analyses performed on the muscles that 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. Additionally, we have conducted infusion studies in knockout mice to determine the origin of citrulline. The dietary and plasma precursors for the synthesis of this non-protein amino acid have been established. Our multitracer approach in conscious mice show that dietary arginine is the main precursor for citrulline synthesis and that when arginine is absent from the diet, plasma arginine and ornithine are the main precursors. The lack of enteral arginine in Arginase II knockout mice reduced the utilization of dietary arginine but increased the contribution of dietary glutamine and proline. Oral ornithine supplementation was mostly channeled to citrulline synthesis.
The ADODR monitors activities for the project by routine site visits, and review of major purchases of supplies/equipment, use of SCA funds for foreign travel, and submission of grant applications by investigators funded through the SCA.
Impact of malnutrition on muscle growth. Malnutrition early in life can result in a permanent deficit in muscle size; however, we don't know if the muscle response differs depending upon the age when the malnutrition occurs. Children's Nutrition Research Center scientists in Houston, Texas, showed that if malnourished mouse models were re-fed when their muscles were still immature, they were able to return to their normal size, but not if the refeeding was delayed until their muscles were more mature. These results indicate that if the growth of an individual is impaired during early life (primarily during the suckling period) because they are not getting sufficient nutrients, the consequences on their muscles will persist throughout life unless nutritional interventions to reverse the effects are implemented before the muscles become completely mature. The results of these studies have implication for both human health and animal production.