2011 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.
The effects of fetal versus postnatal undernutrition on adult muscle mass were compared in Obj. 1, and we showed that when pregnant mouse dams were protein-restricted, the muscle masses and body composition of their offspring at weaning and as adults were the same as for offspring of well-nourished pregnant dams. Thus, effects of maternal dietary protein deficiency during gestation on her offspring's muscles are repaired if the offspring are well fed postnatally. In view of this finding, further study of the muscles of gestationally "undernourished" offspring is not warranted. In contrast, muscles of mice malnourished during the suckling period were smaller at weaning and contained only 77% of the number of satellite cells (muscle stem cells) present in well-nourished controls. However, these satellite cells divided at the same rate as those from well-nourished mice. Measurements are being collected to determine the mechanism responsible for the reduction in satellite cell number in the undernourished mice pups. We also established that when mice malnourished during the suckling period were allowed to recover by eating a complete diet ad libitum from weaning, their muscles were still smaller 4 and 18 months later. On-going evaluations are assessing why they do not recover fully. We have developed the technique of isolating intact muscle fibers with their attached satellite cells and culturing them in vitro. This isolated fiber/satellite system will be used for conducting studies for Obj. 2. We continued to refine surgical procedures for feeding adult mice intravenously for 3 days, with minimum weight loss. We can now do this and have begun to collect data on citrulline production in intravenously fed mice for Obj. 3. Additionally, we conducted studies to determine arginine utilization by the gut and liver in control and arginase II knockout mice. We determined that a lack of (enteral) arginase II reduced arginine extraction by 10%, and consequently more arginine is available to other tissues. Our spf-ash mouse colony developed serious reproductive problems, a well-known fact for this mouse strain. We have imported new breeding pairs and have restarted the colony for Obj. 4. We have also shown that with endotoxemia lack of arginase II activity increases arginine availability and nitric oxide production. Thus, arginase II represents a potential pharmacological target to increase nitric oxide production. The ASL conditional knockout mouse will now be provided by a collaborator, and we have started our studies on the effect of endotoxin in this mutant model. We showed that ornithine supplementation increases citrulline production in arginase II knockout and spf-ash mice; other approaches for manipulating citrulline availability are under investigation.
The ADODR monitors project activities by visits, review of purchases of equipment, review of ARS-funded foreign travel, and review of ARS funds provided through the SCA.
Effects of inadequate maternal protein intake on skeletal muscle growth of offspring. An inadequate intake of nutrients during early development can have long-term effects on the health of the individual. These effects depend on the individual tissue/organ and on the stage of development when the nutrients are limiting; for instance, skeletal muscle is especially vulnerable to an inadequate supply of amino acids. Scientists at the Children's Nutrition Research Center in Houston, TX, conducted a study that showed if maternal protein intake is insufficient during pregnancy, the offspring can develop a normal skeletal muscle mass if they are provided with sufficient nutrients at birth. However, if the offspring are malnourished from birth until they are weaned, and are then provided with a complete diet containing ample amounts of all nutrients, the muscle growth rate attained is insufficient to support complete recovery, and their muscle mass is permanently compromised. These studies emphasize the importance of providing appropriate and adequate amino acids as protein to newborns for skeletal muscle development, and especially in those cases where the mother has been malnourished during pregnancy.