2013 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 overall goal of Objective 2 was to use two different approaches to 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 accelerate satellite cell division. The first approach (Subobjective 2A) was to assess the in vitro function of satellite cells from muscles of mice that had been suckled on normal or protein-restricted dams. We did not do this experiment since we were unable to replace the technical staff who had the capability to conduct this work. Recently, we identified a Baylor College of Medicine faculty member who is routinely isolating satellite cells, and it is our intent to set up a collaboration so that we can perform the experiment as described in the Project Plan next year. As an alternative, we assessed the capacity of voluntary exercise (wheel running) in combination with nutritional rehabilitation to promote muscle growth. In normal young rodents, wheel running has been shown to stimulate satellite cell replication and promotes greater muscle growth. We established the experimental model, and have performed most of the animal work. The preliminary results demonstrated that wheel running in well-nourished controls induced hypertrophy of muscles; this response did not occur in offspring of protein-restricted dams. We established, however, that even after 3 weeks of rehabilitation, the offspring of protein-restricted dams ran only 50% of the distance run by the controls of well-nourished dams suggesting that diet in early postnatal life may influence adult voluntary physical activity. The second approach (Subobjective 2B) was to compare the regeneration of muscles derived from 21-day-old offspring of protein-restricted dams after transplantation into a 10-day-old well-nourished control. This was not successful, and we therefore tested alternative approaches for causing muscle degeneration and regeneration. We developed a method that reproducibly causes injury and necrosis to an entire leg muscle. Over a period of two to four weeks following the injury, the muscle undergoes complete regeneration. We have performed the animal studies to compare the recovery of the offspring of the protein-restricted dams with control offspring. The muscles will be analyzed next year.
For objectives 3 and 4, we continued to work on our novel microdialysis technique for the sampling of amino acid. The ultimate goal of this approach is to have mice that have a permanent cannulae to be able to access and sample peritoneal amino acids at multiple times. The total parenteral nutrition (TPN) of mice presents unique challenges due to the high volume and hyperosmolarity of the solutions infused to provide the animals with enough nutrients. Approximately 50% of the mice fed by TPN develop extravasation and edema in the neck and front leg. This may be due to the removal of lymph nodes during the catheterization of the jugular vein. We are investigating other access points to minimize extravasation and edema. We had reproductive problems in our Spf-ash mouse colony. This mutation is maintained in a C57 BL6 background. Despite replacing the mice with new breeders from Jackson laboratories, we were unable to produce enough animals to conduct the experiments. We have previously passed the mutation to an ICR background that is less affected by the reduction in ornithine transcarbamylase. We generated control, carrier, and mutant females to conduct the experiments on citrulline during pregnancy. However, the rate of timed pregnancy was very low (<20%), and once again we lacked the numbers needed. We have finalized the study of the precursors for citrulline synthesis in mice. We have translated the mathematical models and techniques developed in mice to the piglet model.