2012 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 our objectives 1B and 1C, the average size of muscle fibers in 11- and 22-day-old mouse pups suckled from birth by mothers who were consuming low-protein diets was 73% and 65% of those in well-fed pups, respectively. When undernourished pups were refed at 11 d of age, their muscles returned to the size of those pups that had never been undernourished, whereas those refed at 22 d were still 15% smaller at 18 months of age. To understand the mechanism responsible for the difference in these responses, we measured the number of satellite cells (adult muscle stem cells) and their replicative capacity. We determined that the muscles of the undernourished pups at both ages had fewer satellite cells, and the ability of these cells to divide and generate muscle nuclei was also impaired.
There was a rapid increase in the number of satellite cells with a return to normal values when the 11-d-old pups were refed. However, when the 22-d-old pups were refed, there was no recuperation of the total number of satellite cells; thus, they remained permanently with fewer satellite cells per muscle fiber. Despite this difference, the number of myonuclei added to muscle fibers upon refeeding was greater in the older pups. This indicated that satellite cells in the older pups were dividing, but then they differentiated into myonuclei and did not expand their own population. In contrast, the satellite cells in 11-d-old pups divided, increasing in number first. Of these, only a subset underwent differentiation into myonuclei and thus, they succeeded in both replenishing their own population as well as contributing to the muscle fiber nuclei.
The different fate of satellite cells in 11- vs. 22-d-old pups upon refeeding was substantiated by the differences in the expression of the muscle-specific transcription factors. There was an increase in the expression of MyoD which is expressed in dividing satellite cells only in the younger pups. The expression of Myogenin, which is important for the development of skeletal muscle, in the differentiated myonuclei exhibited smaller differences.
The contrasting responses of the muscles from undernourished 11- vs. 22-d-old pups upon refeeding, together with the concurrent differences in protein synthesis (NIH-funded research), provide clear evidence that when the growth of an organism is compromised because they do not get enough nutrients, their capacity to recover is highly age-dependent. If the duration of undernutrition is brief, so that the recovery occurs when the muscles are still immature, they have a greater potential to recover. These finding have potential long-term implications as it indicates that nutrition in early life can influence the development of sarcopenia (loss of muscle mass with aging) and the capacity of muscle to recover following injury or disuse atrophy.
In objective 3, to study how amino acids are utilized by the body’s organs, we developed a portal vein catheterization technique that can be used when mice are unconscious. However, the conscious model has proved to be very challenging. We have also developed a novel sampling technique, peritoneal microdialysis that can be used in conscious mice as an alternative to arterial blood sampling. This technique allows for the collection of multiple time points without the need to draw blood.
In objective 4A, we studied the internal production of citrulline which, is the immediate precursor for the synthesis of the amino acid, arginine. The contribution of different pathways to this process was quantified. We have also investigated the production of nitric oxide, an important messenger in physiological and pathophysiological processes, in mutant and control mice that were exposed to an endotoxin challenge. Furthermore, arginine supplementation showed a linear increase in the production of nitric oxide.
In objective 4B, we encountered low breeding performance in our spf-ash mouse colony. Thus, new breeders were obtained, but the production of experimental animals was unpredictable, and did not generate the number of animals required to perform a study. To overcome this obstacle, we successfully crossed the mutation into a different genetic background. We are in the process of expanding the colony and expect to have sufficient experimental animals to study within the next several months.