Pepper Pilot studies
Year - 1
Four pilot projects were selected in the first year (6/15/05-4/30/06) of P/EC funding:
1. Transcapillary Insulin Transport and Aging
Project Leader - Elisabet Boersheim, Ph.D.
Department of Surgery
Award Amount: $52,781
The general hypothesis of this study is that delayed transcapillary transport of insulin into the muscle interstitial fluid is a contributing factor to the lack of anabolic effect of insulin on muscle protein in elderly. The general approach involves sampling plasma and interstitial insulin and amino acid concentrations in young and elderly subjects. Muscle protein metabolism will be determined by stable isotopic tracer techniques, muscle biopsies and arterio-venous sampling, and related in time to corresponding insulin concentrations in plasma and interstitial fluid.
A 4-pool model for amino acid metabolism has previously been developed, and analyses of enrichment of stable labeled amino acids and thus concentration of amino acids in the interstitial fluid has been established (Børsheim E. et al. Compartmental Distribution of Amino Acids during Hemodialysis Induced Hypoaminoacidemia. Am J Physiol Endocrinol Metab. 2005 Nov 8; [Epub ahead of print]).
2. Chaperone Modulation of Motor Activity in Aging
Project leader: Henry F. Epstein, M.D.
Neuroscience and Cell Biology
Award Amount: $52,155
Dr. Epstein's laboratory discovered the UNC-45 protein in the model genetic organism, Caenorhabditis elegans, and showed that it can act as a molecular chaperone for the muscle motor protein myosin. In collaboration with the German molecular biologist Torsten Hoppe, it was determined that the level of UNC-45 in muscle is regulated by the ubiquitin-proteasomal system and in turn controls the assembly of myosin into functioning contractile filaments and the resulting motor activity. Further study into cellular basis of muscle weakness and loss of mass in aging is underway.
3. Cyclic Testosterone and Bone Turnover in Older Men
Project Leader: Melinda Sheffield-Moore, Ph.D.
Department of Internal Medicine
Award Amount: $36,240
One subject successfully completed the 5 month protocol. The subject received weekly injections of either testosterone or placebo throughout the study according to the protocol. At each monthly visit, 24-hour urine samples were collected for the analyses of markers of bone turnover in urine. Arterialized venous blood samples were collected at 5 minute intervals during the 1-hour experiments using the heated hand technique. Serum aliquots from the monthly baseline samples will be used for the determination of markers of bone turnover. On the final visit, simultaneous femoral arterial blood samples were collected which will be used to compare to results from the arterialized venous samples and validate the heated hand technique. In addition, two muscle biopsies were performed at each of the six monthly visits for the determination the fractional synthetic and breakdown rates of muscle protein. For this procedure, a stable isotope tracer (13C6-Phe) was injected before the first biopsy was taken and a second tracer (15N-Phe) 30 minutes later. The second biopsy was taken 1 hour after the first biopsy. DEXA scans and 1-RM measurements were obtained at each of the six visits. Three-day dietary records were obtained after the first visit and before the fourth and sixth (final) visit. Analyses of the markers of bone turnover in serum and urine are ongoing. The next subjects will be started on this protocol after validation and review of the heated hand technique and the use of two stable isotope tracers for the simultaneous determination of the fractional rates of synthesis and degradation of muscle protein.
4. Age-dependent Change in Activity of DNA Base Excision Repair Proteins and its Impact on the Functions of Mitochondria in Skeletal Muscle
Project Leader: Bartosz Szczesny, Ph.D.
Sealy Center for Molecular Science
Award Amount: $36,240
In this pilot project, we hypothesize that molecular process responsible for age-dependent muscle wasting, muscle weakness causing frailty and disabilities are the result of chronic oxidative stress generated in mitochondria. During execution of project we were not able to isolate large enough level of intact mitochondria from mouse skeletal muscle, which would be needed for analysis DNA glycosylase and AP enonuclease activities. We therefore decided concentrate our effort on heart muscle tissue instead of skeletal muscle. For comparison we analyze kidney tissue in the same time. So far, we determined age-dependent changes in activity of uracil DNA glycosylase and AP enodonuclease in both tissues. The results clearly suggest that in the heart tissue overall activity of both analyzed enzymes decline with age in contrast to the kidney. Moreover, we find age-dependent increase in the AP endonuclease level from both nucleus and mitochondria but not uracil DNA glycosylase. Currently, we are analyzing oxidative damage in total cell extract as well as in cell subfractions.