Source: UNIVERSITY OF NEBRASKA submitted to
MOLECULAR MECHANISMS REGULATING SKELETAL MUSCLE GROWTH AND DIFFERENTIATION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0157156
Grant No.
(N/A)
Project No.
NEB-13-110
Proposal No.
(N/A)
Multistate No.
NC-131
Program Code
(N/A)
Project Start Date
Oct 1, 2000
Project End Date
Sep 30, 2005
Grant Year
(N/A)
Project Director
Jones, S. J.
Recipient Organization
UNIVERSITY OF NEBRASKA
(N/A)
LINCOLN,NE 68583
Performing Department
ANIMAL SCIENCE
Non Technical Summary
Protein accretion in muscle in animals occur at the cellular level. To improve the effiecency of this physiological event we must understand how this occurs and the mechnism that control the process. The project examines the role that insulin and branched chain amino acids have regulation protein turnover, particularly in fetal porcine muscle. The focus of the project will be to measure the rate of translation of mRNA into protein and the factor that may regulate this process.
Animal Health Component
34%
Research Effort Categories
Basic
66%
Applied
34%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3053320103025%
3053520103075%
Goals / Objectives
The objectives of the NC-131 regional research project are : 1. Characterize extracellular signaling mechanism that regulate skeletal muscle growth and differentiation. 2. Determine molecular mechanisms that control gene expression in skeletal muscle. 3. Characterize mechanism of cytoskeletal protein assembly and degradation in skeletal muscle. This research project belongs under is objective one. The overall objective of this project are: 1. Examine the effect insulin has on mRNA translation and cell proliferation during differentiation of myogenic cells. Questions to be answered: A. What role does insulin have in altering translation in myoblasts and does it effect proliferation? B. How does changes in level of branched chain amino acids effect insulin actions on protein synthesis in myoblasts and myotubes? C. Is translation altered in cultured cells when treated with serum from animals treated to induce diabetes? D. Does insulin's response in cultured cells ameliatored over time?
Project Methods
A. What role does insulin have to alter translation in myoblasts and does it effect proliferation? Cell cultures will grow until that have reached 50 percent confluence. Media will then be changed to media containing DMEM plus Insulin: 0, 1, 10, 100 nano gram per ml. Proliferation and polysome percentages will be determined. Protein, RNA, DNA and creatine kinase activity will be used to characterize the cell cultures. Examination of the changes of initiation factors eIF4 and eIF2 will be accomplished using the western hybridization procedures. Difference in activity and phosphorlyation of the initiation factors will be determined. B. How does changes in level of branched chain amino acids effect insulin actions on protein synthesis in myoblasts and myotubes? Treatment will be applied to myoblasts, 65 percent confluence, and myotubes. At the time of treatment media will be replaced one half the cells receiving insulin as determined in study one and no insulin and level of leucine at 0, 10, 100, 500, and 1000 micro moles per L. All amino acids will be at levels defined in DMEM media. Polysome percent, initiation factor amount, total RNA, DNA and protein will be measured. C. Is translation altered in cultured cells when treated with serum from animals treated to to induce diabetes? Pigs will be bleed and the serum will be isolated by centrifugation. After the each pig is bleed that will allowed to recover for two days. The pigs will then be treated with diazoxide a compound that will reduce the insulin level in the blood. Serum will be prepared from these blood samples. C2C12 or porcine myoblast will be allowed to reach 70 percent confluence in DMEM media with 10 percent fetal bovine serum. At that time the media will be replaced with DMEM with 10 percent porcine serum obtained from the pigs described above. Serum from the pigs prior to treatment will serve as the control for the diaoxide treatment. Cell proliferation, polyribosome percentages, total RNA, DNA and protein will be measured. The second portion the study designed in the a similar manner except the cells that will be used will be differentiated myotubes. The cells will be monitored for fusion by visual examination and also measurement of creatine kinase activity. Once the cells have fused, usually 36 hours, the media will be change to the treatment media described above.D. Does insulin's response in cultured cells ameliatored over time? Myogenic cells, C2C12 or Porcine primary cultures, will be cultured and stimulated to fuse. The experiment will begin after 24 hr after they have been given DMEM plus 2 percent horse serum to stimulate fusion. At 6 hr intervals the group of four plates will be given serum free media containing one of four levels of insulin 0, 1, 10, 100 nano gram per ml. Two hours after insulin media is added the cells will be harvested and polyribosome percent, total protein RNA and DNA will be measured. This process will be repeated on cell taken out of the incubator up until 96 post fusion initiation.

Progress 10/01/00 to 09/30/05

Outputs
Work that has been done jointly with Dr. Michael Zeece has examined the possible mechanisms involved in malignant hypothermia in both pigs and hogs. Microarrays were developed to profile level of proteins in sarcoplasmic reticulum (SR) membranes isolated from porcine Longissimus muscle (LM). The microarrays consisted of SR preparations printed onto to glass slides and probed with monoclonal antibodies to 7 target proteins. Proteins investigated were associated with calcium regulation and included; ryanodine receptor, (RyR), T-Tubule voltage sensing Ca2+ channel (Dihydropyridine receptor, DHPR), triadin (TRI), calsequestrin (CSQ), 90 kDa junctional protein (JP-90), and fast-twitch and slow-twitch SR Ca2+ ATPases (SERCA1 and SERCA2). Signal from a fluorescently-labeled detection antibody was quantitated using a slide reader. The microarray developed here was used to profile SR proteins in LM from halothane (Hal) genotyped animals. Significantly (P<0.05) lower levels of several proteins including RyR, CSQ, TRI, DHPR and SERCA2 were found in Hal positive vs Hal negative animals. The results illustrate the potential of microarrays to perform multiplexed analysis of SR proteins with low sample and reagent requirements. Protein microarray also represents a complimentary approach to other genomic analysis methods for investigations of calcium regulation or its abnormalities in muscle. Another study that is in the preliminary stages is examining the possible mechanism of action of beta agonists on muscle growth. Work will be done to identify the pathways in myogenic cells which are affected by the administration of several different type of beta agonists.

Impacts
Maligant hyperthermia ie PSE has a major impact on meat quality in both pigs and turkeys. This physiological condition has a tremondous impact of both teh hog and turkey industry. Understanding the how maligant hyperthermia affect calcium regulation will provide infomation in possible defects that cause the problem.

Publications

  • Schulz, J.S., Palmerb, N. Steckelberg, J. Jones, S.J. and Zeece, M.B. 2006. Microarray profiling of skeletal muscle sarcoplasmic reticulum proteins. Submitted


Progress 10/01/03 to 09/30/04

Outputs
A study conducted in our lab was to determine if insulin, glucose, and leucine cause an increase in RNA and recruitment of ribosomes for protein synthesis. We hypothesized that all three work independently to increase polyribosome formation in porcine satellite cells (PSC) and in porcine satellite cell derived myotubes (PDM), in vitro. In both PSC and PDM, the addition of insulin or leucine at or above physiological levels or baseline levels, caused RNA and the percentage of polyribosome to increase compared to controls (p<.05). A diminution of insulin or leucine below physiological or baseline levels, caused a decline (p<.05) in total RNA and polyribosome formation. When glucose was excluded from the medium, RNA and polysomes were reduced (p<.05). However, there was no difference in either total RNA or percent polysome formation (p>.05) between glucose added media. These data indicate that 1) insulin and leucine increase total RNA production and percent polysome formation, and 2) the absence of glucose decreases total RNA production and polysome formation. When insulin and leucine were used in combined treatment levels, RNA concentration increased as leucine increased above baseline levels. In PSC cells exposed to baseline leucine levels, as increase in RNA was observed when insulin was at least at physiological levels, and RNA levels tended to plateau at higher levels. Polyribosome formation within PSC cells was not affected by an interaction of the insulin and leucine treatments. Regardless of leucine levels, polyribosome initiation increased with increasing levels of insulin for PSC and PDM. The results for all experiments showed insulin increases RNA synthesis as well as the formation of polyribosomes in both PSC and PDM in vitro. When leucine levels drop below baseline, the production of RNA and polyribosomes significantly decreased. While insulin increased RNA and polyribosomes, it does not appear to work synergistically with leucine to increase RNA production in either cell type.

Impacts
This work will help identify the factor that regulate muscle growtth, an understand will help in more effiecently utilize the resources availble for meat production.

Publications

  • Creamer, B.A., J.M. Scheffler, and S.J. Jones 2004 Effects of insulin and glucose on translation rates in primary porcine satellite cells. J. Anim. Sci 82 (suppl 1) abs T 157.
  • Schulz, J.S., S.J. Jones, and M. Zeece. 2004 Development of a microassay-based ELISA for the assessment of SR-associated calsium regulatory protein in skeletal muscle. J. Anim. Sci. 82 (suppl 1) abs T158


Progress 10/01/02 to 09/30/03

Outputs
There are three projects that we are presently working on in our lab. The following is a short description of each: The objective of our first project is to determine the interaction of insulin, branched-chain amino acids and glucose on protein translation rates in porcine satellite cells. The cell types to be used will be proliferating myoblasts and myotubes as determined by measuring desmin appearance, DNA accumulation and cell fusion. An insulin dose titration curve has been performed for insulin to identify the optimal response of insulin on the initiation of protein translation, as measured by fractionation of polyribosomes by ultracentrifugation. This has been done in defined media with several controls, including a media containing fetal bovine serum, horse serum, and media devoid of serum and insulin. The final portion of the study will examine the interaction of insulin, glucose and the branched chain amino acid lysine on the initiation of translation. Additional data will be collected on the amount and phosphorlyation state of the initiation factor eIF-2alpha. Another project in the beginning processes is a study to quantify the number of beta receptors in different muscles. Procedures will be developed using porcine muscles, then the work will be conducted in beef cattle. Muscles of interest will be the biceps femoris, infraspinatus, psoas major and semimembranosis. Muscle cell membranes will be isolated then a saturation curve will be defined for the compound cyanopindolol. Once this has been developed, competition assays will be conducted using several of the available beta agonists either being used or having been examined, for the effect of improving muscle growth. Nonspecific binding will be determined by co-incubation with isoproterenol. The final study will use microarray techniques to investigate the interactions between the protein calsequestrion and other sarcoplasmic proteins. Relevant proteins for investigation include: RyR1, DHPR, calsequestrin, triadin, junctin, calcineurin, calmodulin, SR-ATPase and others. We have developed a microarray that assesses binding of calsequestrin to RyR1, Triadin and DHPR. In in preliminary work 10 ng of purified calsequestrin was printed onto treated glass slides in 6 x 6 arrays (36 spots in each). The protein was covalently attached to the glass surface via succimidyl ester linked to a spacer molecule. The slide was covered with a mask to isolate each array. The other 6 arrays were incubated with sarcoplasmic reticulum extracts in the presence of EDTA or Calcium. After washing away unbound proteins, each pair of arrays was probed with anti-RyR1, anti-Triadin and anti-DHPR antibodies. The results have shown a strong differential binding of RyR1 to calsequestrin, based on the presence or absence of calcium. Fluorescence intensity values also show that there is less Triadin bound to calsequestrin in the presence of calcium. The value of microarray techniques might be in the identification of altered calcium-dependent binding for RyR1 and other calcium regulatory proteins.

Impacts
This work will help identify the factor that regulate muscle growtth, an understand will help in more effiecently utilize the resources availble for meat production.

Publications

  • Novak, C., S. Scheideler, S. Jones. 2003. Effect of dietary level and TSAA:Lysine ratio on body composition, egg protein and magnum tissue of the laying hen. Poultry Sci. 80: (Suppl. 1) 390.
  • Edeal, James Brad. 2003. Identifying differentially expressed genes in C2C12 myogenic cells using differential display PCR. University of Nebraska.


Progress 10/01/01 to 09/30/02

Outputs
Several projects have begun in our investigation into the factors that regulate protein synthesis in muscle. The first project is to investigate the interaction between insulin and branched chain amino acids on ribosome recruitment for protein synthesis. The work will be done using porcine satellite cells as a model. The cell will be treated in such a way as to have three separate classes of cells, non-fused, non-differentiated; non-fused, differentiated; and fused, differentiated. The cells that will be differentiated but non-fused will be held in this state by removing the calcium from the media. Polyribosome percent, total RNA, total DNA and protein will be quantified. Measurement of several of the initiation factors for protein synthesis will also be determined. Knowledge gained will help in understanding the role of the branched chain amino acids, which can be the limiting amino acid in some swine diets, on protein synthesis in muscle. A second study has been initiated understanding the changes in protein expression in stress susceptible pigs and turkeys. Protocols for isolation of the sarcoplasmic reticulum fraction from skeletal muscle and its separation on 2-dimensional electrophoresis, have been optimized. Additionally, we have developed procedures for in-gel digestion and extraction of resultant peptides. This was a very significant task because many of these proteins have a high percentage of non-polar residues, making trypsin digestion difficult. Additionally, the recovery of peptides with a high degree of hydrophobic residues was very low. We have also mastered procedures for mass spectrometry analysis (MALDI and Q-TOF) of these peptides. These analyses have included learning procedures for searching databases and denovo sequencing of proteins (protein identification). We have succeeded in applying these skills and identified several proteins in the SR fraction (from a 2-D separation) that are involved in calcium regulation.

Impacts
Information on about muscle protein metabolism is important in the both improving the efficiency of muscle growth, ie meat, and the quality of the meat products. The first study examines the factor regulating protein deposition in muscle. The study is focused on the quality aspects the meat from pigs and turkeys.

Publications

  • Gaebler, D.R., S.J. Jones, and R.W. Mandigo. 2002. Measuring oxidative rancidity in meat products with a microplate reader. Meat Sci 62:193-198. Jour. No. 13287.
  • Novak, C., S. Scheideler, S. Jones 2002. Effect of dietary level and TSAA:Lysine ratio on body composition, egg protein and magnum tissue of the laying hen. Poultry Sci. 80: (Suppl. 1) 390.


Progress 10/01/00 to 09/30/01

Outputs
In a study using Differential Display PCR (dd-PCR) with C2C12 murine myogenic cells, the hypothesis which was tested was the differentiation of myoblasts (MB) to form myotubes (MT) is accompanied by significant changes in gene expression. Total RNA of both cell types was reverse transcribed into cDNA followed by amplification by PCR. Samples were run on a 5.8% polyacrylamide gel, bands were excised based on the consistency of banding within cell state versus differences across cell type, reamplified, and sequenced. One hundred and twenty primer combinations (6 Anchor x 20 Arbitraries) were used to identify 149 potential differentially expressed bands of which 28 % were excised and sequenced. Thirty two bands were of a known homology, two were previously identified expressed sequenced tags (EST) and five bands of unknown homology. Sequencing results from five selected bands with homologies to Myosin Light Chain (MLC), Titin, Ribosomal Protein-L3, (RPL3), Ceruloplasmin, and Glutathione Peroxidase were confirmed using Northern hybridizations different cell cultures. It was identified that when compared to 0 h, titin expression was 51.4% at 24 h and 9.2% at 48 h, ceruloplasmin expression was 41% at 24 h and 4.2% at 48 h, RPL3 expression was 84% at 24 h and 79% at 48 h, and MLC expression was 10 fold higher at 24 h and 50 fold higher at 48 h. The goal of the second study was to identify changes in the protein profile (proteome) of skeletal muscle from turkeys affected by the PSE condition. Significant progress has been made toward the objective of optimizing a separation protocol for turkey muscle samples. Work during the past year has focused on development of separations and examination of proteins associated with sarcoplasmic reticulum by two-dimensional electrophoresis. The SR fraction is potentially of greatest relevance to our studies because of its essential role in calcium regulation. Abnormal calcium regulation is widely held to be the underlying cause of stress syndrome and PSE, however, the proteins responsible for this defect are not completely known. Considerable time and effort was spent in developing a 2-D separation protocol that would be useful for analyzing this fraction. Examples of sarcoplasmic reticulum separations made from normal and PSE muscle can be seen in Fig 1a & b. It should be noted, a substantial amount of difficulty was encountered in optimizing the resolution of sarcoplasmic reticulum associated proteins. Comparison of 2-D sarcoplasmic reticulum separations from normal and PSE muscle by image analysis, showed a number of differentially expressed proteins. A group of 20 spots corresponding to a portion of these proteins of interest were excised from the gels and sent for identification to the University of Nebraska Mass Spectrometry facility.

Impacts
This work is in the preliminary stages and will yield results in the near future. The work with differential display will help in explaining the sequence of events in the changes of gene expression during a critical time in muscle growth. The work in protein profiling will help in explaining the cause of stress related conditions in muscle of both turkeys and pigs. It may also be possible from the findings we obtain to develop a procedure to screen animals minutes after slaughter so they can be handled in such a way to minimize the problems.

Publications

  • No publications reported this period


Progress 10/01/99 to 09/30/00

Outputs
To gain insight into the molecular mechanisms of skeletal muscle development, we searched for genes regulated by the differentiation of C2C12 myogenic cells using Differential Display PCR. To date, 120 primer combinations (6 Anchor x 20 Arbitraries) have been used to identify 149 potential differentially expressed bands. Twenty eight percent (45 bands) were excised and sequenced. Thirty two bands had homology to known genes. Two bands had homology to previously identified expressed sequence tags. Five bands have shown no homology to known genes. Three bands were not identified due to inefficient sequencing. Northern blot analysis was performed to validate 12 % of the bands. These bands have shown homologies to Myosin Light Chain-2 (MLC2), AMP deaminase 3, RPL3, and Glutathione Peroxidase 1. Expression of Myosin Light Chain (MLC), one of six polypeptides which comprise myosin the principal component of muscle thick filament, has been shown to only be expressed in myotubes. Its presence in our results provide support for the use of differential display to identify changes in gene expression due to differentiation of myogenic cells. Ribosomal protein L3 (RPL3) sequence is highly conserved across mammalian species, lower eukaryotes, and archaebacteria. RPL3 is the largest protein of the 60s subunit of the ribosome in eukaryotes and acts as the center channel through which new peptides emerge. The results obtained in this study indicate that dd-PCR is able to detect a large number of both up and down regulated genes resulting from the differentiation process. This has allowed us to identify 7 possible ESTs, 5 of which are potentially novel.

Impacts
Muscle growth is the primary reason for livestock production. The events of differentiation can have a profound impact on muscle growth. Using differential display we able to sort through this complex process to determine possible mechanisms of control. To date we have seven gene sequence that have not been identified

Publications

  • Deniz, M., S.J. Jones, S.D. Harn, L. Lippiello, C.A. Hansen, and D.G. Brown. 1999 Effect of 17 -Estradiol (E2) on DNA, protein and glycosaminoglycans (GAG) in the tempromandibular joint knee joint and uterus of the guines pig. Jour. Dent. Res.
  • Edeal, J.B., C.D. Gladney, M.F. Allan, D. Pomp, S.J. Jones. 2000 Identifying differentially expressed genes in C2C12 myogenic cells using differential display PCR. J. Anim. Sci 78 (suppl 1) abs 141.


Progress 10/01/98 to 09/30/99

Outputs
Recent work in our lab has focused on the rate of ribosome recruitment for protein synthesis in myogenic cells during differentiation. We have identified that differentiation reduce initiation rates of ribosomes. This was further verified when C2C12 cell cultures had been treated with fusion media for twenty four hours, then cytosine arabinoside added to eliminate any dividing cells, which also showed a decrease of initiation of ribosome for protein synthesis. A second study in progress in designed to determine changes in mRNA translation in C2C12 myogenic cells that are actively dividing and cells that have fused in to myotubes and been treated with cytosine arabinoside to remove remaining cells that are dividing. Cell will be removed from the plates and ribosomes will be isolated. Ribosomes will be fractionated on a sucrose gradient and the polyribosome fraction will be collected. Ribosomes will be released and the mRNA attached to the ribosome will be identified using differential display method developed by Liang and Pardee (1992) Specific candidate genes that either appear exclusively in myoblast or myotubes will be identify and sequenced to determine what they are. Unknown genes that have not been previously identified will be studied further to determine their possible action.

Impacts
Muscle growth can be controlled by the rate at which proteins are synthesized. Much of the recent work has focused on trnscription of DNA into messenger RNA. This project will focus on the rate of the translation mRNA into protein which has not been fully investigated.

Publications

  • Smith, C.W., J.G. Klaasmeyer, T. L. Woods, and S.J. Jones. 1999. Effects of IGF-I, IGF-II, bFGF, and PDGF on the initiation of mRNA translation in C2C12 myoblasts and differentiating myoblasts. Tissue and Cell 31: 403-412.
  • Smith, C. W., J. G. Klaasmeyer, T. L. Woods, and S. J. Jones. 1999. Effects of serum deprivation, insulin and dexamethasone on polysome percentages in C2C12 myoblasts and differentiating myoblasts. Tissue and Cell 31:451-458.
  • Qingyi, C., S.J. Jones, and M.G. Zeece. 1999. Capillary electrophoretic determination of cathepsin D activity using Oregon green labeled hemoglobin. Electrophoresis. 20:2945-2451


Progress 10/01/97 to 09/30/98

Outputs
Work in our laboratory has continued to focus on changes in ribosome recruitment for protein synthesis. A study was conducted to determine the changes in polysome percentage at the time of differentiation and the effect the media used to stimulate fusion may have on these changes. Cells were stimulated to fuse by changing the concentration of serum from 10% fetal bovine serum (FBS) to 2% horse serum. There was a decline in polysome percentage once the media had been changed and this reduction was maintained for as long as 72 hours. Changes in the total amount of ribosomal RNA were not observed until 72 hours after the stimulation of the fusion. If cells were maintained on media containing 10% fetal bovine serum, the decline in polysome percentage was not realized until 12 hours after media change. Differentiation, as measured by creatine kinase activity, was also delayed due to the presence of the FBS in the media. In a second study, fetal bovine myogenic cells were cultured and induced to fuse by changing to a media containing 2 % fetal bovine serum. Cultures were treated with either insulin or dexamethasone, a synthetic corticoid. Insulin caused an increase in synthesis and a decrease in degradation, whereas dexamethasone caused an increase in degradation and no changes in synthesis. The change in synthesis with insulin was mirrored with changes in polysome percentages. There were no changes in total RNA during the study indicating that the increase in RNA activity caused changes in synthesis rates.

Impacts
(N/A)

Publications

  • Klaasmeyer, J.G., Smith, C.W., Grant, D.L., Woods, T. L., Jones, S. J. 1998. Changes in ribosomal recruitment during translation in differentiating C2C12 myogenic cells. J. Anim. Sci. 76:118 (Suppl. 1).
  • Woods*,T. L., Smith,C. W., Klaasmeyer, J. G., Grant, D. L., Jones, S. J. 1998. Determination of Protein Turnover and Ribosome Activity in Bovine Myotubes: Effects of Serum, Insulin, and Dexamethasone. J. Anim. Sci. 76:117 (Suppl. 1).


Progress 10/01/96 to 09/30/97

Outputs
Molecular mechanisms regulating muscle growth & differentiation work in understanding the role that mRNA translation rate has on protein accretion in muscle was the main focus of research in our lab. Several studies to accomplish this were performed over the past year. The first study examined the differences in the rate of initiation of translation between C2C12 myoblasts prior to fusion & myotubes 24 after stimulation of fusion in the presence or absence of serum. It was observed that initiation rate was reduced when serum was removed from the media. Initiation rates, as measured by polysome percentages, were also lower in myoblasts than myotubes. There were no observed changes in ribosome number as measured by total RNA content of the cell. Initiation rates were determined in the presence of the hormones, insulin or dexamethasone (synthetic glucocorticoid), & the growth factors IGF I, IGF II, FGF, & PDGF. Insulin, IGF I, IGF II, FGF, & PDGF increased the initiation rate in myoblasts & myotubes. Protein synthesis, as measured by tyrosine uptake, demonstrated similar patterns of increases. Dexamethasone demonstrated a decline in initiation rate and protein synthesis. A 2nd study identified that initiation rates decreased within 6 hours after the stimulation of the fusion & continued to decline up until 24 hours after fusion stimulation. There were no differences in RNA amounts indicating those changes in synthesis rates are probably due to changes in the initiation of the mRNA translation.

Impacts
(N/A)

Publications

  • Woods, T.L., Smith, C.W., Zeece, M.G., Jones, S.J. 1997. Conditions for the culture of bovine embryonic myogenic cells. Tiss. Cell.
  • Klaasmeyer, J.G., Smith, C.W., Grant, D., Woods, T.L., Jones, S.J. 1997. Changes in ribosomal recruitment during translation in differentiating C2C12 myogenic cells. J. Anim. Sci. 75:160 (Suppl.
  • Smith, C.W., Klaasmeyer, J. G., Woods, T.L., Jones, S.J. 1997. Insulin, FGF, IGFI, IGFII, and PDGF alter the recruitment of ribosomes to polysomes for protein synthesis in C2C12 myogenic cells.


Progress 10/01/95 to 09/30/96

Outputs
Objective of 1st study was to determine whether serum from other sources might replace fetal bovine serum (FBS) in stimulating cell proliferation in fetal bovine myogenic cells. The different types of serum added to media were horse serum (HS), newborn calf serum (NCS), or iron supplemented calf serum(FeCS)or serum free SF. After 24 hr the cell numbers increase in all treatments; however, the greatest to least growth was SF=FBS>NCS>FeCS=HS (p < .01). After 48 hr, the different treatments continued to alter the cells growth response (FeCS>FBS=NCS>HS>SF)(p < .01). These difference were observed at 60 hr after treatment. The treatments altered the extent of cell differentiation. The greater growth observed when FeCS was used may indicate that this serum could be used as a suitable replacement for FBS. The objective of the 2ND study was to determine the percentage of ribosomes found as polysomes in C2C12 myoblasts & myotubes in the presence or absence of serum. Polysomes were fractionated using 15-60% sucrose gradients. Percent polysomes were determined by measuring the area under the peaks for monosomes and polysomes. The removal of serum in the media decreased (p<.01) the percentage of polysomes in both myoblast & myotubes. Myoblast tended to have a higher (P<.08) polysome percentage than myotubes. Conclusion: Serum removal causes shift from polysomes to monosomes in myoblasts & myotubes, indicating a decrease in initiation of translation of mRNA possibly due to removal of growth factors in serum.

Impacts
(N/A)

Publications

  • Desler, M.M., Jones, S.J., Smith, C.W., Woods, T.L. 1996. Effects of dexamethasone and anabolic agents on proliferation and protein synthesis and degradation in C2C12 myogenic cells J. Anim. Sci. 74:1265-1273.
  • Smith, C.W., Woods, T.L., Blad, K.L., Klopfenstein, T.J., Jones, S.J. 1996. Induced ruminal acidosis in sheep affects protein turnover and proliferation of muscle cells in vitro. J. Anim. Sci. 74:46 (Suppl. 1).
  • Woods, T.L., Smith, C.W., Blad, K., Jones, S.J. 1996. Optimizing the culture conditions for the bovine cloned fetal muscle cell growth in vitro. J. Anim. Sci. 74:46 (Suppl. 1).
  • Woods, T.L., Smith, C.W., Klaasmeyer, J.G., Jones, S.J. 1996. The growth of bovine muscle cells using different sources of serum. J. Anim. Sci. 74:142 (Suppl 1).
  • Smith, C.W., Klaasmeyer, J.G., Woods, T.L., Jones, S.J. 1996. Changes polysome distribution in C2C12 myogenic cells due to differentiation and presence of the serum. J. Anim. Sci. 74:149 (Suppl. 1).


Progress 01/01/95 to 12/30/95

Outputs
The effects of the anabolic compounds estradiol, testosterone and dihydrotestosterone alone and in combination with dexamethasone on proliferation and protein turnover in cultured C2C12 myogenic cells were studied. It was observed that anabolic agents at pharmacological doses do not inhibit the dexamethasone effects on C2C12 myogenic cells, nor do they directly affect proliferation or protein turnover in these cells. A second group of experiments were designed to determine the optimal cell culture conditions for fetal bovine myogenic cells. Different substrates were tested and it was determined that substrata did not affect growth. The optimal medium for the cell to proliferate was Delbecco modified eagle medium (DMEM) M-199. The best fusion medium was DMEM- F12. Another study was to determine if the serum from ruminal acidotic sheep had an affect on myogenic cell growth in culture. The results indicate that acidosis caused a change in the serum of sheep that decreases (P < .05) both protein synthesis and degradation. As the sheep recovered, so did protein turnover.

Impacts
(N/A)

Publications

  • Woods, T.L., Jones, S.J., Zeece, M.G., Smith, C.W. 1995. Protein turnover in primary muscle cells incubated with dexamethasone or insulin. J. Anim Sci. 73(Suppl. 1):110. (Abstr.).
  • Zeece, M.G., Chu, Q., Jones, S.J., Woods, T.L., Reville, W.J. 1995. Determination of 3-methylhistidine by capillary electrophoresis. J. of Capillary Electrophoresis. (Accepted).


Progress 01/01/94 to 12/30/94

Outputs
A study examined the short term responses of muscle to beta-adrenergic agonist (BAA) treatment. Rabbits were given either 7 ppm or 0 ppm BAA. Protein, RNA and DNA were measured in muscle. Muscle RNA concentration and RNA:DNA ratios were increased from day one and remained higher thereafter. Protein:RNA ratios were lower in treated than control rabbits after 4 d and remained lower after 4 d and higher after 16 d; and protein content was higher after 16 d in treated vs control rabbits. These data imply the BAA-induced muscle growth in rabbits occurs through hyperplasia and seems to be related to elevated protein synthetic capacity. A second study was performed to determine the feasibility of using the capillary electrophoresis to determine the release of the 3 MeHis from cultured bovine myotubes. Preliminary results show that this rapid procedure can accurately determine differences in free 3MH levels occurring in response to elevation or suppression of overall protein degradation rates in cultured muscle cells.

Impacts
(N/A)

Publications

  • PRINGLE, T.D., LONERGAN, S.M., CALKINS, C.R., JONES, S.J., MILLER, P.S., KOOHMARAIE, M. 1994. Temporal response of rabbits to beta-adrenergic agonist feeding: tissue weight, calpains and calpastatin activities and nucleic acid an.
  • ZEECE, M.G., KEITH, R., WOODS, T.L., JONES, S.J. 1994. etermination of free 3 methylhistidine in bovine skeletal cell cultures by capillary electrophoresis, 5th Annual Frederick Conf. on Capillary Electrophoresis. Oct. 24-25 1994.


Progress 01/01/93 to 12/30/93

Outputs
The use of beta-adrenergic agonist to increase muscle mass was investigated. Forty wether lambs were used in the experiment with one-half receiving 4 ppm of the beta-adrenergic agonist L-644,969 (BAA) and the other half receiving 0 ppm of the treatment compound. Five animals from each treatment were assigned a slaughter date after 0, 2, 4 and 6 wk of treatment. After slaughter the longissimus (L) and semitendinous (S) muscles were removed and protein, DNA and RNA were measured. Longissimus protein:DNA was higher after 4 wk and 6 wk in BAA treated lambs. The concentrations of RNA and RNA:DNA were higher in L and S muscles of BAA treated lambs after 2 wk and remained higher throughout the study. The S muscle protein was higher after 2, 4, and 6 wk and DNA content was higher after 2 and 6 wk in treated lambs. These data indicate an alter- ation of muscle growth during BAA treatment. A second study examined the short term responses of muscle to BAA treatment. Rabbits were given either 4 ppm or 0 ppm BAA. Protein, RNA and DNA were measured in muscle. Muscle RNA concen- tration and RNA:DNA ratios were increased from day one. These data imply the BAA-induced muscle growth in rabbits occurs through hyperplasia and seems to be related to elevated protein synthetic capacity.

Impacts
(N/A)

Publications

  • PRINGLE, T.D., CALKINS, C.R., KOOHMARAIE, M., JONES, S.J. 1993. Effects over time of feeding a beta-adrenergic agonist to wether lambs on animal performance, muscle growth, endogenous muscle proteinase activities, and meat tender.
  • PRINGLE, T.D., LONERGAN, S.M., CALKINS, C.R., JONES, S.J., MILLER, P.S., KOOHMARAIE, M. 1993. Temporal response of rabbits to beta-adrenergic agonist feeding. J. Anim. Sci. (In press).


Progress 01/01/92 to 12/30/92

Outputs
Response time to cimaterol (CIM), a (beta)-adrenergic agonist, by broiler chickens for carcass characteristics, muscle composition, fiber size catheptic enzyme activity, and tenderness was determined. Measurements were taken at 2, 4, 6, 8, 10, 12 d after treatment diet was given to the birds. Muscles measured were the breast muscle (BM) and leg muscles (LM). Shear force values were higher in BM at d 8 and 10 and higher in LM at d 4, 8, 10, 12, and 14 in CIM fed birds. The BM of CIM-fed birds had higher protein:DNA ratio at d 6 though 14, whereas LM of CIM-fed chickens had higher ratios at d 8, 10, and 14. Fiber size of the BM in CIM-fed chicken tended to be higher at d 8 and 14. Total weight of the BM and as a percentage of final body weight was higher as d 10 and 14. A second study measured the response of animal performance, muscle growth, proteinase activity, and meat tenderness to (beta)-adrenergic agonist treatment in lambs. Lambs were fed a diet containing 4 ppm L-644,969, a (beta)-adrenergic agonist and slaughtered after 0, 2, 4, and 6 wk of treatment. Longissimus protein:DNA was higher after 4 and 6 wk and semitendinosus protein:DNA was higher after 4 wk in treated lambs. Concentrations of RNA and RNA:DNA were higher in both muscles of treated lambs from 2 wk throughout the study. Both studies indicate a rapid alteration of muscle growth and composition when animals are treated with (beta) adrenergic agonists which leads to a change in meat tenderness.

Impacts
(N/A)

Publications