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The Journal of Nutrition Vol. 128 No. 2 February 1998,
pp. 251-256
Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0306
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ABSTRACT |
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Abstract
Introduction
Methods
Results & Discussion
References
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Ruminants have high concentrations of peptide-bound amino acids in the circulation. Earlier studies in our laboratory showed that a myogenic cell line (C2C12) developed from mouse skeletal muscle and a mammary epithelial cell line (MAC-T) developed from bovine mammary epithelial tissues are able to utilize peptides as amino acid sources. In this study, primary cultures of ovine myogenic satellite cells were evaluated for their ability to use peptide-bound methionine as a source of methionine for protein accretion and cell proliferation. The basal medium contained methionine-free Dulbecco's modified Eagle's medium supplemented with 6% desalted fetal bovine serum. Treatment media included the basal medium supplemented with no methionine, methionine or one of 22 methionine-containing peptides. No protein or DNA accretion was observed in the presence of basal medium alone. Growth responses to all of the peptides were obtained, with protein and DNA accretion ranging from 49 to 107% and from 45 to 144% of the corresponding methionine response, respectively. These results indicate that the ovine myogenic satellite cells possess the ability to utilize methionine-containing peptides as methionine sources for protein accretion and cell proliferation.
KEY WORDS: methionine · peptide · ovine · satellite cells · protein accretion · cell proliferation
High concentrations of peptide-bound amino acids were observed in the blood plasma of calves (Koeln et al. 1993 Therefore, in this study, primary cultures of ovine myogenic satellite cells were used to investigate whether ovine skeletal muscle possesses the ability to utilize methionine-containing di- to octapeptides for protein accumulation and cell proliferation.
Methionine-containing peptides.
The twenty-two methionine-containing di- to octapeptides (Sigma Chemical, St. Louis, MO) tested in this study and the hydrophobicity values of the dipeptides are presented in a previous paper (Pan et al. 1996 Medium and vessel preparation.
The preincubation medium (Dodson et al. 1986 Isolation of myogenic satellite cells.
The protocol followed was reviewed and approved by the Virginia Tech Animal Care Committee. Satellite cells were isolated from the semimembranosus and semitendanosus muscles of four lambs (1- to 5-mo old) by a modification of the procedure described by Dodson et al. (1986) Culture procedure for myogenic satellite cells.
A stored cell suspension from one of the four lambs was thawed and then plated on a 80 cm2 culture flask coated with basement membrane matrix. The isolated satellite cells were grown in 80 cm2 flasks for 5-6 d at 37°C, 90% air-10% CO2 in a humidified environment. The growth medium was changed every 12 h within the first 48 h and then every 24 h. The cells were then released by 0.05% trypsin solution, and the enzyme action was stopped by the addition of growth medium. The resulting cells were counted by a hemacytometer, suspended in growth medium, and seeded in 12-well plates (Fisher Scientific) for peptide utilization evaluations. Cell viability was determined by the Trypan blue exclusion test. The viability of the original satellite cells was ~70-80% after the cells were thawed from liquid nitrogen, and the viability of cells used for growth experiments was ~98%.
Myogenic ability of the isolated cells.
To verify their myogenic ability, isolated cells stored in liquid nitrogen were thawed and then cultured in the growth medium for 5-6 d until confluence. The cultures then were incubated with the differentiation medium for 3 d. The myogenic ability of the isolated cells was determined by observing the formation of myotubes.
L-Methionine standard curve for protein accretion.
Experiments were conducted to determine the effect of free L-methionine concentrations in the medium (0-50 µmol/L) on protein accretion in the primary cultures of the ovine skeletal muscle at different cell densities (10,000 and 20,000 cells/well). The response curve generated was used to determine the cell density and the concentration of both L-methionine and methionine-containing peptides to be used in the peptide utilization studies.
Peptide utilization by primary cultures.
The ovine satellite cells obtained from the above propagation were suspended in growth medium, plated at 20,000 cells per well (12-well plate) and incubated at 37°C, 90% air-10% CO2 in a humidified environment for 24 h; then the growth medium was replaced by MFDMEM and the cultured cells were incubated for another 24 h. Subsequently, the MFDMEM was removed from the cultures and the starved cells were incubated with one of the treatment media for 72 h. The treatment media were changed at 24-h intervals.
Harvest and analytical procedures.
After 72 h of incubation, the cultures were washed twice with ice-cold D-PBS. The protein and DNA contents were analyzed either from a sample of the same well or from samples taken from separate wells. When protein and DNA were analyzed from separate samples, cultures for protein assay were dissolved by treatment with 0.4 or 0.8 mL of 0.5 mol/L NaOH overnight. The resulting solutions were neutralized with 0.04 or 0.08 mL of 5 mol/L HCl and the protein contents were determined by the enhanced bicinchoninic acid (BCA, Pierce, Rockford, IL) assay (Smith et al. 1985 Statistical analysis.
Treatments were replicated in four wells. Satellite cells from each lamb were examined separately. The protein and DNA results were analyzed as a randomized complete block design. The model was as follows: Yij = µ + The myogenic ability of the isolated cells was determined by observing the formation of myotubes. Figure 1 shows a representative picture of the mulitnucleated myotubes formed from isolated myogenic cells. Examination of the pictures reveals that there is ~50% differentiation of the cells.
The authors express their appreciation to Don Shaw and Kris Lee for their technical assistance.
INTRODUCTION
Abstract
Introduction
Methods
Results & Discussion
References
, McCormick and Webb 1982, Seal and Parker 1991). Koeln et al. (1993) reported that, in fed calves, ~70% of the amino acid flux across the gastrointestinal tract was in the form of peptides, and that only ~10% of the peptide-bound amino acids entering the portal blood were removed by the liver. On the contrary, most (83%) of the free amino acids entering the portal vein were extracted by the liver. DiRienzo (1990) reported that the flux of peptide-bound amino acids across the stomach region of the gastrointestinal tract accounted for 77% of the total amino acid flux across the portal-drained viscera in both fed sheep and calves. Little is known about the fate of these circulating peptide-bound amino acids. Earlier, we showed that MAC-T cells, a cell line originally developed from bovine mammary epithelial tissue, and C2C12 cells, a myogenic cell line developed from mice, possess the ability to utilize methionine-containing di- to octapeptides for protein accretion and cell proliferation (Pan et al. 1996). Because of the importance of muscle protein synthesis in meat animal production, it is of interest to investigate whether skeletal muscle of ruminants has the ability to utilize methionine-containing di- to octapeptides.
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Fig 3.
Effect of methionine-containing dipeptides on protein accretion in primary cultures of ovine skeletal satellite cells. The cells were incubated in growth medium for 24 h and then in MFDMEM for another 24 h. The cells were then incubated with MFDMEM supplemented with 6% desalted fetal bovine serum and 8 µmol/L L-methionine, or dipeptides (equivalent to L-methionine in methionine content) for 72 h. The treatment media were changed at 24-h intervals. The protein contents of the cultures were determined by the enhanced bicinchoninic assay. Bars (means + SEM, n = 16 wells) with different letters differ (P < 0.05). *Different from L-methionine (P < 0.05). MA, methionylalanine; MF, methionylphenylalanine; MG, methionylglycine; ML, methionylleucine; MP, methionylproline; MV, methionylvaline; MM, methionylmethionine; AM, alanylmethionine; FM, phenylalanylmethionine; GM, glycylmethionine; LM, leucylmethionine; PM, prolylmethionine; SM, serylmethionine; VM, valylmethionine.
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Fig 5.
Effect of methionine-containing tri- to octapeptides on protein accretion in primary cultures of ovine skeletal satellite cells. The cells were incubated in growth medium for 24 h and then in MFDMEM for another 24 h. The cells were then incubated with MFDMEM supplemented with 6% desalted fetal bovine serum and 8 µmol/L L-methionine, or peptides (equivalent to L-methionine in methionine content) for 72 h. The treatmet media were changed at 24-h intervals. The protein contents of the cultures were determined by the enhanced bicinchoninic acid assay. Bars (means + SEM, n = 16 wells) with different letters differ (P < 0.05). *Different from L-methionine (P < 0.05). A, alanine; F, phenylalanine; G, glycine; K, lysine; L, leucine; P, proline; R, arginine; S, serine; V, valine; Y, tyrosine.
MATERIALS AND METHODS
Abstract
Introduction
Methods
Results & Discussion
References
).
) for ovine satellite cells was composed of Dulbecco's modified Eagle's medium (DMEM;4 GIBCO BRL, Grand Island, NY) supplemented with 10% horse serum (HS; GIBCO BRL), 1% antibiotic-antimycotic solution (GIBCO BRL) and 0.1% gentamicin solution (GIBCO BRL). The growth medium for ovine satellite cells contained DMEM supplemented with 15% HS, 1% antibiotic-antimycotic solution and 0.1% gentamicin solution (Dodson et al. 1986). The differentiation medium for the cells contained DMEM supplemented with 1% HS, 1% antibiotic-antimycotic solution and 0.1% gentamicin solution (Dodson et al. 1990). The freezing medium consisted of DMEM supplemented with 20% HS, 10% dimethyl sulfoxide (Sigma Chemical), 1% antibiotic-antimycotic solution and 0.1% gentamicin (Dodson et al. 1990). Before use, culture flasks (80 cm2, Marsh Biomedical Products, Rochester, NY) were coated with basement membrane matrix (Collaborative Biomedical Products, Bedford, MA) to facilitate the attachment of isolated satellite cells. Thawed basement membrane matrix (~1 mL) was added to 9 mL of cold DMEM (2-4°C) supplemented with 1% antibiotic-antimycotic solution and mixed thoroughly. The 1:10 diluted matrix was added to the flasks so that the entire growth area was covered. The flasks were kept in a tissue culture hood (Labconco, Kansas City, MO) for 1 h; then the unbound material was aspirated and the flasks were rinsed gently with serum-free DMEM plus 1% antibiotic-antimycotic solution. The vessels were then ready for use.
. All procedures used in the isolation of muscle and the harvest of cells were conducted aseptically. Semimembranosus and semitendinosus muscles were removed from anesthetized (pentobarbital) lambs immediately after exsanguination. The muscle tissues were immersed in ice-cold Dulbecco's phosphate balanced saline (D-PBS, Sigma Chemical) and transported to a tissue culture hood within 5 min after removal of muscle tissue from the carcass for the isolation of satellite cells. After removal of excessive connective tissue, the muscles were cut into small strips and the strips were passed through a small, sterile meat grinder. The ground muscle was incubated with pronase E (2 g/L D-PBS, Sigma Chemical) for 1 h at 37°C in a water bath with agitation every 10 min. After the incubation, the mixture was centrifuged at 1500 × g for 12 min. The supernatant was discarded. The pellet was suspended in D-PBS and centrifuged at 500 × g for 10 min to pellet tissue debris. The resulting pellet was resuspended in D-PBS and centrifuged again at 500 × g for 10 min. The resultant supernatant was collected and centrifuged at 1500 × g for 10 min to pellet the satellite cells. The resulting pellet was suspended in preincubation medium, and the cell suspension was incubated at 37°C, 90% air-10% CO2 in a humidified environment for 1 h to remove fibroblasts. After preincubation, the cell suspension was centrifuged at 1500 × g for 6 min to pellet the satellite cells again, and the resulting pellet was suspended in freezing medium. Aliquots of 2 mL of the cell suspension were transferred into 2-mL cryogenic vials (Fisher Scientific). The vials were put into a Nalgene Cryo 1°C Freezing Container (Fisher Scientific) and precooled at 
70°C overnight. The precooled vials were stored in liquid nitrogen until used. Satellite cells isolated from the four lambs were stored separately.
). Cultures for DNA determination were harvested, and the DNA concentrations were determined by measuring the fluorescence that resulted from the interaction between DNA and the fluorochrome Hoechst 33258 (Romagnolo et al. 1992).
and the remainder was treated with 400 µL 1 mol/L NaOH overnight (or 18 h). The NaOH-treated sample was neutralized by 0.08 mL of 5 mol/L HCl solution, and the neutralized sample was used for protein assay by the enhanced BCA procedure (Smith et al. 1985).
i + 
j + Eij where Yij is the protein or DNA content obtained from the ith treatment in the jth animal, µ is the overall mean, i is the effect of treatment i, j is the effect of animal j and Eij is the error component associated with the ith treatment in the jth animal.
value of 0.05 (SAS 1989).
RESULTS AND DISCUSSION
Abstract
Introduction
Methods
Results & Discussion
References
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Fig 1.
Representative picture of the multinucleated myotubes formed from cultured satellite cells that were isolated from the semimembranosus and semitendanosus muscles of lambs.
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Fig 2.
Methionine standard curve for protein accretion in primary cultures of ovine skeletal satellite cells. The cells were plated at 10,000 or 20,000 cells per well (12-well plate) and were incubated in growth medium for 24 h. The growth medium was replaced by methionine-free Dulbecco's modified Eagle's medium (MFDMEM) and the cells were incubated for another 24 h. The cells were then incubated with MFDMEM supplemented with 6% desalted fetal bovine serum and various concentrations of L-methionine for 72 h. The protein contents of the cultures were determined by the enhanced bicinchoninic assay. Values are means ± SEM, n = 6.
), we also showed that glycylmethionine and prolylmethionine were poorly utilized by the C2C12 myogenic cell line developed from mouse skeletal muscle and the MAC-T cell line developed from bovine mammary epithelial tissues. It appears that although there is some similarity in the utilization of methionine-containing dipeptides among the three cell types, differences also are obvious. For instance, leucylmethionine, methionylvaline and methionylmethionine were utilized as effectively as methionine by C2C12 myogenic cells and better than methionine by MAC-T mammary epithelial cells, but the growth response of satellite cells to these three dipeptides was ~75% of the methionine response. This suggests that a different ability of peptide utilization may exist among different tissues or even species. This phenomenon was suggested previously. Lochs et al. (1988) investigated the utilization of glycylleucine and glycylglycine by different tissues of dogs in vivo. Their results indicated that all of the tissues examined (liver, muscle, kidney and gut) utilized both peptides. However, tissues differed considerably in the utilization of the same dipeptides. Liver, kidney, muscle and gut extracted 25, 24, 12 and 10% of the infused amount of glycylleucine, respectively. In the case of glycylglycine, kidney played the most important role in the clearance of this peptide (37%), followed by muscle (18%), liver (15%) and gut (11%).
), we showed that 6% desalted FBS could hydrolyze methionine-containing dipeptides to varying degrees, suggesting that hydrolytic enzymes in the serum are at least partially responsible for the observed growth in cultured cells. Hydrolysis of the peptides by plasma, membrane-bound and capillary bed-associated peptidases with the subsequent uptake of the free amino acids has been proposed as the major mechanism for the utilization of peptide-bound amino acids by tissues (Adibi and Morse 1981, Furst et al. 1987, Raghunath et al. 1990). Nutzenadel and Scriver (1976) showed that the diaphragm of rats could take up carnosine via a saturable and Na+-dependent system. Because carnosine has several physiologic functions in muscle (Rodwell 1990), it is not surprising that muscle has a specific transport system for this dipeptide.
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Fig 4.
Effect of methionine-containing dipeptides on DNA accretion in primary cultures of ovine skeletal satellite cells. The cells were incubated in growth medium for 24 h and then in MFDMEM for another 24 h. The cells were then incubated with MFDMEM supplemented with 6% desalted fetal bovine serum and 8 µmol/L L-methionine, or dipeptides (equivalent to L-methionine in methionine content) for 72 h. The treatmet media were changed at 24-h intervals. The DNA contents of the cultures were determined by a fluorescent assay (Romagnolo et al. 1992 ). Bars (means + SEM, n = 16 wells) with different letters differ (P < 0.05). *Different from L-methionine (P < 0.05). See Fig. 3 legend for abbreviations.
) showed that, in the presence of 6% desalted FBS, dipeptides with the same amino acid composition but different molecular structures also affected protein accumulation and/or cell proliferation differently in C2C12 and/or MAC-T cells. Dipeptides with the same amino acid composition but different residue sequences have been shown to affect protein accretion in a number of organisms (Eagle 1955, Kihara and Snell 1952, Naider et al. 1974).
) indicated that C2C12 myogenic and MAC-T mammary epithelial cells were able to utilize tri- to octapeptides as methionine sources for protein accretion. Grahl-Nielsen et al. (1974) showed that, in the presence of 1% dialyzed calf serum, a cell line developed from the small intestine of hamster was able to grow in the presence of di-, tetra-, hepta- and decalysine, but the growth responses decreased with increasing chain length. They observed no detectable peptidase activity against the decalysine. In our earlier study (Pan et al. 1993), we showed that the utilization of methionine-containing tetra- to octaenkephalin segments by MAC-T mammary epithelial cells as a source of methionine was entirely dependent on the presence of 6% desalted FBS in the culture medium; the 6% desalted FBS was able to release all of the methionine residues from the tetra- to octa-methionine-enkephalins within a 24-h incubation in cell-free medium. Although we did not identify the enzyme(s) responsible for the observed hydrolysis of enkephalins, animal sera have been shown to contain enkephalin-hydrolyzing enzymes, including aminopeptidase M, dipeptidyl carboxylpeptidase and angiotensin converting enzyme (Shibanoki et al. 1991 and 1992). Therefore the observed growth of myogenic satellite cells on these methionine-containing enkephalin segments is likely due in part to the utilization of free methionine released from the hydrolysis of these segments by serum-associated hydrolytic enzymes. Protein accretion and cell proliferation rate associated with particular peptides that exceed the rate observed with free methionine are likely not due entirely, if at all, to the rate of hydrolysis of the peptides by peptidases. In these cases, getting methionine to the site of protein synthesis probably also involves transporting the peptides across membranes. It seems reasonable to infer from the data collected with cultured cells that the variation observed in the utilization of the peptides examined may be due to a combination of transport and hydrolytic events.
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Fig 6.
Effect of methionine-containing tri- to octapeptides on DNA accretion in primary cultures of ovine skeletal satellite cells. The cells were incubated in growth medium for 24 h and then in MFDMEM for another 24 h. The cells were then incubated with MFDMEM supplemented with 6% desalted fetal bovine serum and 8 µmol/L L-methionine, or peptides (equivalent to L-methionine in methionine content) for 72 h. The treatmet media were changed at 24-h intervals. The DNA contents of the cultures were determined by a fluorescent assay (Romagnolo et al. 1992 ). Bars (means + SEM, n = 16 wells) with different letters differ (P < 0.05). *Different from L-methionine (P < 0.05). See Fig. 5 legend for abbreviations.
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Fig 7.
Effect of methionine-containing dipeptides on protein:DNA ratio in primary cultures of ovine skeletal satellite cells. Bars (means + SEM, n = 16 wells) with different letters differ (P < 0.05). **Less than L-methionine response (P < 0.05). See Fig. 3 legend for abbreviations.
View this table:
Table 1.
Relationship between methionine dipeptide hydrophobicity and protein accretion in ovine myogenic satellite cells
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Fig 8.
Effect of methionine-containing tri- to octapeptides on protein:DNA ratio in primary cultures of ovine skeletal satellite cells. Bars (means + SEM, n = 16 wells) with different letters differ (P < 0.05). **Less than L-methionine response (P < 0.05). See Fig. 5 legend for abbreviations.
reported that Baco-Peptone or Proteos Peptone in a serum-free medium helped to maintain the beating of chicken embryo heart cells. Two tripeptides (glycylhistidyllysine and glycyllysylhistidine) isolated from human serum have been shown to prolong the survival of normal liver cells and stimulate growth in neoplastic liver (Pickart and Thaler 1973). These findings suggest that, in addition to serving as amino acid sources, circulating peptides may have other physiologic functions.
) showed that, in the presence of desalted FBS, protein accretion in C2C12 and (or) MAC-T cells was related to the hydrophobicity of the methionine-containing dipeptides tested regardless of the methionine position in the dipeptides. Data presented in Table 1 show that, in the primary cultures of ovine myogenic satellite cells, protein accretion and cell proliferation were related to the hydrophobicity of dipeptide with methionine at the N-terminus. This again suggests that hydrophobicity or related properties of the dipeptides tested affects their utilization by cultured animal cells. Hydrophobicity of the dipeptides with methionine at the C-terminus had less effect on the protein accretion in the primary cultures of ovine myogenic cells.
ACKNOWLEDGMENTS
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FOOTNOTES |
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Manuscript received 22 July 1996. Initial reviews completed 19 August 1996. Revision accepted 20 October 1997.
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LITERATURE CITED |
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Abstract
Introduction
Methods
Results & Discussion
References
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-alanine and L-carnosine by rat tissues in vitro: role in nutrition.
Am. J. Physiol.
1976;
230:643-651
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