The Journal of Nutrition Vol. 128 No. 4 April 1998, pp. 744-750

One or More Serum Factors Promote Peptide Utilization in Cultured Animal Cells^1,2,3

Yuanlong Pan, Patrick K. Bender^*, R. Michael Akers, and Kenneth E. Webb Jr.⁴

Departments of Animal and Poultry Sciences, ^* Biochemistry and Anaerobic Microbiology and  Dairy Science, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0306

	ABSTRACT

Abstract Introduction Methods Results & Discussion References

We have shown previously that MACT-T and C₂C₁₂ cells utilize methionine-containing di- to octapeptides as methionine sources for protein accretion and cell proliferation in the presence of 60 mL/L desalted fetal bovine serum. In this study, serum factors that may regulate the use of peptides as amino acid sources in C₂C₁₂ and MAC-T cells were examined. The basal media contained methionine-free Dulbecco's modified Eagle's medium supplemented with 4.0 mL/L bovine serum lipids, 10 mL/L chemically defined lipid concentrate, bovine insulin (1 mg/L), 30 mL/L low protein serum replacement (LPSR-1) or 60 mL/L desalted animal serum. Treatment media included basal media supplemented with no methionine, L-methionine, or one of the methionine-containing peptides. L-Methionine promoted protein and DNA accretion (P < 0.05) in the presence of desalted animal sera, insulin or LPSR-1. Methionine-containing peptides also promoted protein and DNA accretion (P < 0.05) in the presence of desalted animal sera or LPSR-1, but not with insulin, except methionylleucine. In a cell-free medium, fetal bovine serum hydrolyzed peptides to varying degrees. We conclude that animal sera contain one or more factors that regulate utilization of peptides as amino acid sources for C₂C₁₂ and MAC-T cells.

	INTRODUCTION

Abstract Introduction Methods Results & Discussion References

Improving meat and milk production is the ultimate goal of ruminant nutrition research, and optimizing the supply of amino acids is one of the approaches to enhance muscle and milk yield. Absorption of intact small peptides from the gastrointestinal tract appears to constitute a major form by which end products of dietary protein digestion enter the blood. Peptide-bound amino acids account for 52-78% of the total plasma amino acid pool in ruminants (DiRienzo 1990, Koeln et al. 1993, Seal and Parker 1991), 10% in humans (Christensen et al. 1947), 9-51% in rats (Asatoor et al. 1978, Seal and Parker 1991) and 11-14% in guinea pigs (Gardner et al. 1983). Intravenously injected peptides were cleared from the blood and with radiolabeled or hydrolysis-resistant peptides, the clearance of injected peptides from the plasma was accompanied by the appearance of radioactivity or intact hydrolysis-resistant peptides in tissues including liver, muscle, kidney, gut, lung, brain and pancreas (Adibi 1987, Stehle et al. 1991). These results suggest that peptides may serve as sources of free amino acids to meet tissue needs. Certain peptides may have various regulatory effects on cell growth and function. For example, glycylhistidyllysine and glycyllysylhistidine have been shown to prolong the survival of normal liver cells and stimulate growth in neoplastic liver (Pickart and Thaler 1973). Two studies in this laboratory indicated that, in comparison with free methionine, some methionine-containing peptides promoted a greater amount of secreted proteins by cultured tissue explants of mouse mammary glands (Wang et al. 1996) and supported greater protein accretion in cultured C₂C₁₂ myogenic cells and MAC-T mammary epithelial cells in the presence of desalted fetal bovine serum (Pan et al. 1996).

This study was designed to evaluate whether animal serum is required for the observed utilization of methionine-containing peptides and, if so, whether sera from different species facilitate peptide utilization in a similar manner. Insulin has been shown to promote protein and DNA synthesis in the presence of essential free amino acids; therefore, it was also of interest to investigate whether insulin promotes peptide utilization. Because serum-free medium does not contain essential fatty acids, we tested insulin plus lipid preparations in serum-free media so that we could exclude the possibility that the absence of essential fatty acids prevented the cell growth and proliferation in the presence of insulin. Cultured C₂C₁₂ myogenic cells and MAC-T mamary epithelial cells were used in this study.

	MATERIAL AND METHODS

Abstract Introduction Methods Results & Discussion References

Methionine-containing peptides.  The methionine-containing peptides (Sigma Chemical, St. Louis, MO) used in this study and their hydrophobicity values are presented in Table 1.

Media preparation.  The methionine-free Dulbecco's modified Eagle's medium (MFDMEM)⁵ was prepared by adding 584 mg glutamine (Sigma Chemical) per liter of medium and 10 mL/L antibiotic-antimycotic solution (GIBCO BRL, Grand Island, NY) to deficient Dulbecco's modified Eagle medium (GIBCO BRL). The resultant medium was sterilized by filtration (0.2 µm, Nalge Company, Rochester, NY). Adult animal sera (GIBCO BRL), including human serum (HuS), horse serum (HS), porcine serum (PS), rabbit serum (RS) and chicken serum (CS), were desalted by gel filtration chromatography by using a Sephadex G-25M desalting column (Pharmacia LKB Biotechnology, Piscataway, NJ). The basal media contained MFDMEM supplemented with 4 mL/L bovine serum lipids (Sigma Chemical), 10 mL/L chemically defined lipid concentrate (GIBCO BRL), 1 mg/L bovine insulin (Sigma Chemical), 30 mL/L low protein serum replacement (LPSR-1, Sigma Chemical) or 60 mL/L of one of the desalted animal sera.

The treatment media consisted of the appropriate basal medium supplemented with either free L-methionine (6 µmol/L, C₂C₁₂; 15 µmol/L, MAC-T, Sigma Chemical) or one of the methionine-containing peptides at concentrations that were equivalent to L-methionine in methionine content. The growth medium used to maintain and propagate C₂C₁₂ myogenic cells consisted of Dulbecco's modified Eagle's medium (DMEM, GIBCO BRL), 10 mL/L antibiotic-antimycotic solution and 150 mL/Lfetal bovine serum (FBS, GIBCO BRL). The growth medium for MAC-T mammary epithelial cells was composed of DMEM, 10 mL/L antibiotic-antimycotic solution and 100 mL/L FBS.

Cell culture procedure.  The C₂C₁₂ and MAC-T cells were prepared according to the protocols described in Pan et al. (1996).

Harvest and analytical procedures.  Protein and DNA were quantified after 72 h of incubation on either separate or the same culture plate/well. When separate plates/wells were assayed, the cultures were washed twice with ice-cold Dulbecco's phosphate buffered saline (D-PBS, GIBCO BRL) and were prepared and analyzed for protein content by using the enhanced bicinchoninic (Pierce, Rockford, IL) assay of Smith et al. (1985) as previously described (Pan et al. 1996). Cultures for DNA determination were harvested and the DNA concentrations were determined by measuring the fluorescence produced by the interaction between sample DNA and the fluorochrome Hoechst 33258 (Labarca and Paigen 1980).

When protein and DNA were quantified from the same plate/well, the cultures were washed once with D-PBS, and 500 µL of buffer (pH 7.4) containing 0.05 mol/L Na₂HPO₄, 2 mol/L NaCl, and 2 mmol/L EDTA was added to each well; then the cultures were sonicated for 15 s by a Sonic Dismembrator Model 300 (Fisher Scientific, Pittsburgh, PA). Then 100 µL of the sonicated sample was transferred for DNA assay as described by Labarca and Paigen (1980) and the rest of the sonicated sample was treated with 400 µL of 1 mol/L NaOH overnight (or 18 h). The NaOH-treated sample was neutralized with 80 µL of 5 mol/L HCl solution, and the neutralized sample was used for protein assay by the bicinchoninic acid procedure (Smith et al. 1985).

Fetal bovine serum peptidase activity.  The methionine-containing peptides (15 µmol/L) were added to 15-mL sterile centrifuge tubes containing 12 mL of the MFDMEM supplemented with 60 mL/L desalted FBS; the mixed solutions were sealed with caps and incubated at 37°C for 24 h. Before and after the 24-h incubation, samples were taken and filtered through ultrafree MC filters (Waters Millipore, Milford, MA) with a 10,000 Da cut-off to remove large molecules. A 40-µL aliquot of the resulting filtrate was processed for HPLC analysis as described previously (Pan et al. 1996) to detect the appearance of free methionine in the samples.

Statistical analysis.  Treatments were replicated in four wells or dishes. Data were analyzed by the General Linear Models (GLM) procedure of SAS (1989) with culture dish or well as the experimental unit. The ANOVA model was as follows: Y_ij = µ + Â_i + E_ij where Y_ij is the jth protein or DNA response from the ith treatment, µ is the overall mean, Â_i is the effect of the ith treatment and E_ij is the error component associated with the jth protein response from the ith treatment. The model for LPSR-1 supplementation was as follows: Y_ij = µ + Â_i + _j + ã_ij + E_ij where Y_ij is the protein or DNA content obtained from the ith substrate in the presence or absence of LPSR-1, µ is the overall mean, Â_i is the effect of substrate i, _j is the effect of LPSR-1, ã_ij is the interaction between substrates and LPSR-1, and E_ij is the error component. Means of different peptide treatments were compared using Duncan's means separation test at an -value of 0.05 (SAS 1989).

	RESULTS AND DISCUSSION

Abstract Introduction Methods Results & Discussion References

In a previous study, we determined that the supplemented peptides were not contaminated with free methionine and that the desalting process effectively eliminated free methionine from serum (Pan et al. 1996). In the same study, we found that free L-methionine concentrations of 6 and 15 µmol/L in treatment media for C₂C₁₂ and MAC-T cells, respectively, were appropriate for measuring cell responses to the presence of methionine-containing peptides.

Previously, we observed that methionine-containing peptides could serve as sources of methionine to support protein accretion in cultured C₂C₁₂ myogenic and MAC-T mammary epithelial cells when the peptides were added to a medium composed of MFDMEM and supplemented with 60 mL/L desalted FBS (Pan et al. 1996). The data presented in Table 2 show that MAC-T mammary epithelial cell cultures were unable to maintain their initial protein content and cell numbers during 72 h of incubation in serum-free MFDMEM. These cells were able to accumulate small amounts of protein, but did not proliferate, as indicated by similar DNA content, in the serum-free medium containing free methionine. The growth responses to methionine-containing dipeptides were quite different among peptides. The MAC-T cells failed to maintain the initial protein contents in the presence of any of the dipeptides and managed to maintain the cell numbers only in the presence of methionylleucine, leucylmethionine, methionylphenylalanine and phenylalanylmethionine. Methionylproline and prolylmethionine were markedly ineffective as sources of free methionine in the serum-free and methionine-free DMEM. The protein:DNA ratio remained the same as the initial ratio in the presence of free methionine or phenylalanylmethionine. All other treatments resulted in lower (P < 0.05) protein:DNA ratios.

Because it appeared that desalted FBS was necessary for efficient peptide utilization, several other sera were examined for their ability to stimulate the use of methionine-containing peptides as sources of methionine for cell proliferation and protein accumulation by cultured MAC-T cells. Desalted sera from adult pigs, horses, rabbits, humans and chickens were tested. Cultures in MFDMEM supplemented with the desalted sera alone from pigs (Table 3), horses (Table 4) or rabbits (Table 5) resulted in a reduction (P < 0.05) of both protein content and cell number in comparison with initial values. Generally, MAC-T cells cultured in MFDMEM containing 60 mL/L desalted sera from any species and any of the dipeptides accumulated (P < 0.05) protein and increased (P < 0.05) cell number. An exception occurred with prolylmethionine. This dipeptide was not utilized in the presence of pig or horse serum. Only data for protein accumulation were obtained from human and chicken sera; these data are presented in Table 6. As in the other species, MFDMEM supplemented with either human or chicken serum, but with no methionine supplementation, was unable to support protein accumulation in cultured MAC-T cells. Inclusion of an amino acid substrate generally resulted in protein accumulation. Overall, however, the desalted chicken serum supported the least protein accumulation among the various substrates compared with serum from other species. Whatever factor(s) that is responsible for stimulating the utilization of the amino acid substrates appears to be present in lower amounts in adult chicken serum.

We have observed that the location of methionine in dipeptides influences its use by cultured cells (Pan et al. 1996). As in our previous studies, these results indicate that dipeptides with methionine at the N-terminus resulted in a greater utilization than dipeptides with methionine at the C-terminus in the presence of animal sera tested. Among the dipeptides tested, the single exception in the presence of sera from pigs, rabbits, chickens, humans and cattle was with the dipeptide pair, methionylleucine and leucylmethionine. With these dipeptides, methionine in the C-terminus generally was more favorably utilized.

One preliminary experiment indicated that, contrary to the results observed in the presence of 60 mL/L desalted FBS (Pan et al. 1996), both methionylglycine and glycylmethionine failed to serve as methionine sources for MAC-T cells in the presence of bovine insulin, although free methionine promoted protein accretion and cell proliferation in the presence of insulin (data not shown). A subsequent experiment was conducted to investigate the ability of MAC-T cells to utilize other methionine-containing dipeptides in the presence of bovine insulin. The results are shown in Table 7. The MAC-T cells, in the absence of animal serum, had greater protein mass and DNA content in the presence of free methionine and bovine insulin (P < 0.05), indicating that insulin had both cell proliferation and anabolic effects on cultured MAC-T mammary epithelial cells in the presence of this free amino acid. This was consistent with the observation that insulin had a mitogenic effect on mammary epithelial cells (Baumrucker and Stemberger 1989). Kasuga et al. (1981) proposed that insulin exhibits its mitogenic effect via the receptor of insulin-like growth factor-I (IGF-1). Because the insulin concentration used in this experiment was very high (1 mg/L), it is possible that the response elicited by insulin was due to the binding of insulin to the IGF-1 receptor. Contrary to its growth-promoting effects in the presence of free methionine, insulin was essentially ineffective in promoting either protein accumulation or cell proliferation in MAC-T cells in the presence of the methionine-containing dipeptides tested. With the exception of methionylleucine, which resulted in a slight increase (P < 0.05) in protein content and cell number above initial levels, the response to all other peptides in the presence of insulin was either the maintenance or loss of protein and DNA in comparison with starting levels. Leucylmethionine, methionylphenylalanine and phenylalanylmethionine, in the presence of insulin, were able to support the maintenance of initial protein and DNA levels. This means that some protein synthesis was taking place (insulin alone was lower), but cell proliferation was not occurring. A second group of relatively hydrophilic peptides, including methionylproline, prolylmethionine, methionylvaline and valylmethionine, was unable to support maintenance of initial levels of either protein or cell number in the presence of insulin. Insulin exhibits anabolic effects on protein metabolism and stimulates the uptake of neutral amino acids in several tissues, including skeletal muscle and the non-ruminant mammary gland (Anderson and Rilema 1976, Granner 1990). These results also show that MAC-T mammary epithelial cells can grow well on free methionine in the presence of bovine insulin. Differences in protein contents between the absence and presence of insulin were also observed in methionylleucine (25.0 vs. 36.9 µg/well), leucylmethionine (16.3 vs. 27.1 µg/well), methionylphenylalanine (21.6 vs. 28.4 µg/well) MAC-T cells. Insulin had no effect on the growth of MAC-T cells with glycylmethionine, methionylglycine, phenylalanylmethionine, methionylproline, prolylmethionine, methionylvaline or valylmethionine in the culture medium. To the contrary, desalted FBS had stimulatory effects on the growth of MAC-T cells on all of the above-mentioned dipeptides (Pan et al. 1996). Although there was some effect on protein synthesis, bovine insulin seems to have limited stimulating effects on the utilization of the methionine-containing dipeptides tested. Because the insulin concentration used in this study was very high (1 mg/L), it is likely that insulin is not among the serum factors of FBS that promote the utilization of methionine-containing dipeptides.

It appears that there may be some relationship between the observed responses and dipeptide hydrophobicity because less hydrophobic peptides failed to support growth of MAC-T cells in the presence of insulin. The peptides in the second group above are much less hydrophobic than the other dipeptides tested. The specific nature of their relationship is not apparent. These results are consistent with our earlier report that, in MAC-T cells, protein accretion increased with increased hydrophobicity of methionine-containing dipeptides (Pan et al. 1996). Several reports have appeared to suggest that significant amounts of relatively hydrophobic peptides may exist in the circulation as sources of the corresponding free amino acids. First, it was shown that the dipeptide transport system in human jejunum had higher affinity for dipeptides with hydrophobic amino acids. Gardner and Wood (1989) reported that hydrophobic and mucosal hydrolysis-resistant peptides are more likely to be absorbed faster than hydrophilic and hydrolysis-susceptible peptides. Daniel et al. (1992) showed that the oligopeptide/H⁺ symporter in the brush border membrane had a higher affinity for di- and tripeptides with hydrophobic amino acids. The major function of the symporter is to reabsorb peptides of plasmic origin from the glomerular filtrate during the formation of urine. The high affinity for hydrophobic di- and tripeptides may imply that the bulk of the peptides of plasmic origin in the glomerular filtrate are relatively hydrophobic. Therefore, it is reasonable that animal cells such as cultured MAC-T cells have developed the ability to utilize hydrophobic peptides better than hydrophilic ones. In terms of protein contents of the cultures after 72 h of incubation, methionine dipeptides with the methionine residue at the N-terminus resulted in greater (P < 0.05) protein contents than those with methionine residues at the C-terminus in the presence of insulin (Table 7). Dipeptides with the same amino acid compositions had similar effects on DNA content of cultures, indicating comparable cell proliferation rates.

Any culture medium supplemented with animal serum presumably contains essential fatty acids from the animal serum that are necessary for normal cell growth. Because the serum-free DMEM supplemented with bovine insulin may lack essential fatty acids and because cell proliferation appeared to be impaired more than protein synthesis, it was felt that an essential fatty acid deficiency may be responsible for the observed responses. Adding neither 4 mL/L of a bovine serum lipid mixture nor 1 mL/L of a chemically defined lipid concentrate in combination with insulin resulted in protein accumulation above initial levels in the presence of methionylproline or prolylmethionine (Fig. 1). The addition of the bovine serum lipid mixture did result in a lower net protein synthesis in the presence of free methionine. Therefore, it appears that essential fatty acids were not the limiting factor for the growth of MAC-T cells on methionine-containing dipeptides in response to insulin stimulation.

View larger version (36K): [in this window] [in a new window]   Fig 1. Effects of lipid supplementation on protein accretion in MAC-T cells in the presence of methionine-containing dipeptides as the sole source of methionine. The MAC-T cell cultures were prepared as described in Materials and Methods. The cells were then incubated with various treatment media 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 (mean + SEM) lacking common letters differ (P < 0.05); n = 4 wells. Abbreviations used: Initial, protein content of the culture before treatment; M, 15 µmol/L L-methionine; I, methionine-free Dulbecco's modified Eagle's medium supplemented with 1 µg/mL bovine insulin; SL, 4 mL/L bovine serum lipids; CL,10 mL/L chemically defined lipid concentrates; MP, methionylproline containing 15 µmol/L methionine residue; PM, prolylmethionine containing 15 µmol/L methionine residue.

The growth of MAC-T cells on several methionine-containing di-, tri-, tetra- and pentapeptides was evaluated in the presence of LPSR-1, which contains growth-enhancing factors and carrier proteins; its components include albumin, transferrin, insulin, other hormones and growth factors, vitamins, attachment factors and soybean trypsin inhibitor. The actual composition is proprietary information of the manufacturer. Several cell lines including human lung fibroblasts, kidney cells (MDCK), Buffalo Green Monkey (BGM) kidney cells and Vero 76 cells (African green monkey kidney cells) were shown to grow in medium supplemented with LPSR-1 (Candal et al. 1989). Results from this study (Table 8) showed that the inclusion of LPSR-1 in the medium resulted in protein accretion and cell proliferation (P < 0.0001) where substrate was present. The serum factor(s) that facilitates the utilization of methionine-containing peptides appears also to be present in LPSR-1.

The effect of levels of addition of LPSR-1 to the culture medium was examined in a second cell line, C₂C₁₂ myogenic cells (Table 9). There was a direct relationship between level of LPSR-1 and protein accretion. Adding 30 mL/L LPSR-1 to the medium resulted in a greater (P < 0.0001) protein accretion than adding 10 mL/L LPSR-1. Prolylmethionine and the tetra- and pentapeptides were not utilized as well as other substrates.

It may be possible that the serum factor(s) that facilitates the utilization of methionine from methionine-containing peptide is a hydrolase(s). Hydrolytic activity of desalted FBS against a number of methionine-containing peptides was examined in a cell-free incubation. After 24 h of incubation in the cell-free medium, essentially all of the methionine contained in alanylmethionine, methionylphenylalanine and the enkephalin segments was released into the medium (Fig. 2). The released methionine then could be absorbed by the cells and utilized in the same manner as if free methionine was added to the medium. The remaining di- and tripeptides were hydrolyzed less extensively, with methionine release ranging from 42 to 70%. Piez et al. (1960) reported that, after 3-d storage of dialyzed human serum samples at 37°C, the concentrations of valine, methionine, isoleucine, leucine, threonine, phenylalanine, lysine, histidine and arginine were 88, 29, 22, 180, 83, 59, 112, 43 and 106 µmol/L , respectively. At the same time, substantial amounts of nonessential amino acids also accumulated in the serum samples. This clearly shows that animal serum has hydrolytic enzymes that can release amino acids from plasma peptides. Plasma peptidase activities against a number of peptides were reported by Adibi et al. (1986) and Lochs et al. (1988). Because the tetra- to pentapeptides used in this study were methionine-enkephalin segments and because animal sera have been shown to contain aminopeptidase M, dipeptidyl carboxypeptidases and angiotensin-converting enzyme, which are involved in the hydrolysis of enkephalins in plasma (Shibanoki et al. 1991 and 1992), it is not surprising to see this extent of methionine release. The observed serum factor(s) in animal sera that facilitates the utilization of methionine-containing peptides may be the above-mentioned enkephalin-hydrolyzing enzymes and other peptidases.

View larger version (54K): [in this window] [in a new window]   Fig 2. Medium-free methionine concentrations resulting from hydrolysis of methionine-containing di- to octapeptides by 60 mL/L desalted fetal bovine serum in methionine-free Dulbecco's modified Eagle's medium (MFDMEM) after 24 h incubation. Methionine-containing peptides were added to 15-mL sterile centrifuge tubes containing 12 mL of the basal medium (MFDMEM supplemented with 60 mL/L desalted fetal bovine serum). The resulting solutions were then sealed with caps and incubated at 37°C for 24 h. Before and after the incubation, samples were taken and filtered through ultrafree MC filters (10,000 Da cut-off). Free L-methionine concentrations were determined by HPLC analysis. Bars (mean + SEM) with different letters differ (P < 0.05); n = 3 observations. Abbreviations used: M, 15 µmol/L L-methionine; MA, methionylalanine; AM, alanylmethionine; MF, methionylphenylalanine; FM, phenylalanylmethionine; MG, methionylglycine; GM, glycylmethionine; ML, methionylleucine; LM, leucylmethionine; MM, methionylmethionine; MP, methionylproline; PM, prolylmethionine; MS, methionylserine; SM, serylmethionine; MV, methionylvaline; VM, valylmethionine; MAS, methionylalanylserine; MLF, methionylleucylphenylalanine; 4 to 8, tetra- to octa-enkephalin segments. All peptides contained 15 µmol/L methionine residue.

We have observed that FBS contains one or more factors that facilitate the utilization of methionine-containing di- to octapeptides (Pan et al. 1996). Results from this study demonstrate that adult animal sera from humans, horses, chickens, pigs and rabbits all can promote the utilization of methionine-containing dipeptides with few exceptions. In the presence of any of the five sera, methionylphenyalanine and methionylvaline promoted the greatest protein accretion; prolylmethionine was the least utilized dipeptide. There were some differences in the growth responses of the MAC-T cells on the same dipeptides in the presence of different animal sera, suggesting that some species differences may exist. Insulin failed to promote peptide utilization in serum-free medium with or without essential fatty acids, suggesting that insulin is not the serum factor that facilitates peptide utilization. Further studies are required to determine the nature of the serum factor that promotes peptide utilization.

	ACKNOWLEDGMENTS

We appreciate the technical assistance of Don Shaw, Kris Lee and Pat Boyle.

	FOOTNOTES

Manuscript received 19 August 1996. Initial reviews completed 21 October 1996. Revision accepted 9 December 1997.

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