Comparison of protein metabolism and glucose uptake in turkey (Meleagris gallopavo) satellite cells and embryonic myoblasts in vitro

Camp. Biochem. Physiol. Vol. 107A. No. 2, pp. 301-306, 1994 Copyright

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Comparison of protein metabolism and glucose uptake in turkey (Meleagris gallopavo) satellite cells and embryonic myoblasts in vitro Douglas C. McFarland, Jane E. Pesall, Kysa IS. Gilkerson, Neal H. Ferrin, Wenying V. Ye and Todd A. Swenning Department of Animal and Range Sciences, Box 2170, South Dakota State University, Brookings, SD 57007-0392, U.S.A. Protein synthesis, protein degradation, and glucose uptake were compared in clonal-derived turkey myogenic satellite cells (clone DS-SC) and embryonic myoblasts (EM) and between satellite cell cultures from Nicholas (DN) and Merriam’s (WM) turkeys. Protein synthesis rates were higher and degradation rates were lower in myotube cultures of DS-SC compared with EM (P < 0.05). Protein synthesis and degradation rates did not differ between cultures of DN and WM (P 2 0.05). Glucose transport rates were significantly higher in EM than DS-SC cultures and did not differ between DN and WM cultures. Insulin-like growth factors and insulin stimulated protein synthesis, decreased protein degradation, and increased glucose uptake in all cell tines. Key words: Protein metabolism;

Glucose uptake; Meleugris

gallopavo;

Satellite cells; Embryonic

myoblasts. c’omp. Biochem.

Physiol.

107A, 301-306,

1994.

Introduction Insulin and insulin-like growth factors (IGFs) are anabolic compounds which stimulate amino acid uptake, protein synthesis, and glucose transport. Therefore, these hormones are important regulators of tissue growth. Insulin and IGFs have been shown to stimulate protein synthesis and inhibit protein degradation in cultured muscle cells, including rat L6 myoblasts (Ballard et al., 1986) and chick embryonic muscle (Janeczko and Etlinger, 1984). Cultured myoblasts can be induced to differentiate and fuse into multinucleated myotubes, which accumulate muscle-specific proteins (Allen et al., 1979). During both embryonic and postnatal development, increased muscle protein synthesis is critical to skeletal muscle development (Allen et al., 1979; Allen, 1988). Correspondence to: D. C. McFarland, Department of Animal and Range Sciences, Box 2170, South Dakota State University, Brookings, SD 57007-0392, U.S.A. Tel: 605 688-5431; Fax: 605 688-6170. Recieved 14 April 1993; accepted 20 May 1993

Glucose transport occurs through facilitated diffusion mediated by the GLUTI-GLUT5 glucose transporters (Klip and Marette, 1992; Bilan et al., 1992). Glucose transport is regulated to maintain cellular glucose homeostasis under a variety of physiological conditions. Factors affecting the regulation of glucose transport include the IGFs, insulin, glucose, and media conditions. Exposure of muscle cells to insulin or IGFs has been demonstrated to increase glucose transport (Wang et al., 1987; Steele-Perkins et al., 1988; Simpson and Cushman, 1986). IGFs may affect glucose uptake through stimulation of IGF receptors rather than insulin receptors (Cascieri et al., 1986). In the present study, we measured protein synthesis and degradation rates in myotube cultures and glucose uptake in confluent proliferating cultures of turkey muscle cells following stimulation with insulin or IGFs. DS-SC (a satellite cell clone) and EM (an embryonic

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myoblast clone) were used as model systems to compare avian posthatch and embryonic muscle development, respectively. Satellite cells derived from Nicholas (DN) and Merriam’s (WM) turkeys were used as models to compare muscle development in animals with markedly different growth rates. Nicholas turkeys (a commercial variety) have been selected over many generations for rapid whole body and skeletal muscle growth. Merriam’s turkeys are included among the native North American varieties and have not been selected for enhanced growth. Clonal cultures of muscle cells were developed to eliminate interferences from non-myogenic cells in the cultures.

Materials and Methods Materials Media, sera, and antibiotics were purchased from Gibco Laboratories (Grand Island, NY). Cell cultureware were obtained from Corning Glass Works (Corning, NY). Human recombinant IGF-I was obtained from Mallinckrodt (Chesterfield, MO), and IGF-II was obtained from Bachem (Torrance, CA). Human recombinant insulin was a gift from Lilly Research Laboratories (Indianapolis, IN). [ “S]-t_-methionine (107 l-l 190 Ci/mmol) was purchased from ICN (Irvine, CA). 2-Deoxy-D-glucose-[l, 2-3H] (30.2 Ci/mmol) was obtained from New England Nuclear (Boston, MA). Cytochalasin B was obtained from Aldrich (Milwaukee, WI). All other reagents were obtained from Sigma Chemical Co. (St Louis, MO). Muscle cell clones and culture procedures Satellite cells were isolated from the pectoralis major muscle of young tom turkeys (Meleagris gallopavo) by previously described methods (McFarland et al., 1988). Embryonic myoblasts were isolated from 15-day turkey embryos by the procedure described by Konigsberg (1979). Cell clones were developed from secondary cultures using a cloning-ring procedure (Minshall et al., 1990). In the protein synthesis study, sixth-passage clonal-derived satellite cells and embryonic myoblasts were suspended in plating media [DMEM-5% horse serum (HSklO% chicken serum (CS)] and plated in 35-mm diameter cell culture wells pre-coated with 0.1% gelatin (Richler and Yaffe, 1970) and placed in a humidified (5% CO,/95% air) incubator at 38.5”C. Following a 24-hr attachment period, cells were allowed to proliferate for 4 days (cultures were approximately 60% confluent) in growth medium (McCoy’s 5A-15% CS). Cultures were administered fresh media following 48 hr. For

glucose uptake measurements, sixth-passage cells were plated in 16 mm diameter cell culture wells (24-well plates) and grown similarly. Cell plating densities were lo&l25 cells/mm2 for DS-SC and DN clones and 60-80 cells/mm2 for EM and WM clones. Protein synthesis Rates of protein synthesis were measured by the incorporation of [ 35S]methionine into acidinsoluble protein by a modification of the method described by Gulve and Dice (1989). Cultures which were approximately 60% confluent were washed twice with 1.0 ml fusion media (DMEM + 3% HS + 0.01 mg/ml porcine gelatin + 1 mg/ml BSA). Cells were then induced to fuse by administering 5.0 ml fusion medium for 48 hr in a 38.5”C humidified CO, incubator. Following removal of medium, myotube cultures were washed twice with 1.O ml incubation media (DMEM-methioninefree + 0.584 mg/ml glutamine + 3% HS+ 0.01 mg/ml porcine gelatin + 2.0 mg/ml BSA). Myotube cultures were then incubated in 2.0 ml incubation medium containing 2.5 pCi/ml [‘?$I-L-methionine together with hormonal treatments. The incubation media contained basal levels of insulin (0.833 nM) and either 13.1 nM IGF-I, IGF-II, insulin, or no additional factors (controls). In preliminary studies (data not shown) rates of protein synthesis were linear for at least 6 hr under our experimental conditions. In these reported studies, radiolabeled methionine incorporation was terminated after 6 hr by removing media and washing twice with 1.0 ml ice-cold PBS containing 2.0 mM L-methionine. Myotubes were disrupted and protein was precipitated by addition of 1.O ml ice-cold 10% the trichloroacetic acid (TCA). Plates were stored overnight at 4°C. Cells were manually detached from the wells with a cell lifter (Costar Co., Cambridge, MA), and the suspensions were transferred to 1.5 ml microfuge tubes and pooled with a 0.4 ml ice-cold 10% TCA wash of each well. Microtubes were placed on ice for 1 hr to allow complete precipitation. The suspensions were centrifuged at 12,500g for 2 min at 4°C. The supernatants were aspirated and the protein pellets were resuspended again in 1.4 ml 10% TCA and centrifuged under the same conditions. The pellets were dissolved in 1.O ml of 1.0 N NaOH containing 1% sodium deoxycholate. One hundred microliters of the protein solution were neutralized with 100 ~1 1.O N HCI, mixed with 10 ml Ecoliteo (ICN), and counted in a liquid scintillation counter. Protein content in the samples was determined using the bicinchoninic acid (BCA) reagent (Pierce, Rockford, IL).

Protein metabolism and glucose uptake in turkey cells

Protein degradation

Rates of protein degradation were determined by measuring the release of [ 35S]methionine into acid-soluble extracts of pre-labeled cultures by a modification of the method described by Gulve and Dice (1989). Cultures which were approximately 60% confluent were washed twice with 1.Oml labeling medium (minus [ 3SS]-methionine). Labeling medium consisted of a 1: 1 mixture of DMEM (minus methionine) and DMEM, containing 0.292 mg/ml glutamine, 3% HS, 0.01 mg/ml porcine gelatin, 1.0 mg/ml bovine serum albumin (BSA), and 1.OpCi/ml [ ‘*S]-L-methionine. Cells were incubated with 5 ml/well of labeling medium for 48 hr in a 38.5”C humidified CO, incubator. The labeling medium was removed from the cultures and the cells were washed twice with 1.0 ml chase medium (DMEM containing 1.7 mg/ml additional t,-methionine, 3% HS, 0.01 mg/ml gelatin and 2.0 mg/ml BSA). Seven milliliters of chase medium were added to each well and the incubation was continued for 1 hr to allow degradation of short-lived proteins. Following this incubation, media were removed from the wells, washed once with 1 ml chase medium, and replaced with 2.0 ml of chase medium with or without insulin or IGFs and the incubation was continued for 6 hr. In preliminary studies (data not shown) rates of protein degradation were linear for at least 12 hr under our experimental conditions. Two O.&ml aliquots of media were removed from each well and placed in 1.5ml microfuge tubes with 89~1 100% TCA, mixed and placed in an ice-water bath for 30 min. The tubes were then centrifuged at 12,500g for 3 min at 4°C. The supernatants were removed, neutralized with 90 ~1 6 N NaOH, mixed with 10 ml EcolyteQ and counted in a liquid scintillation counter. Cell numbers and fusion percentages were determined in parallel cultures by microscopy (McFarland et al., 1988). G’lucose uptake Glucose uptake was measured using the procedure described by Klip and Ramlal(1987). To measure glucose transport activity, the uptake of the non-metabolizable glucose analogue 2deoxy-[3H]-glucose (2-DOG) was measured. Near-confluent cultures were washed twice with 0.5 ml of McCoy’s 5A medium containing 25 mM D-glucose, 2.0 mg/ml BSA, and 0.01 mg/ml porcine gelatin (McCoy’s SA-Glu) and were preincubated in 1.0 ml McCoy’s 5AGlu medium for 5 hr in a 38.5”C humidified incubator. The medium was removed and the cells were washed twice with 0.4 ml hexose-free uptake solution (HUS; 140 mM NaCl, 20 mM HEPES, 5 mM KCI, 2.5 mM MgSO,, 1.0 mM

303

CaCl,, 2.0 mg/ml BSA, 0.01 mg/ml porcine gelatin, pH 7.4). Five hundred microliters of treatment media (HUS containing IGF-I or insulin [0.013 1 nM, 1.3 1 nM or 13 1.OnM] or no factors [basal]) were added to each well and incubated for 40 min. Culture dishes were floated on a 385°C water bath. Following treatment, the cells were rinsed twice with 0.4 ml HUS and incubated with 0.5 ml HUS in the presence of 2 pCi/ml of 2-DOG for 10 min at room temperature. Non-carrier-mediated uptake was determined in parallel cultures which included 15 PM cytochalasin B and was subtracted from all values. Uptake under our experimental conditions was linear for at least 10 min (data not shown). Glucose uptake was terminated by aspiration of the media, followed by two washings with 0.5 ml ice-cold phosphate buffered saline (PBS). Cells were then lysed with 0.5 ml of 0.05 N NaOH, and suspensions were transferred to scintillation vials and pooled with a 0.5 ml wash of 0.05 N NaOH. This mixture was added to 10 ml of Ecolitea and counted in a liquid scintillation counter. Cell numbers/well were determined in parallel cultures. Cells were detached from the substratum with trypsinEDTA and enumerated using a Model ZI Coulter Counter. Statistical analysis was performed using the one-way analysis of variance procedure by the Statistical Analysis System (SAS, 1985). Stated differences were significant at the 0.05 level of probability (Steel and Torrie, 1960).

Results The effects of IGFs and insulin on protein synthesis were examined in myotubes derived from DS-SC and EM cultures and DN and WM cultures and are summarized in Table 1. Equimolar levels of IGFs or insulin increased protein synthesis rates over basal levels in all but one culture. The exception was that insulin did not increase protein synthesis in EM cells. There did not appear to be a consistent difference in the relative potencies of IGF-I and IGF-II. In D5SC and EM cultures the IGFs stimulated greater rates of protein synthesis than did insulin Synthesis rates were greater in DS-SC than EM cultures regardless of treatment. Conversely, there were no differences between rates of protein synthesis in DN and WM cultures within any treatment. The effects of IGFs and insulin on protein degradation were examined in myotubes derived from DS-SC and EM cultures and DN and WM cultures and are summarized in Table 2. The IGFs and insulin decreased rates of protein degradation over basal levels in all cultures. In DS-SC and EM cultures, the IGFs were more

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Table 1. The effects of IGFs and insulin on protein synthesis rates of turkey

muscle cells in vitro Cell line EM

DS-SC Nuclei per mm2 Fusion (%) Treatments Basal IGF-I (13 nM) IGF-II (13 nM) Insulin (13nM)

787.5 f 52.4” 836.9 + 36” 53 + 2.2” 47.3 f 2.3” DPM/rg protein/hr* 2578 k 41d 3081 & 128’ 3198 k 149bc 3929 & 164” 4118 5 137” 3302 + 83bc 2598 + 77d 3558 f 168b Cell line WM DN

Nuclei per mm’ Fusion (%) Treatments Basal IGF-I ( I3 nM) IGF-II (13 nM) Insulin (13 nM)

328.1 f 20b 28.6 k 2” DPM/pg 2055 k 101’ 3018 * 93ah 2850 k 129”d 2636 f 10lcd

436.1 + 30.2” 41.5k4.3” protein/hr* 2110+69’ 3208 + 93” 2582 k 68d 2929 _+ 121abc

*Measurements of [r5S] methionine incorporated into TCA insoluble protein. Means + SE of six observations. Means with the same superscript are not statistically different (P 2 0.05).

effective in reducing degradation rates than was insulin. As seen with the synthesis measurements, there did not appear to be a consistent difference in the potency of IGF-I and IGF-II in reducing protein degradation rates. Protein degradation rates were lower in DS-SC than in EM cultures and were not different between DN and WM cultures. The effects of IGF-I and insulin on 2-DOG uptake were measured in near-confluent, proliferating cultures of all four cell lines. Rates of 2-DOG uptake are listed in Tables 3 and 4. Uptake was higher (approximately 2-fold) in EM cultures than in DS-SC cultures at all levels of administered IGFs or insulin. Glucose upTable 2. The effects of IGFs and insulin on protein dation rates of turkey muscle cells in vim

degra-

Cell line DS-SC

EM

Nuclei per mm’ Fusion (%) Treatments Basal IGF-I (13 nM) IGF-II (13 nM) Insulin (13nM)

816.4 k 19.5” 42.2 & 1.6b DPM/pg 65 * 1’ 48k 1’ 51 f 1’ 57+ 1e DN

815.1 k 34.7” 59.4 * 3. I” ptotein/hr* 90 * 3” 62 f 2’d 60 + Ide 77* lb WM

Nuclei per mm’ Fusion (%) Treatments Basal IGF-I (13 nM) IGF-II (13 nM) Insulin (13 nM)

279.1 + 7.7b 27.8 + 0.8b DPM/pg 49 * 2”b 40 + I’d 34* I’ 42 + 2cd

414.9 + 13.4” 42 + 2” protein/hr* 50* 1” 39+ Id 41 * 2’d 45 If: 2k

*Measurements of TCA soluble [“Slmethionine from protein. Means + SE of six observations. Means with the same superscript are not statistically ent (P 2 0.05).

release differ-

Table 3. The effect of IGF-I and insulin on 2-deoxy-[‘H]glucose (2-DOG) uptake in satellite cell (DS-SC) and embryonic myoblast (EM) cultures* Treatments

DS-SC

Cells/well

439,020 + 7519 (pmol/lO” 353.8 f 7.4h 340.0 + 8.1h 349.2 k 8.7” 548.6 + 14.8’ 371.0 k 4.2h 495.2 + 5.48 538.4 f. 25.0’

Control Insulin (0.013 1 nM) (1.31 nM) (131 nM) IGF-I(O.013 1nM) (1.31 nM) (131 nM)

EM 417,600 & cells/min) 804.5 + 765.4 + 73 I .3 * 1,003.O k 781.8 + 877.1 + 926.6 &

10,752

I 5.9d 19.3dL 2O.Y 16.6” 8.8d 19.0’ 17.8b

*Means + SE of five observations. Means with the same superscript are not statistically ent (P 2 0.05).

differ-

was significantly higher in cultures administered 13 1 nM IGF-I or insulin than in controls in all four lines. 2-DOG uptake did not differ between DN and WM cultures in all treatments (Table 4). At 131 nM, IGF-I and insulin treatments were equipotent in stimulating 2-DOG uptake. take

Discussion DS-SC and EM clones were used as model systems to compare protein metabolism and glucose uptake in muscles derived from posthatch and embryonic animals. Likewise, DN and WM cell clones were used to compare these properties in SC from rapid and slow-growing animals. Clonal-derived muscle cell cultures were used in these studies to eliminate possible interferences from non-myogenic cells in culture. Using these pure cell culture systems we were able to directly compare cellular metabolic responses induced by IGFs or insulin. In previous studies (McFarland et al., 1991) we demonstrated that there were differences between EM and DS-SC cells in their kinetics of differentiation. When administered low serumcontaining media, differentiation (as measured by fusion) of EM cultures was maximized by Table 4. The effect of IGF-I and insulin on 2-deoxy-[‘HIglucose (2-DOG) uptake in satellite cells derived from Nicholas (DN) and Merriam’s (WM) turkeys* Treatments

DN

WM

Cells/well

283,725 + 5756 (pmol/106 563.9 + 28.9e’ 550.3 + 19.8” 524.9 & 12.1’ 782.6 k 31.3” 519.5 + 8.7’ 640.1 + 23.Td 736.5 ; 23.9”b

200,513 k 5234 cells/min) 543.4 & 27.0” 588.2 + 25.4d” 572.5 k 9. I*” 758.3 & 31.7” 607.0 &- 16.9cd’ 667.9 & 21.0h‘ 735.1 + 3 I .8”b

Control Insulin (0.0131 nM) (1.31 nM) (131 nM) IGF-I (0.013 I nM) (1.31 nM) (131 nM)

*Means + SE of five observations. Means with the same superscript are not statistically ent (P 2 0.05).

differ-

Protein

metabolism

and glucose

36 hr while DS-SC were maximally fused by 72 hr. While EM cultures begin to fuse earlier than DS-SC cultures, the present results suggest that there is no correlation between rates of protein synthesis and fusion. In fact, the reverse was found. Protein degradation rates were lower in DS-SC cultures than EM, which would lead to an even greater level of net protein synthesis in DS-SC myotube cultures. Protein synthesis rates were also compared in SC-derived myotube cultures of DN and WM cells. These cells were derived from turkeys with marked differences in both whole body and skeletal muscle accretion rates. The Nicholas breed, the source of DN satellite cells, grows at approximately a 3-fold greater rate than the Merriam’s turkey, the source of WM satellite cells (McFarland er al., 1993). It was postulated that large differences in whole body growth and skeletal muscle accretion rates would result in significant differences in the rates of protein synthesis of muscle cultures derived from these breeds. However, the results demonstrated that there were no detectable differences in the rates of protein synthesis in satellite cell cultures from these two breeds. These results are similar to those reported by Orcutt and Young (1982) using embryonic myoblasts derived from broiler (fast growing) and layer (slow growing) breeds of chickens. These investigators demonstrated that protein synthesis rates in myotube cultures from these breeds were similar. They concluded from their results that the ability of broiler muscles to accumulate greater levels of protein was primarily due to decreased rates of protein degradation by these cells. In contrast, we did not detect differences in protein degradation rates in cultures derived from the fast- (DN) or slow(WM) growing breeds. Although it is clear that these two breeds do differ greatly in their rates of‘ skeletal muscle protein accretion, this property is apparently not demonstrated, or at least not detected, in vitro. These results agree with previous studies with chick skeletal muscle myotube cultures (Janeczko and Etlinger, 1984) which suggest that both insulin and IGFs stimulate protein synthesis. Studies with rat L8 myotube cultures also indicate that IGFs and insulin stimulate synthesis and inhibit degradation of protein (Gulve and Dice, 1989). Cytochalasin B is a potent competitive inhibitor of carrier-mediated glucose transport in many cell types (Wardzala et al., 1978). In our experiments, cytochalasin B inhibited approximately 94% of the observed 2-DOG uptake. Therefore, glucose uptake is principally carriermediated in these cells. Our studies indicated that high levels (131 nM) of IGF-I or insulin stimulate glucose uptake in the four muscle cell

uptake

in turkey

cells

305

lines examined. These results were consistent with studies using chick embryo fibroblasts (Cynober et al., 1985), human skeletal muscle fiber strips (Dohm et al., 1990) and rat L6 muscle cells (Wang et al., 1987). In conclusion, protein metabolism and 2DOG uptake were compared in four different turkey muscle cell culture systems. Satellite cells and embryonic myoblasts were used as model systems to compare post-hatch and embryonic muscle development, respectively. Satellite cells derived from Nicholas and Merriam’s turkeys were used as models to compare muscle development in animals with markedly different growth rates. Our results demonstrated that rates of protein synthesis were higher, and degradation lower, in satellite cell cultures compared to embryonic myoblast cultures. No differences in either protein synthesis or degradation rates were detected in cultures from turkeys with markedly different growth rates. The results also demonstrated that the uptake of the glucose analog 2-DOG was significantly higher in embryonic myoblasts than in satellite cell cultures and did not differ in satellite cell cultures derived from the fast-growing breed compared with the slow-growing breed. Addition of IGFs or insulin to all four cultures increased the synthesis and decreased the degradation of protein and increased the uptake of 2-DOG. Acknowledgements-This research was funded by the South Dakota Agricultural Experiment Station Project R-390, the National Science Foundation EPSCoR program (No. STI8902066 and EHR-9108773) the South Dakota Future Fund and the South Dakota Poultry Industries Association. The authors thank Pei Ding, Yin Chen, and Lan Jiang for laboratory assistance. Scientific paper number 2724.

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growth factor I/somatomedin C action on 2-deoxyglucose and a-amino isobutyrate uptake in chick embryo fibroblasts. Biochimie 67, 1185-l 190. Dohm G. L., Elton C. W., Raju M. S., Mooney N. D., DiMarchi R., Pories W. J., Fhckinger E. G., Atkinson S. M. Jr. and Caro J. F. (1990) IGF-I-stimulated glucose transport in human skeletal muscle and IGF-I resistance in obesity and NIDDM. Diubefes 39, 1028%1032. Gulve E. A. and Dice J. F. (1989) Regulation of protein synthesis and degradation in L8 myotubes. Effects of serum, insulin and insulin-like growth factors. Biochem. J. 260, 377-387. Janeczko R. A. and Ethnger J. D. (1984) Inhibition of intracellular proteolysis in muscle cultures by multiplication-stimulating activity. J. biol. Chem. 259,62926297. Klip A. and Marette A. (1992) Acute and chronic signals controlling glucose transport in skeletal muscle. J. Cell Biochem. 48, 5140. Klip A. and Ramlal T. (1987) Protein kinase C is not required for insulin stimulation of hexose uptake in muscle cells in culture. Biochem. J. 242, 131-136. Konigsberg I. R. (1979) Skeletal myoblasts in culture. In Methocis in Enzymology (Edited by Jakoby W. B. and Pastan I. H.), Vol. LVIII, pp. 51 l-527. Academic Press, New York. McFarland D. C., Doumit M. E. and Minshall R. D. (1988) The turkey myogenic satellite ceil: optimization of in vitro proliferation and differentiation. Tissue & Cell 20, 899-908. McFarland D. C., Pesall J. E., Gilkerson K. K. and Ferrin N. H. (1991) Comparison of the proliferation and differentiation of myogenic satellite cells and embryonic myoblasts derived from the turkey. Camp. B&hem. Physiol. lOOA, 439443.

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