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Modulation of hexose transport in cultured skeletal muscle
Molecular and Celhdar Endocrinology, 38 (1984) 171-178 Elsevier Scientific Publishers Ireland, Ltd.
171
MCE 01239
Modulation of hexose transport in cultured skeletal muscle Michael F. Shanahan Department of Physiology, University of Wisconsin, Madison, WI 63706 (U.S.A.) (Received
Keywords:
glucose
transport;
insulin action;
31 May 1984; accepted
insulin binding;
5 September
starvation-induced
1984)
transport.
The modulation of hexose transport due to insulin and glucose starvation was investigated in cultures derived from the breast musculature of embryonic quail. Fused myotubes at 37°C exhibited a saturable, stereospecific basal uptake of both D-glucose and 3Omethylglucose which was markedly inhibited by cytochalasin B, a potent inhibitor of hexose transport in other cell systems. In the presence of insulin, 3-O-methylglucose uptake was stimulated relative to untreated controls. Kinetic analysis indicated that insulin increased the V,,, of transport with no significant increase in the apparent K,. Incubation of myotubes in glucose-free medium for 24 h resulted in an increase in D-glucose and 3-O-methylglucose transport activity. Cycloheximide abolished this stimulation effect when it was included during the starvation period, but had no effect on transport in glucose-fed cells. Insulin binding studies on these myotubes indicate that high-affinity insulin receptors are present and continue to increase throughout the life of the culture. This high-affinity binding as well as the capacity to degrade insulin in these cells is characteristically similar to effects observed in other insulin-sensitive cell systems.
Glucose has been demonstrated to enter skeletal muscle via a carrier-mediated process which is accelerated in the presence of insulin (see Clausen, 1975; Elbrink and Bihler, 1975, for reviews). The mechanism of insulin stimulation of hexose transport is unknown, but is presumed to be initiated via an insulin-receptor complex on the outer surface of the plasma membrane. Several investigators have studied the action of insulin on glucose transport (Schudt et al., 1976) as well as insulin binding (Sandra and Przybylski, 1979) during myogenesis in skeletal muscle cell cultures derived from chick embryos. Both these studies have indicated a progressive development of both monosaccharide transport capability and insulin receptor binding during myogenesis. Most of the studies on glucose transport and 0303-7207/84/$03.00
Q 1984 Elsevier Scientific
Publishers
Ireland,
insulin effects have been carried out on muscle cultures derived from embryonic chick. Unfortunately, these preparations often contain a significant proportion of contaminating fibroblasts which are invariably present in chick muscle cell cultures. The obvious disadvantage of having a large number of contaminating fibroblasts in this type of study is that the fibroblasts themselves possess an insulin-sensitive glucose transport system (Vaheri et al., 1972; Shaw and Amos, 1973). Thus, insulin effects on transport kinetic parameters in chick muscle cell cultures may be difficult to evaluate owing to the presence of two different responsive cell populations contributing to the experimental results. In the present study, we have analyzed the insulin-modulated hexose transport system in a Ltd.
172
muscle culture system derived from embryonic quail breast musculature. This system offers a significant advantage in the fact that such cultures provide a virtually pure population of muscle cells with less than 0.25% contaminating fibroblasts (Lipton, 1977). Materials and methods Reagents Powdered culture media and selected horse sera lots were obtained from Gibco (Grand Island, NY), while all other reagents were purchased from Sigma Chemical Co. (St. Louis) with the exception of porcine insulin, which was obtained from Eli Lilly Co. (Indianapolis). Cytochalasin B was solubilized in absolute ethanol and diluted 2000 X following addition to the incubation medium. Radiotracer compounds were obtained from New England Nuclear (Boston). Cell culture Quail muscle cultures were prepared by the method of Konigsberg (1979). Breast musculature from 9-day embryonic Japanese quail (Corturnix corturnix japonica) was excised and minced. The tissue was mechanically dissociated by vortexing in complete medium, and the resulting suspension passed through filters to remove undissociated clumps. Cells were inoculated into gelatin-coated 100 mm plastic tissue culture plates at cell densities of 2 X lo6 cells/plate which precludes fusion during the ensuing 24 h. After 18-24 h of incubation at 37°C in a humidified atmosphere of 5% CO, in air, secondary suspensions were prepared by resuspending the cells with 0.013% trypsin. Secondary cultures were then established by inoculating myoblasts (30000 cells/plate) into 3 ml growth medium in 35 mm gelatin-coated plastic tissue culture dishes. Growth media in all cultures consisted of Eagle’s minimal essential medium supplemented with ‘selected’ (Lipton, 1977) horse serum (IS%), chick embryo extract (10%) and a 1% mixture of penicillin-streptomycin. Up take assays For hexose uptake assays, all incubations and preincubation wash solutions were maintained at
37’C. Secondary cultures were rinsed 4 times with 3 ml of Earle’s salt solution. Cells were then preincubated at 37°C in 4 ml glucose-free Earle’s salts containing 20 mM Hepes buffer (pH 7.4) and other agents at the concentrations described below. Cells were preincubated in the buffer for 15-30 min. Following preincubation, the buffer was removed by aspiration and 4 ml of incubation medium, consisting of the appropriate preincubation medium containing 3 pCi/ml D-[l- 3HIglucose or 3-O-methyl-D-[l-3H]glucose at the concentrations indicated below, was added. The cultures were then placed on a rotating table in an incubator at 37°C for specific incubation periods, after which the buffer was aspirated and cells rinsed 5 times with 4 ml ice-cold Earle’s salt solution per wash. Initial rates measured under control conditions using an ice-cold ‘stopping’ solution contain1.25 mM KI and 0.1 mM ing 1 mM HgCl,, phloretin exhibited no significant difference from rates obtained with ice-cold buffer alone. Timecourse studies were corrected for non-mediated uptake by parallel incubations with cytochalasin B (10e5 M). Cells were solubilized by adding 1.5 ml of 0.2 N NaOH and the plates were scraped with a rubber policeman. The cell solutions were aspirated into test tubes and heated at 50°C for 30 min. Aliquots were then taken for protein determinations (Lowry et al., 1951) and scintillation counting. Plain cell-free gelatin-coated dishes were treated identically to quantify the amount of background protein contributing to the assay. This background usually constituted lo-15% of the total protein determination. 1 ml aliquots were transferred to scintillation vials, neutralized with 30 ~1 of 5 N HCl and counted in 10 ml Aquasol using a Packard Tricarb 5560 liquid scintillation counter. For kinetic studies all assays were performed in triplicate and repeated a minimum of 3 times. Results are presented as the mean points of single representative experiments. In comparison studies differences were significant to at least p I 0.05. Estimates of initial rates (Fig. 1) were obtained by the quadratic regression method described by Jacquez (1978). Calculation of the transport kinetic using the contents K, and V,,, was performed non-linear, iterative regression method of Cleland (1967) modified to correct for non-specific diffusion uptake. Analysis was performed on the
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University of Wisconsin Academic Center’s Sperry 1100 computer.
Computing
Hexose starvation Glucose starvation was initiated by feeding cells complete growth medium lacking glucose. Control cultures were fed ‘standard medium’ consisting of complete medium containing 5.5 mM D-glucose. In addition, in some experiments cycloheximide (10 pg/ml final concentration) was added to triplicate dishes for each group. Both groups were incubated for 24 h after which assays for hexose transport activity were performed as described above. No difference was observed in trace [3H]3O-MeGlc equilibration space between control and treated cells. All assays were performed in triplicate and all experiments were repeated a minimum of 3 times. The results presented are of single representative experiments. Insulin binding These studies were performed using the method of Sandra and Przybylski (1979) with the modifications described below. Cultures were first washed with 3 ml of Earle’s salt solution followed by a 30 min preincubation at 37°C in Eagle’s minimal essential medium supplemented with L-glutamine (0.3 g/l), NaHCO, (1.2 g/l) and bovine serum albumin (5 g/l). Following preincubation, the medium was aspirated and 2 ml of preincubation buffer containing [‘251]insulin at the appropriate concentration was added to each plate. The cells were incubated at 22°C or 37°C on a rotating table in an incubator for the time specified in the figure legends. At the end of this period the buffer was aspirated and the plates were rinsed 6 times with 2 ml ice-cold Earle’s salt wash solution. The cells were solubilized as described above for the uptake assay and aliquots were taken for protein and radioactivity determinations. The latter determinations were performed as described above for liquid scintillation counting. Specific binding is defined as the difference in [‘251]insulin binding in the presence or absence of 80 pgg/ml of unlabeled insulin. Non-specific binding ranged from 20 to 43% of total binding depending on the incubation. Data represent duplicate sets of determinations for both total and non-specific binding at each point.
Results Time-course of uptake Uptake of 3-O-methylglucose (3-O-MeGlc), an analog of D-glucose, was measured in 6-7-day-old cultures of fused, striated muscle cells. Studies were performed on the early time-course of uptake for this sugar to determine if initial rates could be measured under the experimental condition described in Materials and Methods. Since this sugar is neither phosphorylated nor metabolized by cells, it would be expected to accumulate rapidly in the intracellular space. Results are presented in Fig. 1 of an uptake experiment for 3-O-MeGlc (5 mM) in the presence or absence of insulin. In this case uptake was non-linear by 3 min in both control and insulin-treated myotubes. The initial velocity at this sugar concentration for the control experiment was 2.6 nmoles mg protein-’ mini ’ and increased in the presence of insulin to 5.9 nmoles mg protein-’ mm’. In other studies uptake was found to be linear for at least 60 set at methylglucase concentrations up to 25 mM. In subsequent experiments uptake at 1 min was used for determining initial rates. Similar results were obtained for uptake of D-glucose with linearity being maintained for up to 6 min (data not shown).
TIME
(mid
Fig. 1. Uptake of 3-O-methyl-D-glucose into ‘I-day-old myotubes in the presence (0) and absence (0) of insulin. Myotubes (35 mm plates) were preincubated for 30 min in glucose-free Hepes/Earle’s buffer with or without insulin (10 &ml). Cells were then incubated in buffer containing [ 3H]3-O-methylglucase (5 mM) with insulin present in the appropriate cultures during incubation. Uptake in the presence of 5 PM cytochalasin B has been subtracted. The uptake assay was performed as described in Materials and Methods.
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Additional experiments were performed to determine the equilibration times of 3-0-MeGlc in the presence or absence of insulin as well as cytochalasin B, a potent inhibitor of glucose transport in other cell systems (Kletzien and Perdue, 1975; Plagemann et al., 1974; Jung and Rampal, 1977). As shown in Fig. 2, 3-0-MeGlc uptake approaches steady-state levels by 60 min. In the presence of insulin this level is reached by 30 min. Cytochalasin B dramatically inhibits uptake of 3-0-MeGlc to levels generally observed with L-glucose uptake, an indicator of simple diffusion (data not shown). While it may appear that cytochalasin B has altered the steady-state equilibration value of 3-0MeGlc uptake, this is not the case. Rather, it represents a very slow uptake process, since in other experiments in which the time-course was extended beyond 60 min, uptake values in the presence of cytochalasin B continued to increase slowly. This inhibition by cytochalasin B was also observed with D-ghCOSe uptake in other experiments. The low level of sugar uptake observed in the presence of cytochalasin B may be associated with passive diffusion, non-specific glucose binding or a combination of both. Similar insulin responses and cytochalasin B effects have also been observed at other concentrations of sugar and inhibitor. From the steady-state values of 3-0MeGlc uptake in Fig. 2, the methylglucose distribution space is estimated to be 3.8 ~1 mg protein-‘.
In other studies we have performed using tracer [ I4 Clsucrose and [ 3Hlmethylglucose the values for 3-0-MeGlc distribution volume have ranged from 3.5 to 4.2 ~1 mg cell protein-’ (n = 5). Saturation kinetics In order to obtain estimates of the kinetic parameters K, and I’,,, of transport, uptake of 3-0-MeGlc as a function of concentration was examined (Fig. 3). Under these conditions uptake was not linear with concentration, although a complete saturation effect was not observed in the range of concentrations tested. Part of this lack of saturation is probably due to the diffusion component which has not been subtracted in this figure. Insulin stimulation of this uptake occurred at all concentrations of sugar tested (Fig. 3). Non-linear regression analysis of these data (Cleland, 1967) corrected for non-carrier-mediated diffusion, resulted in values for the apparent K, in the presence and absence of insulin of 6.7 and 6.2 mM respectively, which was not a significant difference. Values for V,,, of transport were 2.9 nmoles mg protein-’ rnin-’ for control and 4.4 nmoles mg protein-’ min-’ in the presence of insulin.
[3-O- MeGlc] mM 40
TIME
50
60
(mid
Fig. 2. Uptake of 3-O-methyl-D-glucose (5 mM) in ‘I-day-old mytubes over an extended time-course. Preincubations and incubations were performed as in Fig. 1 but with both incubations containing no additions (o), 10 pg/ml insulin (0), or 5 n M cytochalasin B (0).
Fig. 3. Concentration-dependent uptake (uncorrected for diffusion) of 3-O-methyl-D-glucose into ‘I-day-old myotubes in the presence (0) and absence (0) of insulin. Preincubation conditions were the same as in Fig. 1. 1 min uptakes were performed with [ ‘H]3-0-methylglucose at the concentrations indicated. In experiments with insulin (10 cg/ml) the hormone was present throughout both preincubation and incubation periods.
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Insulin binding Insulin binding studies were performed on 6-14-day-old fused myotubes. The time-course of specific insulin binding was determined in lo-dayold myotubes at both 22°C and 37°C (Fig. 4). At 22°C the binding increased with time and approached saturation by 120 min (Fig. 4A). In contrast, binding at 37°C was a more rapid process and saturation was reached between 30 and 45 min. Beyond this time, however, binding progressively decreased with incubation time (Fig. 4B). This decline has been observed in other binding studies with isolated fat cells (Gliemann and Sonne, 1978), lymphocytes (Gavin et al., 1973) and liver membranes (Kahn et al., 1974) and probably represents both insulin and receptor degradation which is a highly temperature-dependent process (Kahn et al., 1974). In this study after 60 min of incubation at 37°C only 67% of the insulin present in the incubation medium was TCA-precipitable, while in medium which was incubated in a similar
-I
,200~
0
-I
90
30
manner but in the absence of cells, over 95% of the [“‘I]insulin was TCA-precipitable after 60 min. This indicates that significant degradation of insulin occurred under these conditions. Specific binding was found to be linear over a wide range of myotube cell densities for a given age in culture (data not shown). Preliminary studies of [‘2sI]insulin binding as a function of myotube development were also performed. Myoblasts begin to undergo fusion and differentiation between 48 and 65 h after the establishment of secondary cultures. By 144 h most of the cells have fused and myofibrillar development is evident. Fig. 5 illustrates specific binding versus days in culture. It is apparent that insulin binding increased steadily with development between 6 and 14 days. This is consistent with the findings of Sandra and Przybylski (1979) who studied insulin binding in developing chick skeletal muscle cultures between 1 and 6 days. They found a marked increase in insulin binding following fusion of myoblasts to myotubes at around 3 days which increased progressively up to 6 days, the longest time measured. These studies indicate that binding continues to increase well beyond that time. Although the curve appears to level off at around 12 days, further experiments are needed in order to determine if, or when, a plateau level is attained.
120
TIME :kin) Fig. 4. Time-course of specific binding of [‘251]insulin (0.4 &ml) in lo-day-old cultured quail myotubes. The assays were performed in duplicate as described in Materials and Methods. (A) Binding at 22°C. (B) Binding at 37’C.
DAYS IN CULTURE Fig. 5. Specific binding of [ ‘ZSI]insulin to muscle cultures as a function of time in culture. The binding was determined at 37°C after 60 min incubation in the presence of [‘*sI]insulin (0.4 ng/ml).
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Starvation-induced hexose transport It has previously been reported that some cells exhibit non-hormonally induced modulation of the glucose transport system which appears to be regulated by the nutritional state of the cells. Chicken embryo fibroblasts, for example, have been shown to exhibit an enhanced uptake of hexoses following incubation in culture media lacking D-glucose (Martineau et al., 1972; Shaw and Amos, 1973). Furthermore, this effect could be blocked by inhibitors of protein synthesis such as actinomycin D, cordycepin or cycloheximide (Kletzien and Perdue, 1975). In hamster fibroblasts the enhancement of transport by starvation is reversible when cells are re-exposed to medium containing glucose (Christopher et al., 1976). This effect has not pre-
3-0-MaGlc A-Glc
I
I
I
! = E a yw
l-
200-
100 -
Z (z 3 O-
I +Glc
- Glc
Fig. 7. 3-@Methylglucose (50 PM) uptake in glucose-fed or starved cells following treatment with cycloheximide. Cells were incubated for 24 h in the presence or absence of D-glucose (5.5 mM) with (shaded bars) or without (open bars) the inclusion of cycloheximide (10 pg/ml). Uptake of sugars was determined after a 10 min incubation with radiotracer methylglucase under the conditions described in Fig. 2. Each value represents the mean k SEM for 3 plates.
A
5
_ 3oc ._E a# 6 h
I
I
20
25
Fig. 6. A time-course of uptake of 3-O-methyl-D-glucose (5 mM) and D-ghCOSe (5 mM) in 6-day-old myotubes following a 24 h incubation in standard culture medium containing either the normal D-glucose concentration (5.5 mM), or medium from which glucose was omitted. (A) [ ‘H]3-O-methylglucose uptake. (B) [ 3H]D-ghtcose uptake. A, 0, glucose-fed cells; A, 0, starved cells. Uptake was assayed as in Fig. 2 with subtraction of uptake in the presence of 5 pM cytochalasin B.
viously been reported for muscle. Experiments were therefore initiated to investigate whether myotubes in culture were sensitive to this type of glucose transport regulation. 6-day-old myotubes were incubated for 24 h in the presence or absence of 5.5 mM D-glucose. Following this incubation, a time-course of the uptake of both D-glucose and 3-0-MeGlc was measured. Uptake of 3-0-MeGlc (Fig. 6A) was stimulated approximately 40% under starvation conditions and these cells equilibrated with extracellular sugar more rapidly than cells exposed to glucose-containing medium. Similar results were observed for uptake of D-glucose (Fig. 6B). The starvation induction of transport activity was prevented when cycloheximide (10 pg/ml), an inhibitor of protein synthesis, was included in the medium (Fig. 7). No significant effect of cycloheximide on uptake rates was observed in treated fed cells as compared to untreated fed cells. In starved cells, however, the level of 3-0-MeGlc transport was reduced to that of fed cells when cycloheximide was included during the 24 h in-
cubation in glucose-free medium. In other similar experiments, starved cells consistently exhibited higher rates of transport compared to controls, though with some degree of variability between preparations. Discussion Evidence from this study indicates that 6-7day-old fused quail myotubes possess the characteristics of a specific hexose transport system. Both D-glucose and 3-O-methyl-D-glucose enter cells in a rapid manner similar to that observed in other cell types. The rate of entry is saturable at high sugar concentrations and is inhibited between 80 and 90% in the presence of cytochalasin B, a potent competitive inhibitor of glucose transport in other cell systems. In addition, this transport system is stimulated by insulin, and from kinetic constants determined by Lineweaver-Burk analysis, this effect may be characterized as an increase with no significant inin the V,,, of transport crease in the apparent K,. The initial slope of the curve for cytochalasin B-inhibited uptake in Fig. 2 between 0 and 15 min is greater than that of the subsequent portion of the curve (15-60 min). This relatively rapid rise in the early portion of presumably non-mediated uptake may be due to several different factors. A small portion of the increase may be due to some rapid non-specific binding of glucose which persists through the washing phase. A contribution may also be due to the presence of one or more extracellular compartments which equilibrate by diffusion within the first 15 min of incubation, but are incompletely depleted during the rapid washing steps. These compartments could be represented by water spaces such as the trans-tubular network or possibly the space between the cells and the culture dish. Future studies using non-transportable, radiolabeled compounds (such as L-glucose) should help to correct for this phenomenon. These myotubes also exhibit a starvation-induced modulation of sugar transport similar to that observed in cultured fibroblasts. Cells grown in the absence of glucose show an enhanced ability to take up sugars compared to those grown in normal medium. This effect can be abolished when cycloheximide is included in the medium during
the starvation period. Whether or not the starvation- and insulin-induced effects on transport operate via the same mechanism is not addressed in this study, but will be the subject of future investigation. Preliminary insulin binding studies on these cells indicate that they possess high-affinity insulin receptors as well as ‘a significant capability for degrading insulin and/or receptors. This high-affinity binding and degradation activity shows characteristics similar to those associated with insulin receptors in other cells. The decline in binding with increasing time of incubation which we observed in the present studies has also been observed in other binding studies with isolated fat cells (Gliemann and Sonne, 1978), lymphocytes (Gavin et al., 1973) and liver membranes (Kahn et al., 1974). This effect probably represents both insulin and receptor degradation which is a highly temperature-dependent process (Kahn et al., 1974). The fact that supraphysiological concentrations of insulin are required to elicit an uptake response is not unique to the present study. In work on sugar transport (Schudt et al., 1976; Sandra and Przybylski, 1975) and regulation of glycogen synthase (Gaertner et al., 1977) in chick embryonic muscle, levels of insulin similar to those reported here were required to elicit physiological responses. In studies on insulin-stimulated amino acid transport in rat L6 myotubes (Merrill et al., 1977), it was reported that concentrations of insulin 10 to lOOOO-fold higher than physiological levels were necessary .to elicit a response. Thus, some form of insulin ‘resistance’ appears to exist in vitro. The fact that the number of high-affinity insulin receptors in cultured chick muscle appears to be comparable to those in other types of insulin-sensitive tissue (Sandra and Przybylski, 1979) and that a functional glucose transport system is present at this stage of development, as demonstrated in these and other studies (Schudt et al., 1976), suggests that the observed attenuation is related to a post-binding event (Sandra and Przybylski, 1979). This may be a result of the inability to mimic in vivo growth conditions in culture, or it may be due to the age of the cells in culture. No transport studies have been reported on embryonic muscle cells grown in vitro for longer than 7 days; consequently, additional time may be required for
178
the complete manifestation of the insulin response. In this regard, Cowett et al. (1980) have reported that neonatal rats exhibit a blunted response to insulin in the 48 h following birth which is related to a decreased sensitivity of the hexose transport system. Another contributing factor may be the decreased effectiveness of mammalian insulin on avian tissue (Hazelwood et al., 1968). Studies by these investigators on adult chickens indicate that blood glucose levels in these animals are lowered considerably more by physiological levels of avian insulin as compared to using equivalent concentrations of bovine or ovine insulin. In addition, factors such as non-specific insulin binding, the inhibiting influence of cysteine in the medium (Kumegawa et al., 1980), or the potential degradation of insulin may all contribute to lowering the effective concentration of insulin to which the cells are exposed. The present study reveals that cells in cultures derived from embryonic quail breast muscle may be a suitable system for studying physiological regulation of hexose transport in muscle. Cultured muscle provides many advantages over intact muscle systems. Such advantages include the short diffusion distances present between cell membranes and the incubation media, and the absence of a basement membrane, a potential barrier to metabolite diffusion (Lipton, 1977). An additional advantage of the quail culture as compared to other cultured skeletal muscle systems, such as chick embryo or rat, is the virtual absence of contaminating fibroblasts which constitute no greater than 0.25% of the cell population (Lipton, 1977). This obviates the need to use substances such as cytosine arabinoside which is toxic to replicating cells but does not eliminate all fibroblasts (Fischbach, 1972). These attributes of quail culture offer significant advantages in determining kinetic parameters of both glucose transport as well as insulin binding. Although glucose transport and insulin binding in cells of earlier development stages are not reported here, the ontogeny of both glucose transport and insulin sensitivity may provide important clues to the mechanisms of insulin action in muscle and consequently will be the target of future studies in this laboratory.
Acknowledgements The author would like to thank Dr. Bruce H. Lipton and Dr. Edward Schultz for their advice and use of facilities for these studies. I also wish to thank Ms. Nancy Woolf DiBartolomeis for her excellent technical assistance in these studies. This research was supported in part by a grant from the Juvenile Diabetes Foundation. References Christopher, C.W., Ullrey, D., Colby, W. and Kalckar, H.M. (1976) Proc. Natl. Acad. Sci. (U.S.A.) 73, 2429-2433. Clausen, T. (1975) Curr. Topics Membrane Transport 6, 169-226. Cleland, W.W. (1967) Adv. Enzymol. 29, l-32. Cowett, R.M., Czech, M.P., Susa, J.B., Schwartz, R. and Oh, W. (1980) Metabolism 29, 563-567. Elbrink, J. and Bihler, I. (1975) Science 188, 1177-1188. Fischbach, G.D. (1972) Dev. Biol. 28, 407-429. Gaertner, U., Schudt, C. and Pette, D. (1977) Mol. Cell. Endocrinol. 8, 35-46. Gavin, III, J.R., Gordon, P., Roth, J., Archer, J.A. and Buell, D.N. (1973) J. Biol. Chem. 248, 2202-2207. Gliemann, J. and Sonne, 0. (1980) J. Biol. Chem. 253, 7857-7863. Hazelwood, R.L., Kimmel, J.R. and Pollock, H.G. (1968) Endocrinology 83, 1331-1336. Jacquez, J.A. (1978) In: Membrane Physiology, Eds.: T.E. Andreoli, J.E. Hoffman and D.D. Fanestil (Plenum Medical, New York) pp. 157-159. Jung, C.Y. and Rampal, A.L. (1977) J. Biol. Chem. 252, 5456-5463. Kahn, CR., Freychet, P., Roth, J. and Neville, D.M. (1974) J. Biol. Chem. 249, 2249-2257. Kletzien, R.F. and Perdue, J.F. (1975) J. Biol. Chem. 250, 593-600. Konigsberg, I.R. (1979) Methods Enzymol. 58, 511-527. Kumegawa, M., Ikeda, E., Hosada, S. and Takuma, T. (1980) Dev. Biol. 79, 493-499. Liptin, B.H. (1977) Dev. Biol. 61, 135-365. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. Martineau, R., Kohlbacker, M., Shaw, S.N. and Amos, H. (1972) Proc. Natl. Acad. Sci. (U.S.A.) 69, 3407-3411. Merrill, G.F., Florini, J.R. and Dulak, N.C. (1977) J. Cell. Physiol. 93, 173-182. Plagemann, P.G.W. and Richey, D.P. (1974) B&him. Biophys. Acta 344, 264-305. Sandra, A. and Przybylski, R.J. (1975) J. Cell Biol. 67, 318a. Sandra, A. and Przybylski, R.J. (1979) Dev. Biol. 68, 546-556. Schudt, C., Gaertner, U. and Pette, D. (1976) Eur. J. Biochem. 68,103-111. Shaw, S.N. and Amos, H. (1973) B&hem. Biophys. Res. Commun. 53, 357-365. Vaheri, A., Ruoslabti, E. and Nordling, S. (1972) Nature (London) New Biol. 238, 211-212.
171
MCE 01239
Modulation of hexose transport in cultured skeletal muscle Michael F. Shanahan Department of Physiology, University of Wisconsin, Madison, WI 63706 (U.S.A.) (Received
Keywords:
glucose
transport;
insulin action;
31 May 1984; accepted
insulin binding;
5 September
starvation-induced
1984)
transport.
The modulation of hexose transport due to insulin and glucose starvation was investigated in cultures derived from the breast musculature of embryonic quail. Fused myotubes at 37°C exhibited a saturable, stereospecific basal uptake of both D-glucose and 3Omethylglucose which was markedly inhibited by cytochalasin B, a potent inhibitor of hexose transport in other cell systems. In the presence of insulin, 3-O-methylglucose uptake was stimulated relative to untreated controls. Kinetic analysis indicated that insulin increased the V,,, of transport with no significant increase in the apparent K,. Incubation of myotubes in glucose-free medium for 24 h resulted in an increase in D-glucose and 3-O-methylglucose transport activity. Cycloheximide abolished this stimulation effect when it was included during the starvation period, but had no effect on transport in glucose-fed cells. Insulin binding studies on these myotubes indicate that high-affinity insulin receptors are present and continue to increase throughout the life of the culture. This high-affinity binding as well as the capacity to degrade insulin in these cells is characteristically similar to effects observed in other insulin-sensitive cell systems.
Glucose has been demonstrated to enter skeletal muscle via a carrier-mediated process which is accelerated in the presence of insulin (see Clausen, 1975; Elbrink and Bihler, 1975, for reviews). The mechanism of insulin stimulation of hexose transport is unknown, but is presumed to be initiated via an insulin-receptor complex on the outer surface of the plasma membrane. Several investigators have studied the action of insulin on glucose transport (Schudt et al., 1976) as well as insulin binding (Sandra and Przybylski, 1979) during myogenesis in skeletal muscle cell cultures derived from chick embryos. Both these studies have indicated a progressive development of both monosaccharide transport capability and insulin receptor binding during myogenesis. Most of the studies on glucose transport and 0303-7207/84/$03.00
Q 1984 Elsevier Scientific
Publishers
Ireland,
insulin effects have been carried out on muscle cultures derived from embryonic chick. Unfortunately, these preparations often contain a significant proportion of contaminating fibroblasts which are invariably present in chick muscle cell cultures. The obvious disadvantage of having a large number of contaminating fibroblasts in this type of study is that the fibroblasts themselves possess an insulin-sensitive glucose transport system (Vaheri et al., 1972; Shaw and Amos, 1973). Thus, insulin effects on transport kinetic parameters in chick muscle cell cultures may be difficult to evaluate owing to the presence of two different responsive cell populations contributing to the experimental results. In the present study, we have analyzed the insulin-modulated hexose transport system in a Ltd.
172
muscle culture system derived from embryonic quail breast musculature. This system offers a significant advantage in the fact that such cultures provide a virtually pure population of muscle cells with less than 0.25% contaminating fibroblasts (Lipton, 1977). Materials and methods Reagents Powdered culture media and selected horse sera lots were obtained from Gibco (Grand Island, NY), while all other reagents were purchased from Sigma Chemical Co. (St. Louis) with the exception of porcine insulin, which was obtained from Eli Lilly Co. (Indianapolis). Cytochalasin B was solubilized in absolute ethanol and diluted 2000 X following addition to the incubation medium. Radiotracer compounds were obtained from New England Nuclear (Boston). Cell culture Quail muscle cultures were prepared by the method of Konigsberg (1979). Breast musculature from 9-day embryonic Japanese quail (Corturnix corturnix japonica) was excised and minced. The tissue was mechanically dissociated by vortexing in complete medium, and the resulting suspension passed through filters to remove undissociated clumps. Cells were inoculated into gelatin-coated 100 mm plastic tissue culture plates at cell densities of 2 X lo6 cells/plate which precludes fusion during the ensuing 24 h. After 18-24 h of incubation at 37°C in a humidified atmosphere of 5% CO, in air, secondary suspensions were prepared by resuspending the cells with 0.013% trypsin. Secondary cultures were then established by inoculating myoblasts (30000 cells/plate) into 3 ml growth medium in 35 mm gelatin-coated plastic tissue culture dishes. Growth media in all cultures consisted of Eagle’s minimal essential medium supplemented with ‘selected’ (Lipton, 1977) horse serum (IS%), chick embryo extract (10%) and a 1% mixture of penicillin-streptomycin. Up take assays For hexose uptake assays, all incubations and preincubation wash solutions were maintained at
37’C. Secondary cultures were rinsed 4 times with 3 ml of Earle’s salt solution. Cells were then preincubated at 37°C in 4 ml glucose-free Earle’s salts containing 20 mM Hepes buffer (pH 7.4) and other agents at the concentrations described below. Cells were preincubated in the buffer for 15-30 min. Following preincubation, the buffer was removed by aspiration and 4 ml of incubation medium, consisting of the appropriate preincubation medium containing 3 pCi/ml D-[l- 3HIglucose or 3-O-methyl-D-[l-3H]glucose at the concentrations indicated below, was added. The cultures were then placed on a rotating table in an incubator at 37°C for specific incubation periods, after which the buffer was aspirated and cells rinsed 5 times with 4 ml ice-cold Earle’s salt solution per wash. Initial rates measured under control conditions using an ice-cold ‘stopping’ solution contain1.25 mM KI and 0.1 mM ing 1 mM HgCl,, phloretin exhibited no significant difference from rates obtained with ice-cold buffer alone. Timecourse studies were corrected for non-mediated uptake by parallel incubations with cytochalasin B (10e5 M). Cells were solubilized by adding 1.5 ml of 0.2 N NaOH and the plates were scraped with a rubber policeman. The cell solutions were aspirated into test tubes and heated at 50°C for 30 min. Aliquots were then taken for protein determinations (Lowry et al., 1951) and scintillation counting. Plain cell-free gelatin-coated dishes were treated identically to quantify the amount of background protein contributing to the assay. This background usually constituted lo-15% of the total protein determination. 1 ml aliquots were transferred to scintillation vials, neutralized with 30 ~1 of 5 N HCl and counted in 10 ml Aquasol using a Packard Tricarb 5560 liquid scintillation counter. For kinetic studies all assays were performed in triplicate and repeated a minimum of 3 times. Results are presented as the mean points of single representative experiments. In comparison studies differences were significant to at least p I 0.05. Estimates of initial rates (Fig. 1) were obtained by the quadratic regression method described by Jacquez (1978). Calculation of the transport kinetic using the contents K, and V,,, was performed non-linear, iterative regression method of Cleland (1967) modified to correct for non-specific diffusion uptake. Analysis was performed on the
173
University of Wisconsin Academic Center’s Sperry 1100 computer.
Computing
Hexose starvation Glucose starvation was initiated by feeding cells complete growth medium lacking glucose. Control cultures were fed ‘standard medium’ consisting of complete medium containing 5.5 mM D-glucose. In addition, in some experiments cycloheximide (10 pg/ml final concentration) was added to triplicate dishes for each group. Both groups were incubated for 24 h after which assays for hexose transport activity were performed as described above. No difference was observed in trace [3H]3O-MeGlc equilibration space between control and treated cells. All assays were performed in triplicate and all experiments were repeated a minimum of 3 times. The results presented are of single representative experiments. Insulin binding These studies were performed using the method of Sandra and Przybylski (1979) with the modifications described below. Cultures were first washed with 3 ml of Earle’s salt solution followed by a 30 min preincubation at 37°C in Eagle’s minimal essential medium supplemented with L-glutamine (0.3 g/l), NaHCO, (1.2 g/l) and bovine serum albumin (5 g/l). Following preincubation, the medium was aspirated and 2 ml of preincubation buffer containing [‘251]insulin at the appropriate concentration was added to each plate. The cells were incubated at 22°C or 37°C on a rotating table in an incubator for the time specified in the figure legends. At the end of this period the buffer was aspirated and the plates were rinsed 6 times with 2 ml ice-cold Earle’s salt wash solution. The cells were solubilized as described above for the uptake assay and aliquots were taken for protein and radioactivity determinations. The latter determinations were performed as described above for liquid scintillation counting. Specific binding is defined as the difference in [‘251]insulin binding in the presence or absence of 80 pgg/ml of unlabeled insulin. Non-specific binding ranged from 20 to 43% of total binding depending on the incubation. Data represent duplicate sets of determinations for both total and non-specific binding at each point.
Results Time-course of uptake Uptake of 3-O-methylglucose (3-O-MeGlc), an analog of D-glucose, was measured in 6-7-day-old cultures of fused, striated muscle cells. Studies were performed on the early time-course of uptake for this sugar to determine if initial rates could be measured under the experimental condition described in Materials and Methods. Since this sugar is neither phosphorylated nor metabolized by cells, it would be expected to accumulate rapidly in the intracellular space. Results are presented in Fig. 1 of an uptake experiment for 3-O-MeGlc (5 mM) in the presence or absence of insulin. In this case uptake was non-linear by 3 min in both control and insulin-treated myotubes. The initial velocity at this sugar concentration for the control experiment was 2.6 nmoles mg protein-’ mini ’ and increased in the presence of insulin to 5.9 nmoles mg protein-’ mm’. In other studies uptake was found to be linear for at least 60 set at methylglucase concentrations up to 25 mM. In subsequent experiments uptake at 1 min was used for determining initial rates. Similar results were obtained for uptake of D-glucose with linearity being maintained for up to 6 min (data not shown).
TIME
(mid
Fig. 1. Uptake of 3-O-methyl-D-glucose into ‘I-day-old myotubes in the presence (0) and absence (0) of insulin. Myotubes (35 mm plates) were preincubated for 30 min in glucose-free Hepes/Earle’s buffer with or without insulin (10 &ml). Cells were then incubated in buffer containing [ 3H]3-O-methylglucase (5 mM) with insulin present in the appropriate cultures during incubation. Uptake in the presence of 5 PM cytochalasin B has been subtracted. The uptake assay was performed as described in Materials and Methods.
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Additional experiments were performed to determine the equilibration times of 3-0-MeGlc in the presence or absence of insulin as well as cytochalasin B, a potent inhibitor of glucose transport in other cell systems (Kletzien and Perdue, 1975; Plagemann et al., 1974; Jung and Rampal, 1977). As shown in Fig. 2, 3-0-MeGlc uptake approaches steady-state levels by 60 min. In the presence of insulin this level is reached by 30 min. Cytochalasin B dramatically inhibits uptake of 3-0-MeGlc to levels generally observed with L-glucose uptake, an indicator of simple diffusion (data not shown). While it may appear that cytochalasin B has altered the steady-state equilibration value of 3-0MeGlc uptake, this is not the case. Rather, it represents a very slow uptake process, since in other experiments in which the time-course was extended beyond 60 min, uptake values in the presence of cytochalasin B continued to increase slowly. This inhibition by cytochalasin B was also observed with D-ghCOSe uptake in other experiments. The low level of sugar uptake observed in the presence of cytochalasin B may be associated with passive diffusion, non-specific glucose binding or a combination of both. Similar insulin responses and cytochalasin B effects have also been observed at other concentrations of sugar and inhibitor. From the steady-state values of 3-0MeGlc uptake in Fig. 2, the methylglucose distribution space is estimated to be 3.8 ~1 mg protein-‘.
In other studies we have performed using tracer [ I4 Clsucrose and [ 3Hlmethylglucose the values for 3-0-MeGlc distribution volume have ranged from 3.5 to 4.2 ~1 mg cell protein-’ (n = 5). Saturation kinetics In order to obtain estimates of the kinetic parameters K, and I’,,, of transport, uptake of 3-0-MeGlc as a function of concentration was examined (Fig. 3). Under these conditions uptake was not linear with concentration, although a complete saturation effect was not observed in the range of concentrations tested. Part of this lack of saturation is probably due to the diffusion component which has not been subtracted in this figure. Insulin stimulation of this uptake occurred at all concentrations of sugar tested (Fig. 3). Non-linear regression analysis of these data (Cleland, 1967) corrected for non-carrier-mediated diffusion, resulted in values for the apparent K, in the presence and absence of insulin of 6.7 and 6.2 mM respectively, which was not a significant difference. Values for V,,, of transport were 2.9 nmoles mg protein-’ rnin-’ for control and 4.4 nmoles mg protein-’ min-’ in the presence of insulin.
[3-O- MeGlc] mM 40
TIME
50
60
(mid
Fig. 2. Uptake of 3-O-methyl-D-glucose (5 mM) in ‘I-day-old mytubes over an extended time-course. Preincubations and incubations were performed as in Fig. 1 but with both incubations containing no additions (o), 10 pg/ml insulin (0), or 5 n M cytochalasin B (0).
Fig. 3. Concentration-dependent uptake (uncorrected for diffusion) of 3-O-methyl-D-glucose into ‘I-day-old myotubes in the presence (0) and absence (0) of insulin. Preincubation conditions were the same as in Fig. 1. 1 min uptakes were performed with [ ‘H]3-0-methylglucose at the concentrations indicated. In experiments with insulin (10 cg/ml) the hormone was present throughout both preincubation and incubation periods.
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Insulin binding Insulin binding studies were performed on 6-14-day-old fused myotubes. The time-course of specific insulin binding was determined in lo-dayold myotubes at both 22°C and 37°C (Fig. 4). At 22°C the binding increased with time and approached saturation by 120 min (Fig. 4A). In contrast, binding at 37°C was a more rapid process and saturation was reached between 30 and 45 min. Beyond this time, however, binding progressively decreased with incubation time (Fig. 4B). This decline has been observed in other binding studies with isolated fat cells (Gliemann and Sonne, 1978), lymphocytes (Gavin et al., 1973) and liver membranes (Kahn et al., 1974) and probably represents both insulin and receptor degradation which is a highly temperature-dependent process (Kahn et al., 1974). In this study after 60 min of incubation at 37°C only 67% of the insulin present in the incubation medium was TCA-precipitable, while in medium which was incubated in a similar
-I
,200~
0
-I
90
30
manner but in the absence of cells, over 95% of the [“‘I]insulin was TCA-precipitable after 60 min. This indicates that significant degradation of insulin occurred under these conditions. Specific binding was found to be linear over a wide range of myotube cell densities for a given age in culture (data not shown). Preliminary studies of [‘2sI]insulin binding as a function of myotube development were also performed. Myoblasts begin to undergo fusion and differentiation between 48 and 65 h after the establishment of secondary cultures. By 144 h most of the cells have fused and myofibrillar development is evident. Fig. 5 illustrates specific binding versus days in culture. It is apparent that insulin binding increased steadily with development between 6 and 14 days. This is consistent with the findings of Sandra and Przybylski (1979) who studied insulin binding in developing chick skeletal muscle cultures between 1 and 6 days. They found a marked increase in insulin binding following fusion of myoblasts to myotubes at around 3 days which increased progressively up to 6 days, the longest time measured. These studies indicate that binding continues to increase well beyond that time. Although the curve appears to level off at around 12 days, further experiments are needed in order to determine if, or when, a plateau level is attained.
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TIME :kin) Fig. 4. Time-course of specific binding of [‘251]insulin (0.4 &ml) in lo-day-old cultured quail myotubes. The assays were performed in duplicate as described in Materials and Methods. (A) Binding at 22°C. (B) Binding at 37’C.
DAYS IN CULTURE Fig. 5. Specific binding of [ ‘ZSI]insulin to muscle cultures as a function of time in culture. The binding was determined at 37°C after 60 min incubation in the presence of [‘*sI]insulin (0.4 ng/ml).
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Starvation-induced hexose transport It has previously been reported that some cells exhibit non-hormonally induced modulation of the glucose transport system which appears to be regulated by the nutritional state of the cells. Chicken embryo fibroblasts, for example, have been shown to exhibit an enhanced uptake of hexoses following incubation in culture media lacking D-glucose (Martineau et al., 1972; Shaw and Amos, 1973). Furthermore, this effect could be blocked by inhibitors of protein synthesis such as actinomycin D, cordycepin or cycloheximide (Kletzien and Perdue, 1975). In hamster fibroblasts the enhancement of transport by starvation is reversible when cells are re-exposed to medium containing glucose (Christopher et al., 1976). This effect has not pre-
3-0-MaGlc A-Glc
I
I
I
! = E a yw
l-
200-
100 -
Z (z 3 O-
I +Glc
- Glc
Fig. 7. 3-@Methylglucose (50 PM) uptake in glucose-fed or starved cells following treatment with cycloheximide. Cells were incubated for 24 h in the presence or absence of D-glucose (5.5 mM) with (shaded bars) or without (open bars) the inclusion of cycloheximide (10 pg/ml). Uptake of sugars was determined after a 10 min incubation with radiotracer methylglucase under the conditions described in Fig. 2. Each value represents the mean k SEM for 3 plates.
A
5
_ 3oc ._E a# 6 h
I
I
20
25
Fig. 6. A time-course of uptake of 3-O-methyl-D-glucose (5 mM) and D-ghCOSe (5 mM) in 6-day-old myotubes following a 24 h incubation in standard culture medium containing either the normal D-glucose concentration (5.5 mM), or medium from which glucose was omitted. (A) [ ‘H]3-O-methylglucose uptake. (B) [ 3H]D-ghtcose uptake. A, 0, glucose-fed cells; A, 0, starved cells. Uptake was assayed as in Fig. 2 with subtraction of uptake in the presence of 5 pM cytochalasin B.
viously been reported for muscle. Experiments were therefore initiated to investigate whether myotubes in culture were sensitive to this type of glucose transport regulation. 6-day-old myotubes were incubated for 24 h in the presence or absence of 5.5 mM D-glucose. Following this incubation, a time-course of the uptake of both D-glucose and 3-0-MeGlc was measured. Uptake of 3-0-MeGlc (Fig. 6A) was stimulated approximately 40% under starvation conditions and these cells equilibrated with extracellular sugar more rapidly than cells exposed to glucose-containing medium. Similar results were observed for uptake of D-glucose (Fig. 6B). The starvation induction of transport activity was prevented when cycloheximide (10 pg/ml), an inhibitor of protein synthesis, was included in the medium (Fig. 7). No significant effect of cycloheximide on uptake rates was observed in treated fed cells as compared to untreated fed cells. In starved cells, however, the level of 3-0-MeGlc transport was reduced to that of fed cells when cycloheximide was included during the 24 h in-
cubation in glucose-free medium. In other similar experiments, starved cells consistently exhibited higher rates of transport compared to controls, though with some degree of variability between preparations. Discussion Evidence from this study indicates that 6-7day-old fused quail myotubes possess the characteristics of a specific hexose transport system. Both D-glucose and 3-O-methyl-D-glucose enter cells in a rapid manner similar to that observed in other cell types. The rate of entry is saturable at high sugar concentrations and is inhibited between 80 and 90% in the presence of cytochalasin B, a potent competitive inhibitor of glucose transport in other cell systems. In addition, this transport system is stimulated by insulin, and from kinetic constants determined by Lineweaver-Burk analysis, this effect may be characterized as an increase with no significant inin the V,,, of transport crease in the apparent K,. The initial slope of the curve for cytochalasin B-inhibited uptake in Fig. 2 between 0 and 15 min is greater than that of the subsequent portion of the curve (15-60 min). This relatively rapid rise in the early portion of presumably non-mediated uptake may be due to several different factors. A small portion of the increase may be due to some rapid non-specific binding of glucose which persists through the washing phase. A contribution may also be due to the presence of one or more extracellular compartments which equilibrate by diffusion within the first 15 min of incubation, but are incompletely depleted during the rapid washing steps. These compartments could be represented by water spaces such as the trans-tubular network or possibly the space between the cells and the culture dish. Future studies using non-transportable, radiolabeled compounds (such as L-glucose) should help to correct for this phenomenon. These myotubes also exhibit a starvation-induced modulation of sugar transport similar to that observed in cultured fibroblasts. Cells grown in the absence of glucose show an enhanced ability to take up sugars compared to those grown in normal medium. This effect can be abolished when cycloheximide is included in the medium during
the starvation period. Whether or not the starvation- and insulin-induced effects on transport operate via the same mechanism is not addressed in this study, but will be the subject of future investigation. Preliminary insulin binding studies on these cells indicate that they possess high-affinity insulin receptors as well as ‘a significant capability for degrading insulin and/or receptors. This high-affinity binding and degradation activity shows characteristics similar to those associated with insulin receptors in other cells. The decline in binding with increasing time of incubation which we observed in the present studies has also been observed in other binding studies with isolated fat cells (Gliemann and Sonne, 1978), lymphocytes (Gavin et al., 1973) and liver membranes (Kahn et al., 1974). This effect probably represents both insulin and receptor degradation which is a highly temperature-dependent process (Kahn et al., 1974). The fact that supraphysiological concentrations of insulin are required to elicit an uptake response is not unique to the present study. In work on sugar transport (Schudt et al., 1976; Sandra and Przybylski, 1975) and regulation of glycogen synthase (Gaertner et al., 1977) in chick embryonic muscle, levels of insulin similar to those reported here were required to elicit physiological responses. In studies on insulin-stimulated amino acid transport in rat L6 myotubes (Merrill et al., 1977), it was reported that concentrations of insulin 10 to lOOOO-fold higher than physiological levels were necessary .to elicit a response. Thus, some form of insulin ‘resistance’ appears to exist in vitro. The fact that the number of high-affinity insulin receptors in cultured chick muscle appears to be comparable to those in other types of insulin-sensitive tissue (Sandra and Przybylski, 1979) and that a functional glucose transport system is present at this stage of development, as demonstrated in these and other studies (Schudt et al., 1976), suggests that the observed attenuation is related to a post-binding event (Sandra and Przybylski, 1979). This may be a result of the inability to mimic in vivo growth conditions in culture, or it may be due to the age of the cells in culture. No transport studies have been reported on embryonic muscle cells grown in vitro for longer than 7 days; consequently, additional time may be required for
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the complete manifestation of the insulin response. In this regard, Cowett et al. (1980) have reported that neonatal rats exhibit a blunted response to insulin in the 48 h following birth which is related to a decreased sensitivity of the hexose transport system. Another contributing factor may be the decreased effectiveness of mammalian insulin on avian tissue (Hazelwood et al., 1968). Studies by these investigators on adult chickens indicate that blood glucose levels in these animals are lowered considerably more by physiological levels of avian insulin as compared to using equivalent concentrations of bovine or ovine insulin. In addition, factors such as non-specific insulin binding, the inhibiting influence of cysteine in the medium (Kumegawa et al., 1980), or the potential degradation of insulin may all contribute to lowering the effective concentration of insulin to which the cells are exposed. The present study reveals that cells in cultures derived from embryonic quail breast muscle may be a suitable system for studying physiological regulation of hexose transport in muscle. Cultured muscle provides many advantages over intact muscle systems. Such advantages include the short diffusion distances present between cell membranes and the incubation media, and the absence of a basement membrane, a potential barrier to metabolite diffusion (Lipton, 1977). An additional advantage of the quail culture as compared to other cultured skeletal muscle systems, such as chick embryo or rat, is the virtual absence of contaminating fibroblasts which constitute no greater than 0.25% of the cell population (Lipton, 1977). This obviates the need to use substances such as cytosine arabinoside which is toxic to replicating cells but does not eliminate all fibroblasts (Fischbach, 1972). These attributes of quail culture offer significant advantages in determining kinetic parameters of both glucose transport as well as insulin binding. Although glucose transport and insulin binding in cells of earlier development stages are not reported here, the ontogeny of both glucose transport and insulin sensitivity may provide important clues to the mechanisms of insulin action in muscle and consequently will be the target of future studies in this laboratory.
Acknowledgements The author would like to thank Dr. Bruce H. Lipton and Dr. Edward Schultz for their advice and use of facilities for these studies. I also wish to thank Ms. Nancy Woolf DiBartolomeis for her excellent technical assistance in these studies. This research was supported in part by a grant from the Juvenile Diabetes Foundation. References Christopher, C.W., Ullrey, D., Colby, W. and Kalckar, H.M. (1976) Proc. Natl. Acad. Sci. (U.S.A.) 73, 2429-2433. Clausen, T. (1975) Curr. Topics Membrane Transport 6, 169-226. Cleland, W.W. (1967) Adv. Enzymol. 29, l-32. Cowett, R.M., Czech, M.P., Susa, J.B., Schwartz, R. and Oh, W. (1980) Metabolism 29, 563-567. Elbrink, J. and Bihler, I. (1975) Science 188, 1177-1188. Fischbach, G.D. (1972) Dev. Biol. 28, 407-429. Gaertner, U., Schudt, C. and Pette, D. (1977) Mol. Cell. Endocrinol. 8, 35-46. Gavin, III, J.R., Gordon, P., Roth, J., Archer, J.A. and Buell, D.N. (1973) J. Biol. Chem. 248, 2202-2207. Gliemann, J. and Sonne, 0. (1980) J. Biol. Chem. 253, 7857-7863. Hazelwood, R.L., Kimmel, J.R. and Pollock, H.G. (1968) Endocrinology 83, 1331-1336. Jacquez, J.A. (1978) In: Membrane Physiology, Eds.: T.E. Andreoli, J.E. Hoffman and D.D. Fanestil (Plenum Medical, New York) pp. 157-159. Jung, C.Y. and Rampal, A.L. (1977) J. Biol. Chem. 252, 5456-5463. Kahn, CR., Freychet, P., Roth, J. and Neville, D.M. (1974) J. Biol. Chem. 249, 2249-2257. Kletzien, R.F. and Perdue, J.F. (1975) J. Biol. Chem. 250, 593-600. Konigsberg, I.R. (1979) Methods Enzymol. 58, 511-527. Kumegawa, M., Ikeda, E., Hosada, S. and Takuma, T. (1980) Dev. Biol. 79, 493-499. Liptin, B.H. (1977) Dev. Biol. 61, 135-365. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. Martineau, R., Kohlbacker, M., Shaw, S.N. and Amos, H. (1972) Proc. Natl. Acad. Sci. (U.S.A.) 69, 3407-3411. Merrill, G.F., Florini, J.R. and Dulak, N.C. (1977) J. Cell. Physiol. 93, 173-182. Plagemann, P.G.W. and Richey, D.P. (1974) B&him. Biophys. Acta 344, 264-305. Sandra, A. and Przybylski, R.J. (1975) J. Cell Biol. 67, 318a. Sandra, A. and Przybylski, R.J. (1979) Dev. Biol. 68, 546-556. Schudt, C., Gaertner, U. and Pette, D. (1976) Eur. J. Biochem. 68,103-111. Shaw, S.N. and Amos, H. (1973) B&hem. Biophys. Res. Commun. 53, 357-365. Vaheri, A., Ruoslabti, E. and Nordling, S. (1972) Nature (London) New Biol. 238, 211-212.