Activation of Glucose Transport in Muscle by Prolonged Exposure to Insulin

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1 THE JOURNAL OF BOLOGCAL CHEMSTRY hy The American Society of Biological Chemists, nc. Vol. 261, No. 34, ssue of December 5, pp , 1986 Printed in U.S.A. Activation of Glucose Transport in Muscle by Prolonged Exposure to nsulin EFFECTS OF GLUCOSE AND NSULN CONCENTRATONS* (Received for publication, March 7, 1986) Douglas A. Young$, Jennifer J. Uhl, Gregory D. Carteel, and John 0. Holloszy From the Department of Medicine, Washington University School of Medicine, St. Louis, Missouri Glucose transport activity was found to increase from over studies employing the euglycemic insulin clamp that a 5 h in rat epitrochlearis muscle in response to a mod- similar phenomenon occurs in vivo (1). n the euglycemic erate concentration ( microunits/ml) of insulin. clamp procedure, plasma insulin concentration is raised to a This process was examined using 3-methylglucose. The constant level while glucose infusion rate is varied so as to increase in permeability to 3-methylglucose was 2- to maintain plasma glucose concentration constant (2). t has 4-fold greater after 5 h than after 1 h in muscles been reported that during prolonged euglycemic insulin clamp incubated with 50 microunits/ml of insulin and 1 or 8 studies, in which plasma insulin levels were maintained in the mm glucose. The increase in permeability to 3-meth microunits/ml range, the rate of glucose disappearance ylglucose during the period between 1 and 5 h of exincreased for 5 h, attaining a rate nearly 2-fold higher than posure to 50 microunits/ml of insulin and 1 mm glucose that achieved after 2 h (1). Since skeletal muscle is the major was due to an increase in the apparent V,,, of sugar transport. There were two components to this activasite of insulin-stimulated glucose disposal (3), it seems likely tion of glucose transport. One, which was not influthat the response in isolated skeletal muscles is the in vitro enced by inhibition of protein synthesis, resulted in counterpart of the time-dependent increase in insulin action activation of sugar transport to the same extent by 50 seen during the euglycemic clamp. n the present study, we microunits/ml as by 20,000 microunits/ml of insulin; used the isolated rat epitrochlearis muscle to characterize the however, this activation took -20 times longer with progressive increase in sugar transport activity that occurs in 50 microunits/ml insulin. The other, which was response to prolonged exposure to insulin, and to evaluate the blocked by cycloheximide, resulted in a further acti- roles of insulin concentration, glucose concentration, and vation of sugar transport to a level higher than that protein synthesis in this phenomenon. attained in response to 20,000 microunits/ml of insulin. Glucose had no effect on activation of sugar trans- EXPERMENTAL PROCEDURES port during the first hour, but a high concentration Animals and Muscle Preparation-Male specific pathogen-free (20-36 mm) of glucose prevented the further activation Wistar rats (Hilltop Lab Animals, nc., Chatsworth, CA) weighing of glucose transport during prolonged treatment with g were fed Purina chow and water ad libitum. Following an 50 microunits/ml of insulin. t appears from these re- overnight fast, rats were anesthetized with 5 mg/100 g of body weight sults that prolonged exposure a moderate to concentra- of pentobarbital sodium and the epitrochlearis muscles were dissected tion of insulin has previously unrecognized effects that out. The epitrochlearis is a small thin muscle, less than 0.2 mm thick, include: 1) a progressive activation of glucose trans- consisting predominantly (85%) of fast-twitch fibers (4). port over a long time that eventually results as in great ncubation of Muscles with nsulin-the epitrochlearis muscles a response as a supramaximal insulin concentration, and 2) in the presence of low glucose concentration, further activation of glucose transport by an additional, protein synthesis-dependent mechanism. The results also show that a high concentration of glucose can, under some conditions, inhibit stimulation of its own transport. During preliminary studies of the factors involved in the regulation of glycogen synthesis in muscle, we observed that the activation of glucose transport increased markedly over a 5-h period in response to a moderate insulin concentration. This finding seemed of considerable interest relative to the mechanism of insulin action, particularly in view of evidence * This research was supported in part by Research Grant AM18986 from the National nstitutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 2 Supported by nstitutional National Research Service Award AG from the National nstitutes of Health were incubated in a shaking incubator at 37 C for 1-5 h in 2 ml of Krebs-Henseleit buffer (KHB) (5) containing 0.1%bovine serum albumin, 50,100, or 20,000 microunits/ml of regular insulin (Squibb- Novo), and 1,8, 20, or 36 mm glucose in stoppered 25-ml Erlenmeyer flasks with a gas phase of 95% O2 and 5% CO,. To keep osmolarity constant throughout an experiment, sufficient mannitol was included in the incubation medium to keep the sum of the concentration of glucose or 3-methylglucose and mannitol at 40 mm. The KHB was pregassed with 95% O2 and 5% CO,. During prolonged incubations, the medium was changed every hour. nhibition of Protein Synthesis-The effect of protein synthesis on the increase in sugar transport was evaluated by incubating epitrochlearis muscles for 5 h with 1 mm glucose and 50 microunits/ml of insulin, in the presence or absence of20 pg/ml cycloheximide. Following incubation the muscles were rinsed for 10 min in the absence of cycloheximide and sugar transport was evaluated as described below. The effectiveness of 20 pg/ml cycloheximide in inhibiting protein synthesis in epitrochlearis muscle was evaluated by measurement of [3H]leucine incorporation into proteins. Following 2 h of incubation with or without cycloheximide, muscles were incubated in KHB containing 1 mm [3H]leucine (1 pci/ml) and 2 mm [4C]mannitol (0.01 pci/ml) for 1 h (also in the presence or absence of cycloheximide). Muscles were processed for measurement of [3H]leucine incorporation into proteins as described by Goldberg (6). The absence of The abbreviation used is: KHB, Krebs-Henseleit buffer.

2 16050 Prolonged Exposure of Muscle to nsulin [14C]mannitol in the final supernatant and in the protein pellet was used as evidence that the precipitated protein had been adequately washed. Measurement of 3-Methylglucose Transport into Muscle-Sugar transport activity was measured in epitrochlearis muscles using the non-metabolizable glucose analog 3-methylglucose and a modification of the procedure used previously in frog muscle (7-9). Following the incubations with glucose and insulin, the muscles were blotted and then washed by shaking in 2 ml of KHB containing 40 mm mannitol, 0.1% albumin, and the appropriate concentration of insulin at 29 C for 10 min to remove glucose. The muscles were then blotted and transferred to a flask with 2 ml of KHB containing 8 mm 3-0-[3H] methylglucose (437 pci/mmol), 32 mm [ C]mannitol (8 pcilrnmol), 0.1% albumin, and insulin at the same concentration as during the preceding incubation, and incubated at 29 C in a shaking incubator for 10 min. The gas phase in the flasks was 95% O2 and 5% CO,. Following a short period during which the extracellular space equilibrates with the medium, 3-methylglucose accumulation in epitrochlearis muscles is linear until the intracellular concentration reaches -25% of the extracellular concentration (Fig. 1). Following incubation, the muscles were blotted briefly on filter paper dampened with incubation medium, trimmed, and frozen in liquid N,. The samples were weighed, homogenized in 10% trichloroacetic acid, and centrifuged at 1000 X g. Aliquots of the muscle extracts and of the incubation media were placed in scintillation vials containing 10 ml of ScintiVerse (Fisher) and counted in a Packard liquid scintillation counter with channels preset for simultaneous 3H and 14C counting. The amount of each isotope present in the samples was determined and this information was used to calculate the extracellular space and the intracellular concentration of 3-methylglucose (7). The intracellular water content of the muscles was calculated by subtracting the measured extracellular space water from total muscle water. Total muscle water was assumed to be 80% of muscle weight, which is the average value for rat epitrochlearis muscles under our experimental conditions (10). The statistical significance of differences between contralateral muscles was assessed with a paired t test. Differences between groups of nonpaired muscles was assessed by analysis of variance. RESULTS Effect of ncubation Time and nsulin Concentration on 3- Methylglucose Transport-When epitrochlearis muscles were incubated with 50 microunits/ml of insulin and 8 mm glucose for 5 h the increase in permeability to 3-0-methylglucose was 4-fold greater than after 1 h (Table ). Activation of 3- methylglucose transport was also significantly greater after 5 h than after 1 h of incubation with 100 microunits/ml of insulin, but the magnitude of the increase between 1 and 5 h TME OF NCUBATON (min) FG. 1. Accumulation of 3-methylglucose in rat epitrochlearis muscles. Muscles were treated for 1 h with 50 microunits/ml of insulin. ntracellular accumulation of 3-methylglucose was then measured for different time periods during which the muscles were exposed to 8 mm 3-0-[3H]methylgluc~~e 29 at C as described under Experimental Procedures. The dashed line indicates what the intracellular concentration of 3-methylglucose would have been if uptake had continued to be linear. Each point represents the average of 5-10 muscles. 1 TABLE Effects of insulin concentration and duration of exposure to insulin on 3-methylglucose transport activity Rat epitrochlearis muscles were incubated with various concentrations of insulin and 8 mm glucose. One muscle of each pair was incubated for 1 h and the other for 5 h under the same conditions. Glucose transport activity was assessed after the 1- or 5-h incubations by measuring the intracellular accumulation of 3-methylglucose at 29 C for 10 min (for details see Experimental Procedures ). Results are expressed as micromoles of 3-methylglucose taken up per ml of intracellular water in 10 min. Values are means f S.E. for the number of muscles given in parentheses. nsulin concentration uptake 3-Methylglucose 1-h incubation 5-h incubation microunitslml pmol, ml. 10 min f f 0.09 (9) f f 0.19 (5) f (6) 20, f f 0.20 (5) a 5 h versus 1 h, p < w No insulin NCUBATONTME (hours) FG Methylglucose uptake following prolonged incubation with 0, 50, or 20,000 microunits/ml of insulin. Epitrochlearis muscles were incubated with different concentrations of insulin and 1 mm glucose for 1 or 5 h. Permeability to 3-methylglucose was then measured at 29 C as described under Experimental Procedures. Each point is the mean f S.E. of six muscles. was smaller than with 50 microunits/ml. n contrast to the effects of 50 or 100 microunits/ml of insulin, there was no further increase in permeability to sugar after 15 min in muscles incubated with 20,000 microunits/ml of insulin (data not shown; the 15-min value was not significantly different from the 1-h value shown in Table ). As a consequence, 5 h of incubation with 50 or 100 microunits/ml of insulin and 8 mm glucose resulted in as great an activation of sugar transport as did incubation with 20,000 microunits/ml. Furthermore, when the glucose concentration was lowered to 1 mm, activation of 3-0-methylglucose transport after 5 h of exposure to insulin was significantly greater with 50 microunits/ ml than with 20,000 microunits/ml of insulin (Fig. 2). (Following a 1-h incubation with insulin under our conditions half-maximal activation of transport occurs at approximately 65 microunits/ml of insulin, while 20,000 microunits/ml of insulin is well above the concentration needed to produce a maximal effect.)

3 Prolonged Exposure of Muscle to nsulin The progressive increase in permeability to sugar was related to an effect of the 50 or 100 microunits/ml of insulin in the medium, not simply to the long incubation; this is evidenced by the finding that incubation of muscles for 5 h in the absence of insulin had little effect on permeability to 3- methylglucose. Furthermore, the progressive increase in permeability to sugar induced by prolonged incubation with 50 microunits/ml of insulin was mediated specifically by the glucose transporters, since 3-methylglucose uptake was more than 99% inhibited in the presence of 25 pm cytochalasin B (data not shown). Effect of Glucose Concentrution-t appeared that sugar transport was activated to a greater extent by 5 h of incubation with 50 microunits/ml of insulin when glucose concentration in the medium was 1 mm than when it was 8 mm (Table, Fig. 2). To further evaluate a possible role of glucose concentration in this phenomenon, muscles were also incubated for 1 and 5 h with 50 microunits/ml of insulin and either 20 or 36 mm glucose. Glucose concentration had no significant effect on the increase in the 3-methylglucose uptake rate induced by 1 h of incubation with 50 microunits/ml of insulin (Fig. 3). However, at the two higher glucose concentrations (20 and 36mM) there was no further activation of glucose transport between 1 and 5 h (Fig. 3). There was a negative correlation between glucose concentration in the medium (in the 1-20 mm range) during the 5-h incubation and the increase from 1 to 5 h in muscle permeability to 3-methylglucose (R = 0.99; p < 0.05). A high concentration of glucose not only prevented the progressive increase in permeability to sugar during prolonged exposure of muscles to 50 microunits/ml of insulin, but also resulted in a partial reversal of the increase once it had occurred. As shown in Fig. 4, about 80% of the total increase in muscle permeability to 3-methylglucose had occurred after 3 h of incubation with 50 microunits/ml of insulin and 1 mm glucose. When glucose concentration in the medium was increased to 36 mm after 3 h of incubation, a partial reversal of the increase in sugar transport activity occurred over the J GLUCOSECONCENTRATON (mm) FG. 3. Effect of glucose concentration on the activation of sugar transport by prolonged exposure to 50 microunitslml of insulin. Epitrochlearis muscles were incubated with 50 microunits/ml of insulin and either 1, 8, 20, or 36 mm glucose; one muscle of each pair was incubated for 1 h and the other for 5 h. Following the incubations, permeability to 3-methylgluocose was measured at 29 C as described under Experimental Procedures. Results are expressed as micromoles of 3-methylglucose taken up per ml of intracellular water in 10 min. Each point is the mean & S.E. for five to eight muscles. Asterisks indicate a significant difference from 1-h samples (*, p < 0.01; **, p < 0.001) NCUBATON TME (hours) FG. 4. Reversal by a high glucose concentration of the activation of sugar transport during prolonged exposure to insulin. Muscles were incubated for 1,3, or 5 h with 50 microunits/ml of insulin and 1 mm glucose (0). One muscle of each pair incubated for 3 h was transferred to medium containing 50 microunits/ml of insulin and 36 mm glucose and incubated for an additional 2 h (0). The dashed line indicates the decrease in permeability to 3-methylglucose that occurred in response to 2 h of exposure to 36 mm glucose. Results are expressed as micromoles of 3-methylglucose taken up per ml of intracellular water in 10 min. Each point represents the mean & S.E. for six muscles. *, significantly different from 3 h value, p < next 2 h (Fig. 4). These findings provide evidence that the progressive activation of sugar transport in muscles exposed for long periods to moderate insulin concentrations is strongly influenced by the amount of glucose entering the cells. nhibition of Protein Synthesis-The large increase in glucose transport activity in muscles incubated with 50 microunits/ml of insulin and 1 mm glucose occurs progressively over a period of hours. To evaluate the possibility that protein synthesis might beinvolved in this process, we incubated pairs of muscles with 50 microunits/ml of insulin and 1 mm glucose for 5 h with or without 20 pg/ml cycloheximide. This concentration of cycloheximide resulted in a 94% inhibition of [3H]leucine incorporation into protein in epitrochlearis muscles (1,218 uersus 21,529 cpm/g). Cycloheximide prevented -65% of the increase in permeability to 3-methylglucose seen between 1 and 5 h in the presence of 50 microunits/ ml of insulin, but had no effect on 3-methygluocose transport in muscles incubated for 5 h with 20,000 microunits/ml of insulin (Table 11). nterestingly, the rate of 3-methylglucose uptake attained in response to 5 h of incubation of muscles with 50 microunits/ml of insulin in the presence of cycloheximide was not significantly different from that seen in muscles incubated with 20,000 microunits/ml of insulin. Thus, 50 microunits/ml of insulin can, in the absence of protein synthesis, activate sugar transport to the same extent over 5 h as 20,000 microunits/ml does in 1 h. Furthermore, prolonged exposure to 50 microunits/ml insulin, in the presence of a very low glucose concentration, appears to cause an additional, protein synthesis-dependent increase in glucose transport activity. Kinetics of 3-Methylglucose Transport-A number of studies have provided evidence that insulin increases the V,,, of glucose transport without altering the K,,, (7, 11). t appeared of interest to determine whether or not the increase in permeability to 3-methylglucose that occurs between 1 and 5 h in muscles exposed to 50 microunits/ml of insulin and 1 mm glucose is also due to an increase in V,.. Muscles were incubated for either 1 or 5 h, rinsed, and then incubated with

4 16052 Prolonged Exposure of Muscle to nsulin TABLE 1 Effect of cycloheximide on the increase in 3-methylglucose transport activity caused by 5 h of exposure to 50 microunitslml of insulin and 1 mmgluose Epitrochlearis muscles were incubated with the indicated concentration of insulin and 1 mm glucose for 1 h in the absence of cycloheximide or for 5 h in the presence or absence of20 pg/ml cycloheximide. Permeability to 3-methylglucose was then measured as described under Experimental Procedures. Results are expressed as micromoles of 3-methylglucose taken up per ml of intracellular water in 10 min. Values are means & S.E. for the number of muscles given in parentheses. ncubation nsulin Cycloheximide 3-Methylglucose period h microunitslml pmol. ml. 10 min 1 50 No 0.81 * 0.08 (5) 5 50 No 2.16 & 0.09 (5) Yes & O.ll.b (8) 5 20,000 No 1.39 & 0.04 (5) 5 20, Yes k 0.04 (5) 5 h versus 1 h, p < h without cycloheximide versus 5 h with cycloheximide, p < FG. 5. Lineweaver-Burk plot of 3-methylglucose uptake after 1 and 5 h of exposure to 50 microunits/ml of insulin and 1 mm glucose. Muscles were incubated for 1 or 5 h with 50 microunits/ml of insulin and 1 mm glucose. Uptake (v) was then measured at different concentrations of 3-methylglucose (s) at 29 C for 10 min. Each point represents the average of four to six muscles. a range of 3-methylglucose concentrations. As shown by a Lineweaver-Burk plot (12) of the results (Fig. 5), there was a more than 2-fold increase in apparent Vmaxof sugar transport, between 1 and 5 h (7.7 uersus 20.0 gmol/ml/h), while the apparent K,,, for 3-methylglucose was unchanged by prolonged exposure to 50 microunits/ml of insulin and a low glucose concentration (5.3 uersus 5.9 mm). DSCUSSON Our results show that prolonged exposure of rat skeletal muscle to a moderate concentration of insulin causes a progressive increase in permeability to glucose over at least 5 h and that this increase is prevented by a high concentration of glucose. A surprising aspect of this phenomenon is that the magnitude of the increase in sugar transport rate attained after 5 h in response to 50 microunits/ml of insulin in the presence of 1 mm glucose was significantly greater than that caused by a concentration of insulin well above that usually required to produce a maximal effect. To our knowledge, this finding has not been reported previously, probably because studies of the effects of insulin on glucose transport have generally utilized relatively short exposure to insulin. With the treatment periods usually used, i.e min, 50 microunits/ml of insulin brings about an increase in muscle permeability to glucose that is % of that caused by a maximal insulin concentration (13, 14; Table, Fig. 2). The progressive increase in glucose transport activity that occurs in muscles exposed to 50 microunits/ml of insulin and 1 mm glucose has two components. The first appears to involve the same response that occurs on exposure of muscle to a maximal insulin concentration. The major difference is uptake that the activation of sugar transport is completed within 15 min of exposure to 20,000 microunits/ml of insulin, whereas the same increase in permeability to sugar takes hours to develop in the presence of 50 microunits/ml of insulin and 1 mm glucose. This first component of the activation of glucose transport by 50 microunits/ml of insulin does not require protein synthesis. This is in keeping with the results of previous studies, in which relatively short exposures to insulin were used, showing that inhibition of protein synthesis does not prevent activation of glucose transport by insulin (15-17). The second component of the increase in glucose transport activity caused by prolonged exposure of muscle to 50 microunits/ml of insulin and l mm glucose is additive to the first component, resulting in a significantly greater increase in permeability of muscle to sugar after 5 h of exposure to 50 microunits/ml of insulin than to 20,000 microunits/ml. This second process is blocked by inhibition of protein synthesis. Time- and protein synthesis-dependent increases in glucose transport, that may be related to this phenomenon, have been observed in response to other stimuli (18-20). These include (a) glucose deprivation, which causes a large, protein synthesis-dependent increase in the V, of glucose transport in fibroblasts (18) and 3T3 fat cells (19) in culture, and (b) treatment of L6 myoblasts in culture with calf serum (20). Treatment of adipocytes (21-24) and skeletal muscle (25, 26) with insulin results in translocation of glucose transporters from an intracellular storage site into the plasma membrane by a protein synthesis-independent mechanism (17). t has been proposed that this mechanism is responsible for the activation of sugar transport by insulin (21-26). The finding that the V, of sugar transport increased in muscle during the period between 1 and 5 h of exposure to 50 microunits/ ml of insulin and 1 mm glucose (Fig. 5) is compatible with an increase in the number of transporters in the plasma membrane. Thus, viewed in the context of the transporter translocation hypothesis, the first component of the increase in glucose transport activity brought about by 50 microunits/ml of insulin could be due to recruitment over 5 h of the same pool of glucose transporters that is translocated into the plasma membrane in 15 min by 20,000 microunits/ml of insulin. On the other hand, the protein synthesis-dependent second component of the increase in permeability to sugar induced by 50 microunits/ml of insulin could be due to synthesis of new glucose transporters. t is puzzling that 5 h of exposure to 50 microunits/ml of insulin and 1 mm glucose resulted in an additional, protein synthesis-dependent increase in permeability ofmuscle to glucose, while 20,000 microunits/ml of insulin and 1 mm glucose for the same period caused no further activation of glucose transport after the initial rapid (15 min) response. As a consequence of this difference, prolonged exposure of muscle to a moderate concentration of insulin resulted in a greater increase in permeability to sugar than occurred in response

5 Prolonged Exposure of Muscle to nsulin to a very high insulin concentration. This finding suggests 2. DeFronzo, R. A., Tobin, J. D., and Andres, R. (1979) Am. J. that, in addition to activating glucose transport, a very high Physiol. 237, E214-E James, D. E., Burleigh, K. M., and Kraegen, E. W. (1985) Diabetes concentration of insulin may have other effects that limit the 34, magnitude of the increase in permeability of muscle to glucose. 4. Nesher, R., Karl,. E., Kaiser, K. E., and Kipnis, D. M. (1980) The present results provide evidence that glucose can, Am. J. Physiol. 239, E454-E460 under some conditions, play a role in regulating its own 5. Krebs, H. A., and Henseleit, K. (1932) Hoppe-Seyler s 2. Physwl. transport, High concentrations of glucose blocked, while a Chem. 210, low concentration increased, the further activation of sugar 6. Goldberg, A. L. (1968) J. Cell Biol. 36, Narahara, H. T., and Ozand, P. (1963) J. Biol. Chem. 238, 40- transport that occurred during the period between 60 min and 49 5 h of exposure of muscle to 50 microunits/ml of insulin (Fig. 8. Holloszy, J. O., and Narahara, H. T. (1965) J. Biol. Chem. 240, 3). Furthermore, addition of a high concentration of glucose partially reversed the increase in permeability to sugar in- 9. Garthwaite, S. M., and Holloszy, J. 0. (1982) J. Biol. Chem. 257, duced by prolonged exposure to 50 microunits/ml of insulin Wallberg-Henriksson, H., and Holloszy, J. 0. (1985) Am. and 1 mm glucose (Fig. 4). Other evidence for a regulatory J. Physiol. 249, C233-C237 effect of glucose on its own transport has come from studies 11. Klip, A. (1982) Life Sei. 31, showing that glucose deprivation of fibroblasts (18) and 3T3 12. Lineweaver, H., and Burk, D. (1934) J. Am. Chem. SOC. 56,658- adipocytes (19) in culture results in a large increase in perme- 667 ability to sugar. Furthermore, glucose appears to be essential 13. vy, J. L., and Holloszy, J. 0. (1981) Am. J. Physiol. 241, C200- for inactivation of the stimulatory effect of insulin on glucose C Nesher, R., Karl,. E., and Kipnis, D. M. (1985) Am. J. Physiol. transport in adipocytes (27). Similarly, indirect evidence sug- 249, C226-C232 gests that reversal of the activation of glucose transport that 15. Fain, J. N. (1964) Biochim. Biophys. Acta 84, occurs in response to exercise is accelerated by rapid glucose 16. Yu, K. T., and Gould, M. K. (1979) Biochem. Biophys. Res. uptake and slowed when glucose availability and uptake are Commun. 87,9-16 reduced (28). 17. Kono, T., Suzuki, K., Dansey, L. E., Robinson, F. W., and Blevins, Taken together, these findings raise the possibility that T. L. (1981) J. Biol. Chem. 256, Kletzien, R. F., and Perdue, J. F. (1975) J. Biol. Chern. 250,593- glucose may be involved in the internalization or inactivation 600 of glucose transporters. Hypothetically, glucose transporters 19. Van Putten, J. P. M., and Krans, H. M. J. (1985) J. Biol. Chem. may be more susceptible to intracellular sequestration, inac- 260, tivation, or degradation when loaded with glucose than when 20. Klip, A., Li, G., and Logan, W. J. (1984) Am. J. Physiol. 247, empty. t seems possible that when large amounts of glucose E291-E Cushman, S. W., and Wardzala, L. J. (1980) enter the cell, the rate of transporter cycling back to the J. Biol. Chem. 255, intracellular storage site (or of their degradation) is acceler- 22. Suzuki, K., and Kono, T. (1980) Proc. Natl. Acud. Sci. 77,2542- ated, resulting in a lower steady state concentration of trans porters in the plasma membrane. Regardless of its mecha- 23. Karnielli, E. K., Zarnowski, M. J., Hissin, P. J., Simpson,. A., nism, the down regulation of glucose transport activity in Salans, L. B., and Cushman, S. W. (1981) J. Bid. Chem. 256, the presence of a high glucose concentration could serve as a Kono, T., Robinson, F. W., Blevins, T. L., and Ezaki, 0. (1982) protective mechanism against excessive glycogen storage in J. Bwl. Chem. 257, muscle. t could also play a role in the decreased responsive- 25. Wardzala, L. J., and Jeanrenaud, B. (1981) J. Biol. Chem. 256, ness to insulin that develops in diabetes with severe hypergly cermia (10). 26. Wardzala, L. J., and Jeanrenaud, B. (1983) Biochim. Biophys. Acta 730, REFERENCES 27. Ciaraldi, T. P., and Olefsky, J. M. (1980) J. Bid. Chem. 255, Doberne, L., Greenfield, M. S., Schulz, B., and Reaven, G. (1981) 28. Young, J. C., Garthwaite, S. M., Bryan, J. E., Cartier, L.-J., and Diabetes 30, Holloszy, J. 0. (1983) Am. J. Physiol, 245, R684-R688