0013-7227/90/1273-1072$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 127, No. 3 Printed in U.S.A.
Glucagon Inhibits Insulin Activation of Glucose Transport in Rat Adipocytes Mainly through a Postbinding Process NOBUYUKI SATO, MINORU IRIE, HIROSHI KAJINUMA, AND KAZUO SUZUKI Division of Endocrinology and Metabolism, Institute for Adult Diseases, Asahi Life Foundation (N.S., H.K., K.S.), 1-9-14 Nishishinjuku, Shinjuku-ku, Tokyo 160; and the First Department of Internal Medicine, Toho University School of Medicine (N.S., M.I.), 6-11-1 Ohmorinishi, Ohta-ku, Tokyo 143, Japan
ABSTRACT. Incubation of rat adipocytes with 1 ^M glucagon plus adenosine deaminase (5 fig/m\) inhibited maximally insulinstimulated 3-O-methyl-D-glucose (MeGlc) transport by approximately 70%, concomitant with 30% and 55% decreases in insulin binding and cellular ATP, respectively. In contrast, under conditions where cellular ATP levels are well preserved (i.e. high albumin concentration in the medium), the inhibition of transport was reduced to about 30%, but that of insulin binding was not. Because depletion of the cellular ATP level by more than 60% by metabolic inhibitors induced 40% or more inhibition of insulin-stimulated MeGlc transport, the greater inhibition of the transport with the low albumin concentration appears to be
C
ATECHOLAMINES have long been known to aggravate the metabolic derangements of diabetes mellitus by both inhibiting insulin secretion and enhancing gluconeogenesis and glycogenolysis (for review, see Ref. 1). Studies in the past several years have also shown that the hormones exert their diabetogenic effects by counteracting insulin action in peripheral tissues (2-8). Thus, pretreatment of adipocytes with catecholamine resulted in inhibition of both insulin binding (5, 9) and insulin-stimulated glucose transport (2, 4-8) in such cells. The latter effect of the hormone is thought to be caused partly by inhibition of the tyrosine kinase activity of the insulin receptors, presumably through modulation of the phosphorylation state of the receptor by a cAMPdependent process (10-13). In addition, Simpson and coworkers (7, 14, 15) have recently proposed the view that catecholamines inhibit insulin-stimulated glucose transport mainly by directly modulating the intrinsic activity, rather than translocating (16, 17) the glucose transporters. Glucagon, another cAMP-stimulating hormone, has Received February 26, 1990. Address all correspondence and requests for reprints to: Kazuo Suzuki, M.D., Institute for Adult Diseases, Asahi Life Foundation, 19-14 Nishishinjuku, Shinjuku-ku, Tokyo 160, Japan.
caused in part by the secondary effect of ATP loss. The relationship between the amount of cell-bound insulin and hormonestimulated transport activity showed that glucagon does not modulate insulin action at the step of insulin binding to its receptors. Furthermore, glucagon suppressed insulin-stimulated MeGlc transport, mainly through an attenuation of the hormone-induced increase in maximum velocity. The data show that glucagon modulates the process of signal transduction of insulin action. However, the possibility that glucagon directly modulates the process of translocation or the intrinsic activity of the glucose transporters cannot be eliminated. (Endocrinology 127: 1072-1077, 1990)
also been shown to inhibit glucose transport activity in rat adipocytes (18-20). After determining insulin binding and insulin stimulation of the glucose transport activity in glucagon-treated rat adipocytes, Yamuchi et al. (19, 20) recently suggested that glucagon, unlike catecholamines, regulates insulin action by modulating the binding of insulin to its receptors. In this study we assessed the sites at which glucagon modulates insulin-stimulated glucose transport activity in isolated rat adipocytes.
Materials and Methods Chemicals Crystalline porcine insulin and bovine glucagon were purchased from Novo Laboratories (Copenhagen, Denmark). Labeled 3-O-methyl-D-[U-14C]glucose (CFB 141) and [125I]monoiodoinsulin (IM 166) were obtained from Amersham Corp. (Arlington Heights, IL). Labeled L-[1-3H]glucose (NET 456) was purchased from DuPont-New England Nuclear (Boston, MA). Crude bacterial collagenase was obtained from Worthington Biochemical Corp. (Freehold, NJ). 3-O-Methyl-D-glucose (MeGlc), L-glucose (L-G1C), phloretin, and adenosine deaminase (ADA) were obtained from Sigma Chemical Co. (St. Louis, MO). BSA (fraction V) was purchased from Armour (Kankakee, IL). 1072
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MODULATION OF GLUCOSE TRANSPORT BY GLUCAGON Animals and adipocyte preparation Male Wistar rats, weighing 180-220 g, were killed in the morning after an overnight (12-h) fast, and isolated adipocytes were prepared by the collagenase method (21) from epididymal adipose tissues. The buffer used to isolate cells was KrebsRinger-HEPES buffer (KRH), pH 7.4, at 37 C, supplemented with 20 mg BSA/ml and 2 mM glucose. Isolated cells were then washed four times with KRH buffer, supplemented with 20 mg BSA/ml and 2 mM pyruvate as an energy source. MeGlc transport assay The rate of MeGlc transport by adipocytes was determined by method B (22), which is a modification of the equilibrium exchange method of Whitesell and Abumrad (23). Briefly, four parts of a 50% (vol/vol) suspension of adipocytes were mixed with one part of albumin-free KRH buffer containing MeGlc at a concentration 3 times the final concentration desired, and the cells were stabilized by allowing them to stand for 30 min at 37 C. When needed, insulin, glucagon, and/or ADA, which decomposes adenosine released from the cells, were added in a small volume after this preliminary incubation, and the incubation was continued for an additional 15-30 min. Seventyfive microliters of the cell suspension were then pipetted onto 25 n\ albumin-free KRH buffer containing [14C]MeGlc (13.5 MCi/ml) plus unlabeled MeGlc at the desired final concentration and 1 mM L-[3H]Glc (17.6 /xCi/ml) that had been placed in a round bottom polystyrene test tube (10 x 45 mm) and warmed to 37 C. The reaction was continued for 3-15 sec while the tubes were rapidly shaken in a water bath at 37 C and was terminated by adding 200 ^1 albumin-free KRH buffer containing 1 mM phloretin and 0.1% dimethylsulfoxide. Because a high concentration of albumin substantially attenuates the inhibitory effect of phloretin on glucose transport, the concentration of phloretin was increased to 2 mM when a large amount of BSA (5%) was included in the medium. Immediately after phloretin was added, the cells were separated from the medium by the oil flotation method (24), and the radioactivity in the packed cells was counted, as described previously (22). The amount of carrier-mediated uptake of MeGlc was estimated by subtracting the nonmediated uptake of L-[3H]Glc from the total uptake of [14C]MeGle, and the transport rate was expressed as nanomoles of MeGlc per 106 cells/sec. The kinetic parameters were calculated from a Hanes plot, which was drawn by linear regression analysis. Insulin-binding activity Insulin-binding experiments were carried out under the same conditions as those used in the transport assay. Approximately 5.0 x 105 adipocytes that had been treated with glucagon and ADA, as described above, were incubated in 40% suspension (vol/vol) with 25 pM iodoinsulin and various concentrations of unlabeled insulin at 37 C in a total volume of 0.3 ml. Under these conditions, steady state binding of the label was obtained within 15-30 min of incubation. Fifteen minutes after the label was added, the cells were separated from the medium by the oil flotation method (24), and the radioactivity in the packed cells was counted in a 7-spectrometer. Nonspecific binding of the
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label, defined as the amount of cell-bound iodoinsulin in the presence of 16 nM unlabeled hormone, was subtracted from the total. Others To assay ATP, 50 ^1 cell suspension were placed in a boiling water bath for 5 min with 1 ml 20 mM glycine buffer (pH 7.8). The concentration of ATP in the extract was determined by the luciferin- luciferase method (25). The cell number was counted in an improved Neubauer counting chamber. Results were expressed as the mean ± SE. The statistical analysis was performed by Student's t test.
Results The effects of glucagon and ADA on basal and insulinstimulated MeGlc transport activities in adipocytes are shown in Table 1. In the basal cells, ADA (5 Mg/ml) suppressed the transport by 30%, while glucagon (1 JUM) enhanced the activity by 56%. In combination, these agents suppressed the transport by 40%. By contrast, in the presence of a maximal concentration (10 nM) of insulin, glucagon induced a 25% reduction in insulinstimulated transport activity. ADA alone had little effect, if any, on the insulin effect. In combination, however, the two agents caused as much as a 75% reduction in insulin-stimulated MeGlc transport activity. These data are consistent with those of other investigators using cAMP stimulators such as glucagon (18) and catecholamines (4, 7, 8). As has been shown for catecholamines (5, 7-9), glucagon in combination with ADA suppressed not only insulin-stimulated MeGlc transport, but also insulin binding and the cellular ATP level concentration deTABLE 1. Effects of glucagon and ADA on basal and insulin-stimulated MeGlc transport in rat adipocytes MeGlc transport (nmol/106 cells-sec) Additions
None Glucagon (1 MM) ADA (5 Mg/ml) Glucagon (1 MM) + ADA (5 Mg/ml)
„ . Basal 0.055 ± 0.003 0.086 ± 0.007 0.039 ± 0.0056 0.034 ± 0.0056
+Insulin .._ . (10 nM) 0.810 ± 0.611 ± 0.777 ± 0.277 ±
0.222 0.020° 0.011° 0.043°
After 30 min of equilibration in the presence of 5 mM MeGlc, the adipocytes were incubated with the indicated agents for 30 min at 37 C, then incubated further with or without insulin (10 nM) for an additional 15 min. The initial rate of MeGlc transport was determined as described in Materials and Methods. The reaction time was 15 and 3 sec for basal and insulin-stimulated cells, respectively. The final concentration of BSA in the medium was 16 mg/ml. The data show the mean ± SE (n = 4-6). ° P < 0.01 vs. control. 6 P < 0.05 us. control.
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MODULATION OF GLUCOSE TRANSPORT BY GLUCAGON
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pendently over a range of 1 X 10~n to 1 X 10 6 M (Fig. 1A). Maximal inhibition by 1 ixM glucagon of transport, insulin binding, and ATP level was 65%, 20%, and 55%, respectively, of the control value. As insulin activates glucose transport through a process requiring energy (26, 27), it is conceivable that the inhibition of transport in glucagon-treated adipocytes is due at least in part to depletion of cellular ATP. Therefore, we next determined experimental conditions under which glucagon-induced depletion of ATP could be minimized. As shown in Table 2, with the standard concentration of BSA (1.6%) in the medium, an increase in concentration of either pyruvate or glucose (substrates for ATP generation) from 2 to 10 mM was unsuccessful in preventing the loss of ATP, but under B. 5.0% BSA
A. 1.6% BSA
• V* i
o
0.6
c ." 0.4
0
-11 -10
-0
-8
-7
-V--11
-6
0
-10
-9
-8
-7
-6
S 0.6
E 0.4
E
0.4
o-v-o0
-11 -10
-9
-8
-7
-6
0
-11 -10
-9
-8
-7
g 2.0 IV
o 0
-11 -10
-9
-8
-7
Log (Glucagon, M )
-6
0
0 -11 -10 -9 -8 -7
-6
Log (Glucagon, M)
FIG. 1. Effect of glucagon concentration on the inhibition of MeGlc transport, insulin binding, and cellular ATP in rat adipocytes under low (A) and high (B) albumin conditions. After 30 min of equilibrium in the presence of 5 mM MeGlc, the adipocytes were incubated with the indicated concentrations of glucagon in the presence of ADA (5 ng/ ml) for 30 min at 37 C, then incubated with either 1 nM (O) or 10 nM (•) insulin (or with 25 pM iodoinsulin for the insulin binding assay) for an additional 15 min. The final concentration of BSA in the medium was 16 (A) or 50 (B) mg/ml. MeGlc transport, insulin binding, and cellular ATP were determined as described in Materials and Methods. The data show the mean ± SE (n = 4-6).
Endo«1990 Vol 127 • No 3
TABLE 2. Effects of glucagon on cellular ATP and insulin-stimulated MeGlc transport in rat adipocytes
f"1
J»J_'
Glucagon effect (% inhibition)
v_/ o noix l o n s
A) BSA (16 mg/ml) Pyruvate 2 mM 5 mM 10 mM Glucose 2 mM 5 mM 10 mM
Pyruvate (5 mM) + glucose (5 mM) B) BSA (50 mg/ml) Pyruvate (2 mM)
ATP
MeGlc transport
60.1 40.3 23.4
66.6 41.0 38.6
83.1 67.3 35.4 24.9
88.3 65.9 52.7 37.1
3.3
28.6
After 30 min of equilibration in the presence of 5 mM MeGlc, the adipocytes were incubated with glucagon (1 nM) plus ADA (5 jig/ml) in the medium supplemented with a final concentration of either 16 or 50 mg/ml BSA and the indicated concentration of pyruvate, glucose, or both for 30 min at 37 C. The cells were then incubated further in the presence of 10 nM insulin for 15 min, and cellular ATP and MeGlc transport activity were determined as described in Materials and Methods. The data show the means of triplicate determinations of a typical experiment.
the conditions of high BSA concentration (5%), 2 mM pyruvate effectively blocked the loss of ATP. This table also shows that the glucagon-induced inhibition of MeGlc transport is only 28.6% when ATP is maintained at the control level, and the inhibition tends to increase roughly in parallel with the ATP loss. To further characterize the relationship between cellular ATP and the transport activity, we next determined insulin-stimulated MeGlc transport in adipocytes whose ATP levels had been suppressed to various degrees by the metabolic inhibitors KCN and dinitrophenol. The results show that the insulin-stimulated transport activity is inhibited by 40-50% when the cellular ATP level is reduced to approximately 60% of that in control cells, and the activity is nearly abolished when ATP is suppressed by more than 90% (Fig. 2). In addition, in parallel experiments using KCN- or dinitrophenol-treated cells, insulin binding was inhibited by 20% and 50% when the ATP content was suppressed, respectively, to 50% and 5% of the control value. Figure IB shows the reassessment of the effects of glucagon on MeGlc transport, insulin binding, and cellular ATP in the presence of 5% BSA and 2 mM pyruvate. Under these conditions, a physiological concentration of glucagon (10 pM) had little effect on the transport activity stimulated by either 1 or 10 nM insulin. However, 1 /iM glucagon inhibited the activities by 50% and 30%, respectively, indicating that a high concentration of glucagon suppresses not only the response to a maximal
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MODULATION OF GLUCOSE TRANSPORT BY GLUCAGON 100
1075
0.8
eel
'sec
1.0
take, nmol/
0.6
0.4
Q
o O 0.2 (U
20
40
60
80
100
Cellular ATP, % Inhibition FIG. 2. Relationship between cellular ATP and insulin-stimulated MeGlc transport in rat adipocytes. After 30 min of equilibrium in the presence of 5 mM MeGlc, the adipocytes were incubated with various concentrations of KCN (O) or dinitrophenol (A) for 15 min at 37 C. The concentration of each agent was 0.2, 0.3, 0.4, 0.6, or 1.0 mM, from left to right. The cells were incubated further with 10 nM insulin for 15 min, and MeGlc transport and cellular ATP were then determined as described in Materials and Methods. The final concentration of BSA in the medium was 50 mg/ml.
dose of insulin, but also the sensitivity of adipocytes to a submaximal dose of the hormone. Glucagon at this concentration inhibited insulin binding by 35% and 20% when the ligand concentration was 1 and 10 nM, respectively. Because binding of a hormone to its receptor is a crucial step for the subsequent hormone action, we next determined whether the glucagon-induced inhibition of insulin binding could be due to an unresponsiveness of adipocytes to insulin. Figure 3 shows the relationship between the amount of cell-bound insulin and the hormone-stimulated MeGlc transport, which were calculated from the data in Fig. IB. Obviously, the ratio of the insulin effect to the hormone binding was much lower in glucagon-treated cells, showing that glucagon suppresses insulin action mostly through a process(es) distal to the step of insulin binding to its receptors. Table 3 shows the effects of glucagon on the kinetic parameters of MeGlc transport. Consistent with the data previously reported from this laboratory (22), the maximal concentration of insulin reduced the Km to about half of that in basal cells, concomitant with a 9.7-fold increase in the maximum velocity (Vmax). Pretreatment of adipocytes with 1 JIM glucagon plus ADA significantly attenuated the insulin-induced increase in the Vmax (P < 0.05). In contrast, there was no significant inhibition of the effect of insulin on the Km in glucagon-treated cells.
0.1
0.3
0.2
.6
0.4
0.5
Insulin Bound, pmol/10 cells FIG. 3. Relationship between the amount of cell-bound insulin and insulin-stimulated MeGlc transport in rat adipocytes treated with (•) or without (O) glucagon (1 fiM) plus ADA (5 jig/ml). The data are calculated from those in Fig. IB and are presented as the mean ± SE. TABLE 3. Kinetic parameters of MeGlc transport in rat adipocytes Conditions Basal Insulin Glucagon (1 ^M) -t insulin (10 nM)
Vmax
Km (mM)
(nmol/106 cells • sec)
15.9 ± 2.8 7.2 ± 1.0
0.28 ± 0.12 2.72 ± 0.30
9.2 ± 1.6
1.75 ± 0.09
After 30 min of equilibration at 37 C in the presence of 5, 10, 15, or 20 mM MeGlc, the adipocytes were incubated with or without glucagon (1 fiM) for 30 min, then incubated further in the presence or absence of insulin (10 nM) for 15 min. The initial rate of transport at each MeGlc concentration was determined, and the kinetic parameters were estimated by drawing a Hanes plot. ADA (5 fig/ml) was included in the medium throughout the experiment. The data show the mean ± SE (n = 6).
Discussion This study confirmed the reports that glucagon partially inhibits insulin stimulation of glucose transport in rat adipocytes (18-20), and that the effect of the hormone is greatly enhanced when endogenous adenosine, which suppresses adenylate cyclase activity, is degraded by ADA (18). We found also that the magnitude of inhibition varies widely with the experimental conditions. Thus, under conditions where the BSA concentration in the medium is low (1.6%), the maximal stimulation of MeGlc transport by 10 nM insulin was suppressed by 6575% in adipocytes pretreated with 1 fiM glucagon plus ADA (5 /xg/ml); the value is essentially the same as those reported by Green (18) and Yamauchi and Hashizume (19). However, the inhibition was only 30% when the
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MODULATION OF GLUCOSE TRANSPORT BY GLUCAGON
concentration of BSA was high (5%). The wide disparity in the data appears to be due to the difference in the amount of cellular ATP, which is essential for the activation of glucose transport by insulin. Glucagon plus ADA suppressed cellular ATP to about 40-50% of the control level when the BSA concentration was low, but those agents had little effect on cellular ATP when the BSA concentration was high (5%). Presumably, albumin protects against the loss of ATP by binding and thereby decreasing FFA, which inhibit ATP synthesis through uncoupling of oxidative phosphorylation (28). The relationship between the cellular ATP level and the MeGlc transport activity shown in Fig. 2 clearly indicates that the marked inhibition of transport by glucagon plus ADA in the presence of a low concentration of BSA can be explained solely by the secondary effect of a depletion of cellular ATP. Although the cellular ATP content is not reported in the studies of Green (18) and Yamauchi and Hashizume (19), it would be greatly suppressed under the experimental conditions that these workers employed {i.e. glucose-free 1.0% albumin medium). More recently, Yamauchi et al. (20) reported that adipocytes that had been treated with glucagon and then washed free of the hormone showed unimpaired responses of both glucose oxidation and lipogenesis to insulin stimulation. They suggested that glucagon selectively inhibits insulin activation of glucose transport. However, as the glucagon-treated adipocytes in the latter two assays were incubated in medium supplemented with glucose, an apparent lack of effect of glucagon may have been due to the recovery of cellular ATP. Insulin binding was 30% less in adipocytes pretreated with glucagon plus ADA. In contrast to the transport activity, the magnitude of inhibition was not affected by the experimental conditions employed in this study. Our data disagree quantitatively with those of Yamauchi and Hashizume (19), who reported 90% inhibition by 1 /xM glucagon. The greater inhibition observed by these workers could be explained by a severe loss of cellular ATP, since we found that suppression of cellular ATP by 90% or more by metabolic inhibition resulted in a substantial loss of insulin binding. As reported by Yamauchi and Hashizume (19), we also found that a decrease in insulin binding in ATP-depleted cells was due to an apparent loss of the high affinity insulin-binding site (unpublished observation). A slight inhibition of insulin binding by glucagon appears to be responsible for a decrease in the sensitivity of adipocytes to insulin. The relationship between the amount of cell-bound insulin and insulin stimulation of the glucose transport activity suggests that glucagon modulates the insulinsignaling mechanism distal to the step of insulin binding to its receptors. However, the possibility that glucagon
Endo • 1990 Vol 127 • No 3
directly inhibits the intrinsic activity, rather than the process of insulin-induced translocation of the glucose transporters, cannot be eliminated. Such a view has been proposed by Simpson and co-workers (7, 14, 15) based on the following observations: 1) catecholamines in combination with ADA inhibit insulin-stimulated glucose transport by decreasing the transport Vmax without any changes in Km; and 2) a decrease in Vmax is not accompanied by any change in the number of transporters in the plasma membranes, as assessed by either the number of cytochalasin-B-binding sites or the immunoreactivity of the trnasporters in Western blots. Our kinetic data on MeGlc transport showing that glucagon inhibited the transport mainly through the attenuation of an insulin-induced increase in Vmax appear to be consistent with the view of Simpson et al. However, our data might be interpreted otherwise. Thus, we found that the maximal concentration of insulin significantly decreased the Km from that in basal cells. Although not generally accepted at this time (29, 30), the Km-lowering effect of insulin has been reported from this (22) and other laboratories (23, 31, 32). Furthermore, Suzuki (22) reported that the Km tends to be lower as the Vmax increases. A possible explanation for Suzuki's kinetic data is that there are at least two types of glucose transporters in rat adiopcytes, high Km and low Km, and that insulin causes translocation of the mobile low Km transporters from intracellular storage sites to the plasma membranes, where the stationary high Km transporters reside (22). Indeed, Zorzano and co-workers (33) recently demonstrated that rat adipocytes have two immunologically distinct glucose transporters, i.e. erythrocyte (GLUT 1) and muscle/rat (GLUT 4) types, and that the former is distributed on both plasma membranes and low density microsomes, while the latter is mostly found on low density microsomes in the basal state and is preferentially translocatable by insulin. In the light of these findings, it can be noted from our kinetic data that glucagon appears to slightly attenuate the insulin-induced decrease in Km (from 7.2 ± 1.0 to 9.2 ± 1 . 6 HIM), concomitant with an attenuation of an increase in Vmax. This might be explained, although this is highly speculative, by postulating that glucagon modulates the insulin-signaling mechanism to elicit translocation or the process of translocation per se, rather than the intrinsic activity of glucose transporters. Further studies are needed to determine the mechanism by which glucagon modulates insulin-stimulated glucose transport in rat adipocytes.
Acknowledgment We are grateful to Junko Ohno for her excellent technical and secretarial assistance.
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MODULATION OF GLUCOSE TRANSPORT BY GLUCAGON
References 1. Gerich JE, Rizza RA, Verdonk CA, Miles JM, Haymond MW 1981 Role of counterregulatory hormones in diabetes mellitus. In: Martin JM, Ehrkich RM, Holland FJ (eds) Etiology and Pathogenesis of Insulin-Dependent Diabetes Mellitus. Raven Press, New York, pl25 2. Taylor WM, Mak ML, Halperin ML 1976 Effect of 3',5'-cyclic AMP on glucose transport in rat adipocytes. Proc Natl Acad Sci USA 73:4359 3. Chiasson J-L, Shikama H, Chu TW, Exton JH 1981 Inhibitory effect of epinephrine on insulin-stimulated glucose uptake by skeletal muscle. J Clin Invest 68:706 4. Green A 1983 Catecholamines inhibit insulin-stimulated glucose transport in adipocytes, in the presence of adenosine deaminase. FEBS Lett 152:261 5. Pessin JE, Gitomer W, Oka Y, Oppenheimer CL, Czech MP 1983 /9-Adrenergic regulation of insulin and epidermal growth factor receptors in rat adipocytes. J Biol Chem 258:7386 6. Kashiwagi A, Huecksteadt P, Foley JE 1983 The regulation of glucose transport by cAMP stimulators via three different mechanisms in rat and human adipocytes. J Biol Chem 258:13685 7. Smith U, Kuroda M, Simpson IA 1985 Counter-regulation of insulin-stimulated glucose transport by catecholamines in the isolated rat adipose cell. J Biol Chem 259:8758 8. Gliemann J, Bowes SB, Larsen TR, Rees WD 1985 The effect of catecholamines and adenosine deaminase on glucose transport system in rat adiopocytes. Biochim Biophys Acta 845:373 9. Lonnroth P, Smith U 1983 /3-Adrenergic dependent down regulation of insulin binding in rat adiopocytes. Biochem Biophys Res Commun 112:972 10. Haring H, Kirsch D, Obermaier B, Ermel B, Machicao F 1986 Decreased tyrosine kinase activity of insulin receptor isolated from rat adipocytes rendered insulin-resistant by catecholamine treatment in vitro. Biochem J 234:59 11. Stadtmauer L, Rosen O 1986 Increasing the cAMP content of IM9 cells alters the phosphorylation state and protein kinase activity of the insulin receptor. J Biol Chem 261:3402 12. Roth RA, Beaudoin J 1987 Phosphorylation of purified insulin receptor kinase by cAMP kinase. Diabetes 36:123 13. Obermaier B, Ermel B, Kirsch D, Mushack J, Rattenhuber E, Biemer E, Machicao F, Haring HU 1987 Catecholamines and tumor promoting phorbol esters inhibit insulin receptor kinase and induce insulin resistance in isolated human adipocytes. Diabetologia 30:93 14. Kuroda M, Honner RC, Cushman SW, Londos C, Simpson IA 1987 Regulation of insulin-stimulated glucose transport in the isolated rat adipocyte. cAMP-independent effects of lipolytic and antilipolytic agents. J Biol Chem 262:245 15. Joost HG, Weber TM, Cushman SW, Simpson IA 1987 Activity and phosphorylation state of glucose transporters in plasma membranes from insulin-, isoproterenol-, and phorbol ester-treated rat adipose cells. J Biol Chem 262:11261 16. Suzuki K, Kono T 1980 Evidence that insulin causes translocation of glucose transport activity to the plasma membrane from an intracellular storage site. Proc Natl Acad Sci USA 77:2542
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17. Cushman SW, Wardzala LJ 1980 Potential mechanism of insulin action on glucose transport in the isolated rat adipocyte cell: apparent translocation of intracellular transport system to the plasma membranes. J Biol Chem 255:4758 18. Green A 1983 Glucagon inhibition of insulin-stimulated 2-deoxyglucose uptake by rat adipocytes in the presence of adenosine deaminase. Biochem J 212:189 19. Yamauchi K, Hashizume K 1986 Glucagon alters insulin binding to isolated rat epididymal adipocytes: possible role of adenosine 3',5'-monophosphate in modification of insulin action. Endocrinology 119:218 20. Yamauchi K, Hashizume K, Miyamoto T, Otsuka H, Ichikawa K, Nishii Y, Yamada T 1988 Selective alterations of insulin action by glucagon in isolated rat epididymal adipocytes. Endocrinology 123:2800 21. Rodbell M 1961 Metabolism of isolated fat cells. I. Effects of hormones on glucose metabolism and lipolysis. J Biol Chem 239:375 22. Suzuki K 1988 Reassessment of the translocation hypothesis by kinetic studies on hexose transport in isolated rat adipocytes. J Biol Chem 263:12247 23. Whitesell RR, Abumrad NA 1985 Increased affinity predominates in insulin stimulation of glucose transport in the adipocyte. J Biol Chem 260:2894 24. Gliemann J, Osterlind K, Vinten J, Gammeltoft S 1972 A procedure for measurement of distribution spaces in isolated fat cells. Biochim Biophys Acta 286:1 25. McElroy WD 1963 Crystalline firefly luciferase. In: Colowick SI, Kaplan NO (eds) Methods in Enzymology. Academic Press, New York and London, vol 6:445 26. Chandramouli V, Milligan M, Carter RJ 1977 Insulin stimulation of glucose transport in adipose tissues. Biochemistry 16:1151 27. Kono T, Robinson FW, Sarver JA, Vega FV, Pointer RH 1977 Actions of insulin in fat cells. Effects of low temperature, uncouplers of oxidative phosphorylation, and respiratory inhibitors. J Biol Chem 252:2226 28. Angel A, Desai K, Halperin ML 1971 Free fatty acid and ATP levels in adipocytes during lipolysis. Metabolism 20:87 29. Martz A, Mookerjee BD, Jung CY 1986 Insulin and phorbol ester affect the maximum velocity rather than the half-saturation constant of 3-O-methylglucose transport in rat adipocytes. J Biol Chem 261:13606 30. Joost HG, Weber TM, Cushman SW 1988 Qualitative and quantitative comparison of glucose transport activity and glucose transporter concentration in plasma membranes from basal and insulinstimulated rat adipose cells. Biochem J 249:155 31. Toyoda N, Flanagan JE, Kono T 1987 Reassessment of insulin effects on the Vmax and Km values of hexose transport in isolated rat epididymal adipocytes. J Biol Chem 262:2737 32. Okuno Y, Gliemann J 1987 Enhancement of glucose transport by insulin at 37° C in rat adipocytes is accounted for by increased Vmax. Diabetologia 30:426 33. Zorzano A, Wilkinsin W, Kotliar N, Thoidis G, Wadzinkski BE, Ruoho, AE, Pilch PF 1989 Insulin-regulated glucose uptake in rat adipocytes is mediated by two transporter isoforms present in at least two vesicle populations. J Biol Chem 264:12358
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Vol. 127, No. 3 Printed in U.S.A.
Glucagon Inhibits Insulin Activation of Glucose Transport in Rat Adipocytes Mainly through a Postbinding Process NOBUYUKI SATO, MINORU IRIE, HIROSHI KAJINUMA, AND KAZUO SUZUKI Division of Endocrinology and Metabolism, Institute for Adult Diseases, Asahi Life Foundation (N.S., H.K., K.S.), 1-9-14 Nishishinjuku, Shinjuku-ku, Tokyo 160; and the First Department of Internal Medicine, Toho University School of Medicine (N.S., M.I.), 6-11-1 Ohmorinishi, Ohta-ku, Tokyo 143, Japan
ABSTRACT. Incubation of rat adipocytes with 1 ^M glucagon plus adenosine deaminase (5 fig/m\) inhibited maximally insulinstimulated 3-O-methyl-D-glucose (MeGlc) transport by approximately 70%, concomitant with 30% and 55% decreases in insulin binding and cellular ATP, respectively. In contrast, under conditions where cellular ATP levels are well preserved (i.e. high albumin concentration in the medium), the inhibition of transport was reduced to about 30%, but that of insulin binding was not. Because depletion of the cellular ATP level by more than 60% by metabolic inhibitors induced 40% or more inhibition of insulin-stimulated MeGlc transport, the greater inhibition of the transport with the low albumin concentration appears to be
C
ATECHOLAMINES have long been known to aggravate the metabolic derangements of diabetes mellitus by both inhibiting insulin secretion and enhancing gluconeogenesis and glycogenolysis (for review, see Ref. 1). Studies in the past several years have also shown that the hormones exert their diabetogenic effects by counteracting insulin action in peripheral tissues (2-8). Thus, pretreatment of adipocytes with catecholamine resulted in inhibition of both insulin binding (5, 9) and insulin-stimulated glucose transport (2, 4-8) in such cells. The latter effect of the hormone is thought to be caused partly by inhibition of the tyrosine kinase activity of the insulin receptors, presumably through modulation of the phosphorylation state of the receptor by a cAMPdependent process (10-13). In addition, Simpson and coworkers (7, 14, 15) have recently proposed the view that catecholamines inhibit insulin-stimulated glucose transport mainly by directly modulating the intrinsic activity, rather than translocating (16, 17) the glucose transporters. Glucagon, another cAMP-stimulating hormone, has Received February 26, 1990. Address all correspondence and requests for reprints to: Kazuo Suzuki, M.D., Institute for Adult Diseases, Asahi Life Foundation, 19-14 Nishishinjuku, Shinjuku-ku, Tokyo 160, Japan.
caused in part by the secondary effect of ATP loss. The relationship between the amount of cell-bound insulin and hormonestimulated transport activity showed that glucagon does not modulate insulin action at the step of insulin binding to its receptors. Furthermore, glucagon suppressed insulin-stimulated MeGlc transport, mainly through an attenuation of the hormone-induced increase in maximum velocity. The data show that glucagon modulates the process of signal transduction of insulin action. However, the possibility that glucagon directly modulates the process of translocation or the intrinsic activity of the glucose transporters cannot be eliminated. (Endocrinology 127: 1072-1077, 1990)
also been shown to inhibit glucose transport activity in rat adipocytes (18-20). After determining insulin binding and insulin stimulation of the glucose transport activity in glucagon-treated rat adipocytes, Yamuchi et al. (19, 20) recently suggested that glucagon, unlike catecholamines, regulates insulin action by modulating the binding of insulin to its receptors. In this study we assessed the sites at which glucagon modulates insulin-stimulated glucose transport activity in isolated rat adipocytes.
Materials and Methods Chemicals Crystalline porcine insulin and bovine glucagon were purchased from Novo Laboratories (Copenhagen, Denmark). Labeled 3-O-methyl-D-[U-14C]glucose (CFB 141) and [125I]monoiodoinsulin (IM 166) were obtained from Amersham Corp. (Arlington Heights, IL). Labeled L-[1-3H]glucose (NET 456) was purchased from DuPont-New England Nuclear (Boston, MA). Crude bacterial collagenase was obtained from Worthington Biochemical Corp. (Freehold, NJ). 3-O-Methyl-D-glucose (MeGlc), L-glucose (L-G1C), phloretin, and adenosine deaminase (ADA) were obtained from Sigma Chemical Co. (St. Louis, MO). BSA (fraction V) was purchased from Armour (Kankakee, IL). 1072
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MODULATION OF GLUCOSE TRANSPORT BY GLUCAGON Animals and adipocyte preparation Male Wistar rats, weighing 180-220 g, were killed in the morning after an overnight (12-h) fast, and isolated adipocytes were prepared by the collagenase method (21) from epididymal adipose tissues. The buffer used to isolate cells was KrebsRinger-HEPES buffer (KRH), pH 7.4, at 37 C, supplemented with 20 mg BSA/ml and 2 mM glucose. Isolated cells were then washed four times with KRH buffer, supplemented with 20 mg BSA/ml and 2 mM pyruvate as an energy source. MeGlc transport assay The rate of MeGlc transport by adipocytes was determined by method B (22), which is a modification of the equilibrium exchange method of Whitesell and Abumrad (23). Briefly, four parts of a 50% (vol/vol) suspension of adipocytes were mixed with one part of albumin-free KRH buffer containing MeGlc at a concentration 3 times the final concentration desired, and the cells were stabilized by allowing them to stand for 30 min at 37 C. When needed, insulin, glucagon, and/or ADA, which decomposes adenosine released from the cells, were added in a small volume after this preliminary incubation, and the incubation was continued for an additional 15-30 min. Seventyfive microliters of the cell suspension were then pipetted onto 25 n\ albumin-free KRH buffer containing [14C]MeGlc (13.5 MCi/ml) plus unlabeled MeGlc at the desired final concentration and 1 mM L-[3H]Glc (17.6 /xCi/ml) that had been placed in a round bottom polystyrene test tube (10 x 45 mm) and warmed to 37 C. The reaction was continued for 3-15 sec while the tubes were rapidly shaken in a water bath at 37 C and was terminated by adding 200 ^1 albumin-free KRH buffer containing 1 mM phloretin and 0.1% dimethylsulfoxide. Because a high concentration of albumin substantially attenuates the inhibitory effect of phloretin on glucose transport, the concentration of phloretin was increased to 2 mM when a large amount of BSA (5%) was included in the medium. Immediately after phloretin was added, the cells were separated from the medium by the oil flotation method (24), and the radioactivity in the packed cells was counted, as described previously (22). The amount of carrier-mediated uptake of MeGlc was estimated by subtracting the nonmediated uptake of L-[3H]Glc from the total uptake of [14C]MeGle, and the transport rate was expressed as nanomoles of MeGlc per 106 cells/sec. The kinetic parameters were calculated from a Hanes plot, which was drawn by linear regression analysis. Insulin-binding activity Insulin-binding experiments were carried out under the same conditions as those used in the transport assay. Approximately 5.0 x 105 adipocytes that had been treated with glucagon and ADA, as described above, were incubated in 40% suspension (vol/vol) with 25 pM iodoinsulin and various concentrations of unlabeled insulin at 37 C in a total volume of 0.3 ml. Under these conditions, steady state binding of the label was obtained within 15-30 min of incubation. Fifteen minutes after the label was added, the cells were separated from the medium by the oil flotation method (24), and the radioactivity in the packed cells was counted in a 7-spectrometer. Nonspecific binding of the
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label, defined as the amount of cell-bound iodoinsulin in the presence of 16 nM unlabeled hormone, was subtracted from the total. Others To assay ATP, 50 ^1 cell suspension were placed in a boiling water bath for 5 min with 1 ml 20 mM glycine buffer (pH 7.8). The concentration of ATP in the extract was determined by the luciferin- luciferase method (25). The cell number was counted in an improved Neubauer counting chamber. Results were expressed as the mean ± SE. The statistical analysis was performed by Student's t test.
Results The effects of glucagon and ADA on basal and insulinstimulated MeGlc transport activities in adipocytes are shown in Table 1. In the basal cells, ADA (5 Mg/ml) suppressed the transport by 30%, while glucagon (1 JUM) enhanced the activity by 56%. In combination, these agents suppressed the transport by 40%. By contrast, in the presence of a maximal concentration (10 nM) of insulin, glucagon induced a 25% reduction in insulinstimulated transport activity. ADA alone had little effect, if any, on the insulin effect. In combination, however, the two agents caused as much as a 75% reduction in insulin-stimulated MeGlc transport activity. These data are consistent with those of other investigators using cAMP stimulators such as glucagon (18) and catecholamines (4, 7, 8). As has been shown for catecholamines (5, 7-9), glucagon in combination with ADA suppressed not only insulin-stimulated MeGlc transport, but also insulin binding and the cellular ATP level concentration deTABLE 1. Effects of glucagon and ADA on basal and insulin-stimulated MeGlc transport in rat adipocytes MeGlc transport (nmol/106 cells-sec) Additions
None Glucagon (1 MM) ADA (5 Mg/ml) Glucagon (1 MM) + ADA (5 Mg/ml)
„ . Basal 0.055 ± 0.003 0.086 ± 0.007 0.039 ± 0.0056 0.034 ± 0.0056
+Insulin .._ . (10 nM) 0.810 ± 0.611 ± 0.777 ± 0.277 ±
0.222 0.020° 0.011° 0.043°
After 30 min of equilibration in the presence of 5 mM MeGlc, the adipocytes were incubated with the indicated agents for 30 min at 37 C, then incubated further with or without insulin (10 nM) for an additional 15 min. The initial rate of MeGlc transport was determined as described in Materials and Methods. The reaction time was 15 and 3 sec for basal and insulin-stimulated cells, respectively. The final concentration of BSA in the medium was 16 mg/ml. The data show the mean ± SE (n = 4-6). ° P < 0.01 vs. control. 6 P < 0.05 us. control.
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MODULATION OF GLUCOSE TRANSPORT BY GLUCAGON
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pendently over a range of 1 X 10~n to 1 X 10 6 M (Fig. 1A). Maximal inhibition by 1 ixM glucagon of transport, insulin binding, and ATP level was 65%, 20%, and 55%, respectively, of the control value. As insulin activates glucose transport through a process requiring energy (26, 27), it is conceivable that the inhibition of transport in glucagon-treated adipocytes is due at least in part to depletion of cellular ATP. Therefore, we next determined experimental conditions under which glucagon-induced depletion of ATP could be minimized. As shown in Table 2, with the standard concentration of BSA (1.6%) in the medium, an increase in concentration of either pyruvate or glucose (substrates for ATP generation) from 2 to 10 mM was unsuccessful in preventing the loss of ATP, but under B. 5.0% BSA
A. 1.6% BSA
• V* i
o
0.6
c ." 0.4
0
-11 -10
-0
-8
-7
-V--11
-6
0
-10
-9
-8
-7
-6
S 0.6
E 0.4
E
0.4
o-v-o0
-11 -10
-9
-8
-7
-6
0
-11 -10
-9
-8
-7
g 2.0 IV
o 0
-11 -10
-9
-8
-7
Log (Glucagon, M )
-6
0
0 -11 -10 -9 -8 -7
-6
Log (Glucagon, M)
FIG. 1. Effect of glucagon concentration on the inhibition of MeGlc transport, insulin binding, and cellular ATP in rat adipocytes under low (A) and high (B) albumin conditions. After 30 min of equilibrium in the presence of 5 mM MeGlc, the adipocytes were incubated with the indicated concentrations of glucagon in the presence of ADA (5 ng/ ml) for 30 min at 37 C, then incubated with either 1 nM (O) or 10 nM (•) insulin (or with 25 pM iodoinsulin for the insulin binding assay) for an additional 15 min. The final concentration of BSA in the medium was 16 (A) or 50 (B) mg/ml. MeGlc transport, insulin binding, and cellular ATP were determined as described in Materials and Methods. The data show the mean ± SE (n = 4-6).
Endo«1990 Vol 127 • No 3
TABLE 2. Effects of glucagon on cellular ATP and insulin-stimulated MeGlc transport in rat adipocytes
f"1
J»J_'
Glucagon effect (% inhibition)
v_/ o noix l o n s
A) BSA (16 mg/ml) Pyruvate 2 mM 5 mM 10 mM Glucose 2 mM 5 mM 10 mM
Pyruvate (5 mM) + glucose (5 mM) B) BSA (50 mg/ml) Pyruvate (2 mM)
ATP
MeGlc transport
60.1 40.3 23.4
66.6 41.0 38.6
83.1 67.3 35.4 24.9
88.3 65.9 52.7 37.1
3.3
28.6
After 30 min of equilibration in the presence of 5 mM MeGlc, the adipocytes were incubated with glucagon (1 nM) plus ADA (5 jig/ml) in the medium supplemented with a final concentration of either 16 or 50 mg/ml BSA and the indicated concentration of pyruvate, glucose, or both for 30 min at 37 C. The cells were then incubated further in the presence of 10 nM insulin for 15 min, and cellular ATP and MeGlc transport activity were determined as described in Materials and Methods. The data show the means of triplicate determinations of a typical experiment.
the conditions of high BSA concentration (5%), 2 mM pyruvate effectively blocked the loss of ATP. This table also shows that the glucagon-induced inhibition of MeGlc transport is only 28.6% when ATP is maintained at the control level, and the inhibition tends to increase roughly in parallel with the ATP loss. To further characterize the relationship between cellular ATP and the transport activity, we next determined insulin-stimulated MeGlc transport in adipocytes whose ATP levels had been suppressed to various degrees by the metabolic inhibitors KCN and dinitrophenol. The results show that the insulin-stimulated transport activity is inhibited by 40-50% when the cellular ATP level is reduced to approximately 60% of that in control cells, and the activity is nearly abolished when ATP is suppressed by more than 90% (Fig. 2). In addition, in parallel experiments using KCN- or dinitrophenol-treated cells, insulin binding was inhibited by 20% and 50% when the ATP content was suppressed, respectively, to 50% and 5% of the control value. Figure IB shows the reassessment of the effects of glucagon on MeGlc transport, insulin binding, and cellular ATP in the presence of 5% BSA and 2 mM pyruvate. Under these conditions, a physiological concentration of glucagon (10 pM) had little effect on the transport activity stimulated by either 1 or 10 nM insulin. However, 1 /iM glucagon inhibited the activities by 50% and 30%, respectively, indicating that a high concentration of glucagon suppresses not only the response to a maximal
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MODULATION OF GLUCOSE TRANSPORT BY GLUCAGON 100
1075
0.8
eel
'sec
1.0
take, nmol/
0.6
0.4
Q
o O 0.2 (U
20
40
60
80
100
Cellular ATP, % Inhibition FIG. 2. Relationship between cellular ATP and insulin-stimulated MeGlc transport in rat adipocytes. After 30 min of equilibrium in the presence of 5 mM MeGlc, the adipocytes were incubated with various concentrations of KCN (O) or dinitrophenol (A) for 15 min at 37 C. The concentration of each agent was 0.2, 0.3, 0.4, 0.6, or 1.0 mM, from left to right. The cells were incubated further with 10 nM insulin for 15 min, and MeGlc transport and cellular ATP were then determined as described in Materials and Methods. The final concentration of BSA in the medium was 50 mg/ml.
dose of insulin, but also the sensitivity of adipocytes to a submaximal dose of the hormone. Glucagon at this concentration inhibited insulin binding by 35% and 20% when the ligand concentration was 1 and 10 nM, respectively. Because binding of a hormone to its receptor is a crucial step for the subsequent hormone action, we next determined whether the glucagon-induced inhibition of insulin binding could be due to an unresponsiveness of adipocytes to insulin. Figure 3 shows the relationship between the amount of cell-bound insulin and the hormone-stimulated MeGlc transport, which were calculated from the data in Fig. IB. Obviously, the ratio of the insulin effect to the hormone binding was much lower in glucagon-treated cells, showing that glucagon suppresses insulin action mostly through a process(es) distal to the step of insulin binding to its receptors. Table 3 shows the effects of glucagon on the kinetic parameters of MeGlc transport. Consistent with the data previously reported from this laboratory (22), the maximal concentration of insulin reduced the Km to about half of that in basal cells, concomitant with a 9.7-fold increase in the maximum velocity (Vmax). Pretreatment of adipocytes with 1 JIM glucagon plus ADA significantly attenuated the insulin-induced increase in the Vmax (P < 0.05). In contrast, there was no significant inhibition of the effect of insulin on the Km in glucagon-treated cells.
0.1
0.3
0.2
.6
0.4
0.5
Insulin Bound, pmol/10 cells FIG. 3. Relationship between the amount of cell-bound insulin and insulin-stimulated MeGlc transport in rat adipocytes treated with (•) or without (O) glucagon (1 fiM) plus ADA (5 jig/ml). The data are calculated from those in Fig. IB and are presented as the mean ± SE. TABLE 3. Kinetic parameters of MeGlc transport in rat adipocytes Conditions Basal Insulin Glucagon (1 ^M) -t insulin (10 nM)
Vmax
Km (mM)
(nmol/106 cells • sec)
15.9 ± 2.8 7.2 ± 1.0
0.28 ± 0.12 2.72 ± 0.30
9.2 ± 1.6
1.75 ± 0.09
After 30 min of equilibration at 37 C in the presence of 5, 10, 15, or 20 mM MeGlc, the adipocytes were incubated with or without glucagon (1 fiM) for 30 min, then incubated further in the presence or absence of insulin (10 nM) for 15 min. The initial rate of transport at each MeGlc concentration was determined, and the kinetic parameters were estimated by drawing a Hanes plot. ADA (5 fig/ml) was included in the medium throughout the experiment. The data show the mean ± SE (n = 6).
Discussion This study confirmed the reports that glucagon partially inhibits insulin stimulation of glucose transport in rat adipocytes (18-20), and that the effect of the hormone is greatly enhanced when endogenous adenosine, which suppresses adenylate cyclase activity, is degraded by ADA (18). We found also that the magnitude of inhibition varies widely with the experimental conditions. Thus, under conditions where the BSA concentration in the medium is low (1.6%), the maximal stimulation of MeGlc transport by 10 nM insulin was suppressed by 6575% in adipocytes pretreated with 1 fiM glucagon plus ADA (5 /xg/ml); the value is essentially the same as those reported by Green (18) and Yamauchi and Hashizume (19). However, the inhibition was only 30% when the
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MODULATION OF GLUCOSE TRANSPORT BY GLUCAGON
concentration of BSA was high (5%). The wide disparity in the data appears to be due to the difference in the amount of cellular ATP, which is essential for the activation of glucose transport by insulin. Glucagon plus ADA suppressed cellular ATP to about 40-50% of the control level when the BSA concentration was low, but those agents had little effect on cellular ATP when the BSA concentration was high (5%). Presumably, albumin protects against the loss of ATP by binding and thereby decreasing FFA, which inhibit ATP synthesis through uncoupling of oxidative phosphorylation (28). The relationship between the cellular ATP level and the MeGlc transport activity shown in Fig. 2 clearly indicates that the marked inhibition of transport by glucagon plus ADA in the presence of a low concentration of BSA can be explained solely by the secondary effect of a depletion of cellular ATP. Although the cellular ATP content is not reported in the studies of Green (18) and Yamauchi and Hashizume (19), it would be greatly suppressed under the experimental conditions that these workers employed {i.e. glucose-free 1.0% albumin medium). More recently, Yamauchi et al. (20) reported that adipocytes that had been treated with glucagon and then washed free of the hormone showed unimpaired responses of both glucose oxidation and lipogenesis to insulin stimulation. They suggested that glucagon selectively inhibits insulin activation of glucose transport. However, as the glucagon-treated adipocytes in the latter two assays were incubated in medium supplemented with glucose, an apparent lack of effect of glucagon may have been due to the recovery of cellular ATP. Insulin binding was 30% less in adipocytes pretreated with glucagon plus ADA. In contrast to the transport activity, the magnitude of inhibition was not affected by the experimental conditions employed in this study. Our data disagree quantitatively with those of Yamauchi and Hashizume (19), who reported 90% inhibition by 1 /xM glucagon. The greater inhibition observed by these workers could be explained by a severe loss of cellular ATP, since we found that suppression of cellular ATP by 90% or more by metabolic inhibition resulted in a substantial loss of insulin binding. As reported by Yamauchi and Hashizume (19), we also found that a decrease in insulin binding in ATP-depleted cells was due to an apparent loss of the high affinity insulin-binding site (unpublished observation). A slight inhibition of insulin binding by glucagon appears to be responsible for a decrease in the sensitivity of adipocytes to insulin. The relationship between the amount of cell-bound insulin and insulin stimulation of the glucose transport activity suggests that glucagon modulates the insulinsignaling mechanism distal to the step of insulin binding to its receptors. However, the possibility that glucagon
Endo • 1990 Vol 127 • No 3
directly inhibits the intrinsic activity, rather than the process of insulin-induced translocation of the glucose transporters, cannot be eliminated. Such a view has been proposed by Simpson and co-workers (7, 14, 15) based on the following observations: 1) catecholamines in combination with ADA inhibit insulin-stimulated glucose transport by decreasing the transport Vmax without any changes in Km; and 2) a decrease in Vmax is not accompanied by any change in the number of transporters in the plasma membranes, as assessed by either the number of cytochalasin-B-binding sites or the immunoreactivity of the trnasporters in Western blots. Our kinetic data on MeGlc transport showing that glucagon inhibited the transport mainly through the attenuation of an insulin-induced increase in Vmax appear to be consistent with the view of Simpson et al. However, our data might be interpreted otherwise. Thus, we found that the maximal concentration of insulin significantly decreased the Km from that in basal cells. Although not generally accepted at this time (29, 30), the Km-lowering effect of insulin has been reported from this (22) and other laboratories (23, 31, 32). Furthermore, Suzuki (22) reported that the Km tends to be lower as the Vmax increases. A possible explanation for Suzuki's kinetic data is that there are at least two types of glucose transporters in rat adiopcytes, high Km and low Km, and that insulin causes translocation of the mobile low Km transporters from intracellular storage sites to the plasma membranes, where the stationary high Km transporters reside (22). Indeed, Zorzano and co-workers (33) recently demonstrated that rat adipocytes have two immunologically distinct glucose transporters, i.e. erythrocyte (GLUT 1) and muscle/rat (GLUT 4) types, and that the former is distributed on both plasma membranes and low density microsomes, while the latter is mostly found on low density microsomes in the basal state and is preferentially translocatable by insulin. In the light of these findings, it can be noted from our kinetic data that glucagon appears to slightly attenuate the insulin-induced decrease in Km (from 7.2 ± 1.0 to 9.2 ± 1 . 6 HIM), concomitant with an attenuation of an increase in Vmax. This might be explained, although this is highly speculative, by postulating that glucagon modulates the insulin-signaling mechanism to elicit translocation or the process of translocation per se, rather than the intrinsic activity of glucose transporters. Further studies are needed to determine the mechanism by which glucagon modulates insulin-stimulated glucose transport in rat adipocytes.
Acknowledgment We are grateful to Junko Ohno for her excellent technical and secretarial assistance.
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MODULATION OF GLUCOSE TRANSPORT BY GLUCAGON
References 1. Gerich JE, Rizza RA, Verdonk CA, Miles JM, Haymond MW 1981 Role of counterregulatory hormones in diabetes mellitus. In: Martin JM, Ehrkich RM, Holland FJ (eds) Etiology and Pathogenesis of Insulin-Dependent Diabetes Mellitus. Raven Press, New York, pl25 2. Taylor WM, Mak ML, Halperin ML 1976 Effect of 3',5'-cyclic AMP on glucose transport in rat adipocytes. Proc Natl Acad Sci USA 73:4359 3. Chiasson J-L, Shikama H, Chu TW, Exton JH 1981 Inhibitory effect of epinephrine on insulin-stimulated glucose uptake by skeletal muscle. J Clin Invest 68:706 4. Green A 1983 Catecholamines inhibit insulin-stimulated glucose transport in adipocytes, in the presence of adenosine deaminase. FEBS Lett 152:261 5. Pessin JE, Gitomer W, Oka Y, Oppenheimer CL, Czech MP 1983 /9-Adrenergic regulation of insulin and epidermal growth factor receptors in rat adipocytes. J Biol Chem 258:7386 6. Kashiwagi A, Huecksteadt P, Foley JE 1983 The regulation of glucose transport by cAMP stimulators via three different mechanisms in rat and human adipocytes. J Biol Chem 258:13685 7. Smith U, Kuroda M, Simpson IA 1985 Counter-regulation of insulin-stimulated glucose transport by catecholamines in the isolated rat adipose cell. J Biol Chem 259:8758 8. Gliemann J, Bowes SB, Larsen TR, Rees WD 1985 The effect of catecholamines and adenosine deaminase on glucose transport system in rat adiopocytes. Biochim Biophys Acta 845:373 9. Lonnroth P, Smith U 1983 /3-Adrenergic dependent down regulation of insulin binding in rat adiopocytes. Biochem Biophys Res Commun 112:972 10. Haring H, Kirsch D, Obermaier B, Ermel B, Machicao F 1986 Decreased tyrosine kinase activity of insulin receptor isolated from rat adipocytes rendered insulin-resistant by catecholamine treatment in vitro. Biochem J 234:59 11. Stadtmauer L, Rosen O 1986 Increasing the cAMP content of IM9 cells alters the phosphorylation state and protein kinase activity of the insulin receptor. J Biol Chem 261:3402 12. Roth RA, Beaudoin J 1987 Phosphorylation of purified insulin receptor kinase by cAMP kinase. Diabetes 36:123 13. Obermaier B, Ermel B, Kirsch D, Mushack J, Rattenhuber E, Biemer E, Machicao F, Haring HU 1987 Catecholamines and tumor promoting phorbol esters inhibit insulin receptor kinase and induce insulin resistance in isolated human adipocytes. Diabetologia 30:93 14. Kuroda M, Honner RC, Cushman SW, Londos C, Simpson IA 1987 Regulation of insulin-stimulated glucose transport in the isolated rat adipocyte. cAMP-independent effects of lipolytic and antilipolytic agents. J Biol Chem 262:245 15. Joost HG, Weber TM, Cushman SW, Simpson IA 1987 Activity and phosphorylation state of glucose transporters in plasma membranes from insulin-, isoproterenol-, and phorbol ester-treated rat adipose cells. J Biol Chem 262:11261 16. Suzuki K, Kono T 1980 Evidence that insulin causes translocation of glucose transport activity to the plasma membrane from an intracellular storage site. Proc Natl Acad Sci USA 77:2542
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17. Cushman SW, Wardzala LJ 1980 Potential mechanism of insulin action on glucose transport in the isolated rat adipocyte cell: apparent translocation of intracellular transport system to the plasma membranes. J Biol Chem 255:4758 18. Green A 1983 Glucagon inhibition of insulin-stimulated 2-deoxyglucose uptake by rat adipocytes in the presence of adenosine deaminase. Biochem J 212:189 19. Yamauchi K, Hashizume K 1986 Glucagon alters insulin binding to isolated rat epididymal adipocytes: possible role of adenosine 3',5'-monophosphate in modification of insulin action. Endocrinology 119:218 20. Yamauchi K, Hashizume K, Miyamoto T, Otsuka H, Ichikawa K, Nishii Y, Yamada T 1988 Selective alterations of insulin action by glucagon in isolated rat epididymal adipocytes. Endocrinology 123:2800 21. Rodbell M 1961 Metabolism of isolated fat cells. I. Effects of hormones on glucose metabolism and lipolysis. J Biol Chem 239:375 22. Suzuki K 1988 Reassessment of the translocation hypothesis by kinetic studies on hexose transport in isolated rat adipocytes. J Biol Chem 263:12247 23. Whitesell RR, Abumrad NA 1985 Increased affinity predominates in insulin stimulation of glucose transport in the adipocyte. J Biol Chem 260:2894 24. Gliemann J, Osterlind K, Vinten J, Gammeltoft S 1972 A procedure for measurement of distribution spaces in isolated fat cells. Biochim Biophys Acta 286:1 25. McElroy WD 1963 Crystalline firefly luciferase. In: Colowick SI, Kaplan NO (eds) Methods in Enzymology. Academic Press, New York and London, vol 6:445 26. Chandramouli V, Milligan M, Carter RJ 1977 Insulin stimulation of glucose transport in adipose tissues. Biochemistry 16:1151 27. Kono T, Robinson FW, Sarver JA, Vega FV, Pointer RH 1977 Actions of insulin in fat cells. Effects of low temperature, uncouplers of oxidative phosphorylation, and respiratory inhibitors. J Biol Chem 252:2226 28. Angel A, Desai K, Halperin ML 1971 Free fatty acid and ATP levels in adipocytes during lipolysis. Metabolism 20:87 29. Martz A, Mookerjee BD, Jung CY 1986 Insulin and phorbol ester affect the maximum velocity rather than the half-saturation constant of 3-O-methylglucose transport in rat adipocytes. J Biol Chem 261:13606 30. Joost HG, Weber TM, Cushman SW 1988 Qualitative and quantitative comparison of glucose transport activity and glucose transporter concentration in plasma membranes from basal and insulinstimulated rat adipose cells. Biochem J 249:155 31. Toyoda N, Flanagan JE, Kono T 1987 Reassessment of insulin effects on the Vmax and Km values of hexose transport in isolated rat epididymal adipocytes. J Biol Chem 262:2737 32. Okuno Y, Gliemann J 1987 Enhancement of glucose transport by insulin at 37° C in rat adipocytes is accounted for by increased Vmax. Diabetologia 30:426 33. Zorzano A, Wilkinsin W, Kotliar N, Thoidis G, Wadzinkski BE, Ruoho, AE, Pilch PF 1989 Insulin-regulated glucose uptake in rat adipocytes is mediated by two transporter isoforms present in at least two vesicle populations. J Biol Chem 264:12358
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