- Email: [email protected]
Insulin-induced changes in membrane potential and 3-O-methylglucose uptake at various external K concentrations in frog skeletal muscle
Camp. Biochem. F’hysiol.Vol. 86A, No. 2, pp. 331-336, 1987 in Great Britain
0300-9629187 $3.00 + 0.00 ~c 1987 Pergamon Journals Ltd
Printed
INSULIN-INDUCED CHANGES IN MEMBRANE POTENTIAL AND 3-0-METHYLGLUCOSE UPTAKE AT VARIOUS EXTERNAL K CONCENTRATIONS IN FROG SKELETAL MUSCLE YOSHINORI MARUNAKA Department
of Physiology,
and HIROSHI KITASATO
Shiga University
of Medical
Science,
Ohtsu,
520-21 Japan
(Received 6 May 1986) Abstract-l. Insulin induced a hyperpolarization of the membrane and stimulated the 3-0-methylglucose (3-O-MG) uptake in frog skeletal muscle. In the present study, the relationship between the insulininduced changes in the membrane potential and the 3-O-MC uptake was investigated. 2. The stimulatory action of insulin on the 3-O-MC uptake was mediated by two different mechanisms, One of them was dependent on the change in the membrane potential and the other was independent of the change in the membrane potential. 3. Both values of the insulin-induced changes in the membrane potential and the 3-O-MG uptake were diminished by increasing the external K concentration. 4. One of the causes for the diminution of the 3-O-MC uptake with a rise of the external K concentration would be the decrease in the magnitude of the insulin-induced hyperpolarization.
INTRODUCTION Since the stimulatory action of insulin on the glucose transport in heart muscles was reported by Morgan et al. (1959, 1961) the action has been well known. In addition to the stimulatory action on the glucose transport, insulin also has effects on the Na,K-pump in frog skeletal muscle (Kitasato et al., 1980b, c) and on the membrane potential in rat skeletal muscle (Zierler, 1957, 1959); insulin hyperpolarizes the membrane of skeletal muscles by activating the electrogenie Na,K-pump (Moore and Rabovsky, 1979; Marunaka and Kitasato, 1985b). Zierler and Rogus (1980) have reported that a hyperpolarization of the membrane would be a mediator of the stimulatory action of insulin on the glucose uptake in rat skeletal muscle. One of the purposes of the present study is to investigate whether in frog skeletal muscle insulin still has a stimulatory action on the glucose uptake under a condition that the insulin-induced hyperpolarization is abolished. In addition, we have reported that a rise of the external K concentration stimulates the insulininduced increase in the ouabain-sensitive Na efflux from frog skeletal muscle (an index of the Na,Kpump activity; Kitasato ef al., 1980a; Marunaka and Kitasato, 1985a). As mentioned above, insulin hyperpolarizes the membrane of frog skeletal muscles by activating the electrogenic Na,K-pump (Moore and Rabovsky, 1979; Marunaka and Kitasato, 1985b). These observations suggest that the external K concentration would have some effects on the magnitude of the insulin-induced hyperpolarization. In fact, we found that a rise of the external K concentration decreased the magnitude of the insulin-induced hyperpolarization mainly by decreasing the electrogenicity of the insulin-stimulated Na,K-pump 331
(Marunaka et al., 1986; Marunaka, 1986). On the one hand, as mentioned above, Zierler and Rogus (1980) have reported that a hyperpolarization of the membrane would be a mediator of the insulin action on the glucose uptake by rat skeletal muscles. These reports suggest that a change in the external K concentration would modify the insulin action on the glucose uptake. Therefore, we investigated effects of the external K concentration on the glucose uptake in the presence and the absence of insulin in frog skeletal muscle. MATERIALS AND METHODS The composition of bathing solutions used in the present study is summarized in Table 1. Sartorius muscles from bullfrogs (Ram caresbeiana) were carefully dissected under a microscope. The water content in the intracellular space was estimated from the wet weight, the dry weight and the water content in the extracellular space by the same method as that reported in the previous study (Marunaka, 1986). The 3-0-methylglucose (3-O-MG, non-metabolized glucose) uptake was estimated by the similar method to that of Grinstein and Erlij (1977). The 3-O-MC uptake was measured by incubating the muscle for 30 min at 22’C in test solutions containing 2 mM [“‘Cl3-O-MG (0.2 mCi/mmol). Prior to this incubation, the muscle was preincubated for 15 min at 22’C in 3-0-MG-free solutions whose ionic composition was the same as that of test solution for measuring the 3-O-MC uptake. Insulin and/or valinomycin were added at the start of the preincubation and existed during the period of measuring the 3-O-MC uptake. The membrane potential was measured by using a glass microelectrode filled with 3 M-KC1 and with a resistance of about 7 MQ. The microelectrode was connected via a Ag-AgC1 electrode to the input of a preamplifier (ME commercial. model ME-321 1). The muscle was incubated under the same condition as that where the 3-O-MC uptake
332
YOSHINORI MARUNAKAand HIROSHIKITASATO
I.
These observations suggest that a part of the insulin action on the 3-O-MG uptake would be dependent on the membrane potential and that the other part of Solutions NaCl KC1 C&l 3-O-MG the insulin action would be developed by the memI-K solution II0 I 2 2 brane potential-independent mechanism. 2-K solutmn 110 2 2 2 Insulin stimulated the 3-O-MG uptake in I-, 2-, 55-K solution II0 5 2 2 IO-K solution II0 IO 2 2 and IO-K solutions (Fig. IA). The magnitude of the The osmolarlty and pH of the solutions shown in Table I were 3-O-MG uptake in the absence of insulin was inadjusted to 260mOsm/l and 7.4 with Tris-HCI. creased by a rise of the external K concentration (Fig. IA). The insulin-induced increase in the 3-O-MG was measured, and the membrane potential was measured uptake decreased with raising the external K concenat 22 C around 15 min after starting the incubation of tration (Fig. 1A). The ratio of the 3-O-MG uptake in muscle in test solution containing 2 mM 3-O-MC. the presence of insulin to that in the absence of The concentrations of insulin and valinomycin used in the insulin was also decreased by raising the external K present study were 250 mu/ml and 1PM, respectively. concentration (Fig. 1B). Insulin, valinomycin and 3-O-MG were purchased from To investigate the relationship between the insulinSigma. [V]3-O-MC was purchased from New England induced changes in the membrane potential and the Nuclear. 3-O-MG uptake, we observed the effect of the external K concentration on the magnitude of the insulininduced hyperpolarization. The membrane potentials RESULTS were measured in I-, 2-. 5- and 10-K solutions in Insulin and valinomycin hyperpolarized the memthe presence and the absence of insulin. Insulin brane and stimulated the 3-O-MG uptake (Table 2A hyperpolarized the membrane at the external K and B). This observation suggests that a hyperconcentrations of 1, 2, 5 and IO mM (Fig. 2A). The polarization would stimulate the 3-O-MG uptake. magnitude of the insulin-induced hyperpolarization Zierler and Rogus (1980) also suggest that a hyperwas diminished by increasing the external K concenpolarization would be a mediator of the stimulatory tration of I to 10 mM and the magnitude tended action of insulin on the glucose uptake by rat skeletal to reach a steady value around the external K muscles. To clarify whether the suggestion is right, we concentration of 5 mM (Fig. 2B). investigated whether insulin had no stimulatory action on the 3-O-MG uptake under the condition that DISCUSSION insulin had no effect on the membrane potential. In order that the membrane potential would not be In frog skeletal muscles the magnitude of the changed by insulin, we used valinomycin, which is an insulin-induced hyperpolarization reached its maxiionophore of K ion. In the presence of valinomycin, mal value within 20min after addition of insulin although insulin had no significant effect on the (250mU/ml) and little change was observed for the membrane potential, insulin still had a stimulatory following 30 min (Moore and Rabovsky, 1979). Our action on the 3-O-MG uptake (Table 2C). However, preliminary experiments indicate that in frog sarthe magnitude of the stimulatory action on the torius muscle insulin (250-500 mu/ml) shows the 3-O-MG uptake under the condition that insulin had maximal effect on the membrane potential within no effect on the membrane potential was smaller than 3&40 min after addition of insulin and that there is that under the condition that insulin induced a little change in the intracellular Na and K concenhyperpolarization (Table 2A and C). In the presence trations for 60 min after addition of 25&500 mu/ml of insulin, valinomycin depolarized the membrane insulin (our unpublished data). However, around and diminished the 3-O-MG uptake (Table 2D). 2-6 hr after addition of insulin (250-500 mu/ml), the Table
The composition
of solutions
used in the present study
(mM)
Table 2. The relationship
between the membrane potential and the 3.O-MC and the absence of insulin and/or valinomycin Membrane potential (mV)
A.
B.
C.
D.
Valinomycm (-) Insulin (+) Insulin (-) Insulin (-) Valinomycin Valinomycin
(+) (-)
Valinomycin (+) Insulin (+) Insulin (-) Insulin (+) Valinomycin Valinomycin
(+) (-)
(nmol/g
uptake in the presence
3-O-MG uptake muscle water:30 min)
-9X.2 f 0.7’ -94.4 * 0.8
3x1 * Ilt 270 k 6
-96.1 -94.1
kO.6: kO.8
294 * 4t 261 k5
-95.8 -96.0
+ 0.5 + 0.6
321 k 7t 288 ? 3
- 96.0 f 0.5s -98.3 + 0.7
312f7t 375 * 9
Each value of the membrane potential is expressed as the mean value of I I experiments k I SE of the mean value. Each value of the 3.O-MC uptake is expressed as the mean value of six experiments + I SE. of the mean value. In comparison with respective values without Insulin: *. P =0.005; t, P =O.OOl. In comparison with respective values without valinomycin; 1, P = 0.05; 8, P = 0.01; t, P = 0.001.
Hyperpolarization
and insulin
action
333
2.0
I
0 0
I
5
4
4
I
I
I
LO
10
0
CKI, CmM)
I
I
I
I
I
5
10
CKI, CmM)
Fig. I. The effect of the external K concentration on the 3-O-MG uptake in the presence and the absence of insulin. Each symbol expresses the mean value of six experiments. Each vertical line expresses I SE of the mean value. (A) The effect of the external K concentration on the 3-O-MG uptake in the presence and the absence of insulin. Closed and open circles respectively express the magnitude of the 3-O-MG uptake in the presence and the absence of insulin. Closed squares express the insulin-induced increase in the 3-O-MG uptake (the difference between the magnitude of the 3-O-MG uptake in the presence and the absence of insulin). Insulin increased the 3-O-MG uptake (I and 2 mM K, P < 0.001; 5 and IO mM K, P < 0.005). (B) The effect of the external K concentration on the ratio of the 3-O-MG uptake to that in the absence of insulin. The ratio was estimated by using the datum shown in Fig. IA. 6
0
I
0 5 CKlo CmM)
10
0
I
5
I
I
I
I
10
CKlo CmM)
Fig. 2. The effect of the external K concentration on the insulin-induced change in the membrane potential. (A) The effect of the external K concentration on the membrane potential in the presence and the absence of insulin. Closed and open circles represent the magnitude of the membrane potential in the presence and the absence of insulin, respectively. Each symbol expresses the mean value of 15 experiments. Each 1SE of the mean value is smaller than the radius of the symbol. (B) The effect of the external K concentration on the insulin-induced hyperpolarization. Each open square expresses the magnitude of the insulin-induced hyperpolarization. Each symbol expresses the mean value of 15 experiments. Each vertical line expresses 1 SE of the mean value. Insulin induced a hyperpolarization of the membrane at each external K concentration (I and 2mM, P
334
YOSHINORI MARUNAKAand HIROSHIKITASATO
intracellular Na and K concentrations in insulinapplied muscles respectively tend to decrease and increase as compared with insulin-free muscles (our unpublished data). Therefore, the effect of the insulin on the membrane potential and the 3-O-MG uptake must be measured as soon as possible after addition of insulin lest the intracellular Na and K concentrations and other intracellular events are changed by insulin. Therefore, the 3-O-MG uptake and the membrane potential were measured within 45 min after addition of insulin, which is the minimum time required for insulin of 250 mu/ml to develop a maximal effect on the membrane potential in sartorius muscles of bullfrogs. Kipnis and Parrish (1965) reported that 2-deoxyglucose transport into rat diaphragm decreased as the external K concentration was increased equimolarly at the expense of the extenal Na concentration. Zierler et al. (1985) also reported a similar observation about the effect of the external K concentration on 2-deoxyglucose uptake in rat soleus muscles However, this has not been a universal finding (Bihler and Sawh, 1971; Kohn and Clausen, 1972). Kohn and Clausen (1972) reported that an increase in the external K concentration had no significant effect on 3-O-MG release from rat soleus muscles in the absence of insulin. Various observations about effects of the external K concentration on glucose uptake in muscles. The observations shown in Fig. IA indicate that the magnitude of the 3-O-MG uptake in the absence of insulin is increased by raising the external K concentration of 1 to 10 mM and tends to reach a steady value around the external K concentration of 5 mM. This is a contrary result to the observations in rat muscles reported by Kipnis and Parrish (1965) and Zierler et al. (1985). It may be the difference between 3-O-MC and 2-deoxyglucose or/and the difference between frog muscle and rat muscle. 2-deoxyglucose is phosphorylated in the intracellular space, while 3-O-MG is not phosphorylated even if it is incorporated into the intracellular space. Therefore, the effect of the external K concentration on glucose uptake may be related to the intracellular metabolism. However, the cause for these different observations would not be clearly known. As mentioned above, there are various observations about the effect of the external K concentration on glucose uptake in the absence of insulin, however there is widespread agreement that the stimulatory action of insulin on the transport of glucose or its analogues is reduced or inhibited by a rise of the external K concentration (Kipnis and Parrish, 1965; Zierler er al., 1985). The observation shown in Fig. 1A and B also supports this. However, the cause for the diminution of the stimulatory action of insulin on the 3-O-MG uptake with a rise of the external K concentration has not been clarified. Zierler and Rogus (1980) have suggested that the insulin-induced hyperpolarization would be a mediator of the stimulatory action of insulin on glucose uptake. The observations shown in Fig. 2B indicate that the magnitude of the insulin-induced hyperpolarization decreases with a rise of the external K concentration. It is suggested, as follows: (1) insulin induces a hyperpolarization of the membrane; (2) this hyper-
polarization is a mediator of the stimulatory action of insulin on the 3-O-MG uptake; (3) the magnitude of the insulin-induced hyperpolarization decreases with a rise of the external K concentration; (4) as a result of the decrease in magnitude of the insulininduced hyperpolarization, the stimulatory action of insulin on the 3-O-MG uptake decreases with a rise of the external K concentration. However, a rise of the external K concentration depolarizes the membrane (Fig. 2A) and increases the 3-O-MG uptake (Fig. IA). This is a contrary observation to the suggestion that the hyperpolarization is a mediator of the stimulatory action of insulin on the glucose uptake in skeletal muscles. The stimulation of the 3-O-MG uptake induced by a rise of the external K concentration may not be caused by the depolarization, but by the direct action of a rise of the external K concentration in frog skeletal muscle. This must be investigated under the voltage-clamp, and is clarified in the future. A hyperpolarization of the membrane is surely a mediator of the stimulatory action of insulin on glucose uptake in skeletal muscle (Table 2; Zierler and Rogus, 1980). However, it is unclear that the insulin-induced hyperpolarization is only a mediator of the stimulatory action; i.e. insulin still had a stimulatory action on the 3-O-MG uptake even under the condition that insulin had no effect on the membrane potential (Table 2). Zierler et ~11.(1985) have reported that in rat skeletal muscle the magnitude of the insulin-induced hyperpolarization diminishes with increasing the external K concentration to 36 mM, however the stimulatory action of insulin on glucose uptake remains even at the external K concentration of 36mM, furthermore the action of insulin on glucose uptake is smaller than that at low external K concentration. Their study in rat skeletal muscle supports our observation in frog skeletal muscle. Namely, insulin would affect the 3-O-MG uptake by at least two different mechanisms as follows: (1) one is developed by the insulin-induced hyperpolarization; (2) the other is independent of the membrane potential. The latter may be the translocation of the 3-O-MG transport carrier into the plasma membrane (Cushman and Wardzala, 1980; Suzuki and Kono, 1980; Kono et al., 1982; Simpson et al., 1983). If the 3-O-MG transport carrier is translocated into the plasma membrane by insulin, it must be investigated whether the translocated carrier would be dependent on the membrane potential. To clarify this point, the voltage-dependency of the 3-O-MG uptake in the presence and the absence of insulin was estimated. The absolute value of the voltage-dependency was increased 1.9-fold by the presence of insulin (Fig. 3). Figure 3 and the reports mentioned above (Cushman & Wardzala, 1980; Suzuki and Kono, 1980; Kono et al., 1982; Simpson et al., 1983) show that insulin would increase the number of the voltage-sensitive 3-O-MG transport carrier in the plasma membrane. From the observations shown in the present study, it would be concluded as follows: (1) A part of the insulin action on the 3-O-MC uptake is developed through the insulin-induced
Hyperpolarization
-99
-98
-97 Membrane
and insulin
-96 potential
335
action
-95
-94
-93
(mV)
Fig, 3. The relationship between the membrane potential and the 3-O-MG uptake in the presence and the absence of insulin. The data shown in Fig. 3 are the same as those shown in Table 2. Each symbol expresses the mean value. Each vertical or horizontal line expresses 1 SE of the mean value. The condition that each symbol expresses is as follows: closed circle, insulin (+) and valinomycin (-); closed square, insulin (+) and valinomycin (+); open circle, insulin (-) and valinomycin (-); open square, insulin (-) and valinomycin (+). The upper equation expresses the regression line between the membrane potential and the 3-O-MG uptake in the presence of insulin. The lower equation expresses the regression line between the membrane potential and the 3-O-MG uptake in the absence of insulin. The lines were respectively estimated by using the mean values of the membrane potential and the 3-O-MG uptake in the presence and the absence of insulin with the method of least squares. X and Y respectively express the values of the membrane potential and the 3-O-MG uptake, and I is the coefficient of correlation.
hyperpolarization, and the other part of the effect of insulin is independent of the insulin-induced change in the membrane potential; the membrane potentialindependent action of insulin may be developed through increasing the number of the 3-O-MG transport carrier in the plasma membrane. The translocated carrier would be voltage-sensitive. (2) The diminution of the stimulatory action of insulin on the 3-O-MG uptake with a rise of the external K concentration is explained as follows; the rise of the external K concentration induces a decrease in the magnitude of the insulin-induced hyperpolarization and this decrease causes a diminution of the hyperpolarization-stimulated 3-O-MG uptake. Acknowledgement-This work was supported from the Ministry of Education, Science and Japan.
by grants Culture of
REFERENCES
Bihler I. and Sawh P. C. (1971) Regulation of sugar transport in muscle: effect of increased external potassium in vitro. Biochim. Biophys. Acta 241, 302-309. Cushman S. W. and Wardzala L. J. (1980) Potential mechanism of insulin action on glucose transport in the isolated rat adipose cell. Apparent translocation of intracellular transport systems to the plasma membrane. J. biol. Chem. 255, 47584762. Grinstein S. and Erlij D. (1977) Differences between resting and insulin-stimulated amino acid transport in frog skeletal muscle. J. Membr. Biol. 35, 9-28. Kipnis D. M. and Parrish J. E. (1965) The role of Na and K on sugar (2-deoxyglucose) and amino acid (a-aminobutyric acid) transport in striated muscles. Fedn. Proc. Fedn Am. Sots. exp. Biol. 24, 1051-1059.
Kitasato H.. Marunaka Y., Murayama K. and Nishio K. (1980a) Insulin-sensitive Na efflux and the extracellular K concentration. Proc. of the 18th Annual Meeting of Bionhvs. Sot. of Janan, D. 203 (in JaDanese). Kitasatd H., Sato S.,. Maiunaka ‘Y., Murayama K. and Nishio K. (1980b) Effects of ouabain on Na efflux in high internal Na and insulin-preincubated muscles. Jpn. J. Physiol. 30, 59 I-602. Kitasato H., Sato S., Marunaka Y., Murayama K. and Nishio K. (1980~) Anoarent affinitv chances bv insulin of Na-K transport ‘system in frog skeletalmus~le. Jpn. J. Physiol. 30, 603-6 16. Kohn P. G. and Clausen T. (1972) The relationship between the transport of glucose and cations across cell membranes in isolated tissues. VII. The effects of extracellular Na and K on the transoort of 3-0-methvlelucose and glucose in rat soleus muscle. Biochim. Biopi~;. Acta 255, 798-814. Kono T., Robinson F., Blevins T. L. and Ezaki 0. (1982) Evidence that translocation of the glucose transoort activity is the major mechanism of insulin action on glucose transport in fat cells. J. hiol. Chem. 257, 10942-10947. Marunaka Y. (1986) Effects of the external K concentration the electrogenicity of the insulin-stimulated E,K-pump. J. Membr. Biol. 91, 165-172. Marunaka Y. and Kitasato H. (1985a) The sensitivity of the insulin-stimulated ouabain-sensitive Na+ efflux from frog sartorius muscle to internal Na+, external K+ and ouabain. IRCS med. Sci. 13, 445-6. Marunaka Y. and Kitasato H. (1985b) The inhibitory action of insulin-induced hyperpolarization on 3-O-methyl-oglucose efflux from frog sartorius muscles. IRCS med Sci. 13, 637438. Marunaka Y., Murayama K. and Kitasato H. (1986) A rise of the external K concentration decreases the magnitude of the insulin-induced hyperpolarization by diminishing the electrogemclty of the insulin-stimulated electrogenic Na,K-pump in frog skeletal muscles. Proc. of Inter-
336
YOSHINORI MARUNAKA and
national Symposium on Ion Channels and Electrogenic Pump in Biomembranes, pp. 55556. Moore R. D. and Rabovsky J. L. (1979) Mechanism of insulin action on resting membrane potential of frog skeletal muscle. Am. J. Physiol. 236, C2499C254. Morgan H. E., Randles P. .J. and Regen D. M. (1959) Regulation of glucose uptake by muscle. 3. The effects of insulin, anoxia, salicylate and 2:4-dinitrophenol on membrane transport and intracellular phosphorylation of glucose in isolated rat heart. Bioch&. J. $3, 5733579. Morgan H. E.. Henderson M. J.. Reuen D. M. and Park C.-R. (1961) Regulation of glucose-uptake in muscle. I. The effects of insulin and anoxia on glucose transport and phosphorylation in the isolated, perfused heart of normal rats. J. biol. Chem. 236, 253-261. Simpson I. A., Yver D. R., Hissin P. J., Lawrence J., Wardzala L. J., Karnieli E., Salans L. B. and Cushman S. W. (1983) Insulin-stimulated translocation of glucose transporters in the isolated rat adipose cells: character-
HIROSHI KITASATO
ization of subcellular fractions. Biochim. Biophys. Acts 763, 393407. Suzuki K. and Kono T. (1980) Evidence that insulin causes translocation of glucose transport activity to the plasma membrane from an intracellular storage site. Proc. Null Acad. Sci. USA 77, 2542-2545. Zierler K. L. (1957) Increase in resting membrane potential of skeletal muscle produced by insulin. Science 126, 106771068. Zierler K. L. (1959) Effect of insulin on membrane potential and potassium content of rat muscle. Am. J. Physiol. 197, 515-523. Zierler K. and Rogus E. M. (1980) Hyperpolarization as a mediator of insulin action: increased muscle glucose uptake induced electrically. Am. J. Physiol. 239, E21-E29. Zierler K., Rogus E. M., Scherer R. W. and Wu F. (1985) Insulin action on membrane potential and glucose uptake: effects of high potassium. Am. J. Physiol. 249, E17pE25.
0300-9629187 $3.00 + 0.00 ~c 1987 Pergamon Journals Ltd
Printed
INSULIN-INDUCED CHANGES IN MEMBRANE POTENTIAL AND 3-0-METHYLGLUCOSE UPTAKE AT VARIOUS EXTERNAL K CONCENTRATIONS IN FROG SKELETAL MUSCLE YOSHINORI MARUNAKA Department
of Physiology,
and HIROSHI KITASATO
Shiga University
of Medical
Science,
Ohtsu,
520-21 Japan
(Received 6 May 1986) Abstract-l. Insulin induced a hyperpolarization of the membrane and stimulated the 3-0-methylglucose (3-O-MG) uptake in frog skeletal muscle. In the present study, the relationship between the insulininduced changes in the membrane potential and the 3-O-MC uptake was investigated. 2. The stimulatory action of insulin on the 3-O-MC uptake was mediated by two different mechanisms, One of them was dependent on the change in the membrane potential and the other was independent of the change in the membrane potential. 3. Both values of the insulin-induced changes in the membrane potential and the 3-O-MG uptake were diminished by increasing the external K concentration. 4. One of the causes for the diminution of the 3-O-MC uptake with a rise of the external K concentration would be the decrease in the magnitude of the insulin-induced hyperpolarization.
INTRODUCTION Since the stimulatory action of insulin on the glucose transport in heart muscles was reported by Morgan et al. (1959, 1961) the action has been well known. In addition to the stimulatory action on the glucose transport, insulin also has effects on the Na,K-pump in frog skeletal muscle (Kitasato et al., 1980b, c) and on the membrane potential in rat skeletal muscle (Zierler, 1957, 1959); insulin hyperpolarizes the membrane of skeletal muscles by activating the electrogenie Na,K-pump (Moore and Rabovsky, 1979; Marunaka and Kitasato, 1985b). Zierler and Rogus (1980) have reported that a hyperpolarization of the membrane would be a mediator of the stimulatory action of insulin on the glucose uptake in rat skeletal muscle. One of the purposes of the present study is to investigate whether in frog skeletal muscle insulin still has a stimulatory action on the glucose uptake under a condition that the insulin-induced hyperpolarization is abolished. In addition, we have reported that a rise of the external K concentration stimulates the insulininduced increase in the ouabain-sensitive Na efflux from frog skeletal muscle (an index of the Na,Kpump activity; Kitasato ef al., 1980a; Marunaka and Kitasato, 1985a). As mentioned above, insulin hyperpolarizes the membrane of frog skeletal muscles by activating the electrogenic Na,K-pump (Moore and Rabovsky, 1979; Marunaka and Kitasato, 1985b). These observations suggest that the external K concentration would have some effects on the magnitude of the insulin-induced hyperpolarization. In fact, we found that a rise of the external K concentration decreased the magnitude of the insulin-induced hyperpolarization mainly by decreasing the electrogenicity of the insulin-stimulated Na,K-pump 331
(Marunaka et al., 1986; Marunaka, 1986). On the one hand, as mentioned above, Zierler and Rogus (1980) have reported that a hyperpolarization of the membrane would be a mediator of the insulin action on the glucose uptake by rat skeletal muscles. These reports suggest that a change in the external K concentration would modify the insulin action on the glucose uptake. Therefore, we investigated effects of the external K concentration on the glucose uptake in the presence and the absence of insulin in frog skeletal muscle. MATERIALS AND METHODS The composition of bathing solutions used in the present study is summarized in Table 1. Sartorius muscles from bullfrogs (Ram caresbeiana) were carefully dissected under a microscope. The water content in the intracellular space was estimated from the wet weight, the dry weight and the water content in the extracellular space by the same method as that reported in the previous study (Marunaka, 1986). The 3-0-methylglucose (3-O-MG, non-metabolized glucose) uptake was estimated by the similar method to that of Grinstein and Erlij (1977). The 3-O-MC uptake was measured by incubating the muscle for 30 min at 22’C in test solutions containing 2 mM [“‘Cl3-O-MG (0.2 mCi/mmol). Prior to this incubation, the muscle was preincubated for 15 min at 22’C in 3-0-MG-free solutions whose ionic composition was the same as that of test solution for measuring the 3-O-MC uptake. Insulin and/or valinomycin were added at the start of the preincubation and existed during the period of measuring the 3-O-MC uptake. The membrane potential was measured by using a glass microelectrode filled with 3 M-KC1 and with a resistance of about 7 MQ. The microelectrode was connected via a Ag-AgC1 electrode to the input of a preamplifier (ME commercial. model ME-321 1). The muscle was incubated under the same condition as that where the 3-O-MC uptake
332
YOSHINORI MARUNAKAand HIROSHIKITASATO
I.
These observations suggest that a part of the insulin action on the 3-O-MG uptake would be dependent on the membrane potential and that the other part of Solutions NaCl KC1 C&l 3-O-MG the insulin action would be developed by the memI-K solution II0 I 2 2 brane potential-independent mechanism. 2-K solutmn 110 2 2 2 Insulin stimulated the 3-O-MG uptake in I-, 2-, 55-K solution II0 5 2 2 IO-K solution II0 IO 2 2 and IO-K solutions (Fig. IA). The magnitude of the The osmolarlty and pH of the solutions shown in Table I were 3-O-MG uptake in the absence of insulin was inadjusted to 260mOsm/l and 7.4 with Tris-HCI. creased by a rise of the external K concentration (Fig. IA). The insulin-induced increase in the 3-O-MG was measured, and the membrane potential was measured uptake decreased with raising the external K concenat 22 C around 15 min after starting the incubation of tration (Fig. 1A). The ratio of the 3-O-MG uptake in muscle in test solution containing 2 mM 3-O-MC. the presence of insulin to that in the absence of The concentrations of insulin and valinomycin used in the insulin was also decreased by raising the external K present study were 250 mu/ml and 1PM, respectively. concentration (Fig. 1B). Insulin, valinomycin and 3-O-MG were purchased from To investigate the relationship between the insulinSigma. [V]3-O-MC was purchased from New England induced changes in the membrane potential and the Nuclear. 3-O-MG uptake, we observed the effect of the external K concentration on the magnitude of the insulininduced hyperpolarization. The membrane potentials RESULTS were measured in I-, 2-. 5- and 10-K solutions in Insulin and valinomycin hyperpolarized the memthe presence and the absence of insulin. Insulin brane and stimulated the 3-O-MG uptake (Table 2A hyperpolarized the membrane at the external K and B). This observation suggests that a hyperconcentrations of 1, 2, 5 and IO mM (Fig. 2A). The polarization would stimulate the 3-O-MG uptake. magnitude of the insulin-induced hyperpolarization Zierler and Rogus (1980) also suggest that a hyperwas diminished by increasing the external K concenpolarization would be a mediator of the stimulatory tration of I to 10 mM and the magnitude tended action of insulin on the glucose uptake by rat skeletal to reach a steady value around the external K muscles. To clarify whether the suggestion is right, we concentration of 5 mM (Fig. 2B). investigated whether insulin had no stimulatory action on the 3-O-MG uptake under the condition that DISCUSSION insulin had no effect on the membrane potential. In order that the membrane potential would not be In frog skeletal muscles the magnitude of the changed by insulin, we used valinomycin, which is an insulin-induced hyperpolarization reached its maxiionophore of K ion. In the presence of valinomycin, mal value within 20min after addition of insulin although insulin had no significant effect on the (250mU/ml) and little change was observed for the membrane potential, insulin still had a stimulatory following 30 min (Moore and Rabovsky, 1979). Our action on the 3-O-MG uptake (Table 2C). However, preliminary experiments indicate that in frog sarthe magnitude of the stimulatory action on the torius muscle insulin (250-500 mu/ml) shows the 3-O-MG uptake under the condition that insulin had maximal effect on the membrane potential within no effect on the membrane potential was smaller than 3&40 min after addition of insulin and that there is that under the condition that insulin induced a little change in the intracellular Na and K concenhyperpolarization (Table 2A and C). In the presence trations for 60 min after addition of 25&500 mu/ml of insulin, valinomycin depolarized the membrane insulin (our unpublished data). However, around and diminished the 3-O-MG uptake (Table 2D). 2-6 hr after addition of insulin (250-500 mu/ml), the Table
The composition
of solutions
used in the present study
(mM)
Table 2. The relationship
between the membrane potential and the 3.O-MC and the absence of insulin and/or valinomycin Membrane potential (mV)
A.
B.
C.
D.
Valinomycm (-) Insulin (+) Insulin (-) Insulin (-) Valinomycin Valinomycin
(+) (-)
Valinomycin (+) Insulin (+) Insulin (-) Insulin (+) Valinomycin Valinomycin
(+) (-)
(nmol/g
uptake in the presence
3-O-MG uptake muscle water:30 min)
-9X.2 f 0.7’ -94.4 * 0.8
3x1 * Ilt 270 k 6
-96.1 -94.1
kO.6: kO.8
294 * 4t 261 k5
-95.8 -96.0
+ 0.5 + 0.6
321 k 7t 288 ? 3
- 96.0 f 0.5s -98.3 + 0.7
312f7t 375 * 9
Each value of the membrane potential is expressed as the mean value of I I experiments k I SE of the mean value. Each value of the 3.O-MC uptake is expressed as the mean value of six experiments + I SE. of the mean value. In comparison with respective values without Insulin: *. P =0.005; t, P =O.OOl. In comparison with respective values without valinomycin; 1, P = 0.05; 8, P = 0.01; t, P = 0.001.
Hyperpolarization
and insulin
action
333
2.0
I
0 0
I
5
4
4
I
I
I
LO
10
0
CKI, CmM)
I
I
I
I
I
5
10
CKI, CmM)
Fig. I. The effect of the external K concentration on the 3-O-MG uptake in the presence and the absence of insulin. Each symbol expresses the mean value of six experiments. Each vertical line expresses I SE of the mean value. (A) The effect of the external K concentration on the 3-O-MG uptake in the presence and the absence of insulin. Closed and open circles respectively express the magnitude of the 3-O-MG uptake in the presence and the absence of insulin. Closed squares express the insulin-induced increase in the 3-O-MG uptake (the difference between the magnitude of the 3-O-MG uptake in the presence and the absence of insulin). Insulin increased the 3-O-MG uptake (I and 2 mM K, P < 0.001; 5 and IO mM K, P < 0.005). (B) The effect of the external K concentration on the ratio of the 3-O-MG uptake to that in the absence of insulin. The ratio was estimated by using the datum shown in Fig. IA. 6
0
I
0 5 CKlo CmM)
10
0
I
5
I
I
I
I
10
CKlo CmM)
Fig. 2. The effect of the external K concentration on the insulin-induced change in the membrane potential. (A) The effect of the external K concentration on the membrane potential in the presence and the absence of insulin. Closed and open circles represent the magnitude of the membrane potential in the presence and the absence of insulin, respectively. Each symbol expresses the mean value of 15 experiments. Each 1SE of the mean value is smaller than the radius of the symbol. (B) The effect of the external K concentration on the insulin-induced hyperpolarization. Each open square expresses the magnitude of the insulin-induced hyperpolarization. Each symbol expresses the mean value of 15 experiments. Each vertical line expresses 1 SE of the mean value. Insulin induced a hyperpolarization of the membrane at each external K concentration (I and 2mM, P
334
YOSHINORI MARUNAKAand HIROSHIKITASATO
intracellular Na and K concentrations in insulinapplied muscles respectively tend to decrease and increase as compared with insulin-free muscles (our unpublished data). Therefore, the effect of the insulin on the membrane potential and the 3-O-MG uptake must be measured as soon as possible after addition of insulin lest the intracellular Na and K concentrations and other intracellular events are changed by insulin. Therefore, the 3-O-MG uptake and the membrane potential were measured within 45 min after addition of insulin, which is the minimum time required for insulin of 250 mu/ml to develop a maximal effect on the membrane potential in sartorius muscles of bullfrogs. Kipnis and Parrish (1965) reported that 2-deoxyglucose transport into rat diaphragm decreased as the external K concentration was increased equimolarly at the expense of the extenal Na concentration. Zierler et al. (1985) also reported a similar observation about the effect of the external K concentration on 2-deoxyglucose uptake in rat soleus muscles However, this has not been a universal finding (Bihler and Sawh, 1971; Kohn and Clausen, 1972). Kohn and Clausen (1972) reported that an increase in the external K concentration had no significant effect on 3-O-MG release from rat soleus muscles in the absence of insulin. Various observations about effects of the external K concentration on glucose uptake in muscles. The observations shown in Fig. IA indicate that the magnitude of the 3-O-MG uptake in the absence of insulin is increased by raising the external K concentration of 1 to 10 mM and tends to reach a steady value around the external K concentration of 5 mM. This is a contrary result to the observations in rat muscles reported by Kipnis and Parrish (1965) and Zierler et al. (1985). It may be the difference between 3-O-MC and 2-deoxyglucose or/and the difference between frog muscle and rat muscle. 2-deoxyglucose is phosphorylated in the intracellular space, while 3-O-MG is not phosphorylated even if it is incorporated into the intracellular space. Therefore, the effect of the external K concentration on glucose uptake may be related to the intracellular metabolism. However, the cause for these different observations would not be clearly known. As mentioned above, there are various observations about the effect of the external K concentration on glucose uptake in the absence of insulin, however there is widespread agreement that the stimulatory action of insulin on the transport of glucose or its analogues is reduced or inhibited by a rise of the external K concentration (Kipnis and Parrish, 1965; Zierler er al., 1985). The observation shown in Fig. 1A and B also supports this. However, the cause for the diminution of the stimulatory action of insulin on the 3-O-MG uptake with a rise of the external K concentration has not been clarified. Zierler and Rogus (1980) have suggested that the insulin-induced hyperpolarization would be a mediator of the stimulatory action of insulin on glucose uptake. The observations shown in Fig. 2B indicate that the magnitude of the insulin-induced hyperpolarization decreases with a rise of the external K concentration. It is suggested, as follows: (1) insulin induces a hyperpolarization of the membrane; (2) this hyper-
polarization is a mediator of the stimulatory action of insulin on the 3-O-MG uptake; (3) the magnitude of the insulin-induced hyperpolarization decreases with a rise of the external K concentration; (4) as a result of the decrease in magnitude of the insulininduced hyperpolarization, the stimulatory action of insulin on the 3-O-MG uptake decreases with a rise of the external K concentration. However, a rise of the external K concentration depolarizes the membrane (Fig. 2A) and increases the 3-O-MG uptake (Fig. IA). This is a contrary observation to the suggestion that the hyperpolarization is a mediator of the stimulatory action of insulin on the glucose uptake in skeletal muscles. The stimulation of the 3-O-MG uptake induced by a rise of the external K concentration may not be caused by the depolarization, but by the direct action of a rise of the external K concentration in frog skeletal muscle. This must be investigated under the voltage-clamp, and is clarified in the future. A hyperpolarization of the membrane is surely a mediator of the stimulatory action of insulin on glucose uptake in skeletal muscle (Table 2; Zierler and Rogus, 1980). However, it is unclear that the insulin-induced hyperpolarization is only a mediator of the stimulatory action; i.e. insulin still had a stimulatory action on the 3-O-MG uptake even under the condition that insulin had no effect on the membrane potential (Table 2). Zierler et ~11.(1985) have reported that in rat skeletal muscle the magnitude of the insulin-induced hyperpolarization diminishes with increasing the external K concentration to 36 mM, however the stimulatory action of insulin on glucose uptake remains even at the external K concentration of 36mM, furthermore the action of insulin on glucose uptake is smaller than that at low external K concentration. Their study in rat skeletal muscle supports our observation in frog skeletal muscle. Namely, insulin would affect the 3-O-MG uptake by at least two different mechanisms as follows: (1) one is developed by the insulin-induced hyperpolarization; (2) the other is independent of the membrane potential. The latter may be the translocation of the 3-O-MG transport carrier into the plasma membrane (Cushman and Wardzala, 1980; Suzuki and Kono, 1980; Kono et al., 1982; Simpson et al., 1983). If the 3-O-MG transport carrier is translocated into the plasma membrane by insulin, it must be investigated whether the translocated carrier would be dependent on the membrane potential. To clarify this point, the voltage-dependency of the 3-O-MG uptake in the presence and the absence of insulin was estimated. The absolute value of the voltage-dependency was increased 1.9-fold by the presence of insulin (Fig. 3). Figure 3 and the reports mentioned above (Cushman & Wardzala, 1980; Suzuki and Kono, 1980; Kono et al., 1982; Simpson et al., 1983) show that insulin would increase the number of the voltage-sensitive 3-O-MG transport carrier in the plasma membrane. From the observations shown in the present study, it would be concluded as follows: (1) A part of the insulin action on the 3-O-MC uptake is developed through the insulin-induced
Hyperpolarization
-99
-98
-97 Membrane
and insulin
-96 potential
335
action
-95
-94
-93
(mV)
Fig, 3. The relationship between the membrane potential and the 3-O-MG uptake in the presence and the absence of insulin. The data shown in Fig. 3 are the same as those shown in Table 2. Each symbol expresses the mean value. Each vertical or horizontal line expresses 1 SE of the mean value. The condition that each symbol expresses is as follows: closed circle, insulin (+) and valinomycin (-); closed square, insulin (+) and valinomycin (+); open circle, insulin (-) and valinomycin (-); open square, insulin (-) and valinomycin (+). The upper equation expresses the regression line between the membrane potential and the 3-O-MG uptake in the presence of insulin. The lower equation expresses the regression line between the membrane potential and the 3-O-MG uptake in the absence of insulin. The lines were respectively estimated by using the mean values of the membrane potential and the 3-O-MG uptake in the presence and the absence of insulin with the method of least squares. X and Y respectively express the values of the membrane potential and the 3-O-MG uptake, and I is the coefficient of correlation.
hyperpolarization, and the other part of the effect of insulin is independent of the insulin-induced change in the membrane potential; the membrane potentialindependent action of insulin may be developed through increasing the number of the 3-O-MG transport carrier in the plasma membrane. The translocated carrier would be voltage-sensitive. (2) The diminution of the stimulatory action of insulin on the 3-O-MG uptake with a rise of the external K concentration is explained as follows; the rise of the external K concentration induces a decrease in the magnitude of the insulin-induced hyperpolarization and this decrease causes a diminution of the hyperpolarization-stimulated 3-O-MG uptake. Acknowledgement-This work was supported from the Ministry of Education, Science and Japan.
by grants Culture of
REFERENCES
Bihler I. and Sawh P. C. (1971) Regulation of sugar transport in muscle: effect of increased external potassium in vitro. Biochim. Biophys. Acta 241, 302-309. Cushman S. W. and Wardzala L. J. (1980) Potential mechanism of insulin action on glucose transport in the isolated rat adipose cell. Apparent translocation of intracellular transport systems to the plasma membrane. J. biol. Chem. 255, 47584762. Grinstein S. and Erlij D. (1977) Differences between resting and insulin-stimulated amino acid transport in frog skeletal muscle. J. Membr. Biol. 35, 9-28. Kipnis D. M. and Parrish J. E. (1965) The role of Na and K on sugar (2-deoxyglucose) and amino acid (a-aminobutyric acid) transport in striated muscles. Fedn. Proc. Fedn Am. Sots. exp. Biol. 24, 1051-1059.
Kitasato H.. Marunaka Y., Murayama K. and Nishio K. (1980a) Insulin-sensitive Na efflux and the extracellular K concentration. Proc. of the 18th Annual Meeting of Bionhvs. Sot. of Janan, D. 203 (in JaDanese). Kitasatd H., Sato S.,. Maiunaka ‘Y., Murayama K. and Nishio K. (1980b) Effects of ouabain on Na efflux in high internal Na and insulin-preincubated muscles. Jpn. J. Physiol. 30, 59 I-602. Kitasato H., Sato S., Marunaka Y., Murayama K. and Nishio K. (1980~) Anoarent affinitv chances bv insulin of Na-K transport ‘system in frog skeletalmus~le. Jpn. J. Physiol. 30, 603-6 16. Kohn P. G. and Clausen T. (1972) The relationship between the transport of glucose and cations across cell membranes in isolated tissues. VII. The effects of extracellular Na and K on the transoort of 3-0-methvlelucose and glucose in rat soleus muscle. Biochim. Biopi~;. Acta 255, 798-814. Kono T., Robinson F., Blevins T. L. and Ezaki 0. (1982) Evidence that translocation of the glucose transoort activity is the major mechanism of insulin action on glucose transport in fat cells. J. hiol. Chem. 257, 10942-10947. Marunaka Y. (1986) Effects of the external K concentration the electrogenicity of the insulin-stimulated E,K-pump. J. Membr. Biol. 91, 165-172. Marunaka Y. and Kitasato H. (1985a) The sensitivity of the insulin-stimulated ouabain-sensitive Na+ efflux from frog sartorius muscle to internal Na+, external K+ and ouabain. IRCS med. Sci. 13, 445-6. Marunaka Y. and Kitasato H. (1985b) The inhibitory action of insulin-induced hyperpolarization on 3-O-methyl-oglucose efflux from frog sartorius muscles. IRCS med Sci. 13, 637438. Marunaka Y., Murayama K. and Kitasato H. (1986) A rise of the external K concentration decreases the magnitude of the insulin-induced hyperpolarization by diminishing the electrogemclty of the insulin-stimulated electrogenic Na,K-pump in frog skeletal muscles. Proc. of Inter-
336
YOSHINORI MARUNAKA and
national Symposium on Ion Channels and Electrogenic Pump in Biomembranes, pp. 55556. Moore R. D. and Rabovsky J. L. (1979) Mechanism of insulin action on resting membrane potential of frog skeletal muscle. Am. J. Physiol. 236, C2499C254. Morgan H. E., Randles P. .J. and Regen D. M. (1959) Regulation of glucose uptake by muscle. 3. The effects of insulin, anoxia, salicylate and 2:4-dinitrophenol on membrane transport and intracellular phosphorylation of glucose in isolated rat heart. Bioch&. J. $3, 5733579. Morgan H. E.. Henderson M. J.. Reuen D. M. and Park C.-R. (1961) Regulation of glucose-uptake in muscle. I. The effects of insulin and anoxia on glucose transport and phosphorylation in the isolated, perfused heart of normal rats. J. biol. Chem. 236, 253-261. Simpson I. A., Yver D. R., Hissin P. J., Lawrence J., Wardzala L. J., Karnieli E., Salans L. B. and Cushman S. W. (1983) Insulin-stimulated translocation of glucose transporters in the isolated rat adipose cells: character-
HIROSHI KITASATO
ization of subcellular fractions. Biochim. Biophys. Acts 763, 393407. Suzuki K. and Kono T. (1980) Evidence that insulin causes translocation of glucose transport activity to the plasma membrane from an intracellular storage site. Proc. Null Acad. Sci. USA 77, 2542-2545. Zierler K. L. (1957) Increase in resting membrane potential of skeletal muscle produced by insulin. Science 126, 106771068. Zierler K. L. (1959) Effect of insulin on membrane potential and potassium content of rat muscle. Am. J. Physiol. 197, 515-523. Zierler K. and Rogus E. M. (1980) Hyperpolarization as a mediator of insulin action: increased muscle glucose uptake induced electrically. Am. J. Physiol. 239, E21-E29. Zierler K., Rogus E. M., Scherer R. W. and Wu F. (1985) Insulin action on membrane potential and glucose uptake: effects of high potassium. Am. J. Physiol. 249, E17pE25.