W-7 specifically inhibits insulin-induced increase in glucose transport

Diuhetes Research and Clinical Pructice, 6 ( 1989) 109-I 13

109

Elsevier

DRC 00257

W-7 specifically inhibits insulin-induced increase in glucose transport* 0. Ishibashi, M. Kobayashi, T. Sasaoka, M. Sugibayashi and Y. Shigeta ofMedicine.Shiga

Third Department

(Received

Key words: Insulin:

Insulin

25 January

receptor;

Hexose

University of Medical Science, Setu. Ohtsu. 520-2’, Japan

1988, revision

transport;

received 3 August

Calmodulin;

1988, accepted

16 August

1988)

W-7

Summary To elucidate the role of calmodulin in insulin action, we examined the effect of the calmodulin antagonists, W-7 and W-5, on glucose transport in isolated rat adipocytes. W-7 inhibited insulin-stimulated 2-deoxyglucase uptake by 18% at 100 PM, but it did not affect basal uptake levels. W-5, a less potent analogue of W-7, however, had no significant effect at the same concentration, indicating that the effect was specific to calmodulin. Similar results were observed in a 3-O-methylglucose uptake study. Kinetic analysis of 2-deoxyglucose uptake revealed that W-7 affected the insulin-induced increase in Vma,but not Km. These results suggest that calmodulin modifies insulin action in the glucose transport system.

Introduction Calmodulin is a ubiquitous calcium-binding protein that is known to regulate a number of calcium-dependent enzymatic activities [ 11. Recently, evidence has been accumulating that insulin action is regu-

* This study

was supported

from the Ministry Address partment ta. Ohtsu,

in part

of Education,

for correspondence: of Medicine,

by a research

grant-in-aid

Science and Culture, Dr. M. Kobayashi.

Shiga University

of Medical

De-

Science, Se-

520e2r. Japan.

0168-8227/89/$03.50

Japan.

Third

((;j 1989 Elsevier Science Publishers

lated by calmodulin. Insulin is known to increase intracellular Ca’ + [2] and calmodulin binding to adipocyte plasma membranes [3]. Furthermore, the insulin receptor contains a calmodulin-binding domain [4] and it phosphorylates the tyrosine residues of calmodulin [5]. However, the exact role of calmodulin in insulin action is stil not known. Shechter reported that insulin-stimulated glucose transport in isolated fat cells was inhibited by trifluoperazine, a calmodulin antagonist, suggesting that this insulin action depends on calmodulin [6]. Trifluoperazine’s non-specific action [7], however, ob-

B.V. (Biomedical

Division)

110

scures the interpretation of this report. Hidaka et al. introduced N-(6-aminohexyl)-5-chloro- 1-naphthalenesulfonamide (W-7) and N-(6-aminohexyl)- lnaphthalenesulfonamide (W-S), and showed that these calmodulin antagonists produced a highly specific inhibition of cell proliferation [8]. We examined the effect of these specific calmodulin inhib2-deoxy-o-glucose itors on insulin-stimulated (DeGlc) and 3-O-methyl-o-glucose (MeGlc) transport in isolated rat adipocytes, and analyzed the changes they produced in the kinetic parameters of glucose uptake.

Materials and methods Animals

Five-week-old male Sprague-Dawley rats, fed ad libitum, weighing 120&160 g, were used for all experiments. Materials

Single-peak pork insulin was generously supplied by Shimizu Pharmaceutical Co., Ltd., Shimizu, Japan. Collagenase was purchased from Worthington Biochemical Corp., Freehold, NJ, U.S.A. Bovine albumin (fraction V), DeGlc and MeGlc were from Sigma Chemical Company, St. Louis, MO, U.S.A. 2-[3H]deoxy-D-glucose, 5.0 Ci/mmol, and 3-U[methyl-‘4C]methyl-o-g1ucose, 55 mCi/mmol, were from New England Nuclear, Boston, MA. U.S.A. W-7 and W-5 were from Seikagaku Kogyo Co., Ltd., Tokyo, Japan. Plastic tubes were from BlO Plastics Co., Ltd., Osaka, Japan. Silicone oil, 50 CS. was from Shin-Etsu Chemical Co., Ltd., Tokyo, Japan, All other chemicals and reagents were purchased from Nakarai Chemicals Ltd., Kyoto, Japan. Methods

The following working buffer was used throughout all the experiments: 10 mM 4-(2-hydroxyethyl)-lpiperazineethanesulfonic acid (HEPES) buffer. pH 7.4, containing (in mM) Nat, 140; K’, 4.7; Ca”, 2.5; Mg’+, 1.25; Cl-, 142; HzPO4-, 2.5: and SOd2=. 1.25 [9].

Animals were stunned by a blow to the head, decapitated, and their epididymal fat pads were removed and minced. Isolated adipocytes were prepared by shaking specimens at 37°C for 60 min in 3 ml buffer containing glucose (5.5 mM), collagenase (3 mg/ml), and bovine serum albumin (BSA, 40 mg/ ml) [lo]. Cells were passed through nylon mesh to remove tissue debris, and washed twice in the working buffer. A DeGlc uptake study was performed according to our previous method [11,12] with slight modifications. Preliminary studies revealed that less than 5 ng/ml of insulin would exert submaximal stimulation of DeGlc uptake. Therefore, we incubated cells with or without 25 ng/ml of insulin at 24°C for 60 min before the uptake studies, Approximately 2 x lo5 isolated adipocytes were suspended in 500 ~1 buffer containing 10 mg/ml BSA, and were incubated with the same volume of buffer containing 0.4 &i of [3H]DeG1c and unlabeled DeGlc at the concentrations indicated in the Figures at 24°C. At the end of 3 min, two aliquots of 200 ~1each were transferred from the assay mixture to microsampling tubes containing 150 ~1 of silicone oil. The tubes were centrifuged for 30 s, and the assay was considered terminated when centrifugation began. After centrifugation, the tube tops containing packed cells were excised and were submitted to a liquid scintillation count. The cell number of each assay mixture was ultimately confirmed to correct and standardize the results. A MeGlc uptake study was performed according to the method of Whitesell and Gliemann [9]. The assay was conducted at 37°C in an infant incubator. Adipocytes were suspended in the working buffer containing BSA (10 mg/ml) and insulin at the concentrations indicated in the Figures to 40% packed cell volume (approximately 4 x IO6 cells/ml) and were incubated at 37°C for 10 min. Albumin-free buffer (12 pl), containing 0.1 &i of [‘4C]MeGlc with or without W-7, was placed in the bottom of the tube, and 40 ~1 of cell suspension was squirted precisely onto the isotope at time zero. Timing was carried out using a metronome and the incubation was terminated at the end of 2 s by adding 3 ml of albumin-free buffer containing 0.3 mM phloretin,

111 0.12% ethanol (v/v), and 0.05% dimethyl sulfoxide (v/v). Silicone oil was layered on top of the aqueous phase and the cells were separated by centrifugation. Isolated packed cells floating on the oil layer were recovered with a pipe cleaner and were submitted to a liquid scintillation count. For both studies the amount of sugar trapped in the extracellular water space between cell layers was determined by incubating cells with an albumin-free assay mixture containing 0.03 mM of phloretin. This sugar was measured in each experiment, and all sugar uptake data were corrected for this factor. The insulin binding to adipocytes was measured according to our previous method [12]. Isolated rat adipocytes were incubated by shaking with 0.2 ng/ ml of [1251] insulin and unlabeled insulin at the indicated concentration with or without 100 PM W-7 in the working buffer containing 10 mM D-glucose and 10 mg/ml BSA at 24°C for 90 min. The amount of insulin trapped in the extracellular water space between cell layers (non-specific binding) was determined by incubation with an excessive amount of unlabeled insulin (20 pg/ml). Results Effect of W-7 and W-S upon deoxyglucose uptake DeGlc uptake in isolated rat adipocytes was measured in the presence of W-7 or W-5 during a 3-min assay (Fig. 1). After stimulation with 25 ng/ml of insulin (24°C 60 min), the uptake was inhibited by 100-200 PM of W-7, while W-5 showed no significant effect at the same concentration. The effect of W-7 can be shown as statistically more potent than that of W-5 in this range with a paired t-test. Among cells that had not been stimulated with insulin (basal state study), however, neither W-5 nor W-7 demonstrated any significant effect at the same concentration. We next analyzed the kinetics of DeGlc uptake As shown in Fig. 2, insulin stimulated DeGlc uptake by increasing its capacity (I’,,,,,) but not its affinity (l/&J. This effect of insulin was partially reversed when cells were exposed to 200 PM of W-7 during the assay. That is. the insulin-induced increase of V,,, was inhibited by W-7.

s z

3

&

9

0

0

50

P
9

100

200

Antagonist

a

500 (pM)

Fig. 1. The effect of W-7 and W-5 on 2-deoxyglucose uptake in rat adipocytes. After incubation with 25 ng/ml of insulin (24°C 60 mm), the assay (24°C. 3 min) was conducted with 0.1 mM of unlabeled DeGlc in the presence of W-7 (V j or W-5 (0) at the indicated concentrations (dashed lines). Results are the mean f SE of four separate experiments. P values indicate the significance of difference between the results using W-7 and W-S measured by paired r-tests. Basal state levels are shown by solid lines using the same symbols.

Effect of W-7 upon methylglucose uptake Our MeGlc uptake study produced a similar result (Fig. 3). In this experiment, the cells were exposed to the inhibitor for only a 2-s assay period. Uptake was decreased by 100 ,uM of W-7 after stimulation with l-25 ng/ml of insulin (37°C 10 min), although there was no significant change in basal levels.

v (uptake)

0

t

2

5 s (DeGlc

10 cont.)

Fig. 2. Kinetic analysis of 2-deoxyglucose uptake study in rat adipocytes. The result. shown in the inset, was analyzed using a Lineweaver-Burk plot. The assay was conducted as in Fig. I in the presence (V) or absence (0) of 200 nM of W-7 with unlabeled 2-deoxyglucose at the indicated concentrations.

112 0.4

.-

~~

_...-

Discussion

--o

W-5 is an analogue 0.2

/

v

/

0 0

0.2

0.4

0.8

1.2 Insulin

Fig. 3. The effect of W-7 on 3-0-methylglucose representative

uptake

in rat After

a typical

incubation

with 25 ngjml of insulin (37°C.

(37°C 2 s) was conducted

(“!$rnl)

of three experiments.

adipocytes.

in the presence

IO min), the assay

( V) or absence (~0)of

100 FM of W-7.

Efect

of W-7

upon insulin-receptor

binding

The decreased insulin stimulation of glucose uptake may result from decreased insulin binding caused by the calmodulin inhibitors. Therefore, we also examined the effect of W-7 on insulin-receptor binding. Our binding study, however, did not show decreased binding resulting from W-7 (Fig. 4). Rather, it showed a tendency towards more insulin binding in the presence of 100 PM W-7, although the difference was not statistically significant.

1 -

0 10

1

0.1

I 100

Insulin concenlration tnglml) Fig. 4. The effect of W-7 on insulin-receptor

binding in rat adipo-

cytes. The assay (24°C. 90 min) was conducted in the presence (V) or absence ((_)) of 100 pM W-7. The insulin concentration indicdtes

the sum of labeled

the mean

and unlabeled insulin. Results * SE of four separate experiments.

one of W-7’s

demonstrated that W-7 inhibited DeGlc uptake by rat adipocytes, and that it was significantly more

+---Z concentration

of W-7 lacking

chlorine atoms. Our displacement study of [3H]W-7 from calmodulin revealed W-7 to be seven times more potent than W-5 [13]. Considering that their non-specific activities are the same, this difference in their effects should be specific for calmodulin. We

are

potent than W-5 in concentrations

between

100 and

500 PM. The difference between these two analogues indicates the high specificity of organic systems for calmodulin. Moreover, these inhibitors were much less effective in changing basal state glucose transport, suggesting that they affect insulin action but not the glucose transport system directly. To elucidate this point, any future study should include an experimental system using other agents which stimulate glucose transport, such as phorbol esters. We did not expose cells to the inhibitors during prior incubation with insulin, because prolonged incubation with inhibitors may result in some undesirable cytotoxic effects. These inhibitors, therefore, exerted their effects during the assay period, i.e., 3 min during the DeGlc uptake study, and only 2 s during the MeGlc uptake study. This may be the reason why our experimental system did not show as large effect as did Shechter’s study [6]. Still, we cannot conclude that calmodulin is the major mediator of insulin in the stimulation of glucose transport, because the effect of W-7 was only partial. Thus, calmodulin may only modify insulin action in stimulating glucose transport. Besides, the results indicated that these inhibitors acted very rapidly. Although there is still some debatable ground [14], it is generally accepted that insulin stimulates glucose transport by changing its transport capacity or V,,, [15,16]. Our kinetic analysis also revealed that insulin changed V,,, but not the K,,, of glucose transport. Importantly, our data indicate that W-7 inhibited the insulin-stimulated increase of V,,,. Evidence that calmodulin may be involved in insulin action has been reported. The insulin receptor

113 has a calmodulin-binding domain [4], and calmodulin enhances insulin-stimulated phosphorylation of the /? subunit of the insulin receptor [ 171.Conversely, the tyrosine residues of calmodulin are phosphorylated by the insulin receptor [5]. Together these enzymes may provide a feedback regulation system for insulin action, In this regard, we speculate that the insulin stimulation of glucose transport, but not basal glucose transport, is affected by calmodulin inhibitors. The insulin binding was somewhat high in the presence of W-7 during incubation. One possible explanation is that the receptors had been accumulating in the plasma membrane if their internalization was more extensively impaired than was their insertion into the plasma membrane. Majercik and Bourguignon reported that insulin receptor capping following ligand binding was impaired by calmodulin inhibitors by redistributing myosin light chain kinase (MLCK) beneath the insulin caps in IM-9 lymphocytes [18]. By affecting the cytoskeletal system, calmodulin inhibitors may also impede the transmembrane signaling of insulin receptors. In summary, we demonstrated an insulin-induced increase of V,,, of glucose transport was inhibited by W-7. The results suggest that insulin stimulation of glucose transport depends, although only in part, upon calmodulin-related biological action.

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