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Effect of benzyl succinate on insulin receptor function and insulin action in skeletal muscle: Further evidence for a lack of spare high-affinity insulin receptors
Molecular and Cellular Endocrinology, 91 (1993) 29-33 0 1993 Elsevier Scientific Publishers Ireland, Ltd. 0303-7207/93/$06.00
MOLCEL
29
02901
Effect of benzyl succinate on insulin receptor function and insulin action in skeletal muscle: further evidence for a lack of spare high-affinity insulin receptors A. Gum;, F. Vifials, M. Camps, M. Lizarbe, C. Mora, J. Bertran, X. Testar, M. Palacin and A. Zorzano Departament de Bioquimica i Fisiologia, Facultat de Biologia, Unilaersitatde Barcelona, 08028 Barcelona, Spain (Received
Key words: Spare
insulin
receptor;
System A transport;
3 July 1992; accepted
Glucose
transport;
5 October
Insulin
action;
19921
Muscle;
Adipocyte
Summary
Benzyl succinate inhibited insulin binding and tyrosine receptor kinase in a concentration-dependent manner in the partially purified insulin receptor preparation from rat skeletal muscle. Benzyl succinate lowered the apparent number of high-affinity insulin binding sites. We have made use of the inhibitory effect of benzyl succinate to investigate the possible presence of spare high-affinity insulin receptors in muscle. Benzyl succinate inhibited the effect of a supramaximal concentration of insulin on 3-0-methylglucose uptake, 2-(methylaminojisobutyric acid uptake and lactate production by the incubated muscle. Furthermore, the inhibitory effect of benzyl succinate on insulin binding in vitro closely correlated with its inhibitory effect on insulin action in vivo. These findings suggest the absence of spare high-affinity insulin receptors in skeletal muscle. In contrast to data obtained in skeletal muscle, benzyl succinate did not affect the maximally insulin-stimulated glucose transport, although it caused a marked decrease in insulin sensitivity in isolated rat adipocytes, for which the existence of spare insulin receptors is well documented.
Introduction
The initial step in insulin action involves binding to the receptor. This interaction causes autophosphorylation of the p-subunit of the insulin receptor and stimulation of tyrosine kinase activity, triggering the cascade of biochemical events that lead to the biological effects of insulin. The relationship between insulin binding and insulin action is not straightforward. Thus, in isolated adipocytes the existence of spare insulin receptors has been well documented, based on the following findings: (a) the dose-response curve for insulin action is markedly displaced to the left compared to that of insulin binding (Ip et al., 1976; Kahn, 1978; Krupp and Livingston, 1978; Green and Newsholme, 19791, that is, maximal insulin effect is achieved at a submaximal cell surface insulin receptor occupancy, and (b) partial reduction of insulin binding does not modify maximal response to insulin although it causes a rightward dis-
placement of the dose-response curve (Kono and Barham, 1971). Nevertheless, it is far from clear whether the spare receptor is present in all insulin-sensitive tissues. Thus, in isolated rat hepatocytes it has been reported that the stimulation by insulin of a-aminoisobutyric acid uptake parallels high-affinity receptor occupancy (Fehlmann et al., 1981). Furthermore, in rat skeletal muscle it has been reported that tyrosine receptor kinase parallels occupancy of high-affinity binding sites and that there is a good correlation between activation of tyrosine receptor kinase and insulin action (BryerAsh, 1989; Camps et al., 1992). In this report, we provide additional evidence for an absence of spare high-affinity insulin receptors in skeletal muscle, making use of benzyl succinate, a carboxyl compound that contains a hydroxyphenyl moiety and which inhibits insulin binding. Materials
Correspondence to: Antonio Zorzano, Departament de Bioquimica i Fisiologia, Facultat de Biologia, Universitat de Barcelona, Avda. Diagonal 645, 08028 Barcelona, Spain. Tel. 34-3-4021519; Fax 34-3-402-1559.
and methods
Insulin receptor binding and kinase assays
Hind limb muscle was obtained from pentobarbitalanesthetized rats. Muscles were frozen in liquid nitro-
gen and then homogenized and solubilized in 1% Triton X-100 as described (Camps et al., 1990). The solubilized homogenate was centrifuged at 150,000 x g for 90 min at 4°C and the supernatant was subjected to wheat germ agglutinin (WGA) affinity chromatography. Receptors were eluted from the WGA column with buffer containing 25 mM Hepes, 0.1% Triton X-100 and 0.3 M N-acetyl-o-glucosamine (pH 7.4). Insulin binding was measured as in Camps et al. (1990). WGA eluate (20 ~1) was incubated in 30 mM Hepes containing 0.1% bovine serum albumin (BSA), 100 U/ml bacitracin (pH 7.6, 1 h, 22°C 200 pi>, 20,000 cpm [ ‘2sI-TyrA’4 lmonoiodoinsulin (N 60 pM) and increasing concentrations of unlabelled insulin. Receptors were precipitated with 0.5 ml of bovine gamma globulin (1 mg/ml) and 0.5 ml of polyethylene glycol (25%, w/v>. N on-specific binding was estimated as ‘2”I-insulin bound in the presence of 1 PM insulin (5-10% of total binding). Binding data were expressed per pg of protein, with the latter measured using the method of Bradford (1976). Assays of /?-subunit receptor autophosphorylation were performed as described previously (James et al., 1986). Receptor preparations (lo-40 ~1 of WGA eluate) were preincubated for 1 h at 22°C with increasing insulin concentrations in a buffer containing 30 mM Hepes, 4 mM MnCl,, 10 mM MgCl, (pH 7.6). Phosphorylation was initiated by the addition of 50 PM [Y-~~P]ATP (5-10 r*.Ci) in a final volume of 100 ~1. Autophosphorylation reactions were terminated after incubation for 4 min at 22°C by the addition of an equal volume of Laemmli sample buffer containing 0.1 M dithiothreitol. Samples were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Phosphorylation of an exogenous substrate was carried out with receptor preparations which were preincubated for 1 h in 30 mM Hepes buffer (pH 7.6) containing 50 mM magnesium acetate, 4 mM MnCl,, in the absence or presence of 100 nM insulin. The receptor kinase activity was initially activated by addition of 50 PM [Y-~~P]ATP for 10 min. The reaction was initiated by addition of the exogenous substrate (copolymer of Glu/Tyr, 4: 1, 0.25 mg/ml). The reaction was stopped after 30 min by applying samples to filter paper squares and soaking in 10% trichloroacetic acid containing 10 mM sodium pyrophosphate. Papers were washed, dried and counted by Cerenkov radiation. Animals and dissection procedures
Male Wistar rats (50-60 g) from our own colony were fed on Purina Laboratory chow ad libitum and housed in animal quarters at 22°C with a 12 h light/l2 h dark cycle. The dissection and isolation of the extensor digitorum longus (EDL) and soleus muscles were
carried out under anesthesia with pentobarbital (5-7 mg/lOO g body weight, i.p.> as described (Maizels et al., 1977). Muscle incubations
EDL and soleus muscles were incubated in a shaking incubator at 37°C for 3 h in 3 ml of Krebs-Henseleit buffer (pH 7.4) containing 5 mM glucose, 0.20% BSA and 20 mM Hepes, as described (Guma et al., 1988). Muscles were incubated in the absence or presence of insulin (100 nM, added for the last 60 min of incubation) or benzyl succinate (from 0 to 5 mM, for the last 120 min of incubation). Benzyl succinate was dissolved in dimethyl sulfoxide in a final concentration of 0.5%. Amino acid uptake by system A and glucose transport were measured in muscles using the non-metabolizable amino acid analog 2-(methylamino)isobutyric acid (MeAIB) and 30methylglucose, respectively. Following incubation with insulin and benzyl succinate, muscles were transferred to vials with 1.5 ml of KrebsHenseleit buffer (pH 7.4) containing 5 mM glucose, 0.20% BSA, 20 mM Hepes and 0.1 mM [1-‘4C]2-(methylamino)isobutyric acid (800 pCi/mmol), 1 mM [“Hlmannitol (330 pCi/mmol) and insulin or benzyl succinate at the same concentrations as during the preceding incubation period. The vials were stoppered and incubated at 37°C in a shaking incubator for 30 min. The gas phase in the vials was 95% 0, and 5% CO,. In experiments designed to measure 30methylglucose uptake, muscles were incubated in KrebsHenseleit buffer containing 2 mM pyruvate instead of glucose, and during the last 30 min of incubation, the medium contained 0.1 mM [ “C13-O-methylglucose (800 pCi/mmol) and 1 mM [3H]mannitol (330 &i/mmol). Following incubation, the muscles were processed as previously reported (Gum& et al., 1988). Intracellular concentration of [14C]MeAIB or [14C13-O-methylglucase in the extracellular space was calculated from the total label found in tissue as reported (Guma et al., 1988). Lactate release to the incubation media was measured for the last 30 min of incubation as in Maizels et al. (1977). Adipocyte preparation transport acticity
and
measurement
of glucose
Rat adipocytes were obtained from the epididymal fat of male Wistar rats (180-200 g) by collagenase digestion essentially according to the Rodbell procedure (Rodbell, 1964). All incubations were carried out in Krebs-Ringer buffer (pH 7.4) supplemented with 12.5 mM Hepes, 1% fatty acid free BSA and 2 mM sodium pyruvate. Following their isolation, cells were distributed in plastic vials in a final volume of 2 ml; cell concentrations did not exceed 3 x 105/ml. Incubations were conducted at 37°C in a water bath with shaking (120 cycles/min). Throughout the whole incubation
period, cells were incubated in the presence of 2.5 U/ml of adenosine deaminase. Cells were preincubated for 15 min and then were incubated for 45 min in the absence or presence of 2 mM benzyl succinate. For the last 30 min of this period, cells were incubated in the presence of varying concentrations of insulin (ranging from 0 to 10 nM). 2-Deoxyglucose uptake assays were initiated by the addition to 200 ~1 of the cell suspension (about 5 x lo4 cells) of 50 ~1 of Krebs-Ringer-Hepes buffer containing [ 3H]2-deoxyglucose (0.5 PCi) to give a final concentration of 0.1 mM 2-deoxyglucose. The tubes were incubated at 37°C and the reaction was terminated at 2.5 min by addition of 50 ~1 of cytochalasin b (100 PM final concentration). Immediately, 200 ~1 of samples were pipetted to 150 ~1 of silicone oil and centrifuged at 8000 X g in a microcentrifuge. After centrifugation, tubes were cut and the radioactivity of the supernatants counted. Blank tubes were identical to the experimental ones, except that no cells were present. Blanks never exceeded 5-8% of total radioactivity taken up at 0.1 mM [3H12-deoxyglucose.
A
”
0 1
4
;
6
6
Benzylsuccinate
10
(mM)
r
Results and discussion
Benzyl succinate is a carboxyl compound that contains a hydroxyphenyl moiety and which has been reported to inhibit insulin receptor kinase activity (Schechter et al., 1989). In studies designed to assess the specificity of benzyl succinate action, we found that this compound markedly inhibited insulin binding in partially purified insulin receptor preparations from rat skeletal muscle. In these studies we found that benzyl succinate inhibited insulin binding in a dose-response manner (Fig. 1A); thus, at 2 mM benzyl succinate, binding was inhibited by approximately 50% and at 10 mM inhibition reached 80% (Fig. 1A). The inhibition of insulin binding caused by benzyl succinate was independent of the ionic strength of the medium (data not shown). Tyrosine kinase activity of insulin receptors was also assayed by using the same conditions of temperature and incubation time in the presence of insulin than the conditions employed in insulin binding assays. Benzyl succinate inhibited insulin-stimulated insulin receptor kinase activity (Fig. 1A) and, in fact, the dose-response curves for inhibition of binding and kinase activities were superimposable (Fig. 1A). Benzyl succinate also inhibited insulin-dependent P-subunit autophosphorylation (data not shown). Analysis of binding at different concentrations of insulin allowed us to conclude that 2 mM benzyl succinate (a concentration that causes a semi-maximal inhibition of insulin binding) inhibited insulin binding through a decrease in the number of high-affinity binding sites apparently present in the receptor preparation (Fig. 1B). Our results indicate that reduction in high-affinity
0
2
4
Bound
6
(fmol/pg
6
10
1L
protein)
Fig. 1. Effect of benzyl succinate on insulin binding and insulin receptor kinase activity to solubilized insulin receptors from rat skeletal muscle. Partially purified insulin receptors from rat skeletal muscle were incubated in the absence or presence of different concentrations of benzyl succinate dissolved in dimethyl sulfoxide, in a final concentration of 0.5%. (A1 Insulin binding (ml and insulinstimulated tyrosine kinase activity (0 1 were assessed in the absence or presence of different concentrations of benzyl succinate. The data were expressed as percentage of control (no benzyl succinate) values. Each data point is the mean of triplicate determinations, and results are representative of four different experiments. (B) Insulin binding was assessed at different concentrations of insulin (control, H ; 2 mM benzyl succinate, 01. The data shown are from a representative Scatchard plot. Each point is the mean of triplicate determinations,
binding sites causes a parallel drop in receptor kinase activity in a purified insulin receptor preparation from rat skeletal muscle. These findings are in keeping with previous observations in which a parallelism between activation of insulin receptor tyrosine kinase activity and occupancy of high-affinity binding sites was detected in detergent-solubilized partially purified insulin receptor preparations from rat skeletal muscle (Camps et al., 1992). In all, these observations indicate that full receptor kinase activation requires binding to all highaffinity binding sites in insulin receptor preparations from muscle.
32
We next analyzed the impact of a decrease in the number of active high-affinity insulin binding sites on insulin action in skeletal muscle. To this end, we compared the dose-response curve for inhibition of benzyl succinate on insulin binding obtained using the partially purified receptor preparation (experiments done at 22°C) with the dose-response curves of benzyl succinate on different effects of insulin obtained in the incubated muscle (experiments performed at 37°C) (Fig. 2). Benzyl succinate caused a marked inhibition on insulin-stimulated 3-~-methylglucose uptake and lactate production by EDL muscle, and, in fact, the curves of inhibition of 30methylglucose uptake and lactate production paralleled the dose-response curve of inhibition of insulin binding (Fig. 2A). Identical results were obtained when the effect of insulin on system A transport activity was analyzed. Thus, benzyl succinate inhibited the effect of insulin on MeAIB uptake by EDL and soleus muscles in a dose-dependent manner (Fig. 28). Furthermore, the dose-response curves obtained for EDL and soleus muscles again paralleled the dose-response curve for inhibition of binding found in vitro (Fig. ZB). Benzyl succinate did not alter the intracellular concentration of ATP or of creatine phosphate in the incubated muscle (data not shown). These results favor the view that occupancy of all high-affinity insulin binding sites is required to fully express maximal insulin action; that is, there are no spare high-affinity insulin receptors in skeletal muscle. This conclusion is also in keeping with the following previous observations: (a) the curve of insulin-stimulated receptor kinase activity fits closely with the occupancy of high-affinity binding sites in partially purified receptor preparations from skeletal muscle (Camps et al., 1992); (b) there is a high correlation between insulin-stimulated glucose utilization and receptor kinase activity in rat skeletal muscle in vivo (Bryer-Ash, 19891, i.e., there is no spare receptor kinase activity; and cc> maximal insulin action is obtained in rat skeletal muscle, isolated rat hepatocytes, human fibroblasts or isolated rat thymocytes at insulin concentrations which allow full occupancy of high-affinity binding sites (Goldfine et al., 1972; Hollenberg and Cuatrecasas, 1975; Fehlmann et al., 1981; Camps et al., 1992). As a corollary, we have no evidence for any structural role of Iow-affinity insulin receptors either under normal conditions or when there is a specific reduction in high-affinity insulin binding sites in skeletal muscle. To determine whether the pattern obtained in muscle was different compared to cell types in which the spare-receptor concept had been clearly substantiated, we performed experiments in isolated rat adipocytes. These responded to maximal insulin by stimulating 14-fold glucose transport activity (Fig. 3). Under these conditions, maximahy insulin-stimulated glucose transport was not altered by the presence of 2 mM benzyl
0-l 0
1
2
3
Benzylsuccrnate
4
i
(mM)
O-! 0
1
Benzylsuccinate
i
(mM)
Fig. 2. Effect of benzyl succinate on insulin binding to partially purified insulin receptors and on insulin action in the incubated muscle. Partially purified insulin receptors from rat skeletal muscle were incubated in the absence or presence of different concentrations of benzyl succinate and ins&n binding was assessed as explained in Materials and methods. EDL and soleus muscles were incubated for 180 mitt in the absence or presence of 100 nM insulin and different concentrations of benzyl succinate. Insulin action was defined as the increment in uptake or lactate production (insulinbasal) and data were expressed as a percentage of control group (no be& succinate group). Results are means +SE for 6-10 observations per group. Panel A: n , insulin binding; 0, insulin-stimulated 3-O-methylglucose uptake by EDL muscle; A, insulin-stimulated lactate production by EDL muscle. Panel B: n , insulin binding; 0, insuIin-stimulated MeAIB uptake by EDL muscle; A, insulin-stimulated MeAIB uptake by soleus muscle.
succinate (Fig. 3). However, benzyl succinate caused a marked displacement to the right in the dose-response curve of insulin-stimulating glucose transport in the adipocyte (Fig. 3). These data are in agreement with Kono and Barham (1971) who described that reduction in insulin binding down to 90% of normal levels causes a decrease in insulin sensitivity, with no change in maximal insulin responsiveness. Thus, adipocytes behave according to the presence of spare insulin recep-
33
-
I
0
1
10
1
Insulin
This work was supported in part by research grants from the DGICYT (PB86/573 and PB89/0331) and from Fondo de Investigaciones Sanitarias (89/0174), Spain. Porcine monocomponent insulin was a gift from Mr. T.L. Jeatran, Eli Lilly. A.G., F.V., M.C. and J.B. are recipients of predoctoral fellowships from the Ministerio de Education y Ciencia, Spain. F.V. was a recipient of a grant from CIRIT (Generalitat de Catalunya).
(nM)
Fig. 3. Effect of benzyl succinate on insulin-stimulated glucose transport by isolated rat adipocytes. Isolated rat adipocytes were incubated for 45 mitt in the absence ( n 1 or presence (0) of 2 mM benzyl succinate and during the last 30 min were incubated in the presence of varying concentrations of insulin (ranging from 0 to 10 nM). Glucose transport was determined by the 2-deoxyglucose uptake technique (see Materials and methods). Data were expressed as pmol 2-deoxyglucose taken up per lo6 cells/s. Each data point is the mean of triplicate estimations, and the results shown are representative of four different experiments.
tom, that is, a reduction in high-affini~ insulin binding sites does not alter maximal response to insulin although it markedly reduces insulin sensitivity. Based on the fact that the dose-response curves of insulin action in rat adipocytes are displaced to the left compared to the dose-response curve of insulin-stimulated receptor kinase activity obtained both in vivo and in vitro (Arsenis and Livingston, 1986; Klein et al., 1986, 1990, we suggest that the spare-receptor phenomenon of the rat adipocyte is due to post-receptor events. Therefore, differences between isolated rat adipocytes and skeletal muscle might lie at a post-receptor level. In summary, we have shown that benzyl succinate, which causes a decrease in the number of high-affinity insulin binding sites in receptor preparations purified from skeletal muscle, also provokes a reduction in the maximal response to insulin action in the incubated intact muscle. Our data allow us to propose that there are no spare high-affinity insulin binding sites or spare-receptor kinase activity in skeletal muscle, in contrast to what is found in isolated rat adipocytes.
References Arsenis, Ci. and Livingston, J.N. (1986) J. Biol. Chem. 261, 147-153. Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. Bryer-Ash, M. (1989) Diabetes 38, 108-116. Camps, M., Guma, A., Testar, X., Palacin, M. and Zorzano, A. (1990) Endocrinology 127, 2561-2570. Camps, M., Guma, A., Viiials, F., Testar, X., Palacin, M. and Zorzano, A. (39921 Biochem. J. 285, 993-999. Fehlmann, M., Morin, O., Kitabgi, P. and Freychet, P. (1981) Endocrinology 109, 2.53-261. Goldfine, I.D., Gardner, J.D., Neviile, Jr., D.M. (1972) J. Biol. Chem. 247, 6919-6926. Green, A. and Newsholme, E.A. (1979) Biochem. J. 180, 365-370. Gum&, A., Testar, X., Palacin, M. and Zorzano, A. (1988) Biochem. J. 253, 625-629. Hollenberg, M.D. and Cuatrecasas, P. (1975) J. Biol. Chem. 250, 3845-3853. Ip, C., Tepperman, H.M., Holohan, P. and Tepperman, J. (1976) J. Lipid Res. 17, 588-599. James, D.E., Zorzano, A., B&i-Schnetzler, M., Nemenoff, R.A., Powers, A., Pitch, P.F. and Ruderman, N.B. (1986) J. Biol. Chem. 261, 14939-14944. Kahn, CR. (1978) Metabolism 27, 1893-1902. Klein, H.H., Freidenberg, G.R., Kladde, M. and Olefsky, J.M. (1986) J. Biol. Chem. 261,4691-4697. Klein, H.H., Matthaei, S., Drenkhan, M., Ries, W. and Scriba, PC. (1991) Biochem. J. 274, 787-792. Kono, T. and Barham, F.W. (19711 J. Biol. Chem. 246, 6210-6216. Krupp, M.N. and Livingston, J.N. (1978) Proc. Natl. Acad. Sci. USA 75, 2593-2597. Maizels, E.Z., Ruderman, N.B., Godman, M.N. and Lau, D. (1977) Biochem. J. 162, 557-568. Rodbell, M. (1964) J. Biol. Chem. 239, 375-380. Schechter. Y., Yaish, P., Chorev, M., Gilon, C., Braun, S. and Levitski, A. (1989) EMBO J. 8, 1671-1676.
MOLCEL
29
02901
Effect of benzyl succinate on insulin receptor function and insulin action in skeletal muscle: further evidence for a lack of spare high-affinity insulin receptors A. Gum;, F. Vifials, M. Camps, M. Lizarbe, C. Mora, J. Bertran, X. Testar, M. Palacin and A. Zorzano Departament de Bioquimica i Fisiologia, Facultat de Biologia, Unilaersitatde Barcelona, 08028 Barcelona, Spain (Received
Key words: Spare
insulin
receptor;
System A transport;
3 July 1992; accepted
Glucose
transport;
5 October
Insulin
action;
19921
Muscle;
Adipocyte
Summary
Benzyl succinate inhibited insulin binding and tyrosine receptor kinase in a concentration-dependent manner in the partially purified insulin receptor preparation from rat skeletal muscle. Benzyl succinate lowered the apparent number of high-affinity insulin binding sites. We have made use of the inhibitory effect of benzyl succinate to investigate the possible presence of spare high-affinity insulin receptors in muscle. Benzyl succinate inhibited the effect of a supramaximal concentration of insulin on 3-0-methylglucose uptake, 2-(methylaminojisobutyric acid uptake and lactate production by the incubated muscle. Furthermore, the inhibitory effect of benzyl succinate on insulin binding in vitro closely correlated with its inhibitory effect on insulin action in vivo. These findings suggest the absence of spare high-affinity insulin receptors in skeletal muscle. In contrast to data obtained in skeletal muscle, benzyl succinate did not affect the maximally insulin-stimulated glucose transport, although it caused a marked decrease in insulin sensitivity in isolated rat adipocytes, for which the existence of spare insulin receptors is well documented.
Introduction
The initial step in insulin action involves binding to the receptor. This interaction causes autophosphorylation of the p-subunit of the insulin receptor and stimulation of tyrosine kinase activity, triggering the cascade of biochemical events that lead to the biological effects of insulin. The relationship between insulin binding and insulin action is not straightforward. Thus, in isolated adipocytes the existence of spare insulin receptors has been well documented, based on the following findings: (a) the dose-response curve for insulin action is markedly displaced to the left compared to that of insulin binding (Ip et al., 1976; Kahn, 1978; Krupp and Livingston, 1978; Green and Newsholme, 19791, that is, maximal insulin effect is achieved at a submaximal cell surface insulin receptor occupancy, and (b) partial reduction of insulin binding does not modify maximal response to insulin although it causes a rightward dis-
placement of the dose-response curve (Kono and Barham, 1971). Nevertheless, it is far from clear whether the spare receptor is present in all insulin-sensitive tissues. Thus, in isolated rat hepatocytes it has been reported that the stimulation by insulin of a-aminoisobutyric acid uptake parallels high-affinity receptor occupancy (Fehlmann et al., 1981). Furthermore, in rat skeletal muscle it has been reported that tyrosine receptor kinase parallels occupancy of high-affinity binding sites and that there is a good correlation between activation of tyrosine receptor kinase and insulin action (BryerAsh, 1989; Camps et al., 1992). In this report, we provide additional evidence for an absence of spare high-affinity insulin receptors in skeletal muscle, making use of benzyl succinate, a carboxyl compound that contains a hydroxyphenyl moiety and which inhibits insulin binding. Materials
Correspondence to: Antonio Zorzano, Departament de Bioquimica i Fisiologia, Facultat de Biologia, Universitat de Barcelona, Avda. Diagonal 645, 08028 Barcelona, Spain. Tel. 34-3-4021519; Fax 34-3-402-1559.
and methods
Insulin receptor binding and kinase assays
Hind limb muscle was obtained from pentobarbitalanesthetized rats. Muscles were frozen in liquid nitro-
gen and then homogenized and solubilized in 1% Triton X-100 as described (Camps et al., 1990). The solubilized homogenate was centrifuged at 150,000 x g for 90 min at 4°C and the supernatant was subjected to wheat germ agglutinin (WGA) affinity chromatography. Receptors were eluted from the WGA column with buffer containing 25 mM Hepes, 0.1% Triton X-100 and 0.3 M N-acetyl-o-glucosamine (pH 7.4). Insulin binding was measured as in Camps et al. (1990). WGA eluate (20 ~1) was incubated in 30 mM Hepes containing 0.1% bovine serum albumin (BSA), 100 U/ml bacitracin (pH 7.6, 1 h, 22°C 200 pi>, 20,000 cpm [ ‘2sI-TyrA’4 lmonoiodoinsulin (N 60 pM) and increasing concentrations of unlabelled insulin. Receptors were precipitated with 0.5 ml of bovine gamma globulin (1 mg/ml) and 0.5 ml of polyethylene glycol (25%, w/v>. N on-specific binding was estimated as ‘2”I-insulin bound in the presence of 1 PM insulin (5-10% of total binding). Binding data were expressed per pg of protein, with the latter measured using the method of Bradford (1976). Assays of /?-subunit receptor autophosphorylation were performed as described previously (James et al., 1986). Receptor preparations (lo-40 ~1 of WGA eluate) were preincubated for 1 h at 22°C with increasing insulin concentrations in a buffer containing 30 mM Hepes, 4 mM MnCl,, 10 mM MgCl, (pH 7.6). Phosphorylation was initiated by the addition of 50 PM [Y-~~P]ATP (5-10 r*.Ci) in a final volume of 100 ~1. Autophosphorylation reactions were terminated after incubation for 4 min at 22°C by the addition of an equal volume of Laemmli sample buffer containing 0.1 M dithiothreitol. Samples were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Phosphorylation of an exogenous substrate was carried out with receptor preparations which were preincubated for 1 h in 30 mM Hepes buffer (pH 7.6) containing 50 mM magnesium acetate, 4 mM MnCl,, in the absence or presence of 100 nM insulin. The receptor kinase activity was initially activated by addition of 50 PM [Y-~~P]ATP for 10 min. The reaction was initiated by addition of the exogenous substrate (copolymer of Glu/Tyr, 4: 1, 0.25 mg/ml). The reaction was stopped after 30 min by applying samples to filter paper squares and soaking in 10% trichloroacetic acid containing 10 mM sodium pyrophosphate. Papers were washed, dried and counted by Cerenkov radiation. Animals and dissection procedures
Male Wistar rats (50-60 g) from our own colony were fed on Purina Laboratory chow ad libitum and housed in animal quarters at 22°C with a 12 h light/l2 h dark cycle. The dissection and isolation of the extensor digitorum longus (EDL) and soleus muscles were
carried out under anesthesia with pentobarbital (5-7 mg/lOO g body weight, i.p.> as described (Maizels et al., 1977). Muscle incubations
EDL and soleus muscles were incubated in a shaking incubator at 37°C for 3 h in 3 ml of Krebs-Henseleit buffer (pH 7.4) containing 5 mM glucose, 0.20% BSA and 20 mM Hepes, as described (Guma et al., 1988). Muscles were incubated in the absence or presence of insulin (100 nM, added for the last 60 min of incubation) or benzyl succinate (from 0 to 5 mM, for the last 120 min of incubation). Benzyl succinate was dissolved in dimethyl sulfoxide in a final concentration of 0.5%. Amino acid uptake by system A and glucose transport were measured in muscles using the non-metabolizable amino acid analog 2-(methylamino)isobutyric acid (MeAIB) and 30methylglucose, respectively. Following incubation with insulin and benzyl succinate, muscles were transferred to vials with 1.5 ml of KrebsHenseleit buffer (pH 7.4) containing 5 mM glucose, 0.20% BSA, 20 mM Hepes and 0.1 mM [1-‘4C]2-(methylamino)isobutyric acid (800 pCi/mmol), 1 mM [“Hlmannitol (330 pCi/mmol) and insulin or benzyl succinate at the same concentrations as during the preceding incubation period. The vials were stoppered and incubated at 37°C in a shaking incubator for 30 min. The gas phase in the vials was 95% 0, and 5% CO,. In experiments designed to measure 30methylglucose uptake, muscles were incubated in KrebsHenseleit buffer containing 2 mM pyruvate instead of glucose, and during the last 30 min of incubation, the medium contained 0.1 mM [ “C13-O-methylglucose (800 pCi/mmol) and 1 mM [3H]mannitol (330 &i/mmol). Following incubation, the muscles were processed as previously reported (Gum& et al., 1988). Intracellular concentration of [14C]MeAIB or [14C13-O-methylglucase in the extracellular space was calculated from the total label found in tissue as reported (Guma et al., 1988). Lactate release to the incubation media was measured for the last 30 min of incubation as in Maizels et al. (1977). Adipocyte preparation transport acticity
and
measurement
of glucose
Rat adipocytes were obtained from the epididymal fat of male Wistar rats (180-200 g) by collagenase digestion essentially according to the Rodbell procedure (Rodbell, 1964). All incubations were carried out in Krebs-Ringer buffer (pH 7.4) supplemented with 12.5 mM Hepes, 1% fatty acid free BSA and 2 mM sodium pyruvate. Following their isolation, cells were distributed in plastic vials in a final volume of 2 ml; cell concentrations did not exceed 3 x 105/ml. Incubations were conducted at 37°C in a water bath with shaking (120 cycles/min). Throughout the whole incubation
period, cells were incubated in the presence of 2.5 U/ml of adenosine deaminase. Cells were preincubated for 15 min and then were incubated for 45 min in the absence or presence of 2 mM benzyl succinate. For the last 30 min of this period, cells were incubated in the presence of varying concentrations of insulin (ranging from 0 to 10 nM). 2-Deoxyglucose uptake assays were initiated by the addition to 200 ~1 of the cell suspension (about 5 x lo4 cells) of 50 ~1 of Krebs-Ringer-Hepes buffer containing [ 3H]2-deoxyglucose (0.5 PCi) to give a final concentration of 0.1 mM 2-deoxyglucose. The tubes were incubated at 37°C and the reaction was terminated at 2.5 min by addition of 50 ~1 of cytochalasin b (100 PM final concentration). Immediately, 200 ~1 of samples were pipetted to 150 ~1 of silicone oil and centrifuged at 8000 X g in a microcentrifuge. After centrifugation, tubes were cut and the radioactivity of the supernatants counted. Blank tubes were identical to the experimental ones, except that no cells were present. Blanks never exceeded 5-8% of total radioactivity taken up at 0.1 mM [3H12-deoxyglucose.
A
”
0 1
4
;
6
6
Benzylsuccinate
10
(mM)
r
Results and discussion
Benzyl succinate is a carboxyl compound that contains a hydroxyphenyl moiety and which has been reported to inhibit insulin receptor kinase activity (Schechter et al., 1989). In studies designed to assess the specificity of benzyl succinate action, we found that this compound markedly inhibited insulin binding in partially purified insulin receptor preparations from rat skeletal muscle. In these studies we found that benzyl succinate inhibited insulin binding in a dose-response manner (Fig. 1A); thus, at 2 mM benzyl succinate, binding was inhibited by approximately 50% and at 10 mM inhibition reached 80% (Fig. 1A). The inhibition of insulin binding caused by benzyl succinate was independent of the ionic strength of the medium (data not shown). Tyrosine kinase activity of insulin receptors was also assayed by using the same conditions of temperature and incubation time in the presence of insulin than the conditions employed in insulin binding assays. Benzyl succinate inhibited insulin-stimulated insulin receptor kinase activity (Fig. 1A) and, in fact, the dose-response curves for inhibition of binding and kinase activities were superimposable (Fig. 1A). Benzyl succinate also inhibited insulin-dependent P-subunit autophosphorylation (data not shown). Analysis of binding at different concentrations of insulin allowed us to conclude that 2 mM benzyl succinate (a concentration that causes a semi-maximal inhibition of insulin binding) inhibited insulin binding through a decrease in the number of high-affinity binding sites apparently present in the receptor preparation (Fig. 1B). Our results indicate that reduction in high-affinity
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1L
protein)
Fig. 1. Effect of benzyl succinate on insulin binding and insulin receptor kinase activity to solubilized insulin receptors from rat skeletal muscle. Partially purified insulin receptors from rat skeletal muscle were incubated in the absence or presence of different concentrations of benzyl succinate dissolved in dimethyl sulfoxide, in a final concentration of 0.5%. (A1 Insulin binding (ml and insulinstimulated tyrosine kinase activity (0 1 were assessed in the absence or presence of different concentrations of benzyl succinate. The data were expressed as percentage of control (no benzyl succinate) values. Each data point is the mean of triplicate determinations, and results are representative of four different experiments. (B) Insulin binding was assessed at different concentrations of insulin (control, H ; 2 mM benzyl succinate, 01. The data shown are from a representative Scatchard plot. Each point is the mean of triplicate determinations,
binding sites causes a parallel drop in receptor kinase activity in a purified insulin receptor preparation from rat skeletal muscle. These findings are in keeping with previous observations in which a parallelism between activation of insulin receptor tyrosine kinase activity and occupancy of high-affinity binding sites was detected in detergent-solubilized partially purified insulin receptor preparations from rat skeletal muscle (Camps et al., 1992). In all, these observations indicate that full receptor kinase activation requires binding to all highaffinity binding sites in insulin receptor preparations from muscle.
32
We next analyzed the impact of a decrease in the number of active high-affinity insulin binding sites on insulin action in skeletal muscle. To this end, we compared the dose-response curve for inhibition of benzyl succinate on insulin binding obtained using the partially purified receptor preparation (experiments done at 22°C) with the dose-response curves of benzyl succinate on different effects of insulin obtained in the incubated muscle (experiments performed at 37°C) (Fig. 2). Benzyl succinate caused a marked inhibition on insulin-stimulated 3-~-methylglucose uptake and lactate production by EDL muscle, and, in fact, the curves of inhibition of 30methylglucose uptake and lactate production paralleled the dose-response curve of inhibition of insulin binding (Fig. 2A). Identical results were obtained when the effect of insulin on system A transport activity was analyzed. Thus, benzyl succinate inhibited the effect of insulin on MeAIB uptake by EDL and soleus muscles in a dose-dependent manner (Fig. 28). Furthermore, the dose-response curves obtained for EDL and soleus muscles again paralleled the dose-response curve for inhibition of binding found in vitro (Fig. ZB). Benzyl succinate did not alter the intracellular concentration of ATP or of creatine phosphate in the incubated muscle (data not shown). These results favor the view that occupancy of all high-affinity insulin binding sites is required to fully express maximal insulin action; that is, there are no spare high-affinity insulin receptors in skeletal muscle. This conclusion is also in keeping with the following previous observations: (a) the curve of insulin-stimulated receptor kinase activity fits closely with the occupancy of high-affinity binding sites in partially purified receptor preparations from skeletal muscle (Camps et al., 1992); (b) there is a high correlation between insulin-stimulated glucose utilization and receptor kinase activity in rat skeletal muscle in vivo (Bryer-Ash, 19891, i.e., there is no spare receptor kinase activity; and cc> maximal insulin action is obtained in rat skeletal muscle, isolated rat hepatocytes, human fibroblasts or isolated rat thymocytes at insulin concentrations which allow full occupancy of high-affinity binding sites (Goldfine et al., 1972; Hollenberg and Cuatrecasas, 1975; Fehlmann et al., 1981; Camps et al., 1992). As a corollary, we have no evidence for any structural role of Iow-affinity insulin receptors either under normal conditions or when there is a specific reduction in high-affinity insulin binding sites in skeletal muscle. To determine whether the pattern obtained in muscle was different compared to cell types in which the spare-receptor concept had been clearly substantiated, we performed experiments in isolated rat adipocytes. These responded to maximal insulin by stimulating 14-fold glucose transport activity (Fig. 3). Under these conditions, maximahy insulin-stimulated glucose transport was not altered by the presence of 2 mM benzyl
0-l 0
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Benzylsuccrnate
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i
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O-! 0
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Benzylsuccinate
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Fig. 2. Effect of benzyl succinate on insulin binding to partially purified insulin receptors and on insulin action in the incubated muscle. Partially purified insulin receptors from rat skeletal muscle were incubated in the absence or presence of different concentrations of benzyl succinate and ins&n binding was assessed as explained in Materials and methods. EDL and soleus muscles were incubated for 180 mitt in the absence or presence of 100 nM insulin and different concentrations of benzyl succinate. Insulin action was defined as the increment in uptake or lactate production (insulinbasal) and data were expressed as a percentage of control group (no be& succinate group). Results are means +SE for 6-10 observations per group. Panel A: n , insulin binding; 0, insulin-stimulated 3-O-methylglucose uptake by EDL muscle; A, insulin-stimulated lactate production by EDL muscle. Panel B: n , insulin binding; 0, insuIin-stimulated MeAIB uptake by EDL muscle; A, insulin-stimulated MeAIB uptake by soleus muscle.
succinate (Fig. 3). However, benzyl succinate caused a marked displacement to the right in the dose-response curve of insulin-stimulating glucose transport in the adipocyte (Fig. 3). These data are in agreement with Kono and Barham (1971) who described that reduction in insulin binding down to 90% of normal levels causes a decrease in insulin sensitivity, with no change in maximal insulin responsiveness. Thus, adipocytes behave according to the presence of spare insulin recep-
33
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I
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Insulin
This work was supported in part by research grants from the DGICYT (PB86/573 and PB89/0331) and from Fondo de Investigaciones Sanitarias (89/0174), Spain. Porcine monocomponent insulin was a gift from Mr. T.L. Jeatran, Eli Lilly. A.G., F.V., M.C. and J.B. are recipients of predoctoral fellowships from the Ministerio de Education y Ciencia, Spain. F.V. was a recipient of a grant from CIRIT (Generalitat de Catalunya).
(nM)
Fig. 3. Effect of benzyl succinate on insulin-stimulated glucose transport by isolated rat adipocytes. Isolated rat adipocytes were incubated for 45 mitt in the absence ( n 1 or presence (0) of 2 mM benzyl succinate and during the last 30 min were incubated in the presence of varying concentrations of insulin (ranging from 0 to 10 nM). Glucose transport was determined by the 2-deoxyglucose uptake technique (see Materials and methods). Data were expressed as pmol 2-deoxyglucose taken up per lo6 cells/s. Each data point is the mean of triplicate estimations, and the results shown are representative of four different experiments.
tom, that is, a reduction in high-affini~ insulin binding sites does not alter maximal response to insulin although it markedly reduces insulin sensitivity. Based on the fact that the dose-response curves of insulin action in rat adipocytes are displaced to the left compared to the dose-response curve of insulin-stimulated receptor kinase activity obtained both in vivo and in vitro (Arsenis and Livingston, 1986; Klein et al., 1986, 1990, we suggest that the spare-receptor phenomenon of the rat adipocyte is due to post-receptor events. Therefore, differences between isolated rat adipocytes and skeletal muscle might lie at a post-receptor level. In summary, we have shown that benzyl succinate, which causes a decrease in the number of high-affinity insulin binding sites in receptor preparations purified from skeletal muscle, also provokes a reduction in the maximal response to insulin action in the incubated intact muscle. Our data allow us to propose that there are no spare high-affinity insulin binding sites or spare-receptor kinase activity in skeletal muscle, in contrast to what is found in isolated rat adipocytes.
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