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The inhibition of the action of insulin on rat epididymal adipose tissue by sulfhydryl blocking agents
49 °
BIOCHIMICA ET BIOPHYSICA ACTA
BBA
3668
T H E I N H I B I T I O N OF T H E ACTION O F I N S U L I N ON R A T E P I D I D Y M A L A D I P O S E T I S S U E BY S U L F H Y D R Y L B L O C K I N G AGENTS* I. ARTHUR MIRSKY AND GLADYS PERISUTTI Department of Clinical Science, University of Pittsburgh School of Medicine, Pittsburgh, Pa. ( U.S.A .) (Received January 3oth, 1962)
SUMMARY Concentrations of iodoacetate and N-ethylmaleimide which produce no significant effect on the spontaneous oxidation of glucose by rat epididymal adipose tissue in vitro inhibit the action of insulin. These agents do not inhibit the binding of insulin to adipose tissue or to the isolated diaphragm in vitro.
INTRODUCTION The demonstration that synthetic oxytocin and related disulfide peptides with oxytocic activity exert an insulin-like action on rat epididymal adipose tissue 1, ~ is in accord with the evidence that the action of insulin is dependent in part upon its disulfide linkages 3,4. A mechanism whereby the disulfide bridges may be involved is suggested by the cogent evidence that both the binding and the action of vasopressin on the rat kidney and toad bladder are dependent upon an interaction between the disulfide of the hormone and a sulfhydryl group of some specific receptor protein 5-~. Accordingly, it was proposed that the initiation of a series of thiol-disulfide reactions produces alterations in the tertiary structure of the membrane proteins and thereby open channels for the passage of water and specific solutes 7. A similar physical mechanism could account for the accelerated influx of glucose into the cell which occurs in the presence of insulin s. In support of the possibility that the action of insulin is due to some thioldisulfide interchange is the observation that pretreatment of the isolated rat diaphragm with concentrations of iodoacetate or p-chloromercuribenzoate which produce no significant changes in glucose uptake, nevertheless inhibit the action of insulin 9. In accord is the recent preliminary report that the action of insulin on the perfused rat heart is inhibited by N-ethylmaleimide as is also the "binding" of E131Ilinsulin to the hearO °. Likewise, the observation that the total sulfhydryl groups of the isolated rat diaphragm are decreased about 22 % after incubation with insulin n favors the hypothesis that the disulfides of insulin combine with the sulfhydryls of the proteins that comprise the receptor sites on the surface of the cellular membrane. Since the epididymal adipose tissue of the rat exhibits an exquisite sensitivity to the action of insulin in vitro and unlike the isolated diaphragm responds in a * With the technical assistance of D. L. BINLEY. Biochim. Biophys. Acta, 62 (1962) 49o-496
INHIBITION OF INSULIN ACTION
491
similar manner to oxytocin 2, it became pertinent to determine whether the reaction of this tissue is influenced b y sulfhydryl blocking agents. MATERIALS AND METHODS
Fed Carworth rats, weighing lOO-15o g were decapitated, exsanguinated and their epididymal fat pads excised. Each fat pad was cut into two pieces, the pieces from one side were used as controls for those from the other side in a balanced segment design similar to that of SHEPS et al. 12 but employing two instead of three segments from each side. The subsequent treatment of the tissues and the analytical procedures employed were the same as those reported in detail elsewhere 2. The adipose tissues were incubated for 3 h at 37 ° in 2 ml Krebs-Ringer bicarbonate buffer solution (pH 7.4) containing 20/*moles glucose, 0.084/*C ~I-14Clglucose, 0.2 % gelatin in the absence and presence of various concentrations of iodoacetate or N-ethylmaleimide, and in the absence and presence of o.I unit insulin/ml. In other experiments, the adipose tissues were incubated at 37 ° for 5 min in 2 ml K r e b s Ringer buffer without and with the sulfhydryl blocking agents, washed twice for I min in 2 ml fresh buffer solution, blotted, weighed and then incubated for 3 h in the buffer solution containing glucose without and with insulin. At the end of the incubation period the reaction was stopped with o.2 ml IO N H2SO 4 and the C09. collected in Hyamine IO X; the latter was transferred to counting vials containing 15 ml of a liquid scintillator and counted b y means of a Packard Automatic Spectrometer. The initial and final glucose concentrations and their specific activities were determined as described elsewhere ~. All counts were corrected for quenching, background and variations in counting efficiency. The data are expressed as/,moles of glucose oxidized to 14C02/g wet weight of adipose tissue. The "binding" of insulin to adipose tissue and hemidiaphragms was determined b y incubating the tissues for 5 min at 37 ! in the buffer containing 0.2 % gelatin in the absence and presence of lO -3 M iodoacetate or IO-s M N-ethylmaleimide. After two washes for I min each in 2 ml fresh buffer, the tissues were blotted and incubated for 2 rain in I ml buffer containing o.1 unit insulin and I/*C of a trace quantity of very high specific activity [131I]insulin prepared b y the procedure of BERSON et al. ~3. Subsequently, the tissues were washed with 0. 9 % saline as described by STADIE et al. ~, viz., three washings for 30 sec each in 25 ml of saline and one washing for 15 min in 50 ml saline. More extensive washing did not change the results. The tissues were blotted, weighed, placed into the bottom of test-tubes and their radioactivity determined by means of a well-type 7-scintillation counter. The quantity of insulin "bound" to the tissues was expressed as the percentage of the total radioactivity of the incubation mixture. In some instances, the tissues were pretreated as above and then processed b y the procedure described b y SCHWARTZ et al. ~, viz., boiled in water containing io -s M N-ethylmaleimide, washed successively in I M :NaC1, o.15 M NaHCO3 (pH 8.5) and sodium acetate buffer (pH 4.0) and then incubated for 8 h in 3.0 ml of o.I M cysteine, in o.15 M NaHCO 3 (pH 8.5) in a nitrogen atmosphere. In several experiments, the adipose tissue and hemidiaphragm from one side were heated in buffer for 5 min at IOO° while the tissues from the other side were maintained in buffer at 37 °. Subsequently, the heated and unheated tissues were incubated in buffer containing insulin and washed as above. Biochim. Biophys. Acta, 62 (1962) 490-496
492
I . A . MIRSKY, G. PERISUTTI RESULTS
The data summarized in Figs. I and 2 reveal the effect of various concentrations of iodoacetate on the uptake of glucose and the production of 14C0~ from [i-14Clglucose b y the adipose tissue in vitro. I t is apparent that in the presence of a concentration of iodoacetate which has no significant effect on the spontaneous uptake of glucose, the stimulating effect of o.I unit of insulin/ml is partially inhibited. Thus, whereas
'4-11 13 i2
II
IO
7
,-
6
2-
~
I
I nt4.din Number
-- + +J8 5x6 s
-
--
"4"
--
--
+
+
16 fxlO"5
--
+
12-- + ixlO-4
~6
5xl(~ 5
Iodoocetote M
Fig. i. Effect of i o d o a c e t a t e on u p t a k e of glucose b y adipose t i s s u e in a b s e n c e a n d presence of insulin. W h i t e c o l u m n s e q u a l controls i n c u b a t e d in t h e a b s e n c e of i o d o a c e t a t e a n d s h a d e d c o l u m n s equal controls i n c u b a t e d in t h e presence of iodoacetate.
t25
,~
20
5
Insulin 0 Number
-
+
18
5x=o-e
+
-
-I-
-
+
•
-
+
-
+
-
+
-
16
16
12
I x I 0"s
5 x I 0"s
[ x I 0"4
+
Iodoocetote M
Fig. 2. Effect of i o d o a c e t a t e on o x i d a t i o n of [I-14C]glucose to 14CO 2 b y adipose tissue in a b s e n c e a n d presence of insulin. W h i t e c o l u m n s equal controls i n c u b a t e d in t h e a b s e n c e of i o d o a c e t a t e a n d s h a d e d c o l u m n s equal controls i n c u b a t e d in t h e presence of iodoacetate.
10 -5 M iodoacetate exerts no effect on the uptake of glucose by the adipose tissue it produces some inhibition of the action of insulin (Fig. I). In the presence of greater concentrations of iodoacetate there is an appreciable impairment in the uptake of Biochim. Biophys. Acta, 62 (1962) 490-496
INHIBITION
OF
INSULIN
493
ACTION
glucose in the absence of insulin; at such concentration the action of insulin is completely inhibited. The effect of iodoacetate on the formation of 14C0~ from [i-14C]glucose is essentiaUy identical with the effect on the uptake of glucose (Fig. 2). The inhibition of the action of insulin is even more apparent when the data are expressed in terms of the differences between the iodoacetate-untreated and -treated tissues (Fig. 3)-Thus at a concentration of io -5 M iodoacetate the treated tissue produces essentially the +l! I
I
... 1
:I. - 2
~:i:ill¸ ~:!
E
~-6 Wilflout insulin .o_
With insulin
}-9
!
if" - I 0
5 x l O -6
i x l O- 5
5xlO-5
i x l O- 4
Iodoocetote M
Fig.
3.
Effect of iodoacetate on oxidation of [z-laC]glucose to 14CO~ b y adipose tissue. Difference b e t w e e n untreated and treated tissues.
ii io 8 z~ 7
8
i
e
%• 0 Ireufin Number
" ~-
-
' io
÷
-
+
i0-~,
8
-4.
10-3 Iod0ocet0te M
-- +
C -- ÷
--
~ +
-- ~
i0-~ N- Ethylmdeimide M
Effect of pretreatment with iodoacetate and with N-etlaylmaleimide on oxidation o f [I:xaC]glucose to 14COz in absence and presence of insulin. White columns equal no pretreatment with blocking agents; shaded columns equal pretreatment with blocking agents.
Fig.
4.
same amount of 14C0~ as the untreated tissue but the actiort of insulin is partially inhibited. This inhibition is quite evident at higher concentrations even though the spontaneous production of 14C0z from [r-14C]ghicose may not be significantly Biochim. Biophys. Acta,
62 (I962)
490-496
494
I . A . MIRSKY, G. PERISUTTI
diminished. Essentially similar results were obtained when the tissues were incubated with N-ethylmaleimide instead of iodoacetate. Pretreatment of the adipose tissue with iodoacetate results in an inhibition of both the spontaneous utilization of glucose and the increment due to the action of insulin (Fig. 4). The inhibition of the insulin effect, however, occurs in the presence of a smaller concentration of iodoacetate than that necessary to produce an inhibition of the spontaneous oxidation of glucose. Pretreatment with N-ethylmaleimide in concentrations which inhibit the action of insulin also inhibits the spontaneous oxidation of glucose, but to a lesser degree (Fig. 4). TABLE I EFFECT
OF P R E T R E A T M E N T INSULIN
WITH
SULFHYDRYL
TO R A T E P I D I D Y M A L
ADIPOSE
BLOCKING TISSUES
AGENTS
AND
ON " B I N D I N G "
OF
HEMIDIAPHRAGMS
i n each instance five pairs of tissue were incubated for 5 m i n at 37 ° in bicarbonate buffer alone or buffer containing lO -3 M of either blocking agent, washed, blotted and incubated for 2 min in buffer containing o.i unit insulin/ml plus a trace of high specific activity [I31IJinsulin (i /~C). S u b s e q u e n t l y the tissues were processed (A) as described by STADIE et al. 14 or (13) as described b y SCHWARTZ et alfl and their radioactivity m e a s u r e d as described in the text. D a t a expressed as percentage of total radioactivity (mean ± s t a n d a r d error) " b o u n d " to tissue per g w e t wt. Tissue
Adipose tissue Adipose tissue Hemidiaphragm Adipose tissue Hemidiaphragm
Blocking agent
(A) (A) (A) (B) (t3)
Untreated
Iodoacetate N-ethylmaleimide lodoacetate N-ethylmaleimide N-ethylmaleimide
4.I 4.2 2.4 0.7 1. 5
44! i ~
o.I o.4 o.2 o.1 o.i
Treated
4-4 4.8 2.5 0.6 I. 5
± ~ ~ ~ :~
o,4 o,7 o.2 o.I o.I
In order to determine whether the inhibition of the action of insulin by the sulfhydryl blocking agents is due to a decrease in the binding of insulin to the cellular membrane, the adipose tissues were pretreated in the absence and presence of either iodoacetate or N-ethylmaleimide. Table I reveals that pretreatment with a concentration of these agents which inhibits the action of insulin exerts no effect on the "binding" of insulin to adipose tissue. Likewise, pretreatment with iodoacetate does not affect the quantity of insulin bound to rat hemidiaphragms. Incubation of the N-ethylmaleimide-treated and -untreated tissues under conditions which reduce disulfide bonds but presumably do not hydrolyze peptide linkages did not reveal a significant difference in the quantity of labeled insulin bound to the tissues (Table I). Heating at IOO° for 3 min results in a 73 % increase in the quantity of radioactivity retained by the hemidiaphragm and a 66 % increase in that retained b y the adipose tissue. DISCUSSION
In accord with previous observations on muscle% and the more recent observations of others on the perfused rat heart 1°, the data reported herein demonstrate that appropriate concentrations of sulfhydryl blocking agents can inhibit the action of insulin in vitro without affecting the spontaneous utilization of glucose. That this effect is independent of the ~quantity of insulin bound to the tissues is revealed in the lack of any difference between the treated and untreated adipose tissues. Likewise, Biochim. Biophys. Acta, 62 (1962) 490 496
INHIBITION OF INSULIN ACTION
495
a concentration of iodoacetate which inhibits the action of insulin on the isolated rat diaphragm exerts no apparent effect on the binding of insulin to the muscle, Consequently, the binding of insulin as measured in the present study is independent: of the availability of sulfhydryl groups. The dissociation between the binding of insulin a n d the action of insulin is revealed also by the increased quantity of insulin that is bound to boiled adipose tissue and hemidiaphragm. Likewise, conditions such ks anaerobiosis or incubation in a sucrose medium which inhibit the action of insulin on,the isolated hemidiaphragm do not affect the binding of insulin 15. These and other observations support the conclusions by NEWERLY AND BERSONTM that the binding: of insulin to tissues is due to non-specific adsorptive forces and is not related to t h e action of insulin. I t is quite possible that the measure of binding used in this and similar studies is not a true criterion of the quantity of insulin that,reaches the active sites. The insulin that is bound to the diaphragm in vitro is localized: mainly in the fascia covering the muscle while very rapidly after an intravenous injection the insulin is distributed evenly throughout the skeletal muscle 17. If the site at which insulin acts is beyond the fascia and on the membrane of the muscle fibers, then an inhibition of the binding of insulin to the active sites will be masked b y the much greater quantity bound to the fascia. To reveal the influence of an inhibitor on the binding of insulin to the cellular membrane will require more precise techniques than those employed in the present study. The observation that the action of insulin is inhibited by the blocking agents supports, but does not establish, the hypothesis that it is dependent upon an interaction between the disulfide bridge of insulin and the sulfhydryls that comprise the receptor sites of the cellular membrane. Yet, since groups other than sulfhydryl are affected b y both iodoacetate TM and N-ethylmaleimide TM, the inhibition of the action of insulin induced b y these agents m a y be due to some alteration other than or in addition to the blocking of the sulfhydryl groups. Pertinent in this connection is the fact that large concentrations of the sulfhydryl blocking agents inhibit the spontaneous utilization of glucose b y both adipose tissue and muscle in vitro. This m a y be due to the known inhibitory action of these agents on a number of enzymes involved in the metabolism of glucose. It is equally possible that the blocking agents act on some sulfhydryl-dependent system which is responsible for the "transport" of glucose into the cell and which, in turn, is stimulated in the presence of insulin. ACKNOWLEDGEMENTS
This work was aided by grants from the U.S. Public Health Service (A-I4O9) , The Foundations' Fund for Research in Psychiatry, and the Commonwealth of Pennsylvania. REFERENCES 1 I. A. MIRSKY AND G. ]::~ERISUTTI, Biochim. Biophys. Acta, 5o (1961) 603. 2 I. A. MIRSK¥ AND G. •ERISUTTI, Endocrinology, 71 (1962) 158. a O. WINTERSTEINER, J. Biol. Chem., lO2 (1933) 473. * J. LENS AND J. NEOTELINGS, Biochim. Biophys. Acta, 4 (195 o) 5 ol-
Biochim. Biophys. Acta, 62 (1962) 49o-496~
496
I.: A. MIRSKY, G. PERISUTTI
s C. T. O. FONG, L. SILVER, D. R. CHRISTMAN AND I. L. SCHWARTZ, Proc. Natl. Acad. Sci. U.S., 46 (196o) 1273. 6 H. RASMUSSEN, I. L. SCHWARTZ, M. A. SCHOESSLER AND G. HOCHSTER, Proc. Natl. Acad. Sci. U.S., 46 (196o) 1278. 7 I. L. SCHWARTZ, H. RASMUSSEN, M. A. SCHOESSLER, L. SILVER AND C. T. O. FONG, Proc. Natl. Acad. Sci. U.S., 46 (196o) 1288. s K. L. ZIERLER, Bull. Johns Hopkins Hospital, lO9 (1961) 35. 9 D. E. HAFT AND I. A. MIRSKY, J. Pharmacol. Exptl. Therap., lO 4 (1952) 34 o. 10 E. CADENAS, H. I~AJI, C. R. PARK AND H. RASMUSSEN, J. Biol. Chem., 236 (1961) PC63. 11 G. UNGAR AND S. KADIS, Nature, 183 (1959) 49. 12 M. C. SHEPS, R. J. NICKERSON, Y. M. DAGENAIS, J. STEINKE, D. B. MARTIN AND A. E. RENOLD, J. Clin. Invest., 39 (196o) 1499. 13 R. S. YALOW AND S. A. BERSON, J , Clin. Invest., 39 (196o) 1157. 14 W. C. STADIE, N. HAUGAARD AND J. ]3. MARSH, J. Biol. Chem., 188 (1951) 167. is G. ]3HATTACHHARYA,Biochem. J., 79 (1961) 369. 16 K. lqEWERLY AND S. A. BERSON, Proc. Soc. Exptl. Biol. Med., 94 (1957) 751. I 7 0 . STEIN AND J. GRoss, Endocrinology, 65 (1959) 7o7 • xs L. MICHAELIS AND M. P. SCHUBERT, J. Biol. Chem., lO6 (1934) 331. 19 R . BENESCH AND R . E . ]3ENESCI-I,J. Biol. Chem., 236 (1961) 405 .
Biochim. Biophys. Acta, 62 (I96Z) 49o-496
BIOCHIMICA ET BIOPHYSICA ACTA
BBA
3668
T H E I N H I B I T I O N OF T H E ACTION O F I N S U L I N ON R A T E P I D I D Y M A L A D I P O S E T I S S U E BY S U L F H Y D R Y L B L O C K I N G AGENTS* I. ARTHUR MIRSKY AND GLADYS PERISUTTI Department of Clinical Science, University of Pittsburgh School of Medicine, Pittsburgh, Pa. ( U.S.A .) (Received January 3oth, 1962)
SUMMARY Concentrations of iodoacetate and N-ethylmaleimide which produce no significant effect on the spontaneous oxidation of glucose by rat epididymal adipose tissue in vitro inhibit the action of insulin. These agents do not inhibit the binding of insulin to adipose tissue or to the isolated diaphragm in vitro.
INTRODUCTION The demonstration that synthetic oxytocin and related disulfide peptides with oxytocic activity exert an insulin-like action on rat epididymal adipose tissue 1, ~ is in accord with the evidence that the action of insulin is dependent in part upon its disulfide linkages 3,4. A mechanism whereby the disulfide bridges may be involved is suggested by the cogent evidence that both the binding and the action of vasopressin on the rat kidney and toad bladder are dependent upon an interaction between the disulfide of the hormone and a sulfhydryl group of some specific receptor protein 5-~. Accordingly, it was proposed that the initiation of a series of thiol-disulfide reactions produces alterations in the tertiary structure of the membrane proteins and thereby open channels for the passage of water and specific solutes 7. A similar physical mechanism could account for the accelerated influx of glucose into the cell which occurs in the presence of insulin s. In support of the possibility that the action of insulin is due to some thioldisulfide interchange is the observation that pretreatment of the isolated rat diaphragm with concentrations of iodoacetate or p-chloromercuribenzoate which produce no significant changes in glucose uptake, nevertheless inhibit the action of insulin 9. In accord is the recent preliminary report that the action of insulin on the perfused rat heart is inhibited by N-ethylmaleimide as is also the "binding" of E131Ilinsulin to the hearO °. Likewise, the observation that the total sulfhydryl groups of the isolated rat diaphragm are decreased about 22 % after incubation with insulin n favors the hypothesis that the disulfides of insulin combine with the sulfhydryls of the proteins that comprise the receptor sites on the surface of the cellular membrane. Since the epididymal adipose tissue of the rat exhibits an exquisite sensitivity to the action of insulin in vitro and unlike the isolated diaphragm responds in a * With the technical assistance of D. L. BINLEY. Biochim. Biophys. Acta, 62 (1962) 49o-496
INHIBITION OF INSULIN ACTION
491
similar manner to oxytocin 2, it became pertinent to determine whether the reaction of this tissue is influenced b y sulfhydryl blocking agents. MATERIALS AND METHODS
Fed Carworth rats, weighing lOO-15o g were decapitated, exsanguinated and their epididymal fat pads excised. Each fat pad was cut into two pieces, the pieces from one side were used as controls for those from the other side in a balanced segment design similar to that of SHEPS et al. 12 but employing two instead of three segments from each side. The subsequent treatment of the tissues and the analytical procedures employed were the same as those reported in detail elsewhere 2. The adipose tissues were incubated for 3 h at 37 ° in 2 ml Krebs-Ringer bicarbonate buffer solution (pH 7.4) containing 20/*moles glucose, 0.084/*C ~I-14Clglucose, 0.2 % gelatin in the absence and presence of various concentrations of iodoacetate or N-ethylmaleimide, and in the absence and presence of o.I unit insulin/ml. In other experiments, the adipose tissues were incubated at 37 ° for 5 min in 2 ml K r e b s Ringer buffer without and with the sulfhydryl blocking agents, washed twice for I min in 2 ml fresh buffer solution, blotted, weighed and then incubated for 3 h in the buffer solution containing glucose without and with insulin. At the end of the incubation period the reaction was stopped with o.2 ml IO N H2SO 4 and the C09. collected in Hyamine IO X; the latter was transferred to counting vials containing 15 ml of a liquid scintillator and counted b y means of a Packard Automatic Spectrometer. The initial and final glucose concentrations and their specific activities were determined as described elsewhere ~. All counts were corrected for quenching, background and variations in counting efficiency. The data are expressed as/,moles of glucose oxidized to 14C02/g wet weight of adipose tissue. The "binding" of insulin to adipose tissue and hemidiaphragms was determined b y incubating the tissues for 5 min at 37 ! in the buffer containing 0.2 % gelatin in the absence and presence of lO -3 M iodoacetate or IO-s M N-ethylmaleimide. After two washes for I min each in 2 ml fresh buffer, the tissues were blotted and incubated for 2 rain in I ml buffer containing o.1 unit insulin and I/*C of a trace quantity of very high specific activity [131I]insulin prepared b y the procedure of BERSON et al. ~3. Subsequently, the tissues were washed with 0. 9 % saline as described by STADIE et al. ~, viz., three washings for 30 sec each in 25 ml of saline and one washing for 15 min in 50 ml saline. More extensive washing did not change the results. The tissues were blotted, weighed, placed into the bottom of test-tubes and their radioactivity determined by means of a well-type 7-scintillation counter. The quantity of insulin "bound" to the tissues was expressed as the percentage of the total radioactivity of the incubation mixture. In some instances, the tissues were pretreated as above and then processed b y the procedure described b y SCHWARTZ et al. ~, viz., boiled in water containing io -s M N-ethylmaleimide, washed successively in I M :NaC1, o.15 M NaHCO3 (pH 8.5) and sodium acetate buffer (pH 4.0) and then incubated for 8 h in 3.0 ml of o.I M cysteine, in o.15 M NaHCO 3 (pH 8.5) in a nitrogen atmosphere. In several experiments, the adipose tissue and hemidiaphragm from one side were heated in buffer for 5 min at IOO° while the tissues from the other side were maintained in buffer at 37 °. Subsequently, the heated and unheated tissues were incubated in buffer containing insulin and washed as above. Biochim. Biophys. Acta, 62 (1962) 490-496
492
I . A . MIRSKY, G. PERISUTTI RESULTS
The data summarized in Figs. I and 2 reveal the effect of various concentrations of iodoacetate on the uptake of glucose and the production of 14C0~ from [i-14Clglucose b y the adipose tissue in vitro. I t is apparent that in the presence of a concentration of iodoacetate which has no significant effect on the spontaneous uptake of glucose, the stimulating effect of o.I unit of insulin/ml is partially inhibited. Thus, whereas
'4-11 13 i2
II
IO
7
,-
6
2-
~
I
I nt4.din Number
-- + +J8 5x6 s
-
--
"4"
--
--
+
+
16 fxlO"5
--
+
12-- + ixlO-4
~6
5xl(~ 5
Iodoocetote M
Fig. i. Effect of i o d o a c e t a t e on u p t a k e of glucose b y adipose t i s s u e in a b s e n c e a n d presence of insulin. W h i t e c o l u m n s e q u a l controls i n c u b a t e d in t h e a b s e n c e of i o d o a c e t a t e a n d s h a d e d c o l u m n s equal controls i n c u b a t e d in t h e presence of iodoacetate.
t25
,~
20
5
Insulin 0 Number
-
+
18
5x=o-e
+
-
-I-
-
+
•
-
+
-
+
-
+
-
16
16
12
I x I 0"s
5 x I 0"s
[ x I 0"4
+
Iodoocetote M
Fig. 2. Effect of i o d o a c e t a t e on o x i d a t i o n of [I-14C]glucose to 14CO 2 b y adipose tissue in a b s e n c e a n d presence of insulin. W h i t e c o l u m n s equal controls i n c u b a t e d in t h e a b s e n c e of i o d o a c e t a t e a n d s h a d e d c o l u m n s equal controls i n c u b a t e d in t h e presence of iodoacetate.
10 -5 M iodoacetate exerts no effect on the uptake of glucose by the adipose tissue it produces some inhibition of the action of insulin (Fig. I). In the presence of greater concentrations of iodoacetate there is an appreciable impairment in the uptake of Biochim. Biophys. Acta, 62 (1962) 490-496
INHIBITION
OF
INSULIN
493
ACTION
glucose in the absence of insulin; at such concentration the action of insulin is completely inhibited. The effect of iodoacetate on the formation of 14C0~ from [i-14C]glucose is essentiaUy identical with the effect on the uptake of glucose (Fig. 2). The inhibition of the action of insulin is even more apparent when the data are expressed in terms of the differences between the iodoacetate-untreated and -treated tissues (Fig. 3)-Thus at a concentration of io -5 M iodoacetate the treated tissue produces essentially the +l! I
I
... 1
:I. - 2
~:i:ill¸ ~:!
E
~-6 Wilflout insulin .o_
With insulin
}-9
!
if" - I 0
5 x l O -6
i x l O- 5
5xlO-5
i x l O- 4
Iodoocetote M
Fig.
3.
Effect of iodoacetate on oxidation of [z-laC]glucose to 14CO~ b y adipose tissue. Difference b e t w e e n untreated and treated tissues.
ii io 8 z~ 7
8
i
e
%• 0 Ireufin Number
" ~-
-
' io
÷
-
+
i0-~,
8
-4.
10-3 Iod0ocet0te M
-- +
C -- ÷
--
~ +
-- ~
i0-~ N- Ethylmdeimide M
Effect of pretreatment with iodoacetate and with N-etlaylmaleimide on oxidation o f [I:xaC]glucose to 14COz in absence and presence of insulin. White columns equal no pretreatment with blocking agents; shaded columns equal pretreatment with blocking agents.
Fig.
4.
same amount of 14C0~ as the untreated tissue but the actiort of insulin is partially inhibited. This inhibition is quite evident at higher concentrations even though the spontaneous production of 14C0z from [r-14C]ghicose may not be significantly Biochim. Biophys. Acta,
62 (I962)
490-496
494
I . A . MIRSKY, G. PERISUTTI
diminished. Essentially similar results were obtained when the tissues were incubated with N-ethylmaleimide instead of iodoacetate. Pretreatment of the adipose tissue with iodoacetate results in an inhibition of both the spontaneous utilization of glucose and the increment due to the action of insulin (Fig. 4). The inhibition of the insulin effect, however, occurs in the presence of a smaller concentration of iodoacetate than that necessary to produce an inhibition of the spontaneous oxidation of glucose. Pretreatment with N-ethylmaleimide in concentrations which inhibit the action of insulin also inhibits the spontaneous oxidation of glucose, but to a lesser degree (Fig. 4). TABLE I EFFECT
OF P R E T R E A T M E N T INSULIN
WITH
SULFHYDRYL
TO R A T E P I D I D Y M A L
ADIPOSE
BLOCKING TISSUES
AGENTS
AND
ON " B I N D I N G "
OF
HEMIDIAPHRAGMS
i n each instance five pairs of tissue were incubated for 5 m i n at 37 ° in bicarbonate buffer alone or buffer containing lO -3 M of either blocking agent, washed, blotted and incubated for 2 min in buffer containing o.i unit insulin/ml plus a trace of high specific activity [I31IJinsulin (i /~C). S u b s e q u e n t l y the tissues were processed (A) as described by STADIE et al. 14 or (13) as described b y SCHWARTZ et alfl and their radioactivity m e a s u r e d as described in the text. D a t a expressed as percentage of total radioactivity (mean ± s t a n d a r d error) " b o u n d " to tissue per g w e t wt. Tissue
Adipose tissue Adipose tissue Hemidiaphragm Adipose tissue Hemidiaphragm
Blocking agent
(A) (A) (A) (B) (t3)
Untreated
Iodoacetate N-ethylmaleimide lodoacetate N-ethylmaleimide N-ethylmaleimide
4.I 4.2 2.4 0.7 1. 5
44! i ~
o.I o.4 o.2 o.1 o.i
Treated
4-4 4.8 2.5 0.6 I. 5
± ~ ~ ~ :~
o,4 o,7 o.2 o.I o.I
In order to determine whether the inhibition of the action of insulin by the sulfhydryl blocking agents is due to a decrease in the binding of insulin to the cellular membrane, the adipose tissues were pretreated in the absence and presence of either iodoacetate or N-ethylmaleimide. Table I reveals that pretreatment with a concentration of these agents which inhibits the action of insulin exerts no effect on the "binding" of insulin to adipose tissue. Likewise, pretreatment with iodoacetate does not affect the quantity of insulin bound to rat hemidiaphragms. Incubation of the N-ethylmaleimide-treated and -untreated tissues under conditions which reduce disulfide bonds but presumably do not hydrolyze peptide linkages did not reveal a significant difference in the quantity of labeled insulin bound to the tissues (Table I). Heating at IOO° for 3 min results in a 73 % increase in the quantity of radioactivity retained by the hemidiaphragm and a 66 % increase in that retained b y the adipose tissue. DISCUSSION
In accord with previous observations on muscle% and the more recent observations of others on the perfused rat heart 1°, the data reported herein demonstrate that appropriate concentrations of sulfhydryl blocking agents can inhibit the action of insulin in vitro without affecting the spontaneous utilization of glucose. That this effect is independent of the ~quantity of insulin bound to the tissues is revealed in the lack of any difference between the treated and untreated adipose tissues. Likewise, Biochim. Biophys. Acta, 62 (1962) 490 496
INHIBITION OF INSULIN ACTION
495
a concentration of iodoacetate which inhibits the action of insulin on the isolated rat diaphragm exerts no apparent effect on the binding of insulin to the muscle, Consequently, the binding of insulin as measured in the present study is independent: of the availability of sulfhydryl groups. The dissociation between the binding of insulin a n d the action of insulin is revealed also by the increased quantity of insulin that is bound to boiled adipose tissue and hemidiaphragm. Likewise, conditions such ks anaerobiosis or incubation in a sucrose medium which inhibit the action of insulin on,the isolated hemidiaphragm do not affect the binding of insulin 15. These and other observations support the conclusions by NEWERLY AND BERSONTM that the binding: of insulin to tissues is due to non-specific adsorptive forces and is not related to t h e action of insulin. I t is quite possible that the measure of binding used in this and similar studies is not a true criterion of the quantity of insulin that,reaches the active sites. The insulin that is bound to the diaphragm in vitro is localized: mainly in the fascia covering the muscle while very rapidly after an intravenous injection the insulin is distributed evenly throughout the skeletal muscle 17. If the site at which insulin acts is beyond the fascia and on the membrane of the muscle fibers, then an inhibition of the binding of insulin to the active sites will be masked b y the much greater quantity bound to the fascia. To reveal the influence of an inhibitor on the binding of insulin to the cellular membrane will require more precise techniques than those employed in the present study. The observation that the action of insulin is inhibited by the blocking agents supports, but does not establish, the hypothesis that it is dependent upon an interaction between the disulfide bridge of insulin and the sulfhydryls that comprise the receptor sites of the cellular membrane. Yet, since groups other than sulfhydryl are affected b y both iodoacetate TM and N-ethylmaleimide TM, the inhibition of the action of insulin induced b y these agents m a y be due to some alteration other than or in addition to the blocking of the sulfhydryl groups. Pertinent in this connection is the fact that large concentrations of the sulfhydryl blocking agents inhibit the spontaneous utilization of glucose b y both adipose tissue and muscle in vitro. This m a y be due to the known inhibitory action of these agents on a number of enzymes involved in the metabolism of glucose. It is equally possible that the blocking agents act on some sulfhydryl-dependent system which is responsible for the "transport" of glucose into the cell and which, in turn, is stimulated in the presence of insulin. ACKNOWLEDGEMENTS
This work was aided by grants from the U.S. Public Health Service (A-I4O9) , The Foundations' Fund for Research in Psychiatry, and the Commonwealth of Pennsylvania. REFERENCES 1 I. A. MIRSKY AND G. ]::~ERISUTTI, Biochim. Biophys. Acta, 5o (1961) 603. 2 I. A. MIRSK¥ AND G. •ERISUTTI, Endocrinology, 71 (1962) 158. a O. WINTERSTEINER, J. Biol. Chem., lO2 (1933) 473. * J. LENS AND J. NEOTELINGS, Biochim. Biophys. Acta, 4 (195 o) 5 ol-
Biochim. Biophys. Acta, 62 (1962) 49o-496~
496
I.: A. MIRSKY, G. PERISUTTI
s C. T. O. FONG, L. SILVER, D. R. CHRISTMAN AND I. L. SCHWARTZ, Proc. Natl. Acad. Sci. U.S., 46 (196o) 1273. 6 H. RASMUSSEN, I. L. SCHWARTZ, M. A. SCHOESSLER AND G. HOCHSTER, Proc. Natl. Acad. Sci. U.S., 46 (196o) 1278. 7 I. L. SCHWARTZ, H. RASMUSSEN, M. A. SCHOESSLER, L. SILVER AND C. T. O. FONG, Proc. Natl. Acad. Sci. U.S., 46 (196o) 1288. s K. L. ZIERLER, Bull. Johns Hopkins Hospital, lO9 (1961) 35. 9 D. E. HAFT AND I. A. MIRSKY, J. Pharmacol. Exptl. Therap., lO 4 (1952) 34 o. 10 E. CADENAS, H. I~AJI, C. R. PARK AND H. RASMUSSEN, J. Biol. Chem., 236 (1961) PC63. 11 G. UNGAR AND S. KADIS, Nature, 183 (1959) 49. 12 M. C. SHEPS, R. J. NICKERSON, Y. M. DAGENAIS, J. STEINKE, D. B. MARTIN AND A. E. RENOLD, J. Clin. Invest., 39 (196o) 1499. 13 R. S. YALOW AND S. A. BERSON, J , Clin. Invest., 39 (196o) 1157. 14 W. C. STADIE, N. HAUGAARD AND J. ]3. MARSH, J. Biol. Chem., 188 (1951) 167. is G. ]3HATTACHHARYA,Biochem. J., 79 (1961) 369. 16 K. lqEWERLY AND S. A. BERSON, Proc. Soc. Exptl. Biol. Med., 94 (1957) 751. I 7 0 . STEIN AND J. GRoss, Endocrinology, 65 (1959) 7o7 • xs L. MICHAELIS AND M. P. SCHUBERT, J. Biol. Chem., lO6 (1934) 331. 19 R . BENESCH AND R . E . ]3ENESCI-I,J. Biol. Chem., 236 (1961) 405 .
Biochim. Biophys. Acta, 62 (I96Z) 49o-496