Eur. J . Biochem. YS, 607-611 (1979)

Involvement of Calcium in the Inhibition by Insulin of the Glucagon-Stimulated Adenylate-Cyclase Activity Zoltan KISS Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, Szeged (Received September 26, 1978)

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1. The inhibitory effect of adenosine on the glucagon-stimulated adenylate cyclase activity of liver plasma membranes, prepared from PVG/c rats, was potentiated by insulin. In the presence of EGTA, such potentiating effect of insulin was lost. 2. Calcium (10 pM) potentiated the inhibitory effects of both adenosine and insulin on the glucagon-stimulated cyclase activity. The synergestic effect of calcium + insulin required the presence of adenosine as judged from the use of adenosine deaminase. 3. Insulin had no significant inhibitory effect on the glucagon-stimulated cyclase activity of liver plasma membranes, prepared from young Wistar rats, unless both adenosine (50 pM) and calcium (10 pM) were added externally. 4. Results demonstrate an interaction of calcium and insulin at membrane level that, in the presence of adenosine, results in the inhibition of the glucagon-stimulated adenylate cyclase activity.

Insulin has been shown to inhibit the rise in the content of cyclic AMP due to submaximal concentrations of catecholamines or glucagon both in adipose and liver tissues [l-41. This phenomenon is highly reproducible and is most probably due to the inhibition of the hormone-stimulated adenylate cyclase activity by insulin. Using liver cell membrane preparations, the inhibitory effect of insulin on the adenylate cyclase system indeed could be demonstrated in two laboratories [5,6]. Several other laboratories, however, failed to find such effect of insulin [7- 101. The controversy between data suggested that some specific conditions were required in order to demonstrate an inhibitory effect of insulin on the adenylate cyclase activity of cell membrane preparations. In a previous paper it was shown that the inhibition of the glucagon-stimulated adenylate cyclase activity of rat liver plasma membrane by insulin required the presence of adenosine [ 111. There are numerous data in the literature which indicate that calcium is somehow involved in some of the insulin effects (see [12]), although the mechanism for such involvement is essentially unknown. In view Abbreviations. Cyclic AMP, adenosine cyclic 3’:5’-monophosphate; EGTA, ethylene glycol bis(2-aminoethyl ether) N , N , N ’ . N ’ tetraacetic acid. Enzymes. Creatine kinase or ATP :creatine phosphotransferase (EC 2.7.3.2); adenylate cyclase or ATP: pyrophosphate lyase (cyclizing) (EC 4.6.1.1).

of the possibility that the inhibition of the hormonestimulated adenylate cyclase activity may account for some of the effects of insulin in vivo, it was of interest to study whether calcium was involved in this process.

MATERIALS AND METHODS

Muter iuls Crystallized insulin and glucagon as well as ATP, creatine phosphate, creatine phosphokinase and adenosine deaminase were purchased from Sigma (St Louis, Mo., U.S.A.). [u-~’P]ATP (20 Ci/mmol), was obtained from the Radiochemical Centre (Amersham, U.K.). Neutral aluminiumoxid was bought from Merck (Darmstadt, F.R.G.). Methods

Female PVG/c rats (weighing 150- 200 g) that had been starved overnight were used. Liver plasma membranes were prepared according to the method of Neville [ 131. After two washings the remaining sucrose inhibited variably the effect of insulin on the glucagon-stimulated adenylate cyclase activity. Therefore a third washing step was also employed. Plasma membranes were stored in liquid nitrogen and used within 3 - 4 days. After longer storage, the inhibitory

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Inhibition by Insulin of the Adenylate Cyclase Activity

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effects of adenosine + insulin and calcium insulin were significantly decreased. In typical experiments, the assay mixture for the measurement of adenylate cyclase activity contained 0.5 mM [GY-~'P]ATP (20 Ci/mol), 4 mM MgC12, 1 mM cyclic AMP, 25 mM creatine phosphate, 60 pg creatine phosphokinase, 50 mM Tris/HCl pH 7.5 and 45- 55 pg protein of liver plasma membrane in a final volume of 70 p1. Incubations were terminated according to the method of White [I41 and cyclic AMP was separated by column chromatography on neutral alumina [14]. Recovery of cyclic AMP was around 85 % as verified by control runs of cyclic [8-3H]AMP. The formation of cyclic AMP was linear with time up to 10 min. Protein was determined according to the method of Lowry et al. [I51 using bovine serum albumin as standard. RESULTS If otherwise not stated, liver plasma membranes prepared from PVG/c rats, were used for the experi-

ments. Incubation of liver plasma membranes in the presence of EGTA (0.5 mM) resulted in significant increase of the activity of the glucagon stimulated adenylate cyclase (Table 1). The stimulatory effect of glucagon is known to be inhibited by calcium IS]. In independent studies we observed 50 % inhibition by about 0.3 mM calcium. Therefore, the stimulatory effect of EGTA indicated that these membranes contained relatively high concentration of calcium. Inhibition of the cyclase activity by externally added adenosine was not affected by EGTA, however, the potentiating effect of insulin on this inhibition was lost (Table 1). This clearly indicated that calcium, bound to the plasma membrane, was required for the inhibitory effect of insulin. Calcium, added at 10 pM concentration, alone had no significant effect, however, it potentiated the inhibitory effect of adenosine (Table 2). At first look, such effect of calcium seems contradictory to results in Table 1. In the absence of insulin, however, the concentration of the membrane-bound calcium may be too low to affect the inhibitory effect of adenosine.

Table 1. Effect of EGTA on the inhibitory effect of adenosine + insulin on the glucagon-stimulated adenylate cyclase activity The two experiments were performed with two different plasma membrane preparations. Incubations were carried out at 33°C for 10 min. Results are in both experiments the mean k S.E.M. of four incubations. Stimulation by 0.1 pM glucagon over the basal activity was 5.3-5.6-fold. EGTA when added was 0.5 mM Activity

Additions

experiment 2

experiment 1

+ EGTA

- EGTA

- EGTA

+ EGTA

nmol cyclic AMP x (mg protein)-' x (10 min)-' Glucagon 0.1 pM Glucagon 0.1 pM Glucagon 0.1 pM Glucagon 0.1 pM

1.20 k 0.02 0.96 f 0.04 1.20 i 0.03 0.76 f 0.03

+ adenosine 0.1 mM + insulin 1 nM + adenosine 0.1 mM + insulin 1 nM

1.91 k 0.05 1.38 f 0.03 2.15 i 0.05 1.33 f 0.04

1.14 0.05 0.97 f 0.03 1.10 f 0.04 0.78 i 0.02

1.42 f 0.04 1.02 i 0.04 1.36 f 0.03 0.99 f 0.02

Table 2. Potentiation of the inhibitory effect of adenosine by calcium The two experiments were performed with two different plasma membrane preparations. Incubations were carried out at 33 "C for 10 min. Results are in both experiments the mean i S.E.M. of four incubations. Calcium when added was 10 pM Additions

Activity ~~

experiment 1 - calcium

+ calcium

experiment 2 __ - calcium

+ calcium

nmol cyclic AMPx (mg protein)-' x (10 min)-' Glucagon 0.1 pM Glucagon 0.1 pM Glucagon 0.1 pM

+ adenosine 10 pM + adenosine 0.1 mM

1.34 f 0.06 1.27 f 0.03 1.12 i 0.04

1.29 f 0.03 1.06 f 0.02 0.87 f 0.03

1.23 f 0.04 1.15 i 0.02 0.92 f 0.03

1.23 f 0.03 0.96 k 0.03 0.76 0.04

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2 . Kiss

plasma membrane from young (100 g body weight) female Wistar rats were studied for insulin effect. These preparations differed from that of PVG/c rats in some respects, i.e. full stimulation of the cyclase activity was achieved by 0.1 pM glucagon, and EGTA had no stimulatory effect. This latter finding indicated that plasma membranes from Wistar rats had lower calcium content than that from PVG/c rats at least at crucial sites of the cyclase system. The glucagon-stimulated cyclase activity of these membranes was not inhibited by insulin, unless both adenosine (50 pM) and calcium (10 pM) were added (Table 4). It is also an important difference that calcium alone did not potentiate the inhibitory effect of adenosine. Higher than 10 pM concentrations of calcium were not tested. Maximum inhibition by insulin was observed at 1 nM concentration. Insulin at a concentration of 10 nM gave similar rate of inhibition (not shown). In contrast to membrane preparations from PVG/c rats, the effect of 0.1 nM insulin was very low and did not prove to be significant by Student t test (not shown).

Calcium at 0.1 mM was itself inhibitory (20% inhibition) and at this concentration its effect was less synergestic with that of adenosine, especially at higher concentrations (0.1 mM) of the latter (data not shown). In the next experiments mostly 10 pM calcium was used as it occurs in liver at this concentration in vivo. When calcium (10 pM) and insulin (0.1 or 1 nM) were added together, unlike when they were present alone, the glucagon-stimulated cyclase activity was inhibited by about 30 % at both concentrations of insulin (Table 3). The synergestic effect of calcium and insulin disappeared by the addition of adenosine deaminase. This indicated that the presence of adenosine (formed during incubation) was required for the effect of calcium + insulin. Addition of 10 pM of adenosine did not increase further the combined inhibitory effects of calcium + insulin. A sensitization of the cyclase system to the inhibitory effect of adenosine by calcium + insulin would explain this phenomenon. In order to test the reproducibility of the above results, in the next experiments four different preparations of liver

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Table 3. Combined inhibitory eflect of insulin calcium on the glucagon-stimulated adenylate cyclase activity Results are the mean f S.F M. of three experiments each assayed in quadruplicate. Each experiment was performed with different plasma membrane preparations Additions

Activity - insulin

+ insulin 1 nM

+ insulin 0.1 nM

nmol cyclic AMP x (mg protein)-' x (10 min)-' Glucagon 0.1 pM Glucagon 0.1 pM + calcium 10 pM Glucagon 0.1 pM + calcium 10 pM + adenosine deaminase 0.05 U Glucagon 0.1 pM + adenosine 10 pM Glucagon 0.1 pM + calcium 10 pM + adenosine 10 pM a

1.29 f 0.05 1.27 f 0.04

1.32 k 0.07 0.87 & 0.06"

1.23 f 0.09 0.92 f 0.05"

1.34 k 0.10 1.21 f 0.08 0.99 f 0.05

1.26 f 0.1Ib 0.94 f 0.06" 0.92 k 0.03b

1.23 k 0.08 0.96 0.05" 0.91 k 0.06b

Significant by Student's t test, P < 0.01 Not significant.

Table 4. Inhibitory effect of insulin on the glucagon-stimulated adenylate cyclase activity o f liver plasma membranes from Wistar ruts Besides other standard components, the assay mixture contained 1 mM [a3'P]ATP (16 Ci/mol). Stimulation by 50 nM glucagon was about 6-fold. Results are the mean k S.E.M. of four experiments each assayed in triplicate. Each experiment was performed with different preparations of plasma membranes prepared from young (100 g body weight) female Wistar rats Additions

Activity - insulin

P

+ insulin 1 mM

nmol cyclic AMP x (mg protein)-' x (10 min)-' ~~

Glucagon Glucagon Glucagon Glucagon

50 nM 50 nM 50 nM 50 nM

+ adenosine 50 pM + calcium 10 pM + adenosine 50 pM + calcium 10 pM

2.66 0.08 2.36 f 0.11 2.62 f 0.07 2.32 f 0.07

~.

2.61 k 0.06 2.35 i 0.09 2.49 0.08 1.89 & 0.09

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ns. ns. ns. < 0.01

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Inhibition by Insulin of the Adenylate Cyclase Activity

DISCUSSION The present experiments demonstrate that binding of insulin to the liver plasma membrane results in the inhibition of the glucagon-stimulated adenylate cyclase activity if both adenosine and membrane-bound calcium are present. Hepp [161 previously found a similar potentiating effect of calcium on the inhibitory effect of insulin. The present work, however, also provides examples for that membrane preparations from different strains of rats may differ either in their calcium contents or their sensitivity to calcium in promoting the inhibitory effect of adenosine. Thus, with membranes from PVG/c rats 10 pM calcium potentiated the inhibitory effect of adenosine to a similar extent to insulin. On the other hand, with membranes from Wistar rats the addition of both calcium and insulin was required to potentiate the inhibitory effect of adenosine. Clearly, some preliminary work may be required to define the optimum concentrations of adenosine and calcium for insulin effect if membranes from different strains of rats are to be compared. As to the mechanism of insulin action, several possibilities must be considered. It is well known that calcium decreases the fluidity of the biological membranes. The samc sccms true for insulin [17-191. It has also been shown that the stimulatory effect of glucagon on the adenylate cyclase system requires a certain fluidity state of the plasma membrane [20 - 211. Thus, a combined effect of insulin and calcium on the fluidity state of the plasma membrane might result in the inhibition of the glucagonstimulated cyclase activity. The same process may sensitize the cyclase system to the inhibitory effect of adenosine. Sufficiently high concentration of calcium may cause the same effect as a low concentration of calcium insulin. Another possibility can be that insulin increases the binding of calcium to the plasma membrane that in turn would render the cyclase system more sensitive to the inhibitory effect of adenosine. Under the conditions of the adenylate cyclase assay, we have not been able to show a significant effect of insulin on the binding of 45Cato the plasma membrane (unpublished observation). This does not necessarily mean an absence of insulin effect, because calcium may be directed by insulin only to few but specific sites, which could have gone undetected. Calcium, if present at sufficiently high concentration, may saturate these sites even in the absence of insulin. Finally, calcium may be required for an interaction between the insulin-receptor complex and the adenylate cyclase system. This would explain, why membrane-bound calcium is required for the insulin effect, however, would not explain the effect of calcium when insulin is absent.

It is generally assumed that the effects of insulin are triggered exclusively by its interaction with cellsurface protein receptors. This interaction may lead to intracellular effects in principle in two ways, i.e. some signal(s) is(are) generated at membrane level, or the formation of some signal(s) is(are) inhibited by insulin. Formation of cyclic AMP is known to be one of the signals that mediate the effects of hormones, such as glucagon or epinephrine. Insulin in most of the cases opposes the intracellular effects of these hormones. Also, insulin inhibits the rise in cyclic AMP content due to submaximal concentrations of glucagon or catecholamines [l -41. Here, further evidence is presented for under certain conditions (that are present in vivo) insulin inhibits the adenylate cyclase activity stimulated by glucagon. The extent of inhibition might be higher in vivo. In a previous paper it was shown that insulin, although most probably by different mechanism, inhibited the isoproterenol-stimulated cyclase activity as well [I 11 confirming the findings of others [5,6]. All these suggest that inhibition of the hormonestimulated adenylate cyclase activity may be one of the mechanisms that accounts for the effects of insulin in vivo. On the other hand, numerous data in the literature indicated indirectly that calcium is somehow involved in some of the effects of insulin (see [12]). The present work demonstrates a mechanism (inhibition of the glucagon-stimulated adenylate cyclase activity) at membrane level, where insulin and calcium interacts. This observation may help to understand the mechanism of insulin action in vivo and the implication of calcium. I am grateful to Dr M. Wollemann for valuable discussion of this study and to Mrs I. Kasza for skilled technical assistance.

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Z. Kiss, Biokemiai Intezet, M.T.A. Szegedi Biologiai Kozpont, Postafiok 521, H-6701 Szeged, Hungary

Involvement of calcium in the inhibition by insulin of the glucagon-stimulated adenylate-cyclase activity.

Eur. J . Biochem. YS, 607-611 (1979) Involvement of Calcium in the Inhibition by Insulin of the Glucagon-Stimulated Adenylate-Cyclase Activity Zoltan...
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