Rawal & Houslay (1983) FEBS Lett. 154, 87-91; Heyworth, Wallace & ..... latory proteins express different affinities for the catalytic unit or alternatively, as has ...
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Biochem. J. (1983) 214, 547-552 Printed in Great Britain
Insulin exerts actions through a distinct species of guanine nucleotide regulatory protein: inhibition of adenylate cyclase Clare M. HEYWORTH and Miles D. HOUSLAY Department ofBiochemistry and Applied Molecular Biology, University ofManchester Institute of Science and Technology, P.O. Box 88, Manchester M60 I QD, U.K.
(Received 29 March 1983/Accepted 13 May 1983) Insulin failed to exert an effect on the basal and glucagon- and guanosine 5'-[,y-inimid4triphosphate-stimulated adenylate cyclase activities of hepatocyte membranes. In the presence of high GTP (0.1 mM) concentrations, however, insulin was shown to inhibit adenylate cyclase activity. This effect was dose-dependent, exhibiting an EC50 (median effective concentration) of 3,UM for GTP. Elevated glucagon concentrations blocked the inhibitory effect of insulin in a dose-dependent fashion, with an EC50 of 1 nm. The insulin inhibition was dose-dependent (EC50 = 90 pM). The inhibitory effects of insulin were abolished using membranes from either glucagon-desensitized hepatocytes or choleratoxin-treated hepatocytes. If either Mn2+ replaced Mg2+ in adenylate cyclase assays or Na+ was removed from the assay mixtures then insulin failed to exert any inhibitory effect. It is suggested that insulin exerts its action on adenylate cyclase through an inhibitory guanine nucleotide protein. This is integrated with the proposal [Heyworth, Rawal & Houslay (1983) FEBS Lett. 154, 87-91; Heyworth, Wallace & Houslay (1983) Biochem. J. in the press] that insulin mediates a variety of cellular effects through a specific guanine nucleotide regulatory protein and associated protein kinase(s). It is well known that insulin can antagonize the effect of glucagon and catecholamines in elevating intracellular cyclic AMP concentrations in hepatocytes and adipocytes (Johnson et al., 1972; Pilkis et al., 1975; Blackmore et al., 1979; Heyworth et al., 1983a; Houslay et al., 1983a). The dominant mechanism by which this effect appears to be achieved is through the activation of specific membrane-bound cyclic AMP phosphodiesterases (Heyworth et al., 1983a; Houslay et al., 1983a). However, it has been claimed that insulin can exert inhibitory effects on adenylate cyclase (Hepp & Renner, 1972; Illiano & Cuatrecasas, 1972; Kiss, 1978), although others have failed to observe this (Pilkis et al., 1974; Bitensky et al., 1972). The activation of adenylate cyclase by specific hormones and neurotransmitters is well documented (see e.g. Ross & Gilman, 1980; Limbird, 1981). An occupied receptor exerts its stimulatory effect on the catalytic unit of adenylate cyclase through a specific guanine nucleotide regulatory protein (Ross & Gilman, 1980; Rodbell, 1980). Abbreviations used: p[NH]ppG, guanosine 5'-[fy-
imidoltriphosphate; IBMX, 3-isobutyl-1-methylxanthine. Vol. 214
More recently, hormones and neurotransmitters have been identified that actually inhibit adenylate cyclase activity (Ross & Gilman, 1980; Limbird, 1981; Jakobs et al., 1981; Londos et al., 1981; Cooper, 1982). These appear to exert their effects through an inhibitory guanine nucleotide regulatory protein (Londos et al., 1981; Cooper, 1982), which has been demonstrated by genetic analysis to be distinct from the species mediating stimulatory responses (Hildebrandt et al., 1982). Recently, we have demonstrated that insulin can elicit the activation of a liver plasma-membrane cyclic AMP phosphodiesterase (Marchmont & Houslay, 1980b, 1981; Houslay et al., 1983a,b), by a process involving guanine nucleotides (Heyworth et al., 1983b). We have suggested (Heyworth et al., 1983a,b) that insulin exerts its effects on this and certain other cellular processes through a specific guanine nucleotide regulatory protein. This species has properties that bear comparison with the guanine nucleotide regulatory protein identified as mediating inhibitory effects on adenylate cyclase. Here we identify conditions whereupon insulin can elicit inhibition of glucagon-stimulated adenylate cyclase. These are consistent with an effect mediated
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by a specific type of guanine nucleotide regulatory protein with properties resembling those of the so-called inhibitory guanine nucleotide regulatory protein. Materials and methods Collagenase, cyclic AMP, phosphocreatine, creatine kinase, GTP, p[NH]ppG and ATP were all purchased from Boehringer (U.K.), Lewes, East Sussex, U.K. IBMX was from Aldrich Chemical Co., Gillingham, Dorset, U.K. Glucagon was a gift from Dr. W. W. Bromer, Eli Lilly & Co., Indianapolis, IN, U.S.A. Bovine insulin and all other biochemicals were from Sigma Chemical Co., Kingston-upon-Thames, Surrey, U.K. Radiochemicals were all from Amersham International, Amersham, Bucks., U.K. All other chemicals were from BDH Chemicals, Poole, Dorset, U.K. Isolated hepatocytes were prepared from 225250g male Sprague-Dawley rats fed ad libitum as described by Elliott et al. (1976). Cells were pre-incubated in Krebs-Henseleit buffer containing 2.5% (w/v) bovine serum albumin and 2.5 mMCaCl2 at 370C for 20-30min before use as described fully by Smith-et al. (1978). The ATP content of the isolated hepatocytes was determined on an HCl04 extract. After neutralization of the extract, the ATP content was assessed using a luciferase assay (Stanley & Williams, 1969). Viable cells had an ATP content greater than 8.7 nmol/mg dry wt. of cells. Intracellular cyclic AMP concentrations were measured as detailed previously (Whetton et al., 1983). Hepatocytes were treated with cholera toxin (Houslay & Elliott, 1979) or were glucagon-desensitized (Heyworth & Houslay, 1983) as described previously. Briefly cells were incubated with either cholera toxin (lpg/ml) for 35 min or glucagon (10 nM) for 5min before the removal of aliquots of cells. These were rapidly cooled by the addition of an equal volume of ice-cold 1 mM-KHCO3 buffer, pH 7.2 and transfer to an ice-bath. All further procedures were carried out at 40C. A 'low-speed' washed membrane fraction was obtained from these cells as described previously (Houslay & Elliott, 1979). In all cases membranes were kept on ice, resuspended immediately before use and used within 2 h of preparation. Adenylate cyclase activity was assayed as detailed previously (Houslay et al., 1976). In an assay of 100,l final volume the mixture had final concentrations of 5 mM-MgSO4, 10mM-theophylline, 1 mM-EDTA, 1.5 mM-ATP, 1 mM-dithiothreitol, 22mM-disodium phosphocreatine, 1 mg of creatine kinase/ml, 25 mM-triethanolamine/HCI, neutralized to a final pH 7.4 with KOH, and 0.1 mg of membrane protein. We found it essential to use the
C. M. Heyworth and M. D. Houslay more highly purified phosphocreatine from Boehringer to obtain reproducible effects, rather than the product from Sigma. Cyclic AMP phosphodiesterase activity was assayed at 0. 1 gM-cyclic AMP as described in detail by Marchmont & Houslay (1980a). Protein was determined by a modified (Houslay & Palmer, 1978) microbiuret method of Goa (1953). Results Insulin (1 nM) exerted no effect on basal and glucagon (1 nM)-stimulated and p[NH]ppG (0.1 mM)stimulated adenylate cyclase activity (Table 1). However, it achieved (Fig. 1; Table 1) a 32% inhibition of adenylate cyclase activity stimulated by GTP (0.1 mM) alone or an 18% inhibition when glucagon (1 nM) was present together with GTP (0.1 mM). Changes in specific activity are all given in Table 1. Fig. l(a) demonstrates that inhibition of glucagon-stimulated adenylate cyclase activity only occurs in the presence of high GTP concentrations, yielding an EC50 (dose eliciting 50% of the total effect) of 3 ± 2gM-GTP (mean + S.E.M.; n = 5). Elevation of glucagon concentrations in the assay appeared to block the inhibition caused by insulin (Fig. lb), with an EC50 of 1.0+0.2nM-glucagon (mean + S.E.M.; n = 5). The ability of insulin to inhibit glucagon-plus-GTP-stimulated adenylate cyclase activity was clearly dose-dependent (Fig. lc), yielding an EC50 of 90 ± 20pM-insulin (mean + S.E.M., n = 5). Time courses for the production of cyclic AMP
by glucagon-plus-GTP-stimulated adenylate cyclase activity in the isolated membrane fraction were linear over 12 min incubation whether or not insulin (1 nM) was added (Fig. 2a). When membranes were
Table 1. Effect of insulin on adenylate cyclase activity of hepatocyte membranes Assays were carried out for 10min at 300C using a washed membrane fraction from broken hepatocytes (Houslay & Elliott, 1979). Results are means ± S.D. (n = 4) for specific activities and means + S.E.M. (n = 10) for the determination of insulin effects. Here, n refers to different membrane preparations. For any particular membrane preparation adenylate cyclase assays were performed in triplicate. Specific activity Inhibition by (pmol/mg per min) 1 nM-insulin (%) Basal 0.8+0.1 4+2 GTP (0.lmM) 1.6+0.2 32+2 5.1 +0.4 0+3 p[NH]ppG (0.1 mM) Glucagon (1nM) 7.0±0.5 2±3 13.3 +0.9 Glucagon (I nM) 18 + 2 + GTP (0.1 mM)
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-log {[Insulinl (M) -log I lGlucagon I (M) j Fig. 1. Insulin inhibition ofadenylate cyclase activity (a) shows the extent of inhibition elicited by insulin (1 nM) of the glucagon (1 nM)-stimulated adenylate cyclase activity observed in the presence of increasing concentrations of GTP. Results are means ± S.E.M. (n = 5), indicated by the bars. (b) shows the extent of inhibition elicited by insulin (1 nM) of adenylate cyclase activity observed in the presence of GTP (0.1 mM) with increasing concentrations of glucagon. Results are means + S.E.M. (n = 5), indicated by the bars. (c) shows the extent of inhibition of glucagon (1 nM)-plus-GTP (0.1 mM)-stimulated adenylate cyclase activity by increasing concentrations of insulin. Results are means ± S.E.M. (n = 5), indicated by the bars. -log {[GTPI (M) }
prepared from either glucagon-desensitized cells (Heyworth & Houslay, 1983) or cholera-toxintreated cells (Houslay & Elliott, 1979), then, although time courses of glucagon-plus-GTP-stimulated activities were linear, the inhibitory effect of insulin (1nM) was abolished (Fig. 2). Indeed we observed (Fig. 2) no inhibitory effect of insulin on GTP-stimulated adenylate cyclase from either desensitized (
