Jul 24, 1989 - hans by measuring the secretory responses of normal and. PKC-depleted .... Peter-Riesch, B., Fathi, M., Schlegel, W. & Wollheim, C. B.. 15.
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12. Peter-Riesch, B., Fathi, M., Schlegel, W. & Wollheim, C . B. (1988)J. Clin.Invest. 81, 1154-1 I61 13. Persaud, S. J., Jones, P. M., Sugden, D. & Howell, S. L. (1989) FEBS Lett. 245,80-84 14. Wollheim, C. B. Dunne, M. J., Peter-Riesch, B., Bruzzone, R., Pozzan, T. & Petersen, 0.H. (1988)EMBO J. 7,2443-2449 15. Prentki, M., Glennon, M. C., Thomas, A. P., Morris, R. L., Matschinsky, F. M. & Corkey, B. E. ( 1988) J. Uiol. Chem. 263, 11044-11047 16. Wollheim, C. B. & Biden, T. J. ( 1986) J. Uiol. Chern. 261, 8314-83 19 17. Berridge, M. J. (1988) I'roc. R. Soc. London Ser. B 234, 359-378 18. Regazzi, R., Li, G., Ullrich, S., Jaggi, C . & Wollheim, C. B. (1989)J. Uiol.Chem. 264,9939-9944
19. Bell, R. M. ( 1 986) Cell (Cambridge, Muss.} 4 5 , 6 3 1-632 20. Yamatani, T., Chiba, T., Kadowski, S. Hishikawa, R., Yarnaguchi, A., h i , T. & Fujita, S. ( 1988) Endocrinology 122, 2826-2832 21. Yaseen, M. A., Pedley, K. C. & Howell, S. L. (1982) Uiochem. J. 206,81-87 22. Vallar, L., Biden, T. J. & Wollheim, C. B. ( I 987) J. Biol. C'hem. 262,5049-5056 23. Schlegel, W., Winiger, B. P., Mollard, P., Vacher, P.. Wuarin, El, Zahnd, G. R., Wollheim, C. B. & Dufy, B. (1987) Nuture (London)3 2 9 , 7 19-72 I 24. Pralong, W. F., Wollheim, C. B. & Bruzzone, R. (1988) FEHS Lett. 242,79-84 Received 24 July 1989
Protein kinase C and the regulation of insulin secretion presence of the inactive phorbol ester 4a-phorbol 12,13-didecanoate (PDD; 200 nM) to serve as controls. In earlier experiments in which total PKC activity in broken cell preparations was measured, no detectable diolein- or PMAactivated PKC activity remained after 20-24 h treatment Protein kinase C (PKC) is a closely related family of C a 2 + / with PMA, although both diolein and PMA stimulated PKC phospholipid-dependent kinases which have been identified activity in extracts from PDD-treated islets 171. In more recent in a wide range of tissues, including islets of Langerhans and experiments we have measured ion-exchange purified PKC insulin-secreting tumour cells (for review see, [ 11). PKC activity in cytosolic and membrane fractions prepared from activity is thought to be regulated by the availability of di- islets [8].In control islets treated with PDD most of the PKC acylglycerols (DAG) which are generated by synthesis de activity was found in the cytoplasmic fraction (472 k 59 novo, or by the phospholipase C-mediated hydrolysis of fmol/min per islet, r n e a n f s . ~ . ~n. ,= 3 ) and much lower membrane phospholipids in response to glucose, cholinergic levels of activity were associated with a membrane fraction agonists or depolarization (reviewed in [2]). A role for PKC (44 k 6 fmol/min per islet). PMA treatment ( 21 h) virtually in insulin secretion is suggested by the fact that DAG ana- abolished cytoplasmic PKC activity (25 a 4 fmol/min per logues or phorbol esters, which substitute for DAG [3],can islet) without causing significant changes in the low levels of membrane-associated activity (40 f 9 fmol/min per islet). stimulate insulin secretion (for references, see [ 21). As might be expected, given the abolition of PKC activity, Studies of the role of PKC in insulin secretion using inhibitors of PKC activity are often difficult to interpret because of insulin secretion from PMA-treated islets was not stimulated the questionable specificity of many inhibitors [4-61. We have by a subsequent challenge with PMA, as shown in Fig. 1. therefore investigated the functional role of PKC in the regu- However, down-regulation of PKC by PMA treatment did lation of insulin secretion from isolated rat islets of Langer- not detectably inhibit glucose-induced insulin secretion from hans by measuring the secretory responses of normal and intact islets (Fig. l), arguing against a major role for PMA/ PKC-depleted islets to insulin secretagogues, and by corre- diolein-stimulatable PKC as a regulator of glucose-induced lating secretory responses to the activation and redistribution insulin secretion. There is considerable evidence that glucose of PKC within islets. These studies used islets isolated from can stimulate the hydrolysis of membrane phosphoinositides rat pancreas by collagenase digestion and maintained in to produce DAG, which may activate PKC, and inositol tissue culture for 20-24 h [7, 81. Insulin secretion was phosphates, which can release intracellular stores of Ca" measured by radioimmunoassay [ 91 and ion-exchange puri- (see [2]).Whatever the importance of inositol phosphates in fied PKC activity was measured by phosphorylation of signal transduction in the B-cell, the results of our studies, and similar recent reports from other groups [ 51, suggest that exogenous histone, as described in [S]. the activation of PKC is not obligatory for the stimulation of insulin release by glucose, the major physiological insulin Insulin secretion from PKC-depleted islets secretagogue. Phospholipid hydrolysis in B-cells is stimulated by Prolonged exposure to tumour-promoting phorbol esters causes a reduction or abolition of PKC activity, with conse- muscarinic cholinergic agonists, such as carbachol (CCh), quent loss of various cellular functions, in a variety of cell which are known to enhance glucose-induced insulin secretypes (see [lo]).The loss of PKC activity from treated cells tion. Although there are differences between the phosphoprobably reflects an increase in the rate of degradation of the lipid hydrolysis induced by CCh and that induced by glucose kinase within the cells [ 111. This appears to be a relatively (see [2]), there is evidence that cholinergic agonists may specific means of down-regulating PKC, since cellular func- induce rapid increases in DAG concentrations [ 13, 141. It is tions which are lost after such treatment can be restored by therefore feasible that cholinergic stimulation of insulin secretion is mediated by activation of PKC. Fig. 1 shows micro-injection of purified PKC into the treated cells 1121. In our studies, isolated islets were cultured for 20-24 h in the effects of CCh on insulin secretion from PKC-depleted the presence of the PKC activator phorbol 12-myristate 13- islets, and from control (PDD-treated) islets. CCh (500 p ~ ) acetate (PMA; 200 nM) to down-regulate PKC, o r in the caused a marked enhancement of glucose-induced (20 mM) insulin secretion from PDD-treated islets. This response to Abbreviations used: PKC, protein kinase C; DAG, diacylglycerol; CCh was significantly inhibited in PMA-treated (and thus PMA, phorbol 12-myristate 13-acetate; PDD, 4a-phorbol 12, 13- PKC-depleted) islets, although the secretory response to 20 mM-glucose was comparable with control (PDD-treated) didecanoate; CCh, carbachol. SIMON L. HOWELL, PETER M. JONES and SHANTA J. PERSAUD Biomedical Sciences Division, King's College London, Kensington, London W8 7AH, U.K.
1990
NUTRIENT REGULATION OF INSULIN SECRETION islets. T h e reduced secretory response to CCh in PKCdepleted islets cannot be accounted for by a reduction in CCh-induced phospholipase C activity because CCh had similar effects on phospholipase C activity in control (PDD-
115 treated) and PMA-treated islets, as assessed by the generation of radiolabelled inositol phosphates (Table l). These results suggest that the activation of PKC may be responsible, at least in part, for the stimulation of insulin secretion by cholinergic agonists.
PKC translocation in islets
T
Fig. 1. Insulin secretion from I'KC-depleted islets PMA-treated islets ( m ) were unable to respond to a subsequent exposure to PMA (500 nM), and their secretory responses to 500 pM-CCh (in the presence of 20 mMglucose) was significantly reduced when compared with those of control (PPD-treated) islets ( 0 ) . Secretion is expressed as a percentage of the response to 20 mwglucose, but note that the secretory responses to 20 mM-glucose were not affected by the PMA treatment (PDD-treated, 1.7 k 0.4 ng/h per islet; PMA-treated, 2.3k0.4 ng/h per islet; n = 5 ; I>>0.2). Results are means fS.E.M. ( n= 5 ) . Statistical significance: *P< 0.01 versus PDD-treated islets.
A role for PKC in cholinergic control of insulin secretion was supported by measurements of the redistribution of PKC activity within islet cells in response to insulin secretagogues. The activation of PKC is thought to involve translocation of the enzyme from the cytosol to membranes, where it associates with phosphatidylserine forming a catalytically active complex [ 1.51. Exposing islets to PMA (5 min, 37°C) produced a significant decrease in cytosolic PKC activity (control 275 f 19 fmol/min per islet; plus 500 nMPMA, 1 4 0 k 10 fmol/min per islet; 1'
