Biochimica et Biophysica Acta, 1091 (1991) 120-122 © 1991 Elsevier Science Publishers B.V. (Biomedical Division) 0167-4889/91/$03.50 ADONIS 016748899100065V

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BBAMCR 10267

BBA Report

Activation of protein kinase C is essential for sustained insulin secretion in response to cholinergic stimulation Shanta J. Persaud, Peter M. Jones and Simon L. Howell Biomedical Sciences Division, King's College London, Kensington, London (U. K.)

(Received 28 May 1990)

Key words: Islets of Langerhans; Insulin secretion; Cholinergic; Protein kinase C

Insulin secretion from isolated rat islets of Langerhans is enhanced by cholinergic agonists, such as carbachol (CCh), in the presence of a stimulatory concentration of glucose. Depletion of islet protein kinase C activity by prolonged exposure to a tumour-promoting phorbol ester did not prevent the initial secretory response to CCh, but markedly reduced the duration of CCh-induced elevated secretory rates. These results suggest that the major action of PKC is in maintaining rather than initiating the insulin secretory response to cholinergic agonists. Cholinergic muscarinic agonists, such as carbachol (CCh), stimulate insulin secretion from pancreatic Bcells [1-3]. Cholinergic stimulation of iasulin secretion is usually only detected in the presence of a stimulatory concentration of glucose [2,3] and is characterised by an enhancement of both first and second phases of the biphasic secretory response to glucose [2,3]. One mechanism of action of cholinergic agonists appears to be through increasing plasma membrane permeability to Na + ions, resulting in membrane depolarisation and enhanced Ca 2+ influx [4]. In addition, the Ca2+/ phospholipid-dependent protein kinase C (PKC) has been implicated in cholinergic signalling in B-cells. Thus, CCh stimulates the hydrolysis of membrane inositol phospholipids with the consequent generation of diacylglycerol (DAG), which can activate PKC [5,6]; cholinergic agonists promote translocation of PKC activity from cytosol to membrane in both islets [7] and an insulin-secreting cell line [8], consistent with activation of the enzyme; and inhibitors of PKC inhibit cholinergic stimulation of insulin secretion [9]. We have recently shown that cholinergic stimulation of insulin secretion from rat islets of Langerhans was reduced, but not abolished, by phorbol ester-mediated down-regulation of islet PKC activity [3]. These studies confirmed an important role for PKC in cholinergic signalling in islets. However, since cumulative insulin secretion was measured over a fixed time-course in our

Correspondence: S.J. Persaud, Biomedical Sciences Division, King's College London, Campden Hill Road, Kensington, London W8 7AH, U.K.

previous experiments, it was unclear whether PKC activation was necessary for the initiation or maintenance of CCh enhancement of insulin release, or for both of these processes. We have, therefore, measured the effects of CCh on insulin secretion from normal and PKC-depleted islets in a perifusion system to study the effects of PKC-depletion on the pattern of cholinergic stimulation of insulin secretion. To down-regulate PKC, collagenase-isolated rat islets were cultured for 20 h in the presence of the tumourpromoting phorbol ester, phorbol myristate acetate (PMA, 200 nM), as previously described [3]. Control islets were exposed to the inactive analogue 4-a-PMA (200 nM). Treatment with PMA caused a marked (83.3 ± 3.7%, n - 3) reduction in total (cytosolic plus membrane) PKC activity, consistent with enhanced degradation of the enzyme without any change in its rate of synthesis [10]. As expected, insulin release from PKCdepleted islets, measured in static incubation experiments, was not enhanced by a subsequent exposure to PMA (4-a-PMA-treated: 20 mM glucose, 2.57_ 0.3 ng/islet per h; + 500 nM PMA, 5.64 ± 0.25, P < 0.01, n = 9; PMA-treated: 20 mM glucose; 2.67 ± 0.15, + 500 nM PMA, 3.02 ± 0.30 P > 0.2, n = 10). Fig. 1 shows the pattern of insulin secretion from perifused islets in response to 20 mM glucose and to a maximum stimulatory concentration of CCh (500/~M) in the presence of 20 mM glucose. Control (4-a-PMA-treated) islets showed the characteristic biphasic response to a stimulatory concentration of glucose (20 mM). Glucose also stimulated a biphasic release of insulin from PKC-depleted (PMA-treated) islets, but with a significantly enhanced first phase of insulin secretion. Potentiation

121 of the first phase of glucose-induced insulin release following PKC down-regulation has been reported previously [11] but contrary to this earlier study, we found no inhibition of the second phase of secretion from PKC-depleted islets. In control islets CCh (500 /~M) stimulated a rapid enhancement of insulin release which was maintained for the duration of the experiment at levels approximately twice as high as those obtained with 20 mM glucose alone. However, while PKC-depleted islets also responded rapidly to CCh, the secretory response to CCh was not maintained. Thus, the initial secretory response of PKC-depleted islets to CCh was not significantly different to that of control islets during the first 10 min of exposure, but was significantly reduced thereafter (Fig. 1). A number of conclusions can be drawn from the results of these experiments. First, it is clear that downregulation of PKC does not render islets insensitive to cholinergic agonists, since the rapidity and magnitude of the initial response to CCh was similar between control and PKC-depleted islets. The ability of PKC-depleted islets to mount an initial, but unsustained secretory response to CCh explains our previous report [3] of a reduced cumulative secretory response to CCh in PKC-depleted rat islets. Conflicting results of the effect of PKC down-regulation on cholinergic stimulation of insulin secretion have been reported in studies using insulin-secreting tumour cells. In a preliminary study using RINm5F cells, it was reported that PKC depletion markedly enhanced carbachol-induced insulin re-

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lease [12]. However, the recent report of an inhibition of acetylcholine-induced insulin secretion from PKC-depleted HIT-T15 cells is more consistent with our results in rat islets [13]. One possible explanation for apparent differences in the involvement of PKC in secretory responses of islets, HIT or RIN cells could be the differential expression of PKC isozymes in the different cell types [14]. Secondly, the unchanged initial secretory response of PKC-depleted islets to CCh in the present studies implies that activation of PKC is not required for the recognition of, and initiation of secretion by, cholinergic agonists. This in turn suggests that another signalling system, probably Ca 2+ influx following membrane depolarisation [4], initiates CCh-induced insulin secretion. Evidence for the involvement of Ca 2÷ influx, independent of PKC activation, in cholinergic signalling has been provided by studies in HIT-T15 cells where Ca 2÷ influx in response to acetylcholine was not affected by prolonged exposure of the cells to PMA [13]. Further support for our assertion that PKC activation is not responsible for the initial response to CCh are earlier observations that CCh stimulated inositol phosphate (and presumably DAG) generation in the absence of extracellular Ca 2+, but did not affect insulin release [15]. Similarly, we have shown that CCh stimulated the translocation of PKC, but did not stimulate insulin secretion, at concentrations of glucose (2 mM) which do not enhance Ca2+-influx into B-cells [3]. Finally, the transience of the secretory response to CCh in PKC-depleted islets suggests that activation of PKC is essential for the maintenance of a normal secretory response to cholinergic agonists. It was proposed some years ago that the first phase of the biphasic insulin secretory response to glucose is caused by Ca 2+ influx and the second phase by activation of PKC [16]. The results of our own and other [17-19] studies do not support such a mechanism for glucose-induced insulin secretion, but we propose that a similar process may underlie the normal response to cholinergic stimulation. Thus, B-cell membranes already depolarised by glucose or other primary stimuli are further depolarised by cholinergic agonists, leading to enhanced Ca2+-influx and initiation of cholinergic-induced insulin secretion [4]. Thereafter, maintenance of the insulin secretory response to CCh is secondary to DAG-stimulated PKC redistribution.

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Fig. 1. Cholinergic stimulation of insulin secretion from perifused rat islets. Group of 50 islets were perifused [3] with buffer containing 2 mM glucose (0-10 rains) and 20 mM glucose thereafter. After 60 min the buffer (20 mM glucose) was supplemented with 500/,M CCh, as shown. Fractions were collected at 2 min intervals and insulin content measured by radioimmunoassay [20]. Points (O = PMA-treated, 0 -- 4a-PMA-treated) show means+S.E.M. (n--4) of results from four separate experiments, each of two observations. * P < 0.05 comparing PMA-treated vs. 4-a-PMA-treated islets using Student's t-test.

Acknowledgements Financial support from the British Diabetic Association is gratefully acknowledged. S.J.P. is a Wolfson Foundation Research Fellow. P.M.J. is a MRC Senior Research Fellow (Non-Clinical). We also thank Ms. F.E. Mann for expert technical assistance.

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