Effects of cholinergic agonists on diacylglycerol and intracellular calcium levels in pancreatic β-cells

Cellular Signalling Vol. 5, No. 6, pp. 777-786, 1993.

0898-6568/93 $6.00 + 0.00 ~ 1993PergamonPressLtd

Printed in Great Britain.

EFFECTS OF CHOLINERGIC AGONISTS ON DIACYLGLYCEROL AND INTRACELLULAR CALCIUM LEVELS IN PANCREATIC fl-CELLS LING WENG, MARK DAVIES and STEPHENJ. H. ASHCROFT* Nuffield Department of Clinical Biochemistry, John Radcliffe Hospital, Headington, Oxford OX3 9DU, U.K. (Received 1 June 1993; and accepted 21 June 1993)

Abstract--We have studied the effects of cholinergic agonists on the rates of insulin release and the concentrations of diacylglycerol (DAG) and intracellular free Ca 2+ ([Ca2+]i) in the/~-cell line MIN6. Insulin secretion was stimulated by glucose, by glibenclamide and by bombesin. In the presence of glucose, both acetylcholine (ACh) and carbachol (CCh) produced a sustained increase in the rate of insulin release which was blocked by EGTA or verapamil. The DAG content of MIN6 E-cells was not affected by glucose. Both CCh and ACh evoked an increase in DAG.which was maximal after 5 min and returned to basal after 30 min; EGTA abolished the cholinergic-indueed increase in DAG. ACh caused a transient rise in [Ca2+]i which was abolished by omission of Ca 2+ or by addition of devapamil. Thus, cholinergic stimulation of/~-cell insulin release is associated with changes in both [Ca2+]i and DAG. The latter change persists longer than the former and activation of protein kinase C and sensitization of the secretory process to Ca 2+ may underlie the prolonged effects of cholinergic agonists on insulin release. However, a secretory response to CCh was still evident after both [Ca2+]i and DAG had returned to control values suggesting that additional mechanisms may be involved. Key words: Insulin release, acetylcholine, diacylglycerol, intracellular calcium, fl-cell.

INTRODUCTION

diacylglycerol and it has indeed been shown that CCh or ACh elicit breakdown o f p-cell polyphosphinositides and formation of inositol polyphosphates [13-18]. However several pieces of evidence indicate that mobilization of intracellular Ca 2+ by IP 3 is not a sufficient explanation for the stimulatory effect of cholinergic agents on insulin release. Thus, it has been long established that ACh-induced insulin secretion is dependent on extracellular Ca 2+ [11, 19-21]. Both electrophysiological [10, 22-24] and ion flux measurements [12, 21, 25] indicate that there is enhanced influx of Ca 2+ into the//-cell in response to cholinergic stimulation. This has been suggested to be due to depolarization induced by increased sodium permeability of the p-cell membrane [26, 27]. There is general agreement that stimulation o f insulin release by cholinergic agents requires the presence of glucose [10, 28]; it is suggested that only when the p-cell membrane is already depolarized by glucose can ACh elicit sufficient additional depolarization to potentiate insulin release [26].

NEURAL modulation o f glucose-induced insulin secretion is of major importance in vivo [1, 2]. Vagal stimulation causes insulin release [3]; this effect is blocked by atropine [4] and is mediated by muscarinic receptors [5] shown to be of the M 3 type [6, 7]. Cholinergic agents elicit a prolonged potentiation of glucose-stimulated insulin secretion in vitro [8, 9]. The mechanisms responsible have not been fully elucidated although changes in cyclic nucleotide concentrations are not involved [10-12]. Muscarinic cholinergic receptors are frequently coupled to hydrolysis of phosphatidylinositol bisphosphate and generation of inositol trisphosphate and

* Author to whom correspondence should be addressed. ACh--acetylcholine; CCh---carbachol; DAG--sn-l,2-diacylglycerol; D M E M - - D u l b e c c o ' s modified Eagle's medium; BSA--bovine serum albumin; [Ca2 +]i--int racellular free Ca 2+ concentration; DETAPAC--diethylenetriaminepentaacetic acid. Abbreviations:

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778

L. WL~Oet aL

However, it has also been shown that the increase o f Ca 2+ influx brought about by ACh is too small to account fully for the amplification of glucose-induced insulin release [26, 29]. Moreover, in H I T T15 /]-cells we have shown that ACh causes a sustained increase in glucosestimulated insulin release but only a transient rise in intracellular Ca 2+ concentration [9]; we suggested, therefore, that a major mechanism in the response to cholinerglc agents involves an increase in the sensitivity of the secretory system to intracellular Ca 2+, envisaged as arising from activation of protein kinase C. Consistent with this interpretation, pre-treatment of fl-cells with phorbol ester to downregulate protein kinase C did not affect the ability of ACh to increase [Ca2+]i but abolished its potentiation of insulin release. In rat islets of Langerhans, the initial secretory response to carbachol in the presence of 20 mM glucose was not affected by down-regulation of protein kinase C but sustained cholinerglc potentiation of secretion was abolished [30]. Thus, an early increase in [Ca2+]i is important for the initial increase in insulin release induced by ACh but protein kinase C activation is necessary for sustained cholinergic-induced insulin release. It is assumed that diacylglycerol (DAG) mediates the effects on protein kinase C of cholinergic stimulation. However, there are only limited data on D A G content of fl-cells. The aim of the present study was to characterize the response o f D A G to muscarinic cholinergic stimulation in a fl-cell line responsive to glucose and cholinergic agents.

MATERIALS AND METHODS Materials Tissue culture materials were from Gibco Europe (Paisley, U.K.). Guinea-pig anti-insulin serum was a girl from Dr R. Turner (Diabetes Research Laboratory, Radcliffe Infirmary, Oxford, U.K.). Rat insulin standard was a gift from Dr A. J. Moody (Novo Research Laboratories, Gentofte, Denmark). [7-3~]ATP and [nsI]insulin were from Amersham International (Amersham, U.K.). Quln 2 and quln 2/AM were from Aldrich Chemical Co. (Gillingham, U.K.). Bovine serum albumin (BSA) was from BCL

(Lewes, U.K.). Verapamil, carbachol, acetylcholine and protease inhibitors were supplied by the Sigma Chemical Co. (Poole, U.K.). Optifluor was purchased from Packard Instruments (Downers Grove, IL, U.S.A.). Other reagents were from BDH Ltd (Poole, U.K.). MIN6 ~-cells were kindly supplied by Prof. Y. Miyazaki (University of Tokyo) and Escherichia coil strain N4830/pJWI0 was a gift from Dr R. M. Bell (Duke University).

Culture of MIN6 B-cells MIN6 cells were cultured in RPMI 1640 medium containing 10% foetal calf serum and antibiotics at 37°C in a humidified atmmphere of air/CO2 (95:5). Cells were passaged weekly by trypsinization.

Assay of insulin secretion Multiwells were seeded with 5 x 105 MIN6 fl-cells. After 2-3 days, the culture medium was replaced with DMEM medium containing 15% foetal calf serum and 0.5 mM glucose and preincubated for 1 h at 37°C. The medium was replaced with DMEM containing 5% foetal calf serum and glucose and test agents at the concentrations stated in the text and the cells were incubated for the times indicated at 37°C. Samples (0.2 ml) of incubation medium were then centrifuged at 190 g for 5 rain at 4°C to pellet any cells. Insulin in samples of medium was measured by radioimmunoassay as described previously [31]. In some experiments secretion was measured in cell suspensions prepared and incubated as described below for measurement of DAG.

Assay of DAG Cells were harvested by trypsinization, washed twice with DMEM containing 15% foetal calf serum and 0.5 mM glucose by centrifugation at 1500 r.p.m. for 4 rain. Aliquots of 106 cells per tube were preincubated with the same medium for 1 h before incubation for varying times in DMEM medium containing 5% foetal calf serum and glucose and test substances at the concentrations stated in the text. After incubation, the cells were centrifub~d at 1800 r.p.m, for 2 rain, washed with ice-cold phosphatebuffered saline (without Ca 2+ and Mg2+) and the cell pellet resuspended in 0.8 ml of ice-cold 1 M NaCI. Samples were extracted with 3 ml of chloroform/ methanol (1:2 v/v). The monophase was mixed, 1 ml of CHCI 3 and 1 ml 1 M NaCI were added to break phases and the phases were separated by brief centrifugation at 5000 r.p.m. Samples (200 ~tl) of the chloroform phase were dried on a Speedivac and

Diacylglycerol in fl~cells stored at - 2 0 ° C for D A G assay within 72 h. DAG was assayed by the method of Preiss etal. [32]. Membranes containing diacylglyeerol kinase were prepared from E. coil strain N4830/pJWI0, which contains dgkA under control of the 2 left promoter [33] and stored at -70°C. Membranes were diluted to 0.5 mg/ml in l0 mM imidazole/DETAPAC prior to use and then 150 ~tl diluted membranes were added to 750 ttl 0.1 M imidazole buffer (pH 6.6) (containing 0.1 M NaC1, 25 mM MgC12, 2 mM EGTA) and 150 ~tl 2 0 m M dithiothreitol in 10 mM imidazole/DETAPAC. D A G standards (62.5-1000 pmol) were diluted in chloroform and then evaporated to dryness (Speedivac). Samples and standards were dissolved in 20 ttl detergent solution [7.5%(w/v) n-octyl-fl-glucopyranoside, 5 mM cardiolipin in l mM DETAPAC] by vortexing and sonication. To each assay sample was added 70 lal of the membrane mixture and l0 ttl [y-32p]ATP (1 ttCi in 0.1 M imidazole/HCl pH 6.6, 1 mM DETAPAC, 5 mM ATP). Tubes were capped, vortexed and then incubated at 25°C for I h. After incubation, 20 ttl 1% (v/v) perchloric acid and 450 p.l chloroform/methanol (1:2 v/v) were added, the tubes were vortexed and then left at room temperature for l0 rain. After centrifugation at 2000 g for 1 min, 150 lal chloroform and 150 lal perchloric acid (1% v/v) were added and the tubes vortexed and again centrifuged at 2000 g for 1 min. The upper phase was removed and discarded. Perchloric acid 1 ml 1% (v/v) was added, the tubes vortexed (3 x 15 s) and then centrifuged at 2000 g for 1 min. The upper phase was discarded and the washing with perchloric acid repeated. To 50 lal of the lower phase was added 10 ml scintillant and the radioactivity in the samples counted by liquid scintillation spectrometry.

Assay of intracellular calcium MIN6 cells were loaded with quin 2 by incubation for 20 min with 50 aM quin 2/AM and stored on ice at a density of 25 x l06 cells/ml prior to fluorescence measurements carried out in a Perkin Elmer LS5 luminescence spectrometer essentially as described previously for HIT Tl5 fl-cells [34]. Cells (5 x 106) were preincubated at 37°C for 15 rain with continuous stirring in glucose-free Hepes-buffered Krebs medium containing 5 mg/ml BSA before addition of glucose and test agents. Fluorescence measurements were recorded at 30-s or l-rain intervals. At the end of each experiment, quin 2 fluorescence was calibrated as previously described [34] and [Ca2*]i was calculated [35]. The mean intracellular quin 2 concentration for the MIN6 cell suspensions used was calculated to be 1.0_+0.1 mM (mean _-4- S.E.M., n -- 9).

779 RESULTS

Effects of cholinergic and other agents on insulin secretion from MIN6 fl-cell T h e secretory responses o f M I N 6 tic, ells to glucose, C C h and other stimuli were assessed in batch incubations using cells attached to multiwells. Insulin secretion was linear for 4 h; raising extracellular glucose f r o m 0.5 to 5 m M stimulated insulin secretion 3-fold and an increase to 25 m M glucose elicited a further doubling o f secretion rate (Fig. l). In the presence o f 0.5 m M glucose, A C h (10 ttM), C C h (1 m M ) and glibenclamide (10 n M ) m a r k e d l y enhanced insulin secretion (Table 1). In the presence o f 5 m M glucose, insulin release was stimulated by A C h (10 ttM), C C h (0.1 or 1 m M ) and by bombesin (l or l0 n M ) but not by A C h (1 laM), vasopressin (1 or l0 n M ) or neurotensin (l or l0 nM). Neither glucose nor cholinergic agonists stimulated insulin release in the presence o f 50 ~tM verapamil or l0 m M EGTA. Secretion experiments were also conducted over short incubation times to parallel the measurements o f D A G and Ca :+. These experiments were carried out on M I N 6 S0-

40-

20-

O4 0

1

2

3

4

Time (h) FXG. 1 Time course of insulin release from MIN6 fl-cells. Cells were incubated at 37°C in multiwells for the times stated in DMEM containing 0.5 mM (C)), 5 mM (Z~) or 25 mM (Q) glucose. Insulin released into the medium was measured by radioimmunoassay. Data are plotted as mean + S.E.M. for 4 observations.

"780

L. WENG et aL TABLE I. EFFECT OF C X O ~ O I C AND O T m m AGmcrs ON Tim S~"RETXON OF XNSULnq FROM M I N 6 ~-CEL~ Glucose concn (raM) Addition

Insulin release (mU/10 s cells/4h)

0.5

14.7÷ l.l (6) 42.5±2.5 (7)* 42.5±3.1 (6)* II.I±1.5 (3) 12.1± 1.6 (3) 50.3± 1.0 (4)* 20.2±0.7 02)* 23.0± 1.8 (4) 39.7±2.6 (ll)* 62.0± 3.4 (4)* 55.7±4.0 02)* 20.4±2.8 (4) 18.4_ 1.3 (4) 24.8±2.5 (4) 24.7__.2.2(4) 41.I±4.3 (4)* 52.2±2.1 (4)* 25.3± 1.6 (3) 8.25:0.9 (3)* 12.0±0.8 (3)*

5

25

None 10 tiM A C h I mM CCh l mM CCh÷10 mM EGTA I m M C C h + 50 # M veraparnil 0.I p M glibenclamide None I pM ACh 10 p M A C h 0.I m M C C h I mM CCh I n M vasopressin 10 n M vasopressin l n M neurotensin 10 n M neurotensin I n M bombesin 10 n M bombesin None I0 m M E G T A 50 tiM verapamil

MIN6 cells were incubated in multiwells (106 cells/well) for 4 h at 37°C in DMEM containing the additions shown. Insulin released into the medium was measured by radioimmunoassay. *P < 0.01 vs control at the same glucose concentration. E-cells in suspension prepared and incubated under identical conditions to those used for assay of diacylglycerol. The potentiatory effect of CCh on glucose-induced insulin release was shown to be sustained for at least 30 rain (Fig. 2).

Effects of cholinergic and other agents on/S-cell diacylglycerol levels The mean D A G content of MIN6 fl-cells incubated with 0.5 mM glucose for 5 min was 0.254+0.013 fmol/cell (n = 22). An increase in medium glucose concentration to 2, 10 or 25 mM elicited no significant increase in fl-cell D A G (102_+10 ( n = 3 ) , 101_+5 ( n = 3 ) and 100_+6% (n = 4) of the value of 0.5 mM glucose, respectively). The effects of cholinergic agonists on M1N6 fl-cell D A G levels are shown in Table 2. In the presence of 0.5 mM glucose, incubation for 10 rain with CCh (0.1 or 1 raM)

25T 20-

"~ ~

15-

100 5 0

--

.k I

10

I

20

I

30

Time(rain) Fro. 2. Effect of carbachol on insulin release from MIN6 /~ells. Cells were incubated at 37°C in DMEM in multiwells for the times stated in the absence ( O ) or presence ( 1 ) of 1 mM carbachol. Insulin released into the medium was measured by radioimmunoassay. Data are plotted as mean + S.E.M. for 4-8 observations.

Diaeyiglyeerol in fl-eells

TABLE 2. EVICTS OF CHOLINERGICAC~NISTSON THE DAG CONTENTOF MIN6/]-cells

781

TAnLE 3. EFFECTSOF EGTA ON THE n ~ E IN DAG CONTENT OF MIN6/]-CELLS ELICITED BY CHOLINL~GIC AGONISTS

Glucose concn (mM) Addition 0.5

None 10 p,M ACh 0.I mM CCh 1 mM CCh None 10 pM ACh 0.1 mM CCh

25

DAG (fmol/cell)

Agonist

0.154_+ 0.020 (12) 0.269_+0.019(10)* 0.323_+0.032 (6)* 0.550-t-0,120 (2) 0.1304-0.010 (4) 0.3054-0.064 (4)* 0.3504-0.010 (4)*

None (control)

MIN6 /]-cells were incubated in DMEM for 10 min at 37°C with the additions stated. *P < 0.01 vs control at the same glucose concentration.

or ACh (10 ~tM) increased D A G significantly. The magnitude o f the cholinergic-induced increase in ~-cell D A G was not significantly different in the presence of 25 m M as compared with 0.5 m M glucose. The time course of the increase in D A G elicited by CCh is shown in Fig. 3. An elevation

i:o-TT T 100,

, 10

i 20

I 30

1 mM CCh 10 laM ACh

EGTA (10 mM) DAG (fmol/cell)

-

+ + +

0.1404-0.008 (15) 0.1434-0.015 (16) 0.201+0.013 (14)* 0.147+0.014 (14) 0.329_+0.023(3)* 0.120_+0.004 (3)

MIN6//-cells were incubated for 5 min at 37°C in DMEM containing 0.5 mM glucose and the additions stated. *Significantly greater than control, P < 0.001. of D A G could be observed at the earliest time studied (2 min incubation). D A G levels in the presence of CCh remained elevated for 20 min but were not significantly above control values after 30 min. The stimulatory effect of cholinergic agonists on/~-cell D A G was dependent on the presence of extracellular Ca 2+ (Table 3). In the presence of 10 m M E G T A , neither ACh nor CCh elicited a significant effect on /~-cell D A G . CCh was also ineffective in the presence of the Ca-channel blockers devapamil or verapamil ( D A G levels in the presence of 1 m M CCh plus 50 IxM devapamil or verapamil were 81 ___ 7 or 106 _ 13% (n = 4) respectively o f the value in the presence of the channel blocker alone). The effects on ~-cell D A G levels of other modifiers of insulin secretion were also tested. N o significant effect on D A G was observed with 100 nM vasopressin, 100 nM bombesin, 100 nM neurotensin, 100 nM pancreastatin, 100 nM glibenclamide, 100 nM staurosporine or 40 m M K + (Table 4).

Tm~ (min)

FIG. 3. Time course of carbachol-evoked increase of DAG in MIN6 p-cells. Cells were incubated in DMEM in the absence or presence of 1 mM earbachol for the times indicated. After extraction, the DAG content of the cells was measured enzymically. The DAG content of cells incubated with earbachol i s plotted as a percentage of that in control cells at the same time point in the same experiment and given as mean 4- S.E.M. for 4--11 determinations.

Effects of cholinergic and other agents on MIN6 [J-cell intracellular calcium levels As shown in Fig. 4, raising glucose from zero to 0.5 m M caused a marked increase in intracellular Ca 2+ concentration; a further increase of glucose to 2 m M elicited an additional rise in Ca 2+ but no consistent increase was seen when

782

L. WN~Get al.

t

TABLE 4. EvrEcrs OF MODIFIERS OF INSULINSECRETION

ON THE DAG CONTENTOF MIN6/]-CELLS Addition

DAG (% control)

100 nM vasopressin 100 nM bombesin 100 nM neurotensin 100 nM pancreastatin 100 nM glibenclarnide 100 nM staurosporine 40 mM K +

109+ 8 (2) 100+_ 15 (3) 105

TI

+ 20 (3)

91+ 4 (3) 100_ 18 (4) 102+ 6 (8) !17+15 (4)

200

150

MIN6/]-cells were incubated for 5 rain at 37°C in DMEM containing 0.5 mM glucose and the additions stated. Data are expressed as a percentage of the DAG content in control cells (no addition) in the same experiment.

0 5raM

loo 10

I ~ ,

i

15

(

-

20

25

....

i

30

_.

/ " 35

~ i

40

Tune (mira) FIG. 4. Effect of glucose on [Ca2+]i in MIN6/]-cells. Quin 2-loaded MIN6/]-cells (5 x l0 6 cells per tube) were preincubated in glucose-free Hel:~s-buffered Krebs bicarbonate medium for 15 rain at 37°C before sequential additions of glucose to the final conomtrations shown. Data are means for three experiments and representative S.E.M.s are shown.

glucose was subsequently raised from 2 to 10 m M . The effect of ACh on intracellular Ca 2+ was tested in the presence o f 2 m M glucose (Fig. 5). ACh (50 gM) evoked a m a r k e d but transient rise in intraceUular Ca z+ which was completely blocked by the simultaneous presence o f 50 gM devapamil. Ca 2+ levels had returned to their pre-stimulation level within 3 min; however subsequent depolarization of the cells by addition of 40 m M K + was still able to elicit an additional marked rise in Ca 2÷.

Figure 6 shows that the increases in Ca 2+ evoked by 2 m M glucose and by 50 ItM ACh were both abolished in the absence of extracellular Ca 2÷ .

500-

r

+

~oo-

~

~

•s M g l ~

o

,,. . . . . . 15

~ ~

'............ " - . ~ -

ACh

..... - ...... Urr--~'

20

25

4~mM~" III .

i

30

Time (rains) FiG. 5. Effect of acetyleholine on [Ca2+]i in MIN6/]-cells. Quin 2-loaded MIN6/]-cells (5 x l0 6 ceUs per tube) were preincubated in glucose-free Hepes-buffered Krebs bicarbonate medium for 15 min at 37*(2 before sequential addition of 2 mM glucose, 50 I~M aeatylcholine (ACh) and 40 mM K + at the times indicated. For the samples indicated by <>, 50 ~M devapamil was added prior to addition of ACh as indicated. Data are means for three experiments and representative S.E.M.s are shown.

Diacylglycerolin fl-cells 300-

--/

200.

+1~

.s.

EGTA

100.

2ramglem~ 0

;~[ 10

50gMACh II |

i

i

20

25

30

i

35

Time (rains) FIG. 6. Dependence on extracellular Ca2+ of the increase in [Ca2+]i evoked by acetylcholine in MIN6 //-cells. Quin 2-loaded MIN6//-cells (5 x 106 cells per tube) were preincubated in glucose-free Hepesbuffered Krebs bicarbonate medium for 15 min at 37°C before sequential addition of 2 mM glucose and 50 t~M acetylcholine (ACh) at the times indicated. For the samples indicated by <>, Ca2+ was omitted from the incubation medium which also contained 10 mM EGTA. Data are means for three experiments and representative S.E.M.s are shown. DISCUSSION The MIN6 fl-cell line was established from insulinomas obtained by targeted expression of the SV40 T-antigen gene in transgenic mice by Miyazaki etal. [36] who showed that insulin secretion from MIN6 fl-cells measured over a 24 h period was markedly increased by glucose. The present study confirms the glucose sensitivity of MIN6 cells and shows further that a secretory response to glucose can also be demonstrated in much shorter incubations. Essentially linear rates of secretion were obtained in batch incubations of up to 4 h duration. As in other//-cells, glucose-stimulated" insulin release from MIN6 cells was abolished by omission of extracellular Ca 2~ or by addition of Ca2+-channel blockers. We also show here that MIN6 cells retain a secretory response to glibenclamide, to cholinergic agonists and to bombesin. The aim of the present study was to investigate the second messenger systems involved in the eholinergic stimulation of insulin release. It

783

is well established [11, 2-21, 23, 25, 37-39], and confirmed here for MIN6 fl-cells, that ACh-induced insulin release is dependent on the presence of extracellular Ca 2+ and therefore may involve Ca 2+ influx. However, in a previous study using the//-cell line HIT T15 [9], we found that ACh stimulated only a transient increase in [Ca2+]i lasting for approximately 2-3 min; this was in contrast to the effect of ACh on insulin secretion, where the neurotransmitter induced a sustained increase in insulin release. Wollheim and Biden [18] have also shown that CCh stimulated a transient increase in [Ca2+]~ but a sustained increase in insulin secretion in RINm5F cells and a transient increase in Ca 2+ channel activity was elicited by CCh in isolated mouse islets of Langerhans [40]. We further showed in HIT T15 cells that the cholinergicinduced [Ca2+]i transient resulted essentially from Ca 2+ influx, since it was abolished by omission of extracellular Ca 2+ or addition of the Ca 2+ channel blocker verapamil. Similar findings are reported here for MIN6 fl-cells. Hermans and Henquin [29] concluded that extracellular Ca 2+ was essential for sustained insulin release evoked by ACh. It appears therefore that in the fl-cell, muscarinic stimulation has a major effect on Ca 2+ entry rather than primarily mobilizing intracellular Ca 2+. Wollheim and Biden [18] concluded that [Ca2+]i was not a "moment to moment" regulator of cholinerglc-stimulated insulin secretion and other second messenger systems have been considered. In a previous study [9] we investigated the importance of protein kinase C activation in cholinergic-stimulated insulin release in HIT T15 fl-cells. It was found that HIT cells depleted of protein kinase C lost their secretory response to A e h , in agreement with similar findings with CCh in islets of Langerhans [30, 41]. Thus an increase in DAG, the putative modulator of protein kinase C, may play a key role in cholinerglc modulation of insulin secretion. However, prior to the present study, there was only limited information available on D A G levels in fl-cells. Corkey et al. [42] reported that D A G levels were elevated 70% in HIT//-cells

784

L. WENGet al.

exposed for 30 min to 10 mM glucose compared to cells incubated in the absence of exogenous fuel. However, neither the time course nor the specificity of this effect was studied nor were data presented for cholinergic agents. Glucose was also reported to increase DAG levels in rat islets [43]; a peak response was observed at 1 rain and levels did not decrease during the subsequent 10 rain incubation with high glucose. The same group, however, found no effect of glucose on DAG levels in HIT fl-cells [44]. Wolf e t a l . [45] observed no effect of glucose on DAG levels either in rat islets or in human islets although in another publication the same group reported that glucose increased the DAG content of rat islets [46]. To our knowledge only two studies have measured fl-cell DAG in response to carbachol. Peter-Riesch et al. [43] demonstrated a rapid 60% increase in rat islet DAG. Wolf et al. [46] used a mass spectrometric method to assay DAG in rat islets exposed to 0.5 mM CCh. After 2 min no significant change was found, but after 5 rain there was an approximately 50% increase in DAG levels which were still elevated after l0 rain. The dependence of the CCh-induced rise in DAG on glucose or Ca 2+ was not reported. Bombesin has also been shown in one study to elevate DAG in HIT/~cells [44] but the response was not studied in detail. The mean basal DAG in MIN6 fl-cells was 0.254 fmol/cell corresponding to a concentration of approximately 254 I~M. This is similar to the value of 216 I~M reported for human islets [45] and the value of approximately 300 I~M calculable from the data of Wolf et al. [46] for rat islets assuming a mean islet volume of 3 nl. Increasing glucose concentration from 0.5 to 25 raM, which markedly stimulated insulin release in MIN6 fl-cells, had no significant effect on DAG levels. These data do not support the suggestion [47] that an increase in DAG is a coupling factor in glucose-stimulated insulin release. Cholinergic agonists, however, produced rapid increases in B-cell DAG which remained elevated for up to 20 rain but which had returned to basal after 30 min. These find-

ings are consistent with the time course of phosphorylation of an 80,000 M, substrate for protein kinas¢ C in islets of Langerhans exposed to CCh; maximal phosphorylation occurred after 5 min [48]. They are also in agreement with measurements of the effect of CCh on the accumulation of [3H]DAG in rat islets prelabelled for 48 h with [3H]glycerol [46]. Our data show that the cholinergic-induced elevation of DAG is dependent on influx of Ca 2+ since it was abolished by EGTA or by CaE+-channel blockers. However, an increase in [Ca2+]i was not in itself a sufficient trigger to elevation of DAG since neither glibenclamide nor increased extracellular K + led to an increase in DAG. These findings, together with the observation that the increase in [Ca2+]i elicited by ACh was not reduced in protein kinase C-depleted cells [9], support the view that activation of t-cell muscarinic receptors in the t-cell membrane initiates a two-stage process to potentiate glucose-stimulated insulin release. An initial transient increase in Ca 2+ influx stimulates insulin release and primes the secretory mechanism for protein kinase C activation by an increase in DAG content. This in turn sensitizes the secretory machinery to Ca 2+ permitting the sustained enhancement of insulin release shown here and elsewhere to be evoked by cholinergic agonists. In t-cells, protein kinase C activation acts to oppose increases in [Ca2+]i [49], thus accounting for the transient nature of the elevation in [Ca2+]i elicited by cholinergic stimulation. The present study shows in addition that the enhanced secretory rate persists much longer than the observed increase in DAG. Thus, additional mechanisms may play a role in sustaining the secretory response to cholinergic agonists. The source of the increased DAG in response to cholinergic stimulation is not known. Although several studies indicate that cholinergic agonists increase the activity of phospholipase C [13, 17, 38, 50] there is also evidence for activation of phospholipase A2 [51, 52]. Wolf et al. [46] concluded from analysis of the fatty acid composition of DAG in rat islets exposed

Diacylglycerolin/~-eells to CCh that phosphoinositide hydrolysis was a major source o f DAG; however the progressive rise in D A G content o f palmitate suggested that other mechanisms may also contribute. Our studies do not support the view [46] that de novo synthesis o f D A G from glucose represents an important component of glucose-induced insulin release. Thus, they support the growing body o f evidence [9, 30, 53, 54] that protein kinase C is important for cholinerglc modulation o f insulin secretion but does not play a major role in glucose-induced insulin release. Acknowledgements---These studies were supported by grants from the British Diabetic Association and the Medical Research Council. We are very grateful to l~or. Y. MIY^z^KI, University of Tokyo, for providing MIN6 fl-cells and to PROF. R.M. BELL, Duke University, for provision of the DAG kinaseproducing E. coli strain N4830/pJWI0.

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