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Potentiators of insulin secretion modulate Ca2+ sensitivity in rat pancreatic islets
231
Molecular and Cellular Endocrinology, 50 (1987) 231-236 Elsevier Scientific Publishers Ireland, Ltd.
MCE 01629
Potentiators
of insulin secretion modulate in rat pancreatic islets
S.J. Hughes, Nuffield Depurtment
Kq words: Islet of Langerhans;
M.R. Christie and S.J.H. Ashcroft
of Chnicul Blochemisty, (Received
Forskolin;
Ca*’ sensitivity
28 August
Acetyl choline;
John Radcliffe Hospital, Heudmgton, 1986; accepted
Phorbol
4 December
Oxford 0x3
9Dl.4 U.K.
1986)
ester
Summary Insulin secretion stimulated by 10 mM glucose was potentiated by forskolin, an activator of adenyl cyclase, by acetyl choline which may enhance turnover of inositol phospholipids, and by tetradecanoyl phorbol acetate (TPA), an activator of protein kinase C. None of these agents initiated insulin secretion in the presence of 2 mM glucose. Glucose-stimulated insulin secretion was markedly dependent on the concentration of extracellular Ca*+: at or below 10 PM Ca*+ no insulin secretion was evoked by glucose was increased after culture of islets for 44 h. In in freshly isolated islets. The threshold Ca*+ requirement both fresh and cultured islets the presence of a potentiator of secretion produced both a marked increase in the maximum rate of glucose-stimulated insulin secretion and a lowering of the requirement for of insulin release involves an increase in the sensitivity of the B cell extracellular Ca* +. Thus potentiation to Ca*+.
Introduction Agents stimulating insulin release from the pancreatic B cell may be broadly divided into ‘initiators’ and ‘potentiators’ of secretion (Ashcroft, 1980). The former,.which include the major physiological stimulus glucose, are able to elicit insulin release in the absence of other additions. Potentiators, on the other hand, fail to stimulate insulin release when tested alone, but increase the magnitude of the secretory response to glucose or other initiators. Two main categories of potentiators have been identified. The first comprises those agents which elevate B cell cyclic AMP and
Address for correspondence: S.J. Hughes, Nuffield Department of Clinical Biochemistry, John Radcliffe Hospital, Headington, Oxford OX3 9DU, U.K. 0303-7207/87/$03.50
0 1987 Elsevier Scientific
Publishers
Ireland,
includes glucagon and 3-isobutyl-l-methylxanthine (IBMX). The second group, which includes neurotransmitters such as acetyl choline, contains those agents which may primarily enhance turnover of inositol phospholipids; enhanced breakdown of phosphatidyl inositol bisphosphate yields two second messengers, inositol triphosphate, which mobilizes intracellular Ca*+, and diacylglycerol, which activates protein kinase C. The biochemical mechanisms involved in potentiation have not been established. The suggestion (Wollheim and Sharp, 1981) based primarily on measurements of 45Ca fluxes, that an increase in cyclic AMP within the B cell mobilizes intracellular Ca*+ has not been substantiated by direct measurements of cytosolic Ca*+ using Quin2 (Wollheim et al., 1984; Rorsman and AbrahamsLtd.
232
son, 1985). Moreover, in digitonin-permeabilized islets maintained at a fixed intracellular Ca*+ concentration, Tamagawa et al. (1985) showed that both forskolin and TPA were able to evoke insulin release. In platelets made permeable by high-voltage electric discharge, it has been shown that both cyclic AMP (Knight and Scrutton, 1984) and activators of protein kinase C (Knight and Scrutton, 1980; Castanaga et al., 1982) increased the sensitivity of the secretory response to into tracellular Ca 2+ by shifting the dose-response the left. Tetradecanoyl phorbol acetate (TPA) and cyclic AMP have also been reported to cause a similar increase in Ca*+ sensitivity of insulin release from electrically permeabilized islets of Langerhans (Jones et al., 1985, 1986). Such a mechanism could form the basis for a unitary explanation for potentiation of insulin secretion. In the present study we have used intact (non-permeabilized) islets of Langerhans to examine whether potentiation of insulin release can be demonstrated to induce increased sensitivity of the B cell to extracellular Ca2+. Some of these data have been reported previously in abstract form (Christie and Ashcroft, 1985).
using a charcoal separation method (Ashcroft and Crossley, 1975). Rat insulin, kindly supplied by Dr. A.J. Moody, Novo Research Institute, Bagsvaerd, Denmark, was used as standard. Data are given as means -t SEM for the number of batches of islets indicated; the significance of differences was assessed using Student’s t-test. Results In the presence of normal (2.5 mM) extracellular Ca*+, insulin release was increased approximately lo-fold on raising the medium glucose concentration from 2 to 10 mM with a half-maximal response at approximately 6 mM (Table 1). Table 1 also shows that forskolin, TPA and acetyl choline elicited marked potentiation of insulin release in the presence of 6 or 10 mM glucose but did not significantly increase the basal rate of insulin release at 2 mM glucose. The potentiatory effect of acetyl choline could not be demonstrated in freshly prepared islets of Langerhans but was manifest after tissue culture for 48 h. The magni-
TABLE
Methods Islets of Langerhans were prepared by collagenase digestion of pancreases from fed male Wistar rats (Coll-Garcia and Gill, 1969). For measurement of insulin secretion, islets were individually hand-picked under a dissecting microscope, and either incubated directly or cultured for 44 h. The tissue culture medium was RPM1 1640 (Gibco Europe, Paisley, U.K.) containing 10% foetal calf serum, penicillin and streptomycin, and culture was carried out at 37OC in an atmosphere of humidified air/CO, (95/5). The incubation medium for measurement of rates of insulin reaction was a modified Krebs bicarbonate medium containing 20 mM Hepes pH 7.4 and 2 mg/ml bovine serum albumin (Christie and Ashcroft, 1985). The Ca*+ concentration of the medium was varied using EGTA-Ca*+ to give the desired final concentration of free Ca*+. After incubation, aliquots of the medium were removed and stored at - 20” in phosphate buffer containing albumin and merthiolate until assay by radioimmunoassay
1
EFFECTS OF FORSKOLIN, TPA AND ACETYL CHOLINE ON INSULIN RELEASE IN THE PRESENCE OF DIFFERENT GLUCOSE CONCENTRATIONS Batches of five islets were incubated at 37°C for 2 h in Hepes-buffered bicarbonate medium containing 10 mM glucose 2 mg/ml albumin and the additions stated. Freshly isolated islets were used to assess the effects of forskolin and TPA. For study of the effect of acetyl choline, test and control islets had been cultured for 44 h in RPM1 1640 at 37°C in an atmosphere of air: CO, (95 : 5). Insulin released into the medium was measured by radioimmunoassay. Data are given as mean j, SEM for ten observations. Insulin release (,uU/islet
Glucose concentration
(mM)
Conditions Control + Forskolin
(10 PM)
per h)
2
6
10
22k3 20*3
148?12 360*12*
210* 15 465k36 *
Control + TPA (0.1 PM)
13+2 17kl
100+14 252*28*
150?68 377&37
*
Control + acetyl choline (5 PM)
16*3 12*2
92+13 21Oi_40
170?26 330*30
*
* Significantly greater than control centration (P < 0.01).
*
at the same glucose
con-
233
b)
Cc)
300
200
100
’ ok+’
0.1
1.0 Forskolin
YI 1
10
3 0.0 1
(fl)
1.0 TpA”&Yj
+ +-f J’ OY’
0.05 Acetylcholine
0.5
5.0 (Jo!?)
Fig. 1. Potentiation of glucose-stimulated insulin release by forskolin, TPA and acetyl choline. The figure shows the concentration dependence of the stimulatory effects of (u) forskolin, (h) TPA, ( c,) acetyl choline on insulin release stimulated by 10 mM glucose. Batches of five islets were incubated for 2 h. Freshly isolated islets were used to study the effect of forskolin and TPA: islets used to assess the effect of acetyl choline had been cultured for 44 h in RPM1 1640 at 37OC in an atmosphere of humidified air: CO, (95 : 5). Results are shown as mean i SEM for ten observations.
tude of the secretory response to glucose itself was not altered by this period of culture. The concentration dependence of the effects of potentiators on glucose-stimulated insulin release is shown in Fig. 1. Panel (a) shows that as little as 0.5 PM forskolin produced marked potentiation and maximum potentiation was achieved with 10 PM forskolin. Panel (b) demonstrates that TPA at
4oc
ROO-
600 -
concentrations greater than 0.04 PM caused marked potentiation; the maximum effect of TPA had not been achieved up to 2 pM. Panel (c) shows that, above a threshold concentration of 0.05 PM, acetyl choline produced a dose-dependent potentiation of glucose-stimulated insulin release from cultured islets which was essentially maximal at 5 PM.
a” 3oc
2oc
1oc
$6 OF
0.0 1
3.!
1.0
ci
‘+
Cancentwt.icm
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Fig. 2. The effects of forskolin, TPA and acetyl choline on the dependence of glucose-stimulated insulin release on extracellular Ca*+ concentration. The data in panel (a) were obtained from freshly isolated islets whilst those in panel (h) were from islets cultured for 44 h in RPM1 1640. Insulin release rates from batches of five islets were measured in the presence of 10 mM glucose and Ca*+ at the concentrations given (0). Forskolin (m, 10 PM), TPA (0, 0.1 PM) or acetyl choline (0, 5 PM) were present as indicated. Results are plotted as mean + SEM for ten observations.
234
The effects of these potentiators on the dependence of glucose-stimulated insulin release on the extracellular concentration of Ca*+ is shown in Fig. 2. Panel (a) shows the effects of forskolin and TPA using freshly prepared islets. The effects of acetyl choline on cultured islets are shown separately in panel (b) since the culture itself affected the Ca*+ sensitivity of the control islets. In freshly prepared islets, lowering the Ca2+ concentration to 10 PM reduced the insulin secretory response to 10 mM glucose to the basal level seen in the absence of Ca*+. Between 10 and 500 PM Ca*’ there was a progressive increase in the response to glucose with a maximum response at approximately 500 PM. The concentration of Ca*+ giving 50% of the maximum response was 60 PM. Both forskolin and TPA markedly increased the sensitivity to extracellular Ca*+ such that for both drugs there was a significant difference between the rate of insulin release at 10 PM Ca*+ and that in the absence of Ca*+. Both potentiators also increased the maximum response to 10 mM glucose seen at 0.5 or 5 mM extracellular Ca*+. The concentrations of extracellular Ca*+ giving halfmaximal release was reduced by forskolin to 8 PM and by TPA to 35 PM. In the cultured islets (panel (b)), the dependence of glucose-stimulated insulin release on extracellular Ca*+ was altered so that the threshold Ca*+ was in excess of 100 PM and the concentration of Ca*+ required for half-maximal response was markedly increased to 263 PM. Nevertheless in the presence of acetyl choline there was again a leftward shift of the curve which reduced the concentration of Ca*+ required for a half-maximal response to 140 PM; the marked increase in glucose-stimulated insulin release from cultured islets on raising the Ca*+ concentration from 0.5 to 5 mM was no longer present in the presence of acetyl choline. Since cultured islets were always used for assessing the effects of acetyl choline, we also investigated whether cultured islets retained a secretory response to forskolin and TPA. Table 2 shows the roughly equal extent to which acetyl choline, TPA and forskolin potentiate insulin release in the presence of 6 mM glucose in cultured islets. The combined presence of acetyl choline and TPA elicited no greater potentiation than that
TABLE
2
POTENTIATORY EFFECTS OF TPA, ACETYL CHOLINE AND FORSKOLIN ON GLUCOSE-STIMULATED INSULIN RELEASE FROM CULTURED ISLETS Islets were cultured for 44 h in RPM1 1640 at 37 “C in an atmosphere of humidified air: CO, (95 : 5) before incubation with the agents shown for measurement of insulin release. The incubation medium was a Hepes-buffered bicarbonate medium containing 6 mM glucose and 2 mg/ml albumin. In order to combine different batches of cultured islets, data are expressed relative to the control rate seen in the presence of 6 mM glucose alone in the same experiment and are given as mean f SEM for ten observations. All conditions were significantly (P i 0.001) greater than control. The response to TPA + forskolin was significantly (P < 0.001) greater than that to either drug alone. Additions
Insulin release (% of control)
None TPA (0.1 PM) Acetyl choline Forskolin (10 TPA (0.1 PM) TPA (0.1 PM)
100 172 + 16 183+20 215k24 199 f 29 340+39
(5 PM) PM) + acetyl choline (5 PM) + forskolin (10 PM)
seen in the presence of either skolin however augmented potentiation by TPA.
agent alone. significantly
Forthe
Discussion An increase in the intracellular concentration of free Ca*+ is believed to be the key event in the initiation of insulin release in response to an elevation of extracellular glucose. Current evidence favours the view that the enhanced rate of metabolism of glucose by the B cell which occurs on raising the glucose concentration to a stimulatory level is causally related to the increase in intracellular Ca*+; an important component of this process is the inhibition (closing) of a particular class of membrane K+ channel (the G channel) as a consequence of increased metabolic flux (Ashcroft et al., 1984). Those agents (potentiators) which alone give little or no stimulation of insulin release but markedly augment the response to glucose could increase the magnitude of either the change in Ca*+ for a given glucose concentration or the response of the secretory system to a given Ca*+ concentration. Studies in support of the
235
former possibility have suggested two mechanisms for potentiation by cyclic AMP. Firstly, measurements of 45Ca2+ fluxes were interpreted as indicating that cyclic AMP may be mobilize intracellular .calcium (Wollheim and Sharp, 1981). Secondly, studies on the effect of forskolin on the membrane potential of B cells led to the proposal that cyclic AMP may also modulate the permeain the B cell plasma bility of Ca*+ channels membrane (Henquin et al., 1983). However, several findings argue against the view that potentiators of secretion act via an increase in intracellular Ca”. Firstly, studies using fluorescent indicators to measure cytosolic Ca 2+ in mouse islets (Rorsmann and Abrahamsson, 1985) and in a tumoral B cell line (Wollheim et al., 1984) showed that secretory responses to various agents increasing intracellular cyclic AMP were not accompanied by increases in cytosolic Ca*+. Secondly, in Ca2+clamped permeabilized islets, where intracellular and extracellular spaces are in ionic equilibrium and the intracellular Ca2’ is buffered by chelator, both forskolin (Jones et al., 1986) and TPA (Jones et al., 1985) were able to increase insulin secretion. An alternative mode of action of potentiators could be to sensitize the secretory system to Ca*+. Support for such a mechanism for agents acting either via the cyclic AMP system or protein kinase C has been presented for the secretory response of platelets (Knight and Scrutton, 1980, 1984; Castanaga et al., 1982) and digitonin-permeabilized islets (Tamagawa et al., 1985). In electrically permeabilized islets, both TPA (Jones et al., 1985) and forskolin (Jones et al., 1986) also caused a dose-related shift in the Ca2+ activation curve to lower Ca*+ concentrations. In view of the marked dependence of insulin secretion on extracellular calcium such an increase in sensitivity of the secretory system to internal Ca2+ may result in a lowering of the requirement for extracellular Ca2+. In the present study, therefore, we have examined this possibility in intact islets of Langerhans by determining the effects of potentiators on the dependence of glucose-stimulated insulin release on the extracellular Ca*+ concentration. Our data show clearly that all three potentiators tested have qualitatively similar effects, namely (i) a shift to the left of the curve relating the rate of glucose-stimulated insulin release to
extracellular Ca2+; and (ii) an increase in the maximal secretory response at high Ca*+ concentrations. We propose, therefore, as the simplest interpretation of these findings, that potentiators of insulin secretion act, at least in part, by increasing the sensitivity of the secretory response system to Ca*+. Data from several previous studies on intact islets of Langerhans are consistent with this proposal. Devis et al. (1975) showed that theophylline augmented the secretory response to 10 mM Ca2+ of islets pre-treated with EGTA and perifused in the absence of glucose: whether this cyclic AMPmediated effect involved a change in the concentration dependence of Ca*‘-stimulated insulin release was not reported. Evidence that it may do so was provided by Hellman (1976) who demonstrated that IBMX lowered from 20 to 10 mM the concentration of extracellular Ca*+ capable of initiating insulin release in glucose-free medium from islets that had not been pre-exposed to EGTA. Neither of these studies documented the effects of potentiators on the sensitivity of glucose-stimulated insulin release to extracellular Ca2+. Using an app roach more directly comparable to ours, and in agreement with the present findings with TPA, Hii et al. (1985) showed that, in intact islets, the phorbol ester lowered the external Ca2+ concentration necessary for a maximal secretory response to glucose. However, in their study glucose-stimulated insulin release required much higher extracellular Ca2+ levels than in our study; indeed glucose-stimulated insulin release in the study of Hii et al. (1985) was not significantly reduced when extracellular Ca2+ was reduced from normal (2.5 mM) to zero. Two further points are of note. Firstly our data show that TPA is a true potentiator of insulin secretion, eliciting little or no effect at a nonstimulatory glucose concentration. This is in agreement with some earlier reports (Virji et al., 1978) but not others (Malaisse et al., 1980; Yamamoto et al., 1982). We cannot explain this discrepancy but would draw a note of caution about the use of TPA at the high concentrations used by some authors. As our results show, and in agreement with Hii et al. (1986) the response to TPA continues to increase up to the highest concentrations used (2 PM in the present study, 10
236
PM in Hii et al., 1986). The activation of B cell protein kinase C by TPA is however essentially maximal at 0.1 PM (Lord, J.M. and Hughes, S.J., unpublished observation); the possibility that higher concentrations have effects unrelated to protein kinase C should be borne in mind. Secondly, the results with acetyl choline deserve comment. In freshly prepared islets, we could find no consistent potentiatory effects of acetyl choline. This may be related to a possible deleterious effect of collagenase treatment during isolation on the acetyl choline receptor; this interpretation is supported by the finding that a response to acetyl choline was regained after a short period of tissue culture of the islets. Culture for 44 h did not reduce the magnitude of the secretory response to glucose although interestingly the cultured islets did display a decreased sensitivity to extracellular Ca*+; nevertheless acetyl choline induced increased sensitivity to Ca*+. The mechanism responsible for the increased threshold requirement for extracellular Ca*+ after culture of the islets is unknown but could possibly reflect depletion of intracellular Ca*+ because of the low (0.4 mM) Ca*+ content of RPM1 1640 culture medium. Since glucose promotes not only Ca*+ influx but also compensatory uptake into intracellular organelles and outward transport of the ion (Hellman, 1985) a depletion of intracellular calcium could perturb the normal relationship between the magnitude of these fluxes. It is also of note that the potentiatory effect of TPA was not increased further by the simultaneous presence of acetyl choline. This was not attributable to saturation of the release system since forskolin was able to augment significantly the potentiatory effect of TPA. This suggests that, at least in the presence of 6 mM glucose, activation of protein kinase C, via enhanced diacylglycerol formation, may represent the most important component of the potentiatory effect of acetyl choline.
Acknowledgements These studies were supported the Medical Research Council Diabetic Association.
by grants from and the British
Ashcroft, F.M., Harrison, D.E. and Ashcroft, S.J.H. (1984) Nature 312, 446-448. Ashcroft, S.J.H. (1980) Diabetologia 18, S-15. Ashcroft, S.J.H. and Crossly, J.R. (1975) Diabetologia 11, 279-284. Castanaga, M., Takai, Y., Keihuchi, K., Sano, K., Kikkawa, A. and Nishzuka, Y. (1982) J. Biol. Chem. 257, 7847-7851. Christie, M.R. and Ashcroft, S.J.H. (1985) Diabetes Res. Clin. Pratt. Suppl. 1, Abstract No. 260, p. S102. Coil-Garcia, E. and Gill, J.R. (1969) Diabetologia 5, 61-66. Devis, G., Somers, G. and Ma&se, W.J. (1975) Biochem. Biophys. Res. Commun. 67, 525-529. Hellman, B. (1976) FEBS Lett. 63, 125-128. Hellman, B. (1985) Diabetologia 28, 494-501. Henquin, J.-C., Schmeer, W. and Meissner, H.P. (1983) Endocrinology 112, 2218-2220. Hii, C.S.T.. Stutchfield, J. and Howell, S.L. (1986) Biochem. J. 233, 2897-2899. Jones, P.M., Stutchfield. J. and Howell, S.L. (1985) FEBS Lett. 191, 102-106. Jones, P.M., Fyles, J.M. and Howell, S.L. (1986) FEBS Lett. 205, 205-209. Knight. D.E. and Scrutton, M.C. (1980) Thromb. Res. 20, 437-446. Knight, D.E. and Scrutton, M.L. (1984) Nature 309, 66-68. Malaisse, W.J., Lebrun. P., Herchuelz, A., Sener, A. and Malaisse-Lagae, F. (1983) Endocrinology 113, 1870-1877. Rorsman, P. and Abrahamsson, H. (1985) Acta Physiol. Stand. 125, 639-647. Tamagawa, T., Hiki, H. and Niki, A. (1985) FEBS Lett. 1983, 430-433. Virji, M.A.G., Sheffs, M.W. and Estensen, R.D. (1978) Endocrinology 102, 706-711. Wollheim, C.B. and Sharp, G.W.G. (1981) Physiol. Rev. 61, 914-973. Wollheim, C.B., Ullrich, S. and Pozzan, T. (1984) FEBS Lett. 177, 17-22. Yamamoto, S., Nakadate, T., Nakaki, T., Ishii, K. and Kato, R. (1982) Biochem. Biophys. Res. Commun. 105, 759-765.
Molecular and Cellular Endocrinology, 50 (1987) 231-236 Elsevier Scientific Publishers Ireland, Ltd.
MCE 01629
Potentiators
of insulin secretion modulate in rat pancreatic islets
S.J. Hughes, Nuffield Depurtment
Kq words: Islet of Langerhans;
M.R. Christie and S.J.H. Ashcroft
of Chnicul Blochemisty, (Received
Forskolin;
Ca*’ sensitivity
28 August
Acetyl choline;
John Radcliffe Hospital, Heudmgton, 1986; accepted
Phorbol
4 December
Oxford 0x3
9Dl.4 U.K.
1986)
ester
Summary Insulin secretion stimulated by 10 mM glucose was potentiated by forskolin, an activator of adenyl cyclase, by acetyl choline which may enhance turnover of inositol phospholipids, and by tetradecanoyl phorbol acetate (TPA), an activator of protein kinase C. None of these agents initiated insulin secretion in the presence of 2 mM glucose. Glucose-stimulated insulin secretion was markedly dependent on the concentration of extracellular Ca*+: at or below 10 PM Ca*+ no insulin secretion was evoked by glucose was increased after culture of islets for 44 h. In in freshly isolated islets. The threshold Ca*+ requirement both fresh and cultured islets the presence of a potentiator of secretion produced both a marked increase in the maximum rate of glucose-stimulated insulin secretion and a lowering of the requirement for of insulin release involves an increase in the sensitivity of the B cell extracellular Ca* +. Thus potentiation to Ca*+.
Introduction Agents stimulating insulin release from the pancreatic B cell may be broadly divided into ‘initiators’ and ‘potentiators’ of secretion (Ashcroft, 1980). The former,.which include the major physiological stimulus glucose, are able to elicit insulin release in the absence of other additions. Potentiators, on the other hand, fail to stimulate insulin release when tested alone, but increase the magnitude of the secretory response to glucose or other initiators. Two main categories of potentiators have been identified. The first comprises those agents which elevate B cell cyclic AMP and
Address for correspondence: S.J. Hughes, Nuffield Department of Clinical Biochemistry, John Radcliffe Hospital, Headington, Oxford OX3 9DU, U.K. 0303-7207/87/$03.50
0 1987 Elsevier Scientific
Publishers
Ireland,
includes glucagon and 3-isobutyl-l-methylxanthine (IBMX). The second group, which includes neurotransmitters such as acetyl choline, contains those agents which may primarily enhance turnover of inositol phospholipids; enhanced breakdown of phosphatidyl inositol bisphosphate yields two second messengers, inositol triphosphate, which mobilizes intracellular Ca*+, and diacylglycerol, which activates protein kinase C. The biochemical mechanisms involved in potentiation have not been established. The suggestion (Wollheim and Sharp, 1981) based primarily on measurements of 45Ca fluxes, that an increase in cyclic AMP within the B cell mobilizes intracellular Ca*+ has not been substantiated by direct measurements of cytosolic Ca*+ using Quin2 (Wollheim et al., 1984; Rorsman and AbrahamsLtd.
232
son, 1985). Moreover, in digitonin-permeabilized islets maintained at a fixed intracellular Ca*+ concentration, Tamagawa et al. (1985) showed that both forskolin and TPA were able to evoke insulin release. In platelets made permeable by high-voltage electric discharge, it has been shown that both cyclic AMP (Knight and Scrutton, 1984) and activators of protein kinase C (Knight and Scrutton, 1980; Castanaga et al., 1982) increased the sensitivity of the secretory response to into tracellular Ca 2+ by shifting the dose-response the left. Tetradecanoyl phorbol acetate (TPA) and cyclic AMP have also been reported to cause a similar increase in Ca*+ sensitivity of insulin release from electrically permeabilized islets of Langerhans (Jones et al., 1985, 1986). Such a mechanism could form the basis for a unitary explanation for potentiation of insulin secretion. In the present study we have used intact (non-permeabilized) islets of Langerhans to examine whether potentiation of insulin release can be demonstrated to induce increased sensitivity of the B cell to extracellular Ca2+. Some of these data have been reported previously in abstract form (Christie and Ashcroft, 1985).
using a charcoal separation method (Ashcroft and Crossley, 1975). Rat insulin, kindly supplied by Dr. A.J. Moody, Novo Research Institute, Bagsvaerd, Denmark, was used as standard. Data are given as means -t SEM for the number of batches of islets indicated; the significance of differences was assessed using Student’s t-test. Results In the presence of normal (2.5 mM) extracellular Ca*+, insulin release was increased approximately lo-fold on raising the medium glucose concentration from 2 to 10 mM with a half-maximal response at approximately 6 mM (Table 1). Table 1 also shows that forskolin, TPA and acetyl choline elicited marked potentiation of insulin release in the presence of 6 or 10 mM glucose but did not significantly increase the basal rate of insulin release at 2 mM glucose. The potentiatory effect of acetyl choline could not be demonstrated in freshly prepared islets of Langerhans but was manifest after tissue culture for 48 h. The magni-
TABLE
Methods Islets of Langerhans were prepared by collagenase digestion of pancreases from fed male Wistar rats (Coll-Garcia and Gill, 1969). For measurement of insulin secretion, islets were individually hand-picked under a dissecting microscope, and either incubated directly or cultured for 44 h. The tissue culture medium was RPM1 1640 (Gibco Europe, Paisley, U.K.) containing 10% foetal calf serum, penicillin and streptomycin, and culture was carried out at 37OC in an atmosphere of humidified air/CO, (95/5). The incubation medium for measurement of rates of insulin reaction was a modified Krebs bicarbonate medium containing 20 mM Hepes pH 7.4 and 2 mg/ml bovine serum albumin (Christie and Ashcroft, 1985). The Ca*+ concentration of the medium was varied using EGTA-Ca*+ to give the desired final concentration of free Ca*+. After incubation, aliquots of the medium were removed and stored at - 20” in phosphate buffer containing albumin and merthiolate until assay by radioimmunoassay
1
EFFECTS OF FORSKOLIN, TPA AND ACETYL CHOLINE ON INSULIN RELEASE IN THE PRESENCE OF DIFFERENT GLUCOSE CONCENTRATIONS Batches of five islets were incubated at 37°C for 2 h in Hepes-buffered bicarbonate medium containing 10 mM glucose 2 mg/ml albumin and the additions stated. Freshly isolated islets were used to assess the effects of forskolin and TPA. For study of the effect of acetyl choline, test and control islets had been cultured for 44 h in RPM1 1640 at 37°C in an atmosphere of air: CO, (95 : 5). Insulin released into the medium was measured by radioimmunoassay. Data are given as mean j, SEM for ten observations. Insulin release (,uU/islet
Glucose concentration
(mM)
Conditions Control + Forskolin
(10 PM)
per h)
2
6
10
22k3 20*3
148?12 360*12*
210* 15 465k36 *
Control + TPA (0.1 PM)
13+2 17kl
100+14 252*28*
150?68 377&37
*
Control + acetyl choline (5 PM)
16*3 12*2
92+13 21Oi_40
170?26 330*30
*
* Significantly greater than control centration (P < 0.01).
*
at the same glucose
con-
233
b)
Cc)
300
200
100
’ ok+’
0.1
1.0 Forskolin
YI 1
10
3 0.0 1
(fl)
1.0 TpA”&Yj
+ +-f J’ OY’
0.05 Acetylcholine
0.5
5.0 (Jo!?)
Fig. 1. Potentiation of glucose-stimulated insulin release by forskolin, TPA and acetyl choline. The figure shows the concentration dependence of the stimulatory effects of (u) forskolin, (h) TPA, ( c,) acetyl choline on insulin release stimulated by 10 mM glucose. Batches of five islets were incubated for 2 h. Freshly isolated islets were used to study the effect of forskolin and TPA: islets used to assess the effect of acetyl choline had been cultured for 44 h in RPM1 1640 at 37OC in an atmosphere of humidified air: CO, (95 : 5). Results are shown as mean i SEM for ten observations.
tude of the secretory response to glucose itself was not altered by this period of culture. The concentration dependence of the effects of potentiators on glucose-stimulated insulin release is shown in Fig. 1. Panel (a) shows that as little as 0.5 PM forskolin produced marked potentiation and maximum potentiation was achieved with 10 PM forskolin. Panel (b) demonstrates that TPA at
4oc
ROO-
600 -
concentrations greater than 0.04 PM caused marked potentiation; the maximum effect of TPA had not been achieved up to 2 pM. Panel (c) shows that, above a threshold concentration of 0.05 PM, acetyl choline produced a dose-dependent potentiation of glucose-stimulated insulin release from cultured islets which was essentially maximal at 5 PM.
a” 3oc
2oc
1oc
$6 OF
0.0 1
3.!
1.0
ci
‘+
Cancentwt.icm
(71:
Fig. 2. The effects of forskolin, TPA and acetyl choline on the dependence of glucose-stimulated insulin release on extracellular Ca*+ concentration. The data in panel (a) were obtained from freshly isolated islets whilst those in panel (h) were from islets cultured for 44 h in RPM1 1640. Insulin release rates from batches of five islets were measured in the presence of 10 mM glucose and Ca*+ at the concentrations given (0). Forskolin (m, 10 PM), TPA (0, 0.1 PM) or acetyl choline (0, 5 PM) were present as indicated. Results are plotted as mean + SEM for ten observations.
234
The effects of these potentiators on the dependence of glucose-stimulated insulin release on the extracellular concentration of Ca*+ is shown in Fig. 2. Panel (a) shows the effects of forskolin and TPA using freshly prepared islets. The effects of acetyl choline on cultured islets are shown separately in panel (b) since the culture itself affected the Ca*+ sensitivity of the control islets. In freshly prepared islets, lowering the Ca2+ concentration to 10 PM reduced the insulin secretory response to 10 mM glucose to the basal level seen in the absence of Ca*+. Between 10 and 500 PM Ca*’ there was a progressive increase in the response to glucose with a maximum response at approximately 500 PM. The concentration of Ca*+ giving 50% of the maximum response was 60 PM. Both forskolin and TPA markedly increased the sensitivity to extracellular Ca*+ such that for both drugs there was a significant difference between the rate of insulin release at 10 PM Ca*+ and that in the absence of Ca*+. Both potentiators also increased the maximum response to 10 mM glucose seen at 0.5 or 5 mM extracellular Ca*+. The concentrations of extracellular Ca*+ giving halfmaximal release was reduced by forskolin to 8 PM and by TPA to 35 PM. In the cultured islets (panel (b)), the dependence of glucose-stimulated insulin release on extracellular Ca*+ was altered so that the threshold Ca*+ was in excess of 100 PM and the concentration of Ca*+ required for half-maximal response was markedly increased to 263 PM. Nevertheless in the presence of acetyl choline there was again a leftward shift of the curve which reduced the concentration of Ca*+ required for a half-maximal response to 140 PM; the marked increase in glucose-stimulated insulin release from cultured islets on raising the Ca*+ concentration from 0.5 to 5 mM was no longer present in the presence of acetyl choline. Since cultured islets were always used for assessing the effects of acetyl choline, we also investigated whether cultured islets retained a secretory response to forskolin and TPA. Table 2 shows the roughly equal extent to which acetyl choline, TPA and forskolin potentiate insulin release in the presence of 6 mM glucose in cultured islets. The combined presence of acetyl choline and TPA elicited no greater potentiation than that
TABLE
2
POTENTIATORY EFFECTS OF TPA, ACETYL CHOLINE AND FORSKOLIN ON GLUCOSE-STIMULATED INSULIN RELEASE FROM CULTURED ISLETS Islets were cultured for 44 h in RPM1 1640 at 37 “C in an atmosphere of humidified air: CO, (95 : 5) before incubation with the agents shown for measurement of insulin release. The incubation medium was a Hepes-buffered bicarbonate medium containing 6 mM glucose and 2 mg/ml albumin. In order to combine different batches of cultured islets, data are expressed relative to the control rate seen in the presence of 6 mM glucose alone in the same experiment and are given as mean f SEM for ten observations. All conditions were significantly (P i 0.001) greater than control. The response to TPA + forskolin was significantly (P < 0.001) greater than that to either drug alone. Additions
Insulin release (% of control)
None TPA (0.1 PM) Acetyl choline Forskolin (10 TPA (0.1 PM) TPA (0.1 PM)
100 172 + 16 183+20 215k24 199 f 29 340+39
(5 PM) PM) + acetyl choline (5 PM) + forskolin (10 PM)
seen in the presence of either skolin however augmented potentiation by TPA.
agent alone. significantly
Forthe
Discussion An increase in the intracellular concentration of free Ca*+ is believed to be the key event in the initiation of insulin release in response to an elevation of extracellular glucose. Current evidence favours the view that the enhanced rate of metabolism of glucose by the B cell which occurs on raising the glucose concentration to a stimulatory level is causally related to the increase in intracellular Ca*+; an important component of this process is the inhibition (closing) of a particular class of membrane K+ channel (the G channel) as a consequence of increased metabolic flux (Ashcroft et al., 1984). Those agents (potentiators) which alone give little or no stimulation of insulin release but markedly augment the response to glucose could increase the magnitude of either the change in Ca*+ for a given glucose concentration or the response of the secretory system to a given Ca*+ concentration. Studies in support of the
235
former possibility have suggested two mechanisms for potentiation by cyclic AMP. Firstly, measurements of 45Ca2+ fluxes were interpreted as indicating that cyclic AMP may be mobilize intracellular .calcium (Wollheim and Sharp, 1981). Secondly, studies on the effect of forskolin on the membrane potential of B cells led to the proposal that cyclic AMP may also modulate the permeain the B cell plasma bility of Ca*+ channels membrane (Henquin et al., 1983). However, several findings argue against the view that potentiators of secretion act via an increase in intracellular Ca”. Firstly, studies using fluorescent indicators to measure cytosolic Ca 2+ in mouse islets (Rorsmann and Abrahamsson, 1985) and in a tumoral B cell line (Wollheim et al., 1984) showed that secretory responses to various agents increasing intracellular cyclic AMP were not accompanied by increases in cytosolic Ca*+. Secondly, in Ca2+clamped permeabilized islets, where intracellular and extracellular spaces are in ionic equilibrium and the intracellular Ca2’ is buffered by chelator, both forskolin (Jones et al., 1986) and TPA (Jones et al., 1985) were able to increase insulin secretion. An alternative mode of action of potentiators could be to sensitize the secretory system to Ca*+. Support for such a mechanism for agents acting either via the cyclic AMP system or protein kinase C has been presented for the secretory response of platelets (Knight and Scrutton, 1980, 1984; Castanaga et al., 1982) and digitonin-permeabilized islets (Tamagawa et al., 1985). In electrically permeabilized islets, both TPA (Jones et al., 1985) and forskolin (Jones et al., 1986) also caused a dose-related shift in the Ca2+ activation curve to lower Ca*+ concentrations. In view of the marked dependence of insulin secretion on extracellular calcium such an increase in sensitivity of the secretory system to internal Ca2+ may result in a lowering of the requirement for extracellular Ca2+. In the present study, therefore, we have examined this possibility in intact islets of Langerhans by determining the effects of potentiators on the dependence of glucose-stimulated insulin release on the extracellular Ca*+ concentration. Our data show clearly that all three potentiators tested have qualitatively similar effects, namely (i) a shift to the left of the curve relating the rate of glucose-stimulated insulin release to
extracellular Ca2+; and (ii) an increase in the maximal secretory response at high Ca*+ concentrations. We propose, therefore, as the simplest interpretation of these findings, that potentiators of insulin secretion act, at least in part, by increasing the sensitivity of the secretory response system to Ca*+. Data from several previous studies on intact islets of Langerhans are consistent with this proposal. Devis et al. (1975) showed that theophylline augmented the secretory response to 10 mM Ca2+ of islets pre-treated with EGTA and perifused in the absence of glucose: whether this cyclic AMPmediated effect involved a change in the concentration dependence of Ca*‘-stimulated insulin release was not reported. Evidence that it may do so was provided by Hellman (1976) who demonstrated that IBMX lowered from 20 to 10 mM the concentration of extracellular Ca*+ capable of initiating insulin release in glucose-free medium from islets that had not been pre-exposed to EGTA. Neither of these studies documented the effects of potentiators on the sensitivity of glucose-stimulated insulin release to extracellular Ca2+. Using an app roach more directly comparable to ours, and in agreement with the present findings with TPA, Hii et al. (1985) showed that, in intact islets, the phorbol ester lowered the external Ca2+ concentration necessary for a maximal secretory response to glucose. However, in their study glucose-stimulated insulin release required much higher extracellular Ca2+ levels than in our study; indeed glucose-stimulated insulin release in the study of Hii et al. (1985) was not significantly reduced when extracellular Ca2+ was reduced from normal (2.5 mM) to zero. Two further points are of note. Firstly our data show that TPA is a true potentiator of insulin secretion, eliciting little or no effect at a nonstimulatory glucose concentration. This is in agreement with some earlier reports (Virji et al., 1978) but not others (Malaisse et al., 1980; Yamamoto et al., 1982). We cannot explain this discrepancy but would draw a note of caution about the use of TPA at the high concentrations used by some authors. As our results show, and in agreement with Hii et al. (1986) the response to TPA continues to increase up to the highest concentrations used (2 PM in the present study, 10
236
PM in Hii et al., 1986). The activation of B cell protein kinase C by TPA is however essentially maximal at 0.1 PM (Lord, J.M. and Hughes, S.J., unpublished observation); the possibility that higher concentrations have effects unrelated to protein kinase C should be borne in mind. Secondly, the results with acetyl choline deserve comment. In freshly prepared islets, we could find no consistent potentiatory effects of acetyl choline. This may be related to a possible deleterious effect of collagenase treatment during isolation on the acetyl choline receptor; this interpretation is supported by the finding that a response to acetyl choline was regained after a short period of tissue culture of the islets. Culture for 44 h did not reduce the magnitude of the secretory response to glucose although interestingly the cultured islets did display a decreased sensitivity to extracellular Ca*+; nevertheless acetyl choline induced increased sensitivity to Ca*+. The mechanism responsible for the increased threshold requirement for extracellular Ca*+ after culture of the islets is unknown but could possibly reflect depletion of intracellular Ca*+ because of the low (0.4 mM) Ca*+ content of RPM1 1640 culture medium. Since glucose promotes not only Ca*+ influx but also compensatory uptake into intracellular organelles and outward transport of the ion (Hellman, 1985) a depletion of intracellular calcium could perturb the normal relationship between the magnitude of these fluxes. It is also of note that the potentiatory effect of TPA was not increased further by the simultaneous presence of acetyl choline. This was not attributable to saturation of the release system since forskolin was able to augment significantly the potentiatory effect of TPA. This suggests that, at least in the presence of 6 mM glucose, activation of protein kinase C, via enhanced diacylglycerol formation, may represent the most important component of the potentiatory effect of acetyl choline.
Acknowledgements These studies were supported the Medical Research Council Diabetic Association.
by grants from and the British
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