Brropem Joirrnal of Phamaco/ogy, 205 ( 199 I ) 2 I -27 8 1991 Elsevier Science Publishers B.V. All rights reserved OOl4-2999/91/$03.50
21
EJP 52140
Studies on the mechanism by which galanin inhibits insulin secretion in islets Stefan Lindskog ’ and Bo Ahr6n I*’ Departments of I Pharmacology and ’ Surgery. Lund Unicersiry, Lund, Sweden Received 28 February 1991. revised MS received 3 June 1991, accepted 27 August 1991
The mechanism by which the neuropeptide galanin inhibits insulin secretion in normal islets is not yet fully elucidated. Isolated rat or mouse islets were perifused in a medium containing glucose (8.3 mM) and galanin (lob6 M) or the sulphonamide diazoxide (400 PM). In rat islets prelabelled with *Rb+ or “Ca’+, galanin inhibited glucose-induced insulin secretion at the same time as increasing ““Rb+ efflux and reducing ‘%a’+ efflux. The diazoxide-induced X6Rb+ efflux was not affected by galanin, indicating that galanin activates ATP-regulated K+ channels in rat islets. In mouse islets prelabelled with *Rb+, galanin (10e6 M) decreased “Rb+ efflux. These results suggest that galanin inhibits insulin release in isolated islets by increasing K‘ and decreasing Ca’+ permeability. The increased K’ permeability, which is probably regulated differently in rat and mouse islets, is followed by a reduced Ca’+ influx, possibly through voltage-dependent Ca’+ channels. In addition, during a 60-min incubation with isolated islets, galanin inhibited insulin secretion induced by forskolin ( 1 PM). dibutyryl cyclic AMP t 1 mM), or TPA (12-0-tetradecanoylphorbol-lfacetate; 0.1 FM). Galanin also reduced the content of cyclic AMP in islets stimulated by 16.4 mM glucose. We therefore conclude that the inhibitory action of galanin on insulin secretion in normal islets includes increasing K+ permeability as well as interference with the activation of adenylate cyclase and the activity of protein kinase C and cyclic AMP. Galanin; Insulin secretion: Pancreatic islets
1. Introduction The neuropeptide galanin occurs in pancreatic islet nerves (Dunning et al., 1986; Lindskog et al., 1991). It inhibits insulin secretion by an effect that in p-cells from the obese mouse is associated with hyperpolarization of the plasma membrane and a subsequent decrease in intracellular free Ca2+ concentration (AhrCn et al., 1986). In the tumoral insulin-secreting RIN m5F cells, galanin induces hyperpolarization by activating ATP-regulated K+ channels (DeWeille et al., 1988; Dunne et al., 1989). In these cells, galanin also inhibits insulin secretion by inhibiting the activity of adenylate cyclase (Amiranoff et al., 1988) and the action of protein kinase C (Sharp et al., 19891, as well as by directly affecting exocytosis (Ullrich and Wollheim, 19893. In contrast, the mechanism whereby galanin inhibits insulin secretion in normal islet p-cells is not established, since conclusions from studies performed with the glucose-insensitive RIN m5F cells may not be
applicable to normal islets. Only one mechanistic study on the effects of galanin in normal islets has been performed (Drews et al., 1990). In fact, a recent patchclamp study has implied that, in mouse @-cells, galanin does not activate ATP-regulated Kf channels, as it does in RIN m5F cells, but rather that it activates low conductance, sulphonylurea-insensitive K’ channels (Rorsman et al., 1991). The aim of the present investigation was to elucidate the mechanism by which galanis inhibits insulin secretion in normal islets. We therefore investigated the effects of galanin on insulin secretion stimulated by the phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate), forskolin or dibutyryl cyclic AMP. Furthermore, we studied the effects of the neuropeptide on ion permeability, by perifusing islets preloaded with “Rb+ or J5Caz-c, and on the islet content of cyclic AMP.
2. Materials and methods 2. I. Animals
Correspondence to: S. Lindskog, Department of Pharmacology, Lund University. Siilvegatan IO. S-223 62 Lund. Sweden. Tel. 46.46.10.75.86. fax 46.46.10.44.29.
The experiments were performed with pancreatic islets isolated from fed Sprague-Dawley rats or NMRI mice (ALAB, Stockholm, Sweden). The animals had
been given a standard pellet diet and tap water ad ~~~~t~rnbefore the experiments.
lsfets were isolated as described previously (Karlsson and Ah&, 1991). In the ,*Rb’ efflux experiments. the okpernight cultured islets were rinsed three times in KRB medium, then placed for 120 min in 0.5 ml of KRB medium, pH 7.4. with a Ca’+ concentration of I.28 mM. The medium was supplemented with 10 mM HEPES. G.15 huma:: serum a!bumin, 3.3 mM glucose and 40-l 70 pCi/ml ‘“RbCI, added from a stock solution with a specific activity of 1.6-6.4 mCi/mg RbCI. in the “CaZr efflux experiments, the islets were placed for 90 min in 0.4 ml of KRB medium. The medium was supplemented with 0.1% human serum albumin. 8.3 mM glucose and 30-100 PCi ‘“CaCI,, which was added from a stock solution with a specific activity of 16-37 mCi/mg CaC12. In both series of experiments, after labelling and rinsing five times with KRB medium, the islets were divided into four subgroups with 40 or 20 islets in each. Each group was transferred to a column and sandwiched between two thin layers of gel (Bio-gel P-4, mesh. Bio-Rad Lab.. Richmond, CA, U.S.A.). The columns were perifused for 40 min with KRB medium, pH 7.4. supplemented with 0.1% human serum albumin and 8.3 mM glucose. Thereafter, synthetic porcine or rat galanin (lOeh M; Peninsula Lab, Belmonte. CA. U.S.A.) was added to the medium for 20 min. Diazoxide. dissolved in KRB-NaOH (pH 7.4; 400 PM; Schering Corp., Bloomfield. NJ, U.S.A.), was added at the end of the experiments. The medium was gassed continuously with 95% 0, and 5% CO,. The flow rate was 0.1 ml/min and samples were collected at 2-min intervals. From each sample, 150 ~1 were removed: 50 @I were immediately frozen for subsequent analysis of insulin and the rest, 100 ~1, was added to vials containing 5 ml scintillation fluid (Biofluor ’ ~ NEN, Boston, MA, U.S.A.) and radioactivity was counted in a liquid scintillation counter (Packard Instrument Inc., Downers Groove, IL, U.S.A.). The data are presented as the fractional outflow of s6Rb+ or ?a” from islets as calculated for each time point. %RbCI and “CaClz were from Du Pont, Wilmington, DE. U.S.A., and all other chemicals, except when otherwise indicated, were from E Merck, D-6100 Darmstadt, F.R.G. 2.3. Insulin secretion After overnight culture, the islets were rinsed three times in a modified KRB-HEPES medium supplemented with 3.3 mM glucose and 0.1% human serum albumin. The medium consisted of (mM): 114 NaCI,
4.4 KCI, 1.28 CaCI,, 1 MgC12, 29.5 NaHCO, and 10 HEPES. The islets were then preincubated in groups of eight in dishes (Multidish Nunclon”, Nunc, Roskilde, Denmark) containing 1 ml of KRB medium with glucose (3.3 mM) at 37 “C in an incubation box gassed with 95% 0, and 5% CO,. After 45 min, the islets were transferred individually to new dishes, one islet/dish (MicroWell’@ Module F-8, lmmunoquality, medium binding capacity, Nunc, Roskilde, Denmark). The single islets were then incubated for 60 min. Each dish contained 100 E.CIof KRB medium supplemented with glucose at 3.3 mM, to which one of the secretagogues TPA (lo-’ M), forskolin (IO-’ M) or dibutyryl cyclic AMP (IO-” M) was added with or without synthetic porcine galanin (1O-6-1O-x M). In each experiment, control islets were incubated with glucose at 8.3 mM with the different concentrations of galanin. Samples (50 ~1) of the medium were immediately frozen for subsequent analysis of insulin. 2.4. Islet content of cyclic AMP After overnight culture and 45 min preincubation, 20 islets were transferred to glass vials and incubated for 30 min in 200 ~1 of KRB medium (37 a C, pH 7.4) containing 0.1 mM 3-isobutyl-l-methyl-xanthine (Sigma Chemical Co., St. Louis, MO, USA) and glucose (3.3 or 16.7 mM), with or without galanin (10-6-10-9 M). The incubation was stopped by adding 200 ~1 of ice-cold trichloracetic acid. Islets were then sonicated and contrifuged for 15 min at 2000 x g. The supernatant was recovered and washed four times with 2 ml watersaturated diethyl ether. The samples were freeze-dried and diluted in an acetate buffer (0.05 M, pH 5.8) before analysis of cyclic AMP. 2.5. Determination and statistics The concentration of insulin in the incubation medium was determined by radioimmunoassay (Herbert et al., 1965) using a guinea-pig anti-porcine insulin antibody (Milab, MalmG, Sweden), ‘251-1abelled porcine insulin, and porcine insulin as standard (Nova Research lnst, Bagsvaerd, Denmark). The islet content of cyclic AMP was measured by radioimmunoassay (Amersham lnt., Amersham, UK) using a rabbit antisuccinyl cyclic AMP serum, cyclic 2-0-succinyl-3[‘2511methyl ester as tracer, and cyclic AMP as standard. The results are presented as means + S.E.M. Student’s unpaired t-test was used for statistical comparison between means in the static incubations. Pn the perifusion experiments, significant differences between groups were assessed with a two-way analysis of variance (ANOVA).
23
3. Results IO
3.1. Effects of galanin on s6Rb + efflux and insulin
9
secretion from rat islets (fig. 1)
Islets preloaded with “Rb+ were perifused in KRB medium supplemented with 8.3 mM glucose. Galanin increased “Rb+ efflux by 13% above that of control islets (by calculating differences between areas under the curves; AUC, P < 0.001; ANOVA, fig. la). This increased s6Rb + efflux was observed concomitantly with an inhibition of insulin release (P < 0.001; ANOVA; fig. lb). At the end of the perifusion, diazoxide (400 PM) markedly increased &Rb+ efflux approximately 3-fold (P < 0.001; ANOVA) and almost abolished insulin release (P < 0.001; ANOVA). Ti me Imin] -
Galaninlnd)
p--o Control(n=6)
Fig. 2. Effects of galanin (IOM) on “Rb+ efflux induced by 400 PM diazoxide and 8.3 mM glucose from “‘Rb+-loaded isolated rat islets perifused with KRB medium supplemented with 1.28 mM Ca’*. The flow rate was 0.1 ml/min and the perifusate was collected at 2-min intervals. Means f S.E.M. from each group are shown.
3.2. Effects of gaianin combined with diazoxide on ‘“Rb + efflux from rat islets (fig. 2)
Islets preloaded with 8hRb+ were perifused in KRB medium supplemented with 8.3 mM glucose. Galanin (lo-” MI was added 30 min after the introduction of diazoxide (400 PM) to the perifusion medium. Galanin did not affect the “Rb+ cfflux induced by diazoxide. 3.3. Effects of galanin on sbRb + efflu-ufrom mouse islets (fig. 3)
60-
Galanin (IO-’ M) decreased the “Rb+ efflwr from prelabelled mouse islets by 15 % compared to controls (by calculating differences between AUC; P < 0.001; ANOVA). In contrast, diazoxide (400 PM) markedly increased s’Rb+ efflux when added to the perifusion medium.
50 = E
Lr
3 -I
30-
E
2
201 IO i
oil----------22
30
LO
50
60
70
80
control
ln=3)
90
!OO
Tlmelmtn) -
Gainrun
,“=I,,
e-9
Fig. 1. Effects of galanin (10-O M) and diazoxide (400 PM) on “Rb’ efflux (a) and insulin release (b) from “hRbf-loaded isolated rat islets perifused with KRB medium supplemented with 1.28 mM Ca” + and 8.3 mM glucose. The flow rate was 0.1 ml/min and the perifusate was collected at 2-min intervals. MeanskS.E.M. from each group are shown.
.X4. Effects of galanin on ‘“Ca’ + effllur and insulin secretion from rat islets (fig. 4) Rat islets preloaded with 15Ca2+ were perifused with KRB medium supplemented with 8.3 mM glucose. Galanin (10e6 Ml decreased 45Ca2+ efflux and inhibited insulin release when introduced to the perifusion medium (P < 0.001; ANOVA).
‘j_
057
100
Fig. 3. Effects
‘“Rh . KRB
of galanin
(IO- h M)
efths from ““Rb--loaded medium
glucose. collected
The
bupplementrd tlo\\
at J-min
rate
i~lared with
\vas 0.I
intc~,als.
and dinzoxidr
(400
PM)
I 1
on
70
mouse islets perifused with
1.X
mM
ml/min
Ca“
and
Meansk5E.M.
and
the
1
X.3 II~M
perifusate
from each group
601 I
was are
shown.
_
5Oi
E 3 t
LO
I
1
_?..5. Effects of galah on insulin release stimulated 0) ghcose. forskoiin. dibrrtyql cyciic AMP or TPA (table 1) IO i
-r-L-
Oi---r
Isolated islets were incubated in KRB medium supplemented with 3.3 or 8.3 mM glucose. Insulin release was increased almost Z-fold by 8.3 mM glucose, and Z-fold by 1 PM forskolin. 1 mM dibutyql cyclic AMP or 0.1 PM TPA compared to control islets incubated with 3.3 mM glucose. Galanin (lOmh M) significantly inhibited the insulin response induced by glucose, forskolin, dibutyryl cyclic AMP or TPA.
22
30
LO
,
50 Ttme
Fig. 4. Effects ?Ya”
of galanin
I
-T1;6-T.+--60
80
90
100
lmml
(IO-’
M)
and diazoxide
(400
efflux (a) and insulin release (b) from J5Ca”+-loaded
PM)
on
isolated
rat islets perifused with KRB medium supplemented with 1.28 mM Ca? + and 8.3 mM glucose. The flow rate was 0.1 ml/min and the perifusate
was collected
at 2- min
intervals.
Means f S.E.M.
from
each group are shown.
3.6. Effects of galanin on glucose-induced
increase in
islet content of cyclic AMP (table 2)
16.7 mM glucose doubled the islet levels of cyclic AMP (P < 0.09, whereas 8.3 mM glucose had no affect (not shown). Therefore, the effects of galanin on the cyclic AMP response induced by 16.7 mM glucose were examined. This response was completely abolished by
Isolated islets were incubated in KRB medium supplemented with glucose at 3.3 or 16.7 mM. Compared to incubation with 3.3 mM glucose, incubation with
TABLE Effects
I of galanin
on
insulin
release
I?-0-tetrddecanoylphorbol-l3-acetate the insulin assay. Means + S.E.M. Experimental
from (TPA).
isolated
islets induced
are shown for 24 incubations
conditions
by glucose,
Batches of islets were incubated
forskolin
(Forsk),
dibutyryl
cylic AMP
for 60 min. At the end of the incubation.
in each experimental
CAMP).
condition.
Insulin release (pU/ml) Control
Glucose (3.3 mM)
Gal lo- ’ M
Gal lo-’
M
Gal lo-”
27+3
Glucose (8.3 mM)
64 + 3”
51+4h
45*4d
30_13J
Glucose (3.3 mM) + TPA (0.1 ~JM)
s9+3
52+3
36?3d
36*3”
Glucose (3.3 mM)+
76f2
73+4
7252
h4*2’
39+4
44*h
22+3’
20*3r
Forsk (I gM)
Glucose (3.3 mM)+Db
(Db
CAMP (1 mM)
A P < 0.001 vs. glucose 3.3 mM: ” P < O.l)S; r P < 0.01: ’ P < 0.001 vs. corresponding
control.
and
samples were taken for
M
TABLE
2
Cyclic AMP content in isolated rat islets. Batches of 20 islets were incubated
for 30 min in KRB
I-methyl-xanthine. AMP
levels from lev&
individual
experiment
in each experimental Experimental
medium containing
Means+S.E.M.
0.1 mM J-isobutyl-
are shown for changes in cyclic
measured
with 3.3
for n number
mM
glucose
of experimental
in each
sets performed
condition.
conditions
n
Cyclic AMP content (-1 fmol/islet)
Glucose (16.7 mM)
II
X+5”
Glucose (lb.7 mM)+galanin
IO-
M
5
Glucose (16.7 mM;l)+galanin
IO-’
M
5
II+4
Glucose (16.7 mM)+galanin
IO-’
M
h
-3+hh
Glucose (lb.7 mM)igalanin
ItI-”
M
6
- 14k8’
l6*7
” P < 0.001 vs. levels at glucose 3.3 mM; h P < 0.01: ’ P < 0.001 vs. levels at glucose lb.7 mM.
galanin both at IO-’ M (P < 0.001) and at IO-’ M (P < 0.01).
4. Discussion
Galanin is an islet neuropeptide that potently inhibits insulin secretion in a variety of species (AhrCn et al., 1988; Dunning et al., 1986; Hermansen, 1988; Lindskog and Ahren, 1987, 1989; Lindskog et al., 1990; Miralles et al., 1990; Ah& et al., 1991). The neuropeptide has been suggested to function as a sympathetic neurotransmitter in the endocrine pancreas (Dunning and Taborsky, 1988). Several mechanisms in the P-cell seem to be responsible for the inhibitory effect. We show here, in normal islets, that galanin inhibited glucose-induced insulin secretion at the same time as increasing K’ permeability and reducing “Ca” efflux. Furthermore, galanin inhibited insulin secretion stimulated by forskolin, dibutyryl cyclic AMP and TPA. Moreover, galanin decreased the glucose-induced increase in islet content of cyclic AMP. Thus, in normal islets, as previously shown for tumoral RIN m5F cells (Amiranoff et al., 1988; DeWeille et al., 1988; Dunne et al., 1989; Sharp et al., 1989; Ullrich and Wollheim, 1989), galanin interacts with several different steps in the signal transduction pathway. Diazoxide inhibits insulin release by increasing K+ permeability by activating ATP-regulated K+ channels (Henquin and Meissner, 1982; Trube et al., 1986), and consequently increasing HhRb+ efflux (figs. 1, 2, 4; Henquin and Meissner, 1982). Also galanin, in rat islets, increased XhRb+ efflux concomitantly with an inhibition of insulin release (fig. 1). This indicates that galanin increases K+ permeability by activating membraneous K+ channels. The failure of galanin to affect “Rb+ efflux further when the permeability to K’ was already increased by 400 PM diazoxidc (fig. 2) suggests
that galanin and diazoxide affect K’ permeability similarly. Hence, in rat islets, galanin most likely hyperpolarizes the B-cell plasma membrane by activating ATP-regulated K+ channels, which is similar to the proposed action in tumoral RIN m5F cells (DeWeille et al., 1988; Dunne et al., 1989). In mouse islets, the effects of galanin on XhRbC efflux is opposite, since nhRb+ efflux was decreased by galanin (fig. 3). This confirms the results of a previous study in which mouse islets were perifused in the presence of 15 mM glucose (Drews et al., 1990). These authors explained this action of galanin by an effect caused by hyperpolarization, which reduces “‘Rb+ efflux through Ca’+- and voltage-dependent K+ channels. The opposite effect of galanin on “Rb+ efflux in mouse versus rat islets reveals a species difference. This might be explained if galanin activates different subsets of ATP-regulated Kf channels in mouse versus rat p-cells or if the density of ATP-regulated K+ channels in relation to other K’ channels shows species differencies. Alternatively, in the mouse &cell, galanin might activate another type of K+ channel, distinct from the ATP-regulated channel, as implied from the results of a recent patch-clamp study (Rorsman et al., 1991). We also demonstrated that the “Ca” efflux from “Ca’+-preloaded rat islets was depressed by galanin concomitantly with an inhibition of insulin release. This is in agreement with the previously observed galanin-induced reduction in the concentration of cytoplasmic free Cal+ in p-celis (Ahren et al., 1986; Nilsson et al., 1989). Previously, in patch-clamp studies (AhrCn et al., 19891, it has been shown that galanin does not directly affect the Ca’+ current. Moreover, galanin does not affect the intracellular Ca’+ concentration after K+ depolarization (Ah& et al., 1986; Nilsson et al., 1989). Therefore, it is unlikely that the peptide directly affects Ca’+ channel activity. Rather, the observed decrease in ‘5Ca2+ efflux represents the consequences of hyperpolarization, i.e. closure of the voltage-dependent Ca2+ channels, secondary to galanin-induced activation of K’ channels. Activation of K’ channels is apparently not the only mechanism responsible for the inhibitory action of galanin on insulin secI _tion. Previously, it was shown in RIN m5F cells that galanin inhibits the activation of adenylate cyclase as well as the production of cyclic AMP (Amiranoff et al., 1988). We found that, in normal islets, galanin inhibited insulin secretion induced by forskolin (table 11, which increases insulin secretion and activates adenylate cyclase (Wiedenkeller and Sharp, 1983; Seamon et al., 1981). Furthermore, we also observed that galanin reduced the content of cyclic AMP in normal islets (table 2). We also found that galanin interfered with the cellular actions of CYCk AMP in islets, since insulin secretion in the presence of dibutyryl cyclic AMP was inhibited by the neuropep-
tide (tsbfe %I. Moreover. galanin also seems to intere actions of protein kinase C. since insulin ified by TPA (Malaisse et al., 1980). s ~~ote~~ kinase C (Castagna et al., 198% by ga~anin (table I). The inhibitor action OF ga~a~i~ on cyclic AMP- and TPA-activated insulin secretion, as found by us with normal islets. is consistent with its effects in tumoral RIN mSF cells (Sharp et al.. 19S9). Thus, the combined effects of galan~~ in ~o~~~ islets suggest actions not only in the ~~asrna membrane. but are also consistent with galanin interfering with distal steps in the signal transduction pathway, as originally proposed in permeabilized RIN cells by Ulhich and Wollheim (1989). 5 exe. our results suggest that galanin inhibits insulin secretion in normal islets by increasjng K’ permeabi~~ty by an action that seems differently regulated in rat versus in mouse islets. This leads secondarily to closure of 421’” channels. Galanin also reduces the aviation of cyclic AMP and inhibits distal steps in the signal transduction pathway. Thus, several different mecba~~sms have been identified as underlying the ~~bib~to~ action of galanin on insulin secretion in normal islets.
The technical assistance of Lena Kvist. Lilian Bengtsson. and Marie Schwartz is gratefully acknowledged. This study was supported by the Swedish Medical Research Council (Grant No. 14X-68343. Stockholm. Sweden: Nordic Insulin Foundation. Gentofte, Denmark: Swedish Diahetes Association. Stockholm. Sweden: Albert Pihissons and Magnus Bergvaiis Foundations. Maimo and Stockholm. Sweden: frafoordska Stiftelsen, Lund. Sweden; Swedish Hoechst Diabetes Fund. Stockholm. Sweden: Svenska Sliiskapet for Medicinsk Forskning. Stockholm. Sweden: and the Medical Faculty, Lund University, Sweden.
Ahren, B.. P. Arkhammar, P.O. Berggren and T. Niisson, 1986, Gaianin inhibits glucose-stimulated insulin release by a mechanism involving hyperpoiarization and lowering of cytopiasmic free Ca” concentration. Biochem. Biophys. Res. Commun. 140, 1059. Abren, B., P. Rorsman and P.O. Berggren, 19X8. Gaianin and the endocrine pancreas. FEBS Lett. 229, 233. Ahrin, 8.. P.O. Berggren. K. Bokvist and P. Rorsman, 1989, Does gaianin inhibit insulin secretion hy opening of the ATP-regulated K’ channel in th p-ceil?, Peptides 10, 453. Ah&n. 8.. A. Ar’Rajab. G. Biittcher, F. Sundier and BE. Dunning, 1991, Presence of gaianin in human pancreatic nerves and inhibition of insulin secretion from isolated human islets, Ceii Tissue Res. 264, 263. Amiranoff, B.. A.M. Lorinet, 1. Lagny-Pourmir and M. Laburthe, 1988. Mechanism of gaiauin-inhibited insulin release. Occurrence of a pertussis-toxin-sensitive inhibition of adenyiate cyciase, Eur. .I. Biochem. 177, 147. Castagna. M.. Y. Takai, K. Kaibuchi, K. Sano, U. Kikkawa and Y.
Nishizuka, iYX2. Direct activatton of calcium-activated. phospholipid-dependent protein kinase by tumor-promoting phorboi esters. J. Bioi. Chem. 257, 7847. DeWeiiie. J.. H. Schmid-Antomarchi, M. Fosset and M. Lazdunski, 1988, ATP-sensitive K’ channels that are blocked by hypo~iycem~-iliducing suifonyiureas in insulin-secreting ceils are activated by gaianin, a hyperglycemia-inducing hormone, Proc. Nati. Acad. Sci. U.S.A. XS. 1312. Drews. G., A. Debuyser. M. Nenquin and J.C. Henquin, 1990, Gaianin and epinephrine act on distinct receptors to inhibit insulin release by the same mechanisms including an increase in K’ permeability of the B-cell membrane, Endocr~nolo~ 126, 1646. Dunne, M.J., M.J. Buiiett, G. Li. C.B. Woilheim and O.H. i-etersen, lY89, Gaianin activates nucieotide dependent K+ channels in insulin-secreting ceils via a pertussis toxin-sensitive G-protein, EMBO J. 8. 413. Dunning, B.E. and G.J. Taborsky. Jr., 1988, Gaianin - sympathetic neurotransmitter in endocrine pancreas?, Diabetes 37, 1157. Dunning, B.E., 8. Ah&, R.C. Veith, G. Btittcher, F. Sundler and G.J. Taborsky. Jr., i986. Gaianin: a novel pancreatic neuropeptide. Am. J. Physioi. 255, E785. Henquin. J.C. and H.P. Meissner. 1982, Opposite effects of toibutamide and diazoxide on ahRb’ fluxes and membrane potential in pancreatic B ceils, Biochem. Pharmacoi. 31, l407. Herbert. V., KS. Lau, C.W. Gottiieb and S.J. Bieicher, 1965, Coated charcoal immunoassay of insulin, J. Ciin. Endocrinoi. Metab. 25, 1875. Hermansen, K., 1988, Effects of gaianin on the release of insulin, giucagon and somatostatin from the isolated, perfused dog pancreas, Acta Endocrinoi. 119, 91. Karisson, S. and B. Ah&n, 1991, CCK, receptor antagonism inhibits mechanisms underlying CCK-~-stimulated insulin release in isolated rat islets, Eur. J. Pharmacoi. fin press). Lindskog, S. and B. Ah&, 1987, Gaianin: effects on basal and stimulated insulin and giucagon secretion in the mouse, Acta Physioi. Stand. 129, 305. Lindskog, S. and B. Ah&t, 1989, Effects of gaianin on insulin and giucagon secretion in the rat, Int. J. Pancreatoi. 4. 335. Lindskog, S., B.E. Dunning, H. M~rtensson, A. Ar’Rajab, G.J. Taborsky, Jr. and B. Ah&n, 19%. Gaianin of the homologous species inhibits insulin secretion in the rat and the pig, Acta Physioi. Stand. 139, 591. Lindskog, S., B. Ah&, B.E. Dunning and F. Sundler, 1991, Gaianin-immunoreactive nerves in the mouse and rat pancreas, Ceil Tissue Res. 264, 363. Maiaisse, W.J., A. Sener, A. Herchueiz, A.R. Carpineiii, P. Poioczek, J. Winand and M. Castagna, 1980, Insuiinotropic effect of the tumor promoter 12-ii-tetradecanoyiphorboi-13-acetate in rat pancreatic islets, Cancer Res. 40, 3827. Miraiies, P., E. Peiro, P. DCgano, R.A. Siivestre and J. Marco, 1990, Inhibition of insulin and somatostatin secretion and stimulation of glucagon release by homologous gaianin in perfused rat pancreas, Diabetes 39, 996. Niisson, T., P. Arkhammar, P. Rorsman and P.O. Berggren, 1989, Suppression of insulin release by galanin and somatostatin is mediated by a G-protein, J. Bioi. Chem. 264, 973. Rorsman, P., K. Bokvist, C. &nmlil, P. Arkhammar, P.O. Berggren, 0. Larsson and K. Wihiander, 1991, Activation by adrenaline of a low-conductance G protein-dependent K+ channel in mouse pancreatic B ceils, Nature 3, 77. Seamon, K.B., W. Padgett and J.W. Daly. 1981, Forskoiin: unique diterpene activator of adenyiate cyclase in membranes and intact ceils, Proc. Nati. Acad. Sci. U.S.A. 78, 3363. Sharp,G.W.G., Y. Le Marchand-Brustei, T. Yada, L.L. Russo, CR. Bliss, M. Cormont, L. Monges and E. Van Obberghen, 1989, Gaianin can inhibit insulin retease by a mechanism other titan
27 membrane hyperpolarization or inhibition of adenylate cyclase. J. Biol. Chem. 264, 7302. Trube G., P. Rorsman and T. Ohno-Shosaku, 1986, Opposite effects of tolbutamide and diazoxide on the ATP-dependent KC channel in mouse pancreatic p-cells, Pfliigers Arch. 407, 493.
Ullrich, S. and C.B. Wollheim, 1989, Galanin inhibits insulin secretion by direct interference with exocytosis, PEBS Lett. 247, 401. Wiedrnkeller, D.E. and G.W.G. Sharp, 1983, Effects of forskolin on insulin release and cyclic AMP content in rat pancreatic islets, Endocrinology 113, 2311.
21
EJP 52140
Studies on the mechanism by which galanin inhibits insulin secretion in islets Stefan Lindskog ’ and Bo Ahr6n I*’ Departments of I Pharmacology and ’ Surgery. Lund Unicersiry, Lund, Sweden Received 28 February 1991. revised MS received 3 June 1991, accepted 27 August 1991
The mechanism by which the neuropeptide galanin inhibits insulin secretion in normal islets is not yet fully elucidated. Isolated rat or mouse islets were perifused in a medium containing glucose (8.3 mM) and galanin (lob6 M) or the sulphonamide diazoxide (400 PM). In rat islets prelabelled with *Rb+ or “Ca’+, galanin inhibited glucose-induced insulin secretion at the same time as increasing ““Rb+ efflux and reducing ‘%a’+ efflux. The diazoxide-induced X6Rb+ efflux was not affected by galanin, indicating that galanin activates ATP-regulated K+ channels in rat islets. In mouse islets prelabelled with *Rb+, galanin (10e6 M) decreased “Rb+ efflux. These results suggest that galanin inhibits insulin release in isolated islets by increasing K‘ and decreasing Ca’+ permeability. The increased K’ permeability, which is probably regulated differently in rat and mouse islets, is followed by a reduced Ca’+ influx, possibly through voltage-dependent Ca’+ channels. In addition, during a 60-min incubation with isolated islets, galanin inhibited insulin secretion induced by forskolin ( 1 PM). dibutyryl cyclic AMP t 1 mM), or TPA (12-0-tetradecanoylphorbol-lfacetate; 0.1 FM). Galanin also reduced the content of cyclic AMP in islets stimulated by 16.4 mM glucose. We therefore conclude that the inhibitory action of galanin on insulin secretion in normal islets includes increasing K+ permeability as well as interference with the activation of adenylate cyclase and the activity of protein kinase C and cyclic AMP. Galanin; Insulin secretion: Pancreatic islets
1. Introduction The neuropeptide galanin occurs in pancreatic islet nerves (Dunning et al., 1986; Lindskog et al., 1991). It inhibits insulin secretion by an effect that in p-cells from the obese mouse is associated with hyperpolarization of the plasma membrane and a subsequent decrease in intracellular free Ca2+ concentration (AhrCn et al., 1986). In the tumoral insulin-secreting RIN m5F cells, galanin induces hyperpolarization by activating ATP-regulated K+ channels (DeWeille et al., 1988; Dunne et al., 1989). In these cells, galanin also inhibits insulin secretion by inhibiting the activity of adenylate cyclase (Amiranoff et al., 1988) and the action of protein kinase C (Sharp et al., 19891, as well as by directly affecting exocytosis (Ullrich and Wollheim, 19893. In contrast, the mechanism whereby galanin inhibits insulin secretion in normal islet p-cells is not established, since conclusions from studies performed with the glucose-insensitive RIN m5F cells may not be
applicable to normal islets. Only one mechanistic study on the effects of galanin in normal islets has been performed (Drews et al., 1990). In fact, a recent patchclamp study has implied that, in mouse @-cells, galanin does not activate ATP-regulated Kf channels, as it does in RIN m5F cells, but rather that it activates low conductance, sulphonylurea-insensitive K’ channels (Rorsman et al., 1991). The aim of the present investigation was to elucidate the mechanism by which galanis inhibits insulin secretion in normal islets. We therefore investigated the effects of galanin on insulin secretion stimulated by the phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate), forskolin or dibutyryl cyclic AMP. Furthermore, we studied the effects of the neuropeptide on ion permeability, by perifusing islets preloaded with “Rb+ or J5Caz-c, and on the islet content of cyclic AMP.
2. Materials and methods 2. I. Animals
Correspondence to: S. Lindskog, Department of Pharmacology, Lund University. Siilvegatan IO. S-223 62 Lund. Sweden. Tel. 46.46.10.75.86. fax 46.46.10.44.29.
The experiments were performed with pancreatic islets isolated from fed Sprague-Dawley rats or NMRI mice (ALAB, Stockholm, Sweden). The animals had
been given a standard pellet diet and tap water ad ~~~~t~rnbefore the experiments.
lsfets were isolated as described previously (Karlsson and Ah&, 1991). In the ,*Rb’ efflux experiments. the okpernight cultured islets were rinsed three times in KRB medium, then placed for 120 min in 0.5 ml of KRB medium, pH 7.4. with a Ca’+ concentration of I.28 mM. The medium was supplemented with 10 mM HEPES. G.15 huma:: serum a!bumin, 3.3 mM glucose and 40-l 70 pCi/ml ‘“RbCI, added from a stock solution with a specific activity of 1.6-6.4 mCi/mg RbCI. in the “CaZr efflux experiments, the islets were placed for 90 min in 0.4 ml of KRB medium. The medium was supplemented with 0.1% human serum albumin. 8.3 mM glucose and 30-100 PCi ‘“CaCI,, which was added from a stock solution with a specific activity of 16-37 mCi/mg CaC12. In both series of experiments, after labelling and rinsing five times with KRB medium, the islets were divided into four subgroups with 40 or 20 islets in each. Each group was transferred to a column and sandwiched between two thin layers of gel (Bio-gel P-4, mesh. Bio-Rad Lab.. Richmond, CA, U.S.A.). The columns were perifused for 40 min with KRB medium, pH 7.4. supplemented with 0.1% human serum albumin and 8.3 mM glucose. Thereafter, synthetic porcine or rat galanin (lOeh M; Peninsula Lab, Belmonte. CA. U.S.A.) was added to the medium for 20 min. Diazoxide. dissolved in KRB-NaOH (pH 7.4; 400 PM; Schering Corp., Bloomfield. NJ, U.S.A.), was added at the end of the experiments. The medium was gassed continuously with 95% 0, and 5% CO,. The flow rate was 0.1 ml/min and samples were collected at 2-min intervals. From each sample, 150 ~1 were removed: 50 @I were immediately frozen for subsequent analysis of insulin and the rest, 100 ~1, was added to vials containing 5 ml scintillation fluid (Biofluor ’ ~ NEN, Boston, MA, U.S.A.) and radioactivity was counted in a liquid scintillation counter (Packard Instrument Inc., Downers Groove, IL, U.S.A.). The data are presented as the fractional outflow of s6Rb+ or ?a” from islets as calculated for each time point. %RbCI and “CaClz were from Du Pont, Wilmington, DE. U.S.A., and all other chemicals, except when otherwise indicated, were from E Merck, D-6100 Darmstadt, F.R.G. 2.3. Insulin secretion After overnight culture, the islets were rinsed three times in a modified KRB-HEPES medium supplemented with 3.3 mM glucose and 0.1% human serum albumin. The medium consisted of (mM): 114 NaCI,
4.4 KCI, 1.28 CaCI,, 1 MgC12, 29.5 NaHCO, and 10 HEPES. The islets were then preincubated in groups of eight in dishes (Multidish Nunclon”, Nunc, Roskilde, Denmark) containing 1 ml of KRB medium with glucose (3.3 mM) at 37 “C in an incubation box gassed with 95% 0, and 5% CO,. After 45 min, the islets were transferred individually to new dishes, one islet/dish (MicroWell’@ Module F-8, lmmunoquality, medium binding capacity, Nunc, Roskilde, Denmark). The single islets were then incubated for 60 min. Each dish contained 100 E.CIof KRB medium supplemented with glucose at 3.3 mM, to which one of the secretagogues TPA (lo-’ M), forskolin (IO-’ M) or dibutyryl cyclic AMP (IO-” M) was added with or without synthetic porcine galanin (1O-6-1O-x M). In each experiment, control islets were incubated with glucose at 8.3 mM with the different concentrations of galanin. Samples (50 ~1) of the medium were immediately frozen for subsequent analysis of insulin. 2.4. Islet content of cyclic AMP After overnight culture and 45 min preincubation, 20 islets were transferred to glass vials and incubated for 30 min in 200 ~1 of KRB medium (37 a C, pH 7.4) containing 0.1 mM 3-isobutyl-l-methyl-xanthine (Sigma Chemical Co., St. Louis, MO, USA) and glucose (3.3 or 16.7 mM), with or without galanin (10-6-10-9 M). The incubation was stopped by adding 200 ~1 of ice-cold trichloracetic acid. Islets were then sonicated and contrifuged for 15 min at 2000 x g. The supernatant was recovered and washed four times with 2 ml watersaturated diethyl ether. The samples were freeze-dried and diluted in an acetate buffer (0.05 M, pH 5.8) before analysis of cyclic AMP. 2.5. Determination and statistics The concentration of insulin in the incubation medium was determined by radioimmunoassay (Herbert et al., 1965) using a guinea-pig anti-porcine insulin antibody (Milab, MalmG, Sweden), ‘251-1abelled porcine insulin, and porcine insulin as standard (Nova Research lnst, Bagsvaerd, Denmark). The islet content of cyclic AMP was measured by radioimmunoassay (Amersham lnt., Amersham, UK) using a rabbit antisuccinyl cyclic AMP serum, cyclic 2-0-succinyl-3[‘2511methyl ester as tracer, and cyclic AMP as standard. The results are presented as means + S.E.M. Student’s unpaired t-test was used for statistical comparison between means in the static incubations. Pn the perifusion experiments, significant differences between groups were assessed with a two-way analysis of variance (ANOVA).
23
3. Results IO
3.1. Effects of galanin on s6Rb + efflux and insulin
9
secretion from rat islets (fig. 1)
Islets preloaded with “Rb+ were perifused in KRB medium supplemented with 8.3 mM glucose. Galanin increased “Rb+ efflux by 13% above that of control islets (by calculating differences between areas under the curves; AUC, P < 0.001; ANOVA, fig. la). This increased s6Rb + efflux was observed concomitantly with an inhibition of insulin release (P < 0.001; ANOVA; fig. lb). At the end of the perifusion, diazoxide (400 PM) markedly increased &Rb+ efflux approximately 3-fold (P < 0.001; ANOVA) and almost abolished insulin release (P < 0.001; ANOVA). Ti me Imin] -
Galaninlnd)
p--o Control(n=6)
Fig. 2. Effects of galanin (IOM) on “Rb+ efflux induced by 400 PM diazoxide and 8.3 mM glucose from “‘Rb+-loaded isolated rat islets perifused with KRB medium supplemented with 1.28 mM Ca’*. The flow rate was 0.1 ml/min and the perifusate was collected at 2-min intervals. Means f S.E.M. from each group are shown.
3.2. Effects of gaianin combined with diazoxide on ‘“Rb + efflux from rat islets (fig. 2)
Islets preloaded with 8hRb+ were perifused in KRB medium supplemented with 8.3 mM glucose. Galanin (lo-” MI was added 30 min after the introduction of diazoxide (400 PM) to the perifusion medium. Galanin did not affect the “Rb+ cfflux induced by diazoxide. 3.3. Effects of galanin on sbRb + efflu-ufrom mouse islets (fig. 3)
60-
Galanin (IO-’ M) decreased the “Rb+ efflwr from prelabelled mouse islets by 15 % compared to controls (by calculating differences between AUC; P < 0.001; ANOVA). In contrast, diazoxide (400 PM) markedly increased s’Rb+ efflux when added to the perifusion medium.
50 = E
Lr
3 -I
30-
E
2
201 IO i
oil----------22
30
LO
50
60
70
80
control
ln=3)
90
!OO
Tlmelmtn) -
Gainrun
,“=I,,
e-9
Fig. 1. Effects of galanin (10-O M) and diazoxide (400 PM) on “Rb’ efflux (a) and insulin release (b) from “hRbf-loaded isolated rat islets perifused with KRB medium supplemented with 1.28 mM Ca” + and 8.3 mM glucose. The flow rate was 0.1 ml/min and the perifusate was collected at 2-min intervals. MeanskS.E.M. from each group are shown.
.X4. Effects of galanin on ‘“Ca’ + effllur and insulin secretion from rat islets (fig. 4) Rat islets preloaded with 15Ca2+ were perifused with KRB medium supplemented with 8.3 mM glucose. Galanin (10e6 Ml decreased 45Ca2+ efflux and inhibited insulin release when introduced to the perifusion medium (P < 0.001; ANOVA).
‘j_
057
100
Fig. 3. Effects
‘“Rh . KRB
of galanin
(IO- h M)
efths from ““Rb--loaded medium
glucose. collected
The
bupplementrd tlo\\
at J-min
rate
i~lared with
\vas 0.I
intc~,als.
and dinzoxidr
(400
PM)
I 1
on
70
mouse islets perifused with
1.X
mM
ml/min
Ca“
and
Meansk5E.M.
and
the
1
X.3 II~M
perifusate
from each group
601 I
was are
shown.
_
5Oi
E 3 t
LO
I
1
_?..5. Effects of galah on insulin release stimulated 0) ghcose. forskoiin. dibrrtyql cyciic AMP or TPA (table 1) IO i
-r-L-
Oi---r
Isolated islets were incubated in KRB medium supplemented with 3.3 or 8.3 mM glucose. Insulin release was increased almost Z-fold by 8.3 mM glucose, and Z-fold by 1 PM forskolin. 1 mM dibutyql cyclic AMP or 0.1 PM TPA compared to control islets incubated with 3.3 mM glucose. Galanin (lOmh M) significantly inhibited the insulin response induced by glucose, forskolin, dibutyryl cyclic AMP or TPA.
22
30
LO
,
50 Ttme
Fig. 4. Effects ?Ya”
of galanin
I
-T1;6-T.+--60
80
90
100
lmml
(IO-’
M)
and diazoxide
(400
efflux (a) and insulin release (b) from J5Ca”+-loaded
PM)
on
isolated
rat islets perifused with KRB medium supplemented with 1.28 mM Ca? + and 8.3 mM glucose. The flow rate was 0.1 ml/min and the perifusate
was collected
at 2- min
intervals.
Means f S.E.M.
from
each group are shown.
3.6. Effects of galanin on glucose-induced
increase in
islet content of cyclic AMP (table 2)
16.7 mM glucose doubled the islet levels of cyclic AMP (P < 0.09, whereas 8.3 mM glucose had no affect (not shown). Therefore, the effects of galanin on the cyclic AMP response induced by 16.7 mM glucose were examined. This response was completely abolished by
Isolated islets were incubated in KRB medium supplemented with glucose at 3.3 or 16.7 mM. Compared to incubation with 3.3 mM glucose, incubation with
TABLE Effects
I of galanin
on
insulin
release
I?-0-tetrddecanoylphorbol-l3-acetate the insulin assay. Means + S.E.M. Experimental
from (TPA).
isolated
islets induced
are shown for 24 incubations
conditions
by glucose,
Batches of islets were incubated
forskolin
(Forsk),
dibutyryl
cylic AMP
for 60 min. At the end of the incubation.
in each experimental
CAMP).
condition.
Insulin release (pU/ml) Control
Glucose (3.3 mM)
Gal lo- ’ M
Gal lo-’
M
Gal lo-”
27+3
Glucose (8.3 mM)
64 + 3”
51+4h
45*4d
30_13J
Glucose (3.3 mM) + TPA (0.1 ~JM)
s9+3
52+3
36?3d
36*3”
Glucose (3.3 mM)+
76f2
73+4
7252
h4*2’
39+4
44*h
22+3’
20*3r
Forsk (I gM)
Glucose (3.3 mM)+Db
(Db
CAMP (1 mM)
A P < 0.001 vs. glucose 3.3 mM: ” P < O.l)S; r P < 0.01: ’ P < 0.001 vs. corresponding
control.
and
samples were taken for
M
TABLE
2
Cyclic AMP content in isolated rat islets. Batches of 20 islets were incubated
for 30 min in KRB
I-methyl-xanthine. AMP
levels from lev&
individual
experiment
in each experimental Experimental
medium containing
Means+S.E.M.
0.1 mM J-isobutyl-
are shown for changes in cyclic
measured
with 3.3
for n number
mM
glucose
of experimental
in each
sets performed
condition.
conditions
n
Cyclic AMP content (-1 fmol/islet)
Glucose (16.7 mM)
II
X+5”
Glucose (lb.7 mM)+galanin
IO-
M
5
Glucose (16.7 mM;l)+galanin
IO-’
M
5
II+4
Glucose (16.7 mM)+galanin
IO-’
M
h
-3+hh
Glucose (lb.7 mM)igalanin
ItI-”
M
6
- 14k8’
l6*7
” P < 0.001 vs. levels at glucose 3.3 mM; h P < 0.01: ’ P < 0.001 vs. levels at glucose lb.7 mM.
galanin both at IO-’ M (P < 0.001) and at IO-’ M (P < 0.01).
4. Discussion
Galanin is an islet neuropeptide that potently inhibits insulin secretion in a variety of species (AhrCn et al., 1988; Dunning et al., 1986; Hermansen, 1988; Lindskog and Ahren, 1987, 1989; Lindskog et al., 1990; Miralles et al., 1990; Ah& et al., 1991). The neuropeptide has been suggested to function as a sympathetic neurotransmitter in the endocrine pancreas (Dunning and Taborsky, 1988). Several mechanisms in the P-cell seem to be responsible for the inhibitory effect. We show here, in normal islets, that galanin inhibited glucose-induced insulin secretion at the same time as increasing K’ permeability and reducing “Ca” efflux. Furthermore, galanin inhibited insulin secretion stimulated by forskolin, dibutyryl cyclic AMP and TPA. Moreover, galanin decreased the glucose-induced increase in islet content of cyclic AMP. Thus, in normal islets, as previously shown for tumoral RIN m5F cells (Amiranoff et al., 1988; DeWeille et al., 1988; Dunne et al., 1989; Sharp et al., 1989; Ullrich and Wollheim, 1989), galanin interacts with several different steps in the signal transduction pathway. Diazoxide inhibits insulin release by increasing K+ permeability by activating ATP-regulated K+ channels (Henquin and Meissner, 1982; Trube et al., 1986), and consequently increasing HhRb+ efflux (figs. 1, 2, 4; Henquin and Meissner, 1982). Also galanin, in rat islets, increased XhRb+ efflux concomitantly with an inhibition of insulin release (fig. 1). This indicates that galanin increases K+ permeability by activating membraneous K+ channels. The failure of galanin to affect “Rb+ efflux further when the permeability to K’ was already increased by 400 PM diazoxidc (fig. 2) suggests
that galanin and diazoxide affect K’ permeability similarly. Hence, in rat islets, galanin most likely hyperpolarizes the B-cell plasma membrane by activating ATP-regulated K+ channels, which is similar to the proposed action in tumoral RIN m5F cells (DeWeille et al., 1988; Dunne et al., 1989). In mouse islets, the effects of galanin on XhRbC efflux is opposite, since nhRb+ efflux was decreased by galanin (fig. 3). This confirms the results of a previous study in which mouse islets were perifused in the presence of 15 mM glucose (Drews et al., 1990). These authors explained this action of galanin by an effect caused by hyperpolarization, which reduces “‘Rb+ efflux through Ca’+- and voltage-dependent K+ channels. The opposite effect of galanin on “Rb+ efflux in mouse versus rat islets reveals a species difference. This might be explained if galanin activates different subsets of ATP-regulated Kf channels in mouse versus rat p-cells or if the density of ATP-regulated K+ channels in relation to other K’ channels shows species differencies. Alternatively, in the mouse &cell, galanin might activate another type of K+ channel, distinct from the ATP-regulated channel, as implied from the results of a recent patch-clamp study (Rorsman et al., 1991). We also demonstrated that the “Ca” efflux from “Ca’+-preloaded rat islets was depressed by galanin concomitantly with an inhibition of insulin release. This is in agreement with the previously observed galanin-induced reduction in the concentration of cytoplasmic free Cal+ in p-celis (Ahren et al., 1986; Nilsson et al., 1989). Previously, in patch-clamp studies (AhrCn et al., 19891, it has been shown that galanin does not directly affect the Ca’+ current. Moreover, galanin does not affect the intracellular Ca’+ concentration after K+ depolarization (Ah& et al., 1986; Nilsson et al., 1989). Therefore, it is unlikely that the peptide directly affects Ca’+ channel activity. Rather, the observed decrease in ‘5Ca2+ efflux represents the consequences of hyperpolarization, i.e. closure of the voltage-dependent Ca2+ channels, secondary to galanin-induced activation of K’ channels. Activation of K’ channels is apparently not the only mechanism responsible for the inhibitory action of galanin on insulin secI _tion. Previously, it was shown in RIN m5F cells that galanin inhibits the activation of adenylate cyclase as well as the production of cyclic AMP (Amiranoff et al., 1988). We found that, in normal islets, galanin inhibited insulin secretion induced by forskolin (table 11, which increases insulin secretion and activates adenylate cyclase (Wiedenkeller and Sharp, 1983; Seamon et al., 1981). Furthermore, we also observed that galanin reduced the content of cyclic AMP in normal islets (table 2). We also found that galanin interfered with the cellular actions of CYCk AMP in islets, since insulin secretion in the presence of dibutyryl cyclic AMP was inhibited by the neuropep-
tide (tsbfe %I. Moreover. galanin also seems to intere actions of protein kinase C. since insulin ified by TPA (Malaisse et al., 1980). s ~~ote~~ kinase C (Castagna et al., 198% by ga~anin (table I). The inhibitor action OF ga~a~i~ on cyclic AMP- and TPA-activated insulin secretion, as found by us with normal islets. is consistent with its effects in tumoral RIN mSF cells (Sharp et al.. 19S9). Thus, the combined effects of galan~~ in ~o~~~ islets suggest actions not only in the ~~asrna membrane. but are also consistent with galanin interfering with distal steps in the signal transduction pathway, as originally proposed in permeabilized RIN cells by Ulhich and Wollheim (1989). 5 exe. our results suggest that galanin inhibits insulin secretion in normal islets by increasjng K’ permeabi~~ty by an action that seems differently regulated in rat versus in mouse islets. This leads secondarily to closure of 421’” channels. Galanin also reduces the aviation of cyclic AMP and inhibits distal steps in the signal transduction pathway. Thus, several different mecba~~sms have been identified as underlying the ~~bib~to~ action of galanin on insulin secretion in normal islets.
The technical assistance of Lena Kvist. Lilian Bengtsson. and Marie Schwartz is gratefully acknowledged. This study was supported by the Swedish Medical Research Council (Grant No. 14X-68343. Stockholm. Sweden: Nordic Insulin Foundation. Gentofte, Denmark: Swedish Diahetes Association. Stockholm. Sweden: Albert Pihissons and Magnus Bergvaiis Foundations. Maimo and Stockholm. Sweden: frafoordska Stiftelsen, Lund. Sweden; Swedish Hoechst Diabetes Fund. Stockholm. Sweden: Svenska Sliiskapet for Medicinsk Forskning. Stockholm. Sweden: and the Medical Faculty, Lund University, Sweden.
Ahren, B.. P. Arkhammar, P.O. Berggren and T. Niisson, 1986, Gaianin inhibits glucose-stimulated insulin release by a mechanism involving hyperpoiarization and lowering of cytopiasmic free Ca” concentration. Biochem. Biophys. Res. Commun. 140, 1059. Abren, B., P. Rorsman and P.O. Berggren, 19X8. Gaianin and the endocrine pancreas. FEBS Lett. 229, 233. Ahrin, 8.. P.O. Berggren. K. Bokvist and P. Rorsman, 1989, Does gaianin inhibit insulin secretion hy opening of the ATP-regulated K’ channel in th p-ceil?, Peptides 10, 453. Ah&n. 8.. A. Ar’Rajab. G. Biittcher, F. Sundier and BE. Dunning, 1991, Presence of gaianin in human pancreatic nerves and inhibition of insulin secretion from isolated human islets, Ceii Tissue Res. 264, 263. Amiranoff, B.. A.M. Lorinet, 1. Lagny-Pourmir and M. Laburthe, 1988. Mechanism of gaiauin-inhibited insulin release. Occurrence of a pertussis-toxin-sensitive inhibition of adenyiate cyciase, Eur. .I. Biochem. 177, 147. Castagna. M.. Y. Takai, K. Kaibuchi, K. Sano, U. Kikkawa and Y.
Nishizuka, iYX2. Direct activatton of calcium-activated. phospholipid-dependent protein kinase by tumor-promoting phorboi esters. J. Bioi. Chem. 257, 7847. DeWeiiie. J.. H. Schmid-Antomarchi, M. Fosset and M. Lazdunski, 1988, ATP-sensitive K’ channels that are blocked by hypo~iycem~-iliducing suifonyiureas in insulin-secreting ceils are activated by gaianin, a hyperglycemia-inducing hormone, Proc. Nati. Acad. Sci. U.S.A. XS. 1312. Drews. G., A. Debuyser. M. Nenquin and J.C. Henquin, 1990, Gaianin and epinephrine act on distinct receptors to inhibit insulin release by the same mechanisms including an increase in K’ permeability of the B-cell membrane, Endocr~nolo~ 126, 1646. Dunne, M.J., M.J. Buiiett, G. Li. C.B. Woilheim and O.H. i-etersen, lY89, Gaianin activates nucieotide dependent K+ channels in insulin-secreting ceils via a pertussis toxin-sensitive G-protein, EMBO J. 8. 413. Dunning, B.E. and G.J. Taborsky. Jr., 1988, Gaianin - sympathetic neurotransmitter in endocrine pancreas?, Diabetes 37, 1157. Dunning, B.E., 8. Ah&, R.C. Veith, G. Btittcher, F. Sundler and G.J. Taborsky. Jr., i986. Gaianin: a novel pancreatic neuropeptide. Am. J. Physioi. 255, E785. Henquin. J.C. and H.P. Meissner. 1982, Opposite effects of toibutamide and diazoxide on ahRb’ fluxes and membrane potential in pancreatic B ceils, Biochem. Pharmacoi. 31, l407. Herbert. V., KS. Lau, C.W. Gottiieb and S.J. Bieicher, 1965, Coated charcoal immunoassay of insulin, J. Ciin. Endocrinoi. Metab. 25, 1875. Hermansen, K., 1988, Effects of gaianin on the release of insulin, giucagon and somatostatin from the isolated, perfused dog pancreas, Acta Endocrinoi. 119, 91. Karisson, S. and B. Ah&n, 1991, CCK, receptor antagonism inhibits mechanisms underlying CCK-~-stimulated insulin release in isolated rat islets, Eur. J. Pharmacoi. fin press). Lindskog, S. and B. Ah&, 1987, Gaianin: effects on basal and stimulated insulin and giucagon secretion in the mouse, Acta Physioi. Stand. 129, 305. Lindskog, S. and B. Ah&t, 1989, Effects of gaianin on insulin and giucagon secretion in the rat, Int. J. Pancreatoi. 4. 335. Lindskog, S., B.E. Dunning, H. M~rtensson, A. Ar’Rajab, G.J. Taborsky, Jr. and B. Ah&n, 19%. Gaianin of the homologous species inhibits insulin secretion in the rat and the pig, Acta Physioi. Stand. 139, 591. Lindskog, S., B. Ah&, B.E. Dunning and F. Sundler, 1991, Gaianin-immunoreactive nerves in the mouse and rat pancreas, Ceil Tissue Res. 264, 363. Maiaisse, W.J., A. Sener, A. Herchueiz, A.R. Carpineiii, P. Poioczek, J. Winand and M. Castagna, 1980, Insuiinotropic effect of the tumor promoter 12-ii-tetradecanoyiphorboi-13-acetate in rat pancreatic islets, Cancer Res. 40, 3827. Miraiies, P., E. Peiro, P. DCgano, R.A. Siivestre and J. Marco, 1990, Inhibition of insulin and somatostatin secretion and stimulation of glucagon release by homologous gaianin in perfused rat pancreas, Diabetes 39, 996. Niisson, T., P. Arkhammar, P. Rorsman and P.O. Berggren, 1989, Suppression of insulin release by galanin and somatostatin is mediated by a G-protein, J. Bioi. Chem. 264, 973. Rorsman, P., K. Bokvist, C. &nmlil, P. Arkhammar, P.O. Berggren, 0. Larsson and K. Wihiander, 1991, Activation by adrenaline of a low-conductance G protein-dependent K+ channel in mouse pancreatic B ceils, Nature 3, 77. Seamon, K.B., W. Padgett and J.W. Daly. 1981, Forskoiin: unique diterpene activator of adenyiate cyclase in membranes and intact ceils, Proc. Nati. Acad. Sci. U.S.A. 78, 3363. Sharp,G.W.G., Y. Le Marchand-Brustei, T. Yada, L.L. Russo, CR. Bliss, M. Cormont, L. Monges and E. Van Obberghen, 1989, Gaianin can inhibit insulin retease by a mechanism other titan
27 membrane hyperpolarization or inhibition of adenylate cyclase. J. Biol. Chem. 264, 7302. Trube G., P. Rorsman and T. Ohno-Shosaku, 1986, Opposite effects of tolbutamide and diazoxide on the ATP-dependent KC channel in mouse pancreatic p-cells, Pfliigers Arch. 407, 493.
Ullrich, S. and C.B. Wollheim, 1989, Galanin inhibits insulin secretion by direct interference with exocytosis, PEBS Lett. 247, 401. Wiedrnkeller, D.E. and G.W.G. Sharp, 1983, Effects of forskolin on insulin release and cyclic AMP content in rat pancreatic islets, Endocrinology 113, 2311.
