- Email: [email protected]
Helodermin stimulates glucagon secretion in the mouse
Peptides, Vol. 10, pp. 709-711. ©Pergamon Press plc, 1989. Printed in the U.S.A.
0196-9781/89 $3.00 + .00
BRIEF COMMUNICATION
Helodermin Stimulates Glucagon Secretion in the Mouse B O AHRI~N 1
Departments of Pharmacology, and Surgery, Lund University, Lund, Sweden R e c e i v e d 17 O c t o b e r 1988
AHRt~N, B. Heloderminstimulates glucagon secretion in the mouse. PEPTIDES 111(3) 709-711, 1989.--Helodermin is structurally similar to VIP (vasoactive intestinal peptide) and PHI (peptide histidine isoleucine). Since VIP and PHI both stimulate insulin and glucagon secretion, we investigated the effects of helodermin on insulin and glucagon secretion in the mouse, both in the basal state and during administration of glucose and the cholinergic agonist carbachol. After intravenous injection at dose levels between 0.5 and 8.0 nmol/kg, helodermin markedly enhanced basal plasma glucagon levels, for example at 8 nmol/kg from 139 --- 14 to 421 -+86 pg/ml (p<0.001) after 6 minutes, without affecting basal plasma insulin levels. Together with glucose (2.8 mmol/kg), helodermin (2 and 8 nmol/kg) augmented plasma glucagon levels but had no effect on plasma insulin levels. When injected together with the cholinergic agonist carbachol (0.16 p.mol/kg), helodermin markedly potentiated the increase in plasma glucagon levels (more than three-fold; p<0.001), again without affecting the plasma insulin levels. Combined ~- and 13-adrenoceptorblockade (yohimbine + L-propranolol) reduced the augmenting effect of helodermin on glucagon secretion by approximately 60%. It is concluded helodermin stimulates glucagon secretion in the mouse by an effect that is partially antagonized by combined ix- and fl-adrenoceptor antagonism. Helodermin
Glucagon secretion
Insulin secretion
Glucose
HELODERMIN is a peptide consisting of a 35 amino acid residue initially isolated from the venom of the Gila monster lizard (15). Immunocytochemistry has demonstrated helodermin-like immunoreactivity in salivary glands of several species, including man, and in the brain and gut in the rat and in the human thyroid (12,14). Helodermin is structurally related to VIP (vasoactive intestinal peptide) and PHI (peptide histidine isoleucine) (12). These two latter peptides have previously been demonstrated to stimulate the secretion of insulin and glucagon in several species, including the mouse (2, 5, 8). Therefore, we investigated the possible influences of helodermin on insulin and glucagon secretion in the mouse, both under basal conditions and during administration of glucose and the cholinergic agonist carbachol.
Carbachol
In vivo
Mouse
injected intravenously in a tail vein with synthetic helodermin (Peninsula Ltd., Belmont, CA) at various dose levels ranging from 0.5 to 8.0 nmol/kg. Controls were injected with saline; the volume load was 10 Ixl/g. Blood samples were drawn from the retroorbital plexus at 2, 6, or 10 min after the injection. In the second series of experiments, the mice were injected with helodermin at 2.0 or 8.0 nmol/kg alone or together with each of the secretagogues D-glucose (2.8 mmol/kg) or the cholinergic agonist carbachol (0.16 ixmol/kg). Blood samples were taken 2 min after the intravenous injections, which is the time point when the increase in plasma levels of insulin or glucagon after the injection of glucose or carbachol in mice is maximal (3,7). In the third experimental series, yohimbine hydrochloride (2.6 Ixmol/kg; Serva, Heidelberg, Fed. Rep. Germany) and L-propranolol hydrochloride (9.6 txmol/kg; ICI Ltd., Macclesfield, U.K.) were injected intraperitoneally 10 min prior to intravenous injection of helodermin (8.0 nmol/kg) and/or carbachol (0.16 Ixmol/kg), and blood samples were taken 2 min later. Dose levels of glucose, carbachol, yohimbine and L-propranolol were selected from previous studies (3, 4, 13). Controls were injected with each of the secretagogues alone, or with saline. Glucose and carbachol were from British Drug Houses Ltd., Poole, England. Helodermin was dissolved in saline with the addition of 0.1% gelatine to avoid adsorption to tubes.
METHOD
Animals Female mice of the NMRI strain (Anticimex, Stockholm, Sweden), weighing 25-30 g, were used. The animals were fed a standard pellet diet (Astra-Ewos, Stidert~lje, Sweden) and tap water ad lib.
Experiments In the first experimental series, unanaesthetized animals were
~Requests for reprints should be addressed to Dr. Bo Ahr6n, Department of Pharmacology, SiSlvegatan 10, S-223 62 Lund, Sweden.
709
710
AHRI~N
6O
GLUCOSE
BASAL
6oj
BASAL
CARBACHOL-
E ~" ::)
40
4o e-
t'-
•~
20-
20
t"
C
0
5001
..
."
.EE 2500.
t
t
•
T
Q.. ¢-
¢0
0
250
o'~ 1250-
0
o
~r
t_)
_20]
-6 E
12
o
8
--
0
E
16
///
E
_J_
FIG. 1. Plasma levels of insulin (upper panel), glucagon (middle panel) and glucose (lower panel) 2 min after the intravenous injection of glucose (2.8 mmol/kg) alone or together with helodermin (2 or 8 nmol/kg). Controls were given saline. There were 20 animals in each group. Means-+SEM are shown. Asterisks indicate the probability levels of random difference versus controls. ***p<0.001. Open columns: control, striped columns: helodermin 2 nmol/kg, solid columns: helodermin 8 nmol/kg.
Determinations of Insulin, Glucagon, and Glucose Plasma levels of insulin and glucagon were determined with radioimmunoassay (6,10). The antiserum used for the determination of plasma glucagon was specific for pancreatic glucagon (Milab AB, Maim6, Sweden). Plasma glucose was determined by the glucose oxidase method.
FIG. 2. Plasma levels of insulin (upper panel), glucagon (middle panel) and glucose (lower panel) 2 miu after the intravenous injection of carbachol (0.16 ~.mol/kg) alone or together with helodermin (2 or 8 nmol/kg). Controls were given saline. There were 20 animals in each group. Means---SEM are shown. Asterisks indicate the probability levels of random difference versus controls. ***p<0.001. Open columns: control, striped columns: helodermin 2 nmol/kg, solid columns: helodermin 8 nmol/kg.
For example, after injection of helodermin at 8.0 nmol/kg, plasma glucagon levels at 2 min were 372 ± 49 pg/ml [controls 156 ± 31 pg/ml; F(20,20)=4.15, p<0.001], at 6 min 4 2 1 ± 8 6 pg/ml [controls 139± 14 pg/ml; F(20,20)=4.32, p<0.001], and at 10 min 296 ± 64 pg/ml [controls 140 ± 18 pg/ml; F(20,20) = 3.02, p<0.01]. Plasma glucose levels were not altered during the first 2 min after injection of helodermin, but at 6 min [12.1 ± 0 . 2 mmol/1 vs. in controls 10.1 ±0.1 mmol/1; F(20,20)=4.29, p<0.001] and at 10 min after injection [12.1±0.3 mmol/1 vs. in controls 10.9±0.2 mmol/1; F(20,20)=4.52, p<0.001] they were elevated.
Effects of Helodermin on Plasma Levels of Insulin, Glucagon and Glucose After Injection of Glucose (Fig. 1)
Statistics The results are expressed as means ± SEM. When increases above basal were determined, the weighted SEM was calculated taking into account SEM of both groups. Two-way analysis of variance (ANOVA) and multiple comparisons test were used to test the degree of significance. RESULTS
Effects of Helodermin on Basal Levels of Insulin, Glucagon, and Glucose Helodermin did not affect the basal plasma levels of insulin in samples taken at 2, 6, or 10 min after its intravenous injection at dose levels ranging from 0.5 to 8.0 nmol/kg. In contrast, basal plasma glucagon levels were markedly elevated by helodermin.
Glucose enhanced plasma insulin levels and this increase was not affected by helodermin at 2 or 8 nmol/kg. Glucose slightly lowered basal plasma glucagon levels [from 162---28 pg/ml to 107 - 22 pg/ml; F(20,20) = 2.98, p<0.01]. Helodermin elevated basal plasma glucagon levels in spite of the concomitant injection of glucose. The glucose-induced increase in plasma glucose levels was not affected by helodermin during the 2 min period studied.
Effects of Helodermin on Plasma Levels of Insulin, Glucagon, and Glucose After Injection of Carbachol (Fig. 2) Carbachol enhanced the plasma levels of both insulin and glucose. Helodermin did not affect the carbachol-induced increase in plasma insulin levels. Carbachol elevated plasma glucagon
HELODERMIN STIMULATES GLUCAGON SECRETION
levels to 712 - 45 pg/ml, F(20,20) = 7.38, p<0.001, and helodermin at 8 nmol/kg potentiated this increase to 2376 ± 204 pg/ml, F(20,20)= 11.39, p<0.001. Helodermin did not affect plasma glucose levels after injection of carbachol during the 2 min period studied. Pretreatment with yohimbine + L-propranolol reduced the potentiation by helodermin of carbachol-induced glucagon secretion. Thus, without yohimbine + propranolol, plasma glucagon levels were enhanced by carbachol from 233 --- 18 pg/ml to 6 2 8 ± 5 9 pg/ml, by helodermin (8.0 nmol/kg) to 1063±119 pg/ml, and by carbachol + hetodermin to 2528 ± 217 pg/ml. After injection of yohimbine + L-propranolol, basal plasma glucagon levels were lowered to 145 --- 34 pg/ml, F(10,10) = 3.83, p < 0 . 0 5 , and these values were increased by carbachol to 678 ± 162 pg/ml, by helodermin to 396 ± 51 pg/ml, and by carbachol + helodermin to 1 0 8 2 - 2 1 2 pg/ml (n = 10 in each group). Hence, the elevation above basal by carbachol + helodermin was reduced by yohimbine + propranolol from 2295 ± 68 pg/ml to 937 ± 67 pg/ml, i.e., by 60%, F(10,10)=6.49, p<0.001. DISCUSSION Helodermin has earlier been shown to stimulate amylase secretion from rat pancreatic acini (9), prolactin secretion in the rat (11), and thyroid hormone secretion in the mouse (1). We present here evidence for a stimulatory action of helodermin on glucagon secretion in the mouse. The potentiating effect by helodermin on carbachol-induced glucagon secretion was very impressive: it enhanced the response to carbachol more than three-fold. The potency of helodermin to stimulate glucagon secretion is illustrated also by its capability to increase plasma glucagon levels despite a concomitant glucose injection. In contrast, plasma
711
insulin levels were not altered by helodermin. Basal plasma glucose levels were elevated by helodermin, probably as a consequence of the increased plasma glucagon levels. Combined o~- and 13-adrenoceptor blockade reduced the helodermin-induced potentiation of glucagon secretion. This implies either that intact adrenoceptors within the islets are prerequisites for the action of helodermin, or that the stimulatory effect on glucagon secretion is indirectly mediated by enhanced sympathetic activity. An indirect action could be expected if helodermin, like VIP, reduces blood pressure by a vasodilatory action. On the other hand, if the action of helodermin were mediated solely by hypotension-induced sympathetic discharge, inhibited glucose-stimulated insulin secretion would be expected, and this was not observed. Previously, VIP has been shown in the mouse to markedly potentiate carbachol-induced glucagon secretion but to exert only a very mild insulinotropic action (2,5). Also PHI has, like helodermin, been shown to potentiate carbachol-induced glucagon secretion in the mouse and to be without effect on carbacholinduced insulin secretion (8). Hence, at least in the mouse, this family of peptides (hetodermin, VIP and PHI) seems to potentiate carbachol-induced glucagon secretion preferentially to carbacholinduced insulin secretion. In other species, the effects of helodermin on the endocrine pancreas have not yet been studied. ACKNOWLEDGEMENTS The technical assistance of Lena Kvist is gratefully acknowledged. This study was supported by the Swedish Medical Research Council (Grants No. 14X-6834 and 17P-8453), by Nordisk Insulinfond, by Svenska Diabetesftirbundet, by Diabetesft~reningen i Maim6, by Svenska Hoechst AB, by Albert P~lssons and Magnus Bergvalls Stiftelser, and by the Medical Faculty, Lund University.
REFERENCES 1. Ahr6n, B.; Hedner, P. Effects of VIP and helodermin on thyroid hormone secretion in the mouse. Neuropeptides 13:59-64; 1989. 2. Ahr6n, B.; Lundquist, I. Effects of vasoactive intestinal polypeptide (VIP), secretin and gastrin on insulin secretion in the mouse. Diabetologia 20:54-59; 1981. 3. Ahr6n, B.; Lundquist, I. Effects of selective and non-selective I~-adrenergic agents on insulin secretion in vivo. Eur. J. Pharmacol. 71:93-104; 1981. 4. Ahr6n, B.; Lundquist, I. Effects of autonomic blockade by methylatropine and optical isomers of propranolol on plasma insulin levels in the basal state and after stimulation. Acta Physiol. Scand. 112:57-63; 1981. 5. Ahr6n, B.; Lundquist, I. Interaction of vasoactive intestinal peptide (VIP) with cholinergic stimulation of glucagon secretion. Experientia 38:405-406; 1982. 6. Ahr6n, B.; Lundquist, I. Glucagon immunoreactivity in plasma from normal and dystrophic mice. Diabetologia 22:258-263; 1982. 7. Ahr6n, B.; Lundquist, I. Secretin potentiates cholinergically induced glucagon secretion in the mouse. Acta Physiol. Scand. 128:575-578; 1986. 8. Ahr6n, B.; Lundquist, I. Effects of peptide HI on basal and stimulated insulin and glucagon secretion in the mouse. Neuropeptides 11: 159-162; 1988. 9. Dehaye, J. P.; Winand, J.; Damien, C.; Gomez, F.; Poloczek, P.; Robberecht, P.; Vandermeers, A.; Vandermeers-Piret, M.C.; Stieve-
10. 11. 12.
13. 14.
15.
nart, M.; Christophe, J. Receptors involved in helodermin action on rat pancreatic acini. Am. J. Physiol. 251:G602--G610; 1986. Heding, L. A simplified insulin radioimmunoassay method. In: Donato, L.; Milhaud, G.; Sirchis, J., eds. Labelled proteins in tracer studies. Brussels: Euratom; 1966:345-350. Koshiyama, H.; Kato, Y.; Inoue, T.; Christophe, J.; Yanaihara, N.; Imura, H. Helodermin stimulates prolactin secretion in the rat. Eur. J. Pharmacol. 141:319-321; 1987. Robberecht, P.; De Neef, P.; Vandermeers, A.; Vandermeers-Piret, M. C.; Sveboda, M.; Meuris, S.; De Graef, J.; Woussen-Colle, M. C.; Yanaihara, C.; Yanaihara, N.; Christophe, J. Presence ofheloderminlike peptides of the VIP-secretin family in mammalian salivary glands and saliva. FEBS. Lett. 190:142-146; 1985. Skoglund, G.; Lundquist, I.; Ahr6n, B. Effects of oq- and ct2adrenoceptor stimulation and blockade on plasma insulin levels in the mouse. Pancreas 1:415-420; 1986. Sundler, F.; Christophe, J.; Robberecht, P.; Yanaihara, N.; Yanaihara, C.; Grunditz, T.; H~tkanson, R. Is helodermin produced by medullary thyroid carcinoma cells and normal C-cells? Immunocytochemical evidence. Regul. Pept. 20:83-89; 1988. Vandermeers, A.; Vandermeers-Piret, M. C.; Robberecht, P.; Wael° broeck, M.; Dehaye, J. P.; Winand, J.; Christophe, J. Purification of a novel pancreatic secretory factor (PSF) and a novel peptide with VIP- and secretin-like properties (helodermin) from Gila monster venom. FEBS Lett. 166:273-276; 1984.
0196-9781/89 $3.00 + .00
BRIEF COMMUNICATION
Helodermin Stimulates Glucagon Secretion in the Mouse B O AHRI~N 1
Departments of Pharmacology, and Surgery, Lund University, Lund, Sweden R e c e i v e d 17 O c t o b e r 1988
AHRt~N, B. Heloderminstimulates glucagon secretion in the mouse. PEPTIDES 111(3) 709-711, 1989.--Helodermin is structurally similar to VIP (vasoactive intestinal peptide) and PHI (peptide histidine isoleucine). Since VIP and PHI both stimulate insulin and glucagon secretion, we investigated the effects of helodermin on insulin and glucagon secretion in the mouse, both in the basal state and during administration of glucose and the cholinergic agonist carbachol. After intravenous injection at dose levels between 0.5 and 8.0 nmol/kg, helodermin markedly enhanced basal plasma glucagon levels, for example at 8 nmol/kg from 139 --- 14 to 421 -+86 pg/ml (p<0.001) after 6 minutes, without affecting basal plasma insulin levels. Together with glucose (2.8 mmol/kg), helodermin (2 and 8 nmol/kg) augmented plasma glucagon levels but had no effect on plasma insulin levels. When injected together with the cholinergic agonist carbachol (0.16 p.mol/kg), helodermin markedly potentiated the increase in plasma glucagon levels (more than three-fold; p<0.001), again without affecting the plasma insulin levels. Combined ~- and 13-adrenoceptorblockade (yohimbine + L-propranolol) reduced the augmenting effect of helodermin on glucagon secretion by approximately 60%. It is concluded helodermin stimulates glucagon secretion in the mouse by an effect that is partially antagonized by combined ix- and fl-adrenoceptor antagonism. Helodermin
Glucagon secretion
Insulin secretion
Glucose
HELODERMIN is a peptide consisting of a 35 amino acid residue initially isolated from the venom of the Gila monster lizard (15). Immunocytochemistry has demonstrated helodermin-like immunoreactivity in salivary glands of several species, including man, and in the brain and gut in the rat and in the human thyroid (12,14). Helodermin is structurally related to VIP (vasoactive intestinal peptide) and PHI (peptide histidine isoleucine) (12). These two latter peptides have previously been demonstrated to stimulate the secretion of insulin and glucagon in several species, including the mouse (2, 5, 8). Therefore, we investigated the possible influences of helodermin on insulin and glucagon secretion in the mouse, both under basal conditions and during administration of glucose and the cholinergic agonist carbachol.
Carbachol
In vivo
Mouse
injected intravenously in a tail vein with synthetic helodermin (Peninsula Ltd., Belmont, CA) at various dose levels ranging from 0.5 to 8.0 nmol/kg. Controls were injected with saline; the volume load was 10 Ixl/g. Blood samples were drawn from the retroorbital plexus at 2, 6, or 10 min after the injection. In the second series of experiments, the mice were injected with helodermin at 2.0 or 8.0 nmol/kg alone or together with each of the secretagogues D-glucose (2.8 mmol/kg) or the cholinergic agonist carbachol (0.16 ixmol/kg). Blood samples were taken 2 min after the intravenous injections, which is the time point when the increase in plasma levels of insulin or glucagon after the injection of glucose or carbachol in mice is maximal (3,7). In the third experimental series, yohimbine hydrochloride (2.6 Ixmol/kg; Serva, Heidelberg, Fed. Rep. Germany) and L-propranolol hydrochloride (9.6 txmol/kg; ICI Ltd., Macclesfield, U.K.) were injected intraperitoneally 10 min prior to intravenous injection of helodermin (8.0 nmol/kg) and/or carbachol (0.16 Ixmol/kg), and blood samples were taken 2 min later. Dose levels of glucose, carbachol, yohimbine and L-propranolol were selected from previous studies (3, 4, 13). Controls were injected with each of the secretagogues alone, or with saline. Glucose and carbachol were from British Drug Houses Ltd., Poole, England. Helodermin was dissolved in saline with the addition of 0.1% gelatine to avoid adsorption to tubes.
METHOD
Animals Female mice of the NMRI strain (Anticimex, Stockholm, Sweden), weighing 25-30 g, were used. The animals were fed a standard pellet diet (Astra-Ewos, Stidert~lje, Sweden) and tap water ad lib.
Experiments In the first experimental series, unanaesthetized animals were
~Requests for reprints should be addressed to Dr. Bo Ahr6n, Department of Pharmacology, SiSlvegatan 10, S-223 62 Lund, Sweden.
709
710
AHRI~N
6O
GLUCOSE
BASAL
6oj
BASAL
CARBACHOL-
E ~" ::)
40
4o e-
t'-
•~
20-
20
t"
C
0
5001
..
."
.EE 2500.
t
t
•
T
Q.. ¢-
¢0
0
250
o'~ 1250-
0
o
~r
t_)
_20]
-6 E
12
o
8
--
0
E
16
///
E
_J_
FIG. 1. Plasma levels of insulin (upper panel), glucagon (middle panel) and glucose (lower panel) 2 min after the intravenous injection of glucose (2.8 mmol/kg) alone or together with helodermin (2 or 8 nmol/kg). Controls were given saline. There were 20 animals in each group. Means-+SEM are shown. Asterisks indicate the probability levels of random difference versus controls. ***p<0.001. Open columns: control, striped columns: helodermin 2 nmol/kg, solid columns: helodermin 8 nmol/kg.
Determinations of Insulin, Glucagon, and Glucose Plasma levels of insulin and glucagon were determined with radioimmunoassay (6,10). The antiserum used for the determination of plasma glucagon was specific for pancreatic glucagon (Milab AB, Maim6, Sweden). Plasma glucose was determined by the glucose oxidase method.
FIG. 2. Plasma levels of insulin (upper panel), glucagon (middle panel) and glucose (lower panel) 2 miu after the intravenous injection of carbachol (0.16 ~.mol/kg) alone or together with helodermin (2 or 8 nmol/kg). Controls were given saline. There were 20 animals in each group. Means---SEM are shown. Asterisks indicate the probability levels of random difference versus controls. ***p<0.001. Open columns: control, striped columns: helodermin 2 nmol/kg, solid columns: helodermin 8 nmol/kg.
For example, after injection of helodermin at 8.0 nmol/kg, plasma glucagon levels at 2 min were 372 ± 49 pg/ml [controls 156 ± 31 pg/ml; F(20,20)=4.15, p<0.001], at 6 min 4 2 1 ± 8 6 pg/ml [controls 139± 14 pg/ml; F(20,20)=4.32, p<0.001], and at 10 min 296 ± 64 pg/ml [controls 140 ± 18 pg/ml; F(20,20) = 3.02, p<0.01]. Plasma glucose levels were not altered during the first 2 min after injection of helodermin, but at 6 min [12.1 ± 0 . 2 mmol/1 vs. in controls 10.1 ±0.1 mmol/1; F(20,20)=4.29, p<0.001] and at 10 min after injection [12.1±0.3 mmol/1 vs. in controls 10.9±0.2 mmol/1; F(20,20)=4.52, p<0.001] they were elevated.
Effects of Helodermin on Plasma Levels of Insulin, Glucagon and Glucose After Injection of Glucose (Fig. 1)
Statistics The results are expressed as means ± SEM. When increases above basal were determined, the weighted SEM was calculated taking into account SEM of both groups. Two-way analysis of variance (ANOVA) and multiple comparisons test were used to test the degree of significance. RESULTS
Effects of Helodermin on Basal Levels of Insulin, Glucagon, and Glucose Helodermin did not affect the basal plasma levels of insulin in samples taken at 2, 6, or 10 min after its intravenous injection at dose levels ranging from 0.5 to 8.0 nmol/kg. In contrast, basal plasma glucagon levels were markedly elevated by helodermin.
Glucose enhanced plasma insulin levels and this increase was not affected by helodermin at 2 or 8 nmol/kg. Glucose slightly lowered basal plasma glucagon levels [from 162---28 pg/ml to 107 - 22 pg/ml; F(20,20) = 2.98, p<0.01]. Helodermin elevated basal plasma glucagon levels in spite of the concomitant injection of glucose. The glucose-induced increase in plasma glucose levels was not affected by helodermin during the 2 min period studied.
Effects of Helodermin on Plasma Levels of Insulin, Glucagon, and Glucose After Injection of Carbachol (Fig. 2) Carbachol enhanced the plasma levels of both insulin and glucose. Helodermin did not affect the carbachol-induced increase in plasma insulin levels. Carbachol elevated plasma glucagon
HELODERMIN STIMULATES GLUCAGON SECRETION
levels to 712 - 45 pg/ml, F(20,20) = 7.38, p<0.001, and helodermin at 8 nmol/kg potentiated this increase to 2376 ± 204 pg/ml, F(20,20)= 11.39, p<0.001. Helodermin did not affect plasma glucose levels after injection of carbachol during the 2 min period studied. Pretreatment with yohimbine + L-propranolol reduced the potentiation by helodermin of carbachol-induced glucagon secretion. Thus, without yohimbine + propranolol, plasma glucagon levels were enhanced by carbachol from 233 --- 18 pg/ml to 6 2 8 ± 5 9 pg/ml, by helodermin (8.0 nmol/kg) to 1063±119 pg/ml, and by carbachol + hetodermin to 2528 ± 217 pg/ml. After injection of yohimbine + L-propranolol, basal plasma glucagon levels were lowered to 145 --- 34 pg/ml, F(10,10) = 3.83, p < 0 . 0 5 , and these values were increased by carbachol to 678 ± 162 pg/ml, by helodermin to 396 ± 51 pg/ml, and by carbachol + helodermin to 1 0 8 2 - 2 1 2 pg/ml (n = 10 in each group). Hence, the elevation above basal by carbachol + helodermin was reduced by yohimbine + propranolol from 2295 ± 68 pg/ml to 937 ± 67 pg/ml, i.e., by 60%, F(10,10)=6.49, p<0.001. DISCUSSION Helodermin has earlier been shown to stimulate amylase secretion from rat pancreatic acini (9), prolactin secretion in the rat (11), and thyroid hormone secretion in the mouse (1). We present here evidence for a stimulatory action of helodermin on glucagon secretion in the mouse. The potentiating effect by helodermin on carbachol-induced glucagon secretion was very impressive: it enhanced the response to carbachol more than three-fold. The potency of helodermin to stimulate glucagon secretion is illustrated also by its capability to increase plasma glucagon levels despite a concomitant glucose injection. In contrast, plasma
711
insulin levels were not altered by helodermin. Basal plasma glucose levels were elevated by helodermin, probably as a consequence of the increased plasma glucagon levels. Combined o~- and 13-adrenoceptor blockade reduced the helodermin-induced potentiation of glucagon secretion. This implies either that intact adrenoceptors within the islets are prerequisites for the action of helodermin, or that the stimulatory effect on glucagon secretion is indirectly mediated by enhanced sympathetic activity. An indirect action could be expected if helodermin, like VIP, reduces blood pressure by a vasodilatory action. On the other hand, if the action of helodermin were mediated solely by hypotension-induced sympathetic discharge, inhibited glucose-stimulated insulin secretion would be expected, and this was not observed. Previously, VIP has been shown in the mouse to markedly potentiate carbachol-induced glucagon secretion but to exert only a very mild insulinotropic action (2,5). Also PHI has, like helodermin, been shown to potentiate carbachol-induced glucagon secretion in the mouse and to be without effect on carbacholinduced insulin secretion (8). Hence, at least in the mouse, this family of peptides (hetodermin, VIP and PHI) seems to potentiate carbachol-induced glucagon secretion preferentially to carbacholinduced insulin secretion. In other species, the effects of helodermin on the endocrine pancreas have not yet been studied. ACKNOWLEDGEMENTS The technical assistance of Lena Kvist is gratefully acknowledged. This study was supported by the Swedish Medical Research Council (Grants No. 14X-6834 and 17P-8453), by Nordisk Insulinfond, by Svenska Diabetesftirbundet, by Diabetesft~reningen i Maim6, by Svenska Hoechst AB, by Albert P~lssons and Magnus Bergvalls Stiftelser, and by the Medical Faculty, Lund University.
REFERENCES 1. Ahr6n, B.; Hedner, P. Effects of VIP and helodermin on thyroid hormone secretion in the mouse. Neuropeptides 13:59-64; 1989. 2. Ahr6n, B.; Lundquist, I. Effects of vasoactive intestinal polypeptide (VIP), secretin and gastrin on insulin secretion in the mouse. Diabetologia 20:54-59; 1981. 3. Ahr6n, B.; Lundquist, I. Effects of selective and non-selective I~-adrenergic agents on insulin secretion in vivo. Eur. J. Pharmacol. 71:93-104; 1981. 4. Ahr6n, B.; Lundquist, I. Effects of autonomic blockade by methylatropine and optical isomers of propranolol on plasma insulin levels in the basal state and after stimulation. Acta Physiol. Scand. 112:57-63; 1981. 5. Ahr6n, B.; Lundquist, I. Interaction of vasoactive intestinal peptide (VIP) with cholinergic stimulation of glucagon secretion. Experientia 38:405-406; 1982. 6. Ahr6n, B.; Lundquist, I. Glucagon immunoreactivity in plasma from normal and dystrophic mice. Diabetologia 22:258-263; 1982. 7. Ahr6n, B.; Lundquist, I. Secretin potentiates cholinergically induced glucagon secretion in the mouse. Acta Physiol. Scand. 128:575-578; 1986. 8. Ahr6n, B.; Lundquist, I. Effects of peptide HI on basal and stimulated insulin and glucagon secretion in the mouse. Neuropeptides 11: 159-162; 1988. 9. Dehaye, J. P.; Winand, J.; Damien, C.; Gomez, F.; Poloczek, P.; Robberecht, P.; Vandermeers, A.; Vandermeers-Piret, M.C.; Stieve-
10. 11. 12.
13. 14.
15.
nart, M.; Christophe, J. Receptors involved in helodermin action on rat pancreatic acini. Am. J. Physiol. 251:G602--G610; 1986. Heding, L. A simplified insulin radioimmunoassay method. In: Donato, L.; Milhaud, G.; Sirchis, J., eds. Labelled proteins in tracer studies. Brussels: Euratom; 1966:345-350. Koshiyama, H.; Kato, Y.; Inoue, T.; Christophe, J.; Yanaihara, N.; Imura, H. Helodermin stimulates prolactin secretion in the rat. Eur. J. Pharmacol. 141:319-321; 1987. Robberecht, P.; De Neef, P.; Vandermeers, A.; Vandermeers-Piret, M. C.; Sveboda, M.; Meuris, S.; De Graef, J.; Woussen-Colle, M. C.; Yanaihara, C.; Yanaihara, N.; Christophe, J. Presence ofheloderminlike peptides of the VIP-secretin family in mammalian salivary glands and saliva. FEBS. Lett. 190:142-146; 1985. Skoglund, G.; Lundquist, I.; Ahr6n, B. Effects of oq- and ct2adrenoceptor stimulation and blockade on plasma insulin levels in the mouse. Pancreas 1:415-420; 1986. Sundler, F.; Christophe, J.; Robberecht, P.; Yanaihara, N.; Yanaihara, C.; Grunditz, T.; H~tkanson, R. Is helodermin produced by medullary thyroid carcinoma cells and normal C-cells? Immunocytochemical evidence. Regul. Pept. 20:83-89; 1988. Vandermeers, A.; Vandermeers-Piret, M. C.; Robberecht, P.; Wael° broeck, M.; Dehaye, J. P.; Winand, J.; Christophe, J. Purification of a novel pancreatic secretory factor (PSF) and a novel peptide with VIP- and secretin-like properties (helodermin) from Gila monster venom. FEBS Lett. 166:273-276; 1984.