Br. J. Pharmacol. (1991), 102, 916-918
(D Macmillan Press Ltd, 1991
Effects of helospectin I on insulin and glucagon secretion in the mouse Bo Ahren Departments of Pharmacology and Surgery, Lund University, Lund, Sweden 1 The helospectins are peptides structurally related to helodermin, vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI) and secretin, which all potently stimulate glucagon secretion in the mouse. Therefore, the effects of helospectin I (0.1-0.8nmolkg-1) on insulin and glucagon secretion under basal conditions and after stimulation with glucose (2.8mmolkg-1) or the cholinoceptor agonist, carbachol (0.16 umol kg- 1), were examined in vivo in the mouse. 2 Helospectin I potently increased plasma levels of glucagon after its intravenous injection in mice. The increase was observed after only 2 min, and was evident also after 6 min. 3 In contrast, plasma insulin levels were not altered by helospectin I after 2 min, but slightly increased after 6 min. Plasma glucose levels were not altered by the peptide. 4 Carbachol-induced glucagon secretion was markedly potentiated by helospectin I. In contrast, glucose- or carbachol-stimulated insulin secretion was not affected by the peptide. 5 In conclusion, helospectin I markedly stimulates glucagon secretion in the mouse whereas the peptide has no direct action on insulin secretion. This pattern of effect of helospectin I is similar to that previously reported for helodermin, VIP, PHI and secretin in the mouse, i.e., for all peptides belonging to this superfamily of peptides. Keywords: Helospectin; glucagon; insulin secretion in vivo in mouse
Introduction Helospectin I and helospectin II are two helodermin-like peptides isolated from the venom of the Gila monster (Parker et al. 1984). They show structural relationship with vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI), secretin, and glucagon, i.e., to peptides belonging to the glucagon peptide superfamily (Parker et al., 1984; Robberecht et al., 1985; Table 1). Furthermore, the helospectins and helodermin have been demonstrated to occur also in mammalian tissue (Robberecht et al., 1985; Grunditz et al., 1989; Bjartell et al., 1989). The helospectins have previously been demonstrated to produce relaxation in rat isolated femoral arteries and to reduce blood pressure when injected intravenously in rats (Grundemar & Hogestatt, 1990). Since similar effects were induced by helodermin and VIP, it was proposed that helospectin I and helospectin II act on the same receptors as these peptides (Grundemar & Hogestatt, 1990). This raised the possibility that all the peptides belonging to the VIP/PHI/ secretin/glucagon peptide family have the same profile of effects. A marked stimulation of glucagon secretion when hel-
odermin, VIP, PHI or secretin was injected intravenously into mice was demonstrated previously, whereas insulin secretion was stimulated only weakly or not at all (Ahren & Lundquist 1982a; 1986; 1988; Ahren 1989). To discover whether the helospectins also potently stimulate glucagon secretion, the present study was carried out and the effects of helospectin I on basal and stimulated glucagon secretion in the mouse were studied. The cholinoceptor agonist carbachol was used as a glucagonotropic substance. The effects of helospectin I on glucose- and carbachol-stimulated insulin secretion were also studied.
Methods Animals Female mice of the NMRI strain (Anticimex, Stockholm, Sweden) weighing 25-30g, were used throughout the experiments. The animals were given a standard pellet diet (Astra-
Table 1 Amino acid sequence of peptides belonging to the helodermin/vasoactive intestinal polypeptide (VIP)/peptide histidine isoleucine (PHI)/secretin peptide superfamily 1
2
3
Helospectin II Helodermin VIP (rat) PHI (rat) Secretin (pig) Glucagon (human)
His His His His His His His
Ser Ser Ser Ser Ala Ser Ser
Asp Asp Asp Asp Asp Asp
20 Gln Gln Gln Lys Lys Gln Gln
21
22
Halospectin I Helospectin II Helodermin VIP (rat) PHI (rat) Secretin (pig) Glucagon (human)
Lys Lys Lys Lys Lys Arg Asp
Tyr Tyr Tyr Tyr Tyr Leu Phe
Helospectin I
Gln
4
5
Ala Thr Ala Thr Ala Ile Ala Val Gly Val Gly Thr Gly Thr 24 23 Leu Glu Leu Glu Leu Ala Leu Asn Leu Glu Leu Gln Val GIn
6
7
Phe Phe Phe Phe Phe Phe Phe 25 Ser Ser Ser Ser Ser
Thr Thr Thr Thr Thr Thr Thr
Gly Trp
8
Ala Ala Gln Asp Ser Ser Ser 27 26 Ile Leu Ile Leu Ile Leu Ile Leu Leu Ile Leu Val Leu Met
9
10
11
12
13
Glu Tyr Glu Tyr Glu Tyr Asn Tyr Asp Tyr Glu Leu Asp Tyr
Ser
Lys Lys
Leu Leu Leu Leu Leu Leu Tyr 32 Ser Ser Ser
28
29
Gly Ser Gly Ser Gly Ser Asn Asn Thr
Ser
Ser Thr Ser Ser Ser 30 Ser Ser
Lys Arg
Arg Arg Lys
31 Thr Thr Arg Thr
14
15
Leu Ala Leu Ala Leu Ala Arg Lys Leu Gly Arg Asp Leu Asp 33 34 Pro Arg Pro Arg Pro Pro
16
17
18
Lys
Leu
Ala
Lys
Leu Ala Leu Leu Ala Leu Met Ala Val Ile Ser Ala Ala Arg Leu Arg Arg Ala Leu
Lys Gln Gln Ser Ser 35 36 Pro Pro Pro Pro Pro
19
37 Ser Ser
38 Ser
HELOSPECTIN I AND ISLET HORMONE SECRETION a
Ewos, Sodertaije, Sweden) and tap water ad libitum before and during the experiments.
Experiments Unanaesthetized mice were injected intravenously in a tail vein with synthetic helospectin I (Peninsula Labs, Belmonte, Ca, U.S.A.; dissolved in saline + 0.1% gelatine) alone (0.10.8 nmol kg- ') or in combination with D-glucose (British Drug Houses Ltd., Poole, England; 2.8mmolkg-1) or the cholinoceptor agonist, carbachol (British Drug Houses Ltd., Poole, England; 0.164umolkg-1). The volume load was 10u1g`1. Controls were given saline/gelatine. Blood samples were taken from the retrobulbar plexus at 2 or 6 min after the intravenous injection. At 2 min, the maximal increases in plasma insulin levels after injection of glucose or carbachol and in plasma glucagon levels after intravenous injection of carbachol are seen (Ahren & Lundquist 1981; 1986).
en
917
2 min
E
b Ca 0
E
m
-
c
10-I Determinations Immediately after blood sampling, plasma was separated and stored at -20°C until analyses. Plasma levels of insulin and glucagon were determined radioimmunochemically (Herbert et al., 1965; Ahren & Lundquist, 1982b). For insulin radioimmunoassay, guinea-pig anti-porcine insulin antiserum (MILAB, Malmo, Sweden), '25I-labelled porcine insulin and, as standard, porcine insulin (Novo Res, Bagsvaerd, Denmark) were used. For glucagon radioimmunoassay, a rabbit antiporcine glucagon antiserUm specific for pancreatic glucagon and 125I-labelled porcine glucagon (MILAB, Malmo, Sweden) and, as standard, human glucagon were used. The insulin and glucagon antibodies do not cross-react with helospectin I. The separation of bound and free radioactivity was performed, in both assays, by the dextran-coated charcoal technique. In the insulin assay, a change of 0.3 puu/tube ( = 3 uu ml- ') is detected with 95% confidence, and 4 uu/tube (=40yuuml-1) displaces 50% specific tracer activity. In the glucagon assay, a change of 0.75 pg/tube (= 7.5 pg ml -1) is detected with 95% confidence, and 12.5pg/tube (=125pgml-1) displaces 50% of specific tracer activity. Finally, plasma glucose levels were determined with the glucose oxidase technique.
8-i
-
00
0.2 0.4 0.8 nmol kg-' Figure 1 Changes in plasma levels of insulin (a), glucagon (b) and plasma levels of glucose (c) at 2min after the intravenous injection of helospectin I at 0.1-0.8nmolkg-' in mice. Plasma levels in salineinjected controls were: insulin: 12 + 3 uuml -, glucagon 389 ± 86 pgml- 1, glucose 8.0 + 0.2 mmoll- '. There were 20 animals in each group. Means are shown with s.e.mean indicated by vertical bars. Asterisks indicate the probability level of random difference versus the controls: ** P < 0.01; *** P < 0.001. 0.1
a
C._cc E
-
20
-
10
-
A*
6 min
:
O-
-10-i
LrJ
b
Statistics Means + s.e.mean are given. Student's unpaired t test was used as a test of significance. When calculating the increase in plasma insulin and glucagon levels above basal, as demonstrated in the figures, the plasma insulin and glucagon levels in the saline-injected group were subtracted from those in the experimental group and the weighted s.e.mean was calculated, taking into account the s.e.mean of both groups. Therefore, saline-injected controls were included in all individual experiments.
30
4000-1 c
o_ CMI
H*
3000
-rl
-
OEE
F0 (3
2000 ***
c
a
1000 ld 0 C
14a)I In-
0-
12-
oE E
10{
00 =
Results
ElifIf
0
0.1
Basal levels ofglucagon, insulin, and glucose Basal plasma glucagon levels were significantly enhanced after the intravenous injection of helospectin I after 2min (Figure 1). In contrast, plasma insulin of glucose levels were not altered. At 6min after intravenous injection of helospectin I, plasma glucagon levels were markedly elevated, and, at this time, plasma insulin levels were also increased (Figure 2). In contrast, plasma glucose levels were, again, not altered.
0.2
0.4
0.8
nmol kg 1 Figure 2 Changes in plasma levels of insulin (a), glucagon (b) and plasma levels of glucose (c) at 6min after the intravenous injection of helospectin I at 0.1-0.8nmolkg-' in mice. Plasma levels in salineinjected controls were: insulin: 18 + 4uu ml ', glucagon 495 + 106pgml-1, glucose 10.2 + 0.3mmoll-1. There were 20 animals in each group. Means are shown with s.e.mean indicated by vertical bars. Asterisks indicate the probability level of random difference versus the saline-injected controls: * P < 0.05; ** P < 0.01; *** P < 0.001.
B. AHRIN
918
a
Glucose
Carbachol
80
(Figure 3b). Plasma glucose levels after injection of glucose or carbachol were not altered by helospectin I (Figure 3c).
60
-60
40 n-I*I*
Discussion
Glucose- and carbachol-stimulated levels of glucagon, insulin, and glucose
Helospectin I was found to stimulate markedly basal glucagon secretion and to potentiate carbachol-stimulated glucagon secretion in mice. The marked stimulation of glucagon secretion is also evident from its potent stimulatory action during hyperglycaemia. In contrast, its stimulatory effect on insulin secretion was weak and evident only at a late time point when it is likely to be mediated by glucagon and not directly by helospectin I. Helospectin I is thus similar to VIP, secretin, PHI and helodermin in markedly stimulating glucagon secretion but affecting insulin secretion only marginally (Ahren & Lundquist, 1981; 1986; 1988; Ahren, 1989). This suggests that the peptides affect the same mechanisms in the islet A-cells. A similar identity of mechanism for the helospectins I and II, helodermin and VIP was recently suggested for their vascular effects (Grundemar & HMgestatt, 1990). It is known that carbachol activates cholinoceptors which initiate phosphoinositide hydrolysis in the islets (Ahren et al., 1990). However, whether helospectin I and II, helodermin, and VIP augment this process is not known. Nonetheless, these peptides show a high degree of structural similarities (Table 1), which suggests that they activate the same receptors. Previously, both VIP and helodermin have been shown to activate exocrine pancreatic adenylate cyclase (Vandermeers et al., 1984). Whether a similar action is exerted in the islets is not known. In any case, the present results suggest that helospectin I initiates the same islet mechanisms as the other peptides belonging to the helodermin/VIP/PHI/secretin peptide family with marked stimulation of glucagon secretion as the predominant action.
Glucose and carbachol elevated plasma levels of insulin (P < 0.001). Helospectin I did not affect these increases (Figure 3a). Carbachol also increased plasma levels of glucagon (P < 0.001). Helospectin I, both at 0.2 and 0.8 nmol kg- ', markedly potentiated this increase. Helospectin I also increased plasma glucagon levels after injection of glucose
The author is grateful to Lena Kvist and Lilian Bengtsson for expert technical assistance. The study was supported by the Swedish Medical Research Council (Grants No 14x-6834 and 12x-712), Nordisk Insulinfond, Swedish Diabetes Association, Albert Pahlssons, Crafoordska and Magn Bergvalls Foundations, Sw Hoechst Diabetes Fund, and The Faculty of Medicine, Lund.
20
b
4000C
3000--
1200
-
0-1000
D6 0
100 30 2
-~E
1001
**|*
l
n
11
Figure 3 Changes in plasma levels of insulin (a), glucagon (b) and plasma levels of glucose (c) at 2min after the intravenous injection of glucose (2.8 mmol kg- 1) or the cholinoceptor agonist, carbachol (0.16 ymol kg- ) alone (control, open columns) or together with helospectin I, 0.2 (hatched columns) or 0.8nmol kg-1 (solid columns) in mice. Plasma levels in saline-injected controls were: insulin 9 + 2puml-', 355 + 46pg ml-', and glucose glucagon 8.1 + 0.2 mmol 11. Asterisks indicate the probability level of random difference versus the glucose- or carbachol-injected controls. *** P < 0.001.
References AHREN, B. (1989). Helodermin stimulates glucagon secretion in the mouse. Peptides, 19, 709-711. AHREN, B. & LUNDQUIST, 1. (1981). Effects of selective and nonselective fl-adrenergic agents on insulin secretion in vivo. Eur. J. Pharmacol., 71, 93-104. AHREN, B. & LUNDQUIST, I. (1982a). Interaction of vasoactive intestinal peptide (VIP) with cholinergic stimulation of glucagon secretion. Experientia, 38, 405-406. AHREN, B. & LUNDQUIST, 1. (1982b). Glucagon immunoreactivity in plasma from normal and dystrophic mice. Diabetologia, 22, 258263. AHREN, B. & LUNDQUIST, 1. (1986). Secretin potentiates cholinergically induced glucagon secretion in the mouse. Acta Physiol. Scand., 128, 575-578. AHREN, B. & LUNDQUIST, I. (1988). Effects of PHI on basal and stimulated insulin and glucagon secretion in the mouse. Neuropeptides, 11, 159-162. AHRtN, B., KARLSSON, S. & LINDSKOG, S. (1990). Cholinergic regulation of the endocrine pancreas. In Progress in Brain Research, ed. Aquilonius, S. & Gillberg, P.G. pp. 209-218. Amsterdam: Elsevier. BIARTELL, A., PERSSON, P., ABSOOD, A., SUNDLER, F. & HAKANSON, R. (1989). Localization and characterization of helodermin-like material in the mammalian adrenal gland. Regul. Pept., 26, 27-34. GRUNDEMAR, L. & HOGESTATT, E.D. (1990). Vascular effects of helo-
dermin, helospectin I and helospectin II: a comparison with vasoactive intestinal peptide (VIP). Br. J. Pharmacol., 99, 526-528. GRUNDITZ,
T.,
PERSSON,
P., HAKANSON,
R.,
ABSOOD,
A.,
BOTrCHER, G., RERUP, C. & SUNDLER, F. (1989). Helodermin-like peptides in thyroid C cells. Stimulation of thyroid hormone secretion and suppression of calcium incorporation into bone. Proc. Natl. Acad. Sci., U.S.A., 86, 1357-1361. HERBERT, V., LAU, K.S., GOTTLIEB, C.W. & BLEICHER, S.J. (1965). Coated charcoal immunoassay of insulin. J. Clin. Endocrinol. Metab., 25, 1375-1384. PARKER, D., RAUFMAN, J.-P., O'DONOHUE, T., BLEDSOE, M., YOSHIDA, H. & PISANO, J. (1984). Amino acid sequences of helo-
spectins, new members of the glucagon superfamily, found in Gila monster venom. J. Biol. Chem., 259, 11751-11755. ROBBERECHT, P., DE NEEF, P., VANDERMEERS, A., VANDERMEERSPIRET, M.C., SVOBODA, M., MEURIS, S., DE GRAEF, J., WOUSSENCOLLE, M.C., YANAIHARA, C., YANAIHARA, N. & CHRISTOPHE, J.
(1985). Presence of helodermin-like peptides of the VIP-secretin family in mammalian salivary glands and saliva. FEBS Lett., 190, 142-146. VANDERMEERS, P., VANDERMEERS, M.C., ROBBERECHT, P., VAEGELBROECK, M., DEHAYE, J.-P. & CHRISTOPHE, J. (1984). Purifi-
cation 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.
(Received July 11, 1990 Revised November 20, 1990 Accepted November 26, 1990)
(D Macmillan Press Ltd, 1991
Effects of helospectin I on insulin and glucagon secretion in the mouse Bo Ahren Departments of Pharmacology and Surgery, Lund University, Lund, Sweden 1 The helospectins are peptides structurally related to helodermin, vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI) and secretin, which all potently stimulate glucagon secretion in the mouse. Therefore, the effects of helospectin I (0.1-0.8nmolkg-1) on insulin and glucagon secretion under basal conditions and after stimulation with glucose (2.8mmolkg-1) or the cholinoceptor agonist, carbachol (0.16 umol kg- 1), were examined in vivo in the mouse. 2 Helospectin I potently increased plasma levels of glucagon after its intravenous injection in mice. The increase was observed after only 2 min, and was evident also after 6 min. 3 In contrast, plasma insulin levels were not altered by helospectin I after 2 min, but slightly increased after 6 min. Plasma glucose levels were not altered by the peptide. 4 Carbachol-induced glucagon secretion was markedly potentiated by helospectin I. In contrast, glucose- or carbachol-stimulated insulin secretion was not affected by the peptide. 5 In conclusion, helospectin I markedly stimulates glucagon secretion in the mouse whereas the peptide has no direct action on insulin secretion. This pattern of effect of helospectin I is similar to that previously reported for helodermin, VIP, PHI and secretin in the mouse, i.e., for all peptides belonging to this superfamily of peptides. Keywords: Helospectin; glucagon; insulin secretion in vivo in mouse
Introduction Helospectin I and helospectin II are two helodermin-like peptides isolated from the venom of the Gila monster (Parker et al. 1984). They show structural relationship with vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI), secretin, and glucagon, i.e., to peptides belonging to the glucagon peptide superfamily (Parker et al., 1984; Robberecht et al., 1985; Table 1). Furthermore, the helospectins and helodermin have been demonstrated to occur also in mammalian tissue (Robberecht et al., 1985; Grunditz et al., 1989; Bjartell et al., 1989). The helospectins have previously been demonstrated to produce relaxation in rat isolated femoral arteries and to reduce blood pressure when injected intravenously in rats (Grundemar & Hogestatt, 1990). Since similar effects were induced by helodermin and VIP, it was proposed that helospectin I and helospectin II act on the same receptors as these peptides (Grundemar & Hogestatt, 1990). This raised the possibility that all the peptides belonging to the VIP/PHI/ secretin/glucagon peptide family have the same profile of effects. A marked stimulation of glucagon secretion when hel-
odermin, VIP, PHI or secretin was injected intravenously into mice was demonstrated previously, whereas insulin secretion was stimulated only weakly or not at all (Ahren & Lundquist 1982a; 1986; 1988; Ahren 1989). To discover whether the helospectins also potently stimulate glucagon secretion, the present study was carried out and the effects of helospectin I on basal and stimulated glucagon secretion in the mouse were studied. The cholinoceptor agonist carbachol was used as a glucagonotropic substance. The effects of helospectin I on glucose- and carbachol-stimulated insulin secretion were also studied.
Methods Animals Female mice of the NMRI strain (Anticimex, Stockholm, Sweden) weighing 25-30g, were used throughout the experiments. The animals were given a standard pellet diet (Astra-
Table 1 Amino acid sequence of peptides belonging to the helodermin/vasoactive intestinal polypeptide (VIP)/peptide histidine isoleucine (PHI)/secretin peptide superfamily 1
2
3
Helospectin II Helodermin VIP (rat) PHI (rat) Secretin (pig) Glucagon (human)
His His His His His His His
Ser Ser Ser Ser Ala Ser Ser
Asp Asp Asp Asp Asp Asp
20 Gln Gln Gln Lys Lys Gln Gln
21
22
Halospectin I Helospectin II Helodermin VIP (rat) PHI (rat) Secretin (pig) Glucagon (human)
Lys Lys Lys Lys Lys Arg Asp
Tyr Tyr Tyr Tyr Tyr Leu Phe
Helospectin I
Gln
4
5
Ala Thr Ala Thr Ala Ile Ala Val Gly Val Gly Thr Gly Thr 24 23 Leu Glu Leu Glu Leu Ala Leu Asn Leu Glu Leu Gln Val GIn
6
7
Phe Phe Phe Phe Phe Phe Phe 25 Ser Ser Ser Ser Ser
Thr Thr Thr Thr Thr Thr Thr
Gly Trp
8
Ala Ala Gln Asp Ser Ser Ser 27 26 Ile Leu Ile Leu Ile Leu Ile Leu Leu Ile Leu Val Leu Met
9
10
11
12
13
Glu Tyr Glu Tyr Glu Tyr Asn Tyr Asp Tyr Glu Leu Asp Tyr
Ser
Lys Lys
Leu Leu Leu Leu Leu Leu Tyr 32 Ser Ser Ser
28
29
Gly Ser Gly Ser Gly Ser Asn Asn Thr
Ser
Ser Thr Ser Ser Ser 30 Ser Ser
Lys Arg
Arg Arg Lys
31 Thr Thr Arg Thr
14
15
Leu Ala Leu Ala Leu Ala Arg Lys Leu Gly Arg Asp Leu Asp 33 34 Pro Arg Pro Arg Pro Pro
16
17
18
Lys
Leu
Ala
Lys
Leu Ala Leu Leu Ala Leu Met Ala Val Ile Ser Ala Ala Arg Leu Arg Arg Ala Leu
Lys Gln Gln Ser Ser 35 36 Pro Pro Pro Pro Pro
19
37 Ser Ser
38 Ser
HELOSPECTIN I AND ISLET HORMONE SECRETION a
Ewos, Sodertaije, Sweden) and tap water ad libitum before and during the experiments.
Experiments Unanaesthetized mice were injected intravenously in a tail vein with synthetic helospectin I (Peninsula Labs, Belmonte, Ca, U.S.A.; dissolved in saline + 0.1% gelatine) alone (0.10.8 nmol kg- ') or in combination with D-glucose (British Drug Houses Ltd., Poole, England; 2.8mmolkg-1) or the cholinoceptor agonist, carbachol (British Drug Houses Ltd., Poole, England; 0.164umolkg-1). The volume load was 10u1g`1. Controls were given saline/gelatine. Blood samples were taken from the retrobulbar plexus at 2 or 6 min after the intravenous injection. At 2 min, the maximal increases in plasma insulin levels after injection of glucose or carbachol and in plasma glucagon levels after intravenous injection of carbachol are seen (Ahren & Lundquist 1981; 1986).
en
917
2 min
E
b Ca 0
E
m
-
c
10-I Determinations Immediately after blood sampling, plasma was separated and stored at -20°C until analyses. Plasma levels of insulin and glucagon were determined radioimmunochemically (Herbert et al., 1965; Ahren & Lundquist, 1982b). For insulin radioimmunoassay, guinea-pig anti-porcine insulin antiserum (MILAB, Malmo, Sweden), '25I-labelled porcine insulin and, as standard, porcine insulin (Novo Res, Bagsvaerd, Denmark) were used. For glucagon radioimmunoassay, a rabbit antiporcine glucagon antiserUm specific for pancreatic glucagon and 125I-labelled porcine glucagon (MILAB, Malmo, Sweden) and, as standard, human glucagon were used. The insulin and glucagon antibodies do not cross-react with helospectin I. The separation of bound and free radioactivity was performed, in both assays, by the dextran-coated charcoal technique. In the insulin assay, a change of 0.3 puu/tube ( = 3 uu ml- ') is detected with 95% confidence, and 4 uu/tube (=40yuuml-1) displaces 50% specific tracer activity. In the glucagon assay, a change of 0.75 pg/tube (= 7.5 pg ml -1) is detected with 95% confidence, and 12.5pg/tube (=125pgml-1) displaces 50% of specific tracer activity. Finally, plasma glucose levels were determined with the glucose oxidase technique.
8-i
-
00
0.2 0.4 0.8 nmol kg-' Figure 1 Changes in plasma levels of insulin (a), glucagon (b) and plasma levels of glucose (c) at 2min after the intravenous injection of helospectin I at 0.1-0.8nmolkg-' in mice. Plasma levels in salineinjected controls were: insulin: 12 + 3 uuml -, glucagon 389 ± 86 pgml- 1, glucose 8.0 + 0.2 mmoll- '. There were 20 animals in each group. Means are shown with s.e.mean indicated by vertical bars. Asterisks indicate the probability level of random difference versus the controls: ** P < 0.01; *** P < 0.001. 0.1
a
C._cc E
-
20
-
10
-
A*
6 min
:
O-
-10-i
LrJ
b
Statistics Means + s.e.mean are given. Student's unpaired t test was used as a test of significance. When calculating the increase in plasma insulin and glucagon levels above basal, as demonstrated in the figures, the plasma insulin and glucagon levels in the saline-injected group were subtracted from those in the experimental group and the weighted s.e.mean was calculated, taking into account the s.e.mean of both groups. Therefore, saline-injected controls were included in all individual experiments.
30
4000-1 c
o_ CMI
H*
3000
-rl
-
OEE
F0 (3
2000 ***
c
a
1000 ld 0 C
14a)I In-
0-
12-
oE E
10{
00 =
Results
ElifIf
0
0.1
Basal levels ofglucagon, insulin, and glucose Basal plasma glucagon levels were significantly enhanced after the intravenous injection of helospectin I after 2min (Figure 1). In contrast, plasma insulin of glucose levels were not altered. At 6min after intravenous injection of helospectin I, plasma glucagon levels were markedly elevated, and, at this time, plasma insulin levels were also increased (Figure 2). In contrast, plasma glucose levels were, again, not altered.
0.2
0.4
0.8
nmol kg 1 Figure 2 Changes in plasma levels of insulin (a), glucagon (b) and plasma levels of glucose (c) at 6min after the intravenous injection of helospectin I at 0.1-0.8nmolkg-' in mice. Plasma levels in salineinjected controls were: insulin: 18 + 4uu ml ', glucagon 495 + 106pgml-1, glucose 10.2 + 0.3mmoll-1. There were 20 animals in each group. Means are shown with s.e.mean indicated by vertical bars. Asterisks indicate the probability level of random difference versus the saline-injected controls: * P < 0.05; ** P < 0.01; *** P < 0.001.
B. AHRIN
918
a
Glucose
Carbachol
80
(Figure 3b). Plasma glucose levels after injection of glucose or carbachol were not altered by helospectin I (Figure 3c).
60
-60
40 n-I*I*
Discussion
Glucose- and carbachol-stimulated levels of glucagon, insulin, and glucose
Helospectin I was found to stimulate markedly basal glucagon secretion and to potentiate carbachol-stimulated glucagon secretion in mice. The marked stimulation of glucagon secretion is also evident from its potent stimulatory action during hyperglycaemia. In contrast, its stimulatory effect on insulin secretion was weak and evident only at a late time point when it is likely to be mediated by glucagon and not directly by helospectin I. Helospectin I is thus similar to VIP, secretin, PHI and helodermin in markedly stimulating glucagon secretion but affecting insulin secretion only marginally (Ahren & Lundquist, 1981; 1986; 1988; Ahren, 1989). This suggests that the peptides affect the same mechanisms in the islet A-cells. A similar identity of mechanism for the helospectins I and II, helodermin and VIP was recently suggested for their vascular effects (Grundemar & HMgestatt, 1990). It is known that carbachol activates cholinoceptors which initiate phosphoinositide hydrolysis in the islets (Ahren et al., 1990). However, whether helospectin I and II, helodermin, and VIP augment this process is not known. Nonetheless, these peptides show a high degree of structural similarities (Table 1), which suggests that they activate the same receptors. Previously, both VIP and helodermin have been shown to activate exocrine pancreatic adenylate cyclase (Vandermeers et al., 1984). Whether a similar action is exerted in the islets is not known. In any case, the present results suggest that helospectin I initiates the same islet mechanisms as the other peptides belonging to the helodermin/VIP/PHI/secretin peptide family with marked stimulation of glucagon secretion as the predominant action.
Glucose and carbachol elevated plasma levels of insulin (P < 0.001). Helospectin I did not affect these increases (Figure 3a). Carbachol also increased plasma levels of glucagon (P < 0.001). Helospectin I, both at 0.2 and 0.8 nmol kg- ', markedly potentiated this increase. Helospectin I also increased plasma glucagon levels after injection of glucose
The author is grateful to Lena Kvist and Lilian Bengtsson for expert technical assistance. The study was supported by the Swedish Medical Research Council (Grants No 14x-6834 and 12x-712), Nordisk Insulinfond, Swedish Diabetes Association, Albert Pahlssons, Crafoordska and Magn Bergvalls Foundations, Sw Hoechst Diabetes Fund, and The Faculty of Medicine, Lund.
20
b
4000C
3000--
1200
-
0-1000
D6 0
100 30 2
-~E
1001
**|*
l
n
11
Figure 3 Changes in plasma levels of insulin (a), glucagon (b) and plasma levels of glucose (c) at 2min after the intravenous injection of glucose (2.8 mmol kg- 1) or the cholinoceptor agonist, carbachol (0.16 ymol kg- ) alone (control, open columns) or together with helospectin I, 0.2 (hatched columns) or 0.8nmol kg-1 (solid columns) in mice. Plasma levels in saline-injected controls were: insulin 9 + 2puml-', 355 + 46pg ml-', and glucose glucagon 8.1 + 0.2 mmol 11. Asterisks indicate the probability level of random difference versus the glucose- or carbachol-injected controls. *** P < 0.001.
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(Received July 11, 1990 Revised November 20, 1990 Accepted November 26, 1990)
