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Adreneric control of insulin release from isolated islet tissue in the rainbow trout, Salmo gairdneri R
GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
59, 460-467 (1985)
Adrenergic Control of insulin Release from Isolated Islet Tissue in the Rainbow Trout, Salmo gairdneri R. JOHN E TILZEY,~ VERINA WAIGHTS, AND RICHARD HOLMES Department
of Biology,
The Open
University,
Milton
Keynes,
United
Kingdom
Accepted January 14, 1985 Immunoreactive insulin levels (IRI) were measured by a homologous fish insulin radioimmunoassay. An in vitro pancreatic islet superfusion technique was employed to monitor the changes in IRI in the presence and absence of specific adrenergic agonists and antagonists. Exogenous adrenaline at low concentrations (lo- lo M) inhibited IRI release but evoked an IRI stimulation at high concentrations (10e6 M). The stimulation of IRI by adrenaline is thought to involve B-adrenoceptors located postsynaptically on the B-cell membrane as the effect of adrenaline was mimicked by the B-agonist, isoproterenol, and abolished by the pantagonist, propranolol. Phentolamine (an a-antagonist) potentiated the adrenergic stimulation of IRI, whereas yohimbine (an a2-antagonist) was without effect. Phenylephrine (a,adrenoceptor agonist) inhibited IRI release suggesting the presence of a,-inhibitory adrenoceptors which exert a modulatory influence on adrenaline-stimulated insulin release. 8 1985;Academic Press, Inc.
al., 1966; Porte and Williams, 1966; Woods and Porte, 1974). However, the work of Loubatieres-Mairani et al., (1977, 1980) on the dog in vivo and isolated perfused dog pancreas, respectively, has demonstrated that during continuous infusion of adrenaline there is a stimulation of basal insulin release. Stress conditions which are typically associated with hyperactivity of the sympathoadrenal axis in mammals are characterized by an absolute or relative insulin deficiency and hyperglucagonemia. Further to this, Ribes et al. (1984) have indicated that whether adrenaline has a mainly stimulatory or inhibitory effect on insulin secretion depends on such variables 1983). as species, age of animal, dose of adrenaIt has generally been accepted that the line, and the prevailing glucose concentration (Campfield and Smith, 1983; Wollheim predominant effect of the catecholamines adrenaline and noradrenaline is an inhibi- and Sharp, 1981). The work of Ince and Thorpe (1977) on tion of insulin release from mammalian /3the eel, Anguilla, with an in vivo applicacells (Coore and Randle, 1964; Loubatieres et al., 1970; Malaisse et al., 1967; Porte et tion of exogenous adrenaline, reported an inhibitory effect on plasma insulin levels followed 3 to 6 hr postinjection by a signif’ Present address: Department of Biochemistry, icant increase in plasma insulin. During peUniversity of Massachusetts Medical Center, 55 Lake Avenue North, Worcester, Mass. 01605. riods of high energy utilization such as
Insulin release from the pancreas of higher vertebrates is modified by a number of factors including dietary metabolites, neurotransmitters, inorganic ions, steroid hormones, changes in vascular tension, prostaglandins, cyclic nucleotides, and paracrine islet hormones (Campfield and Smith, 1983). However, in the lower vertebrates the control of insulin release remains poorly understood. The activity of fish p-cells can be modified by such stimuli as glucose, amino acids, reproductive activity, and seasonal changes as well as by catecholamines and the islet hormones glucagon, somatostatin, and pancreatic polypeptide (Ince and Thorpe, 1977; Ince,
460 0016~6480/85 $1.50 Copyright 6 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.
CONTROL
OF INSULIN
spawning and migration, a rapid mobilisation of metabolic fuels following the inhibition of insulin would be adaptive. Amongst the lower vertebrates, and in particular the teleosts, there appears to be a degree of sympathetic innervation of the macroscopic principle islet or Brockmann body. Electron microscopy studies of the trout Brockmann body have indicated the presence of adrenergic nerve endings that penetrate the endocrine core (see Brinn, 1973; Epple and Brinn, 1975). However, as yet there has been little characterisation of the sympathetic and parasympathetic receptors controlling insulin fluxes in fish. The present study employs a homologous teleost insulin radioimmunoassay (Tilzey et al., in press) which has been developed to assess accurate, reproducible IRI levels, and an in vitro isolated fish islet superfusion technique developed in this laboratory, in an attempt to clarify the role and control of adrenaline on insulin output from trout islet B-cells, superimposed on normal intermediary metabolic insulin release in response to dietary components supplied by the superfusion medium. The results suggest that high levels of adrenaline activating via Breceptors stimulate insulin release, whereas low levels of adrenaline inhibit insulin release via cY-receptors. MATERIALS
RELEASE
461
IN VITRO
was 320 mOs. The dissection was carried out under a dissecting microscope in which the exocrine cortex of the Brockmann body and connective tissue was carefully teased away. Initial incubation was for 25 min and the diced islet tissue was then washed and introduced by suction into the superfusion chamber (see Fig. 1) composed of a Sartorius filter holder enclosing a tine nylon mesh. The chamber was secured from leakage using PTFE tape. Islet tissue was gassed with 95% 0,:5% CO, and superfused at a rate of 1 ml medium/2 min. Two-minute fractions were collected and stored at - 20” for radioimmunoassay. All exogenous drugs were administered via a system of three-way taps. Each experiment consisted of a 6min control period followed by a 30-min infusion period and finally a IO-min recovery period. Perfusate concentrations of IRI were expressed as picograms per millilitre per milligram dry Brockmann body. Ascorbic acid was added (1 mh4) to prevent the oxidation of added catecholamines. Adrenaline bitartrate, propranolol HCI, yohimbine HCl, phenylephrine HCl, and isoproterenol HCl were purchased from Sigma and phentolamine mesylate (Rogitine) was a gift from Ciba-Geigy. Insulin
radioimmunoassay:
antibody
production.
Bonito (Katsuwonas pelamis) insulin antibody was produced in rabbits. Each rabbit was immunized initially according to a regime used by Sumpter (personal communication). Rabbit anti-bonito insulin (RABI) was precipitated from serum at room temperature using ammonium sulphate (0.31 g/ml). After being left to stand for 2 hr the mixture was centrifuged at 60,OOOg
superfuslan medlm
AND METHODS
Normal rainbow trout (mean body wt 425 g) were starved for 24 hr and then killed by a blow to the head. The Brockmann body (BB) was removed (wet wt ranged from 0.6 to 5 mg) from a position close to the gall bladder and bile duct and in close association with the coeliacomesenteric artery, splenic artery, and hepatic portal vein. After excision the islet tissue was placed in 5 ml medium consisting of 2.5 mg collagenase (Worthington) in freshwater teleost Krebs-Ringer (isotonic to trout plasma) superfusion medium, consisting of 14.4 mA4 NaCl; 26.2 mM NaHCO,; 1.6 r&4 Na,HPO,; 2.0 mM KCl; 0.3 mM KH,PO,; 1.3 mM CaCl,; 2.0 mM MgSO, * 7H,O; 0.4 mJ4 (NH&SO,; and 0.3% BSA (grade V, Sigma). The following was also added: 50 mg/lOO ml glucose, 6 mg/lOO ml lysine, 7 mg/lOO ml, leucine 7 mg/lOO ml, arginine 1.7 mg/lOO ml, 1.7 mg/lOO ml, phenol red, pH 7.35. Osmolarity
drug
I
l--l
perlstaltlc
DwlD
fraction collectIon
FIG. 1. Diagram of superfusion apparatus. Rainbow trout isolated islet tissue is contained in the tissue chamber, consisting of a modified Sartorius filter holder and nylon mesh tissue trap. Fractions are collected at 2-min intervals. The superfusion period consists of an initial basal 6-min period followed by a 30min drug infusion period and a further 6-min control recovery period.
462
TILZEY,
WAIGHTS
for 30 min at 30”. The supernatant was decanted and the pellet was resuspended to the original volume with 50 mM sodium phosphate buffer, pH 7.4, containing 0.31 g/ml ammonium sulphate, left to stand for 2 hr and again centrifuged at 60,OOOgfor 30 min. The pellet was resuspended in 50 mM sodium phosphate buffer and the antisera was stored in working aliquots of titre 1:120 at -20”. Zodination. The iodination of the bonito insulin was based on the method of Hunter and Greenwood (1962). Greenwood cr al. (1963), and Bolton et al. (1979). To the reaction vial containing 1 mCi sodium iodide-125 (Amersham, UK, sp act 13.2 mCi, i151/mg of iodine). 10 ug bonito insulin (gift from Kadoma, Tokyo) was added in 100 ~140 mM sodium phosphate buffer containing 5 mM HCl and 60 kg chloramine-T. The oxidative iodination reaction was run for 20 set and then stopped by adding 60 p.g of sodium metabisulphate together with 100 ~1 of 10% bovine serum albumin (BSA)/KI (10 mg/ml). The reaction mixture was placed on top of a Sephadex G-25 medium column (0.5 x 10 cm), presaturated with 1% BSA in 40 mM phosphate buffer containing 0.1% BSA and 200~ul fractions were collected. Fractions used for radioimmunoassay were from the second elution profile peak (Ca. 1400 ul elution volume). The working dilution of the iodinated insulin was 1:lOOO of the neat fraction (ca. 2000 cpm). Radioimmunoassay procedure. Immunoreactive insulin in the superfusion medium was measured by a double antibody precipitation technique of Morgan and Lazerow (1963) and Hales and Randle (1963). Bonito insulin standards, ‘?SI-insulin, and the RABl were all diluted with RIA buffer (40 mM sodium phosphate containing 0.5% BSA and 0.5% NaCl). Each assay tube contained 25 ~1 of all reagents and was incubated for 24 hr at 4”. The second antibody, donkey anti-rabbit 1gG (Wellcome) was added and incubated for a further 24 hr, after which 500 ul of buffer was added. All tubes were vortex mixed and centrifuged at 2500~ for 25 min at 4”. The supernatant was decanted and the resulting precipitate was counted in a Beckman 5.500 Gamma counter. RIA results were expressed as a semi-log plot of percentage relative bound counts against log insulin concentration in nanograms per millilitre.
RESULTS
Spontaneous IRI output was measured over a 42-min superfusion period. The initial IRI release rate was 241 pg/ml/mg BBI min but declined slowly over the experimental period reaching 92 pg/ml/mg BB/min at 42 min. Thus although there was an overall decrease in IRI throughout the superfusion period, basal IRI output re-
AND HOLMES
mained relatively be reproducible ments.
stable and was found to in a number of experi-
Effect of Adrenaline on IRI Release Exogenous adrenaline at 10e6 M produced both an initial (+ 6 min) and a late (+ 24 min) stimulation of IRI release. However, at low concentration (lo-to A&) adrenaline evoked a near total inhibition of basal IRI release (see Fig. 2). To determine whether the stimulation in IRI release evoked by adrenaline at lop6 M was the result of an interaction with B-adrenoceptors, we investigated the effects of the B-adrenoceptor antagonist, propranolol, and the B-adrenoceptor agonist, isoproterenol, on IRI output. Propranolol, when infused with adrenaline, abolished the stimulation of IRI release (see Fig. 3). Moreover, isoproterenol also stimulated IRI release and was found to be more potent in this respect than an equimolar concentration of adrenaline. Isoproterenol-evoked stimulation of IRI release was also abolished by propranolol (see Fig. 4), suggesting that the stimulation of IRI release by adrenergic agonists is mediated via B-adrenoceptors. In contrast to the inhibition of adrenaline-stimulated IRI release induced by the P-receptor antagonist propranolol, the (Yadrenoceptor antagonist phentolamine produced a marked potentiation in the increase of IRI release produced by adrenaline at 10m6 M (see Fig. 5). Yohimbine (a specific olz-antagonist) when super-fused with adrenaline at 1O-6 M produced an IRI release curve similar to that produced by adrenaline alone (see Fig. 5). This suggests that ol,-adrenoceptors are involved in the inhibition of IRI release by exogenous adrenaline. This is further supported by the fact that phenylephrine (a specific at-agonist) markedly inhibited IRI output to below basal release (see Fig. 5). An infusion of a combination of phentolamine, propranolol, and adrenaline produced no significant dif-
CONTROL
OF INSULIN
minutes
RELEASE
IN VZTRO
463
superfusion
FIG. 2. Effect of adrenaline at high concentration (10m6 M, n = 5,O) and a low concentration (lo-i0 M, n = 4, N) on IRI release during period 0 to 30 min (as indicated by arrows) against control IRI release (0, n = 6). Values are means + SE.
ference from control IRI release throughout the superfusion (data not shown). Therefore with an increase in exogenous adrenaline to produce local islet concentrations of lop6 M, P-adrenoceptors are activated to effect an IRI stimulation which appears to be under the control of inhibitory ol,-adrenoceptors. DISCUSSION
The development of a homologous fish insulin radioimmunoassay to measure ac-
adrenaline adrenaline
curately changes in insulin flux under varying conditions was essential in this study. The available mammalian radioimmunoassays have not proved to be of any value in fish insulin studies because of weak cross-reactivities between many fish and mammalian antibodies (Falkmer and Wilson, 1967; Thorpe and Ince, 1976). We found it was possible to produce anti-bonito insulin antiserum in rabbits which crossreacted well with trout immunoreactive insulin to provide a reliable and reproduc-
+ Dropronoloi I
10 mlnutes
20 superfusion
FIG. 3. Effect of g-antagonist propranolol on adrenaline modified IRI release. Adrenaline (10e6 M) and propranolol (10e6 M, n = 4, n ) added during period 0 to 30 min (indicated by arrows) against adrenaline at 10e6 M (0, n = 5) and control (0, n = 6) superfusion curves. Values are means 5 SE.
464
TILZEY,
WAIGHTS
minutes
AND HOLMES
superfusion
4. Effect of g-agonist isoproterenol and P-antagonist propranolol on IRI release. Isoproterenol (10m5 M, n = 5, 0); isoproterenol (10e5 J&! and propranolol (10m6 M) (0, n = 4) added during period 0 to 30 min (as indicated by arrows) against control superfusion IRI release 01). Values are means f SE (SE < 10% not indicated). FIG.
ible radioimmunoassay. The double-antibody precipitation technique was found to be economical and of relatively short assay time span. The in vitro superfusion technique was developed as an aiternative to in vivo whole animal preparations, where the effects of local applications of specific insulin influencing agents is difficult to achieve due to the complex metabolic nature of insulin release and the interaction of other endogenous insulin secretagogues and antagonists. The decrease of basal IRI output
minutes
throughout the superfusion period may in part be due to the presence of proteolytic enzymes in the collagenase preparation that are cytotoxic to islet cells (see Schwizer, 1984). Current work using aprotinin in the superfusion medium is being undertaken. However, the in vitro super-fusion of trout islet tissue was reproducible in basal insulin release, and provided a useful tool in studying the adrenergic control of insulin release. Insulin secretion can be considered to be the result of two classes of signals acting
superfusion
FIG. 5. Effect of a-antagonists yohimbine (a,) and phentolamine on adrenaline modified IRI release and the a,-agonist phenylephrine on basal IRI release. Adrenaline (10m6 &f) and phentoiamine (low6 W, 0, n = 4); adrenaline (10e6 M) and yohimbine (10m6 M, 0, n = 4); phenylephrine (10m6 M. n = 4, n ) added during period 0 to 30 min (as indicated by arrows). Values are means 2 SE (SE < 10% not indicated).
CONTROL
OF
INSULIN
RELEASE
IN
VITRO
465
on the B-cell: metabolic (amino acids and the activation of p-adrenoceptors probably glucose) and neural (sympathetic and para- located on the P-cell membrane. sympathetic) (Campfield and Blocker, This B-mediated stimulation of IRI by 1979; Campfield and Smith, 1980, 1983; high concentrations of exogenous adrenaGirardier et al., 1976; Sharp et al., 1974). line appears to be under the control of an Therefore, by achieving a constant meta- inhibitory o-receptor. This was determined bolic input via the medium superfusing the by the fact that the o-adrenergic antagonist, phentolamine, potentiated the effect of islet tissue, precise modifications of insulin secretion can be monitered with the appli- -adrenaline at 10m6 M causing a marked increase in insulin release. This inhibitory cation of exogenous neurotransmitters. receptor seems to be an ol,-adrenoceptor The results of these experiments indicate as the cw,-specific antagonist yohimbine that low concentrations of exogenous adrenaline (lop7 M) induce a suppression showed no potentiation of the adrenalineof insulin release. Given that normal cir- stimulated IRI release. The presence of an a-receptor is further substanculating levels of adrenaline in the trout are inhibitory of the order 2 x low9 M (Ristori et al., tiated by the fact that the a-agonist phenylephrine (lop6 M) also inhibited insulin re1979), under normal conditions adrenaline exerts an inhibitory tone on the islet B-cell. lease. Therefore it would appear that cr-adHowever, higher concentrations of adren- renoceptors ((Y,) evoke an inhibitory tone aline (low6 M), in the presence of interon the adrenergic control of insulin release. mediary levels of glucose and amino acids, Whether the a-inhibitory receptors are loproduce an increase in insulin secretion. cated on the B-cells is not known. It is conWith an increase in circulating catecholceivable that they may be located presynamines to specific activating concentrations aptically on sympathetic fibres present (in the trout of the order of 10e6 M, within the endocrine core of islet tissue where they may modulate endogenous Mazeaud and Mazeaud, 1981), the normal (resting) inhibition of IRI release is over- adrenaline release by negative feedback ridden producing an increase in insulin se- mechanism. cretion. Insulin release from the trout B-cell Electron microscopy shows the presence in vitro can thus be modified by an exogeof both sympathetic and parasympathetic nous adrenergic sensitivity gradient, similar tibres in islet tissue (Brinn, 1973; Epple and to that found in vivo in the dog (LoubaBrinn, 1975), thus an interaction with chotieres-Mariani et al., 1977). linergic terminals is also possible. Further The use of specific CY-and p-adrenergic experimentation is required to elucidate agonists and antagonists reveal the sub- these possibilities. types of adrenoceptors involved in the In summary, our results show that isoneural control of insulin release from trout lated trout islet tissue responds to exogenous adrenaline with modifications in inB-cells. In the presence of high concentrasulin output. At concentrations of the order tions of exogenous adrenaline the resultant of normal circulating levels of adrenaline IRI stimulation is blocked by the B-antag(ca. lo-i0 M) there is an inhibition of inonist, propranolol. This adrenergic stimulation of insulin release was mimicked by sulin release, but at higher concentrations the P-specific agonist, isoproterenol, the ef- (lop6 M) insulin release is stimulated. The use of specific (Y-and B-adrenergic agonists fect of which was also blocked by propranand antagonists show that the stimulation 0101. Therefore the resultant insulin stimuof insulin release from isolated islet tissue lation from B-cells as a result of a high catis due to adrenaline acting on P-adrenergic echolamine concentration (lop6 M) is via
TILZEY,
466
WAIGHTS
receptors probably located postsynaptitally on the P-cell membrane. However, these studies also reveal the presence of inhibitory a-adrenoceptors which appear to control the adrenaline-evoked release of insulin. The precise location of these receptors is yet to be determined. ACKNOWLEDGMENTS We thank Dr. S. Murphy and Dr. B. Pierce of the Brain Research group, The Open University for their help in the iodinatin of bonito insulin (S.M.) and for their constructive critisism of this manuscript, Dr. J. Sumpter, Brunel University, for his help in the RIA; and Miss Elaine Dale for the typing of the manuscript.
REFERENCES Bolton, A. E., Lee-Own, V., Kramer, C., McLean, R., and Challard, G. S. (1979). Three different radioiodination methods from human spleen ferritin compared. C&z. Chem. 25, 1826- 1830. Brinn, J. (1973). The pancreatic islets of bony fishes. Amer. Zool. 13(3), 653-665. Campfield, L. A., and Blocker, D. C. (1979). Simulation of the autonomic neural control of insulin secretion. Comput. Biol. Med. 9, 191-203. Campfield, L. A., and Smith, E J. (1980). Modulation of insulin secretion by the autonomic nervous system. Bruin Res. Bull. S(Suppl. 4) 103-107. Campfield, L. A.. and Smith, F. J. (1983). Neural control of insulin secretion: interaction of norepinephrine and acetylcholine. Amer. J. Physiol. 244 (Regul. Integrative Comp. Physiol. 13, R629R634. Coore, H. G., and Randle, P. J. (1964). Regulation of insulin secretion studied with pieces of rabbit pancrease incubated in vitro. Biochem. J. 88, 137146. Epple, A., and Brinn, J. (1975). Islet histophysiology: Evolutionary correlation. Gen. Comp. Endocrinol.
27, 320-349.
Falkmer, S., and Wilson, S. (1967). Comparative aspects of the immunology and biology of insulin. Diabetologia
3, 519-528.
Girardier, L., Seydoux, J., and Campfield, L. A. (1976). Control of A and B cells in vivo by sympathetic nervous input and selective hyper- or hypoglycemia in dog pancreas. J. Physiol. (Paris) 72, 801-814.
Greenwood, F. C., Hunter, W. M., and Clover, J. S. (1963). The preparation of i31-labeled human growth hormone of high specific radioactivity. Biochem. J. 89, 114-123. Hales, C. N., and Randle, P. J. (1963). Immunoassay of insulin with insulin-antibody precipitate. Biochem.
J. 88,
137-146.
Hunter, W. M., and Greenwood, F. C. (1962). Preparation of iodine-131 labelled human growth hor-
AND HOLMES mone of high specific activity. Nature
(London)
194, 495.
Ince, B. W. (1983). Pancreatic control of metabolism. In “Control Processes in Fish Physiology” (Rankin, Pitcher, and Duggan, eds.). Croom Helm. Ince, B. W., and Thorpe, A. (1977). Plasmainsulin and glucose responses to glucagon and catecholamines in the European silver eel, Anguillu anguilla.
Gen.
Comp.
Endocrinol.
33, 453-459.
Loubatieres, A., Mariani, M. M., and Chapal. (1970). lnsulino-secretion etudiee sur le pancreas isole et perfuse du rat. II. Action des catecholamines et des substances bloquant les recepteurs adrenergiques. Diabetologia 6, 533-541. Loubatieres-Mariani, M. M., Chapal, J., Ribes, G., and Loubatieres, A. L. (1977). Discrepdnces in the response of the insulin-secreting cells of the dog and rat to different adrenergic stimulating agents. Acta Diabetol. Lat. 14, 144-155. Loubatieres-Mariani, M. M., Ribes, G., Blayoc, J. P., and Campo, P (1980). Insulin secretory effect of a low dose of adrenaline in the dog. Horm. Metab. Res.
12, 126-127.
Malaisse, W. J., Mailaisse-Lagae, F., Wright, P. H., and Ashmore, J. (1967). Effects of adrenergic and cholinergic agents upon secretion in vitro. Endocrinology
30, 975-978.
Mazeaud, M. M. (1971). “Recherches sur la biosynthese, la secretion et le catabolism de l’adrenaline et de la noradrenaline chez quelques especes de cyclastomes et de poissons.” PhD Thesis, es Sciences. Paris. Mazeaud, M. M., and Mazeaud, F. (1981). In “Stress and Fish” (A. D. Pickering, ed.), pp. 49-67. Academic Press, London/New York. Morgan, C. R., and Lazarow, A, (1963). Immunoassay of insulin: Two antibody system. Diabetologia 12, 115-126. Nakano, T., and Tomlinson, N. (1967). Catecholamines and carbohydrate concentrations in rainbow trout, Salmo gairdneri, in relation to physical disturbances. J. Fish. Res. Board Canad. 24, 1701-1715. Porte, D., Jr., Graber, A. L., Kuzuya, T., and Williams, R. H. (1966). The effect of epinephrine on immunoreactive insulin levels in man. J. Clin. Znvest.
45, 228-236.
Porte, D., Jr., and Williams, R. H. (1966). inhibition of insulin release by norepinephrine in man. Science
152,
1248-1250.
(Washington,
D.C.)
Ribes, G., Trimble, E. R., Blayac, J. P., Wollheim, C. B., and Loubatieres-Mariani, M. M. (1984). In viva stimulation of pancreatic hormone secretion by norepinephrine infusion in the dog. Amer. J. Physiol. E343.
246,
(Endocrinol.
Metab.
9),
E239-
Ristori, M-Th., Rhehm, J-Cl., and Laurent, P. (1979). Dosages des catecholamines plasmatiques chez la
CONTROL
OF INSULIN
truite au tours de l’hypoxie controlee. J. Physiol. (Paris) 75, 67A. Schwizer, R. W. (1984). Effects of dissociation and culture on the maintenance of insulin, glucagon and somatostatin release by neonatal rat islets of Langerhans. Life Sci. 35, 783-788. Sharp, R., Culbert, S., Cook, J., Jennings, A., and Burr, I. M. (1974). Cholinergic modification of glucose-induced biphasic insulin release in vitro. J. Clin. Invest. 53, 710-716.
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IN VITRO
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Thorpe, A., and Ince, B. W. (1976). Plasma insulin levels in teleosts determined by a charcoal-separation radioimmunoassay technique. Cm. Camp. Endocrinol.
30, 332-339.
Wollheim, C. B., and Sharp, G. W. G. (1981). Regulation of insulin release by calcium. Physiol. Rev. 61, 914-973. Woods, S. C., and Porte, D., Jr. (1974). Neural control of the endocrine pancreas. Physiol. Rev. 54, 596619.
AND
COMPARATIVE
ENDOCRINOLOGY
59, 460-467 (1985)
Adrenergic Control of insulin Release from Isolated Islet Tissue in the Rainbow Trout, Salmo gairdneri R. JOHN E TILZEY,~ VERINA WAIGHTS, AND RICHARD HOLMES Department
of Biology,
The Open
University,
Milton
Keynes,
United
Kingdom
Accepted January 14, 1985 Immunoreactive insulin levels (IRI) were measured by a homologous fish insulin radioimmunoassay. An in vitro pancreatic islet superfusion technique was employed to monitor the changes in IRI in the presence and absence of specific adrenergic agonists and antagonists. Exogenous adrenaline at low concentrations (lo- lo M) inhibited IRI release but evoked an IRI stimulation at high concentrations (10e6 M). The stimulation of IRI by adrenaline is thought to involve B-adrenoceptors located postsynaptically on the B-cell membrane as the effect of adrenaline was mimicked by the B-agonist, isoproterenol, and abolished by the pantagonist, propranolol. Phentolamine (an a-antagonist) potentiated the adrenergic stimulation of IRI, whereas yohimbine (an a2-antagonist) was without effect. Phenylephrine (a,adrenoceptor agonist) inhibited IRI release suggesting the presence of a,-inhibitory adrenoceptors which exert a modulatory influence on adrenaline-stimulated insulin release. 8 1985;Academic Press, Inc.
al., 1966; Porte and Williams, 1966; Woods and Porte, 1974). However, the work of Loubatieres-Mairani et al., (1977, 1980) on the dog in vivo and isolated perfused dog pancreas, respectively, has demonstrated that during continuous infusion of adrenaline there is a stimulation of basal insulin release. Stress conditions which are typically associated with hyperactivity of the sympathoadrenal axis in mammals are characterized by an absolute or relative insulin deficiency and hyperglucagonemia. Further to this, Ribes et al. (1984) have indicated that whether adrenaline has a mainly stimulatory or inhibitory effect on insulin secretion depends on such variables 1983). as species, age of animal, dose of adrenaIt has generally been accepted that the line, and the prevailing glucose concentration (Campfield and Smith, 1983; Wollheim predominant effect of the catecholamines adrenaline and noradrenaline is an inhibi- and Sharp, 1981). The work of Ince and Thorpe (1977) on tion of insulin release from mammalian /3the eel, Anguilla, with an in vivo applicacells (Coore and Randle, 1964; Loubatieres et al., 1970; Malaisse et al., 1967; Porte et tion of exogenous adrenaline, reported an inhibitory effect on plasma insulin levels followed 3 to 6 hr postinjection by a signif’ Present address: Department of Biochemistry, icant increase in plasma insulin. During peUniversity of Massachusetts Medical Center, 55 Lake Avenue North, Worcester, Mass. 01605. riods of high energy utilization such as
Insulin release from the pancreas of higher vertebrates is modified by a number of factors including dietary metabolites, neurotransmitters, inorganic ions, steroid hormones, changes in vascular tension, prostaglandins, cyclic nucleotides, and paracrine islet hormones (Campfield and Smith, 1983). However, in the lower vertebrates the control of insulin release remains poorly understood. The activity of fish p-cells can be modified by such stimuli as glucose, amino acids, reproductive activity, and seasonal changes as well as by catecholamines and the islet hormones glucagon, somatostatin, and pancreatic polypeptide (Ince and Thorpe, 1977; Ince,
460 0016~6480/85 $1.50 Copyright 6 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.
CONTROL
OF INSULIN
spawning and migration, a rapid mobilisation of metabolic fuels following the inhibition of insulin would be adaptive. Amongst the lower vertebrates, and in particular the teleosts, there appears to be a degree of sympathetic innervation of the macroscopic principle islet or Brockmann body. Electron microscopy studies of the trout Brockmann body have indicated the presence of adrenergic nerve endings that penetrate the endocrine core (see Brinn, 1973; Epple and Brinn, 1975). However, as yet there has been little characterisation of the sympathetic and parasympathetic receptors controlling insulin fluxes in fish. The present study employs a homologous teleost insulin radioimmunoassay (Tilzey et al., in press) which has been developed to assess accurate, reproducible IRI levels, and an in vitro isolated fish islet superfusion technique developed in this laboratory, in an attempt to clarify the role and control of adrenaline on insulin output from trout islet B-cells, superimposed on normal intermediary metabolic insulin release in response to dietary components supplied by the superfusion medium. The results suggest that high levels of adrenaline activating via Breceptors stimulate insulin release, whereas low levels of adrenaline inhibit insulin release via cY-receptors. MATERIALS
RELEASE
461
IN VITRO
was 320 mOs. The dissection was carried out under a dissecting microscope in which the exocrine cortex of the Brockmann body and connective tissue was carefully teased away. Initial incubation was for 25 min and the diced islet tissue was then washed and introduced by suction into the superfusion chamber (see Fig. 1) composed of a Sartorius filter holder enclosing a tine nylon mesh. The chamber was secured from leakage using PTFE tape. Islet tissue was gassed with 95% 0,:5% CO, and superfused at a rate of 1 ml medium/2 min. Two-minute fractions were collected and stored at - 20” for radioimmunoassay. All exogenous drugs were administered via a system of three-way taps. Each experiment consisted of a 6min control period followed by a 30-min infusion period and finally a IO-min recovery period. Perfusate concentrations of IRI were expressed as picograms per millilitre per milligram dry Brockmann body. Ascorbic acid was added (1 mh4) to prevent the oxidation of added catecholamines. Adrenaline bitartrate, propranolol HCI, yohimbine HCl, phenylephrine HCl, and isoproterenol HCl were purchased from Sigma and phentolamine mesylate (Rogitine) was a gift from Ciba-Geigy. Insulin
radioimmunoassay:
antibody
production.
Bonito (Katsuwonas pelamis) insulin antibody was produced in rabbits. Each rabbit was immunized initially according to a regime used by Sumpter (personal communication). Rabbit anti-bonito insulin (RABI) was precipitated from serum at room temperature using ammonium sulphate (0.31 g/ml). After being left to stand for 2 hr the mixture was centrifuged at 60,OOOg
superfuslan medlm
AND METHODS
Normal rainbow trout (mean body wt 425 g) were starved for 24 hr and then killed by a blow to the head. The Brockmann body (BB) was removed (wet wt ranged from 0.6 to 5 mg) from a position close to the gall bladder and bile duct and in close association with the coeliacomesenteric artery, splenic artery, and hepatic portal vein. After excision the islet tissue was placed in 5 ml medium consisting of 2.5 mg collagenase (Worthington) in freshwater teleost Krebs-Ringer (isotonic to trout plasma) superfusion medium, consisting of 14.4 mA4 NaCl; 26.2 mM NaHCO,; 1.6 r&4 Na,HPO,; 2.0 mM KCl; 0.3 mM KH,PO,; 1.3 mM CaCl,; 2.0 mM MgSO, * 7H,O; 0.4 mJ4 (NH&SO,; and 0.3% BSA (grade V, Sigma). The following was also added: 50 mg/lOO ml glucose, 6 mg/lOO ml lysine, 7 mg/lOO ml, leucine 7 mg/lOO ml, arginine 1.7 mg/lOO ml, 1.7 mg/lOO ml, phenol red, pH 7.35. Osmolarity
drug
I
l--l
perlstaltlc
DwlD
fraction collectIon
FIG. 1. Diagram of superfusion apparatus. Rainbow trout isolated islet tissue is contained in the tissue chamber, consisting of a modified Sartorius filter holder and nylon mesh tissue trap. Fractions are collected at 2-min intervals. The superfusion period consists of an initial basal 6-min period followed by a 30min drug infusion period and a further 6-min control recovery period.
462
TILZEY,
WAIGHTS
for 30 min at 30”. The supernatant was decanted and the pellet was resuspended to the original volume with 50 mM sodium phosphate buffer, pH 7.4, containing 0.31 g/ml ammonium sulphate, left to stand for 2 hr and again centrifuged at 60,OOOgfor 30 min. The pellet was resuspended in 50 mM sodium phosphate buffer and the antisera was stored in working aliquots of titre 1:120 at -20”. Zodination. The iodination of the bonito insulin was based on the method of Hunter and Greenwood (1962). Greenwood cr al. (1963), and Bolton et al. (1979). To the reaction vial containing 1 mCi sodium iodide-125 (Amersham, UK, sp act 13.2 mCi, i151/mg of iodine). 10 ug bonito insulin (gift from Kadoma, Tokyo) was added in 100 ~140 mM sodium phosphate buffer containing 5 mM HCl and 60 kg chloramine-T. The oxidative iodination reaction was run for 20 set and then stopped by adding 60 p.g of sodium metabisulphate together with 100 ~1 of 10% bovine serum albumin (BSA)/KI (10 mg/ml). The reaction mixture was placed on top of a Sephadex G-25 medium column (0.5 x 10 cm), presaturated with 1% BSA in 40 mM phosphate buffer containing 0.1% BSA and 200~ul fractions were collected. Fractions used for radioimmunoassay were from the second elution profile peak (Ca. 1400 ul elution volume). The working dilution of the iodinated insulin was 1:lOOO of the neat fraction (ca. 2000 cpm). Radioimmunoassay procedure. Immunoreactive insulin in the superfusion medium was measured by a double antibody precipitation technique of Morgan and Lazerow (1963) and Hales and Randle (1963). Bonito insulin standards, ‘?SI-insulin, and the RABl were all diluted with RIA buffer (40 mM sodium phosphate containing 0.5% BSA and 0.5% NaCl). Each assay tube contained 25 ~1 of all reagents and was incubated for 24 hr at 4”. The second antibody, donkey anti-rabbit 1gG (Wellcome) was added and incubated for a further 24 hr, after which 500 ul of buffer was added. All tubes were vortex mixed and centrifuged at 2500~ for 25 min at 4”. The supernatant was decanted and the resulting precipitate was counted in a Beckman 5.500 Gamma counter. RIA results were expressed as a semi-log plot of percentage relative bound counts against log insulin concentration in nanograms per millilitre.
RESULTS
Spontaneous IRI output was measured over a 42-min superfusion period. The initial IRI release rate was 241 pg/ml/mg BBI min but declined slowly over the experimental period reaching 92 pg/ml/mg BB/min at 42 min. Thus although there was an overall decrease in IRI throughout the superfusion period, basal IRI output re-
AND HOLMES
mained relatively be reproducible ments.
stable and was found to in a number of experi-
Effect of Adrenaline on IRI Release Exogenous adrenaline at 10e6 M produced both an initial (+ 6 min) and a late (+ 24 min) stimulation of IRI release. However, at low concentration (lo-to A&) adrenaline evoked a near total inhibition of basal IRI release (see Fig. 2). To determine whether the stimulation in IRI release evoked by adrenaline at lop6 M was the result of an interaction with B-adrenoceptors, we investigated the effects of the B-adrenoceptor antagonist, propranolol, and the B-adrenoceptor agonist, isoproterenol, on IRI output. Propranolol, when infused with adrenaline, abolished the stimulation of IRI release (see Fig. 3). Moreover, isoproterenol also stimulated IRI release and was found to be more potent in this respect than an equimolar concentration of adrenaline. Isoproterenol-evoked stimulation of IRI release was also abolished by propranolol (see Fig. 4), suggesting that the stimulation of IRI release by adrenergic agonists is mediated via B-adrenoceptors. In contrast to the inhibition of adrenaline-stimulated IRI release induced by the P-receptor antagonist propranolol, the (Yadrenoceptor antagonist phentolamine produced a marked potentiation in the increase of IRI release produced by adrenaline at 10m6 M (see Fig. 5). Yohimbine (a specific olz-antagonist) when super-fused with adrenaline at 1O-6 M produced an IRI release curve similar to that produced by adrenaline alone (see Fig. 5). This suggests that ol,-adrenoceptors are involved in the inhibition of IRI release by exogenous adrenaline. This is further supported by the fact that phenylephrine (a specific at-agonist) markedly inhibited IRI output to below basal release (see Fig. 5). An infusion of a combination of phentolamine, propranolol, and adrenaline produced no significant dif-
CONTROL
OF INSULIN
minutes
RELEASE
IN VZTRO
463
superfusion
FIG. 2. Effect of adrenaline at high concentration (10m6 M, n = 5,O) and a low concentration (lo-i0 M, n = 4, N) on IRI release during period 0 to 30 min (as indicated by arrows) against control IRI release (0, n = 6). Values are means + SE.
ference from control IRI release throughout the superfusion (data not shown). Therefore with an increase in exogenous adrenaline to produce local islet concentrations of lop6 M, P-adrenoceptors are activated to effect an IRI stimulation which appears to be under the control of inhibitory ol,-adrenoceptors. DISCUSSION
The development of a homologous fish insulin radioimmunoassay to measure ac-
adrenaline adrenaline
curately changes in insulin flux under varying conditions was essential in this study. The available mammalian radioimmunoassays have not proved to be of any value in fish insulin studies because of weak cross-reactivities between many fish and mammalian antibodies (Falkmer and Wilson, 1967; Thorpe and Ince, 1976). We found it was possible to produce anti-bonito insulin antiserum in rabbits which crossreacted well with trout immunoreactive insulin to provide a reliable and reproduc-
+ Dropronoloi I
10 mlnutes
20 superfusion
FIG. 3. Effect of g-antagonist propranolol on adrenaline modified IRI release. Adrenaline (10e6 M) and propranolol (10e6 M, n = 4, n ) added during period 0 to 30 min (indicated by arrows) against adrenaline at 10e6 M (0, n = 5) and control (0, n = 6) superfusion curves. Values are means 5 SE.
464
TILZEY,
WAIGHTS
minutes
AND HOLMES
superfusion
4. Effect of g-agonist isoproterenol and P-antagonist propranolol on IRI release. Isoproterenol (10m5 M, n = 5, 0); isoproterenol (10e5 J&! and propranolol (10m6 M) (0, n = 4) added during period 0 to 30 min (as indicated by arrows) against control superfusion IRI release 01). Values are means f SE (SE < 10% not indicated). FIG.
ible radioimmunoassay. The double-antibody precipitation technique was found to be economical and of relatively short assay time span. The in vitro superfusion technique was developed as an aiternative to in vivo whole animal preparations, where the effects of local applications of specific insulin influencing agents is difficult to achieve due to the complex metabolic nature of insulin release and the interaction of other endogenous insulin secretagogues and antagonists. The decrease of basal IRI output
minutes
throughout the superfusion period may in part be due to the presence of proteolytic enzymes in the collagenase preparation that are cytotoxic to islet cells (see Schwizer, 1984). Current work using aprotinin in the superfusion medium is being undertaken. However, the in vitro super-fusion of trout islet tissue was reproducible in basal insulin release, and provided a useful tool in studying the adrenergic control of insulin release. Insulin secretion can be considered to be the result of two classes of signals acting
superfusion
FIG. 5. Effect of a-antagonists yohimbine (a,) and phentolamine on adrenaline modified IRI release and the a,-agonist phenylephrine on basal IRI release. Adrenaline (10m6 &f) and phentoiamine (low6 W, 0, n = 4); adrenaline (10e6 M) and yohimbine (10m6 M, 0, n = 4); phenylephrine (10m6 M. n = 4, n ) added during period 0 to 30 min (as indicated by arrows). Values are means 2 SE (SE < 10% not indicated).
CONTROL
OF
INSULIN
RELEASE
IN
VITRO
465
on the B-cell: metabolic (amino acids and the activation of p-adrenoceptors probably glucose) and neural (sympathetic and para- located on the P-cell membrane. sympathetic) (Campfield and Blocker, This B-mediated stimulation of IRI by 1979; Campfield and Smith, 1980, 1983; high concentrations of exogenous adrenaGirardier et al., 1976; Sharp et al., 1974). line appears to be under the control of an Therefore, by achieving a constant meta- inhibitory o-receptor. This was determined bolic input via the medium superfusing the by the fact that the o-adrenergic antagonist, phentolamine, potentiated the effect of islet tissue, precise modifications of insulin secretion can be monitered with the appli- -adrenaline at 10m6 M causing a marked increase in insulin release. This inhibitory cation of exogenous neurotransmitters. receptor seems to be an ol,-adrenoceptor The results of these experiments indicate as the cw,-specific antagonist yohimbine that low concentrations of exogenous adrenaline (lop7 M) induce a suppression showed no potentiation of the adrenalineof insulin release. Given that normal cir- stimulated IRI release. The presence of an a-receptor is further substanculating levels of adrenaline in the trout are inhibitory of the order 2 x low9 M (Ristori et al., tiated by the fact that the a-agonist phenylephrine (lop6 M) also inhibited insulin re1979), under normal conditions adrenaline exerts an inhibitory tone on the islet B-cell. lease. Therefore it would appear that cr-adHowever, higher concentrations of adren- renoceptors ((Y,) evoke an inhibitory tone aline (low6 M), in the presence of interon the adrenergic control of insulin release. mediary levels of glucose and amino acids, Whether the a-inhibitory receptors are loproduce an increase in insulin secretion. cated on the B-cells is not known. It is conWith an increase in circulating catecholceivable that they may be located presynamines to specific activating concentrations aptically on sympathetic fibres present (in the trout of the order of 10e6 M, within the endocrine core of islet tissue where they may modulate endogenous Mazeaud and Mazeaud, 1981), the normal (resting) inhibition of IRI release is over- adrenaline release by negative feedback ridden producing an increase in insulin se- mechanism. cretion. Insulin release from the trout B-cell Electron microscopy shows the presence in vitro can thus be modified by an exogeof both sympathetic and parasympathetic nous adrenergic sensitivity gradient, similar tibres in islet tissue (Brinn, 1973; Epple and to that found in vivo in the dog (LoubaBrinn, 1975), thus an interaction with chotieres-Mariani et al., 1977). linergic terminals is also possible. Further The use of specific CY-and p-adrenergic experimentation is required to elucidate agonists and antagonists reveal the sub- these possibilities. types of adrenoceptors involved in the In summary, our results show that isoneural control of insulin release from trout lated trout islet tissue responds to exogenous adrenaline with modifications in inB-cells. In the presence of high concentrasulin output. At concentrations of the order tions of exogenous adrenaline the resultant of normal circulating levels of adrenaline IRI stimulation is blocked by the B-antag(ca. lo-i0 M) there is an inhibition of inonist, propranolol. This adrenergic stimulation of insulin release was mimicked by sulin release, but at higher concentrations the P-specific agonist, isoproterenol, the ef- (lop6 M) insulin release is stimulated. The use of specific (Y-and B-adrenergic agonists fect of which was also blocked by propranand antagonists show that the stimulation 0101. Therefore the resultant insulin stimuof insulin release from isolated islet tissue lation from B-cells as a result of a high catis due to adrenaline acting on P-adrenergic echolamine concentration (lop6 M) is via
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receptors probably located postsynaptitally on the P-cell membrane. However, these studies also reveal the presence of inhibitory a-adrenoceptors which appear to control the adrenaline-evoked release of insulin. The precise location of these receptors is yet to be determined. ACKNOWLEDGMENTS We thank Dr. S. Murphy and Dr. B. Pierce of the Brain Research group, The Open University for their help in the iodinatin of bonito insulin (S.M.) and for their constructive critisism of this manuscript, Dr. J. Sumpter, Brunel University, for his help in the RIA; and Miss Elaine Dale for the typing of the manuscript.
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