Nervous regulation of insulin release by the intestinal vagal glucoreceptors

Journal o f the Autonomic Nervous System, 4 (1981) 351--363

351

Elsevier/North-Holland Biomedical Press

NERVOUS REGULATION OF INSULIN RELEASE BY THE INTESTINAL VAGAL GLUCORECEPTORS NO~ILMEI, AURORE ARLHAC and ANDR~ BOYER C.N.R.S. - Ddpartment de Neurophysiologie vdgdtative (I.N.P.OI ) 3 I Chemin Joseph Aiguier, B.P. 71, 13277 Marseille Cedex 9 (France)

(Received December 12th, 1980) (Accepted March l l t h , 1981)

Key words: nervous insulin release -- intestinal glucoreceptors -- vagus nerves splanchnic nerves -

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ABSTRACT

In anesthetized cats and rats, it is demonstrated that glucose per~asion of the small intestine produces a rapid increase of insulin secretion (IRI} which precedes glycemia variation. This mechanism involves the auton~mic nervous system and originates from intestinal glucoreceptors, the existe:~ce of which was recently reported. The nervous pathways are described in this study: (1) the afferent pathway is represented by vagal fibers coming from the intestinal glucoreceptors; (2} the efferent pathway involves both sympathetic fibers (splm~chnic nerves) and chiefly parasympathetic fibers (vagal nerves). These results are established after surgical suppression of afferent and efferent vagal fibers, and pharmacological exclusion of parasympathetic or ~ympathetic fibers. The role of this nervous regulatioz~ of insulin secretion is discussed with special reference to other already known mechanisms.

INTRODUCTION

The possible involvement of the nervous system in the regulation of insulin release, generally accepted now [ 41], is mainly supported by 4 types of data: (1) histological investigation ~udicates ~hat nervous fibers are present is the Langerhans islets and especially close to the beta cells [40]; this is c o ~ m e d by histochemical and auto'radiographic methods which provide evidence f o r both adrene~ic and cho~nergic fibers at this level [4,9]; ( 2 ) e l e c t r i ~ stimulation of the peripher~ vagus nerve at the cervical level induces a marked incxea~e in insulin secretion. This result first obtained 0165-1838/81/0000--0000/$02.7 5 © 1981 Elsevier/North-Holland Biomedical Press

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from estimation cf the blood glucose by Clark [3] and La Barre [22] was completely confirmed with the insulin radioimmunoa~say technique [16, 21]. Stimulation of the abdominal vagal nerve [5] or of the pancreatic net. vous branches [36] produces similareffects;(3) the use of sympathetico- or psrasympatheticomimetic substances and of alpha- or beta-block~ agents demonstrates that electricalsthnulation of: (1)the parasympathetic fibersto pancreas (vagal nerve) increases the insulin release; (2) the sympathetic fibers/to pancreas (splanchnicnerves)induces either an increase (beta effect) or a decrease (alpha effect) of insulin(seein particu~ Porte et al. [36 ]); (4) the preabsorptive insulh~ release i n d u ~ by oral glucose administration is vagal
Experiments were conducted o n 81 Wistar rats (180--320 g) and on 30 cats (2.4--3 kg) of either sex, which had fasted since the day before. All animals were anesthetized firstwith halothane and then with chlomlose (75 mg/kg, i.v.) of Nesdonal (sodium thiopentone, 15 mg/kg, i.v.) for cats, and urethane (75 mg/kg, subcutaneously) for rats. During experiments, the depth of anesthesia was controlled by observing the heart rate monitored with a loudspeaker and by testing the corneal reflex.

Surgical preparations In tracheotomized animals the right "jugular vein and the left external saphenous vein were catheterized in rats.and cats respectively in order to inject anesthetics or dnlgs. One carotid artery was isolated and catheterized in both species to sample blood; carefully under an operating microscope to avoid damsging the vagal nerve. The proximal part of small inte~ine including duodenum and upper jejunum was isolateda~era median laparotomy, performed as usual [31].

353

Intestinal perfusions Glucose solutions (I00 g glucose diluted in one literof distilledwater, i.e. 550 m Osmol/l were routinely employed. Gene:ally this solution (20 ral for cats and 2 ml for rats} was injected in the small intes*~ine with a syringe {,.injectionduration: 10 s for cats and 5 s for rats).

Blood samplings and dosage of insulin and glucose Sham-operared animals. In 5 rats and 4 cats, blood samples w~re taken every 5 rain for 20 min, which represents mo~e than the duration of an experiment (5 rain, see below). Animals were prepared as usual (hparotomy and isolation of duodenum), but no perfusion was effected. Stimulation experiments. In a first series of experiments (11 rats a.~d 9 cats), samples were taken every 20 s during the first minute follo~ring t!~e intestinal glucose perfusion, and at the second, third and fifth minutes. This series was performed in order to determine when blood insulin and glucose increased. From it, a second series of experiments was cvxried out on 20 rats and 14 cats. Distension of the small intestine. In order to determine the effect of distension on blood insulin, 2 ml saline solution (NaC1 9%c,) or distilled water was injected in the small intestine of 8 rats. In these experiments, the inferior part of isolated intestine was closed. Dosages of insulin and glucose. Samples of 200/~7 of arterial blood were taken. Heparin was added to prevent clotting and ~he samples were immediately put in ice. At the end of experiments, s e ~ m was obtained after centrifugation at 1500 rpm for 15 rain. Then samples were frozen and stored at --20°C until serum insu,in and gluc~se assays were performed. Serum IRI (Immune Reactiv Insulin) was det~rmined by double antibody radioimmunoassay (~2sI-Insuline RIA Kit from Biom~rieux). This material devoted to human insulin has a specificity o:.~ 90% in rats and 30% in cats; this was established with rat insulin (Biom~rieux) or cat insulin (extracted especially for this work by NOVO Research Institute). The blood glucose level was determined by the glucose oxidase method.

Surgical or pharmacological suppression of the pancreas innervation The suppression of both afferents and efferents of the vagus was obtained by a bilateral cervical vagotomy (10 rats). The pharmacological exclusion of vagal efferents only was obtained with atropine (100 pg/kg, i.v.) (12 rat~). The sympathetic fibers which are included in the splanchnic nerves were eliminated by a beta&locking agent (propranolol, 100 ~g/kg, i.v.)and by an alpha-blocking agent (phentolamine, 100/lg/kg, i.v.) (8 rats).

Electrical stimulation of the splanchnic efferents The left splanchnic trunk was reached through a lateral abdominal access; the nerve was stimulated above the coeliac ganglion and cut centrally (duration of each stimulu,s: I ms; voltage: 10 V; frequency: 20 impulses/s for 5 min). Blood sa.,vples were taken immediately before and after stimulation

354

(7 rats). In supplemen~ry rats, the stimulation was repeated after injection of an alpha-blocking agent (4 cases) or a beta-blocking agent (4 cases), with doses equal to these indicated above. Statistical calculations The student's r-test was used for pmxed values in order to compare the insulin changes. RESULTS

Normal values of blood insulin and glucose Rats. In sham-operated Subjects, the values of blood insulin ranged between 9 and 28/~U/ml (mean 20 +- 4). But for each animal, the vaxiations between 2 samples (5 rain intervals) were not significantly different; the same observations were made for glucose levels which appar,mtly very stable, except for a slight increase at the twentieth minute (Fig. 1A). On the other hand, when 2 control samples were taken before stimulaton (5 rain intervals, see F~g. 2), there was no significant change in glucose and insulin levels. Ca:s In sham-operated subjects, the values of blood insulin were variable (Fig. 1B) and for each animal differences between two consecutive samp;es were not significant, as in rats. Nevertheless the mean values were

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Fig. 2. Elevation of insulin and glucose blood level after intestinal perfusion with glucose in rats (A) and cat8 (B). In rats, there is a significant increase of insulin (IRD at 20 s (P < 0.05) and at 40 s (P < 0.01). In cats the increase is also present at 20 s, but the difference is significant after 40 s only (P < 0.01). The glucose begins to rise slightly at the second minute in rats and at the first minute in cats. C1 and C2: control values; the arrow indicates the end of injection.

weaker (8.0 ± 2). The control samplings before stimulation (see Fig. 2) did not either show any signifi~t difference. In conclusion, w e could assess that the spontaneous changes in blood insulin and glucose durir~ the time of one test (5 rain) were not significantly different both in rats and: cats.

Elevation of insulin and glucose blood levels after intestinal perfusion with glucose Rats. Insulin started to increase 20 s after the end of glucose peffusion. At 60 s, the insulin was comparable. At these times, the corresponding values of glucose did not change: the increase began to occur in the rat after 2 rain (Fig. 2A). Cats. The glucose increase oc~Jxted earlier than in rats since it was present at the first minute. In contrast, the insuUn changes were revealed at " 2 B) ~ the t w e n t i e t.h.~.~. (Fig. rats. m.g the time interral r ~

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356

experiments, we studied on 34 supplementary rats and cats the effect of a 100g/1 glucose solution on inmflir, levels after 1 rain in rats and 40 s in cats.

Blood insulin variations prodpmed by intestinal perfusion with glucose: complementary data In 20 rats and 14 cats, the "pure" effect o f intestinal glucose on blood insulin was on the average a significant increase (77% in rats and 44% in cats, Fig. 3A and Table IA). But in 3 other cases (2 rots and 1 cat) we noted a discre~, decrease respectively of 1 8 , 1 5 and 12%.

Effect of distension on insulin release One might think that the negative results reported above were due to the mechanical effect of perfusion. In order to verify this point, a special series o f e~:periments (cf. M ~ h ( x i s ) were performed in 8 rats.

TABLE I BLOOD IRI VARIATION8 Mean values ,+ S.E. Note that the control values in bivagotomized rats before electrical stimulation of splanchnic nerves were different from usual values. These differences might be explained respectively by ~mction and dissection of nervous trunks, n, animal number; N.S., not significant. Experiments

Control values

(A) After glucose per?us±on of the small intestine Normal rats (n = 20) 19.0 '+ 3.0 Normal cats (n = 14)

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(P< 0.01) (C) .After peripheral stimulatiot~ of the left 8planchnic ner~ ~s Normal ratg (n = 7) 9.5 + 3.0 ,5.0 4" 2.0 (1' < 0.05) Alpha-blocked rats (n ffi 4) 18.0 -+ 3.5 215.0 4" ,LO (t' < 0.05) Bet~-blocked rats (n = 4) 14.0 -+ 2.5 ~.0 -+ 2.0 (t' < 0.05)

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In all experiments, we noted a significant decrease n insulin level after intestinal distension (34% in mean, Fig. 3B and Table IB).

Effects of complete bivagotomy, i.v. administration of ~=~:ropine,propranolol and phentolamine complete cervical bivagotomy, gluco~.• perfusion did not elicit significant changes in blood insulin (Table IA and ~ig. 4A). After pharmacological exclusion of pm:a~ympathetic vagal fibers (i.v. administration of 100/lg/kg atropine) no eff,~.ct on blood insulin was noted (Table IA and Fig. 4B). After pharmacological exclusion of the syrr~)ath,~tic fibers (i.v. admini,.Itration of 100/~g/kg propranolol and 300/~g/kg phentolamine) glucose perfusion produced a significant increase in insulin, ~lightly inferior to the control effect (Table IA and Fig. 4C).

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Electrical stimulation o f the peripheral end o f the splanchnic nerve The ~ ~ v e electrical stimulation of the p e r i p h ~ end of the left splanchnic nerve induced a decrease of blood ~ . This effect was reversed after administration of phentol~nme and ~ t l y r e d u ~ after propar~lol administration. Note that in these exl~riments the control valu~ were low, in c o m ~ n with the ~ ones {Table IC), DISCUSSION ~ e present work establishesthat dt~odenum peffu~on with glucose solutions elicitsa rapid increaseof the blood insui~; this effect isalready present after the first20 s following the end of inte~t~ perfasion. This phenomenon is more marked in rats than in cats. Such a difference might be attributed to technical reasons (the radioimm~noasmy technique used gives better remits in rats than in cats) or to specificones (the normal values of basal insulin appear more ~ l e in the lat#~erthan in the former anLmals). However, this mechanism is not related t~ the direc~ action of glucose on beta cells,because it clearly precedes the beginning of glucose ~ e n t a t i o n in the systemic circulation.On the other hand, it doe~ not depend eitheron intestinalhormones released by glucose whose involvement in i n ~ secretion was expected [17~4~6]. This ~sessment is based on the 2 foUowing main reasons: (1) the stimulation of v~.~Jsnerve to ~ artificiallypeffused pancreas [10] and the stimulat~n of pancreatic nerves [36] causes in both cases an insulin secretion; (2) from the l i t ~ t u r e data [1~24], it s ~ m s that the release -- and chiefly ~he transport to p~mcreas- of intestin~ ~.~onnones need a longer t ~ e than the latencies ~eported here (20 s after ~h~nulation the insulinaugmentation isalre~y present). Therefore, the rapid insulin secretion is rledlated through the autonomic nervous system. Thus we must examine tl~¢ origin, ~he pathways and the centres implicated.

Origin of the rapid nervous regulation o f ir~sz~linrele~e It is clear that the vagal glucoreceptors recently desc~bed in the duode. num [31] are mainly involved in this mechanism, i~ndeed their functional properties (activation by the same glucose concentrations in intestinal perfusion, short htencies of responses in particular) are c o n s e n t with the characteristics of ~ e neurogenic insulin response. However, other receptors might also be implicated: (1) Other intestw~ glucoreceptors: ghcoreceptors whose afferents course in the splanchnic r~erves were identified by Sharma and Nasset [38] and Hardcastle et al. t115], but they seem to be less numerous than the afferents ascending in the vagtt~ nerves and are chiefly located in the lower part of ~ e intestin~ glucoreceptors: the hel~ do not ~eem to ir,tervene in the phenomenon d ~ b e d here because of its

359 short latency; however, these receptors may act in the rapid insulin release recently described in dogs, after i.v. injection of gluc~)se [ 16]; (3) vagat mechanoreceptors: these endings ~ e ~equently found in the intestinal region [7e23,29,35]; they are discontinuously activated by digestion during the passage of peristaltic waves; they may also be stimulated by a maintained local distension [29], b u t under our experirnent~ cond~:tions (distension of the whole duodenum), an opposite effect on blood insulin was noted.

Pathways o f the neural insulin regulation Afferent pathway. The above results indicate clearly that the afferent pathway of the neural regulation of insulin is probably entirely nervous and represented by the vagal nerves. This is confirmed by the fact that the central stimulation of a vagal nerve may induce effects similar to those produced by the peripheral stimulation (N. Mei, unpublished ob~rvations). Additionally, the total bivagotomy abolished completely the insulin changes. Efferent pathway. The neural mechanism of insulin secretion must be mediated mainly by parasympathetic fibers included in the vagus nerves. Actually, there is no significant change in insulin level after bivagotomy (suppression of both parasympathetic and sentry vagal fibers) and after atropine administration (suppression cf parasympathetic fibersonly). O n the contrary, the exclusions of sympathetic fibers included in the splanchnic nerves (i.v. injection of alpha- and beta-blocking agents) do not provoke great modifications in comparison wi~h the control values.Nevertheless,the mean decrease observed (increase of 62% in mean instead of 77%, see Table I) and some individual data do not allow us to exclude completely the sympathetic fibers. The results of the peripheral stimulation of splanchnic nerve after injection of beta- or alpha-blocking agents, show that these fibers, if they intervene, elicit the insulin reiease through a beta effect, as in other species including man [ 41]. Centres involved in the neural insulin regulation The central pathways of the nervous regulation of insulin are not yet known. However, numerous experiments i n v o l ~ destruction of brain areas have suggested for a long time [ 4 i ] that the hypothalamic area may be implicated. This was recently confirmed by electrophysiological data showing that the whole splanchnic and vagal afferents, including those originating from intestinal glucoreceptors, project on the VHM and LH regions [ 19,20,27 ]. Fig. 5. summarizes the modem data on the nervous system pathways of insulin regulation.

Role of the nervous system regulation of insulin release from ~,agal intestinal glucoreceptors F r o m the type of ~hnuhtion u ~ in th~ work (intest~l peffusion with gtucose ~ ~ i o n ) , it ~ be ~ m e d that the nervous r ~ t i o n of the

360 insulin release must occur in p h y s i o ~ c o ~ o n s , i~. d ~ digestion. ~es the~ probable role in coozdi~tion of the gastrointestinalmotility [7,37J,and their general involvement in food i n ~ e regulation [2,34], the int~ glucoreceptors piay a specific zole in insulin zeguia~n. They elicit a m p ~ stimulation of b ~ cellsw ~ h precedes the d ~ activationdue to glucose. Thus the mechanism studied here is c o m p a m b l e ~ preabsorptive reflexes, induced from the oral sphere [ 1 1 J 2 ~ 3 2 ] oz gastrointestinalregion [ 1 8 ~ 6 ] , but it dithers in its ~ h w a y which is entirely nervous. One can think that the different pmabsorptive and ~ r p t i v e reflexes act successively in order to Imve the optimal effects and to potentialize their mutual actions. Here one can mention: (1) the pmabsorptive mechanisms: the entire neural regulation described here; the neuro-humoral regulations mediated by the afferent~ pathway and the efferenth u m o m l pathway represented by either intestinal hormones [17~25] or a hypothaiamic factor [28]; (2) the post absorpti~ mechanisms: the neurohumoral regulationinitiatedby peripheralreceptors (such as hepatic ones) or by centralreceptors [7 ];the direct act on of glucose on beta cells.

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Fig. 5. Schematic representation of the n~rvous pathways involved in the nervo~ regu~tion on insulin release, a, afferent vagal )athway; b~ efferent vagal pathway; c, efferent splanchnie pathway; 1 and 2, direct eff4~ct of glucose on ~ c r e a s . ~ e o~ez putative pathways are not represen~ here (see Di ;cuuion).

361

~ n c e r n ~ the respective r o l ~ of vagal and splm-~chnic nerves we have shown that the former were predominant in our experimental conditions, i.e. during the beginning of glucose absorption at the duodenal level.But it is poss~le that the sympathetic fibers become more active ha other cho~ , for example at the end of a meal when glucose and ir~ulin are bothl b2gh [36], during moderate stress,or d ~ maintain~ distension of duodenum (which appears in abnonn~ or pathological conditions). ACKNOWLEDGEMENTS

We are greatly indebted to Mr. P. ~ e and Mrs. S. Monnier (Service commun ~ i o ~ l ~ m e n t s de I'IN~P.) for the dose of insulin, to Dr. F. Lucciani (C.R.E.P.A~.) for breeding cats and to Dr. Schlichtkrull (NOVO Research Institute) .who mpplied the cat Insulin. This work was supported by Grant ATP~omportement alimentaire humain, C ~.R.S. We thank Mrs. Famarier for her help with the English translation of this manuscript. REFERENCES

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