J. Physiol. (1978), 280, pp. 9-23 9With 8 text-figuresPrinted in Great Britain

THE ROLE OF THE AUTONOMIC NERVOUS SYSTEMIN THE CONTROL OF GLUCAGON, INSULIN AND PANCREATIC

POLYPEPTIDE RELEASE FROM THE PANCREAS

BY S. R. BLOOM,* A. V. EDWARDSAND R. N. HARDY

From the Physiological Laboratory,University of Cambridge, Cambridge CB2 3EG and the

*Department of Medicine, Royal Postgraduate Medical School,Hammersmith Hospital, London W12

(Received 6 January 1978)

SUMMARY

1. The mechanisms of release of pancreatic glucagon, insulin and pancreaticpolypeptide (PP) in response to hypoxia and to 2-deoxyglucose have been investi-gated in conscious calves 3-5 weeks after birth.

2. A single injection of 2-deoxyglucose (200 mg/kg i.v.) produced an abrupt risein the concentrations of pancreatic glucagon, insulin and PP in the arterial plasma.The changes in plasma insulin and PP concentration were unaffected by priorsection of the splanchnic nerves but were effectively abolished by atropine (0.2 mg/kg i.v.). The rise in plasma pancreatic glucagon concentration was prevented incalves with cut splanchnic nerves that were given atropine but neither procedurealone suppressed the response.

3. 2-deoxyglucose also caused a substantial increase in the output of glucocorti-coids from the right adrenal gland together with a pronounced rise in adrenalblood flow. There was also a small but significant increase in catecholamine outputfrom the adrenal medullae in these animals.

4. Intense hypoxia caused a pronounced increase in the concentration of PP inthe arterial plasma. This was found to resemble the glucagon response to intensehypoxia in that it persisted in animals with cut splanchnic nerves that were givenatropine. Less intense hypoxia caused a rise in plasma pancreatic glucagon con-centration (but not PP) that was abolished by section of the splanchnic nerves.The changes in plasma insulin concentration in these experiments were consistentwith the conclusion that they were secondary to changes in plasma glucose concen-tration.

5. It is concluded that pancreatic endocrine responses to both moderate hypoxiaand 2-deoxyglucose are mediated by the autonomic innervation.

INTRODUCTION

It is now generally accepted that both adrenergic and cholinergic stimulationmodify the rates at which the pancreatic hormones are released (see for instanceWoods & Porte, 1974). Stimulation of either the sympathetic or parasympathetic

S. R. BLOOM, A. V. EDWARDS AND R. N. HARDY

innervation to the gland provokes release of glucagon (Bloom, Edwards & Vaughan,1973, 1974; Bloom & Edwards, 1975; Kaneto, Miki & Kosaka, 1974) whereasinsulin release appears to be promoted by vagal, cholinergic or f-adrenoceptorstimulation (Frohman, Ezdinli & Javid, 1967; Kaneto, Kosaka & Nakao, 1967;Daniel & Henderson, 1967; Porte, 1967; Alric, Loubatieres-Mariani, Loubatieres &Puech, 1972; Iversen, 1973), but is inhibited by a-adrenoceptor or sympatheticstimulation (Coore & Randall, 1964; Malaisse, 1971; Bloom et al. 1973; Bloom &Edwards, 1975). More recently, evidence has been obtained suggesting that theparasympathetic innervation is implicated in the release of pancreatic polypeptide(PP) from the gland (Adrian, Bloom, Besterman, Barnes, Cook, Russell & Faber,1977; Schwartz, Holst, Fahrenkrug, Kuhl, Lindkaer-Jensen, Rehfield, Schaffalitskyde Muckadell, Stadil & Vagn-Nielsen, 1977; Bloom, Edwards & Hardy, 1978).The present paper describes experiments which were devised to assess the extent

to which these efferent autonomic pathways are implicated in the pancreatic endocrineresponses to hypoxia and hypoglycaemia in the calf. Insulin has been found toinhibit glucagon release under certain circumstances (Marks & Samols, 1968), forwhich reason 2-deoxyglucose has been employed to mimic insulin hypoglycaemiaand yet provide an effective stimulus to the glucoreceptors in the central nervoussystem (Hokfelt & Bydgeman, 1961; Himsworth, 1968). The results show that thepancreatic endocrine responses to both moderate hypoxia and 2-deoxyglucosedepend on the integrity of the autonomic innervation in this species. The responseto moderate hypoxia is mediated by the splanchnic nerves, whereas release of bothinsulin and PP in response to 2-deoxyglucose is blocked by atropine. Both sympatheticand parasympathetic mechanisms are involved in the release of pancreatic glucagonthat occurs in response to 2-deoxyglucose.Some of these results have been published previously in a preliminary form

(Edwards & Bloom, 1978).METHODS

Animals

The experiments were carried out on pedigree Jersey and Jersey x Charollais calves whichwere obtained from local farms shortly after birth and used at ages ranging between 15 and46 days (24.7-39-4 kg body weight). The animals were kept in individual pens in the laboratoryanimal house and maintained on a diet of milk (6-7 pints/day). Food was withheld for at least6 hr before surgery and for a period of between 12 and 14 h before each experiment.

Experimental procedures

Preparatory surgery involved the insertion of narrow bore catheters into the saphenousarteries so that the tips lay in the abdominal aorta. These catheters were used subsequentlyto monitor aortic blood pressure and for collection of arterial samples. When required, a braunulacannula was placed in the right jugular vein and both splanchnic nerves were cut immediatelybehind the diaphragm. In addition, animals were fitted with an 'adrenal clamp' using a techniquethat has been described in detail previously (Edwards, Hardy & Malinowska, 1974), in orderthat adrenal responses to 2-deoxyglucose could be quantified. Procaine penicillin (600,000 i.u.)and dihydrostreptomycin (0.5 g) (May & Baker Ltd, Dagenham) were administered routinelybefore surgery.

Experiments were carried out 24 hr after surgery, and at the same time of day in order tominimize diurnal variation. Animals destined to be exposed to hypoxia were habituated towear a light, transparent, loosely-fitting, 'helmet' which was constructed of aluminium andpolyethylene. This fabrication measured 29 x 29 x 54 cm and was perfused with air at a rate

10

NEURAL CONTROL OF PANCREATIC HORMONESof 15 litres/min. In one series of experiments intense hypoxia was induced by perfusing nitrogenalone through the 'helmet' at a rate of 15 litres/min for periods of up to 10 min. Otherwisecomparatively mild hypoxia was induced by perfusing a mixture of equal volumes of nitrogenand air at the same rate for 30 min.When required, a 20% solution of 2-deoxyglucose (w/v) was administered by rapid i.v.

injection at a dose of 200 mg/kg. In some experiments atropine (atropine sulphate, B.D.H.)was given by i.v. injection (0-9% (w/v) NaCi, 0 1 g/100 ml) at a dose of 0-2 mg/kg 10 minbefore either hypoxia was initiated or 2-deoxyglucose administered.Heart rate and aortic blood pressure were monitored continuously, by means ofa Devices L221

pressure transducer connected to a Devices M19 or M2 recorder. Only one experiment wascarried out on any individual calf.

E8timationsSamples of arterial blood were collected anaerobically for blood gas and pH estimations and

into heparinized tubes containing aprotonin (Trasylol, Bayer; 1000 K.i.u./ml blood) for otheranalyses. These tubes were centrifuged at 4 0C immediately and the plasma then stored at-20 0C.Pancreatic glucagon was measured by a radioimmunoassay using an antiserum relatively

specific for pancreatic glucagon which was C terminal reacting (Assan & Slusher, 1972); thisassay reacted less than 5% with glucagon-like immunoreactivity of ileal origin (enteroglucagon).Insulin and PP were also measured by radioimmunoassay (Albano, Ekins & Turner, 1972;Adrian, Bloom, Bryant, Polak, Heitz & Barnes, 1977). 2-Deoxyglucose was estimated colori-metrically using the malonaldehyde-thiobarbituric acid reaction (Waravdekar & Saslaw, 1959),and glucose by means of a Beckman Glucose Analyser, correcting for interference by 2-deoxy-glucose when necessary. Adrenal blood flow was measured gravimetrically and glucocorticoidswere estimated by competitive protein binding after preliminary separation using SephadexLH20(Malinowska, Hardy & Nathanielsz, 1972). Adrenaline and noradrenaline were measured bya modification of the method of Euler & Floding (1955) as described previously (Bloom,Edwards, Hardy, Malinowska & Silver, 1975a). Blood P02, Pco2 and pH were measured,immediately after samples had been collected, using standard Radiometer equipmentequilibrated at 38-5 0C.At the end of each experiment small pieces of liver were removed for glycogen analysis

(Edwards, 1971). These estimations established that the concentration of glycogen in the liverexceeded 10 mg/g in each animal in this series.

Statistical analyses were made according to the methods of Snedecor & Cochran (1967).

RESULTS

Pancreatic endocrine responses to 2-deoxyglucoseEndocrine responses to 2-deoxyglucose were examined in five conscious, un-

restrained 3-5 week old calves. It was administered as a single dose (11 mmol/kg)by rapid I.v. injection at time = 0 (Fig. 1). The arterial plasma 2-deoxyglucoseconcentration was 4-20 + 0-23 mmol at 5 min and fell approximately exponentiallythereafter to 0-58 + 0 04 mmol at 120 min. The concentrations of plasma pancreaticglucagon, glucose and insulin rose as shown in Fig. 1. The most rapid and pronouncedof these changes was that in mean pancreatic glucagon concentration, whichincreased steadily from 42-9 + 10.0 pmol/l to a peak incremental value of 151-7 + 15.5at 20 min and had returned to normal at 90 min. This is roughly equivalent to thechange in plasma glucagon that occurs in response to stimulation of the sympatheticinnervation to the pancreas at 2-0 Hz for 10 min in the calf at this age (Bloom et al.1973). By comparison the changes in mean plasma glucose and insulin concentrationwere more sluggish but also more persistent. In each case a significant increase wasapparent after 10 min. The peak incremental values for glucose were achieved at

11

12 S. R. BLOOM. A. V. EDWARDS AND R. N. HARDY75 min (1.60 + 0-17 mmol/l) and for insulin at 90 min (522 + 120 pmol/l) and theinitial concentration was restored between 210 and 240 min after administration of2-deoxyglucose (Fig. 1).The extent to which these responses depend on the integrity of the autonomic

nervous system was examined in six calves of the same age in which both splanchnic

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2

800BE0

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Fig. 1. Comparison of the changes in plasma 2-deoxryglucose, glucose, glucagon andinsulin concentration in normal calves (O. n = 5) with those in calves with cutsplanchnic nerves given atropine (@ n = 6) in response to 2-deoxyglucose (200 mg/kg).Vertical bars: s.c. of each mean value. 2-Deoxyglucose was administered by rapid I.v.injection at time = 0 (vertical arrow).

nerves had been cut and atropine (0.2 mg/kg) was given by rapid i.v. injection10 min before administration of 2-deoxyglucose. The subsequent changes in theconcentration of this glucose analogue in the arterial plasma were closely similarto those in the control group (Fig. 1) showing that the characteristics of the stimuluswere unaffected by autonomic blockade. Mean basal plasma insulin concentrationwas significantly lower in the calves with cut splanchnic nerves given atropinethan in the control group (P < 0@002; Table 1) whereas mean plasma glucose and

NEURAL CONTROL OF PANCREATIC HORMONES 13

glucagon concentrations were apparently unaffected by these procedures. The risein plasma pancreatic glucagon concentration was effectively abolished by autonomicblockade. Comparison of the mean increments at 20 min shows that there is a highlysignificant difference between the two groups (control: 151P7 + 15-5 pmol/l; experi-

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100

E E 50

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1000

0f 800-~S600-

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Fig. 2. Comparison of the changes in plasma 2-deoxyglucose, glucose, glucagon andinsulin concentration in calves with cut splanchnic nerves (O. n = 6) with those incalves given atropine (@, n = 6) in response to 2-deoxyglucose (200 mg/kg). Verticalbars: s.E. of each mean value. 2-Deoxyglucose was administered by rapid i.v. injectionat time = 0 (vertical arrow).

mental: 40 0 + 18 0 pmol/l: P < 0 002) and that the mean values for calves withcut splanchnic nerves, given atropine, did not rise significantly above normal atany stage during the experiments.The hyperglycaemic response was reduced but not abolished by autonomic

S. R. BLOOM, A. V. EDWARDS AND R. N. HARDY

blockade, presumably due to a reduction of glucose exit rate in the presence of2-deoxyglucose, but it should be noted that differences between the mean valueswere not statistically significant in these small groups. The initial rise in plasmainsulin concentration was absent in the experimental group, in which the levelrose slowly at a gradually increasing rate to a maximum at 90 min, after which itslowly declined. This slow rise and fall would be consistent with a delayed responseof the pancreatic fi cells to the observed change in plasma glucose concentration.

TABLE 1. Basal plasma glucose, glucagon, insulin and PP concentration in 3-5 week oldcalves immediately before administration of 2-deoxyglucose

Glucose Glucagon Insulin PP(mmol/l) (pmol/l) (pmol/l) (pmol/l)

Normal controls(n = 5) 4.1+±03 42-9+10-0 90-7+14-4 408±5-0

Cut splanchnic nervesand atropine (n = 6) 46+0-3 53-0+8-2 22-5+4±4 26-8+9±1

Atropine alone(n = 6) 4-3+0±2 44*1+7*3 42-6+6.6 21-8+10-4

Cut splanchnic nervesalone (n = 6) 45 + 0-2 25-5 ± 7-8 78.5 + 29-2 45-3 + 14-9

The responses to 2-deoxyglucose of calves given atropine alone, or with cutsplanchnic nerves without atropine, are illustrated in Fig. 2 and the mean basalconcentrations of glucose and the pancreatic hormones are tabulated in Table 1.Mean plasma insulin concentration was significantly lower than that of the controlgroup in calves given atropine, but not in animals with cut splanchnic nerves,whereas neither the plasma glucose nor glucagon concentration was significantlyaffected by either procedure. Atropine also caused an abrupt fall in mean plasmaPP concentration both in calves with cut splanchnic nerves (43.8 + 13-5 to 26-9 + 9.1pmol/l in 10 min; n = 6) and in intact calves (49.3 + 15-0 to 21-8 + 10-4 pmol/l in10 min; n = 6). These differences are not statistically significant for these smallgroups, but provide a complete explanation for the comparatively low plasma PPconcentration in the atropinized groups immediately before 2-deoxyglucose wasgiven (Table 1).The changes in the concentration of 2-deoxyglucose in the plasma were closely

similar to those observed in normal animals (Fig. 1) but the results show that therise in plasma glucagon concentration was not prevented by blocking either thesplanchnic sympathetic, or the parasympathetic innervation alone. In animals withcut splanchnic nerves rather wide variations between individuals were encounteredbut the mean concentration rose steadily to a peak incremental value of 211-2 + 79-1pmol/l at 60 min and declined slowly thereafter. This rise was substantially greaterthan that in normal animals, which might be related to the difference in the hyper-glycaemic responses of the two groups. Thus, in the normal controls, administrationof 2-deoxyglucose caused an abrupt rise in plasma glucose concentration (Fig. 1),which would tend to inhibit the pancreatic cells directly, whereas there was noappreciable hyperglycaemic response in the calves with cut splanchnic nerves(Fig. 2). These results strongly suggest that the difference between the hyperglyeaemic

14

NEURAL CONTROL OF PANCREATIC HORMONES

responses of calves with cut splanchnic nerves (Fig. 2) and those given atropine inaddition (Fig. 1) is related to insulin release. Complete autonomic blockade effectivelyinhibited the release of both glucagon and insulin from the pancreas in response to2-deoxyglucose for about 30 min, by which time the full extent of the rise in plasmaglucose concentration was realized. Administration of 2-deoxyglucose in calves withcut splanchnic nerves, which were not given atropine, produced an abrupt increasein plasma insulin concentration, which had risen by 354 + 147 pmol/l at 10 minand was associated with a slight fall in mean plasma glucose concentration (Fig. 2).

80

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CD-

80-

40

0=

-30 0 30 60 90 120 150 180 210Time (min)

Fig. 3. Comparison of the changes in plasma pancreatic polypeptide concentrationin groups of 35 week old calves given 2-deoxyglucose (200 mg/kg). 0O normalcontrols (n = 5). 0, cut splanchnic nerves and atropine (n = 6). O. cut splanchnicnerves alone (n = 6). *, atropine alone (n = 6). Horizontal bars: s.E. of each mean

value. 2-Deoxyglucose was administered by rapid i.v. injection at time = 0 (verticalarrow).

It is therefore concluded that the rise in plasma glucose concentration in responseto 2-deoxyglucose, observed in calves with cut splanchnic nerves given atropine,represents a base line response uncomplicated by any rapid change in pancreaticendocrine activity. In the absence of atropine it is restrained by simultaneous insulinrelease (in spite of the associated release of pancreatic glucagon; Fig. 2), whereasit is enhanced in intact animals, presumably due to hepatic glycogenolysis consequentupon release of pancreatic glucagon and increased activity of the sympatheticinnervation to the liver (Edwards, 1972) but not to release of catecholamines fromthe adrenal medullae (see Figs. 4 and 5).The changes in plasma insulin concentration in these four groups of animals

were entirely consistent with the view that there is a rapid release of insulin inresponse to 2-deoxyglucose that is abolished by atropine and is therefore mediatedvia the parasympathetic innervation. This is followed by a later phase of secretion,the extent and time course of which depend on the rise in plasma glucose con-centration.

15

16 S. R. BLOOM, A. V. EDWARDS AND R. N. HARDY

2-Deoxyglucose also leads to a rapid rise in the concentration of PP in the arterialplasma. In normal calves and calves with cut splanchnic nerves the mean PPconcentration had risen by between 52-8 + 18-9 and 87-8 + 34 0 pmol/l to a peak at20 min (Fig. 3). This response was completely abolished by the administration ofatropine.Adrenal responses to 2-deoxyglucose were examined in five 3-5 week old calves

that had been fitted with an 'adrenal clamp' (Edwards et al. 1974) the previous

~~:, 200 ~A 4

7 0

20 E- ~ ~~ ~ ~ ~ ~ ~ ~ ~ ~ ~

E- 10 -

E~ 1-0

0 0

500-

400-a~Eo 300-

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a100

00

-30 0 30 60 90 120 150 180 210 240Time (min)

Fig. 4. Mean responses to 2-deoxyglucose (200 mg/kg) in five calves 3-5 weeks afterbirth. Above: changes in haematocrit (-) and right adrenal blood flow (0). Centre:changes in plasma cortisol (Q) and corticosterone concentration (@). Below: changesin cortisol (0), corticosterone (@) and catecholamine (A) output from the rightadrenal gland. Vertical bars: s.E. of each mean value. 2-Deoxyglucose was administeredby rapid i.v. injection at time = 0 (vertical arrow).

day. The outputs of both cortisol and corticosterone rose rapidly in response to2-deoxyglucose and peak values of 403 + 83 and 196 + 54 ng . kg-1 min-' respectivelywere observed at 20 min, after which the output subsided steadily towards normalduring the next 100 min. The concentration of cortisol, but not corticosterone, alsorose significantly in the peripheral plasma, reflecting the increase in output of thissteroid from the gland (Fig. 4B) and there was an associated rise in the rightadrenal blood flow (Fig. 4A). The changes in mean aortic blood pressure duringthese experiments were comparatively small, indicating that the rise in adrenal

NEURAL CONTROL OF PANCREATIC HORMONES 17

30r

> Lo10~ 01

-30 0 30 60 90 120 150 180 210 240Time (min)

Fig. 5. Comparison of the changes in adrenaline (0) and noradrenaline (0) outputfrom the right adrenal gland in five calves 3-5 weeks after birth in response to 2-deoxy-glucose (200 mg/kg). Vertical bars: s.E. of each mean value. 2-Deoxyglucose wasadministered by rapid i.v. injection at time = 0 (vertical arrow).

20C 0c-20

608o-480 A I /

Q~-80

200 B

150 -

C-

0 0

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100

_ 50EFEOC 0

0 - 0E -05 -

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.~. 200 -

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-10 0 10 20 30 40 50 60Time (min)

Fig. 6. Comparison of the changes in arterial Po2, plasma glucagon, glucose andinsulin concentration in 3-5 week old calves in response to moderate hypoxia. 0,normal calves (n = 5). *, cut splanchnic nerves (n = 6). Vertical bars: s.E. of eachmean value. Horizontal bar: duration of hypoxia.

S. R. BLOOM, A. V. EDWARDS AND R. N. HARDYblood flow was due to vasodilatation of adrenal blood vessels. There was also anabrupt increase in mean haematocrit (Fig. 4A), the latency and duration of whichwere closely similar to the changes in adrenal cortical activity.A small but significant increase in the outputs of adrenaline and noradrenaline from

the adrenal medulla was observed, peaking at 10-20 min (Fig. 5). The absolute amountsof the two amines that were released (adrenaline 24 + 6 ng . kg-1 min-'; noradrenaline7 + 3 ng. kg-' min-' at 20 min) were too small to produce any detectable physio-logical effect. However, the adrenal medullary response to 2-deoxyglucose resembledthat to hypoglycaemia in the conscious calf in that adrenaline was the predominantamine (Bloom et al. 1975).

20

C-20C.0N -60-80

C 40OE

0.- -40-10 0 10 20 30 40 50 60

Time (min)

Fig. 7. Mean plasma PP concentration and changes in arterial Po2 in five normal3-5 week old calves exposed to moderate hypoxia. Vertical bars: S.E. of each meanvalue. Horizontal bar, duration of hypoxia.

Pancreatic endocrine responses to hypoxiaPancreatic endocrine responses to hypoxia were investigated in calves habituated

to wear a light helmet through which the desired gas mixture was perfused (seeMethods). Moderate hypoxia was induced by perfusing the 'helmet' with a mixtureof equal parts of air and nitrogen for 30 min, which had the effect of reducing thePo, of the arterial blood by roughly 70% over this period (Fig. 6A). In normalcalves this procedure causes a substantial rise in plasma glucagon concentration(Bloom, Edwards & Hardy, 1977). This response was unaffected by administrationof atropine but was completely abolished by section of both splanchnic nerves whichalso reduced the rise in mean plasma glucose concentration (Fig. 6B, C). In eachgroup there was a delayed rise in mean plasma insulin concentration, the extent ofwhich was related to the rise in plasma glucose concentration (Fig. 6C, D). Noevidence was obtained to support the contention that insulin is released as a directconsequence of hypoxia.No significant change in plasma PP concentration was observed in response to

18

NEURAL CONTROL OF PANCREATIC HORMONES 19

moderate hypoxia (Fig. 7) but a more intense hypoxic stimulus, imposed for ashorter period, produced an abrupt rise in plasma PP concentration (Fig. 8). Closelysimilar changes in plasma PP concentration were found to occur in calves withcut splanchnic nerves that were given atropine (0-2 mg/kg) and exposed to hypoxiaof the same intensity. It is therefore concluded that release of PP under theseconditions is not mediated by the autonomic innervation.

40-20 -

Cvo.5-20-~e-40-

, -60 --80 -

60

E40

E 20-

0-

E

0 30 60 90 120Time (min)

Fig. 8. Comparison of the changes in arterial P02 and plasma PP concentration inresponse to intense hypoxia in 3-5 week old calves. 0O normal controls; n = 6. *,cut splanchnic nerves and atropine (0.2 mg/kg); n = 4. Vertical bars, s.E. of eachmean value. Horizontal bar, duration of hypoxia.

DISCUSSION

The extent to which either parasympathetic or sympathetic pathways are impli-cated in pancreatic neuroendocrine responses appears to depend on both the modalityand the intensity of the stimulus.The results of the experiments described in this paper show that the release of

insulin and PP in response to intravenous injections of 2-deoxyglucose in 3-5 week.old calves is completely blocked by atropine. In contrast, release of pancreaticglucagon, in response to the same stimulus, can only be blocked by the administrationof atropine in calves in which both splanchnic nerves have been cut. This is inaccordance with the known stimulator effect of both the parasympathetic andsympathetic innervation on pancreatic glucagon release (Bloom et al. 1973, 1974)and indicates that both pathways were activated in these experiments.The sympathetic system is relatively insensitive to hypoglyeaemia; section of

both splanchnic nerves has no effect on subsequent glucagon release during moderate

S. R. BLOOM, A. V. EDWARDS AND R. N. HARDY

transient hypoglyeaemia (0.1 u. insulin/kg) in the calf (Bloom et al. 1974) and inman the glucagon response to insulin hypoglyeaemia is unaffected by adrenergicblockade (Walter, Dudl, Palmer & Ensinck, 1974). However, the magnitude of theadrenal responses in calves with 'adrenal clamps' in the present experiments suggeststhat this dose of 2-deoxyglucose represents a relatively intense hypoglycaemicstimulus. The rise in mean glucocorticoid output from the right adrenal gland andthe increase in adrenal blood flow were roughly equivalent to those observed inresponse to an infusion of corticotrophin at a dose of 5 0 ng. kg-' min-' (Edwards,Hardy & Malinowska, 1975). Furthermore, the rise in glucocorticoid output fromthe gland greatly exceeded, and that of catecholamine approximately equalled, thatobserved during the intense hypoglycaemia that develops over the first 60 minafter a large dose of i.v. insulin (4-0 u./kg) in calves of the same age (Bloom et al.

1975).Activation of the sympathetic system in response to 2-deoxyglucose has been

reported in various species including rodents, dogs and primates (HMkfelt & Bydge-man, 1961; Altszuler, Dunn, Steele, Bishop & de Bodo, 1963; Lipman, Ulvedal,Bradley & Lecocq, 1970; Smith, Gibbs, Strohmayer & Stokes, 1972; Muller, Froh-man & Cocchi, 1973; Niijima, 1975).As during feeding (Bloom, Edwards, Hardy, Malinowska & Silver, 1975b), or

exposure to moderate hypoxia or hypercapnia (Bloom et al. 1977), sympatheticactivation leads to the release of quite trivial, physiologically ineffective, amounts ofadrenaline and noradrenaline from the adrenal medulla in the calf. Hitherto, thehyperglyeaemic response to 2-deoxyglucose has generally been attributed to reflexrelease of adrenaline (Sakata, Hayama & Sloviter, 1963; Muller et al. 1973; Woods& Porte, 1974). This was evidently not so in the present experiments, in which therise in plasma glucose concentration is attributable to several separate effects: first,direct interference by 2-deoxyglucose with glucose uptake; secondly, the glyco-genolytic effect of glucagon released from the pancreas as a result of both vagaland sympathetic activation; thirdly, hepatic glycogenolysis stimulated via thesympathetic innervation to the liver (Edwards, 1972) and finally it is likely to havebeen restrained by the hypoglycaemic effect of insulin released in response to increasedvagal stimulation. We therefore conclude that it is no longer justified to ascribe thehyperglycaemic response to 2-deoxyglucose solely to release of adrenaline fromthe adrenal medulla.

It seems that the PP secreting cell resembles thea cell in that it is directlysensitive to hypoxia and responds by release of hormone if the stimulus is sufficientlyintense (Bloom et al. 1977) even after complete autonomic blockade. Both types ofcell appear to be more sensitive to the direct effect of hypoxia than the chromaffincells of the adrenal medulla, from which release of catecholamines is effectivelyabolished by section of the splanchnic nerves in the calf at this age (Bloom, Edwards,Hardy & Silver, 1976; Bloom et al. 1977). The changes in plasma insulin concen-

tration which occurred during and after hypoxia were consistent with the conclusionthat release of insulin from the pancreas under these conditions was invariablysecondary to hyperglyeaemia, as found previously (Bloom et al. 1977).

Less intense hypoxia, such as might be encountered under physiologicalconditions at altitude, caused release of pancreatic glucagon, but not of PP,

20

NEURAL CONTROL OF PANCREATIC HORMONES

and the glucagon response was mediated by the splanchnic sympathetic innervation.There is virtually no release of adrenal catecholamines in the calf in responseto this level of hypoxia (Bloom et al. 1977) showing that release of pancreaticglucagon represents much the more sensitive of these sympathetic neuroendocrineresponses to hypoxia, at least in this species. The sensitivity of the a cell to stimu-lation via the sympathetic innervation is such (Bloom et al. 1973; Bloom & Edwards,1975) that any stimulus that causes an increase in the tonic activity of these nervefibres must stimulate secretion of pancreatic glucagon, as during moderate hypoxiaor after 2-deoxyglucose in the present experiments. Glucagon release in responseto other non-specific stimuli such as haemorrhagic hypotension, denervation ofcarotid baroreceptors in anaesthetized cats (Jarhult, 1975; JArhult & Holst, 1977)or exercise in man (JArhult, Holst & Ingemansson, 1977) appear to be mediatedby the sympathetic innervation and glucagon levels in the plasma are elevated ina wide variety of stressful situations (Bloom, 1973; Woods & Porte, 1974).

It seems likely that both divisions of the autonomic innervation to the pancreascontribute to the maintenance of glucagon output at rest. Insulin is tonicallyinhibited by sympathetic and adrenergic activity (Woods & Porte, 1974; Bloom &Edwards, 1975; Girardier, Seydoux & Campfield, 1976) and the present resultssuggest that the parasympathetic system is implicated in maintaining a basalsecretion of both insulin and PP. The evidence now available indicates that enhancedrelease ofglucagon, insulin and PP in response to such 'glyeaemic' stimuli as moderatehypoglycaemia, 2-deoxyglucose and food ingestion is mediated exclusively via thevagal innervation in the calf (Bloom et al. 1974; Bloom et al. 1978). These findingssupport the contention that, under physiological conditions, the autonomic nervoussystem plays an important part in controlling the activity of pancreatic endocrinecells (Bloom et al. 1975).

This work has been supported by grants from the Agricultural Research Council, the BritishDiabetic Association, the Medical Research Council and the Wellcome Trust. It is necessaryto acknowledge the important part played in this work by Miss V. Kenyon and Messrs T. E.Adrian and P. M. M. Bircham.

REFERENCES

ADRIAN, T. E., BLOOM, S. R., BESTERMAN, H. S., BARNEs, A. J., CooK, J. V. C., RUSSELL,R. C. G. & FABER, R. G. (1977). Mechanism of pancreatic polypeptide release in man.Lancet i, 161-163.

ADRIAN, T. E., BLOOM, S. R., BRYANT, M. G., POLAK, J. M., HEITZ, P. H. & BARNES, A. J.(1976). Distribution and release of human pancreatic polypeptide. Gut 17, 940-944.

ALBANO, J. D. M., EKINS, R. P. & TURNER, R. C. (1972). Sensitive precise radioimmunoassayof serum insulin relying on charcoal separation of bound and free hormone moieties. Actaendocr., Copenh. 70, 487-509.

ALRic, R., LOUBATIS:RES-MARIANI, M. M., LOUBATItRES, A. L. & PUECH, R. (1972). R6cepteurscholinergiques et s6cr6tion de glucagon. Etude sur le pancreas isol6 et perfus6 du rat.C. r. Seanc. Soc. Biol. 166, 1030-1033.

ALTSZULER, N., DuNN, A., STEELE, R., BISHOP, J. S. & DE BODO, R. C. (1963). Effect of 2-deoxy-glucose on glucose turnover in normal and adrenalectomized dogs. Am. J. Physiol. 204,1008-1012.

AsSAN, R. & SLUSHER, N. (1972). Structure/function and structure/immunoreactivity relation-ships of the glucagon molecule and related synthetic peptides. Diabetes 21, 843-855.

BLOOM, S. R. (1973). Glucagon, a stress hormone. Postgrad. Med. J. 49, 607-614.

21

S.R. BLOOM, A. V. EDWARDS ANDR. N. HARDYBLOOM, S. R. & EDWARDS, A. V. (1975). The release of pancreatic glucagon and inhibition of

insulin in response to stimulation of the sympathetic innervation. J. Physiol. 253, 157-173.BLOOM, S. R., EDWARDS, A. V. & HARDY, R. N. (1977). Adrenal and pancreatic endocrine

responses to hypoxia and hypercapnia in the calf. J. Physiol. 269, 131-154.BLOOM, S. R., EDWARDS, A. V. & HARDY, R. N. (1978). The role of the autonomic nervoussystem in the control of pancreatic endocrine responses to milk ingestion in the calf. J.Physiol. 280, 37-53.

BLOOM, S. R., EDWARDS, A. V., HARDY, R. N., MALiNOWSKA, K. W. & SILVER, M. (1975a).Endocrine responses to insulin hypoglycaemia in the young calf. J. Phy8iol. 244, 783-803.

BLOOM, S. R., EDWARDS, A. V., HARDY, R. N. & SILVER, M. (1975b). Cardiovascular andendocrine responses to feeding in the young calf. J. Physiol. 253, 135-155.

BLOOM, S. R., EDWARDS, A. V., HARDY, R. N. & SILVER, M. (1976). Adrenal and pancreaticendocrine responses to hypoxia in the conscious calf. J. Physiol. 261, 271-283.

BLOOM, S. R., EDWARDS, A. V. & VAUGHAN, N. J. A. (1973). The role of the sympatheticinnervation in the control of plasma glucagon concentration in the calf. J. Physiol. 233,457-466.

BLOOM, S. R., EDWARDS, A. V. & VAUGHAN, N. J. A. (1974). The role of the autonomicinnervationin the control of glucagon release in the calf. J. Physiol. 236, 611-623.

Coops;, H. G. & RANDLE, P. J. (1964). Regulation of insulin secretion studied with pieces ofrabbit pancreas incubated in vitro. Biochem. J. 93, 66-78.

DANIEL, P. M. & HENDERSON, J. R. (1967). The effect of vagal stimulation on plasma insulinand glucose levels in the baboon. J. Physiol. 192, 317-326.

EDWARDS, A. V. (1971). The glycogenolytic response to stimulation of the splanchnic nerves inadrenalectomized calves, sheep, dogs, cats and pigs. J. Physiol. 213, 741-759.

EDWARDS, A. V. (1972). The sensitivity of the hepatic glycogenolytic mechanism to stimulationof the splanchnic nerves. J. Physiol. 220, 315-334.

EDWARDS, A. V. & BLOOM, S. R. (1978). Neural control of pancreatic hormones. In Gut Hormones,ed. BLOOM, S.R. Edinburgh: Churchill Livingstone. (In the Press.)

EDWARDS, A. V., HARDY, R. N. & MALINOWSKA, K. W. (1974). The effects of infusions ofsynthetic adrenocorticotrophin in the conscious calf. J. Physiol. 239, 477-498.

EDWARDS, A. V., HARDY, R. N., MALINOWSKA, K. W. (1975). The sensitivity of adrenalresponses to synthetic adrenocorticotrophin in the conscious unrestrained calf. J. Physiol.245, 639-653.

EULER, U. S. VON & FLODING, I. (1955). A fluorimetric micromethod for differential estimationof adrenaline and noradrenaline. Acta phy8iol. 8cand. 33, suppl. 118, 45-56.

FROHMAN, L. A., EZDINLI, E. Z. & JAVID, R. (1967). Effect of vagotomy and vagal stimulationon insulin secretion. Diabetes 16, 443-448.

GIRARDIER, L., SEYDOUX, J. & CAMPFIELD, L. A. (1976). Control of A and B cells in vivo bysympathetic nervous input and selective hyper or hypoglycaemia in dog pancreas. J. Physiol.,Paris 72, 801-814.

HImSWORTH, R. L. (1968). Compensatory reactions to a lack of metabolizable glucose. J. Physiol.198, 451-465.

HO6EELT, B. & BYDGEMAN, S. (1961). Increased adrenaline production following administrationof 2-deoxy-D-glucose in the rat. Proc. Soc. exp. Biol. Med. 106, 537-539.

IVERsEN, J. (1973). Effect of acetylcholine on the secretion of glucagon and insulin from theisolated, perfused canine pancreas. Diabetes 22, 381-387.

JARHULT, J. (1975). Role of the sympatho-adrenal system in haemorrhagic hypoglycaemia.Acta physiol. scand. 93, 25-33.

JARULT, J. & HOLST, J. J. (1977). Stimulation of glucagon and inhibition of insulin secretionevoked from carotid baroreceptors. Experientia 33, 326-327.

JARHULT, J., HOLST, J. J. & INGEMANSSON, S. (1977). Physiological evidence for direct sympa-thetic nerves to the endocrine pancreas in man. Acta endocr., Copenh. suppl. 212, Abstract 154,p. 102.

KANETO, A., KosARA, K. & NAKAO, K. (1967). Effects of stimulation of the vagus nerve oninsulin secretion. Endocrinology 80, 530-536.

KANETO, A., MIKI, E. & KosBAA, K. (1974). Effects of vagal stimulation on glucagon andinsulin secretion. Endocrinology 95, 1005-1010.

22

NEURAL CONTROL OF PANCREATIC HORMONES 23

LiPrmA, R. L., ULVEDAL, F., SCHNURE, J. L., BRADLEY, E. M. & LEcOCQ, F. R. (1970). Gluco-regulatory hormone response to 2-deoxy-D-glucose infusion in normal subjects at bedrest.Metabolism 19, 980-987.

MALAISSE, W. J. (1972). Hormonal and environmental modification of islet activity. In Hand-book of Physiology, Endocrine Pancreas, sect. 7, vol. 1, pp. 237-260. Washington D.C.: Amer.Physiol. Soc.

MALNowsEA, K. W., HARDY, R. N. & NATHANIELSZ, P. W. (1972). Neonatal adrenocorticalfunction and its possible relation to the uptake of macromolecules by the small intestine ofthe guinea pig and rabbit. J. Endocr. 55, 397-404.

MARYs, V. & SAmoLs, E. (1968). Glucose homeostasis. In Recent Advances in Endocrinology,ed. JAMES, V. H. T. chap. 4, pp. 111-138. London: Churchill.

MULLER, E. E., FROHMAN, L. A. & Coccm, D. (1973). Drug control of hypoglycaemia andinhibition of insulin secretion due to centrally administered 2-deoxy-D-glucose. Am. J.Physiol. 224, 1210-1217.

NIIJIMA, A. (1975). The effect of 2-deoxy-D-glucose and D-glucose on the efferent dischargerate of sympathetic nerves. J. Physiol. 251, 231-243.

PORTE, D. JNR. (1967). Beta adrenergic stimulation of insulin release. Diabetes 16, 150-155.SAxATA, K., HAYANA, S. & SLOVITER, H. A. (1963). Effect on blood glucose concentration of

changes in availability of glucose to the brain. Am. J. Physiol. 204, 1127-1132.SCHWARTZ, T., HOLST, J. J., FAHRENKRUG, J., KUHL, C., LINDxAER-JENSEN, S., REHFELD,

J. F., SCAFFALITSKY DE MUCKADgLL, 0. B., STADIL, F. & VAGN-NIELSEN, 0. (1977). Vagal,cholinergic regulation of pancreatic polypeptide secretion. Acta endocr. Copenh. suppl. 212,Abstract 161, p. 106

SNEDECOR, G. W. &-CocHRN, WV. G. (1967). Statistical Methods, 6th edn. Ames: Iowa StateCollege Press.

SmITE, G. P., GIBBS, J., STROHMAYER, A. J. & STOKES, P. E. (1972). Threshold doses of 2-deoxy-D-glucose for hyperglycaemia and feeding in rats and monkeys. Am. J. Physiol. 222, 77-81.

WALTER, R. M., DUDL, R. J., PALMER, J. P. & ENSINCK, J. W. (1974). The effect of adrenergicblockade on the glucagon responses to starvation and hypoglycaemia in man. J. clin. invest.54, 1214-1220.

WARAVDEKAR, V. S. & SASLAw, L. D. (1959). A sensitive calorimetric method for the estimationof 2-deoxy sugars with the use of the malonaldehydethiobarbituric acid reaction. J. biol.Chem. 234, 1945-1949.

WOODS, S. C. & PORTE, D. JNR. (1974). Neural control of the endocrine pancreas. Physiol. Rev.54, 596-619.