Since, for many months after section of the right vagus in the neck,

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1 THE INFLUENCE OF THE VAGUS ON THE ISLETS OF LANGERHANS. Part II. The effect of cutting the vagus upon sugar tolerance. BY G. A. CLARK. (From the Physiological Laboratory, Sheffield University.) IN Part I of this paper (1) experimental evidence was given, which supported the view of de Corral(2) and of Macleod and his co-workers(3), that the right vagus contains secretory fibres to the islets of Langerhans. Britton(4) has recently confirmed the earlier observations, using methods which eliminate sources of error that were possible in the experiments of de Corral and Macleod. The significance of these secretory fibres, however, remains obscure. Since, for many months after section of the right vagus in the neck, rabbits appear perfectly healthy, gain in weight and show no obvious change in blood-sugar level, it is clear that the essential function of the islets in the metabolism of sugar is carried on in spite of the nerves having been cut. The production of insulin must continue, and the islets themselves apparently respond to variations in the composition of the blood with suitably varying activity, this local adjustment being sufficient at any rate for the needs of animals leading a placid life in confinement. In other circumstances the rich nerve supply which de Castro(5), Gentes (6) and Pensa (7) have traced from the right vagus to the islets may intervene. When, for instance, a relatively large amount of glucose is rapidly introduced into the circulation, it seems possible that a reaction might be set up somewhere that leads to such intervention. The methods employed and precautions adopted in the present investigation are similar to those previously described (1). When the vagus was cut in the neck, the section was always made below the origin of the cardiac branches, and a piece of the nerve removed to prevent regeneration, which might otherwise have occurred in those experiments of long duration. If secretory nerves to the islets play a part in the normal secretion of insulin, a decreased sugar tolerance would be expected after section of those nerves, for it is evident that an increased supply of insulin is necessary to deal with the hyperglyctemia resulting from the intravenous injection of glucose. Tests, in which 1 grm. of pure glucose per kilo of body weight was injected intravenously in 10 p.c. solution

2 VAGUS AND SUGAR TOLERANCE. into rabbits, gave no indication of decreased tolerance following right vagotomy within a period of about two months. On the contrary, in a fewcases an increase in tolerance was suggested, and in order to investigate this, a more severe test was imposed. The animal was given four intravenous injections of 1 grm. of glucose per kilo at two-hourly intervals and the blood-sugar estimated every half-hour after the last injection. In the normal rabbit thus treated the blood-sugar does not return to the fasting level for 3 hours or more (Table I and Fig. 1), but about 14 days after section of the right vagus the fasting level is reached within 1 or 1 hours (Table I and Fig. 3). In Table I the initial blood-sugar levels in all cases before vagotomy appear high, because, at the time of observation, the hyperglyceamia due to the third injection had not passed off: after vagotomy, the normal fasting level had already been reached when the fourth injection was given, two hours after the third. The fasting sugar level is given in a separate column. No tolerance test was done earlier than 10 days after operation. Hours after last Rabbit injection 1 Before vagotomy 2 Before 3 BefOre, 4 BefOre, Av. *25 K 120 *15 SBefore,, 6 Before (, ibefore 0 *140 *102 *121 *120 *138 *113 *131 *244 '195 *224 *206 *308 '230 *221 *229 *212 TABLE I. 1 *183 *122 '202 *117 *261 *181 *100 *184 1* *144 *112 *175 *120 *210 *123 *160 *121 *171 2 *138 *102 *165 *108 *177 *124 *152 *104 * *133 *113 *145 *123 *150 *120 *148 *113 *138 3 *131 *137 *136 *134 '116 *125 *114 *219 *106 *112 *094 '100 *115 *134 *242 '202 *172 *156 *143 *133 '114 *216 '115 '112 *106 *114 ('116) ' '20 '20 '15 '15 * Fasting B.-S. *105 ' *115 *118 I112 *112 '106 ' 117 (.126) ('106).101 I,.J, Hours Fig. 1. PH. LXI. '10 L, I 10 Hours Hours Fig. 2. Fig

3 578 G. A. CLARK. A striking condition of increased tolerance is thus found in every one of the five rabbits on which the test was successfully carried out. The comparison of the average values at the different times before and after vagotomy shows this clearly. The result in each case conforms to these averages. This condition, however, was found to pass gradually off, and after about two months the hyperglycaemia following a single injection of glucose (1 grm. per kilo) began to last longer than is the case in normal animals, that is to say, the increased tolerance regularly found during the first weeks after vagotomy is succeeded by diminished tolerance. This is still, in the four rabbits that have been studied longest, evident after 21, 25, 33 and 55 weeks respectively; this second phase of the changes induced by vagotomy, a condition of diminished tolerance, seems, therefore, to be permanent. Table II gives the response of one of these rabbits to 1 grm. of glucose per kilo at different intervals after operation and the figures are typical of those found in the other cases. It will be noticed that there is a tendency for the fasting sugar level to rise to a maximum at about the fourth month after vagotomy, thereafter returning to a lower level. In the last two tests given it will be seen that the return of the curve to the normal level is interrupted by a secondary rise; this was also seen in the other cases. TABLE II. Hours after glucose 0 i 1 lj 2 2j 3 Before vagotomy *116 * *100 *106 *118 *120 '245 *132 *106 *115 *120,, 5 weeks *115 '208 *116 '103 *120 *116 11,, *123 *240 '159 *136 '124 16, 126 '252 *150 *138 '135 '130 20,, '121 '236 '177 '135 *122 * '114 *262 *145 '149 '130 '115 41,, *106 *232 '145 *150 *127 *117 55,, *105 *213 '137 '140 '135 '115 These two stages in the tolerance changes are, therefore, definite and distinict. It is evident that section of secretory nerves to the islets of Langerhans will not explain the first of them, the increased tolerance during the first weeks after the operation: the section must interfere with other influences affecting the sugar in the blood. Some of the ways in which the phenomenon might be accounted for have been examined. 1. As the recurrent laryngeal fibres were in all cases severed in the vagus trunk, it was necessary to eliminate any possible effect of this

4 VAGUS AND SUGAR TOLERANCE. 579 section on the thyroid gland bearing on the result. In three rabbits, therefore, the right recurrent laryngeal nerve alone was cut after the normal response to a fourth glucose injection had been determined as described above. Two weeks later the response to the test was unaltered (Table III and Fig. 2), but two weeks after subsequent division of the right vagus trunk two animals showed the typical increased tolerance (Table III and Fig. 3). Rabbit 9 died under the aneesthetic during section of the vagus. TABLE III. Rabbit Hours after last injection 0 i 1 1* 2 2i 3 (Normal *308 *261 *210 *177 *150 *136 3 Sect. rt. rec. laryng. n. *142 *243 *173 *160 *152 *142 *133 (,, vagus 4120 * Normal 4138 *221 *181 *160 *152 *148 *134 4 Sect. rt. rec. laryng. n. * * ,, vagus *113 *229 *100 *121 * *116 9 s Normal * *152 *144 *140 *132 Sect. rt. rec. laryng. n. *136 *234 * *166 *173 * The increased tolerance following section of the right vagus might be due to a diminution of the normal rate at which sugar leaves the liver; this hypothesis involving in turn the supposition that in the right vagus are contained, either fibres to the liver that directly stimulate the breakdown of glycogen into sugar, or, alternatively, fibres to the suprarenal which by increasing the discharge of adrenaline would produce the same effect indirectly. It seemed that it might be possible to test this conjecture by the reaction to insulin. If cutting the right vagus arrests the normal delivery of sugar into the blood-stream by the liver, the fall in blood-sugar, brought about in the reaction to insulin injected after this *operation, might be more rapid than in the normal animal. Two experiments were, therefore, done in which the blood-sugar was estimated -every half-hour after *1 c.c. insulin (B.D.H.), diluted 1 in 10, was given intravenously before, and about 14 days after section of the right vagus. It will be seen from Table IV that in neither case was a greater degree of TABLE IV. Hours after insulin 0 i i i Rabbit 10 Normal *108 *081 -~104 *073 * *076 * vagotomy * * *103 *106 *093 * *099 *102 *103 Rabbit 11 Normal *093 - * * * *062 - *059 *070 *082 *091 vagotomy * '060 * A *

5 580 G. A. ClARK. hypoglycsemia observed after vagotomy. All the tests were made under identical conditions, and each was repeated at an interval of 2 or 3 days. 3. Or, thirdly, since it appears that in some conditions, the placid life of a hutch rabbit for instance, no gross disturbance of the regulation of the sugar content of the blood is to be detected, after cutting the right vagus, and the islets must, therefore, be still functioning and acted on by variations in the composition of the blood, it is not impossible that as the acini of the pancreas are stimulated to secretion by something brought to them in the blood, and, nevertheless, receive a supply from the vagus of augmentor and also inhibitory nerves, so, too, the islets may receive inhibitory as well as augmentor fibres from the vagus. If this were so, the fact that departures from the normal level of blood-sugar are almost always, if not always, in the direction of hyperglyceemia would indicate that inhibition of the islets was the rule and augmentation the exception. Cutting the vagus in that case might increase the reaction of the islets to a sudden rise of blood-sugar, and in this way the increased tolerance during the first weeks after the operation might receive an explanation. A test of this hypothesis was planned as follows: since excess of sugar in the blood is more quickly removed after section of the right vagus, it might be expected that the hyperglyceemia, resulting from injection of adrenaline, would in that case also be of shorter duration. Two experiments were, therefore, done in which *5 c.c. adrenaline (P. D. and Co.) diluted 1 in 4 was injected subcutaneously before, and 2 weeks after cutting the right vagus, and the blood-sugar estimated every half-hour for 5 hours. It will be seen from Table V that the average hyperglyceemia is essentially the same in the two conditions. At first sight this may appear to oppose the theory of tonic inhibitory fibres to the islets, for, after section of the vagus, the readier supply of insulin would be expected to combat more efficiently the hyperglycaemia following adrenaline. It is obvious, however, that the secretion of insulin depends on an adequate blood-supply to the islets, and there is no evidence that adrenaline has not the same constrictor action on the vessels of the pancreas that it has elsewhere. The greater secretion of insulin, therefore, that is otherwise available after right vagotomy may be held in check by diminution of TABLE V. Rabbit Hours afteradrenaline 0 i 1 lj 2 2j j 5 increase,- 12 Before vagotomy *100 * *237 *226 * *188 *135 *114 * *114 *170 *229 *234 *230 *234 *230 *196 *163 *122 *103 * Before *115 *185 *239 * * *236 *198 *161 *134 M,, *258 *301 *310 *325 * *233 *166 *132 *140 Av.

6 VAGUS AND SUGAR TOLERANCE. the blood supply to the islets due to the actioh of adrenaline on the vessels. Incidentally it may be noted that the results given above do not support the view of Snyder, Wells and Culley(s) that the vagus contains fibres which exert an inhibitory influence on glycogenolysis, for, if their suggestion is correct, an increased hyperglyca3mic response to adrenaline would be expected after section of the vagus. The presence in the right vagus of both inhibitory and secretory fibres to the islets of Langerhans would offer an explanation of the failure of earlier workers(2,3) to produce consistently a hypoglycwemia on stimulating this nerve. B ritton(4) was able in every case to cause a lowering of blood-sugar by electrical stimulation, but observes that "in a few experiments a brief latent period with slight or no decline in the bloodsugar level following stimulation was sometimes observed." Moreover, in his experiments, the initial sugar level was abnormally high, and it is to be expected, therefore, that any agency tending to reduce this level to normal (i.e. stimulation of secretory fibres) would predominate over one which might raise it still further (i.e. stimulation of inhibitor fibres). That the two sets of fibres are not equally stimulated by drugs is suggested by a comparison of the action of pilocarpine and of guanidine, both of which produce a fall in blood-sugar by vagal stimulation if ergotamine has been previously given to paralyse the sympathetic (1). If degree of salivation be taken as an index of parasympathetic stimulation, it has repeatedly been observed that 1 mg. per kilo of pilocarpine will produce profuse salivation but a moderate fall in blood-sugar, while *1 grm. per kilo guanidine hydrochloride causes little or moderate salivation but a considerable fall in blood-sugar. These facts suggest that pilocarpine may stimulate both sets of fibres. It has previously been shown (9) that atropine will paralyse the secretory fibres to the islets, and the results given in Table VI suggest that this drug also paralyses the inhibitory. The figures given indicate the response of two animals to the last of four glucose injections given at two-hourly intervals, in one case each injection being preceded by *25 mg. atropine sulphate intravenously. It will be seen that where atropine has been given the Hours after fourth TABLE VI. Fasting Rabbit injection 0 i 1 lj B.-S. 14 j Without atropine *126 *227 *155 *134 *127 *122 * With,, *104 *211 *147 *102 *100 *100 * Without,, *127 *242 *166 *153 *145 *131 *125 )103 With,, *103 '233 ' '102 *100 _ 1 581

7 582 G. A. CLARK. rabbit shows increased sugar tolerance. The experiments indicate that normally the inhibitor fibres play a more important part than the secretory. Experiments are being carried out on other lines from which a decision may be obtained. For the second and later result of section of the vagus when the quickened recovery from hyperglycaemia gives place to one that is unduly delayed, some other factor must be responsible. Observations that point to the influence of the pituitary being here concerned will form the subject of another paper. SUMMARY. 1. vagotomy the sugar tolerance is for some weeks increased, and not diminished. If the nerve fibres to the islets in the vagus were entirely secretory nerves this would not be expected. The possibility of the vagus containing also fibres that exert a tonic inhibition upon the islets has been considered. 2. This increased tolerance after vagotomy gradually declines and is later followed by diminished tolerance. The explanation for this phenomenon is beiug investigated. I wish to express my thanks to Professor Lea the s for much helpful criticism in this work. The expenses of this research have been defrayed by a grant from the Medical Research Council. REFERENCES. 1. Clark. This Journ. 59. p de Corral. Ztschr. f. Biol. 68. p McCormick, Macleod and O'Brien. Trans. Roy. Soc. Can. 17. p Britton. Amer. Journ. Physiol. 74. p de Castro. Rech. Biol. de l'universit6 de Madrid, 21. p Gentes. C. R. Soc. Biol. 54. p Pensa. Arch. Ital. de Biol. 44. p Snyder, Wells and Culley. Amer. Journ. Physiol. 66. p Clark. This Journ. 58. p