D E P A R T M E N T OF MEDICINE, K O L D I N G S Y G E H U S , KOLDING, D E N M A R K

BLOOD SUGAR AND GLUCOSE TOLERANCE IN ARTIFICIAL DIPHTHERIA TOXEMIA

by B . Fr i i s -Hansen, Ole Mor tensen and Niels A. Nielsen.') (Received for publication, February 8th, 1947).

In rabbits, artificially intoxicated with diphtheria toxin, pronounced changes may be demonstrated in the blood-sugar and blood-sugar curve after administration of glucose.

Mikami (1925) found that in well-fed 'rabbits a considerable hyper- glycaemia may be ascertained after the injection of diphtheria toxin in doses which will kill the animals within a day. The hyperglycaemia commences soon after the injection and reaches maximum in the course of 3-5 hours, whereupon the blood-sugar falls again. The hyperglycaemia fails to appear, if the toxin has been neutralized before injection. Lithander ( 1945) also iound a considerable hyperglycaemia four hours after injecting rabbits intravenously with 50 d. m. 1. diph- theria toxin per kilogram, a dose which kills the animals within 8-12 hours. Shortly before the death of the animals the blood-sugar falls to normal.

After a somewhat smaller dose, which would kill the rabbits in 30-48 hours, Rosenthal (1914) found a terminal hypoglycaemia. Mika- mi (1925) demonstrates hypoglycaemia 2 G 2 9 hours after the injection of diphtheria toxin. At that time rabbits are stated to be weakened or even already quite moribund. Lithander (1945) who gave rabbits 0.6 d. m. 1. diphtheria toxin per kilogram, which kills the animals in 24-36 hours found, in some cases, a slight increase in blood-sugar 8-12 hours after the injection, and in almost all animals hypogly- caemia is found 24 hours after the injection.

Following an injection of a dose of diphtheria toxin which would kill the rabbits only in the course of 3-5 days Sweeny and Lackey (1928) demonstrated that the blood-sugar had not changed. In most cases a slight terminal hyperglyczmia was found by Lawrence and Buckley (1927) , Schwentker and Noel (1930) and Yannet and Darrow

*) Aided by a grant to N. A. N. from ))Nordisk Insulinfondu.

156

(1933) in those rabbits which survived corresponding doses for more than four days, while otherwise the blood-sugar remained normal.

The glucose tolerance was examined by Sweeny and Lackey ( 19281, who found decreased tolerance in rabbits which had received a dose of diphtheria toxin that would kill the animals in 3-5 days.

In the following a series of experiments is presented, confirming the findings of earlier investigators and showing the pathological course of the glucose tolerance curve at a time when hypoglycaemia due to diphtheria intoxication has been demonstrated.

M e t hods. Rabbits of about 2 kilograms weight were used for the experiments. The

diphtheria toxin used was placed at our disposal by the State Serum Institute, Copenhagen. In all cases the toxin was administered intravenously, diluted with physiological saline.

About 24 hours before the glucose tolerance test was performed, the food was removed from the rabbits’ cage. 1 gram glucose per kilogram was given in 50 per cent solution. The solution was injected in an ear vein in the course of about one minute. The blood-samples were taken from the marginal vein of the other ear. The blood-sugar values stated were the means of duplicate analyses (Hagedorn-Norman Jensen’s method).

Results. Large doses of toxin. The effect of very large doses of diphtheria

toxin was examined in two rabbits, weighing 2.1 kilograms. 100 and 125 d. m. l., respectively, were administered. In both animals hyper- glycaemia was subsequently found, commencing about two hours after the injection and reaching maximum values of 221 and 374 mg. per 100 ml. after four and five hours, respectively, whereupon the blood- sugar showed a downward tendency. One rabbit died 8-9 hours after the injection. The other one was killed seven hours after the injection, as it was very limp and short of breath.

Medium doses of toxin. 25 d. m. 1. were administered to four rab- bits, whose glucose tolerance had previously been tested and found normal. They became hypoglycaemic 13-18 hours later, and they had either been lying slackly on their side or had convulsions. At this point a glucose tolerance test was performed, which showed that the injected glucose disappeared more slowly from the blood than normally (Fig. 1 and Table 1 ) . In immediate connection with the glucoseinjection the slackness and the convulsions vanished, enabling the rabbits to skip about in a natural way. One of the rabbits died 2% hours after the glucose injection, without any signs of hypoglycaemia, another presented hypoglycemic convulsions 2% hours after injection, while the two remaining animals showed no signs of hypoglycaemia after four and six hours. The two first rabbits mentioned, which were observed till death, survived the injection of diphtheria toxin for 20 and 20% hours respectively.

157

2

3

4

mj %

206

ioo

0

45 I 5 0 18 1 20 I 35 1 45 I 50 I 55 I 70 80 90 100 110

56 482 208 206 172 164 148

60 352 334 330 202 230 158

49 242 164 126 120 128

30 0 30 60 90 min F i g . d .

The glucose tolerance curve for rabbit No. 1 before (O---O) and 17 hours after (0-0) intravenous injection of 25 d.m.l. diphtheria toxin. At 0 in-

travenous injection of 1 gram glucose per kilogram.

Table d . Blood-Fugar in mg. per 100 ml. before and after intravenous injection of 1 gram glucose per kilogram to a rabbit, to which diphtheria toxin 25 d. m. 1.

intravenously has been administered 13-18 hours previously.

Minutes after glucose inject’on I Minutes N ~ . I before

158

s mj 4

_I 30 30 60 9Omin

Fig. 2. The glucose tolerance curve of a rabbit 24 hours after the injection of 1 d. m. 1. diphtheria toxin intravenously. At 0 intravenous injection of 1 gram glucose

per kilogram.

Small doses of toxin. In these experiments 1 d. m. 1. diphtheria toxin was administered. In two rabbits the glucose tolerance was tested 24 hours after the injection of toxin, whereupon they were killed. The curves showed reduced tolerance to glucose (Fig. 2 ) . In two other rabbits, who had received the same dose of toxin, the glucose tolerance was tested daily after the toxin injection. Daily administration of glucose to normal rabbits gave identical curves on the different days. In the two intoxicated rabbits diabetic curves were ascertained, and on the last days a slight increase in the fasting blood-sugar values (Table 2 ) .

159

Before glucose

injection

Table 2. Blood-sugar in mg. per 100 ml. before and after intravenous injection of 1 gram glucose per kilogram to a rabbit, to which diphtheria toxin 1 d. m. 1.

intravenously has been administered.

15

Hours after toxin

injection

24

48

72

96

120

30

240

290

304

336

50 70 90 110

179 165 152 142

210 165 156 141

290 242 242

287

94

117

165

152

402

376

Discussion. Comparison of the above results with those of earlier investigators,

as stated in the introduction, shows the following: Injection of such a quantity of diphtheria toxin, as kills a rabbit

within 12 hours (e. g. 100 d. m. 1.) gives pronounced hyperglycaemia. If the dose is of such a size, that the animal survives for more than

12 hours, but dies within 36 hours (e. g. 25 d. m. l.), at first a slight increase in blood-sugar appears, in some cases. Terminally a pro- nounced hypoglycaemia is found. Administration of glucose at this stage shows reduced tolerance to glucose.

After a dose, which is just sufficiently large to kill the animal in the course of a few days ( 1 d. m. I . ) , normal fasting blood-sugar with decreased glucose tolerance is found. Terminal hyperglycaemia is de- monstrated in the majority of those rabbits, that survive for more than four days.

It has been tried in various ways to explain these changes in blood- sugar regulation. One alleged cause is the altered function of the endocrine glands. E. g. Mikami ( 1925) assumes that the hyperglycaemia is due to increased secretion of adrenalin, while Lithander (1945) explains the terminal hypoglycaemia as a link in a suprarenal insuf- ficiency. But also changes in the function of the thyroid gland (Buck- ley and Lawrence, 1927) or of the pituitary (Holmes, 1939) have been mentioned, and finally a direct impairment of the liver has been alleged as the cause (Soskin, Allweis and Mirsky, 1935).

The experimental results published in this paper can not decide this question. It should be mentioned however, that the terminal hypo- glycaemia can not solely be due to suprarenal insufficiency, when, as demonstrated, it is combined with a reduction of the glucose tolerance, and that this combination of low blood-sugar and reduced glucose tolerance has also been ascertained in partially hepatectomised rabbits (Friis-Hansen, Mortensen and Nielsen, 1947).

160

Summary. Injection of diphtheria toxin in quantities, which kill the rabbits

within 12 hours, causes pronounced hyperglycaemia. After a somewhat smaller dose, which the animal survives for more

than 12 hours, but which kills it in less than 36 hours, in some cases at first a slight increase of the blood-sugar appears. Terminally hypo- glycaemia combined with decreased glucose tolerance is found.

After injection of 1 d. m. 1. diphtheria toxin normal blood-sugar and reduced glucose tolerance is ascertained. Finally hyperglyczmia is found in most of the rabbits, which survive for more than 4 days.

REFERENCES Friis-Hansen, B., 0. Mortensen & N . A. Nielsen: Acta physiol. Scand. 1947, 43,

Holmes, E.: Physiol. Rev. 1939, 49, 439. Lawrence, R. D . & 0. B. Buckley: Brit. J. exp. Path. 1927, 8, 58. Lithander, A.: Acute adrenal insufficiency in rabbits produced by some bac-

Mikami, S.: Tohoku. J. exp. Med. 1925, 6, 299. Rosenthal, F.: Arch. exp. Path. u. Pharm. 1914, 78, 99. Schwentker, F . F. & W. W. Noel: Bull. Johns Hopkins Hosp. 1930, 46, 2-59. Soskin, S., M. D . Allweis & I . A. Mirsky: Arch. int. Med. 1935, 56, 927. Sweeny, J . S. & R . W . Lackey: Arch. int. Med. 1928, 41, 257. Yannet , H . & D. C . Darrow: J. elin. Invest. 1933, 12, 767.

291.

terial toxins. Acta med. Scand. Suppl. 160. Stockholm 1945, p. 67-71.