Effect of Insulin and Epinephrine on the Eosinophil and Blood Glucose Levels in Sheep; Lack of Diurnal Rhythm

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1 I T Effect of Insulin and Epinephrine on the Eosinophil and Blood Glucose Levels in Sheep; Lack of Diurnal Rhythm M. X. ZARROW, M. E. DENISON, B. ROSENBERG, D. E. MANN, JR. AND G. M. NEHER From the Department of Biological Sciences, Purdue University, Lafayette, Indiana IS well known that the blood glucose levels of such species as the cow, sheep and goat are lower than those found in the carnivores and omnivores. Blood sugar values of 35 to 65 mg. per cent have been reported for a wide variety of adult ruminants although concentrations approaching the carnivore levels have been found in the suckling young (I). Neither pancreatectomy (2) nor alloxan injections (3) produce complete experimental diabetes; however, Jarret (4) has reported the same sequence of events in sheep as in other animals after treatment with the drug. Following alloxan injection, the initial hyperglycemic phase does not occur in the calf but a rapid and severe hypoglycemia does develop (3). In this respect the normal calf may be compared with the hypophysectomized rat (5) and the adrenalectomized rat (5) and rabbit (6). F rom these and other observations, McCandless and Dye (I, 3) concluded that the calf and possibly all ruminants possess a relative deficiency of the pituitary-adrenal gland mechanism. Such a concept is extremely attractive and could explain the low blood sugar levels in ruminants. The present invertigation is part of a study to determine whether the pituitaryadrenocortical system is relatively deficient in this group of animals, i.e. the ruminants, and which of the two glands is responsible for the deficiency. In the present investigation adult sheep were used and the eosinophil and blood glucose levels determined for the presence of a diurnal rhythm and after such stimuli as handling, epinephrine and insulin. MATERIAL AND METHODS Young adult sheep aged I to 3 years and belonging to the Dorset strain were used in this investigation. The animals were kept either in runways or on a farm and received water, hay and oats ad libitum. The animals were injected intramuscularly and blood obtained from the ear veins. Precautions were taken to insure free flowing blood and the first three or four drops were discarded. The epinephrine (Parke, Davis) was injected at a concentration of I to 1,000 and the insulin (Lilly) at a concentration of 40 units/ml. Blood glucose levels were determined by the micro method of Nelson (7) including certain modifications by Somogyi (8) and eosinophils by the method of Pilot (9). In the latter instance the blood was diluted I to IO and the eosinophils counted in 2 entire fields of a Neubauer hemocytometer. RESULTS In order to test the action of insulin and epinephrine on the blood sugar and eosinophils of the sheep, control experiments were first carried out on the effect of handling the animals. In these experiments the only stimulus involved was that of Received for publication August 25,

2 December I 952 EOSINOPHILS AND BLOOD SUGAR IN SHEEP 637 catching and holding the animal and pricking the ear. The animals were bled at o, 2, 4, 6 and 12 hours and eosinophils levels were determined. In figure I the data obtained from 5 sheep are averaged and plotted as the percentage change from the initial level. A maximum drop of 17 per cent was observed on the 6th hour and a rise of 18 per cent on the 12th hour. It would appear that the stimulus of handling had no effect on the circulating eosinophils of the blood. Two sheep were injected intramuscularly with 25 units of insulin and the blood sugar and eosinophils followed for 12 hours. Figure 2 contains the data from this experiment. It is apparent that the insulin produced a hypoglycemia within a half hour and a maximum depression of 52 per cent within 2 hours as compared with the in- Fig. I. FAILURE OF HANDLl cause an eosinopenia in sheep. :NG to!j loo- I /n 12 *so *30* +20- *lo EOSINOPHILS X-BLOOD SUGAR.* Fig. 2. BLOOD GLUCOSE and eosinophi1 levels in sheep after intramuscular injection of 25 units of insulin , / lOO- I 2 3 * w I2 itial value. Thereafter the blood sugar began to increase, returning to normal by 6 hours. The eosinophils showed a normal response to the insulin, reaching a maximum decrease of g2 per cent by the 6th hour and returning to a minus 24 per cent by 12 hours. The response to epinephrine was a bit more variable. At 250,ug. of epinephrine the eosinophils showed no decrease but when the dose was increased to 2 mg. the eosinophils showed an 80 per cent decrease in 6 hours. Both IOO and 250 pg. of epinephrine caused an increase of approximately IOO per cent in the blood glucose. Two milligrams of epinephrine caused an 80 per cent decrease in eosinophils and a rise in blood sugar from a normal of 32 mg. per cent to 178 mg. per cent, an increase of approximately fivefold (fig. 3). Blood glucose levels and eosinophils were determined in seven sheep at I I : oo A.M. and I I :oo P.M. The data in table I show the individual values for both the blood

3 638 IN. X. ZARROW ET AL. volz6nle I?I glucose and the eosinophils. It is apparent that no diurnal fluctuation exists for either the eosinophils or the blood glucose. The average blood glucose levels for the seven sheep was 52.7 mg. per cent at ICOO A.M. and 56.7 mg. per cent at II:OO P.M., an increase of 7 per cent which is certainly not significant. Examination of the data for the individual sheep indicates that four of the seven animals showed a slight increase for the night value, two showed a drop and one showed no change at all. The seven sheep showed an average eosinophil count of 197 cells per cu. mm. at I I : oo A.M. and 219 cells at 11:00 P.M., an increase of I I per cent which is not significant. Three of the animals showed no change at all and four showed a slight increase. Figure 4 presents the data obtained from two sheep that were bled morning and night for 2 consecutive days. While a slight increase occurred on the second day in both the blood % I- 2 O-EOSINOPHILS -,& 5 X-BLOOD SUGAR : 0 I- Id 5 +4O -140a u +arf ' -2o- z -4Q g -6O- u, loo- I AA " l2+% Fig. 3. BLOOD GLUCOSE and eosinophil levels in sheep following the intramuscular injection of 2 mg. of epinephrine. TABLE I. LACK OFDIURNAL RHYTHM IN EOSINOPHIL AND BLOOD GLUCOSE LEVELS OF SHEFX SHEEP NO. BLOOD GLUCOSE, MG. % EOSINOPHlLS PER CU. MM. 11 A.M. 11. P.M. 11 A.M. 11 P.M. I Average 17yo increase s3* I I 1% increase. so* I33 so-4 I I64 49-I I97 219~ sugar and to a parent. somewhat greater extent in the eosinophils, no diurnal rhythm is ap- DISCUSSION Treatment either with epinephrine or insulin prod uces an eosinope nia in the sheep and a hyperglycemia following epinephrine and a hypoglycemia fol lowing insulin. Recently both the hypoglycemic and eosinopenic response to insulin (IO) and the eosinopenic response to epinephrine (I I) have been suggested as tests for adrenocortical function in man. However, the validity of these tests is still open to question since a) eosinophils may fluctuate in the Addisonian patient (12), b) eosinopenia may be due to a nonspecific stress and not due to adrenocortical activity (13) and c) the eosinopenic reaction to insulin is not a direct one but secondary to the hypoglycemia (14). Consequently, it is impossible to draw any defin ite con elusions from t he experiments involving insulin and epinephrine except that these hormon es act in

4 i December 1952 EOSINOPHILS AND BLOOD SUGAR IN SHEEP 639 sheep as they do in the nonruminants. Such tests do not eliminate the possibility of a relative deficiency on the part of the pituitary-adrenocortical axis. It is apparent however from these results that neither the eosinophils nor the blood glucose levels of the sheep show any diurnal fluctuations. This is in agreement with Allcroft (15) who reported a diurnal variation in the blood sugar of the lactating cow but no diurnal variation in the nonlactating cow or ewe. On the other hand, diurnal rhythms have been reported for I 7-ketosteroid excretion in man (16), eosinophi1 levels in man (I 7) and mice (IS) and blood sugar levels in rats (19). These rhythms are considered as indicative of fluctuations in adrenocortical secretion and dependent on the ah-hour activity rhythm of the species involved. The greatest activity occurs during the daytime in man whereas the reverse is true of the rodent. Hence the diurnal rhythms of the two species are opposite. In both sheep and cows the lack of diurnal rhythm would tend to indicate a sluggish pituitary-adrenocortical system, insensitive to the stimulus of daytime activity. Further evidence for this concept may be seen in the failure to observe an eosinopenia in the sheep after handling Fig. 4. LACK OF A DIURNAL RHYTHM in the blood sugar and eosinophils of sheep. Determinations were made every 12 hours for 2 days. i. I- 0-EOSINOPHIL S ; X-BLOOD SUGAR (L < k! IIAM Wki IfAM DAY I DAY 2 Tl ME IPM whereas such a stimulus is sufficient to produce a marked eosinophil decrease in the normal mouse (20). The failure to obtain an eosinopenia after the handling stimulus and the failure to observe any diurnal rhythm would seem to point to a deficiency in one of these two glands. The positive response obtained with insulin and epinephrine may simply indicate that the dosage was extremely high and that the gland or glands can respond at such levels. White (2 I) has reported the presence of the adaptation syndrome in sheep following injections with formalin. This does not necessarily indicate a sensitive pituitary-adrenocortical system but might be due to the severity of the stimulus. It is of interest to note that the cortisone can produce hyperglycemia in sheep (2 2) indicating that the end organs will respond to the adrenocortical secretions. CONCLUSIONS Studies on blood glucose and eosinophil levels indicate a lack of diurnal rhythm in the sheep. Treatment with insulin or epinephrine produces the expected effects on the blood sugar and a definite eosinopenia. It is suggested that the failure to find a diurnal rhythm or to obtain any response with the stimulus of handling is added evidence for the theory that sheep possess a relative deficiency in the pituitary-adrenal mechanism. REFERENCES I. MCCANDLESS, E. L. AND J. A. DYE. Am. J. Physiol. 162: 434, COOK, E. T., J. A. DYE AND E.L. MCCANDLESS. Am.J.PlzysioZ. 156: 349, MCCANDLESS, E. L. AND J. A. DYE. Am. J. Physiok 156: 355, 1949.

5 640 M. X. ZARROW ET AL. Volume r * JARRETT, I. G. Atistralian J. Exper. Biol. & M. SC. 24: 95, KIRSCHBAUM, A., L. J. WELLS AND D. MOLANDER. Proc. Sot. Exper. Biol. & Med. 58: 294, GOLDNER, M. G. AND G. GOMORI. Endocrinology 35: 241, NELSON, N. J. Biol. Chew. 153 : 375, SOMOGYI, M. J. Biol. Chem. 195: 19, * PILOT, M. L. Am. J. C&z. Path. 20: 850, IO. PERLMUTTER, M. AND M. MUFSON. J. C&z. Endocrinol. II: 277, II. RECANT, L., D. M. HUME, P. H. FORSHAM AND G. W. THORN. J. C&z. Endocrinol. IO: I 87, 12. BONNER, C. D. J.A.M.A. 148: 634, SPIERS, R. S. AND R. K. MEYER. Endocrinology 48: 316, TSAI, S. Y., A. BENNET, L. G. MAY AND R. L. GREGORY. Proc. Sot. Exper. Biol. & Med. 74: 782, ALLCROFT, A. M. Biochem. J. 27: 1820, PrNCUS, G. J. Cl&. Endocrinol. 3: 195, =7- FLINK, E. B. AND F. HALBERG. 34th Meeting of the Endocrine Society, HALBERG, F. AND M. B. VISSCHER. Proc. sot. Exper. BioZ. & Med. 75: 846, =9* PITTS, G. E. Am. J. Physiol. 139 : 109, SPIERS, R. S. AND R. K. MEYER. Endocrinology 45: 403, WHITE, I. G. J. Endocrinol. 7: 143, 195 I. 22. HOLM, L. W., J. A. HOWARTH AND J. KELLEHER. Am.J. Vet. Research 12:314, 1951.