Catecholamine response of chickens to exogenous insulin and tolbutamide

Catecholamine response of chickens to exogenous insulin and tolbutamide

Comp. Biochem. Physiol., 1973, VoL 45A, pp. 141 to 147. Pergamon Press. Printed in Great Britain CATECHOLAMINE RESPONSE OF CHICKENS TO EXOGENOUS INSU...

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Comp. Biochem. Physiol., 1973, VoL 45A, pp. 141 to 147. Pergamon Press. Printed in Great Britain

CATECHOLAMINE RESPONSE OF CHICKENS TO EXOGENOUS INSULIN AND TOLBUTAMIDE ROBERT P. PITTMAN and ROBERT L. HAZELWOOD Depa, tment of Biology, University of Houston, Houston, Texas 77004 (Received 3 August 1972)

Abstract--1. An investigation was made of what role adrenomedullary secretions play in "protecting" chickens from the hypoglycemic effects of exogenous insulin. 2. Adult single-comb White Leghorn chickens were injected either with saline, beef insulin, chicken insulin or sodium tolbutamide. Doses of the hypoglycemic agents were sdected to produce equivalent nadirs and durations of hypoglycemia over a 60-min observation period. 3. Plasma norepinephrine levels were depressed consistently only in the group injected with chicken insulin (0"74__.0"23 vs. 0"10_+0 09/~g/l.) while epinephrine levels increased eighffold (3.4 vs. 27.3/zg/1.). Beef insulin injections increased plasma epinephrine levels fourfold (3.4 vs. 14-1 gg/l.) but were unchanged (3"4 vs. 4"1 I~g/l.) in both tolbutamide and saline groups. 4. It was concluded that catecholamine release may afford adult chickens some resistance to pharmacological doses of insulin but hypoglycemia per se is not a major determining factor in evoking epinephrine secretion. The distinction between hypoglycemic action and insulin release by tolbutamide is suggested in Ayes. INTRODUCTION

sheep or pork insulin has been described particularly for the chicken and, to a certain extent, has been attributed to plasma factors which interfere with the cellular expression of hormone action (Chen et al., 1945; Hazelwood, 1965; Pittman, 1970; Hazelwood et al., 1971). Prominent among the differences between Ayes and Mammalia in responding to injected mammalian insulin(s) is the degree of hypoglycemia and, in Ayes, the relative absence of hypoglycemic convulsions or central nervous system (CNS) dysfunction (Hazelwood, 1965, 1971). Studies of the role which cerebrospinal fluid may play in protecting the CNS of chickens from hypoglycemic crises have not explained this avian "resistance" to exogenous insulin (Anderson & Hazelwood, 1969; Hunzicker & Hazelwood, 1970); furthermore, cardiovascular alterations or shifts of body fluid volumes do not appear of sufficient magnitude to explain further this tolerance of the hormone (Pittman, 1970; Pittman & Hazelwood, 1971). However, during the latter studies, a consistent tachycardia was observed soon (less than 15 rain) after insulin injections of 1, 10 or 50 U/kg body weight. Additionally, alterations of other cardiovascular parameters indicated the possibility of

AVlA.~ "resistance" to pharmacological doses of beef,




compensatory catecholamine release during insulin-induced hypoglycemia, a possibility which appeared to merit investigation (Pittman, 1970; Pittman & Hazelwood, 1971). Such adrenomedullary secretion could very well "buffer" the rapid glycemic nadir usually effected by insulin injection through action of the catecholamines as hepatic glycogenolytic agents. The purpose of this report is to communicate results of an investigation on the adrenomedullary response of chickens to equivalent degrees of hypoglycemia produced by injections of beef insulin, chicken insulin and a sulfonylurea, sodium tolbutamide. MATERIALS AND METHODS Female single-comb White Leghorn chickens (1"2-1"8 kg each) used in this experiment were maintained at 24+ I°C and were kept under a periodic light schedule of 12 hr light per day; Purina feed (growing mash) and water were given ad lib. Glucose was determined by Nelson's modification of the Somogyi method (1952) with equal volumes of 10% NaWO, and ~ N H,SO, used as deproteinization agents. Estimation of norepinephrine and epinephrine was accomplished as described by Anton & Sayre (1962) with the developed fluorescense measured on an Amines-Bowman Fluorometer (Model 4-8100). The activatlon-fluorescence wavelengths were 400-519 m/~ for norepinephrine and 422-529 m/z for epinephrine. Saline, beef insulin (Iletin, Regular U-40, Eli Lilly), 10 units/kg body weight, chicken insulin 2 (lot Ch-I-36), 8"16 units/kg body weight or sodium tolbutamide (Orin~e Diagnostic, Upjohn), 40 mg/kg body weight, was injected into a femoral vein exposed under sodium pentobarbitot anesthesia. Lidocaine (1% solution) was injected (i.m.) into the area of surgery. Blood samples for glucose determination were drawn immediately before saline, insulin or tolbutamide injection and again at termination of the experiment. Terminal blood samples of 12.5 ml for catecholamine determination were taken from the cannulated femoral artery of two similarly prepared birds and pooled. Because of the large amount of blood required to determine circulating avian catecholamines, chickens were sacrificed at different times after injection in repeated experiments to obtain sufficient data to analyze and evaluate. All experiments were carried out in controlled temperature, humidity, light and sound conditions. Each "run" consisted of at least one saline control bird and one each of the three experimental birds. Terminal blood samples were drawn 7"5, 15, 30 and 60 rain following insulin or tolbutamide injection and 0, 15 and 60 rain following saline injection. Statistical analysis of significance for the plasma norepinephrine data was calculated using single classification analysis of variance (ANOVA). The statistical analyses of plasma epinephrine and plasma glucose levels were performed using a least square regression analysis method with the test for significance of the regression coefficient as described by Sokal & Rohlf (1952). Test of homogeneity of slope between two regression lines was performed to determine whether or not two experimental procedures resulted in similar rates of change of plasma epinephrine or glucose. RESULTS

The doses of insulin and tolbutamide selected in this study were based on pilot studies which indicated that equal hypoglycemic nadirs would result. I n this manner it was hoped that a distinction could be made between a possible catecholamine effect in response to the magnitude and/or duration of hypoglycemic conditions from that response which might occur in response to the presence of the non-homologous (beef) hormone. As observed in Fig. 1, doses of 10.0 U beef








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FIG. 1. Adult single-comb White Leghorn chickens were injected at zero time either with saline, chicken insuhn, beef insulin or sodium tolbutamide and sacrificed at varying times over the 60-min observation period. Each point represents the mean of four to six replicate rims, each on pooled (two birds) samples; a total of sixty-eight chickens were employed. Vertical bars are S.E.M. and simultaneous plasma norepinephrine data are presented in Table 1.

insulin, 8-2 U chicken insulin and 40 mg sodium tolbutamide/kg body weight, respectively, caused equal degrees and durations of hypoglycemia in adult chickens, there being no significant (ANOVA) differences among the three experimental groups. Regression equations representing plasma glucose depression after injection of beef and chicken insulin were: Y = 42-36-75.92X and Y = 25.36-57.80X, respectively. Slopes of these two plasma glucose regression equations were found to be homogeneous (Fe~ao= 0.59 < < F0.05(1.eo) = 4.00). Simultaneous with plasma glucose depression, consistent alteration of plasma norepinephrine levels was observed only after injection of chicken insulin (Table 1), there being no statistically (ANOVA) significant differences among the other three groups throughout the observation period. Linear equations representing plasma epinephrine increases (Fig. 1) after beef and chicken insulin injections, however, were calculated and found to be: Y-- 1.18+6.93X and Y = -3.82+15.73X, respectively. Slopes of these two regression equations representing epinephrine





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release to plasma were significantly different (Foao = 16.87>F0.05u,6o~= 4.00) indicating that there was a greater release of epinephrine after chicken insulin injection than after injection of the beef hormone. Tolbutamide-induced hypoglycemia (Fig. 1) did not increase epinephrine levels above control values even though the plasma glucose decrement was indistinguishablefrom the hypoglycemia produced by the two insulin preparations. DISCUSSION The presence of increased epinephrine release in response to insulin injection is well documented in many mammals (e.g. Goldfein et al., 1958; Sarcione et al., 1963; Alexander et aI., 1969). Furthermore, marked alterations in cardiovascular parameters (including heart rate, cardiac output and blood pressures) occur concomitant with the period of hypoglycemia (Arner et al., 1963; Werk et al., 1961; Hiatt et al., 1970; Pittman, 1970). In the only report employing birds Leibson (1965-66) reported a marked decrease in epinephrine concentration in the adrenal gland (histochemical evidence) of 8-day-old chicks previously injected with insulin, suggesting that plasma levels may have increased simultaneously. Very few reports exist in the literature which are concerned with circulating catecholamines in Aves. However, the possibility that involvement of the adrenal gland might afford protection to the bird against the physiological impact of large doses of insulin was indicated by workers who reported the abolishment of an hyperglycemic rebound to chronic insulin injections by adding a blocking agent, dibenzyline hydrochloride, to chicken feed (Hazelwood & Lorenz, 1959). More recently, indications that adrenomedullary secretions may be increased during the early minutes following insulin injection were observed as marked changes occurred in heart rate and, to a lesser extent, in other cardiovascular indices (Pittman & Hazelwood, 1971). Finally, since avian plasma factors, though reducing effective non-avian insulin activity, account only for slightly more than half of the so-called "avian resistance" to non-avian insulin (Hazelwood, 1971; Hazelwood et al., 1971), the possibility of adrenomedullary compensation during insulin-induced hypoglycemia appeared even more appealing. The present experimental design did not allow for a comparison of the rate of catecholamine release from the adrenal gland with that of plasma epinephrine levels; in the latter regard it is known that body fluid volumes are not altered significantly by insulin (Pittman & Hazelwood, 1971). However, if the rate of plasma glucose decrement, or the magnitude (or duration) of hypoglycemia after insulin injection, determines the magnitude of catecholamine release to plasma, equal hypoglycemic responses in chickens might be expected to be attended by equal plasma catecholamine elevations. Pilot studies indicated that the doses of insulin and tolbutamide employed herein would produce equivalent rate, depth and duration of blood glucose decreases; statistical analysis of these glucose data verified that such indeed occurred in the present study. Thus, the difference in epinephrine release to plasma in response to three different (though equal in hypoglycemic potential) agents suggests that any explanation must be based upon the



nature of each hypoglycemic substance. Since chicken insulin encouraged greater epinephrine release than did beef insulin, the presence of the heterologous protein (hormone) per se does not explain the difference in catecholamine response. The present observations may well indeed indicate the greater sensitivity of the chicken adrenomedullary tissue to the homologous insulin molecule and suggest future investigation of the effects of insulin on enzymes regulating catecholamine synthesis within the avian adrenal gland. Also, interfering avian "plasma factors" possibly could explain the difference in adrenal catecholamine response to the two insulin molecules, the difference being due to the complexed or inactive moiety of beef insulin as compared with the uninhibited chicken hormone (Hazelwood et al., 1971). Tolbutamide was employed in this study to parallel in effect the injection of chicken insulin. Despite the marked hypoglycemia the failure of this sulfonylurea to alter the level of either catecholamine was unexpected and again possibly emphasizes the specificity of avian adrenomedullary response to hypoglycemic agents. Certainly, hypoglycemia per se is obviated as a predisposing condition to catecholamine release in chickens by these observations. While tolbutamide has been found to be hypoglycemic in depancreatized and enterectomized birds (except geese), suggesting a possible non-pancreatic insulin source in Ayes (Mirsky & Gitelson, 1957, 1958; Hazelwood, 1958, 1971), the lack of catecholamine response observed here during a profound tolbutamide hypoglycemia may indicate that this sulfonylurea, or its major metabolites (carboxytolbutamide and hydroxymethyltolbutamide), is hypoglycemic independent of its insulin release potential. Such a divorce of sulfonylurea actions (insulin release vs. hypoglycemic activity) has been established in mammalian systems (Feldman & Lebovitz, 1969a, b). Certainly, this possibility merits further investigation. In conclusion, avian "resistance" to large doses of mammalian insulin appears to be due not only to the previously reported plasma factor(s) but also to compensatory adrenomedullary discharge of epinephrine. Release of the cateeholamine appears independent of hypoglycemia per se. Like the previously reported sensitivity of chickens to the hypoglycemic potential of the homologous hormone, chicken insulin is more effective in elevating eatecholamine levels than is the bovine counterpart. Acknowledgements---This work was supported by N.S,F. Grants Nos. GB-6012and GB, 8457. The hormone, generously donated by Dr. J. R. Kimmel, was assayed to have 19.6 IU/mg by the mouse convulsion test and contained less than 0.5% glueagon as a contaminant.


R. W., KUGF~AL., KERTH W. J., HARRISON J. • GERBODE F. (1969)Adrenal

catecholamine and cortisol secretion during extracorporeal circulation in dogs..7. Thoracic Cardiovas. Surg. ~ , 250-258. Ar~m~soN D. K. & HAZELWOODR. L. (1969) Chicken eerebroapinalfluid: normal composition and response to insulin administration, ft, Physiol,, Land. 202, 83-95.



ANTON H. A. & SAYR~D. F. (1962) A study of the factors affecting the aluminum oxidetrihydroxyindole procedure for the analysis of catecholamines. J. Pharmac. exp. Thee. 138, 360-375. ARN~ B., I-tm)Nga P., KARL~ORS T. & WESTLXNGH. (1963) Haemodynamic changes and adrenal function in man during induced hypoglycemia. Acta Endocrinol. 44, 430 AA.2. CHXN K. K., ANDF_~SONR. C. & MAZ~ N. (1945) Susceptibility of birds to insulin as compared with mammals. J. Pharmac. exp. Thee. 84, 74-77. FELDMANJ. M. & L~SOVITZH. E. (1969a) Appraisal of the extrapancreatic actions of sulfonylureas. Archs Intern. Med. 123, 314-322. FELDMAN J. M. & LESOVITZ H. E. (1969h) Biological activities of tolbutamide and its metabolites. Diabetes 18, 529-537. GOLDFI~ A., ZIL~LI M. S., DmPOINTES R. H. & ]3grHUNEJ. E. (1958) The effect of hypoglycemia on the adrenal secretion of epinephrine and nor-epinephrine in the dog. Endocrinol. 62, 749-757. HAZlZLWOODR. L. (1958) The peripheral action of tolbutamide in domestic fowl. Endocrlnol. 63, 611-618. HAZF.LWOOD R. L. (1965) Carbohydrate metabolism. In Avian Physiology (Edited by STURKm P. D.), pp. 313-357. Comstock Press, New York. HAZELWOODR. L. (1971) Endocrine control of avian carbohydrate metabolism. Poult. S d . 50, 9-18. HAZELWOODR. L., KIMMEL J. R. & POLLOCKH. G. (1971) Influence of chicken and rat plasma on in vitro activity of chicken and beef insulin. Comp. Biochem. Physiol. 39B, 267-278. HAZELWOODR. L. & LORENZ F. W. (1959) Effects of fasting and insulin on carbohydrate metabolism of the domestic fowl. Am. J. Physiol. 197, 47-51. HXATT N., KATZ J. & SHEINKOPFJ. A. (1970) Insulin bIockade of epinephrine. Endocrinol. 87, 186-191. HLR~ZlCKER M. E. & HaZ~LWOOD R. L. (1970) Chicken cerebrospinal fluid: insulin-like activity. Comp. Biochem. Physiol. 36, 795-801. LEmSON L. (1965-66) The endocrine factors in the regulation of carbohydrate metabolism in the developing chick embryo. Biol. Neonat. 9, 249-262. MmsKY I. A. & GITELSONS. (1957) Comparison of the hypoglyeemic action of tolbutamide in the fowl and other species. Endocrinology 61, 148-152. MmsKv I. A. & GIT~LSON S. (1958) The diabetic response of geese to pancreatectomy. Endocrinology 63, 345-348. PITTMAN R. P. (1970) Possible mechanisms which mediate avian resistance to exogenous insulin. Doctoral dissertation, University of Houston, Houston, Texas. PITTMAN R. P. & HAZELWOODR. L. (1971) Cardiovascular response of chickens to administration of non-avian insulin. Peoc. Soc. exp. Biol. Med. 137, 1060-1065. SARCIONE E. J., BACK N., SOKALJ. E., MEHLMAN]3. & KNOBLOCKE. (1963) Elevation of plasma epinephrine levels produced by glucagon in vivo. Endocrinology 72, 523-526. SOKAL R. R. & ROHLF F. J. (1952) Biometry. W . H . Freeman, San Francisco. SOMOGYI M. (1952) Notes on sugar determination. J. biol. Chem. 195, 19-23. WERK E. E., GARBERS. & SHOLXTONL. J. (1961) Effect of sympathetic blockage on changes in blood ketones and non-esterified fatty acids following hypoglycemia in man. Metabolism 10, 115-125. Key Word Index---Catecholamines; chicken; insulin; hypoglycemia; sulfonylurea.