Effect of vasotocin and glucagon on plasma growth hormone levels in the pigeon

Camp. Biochem. Physiol., 1974, Vol. 48A, pp. 521 to 526. Pergamon Press. Printed in Great Britain

EFFECT OF VASOTOCIN AND GLUCAGON ON PLASMA GROWTH HORMONE LEVELS IN THE PIGEON* T. M. JOHN,

B. A. McKEOWN

and J. C. GEORGE

Department of Zoology, University of Guelph, Guelph, Ontario, Canada (Received 23kly 1973) Abstract-l. Injection of vasotocin did not have a significant effect on the plasma growth hormone (GH) level in the pigeon, at 15 or 30 min post-injection, when compared with isotonic saline injection. 2. GH levels at 30 min post-injection of vasotocin as well as of saline were significantly higher than the respective levels at 15 min post-injection. 3. Glucagon produced an elevation in plasma GH at 5 min post-injection. 4. Injection of vasotocin 5 min after the glucagon injection produced a further significant increase in plasma GH when tested 15 min after the vasotocin injection (i.e. 20 min after glucagon injection). No such increase was noticed when saline alone was injected instead of vaaotocin. 5. A synergistic effect of glucagon and vasotocin on plasma GH level has been suggested. INTRODUCTION

IN A RECENTstudy on the effect of certain neurohypophysial hormones and glucagon on the level of plasma free fatty acids (FFA) in the pigeon, it was observed that vasotocin, which actually brought about a decline in FFA at 15 min post-injection, produced an elevation at 30 min post-injection (John & George, 1973). Glucagon, which caused a significant increase in FFA at 5 min post-injection, when followed by vasotocin produced a synergistic effect with the latter by registering a further increase in FFA at 20 min post-injection. However, the exact mode of action of these hormones in effecting FFA elevation remains to be understood. The existence of a circadian rhythm of plasma FFA in the pigeon has been shown (John & George, 1972) and it was found to synchronize with a corresponding rhythm in the level of the plasma growth hormone (GH) in the pigeon (McKeown et al., 1973). Since GH is known to be a lipolytic hormone in the pigeon (John et al., 1973), it was thought necessary to investigate the effect of vasotocin and glucagon on plasma GH in the pigeon in order to see if the lipolytic action of these hormones was direct or mediated through GH. MATERIALS

AND METHODS

The present experiments were conducted under exactly identical conditions as those of the earlier experiments of John & George (1973) in which they studied the effect of glucagon * This study was supported by the National Research Council of Canada. Series 077. 521

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T. M. JOHN, B. A. MCKEOWN AND J. C. GEORGE

and certain neurohypophysial hormones on the plasma FFA level in the pigeon, so that the effects of the test hormones on the plasma levels of GH and FFA could be tallied. Sixty healthy adult pigeons (Columba livia), maintained in outdoor pens, were obtained from a commercial supplier in early February (1972) and were transferred into a controlled environmental room. The temperature of the room was maintained at 25 _+1°C and the relative humidity at 30%. The lighting (about 200 lx at cage level supplied by “cool white fluorescence”) was set on a 12-hr daily photoperiod to be “on” at 0600 hours and ‘off” at 1800 hours. The birds were fed commercial pigeon feed (Purina Chow) and water ad lib. The weight of the birds ranged between 325 and 350 g. An acclimation period of 2 weeks was allowed before the commencement of the experiment. Since it was known that the plasma GH (McKeown et al., 1973) as well as FFA (John & George, 1972) levels are known to have the minimal fluctuation between 1200 hours and 1800 hours, the afternoon (1400-1600 hours) was selected as a suitable period for experimentation. The brachial vein was used as the site for intravenous injection of test solutions as well as for withdrawing blood samples. One ml of blood was drawn for GH assay as “pre-treatment control” from each bird on the fifteenth day of acclimation in the environmental room. On the twenty-fifth day the birds were divided into eight groups each consisting of seven or eight birds which included more or less equal number (three or four) of males and females. Birds of Group 1 did not receive any injection and were bled during the experimentation time (between 1400 and 1600 hours) on Day 25. Group 2 received injection of saline (0~5 ml of 0.85% saline/ pigeon) and Group 3 arginine-vasotocin (400 mU in 0.5 ml of 0.85% saline/pigeon). Blood samples for GH assay were taken exactly 15 min after the injection. Groups 4 and 5 were also given doses of the same injections (saline and vasotocin respectively) but were bled 30 min after the injection. Birds of Group 6 received injections of glucagon (60 pg/O.25 ml saline per pigeon) and were bled 5 min later. Group 7 was first given an injection of glucagon (60 pg/O.25 ml of 0.85% saline/pigeon) and 5 min later saline (0.25 ml of 0.8576 saline/ pigeon) and bled 15 min after the saline injection. Group 8 was given glucagon as in Group 7, but was followed by vasotocin (400 mu/O-25 ml of 0.85 “/osaline/pigeon) and bled 15 min after the vasotocin injection. All blood samples for GH determination were collected in oxalated tubes, centrifuged and stored at - 70°C until assayed (less than a week). GH was analyzed by radio-immunoassay using the solid-phase method of Catt & Tregear (1967), with slight modifications in the radio-iodination and assay procedures as described previously (McKeown, 1972). Antiserum to ovine growth hormone (NIH-GH-S9, 1.09 I.U./mg) was produced in rabbits for the radio-immunoassay. The procedures for immunization and testing specificity of the antisera have been described earlier (McKeown & van Overbeeke, 1971). The same GH used for immunization was radio-iodinated with lz51 (Amersham) according to the method of Greenwood et al. (1963) for use as a tracer antigen. Each pigeon plasma sample was assayed at three dilutions (1 : 200, 1 : 400 and 1 : 800) in 0.15 M NaCI. The antiserum was diluted 1 : 3000 for all assays. Since purified pigeon GH was not available, standard curves could not be produced and, therefore, the absolute concentrations of the hormone could not be determined. However, a series dilution of a plasma sample was used to produce a type of “standard curve” (John et al., 1973 ; McKeown et al., 1973) and by assigning arbitrary units of concentration to this curve, relative concentration differences could be determined.

RESULTS

The results obtained are presented in Fig. 1, in terms of percentage difference of plasma GH concentration from the pre-treatment level. Group 1 which received

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PLASMA GROWTH HORMONE LEVELS IN THE PIGEON

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I 4.

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>

FIG. 1. Mean percentage difference in plasma growth hormone from pre-treatment level (100 per cent line) + standard error (S.E.).

no injection showed no significant difference in the GH level from that of the pretreatment level (i.e. the first bleeding) (P> 0.1). Groups 2 and 4 which were given injections of saline and bled after 15 and 30 min respectively neither showed any significant variation in GH level between the groups (P > 0.1) nor did they vary significantly from the group that received no injection (P>O*l). Birds which received injection of vasotocin showed an apparent reduction in plasma GH at 15 min post-injection (Group 3) and an increase 30 min after the administration (Group 5), but these changes were not statistically significant (P> 0.1) when compared with the respective saline-injected controls. The GH levels at 30 min postinjection of vasotocin as well as of saline were significantly higher than the respective levels at 15 min post-injection (P-C 0.01 and P-c 0.05 respectively). However, the difference between 15 min post-injection and 30 min post-injection of vasotocin was greater than the difference noted in the case of saline injection. Glucagon (Group 6) produced an elevated plasma GH level 5 min after administration. In testing the effect of glucagon at 5 min post-injection, no specific control was run with saline injection. Injection of saline, however, was found to have no significant effect on plasma GH at 15 or 30 min post-injection. The GH level observed 5 min after glucagon injection was hence tested against that observed at 15 min after saline injection, for statistical evaluation (P-KO-05). In Group 7, which received saline injection 5 min after glucagon, the GH.level was about normal when examined at 15 min after saline administration (i.e. 20 min after the glucagon injection). However, injection of vasotocin 5 min after glucagon (Group 8) produced a significantly high level of plasma GH (P-e 0.05) when examined at 15 min after the vasotocin injection (i.e. 20 min after glucagon injection). No significant sex differences in plasma GH level were noticed.

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T. M. JOHN, B. A. MCKJXOWNANDJ. C. GEORGE DISCUSSION

Although until a few years ago glucagon was considered as a hormone having only the glycogenolytic-hyperglycemic effect, in recent years a bewildering array of physiological effects such as lipolytic stimulation, insulin secretion, release of catecholamines and GH secretion has been attributed to glucagon (for review see Unger & Lefebvre, 1972). However, reports regarding GH (especially human GH) response to glucagon have presented conflicting results (see Cain et al., 1970). The time and route of the administration of glucagon varied in the investigations reported. Some recent studies on the effect of glucagon administration tend to indicate that glucagon raises GH level in the blood (Cain et al., 1970 ; Tiengo et al., 1970 ; Weber et al., 1970). In the present study too an increase in plasma GH at 5 min post-injection of glucagon was observed. However, glucagon administration when followed by saline injection after 5 min did not show significant increase in GH level when assayed 20 min after the glucagon injection. This probably suggests that in the pigeon the effect of glucagon on GH release is immediate and diminished in the 20 min post-injection period or that the injection of saline after glucagon has an inhibitory effect on the release of GH. It should, however, be pointed out that saline injection per se did not have any significant effect on plasma GH (Fig. 1). Injection of vasotocin 5 min after glucagon administration produced a marked increase in GH when assayed 20 min after the glucagon injection indicating a GHstimulating “synergistic” effect of glucagon and vasotocin (Fig. 1). In a recent study of the effect of neurohypophysial hormones and glucagon on plasma FFA level in the pigeon under exactly identical conditions, John & George (1973) observed an increase in plasma FFA level with glucagon at 5 min postinjection. Injection of vasotocin 5 min after glucagon administration produced a further increase in FFA level when assayed 20 min after the glucagon injection (John & George, 1973). In the present investigation we have observed a concomitant rise in plasma GH (Fig. 1). The synchronous rise in the levels of plasma FFA and GH after the administration of glucagon alone, or glucagon followed by vasotocin, tends to suggest the mediation of GH in the release of FFA in pigeons since a synchronizing relationship between the circadian rhythms of plasma GH and FFA levels in the pigeon has been demonstrated (John & George, 1972; McKeown et al., 1973). Also, our finding that injection of GH causes elevation of plasma FFA level in the pigeon (John et al., 1973) lends further support to this inference. Although vasotocin seemed to produce an apparent reduction in plasma GH at 15 min post-injection and an increase at 30 min post-injection, these differences were not statistically significant, thereby indicating that vasotocin alone had no significant effect on plasma GH level. This is in agreement with the findings of Hertelendy et al. (1971) who found that neither hypothalamic extracts nor synthetic lysine-vasopressin had any effect on GH release from rat pituitaries in vitro. Chronic electric stimulation of the hypothalamus in conscious cats was also found to have no effect on plasma GH level (Kokka et al., 1972).

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However, investigations to study the effect of hypothalamic extracts (which presumably contain the neurohypophysial hormones as well) on GH have yielded conflicting results. In a recent study, Steiner et al. (1970) observed that three out of four stalk median eminence preparations stimulated GH release from rat pituitaries in oitro. On the other hand, Hertelendy et al. (1971) found that crude and purified hypothalamic extracts failed to stimulate GH release in vitro. However, Frohman et al. (1971) obtained positive results in rats, following intrapituitary injections of a purified sheep hypothalamic extract, though a somewhat less retarded fraction of the same preparation used by Hertelendy et al. (1971) was found to be ineffective. Injection of vasotocin has been shown to elevate plasma FFA level at 30 min post-injection in pigeons kept under exactly identical conditions as in the present investigation (John & George, 1973). Since the apparent increase in GH at 30 min post-injection seen in the present experiments was not statistically significant, it is not possible to attribute the elevation in plasma FFA noticed 30 min after the vasotocin injection to GH. The similarity in plasma GH response to injections of saline and vasotocin tends to suggest the involvement of common factor(s) stimulating GH release as a stress syndrome initiated by the injection itself and also the bleeding process for obtaining the blood sample. The significantly greater post-injection (15 and 30 min) GH release with vasotocin over saline suggests a higher potency for vasotocin in stimulating ACTH secretion. Vasotocin is known to be capable of stimulating ACTH secretion in birds (Salem et al., 1970) as well as mammals (Hiroshige et al., 1968). A synergistic effect of the exogenous vasotocin and the endogenous ACTH may also be considered as a possibility for greater GH release noted after vasotocin injection. Acknowledgements-It is a pleasure to express our thanks to Mrs. S. Hoover, Mrs. E. Johnson, Mrs. G. Wilson, Mrs. M. John and Mr. T. Hwang for technical assistance.

REFERENCES CAIN J. P., WILLIAMSG. H. & DLUHY R. G. (1970) Glucagon stimulation of human growth hormone. J. clin. Endocr. 31, 222-224. CATT K. & TREGFAR G. W. (1967) Solid-phase radio-immunoassay in antibody-coated tubes. Science, Wash. 158, 1570-1572. FROHMANL. A., MARANJ. W., YATES F. E. & DHARIWALA. P. S. (1971) Growth hormone responses to intrapituitary injection of growth hormone releasing factor in the rat as measured by radioimmunoassay. Fedn PYOC.Fedn Am. Socs exp. Biol. 30,198 (Abstract). GREENWOOD F. C., HUNTFB W. H. & GLOVERJ. S. (1963) The preparation of lSII-labelled human growth hormone of high specific radio-activity. Biochem. J. 89, 114-123. HWTELENDYF., TODD H., PEAKEG. T., MACHLINL. J., JOHNSTONG. & POUNDSG. (1971) Studies on growth hormone secretion : 1. Effects of dibutyryl cyclic AMP, theophylline epinephrine, ammonium ion and hypothalamic extracts on the release of growth hormone from rat anterior pituitaries in vitro. Endocrinology 89, 1256-1262. HIROSHIGET., KUNITAH., OGURAC. & ITOH S. (1968) Effects on ACTH release of intrapituitary injections of posterior pituitary hormones and several amines in the hypothalamus. Ju9.J. Physiol. 18, 609-619.

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T. M. & GEORGEJ. C. (1972) Circadian rhythm of free fatty acid levels in plasma and pectoralis muscle of the pigeon. 3’. interdiscipl. Cycle Res. 3, 33-37. JOHN T. M. & GEORGEJ. C. (1973) Influence of glucagon and neurohypophysial hormones on plasma free fatty acid levels in the pigeon. Comp. Biochem. Physiol. 35A, 541-547. JOHN T. M., MCKEOWN B. A. & GEORGEJ. C. (1973) Influence of exogenous growth hormone and its antiserum on plasma free fatty acid level in the pigeon. Comp. Biochem. Physiol. 46A, 497-504. KOKKAN., EISENBERGR. M., GARCIAJ. & GEORGER. (1972) Blood glucose, growth hormone, and cortisol levels after hypothalamic stimulation. Am. J. Physiol. 222, 296-301. MCKEOWN B. A. (1972) Prolactin and growth hormone concentrations in the plasma of the toad Bufo bufo (L.) following ectopic transplantations of the pars distalis. Gen. & compar. Endocr. 19, 167-l 74. MCKEO~N B. A. & VAN OVERBEEKEA. P. (1971) Immunohistochemical identification of pituitary hormone producing cells in the sockeye salmon (Oncorhynchus nerka, Walbaum). 2. Zellforsch. mikrosk. Anat. 112, 350-362. MCKEOWN B. A., JOHN T. M. & GEORGEJ. C. (1973) Circadian rhythm of plasma growth hormone levels in the pigeon. J. interdiscipl. Cycle lies. 4. (In press.) SALEM M. H. M., NORTONH. W. & NALBANDOVA. V. (1970) The role of vasotocin and of CRF in ACTH release in the chicken. Gen. & compar. Endocr. 14, 281-289. STEINER A. L., PEAKEG. T., UTIGER R. D., KARL I. E. & KIPNIS D. M. (1970) Hypothalamic stimulation of growth hormone and thyrotropin release in vitro and pituitary 3’5adenosine cyclic monophosphate. Endocrinology 86, 1354-1360. TIENGO A., MUGGEO M., FEDELE D., FELLIN R. & CREPALDI G. (1970) The effects of glucagon on growth hormone production. Acta isotop. 10,359-370. UNGER R. H. & LEFEBVRE P. J. (Eds.) (1972) Glucagon. Pergamon Press, Oxford. WEBER B., HELGE H. & QUABBEH.-J. (1970) Glucagon-induced growth hormone release in children. Acta endocr. 65, 323-341. JOHN

Key Word Index-Pigeon; fatty acids ; ACTH.

vasotocin;

glucagon;

plasma growth hormone;

plasma free