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Some biological activities of recombinant DNA-derived growth hormone on plasma metabolite concentrations in domestic fowl
Comp. Biochem. Physiol. Vol. 86A, No. 1, pp. 2%34, 1987 Printed in Great Britain
0300-9629/87 $3.00 + 0.00 Pergamon Journals Ltd
SOME BIOLOGICAL ACTIVITJES OF RECOMBINANT DNA-DERIVED GROWTH HORMONE ON PLASMA METABOLITE CONCENTRATIONS IN DOMESTIC FOWL T. R. HALL,* A. CHEUNG and S. HARVEY Wolfson Institute, University of Hull, Hull HU6 7RX, UK (Received 10 March 1986) Abstract-l. The biological activity of recombinant-DNA-derived chicken growth hormone (rcGH) has been examined in young broiler cockerels, by determining its effects on plasma concentrations of glucose, free fatty acids and a-amino nitrogen. 2. A single injection of rcGH increased plasma glucose, which remained high for several hours, whereas daily treatment with rcGH for 1 week had no effect on basal plasma glucose concentrations but blunted the glucose response to a further rcGH challenge. 3. Plasma free fatty acids were also promptly increased following acute rcGH treatment, and chronic exposure to rcGH again attenuated this response. 4. The effects of rcGH on plasma a-amino nitrogen were more variable. The stress of repeated blood sampling tended to reduce a-amino nitrogen, and after rcGH, an increase relative to vehicle-injected controls was seen in both acute and chronically-treated birds. 5. These data suggest that rcGH has both hyperglycaemic and lipolytic activity in chickens, and may also increase amino-acid availability.
INTRODUCTION
Although
highly
purified
avian
growth
pose tissue in vitro (Goodman, 1984). Recombinant cGH does not Seem to be lipolytic with rat epidymal fat in vitro (Hart et al., 1984), though this is disputed by Cambell and Scanes (1985). The present experiments examine the effects of rcGH on metabolic parameters in the chicken in vivo.
hormones
(GHs) were isolated some time ago (e.g. Farmer et al., 1974; Harvey and Scanes, 1977), their physiological effects are largely unknown because of the limited qualities available of the GH preparations. Mammalian GHs are more freely available and many of the physiological effects of GH in birds have been elucidated using mammalian GH preparations, though results must be interpreted cautiously because of possible species-specific responses. Bovine GH increases plasma glucose, free fatty acids and amino acid concentrations in hypophysectomized ducks (Foltzer et al., 1975; Foltzer and Mialhe, 1976). Harvey et al., (1977), Scanes and Lauterio (1984) and Cameron et al., (1985) have reported on the growth-promoting, hyperglycaemic and lipolytic properties of avian GHs in birds and mammals. However, there have been suggestions that some properties of purified GHs may not be intrinsic to the GH molecule, but rather be produced by trace contaminants (Hart et al., 1984). The production of GHs using recombinant DNA techniques has made available considerable quantities of pure hormones for physiological testing, including recombinantderived bovine (rbGH), human (rhGH) and chicken (rcGH) GHs (Goodman, 1984; Hart et al., 1984; Skottner et al., 1984; Souza et al., 1984). Recombinant bGH has lipolytic and hyperglycaemic actions in rat, mouse and sheep (Hart et al., 1984) and rhGH has both lipolytic and insulin-like actions on rat adi-
MATERIALS
One-day-old Ross broiler cockerel chicks were obtained commercially and reared under standard laboratory conditions, with food and water available ad libitum, until they were 5-6 weeks old. Three experiments were performed. In the first, 6-week-old birds were injected with 2OOyg/kg recombinant-derived chicken GH(rcGH) or with 1 ml/kg vehicle (0.25% NaHCO,, 0.2% mannitol, pH 8.5) into a wing vein. Blood samples were withdrawn from a wing vein lo,30 and 120 min after injection. In the second experiment, 5-week-old birds were injected with 10 or 100 pg/kg rcGH or vehicle, into the pectoral muscle (i.m.) once daily, between 0100 and 1000 hr for 8 days. Immediately before the last injection, a venous blood sample was removed, and trunk blood was collected following decapitation 2 hr later. A further three groups of birds, aged 6 weeks, were injected once only with the same treatments. In the third experiment, 5-week-old chickens were given i.m. injections of vehicle or 2OOpg/kg rcGH daily for 7 days. At the time of the last injection, further groups of birds were given a single injection of vehicle or rcGH. Blood samples were withdrawn from a wing vein at various time intervals up to 240min after injection. Plasma was separated from all blood samples and stored at - 20°C to await analysis. Assays and a&a analysis
Plasma glucose was measured enzymatically using the Sigma glucose assay kit, which utilizes glucose oxidase and o-dianisidine. Plasma free fatty acids were measured spectrophotometrically by the method of Laurel1 and Tibling (1967) using palmitic acid as standard. Plasma a-amino nitrogen
*Present address: Biovet Unit, Ciba-Geigy SA, Centre de Recherches Agricoles, CH- 1566 St-Aubin/FR, land. (Telephone: 037-771822).
AND METHODS
Switzer29
T. R. HALL et al.
30
was estimated, in urotein-free suuernant. bv the method of Matthews er aL (l&4) using gly&e as &&lard. All results show means + SEM (N = 8-16). Differences between experimentally-treated g&ups and iheir respective controls were evaluated using the Student’s t-test, with a level of
significance of P < 0.05. RESULTS
Table 1 shows plasma free fatty acid concentrations after acute and chronic rcGH treatment. No ,significant variations in free fatty acids were seen after acute or chronic vehicle treatment. Following a single injection of rcGH, plasma concentrations of free fatty acids increased over the fust 40 min, and were declining at 180 min after injection. After the
Figure 1 shows plasma metabolites 10, 30 and 120min after a single injection of vehicle or rcGH. Plasma glucose was significantly elevated (P c 0.01) at 10min and remained high throughout the experient, though by 120min the glucose concentrations were only 107.8 + 25% of the control, compared with 116.9 +3.7% at 1Omin and 118.4f2.8% of the control at 30min. Plasma free fatty acids were also elevated, by 140.8 + 9.2% (P < 0.05) at 10 min, 154.8 f 17.0% (P c 0.05) at 30 min, and by 183.4 + 13.9% (P < 0.01) at 120 min. On the other hand, plasma a-ammo nitrogen was not significantly affected at 10 or 30min, but increased (P c 0.05) 120min after injection. Figure 2 shows plasma metabolites 2 hr after acute or chronic treatment with rcGH. Plasma glucose was significantly (P < 0.01) elevated by 109.2 + 1.9% and 0.7118.7 + 2.3% following a single injection of 10 and lOOpgg/kg respectively of rcGH, but was increased by only 104.1 f 1.6% (P c 0.05) and 108.5 + 2.7% (P < 0.01) 2 hr following the eighth injection of 10 and 100 pg/kg rcGH. Vehicle-injected controls showed no significant changes in plasma glucose concentrations. Plasma free fatty acids also increased after rcGH treatment though the absolute levels of free fatty acids were less following chronic rcGH treatment compared with acute effects. The relative increases were comparable (151.8 + 11.8%, P < 0.05, and 163.1 + 9.9%, P < 0.01, acutely and 146.0 + 13.4%, P ~0.05,and 161.9+ 8.1%, P < 0.05, chronically with 10 and 100 pg/kg rcGh respectively). Plasma a-amino nitrogen was significantly increased (P < 0.01) only after a single injection of 100 pg/kg rcGH. Following chronic treatment with 100 fig/kg, but not 10 pg/kg rcGH nor vehicle, the preinjection concentration of a-amino nitrogen was high (120.5% 0 -3 of vehicle-injected control) and an injection of rcGH had no further effect. Figure 3 shows plasma glucose changes in response l to an injection of rcGH and also the response to the s eighth daily injection. The vehicle-injected controls El 120showed a small, but nevertheless significant (P < 0.05) increase in plasma glucose concentrations over .! 110 the first 20 min after acute injection. However, after .E rcGH there was a prompt and large rise in plasma k IOOglucose, which peaked at l@-20min after injection. Thereafter, glucose concentrations gradually decline, d but were still elevated (P < 0.05)180 min after E" 90" injection. Following daily treatment with vehicle for a week, no significant changes in plasma glucose were 3 0. .._I seen in the period up to 24Omin after the final 20 mtn Or nin injection. After chronic rcGH, the preinjection concentration of glucose was not different from the Tbme after injectton control. Plasma glucose increased after the final Fig. 1. Effects of an i.v. injection of 200 pg/lcg recombinantrcGH injection, but to a much lower level than derived chicken GH (rcGH) or vehicle on Dlasma metabolite following a single rcGH treatment. Plasma glucose concentrations in b-wee&old cockereis. Results show remained significantly (P < 0.05) elevated for 120 means f SEM (N = 8) *P ~0.05, **p < 0.01 compared min after rcGH injection. with vehicle.
““1
F
b
II
19
.o
31
Effect of GH on fowl Dlasma metabolites
1
**
T
a
PCS-InJCCllOn
Zhr after
,n~e~t,on
1~* l *
lb.0
**
0.8
T
1
0.7
0 0.2
E !? 0 0.1 0
I-
I Vehicle
lorslks Acute
“‘o/.m/kg
treotment
Fig. 2. Changes in plasma metabolites after acute administration of vehicle or rcGH. Blood samples into 6-week-old cockerels. Results show means + preinjection
I
.1 Chronic
GH 10 I.rdkg 100 rdks
treatment
(single injection) or chronic (eight daily injections) i.p. were taken immediately before and 2 hr after injection SEM (N = 16) *P c 0.05, **P c 0.01 compared with concentration.
32
Fig. 3. Effects af a single injection (a, acute response) or the seventh injection (b. chronic treatment) of vehicle or 200 g&/kg r&H i.p. into 6--week-old cockerels on plasma glucose concentrations. Re&s show means f SEM (N = 8).
last of a series of rcGH injections, plasma free fatty acids increased, but to a much lesser degree, and rapidly returned to preinjection levels. Table 2 shows plasma rr-amino r&rogen ConCentrations after acute and chronic ffiH treatment. After a single injection of vehicle, plasma a-amino nitrogen showed a gradual decline on repeated blood sampling. F&awing chronic vehicle treatment, plasma a-amino nitrogen was considerably elevated (P < O.Ol), and showed a significant decline (P c 0.05) with serial sampling. After acute or chronic r&H, plasma a-amino nitsogen concentrations were higher than their respective ~ntrois. ‘fable 1. Effects
DISCUSSION The importance of GH in the regulation of carbohydrate, lipid and protein metabolism in domestic animats has bean emphasized a number of times (e.g, Foitzer and Mialhe, 19%; Seanes and Harvey, 1982; Scanes and Lauterio, 1984; Chung et al., 1985). Mammalian (bovine) GH increases plasma glucose levels in hypophysectomized ducks (Foltzer et al., 1975) while, similarly, avian (ostrich) GH increases plasma glucase concentations in the mouse (Cameron et al,, 19g5). Avian GH inhibits the in vitro ~ns~jn-~ndu~ l&genesis whib stimulating lipoiysis
of singte injection (acute)
or the seventh daily in$ction (chronic) of rccombinaet DNAdcrived chicken growth hormone frcGH, 2OO@kg) or vehicle on plasma free f&ty acid concentrations (mmole/l). Results show means p SEM. (N = 8) Time fallowing final injection (min) 0
5
10
20
40
80
VEtiIiXE
0.76
0.83
0.69
0.75
0.76
0.77
(acute)
+O.OS
+o.r2
to.10
+a.ro
0.65
0.92
0.93
0.99
+O.OS
50.04
+0.07
+0.06
0.63
0.63
0.76
Treatment
rcGH
(acute) \rEHICtE (Chronic) rcGH (chronic)
20.05
+o.orJ
_tO.lO
1.04*
+cr.os
120
180
0.78
0.83
$.lO
+0.09 -
0.89
1.03
1.01
kO.07
+0.09 -
20.14
-+O.lO
0.70
0.71
0.68
0.69
0.61
+o.os
TO.07
+0.04
+0.06 -
kO:p.02
-+0.03
0.66
0.75
0.87
0.91
0.80
0.79
0.74
0.72
kO.06
+0.05
50.06
50.07
+a.06
50.06
+o.as
LO.05
*P eQ.05 compared with vehicie at same time.
Effect of GH on fowl plasma metabolites
33
Fable2. Effects of a single injection (acute) or the seventh daily injection (chronic) of rcGH or vehicleon plasma a-amino nitrogen concentrations (mg/l) Time following final injection (min) Treatment
0
5
10
20
40
80
Vehicle
77.3
71.4
61.4
68.8
63.2
(acute)
-t7.2
t5.3 -
23.4
-t3.0
rcGH
80.3
82.1
83.2
(acute)
t4.5
t7.6 -
Vehicle
103.9
(chronic) rcGH (chronic)
120
180
240
63.4
60.6
69.8
65.1
-t2.8
53.0
23.6
23.3
_t4.3
80.9
71.6
69.1
62.9
71.0
82.5
214.8
212.0
23.7
_t3.6
52.9
-t4.3
-t7.0
95.8
93.4
87.3
89.0
86.4
86.2
83.1
86.1
22.6
55.0
~5.1
56.5
55.7
57.1
55.2
53.2
24.3
104.7
112.2
104.5
101.5
106.4
104.3
96.4
106.0
106.5
54.6
57.8
25.2
57.3
k6.4
t3.5
25.5
24.5
t6.0
Results show means f SEM. (N = 8).
(Scanes et al., 1984; Scanes and Lauterio, 1984), though chicken GH apparently glucose uptake into fat cells (Rudas and Scanes, 1983). Interestingly the different functions of GH may be produced by different parts of the GH molecule (Bulatov et al., 1983). The production of recombinant-DNA-derived GHs allows the investigation into the question of whether the metabolic effects of GH are intrinsic to the molecule or are due to trace pituitary contaminants (Hart et al., 1984). Recombinant cGH has been manufactured and its primary sequence determined. The bacterial-derived GH is identical to the 191-amino acid native GH except for the Nterminal amino acid (Souza et al., 1984; Lai et al., 1984). The present data show that this small difference does not preclude full biological activity. Injection of rbGH into sheep elevates plasma glucose, but only after 3 hr, and the levels remain high for at least 6 hr subsequently (Hart et al., 1984). In our experiments, plasma GH was significantly increased within 5 min and, though the glucose concentrations remained high for several hours, they started to fall after 20min. These results illustrate a potentially important difference between mammals and birds in the regulation of plasma glucose levels. The small, transient response seen in vehicle-injected birds probably represents a stress effect of repeated bleeding (Davidson, 1975). Interestingly, the response to rcGH was blunted after a week of daily treatment, suggesting an adaptation of the hyperglycaemic response. When mice were injected with various GH preparations for 3 days, fasting blood glucose levels were significantly elevated (Cameron et al., 1985). In our experiments, though, repeated GH injections did not affect resting glucose concentrations. Growth hormone is also involved in the regulation of lipid metabolism (Scanes et al., 1984). Whereas
pituitary GHs can stimulate the liberation of glycerol from fat pad in vitro (Duquette et al., 1984), it was suggested by Hart et al., (1984) that recombinant GHs are devoid of activity though they increase free fatty acid levels in vim (Knudtzon et al., 1985) and they proposed the idea that the lipolytic action of GH is due to modification (cleavage?) of the molecule in uivo or to production of a lipolytic (hepatic?) intermediate. However, Goodman (1984) and Cambell and Scanes (1985) have shown that native GHs and several recombinant-derived GHs all stimulate in oitro lipolysis in fat cells. Both native and rcGH have equipotent lipolytic activity (Cambell and Scanes, 1985). Bovine GH increases free fatty acids in hypophysectomized ducks (Foltzer and Mialhe, 1976), and anti-GH antiserum reduces plasma free fatty acid concentrations in pigeons (John et al., 1973). Recombinant cGH increases free fatty acids in the plasma of broiler cockerels and, as with glucose, the response occurs rapidly and is blunted following a week of daily treatment with rcGH. The attenuation of the hyperglycaemic and lipolytic responses following repeated rcGh therapy may be due to desensitization or “down regulation” of the GH receptors, or a reduced intracellular responsiveness to stimulation of the key metabolic enzymes, or other factors, These possibilities deserve further investigation. Whereas GH decreases plasma amino acid concentrations in mammals, in line with its stimulation of protein synthesis, bovine GH increases plasma amino acid concentrations in hypophysectomized ducks (Foltzer and Mialhe, 1976). Our results show that, similarly, rcGH increases amino acid concentrations in the plasma of intact cockerels. The increased plasma amino acids may be a result of a decreased catabolic utilization (Scanes et al., 1984). Compared with the glucose and free fatty acid responses, the
T. R. HALL etal.
34
changes in amino acid concentrations occurred slowly and were more variable. In one experiment, chronic treatment with vehicle or rcGH increased resting amino acid levels, which may represent a stress reponse to the repeated handling and injections. In summary, these results show that rcGH has biological activity in the domestic fowl, increasing plasma concentrations of glucose, free fatty acids and amino acids. Chronic administration of rcGH blunts the glycolytic and lipolytic responses.
Goodman H. M. (1984) Biological activity of bacterial derived human growth hormone in adipose tissue of hypophysectomixed rats. Endocrinology 114, 13 l-l 35. Hart I. C., Chadwick P. M. E., Boone T. C., Langley K. E., Rudman C. and Souza L. M. (1984) A comparison of the growth-promoting, lipolytic, diabetogenic and immunological properties of pituitary and recombinant-DNAderived bovine growth hormone (somatotropin). Biothem. J. 224, 93-100. Harvey S. and Scanes C. G. (1977) Purification and radioimmunoassay of chicken growth hormone. J. Endocr. 73,
Acknowledgemenrs-This work was supported by a grant from Imperial Chemical Industries. The authors are grateful to Amgen (Thousand Oaks, California) for the generous gift of r&H.
Harvey S., Scanes C. G. and Howe T. (1977) Growth hormone effects on in vitro metabolism of avian adipose and liver tissue. Gen. Comp. Endocr. 33, 322-328. John T. M., McKeown B. A. and George J. C. (1973) Influence of exogenous growth hormone and its antiserum on plasma free fatty acid level in the pigeon. Comp.
321 l-329.
REFERENCES Bulatou A. A., Osipova T. A., Terekhov S. M., Saxlna E. T. and Pankov Y. A. (1983) New data on biological activity of fragment 77-107 of hypophyseal somatropin Biokhimiya 48, 1305-l 3 10. Cambell R. M. and Scanes C. G. (1985) Lipolytic activity of purified pituitary and bacterially derived growth hormone on chicken adipose tissue in vitro. Proc. Sot. exp. Biol. Med. 188, 513-517. Cameron C. M.. Kostio J. L.. and Pankoff H. (1985) Nonmammalian growth hormones have diabetogenic and insulin-like activities. Endocrinology 116, 1501-1505 Chung C. S., Etherton T. D. and Wiggins J. P. (1985). Stimulation of swine growth by porcine growth hormone. J. Anim. Sci. 68, 118-130. Davison T. F. (1975) The effects of multiple sampling by cardiac puncture and diurnal rhythm on plasma glucose and hepatic glycogen of the immature chicken. Comp. Biochem. Physiol. S8A, 569-573. Duquette P. F., Scanes C. G. and Muir L. A. (1984) Effects of ovine growth hormone and other anterior pituitary hormones on lioolvsis of rat and ovine adiuose tissue in vitro. J. An& Sii. 58, 1191-1197. _ Farmer S. W.. Pankoff H. and Havashida T. 11974) Purification and properties of avian -growth hormones. Endocrinology 95, 1560-1565. Foltxer C., Leclerq-Meyer V. and Mialhe P. (1975) Pituitary and adrenal control of pancreatic endocrine function in the duck. I. Plasma glucose and pancreatic ghrcagon variations following hypophysectomy and replacement therapy by growth hormone and corticosterone. Diabere et Metabolirme, Paris 1, 3944. Foltzer C. and Mialhe P. (1976) Pituitary and adrenal control of uancreatic endocrine function in the duck. II. Plasma fre;! fatty acids, amino acids, and insulin variations following hypophysectomy and replacement therapy with growth hormone and corticosterone. Diabete et Metabolisme, Paris 2, 101-105.
Biochem. Physiol. 46A, 497-504.
Knudtzon J., Edminson P. D. and Reichelt K. L. (1985) Different acute in vieo effects of bacterial derived and pituitary growth hormone preparation on plasma levels of glucagon, insulin and free fatty acids in rabbits. Hormone Res. 21, 10-18. Lai P. H., Dupka D. R., Souza L. M. and Scanes C. G. (1984) Purification and properties of chicken growth hormone. ZRCS Med. Sci. 12. 1077-1078. Laurel1 S. and Tibbling G. (1967) Calorimetric microdetermination of free fatty acids in plasma. C/in. Chim. Acta 16, 57-62. Matthews D. M., Muir G. G. and Baron D. N. (1964) Estimation of alpha-amino nitrogen in plasma and urine by the calorimetric ninhydrin reaction. J. clin. Path. 17, 150-153.
Rudas P. and Scanes C. G. (1983) Influences of growth hormone on glucose uptake by avian adipose tissue. Poultry Sci. 62, 18381845.
Scanes C. G. and Harvey S. (1982) Hormones, nutrition and metabolism in birds. In Aspects of Avian Endocrinology: Practical and Theoretical Imp&ions (Edited by Scanes C. G.), pp. 173-184. Grad Studies Texas Tech Univ., Lubbock. Scanes C. G. and Lauterio T. S. (1984) Growth hormone: its physiology and control. J. exp. Zool. 222, 443-452. Scanes C. G., Stockell Hat-tree A. and Cunningham F. J. (1984) The Pituitary gland. In Physiology and Biochemistry of the Domestic Fowl (Edited by Freeman B. M.), pp. 39-84. Academic Press, London. Skottner A., Forsman A., LGfberg E. and Thomgren G. (1984) Comparison of methionyl human growth hormone and pituitary growth hormone on somatic growth of hypophysect&&d rats. Acta Endocr. 107, l&198. Souxa L. M. Boone T. C., Murdock D.. Lanalev K.. Wypych J., Fenton D., Johnson S., Lai P..J., E&&t R.1 Hsu R.-Y. and Bossehnan R. (1984) Application of recombinant DNA technologies to studies on chicken growth hormone. J. exp. Zool. 232, 455473.
0300-9629/87 $3.00 + 0.00 Pergamon Journals Ltd
SOME BIOLOGICAL ACTIVITJES OF RECOMBINANT DNA-DERIVED GROWTH HORMONE ON PLASMA METABOLITE CONCENTRATIONS IN DOMESTIC FOWL T. R. HALL,* A. CHEUNG and S. HARVEY Wolfson Institute, University of Hull, Hull HU6 7RX, UK (Received 10 March 1986) Abstract-l. The biological activity of recombinant-DNA-derived chicken growth hormone (rcGH) has been examined in young broiler cockerels, by determining its effects on plasma concentrations of glucose, free fatty acids and a-amino nitrogen. 2. A single injection of rcGH increased plasma glucose, which remained high for several hours, whereas daily treatment with rcGH for 1 week had no effect on basal plasma glucose concentrations but blunted the glucose response to a further rcGH challenge. 3. Plasma free fatty acids were also promptly increased following acute rcGH treatment, and chronic exposure to rcGH again attenuated this response. 4. The effects of rcGH on plasma a-amino nitrogen were more variable. The stress of repeated blood sampling tended to reduce a-amino nitrogen, and after rcGH, an increase relative to vehicle-injected controls was seen in both acute and chronically-treated birds. 5. These data suggest that rcGH has both hyperglycaemic and lipolytic activity in chickens, and may also increase amino-acid availability.
INTRODUCTION
Although
highly
purified
avian
growth
pose tissue in vitro (Goodman, 1984). Recombinant cGH does not Seem to be lipolytic with rat epidymal fat in vitro (Hart et al., 1984), though this is disputed by Cambell and Scanes (1985). The present experiments examine the effects of rcGH on metabolic parameters in the chicken in vivo.
hormones
(GHs) were isolated some time ago (e.g. Farmer et al., 1974; Harvey and Scanes, 1977), their physiological effects are largely unknown because of the limited qualities available of the GH preparations. Mammalian GHs are more freely available and many of the physiological effects of GH in birds have been elucidated using mammalian GH preparations, though results must be interpreted cautiously because of possible species-specific responses. Bovine GH increases plasma glucose, free fatty acids and amino acid concentrations in hypophysectomized ducks (Foltzer et al., 1975; Foltzer and Mialhe, 1976). Harvey et al., (1977), Scanes and Lauterio (1984) and Cameron et al., (1985) have reported on the growth-promoting, hyperglycaemic and lipolytic properties of avian GHs in birds and mammals. However, there have been suggestions that some properties of purified GHs may not be intrinsic to the GH molecule, but rather be produced by trace contaminants (Hart et al., 1984). The production of GHs using recombinant DNA techniques has made available considerable quantities of pure hormones for physiological testing, including recombinantderived bovine (rbGH), human (rhGH) and chicken (rcGH) GHs (Goodman, 1984; Hart et al., 1984; Skottner et al., 1984; Souza et al., 1984). Recombinant bGH has lipolytic and hyperglycaemic actions in rat, mouse and sheep (Hart et al., 1984) and rhGH has both lipolytic and insulin-like actions on rat adi-
MATERIALS
One-day-old Ross broiler cockerel chicks were obtained commercially and reared under standard laboratory conditions, with food and water available ad libitum, until they were 5-6 weeks old. Three experiments were performed. In the first, 6-week-old birds were injected with 2OOyg/kg recombinant-derived chicken GH(rcGH) or with 1 ml/kg vehicle (0.25% NaHCO,, 0.2% mannitol, pH 8.5) into a wing vein. Blood samples were withdrawn from a wing vein lo,30 and 120 min after injection. In the second experiment, 5-week-old birds were injected with 10 or 100 pg/kg rcGH or vehicle, into the pectoral muscle (i.m.) once daily, between 0100 and 1000 hr for 8 days. Immediately before the last injection, a venous blood sample was removed, and trunk blood was collected following decapitation 2 hr later. A further three groups of birds, aged 6 weeks, were injected once only with the same treatments. In the third experiment, 5-week-old chickens were given i.m. injections of vehicle or 2OOpg/kg rcGH daily for 7 days. At the time of the last injection, further groups of birds were given a single injection of vehicle or rcGH. Blood samples were withdrawn from a wing vein at various time intervals up to 240min after injection. Plasma was separated from all blood samples and stored at - 20°C to await analysis. Assays and a&a analysis
Plasma glucose was measured enzymatically using the Sigma glucose assay kit, which utilizes glucose oxidase and o-dianisidine. Plasma free fatty acids were measured spectrophotometrically by the method of Laurel1 and Tibling (1967) using palmitic acid as standard. Plasma a-amino nitrogen
*Present address: Biovet Unit, Ciba-Geigy SA, Centre de Recherches Agricoles, CH- 1566 St-Aubin/FR, land. (Telephone: 037-771822).
AND METHODS
Switzer29
T. R. HALL et al.
30
was estimated, in urotein-free suuernant. bv the method of Matthews er aL (l&4) using gly&e as &&lard. All results show means + SEM (N = 8-16). Differences between experimentally-treated g&ups and iheir respective controls were evaluated using the Student’s t-test, with a level of
significance of P < 0.05. RESULTS
Table 1 shows plasma free fatty acid concentrations after acute and chronic rcGH treatment. No ,significant variations in free fatty acids were seen after acute or chronic vehicle treatment. Following a single injection of rcGH, plasma concentrations of free fatty acids increased over the fust 40 min, and were declining at 180 min after injection. After the
Figure 1 shows plasma metabolites 10, 30 and 120min after a single injection of vehicle or rcGH. Plasma glucose was significantly elevated (P c 0.01) at 10min and remained high throughout the experient, though by 120min the glucose concentrations were only 107.8 + 25% of the control, compared with 116.9 +3.7% at 1Omin and 118.4f2.8% of the control at 30min. Plasma free fatty acids were also elevated, by 140.8 + 9.2% (P < 0.05) at 10 min, 154.8 f 17.0% (P c 0.05) at 30 min, and by 183.4 + 13.9% (P < 0.01) at 120 min. On the other hand, plasma a-ammo nitrogen was not significantly affected at 10 or 30min, but increased (P c 0.05) 120min after injection. Figure 2 shows plasma metabolites 2 hr after acute or chronic treatment with rcGH. Plasma glucose was significantly (P < 0.01) elevated by 109.2 + 1.9% and 0.7118.7 + 2.3% following a single injection of 10 and lOOpgg/kg respectively of rcGH, but was increased by only 104.1 f 1.6% (P c 0.05) and 108.5 + 2.7% (P < 0.01) 2 hr following the eighth injection of 10 and 100 pg/kg rcGH. Vehicle-injected controls showed no significant changes in plasma glucose concentrations. Plasma free fatty acids also increased after rcGH treatment though the absolute levels of free fatty acids were less following chronic rcGH treatment compared with acute effects. The relative increases were comparable (151.8 + 11.8%, P < 0.05, and 163.1 + 9.9%, P < 0.01, acutely and 146.0 + 13.4%, P ~0.05,and 161.9+ 8.1%, P < 0.05, chronically with 10 and 100 pg/kg rcGh respectively). Plasma a-amino nitrogen was significantly increased (P < 0.01) only after a single injection of 100 pg/kg rcGH. Following chronic treatment with 100 fig/kg, but not 10 pg/kg rcGH nor vehicle, the preinjection concentration of a-amino nitrogen was high (120.5% 0 -3 of vehicle-injected control) and an injection of rcGH had no further effect. Figure 3 shows plasma glucose changes in response l to an injection of rcGH and also the response to the s eighth daily injection. The vehicle-injected controls El 120showed a small, but nevertheless significant (P < 0.05) increase in plasma glucose concentrations over .! 110 the first 20 min after acute injection. However, after .E rcGH there was a prompt and large rise in plasma k IOOglucose, which peaked at l@-20min after injection. Thereafter, glucose concentrations gradually decline, d but were still elevated (P < 0.05)180 min after E" 90" injection. Following daily treatment with vehicle for a week, no significant changes in plasma glucose were 3 0. .._I seen in the period up to 24Omin after the final 20 mtn Or nin injection. After chronic rcGH, the preinjection concentration of glucose was not different from the Tbme after injectton control. Plasma glucose increased after the final Fig. 1. Effects of an i.v. injection of 200 pg/lcg recombinantrcGH injection, but to a much lower level than derived chicken GH (rcGH) or vehicle on Dlasma metabolite following a single rcGH treatment. Plasma glucose concentrations in b-wee&old cockereis. Results show remained significantly (P < 0.05) elevated for 120 means f SEM (N = 8) *P ~0.05, **p < 0.01 compared min after rcGH injection. with vehicle.
““1
F
b
II
19
.o
31
Effect of GH on fowl Dlasma metabolites
1
**
T
a
PCS-InJCCllOn
Zhr after
,n~e~t,on
1~* l *
lb.0
**
0.8
T
1
0.7
0 0.2
E !? 0 0.1 0
I-
I Vehicle
lorslks Acute
“‘o/.m/kg
treotment
Fig. 2. Changes in plasma metabolites after acute administration of vehicle or rcGH. Blood samples into 6-week-old cockerels. Results show means + preinjection
I
.1 Chronic
GH 10 I.rdkg 100 rdks
treatment
(single injection) or chronic (eight daily injections) i.p. were taken immediately before and 2 hr after injection SEM (N = 16) *P c 0.05, **P c 0.01 compared with concentration.
32
Fig. 3. Effects af a single injection (a, acute response) or the seventh injection (b. chronic treatment) of vehicle or 200 g&/kg r&H i.p. into 6--week-old cockerels on plasma glucose concentrations. Re&s show means f SEM (N = 8).
last of a series of rcGH injections, plasma free fatty acids increased, but to a much lesser degree, and rapidly returned to preinjection levels. Table 2 shows plasma rr-amino r&rogen ConCentrations after acute and chronic ffiH treatment. After a single injection of vehicle, plasma a-amino nitrogen showed a gradual decline on repeated blood sampling. F&awing chronic vehicle treatment, plasma a-amino nitrogen was considerably elevated (P < O.Ol), and showed a significant decline (P c 0.05) with serial sampling. After acute or chronic r&H, plasma a-amino nitsogen concentrations were higher than their respective ~ntrois. ‘fable 1. Effects
DISCUSSION The importance of GH in the regulation of carbohydrate, lipid and protein metabolism in domestic animats has bean emphasized a number of times (e.g, Foitzer and Mialhe, 19%; Seanes and Harvey, 1982; Scanes and Lauterio, 1984; Chung et al., 1985). Mammalian (bovine) GH increases plasma glucose levels in hypophysectomized ducks (Foltzer et al., 1975) while, similarly, avian (ostrich) GH increases plasma glucase concentations in the mouse (Cameron et al,, 19g5). Avian GH inhibits the in vitro ~ns~jn-~ndu~ l&genesis whib stimulating lipoiysis
of singte injection (acute)
or the seventh daily in$ction (chronic) of rccombinaet DNAdcrived chicken growth hormone frcGH, 2OO@kg) or vehicle on plasma free f&ty acid concentrations (mmole/l). Results show means p SEM. (N = 8) Time fallowing final injection (min) 0
5
10
20
40
80
VEtiIiXE
0.76
0.83
0.69
0.75
0.76
0.77
(acute)
+O.OS
+o.r2
to.10
+a.ro
0.65
0.92
0.93
0.99
+O.OS
50.04
+0.07
+0.06
0.63
0.63
0.76
Treatment
rcGH
(acute) \rEHICtE (Chronic) rcGH (chronic)
20.05
+o.orJ
_tO.lO
1.04*
+cr.os
120
180
0.78
0.83
$.lO
+0.09 -
0.89
1.03
1.01
kO.07
+0.09 -
20.14
-+O.lO
0.70
0.71
0.68
0.69
0.61
+o.os
TO.07
+0.04
+0.06 -
kO:p.02
-+0.03
0.66
0.75
0.87
0.91
0.80
0.79
0.74
0.72
kO.06
+0.05
50.06
50.07
+a.06
50.06
+o.as
LO.05
*P eQ.05 compared with vehicie at same time.
Effect of GH on fowl plasma metabolites
33
Fable2. Effects of a single injection (acute) or the seventh daily injection (chronic) of rcGH or vehicleon plasma a-amino nitrogen concentrations (mg/l) Time following final injection (min) Treatment
0
5
10
20
40
80
Vehicle
77.3
71.4
61.4
68.8
63.2
(acute)
-t7.2
t5.3 -
23.4
-t3.0
rcGH
80.3
82.1
83.2
(acute)
t4.5
t7.6 -
Vehicle
103.9
(chronic) rcGH (chronic)
120
180
240
63.4
60.6
69.8
65.1
-t2.8
53.0
23.6
23.3
_t4.3
80.9
71.6
69.1
62.9
71.0
82.5
214.8
212.0
23.7
_t3.6
52.9
-t4.3
-t7.0
95.8
93.4
87.3
89.0
86.4
86.2
83.1
86.1
22.6
55.0
~5.1
56.5
55.7
57.1
55.2
53.2
24.3
104.7
112.2
104.5
101.5
106.4
104.3
96.4
106.0
106.5
54.6
57.8
25.2
57.3
k6.4
t3.5
25.5
24.5
t6.0
Results show means f SEM. (N = 8).
(Scanes et al., 1984; Scanes and Lauterio, 1984), though chicken GH apparently glucose uptake into fat cells (Rudas and Scanes, 1983). Interestingly the different functions of GH may be produced by different parts of the GH molecule (Bulatov et al., 1983). The production of recombinant-DNA-derived GHs allows the investigation into the question of whether the metabolic effects of GH are intrinsic to the molecule or are due to trace pituitary contaminants (Hart et al., 1984). Recombinant cGH has been manufactured and its primary sequence determined. The bacterial-derived GH is identical to the 191-amino acid native GH except for the Nterminal amino acid (Souza et al., 1984; Lai et al., 1984). The present data show that this small difference does not preclude full biological activity. Injection of rbGH into sheep elevates plasma glucose, but only after 3 hr, and the levels remain high for at least 6 hr subsequently (Hart et al., 1984). In our experiments, plasma GH was significantly increased within 5 min and, though the glucose concentrations remained high for several hours, they started to fall after 20min. These results illustrate a potentially important difference between mammals and birds in the regulation of plasma glucose levels. The small, transient response seen in vehicle-injected birds probably represents a stress effect of repeated bleeding (Davidson, 1975). Interestingly, the response to rcGH was blunted after a week of daily treatment, suggesting an adaptation of the hyperglycaemic response. When mice were injected with various GH preparations for 3 days, fasting blood glucose levels were significantly elevated (Cameron et al., 1985). In our experiments, though, repeated GH injections did not affect resting glucose concentrations. Growth hormone is also involved in the regulation of lipid metabolism (Scanes et al., 1984). Whereas
pituitary GHs can stimulate the liberation of glycerol from fat pad in vitro (Duquette et al., 1984), it was suggested by Hart et al., (1984) that recombinant GHs are devoid of activity though they increase free fatty acid levels in vim (Knudtzon et al., 1985) and they proposed the idea that the lipolytic action of GH is due to modification (cleavage?) of the molecule in uivo or to production of a lipolytic (hepatic?) intermediate. However, Goodman (1984) and Cambell and Scanes (1985) have shown that native GHs and several recombinant-derived GHs all stimulate in oitro lipolysis in fat cells. Both native and rcGH have equipotent lipolytic activity (Cambell and Scanes, 1985). Bovine GH increases free fatty acids in hypophysectomized ducks (Foltzer and Mialhe, 1976), and anti-GH antiserum reduces plasma free fatty acid concentrations in pigeons (John et al., 1973). Recombinant cGH increases free fatty acids in the plasma of broiler cockerels and, as with glucose, the response occurs rapidly and is blunted following a week of daily treatment with rcGH. The attenuation of the hyperglycaemic and lipolytic responses following repeated rcGh therapy may be due to desensitization or “down regulation” of the GH receptors, or a reduced intracellular responsiveness to stimulation of the key metabolic enzymes, or other factors, These possibilities deserve further investigation. Whereas GH decreases plasma amino acid concentrations in mammals, in line with its stimulation of protein synthesis, bovine GH increases plasma amino acid concentrations in hypophysectomized ducks (Foltzer and Mialhe, 1976). Our results show that, similarly, rcGH increases amino acid concentrations in the plasma of intact cockerels. The increased plasma amino acids may be a result of a decreased catabolic utilization (Scanes et al., 1984). Compared with the glucose and free fatty acid responses, the
T. R. HALL etal.
34
changes in amino acid concentrations occurred slowly and were more variable. In one experiment, chronic treatment with vehicle or rcGH increased resting amino acid levels, which may represent a stress reponse to the repeated handling and injections. In summary, these results show that rcGH has biological activity in the domestic fowl, increasing plasma concentrations of glucose, free fatty acids and amino acids. Chronic administration of rcGH blunts the glycolytic and lipolytic responses.
Goodman H. M. (1984) Biological activity of bacterial derived human growth hormone in adipose tissue of hypophysectomixed rats. Endocrinology 114, 13 l-l 35. Hart I. C., Chadwick P. M. E., Boone T. C., Langley K. E., Rudman C. and Souza L. M. (1984) A comparison of the growth-promoting, lipolytic, diabetogenic and immunological properties of pituitary and recombinant-DNAderived bovine growth hormone (somatotropin). Biothem. J. 224, 93-100. Harvey S. and Scanes C. G. (1977) Purification and radioimmunoassay of chicken growth hormone. J. Endocr. 73,
Acknowledgemenrs-This work was supported by a grant from Imperial Chemical Industries. The authors are grateful to Amgen (Thousand Oaks, California) for the generous gift of r&H.
Harvey S., Scanes C. G. and Howe T. (1977) Growth hormone effects on in vitro metabolism of avian adipose and liver tissue. Gen. Comp. Endocr. 33, 322-328. John T. M., McKeown B. A. and George J. C. (1973) Influence of exogenous growth hormone and its antiserum on plasma free fatty acid level in the pigeon. Comp.
321 l-329.
REFERENCES Bulatou A. A., Osipova T. A., Terekhov S. M., Saxlna E. T. and Pankov Y. A. (1983) New data on biological activity of fragment 77-107 of hypophyseal somatropin Biokhimiya 48, 1305-l 3 10. Cambell R. M. and Scanes C. G. (1985) Lipolytic activity of purified pituitary and bacterially derived growth hormone on chicken adipose tissue in vitro. Proc. Sot. exp. Biol. Med. 188, 513-517. Cameron C. M.. Kostio J. L.. and Pankoff H. (1985) Nonmammalian growth hormones have diabetogenic and insulin-like activities. Endocrinology 116, 1501-1505 Chung C. S., Etherton T. D. and Wiggins J. P. (1985). Stimulation of swine growth by porcine growth hormone. J. Anim. Sci. 68, 118-130. Davison T. F. (1975) The effects of multiple sampling by cardiac puncture and diurnal rhythm on plasma glucose and hepatic glycogen of the immature chicken. Comp. Biochem. Physiol. S8A, 569-573. Duquette P. F., Scanes C. G. and Muir L. A. (1984) Effects of ovine growth hormone and other anterior pituitary hormones on lioolvsis of rat and ovine adiuose tissue in vitro. J. An& Sii. 58, 1191-1197. _ Farmer S. W.. Pankoff H. and Havashida T. 11974) Purification and properties of avian -growth hormones. Endocrinology 95, 1560-1565. Foltxer C., Leclerq-Meyer V. and Mialhe P. (1975) Pituitary and adrenal control of pancreatic endocrine function in the duck. I. Plasma glucose and pancreatic ghrcagon variations following hypophysectomy and replacement therapy by growth hormone and corticosterone. Diabere et Metabolirme, Paris 1, 3944. Foltzer C. and Mialhe P. (1976) Pituitary and adrenal control of uancreatic endocrine function in the duck. II. Plasma fre;! fatty acids, amino acids, and insulin variations following hypophysectomy and replacement therapy with growth hormone and corticosterone. Diabete et Metabolisme, Paris 2, 101-105.
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Knudtzon J., Edminson P. D. and Reichelt K. L. (1985) Different acute in vieo effects of bacterial derived and pituitary growth hormone preparation on plasma levels of glucagon, insulin and free fatty acids in rabbits. Hormone Res. 21, 10-18. Lai P. H., Dupka D. R., Souza L. M. and Scanes C. G. (1984) Purification and properties of chicken growth hormone. ZRCS Med. Sci. 12. 1077-1078. Laurel1 S. and Tibbling G. (1967) Calorimetric microdetermination of free fatty acids in plasma. C/in. Chim. Acta 16, 57-62. Matthews D. M., Muir G. G. and Baron D. N. (1964) Estimation of alpha-amino nitrogen in plasma and urine by the calorimetric ninhydrin reaction. J. clin. Path. 17, 150-153.
Rudas P. and Scanes C. G. (1983) Influences of growth hormone on glucose uptake by avian adipose tissue. Poultry Sci. 62, 18381845.
Scanes C. G. and Harvey S. (1982) Hormones, nutrition and metabolism in birds. In Aspects of Avian Endocrinology: Practical and Theoretical Imp&ions (Edited by Scanes C. G.), pp. 173-184. Grad Studies Texas Tech Univ., Lubbock. Scanes C. G. and Lauterio T. S. (1984) Growth hormone: its physiology and control. J. exp. Zool. 222, 443-452. Scanes C. G., Stockell Hat-tree A. and Cunningham F. J. (1984) The Pituitary gland. In Physiology and Biochemistry of the Domestic Fowl (Edited by Freeman B. M.), pp. 39-84. Academic Press, London. Skottner A., Forsman A., LGfberg E. and Thomgren G. (1984) Comparison of methionyl human growth hormone and pituitary growth hormone on somatic growth of hypophysect&&d rats. Acta Endocr. 107, l&198. Souxa L. M. Boone T. C., Murdock D.. Lanalev K.. Wypych J., Fenton D., Johnson S., Lai P..J., E&&t R.1 Hsu R.-Y. and Bossehnan R. (1984) Application of recombinant DNA technologies to studies on chicken growth hormone. J. exp. Zool. 232, 455473.