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Effect of Synthetic Human Pancreatic Growth Hormone-Releasing Factors on Plasma Growth Hormone Concentrations in Lactating Cows1
Effect of Synthetic Human Pancreatic Growth Hormone-Releasing Factors on Plasma Growth Hormone Concentrations in Lactating Cows 1
S T U A R T N. McCUTCHEON 2 and DALE E. B A U M A N 3 Department of Animal Science Cornell University Ithaca, NY 14853-0281 W I L L I A M A. M U R P H Y , V A L E N T I N E A. LANCE, and D A V I D H. COY Department of Medicine Section of Endocrinology and Metabolism Tulane University School of Medicine New Orleans, LA 70112
ABSTRACT
result from intravenous injection of up to 2000 /ag of growth hormone-releasing factor (1-29) would not be sufficient to increase milk yield of cows.
Two fragments of human pancreatic growth hormone-releasing factor, hpGRF(1-24)-NH2 and hpGRF(1-29)-NH2, were administered to lactating dairy cows in separate experiments. Each experiment involved a 4 x 4 Latin square with treatments being intravenous administration of saline (controls) or three doses of releasing factor (500, 1000, and 2000/~g). Administration of the 24-amino acid fragment increased plasma growth hormone concentration from baseline 3 ng/ml to peak 14 ng/ml within 10 rain. Growth hormone concentrations returned to baseline by 60 min post treatment. The 29amino acid fragment resulted in greater responses in both peak concentration (30 ng/ml) and persistency (>240 min). Relationships between dose of releasing factor and response in growth hormone release (as measured by area under the response curve) were not consistent in most cows. Responses of concentrations of growth hormone in blood plasma that
INTRODUCTION
Received April 30, 1984. 1Supported in part by Cornell University Agricultural Experiment Station and American Cyanamid Company. The authors gratefully acknowledge the assistance of C. A. Ricks (American Cyanamid Company) and R. C. Gorewit, B. Palhof, and W. English (Cornell University). 2On leave from Department of Animal Science, Massey University, Palmerston North, New Zealand. 3 Reprint requests. 1984 J Dairy Sci 67:2881--2886
Human pancreatic growth hormone-releasing factor (hpGRF) first was isolated as a 40- or 44-amino acid peptide from pancreatic tumors in two human acromegalics (7, 11). This factor, its synthetic replicates, and certain analogs, promoted secretion of growth hormone by rat pituitary cells in culture (7, 11, 14). When administered intravenously, hpGRF acted specifically to elevate circulating concentrations of growth hormone in ruminant (1, 8, 10) and nonruminant species (5, 7, 11, 12). Administration of bovine growth hormone (bGH) to lactating dairy cows substantially increases yields of milk and milk components (see review by Collier et al. (2)). Although the magnitude of this response is a function of bGH dose (3), it is independent of the pattern in which bGH concentration in plasma is elevated by treatment. For example, Fronk et al. (4) administered 51.5 IU/day of bGH by three methods (single daily subcutaneous injection, continuous subcutaneous infusion or pulsatile intravenous injection at 4-h intervals). These methods produced different patterns of elevation of growth hormone in blood plasma, but the mean elevation (as measured by area under the response curve) was identical. All three treatments increased milk yield by 31% over control. Thus, elevation of circulating growth hormone by administration of synthetic hpGRF, rather than by administration of bGH
2881
2882
McCUTCHEON ET AL.
per se, potentially offers an alternative means of achieving the lactational response. For this reason we examined effects of two fragments of hpGRF on bGH secretion in lactating dairy COWS.
MATERIALS AND METHODS
place. The hpGRF(1-24)-NH2 was preweighed into gelatin-lined vials, solubilized in sterile saline, and administered via cannulae at time zero (1000 h). Controls received 4 ml of saline, also per cannula. Blood samples were withdrawn at - 3 0 , --15, 0, 2, 4, 6, 8, 10, 15, 20, 25, 30, 45, and 60 rain postadministration, then every 60 min for a further 7 h.
Peptide Synthesis
The hpGRF(1-24)-NH2 a and hpGRF(1-29)NH2 were prepared synthetically by a solid phase approach (13). Crude peptides were purified by gel filtration on Sephadex G-50 followed by preparative high performance liquid chromatography on Vydac C-18 silica eluted with an acetonitrile- .1% trifluoroacetic acid system, Homogeneity of the final product was confirmed by analytical high performance liquid chromatography on ph~nyl and C-18 silica (Vydac, 30 nm pore size), thin layer chromatography in several systems, and amino acid analysis of enzyme and acid hydrolysates. Yields of 12% with >95% peptide purity were achieved by this methodology. Amino acid ratios from the analyses were within expected ranges for respective peptides. Experiment 1 : hpGRF(1-24)-NH 2
Four Holstein cows in second lactation were assigned to treatments in a balanced Latin square design. At commencement of the study, cows averaged 177 days postpartum and were producing 18 to 25 kg milk per day. Treatments were 0, 500, 1000, and 2000/ag hpGRF(1-24)-NH2 and were administered every 2nd day. Body weights averaged 549 kg so that doses of hpGRF(1-24)-NH2 averaged 0, .91, 1.82, and 3.64 /ag/kg body weight. Jugular cannulae were inserted 24 h before first administration, and prophylactic antibiotic treatment was maintained while cannulae were in
4The abbreviations hpGRF(1-24)-NH2, hpGRF(1-27)-NH2, hpGRF(1-29)-NH2, and hpGRF(1-44)NH 2 refer to the amidated forms of human pancreatic growth hormone-releasing factor containing 24, 27, 29, and 44 amino acids, respectively. The hpGRF(140)-OH is human pancreatic growth hormone-releasing factor consisting of 40 amino acids and having a free earboxy terminal. Journal of Dairy Science Vol. 67, No. 12, 1984
Experiment 2: hpGRF(1-29)-NH 2
Four additional Holstein cows, also in second lactation and an average 132 days postpartum, were used. Milk production was 15 to 35 kg per day at commencement of the study. Treatments were 0, 500, 1000, and 2000 gtg hpGRF(1-29)-NH2 and were administered as described. Cows averaged 609 kg body weight, and mean doses were, therefore, 0, .82, 1.64, and 3.28 gtg/kg body weight. Blood sampling followed a protocol similar to that for Experiment 1 except that sampling did not continue beyond 300 min postadministration. At the end of this experiment, two cows received 1000 gig hpGRF(1-29)-NH2 each by intramuscular and subcutaneous injection. Jugular blood was sampled at - 3 0 , - 1 5 , and 0 min relative to injection and thereafter for 2 h at 15-min intervals and a further 8 h at 30-rain intervals. In each experiment cows were in tie stalls and were offered a complete mixed diet at a rate calculated to meet requirements (9). Cows were fed and milked at 0600 and 1800 h daily. At each milking, milk yield was recorded, and samples were taken for determination of milk fat, protein, and lactose content (3). There was no evidence that milk yield or composition was influenced by treatment and, accordingly, results are not presented. Growth hormone concentration in blood plasma was quantified (6) with bovine growth hormone (bGH; Miles Lot 1 2 ) f o r iodination and reference standards. The linear range of the assay was .5 to 7.0 rig/assay tube. Intraassay and interassay coefficients of variation averaged 8.3 and 15.3%. Treatment effects on the response in bGH concentrations in plasma were determined within each experiment by calculated area under the response curve. Area data were corrected for differences in basal bGH concentration (from samples at - 3 0 , - 1 5 , and 0 min) and subjected to analysis of variance.
GROWTH HORMONE-RELEASINGFACTORS IN COWS
=
9-
in Figure 1. Doses of 500 and 1000 #g elevated bGH in plasma to approximately 10 ng/ml whereas peak concentration following administration of 2000 #g was close to 14 ng/ml. Plasma bGH concentration had essentially returned to baseline by 60 mitt postadministration, and subsequent concentrations are not presented. For this experiment, response areas were calculated over the interval 0 to 60 min postadministration (Table 1). Average response areas increased with dose of hpGRF(1-24)-NH2. There was, however, considerable variability between cows in response to each dose. Only two of the cows exhibited elevation of plasma bGH concentration which was linearly related to dose. In the remaining cows, responses were less marked and not dose-dependent.
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Figure 1. Responses in plasma concentrations of 8~owth hormone to varying doses of hpGRF(1-24)NHz (the amidated form of human pancreatic growth hormone-releasing factor containing 24 amino acids) administered intravenously at time zero. Standard errors averaged 16% of the mean.
RESULTS Experiment 1 : hpGR F(1-24)-NH2
Profiles of bGH concentration in plasma following administration of hpGRF(1-24)-NH2 are
Experiment 2: hpGR F(1-29)-NH 2
Responses of bGH concentration in plasma following intravenous administration of hpGRF(1-29)-NH2 were much greater than those with the 1-24 fragment in terms of both peak concentration and persistency of response. Thus, mean peak concentrations were up to 30 ng/ml, and concentrations in plasma generally did not return to baseline until 4 to 5 h after administration (Figure 2). As in Experiment 1, differences were marked between cows in magnitude and pattern of response to each dose of hpGRF(1-29)-NH2. Response areas were calculated over the entire postadministration sampling period (0 to 300 min), and results are in Table 1. There was
TABLE 1. Plasma growth hormone (bGH) response to varying doses of human pancreatic growth hormonereleasing factors (hpGRF(I-24)-NH 2 and hpGRF(1-29)-NH~).
Treatment
hpGRF(1-24)-NH22 hpGRF(1.29)-NH22
0
hp GRF dose (#g) ~ 500 1000
2000
Pooled SE
-11.3a 173.3a
Area response (ng.min-m1-1) 52.9ab 129.3ab 2170.3 b 2394.8 b
187.4b 2435.3 b
47.2 479.2
a'bwithin each row, treatment means with different superscripts differ (P<.05). t Doses were O, .91, 1.82, and 3.64 #g/kg, respectively, for hpGRF(1-24)-NH 2 and 0, .82, 1.64, and 3.28 #g/kg respectively, for hpGRF(1-29)-NH 2. Amidated forms of human pancreatic growth hormone-releasing factor containing 24 or 29 amino acids. Journal of Dairy Science Vol. 67, No. 12, 1984
2884
McCUTCHEON ET AL.
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Time (min) Figure 2. Responses in plasma concentrations of growth h o r m o n e to varying doses of hpGRF(1-29)-NH 2 (the amidated form of h u m a n pancreatic growth hormone-releasing factor containing 29 amino acids) administered intravenously at time zero. Standard errors averaged 26% of the mean.
Journal o f Dairy Science Vol. 67, No. 12, 1984
GROWTH HORMONE-RELEASING FACTORS IN COWS difference between experiments in calculated "response" of the controls (--11.3 vs. 173.3 ngm i n . m l - 1 ) , but this largely reflects errors associated with estimation of baseline. Neither "response" was significantly different from zero by t test. All doses of hpGRF(1-29)-NH2 gave similar responses that were significantly different from controls. Administration of 1000/~g of hpGRF(1-29)NH2 to two cows by subcutaneous and intramuscular routes gave similar responses (Table 2). However, these responses were tess persistent than those achieved by intravenous injection of the same dose, and as a result, response areas were lower. DISCUSSION
This is the first study to examine effects of h p G R F on plasma concentrations of growth hormone in lactating cows. The N-terminal 1-24 and 1-29 fragments of h p G R F both stimulated growth hormone release in lactating dairy cows. Responses to hpGRF(1-24)-NH2 tended to increase across the dose range (500 to 2000/~g), whereas with the 1-29 fragment responses to a dose of 500 /ag were similar to those at 1000 and 2000/~g. Although the two fragments were not compared directly in the same cows, hpGRF(1-24)-NH2 had approximately 5 to 7% the potency of hpGRF(1-29)-NH2 (based on area responses at the 1000 and 2000/~g doses). When assayed in anaesthetized rats, hpGRF(124)-NH2 had 4% the potency of hpGRF(1-29)NH2 (Lance, Murphy, and Cox, unpublished). Rivier et al. (11) reported that hpGRF(1-29)NH2 had similar potency (within a factor of two) to hpGRF(1-40)-OH when tested in vitro with rat pituitary cell cultures. However, potency o f hpGRF(1-27)-NH2 was only 10 to 20% that of hpGRF(1-40)-OH. Administration of 500 to 2000/~g h p G R F (1-24)-NH2 to these cows produced detectable increases of bGH concentration in plasma within 5 rain and a return to baseline at 60 min. This pattern is similar to that for heifers given .01 to .10 /ag hpGRF(1-40)-OH per kg body weight (10) and for steers given .03 to .30 /lg hpGRF(1-44)-NHz per kg body weight (8). In our study, intravenous injection of hpGRF(1-29)-NH2 caused much more persistent responses with few cows returning to baseline before 240 min postadministration. Moseley
2885
et al. (8) observed a biphasic release of bGH in steers administered 1000 /ag hpGRF(1-44)NH2. A similar pattern was evident when we administered hpGRF(1-29)-NH2 to the cows in Experiment 2, particularly at the 2000/ag dose (Figure 2). However, cows were not consistent in timing or magnitude of the second bGH spike, and more extensive sampling would be required to characterize it adequately. With respect to our original hypothesis, we conclude that attempts to elevate plasma bGH concentrations by administration of h p G R F , and thus induce a lactational response, are likely to be limited by a number of factors. That cows do not exhibit consistent relationships between dose of h p G R F and bGH response (area under the curve) is obviously a major problem. Our preliminary data suggest that, in contrast to administration of exogenous bGH (4), the method by which h p G R F is delivered influences the response of circulating concentrations of bGH. In the two cows that we examined, intravenous injection gave responses three times as great as those arising from alternative routes of administration. The possibility exists that, when administered by intramuscular or subcutaneous injection, hpGRF(1-29)-NHz is degraded partially before absorbed into the bloodstream. Finally, responses of bGH concentration in plasma that result from i.v. injection of up to 2000 /~g hpGRF(1-29)-NHz would not be sufficient to generate substantial increases of milk yield. For example, Fronk et al. (4) administered 39.6 mg/day (51.5 IU/day) of exogenous bGH to dairy cows and increased daily milk production by 4.2 kg (31%). Plasma bGH concentration
TABLE 2. Plasma growth hormone (bGH) response in two cows following administration of 1000 ug hpGRF(1-29)-NH2 ~ by various routes.
Route of administration
Area response (ng.min'm1-1 ) Cow A Cow B
Intravenous Subcutaneous Intramuscular
3665 1325 1415
2230 780 760
Amidated form of human pancreatic growth hormone-releasing factor containing 29 amino acids. Journal of Dairy Science Vol. 67, No. 12, 1984
2886
McCUTCHEON ET AL.
was elevated b y 20 n g / m l w h i c h over 24 h r e p r e s e n t s an area r e s p o n s e of 2 8 , 8 0 0 n g ' m i n " m1-1. We c o m p l e t e d a similar s t u d y in w h i c h 17.9 m g / d a y (25 I U / d a y ) b G H was a d m i n i s tered (McCutcheon and Bauman, unpublished). Milk yield i n c r e a s e d b y 4.9 kg (27%) over controls a n d p l a s m a b G H b y 9 3 0 0 n g ' m i n ' m 1 - 1 over a w h o l e day. T h e s e responses in area u n d e r t h e b G H c o n c e n t r a t i o n curve are, respectively, 11.8 a n d 3.8 t i m e s as great as t h o s e f o l l o w i n g injection of 2000/ag hpGRF(1-29)-NH2. Thus, t o o b t a i n a l a c t a t i o n a l response, m u l t i p l e doses of h p G R F w o u l d n e e d to b e a d m i n i s t e r e d , a n d q u e s t i o n s o f r e f r a c t o r i n e s s to t h e releasing factor become of particular importance. REFERENCES
1 Baile, C. A., M. A. Della-Fera, and F. C. Buonomo. 1983. Growth hormone releasing factor (GRF) injections in sheep. J. Anita. Sci. 57(Suppl. 1):188. (Abstr.) 2 Collier, R. J., J. P. MeNamara, C. R. Wallace, and M. H. Dehoff. 1984. A review of endocrine regulation of metabolism during lactation. J. Anim. Sci. 59:498. 3 Eppard, P. J., D. E. Bauman, and S. N. McCutcheon. 1984. Effect of varying doses of bovine growth hormone on lactational performance of dairy cows. J. Dairy Sci. (submitted). 4 Fronk, T. J., C. J. Peel, D. E. Bauman, and R. C. Gorewit. 1983. Comparison of different patterns of exogenous growth hormone administration on milk production in Holstein cows. J. Anita. Sci. 57:699. 5 Gelato, M. C., O. Pescovitz, F. Cassorla, D. L. Loriaux, and G. R. Merriam. 1983. Effects of a growth hormone releasing factor in man. J. Clin.
Journal of Dairy Science Vol. 67, No. 12, 1984
Endocrinol. Metab. 57:674. 6 Gorewit, R. C. 1981. Pituitary, thyroid and adrenal response to clonidine in dairy cattle. J. Endocrinol. Invest. 4:135. 7 Guillemin, R., P. Brazeau, P. Bohlen, F. Eseh, N. Ling, and W. B. Wehrenberg. 1982. Growth hormone-releasing factor from a human pancreatic tumor that caused acromegaly. Science 218:585. 8 Moseley, W. M., L. F. Krabill, A. R. Friedman, and R. F. Olsen. 1984. Growth hormone response of steers injected with synthetic human pancreatic growth hormone-releasing factors. J. Anim. Sci. 58:430. 9 National Research Council. 1978. Nutrient requirements of domestic animals. No. 3. Nutrient requirements of dairy cattle. 5th rev. ed. Natl. Acad. Sci., Washington, DC. 10 Plouzek, C. A., L. L. Anderson, D. L. Hard, J. R. Molina, A. Trenkle, W. Vale, and J. Rivier. 1983. Effect of intravenous injection of a growth hormone-releasing factor on plasma growth hormone concentrations in cattle. J. Anim. Sci. 57(Suppl. 1):203. (Abstr.) 11 Rivier, J., J. Spiess, M. Thorner, and W. Vale. 1982. Characterization of a growth hormone-releasing factor from a human pancreatic islet tumor. Nature 300:276. 12 Rosenthal, S. M., E. A. Schriock, S. L. Kaplan, R. Guillemin, and M. M. Grumbach. 1983. Synthetic human pancreas growth hormone-releasing factor (hpGRF144-NH~) stimulates growth hormone secretion in normal men. J. Clin. Endocrinol. Metab. 57:677. 13 Sueiras-Diaz, J., V. A. Lance, W. A. Murphy, and D. H. Coy. 1984. Structure-activity studies on the N-terminal region of glucagon. J. Med. Chem. 27: 310. 14 Vale, W., J. Vaughan, G. Yamamoto, J. Spiess, and J. Rivier. 1983. Effects of synthetic human pancreatic (tumor) GH releasing factor and somatostatin, triiodothyronine and dexamethazone on GH secretion in vitro. Endocrinology 112:1553.
S T U A R T N. McCUTCHEON 2 and DALE E. B A U M A N 3 Department of Animal Science Cornell University Ithaca, NY 14853-0281 W I L L I A M A. M U R P H Y , V A L E N T I N E A. LANCE, and D A V I D H. COY Department of Medicine Section of Endocrinology and Metabolism Tulane University School of Medicine New Orleans, LA 70112
ABSTRACT
result from intravenous injection of up to 2000 /ag of growth hormone-releasing factor (1-29) would not be sufficient to increase milk yield of cows.
Two fragments of human pancreatic growth hormone-releasing factor, hpGRF(1-24)-NH2 and hpGRF(1-29)-NH2, were administered to lactating dairy cows in separate experiments. Each experiment involved a 4 x 4 Latin square with treatments being intravenous administration of saline (controls) or three doses of releasing factor (500, 1000, and 2000/~g). Administration of the 24-amino acid fragment increased plasma growth hormone concentration from baseline 3 ng/ml to peak 14 ng/ml within 10 rain. Growth hormone concentrations returned to baseline by 60 min post treatment. The 29amino acid fragment resulted in greater responses in both peak concentration (30 ng/ml) and persistency (>240 min). Relationships between dose of releasing factor and response in growth hormone release (as measured by area under the response curve) were not consistent in most cows. Responses of concentrations of growth hormone in blood plasma that
INTRODUCTION
Received April 30, 1984. 1Supported in part by Cornell University Agricultural Experiment Station and American Cyanamid Company. The authors gratefully acknowledge the assistance of C. A. Ricks (American Cyanamid Company) and R. C. Gorewit, B. Palhof, and W. English (Cornell University). 2On leave from Department of Animal Science, Massey University, Palmerston North, New Zealand. 3 Reprint requests. 1984 J Dairy Sci 67:2881--2886
Human pancreatic growth hormone-releasing factor (hpGRF) first was isolated as a 40- or 44-amino acid peptide from pancreatic tumors in two human acromegalics (7, 11). This factor, its synthetic replicates, and certain analogs, promoted secretion of growth hormone by rat pituitary cells in culture (7, 11, 14). When administered intravenously, hpGRF acted specifically to elevate circulating concentrations of growth hormone in ruminant (1, 8, 10) and nonruminant species (5, 7, 11, 12). Administration of bovine growth hormone (bGH) to lactating dairy cows substantially increases yields of milk and milk components (see review by Collier et al. (2)). Although the magnitude of this response is a function of bGH dose (3), it is independent of the pattern in which bGH concentration in plasma is elevated by treatment. For example, Fronk et al. (4) administered 51.5 IU/day of bGH by three methods (single daily subcutaneous injection, continuous subcutaneous infusion or pulsatile intravenous injection at 4-h intervals). These methods produced different patterns of elevation of growth hormone in blood plasma, but the mean elevation (as measured by area under the response curve) was identical. All three treatments increased milk yield by 31% over control. Thus, elevation of circulating growth hormone by administration of synthetic hpGRF, rather than by administration of bGH
2881
2882
McCUTCHEON ET AL.
per se, potentially offers an alternative means of achieving the lactational response. For this reason we examined effects of two fragments of hpGRF on bGH secretion in lactating dairy COWS.
MATERIALS AND METHODS
place. The hpGRF(1-24)-NH2 was preweighed into gelatin-lined vials, solubilized in sterile saline, and administered via cannulae at time zero (1000 h). Controls received 4 ml of saline, also per cannula. Blood samples were withdrawn at - 3 0 , --15, 0, 2, 4, 6, 8, 10, 15, 20, 25, 30, 45, and 60 rain postadministration, then every 60 min for a further 7 h.
Peptide Synthesis
The hpGRF(1-24)-NH2 a and hpGRF(1-29)NH2 were prepared synthetically by a solid phase approach (13). Crude peptides were purified by gel filtration on Sephadex G-50 followed by preparative high performance liquid chromatography on Vydac C-18 silica eluted with an acetonitrile- .1% trifluoroacetic acid system, Homogeneity of the final product was confirmed by analytical high performance liquid chromatography on ph~nyl and C-18 silica (Vydac, 30 nm pore size), thin layer chromatography in several systems, and amino acid analysis of enzyme and acid hydrolysates. Yields of 12% with >95% peptide purity were achieved by this methodology. Amino acid ratios from the analyses were within expected ranges for respective peptides. Experiment 1 : hpGRF(1-24)-NH 2
Four Holstein cows in second lactation were assigned to treatments in a balanced Latin square design. At commencement of the study, cows averaged 177 days postpartum and were producing 18 to 25 kg milk per day. Treatments were 0, 500, 1000, and 2000/ag hpGRF(1-24)-NH2 and were administered every 2nd day. Body weights averaged 549 kg so that doses of hpGRF(1-24)-NH2 averaged 0, .91, 1.82, and 3.64 /ag/kg body weight. Jugular cannulae were inserted 24 h before first administration, and prophylactic antibiotic treatment was maintained while cannulae were in
4The abbreviations hpGRF(1-24)-NH2, hpGRF(1-27)-NH2, hpGRF(1-29)-NH2, and hpGRF(1-44)NH 2 refer to the amidated forms of human pancreatic growth hormone-releasing factor containing 24, 27, 29, and 44 amino acids, respectively. The hpGRF(140)-OH is human pancreatic growth hormone-releasing factor consisting of 40 amino acids and having a free earboxy terminal. Journal of Dairy Science Vol. 67, No. 12, 1984
Experiment 2: hpGRF(1-29)-NH 2
Four additional Holstein cows, also in second lactation and an average 132 days postpartum, were used. Milk production was 15 to 35 kg per day at commencement of the study. Treatments were 0, 500, 1000, and 2000 gtg hpGRF(1-29)-NH2 and were administered as described. Cows averaged 609 kg body weight, and mean doses were, therefore, 0, .82, 1.64, and 3.28 gtg/kg body weight. Blood sampling followed a protocol similar to that for Experiment 1 except that sampling did not continue beyond 300 min postadministration. At the end of this experiment, two cows received 1000 gig hpGRF(1-29)-NH2 each by intramuscular and subcutaneous injection. Jugular blood was sampled at - 3 0 , - 1 5 , and 0 min relative to injection and thereafter for 2 h at 15-min intervals and a further 8 h at 30-rain intervals. In each experiment cows were in tie stalls and were offered a complete mixed diet at a rate calculated to meet requirements (9). Cows were fed and milked at 0600 and 1800 h daily. At each milking, milk yield was recorded, and samples were taken for determination of milk fat, protein, and lactose content (3). There was no evidence that milk yield or composition was influenced by treatment and, accordingly, results are not presented. Growth hormone concentration in blood plasma was quantified (6) with bovine growth hormone (bGH; Miles Lot 1 2 ) f o r iodination and reference standards. The linear range of the assay was .5 to 7.0 rig/assay tube. Intraassay and interassay coefficients of variation averaged 8.3 and 15.3%. Treatment effects on the response in bGH concentrations in plasma were determined within each experiment by calculated area under the response curve. Area data were corrected for differences in basal bGH concentration (from samples at - 3 0 , - 1 5 , and 0 min) and subjected to analysis of variance.
GROWTH HORMONE-RELEASINGFACTORS IN COWS
=
9-
in Figure 1. Doses of 500 and 1000 #g elevated bGH in plasma to approximately 10 ng/ml whereas peak concentration following administration of 2000 #g was close to 14 ng/ml. Plasma bGH concentration had essentially returned to baseline by 60 mitt postadministration, and subsequent concentrations are not presented. For this experiment, response areas were calculated over the interval 0 to 60 min postadministration (Table 1). Average response areas increased with dose of hpGRF(1-24)-NH2. There was, however, considerable variability between cows in response to each dose. Only two of the cows exhibited elevation of plasma bGH concentration which was linearly related to dose. In the remaining cows, responses were less marked and not dose-dependent.
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Figure 1. Responses in plasma concentrations of 8~owth hormone to varying doses of hpGRF(1-24)NHz (the amidated form of human pancreatic growth hormone-releasing factor containing 24 amino acids) administered intravenously at time zero. Standard errors averaged 16% of the mean.
RESULTS Experiment 1 : hpGR F(1-24)-NH2
Profiles of bGH concentration in plasma following administration of hpGRF(1-24)-NH2 are
Experiment 2: hpGR F(1-29)-NH 2
Responses of bGH concentration in plasma following intravenous administration of hpGRF(1-29)-NH2 were much greater than those with the 1-24 fragment in terms of both peak concentration and persistency of response. Thus, mean peak concentrations were up to 30 ng/ml, and concentrations in plasma generally did not return to baseline until 4 to 5 h after administration (Figure 2). As in Experiment 1, differences were marked between cows in magnitude and pattern of response to each dose of hpGRF(1-29)-NH2. Response areas were calculated over the entire postadministration sampling period (0 to 300 min), and results are in Table 1. There was
TABLE 1. Plasma growth hormone (bGH) response to varying doses of human pancreatic growth hormonereleasing factors (hpGRF(I-24)-NH 2 and hpGRF(1-29)-NH~).
Treatment
hpGRF(1-24)-NH22 hpGRF(1.29)-NH22
0
hp GRF dose (#g) ~ 500 1000
2000
Pooled SE
-11.3a 173.3a
Area response (ng.min-m1-1) 52.9ab 129.3ab 2170.3 b 2394.8 b
187.4b 2435.3 b
47.2 479.2
a'bwithin each row, treatment means with different superscripts differ (P<.05). t Doses were O, .91, 1.82, and 3.64 #g/kg, respectively, for hpGRF(1-24)-NH 2 and 0, .82, 1.64, and 3.28 #g/kg respectively, for hpGRF(1-29)-NH 2. Amidated forms of human pancreatic growth hormone-releasing factor containing 24 or 29 amino acids. Journal of Dairy Science Vol. 67, No. 12, 1984
2884
McCUTCHEON ET AL.
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=
= :>000/.~g
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0
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I
-40
I
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i
40
80
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120
l
~
160
200
,
240
280
Time (min) Figure 2. Responses in plasma concentrations of growth h o r m o n e to varying doses of hpGRF(1-29)-NH 2 (the amidated form of h u m a n pancreatic growth hormone-releasing factor containing 29 amino acids) administered intravenously at time zero. Standard errors averaged 26% of the mean.
Journal o f Dairy Science Vol. 67, No. 12, 1984
GROWTH HORMONE-RELEASING FACTORS IN COWS difference between experiments in calculated "response" of the controls (--11.3 vs. 173.3 ngm i n . m l - 1 ) , but this largely reflects errors associated with estimation of baseline. Neither "response" was significantly different from zero by t test. All doses of hpGRF(1-29)-NH2 gave similar responses that were significantly different from controls. Administration of 1000/~g of hpGRF(1-29)NH2 to two cows by subcutaneous and intramuscular routes gave similar responses (Table 2). However, these responses were tess persistent than those achieved by intravenous injection of the same dose, and as a result, response areas were lower. DISCUSSION
This is the first study to examine effects of h p G R F on plasma concentrations of growth hormone in lactating cows. The N-terminal 1-24 and 1-29 fragments of h p G R F both stimulated growth hormone release in lactating dairy cows. Responses to hpGRF(1-24)-NH2 tended to increase across the dose range (500 to 2000/~g), whereas with the 1-29 fragment responses to a dose of 500 /ag were similar to those at 1000 and 2000/~g. Although the two fragments were not compared directly in the same cows, hpGRF(1-24)-NH2 had approximately 5 to 7% the potency of hpGRF(1-29)-NH2 (based on area responses at the 1000 and 2000/~g doses). When assayed in anaesthetized rats, hpGRF(124)-NH2 had 4% the potency of hpGRF(1-29)NH2 (Lance, Murphy, and Cox, unpublished). Rivier et al. (11) reported that hpGRF(1-29)NH2 had similar potency (within a factor of two) to hpGRF(1-40)-OH when tested in vitro with rat pituitary cell cultures. However, potency o f hpGRF(1-27)-NH2 was only 10 to 20% that of hpGRF(1-40)-OH. Administration of 500 to 2000/~g h p G R F (1-24)-NH2 to these cows produced detectable increases of bGH concentration in plasma within 5 rain and a return to baseline at 60 min. This pattern is similar to that for heifers given .01 to .10 /ag hpGRF(1-40)-OH per kg body weight (10) and for steers given .03 to .30 /lg hpGRF(1-44)-NHz per kg body weight (8). In our study, intravenous injection of hpGRF(1-29)-NH2 caused much more persistent responses with few cows returning to baseline before 240 min postadministration. Moseley
2885
et al. (8) observed a biphasic release of bGH in steers administered 1000 /ag hpGRF(1-44)NH2. A similar pattern was evident when we administered hpGRF(1-29)-NH2 to the cows in Experiment 2, particularly at the 2000/ag dose (Figure 2). However, cows were not consistent in timing or magnitude of the second bGH spike, and more extensive sampling would be required to characterize it adequately. With respect to our original hypothesis, we conclude that attempts to elevate plasma bGH concentrations by administration of h p G R F , and thus induce a lactational response, are likely to be limited by a number of factors. That cows do not exhibit consistent relationships between dose of h p G R F and bGH response (area under the curve) is obviously a major problem. Our preliminary data suggest that, in contrast to administration of exogenous bGH (4), the method by which h p G R F is delivered influences the response of circulating concentrations of bGH. In the two cows that we examined, intravenous injection gave responses three times as great as those arising from alternative routes of administration. The possibility exists that, when administered by intramuscular or subcutaneous injection, hpGRF(1-29)-NHz is degraded partially before absorbed into the bloodstream. Finally, responses of bGH concentration in plasma that result from i.v. injection of up to 2000 /~g hpGRF(1-29)-NHz would not be sufficient to generate substantial increases of milk yield. For example, Fronk et al. (4) administered 39.6 mg/day (51.5 IU/day) of exogenous bGH to dairy cows and increased daily milk production by 4.2 kg (31%). Plasma bGH concentration
TABLE 2. Plasma growth hormone (bGH) response in two cows following administration of 1000 ug hpGRF(1-29)-NH2 ~ by various routes.
Route of administration
Area response (ng.min'm1-1 ) Cow A Cow B
Intravenous Subcutaneous Intramuscular
3665 1325 1415
2230 780 760
Amidated form of human pancreatic growth hormone-releasing factor containing 29 amino acids. Journal of Dairy Science Vol. 67, No. 12, 1984
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McCUTCHEON ET AL.
was elevated b y 20 n g / m l w h i c h over 24 h r e p r e s e n t s an area r e s p o n s e of 2 8 , 8 0 0 n g ' m i n " m1-1. We c o m p l e t e d a similar s t u d y in w h i c h 17.9 m g / d a y (25 I U / d a y ) b G H was a d m i n i s tered (McCutcheon and Bauman, unpublished). Milk yield i n c r e a s e d b y 4.9 kg (27%) over controls a n d p l a s m a b G H b y 9 3 0 0 n g ' m i n ' m 1 - 1 over a w h o l e day. T h e s e responses in area u n d e r t h e b G H c o n c e n t r a t i o n curve are, respectively, 11.8 a n d 3.8 t i m e s as great as t h o s e f o l l o w i n g injection of 2000/ag hpGRF(1-29)-NH2. Thus, t o o b t a i n a l a c t a t i o n a l response, m u l t i p l e doses of h p G R F w o u l d n e e d to b e a d m i n i s t e r e d , a n d q u e s t i o n s o f r e f r a c t o r i n e s s to t h e releasing factor become of particular importance. REFERENCES
1 Baile, C. A., M. A. Della-Fera, and F. C. Buonomo. 1983. Growth hormone releasing factor (GRF) injections in sheep. J. Anita. Sci. 57(Suppl. 1):188. (Abstr.) 2 Collier, R. J., J. P. MeNamara, C. R. Wallace, and M. H. Dehoff. 1984. A review of endocrine regulation of metabolism during lactation. J. Anim. Sci. 59:498. 3 Eppard, P. J., D. E. Bauman, and S. N. McCutcheon. 1984. Effect of varying doses of bovine growth hormone on lactational performance of dairy cows. J. Dairy Sci. (submitted). 4 Fronk, T. J., C. J. Peel, D. E. Bauman, and R. C. Gorewit. 1983. Comparison of different patterns of exogenous growth hormone administration on milk production in Holstein cows. J. Anita. Sci. 57:699. 5 Gelato, M. C., O. Pescovitz, F. Cassorla, D. L. Loriaux, and G. R. Merriam. 1983. Effects of a growth hormone releasing factor in man. J. Clin.
Journal of Dairy Science Vol. 67, No. 12, 1984
Endocrinol. Metab. 57:674. 6 Gorewit, R. C. 1981. Pituitary, thyroid and adrenal response to clonidine in dairy cattle. J. Endocrinol. Invest. 4:135. 7 Guillemin, R., P. Brazeau, P. Bohlen, F. Eseh, N. Ling, and W. B. Wehrenberg. 1982. Growth hormone-releasing factor from a human pancreatic tumor that caused acromegaly. Science 218:585. 8 Moseley, W. M., L. F. Krabill, A. R. Friedman, and R. F. Olsen. 1984. Growth hormone response of steers injected with synthetic human pancreatic growth hormone-releasing factors. J. Anim. Sci. 58:430. 9 National Research Council. 1978. Nutrient requirements of domestic animals. No. 3. Nutrient requirements of dairy cattle. 5th rev. ed. Natl. Acad. Sci., Washington, DC. 10 Plouzek, C. A., L. L. Anderson, D. L. Hard, J. R. Molina, A. Trenkle, W. Vale, and J. Rivier. 1983. Effect of intravenous injection of a growth hormone-releasing factor on plasma growth hormone concentrations in cattle. J. Anim. Sci. 57(Suppl. 1):203. (Abstr.) 11 Rivier, J., J. Spiess, M. Thorner, and W. Vale. 1982. Characterization of a growth hormone-releasing factor from a human pancreatic islet tumor. Nature 300:276. 12 Rosenthal, S. M., E. A. Schriock, S. L. Kaplan, R. Guillemin, and M. M. Grumbach. 1983. Synthetic human pancreas growth hormone-releasing factor (hpGRF144-NH~) stimulates growth hormone secretion in normal men. J. Clin. Endocrinol. Metab. 57:677. 13 Sueiras-Diaz, J., V. A. Lance, W. A. Murphy, and D. H. Coy. 1984. Structure-activity studies on the N-terminal region of glucagon. J. Med. Chem. 27: 310. 14 Vale, W., J. Vaughan, G. Yamamoto, J. Spiess, and J. Rivier. 1983. Effects of synthetic human pancreatic (tumor) GH releasing factor and somatostatin, triiodothyronine and dexamethazone on GH secretion in vitro. Endocrinology 112:1553.