DOMESTIC ANIMAL ENDOCRINOLOGY
Vol. 8(2):307-321, 1991
EFFECT OF GROWTH HORMONE-RELEASING FACTOR INFUSION ON SOMATOTROPIN, PROLACTIN, THYROXINE, INSULIN, INSULIN-LIKE GROWTH FACTOR I AND BLOOD METABOLITES IN CONTROL AND SOMATOSTATIN-IMMUNIZED GROWING PIGSt PoDubreuil*, D. Petitclerc, P. Gaudreau, P. Brazeau and G. Pelletier Agriculture Canada, Research Station, Lennoxville, Quebec, Canada, J 1M 1Z3 and Notre-Dame Hospital, University of Montreal, Montreal, Qu6bec, Canada H2L 4MI Received January 29, 1990
ABSTRACT The aim of this study was to characterize the effects of prolonged infusion of growth hormonereleasing factor (1-29)NH2 (GRF) on plasma concentrations of hormones and metabolites when administered to control pigs and pigs immunized against somatostatin (SRIF). In the first experiment, eight purebred Yorkshire boars averaging 113 _+ 2 kg BW were immunized against SRIF conjugated to bovine serum albumin (BSA) (n = 4) or BSA alone (n = 4). Somatotropin (ST) response to four rates of GRF infusion (0, 1.66, 5 and 15 ng/min/kg BW) for 6 hr was evaluated using a double balanced 4 x 4 Latin square design. During the 4 hr before infusion, SRIF-immunized animals tended (P = 0.06) to have a higher ST release (613 vs 316 ng • min/ml, SE = 232) than controls. During infusion, GRF elicited a dose-dependent increase in ST release in both squares; the ST response was not better in SRIF-immunized animals than in controls (P > 0.05) (1435 vs 880 ng• mirdml; SE = 597). In the second experiment, ten purebred Yorkshire boars (5 controls and 5 SRIF-immunized animals) averaging 69 _+2 kg BW were continuously infused with GRF at the rate of 15 ng/min/kg BW for six consecutive d. Under GRF infusion, ST concentrations increased (P < 0.05) from 805 to 4768 ng • min/ml (SE = 507) from day 1 to day 6 in both SRIF-immunized and control animals. Prolactin levels increased (P < 0.05) with GRF infusion; pattern of increase was different (P 0.10) for this period over the four consecutive d of sampling. No interaction between immunization status and dose of GRF infused was observed (P > 0.10). During the six hr of GRF infusion, immunized animals did not release (P > 0.10) more ST than controls (1435 v s 880 ng.min/ml). In both squares, ST concentrations increased in relation to the dose of GRF infused (P = 0.04) from 669 to 2137 ng°min/ml. During the 4-hr period following the infusion, SI animals displayed higher ST concentrations than CI animals (2678 v s 1067 ng.min/ml, P < 0.05) and this effect was related (P = 0.04) to the dose of GRF infused (Table 1 and Fig. 1). TABLE 1. AREASUNDERTHE ST CURVESFORPERIODSOF 4-HR PRE, 6-HR DURINGAND4-HR FOLLOWING /V PGRF(I-29)NH 2 INFUSIONIN CONTROLSAND SRIF-IMMUN1ZEDPIGS
Item
GRF (n~,/ks/min)
P value
0
1.66
5.0
15.0
control
158
482
206
422
immunized
612
502
531
807
control
468
582
883
1587
immunized
870
910
1275
2687
control Post-infusion (4 hr) immunized
162
340
202
363
466
629
513
1070
Pre-infusion (4 hr) During infusion (6 hr)
SE
lmmunizaton Linear effect of dose
232 .06
.7
597
.21
.04
146
.01
.04
Values are least squares means of the AUC for ST (ng.min/ml). P values represent: 1° the difference between control and immunized pigs, and 2 ° the original polynomial contrast of dose effect; the quadratic and cubic contrasts were not significant. The interaction immunization X dose was not significant (P > 0.10).
GRF INFUSION ON HORMONE AND BLOOD VARIABLES
18
311
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Figure 1. Effectsof pGRF (1-29)NH2infusionin 4 controland 4 SRIF-immunizedgrowingpigs over 4 consecutive d usingtwo 4 x 4 Latin squaredesign. The rate of GRF infusionwas 0, 1.66,5.0 and 15.0ng/min/kgBW. Each point is the mean ST concentrationsof 4 animals. Bloodsamplingprocedurewas for 14 hr (0800to 2200 hr) and the infusion lasted for 6 hr (1200to 1800hr). Experiment 2. Binding titers of CI animals five d prior to sampling averaged 1.3 + 0.2% and 1.1 + 0.2% compared to 55.2 + 10.5% and 41.4 + 11.3% in SI animals at 1:150 and 1:1500 serum dilution, respectively. Growth hormone-releasing factor increased (P < 0.05) the amplitude of ST peak as well as the AUC between 2 and 6 hr after the beginning of the infusion in CI and SI pigs. Over the six d of infusion, the ST AUC increased from 805 ng-min/ml during the pre-infusion period to 4768 ng.min/ml on day 6 (Table 2, Fig. 2). Prolactin concentrations were increased (P < 0.05) by chronic GRF exposure; however, pattern of increase was different depending if the animals were immunized or not against SRIF as indicated by the interaction day X treatment (P < 0.01) (Table 2). Thyroxine and IGF-I concentrations were increased during GRF infusion and this effect reached a plateau (P < 0.05) after three d of infusion. For both hormones, SI animals exhibited higher (P < 0.05) hormone concentrations than CI (Table 3). Somatostatin-immunized and CI animals had similar serum glucose and insulin concentrations. A rapid increase (P < 0.05) in serum glucose was observed as soon as 6 hr after the beginning of the infusion without change (P > 0.05) in insulin concentrations. After two d of infusion, glucose and insulin concentrations reached maximal values to decline gradually during the three following d (Table 3).
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TABLE2. EFFEC'rOF PGRF(I-29)NHz INFUSIONDURING6 CONSECUTIVEDAYSON DWFERENTPARAMETERSOF SOMATOTROPIN(ST) ANDPROLACTIN(PRL) RELEASEIN GROWINGPIGS Item
Day 1
2
4hr before infusion
2-6hr after infusion
3
P value 4
5
6 SE
ST peak amplitude (ng/ml)
control
10.4
25.1
13.0
38.6
35.1
64.3
48.3
immunized
6.4
32.7
19.3
66.9
49.1
57.6
60.6
Area under the ST curve (ng.min/ml)
control
844
1584
1257
2700
3091
4620
4461
immunized
766
2180
1560
4279
3451
4372
5074
Area under the Prl curve (ngomin/ml)
control
945
939
1170
1372
1441
1224
1453
immunized
979
1076
SI
1304
1328
1467
1165
Day X SI
1 vs 1
Day effect L
Q
C
8.4
NS
NS
.02
.001
.01
NS
507
NS
NS
.04
.001
NS
NS
39
NR
.01
NS NS
.001 .001
.01 .001
.02 .001
1311
Values are least squares means of 5 animals. P values represent: 1° the difference observed between control and SRIF-immunized (SI) animals; 2° the interaction day X SI; 3 ° comparison of 4 hr before to 2 to 6 hr after the beginning of the infusion (1 v s 1); and, 4 ° the orthogonal polynomial contrasts of day effect. L: linear, Q: quadratic, C: cubic. NS: not significant (P > 0.05). NR: not reported because of the significant day X SI interaction. t~ C m C I--"
m
GRF INFUSION ON HORMONE AND BLOOD VARIABLES
mI 40
313
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TIME (h) Figure 2. Effects pGRF (1-29)NH~ infusion at the rate of 15 ng/min/kg BW on ST release in five control and 5 SRIFimmunized pigs over 6 consecutive d. Blood samples were taken daily at 20-min intervals for 4 hr (0900 to 1300 hr).
4.2
1
5.5
1.70
--
--
3.60
2.52
2-6hr after infusion
4.4
3.53
169
129
3.99
2.80
2
4.7
3.99
200
158
4.50
3.53
3
Day
4.2
3.34
254
197
4.68
3.58
4
4.2
3.02
240
219
4.48
3.31
5
3.7
3.00
248
224
3.94
2.96
6
.2
.43
11
.16
SE
NS
NS
.03
.008
SI
NS
NS
NS
NS
Day X SI
.001
NS
NA
.001
1 vs 1
P value
.01
NS
.001
.001
L
.01
.001
.01
.001
Q
Day effect
NS
NS
NS
NS
C
Values are least squares means of 5 animals. P values represent: 1° the difference observed between control and SRIF-immunized animals (SI); 2 ° the interaction day X SI; 3° comparison of values before and 2-6 hr after the beginning of the infusion (1 v s 1); and, 4 ° the orthogonal polynomial contrasts of day effect. L: linear, Q: quadratic, C: cubic. NS: not significant (P > 0.05). NA: not available.
Glucose (retool/l)
139
immunized
1.84
90
2.76
immunized
control
2.29
control
4hr before infusion
Insulin (ng/ml)
IGF-I (ng/ml)
T4 (I.tg/dl)
Item
TABLE3. EFFECTOFPGRF(1-29)NH 2 INFUSIONDURING6 CONSECUTIVEDAYSONTHYROXINE(T4), INSULrN-L]KEGROWTHFACTORI (IGF-I), INSULINANDGLUCOSECONCENTRATIONSIN GROWINGPIGS
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GRF INFUSION ON HORMONE AND BLOOD VARIABLES
315
Table 4 reports blood variables which were not influenced by immunization and did not show any significant day X immunization interaction. As early as 6 hr after the beginning of the infusion, values of K and triglyceride increased (P < 0.05) and creatinine and P decreased (P < 0.05). A steady increase with days of infusion was observed with Na, anion gap and FFA. A transitory increase was observed with Ca, P, alkaline phosphatase, triglyceride, K and total bilirubin and a transitory decrease was notified with GOT and creatinine. Table 5 reports blood parameters from which an interaction day X immunization was significant (P < 0.05). Chlorine and cholesterol values increased (P < 0.05) gradually and albumin and COz increased transitory in SI and CI pigs, respectively. The other variables did not show any variation due to treatment over time. DISCUSSION The present data indicates that both active immunization against SRIF and GRF infusion increase ST concentrations in growing pigs. In the first experiment, before the beginning of the infusion, SI pigs had higher ST concentrations than controls, indicating the presence of an inhibitory action by hypothalamic SRIF on ST release in pigs (5,51) as reported in rats (4) and ruminants (27,28). However, even if GRF induced a linear increase in ST release with increasing rates of GRF infusion, SI animals did not respond better than CI during this period. Similar observations were reported in dairy heifers (28) indicating that endogenous SRIF exerts a negative effect on basal ST release but, during ST peak mediated by an increase of exogenous GRF, the presence of residual SRIF does not seem to influence the magnitude of the ST response. In normal adult men infused with hGRF(1-44)NH2, at the rate of 1.0, 3.3, 10 and 33 ng/min/kg BW, an increase in ST release was also observed in a dose response manner for the three lowest doses (14). During the 4-hr period following the end of the GRF infusion, SI pigs exhibited higher ST concentrations indicating that SRIF could actively participate in the return of ST to basal level after GRF infusion. Because the highest rate of GRF infusion in experiment 1 elicited the highest ST release in CI and SI pigs without evidence of desensitization, this dose (15 ng/min/kg BW) was infused over 6 d. As observed in experiment 1, SI animals did not release more ST than CI under GRF stimulation but the area under the ST curve increased with increasing days of infusion in both groups. In short term studies, this observation has never been reported: normal healthy adults infused with hGRF(I-44)NH 2 at the rate of 100 I.tg/h (approximately 25 ng/min/kg BW) for 2 to 5 hr did not sustain elevated levels of ST (15). The same observation was reported in boys (12 + 1.7 year of age) infused for 3 hr at the rate of 2500 ng/min (13). However, when six healthy young men were infused with hGRF(1-40)NH 2 at the rate of 2 ng/min/kg BW, an increase in the natural occurrence of ST pulses was noted (16). In human, it seems that high rates of GRF infusion over short periods of time produces a reduction of ST release over time and that longer infusion periods at a lesser rate of infusion maintained and even increased ST level. In conscious freely moving rats passively immunized against SRIF and infused over 24 hr with hGRF(1-44)NH 2 at the rate of 750 ng/min/kg BW, a decrease in ST release was observed after the first 6 hr of infusion. This decrease was associated with a depletion of the ST pituitary store (12). Since SRIF might exert a role to maintain somatotroph responsiveness to GRF (30), SRIF-neutralization may worsen the desensitization. In rat anterior pituitary cells exposed for 24 hr to rat GRF, a depletion of cellular ST store and a decrease sensitivity to rat GRF were observed (29). In cattle, 5 d of microinjections of hGRF( 1-44)NH2 at 3.75-min intervals at a dose close to 14 ng/min/kg BW sustained over time ST release (17). In steers and wethers implanted with osmotic pumps, a sustained increase in ST levels for 7 d was also observed (31). In our experiment, ST release increased with number of days of infusion. To our knowl-
144.2 5.4 2.45 2.86 17.6 4.1 42.5 213 70.1 157 103 .443 1.70
4 hr before infusion
1
145.3 5.8 2.42 2.76 16.5 4.5 34.3 230 66.5 125 94 .538 1.66
2-6 hr after infusion
148.1 6.0 2.58 3.05 19.3 3.6 34.2 221 70.0 180 99 .520 2.17
2
145.3 5.8 2.62 3.13 17.6 3.8 29.2 244 71.0 136 l 01 .508 2.20
3
Day
147.8 6.2 2.64 3.29 18.4 4.3 30.6 289 72.3 176 101 .479 2.08
4
147.8 6.3 2.49 3.01 19.0 3.9 34.5 294 69.3 179 105 .416 2.30
5
148.3 5.7 2.41 3.01 20.3 3.5 37.3 251 71.0 204 104 .389 1.88
6
.1 .1 .02 .03 .7 .2 3.1 16 .7 I1 1 .027 .09
SE NS .001 NS .02 NS NS NS NS NS NS .001 .01 NS
1 vs 1
.001 .001 NS .001 .02 NS NS .001 NS .01 .02 .01 NS
L NS .001 .001 .001 NS NS .002 .03 NS NS .02 .01 .001
Q
Day effect
P value
NS .001 NS NS NS .001 NS .02 .01 NS .02 NS NS
C
Values are least squares means of 5 animals. Effect of SI and the interaction days X SI were not significant (P > 0.05). P values represent: 1° comparison of values 4 hr before and 2-6 hr after the beginning of the infusion (1 vs 1); and, 2 ° the orthogonal polynomial contrasts of day effect. L: linear, Q: quadratic, C: cubic.
Na (mmol/1) K (mmolB) Ca (mmol/l) P (mg/dl) Anion Gap BUN (mmol) GOT (IU/1) Alk. Phosp. (IU/L) Tot. Prot. (g/l) FFA (I.tEq/1) C reatinine (lamol/l) Triglyceride (mmol/l) Total Bilirubin (I.tmol/l)
Item
TABLE 4. EFFECT OF PGRF( 1-29)NH 2 INFUSION DURING 6 CONSECUTIVE DAYS ON DIFFERENT SERUM PARAMETERSIN GROWING PIGS
C F m --I
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~0
w
C
co
2.75
2.37
Control
Immunized
35.6
36.6
Control
Immunized
23.2
22.3
Control
Immunized
103.2
104.4
Immunized
Control
4hr before infusion
1
2.49
2.26
34.9
35.6
24.1
25.2
104.4
103.9
2-6hr after infusion
2.68
2.36
38.1
35.4
24.0
24.2
104.6
104.8
2
2.74
2.41
37.5
36.5
23.4
25.5
104.0
102.5
3
Day
2.85
2.79
36.8
37.9
23.5
26.2
105.0
104.1
4
2.91
2.63
36.9
34.9
23.9
25.3
105.1
103.4
5
2.84
2.86
36.1
35.7
22.1
23.4
105.3
105.1
6
.07
.5
.5
.4
SE
.03
.001
.005
.01
X SI
Day
NS
NS
NS
NS
NS
.001
NS
NS
1 vs 1
NS
.001
NS
NS
NS
.04
.01
NS
L
P value
NS
NS
.02
NS
NS
.001
NS
NS
Q
Day effect
NS
NS
NS
NS
NS
NS
NS
NS
C
Values are least squares means of 5 animals. No effect of immunization was observed (P > 0.05). P values represent: 1° the interaction day X immunization treatment (SI); 2° the comparison of values 4 hr before and 2-6 hr after the beginning of the infusion ( 1 v s 1); and, 3° the orthogonal polynomial contrasts of day effect: L: linear, Q: quadratic, C: cubic. NS: not significant (P > 0.05).
Cholesterol (mmol/l)
Albumin (g/l)
CO 2 (mmol/l)
Chlorine (retool/l)
Item
TABLE5. EFFECTOFPGRF(1-29)NH 2 INFUSIONDURING6 CONSECUTIVEDAYSON DIFFEREm"SERUMPARAMETERSINFLUENCEDBY IMMUNIZATIONANDDAYSOF INFUSIONIN GROWINGPIGS
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318
DUBREUIL ET AL
edge, only one experiment (33) using infusion in young bull calves reported similar observations. However, injection of GRF in rats with the pituitary autotransplanted to the kidney capsule (35) and in ST deficient children (36), it was observed that multiple administration of GRF enhanced the ST response to subsequent GRF injection. In lactating sows (37) and in growing pigs (19) injected with pGRF(I-29)NH 2 at doses of 20 and 15 ~tg/kg BW, respectively, there were increases in ST release with increasing days of injection (20 d in sows and 85 d in growing pigs). These results indicate that GRF could enhance ST synthesis (38,39) or somatotroph proliferation (40). Somatotropin surges were evident within each daily 4-hr period of blood collection in both CI and SI pigs. Similar surge occurred in rats (32), human (14,16) and cattle (33,34) receiving GRF infusion. A cyclic SRIF and GRF secretion has been postulated to be responsible of the occurrence of ST peaks in rats (3) and pigs (5) and SRIF has been reported to control ST baseline level (5). Because SI animals also exhibited ST peak, this suggests that GRF in pigs could also be released in a cyclic manner. Moreover, GRF infusion added to the cyclic endogenous GRF produced an increase in ST peak. In this model, it seems that SRIF has only a minor role to play to control the amplitude of ST peak since CI and SI animals did not show any significant difference. Immunization against SRIF did not have any effect on basal Prl release in rats (4) which is in accordance with the fact that SRIF exerts no effect on basal Prl secretion (41). Growth hormone-releasing factor was also reported to have no effect on Prl release in rats (42) and bull calves (34) after a single injection and in man after 4 hr of GRF infusion (15). In the present study, an increase in Prl concentrations was observed 24 hr after the beginning of the infusion. Immunized pigs showed an increase in Prl concentrations which was 24 hr sooner than controls, indicating that SRIF might exert a negative effect on the GRF-induced Prl release (41). In a previous experiment (53) using growing pigs injected twice daily for 12 weeks with pGRF(1-29)NH 2, we observed that injected animals had constant Pri concentrations over time when compared to controls which showed a decrease in Prl levels over time. Since immunoreactive ST and Prl have been isolated in the same adenohypophysial cell, it is possible that chronic ST stimulation leads to Prl secretion. Indeed, serum Prl concentrations are elevated in 40% of acromegalic women (43). On the other hand, GRF might directly affect the lactotroph cells (45). Since SRIF regulates thyrotropin (TSH) levels (43), SI animals might have higher TSH levels (4) and consequently higher T4 levels. However, the transient increase in T 4 observed as soon as 6 hr after the beginning of the infusion is not clear since acromegalic patients have normal T4 concentrations (43) as well as lactating cows infused for 20 d with GRF (44). Insulin and glucose concentrations demonstrated a transient increase. An increase in glucose levels over an infusion period of 20 d with GRF has been reported in dairy cows (44) and in growing pigs injected directly with ST or GRF (6) and could be associated with a concurrent increase in hepatic glucose production and/or impaired glucose clearance by peripheral tissues (46). Insulin-like growth factor-1 was higher in SI animals, which is in agreement with the observed increase in ST concentrations in these animals. Somatostatin-immunized sheep have also increased ST levels (47) and IGF-I levels (48). The rate of increase in IGF-I concentrations is slower after 4 or 5 d of GRF infusion, but pigs injected for 57 d (54) with GRF have a constant increase in IGF-I indicating that the production of this hormone does not seem to be desensitized. Several blood parameters are influenced by GRF infusion and high ST levels. However, the fairly short period of time (6 d) probably did not permit an equilibration of these parameters. The most recognized parameters influenced by GRF are a decrease in BUN (6), and an increase in FFA (44) and cholesterol as observed in experiment 2 (49). Baile et al. (50) re-
GRF INFUSION ON HORMONE AND BLOOD VARIABLES
319
ported some changes in blood Na, C1, cholesterol, BUN, albumin, globulin and GOT 4 hr following human ST injections on the fast and last day of the growing period. The results of the present study show that active immunization against SRIF increases ST and IGF-I concentrations in growing pigs and constant pGRF(1-29)NH2 infusion at the rate of 15 ng/min/kg BW over 6 d induced an increase in ST concentrations with days of infusion. An increase in Prl, T 4 and IGF-I concentrations and a transient increase in glucose and insulin concentrations were also observed. Long-acting delivery systems hold promise for future use of GRF in production trials. ACKNOWLEDGMENTS/FOOTNOTES We wish to acknowledge the technical assistance of I. Blanchet, J. Brochu, L. Delorme, L. St-James, L. Brousseau,G. Bertrandand M. Dumas,the secretarialassistanceof L. C6t~and editingassistanceof E Lowry. tThis study was supported by Agriculture Canada Contribution #315, NSERC grant A2312 and Sanofi Recherche, Montpellier,France. *Fellow of the MedicalResearch Councilof Canada. Presentaddress: Dept. of Medicine,Collegeof Veterinary Medicine,Universityof Montreal,Box 5000 St-Hyacinthe,Qu6bec,CanadaJ2S 7C6. From Dr. G. Descotes, Sanofi,Montpellier,France From Dr. T.E Mowles,HoffmanLaRoche,Nutley,N.J. 3 Kindlydonated by the USDA AnimalHormoneProgram and the NationalHormoneand PituitaryProgram, Beltsville,MD, USA 4ResearchProductInternationalCorp.,410 N. BusinessCenterDrive,MountProspect, Illinois60056. 5DaymarLaboratoriesInc.,229 YongeSt., Suite207, Toronto,OntarioM5B 1N9. 6SigmaChemicalCompany,P.O.Box 14508,St-Louis,Missouri63178. 7LincoResearch Inc.,P.O. Box 641, Eureka,Missouri63025. s Antiserumprovided by Drs. J. Underwoodand J.J. Van Wyk, 509 Clinical Sciences Bldg 229H, School of Medicine,Universityof North Carolina,North Carolina,ChappelHill,North Carolina27514 and distributedby the HormoneDistributionprogramof the NIDDKthroughthe NationalHormoneand PituitaryProgram. 9Antibodyprovidedby Drs. A. Belangerand S. Caron, Laboratoirede Neuroendocrinologie,Centre Hospitalier Universitaire,Universit6Laval,2705 Blvd.Laurier,Qu6bec,Qu6becG1V 4G2. ~oBiomegaDiagnosticInc.,Montr6al,Qu6bec,Canada. HBoehringerMannheim,11450C6t6de Liesse Dorval,Qu6bec,Canada. ~2BeckmanInstrumentsInc.,52-54ch. des Bourdons,Gagny,France. ~3BoehringerMannheim,38240Meylan,France. ~4BoehringerMannheim,9115 HagueRd., Indianopolis,IN, USA. ,5BoehringerMannheim,Touanvmon,Min Ato-Ky,Tokyo,Japan. ~6WellcomeFoundationLtd., TempleHill,Dartford, England. REFERENCES 1. Brazcau P, Vale W, Burgus R. Hypothalamic peptide that inhibits the secretion of immunoreactive pituitary GH. Science 179:77-79, 1973. 2. Guillemin R, Brazeau P, Bohlen P, Esch E Ling N, Wehrenberg WB. Growth hormone-releasing factor from a human pancreatic tumor that caused acromegaly. Science 218:585-587, 1982. 3. Tannenbaum GS, Ling N. The interrelationship of growth hormone- releasing factor and somatostatin in the generation of the ultradian rhythm of growth hormone secretion. Endocrinology 115:1952-1957, 1984. 4. Terry LC, Martin JB. The effects of lateral hypothalamic medial forebrain stimulation and somatostatin antiserum on pulsatile growth hormone secretion in freely behaving rats. Endocrinology 109:622-627, 1981. 5. Klindt J, Ford J, Berardinelli JG, Anderson LL. Growth hormone secretions after hypophysial stalk transection in pigs. Proc Soc Exp Bioi Meal 172:508-513, 1983. 6. Etherton TD, Wiggins JP, Chung CS, Evock CM, Rebhun JF, Walton PE. Stimulation of pig growth performance by porcine growth hormone and growth hormone-releasing factor. J Anita Sci 63:1389-1399, 1986. 7. Della-Fera MA, Buonomo FC, Baile CA. Growth hormone-releasing factors and secretion of growth hormone in sheep, calves and pigs. Domest Anita Endocrinol 3:165-176, 1986. 8. Petitclere D, Pelletier (3, Lapierr¢ H, Gaudreau P, Couture Y, Dubreuil P, Morisset J, Brazeau P. Dose-response of two synthetic human growth hormone-releasing factors on growth hormone release in heifers and pigs. J Anim Sci 65:996-1105, 1987. 9. Dubreuil P, Pelletier G, Petitclerc D, Lapierre H, Couture Y, Gaudreau P, Morisset J, Brazeau P. Influence of growth hormone-releasing factor and(or) thyrotropin-releasing factor on somatotropin, prolactin, triiodothyronine and thyroxine release in growing pigs. Can J Anim Sci 68:699-710, 1988. 10. Dubreuil P, Lapierre H, Pelletier G, Petitclerc D, Couture Y, Gaudreau P, Morisset J, Brazeau P. Serum growth hormone release in response to a growth hormone-releasing factor analog during
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DUBREUIL ET AL
and after anesthesia in pigs. Can J Vet Res 52:304-307, 1988. 11. Dubreuil P, Pelletier G, Petitclerc D, Lapierre H, Couture Y, Brazeau P, Gaudreau P, Morisset J. Influence of age and sex on basal secretion of growth hormone (GH) and on GH-induced release by porcine GH-releasing factor p GRF(1-29)NH 2 in growing pigs. Domest Anim Endocrinol 4:299307, 1987. 12. Wehrenberg WB, Brazeau P, Ling N, Textor G, Guillemin R. Pituitary growth hormone response in rats during a 24-hour infusion of growth hormone-releasing factor. Endocrinology 114:1613-1616, 1984. 13. Takano K, Hizuka N, Shizume K, Hirose N, Ling N. Plasma growth hormone secretion during constant growth hormone-releasing factor infusion. Endocrinol Jpn 31:681-685, 1984. 14. Webb CB, Vance ML, Thomer MD, Perisutti G, Thominet J, Rivier J, Vale W, Frohman LA. Plasma growth hormone responses to constant infusions of human pancreatic growth hormone releasing factor. J Clin Invest 74:96-103, 1984. 15. Losa M, Bork Lilian, Schopahl J, Stalla GH, Miller OA, yon Werber K. Growth hormone-releasing factor infusion does not sustain elevated GH-levels in normal subjects. Acta Endocrinol 107:462470, 1984. 16. Vance ML, Kaiser DL, Evans WS, Furlanetto R, Vale W, Rivier J, Thorner MO. Pulsatile growth hormone secretion in normal man during a continuous 24 hr infusion of human growth hormonereleasing factor. J Clin Invest 75:1584-1590, 1985. 17. Moseley WM, Krabill LF, Freedman AR, Olsen RE Administration of synthetic human pancreatic growth hormone-releasing factor for five days sustains raised serum concentrations of growth hormone in steers. J Endocrinol 104:433-439, 1985. 18. Dubreuil P, Pelletier G, Petitclerc D, Lapierre H, Couture Y, Morisset J, Gaudreau P, Brazeau P. Effect of growth hormone-releasing factor ( 1-29)NH2 and thyrotropin-releasing factor on growth hormone release in lactating sows. Reprod Nutr Dev 27:97-99, 1987. 19. Dubreuil P, Couture Y, Pelletier G, Petitclerc D, Lapierre H, Gaudreau P, Morisset J, Brazeau P. Effect of long term administration of porcine growth hormone-releasing factor and(or) thyrotropinreleasing factor on growth hormone and thyroxine release in growing pigs. J Anita Sci 65 [Suppl. 1]:389, 1987. (Abstract) 20. Ford JJ, Maurer RR. Simple technique for chronic venous catheterization of swine. Lab Anim Sci 28:615-618, 1978. 21. Merrifield RD. Solid phase peptide synthesis 1. The synthesis of tetrapeptide. J Am Chem Soc 85:2149-2154, 1963. 22. Gaudreau P, Baillet M, Lrpine-Frenette C, Gorys V, Brazeau P. Preparative high performance liquid chromatography purification of mammalian somatocrinin. Fifth International Symposium of HPLC of Proteins, Peptides and Polynucleotides, Toronto, Canada, abstract #709, 1985. 23. Elsasser TH, Rumsey TS, Hammond AC, Frazer R. Influence of parasitism on plasma concentrations of growth hormone, somatomedin-C and somatomedin binding proteins in calves. J Endocrinol 116:191-200, 1988. 24. Statistical Analysis System Institute, Inc. SAS user's guide. SAS Institute, Inc. Carg. NC, 1982. 25. Gill JL. Design and analysis of experiments in the animal and medical sciences. Vol. 1, Iowa State University Press, Ames, IA, 1978. 26. Abramovitz M, Slegun IA. Handbook of Mathematical Functions, Dover Publications Inc., New York, pp. 885, 1972. 27. Varner MA, Davis SL, Reeves JJ. Temporal serum concentrations of growth hormone, thyrotropin, insulin and glucagon in sheep immunized against somatostatin. Endocrinology 106:1027-1032, 1980. 28. Petitclerc, D, Pelletier G, Dubreuil E Lapierre H, Farmer C, Brazeau E Effects of active immunization against somatostatin and growth hormone releasing factor infusion on growth hormone secretion in dairy heifers. J Anim Sci 66 [Suppl. 1]:389, 1988. (Abstract) 29. Bilezikjian LM, Vale WW. Long term exposure of cultured rat anterior pituitary cells to rat hypothalamic GRF alters somatotropin responsiveness to GRF. Endocrinology 115:2032-2034, 1984. 30. Clayton RN, Bailey LC. Somatostatin partially reverses desensitization of somatotrophs induced by growth hormone-releasing factor. J Endocrinol 112:69-76, 1987. 31. Wheaton JE, A1-Raheen SN, Godfredson JA, Dorm JM, Wong EA, Vale W, Rivier J, Mowles TF, Heimer ER Felix AM. Use of osmotic pumps for subcutaneous infusion of growth hormone-releasing factors in steers and wethers. J Anim Sci 66:2876-2885, 1988. 32. Wehrenberg WB. Continuous infusion of growth hormone-releasing factor: Effects on pulsatile growth hormone secretion in normal rats. Neuroendocrinology 43:391-393, 1986. 33. Moseley WM, Huisman J, Van Weerden EJ. Serum growth hormone and nitrogen metabolism responses in young bull calves infused with growth hormone-releasing factor for 20 days. Domest
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Anim Endocrinol 4:51-59, 1987. 34. Enright WJ, Zinn SA, Chapin LT, Tucker HA. Growth hormone response of bull calves to growth hormone-releasing factor. Endocrinology 184:483-488, 1987. 35. Jansson JO, Carlsson L, Isaksson OGP. Growth hormone-releasing factor pretreatment enhances the GRF-induced GH secretion in rats with the pituitary autotransplanted to the kidney capsule. Endocrinology 116:95-98, 1984. 36. Takano K, Hizuka N, Shizume K, Honda W, Ling NC. Plasma growth hormone responses to single and repetitive sc administration of hpGRF(1-44) in normal and GH deficient children. Acta Endocrinol 108: l 1- 19, 1985. 37. Epelbaum J, Brazeau P, Tsang D, Brawer J, Martin JB. Subcellular distribution of radioimmuoassayable SRIF in rat brain. Brain Res 126:309-323, 1977. 38. Cella SG, Locatelli V, Gennaro V, Puggioni R, Pintor C, MiJeller E. Human pancreatic growth hormone-releasing factor stimulates GH synthesis and release in infant rats. Endocrinology 116:575577, 1985. 39. Fukato J, Diamond DJ. Martin JB. Effects of rat GRF and somatostatin on the release and synthesis of rat GH in dispersed pituitary cells. Endocrinol I 17:457-467, 1985. 40. Billestrup N, Swanson LW, Vale W. Growth hormone-releasing factor stimulates proliferation of somatotrophs in vitro. Proc Natl Acad Sci USA 83:6854-6857, 1986. 41. Drouin J, De Lean A, Rainville D, Lachance R, Labrie E Characteristics of the interaction between TRH and somatostatin for thyrotropin and prolactin release. Endocrinology 98:514-517, 1976. 42. Guillemin R, Zeytin E Ling N, Bohlen P, Esch E Brazeau, E Bloch B, Wehrenberg WB. Growth hormone-releasing factor: chemistry and physiology. Proc Soc Exp Biol Med 175:407-413, 1984. 43. Wilson JD, Foster DW. Textbook of Endocrinology 7th Ed. WB Saunders Co, Philadelphia pp. 1413, 1985. 44. Enright WJ, Chapin LT, Moseley WM, Zinn SA, Tucker HA. Effect of 20-day infusions of GRF on blood hormones and metabolites in Holstein cows. J Anim Sci 66:390, 1988. (Abstract) 45. Stachura ME, Tyler JM, Farmer PK. Human pancreatic GRF differentially stimulates release of stored and newly synthesized rat GH in vitro. Endocrinology 116:698-706, 1985. 46. Gopinath R, Etherton TD. Porcine GH increases hepatic glucose production rate and impairs glucose clearance. J Anita Sci 66:292, 1988. (Abstract) 47. Varner MA, Davis SL, Reeves JJ. Temporal serum concentrations of GH, TSH, insulin and glucagon in sheep immunized against somatostatin. Endocrinology 106:1027-1032, 1980. 48. Bass JJ, Gluckman PD, Fairclough RJ, Peterson AJ, Davis SR, Carter WD. Effect of nutrition and immunization against somatostatin on growth and insulin-like growth factors in sheep. J Endocrinol 112:27-31, 1987. 49. Pelletier G, Dubreuil P, Gaudreau E Farmer C, Petitclerc D, Pommier S, Lapierre H, Mowles TF. Effect of dose and frequency of a potent analog of human GRF on serum components of growing pigs. J Anim Sci 66 [Suppl. l]: 295, 1988. (Abstract) 50. Baile CA, Della-Fera MA, McLaughlin C. Performance and carcass quality of swine injected with bacterially-synthesizedhuman GH. Growth 47:225-236, 1983. 51. Dubreuil P, Pelletier G, Petitclerc D, Lapierre H, Gaudreau E Brazeau E Effects of active immunization against somatostatin on serum growth hormone concentration in growing pigs: influence of fasting and repetitive somatocrinin injections. Endocrinology 125:1378-1384, 1989. 52. Purchas RW, Zinn SA, Tucker HA. A simple method of separating unbound and bound cortisol in radioimmunoassay. Analytical Biochem 149:399-403, 1985. 53. Dubreuil P, Couture Y, Pelletier G, Petitclerc D, Delorme L, Lapierre H, Gaudreau E Morisset J, Brazeau E Effect of long-term administration of porcine growth hormone-releasing factor and(or) thyrotropin-releasingfactor on growth hormone, prolactin and thyroxine concentrations in growing pigs. J Anita Sci 68:95-107, 1990. 54. Dubreuil E Petitclerc D, Pelletier G, Gaudreau P, Farmer C, Mowles TF, Brazeau E Effect of dose and frequency of administration of a potent analog of human growth hormone-releasing factor on hormone secretion and growth in pigs. J Anim Sci 68: 1254-1268, 1990.
Vol. 8(2):307-321, 1991
EFFECT OF GROWTH HORMONE-RELEASING FACTOR INFUSION ON SOMATOTROPIN, PROLACTIN, THYROXINE, INSULIN, INSULIN-LIKE GROWTH FACTOR I AND BLOOD METABOLITES IN CONTROL AND SOMATOSTATIN-IMMUNIZED GROWING PIGSt PoDubreuil*, D. Petitclerc, P. Gaudreau, P. Brazeau and G. Pelletier Agriculture Canada, Research Station, Lennoxville, Quebec, Canada, J 1M 1Z3 and Notre-Dame Hospital, University of Montreal, Montreal, Qu6bec, Canada H2L 4MI Received January 29, 1990
ABSTRACT The aim of this study was to characterize the effects of prolonged infusion of growth hormonereleasing factor (1-29)NH2 (GRF) on plasma concentrations of hormones and metabolites when administered to control pigs and pigs immunized against somatostatin (SRIF). In the first experiment, eight purebred Yorkshire boars averaging 113 _+ 2 kg BW were immunized against SRIF conjugated to bovine serum albumin (BSA) (n = 4) or BSA alone (n = 4). Somatotropin (ST) response to four rates of GRF infusion (0, 1.66, 5 and 15 ng/min/kg BW) for 6 hr was evaluated using a double balanced 4 x 4 Latin square design. During the 4 hr before infusion, SRIF-immunized animals tended (P = 0.06) to have a higher ST release (613 vs 316 ng • min/ml, SE = 232) than controls. During infusion, GRF elicited a dose-dependent increase in ST release in both squares; the ST response was not better in SRIF-immunized animals than in controls (P > 0.05) (1435 vs 880 ng• mirdml; SE = 597). In the second experiment, ten purebred Yorkshire boars (5 controls and 5 SRIF-immunized animals) averaging 69 _+2 kg BW were continuously infused with GRF at the rate of 15 ng/min/kg BW for six consecutive d. Under GRF infusion, ST concentrations increased (P < 0.05) from 805 to 4768 ng • min/ml (SE = 507) from day 1 to day 6 in both SRIF-immunized and control animals. Prolactin levels increased (P < 0.05) with GRF infusion; pattern of increase was different (P 0.10) for this period over the four consecutive d of sampling. No interaction between immunization status and dose of GRF infused was observed (P > 0.10). During the six hr of GRF infusion, immunized animals did not release (P > 0.10) more ST than controls (1435 v s 880 ng.min/ml). In both squares, ST concentrations increased in relation to the dose of GRF infused (P = 0.04) from 669 to 2137 ng°min/ml. During the 4-hr period following the infusion, SI animals displayed higher ST concentrations than CI animals (2678 v s 1067 ng.min/ml, P < 0.05) and this effect was related (P = 0.04) to the dose of GRF infused (Table 1 and Fig. 1). TABLE 1. AREASUNDERTHE ST CURVESFORPERIODSOF 4-HR PRE, 6-HR DURINGAND4-HR FOLLOWING /V PGRF(I-29)NH 2 INFUSIONIN CONTROLSAND SRIF-IMMUN1ZEDPIGS
Item
GRF (n~,/ks/min)
P value
0
1.66
5.0
15.0
control
158
482
206
422
immunized
612
502
531
807
control
468
582
883
1587
immunized
870
910
1275
2687
control Post-infusion (4 hr) immunized
162
340
202
363
466
629
513
1070
Pre-infusion (4 hr) During infusion (6 hr)
SE
lmmunizaton Linear effect of dose
232 .06
.7
597
.21
.04
146
.01
.04
Values are least squares means of the AUC for ST (ng.min/ml). P values represent: 1° the difference between control and immunized pigs, and 2 ° the original polynomial contrast of dose effect; the quadratic and cubic contrasts were not significant. The interaction immunization X dose was not significant (P > 0.10).
GRF INFUSION ON HORMONE AND BLOOD VARIABLES
18
311
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Figure 1. Effectsof pGRF (1-29)NH2infusionin 4 controland 4 SRIF-immunizedgrowingpigs over 4 consecutive d usingtwo 4 x 4 Latin squaredesign. The rate of GRF infusionwas 0, 1.66,5.0 and 15.0ng/min/kgBW. Each point is the mean ST concentrationsof 4 animals. Bloodsamplingprocedurewas for 14 hr (0800to 2200 hr) and the infusion lasted for 6 hr (1200to 1800hr). Experiment 2. Binding titers of CI animals five d prior to sampling averaged 1.3 + 0.2% and 1.1 + 0.2% compared to 55.2 + 10.5% and 41.4 + 11.3% in SI animals at 1:150 and 1:1500 serum dilution, respectively. Growth hormone-releasing factor increased (P < 0.05) the amplitude of ST peak as well as the AUC between 2 and 6 hr after the beginning of the infusion in CI and SI pigs. Over the six d of infusion, the ST AUC increased from 805 ng-min/ml during the pre-infusion period to 4768 ng.min/ml on day 6 (Table 2, Fig. 2). Prolactin concentrations were increased (P < 0.05) by chronic GRF exposure; however, pattern of increase was different depending if the animals were immunized or not against SRIF as indicated by the interaction day X treatment (P < 0.01) (Table 2). Thyroxine and IGF-I concentrations were increased during GRF infusion and this effect reached a plateau (P < 0.05) after three d of infusion. For both hormones, SI animals exhibited higher (P < 0.05) hormone concentrations than CI (Table 3). Somatostatin-immunized and CI animals had similar serum glucose and insulin concentrations. A rapid increase (P < 0.05) in serum glucose was observed as soon as 6 hr after the beginning of the infusion without change (P > 0.05) in insulin concentrations. After two d of infusion, glucose and insulin concentrations reached maximal values to decline gradually during the three following d (Table 3).
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TABLE2. EFFEC'rOF PGRF(I-29)NHz INFUSIONDURING6 CONSECUTIVEDAYSON DWFERENTPARAMETERSOF SOMATOTROPIN(ST) ANDPROLACTIN(PRL) RELEASEIN GROWINGPIGS Item
Day 1
2
4hr before infusion
2-6hr after infusion
3
P value 4
5
6 SE
ST peak amplitude (ng/ml)
control
10.4
25.1
13.0
38.6
35.1
64.3
48.3
immunized
6.4
32.7
19.3
66.9
49.1
57.6
60.6
Area under the ST curve (ng.min/ml)
control
844
1584
1257
2700
3091
4620
4461
immunized
766
2180
1560
4279
3451
4372
5074
Area under the Prl curve (ngomin/ml)
control
945
939
1170
1372
1441
1224
1453
immunized
979
1076
SI
1304
1328
1467
1165
Day X SI
1 vs 1
Day effect L
Q
C
8.4
NS
NS
.02
.001
.01
NS
507
NS
NS
.04
.001
NS
NS
39
NR
.01
NS NS
.001 .001
.01 .001
.02 .001
1311
Values are least squares means of 5 animals. P values represent: 1° the difference observed between control and SRIF-immunized (SI) animals; 2° the interaction day X SI; 3 ° comparison of 4 hr before to 2 to 6 hr after the beginning of the infusion (1 v s 1); and, 4 ° the orthogonal polynomial contrasts of day effect. L: linear, Q: quadratic, C: cubic. NS: not significant (P > 0.05). NR: not reported because of the significant day X SI interaction. t~ C m C I--"
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GRF INFUSION ON HORMONE AND BLOOD VARIABLES
mI 40
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TIME (h) Figure 2. Effects pGRF (1-29)NH~ infusion at the rate of 15 ng/min/kg BW on ST release in five control and 5 SRIFimmunized pigs over 6 consecutive d. Blood samples were taken daily at 20-min intervals for 4 hr (0900 to 1300 hr).
4.2
1
5.5
1.70
--
--
3.60
2.52
2-6hr after infusion
4.4
3.53
169
129
3.99
2.80
2
4.7
3.99
200
158
4.50
3.53
3
Day
4.2
3.34
254
197
4.68
3.58
4
4.2
3.02
240
219
4.48
3.31
5
3.7
3.00
248
224
3.94
2.96
6
.2
.43
11
.16
SE
NS
NS
.03
.008
SI
NS
NS
NS
NS
Day X SI
.001
NS
NA
.001
1 vs 1
P value
.01
NS
.001
.001
L
.01
.001
.01
.001
Q
Day effect
NS
NS
NS
NS
C
Values are least squares means of 5 animals. P values represent: 1° the difference observed between control and SRIF-immunized animals (SI); 2 ° the interaction day X SI; 3° comparison of values before and 2-6 hr after the beginning of the infusion (1 v s 1); and, 4 ° the orthogonal polynomial contrasts of day effect. L: linear, Q: quadratic, C: cubic. NS: not significant (P > 0.05). NA: not available.
Glucose (retool/l)
139
immunized
1.84
90
2.76
immunized
control
2.29
control
4hr before infusion
Insulin (ng/ml)
IGF-I (ng/ml)
T4 (I.tg/dl)
Item
TABLE3. EFFECTOFPGRF(1-29)NH 2 INFUSIONDURING6 CONSECUTIVEDAYSONTHYROXINE(T4), INSULrN-L]KEGROWTHFACTORI (IGF-I), INSULINANDGLUCOSECONCENTRATIONSIN GROWINGPIGS
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GRF INFUSION ON HORMONE AND BLOOD VARIABLES
315
Table 4 reports blood variables which were not influenced by immunization and did not show any significant day X immunization interaction. As early as 6 hr after the beginning of the infusion, values of K and triglyceride increased (P < 0.05) and creatinine and P decreased (P < 0.05). A steady increase with days of infusion was observed with Na, anion gap and FFA. A transitory increase was observed with Ca, P, alkaline phosphatase, triglyceride, K and total bilirubin and a transitory decrease was notified with GOT and creatinine. Table 5 reports blood parameters from which an interaction day X immunization was significant (P < 0.05). Chlorine and cholesterol values increased (P < 0.05) gradually and albumin and COz increased transitory in SI and CI pigs, respectively. The other variables did not show any variation due to treatment over time. DISCUSSION The present data indicates that both active immunization against SRIF and GRF infusion increase ST concentrations in growing pigs. In the first experiment, before the beginning of the infusion, SI pigs had higher ST concentrations than controls, indicating the presence of an inhibitory action by hypothalamic SRIF on ST release in pigs (5,51) as reported in rats (4) and ruminants (27,28). However, even if GRF induced a linear increase in ST release with increasing rates of GRF infusion, SI animals did not respond better than CI during this period. Similar observations were reported in dairy heifers (28) indicating that endogenous SRIF exerts a negative effect on basal ST release but, during ST peak mediated by an increase of exogenous GRF, the presence of residual SRIF does not seem to influence the magnitude of the ST response. In normal adult men infused with hGRF(1-44)NH2, at the rate of 1.0, 3.3, 10 and 33 ng/min/kg BW, an increase in ST release was also observed in a dose response manner for the three lowest doses (14). During the 4-hr period following the end of the GRF infusion, SI pigs exhibited higher ST concentrations indicating that SRIF could actively participate in the return of ST to basal level after GRF infusion. Because the highest rate of GRF infusion in experiment 1 elicited the highest ST release in CI and SI pigs without evidence of desensitization, this dose (15 ng/min/kg BW) was infused over 6 d. As observed in experiment 1, SI animals did not release more ST than CI under GRF stimulation but the area under the ST curve increased with increasing days of infusion in both groups. In short term studies, this observation has never been reported: normal healthy adults infused with hGRF(I-44)NH 2 at the rate of 100 I.tg/h (approximately 25 ng/min/kg BW) for 2 to 5 hr did not sustain elevated levels of ST (15). The same observation was reported in boys (12 + 1.7 year of age) infused for 3 hr at the rate of 2500 ng/min (13). However, when six healthy young men were infused with hGRF(1-40)NH 2 at the rate of 2 ng/min/kg BW, an increase in the natural occurrence of ST pulses was noted (16). In human, it seems that high rates of GRF infusion over short periods of time produces a reduction of ST release over time and that longer infusion periods at a lesser rate of infusion maintained and even increased ST level. In conscious freely moving rats passively immunized against SRIF and infused over 24 hr with hGRF(1-44)NH 2 at the rate of 750 ng/min/kg BW, a decrease in ST release was observed after the first 6 hr of infusion. This decrease was associated with a depletion of the ST pituitary store (12). Since SRIF might exert a role to maintain somatotroph responsiveness to GRF (30), SRIF-neutralization may worsen the desensitization. In rat anterior pituitary cells exposed for 24 hr to rat GRF, a depletion of cellular ST store and a decrease sensitivity to rat GRF were observed (29). In cattle, 5 d of microinjections of hGRF( 1-44)NH2 at 3.75-min intervals at a dose close to 14 ng/min/kg BW sustained over time ST release (17). In steers and wethers implanted with osmotic pumps, a sustained increase in ST levels for 7 d was also observed (31). In our experiment, ST release increased with number of days of infusion. To our knowl-
144.2 5.4 2.45 2.86 17.6 4.1 42.5 213 70.1 157 103 .443 1.70
4 hr before infusion
1
145.3 5.8 2.42 2.76 16.5 4.5 34.3 230 66.5 125 94 .538 1.66
2-6 hr after infusion
148.1 6.0 2.58 3.05 19.3 3.6 34.2 221 70.0 180 99 .520 2.17
2
145.3 5.8 2.62 3.13 17.6 3.8 29.2 244 71.0 136 l 01 .508 2.20
3
Day
147.8 6.2 2.64 3.29 18.4 4.3 30.6 289 72.3 176 101 .479 2.08
4
147.8 6.3 2.49 3.01 19.0 3.9 34.5 294 69.3 179 105 .416 2.30
5
148.3 5.7 2.41 3.01 20.3 3.5 37.3 251 71.0 204 104 .389 1.88
6
.1 .1 .02 .03 .7 .2 3.1 16 .7 I1 1 .027 .09
SE NS .001 NS .02 NS NS NS NS NS NS .001 .01 NS
1 vs 1
.001 .001 NS .001 .02 NS NS .001 NS .01 .02 .01 NS
L NS .001 .001 .001 NS NS .002 .03 NS NS .02 .01 .001
Q
Day effect
P value
NS .001 NS NS NS .001 NS .02 .01 NS .02 NS NS
C
Values are least squares means of 5 animals. Effect of SI and the interaction days X SI were not significant (P > 0.05). P values represent: 1° comparison of values 4 hr before and 2-6 hr after the beginning of the infusion (1 vs 1); and, 2 ° the orthogonal polynomial contrasts of day effect. L: linear, Q: quadratic, C: cubic.
Na (mmol/1) K (mmolB) Ca (mmol/l) P (mg/dl) Anion Gap BUN (mmol) GOT (IU/1) Alk. Phosp. (IU/L) Tot. Prot. (g/l) FFA (I.tEq/1) C reatinine (lamol/l) Triglyceride (mmol/l) Total Bilirubin (I.tmol/l)
Item
TABLE 4. EFFECT OF PGRF( 1-29)NH 2 INFUSION DURING 6 CONSECUTIVE DAYS ON DIFFERENT SERUM PARAMETERSIN GROWING PIGS
C F m --I
ITI
~0
w
C
co
2.75
2.37
Control
Immunized
35.6
36.6
Control
Immunized
23.2
22.3
Control
Immunized
103.2
104.4
Immunized
Control
4hr before infusion
1
2.49
2.26
34.9
35.6
24.1
25.2
104.4
103.9
2-6hr after infusion
2.68
2.36
38.1
35.4
24.0
24.2
104.6
104.8
2
2.74
2.41
37.5
36.5
23.4
25.5
104.0
102.5
3
Day
2.85
2.79
36.8
37.9
23.5
26.2
105.0
104.1
4
2.91
2.63
36.9
34.9
23.9
25.3
105.1
103.4
5
2.84
2.86
36.1
35.7
22.1
23.4
105.3
105.1
6
.07
.5
.5
.4
SE
.03
.001
.005
.01
X SI
Day
NS
NS
NS
NS
NS
.001
NS
NS
1 vs 1
NS
.001
NS
NS
NS
.04
.01
NS
L
P value
NS
NS
.02
NS
NS
.001
NS
NS
Q
Day effect
NS
NS
NS
NS
NS
NS
NS
NS
C
Values are least squares means of 5 animals. No effect of immunization was observed (P > 0.05). P values represent: 1° the interaction day X immunization treatment (SI); 2° the comparison of values 4 hr before and 2-6 hr after the beginning of the infusion ( 1 v s 1); and, 3° the orthogonal polynomial contrasts of day effect: L: linear, Q: quadratic, C: cubic. NS: not significant (P > 0.05).
Cholesterol (mmol/l)
Albumin (g/l)
CO 2 (mmol/l)
Chlorine (retool/l)
Item
TABLE5. EFFECTOFPGRF(1-29)NH 2 INFUSIONDURING6 CONSECUTIVEDAYSON DIFFEREm"SERUMPARAMETERSINFLUENCEDBY IMMUNIZATIONANDDAYSOF INFUSIONIN GROWINGPIGS
..A
fm
W
8
Z 0 Oo
Z m
O
2O
O
"I-
Z
O
Z
C Or)
T~
2O "rl
318
DUBREUIL ET AL
edge, only one experiment (33) using infusion in young bull calves reported similar observations. However, injection of GRF in rats with the pituitary autotransplanted to the kidney capsule (35) and in ST deficient children (36), it was observed that multiple administration of GRF enhanced the ST response to subsequent GRF injection. In lactating sows (37) and in growing pigs (19) injected with pGRF(I-29)NH 2 at doses of 20 and 15 ~tg/kg BW, respectively, there were increases in ST release with increasing days of injection (20 d in sows and 85 d in growing pigs). These results indicate that GRF could enhance ST synthesis (38,39) or somatotroph proliferation (40). Somatotropin surges were evident within each daily 4-hr period of blood collection in both CI and SI pigs. Similar surge occurred in rats (32), human (14,16) and cattle (33,34) receiving GRF infusion. A cyclic SRIF and GRF secretion has been postulated to be responsible of the occurrence of ST peaks in rats (3) and pigs (5) and SRIF has been reported to control ST baseline level (5). Because SI animals also exhibited ST peak, this suggests that GRF in pigs could also be released in a cyclic manner. Moreover, GRF infusion added to the cyclic endogenous GRF produced an increase in ST peak. In this model, it seems that SRIF has only a minor role to play to control the amplitude of ST peak since CI and SI animals did not show any significant difference. Immunization against SRIF did not have any effect on basal Prl release in rats (4) which is in accordance with the fact that SRIF exerts no effect on basal Prl secretion (41). Growth hormone-releasing factor was also reported to have no effect on Prl release in rats (42) and bull calves (34) after a single injection and in man after 4 hr of GRF infusion (15). In the present study, an increase in Prl concentrations was observed 24 hr after the beginning of the infusion. Immunized pigs showed an increase in Prl concentrations which was 24 hr sooner than controls, indicating that SRIF might exert a negative effect on the GRF-induced Prl release (41). In a previous experiment (53) using growing pigs injected twice daily for 12 weeks with pGRF(1-29)NH 2, we observed that injected animals had constant Pri concentrations over time when compared to controls which showed a decrease in Prl levels over time. Since immunoreactive ST and Prl have been isolated in the same adenohypophysial cell, it is possible that chronic ST stimulation leads to Prl secretion. Indeed, serum Prl concentrations are elevated in 40% of acromegalic women (43). On the other hand, GRF might directly affect the lactotroph cells (45). Since SRIF regulates thyrotropin (TSH) levels (43), SI animals might have higher TSH levels (4) and consequently higher T4 levels. However, the transient increase in T 4 observed as soon as 6 hr after the beginning of the infusion is not clear since acromegalic patients have normal T4 concentrations (43) as well as lactating cows infused for 20 d with GRF (44). Insulin and glucose concentrations demonstrated a transient increase. An increase in glucose levels over an infusion period of 20 d with GRF has been reported in dairy cows (44) and in growing pigs injected directly with ST or GRF (6) and could be associated with a concurrent increase in hepatic glucose production and/or impaired glucose clearance by peripheral tissues (46). Insulin-like growth factor-1 was higher in SI animals, which is in agreement with the observed increase in ST concentrations in these animals. Somatostatin-immunized sheep have also increased ST levels (47) and IGF-I levels (48). The rate of increase in IGF-I concentrations is slower after 4 or 5 d of GRF infusion, but pigs injected for 57 d (54) with GRF have a constant increase in IGF-I indicating that the production of this hormone does not seem to be desensitized. Several blood parameters are influenced by GRF infusion and high ST levels. However, the fairly short period of time (6 d) probably did not permit an equilibration of these parameters. The most recognized parameters influenced by GRF are a decrease in BUN (6), and an increase in FFA (44) and cholesterol as observed in experiment 2 (49). Baile et al. (50) re-
GRF INFUSION ON HORMONE AND BLOOD VARIABLES
319
ported some changes in blood Na, C1, cholesterol, BUN, albumin, globulin and GOT 4 hr following human ST injections on the fast and last day of the growing period. The results of the present study show that active immunization against SRIF increases ST and IGF-I concentrations in growing pigs and constant pGRF(1-29)NH2 infusion at the rate of 15 ng/min/kg BW over 6 d induced an increase in ST concentrations with days of infusion. An increase in Prl, T 4 and IGF-I concentrations and a transient increase in glucose and insulin concentrations were also observed. Long-acting delivery systems hold promise for future use of GRF in production trials. ACKNOWLEDGMENTS/FOOTNOTES We wish to acknowledge the technical assistance of I. Blanchet, J. Brochu, L. Delorme, L. St-James, L. Brousseau,G. Bertrandand M. Dumas,the secretarialassistanceof L. C6t~and editingassistanceof E Lowry. tThis study was supported by Agriculture Canada Contribution #315, NSERC grant A2312 and Sanofi Recherche, Montpellier,France. *Fellow of the MedicalResearch Councilof Canada. Presentaddress: Dept. of Medicine,Collegeof Veterinary Medicine,Universityof Montreal,Box 5000 St-Hyacinthe,Qu6bec,CanadaJ2S 7C6. From Dr. G. Descotes, Sanofi,Montpellier,France From Dr. T.E Mowles,HoffmanLaRoche,Nutley,N.J. 3 Kindlydonated by the USDA AnimalHormoneProgram and the NationalHormoneand PituitaryProgram, Beltsville,MD, USA 4ResearchProductInternationalCorp.,410 N. BusinessCenterDrive,MountProspect, Illinois60056. 5DaymarLaboratoriesInc.,229 YongeSt., Suite207, Toronto,OntarioM5B 1N9. 6SigmaChemicalCompany,P.O.Box 14508,St-Louis,Missouri63178. 7LincoResearch Inc.,P.O. Box 641, Eureka,Missouri63025. s Antiserumprovided by Drs. J. Underwoodand J.J. Van Wyk, 509 Clinical Sciences Bldg 229H, School of Medicine,Universityof North Carolina,North Carolina,ChappelHill,North Carolina27514 and distributedby the HormoneDistributionprogramof the NIDDKthroughthe NationalHormoneand PituitaryProgram. 9Antibodyprovidedby Drs. A. Belangerand S. Caron, Laboratoirede Neuroendocrinologie,Centre Hospitalier Universitaire,Universit6Laval,2705 Blvd.Laurier,Qu6bec,Qu6becG1V 4G2. ~oBiomegaDiagnosticInc.,Montr6al,Qu6bec,Canada. HBoehringerMannheim,11450C6t6de Liesse Dorval,Qu6bec,Canada. ~2BeckmanInstrumentsInc.,52-54ch. des Bourdons,Gagny,France. ~3BoehringerMannheim,38240Meylan,France. ~4BoehringerMannheim,9115 HagueRd., Indianopolis,IN, USA. ,5BoehringerMannheim,Touanvmon,Min Ato-Ky,Tokyo,Japan. ~6WellcomeFoundationLtd., TempleHill,Dartford, England. REFERENCES 1. Brazcau P, Vale W, Burgus R. Hypothalamic peptide that inhibits the secretion of immunoreactive pituitary GH. Science 179:77-79, 1973. 2. Guillemin R, Brazeau P, Bohlen P, Esch E Ling N, Wehrenberg WB. Growth hormone-releasing factor from a human pancreatic tumor that caused acromegaly. Science 218:585-587, 1982. 3. Tannenbaum GS, Ling N. The interrelationship of growth hormone- releasing factor and somatostatin in the generation of the ultradian rhythm of growth hormone secretion. Endocrinology 115:1952-1957, 1984. 4. Terry LC, Martin JB. The effects of lateral hypothalamic medial forebrain stimulation and somatostatin antiserum on pulsatile growth hormone secretion in freely behaving rats. Endocrinology 109:622-627, 1981. 5. Klindt J, Ford J, Berardinelli JG, Anderson LL. Growth hormone secretions after hypophysial stalk transection in pigs. Proc Soc Exp Bioi Meal 172:508-513, 1983. 6. Etherton TD, Wiggins JP, Chung CS, Evock CM, Rebhun JF, Walton PE. Stimulation of pig growth performance by porcine growth hormone and growth hormone-releasing factor. J Anita Sci 63:1389-1399, 1986. 7. Della-Fera MA, Buonomo FC, Baile CA. Growth hormone-releasing factors and secretion of growth hormone in sheep, calves and pigs. Domest Anita Endocrinol 3:165-176, 1986. 8. Petitclere D, Pelletier (3, Lapierr¢ H, Gaudreau P, Couture Y, Dubreuil P, Morisset J, Brazeau P. Dose-response of two synthetic human growth hormone-releasing factors on growth hormone release in heifers and pigs. J Anim Sci 65:996-1105, 1987. 9. Dubreuil P, Pelletier G, Petitclerc D, Lapierre H, Couture Y, Gaudreau P, Morisset J, Brazeau P. Influence of growth hormone-releasing factor and(or) thyrotropin-releasing factor on somatotropin, prolactin, triiodothyronine and thyroxine release in growing pigs. Can J Anim Sci 68:699-710, 1988. 10. Dubreuil P, Lapierre H, Pelletier G, Petitclerc D, Couture Y, Gaudreau P, Morisset J, Brazeau P. Serum growth hormone release in response to a growth hormone-releasing factor analog during
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DUBREUIL ET AL
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