Growth hormone increases insulin-like growth factor-I (IGF-I) and decreases IGF-II in plasma of growing pigs P. C.
Owens, R. J. Johnson, R. G. Campbell and F. J. Ballard
CSIRO Division of Human Nutrition, Kintore Avenue, Adelaide, South Australia 5000, Australia *Victorian Department of Agriculture and Rural Affairs, Animal Research Institute, Werribee, Victoria 3030, Australia received
6 June 1989
ABSTRACT
Insulin-like growth factor-I (IGF-I) and IGF-II have been measured in plasma obtained from male and female pigs of two strains during daily administration of pituitary-derived porcine GH (pGH; 100 \g=m\g/kg) from 60 to 90 kg body weight. Each plasma sample was first chromatographed to separate the IGF from binding proteins in order to obtain reliable measurements. IGF-I concentrations showed no differences between strains, but were higher in untreated males (497 \m=+-\43 (s.e.m.) \g=m\g/l) than females (299\m=+-\15 \g=m\g/l). GH-treated animals had two-fold higher concentrations of IGF-I. IGF-II concentrations were not
different between sexes or strains, but decreased in pigs treated with pGH (299 \m=+-\28 \g=m\g/ 1) compared with controls (431 \m=+-\32\g=m\g/l).Binding protein concentrations, measured as interference in the IGF-I and IGF-II assays, were not different between sexes or strains, but were increased in pGH\x=req-\ treated animals. Taken together, these results indicate that in addition to the expected increase in IGF-I concentrations, exogenous administration of pGH to pigs leads to an increase in IGF-binding protein and a depression in IGF-II concentrations. Journal of Endocrinology (1990) 124, 269\p=n-\275
INTRODUCTION
animals from two strains with different
significantly were
growth
rates.
pigs with porcine growth hor¬ improved growth performance concomitant with increases in insulin-like growth factor-I (IGF-I) concentrations (Etherton, Wiggins, Treatment of young mone
(pGH) leads
to
Chung et al. 1986; Buonomo, Lauterio, Baile & Campion, 1987; Etherton, Wiggins, Evock et al. 1987; Campbell, Steele, Caperna et al. 1988). However, this apparent relationship must be questioned because the IGF-I assays used did not involve rigorous removal of IGF-binding proteins, while IGF-binding proteins are detected as IGF-I or IGF-II in radioimmunoassays or radioreceptor assays, since the binding proteins com¬ pete for the radioligand. The situation is exacerbated because the major also increased
was
Etherton, 1989).
IGF-binding protein in pig serum by the pGH treatment (Walton &
In this investigation of pGH effects in pigs, we have included measurements of IGF-II using a recentlycharacterized assay (Francis, Owens, McNeil et al. 1989), ensured removal of IGF-binding proteins before measurement of concentrations of IGF, and extended the analysis to compare male and female
MATERIALS AND METHODS
Materials
Pituitary-derived
pGH
(USDA-pGH-B-1)
was
obtained from Dr D. J. Bolt through the USDA Animal Hormone Program administered by the Animal Sciences Institute, Beltsville, MD, U.S.A. Recombinant human IGF-I was kindly provided by Drs H. H. Peter and K. Scheibli, CIBA-Geigy, Basle, Switzerland and recombinant human IGF-II by Lilly Research Laboratories, Indianapolis, IN, U.S.A. Porcine IGF-I and IGF-II were purified to homogen¬ eity from serum (Francis et al. 1989). Antiserum to IGF-I was obtained by immunizing rabbits with an ovalbumin-bovine IGF-I conjugate (Dawe, Francis, McNamara et al. 1988), and antiserum against rabbit -globulin was raised in goats. Recombinant human IGF-I and IGF-II were iodinated with Na l25I (Amersham Australia Pty Ltd, North Ryde, New South Wales, Australia) to specific activities between
30 and 80 Ci/g using chloramine , and separated from reaction components by chromatography on Sephadex G-50 (Pharmacia, North Ryde, New South Wales, Australia) as described previously (Read, Ballard, Francis et al. 1986). Ovine placental mem¬ branes were isolated as reported by Baxter & DeMellow (1986). The Protein-Pak 125 columns used to separate IGF from binding proteins were obtained from Waters, Lane Cove, New South Wales, Australia. Freon (1,1,2trichloro-l,2,2-trifluoroethane), AR grade, was pur¬ chased from Mallinckrodt, Paris, KY, U.S.A. and polyethylene glycol 6000 from Sigma Chemical Co., St Louis, MO, U.S.A. Animals and their management Details of the animals, their management, adminis¬ tration of pGH, bleeding, carcass and production data have been described in detail elsewhere (Campbell, King, Taverner & Johnson, 1989). Briefly, 48 crossbred (Large White X Landrace) pigs comprising equal numbers ofintact males and females of two strains were used in the experiment. Pigs were allocated to treat¬ ments when a body weight of 60 kg was attained. There 2 2 factorial with were six treatments arranged in a 2 six replicates. Factors were strain (A and B), sex and exogenous pGH administration (control and pGH at 100µg/kg body weight per day). Strain A was faster growing than strain B, and the growth and carcass characteristics of both strains have been described previously (Campbell & Taverner, 1988). Pigs were kept in individual pens and fed ad libitum until each reached 90 kg body weight. Pigs were injected i.m. daily with 100 µg pGH/kg body weight, solubilized in NaCl ( 154 mmol/1) buffered to pH 9-4 with (25 mmol/1) and NaHC03 (25 mmol/1). Control pigs received an equivalent volume of buffer. At 90 kg body weight, blood samples were collected from each pig by venipuncture immediately before injection of pGH or buffer. Plasma was recovered by centrifugation and stored at 20 °C before analysis.
Na2CO?
—
Chromatography of plasma samples Plasma samples (70 µ ) were diluted with water and concentrated buffer to obtain 350 µ of solution con¬ taining acetic acid (200 mmol/1) and trimethylamine (50 mmol/1) at pH 2-8. Each solution was mixed thoroughly with an equal volume of Freon, and the aqueous layer collected after centrifugation at
10 000 g for 10 min. The defatted solution was on a Protein-Pak 125 molecular sieve column that was equilibrated with the above
chromatographed
buffer (mobile phase)
using an autoinjector calibrated
apply 200 µ , high performance liquid chromatog¬ raphy pump, A280 detector and fraction collector. For to
analysis, 0-5 ml samples were collected. Subsequent chromatography involved the collection of pools eluting between 6 and 8 ml (binding protein region), 8 and 9 ml (intermediate fraction), 9 and 12-5 ml (IGF region) and 12-5 and 13 ml. the initial
IGF-I and IGF-II measurements Measurement of IGF-I by radioimmunoassay involved the addition of 50 µ of the pooled IGF region of the column eluate, the mobile phase alone or standards in mobile phase to 30 µ Tris base (400 mmol/1), followed by 200 µ assay buffer (30 mmol NaH2P04/l; 0-2% (w/v) protamine sulphate; 10 mmol disodium EDTA/1; 0-2% (w/v) NaN3; 0-05% (w/v) Tween-20; pH 7-5), 50 µ l25I-labelled IGF-I (approx. 10 000 c.p.m.) in assay buffer and 50 µ of a sufficient anti-IGF-I antiserum in assay buffer (final dilution 1 : 10 000) to bind 30-40% of the added radioactivity in the absence of competing ligand. The tubes were incubated at 4 °C for 16 h and 10 µ excess goat antirabbit -globulin in assay buffer and 50 µ of 5% (v/v) normal rabbit serum were subsequently added. After a further incubation for 30 min at 4 °C, 1 ml cold 6% (w/v) polyethylene glycol 6000 in NaCl (150 mmol/1) was added and the tubes were centrifuged for 30 min at 4000 g-. After removal of the supernatants by aspi¬ ration, the radioactivity in the pellet was measured. Radioactivity in the tubes in the absence of IGF-I antibody was subtracted from each value. Unknowns and standards were measured in triplicate. The amount of IGF-I that inhibited radioligand binding by 50% averaged 180pg, while the C.V. for the same plasma sample extracted, chromatographed and assayed on different occasions was 9-9%. The crossreactivity of porcine IGF-II was 0-8% (Francis et al.
1989).
Insulin-like growth factor-I was also measured in acid-ethanol extracted plasma by a method (Johnson, McMurtry & Ballard, 1989) modified by Dr P. D. Gluckman, University of Auckland, New Zealand, from the procedure described originally by Daughaday, Parker, Dorowsky et al. (1982). Measurement of IGF-II in column fractions by radioreceptor assay involved the addition of 50 µ of the pooled IGF region of the column eluate, the mobile phase alone or standards in mobile phase to 30 µ Tris base (400 mmol/1), followed by 200 µ buffer (10 mmol Tris/1; 0-5% (w/v) bovine serum albumin; 10 mmol CaCl2/l; pH 74), 50 µ 125I-labelled IGF-II (approx. 10000c.p.m.) and 100µ of sufficient ovine placental membranes (final concentration 0-2 mg protein/ml) in assay buffer to bind 30-40% of the added radioactivity in the absence of competing ligand. The tubes were incubated for 16 h at 4°C, after which 1 ml of a solution containing Tris
(10 mmol/1), bovine serum albumin (0-1% w/v) and CaCl2 (100 mmol/1) at pH 74 was added. The tubes were centrifuged at 4000 g for 30 min, supernatants were aspirated and the pelleted radioactivity was
measured. Radioactivity in the tubes in the absence of membranes was subtracted from each value. Unknowns and standards were assayed in triplicate. The amount of IGF-II that inhibited radioligand binding by 50% averaged 1 ng, while the C.V. for the same sample extracted, chromatographed and assayed on different occasions was 21%. The crossreactivity of IGF-I in this assay was 0-05% (Francis etal. 1989). Insulin-like growth factor-II was also measured in acid-ethanol extracted plasma obtained as described for chicken plasma by Johnson et al. (1989). The procedure was similar to that given above for acid column fractions except that neutralization of extracts occurred before the assay (Johnson et al. 1989). IGF-I and IGF-II
binding protein measurements The high molecular weight regions from the molecular sieve chromatography of plasma samples were pooled
and assayed in the IGF-I radioimmunoassay and the IGF-II radioreceptor assay using the same volumes and procedures as described for analysis of the IGF pools. Since the measurements represent interference caused by the binding proteins competing with anti¬ sera or receptors for the radioligand in the respective IGF assays, quantitation is expressed as ng IGF-I or IGF-II.
Statistics Values are reported as means + s.e.m. for each group of animals. Significance between groups was assessed
by unpaired
i-tests.
RESULTS
Molecular sieve acid conditions
chromatography of pig plasma under
The elution volumes of IGF-I, IGF-II and binding proteins were determined by IGF-I immunoassay and IGF-II receptor assay after plasma samples from
I II
1-0
0-8
0-8
0-6
0-6 ta
co
0-4
0-4
0-2
o-:
BP
O
13
15 5 Elution volume (ml)
IGF
II
13
15
O
1. Measurement of (a) insulin-like growth factor-I (IGF-I) and (b) IGF-II in 0-5 ml fractions following molecular sieve chromatography under acid conditions of plasma samples from one control (O) and one GHtreated (·) female pig of strain A. Values are expressed as B/B0, the ratio between binding in the absence of competing ligand and the binding observed with 50 µ of each fraction. The bars represent IGF and binding protein (BP) regions collected as pools for subsequent analysis. figure
3. Insulin-like growth factor-II (IGF-II) measured in (a) IGF and (b) binding protein regions (Fig. 1) of chromatographed plasma samples from control (open bars) and GH-treated pigs (stippled bars). Values are means ± s.e.m.; figure
2. Immunoreactive insulin-like growth factor-I (IGF-I) measured in (a) IGF and (b) binding protein regions (Fig. 1) of chromatographed plasma samples from control (open bars) and GH-treated pigs (stippled bars), Values are means + s.e.m.; numbers of animals in each group are given inparentheses. *
Owens, R. J. Johnson, R. G. Campbell and F. J. Ballard
CSIRO Division of Human Nutrition, Kintore Avenue, Adelaide, South Australia 5000, Australia *Victorian Department of Agriculture and Rural Affairs, Animal Research Institute, Werribee, Victoria 3030, Australia received
6 June 1989
ABSTRACT
Insulin-like growth factor-I (IGF-I) and IGF-II have been measured in plasma obtained from male and female pigs of two strains during daily administration of pituitary-derived porcine GH (pGH; 100 \g=m\g/kg) from 60 to 90 kg body weight. Each plasma sample was first chromatographed to separate the IGF from binding proteins in order to obtain reliable measurements. IGF-I concentrations showed no differences between strains, but were higher in untreated males (497 \m=+-\43 (s.e.m.) \g=m\g/l) than females (299\m=+-\15 \g=m\g/l). GH-treated animals had two-fold higher concentrations of IGF-I. IGF-II concentrations were not
different between sexes or strains, but decreased in pigs treated with pGH (299 \m=+-\28 \g=m\g/ 1) compared with controls (431 \m=+-\32\g=m\g/l).Binding protein concentrations, measured as interference in the IGF-I and IGF-II assays, were not different between sexes or strains, but were increased in pGH\x=req-\ treated animals. Taken together, these results indicate that in addition to the expected increase in IGF-I concentrations, exogenous administration of pGH to pigs leads to an increase in IGF-binding protein and a depression in IGF-II concentrations. Journal of Endocrinology (1990) 124, 269\p=n-\275
INTRODUCTION
animals from two strains with different
significantly were
growth
rates.
pigs with porcine growth hor¬ improved growth performance concomitant with increases in insulin-like growth factor-I (IGF-I) concentrations (Etherton, Wiggins, Treatment of young mone
(pGH) leads
to
Chung et al. 1986; Buonomo, Lauterio, Baile & Campion, 1987; Etherton, Wiggins, Evock et al. 1987; Campbell, Steele, Caperna et al. 1988). However, this apparent relationship must be questioned because the IGF-I assays used did not involve rigorous removal of IGF-binding proteins, while IGF-binding proteins are detected as IGF-I or IGF-II in radioimmunoassays or radioreceptor assays, since the binding proteins com¬ pete for the radioligand. The situation is exacerbated because the major also increased
was
Etherton, 1989).
IGF-binding protein in pig serum by the pGH treatment (Walton &
In this investigation of pGH effects in pigs, we have included measurements of IGF-II using a recentlycharacterized assay (Francis, Owens, McNeil et al. 1989), ensured removal of IGF-binding proteins before measurement of concentrations of IGF, and extended the analysis to compare male and female
MATERIALS AND METHODS
Materials
Pituitary-derived
pGH
(USDA-pGH-B-1)
was
obtained from Dr D. J. Bolt through the USDA Animal Hormone Program administered by the Animal Sciences Institute, Beltsville, MD, U.S.A. Recombinant human IGF-I was kindly provided by Drs H. H. Peter and K. Scheibli, CIBA-Geigy, Basle, Switzerland and recombinant human IGF-II by Lilly Research Laboratories, Indianapolis, IN, U.S.A. Porcine IGF-I and IGF-II were purified to homogen¬ eity from serum (Francis et al. 1989). Antiserum to IGF-I was obtained by immunizing rabbits with an ovalbumin-bovine IGF-I conjugate (Dawe, Francis, McNamara et al. 1988), and antiserum against rabbit -globulin was raised in goats. Recombinant human IGF-I and IGF-II were iodinated with Na l25I (Amersham Australia Pty Ltd, North Ryde, New South Wales, Australia) to specific activities between
30 and 80 Ci/g using chloramine , and separated from reaction components by chromatography on Sephadex G-50 (Pharmacia, North Ryde, New South Wales, Australia) as described previously (Read, Ballard, Francis et al. 1986). Ovine placental mem¬ branes were isolated as reported by Baxter & DeMellow (1986). The Protein-Pak 125 columns used to separate IGF from binding proteins were obtained from Waters, Lane Cove, New South Wales, Australia. Freon (1,1,2trichloro-l,2,2-trifluoroethane), AR grade, was pur¬ chased from Mallinckrodt, Paris, KY, U.S.A. and polyethylene glycol 6000 from Sigma Chemical Co., St Louis, MO, U.S.A. Animals and their management Details of the animals, their management, adminis¬ tration of pGH, bleeding, carcass and production data have been described in detail elsewhere (Campbell, King, Taverner & Johnson, 1989). Briefly, 48 crossbred (Large White X Landrace) pigs comprising equal numbers ofintact males and females of two strains were used in the experiment. Pigs were allocated to treat¬ ments when a body weight of 60 kg was attained. There 2 2 factorial with were six treatments arranged in a 2 six replicates. Factors were strain (A and B), sex and exogenous pGH administration (control and pGH at 100µg/kg body weight per day). Strain A was faster growing than strain B, and the growth and carcass characteristics of both strains have been described previously (Campbell & Taverner, 1988). Pigs were kept in individual pens and fed ad libitum until each reached 90 kg body weight. Pigs were injected i.m. daily with 100 µg pGH/kg body weight, solubilized in NaCl ( 154 mmol/1) buffered to pH 9-4 with (25 mmol/1) and NaHC03 (25 mmol/1). Control pigs received an equivalent volume of buffer. At 90 kg body weight, blood samples were collected from each pig by venipuncture immediately before injection of pGH or buffer. Plasma was recovered by centrifugation and stored at 20 °C before analysis.
Na2CO?
—
Chromatography of plasma samples Plasma samples (70 µ ) were diluted with water and concentrated buffer to obtain 350 µ of solution con¬ taining acetic acid (200 mmol/1) and trimethylamine (50 mmol/1) at pH 2-8. Each solution was mixed thoroughly with an equal volume of Freon, and the aqueous layer collected after centrifugation at
10 000 g for 10 min. The defatted solution was on a Protein-Pak 125 molecular sieve column that was equilibrated with the above
chromatographed
buffer (mobile phase)
using an autoinjector calibrated
apply 200 µ , high performance liquid chromatog¬ raphy pump, A280 detector and fraction collector. For to
analysis, 0-5 ml samples were collected. Subsequent chromatography involved the collection of pools eluting between 6 and 8 ml (binding protein region), 8 and 9 ml (intermediate fraction), 9 and 12-5 ml (IGF region) and 12-5 and 13 ml. the initial
IGF-I and IGF-II measurements Measurement of IGF-I by radioimmunoassay involved the addition of 50 µ of the pooled IGF region of the column eluate, the mobile phase alone or standards in mobile phase to 30 µ Tris base (400 mmol/1), followed by 200 µ assay buffer (30 mmol NaH2P04/l; 0-2% (w/v) protamine sulphate; 10 mmol disodium EDTA/1; 0-2% (w/v) NaN3; 0-05% (w/v) Tween-20; pH 7-5), 50 µ l25I-labelled IGF-I (approx. 10 000 c.p.m.) in assay buffer and 50 µ of a sufficient anti-IGF-I antiserum in assay buffer (final dilution 1 : 10 000) to bind 30-40% of the added radioactivity in the absence of competing ligand. The tubes were incubated at 4 °C for 16 h and 10 µ excess goat antirabbit -globulin in assay buffer and 50 µ of 5% (v/v) normal rabbit serum were subsequently added. After a further incubation for 30 min at 4 °C, 1 ml cold 6% (w/v) polyethylene glycol 6000 in NaCl (150 mmol/1) was added and the tubes were centrifuged for 30 min at 4000 g-. After removal of the supernatants by aspi¬ ration, the radioactivity in the pellet was measured. Radioactivity in the tubes in the absence of IGF-I antibody was subtracted from each value. Unknowns and standards were measured in triplicate. The amount of IGF-I that inhibited radioligand binding by 50% averaged 180pg, while the C.V. for the same plasma sample extracted, chromatographed and assayed on different occasions was 9-9%. The crossreactivity of porcine IGF-II was 0-8% (Francis et al.
1989).
Insulin-like growth factor-I was also measured in acid-ethanol extracted plasma by a method (Johnson, McMurtry & Ballard, 1989) modified by Dr P. D. Gluckman, University of Auckland, New Zealand, from the procedure described originally by Daughaday, Parker, Dorowsky et al. (1982). Measurement of IGF-II in column fractions by radioreceptor assay involved the addition of 50 µ of the pooled IGF region of the column eluate, the mobile phase alone or standards in mobile phase to 30 µ Tris base (400 mmol/1), followed by 200 µ buffer (10 mmol Tris/1; 0-5% (w/v) bovine serum albumin; 10 mmol CaCl2/l; pH 74), 50 µ 125I-labelled IGF-II (approx. 10000c.p.m.) and 100µ of sufficient ovine placental membranes (final concentration 0-2 mg protein/ml) in assay buffer to bind 30-40% of the added radioactivity in the absence of competing ligand. The tubes were incubated for 16 h at 4°C, after which 1 ml of a solution containing Tris
(10 mmol/1), bovine serum albumin (0-1% w/v) and CaCl2 (100 mmol/1) at pH 74 was added. The tubes were centrifuged at 4000 g for 30 min, supernatants were aspirated and the pelleted radioactivity was
measured. Radioactivity in the tubes in the absence of membranes was subtracted from each value. Unknowns and standards were assayed in triplicate. The amount of IGF-II that inhibited radioligand binding by 50% averaged 1 ng, while the C.V. for the same sample extracted, chromatographed and assayed on different occasions was 21%. The crossreactivity of IGF-I in this assay was 0-05% (Francis etal. 1989). Insulin-like growth factor-II was also measured in acid-ethanol extracted plasma obtained as described for chicken plasma by Johnson et al. (1989). The procedure was similar to that given above for acid column fractions except that neutralization of extracts occurred before the assay (Johnson et al. 1989). IGF-I and IGF-II
binding protein measurements The high molecular weight regions from the molecular sieve chromatography of plasma samples were pooled
and assayed in the IGF-I radioimmunoassay and the IGF-II radioreceptor assay using the same volumes and procedures as described for analysis of the IGF pools. Since the measurements represent interference caused by the binding proteins competing with anti¬ sera or receptors for the radioligand in the respective IGF assays, quantitation is expressed as ng IGF-I or IGF-II.
Statistics Values are reported as means + s.e.m. for each group of animals. Significance between groups was assessed
by unpaired
i-tests.
RESULTS
Molecular sieve acid conditions
chromatography of pig plasma under
The elution volumes of IGF-I, IGF-II and binding proteins were determined by IGF-I immunoassay and IGF-II receptor assay after plasma samples from
I II
1-0
0-8
0-8
0-6
0-6 ta
co
0-4
0-4
0-2
o-:
BP
O
13
15 5 Elution volume (ml)
IGF
II
13
15
O
1. Measurement of (a) insulin-like growth factor-I (IGF-I) and (b) IGF-II in 0-5 ml fractions following molecular sieve chromatography under acid conditions of plasma samples from one control (O) and one GHtreated (·) female pig of strain A. Values are expressed as B/B0, the ratio between binding in the absence of competing ligand and the binding observed with 50 µ of each fraction. The bars represent IGF and binding protein (BP) regions collected as pools for subsequent analysis. figure
3. Insulin-like growth factor-II (IGF-II) measured in (a) IGF and (b) binding protein regions (Fig. 1) of chromatographed plasma samples from control (open bars) and GH-treated pigs (stippled bars). Values are means ± s.e.m.; figure
2. Immunoreactive insulin-like growth factor-I (IGF-I) measured in (a) IGF and (b) binding protein regions (Fig. 1) of chromatographed plasma samples from control (open bars) and GH-treated pigs (stippled bars), Values are means + s.e.m.; numbers of animals in each group are given inparentheses. *
