Exogenous growth hormone stimulates somatotropic axis function
and growth in neonatal pigs
Timothy J.
Wester,
Teresa A.
Davis,
Marta L.
Fiorotto, and
Douglas
G.
Burrin
United States Department of Agriculture/Agricultural Research
Service Children's Nutrition Research Center, Department of
Pediatrics, Baylor College of Medicine, Houston, Texas 77030
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ABSTRACT |
We studied the effects of exogenous porcine growth hormone (pGH)
administration on circulating insulin-like growth factor I (IGF-I)
concentration, IGF-binding proteins (IGFBP), tissue growth, and protein
synthesis in neonatal pigs. One-day-old pigs were given daily
intramuscular injections of either pGH (1 mg/kg body wt)
(n = 6) or saline
(n = 5) for 7 days, after
which time we measured in vivo protein synthesis using a bolus of
[3H]-phenylalanine. Mean plasma pGH
concentration in pGH-treated pigs measured on
day 7 was 22-fold higher than in controls. The plasma IGF-I concentration in
pGH-treated pigs was significantly greater than in controls after 1 day
of treatment and plateaued at 285% of control values after 4 days.
After 7 days of treatment, plasma IGFBP-3 concentrations and the plasma
glucose response to a meal were also greater in pGH-treated than
control pigs. pGH treatment significantly increased body weight gain
and food conversion efficiency and the protein synthesis rate in
several visceral organs. Our results demonstrate that exogenous pGH
increases circulating IGF-I and IGFBP-3 concentrations and visceral
organ growth in neonatal pigs, suggesting that the somatotrophic axis is functional in the neonate.
insulin-like growth factor I; protein synthesis; insulin; insulin-like growth factor-binding protein-3
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INTRODUCTION |
POSTNATAL SOMATIC GROWTH is considered to be largely
dependent on direct effects of growth hormone (GH) and its indirect
anabolic effects, which are mediated via insulin-like growth factor I
(IGF-I) (12). However, the prevailing dogma, that neonatal growth is GH
independent, is based on limited evidence of normal birth weight in
anencephalic infants and Laron-type dwarfs and also on relatively low
hepatic GH binding and circulating IGF-I concentrations in neonates
(8). These findings have raised doubts as to whether neonatal growth is
GH responsive or if indeed the GH-IGF-I somatotrophic axis is
functional in the neonate.
However, recent studies suggest that GH responsiveness begins to
develop before birth and is partially functional during the early
neonatal period. Evidence from the rat indicates an upregulation of
GH-receptor mRNA with increasing fetal age to 10% of adult levels at
birth with concurrent activation of GH-responsive mRNAs (2, 20).
Furthermore, other recent studies have shown that somatic growth in
neonatal rats, reduced 50% after hypophysectomy, can be restored with
GH replacement therapy (22), and spontaneous dwarf rats, which exhibit
isolated GH deficiency, are born with significantly reduced birth
weight relative to control rats (28). In addition, Lumpkin et al. (32)
demonstrated that ablation of GH secretion by a GH-releasing factor
receptor antagonist resulted in cessation of somatic growth in weanling
rats despite maintenance of normal nutrient intake.
Numerous studies have shown that exogenous administration of GH to
adult pigs significantly increases weight gain and the efficiency of
dietary nutrient utilization for protein deposition (15, 16, 17, 29).
GH administration to adult pigs is also associated with increases in
circulating glucose and insulin concentrations, suggesting insulin
resistance of glucose metabolism (14, 23, 43). However, the few studies
in neonatal pigs have demonstrated a lack of growth response to
exogenous GH (1, 25). This apparent lack of effect may be attributable
to the relatively low plasma and hepatic GH binding capacity of the
neonatal pig (3, 37), which would reduce both the responsiveness and
sensitivity to exogenous GH compared with adult pigs. The dose of GH
administered in these studies was similar to that used previously in
adult pigs (25). The dose used in adult pigs, however, may have been insufficient to elicit a biological response in the neonate if the
sensitivity of the tissues to GH is reduced, and/or if GH is
cleared more rapidly from the circulation due to lower plasma binding
capacity.
The objective of this study was to determine whether neonatal pigs are
responsive to exogenous porcine GH (pGH). To maximize the likelihood of
inducing a response, we administered pGH at a dose that was higher than
is typically used for adult pigs, and which was given in three daily
doses rather than as a single injection. We have determined the effect
of exogenous pGH on the circulating IGF-I concentration to test whether
the somatotropic axis is functional in neonatal pigs. In addition, we
have characterized tissue growth and metabolism to establish whether,
in the neonatal pig, pGH can induce a biological response, either
directly or indirectly via IGF-I.
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MATERIALS AND METHODS |
Animals and design.
Two litters of conventional crossbred newborn pigs (Texas A&M
University, College Station, TX) were obtained immediately after birth
and weighed. Before the start of the experiment and after each pig had
been ~12 h with the sow, a 20-gauge Silastic catheter was surgically
inserted into the jugular vein of each pig, with the animals under
general isoflurane anesthesia. Pigs were allowed to recover for at
least 1 h before the start of the experiment. Pigs were housed two to
three per cage at an ambient temperature of ~29°C. Pigs were
assigned randomly to receive either recombinant pGH (Monsanto, St.
Louis, MO) at a daily dose of 1 mg/kg body weight
(n = 6) or saline
(n = 5) in three equal, intramuscular injections daily. To eliminate any possible confounding effects due to
differences in voluntary nutrient intake between control and
pGH-treated groups, pigs were gavage-fed an equal amount of food per
unit of body weight during the 7-day period. Pigs were gavage-fed
colostrum six times daily (195 ml/kg body wt) for the first 24 h and
then commercial sow milk replacer (240 ml/kg body wt; Soweena,
Merrick's, Middleton, WI) for the rest of the experiment. The
colostrum was a pooled sample taken from conventional sows within 24 h
postpartum. The volume of milk replacer fed provided each animal 15 g
of protein · kg body
wt
1 · day
1
and 544 kJ · kg body
wt
1 · day
1.
Blood sampling protocol.
Venous blood (2 ml) was sampled daily for analysis of plasma IGF-I and
urea nitrogen (PUN) concentrations. After 7 days of treatment, serial
blood samples were taken from all pigs to determine the plasma GH
secretory profile and the glucose and insulin response to feeding. The
morning of day
7 of treatment, after an overnight fast, blood samples (1 ml) were obtained. Immediately thereafter, the
pigs received a formula feeding (40 ml/kg body wt) and either a saline
or pGH (333 µg/kg body wt) injection, and then blood sampling was
continued every 20 min for 4 h. All samples were taken with the use of
heparinized syringes, and plasma was harvested and stored at
70°C until analysis.
Plasma IGF-I, insulin, GH, PUN, and glucose.
Plasma IGF-I concentration was measured by radioimmunoassay after
acidification and chromatography to remove binding proteins (7). Plasma
insulin and glucose concentrations were measured in the first six
serial samples. Plasma insulin was measured by radioimmunoassay (Linco
Research, St. Louis, MO). The human-specific antibody used in this
assay exhibited 100% cross-reactivity with porcine insulin. Intra- and
interassay coefficients of variation were 2.8 and 2.9%, respectively.
All samples were measured in one assay, and the intra-assay coefficient
of variation was 6.9%. Plasma GH concentration was measured by
double-antibody homologous radioimmunoassay (42). All samples were
measured in one assay, and the intra-assay coefficient of variation was
4.4%. The secretory profile of GH was analyzed using the Pulsar
peak-fitting program to estimate the mean GH concentration. Plasma
glucose was measured by an automated glucose oxidase procedure (Yellow
Springs Instruments, Yellow Springs, OH). PUN concentration was
measured using an endpoint enzymatic assay (Roche, Sommerville, NJ).
Ligand blot analysis of plasma IGF-binding proteins.
Analysis of IGF-binding proteins (IGFBP) was performed using a modified
Western ligand blot procedure (26). Equal volumes of plasma from each
animal were diluted 1:10 in sample buffer [62.5 mM
tris(hydroxymethyl)aminomethane (Tris), 23 g/l sodium dodecyl sulfate,
100 ml/l glycerol, and 0.5 g/l bromophenol blue, pH 6.8], placed
in boiling water for 5 min, and electrophoresed at 110 V overnight at
4°C through a 3% stacking gel on a 12.5% resolving gel.
Approximately 1-3 µl of plasma were run in each lane. Proteins
were electrotransferred to a nitrocellulose membrane at 200 mA for 3 h
at room temperature. Before ligand binding, blots were incubated for 30 min in Tris-buffered saline (TBS; 10 mM Tris, 150 mM NaCl, pH 7.4)
containing 30 ml/l Triton X-100 followed by blocking buffer (TBS
containing 70 g/l nonfat dry milk, pH 7.4) for 2 h. Blots were then
returned to the first buffer for 10 min. Incubations were performed
overnight with gentle shaking. The activity of the tracer buffer (TBS
containing 1 ml/l Tween-20, 20 g/l nonfat dry milk, and radioligand, pH
7.4) was 130 kBq 125I-labeled
IGF-I per blot in 50 ml of solution. Blots were washed at room
temperature twice for 15 min in a buffer containing TBS containing 1 ml/l Tween-20, pH 7.4, and three times for 15 min in TBS. Blots were
dried at room temperature and then exposed to X-ray film in the
presence of intensifying screens at
70°C for 7 days. The
band intensities on the autoradiographs were quantified using laser
densitometry (Pharmacia LKB Biotechnology, Piscataway, NJ). Relative
molecular weights of IGFBPs were estimated by comparison with Coomassie
blue-stained protein standards run under identical conditions.
Immunoblotting and immunoradiometric analysis of plasma IGFBP.
For Western blotting, plasma samples were diluted 1:4 in sample buffer
(see above), placed in boiling water for 5 min, and electrophoresed at
200 V for 2 h at 4°C through a 3% stacking on a 12% resolving
gel. Approximately 2.5 µl of plasma were loaded in each lane.
Proteins were electrotransferred from the gel to a nitrocellulose
membrane at 100 V for 3 h at 4°C. Immunoblotting was performed
using the following dilutions of antibodies: 1:2000 dilution of
anti-bovine IGFBP-2 polyclonal rabbit antiserum (Upstate Biotechnology,
Lake Placid, NY) and 1:1,000 dilution of anti-human IGFBP-3 polyclonal
rabbit antiserum (Diagnostic Systems Laboratories, Webster, TX). For
direct comparison of proteins identified by ligand blotting and
immunoblotting, sections of Western blots were cut and developed using
both ligand blotting (see above) and immunblotting procedures.
Immunoblots were developed by incubating in blocking buffer (TBS
containing 30 g/l nonfat dry milk, pH 7.4) for 1 h, and then antibodies
were added and the blots were incubated overnight at 4°C. Blots
were rinsed twice for 7 min at room temperature in TBS containing 1 ml/l Tween-20 and then incubated for 1 h at room temperature with
biotinylated goat anti-rabbit immunoglobulin G (Dako, Carpinteria, CA)
diluted 1:6,000 in TBS containing 30 g/l nonfat dry milk, pH 7.4. Blots
were rinsed six times for 5 min at room temperature in TBS containing 1 ml/l Tween-20 and then incubated for 1 h at room temperature with
neutravidin-conjugated horseradish peroxidase (Pierce, Rockford, IL)
diluted 1:20,000 with TBS containing 1 ml/l Tween-20. Blots were rinsed
six times for 5 min at room temperature in TBS containing 1 ml/l
Tween-20 and incubated for 5 min with a chemiluminescent substrate as
described by the manufacturer (Super-Signal, Pierce). Immunoreactive
proteins were visualized and detected by autoradiography using brief
(30- to 60-s) exposures to X-ray film. Relative molecular weights of IGFBP were estimated by comparison to Coomassie blue-stained protein standards run on the same gel. Plasma IGFBP-3 was also measured by
immunoradiometric assay (Diagnostic Systems Laboratories). This is a
heterologous assay that uses the same anti-human IGFBP antibody used in
the immunoblotting procedure described above.
Tissue collection and measurement of in vivo protein synthesis.
As indicated above, on the 7th day of treatment, pigs were fed formula
(40 ml/kg body wt) before serial blood sampling and again 1 h
before radioisotope administration to ensure that pigs were in a fed state. In vivo fractional tissue protein synthesis rates
were measured by flooding-dose methodology (5, 21). Briefly, pigs were
injected via the jugular catheter with a bolus dose of
L-[4-3H]phenylalanine
(37 MBq/kg body wt; Amersham, Arlington Heights, IL) in a 150 mM
phenylalanine solution at a dose of 10 ml/kg body weight. Blood samples
were taken at 5, 15, and 30 min from the midpoint of the injection for
measurement of the specific radioactivity of the extracellular free
pool of phenylalanine. Immediately after the 30-min blood sampling,
pigs were anesthetized with an intravenous dose of pentobarbital sodium
(50 mg/kg body wt) and exsanguinated. The abdomen was opened, and
organs were quickly dissected and weighed. Tissue subsamples were taken
rapidly and frozen in liquid nitrogen. All sampled organs were
removed and frozen within 6 min of exsanguination.
Tissue samples (100-200 mg) were homogenized in water, and
aliquots were removed for analysis of protein and DNA using
bicinchoninic acid (41) and
bis-benzimide (30), respectively. The
specific radioactivity of
[3H]phenylalanine was
determined in whole blood samples and in the tissue samples as
described previously (3). The tissue homogenate was acidified with 2 M
perchloric acid (PCA) and processed as described previously (6). In
brief, homogenate supernatants containing the tissue free amino acid
pool were separated from PCA-insoluble precipitates and neutralized.
PCA-insoluble precipitates were washed and solubilized. The solution
was reacidified, and the acid-soluble fraction was assayed for RNA
(35). The protein-containing pellet was washed and hydrolyzed for 24 h
in 6 M HCl. The protein hydrolysate, homogenate supernatant, and blood
supernatant were vacuum-dried and resuspended in water for
determination of phenylalanine specific activity. Phenylalanine was
separated from other amino acids using anion exchange chromatography.
Radioactivity associated with the collected phenylalanine fraction was
measured using liquid scintillation counting.
Calculations.
Protein synthesis was calculated as a fractional rate (FSR, %/day)
from the equation described by Garlick et al.
(21)
where
Sb is the specific activity of the
PCA-insoluble or protein-bound phenylalanine pool (Bq/µmol),
Sa is the specific activity of the
PCA-soluble or tissue free phenylalanine pool (Bq/µmol), and
t is time of labeling in minutes. The
value used for Sa was corrected to
represent the average tissue free phenylalanine specific activity at
the midpoint (t1/2) of the 30-min labeling period. The corrected Sa for each
pig was calculated by adding individual tissue
Sa values (Bq/µmol) after time
(t) and the rate of change in blood
Sa
(Bq · µmol
1 · min
1)
estimated from the regression of 5-, 15-, and 30-min blood samples of
all pigs within a treatment group as described previously
(5)
Absolute
synthesis rate (ASR, g protein/day) was calculated as the fractional
rate times the tissue protein content. Protein synthetic efficiency (mg
protein · day
1 · mg
RNA
1) was calculated as
total daily protein synthesized per total RNA. Protein synthetic
capacity (g RNA/mg protein) was estimated by the RNA to protein ratio.
Statistical analysis.
Data consisting of single observations in time, e.g., tissue weights,
substrate dissociation constant, and plasma GH profile, were analyzed by one-way analysis of variance (ANOVA) with treatment as
the main effect. Observations taken across time, e.g., daily plasma
hormone and metabolite concentrations, were analyzed by repeated-measures ANOVA, with treatment and sampling time as main effects. Differences with P < 0.05 were considered significant. Results are presented as means with pooled
SD from the one-way ANOVA or as means ± SE.
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RESULTS |
Plasma GH, IGF-I, and IGFBP.
Measurements taken after 7 days of treatment indicated that
administration of exogenous pGH on average resulted in a 22-fold increase in plasma GH concentration above the mean GH level of controls
(Fig. 1). Within 1 h of dosing, the
exogenous pGH completely obliterated any endogenous secretory pulses
and resulted in mean plasma GH levels nearly 40 times those of controls
(14 vs. 539 ng/ml, control and pGH-treated, respectively;
P < 0.01), which then decreased to
11 times that of controls (14 vs. 151 ng/ml, control and pGH-treated,
respectively; P < 0.01) by the end
of the sampling period, 4 h after dosing.

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Fig. 1.
Plasma growth hormone (GH) concentrations during 240 min after a single
injection of either saline (control, n = 5) or porcine GH (pGH, n = 5) at end
of 7-day treatment period. Plasma GH concentrations in pGH-treated pigs
were significantly (P < 0.01)
greater than control pigs at all time points. Values are means ± SD.
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After 1 day of pGH administration, plasma IGF-I was elevated above
controls (32.1 vs. 54.2 ng/ml, control and pGH-treated, respectively;
P < 0.01) and continued to increase
until it reached a plateau value ~300% of that of controls on
day 4 of treatment (Fig. 2). In ligand blots of
plasma samples collected after 7 days of treatment, we identified five
bands of IGF binding that corresponded to apparent molecular weights of
43, 39, 33, 29, and 24 kDa (Fig. 3). These
bands were putatively identified by comparing them with bands in
published reports that used immunological methods (31, 34, 40) as
differentially glycosolated forms of IGFBP-3 for the 43- and 39-kDa
bands, IGFBP-2 for the 34-kDa band, IGFBP-1 for the 29-kDa band, and
IGFBP-4 for the 24-kDa band. Commensurate with these previous reports,
we found, on the basis of immunoblotting, that the anti-human IGFBP-3
antiserum recognized only the 43- and 39-kDa proteins and the
anti-human IGFBP-2 antiserum recognized only a 34-kDa protein (Fig. 3).
Although we did not verify immunologically the 29- and 24-kDa bands
observed by ligand blotting, we believe these to be the same proteins
previously identified as IGFBP-1 in neonatal pig serum (34) and
IGFBP-4 (40), respectively. Moreover, because the 29- and
24-kDa bands were not detected by immunoblotting with IGFBP-2 and -3 antibodies, it is unlikely that they are proteolytic products of either
IGFBP-2 or IGFBP-3 degradation. On the basis of ligand blotting, when expressed as a percentage of the total plasma IGFBP abundance, the
proportions of IGFBP-1 and IGFBP-2 were lower
(P < 0.05) and those of IGFBP-3 and
IGFBP-4 were higher (P < 0.05) in
pGH-treated than in control pigs (Fig. 4).
The abundance of plasma IGFBP-3 measured by immunoblotting was
approximately threefold higher in pGH-treated than in control pigs;
however, there was no difference in the abundance of IGFBP-2 (Fig.
5). When measured by the immunoradiometric assay, IGFBP-3 was nearly twofold higher in pGH-treated than in control
pigs (88.7 ± 4.3 vs. 149.2 ± 5.5 ng/ml, control and
pGH-treated, respectively; P < 0.01).

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Fig. 2.
Daily plasma insulin-like growth factor I (IGF-I) concentration in
neonatal pigs administered either saline (control,
n = 5) or pGH
(n = 6) for 7 days after birth. Values
are means ± SD.
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Fig. 3.
Comparison of plasma IGF-binding proteins (IGFBPs) as determined by
either 125I-labeled IGF-I ligand
blotting (lane 1) or immunoblotting using
antibodies to IGFBP-3 (BP-3 Ab; lane 2) and IGFBP-2 (BP-2 Ab;
lane 3). Apparent molecular weights (MW)
of IGFBPs were determined from comparison with prestained standards run
on same gel.
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Fig. 4.
Relative abundance of plasma IGFBPs determined by
125I-IGF-I ligand blotting
procedure in neonatal pigs administered either saline (control,
n = 5) or pGH
(n = 6) for 7 days after birth.
Results are expressed as a percentage of total IGF binding as
determined by densitometric scanning of autoradiogram from
Western-ligand blot. Values are means ± SD.
* P < 0.05.
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Fig. 5.
A: Western immunoblot of plasma
samples from neonatal pigs administered either saline (control,
lanes 1-4)
or pGH (lanes 5-8)
for 7 days after birth. Blots were coincubated with antiserums to
IGFBP-2 and IGFBP-3. B: quantitative
results from immunoblot are expressed as arbitrary densitometric units
of either IGFBP-3 or IGFBP-2 as determined by laser densitometric
scanning. See MATERIALS AND METHODS
for details of immunoblotting procedure. Values are means ± SD. * P < 0.05.
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PUN, glucose, and insulin.
During the 7-day treatment period, PUN concentrations were not
statistically different (P > 0.05)
between control and pGH-treated pigs (Fig.
6). However, with the exception of
day
4, PUN tended to be lower (ranging
from 15 to 48%) in pGH-treated than in control pigs. To determine
whether 7 days of pGH treatment resulted in insulin resistance, plasma
insulin and glucose concentrations were measured in response to feeding
after an overnight fast. Although the plasma insulin response in the
first 60 min after feeding appeared to be blunted in the pGH-treated
compared with control pigs, there was no difference in the area under
the plasma insulin curve between treatments (4,423 ± 812 vs. 3,646 ± 378 µU · ml
1 · min
1,
control and pGH treated, respectively) (Fig.
7). The plasma glucose concentration after
feeding was greater at all time points in pGH treated pigs compared
with controls. Thus the area under the plasma glucose curve in response
to feeding was greater in pGH-treated than control pigs (843 ± 50 vs. 1,084 ± 63 mmol · l
1 · min
1,
control and pGH-treated, respectively;
P < 0.05) (Fig. 7).

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Fig. 6.
Daily plasma urea nitrogen (PUN) concentration in neonatal pigs
administered saline (control, n = 5)
or pGH (n = 6) for 7 days after birth.
Values are means ± SD.
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Fig. 7.
Plasma glucose (A) and insulin
(B) concentrations in response to
feeding in neonatal pigs after 7 days of either saline (control,
n = 5) or pGH
(n = 6) administration. Values are
means ± SD. *P < 0.05.
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Body weight gain, organ growth, and protein synthesis.
Body weights of pGH-treated pigs were 10% greater than those of
control pigs after 7 days of treatment. During the 7 days of treatment,
the rate of body weight gain was faster (+15%) and weight gain per
unit of food intake, commonly referred to as food conversion
efficiency, was greater (+16%) in pGH-treated pigs than control pigs
(P < 0.05, Table
1). Among the various organs measured,
liver (+24%), kidney (+16%), jejunum (+22%), and heart (+18%)
weights were significantly (P < 0.05) greater after 7 days of pGH treatment. When organ weights were
normalized to body weight (g/kg body wt), the liver (45.2 vs. 36.5, SD = 2.7, P < 0.01) and kidney (8.81 vs. 7.62, SD = 0.54, P < 0.01) were
still greater in pGH-treated than in control pigs, respectively.
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Table 1.
Body weight gain and tissue weights in neonatal pigs administered
saline (control) or pGH for 7 days after birth
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The tissue protein contents of the kidney (+41%), stomach (+36%), and
jejunum (+25%) were significantly greater in pGH-treated than control
pigs (Table 2); the protein content of the
liver and heart also tended to be greater
(P < 0.10) in pGH-treated than
control pigs. There were no differences between treatments for tissue
concentrations of protein, RNA, and DNA (data not shown).
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Table 2.
Tissue content, FSR, and ASR of protein in neonatal pigs administered
saline (control) or pGH for 7 days after birth
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The FSR values in all of the tissues measured were similar
(P > 0.05) in control and
pGH-treated groups (Table 2). However, the ASR values for liver
(+39%), kidney (+49%), stomach (+41%), jejunum (+47%), and heart
(+43%) were greater in pGH-treated pigs than in controls
(P < 0.05). The ASR of the soleus
muscle (+18%) and lung (+21%) tended to be greater in pGH-treated vs.
control pigs (P < 0.10). In
addition, protein synthetic capacity was increased (+14%,
P < 0.05) in liver, and the protein
synthetic efficiency was increased (+31%,
P < 0.05) in semitendinosus of
pGH-treated pigs compared with controls (data not shown).
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DISCUSSION |
It is widely believed that neonates are unresponsive to GH and that
neonatal growth, therefore, is relatively GH independent. However, few
studies have examined the effects of exogenous GH on growth or the
function of the somatotropic axis in neonates. Our current study has
demonstrated that pGH administered to neonatal pigs results in
increased plasma concentrations of IGF-I, IGFBP-3, and glucose and
elevated body weight gain and visceral organ protein synthesis. These
changes in neonatal pigs are characteristic of the metabolic and
anabolic response to GH treatment, although the magnitude of the
responses is considerably lower than observed previously in more mature
pigs. Nevertheless, the results indicate that the somatotropic axis in
neonatal pig is, indeed, responsive to exogenous pGH.
Endocrine effects of GH.
Our results demonstrate that pGH administration to neonatal pigs
produced the characteristic stimulation of the somatotropic axis as
evidenced by the marked increase in the circulating IGF-I concentration
compared with control pigs. The temporal increase in circulating IGF-I
noted in the initial 4 days of pGH treatment may reflect a gradual
increase in GH responsiveness and is supported by evidence of
upregulation of both hepatic GH-binding and GH-receptor mRNA abundance
in response to chronic pGH treatment in pigs (1, 4, 9). Previous
studies of GH administration to neonatal pigs have reported variable
effects on circulating IGF-I concentrations, due to the differences in
the GH dose and frequency of administration used (1 vs. 3 times per
day) compared with our study (1, 25, 33). However, the two- to
threefold increase in circulating IGF-I concentration that we observed
in pGH-treated neonatal pigs is consistent with values reported
recently (33) in neonatal pigs at a slightly lower dose (~0.5 mg/d)
than we used and in mature pigs, albeit at much lower doses (11, 16,
29, 43).
At least two possible mechanisms are responsible for the increase in
circulating IGF-I in pGH-treated neonatal pigs. The first is increased
endogenous IGF-I production and several tissues, including liver,
adipose tissues, and certain skeletal muscles, have been shown to be GH
responsive, as measured by increased IGF-I mRNA expression (4, 11).
Therefore, the increased circulating IGF-I concentration in pGH-treated
neonatal pigs in our study was likely a result of increased tissue
expression, and, indeed, recent evidence indicates that administration
of pGH increases hepatic IGF-I expression in neonatal pigs (33). Our
results suggest that a second mechanism responsible for the increased circulating IGF-I was an increase in plasma IGFBP-3 concentration, which may have prolonged the half-life of IGF-I. In adults, IGFBP-3 is
the most abundant IGFBP and has the greatest affinity for both IGF-I
and -II (27). In neonates, however, the plasma levels of IGFBP-2 are
higher and those of IGFBP-3 are lower compared with those of adults
(13). On the basis of measurements of ligand binding, immunoblotting,
and immunoradiometric assay, our results indicate that pGH treatment
increased both the absolute concentration and relative abundance of
plasma IGFBP-3. The changes in the abundance of plasma IGFBP-3 induced
by pGH treatment in our study were similar to those observed in
14-wk-old pigs (10). Thus, given that most of the circulating IGF-I is
bound to IGFBP-3, which does not leave the vascular compartment, we
would postulate that the increased circulating IGF-I in the pGH-treated
pigs was partially a result of a longer half-life. The increased plasma
IGFBP-3 was likely a result of the increase in circulating IGF-I
concentrations in response to pGH treatment (27, 46). Thus, on the
basis of the changes in both circulating IGF-I and IGFBP
concentrations, our results demonstrate that the somatotropic axis in
neonatal pigs, even as early as 2 days of age, is responsive but
considerably less sensitive to pGH treatment than that of adult pigs,
given the higher dose of exogenous pGH required to attain the similar changes.
Metabolic effects of GH.
One of the classic metabolic effects of GH treatment is the
diabetogenic effect that is manifested as insulin resistance (36). Insulin resistance in pGH-treated adult pigs is characterized by
increased circulating concentrations of both glucose and insulin; the
hyperglycemia results from both increased hepatic production and
decreased peripheral glucose clearance (14, 23). Our results, based on
the meal-tolerance test after 7 days of pGH treatment, indicated that
these pigs were becoming insulin resistant. The presence of
hyperglycemia, despite equal circulating insulin concentrations, in
pGH-treated pigs is consistent with either increased hepatic production
or decreased glucose clearance, indicating insulin resistance of
glucose metabolism. The diabetogenic effect of pGH on glucose
metabolism, which we observed in the neonate, was, however, somewhat
blunted compared with the responses reported in more mature pigs (23,
44). This more blunted hyperglycemic effect suggests that the neonatal
pig may be less sensitive to pGH compared with the adult pig.
Alternatively, our previous observations that the insulin
responsiveness of glucose disposal is elevated in early neonatal
compared with late neonatal or adult pigs (43, 44) may perhaps explain
why this diabetogenic effect of pGH treatment on glucose metabolism was
somewhat blunted compared with the adult pig. It has been shown that
the diabetogenic effect of pGH on glucose metabolism is mediated
particularly through decreased glucose utilization by adipose tissue
(14, 22). Therefore, neonatal pigs may be less sensitive to the
diabetogenic effects of pGH on glucose metabolism because they have a
considerably lower body fat content than adult pigs (39).
Another characteristic metabolic effect of GH treatment is reduced PUN
concentrations, an effect that has been shown to be dependent on dose
and frequency of administration (16, 17, 45). Recent studies in
neonatal pigs given much lower doses than in the current study suggest
that the reduction in PUN after pGH treatment does not become
responsive until ~3-4 wk of age, concurrent with the increase in
hepatic GH binding (25). Consistent with this previous report, we found
no significant PUN response, which may have been because the neonatal
pig is less sensitive to pGH compared with the adult pig, as well as an
inherently large between-animal variability. Moreover, in our study,
pigs were fed dietary protein in excess of their requirement to ensure
that any growth-promoting effects of pGH could be achieved. This may have masked any subtle changes in the efficiency of protein
utilization, as reflected by PUN.
Body weight gain, tissue growth, and protein synthesis.
Two of the fundamental anabolic effects of GH treatment in pigs are
increased body weight gain and food conversion efficiency that result
from the increased deposition of lean and reduction of adipose tissue
(15, 16). We observed pGH responses in both body weight gain and food
conversion efficiency in neonatal pigs that were similar to those
reported for older pigs given exogenous GH (15, 17, 38). It should be
noted that the increases in organ protein content in response to pGH
treatment were not associated with a decrease in the protein
concentration (i.e., mg/g tissue) in any tissue measured, indicating
that the increase in tissue weight was not due to changes in tissue
hydration but to a net increase in protein deposition. The combined
protein content of all the tissues or organs measured was increased
~17% by pGH treatment, similar to the increase in body weight gain,
suggesting that the growth response was allometric. However, the sum of
all the tissues measured, mostly internal organs, represents a
relatively small proportion (13-14%) of body weight. Thus an
unanswered question is whether the growth response occurred in the
proportionally larger mass of carcass tissues composed of skeletal
muscle, bone, adipose, and skin. It is unlikely that changes in adipose
tissue mass contributed to the differences in body weight gain in
response to pGH treatment, since the body fat content of the neonatal
pig is relatively low (1-2%) (39). Although we did not measure
body composition, we found only modest increases in the protein content of both soleus (+10%) and semitendinosus (+3%) muscles, indicating that skeletal muscle protein deposition was minimally responsive to pGH
treatment in neonatal pigs.
In the present study, we did not observe any statistically significant
increase in the FSR for any tissue measured. However, we observed that
the ASR of protein was significantly greater in several tissues, which
reflected the higher tissue protein content in pGH-treated than control
pigs. The tissues in which pGH treatment produced the largest
stimulation of protein ASR were the kidney, jejunum, stomach, and
liver. Although several reports have noted enhanced skeletal muscle
protein synthesis in response to administration of either GH or IGF-I,
we observed only minimal effects (18, 19, 38). The fact that we did not
detect any significant increases in FSR suggests that the increased
protein content observed in several tissues from pGH-treated pigs
resulted from either a decrease in the fractional rate of protein
degradation or that an increase in the FSR occurred earlier in the
treatment period. It was notable, however, that, despite a threefold
increase in circulating IGF-I concentration, protein synthesis and
weight gain were only modestly increased compared with the responses
reported in more mature animals (15, 17, 18, 38). Thus, whereas the
somatotropic axis is functional in the neonatal pig, an increase in the
circulating IGF-I concentration above normal resulted in only a modest
stimulation of growth. This relatively modest response to increased
circulating IGF-I may be a consequence of the existing high rates of
growth and anabolism in the neonatal pig that cannot be increased
further.
Perspectives.
The results indicate that the somatotrophic axis in neonatal pigs is
functional and responsive to exogenous pGH. Although we found that the
proportional increases in circulating IGF-I and IGFBP-3 concentrations
in response to exogenous pGH were similar to those observed in previous
studies with adult pigs, the pGH dose we used was severalfold greater.
This suggests that the somatotropic axis in neonatal pigs is less
sensitive to exogenous pGH than that in adult pigs. Despite the
pharmacological dose of pGH and the nearly threefold increase in
circulating IGF-I, the metabolic and anabolic growth responses we
observed were relatively modest, suggesting that neonatal pigs are less
sensitive not only to exogenous pGH but also to circulating IGF-I,
compared with adult pigs.
 |
ACKNOWLEDGEMENTS |
The authors express appreciation for the technical assistance of X. Chang, H. Nguyen, D. Lin, C. Lerner, and S. Ziari. The authors also
acknowledge Drs. L. Underwood and J. J. Van Wyk, the National Hormone
and Pituitary Program, University of Maryland School of Medicine, and
the National Institute of Diabetes and Digestive and Digestive
Diseases, for providing antiserum UB3-189 for human IGF-I and Dr.
J. McMurtry and the US Department of Argiculture (USDA) Animal Hormone
Program for providing antiserum JM44A3 for pGH and immunochemical grade
pGH USDA-pGH-I-2. The authors sincerely thank Monsanto Agricultural
Products Co., St. Louis, MO, for providing the recombinant pGH used in
this study.
 |
FOOTNOTES |
T. J. Wester was supported by National Institutes of Health Training
Grant T32-HD-07445-02. This work was supported in part by federal
funds from the USDA, Agricultural Research Service under Cooperative
Agreement no. 58-6250-1-003.
The contents of this publication do not necessarily reflect the views
or policies of the USDA, nor does mention of trade names, commercial
products, or organizations imply endorsement from the US government.
Current address of T. J. Wester: Dept. of Agriculture, Univ. of
Aberdeen, MacRobert Building, 581 King Street, Aberdeen, AB24 5UA,
Scotland, UK.
Address for reprint requests: D. G. Burrin, Children's Nutrition
Research Center, 1100 Bates St., Houston, TX 77030.
Received 22 May 1997; accepted in final form 18 September 1997.
 |
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