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Division of Nutritional Sciences and the Department of Food Science and Human Nutrition, University of Illinois, Urbana, IL 61801
3To whom correspondence should be addressed at 439 Bevier Hall, 905 South Goodwin, Urbana, Illinois 61801.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The efficacy of recombinant human growth hormone (GH) and/or a diet enriched in protein and energy to improve growth recovery following prolonged malnutrition was examined in male rats food-restricted from birth until 120 d of age. At d 121, restricted rats were randomly assigned to recovery groups receiving either a control or enriched diet with or without daily subcutaneous injections of GH. Rats were killed after 16 or 47 d of recovery. At d 16, GH treatment stimulated liver, heart, plantaris, soleus, carcass and body weight gain and inhibited fat gain when compared to recovery controls. Rats receiving GH also exhibited the highest serum insulin-like growth factor-I (IGF-I) concentrations and total muscle protein. At d 47, GH effects on body and muscle recovery were minimal, and differences among recovery groups in serum IGF-I concentration and total muscle protein were no longer present. Consumption of an enriched diet increased fat pad and liver mass, but did not promote muscle recovery. There were no differences among treatment groups in skeletal muscle IGF-I mRNA levels at d 16 or 47. In summary, GH had positive effects on somatic and skeletal muscle growth early in the recovery process, possibly via endocrine IGF-I-stimulated protein accretion. In contrast, the enriched diet promoted fat deposition with no impact on skeletal muscle growth recovery.
KEY WORDS: • growth hormone • growth recovery • insulin-like growth factor-I • protein-energy malnutrition • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; MacLean and Graham, 1980
). It is assumed that proper nutrition is essential
for optimal muscle growth recovery; however, realimentation alone does
not achieve complete growth recovery of lean body mass following
prolonged undernutrition (Glore and Layman 1987
).
Normal growth is controlled in part by the growth hormone
(GH)4
insulin-like growth factor-I (IGF-I) axis. Many of the growth-promoting
actions of GH are mediated by the synthesis and secretion of IGF-I, a
single-chain polypeptide produced by most tissues of the body and
abundant in the circulation. IGF-I is a regulator of cell growth and
differentiation and has the potential to act via endocrine as well as
autocrine and/or paracrine mechanisms (Cohick and Clemmons 1993
). The biological activity of IGF-I in serum and other
biological fluids is modulated by at least six different high-affinity
insulin-like growth factor binding proteins (IGFBP) (Bach and Rechler 1995
). IGFBP in extracellular fluids act to increase
the half-life of IGF-I and/or block IGF activity, whereas IGFBP on the
cell surface may play a role in enhancing IGF-I activity (Bach and Rechler 1995
). Both IGF-I and IGFBP are nutritionally and
hormonally regulated (Sara and Hall 1990
). Although the
response of the IGF-I system following short-term food deprivation and
refeeding was described (Strauss and Takemoto 1990
;
Zhao et al. 1995
), the response of the IGF-I system
during growth recovery following prolonged undernutrition was not
assessed.
Previously, treatment with GH or GH plus IGF-I after neonatal
malnutrition supported complete growth recovery in young rats by
increasing body weight and enhancing lean body mass accretion
(Zhao et al. 1995
; Zhao and Donovan 1995
). In these studies, the food-restriction and refeeding
periods were relatively short-term. Few studies have examined growth
recovery in older rats subjected to chronic malnutrition, and very few
data in the literature address whether hormonal therapy following
prolonged undernutrition could improve gains obtained by nutrition
alone. Furthermore, it is unclear if extending the period of GH
administration would result in further anabolic gains beyond the
initial phase of rapid catch-up growth.
The present investigation evaluates the efficacy of administering exogenous GH to male rats following prolonged food restriction. Moreover, this study describes the response of the IGF-I system during growth recovery to elucidate the role of IGF-I and the IGFBP in mediating anabolic catch-up growth. It was hypothesized that GH administration would enhance somatic and skeletal muscle growth recovery, and further, that refeeding a diet enriched in protein and energy would improve growth recovery by nutritionally supporting GH-induced anabolic gains. This study demonstrates that GH therapy produces significant gains in somatic and skeletal muscle mass during the initial period of growth recovery. Conversely, refeeding a protein and energy-dense diet increases adipose deposition and has little impact on muscle growth recovery.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All chemicals and standards were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise stated. Dessicated recombinant human growth hormone was generously provided by Genentech (South San Francisco, CA). 3-[125I]Iodotyrosyl insulin-like growth factor-I and [32P]-dCTP were purchased from Amersham (Arlington Heights, IL).
Animals and diets.
The animal protocol used in this study was approved by the Laboratory Animal Care Advisory Committee of the University of Illinois at Urbana-Champaign. All animals were provided free access to tap water throughout the study and were maintained in a temperature-controlled environment (22–24°C) with a 12 h/12 h light/dark cycle. Thirty-one timed-pregnant (d 15 gestation) Sprague-Dawley rats (285.8 ± 3.37 g) were purchased from Harlan Industries (Indianapolis, IN) and housed individually in polypropylene box cages. Dams were allowed free access to a pelleted diet (Harlan Teklad Laboratory Diets, Madison, WI) until parturition. All pups were delivered within 48 h of predicted 21 d gestation.
Following parturition, dams were switched to a semi-purified control
powder diet (Table 1
)that they consumed freely until d 2 postpartum. On d 2 postpartum,
female pups were removed from the litters to standardize each litter to
eight pups per dam. Dams were then randomly designated as either
freely-fed controls (n = 8) or food-restricted
(n = 23). After d 2 postpartum, control dams continued
freely consuming the control diet, while the food-restricted dams were
switched to the restricted diet (Table 1)
and fed 50% the mean food
intake consumed by the control dams over the previous 24-h period. A
50% food restriction to dams during lactation was reported to produce
~50% reduction in milk yield on d 14 of lactation and ~50%
reduction in pup growth (Kliewer and Rasmussen 1987
).
The restricted diet contained the same concentrations of protein and
energy as the control diet, but contained twice the concentrations of
vitamins and minerals. This was to prevent confounding the ensuing
protein-energy malnourishment with an additional vitamin and/or mineral
deficiency. Dams were weighed daily and litters were weighed every
other day until weaning (d 21 postpartum).
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).
At d 121 postpartum (d 1 of recovery), food-restricted rats were
randomly assigned to one of four growth recovery treatments: RC, refed
the control diet (serving as recovery controls); E, refed an enriched
diet (Table 1)
; GHC, refed the control diet plus received twice daily
subcutaneous injections of recombinant human GH (rhGH) at a dosage
level of 1.0 mg · kg-1 · d-1;
GHE, refed an enriched diet plus received twice daily subcutaneous rhGH
injections. Animals with free access to the control diet from birth
were designated as normal growth age controls (NG) and continued to
have free access to food throughout the recovery period. Based on the
Nutrient Requirements for the Laboratory Rat (1978)
, the
control diet was designed to promote the healthy growth of rats, while
the enriched diet provided twice the concentrations of protein,
minerals and vitamins and three times the concentration of fat. The GH
dose used in the present study was chosen based on its ability to
restore musculoskeletal growth in hypophysectomized rats
(Grindeland et al. 1994
). Rats that did not receive rhGH
injections were injected with an equal volume of excipient (9 g/L
saline). Rats were allowed to recover for 47 d, with body weight
and food intake recorded daily.
Biological samples.
Rats (n = 12 per group) from each of the four growth recovery treatment groups) were killed by carbon dioxide asphyxiation followed by decapitation on recovery d 0 (baseline), d 16 (designated as early growth recovery), and d 47 (designated as late growth recovery). Normal growth controls were killed on d 0 and d 47. Animals were not food-deprived at any point before collection of biological samples. Blood samples were collected from the cervical stump and allowed to clot on ice for 30 min. Serum was obtained by centrifugation at 1,800 x g for 10 min and then stored at -80°C. Three muscles, the gastocnemius, plantaris, and soleus were extracted from both hindlimbs, blotted and weighed after all visible connective tissue was removed. The heart was removed, blotted to remove blood and then trimmed of any visible remnants of the superior vena cava, aorta or pulmonary artery before weighing. The liver was dissected and blotted, and interior and exterior sections of the left, median, right and caudate lobes were sampled after weighing entire organ. The inguinal and perirenal fat pads were collected, pooled and weighed as a measure of body fat content, and the eviscerated carcass weight was recorded. All organs and muscles were quickly frozen in liquid nitrogen and stored at -80°C.
Serum measurements.
Serum IGF-I was measured by a specific radioimmunoassay (RIA) following
acid chromatography as previously described (Zhao et al. 1995
). Serum insulin was measured in duplicate (100 µL) using
a commercial RIA kit for rat insulin (Linco Research Inc., St. Louis,
MO). Serum samples were analyzed within a single assay with intraassay
coefficients of variation of 6 and 6.5% for IGF-I and insulin,
respectively. Sample binding was determined using a gamma counter
(COBRA AutoGamma 5000; Packard Instrument, Meriden, CT).
Serum IGFBP profiles were determined in six randomly chosen rats from
each treatment group at d 16 and d 47 of growth recovery by SDS-PAGE
and western ligand blotting as previously described (Zhao et al. 1995
). Briefly, serum samples (3 µL) were separated on a 12%
SDS-polyacrylamide gel, the proteins were transferred to nitrocellulose
and the nitrocellulose was incubated overnight with
[125I]IGF-I. IGFBP were visualized by autoradiography for
7 d at -80°C.
Total cellular DNA and protein content.
DNA and protein content were determined for the gastrocnemius. Frozen
samples were homogenized in 8 mL of distilled water, and duplicate
aliquots of the homogenate were processed according to the method of
Schmidt and Thannhauser (1945)
as modified by
Munro and Fleck (1969)
. DNA was determined by the indole
procedure as described by Cerotti (1952)
. Protein was
determined by the bicinchoninic acid assay as described by Smith et al. (1985)
(Pierce, Rockford, IL). Calf thymus DNA and
bovine serum albumin (BSA) were used for standards.
RNA preparation and tissue mRNA analysis.
Total cellular RNA was isolated from 75-mg samples of liver and
gastrocnemius muscle (Chomczynkski and Sacchi 1987
). RNA
quantity and purity were determined by the ratio of absorbance at 260
and 280 nm. Liver and gastrocnemius IGF-I and IGFBP mRNA expression
were determined by slot blot (Sambrook et al. 1989
). For
slot blotting, liver (15 µg) and gastrocnemius (20 µg) total RNA
were blotted onto a 0.45-µm nylon membrane using a Bio-Dot SF
Microfiltration Apparatus (Bio-Rad Laboratories, Richmond, CA). Prior
to hybridization, membranes were incubated in 225-mm bottles in a
hybridization oven (Bellco, Vineland, NJ) for 2 h at 65°C in a
solution containing 10 g/L of BSA, 7 g/L of sodium dodecyl sulfate
(SDS), 0.45 mol/L of sodium phosphate, 0.25 mol/L of NaCl and 0.001
mol/L of EDTA (pH 7.2). Hybridizations were performed in this same
buffer at 65°C for 12–24 h, and membranes were probed with
random-prime [32P]-dCTP-labeled rat cDNA for IGFBP-1
(Murphy et al. 1990
), IGFBP-2 (Margot et al. 1989
) and IGFBP-4 (Shimasaki et al. 1990
).
Hepatic and gastrocnemius IGF-I was probed using a
[32P]-labelled RNA sequence corresponding to Exon 4 of
the rat IGF-I gene, kindly provided by Dr. Keith Kelley, Department of
Animal Sciences, University of Illinois (Biragyn et al. 1994
). Membranes were also probed with human 28S ribosomal RNA
to control for loading variation. After hybridization, membranes were
rinsed twice at room temperature with 1 g/L of SDS in 0.2x SSC, (0.03
mol/L NaCl, 0.003 mol/L sodium citrate) followed by two 30-min
incubations at 65°C. Membranes were wrapped in plastic wrap and
visualized by autoradiography at -80°C for 2–3 d.
Densitometry.
Audioradiographs of Western ligand blots, and slot blots were quantitated using relative pixel intensities determined by a FotoAnalyst II Imager System and Collage software (Fotodyne Inc., New Berlin, WI). For slot blots, density of IGF-I and IGFBP mRNA were normalized to ribosomal RNA.
Statistical analysis.
Data were analyzed by ANOVA using the General Linear Model procedure of SAS (Version 6.08; SAS Institute, Cary, NC) with diet and GH treatment as independent variables. If overall significance was demonstrated, Duncan's Multiple Range was used to compare main treatment (diet, GH treatment) and interaction effects. All data are expressed as mean ± SEM. The level of significance was set at P < 0.05 for all statistical tests.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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During early growth recovery, rats administered GH (GHC and GHE) gained
more body weight than those injected with saline (RC and E), and at d
16 were 110 and 111% of recovery controls, respectively (Fig. 1
and Table 2
).Conversely, rats consuming the enriched diet (E and GHE) did not
exhibit increased body weight as compared to those consuming the
control diet (RC and GHC) (P < 0.09 for main effect of
diet). Body weight gain during late growth recovery slowed markedly in
all recovery treatment groups (Fig. 1
and Table 2
). At d 47, body
weights of E, GHC and GHE animals were 105, 106 and 108% of recovery
controls, respectively. Animals consuming the enriched diet (E and GHE)
tended to gain more weight (P < 0.052) than those
consuming the control diet (RC and GHC) during late growth recovery.
Overall, GH treatment promoted whole body growth recovery early in the
recovery process, while the enriched diet tended to increase body
weight gain during late growth recovery.
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In all refed rats, energy consumed per day was higher during early
recovery vs. late recovery (Table 2)
, due in part to all recovery
animals consuming a disproportionately large amount of energy relative
to their body weight during the first few days of growth recovery.
Average daily energy intake of the NG group was not different between
early and late growth recovery. The energy intake of the E group was
significantly higher than all other treatment groups during the early
recovery period. During early growth recovery, animals administered GH
(GHC and GHE) displayed greater efficiency of growth than animals
injected with excipient (RC and E). Conversely, during late growth
recovery, the growth efficiency of animals treated with GH was similar
to recovery controls. Further, the growth efficiency of rats receiving
GH and consuming the control diet was not different from that of the NG
group.
Tissue and organ growth during refeeding following food restriction.
During early growth recovery, GH treatment increased carcass, liver,
heart and skeletal muscle mass and inhibited fat deposition compared to
rats not receiving GH (Table 3
).While lean mass increased in both the GHC and GHE groups, a reduction
in fat deposition was most obvious in the GHC group, which had 29%
less fat pad mass compared to recovery controls. In contrast, rats
refed the enriched diet (E and GHE) had more body fat than those refed
the control diet (RC and GHC). Specifically, animals fed the enriched
diet during recovery (E) gained 34% more fat than recovery controls
and had the most fat pad mass per 100 g of body weight.
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).Additionally, GH treatment was unable to suppress fat deposition.
Between recovery d 16 and d 47, rats treated with GH (GHC and GHE)
gained 2- to 3-fold more fat pad mass than animals injected with
excipient (RC and E). Moreover, the enriched diet continued to promote
gains in fat and liver mass while having no impact on muscle growth. As
a result, the combination of GH plus the enriched diet resulted in
animals with the greatest gains in liver and adipose between d 16 and d
47. Thus, treatment with GH increased lean body mass while limiting fat
deposition during early growth recovery; however, continued treatment
did not enhance these effects. Conversely, refeeding a diet enriched in
protein and energy increased fat deposition while having minimal
effects on muscle growth recovery.
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Total DNA and protein in the gastrocnemius muscle during early and late
growth recovery were used as a measure of cellular growth recovery
(Table 5
).There were no differences in DNA content or in the protein/DNA ratio
among the treatment groups during early growth recovery. However, total
muscle protein in the GHC group was higher than recovery controls at d
16. There were no differences in DNA content or in the protein/DNA
ratio in the gastrocnemius among the recovery treatment groups nor
between the recovery groups and the normal growth controls at d 47.
There were also no differences among recovery groups in total muscle
protein, but all were lower than the normal growth controls.
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At d 16 of growth recovery, serum IGF-I and insulin concentrations of
all treatment groups were greater than d 0 (d 120 postpartum) values of
25.7 ± 3.66 nmol/L and 0.66 ± 0.003 nmol/L, respectively
(Fig. 2
).Further, serum IGF-I concentrations in the GHC animals were
significantly greater than all recovery treatment groups. At d 47,
there were no differences in serum IGF-I among recovery treatment
groups, but the GHE animals had significantly higher serum IGF-I than
normal growth controls. There were no differences in serum insulin
among recovery groups, nor between the recovery groups and the normal
growth controls at either d 16 or d 47.
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Representative IGFBP profiles of animals at d 16 and d 47 are displayed
in Figure 3.
In these autoradiographs, serum from all animals showed three regions
of [125I]IGF-I binding, (i) a cluster of several bands
with an apparent molecular retention (Mr) of 39–48 kDa
which corresponds to IGFBP-3, (ii) bands with an apparent
Mr of 29–31 kDa, which contains IGFBP-1, IGFBP-2 and
IGFBP-3 fragments and (iii) a 24-kDa band which corresponds to IGFBP-4.
Densitometric measurement of these binding regions showed that at both
d 16 and d 47 (Table 6
),GHC and GHE animals had higher IGFBP-3 (39–48 kDa) levels than E
animals, but these levels were not different from recovery controls or
normal growth controls. The 29–31 kDa IGFBP at both d 16 and d 47 was
lower in the E group as compared to recovery controls, but not
different from the GHC, GHE and NG groups. The 24 kDa IGFBP at d 16 was
lower in the E group as compared to RC and GHC groups, but was not
different from the GHE group. There were no differences among treatment
groups in the 24 kDa IGFBP at d 47.
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There were no differences among recovery treatment groups in hepatic
IGF-I mRNA at d 16 (Table 7
).At d 47, hepatic IGF-I mRNA expression was higher in the GHC treatment
group when compared to other recovery treatment groups and not
different from normal growth controls. In contrast, gastrocnemius IGF-I
mRNA expression did not differ among recovery groups or between any of
the recovery groups and the normal growth controls at d 16 or d 47.
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).There were no differences among recovery groups in IGFBP-4 mRNA
expression. At d 47, there were no differences in IGFBP-1, -2, or -4
mRNA expression among the recovery treatment groups or between any of
the recovery groups and the normal growth controls.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Animals and
humans recovering from chronic malnutrition possess substantial
capacity for catch-up growth, although limitations in the ability to
fully recover seem to exist (Glore and Layman 1987
). In
the present investigation, the potential for exogenous GH alone or in
combination with a diet enriched in protein and energy to enhance
growth recovery of lean body mass in older rats following prolonged
undernutrition was explored. The length of administration (47 d) was
chosen to investigate longer-term effects of GH, for the majority of
previous reports used shorter-term administration (Zhao et al. 1995
; Zhao and Donovan 1995
). Our hypothesis was
two-fold: first, GH treatment would activate the IGF-I system and
increase whole body and skeletal muscle growth recovery. Second, freely
feeding a protein and energy-dense diet would support GH-induced
increases in plasma IGF-I concentrations, leading to improved growth
recovery.
Overall, whole body growth recovery was improved by GH treatment.
Animals injected with GH recovered to more than 80% of the normal
growth controls in 47 d. Previously, Glore and Layman (1987)
using a similar experimental design reported 76%
recovery of body weight in rats refed for 196 d following
long-term malnutrition. In the present study, nutrition alone increased
body size to 75% of controls, in accordance with the earlier study.
The current findings demonstrating a significantly higher final body
weight with GH treatment suggest that growth recovery can be improved
by GH treatment following chronic undernutrition.
Perhaps more important than the absolute body weight gain, GH treatment
during early growth recovery partitioned available energy toward
protein accretion while limiting fat deposition. Studies by Zhao et al. (1995)
and Zhao and Donovan (1995)
determined GH treatment during recovery from neonatal malnutrition to
promote lean body mass accretion. Additionally, Bates et al. (1993)
found that in hypophysectomized rats, GH caused
repartitioning of energy into protein and away from fat. In the current
study, animals receiving GH gained more body, carcass, liver and muscle
weight and deposited less fat during the first 16 d of recovery
than animals injected with excipient. Thus, GH administration has a
positive role in promoting body weight gain and body protein mass while
diminishing fat deposition following prolonged undernutrition in rats.
A potential mechanism of GH action during growth recovery is via
IGF-I-stimulated protein accretion. DelBarrio et al. (1993)
found that a single injection of rat GH (1 µg/g body
weight) to normal adult rats produced immediate increases in muscle and
bone protein synthesis. Additionally, other studies showed GH has a
major protein anabolic effect in skeletal muscle (Boyd and Bauman 1989
; Clemmons et al. 1993). Furthermore,
Bates et al. (1993)
showed that interactions between
dietary protein and GH administration on the body composition of
hypophysectomized rats were at least partly due to changes in plasma or
tissue IGF-I levels. In the present study, GH combined with the control
diet increased both serum IGF-I concentrations and total muscle protein
content during the first 16 d of recovery. Together, these data
support a protein anabolic role for the GH/IGF-I axis during early
growth recovery.
It was established nearly two decades ago that feeding a high-fat or
highly-palatable diet during growth recovery causes weight gain and
obesity in rats (Harris and Widdowson 1978
;
Ozelci et al. 1978
). However, feeding a nutrient dense
diet in combination with hormone therapy was not considered. In this
study, feeding an energy-dense diet had no effect on growth recovery of
lean body mass. Rather, rats fed the enriched diet gained more fat pad
mass during growth recovery than their control diet counterparts.
Further, the enriched diet counteracted the repartitioning effects of
the GH treatment. Dawson et al. (1998)
reported that
stimulation of lipolysis following a bolus injection of bovine GH is
markedly less in cattle well-fed (2.65 x maintenance energy
requirement) as compared to animals fed on lower planes of nutrition.
The authors suggest that a high protein intake may augment this effect,
for Brameld et al. (1996)
demonstrated GH receptor mRNA
expression to be decreased in adipose tissue of GH-treated pigs fed a
high protein diet. These results suggest that the macronutrient
composition of the recovery meal markedly influences the effectiveness
of exogenous GH in rats.
There are several possible explanations for the decline in GH activity
over time. Formation of anti-rhGH antibodies may explain the declining
effect of rhGH on somatic and skeletal muscle growth during late growth
recovery, for hGH is antigenic in rats (Groesbeck and Parlow 1987
). Moreover, the probability of an immune response is
increased when an antigen is given subcutaneously or when given
multiple times over a several-week period (Janeway and Travers 1994
). Fielder et al. (1996)
reported positive
anti-rhGH titers in 88% of hypophysectomized rats receiving daily
subcutaneous injections of rhGH for 28 d. Additionally, both
Fielder et al. (1996)
and Froesch et al. (1988)
found the growth-promoting effects of rhGH to diminish
in adult hypophysectomized rats after 2–3 wk of rhGH treatment. The
available data suggest that despite early gains in growth and body
protein with rhGH administration, the development of anti-rhGH
antibodies in our animals may have prevented further gains during
prolonged recovery.
A second possibility for diminished GH effects over time may involve
down-regulation of GH receptors. Maiter et al. (1988)
found that in hypophysectomized and intact rats a surge of exogenously
administered GH in the serum acutely down-regulated hepatic GH
receptors in a dose- and time-dependent fashion. Additionally,
Singh et al. (1992)
determined that GH-deficient rats
treated with GH had reduced hepatic GH receptor mRNA as compared to
saline controls. Therefore, it is possible that chronic administration
of GH may have contributed to some degree of GH receptor
down-regulation.
A third possible explanation for why skeletal muscle growth recovery
slowed over time, despite continued treatment with GH, involves
attainment of maximal growth potential. Previous work showed that
proliferation of myonuclei slows considerably around 100 d of age
in the rat (Layman et al. 1980
). Therefore, by waiting
until 120 d of age to begin recovery, the recovery potential of
skeletal muscle may be more limited. This idea is supported by work
from Zhao and Donovan (1995)
that demonstrated GH
treatment to stimulate complete body weight recovery in malnourished
neonatal rats. Further, in the current study, total muscle DNA was not
increased by GH treatment during growth recovery. Therefore, it is
possible that physiologic limitations in muscle recovery potential
contributed to the diminished effect of GH over time.
Skeletal muscle IGF-I mRNA levels were analyzed to determine if the
anabolic effects of GH on skeletal muscle recovery were associated with
increased local expression of IGF-I. Contrary to this notion, skeletal
muscle IGF-I mRNA levels were not different among the growth recovery
groups or between the normal growth group and the growth recovery
groups. Svanberg et al. (1996)
determined in mice that
18 h of food-deprivation caused a rapid 30% reduction in skeletal
muscle IGF-I mRNA expression which was immediately restored to normal
levels upon refeeding. This suggests that refeeding normalizes local
expression of IGF-I, but does not necessarily stimulate the local
production of IGF-I above that of freely-fed animals. Alternatively,
increased expression of IGF-I mRNA in skeletal muscle may have occurred
during early growth recovery, but the timing of the measurements may
have been too late to detect changes, as up-regulation of skeletal
muscle IGF-I mRNA expression occurred within hours of GH administration
in previous studies (Svanberg et al. 1996
).
GH treatment tended to increase serum IGFBP-3 over that observed in
animals not treated with GH. Studies by Lemmey et al. (1997)
and Fielder et al. (1996)
demonstrated
that a single GH injection or 28 d of GH treatment, respectively,
significantly increased IGFBP-3 concentrations in rats. An increase in
IGFBP-3 in the serum may be beneficial for growth recovery by extending
the half-life of IGF-I and therefore potentiating the growth-promoting
effects of IGF-I.
Refeeding the enriched diet without receiving GH resulted in a 43%
decline in hepatic IGF-I mRNA expression as compared to recovery
controls and suppression of serum 29–31 kD binding proteins and
hepatic IGFBP-1 and -2 mRNA expression at d 16 of growth recovery.
While it is unclear as to why these effects occurred, it is speculated
that the high energy content of the diet may have caused some hepatic
dysfunction. Research by Ney et al. (1995)
investigated
the effect of 8 d of high-energy total parenteral nutrition in
rats and found that both hepatic IGF-I and IGFBP-1 mRNA expression were
reduced compared to control rats. The authors attribute their results
to the development of hepatic steatosis. While the liver weights of
animals refed the enriched diet were increased as compared to rats
consuming the control diet, the fat content of the livers were not
analyzed. Therefore, the suggestion that the potential development of
steatosis in our rats remains purely speculatory.
In conclusion, administration of rhGH promoted early growth recovery of lean body mass by limiting fat deposition and increasing lean body mass. GH treatment increased serum IGF-I concentrations and total muscle protein during this period, suggesting that GH therapy supports skeletal muscle growth by enhancing protein accretion. The anabolic and repartitioning effects of GH were not further enhanced with extended treatment. Possible causes for this effect include the formation of anti-rhGH antibodies, GH receptor down-regulation or a physiologic limit on growth recovery which could not be overcome by GH. Consumption of a diet enriched in protein, lipid, vitamins and minerals promoted gains in liver and fat mass. While refeeding the enriched diet did not negatively impact lean tissue growth, it also produced no apparent benefit to the growth recovery of skeletal muscle. Further, the inhibitory effects of GH on fat deposition were counteracted by the enriched diet. Taken together, these results suggest that refeeding an energy-dense diet is counterproductive to the growth recovery process and may be detrimental to the functioning of the GH/IGF-I axis.
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FOOTNOTES |
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2 This work was supported by the National
Institutes of Health (HD-27558).
3 Abbreviations used: BSA, bovine serum albumin;
E, refed an enriched diet; GH, growth hormone; GHC, refed the control
diet plus human growth hormone; GHE, refed an enriched diet plus
recombinant/human growth hormone injections; IGF-I, insulin-like growth
factor-I; IGFBP, insulin-like growth factor binding proteins;
Mr, molecular retention; NG, normal growth age controls;
PEM, protein-energy malnutrition; rhGH, recombinant human growth
hormone; RC, refed the control diet; RIA, radioimmunoassay; SDS, sodium
dodecyl sulfate.
Manuscript received July 16, 1998. Initial review completed September 10, 1998. Revision accepted January 5, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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