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Institut National de la Recherche Agronomique, Station de Recherches Porcines, 35590 Saint Gilles, France
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGMENTS
FOOTNOTES
LITERATURE CITED
ABSTRACT 
The present study was conducted to determine the influence of food restriction on growth hormone receptor (GHR) in porcine skeletal muscle (longissimus dorsi and trapezius) and liver in relationship to plasma growth hormone binding protein (GHBP). At 76 d of age, pigs were allotted to one of three groups. In Group R100kg-196d, pigs were fed 70% of control food intake and slaughtered at 100 kg. Control pigs had free access to food and were slaughtered either at the same weight (Group C100kg-161d) or at the same age (Group C130kg-196d) as R100kg-196d pigs. Plasma insulin-like growth factor-I concentrations tended to be lower in food-restricted pigs than in control pigs at 40 kg (P < 0.1) and 70 kg (P < 0.05). At slaughter, there were no significant differences among the three groups. In liver, 125I-labeled bovine GH specific binding was higher (P < 0.05) in food-restricted pigs than in control pigs, and GHR mRNA level was higher (P < 0.05) in food-restricted pigs than in C100kg-161d pigs. In trapezius, 125I-labeled bovine GH specific binding was lower (P < 0.05) in food-restricted pigs than in C130kg-196d pigs, and the level of GHR mRNA was higher (P < 0.01) in food-restricted pigs than in control pigs. The levels of GHR in longissimus dorsi were not affected by food restriction. The level of plasma GHBP was lower (P < 0.05) in food-restricted pigs than in C130kg-196d pigs. These data indicate that nutritional status regulates GHR in a tissue-specific manner and that there is no simple relationship between plasma GHBP and hepatic GHR in pigs.
KEY WORDS: growth · growth hormone receptor · food restriction · muscle · pigsINTRODUCTION
Growth hormone (GH)6 and insulin-like growth factor-I (IGF-I) play a major role in growth regulation and direct substrate availability toward lean body mass. In pigs, GH administration markedly increases muscle growth (Campbell et al. 1989
, Chung et al. 1985). The mechanisms involved in GH action remain unclear. Because the first step in GH action is binding to a specific cell surface receptor, changes in GH receptor (GHR) levels may provide some insight into the importance of GH actions. Numerous studies indicate that nutritional status influences the GH/IGF-I axis by affecting hormonal and/or receptor level. Food restriction and deprivation are known to decrease plasma IGF-I concentrations in several species, including pigs (Buonomo and Baile 1991, Dauncey et al. 1993 and 1994). A 50-66% restriction of food intake decreased hepatic GH binding in steers (Breier et al. 1988) and GHR mRNA levels in young pigs (Dauncey et al. 1994). Similarly 48-72 h of food deprivation decreased both GH binding (Baxter et al. 1981, Maes et al. 1983) and GHR mRNA levels (Bornfeldt et al. 1989, Straus and Takemoto 1990) in rat liver. The regulation of GHR has been studied more in liver than in other tissues and especially in skeletal muscle, which is a potentially important target tissue for GH. Indeed, the presence of GH binding sites has been demonstrated from the last third of gestation to the adult stage in pig skeletal muscle (Schnoebelen-Combes et al. 1996). However, it is not known whether the change in growth induced by food restriction is associated with change in GH binding level in that tissue. To our knowledge, there is only a recent study that demonstrates that a 50% restriction of food intake increases the level of GHR mRNA in skeletal muscle (Dauncey et al. 1994).
In addition to GHR in tissues, a plasma GH binding protein (GHBP) has been identified in several species, including pigs (Davis et al. 1992). It is believed that GHBP results from a proteolytic cleavage of GHR in pigs, as in humans and rabbit. Serum GHBP measurement may be useful as a simple indicator of hepatic GHR status because liver is the major source of GHBP mRNA in rats (Tiong and Herington 1991). To our knowledge, only one study reports a positive correlation between these two variables in food-restricted rats (Villares et al. 1994). It remains to be determined whether plasma GHBP and hepatic GHR levels also vary in a parallel manner in pigs.
70°C. Results of densitometric analyses are shown in Figure 6.
In all studies performed to date, extreme changes in nutritional status for a short time were investigated. The present study was undertaken to examine the influence of 30% food intake restriction on GHR levels in two skeletal muscles (one white and one red) compared with liver, considering GHR mRNA and also GH-binding sites. Plasma IGF-I and GHBP levels were also determined. A moderate intensity but long food restriction was chosen because the growth rates obtained are close to those observed in pig production.
MATERIALS AND METHODS
20°C; tissues for mRNA analysis were frozen in liquid nitrogen and stored at 70°C until assayed.
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Table 1. Animal characteristics and effect of a 30% restriction of food intake on growth performance of pigs1,2 |
). The assay was performed using recombinant IGF-I (human recombinant IGF-I, receptor quality, Mallinckrodt, St Louis, MO) as a tracer with a specific radio-activity of 5.3 MBq/µg and as a standard. A polyclonal antibody raised in rabbit (Claus et al. 1992) was used at a final dilution of 1:40,000. All the samples were analyzed within a single assay. The intraassay CV for a plasma sample containing 5.4 and 63.6 nmol/L of IGF-I were 8.8 and 12.9%, respectively. As recommended by Bang et al. (1995), a validation of the assay was performed (Louveau and Bonneau 1996). The data indicated that although acid-ethanol extraction lead to IGF-I levels that differed from those obtained for glycyl-glycine/G-50 extracted plasma, there was a close relationship (r = 0.97, P < 0.001) between the values obtained by these two methods.
Membrane preparation.
Microsomal membranes were prepared as previously described (Meserole and Etherton 1984). In brief, frozen tissues were cut into small pieces and homogenized (1:5, wt/v) in ice-cold 50 mmol/L Tris-HCl buffer (pH 7.4) containing 250 mmol/L sucrose, 1 mmol/L EDTA and 1 mmol/L phenylmethylsulfonyl fluoride, using a Polytron homogenizer. After centrifugation, the final pellet was resuspended in 50 mmol/L Tris-HCl buffer (pH 7.4), and the microsomal membrane preparations were stored at 20°C until binding studies were performed. Protein concentration was estimated using the bicinchoninic acid assay (Pierce, Rockford, IL) with bovine serum albumin as a standard.
Growth hormone binding assays.
All GH binding assays were performed in triplicate as previously described (Schnoebelen-Combes et al. 1996). Briefly, microsomal membrane proteins (400 µg for liver and 500 µg/tube for muscle) were incubated with 125I-labeled bovine GH (bGH, 0.6 kBq/tube or 0.6 µg/L) in 25 mmol/L Tris-HCl (pH 7.4) containing 10 mmol/L CaCl2 , 5 g/L bovine serum albumin, 0.2 g/L NaN3 (binding buffer). Nonspecific binding was determined by addition of an excess of unlabeled bGH (250 ng/tube). After a 48-h incubation at room temperature, 2 mL of ice-cold binding buffer was added to stop the reaction. Bound hormone and free hormone were separated by centrifugation at 4000 × g for 20 min at 4°C.
Growth hormone binding protein determination.
The GHBP level was determined using HPLC gel filtration as previously described (Tar et al. 1990). After filtration through a 0.45-µm Millipore minifilter, plasma (100 µL) was incubated overnight at 4°C with 100 µL of buffer [0.1 mol/L KH2PO4 (pH 7.0), 1 g/L bovine serum albumin] containing 125I-labeled human GH (hGH Serono Laboratories, Genova, Switzerland) with a specific radioactivity of 3.7-5.6 MBq/µg. A parallel incubation was performed in the presence of an excess of unlabeled hGH (2 µg) to evaluate nonspecific binding. The entire incubation mixture was placed onto a HPLC Protein Pak 300sw column (Waters, Milford, MA; 0.75 × 30 cm). Elution was performed using a degassed buffer (0.1 mol/L Na2SO4 , 0.1 mol/L KH2PO4 , pH 7.0). The binding of GH is expressed as the radioactivity in the GHBP peak divided by the total radioactivity (GH and the GHBP peaks). The somatogenic specificity of hGH binding to plasma GHBP in pigs was previously demonstrated (Schnoebelen-Combes et al. 1996).
RNA isolation and RNAse protection assays.
Total RNA was isolated from tissues using the guanidium thiocyanate method (Chomczynski and Sacchi 1987). Quantity and quality of isolated total RNA were evaluated spectrophotometrically and confirmed with horizontal gel electrophoresis. The abundance of GHR mRNA was quantified using a sensitive solution hybridization-RNAse protection assay. Porcine GHR cDNA in pGEM 3Zf- (Promega, Madison, WI) was kindly provided by Terry D. Etherton (Penn State University, University Park, PA). To generate antisense riboprobe, the plasmid was linearized by digestion with Hind II and transcribed with Promega kit using T7 polymerase (Promega) in the presence of [
32P]CTP (Du Pont de Nemours, Les Ulis, France). The RNA probe consisted of 375 nucleotides, of which 368 were the complementary sequence +654 to +1021 of the GHR. To check for possible differences in quantification and/or loading, samples were also assayed for 18S RNA using human 18S cDNA (pT7 RNA 18S) obtained from Ambion (Austin, TX). To obtain a low specific activity probe, the linearized plasmid was transcribed with RiboMax large scale system kit using T7 polymerase (Promega). The 18S RNA probe consisted of 116 nucleotides and protected an RNA doublet of 80 and 70 bp.
70°C. Unhybridized probe digested or undigested by RNAses was used as control. The relative intensities of the protected bands were quantified using Densylab software (Microvision Instruments, Evry, France). The data were not normalized to the abundance of 18S RNA. However, when the 18S RNA was barely detectable because of loss of the sample, the value of GHR was not considered.
RESULTS
Plasma insulin-like growth factor-I concentrations. Plasma IGF-I concentrations (Fig. 2) were lower in food-restricted pigs (R100kg-196d) than in control pigs (C100kg-161d and C130kg-196d) at 40 kg (P < 0.1) and 70 kg (P < 0.05). At slaughter, there were no significant differences among the three groups.
Growth factor binding in liver, skeletal muscle and plasma. In liver, 125I-labeled bGH specific binding was higher (P < 0.05) in restricted pigs (R100kg-196d) than in control pigs (C100kg-161d and C130kg-196d) (Fig. 3). In TR, 125I-labeled bGH specific binding was lower (P < 0.05) in food-restricted pigs (R100kg-196d) than in C130kg-196d pigs (Fig. 3). The effect of food restriction on 125I-labeled bGH specific binding was not significant in LD (Fig. 3). 125I-labeled hGH binding to plasma GHBP was higher (P < 0.05) in C130kg-196d pigs than in R100kg-196d or C100kg-161d pigs (Fig. 4). No difference was observed between C100kg-161d and R100kg-196d pigs.
Expression of growth hormone receptor mRNA in liver and skeletal muscle. Growth hormone receptor mRNA expression was assessed by RNAse protection assay (Fig. 5). In liver, the level of GHR mRNA was higher (P < 0.05) in restricted pigs (R100kg-196d) than in C100kg-161d pigs (Fig. 6). In TR, GHR mRNA level was higher (P < 0.01) in restricted pigs (R100kg-196d) than in control pigs (C100kg-161d and C130kg-196d) (Fig. 6). In LD, there was a wide individual variation, and GHR mRNA level did not differ significantly among the three groups (Fig. 6).
DISCUSSION
, Dauncey et al. 1993 and 1994). However, differences in plasma IGF-I concentrations between food-restricted and control pigs were low. At slaughter, the lack of a significant difference among the groups may be related to a greater variability among pigs. The observation that food restriction induced an elevation in both GH binding and GHR mRNA levels in liver is not consistent with data reported previously. A reduction of GH binding in liver has been observed in rats (Villares et al. 1994) and cattle (Breier et al. 1988). It has also been reported that food restriction decreased GHR mRNA levels in the liver of pigs (Dauncey et al. 1994) but had no effect on GHR mRNA levels in the liver of rats (Villares et al. 1994). In the present study, food restriction induced a slight reduction in GH binding associated with a slight increase in GHR mRNA level in TR muscle. Food restriction had no significant effect on GH binding and GHR mRNA levels in LD muscle, whereas an increase in the GHR mRNA content in that tissue was observed previously (Dauncey et al. 1994). To our knowledge, there are no other available data in skeletal muscle.
. The importance of age in IGF-I and GHR regulation has been suggested in other situations. It has been observed that GH-induced increase in GH binding to liver is much higher in 30-d-old pigs (81%) than in 120-d-old pigs (15%) (Ambler et al. 1992). Similarly, it has been shown that the effect of a dietary protein restriction on plasma IGF-I and hepatic GHR is age-dependent in rats (Fliesen et al. 1989). Protein restriction in 3-wk-old rats induced a decrease in plasma IGF-I (66%) and in hepatic GH binding. These effects were progressively attenuated with increasing age. At 12 wk of age, there was a slight reduction in plasma IGF-I (17%) and a slight increase in hepatic GH binding. Our findings are in accordance with these latter observations and support the hypothesis that the influence of food restriction on the GH/IGF-I axis is also age dependent. In contrast to results for several other studies involving development (Breier et al. 1989), GH administration (Chung and Etherton 1986), 50% restriction of food intake (Dauncey et al. 1994) in pigs or food deprivation in rats (Maes et al. 1983), hepatic GHR level and plasma IGF-I concentration did not vary in a parallel manner in the current study. This lack of parallelism is consistent with the hypothesis of a post-receptor defect as suggested during protein restriction in rats (Fliesen et al. 1989, Maiter et al. 1989, Thissen et al. 1990).
). Our results also demonstrate that the tissue specificity is expressed at the GH binding level. Indeed, although a clear increase in GH binding level was observed in liver, there was either no change or a slight reduction in GH binding in skeletal muscle of food-restricted pigs. Our results also suggest that LD and TR muscles may not respond similarly to food restriction. The differences are too small to draw any conclusions. Therefore, further studies are needed to determine whether the effect of food restriction on GHR is muscle specific.
). To our knowledge, the effect of food restriction on plasma GHBP levels has been examined only in rats, and the results of the two studies are controversial. Although a 10-20% restriction of food intake for 10 d increased plasma GHBP level (Tönshoff et al. 1994), a 50% restriction of food intake for 9 d induced a decrease in plasma GHBP level (Villares et al. 1994). As discussed above, these studies suggest that plasma GHBP level might change with the extent of food restriction. It is believed that measurement of GHBP plasma level may be an indicator of hepatic GHR level. To our knowledge, there is only one study that has shown a positive correlation between plasma GHBP and GH binding to liver under food restriction in rats (Villares et al. 1994). In the present study, food restriction did not influence plasma GHBP and hepatic GHR levels in a similar manner. Taken together, these data indicate that there is no simple relationship between these two variables and support the hypothesis that other tissues such as skeletal muscle may contribute to the generation of GHBP (Schnoebelen-Combes et al. 1996).
ACKNOWLEDGMENTS
FOOTNOTES
Manuscript received 7 April 1997. Initial reviews completed 16 May 1997. Revision accepted 30 June 1997.
LITERATURE CITED
on hepatic high and low-affinity somatotrophic binding sites.
J. Endocrinol.
1988;
116:169-177
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