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The Role of the Growth Hormone/Insulin-Like Growth Factor I Axis in Stimulation of Protein Synthesis in Skeletal Muscles Following Oral Refeeding

Department of Surgery and Internal Medicine
¹ , University of Göteborg, Sweden and Genentech, Inc., South San Francisco, California

Address all correspondence and requests for reprints to: Professor Kent Lundholm, Department of Surgery, Sahlgrenska University Hospital, S-413 45 Göteborg, Sweden.

	Abstract

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The mechanisms behind stimulation of protein synthesis in skeletal muscles following oral feeding are not well understood. Previous research has not confirmed that insulin is a major factor behind this stimulation. In the present study we have used genetically altered mice, with either a lack of GH secretion due to a mutational gene inactivation [GH (-/-) dwarf, DW/JOrlBom-dw] or mice with a homozygous site-specific insertion mutation in the insulin-like growth factor-1 gene [IGF-I (m/m)], leading to a deficient IGF-I production. These gene knock-outs were used in comparison to their normal wild types for evaluation of the role that the GH/IGF-I axis may have in activation of nutritionally induced stimulation of protein synthesis in skeletal muscles during oral refeeding. Weight stable adult C57Bl6 mice served as an additional normal control group. Protein synthesis was measured by a modified flooding dose technique with radioactive L-[¹⁴C-U]phenylalanine incorporation into acid precipitated muscle proteins.

Fractional protein synthesis in skeletal muscles after an overnight fast was comparable among C57Bl6 (0.076 ± 0.009%/h), wild-type IGF-I(+/+) (0.061 ± 0.008) and IGF-I(m/m) deficient mice (0.068 ± 0.006%/h), whereas GH(-/-) incompetent mice had a lower fractional synthesis rate compared with GH(+/+) competent mice (0.045 ± 0.006 vs. 0.068 ± 0.007, P < 0.05). Refeeding with standard chow diet stimulated protein synthesis in muscles by more than 60% in all animal groups. This response was independent of circulating GH, total IGF-I concentrations in blood, as well as up-regulation of locally produced IGF-I messenger RNA (mRNA) in skeletal muscles.

	Introduction

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IT IS NOT well understood how protein synthesis is regulated in response to oral feeding for maintenance of protein balance in skeletal muscles. The classical view is that insulin represents a key factor in activation of peptide formation as demonstrated in simplified experimental models such as perfused organs, isolated muscle preparations, and cell cultures exposed to medium deficient in growth factors (1). However, a clear cut stimulatory effect by exogenously provided insulin during in vivo experiments has been difficult to demonstrate (2, 3), although such effects have been reported (4). It has been speculated that insulin-like growth factor-1 (IGF-I) could be more effective than insulin to stimulate protein synthesis in skeletal muscles based on findings that provision of recombinant IGF-I to animals (5) and man (6, 7, 8, 9) improves muscle protein synthesis. The role of IGF-I for stimulation of protein synthesis during feeding is, however, unclear in light of the complex situation with circulating IGF-I and related plasma binding proteins (9, 10), as well as local production of IGF-I in various tissues (9, 11). This complex situation is illustrated by recent findings of up- and down-regulation of IGF-I messenger RNA (mRNA) in skeletal muscles following starvation and oral refeeding (5) as reported for work-induced skeletal muscle growth (12). The aim of the present study was therefore to evaluate to what extent the GH/IGF-I axis is a prerequisite for the normal activation of protein synthesis in response to feeding and whether up-regulation of IGF-I mRNA production in skeletal muscles is necessary for this stimulation.

	Materials and Methods

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Animals
Adult weight stable female C57Bl6 mice (Bomholt Gård, Ry, Denmark), were used as healthy controls. Weight stable IGF-I (m/m) deficient mice were kindly provided by Dr. Powell-Braxton (Genentech, Inc., San Francisco, CA). A targeting mutation of the IGF-I gene in this strain resulted in a site-specific insertional mutation that in the homozygous state reduced, but did not eliminate IGF-I expression in surviving animals. These animals are viable, fertile, and profoundly growth retarded (<60% of normal body weight) with muscle dystrophy due to an alternate splicing of the IGF-I mRNA (13). Therefore, they have significantly lower plasma IGF-I (30%) compared with wild sibling controls. Their fasting plasma insulin levels were 0.82 ± 0.22 ng/ml vs. 0.46 ± 0.13 ng/ml in the wild-type. Corresponding plasma GH levels were 7.6 ± 0.5 ng/ml vs. 1.6 ± 0.5 ng/ml, respectively (measured at Genentech, Inc.). Skeletal muscles in IGF-I (m/m) mice appeared less organized and had a decreased amount of myofibrils, whereas the degree of cellularity did not appear to be decreased. Attempts to further characterize surviving IGF-I deficient mice by measuring IGF-I protein in skeletal muscles tissue were inconclusive with intracellular concentrations below background levels due to contamination of plasma IGF-I in the extracellular space. IGF-I (m/m) mice will be referred to as IGF-I deficient mice (IGF-I -/-) because they have a partial production of IGF-I.

Genetic Snell dwarf mice (DW/JOrlBom-dw) were purchased from Bomholt Gård. These animals have a defect anterior pituitary gland due to a mutation on chromosome 6 (14). They lack GH production due to the absence of acidophilic cells, and consequently cannot produce GH at all, which has been demonstrated by others (15, 16) Dwarf Snell mice reach only one fourth to one third of the wild-type body size. They will be referred to as GH incompetent mice because GH production is undetectable both at the pituitary and plasma level (15, 16).

All animals were of similar age and kept on a diurnal 12 h light cycle and provided with standard rodent chow (containing 16.5% protein, 4.0% fat and 58% carbohydrates; ALAB AB, Stockholm, Sweden and Genentech, Inc.) and tap water ad libitum for at least 2 weeks before the experiments. Animals were starved 18 h before experimentation and were then refed for 3 h, when the animals were killed by cervical dislocation and blood and muscle specimens were taken for analyzes. Animals in the various groups ate comparable amounts of chow per unit body weight on refeeding confirmed by weighing the animals. Plasma measurements were focused on IGF-I and IGF-I binding proteins only for the following reasons: genetically deficient IGF-I mice were only available in limited numbers to us which made blood samples a limiting factor; isolated observations of GH in blood is of minimal physiological value; previous studies have demonstrated that dwarf Snell mice lack any production of GH (15, 16). Insulin was not evaluated in the present study because we have reported that postprandial stimulation of protein synthesis in skeletal muscles following oral feeding does not require insulin (5, 17). It was not possible to evaluate how muscle protein synthesis relates to increases in plasma cortisol after overnight starvation because completely adrenalectomized mice did not survive overnight starvation. Experiments in IGF-I-deficient mice were carried out by ourselves at Genentech, Inc., while the experiments on the other animal groups were performed in Sweden on a later occasion.

Analytical methods
Skeletal muscle protein synthesis was measured by the flooding dose technique (18, 19). A single-dose injection of L-[U-¹⁴C] phenylalanine (0.4 µCi/g) in 150 mmol/liter phenylalanine was provided ip 30 min before decapitation. Blood samples were collected in heparinized syringes by puncture of the heart and frozen until analysis. The specific radioactivity of phenylalanine and tyrosine in plasma was determined by HPLC chromatography on a reversed phase column (Waters Associates Liquid Chromatographic System, Millford, MA), (19, 20), with a variation coefficient less than 3%. Hind limb mixed muscles (mainly quadriceps) were rapidly excised and frozen until analysis. Muscle tissue was homogenized (10% wt/vol) and proteins were isolated by acid precipitation and lipid extraction as described elsewhere (19). Isolated muscle proteins were solubilized in Soluene (Packard Inc., Groeningen, The Netherlands), and the protein bound radioactivity was counted by liquid scintillation with a variation coefficient less than 5%. Analyzes of acid hydrolyzed muscle proteins in previous experiments have demonstrated that protein bound radioactivity represented phenylalalanine (80%) and tyrosine (20%) in skeletal muscles when ¹⁴C-phenylalanine (1 µCi/g) was provided ip (unpublished). This demonstrates that other pathways than the phenylalanine hydroxylase activity were negligible.

Standard determination of the specific radioactivity of phenylalanine in proteins, based on enzymatic conversion of phenylalanine into ß-phenetylamine following acid hydrolysis of muscle proteins, was not feasible in this model due to limited amount of skeletal muscle tissue in genetically defect individual mice. Also, determination of the specific radioactivity of protein bound phenylalanine isolated by HPLC was imprecise due to insufficiently low incorporated radioactivity above background in the muscle samples from mice subjected to saturating doses of phenylalanine containing ¹⁴C-phenylalanine. Therefore, compared with the original formula (19) a modified model was applied, where the fractional synthesis rate (ks, %/30 min) of muscle proteins was calculated as: ks = SBt/SAt; where SBt is the protein bound radioactivity (phenylalanine and tyrosine, dpm/µg) at 30 min, and SAt is the sum of the specific radioactivity of plasma phenylalanine and tyrosine (dpm/nmol) at 30 min (t) given that the kinetics of phenylalanine and tyrosine labeling in muscle are the same during the 30 min period, which has been demonstrated elsewhere (19). The plateau in phenylalanine and tyrosine specific radioactivities in plasma was reached within 3–5 min following ip injections of a flooding dose of phenylalanine in both overnight starved and freely fed mice, and the specific radioactivity of phenylalanine in plasma was only 16–18% higher than for tyrosine at 5, 10, and 30 min following ip injections. Thus, the fractional decline of plasma phenylalanine and tyrosine specific radioactivities were similar and strictly linear (19), giving the same rate of appearance of label in protein-bound phenylalanine and tyrosine in skeletal muscles (unpublished); i.e. fractional synthesis gave corresponding results (within the variation of coefficient for the entire procedure) when calculated from either phenylalanine or tyrosine evaluated in separate experiments. This method quantifies the overall complete synthesis of mixed muscle proteins (globular and myofibrillar) translated during the 30 min where incorporation takes place (18, 19).

The specific radioactivity in plasma, in complete equilibration with intracellular pools, was used to reflect the specific radioctivity in the true precursor pool for incorporation of amino acids into proteins. Previous experiments in our laboratory have demonstrated that the immediate precursor pool of amino acids (phenylalanine, tyrosine, leucine) for protein synthesis is not equal to a defined amino acid pool neither in plasma, acid soluble tissue amino acids, nor is it equal to the total pool of extractable tissue tRNA (19). Therefore, the precursor specific radioactivity determined in amino acids isolated from either plasma, whole tissue or tRNA remains only approximations of the true specific radioactivity of the precursor pool (19). Therefore, kinetics of protein synthesis in vivo represents only estimates of the true rate, but the flooding dose technique is presently the best choice considering cost, reproducibility, simplicity, and theoretical concept for completely unrestrained mice. The present modified flooding method to estimate changes in protein synthesis during starvation/refeeding has been qualitatively validated by comparing the results obtained by a totally independent method for measurements of translation initiation of protein synthesis (17).

Acid extracted total amounts of plasma IGF-I were determined by RIAs (Nicholls Institute, San Juan, Capistrano, CA) (21). The IGF-I RIA has been validated for rat samples with a variation coefficient of 6–7% (22, 23), although the assay was not specifically validated for mice. IGF-I sequence homology between mouse and rat is close to 97%, and the acid extraction procedure makes possible influences of plasma binding proteins nonsignificant as confirmed for other murine samples (24).

Solution hybridization assay
Concentration of IGF-I mRNA was determined by hybridization in solution from muscle extracts from starved, refed, and freely fed mice. Antisense IGF-I [³⁵S]-UTP labeled RNA was synthesized from an EcoRI linearized pSP64 plasmid carrying a 153 bp mouse genomic subclone corresponding to exon 3, by analogy to the human IGF-I gene and thus recognizing all reported variants of IGF-I mRNAs (26). This probe gives a 147 bp protected band in RNase protection assays (27). Total nucleic acids (TNA) for the solution hybridization assay were prepared by homogenizing tissues with a polytrone in a buffer containing 1% (weight/vol) SDS, 20 mM Tris-HCl (pH 7.5), and 4 mM EDTA. The homogenized tissue was digested by overnight proteinase-K treatment and the TNA was prepared by subsequent phenol-chloroform extraction according to Durnam and Palmiter (28). The RNase protection solution hybridization assay was carried out according to protocol described by Mathews et al. (26). Protected hybrids were precipitated with TCA, collected on glass-fiber filters, and counted in a scintillation counter. The signal was compared with a standard curve, obtained by hybridization to known amounts of IGF-I mRNA. The results were correlated to the DNA content as measured according to the method of Labarca and Paigen (29). The coefficient of variation is less than 20% by this method (30).

Statistics
Results are presented as mean ± SEM. Statistical comparisons among several groups were performed by one-factor ANOVA. P < 0.05 was considered statistically significant.

	Results

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The mean body weight was 21.5 ± 0.6 g in normal C57Bl6 control mice before starvation/refeeding experiments started; 21.3 ± 0.5 g in IGF-I (+/+) mice; 15.7 ± 0.5 g in IGF-I(-/-) mice; 21.5 ± 0.7 g in GH(+/+) mice and 7.5 ± 0.9 g in GH(-/-) incompetent mice. The muscle mass among these animals was essentially proportional to body weight assessed by weighing muscles following a complete dissection in representative animals.

Protein synthesis in skeletal muscles after an overnight fast was comparable among C57Bl6, wild-type GH (+/+) wild-type IGF-I(+/+) and IGF-I (-/-) deficient mice, whereas GH (-/-) incompetent mice had a lower fractional synthesis rate (Table 1). Refeeding with standard chow stimulated the fractional protein synthesis by 60% or more in all animal groups independently of genetic competence for either GH or IGF-I (Table 1).

	Discussion

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It appeared in the fasted state that muscle protein synthesis was lower in the GH -/- than in GH +/+ mice. This indicates that either circulating GH or IGF-I controls the total capacity for protein synthesis in muscles. However, the fractional synthesis rate, which is a relevant measure in studies on acute activation during refeeding, was not related to either GH incompetence or IGF-I deficiency. These results should be seen in the light of previous findings that provision of recombinant GH and IGF-I improved protein synthesis and protein balance in skeletal muscles in both animal (5) and human models (6, 7, 8, 35). Therefore, such therapeutic effects may not be entirely reflect physiological effects by these hormones. In this context, it is important to distinguish between metabolic effects exerted by GH and IGF-I for development of muscles and catch up growth (36); effects that may prevent involution of matured skeletal muscles (37) vs. the physiologic effect to stimulate resynthesis of diurnally turned over proteins (38).

The evidence that the IGF-I knockouts are deficient in IGF-I is the low concentration of circulating IGF-I, which was 30% of the wild-type. However, the mRNA expression in skeletal muscles was not different from wild-type, which suggests that surviving pups were selected from completely IGF-I deficient pups with respect to IGF-I mRNA expression in skeletal muscles, which is probably necessary to allow muscle development above the lethal threshold. Experiments were performed to demonstrate the presence of IGF-I protein within skeletal muscle cells from IGF-I deficient mice, but discrimination above background levels from circulating IGF-I was not resolved. Therefore, our results do not address the question as to where IGF-I is expressed in skeletal muscle tissues. Considering the possibility that IGF-I is expressed in muscle cells in surviving pups, our results may be consistent with a promoter that fails to respond to increased substrate, but has a relatively normal constitutive expression. A definite answer to these questions must await a more precise genetic definition of the surviving IGF-I (-/-) mice than is presently available from previous descriptions of the homozygotes (13).

It is well known that deficiency of either GH or IGF-I leads to both functional and compositional changes in skeletal muscles (39). Altered fiber type composition may partly explain the unexpected results of intact stimulation of protein synthesis in GH incompetent and IGF-I deficient mice. It is well known that muscles with a dominant white fiber composition are more vulnerable to undernutrition compared with muscles with predominantly red fibers (39). Hypothetically, lack of GH and IGF-I deficiency could lead to a predominant loss of fast white fibers, leaving the slow, fatigue-resistant, and nutritionally more resistant red fibers (40). If so, red fibers could have maintained their ability to respond with normal stimulation of protein synthesis during feeding despite exposure to overnight fast. However, previous reports on muscle fiber type composition have rather documented a relative loss of red fibers during GH deficiency, sparing a relatively larger number of the nutritionally vulnerable white fibers (39). This question is now under investigation by determination of fiber type composition in GH and IGF deficient mice. Further research is needed to identify the mechanisms behind food-induced stimulation of peptide formation because neither insulin (2, 17), GH or IGF-I alone (41), nor in combination seem to explain the entire phenomenon. Nutrients, particularly the effect of amino acids (41, 42), perhaps in combination with small but permissive changes in tissue IGF-I expression, is at present the most likely explanation. In line with this concept, recent evidence has demonstrated that muscle protein synthesis after prolonged exercise is associated with increased levels of muscle IGF-I protein, whereas circulating IGF-I was not affected (43).

Received March 6, 1998.

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