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Endocrinology Vol. 146, No. 4 1772-1779
Copyright © 2005 by The Endocrine Society

Intact Insulin and Insulin-Like Growth Factor-I Receptor Signaling Is Required for Growth Hormone Effects on Skeletal Muscle Growth and Function in Vivo

Hyunsook Kim, Elisabeth Barton, Naser Muja, Shoshana Yakar, Patricia Pennisi and Derek LeRoith

Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases (H.K., S.Y., P.P., D.L.), and National Institute of Neurological Disorders and Stroke (N.M.), National Institutes of Health, Bethesda, Maryland 20892; and Anatomy and Cell Biology (E.B.), University of Pennsylvania School of Dental Medicine, Philadelphia, Pennsylvania 19104

Address all correspondence and requests for reprints to: Derek LeRoith, Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 9000 Rockville Pike, Building 10, Room 8D12, Bethesda, Maryland 20892-1758. E-mail: Derek{at}helix.nih.gov.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
GH and IGF-I are potent regulators of muscle growth and function. Although IGF-I is known to mediate many of the effects of GH, it is not yet clear whether all effects of GH are completely dependent on the IGF-I system. To evaluate the biological effects of the GH/IGF-I axis on muscle growth, we administrated recombinant human GH to mice, which lack IGF-I function specifically in skeletal muscle, due to the overexpression of a dominant-negative IGF-I receptor in this tissue (MKR mice). GH treatment significantly increased the levels of hepatic IGF-I mRNA and serum IGF-I levels in both wild-type (WT) and MKR mice. These GH-induced effects were paralleled by increases in body weight and in the weights of most GH-responsive organs in both groups of mice. Interestingly, unlike WT mice, GH treatment had no effect on skeletal muscle weight in MKR mice. GH treatment failed to reverse the impaired muscle function in MKR mice. Furthermore, MKR mice exhibited no effects of GH on the cross-sectional area of myofibers and the proliferation of satellite cells. Taken together, these data suggest that the in vivo effects of GH on muscle mass and strength are primarily mediated by activation of the IGF-I receptor.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ACCORDING TO THE somatomedin hypothesis (1), the effects of GH on somatic growth are mediated by the endocrine form of IGF-I, which is mostly derived from the liver and secreted into the circulation. GH-induced somatic growth is also mediated by local production of IGF-I, which acts in an autocrine/paracrine manner (2). The administration of GH restores muscle mass and function in hypophysectomized animals and GH-deficient patients (3, 4). The effects of GH on skeletal muscle are thought to be mediated by IGF-I. Several studies have shown that GH treatment increases IGF-I mRNA in skeletal muscle in animals as well as in cell line (C2C12 myoblasts) (5, 6, 7). However, IGF-I-mediated effects of GH on skeletal muscle in vivo have not been precisely elucidated.

IGF-I plays a critical role in the formation, maintenance, and regeneration of skeletal muscle. IGF-I receptor knockout mice exhibited significant muscle hypoplasia and died shortly after birth (8). On the other hand, overexpression of IGF-I in muscle using skeletal {alpha}-actin promoter results in muscle hypertrophy (9). Viral overexpression of IGF-I in muscle via a myosin light chain promoter increased muscle mass and strength in the extensor digitorum longus (EDL) of young adult mice (10). Expression of endogenous IGF-I was associated with muscle regeneration in hypophysectomized rats (11). IGF-I has been also shown to induce proliferation and differentiation of myoblasts. The MAPK pathway is involved in IGF-I-induced cellular proliferation, whereas the phosphoinositide 3'-kinase pathway mediates differentiation (12).

Multiple steps are involved in the development of skeletal muscle in vertebrates. After pluripotent mesodermal stem cells become committed to the myoblast lineage, myoblasts exit the cell cycle and express muscle-specific genes that lead to the fusion of myoblasts into multinucleated differentiated myotubes. Proliferating myoblasts express MyoD and Myf5, which are muscle-specific basic helix-loop-helix transcription factors. Myoblasts withdrawn from cell cycle express other basic helix-loop-helix regulatory transcription factors, such as myogenin and muscle regulatory factor 4, and enter the terminal differentiation stage (13). Adult skeletal muscle includes two distinct types of muscle progenitor cells, satellite cells and muscle-derived side population cells that have been suggested as the progenitors of satellite cells (14). Satellite cells, located between the sarcolemma and the basal lamina of a muscle fiber, contribute to adult muscle growth by providing additional nuclei. During adult muscle growth, satellite cells differentiate into myoblasts and become incorporated into myofibers (15).

Transgenic MKR mice overexpress a dominant-negative IGF-I receptor (DN-IGF-IR) specifically in skeletal muscle under the muscle creatine kinase promoter (16). We previously showed that these mice exhibit significantly lower levels of muscle mass and hypoplasia from birth to 3 wk of age (17). In adulthood, the wet weight of hindlimb muscles was an average of 10% less in MKR mice compared with WT mice, whereas 30% less was observed from birth to 3 wk of age. This increased muscle mass in adulthood was correlated with compensatory hyperplasia (17). This study was aimed to determine whether the in vivo effects of GH on skeletal muscle growth are mediated through IGF-I receptor (and the insulin receptor because the DN-IGF-IR also forms hybrids with the endogenous insulin receptor). MKR mice were treated with recombinant human (rh) GH for 4 wk. Here we show that MKR mice failed to exhibit the GH-induced increases in muscle mass and function observed in WT mice. Unlike WT mice, MKR mice did not exhibit hypertrophy of muscle fibers and proliferation of satellite cells in response to GH treatment. GH treatment increased myogenin and myoD levels in WT mice but had no effect on the levels of these transcription factors in the skeletal muscle of MKR mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
The generation and genotyping of MKR mice have been previously described (16). Mice were maintained on a 12-h light, 12-h dark cycle and provided with the NIH-07 rodent food diet (Zeigler Brothers, Gardens, PA). Three-week-old male wild-type (WT, FVB/N background) and MKR (homozygous, FVB/N background) mice were injected ip with 3 mg/kg·d of rhGH (Genentech Inc., South San Francisco, CA) or with an equivalent volume of sterile saline twice daily (0800 and 1730 h) for 4 wk. At the end of the study, mice were anesthetized using Avertin (0.5 g of tribromoethanol and 0.25 g of tert-amyl alcohol in 39.5 ml of water; 0.02 ml/g of body weight) and blood was collected from the retro-orbital vein in the fed state between 1000 and 1200 h. The body length (from nose to anus) of anesthetized mice was measured. Liver, kidneys, heart, lungs, spleen, and hindlimb skeletal muscles were dissected. Organs were weighed and immediately frozen in liquid nitrogen for RNA and protein extraction. All experiments were performed according to guidelines of the National Institutes of Health and were approved by the Animal Care and Use Committee of the National Institute of Diabetes and Digestive and Kidney Diseases.

Ribonuclease (RNase) protection assay
Total RNA was extracted using TRIzol reagent (Invitrogen Life Technologies, Rockville, MD) according to the manufacturer’s instructions. RNase protection assays were then performed to measure the levels of IGF-I and GH receptor mRNA using riboprobes derived from mouse IGF-I exon 4, GH receptor exon 4, and ß-actin (Ambion, Inc., Austin, TX), as described previously (18, 19, 20). The levels of mRNA in protected bands were quantified by phosphorimager (BAS-1800 II, Fujifilm, Tokyo, Japan) and were normalized to ß-actin mRNA levels.

Serum IGF-I levels
Serum concentrations of IGF-I were determined by RIA, as described previously (21).

Functional measurement of muscle
Three-week-old WT and MKR mice were treated with GH or vehicle for 4 wk and isolated whole muscle mechanics were performed on the EDL muscles, as previously described (10). Briefly, the animals were anesthetized with Avertin as described above and EDL muscles were removed, retaining the tendons intact. The tendons were attached to a rigid post and to an isometric force transducer in a bath of Ringers solution equilibrated with 95% O2/5% CO2. The optimum length (Lo) of the treated muscle was determined by twitch force from supramaximal stimulation. Maximal tetanic force was determined at 120 Hz by a 500-msec pulse delivered via two parallel platinum plate electrodes. At the completion of the mechanical measurements, the muscles were removed from the bath, blotted, and weighed. The muscles were then surrounded in embedding medium (Tissue-Tek, Torrance, CA) and rapidly frozen in liquid nitrogen-cooled isopentane. Muscles were stored at –80 C for subsequent analysis. The cross-sectional area (CSA) was determined using the following formula (22): muscle mass (mg)/ [1.06 mg/mm3 x L/Lo x Lo (mm)], where L is fiber length, and L/Lo is 0.45 for the EDL. Specific force was calculated from the maximal tetanic force divided by the CSA of each muscle.

Muscle morphology
Quadriceps (quad) muscles were removed from anesthetized mice, fixed in 4% paraformaldehyde, and embedded in paraffin for the morphological study. Three consecutive cross-sections (5 µm) of muscle tissues from four to five animals in each group were used for hematoxylin and eosin staining. A video camera attached to a Zeiss Axiovert S100 TV microscope (Carl Zeiss Microimaging Inc., Thornwood, NY) at x20 magnification was used to analyze three different areas within each section. Adobe Photoshop version 5.0 software (Adobe Systems Inc., San Jose, CA) was used to determine the size of each individual myofiber, the number of myofibers per given area, and number of nuclei per myofiber.

Immunohistochemistry
Mice received daily ip injections of bromo-deoxyuridine (BrdU) (Sigma Chemical Inc., St. Louis, MO) (50 mg/kg of body weight) for the last 15 d of the 4-wk treatment period. Cross-sections (5 µm) of quad muscle were stained with BrdU using a cell proliferation kit (Amersham Biosciences, Piscataway, NJ), as described previously (17). BrdU-stained cells were identified with a x20 objective (Leica Microsystems Nussloch GmGH, Nussloch, Germany) and were counted in three areas of each section from three animals in each group. The percentage of BrdU-positive cells was expressed as the number of cells containing BrdU-stained nuclei relative to total number of cells in each area.

Northern blot analysis
Total RNA (30 µg) was subjected to Northern blot analysis as described previously (23). cDNA probes were prepared from myogenin and myoD plasmid DNAs, generously provided by P. Rotwein, Oregon Health Sciences University (Portland, OR).

Western blot analysis
Protein (150 µg) extracted from livers was used for Western blot analysis as previously described (23), using phospho (p)-signal transducer and activator of transduction (STAT5) antibody (Upstate, Charlottesville, VA) and STAT5 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Statistical analysis
Data are expressed as mean ± SE. Statistical differences were determined using a one-factor ANOVA followed by a t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Hepatic IGF-I and GH receptor mRNA and serum IGF-I levels were increased in both WT and MKR mice after GH administration
The liver is a major target for GH action and the production of hepatic IGF-I in response to GH is the primary source of endocrine IGF-I levels. To evaluate the effect of exogenously administrated GH, we measured hepatic IGF-I and GH receptor mRNA levels, serum IGF-I levels, and STAT5 phosphorylation. GH treatment significantly increased the levels of hepatic IGF-I and GH receptor mRNA in both WT and MKR mice (Fig. 1AGo). GH treatment had no effect on IGF-I receptor or GH receptor mRNA levels in skeletal muscle of either WT or MKR mice (Fig. 1BGo). Indeed, GH receptor levels were similar in MKR mice compared with controls. GH treatment significantly increased serum IGF-I levels in both groups (WT, 350 ± 21.6 to 501 ± 11.9 ng/ml; MKR, 357 ± 12.3 to 463 ± 12.4 ng/ml) (Fig. 1CGo). The phosphorylation of STAT5, a downstream target of the GH-signaling cascade, was robustly induced in the liver 1 h after GH treatment, and this persisted for at least 3 h in WT mice (Fig. 1DGo), showing the effectiveness of the GH treatment regimen.



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  		FIG. 1. Effect of GH on expression of hepatic IGF-I and GH receptor mRNA, muscle IGF-I receptor and GH receptor mRNA, serum IGF-I levels, and phosphorylation of STAT5. Liver, muscle, and serum were collected from WT and MKR mice treated with either vehicle ({blacksquare}) or rhGH ({square}) for 4 wk, as described in Materials and Methods. Levels of hepatic IGF-I and GH receptor mRNA (A) and levels of muscle IGF-I receptor and GH receptor (B) were measured by RNase protection assay and are expressed relative to ß-actin mRNA levels. C, Levels of serum IGF-I. D, WT mice were injected with either vehicle or rhGH and livers were removed 1 h and 3 h after injection. Samples were subjected to p-STAT5 and STAT5 Western blot analysis; representative blots are shown. Data are expressed as mean ± SE (n = 6–7 in each group). *, P < 0.05 vs. vehicle-treated mice.

 
These findings suggest that the action of GH in the liver is the primary mechanism by which circulating IGF-I levels are increased in response to GH treatment in both WT and MKR mice.

Body weight and length were increased in both WT and MKR mice after GH administration
Four weeks of treatment with GH, from the age of 3–7 wk, significantly increased total body weight in both WT and MKR mice by 23.8% and 17.7%, respectively (Fig. 2AGo). Additionally, both WT and MKR mice exhibited a significant increase in body length after GH treatment (WT, from 8.93 ± 0.14 to 9.8 ± 0.14 cm; MKR, from 8.72 ± 0.09 to 9.22 ± 0.15 cm) (Fig. 2BGo). Liver weight was significantly increased in response to GH treatment in WT and MKR mice, by 27.6% and 21.8%, respectively (Fig. 2CGo). There were significant increases in the weights of other GH-responsive organs, including kidney, lung, spleen, and heart in both WT and MKR mice in response to GH treatment (Table 1Go).



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  		FIG. 2. Effect of GH on growth in WT and MKR mice. Three-week-old MKR and WT mice were injected with either vehicle or rhGH twice daily for 4 wk. A, Body weight was measured weekly. B, Body length was measured from nose to anus after 4 wk of GH treatment with either vehicle ({blacksquare}) or rhGH ({square}). C, Liver weight was measured after 4 wk of treatment with either vehicle ({blacksquare}) or rhGH ({square}). Results are expressed as mean ± SE (n = 6–8 in each group). *, P < 0.05 vs. vehicle-treated mice.

 

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  		TABLE 1. The effect of GH on organ weight in WT and MKR mice

 
GH did not increase muscle mass in MKR mice
At 7 wk of age, the weight of three muscle groups was lower in MKR mice compared with WT mice (Fig. 3AGo). GH induced a significant increase in the wet weight of the EDL, combined gastrocneminus and soleus, and quad muscles in WT mice by 17.7%, 17.2%, and 22.1%, respectively, (P < 0.05), compared with the control group (Fig. 3AGo). However, GH treatment had no effect on the weight of these muscles in MKR mice (Fig. 3AGo). The level of IGF-I mRNA was increased by 15% (P = 0.07) in WT mice after GH treatment but not in MKR mice (Fig. 3BGo). The phosphorylation of STAT5 was significantly (P < 0.05) enhanced in muscle of both WT and MKR mice after GH treatment (Fig. 3CGo). However, the stimulation of p-STAT5 in MKR mice was significantly less than that seen in WT mice (Fig. 3CGo). The data further suggest that the increased total body weight of MKR mice after GH treatment is associated with increases in the weights of GH-responsive organs other than skeletal muscle.



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  		FIG. 3. Effect of GH on EDL, combined gastrocneminus (GC) plus soleus (SO), and quad muscle weight (A), on levels of IGF-I mRNA (B) and on STAT5 phosphorylation levels in hindlimb muscle (C). Muscle weight was measured after 4 wk of vehicle (squlf]) or rhGH ({square}) treatment in WT and MKR mice. Hindlimb muscles were removed and subjected to RNase protection assay and Western Blot as described in Materials and Methods. The levels of mRNA were normalized to ß-actin mRNA levels. Levels of p-STAT5 were normalized to total levels of STAT5 protein. Results are expressed as mean ± SE (n = 6–8 in each group). *, P < 0.05 vs. vehicle-treated mice. #, P < 0.05 vs. WT mice. {dagger}, P < 0.05 vs. WT GH-treated mice.

 
GH treatment did not normalize the functional defect in skeletal muscle of the MKR mice
To determine whether the effect of GH on functional changes in skeletal muscle is mediated through the IGF-I receptor-signaling pathway, we next measured force production in isolated EDL muscles after GH treatment. EDL muscle mass and CSA were significantly reduced in MKR mice compared with WT mice (Table 2Go), which is consistent with our previous findings (17). Muscle mass and CSA were 30.2% and 21% lower, respectively, in MKR mice than in WT mice (Table 2Go). Specific force and tetanic force were also significantly lower in muscles of MKR mice, compared with WT mice (Table 2Go). After GH treatment, muscle mass and CSA in the EDL muscles of WT mice were significantly increased (Table 2Go). GH treatment induced a similar increase in the maximum tetanic force production in WT mice. However, it did not reach statistical significance. This led to similar overall levels of specific force in the EDL muscles of treated and untreated WT mice (Table 2Go). However, GH treatment did not increase muscle mass, CSA, or tetanic force in EDL muscles from MKR mice (Table 2Go). Specific force of these muscles also remained lower after GH treatment than in muscle from WT mice (Table 2Go).


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  		TABLE 2. Effect of GH on muscle weight, CSA, tetanic force, and specific force in WT and MKR mice

 
GH administration did not restore defective muscle development in MKR mice
We next asked whether the lack of IGF-I-mediated GH effects on skeletal muscle mass in MKR mice was associated with changes in cell size or number. To this end, we quantified muscle size, fiber size, number of fibers per given area, and number of nuclei per fiber. The CSA of EDL muscle was significantly increased in WT mice in response to GH treatment, whereas GH treatment had no effect on CSA in MKR mice (Table 1Go). GH treatment significantly increased the size of individual myofibers in WT mice (843.9 ± 25.3 to 1136.6 ± 32.2 µm2) but not in MKR mice (Fig. 4AGo). Due to the increase in myofiber size, the number of myofibers per given area was slightly reduced in response to GH treatment in WT mice (from 134.6 ± 9.64 to 109.8 ± 11.4) but was unchanged in MKR mice (Fig. 4BGo). The number of nuclei per myofiber was also significantly increased in WT mice treated with GH (from 2.57 ± 0.10 to 3.04 ± 0.09), but not in MKR mice (Fig. 4CGo).



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  		FIG. 4. Effects of GH treatment on myofiber properties in WT and MKR. WT and MKR mice were treated for 4 wk with either with vehicle ({blacksquare}) or with rhGH ({square}), as described in Materials and Methods. Quad muscles were collected and fiber size (individual myofiber area, in µm2) (A), number of myofibers per given area (B), and number of nuclei per myofiber (C) were determined. All results are expressed as mean ± SE (n = 4–5 in each group). *, P < 0.05 vs. vehicle-treated WT. #, P < 0.05 vs. vehicle-treated WT.

 
The proliferation of satellite cells is associated with increased myofiber proliferation in adult muscle and the percentage of cells stained with BrdU is closely correlated with satellite cell proliferation (24). GH treatment significantly increased the percentage of cells labeled by BrdU in WT mice (from 10.94 ± 1.04 to 15.1 ± 1.73) (Fig. 5Go). However, GH treatment had no effect on the level of BrdU incorporation in MKR mice (Fig. 5Go). IGF-I has been shown to induce the expression of genes specific to muscle differentiation, such as the myogenin and myo D transcription factors (25). Myogenin and myo D mRNA levels were significantly increased after GH treatment in WT mice, but their levels were unchanged in response to GH treatment in MKR mice (Fig. 6Go).



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  		FIG. 5. Effect of GH on BrdU-stained nuclei in WT and MKR mice. WT and MKR mice were treated for 4 wk with either with vehicle ({blacksquare}) or with rhGH ({square}), as described in Materials and Methods. Mice received daily injections of BrdU for the last 15 d of the 4-wk treatment and quad muscles were collected for immunohistochemistry. Upper panels, Representative BrdU-stained nuclei, indicated by arrows. Lower panel, Average percentage of cells containing BrdU-stained nuclei (cells) per total fiber number per given area. Data are expressed as mean ± SE (n = 5–6 in each group). *, P < 0.05 vs. vehicle-treated WT. #, P < 0.05 vs. vehicle-treated WT.

 


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  		FIG. 6. Comparison of myogenin and myoD mRNA levels in WT and MKR mice after GH treatment. Mice were treated with either vehicle ({blacksquare}) or rhGH ({square}) for 4 wk, as indicated. Hindlimb muscles were removed and subjected to Northern blot analysis. Upper panel, Levels of myogenin and myoD mRNA expressed relative to 18S RNA levels. Lower panel, Representative northern blots. All results are expressed as mean ± SE (n = 6 in each group). *, P < 0.05 vs. vehicle-treated WT. #, P < 0.05 vs. vehicle-treated WT.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The anabolic effects of GH, acting either directly or through IGF-I, play an important role in regulating whole body and skeletal muscle growth and development. It has not yet been clearly established whether GH has direct effects on the mass, fiber composition, and function of skeletal muscle. It has been shown that chronic GH treatment increases muscle mass and function in GH-deficient patients and animals (26, 27, 28, 29). These effects of GH correlated with hypertrophy of existing muscle fibers and proliferation of satellite cells (30, 31, 32). GH administration significantly increased serum and tissue levels of IGF-I, which acts in an endocrine or autocrine/paracrine manner and regulates muscle growth and function (10, 33, 34). Nevertheless, the extent to which IGF-I-mediated effects of GH on skeletal muscle growth and function has not been fully elucidated.

In a previous study, we generated MKR mice, which lack IGF-I receptor function and have a major reduction in insulin receptor function specifically in skeletal muscle (17). Thus, both the endocrine and the autocrine/paracrine actions of IGF-I in response to GH administration are defective in skeletal muscle of MKR mice. We now show that GH treatment has no effect on muscle mass in MKR mice. GH treatment also failed to improve physiological muscle function in these mice, as measured by force production. Furthermore, GH-induced hypertrophy and stimulation of proliferation of satellite cells were attenuated in MKR mice, compared with WT mice. GH treatment failed to induce the expression of myogenin and myoD, two muscle-specific transcription factors, in MKR mice. Taken together, our findings demonstrate that the increases in skeletal muscle mass and function, the induction of muscle fiber hypertrophy, and the proliferation and differentiation of satellite cells induced by GH are mediated through the IGF-I receptor (and insulin receptor).

Exogenous administration of GH significantly increased skeletal muscle weight in WT mice. This increase in muscle weight paralleled the increase in total body weight. Skeletal muscle weight in MKR mice was approximately 30% lower than that in WT mice, which is consistent with the findings of our previous study (17). GH treatment had no effect on skeletal muscle weight in MKR mice, whereas it substantially increased total body weight, body length, and the weights of other GH-responsive organs in these mice. These findings suggest that IGF-I receptor signaling is involved in the GH-induced increase in skeletal muscle mass, but that GH may have an IGF-I receptor-independent effect in liver where IGF-I receptors are not expressed postnatally. The positive effects of GH on skeletal muscle mass are related to hypertrophy of muscle fibers under normal or GH-deficient conditions (3, 32, 35, 36). The CSA of skeletal muscle and the areas of individual myofibers were decreased in MKR mice by 30% and 40%, respectively, compared with WT mice. GH induced muscle hypertrophy in WT mice, as demonstrated by an increase in CSA and individual fiber area by 11% and 30%, respectively. However, GH did not produce these effects in the skeletal muscle of MKR mice. Therefore, the GH-induced hypertrophy of skeletal muscle requires the IGF-I receptor signaling pathway.

GH treatment increases the relative proportion of type 1 muscle fibers under GH-deficient conditions (37), yet it does not affect the muscle fiber type composition in normal animals (38). We observed no changes in the proportion of muscle fiber type in either WT or MKR mice in response to GH administration (data not shown).

It has been well documented that the majority of the circulating IGF-I released in response to GH administration is derived from the liver (21) (2). Consistent with these data, we found that 4 wk of GH treatment significantly increased hepatic IGF-I and GH receptor mRNA and serum IGF-I levels in both WT and MKR mice. However, 15% increase in IGF-I mRNA level did not reach statistical significance (P = 0.07) in skeletal muscle of WT after GH treatment. Furthermore, GH receptor and IGF-I receptor mRNA levels were not changed in skeletal muscle of either WT or MKR mice after GH administration, although WT mice exhibited a significant increase in skeletal muscle mass. This lack of effect of exogenously administrated GH on these genes in skeletal muscle of WT mice contrasts with what is observed in liver. This may reflect a dominant somatotrophic function of GH in liver.

We showed that GH administration significantly increased the weight and CSA of EDL muscle in WT mice by 11%. These GH-induced changes were accompanied by proportional increases in tetanic force production. Specific force normalized to muscle CSA was similar in both GH-treated and untreated WT mice, indicating that muscle strength per muscle unit was not affected by GH administration. In contrast, MKR mice did not exhibit changes in these parameters (CSA, muscle weight, and tetanic force in EDL muscle) in response to GH treatment, and the levels of specific force were significantly lower in MKR mice than in WT mice. Overexpression of IGF-I in EDL muscle via an adeno-associated virus with a myosin light chain promoter increased muscle mass and strength in both young and old adult mice, indicating that IGF-I plays a significant role in skeletal muscle function (10). Overall, the impaired physiological function of MKR mice was not restored by GH treatment, suggesting that IGF-I receptor signaling pathways are critical for GH-induced functional improvement in skeletal muscle.

It has not been conclusively established whether GH has a direct effect on proliferation and differentiation of satellite cells (39). However, several lines of evidence suggest that IGF-I induces satellite cells to proliferate and to differentiate into myoblasts (40). GH treatment significantly increased the percentage of BrdU-stained cells in EDL muscles of WT mice, indicating that the proliferation of satellite cells was increased. However, GH treatment did not exert this effect in MKR mice, suggesting that activation of the GH/IGF-I axis is critical for the proliferation of satellite cells. In a previous study, we observed muscle hypoplasia in MKR mice from birth to the age of 3 wk, whereas adult mice exhibited compensatory hyperplasia (17). In the present study, MKR mice at the age of 7 wk exhibited a significant increase in the percentage of BrdU-labeled cells in EDL muscle compared with WT mice, which reflects the level of proliferation of satellite cells. Thus, we cannot exclude the possibility that GH may have an attenuated effect on the proliferation of satellite cells in MKR mice, possibly due to the already higher levels of BrdU-stained cells.

IGF-I is known to increase the mRNA levels of myogenin and myoD, which act as transcription factors during muscle differentiation (41). GH treatment, in contrast, decreases myogenin and myoD mRNA levels (42). Therefore, the increased levels of myogenin and myoD in response to GH treatment in WT mice could be due to an IGF-I-mediated GH effect. In contrast, GH did not induce these changes in MKR mice, suggesting that the effects of GH on differentiation of satellite cells during adult muscle myogenesis are IGF-I dependent.

Muscle growth is regulated by the processing of protein turnover (protein synthesis and protein degradation). Muscle protein degradation is increased by insulin resistance, GH resistance, and insulin deprivation under catabolic states such as sepsis, chronic renal failure, and insulin-dependent diabetes mellitus (43, 44, 45, 46). Previous studies (17) showed that MKR mice exhibited impaired signaling of both IGF-I and insulin receptor and metabolic alterations such as hyperinsulinemia, hyperlipidemia, and hyperglycemia resulting in the development of type 2 diabetes. Therefore, insulin resistance and/or other metabolic alterations could possibly contribute to the defective muscle growth in MKR mice. Furthermore, there are possibilities that lack of GH response on muscle mass is attributed to insulin resistance, metabolic alterations, or GH resistance in skeletal muscle of MKR mice. However, hyperglycemia, which develops in adulthood of MKR mice, is uncoupled with compensatory increases in muscle mass observed upon reaching adulthood, suggesting that uncontrolled diabetes or insulin resistance is unlikely responsible for delayed muscle growth and lack of GH response on muscle mass in MKR mice. However, it would require the development of a specific IGF-I receptor gene deletion in skeletal muscle using the cre-lox/P system to totally exclude the effect of the insulin receptor in the process described in this study. The possible role of muscle GH resistance in defective GH effect on muscle mass is not clear even though, in this study, the level of muscle GH receptor was not reduced in MKR mice compared with WT mice (data not shown). The finding that p-STAT was enhanced by GH treatment suggests that GH is able to stimulate muscle function in MKR mice. However, there seemed to be some impairment in this response, suggesting that perhaps the GH receptor signaling maybe affected by the DN-IGF-IR expression. Further studies on several signaling proteins such as suppressors of cytokine signaling proteins will be required to elucidate GH resistance and muscle growth in MKR mice after GH treatment.

In summary, GH treatment significantly increased muscle mass and stimulated the proliferation of satellite cells and myofiber hypertrophy in skeletal muscle of WT mice. However, GH failed to exert these effects in MKR mice, which lack functional IGF-I and insulin receptors. Furthermore, GH was not able to restore impaired muscle function in MKR mice. The levels of myogenin and myoD remained unchanged in MKR mice in response GH treatment. Thus, IGF-I receptor signaling pathways are critical for GH-induced increases in muscle mass and strength.

GH has been extensively used to induce therapeutic muscle growth and function in GH-deficient subjects. The use of GH therapy in frail and elderly individuals is also currently being investigated. Aging skeletal muscle has been reported to show a down-regulation of the IGF-I system, in particular, a decrease in IGF-I receptor numbers (47). Our study strongly suggest that the potential use of GH therapy in elderly patients should be reevaluated.


   Footnotes
 
First Published Online December 23, 2004

Abbreviations: BrdU, Bromo-deoxyuridine; CSA, cross-sectional area; DN-IGF-IR, dominant-negative IGF-I receptor; EDL, extensor digitorum longus; Lo, optimum length; p, phospho; quad, quadriceps; rh, recombinant human; RNase, ribonuclease; STAT, signal transducer and activation of transcription; WT, wild-type.

Received July 14, 2004.

Accepted for publication December 3, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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