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Am J Physiol Regul Integr Comp Physiol 285: R34-R43, 2003. First published April 10, 2003; doi:10.1152/ajpregu.00177.2002
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TRANSLATIONAL PHYSIOLOGY

Insulin-like growth factor I prevents corticosteroid-induced diaphragm muscle atrophy in emphysematous hamsters

Mario Fournier,1,2 Zhi-Shen Huang,1 Hongyan Li,3 Xiaoyu Da,1 Bojan Cercek,2,3 and Michael I. Lewis1,2

Divisions of 1Pulmonary/Critical Care Medicine and 3Cardiology, The Burns and Allen Research Institute, Cedars-Sinai Medical Center, and 2The David Geffen School of Medicine at University of California Los Angeles, Los Angeles, California 90048

Submitted 21 March 2002 ; accepted in final form 1 April 2003


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The aim of this study was to evaluate whether recombinant human insulin-like growth factor I (rhIGF-I) could attenuate or prevent diaphragm (DIA) fiber atrophy with corticosteroid (CS) administration to emphysematous (EMP) hamsters. DIA muscle IGF-I responses to CS administration with and without exogenous rhIGF-I administration were evaluated. Three groups were studied: 1) EMP; 2) EMP + triamcinolone (T; 0.4 mg·kg-1·day-1 im); and 3) EMP + T + IGF-I (600 µg/day by constant infusion). After 4 wk, the DIA was analyzed histochemically and biochemically (IGF-I mRNA levels by RT-PCR and endogenous and exogenous IGF-I peptide levels immunochemically). Body weights of EMP-T progressively decreased, while those of EMP and EMP-T-IGF-I remained stable despite similarly reduced food intake in both T groups. DIA weight was reduced with T but preserved with rhIGF-I infusion. DIA fiber proportions were similar among the groups. The cross-sectional areas of types I, IIa, and IIx fibers were reduced (17 to 31%) with T administration but unchanged with rhIGF-I infusion. DIA IGF-I mRNA levels were similar across all groups. By contrast, the endogenous DIA IGF-I levels were reduced (41%) in the EMP-T-IGF-I animals. Total DIA IGF-I levels (endogenous + exogenous) were still significantly reduced. IGF-I immunoreactivity confirmed this reduction in all DIA fibers. We conclude that DIA fiber atrophy with T was completely prevented by exogenous rhIGF-I administration. This effect was likely mediated by the pharmacological influences of exogenously administered rhIGF-I. We speculate that this results from increased bioavailability of free IGF-I to react with muscle receptors. Reduced endogenous IGF-I levels in the DIA likely reflect a negative-feedback influence. These results may have important clinical implications for treatment options to offset the adverse effects of CS on the respiratory muscles in patients with chronic lung disorders.

triamcinolone; respiratory muscles; insulin-like growth factor I; autocrine/paracrine effects; diaphragm fiber cross-sectional areas

CORTICOSTEROIDS (CS) ARE COMMONLY used in patients with chronic obstructive pulmonary disease (COPD), often in high dosage, during acute exacerbations of their condition (5, 52, 66) or as maintenance therapy in the subset of patients deemed to be steroid responsive (9). More recently, it has been reported that CS may produce adverse effects on skeletal muscle structure and function in patients with COPD. For example, Decramer and coworkers (17) reported severe steroid myopathy in patients with COPD or asthma treated with high doses of CS, while prolonged therapy with relatively low doses of CS in patients with COPD was associated with respiratory and limb muscle weakness (15, 16). Quadriceps biopsy revealed atrophic and necrotic muscle fibers, and survival of COPD patients with steroid myopathy was reduced compared with those without myopathy, despite similar degrees of airflow obstruction (15).

In animal studies, CS (particularly the fluorinated compounds) have been shown to produce diaphragm and limb muscle wasting characterized by atrophy of type II fibers (particularly IIx, IIb) with less or no impact observed in type I fibers (18, 20, 47, 51, 56, 70). In animal models, recovery of steroid-induced fiber atrophy may be incomplete or markedly delayed. For example, Dekhuijzen and colleagues (19) reported type IIx/b fiber atrophy in the diaphragm and gastrocnemius muscles of the rat 2 mo after cessation of triamcinolone therapy.

The above studies provide a rationale for the use of anabolic agents to prevent or ameliorate the adverse effects of CS on skeletal muscle structure and function or accelerate recovery. Growth hormone, however, failed to reverse triamcinolone-induced diaphragm fiber atrophy or alterations in contractile properties of the diaphragm in rats (54), and clenbuterol, a {beta}-agonist anabolic agent, failed to improve reduced diaphragmatic strength in rabbits, despite an attenuating effect on dexamethasone-induced diaphragm fiber atrophy (34).

The provision of insulin-like growth factor I (IGF-I), a polypeptide growth factor, to offset the adverse effects of CS on skeletal muscle, is attractive for several reasons. 1) CS exert adverse effects on protein turnover in skeletal muscle by both reducing synthesis of myofibrillar proteins and by enhancing their proteolysis (31). While growth hormone mediates anabolism by enhancing protein synthesis in skeletal muscle, IGF-I stimulates both protein synthesis and inhibits proteolysis, a major mechanism contributing to muscle atrophy with CS administration (41). 2) In a rat model of acute nutritional deprivation, we were able to demonstrate that recombinant human (rh) IGF-I was able to attenuate diaphragm fiber atrophy, while growth hormone had no impact, suggesting that IGF-I "bypassed" the peripheral resistance to growth hormone action (46). 3) A number of recent studies have reported on the preventive effects of IGF-I on CS-induced nitrogen wasting or limb muscle fiber atrophy (36, 50, 67), while growth hormone had no effect (50).

The aim of the present study was to evaluate whether IGF-I could attenuate or prevent diaphragm fiber atrophy during CS administration to emphysematous (EMP) hamsters. We also evaluated local diaphragm muscle IGF-I responses to CS administration with and without exogenous rhIGF-I administration.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Induction and Verification of EMP

EMP was induced in male golden Syrian hamsters (n = 24; initial body wt ~ 100 g; Simonsen Laboratories). Induction was performed under general anesthesia [ketamine (200 mg/kg) and xylazine (10 mg/kg) ip] by the intratracheal instillation of pancreatic porcine elastase (Sigma Chemicals; 40 IU/100 g body wt in 0.3 ml 0.9% saline). After the conclusion of all experimental paradigms (see below), EMP was verified by measurement of static pressure-volume relationships of the lungs, with the volume at 25 cmH2O being defined as maximum lung volume (MLV; Ref. 58).

Animal Groups

Nine to ten months after the induction of EMP, the animals were divided into three groups: 1) EMP, 2) an EMP group in whom triamcinolone (T) was administered (EMP-T), and 3) an EMP group in whom both T and rhIGF-I were given (EMP-T-IGF-I).

Pharmacological Agents

T diacetate (Steraloids, Newport, RI) was administered by daily intramuscular injection over a period of 4 wk (dose 0.4 mg·kg-1·day-1). rhIGF-I (a generous gift from Genentech, South San Francisco, CA) was provided as a continuous infusion via a subcutaneously implanted miniature osmotic pump (Alzet, model 2001) with the total daily dose being 600 µg. The rhIGF-I was administered over the same 4-wk period. EMP animals received saline injection (intramuscular) daily, and sham surgery was performed on EMP and EMP-T animals. All animals were provided with water and food ad libitum. The research protocol was reviewed and approved by the Burns and Allen Research Institute Animal Care and Use Committee of Cedars-Sinai Medical Center.

Histochemical Procedures

Muscle fiber-type proportions. Under general anesthesia (pentobarbital sodium, 6 mg/100 g ip), the diaphragm was rapidly excised, and a segment of the midcostal region was mounted on cork at its determined resting length and rapidly frozen in isopentane (which had been cooled to its melting point by liquid nitrogen). The remaining portion of the diaphragm to be used for biochemical studies was rapidly frozen in liquid nitrogen and stored at -80°C until analysis. Serial cross-sections of the diaphragm were cut at 10-µm thickness using a cryostat (model 2800 E, Reichert-Jung) kept at -20°C. Diaphragm muscle fibers were classified based on difference in staining intensity for myofibrillar ATPase (mATPase) after alkaline (pH 9.0) and acid (pH 4.3 and 4.55) preincubations (24). One additional serial section was fixed in 2% paraformaldehyde at pH 7.4 for 2 min at room temperature and then preincubated at pH 10.4 (modification of method by Guth and Samaha; Ref. 30). These various staining procedures allow the classification of fibers into several types, i.e., types I, IIa, IIb, IIx, and IIc (24, 25). Fiber-type proportions were determined from a sample of 200–300 fibers from each muscle. In previous studies, in both hamsters and rats, we verified diaphragm muscle fiber type immunohistochemically, with 95% or more correspondence between the mATPase-based classification and the major isoform of myosin heavy chain in single diaphragm fibers (24).

Fiber cross-sectional areas. Muscle fiber cross-sectional area was determined from microscopic images of digitized muscle sections, using a computer-based imaging processing system. The latter is composed of a Leitz Laborlux S (Leica) microscope, CCD video camera system (model VI-470; Optronics Engineering, Goleta, CA), high-resolution Trinitron color video monitor (model PVM-1343MD; Sony, Japan), 486 DX-50 MHz PC with a Targa+ imaging board (Truevision,) and Mocha image analysis software (v 1.20; Jandel, San Rafael, CA). A microscope stage micrometer was used to calibrate the imaging system for morphometry. The cross-sectional area of 200–300 individual fibers (i.e., sampled from those used in the analysis of fiber proportions) was determined from the number of pixels within manually outlined fiber boundaries.

Immunohistochemical Studies: IGF-I

The identification of total (i.e., endogenous and exogenous) IGF-I in the hamster diaphragm was obtained by indirect immunoperoxidase technique. Serial muscle cryosections were dried at room temperature, fixed in cold acetone for 5 min, washed with PBS for 5 min, and incubated in normal goat serum for 15 min at room temperature. Sections were incubated for 2 h at room temperature in rabbit polyclonal antiserum AFP4892898 (1:10) specific for IGF-I [provided by Dr. A. F. Parlow, Scientific Director of the National Hormone and Pituitary Program (NHPP) of the National Institute of Diabetes and Digestive and Kidney Diseases, Torrance, CA]. Cross-reactivity with IGF-II has been reported to be <1% (technical data provided by the NHPP). Sections were rinsed with PBS and exposed to a biotinylated secondary antibody for 30 min at room temperature. Control sections were exposed to a normal rabbit serum and a secondary antibody. Sections were rinsed with PBS and exposed to the avidinbiotinylated enzyme complex (ABC: Elite PK-6100; Vector) reagent for 20 min at room temperature. Sections were rinsed again with PBS, and visualization was obtained after exposure to the peroxidase substrate 3-amino-9-ethylcarbazole (AEC peroxidase substrate kit) for 10 min. Sections were washed for 5 min and mounted with glycerin jelly mounting medium. To determine relative differences in the expression of IGF-I in different fiber types, microdensitometric measurements of the intensity of IGF-I immunoreactivity within type-identified fibers (from serial sections stained for mATPase) were performed using the image-processing system described above. Final densitometric measurements took into account the subtraction of nonspecific background from blank serial sections treated with normal rabbit serum and a secondary antibody.

Protein Studies: IGF-I

Serum IGF-I. Serum total IGF-I concentration was determined using kits for both endogenous (rodent) and exogenous (human) IGF-I. Rodent IGF-I was measured by RIA from blood samples obtained at the terminal experiments. The rodent-specific IGF-I antibodies exhibit no cross-reactivity with hIGF-I. The rat IGF-I antibody used in the RIA kit appears to have high level cross-reactivity with hamster IGF-I compared with reported data by the Diagnostic Systems Laboratories (DSL, Webster, TX) for most other species, including guinea pig and rabbit, which have virtually undetectable serum IGF-I levels (also personal communication with Dr. S. K. Durham, DSL). There is unfortunately no recombinant hamster IGF-I available to validate more accurately our robust findings. Human IGF-I was measured by immunoradiometric assay (IRMA) from the same samples. Similarly, we have demonstrated that the human-specific IGF-I antibodies do not react with hamster IGF-I. Before assays, IGF-I was extracted from the serum, and insulin-like growth factor binding proteins (IGFBPs) were precipitated by incubation in acid-ethanol (14). Supernatants were neutralized and assays performed. For rodent IGF-I, a commercial double-antibody system kit (DSL-2900; DSL) was used according to manufacturer's protocol. This assay shows high cross-reactivity for hamster, mouse, and rat antigens (35). The intra-assay coefficient of variation is 5.9%, and the interassay coefficient of variation is 9.7%. The sensitivity of the assay allows the detection of IGF-I peptide levels of >21 ng/ml. For human IGF-I, a commercial two-site IRMA kit (DSL-5600 ACTIVE; DSL) was used according to manufacturer's protocol. The intra-assay coefficient of variation is 1.5%, and the inter-assay coefficient of variation is 3.7%. The sensitivity of the assay allows the detection of IGF-I peptide levels of >0.8 ng/ml.

Diaphragm muscle IGF-I. Frozen diaphragm tissue was pulverized in liquid nitrogen, IGF-I was extracted twice in acetic acid (1 mg/10 µl), and 100-µl aliquots of the supernatant were lyophilized and stored frozen overnight (21). Aliquots were resuspended in assay buffer and assayed using the same RIA and IRMA kits as described above for the determination of serum IGF-I. Results obtained with these RIA and IRMA methods have been shown to be highly correlative with an independent acid-chromatography separation study performed at Stanford University (rat: r = 0.99, see Ref. 42 for details; human: r = 0.9, technical data provided by Dr. M. Nicar, DSL).

Critique of methodology for IGF-I protein studies. The protein extraction procedures used for serum are standard methodologies with reagents provided by the individual kits (i.e., rat and human), while for muscle protein extraction, the D'Ercole et al. (21) method was used. This method has been employed in the extraction of IGF-I in tissues of many different species. Extraction procedures, however, have been criticized for failure to remove all IGFBPs, particularly the low-molecular-mass IGFBPs (12). Nevertheless, it has been reported that the remaining IGFBPs do not significantly interfere with the subsequent RIA procedures (12). Despite different extraction procedures reported in the literature, the levels of serum and muscle IGF-I we report for both rat (45) and hamster are well within recently published ranges using rat-specific and other RIA methods (1, 2, 3, 13, 32, 49, 62). Furthermore, in our experience, there is no cross-reactivity between rat (see also 42) and hamster tissues and the human-specific IGF-I antibody of the DSL IRMA kit. By contrast, significant reactivity occurs in other rodent tissues such as hamster and mice (13) using the rat-specific IGF-I antibody. This provides strong inference that the sensitivity and specificity of this DSL kit for identifying a rat-like IGF-I in other rodent tissues is relatively high. While validation of our hamster IGF-I assays after acid-chromatography separation would be optimal, no purified hamster IGF-I and IGFBPs are available to validate such measurements. We believe, however, that the relative magnitude and direction of change using the methods of the present study are unlikely to differ significantly from those using acid-chromatography separation were this readily available. In addition, all of our samples were analyzed simultaneously for either endogenous hamster IGF-I or rhIGF-I using the same reagents provided with the species-specific kits, thus allowing direct comparison across experimental groups.

Muscle protein concentrations. Muscle protein concentrations were determined for both the soluble and myofibrillar fractions using a commercial protein assay kit (Bio-Rad, Hercules, CA) based on the Bradford (7) method and measured with a spectrophotometer (SmartSpec 3000; Bio-Rad).

mRNA Studies: IGF-I

Total RNA extraction. Total RNA was extracted from 50 mg samples of the costal diaphragm with TRIZOL reagent (Life Technologies, Rockville, MD) according to manufacturer's protocol. Quality and concentrations of total RNA were determined with a spectrophotometer (SmartSpec 3000). Samples were stored at -80°C in RNase-free water until analysis.

Oligonucleotides. The primers for IGF-I and {beta}-actin were designed based on published rat cDNA sequences because hamster sequences are not available. Primer sequences for IGF-I were the following: upstream (5' to 3') AAG CCT ACA AAG TCA GCT CG and downstream (5' to 3') GGT CTT GTT TCC TGC ACT TC. Primer sequences for {beta}-actin were the following: upstream (5' to 3') TGA CGT TGA CAT CCG TAA AG and downstream (5' to 3') ACA GTG AGG CCA GGA TAG AG. The expected lengths of the RT-PCR products were 114 bp for IGF-I and 194 bp for {beta}-actin. {beta}-Actin served as a housekeeping gene for sample normalization because it is not affected in catabolic states such as malnutrition and CS treatments. We have previously verified the stability of {beta}-actin against 18S by Northern analysis.

Semiquantitative RT-PCR. cDNA was synthesized from 1 µg total RNA in 40 µl of reaction buffer comprised of 250 µM dNTPs, 1.25 µM random hexamer primers, 10 U RNase inhibitor (Boehringer Mannheim, Indianapolis, IN), and 100 U Superscript II reverse transcriptase (Life Technologies). First, the reaction was carried out for 10 min at 25°C, then for 30 min at 42°C, followed by 5 min at 95°C and cooling at 4°C. PCR was carried out with 5–25 ng of reverse-transcribed RNA Taq polymerase buffer (Promega Biotech, Madison, WI) containing 200 µM dNTPs, 1.25 U Taq polymerase, 250 nM sense and anti-sense primers, and 2 mM Mg2+, in a total volume of 50 µl by using the thermal cycler (PTC-100; MJ Research, Watertown, MA). Each cycle consisted of 1 min denaturation at 95°C, 45 s annealing at 65°C, and 2 min elongation at 72°C. Samples normalized by {beta}-actin amplification were amplified in a linear range established using serial cDNA dilutions and varying the number of cycles. A total of 26 cycles was used for IGF-I and 28 cycles for {beta}-actin. Amplified products were separated by electrophoresis in 4% agarose gels and visualized under UV light after staining with ethidium bromide. Routine RT-PCR controls without reverse transcriptase were negative. The relative amounts of the PCR products were measured by densitometry (Kodak Electrophoresis Documentation and Analysis System 120).

Statistical Analysis

The distribution of all data was tested for normality. Statistical analysis was then performed by using an ANOVA, with the experimental factors being the administration of T with or without IGF-I. If a significant interaction was found, post hoc analysis (Newman-Keuls test) was used to compare differences in independent groups. An {alpha}-level of 0.05 was used to compare differences in independent groups and to determine overall significance. Values are means ± SE.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Body Weights and Food Intake

A significant and progressive decline in body weight was observed over the 4-wk experimental period in the EMP-T animals (Fig. 1A). The provision of rhIGF-I to EMP animals receiving T prevented any significant decline in body weight compared with EMP animals, in whom body weight was relatively stable during the experimental period (Fig. 1A). It is of interest that the administration of rhIGF-I exerted its positive impact in EMP animals receiving T despite similar declines in food intake in both EMP-T and EMP-T-IGF-I animals compared with the EMP group (Fig. 1B).



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  		Fig. 1. A: relative body weight changes during the 28-day experimental period in emphysematous (EMP) hamsters, EMP hamsters treated with triamcinolone (EMP-T), and EMP-T hamsters with recombinant human insulin-like growth factor I (rhIGF-I) infusion (EMP-T-IGF). Note the significant loss (15%) in body weight in EMP-T animals, which was prevented by rhIGF-I infusion. B: mean daily food intake during the 28-day experimental period in EMP, EMP-T, and EMP-T-IGF hamsters. Note the significantly lower food intake with T treatment, which was not prevented by rhIGF-I infusion. *Significantly different from EMP. Values are means ± SE.

 

Lung Volumes

All EMP animals demonstrate obvious macroscopic evidence of EMP changes. Evaluation of pressure-volume relationships of the lungs revealed similar MLVs in all groups (EMP: 18.7 ± 0.7 ml; EMP-T: 18.2 ± 0.6 ml; EMP-T-IGF-I: 18.5 ± 0.6 ml), which were 180–200% of those observed in control non-EMP animals (10.2 ± 0.2 ml; see Ref. 25).

Muscle Weights

Final diaphragm muscle mass was significantly reduced in the EMP-T animals compared with EMP and EMP-T-IGF-I groups (Fig. 2). Muscle protein concentrations, however, were not reduced with T administration. As muscle proteins comprise the major component of muscle mass, there is strong inference for reduction in protein mass in view of the reduction in total diaphragm mass in EMP-T animals. Diaphragm muscle mass was similar between EMP and EMP-T-IGF-I groups (Fig. 2).



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  		Fig. 2. Diaphragm muscle mass in EMP, EMP-T, and EMP-T-IGF hamsters. Note the significantly lower diaphragm muscle weight in EMP-T animals, which was prevented by rhIGF-I infusion. *Significantly different from EMP; +significantly different from EMP-T-IGF. Values are means ± SE.

 

Diaphragm Fiber-Type Proportions and Cross-Sectional Areas

No differences in fiber-type proportions were observed between the groups (Fig. 3A). T resulted in significant atrophy of types IIa and IIx fibers of EMP animals (P < 0.05; Fig. 3B). The provision of rhIGF-I completely prevented atrophy of types IIa and IIx diaphragm fibers in EMP animals receiving the CS and in addition significantly increased the CSA of type I diaphragm fibers compared with EMP-T animals (P < 0.05; Fig. 3B).



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  		Fig. 3. Diaphragm fiber (types I, IIa, and IIx) proportions (A) and fiber cross-sectional areas (CSA; B) in EMP, EMP-T, and EMP-T-IGF hamsters. Note in B the significant atrophy of type IIa and IIx diaphragm fibers in EMP-T animals, which was prevented by rhIGF-I infusion. *Significantly different from EMP; +significantly different from EMP-T-IGF. Values are means ± SE.

 

Blood and Serum Studies

Blood glucose was similar across the groups (EMP: 166.4 ± 22.8 mg/dl; EMP-T: 143.4 ± 21.9 mg/dl; EMP-T-IGF-I: 142.4 ± 21.6 mg/dl). No hypoglycemia was observed with the use of rhIGF-I or hyperglycemia with T.

Serum endogenous IGF-I studies revealed a decline in serum IGF-I with the provision of T (P < 0.05), while the combination of T and exogenous rhIGF-I resulted in a further significant decrement in endogenous levels of circulating IGF-I (P < 0.0001; Fig. 4A). Serum levels of exogenous rhIGF-I were undetectable in EMP and EMP-T animals, while levels of 473 ± 74 ng/ml were noted in EMP-T-IGF-I hamsters (Fig. 4A). Total IGF-I, however, was still significantly less than those in EMP animals (P < 0.01).



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  		Fig. 4. Total serum (A) and muscle (B) IGF-I levels (i.e., endogenous and exogenous) in EMP, EMP-T, and EMP-T-IGF hamsters. Note in A the significantly lower (21%) endogenous serum IGF-I levels with T treatment, and the further reduction (70%) with rhIGF-I infusion. Total serum IGF-I levels were similar in EMP-T and EMP-T-IGF-I groups but still reduced (30%) compared with EMP. Note also in B the significantly lower muscle endogenous IGF-I levels in animals receiving rhIGF-I infusion compared with EMP and EMP-T. Total muscle IGF-I levels in EMP-T-IGF-I animals remained significantly less (30%) than in EMP hamsters *Significantly different from EMP; +significantly different from EMP-T. Values are means ± SE.

 

Diaphragm Muscle IGF-I

Mean diaphragm muscle endogenous IGF-I levels were unaffected by the administration of T but significantly reduced when exogenous rhIGF-I infusion was combined with T administration (P < 0.001; Fig. 4B). Exogenous rhIGF-I in diaphragm muscle was detectable only in animals receiving infusion of rhIGF-I in rather low concentrations (32 ± 12 ng/g; Fig. 4B). Total muscle IGF-I in EMP-T-IGF-I animals was still significantly reduced compared with the other groups (P < 0.01; Fig. 4B).

Immunohistochemical studies for IGF-I were performed on diaphragm muscle sections to localize total IGF-I (i.e., staining of both endogenous and exogenous IGF-I) within specific fiber types. These studies showed that IGF-I immunoreactivity was reduced in the EMP-T-IGF-I animals across all fiber types, compared with EMP hamsters (P < 0.01; Fig. 5). The intensity of IGF-I immunoreactivity was also reduced in the EMP-T-IGF-I group for type IIa (P < 0.01) and IIx (P < 0.5) fibers compared with EMP-T animals (Fig. 5).



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  		Fig. 5. Mean gray level intensity of IGF-I immunoreactivity of individual diaphragm fibers in EMP, EMP-T, and EMP-T-IGF hamsters. Note the significantly lower intensity of IGF-I immunoreactivity in all 3 types of diaphragm fibers in animals receiving rhIGF-I infusion. *Significantly different from EMP; +significantly different from EMP-T. Values are means ± SE.

 

IGF-I mRNA

Diaphragm muscle IGF-I mRNA abundance was unaffected by the provision of the CS or when T was combined with rhIGF-I infusion (Fig. 6).



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  		Fig. 6. IGF-I and {beta}-actin mRNA determined by RT-PCR in the diaphragm of EMP, EMP-T, and EMP-T-IGF hamsters. Electrophoresis of RT-PCR products (A) and ratio of IGF-I to {beta}-actin (B) demonstrate no differences between the groups. Values are means ± SE.

 


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study demonstrated a number of important findings. 1) The exogenous administration of rhIGF-I for 4 wk attenuated CS-induced loss of body weight in EMP hamsters. 2) rhIGF-I prevented loss of diaphragm muscle mass and atrophy of all diaphragm fiber types. While muscle protein concentration was unchanged with T administration, the reduction in diaphragm mass and fiber atrophy reflects a loss in total protein mass with T and recovery of total protein mass with rhIGF-I rescue. 3) rhIGF-I achieved these effects despite CS-induced anorexia in EMP animals receiving T. 4) Exogenous rhIGF-I reduced diaphragm muscle IGF-I levels (for all fiber types) while diaphragm muscle IGF-I mRNA abundance was preserved at 4 wk. Total muscle IGF-I levels (i.e., endogenous and exogenous) remained significantly lower than those groups not receiving rhIGF-I. By contrast, total serum IGF-I levels were similar between EMP-T-IGF-I and EMP-T animals, although still significantly lower than in EMP hamsters. CS-induced decrement in serum IGF-I has previously been reported by a number of other groups (39, 53).

Diaphragm Fiber Cross-Sectional Area

In the present study, the exogenous administration of rhIGF-I in the presence of concomitant CS use had a distinct impact on the cross-sectional areas of all diaphragm fiber types and prevented the atrophy of particularly types IIa and IIx fiber. Glucocorticoids may produce atrophy of skeletal muscle fibers via a number of influences on protein turnover. For example, the rate of protein synthesis in the gastrocnemius muscle of the rat was reduced by 60% after 5 days of cortisone treatment (37). The reduced protein synthesis is likely related to translational abnormalities (57). More recently it has been established that CS-induced translational defects are in large part related to the process of translation initiation whereby key factors such as eukaryotic initiation factor 4E are sequestered from the translational machinery or ribosomal protein S6 kinase is inhibited (59, 60). CS may also promote protein degradation in skeletal muscle in large part via the ubiquitin-proteasome proteolytic pathways (4, 11, 71).

IGF-I has the potential to offset the adverse effects of CS on protein turnover in skeletal muscle. IGF-I has been demonstrated to both augment protein synthesis and inhibit proteolysis (27, 28). The latter may be due to influences on the ubiquitin proteasome proteolytic pathways, as IGF-I has been shown to inhibit E2–14k, a key ubiquitin-conjugating enzyme (71). In the present study, food intake was reduced to a similar degree in both groups receiving T. This reduction in protein and calorie intake did not, however, impair the ability of IGF-I to prevent loss of muscle mass and diaphragm fiber-specific atrophy. This is in contrast to the report by Petrof et al. (54), whereby growth hormone failed to offset the adverse effects of CS in rats. This negative result may have been due to peripheral resistance to the action of growth hormone in the presence of anorexia-induced caloric deficit (65) as well as the lack of growth hormone effects on CS-induced proteolysis (31). In a model of severe nutritional deprivation, we have also previously reported a positive effect of rhIGF-I on diaphragm fiber size, while growth hormone had no impact (46). IGF-I has also been reported in recent studies to prevent CS-induced nitrogen wasting or limb muscle fiber atrophy in several animal models (36, 50, 67). It is also of interest that nandrolone, a synthetic anabolic steroid, has been reported to reverse glucocorticoid-induced diaphragm fiber atrophy in rats (68) as we have recently reported that diaphragm muscle fiber hypertrophy induced by nandrolone is, in part, mediated by increased muscle IGF-I expression (45).

Diaphragm Muscle IGF-I

In the present study, local diaphragm IGF-I responses were evaluated 1) to characterize possible changes in muscle IGF-I to relatively low-dose CS administration, and 2) to gain insight into the effects of exogenously administered rhIGF-I on the local milieu of IGF-I in diaphragm muscle as well as the possibility of exogenous rhIGF-I transfer. With the administration of T, diaphragm muscle IGF-I mRNA abundance and protein levels were preserved. This contrasts with the report of Gayan-Ramirez et al. (29) in which significant decrements in IGF-I mRNA were observed after massive doses of T (80 mg/kg) over 5 days. Using doses of T similar to that used in the present study, we have also demonstrated transient reduction in diaphragm IGF-I mRNA abundance, which normalized at 3–7 days of administration (23). Thus, with 4 wk of administration, our present results were not unexpected. Discordance between tissue IGF-I mRNA and protein levels has previously been reported in a number of different models, including the administration of dexamethasone (22, 39). In our study, the preservation of IGF-I mRNA in the diaphragm, yet decreased endogenous IGF-I levels, suggests translational or post-translational influences with T administration. The further decrement in diaphragm IGF-I levels with the infusion of rhIGF-I is more difficult to explain (see speculation below).

New ideas related to IGF-I physiology have recently emerged. It has become evident that IGF-I is produced in most body tissues, including skeletal muscle, implying important local autocrine/paracrine effects separate from those elaborated by the circulating form, which is produced mainly by the liver (41). This concept is supported by the liver-specific IGF-I gene-deleted mouse model (61, 72) in which normal growth and muscle mass was observed despite reduction in circulating levels of IGF-I by 75–80% compared with wild-type animals (61, 72). This contrasts with the full IGF-I gene-deleted mouse model in which a markedly attenuated growth profile is evident (6, 26, 48, 55), together with a significant reduction in diaphragm muscle mass and fiber size and number in surviving adults (26). Thus it has been proposed that the growth-promoting properties of IGF-I are due mainly to autocrine/paracrine influences, while circulating IGF-I has an important role in glucose homeostasis (8). There is also evidence supporting a dynamic local IGF-I milieu in muscle. This includes the increase in IGF-I mRNA abundance in muscle with growth hormone administration (33, 43) and the muscle expression of a variety of IGFBPs, which may modulate the ability of muscle IGF-I to bind to its receptor in an autocrine/paracrine fashion (35, 45).

The combination of T and exogenous rhIGF-I resulted in suppressed endogenous levels of circulating IGF-I as well as endogenous diaphragm muscle IGF-I. This suggests that the preservation of diaphragm muscle mass and fiber size was mediated pharmacologically by the administered rhIGF-I acting on muscle IGF-I receptors. This does not imply that rhIGF-I is more potent than hamster IGF-I. Rather, the anabolic potency of IGF-I in muscle depends on its bioavailability to its receptors (35). We postulate that the availability of free IGF-I to muscle tissue receptors with the rhIGF-I infusion is enhanced with a positive anabolic effect. This in part may be due to reduced circulating and tissue levels of IGFBPs, such as IGFBP-3, as a result of the effect of the administered glucocorticoid (38, 69) and the lowered endogenous IGF-I levels. It has also been shown in dexamethasone-treated rats that an infusion of rhIGF-I had greater anabolic potency compared with an infusion of equimolar amounts of rhIGF-I bound to rhIGFBP-3 (38). It is also of interest that treatment of porcine myogenic cultures with IGF-I reduces the level of IGFBP-3 protein and mRNA produced by muscle cells (73). Furthermore, the exogenous rhIGF-I appeared to exert a negative-feedback influence on both circulating endogenous IGF-I as well as diaphragm muscle endogenous IGF-I. The mechanisms responsible for this negative-feedback effect are not known. It certainly is possible that negative feedback on growth hormone secretion could have influenced liver IGF-I production and thus circulating IGF-I, as well as reduced stimulation of local muscle IGF-I. It is of interest that in the liver-specific IGF-I gene-deleted mouse model, serum growth hormone levels were increased (61, 72). We also speculate that sustained stimulation of the IGF-I receptor by exogenous rhIGF-I suppressed local IGF-I production in muscle possibly via an impact on IGFBPs or through as yet ill-defined regulatory pathways. The signaling pathways responsible for these speculations, however, are unclear. In preliminary data from our laboratory, the administration of rhIGF-I did not appear to alter the abundance of IGFBP-4 and IGFBP-5 mRNA in diaphragm muscle (Fournier, unpublished data). The fact that muscle fiber atrophy was prevented in EMP-T-IGF-I hamsters compared with T-treated animals, despite significantly reduced total muscle IGF-I levels, highlights the effectiveness of rhIGF-I on muscle. The presence of small amounts of rhIGF-I in the diaphragm muscle of treated animals suggests either rhIGF-I trapped in muscle blood vessels as reported by D'Ercole et al. (21) or transfer of the growth factor from serum to muscle. By contrast, it has been suggested that tissues such as muscle, kidney, spleen, fat, and bone may contribute to circulating IGF-I levels (72).

Clinical Implications

CS may have serious adverse consequences on diaphragm and/or limb muscle structure and function when administered in high dosage with acute exacerbation of the condition or with prolonged maintenance regimens as outlined in the introduction (5, 9, 15, 16, 17, 52, 66). Furthermore, a significant proportion of ambulatory patients with COPD are nutritionally depleted (one-quarter to one-third; Ref. 44) with even higher prevalence rates in those admitted to hospital after acute exacerbations (40). This may compound the problem and result in a permanent further reduction in muscle mass (10), which could profoundly affect respiratory muscle function and contribute to serious sarcopenia (10).

In the present study, clinically relevant dosage schedules were used, simulating modest maintenance regimens. This allows for strong inferences to be made in terms of the clinical implication of our findings. Furthermore, the infusion of rhIGF-I was at a dosage that did not induce hypoglycemia. Our study data suggest that IGF-I or similar growth factors may be of value in offsetting the adverse effects of CS in the short term. Possible future alternative routes of administration that would be more logistically feasible include twice-daily injections of an IGF-I-IGFBP-3 binary complex that was shown to augment protein synthesis in animal models of undernutrition and sepsis (63, 64).

In summary, low-dose T administered to EMP hamsters over 4 wk resulted in significant reductions in diaphragm muscle mass and diaphragm fiber atrophy despite preservation of local diaphragm IGF-I mRNA abundance and protein expression. The concomitant infusion of rhIGF-I completely prevented loss of diaphragm mass or fiber atrophy, an effect possibly mediated by a greater bioavailability of free IGF-I to muscle tissue receptors. Endogenous IGF-I expression in the diaphragm muscle was significantly reduced likely secondary to a negative-feedback influence. These results may have important potential implications for possible treatment options to offset the effects of CS on muscle structure and function in patients with COPD.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge the assistance of Drs. D. R. Clemmons, S. K. Durham, and P. D. K. Lee, as well as the secretarial help of D. Craig.

This research was supported by funds from the National Institutes of Health (Grant HL-47537) and the California Tobacco-Related Disease Research Program of the University of California (Grants 6RT-0144 and 7RT-0161).


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Fournier, Cedars-Sinai Medical Center, 8700 Beverly Blvd., Rm. 6732, Los Angeles, CA 90048 (E-mail: mario.fournier{at}cshs.org).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 METHODS
 RESULTS
 DISCUSSION
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