Ghrelin receptor agonist GHRP-2 prevents arthritis-induced increase in E3 ubiquitin-ligating enzymes MuRF1 and MAFbx gene expression in skeletal muscle

Miriam Granado,1 Teresa Priego,1 Ana I. Martín,2 Mª Ángeles Villanúa,1 and Asunción López-Calderón1

1Facultad de Medicina, Departamento Fisiología, Universidad Complutense y 2Departamento Ciencias Morfológicas y Fisiología, Universidad Europea, Madrid, Spain

Submitted 11 March 2005 ; accepted in final form 14 July 2005


    ABSTRACT
 TOP
 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chronic arthritis is a catabolic state associated with an inhibition of the IGF system and a decrease in body weight. Cachexia and muscular wasting is secondary to protein degradation by the ubiquitin-proteasome pathway. The aim of this work was to analyze the effect of adjuvant-induced arthritis on the muscle-specific ubiquitin ligases muscle ring finger 1 (MuRF1) and muscle atrophy F-box (MAFbx) as well as on IGF-I and IGF-binding protein-5 (IGFBP-5) gene expression in the skeletal muscle. We also studied whether the synthetic ghrelin receptor agonist, growth hormone releasing peptide-2 (GHRP-2), was able to prevent arthritis-induced changes in the skeletal muscle. Arthritis induced an increase in MuRF1, MAFbx (P < 0.01), and tumor necrosis factor (TNF)-{alpha} mRNA (P < 0.05) in the skeletal muscle. Arthritis decreased the serum IGF-I and its gene expression in the liver (P < 0.01), whereas it increased IGF-I and IGFBP-5 gene expression in the skeletal muscle (P < 0.01). Administration of GHRP-2 for 8 days prevented the arthritis-induced increase in muscular MuRF1, MAFbx, and TNF-{alpha} gene expression. GHRP-2 treatment increased the serum concentrations of IGF-I and the IGF-I mRNA in the liver and in the cardiac muscle and decreased muscular IGFBP-5 mRNA both in control and in arthritic rats (P < 0.05). GHRP-2 treatment increased muscular IGF-I mRNA in control rats (P < 0.01), but it did not modify the muscular IGF-I gene expression in arthritic rats. These data indicate that arthritis induces an increase in the activity of the ubiquitin-proteasome proteolytic pathway that is prevented by GHRP-2 administration. The parallel changes in muscular IGFBP-5 and TNF-{alpha} gene expression with the ubiquitin ligases suggest that they can participate in skeletal muscle alterations during chronic arthritis.

growth hormone-releasing peptide-2; atrogenes; insulin-like growth factor I; insulin-like growth factor-binding protein-5; tumor necrosis factor; arthritic rats


ADJUVANT-INDUCED ARTHRITIS is a well-established model of rheumatoid arthritis that can be induced in rats by an intradermal injection of Freund's adjuvant. Between 10 and 14 days after adjuvant injection, rats start to develop external signs of inflammation and have a dramatic decrease in body weight gain and cachexia (48). Similarly, rheumatoid arthritis patients have cachexia hypermetabolism and accelerated protein breakdown (47). Muscle wasting results in weakness and in an increase in the risk of many alterations, such as inadequate gas exchange or vein thrombosis in lower limbs. Accordingly, it has been postulated that rheumatoid cachexia is an important contributor in increasing morbidity and premature mortality in rheumatoid arthritis patients (59).

Skeletal muscle atrophy is a debilitating response that occurs in many chronic diseases, such as cancer, sepsis, heart or renal failure, and diabetes. Although adjuvant arthritis decreases food intake, cachexia is not the result of the decrease in caloric intake (48). Chronic arthritis decreases serum concentrations of IGF-I and its mRNA in the liver (37). This decrease is not secondary to modifications in food intake, since pair-fed rats have normal serum IGF-I and IGF-I gene expression in the liver (38). In addition, growth hormone (GH) administration to arthritic rats results in an increase in body weight gain along with an increase in IGF-I gene expression in the liver without modifying food intake (28, 39). These data suggest that the weight loss during chronic inflammation may result, at least in part, from disturbances in anabolic hormones such as IGF-I and GH.

The effect of IGF-I on cellular growth and differentiation can be exerted through endocrine or paracrine/autocrine pathways. In chronic heart failure-induced cachexia, the disturbances in skeletal muscle are associated with a decrease in local expression of IGF-I, whereas serum concentrations of IGF-I are not modified (50). The actions of IGF-I are modulated locally by the IGF-binding protein IGFBPs. A downregulation of IGFBP-5 has been reported by microarray studies in several models of muscular wasting (33). However, skeletal muscle hypertrophy induced by overloading is associated with an increase in IGF-I and with a decrease in IGFBP-5 gene expression, whereas unloading is associated with muscular atrophy and an increase in IGFBP-5 expression (2). These data indicate that modifications in local IGF-I and/or IGFBP-5 may be one of the mechanisms of skeletal muscle dysfunction.

Regardless of the illness, skeletal muscle atrophy is associated with an increase in protein degradation (for review, see Ref. 21). Most of the proteolysis in cachexia-induced skeletal muscle atrophy seems to be because of activation of the ATP-dependent ubiquitin-proteasome proteolytic pathway (30). Ubiquitin is a short peptide that can be conjugated to specific substrates. Conjugation of ubiquitin to proteins occurs in a series of steps involving several enzymes, E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme, and E3 ubiquitin-ligating enzyme. The key enzymes in this process are E3 ubiquitin ligases, and they confer substrate specificity. Among these, two are increased during skeletal muscle atrophy [muscle ring finger 1 (MuRF1) and muscle atrophy F-box (MAFbx) or Atrogin-1 (5, 23)]. The gene expressions of these E3 ubiquitin ligases in the skeletal muscle are upregulated in several types of cachexia, including sepsis (33, 58). Therefore, it has been proposed that mRNA for these E3 ubiquitin-ligating enzymes are the most sensitive markers for muscular atrophy. Taking into account that administration of an inhibitor of the proteasome activity has an anti-inflammatory effect in arthritic rats (42), it is possible that in arthritic rats the ubiquitin-proteasome proteolytic pathway is upregulated as a result of inflammatory stimuli.

Ghrelin, a 28-amino acid endocrine peptide, is mainly secreted by the stomach and has been identified as the endogenous ligand of the GH secretagogues receptor. Ghrelin has a potent GH-releasing effect but stimulates food intake and promotes adiposity by a GH-independent action (53). We have recently reported that, during the active phase of arthritis, administration of growth hormone-releasing peptide-2 (GHRP-2), a synthetic ghrelin receptor agonist, reduced the symptoms of arthritis and the serum concentration of IL-6 (24). The anti-inflammatory effect of ghrelin has also been reported in human and rodent macrophages and lymphocytes (18, 61). In addition, ghrelin has been suggested as a treatment to prevent cachexia resulting from different illnesses (29). Moreover, peripheral administration of ghrelin attenuated body weight loss in several models of cachexia, such as chronic heart failure (40), lipopolysaccharide (LPS) injections (26), and cancer (25).

The aim of this work was to study the effect of adjuvant-induced arthritis on IGF-I and IGFBP-5 and the ubiquitin ligases, MuRF1 and MAFbx gene expression in skeletal muscle, as well as to examine whether these changes can be affected by administration of the ghrelin receptor agonist GHRP-2.


    MATERIAL AND METHODS
 TOP
 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
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Animals and experimental protocol. Control and arthritic male Wistar rats were purchased from Charles River (Barcelona, Spain). Arthritis was induced by an intradermal injection of a suspension of 1 mg Freud's adjuvant in the right paw, under ketamine (75 mg/kg) plus diazepam (5 mg/kg) anesthesia. They were housed 3–4/cage, under controlled conditions of temperature (22°C) and light (lights on from 0730 to 1930). Food and water were available ad libitum. The procedures followed the guidelines recommended by the European Union for the care and use of laboratory animals and were approved by the animal care committee of the Universidad Complutense.

On day 15 after adjuvant injection, 24 arthritic and 20 control rats were randomly divided into the following two groups: one group was subcutaneously injected daily with 100 µg/kg GHRP-2 (Bachem, Bubendorf, Switzerland) from days 15 to 22, and the second group received 250 µl of saline. GHRP-2 administration had an anti-inflammatory effect, since the paw volume and the arthritis score were lower in the arthritic rats injected with GHRP-2 than in the arthritic rats injected with saline (24).

All rats were killed by decapitation 22 days after adjuvant or vehicle injection and after 8 days of GHRP-2 treatment, 2.5 h after the last injection in a separated room, within 30 s after being removed from their cages. Trunk blood was collected in cooled tubes, and the serum was stored at –20°C until IGF-I analysis was performed. Immediately after decapitation, the liver, heart, and gastrocnemius muscle were removed and stored at –80°C.

RNA extraction. RNA was extracted by the guanidine thiocyanate method using a commercial kit (Ultraspec RNA; Biotecx Laboratories, Houston, TX). The integrity and the concentration of the RNA were confirmed using agarose gel electrophoresis.

Northern blot analysis. RNA (20 µg) from the skeletal and cardiac muscles and 30 µg from the liver were separated by formaldehyde-agarose gel electrophoresis and transferred to nylon membranes (Hybond-N+; Amersham).

The rat growth hormone receptor (GHR) and IGF-I probes (4, 46) were derived from a HindIII fragment of the pGEM-3 plasmid vector (Promega, Madison, WI). 32P-labeled RNA antisense probes were generated from linearized plasmid with [{alpha}-32P]cytidine triphosphate (Nuclear Ibérica, Madrid, Spain) and T7 RNA polymerase (Roche Molecular Biochemicals, Barcelona, Spain). Prehybridization was performed for 30 min at 68°C in ULTRAhyb buffer (Ambion, Austin, TX) followed by hybridization for 16 h at the same temperature with IGF-I- and GHR-labeled riboprobes. To verify loading, control hybridization was performed with a 28S DNA probe labeled with [32P]deoxycytidine triphosphate by random primer.

Autoradiographs were analyzed by densitometric scanning using a Gengenius (Syngene, Cambridge, UK). The rat IGF-I gene gives different IGF-I mRNA transcripts that can be visualized by Northern blot analysis and consist of a group of transcripts ranging from 7.5 to 0.9 kb. Because all these transcripts may potentially be translated to IGF-I, the densitometric results correspond to the sum of the IGF-I transcripts 0.9, 1.7, and 7.5 kb. The rat GHR gene encodes the GHR and the GHBP mRNA of 4.5 and 1.2 kb; both transcripts were quantified by densitometric analysis, and results refer to the total GHR mRNA. The intensities of autoradiogram signal levels were normalized for 28S ribosomal RNA levels.

Real-time PCR. For RT-PCR analysis, 10 µg of cardiac or skeletal muscle total RNA were reverse transcribed in a total volume of 30 µl at 37°C for 60 h with 125 units of Moloney murine leukemia virus RT (Maxim Bioch, San Francisco, CA). Each RT-PCR reaction consisted of 312 ng total RNA equivalents, 1x QuantiTect SYBR Green Master Mix (Qiagen, Valencia, CA), and 300 nM forward and reverse primers in a reaction volume of 25 µl. Reactions were carried out on a SmartCycler (Cepheid, Sunnyvale, CA). Primers for PCR (Table 1) were obtained from previously published sequences tumor necrosis factor-{alpha} (TNF-{alpha}) and MuRF1 (14) r18S (3) or by using the rat GenBank and the EXIQON ProbeLibrary IGFBP-5 and MAFbx. Primers were designed to span a single sequence derived from two exons (i.e., separated by an intron in genomic DNA and primary RNA transcripts to minimize amplification). Parameters included an initial activation of hotStar Taq DNA polymerase at 95°C for 15 min, followed by 40 cycles of denaturation at 94°C for 15 s, annealing at 53°C (MuRF1), 57°C (TNF-{alpha}), 52°C (MAFbx), 53°C (IGFBP-5), and 60°C (r18S) for 30 s, and extension at 72°C for 30 s. Specific amplification was confirmed by the presence of one single peak in the melting curve plots. In addition, the PCR products were analyzed in agarose gel electrophoresis. Results were calculated as percentage of control rats injected with saline, using the {Delta}{Delta}CT method (36) with r18S as the control gene.


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Table 1. Primers for real-time PCR

 
RIA. IGF-I concentrations were measured by a double-antibody RIA using the antibody National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) UB2–495 distributed by the Hormone Distribution Program of NIDDK through the National Hormone and Pituitary Program, which was a gift from Drs. L. Underwood and J. Van Wyk. Levels of IGF-I were expressed in terms of IGF-I from Gropep (Adelaide, Australia). The intra-assay coefficient of variation was 8%. All samples were run in the same assay.

Protein content. Tissues were homogenized in 1 N acetic acid, and protein content was measured by the bicinchoninic acid method (Pierce, Rockford, IL).

Statistical analysis. Statistics were computed using the statistics program STATGRAPHICS plus for Windows. Statistical significance was calculated by multifactorial ANOVA with arthritis and GHRP-2 administration as factors. Post hoc comparisons were made by using the unpaired Student's t-test. Correlation between different variables was calculated by linear regression. P values <0.05 were considered significant.


    RESULTS
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 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
As shown in Table 2, arthritis induced a marked decrease in gastrocnemius weight (P < 0.01) and also decreased the protein concentration in this muscle (P < 0.05), whereas GHRP-2 administration to arthritic rats increased the weight and protein concentration of this skeletal muscle (P < 0.05).


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Table 2. Effect of arthritis and 8-day administration of GHRP-2 on gastrocnemius weight and protein concentration in gastrocnemius, heart, and liver

 
Arthritis induced a significant increase in the two ubiquitin ligases MAFbx and MuRF1 gene expression in the skeletal muscle (P < 0.01; Fig. 1), and GHRP-2 administration to arthritic rats prevented the effect of arthritis on both mRNAs. There was also a significant increase (P < 0.05) in TNF-{alpha} mRNA in the skeletal muscle in the arthritic rats injected with saline, whereas GHRP-2 administration reverted the effect of arthritis on TNF-{alpha} mRNA levels in the skeletal muscle, reaching levels similar to those observed in control rats (Fig. 1).



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Fig. 1. Muscle ring finger 1 (MuRF1), muscle atrophy F-box (MAFbx), and tumor necrosis factor-{alpha} (TNF-{alpha}) gene expression in skeletal muscle of control or arthritic (AA) rats injected with saline or 100 µg/kg growth hormone-releasing peptide-2 (GHRP-2) for 8 days. MuRF1, MAFbx, and TNF-{alpha} mRNA were quantified using real-time RT-PCR and are presented as a percentage of the mean value in control rats treated with saline by analyzing the critical threshold (CT) numbers corrected by CT readings of corresponding internal 18S rRNA controls. There was an interaction between the effects of arthritis and GHRP-2 administration on MuRF1 [F(1,36) = 6.2, P < 0.05] and MAFbx [F(1,33) = 7.2, P < 0.05] gene expression in the skeletal muscle, since arthritis increased MuRF1 and MAFbx mRNA in saline but not in GHRP-2-treated rats. Arthritis increased the TNF-{alpha} mRNA in skeletal muscle [F(1,30) = 4.36 P < 0.05], whereas GHRP-2 administration decreased the TNF-{alpha} mRNA [F(1,30) = 3.91 P < 0.05]. Results represent means ± SE for 7–10 rats/group. *P < 0.05 and **P < 0.01 vs. control. °P < 0.05 and °°P < 0.01 vs. AA-saline.

 
As shown in Fig. 2, arthritis decreased the serum concentrations of IGF-I (P < 0.01), and GHRP-2 administration increased the serum concentrations of IGF-I both in control (P < 0.05) and in arthritic (P < 0.05) rats. Serum concentration of GH was not significantly modified by arthritis or by GHRP-2 administration (data not shown). Arthritis decreased the IGF-I mRNA in the liver (P < 0.01; Fig. 2), and GHRP-2 administration increased the IGF-I gene expression in the liver in control and in arthritic rats (P < 0.05). There was a positive correlation between the serum concentrations of IGF-I and the liver IGF-I mRNA (r = 0.68, P < 0.01), whereas there was no correlation between the serum concentrations of IGF-I and the IGF-I mRNA in cardiac or in skeletal muscle. The GHR mRNA in the liver was not modified by arthritis or by GHRP-2 treatment (data not shown). Both arthritis and GHRP-2 administration modified the protein content in the liver (Table 2). Arthritis increased the protein concentration in the liver (P < 0.01), whereas GHRP-2 administration decreased it (P < 0.01).



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Fig. 2. Serum concentrations of IGF-I (A) and IGF-I mRNA in the liver (B) in control or arthritic rats injected with saline or 100 µg/kg GHRP-2 for 8 days. D: representative Northern blot of IGF-I mRNA hybridization of total liver RNA. The size of the hybridization band (in kb) is indicated on left; 28S, 28S ribosomal RNA; C, control, S, saline; G, GHRP. Quantitative analyses are expressed as percentages of control rats injected with saline. 28S mRNA expression was used as a control for mRNA. Arthritis decreased the serum concentrations of IGF-I [F (1,35) = 37, P < 0.01] and the IGF-I mRNA in the liver [F(1,37) = 16.8, P < 0.01]. GHRP-2 administration increased IGF-I levels in serum [F(1,35) = 7.4, P < 0.01] and IGF-I mRNA in liver [F (1,37) = 7.33, P < 0.01]. There was a significant correlation [r = 0.68; F (1,32) = 27, P < 0.01] between serum concentrations of IGF-I and liver IGF-I mRNA (C). Values shown are means ± SE of 9–11 rats/group. **P < 0.01 and *P < 0.05 vs. control-saline. ++P < 0.01 vs. control-GHRP-2. °P < 0.05 vs. AA-saline.

 
The effects of arthritis and GHRP-2 on IGF-I mRNA in the cardiac muscle are shown in Fig. 3. Arthritis did not modify the IGF-I expression in the cardiac muscle. In contrast, GHRP-2 administration increased (P < 0.05) the IGF-I mRNA in the cardiac muscle, but this increase was not significant when the individual means were analyzed separately. Neither arthritis nor GHRP-2 administration modified the GHR or MuRF1 gene expression in the cardiac muscle (data not shown).



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Fig. 3. Effect of 8-day administration of GHRP-2 (100 µg/kg sc) or saline (250 µl) to control or arthritic rats on IGF-I mRNA in cardiac muscle. A representative Northern blot analysis showing the 7.5, 1.7, and 0.9 kb IGF-I transcripts and the 28S ribosomal RNA in each sample is shown on bottom. Data from 9–10 individual rats were quantified by densitometry and expressed as a percentage of the mean value in control rats treated with saline. Arthritis did not modify IGF-I gene expression in the cardiac muscle. GHRP-2 administration increased IGF-I mRNA in cardiac muscle [F(1,35) = 4.8, P < 0.05].

 
The effect of arthritis on IGF-I mRNA in skeletal muscle was opposite to that in the liver, since the IGF-I mRNA in the skeletal muscle was higher in arthritic than in control rats (P < 0.01; Fig. 4). GHRP-2 administration increased the IGF-I mRNA in skeletal muscle in control rats (P < 0.01). Neither arthritis nor GHRP-2 treatment induced significant modifications in GHR gene expression in the skeletal muscle (Fig. 4). Arthritis induced an increase in IGFBP-5 mRNA (P < 0.01) in the skeletal muscle, whereas GHRP-2 administration decreased the IGFBP-5 mRNA both in control (P < 0.05) and in arthritic (P < 0.05; Fig. 4) rats.



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Fig. 4. Growth hormone receptor (GHR), IGF-I, and IGF-I-binding protein-5 (IGFBP-5) gene expression in skeletal muscle of control or arthritic rats injected with saline or 100 µg/kg GHRP-2 for 8 days. A: IGF-I mRNA levels; data from 8–11 individual rats were quantified by densitometry and expressed as a percentage of the mean value in control rats treated with saline. A representative Northern blot analysis showing the 7.5-, 1.7-, and 0.9-kb IGF-I transcripts and the 28S ribosomal RNA in each sample is shown on bottom. B: GHR mRNA levels; data from 6–10 individual rats were quantified by densitometry and expressed as a percentage of the mean value in control rats treated with saline. A representative Northern blot analysis showing the 4.5- and 1.2-kb GHR transcripts and the 28S ribosomal RNA in each sample is shown on bottom. C: IGFBP-5 mRNA; data from 8–11 rats were quantified using real-time RT-PCR and are presented as a percentage of the mean value in control rats treated with saline by analyzing the CT numbers corrected by CT readings of the corresponding internal 18S rRNA controls. Both arthritis and GHRP-2 administration induced an increase in IGF-I mRNA [F(1,38) = 10, P < 0.01; F (1,38) = 4.5, P < 0.05, respectively]. Arthritis increased IGFBP-5 mRNA [F (1,35) = 13.3, P < 0.01], whereas GHRP-2 decreased IGFBP-5 mRNA [F(1,35) = 6.5, P < 0.05]. *P < 0.05 and **P < 0.01 vs. control-saline. #P < 0.05 vs. control-GHRP-2. °P < 0.05 vs. AA-saline.

 

    DISCUSSION
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 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
In the present study, chronic arthritis resulted in an increase in gene expression of the ubiquitin ligases E3, MuRF1, and MAFbx as well as in TNF-{alpha} and IGFBP-5 in the skeletal muscle, and these upregulations were blocked by treatment with the ghrelin analog GHRP-2. These findings suggest that, in addition to its anti-inflammatory effect in arthritic rats, the synthetic ghrelin analog is also able to decrease arthritis-induced muscle proteolysis.

Chronic arthritis induced a decrease in the weight and protein concentration of the skeletal muscle along with an increase in the expression of ubiquitin ligases MuRF1 and MAFbx. These data suggest that the decrease in skeletal muscle weight is secondary to an enhanced protein degradation pathway, the ubiquitin-proteasome proteolytic pathway. MuRF1 and MAFbx belong to a common set of genes, called atrogenes, that are induced in muscles as a result of a common transcriptional program that is activated in many systemic diseases, leading to a catabolic state and muscle atrophy (33). However, arthritis did not modify the protein concentration or MuRF1 gene expression in the cardiac muscle. Similarly, fasting increases MAFbx in the gastrocnemius but not in the cardiac muscle (23).

The effect of chronic arthritis on IGF-I gene expression is different depending on the tissue analyzed. The correlation between serum IGF-I and IGF-I mRNA in the liver along with the lack of correlation between the IGF-I mRNA in the cardiac or the skeletal muscles are in accordance with previous studies demonstrating that circulating IGF-I is mostly derived from the liver (62). Moreover, IGF-I-induced somatic growth is also mediated by local production of IGF-I, which acts in an autocrine/paracrine manner (34). One mechanism that can increase IGF-I gene expression in muscle is work overload (22). This is not the case in arthritic rats, since their motor activity is not increased; on the contrary, it seems to be decreased.

The role of IGF-I in muscle atrophy is not well known. A decrease in IGF-I mRNA in the skeletal muscle has been detected in muscular wasting induced by hindlimb suspension (2), sepsis (14), and chronic heart failure (50). However, an increase has also been reported in muscular atrophy induced by denervation, chronic human disuse, or chronic skeletal muscle ischemia in humans (9, 45, 55). In addition, no changes in IGF-I expression in the skeletal muscle were detected by microarray analysis in skeletal muscle atrophy induced by cancer, diabetes, or uremia (33). Furthermore, IGF-I overexpression in transgenic mice did not prevent muscle atrophy induced by hindlimb unloading (12). It is possible that the increased muscular IGFBP-5 binds IGF-I and inhibits IGF-I action by preventing its interaction with the type I receptor, as it has been previously reported (31).

Different IGFBP-5 effects on cell proliferation have been reported depending on the tissue and the circumstances. Transgenic mice overexpressing IGFBP-5 are growth retarded and have a decreased skeletal muscle weight relative to body weight (49). Moreover, local application of IGFBP-5 to paralyzed muscle prevents the stimulation of interstitial cell proliferation (9). In contrast, antiapoptotic stimulation of cell survival effects of IGFBP-5 have also been reported in myoblasts and chondrocytes (10, 32). Both an increase and a decrease in muscular IGFBP-5 mRNA in muscular atrophy have been reported (2, 33). Similarly to our data, in chondrocytes isolated from osteoarthritic cartilage, IGF-I and IGFBP-5 gene expressions are increased (41). In arthritis, the increase in IGFBP-5 can be secondary to the increase in cytokine release, since IL-1 and TNF-{alpha} are able to stimulate IGFBP-5 in articular chondrocytes (51).

Cytokines also modulate the activity of the ubiquitin pathways in situations such as cancer and sepsis (1). Taking into account that TNF-{alpha} activates nuclear factor-{kappa}B, and this activation promotes muscle atrophy through MuRF1 upregulation (8), the increased MuRF1 and MAFbx gene expressions in the arthritic rats can be because of the marked increase in TNF-{alpha} gene expression in the skeletal muscle.

The gene expression of the proinflammatory cytokine TNF-{alpha} in the skeletal muscle increases in several skeletal muscle alterations, such as diabetes, sepsis, and chronic heart failure (14, 15, 50). It has been postulated that TNF-{alpha} exerts its catabolic effects in skeletal muscle in a paracrine/autocrine manner (20). TNF-{alpha} influences both muscle protein synthesis and degradation (58). TNF-{alpha} blockade prevents muscle protein breakdown by suppressing the activation of the ubiquitin-proteasome-dependent proteolysis in cancer and septic rats (11, 37, 58).

A possible cause of the IGF-I resistance in the muscle of arthritic rats, in addition to IGFBP-5, can be the increase in TNF-{alpha} gene expression in the skeletal muscle. TNF-{alpha} is able to impair IGF-I-induced protein synthesis in myoblasts. This is not done at the level of receptor but rather by targeting some signaling proteins associated with the IGF-I receptor (6). IGF-I resistance in the muscle at the postreceptor level has also been described in septic rats, in which IGF-I is not able to reduce muscle proteolysis in vivo or in vitro (19, 27).

The IGF-I response to GHRP-2 is small and more evident in control rats than in arthritic rats. Similarly, ghrelin administration two times a day for 5 days was unable to increase serum concentrations of GH or IGF-I in control or in LPS-injected rats (26). After GHRP-2 administration (1 h), no significant increases in serum concentration of IGF-I or insulin have been detected in normal mice (54). These data can be explained by the fact that the stimulatory effect of ghrelin or ghrelin receptor agonists on the GH-IGF-I system does not last very long. It has been postulated that ghrelin is not physiologically involved in the regulation of GH, since circulating levels of ghrelin are not correlated with those of GH either in physiological or in pathological conditions (52).

Arthritic rats showed an increase in the weight and protein concentration of gastrocnemius muscle after GHRP-2 administration. This suggests a reduction in proteolysis related to a reduction in MuRF1 and MAFbx gene expression. The small increase in serum concentrations in IGF-I along with the decrease in muscular IGFBP-5 and TNF-{alpha} after GHRP-2 administration in arthritic rats can contribute to the decrease in MuRF1 and MAFbx gene expression and the increase in skeletal muscle protein.

A wide variety of ghrelin effects has been described as GH independent. Chronic GHRP-2 administration does not modify serum levels of IGF-I, insulin, and glucose, although it increases body weight gain and increases caloric intake (53). Treatment with ghrelin receptor agonists protects the heart from ischemia reperfusion damage (13) through a GH-independent action (38). A protective effect of GHRP-2 administration on proteolysis during critical illness has been previously reported in humans and was not related to the changes observed within the somatotropic axis (57).

The possibility exists that GHRP-2 has a direct effect on the skeletal muscle. Binding of ghrelin agonists in the skeletal muscle have been reported (43). In vitro application of ghrelin agonists or ghrelin modulates the chloride and potassium conductances in rat skeletal muscle, indicating that ghrelin directly affects skeletal muscle function and the presence of ghrelin receptors in this tissue (44). These effects of ghrelin on skeletal muscle are not mediated through the GH/IGF-I axis.

The inhibitory effect of GHRP-2 on TNF-{alpha} gene expression in the skeletal muscle is in accordance with other anti-inflammatory effects previously described for ghrelin and synthetic ghrelin receptor agonists. Ghrelin is able to prevent the LPS-induced increase in TNF-{alpha} gene expression in the spleen and liver (18). Ghrelin decreases circulating cytokines in pancreatitis (16), in arthritis (24), and in sepsis induced by LPS injection (35). This anti-inflammatory effect is independent of pituitary GH secretion, since ghrelin or ghrelin receptor agonists are able to decrease cytokine production in endothelial or immune cells in culture (18, 24, 35, 61). These inhibitory effects of cytokine production in vitro do not seem to be mediated by GH, since this hormone has a stimulatory effect on cytokine activity (17, 56).

In conclusion, this study demonstrates that arthritis induces a marked decrease in skeletal muscle weight along with an activation of the ubiquitin-proteasome proteolytic pathway, whereas administration of GHRP-2, the synthetic ghrelin analog, prevents these effects. Both the effect of arthritis and the GHRP-2 administration on the skeletal muscle can be mediated by changes in muscular IGFBP-5 and TNF-{alpha} gene expression.


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 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
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This work was supported by Programa Nacional de Promoción General del Conocimiento, Plan Nacional de Investigación Científica, from Ministerio de Educación Ciencia (BFI-2003–02149) and a Fellowship from Ministerio de Educación y Ciencia to M. Granado (Formación del Profesorado universitario, AP2003–2564).


    ACKNOWLEDGMENTS
 
We thank Dr. D Sanz-Rosa for help in the setting of the PCR. We are indebted to A. Carmona for technical assistance and to C. Bickart for the English correction of the manuscript. We are grateful to the U.S. National Institute of Diabetes, Digestive, and Kidney Diseases, National Hormone and Pituitary Program for the IGF-I antibody.


    FOOTNOTES
 

Address for reprint requests and other correspondence: A. López-Calderón, Dept Fisiología, Fac Medicina, Univ Complutense, 28040, Madrid (e-mail: ALC{at}med.ucm.es)

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.


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