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Am J Physiol Regul Integr Comp Physiol 284: R1277-R1286, 2003. First published December 19, 2002; doi:10.1152/ajpregu.00644.2002
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Vol. 284, Issue 5, R1277-R1286, May 2003

Prolonged fasting and cortisol reduce myostatin mRNA levels in tilapia larvae; short-term fasting elevates

Buel D. Rodgers1, Gregory M. Weber2, Kevin M. Kelley3, and Michael A. Levine1

1 Department of Pediatrics, Division of Endocrinology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21208; 2 US Department of Agriculture/Agricultural Resource Service, National Center for Cool and Cold Water Aquaculture, Kearneysville, West Virginia 25430; and 3 Department of Biological Sciences, California State University at Long Beach, Long Beach, California 90840


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Myostatin negatively regulates muscle growth and development and has recently been characterized in several fishes. We measured fasting myostatin mRNA levels in adult tilapia skeletal muscle and in whole larvae. Although fasting reduced some growth indexes in adults, skeletal muscle myostatin mRNA levels were unaffected. By contrast, larval myostatin mRNA levels were sometimes elevated after a short-term fast and were consistently reduced with prolonged fasting. These effects were specific for myostatin, as mRNA levels of glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphatase were unchanged. Cortisol levels were elevated in fasted larvae with reduced myostatin mRNA, whereas in addition immersion of larvae in 1 ppm (2.8 µM) cortisol reduced myostatin mRNA in a time-dependent fashion. These results suggest that larval myostatin mRNA levels may initially rise but ultimately fall during a prolonged fast. The reduction is likely mediated by fasting-induced hypercortisolemia, indicating divergent evolutionary mechanisms of glucocorticoid regulation of myostatin mRNA, since these steroids upregulate myostatin gene expression in mammals.

muscle growth and development; Oreochromis mossambicus; growth/differentiating factor-8


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MEMBERS OF THE TRANSFORMING GROWTH FACTOR-beta (TGFbeta ) superfamily of hormones and cytokines help to regulate the growth and development of many diverse tissues and cell types, including skeletal muscle (18, 57). The biological actions of these extracellular peptides are equally diverse and can either activate or suppress cellular growth or differentiation, depending on the specific factor, the time of its release, and the differentiation status of the affected cell. Myostatin, a member of this superfamily, specifically inhibits myoblast proliferation via cell-cycle arrest within the G1 and G2 phases and may therefore initiate myoblast differentiation (39, 52). This explains, in part, the markedly increased skeletal muscle mass observed in myostatin-null mice and in cattle that possess mutant alleles for this cytokine (13, 14, 22, 29, 31, 49).

Since the initial characterization of mouse myostatin (29), dozens of additional cDNA isoforms have been cloned from different vertebrates, including several mammalian, avian, and fish species (10, 12, 22, 30, 31, 41, 44, 45). Most of these orthologs were isolated from commercially important species, because the successful manipulation of the cytokine's actions or its availability has the potential to profoundly impact the agricultural and economic communities that rely on these commodities. Comparative and applied scientists with interests in aquaculture appear to be particularly interested, as 17 of the 21 most recent myostatin sequences submitted to GenBank were isolated from commercially important fish, including that of the tilapia, Oreochromis mossambicus, which was the first such ortholog to be identified (45).

Unlike in mammals, where its expression occurs primarily in skeletal muscle, myostatin is expressed in a variety of fish tissues (41, 45). Although little is known regarding the regulation of its expression independent of the tissue or the species, it appears to be developmentally sensitive in both mammals and fish (21, 29, 45). Furthermore, myostatin production increases with hindlimb unloading and in regenerating rodent skeletal muscle (4, 55, 56). Yamanouchi et al. (56) further showed that extracts from bupivacaine-treated (regenerating) muscle stimulated a dose-dependent increase of myostatin mRNA levels in primary fibroblasts isolated from adult skeletal muscle biopsies. Whether or not increased myostatin production contributes to skeletal muscle atrophy during hindlimb unloading cannot be determined, as the rise could be a regenerative response as well. Nevertheless, its expression appears to be influenced by factors and/or signals that are related to skeletal muscle conditioning, possibly including cytokines and hormones.

Nutritional status has profound effects on the growth and development of somatic tissues, particularly skeletal muscle. We therefore sought to determine whether myostatin mRNA levels were altered in fasting adult and larval tilapia. The results presented herein indicate that prolonged fasting simultaneously reduces somatic growth and myostatin mRNA levels in larvae, albeit after an initial rise in myostatin mRNA. The exogenous administration of cortisol, a stress hormone whose circulating concentrations are known to rise in many different fasting vertebrates, including in the fasted larvae of these studies, also reduced whole larval myostatin mRNA, suggesting that hypercortisolemia could contribute to the effects of a prolonged fast.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animal Studies

The tilapia used in these studies were originally obtained from Oahu, Hawaii. All experiments were performed in accordance with the Animal Care and Use Committee of the Johns Hopkins University School of Medicine under a preapproved protocol. Fish were maintained in recirculating fresh-water systems at 26.5°C, exposed to a 12:12-h light-dark photoperiod, and fed to satiation once daily with Southern States 8500, protein 38%. Tilapia larvae were sampled from stocks used to generate animals for research. Once a brood was detected, it was removed from the females and maintained in a 4-liter tank at 26.5°C and fed Tetramin tropical fish food.

Experiment 1: effects of fasting on adult tilapia. Adult male fish weighing 39.7 ± 2.0 g (mean ± SE) were divided into two groups of five fish each and were either fed or fasted for 28 days. At this time, fish were anesthetized by submersion in 100 mg/l 3-aminobenzoic acid ethyl ester methanesulfonate, (Tricaine), weighed, measured (standard length), and decapitated. Liver and gonads were removed to determine the hepatosomatic and gonadosomatic indexes, respectively, and a single biopsy of white axial skeletal muscle was obtained and snap-frozen in liquid nitrogen. Skeletal muscle myostatin mRNA levels were determined by RT-PCR, as described in Semiquantitative RT-PCR.

Experiment 2a and 2b: effects of fasting on tilapia larvae. For experiment 2a, two separate clutches of larvae were then divided in half at yolk sac absorption (YSA), producing four groups (see outline of experimental design in Fig. 1). Starting 5 days post-YSA, a group from each clutch was then either fed or fasted for 6 days. At least five larvae from each group were removed after 0, 3, and 6 days, anesthetized with Tricaine, weighed individually, and frozen in liquid nitrogen. Before being frozen, all individuals from a group were pooled, and myostatin mRNA levels were eventually measured on the pooled sample, as tank effect would be shared by all individuals within a particular tank. Individual variance was therefore diluted within the group. Treatment groups from each clutch were analyzed separately to account for variance between the groups. Larvae were also removed at these time points for cortisol measurements (3 larvae/clutch = 6 total for each time point in each fed and fasted group), although these animals were not pooled. An additional three larvae were also removed from the 6-day groups, decapitated, gutted, and filleted before being frozen to measure myostatin mRNA levels in the heads and bodies of fed and fasted larvae. In each experiment, fed animals were not fed within 4 h of sampling. Experiment 2b was a repeat of experiment 2a, with a third clutch of larvae; however, individuals were separated so that myostatin mRNA levels could be measured in each larva rather than on pooled samples.


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  		Fig. 1.   Experimental design of larval fasting experiments 2a and 2b. A: 1st larval fasting experiment 2a with clutches 1 and 2. Each arrow represents a different group of larvae. Each clutch was divided in half and was either fed or fasted for 3 or 6 days. Body weights, pooled whole larvae (WL) myostatin (MSTN) mRNA, and individual WL cortisol levels were determined on days 3 and 6, and MSTN mRNA levels were additionally measured in larval heads and in gutted bodies on day 6. B: larval fasting experiment 2b, with clutch 3. Larvae were divided and treated as described above, although the experimental period was extended from 6 to 9 days. Body weights were measured at both time points, as were individual mRNA levels for MSTN, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and glucose-6-phosphatase (G-6-Pase), all of which were normalized to 18S rRNA levels.

Experiment 3: effects of cortisol treatment on myostatin message in larvae. In parallel experiments, larvae were also placed into four 0.5-liter nonrecirculating and aerated tanks (n = 5/tank) containing 0, 0.01, 0.1, and 1 ppm (2.8 µM) cortisol (initially dissolved in ethanol) for periods of 3 and 6 h. The control group (0 ppm) received ethanol alone. This experiment was repeated two additional times with two and three larvae per group. Whole larvae were then anesthetized with Tricaine and snap-frozen. Myostatin mRNA levels in whole larvae and in larval heads and gutted bodies were determined by semiquantitative RT-PCR as described in Semiquantitative RT-PCR.

Isolation of Partial cDNA Clones for Tilapia "Housekeeping" Gene

In tilapia, genes suitable for normalization purposes have not been identified to date. Therefore, partial clones were isolated by using degenerate and conserved primers in an RT-PCR assay. A multiple-sequence alignment of several known vertebrate homologs of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glucose-6-phosphatase (G-6-Pase), and 18S ribosomal RNA was performed using MacVector 7.0. The resulting primer sequences and their corresponding annealing temperatures are included in Table 1. Tilapia skeletal muscle cDNA was amplified for 30 cycles of 94°C for 30 s, 52°C for 30 s, and 72°C for 1 min. The GAPDH, G-6-Pase, and 18S amplicons were then gel purified, subcloned into the pCR4-TOPO TA cloning vector (Invitrogen), and sequenced at the Johns Hopkins University School of Medicine's CORE facility. Contig assembly, BLAST analysis, and multiple-sequence comparisons of the novel clones were performed using MacVector 7.0. 

                              
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  		Table 1.   GAPDH, G-6-Pase, 18S and myostatin primer pairs

Semiquantitative RT-PCR

Total RNA was isolated from previously frozen adult skeletal muscle biopsies or from whole larvae by using Trizol reagent (GIBCO-BRL Life Technology). First-strand cDNA was reverse transcribed using 5 µg of total RNA from the biopsies and 1 µg from the larval pools by use of random hexamer primers. The linear portion of the amplification curves for myostatin, GAPDH, and G-6-Pase were simultaneously determined by cycle titration PCR with pooled first-strand cDNA from both fed and fasted groups. Aliquots of 10 µl were removed after 30, 35, 40, and 45 cycles of 94°C for 30 s, 54°C for 30 s, and 72°C for 1 min. The linear portion of the amplification curve for 18S was similarly defined at 10, 15, 20, and 25 cycles. The optimal number of cycles used for the myostatin, GAPDH, and G-6-Pase reactions was 40, whereas the optimal number for the 18S reaction was 20. Amplicons were isolated by electrophoresis through 1.5% Tris-acetate-EDTA-agarose gels containing Vistagreen (Amersham Life Science), and band intensities were quantified electronically with a fluorescence imager (Bio-Rad).

Because of primer incompatibility, a PCR mastermix was constructed that lacked primers. From this mix, 94 µl were first aliquoted, and 4 µl of cDNA from each sample were then added to ensure equal cDNA loading. Individual aliquots were then divided in half, and primers were subsequently added (1 µl of a primer pair at a stock concentration of 10 µM into 50 µl of total reaction volume). To this end, cDNA from each sample was then amplified for an appropriate number of cycles, and myostatin, GAPDH, and G-6-Pase values were normalized to those of 18S, thus limiting intra-assay variability due to loading errors and RNA quality.

Cortisol ELISA and Sample Extraction

The cortisol 3-carboxymethyloxime (CMO) antiserum and the enzyme conjugate, cortisol-3-CMO-horseradish peroxidase (HRP), were purchased from Dr. Coralie Munro of the University of California at Davis (Dept. of Reproduction, School of Veterinary Medicine). The cortisol ELISA based on these reagents has been previously validated for use on fish sera (2). The ELISA was conducted following the procedures described by Barry et al. (2) with the following modifications. The tilapia larvae were extracted as described below, the extracts were diluted in cortisol-HRP conjugate solution, and the antiserum was diluted 1:8,500 with coating buffer.

The frozen tilapia were extracted by following procedures similar to those previously described for extracting thyroid hormones from tilapia larvae (34) and for extracting cortisol from Japanese flounder larvae (16). Briefly, two frozen larvae weighing ~2 mg were weighed, placed in a 1.5-ml microcentrifuge tube with 100 µl of deionized water, and homogenized on ice with a Con-torque tissue grinder (Eberbach, Ann Arbor, MI) outfitted with a Teflon pellet pestle (Kontes Glass, Vineland, NJ) for 30 s. The sample was then sonicated on ice two times at force 2, 70% duty cycle, for 10 pulses with 15 s in ice between pulse sets with a Sonifier 450 (Branson Ultrasonics, Danbury, CT) outfitted with a microtip. [3H]cortisol (10,000 dpm; specific activity 69.0 Ci/mmol; Amersham Pharmacia Biotech, Piscataway, NJ) was added to each sample for the determination of extraction efficiency. The samples were vortexed and allowed to incubate at 4°C for 3 h. The samples were extracted three times with 0.5 ml of ice-cold absolute ethanol (Sigma-Aldrich). The ethanol supernatants were collected after centrifugation at 4°C at 3,110 g for 10 min. The combined supernatants were evaporated under a stream of air at room temperature and then reconstituted with 350 µl of carbon tetrachloride (CCl4; Sigma-Aldrich). The cortisol was recovered by adding 400 µl of assay buffer (PBS) without BSA to the CCl4, vortexing for 10 min, and then centrifuging as above. Extraction efficiencies averaged 83%.

The assay was validated for use with the tilapia extract. Tilapia larva extract diluted parallel to the standard curve (Fig. 2). Recoveries of hormone added to an extract pool were 98, 99, and 90% for 10, 25, and 100 pg, respectively (n = 3). One or nine microliters of CCl4 were added to two wells each of the assay and did not alter the measured cortisol concentration of the sample; 24.6 ± 2.8 ng/ml (mean ± SE) for extract pool, 22.2 ± 0.3 ng/ml for extract plus 1 µl of CCl4, and 23.1 ± 1.1 ng/ml for extract plus 9 µl of CCl4.


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  		Fig. 2.   Displacement curves for cortisol standards and serial dilutions of larval extract. Each point is the mean of triplicate determinations. Frozen tilapia were extracted by following procedures similar to those previously described (16, 34). Extraction efficiencies were determined by addition of [3H]cortisol to each sample and averaged 83%. Shown are tilapia larvae extracts diluted in parallel to the standard curve.

Statistical Analysis

After normalization, the arbitrary mRNA values were expressed as a percentage of the fed control values, because the actual values varied considerably between experiments and between gels. When appropriate, data were expressed as mean values ± SE, and differences between the means were determined either by a Student's t-test or by analysis of variance and the Bonferroni-Dunn post hoc test for multiple mean comparison with StatView 5.0 for the Macintosh.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Isolation of Tilapia GAPDH, G-6-Pase, and 18S cDNA Clones

As expected of housekeeping genes, each of the novel clones identified was highly homologous to related genes from other species (data not shown; see GenBank accession nos. below). BLAST analysis indicated that the partial tilapia 18S rRNA described (GenBank no. AF497908) is identical to that of another tilapian species, O. esculentus and is 99% homologous to red seabream, longspine thornyhead, and Atlantic salmon rRNA. The tilapia G-6-Pase sequence described (AY094487) is 98% homologous to that of a related cichlid, the fulu Haplochromis nubilus, and is 63% identical and 79% similar to the human amino acid sequence. Although GAPDH sequences from species closely related to O. mossambicus have yet to be described, tilapia GAPDH (AY140649) is highly homologous to many other vertebrate orthologs, including that of the rainbow trout, Onchorynchus mykiss. These sequences are, in fact, 85% homologous at the nucleic acid level, and their amino acid sequences are 92% identical and 97% similar.

Myostatin mRNA Levels and Somatic Growth in Fasting Tilapia

Among adults (experiment 1), fed fish gained an average of 3.75 g during the 28-day experimental period, whereas the fasted fish lost 4.44 g. This accounted for an ~20% difference in body weight between fed and fasted fish (Fig. 3). The condition factor (body wt/standard length3 × 100) and the hepatosomatic index (liver weight/body wt × 100) were both significantly reduced by fasting, although the body length and gonadosomatic index (gonad weight/body wt × 100) were not. Skeletal muscle myostatin mRNA levels were similarly unaffected by fasting and suggest that, in adult tilapia skeletal muscle, myostatin gene expression and/or mRNA turnover are insensitive to a moderate fast.


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  		Fig. 3.   Effects of fasting on adult tilapia (experiment 1). Body weight (BW), standard length (L), condition factor (CF), gonadosomatic index (GSI), hepatosomatic index (HSI), and skeletal muscle myostatin (MSTN) mRNA levels (via RT-PCR, as described in MATERIALS AND METHODS) of fed and 28-day-fasted fish were determined. Differences between means (±SE; n = 5) were determined by a t-test (*P <=  0.05).

The effects of fasting on larval growth and myostatin mRNA levels were determined in two separate sets of experiments with three distinct clutches of larvae originating from different breeding pairs. Quantifying individual responses to a particular stimulus ignores any unapparent effects that would presumably be shared by all fish within the same tank (i.e., tank effect). However, pooling samples similarly masks individual variance within a group. Therefore, changes in whole larval myostatin mRNA levels were first measured in total RNA extracted from pooled larvae (experiment 2a). The experiment was subsequently repeated by measuring myostatin mRNA levels individually (experiment 2b). In experiment 2a, the body weights of larvae fed for 3 days increased by 17%, whereas fasted weights decreased by 14% (Fig. 4). After 6 days, fed larval body weights increased by 86% and fasted weights decreased by 31%. Therefore, the inhibitory effects of fasting on somatic growth were much more severe in larvae than they were in adult fish. Fasting reduced myostatin mRNA levels from pooled larvae in a time-dependent manner that was comparable to the changes in body weight (Fig. 5). After 3 days of fasting, myostatin mRNA levels were markedly reduced compared with those in the fed fish, whereas after 6 days they were not detected (Fig. 5). Therefore, the degree by which myostatin mRNA was reduced appeared to directly reflect the magnitude of nutritional insult. This reduction was evident whether 18S or GAPDH mRNA levels (in separate RT-PCR reactions) were used for normalization (data not shown).


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  		Fig. 4.   Body weights of fed and fasted larval tilapia. Individual larvae from 2 separate experiments (experiment 2a with clutches 1 and 2, experiment 2b with clutch 3) were either fed or fasted for 3, 6, or 9 days. Larvae were weighed at the beginning (day 0) and on termination. Mean body weights (±SE) of all larvae weighed at 3, 6, and 9 days are significantly different from their corresponding day 0 weights and between fed and fasted groups (P <=  0.05).



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  		Fig. 5.   Myostatin mRNA levels and body weight differences in fed and fasted larval tilapia (experiment 2a). A: whole larva myostatin mRNA levels, in 2 separate clutches of fed and fasted (3 or 6 days) larvae, were determined by semiquantitative RT-PCR (see MATERIALS AND METHODS) on pooled larvae from clutches 1 and 2. Interassay variation was controlled by normalizing the myostatin values to those of 18S rRNA. B: differences between mean (±SE) body weights (n = 5) were determined by a t-test (*P <=  0.05). The myostatin values on the histogram represent means ± range (n.d., not detected).

In experiment 2b, the body weights of 3-day-fed and -fasted larvae changed by 9 and -17%, respectively, whereas after 9 days they changed by 95 and -26% (Fig. 4). Although the age and developmental status of the larvae used in this experiment were similar to those discussed above, the effects of fasting were much less pronounced in these animals and are likely associated with a 29% (3.8 mg) greater body mass before the study began. In contrast to the previous experiments, individually measured myostatin mRNA levels were almost twofold higher than those of fed larvae after 3 days (Fig. 6). However, fasting for 9 days markedly reduced myostatin mRNA levels (-59%) as in the previous experiments. Neither GAPDH nor G-6-Pase mRNA levels were affected by fasting after either time point (Fig. 6). These data indicate that myostatin mRNA levels rise during a short-term fast but are ultimately reduced with prolonged fasting. These changes appear to be specific for myostatin and are not generic cellular responses to nutritional insult, as GAPDH and G-6-Pase mRNA levels were not affected by fasting.


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  		Fig. 6.   Individual measurements of myostatin, GAPDH, and G-6-Pase mRNA levels in fed and fasted larvae (experiment 2b). A: Vistagreen staining of amplicons generated by semiquantitative RT-PCR assay (see MATERIALS AND METHODS) on fed or fasted larvae (clutch 3) for 3 and 9 days. Transcript levels were determined on individual larvae, which are numbered above. B: histogram of semiquantitative RT-PCR results. Interassay variation was controlled by normalizing the myostatin values to those of 18S rRNA. Differences between means (±SE) were determined by a t-test (*P <=  0.05) between fed and fasted groups for each transcript.

Systemic Responses in Fasting Larvae

Myostatin expression occurs in several different fish tissues and is not limited primarily to skeletal muscle as it is in mammals (41, 45). Therefore, to determine whether the fasting-induced suppression of whole larval myostatin mRNA was due to changes in the muscle levels alone or to changes in other tissues as well, mRNA levels were also measured independently in the heads and gutted bodies of larvae that were fed or fasted for 6 days (from clutches 1 and 2, experiment 2a). The gutted body consists predominantly of skeletal muscle and to a much lesser degree of bone, kidney, and integument. Thus most, if not all, of the myostatin message detected in the bodies will have occurred in skeletal muscle, as myostatin expression has not been shown to occur in either bone or skin and it does not occur in the kidney (45). Although GAPDH mRNA levels were equal in all groups, those of myostatin were not detected in either the heads or bodies of 6-day-fasted larvae (Fig. 7A). Moreover, myostatin mRNA levels were similar in both heads and bodies of fed larvae and thus were similarly suppressed by fasting. These results suggest that the inhibitory effects of fasting on myostatin mRNA occur in different tissues and are not due to changes in skeletal muscle expression alone.


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  		Fig. 7.   Systemic responses to fasting. A: Vistagreen staining of amplicons generated by semiquantitative RT-PCR assay (see MATERIALS AND METHODS). Myostatin and GAPDH mRNA levels in pooled heads and bodies (gutted) of 6-day-fed and -fasted larvae pooled from experiment 2a (clutches 1 and 2). B: whole animal cortisol levels from experiment 1 larvae (n = 6/group) as determined by an ELISA. Significant differences (P <=  0.05) are indicated by different letters, whereas the same letter indicates no difference.

Nutritional stress in fish and other vertebrates is associated with a concomitant rise in glucocorticoid synthesis and circulating concentrations, which antagonizes the glucose-clearing and anabolic effects of insulin and other growth promoters (8, 23, 33, 42, 53). Although the hypothalamo-pituitary-interrenal axis is active in tilapia larvae post-YSA, the typical corticotropic response that occurs in many adult vertebrates during a fast has not been described in larvae before this study. Fasting significantly elevated whole larval cortisol levels approximately three- and fivefold after 3 and 6 days, respectively (Fig. 7B). The 6-day fasting cortisol levels were significantly higher than those after 3 days, suggesting that the corticotropic response to fasting is proportional to the degree of nutritional stress.

Cortisol-Induced Suppression of Myostatin mRNA Levels in Whole Larvae

Because whole larval cortisol levels were elevated in fasted larvae, we sought to determine whether cortisol could mimic the effects of prolonged fasting on myostatin mRNA levels (experiment 3). Although myostatin mRNA levels were unaffected by the lower doses tested (data not shown), they were reduced by 66 and 75% after immersion of larvae in water containing 1 ppm cortisol for 3 and 6 h, respectively (Fig. 8). Thus cortisol is capable of reducing myostatin mRNA levels to a degree comparable to that achieved by 3 days of fasting. This suppressive effect may be time dependent, since there was a trend toward a reduction in absolute levels of myostatin message, as well the variance, over time.


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  		Fig. 8.   Effects of cortisol (F) on myostatin mRNA levels in larval tilapia (experiment 3). Whole larvae were immersed in 0.01, 0.1, and 1 ppm cortisol for 3 and 6 h. The 2 lower doses of cortisol had no effect (data not shown). A: Vistagreen staining of amplicons generated by semiquantitative RT-PCR assay (see MATERIALS AND METHODS) on individual larvae. B: histogram of semiquantitative RT-PCR results. Interassay variation was controlled by normalizing the myostatin values to those of GAPDH. Significant differences between mean (±SE) MSTN/GAPDH values of 1 ppm cortisol-treated larvae vs. controls were determined by a t-test (*P <=  0.05).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In adult tilapia, fasting for 28 days failed to alter skeletal muscle myostatin mRNA levels. Ji et al. (21) determined that a 3-day fast in juvenile pigs was similarly ineffective. Although these limited data appear to suggest that fasting in adults does not impact myostatin mRNA levels, it is also possible that the experimental designs used thus far have not generated the magnitude of nutritional stress necessary to alter myostatin gene expression or mRNA half-life. In fact, it is doubtful that either of these animal models was unduly stressed, as two of the five growth indexes examined in the present study were not affected by the fast, whereas the remaining three were only minimally reduced. Adult tilapia are notably capable of tolerating fasting conditions, especially compared with mammals (43, 54). The duration of the adult fast discussed herein was determined in a previous study, which indicated that circulating growth hormone levels were significantly elevated after 31 days of fasting but not after 21 (54). This is in contrast to the rapid rise in the synthesis and release of many counterregulatory hormones, including growth hormone, that occurs in fasting mammals (8, 42). The differential responses to a catabolic insult in fish vs. mammals is likely due to the reduced caloric needs of the poikilotherm, since it lacks the energy requirements necessary to maintain the endothermic physiology of mammalian and avian species (17, 50). Thus the observation that adult skeletal muscle myostatin mRNA was unresponsive to fasting may not necessarily be surprising, since, like the juvenile pigs, it can be argued that the adult tilapia were only moderately compromised under these conditions. The impact of chronic nutritional stress from a more substantial fast on adult myostatin mRNA levels remains to be fully characterized.

By contrast, fasting significantly altered myostatin mRNA levels in whole larvae, as they were reduced after both short- and long-term fasts in experiment 2a. In a separate experiment (experiment 2b), the levels rose initially but were eventually reduced to levels well below those of the fed controls as the animals continued to fast. This reduction in message appeared to be dependent not only on the time spent fasting but also on the degree of catabolism, as larval body weights and myostatin mRNA levels were similarly affected. When the experiment began, the larvae used in experiment 2b were considerably larger (29%) than those in experiment 2a, although the age and days after YSA were the same. This suggests that body energy reserves, and therefore potential resistance to the impact of fasting, were greater in the larvae of experiment 2b. In fact, myostatin message was not detected in the 6-day-fasted larvae of experiment 2a but was still measurable on day 9 in experiment 2b. We conclude that myostatin message may rise initially in the early stages of a fast, when the degree of catabolism is not yet severe, but that it consistently decreases with prolonged nutritional insult.

It is unknown whether fasting induced similar changes in myostatin message in nonskeletal muscle tissues of adult fish, as this was the only tissue analyzed. However, the fasting-induced reduction in myostatin mRNA was not exclusive to larval skeletal muscle, as the levels were equally suppressed in bodies as they were in heads. Although a minimal amount of skeletal muscle is present in larval heads, this represents only a minor fraction of the available tissue mass and total cell number. Thus it is highly unlikely that the muscle present in a decapitated larval head is the primary source of myostatin message, since brain, gills, and eyes are all known to express myostatin in tilapia. This is particularly true for brain, as the levels in this tissue are comparable to those found in skeletal muscle (45). Nevertheless, these results suggest that the effects of prolonged fasting were mediated systemically by a common intracellular response either to nutrient deprivation, to a circulating factor that rises in response to fasting, or to both. Overall, these results are consistent with those presented by Rescan et al. (38), who demonstrated that rainbow trout myostatin (myostatin-2; see below) mRNA levels decrease with maturation-induced muscle wasting.

In many different species of fish larvae, including O. mossambicus, cortisol biosynthesis, in vivo secretion, and tissue sensitivity, as well as an intact cortisol stress response, have all been documented (3, 6, 19, 20, 47, 51). In addition, circulating concentrations of this stress hormone rise in fasting and catabolic adult fishes (23, 53). In the present study, we have similarly shown that increased cortisol synthesis in tilapia larvae (experiment 2a) is also associated with fasting, as whole larval cortisol levels were significantly elevated in a time-dependent manner. In the same experiment, myostatin mRNA levels appeared to be inversely related to cortisol, since they were lowest in larva groups with the highest cortisol levels. Immersion of fed larvae into water containing 1 ppm cortisol similarly reduced myostatin mRNA levels, which is supportive of a direct inverse relationship between cortisol and myostatin message. Thus hypercortisolemia may contribute to the fasting-induced reductions in tilapia larval myostatin mRNA levels; this may also apply to the reduced myostatin levels in maturing skeletal muscle of rainbow trout (38), as cortisol levels rise substantially during salmonid maturation as well (5, 7, 36). In contrast to our data, glucocorticoids upregulate myostatin gene expression in mice. This occurs through direct interactions between the activated glucocorticoid receptor and a glucocorticoid response element within the myostatin gene promoter (25, 27). Thus mammalian and tilapia myostatin mRNA appear to be differentially regulated by these stress hormones.

The extreme gains in skeletal muscle mass of myostatin knockout mice are due to both myocyte hyperplasia and hypertrophy (29). Thomas et al. (52) and Rios et al. (39) have determined that myostatin inhibits myoblast proliferation through G1 and G2 growth arrest of the cell cycle. However, two recent studies (26, 40) have determined that myostatin can prevent the in vitro differentiation of myoblasts under low-serum conditions via phospho-Smad 3 squelching of the myogenic transcription factor MyoD. Thus myostatin appears to simultaneously initiate and inhibit myoblast differentiation, as cell cycle withdrawal is a prerequisite to differentiation. It is possible that, under normal physiological states, myostatin may not necessarily be a growth inhibitor per se but may contribute to the normal formation of mature muscle fibers by initiating cell cycle withdrawal, whereas under conditions such as a short-term fast in tilapia larvae, both myoblast proliferation and differentiation would be inhibited by elevated myostatin production. The etiological consequences of such changes in catabolic larvae would prevent nutrient utilization for nonessential and expensive anabolic processes (i.e., muscle growth and development) until such nutrients are more plentiful. In the presence of reduced myostatin production, as occurs with the prolonged fasting of tilapia larvae, myocyte hyperplasia is likely prevented by growth factor resistance, which occurs in many vertebrates under catabolic duress and is often mediated by glucocorticoids (11, 15, 18, 42). In fact, excessive glucocorticoids inhibit muscle cell growth and development (9, 48), which may have contributed indirectly to the reduced myostatin message during the prolonged fast in addition to the direct effects of cortisol on tilapia myostatin mRNA levels.

Myostatin production has been shown to increase (32, 56) as well as decrease (24, 46) in response to muscle injury. These apparently conflicting results are likely due to the multifaceted processes involved in muscle regeneration. These include both myoblast proliferation with concomitant decreases in myostatin production (32) and differentiation with concomitant increases. The previously described regenerative actions of myostatin, the cytokine's ability to initiate myoblast differentiation, and the results presented herein all suggest that the common characterization of the cytokine's actions as purely growth inhibitory may be limited. Rather, myostatin appears to be an integral regulator of skeletal muscle growth and development. Such hypotheses assume that the changes in myostatin mRNA are mechanistically related to muscle. However, the differential changes in larval myostatin mRNA levels, in response to short- vs. long-term fasting, may help to mediate other processes independently of muscle growth and development altogether. Whereas the rise in myostatin mRNA after a short fast may ultimately inhibit myoblast proliferation, the reduction after a prolonged fast could prevent other processes unrelated to muscle, as this cytokine is expressed in a variety of fish tissues (41, 45).

Although myostatin is expressed from only a single allele in tilapia and zebrafish (31, 45), salmonids possess an additional myostatin (myostatin-2), whose expression is much more limited and occurs primarily in skeletal muscle (35, 38, 41). Although often proposed, it is highly unlikely that the second myostatin gene arose from the tetraploidization of the salmonid genome, since an alternative myostatin gene (with a similar expression pattern) has also been identified in a Perciforme, the gilthead seabream Sparus aurata (28). These additional alleles likely arose via an earlier duplication event that occurred well before the radiation of Salmoniformes and other teleosts (1, 37) and subsequently diverged or were lost altogether as more recent vertebrates evolved. Mammals express a highly related cytokine, growth/differentiating factor (GDF)-11, which also belongs to the TGFbeta superfamily and which is believed to recognize similar, if not identical, receptors, as the bioactive domains of these TGFbeta siblings are nearly identical (30). The expression pattern of mammalian GDF-11 is similar to that of tilapia myostatin and salmonid myostatin-1, raising the possibility that myostatin-1 is ancestral to tilapia myostatin and quite possibly to mammalian GDF-11, as salmonids evolved much earlier than the tilapian species. Although the functional analysis and sequence of the GDF-11 promoter remains uncharacterized, it should be interesting to determine whether the differential effects of cortisol on myostatin mRNA in tilapia vs. rodents could be related to a more direct relationship between tilapia myostatin and mammalian GDF-11.

Our results indicate that prolonged fasting significantly reduces somatic growth and myostatin mRNA levels in larval tilapia, whereas cortisol synthesis rises. Because cortisol treatment in vivo similarly affects myostatin mRNA, this steroid represents a potentially important endocrine link between catabolic shifts in skeletal muscle (and possibly other fish tissues) and reduced myostatin mRNA in the larvae of tilapia and possibly other fishes. Other corticotropic stressors in addition to fasting may have similar effects on myostatin mRNA and thus on myogenesis in developing tilapia. By contrast, cortisol increases myostatin gene expression in rodents. This suggests that the mechanisms involved in the regulation of myostatin gene expression or mRNA degradation differ in tilapia and rodents, which may be related to the expression of a single myostatin-GDF-11 cytokine in this neoteleost or, more simply, to the inherent physiological differences between fish and mammals.


    ACKNOWLEDGEMENTS

We thank Jill Hoover for assistance with the cortisol measurements.


    FOOTNOTES

These studies were supported by a grant from the US Department of Agriculture (2001-35206-10078) to B. D. Rodgers.

Address for reprint requests and other correspondence: B. D. Rodgers, Dept. of Pediatrics, Div. of Endocrinology, Johns Hopkins Univ. School of Medicine, 600 N. Wolfe St., Park 211, Baltimore, MD 21287 (E-mail: drodgers{at}jhmi.edu).

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.

First published December 19, 2002;10.1152/ajpregu.00644.2002

Received 18 October 2002; accepted in final form 18 December 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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