The IGF-binding proteins (IGFBPs) play a dual role in the regulation of the activity and bioavailability of IGFs in different tissues. Diverse evidence has shown that IGFBPs can inhibit and/or potentiate IGF actions. In this study, igfbp1, 2, 3, 4, 5, and 6 were isolated in the fine flounder, a flat fish species that shows slow growth and inherent Gh resistance in muscle. Subsequently, the expression of all igfbps was assessed in the skeletal muscle of flounder that underwent different nutritional statuses. igfbp1 was not expressed in muscle during any of the nutritional conditions, whereas igfbp3 and igfbp5 were the lowest and the highest igfbps expressed respectively. A dynamic expression pattern was found in all the igfbps expressed in skeletal muscle, which depended on the nutritional status and sampling period. During the fasting period, igfbp2, 4, and 5 were downregulated, whereas igfbp3 was upregulated during part of the fasting period. The restoration of food modulated the expression of the igfbps dynamically, showing significant changes during both the long- and short-term refeeding. igfbp3 and igfbp6 were downregulated during short-term refeeding, whereas igfbp5 was upregulated, and igfbp2 and igfbp4 remained stable. During long-term refeeding, the expression of igfbp2, 4, 5, and 6 increased, while igfbp3 remained unchanged. In conclusion, this study shows for the first time the isolation of all igfbps in a single fish species, in addition to describing a dynamic nutritional and time-dependent response in the expression of igfbps in the skeletal muscle of a nonmammalian species.
The insulin-like growth factor (IGF) system regulates somatic growth in vertebrates and is integrated by the IGFs (IGF1 and IGF2), the IGF receptors (IGF1R and IGF2R), and the IGF-binding proteins (IGFBPs; Duan et al. 2010). IGFs are mainly synthesized by the liver; however, they can be locally produced in numerous tissues, including skeletal muscle acting in an autocrine/paracrine fashion (Velloso 2008). In particular, muscle-derived IGF1 has been proposed as the main regulator of growth in mammals (Sjögren et al. 1999, Yakar et al. 1999). As in mammals, some recent evidence has shown that in fish, local Igf1 is of importance in muscle growth (Eppler et al. 2007, Fox et al. 2010, Fuentes et al. 2012a). The dynamic of the biological actions of IGFs is modulated by the binding of these growth factors with either specific membrane receptors and/or the IGFBPs. The IGFBPs are a family of proteins that show high affinity for IGFs and are composed of six members, denominated IGFBP1–6 (Jones & Clemmons 1995). In circulation, the IGFBPs bind to IGFs, transporting and increasing their half-life by preventing their proteolytic degradation (Hwa et al. 1999). The IGFBPs have a higher affinity for IGF than IGF receptors; thus, they are implicated in the modulation of the activity and bioavailability of IGFs in different tissues (Clemmons 1998). Numerous evidences indicate that IGFBPs can inhibit and/or potentiate IGF actions depending on the physiological context (Duan & Xu 2005, Duan et al. 2010). IGFBPs can also be synthesized in different cell types and tissues, exerting an important role in the local regulation of IGFs (Jones & Clemmons 1995, Firth & Baxter 2002, Duan et al. 2010). Particularly, in skeletal muscle, the igfbps are expressed differentially during myogenesis and are implicated in the modulation of muscle growth, showing ligand-dependent and -independent actions (James et al. 1993, Ewton & Florini 1995, Bayol et al. 2000, Awede et al. 2002, Foulstone et al. 2003, Ren et al. 2008).
In fish, several studies have described the isolation of individual igfbps (Duan et al. 1999, Maures & Duan 2002, Chen et al. 2004, Kamangar et al. 2006, Bower et al. 2008, Li et al. 2009, Pedroso et al. 2009a, Peterson & Waldbieser 2009, Wang et al. 2009, Dai et al. 2010, Rahman & Thomas 2011). However, until now, the isolation and assessment of the expression patterns of all six igfbps in the same species have not been achieved. Previous studies on the biology of Igfbps in fish have shown that Igfbps regulate embryonic development (Duan et al. 1999, Kajimura et al. 2005, Li et al. 2009, Wang et al. 2009) and exert ligand-dependent and -independent mechanisms (Dai et al. 2010, Zhong et al. 2011), as well as showing that Igfbps are modulated by Gh treatment (Duan et al. 1999, Cheng et al. 2002, Chen et al. 2004, 2010, Pedroso et al. 2009b), oxygen availability (Kajimura et al. 2005, Kamei et al. 2008, Rahman & Thomas 2011, Sun et al. 2011), reproduction (Kamangar et al. 2006, Chen et al. 2010), and nutrition (Gabillard et al. 2006, Bower et al. 2008, Li et al. 2009, Pedroso et al. 2009a, Peterson & Waldbieser 2009, Amaral & Johnston 2011, Macqueen et al. 2011). Nevertheless, studies on local muscle-derived igfbps (autocrine/paracrine) have been scarce and have recently shown contrasting results, reflecting a complex physiology of Igfbps and suggesting that intricate patterns of regulation have evolved between and within teleost lineages (Gabillard et al. 2006, Bower et al. 2008, Amaral & Johnston 2011, Macqueen et al. 2011).
The fine flounder (Paralichthys adspersus (P. adspersus)) is an endemic flatfish species of the southern East Pacific that shows a slow growth rate and poor food intake even under favorable nutritional conditions (Fuentes et al. 2012a,b). Previously, we have described part of the molecular and endocrine basis accounting for the slow growth of this teleost species, showing that fine flounder present an inherent Gh resistance in skeletal muscle, which results in low basal levels of muscle-derived Igf1 (Fuentes et al. 2012a,b). Thus, the low production of autocrine/paracrine Igf1 was the responsible mechanism for growth deficit in this species; however, the regulation of the main modulators of Igf1 availability and actions, such as the Igfbps, remained unknown as did their possible consequences on growth in this species. Thus, use of this intriguing fish model will allow us to go further in the understanding of igfbps' physiology in fish, particularly in the understanding of local igfbps produced in the muscle. In this context, the aim of this work was to assess a detailed time course changes in the expression pattern of all six igfbps in skeletal muscle in the fine flounder, evaluating two contrasting nutritional periods, fasting and refeeding (short and long term). In order to achieve this goal, six igfbps were previously isolated.
Materials and Methods
Animals, sampling, and experimental design
Three-year-old juvenile fine flounder (P. adspersus) with an average weight of 300±10 g and length of 27±2 cm were maintained under natural temperature and photoperiod conditions corresponding to the geographic location of CIMARQ (33°13′S; 71°38′W) during the spring season of 2009 in the southern hemisphere. Fish were randomly divided into two tanks and acclimatized for 2 weeks under satiety feeding conditions. At the beginning of the experiment (week 0), food was withheld from one group for 3 weeks (experimental (EXP)), inducing a nutritionally catabolic state of fasting. Then, fish were subjected to a 4-week satiety refeeding period, returning the fish to an anabolic state. The other group (control (CTRL)) was fed until satiety throughout the 7-week study. Samples were obtained weekly over the trial to study long-term regulation and dynamics of the igfbps' expression in skeletal muscle. At the beginning of the refeeding period, fish were sampled at 2, 4, and 24 h in order to assess short-term changes in the expression of igfbps. In order to study the expression of the igfbps in different tissues (e.g. tissues from gills, intestine, stomach, spleen, heart, liver, and red and white muscle) of the fine flounder, tissues were collected from nonmanipulated flounder maintained in the conditions mentioned earlier. For each sampling point, three individuals were sampled (n=3). Sampling was performed under anesthesia (3-aminobenzoic acid ethyl ester, 100 mg/l) and tissue was subsequently collected. Tissue was frozen immediately in liquid nitrogen and stored at −80 °C until processing. The study adhered to animal welfare procedures and was approved by the Bioethical Committees of the Universidad Andres Bello and the National Commission for Scientific and Technological Research of the Chilean government.
RNA extraction and cDNA synthesis
Total RNA was extracted from tissue using the RNeasy Mini Kit (Qiagen) following the manufacturer's recommendations. RNA was quantified using NanoDrop technology with the Epoch Multi-Volume Spectrophotometer System (BioTek, Winooski, VT, USA). Assessment of RNA quality was performed by electrophoresis on a 1.2% formaldehyde agarose gel containing ethidium bromide. Only RNAs with an A260:A280 ratio between 1.9 and 2.1 were used for cDNA synthesis. Residual genomic DNA was removed using the genomic DNA wipeout buffer included in the Quantitect RT kit (Qiagen). Subsequently, 800 ng RNA was reverse transcribed into cDNA for 30 min at 42 °C using the manufacturer's recommendations.
Isolation and identification of igfbps in fine flounder
Once high-quality cDNA was obtained from the liver as described previously, igfbp1, 2, 3, 4, 5, and 6 were isolated and deposited in the GenBank (Table 1). Primers used for obtaining the sequences of igfbps were designed by multiple alignments of sequences from fish species using ClustalW, identifying evolutionary conservative regions (Table 1). PCR was performed using 1 μl cDNA template, 10 μl PCR buffer 10×, 200 μM each dNTP, 500 nM each primers, 0.3 μl Taq DNA polymerase (12 U/μl) (Promega), and RNAse-free water to a final volume of 50 μl. Primers used for isolating the six igfbps in the fine flounder are listed in Table 1. Thermal cycling conditions were as follows: initial denaturation of 10 min at 95 °C, followed by 40 cycles of 30 s at 95 °C; 30 s at 53 °C (igfbp3), 56 °C (igfbp1 and igfbp2), or 58 °C (igfbp4–6); 30 s at 72 °C; and a final extension of 10 min at 72 °C. PCR products were loaded on a 1.5% agarose gel and then isolated and purified using the Wizard SV Gel and PCR Clean-Up System (Promega).
Primer sequences for cloning and quantitative PCR (qPCR) assay of the igfbps of the fine flounder (M=A/C, R=A/G, Y=C/T, S=G/C, and K=G/T). Amplicon size (pb), qPCR efficiencies (E (%)), coefficient of standard curves (R2), and GenBank accession number are also shown
|Gene||Primer||Sequence (5′–3′)||Size (bp)||E (%)||R2||Accession number|
Specific PCR products were cloned into a TOPO TA Cloning system (Invitrogen) using the manufacturer's recommendations. In short, PCR products were ligated into the T/A pCR4-TOPO vector (Invitrogen) and subsequently One Shot TOP10 competent Escherichia coli (Invitrogen) was transformed with the vector. Individual colonies were cultured and plasmids were isolated and purified using the Qiagen Plasmid Purification (Qiagen) and subsequently sequenced.
Phylogenetic analyses and logos alignment of Igfbps in fish
In order to further prove that the partial sequences isolated corresponded to six different Igfbps in the fine flounder sequence, alignment and construction of the phylogenetic tree using ClustalW and MEGA 5 were performed (Tamura et al. 2011). Owing to the lack of full-length sequences, phylogenetic or evolutionary relationships were not evaluated in this phylogenetic tree. For distance analysis, the neighbor-joining approach with the p-distance method and bootstraping (1000 replicates) as support for subgroup on the phylogenetic tree was used.
Logos alignment was performed using the variable central linker (L) domain. The alignment was carried out using LogoBar, a program that allows graph visualization of protein Logos alignment with gaps (Pérez-Bercoff et al. 2006).
Assessment of igfbps expression
Primer designs for quantitative PCR (qPCR) of igfbps were based on partial sequences previously obtained and are listed in Table 1. In order to get high-quality primers and avoid secondary structure (hairpins, homo-, and cross-dimers), Amplifx 1.5.4 (
Quantitative real-time PCR
All the procedures were carried out according to the protocol outlined by Bower & Johnston (2010), with minor modification. All qPCR assays were carried out to comply with the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines (Bustin et al. 2009).
Total RNA extraction and cDNA synthesis from different tissues (i.e. igfbps expression in different tissue analysis) and skeletal muscle of all sampling points (i.e. igfbps during fasting and refeeding analysis) were performed as described previously (See RNA extraction and cDNA synthesis). qPCR was performed using the Stratagene MX3005P QPCR system (Stratagene, La Jolla, CA, USA). Each qPCR reaction mixture contained 7.5 μl Brilliant II SYBR green master mix (Agilent Technologies, Stratagene), 6 μl cDNA (40-fold dilution), 250 mM each primers, 5 μM ROX, and RNAse-free water to a final volume of 15 μl. Amplifications were performed in triplicate with the following thermal cycling conditions: initial activation at 95 °C for 10 min, followed by 40 cycles of 30 s at 95 °C, 30 s at 58 °C, and 30 s at 72 °C. CTRL reactions included a no template control (NTC) and a CTRL without reverse transcriptase (−RT). SYBR green fluorescence was always recorded during the linear phase of cycling. In order to confirm the presence of a single PCR product, dissociation curve analysis of the PCR products was performed. Products were also evaluated by electrophoresis on a 2% agarose gel to confirm that a single product was amplified. With the purpose of estimating the assay's efficiency, twofold dilution series were created from a cDNA pool. Efficiency values were estimated from the slope of the curve following the equation efficiency: E=10(−1/slope)−1 (Table 1). qPCR data were analyzed using Ct values, which were then exported and processed into a Microsoft Excel-based Software application (Q-Gene; Muller et al. 2002). For the analysis of igfbps expression in different tissue, graphs were expressed as arbitrary units (A.U.) using as a reference gene 18S rRNA (18S). For the analysis of igfbps expression during fasting and refeeding, graphs are expressed as a fold change over basal levels found at the beginning of the trial (week 0) for long-term changes. Short-term changes are expressed as fold changes over fasting levels at the end of this period (third week). 40S ribosomal protein S30 (FAU) was used as the most stable reference gene (Fuentes EN, Safian D, Valdes JA & Molina A 2012, unpublished results).
Heat map summary and hierarchical clustering
In order to establish relationships among relative expression profiles of the igfbps with the rest of the components of the Igf system, a heat map summary and hierarchical clustering analysis of gene expression were performed using Permutmatrix (Caraux & Pinloche 2005). In order to place the expression pattern of igfbps into a global context into the Igf system in muscle, we used the expression of previously published igf1, igf2, and igf1r using the same sampling points (Fuentes et al. 2012a,b). Clustering and seriation were based on Pearson's correlation coefficient of z-score-normalized relative transcript abundance values (scaled from 0 to 1). McQuitty's method of hierarchical clustering was used.
Statistical analysis used to study differences in gene expression was based on an advanced linear model. This model was the general linear model (GLM) followed by Tukey's analyses as post-test. All statistical analyses were performed using the STATISTICA 7 Software (Tulsa, OK, USA).
Isolation of igfbps
Logos alignment showed a low identity when the L-domain of all Igfbps of the fine flounder (ffIGFBPs) was compared among them (Tables 2 and 3). The Logos alignment showed that Igfbp1, 2, and 3 are very variable among teleost fish whereas Igfbp4, 5, and 6 are highly conserved among teleost fish (Fig. 1). Using the partial sequence of all Igfbps, including part of the N- and C-domains, equal results were found (data not shown). In order to further corroborate the identity of all six ffIGFBPs, a phylogenetic tree using the L-domain from all Igfbps was constructed, showing that all Igfbps are clustered in different branches with a respective Igfbp (
Amino acid sequence identity between the L-domain of the Igfbps of rainbow trout (rtigfbp). Accession number for the rtigfbps: igfbp1 (NM_001124561), igfbp2 (NM_001124649), igfbp3 (NM_001124557), igfbp4 (DQ146967), igfbp5 (NM_001124652), and igfbp6 (NM_001124560)
Numbers in bold indicate comparison between respective orthologs sequences.
Amino acid sequence identity between the L-domain of the Igfbps of the fine flounder (ffIgfbp)
Expression of igfbps in different tissues of the fine flounder
igfbp1 and igfbp2 were predominantly expressed in the liver, showing low expression levels in the other tissues (Fig. 2A and B). Particularly, igfbp1 was not expressed in red and white muscle, whereas igfbp2 was not detected in the stomach (Fig. 2A and B). igfbp3 and igfbp4 were detected in all analyzed tissues, but igfbp3 was predominantly expressed in the liver, whereas igfbp4 was highly expressed in the intestine, stomach, and spleen (Fig. 2C and D). igfbp5 expression was also ubiquitous, but particularly highly expressed in the stomach and heart (Fig. 2E). Likewise, igfbp6 was detected in all tissues assessed, showing high expression in gill, intestine, stomach, and heart and low expression in spleen, red muscle, white muscle, and liver (Fig. 2F).
Expression of igfbps in skeletal muscle during fasting and refeeding
Different results in the gene expression of fine flounder igfbps in skeletal muscle were found, which depended on the feeding status. igfbp1 was not detected in the skeletal muscle after 40 cycles.
igfbp2 mRNA levels decreased during fasting in the EXP group, showing almost three and 12-fold lower expression than basal levels (0 week) after 2–3 weeks of food withdrawal respectively (Fig. 3A,
igfbp3 mRNA levels in the EXP group increased significantly during 1 and 2 weeks of fasting (four- and three fold respectively), but, interestingly, the expression of igfbp3 declined and then returned to basal levels at the end of fasting (Fig. 3B,
igfbp4 mRNA levels in the EXP group were significantly downregulated during the third week of fasting (sevenfold lower mRNA levels; Fig. 3C,
igfbp5 mRNA levels decreased after 3 weeks of fasting in the EXP group (fourfold lower expression than 0 week; Fig. 3D,
igfbp6 mRNA levels in the EXP group did not change significantly during fasting (Fig. 3E,
High differences were observed when analyzing the differences in the mRNA contents among all igfbps in skeletal muscle of the fine flounder during basal, fasting, and refeeding conditions. igfbp1 was not detected in skeletal muscle in any of the nutritional conditions assessed. igfbp2 and igfbp6 mRNA contents were similar in basal, fasting, and refeeding conditions (Fig. 3F). igfbp3 expression was the lowest of all igfbps (except igfbp1; Fig. 3F). igfbp4 contents were not significantly different in basal and fasting conditions but were significantly different during refeeding. igfbp5 was always the most abundant of all igfbps, independent of nutritional status and sampling period (Fig. 3F). At the beginning of the short-term refeeding (0 h, fasting), igfbp2, 3, 4, and 6 contents were not significantly different, with the exception of igfbp5, which was the most abundant (Fig. 4F). After 24 h of refeeding, igfbp2, 4, and 6 expression was similar, whereas igfbp3 and igfbp5 expression was the lowest and the highest respectively (Fig. 4F).
Heat map summary and hierarchical clustering
Hierarchical clustering of Igf system expression during long-term fasting and refeeding showed different variations among different components of the Igf system. During long-term refeeding, two clades were found; the first clade showed a close relation and covariation in the expression of igf1 and igfbp2, 4, 5, and 6 (Fig. 5A), whereas igf1 showed more similitude with igfbp5 and igfbp6 than with igfbp2 and igfbp4. The second clade clustered igf2, igf1r, and igfbp3. During short-term refeeding, two clades were found. The first clade showed two subgroups; the first subgroup clustered igf2, igf1r, igfbp2, and igfbp6 while the second subgroup clustered igfbp3 and igfbp4. The second clade clustered igf1 and igfbp5 (Fig. 5B).
Isolation of igfbp1, 2, 3, 4, 5, and 6 in the fine flounder
In fish, the igfbps have been isolated from different models (Bower et al. 2008, Li et al. 2009, Pedroso et al. 2009a, Peterson & Waldbieser 2009, Rahman & Thomas 2011); however, the isolation and assessment of gene expression of the six igfbps in a single teleost had not been achieved until now. Kamangar et al. (2006) claimed the isolation of the six igfbps in rainbow trout. Nevertheless, in this publication, igfbp3 was misnamed, with the igfbp2 paralog 2 being the real igfbp isolated (Bower et al. 2008, Rodgers et al. 2008, Ocampo Dasa et al. 2011). The similarity among different Igfbps and/or other related proteins has been a common problem in the correct identification of Igfbps (Rodgers et al. 2008). All Igfbps have a common structure with a highly conserved N-terminal (also known as IGFBP domain), a conserved C-terminal domain (also known as thyroglobulin type-1 domain), and an internal variable L-domain (Duan & Xu 2005). Moreover, whole genome duplication of teleost has triggered that teleosts often have two copies of each igfbps, which is shown to have different expression pattern (Kamei et al. 2008, Zhou et al. 2008, Wang et al. 2009, Bower & Johnston 2010, Dai et al. 2010). In this study, we were unable to isolate paralogs for the igfbps in the fine flounder. Genomic resources would be a helpful methodology to try to isolate paralogs in this species, considering that until now all paralogs for igfbps have been identified in model fish models either with genome-sequenced (e.g. zebra fish and takifugu) or with EST sequence database (e.g. Atlantic salmon or rainbow trout), whereas in nonmodel fish, just one copy of the igfbps have been found (Atlantic croaker, carp, yellowtail, etc.; Cheng et al. 2002, Pedroso et al. 2009a, Peterson & Waldbieser 2009, Rahman & Thomas 2011, Sun et al. 2011). Comparing the ffgfbps, a very low identity was found, corroborating that they correspond to different Igfbps. In general, the six individual ffIgfbps have a high identity with their orthologs. However, the ffIgfbp3 showed a very low identity with the rtIgfbp3, highlighting that the ffIgfbp3 is not the Igfbp2 paralog (Kamangar et al. 2006). In fact, when ffIgfbp3 is compared with other orthologs, a higher identity is found. Also by phylogenetic analysis, the ffIgfbps are clustered separately in different branches, grouping each of the ffIgfbps with their respective orthologs.
Expression of Igfbp in different tissues of the fine flounder
IGFBPs are synthesized mainly in the liver; however, they can also be synthesized in different tissues, regulating the IGF actions in an autocrine/paracrine fashion (Jones & Clemmons 1995, Firth & Baxter 2002, Duan et al. 2010). Fine flounder igfbp1 and igfbp2 are predominantly expressed in the liver with lower expression in other tissue, which is consistent with the previous findings in other fish species (Maures & Duan 2002, Kamangar et al. 2006, Kamei et al. 2008, Pedroso et al. 2009a). Igfbp1 is not detected in red and white muscles, similar to the previous reports in other fish species (Maures & Duan 2002, Gabillard et al. 2006, Bower et al. 2008, Kamei et al. 2008, Pedroso et al. 2009a). On the other hand, igfbp2 is highly expressed in the liver of fine flounder. Recently, in Chinook salmon, Igfbp2 was shown to be the most abundant Igfbp into circulation system (Shimizu et al. 2011); this coincides with our results pointing out that liver is the major organ for igfbp2 synthesis. Likewise, igfbp3 is highly expressed in the liver, but it is also detected in all the tissues assessed, according to the previous reports on tilapia (Cheng et al. 2002) and zebra fish (Chen et al. 2004). Similar to other fish species, igfbp4, 5, and 6 are ubiquitously expressed in different tissues of fine flounder; interestingly, these Igfbps are expressed low in liver, despite this tissue being considered the main source of all Igfbps in fish (Kamangar et al. 2006, Li et al. 2009, Pedroso et al. 2009a, Wang et al. 2009). Altogether, these results suggest that Igfbps could be regulating the actions of local Igfs in the fine flounder.
Transcriptional regulation of igfbps in the skeletal muscle during fasting and refeeding
Igfbps regulate the half-life of circulating Igfs as well as modulating their availability and biological activity, either inhibiting or potentiating Igf's actions depending on the EXP conditions, cell type, tissue, species, and physiological context (Wood et al. 2000, Zhou et al. 2004, Ning et al. 2006, 2008, Ren et al. 2008, Dai et al. 2010, Amaral & Johnston 2011). Therefore, in the present work, the results were analyzed according to the physiological context of using two contrasting nutritional conditions: a catabolic period of fasting that leads to muscle atrophy followed by an anabolic period of refeeding that leads to hypertrophy of muscle. The fine flounder was used as a model, which is a fish that presents an inherent low production of muscle-derived Igf1 in muscle, which is responsible for growth deficiency in this species (Fuentes et al. 2012a,b).
The igfbp1 is not detected in skeletal muscle, although it is highly expressed in the liver, which is concordant with the previous studies on fish (Maures & Duan 2002, Gabillard et al. 2006, Bower et al. 2008, Kamei et al. 2008, Pedroso et al. 2009a). Nevertheless, a recent report by Amaral & Johnston (2011) described that igfbp1 is expressed in skeletal muscle of zebra fish and is regulated by nutrition, increasing during fasting and rapidly returning to basal levels during refeeding. The fact that igfbp1 is upregulated during fasting has been associated with a negative role in growth in both fish and mammals (Maures & Duan 2002, Kajimura et al. 2005, Kajimura & Duan 2007). However, the effects caused by the absence of igfbp1 expression in muscle of the fine flounder and other teleost fish are unknown and may be indicating a mechanism of fine regulation of local Igfs, independent of Igfbp1.
Mammalian IGFBP2 has been shown to both inhibit and potentiate IGF actions depending on cellular context (Duan & Xu 2005). Studies on Igfbp2 in fish have pointed out that this binding protein is a growth inhibitory protein, regulating Igf1 bioavailability (Duan et al. 1999, Wood et al. 2005). Few reports have described the dynamic of muscle-derived igfbp2. In C2C12 cell cultures, igfbp2 mRNA levels decrease with serum reduction (Bayol et al. 2000). In the muscle of fine flounder, igfbp2 mRNA levels decrease during fasting and return to basal levels during long-term refeeding, in agreement with a previous report on the muscle of rainbow trout (Gabillard et al. 2006). In contrast, other recent reports have shown that igfbp2 expression increases during fasting, supporting the negative role of this binding protein (Bower et al. 2008, Macqueen et al. 2011).
In mammals, IGFBP3 is the major circulating IGFBP in plasma, transporting and extending the half-life of IGF1 (Firth & Baxter 2002, Yamada & Lee 2009, Domené et al. 2011). Locally produced IGFBP3 shows a different dynamic in muscle. This binding protein is not the most abundant IGFBP in muscle and shows both IGF-dependent actions by stimulating myoblast differentiation (Foulstone et al. 2003), IGF-independent actions, and suppressing cell proliferation (Pampusch et al. 2003). In fish, the role of Igfbp3 in skeletal muscle has not been defined, and no studies have investigated its expression during fasting and refeeding. The ffigfbp3 was the least expressed of all the six igfbps. This coincides with the inherent Gh resistance and low basal production of local igf1 (Fuentes et al. 2011). igfbp3 in fish is regulated by Gh and has been shown to decrease in hypophysectomized striped bass (Siharath et al. 1995) and increase with Gh injections in tilapia and yellowtail (Cheng et al. 2002, Pedroso et al. 2009b); therefore, low levels of igfbp3 in skeletal muscle of the fine flounder may be associated with an impairment in Gh actions, preventing its expression in this tissue. Interestingly, igfbp3 was the only binding protein upregulated during fasting, suggesting a negative role of this binding protein in muscle.
In mammals, IGFBP4 is considered a strong inhibitor of the anabolic action of IGFs (Ning et al. 2008); however, this binding protein also shows positive effects (Ning et al. 2008). Autocrine/paracrine IGFBP4 in muscle has also been associated with both negative actions, by inhibiting IGF1- and IGF2-stimulated proliferation and differentiation (Damon et al. 1998, Ewton et al. 1998), and positive actions, by being upregulated concomitant with igf1 during muscle hypertrophy (Awede et al. 1999, 2002) and myoblast proliferation (Ewton & Florini 1995). In teleost fish, an elevated correlation between igfbp4, which is highly expressed during proliferation, and myog, myoD, and myf5 in myogenic cell has been described (Bower & Johnston 2010). Also in this study, both Igf1 and amino acids synergistically stimulate the expression of igfbp4 (Bower & Johnston 2010). In vivo studies on fish skeletal muscle have shown that igfbp4 is constitutively upregulated during nutritionally favorable anabolic periods (Gabillard et al. 2006, Bower et al. 2008, Macqueen et al. 2011), coinciding with the present results. Interestingly, when different populations of the Arctic charr were assessed, dwarf fish expresses less igfbp4 in muscle in basal conditions than normal-sized fish, and during refeeding, the expression of this binding protein was twofold lower in dwarfs than in normal-sized Arctic charr (Macqueen et al. 2011). In the fine flounder, igfbp4 expression is also similar to that of igf1 long-term fasting and refeeding but not during short-term refeeding, suggesting that Igfbp4 may be a pro-myogenic molecule, as has been suggested previously in fish (Bower et al. 2008, Macqueen et al. 2011).
Igfbp5 is the most conserved gene of the IGFBP family and is predominantly synthesized in skeletal muscle, similar to the present results (James et al. 1993, Kamangar et al. 2006, Dai et al. 2010). Despite some reports that have shown that IGFBP5 inhibits cell proliferation and differentiation in muscle (Ewton et al. 1998), this negative role has been questioned (Duan et al. 2010), showing abundant evidence that points out a positive role in muscle growth (Awede et al. 2002, Gabillard et al. 2006, Lang et al. 2006, Bower et al. 2008, Ren et al. 2008, Bower & Johnston 2010, Macqueen et al. 2011). Particularly, IGFBP5 promotes muscle differentiation; its absence impairs myogenesis (Ren et al. 2008), and its expression increases with muscle hypertrophy and decreases during muscle atrophy (Awede et al. 2002, Lang et al. 2006). In fish muscle, only positive effects of igfbp5 have been described, showing that amino acids alone, as well as the combination of amino acids and Igf1 or Igf2, promote igfbp5 expression in differentiated myogenic cells (Bower & Johnston 2010), supporting the fact that pro-anabolic molecules (i.e. Gh and Igf1) increase igfbp5 expression, as was previously shown in mammals (Lang et al. 2006). In the fine flounder, igfbp5 expression decreases under nutritional catabolic periods of fasting and increases during favorable nutritional anabolic periods of refeeding, similar to previous reports in other fish species, supporting the idea that Igfbp5 has a positive role in muscle (Gabillard et al. 2006, Bower et al. 2008, Macqueen et al. 2011).
IGFBP6 is unique among IGFBPs for its IGF2-binding specificity, showing 50- to 100-fold higher affinity for IGF2 than IGF1 (Bach 2005). This binding protein inhibits the actions of both IGF2 and IGF1, with no evidence of potentiating IGF actions (Bach 2005). In fish, similar findings have been reported, showing that the overexpression of igfbp6 generates different growth disorders, associated with a delay in corporal size and normal development of somites in zebra fish embryos (Wang et al. 2009). Studies on muscle-derived igfbp6 have been very limited and show that igfbp6 is present in catabolic periods and in quiescent cells (Ewton & Florini 1995). In fish, similar results have been observed, showing that igfbp6 is highly expressed in quiescent satellite cells and its expression decreases throughout myogenesis (Bower & Johnston 2010). Interestingly, in the fine flounder, igfbp6 expression remains stable during fasting and decreases during short-term refeeding but not during long-term refeeding. Previous reports on skeletal muscle of fish show that igfbp6 expression also decreases during refeeding, but this change was observed during long-term refeeding (Gabillard et al. 2006, Bower et al. 2008). Thus, although igfbp6 expression in fish muscle decreases during refeeding, this occurs in different periods, showing that igfbp6 shows a different temporal expression in different fish species and that this Igfbp is acting as a negative regulator of Igf1 actions in muscle.
Integration of the Igf system in fish skeletal muscle during fasting and refeeding
The components of the Igf system are in a dynamic and organized balance in skeletal muscle. Considering that Igfbps present a complex physiology, an integrative approach of the results, rather than individual interpretation of them, is more appropriate. In this context, the present results of igfbps were placed into an endocrine framework of growth in muscle, compiling the present information with our previous reports in order to have an integrative perspective of the endocrine system of growth in the skeletal muscle of a nonmammalian species. We suggest that the absence of Igfbp1 in the muscle of the fine flounder may be compensated by the rest of the Igfbps, as has been previously observed when some of the Igfbps are absent (Wood et al. 2000, Ning et al. 2006). During fasting, the low basal production of Igf1 in the fine flounder decreases even further (Fuentes et al. 2012a,b). During this period, the igfbp4 and igfbp5 decreased concomitantly with an increase in Igfbp3 (Fig. 5C). Interestingly, different studies on fish have previously suggested that both Igfbp4 and Igfbp5 may be positive regulators of Igf1, being diminished in catabolic conditions (Gabillard et al. 2006, Bower et al. 2008, Bower & Johnston 2010, Macqueen et al. 2011). On the other hand, Igfbp3 in fish skeletal muscle has not been evaluated until now. We suggest that Igfbp3 could be acting as a negative modulator of Igf1 in skeletal muscle. Owing to low levels of available Igf1, the PI3K/Akt and MAPK/ERK signaling pathways become inactivated, reducing growth in the fine flounder (Fuentes et al. 2011). During short-term refeeding, muscle-derived Igf1 increases drastically in this species (Fuentes et al. 2012a,b). During this period, igfbp2, 3, and 6 decrease, suggesting inhibitory actions of these Igfbps, as has been previously pointed out in fish (Duan et al. 1999, Gabillard et al. 2006, Bower et al. 2008, Wang et al. 2009, Macqueen et al. 2011). Interestingly, during this period, igfbp5 increases significantly. Therefore, we suggest that Igf1 would be more available to Igf1r, and PI3K/Akt and MAPK/ERK become strongly reactivated, restoring myogenic activity as we previously reported (Fuentes et al. 2011). Thus, in this stage, positive signals may be exceeding negative signals, promoting a strong catchup growth, as is observed during the first stages of refeeding in the fine flounder (Fuentes et al. 2011, 2012a,b). During long-term refeeding, low production of local igf1 is restored (Fuentes et al. 2012a,b). igfbp4 and igfbp5 increase even further than basal levels during this period, concomitant with an increase in igfbp2 and igfbp6 (Fig. 5C). Interestingly, contents of igfbp4 and igfbp5 are higher than igfbp2 and igfbp6 at the end of long-term refeeding. These results suggest that different Igfbps might be competing by Igf1, promoting growth, but more attenuated, as has been previously observed in this species full-compensating growth at the end of refeeding (Fuentes et al. 2011, 2012a,b).
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Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
This work was supported by Fondo Nacional de Desarrollo Científico y Tecnologico (FONDECYT) Grant 1090416 (to A M), Universidad Andres Research Bello Fund DI-14-11/I (to E N F).
The authors thank Juan Manuel Estrada for technical assistance in the Centro de Investigacion Marina de Quintay (Chile); Dr Neil I Bower and Dr Daniel J Macqueen (University of St Andrews, St Andrews, Scotland) for offering advice on the quantitative PCR assays; and Ashley VanCott, BA (The University of Nevada, Reno, USA) for improving and correcting the English of the manuscript.
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(D Safian and E N Fuentes contributed equally to this work)