IGF1 Gilthead Seabream

What is the molecular structure of gilthead seabream IGF1?

Gilthead seabream IGF1 is a single, non-glycosylated polypeptide chain containing 68 amino acids with a molecular mass of 7545.4 Dalton and a predicted isoelectric point (pI) of 7.72. Its amino acid sequence is MSPETLCGAELVDTLQFVCGERGFYFSKPGYGPNARRSRGIVDECCFQSCELRRLEMYCAPAKTSK . The protein belongs to the somatomedin family, which includes three main types: somatomedin C (IGF1), somatomedin A (IGF2), and somatomedin B . This growth factor mediates many of the growth-promoting effects of growth hormone (GH) and plays critical roles in the regulation of fish metabolism and development .

How do IGF system components differ between gilthead seabream and mammals?

The IGF system in gilthead seabream shares fundamental components with mammals but exhibits important differences resulting from teleost-specific genome duplication events. Unlike mammals, gilthead seabream possesses two distinct IGF-I receptor paralogues (IGF-IRa and IGF-IRb) that have been characterized from embryonic tissue . Additionally, gilthead seabream exhibits a full repertoire of GH receptors, IGFs, and IGF-binding proteins with tissue-specific expression patterns that evolve through development . The IGF-binding protein system in gilthead seabream shows evolutionary divergence into distinct clades (Igfbp1/2/4 and Igfbp3/5/6) that function predominantly as hepatic and muscle subtypes, respectively . These distinctions have important implications for experimental design when using this species as a model for growth and metabolism studies.

What are the optimal methods for reconstituting and storing recombinant gilthead seabream IGF1?

Recombinant gilthead seabream IGF1 requires specific handling procedures to maintain stability and bioactivity. Lyophilized IGF1 should be reconstituted in sterile 0.4% NaHCO3 adjusted to pH 8-9 at a concentration not less than 100μg/ml . While the lyophilized form remains stable at room temperature for approximately three weeks, long-term storage requires desiccation below -18°C . After reconstitution, IGF1 should be stored at 4°C if used within 2-7 days or below -18°C for future use . For extended storage periods, it is recommended to add a carrier protein (0.1% HSA or BSA) to prevent protein degradation . Repeated freeze-thaw cycles should be strictly avoided as they significantly reduce bioactivity . Purity analysis should be performed using SEC-HPLC and SDS-PAGE techniques, while protein content can be accurately quantified through RP-HPLC using a calibrated solution of IGF1 as a reference standard .

How can researchers effectively establish and utilize primary cultures of gilthead seabream myocytes for IGF1 studies?

Primary cultures of gilthead seabream myocytes provide an invaluable in vitro system for investigating the metabolic effects of IGF1. These cultures should be established according to previously described protocols (such as Montserrat et al., 2007a) which allow for the study of both myocytes (day 4) and small myotubes (day 9) . When conducting uptake studies, researchers should consider the differential responses of cells at different stages of differentiation, as 2-deoxyglucose (2-DG) uptake increases along with muscle cell differentiation, while L-alanine uptake shows an inverse pattern with higher uptake in small myocytes than in large myotubes . The experimental design should include appropriate time points for measurements, as insulin and IGFs stimulate glucose uptake in a time-dependent manner . To investigate signaling pathways, specific inhibitors such as PD-98059 (for MAPK pathway), wortmannin (for PI3K-Akt pathway), and cytochalasin B should be incorporated as pretreatments before stimulation with IGF1 . Additionally, researchers should consider measuring GLUT4 protein synthesis, which is differentially stimulated by insulin and IGFs at various stages of cell culture development .

Which signaling pathways mediate the metabolic effects of IGF1 in gilthead seabream muscle cells?

The metabolic effects of IGF1 in gilthead seabream muscle cells are mediated through at least two main signaling pathways: the mitogenesis activator protein kinase (MAPK) pathway and the PI3K-Akt transduction pathway . Experimental evidence using specific inhibitors (PD-98059 for MAPK and wortmannin for PI3K-Akt) has demonstrated that both pathways are essential for insulin and IGF-stimulated glucose uptake in these cells . The involvement of these pathways has been confirmed through 2-deoxyglucose (2-DG) uptake studies, which showed that inhibition of either pathway significantly reduces the stimulatory effects of IGF1 . Additionally, cytochalasin B inhibition studies have revealed that glucose transport occurs through specific glucose transporters, with GLUT4 being particularly important in mediating IGF1-stimulated glucose uptake . Both insulin and IGFs stimulate GLUT4 protein synthesis in gilthead sea bream muscle cells, though the pattern of stimulation varies depending on the stage of cell differentiation (day 4 versus day 9 of culture) .

How do IGF1 and IGF2 differ in their metabolic effects on gilthead seabream compared to insulin?

IGF1 and IGF2 exhibit greater efficacy than insulin in stimulating both glucose and amino acid uptake in gilthead seabream muscle cells, though their relative potency varies depending on the metabolic substrate and stage of cell differentiation . For glucose uptake, studies using 2-deoxyglucose have shown that both IGFs are more effective than insulin in stimulating uptake in both myocytes and large myotubes, with the stimulatory effect increasing along with muscle cell differentiation in vitro . Conversely, for amino acid uptake (measured using L-alanine), both IGF1 and IGF2 again demonstrate higher potency than insulin, but the effect is more pronounced in early stages of cell culture (day 4) compared to more differentiated cells (day 9) . This suggests that amino acid requirements are higher during proliferative stages than during muscle cell development . These differential effects reflect the specialized roles of IGFs versus insulin in fish metabolism, with IGFs playing particularly important roles in both growth and metabolic regulation . The biological effects of both IGFs appear to be mediated by the type I IGF receptor based on studies in mammalian systems, though the presence of IGF-II receptor has been identified in some fish species .

How does the expression of IGF1 system components change during gilthead seabream development?

The expression of IGF1 system components undergoes significant temporal changes during gilthead seabream development, with distinct tissue-specific patterns emerging over time . Transcriptomic profiling of the Gh/Igf system during early life stages (60-127 days post-hatching) has revealed that almost all genes in this system vary in expression with age . Liver and muscle tissues show prompted tissue-specific differentiation with temporal changes in the relative expression of the full repertoire of GH receptors, IGFs, and IGF-binding proteins . Studies support a differential contribution of GH receptor (ghr) and IGF subtypes through development, affecting whether GH acts via systemic or direct local tissue effects . The IGF-binding proteins also show clear developmental evolution, with the Igfbp1/2/4 clade predominantly emerging as hepatic subtypes and the Igfbp3/5/6 clade as muscle subtypes . This developmental trade-off is highly plastic and can be modulated by environmental factors, as demonstrated by the identification of ghr1 and igfbp1/3/4/5 as hypoxic imprinting genes during critical early developmental windows .

How does nutritional status affect IGF1 function and expression in gilthead seabream?

Nutritional status exerts profound effects on IGF1 function and expression in gilthead seabream, establishing a link between feeding, growth, and metabolic regulation. In vivo studies on protein/energy ratio have demonstrated nutritional regulation of the IGF-I axis in this species . Research has revealed a positive correlation between plasma IGF-I levels and changes in ration size, indicating direct responsiveness to feeding level . The role of Growth Hormone (GH) and IGF-I in seasonal growth has been documented, along with the effects of plant protein diets on nitrogen metabolism and the GH-liver axis . During compensatory growth periods following feed restriction, insulin and IGF-I show distinct actions in response to nutritional status, highlighting their specialized roles . The interplay between amino acid metabolism and glucose homeostasis has been demonstrated through studies showing that intraperitoneal arginine injection increases glucose, glucagon, and insulin plasma levels . Furthermore, experimental diets can modulate the expression of glucose transporters, with intraperitoneal arginine administration increasing GLUT4 protein levels in white skeletal muscle . These findings highlight the adaptability of the IGF system in responding to changing nutritional conditions and its importance in maintaining metabolic homeostasis during periods of nutrient fluctuation.

How can the hypoxia response of the IGF system be utilized in aquaculture research?

The hypoxia response of the IGF system in gilthead seabream provides a valuable research tool for aquaculture applications, particularly for identifying metabolic phenotypes and improving selection methods. Transcriptional reprogramming by mild hypoxia can be assessed in fingerling fish with different historical trajectories of oxygen availability . Research has identified specific genes within the GH/IGF system that function as hypoxic imprinting genes during critical early developmental windows, including ghr1 and igfbp1/3/4/5 . These molecular markers allow researchers to recognize individuals with different history trajectories of oxygen availability and, consequently, different metabolic capabilities later in life . This knowledge can be applied in practical aquaculture settings to develop improved protocols for fish selection and breeding programs focused on hypoxia tolerance. Experimental approaches should include controlled hypoxia challenges during early development followed by molecular profiling of the IGF system components. The plastic nature of this system makes it particularly valuable for understanding how early environmental conditions can shape long-term phenotypic outcomes in farmed fish populations, potentially leading to more resilient stocks with improved growth performance under suboptimal oxygen conditions.

What methodological approaches can resolve contradictions in IGF1 receptor signaling data between in vitro and in vivo studies?

Resolving contradictions between in vitro and in vivo IGF1 receptor signaling data in gilthead seabream requires integrated methodological approaches that bridge cellular and organismal contexts. A primary challenge stems from the presence of two distinct IGF-I receptor paralogues (IGF-IRa and IGF-IRb) with potentially different functions and expression patterns . Researchers should employ tissue-specific and developmental stage-specific analysis of both receptors using quantitative PCR techniques to establish their relative abundance across contexts . The differential effects of IGF-I and IGF-II on receptor regulation during in vitro myogenesis should be compared with expression patterns during ontogeny in muscle tissue . When discrepancies arise, researchers should consider the following methodological refinements: (1) implement ex vivo tissue culture systems that preserve tissue architecture while allowing controlled manipulation of IGF1 levels; (2) utilize CRISPR-Cas9 technology for paralog-specific knockout or knockdown to determine the relative contribution of each receptor type; (3) employ phosphoproteomic approaches to map downstream signaling events in both contexts; (4) develop computational models that integrate both in vitro and in vivo data to predict system-level responses. Additionally, researchers must consider that the evolutionary divergence of the two receptor paralogues may reflect functional specialization that is context-dependent, requiring experimental designs that specifically account for environmental factors, developmental stage, and nutritional status when interpreting results.

How does the IGF1 system in gilthead seabream compare to other teleost species and vertebrates?

The IGF1 system in gilthead seabream exhibits both conserved and divergent features when compared to other teleosts and vertebrates, reflecting evolutionary adaptations specific to different lineages. Phylogenetic analysis of IGF-IR orthologues from different vertebrates places gilthead seabream receptors within the broader evolutionary context of this system . Like other teleosts, gilthead seabream possesses two IGF-I receptor paralogues (IGF-IRa and IGF-IRb) resulting from the teleost-specific genome duplication event, whereas mammals have a single receptor . The amino acid sequences of these receptors show significant conservation of functional domains across vertebrates, but with teleost-specific modifications that may relate to the unique physiology of fish . Similarly, the IGF-binding protein system in gilthead seabream shows evolutionary divergence into distinct clades (Igfbp1/2/4 and Igfbp3/5/6) , which parallels patterns seen in other fish species but with species-specific expression profiles. While mammals and fish share core IGF signaling pathways (MAPK and PI3K-Akt), the relative importance and regulation of these pathways may differ, as evidenced by the distinct metabolic roles of IGF1 in fish compared to mammals . These differences likely reflect adaptations to different environmental challenges and metabolic demands between aquatic and terrestrial vertebrates, as well as between different fish lineages adapted to varying ecological niches.

What insights does the duplicated IGF1 receptor system in gilthead seabream provide about the evolution of growth regulation in vertebrates?

The duplicated IGF1 receptor system in gilthead seabream provides valuable insights into the evolution of growth regulation mechanisms following whole genome duplication events in the vertebrate lineage. The presence of two distinct IGF-I receptor paralogues (IGF-IRa and IGF-IRb) in this species represents a classic case of gene duplication followed by potential sub-functionalization or neo-functionalization . Comparative analysis of the coding sequences and protein architecture of these receptors, coupled with their expression patterns across tissues, reveals how duplicated genes can evolve distinct but complementary roles in regulating growth and metabolism . The differential expression of these receptors during ontogeny in muscle tissue, along with their distinct responses to IGF-I and IGF-II during in vitro myogenesis, suggests specialized functions that may allow for more nuanced regulation of growth processes . This duplication parallels the expanded repertoire of GH receptors, IGFs, and IGF-binding proteins in teleosts compared to tetrapods , collectively creating a more complex regulatory network. The evolutionary divergence of IGF-binding proteins into primarily hepatic (Igfbp1/2/4) and muscle (Igfbp3/5/6) subtypes further illustrates how gene duplication events can lead to tissue-specific refinement of signaling systems . These observations support the hypothesis that genome duplication events have facilitated the evolution of more sophisticated growth regulation mechanisms in vertebrates, potentially contributing to their remarkable diversity in body size and metabolic adaptations.