Alfredo Molina

Regulation of skeletal muscle growth in fish by the growth hormone - Insulin-like growth factor system

The growth hormone (GH)-insulin-like growth factor (IGF) system is the key promoter of growth in vertebrates; however, how this system modulates muscle mass in fish is just recently becoming elucidated. In fish, the GH induces muscle growth by modulating the expression of several genes belonging to the myostatin (MSTN), atrophy, GH, and IGF systems as well as myogenic regulatory factors (MRFs). The GH controls the expression of igf1 via Janus kinase 2 (JAK2)/signal transducers and activators of the transcription 5 (STAT5) signaling pathway, but it seems that it is not the major regulator. These mild effects of the GH on igf1 expression in fish muscle seem to be related with the presence of higher contents of truncated GH receptor1 (tGHR1) than full length GHR (flGHR1). IGFs in fish stimulate myogenic cell proliferation, differentiation, and protein synthesis through the MAPK/ERK and PI3K/AKT/TOR signaling pathways, concomitant with abolishing protein degradation and atrophy via the PI3K/AKT/FOXO signaling pathway. Besides these signaling pathways control the expression of several genes belonging to the atrophy and IGF systems. Particularly, IGFs and amino acid control the expression of igf1, thus, suggesting other of alternative signaling pathways regulating the transcription of this growth factor. The possible role of IGF binding proteins (IGFBPs) and the contribution of muscle-derived versus hepatic-produced IGF1 on fish muscle growth is also addressed. Thus, a comprehensive overview on the GH-IGF system regulating fish skeletal muscle growth is presented, as well as perspectives for future research in this field.

IGF-I/PI3K/Akt and IGF-I/MAPK/ERK pathways in vivo in skeletal muscle are regulated by nutrition and contribute to somatic growth in the fine flounder

The insulin-like growth factor-I (IGF-I) is a key regulator of skeletal muscle growth in vertebrates, promoting mitogenic and anabolic effects through the activation of the MAPK/ERK and the PI3K/Akt signaling pathways. Nutrition also affects skeletal muscle growth, activating intracellular pathways and inducing protein synthesis and accretion. Thus, both hormonal and nutritional signaling regulate muscle mass. In this context, plasma IGF-I levels and the activation of both pathways in response to food were evaluated in the fine flounder using fasting and refeeding trials. The present study describes for the first time in a nonmammalian species that the MAPK/ERK and PI3K/Akt are activated by exogenous circulating IGF-I, as well as showing that the MAPK/ERK pathway activation is modulated by the nutritional status. Also, these results show that there is a time-dependent regulation of IGF-I plasma levels and its signaling pathways in muscle. Together, these results suggest that the nutritionally managed IGF-I could be regulating the activation of the MAPK/ERK and the PI3K/Akt signaling pathways differentially according to the nutritional status, triggering different effects in growth parameters and therefore contributing to somatic growth in fish. This study contributes to the understanding of the nutrient regulation of IGF-I and its signaling pathways in skeletal muscle growth in nonmammalian species, therefore providing insight concerning the events controlling somatic growth in vertebrates.

Plasma leptin and growth hormone levels in the fine flounder (Paralichthys adspersus) increase gradually during fasting and decline rapidly after refeeding.

In fish, recent studies have indicated an anorexigenic role of leptin and thus its possible involvement in regulation of energy balance and growth. In the present study, the effects of fasting and refeeding periods on plasma leptin levels were studied in the fine flounder, a flatfish with remarkably slow growth. To further assess the endocrine status of the fish during periods of catabolism and anabolism, plasma growth hormone (GH) levels were also analyzed. Under normal feeding condition, plasma leptin and GH levels remained stable and relatively high in comparison with other teleost species. For the three separate groups of fish, fasted for 2, 3, and 4 weeks, respectively, plasma leptin levels increase gradually, becoming significantly elevated after 3 weeks, and reaching highest levels after 4-week fasting. Plasma GH levels were significantly elevated after 2-week fasting. At the onset of refeeding, following a single meal, leptin levels decline rapidly to lower than initial levels within 2 h, irrespective of the length of fasting. Plasma GH also decline, the decrease being significant after 4, 24 and 2 h for the 2, 3 and 4-week fasted groups, respectively. This study shows that plasma leptin levels in the fine flounder are strongly linked to nutritional status and suggests that leptin secretion is regulated by fast-acting mechanisms. Elevated leptin levels in fasted fish may contribute to a passive survival strategy of species which experience natural food shortage periods by lowering appetite and limiting physical foraging activity.

Nutritional status modulates plasma leptin, AMPK and TOR activation, and mitochondrial biogenesis: Implications for cell metabolism and growth in skeletal muscle of the fine flounder.

Insight of how growth and metabolism in skeletal muscle are related is still lacking in early vertebrates. In this context, molecules involved in these processes, such as leptin, AMP-activated protein kinase (AMPK), target of rapamicyn (TOR), peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α, and oxidative phosphorylation complexes (OXPHOS), were assessed in the skeletal muscle of a fish species. Periods of fasting followed by a period of refeeding were implemented, using the fine flounder as a model (Paralichthys adspersus). This species exhibits remarkably slow growth and food intake, which is linked to an inherent growth hormone (GH) resistance and high circulating levels of leptin. Leptin increased during fasting concomitantly with AMPK activation, which was inversely correlated with TOR activation. On the other hand, AMPK was directly correlated with an increase in PGC-1α and OXPHOS complexes contents. Dramatic changes in the activation and content of these molecules were observed during short-term refeeding. Leptin, AMPK activation, and PGC-1α/OXPHOS complexes contents decreased radically; whereas, TOR activation increased significantly. During long-term refeeding these molecules returned to basal levels. These results suggest that there is a relation among these components; thus, during fasting periods ATP-consuming biosynthetic pathways are repressed and alternative sources of ATP/energy are promoted, a phenomenon that is reversed during anabolic periods. These results provide novel insight on the control of metabolism and growth in the skeletal muscle of a non-mammalian species, suggesting that both processes in fish muscle are closely related and coordinated by a subset of common molecules.

Catabolic Signaling Pathways, Atrogenes, and Ubiquitinated Proteins Are Regulated by the Nutritional Status in the Muscle of the Fine Flounder

A description of the intracellular mechanisms that modulate skeletal muscle atrophy in early vertebrates is still lacking. In this context, we used the fine flounder, a unique and intriguing fish model, which exhibits remarkably slow growth due to low production of muscle-derived IGF-I, a key growth factor that has been widely acknowledged to prevent and revert muscle atrophy. Key components of the atrophy system were examined in this species using a detailed time-course of sampling points, including two contrasting nutritional periods. Under basal conditions high amounts of the atrogenes MuRF-1 and Atrogin-1 were observed. During fasting, the activation of the P38/MAPK and Akt/FoxO signaling pathways decreased; whereas, the activation of the IκBα/NFκB pathway increased. These changes in signal transduction activation were concomitant with a strong increase in MuRF-1, Atrogin-1, and protein ubiquitination. During short-term refeeding, the P38/MAPK and Akt/FoxO signaling pathways were strongly activated, whereas the activation of the IκBα/NFκB pathway decreased significantly. The expression of both atrogenes, as well as the ubiquitination of proteins, dropped significantly during the first hour of refeeding, indicating a strong anti-atrophic condition during the onset of refeeding. During long-term refeeding, Akt remained activated at higher than basal levels until the end of refeeding, and Atrogin-1 expression remained significantly lower during this period. This study shows that the components of the atrophy system in skeletal muscle appeared early in the evolution of vertebrates and some mechanisms have been conserved, whereas others have not. These results represent an important achievement for the area of fish muscle physiology, showing an integrative view of the atrophy system in a non-mammalian species and contributing to novel insights on the molecular basis of muscle growth regulation in earlier vertebrates.

Dynamic transcriptional regulation of autocrine/paracrine igfbp1, 2, 3, 4, 5, and 6 in the skeletal muscle of the fine flounder during different nutritional statuses

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.

Inherent growth hormone resistance in the skeletal muscle of the fine flounder is modulated by nutritional status and is characterized by high contents of truncated GHR, impairment in the JAK2/STAT5 signaling pathway, and low IGF-I expression.

A detailed understanding of how the GH and IGF-I regulate muscle growth, especially in early vertebrates, is still lacking. The fine flounder is a flatfish species exhibiting remarkably slow growth, representing an intriguing model for elucidating growth regulatory mechanisms. Key components of the GH system were examined in groups of fish during periods of feeding, fasting, and refeeding. Under feeding conditions, there is an inherent systemic and local (muscle) GH resistance, characterized by higher levels of plasma GH than of IGF-I, skeletal muscle with a greater content of the truncated GH receptor (GHRt) than of full-length GHR (GHRfl), an impaired activation of the Janus kinase 2 (JAK2)-signal transducers and activators of transcription 5 (STAT5) signaling pathway, and low IGF-I expression. Fasting leads to further elevation of plasma GH levels concomitant with suppressed IGF-I levels. The ratio of GHRfl to GHRt in muscle decreases during fasting, causing an inactivation of the JAK2/STAT5 signaling pathway and suppressed IGF-I expression, further impairing growth. When fish are returned to nutritionally favorable conditions, plasma GH levels decrease, and the ratio of GHRfl to GHRt in muscle increases, triggering JAK2/STAT5 reactivation and local IGF-I expression, concomitant with increased growth. The study suggests that systemic IGF-I is supporting basal slow growth in this species, without ruling out that local IGF-I is participating in muscle growth. These results reveal for the first time a unique model of inherent GH resistance in the skeletal muscle of a nonmammalian species and contribute to novel insights of the endocrine and molecular basis of growth regulation in earlier vertebrates.

A transformed fish cell line expressing a green fluorescent protein-luciferase fusion gene responding to cellular stress

We obtained a stable transformed fish (EPC) cell line containing a reporter gene under the control of the tilapia HSP70 promoter. Expression of the reporter gene, coding for a green fluorescent protein (GFP)-luciferase fusion protein, was assessed by measuring the luciferase enzymatic activity by luminometry and the GFP expression by fluorescence microscopy and flow cytometry. The clone was characterized for its capacity to respond to heat shock treatment. The results show high induction after 1 h at 37 degrees C of treatment, up to 500-fold. In addition, its convenience to detect a large range of cellular stressors was evaluated. We observed high induction when Cd2+, Zn2+, Hg2+ or Cu2+ was added, but not Pb2+. In addition, activation of the reporter gene was observed in the presence of other compounds such as acetyl chloride, tetrachlorophenol, chloroacetamide and sodium arsenite. In conclusion, this cell line can be used as a rapid, cheap and easy biological test to determine cellular stress induced by environmental pollutants, alone or in conjunction with other, more specific assays.

Molecular cloning of IGF-1 and IGF-1 receptor and their expression pattern in the Chilean flounder (Paralichthys adspersus)

Insulin-like growth factor-1 and insulin-like growth factor-1 receptor (IGF-1 and IGF-1R) play main roles in vertebrate growth and development. In fish, besides contributing to somatic growth, both molecules exhibit pleiotropic functions. We isolated complete cDNAs sequences encoding for both IGF-1 and IGF-1R in the Chilean flounder by using RT-PCR and rapid amplification of cDNAs ends (RACE) techniques. We analyzed gene expression in pre-metamorphic larvae and different organs of juvenile fish through whole mount in situ hybridization and RT-PCR, respectively. In addition, we studied the presence of calcified skeletal structures in pre-metamorphic larvae through the fluorescent chromophore calcein. The IGF-1 cDNA sequence displays an open reading frame of 558 nucleotides, encoding a 185 amino acid preproIGF-1. Moreover, IGF-1R contains an open reading frame spanning 4239 nucleotides, rendering a 702 amino acid subunit alpha and a 676 amino acid subunit beta. The deduced mature IGF-1 and IGF-1R exhibited high sequence identities with their corresponding orthologs in fishes, especially those domains involved in biological activity. RT-PCR showed expression of IGF-1 and IGF-1R transcripts in all studied tissues, consistent with their pleiotropic functions. Furthermore, we observed IGF-1 expression in notochord and IGF-1R expression in notochord, somites and head in larvae of 8 and 9 days post fertilization. Complementarily, we detected in larvae of 8 days post fertilization the presence of calcified skeletal structures in notochord and head. Interestingly, both mRNAs and calcified structures were found in territories such as notochord, an embryonic midline structure essential for the pattern of surrounding tissues as nervous system and mesoderm. Our results suggest that IGF-1 and its receptor play an important role in the development of the nervous system, muscle and bone-related structures during larval stages.

An increased expression of nucleolin is associated with a physiological nucleolar segregation.

Nucleolar segregation is the most striking cellular phenotypic feature of cold-acclimatized carp and depicts the cyclical reprogramming that the physiology of the fish undergoes between summer and winter, where a clear differential expression of some nucleolar related genes occurs. We characterized carp nucleolin, a nucleolar protein involved in multiple steps of ribosome biogenesis, and evaluated its expression upon fish acclimatization. We show that the carp cDNA deduced amino acid sequence exhibits the same tripartite structural organization found in other species. Nevertheless, we observed that nucleolin mRNA expression was strongly induced in the cold-adapted carp as was the nuclear protein content, assessed by immunocytochemistry in liver sections. The physiological up-regulation of nucleolin in the cold-acclimatized carp, where rRNA transcription and processing are depressed concomitantly with the nucleolus segregation, is consistent with the notion that nucleolin plays a fundamental role in repressing rRNA synthesis.