Effects of sustained swimming on the red and white muscle transcriptome of rainbow trout (Oncorhynchus mykiss) fed a carbohydrate-rich diet

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Abstract

Training at sustainable swimming speeds can produce changes in fish skeletal muscle that are important for aquaculture due to their growth-potentiating effects. Such changes may be even more relevant when fish are fed diets containing an increasing proportion of carbohydrates as an energy source. We evaluated the effects of moderate-intensity sustained swimming on the transcriptomic response of red and white muscle in rainbow trout fed a carbohydrate-rich diet using microarray and qPCR. Analysis of the red and white muscle transcriptome in resting or swimming (1.3 body lengths/s) fish for 30 days revealed significant changes in the expression of a large number of genes (395 and 597, respectively), with a total of 218 differentially expressed genes (DEGs) common for both muscles. A large number of the genes involved in glucose use and energy generation, contraction, development, synthesis and catabolism of proteins were up-regulated in red and white muscle. Additionally, DEGs in both muscles were involved in processes of defense response and apoptosis. Skeletal muscle contraction activates a transcriptional program required for the successful adaptation of both muscles to the changing demands imposed by swimming conditions. Future studies should further clarify the mechanisms involved in the adaptation of both tissues to exercise and assess possible benefits of such conditions for cultured fish.

Introduction

The optimization of culture conditions to maximize fish growth is an aspect of major importance for aquaculture's commercial production. Because fish growth is strongly influenced by environmental and nutritional factors (Haard, 1992, Lie, 2001, Palstra and Planas, 2011), it is essential to understand how swimming activity and the use of an alternative diet may affect the physiology of fish, particularly with regard to their skeletal muscle.

Skeletal muscle represents more than 60% of the fish body mass and plays an important role during swimming as well as in whole-body metabolic homeostasis. In fish, skeletal muscle is spatially segregated and consists of deep white fibers than constitute approximately 90% of the total muscle mass, covered by a thin superficial layer of red muscle fibers. Red and white muscle fibers differ in their structure and function as they are recruited in different proportions at different swimming velocities (Bone, 1979). Red fibers are active during sustained swimming and contain high numbers of lipid droplets and mitochondria, relying on aerobic metabolism. On the other hand, white fibers are recruited in an increasing proportion as swimming velocity increases, and they contain very few mitochondria, relying mainly on anaerobic glycolysis (Johnston, 1981, Altringham and Ellerby, 1999).

Skeletal muscle growth in fish involves a complex system of regulation influenced by several factors including the genotype and the environment (Kiessling et al., 2006, Johnston et al., 2011). Swim training has growth-potentiating effects in both white and red muscle by increasing their number of fibers and/or their size (Greer Walker and Emerson, 1978, Davison, 1997, Johnston, 1999, Ibarz et al., 2011). In addition to these modifications, swim training at sustainable speeds produces important metabolic adjustments in skeletal muscle (Davison and Goldspink, 1977, Farrell et al., 1991, Magnoni and Weber, 2007, Anttila et al., 2008, LeMoine et al., 2010).

Fish are able to cover the increased demands of ATP required during muscle contraction through hydrolysis of phosphocreatine, glycolysis and oxidative phosphorylation (Richards et al., 2002). Although there can be great variability in the source and quantity of energy available, this energy must be derived from the catabolism of lipids, carbohydrates or proteins (Weber, 2011), that ultimately have to be obtained from the diet. In terms of whole-body energy metabolism, carbohydrates and lipids appear to have preponderant roles as metabolic fuels, particularly at low to moderate-intensity swimming speeds (Moyes and West, 1995). In particular, sustainable swimming in rainbow trout at 1–2 body lengths per second (BL/s) is supported by approximately equal contributions of carbohydrate and lipid oxidation (Richards et al., 2002). Therefore, the metabolic changes produced in both red and white muscles by swimming activity are relevant, particularly when fish are fed diets containing an increased proportion of energy in the form of highly digestible carbohydrates. An increasing proportion of dietary carbohydrates can provide the energy needed for maintenance and to sustain swimming, so that a greater proportion of proteins can be spared to build up muscle tissue (Houlihan et al., 1995). In addition, supplementing the diet of fish with carbohydrates would improve the sustainability and reduce the environmental impact of the fish farming industry. Hence, there is a continuing interest to understand how the metabolic phenotype of fish can adapt to increased dietary carbohydrate availability. Nevertheless, rainbow trout (Oncorhynchus mykiss) are unable to use high levels of dietary carbohydrates because feeding the fish such a diet results in prolonged postprandial hyperglycemia (Moon, 2001, Hemre et al., 2002). However, we recently showed that when trout are fed a carbohydrate-rich diet (composed of 30% gelatinized starch) the capacity of glucose uptake and utilization by red and white muscle is increased during swimming (1.3 BL/s), decreasing the duration of hyperglycemia and improving the deposition of dietary protein for muscle growth (Felip et al., 2012).

In mammals, exercise has a clear effect in lowering plasma glucose (Hayashi et al., 1997), with skeletal muscle playing a key role in glucose homeostasis (Jessen and Goodyear, 2005). The rate-limiting step in glucose utilization by mammalian skeletal muscle appears to be its movement across the sarcolemma by carrier facilitative transport (Kubo and Foley, 1986), with glucose transporters (GLUT) 1 and 4 being the most preponderant in this tissue. Exercise increases glucose utilization in skeletal muscle by, at least in part, increasing the transcription of the GLUT4 gene (Neufer and Dohm, 1993, MacLean et al., 2002). Salmonid fish possess GLUT homologs that are expressed and regulated in response to hormonal treatment and feeding status in red and white muscle (Planas et al., 2000, Capilla et al., 2002, Díaz et al., 2007a, Díaz et al., 2007b, Díaz et al., 2009). Therefore, it is possible that the effects of swimming on glucose metabolism could be mediated by the up-regulation of GLUT1 and/or GLUT4 in rainbow trout skeletal muscle. The transcriptome of fish skeletal muscle is also altered by changing feed composition, resulting in modifications in energy use, protein metabolism, cell proliferation, apoptosis and immune response mechanisms (Tacchi et al., 2011, Tacchi et al., 2012) (Table 1).

In this study, we used microarray technology and quantitative real-time PCR (qPCR) to assess overall gene expression and to determine traits that may be modified in the red and white muscle of trout with exercise. Because this study was part of a broader study, our goal was to investigate the transcriptomic response of red and white muscles to swimming activity when using an energy-rich diet, integrating these results with those previously published on the use of nutrients as energy fuels for swimming (Felip et al., 2012). For that purpose, rainbow trout were fed a carbohydrate-rich diet and were swum or kept under a low speed current for 30 days. After that period, we analyzed changes in the expression of structural, regulatory, metabolic and immune related genes in the red and white muscle of trout.

Section snippets

Animals and experimental conditions

Juvenile rainbow trout (O. mykiss) purchased from a local fish farm (Truites del Segre, Lleida, Spain) were held in the facilities of the School of Biology (University of Barcelona, Barcelona, Spain) in 1000 L tanks with fresh water in a semi-closed system (10% of water renovation daily) with physical and biological filters, ozone skimmers, continuous aeration and optimal water quality parameters at 15 °C and a 12 h light–12 h dark photoperiod. Fish with an average mass of 60 g were randomly

Results

This study was undertaken to understand the effects of sustained swimming in rainbow trout fed a diet containing a high proportion of digestible carbohydrates as an important source of metabolizable energy. In the first part of this study, we previously evaluated the changes produced by swimming activity on the relative metabolic use of carbohydrates and proteins by using stable isotopes as dietary tracers (Felip et al., 2012). To further characterize these observed metabolic effects, we

Discussion

This is the first study to evaluate the effect of moderate-intensity sustained swimming on the transcriptomic response of red and white muscle in rainbow trout fed a carbohydrate-rich diet. A swimming regime of 1.3 BL/s for 1 month was used as a metabolic promoter to increase glucose utilization by the skeletal muscle of trout. We had previously reported that, in this same group of fish, swimming 1) increases food intake, 2) decreases plasma glucose levels and 3) increases deposition of glycogen

Conclusions

Overall, transcriptomic profiling of skeletal muscle from trout subjected to sustained swimming provides molecular evidence that supports the physiological changes experienced by trout under these conditions. Namely, the increase in fiber hypertrophy that is the basis of the growth-promoting effects of exercise, the increase in the uptake and use of carbohydrates as fuel, the increase in protein deposition and the resulting protein-sparing effect as previously suggested by Felip et al. (2012).

Acknowledgments

This study was supported by grants from the Ministerio de Ciencia e Innovacion, Spain (CSD2007-0002 and AGL2009-07006 to J. V. Planas). L. J. Magnoni was supported by a FP7-PIIF-2009 fellowship (Marie Curie Action) from the European Commission (GLUCOSE USE IN FISH) with grant agreement number 235581. We would like to thank the Turku Centre of Biotechnology (Finland) for the preparation of microarrays and Dr. Aleksei Krasnov (Nofima, As, Norway) for his assistance with microarray analysis.

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    Present address: Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). Instituto de Investigaciones Biotecnológicas — Instituto Tecnológico de Chascomús (IIB-INTECH), Av. Intendente Marino Km. 8,2 (B7130IWA), Chascomús, Argentina.

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