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Carbohydrate utilization by the working muscle of rainbow trout

Final Report Summary - GLUCOSE USE IN FISH (Carbohydrate utilization by the working muscle of rainbow trout)

Carbohydrate utilization by the working muscles of rainbow trout (GLUCOSE USE IN FISH).
Fish commercial diets may contain high content of carbohydrates which appears to be in discrepancy with their low natural capacity of carnivorous species to utilize it. This is an important area for research, because a high proportion of carbohydrates in the diet can provide readily available glucose to supply most of the energy needed for maintenance, so that a greater proportion of protein can be reserved to build up muscle tissue. We show that exercise increase carbohydrate utilization in fish, using trout, a carnivorous group of fish cultured world-wide as the experimental model. To study the effects of exercise on trout we used a multi-tissue genomic approach, a whole-organism physiological assessment and a cellular mechanistic approach, placing emphasis on candidate targets of exercise including glucose transporters and AMP-activated protein kinase in fish muscles.
1. Evaluating physiological changes related to sustain swimming in trout.
We divided this study in two parts, the first studying the general physiological changes occurring in trout during long term swimming and the effects of this condition on several metabolic parameters, changes in specific enzyme activities and gene expression. On the second part we use an in depth-study on the response of trout red and white muscle transcriptome to the same conditions. For this purpose we used spawning female rainbow trout (Oncorhynchus mykiss) rested (n=10) or exercised (n=10) for 1176 km at 0.75 body-lengths per second in a 6,000-L swim-flume. The results summarized in this section will be submitted as two separate publications in two international journals and are currently in the last stage of preparation. We found that after 40 days of exercise the rainbow trout, the expression of glucose transporters in red and white muscles of rainbow trout was unaltered. During this metabolic condition that recreates the spawning migration in trout, levels of glucose, lactate and lipids in the circulation were unaltered. Similarly, glycogen and lipid reserves were conserved in both types of muscles, which may suggest the importance of mechanisms controlling energy use in these tissues, as is suggested by the increase in AMPK activity in both muscles. In this work we identified and measured gene expression of the different AMPK subunits in muscle of trout for the first time in this species. Additionally, we analyzed changes occurring at the gene expression level of enzymes and transporters related with fatty acid use and aerobic metabolism. Results indicate that in this metabolic condition trout mobilize lipid reserves that are used to sustain the metabolic demands of endurance exercise. We have used Next-Generation Sequencing (NGS, Solexa/Illumina GA2 sequencing system) to provide an in-depth view of the transcriptome of red and white skeletal muscle of exercised and non-exercised rainbow trout with the specific objective to quantify transcriptomic effects of exercise. Red and white muscle RNA of exercised and non-exercised fish was sequenced and resulted in 15-17 million reads per lane that after de novo assembly yielded 149,159 red muscle contigs and 118,572 white muscle contigs. Most contigs were annotated by iteratively blasting against salmonid ESTs, zebrafish genome and Metazoan genes. In red muscle of exercised compared to rested fish large contigs were up- (51) or down-regulated (118), whereas in white muscle large contigs were up- (29) or down-regulated (71). Transcriptomic analysis revealed that the red muscle is more involved in the induced sustained swimming than the white muscle although the exercised white muscle shows increased transcriptional activity and may switch to a more aerobic phenotype. The unique collection of transcript sequences in red and white muscle that was obtained in this study is a valuable database that will greatly contribute to the understanding of red and white muscle functioning.
2. Evaluating the ability of exercised trout to utilize a high-carbohydrate diet.
After 30 days of exercising and feeding the rainbow trout with a high-carbohydrate diet, the expression of GLUT1 in red muscle of rainbow trout was up-regulated with respect to the control fish. A similar analysis of the expression of GLUT4 in the muscle of rainbow trout shows that this transporter is up-regulated during the high-carbohydrate diet/exercise condition. Our results confirm that the expression of both GLUT transporters in the sarcolemma of rainbow trout is regulated by the combination of high dietary carbohydrates and exercise, as it has been previously demonstrated in trout during fasting or insulin administration. Moreover, this study is the first to show that GLUT1 and GLUT4 transporters can be up-regulated when the supply of glucose (i.e. with carbohydrate-rich diets) and the metabolic disposal of this metabolite by red and white muscle (i.e. by sustained swimming) are increased.
Mircoarray analysis of red and white muscle evidenced changes in the expression of genes related with the immune response and muscle development. These changes suggest an important restructuration and a boost of the immune response of the fish. Future research should be directed toward this area to find the exact mechanism in which this process operates, which may produce associated benefits of exercise in farmed fish, in addition to the increase in carbohydrate utilization. The results summarized in this section will be submitted for publication in an international journal, and are currently in the last stage of preparation.
3. Studying the effects of the activation of AMP-dependent protein kinase (AMPK) on differentiated trout muscle cells using exercise mimetic compounds.
AMP-activated protein kinase (AMPK), a master metabolic switch, mediates the observed increase of glucose uptake in locomotory muscle of mammals during exercise. AMPK is activated by changes in the intracellular ATP:AMP ratio but also by synthetic compounds such as AICAR and the anti-diabetic drugs metformin and phenformin, that have been shown to increase glucose transport in mammalian muscle cells. Despite the physiological importance of exercise as a modulator of glucose disposal by locomotory muscle, the possible role of AMPK mediating the effects of muscle contraction on glucose uptake has not been fully investigated in other vertebrates, including fish. We investigated the role of AMPK in the regulation of glucose transport in fish muscle cells by studying the direct effects of AMPK activators on glucose uptake, cell surface levels and expression of GLUT4 and AMPK activity in trout myotubes. Our results show that AICAR, metformin and phenformin significantly stimulate glucose uptake by trout myotubes after a 24 h incubation and addition of compound C, a selective inhibitor of AMPK, completely abrogates the stimulatory effects of all AMPK agonists. Interestingly, the combination of insulin and AMPK-agonists does not result in additive nor synergistic effects on glucose uptake in trout myotubes. Moreover, exposure of trout myotubes to AICAR and metformin results in an increase in AMPK activity. Finally, we also show that AMPK-agonist stimulation of glucose uptake in trout myotubes may take place, at least in part, by significantly increasing the cell surface levels of trout GLUT4 and GLUT4 mRNA expression, Therefore, we provide direct evidence for the first time in non-mammalian vertebrates for an important role of AMPK in stimulating glucose uptake in muscle cells. The results summarized in this section are submitted for publication in the American Journal of Physiology.
4. Studying the effects of electrical stimulation on differentiated trout muscle cells.
We developed an in vitro model that mimics an exercise condition as a tool that allows us to further investigate the involvement of fish muscle in the possible mechanism of glucose homeostasis.
We developed a protocol under specific conditions to deliver electric pulses to a preparation of trout myotube and found that the stimulus applied to them produces a significant increase in their glucose uptake. Such process is mediated by AMPK because the addition of compound C, a selective inhibitor of AMPK, completely abrogates the stimulatory effects of the electrical stimulation. We have confirmed this mechanism by studying changes occurring in the activity of AMPK and in GLUT1 and GLUT4 gene expression of trout myotubes under electric stimulation. The results summarized here will be prepared for scientific communication within the next 2 months.
This project has provided important evidence for swimming-enhanced carbohydrate utilization in fish, which may contribute to improve growth. These conclusions contribute to our integral understanding of the exercise effects in fish and their application for farming a fitter fish in sustainable aquaculture. Our integrative study involving the effects of exercise in vivo and using in vitro models to find mechanisms responsible for the carbohydrate utilization by the trout muscle showed that, in this species, sustained exercise could be used to enhance the use of carbohydrate-rich diets. Especially for this purpose, the 1st International FitFish Workshop on the Swimming Physiology of Fish (http://www.ub.edu/fitfish2010) has been organized to bring together a multidisciplinary group of scientists using exercise models, industrial partners and policy makers and for setting directions for policy and future studies.