**5.1. Regulation of GLUT4 mRNA levels in fish**

In mammals, GLUT4 is mainly expressed in insulin-sensitive tissues, namely skeletal and cardiac muscle and adipose tissue. In skeletal muscle, GLUT4 is the main transporter expressed and it has been estimated that it accounts for approximately 70% of the basal glucose transport in this tissue (reviewed in [29]). In fish, the pattern of the tissue expression of GLUT4 at the mRNA level has only been examined in two different species: the brown trout [26] and the Atlantic cod [56]. In these two fish species, the level of GLUT4 mRNA was shown to be highest in red (slow) and white (fast) skeletal muscle. In Atlantic cod, the heart also showed high levels of GLUT4 mRNA but not in brown trout. Lower levels of GLUT4 mRNA were observed in adipose tissue, gill, kidney and other tissues in these two species. In rainbow trout, GLUT4 mRNA transcripts have also been detected in white skeletal muscle [57]. Overall, the pattern of tissue expression of GLUT4 transcripts in fish agrees well with the reported main expression of GLUT4 in the mammalian skeletal muscle, coinciding with this tissue as the primary insulin target and major site for glucose disposal. The presence of the GLUT4 protein in skeletal muscle and adipose tissue of trout has been confirmed by immunoblotting [31,32].

The expression of the GLUT4 gene is known to be subjected to an important transcriptional regulation that determines the protein levels of GLUT4 in skeletal and cardiac muscle and in adipose tissue in mammals. In mammals, a number of factors are known to influence GLUT4 expression, most notably the nutritional and dietary status and hormones (e.g. insulin, insulinlike growth factor I (IGF-I) and thyroid hormones) (reviewed in [28,58]). In fish, most current evidence regarding the regulation of GLUT4 expression is at the mRNA level and in skeletal muscle, due to the importance of this tissue in glucose homeostasis. As in mammals, the expression of GLUT4 mRNA levels increase during muscle cell differentiation in trout, as was demonstrated by the gradual increase in GLUT4 mRNA levels during the differentiation process of trout muscle cells in culture from myoblasts to myotubes [59]. In addition, the amount of immunoreactive GLUT4 was observed to be higher in trout myotubes than in myoblasts [32], strongly suggesting that GLUT4 can be considered a marker of muscle differentiation also in fish.

*5.1.2. Regulation of GLUT4 mRNA levels by contractile activity in fish*

through the induction of AMPK activity.

**5.2. Regulation of the activity of the fish GLUT4 promoter**

In mammals, exercise is known to increase the transcription of the GLUT4 gene and, conse‐ quently, to increase glucose utilization in skeletal muscle [61,62]. The exercise-induced GLUT4 mRNA expression in the mammalian skeletal muscle is believed to be mediated largely by AMP-dependent protein kinase (AMPK), an energy sensor that is activated when increases in the AMP/ATP ratio occur, as in response to exercise [63]. In trout, swimming-induced exercise was also recently shown to promote glucose uptake and utilization in skeletal muscle [64]. Importantly, swimming-induced exercise increased the mRNA levels of GLUT4 in red and white skeletal muscle in trout, as in mammals [65], supporting the notion that the increase in GLUT4 in skeletal muscle may have been responsible, at least in part, for the decrease in circulating glucose levels and increased uptake and utilization of glucose by skeletal muscle of exercised trout [64]. Furthermore, pharmacological activation of AMPK by AICAR or metformin in trout skeletal muscle cells in culture caused an increase in GLUT4 mRNA levels [66]. Given that swimming-induced exercise increased AMPK activity in red and white skeletal muscle in trout (Magnoni and Planas, unpublished observations), there is strong evidence to believe that swimming-induced exercise increases GLUT4 mRNA levels in skeletal muscle

Structural and Functional Evolution of Glucose Transporter 4 (GLUT4): A Look at GLUT4 in Fish

http://dx.doi.org/10.5772/58094

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It is known that changes in the mRNA levels of GLUT4 in skeletal muscle in mammals (i.e. increases during exercise and decreases during states of insulin deficiency) are due to altera‐ tions in the transcription rate of the GLUT4 gene [29]. In mammals, the cis-regulatory region of the GLUT4 gene is relatively well characterized and is known to contain motifs that are important for the tissue-specific expression of the GLUT4 gene and its regulation. As indicated above (section 5.1.1), one of the most effective inducers of GLUT4 mRNA and protein expres‐ sion in mammals is insulin. However, the regulation of the transcription of the GLUT4 gene by insulin in mammals is not well understood, particularly in the light of published data indicating that, paradoxically, insulin inhibits the transcription of the GLUT4 gene [67,68]. Interestingly, a recent study reported that the activity of a fish (i.e. Fugu) GLUT4 promoter, when expressed in mammalian muscle L6 cells, is inhibited by insulin [30]. Although the mechanism by which insulin represses the activity of the GLUT4 gene is not known in mammals, deletion analyses of the Fugu GLUT4 promoter have indicated that the region of the Fugu GLUT4 gene that is downstream of the main transcription start site may be sufficient for mediating the inhibitory effects of insulin on GLUT4 transcription [30]. Further studies are clearly needed to resolve the question of the paradoxical effects of insulin on GLUT4 gene transcription. Despite the inhibition of the activity of the Fugu GLUT4 gene promoter by insulin, other stimuli known to increase GLUT4 mRNA levels have been shown to cause an induction of Fugu GLUT4 promoter activity. First, ligand activation of PPARγ, which in mammals results in an increase of GLUT4 mRNA levels [69], increased the activity of the Fugu GLUT4 promoter expressed in L6 cells [30]. Second, electrical stimulation of mouse C2C12 myotubes expressing the Fugu GLUT4 promoter resulted in an increase in the activity of the Fugu GLUT4 promoter. Given the recent demonstration that swimming-induced skeletal

#### *5.1.1. Regulation of GLUT4 mRNA levels by insulin in fish*

In mammals, insulin exerts its hypoglycemic action in part by increasing the expression of GLUT4 in skeletal muscle. Data in fish also indicates that the expression of GLUT4 mRNA in skeletal muscle appears to be regulated by circulating insulin levels in a muscle type specific manner. As it is well known, fish skeletal muscle can be differentiated into two anatomically and functionally different types of muscle: white muscle, that is a fast, anaerobic muscle that permits sudden bursts of motion, and red muscle, that is a slow, aerobic muscle that permits sustained locomotion. Although red muscle only comprises 5-10% of the body weight in fish (in contrast to > 50% for white muscle), it has a higher glucose uptake rate and insulin receptor density than white muscle. Interestingly, the *in vivo* regulation of GLUT4 mRNA levels by circulating insulin in trout appears to take place only in red skeletal muscle. Decreases in circulating insulin levels caused by fasting or by feeding a diet containing low protein and high carbohydrate levels were associated with a decrease in GLUT4 mRNA levels in red but not white skeletal muscle in trout [60]. In contrast, increases in circulating insulin levels caused by insulin or arginine (an insulin secretagogue in fish; [24]) administration were accompanied by an increase in GLUT4 mRNA levels in red but not white skeletal muscle in trout [60]. More recently, it was shown that GLUT4 expression in white skeletal muscle of trout fed a diet rich in carbohydrates was not affected [57]. Therefore, there is strong evidence suggesting that GLUT4 mRNA levels in red skeletal muscle may be regulated *in vivo* by circulating insulin in trout, as in mammals. However, these observations raised the question as to whether the expression of GLUT4 could be regulated in white skeletal muscle, given that it accounts for the bulk of glucose taken up by skeletal muscle. In contrast to trout, GLUT4 mRNA levels in the white muscle of Atlantic cod increased after fasting and decreased after refeeding [56], suggesting the possibility of species-specific differences in the regulation of GLUT4 in this tissue.

*In vitro* studies using a primary culture of trout skeletal muscle cells have assessed the effects of insulin and IGF-I on GLUT4 mRNA levels. The results obtained from these studies clearly showed that insulin and IGF-I increased the GLUT4 mRNA content in myoblasts and in myotubes [59] and support the notion that insulin can indeed regulate GLUT4 mRNA levels in trout skeletal muscle by acting directly on muscle cells. Since insulin is known to stimulate the uptake of glucose by trout skeletal muscle cells *in vitro* [32], it has been hypothesized that this effect of insulin may have been due, at least in part, to its effects on GLUT4 expression. Therefore, it appears that the hypoglycemic effects of insulin in fish, as in mammals, may involve the stimulation of GLUT4 mRNA expression in skeletal muscle.
