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

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

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

*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.

differentiation also in fish.

48 Glucose Homeostasis

tissue.

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

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 through the induction of AMPK activity.

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

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 muscle activity in trout increased the mRNA levels of GLUT4 in trout skeletal muscle [65], these results suggest that induction of contractile activity in skeletal muscle cells results in the transcriptional activation of the GLUT4 gene, resulting in increased GLUT4 mRNA levels that, in turn, may increase the amount of GLUT4 and, consequently, the entry and utilization of glucose in skeletal muscle in fish.

total amount of GLUT4 protein in skeletal muscle and, more importantly, the cell surface levels of GLUT4 in skeletal muscle cells are similar between fish and mammals, evidencing a remarkable degree of conservation of the mechanism(s) by which insulin exerts its hypogly‐

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

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

51

In mammals, the main feature that characterizes GLUT4 in skeletal muscle and adipose tissue and makes it unique is its ability to translocate to the PM in response to insulin [15,73]. This greatly increases the capacity of the cells to uptake glucose during the postprandial state, which is crucial to properly maintain glucose homeostasis. Notwithstanding, evidence in mammalian cells clearly indicates that in the basal state GLUT4 is not static; instead, GLUT4 circulates among numerous intracellular compartments, such as the trans-Golgi network (TGN), early and late endosomes, a specialized insulin responsive compartment (IRC), as well as the PM [71,74-75]. The amount of GLUT4 present at the PM in the basal state corresponds to about only 5-10% of the total GLUT4 protein, whereas the remaining 90-95% is sequestered intra‐

The intracellular trafficking characteristics of the two glucose transporters identified in salmonids (btGLUT4 and okGLUT4) have been studied in comparison with mammalian GLUT4 mainly when expressed in heterologous systems (mammalian adipocytic or myoblastic cell lines), but also as the endogenous GLUT4 in primary cultured trout myocytes. In 3T3-L1 adipocytes transiently expressing separately btGLUT4 or okGLUT4 under steady-state conditions, btGLUT4 exhibited significantly higher protein levels at the PM (30-40%), also okGLUT4 but to a lesser extent (15-20%), than rat GLUT4 (10-15%) [31,79]. This was not only observed in adipocytes, since btGLUT4 was present also at the PM at higher levels (20-25%) than rat GLUT4 (10-15%) when stably-expressed in L6 muscle cells [32]. Importantly, the basal localization of endogenous GLUT4 at the PM in trout myocytes in culture was also relatively high [32]. Therefore, under basal or unstimulated conditions fish GLUT4 appears to be less efficiently retained in the cytosol in adipocytes and myocytes than mammalian GLUT4, suggesting differences in the mechanisms responsible for the intracellular retention of GLUT4 between fish and mammals (see below). Furthermore, based on the observed differences in PM localization between fish GLUT4s under basal conditions, with okGLUT4 being more similar to its mammalian counterparts than btGLUT4, it has been suggested that the different traffic behavior of these two fish GLUT4 protein variants may be related to differences in characteristic regulatory features in the GLUT4 protein sequence (i.e. N-and C-terminal protein

Moreover, the ability of fish GLUT4s to respond to insulin has been also evaluated. The first studies trying to demonstrate that a fish GLUT4 translocates to the PM upon insulin stimula‐ tion were performed in *Xenopus* oocytes [31]. Nevertheless, the system was not appropriate to study the translocation of GLUT4 and oocytes expressing okGLUT4 or a rat GLUT4 did not show differences in transporter localization within the cell in response to insulin [31]. Instead,

cemic effects on skeletal muscle.

**6. Regulation of the traffic of fish GLUT4**

cellularly in the IRC compartment [76-78].

motifs) (see section 3; [79]).
