**4. Sugar transport properties of fish GLUT4**

STS sequence in the extracellular segment between TMVII and TMVIII, known to be important for the conformational change of GLUT4 during the transport of glucose and (3) several proline (P) residues in TMVI and TMX that are known to be important for transport activity [33-35]

**Figure 6.** Multiple alignment of GLUT4 protein sequences. Identical residues identified using ClustalW are indicated as dots. Grey boxes: Transmembrane domains. Red boxes: Important motives for GLUT4 trafficking. Dashed box: Impor‐ tant motive for glucose transport. Gene IDs and accession numbers were retrieved from public databases-Human: ENSG00000181856; Fugu: ENSTRUG00000011935; Tetraodon: ENSTNIG00000010138; Tilapia: ENSO‐ NIG00000018958; Stickleback: ENSGACG00000019384; Medaka: ENSORLG00000006341; Platyfish: ENSX‐

MAG00000015723; Atlantic cod: AAZ15731.1; Brown trout: AAG12191.1; Coho Salmon: AAM22227.1.

(Figure 6).

44 Glucose Homeostasis

The functionality of GLUT4 in fish has been investigated for okGLUT4 using the *Xenopus laevis* oocyte system [31]. The *Xenopus* oocyte system was extensively used in the 90s to functionally characterize the different GLUT isoforms in mammals [7,8,42,43], since it presents a series of advantages with respect to other *in vitro* systems: 1) the oocyte contains all the machinery necessary to properly express heterologous proteins; 2) it has very low endogenous levels of glucose transport, which avoids interferences in the measurements; and 3) it allows to analyze separately GLUTs that *in vivo* may be present together in the same tissue. Therefore, using this system, okGLUT4 was demonstrated to be a functional glucose transporter, with characteristics similar to those of its mammalian counterparts [31].

transporter [45]. Furthermore, in L6 muscle cells stably expressing btGLUT4, a dose-dependent inhibition in response to cytochalasin B was also reported [32]. In this same study, the effect of indinavir, a known GLUT4 mammalian inhibitor [53-55] was analyzed, causing also a concentration-dependent decrease on 2-DG uptake. Overall, these results confirm that okGLUT4 presents similar biochemical properties as mammalian GLUT4. In summary, all these studies have demonstrated that fish GLUT4 is a functional GLUT that is a structural and functional homolog of mammalian GLUT4 but with the important difference that has lower

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

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

47

As all membrane solute carriers, GLUT4 exerts its glucose transport properties when is present in the PM, allowing the flux of glucose across a concentration gradient. Therefore, the GLUT4 mediated transport of glucose can be determined, at least in great part, by the number of transporter molecules present at the PM at any given time. In mammals, the abundance of GLUT4 at the PM of skeletal muscle or adipose cells is, in turn, dependent on the traffic mechanisms that translocate pre-existing, vesicle-bound GLUT4 to the PM but, ultimately, on the synthesis of GLUT4 proteins (reviewed in [29]). The following section reviews available data on the regulation of fish GLUT4 at the mRNA, protein and promoter activity levels.

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

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

and adipose tissue of trout has been confirmed by immunoblotting [31,32].

affinity for glucose and wider substrate specificity than mammalian GLUT4.

**5. Regulation of the expression of GLUT4 in fish**

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

Regarding the kinetics of fish GLUT4, studies were performed using zero-trans and equilibri‐ um exchange conditions with 2-deoxy-glucose (2-DG) and the non-metabolizable glucose analogue, 3-O-methylglucose (3-OMG), respectively [31]. From the zero-trans kinetic analyses, the transport of glucose by the oocyte was demonstrated to be saturable, with a Km value on average of 7.6 mM. Using the equilibrium exchange assay and, assuming the transport follows first-order kinetics, the average Km value calculated for 3-OMG uptake by okGLUT4-express‐ ing oocytes was of 14.4 mM. These Km values were higher than those reported using the same oocyte system for mammalian GLUT4s: 4.6 and 1.8-4.3 mM for zero-trans and equilibrium exchange kinetics, respectively [7,8,44], suggesting that the fish transporter has less affinity for glucose. Nevertheless, when compared with kinetics of the Glut1 isoform identified in trout (OnmyGlut1), also studied in *Xenopus* oocytes, we observed that the zero-trans Km value of okGLUT4 was lower than that of OnmyGlut1 (8.3-14.9 mM) [45]. Moreover, the Km value reported in American eel erythrocytes, that primarily express Glut1, was also higher (10.4 mM) than that of okGLUT4 [46]; thus, indicating that the relative affinity for glucose of fish GLUTs parallels that of mammals, since the ubiquitous Glut1 has also lower affinity for glucose (21.3-26.2 mM) than GLUT4 [7,44]. On the other hand, the lower affinity for glucose of the fish GLUTs in comparison to mammals, may explain the reduced tolerance presented by the former to dietary carbohydrates [21,22].

To further characterize the fish GLUT4 transporter, stereoselectivity and substrate specificity were also analyzed in *Xenopus* oocytes expressing okGLUT4, using different sugars as transport competitors of 2-DG [31]. okGLUT4 was demonstrated to be stereospecific as its mammalian counterparts [8,31], since all the D-monosaccharides tested (glucose, mannose and galactose), but not the corresponding L-isomers, reduced the uptake of 2-DG into the oocyte. Interestingly, okGLUT4 transported all the various D-sugars, although with different affinities, as shown by the degree of competition towards 2-DG uptake observed for each monosacchar‐ ide. In this sense, D-glucose and D-mannose were primarily transported by okGLUT4 and, to a lesser extent, D-galactose and also D-fructose, albeit with even lower affinity. Overall, these data indicate that fish GLUT4 shows lower substrate specificity than mammalian GLUT4, suggesting that they may have a broader range of functions in fish controlling glucose metabolism. In this sense, it is interesting to note that okGLUT4 is able to uptake fructose, a role that is attributed to Glut2 in mammals [8,31]. Therefore, together with the fact that fish GLUT4 has a wider tissue distribution, the transport characteristics of fish GLUT4 support the idea that GLUT4 may play a role in the postprandial absorption of dietary glucose.

Moreover, okGLUT4 transport activity was shown to be suppressed by different transport inhibitors; thus, corroborating that this fish transporter belongs to the family of facilitative GLUTs [31]. 2-DG uptake in *Xenopus* oocytes expressing okGLUT4 was dose-dependently decreased by the well-known intracellular inhibitor cytochalasin B [47-49] as well as by the extracellular inhibitor, ethylidene glucose [34]. Cytochalasin B was previously shown to inhibit glucose transport in fish erythrocytes, suggesting the existence of GLUT transporters in fish before their discovery [46,50-52], and later also in *Xenopus* oocytes expressing the OnmyGlut1 transporter [45]. Furthermore, in L6 muscle cells stably expressing btGLUT4, a dose-dependent inhibition in response to cytochalasin B was also reported [32]. In this same study, the effect of indinavir, a known GLUT4 mammalian inhibitor [53-55] was analyzed, causing also a concentration-dependent decrease on 2-DG uptake. Overall, these results confirm that okGLUT4 presents similar biochemical properties as mammalian GLUT4. In summary, all these studies have demonstrated that fish GLUT4 is a functional GLUT that is a structural and functional homolog of mammalian GLUT4 but with the important difference that has lower affinity for glucose and wider substrate specificity than mammalian GLUT4.
