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

using this system, okGLUT4 was demonstrated to be a functional glucose transporter, with

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 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

characteristics similar to those of its mammalian counterparts [31].

to dietary carbohydrates [21,22].

46 Glucose Homeostasis

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.
