**7. Conclusions and perspectives**

effects through two different intracellular signaling pathways [15,93]. The first is the wellknown pathway of phosphatidylinositol-3-kinase (PI3K)/protein kinase B (PKB), also known as Akt, that is necessary for GLUT4 translocation to occur [94,95] but not sufficient [96]. The second pathway is that including the Cbl associated protein (CAP), which binds the insulin receptor and activates a small GTPase from the Rho family named TC10, and that was described in adipocytes [97]. However, the TC10 pathway appears not to be involved in muscle cells, in which another Akt-independent input was shown to contribute to the cytoskeleton remodeling required for complete GLUT4 translocation [98,99]. Downstream of Akt, a protein named Akt substrate of 160 KDa (AS160) or TCB1D4, has been found to be the key to com‐ municate the phosphorylation cascade initiated by insulin with the vesicle trafficking machi‐ nery [100,101]. AS160 has 6 residues of threonine/serine that can be phosphorylated to inhibit its activity, and contains a GTPase-activating protein (GAP) domain that in the basal state inactivates a series of Rab proteins, small GTPases from the Ras superfamily responsible of membrane trafficking [100]. Phosphorylation of AS160 by Akt inhibits its GAP activity, allowing the activation of the Rab proteins; thus, causing the translocation of GLUT4 to the PM [101,102]. The Rab members identified as responsible for GLUT4 trafficking in mammals are Rab8a, Rab10 and Rab14 [103-106]. In addition to regulating the movement of GLUT4 vesicles, AS160 has been demonstrated to be required for fully retaining GLUT4 into the IRC [107]. To demonstrate that AS160 phosphorylation is critical for GLUT4 translocation in mammals, a dominant-inhibitory form of AS160 was created by mutating 4 of its 6 phosphor‐ ylation sites (AS160-4P) [101]. When co-expressed in a cellular system together with rat GLUT4, the translocation of this molecule to the PM was blocked, as well as the increase in glucose uptake observed after insulin incubation [40,108]. Regarding their sensitivity towards AS160-4P, clear differences were observed between okGLUT4 and btGLUT4, with the former showing similar properties as those of mammalian GLUT4, and the latter being unaffected [79]. These results were in agreement with the differences observed between the two fish GLUT4 proteins in terms of their intracellular retention and support the hypothesis that AS160 may sequester okGLUT4, but not btGLUT4, in the IRC and that btGLUT4 may be more widely distributed inside the cell than the other GLUT4 transporters. Moreover, since the results obtained for btGLUT4 towards AS160 sensitivity agreed with those reported previously for a GLUT4-F5A mutant [40,73], the faster exocytic rate of btGLUT4 was suggested to be due to

In summary, by investigating the trafficking characteristics of the two fish GLUT4 proteins (btGLUT4 and okGLUT4) compared with mammalian GLUT4 and Glut1, it is clear that important differences exist between these transporters (Figure 7). In this regard, okGLUT4 behaves in many aspects similarly to mammalian GLUT4 due to its sensitivity towards GGA and AS160; thus, supporting a role for these molecules in the regulation of the traffic of okGLUT4 synthesized *de novo* from the TGN into the IRC, and from the TGN into the PM in response to insulin, respectively. In contrast, btGLUT4 appears to be less regulated, trafficking to the IRC independently of GGA, as well as being retained in the IRC and exiting to the PM only in part under the control of AS160; therefore, moving towards the cell surface possibly,

the lack of a conserved FQQI motif [32,79].

56 Glucose Homeostasis

in part, through a constitutive pathway as that used by Glut1.

All the evidence accumulated to date on the function and regulation of GLUT4 in fish indicates that the various molecular and cellular mechanisms regulating the amount of GLUT4 that is present at the cell surface in skeletal muscle and adipose tissue cells and that determine the amount of glucose uptake have been relatively well conserved during evolution from fish to mammals. Importantly, GLUT4 in fish is regulated by insulin at the level of mRNA and total protein amount as well as at the level of its abundance at the PM. In fish, like in mammals, GLUT4 responds to the effects of insulin by facilitating the uptake of glucose in insulinsensitive tissues such as skeletal muscle. Therefore, GLUT4 plays an important role in mediating the hypoglycemic effects of insulin from fish to mammals and underscores the importance of the maintenance of glucose homeostasis, and the role of GLUT4 in this process, throughout vertebrates. However, the presence of a seemingly well conserved insulinregulated mechanism of glucose transport involving GLUT4 contrasts with the relative glucose intolerance of teleost fish, that is evidenced by the lower ability of fish to clear a glucose load, when compared to mammals. It was initially hypothesized that the persistent hyperglycemia in fish may have been due to the possible lack of an insulin-regulated GLUT [25], given that fish have functional insulin receptors [109] and insulin is involved in the postprandial regulation of blood glucose levels [24]. The demonstration of the participation of an insulinregulatable glucose transport system involving GLUT4 in skeletal muscle and adipose tissue of fish [26,31-32,59-60,70,79] rules out that fish may experience peripheral resistance to insulin. What today appears as a likely contribution to explain the poor regulation of glucose plasma levels in fish, when compared to mammals, are the particular transport characteristics and intracellular trafficking behavior of fish GLUT4. Functionally, fish GLUT4 differs from mammalian GLUT4 in that it has a lower affinity for glucose and a wider substrate specificity. In addition, the intracellular traffic of fish GLUT4 is somewhat different than that of mamma‐ lian GLUT4. Although insulin stimulates the translocation of fish GLUT4 to the PM, the intracellular route(s) used by fish GLUT4 to reach the PM are not as dependent on proteins required for the intracellular sorting and retention of mammalian GLUT4, which leads to the proportionally higher levels of fish GLUT4 at the PM under basal conditions. Differences in the intracellular traffic behavior of fish GLUT4, when compared to mammalian GLUT4, are likely due to differences in key protein motifs in GLUT4. Therefore, we propose that during evolution from fish to mammals, the control of glucose homeostasis has improved possibly due to the increase in the affinity of GLUT4 for glucose and to the improvement of the intracellular sorting and retention mechanisms of GLUT4 in insulin-sensitive cells.

Interestingly, GLUT4 from different fish species that contain slightly different amino acid sequences in key trafficking motifs can be considered natural mutants of mammalian GLUT4 and used to identify and further characterize amino acid motifs or protein domains in mammalian GLUT4 that are important for the regulation of the traffic of mammalian GLUT4. Our studies on the traffic behavior of chimeric GLUT4 proteins incorporating fish GLUT4 protein motifs into a mammalian GLUT4 backbone represent a first step in that direction. Given that the traffic of GLUT4 is dependent on the binding of GLUT4 to sorting and trafficking proteins that are not fully characterized in mammals, studies comparing the traffic of fish and mammalian GLUT4 could potentially identify important binding partners of mammalian GLUT4 that do not interact or interact poorly with fish GLUT4 motifs. Consequently, the comparative study of GLUT4 from evolutionarily distant species could contribute to our understanding of the biology of GLUT4 in health and disease.

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