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

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

To date, studies on the regulation of GLUT4 protein levels in fish are limited to salmonid species. The availability of a polyclonal antibody against okGLUT4 made possible the locali‐ zation and the quantification of GLUT4 in trout skeletal muscle. By performing immunoloc‐ alization studies of GLUT4 in trout skeletal muscle cells in culture, an increase in the amount of total GLUT4 protein was observed during the differentiation of myoblasts into myotubes [32]. Subsequent studies showed that the total content of GLUT4 differs between the two types of skeletal muscle in trout, with red muscle containing a higher amount of GLUT4 than white muscle [70]. In agreement with changes in the expression of GLUT4 at the mRNA level, the total amount of GLUT4 protein in red muscle increased in trout stimulated with insulin *in vivo* and decreased after fasting. On the other hand, insulin administration failed to increase GLUT4 protein content in trout white muscle [32], supporting the lack of changes of GLUT4 mRNA levels previously described [60]. Interestingly, nutritional factors regulate GLUT4 mRNA and protein levels in white muscle in different manners. In trout, fasting decreased the amount of GLUT4 protein in white muscle [32], whereas no changes in mRNA levels were observed in the same condition [60], suggesting that post-transcriptional regulation of GLUT4 expression may take place in white skeletal muscle in fish. Therefore, it appears that insulin plasma levels may regulate the amount of GLUT4 present in red skeletal muscle in fish and strongly suggest that insulin may stimulate the de novo synthesis of GLUT4, at least in red skeletal muscle, by increasing the mRNA levels of GLUT4. The lack of effects after insulin administration *in vivo* on GLUT4 mRNA and protein levels in white muscle in trout are puzzling in the light of data showing that glucose uptake increases in white muscle after a glucose load in trout and that this tissue contributes about five times more than red muscle to the total glucose uptake when expressed as percent of the total body mass [23]. Further studies are required to understand the factors and mechanisms involved in the regulation of glucose

As part of the complex regulation of GLUT4, the translocation of this glucose transporter to the PM from intracellular vesicles is highly dynamic and is regulated by a number of factors [71], representing an efficient mechanism that allows a fast equilibration of glucose levels at either side of the PM in response to a hypoglycemic stimulus. In fish, insulin has been shown to increase the PM levels of GLUT4 in *in vitro* stimulated trout muscle cells in culture [32], demonstrating that insulin stimulates glucose uptake in fish skeletal muscle cells by increasing the levels of the GLUT4 protein at the PM, as in mammals. Other stimuli that have been shown to increase the uptake of glucose by trout myocytes and that also increase the cell surface levels of GLUT4 are AMPK activators (i.e. AICAR and metformin) [66] and the pro-inflammatory cytokine tumor necrosis factor α (TNFα) [72]. These results indicate that the regulation of the

glucose in skeletal muscle in fish.

50 Glucose Homeostasis

uptake in white skeletal muscle in fish.

**5.3. Regulation of GLUT4 protein levels in fish**

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‐ cellularly in the IRC compartment [76-78].

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

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, the 3T3-L1 adipocyte cell system was used successfully to demonstrate that both okGLUT4 and btGLUT4 were able to significantly translocate to the PM after insulin treatment [31,79], as it occurs in mammals. Moreover, insulin-stimulated translocation to the PM of btGLUT4 was demonstrated in L6 myoblasts and differentiated myotubes stably-expressing the fish GLUT4 transporter [32]. Therefore, the fish homologs of GLUT4 were shown to be insulin responsive like their mammalian counterpart, despite their higher PM localization at steadystate.

independent pathway. In agreement with the different intracellular distribution observed between btGLUT4 and rat GLUT4 [32], the traffic of btGLUT4 to the PM may be occurring via the constitutive pathway used by Glut1 or the transferrin receptor [37,73] (Figure 7). It is known that GLUT4, upon arriving in the IRC, acquires the capability to respond to insulin and to translocate to the PM. When a plasmid coding rat GLUT4 is transfected into 3T3-L1 adipocytes, the cells require 6 to 9 hours to produce the new protein and to target it to the IRC [73,85]. In contrast, both okGLUT4 and btGLUT4, when expressed in the same cellular system, undergo insulin-stimulated translocation only 3 hours after transfection [79], suggesting that fish GLUT4 undergoes faster synthesis, processing or traffic. Interestingly, okGLUT4 showed a temporal response that was intermediate between rat GLUT4 and btGLUT4, but closer to the

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latter, despite showing a similar sensitivity towards GGA as the mammalian GLUT4.

**Figure 7.** Schematic model illustrating differences in traffic between fish GLUT4s and rat GLUT4 and Glut1 during the basal state in adipocytes. In (A), the traffic of newly synthesized rat GLUT4 is shown to take place through the Golgi and the trans-Golgi network (TGN) and into the insulin-responsive compartment (IRC) in a sorting process that is de‐ pendent upon the adaptor protein GGA. In the basal state, rat GLUT4 is mainly sequestered into the IRC, a process that is regulated by AS160 and that requires the F5QQI8 amino terminal motif of GLUT4. In contrast, after biosynthesis, Glut1 directly travels from the TGN to the plasma membrane (PM) in a GGA-independent process. In (B), the traffic of newly synthesized trout (btGLUT4) or salmon (okGLUT4) GLUT4 is shown. We postulate that the high levels of btGLUT4 at the PM appear to be due to an increased exocytic rate, as a result of btGLUT4 following a GGA-independ‐ ent route from the TGN to the IRC and by showing less AS160-regulated sequestration at the IRC than rat GLUT4. We hypothesize that the different trafficking behavior of btGLUT4 may be related to the different sequence in its N-termi‐ nal motif (F5QHL8). The traffic behavior of okGLUT4 (F5QQL8) appears to be intermediate between that of rat GLUT4

As previously mentioned in section 3, among the different regulatory amino acid motifs found

important for GLUT4 sequestration into the IRC and insulin-stimulated translocation to the PM [85-88]. Interestingly, in addition to the fact that both fish GLUT4 transporters have a

targeting motif in the N-terminus has been shown to be

QQI8 motif shows one conserved amino acid substitution in

QQI8

and btGLUT4. ER: endoplasmic reticulum.

in GLUT4 in mammals, the F5

shorter N-terminal domain, the F5

As previously mentioned, GLUT4 in mammals is distributed inside the cells in two major storage compartments, the IRC and the endosomal system [75,80]. Interestingly, btGLUT4 showed only partial co-localization with rat GLUT4 when both were co-expressed either in 3T3-L1 adipocytes [79] or in L6 muscle cells [32]. This observation, together with the fact that btGLUT4 showed lower levels of retention in intracellular compartments during basal conditions although it still responded to insulin stimulation in both cell types [32,79], suggested that btGLUT4 is equally distributed between the specialized IRC and the endosomal com‐ partment, from where it cycles continuously with the PM. Moreover, both in 3T3-L1 adipocytes and L6 muscle cells, the higher PM levels observed for btGLUT4 were shown to be due to a faster externalization rate rather than to a decrease in the rate of endocytosis [32,79].

In mammals, several proteins have been described to interact with GLUT4 to regulate its intracellular traffic and to maintain the proteins sequestered in the IRC. For example, the Golgilocalized γ-ear-containing Arf-binding protein (GGA) has been described to function as a traffic controller of newly synthesized GLUT4 from the TGN to the IRC [73,81]. In this step, sortilin has been described also to have a key role, as GLUT4 does not contain the specific targeting motif to be recognized by GGA as a cargo molecule [82]. More recently, the insulinregulated aminopeptidase (IRAP), which co-localizes with GLUT4 in intracellular vesicles, has been shown to play a role in the sorting of GLUT4 from endosomes into the IRC [83]. Moreover, a protein named TUG (tether containing a UBX domain for GLUT4), has been reported to interact with the large intracellular GLUT4 loop present between TMVI and TMVII and to tether GLUT4 to intracellular vesicles through its interaction, via its UBX domain, with cellular membranes [84]. The possible roles of several GLUT4-interacting proteins in the regulation of the traffic of the fish GLUT4 isoforms have been explored in 3T3-L1 adipocytes expressing the corresponding mammalian orthologs. In particular, the adaptor protein GGA has been reported to be involved in the early sorting steps of GLUT4 from the TGN. The expression in 3T3-L1 adipocytes of rat GLUT4 together with wild-type GGA or a dominant-interfering form of GGA (GGA-DN) demonstrated that GGA is required for GLUT4 to reach the IRC because the insulin-stimulated translocation of rat GLUT4 to the PM was completely blunted in the presence of GGA-DN [40,73]. Interestingly, the intracellular traffic of btGLUT4 and okGLUT4 in 3T3-L1 adipocytes showed differences with regards their sensitivities to GGA because the traffic of btGLUT4 to the PM either under basal or insulin-stimulated conditions was only partially suppressed by co-expression with GGA-DN whereas okGLUT4 showed an identical response as that of rat GLUT4 [79]. These results suggested that okGLUT4 may traffic in adipocytes through the same pathway as mammalian GLUT4, but that btGLUT4 may be in part escaping the regulated biosynthetic traffic route, moving to the PM following a GGA- independent pathway. In agreement with the different intracellular distribution observed between btGLUT4 and rat GLUT4 [32], the traffic of btGLUT4 to the PM may be occurring via the constitutive pathway used by Glut1 or the transferrin receptor [37,73] (Figure 7). It is known that GLUT4, upon arriving in the IRC, acquires the capability to respond to insulin and to translocate to the PM. When a plasmid coding rat GLUT4 is transfected into 3T3-L1 adipocytes, the cells require 6 to 9 hours to produce the new protein and to target it to the IRC [73,85]. In contrast, both okGLUT4 and btGLUT4, when expressed in the same cellular system, undergo insulin-stimulated translocation only 3 hours after transfection [79], suggesting that fish GLUT4 undergoes faster synthesis, processing or traffic. Interestingly, okGLUT4 showed a temporal response that was intermediate between rat GLUT4 and btGLUT4, but closer to the latter, despite showing a similar sensitivity towards GGA as the mammalian GLUT4.

the 3T3-L1 adipocyte cell system was used successfully to demonstrate that both okGLUT4 and btGLUT4 were able to significantly translocate to the PM after insulin treatment [31,79], as it occurs in mammals. Moreover, insulin-stimulated translocation to the PM of btGLUT4 was demonstrated in L6 myoblasts and differentiated myotubes stably-expressing the fish GLUT4 transporter [32]. Therefore, the fish homologs of GLUT4 were shown to be insulin responsive like their mammalian counterpart, despite their higher PM localization at steady-

As previously mentioned, GLUT4 in mammals is distributed inside the cells in two major storage compartments, the IRC and the endosomal system [75,80]. Interestingly, btGLUT4 showed only partial co-localization with rat GLUT4 when both were co-expressed either in 3T3-L1 adipocytes [79] or in L6 muscle cells [32]. This observation, together with the fact that btGLUT4 showed lower levels of retention in intracellular compartments during basal conditions although it still responded to insulin stimulation in both cell types [32,79], suggested that btGLUT4 is equally distributed between the specialized IRC and the endosomal com‐ partment, from where it cycles continuously with the PM. Moreover, both in 3T3-L1 adipocytes and L6 muscle cells, the higher PM levels observed for btGLUT4 were shown to be due to a

faster externalization rate rather than to a decrease in the rate of endocytosis [32,79].

In mammals, several proteins have been described to interact with GLUT4 to regulate its intracellular traffic and to maintain the proteins sequestered in the IRC. For example, the Golgilocalized γ-ear-containing Arf-binding protein (GGA) has been described to function as a traffic controller of newly synthesized GLUT4 from the TGN to the IRC [73,81]. In this step, sortilin has been described also to have a key role, as GLUT4 does not contain the specific targeting motif to be recognized by GGA as a cargo molecule [82]. More recently, the insulinregulated aminopeptidase (IRAP), which co-localizes with GLUT4 in intracellular vesicles, has been shown to play a role in the sorting of GLUT4 from endosomes into the IRC [83]. Moreover, a protein named TUG (tether containing a UBX domain for GLUT4), has been reported to interact with the large intracellular GLUT4 loop present between TMVI and TMVII and to tether GLUT4 to intracellular vesicles through its interaction, via its UBX domain, with cellular membranes [84]. The possible roles of several GLUT4-interacting proteins in the regulation of the traffic of the fish GLUT4 isoforms have been explored in 3T3-L1 adipocytes expressing the corresponding mammalian orthologs. In particular, the adaptor protein GGA has been reported to be involved in the early sorting steps of GLUT4 from the TGN. The expression in 3T3-L1 adipocytes of rat GLUT4 together with wild-type GGA or a dominant-interfering form of GGA (GGA-DN) demonstrated that GGA is required for GLUT4 to reach the IRC because the insulin-stimulated translocation of rat GLUT4 to the PM was completely blunted in the presence of GGA-DN [40,73]. Interestingly, the intracellular traffic of btGLUT4 and okGLUT4 in 3T3-L1 adipocytes showed differences with regards their sensitivities to GGA because the traffic of btGLUT4 to the PM either under basal or insulin-stimulated conditions was only partially suppressed by co-expression with GGA-DN whereas okGLUT4 showed an identical response as that of rat GLUT4 [79]. These results suggested that okGLUT4 may traffic in adipocytes through the same pathway as mammalian GLUT4, but that btGLUT4 may be in part escaping the regulated biosynthetic traffic route, moving to the PM following a GGA-

state.

52 Glucose Homeostasis

**Figure 7.** Schematic model illustrating differences in traffic between fish GLUT4s and rat GLUT4 and Glut1 during the basal state in adipocytes. In (A), the traffic of newly synthesized rat GLUT4 is shown to take place through the Golgi and the trans-Golgi network (TGN) and into the insulin-responsive compartment (IRC) in a sorting process that is de‐ pendent upon the adaptor protein GGA. In the basal state, rat GLUT4 is mainly sequestered into the IRC, a process that is regulated by AS160 and that requires the F5QQI8 amino terminal motif of GLUT4. In contrast, after biosynthesis, Glut1 directly travels from the TGN to the plasma membrane (PM) in a GGA-independent process. In (B), the traffic of newly synthesized trout (btGLUT4) or salmon (okGLUT4) GLUT4 is shown. We postulate that the high levels of btGLUT4 at the PM appear to be due to an increased exocytic rate, as a result of btGLUT4 following a GGA-independ‐ ent route from the TGN to the IRC and by showing less AS160-regulated sequestration at the IRC than rat GLUT4. We hypothesize that the different trafficking behavior of btGLUT4 may be related to the different sequence in its N-termi‐ nal motif (F5QHL8). The traffic behavior of okGLUT4 (F5QQL8) appears to be intermediate between that of rat GLUT4 and btGLUT4. ER: endoplasmic reticulum.

As previously mentioned in section 3, among the different regulatory amino acid motifs found in GLUT4 in mammals, the F5 QQI8 targeting motif in the N-terminus has been shown to be important for GLUT4 sequestration into the IRC and insulin-stimulated translocation to the PM [85-88]. Interestingly, in addition to the fact that both fish GLUT4 transporters have a shorter N-terminal domain, the F5 QQI8 motif shows one conserved amino acid substitution in okGLUT4 (F5 QQL8 ), and it is less conserved in btGLUT4 (F5 QHL8 ), where the double residue substitution causes important size and charge changes (Figure 8A). The possibility that these sequence differences were able to account for the increased basal cell surface levels observed for btGLUT4 was investigated (Capilla and Planas, unpublished data). Figure 8B shows that mutation of the btGLUT4 motif F5 QHL8 to F5 QQI8 caused a slight decrease in basal PM levels; however, mutation of the F5 QHL8 motif to F5 QQL8 significantly reduced the cell surface levels of btGLUT4 to levels comparable to those of okGLUT4 or the mammalian GLUT4. These results indicate that specific amino acid motifs as well as the folding of the molecule appear to be important for the intracellular domains of the GLUT4 molecule to interact with the different regulatory proteins for proper traffic and specific compartment localization and/or retention.

An earlier study in mammals using chimeras between GLUT4 and Glut1 demonstrated that substituting the N-terminus and the intracellular loop of Glut1 for those of GLUT4 is sufficient to confer to the chimeric Glut1 protein the characteristics of GLUT4 in 3T3-L1 adipocytes [89]. Thus, in order to identify the protein domains in trout GLUT4 that confer its particular traffic characteristics (i.e. lower intracellular retention; higher PM levels under basal conditions), chimeric proteins were created that have the N-terminus (btN) or the intracellular loop (btL) of btGLUT4 in a rat GLUT4 backbone and were named btN-GLUT4 or btL-GLUT4, respec‐ tively. These constructs were then stably expressed in 3T3-L1 cells and their capacity to be retained in the cytosol under basal conditions and to respond to insulin were analyzed (Simoes, Planas and Camps, unpublished results). The results obtained indicated that all constructs were able to translocate to the PM in response to insulin but with certain differences among them (Figure 9). First, the insulin-stimulated translocation of btGLUT4 was lower than that of rat GLUT4. Second, btN-GLUT4 had the weakest response to insulin, suggesting a role for the N-terminus in the correct targeting of GLUT4 to the IRC or in the translocation of GLUT4 to the PM. Third, the substitution of the cytoplasmic loop in btL-GLUT4 caused a reduction in the response of rat GLUT4 to insulin comparable to that of btGLUT4. These preliminary results support the idea that the N-terminus and the cytoplasmic loop of GLUT4 are responsible for

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**Figure 9.** Cell surface levels of various GLUT4 constructs in the presence of insulin. N-GLUT4 and L-GLUT4 represent constructs with the amino terminus (N) or the intracellular loop (L) of btGLUT4 in a rat GLUT4 backbone, respectively. Differentiated 3T3-L1 adipocytes expressing the various GLUT4 constructs were incubated in the absence or presence of insulin (100nM) for 30 min and the determination of surface GLUT4 levels was performed as described in [32]. Cell surface GLUT4 is expressed relative to the unstimulated control for each cell line. Different letters indicate statistically

Following insulin stimulation, mammalian GLUT4 traffics and fuses with the PM, increasing its presence in the cell surface up to 10-fold; thus, supporting the increase in glucose uptake observed after feeding. Insulin increases the number of transporters at the PM not only by enhancing exocytosis but also by decreasing the rate of endocytosis [90-92]. Insulin exerts its

some of the trafficking differences between btGLUT4 and rat GLUT4.

significant differences (p < 0.05).

**Figure 8.** The elevated basal cell surface levels of btGLUT4 are reduced by mutating the F5QHL8 motif. (A) Amino acid sequence alignment of the N-terminal region of rat, brown trout (btGLUT4, AF247395) and coho salmon (okGLUT4, AF502957) GLUT4 molecules. The box encloses the important trafficking motif F5QQI8 partially conserved in the fish species. (B) Differentiated 3T3-L1 adipocytes expressing either btGLUT4 or any of the two point mutants (btGLUT4- FQQI or btGLUT4-FQQL) were incubated with or without insulin (100 nM, 30 min). Data is presented as percentage (mean ± SEM) of cells showing a complete plasma membrane (PM) rim obtained by counting 100 cells per condition in 3 independent experiments. Statistical analysis was performed by unpaired t-test against the wild type btGLUT4 at basal or insulin-stimulated conditions respectively (\* denotes significance at p < 0.05).

An earlier study in mammals using chimeras between GLUT4 and Glut1 demonstrated that substituting the N-terminus and the intracellular loop of Glut1 for those of GLUT4 is sufficient to confer to the chimeric Glut1 protein the characteristics of GLUT4 in 3T3-L1 adipocytes [89]. Thus, in order to identify the protein domains in trout GLUT4 that confer its particular traffic characteristics (i.e. lower intracellular retention; higher PM levels under basal conditions), chimeric proteins were created that have the N-terminus (btN) or the intracellular loop (btL) of btGLUT4 in a rat GLUT4 backbone and were named btN-GLUT4 or btL-GLUT4, respec‐ tively. These constructs were then stably expressed in 3T3-L1 cells and their capacity to be retained in the cytosol under basal conditions and to respond to insulin were analyzed (Simoes, Planas and Camps, unpublished results). The results obtained indicated that all constructs were able to translocate to the PM in response to insulin but with certain differences among them (Figure 9). First, the insulin-stimulated translocation of btGLUT4 was lower than that of rat GLUT4. Second, btN-GLUT4 had the weakest response to insulin, suggesting a role for the N-terminus in the correct targeting of GLUT4 to the IRC or in the translocation of GLUT4 to the PM. Third, the substitution of the cytoplasmic loop in btL-GLUT4 caused a reduction in the response of rat GLUT4 to insulin comparable to that of btGLUT4. These preliminary results support the idea that the N-terminus and the cytoplasmic loop of GLUT4 are responsible for some of the trafficking differences between btGLUT4 and rat GLUT4.

okGLUT4 (F5

54 Glucose Homeostasis

QQL8

mutation of the btGLUT4 motif F5

however, mutation of the F5

), and it is less conserved in btGLUT4 (F5

to F5

QHL8

QHL8 motif to F5

substitution causes important size and charge changes (Figure 8A). The possibility that these sequence differences were able to account for the increased basal cell surface levels observed for btGLUT4 was investigated (Capilla and Planas, unpublished data). Figure 8B shows that

of btGLUT4 to levels comparable to those of okGLUT4 or the mammalian GLUT4. These results indicate that specific amino acid motifs as well as the folding of the molecule appear to be important for the intracellular domains of the GLUT4 molecule to interact with the different regulatory proteins for proper traffic and specific compartment localization and/or retention.

**Figure 8.** The elevated basal cell surface levels of btGLUT4 are reduced by mutating the F5QHL8 motif. (A) Amino acid sequence alignment of the N-terminal region of rat, brown trout (btGLUT4, AF247395) and coho salmon (okGLUT4, AF502957) GLUT4 molecules. The box encloses the important trafficking motif F5QQI8 partially conserved in the fish species. (B) Differentiated 3T3-L1 adipocytes expressing either btGLUT4 or any of the two point mutants (btGLUT4- FQQI or btGLUT4-FQQL) were incubated with or without insulin (100 nM, 30 min). Data is presented as percentage (mean ± SEM) of cells showing a complete plasma membrane (PM) rim obtained by counting 100 cells per condition in 3 independent experiments. Statistical analysis was performed by unpaired t-test against the wild type btGLUT4 at

basal or insulin-stimulated conditions respectively (\* denotes significance at p < 0.05).

QHL8

QQI8 caused a slight decrease in basal PM levels;

QQL8 significantly reduced the cell surface levels

), where the double residue

**Figure 9.** Cell surface levels of various GLUT4 constructs in the presence of insulin. N-GLUT4 and L-GLUT4 represent constructs with the amino terminus (N) or the intracellular loop (L) of btGLUT4 in a rat GLUT4 backbone, respectively. Differentiated 3T3-L1 adipocytes expressing the various GLUT4 constructs were incubated in the absence or presence of insulin (100nM) for 30 min and the determination of surface GLUT4 levels was performed as described in [32]. Cell surface GLUT4 is expressed relative to the unstimulated control for each cell line. Different letters indicate statistically significant differences (p < 0.05).

Following insulin stimulation, mammalian GLUT4 traffics and fuses with the PM, increasing its presence in the cell surface up to 10-fold; thus, supporting the increase in glucose uptake observed after feeding. Insulin increases the number of transporters at the PM not only by enhancing exocytosis but also by decreasing the rate of endocytosis [90-92]. Insulin exerts its 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 the lack of a conserved FQQI motif [32,79].

**7. Conclusions and perspectives**

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

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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

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