**3. Structural characteristics of the GLUT4 protein in fish**

In fish, the deduced amino acid sequence of GLUT4 is known in approximately 10 different species as a result of either conventional cloning techniques or available sequences in data‐ bases. Similar to mammalian GLUT4, a protein of 509 amino acids in length, known fish GLUT4 proteins range between 503 and 511 amino acids in length. Also like mammalian GLUT4, fish GLUT4 proteins have a predicted molecular mass of approximately 55 kDa and an isoelectric point of 6.7. Western blotting using polyclonal antibodies against coho salmon GLUT4 (okGLUT4) confirmed that the molecular weight of native GLUT4 in adipose tissue and skeletal muscle cells from salmonid species was approximately 50 kDa [31,32]. Comparison of human GLUT4 and fish GLUT4 proteins evidences a relatively high degree of conservation at the amino acid level, with fish GLUT4 proteins showing a 79% sequence homology to human GLUT4. However, fish GLUT4 proteins show more than 90% homology amongst themselves at the amino acid level, even when considering species that are phylogenetically distant such as coho salmon and tilapia. Phylogenetic analyses of fish GLUT4 proteins in relation to human GLUT4 reveal that all fish GLUT4 proteins are evolutionarily related to human GLUT4 and that they cluster according to their phylogenetic position within the fish evolutionary tree (Figure 5).

which is an important cassette placed in an enhancer region of the promoter of the GLUT4 gene in mammals [29], and the core promoter, essential for the basal expression of the GLUT4 gene. These observations highlight the high degree of conservation of the GLUT4 gene and its

**Figure 4.** Conservation profile of the promoter region of known fish GLUT4 genes. Sequence elements of significant length (≥ 100 nucleotides) that show higher than 60% of homology are highlighted in red and depicted with the dark-red rectangles on the top of each graph. The sequence comparison between the Fugu and Tetraodon (A), Fugu and Stickleback (B) and Stickleback and Medaka (C) GLUT4 promoters is shown. The horizontal axis represents the po‐ sition of the nucleotides within the 1314 bp sequence compared, starting at the 5' end. The vertical axis represents the percent of identity between the aligned genomes. In the bottom, a schematic representation of the-1132 Fugu GLUT4 gene promoter highlighting the most relevant predicted binding sites is shown. The open boxes delineating the regions comprised between-786/-334 and-234/+182 nucleotides represent conserved areas in fish GLUT4 gene

In fish, the deduced amino acid sequence of GLUT4 is known in approximately 10 different species as a result of either conventional cloning techniques or available sequences in data‐ bases. Similar to mammalian GLUT4, a protein of 509 amino acids in length, known fish GLUT4 proteins range between 503 and 511 amino acids in length. Also like mammalian GLUT4, fish GLUT4 proteins have a predicted molecular mass of approximately 55 kDa and an isoelectric

**3. Structural characteristics of the GLUT4 protein in fish**

regulatory region during evolution from fish to mammals.

42 Glucose Homeostasis

promoters. Adapted from [30].

**Figure 5.** Unrooted phylogenetic tree of GLUT4 amino acid sequences. The tree was created by the UPGMA method using ClustalW multiple alignment and bootstrapped 5000 times. The scale for the given branch length indicates 0.09 amino acid substitutions per site. 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.

Importantly, the structural characteristics of all the known fish GLUT4 proteins correspond to those of the facilitated glucose transporter family and, specifically, to those of mammalian GLUT4. The structural conservation of GLUT4 from fish to mammals can clearly be observed when an alignment of the deduced amino acid sequence of fish GLUT4 proteins with human GLUT4 is performed (Figure 6). Like human GLUT4, all fish GLUT4 proteins contain the typical 12 (I-XII) hydrophobic transmembrane domains (TMs) of 21 amino acids that have also been revealed by hydropathy plots [26,31]. Furthermore, all fish GLUT4 proteins contain four major hydrophylic regions corresponding to the amino (N) terminus, the carboxy (C) terminus and the two main extracellular and intracellular domains. The main extracellular domain corresponds to a loop of approximately 30 amino acids located between TMI and TMII and contains a predicted glycosylation site (K50) that is present in all mammalian and avian GLUT proteins. The large intracellular domain corresponds to a cytoplasmic loop of 65 amino acids located between TMVI and TMVII. Other structural characteristics of functional GLUT proteins that are found in fish GLUT4 include the presence of (1) the QLS motif in TMVII, known to be important for the high-affinity recognition of the transported substrate, (2) the 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).

In addition to having sequence features typical of GLUTs, fish GLUT4 proteins also share certain sequence motifs that are characteristic of GLUT4 and that confer its particular intra‐ cellular traffic behavior. Specifically, the N-terminus of human GLUT4 contains the F5

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

motif that has been shown to be important for its internalization from the cell surface [36]. Furthermore, the C-terminus of human GLUT4 contains the acidic cluster T498ELEY502 that has been shown to be important for the intracellular retention of GLUT4 under basal conditions [37] (see below). Mutations of either these two motifs in GLUT4 lead to alterations in the intracellular traffic of GLUT4 [37]. Importantly, fish GLUT4 proteins contain these two essential motifs, although with some differences in their sequence. For example, fish GLUT4 proteins show similarities to human GLUT4 in terms of the sequence of these two motifs. The

fish species. In addition, the T498ELEY502 motif is present in fish GLUT4 proteins, with medaka GLUT4 having an identical motif but with small substitutions in other fish GLUT4 proteins, such as D501 for E501 in six species (i.e. salmon, trout, cod, platyfish, tilapia and stickleback) and such as M500 for L500 in only two species (tetraodon and tilapia). The C-terminal residue Y502, thought to be important for regulating the release of GLUT4 from its intracellular storage compartments [38], was present in five of the fish species examined (tilapia, medaka, platyfish, trout and salmon). However, despite these similarities between human and fish GLUT4 proteins, the motif L489L490 that is thought to be important for the regulation of GLUT4 translocation from cytosolic compartments to the PM and for its endocytosis [39-41], is not present in fish GLUT4 proteins. Therefore, although fish GLUT4 proteins are clearly homolo‐ gous to mammalian GLUT4 and contain protein motifs important for the function, regulation and traffic of GLUT4, they show differences in the sequence of these important motifs that may account for slightly different properties. One interesting possibility is that the abovementioned differences in the primary structure between fish and mammalian GLUT4 could cause differences in the structure/conformation of the transporter, which could affect its ability to transport glucose and/or to bind factors that interact with GLUT4 and, consequently, alter its intracellular traffic. For this reason, studies have been performed with fish GLUT4 as a natural mutant of mammalian GLUT4 to contribute to our understanding of the role of the

specific domains of GLUT4 that are responsible for its traffic behavior (see Section 6).

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,

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

may have functional consequences for the traffic of GLUT4 among the different

QQL8 in all fish species examined, except for brown

motif (Figure 6). As will be discussed below, substitution

F5

of H7

for Q7

QQI8 motif is present in the form of F5

QHL8

trout (btGLUT4) that has a F5

QQI8

45

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


**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.

In addition to having sequence features typical of GLUTs, fish GLUT4 proteins also share certain sequence motifs that are characteristic of GLUT4 and that confer its particular intra‐ cellular traffic behavior. Specifically, the N-terminus of human GLUT4 contains the F5 QQI8 motif that has been shown to be important for its internalization from the cell surface [36]. Furthermore, the C-terminus of human GLUT4 contains the acidic cluster T498ELEY502 that has been shown to be important for the intracellular retention of GLUT4 under basal conditions [37] (see below). Mutations of either these two motifs in GLUT4 lead to alterations in the intracellular traffic of GLUT4 [37]. Importantly, fish GLUT4 proteins contain these two essential motifs, although with some differences in their sequence. For example, fish GLUT4 proteins show similarities to human GLUT4 in terms of the sequence of these two motifs. The F5 QQI8 motif is present in the form of F5 QQL8 in all fish species examined, except for brown trout (btGLUT4) that has a F5 QHL8 motif (Figure 6). As will be discussed below, substitution of H7 for Q7 may have functional consequences for the traffic of GLUT4 among the different fish species. In addition, the T498ELEY502 motif is present in fish GLUT4 proteins, with medaka GLUT4 having an identical motif but with small substitutions in other fish GLUT4 proteins, such as D501 for E501 in six species (i.e. salmon, trout, cod, platyfish, tilapia and stickleback) and such as M500 for L500 in only two species (tetraodon and tilapia). The C-terminal residue Y502, thought to be important for regulating the release of GLUT4 from its intracellular storage compartments [38], was present in five of the fish species examined (tilapia, medaka, platyfish, trout and salmon). However, despite these similarities between human and fish GLUT4 proteins, the motif L489L490 that is thought to be important for the regulation of GLUT4 translocation from cytosolic compartments to the PM and for its endocytosis [39-41], is not present in fish GLUT4 proteins. Therefore, although fish GLUT4 proteins are clearly homolo‐ gous to mammalian GLUT4 and contain protein motifs important for the function, regulation and traffic of GLUT4, they show differences in the sequence of these important motifs that may account for slightly different properties. One interesting possibility is that the abovementioned differences in the primary structure between fish and mammalian GLUT4 could cause differences in the structure/conformation of the transporter, which could affect its ability to transport glucose and/or to bind factors that interact with GLUT4 and, consequently, alter its intracellular traffic. For this reason, studies have been performed with fish GLUT4 as a natural mutant of mammalian GLUT4 to contribute to our understanding of the role of the specific domains of GLUT4 that are responsible for its traffic behavior (see Section 6).
