**2.1. Structural characteristics of the GLUT4 gene in fish**

mammals, particularly with respect to their tolerance of hyperglycemia (reviewed in [20]). A large body of research has been devoted to the study of glucose metabolism in fish species, mostly due to their phylogenetic position and their economic interest (i.e. fisheries and aquaculture). As a result of their lower ability to return to normoglycemia after feeding or after a glucose load, when compared to mammals, fish have been considered to be "glu‐ cose intolerant" [21]. This conclusion was based on the demonstrated lower ability of fish peripheral tissues (e.g. mostly the skeletal muscle, representing more than 50% of the body weight) to utilize glucose [22]. However, despite the low glucose uptake in the fish skele‐ tal muscle, when compared to mammals, there was evidence indicating that skeletal muscle represents not only the main site of glucose uptake but that it was the only tissue in which the rate of glucose uptake was increased after a glucose load [23], suggesting that glucose uptake in skeletal muscle could be regulated. Given that insulin had been shown to lower blood glucose levels in fish by, at least in part, stimulating the *in vivo* uptake and utiliza‐ tion of glucose mostly by the skeletal muscle [24] and given that the blood levels of insulin were shown to be increased by administration of a glucose load in trout [23], it was hypothesized that insulin may exert its hypoglycemic effects in fish through the regulation of glucose transporters, possibly GLUT4. Initially it was claimed that GLUT4 did not exist in fish based on the inability of antibodies against mammalian GLUT4 to recognize a putative fish GLUT4 protein [25]. However, the identification of a true GLUT4 homolog in fish in 2000 by molecular cloning [26] paved the way for investigating the possible regulation of glucose homeostasis in fish through the regulation of an insulin-regulatable glucose transporter homologous to GLUT4. Here, we review the accumulated evidence to date on

the functional characteristics and regulation of the GLUT4 homolog in fish.

**2. Evolution of the structural characteristics of the GLUT4 gene**

their emergence.

38 Glucose Homeostasis

Among the sequenced genomes of vertebrate species, the GLUT4 gene (named SLC2A4) is found in mammals (41 species), reptiles (2 species) and ray-finned fishes (10 species) (Figure 1), suggesting that the GLUT4 gene has been conserved throughout evolution. Surprising‐ ly, the GLUT4 gene is absent among birds and amphibians. The lack of GLUT4 in birds, but the presence of other GLUT family members (Glut1, Glut2 and Glut3), has recently been confirmed experimentally [27]. Moreover, no GLUT4 gene is present in the only amphib‐ ian genome available to date (i.e. *Xenopus tropicalis*) and searches for GLUT4 in Xenopus EST databases were unable to yield any transcript with homology for GLUT4. At this time, it is not known if the lack of the GLUT4 gene is general of the avian and amphibian classes, particularly since only a very reduced number of species have been examined, and, more importantly, if these two groups of vertebrates have subsequently lost the GLUT4 gene after Fish, after mammals, are the vertebrate group for which more genomic information is available mainly due to their importance as research model species (e.g. medaka, zebrafish, stickleback) and for their economic importance in fisheries and aquaculture (e.g. cod, tilapia). Therefore, fish are only second to mammals in the number of species for which there is genomic infor‐ mation corresponding to the GLUT4 gene. In mammals, the GLUT4 gene is located in chro‐ mosome 17 and consists of 11 coding exons, spanning 6.6 kb. Despite the fact that the fish GLUT4 protein is well conserved among the different fish species (see section 3), certain structural differences among the fish GLUT4 genes can be observed. For example, the number of exons coding for GLUT4 and the length of the GLUT4 gene varies among the different species: 12 exons in Fugu, 13 in Tetraodon, 13 in Tilapia, 11 in Stickleback, 12 in Medaka and 15 in Platyfish, spanning 4.8 kb, 4.5 kb, 10.2 kb, 4.9 kb, 12.7 kb and 14.1 kb, respectively (Figure 2). Moreover, it is worth mentioning that exons 6 to 10 of the human GLUT4 gene appear to be highly conserved in fish (Figure 2). The only fish species with a sequenced genome for which the GLUT4 gene appears to be absent is the zebrafish. The lack of a GLUT4 gene in the zebrafish genome sequence database (Ensembl) is related to the lack of transcripts for GLUT4 in zebrafish EST dabases. These observations support the notion that zebrafish may have lost the GLUT4 gene.

In addition to the gene structure characteristics of GLUT4 in fish, another aspect of interest with regards to the evolution of GLUT4 is whether the genes flanking the GLUT4 loci in the genome of the various fish species have also been conserved. A synthetic analysis of the GLUT4 loci in different fish species evidences that a number of the genes flanking the fish GLUT4 genes are homologs to those flanking the human GLUT4 gene (e.g. YBX2, EIF5AL1, GPS2, GABARAP, CTDNEP1, NEURL4, TNK1, PLSCR3) (Figure 3A). Furthermore, the nature and the genomic position of the genes flanking the GLUT4 gene in fish are highly conserved across the different fish species (Figure 3B).

**Figure 3.** Comparison of genomic regions carrying GLUT4 loci. A. Synteny in relation to the human GLUT4 gene. B. Synteny of fish GLUT4 genes in relation to the Fugu GLUT4 gene. Boxes of the same color indicate the position of the ortholog in the different species. The coding direction of the genes is indicated by the pointed end. Pale blue symbols indicate nonsyntenic genes. Location in the Genome of each species is indicated at the left side of the diagram. Gene IDs were retrieved from public databases-Human: ENSG00000181856; Fugu: ENSTRUG00000011935; Tetraodon: ENSTNIG00000010138; Tilapia: ENSONIG00000018958; Stickleback: ENSGACG00000019384; Medaka: EN‐ SORLG00000006341; Platyfish: ENSXMAG00000015723; Atlantic cod: ENSGMOG00000007757. The diagram was

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

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41

As is known in mammals, the GLUT4 gene undergoes an important regulation at the tran‐ scriptional level that takes place in the upstream regulatory regions of the gene (i.e. promoter region) [29]. Recently, the promoter region of the GLUT4 gene in a fish species (i.e. Fugu) was characterized for the first time [30]. A 1.3 kb 5´-flanking region of the Fugu GLUT4 gene was characterized with 3 possible transcription start sites, a conserved cluster of CpG dinucleoties (i.e. CpG island) and several transcription factor binding sites known to be important for the transcriptional regulation of the mammalian GLUT4 gene such as MEF2, SREBP, KLF, SP1/GC-box, NF-Y, E-box, PPARγ, PPAR-RXR and HIF-1 (Figure 4) [30]. In addition, like in mammals, the Fugu GLUT4 gene promoter lacks TATA-box elements [30]. *In silico* comparison of the 1.3 kb genomic DNA region upstream of the GLUT4 gene in different fish species revealed the presence of two highly conserved regions containing most of the above cited binding motifs (Figure 4). Interestingly, these two regions contain the E-box/MEF2/Klf cassette,

generated using Genomicus genome browser [28].

**Figure 2.** Genomic structure and organization of GLUT4 in fish. Exons are represented by solid boxes and introns are represented by connecting lines. The sizes of exons are shown in base pairs on the top of the boxes. Exons are num‐ bered with roman numbers beneath the boxes. Exons conserved in all the species are highlighted in green. Gene IDs were retieved from public databases-Human: ENSG00000181856; Fugu: ENSTRUG00000011935; Tetraodon: ENST‐ NIG00000010138; Tilapia: ENSONIG00000018958; Stickleback: ENSGACG00000019384; Medaka: EN‐ SORLG00000006341; Platyfish: ENSXMAG00000015723.

Structural and Functional Evolution of Glucose Transporter 4 (GLUT4): A Look at GLUT4 in Fish http://dx.doi.org/10.5772/58094 41

**Figure 3.** Comparison of genomic regions carrying GLUT4 loci. A. Synteny in relation to the human GLUT4 gene. B. Synteny of fish GLUT4 genes in relation to the Fugu GLUT4 gene. Boxes of the same color indicate the position of the ortholog in the different species. The coding direction of the genes is indicated by the pointed end. Pale blue symbols indicate nonsyntenic genes. Location in the Genome of each species is indicated at the left side of the diagram. Gene IDs were retrieved from public databases-Human: ENSG00000181856; Fugu: ENSTRUG00000011935; Tetraodon: ENSTNIG00000010138; Tilapia: ENSONIG00000018958; Stickleback: ENSGACG00000019384; Medaka: EN‐ SORLG00000006341; Platyfish: ENSXMAG00000015723; Atlantic cod: ENSGMOG00000007757. The diagram was generated using Genomicus genome browser [28].

As is known in mammals, the GLUT4 gene undergoes an important regulation at the tran‐ scriptional level that takes place in the upstream regulatory regions of the gene (i.e. promoter region) [29]. Recently, the promoter region of the GLUT4 gene in a fish species (i.e. Fugu) was characterized for the first time [30]. A 1.3 kb 5´-flanking region of the Fugu GLUT4 gene was characterized with 3 possible transcription start sites, a conserved cluster of CpG dinucleoties (i.e. CpG island) and several transcription factor binding sites known to be important for the transcriptional regulation of the mammalian GLUT4 gene such as MEF2, SREBP, KLF, SP1/GC-box, NF-Y, E-box, PPARγ, PPAR-RXR and HIF-1 (Figure 4) [30]. In addition, like in mammals, the Fugu GLUT4 gene promoter lacks TATA-box elements [30]. *In silico* comparison of the 1.3 kb genomic DNA region upstream of the GLUT4 gene in different fish species revealed the presence of two highly conserved regions containing most of the above cited binding motifs (Figure 4). Interestingly, these two regions contain the E-box/MEF2/Klf cassette,

**Figure 2.** Genomic structure and organization of GLUT4 in fish. Exons are represented by solid boxes and introns are represented by connecting lines. The sizes of exons are shown in base pairs on the top of the boxes. Exons are num‐ bered with roman numbers beneath the boxes. Exons conserved in all the species are highlighted in green. Gene IDs were retieved from public databases-Human: ENSG00000181856; Fugu: ENSTRUG00000011935; Tetraodon: ENST‐ NIG00000010138; Tilapia: ENSONIG00000018958; Stickleback: ENSGACG00000019384; Medaka: EN‐

SORLG00000006341; Platyfish: ENSXMAG00000015723.

40 Glucose Homeostasis

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 regulatory region during evolution from fish to mammals.

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

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**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‐

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

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

(Figure 5).

**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 promoters. Adapted from [30].
