Glucose transporters (Gluts), Glucose transporter -1 (Glut-1)

The family of facilitative glucose transporters includes thirteen isoforms (Glut 1-12 and the myo-inositol transporter [HMIT1]), all of which have common structural features. The protein structure consists of 12 transmembrane-spanning helices which contain abundant hydroxyl and amide side chains that participate in glucose binding, seven conserved glycine residues in the helices, several basic and acidic residues at the intracellular surface of the proteins, two conserved tryptophan residues, two conserved tyrosine residues, an intracellular loop, an extracellular segment and an intracellularly located N (amino) and C (carboxyl terminus) (JOOST et al. 2002;

JOOST and THORENS 2001; MUECKLER et al. 1985). The different isoforms show a high degree of sequence homology but vary in their tissue specifity (JOOST et al.

2002). In adult tissue, Glut-1 is the most abundant isoform and is expressed and localized in the basolateral membranes of polarized epithelial cells (FUKUMOTO et al. 1989; PASCOE et al. 1996).

The expression pattern of Glut-1 during preimplantation development has been studied in several species. Glut-1 was found in oocytes and all preimplantation stages studied in the mouse (HOGAN et al. 1991; MORITA et al. 1992), rabbit (ROBINSON et al. 1990), human (DAN-GOOR et al. 1997) and bovine (WRENZYCKI

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et al. 1999; AUGUSTIN et al. 2001). Similarly, messenger RNA expression for Glut-3 and Glut-8 has been determined in oocytes and preimplantation bovine embryos (AUGUSTIN et al. 2001). In mouse embryos, Glut-3 is first expressed around compaction (PANTALEON et al. 1997a), while Glut-2 and Glut-8 are first detected at the blastocyst stage (AGHAYAN et al. 1992; CARAYANNOPOULOS et al. 2000).

Glut-9 is expressed from the 2-cell stage onwards and persists up to blastocyst stage (CARAYANNOPOULOS et al. 2003).

In non-embryonic cells, Glut-6 has been found in spleen, leucocytes and brain, Glut 7 in muscle, fat and heart, Glut-10 in liver and pancreas, Glut-11 in heart and skeletal muscle, Glut-12 in heart, prostate, placenta and Xenopus laevis oocytes, and HMIT1 in brain (JOOST et al. 2002; JOOST and THORENS 2001; ROGERS et al. 2003).

In mouse blastocysts, Glut-1 protein has been localized in the apical, basolateral and intercellular membranes of the trophectoderm (TE) and ICM cells (AGHAYAN et al.

1992). Other studies found a specific basolateral distribution in outer morula cells and trophectoderm and a uniform distribution on the plasmatic membrane of ICM cells (PANTALEON et al. 1997a; PANTALEON et al. 2001). In bovine blastocysts, Glut-1 was specifically localized in the lateral membrane of the trophectoderm and heterogeneously distributed in ICM cells (AUGUSTIN et al. 2001). On the other hand, Glut-3 has been localized in apical membranes of polarized cells of the compacted morula and on the apical membranes of TE cells (PANTALEON et al. 1997a). This specific localization of Glut-1 and Glut-3 protein makes it possible for apical Glut-3 in trophectoderm cells to take up glucose from the external medium and for the basolateral Glut-1 to deliver glucose into the blastocoel for subsequent uptake by the ICM cells via Glut-1 (Fig. 14). This model is consistent with metabolic studies in mice and bovine embryos which have demonstrated that the ICM has higher glycolytic activity than that of the trophectoderm, indicating that TE acts as a transporting epithelium, saving nutrients for metabolism of the ICM (HEWITSON and LEESE 1993; GOPICHANDRAN and LEESE 2003).

Differences in the expression of Glut-1 mRNA during embryonic development have been observed. Hatched blastocysts express Glut-1 mRNA at higher levels than immature and mature oocytes, zygotes, 2-4-cell and 8-16-cell bovine embryos

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(WRENZYCKI et al. 1999). Similarly, in mouse blastocysts levels of Glut-1 protein are 20-fold greater than in unfertilized oocytes (MORITA et al. 1992).

In cattle, glucose transporter -4 (Glut-4) has been detected in expanded and hatched blastocysts, while Glut-2 transcripts have been detected in 14 days old elongated blastocysts. Glut-5 is transcribed from the 8-16-cell stage onwards (AUGUSTIN et al.

2001). Table 3 shows a summary of mRNA expression for Gluts in oocytes and embryos from three different species.

Nucleus

TE

ICM Nucleus

Nucleus

TE

ICM

Fig: 14. Schematic representation of glucose transports localization in the mouse blastocyst. Apically localized GLUT3 on trophectoderm (TE) cells is responsible for uptake of maternal glucose while, at basolateral surfaces, transport is mediated by GLUT1. The inner cell mass (ICM) also relies on GLUT1 for provision of glucose from the blastocoel. The importance of GLUT2 also reported to be present on basolateral TE and membranes of the ICM, is unclear. Adapted from PANTALEON and KAYE 1998.

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Blastocyst + ⎯ ⎯ ⎯ ? ? ?

8-16-cell + ⎯ ⎯ ⎯ ? ? ?

2-4-cell + ⎯ ⎯ ⎯ ? ? ?

Human Oocyte + ⎯ ⎯ ⎯ ? ? ? DAN-GOOR et al. (1997)

Blastocyst + + + + + + ?

8-16-cell + ⎯ + ⎯ + + ?

2-4-cell + ⎯ + ⎯ ⎯ + ?

Bovine Oocyte + ⎯ + ⎯ ⎯ + ? WRENZYCKI et al. (1999) AUGUSTIN et al. (2001) + = transcript detected ⎯= transcript not determined ? = no data

Blastocyst + + + ⎯ ? + +

8-16-cell + + + ⎯ ? ⎯ +

2-4-cell + ⎯ ⎯ ⎯ ? ⎯ +

Mouse Oocyte + ⎯ ⎯ ⎯ ? ⎯ ? MORITA et al. (1992) PANTALEON et al. (1997a) AGHAYAN et al. (1992) CARAYANNOPOULOS et al. (2000), CARAYANNOPOULOS et al. (2003)

Messenger RNA expression for Gluts in oocytes and embryos from three different species Specie

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Glucose is taken up into the cell by a mechanism which requires a sodium gradient generated by Na⁺–K⁺ ATPases and is in that sense energy dependent. The actual uptake of glucose is accomplished by Na⁺–glucose cotransporters (e.g. Sodium-Glucose co-transporter SGLT1), which couple glucose uptake to the influx of Na⁺ along that concentration gradient (Fig. 15). Additional glucose is transported by facilitative glucose transporters (Gluts 1-12) [Fig. 16] (PANTALEON and KAYE 1998;

AUGUSTIN et al. 2001; JOOST et al. 2002). Expression of SGLT mRNA has been observed in oocytes and at all stages of early bovine embryonic development (AUGUSTIN et al. 2001), but its importance for glucose uptake during early embryonic development is still controversial (PANTALEON and KAYE 1998).

(SGLT)

(Gluts) (SGLT)

(Gluts)

Fig: 15. Integrated multiple transport systems allow glucose to be transported from the intestine to the blood through an intestinal epithelial cell.

Glucose is actively transported into the cell by a sodium ion driven cotransport system located only on the part of the plasma membrane in contact with the intestinal lumen. The sodium ion gradient across the plasma membrane is maintained by sodium-potassium pumps, which keep the intracellular sodium ion concentration low by actively transporting it from the cytoplasm into the blood. The active transport of glucose keeps its intracellular concentration high, so that it can enter the blood by facilitated diffusion. The carrier proteins responsible for the facilitated diffusion of glucose are located only on the regions of the plasma membrane in contact with the capillary. Adapted from SOLOMON et al. 1996d.

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Fig: 16. Schematic representation of glucose uptake through the Glut-4 channel.

Insulin binding to its receptor on the cell membrane activates translocation of the Glut-4 channel from intracellular vesicles to the cell membrane. Glut-4 allows entry of glucose into the cell in two steps: a) When the external face of the glucose transporter channel is open, a glucose molecule becomes bound within the channel; b) the outer portion of channel then closes and the inner face opens, allowing glucose release into the cell. No energy is required for this process which depends only on the glucose gradient. Adapted from www.zonamedica.com.ar/categorias/medicinailustrada/insulina/glucosa.htm

As in most cells, bovine pre-compaction embryos are highly dependent on oxidative phosphorylation (formation of adenosine triphosphate [ATP] by electron transport) as the primary energy production pathway, during which it is estimated that approximately 90% of all ATP is derived from oxidation of pyruvate as the preferred substrate (THOMPSON et al. 1996; KHURANA and NIEMANN 2000). During compaction and blastulation, the demand for ATP increases to allow increases in protein synthesis (THOMPSON et al. 1998) and concomitantly the activity of the Na⁺– K⁺-ATPase necessary for formation and maintenance of the blastocoel (RIEGER et al. 1992a; WATSON et al. 1999). In a recent study, it was estimated that in bovine embryos approximately 36% of the ATP is used by the sodium pump during blastocoel expansion (HOUGHTON et al. 2003). The increased demand for ATP causes increased consumption of the major substrates, including oxygen, pyruvate, glucose (THOMPSON et al. 1996; KHURANA and NIEMANN 2000) and amino acids (PARTRIDGE and LEESE 1996). It has been observed that glucose metabolism

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increases steadily from the 1-cell stage to the blastocyst stage and that there is a marked increase from the 8-16-cell stage onwards (RIEGER et al. 1992a; KHURANA and NIEMANN 2000). This increase in glucose consumption is such that the contribution of glycolysis alone to ATP production increases from approximately 4–

8% to 15–18% between pre- and post-compaction stages, respectively, in an environment in which O2 is abundant (THOMPSON et al. 1996). Moreover, employing inhibitors and uncouplers of oxidative phosphorylation during peri-compaction of in vitro produced bovine embryos demonstrated that ATP production via oxidative phosphorylation is essential for bovine embryo development in vitro.

However, transient inhibition caused by the addition of 10 or 100 µmol of 2,4-Dinitrophenol increases the percentage of embryos reaching the expanded blastocyst stage and the total number of cells per blastocyst (THOMPSON et al.

2000). A study in monkeys, hamsters and rabbits indicated that O2 tension in the reproductive tract decreases as embryos travel from the oviduct to the uterine cavity (FISCHER and BAVISTER 1993). These findings indicate that there is a shift in metabolic pathways preference for embryonic ATP production from oxidative phosphorylation to glycolysis, this corresponds with development within the uterine cavity, where the O2 availability is limited. A shift in pathway preference has been also reported for human (GOTT et al. 1990) and rat (LEESE 1991) embryos.

Recently this shift in metabolic preference has been associated with a change in the reduction-oxidation (REDOX) state within the embryo, which would affect not only energy production required for development, but also the activity of REDOX-sensitive transcription factors, which may alter gene expression patterns (HARVEY et al.

2002).

2.4.3.2.2 Eukaryotic translation initiation factor (eIFs), eukaryotic initiation factor 1A (eIF1A)

Initiation of translation is a complex process in which initiator tRNA, 40S and 60S ribosomal subunits are assembled by eukaryotic initiation factors (eIFs) into an 80S ribosome at the initiation codon of mRNA. A eukaryotic cell uses more than 12

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initiation factors (all together comprising 27 polypeptides chains) to assemble an 80S ribosome that is competent for protein synthesis (PESTOVA et al. 2001).

Eukaryotic translation initiation factor 1A [(eIF1A), formerly called eIF-4C], is a 17 kDa protein universally conserved throughout all biological kingdoms (ROLL-MECAK et al. 2001). Amongst others, eIF1A is involved in the initiation of translation in eukaryotic cells (CHAUDHURI et al. 1997) mainly by catalyzing the transfer of Met-tRNA-eIF2-GTP complex to the 40S ribosomal subunits to form a stable 40S preinitiation complex thereby accomplishing 60S subunits during translation (CHAUDHURI et al. 1999).

Amino acid sequence comparisons of translation initiation factors in bacteria and eukaryotes revealed conservation of two translation initiation factors throughout evolution: IF1/eIF1A and IF2/eIF5B (KYRPIDES and WOESE 1998a; KYRPIDES and WOESE 1998b). Messenger RNA transcripts for eIF1A, eIF4A, eIF5, eIF4E and eIF2α have been detected in in vivo-developed mouse embryos (DE SOUSA et al.

1998b). Using semi-quantitative RT-PCR, a transient increase of expression for eIF1A mRNA and protein in 2-cell stages in the mouse and 8-16-cell stages in cows was detected, emphasizing that it is a conserved endogenous marker of genome activation (DAVIS, JR. et al. 1996; DE SOUSA et al. 1998b).