PARENT SESSION
Preimplantation Embryo/Fetal Development

CLONING AND MOLECULAR CHARACTERIZATION OF MOUSE GLUT9, A NEW EMBRYONICALLY EXPRESSED GLUCOSE TRANSPORTER.

Carayannopoulos, Mary¹, Phay, John², Cui, Ying¹, Moley, Jeffrey², Moley, Kelle¹, ^{1 2}

ABSTRACT- During preimplantation development in the mouse, it is critical that glucose metabolism is not compromised. Previous work from our laboratory has shown that any decrease in glucose uptake at this critical stage in development can result in embryo dysmorphogenesis or embryo demise. Therefore, we are interested in elucidating what molecules expressed in the preimplantation blastocyst are responsible for glucose delivery. At this time, four members of the facilitative glucose transporter family are known to be expressed at this stage of development. These include GLUT1, GLUT2, GLUT3, and GLUT8. We have recently cloned another member of this family, which is expressed embryonically. This transporter, GLUT9, was originally cloned in human. Based on our studies with the human homolog, GLUT9 is expressed predominantly in adult liver, kidney, and placenta. Using a Xenopus laevis oocyte overexpression system, we were able to show that human GLUT9 transports glucose at a rate significantly greater than sham injected controls but not significantly different from another glucose transporter, GLUT4. In addition, we were able to show that human GLUT9 colocalizes to the endoplasmic reticulum (ER) with the ER specific protein Bip and with the ER specific dye, DiOC6. These data suggest that GLUT9 may be the putative ER glucose transporter associated with the glucose 6-phosphatase system. In order to clone mouse GLUT9, we screened the mouse EST database for clones having homology to the human sequence. A clone corresponding to the 5' end of human GLUT9 was identified. This sequence was used to design primers and perform 5' and 3' RACE using mouse liver cDNA as a template to identify the remainder of the clone. A full-length cDNA was PCR amplified from liver cDNA, cloned and sequenced in both directions. Mouse GLUT9 encodes a 539 amino acid protein which shares 83% sequence identity with human GLUT9. Using an RNase protection assay, mouse GLUT9 is detected in adult kidney, liver and the gastrointestinal tract and is expressed as early as embryonic day 13, also in the liver, kidney and gastrointestinal tract. By RT-PCR, mouse GLUT9 is detected in 2-cell and blastocyst stage embryos. Because of its expression at this early stage of development, we are interested in further characterization of this new facilitative glucose transporter.

KEY WORDS: embryonic glucose transporter, blastocyst development, endoplasmic reticulum