The recently cloned human GLUT9 gene, which maps to chromosome 4p15.3-p16, consists of 12 exons coding for a 540-amino acid protein. Based on a sequence entry (NCBI accession number BC018897) and screening of expressed sequence tags, we have cloned an alternative splice variant of GLUT9 from human kidney cDNA. The RNA of this splice variant consists of 13 exons and codes for a putative protein of 512 amino acids (GLUT9⌬N). The predicted proteins differ only in their N terminus, suggesting a different subcellular localization and possible physiological role. Screening human tissue RNA by reverse transcription-PCR showed that GLUT9 is expressed mainly in kidney, liver, placenta, and leukocytes, whereas GLUT9⌬N was detected only in kidney and placenta. The GLUT9 protein localized by immunohistochemistry to human kidney proximal tubules, and subcellular fractionation of human kidney revealed the GLUT9 protein in plasma membranes and high density microsomal membranes. Treatment of kidney membrane proteins with peptide Nglycosidase F showed that GLUT9 and GLUT9⌬N are expressed in vivo. Localization of GLUT9 and GLUT9⌬N in three kidney-derived cell lines revealed a plasma membrane distribution for GLUT9 in COS-7 and HEK293 cells, whereas GLUT9⌬N showed a perinuclear pattern and plasma membrane staining in COS-7 and HEK293 cells, respectively. In polarized Madin-Darby canine kidney cells, GLUT9 trafficked to the basolateral membrane, whereas GLUT9⌬N localized to the apical membrane. Using heterologous expression of GLUT9 in Xenopus oocytes, GLUT9 appears to be a functional isoform with low affinity for deoxyglucose. Deoxyglucose transport mediated by GLUT9 was not inhibited by cytochalasin B. GLUT9 did not bind cytochalasin B as shown by a cytochalasin B binding assay, indicating a similar behavior of GLUT9 compared with GLUT5.

Mammalian preimplantation blastocysts exhibit insulin-stimulated glucose uptake despite the absence of the only known insulinregulated transporter, GLUT4. We describe a previously unidentified member of the mammalian facilitative GLUT superfamily that exhibits Ϸ20 -25% identity with other murine facilitative GLUTs. Insulin induces a change in the intracellular localization of this protein, which translates into increased glucose uptake into the blastocyst, a process that is inhibited by antisense oligoprobes. Presence of this transporter may be necessary for successful blastocyst development, fuel metabolism, and subsequent implantation. Moreover, the existence of an alternative transporter may explain examples in other tissues of insulin-regulated glucose transport in the absence of GLUT4.

The PI3K/Akt signal transduction pathway is a well-known mediator of growth promoting and cell survival signals. While the expression and function of this pathway have been documented during early and late stages of the reproductive process, currently, there is no evidence demonstrating either the presence or function of the PI3K/Akt pathway in murine preimplantation embryos. We found, using confocal immunofluorescent microscopy and Western blot analysis, that the p 85 and p110 subunits of PI3K and Akt are expressed from the 1-cell through the blastocyst stage of murine preimplantation embryo development. These proteins were localized predominantly at the cell surface from the 1-cell through the morula stage. At a blastocyst stage, both PI3K and Akt exhibited an apical staining pattern in the trophectoderm cells. Interestingly, phosphorylated Akt was detected throughout murine preimplantation development, and its presence at the plasma membrane is a reflection of its activation status. Inhibition of Akt activity had significant effects on the normal physiology of the blastocyst. Specifically, inhibition of this pathway resulted in a reduction in insulin-stimulated glucose uptake. In addition, inhibiting Akt activity resulted in a significant delay in blastocyst hatching, a developmental step facilitating implantation. Finally, we established the presence of this pathway in trophoblast stem (TS) cells, a potentially useful in vitro model to study this signaling cascade. Taken together, these data are the first to demonstrate the presence and function of the PI3K/Akt pathway in mammalian preimplantation embryos.

We report that a decrease in facilitative glucose transporter (GLUT1) expression and reduced glucose transport trigger apoptosis in the murine blastocyst. Inhibition of GLUT1 expression either by high glucose conditions or with antisense oligodeoxynucleotides significantly lowers protein expression and function of GLUT1 and as a result induces a high rate of apoptosis at the blastocyst stage. Similar to wild-type mice, embryos from streptozotocin-induced diabetic Bax ؊/؊ mice experienced a significant decrease in glucose transport compared with embryos from non-diabetic Bax ؊/؊ mice. However, despite this decrease, these blastocysts demonstrate significantly fewer apoptotic nuclei as compared with blastocysts from hyperglycemic wild-type mice. This decrease in preimplantation apoptosis correlates with a decrease in resorptions and malformations among the infants of the hyperglycemic Bax ؊/؊ mice versus the Bax ؉/؉ and ؉/؊ mice. These findings suggest that hyperglycemia by decreasing glucose transport acts as a cell death signal to trigger a BAX-dependent apoptotic cascade in the murine blastocyst. This work also supports the hypothesis that increased apoptosis at a blastocyst stage because of maternal hyperglycemia may result in loss of key progenitor cells and manifest as a resorption or malformation, two adverse pregnancy outcomes more common in diabetic women.In prior studies, it has been shown that maternal hyperglycemia results in down-regulation of the embryonic facilitative glucose transporters (GLUT), 1 GLUT1, GLUT2, and GLUT3, at the blastocyst stage of mouse development (1). Culturing two-cell embryos for 72 h in high concentrations of glucose (30 or 52 mM) likewise causes a decrease in the expression of these facilitative transporters at the mRNA and protein levels. This decrease in transporter expression leads to a significant drop in intraembryonic free glucose levels in blastocysts obtained from mice made hyperglycemic by streptozotocin injection or after culturing two-cell embryos from normal mice in high glucose. Blastocysts cultured under similar conditions also experience a 6-fold increase in expression of the proapoptotic protein BAX, as compared with controls and undergo increased apoptosis (2). Approximately 40% of all nuclei from embryos from hyperglycemic mothers showed evidence of terminal dUTP nick-end labeling or TUNEL-positive staining compared with less than 10% among controls. This apoptotic event requires BAX expression because blastocysts recovered from diabetic Bax Ϫ/Ϫ mice are resistant to the hyperglycemia-induced apoptosis. Similarly, the hyperglycemia-induced event is inhibited partially with either the caspase inhibitor z-Val-Ala-Asp-fluoromethylketone (zVAD-FMK), or the ceramide synthase inhibitor, fumonisin B1, strongly suggesting that these apoptosis-associated pathways are involved. Apoptosis at this developmental stage may manifest later in pregnancy as a malformation or, if a significant cell loss occurs, as a miscarriage. Both these adverse pregnancy outcomes occur mor...

A novel glucose transporter (GLUT), mouse GLUT9 (mGLUT9), was recently cloned from mouse 7-d embryonic cDNA. Several splice variants of mGLUT9 were described, two of which were cloned (mGLUT9a and mGLUT9a Delta 209-316). This study describes the cloning and characterization of another splice variant, mGLUT9b. Cloned from adult liver, mGLUT9b is identical to mGLUT9a except at the amino terminus. Based on analysis of the genomic structure, the different amino termini result from alternative transcriptional/translational start sites. Expression and localization of these two mGLUT9 splice variants were examined in control and diabetic adult mouse tissues and in cell lines. RT-PCR analysis demonstrated expression of mGLUT9a in several tissues whereas mGLUT9b was observed primarily in liver and kidney. Using a mGLUT9-specific antibody, Western blot analysis of total membrane fractions from liver and kidney detected a single, wide band, migrating at approximately 55 kDa. This band shifted to a lower molecular mass when deglycosylated with peptide-N-glycosidase F. Both forms were present in liver and kidney. Immunohistochemical localization demonstrated basolateral distribution of mGLUT9 in liver hepatocytes and the expression of mGLUT9 in specific tubules in the outer cortex of the kidney. To investigate the alternative amino termini, mGLUT9a and mGLUT9b were overexpressed in kidney epithelium cell lines. Subcellular fractions localized both forms to the plasma membrane. Immunofluorescent staining of polarized Madin Darby canine kidney cells overexpressing mGLUT9 depicted a basolateral distribution for both splice variants. Finally, mGLUT9 protein expression was significantly increased in the kidney and liver from streptozotocin-induced diabetic mice compared with nondiabetic animals.

GLUT1 is essential for human brain development and function, as evidenced by the severe epileptic encephalopathy observed in children with GLUT1 deficiency syndrome resulting from inherited loss-of-function mutations in the gene encoding this facilitative glucose transporter. To further elucidate the pathophysiology of this disorder, the zebrafish orthologue of human GLUT1 was identified, and expression of this gene was abrogated during early embryonic development, resulting in a phenotype of aberrant brain organogenesis consistent with the observed expression of Glut1 in the embryonic tectum and specifically rescued by human GLUT1 mRNA. Affected embryos displayed impaired glucose uptake concomitant with increased neural cell apoptosis and subsequent ventricle enlargement, trigeminal ganglion cell loss, and abnormal hindbrain architecture. Strikingly, inhibiting expression of the zebrafish orthologue of the proapoptotic protein Bad resulted in complete rescue of this phenotype, and this occurred even in the absence of restoration of apparent glucose uptake. Taken together, these studies describe a tractable system for elucidating the cellular and molecular mechanisms of Glut1 deficiency and provide compelling in vivo genetic evidence directly linking nutrient availability and activation of mitochondria-dependent apoptotic mechanisms during embryonic brain development.The cellular uptake of glucose is dependent in part upon the GLUT family of polytopic membrane transporters that facilitate the passive diffusion of this essential nutrient across membranes (1). GLUT1, the prototypic member of this protein family, is highly expressed in erythrocytes and the central nervous system and is believed to play an essential role in the homeostasis of brain glucose in the developing human infant (2). In support of this concept, infants with GLUT1 deficiency syndrome, a rare genetic disorder resulting from inherited heterozygous loss-of-function mutations in the gene encoding GLUT1, develop seizures, acquired microcephaly and profound developmental delay in association with profound hypoglycorrhachia (3). Despite considerable study of these patients, the neurochemical and neuropathologic consequences of GLUT1 deficiency during development are not well understood, and treatment of affected patients remains challenging.Although abundant in the extracellular milieu, the cellular uptake of glucose is precisely regulated by specific growth factors and signaling pathways (4). Cell culture studies reveal that inhibition of cellular glucose uptake dramatically increases apoptosis under conditions of growth factor restriction and that cell survival under such circumstances is dependent upon the regulation of glucose uptake and metabolism by the proto-oncogene Akt (5-7). A biochemical link between glucose homeostasis and apoptosis was further suggested by studies demonstrating an interaction between the proapoptotic protein, Bad, and the glycolytic enzyme, glucokinase (8). In support of these findings, a recent study now demonstrates a...

The phosphatidylinositol 3-kinase (PI3K) signal transduction pathway is a well known mediator of cell growth, proliferation, and survival signals. Whereas the expression and function of this pathway has been documented during mammalian development, evidence demonstrating the physiologic importance of this pathway in murine preimplantation embryos is beginning to emerge. This study demonstrates that inhibition of the PI3K pathway leads to the induction of apoptosis in both murine blastocysts and trophoblast stem cells. The apoptosis induced in both model systems correlates with a decrease in the expression of the glucose transporter GLUT1 at the plasma membrane. In addition, blastocysts cultured in the presence of the PI3K inhibitor LY-294002 display a decrease in both 2-deoxyglucose uptake and hexokinase activity as compared with control blastocysts. To determine the impact of PI3K inhibition on pregnancy outcome, embryo transfer experiments were performed. Blastocysts cultured in the presence of LY-294002 demonstrate a dramatic increase in fetal resorptions as compared with control embryos. Finally, we demonstrate that impairment of glucose metabolism via iodoacetate, a glyceraldehyde-3-phosphate dehydrogenase inhibitor, is sufficient to induce apoptosis in both blastocysts and trophoblast stem cells. Moreover, blastocysts treated with iodoacetate result in poor pregnancy outcome as determined by embryo transfer experiments. Taken together these data demonstrate the critical importance of the PI3K pathway in preimplantation embryo survival and pregnancy outcome and further emphasize the importance of glucose utilization and metabolism in cell survival pathways.The blastocyst stage of murine preimplantation development occurs approximately 4 days post-fertilization. At this stage of embryonic development the first cell differentiation step has occurred. The blastocyst is comprised of the epithelial trophectoderm, which is the layer of cells that develop into the placenta, and the inner cell mass, which consists of the pluripotent cells that gives rise to the embryo proper. Prior to implantation, the developing embryo is dependent on signals generated by growth factors that are either made by the embryo itself or are present in the maternal environment. These growth factors are known to regulate cellular proliferation and differentiation during mammalian preimplantation development (1, 2). Importantly, the preimplantation embryo expresses a number of growth factor receptors known to activate the phosphatidylinositol 3-kinase (PI3K) 2 pathway including the insulin and insulin-like growth factor-I receptors (3, 4).PI3K is a lipid kinase that phosphorylates the D-3 position on the inositol ring in phosphoinositides (5-7). It is a heterodimeric enzyme that consists of an 85-kDa regulatory subunit and a 110-kDa catalytic subunit. A number of extracellular signals activate PI3K including insulin and other growth factors. Insulin and insulin-like growth factor-I have been shown to have both mitogenic and anti-apoptotic ...

Glucose transporter (GLUT) 8 is an insulin-responsive facilitative glucose transporter expressed predominantly in the murine blastocyst. To determine the physiologic role of GLUT8, two-cell embryos were cultured to a blastocyst stage in antisense or sense oligonucleotides to GLUT8. Apoptosis was assessed using the TUNEL techniques and recorded as the percentage of TUNEL-positive nuclei/total nuclei. Embryos cultured in GLUT8 antisense experienced increased TUNEL-positive nuclei, whereas sense embryos did not. Embryos cultured in a control AS oligonucleotide, specific for heat shock protein 70-2, showed a rate of apoptosis similar to sense. To determine the outcome of these apoptotic embryos, blastocysts exposed to sense vs. antisense were transferred back into foster mice and the pregnancy continued until Day 14.5, at which time the uteri were examined for normal gestational sacs and resorptions. Embryos exposed to GLUT8 antisense experienced higher rates of resorptions and lower normal pregnancy rates compared to embryos cultured in GLUT8 sense. To examine the insulin growth factor (IGF)-1/insulin intracellular signaling pathways involved in GLUT8 translocation, IGF-1 receptor (IGF-1R) expression was decreased in the blastocysts with antisense oligonucleotides. Using confocal immunofluorescent microscopy, GLUT8 translocation in response to insulin was observed. Exposure to insulin in the embryos exposed to IGF-1R sense induced translocation of GLUT8 from intracellular compartments to the plasma membrane. Blastocysts exposed to IGF-1R antisense, however, failed to demonstrate any change in the intracellular location of GLUT8 with insulin treatment. The IGF-1R antisense embryos also displayed significantly greater TUNEL staining compared to sense embryos. These data suggest that GLUT8 expression and translocation in response to insulin are critical for blastocyst survival.