Syntaxin 4 Expression Affects Glucose Transporter 8 Translocation and Embryo Survival
Amanda Hoehn Wyman,
Maggie Chi,
Joan Riley,
Mary O. Carayannopoulos,
Chunmei Yang,
Kenneth J. Coker,
Jeffrey E. Pessin and
Kelle H. Moley
Department of Obstetrics and Gynecology (A.H.W., M.C., J.R., M.O.C., K.H.M.) and Department of Cell Biology and Physiology (K.H.M.),Washington University School of Medicine, St. Louis, Missouri 63110; Lexicon Genetics, Inc. (C.Y., K.J.C.), The Woodlands, Texas 77381; and The University of Iowa (J.E.P.), Iowa City, Iowa 52242
Address all correspondence and requests for reprints to: Kelle H. Moley, 4911 Barnes-Jewish Hospital Plaza, 6th Floor Maternity, St. Louis, Missouri 63110. E-mail: moleyk{at}msnotes.wustl.edu.
 |
ABSTRACT
|
---|
Target-soluble N-ethylmaleimide-sensitive factor attachment protein receptors (t-SNAREs) are receptors that facilitate vesicle and target membrane fusion. Syntaxin 4 is the t-SNARE critical for insulin-stimulated glucose transporter 4 (GLUT4)-plasma membrane fusion in adipocytes. GLUT8 is a novel IGF-I/insulin-regulated glucose transporter expressed in the mouse blastocyst. Similar to GLUT4, GLUT8 translocates to the plasma membrane to increase glucose uptake at a stage in development when glucose serves as the main substrate. Any decrease in GLUT8 cell surface expression results in increased apoptosis and pregnancy loss. Previous studies have also shown that disruption of the syntaxin 4 (Stx4a) gene results in early embryonic lethality before embryonic d 7.5. We have now demonstrated that syntaxin 4 protein is localized predominantly to the apical plasma membrane of the murine blastocyst. Stx4a inheritance, as detected by protein expression, occurs with the expected Mendelian frequency up to embryonic d 4.5. In parallel, 22% of the blastocysts from Stx4a+/- matings had no significant insulin-stimulated translocation of GLUT8 whereas 77% displayed either partial or complete translocation to the apical plasma membrane. This difference in GLUT8 translocation directly correlated with one-third of blastocysts from Stx4a+/- mating having reduced rates of insulin-stimulated glucose uptake and 67% with wild-type rates. These data demonstrate that the lack of syntaxin 4 expression results in abnormal movement of GLUT8 in response to insulin, decreased insulin-stimulated glucose uptake, and increased apoptosis. Thus, syntaxin 4 functions as the necessary t-SNARE protein responsible for correct fusion of the GLUT8-containing vesicle with the plasma membrane in the mouse blastocyst.
 |
INTRODUCTION
|
---|
FACILITATIVE GLUCOSE UPTAKE is mediated by a family of related glucose transporter proteins with distinct but overlapping tissue-specific expression, enzymatic properties, and hormonal/metabolic regulation (1, 2, 3, 4). Within this family, glucose transporter 8 (GLUT8) is a recently described facilitative glucose transporter that is predominantly expressed during mammalian blastocyst development and is essential for cellular glucose uptake during development (5, 6). This protein resides in intracellular compartments and, upon insulin or IGF-I stimulation via IGF-I receptor binding, translocates to the plasma membrane, thereby increasing cellular glucose uptake necessary for the maintenance of blastocyst viability. This process is analogous to the well established insulin-stimulated translocation of GLUT4 from intracellular storage sites to the cell surface membrane in skeletal muscle and adipose tissue (3). However, the GLUT4 protein is not expressed in mouse blastocysts and, to date, none of the other facilitative glucose transporter proteins have been found to display any significant intracellular sequestration with translocation in response to insulin (7, 8, 9).
Studies examining the translocation of GLUT4 have identified several of the key proteins involved in the docking and/or membrane fusion process. Vesicle-associated membrane protein-2, or VAMP2, appears to function as the v-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein that directly interacts with the plasma membrane t-SNARE proteins, syntaxin 4 and synaptosome-associated protein 23 in the fusion process of GLUT4 (10, 11). These proteins generate a high energy four-helical bundle that is thought to provide the energy necessary for the membrane fusion event (12, 13). Although the specific SNARE isoform responsible for the insulin-stimulated translocation of GLUT8 in blastocysts has not been examined, homozygotic syntaxin 4 null mice are not viable and are lost during early development, before embryonic d 7.5 (E7.5) (14).
Glucose transport and metabolism are critical for mammalian blastocyst formation and further development (15, 16, 17). At this stage, a switch occurs from oxidative phosphorylation and the metabolism of pyruvate and lactate to glycolysis and the use of glucose as the main substrate (18, 19). As a result, the blastocyst exhibits extreme sensitivity to glucose deprivation. We have previously shown that any decrease in blastocyst glucose transport, basal or insulin stimulated, results in enhanced apoptosis at this stage, which manifests later in pregnancy as a malformation or miscarriage (16, 20). Because glucose uptake through GLUT8 function is essential for early embryo survival (6), we speculate that syntaxin 4 may function as the necessary t-SNARE for insulin-stimulated GLUT8 translocation in the blastocyst. In this study we demonstrate that syntaxin 4 null blastocysts are unable to translocate GLUT8 in response to insulin. This loss of glucose uptake results in a higher percentage of apoptotic nuclei that directly accounts for the early embryonic lethality in these animals.
 |
RESULTS
|
---|
Syntaxin 4, VAMP2, and VAMP3 Proteins Are Expressed in the Mouse Blastocyst
To determine whether the SNARE protein isoform syntaxin 4, VAMP2, and VAMP3 were expressed in mouse blastocysts, we used immunofluorescent microscopy (Fig. 1
). Individual cells were identified by propidium iodide labeling of nuclei (red channel), and colabeling with syntaxin 4 demonstrated that this SNARE protein was primarily confined to the outer surface of the blastocyst (green channel) indicative of apical plasma membrane localization (Fig. 1
). Syntaxin 4 protein expression was seen predominantly in trophectoderm cells with little or no expression in the inner cell mass. VAMP2 and VAMP3 were primarily localized to intracellular vesicular compartments in both trophectoderm cells and inner cell mass, with only a small extent of apical plasma membrane labeling (Fig. 1
). The predominant intracellular localization of VAMP2/3 and apical plasma membrane localization of syntaxin 4 are consistent with their distribution in other cell types. As controls, there was no specific labeling to mouse blastocyst using preimmune sera (data not shown).

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 1. t-SNARE and v-SNARE Proteins Are Expressed in the Mouse Blastocyst
Blastocysts fixed and stained with the nuclear dye, propidium iodide (red channel) were then immunolabeled with primary antibody to syntaxin 4, VAMP2, or VAMP3. The secondary antibody was FITC conjugated and appears in the green channel. A minimum of 15 blastocysts were stained for each group.
|
|
Syntaxin 4 Expression in Embryos from Heterozygote Matings Follows Mendelian Ratios
Because matings of syntaxin 4 heterozygotic mice (Stx4a+/-) never produce syntaxin 4 null (Stx4a-/-) live births, we next examined the inheritance of the syntaxin 4 alleles in blastocysts. Individual embryo genotyping using PCR was unsuccessful, and therefore genotyping was done by protein expression using immunofluorescent microscopy. In an analysis of three independent litters of Stx4a+/- matings, eight of 35 blastocysts lacked syntaxin 4 expression whereas 21 of 35 had an intermediate level, and six of 35 had a relatively high level of expression (Fig. 2
). This results in a ratio of 23:60:17, similar to the 25:50:25 expected for Mendelian inheritance of heterozygotic matings. Similar identification of Mendelian expression using immunofluorescent microscopy has been performed previously in genetic models that experience embryo lethality on E5.5 or above (16, 20, 21).

View larger version (10K):
[in this window]
[in a new window]
|
Fig. 2. Syntaxin 4 Expression in Blastocysts Obtained from Heterozygote Matings Follows Mendelian Ratios
Blastocysts from five litters were fixed and immunostained for syntaxin 4 expression. These blastocysts fell into three groups based on Stx4a expression. High-level expression (wild-type) was seen in 17%, intermediate-level expression (Stx4a+/-) was seen in 60%, and no expression (Stx4a-/-) was seen in 23%.
|
|
Although the Stx4a+/+ and Stx4a+/- embryos displayed predominantly apical plasma membrane staining, both also demonstrated a small amount of cytoplasmic staining at a blastocyst stage. Similar cytoplasmic localization of proteins normally found at the plasma membrane in differentiated somatic cells has been described previously in murine blastocysts and may be associated with developmental expression of the protein (22, 23).
Apoptosis Is Higher in Syntaxin 4 Null Embryos
To determine the time at which the Stx4a-/- embryos are lost during development, we next isolated blastocysts at E3.5 that were cultured for 48 h. Embryos lacking syntaxin 4 expression were still present at E4.5 and represented seven of 25 embryos examined. There was a complete loss of syntaxin 4 null embryos by E5.5. This was represented by nine of 31 embryos demonstrating intermediate staining of syntaxin 4 labeling, and the remaining 22 demonstrating a high level of expression. Based upon these data we isolated blastocysts at E3.5 and examined the relationship between syntaxin 4 expression and apoptosis (Fig. 3
). This was accomplished by first performing the TUNEL assay and dividing the percent TUNEL-positive nuclei per total nuclei per embryo into three groups. These includes embryos demonstrating more than 50% TUNEL-positive nuclei, 4050% TUNEL positive, and less than 40% TUNEL positive. Embryos from wild-type controls have been shown in previous reports to have less than 40% TUNEL-positive nuclei. The distribution of embryos from three different litters into these three groups also followed a Mendelian pattern. Seven of 33 or 21% fell into the highest apoptotic category (Fig. 3A
, panel C), 15 of 33 or 45% fell into the middle range (Fig. 3A
, panel B), and 11 of 33 or 33% fell into the lowest category (Fig. 3A
, panel A). Triple staining with TUNEL and syntaxin 4 antibody was also performed to confirm that the higher percentage of apoptosis was present among those embryos demonstrating no syntaxin 4 expression. From three separate litters, seven of 30 embryos revealed less than 40% TUNEL-positive nuclei and high syntaxin 4 expression; 18 of 30 had 4050% TUNEL-positive nuclei and intermediate syntaxin 4 staining; and five of 30 had more than 50% TUNEL-positive nuclei and no syntaxin 4 staining (Fig. 3B
).

View larger version (67K):
[in this window]
[in a new window]
|
Fig. 3. Increased Apoptosis in Stx4a Null Embryos
A, Blastocysts from six heterozygote crossed with heterozygote litters were fixed and subjected to TUNEL assay. These blastocysts fell into three groups based on percentage of TUNEL-positive nuclei (yellow channel)/total nuclei per embryo (red channel). Less than 40% TUNEL (wild-type) was seen in 33% (A), between 40 and 50% TUNEL (Stx4a+/-) was seen in 45% (B), and greater than 50% TUNEL (Stx4a-/-) was seen in 21% (C). B, Triple staining with TUNEL, nuclear dye, and syntaxin 4 protein antibody confirmed that higher syntaxin 4 protein expression (green channel) occurred simultaneously with decreased TUNEL-positive nuclei (pink channel)/total nuclei per embryo (blue channel). Similarly, decreased syntaxin 4 expression correlated with increased TUNEL-positive nuclei per total nuclei.
|
|
To ensure that superovulation did not affect the incidence of TUNEL staining, embryos from natural matings were examined. There was no difference in the incidence of TUNEL staining among these embryos as compared with superovulated embryos. Zero of seven wild-type embryos from natural matings displayed TUNEL more than 40% as compared with zero of 13 superovulated, wild-type embryos.
GLUT8 Translocation in Response to Insulin Does Not Occur in Syntaxin 4 Null Embryos
Because syntaxin 4 is a necessary t-SNARE for insulin-stimulated GLUT4 translocation in adipocytes (3, 14), E3.5 embryos from Stx4a+/- matings were stimulated with insulin and examined for GLUT8 translocation (Fig. 4
). In the absence of insulin, GLUT8 was exclusively localized intracellularly and was not detected on the plasma membrane (Fig. 4A
, panels B, D, and F). However, after insulin stimulation, nine of 39 or 22% had no change in GLUT8 location and 30 of 39 embryos or 77% displayed either partial or complete translocation to the apical plasma membrane (Fig. 4A
, panels A, C, and E). In parallel, embryos that did not display insulin-stimulated GLUT8 translocation were also negative for syntaxin 4 expression (Fig. 4C
, panels A and B).

View larger version (40K):
[in this window]
[in a new window]
|
Fig. 4. Embryos Lacking Syntaxin 4 Expression Do Not Undergo GLUT8 Translocation in Response to Insulin
A, Blastocysts, from heterozygote crossed with heterozygote matings, were fixed under basal conditions or exposed to insulin and fixed; 77% of embryos demonstrated either complete (A) or partial (B) translocation of GLUT8 in response to insulin as compared with GLUT8 staining under basal conditions (D and E). GLUT8 remained intracellularly in 22% (C) and was unchanged as compared with basal conditions (F). B, Wild-type blastocysts stained with antibody to GLUT3 showing apical staining (A), or with antibody to GLUT1 showing basolateral staining (B). C, Triple staining with nuclear dye (blue channel), GLUT8 antibody (green channel), and syntaxin 4 antibody (red channel) confirmed no syntaxin 4 staining in 25% of blastocysts under basal conditions (A) and no syntaxin 4 staining in the blastocysts, demonstrating no translocation of GLUT8 to the plasma membrane in response to insulin (B). Panels A and B in Fig. 4C correspond to panels C and F in Fig. 4A.
|
|
To test whether the GLUT8 staining in the wild-type embryos was located at the apical plasma membrane, immunostaining with peptide-purified antibody to GLUT3 was done (Fig. 4B
, panel A). As reported previously (17), this transporter is expressed at the apical surface in the blastocyst, and this distribution pattern is similar to that seen with GLUT8 in an insulin-stimulated state. In contrast, GLUT1 is expressed in the basolateral plasma membrane (Fig. 4B
, panel B), and this distribution is not similar to that seen with any GLUT8 immunostaining.
To confirm that the three groups with differences in GLUT8 translocation were reflective of the three different genotypes, the experiments were repeated with embryos from Stx4a+/+ x Stx4a+/+ matings. In these experiments, the known +/+ embryos responded similarly to the high syntaxin 4-expressing, presumed wild-type group. Specifically, GLUT8 translocated to the plasma membrane with insulin stimulation in all the embryos, 11 of 11, to the same degree as did the group we classified as wild type in Fig. 4
.
Insulin-Stimulated Glucose Transport Is Significantly Lower in Syntaxin 4 Null Embryos
We have reported that insulin-stimulated GLUT8 translocation accounts for the majority of insulin-stimulated glucose uptake in mouse blastocysts (5, 6). As previously observed, insulin stimulation of wild-type blastocysts obtained from Stx4a+/+ x Stx4a+/+ resulted in 0.15 mmol/kg/15 min of glucose uptake (Fig. 5
). Compared with the wild-type blastocysts, one third of the embryos from Stx4a+/- matings (group 1) demonstrated an uptake that was significantly lower than that of Stx4a+/+ blastocysts. The remaining two thirds experienced 73 ± 3% of the control uptake (group 2). This was not significantly different from Stx4a+/+ blastocysts but was significantly higher than group 1.

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 5. Insulin-Stimulated Glucose Transport Is Significantly Lower in Syntaxin 4 Null Embryos
Blastocysts from heterozygote crossed with heterozygote matings were measured for insulin-stimulated glucose uptake and compared with embryos from syntaxin 4 wild-type mice. Among those blastocysts from the heterozygote matings, group 1 contained 11 of 33 blastocysts that demonstrated significantly lower glucose uptake (30 ± 4% lower than wild-type). The remaining 22 blastocysts classified as group 2 demonstrated 73 ± 3% lower uptake, which was not significantly different than wild-type blastocysts. *, P < 0.01 vs. wild-type; **, P < 0.01 vs. group 1.
|
|
 |
DISCUSSION
|
---|
Our previous studies have shown that decreased glucose transport via any of the facilitative glucose transporters at this critical blastocyst stage results in increased apoptosis (6, 16, 20). Crossing this threshold of transport leads not only to exaggerated apoptosis but also manifests later in pregnancy as a miscarriage or malformation (6, 16). We hypothesized that the lethality in embryos lacking syntaxin 4 expression was, in fact, due to decreased GLUT8 translocation and fusion of GLUT8-containing vesicles with the plasma membrane in the embryos. This model was based upon the studies demonstrating that matings of Stx4a+/- mice never produced any Stx4a-/- offspring and that Stx4a+/- mice displayed impaired translocation of GLUT4 to the adipocyte plasma membrane, resulting in decreased insulin-stimulated glucose uptake and insulin resistance (14).
In the present manuscript, we have determined that the syntaxin 4 null mouse is embryonic lethal at approximately E4.5 to E5.5 after fertilization and that this death is due in part to exaggerated apoptosis. Increased apoptosis is detected as early as the blastocyst stage, or E3.5 in those embryos lacking syntaxin 4 expression as compared with other embryos. Importantly, in the blastocysts lacking syntaxin 4 expression, GLUT8 translocation fails to occur in response to insulin stimulation, and GLUT8 remains localized in intracellular vesicles. Using single-embryo glucose uptake assays, this study further correlates this lack of translocation with decreased insulin-stimulated glucose uptake. Recent studies have shown that decreased expression of GLUT8 using antisense oligonucleotides results in decreased insulin-stimulated glucose uptake and increased apoptosis within the blastocyst (5, 6). As with GLUT4, it is presumed that GLUT8-containing vesicles recycle constitutively from an intracellular location to the plasma membrane and that this pathway accelerates with insulin or IGF-I stimulation. Thus, a decrease in GLUT8 expression or GLUT8 translocation would drop net glucose transport rates below necessary threshold levels and as a result this would trigger an apoptotic cascade. These embryos exposed to GLUT8 antisense exhibit higher rates of resorptions and pregnancy losses when transferred back into donor mice (6). It is presumed that the fate of the syntaxin 4 null embryos is the same. However, the levels of apoptosis are higher, perhaps due to complete loss of syntaxin 4 expression with the knockout model as compared with antisense technology, and thus the blastocysts die before implantation and do not progress to a resorption state.
Recent studies have shown that cell death caused by reduced availability of glucose, as seen with growth factor withdrawal or in our models of decreased glucose transport, is initiated by mitochondrial changes that result in cytochrome c release (24, 25, 26). Overexpression of GLUT1 can prevent this onset of apoptosis (24). In addition, the regulation of outer mitochondrial membrane integrity via the voltage-dependent anion channel appears to depend on cellular metabolic changes associated with glycolysis (27). Perturbations in glucose metabolism, leading to altered pyruvate and nicotinamide adenine dinucleotide+ levels, have been shown to trigger voltage-dependent anion channel closure, thus limiting ATP/ADP exchange and leading to a stall in the electron transport chain. Although these metabolic irregularities may link decreased glucose transport to apoptosis, the mechanisms responsible for this phenomenon are still not clear.
The only v-SNAREs examined in this study, VAMP2 (or synaptobrevin) and VAMP3 (or cellubrevin), were present but expressed predominantly in the cytoplasm, not the plasma membrane where they would be expected to move following insulin stimulation. In insulin-responsive tissues such as skeletal muscle and adipocytes, VAMP2 and, to a lesser extent, VAMP3 form a complex at the plasma membrane with syntaxin 4 and synaptosome-associated protein 23 to allow fusion of the GLUT4 containing vesicles (13). Because this is a constitutive process, it is predicted that if either of these two v-SNAREs were involved in GLUT8 translocation that some plasma membrane staining would be detected. Although the level of expression may have been too low to be detected, it is also possible that some other v-SNARE is present in GLUT8-containing vesicles in the embryonic cells. More than 12 different v-SNARES have been identified, and each appears to have different specificity depending on cell type and vesicle contents (28). This specificity is thought to provide some degree of control within the cell and prevent nonspecific fusion of vesicles with intracellular compartments or plasma membrane. The existence of an embryonic v-SNARE responsible for GLUT8 translocation may also explain why this translocation step is not seen in other cell types such as primary adipocytes (29).
 |
MATERIALS AND METHODS
|
---|
Embryo Recovery and Culture
All mouse studies were approved by the Animal Studies Committee at Washington University School of Medicine. Embryos were recovered as previously described (16, 30). In brief, female mice (B6 x SJL F1, Jackson Laboratories, Bar Harbor, ME) of 46 wk of age were superovulated with an ip injection of 10 IU/animal of pregnant mare serum gonadotropin (Sigma Chemical Co., St. Louis, MO) followed later by 10 IU/animal of human chorionic gonadotropin (Sigma Chemical Co.). Mating was confirmed by identification of a vaginal plug. Animals were killed on E3.5, 96 h after human chorionic gonadotropin administration and mating. Blastocysts were obtained by flushing dissected uterine horns and ostia as described previously. The embryos were then immediately placed in potassium-rich simplex optimization media (KSOM) (Specialty Media, Phillipsburg, NJ) and cultured at 37 C in an atmosphere of 5% CO2, 5% O2, and 90% N2. The syntaxin 4 mice were genotyped and maintained as previously described (14). For the Stx4a-/- embryos, Stx4a+/- females were superovulated and mated as above with Stx4a+/- males of proven fertility. To test whether superovulation affected apoptosis, natural matings were also performed.
Localization and Expression of t-SNARE and v-SNARE Proteins in the Mouse Blastocysts Obtained from Wild-Type and Syntaxin Heterozygote Matings
Blastocysts were fixed on glass slides with 3% paraformaldehyde and permeabilized with 0.1% Tween. The embryos were then washed and incubated with a primary sheep antimouse syntaxin 4 antibody, a primary sheep antimouse VAMP2 antibody, or a primary sheep antimouse VAMP3 antibody for 1 h at room temperature (1:500). All antibodies were peptide purified, and peptide-purified sheep preimmune serum was used at the same concentration as a negative control. The embryos were then washed and incubated with a secondary antibody, goat antisheep FITC-labeled antibody, for 1 h. Nuclear staining was then performed by incubating the embryos in either propidium iodide at a concentration of 0.01 mg/ml or To-Pro-3 iodide (Molecular Probes, Inc., Eugene, OR) at a concentration of 4 µM. After extensive washing, confocal immunofluorescent microscopy (Bio-Rad MRC-600, Bio-Rad Laboratories, Hercules, CA) was then use to detect fluorescence as previously described (20). Syntaxin 4 expression was recorded as 0 for none, 1+ for intermediate expression, and 3+ for high expression. All embryos from the syntaxin 4 heterozygote matings and wild-type matings were graded using this system. Embryos were graded for syntaxin 4 staining intensity by two different blinded observers.
Apoptosis by TUNEL Assay in the Blastocysts from Matings Between Syntaxin Heterozygote Mice
Blastocysts were fixed in 3% paraformaldehyde, permeabilized with 0.1% Tween-20, and then incubated in fluorescein-labeled dUTP and terminal transferase in the dark for 1 h at 37 C to label fragmented 3'-DNA (TUNEL, Cell Death In Situ Kit, Roche Molecular Biochemicals, Indianapolis, IN) as previously described (20, 30). Counterstaining of nuclear DNA was achieved by incubating the embryos in propidium iodide (0.01 mg/ml, red channel) or To-Pro-3 (4 µM, blue channel) for 20 min. Embryos were visualized using confocal immunofluorescent microscopy (Bio-Rad MRC-600) at x63 magnification. A Z-series was performed on each blastocyst to determine the total number of nuclei and the number of apoptotic or TUNEL-positive nuclei. Apoptosis was expressed as percent TUNEL-positive nuclei per total nuclei per embryo. In the triple-staining experiments, the TUNEL assay was performed first, followed by the syntaxin 4 immunoblotting as described above.
Translocation of GLUT8 in Response to Insulin in Blastocysts from Matings Between Syntaxin Heterozygote Mice
As described previously (6), blastocysts were recovered from Stx4a +/- x Stx4a +/- matings or wild-type x wild-type matings, and moved to KSOM media at a final glucose concentration of 5.6 mM with or without 500 nM insulin (Bovine Pancreas, Sigma) for 30 min. The blastocysts were immediately fixed on glass slides with 3% paraformaldehyde and permeabilized with 0.1% Tween. The embryos were then washed and incubated with a primary rabbit antimouse antibody to GLUT8 for 1 h at room temperature. The GLUT8 rabbit antisera for this technique was peptide purified by high-efficiency immunoaffinity purification on thiopropyl Sepharose. This purified antibody was then used at a final concentration of 10 µg/ml. The embryos were washed and incubated with a secondary antibody, donkey antirabbit FITC-labeled antibody for 1 h. Nuclear staining was then performed by incubating the embryos in propidium iodide at a concentration of 0.01 mg/ml. After extensive washing, confocal immunofluorescent microscopy (Bio-Rad MRC-600) was then used to detect fluorescence. Two independent, blinded observers recorded the GLUT8 localization as predominantly intracellular or plasma membrane staining.
For triple staining of GLUT8, syntaxin 4, and nuclei, embryos were incubated with both primary rabbit antimouse GLUT8 antibody and primary sheep antimouse syntaxin 4 antibody. The concentrations used are described above. Secondary antibodies, goat antirabbit FITC-labeled antibody and goat antisheep Alexa-564 antibody, were used to detect GLUT8 and syntaxin 4 protein, respectively.
Insulin-Stimulated 2-Deoxyglucose Uptake in Blastocyst from Matings Between Syntaxin Heterozygote Mice
Blastocysts were recovered as above, and then preincubated for 30 min in KSOM media containing 0 or 170 nM insulin. As previously described (16, 30), embryos were directly placed in 200 µM 2-deoxyglucose for 15 min, washed in deoxyglucose-free, BSA-free buffer for 1 min, and then quick frozen on a glass slide. After freeze drying overnight, the embryos were extracted in microliter volumes under oil, and single embryos were assayed for deoxyglucose and 2-deoxyglucose-6-phosphate as described previously (16, 23). The final measurements are expressed as millimoles per kilogram of wet weight. These values can be converted to picomoles per embryo per 15 min by multiplying by 0.16. Experiments were performed in duplicate on 1015 individual embryos per group for each experiment.
Statistical Analysis
Differences experienced by the three different groups of embryos for TUNEL values and glucose uptake were compared by one-way ANOVA coupled with Fishers test (by using STATVIEW 4.5). All data are expressed as means ± SEM. The immunofluorescent microscopy studies, TUNEL, and transport assays were performed in triplicate. Significance was defined as P < 0.05.
 |
FOOTNOTES
|
---|
This work was supported by an American Diabetes Association Research grant, a Burroughs Wellcome Fund Career Development Award, and by grants from NIH (HD-38061 and HD40390 to K.H.M. and DK55811 to J.E.P.).
Abbreviations: E, Embryonic day; FITC, fluorescein isothiocyanate; GLUT, glucose transporter; KSOM, potassium-rich simplex optimization media; t-SNARE, target-soluble N-ethylmaleimide-sensitive factor attachment protein receptor; TUNEL, terminal dUTP nick end lableling; v-SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor.
Received for publication July 10, 2002.
Accepted for publication June 20, 2003.
 |
REFERENCES
|
---|
- Mueckler M 1994 Facilitative glucose transporters. Eur J Biochem 219:713725[Abstract]
- Hruz PW, Mueckler MM 2001 Structural analysis of the GLUT1 facilitative glucose transporter. Mol Membr Biol 18:183193[CrossRef][Medline]
- Watson RT, Pessin JE 2001 Intracellular organization of insulin signaling and GLUT4 translocation. Recent Prog Horm Res 56:175193[Abstract/Free Full Text]
- Saltiel AR, Pessin JE 2002 Insulin signaling pathways in time and space. Trends Cell Biol 12:6571[CrossRef][Medline]
- Carayannopoulos M, Chi M, Cui Y, Pingsterhaus J, Moley K 2000 GLUT8, a glucose transporter responsible for insulin-stimulated uptake in the blastocyst. Proc Natl Acad Sci USA 97:73137318[Abstract/Free Full Text]
- Pinto A, Carayannopoulos M, Hoehn A, Dowd L, Moley K 2002 GLUT8 expression and translocation are critical for murine blastocyst survival. Biol Reprod 66:17291733[Abstract/Free Full Text]
- Aghayan M, Rao LV, Smith RM, Jarett L, Charron MJ, Thorens B, Heyner S 1992 Developmental expression and cellular localization of glucose transporter molecules during mouse preimplantation development. Development 115:305312[Abstract]
- Hogan A, Heyner S, Charron MJ, Copeland NG, Gilbert DJ, Jenkins NA, Thorens B, Schultz GA 1991 Glucose transporter gene expression in early mouse embryos. Development 113:363372[Abstract]
- Joost HG, Bell GI, Best JD, Birnbaum MJ, Charron MJ, Chen YT, Doege H, James DE, Lodish HF, Moley KH, Moley JF 2002 Nomenclature of the GLUT/SLC2A family of sugar/polyol transport facilitators. Am J Physiol 282:E974E976
- Volchuk A, Wang Q, Ewart HS, Mueckler M, Rogers S, Schurmann A, Seino S, Thorens B 1996 Syntaxin 4 in 3T3-L1 adipocytes: regulation by insulin and participation in insulin-dependent glucose transport. Mol Biol Cell 7:10751082[Abstract]
- Cheatham B, Volchuk A, Kahn CR, Wang L, Rhodes CJ, Klip A 1996 Insulin-stimulated translocation of GLUT4 glucose transporters requires SNARE-complex proteins. Proc Natl Acad Sci USA 93:1516915173[Abstract/Free Full Text]
- Parlati F, McNew JA, Fukuda R, Miller R, Sollner TH, Rothman JE 2000 Topological restriction of SNARE-dependent membrane fusion. Nature 407:194198[CrossRef][Medline]
- Foster L, Kilp A 2000 Mechanism and regulation of GLUT-4 vesicle fusion in muscle and fat cells. Am J Physiol 279:C877C890
- Yang C, Coker K, Kim J, Mora S, Thurmond DC, Davis AC, Yang B, Williamson RA, Schulman GI, Pessin JE 2001 Syntaxin 4 heterozygous knockout mice develop muscle insulin resistance. J Clin Invest 107:13111318[Abstract/Free Full Text]
- Gardner DK, Leese HJ 1988 The role of glucose and pyruvate transport in regulating nutrient utilization by preimplantation mouse embryos. Development 104:423429[Abstract]
- Chi MM, Pingsterhaus J, Carayannopoulos M, Moley KH 2000 Decreased glucose transporter expression triggers BAX-dependent apoptosis in the murine blastocyst. J Biol Chem 275:4025240257[Abstract/Free Full Text]
- Pantaleon M, Harvey MB, Pascoe WS, James DE, Kaye PL 1997 Glucose transporter GLUT3: ontogeny, targeting and role in the mouse blastocyst. Proc Natl Acad Sci 94:37953800[Abstract/Free Full Text]
- Leese HJ, Barton AM 1984 Pyruvate and glucose uptake by mouse ova and preimplantation embryos. J Reprod Fertil 72:913[Abstract]
- Gardner DK, Leese HJ 1986 Non-invasive measurement of nutrient uptake by single cultured pre-implantation mouse embryos. Hum Reprod 1:2527[Abstract]
- Moley KH, Chi MM-Y, Knudson CM, Korsmeyer SJ, Mueckler MM 1998 Hyperglycemia induces apoptosis in preimplantation embryos via cell death effector pathways. Nat Med 12:14211424[CrossRef]
- Keim AL, Chi MM, Moley KH 2001 Hyperglycemia-induced apoptotic cell death in the mouse blastocyst is dependent on expression of p53. Mol Reprod Dev 60:214224[CrossRef][Medline]
- Pantaleon M, Ryan JP, Gil M, Kaye PL 2001 An unusual subcellular localization of GLUT1 and link with metabolism in oocytes and preimplantation mouse embryos. Biol Reprod 64:12471254[Abstract/Free Full Text]
- Moley KH, Chi M, Mueckler M 1998 Maternal hyperglycemia alters glucose transport and utilization in mouse preimplantation embryos. Am J Physiol 275:E38E47
- Vander Heiden MG, Plas DR, Rathmell JC, Fox CJ, Harris MH, Thompson CB 2001 Growth factors can influence cell growth and survival through effects on glucose metabolism. Mol Cell Biol 21:58995912[Abstract/Free Full Text]
- Harris MH, Thompson CB 2000 The role of the Bcl-2 family in the regulation of outer mitochondrial membrane permeability. Cell Death Differ 7:11821191[CrossRef][Medline]
- Chi M-Y, Hoehn A, Moley K 2002 Metabolic changes in the glucose-induced apoptotic blastocyst suggest alterations in mitochondrial physiology. Am J Physiol 283:E226E232
- Vander Heiden MG, Li XX, Gottleib E, Hill RB, Thompson CB, Colombini M 2001 Bcl-xL promotes the open configuration of the voltage-dependent anion channel and metabolite passage through the outer mitochondrial membrane. J Biol Chem 276:1941419419[Abstract/Free Full Text]
- Scales S, Cen Y, Yoo B, Patel S, Doung Y, Scheller R 2000 SNAREs contribute to the specificity of membrane fusion. Neuron 26:457464[Medline]
- Lisinski I, Schurmann A, Joost H-G, Cushman S, Al-Hasani H 2001 Targeting of GLUT8 and GLUT6 in rat adipocytes. Biochem J 358:517522[CrossRef][Medline]
- Chi MM, Schlein AL, Moley KH 2000 High insulin-like growth factor 1 (IGF-1) and insulin concentrations trigger apoptosis in the mouse blastocyst via down-regulation of the IGF-1 receptor. Endocrinology 141:47844792[Abstract/Free Full Text]