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Glucose Transporter 8 Expression and Translocation Are Critical for Murine Blastocyst Survival¹

^a Department of Obstetrics and Gynecology and ^b Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
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.

apoptosis, early development, embryo, insulin, insulin-like growth factor receptor

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Glucose transport and metabolism are critical for mammalian blastocyst formation and further development [1–7]. At this stage, the embryo switches from using pyruvate and lactate as its main energy substrates via oxidative phosphorylation, to using glucose via glycolysis, almost exclusively [8, 9]. As a result, the blastocyst exhibits extreme sensitivity to glucose deprivation. We have previously shown that a decrease in glucose transport, either basal or insulin-stimulated, results in enhanced apoptosis at this stage, which manifests later in pregnancy as a malformation or miscarriage [10–12]. This decrease can occur either in basal transport or insulin-stimulated transport. Such decreases in transport occur in conditions of maternal hyperglycemia and hyperinsulinemia, respectively.

In a previous study, we cloned a novel, insulin-regulated glucose transporter, glucose transporter (GLUT) 8, which is expressed at high levels in the murine blastocyst and may represent an embryonic transporter [13]. Unlike the other glucose transporters present, GLUT8 is localized intracellularly in a basal state, but upon insulin or IGF-1 treatment, this transporter is translocated to the plasma membrane. This movement corresponds to an increase in glucose uptake, and its signal occurs via the insulin-like growth factor (IGF)-1 receptor and not the insulin receptor. Transgenic and null models of the IGF-1 ligand/receptor system all have developmental manifestations [14]. Although most of these are postimplantation effects and our event is preimplantation, compensatory adaptations may occur in these genetically altered models. It is possible, due to the redundancy of the insulin/IGF-1/IGF-2 system, that the insulin receptor substitutes for the IGF-1 receptor in these genetic embryo models. In previous studies, we have shown that this phenomenon does not occur in the wild-type preimplantation period [15].

In these studies we investigate the physiological role of this novel insulin-regulated glucose transporter, which is expressed predominantly in preimplantation development. In addition, the IGF-1/insulin intracellular signaling pathways involved in translocation of GLUT8 are examined.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Embryo Recovery and Culture Conditions

Two-cell embryos were obtained from superovulated B6XSJL F1 female mice (Jackson Laboratories, Bar Harbor, ME) as previously described [16]. The embryos were cultured for 72 h in either sense or antisense oligonucleotides to GLUT8 as previously described [13]. In brief, 5 µM GLUT8 sense (5'-ATGAGTCCCGAGGACCCCCAG-3') or GLUT8 antisense (5'-GTGGGGGTCCTCGGGACTCAT-3') phosphorothioate oligonucleotide (Oligos Etc. Inc.; Wilsonville, OR) was added to human tubal fluid (HTF) media (Irvine Scientific, Irvine, CA) and the embryos were cultured to a blastocyst stage in 5% O₂, 5% CO₂, and 90% N₂ at 37°C. As a negative control, embryos were also cultured for the same time period in an antisense oligonucleotide to heat shock protein 70-2 (hsp 70-2; 5'-GCCGCGGGCAGACAT-3'). This protein had been shown previously to have no effect on preimplantation blastocyst development by null mutation studies [17].

Evaluation of Apoptosis by TUNEL Assay

The blastocysts exposed to GLUT8 sense, antisense, or hsp 70-2 antisense 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 Boehringer-Mannheim, Mannheim, Germany) as previously described [11]. Counterstaining of all nuclear DNA was achieved by incubating the embryos in propidium iodide (0.01 mg/ml, red channel) for 20 min. Embryos were visualized using confocal immunofluorescent microscopy (MRC-600; Bio-Rad, Hercules, CA) at 63x 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 the percentage of TUNEL-positive nuclei per total nuclei per embryo. TUNEL was performed on 20–25 blastocysts in each group.

Evaluation of Apoptosis by Caspase-3 Activity

The blastocysts exposed to GLUT8 sense, antisense or hsp 70-2 antisense were cultured for 1 additional hour in 180 nM PhiPhiLux (OncoImmunin, Inc., Gaithersburg, MD) to detect apoptosis as described previously [18]. This substrate reagent mix contains a fluorophore-labeled peptide that acts as a substrate for caspase-3. When cleaved by this enzyme, the peptide substrate fluoresces. The blastocysts were then fixed, permeabilized, and counterstained as described above. Embryos were visualized using confocal microscopy (Bio-Rad MRC-600) at 63x magnification. Whole embryo fluorescence was then quantitated using NIH Image (version 1.6).

Blastocyst Transfer after Exposure In Vitro to GLUT8 Antisense or GLUT8 Sense Oligonucleotides

Following the incubation of two-cell embryos in the oligonucleotides for 72 h as described above, a total of eight blastocysts were transferred into recipient pseudopregnant ICR female mice (Harlan Sprague Dawley Inc., Indianapolis, IN) as described by Hogan [19]. The mice were killed on Day 14.5, and the numbers of normal implantation versus resorption sites were recorded as previously described [20]. Implantation rate represents normal gestational sacs divided by total number of sacs including resorptions. Distinctions were not made between abnormalities in decidualization, placentation, or fetal development because most resorptions appeared to have occurred much earlier, and the contents of the sacs were necrotic. Six experiments were performed for each group, both sense-treated and antisense-treated embryos. A total of 8 foster mice and 64 embryos were transferred for the sense group, and 13 foster mice and 104 embryos were transferred for the antisense group. Only foster mice that had at least one successful implantation in either the sense or the antisense embryo groups were used in the data collected.

Insulin-Stimulated GLUT8 Translocation in Embryos Exposed to IGF-1R Oligonucleotide

Embryos were cultured from a two-cell to a blastocyst stage in 0.5 µM oligonucleotide to either 1) IGF-1 receptor sense or 2) IGF-1 receptor antisense as previously described [15]. Blastocysts from each condition were incubated in HTF media at a final glucose concentration of 5.6 mM with or without 500 nM insulin (bovine pancreas; Sigma Chemical Company, St. Louis, MO) 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 anti-mouse 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 anti-rabbit fluorescein isothiocyanate (FITC)-labeled antibody (Molecular Probes Inc., Eugene, OR) for 1 h. Nuclear staining was then performed by incubating the embryos in propidium iodide at a concentration of 0.01 mg/ml. Following extensive washing, confocal immunofluorescent microscopy (Bio-Rad MRC-600) was then used to detect fluorescence as previously described. Whole embryo fluorescence was then quantitated using NIH Image (version 1.6). In order to confirm down-regulation of the IGF-1R, insulin-stimulated deoxyglucose uptake was measured in embryos exposed to sense and antisense oligonucleotides as previously described [15]. In addition, TUNEL assay was performed on the sense and antisense oligonucleotides. Increased TUNEL staining had been shown previously to occur in embryos exposed to IGF-1R antisense oligonucleotide.

Statistical Analysis

Differences between TUNEL values experienced by sense, antisense, and control antisense-treated embryos were compared by one-way ANOVA coupled with the Fisher test (by using Statview 4.5). Differences between resorption rates and normal pregnancy rate in sense- and antisense-treated embryos were compared by an unpaired Student t-test (Statview 4.5). All data are expressed as means ± SEM. The TUNEL and caspase assays were performed in triplicate and duplicate, respectively. Six embryo-transfer studies were completed. Significance was defined as P < 0.05.

	RESULTS

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Decreasing GLUT8 Expression During Preimplantation Development Induces Increased Apoptosis in the Murine Blastocyst

Both TUNEL-positive nuclei and caspase-3 activity were increased in embryos exposed to GLUT8 antisense. Embryos cultured in GLUT8 antisense (n = 25) experienced 39% ± 7% TUNEL-positive nuclei per total nuclei per embryo, compared with 6% ± 3% in the control embryos cultured in GLUT8 sense (n = 20; P < 0.001). Embryos cultured in the control antisense had a rate of apoptosis that was not significantly different from that of sense (8% ± 4%; n = 21; Fig. 1, A and B). Embryos cultured in GLUT8 antisense (n = 6) also experienced increase caspase-3 activity as detected by PhiPhiLux and were quantitated (Fig. 2). Embryos in GLUT8 sense (n = 8) and control antisense (n = 6) had minimally detectable levels of caspase-3 activity.

View larger version (35K): [in this window] [in a new window]   FIG. 1. Antisense oligoprobe results in increased TUNEL-positive nuclei. A) Embryos cultured from a two-cell to a blastocyst stage in 5 µM GLUT8 antisense, GLUT8 sense, or a control antisense oligoprobe were examined for TUNEL staining. The red channel represents nuclear staining by propidium iodide, the green channel represents FITC-labeled 3'-fragmented DNA. The figure shows one of a Z-series representative of the results. B) Percentage of TUNEL-positive nuclei demonstrating DNA fragmentation per total embryonic nuclei in each group

View larger version (27K): [in this window] [in a new window]   FIG. 2. Antisense oligoprobe results in increased caspase-3 activity. Embryos cultured from a two-cell to a blastocyst stage in 5 µM GLUT8 antisense, GLUT8 sense, or a control antisense oligoprobe were examined for caspase-3 activity by fluorescence of a cleaved substrate. The red channel represents nuclear staining by propidium iodide; the green channel represents cleaved caspase-3 substrate and is a measure of caspase-3 activity

Decreasing GLUT8 Expression During Preimplantation Development Results in Poor Pregnancy Outcome

Transferred embryos exposed to GLUT8 antisense (n = 51) experienced higher rates of resorptions (86.5% ± 5.2%) compared with embryos exposed to GLUT8 sense (40.0% ± 8.5%; n = 18; P = 0.0002; Fig. 3). GLUT8 antisense embryos (n = 6) also demonstrated lower normal pregnancy rates (10.5% ± 4.3%) compared with the GLUT8 sense embryos (59.6% ± 8.5%; n = 27; P < 0.0001).

View larger version (11K): [in this window] [in a new window]   FIG. 3. Down-regulation of GLUT8 by antisense oligonucleotides adversely affects pregnancy outcome. Two-cell embryos cultured for 72 h in 5 µM of either sense or antisense oligonucleotides to GLUT8 were transferred into pseudopregnant recipient females and the pregnancies were evaluated on Day 14.5 of gestation

Translocation of GLUT8 Occurs via Activation of the IGF-1 Receptor Signaling Pathway

Exposure to insulin in the sense-treated embryos (n = 12) induced translocation from intracellular compartments to the plasma membrane as previously described. Blastocysts exposed to IGF-1R antisense (n = 10), however, failed to demonstrate any change in intracellular location with insulin treatment (Fig. 4). The IGF-1R antisense embryos also displayed significantly greater TUNEL staining compared with the sense embryos, as previously shown (data not shown). Similarly, insulin-stimulated glucose uptake was significantly lower in these embryos exposed to IGF-1R antisense as previously shown [15].

View larger version (27K): [in this window] [in a new window]   FIG. 4. Down-regulation of the IGF-1 receptor by antisense oligoprobes results in lack of GLUT8 translocation to the plasma membrane. Two-cell embryos were cultured for 72 h in 0.5 µM of either sense or antisense oligonucleotides to IGF-1 receptor. The blastocysts were then transferred to media with or without 500 nM insulin for 30 min, and were immediately fixed and immunostained for GLUT8 and counterstained with propidium iodide. The green channel represents the GLUT8 protein expression and the red channel reflects nuclear staining. A) Embryos exposed to IGF-1 receptor sense under basal conditions. B) Embryos exposed to IGF-1 receptor sense under insulin-stimulated conditions. C) Embryos exposed to IGF-1 receptor antisense under basal conditions. D) Embryos exposed to IGF-1 receptor antisense under insulin-stimulated conditions.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The blastocyst stage marks a new peak in cellular proliferation and growth as the first epithelial layer, the trophectoderm, is formed. These changes create new energy demands on the embryo and GLUT8 may be expressed at this stage to meet these demands. Maintenance of a high rate of glycolysis is believed to be important for providing metabolic intermediates for biosynthetic pathways [21]. It has also been suggested that increasing amounts of glucose are converted to lactate at the blastocyst stage in species such as humans and rodents, in which the embryo resides in the uterus for a relatively short time before implantation [22]. The reason for this anaerobic type of metabolism is a lack of adequate vascularization and oxygenation at the implantation sites or decidual zones. The only source of ATP for the embryo at this point would be conversion of glucose to lactate via glycolysis. Thus, the embryo, in preparation for the lack of oxygen, may be maximizing glucose uptake at this stage by expressing GLUT8 along with the other facilitative transporters in order to convert glucose to lactic acid by glycolysis.

The mouse blastocyst appears to have adapted to this demand for glucose by increasing the number of transporters. A critical factor, however, may not be the absolute amounts of the transporters, but their locations. Each is specialized to different compartments and thus have their own individual functions. GLUT3 is expressed at the apical plasma membrane of trophectoderm [3, 10], GLUT1 is expressed on all surfaces of both trophectoderm and inner cell mass cells [10, 23, 24], and GLUT2 is present in the basolateral plasma membrane of trophectoderm only, whereas GLUT8 is retained intracellularly and translocates to the plasma membrane [13]. Expression of GLUT8 appears concurrently with expression of the IGF-1 receptor in the mammalian blastocyst and coincides with movement of the embryo from the oviduct to the uterus, which contains 10-fold higher concentrations of IGF-1 and insulin [25]. This specialized transporter appears to translocate in response to increasing IGF-1 levels and functions only at this peri-implantation time of maximal glucose need. For these physiologic reasons, evidence exists to support the functional need for GLUT8.

This study extends our investigation of GLUT8 and demonstrates that GLUT8 expression affects blastocyst survival. A decrease in GLUT8 expression by antisense oligonucleotides leads to an increase in apoptosis at the blastocyst stage as detected by TUNEL assay and caspase-3 activity. These antisense embryos, when transferred back to foster mice, experience a higher rate of pregnancy failure and resorption compared with sense-treated embryos. In addition, inhibition of the insulin/IGF-1 signaling pathway, which we have previously shown, results in increased blastocyst apoptosis and embryo resorption, and prevents translocation of GLUT8 to the plasma membrane. We propose that this overall decrease in glucose transport, in this case due to retention of GLUT8 intracellularly, is responsible for the apoptosis experienced by these embryos. This hypothesis is supported by previous studies demonstrating that a threshold of glucose transport is required for blastocyst survival [10, 12]. A similar reliance on glucose transport to avoid triggering the apoptotic cascade has been demonstrated in other cell types [26–29]. GLUT8 may have evolved as an additional antiapoptotic mechanism in the blastocyst to maximize glucose utilization at this critical turning point in development when the embryo switches abruptly from using pyruvate as its main energy substrate to using glucose [30].

In addition, this work proposes that the mammalian blastocyst represents the earliest insulin-sensitive tissue. In glucose uptake studies with mouse blastocysts, it has been shown that insulin and IGF-1 stimulate glucose uptake via binding to the IGF-1 receptor, not the insulin receptor [31]. We postulate that signaling via the IGF-1 receptor pathway within the trophectoderm cells may trigger translocation of GLUT8 to the apical plasma membranes and facilitate glucose transport, possibly using the same signaling pathways as GLUT4 translocation (Fig. 5). Insulin-stimulated translocation of GLUT4 in muscle and adipocytes occurs via binding to and autophosphorylation of the insulin receptor. This event then triggers the phosphorylation of insulin receptor substrate-1 (IRS-1) and other IRS proteins, leading to docking and binding of the p85 subunit of p110-type phosphatidyl-inositol 3-kinase (PI 3-kinase.) Downstream activation of Akt/protein kinase B is then believed to trigger translocation of GLUT4 from specialized intracellular compartments known as GLUT4 storage vesicles (GSVs) to the plasma membrane. In the mouse preimplantation embryo, expression of IRS-1 has recently been reported at both the mRNA and protein levels, and these levels increase significantly at the blastocyst stage of development [32]. Although this expression has not been localized to a particular cell type, this finding suggests that activity of this IGF-1/insulin–IGF-1R–IRS-1–GLUT8 pathway may be functional within the blastocyst, possibly within the trophectoderm and inner cell mass. Constitutive recycling of this transporter to the plasma membrane must occur because decreased expression by antisense oligonucleotides is enough to decrease basal transport and induced apoptosis. Further investigations are necessary to support these conclusions.

View larger version (65K): [in this window] [in a new window]   FIG. 5. Schematic representation of the insulin-stimulated pathways proposed for the blastocyst. GLUT8 resides in an intracellular compartment, which upon activation of the IGF-1 receptor by insulin or IGF-1, triggers a signaling pathway that results in translocation of GLUT8 to the plasma membrane. Any disruption of the upstream components of this pathway results in failure of GLUT8 to move to the plasma membrane, no increase in glucose transport, and thus increased apoptosis. IRS-1 and IRS-2, Insulin-receptor substrate proteins; p85/p110, subunits for PI 3-kinase or phosphatidylinositol 3 kinase; Akt/PKB, protein kinase B

Finally, this work suggests that establishment of this distinct glucose transport system is critical to this stage of development and that its dysregulation may be responsible for increased apoptosis experienced under conditions of hyperinsulinemia, as seen in polycystic ovary syndrome. Women with polycystic ovary syndrome experience higher rates of spontaneous miscarriages [33–35]. We have shown previously that the hyperinsulinemia and high IGF-1 levels associated with this endocrine disorder lead to increased apoptosis at a blastocyst stage and decreased insulin-stimulated glucose transport via the IGF-1 receptor [15]. These embryos, when transferred into pseudopregnant mice, have a much lower successful pregnancy rate [20]. This study suggests that disorders that affect insulin-signaling in the blastocyst, such as high maternal insulin and IGF-1 levels, may adversely affect GLUT8 translocation and function, and that this decrease in glucose transport may be directly responsible for blastocyst apoptosis and the increased pregnancy loss rates experienced by these patients. GLUT8 dysregulation and resulting apoptosis may account for the increased rate of pregnancy losses experienced by these women.

	FOOTNOTES

 
First decision: 10 December 2001.

¹ Supported by an ADA Research grant, a Burrough Wellcome Fund Career Development Award, and by HD38061-01 from the National Institutes of Health to K.H.M.

² Correspondence: Kelle H. Moley, 4566 Scott Avenue, St. Louis, MO 63110. FAX: 314 747 4150; moleyk{at}msnotes.wustl.edu

Accepted: January 4, 2002.

Received: November 27, 2001.

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TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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