Maternal hyperglycemia alters glucose transport and
utilization in mouse preimplantation embryos
Kelle H.
Moley1,
Maggie M.-Y.
Chi1, and
Mike M.
Mueckler2
Departments of 1 Obstetrics and
Gynecology and of 2 Cell Biology
and Physiology, Washington University School of Medicine, St.
Louis, Missouri 63110
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ABSTRACT |
Glucose
utilization was studied in preimplantation embryos from normal and
diabetic mice. With use of ultramicrofluorometric enzyme assays,
intraembryonic free glucose in single embryos recovered from
control and streptozotocin-induced hyperglycemic mice was measured at
24, 48, 72, and 96 h after mating. Free glucose concentrations dropped
significantly in diabetics at 48 and 96 h, corresponding to the
two-cell and blastocyst stages (48 h: diabetic 0.23 ± 0.09 vs.
control 2.30 ± 0.43 mmol/kg wet wt;
P < 0.001; 96 h: diabetic 0.31 ± 0.29 vs. control 5.12 ± 0.17 mmol/kg wet wt;
P < 0.001). Hexokinase activity was
not significantly different in the same groups. Transport was
then compared using nonradioactive 2-deoxyglucose uptake and
microfluorometric enzyme assays. The 2-deoxyglucose uptake was
significantly lower at both 48 and 96 h in embryos from
diabetic vs. control mice (48 h diabetic, 0.037 ± 0.003; control,
0.091 ± 0.021 mmol · kg wet
wt
1 · 10 min
1,
P < 0.05; 96 h diabetic, 0.249 ± 0.008; control, 0.389 ± 0.007 mmol · kg wet
wt
1 · 10 min
1,
P < 0.02). When competitive
quantitative reverse transcription-polymerase chain reaction was used,
there was 44 and 68% reduction in the GLUT-1 mRNA at 48 h
(P < 0.001) and 96 h
(P < 0.05), respectively, in
diabetic vs. control mice. GLUT-2 and GLUT-3 mRNA values were decreased
63 and 77%, respectively (P < 0.01, P < 0.01) at 96 h. Quantitative
immunofluorescence microscopy demonstrated 49 ± 6 and 66 ± 4% less
GLUT-1 protein at 48 and 96 h and 90 ± 5 and 84 ± 6%
less GLUT-2 and -3 protein, respectively, at 96 h in diabetic embryos.
These findings suggest that, in response to a maternal diabetic state,
preimplantation mouse embryos experience a decrease in glucose
utilization directly related to a decrease in glucose transport at both
the mRNA and protein levels.
glucose toxicity; cleavage-stage embryos; glucose transport
downregulation; maternal diabetes
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INTRODUCTION |
WOMEN WITH POORLY CONTROLLED insulin-dependent diabetes
mellitus (IDDM) have a much higher incidence of early pregnancy
complications (17, 30, 42). These include spontaneous miscarriages,
early growth delay, and congenital malformations. The incidence of
these complications decreases somewhat with improved maternal glycemic control during the period of fetal organogenesis. This reduced incidence, however, still remains three to four times higher than control. Because of this persistent elevation, it is possible that the
metabolic insult leading to these complications may be occurring
earlier in development, before organogenesis, specifically during the
preimplantation period.
Maternal hyperglycemia adversely affects preimplantation progression
from a one-cell to a blastocyst stage in a streptozotocin-induced or a
nonobese diabetic (NOD) mouse model (11, 32, 33). In the NOD model at
96 h after superovulation and mating, only 20% of the recovered
embryos reach a blastocyst stage compared with 90% among the
nondiabetic mice. This developmental delay is reversible by treating
the mothers with insulin before superovulation and mating and during
the first 96 h of gestation. These findings suggested that some
metabolite, elevated during periods of poor glycemic control, is
responsible for the developmental retardation. This early
preimplantation delay may be manifested later in gestation as a fetal
loss or early growth delay. Alternatively, this early perturbation in
development may predispose the fetus to a congenital malformation.
The purpose of this work, therefore, was to compare glucose utilization
as defined by the combination of glucose uptake and subsequent
metabolism in preimplantation embryos from control and
streptozotocin-induced hyperglycemic mice. We hypothesized that
maternal hyperglycemia or other metabolic factors may alter the
preimplantation embryo's ability to utilize glucose and that this may
lead to impaired development, akin to fuel-mediated teratogenesis (14).
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MATERIALS AND METHODS |
Induction of hyperglycemia and recovery of embryos.
The study was conducted using B6 × SJL F1 female mice, 4-6
wk of age (Jackson Laboratories, Bar Harbor, ME). Mice were given free
access to food and water and were maintained on a 12:12-h light-dark
cycle. Hyperglycemia was induced by a single intraperitoneal injection
of streptozotocin (Sigma Chemical, St. Louis, MO) dissolved in sodium
acetate, pH 4.4, at a dose of 190 mg/kg. Blood glucose levels were
checked with tail blood by use of a Hemocue B glucose analyzer
(Angelholm, Sweden)
4 days after the injection. Blood glucose levels
of >250 mg/dl were considered hyperglycemic. In all mice,
superovulation was achieved with an intraperitoneal injection of 10 IU/animal of pregnant mare serum gonadotropin (Sigma Chemical),
followed 48 h later by 5 IU/animal of human chorionic gonadotropin
(hCG; Sigma Chemical). Female mice were mated with males of proven
fertility overnight after hCG injection. Mating was confirmed by
identification of a vaginal plug.
Animals were killed by cervical dislocation at either 24, 48, 72, or 96 h after hCG administration and mating. The uterine horns with ostia
intact were dissected free and placed in M2 media that had been
equilibrated overnight at 37°C in an atmosphere of 5%
CO2. Preimplantation embryos were
flushed immediately from the horns with the dissecting microscope by
introducing a 30-gauge needle (Becton-Dickinson, Rutherford, NJ) at the
tubal ostia and flushing out the embryos into the M2 media.
General analytic procedures.
Embryos were recovered from control vs. diabetic mice at 24, 48, 72, or
96 h after hCG and immediately freeze-dried by a procedure described
elsewhere (31). Each embryo was washed twice in a simple salt solution
containing no glucose and then was transferred with 0.5-1 µl of
the same salt solution onto a glass slide with a braking pipette. The
embryo was then quick-frozen immediately by dipping the slide into
Freon-12
(CCl2F2)
brought to its freezing point (
170°C) with liquid
N2. The specimens were
freeze-dried on the slide at
35°C in a glass vacuum tube at
a vapor pressure of
0.01 mmHg. The slides were then stored at
20°C under reduced pressure. The general microanalytic
procedures have been described for the analysis of single mouse ova and
embryos (31, 38). All enzymes and reagents were obtained from Sigma
Chemical unless otherwise noted. First, the freeze-dried embryo is
transferred with a specially shaped hair point into a microliter
droplet of the extraction reagent. For glucose, 2-deoxyglucose, and
glucose 6-phosphate assays, the embryos were extracted for 20 min at
room temperature in 0.5 µl of a weak acid (0.02 N HCl). For
hexokinase activity assays, the embryos were extracted for 120 min at
room temperature in 1 µl of a phosphate buffer consisting of 20 mM sodium phosphate, pH 7.4, 0.02% BSA, 0.5 mM EDTA, 5 mM
mercaptoethanol, 25% glycerol, and 0.5% Triton X-100. These droplets
are placed in wells 5 mm deep by 2 mm wide drilled in a piece of Teflon
and covered with a layer of oil. For the metabolite assays, the extract is then heated to 80°C for 20 min. Enzymes and preformed reduced pyridine nucleotides, which might interfere with later measurements, are destroyed by heating in acid. The extract is returned to pH 8.1, 28 mM Tris HCl, by adding 0.2 µl of 0.1 M Tris base before proceeding to
the assay. The final volume of this extraction aliquot is ~0.7 µl.
At this point the extraction aliquots are either assayed immediately or
stored at
70°C in the oil well in a vacuum tube placed under
reduced pressure to about one-fourth atmosphere. For the microanalytic
assays (see Fig. 1) in the first step, a 0.1- to 0.2-µl aliquot is
removed from the acid extraction and used in the specific reaction
sequence, ending in oxidation or reduction of a pyridine nucleotide.
The excess pyridine nucleotide from the first step is destroyed with
acid if it is in the reduced form or with alkali if it is in the
oxidized form. The second step is the enzymatic cycling or
amplification step. The principle of these reactions is illustrated in
Fig. 1. NADPH is alternatively oxidized and
reduced. In each oxidation/reduction cycle, 1 mol each of
6-phosphogluconate and glutamate is produced. Cycling rates up to
200,000 are achieved depending on the temperature and concentration of
the two enzymes. After the desired multiple of amplification, the
enzymes are inactivated with heat, and in the third step, the
6-phosphogluconate is measured by the fluorescence of the NADPH
generated in the conversion of 6-phosphogluconate to ribulose
5-phosphate via 6-phosphogluconate dehydrogenase (EC 1.1.1.43).
Fluorescence is measured directly in a 1-ml volume in 10 × 75-mm
fluorometer tubes by use of a Farrand fluorometer. Calculations are
based on internal standards and are therefore independent of variation
in enzyme activities, temperature, or incubation time. Exact
proportionality between readings and NADP concentration is usually
achieved, because the nucleotide concentrations are kept far below the
Michaelis constants for the enzymes. The general methodology and each
of the specific assays for measuring glucose, glucose 6-phosphate, and
hexokinase used in these methods have been described (38).

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Fig. 1.
General microanalytic procedures. By use of the example of glucose in
step I, the metabolite to be measured
is linked to a reaction, generating an oxidized or reduced pyridine
nucleotide. In step II, the pyridine
nucleotide is amplified by adding a reaction mixture containing
-ketoglutarate, ammonia chloride, and glucose 6-phosphate
(G-6-P), with enzymes glutamate
dehydrogenase (GDH) and glucose-6-phosphate dehydrogenase (G-6-PDH). By
variation of the concentration of these enzymes and the temperature,
reactions were cycled to obtain amplification of as much as 20,000- to
250,000-fold (see MATERIALS AND
METHODS for details). Finally, in step
III, one of the cycling reaction products,
6-phosphogluconate (6-P-gluconate), is measured by a simple
fluorometric assay using 6-phosphogluconate dehydrogenase (6-PGDH).
With the use of internal standards, the original concentration of the
metabolite can be calculated. HK, hexokinase;
ribulose-5-P, ribulose 5-phosphate.
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All metabolite measurements are expressed as millimoles per kilogram
wet weight, with the value of 160 ng or 160 pl per embryos used in the
calculation. These values are the means reported by Lewis and Wright
(28) for mouse preimplantation one-cell, two-cell, and eight-cell
embryos and are similar to those reported by Barbehenn et al. (3) for
two-cell, eight-cell, morula, and blastocyst preimplantation mouse
embryos. Moreover, dry weight has been shown not to change
significantly over this period of development (2, 6). Thus expressing
the data as a factor of wet or dry weight should not change the
results. Because of these findings, the values of 160 pl and 160 ng
have been used as wet weights in the calculations. To express the data
presented here as picomole per embryo, the values can be multiplied by
0.16.
2-Deoxyglucose uptake assays.
Embryos at different embryonic stages were incubated at 25°C in 200 µM 2-deoxyglucose (DG) for several different time points ranging from
10 s to 60 min. After the incubation times, the embryos were washed in
a DG-free, BSA-free buffer for 1 min and then quick-frozen on a glass
slide as described in General analytic
procedures. The embryos were then freeze-dried
overnight and extracted as described above. To assay the embryonic DG,
the following enzymatic steps were performed (Fig.
2). These assays have been described for
other systems and embryos elsewhere (9, 10). First, in the
oil well apparatus, a 0.1-µl aliquot of the extraction sample was
added to a 0.1-µl aliquot of a 2× 6-phosphate removal reagent [60 mM Tris acetate, 0.04% BSA, 6 mM
MgCl2, 100 µM
NADP+, and 100 µg/ml of
glucose6-phosphate dehydrogenase (G-6-PDH)], which converted
all the 6-phosphate compounds [2-deoxyglucose-6-phosphate (DG-6-P) and glucose 6-phosphate
(G-6-P)] to
6-phosphogluconates via an excess of the enzyme glucose-6-phosphate
(Leuconostoc mesenteroides) dehydrogenase. This reaction occurred at room temperature over 20 min.
After completion of the first reaction, 0.05 µl of 0.2 N HCl was
added and the reaction mix was heated to 80°C for 20 min to destroy
the formed NADPH to avoid interference with subsequent steps. NaOH (0.2 N, 0.05 µl) was then added to neutralize the solution. In the third
step, 0.1 µl of a 4× removal reagent (50 mM Tris acetate,
0.04% BSA, 200 mM potassium acetate, 1.2 mM ATP, 4 mM
phosphoenolpyruvate, 40 µg/ml
phosphoglucosisomerase, 65 µg/ml phosphofructokinase, 20 µg/ml
pyruvate kinase, and 40 µg/ml hexokinase) was added to the reaction,
and the reaction was allowed to occur over 20 min at room temperature.
In this two-step reaction, hexokinase is added to convert the remaining
free glucose and free DG to the 6-phosphate compounds.
Phosphoglucosisomerase then selectively converts the formed
G-6-P to fructose 6-phosphate but does
not convert DG-6-P. This phosphoglucosisomerase reaction is then driven
to completion by adding phosphofructokinase to convert the fructose
6-phosphate to fructobisphosphate. ATP, pyruvate kinase, and
phosphoenolpyruvate are added to drive
both the phosphofructokinase and hexokinase reactions to completion by
replenishing ATP levels. After 20 min at room temperature, the reaction
is heated to 80°C for 20 min to destroy the enzymes and prevent the
back reactions. In the next step, 0.1 µl of a 5×
DG-6-P reagent (50 mM Tris acetate, 0.04% BSA, 10 mM MgCl2, 20 µm
NADP+, and 250 µg/ml G-6-PDH) is
added to convert the remaining DG-6-P from the previous step to deoxyglucose-6-phosphogluconate, generating an equimolar amount of NADPH. This reaction is performed at room temperature for 40 min. In the final step, 0.1 µl of 0.3 N NaOH is
added, and the reaction is heated to 80°C for 20 min to destroy the
enzymes. The NADPH generated in the final enzymatic reaction is then
cycled overnight for 150,000 cycles as has been described. As
previously reported, mouse blastocysts accumulate DG above equilibrium
values (9). This phenomenon, which is not seen at any other
preimplantation developmental stage, is thought to be due to the
presence of a putative active hexose transporter.

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Fig. 2.
2-Deoxyglucose (DG) assay for measuring glucose uptake in embryos (see
MATERIALS AND METHODS for details and
definitions). Step 1: 6-phosphate
removal step. Step 2: glucose removal
step/deoxyglucose conversion to deoxyglucose-phosphate step.
Step 3: desired substrate reaction
generating NADPH. Step 4:
amplification step.
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Competitive reverse transcription-polymerase chain reaction
techniques.
Groups of 100-150 embryos were collected, pooled, and homogenized
by centrifugation over a QIAshredder (Qiagen, Chatsworth, CA). Total
RNA was extracted from the homogenate by denaturing with a
-mercaptoethanol buffer, adjusting the sample to the appropriate binding conditions with 70% ethanol, and then applying the samples to a silica gel column (RNeasy total RNA kit, Qiagen). Successive washes and centrifugation were then used to eliminate contaminants, and the isolated RNA was eluted in 30 µl of diethyl
pyrocarbonate-treated water. An aliquot of this embryonic RNA (11.2 µl) was then added to 500 ng of random hexamers (GIBCO, Gaithersburg,
MD), heated to 65°C for 10 min, and placed on ice for 5 min. A
master mix was then added to produce a 20-µl reaction with the
following final concentrations: 50 mM Tris · HCl, pH
8.3, 50 mM KCl, 1 mM dNTP, 10 mM dithiothreitol (DTT), 1 U/ml RNAsin
(Promega), and 10 U/ml M-MLV reverse transcriptase (Superscript,
GIBCO). The reaction was incubated for 50 min at 42°C and
terminated by heating at 70°C for 10 min.
For the competitive quantitative polymerase chain reaction techniques,
an internal standard for each transporter was generated from the mouse
cDNAs for GLUT-1, GLUT-2, and GLUT-3. By use of site-directed
mutagenesis, 35-bp deletions were made in each of the cDNAs: GLUT-1
base pairs 1,440-1,475; GLUT-2 base pairs 1,402-1,432, GLUT-3
base pairs 1,521-1,541. Polymerase chain reactions (PCR) were then
set up by adding an unknown amount of embryonic cDNA, a known amount of
internal standard, and intron spanning primers that included the 35-bp
deletion (Table 1). All PCR techniques were
performed with a Perkin-Elmer Cetus 480 DNA thermal cycler. Cycling
parameters included denaturing for 1 min at 94°C, annealing for 1 min at 62°C, and elongating for 1.5 min at 72°C for a total of
40 cycles. The final PCR buffer included 20 mM Tris, pH 8.3, 50 mM KCl,
1.75 mM MgCl, and 0.001% gelatin. Primers were added at a
concentration of 0.6 µM and dNTP at 0.2 mM. In these competitive reactions, two bands would appear when run out on a 4% Nu-Sieve (FMC
products, Rockland, ME) agarose gel. The upper band is the endogenous
embryo cDNA, and the lower band corresponds to the internal standard or
smaller fragment. By variation of the amount of known internal standard
to the same amount of unknown embryonic cDNA, a point of equivalency
was reached in which the intensity of the bands by ethidium bromide
staining became equal, and thus the concentration in the internal
standard equaled that in the embryo extract (13, 21). For each time
point, three different groups of embryos were assayed for mRNA. For
each group, a standard curve was constructed in which the amount of the
internal standard that had been added to each PCR reaction tube was
plotted against the ratio of the internal standard DNA to endogenous
target cDNA template-generated PCR products. The reproducibility of the
measurements was checked by amplifying the same sample of cDNA on three
separate occasions. The coefficient of variation ranged from 10 to
13.7%.
Immunofluorescent labeling to quantitate protein expression.
Immunofluorescence staining techniques have been described in embryo
preparations previously (19). All labeling was performed in
microdroplets. The embryos at different stages were fixed in 3%
neutral buffered formaldehyde for 30 min and then permeabilized with
0.1% Tween 20 for 10 min. The embryos were then blocked by incubating
for 60 min in 20% donkey serum in PBS containing 2% BSA.
Embryos were then washed three times for 10 min each in PBS-BSA and
incubated in the affinity-purified primary antibody (polyclonal mouse GLUT-1, GLUT-2, or GLUT-3) at dilutions ranging from 6 to 15 µg/ml for 30 min. The GLUT-2 antibody was a generous gift from Dr.
Bernard Thorens, University of Lausanne (44). For negative controls,
embryos were incubated in preimmune serum at a dilution of
1:200-1:500, or, as for GLUT-3, the original peptide was used to
generate the antibody at a concentration of 20 µg/ml. The embryos were then washed three times for 10 min each in PBS-BSA and incubated with the secondary antibody, FITC-labeled goat anti-rabbit IgG (Chemicon, Temecula, CA) at a concentration of 1:80, and
rhodamine-conjugated phalloidin (Molecular Probes, Eugene, OR), to
stain cytoskeletal structures, at a concentration of 1:80. Finally, the
embryos were washed three times for 10 min each in PBS-BSA and mounted
in drops of Vectoshield (Vecto Laboratories, Burlingame, CA) under a
supported coverslip. Fluorescence was detected with a Bio-Rad MRC-600
laser-scanning confocal microscope. Confocal images were taken at
×63 magnification. Total fluorescence per embryo was expressed as
a number per area with NIH Image (version 1.60). Similar fluorescence
ratios were derived for preimmune serum images and subtracted from the
GLUT images to generate a total fluorescence value. The average of three of these mean values was then compared between control and diabetic embryos.
Statistical analysis.
Unpaired t-tests were used to compare
diabetic and control groups. The Statview 4.0 (Abacus Concepts,
Berkeley, CA) statistical package was used for all analyses.
Significance was defined as P < 0.05. Error bars represent ± SE.
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RESULTS |
Intracellular free glucose and G-6-P.
Because previous studies had found negligible hexokinase activity (2,
40) and no glycolytic activity until the blastocyst stage in mouse
preimplantation embryos, intraembryonic free glucose levels were
measured at different embryonic stages. These experiments were done to
determine whether, under hyperglycemic conditions, free glucose was
increasing in these embryos or, alternatively, whether the transported
glucose was being metabolized by some other metabolic pathway that
could then be investigated. Free glucose and
G-6-P were measured using the
microanalytic techniques described in MATERIALS AND
METHODS, as seen in Fig. 3,
and expressed as millimoles per kilogram wet weight. These results
represent three different sets of embryos with 5-7 embryos for
each time point in each experiment. Free glucose levels among the
embryos from diabetic mice were consistently higher at 24 and 72 h,
corresponding to the one-cell and morula stages (24 h: diabetic, 2.59 ± 0.25 vs. control, 1.6 ± 0.35 mmol/kg wet wt;
P < 0.05; 72 h: diabetic, 3.59 ± 0.1 vs. control, 1.32 ± 0.27 mmol/kg wet wt;
P < 0.01). Conversely, free glucose
levels consistently dropped to barely detectable levels at 48 and 96 h,
corresponding to two-cell and blastocyst stages (48 h: diabetic, 0.23 ± 0.09 vs. control, 2.30 ± 0.43 mmol/kg wet wt;
P < 0.001; 96 h: diabetic, 0.31 ± 0.29 vs. control, 5.12 ± 0.17 mmol/kg wet wt;
P < 0.001).
G-6-P levels were measured at the same
intervals and were found to be barely detectable and not significantly
different between embryos from control vs. diabetic mice (data not
shown). This significant drop in free glucose at 48 and 96 h could
represent either a decrease in glucose transport or an increase in
hexokinase activity in response to the hyperglycemic
conditions.

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Fig. 3.
Intraembryonic free glucose concentration in embryos from diabetic vs.
control mice. Free glucose levels are expressed as mmol/kg wet weight
in embryos recovered at different time points after human chorionic
gonadotropin (hCG) and mating from control mice ( ) vs.
streptozotocin-induced hyperglycemic mice ( ). Values are mean ± SE
data from ~15-20 individual embryos from each time point of
embryo recovery. Significance at 48 and 96 h,
* P < 0.001; at 72 h,
+ P < 0.01; at 24 h, # P < 0.05.
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Hexokinase activity.
As seen in Fig. 4, hexokinase activity was
not significantly different at any stage in embryos from control vs.
diabetic mice. The overall pattern of activity, low levels at the early
cleavage stages with an exponential increase at the morula and
blastocyst stages, has been reported elsewhere in the literature and is
thought to parallel the increase in glycolytic capacity experienced by these embryos at the blastocyst stage (2, 40).

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Fig. 4.
Intraembryonic hexokinase activity in embryos from diabetic vs. control
mice. Hexokinase activity expressed as
pmol · embryo 1 · min 1
in embryos recovered at different time points after hCG and mating from
control mice ( ) vs. streptozotocin-induced hyperglycemic mice ( ).
Results are not significant at any time point.
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Glucose transport.
To determine whether the decrease in glucose utilization was
attributable to a decrease in uptake, glucose transport into single
embryos at different developmental stages from diabetic vs. control
mice was measured. 2-DG uptake experiments were conducted in three
separate experiments for each of the different embryonic stages.
Approximately 7-10 single embryos were assayed at each time point
from each stage. Ten minutes was chosen as the representative time
point because the uptake curve was still linear at this time. There was
a significant difference in the DG uptake between the control and
diabetic embryos at 48 and 96 h, corresponding temporally to the
decrease in intraembryonic free glucose levels (48 h: diabetic, 0.037 ± 0.003 vs. control, 0.091 ± 0.021 mmol · kg wet
wt
1 · 10 min
1;
P < 0.05; 96 h: diabetic, 0.249 ± 0.008 vs. control 0.389 ± 0.007 mmol · kg wet
wt
1 · 10 min
1;
P < 0.02; Fig.
5). Because hexokinase activity was
unchanged at 48 and 96 h, whereas transport dropped significantly, the
change in free glucose levels and thus glucose utilization at these
time points can be attributed to the decrease in transport activity.

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Fig. 5.
DG uptake in embryos from diabetic vs. control mice. Intraembryonic
uptake of DG per 10 min in embryos from control mice (open bars) and
diabetic mice (closed bars). Significance at 48 h,
* P < 0.05; at 96 h,
# P < 0.02.
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To explain the decrease in glucose transport, the following hypothesis
is presented. Heyner and co-workers [Aghayan et al. (1) and Hogan
et al. (19)] previously showed that GLUT-1 is expressed at the
protein and mRNA levels throughout the preimplantation period and that
GLUT-2 is first seen at the early morula stage. As recently shown by
others (37), GLUT-3, like GLUT-2, is first expressed at the early
morula stage, corresponding to ~68 h after hCG. We hypothesize that,
in these hyperglycemic mice, the oocyte is ovulated and fertilized in
the fallopian tube and exists for the first 24 h in a relatively
hyperglycemic milieu. In mouse oviduct fluid, the concentration of
glucose parallels the serum glucose concentration, although it is lower
in absolute value (15), and thus, in a hyperglycemic mouse, this tubal
environment should likewise reflect the hyperglycemia. Because of the
expression of GLUT-1, free glucose is transported into the embryo and
reaches high intracellular levels, as seen here. We then speculate that GLUT-1 is downregulated in these embryos over the next 24 h in response
to the high glucose. As a result, intraembryonic glucose levels fall.
Expression of GLUT-2 and GLUT-3 is first evident at 68 h, and, by 72 h,
free intraembryonic glucose is elevated again. In response to the high
glucose load, the blastocyst, either by the same or different
mechanisms as the two-cell embryo, downregulates GLUT-2 and GLUT-3,
leading to low levels of intracellular glucose. To test this
hypothesis, the expression of GLUT-1, GLUT-2, and GLUT-3 was measured
at both the protein and mRNA levels in embryos from diabetic and
control mice at different embryonic stages.
Competitive reverse transcription-PCR.
The levels of mRNAs for GLUT-1, GLUT-2, and GLUT-3 were determined by
quantitative PCR using three different groups of embryos for each time
point. The mean values ± SE were compared among the three groups of
embryos. The quantitative PCR data indicate that the levels of GLUT-1
mRNA in control embryos were 1.8-fold greater than those of diabetic
embryos recovered at 48 h, or a 44% reduction in the GLUT-1 mRNA (48 h: diabetic, 0.105 ± 0.006 vs. control, 0.189 ± 0.012 fg/embryo;
P < 0.001; Fig.
6). At 96 h, control blastocysts expressed
3.2-fold more GLUT-1 mRNA, or a 68% reduction in the diabetic embryos
(96 h: diabetic, 0.125 ± 0.021 vs. control, 0.394 ± 0.033 fg/embryo; P < 0.005). Expression of
GLUT-2 and GLUT-3 mRNA was not detected until ~68 h after hCG. The
levels of GLUT-2 and GLUT-3 mRNA were not significantly different at 72 h after hCG; however, at 96 h, control blastocysts expressed 2.7- and
4.3-fold more GLUT-2 and GLUT-3 mRNA, respectively, than blastocysts
recovered from diabetic mice, corresponding to a 63 and 77% reduction
in mRNA, respectively (GLUT-2 diabetic, 0.091 ± 0.009 vs. control,
0.243 ± 0.01 fg/embryo; GLUT-3 diabetic, 0.05 ± 0.009 vs. control,
0.213 ± 0.031 fg/embryo; P < 0.001, GLUT-2 and GLUT-3; Fig. 7). These
data support the hypothesis that the facilitative glucose transporters
are downregulated at the mRNA level in the preimplantation embryo from
diabetic hyperglycemic mice.

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Fig. 6.
GLUT-1 mRNA levels in embryos from control vs. diabetic mice. Embryonic
amount of GLUT-1 mRNA in fg/embryo in embryos recovered at different
time points from control (open bars) vs. hyperglycemic (closed bars)
mice. Significance at 48 h, * P < 0.001; at 96 h,
+ P < 0.005.
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Fig. 7.
GLUT-2 (A) and GLUT-3
(B) mRNA levels in embryos from
control vs. diabetic mice. Embryonic amount of GLUT-2 or GLUT-3 mRNA in
fg/embryo in embryos recovered at different time points from control
(open bars) vs. hyperglycemic (closed bars) mice. Significance: 96 h,
* P < 0.001.
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Immunofluorescence microscopy.
To determine whether this downregulation was also seen at a protein
level, quantitative immunofluorescence microscopy was used to measure
GLUT protein in single embryos from hyperglycemic vs. control mice.
Two-cell embryos recovered from diabetic mice at 48 h post-hCG
expressed 49 ± 6% less GLUT-1 protein than control embryos
at the equivalent stage. Embryos recovered at 96 h expressed a 66 ± 4% reduction (Fig. 8). Embryos recovered
at 72 h post-hCG from diabetic mice expressed equivalent amounts of
GLUT-2 and GLUT-3 protein; however, 24 h later at 96 h post-hCG, the
diabetic embryos demonstrated a 90 ± 5 and 84 ± 6% reduction in
the amount of GLUT-2 and GLUT-3 protein (Fig.
9). These quantitative protein data confirm
that the downregulation of the facilitative glucose transporters seen
at the mRNA level was also seen at the protein level and thus could
account for the decrease in glucose transport and free glucose seen in
the embryos from hyperglycemic mice at 48 and 96 h after mating.

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Fig. 8.
Confocal immunofluorescent labeling to quantitate GLUT-1 protein
expression. Embryos recovered 48 h after hCG and mating from
control mice (A) vs. diabetic mice
(B) labeled with polyclonal GLUT-1
antibody. Embryos recovered 48 h after hCG and mating from control mice
(C) vs. diabetic mice
(D) labeled with preimmune serum
antibody. Embryos recovered 96 h after hCG and mating from control mice
(E) vs. diabetic mice
(F) labeled with polyclonal GLUT-1
antibody.
|
|

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[in this window]
[in a new window]
|
Fig. 9.
Confocal immunofluorescent labeling to quantitate GLUT-2 and GLUT-3
protein expression. Embryos recovered 72 h after hCG and mating from
control mice (A,
E) vs. diabetic mice
(B,
F) labeled with polyclonal GLUT-2
(A,
B) or GLUT-3
(E,
F) antibody, respectively. Embryos
recovered 96 h after hCG and mating from control mice
(C,
G) vs. diabetic mice
(D,
H) labeled with polyclonal GLUT-2
(C,
D) or GLUT-3
(G,
H) antibody, respectively.
|
|
 |
DISCUSSION |
This study suggests that maternal hyperglycemia induces a decrease in
intracellular free glucose in preimplantation embryos, specifically
at 48 and 96 h postmating. This decrease was due to a decrease in
glucose transport and not an increase in hexokinase activity. DG uptake
in single embryos was decreased in those from diabetic mothers compared
with those from controls at 48 and 96 h after the hCG time points. This
decrease in transport was reflected by the simultaneous decrease in
facilitative glucose transporters at both the mRNA and protein levels,
suggesting downregulation of these transporters due to the
hyperglycemic state.
Two questions need to be investigated. First, how does glucose regulate
the transporter expression? Second, how does a decrease in glucose
transport lead to abnormal embryonic development, eventually manifested
as growth delay, congenital malformations, or spontaneous miscarriage?
To address the first question, it is important to note that similar
downregulation of the facilitative glucose transporters in response to
high ambient glucose is seen in vivo and in vitro in many different
cell types (16, 25, 39). In some cell culture systems, glucose
deprivation causes a rapid and sustained increase in GLUT-1 mRNA and
protein, reflected in the acute increase in glucose transport
(18, 45). Conversely, high glucose conditions induce an acute
downregulation of transport (26, 41). The mechanisms for the regulation
of GLUT-1 by glucose availability are not clear and differ depending on
the cell type. The metabolic control of glucose transport can occur at
multiple levels, including intrinsic activity of the transporter,
localization and content of the transporter protein, and
transcriptional and pre- and posttranslational regulation. The control
of GLUT-1 expression in preimplantation embryos seen in response to
maternal hyperglycemia in this study occurs at the mRNA level and thus
may reflect a complex series of events orchestrated by transcription
factors acting on upstream DNA sequences of the GLUT-1 gene. This may
occur directly or indirectly via posttranscriptional modulation of the
transcription factors. Such speculation is founded, given that glucose
regulates the expression of the insulin receptor in a similar fashion
(12).
Next, further investigation is needed to determine whether a decrease
in glucose transport at these critical time points in preimplantation
development, namely the two-cell and blastocyst stages, leads to
abnormalities in further development. Mouse preimplantation embryos
preferentially metabolize pyruvate until the late morula/early blastocyst stage, at which time glucose becomes the predominant energy
substrate (5). Glucose, however, is required earlier than the
blastocyst stage for optimal development (7, 8, 29). Embryos from F1
hybrid strains need to be exposed to glucose for a period of time as
short as 22 h between 24 and 72 h post-hCG to develop to blastocysts in
vitro (29). Clearly, preimplantation embryos require some degree of
glucose exposure at ~48 h post-hCG, the same time the diabetic
embryos in this study are experiencing a decrease in glucose transport,
leading to significantly lower intraembryonic levels of free glucose.
Glucose deprivation at this critical time of glucose need may be
responsible for the later problems these embryos experience.
These findings raise the question, what role does glucose play at this
early stage in development, before the activation of anaerobic
metabolism? Glycogen synthesis occurs during this period. Glycogen
levels rise 10-fold between the one- and two-cell stages in mouse
embryos (20, 35). Adequate glycogen stores may be necessary to meet the
embryos' energy needs later during compaction or implantation.
Alternatively, glucose may be required for the glycosylation of key
cellular glycoproteins. Previous studies have shown that, in the
presence of tunicamycin, an inhibitor of glycosylation, embryos can
undergo compaction but cannot maintain compaction and thus degenerate
(46). It has been postulated that the preimplantation embryo relies on
a Ca+-independent mechanism of
cell adhesion to maintain compaction (34). Furthermore, fucosylated
cell surface glycoproteins are required for this
Ca+-independent stabilization of
the compacted state (24). Thus deprivation of glucose at this stage
could impair later development of the embryo by impairing surface
glycoprotein synthesis. Such an early insult in the development of the
embryo most likely would lead to embryo loss before implantation.
In those embryos reaching a blastocyst stage, the next question is, how
would the decrease in glucose transport affect further development?
Several recent studies have linked a decrease in glucose transport to
the initiation of apoptosis (4, 23). In models of neuronal development
and trophic factor deprivation, a reduction in glucose uptake is one of
the earliest changes observed in the cascade of apoptotic events
leading to programmed cell death (PCD) (22). Moreover, previous studies
have shown that maternal diabetes and hyperglycemia per se adversely
affect single cell viability at the blastocyst stage. Pampfer et al.
(36) reported a decrease in the number of inner cell mass (ICM) and trophectoderm (TE) cells in blastocysts from diabetic rats. The mechanism behind this decrease and the timing of this cellular loss
were not pursued. Similarly, Lea et al. (27) found a 20% reduction in
the number of cells in the ICM of blastocysts recovered from the
spontaneous IDDM rat model, the BB/E rat. These embryos were
morphologically characterized as having cellular blebbing and nuclear
condensation, suggestive of apoptosis. It is possible that the
hyperglycemia-induced downregulation of the GLUTs and subsequent fall
in free glucose as seen at the blastocyst stage in this study trigger
the premature onset of an increase in PCD, leading to loss of key
progenitor ICM and TE cells. Because a critical threshold in the number
of ICM cells is required for normal postimplantation development (43),
such an apoptotic event may predispose these diabetic embryos to
dysmorphogenesis, fetal loss, or early growth delay, all events
occurring at an increased incidence in women with IDDM.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Bernard Thorens for the mouse GLUT-2 antibody and
cDNA, Dr. Chuck Burant for the mouse GLUT-3 cDNA, and Dr. Daniel Lane
for the mouse GLUT-1 cDNA. We also thank Dr. Harry Heimberg for expert
advice and Joyce Pingsterhaus for excellent technical assistance.
 |
FOOTNOTES |
This work was supported by National Institutes of Health Grants RO1
DK-38495 and DK-50322 (to M. M. Mueckler) and K12 HD-00849-06 and RO3
HD-34693-01 (to K. H. Moley) and by the Berlex Foundation (K. H. Moley).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: K. H. Moley, 660 S. Euclid, Dept. of Cell
Biology and Physiology, Campus Box 8228, St. Louis, MO 63110.
Received 26 January 1998; accepted in final form 17 March 1998.
 |
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