Departments of 1 Obstetrics and Gynecology and 2 Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110
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ABSTRACT |
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Mammalian preimplantation embryos experience a critical switch from an oxidative to a predominantly glycolytic metabolism. In this study, the change in nutrient metabolism between the 2-cell and blastocyst stages was followed by measuring single embryo concentrations of tricarboxylic acid (TCA) cycle and glycolytic metabolites with microfluorometric enzymatic cycling assays. When the normal values were established, further changes that occur as a result of the induction of apoptosis by exposure to high-glucose conditions were examined. From the 2-cell to the blastocyst stage, the embryos experienced an increase in TCA metabolites and a dramatic increase in fructose 1,6-bisphosphate (FBP). The high TCA metabolites may result from accumulation of substrate due to a slowing of TCA cycle metabolism as glycolysis predominates. Embryos exposed to elevated glucose conditions experienced significantly lower FBP, suggesting decreased glycolysis, significantly higher pyruvate, suggesting increased pyruvate uptake by the embryos in response to decreased glycolysis, and increased TCA metabolites, suggesting an inability to oxidize the pyruvate and a slowing of the TCA cycle. We speculate that the glycolytic changes lead to dysfunction of the outer mitochondrial membrane that results in the abnormal TCA metabolite pattern and triggers the apoptotic event.
tricarboxylic acid cycle; glycolysis; preimplantation embryo; programmed cell death
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INTRODUCTION |
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GLUCOSE TRANSPORT AND METABOLISM are critical for mammalian blastocyst formation and further development (8, 20). At this stage, the switch occurs from oxidation of pyruvate via the tricarboxylic acid (TCA) cycle to the use of glucose as the main substrate via glycolysis (7, 14). As a result, the blastocyst exhibits extreme sensitivity to glucose deprivation. We have previously shown that any decrease in glucose transport, basal or insulin stimulated, results in enhanced apoptosis at this stage, which manifests later in pregnancy as a malformation or miscarriage (3, 4, 21, 22). This decrease in blastocyst glucose transport and resulting apoptosis occur in conditions of maternal hyperglycemia and hyperinsulinemia.
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 biosynthetic demands on the embryo. Maintenance of a high rate of glycolysis is thought to be important for providing a "dynamic buffer" of metabolic intermediates for the biosynthesis of macromolecules (23). For example, glucose 6-phosphate is used in the formation of the ribose 5-phosphate required for DNA and RNA synthesis. Another reason for the increased glucose demand may be that increasing amounts of glucose are converted to lactate at the blastocyst stage in humans and rodents (14). In these species in particular, the embryo resides in the uterus for a relatively short time before implantation, and the switch to an anaerobic metabolism is in response to the 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 pyruvate and lactate via glycolysis.
Recent studies have shown that cell death caused by reduced availability of glucose, as seen with growth factor withdrawal, is initiated by mitochondrial changes that result in cytochrome c release (10, 27). Overexpression of GLUT1 can prevent this onset of apoptosis (27), and the regulation of outer mitochondrial membrane integrity via the voltage-dependent anion channel (VDAC) appears to depend on cellular metabolic changes associated with glycolysis (26).
In this study, we attempted to track the changes in nutrient metabolism in the blastocyst by measuring single embryo concentrations of TCA metabolites and glycolytic metabolites by use of microfluorometric enzymatic cycling assays. We postulate that perhaps some of the metabolic alterations experienced by the blastocyst exposed to hyperglycemia may be responsible for perturbations in mitochondrial physiology and thus may trigger an apoptotic cascade.
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MATERIALS AND METHODS |
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Embryo collection and culturing. Embryos were recovered as described previously (21) from superovulated female mice (B6 × SJL F1, Jackson Laboratories). In vivo retrieved embryos were then flushed from the oviducts 48, 72, and 96 h later, corresponding to 2-cell, morula, and blastocyst stages, respectively. In vitro cultured embryos were flushed at the 2-cell stage and cultured in KSOM mouse embryo culture media (Specialty Media, Phillipsburg, NJ) containing either 0.2 mM D-glucose, the control concentration of glucose, or 5.6 or 50 mM D-glucose as the test conditions. The embryos were cultured at 37°C in 5% CO2-5% O2-90% N2.
Embryo extraction for metabolite assays.
At each stage, embryos were washed in BSA-free medium for 1 min and
then quickly frozen on a glass slide by dipping in cold isopentane
equilibrated with liquid N2. After freeze-drying overnight in a vacuum at 35°C, the embryos were extracted in nanoliter volumes under oil, as previously described (21). Citrate,
-ketoglutarate, aspartate, glycerol 3-phosphate, and ATP were
measured in alkaline-extracted embryos. These embryos were extracted in
1 µl of 0.1 N NaOH at room temperature for 20 min, 0.5 µl
of the extract was heated to 80°C for 20 min, and a 0.2-µl mixture
of 0.2 N HCl and 0.1 M Tris · HCl (pH 6.8) was added. Malate,
fumarate, and glutamate were measured in acid-extracted embryos. These
embryos were extracted in 1 µl of 0.1 N NaOH at room temperature for
20 min, 0.5 µl of the extract was added to 0.1 µl of 0.6 N HCl and
heated to 80°C for 20 min, and finally the extract was neutralized
with 0.1 µl of 0.2 M Tris base. Both treated extracts were stored at
70°C.
Metabolite microanalytic assays.
Separate assays were developed for each metabolite measured and were
designed to link to reactions using NADH or NADPH (Tables 1 and 2,
steps 1 and 2). The NADPH/NADH by-product is then
enzymatically amplified in a cycling reaction (Table 1, steps
3 and 4), and a byproduct of the amplification step is
measured in a fluorometric assay (Table 1, step 5). All
metabolites except fructose 1,6-bisphosphate (FBP) are expressed as
millimoles per kilogram wet weight based on the wet weight of 160 pg
per embryo. FBP levels were extremely low in individual embryos and are
expressed as micromoles per kilogram wet weight. Absolute
concentrations of metabolites can be calculated in picomoles by
multiplying by 0.16. ATP was used as a marker of viability, and if any
embryos had abnormally low levels, the entire set of experiments was
discarded. Previous studies have shown that cells with low ATP undergo
necrosis rather then apoptosis, because apoptosis
requires energy (6, 15). ATP levels are placed at the
outset of each table or figure to demonstrate the equality of the
embryos tested.
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Assay conditions.
All assays for a particular metabolite in a given experiment were made
at the same time and conducted at ambient temperatures. Metabolites
were measured according to the protocols of Table 1 and reagents of
Table 2. Standards were carried throughout the entire procedure. Most
of the enzymes were purchased in a suspension of ammonia sulfate, spun
down, and reconstituted with 20 mM imidazole HCl (pH 7.0) and 0.02%
BSA. All experiments were completed a minimum of three times. For each
metabolite measurement under each condition, at least 16 individual
embryos were used. The NADP+ cycling reagent, used only for
the ATP assay, contains 100 mM imidazole HCl (pH 7.0), 7.5 mM
-ketoglutarate, 5 mM glucose 6-phosphate, 25 mM NH4Ac,
0.02% BSA, 100 µM ADP, 100 µg/ml beef liver glutamate dehydrogenase, and 10 µg/ml Leuconostoc mesenteroides
glucose 6-phosphate dehydrogenase. This enzyme ratio gives
~100,000-fold amplification, as detailed in Table 1 for ATP. The
indicator reaction for the NADP+ cycling step, or
step 5 in Table 1, involves adding 10 µl of the reaction
to 1 ml of the indicator, 6-phosphogluconate reagent, containing 50 mM
imidazole HAc (pH 7.0), 1 mM EDTA, 30 mM NH4Ac, 5 mM
MgCl2, 100 µM NADP+, and 2 µg/ml yeast
6-phosphogluconate dehydrogenase. The NAD+ cycling reagent,
which is used for all other metabolite assays in Table 1, contains 100 mM Tris · HCl (pH 8.1), 2 mM
-mercaptoethanol, 2 mM
oxaloacetate, 300 mM ethanol, 0.02% BSA, 15 µg/ml alcohol dehydrogenase, and 1.5 µg/ml heart malate dehydrogenase. This enzyme
ratio gives ~15,000-fold amplification overnight at room temperature
and is what was used to measure citrate and pyruvate. This ratio was
altered accordingly to achieve the number of cycles detailed in Table
1. The indicator reaction for the NAD+ cycling step, or
step 5 in Table 1, involves adding 10 µl of the reaction
to 1 ml of the indicator, malate reagent, containing 20 mM
2-amino-2-methylpropanol HCl (pH 9.9), 10 mM L-glutamate, 200 µM NAD+, 5 µg/ml malate dehydrogenase, and 2 µg/ml glutamic-oxaloacetic transaminase.
Statistical analysis. Data are expressed as means ± SE. Differences in metabolites between 2-cell and blastocyst stage embryos recovered either in vivo or in vitro were analyzed using Student's t-test. FBP and pyruvate measurements were the exception. Because of the large number of embryos required for the measurement of these two metabolites, the 2-cell embryos, in vivo blastocyst, and in vitro blastocyst assays were grouped together and the results analyzed using ANOVA with the Bonferroni/Dunn post hoc test. Differences in metabolites from embryos cultured in different concentrations of glucose were analyzed using ANOVA with the Bonferroni/Dunn post hoc analysis. Differences were considered significant at P < 0.05. StatView 4.5 (Abacus Concepts, Berkeley, CA) was used for statistical analyses.
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RESULTS |
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Progression from 2-cell to blastocyst stage in vivo or in vitro
results in an accumulation of TCA cycle metabolites and a decrease in
glycolytic metabolites.
The majority of TCA cycle substrates demonstrated a significant
increase in concentration as the embryo developed from a 2-cell- to a
blastocyst-stage embryo (see Table 3 and
Fig. 1). This increase, comparing
2-cell to blastocyst, occurred both in embryos cultured in vitro and in
embryos obtained directly in vivo. Similarly, the FBP increased
significantly, whereas glycerol 3-phosphate, a glycolytic intermediate,
dropped during the same period in both conditions. This may reflect the
embryo's adaptation to an anoxic peri-implantation existence, as well
as the embryo's switch at the blastocyst stage to a predominantly
glycolytic metabolism. The low level of FBP in early cleavage-stage
embryos agrees with previous studies (1).
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Blastocysts exposed to moderate glucose concentrations (5.6 mM) compared with normal glucose conditions (0.2 mM) experience a
decrease in TCA cycle metabolites and an increase in glycolytic
metabolites.
Except for citrate and malate, the TCA cycle metabolites in
embryos cultured in 5.6 mM glucose were only slightly lower than those
in embryos cultured in a normal glucose concentration of 0.2 mM. In
addition, only glycerol 3-phosphate levels were significantly higher in
the blastocysts in 5.6 mM glucose (See Table
4 and Fig.
2), and pyruvate and FBP were only
slightly higher. Because the prior 2-cell to blastocyst studies
suggested that a slowing of flux via the TCA cycle corresponds to an
increase in TCA metabolites and a decrease in glycerol 6-phosphate
levels, these results of 5.6 mM glucose compared with 0.2 mM glucose
suggest the opposite. The mildly elevated glucose concentration appears
to be causing increased flux through the TCA cycle with lowering of the
levels, and perhaps a saturation of the glycolytic pathway, and with an increase in one of the byproducts used to make glycolipids, glycerol 3-phosphate.
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Blastocysts exposed to high glucose concentrations (50 mM) compared with normal glucose concentrations (0.2 mM) experience an increase in TCA cycle metabolites, increased pyruvate, and a decrease in glycolytic metabolites. Blastocysts cultured in 50 mM glucose compared with 0.2 mM glucose had a predominantly higher number of TCA cycle metabolites, significantly higher pyruvate and glycerol 6-phosphate levels, and significantly lower FBP levels (See Table 4 and Fig. 2). The discrepancy between citrate and pyruvate levels suggests strongly that a block to pyruvate oxidation via the TCA cycle exists in these embryos. Moreover, significantly decreased FBP levels suggest that glycolysis is also blocked, perhaps due to the low free glucose levels that are known to be present in these embryos exposed to high glucose conditions (21). The elevated pyruvate levels may occur because of increased pyruvate uptake, which occurs in response to glucose deprivation (7, 8).
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DISCUSSION |
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Progression from the 2-cell to the blastocyst stage was associated with a significant increase in TCA cycle metabolites, a dramatic increase in FBP, and a decrease in glycerol 3-phosphate, as seen in this analysis. These results give a profile of a normal transition from a metabolism based on pyruvate oxidation via TCA cycle to a metabolism based on glucose metabolism via glycolysis. TCA cycle metabolites may accumulate because of blastocyst reliance on glycolytic metabolism, slowing of the TCA cycle, and accumulation of substrates. Less than 1% of glucose consumed at a blastocyst stage is oxidized via the TCA cycle (5), and this finding of an increase in TCA cycle metabolites corresponds with previous reports that have used techniques similar to those used in this study (1).
Comparing blastocysts cultured in 0.2 mM glucose with those in 5.6 mM glucose demonstrates the effect of a moderate increase in glucose on the metabolic products. The decrease in TCA cycle metabolites suggests increased flux through this pathway, with increased substrate utilization. Regulation of TCA cycle flux depends in part on increased availability of pyruvate and NAD+. Increasing glucose availability by incubating in 5.6 mM glucose would increase pyruvate production and decrease NADH levels as increased pyruvate is converted to lactate. Moreover, increased FBP levels would also stimulate TCA cycle flux and perhaps slow glycolysis. Previous studies have shown that glucose consumption rate is saturated at 0.29 mM but that a further 10% increase is obtained at 3 mM, which is similar to the 5.6 mM used in this study (5). All of our findings are consistent with a slight but measurable increase in glucose consumption. Importantly, none of these metabolic changes at this moderate glucose concentration induce apoptosis in the embryo, as shown in previous studies (3, 22, 24).
In contrast, maternal diabetes or in vitro hyperglycemia, at 50 mM glucose, does lead to apoptosis in the mouse embryo, requiring expression of the proapoptotic Bcl-2 family member BAX (13, 22). Our previous studies show that this apoptotic event is triggered by decreased GLUT1 expression and glucose transport at the blastocyst stage (3, 21). The present study demonstrates that this drop in transport leads to a decrease in glycolysis, resulting in lower FBP levels in the individual blastocysts. Other studies have linked inhibition of glycolysis to initiation of an apoptotic cascade (18). Likewise, overexpression of glucose transporters prevents hypoxia-induced programmed cell death (16). Glucose metabolism and glucose uptake also exhibit a protective effect against apoptosis induced by growth factor withdrawal in different cell types (2, 12, 19). We postulate that the decrease in glycolysis in these blastocysts, as demonstrated by decreased FBP levels, and the embryos' attempts to compensate by increasing pyruvate uptake, lead to severe alteration in mitochondrial physiology that results in the triggering of the apoptotic cascade.
As shown in the growth factor withdrawal model (27), a depletion of glycolytic byproducts leads to decreased electron transport substrates and a resulting decrease in outer mitochondrial membrane potential (9) due to formation of an electron gradient and closure of the VDAC. VDAC is a large conductance channel that, when open, is the major pathway for metabolite transport across the outer mitochondrial membrane (28). Closure of this channel occurs under conditions of altered intracellular NADH and pyruvate resulting from glycolytic changes (17, 29) and has been shown to trigger apoptosis in growth factor withdrawal models due to hyperpolarization, loss of outer mitochondrial integrity, and cytochrome c release (25, 26).
Previous data show that VDAC closure leads to severe perturbation in
mitochondrial physiology, leading to lack of mitochondrial availability
of malate and ADP, slowing down of the TCA cycle and
NAD+/NADH shuttles, and accumulation of TCA cycle
metabolites as seen in this study (11). This lack of
pyruvate oxidation would also support the elevated pyruvate levels.
Although the citrate and -ketoglutarate levels are lower in this
report, unlike all the other TCA cycle components, these two
metabolites can readily be converted to glutamate and glutamine and may
serve as alternative energy sources under these stress conditions.
Because of the anticipated problems with the mitochondrial matrix, the
NADH shuttle would not be functioning and thus might result in the
increased glycerol 3-phosphate levels. Most of the changes in TCA cycle
metabolites seen here can be explained by perturbations in
mitochondrial physiology due to a closure of VDAC.
In summary, hyperglycemia, by causing a decrease in glucose transport, results in a decrease in glycolysis and a decrease in FBP in the mouse blastocyst as measured in this study. This depletion of glycolytic substrates, in combination with the embryo's attempts to increase pyruvate uptake as reflected here, may be responsible for loss of integrity of the outer mitochondrial membrane as seen in other cell types. An inability of the mitochondria to successfully complete pyruvate oxidation would explain the increase in TCA cycle metabolites seen in these embryos. Recent studies provide strong evidence to support the mitochondria as the site of apoptosis initiation in response to growth factor withdrawal (25-27). These apoptotic effects are believed to be due to metabolic alterations linked to changes in glycolysis, as seen here with the blastocysts undergoing alterations in response to high-glucose conditions. Data from this study suggest that hyperglycemia-induced apoptosis in the mouse blastocyst may also involve metabolic alterations, leading to problems with outer mitochondrial membrane permeability.
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ACKNOWLEDGEMENTS |
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This work was supported by grants to K. H. Moley from the National Institute of Child Health and Human Development (NICHD; RO1 HD-38061) and from NICHD and National Institute of Diabetes and Digestive and Kidney Diseases (RO1 HD/DK-40390) and a research grant from the American Diabetes Association.
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FOOTNOTES |
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Address for reprint requests and other correspondence: K. H. Moley, Dept. of OB/GYN, Washington Univ. School of Medicine, 4911 Barnes-Jewish Hospital Plaza, 2nd Floor Maternity, St. Louis, MO 63110 (E-mail: moleyk{at}msnotes.wustl.edu).
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. Section 1734 solely to indicate this fact.
March 27, 2002;10.1152/ajpendo.00046.2002
Received 1 February 2002; accepted in final form 18 March 2002.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Barbehenn, EK,
Wales RG,
and
Lowry OH.
Measurement of metabolites in single preimplantation embryos: a new means to study metabolic control in early embryos.
J Embryol Exp Morphol
43:
29-46,
1978[ISI][Medline].
2.
Berridge, MV,
Tan AS,
McCoy KD,
Kansara M,
and
Rudert F.
CD95 (Fas/Apo-1)-induced apoptosis results in loss of glucose transporter function.
J Immunol
156:
4092-4099,
1996[Abstract].
3.
Chi, MM,
Pingsterhaus J,
Carayannopoulos M,
and
Moley KH.
Decreased glucose transporter expression triggers BAX-dependent apoptosis in the murine blastocyst.
J Biol Chem
275:
40252-40257,
2000
4.
Chi, MM,
Schlein AL,
and
Moley KH.
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:
4784-4792,
2000
5.
Dufrasnes, E,
Vanderheyden I,
Robin D,
Delcourt J,
Pampfer S,
and
De Hertogh R.
Glucose and pyruvate metabolism in preimplantation blastocysts from normal and diabetic rats.
J Reprod Fertil
98:
169-177,
1993[Abstract].
6.
Eguchi, Y,
Shimizu S,
and
Tsujimoto Y.
Intracellular ATP levels determine cell death fate by apoptosis or necrosis.
Cancer Res
57:
1835-1840,
1997[Abstract].
7.
Gardner, DK,
and
Leese HJ.
Non-invasive measurement of nutrient uptake by single cultured pre-implantation mouse embryos.
Hum Reprod
1:
25-27,
1986[Abstract].
8.
Gardner, DK,
and
Leese HJ.
The role of glucose and pyruvate transport in regulating nutrient utilization by preimplantation mouse embryos.
Development
104:
423-429,
1988[Abstract].
9.
Hackenbrock, CR.
Ultrastructural bases for metabolically linked mechanical activity in mitochondria. I. Reversible ultrastructural changes with change in metabolic steady state in isolated liver mitochondria.
J Cell Biol
30:
269-297,
1966
10.
Harris, MH,
and
Thompson CB.
The role of the Bcl-2 family in the regulation of outer mitochondrial membrane permeability.
Cell Death Differ
7:
1182-1191,
2000[ISI][Medline].
11.
Hodge, T,
and
Colombini M.
Regulation of metabolite flux through voltage-gating of VDAC channels.
J Membr Biol
157:
271-279,
1997[ISI][Medline].
12.
Kan, O,
Baldwin SA,
and
Whetton AD.
Apoptosis is regulated by the rate of glucose transport in an interleukin 3 dependent cell line.
J Exp Med
180:
917-921,
1994[Abstract].
13.
Keim, AL,
Chi MM,
and
Moley KH.
Hyperglycemia-induced apoptotic cell death in the mouse blastocyst is dependent on expression of p53.
Mol Reprod Dev
60:
214-224,
2001[ISI][Medline].
14.
Leese, HJ,
and
Barton AM.
Pyruvate and glucose uptake by mouse ova and preimplantation embryos.
J Reprod Fertil
72:
9-13,
1984[Abstract].
15.
Leist, M,
Single B,
Castoldi AF,
Kuhnle S,
and
Nicotera P.
Intracellular adenosine triphosphate (ATP) concentration: a switch in the decision between apoptosis and necrosis.
J Exp Med
185:
1481-1486,
1997
16.
Lin, Z,
Weinberg J,
Malhotra R,
Merritt S,
Holzman L,
and
Brosius F, III.
GLUT-1 reduces hypoxia-induced apoptosis and JNK pathway activation.
Am J Physiol Endocrinol Metab
278:
E958-E966,
2000
17.
Liu, MY,
and
Colombini M.
Regulation of mitochondrial respiration by controlling the permeability of the outer membrane through the mitochondrial channel, VDAC.
Biochim Biophys Acta
1098:
255-260,
1992[ISI][Medline].
18.
Malhotra, R,
and
Brosius FC, III.
Glucose uptake and glycolysis reduce hypoxia-induced apoptosis in cultured neonatal rat cardiac myocytes.
J Biol Chem
274:
12567-12575,
1999
19.
Malhotra, R,
Lin Z,
Vincenz C,
and
Brosius FC, III.
Hypoxia induces apoptosis via two independent pathways in Jurkat cells: differential regulation by glucose.
Am J Physiol Cell Physiol
281:
C1596-C1603,
2001
20.
Martin, KL,
and
Leese HJ.
Role of glucose in mouse preimplantation embryo development.
Mol Reprod Dev
40:
436-443,
1995[ISI][Medline].
21.
Moley, KH,
Chi MM,
and
Mueckler MM.
Maternal hyperglycemia alters glucose transport and utilization in mouse preimplantation embryos.
Am J Physiol Endocrinol Metab
275:
E38-E47,
1998
22.
Moley, KH,
Chi MMY,
Knudson CM,
Korsmeyer SJ,
and
Mueckler MM.
Hyperglycemia induces apoptosis in preimplantation embryos via cell death effector pathways.
Nature Med
12:
1421-1424,
1998.
23.
Newsholme, P,
and
Newsholme EA.
Rates of utilization of glucose, glutamine and oleate and formation of end-products by mouse peritoneal macrophages in culture.
Biochem J
261:
211-218,
1989[ISI][Medline].
24.
Pampfer, S,
Vanderheyden I,
McCracken JE,
Vesela J,
and
De Hertogh R.
Increased cell death in rat blastocysts exposed to maternal diabetes in utero and to high glucose or tumor necrosis factor-alpha in vitro.
Development
124:
4827-4836,
1997
25.
Vander Heiden, MG,
Chandel NS,
Li XX,
Schumacker PT,
Colombini M,
and
Thompson CB.
Outer mitochondrial membrane permeability can regulate coupled respiration and cell survival.
Proc Natl Acad Sci USA
97:
4666-4671,
2000
26.
Vander Heiden, MG,
Li XX,
Gottleib E,
Hill RB,
Thompson CB,
and
Colombini M.
Bcl-xL promotes the open configuration of the voltage-dependent anion channel and metabolite passage through the outer mitochondrial membrane.
J Biol Chem
276:
19414-19419,
2001
27.
Vander Heiden, MG,
Plas DR,
Rathmell JC,
Fox CJ,
Harris MH,
and
Thompson CB.
Growth factors can influence cell growth and survival through effects on glucose metabolism.
Mol Cell Biol
21:
5899-5912,
2001
28.
Zalman, LS,
Nikaido H,
and
Kagawa Y.
Mitochondrial outer membrane contains a protein producing nonspecific diffusion channels.
J Biol Chem
255:
1771-1774,
1980
29.
Zizi, M,
Forte M,
Blachly-Dyson E,
and
Colombini M.
NADH regulates the gating of VDAC, the mitochondrial outer membrane channel.
J Biol Chem
269:
1614-1616,
1994