REPRODUCTION-DEVELOPMENT
Insulin-Stimulated Glucose Uptake Occurs in
Specialized Cells within the Cumulus Oocyte Complex
Scott H. Purcell, Maggie M. Chi, and Kelle H. Moley
Department of Obstetrics and Gynecology, Washington University in St. Louis, St. Louis, Missouri 63110
The oocyte exists within the mammalian follicle surrounded by somatic cumulus cells. These cumulus cells metabolize the majority of the glucose within the cumulus oocyte complex and provide
energy substrates and intermediates such as pyruvate to the oocyte. The insulin receptor is present
in cumulus cells and oocytes; however, it is unknown whether insulin-stimulated glucose uptake
occurs in either cell type. Insulin-stimulated glucose uptake is thought to be unique to adipocytes,
skeletal and cardiac muscle, and the blastocyst. Here, we show for the first time that many of the
components required for insulin signaling are present in both cumulus cells and oocytes. We
performed a set of experiments on mouse cumulus cells and oocytes and human cumulus cells using
the nonmetabolizable glucose analog 2-deoxy-D-glucose to measure basal and insulin-stimulated
glucose uptake. We show that insulin-stimulated glucose uptake occurs in both compact and
expanded cumulus cells of mice, as well as in human cumulus cells. Oocytes, however, do not display
insulin-stimulated glucose uptake. Insulin-stimulated glucose uptake in cumulus cells is mediated
through phosphatidylinositol 3-kinase signaling as shown by inhibition of insulin-stimulated glucose uptake and Akt phosphorylation with the specific phosphatidylinositol 3-kinase inhibitor,
LY294002. To test the effect of systemic in vivo insulin resistance on insulin sensitivity in the cumulus
cell, cumulus cells from high fat-fed, insulin-resistant mice and women with polycystic ovary syndrome were examined. Both sets of cells displayed blunted insulin-stimulated glucose uptake. Our
studies identify another tissue that, through a classical insulin-signaling pathway, demonstrates
insulin-stimulated glucose uptake. Moreover, these findings suggest insulin resistance occurs in
these cells under conditions of systemic insulin resistance. (Endocrinology 153: 2444 –2454, 2012)
ammalian oocytes are surrounded by a layer of specialized granulosa cells called “cumulus cells” that
differentiate at the time of antral follicle formation. Cumulus cells differ from the mural granulosa cells that line
the follicle in function as well as gene and protein expression (1–3), including genes involved in glycolytic metabolism (4). The cumulus-oocyte-complex (COC) exists
within the ovarian follicle from the antral follicle stage
until after ovulation when, after the LH surge cumulus
cells expand and secrete hyaluronic acid. Bidirectional
communication through gap junctions, connexins, and
paracrine signaling between the oocyte and surrounding
cumulus cells is necessary for normal oocyte development,
including oocyte meiotic maturation (5, 6). Additionally,
M
the cumulus cells are particularly important for metabolism within the COC. The cumulus cells metabolize the
majority of the glucose within the COC and provide metabolic intermediates to the oocyte, which has a poor capacity to metabolize glucose on its own, and preferentially
metabolize pyruvate from the cumulus cells (7–10). It has
been shown that oocytes denuded of their surrounding
cumulus cells have low glycolytic activity (11) mediated, in
part, by low phosphofructokinase activity (12).
Recently, the insulin receptor (IR) was identified in
mouse oocytes and cumulus cells (13). Prolonged culture
(10 d) of preantral mouse follicles with insulin resulted in
phosphorylation of glycogen synthase kinase 3B in the
oocyte, indicating that some activation of the insulin-sig-
ISSN Print 0013-7227 ISSN Online 1945-7170
Printed in U.S.A.
Copyright © 2012 by The Endocrine Society
doi: 10.1210/en.2011-1974 Received November 10, 2011. Accepted February 13, 2012.
First Published Online March 9, 2012
Abbreviations: BMI, Body mass index; COC, cumulus-oocyte-complex; 2-DG, 2-deoxyglucose; DMSO, dimethylsulfoxide; GLUT, glucose transporter; GV, germinal vesicle; HTF,
human tubal fluid; IR, insulin receptor; IRS, IR substrate; PCOS, polycystic ovarian syndrome;
PI3K, phosphatidyl-inositol-3 kinase; qRT-PCR, quantitative RT-PCR; SSS, synthetic serum
substitute.
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Endocrinology, May 2012, 153(5):2444 –2454
naling cascade occurs in oocytes (13). Previous work had
identified IR mRNA in oocytes of the human, bovine, and
rat (14 –16); and IR protein in pig (17) and human oocytes
(18). Insulin can affect cell growth, apoptosis, and metabolism in a variety of tissues (19); however, insulin-stimulated glucose uptake occurs predominantly in muscle,
heart, and fat, as well as the blastocyst-stage embryo (20)
and takes only minutes to detect, as opposed to hours as
previously measured in the ovarian follicle. The primary
metabolic function of insulin is to increase rapidly glucose
uptake in the target tissues, primarily skeletal muscle and
fat, in the postprandial state. A family of facilitative glucose transporters known as glucose transporters (GLUT)
mediates glucose uptake into tissues. There are 14 members of the GLUT family that differ in their substrate specificity, kinetic characteristics, and subcellular distribution
(21, 22). Insulin-stimulated glucose uptake into peripheral
tissues is mediated through GLUT4 (23). However, the
GLUT4 knockout mouse does not develop hyperglycemia
(24), and soleus muscle from GLUT4 knockout mice can
increase glucose uptake in response to insulin (25), indicating that other GLUT such as GLUT8 (20) and GLUT12
(26, 27) may also be insulin responsive in key target
tissues.
As described above, the cumulus cells are responsible
for the majority of glucose metabolism within the COC,
and both cumulus cells and the oocyte express the IR. It is
unknown however, whether either component of the COC
is capable of insulin-stimulated glucose uptake. The measurement of lactate accumulation in media of primary cultures of human granulosa cells cultured with or without
insulin has only provided indirect evidence that insulin
may affect glucose metabolism in these cells (28, 29). Our
aim was to determine whether the oocyte, cumulus cells,
or both exhibit insulin-stimulated glucose uptake through
classical insulin-signaling pathways, and if so, whether
peripheral insulin resistance would impair this process.
These studies are particularly relevant in light of the increasing incidence of obesity and insulin resistance in
women of reproductive age, which appears to negatively
impact fertility (30 –34). Specifically, the impact of obesity
on the oocyte, subsequent embryonic development, and
pregnancy outcome is an active area of research (35–39).
Materials and Methods
Animal care and use
All mouse studies were approved by the Animal Studies Committee at Washington University School of Medicine and conform to the Guide for the Care and Use of Laboratory Animals
published by the National Institutes of Health. Female ICR (Har-
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lan, Indianapolis, IN) mice, 3– 4 wk, of age were used for all
experiments. For high-fat experiments, mice (n ⫽ 40) were fed a
high-fat diet containing 35.8% fat, 20.7% protein, and 35%
carbohydrates (58% Fat energy-A1N-76A; TestDiet, Richmond, IN) from ages 3–7 wk before experiments; age-matched
mice (n ⫽ 40) were fed standard mouse chow containing 4.8%
fat, 73.9% carbohydrate, and 14.8% protein (12% Fat energy;
TestDiet). After 4 wk on the diet measurements of fasting weight,
blood glucose, and serum insulin were taken, and a glucose tolerance test was performed. Mice had access to food and water ad
libitum.
Human cumulus cells
The Washington University Human Research Protection Office and Institutional Review Board approved all human studies.
Cumulus cells were obtained from women undergoing infertility
treatment at Washington University School of Medicine’s Reproductive Endocrinology and Infertility Clinic using standard
procedures in preparation of human oocytes for in vitro fertilization. Control cumulus cells were obtained from patients (n ⫽
7) with a male factor-only infertility diagnosis with an average
body mass index (BMI) of 27.4 and age of 29.6 ⫾ 1.0 yr. Cumulus cells were also obtained from women with polycystic
ovarian syndrome (PCOS) (n ⫽ 4) (based on Rotterdam criteria)
with an average BMI of 34.8 and age of 28 ⫾ 0.6 yr. Cumulus
cells were removed from oocytes approximately 1–3 h after retrieval from the ovaries. One to three oocytes were placed into
200-␮l drops of 80 U/ml hyaluronidase (Sigma Chemical Co., St.
Louis, MO) in HEPES-buffered human tubal fluid (HEPESHTF; Irvine Scientific, Santa Ana, CA) supplemented with 10%
Synthetic Serum Substitute (SSS; Irvine Scientific). To effect removal, the oocytes underwent rapid pipetting through a 150-␮m
bore pipette tip (MidAtlantic Diagnostics, Mt. Laurel, NJ) for 10
sec before transfer to 100 ␮l wash drops of HEPES-HTF ⫹ 10%
SSS. Rapid pipetting was continued in fresh wash drops until all
cumulus cells were removed and the denuded oocytes were transferred to culture for clinical use. The spent drops of hyaluronidase and wash droplets containing the cumulus cells were collected and diluted 1:2 in HEPES-HTF ⫹ 10% SSS and pelleted.
Samples were either used fresh or washed in PBS and stored at
⫺80 C until use.
Isolation of mouse oocytes and cumulus cells
For compact cumulus cells and germinal vesicle (GV) stage
oocytes, mice were injected with 10 IU PMSG (National Hormone and Peptide Program, Torrance, CA) and euthanized 48 h
later. Ovaries were isolated and placed under M2 media (Sigma).
Follicles were manually punctured and COC were separated and
placed in a clean dish of M2 media. Cumulus cells were then
removed from isolated COC by manual pipetting using pulled
glass pipettes. For collection of expanded cumulus cells, mice
were injected with 10 IU PMSG and 48 h later with 10 IU human
chorionic gonadotropin (Sigma). Mice were euthanized 13 h after human chorionic gonadotropin injection, and oviducts were
dissected out and placed in M2 media. Expanded COC were then
isolated by dissecting oviducts and placing COC in a clean dish
of M2 media containing 1 mg/ml hyaluronidase (Sigma) for 5
min. Oocytes were removed and cumulus cells were washed
twice in PBS before use. Oocytes and cumulus cells were either
used fresh or washed in PBS and stored at ⫺80 C before use.
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Purcell et al.
Insulin and the Cumulus Oocyte Complex
Western blot analysis
Isolated cumulus cells were lysed in 30 ␮l protein lysis buffer,
50 mM Tris HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 0.5%
Nonidet P-40, and complete Mini protease inhibitor cocktail
(Roche Applied Sciences, Indianapolis, IN). Other tissues used as
positive controls (soleus muscle, fat, endometrial stromal cells,
and testis) were lysed in 150 ␮l lysis buffer. Protein concentration
was quantified using the BCA Protein Assay Kit (Pierce Chemical
Co., Rockford, IL). Denuded GV-stage oocytes were used in all
Western blot experiments in 100 oocyte aliquots per lane. After
addition of 5% ␤-mercaptoethanol and Laemmli buffer, wholecell lysates or oocytes were separated by SDS-PAGE and transferred to nitrocellulose. Nonspecific antibody binding was
blocked in 5% nonfat dry milk powder in Tris-buffered saline for
1 h. All blots were then incubated overnight at 4 C in 5% nonfat
dry milk or 5% BSA, according to the manufacturer’s instructions with the following antibodies: IR (1:200; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), IGF-1R (1:1000; Cell Signaling Technology, Danvers, MA), IR substrate-1 (IRS-1) (1:1000;
Cell Signaling Technology), IRS-2 (1:1000; Cell Signaling Technology), Phosphatidyl-inositol-3 kinase (PI3K) p85 subunit (1:
000; Cell Signaling Technology), PI3K p110␣ subunit (1:1000;
Cell Signaling Technology), Akt (pan) (1:1000; Cell Signaling
Technology), Phospho(p)-Akt (1:1000; Cell Signaling Technology), GLUT1 (1:3000; kindly provided by Dr. Michael Mueckler, Washington University) (40); GLUT4 (1:1000; kindly provided by Dr. Michael Mueckler, Washington University) (40);
GLUT8 (1:1000; previously generated in our laboratory) (20);
GLUT12 (1:1000; previously generated in our laboratory) (41);
␤-actin (1:5000; Millipore Corp., Bedford, MA). After primary
antibody incubation, blots were incubated with goat-antirabbit
IRDye 800 (1:10,000) or goat-antimouse IRDye 680 (1:10,000)
(LI-COR Biosciences, Lincoln, NE) for 1 h at room temperature.
Membranes were then scanned and analyzed using the Odyssey
fluorescent imager (LI-COR Biosciences) and normalized to
␤-actin.
Insulin-stimulated 2-deoxyglucose (2-DG) uptake
assay
For mice, isolated nonfrozen cumulus cells from approximately 10 mice were used per replicate. Human cumulus cells
isolated from one patient were used per replicate. Cumulus cells
were washed in PBS, centrifuged at 4500 rpm, and resuspended
in 50 ␮l M2 media (Sigma) containing 5.6 mM D-glucose with or
without 500 nM bovine insulin (Sigma) for 15 min at 37 C. Cells
were then centrifuged and washed once in 50 ␮l 1 ⫻ Krebs-ringer
solution (125 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 2.4 mM
MgSO4, 25 mM NaHCO3, 1.2 mM K2HPO4, and 1% BSA) without glucose, and then incubated in 50 ␮l 1 ⫻ Krebs-ringer solution without glucose for 15 min at 37 C in 5% CO2. Cells were
then centrifuged and resuspended for 1 min with 1 ⫻ Krebsringer containing 0.1 mM 2-deoxy-D-glucose and 1.2 ␮M (2 ␮Ci)
of [1,2-3H]-2-deoxy-D-glucose ([3H]-2-DG; MP Biomedicals,
Solon, OH). The 2-DG uptake reaction was stopped by the addition of 50 ␮M ice-cold cytochalasin B. Samples were then
washed twice in PBS and resuspended in 25 ␮l protein lysis buffer. A 10-␮l sample was used to quantify protein concentration
by BCA assay (Pierce, Thermo Scientific, Rockford, IL). The
remaining sample was added to 20 ml scintillation fluid and
[3H]-2-DG was counted and normalized to protein concentra-
Endocrinology, May 2012, 153(5):2444 –2454
tion of each sample. For calculation of nonradioactive 2-DG
uptake in oocytes and blastocysts, fresh GV-stage denuded
oocytes or expanded blastocysts were collected from 3-wk-old
ICR mice, and basal or insulin-stimulated 2-DG uptake was measured in individual oocytes or blastocysts using enzymatic cycling assays previously developed and validated in our laboratory (20, 42). For all 2-DG uptake assays, uptake was calculated
as the fold change in insulin-stimulated 2-DG uptake compared
with the average basal 2-DG uptake.
Inhibition of PI3K signaling
A set of 2-DG uptake experiments was performed on a separate group of compact cumulus cells and human cumulus cells
as described above with the following modifications: a 20-min
preincubation period in M2 media containing 250 ␮M
LY294002 (Sigma) or dimethylsulfoxide (DMSO) vehicle control was performed before 15 min treatment in M2 media containing 5.6 mM D-glucose with or without 500 nM insulin and 250
␮M LY294002 or DMSO vehicle control. For Western blots of
p-Akt and total Akt, compact cumulus cells, human cumulus
cells, and GV-stage oocytes were collected and placed in glucosefree Krebs-Ringer solution with 250 ␮M LY294002 or DMSO
vehicle control for 30 min. Samples were then treated for 15 min
in Krebs-Ringer with 2.7 mM D-glucose, supplemented with or
without 500 nM insulin and 250 ␮M LY294002 or DMSO vehicle
control. Samples were then washed in PBS and frozen at ⫺80 C
before use.
Quantitative RT-PCR (qRT-PCR)
All qRT-PCR were performed on aliquots of compact cumulus cells collected from five to 10 mice, or from individual human
patients. All oocyte samples were GV-stage oocytes collected in
200-oocyte aliquots. Total RNA was isolated from cumulus cell
(n ⫽ 5– 6) or oocyte samples (n ⫽ 5) using the Arcturus PicoPure
RNA kit (Applied Biosystems, Foster City, CA). For positive
control of GLUT4 mRNA expression, total RNA was isolated
from soleus muscle or epigonadal fat (n ⫽ 4 each) from 3-wk-old
ICR female mice using the RNeasy Mini kit (QIAGEN, Valencia,
CA). cDNA was generated from 150 –200 ng RNA using the
Quantitect RT kit (QIAGEN). Quantitative RT-PCR was performed using the 7500 Fast Real-Time PCR System (Applied
Biosystems). Each reaction was run in triplicate and included a
control sample with no reverse transcription and consisted of 10
ng cDNA for cumulus cells, soleus muscle, or fat, or 7 ng cDNA
for oocytes, 1 ⫻ Fast Power SYBR Green PCR System (Applied
Biosystems), and 300 nM validated mouse or human primers for
GLUT1, GLUT4, GLUT8, GLUT12, or ␤-actin mRNA (43).
Quantifications were performed with standard curves generated
with pCR 2.1 TOPO plasmids containing specific GLUT cDNA
amplicons using the ⌬⌬ Ct method as previously described for
these primer sets (43). Product specificity was confirmed by melt
curve analysis and by electrophoresis and visualization of qRTPCR products on a 2% agarose gel.
Statistical analysis
Experimental results are shown as means ⫾ SE. Results from
insulin-stimulated glucose uptake assays were compared using
Student’s t test.
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that IRS-1 is usually associated with glucose metabolism
and insulin-stimulated glucose uptake, whereas IRS-2 is
linked to lipid metabolism (44). We observed consistent
increases in glucose uptake as measured by 2-DG after
insulin treatment in both compact and expanded cumulus
cells (Fig. 2, A and B). This was somewhat surprising because only a handful of tissues display this metabolic property in response to insulin. We measured insulin-stimulated glucose uptake in individual mouse GV-stage
oocytes and in the same experiment used mouse blastocyst-stage embryos as a positive control that is known to
display insulin-stimulated glucose uptake using this assay
(20, 42). We observed no significant change in glucose
uptake in the oocytes (Fig. 2C) and an increase in insulinstimulated glucose uptake in blastocysts (Fig. 2D), similar
to the fold change in glucose uptake that we measure in
cumulus cells (Fig. 2, A and B).
FIG. 1. Representative Western blots showing components of the
insulin-signaling pathway in mouse cumulus cells (cc) and denuded
oocytes (do), and in human cumulus cells. Each lane contains a
separate sample of either 5 ␮g of cumulus cell protein or 100 oocytes.
Each protein was tested in four separate samples of cc or do and
normalized to ␤-actin. Two samples of each tissue are shown. p85,
PI3K-regulatory subunit; p110, PI3K catalytic subunit; Akt, murine
thymoma viral oncogene homolog 1.
Results
Insulin-signaling pathway
Many of the insulin-signaling components necessary
for insulin-stimulated glucose uptake are found at the protein level in both cumulus cells and oocytes (Fig. 1). All of
the proteins found in mouse cumulus cells were also present in human cumulus cells. Most notably, whereas IRS-1
is present in cumulus cells of mice and humans, we did not
detect IRS-1 in oocytes; and the inverse was true for IRS-2.
The IRS-2 detected in the oocyte appeared to be heavily
glycosylated (Fig. 1).
Insulin-stimulated glucose uptake occurs in mouse
cumulus cells but not oocytes
Based on previous evidence of greater glucose metabolism in cumulus cells compared with oocytes and the
observed lack of IRS-1 in oocytes in these studies, we hypothesized that if insulin-stimulated glucose uptake was
present, it may only occur in cumulus cells and be absent
in the oocyte. We came to this conclusion, based on the fact
Inhibition of PI3K pathway or in vivo insulin
resistance blunts insulin-stimulated glucose uptake
in cumulus cells
We had observed that both the p85 and p110 subunits
of PI3K are present in mouse and human cumulus cells, as
well as mouse oocytes (Fig 1). The addition of a specific
PI3K inhibitor, LY294002, was able to significantly decrease insulin-stimulated glucose uptake in mouse (Fig.
3A), and in human cumulus cells there was a trend (P ⬍
0.1) for decreased glucose uptake (Fig. 3C), indicating that
the canonical PI3K pathway is used in these cells. A downstream target of PI3K is Akt, which is phosphorylated in
response to IR activation. We show that the use of
LY294002 also prevents Akt phosphorylation by insulin
in mouse (Fig. 3B) and human (Fig. 3D) cumulus cells. In
contrast, insulin had no effect on Akt phosphorylation in
oocytes, whereas LY294002 decreased Akt phosphorylation both with and without insulin treatment (Supplemental Fig. 1 published on The Endocrine Society’s Journals
Online web site at http://endo.endojournals.org). Mice fed
a high fat diet for 4 wk before measurement of insulinstimulated glucose uptake displayed hyperinsulinemia
and impaired glucose tolerance, as well as moderate
weight gain but did not become hyperglycemic to the point
of being considered diabetic (⬎250 mg/dl; Supplemental
Table 1 and Supplemental Fig. 2). Insulin-stimulated glucose uptake in the cumulus cells of these mice was significantly blunted (Fig. 4A). Similarly, women with PCOS
who also had a higher average BMI than control women
without PCOS also had cumulus cells that displayed
numerically, but not statistically, significant decreased
insulin-stimulated glucose uptake (Fig. 4B). Despite hyperinsulinemia, the cumulus cells from mice fed a high
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Insulin and the Cumulus Oocyte Complex
Endocrinology, May 2012, 153(5):2444 –2454
cDNA in the reaction and observing no
difference in ␤-actin mRNA across
these three tissues. Furthermore, we did
not observe GLUT4 protein in mouse
cumulus cells or oocytes and show soleus muscle as a positive control for
our GLUT4 antibody (Fig. 5E). After
GLUT1, the most abundantly expressed
GLUT in cumulus cells were GLUT8
and GLUT12, and this pattern of gene
expression was similar in both mice and
human cumulus cells (Fig. 5, A and B).
Oocytes also expressed high concentrations of GLUT1 mRNA; however, here
GLUT12 mRNA was more abundant
than GLUT8 mRNA, and total mRNA
expression of any of these transcripts
was more than 2-fold lower than expression in cumulus cells (Fig. 5C).
Western blotting with a positive control for each antibody showed the presence of GLUT1, 8, and 12 in mouse cumulus cells, recapitulating what was
observed from qRT-PCR (Fig. 5E).
Oocyte Western blots showed GLUT1
FIG. 2. Metabolic assays showing insulin-stimulated glucose uptake occurs in cumulus cells,
3
and GLUT12 to be present, similar to
but not in denuded oocytes in mice. Insulin-stimulated uptake of 1,2- H-radiolabeled 2-DG
the qRT-PCR data. However, a band
uptake measured in compact cumulus cells (A) and expanded cumulus cells (B) of mice.
Insulin-stimulated nonradiolabeled 2-DG uptake was also measured in denuded oocytes (C)
around the size of GLUT8 was present
with blastocyst-stage embryos (D) measured in the same experiment as a positive control for
in the oocyte at oversaturated concenthe assay. Uptake for all experiments is expressed as fold change from basal in 2-DG uptake
trations and did not appear as a highly
after 500 nM insulin treatment. For panels A and B, values are means ⫾ SE of four to five
separate experiments; For B and C, values are means ⫾ SE of 30 –35 individual oocytes or
glycosylated smear typical of GLUT
blastocysts. *, P ⬍ 0.05 compared with average basal uptake.
(41) and was not similar to the positive
control. Based on this and the relatively
low abundance of GLUT8 mRNA we
fat diet did not display down-regulation of the IR or
do not believe this band in the oocyte lane to be GLUT8,
IGF-1R (Fig. 4C).
but rather nonspecific binding.
Insulin-stimulated glucose uptake in cumulus cells
occurs without GLUT4
Quantitative real-time PCR and Western blotting were Discussion
used to determine which GLUT may be mediating glucose
uptake in oocytes and cumulus cells. The ubiquitously These are the first studies to show that the cumulus cells
present GLUT1, as well as the putative insulin-sensitive that surround the mammalian oocyte display insulin-stimGLUT4, GLUT8, and GLUT12 were analyzed in cumulus ulated glucose uptake. This is a unique finding; although
cells as well as oocytes. Despite the observed increase in the mitogenic and antiapoptotic effects of insulin are seen
glucose uptake after insulin treatment (Fig. 2), GLUT4 in many cells, the metabolic effect of increased rapid glumRNA was not detected in cumulus cells of mice (Fig. 5A) cose uptake is primarily observed only in muscle and ador humans (Fig. 5B) or in mouse oocytes (Fig. 5C). Due to ipose tissue. A notable exception is the blastocyst stage
this lack of detection, we used these GLUT4 primers on embryo that also displays insulin-stimulated glucose uptissues known to have abundant GLUT4 mRNA expres- take (20). Insulin can signal through its own receptor as
sion, soleus muscle, and fat. In these tissues we observed well as the IGF-1R, although at a lower affinity (45). We
robust GLUT4 mRNA expression and no GLUT4 mRNA detected both the IR and the IGF-1R in cumulus cells and
in cumulus cells (Fig. 5D) when loading equal amounts of in oocytes. Previous work had also detected the IR in
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PI3K, LY294002, blocks insulin-stimulated glucose uptake and GLUT4
translocation to the cell surface (50 –
53), whereas expression of constitutively active PI3K stimulates insulinstimulated glucose uptake (54, 55).
PI3K consists of the regulatory p85 and
catalytic p110 subunits. We show that
both the p85 and p110 subunits are also
present in oocytes and cumulus cells,
and that blocking this insulin-signaling
pathway with LY294002 inhibits insulin-stimulated glucose uptake in cumulus cells of both mice and humans. Similar results are also reported in the
blastocyst stage embryo (56, 57). Akt
phosphorylation by PI3K is required
for insulin-stimulated glucose uptake
(23, 58). In our study, Akt phosphorylation by insulin in cumulus cells
could be inhibited by the addition of
FIG. 3. Insulin-stimulated glucose uptake and Akt phosphorylation are inhibited in mouse
LY294002, showing that insulin sigand human cumulus cells by LY294002, a specific inhibitor of PI3K activity. Mouse (A) or
human (C) cumulus cells were preincubated with either 250 ␮M LY294002 or DMSO vehicle
naling through this pathway occurs.
control before measurement of insulin (500 nM)-stimulated 2-DG uptake. For panels A and C,
We also observed a decrease in Akt
values are means ⫾ SE of four separate experiments. Western blots for phospho-Akt and total
phosphorylation in denuded oocytes
Akt in mouse (B) or human (D) cumulus cells preincubated with 250 ␮M LY294002 or DMSO
control before treatment with or without 500 nM insulin. Western blot experiments were
after treatment with LY294002; howreplicated three times. *, P ⬍ 0.05; †, P ⬍ 0.1 compared with fold change in DMSO-treated
ever, because insulin had no effect on
cells.
glucose uptake in the oocyte, we did not
measure the effect of PI3K inhibition on
mouse cumulus cells and oocytes (13) and the IGF-1R in glucose uptake in the oocyte. The presence of Akt and
granulosa cells of mice and human follicles (46). In both glycogen synthase kinase 3A/B was previously reported in
these studies, however, IR and IGF-1R stimulation was mouse oocytes (13); however, Akt phosphorylation in reonly tested after chronic exposure, not acute activation. sponse to insulin was not assessed. During oocyte meiotic
The activated IR can tyrosine phosphorylate adaptor pro- maturation Akt mRNA increases and is localized along the
teins such as the IRS family, which recruit and phosphor- meiotic spindle (59). Treatment with LY294002 appears
ylate downstream effectors. There are four IRS isoforms, to alter localization of phosphorylated Akt during oocyte
but IRS1 and IRS2 are the most important to glucose me- maturation and impairs polar body extrusion (60). The
tabolism (19) and were studied here, and interestingly effects of LY294002 on Akt in the oocyte could be due to
showed cell-specific expression. We saw IRS1 only in cu- FSH signaling as opposed to insulin.
mulus cells of mice and humans, and IRS2 only in mouse
Both insulin and FSH use components of the PI3K pathoocytes. However, others have detected IRS-2 mRNA in way, and a number of studies have investigated the effect
cumulus cells of women (47) as well as IRS-1 mRNA and of FSH on COC metabolism. In mice, FSH-induced matprotein in rat granulosa cells (48). It is known that IRS1 uration of COC is also associated with increased glucose
and IRS2 can display distinct functions in different cell uptake and lactate production (61). Use of LY294002 intypes (19, 49). Our data would indicate that IRS1 has a hibited FSH-induced glucose uptake and lactate producgreater role in insulin-stimulated glucose uptake in the tion in culture media (61). The interaction of gonadotrocumulus cells because IRS2 was not detected.
pins and cumulus cell metabolism has been noted by others
PI3K is a downstream target of IRS proteins, and sig- that reported increased glucose consumption in cumulus
naling through this pathway is required for translocation cells (62, 63) and increased hexokinase activity in cumulus
of GLUT to the plasma membrane and insulin-stimulated cells after FSH treatment (64). The impact of insulin on
glucose uptake to occur (23, 50). Treatment of adipose or glucose metabolism has been noted in primary cultures of
muscle in vivo and in vitro with the selective inhibitor of human granulosa cells from normal women or women
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Insulin and the Cumulus Oocyte Complex
FIG. 4. A high-fat diet in mice (A) or PCOS in women (B) blunts
insulin-stimulated 2-DG uptake in cumulus cells. A, Mice were fed a
high-fat or control diet for 4 wk before measuring insulin-stimulated
2-DG uptake in cumulus cells. B, Cumulus cells were obtained from
women with PCOS or male factor-only infertility diagnosis, and 2-DG
uptake was measured. C, Western blots of IR and IGF-1R in cumulus
cells of mice fed a high-fat or control diet. Data for 2-DG uptake are
means ⫾ SE of four to five separate experiments. *, P ⬍ 0.05
compared with fold change in control cells. cc, Cumulus cells; Con,
control; HF, high fat.
with PCOS. Two studies reported that glucose uptake and
lactate production in culture media were increased by insulin in a dose-dependent fashion (28, 29) and that granulosa cells from women with PCOS who were also anovulatory (28), or had peripheral insulin resistance (29),
had decreased lactate production in response to insulin.
Differences between the above-mentioned studies and
ours are noteworthy. First, these studies measured glucose
consumption by COC or cultured granulosa cells over a
Endocrinology, May 2012, 153(5):2444 –2454
15- to 48-h time period, and second, they used D-glucose
or assays for D-glucose or lactate production in the media
(61– 63). Glucose is continually metabolized as it enters
the cell and thus cannot be used to accurately measure
uptake or transport alone. In our studies we use the glucose analog 2-DG, which enters the cell, and then is phosphorylated by hexokinase, but not further metabolized,
thus can accurately measure transport and not subsequent
steps in glucose metabolism. However, glucose transport
into the cell slows or stops once intracellular glucose
reaches a certain concentration (65). In our study, we used
a relatively low concentration of 2-DG in our studies (0.1
mM 2-DG and 1.2 ␮M [3H]2-DG) and a short time period
(1 min) to measure uptake during the linear phase and
avoid saturating the intracellular concentration of glucose. Similarly, in isolated adipocytes, insulin-stimulated
2-DG uptake is measured over 1 min (66, 67), and in
blastocyst-stage embryos over 10 min (65).
Glucose uptake is mediated through GLUT, and insulin-stimulated glucose uptake in muscle and fat is mediated through GLUT4 (23). At the transcript level, GLUT4
mRNA has been detected in granulosa cells of the human
(47), rat (48), sheep, and cattle (68, 69). Whereas GLUT4
has been detected in mouse granulosa cells by immunohistochemistry (61) and in rat granulosa cells by Western
blot (48), others failed to detect GLUT4 in rat granulosa
cells by Western blot (70). Here, we used qRT-PCR, with
validated primers that run at a high efficiency in the qRTPCR (43) with a positive control tissue for these primers,
as well as Western blotting with an established antibody
(71) and positive control for that antibody. We did not
detect GLUT4 mRNA or GLUT4 protein in cumulus cells
or oocytes. There are differences in function and gene expression between cumulus and granulosa cells (1–3), including differences in genes involved in glucose metabolism (4), which may explain some of the differences
compared with studies that used only granulosa cells. Additionally, the two studies that did detect GLUT4 protein
(48, 61) used an antibody different from the one used in
our study. One study that did use qRT-PCR to detect
GLUT4 mRNA failed to span an intron (48) and thus
could have detecting genomic DNA, not mRNA. Based on
these observations, insulin-stimulated glucose uptake in
cumulus cells is likely mediated through a GLUT other
than GLUT4. Despite a lack of GLUT4 in cumulus cells,
earlier studies from the GLUT4 knockout mouse indicated
that other insulin-sensitive GLUT may exist (24, 25). Indeed, previous work in our laboratory has shown that
GLUT8 can mediate insulin-stimulated glucose uptake in
blastocyst-stage embryos (20), and recent work from our
laboratory and others indicates that GLUT12 may also be
an insulin sensitive glucose transporter (26, 27). We show
Endocrinology, May 2012, 153(5):2444 –2454
endo.endojournals.org
2451
FIG. 5. qRT-PCR and Western blot analysis of GLUT1, 4, 8, and 12 in cumulus cells of mice and humans, and oocytes of mice. qRT-PCR was
performed in mouse (A) and human (B) cumulus cells; as well as in mouse denuded oocytes (C). An additional GLUT4 qRT-PCR was conducted
with cumulus using soleus muscle and fat as positive controls (D) for the GLUT4 primers. For qRT-PCR, values are means ⫾ SE for five separate
samples. Western blots (E) of cumulus cells (cc) and denuded oocytes (do) with the following positive control samples (⫹): GLUT1, endometrial
stromal cells (esc); GLUT4, soleus muscle; GLUT8, testes from transgenic mice that overexpress GLUT8; GLUT12, soleus muscle from transgenic
mice that overexpress GLUT12. All samples were normalized to ␤-actin.
that both GLUT8 and GLUT12 are present in cumulus
cells of mice and humans and may be responsible for insulin-stimulated glucose uptake in these cells.
Insulin-stimulated glucose uptake into peripheral tissues is impacted by obesity and insulin resistance. Maternal obesity is increasing and affects almost a quarter of US
women (31). A recent study examining 43,163 embryo
transfers from infertility clinics reported that obesity was
associated with a significant decrease in clinical pregnancy
rate with the use of autologous, but not donor, oocytes,
indicating that influence of the oocyte alone can impact
pregnancy outcome (34). Our studies show that peripheral
2452
Purcell et al.
Insulin and the Cumulus Oocyte Complex
insulin sensitivity also has metabolic consequences for the
cumulus cells surrounding the oocyte. We fed mice a high
fat diet for 4 wk, which resulted in moderate weight gain,
impaired glucose tolerance, and hyperinsulinemia. This
diet caused a reduction in insulin-stimulated glucose uptake in the cumulus cells from these mice. In women,
PCOS is a common metabolic disorder associated with
insulin resistance, and anovulation (35, 72). Women with
PCOS are not always obese; however, obesity is more common (73), and the BMI of women with PCOS was greater
than the BMI of control women in our study (34.8 vs. 27.4,
respectively) and consistent with obesity. We did not observe significantly decreased insulin-stimulated glucose
uptake in cumulus cells of women with PCOS compared
with controls; however, our number of women with PCOS
used for this study (n ⫽ 4) was relatively low. We also did
not observe down-regulation of the IR or the IGF-1R in
mice fed a high fat diet; others have observed IR expression
is down-regulated in granulosa cells of women with PCOS
who were also insulin resistant (29). These results from
our high fat-fed mice indicate that the same conditions that
lead to peripheral insulin resistance can also impair insulin-stimulated glucose utilization in the cumulus cells. Alternatively, the blunted response to insulin in the cumulus
cells of high fat-fed mice could be an adaptation to protect
the germ line from elevated insulin concentrations.
In conclusion, these studies identify another tissue that
through a classical insulin-signaling pathway demonstrates insulin-stimulated glucose uptake, albeit through a
nonclassical insulin-sensitive glucose transporter. This
metabolic action of insulin was shown in both a mouse
model and in human cells. Moreover, under in vivo conditions of insulin resistance and/or obesity, this insulin
sensitivity in the cumulus cells is impaired. The cumulus
cells are critically important to oocyte growth, maturation, and metabolism, and thus the oocyte’s ability to develop into a viable embryo. Possibly, the insulin responsiveness of cumulus cells could be used as a biomarker of
oocyte competence in clinical assisted reproduction. Obesity-associated oocyte abnormalities may be due, in part,
to this impaired insulin action in the cumulus cells. Future
studies on the mechanisms of insulin resistance in cumulus
cells will be beneficial.
Acknowledgments
We thank the staff at the Washington University Reproductive
Endocrinology and Infertility in Vitro Fertilization Laboratory,
including Susan Lanzendorf, for collection and processing of
human cumulus cells.
Endocrinology, May 2012, 153(5):2444 –2454
Address all correspondence and requests for reprints to: Kelle
H. Moley, M.D., Department of Obstetrics and Gynecology,
Washington University in St. Louis, 660 South Euclid Avenue, St.
Louis, Missouri 63110. E-mail: [email protected].
This work was supported by National Institutes of Health
Grant R01HD040390 – 07 (to K.H.M.).
This work was funded by T32 HD49305 (to S.H.P.) and R01
HD065435 (to K.H.M.).
Disclosure Summary: The authors have nothing to disclose.
References
1. Latham KE, Bautista FD, Hirao Y, O’Brien MJ, Eppig JJ 1999 Comparison of protein synthesis patterns in mouse cumulus cells and
mural granulosa cells: effects of follicle-stimulating hormone and
insulin on granulosa cell differentiation in vitro. Biol Reprod 61:
482– 492
2. Latham KE, Wigglesworth K, McMenamin M, Eppig JJ 2004 Stagedependent effects of oocytes and growth differentiation factor 9 on
mouse granulosa cell development: advance programming and subsequent control of the transition from preantral secondary follicles
to early antral tertiary follicles. Biol Reprod 70:1253–1262
3. Joyce IM, Pendola FL, Wigglesworth K, Eppig JJ 1999 Oocyte regulation of kit ligand expression in mouse ovarian follicles. Dev Biol
214:342–353
4. Sugiura K, Pendola FL, Eppig JJ 2005 Oocyte control of metabolic
cooperativity between oocytes and companion granulosa cells: energy metabolism. Dev Biol 279:20 –30
5. Albertini DF, Combelles CM, Benecchi E, Carabatsos MJ 2001 Cellular basis for paracrine regulation of ovarian follicle development.
Reproduction 121:647– 653
6. Matzuk MM, Burns KH, Viveiros MM, Eppig JJ 2002 Intercellular
communication in the mammalian ovary: oocytes carry the conversation. Science 296:2178 –2180
7. Leese HJ, Barton AM 1984 Pyruvate and glucose uptake by mouse
ova and preimplantation embryos. J Reprod Fertil 72:9 –13
8. Harris SE, Leese HJ, Gosden RG, Picton HM 2009 Pyruvate and
oxygen consumption throughout the growth and development of
murine oocytes. Mol Reprod Dev 76:231–238
9. Biggers JD, Whittingham DG, Donahue RP 1967 The pattern of
energy metabolism in the mouse oocyte and zygote. Proc Natl Acad
Sci USA 58:560 –567
10. Leese HJ, Barton AM 1985 Production of pyruvate by isolated
mouse cumulus cells. J Exp Zool 234:231–236
11. Harris SE, Adriaens I, Leese HJ, Gosden RG, Picton HM 2007 Carbohydrate metabolism by murine ovarian follicles and oocytes
grown in vitro. Reproduction 134:415– 424
12. Cetica P, Pintos L, Dalvit G, Beconi M 2002 Activity of key enzymes
involved in glucose and triglyceride catabolism during bovine oocyte
maturation in vitro. Reproduction 124:675– 681
13. Acevedo N, Ding J, Smith GD 2007 Insulin signaling in mouse
oocytes. Biol Reprod 77:872– 879
14. Lighten AD, Hardy K, Winston RM, Moore GE 1997 Expression of
mRNA for the insulin-like growth factors and their receptors in
human preimplantation embryos. Mol Reprod Dev 47:134 –139
15. Zhang X, Kidder GM, Watson AJ, Schultz GA, Armstrong DT 1994
Possible roles of insulin and insulin-like growth factors in rat preimplantation development: investigation of gene expression by reverse transcription-polymerase chain reaction. J Reprod Fertil 100:
375–380
16. Watson AJ, Hogan A, Hahnel A, Wiemer KE, Schultz GA 1992
Expression of growth factor ligand and receptor genes in the preimplantation bovine embryo. Mol Reprod Dev 31:87–95
Endocrinology, May 2012, 153(5):2444 –2454
17. Quesnel H 1999 Localization of binding sites for IGF-I, insulin and
GH in the sow ovary. J Endocrinol 163:363–372
18. Samoto T, Maruo T, Ladines-Llave CA, Matsuo H, Deguchi J, Barnea ER, Mochizuki M 1993 Insulin receptor expression in follicular
and stromal compartments of the human ovary over the course of
follicular growth, regression and atresia. Endocr J 40:715–726
19. Thirone AC, Huang C, Klip A 2006 Tissue-specific roles of IRS
proteins in insulin signaling and glucose transport. Trends Endocrinol Metab 17:72–78
20. Carayannopoulos MO, Chi MM, Cui Y, Pingsterhaus JM, McKnight RA, Mueckler M, Devaskar SU, Moley KH 2000 GLUT8 is a
glucose transporter responsible for insulin-stimulated glucose uptake in the blastocyst. Proc Natl Acad Sci USA 97:7313–7318
21. Joost HG, Bell GI, Best JD, Birnbaum MJ, Charron MJ, Chen YT,
Doege H, James DE, Lodish HF, Moley KH, Moley JF, Mueckler M,
Rogers S, Schürmann A, Seino S, Thorens B 2002 Nomenclature of
the GLUT/SLC2A family of sugar/polyol transport facilitators. Am J
Physiol Endocrinol Metab 282:E974 –E976
22. Wu X, Freeze HH 2002 GLUT14, a duplicon of GLUT3, is specifically expressed in testis as alternative splice forms. Genomics 80:
553–557
23. Leney SE, Tavaré JM 2009 The molecular basis of insulin-stimulated glucose uptake: signalling, trafficking and potential drug targets. J Endocrinol 203:1–18
24. Katz EB, Stenbit AE, Hatton K, DePinho R, Charron MJ 1995 Cardiac and adipose tissue abnormalities but not diabetes in mice deficient in GLUT4. Nature 377:151–155
25. Stenbit AE, Burcelin R, Katz EB, Tsao TS, Gautier N, Charron MJ,
Le Marchand-Brustel Y 1996 Diverse effects of Glut 4 ablation on
glucose uptake and glycogen synthesis in red and white skeletal
muscle. J Clin Invest 98:629 – 634
26. Purcell SH, Aerni-Flessner LB, Willcockson AR, Diggs-Andrews
KA, Fisher SJ, Moley KH 2011 Improved Insulin Sensitivity by
GLUT12 Overexpression in Mice. Diabetes 60:1478 –1482
27. Stuart CA, Howell ME, Zhang Y, Yin D 2009 Insulin-stimulated
translocation of glucose transporter (GLUT) 12 parallels that of
GLUT4 in normal muscle. J Clin Endocrinol Metab 94:3535–3542
28. Rice S, Christoforidis N, Gadd C, Nikolaou D, Seyani L, Donaldson
A, Margara R, Hardy K, Franks S 2005 Impaired insulin-dependent
glucose metabolism in granulosa-lutein cells from anovulatory
women with polycystic ovaries. Hum Reprod 20:373–381
29. Fedorcsák P, Storeng R, Dale PO, Tanbo T, Abyholm T 2000 Impaired insulin action on granulosa-lutein cells in women with polycystic ovary syndrome and insulin resistance. Gynecol Endocrinol
14:327–336
30. Robker RL 2008 Evidence that obesity alters the quality of oocytes
and embryos. Pathophysiology 15:115–121
31. Vahratian A 2009 Prevalence of overweight and obesity among
women of childbearing age: results from the 2002 National Survey
of Family Growth. Matern Child Health J 13:268 –273
32. Gesink Law DC, Maclehose RF, Longnecker MP 2007 Obesity and
time to pregnancy. Hum Reprod 22:414 – 420
33. Rich-Edwards JW, Goldman MB, Willett WC, Hunter DJ, Stampfer
MJ, Colditz GA, Manson JE 1994 Adolescent body mass index and
infertility caused by ovulatory disorder. Am J Obstet Gynecol 171:
171–177
34. Luke B, Brown MB, Stern JE, Missmer SA, Fujimoto VY, Leach R
2011 Female obesity adversely affects assisted reproductive technology (ART) pregnancy and live birth rates. Hum Reprod 26:245–
252
35. Jungheim ES, Schoeller EL, Marquard KL, Louden ED, Schaffer JE,
Moley KH 2010 Diet-induced obesity model: abnormal oocytes and
persistent growth abnormalities in the offspring. Endocrinology
151:4039 – 4046
36. Wu LL, Dunning KR, Yang X, Russell DL, Lane M, Norman RJ,
Robker RL 2010 High-fat diet causes lipotoxicity responses in cu-
endo.endojournals.org
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
2453
mulus-oocyte complexes and decreased fertilization rates. Endocrinology 151:5438 –5445
Yang X, Dunning KR, Wu LL, Hickey TE, Norman RJ, Russell DL,
Liang X, Robker RL 2010 Identification of perilipin-2 as a lipid
droplet protein regulated in oocytes during maturation. Reprod Fertil Dev 22:1262–1271
Igosheva N, Abramov AY, Poston L, Eckert JJ, Fleming TP, Duchen
MR, McConnell J 2010 Maternal diet-induced obesity alters mitochondrial activity and redox status in mouse oocytes and zygotes.
PLoS One 5:e10074
Minge CE, Bennett BD, Norman RJ, Robker RL 2008 Peroxisome
proliferator-activated receptor-␥ agonist rosiglitazone reverses the
adverse effects of diet-induced obesity on oocyte quality. Endocrinology 149:2646 –2656
Marshall BA, Ren JM, Johnson DW, Gibbs EM, Lillquist JS, Soeller
WC, Holloszy JO, Mueckler M 1993 Germline manipulation of
glucose homeostasis via alteration of glucose transporter levels in
skeletal muscle. J Biol Chem 268:18442–18445
Frolova A, Flessner L, Chi M, Kim ST, Foyouzi-Yousefi N, Moley
KH 2009 Facilitative glucose transporter type 1 is differentially regulated by progesterone and estrogen in murine and human endometrial stromal cells. Endocrinology 150:1512–1520
Moley KH, Chi MM, Mueckler MM 1998 Maternal hyperglycemia
alters glucose transport and utilization in mouse preimplantation
embryos. Am J Physiol 275:E38 –E47
Frolova AI, Moley KH 2011 Quantitative analysis of glucose transporter mRNAs in endometrial stromal cells reveals critical role of
GLUT1 in uterine receptivity. Endocrinology 152:2123–2128
Taniguchi CM, Ueki K, Kahn R 2005 Complementary roles of IRS-1
and IRS-2 in the hepatic regulation of metabolism. J Clin Invest
115:718 –727
Saltiel AR, Kahn CR 2001 Insulin signalling and the regulation of
glucose and lipid metabolism. Nature 414:799 – 806
Zhou J, Bondy C 1993 Anatomy of the human ovarian insulin-like
growth factor system. Biol Reprod 48:467– 482
Robker RL, Akison LK, Bennett BD, Thrupp PN, Chura LR, Russell
DL, Lane M, Norman RJ 2009 Obese women exhibit differences in
ovarian metabolites, hormones, and gene expression compared with
moderate-weight women. J Clin Endocrinol Metab 94:1533–1540
Dorfman M, Ramirez VD, Stener-Victorin E, Lara HE 2009 Chronic-intermittent cold stress in rats induces selective ovarian insulin
resistance. Biol Reprod 80:264 –271
Karlsson HK, Zierath JR 2007 Insulin signaling and glucose transport in insulin resistant human skeletal muscle. Cell Biochem Biophys 48:103–113
Shepherd PR, Navé BT, O’Rahilly S 1996 The role of phosphoinositide 3-kinase in insulin signalling. J Mol Endocrinol 17:175–184
Cheatham B, Vlahos CJ, Cheatham L, Wang L, Blenis J, Kahn CR
1994 Phosphatidylinositol 3-kinase activation is required for insulin
stimulation of pp70 S6 kinase, DNA synthesis, and glucose transporter translocation. Mol Cell Biol 14:4902– 4911
Clarke JF, Young PW, Yonezawa K, Kasuga M, Holman GD 1994
Inhibition of the translocation of GLUT1 and GLUT4 in 3T3–L1
cells by the phosphatidylinositol 3-kinase inhibitor, wortmannin.
Biochem J 300:631– 635
Okada T, Kawano Y, Sakakibara T, Hazeki O, Ui M 1994 Essential
role of phosphatidylinositol 3-kinase in insulin-induced glucose
transport and antilipolysis in rat adipocytes. Studies with a selective
inhibitor wortmannin. J Biol Chem 269:3568 –3573
Frevert EU, Kahn BB 1997 Differential effects of constitutively active phosphatidylinositol 3-kinase on glucose transport, glycogen
synthase activity, and DNA synthesis in 3T3–L1 adipocytes. Mol
Cell Biol 17:190 –198
Katagiri H, Asano T, Ishihara H, Inukai K, Shibasaki Y, Kikuchi M,
Yazaki Y, Oka Y 1996 Overexpression of catalytic subunit p110␣
of phosphatidylinositol 3-kinase increases glucose transport activity
2454
56.
57.
58.
59.
60.
61.
62.
63.
Purcell et al.
Insulin and the Cumulus Oocyte Complex
with translocation of glucose transporters in 3T3–L1 adipocytes.
J Biol Chem 271:16987–16990
Riley JK, Carayannopoulos MO, Wyman AH, Chi M, Ratajczak
CK, Moley KH 2005 The PI3K/Akt pathway is present and functional in the preimplantation mouse embryo. Dev Biol 284:377–386
Riley JK, Carayannopoulos MO, Wyman AH, Chi M, Moley KH
2006 Phosphatidylinositol 3-kinase activity is critical for glucose
metabolism and embryo survival in murine blastocysts. J Biol Chem
281:6010 – 6019
Khan AH, Pessin JE 2002 Insulin regulation of glucose uptake: a
complex interplay of intracellular signalling pathways. Diabetologia 45:1475–1483
Cecconi S, Rossi G, Santilli A, Stefano LD, Hoshino Y, Sato E,
Palmerini MG, Macchiarelli G 2010 Akt expression in mouse
oocytes matured in vivo and in vitro. Reprod Biomed Online 20:
35– 41
Hoshino Y, Yokoo M, Yoshida N, Sasada H, Matsumoto H, Sato
E 2004 Phosphatidylinositol 3-kinase and Akt participate in the
FSH-induced meiotic maturation of mouse oocytes. Mol Reprod
Dev 69:77– 86
Roberts R, Stark J, Iatropoulou A, Becker DL, Franks S, Hardy K
2004 Energy substrate metabolism of mouse cumulus-oocyte complexes: response to follicle-stimulating hormone is mediated by the
phosphatidylinositol 3-kinase pathway and is associated with
oocyte maturation. Biol Reprod 71:199 –209
Hillier SG, Purohit A, Reichert Jr LE 1985 Control of granulosa cell
lactate production by follicle-stimulating hormone and androgen.
Endocrinology 116:1163–1167
Zuelke KA, Brackett BG 1992 Effects of luteinizing hormone on
glucose metabolism in cumulus-enclosed bovine oocytes matured in
vitro. Endocrinology 131:2690 –2696
Endocrinology, May 2012, 153(5):2444 –2454
64. Downs SM, Humpherson PG, Martin KL, Leese HJ 1996 Glucose
utilization during gonadotropin-induced meiotic maturation in cumulus cell-enclosed mouse oocytes. Mol Reprod Dev 44:121–131
65. Hansen PA, Gulve EA, Holloszy JO 1994 Suitability of 2-deoxyglucose for in vitro measurement of glucose transport activity in skeletal muscle. J Appl Physiol 76:979 –985
66. Gustavsson J, Parpal S, Strålfors P 1996 Insulin-stimulated glucose
uptake involves the transition of glucose transporters to a caveolaerich fraction within the plasma membrane: implications for type II
diabetes. Mol Med 2:367–372
67. Turban S, Beardmore VA, Carr JM, Sakamoto K, Hajduch E, Arthur JS, Hundal HS 2005 Insulin-stimulated glucose uptake does not
require p38 mitogen-activated protein kinase in adipose tissue or
skeletal muscle. Diabetes 54:3161–3168
68. Nishimoto H, Matsutani R, Yamamoto S, Takahashi T, Hayashi
KG, Miyamoto A, Hamano S, Tetsuka M 2006 Gene expression of
glucose transporter (GLUT) 1, 3 and 4 in bovine follicle and corpus
luteum. J Endocrinol 188:111–119
69. Williams SA, Blache D, Martin GB, Foot R, Blackberry MA, Scaramuzzi RJ 2001 Effect of nutritional supplementation on quantities
of glucose transporters 1 and 4 in sheep granulosa and theca cells.
Reproduction 122:947–956
70. Kodaman PH, Behrman HR 1999 Hormone-regulated and glucosesensitive transport of dehydroascorbic acid in immature rat granulosa cells. Endocrinology 140:3659 –3665
71. Marshall BA, Murata H, Hresko RC, Mueckler M 1993 Domains
that confer intracellular sequestration of the Glut4 glucose transporter in Xenopus oocytes. J Biol Chem 268:26193–26199
72. Dumesic DA, Abbott DH 2008 Implications of polycystic ovary
syndrome on oocyte development. Semin Reprod Med 26:53– 61
73. Norman RJ, Dewailly D, Legro RS, Hickey TE 2007 Polycystic
ovary syndrome. Lancet 370:685– 697
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