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POLYCYSTIC ovary syndrome (PCOS) affects
approximately 7-12% of women, and is the most com-
mon cause of infertility in reproductive aged women
[1]. Reproductive endocrine abnormalities in PCOS
include amenorrhea or oligomenorrhea, infertility,
hirsutism, and acne resulting from increased ovar-
ian androgen production [2-6]. Although there have
been reports suggesting as high of 50% of women with
PCOS have hyperinsulinemia and peripheral insulin
resistance in adipose and skeletal muscle, ovarian theca
and granulosa cells have been reported to be exquisitely
sensitive to insulin and are not insulin resistant. Thus,
a dichotomy is present with peripheral tissue insulin
resistance and ovarian insulin sensitivity.
We have proposed an inositol imbalance consist-
ing of excess myo-inositol and decient chiro-inositol
as a measure of insulin resistance [7]. This imbalance
Endocrine Journal 2014, 61 (2), 111-117
Decreased myo-inositol to chiro-inositol (M/C) ratios and
increased M/C epimerase activity in PCOS theca cells
demonstrate increased insulin sensitivity compared to controls
Douglas Heimark 1), Jan McAllister 2) and Joseph Larner 1)
1) Department of Pharmacology, University of Virginia, Charlottesville, VA 22903 USA
2) Department of Pathology, The Pennsylvania State University College of Medicine, Hershey, PA, 17033 USA
Abstract. Previous studies from our and other labs have shown that insulin resistance is associated with an inositol
imbalance of excess myo-inositol and decient chiro-inositol together with a deciency of myo-inositol to chiro-inositol
epimerase in vivo and in vitro. In this report, we utilized well characterized theca cells from normal cycling women, with
normal insulin sensitivity, and theca cells from women with polycystic ovary syndrome (PCOS), with increased insulin
sensitivity to examine the myo-inositol to chiro-inisitol (M/C) ratio and the myo-inositol to chiro-inositol epimerase
activity. PCOS theca cells with increased insulin sensitivity were specically used to investigate whether the inositol
imbalance and myo-inositol to chiro-inositol epimerase are regulated in a similar or the opposite direction than that observed
in insulin resistant cells. The results of these studies are the rst to demonstrate that in insulin sensitive PCOS theca cells
the inositol imbalance goes in the opposite direction to that observed in insulin resistant cells, and there is a decreased M/C
ratio and an increased myo-inositol to chiro-inositol epimerase activity. Further biochemical and genetic studies will probe
the mechanisms involved.
Key words: Chiro-Inositol, Myo-Inositol, Epimerase, Polycystic ovary syndrome, Theca
has been demonstrated in urine of type 2 diabetics [8],
Rhesus monkeys progressing from normal to obese to
diabetic [8], in muscle biopsies of type 2 diabetics dur-
ing an insulin clamp [8], as well as in autopsy muscle
specimens of type 2 diabetics [9]. In addition, de-
ciency of chiro-inositol in urine of Japanese subjects
has been demonstrated to be linearly related to insulin
resistance in type 2 diabetics, subjects with impaired
glucose tolerance and normal subjects [10]. Further,
lack of appearance of chiro-inositol glycan bioactiv-
ity (measured as PDH phosphatase (PDHP) activity)
in blood of type 2 diabetic subjects during a glucose
tolerance test, lack of chiro-inositol glycan bioactiv-
ity in women with PCOS during an insulin clamp, and
lack of appearance of chiro-inositol glycan bioactivity
release from placental membranes with insulin admin-
istration in vitro in women with preeclampsia have
also been shown [11-13]. One glycan isolated from
beef liver, named INS-2, has been identied, struc-
ture determined and chemically synthesized. Its struc-
ture is galactosamine-β-1,4 pinitol (3-O-methyl ether
of D-Chiro-Inositol). It is insulin mimetic and insulin
Submitted Jun. 25, 2013 as EJ13-0265; Accepted Oct. 16, 2013 as EJ13-0423
Released online in J-STAGE as advance publication Nov. 2, 2013
Correspondence to: Jan McAllister, Professor Pathology/OB/GYN,
Hershey College of Medicine, The Pennsylvania State University,
Hershey, PA 17033 USA. E-mail: jxm63@psu.edu
Or i g i n a l
©The Japan Endocrine Society
112 Heimark et al.
ing previously described growth medium (1:1 mixture
of Dulbecco’s Eagles Medium (DME) and Hams F-12
medium containing 5% FBS, 5% horse serum (HS),
2% UltroSer G, 20 nM insulin, 20 nM selenium, 1 µM
vitamin E and antibiotics). The cells were grown in
reduced oxygen tension (5% O2, 90% N2, and 5% CO2)
and given supplemental antioxidants (vitamin E and
selenium) to prevent oxidative damage.
The theca cell cultures utilized in these studies
were described and functionally characterized previ-
ously [18-20]. Experiments comparing PCOS and nor-
mal theca were performed utilizing 4th-passage (31-38
population doublings) theca cells isolated from size-
matched follicles obtained from age-matched subjects.
The use of fourth passage cells allowed us to perform
multiple experiments from the same patient population,
and were propagated from frozen stocks of second pas-
sage cells in the media described above. For all stud-
ies, theca cell cultures obtained from at least 5 inde-
pendent normal and 5 independent PCOS patients were
examined. The passage conditions and split ratios for
all normal and PCOS cells were identical. For each of
the experiments outlined in these studies fourth pas-
sage theca cells cells were grown to subconuence
and transferred into serum-free medium, containing
DMEM/F12 1.0 mg/mL BSA, 100 µg/mL transferrin,
20 nM insulin, 20 nM selenium, 1.0 µM vitamin E and
antibiotics, 48 h prior to being rinsed in PBS, ash fro-
zen and processed for assay of myo-inositol to chiro-
inositol epimerase.
The PCOS and normal ovarian tissue came from
age-matched women, 28-40 years old. The diagno-
sis of PCOS was made according to established guide-
lines [21], including hyperandrogenemia, oligoovula-
tion, and the exclusion of 21α-hydroxylase deciency,
Cushing’s syndrome, and hyperprolactinemia. All of
the PCOS theca cell preparations studied came from
ovaries of women with fewer than six menses per
year and elevated serum total testosterone or bioavail-
able testosterone levels, as previously described [18,
22, 23]. Each of the PCOS ovaries contained multi-
ple subcortical follicles of less than 10 mm in diame-
ter. The control (normal) theca cell preparations came
from ovaries of fertile women with normal menstrual
histories, menstrual cycles of 21-35 days, and no clini-
cal signs of hyperandrogenism. Neither PCOS nor nor-
mal subjects were receiving hormonal medications at
the time of surgery. Indications for surgery were dys-
functional uterine bleeding, endometrial cancer, and/or
sensitizing in vivo and in vitro [14].
Based on these studies of inositol imbalance; i.e.
increased M/C ratio and of chiro-inositol glycan de-
ciency associated with insulin resistance, we hypothe-
sized a defective epimerization of myo-inositol to chi-
ro-inositol, an inversion of carbon 3 hydroxyl [7], as a
cause of the inositol imbalance and chiro-inositol gly-
can deciency. We next demonstrated in vivo in the
GK type 2 diabetic rat that in the insulin sensitive tis-
sues, muscle, liver and fat [3H]myo-inositol conversion
to [3H]chiro-inositol was reduced from about 20–30%
to under 5% [15]. We partially puried the myo-inos-
itol to chiro-inositol epimerase from rat liver and dem-
onstrated its absolute requirement for nucleotide, indi-
cating that it acted via an oxido-reductive mechanism
[16]. Analyses of tissue extracts from type 2 diabetic
GK rat tissues compared to control Wistars demon-
strated reduced epimerase enzyme activity [16].
As mentioned above, it is well established that ovar-
ian cells from PCOS subjects are insulin sensitive com-
pared to peripheral tissues, which are insulin resistant.
In view of the above mentioned results demonstrating
increased M/C ratios and decreased myo-inositol to
chiro-inositol epimerase activity associated with insu-
lin resistance in type 2 diabetes, GK type 2 diabetic rat
and the lack or deciency of chiro-inositol glycan in
type 2 diabetes, PCOS and preeclampsia, we wished
to determine whether ovarian theca cells from women
with PCOS with increased ovarian insulin sensitivity
demonstrated the opposite, i.e. decreased M/C ratios
and increased epimerase activity. We now show this
is in fact the case.
Research Design and Methods
Human theca interna tissue was obtained from fol-
licles of women undergoing hysterectomy, following
informed consent under a protocol approved by the
Institutional Review Board of the Pennsylvania State
University College of Medicine. Individual follicles
were dissected away from ovarian stroma, dissected,
and dispersed with 0.05% collagenase I, 0.05% colla-
genase IA, and 0.01% deoxyribonuclease, in medium
containing 10% fetal bovine serum (FBS), as previ-
ously described [17]. The isolated follicles were size-
selected for diameters ranging from 3-5 mm so that
theca cells derived from follicles of similar size from
normal and PCOS subjects could be compared. Theca
cells were cultured on bronectin coated dishes utiliz-
113
Dec M/C incr epimerase in theca cells
and dried in vacuo in order to remove excess HCl.
Samples were reconstituted in 1 mL H2O and
then loaded onto a 3 mL bed volume mixed-bed ion
exchange column and the pass-through collected.
Column was washed with an additional aliquot of 19
mL H2O. Samples were then dried in vacuo prior to
analysis.
Samples reconstituted in 400 µL H2O were centri-
fuged through a 0.2 μm lter and the ltrate injected
onto Dionex MA-1 HPLC column. Myo-inositol and
chiro-inositol were detected with an electrochemical
detector consisting of a Au electrode and a Ag/AgCl
reference electrode. The solvent was 100 mM NaOH
running at 0.4 mL/min. The detected peaks were inte-
grated and quantitated using Dionex PeakNet 6.4 soft-
ware by comparing the area of the unknown to the area
of the known standard (D-chiro-inositol was obtained
from Cyvex, Inc. and myo-inositol from Sigma-Aldrich
Chemical Company). The amounts of myo-inositol and
chiro-inositol were expressed as nmoles and the ratio
of myo-inositol to chiro-inositol (M/C) calculated.
For all the above experiments, Sera and growth fac-
tors were obtained from the following sources: FBS
and DME/F12 (Irvine Scientic, Irvine, CA): horse
serum (Life Technologies, Grand Island, NY); UltroSer
G (Reactifs IBF, Villeneuve-la-Garenne, France): other
compounds were purchased from Sigma (St. Louis,
MO). All other chemicals were analytical grade or bet-
ter and purchased from Cyvex Inc., Fisher Scientic
and Sigma-Aldrich Chemical Company.
Statistical analysis
Unpaired t-test was performed using GraphPad
Prism version 5.0f for Mac, GraphPad Software, San
Diego California USA, www.graphpad.com.
Results
The myo-inositol to chiro-inositol epimerase activi-
ties and M/C ratios for both normal and PCOS ovar-
ian theca cells are plotted separately as shown in Figs
1 and 2. Fig. 1 shows the epimerase values in a scat-
tergram with mean ± SE as shown by horizontal lines.
The myo-inositol to chiro-inositol epimerase specic
activity (Units/µg protein) mean value for PCOS is 3
times as high as that for the normals (0.017 ± 0.003
(n=11) vs. 0.006 ± 0.002 (n=10), resp.). There is also
more scatter for the PCOS ovarian theca cells values
than for the normal ovarian theca cells (0.002 → 0.034
pelvic pain.
Ovarian theca cells from women with polycystic
ovarian syndrome and control normals were cultured,
scraped, processed and analyzed for myo-inositol con-
tent, chiro-inositol content (plotted as a M/C ratio) and
a myo-inositol to chiro-inositol epimerase assay was
performed. Data obtained from these assays were plot-
ted as scattergrams. The mean ± SE was determined
and plotted as horizontal lines for normal and PCOS
tissues. Unpaired t-tests were performed.
Myo-inositol to chiro-inositol epimerase
Plates were partially thawed at 4ºC. 500 μL of 10
mM HEPES pH 7 with protease inhibitor cocktail con-
taining 1 mM AEBSF, 1:200 of 1.4 mg/mL protein
stock aprotinin, 10 μM leupeptin, 10 μM pepstatin, 10
μM E64 and 1 mM mercaptoethanol was added (10
HPic). Cells were scraped, transferred to microcentri-
fuge tubes and homogenized by hand using a microcen-
trifuge tube homogenizer. Samples were centrifuged
for 5 min at 4ºC. Assay tubes containing ± nucleotides
(1 mM ea NAD+, NADP, NADH and NADPH), 1 mM
nicotinamide, 1 mM MgCl2, 1 mM myo-inositol, 10
HPic were set up in total volume of 450 μL. Fifty
μL of the cell supernatant from above centrifugation
was added at timed intervals, vortexed, covered and
placed in a 37°C incubator for 6 hours with gentle mix-
ing. To stop the reaction 2 mL of ice-cold abs. ethanol
was added, vortexed and incubated on ice for 20 min.
Samples were dried in vacuo.
Samples were then processed as described below
for chiro-inositol content. Total Units were calculated
by subtracting the chiro-inositol content in the minus
nucleotides tube from the chiro-inositol content in the
plus nucleotides tube. Protein was analyzed by modi-
ed Bradford Protein Assay (Pierce® 660 nm Protein
Assay kit). The Specic Activity (Units/μg protein)
was calculated by dividing the Total Units by the Total
Protein.
Myo-inositol and chiro-inositol content
Centrifugation lters were purchased from Fisher.
An aliquot from the ovarian theca cell homogenate was
transferred to a ame-seal ampoule, diluted to 6N HCl
from 12N HCl, ame sealed and hydrolyzed at 100ºC
for 48 hours. Samples were then transferred to centri-
fuge tubes, with the original vial washed three times
with HPLC grade H2O and dried in vacuo. Several 400
µL aliquots of H2O were added to the centrifuge vial
114 Heimark et al.
CYP11 mRNA abundance results from both increased
transactivation of the promoter and augmented mRNA
stability in PCOS cells [20, 29] [23]. Moreover the
5’ untranslated region of CYP17A1 and CYP11A1
mRNA have been shown to confer increased mRNA
half life in PCOS theca cells as compared to normal
theca cells, thus increasing CYP17A1 and CYP11A1
expression and androgen production in PCOS theca
cells [23, 29].
Insulin acting through the insulin receptor stimu-
lates androgen (i.e., testosterone) production in theca
cells, as does LH acting via cAMP [26]. Antibody
blockade of the insulin receptor abolished insulin’s
stimulatory action, whereas effective antibody block-
ade of the insulin-like growth factor 1 receptor did
not alter insulin’s stimulation of theca cell testoster-
one biosynthesis [26]. Insulin’s action on PCOS theca
cells to produce testosterone is massively greater than
on control theca cells [26], a measure of markedly
increased insulin sensitivity. Nestler et al. have ele-
gantly shown that chiro-inositol glycans are the sig-
nal transduction system for theca cell testosterone
synthesis. A chiro-inositol containing glycan (INS-2)
increased theca testosterone biosynthesis similarly to
insulin [30]. Further, an INS-2, anti inositol glycan
antibody, abolished insulin’s stimulatory effect, but
not that of hCG [30]. These ndings suggest that inos-
itol glycans serve as the signal transduction system
for insulin’s stimulation of human theca cell testos-
terone biosynthesis. Specically, they demonstrated
that external INS-2 activates theca cell testosterone
production dose dependently as effectively as insulin.
vs. 0.0005 → 0.019, resp.).
Fig. 2 shows the M/C ratio in a scattergram with
mean ± SE as shown by horizontal lines. The mean
value for the M/C ratio of normals is about 4 times as
high as that for PCOS (18 ± 3 (n=6) vs. 5 ± 2 (n=7),
resp. and a range of (7 → 24 vs. 2 → 15, resp.). These
results are in keeping what is seen with the epimerase
results, and demonstrate that the PCOS ovary is insulin
sensitive compared to control by these two parameters.
Two-tailed unpaired t-test (p < 0.05 was considered
statistically signicant) was performed resulting in p <
0.01 vs. PCOS for the epimerase and p < 0.002 vs. nor-
mals for the M/C ratios.
Discussion
Ovarian theca cells are recognized as one of the pri-
mary sources of excess androgen biosynthesis in the
PCOS ovary [3, 24-26]. Using long-term cultures of
normal and PCOS theca cells grown for successive
population doublings in long-term culture, we have
demonstrated that androgen production is elevated in
theca cells isolated from the ovaries of women with
PCOS, as compared to theca cells from the ovaries
of normal cycling women [18, 27]. This increase in
androgen production in PCOS theca cells results from
increased mRNA accumulation of several steroido-
genic enzymes, including cholesterol side chain cleav-
age (CYP11A1), 17α-hydroxylase (CYP17A1), and
HSD3B2 [18, 28]. Extensive examination of CYP17A1
and CYP11A1 gene expression in normal and PCOS
theca cells has revealed that increased CYP17 and
Fig. 1 Myo-inositol to chiro-inositol epimerase assay
Data points for PCOS and normals shown in scatter plot
with mean ± SE shown as horizontal bars. ***, p < 0.01 vs.
PCOS.
Fig. 2 Myo-inositol to chiro-inositol ratios
Data points for PCOS and Normals shown in scatter plot
with mean ± SE shown as horizontal bars. *, p < 0.002 vs.
normals.
115
Dec M/C incr epimerase in theca cells
myo-inositol to chiro-inositol epimerase activity and
a decreased myo-inositol to chiro-inositol ratio. In
all previous papers, we have provided evidence for
decreased epimerase activity and increased myo-inosi-
tol to chiro-inositol ratios in cases of insulin resistance
[7-16].
Our experiments do not shed light on the continu-
ing enigma of the primacy of increased testosterone
versus the peripheral insulin resistance as the initiating
event in PCOS. They do however provide evidence
for the utility of both myo-inositol and chiro-inositol
as effective agents in treatment. Certainly, a balance
between the two inositols is required for normal phys-
iological function and regulation of the myo-inositol
to chiro-inositol epimerase opens a new avenue for
future studies. Thus, IP3 [41], IP2 [42] and IP7 [43]
as well as INS-2 [44] all inositol-containing molecules
have been shown to act to allosterically control insu-
lin signaling.
In this connection it is important to point out that
the presence of the myo-inositol to chiro-inositol
epimerase has been questioned in a recent paper by
Lin, Gopalan and Ostlund entitled, “D-chiro-inositol
is Absorbed but not Synthesized” [45]. The authors
fed rats a chiro-inositol free diet for 10 or 12 weeks
and then tested for chiro-inositol synthesis with heavy
water or labeled myo-inositol. They conclude that
there is no synthesis of chiro-inositol. Their fail-
ure in logic derives from the omission of the phrase
“under our conditions”; i.e. the prolonged chiro-inos-
itol free diet. The authors further fail to cite our in
vivo epimerization of [3H]myo-inositol to [3H]chiro-
inositol [15] and our partial purication, characteriza-
tion with a demonstration of an absolute nucleotide
requirement for a rat liver epimerase [16]. As stated
above, in both in vivo and in vitro experiments, epim-
erase enzyme activity [16] was reduced in vivo in dia-
betic animals and in diabetic tissue extracts [15].
The present data on the decreased M/C ratios and
the increased myo-inositol to chiro-inositol epimerase
activity in PCOS ovarian theca cells further strength-
ens our argument that these two parameters are associ-
ated with insulin resistance and sensitivity. They open
a new area of insight into this much-studied area.
Conict of Interest
No conict of interest for all authors of this manuscript.
Extracellular inositol glycan generation [31] as well as
an ATP dependent inositol glycan transporter in liver
have been demonstrated [32].
Our new data demonstrating increased M/C epim-
erase and decreased M/C ratios in PCOS theca cells
provides a mechanistic explanation of how the PCOS
ovary is more insulin sensitive. Increased M/C epi-
merase would provide increased chiro-inositol to be
incorporated into precursor GPI-phospholipid and or
precursor GPI-protein, which could then be cleaved
into INS-2, thus enhancing insulin sensitivity by
increasing glucose disposal [14]. INS-2 allosterically
activates protein phosphatase PP2Cα to activate GS
and mitochondrial PDHP to activate pyruvate dehy-
drogenase (PDH), both Mg2+ or Mn2+ requiring phos-
phatases (PPM family) leading to intracellular non-ox-
idative and oxidative glucose disposal and reduction of
hyperglycemia [33]. This increased insulin sensitiv-
ity would also increase testosterone supply to induce
peripheral insulin resistance. Mechanisms of testos-
terone inducing peripheral insulin resistance are not
fully understood, but in an animal model, decreased
GLUT4 and decreased glucose transport was observed
[34]. In other studies, increased insulin sensitivity with
administered testosterone has been observed [35, 36].
How the ovary is induced to increased insulin sensi-
tivity via ovarian upstream mechanisms is unclear and
requires further experimentation.
It is abundantly clear that there is marked periph-
eral insulin resistance in vivo in PCOS subjects [37].
However, when peripheral tissue cell lines from PCOS
subjects are tested for the stability of the insulin resis-
tance in vitro, variable results are seen [38, 39]. Thus
at present there is not yet an agreed upon cell line
that demonstrated stable insulin resistance in PCOS
in vitro. For this reason we have not studied inosi-
tols and epimerase in peripheral cell lines from PCOS
subjects.
In a paper entitled “The D-Chiro-Inositol Paradox
in the Ovary” [40], the authors speculate that “PCOS
patients with hyperinsulinemia likely present an
enhanced MI to DCI epimerization in the ovary; this
would result in an increased DCI/MI ratio (i.e. over-
production of DCI), which would in turn would lead to
a MI deciency in the ovary.”
The present data with theca cells from PCOS sub-
jects and controls demonstrates that this is indeed the
case. This is the rst instance in which a cell with
increased insulin sensitivity manifests an increased
116 Heimark et al.
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