Obesity induced by high-fat diet promotes insulin resistance in the ovary

65

Obesity induced by high-fat diet p

Eliana H Akamine1,2, Anderson C Marcal1, Joao Paulo Camporez1, Mara S Hoshida3,

Luciana C Caperuto4, Estela Bevilacqua3 and Carla R O Carvalho1

Departments of 1Physiology and Biophysics, Institute of Biomedical Sciences, 2Pharmacology, Institute of Biomedical Sciences and 3Cell and Development ofBiology, University of Sao Paulo, 05508-900 Sao Paulo/SP, Brazil

4Department of Biological Sciences, Federal University of Sao Paulo, 04023-900 Diadema/SP, Brazil

(Correspondence should be addressed to C R O Carvalho; Email: [email protected])

Abstract

Besides the effects on peripheral energy homeostasis, insulin

also has an important role in ovarian function. Obesity has a

negative effect on fertility, and may play a role in the

development of the polycystic ovary syndrome in susceptible

women. Since insulin resistance in the ovary could contribute

to the impairment of reproductive function in obese women,

we evaluated insulin signaling in the ovary of high-fat

diet-induced obese rats. Female Wistar rats were submitted to

a high-fat diet for 120 or 180 days, and the insulin signaling

pathway in the ovary was evaluated by immunoprecipitation

and immunoblotting. At the end of the diet period, we

observed insulin resistance, hyperinsulinemia, an increase in

Journal of Endocrinology (2010) 206, 65–740022–0795/10/0206–065 q 2010 Society for Endocrinology Printed in Great

progesterone serum levels, an extended estrus cycle, and

altered ovarian morphology in obese female rats. Moreover,

in female obese rats treated for 120 days with the high-fat diet,

the increase in progesterone levels occurred together with

enhancement of LH levels. The ovary from high-fat-fed

female rats showed a reduction in the insulin receptor

substrate/phosphatidylinositol 3-kinase/AKT intracellular

pathway, associated with an increase in FOXO3a, IL1B, and

TNFa protein expression. These changes in the insulin

signaling pathway may have a role in the infertile state

associated with obesity.

Journal of Endocrinology (2010) 206, 65–74

Introduction

Insulin signaling begins when insulin binds and activates its

receptor, resulting in tyrosine phosphorylation of several

substrates, including the insulin receptor substrate (IRS) 1–4.

IRS proteins, in turn, bind and activate the enzyme

phosphatidylinositol 3-kinase (PI3K; Backer et al. 1992,

Cheatham & Kahn 1995, Patti et al. 1995). AKT is a key

downstream target of PI3K, activated by serine and threonine

phosphorylation (Kohn et al. 1996, Bandyopadhyay et al.

1997). The PI3K/AKT pathway has an important role in the

metabolic effects of insulin. AKT also phosphorylates and

inactivates the members of the forkhead transcription factor

subfamily (FOXO) and glycogen synthase kinase 3B

(GSK3B). Upon insulin receptor autophosphorylation,

there is also recruitment of the SHC protein and GRB2,

leading to the activation of the extracellular signal-regulated

kinase (ERK) pathway (Skolnik et al. 1993, Giorgetti et al.

1994, Saltiel & Pessin 2002).

The ability of insulin to stimulate steroidogenesis in ovarian

cells in vitro (Barbieri et al. 1983) and the presence of insulin

receptor in both stromal and follicular compartments of

human ovary (Poretsky et al. 1984) have established the ovary

as a target organ for insulin action. Indeed, insulin signaling

has been shown to have a role in ovarian function, including

the regulation of ovarian steroidogenesis, follicular develop-

ment, and granulosa cell proliferation (Willis et al. 1996,

Adashi et al. 1997, Poretsky et al. 1999).

The prevalence of obesity is constantly on the rise and

constitutes a major worldwide epidemic. Obesity increases

the risk of type 2 diabetes mellitus and cardiovascular disease,

and has also a negative effect on fertility. The Nurses’ Health

Study reported that the risk of infertility in women increases

with increasing body mass index value (Rich-Edwards et al.

1994). Polycystic ovary syndrome is a condition commonly

associated with anovulatory infertility, and obesity may play a

role in the development of the syndrome in susceptible

women (Pasquali & Casimirri 1993, Legro 2000, Metwally

et al. 2007).

The major factor underlying the adverse metabolic

consequences of obesity is believed to be insulin resistance.

The reduction in the sensitivity to the biological actions of

insulin affects not only glucose metabolism, but also all aspects

of insulin action. However, the obesity effect on insulin

signaling in the ovary has not yet been evaluated.

The aim of the present study was to analyze the effect of

high-fat diet-induced obesity on the insulin signaling in the

ovary. We also verified if insulin signaling impairment is time

dependent in relation to the period during which the ovary

was submitted to adverse effects of obesity. We demonstrated

DOI: 10.1677/JOE-09-0461Britain Online version via http://www.endocrinology-journals.org

E H AKAMINE and others . Obesity and insulin signaling in the ovary66

that insulin resistance in the ovary occurred in a similar

fashion in relation to the classical target tissues of insulin,

providing evidence for intracellular mechanisms that are

possibly involved in the impaired reproductive function in the

obese female model.

Materials and Methods

The present experimental protocols were approved and

performed in accordance with the guidelines of the

Committee for Ethics in Animal Research of the Institute

of Biomedical Sciences, University of Sao Paulo, Brazil.

Animals

Female Wistar rats (8–10 weeks of age, weighing 150–180 g

at the beginning of the experiments) were obtained from our

breeding colony at the Institute. The animals were

randomized into two matching weight groups and assigned

to receive two types of diet: a standard rat chow (control rats;

3.8 kcal/g – 63.4% carbohydrate, 25.6% proteins, and 11.0%

fat), or a high-fat diet (obese rats; 5.4 kcal/g – 25.9%

carbohydrate, 14.9% proteins, and 59.0% fat; Rhoster

Industria e Comercio Ltda, Sao Paulo, SP, Brazil), for 120

or 180 days. The animals were housed with constant room

temperature, 12 h light:12 h darkness cycle, 60% humidity,

diet, and water made available ad libitum. All experiments

were performed with female rats in the estrous phase.

Characterization of high-fat diet-induced obese female rats

At the end of the diet period, the body weight, retro-

peritoneal and gonadal fat pad, and HOMA index ((blood

glucose (mM)!serum insulin (mU/ml))/22.5) were

determined. After 5 h of food withdraw, female rats were

anesthetized with thiopental (5 mg/100 g, i.p.; Cristalia, Sao

Paulo, SP, Brazil), and a tail vein blood sample was obtained

for determination of glucose concentration using a blood

glucose monitor (Roche). Blood samples were taken from the

abdominal aorta of anesthetized rats for the determination of

Table 1 Characteristics of high-fat diet-induced obese female rats. The

120 days

Control High-fat diet

Body weight (g) 238.00G3.00 (nZ10) 283.69G7.85‡ (nRetroperitoneal fat

(g/100 g body weight)0.85G0.15 (nZ4) 3.48G0.54† (n

Gonadal fat (g/100 gbody weight)

1.59G0.16 (nZ7) 3.82G0.30‡ (n

Blood glucose (mM) 5.68G0.32 (nZ10) 5.96G0.17 (nSerum insulin (mU/ml) 15.23G2.08 (nZ9) 32.42G3.27‡ (nHOMA-IR 3.90G0.71 (nZ9) 8.79G0.93‡ (n

*P!0.05, †P!0.01, and ‡P!0.001 in comparison with female age-matched con

Journal of Endocrinology (2010) 206, 65–74

serum concentrations of insulin, estradiol, progesterone, FSH,

LH, and testosterone using RIAs.

Estrous cycle analysis and ovarian morphology

Estrous cycle was determined once a day by cytological

examination of vaginal smears obtained for two consecutive

weeks before the end of the period of treatment with the

high-fat diet. Ovaries were collected from control and high-

fat diet-treated rat females (120 and 180 days), anesthetized

with thiopental (5 mg/100 g, i.p.), and fixed in 4%

paraformaldehyde in 0.1 M phosphate buffer, pH 7.2, for

morphological analysis. The specimens were routinely

processed for Histosec (Merck) embedding. Sections of

5 mm were used for hematoxylin and eosin staining and for

histological analysis with standard light microscopy.

Immunoprecipitation and immunoblotting analysis

The abdominal cavity of rats fed high-fat diet for 120 and

180 days was opened, and the portal vein was exposed under

anesthetization with thiopental (5 mg/100 g, i.p.). After

2 min of an injection of 0.2 ml saline (0.9% NaCl) with or

without insulin, the ovaries were removed and homogenized

in w1 ml of ice-cold extraction buffer containing 100 mM

Tris (pH 7.4), 10 mM EDTA, 1% Triton-X 100, 100 mM

sodium fluoride, 10 mM sodium pyrophosphate, 10 mM sodium

vanadate, 2 mM phenylmethylsulfonylfluoride, and 0.01 mg

aprotinin. Insoluble material was removed by centrifugation

for 40 min at 15 000 g at 4 8C. The supernatants of the

samples were used for immunoprecipitation with anti-insulin

receptor, anti-IRS1 and anti-IRS2 antibodies, and protein

A-Sepharose 6MB (GE Healthcare, Little Chalfont,

Buckinghamshire, UK). The precipitated proteins and

whole-tissue extract (85 mg) were treated with Laemmli’s

sample buffer (Laemmli 1970) containing 100 mM dithio-

threitol and run on SDS-PAGE (6.5–10% bis-acrylamide) in a

Bio-Rad miniature gel apparatus (Bio-Rad).

Electrotransfer of proteins from the gel to nitrocellulose

membrane (Bio-Rad) was performed for 90 min at 120 V

(constant) in a miniature transfer apparatus (Bio-Rad).

values were expressed as meanGS.E.M.

180 days

Control High-fat diet

Z15) 245.81G9.43 (nZ11) 351.70G20.67‡ (nZ15)Z7) 1.09G0.18 (nZ5) 3.35G0.67* (nZ5)

Z12) 2.74G0.37 (nZ5) 5.13G0.47† (nZ5)

Z15) 5.37G0.40 (nZ10) 6.27G0.23* (nZ14)Z12) 19.56G3.61 (nZ7) 48.28G4.26‡ (nZ14)Z12) 5.20G1.34 (nZ7) 13.86G1.59† (nZ14)

trol rats.

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Table 2 Serum sex hormone levels in the estrous phase. The values were expressed as meanGS.E.M.

120 days 180 days

Control High-fat diet Control High-fat diet

Estradiol (pg/ml) 17.60G2.96 (nZ10) 18.65G4.16 (nZ8) 22.43G5.75 (nZ6) 33.75G10.07 (nZ5)Progesterone (ng/ml) 41.06G2.22 (nZ10) 59.32G7.52* (nZ10) 52.47G3.14 (nZ6) 76.35G4.88† (nZ5)Testosterone (pg/ml) 161.00G26.84 (nZ6) 145.40G28.49 (nZ10) 155.00G14.30 (nZ6) 162.00G20.21 (nZ5)LH (ng/ml) 14.26G1.79 (nZ10) 38.06G4.85† (nZ10) 9.37G0.55 (nZ6) 8.30G1.39 (nZ5)FSH (ng/ml) 3.96G0.44 (nZ10) 4.14G0.71 (nZ10) 5.13G1.20 (nZ6) 3.10G0.12 (nZ5)

*P!0.05 and †P!0.001 in comparison with female age-matched control rats.

Obesity and insulin signaling in the ovary . E H AKAMINE and others 67

To reduce nonspecific protein binding to nitrocellulose, the

membrane was pre-incubated overnight at 4 8C in blocking

buffer (5% nonfat dry milk, 10 mM Tris, 150 mM NaCl, and

0.02% Tween 20). The nitrocellulose blots were incubated

with specific antibodies diluted in blocking buffer (3% nonfat

dry milk) overnight at 4 8C. The nitrocellulose membranes

were treated with Ponceau staining immediately after transfer

as a quick visual control of the amount of protein in each lane.

To visualize the autoradiogram, commercial enhanced

chemiluminescence reagents (GE Healthcare) applied to

photographic film were used. The band intensities were

quantified by optical densitometry (Scion Image Software,

Frederick, MD, USA).

Antibodies

Anti-insulin receptor, anti-IRS1, anti-IRS2, anti-phospho-

tyrosine, anti-pAKT, and anti-GSK3 antibodies were pur-

chased from Santa Cruz Biotechnology (Santa Cruz, CA,

USA). Anti-PI3K, anti-AKT, anti-FOXO3a, anti-ERK, and

anti-pERK antibodies were purchased from Cell Signaling

Technology (Beverly, MA, USA). Anti-TNFa and anti-IL1B

were purchased from Biolegend (San Diego, CA, USA).

Statistical analysis

The results were expressed as meansGS.E.M. Statistical

analyses were performed using t-test or one-way ANOVA

followed by Bartlett’s test for homogeneity of variances and

Tukey–Kramer multiple comparisons test when appropriate.

The estrous cycle was analyzed by the unpaired t-test with

Welch’s correction. The minimum acceptable level of

significance was P!0.05.

Table 3 Estrous cycle: duration of the different phases in days. The valu

120 days

Control (nZ16) High-fat diet (n

Diestrus 0.8G0.1 0.8G0.2Proestrus 1.3G0.1 2.5G0.3‡

Estrus 1.0G0.0 1.0G0.0Metestrus 1.0G0.1 0.9G0.2Total duration 4.1G0.2 5.1G0.4*

*P!0.05, †P!0.01, and ‡P!0.001 in comparison to female age-matched contro

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Results

Characteristics of the animals

Female rats submitted to high-fat diet for 120 and 180 days

displayed greater body weight, retroperitoneal and gonadal fat

pad, serum insulin levels, and HOMA index (Table 1).

Sex steroid levels, estrus cycle, and ovarian morphology in femalessubmitted to high-fat diet

In the estrous phase, the serum levels of estradiol, testosterone,

and FSH did not change after treatment with high-fat diet for

120 or 180 days (Table 2). On the other hand, progesterone

levels were increased in both obese groups, but LH levels

were increased only in females fed with high-fat diet for

120 days (Table 2).

Female rats submitted to high-fat diet for 120 and 180 days

exhibited an extended estrous cycle due to a prolonged

proestrus phase (Table 3). Females fed high-fat diet for

180 days also exhibited a prolonged diestrus phase, and

reduced estrus and metestrus phase (Table 3).

Treatment with the high-fat diet for 120 days did not

promote significant changes in the ovarian morphology (data

not shown). However, morphological differences were

found between the ovaries from female rats submitted to

high-fat diet for 180 days and their controls. In the control

group, the ovaries exhibited morphologically normal antral

and pre-antral follicles and corpora lutea. The granulosa

layer showed a typical epithelioid arrangement, and were

completely embraced by thecal cells. Interstitial cells were

occasionally found throughout the cortical stroma, among

es are expressed as meanGS.E.M.

180 days

Z14) Control (nZ10) High-fat diet (nZ12)

0.7G0.1 4.6G1.4*1.5G0.2 4.5G1.1*1.0G0.0 0.7G0.1*1.3G0.1 0.4G0.2†

4.5G0.3 10.2G0.9‡

l rats.

Journal of Endocrinology (2010) 206, 65–74

CL

tc

tc

tc

s

gc

gc

gcgc

tcs

C

D

E

A

B

CL

m

Figure 1 Ovarian sections from control (180-day period) femalerats. (A) Antral follicles (*) and corpora lutea (CL), normal inappearance, are seen in the cortical ovarian zone. (m), ovarianmedulla. (B–D) The micrographs show the typical follicular wall, inwhich the granulosa cells (gc) assume an epithelioid aspect andare completely embraced by thecal cells (tcs). (E) In the ovarianstroma (S), interstitial cells (arrows) are frequent among fibroblastcells. Hematoxylin–eosin staining. The bar represents 500 mm inA, 80 mm in B, 25 mm in C, and 40 mm in D and E.

E H AKAMINE and others . Obesity and insulin signaling in the ovary68

fibroblast and blood vessels (Fig. 1). In ovaries from rats of

the high-fat diet group, pre-antral and antral follicles share

the cortical ovarian area with abundant polygonal cells with

clear vacuolated cytoplasm, filled with abundant lipid

droplets. These cells were widely distributed in the stroma,

organized as columns, solid groups of cells, or as cysts

containing liquid. Cells belonging to the granulosa layer also

occasionally exhibited a similar morphology (Fig. 2). The

weight of ovaries from females treated by 180 days was

higher than that from controls (control: 50.93G1.89 mg;

obese: 60.80G1.22 mg, P!0.05).

Insulin signaling pathways on ovaries from females submitted tohigh-fat diet for 120 days

The protein expression of AKT and FOXO3a in the whole

ovary homogenates was modified by 120 days of high-fat diet

feeding, whereas the insulin receptor, IRS1, IRS2, ERK1-2,

and GSK3 expression were similar to that detected in control

rats (Fig. 3A). The AKT and FOXO3a protein levels were

enhanced by 69 and 67% respectively above those detected in

control rats (Fig. 3B and C respectively).

Acute insulin infusion induced a sixfold increase in the

tyrosine phosphorylation degree of the insulin receptor above

the baseline, which was similar in the ovaries of control and

Journal of Endocrinology (2010) 206, 65–74

obese rats (Fig. 4A). There was also a twofold enhancement in

the insulin-induced IRS1 association with PI3K above the

baseline in ovaries of both groups (Fig. 4B). On the other

hand, in the ovary from control rats, there was a sixfold

increase in the IRS2 association with PI3K after insulin

infusion, whereas in the ovary from obese rats there was only a

twofold increase above the baseline (Fig. 4C).

Although the degree of insulin-induced AKT serine

phosphorylation was similar in both groups, the phosphoryl-

ation on the basal condition was higher in ovaries from obese

rats (Fig. 4D). In this regard, insulin promoted a threefold

increase in AKT serine phosphorylation in obese female rats,

whereas in control female rats that increase was sixfold.

Despite the fact that ERK1-2 protein expression was similar

in ovaries from both groups (Fig. 3A), the acute insulin

infusion-induced ERK1-2 phosphorylation was w50% lower

in obese rats (Fig. 4E).

Insulin signaling pathways in ovaries from females submitted tohigh-fat diet for 180 days

The maintenance of the high-fat diet for 180 consecutive

days had no impact on the protein levels of IRS1, AKT, and

GSK3 in the ovary (Fig. 5A). However, there was a

reduction in the IRS2 protein level by w40% (Fig. 5B)

and an enhancement of w30% in the FOXO3a protein level

(Fig. 3C) in the ovary from diet-induced obese rats when

compared with controls. The acute insulin infusion-induced

increase in the IRS1 and IRS2 association with PI3K was

detected only in the ovaries from control female rats (Fig. 6A

and B). There was a twofold increase above the baseline in

the insulin-induced AKT serine phosphorylation in the

control female rats, but insulin did not promote any effect in

the ovaries from obese rats (Fig. 6C).

Cytokine protein expression in the ovaries from females submittedto high-fat diet

IL1B (Fig. 7A) and TNFa (Fig. 7B) protein expression in

the ovary did not change after 120 days of high-fat

diet. However, treatment with high-fat diet for 180 days

promoted a fourfold increase in IL1B (Fig. 7C) and TNFa(Fig. 7D) protein levels in the ovary when compared

with controls.

Discussion

Reproductive health and female fertility are compromised

by overweight and obesity (Shaw et al. 1997, Gesink Law et al.

2007). The role of systemic insulin resistance in obesity-

impaired reproductive performance is shown by the positive

effects of the insulin-sensitizing agents (Nestler et al. 1998,

Tang et al. 2006). In the present study, we demonstrated that

systemic insulin resistance and hyperinsulinemia occur

together with impairment of insulin signaling in the ovary,

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s

S

S

gc

EA

B

C

D

G

F

Figure 2 Ovarian sections from high-fat diet (180-day period)-induced obese female rats.(A) The panoramic view of the ovary shows antral follicles (*) and a stroma (S) of vacuolatedappearance. Details of this peculiar morphology are depicted on micrographs B through F.Antral (*) and pre-antral (**) follicles are shown in C–E. In D and E, note the vacuolatedaspect (double arrows) also in the granulosa layer (gc) of the antral follicle; in F and G, cellswith a vacuolated cytoplasm filled with lipid droplets are seen. Hematoxylin–eosin staining.The bar represents 500 mm in A, 300 mm in B, 100 mm in C, 400 mm in D, 125 mm in E and F,and 25 mm in G.

Obesity and insulin signaling in the ovary . E H AKAMINE and others 69

enhancement of progesterone serum levels, and alteration of

estrous cycle and of ovarian morphology in high-fat diet-

induced obese female rats.

Insulin signaling has been implicated in the regulation of

female reproductive function by acting in both central

nervous system and ovaries. The hyperinsulinemia can

potentiate gonadotropin-stimulated steroidogenesis in

granulosa and thecal cells, by increasing the low-density

lipoprotein (LDL) receptor, 3b-hydroxysteroid dehydro-

genase, 17a-hydroxylase, and 17,20 lyase expression (Nestler

& Strauss 1991, McGee et al. 1995, Franks et al. 1999,

Poretsky et al. 1999, Zhang et al. 2000). In addition, insulin

may act on the pituitary to increase LH release (Adashi et al.

1981, Weiss et al. 2003, Dorn et al. 2004, Moret et al. 2009). In

agreement with these data, we observed both hyperinsuline-

mia and enhancement of progesterone serum levels in obese

female rats. Moreover, the LH levels were also enhanced in

female obese rats receiving the high-fat diet for 120 days,

which could explain the progesterone increase. Surprisingly,

the LH levels in obese females treated for 180 days with

high-fat diet were similar to controls. GNRH stimulates the

secretory response of LH by a calcium-dependent signaling,

and increases mRNA and protein synthesis of LH through the

MAPK signaling pathway. In the gonadotropes, both GNRH

and insulin activate the MAPK pathway, which is the point of

signaling interaction and may be involved with the

potentiation of gonadotropin release by insulin. It was

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postulated that although insulin acts cooperatively with

GNRH, chronic hyperstimulation may induce a refractory

state ultimately leading to a decreased biosynthetic capacity

(Lawson et al. 2008). Such a mechanism would explain the

similar LH levels in control and obese females treated for

180 days with high-fat diet, since the hyperinsulinemia

persisted at 180 days of high-fat diet treatment and the insulin

level was higher than in female rats that received the high-fat

diet for 120 days. Moreover, the accumulation of lipid

droplets in the stromal and granulosa cells may be a

consequence of hyperinsulinemia on the expression of LDL

receptor, which would contribute to increase steroidogenesis

after 180 days of high-fat diet treatment.

Both low and high progesterone levels have a negative

impact on pregnancy outcome (Check et al. 2009). An

extended estrous cycle was observed with both periods of

high-fat diet treatment, but the 180-day period promoted the

alterations that were more pronounced than after 120 days,

with a prolonged diestrus phase and short estrus and metestrus

phases, which could result in reduced reproductive capacity.

Female mice that lack crucial components of the insulin

signaling pathway, such as IRS2, are infertile and display

ovarian and hypothalamic function deficits, suggesting that

the IRS2 pathway mediates the insulin effects upon the

reproductive function (Burks et al. 2000). In insulin resistance

conditions, such as the polycystic ovary syndrome, the ovary

remains sensitive to insulin, whereas other organs exhibit a

Journal of Endocrinology (2010) 206, 65–74

200

150

100

50

0

IB: IR-β

IB: IRS1

IB: IRS2

IB: AKT

IB: ERK1–2

IB: FOXO3a

IB: GSK3

Control Obese

Control Obese

% o

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trol

200

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50

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% o

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trol

*

*

A B

C

Figure 3 Insulin signaling in the ovary from control and high-fat diet(120 days)-induced obese female rats. (A) Representative blots ofinsulin receptor b-subunit, IRS1, IRS2, AKT, ERK1–2, FOXO3a, andGSK3 protein expression. (B) Densitometric analyses of the AKTprotein levels in ovaries from both control and high-fat diet-inducedobese rats. (C) Densitometric analyses of the FOXO3a protein levelsin ovaries from both control and high-fat diet-induced obese rats.Data are expressed as meansGS.E.M. obtained from six animals.*P!0.05 versus control group.

IP: IR-β

IB: pY

800

600

400

200

0

Control Obese– + – +

# #

% o

f con

trol

A

IP: IRS1

IB: pAKT

IP: IRS2

IB: p85 PI3-K

IB: p85 PI3-K IB: pERK1–2

250 1500

1000

500

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0

0

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0

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Control Obese

– + – +

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#

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###

##*

*

% o

f con

trol

% o

f con

trol

% o

f con

trol

% o

f con

trol

B D

C E

Figure 4 Insulin signaling in the ovary from control and high-fat diet(120 days)-induced obese female rats. Ovary homogenates wereimmunoprecipitated with anti-insulin receptor subunit b (IR-b) (A),anti-IRS1 (B), and anti-IRS2 (C) antibodies, followed by immuno-blotting with antiphosphotyrosine antibody (A) and anti-p85 kDasubunit of the PI3-kinase (B and C). The membranes containingwhole extract of ovary homogenates were incubated with anti-phospho-Ser473-AKT (D) and anti-phospho-Thr185/Tyr187-ERK1–2 (E) antibodies. The phosphorylation and association degree weredetermined in basal (K) and insulin-stimulated (C) conditions.Data are expressed as meansGS.E.M. obtained from six animals.#P!0.05 versus basal condition of respective group. *P!0.05versus respective condition of control group.

E H AKAMINE and others . Obesity and insulin signaling in the ovary70

significant reduction in insulin action (Poretsky 1991, Lima

et al. 2006). Since deficits of insulin receptor signaling in the

muscle, liver, and adipose tissue are related to obesity-

associated insulin resistance, and as it is known that these

deficits occur through tissue- and pathway-specific factors, it

is interesting to evaluate if the ovary from obese female rats

remains sensitive to insulin concomitantly with enhancement

of progesterone levels, and alteration of estrous cycle and of

ovarian morphology.

In the present study, the ovaries from obese females that

received the high-fat diet for 120 days presented AKT

phosphorylation similar to controls, despite a reduction in

IRS2/PI3K association, probably by a compensatory increase

in AKT protein expression. On the other hand, in the ovary

from obese females treated for 180 days with the high-fat diet,

both IRS1 and IRS2/PI3K association were reduced,

concomitantly with a reduction in AKT phosphorylation.

These results suggest that the degree of impairment of the

insulin signaling pathway exhibits a time-dependent relation

to the exposure to obesity-generated deleterious effects. The

synergistic action of insulin with LH in the ovary may be due

to a positive crosstalk of the intracellular signaling pathway of

both hormones. Carvalho et al. (2003) demonstrated that

simultaneous infusion of insulin and LH induced higher

phosphorylation of AKT in the ovary than each hormone

alone. Moreover, PI3K, an upstream protein of AKT in

the insulin signaling pathway, is involved in the LH- and

insulin-induced upregulation of the LDL receptor expression

Journal of Endocrinology (2010) 206, 65–74

(Sekar & Veldhuis 2001). Our data could indicate that the

IRS/PI3K/AKT pathway may not be involved, at least

directly, in the enhancement of steroidogenesis or in the

accumulation of lipid droplets observed in ovarian cells of

obese female rats.

A balance between IRS/PI3K/AKT pathway-stimulated

and MAPK pathway-inhibited steroidogenesis coordinates

the ovarian function. Indeed, it was demonstrated that

stimulation of ERK1-2 pathway inhibits steroidogenesis in

the ovary (Nelson-Degrave et al. 2005). In this regard, the

reduced ERK1–2 phosphorylation detected after 120 days

of high-fat diet could have a role in the increase in

progesterone levels.

Beyond the effect on the ovarian steroidogenesis, insulin is

involved in follicular development and granulosa cell

proliferation (Willis & Franks 1995, Nestler et al. 1998,

Poretsky et al. 1999). In fact, the ovaries from IRS2-null

female mice present reduced follicle size, impaired oocyte

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IB: IRS1

IB: IRS2

IB: AKT

IB: FOXO3a

IB: GSK3

150

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0Control Obese

Control Obese

200

100

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*

*

% o

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% o

f con

trol

A B

C

Figure 5 Insulin signaling in the ovary from control and high-fat diet(180 days)-induced obese female rats. (A) Representative blotsof IRS1, IRS2, AKT, FOXO3a, and GSK3 protein expression.(B) Densitometric analyses of the IRS2 protein levels in ovaries fromboth control and high-fat diet-induced obese rats. (C) Densitometricanalyses of the FOXO3a protein levels in ovaries from both controland high-fat diet-induced obese rats. Data are expressed asmeansGS.E.M. obtained from six animals. *P!0.05 versuscontrol group.

IP: IRS1

IB: p85 PI3-K

% o

f con

trol

% o

f con

trol

% o

f con

trol

300

200

100

0– + – +

#

*

Control

IP: IRS2

IB: p85 PI3-K

IB: pAKT

Obese

– + – +

Control Obese

– + – +

Control Obese

250

200

150

100

50

0

250

200

150

100

50

0

A

B

C

#

#

Figure 6 Insulin signaling in the ovary from control and high-fat diet(180 days)-induced obese female rats. Ovary homogenates wereimmunoprecipitated with anti-IRS1 (A) and anti-IRS2 (B) antibodies,followed by immunoblotting with anti-p85 kDa subunit of the PI3-kinase. The membranes containing the whole extract of ovaryhomogenates were incubated with anti-phospho-Ser473-AKT (C).The phosphorylation and association degree were determined inbasal (K) and insulin-stimulated (C) conditions. Data areexpressed as meansGS.E.M. obtained from six animals. #P!0.05versus basal condition of respective group. *P!0.05 versusrespective condition of control group.

Obesity and insulin signaling in the ovary . E H AKAMINE and others 71

growth, defective granulosa cell proliferation, as well as

reduced insulin-stimulated AKT phosphorylation (Neganova

et al. 2007). Several proteins that regulate cell survival and

proliferation are downstream effectors of AKT. Members of

the FOXO forkhead transcription factor subfamily and

GSK3 are phosphorylated and thus inactivated by AKT.

Female mice that lack FOXO3a exhibit early depletion of

ovarian follicles and secondary infertility due to follicular

activation, indicating that this factor functions as a suppressor

of the earliest stages of follicular growth (Castrillon et al.

2003). The cascade from granulosa cell-derived AKT/GSK3

pathway also regulates the early ovarian follicular develop-

ment (Liu et al. 2007). Despite the increase in AKT

expression after 120 days of high-fat diet, its phosphorylation

degree induced by insulin is similar to the control, which

could indicate a lack of effect of the AKT increase upon

FOXO3a. On the other hand, the ovary from female rats

treated with high-fat diet displayed increased FOXO3a

expression and reduced AKT phosphorylation after 180 days

of high-fat diet, which could be implicated in obesity-

induced impaired reproductive function.

Despite the finding of pre-antral and antral follicles in

ovaries of both control and high-fat diet (180-day period)

groups, which could indicate that follicular development was

not affected, other parameters of the reproductive function

were altered in the experimental group. As discussed above, a

decrease in the IRS/PI3K/AKT pathway does not seem

implicated in the enhancement of steroidogenesis or with

accumulation of lipid droplets observed in ovarian cells of

www.endocrinology-journals.org Journal of Endocrinology (2010) 206, 65–74

150

100

50

0

150

200

100

50

0

% o

f con

trol

% o

f con

trol

% o

f con

trol

% o

f con

trol

Control Obese Control Obese

Control Obese Control Obese

600

400

200

0

600

400

200

0

IB: IL1B IB: TNFα

IB: IL1B IB: TNFα

**

C D

A B

Figure 7 IL1B and TNFa protein expression in the ovary from obesefemale rats treated with the high-fat diet for 120 days (A and Brespectively) and 180 days (C and D respectively). Data areexpressed as meansGS.E.M. obtained from six animals. *P!0.05versus respective female control rats.

E H AKAMINE and others . Obesity and insulin signaling in the ovary72

obese female rats. On the other hand, progesterone could

decrease the IRS/PI3K/AKT pathway. Progesterone is

implicated in insulin resistance during pregnancy by

inhibiting the PI3K pathway in adipocytes (Wada et al.

2010). Therefore, ovarian insulin resistance would not permit

the ovaries of obese females to respond appropriately to

metabolic demands required for sustaining the oocyte during

the periovulatory period (Kol et al. 1997), possibly resulting in

a reduced estrus phase.

During the ovulation process, expression of inflammatory

factors and molecules related to the innate immune response

is observed, such as prostaglandins, cytokines, and Toll-like

receptors. While regulated synthesis and release of cytokines is

essential for follicular development and ovulation (Machelon

& Emilie 1997, Bornstein et al. 2004, Gerard et al. 2004),

enhanced production can lead to infertility (Adashi et al. 1989,

Ghersevich et al. 2001, Herath et al. 2007). Blood levels of

IL6 are elevated in patients with endometriosis, and could

lead to infertility (Odukoya et al. 1997, Bedaiwy et al. 2002,

Umezawa et al. 2008). The enhancement of white adipose

tissue increases the production of factors related with immune

cells, cytokines, and free fatty acids, which could contribute

to the installation of infertility in the obese condition

(Schaffler et al. 2007). In the present study, the pro-

inflammatory cytokine expression was similar in the ovary

from control and obese female rats receiving the high-fat diet

for 120 days, but it was increased in the ovary from female rats

treated for 180 days. This cytokine enhancement could be

involved in the insulin signaling reduction in the ovary after

Journal of Endocrinology (2010) 206, 65–74

180 days of a high-fat diet, which could contribute to

infertility in obese females.

In summary, our results show that insulin resistance in the

ovary occurs in a way which is similar to that observed in the

classical target tissues of insulin, and that the insulin signaling

alterations is time dependent in relation to the period of

exposure to obesity-related deleterious effects. These data

suggest that the positive effects of insulin sensitizer agents

upon the reproductive function could actually correct insulin

signaling directly in the ovary.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived

as prejudicing the impartiality of the research reported.

Funding

This work was supported by grants from Fundacao de Amparo a Pesquisa do

Estado de Sao Paulo – FAPESP (2006/52163-7; 2006/60215-7) and

Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico – CNPq

(477906/2006-0). ACM and JPC were supported by fellowships from

FAPESP, and EHA was the recipient of fellowships from FAPESP and CNPq

in different phases of the project.

Acknowledgements

We gratefully acknowledge Dr Celso Franci for hormone measurement and

Mrs Luciene M Ribeiro for technical assistance.

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Received in final form 17 April 2010Accepted 6 May 2010Made available online as an Accepted Preprint7 May 2010

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