Knockdown of prohibitin expression promotes glucose metabolism in eutopic endometrial stromal cells from women with endometriosis

Reproductive BioMedicine Online (2014) 29, 761–770

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ARTICLE

Knockdown of prohibitin expression promotes glucose metabolism in eutopic endometrial stromal cells from women with endometriosis Xiaochen Qi a,b,1, Yanxin Zhang a,c,1, Hui Ji a,d, Xiadi Wu a, Fuxin Wang e, Manxin Xie f, Li Shu a, Shiwen Jiang g,h, Yundong Mao a,*, Yugui Cui a, Jiayin Liu a a

Center of Clinical Reproductive Medicine, State Key Laboratory of Reproductive Medicine, The First Affiliated Hospital of Nanjing Medical University/Jiangsu Province Hospital, Nanjing, China; b Department of Obstetrics and Gynecology, The First Clinical Medical College of Three Gorges University, Yichang Central People’s Hospital, Yichang, China; c Department of Obstetrics and Gynecology, Clinical Medical College of Yangzhou University, Subei People’s Hospital of Jiangsu, Yangzhou, China; d Department of Obstetrics and Gynecology, Nanjing Maternal and Child Health Hospital, Nanjing Medical University, Nanjing, China; e Center of Reproductive and Genetic Medicine, Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou Municipal Hospital, Suzhou, China; f Department of Obstetrics and Gynecology, The First Affiliated Hospital of Nanjing Medical University/Jiangsu Province Hospital, Nanjing, China; g Department of Biological Science, Mercer University School of Medicine; Savannah, USA; h Department of Obstetrics and Gynecology, Memorial Health Hospital, Savannah, USA * Corresponding author.

E-mail address: [email protected] (Y Mao).

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These authors contributed equally to this study and share first

authorship. Mao Yundong is currently Associate Professor at the Department of Obstetrics and Gynaecology of the First Affiliated Hospital of Nanjing Medical University, where he has been researching and working for more than two decades. He obtained his PhD degree at Nanjing Medical University in 2007, specializing in human assisted reproduction. His research interests are mostly focused on reproductive endocrinology, unfertility, assisted reproductive technology, endometriosis, laparoscopy and ultrasonography.

Abstract In this in-vitro study, the effect of prohibitin (PHB) on glucose metabolism in eutopic endometrial stromal cells from women with endometriosis was investigated. Endometrial stromal cells were isolated from endometrium in women with endometriosis, in women without endometriosis, or from endometrioma tissues. Glucose metabolic phenotype of stromal cells were examined in vitro. Quantitative polymerase chain reaction was used to measure the mRNA expression of glycolysis-related genes. Glucose consumption and lactate production were examined after knockdown of PHB expression in women with endometriosis with siRNA. In endometrioma tissue, significantly increased glucose consumption, lactate production and aberrant expression of glycolysis-related enzymes were found in women with endometriosis compared with women who do not have endometriosis (P < 0.05 versus P < 0.001). In women

http://dx.doi.org/10.1016/j.rbmo.2014.09.004 1472-6483/© 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

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with endometriosis, PHB mRNA and protein were under-expressed in endometrioma tissue; in women without endometriosis, PHB mRNA and protein were over-expressed. Knockdown of PHB expression in women with endometriosis increased glucose consumption, although it had no effect on lactate production. This study suggests that aberrant expression of glycolysis-related enzymes in endometrioma tissue is associated with enhanced glycolytic metabolism. The malignant-like feature may be partially caused by lowexpression of PHB gene in endometriotic stromal cells. © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: endometriosis, glucose metabolism, glycolysis, prohibitin

Introduction Endometriosis is defined as the presence of active endometrial glands and stroma outside the uterus. Although its exact mechanism is poorly understood, retrograde menstruation is considered a key pathogenic cause (Giudice, 2010). It is believed that enhanced migration, apoptosis escape, and increased invasion and angiogenic activities of endometrial cells contribute to the development of the disease (Moggio et al., 2012; Taylor et al., 2009). Accordingly, endometrial cells from endometriosis may share some features with cancers. One characteristic feature of cancer is the ‘aerobic glycolysis’, or ‘Warburg effect’, first described by Warburg in the early 1930s (Kroemer and Pouyssegur, 2008). Cancer cells preferentially use glycolysis as a main energy source rather than oxidative phosphorylation (OXPHOS) even in the presence of abundant oxygen, which leads to increased glucose consumption and accumulated lactate levels (Vander Heiden et al., 2009). The altered metabolism has been linked to several malignant phenotypes, such as unlimited cell proliferation, apoptosis escape, sustained angiogenesis, tissue invasion and metastasis (Kroemer and Pouyssegur, 2008). Multiple crosstalk signalling pathways and cancer-related mutations are implicated in the alterations in the energy mechanism of cancer cells, including p53, PTEN, MYC, HIF mutations and PI3K, AMPactivated protein kinase pathways (Cairns et al., 2011; Ward and Thompson, 2012). Although transcriptomic and proteomic profiling allows identification of various gene expression alterations in endometriotic lesions that might be potentially related to energy metabolism (Kobayashi et al., 2009; Mao, 2007), it remains unclear how aberrations in energy metabolism may contribute to the development of endometriosis. Some clinical observational studies have suggested that endometriotic lesions could give positive results on fluorine-18 fluorodeoxyglucose (18F-FDG) positron emission tomography scans, which has been highly accurate in distinguishing malignant lesions from benign tissues, based on an increased uptake of 18F-FDG in malignancies (Fastrez et al., 2011; Jeffry et al., 2004). It has been debated, however, whether the increased uptake of 18F-FDG is caused by the endometriosis itself or by a false-positive scan from inflammatory reactions, which are often associated with the endometriomas (Jeffry et al., 2004). In endometriosis, stromal cells are known to be involved in early menstrual endometrium attachment and be responsible for further growth of glandular epithelial cells, leading to endometrial gland formation at endometriotic lesions (Arnold et al., 2001; Nisolle et al., 2000). In the culture used in the present study, primary glandular epithelial cells were observed as colony growth and showed a lower proliferation than stromal cells

(unpublished observations). The hypothesis that the metabolic phenotype of endometrial stromal cells from women with endometriosis is similar to that of cancer cells was tested. Prohibitin-1 (PHB) is a chaperone protein that is highly conserved evolutionarily and present in different cellular compartments. It has been shown that PHB is differentially expressed in eutopic endometrium from women with endometriosis and those without (Fowler et al., 2007; Mao, 2007). Recent studies have provided strong evidence for an important biological role of PHB in mitochondrial function, cell proliferation and embryo development (Artal-Sanz and Tavernarakis, 2009). It regulates glucose metabolism in RINm5Fβ-cells through insulin receptor activation, PI3K/ AKT pathway and its O-GlcNAc modification (Ande et al., 2009; Mishra et al., 2010). Potential involvement of PHB in the pathogenesis of endometriosis, however, has not been investigated. The aim of this study was to evaluate the glucose metabolic phenotype of endometrial stromal cells from endometrium in women with endometriosis, without endometriosis, and endometrial stromal cells from ovarian endometrioma tissues. As an initial attempt to reveal the mechanistic change underlying the observed energy changes associated with endometrisois, the role of PHB on glucose metabolism in eutopic stromal cells from women with endometriosis was also examined.

Materials and methods Patients and sample collection Ethical approval was obtained from the First Affiliated Hospital of Nanjing Medical University Ethical Committee on 22 February 2013 (reference number 2013-SRFA-093). Thirtyfive patients aged between 23 and 43 years who underwent laparoscopic examination from February 2013 to November 2013 in the First Affiliated Hospital of Nanjing Medical University, donated their endometrium and ovarian endometrioma and provided informed consent. All samples were collected during the proliferative phase of menstrual cycle according to the date of the last menstrual period. Twentythree of them were histologically confirmed as endometriosis (stromal cells from the endometrium in women with endometriosis n = 11); stromal cells isolated from endometrioma tissues: n = 12). Endometriosis was staged as III-IV according to the revised American Fertility Society classification. Endometrial tissues obtained from other patients diagnosed with other benign gynaecological disease, including nine cases of hydrosalpinx and three cases of benign ovarian teratomas, were enrolled as normal controls (i.e. stromal cells from endometrium in women without endometriosis: n = 12).

Glucose metabolism and endometriosis Women who had irregular menstrual cycles and had received anti-inflammatory or hormonal therapies less than 3 months before surgery were excluded from the study.

Endometrial stromal-cell dissociation and cell culture Immediately after dissection, the tissues were placed into culture medium at 4°C for less than 4 h. Endometrial stromal cells were separated by digesting the minced tissues as previously described with a minor modification (Hou et al., 2008). Two types of tissues were cut into 1–2 mm3 pieces and incubated in serum free Dulbecco’s Modified Eagle’s Medium (DMEM)/F12 (Thermo, USA) containing 0.125% collagenase IV (Sigma Company, USA) at 37°C for 70–90 min. Then, the suspension was filtrated through 80- and 40-µm wire sieves, allowing the separation of stromal cells and epithelial cells. After being washed and resuspended in DMEM/F12 containing 10% fetal bovine serum and 1% penicillin and streptomycin (Huabei Pharmacy, Shijiazhuang, China), the stromal cells were plated into 10-cm cell culture dishes and grown in a humid atmosphere at 37°C, 5% carbon dioxide. After 1–2 h, endometrial stromal cells were attached to the bottom of the plate and the culture medium was replaced to discard the epithelial glands.

Measurements of glucose and lactate At 80–90% confluency, primary stromal cells were trypsinized and seeded in 12-well plate at the total of 8~12×104 cells per well. The culture medium was replaced at 50–60% cell confluency and collected after 12, 24 and 36 h, respectively. One sample at each time point was evaluated from triplicate wells with three independent cell counts per well by using haemacytometer cell counts. Triple blank wells without cells were included in the experiment as background. The medium were stored at –80°C and were tested at the Clinical Laboratory, The First Affiliated Hospital Of Nanjing Medical University for glucose levels. Glucose levels were tested using a kit (Glucose HK 125 kit, ABX diagnostics, France) by hexokinase method. Lactate levels were measured using a microplate reader (Bio-rad model680, USA) with a measurement kit (Jiancheng Bio-technique Institute, Nanjing, China). Aliquots of 1 µl of samples were prepared in duplicate along with a standard curve of known concentrations of lactate in the range 0–6 mmol/L. Glucose consumption and lactate production per sample were determined by average levels after normalization to cell count. Results were expressed as mmol/L per 106 cells. As described below, PHB was knocked down in eutopic endometrial stromal cells from women with endometriosis. Twenty-four hours after cell transfection, cells were cultured in fresh culture medium on 12-well plates for 12, 24 and 36 h. The culture medium was collected and tested as described above.

Real-time fluorescent quantitative polymerase chain reaction Total cellular RNA was extracted with Trizol reagent (Takara, Japan) according to the manufacturer’s recommendations.

763 Quality of RNA was identified by 1% agarose gel, and its concentration was determined by spectrophotometry (Eppendorf, Germany). DNAse (Invitrogen) was added to RNA to avoid contamination of any DNA. Total RNA 800 ng was reverse transcribed into cDNA with Primer-Script RT reagent Kit (Takara, Japan) according to the protocols in a total volume of 20 µl. Seventeen target genes were analysed by real-time quantitative polymerase chain reaction (PCR) using the Step-one system with SYBR Premix Ex Taq (Takara, Japan). The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase was used for normalization. Primers were designed using Primer Premier 5 software (PREMIER Biosoft, Palo Alto, CA), and were synthesized by Invitrogen. The PCR protocol consisted of an initial denaturation step of 1 min at 95°C followed by 40 cycles of 10 s at 95°C, 31 s at 60°C, 10 s at 72°C, and a finally melting curve. The relative expression levels of target genes to internal gene were determined by the formula 2-ΔΔCt. –ΔΔ Ct = – [(CT target gene–CT housekeeping gene) –(Avg. CT target gene– Avg. CT housekeeping gene)] (Arnold et al., 2001). Each sample was run in triplicate. Primers used for real-time quantitative PCR are shown in Table S1.

Western blot analysis Total cellular protein, mitochondrial protein and nulcear protein were obtained from cultured cells by using protein extraction reagents (KeyGene, Nanjing, China) according to the manufacturer’s instructions. Total tissue protein was extracted from frozen endometrial tissues with radioimmunoprecipitation assay buffer (50 mm tris(hydroxymethyl)aminomethane pH 7.4, 150 mm sodium chloride, 0.5% sodium deoxycholate, 1% tergitol-type NP-40, 0.1% sodium dodecyl sulfate, 1 mm ethylene-diaminetetraacetic acid, 2 mm sodium orthovanadate, 50 mm sodium fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mm phenylmethanesulfonyl fluoride). Protein was quantified with BCA protein detection kit (Bioworld, Shanghai). After boiling with loading buffer (Bioworld, Shanghai), an equal amount of protein (15–40 µg) was separated on 10% sodium dodecyl sulfate polyacrylamide gel and transferred to a 0.45-µm pore polyvinylidene difluoride membrane (Millipore, USA). The membranes were blocked with 5% fat-free milk in tris-buffered saline (10 mm tris(hydroxymethyl)aminomethane [pH7.4], 0.5M sodium choloride, 0.1% Tween 20) at room temperature and incubated with primary antibodies at 4°C overnight. The antiPHB (Abcam, USA) antibody was diluted 1:1000 in 5% fat-free milk in TBS-T (tris-buffered saline and Tween 20), and anti-PGK1 antibody (Abcam, ab38007) 1:500, and antilactate dehydrogenase A antibody (Abcam, ab47010) 1:1000, and anti-aldolase antibody (Abcam, ab54770) 1:1000 and anti-pyruvate dehydrogenase E1 beta subunit antibody (Abcam ab110331) 1:1000. Anti-GAPDH (Abcam, USA), anti-β-tubulin (Bio-world, Shanghai) and anti-lamin A/C (Bio-world, Shanghai) antibody were used as a loading control at 1:2000, 1:500 and 1:1000 dilution, respectively. Then, membranes were washed and incubated with the appropriate secondary antibodies for 1 h at 37°C. Protein bands were detected by a chemiluminescence system (Alpha Innotech, USA) and quantified by using quantity one software (Bio-rad, USA).

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Immunofluorescence T-cells from human embryonic stem cells (T-hESC) lines (Human Endometrial Stromal Cells, ATCC, USA) were plated on the poly-lysine-coated culture slides for immunefluorescence analysis. At appropriate cell confluence, cells on the slides were pre-incubated with 500 nM Mito-Tracker Red CMVRos (Invitrogen, Shanghai) for 10 min at 37°C. The cells were fixed and permeabilized with 4% paraformaldehyde for 15 min and 0.2% Triton X-100 for 5 min, and then blocked with 1% bovine serum albumin in phosphate buffered saline for 1 h at room temperature. The slides were then incubated with anti-PHB antibody (Abcam, ab75771, USA) at a dilution of 1:100 in 1% bovine serum albumin overnight at 4°C. Anti-PHB antibody binding was visualized by incubation with a fluorescein isothiocyanate tagged goat antirabbit immunoglobulin G secondary antibody (1:100, Zhongshanjinqiao, Beijing) for 1 h at 37°C. Hoechst33342 (5 µg/mL) was used for nucleus counterstain. Cells were observed on a Zeiss LSM 700 confocal scanning microscope (Carl Zeiss, Jena, Germany).

Knockdown of prohibitin expression in eutopic endometrial stromal cells from women with endometriosis stromal cells Eutopic endometrial stromal cells from women with endometriosis were plated into 6-well plates and cultured to 70– 80% confluency before cell transfection. Short interfering RNA (siRNA) specifically against prohibitin (Invitrogen, PHBHSS182281) and the negative control siRNA (Invitrogen, lot:789324) were purchased from invitrogen biotechnology. Transfection was carried out with lipofectamine 2000 kit (Invitogen) at a ratio for 100 pmol siRNA to 5 µl lipofectamine 2000 according to the manufacturer’s instruction. Six hours after that, the culture medium was removed and replaced with fresh DMEM/F12 containing 10% fetal bovine serum.

Statistical analysis Analysis of variance and paired Student t-test were used for statistical comparison. Multiple comparison between the groups was carried out using the Student–Newman-Keuls method (post-hoc test). All data were presented as mean ± SEM using GraphPad Prism 5 (GraphPad Software, Inc, La Jolla, CA). P < 0.05 was considered significant.

Results Increased glucose consumption and lactate production in stromal cells from endometrioma tissue To evaluate the metabolism feature of cultured primary stromal cells, glucose consumption and lactate production were tested by collecting medium at 12 h, 24 h, and 36 h time point after transferring to fresh medium for each sample. Compared with stromal cells from women with and without with endometriosis, the stromal cells isolated from endometrial tissues showed increased glucose uptake (Figure 1 A) and lactate production (Figure 1B). No significant differences, however, were found between stromal cells from women with and without endometriosis. These results showed that stromal cells isolated from endometrioma tissues, cultured in vitro, required a higher glucose consumption for survival compared with stromal cells from women with and without endometriosis, followed by elevated lactate production.

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Figure 1 Glucose consumption and lactate production in cultures of primary stromal cells isolated from eutopic endometrium with stromal endometrial cells from women with endometriosis (EU), women without endometriosis (EN) or ovarian-endometrioma tissues (EC). After transfer to fresh medium, medium were collected at 12, 24 and 36 h, respectively, for testing (A) glucose consumption and (B) lactate production. Data represented as mean ± SEM, relative to normal controls (endometrial stromal cells isolated from the endometrium in women without endometriosis). *P < 0.05, **P < 0.001.

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Aberrant expression of glycolysis related enzymes in primary stromal cells from endometrioma tissue As the above results point to significant aberrations in the glucose metabolism in the stromal cells from endometrioma tissues from women with endometriosis, we investigated whether the genes involved in glucose consumption and lactate production were differentially expressed in endometrial stromal cells of women with endometriosis, those without and in cells isolated from endometrioma tissues. The mRNA expression of the glycolysis related genes between the three groups was first evalauted. Of the 14 target genes analysed (Table S1) using GAPDH as an internal reference gene, 10 genes were observed aberrantly expressed in cells isolated from endometrioman tissues, including seven genes (GLUT1, GLUT3, HK1, PKM, PDHA1, PDHB, and SDH) subunits A, B, C (versus endometrial cells from women with endometriosis only), D and down-regulated, and three genes (LDHA, HIF1α and PDK1) up-regulated. No significant differences were found among the groups in ALDOA, ALDOC, PGK1 and ENO1α mRNA

expression (Figure 2). These results suggest that altered expression of glycolysis related enzymes in cells from endometrioma tissues may contribute to the aberrant glucose metabolism.

Prohibitin is differentially expressed in endometrial stromal cells of women with endometriosis and in stromal cells of women without endometriosis In this study, endometrial stromal cells from women with endometriosis, women without endometrosis, and from endometrioma tissue were isolated and verified that the expression of PHB in stromal cells from endometrioma tissue is significantly down-regulated (P < 0.05 versus cells from women without endometriosis and P < 0.001 versus those with endometriosis) and in stromal endometrial cells from with with endometriosis up-regulated (P < 0.05 versusendometrial stromal cells in women without

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Figure 2 Real-time polymerase chain reaction data for genes related to glucose metabolism. (A) Glucose transporters, GLUT1 and GLUT3, were found down-regulated in stromal endometrial cells isolated from endometrioma tissues. (B) HIF1α and its target gene PDK1 showed up-regulated in stromal cells from endometrioma tissues, whereas PDHA1 and PDHB were down-regulated. (C) Some glycolytic enzymes, including HK1, PKM and LDHA, are aberrantly expressed in endometrial stromal cells from endometrioma tissues at mRNA level. (D) The mRNA expression of SDHA, SDHB, SDHC, SDHD (four units of SDH enzyme) were decreased in stromal cells rom endometrioma tissues, although for SDHC this was only significant when compared with endometrial stromal cells form women with endometriosis. Data represented as mean ± SEM. *P < 0.05, **P < 0.001.

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Figure 3 Prohibitin (PHB) mRNA expression levels in stromal cells and its sub-cellular localization in T-hESC cell line. (A) PHB was expressed at a low mRNA level in endometrial stromal cells from endometrioma tissues compared with endometrial cells in women with endometriosis and those without. Data represented as mean ± SEM. *P < 0.05, **P < 0.001. (B) Images obtained with Zeiss LSM 700 confocal microscopy. PHB (FITC/green) co-localized with the Mito-tracker Red. Nuclei were stained with Hoechst33252 (blue). Scale bar = 10 µm. (C) Western blots showing nuclear (nuc), cytoplasmic (cyto) and mitochondrial (mito) proteins extracted from T-hESC cells. Cyto* represented cytoplasmic protein excluding mitochondria. The extracts were immunoblotted for PHB; Lamin A/C, a nuclear antigen; β-tubulin is used to access the purity of the mitoplast preparation.

endometriosis) (Figure 3A). To determine the cell location of PHB, a double immune-fluorescence was carried out on T-hESC line which is a human endometrial stromal cell line immortalized with telomerase human telomerase reverse transcriptase. A rabbit monoclonal anti-PHB antibody and a fluorescein isothiocyanate tagged secondary antibody was used to detect PHB protein. Mito-Tracker Red was used to localize mitochondria. The co-localization of the green fluorescein isothiocyanate with Mito-Tracker Red resulted in a yellow colour on merged image (Figure 3B). In T-hESC cells, most of the PHB was localized in mitochondria. As a confirmatory procedure, proteins were extracted from mitochondria, and nuclei from T-hESC cells and Western blotting analyses was carried out. The results showed that PHB protein was detected predominantly in mitochondria and a little in nuclei, not in cytoplasm (Figure 3C).

Knockdown of prohibitin expression led to increased glucose consumption in endometrial stromal cells of women with endometriosis To investigate how PHB down-regulation might contribute to the increased glucose consumption in stromal cells isolated from endometrioma tissues, we conducted siRNA-mediated knockdown experiments. Stormal cells of women with endometriosis were transfected with PHB-specific siRNA oligonucleitides. As negative control siRNA with scrambled sequences was used for cell transfection 24 and 48 h after transfection, the expression of PHB mRNA and protein levels were analysed by quantitative PCR and immunoblotting (Figure 4A). The culture medium was collected at 12 h, 24 h, 36 h time point after 24 h after transfection for testing glucose consumption and lactate production. As shown in Figure 4B,

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Prohibitin regulated the expression of glycolysis related enzymes To determine whether PHB regulated glucose metabolism by regulating the expression of glycolysis related enzymes in EU, the mRNA expression of PKM, ENO1α, ALDOA, ALDOC, LDHA, HIF1α, PDK1, PDHA1 and PDHB were evaluated by quantitative PCR 24 h after transfection in PHB knockdown and control group. Compared with the control group, the mRNA expression of PKM, ALDOC, LDHA, PDHA1 and PDHB in the PHB knockdown group were significantly increased (P < 0.05), whereas HIF1α, ENO1α and ALDOA had no change (Figure 5).

Discussion In this study, it was observed that stromal cells isolated from endometrioma tissues and cultured in vitro had increased

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knockdown of PHB led to a significant increase in glucose consumption in EU at 24 h, 36 h time point 24 h after transfection (P < 0.05). At 24 h and 36 h time point the lactate levels were marginally increased, but the change did not reach a statistically significant level.

Relative expression of mRNA levels

Figure 4 Evaluation of glucose consumption and lactate production after knocking down prohibitin (PHB) expression. (A) For quantitative polymerase chain reaction and immunoblot identification of transfection efficiency, PHB mRNA and protein levels decreased by more than 50% 24 and 48 h after transfection, respectively. PHB mRNA data was represented as mean ± SEM, which was from experiment cells from nine paired different individuals. *P < 0.05. (B) At 24 and 36 h time point 24 h after transfection, knockdown of PHB in endometrial stromal cells in women with endometriosis increased glucose consumption. (C) Knockdown of PHB did not affect lactate production. Results were from experiments using cells from at least 4 women. siRNA-Ctl, negative control; siRNAPHB, siRNA against PHB; *P < 0.05; NS, non-significant.

Figure 5 Real-time polymerase chain reaction data for genes related to glucose metabolism after knocking down prohibitin expression. Knockdown of prohibitin in stromal endometrial cells rom women with endometriosis caused increased expression of PKM, ALDOC, LDHA, PDHA1 and PDHB at a ratio of 1.42, 1.23, 1.26, 1.26 and 1.19, respectively, compared with negative controls. HIF1α, ENO1α and ALDOA were not affected. Results were from experiment cells from 9 different women. *P < 0.05 (Supplementary Figure 1).

768 glucose consumption and lactate production compared with stromal cells islated from the endometrium in women with endometriosis and those without. No difference, however, was found between ensometrial stromal cells in women with endometriosis and those without. The increased glucose uptake by stromal cells from endometrioma tissue is largely supported by findings from Dutta et al. (2012), which show that serum samples from endometriosis patients have increased levels of lactate as well as decreased levels of glucose compared with controls. Furtherhigher levels of glycolytic intermediate pyruvate, tricarboxylic acid (TCA) cycle product succinate, reactive oxygen species and insufficient antioxidant capacity were observed in endometriosis woman by using metabonomics method based on serum metabolite profiles (Jana et al., 2013). The author, however, did not show the data if glucose levels correlated with disease severity. In the present study, two reasons may explain no difference between endometrial stromal cells from women with endometriosis and those without. The first is that a minor difference between the two on glucose metabolism is hardly detected by our experimental system. Secondly, in-vitro environment may attenuate differences of the groups. Using quantitative PCR, the glycolytic genes mRNA expression were compared among the groups and 10 genes differentially expressed in stromal cells from emdometrioma tissues were found, with seven genes (GLUT1, GLUT3, HK1, PKM, PDHA1, PDHB and SDH subunits A, B, C [versus stromal cells from women with endometriosis] D and down-regulated and three genes (LDHA, HIF1α and PDK1) up-regulated (Figure 2). The altered glucose consumption and lactate production may be caused by differentially expressed glycolysisrelated genes in stromal cells isolated from endometrioma tissues, which may be associated with inflammatory response (Giudice, 2010), hormonal alteration (Huhtinen et al., 2012), immunological changes (Kyama et al., 2009), decreased oxygen tension (Wu et al., 2007), high concentration of free iron (Kobayashi et al., 2009), which endometriotic lesions are reportedly exposed to. For example, HIF1α, a key transcription activator that responds to hypoxia, is previously found up-regulated in stromal cells from endometrioma tissue (Wu et al., 2007). HIF1α is important to promote glycolysis in cancer cells, and its target genes include glucose transporters (GLUT1, GLUT3), glycolytic enzymes (HK1, ALDOA, ALDOC, PGK1, ENO1α, PKM, LDHA) (Marin-Hernandez et al., 2009). It promotes glucose influx and conversion to pyruvate and lactate, providing a carbonic source for energy generation. PDK1, another HIF1α inducible gene, inhibits the activity of pyruvate dehydrogenase complex that modulates the entry of pyruvate to TCA cycle (Kroemer and Pouyssegur, 2008; McFate et al., 2008; Wigfield et al., 2008). Ultimately, cancer cells perform enhanced glycolysis and inactivating OXPHOS. Similarly to cancer cells, the ‘Warburg effect’ in stromal cells from endometroma tissue can occur because of decreased PDHA1, PDHB and increased LDHA mRNA expression (Wigfield et al., 2008). LDHA, previously shown to be regulated by HIF1α and c-Myc at transcriptional level, is critical for a shift of glycolysis to OXPHOS, which is essential for cell proliferation (Fantin et al., 2006). Pyruvate dehydrogenase complex consist three component enzymes: E1, E2 and E3. Pyruvate dehydrogenase E1 component subunits are PDHA1 and PDHB. Pyruvate dehydrogenase complex activity is inhibited by PDK1 whereas it is directly up-regulated by HIF1α

X Qi et al. transcriptional activity (McFate et al., 2008). Pyruvate dehydrogenase complex is identified as a key player in the glycolytic pathway as it controls the conversion of pyruvate to acetyl coenzyme A, which can then enter the TCA cycle for glucose oxidation. We showed the mRNA expression of glucose transporters GLUT1 and GLUT3 were down-regulated in endometriotic stromal cell from endometrioma tissues. GLUT1 mRNA was identified as the most abundant and GLUT3 the third most abundant among the GLUTs in human stromal cells (Frolova and Moley, 2011). Both of them may play important roles in glucose uptake and GLUT1 is likely the most influential factor in stromal cell decidualization that is related to increased glucose uptake (Frolova et al., 2009; Frolova and Moley, 2011). Moreover, GLUT1 protein level is positively correlated with glucose consumption activity (Frolova et al., 2009). It is unclear, however, why GLUT1 and GLUT3 mRNA in endometrial stromal cells from endometrioma tissue are decreased, whereas glucose consumption increased compared with endometrial stromal cells in women with endometriosis and women without endometriosis. At the same time, GLUT1 protein expression was not detected in stromal cells. Baban et al. (2001) reported that GLUT1 mRNA expression was up-regulated in endometriotic epithelial cell in endometriosis patients staged as III or IV during proliferative and secretary phases of the menstrual cycle. Strowitzki et al. (2001) detected that eutopic endometrium had increased GLUT1 protein expression than endometriotic lesions, in which GLUT1 mainly located in epithelial cells. In mouse models established by Becker et al. (2008), endometriosis-like lesions showed active HIF1α expression immediately after transplantation, with HIF1α target genes (GLUT1, PGK and VEGF) up-regulated and subsequently decreased over time. Accordingly, we speculate the following: that other GLUTs, not GLUT1 or GLUT3, may contribute to the glucose uptake in endometriotic stromal cells; that GLUT1 may be mainly expressed in epithelial cells therefore its protein expression in stromal cells at proliferative phase of menstruation is very low for detection; and that GLUT1 and other glycolytic enzymes (HK1, PKM) may be differentially expressed at early or advanced stage of the disease. The mRNA expression for ALDOA and ALDOC, members of aldolase family, showed no difference between the endometriotic and eutopic endometrial stromal cells. In our previous study, ALDOA was found to be highly expressed in endometrioma tissue at protein level (Mao, 2007), and this protein was up-regulated by IL-1β in human endometrial stromal cells (Hou et al., 2008). The results supported that the expression of glycolysis associated genes in cells were determined by cell type, hormonal regulation and local microenvironment, which may be implicated at different stages of the disease (Frolova et al., 2009; Machado-Linde et al., 2012). Four subunits of succinate dehydrogenase (SDH), SDHA, SDHB, SDHC and SDHD, consist of mitochondrial complex II (succinate-ubiquinone oxidoreductase), which is involved in electron transition from succinate, SDHA, iron-sulphur centers (SDHB) to ubiquinone (coenzyme Q, coQ) (Bardella et al., 2011). Mettler et al. (2007) used c-DNA microarray analysis and found SDHD expression was decreased in endometriotic tissues. Decreased expression of SDHD may contribute to mitochondrial deficicency and accumulation of succinate, an important component of the glycolytic pathway (Puissegur et al., 2011). When combined with other evidence of the deficiency of mitochon-

Glucose metabolism and endometriosis drial enzyme in endometriosis, such as ATP synthase beta subunit (Zhang et al., 2006) and succinate-CoA ligases (Meola et al., 2010), cytochrome c oxidase subunits VIc (Mettler et al., 2007), our findings support the thought that endometriosis seems to be a disease with mitochondrial dysfunction at the cellular level (Ding et al., 2010). All the results above may indicate that aberrant glycolytic enzyme expression and mitochondrial dysfunction contribute to the enhanced glycolysis in stromal cells derived from endometrioma tissue. Our results also show a low level of PHB expression in endomtrial stromal cells isolated from endometrioma tissues, and PHB was mainly located in the mitochondria. In the study, an in-vitro model with down-regulated PHB expression in eutopic stromal cells to determine whether down-regulated PHB could induce increased glucose uptake as well as elevated lactate production, which were observed in EC cells in vitro. Then, it was proposed that PHB deficiency may be responsible for increased glucose metabolism in endometrial cells isolated from endometrioma tissues in vitro. It was shown that when PHB was knocked down in endometrial cells from endometrioma tissues, increased mRNA expression of PKM, ALDOC, LDHA, PDHA1 and PDHB were noted, suggesting that PHB deficiency may promote glucose consumption. The no change of lactate production seems to contradict the results of glucose consumption. A reasonable explanation may be that eutopic and ectopic stromal cells have different gene expression profiles (Wu et al., 2006). Prohibitin deficiency may contribute to the increased entry of pyruvate to TCA cycle, as can be speculated from up-regulated expression of PDHA1 and PDHB. This may lead to higher activity of TCA cycle , which is quite different from stromal cells from endometrioma tissues because of its mitochondrial dysfunction, as mentioned above. We hypothesize that PHB deficiency may play an important role in aberrant glucose metabolism of stromal cells from endometrioma tissues, especially in exposure of their local environment, such as decreased oxygen tension. The regulation of glucose metabolism by PHB deficiency in stroma cells from endometrioma tissues need further study. Vessal et al. (2006) provided evidence that exogenous PHB-1 suppressed glucose oxidation in adipocytes via inhibiting pyruvate carboxylase enzyme activity. Ande and Mishra (2009) showed that phosphorylation of PHB and its interaction with PIP may be involved in insulin signalling. It may also play a role in the PI3K/ AKT pathway pathway as a substrate for insulin and insulinlike growth factor-1 receptors, involved in glucose metabolism (Mishra et al., 2010). Deficiency in PHB may activate a retrograde communication that is associated with mitochondrial proliferation and energy production (Artal-Sanz and Tavernarakis, 2009). At this time, whether mitochondrial PHB to function through these pathways in endometriosis stromal cells remains unclear, and further mechanistic studies are required to better understanding of the mechanisms. In this study, an attempt was made to determine the differential expression of glycolytic enzymes that contributed to aberrant glucose metabolism in endometriosis in vitro. The first limitation in the study is our inability to test glucose uptake and lactate production to investigate the glucose metabolism in vivo. The second limitation is that we only showed mRNA levels of glycolytic enzymes (Figure 2 and Figure S1) and protein levels (Figure S2) for some of the enzymes of the glycolytic pathway because of limited quantities of stromal cells available and the activity of the enzymes was not assessed.

769 In conclusion, our findings support that glycolytic genes differential expression and altered glucose metabolism may be involved in the process of endometriosis. Prohibitin might serve as an upstream regulator for the enzymes involved in the alteration of glucose metabolism in the eutopic endometrium from endometriosis woman. These findings have provided new insights into the pathophysiology of endometriosis. The significance and exact role of PHB in glucose metabolism and its involvement in different stages of endometriosis should be investigated in future studies.

Acknowledgements Thanks is due to Professor Li Da-Jin (Dean & Professor, Laboratory for Reproductive Immunology, Hospital and Institute of Obstetrics and Gynecology, Fudan University Shanghai Medical College, Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Shanghai, China) for his scientific guidance in the research. This work was supported by grants from the Major State Basic Research Development Program of China (2012CB944902, 2012CB944703), the National Natural Science Foundation of China (81200424), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and the Jiangsu Province Maternal and Child Health Program for Talented Person (No. FRC201215).

Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.rbmo.2014.09.004.

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Declaration: The authors report no financial or commercial conflicts of interest.

Received 7 April 2014; refereed 31 August 2014; accepted 4 September 2014.