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omental/visceral and subcutaneous fat depots: a comparison from pregnant and obese women. Suman Rice, Ph.D.,a Bijal Patel, B.Sc.,a Gul Bano, M.D., ...
ORIGINAL ARTICLE: REPRODUCTIVE ENDOCRINOLOGY

Aromatase expression in abdominal omental/visceral and subcutaneous fat depots: a comparison from pregnant and obese women Suman Rice, Ph.D.,a Bijal Patel, B.Sc.,a Gul Bano, M.D., F.R.C.P.,b Austin Ugwumadu, Ph.D., F.R.C.O.G.,c and Saffron A. Whitehead, Ph.D.a a Division of Biomedical Sciences, b Thomas Addison Endocrinology Unit, and University of London, London, United Kingdom

c

Department of Obstetrics and Gynaecology, St. Georges

Objective: To investigate total and promoter expression of aromatase in subcutaneous and omental (visceral) fat and compare this expression in pregnant and obese women. Design: Cross-sectional study. Setting: Academic hospital. Patient(s): Six women undergoing elective cesarean section and three women undergoing bariatric surgery. Intervention(s): Subcutaneous and omental fat obtained during surgery. Main Outcome Measure(s): Total aromatase and promoter expression was measured by polymerase chain reaction and protein levels by Western blotting. Result(s): Total aromatase expression was significantly higher in omental compared with subcutaneous fat from pregnant women; this pattern was reversed in obese women. Aromatase messenger RNA in omentum was significantly higher in pregnancy than obesity, and this was linked to an upregulation of promoter II (PII). Promoter 1.4 (P1.4) expression was lower than PII, and there was no difference in P1.4 expression between the two fat depots from pregnant subjects. In obese women both P1.4 and PII were up-regulated in subcutaneous compared with omental depots, with P1.4 expression greater than that of PII. Aromatase protein levels were extremely low in fat depots of pregnant women and undetectable in obese women. Conclusion(s): There are differences between total aromatase and promoter expression in subcutaneous and omental fat from pregnant compared with obese women. These differences support the evidence that the fat depots are derived from different cell lineages and that the promoter-derived aromatase translation varies according to physiologic/pathophysiologic status. (Fertil SterilÒ 2012;-:-–-. Ó2012 by American Society for Reproductive Medicine.) Key Words: Adipose tissue, omental/visceral fat, aromatase, promoters

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t is now well recognized that adipose tissue is an endocrine organ, not only secreting a range of adipokines that can regulate food intake and responses to insulin, but also synthesizing active steroids from circulating precursors (1). A major steroid produced by adipose tissue is E2, and its local production is important in increasing proliferation of preadipocytes, the size of mature adipocytes, and thus adipose mass (2).

There are two types of adipose tissue, subcutaneous (SC) and visceral/ omental (OM), which are derived from different cell lineages (3) and may have different functions. Approximately 80% of all body fat is in the SC area, whereas visceral fat accounts for up to 10%–20% of total fat in men and 5%–8% in women (4). Both types of adipose tissue are dynamic structures, and their mass varies according to sex, different physiologic states,

Received August 11, 2011; revised February 15, 2012; accepted March 6, 2012. S.R. has nothing to disclose. B.P. has nothing to disclose. G.B. has nothing to disclose. A.U. has nothing to disclose. S.A.W. has nothing to disclose. Funding was received from St. George's University of London. Reprint requests: Suman Rice, Ph.D., Division of Biomedical Sciences, St. Georges University of London, Jenner Wing, Cranmer Terrace, London SW17 ORE, United Kingdom (E-mail: [email protected]). Fertility and Sterility® Vol. -, No. -, - 2012 0015-0282/$36.00 Copyright ©2012 American Society for Reproductive Medicine, Published by Elsevier Inc. doi:10.1016/j.fertnstert.2012.03.008 VOL. - NO. - / - 2012

and nutritional status (5–8). Visceral fat mass is known to increase in generalized obesity and is more strongly associated with an abnormal metabolic profile than upper body SC fat (5, 9). Thus it is important to study differences in visceral and SC fat to understand the important contribution made by visceral fat in the etiology of various disorders, including the metabolic syndrome and polycystic ovary syndrome (PCOS) (10–12). Estradiol synthesis is determined by the expression of P450 aromatase (13), which converts androstenedione (A) to estrone and T to E2. Transcriptional regulation of the CYP19 gene that encodes for aromatase expression occurs by alternative splicing of a number of untranslated first exons onto a common splice site upstream of the 1

ORIGINAL ARTICLE: REPRODUCTIVE ENDOCRINOLOGY coding region of CYP19. The various first exons are regulated by tissue-specific promoters containing different cohorts of response elements. This then allows for the biosynthesis of estrogen (E), via the aromatase gene expression, to be uniquely regulated in each tissue by specific hormones and secondmessenger pathways (14). In adipose tissue the main promoter used is promoter 1.4 (P1.4), although transcripts specific for promoter II (PII) and 1.3 (a splice variant of PII) have also been identified (13). This contrasts the major promoters expressed in the ovary (PII), normal breast tissue (1.4), and placenta (1.1). These promoters can be activated by different signaling pathways, and in adipose tissue glucocorticoids and class 1 cytokines, including interleukin-6 and -11 acting via the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway, increase 1.4 transcripts (15). In contrast, increased transcriptional activity of P1.3/II can occur through activators of cyclic adenosine 30 :50 monophosphate signaling (e.g., prostaglandin E2 and FSH) (16). Although there are clear differences in the metabolic profile between OM and SC fat (4), very few studies have addressed whether the expression and activity of steroidogenic enzymes varies between OM and SC fat, and to date no studies have investigated whether there are any differences in aromatase promoter usage between visceral and SC fat, as has been observed in several tissues, including ovaries, bone, breast tissue, placenta, vascular endothelium, skin, and brain (17). To this end, we hypothesized that there were differences between visceral and SC fat in aromatase promoter usage, resulting in differences in total aromatase production between the two fat depots and ultimately local and/or circulating E levels. We were also interested to investigate whether the expression of aromatase gene transcripts is different in pregnant women compared with nonpregnant women, given that pregnancy is associated with a dramatic rise in E levels and other hormones and a gain in fat mass to support the developing fetus (6).

MATERIALS AND METHODS Subjects and Tissue Samples After approval by the Wandsworth Local Research Ethics Committee (08/H0803/134), six pregnant subjects attending the Day Assessment Unit at St George's Hospital, Tooting on the day before elective cesarean sections were recruited. Written consent was obtained from all patients. All the pregnant women were at R39 weeks' gestation, and all were singleton pregnancies. The mean (SEM) age of the women was 33.7  2.5 years, and average body mass index (BMI) (at first antenatal check at approximately 10 weeks' pregnant) was 24.9  1.4 kg/m2. All but one of the pregnant subjects had conceived spontaneously, the other by IVF (Supplemental Table 1, available online). Samples of SC and OM fat (taken from the free edge of the omentum) were collected during cesarean section by the same surgeon, to minimize variation in sampling sites and technique, along with placenta from one patient. In addition, samples were taken from seven nonpregnant subjects. Four were obtained during laparoscopic cholecystectomies, and three during bariatric surgery for the treatment of obesity. Biopsy samples obtained from the laparoscopic 2

procedures were too small for effective extraction of RNA/ protein, leaving us with three nonpregnant controls. The mean age of the obese women was 34  3.5 years, with an average BMI of 47.4  1.3 kg/m2. These women had regular periods with no features of PCOS, diabetes, or metabolic syndrome. All three had conceived spontaneously (Supplemental Table 1). Samples (SC and OM) were collected on the day of their bariatric surgery. All samples obtained were placed in RNase-DNase-free Eppendorf tubes and immediately snap-frozen in liquid nitrogen and stored at 80 C before RNA or protein extraction.

RNA Extraction and Complementary DNA Synthesis Initially tissue samples were defrosted on ice, and up to 70 mg of each fat sample was cut and total RNA extracted according to the manufacturer's protocol using the RNeasy minikit (Qiagen). However, the yield of RNA was extremely low owing to the nature of adipose tissue. Subsequent samples were homogenized in TRIzol reagent (Sigma-Aldrich), and the extracted RNA was then cleaned up and treated with deoxyribonuclease (to eliminate contaminating DNA) using the RNeasy minikit. Ribonucleic acid quantity was measured using a Nanodrop spectrophotometer (Agilent Technologies), and where possible equal amounts of RNA from each OM and SC sample were reverse transcribed as previously described (18). Controls to check for genomic contamination were generated by omitting Moloney murine leukemia virus reverse transcriptase (MMLV-RTase), and positive controls used sample homogenates from ovarian stroma, granulosa-luteal cells, or KGN (granulosa-tumor cell line).

Total Aromatase Real-Time PCR Assay Protocol Real-time polymerase chain reaction (PCR) analysis for total aromatase messenger RNA (mRNA) expression was performed as previously described, with normalization to b-actin as the reference gene (17). Data are expressed as the mean of the gene expression ratio  SEM.

Aromatase Exon-Specific Promoter PCR Protocol To determine the expression of specific aromatase promoters II (PII), 1.4 (P1.4), and 1.3 (P1.3) in exon 1, analysis of untranslated first exons was performed using standard PCR assays as previously described (19). Briefly, promoter-specific forward primers were designed to be located in exon 1 with a common reverse primer in exon III. Semiquantification of amplified PCR products was performed by densitometry analysis of the ratio of the promoter against b-actin mRNA expression (as a loading control).

Protein Extraction and Western Blotting Samples were defrosted on ice and then homogenized with a polytron homogenizer in lysis buffer of 100 mM Tris (trishydroxymethylamine), 250 mM NaCl, and 1 mM ethylenediaminetetraacetic acid along with a cocktail of protease inhibitors (Sigma) and phenylmethylsulfonyl fluoride. After VOL. - NO. - / - 2012

Fertility and Sterility® homogenization, 1% NP-40 was added to the lysates and the samples rotated at 4 C for 30 minutes followed by centrifugation. The supernatant containing extracted protein was then resolved by Western blotting and visualized on the Odyssey Imaging System (Li-cor Biosciences) as previously described (20) using a fluorescently conjugated secondary goat antimouse antibody IR700 (1:5,000) against anti-aromatase (1:250, MCA2077S; Serotec) and anti-b-actin (1:1,000, ab3280; Abcam), which was used as a loading control. Placental tissue served as a positive control of aromatase expression.

FIGURE 1

Statistical Evaluation The fold change in total aromatase mRNA expression in each fat depot (either SC or OM) was assessed by the 2DCt method, whereby the cycle threshold (Ct) values from the real-time PCR analysis for aromatase were normalized to Ct values for b-actin (21). Data represent the mean of the gene expression ratio  SEM. Differences were analyzed statistically using one-way analysis of variance (ANOVA; Kruskal-Wallis test), followed by post hoc Mann-Whitney t test and F test for variance. The densitometry measurements of promoter expression from each fat depot was averaged for all the subjects and expressed as mean  SEM. Statistical significance between the two promoters and two depots was determined by two-way ANOVA, followed by unpaired t test with Welch's correction. Significance was set at P%.05.

RESULTS Total Aromatase mRNA Expression from Fat Depots Total aromatase mRNA levels were more than 10-fold higher in the OM depot compared with SC depot from pregnant women. This depot-specific difference was significant using the F test for variance (P¼ .03), which indicated that the two populations are different, and just under the borderline for significance using Mann-Whitney (P=.057). However, in contrast, in the tissue depots from obese women, total aromatase was approximately fourfold higher in the SC compared with OM depot (P¼ .05), with virtually no aromatase expression in OM fat. Aromatase levels in OM tissue from pregnant women were significantly up-regulated compared with those in obese women (P¼ .03) (Fig. 1).

Exon-Specific Aromatase Promoter Expression Pregnant samples. To characterize the promoter usage in individual SC and OM samples, reverse transcription (RT)-PCR analysis was performed using exon-specific primers corresponding to PII, P1.4, and P1.3 in exon 1. Complementary DNA from granulosa-luteal cells and ovarian cortex was used as positive control, having been shown to express these promoters (22). All samples expressed b-actin (housekeeping gene), indicating adequate amounts of RNA and effective RT (Fig. 2A, representative gel, bottom). The level of PII expression was significantly higher in OM samples from all subjects compared with SC, and in some instances there was negligible mRNA transcription of PII in SC depot (Fig. 2A, representative gel, top). The level of P1.4 compared with that of PII was lower in all subjects, and there was no difference in expression VOL. - NO. - / - 2012

Column graph showing total levels of CYP19 mRNA measured in SC and OM adipose tissue samples from pregnant (n ¼ 4) and obese women (n ¼ 3). Aromatase mRNA was measured by real-time quantitative PCR and expressed as a fold change in expression relative to b-actin. There was a more than 10-fold increase in total aromatase mRNA in OM compared with SC in samples from pregnant women (P¼.057, Mann-Whitney), and this depot-specific difference was significant using the F test for variance (*P¼.03). In samples from obese women, there was a fourfold higher increase in total aromatase mRNA in the SC compared with OM depot (*P¼.05, Mann-Whitney). Expression of aromatase was significantly up-regulated in OM tissue from pregnant women compared with obese (*P¼.03, Mann-Whitney). Rice. Aromatase promoters in fat depots. Fertil Steril 2012.

levels of P1.4 between the two types of fat depots (Fig. 2A, representative gel, second panel from top). There was no expression of P1.3 in either fat depot of all samples (Fig. 2A, representative gel, third panel from top). Densitometry analysis of the mean expression ratio confirmed these results (Fig. 2B) and demonstrated that not only was PII expression significantly higher in OM compared with SC (P¼ .004), it was also expressed at an appreciably higher level than P1.4 in both OM and SC (P¼ .03). Two-way ANOVA of the source of variation confirmed that the interaction of both types of fat depot with expression of both promoters is significant (P¼ .048). Obese samples. As for the pregnant samples, all tissues expressed b-actin mRNA (Fig. 3, bottom), but there were greater expression levels of P1.4 (Fig. 3, second panel from top) rather than PII (Fig. 3, top), and this was more apparent in the SC compared with OM depot. There was no expression of P1.3 (Fig. 3, third panel from top) in any of the fat samples. Complementary DNA from KGN cells were used as a positive control. Though sample OB3 did not express any of the promoters assayed in either SC or OM, it did express total aromatase mRNA, indicating the probable use of alternative promoters.

Aromatase Protein Expression in Adipose Tissue Samples Protein expression of aromatase was only detected in samples from pregnant women (Fig. 4A) and surprisingly not in fat samples from obese women (Fig. 4B), despite there being abundant expression of b-actin protein in all tissue samples. There was no discernible difference in aromatase protein levels between the depots and no correlation between 3

ORIGINAL ARTICLE: REPRODUCTIVE ENDOCRINOLOGY

FIGURE 2

(A) Representative picture of agarose gels (2%) showing the mRNA expression of various aromatase promoters in SC and OM adipose samples from pregnant subjects 1, 2, 5, and 6. Standard PCR analysis was performed using promoter-specific primers to investigate the difference between the two depots of mRNA expression of PII, P1.4, and P1.3. Messenger RNA from ovary (OC) and granulosa-luteal cells were included as a positive control for the promoters. Also included was a no-RT control for sample 6 (OM and SC), which indicated that there was no genomic contamination of RNA extracted. Beta-actin mRNA expression was used as a reference gene and was expressed in all tissue samples. Promoter 1.3 was not expressed in any adipose tissue samples. (B) Graph of densitometry values of PCR analysis of PII and P1.4 in all SC and OM samples from pregnant women (mean  SEM). The PII expression was significantly higher in OM compared with SC (**P¼.004) and also higher than P1.4 in either SC (*P¼.03) or OM (*P¼.03) depots (unpaired t test with Welch's correction). Rice. Aromatase promoters in fat depots. Fertil Steril 2012.

mRNA and protein expression in the tissue samples from the pregnant subjects. Placenta was used as a positive control for aromatase protein and, as expected, showed considerable expression, confirming validity of the antibody.

DISCUSSION The results have shown that the total aromatase mRNA expression was significantly higher in OM vs. SC fat in pregnant women and that this was primarily due to the increased expression of PII promoter expression in OM compared with SC fat. This shows a clear-cut difference in aromatase promoter usage between OM vs. SC fat and highlights the differences between the two fat populations. However, there was no difference in total protein expression between OM and SC fat of pregnant women and, compared with the placenta, the 4

aromatase protein was weakly expressed. Interestingly there were measurable levels of aromatase mRNA in the fat from control subjects, with virtually no expression of protein in either depot. McTiernan et al. (23) investigated the differences in the activity and/or expression of aromatase in SC vs. OM fat. Basal aromatase activity in preadipocytes was higher in SC fat compared with OM fat, but in mature adipocytes aromatase activity was similar in both SC and OM fat but at least 10-fold lower than that observed in preadipocytes (23). They also showed that glucocorticoid-induced aromatase activity was higher in OM fat of postmenopausal women compared with premenopausal women but similar in SC fat from pre- and postmenopausal women. This indicates an increase in glucocorticoid-induced P1.4 expression (14, 16) in OM fat in postmenopausal women. VOL. - NO. - / - 2012

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FIGURE 3

Representative picture of agarose gels (2%) showing the mRNA expression of various aromatase promoters in SC and OM adipose samples from obese subjects 1, 2, and 3. Standard PCR analysis was performed using promoter-specific primers to investigate the difference between the two depots of mRNA expression of PII, P1.4, and P1.3. Messenger RNA from KGN was included as a positive control for the promoters. Also included was a no-RT control for sample 3 (OM), which indicated that there was no genomic contamination of RNA extracted. Beta-actin mRNA expression was used as the reference gene and was expressed in all tissue samples. Promoter 1.3 was not expressed in any adipose tissue samples. Rice. Aromatase promoters in fat depots. Fertil Steril 2012.

In a further study the expression of several steroidogenic enzymes was investigated in SC (abdominal) and OM fat of eight women undergoing cesarean section (24). Using realtime PCR, the investigators found that there was a significant increase in aromatase expression in OM vs. SC fat, as well as in the expression of StAR (steroidogenic acute regulatory protein), 11b-hydoroxysteroid dehydrogenase (HSD) type 1,

which converts inactive cortisone to active cortisol, 21-hydroxylase, converting P to deoxycortisol, and 17b-HSD type 7 that converts A to T. Our results confirm the findings of an increase in aromatase expression in OM fat in pregnancy and show that the mechanism is via an up-regulation of PII transcription. A few other studies comparing enzyme activity and expression in SC vs. OM fat have been undertaken. Courbold et al. (25) reported that both SC and OM preadipocytes converted A to T, but there was little conversion of A to estrone. This agrees with our findings of low expression of aromatase in both OM and SC fat in pregnant women. They further showed that the ratio of the levels of 17b-HSD type 3 mRNA to aromatase mRNA was positively correlated with BMI in OM fat, whereas the ratio in SC fat was negatively correlated. In this respect it is interesting to note that generalized obesity has been associated with increased aromatase mRNA, whereas central obesity (defined as waist/hip ratio) was associated with increased mRNA for the predominant aldoketoreductase isoforms (AKR1C2 and AKR1C3) that account for 3a-HSD and 17b-HSD activity involved in T synthesis and metabolism (26). Our studies show that aromatase protein expression was absent in obese subjects, although mRNA expression, notably in the SC fat, was readily detected. We do not believe that this is due to small sample size of our study because the real-time PCR assay results were extremely consistent and produced statistically significant results, but they do highlight the importance of adequate tissue weight from biopsy samples to extract sufficient quantities of mRNA and protein. It is well documented that in many tissues, aromatase protein is expressed at low levels, and its detection is limited by sensitivity of the antibody (27). The antibody used in this study successfully detected aromatase in granulosa-luteal cells, ovarian KGN, and in placenta,

FIGURE 4

(A) Representative picture of Western blots showing the protein expression of aromatase and b-actin in SC and OM tissue samples from pregnant women (samples P1, P2, P5, and P6), with protein from placenta (plac) as a positive control from aromatase expression. There was expression of b-actin in all samples. Aromatase was detected in all samples, albeit at a much lower level than placenta, with no discernible differences between the depots. L ¼ protein marker. (B) Representative picture of Western blots showing the protein expression of aromatase and b-actin in SC and OM tissue samples from obese women (samples Ob1, Ob2, and Ob3). There was barely any detectable aromatase protein present despite adequate amounts of b-actin. Rice. Aromatase promoters in fat depots. Fertil Steril 2012.

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ORIGINAL ARTICLE: REPRODUCTIVE ENDOCRINOLOGY and these levels correlated well with aromatase mRNA (19, 20). In contrast, we were unable to detect aromatase protein in MCF-7 breast cancer cells, though aromatase mRNA was readily measurable (unpublished observation). Hence, in numerous studies aromatase mRNA abundance is used as a surrogate readout for protein expression (27). Another possible reason for the differences between aromatase mRNA and protein expression in adipose tissue could be the important role of the alternative exon 1s in posttranscriptional regulation (27). Using a common heterologous promoter to drive the expression of multiple aromatase complementary DNA sequences, which differ only in their first untranslated exon, Wang et al. (27) showed that there were huge differences in the levels of aromatase mRNA and protein. This was due to negative regulation by several exon 1 sequences on protein translation and RNA stability. Agarwal et al. (28) studied alternatively spliced transcripts of the aromatase gene, CYP 19, in SC adipose tissue obtained from buttocks, thighs, and abdomen of 11 women aged 23–61 years. They showed that the exon 1.4–specific transcripts predominated, regardless of the tissue site, with lower expression of PII and 1.3 transcripts. Both exonspecific and total aromatase expression increased proportionately with age, with levels being highest in buttocks, followed by thighs, and lowest in the abdomen. In contrast to this study, we did not find any expression of P1.3 transcripts in SC and OM abdominal fat depots of pregnant women, which may be due to differences in techniques used or because pregnancy induces a different promoter usage. Taken together, these findings agree with the fact that abdominal SC and visceral fat originate from distinct populations, as shown by genome-wide profiling of primary adipocytes cultured in parallel from abdominal SC and visceral fat (3). Our own data support this, indicating that there are population differences between the two depots. Furthermore, because the transcriptional activity of different promoters is regulated by different signaling molecules (i.e., hormones, growth factors, and cytokines [14, 16, 18]), this would suggest that the two depots are under different regulatory mechanisms. Pregnancy is associated with a preferential accumulation of adipose tissue in the abdominal visceral compartment relative to abdominal SC compartment, as shown by a longitudinal prospective study carried out before conception and after delivery in 122 women (29). This may be due to the induction of different promoter usage induced by hormonal changes in pregnancy and the consequent preferential differentiation of visceral preadipocytes compared with SC preadipocytes (2). It is clear from this study that further investigations are required to identify the significance of different promoter usage in SC vs. OM fat, the signals that control different promoter transcript expression, and whether this has any significance in the differentiation of central vs. SC adiposity and in the etiology of obesity, the metabolic syndrome, and PCOS.

advice on protein extraction from fat; Ms. Heather Nash for her help with collecting information on the pregnant subjects; and the Biomics Department, St. George's University of London for access to the real-time cycler.

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Acknowledgments: The authors thank the patients at St. George's Hospital for donation of the tissues used in this study, and the surgical staff for their help in collecting it; Dr. Fu Liang Ng for consenting and collecting adipose tissue samples from the obese patients; Dr. Mark Christian for his 6

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SUPPLEMENTAL TABLE 1 Details of patients involved in this study. Subject P1 P2 P3 P4 P5 P6 P, mean  SEM Ob1 Ob2 Ob3 Ob, mean  SEM

Age (y)

BMI

Conception

PCOS

32 41 38 34 34 23 33.7  2.5 27 37 38 34  3.5

25 25.4 31.1 23.6 20.9 23.2 24.9  1.4 48 45 49.2 47.4  1.3

Spontaneous IVF Spontaneous Spontaneous Spontaneous Spontaneous

Yes No No No No No

Spontaneous Spontaneous Spontaneous

No No No

Note: BMI ¼ body mass index; PCOS ¼ polycystic ovary syndrome; P ¼ pregnant; Ob ¼ obese. Rice. Aromatase promoters in fat depots. Fertil Steril 2012.

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