J Clin Endocrin Metab. First published ahead of print November 11, 2013 as doi:10.1210/jc.2013-2017
ORIGINAL E n d o c r i n e
ARTICLE R e s e a r c h
Expression and Subcellular Localization of Estrogen Receptors ␣ and ␤ in Human Fetal Brown Adipose Tissue Ksenija Velickovic, Aleksandra Cvoro, Biljana Srdic, Edita Stokic, Milica Markelic, Igor Golic, Vesna Otasevic, Ana Stancic, Aleksandra Jankovic, Milica Vucetic, Biljana Buzadzic, Bato Korac, and Aleksandra Korac University of Belgrade (K.V., M.M., I.G., A.K.), Faculty of Biology, Center for Electron Microscopy, and Department of Physiology (V.O., A.S., A.J., M.V., B.B., B.K.), Institute for Biological Research “Sinisa Stankovic,” University of Belgrade, 11000 Belgrade, Serbia; Department of Genomic Medicine (A.C.), The Methodist Hospital Research Institute, Houston, Texas; Department of Anatomy (B.S.), Faculty of Medicine, University of Novi Sad, Novi Sad, Serbia; and Department of Endocrinology (E.S.), Institute of Internal Disease, Clinical Center Vojvodina, Novi Sad, Serbia Context: Brown adipose tissue (BAT) has the unique ability of generating heat due to the expression of mitochondrial uncoupling protein 1 (UCP1). A recent discovery regarding functional BAT in adult humans has increased interest in the molecular pathways of BAT development and functionality. An important role for estrogen in white adipose tissue was shown, but the possible role of estrogen in human fetal BAT (fBAT) is unclear. Objective: The objective of this study was to determine whether human fBAT expresses estrogen receptor ␣ (ER␣) and ER␤. In addition, we examined their localization as well as their correlation with crucial proteins involved in BAT differentiation, proliferation, mitochondriogenesis and thermogenesis including peroxisome proliferator-activated receptor ␥ (PPAR␥), proliferating cell nuclear antigen (PCNA), PPAR␥-coactivator-1␣ (PGC-1␣), and UCP1. Design: The fBAT was obtained from 4 human male fetuses aged 15, 17, 20, and 23 weeks gestation. ER␣ and ER␤ expression was assessed using Western blotting, immunohistochemistry, and immunocytochemistry. Possible correlations with PPAR␥, PCNA, PGC-1␣, and UCP1 were examined by double immunofluorescence. Results: Both ER␣ and ER␤ were expressed in human fBAT, with ER␣ being dominant. Unlike ER␤, which was present only in mature brown adipocytes, we detected ER␣ in mature adipocytes, preadipocytes, and mesenchymal and endothelial cells. In addition, double immunofluorescence supported the notion that differentiation in fBAT probably involves ER␣. Immunocytochemical analysis revealed mitochondrial localization of both receptors. Conclusion: The expression of both ER␣ and ER␤ in human fBAT suggests a role for estrogen in its development, primarily via ER␣. In addition, our results indicate that fBAT mitochondria could be targeted by estrogens and pointed out the possible role of both ERs in mitochondriogenesis. (J Clin Endocrinol Metab 98: 0000 – 0000, 2013)
rown adipose tissue (BAT) is a powerful thermogenic organ specialized for heat production and energy expenditure. These specialized functions are due to high mitochondrial content and the ability to uncouple cellular
respiration through the action of uncoupling protein 1 (UCP1) (1–3). Until recently, it was commonly thought that BAT was found only in rodents and human infants. However, a recent discovery of functional BAT in hu-
ISSN Print 0021-972X ISSN Online 1945-7197 Printed in U.S.A. Copyright © 2013 by The Endocrine Society Received April 22, 2013. Accepted October 9, 2013.
Abbreviations: BAT, brown adipose tissue; EM, electron microscopy; ER, estrogen receptor; fBAT, fetal BAT; MSC, mesenchymal stem cell; PCNA, proliferating cell nuclear antigen; PGC-1␣, PPAR␥-coactivator-1␣; PPAR␥, proliferator-activated receptor ␥; TBS, Tris-buffered saline; UCP1, uncoupling protein 1.
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Copyright (C) 2013 by The Endocrine Society
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Estrogen Receptors in Human Fetal Brown Adipose Tissue
man adults, as a possible therapeutic target in treating obesity and associated metabolic disorders, brought this tissue into the focus of scientific interest (4, 5). Now, when the presence of metabolically active BAT in adults is shown, fetal BAT (fBAT) has become an important model for studying the mechanisms involved in its development. In humans, the development of BAT begins at the embryonic stage, and there is evidence that it is already wellestablished in the axillary, cervical, perirenal, and periadrenal regions of the fetus in the fifth month of gestation (6). During BAT adipogenesis, the multipotent mesenchymal stem cells (MSCs) are initially committed toward the adipocyte lineage, and this is generally described as a 2-step process. The first step includes the generation of preadipocytes, which have the capacity for proliferation and differentiation. The second step involves the terminal differentiation of these cells into mature brown adipocytes (7, 8). Adipogenesis is closely related to the process of angiogenesis, ensuring the coordinated development of both adipocytes and the capillary network in BAT (9). All of the above-mentioned changes are regulated by a network of transcription factors that coordinate the expression of numerous proteins that determine the ultimate phenotype of brown adipocytes. Of particular relevance are peroxisome proliferator-activated receptor ␥ (PPAR␥), PPAR␥-coactivator-1␣ (PGC-1␣) and UCP1, because of their role in the regulation of fatty acid metabolism and brown adipocyte differentiation, mitochondriogenesis, and uncoupling (10). Estrogen receptors (ERs) are involved in the regulation of growth and differentiation in a broad range of tissues and also play an important role in adipogenesis. Traditionally associated with female reproduction, the importance of estrogen signaling, including metabolic homeostasis and lipid metabolism in both sexes, was later established (11–13). Two major ERs, ER␣ and ER␤, are members of the nuclear hormone receptor family and act as ligand-dependent transcription factors that regulate the expression of specific genes (14, 15). The fact that estrogen is an important factor in the determination of white adipocyte numbers indicates the possibility that it regulates key developmental events in BAT adipogenesis (16). Although estrogens are present in the fetal circulation and amniotic fluid and the presence of both ER␣ and ER␤ are shown in numerous human fetal tissues (17, 18), the data of ER expression in human fetal BAT are still missing. Because ERs regulate numerous functions in adult BAT (1), their roles in embryonic BAT may be extremely important. Apart from several microscopic studies performed on human fBAT, little information is available regarding its
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development. Because current research aims to understand the regulation of human adult BAT mass and activity, the investigation of its adipogenesis during development in fetal life could shed new light on understanding the complex regulatory mechanisms involved in BAT recruitment and activation. Thus, the aim of this study was to examine whether human fBAT expresses ERs and their possible correlations with crucial proteins involved in BAT adipogenesis.
Materials and Methods Tissue preparation Human BAT was collected from four male fetuses aged 15, 17, 20, and 23 weeks gestation after medical or surgical termination of pregnancy in healthy women. The tissue was obtained under informed consent, and the protocol was conducted in compliance with state guidelines and approved by the Medical Faculty, University of Novi Sad, Serbia. Gestational age was determined by crown-rump length, and BAT portions from the axillary region were prepared for Western blot analysis and light and transmission electron microscopy (EM).
SDS-PAGE and Western blotting Western blot was carried out as described previously (19) using antibodies against ER␣ (1:200; sc-543; Santa Cruz Biotechnology), ER␤ (1:1500; GTX70174 [14C8]; GeneTex) showing no cross-reaction with ER␣ (20), PPAR␥ (1:400; ab19481; Abcam), proliferating cell nuclear antigen (PCNA) (1:300; sc-7907; Santa Cruz Biotechnology), and ␤-actin (1: 1000; ab8226; Abcam). Quantitative analysis of immunoreactive bands was conducted using ImageQuant software. The volume was the sum of all the pixel intensities within a band; 1 pixel ⫽ 0.007744 mm2. We averaged the ratio of dots per band for the target protein and actin in corresponding time periods from 3 similar independent experiments and expressed them relative to the group aged 15 weeks gestation, which was standardized as 100%. Data were then statistically analyzed.
Immunohistochemistry Five-micrometer sections of BAT from fetus samples aged 20 weeks gestation were immunostained with biotin/streptavidinperoxidase according to the manufacturer’s protocol (Dako Scientific LSAB Universal Kit) as described previously (21). The primary antibodies were anti-ER␣, anti-ER␤, and anti-UCP1 antibody (1:800; ab10983; Abcam). The sections were rinsed in distilled water, counterstained with hematoxylin, and examined with a Leica DMLB microscope (Leica Microsystems).
Double-immunofluorescence staining We used a colocalization assay to determine the presence of ERs and protein master regulators of adipogenic/thermogenic programs in the same cell compartment. Mutual colocalizations of ER␣ and ER␤, as well as their colocalizations with PPAR␥, PCNA, UCP1, and PGC-1␣ (1:300; ab54481; Abcam), respectively, were detected by confocal microscopy. Paraffin-embedded 5-m-thick sections of human fBAT sam-
ples aged 20 weeks gestation were deparaffinized and rehydrated. After antigen retrieval in 10mM citrate buffer (5 minutes in the microwave oven) and washing in PBS, sections were incubated with normal goat serum (1:100; ab7481; Abcam) for 1 hour. This was followed by an overnight incubation at 4°C with a mixture of antibodies to ER␣ and ER␤, ER␣ and PPAR␥, ER␣ and PCNA, ER␣ and UCP1, ER␣ and PGC-1␣, ER␤ and PPAR␥, ER␤ and PCNA, ER␤ and UCP1, or ER␤ and PGC-1␣. After rinsing in PBS, sections were labeled with the appropriate fluorochrome-conjugated secondary antibody mixture. ER␣ and ER␤ were labeled with appropriate fluorescein isothiocyanate-conjugated secondary antibody (1: 200; ab6717 and ab6785; Abcam). For PPAR␥, PCNA, UCP1, and PGC-1␣ staining, the slides were incubated with the appropriate tetramethylrhodamine isothiocyanate-conjugated secondary antibody (1:300; ab6718 and ab7065; Abcam). After washing with PBS for 20 minutes, the slides were mounted with Mowiol (Sigma Aldrich). Confocal images were acquired with a Leica TCS SP5 confocal laser scanning microscope (Leica Microsystems) in sequential mode to avoid cross talk between channels, and the same conditions were used to measure colocalization. The double-stained samples were excited with 488- and 543-nm light, respectively. The specificity of immunostaining, for both immunofluorescence and routine immunohistochemistry, was tested by the omission of primary antibodies.
phate buffer (pH 7.2), postfixed in 2% osmium tetroxide in the same buffer, dehydrated through a series of alcohol solutions of increasing concentration, and embedded in Araldite (Fluka). Tissue blocks were trimmed and cut with diamond knives (Diatome) on UC6 Leica ultramicrotome, mounted on grids, counterstained with Leica EM stain and uranyl acetate/lead citrate (Leica Microsystems) and examined using a Philips CM12 transmission EM (Philips/FEI). Immunogold staining was performed to determine the subcellular distribution pattern of ER␣ and ER␤ in human BAT. After antigen retrieval in 10mM citrate buffer, ultrathin tissue sections (70 nm) mounted on nickel grids were blocked with 1% BSA in Tris-buffered saline (TBS) for 1 hour at ambient temperature and incubated with primary antibodies overnight at 4°C against ER␣ (1:20) and ER␤ (1:100), respectively. After washing in TBS, sections were incubated with appropriate 10-nm gold-conjugated secondary antibodies for 1 hour at ambient temperature (1:20; Abcam), rinsed in TBS and distilled water, dried, and examined with a Philips CM12 transmission EM. The specificity of the immune reactions was tested by replacing the primary antibody with TBS. Determination of mesenchymal cells, preadipocytes, and mature brown adipocytes was based on their ultrastructural characteristics and UCP1 presence (22).
Additional assays and statistical analysis Transmission EM and immunogold staining Human fBAT samples aged 20 weeks gestation were cut into small pieces, fixed in 2.5% glutaraldehyde in a 0.1 mol/L phos-
Protein content was estimated using BSA as a reference (23). ANOVA was used to test within-group comparisons. If the F test indicated an overall difference, Tukey’s test was applied to eval-
Figure 1. Band density and mutual colocalization of ER␣ and ER␤ in human fBAT. A, The presence of ER␣ and ER␤ was confirmed by Western blotting and showed time-dependent changes in ER␣ and ER␤ expression in human fBAT. B and C, Data were obtained after quantification of specific bands, are expressed as percentage of the 15-week gestation group, which was taken as 100%, and are presented as the mean ⫾ SEM. D, Representative images of mutual colocalization of ER␣ and ER␤ by double immunofluorescence in human fBAT. ER␣ and ER␤ staining appears as green and red fluorescence, respectively. Colocalization results appear yellow in the merged image. Inset in D is an enlarged area of image; nuclei of brown adipocytes are marked with arrows. Scale bar, 20 m.
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Estrogen Receptors in Human Fetal Brown Adipose Tissue
uate the significance of the differences. Statistical significance was set at P ⬍ .05. Quantification of colocalization was performed using Pearson’s correlation coefficient (r). Each pairwise comparison was performed on 10 sets of images acquired with the same optical settings and excluding the consideration of autofluorescence of erythrocytes in the tissue. Student’s unpaired t test was used for statistical analysis, and the data are presented as mean r ⫾ SEM. Pearson’s correlation coefficients greater than ⬃0.5 were interpreted as indicative of reliable colocalization between 2 spectrally separated fluorophores (24).
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Results Expression of ER␣ and ER␤ in human fBAT The level of ER␣ and ER␤ protein expression during 15, 17, 20, and 23 weeks of human fBAT development was determined by Western blot analysis (Figure 1). ER␣ protein content progressively increased in all examined groups (at 17 weeks gestation, P ⬍ .025; at 20 and 23 weeks gestation, P ⬍ .005) compared with week 15 (Figure 1, A and B). On the other hand, ER␤ protein expression (Figure 1, A and C) was detectable only at weeks 17 and 20 (P ⬍ .025). ER␣ and ER␤ RNA levels were determined by qRT-PCR at 20 weeks gestation (Supplemental Figure 1, published on The Endocrine Society’s Journals Online website at http://jcem.endojournals.org). Double-immunofluorescence analysisofER␣ andER␤ colocalizationinhuman fBAT (Figure 1D) revealed weak nuclear and/or cytoplasmic colocalization in some mature brown adipocytes. Pearson’s correlation coefficient of colocalization confirmed our microscopic observationsandwasbelow0.5(0.32⫾ 0.07).
Figure 2. Immunohistochemical localization of ER␣ and ER␤ in human fBAT. A and B, Unlike ER␤, light microscopy revealed a strong immunopositivity for ER␣. At an ultrastructural level (C–J), ER␣ was detected in mesenchymal cells (C), preadipocytes (E), brown adipocytes (G), and endothelial cells (I), whereas ER␤ was detected only in brown adipocytes (H). Also, immunogold labeling revealed mitochondrial localization for both ER␣ and ER␤ (G and H, lower insets). The insets (C, E, G, H, I) are enlargements of the areas in rectangles. Insets in A and B represent negative controls. Scale bars, 25 m (A and B) and 500 nm (C–J). Abbreviations: En, endothelial cell; Er, erythrocyte; Mt, mitochondria; Nu, nucleus.
ER␣ and ER␤ immunohistochemistry and immunocytochemistry As seen in Figure 2, immunohistochemical staining showed that human fBAT at 20 weeks gestation expressed both ER␣ and ER␤, especially ER␣. ER␣ immunostaining showed a strong cytoplasmic reaction in both brown adipocytes and endothelial cells (Figure 2A). In addition, a strong reaction for ER␣ was seen in connective tissue stroma. A weak positive reaction for ER␤ was seen only in mature brown adipocytes (Figure 2B). The results of immunocytochemical analyses are presented in Figure 2. Unlike ER␤ (Figure 2, D, F, H, and J), which was observed only in mature brown adipocytes, the presence of ER␣ was detected in the nuclei and cytoplasm of mature brown adipocytes, preadipocytes, MSCs, and endothelial cells of human fBAT
Figure 3. Heterogeneous immunoexpression of UCP1 in human fBAT. A, Low magnification of fBAT tissue, surrounded by the stromalvascular fraction. B and C, Enlarged area of the section shows UCP1 immunoexpression in mature brown adipocytes (B, solid rectangle in A), both in the nuclei and in cytoplasm, as well as in unilocular adipocytes (C, dashed-line rectangle in A). Inset in A represents negative control. Scale bars, 100 m (A) and 10 m (B and C).
(Figure 2, C, E, G, and I). In the nuclei, ER␣ was associated with both euchromatin and heterochomatin. Furthermore, immunogold labeling revealed mitochondrial and plasma membrane localization of both ER␣ and ER␤ (insets in Figure 2, G and H). Expression of UCP1 in human fBAT At 20 weeks gestation, human fBAT shows heterogeneous UCP1 immunoexpression, known as the Harlequin phenomenon, as part of a BAT morphological signature. Immunohistochemical analysis (Figure 3A) revealed strong nuclear and cytoplasmic UCP1 immunoexpression in both mature brown adipocytes and preadipocytes. In addition to immunopositive multilocular cells (Figure 3B), unilocular adipocytes also showed a UCP1 reaction (Figure 3C). Expression of PPAR␥ and PCNA in human fBAT The expression of PPAR␥ and PCNA proteins in human fBAT is summarized in Figure 4, A–C. The content of PPAR␥ proteins at 17 (P ⬍ .025), 20, and 23 (P ⬍ .005) weeks gestation was higher than that at 15 weeks. Similarly, PCNA protein was higher in fBAT samples at 17 (P ⬍ .005), 20 (P ⬍ .025), and 23 (P ⬍ .005) weeks gestation compared with the fBAT sample at 15 weeks. Ultrastructural analysis of human fBAT showed numerous adipogenic-lineage cells at different stages of differentiation (Figure 4D). Most cells were multilocular mature brown adipocytes; however, numerous MSCs and preadipocytes were also seen. MSCs, which possess a small amount of cytoplasm, are most often observed near
Figure 4. PPAR␥ and PCNA protein expression increases during human fBAT development. A, The presence of PPAR␥ and PCNA was confirmed by Western blotting and showed time-dependent changes in PPAR␥ and PCNA expression in human fBAT. B and C, Data were obtained after quantification of specific bands, expressed as percentage of the 15-week gestation group which was taken as 100%, and are presented as the mean ⫾ SEM (B, C). Transmission EM (D) showed numerous precursor cells in fBAT demonstrating tissue intensive process of proliferation and differentiation and confirmed the ultrastructural features of adipogenic lineage cells: mesenchymal cells, preadipocytes and mature brown adipocyte. Scale bars, 10 m. Abbreviations: BA, brown adipocytes; EN, endothelial cells; PA, preadipocytes.
newly established capillaries. Preadipocytes, fibroblastlike cells with increased volume, contain small lipid droplets and mitochondria. The cytoplasm of fully differentiated brown adipocytes mainly contained numerous lipid droplets and mitochondria with well-packed, parallel cristae. Colocalization of ERs with PPAR␥ and PCNA The results of double-immunofluorescence analysis of ER␣ and ER␤ with PPAR␥ and PCNA, respectively, are presented in Figure 5. Pearson’s correlation coefficient implies significant colocalization of ER␣ and PPAR␥ (0.58 ⫾ 0.07) in human fBAT, in contrast to ER␤ and PPAR␥, where Pearson’s correlation coefficient was below 0.5 (0.32 ⫾ 0.03). Based on the same coefficient, there was significant colocalization between ER␣ and PCNA
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Figure 5. A–D, Colocalization of ERs with PPAR␥ (A) and PCNA (C) by immunofluorescence and quantification of colocalization (B and D) in human fBAT. Staining ER␣ and ER␤ staining appears as green fluorescence, whereas PPAR␥ and PCNA staining appears as red fluorescence. Colocalization results appear yellow in the merged image. Insets in A and C are enlarged areas of images; nuclei of brown adipocytes are marked with arrows. Quantification of ERs with PPAR␥ and PCNA was carried out using Pearson’s correlation coefficient r. The minimal r value for significant colocalization is 0.5 (horizontal line). Data are presented as the mean ⫾ SEM. Scale bars, 50 m.
(0.52 ⫾ 0.02) as well as between ER␤ and PCNA (0.68 ⫾ 0.02) in human fBAT. Colocalization of ERs with UCP1 and PGC-1␣ Figure 6 shows representative images obtained after double-immunofluorescence analysis of ER␣ and ER␤, respectively, with UCP1 and PGC-1␣. Pearson’s correlation coefficient indicated significant colocalization between ER␣ and UCP1 (0.69 ⫾ 0.01) as well as between ER␤ and UCP1 (0.58 ⫾ 0.02). Moreover, ER␣ colocalized with PGC-1␣ (0.62 ⫾ 0.04) in human fBAT as well as ER␤ (0.60 ⫾ 0.03).
Discussion To date, the expression of ERs in adipose tissues has been studied only in rodent BAT and human white adipose tissue (25, 26). In the present study, we demonstrated, for the
first time, that human fBAT expressed both ER␣ and ER␤, suggesting a role for estrogen in its development. Using Western blotting, we demonstrated that the level of ER␣ gradually increases during human fBAT development, whereas ER␤ showed weaker and transient expression. This expression pattern for both ERs was also confirmed by immunohistochemistry and immunocytochemistry and clearly showed that ER␣ is the dominant isoform expressed in human fBAT. However, our results indicate that during a particular time in human fBAT development, estrogens can exert their effects through both ER isoforms. Although ER␣ and ER␤ basically have opposite transcription functions, this does not exclude the possibility that they also have a common function (20). Moreover, a similar ratio of ER␣ and ER␤ expression was detected in human adult white adipose tissue (26). To determine the subcellular distribution of ERs in human fBAT, immunocytochemistry was performed. The
Figure 6. Colocalization of ERs with UCP1 (A) and PGC-1␣ (C) by immunofluorescence and quantification of colocalization (B and D) in human fBAT. ER␣ and ER␤ staining appears as green fluorescence, whereas UCP1 and PGC-1␣ staining appears as red fluorescence. Colocalization results appear yellow in the merged image. Insets A and C are enlarged areas of images; nuclei of brown adipocytes are marked with arrows. Quantification of ERs with UCP1 and PGC-1␣ was carried out using Pearson’s correlation coefficient r. The minimal r value for significant colocalization is 0.5 (horizontal line). Data are presented as the mean ⫾ SEM. Scale bars, 50 m.
presence of ER␣ in adipocyte-lineage cells (MSCs, preadipocytes, and mature brown adipocytes) suggests a possible involvement of this receptor in the adipogenesis of fBAT. Taking into account the presence of ER␤ immunogold particles only in mature brown adipocytes, the differentiation of brown adipocytes, as an integral part of human fBAT development, could occur mainly through ER␣. An increase in ER␣ expression during adipogenesis of human fBAT from 15 to 23 weeks gestation additionally supports its role in differentiation. Although reports on this topic are not entirely consistent (27), our results are in line with studies that indicated that estrogen stimulates differentiation in adipocytes through the regulation of PPAR␥ expression, which is the master regulator of BAT adipogenesis (28).In addition, detailed immunocytochemical analysis of human fBAT in this study demonstrated the presence of both ER isoforms in the nucleus, cytoplasm,
and mitochondria of mature brown adipocytes and at the level of the cell membrane. Although estrogens exert their effects mainly by directly regulating the expression of genes in the nucleus, it is known that a small number of these receptors may be associated with the cell membrane and initiate rapid, nongenomic effects (29). Moreover, Paruthiyil et al (30) recently showed that some ER actions were ligand-independent. In the present study, we also demonstrated ER␣ and ER␤ localization in the mitochondria of human fBAT, which suggests that these organelles are direct targets of estrogen action. Considering that estrogen response elements were detected in the mitochondrial genome of mice and humans (31), it is possible that estrogen can regulate mitochondrial genes in human fBAT through both ERs. In addition, the nuclear and mitochondrial localization of ERs shown in this study indicates a coordinated regulation
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of gene expression between the 2 cell compartments in human fBAT, which is essential for mitochondrial biogenesis in BAT (32). In accordance with the well-known roles of PPAR␥ and PCNA in BAT differentiation and proliferation, we were interested to determine whether changes in the level of these proteins during fBAT development were related to the observed changes in the protein levels of ERs. We demonstrated a time-dependent increase in PPAR␥ and PCNA protein content in this study, which is in accordance with BAT hyperplasia during gestation, which leads to a progressive increase in fBAT mass and UCP1 expression reaching a maximum at birth (33). The ultrastructural study of human fBAT tissue showed all the important cytological characteristics of hyperplastic tissue: a large number of precursor cells (MSCs and preadipocytes) at different stages of differentiation as well as preadipocytes at stages of cell division. The ER-mediated proliferative effects of estrogen were previously reported in various cell types (34). However, the role of ERs in the proliferation of mature adipocytes in human fBAT remains unclear. It is possible that this effect could be mediated through a paracrine mechanism as was noted in normal breast epithelial cells (35). Based on the results of ER␣ and ER␤ double immunofluorescence and immunocytochemistry, which showed that ER␣ was present in all cells of the adipogenic line in human fBAT, we assumed that ER isoforms may be responsible for different biological functions during human fBAT development. The finding that ER␣ and ER␤ rarely colocalize in brown adipocytes supports the notion that the function of each receptor is largely unique. This was also supported by the double-immunofluorescence results of ER␣ and ER␤ with PPAR␥ and PCNA, respectively in fBAT. The strong colocalization of ER␣ with both PPAR␥ and PCNA as well as ER␤ with PCNA suggests that proliferation in human fBAT may occur through both ERs, whereas the process of differentiation occurs through ER␣. An observation that requires further elucidation is the strong colocalization observed between both ERs and PGC-1␣ in fBAT. Bearing in mind that PGC-1␣ binds to PPAR-␥ and coactivates PPAR-␥ to stimulate the transcription of genes involved in the brown adipocyte differentiation process (10), it is possible that ER␤ is also indirectly involved in the process of differentiation. In addition, PGC-1␣ is also involved in the control of mitochondrial biogenesis (36) and affects oxidative metabolism in BAT (37). Thus, localization of both ER␣ and ER␤ in mitochondria shown in this study and their colocalization with PGC-1␣ could be associated with mitochondrial biogenesis. This hypothesis is supported by other studies
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that demonstrated that ER␣ can modulate the transcriptional activity of PGC-1␣ (38). Although a number of studies have shown the presence of ERs in mitochondria (39), this is the first evidence of their localization in human fBAT mitochondria. Safdar et al (40) reported that PGC-1␣ is localized in nuclear and mitochondrial compartments where it functions as a transcriptional coactivator for both nuclear and mitochondrial DNA transcription factors. Thus, it seems that ERs have both mutual and separate biological roles during the adipogenesis of human fBAT. Depending on the localization of ERs in different cell compartments shown in this study, multiple roles for ERs could be achieved both by the binding of estrogen to ERs and via unliganded ERs. In summary, this study demonstrated, for the first time, that human fBAT expresses both ER␣ and ER␤, suggesting a role for estrogen in its development, presumably controlling differentiation mainly via ER␣ and proliferation via both ERs. Our results also suggested possible involvement of both ER␣ and ER␤ in mitochondriogenesis in fBAT. Future investigations should explore the underlying mechanisms by which ERs may regulate differentiation, proliferation, and mitochondriogenesis in human fBAT. However, further work is required to determine which of the ER splice variants are expressed in human fBAT as well as their possible roles in its development.
Acknowledgments Address all correspondence and requests for reprints to: Prof Aleksandra Korac, University of Belgrade, Faculty of Biology, Studentski trg 16, 11000 Belgrade, Serbia. This research was supported by Grants 173055 and 173054 from the Serbian Ministry of Education, Science, and Technological Development. Disclosure Summary: The authors have nothing to disclose.
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