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1 Division of Medical Sciences, The Medical School, Institute of Biomedical Research, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
2 Diabetes Drug Discovery, AstraZeneca, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK
3 Clinical Endocrinology, Campus Mitte, Charité Universitätsmedizin Berlin, Berlin, Germany
(Requests for offprints should be addressed to P M Stewart; Email: p.m.stewart{at}bham.ac.uk)
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDiscussionSummaryReferences |
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, C/EBPß, IL-6, FABP4, APOD, IRS2, AGTR1 and GHR. One of the most upregulated genes in OM but not in SC cells was HSD11B1. The GR was similarly expressed and not regulated by glucocorticoids in SC and OM human preadipocytes. C/EBP was expressed in SC preadipocytes and upregulated by F, but was below the detection level in OM cells. C/EBPß was highly expressed both in SC and in OM preadipocytes, but was not regulated by F. Our results provide insight into the genes involved in the regulation of adipocyte differentiation by cortisol, highlighting the depot specifically in human adipose tissue.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSummaryReferences |
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Clinical observations in patients with Cushing’s syndrome who develop reversibly central obesity highlight the importance of glucocorticoids in this regard (Rebuffe-Scrive 1988). Glucocorticoids (GCs) induce adipocyte differentiation (Hauner et al. 1987) preferentially within visceral adipose tissue due to increased glucocorticoid receptor (GR) expression (Bronnegard et al. 1990), although other tissue-specific factors may be important.
In human adipose tissue, cortisol availability to bind and activate GR is controlled by 11ß-hydroxysteroid dehydrogenase type-1 (11ß-HSD1). 11ß-HSD1 amplifies glucocorticoid action by reducing inactive cortisone (E) to active cortisol (F) (Lakshmi & Monder 1988) and is highly expressed in omental (OM) compared with subcutaneous (SC) fat. Furthermore in primary adipose cultures, 11ß-HSD1 expression and activity are induced by F itself (Bujalska et al. 1997), thus representing a novel mechanism whereby cells can generate F locally independent of circulating concentrations. Several factors are known to regulate 11ß-HSD1 expression and activity in the liver (Jamieson et al. 1995) and adipocytes; the most potent inducers are glucocorticoids, cytokines (leptin, tumour necrosis factor (TNF), interleukin (IL)-1ß, IL-4, IL-6 and IL-14; Handoko et al. 2000, Tomlinson et al. 2001) and peroxisome proliferator activated receptor (PPAR
) agonists (Berger et al. 2001, Laplante et al. 2003). Additionally, a significant role for C/EBP transcription factors in regulating rat hepatic 11ß-HSD1 has been reported (Williams et al. 2000). Amongst the factors suppressing 11ß-HSD1 activity are: growth hormone, insulin-like growth factor-I (IGF-I) (Moore et al. 1999, Tomlinson et al. 2001), liver X receptors (Stulnig et al. 2002), PPAR agonists (Hermanowski-Vosatka et al. 2000) and pituitary hormones like corticotrophin-releasing hormone and adrenocorticotrophin hormone (Friedberg et al. 2003).
Microarray approaches have been increasingly used to study gene profiling during adipocyte differentiation; however, most of these studies were carried out on mouse cell lines and not on human primary preadipocyte cultures (Burton et al. 2002, Jessen & Stevens 2002, Wang et al. 2004). On this background, we aimed to identify factors that might regulate 11ß-HSD1 gene transcription in human OM versus SC adipose tissue. We have also hypothesised that the identification of GR-induced target genes in human OM versus SC adipose tissue will increase our knowledge base of the predilection of glucocorticoids to increase visceral fat mass and might reveal exciting candidate targets involved in the pathogenesis of central obesity.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSummaryReferences |
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Paired SC and OM abdominal adipose tissue was obtained from five females (mean age 44.8 years, mean body mass index 29.4 kg/m2; patient details shown in Table 1
). All patients were undergoing hysterectomy and not on any hormonal treatment. Tissues were dispersed with collagenase 1 as described earlier (Bujalska et al. 1999). Preadipocytes were seeded in Dulbecco’s Modified Eagle’s Medium/Ham’s Nutrient Mixture F-12 (DMEM/F12) media with 10% FCS into six-well tissue culture dish and cultured to confluence for 4–6 days. Media were changed every other day. The study had the approval of the local research ethics committee, and all patients had given written informed consent prior to study inclusion.
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Confluent SC and OM preadipocytes were treated with 100 nM F for 24 h in serum-free DMEM/F12 media. In control cells, F was omitted.
RNA isolation
Total RNA was extracted from primary cultures using TriReagent (Sigma). Total RNA was extracted from five confluent primary cultures of human SC and OM preadipocytes treated with or without 100 nM F. Total RNA was treated with DNaseI to remove any genomic DNA contamination (DNaseI, Invitrogen) and its quantity and quality were assessed spectrophotometrically at OD260/280 and by electrophoresis on 1% agarose gel.
Affymetrix microarray experiments
All experiments were performed using Affymetrix human HgU133A and HgU133B oligonucleotide array set, as described at http://www.affymetrix.com/products/arrays/specific/hgu133.affyx and complied with Minimum Information About a Microarray Experiment (MIAME) standard. Total RNA was used to prepare biotinylated-target RNA with minor modifications from the manufacturer’s recommendations (http://www.affymetrix.com/support/technical/manual/expression_manual.affx). Briefly, 10 µg mRNA (pool of 2 µg total RNA from each cell preparation) were used to generate first-strand cDNA by using a T7-linked oligo(dT) primer (SuperScirpt Double Stranded cDNA Synthesis Kit, Invitrogen). After second-strand synthesis, in vitro transcription was performed with biotinylated UTP and CTP (Enzo Life Sciences, Farmingdale, NY, USA), resulting in approximately 100-fold amplification of RNA. A complete description of procedures is available at http://bioinf.picr.man.ac.uk/mbcf/downloads/GeneChip_Target_Prep_Protocol_CRUK_v_2.pdf. The target cDNA generated from each sample was processed as per manufacturer’s recommendation using an Affymetrix GeneChip Instrument System (http://www.affymetrix.com/support/technical/manual/expression_manual.affx). Briefly, spike controls were added to 15 µg fragmented cDNA before overnight hybridisation. Arrays were then washed and stained with streptavidin–phycoerythrin, before being scanned on an Affymetrix GeneChip scanner. A complete description of these procedures is available at http://bioinf.picr.man.ac.uk/mbcf/downloads/GeneChip_Hyb_Wash_Scan_Protocol_v_2_web.pdf. Additionally, quality and amount of starting RNA were confirmed using an agarose gel. After scanning, array images were assessed by eye to confirm scanner alignment and the absence of significant bubbles or scratches on the chip surface. 3'/5' ratios for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ß-actin were confirmed to be within acceptable limits (0.96–1.14), and BioB spike controls were found to be present on all chips, with BioC, BioD and CreX also present in increasing intensity. When scaled to a target intensity of 100, scaling factors for all arrays were within acceptable limits (0.8–1.1), as were background, Q values and mean intensities.
Microarray data analysis
Data from absolute and comparison files (F treated versus control) in SC and OM preadipocytes were normalised and analysed using Gene Expression Pattern Analysis Suite 2.0 software (http://gepas.bioinfo.cipf.es). Methods used: background correction: Robust Multi-array Analysis (RMA); normalisation: quantiles; Perfect Match (PM) correction: pmonly; summary: medianpolish (http://gepas.bioinfo.cipf.es/cgi-bin/tools). A total of 22 283 (U133A) and 22 645 (U133B) entries in comparison analysis were sorted out according to criteria: (1) genes with absent call in control and treated cell arrays were deleted; (2) genes with difference call of no change were deleted; (3) genes with absolute signal below 100 were eliminated; (4) genes with signal log ratio (SLR) below 1 for I (increase) or above –1.0 for D (decrease) were deleted (arbitrary cut-off point of twofold change) and (5) expressed sequence tags (ESTs) and hypothetical protein entries were not analysed.
This filtering procedure generated two datasets: SC preadipocytes treated with F versus control and OM preadipocytes treated with F versus control. Genes were analysed according to their metabolic function, such as glucocorticoid metabolism, transcription factors, genes involved in growth arrest, extracellular matrix (ECM) modulation, adipocyte-specific genes, immune responses, glucose metabolism and metabolic enzymes using NetAffx Analysis Center (www.affymetrix.com).
Reverse transcription
Total RNA (1 µg) in total volume of 20 µl was reverse transcribed using avian myeloblastosis virus reverse transcriptase (AMV-RT) and random hexamers according to manufacturer’s protocol (Promega). Fifty nanograms of cDNA (1 µl reverse transcriptase (RT) reaction) were used for real-time PCR to confirm the expression of ten genes.
Real-time PCR
To confirm that the Affymetrix DNA microarray accurately identified the gene expression changes, we carried out quantitative RT-PCR of ten genes with varying degree of change, using the Applied Biosystems 7700 Real-Time PCR System (PE Applied Biosystems, UK). Briefly, PCRs were carried out in 25 µl volumes on 96-well plates, in a reaction buffer containing 2x TaqMan Universal PCR Master Mix. All reactions were singleplexed with the housekeeping gene 18S rRNA on the same plate, provided as a preoptimised control probe (PE Biosystems, Warrington, UK), enabling data to be expressed in relation to an internal reference, to allow for differences in RT efficiency. Measurements were carried out at least three times for each sample. All target gene probes were labelled with FAM, and the housekeeping gene with VIC. Reactions were as follows: 50 °C for 2 min, 95 °C for 10 min; then 44 cycles of 95 °C for 15 s and 60 °C for 1 min. According to the manufacturer’s guidelines, data were expressed as Ct values (the cycle number at which logarithmic PCR plots cross a calculated threshold line) and used to determine 
Ct values (Ct=Ct of the target gene minus Ct of the housekeeping gene). To exclude potential bias due to averaging data, which had been transformed through the equation 2– Ct to give a fold change in gene expression, all statistics were performed with Ct values. Five sequences of primers and probes used for real-time PCR were designed using PrimerEx-press software (Applied Biosystems, UK) and shown in Table 2. Five other genes; IL-6, IRS2, AGTR1, APOD and FABP4 were validated using commercially available Assay on Demand (Applied Biosystems, Applera, UK). Inter-patient and site differences of gene expressions were tested using individual cDNA preparation. These confirmed the same pattern of change as pooled cDNA used for Affymetrix arrays (data not shown).
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In comparison analysis where baseline array were the control cells, statistical significance of signal log ratio (SLR; quantitative measure of the relative change in transcript abundance) was calculated. The Wilcoxon’s signed rank test was used to compute change P value for each probe set. The P values range in scale from 0.0 to 1.0 and provide the likelihood of change and direction. P values close to 0.0 (<0.001) and 1.0 (>0.998) were used for significant increase and significant decrease respectively (detailed information can be found in GeneChip Expression Analysis, Data Analysis Fundamentals, Affymetrix). For quantitative RT-PCR of the gene validation, statistical software (SPSS for Windows 11.5, SPSS, Inc., UK) was used for statistical analysis. Data are presented as mean values with standard error and P value of <0.05 was accepted as statistically significant.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSummaryReferences |
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): (a) SC specific – 43 upregulated and 21 downregulated; (b) OM specific – 27 upregulated and 10 downregulated and (c) adipose specific – 29 upregulated and 9 downregulated in both SC and OM depots. The list of genes with their symbol, GeneBank accession number, description, fold change and function is shown in Table 3 (for increased expression) and Table 4 (for decreased expression).
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Genes that were upregulated by cortisol included those associated with negative regulation of cell growth (BTG1, PMP22, CD9, RAI2 and CUGBO2) and apoptosis-related proteins (e.g. CDO1, AMIGO2, CFLAR and CUGBP2) as well as genes involved in cell matrix rearrangements (e.g. CHL1, DPT, ABMLIM, PRELP and KIAA1011; Table 2
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Three transcription factors linked to cell growth, C/EBP, FOS (part of the AP-1 complex) and GFB were upregulated 2.0-, 2.2- and 2.1-fold (respectively). Additionally, a number of genes linked to lipid metabolism were upregulated – FACL2, LPL and APOB. Significant upregulation of DF (adipsin), FABP4 and AGTR1 was also seen in SC preadipocytes, changes that were absent in OM cells. Expression levels of three of these genes (C/EBP, FABP4 and AGTR1) as well as C/EBPß and glucocorticoid receptor (GR) were validated by real-time PCR (Figs 2–4).
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).
Genes that were specifically decreased in SC adipose stromal cells following incubation with cortisol (Table 4) included those positively regulating cell proliferation (VEGF and WARS), oncogenes (ENPP2), anti-apoptotic (OXTR). Interestingly, in SC preadipocytes F-treatment decreased expression of two genes associated with Wnt-signalling pathways (FZD7 and SFRP4; 3.5- and 3.3-fold respectively; Fig. 3).
F-treatment significantly decreased IL-6 expression only in SC preadipocytes (2.3-fold), however, the absolute signal of IL-6 was much higher in OM preadipocytes. This finding was confirmed and validated by real-time PCR (Fig. 3).
The expression levels of two transcription factors, LDB2 and GATA6, were lowered by cortisol (5.0- and 2.0-fold respectively). Additionally, SGNE1 (associated with active transport from the ER) and IGFBP5 (IGF-signalling pathway) were downregulated by F (2.1- and 2.4-fold).
OM preadipocytes
Several genes were specifically upregulated by cortisol but only in OM depots: GAS1, PDGFD and ID1 negatively associated with cell growth (2.4-, 2.1- and 2.6-fold respectively), positively with cell differentiation (DIAPH2, 2.0-fold), and ECM proteins (LAMA2, AKAP2, ACKLAM, SPRX and KIAA0992). A number of metabolic enzymes were upregulated in OM cells only: ALDH1 (2.4-fold), AOX1 (3.0-fold) and AKR1C1 (2.1-fold). Interestingly, among metabolic enzymes involved in glucocorticoid metabolism, HSD11B1 was upregulated in OM preadipocytes (3.3-fold), encoding 11ß-HSD1 enzyme. This was confirmed using real-time PCR (Fig. 2). The gene encoding adrenomedullin (ADM) was upregulated 2.0-fold. Fibronectin (FN) involved in the immune response was upregulated in OM cells (2.3-fold) but not in SC cells. One member of Wnt-signalling pathway, WISP1, was upregulated in OM cells (2.1-fold) as well as two transcription factors, CITED2 and FOXO1A (2.1- and 2.3-fold respectively).
Downregulated OM-specific genes included PRAD1 (controlling G1/S transition) and ECM proteins (HAS2 and MMP1). A gene associated with transforming growth factorß signalling (SMURF2) was 2.8-fold. Small inducible cytokine subfamily A, member 11 (CCL11) and leukaemia inhibitory factor involved in immune responses were downregulated 4.5- and 2.2-fold respectively in OM cells. Interestingly, natriuretic peptide B (NPB), gene involved in electrolyte homeostasis and lipolysis in human adipocytes was decreased 2.7-fold by cortisol and was below detection levels in SC cells.
Common adipose-specific genes
Twenty-eight genes were upregulated by cortisol in both SC and OM depots, including those involved in cell cycle regulation and growth (CDKN1C, RGC32 and ADAMTS1), oneassociated withcell adhesion (NRCAM)and rearrangement of cell matrix (FBN2 and MGP). A number of transcription factors were upregulated in cells from both depots: HXL1, LDO3, TSC22D3, REV3L and ZFP145. Components of insulin-signalling pathway: IRS2, GHR, PDK4 and glucose-regulated chaperone protein (HSPA2) were significantly upregulated by cortisol in SC and OM preadipocytes as apolipoprotein D (ApoD) implicated in lipid metabolism (Table 3). IRS2 and GHR expression profiles were validated by real-time PCR (Figs 3 and 4).
In terms of genes related to the immune response, expression of CD163, CHST2 and CPM all increased in both depots. Plasma glutathione peroxidase 3 precursor (GPX3), catalyses the reduction of hydrogen peroxide, organic hydroperoxide and lipid peroxides, protecting cells against oxidative damage. It was expressed at very high levels in control SC cells and was significantly upregulated in both SC and OM depots by F. Additionally, genes encoding many metabolic enzymes like GLUL (glutamate/nitrogen metabolism), ADH2 (ethanol metabolism) and MAOA (neurotransmitters catabolism) were upregulated in preadipocytes from both depots (Table 3).
Most of the genes downregulated by cortisol in both SC and OM preadipocytes were associated with cell cycle control and proliferation/growth processes; e.g. FGF2, TRIB3 and oncogene (C1orf24). As expected, pro-inflammatory gene such as TNF induced protein 6 (TNFA1P6) was decreased by cortisol in both depots. Transcription factor DSCR1 was decreased in SC and OM cells (3.1- and 2.7-fold respectively). Cortisol treatment decreased expression of one component of Wnt-signalling pathway (DACT1) in SC and OM preadipocytes (2.6-and 2.1-fold respectively), Table 4.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSummaryReferences |
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In terms of the GR itself, earlier studies had reported increased expression in OM adipose tissue when compared with SC fat (Bronnegard et al. 1990). Here, we show very similar levels of GR mRNA in preadipocytes of both depots and report that incubation with F for 24 h had no significant effect on GR expression in human SC or OM preadipocytes. By contrast, the study does confirm our earlier reports of increased expression of the ‘prereceptor’ regulator of GR action, the HSD11B1 gene which encodes the enzyme 11ß-hydroxysteroid dehydrogenase type-1 (11ß-HSD1) in OM compared with SC preadipocytes and significant upregulation of its expression by F only in OM sites (Bujalska et al. 1997). The enzyme 11ß-HSD2 was absent confirming previous observations (Bujalska et al. 1997). The explanation for the increased HSD11B1 expression in OM versus SC sites remains unclear. Rodent studies had indicated marked induction of expression by the transcription factors CCAAT enhancer-binding protein (C/EBP; Williams et al. 2000). However, in this human-based study C/EBP expression was absent in OM preadipocytes and C/ EBPß, whilst being highly expressed in SC and OM preadipocytes, was not regulated by F. It seems unlikely based on these data that C/EBPs play a major role in the site-specific transcriptional regulation of HSD11B1 or GR in human preadipocytes.
Growth arrest/extracellular matrix
Whilst GCs provide a crucial signal for preadipocyte differentiation (Hauner et al. 1987), the first step defining adipogenesis is cell growth arrest (Ailhaud et al. 1989). This study looked comprehensively on the effect of GCs on genes associated with cell cycle control. We identified a number of genes such as CDKN1C, which overexpression caused complete cell arrest in G1 phase (Lee et al. 1995), and RGC32 negatively controlling cell growth (Badea et al. 1998) as novel GC-target genes in SC and OM preadipocytes. Furthermore, we found ADAMTS1, gene which disruption in knockout mice caused adipose tissue malformation (Shindo et al. 2000), being upregulated by GC-target in human preadipocytes together with downregulation of FGF2 providing positive proliferation signal (Kurokawa et al. 1987). We identified different sets of genes regulating cell growth in depot-specific manner. Only in OM cells, we see increased expression of GAS1 (involved in growth suppression) and decreased of PRAD1 (a G1 marker gene decreased by dexamethasone in airway smooth muscle cells (Fernandes et al. 1999)), but increased expression of BTG1 in SC cells. Our findings imply site-specific pathway of cell arrest in human preadipocytes.
Interestingly, downregulation of genes, FZD7 and SFRP4 associated with the Wnt-signalling pathway was observed only in SC preadipocytes while upregulation of WISP1, down-stream target of Wnt pathway in hepatocellular carcinoma cells (Cervello et al. 2004), was seen only in OM cells. Downregulation of Wnt pathway was recently shown to be crucial for the adipogenesis in mouse cell line (3T3L1; Bennett et al. 2002), however, its role in human preadipocytes still needs to be evaluated.
Preadipocyte growth arrest is accompanied by reorganisation of extracellular matrix (ECM; Gregoire et al. 1991). We found several novel GC-target genes associated with cell–cell adhesion proteins in human preadipocytes, e.g. FBN2, MGP and NRCAM. However, several ECM GC-target genes were site specific and their role in site-specific adipogenesis remains to be elucidated. For example, in SC cells, CLEC3B (tetranectin) was highly expressed and upregulated by F but below detection levels in OM cells. CHL1 ABLIM, dermatopontin (DPT) and PRELP were GC-target genes of the ECM upregulated only in SC cells. DPT, cartilage matrix component, was recently shown to be upregulated by dexamethasone in chondrogenic differentiation from human bone marrow mesenchymal stem cells (Derfoul et al. 2006). Conversely, expression of ALCAM, KIAA0992 (identified as risk factor for myocardial infarction; Shiffman et al. 2005) and SRPX was upregulated in OM cells.
Hereby, we demonstrate that GCs induce a site-specific block on the cell cycle progression together with site-specific differences in ECM remodelling. These would indicate unique pathways of preadipocyte commitment to adipogenesis in these two human adipose tissue depots.
Metabolic enzymes
Human alcohol dehydrogenase 2 (ADH2) exhibits high activity for unsaturated and aromatic alcohols and aldehydes in the metabolism of noradrenaline, retinoids and lipid peroxidation thus plays an important role in detoxification in the liver, reviewed by Higuchi et al.(2004). This study revealed high ADH2 expression in human preadipocytes (much higher in SC than OM) and positive regulation by GCs in both adipose depots. Dexamethasone has been shown to upregulate ADH2 expression in the HepG2 cell line (Dong et al. 1988); however, ADH2 expression and its role in the human adipose tissue have not yet been elucidated. On the other hand, aldo–keto reductase family 1, member C1 (AKR1C1), a major enzyme in progesterone metabolism was also highly expressed in SC and OM preadipocytes (again more in SC than OM), but was only upregulated by GCs in OM preadipocytes. AKR1C1 has been shown to be expressed in human SC and OM adipose tissue (Blanchette et al. 2004) and positively correlated with visceral obesity. Our findings may provide an explanation for this correlation, since more cortisol is generated in OM tissue due to increased expression of 11ß-HSD1.
Adipose-specific genes
In keeping with previous findings (Masuzaki et al. 1995), leptin expression was undetectable in SC and OM preadipocytes; GCs increased its expression only in SC preadipocytes. It has been shown that C/EBP upregulates human (Miller et al. 1996) leptin mRNA expression and we confirmed a parallel induction of leptin and C/EBP by GCs in SC cells. Lipoprotein lipase, an early differentiation marker of adipogenesis has been shown to be expressed at higher levels and upregulated by GCs in SC human female adipose tissue (Fried et al. 1993), a pattern that was confirmed in preadipocytes in this study. In rat, insulin is required for the induction of early differentiation genes such as LPL (Gregoire et al. 1991), but we showed that a relatively short exposure to GCs to SC preadipocytes induced LPL expression without the requirement of insulin.
FABP4, product of the aP2 gene, is regulated by dexamethasone in mouse 3T3L1 cells (Cook et al. 1988) as well as in differentiating preadipocytes from rat bone marrow (Atmani et al. 2003). This study verified FABP4 gene as a glucocorticoid-target gene but only in SC preadipocytes. Upregulation of aP2 gene in response to GCs as well as transcription factors-binding sites for C/ EBP and c-fos (forming AP-1 complex) in the promoter region of mouse aP2 gene was shown previously (Ross et al. 1990). Our findings, demonstrating increased expression of these two transcription factors together with FABP4 expression in SC preadipocytes, might reflect different sensitivity of human SC and OM preadipocytes to adipogenic effect of GCs.
ApoD is involved in cellular transport of small hydrophobic ligands, such as progesterone, cholesterol, pregnenolone, bilirubin and arachidonic acid but its exact role is still not known. ApoD was expressed at similar levels in SC and OM preadipocytes but these fat compartments responded differently to GC (upregulation SC>OM). On the other hand, ApoB, component for VLDL associated with type-2 diabetes and insulin resistance reviewed by Ginsberg et al.(2005) and increased risk of coronary heart disease (Despres 1998), was positively regulated only in SC cells. Although our finding provides evidence for glucocorticoid regulation of ApoB in human SC preadipocyte, its significance needs to be evaluated.
Natriuretic peptides have been shown to have a potent lipolytic effect in human adipocytes (Sengenes et al. 2000). Moreover, significantly reduced levels of NPB have been found in obese patients with heart failure (Mehra et al. 2004). Our finding shows NPB expression in OM preadipocytes as well as GC-target gene (but absent in SC) could provide a novel mechanism explaining the relationship between glucocorticoids, central obesity and heart failure. Moreover, increased expression of ADM has been positively correlated with cardiovascular disease and hypertension reviewed in Nikitenko et al.(2002). Here, we found ADM as a novel GC-target gene only in OM preadipocytes; its angiogenic and antilipolytic effects (Harmancey et al. 2005) might be an important factor explaining depot specificity of GC action in human adipose tissue.
Genes involved in glucose metabolism/insulin signalling
Many genes were similarly expressed and regulated by GCs in preadipocytes from both depots. Among them was the growth hormone receptor (GHR). GH has an anti-insulin activity (Roupas et al. 1991) and the upregulation of its receptor by GCs may be one mechanism underpinning GC-induced insulin resistance.
Insulin receptor substrate (IRS) plays a crucial role in adipogenesis; IRS2 (–/–) embryonic mouse fibroblasts retain only 15% of their ability to differentiate to adipocytes (Miki et al. 2001). IRS2 knockout mice develop progressive deterioration in glucose homeostasis through impaired insulin signalling in the liver and skeletal muscle leading to type-2 diabetes (Withers et al. 1998). IRS2 expression was higher in OM than SC preadipocytes, but was induced to a similar extent by GCs in both depots.
PDK4, an enzyme involved in glucose metabolism and fatty acid synthesis is known to be expressed in skeletal muscle and adipose tissue (Rowles et al. 1996). Decreased PDK4 in muscle has been associated with increased insulin sensitivity (Rosa et al. 2003) and increased expression with insulin resistance, diabetes and hyperthyroidism reviewed by Sugden (2003). The relationship between PDK4 and glucocorticoids has been studied in HepG2 cells but not in human adipose tissue. Elevation of PDK4 expression in human preadipocytes might be an additional factor in GC-induced insulin resistance.
Anti-inflammatory
In general, primary preadipocyte cultures isolated from human adipose tissue represent heterogeneous cell populations, some of which can be a part of the immune system (Kershaw & Flier 2004). In this study, we observed GC effects on genes associated with immune responses, such as IL-6, TNFAIP6 and CD163. Activation of anti-inflammatory protein, TNFAIP6 (TNF-stimulated gene 6) gene by NF/IL-6 was previously demonstrated (Klampfer et al. 1995). The simultaneous GC-induced downregulation of the TNFAIP6 and IL-6 in human preadipocytes might reflect interaction between these two genes in adipose tissue inflammation. GPX3, plasma glutathione peroxidase 3 precursor, catalyses the reduction of hydrogen peroxide, organic hydroperoxide and lipid peroxides, thus protecting cells against oxidative damage. GPX3 was reported to be present in human adipose tissue (Maeda et al. 1997), and here we identified GPX3 expression as being one of the most highly expressed genes in preadipocyte compartment of human adipose tissue as well as a novel GC-target gene.
Recently, adipose tissue has been identified as a major production site of serum amyloid A (ASSA; Sjoholm et al. 2005), a known risk factor for coronary artery disease (Johnson et al. 2004). In this study, we have shown that ASSA is a GC-target gene in OM preadipocytes. Together, these findings contribute to the role of OM tissue as a potential link between an inflammatory response and coronary disease.
Transcription factors
Several transcription factors are known to be key regulators of the adipogenic processes, reviewed by MacDougald & Mandrup (2002). In this study, we identified distinctively different as well as common sets of transcription factors involved in early glucocorticoid-induced adipogenesis in human SC and OM adipose tissues. Downregulation of Wnt-signalling pathway during adipogenesis has been shown in 3T3 cells (Bennett et al. 2002). Here, we demonstrate that the downregulation of Wnt-signalling pathway, as well as the induction of C/EBP occurs only in SC preadipocytes. Different transcription factors, FOXO1A (upregulated in OM cells) and FOS (upregulated in SC cells), were the depot-specific early GC-targets genes. FOXO1 is induced in early stages of adipocyte differentiation (Nakae et al. 2003); our finding confirmed this but only in OM depots. Recently, involvement of two members of the zinc finger proteins family of the transcription factors, KLF2 and KLF15 has been reported in adipogenesis (Gray et al. 2002, Banerjee et al. 2003). Our study identified another member of this family, zinc finger protein 145 (ZFP145) as a novel, common, GC-target in human preadipocytes. Moreover, the pattern of induction from undetectable levels suggests its important role in GCs-induced adipogenesis; however, the functional role of ZFP145 in adipose tissue needs to be further evaluated.
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Summary |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSummaryReferences |
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Acknowledgements |
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSummaryReferences |
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Received 5 June 2006
Accepted 29 June 2006
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twofold) after 24 h cortisol (F) treatment in SC, OM preadipocytes or in both adipose depots (adipose specific).
