Dehydroepiandrosterone (DHEA) treatment in vitro inhibits adipogenesis in human omental but not subcutaneous adipose tissue

Molecular and Cellular Endocrinology 320 (2010) 51–57

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Dehydroepiandrosterone (DHEA) treatment in vitro inhibits adipogenesis in human omental but not subcutaneous adipose tissue S.P.L. Rice, L. Zhang, F. Grennan-Jones, N. Agarwal, M.D. Lewis, D.A. Rees, M. Ludgate ∗ Centre for Endocrine and Diabetes Sciences, School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK

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Article history: Received 27 August 2009 Received in revised form 7 January 2010 Accepted 10 February 2010 Keywords: DHEA Proliferation 3T3-L1 Adipogenesis Preadipocytes

a b s t r a c t Dehydroepiandrosterone (DHEA), a precursor sex steroid, circulates in sulphated form (DHEAS). Serum DHEAS concentrations are inversely correlated with metabolic syndrome components and in vivo/in vitro studies suggest a role in modulating adipose mass. To investigate further, we assessed the in vitro biological effect of DHEA in white (3T3-L1) and brown (PAZ6) preadipocyte cell lines and human primary preadipocytes. DHEA (from 10−8 M) caused concentration-dependent proliferation inhibition of 3T3-L1 and PAZ6 preadipocytes. Cell cycle analysis demonstrated unaltered apoptosis but indicated blockade at G1/S or G2/M in 3T3-L1 and PAZ6, respectively. Preadipocyte cell-line adipogenesis was not affected. In human primary subcutaneous and omental preadipocytes, DHEA significantly inhibited proliferation from 10−8 M. DHEA 10−7 M had opposing effects on adipogenesis in the two fat depots. Subcutaneous preadipocyte differentiation was unaffected or increased whereas omental preadipocytes showed significantly reduced adipogenesis. We conclude that DHEA exerts fat depot-specific differences which modulate body composition by limiting omental fat production. © 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Dehydroepiandrosterone (DHEA) is a precursor sex steroid which, in common with all adrenal steroids, is derived from the parent molecule cholesterol. It was initially identified in its sulphated form, dehydroepiandrosterone sulphate (DHEAS) (Migeon and Plager, 1954) and was the first hormone with androgenic activity to be isolated from the human circulation. Subsequent studies have shown that DHEA and DHEAS are present in the human and higher primate circulations in concentrations greater than any other steroid hormone (Parker, 1989) although levels decline dramatically after middle age (Bonny et al., 1984; Orentreich et al., 1984). The age-related fall in concentration has led to speculation that this physiological reduction in DHEA/DHEAS may be causally related to disease processes normally associated with aging. However, the precise role of DHEA remains uncertain despite a variety of epidemiological studies demonstrating an inverse correlation with some features of the metabolic syndrome (central obesity and insulin resistance) and cardiovascular morbidity and mortality (Trivedi and Khaw, 2001; Berr et al., 1996).

∗ Corresponding author. Tel.: +44 2920 745457. E-mail address: [email protected] (M. Ludgate). 0303-7207/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2010.02.017

Adipose tissue contains significant quantities (present at levels 10 times greater than in the circulation) of DHEA (Fehér and Bodrogi, 1982; Szymczak et al., 1998) and possesses the appropriate enzymatic machinery for its metabolism (Bleau et al., 1974; Dalla Valle et al., 2006). Thus investigators have examined a role for DHEA in adipose tissue biology, and despite the low circulating levels of DHEA/DHEAS in rodents, many have used murine cell lines or in vivo rodent models as their experimental systems. DHEA has been reported to inhibit 3T3-L1 adipogenesis (Gordon et al., 1986) and proliferation (Lea-Currie et al., 1998). DHEAS supplementation in rats fed a high fat diet reduced carcass lipid, fat depot mass and adipocyte number (Lea-Currie et al., 1997); a further study showed that DHEA protects against both increased visceral fat accumulation and reductions in insulin-stimulated glucose uptake (Hansen et al., 1997). DHEA has also been shown to modulate adipocytokine expression, including upregulation of resistin (Kochan and Karbowska, 2004). An in vitro study of human adipose tissue samples cultured in DHEAS for 24 h demonstrated upregulation of adiponectin gene expression in omental adipocytes (Hernandez-Morante et al., 2006). However, in contrast to most in vitro and animal model studies, which have demonstrated an inhibitory action of DHEA on adipocyte mass, many interventional studies in human subjects, have failed to demonstrate such convincing results (Bergthorsdottir et al., 2006; Tchernof and Labrie, 2004).

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In light of this confusion, the aim of this study was to assess the biological effect of DHEA on adipose tissue in vitro by examining the influence of DHEA on preadipocyte cell lines, including a human brown adipose tissue (BAT) cell line, and primary cultures (human subcutaneous and omental) and to investigate the mechanism behind any observed effects. 2. Materials and methods 2.1. Patients studied We collected subcutaneous and omental adipose tissues from 12 individuals undergoing elective open abdominal surgery for non-metabolic conditions, e.g. hernia repair. All samples were obtained with informed consent. The study was performed with appropriate Ethics Committee approval and was conducted in accordance with the principles of ‘The Declaration of Helsinki’. The group comprised 6 women and 6 men, whose ages ranged from 49 to 77 years and having a BMI ranging from 20 to 28. 2.2. Reagents DHEA, DHEAS, finasteride, ␤-estradiol, testosterone and androstenedione were purchased from Sigma–Aldrich Company Ltd. (Dorset, UK). Trilostane was kindly donated by Bioaccelerate Ltd. (Savannah House, 11-12 Charles II Street, London, UK) and pioglitazone was kindly provided by Takeda Chemical Industries Co., Ltd., Osaka, Japan. All reagents were dissolved in dimethyl sulphoxide (DMSO) [Sigma–Aldrich Company Ltd., Dorset, UK] to produce stock solutions of 10−2 molar, a maximum concentration of DMSO of 0.1% was present in any one experiment; furthermore 0.1% DMSO was included in the control in all experiments. 2.3. Adipose tissue collection and preparation Preadipocytes from subcutaneous and omental depots were obtained by collagenase digest followed by centrifugation through phthalic acid dionyl ester, as previously described (Crisp et al., 2000). Since human primary preadipocytes of low passage number (<2) were used it was not possible to analyse every sample in all of the different protocols employed. 2.4. Cell culture and adipogenesis protocols The two populations of human preadipocytes and the 3T3-L1 murine preadipocyte cell line (obtained from the ATCC) were routinely cultured in complete medium (CM), DMEM/F12 with 10% FCS. The human BAT cell line, PAZ6 (Zilberfarb et al., 1997), was kindly provided by Vladimir Zilberfarb (Université Paris Descartes); it was maintained in CM, DMEM/F12 with 8% heat-inactivated FCS. In all cases adipogenesis was induced in confluent cells by replacing with the same differentiation medium (DM) containing 5% FCS (4% for PAZ6), biotin (33 ␮M), panthothenate (17 ␮M), T3 (1 nM), dexamethasone (100 nM), thiazolidinedione (1 ␮M) and insulin (500 nM), throughout the adipogenic process, i.e. for 9–15 (cell lines) and 21 (primary preadipocytes) days, as previously described (Starkey et al., 2003; Zhang et al., 2006). 2.5. Effect on proliferation; cell cycle analysis The primary preadipocytes and cell lines were plated at 1 × 103 per well in CM in 24 well plates. Once attached (24 h later) the cells were incubated in DHEA at final concentrations of 10−6 to 10−8 M; the 3T3-L1 and PAZ6 cell lines were trypsinized 24 and 48 h later and cell numbers measured using a Coulter particle counter [Z2 , Beckman Coulter Gmbh, Germany] with particle size set between 3.8 and 9.0 nm. To determine whether any effects were due to DHEA itself or a metabolite, the 3T3L1 cell line was incubated in 10−6 or 10−7 M DHEA alone or in combination with inhibitors of 3-␤-hydroxysteroid dehydrogenase (3-␤HSD) or 5-␣-reductase (5-␣R). These were trilostane, finasteride or pioglitazone all at 10−6 or 10−7 M. The human primary preadipocytes were examined in the same way but after 72 and 168 h, because of their lower proliferation rate. For the cell cycle analysis, 3T3-L1 and PAZ6 cells (104 per well in CM) were plated in 6 well plates; 24 h later DHEA was added to a final concentration of 10−6 M. Incubation continued for a further 48 h when the cells were trypsinized, fixed in ice-cold 70% ethanol, washed twice in PBS and incubated for 20 min at 37 ◦ C with propidium iodide (50 ␮g/mL in PBS) and RNAse A (50 ␮g/mL). The resulting samples were then analyzed using a FACSCalibur flow cytometer [Dakocytomation, Cambridge, UK]. 2.6. Effect on metabolite production Conditioned medium (from 3T3-L1 cells exposed to 10−6 M DHEA for 48 h) was assayed for testosterone (Siemens Healthcare Diagnostics Immunoassay Analyzer, COV 3.2–15.6%, LLD 0.35 nmol/L), ␤-estradiol, androstenedione (coat-a-count system, Diagnostics Products Corporation, Llanberis, Wales, COV 3.2–15.6%, LLD

0.14 nmol/L) and DHEAS (Roche Codas Immunoassay Analyzer, COV 2.3–3.3%, LLD 0.1 ␮mol/L). 2.7. Effect on spontaneous and PPAR induced adipogenesis and adiponectin production The various cell populations (in 12 well plates) were examined in complete and differentiation medium. Microscopic examination provided a means of determining whether morphological changes, e.g. rounding-up of cells and/or acquisition of lipid filled droplets, had occurred. Foci of differentiation (groups of cells with lipid droplets) were counted in ten different fields for each experimental condition. In addition, the cells were fixed in 60% isopropanol, stained with 0.3% oil red O, followed by extraction of the absorbed dye with 100% isopropanol and measurement of the OD490 . Human adiponectin was assayed in neat culture supernatants from the human primary preadipocyte cultures using the adiponectin Duoset® ELISA development system [R&D systems, Oxford, UK] according to the manufacturer’s instructions. 2.8. QRT-PCR of transcript for markers of terminal adipogenesis The various cell populations were plated in 6 well plates in CM or DM for 15–20 days. RNA was extracted, reverse transcribed and transcript copy numbers for mouse glucose-6-phosphate dehydrogenase (GPDH, forward: ATG CTC GCC ACA GAA TCC ACAC, reverse: AAC CGG CAG CCC TTG ACTTG) and housekeeping gene acidic ribosomal phosphoprotein (ARP forward: GAG GAA TCA GAT GAG GAT ATG GGA, reverse: AAG CAG GCT GAC TTG GTT GC) or human lipoprotein lipase (LPL forward: GAG ATT TCT CTG TAT GGA CC, reverse: CTG CAA ATG AGA CAC TTT CTC), uncoupling protein 1 (UCP1, forward: CGG ATG AAA CTC TAC AGC GG, reverse: CAC TTT TGT ACT GTC CTG GTG G) and housekeeping gene adenosine phosphoribosyl-transferase (APRT forward: GCT GCG TGC TCA TCC GAA AG, reverse: CTT TAA GCG AGG TCA GCT GC) were measured using Sybr green and a Stratagene MX3000 light cycler, all as previously described (Zhang et al., 2006). Standard curves (the PCR amplicon subcloned into pGEM-T at 106 to 102 copies) were included for each gene and results are expressed as an absolute value, either per microgram of input mRNA or relative to the appropriate housekeeping gene. 2.9. Statistical analysis The SPSS statistical software package was used. The Friedman test was performed on each experiment and Wilcoxon Signed Ranks test was performed on individual treatments relative to control. A p value < 0.05 was considered significant in all cases.

3. Results 3.1. DHEA inhibits the proliferation of preadipocyte cell lines In our experimental model, doubling times were found to be approximately 22 h for 3T3-L1 cells and 28 h for the PAZ6 cells. Significant reductions in proliferation were seen, in both cell types, following 24 h exposure at DHEA concentrations of 10−8 to 10−5 M (data not shown) and similarly after 48 h incubation (from 10−7 M in 3T3-L1 cells) as shown in Fig. 1. Circulating concentrations of DHEA are in the nM range but as previously mentioned, intraadipose concentrations may be in an order of magnitude higher still (Fehér and Bodrogi, 1982; Szymczak et al., 1998). The variation in proliferation rate made it impossible to determine whether one cell type is more proliferation-sensitive to DHEA than the other. Experiments using trypan-blue exclusion eliminated toxicity as an explanation for the observed reduced growth and DHEAS did not significantly inhibit the proliferation of 3T3-L1 or PAZ6 preadipocytes (except at 10−6 molar in 3T3-L1 cells). A further indicator of specificity was the lack of effect of DHEA on the proliferation of two other cell lines, FRTL5 (rat thyroid) and HaCat (human keratinocyte) with DHEA concentrations of 10−6 to 10−9 molar. Aware that the effects may not be due to DHEA, but to a downstream metabolite, we attempted to inhibit 3-␤HSD and 5-␣R, the enzymes which catalyze the conversion of DHEA to androstenedione and other metabolites, using the specific inhibitors trilostane, finasteride and pioglitazone (Cooke, 1996; Faller et al., 1993; Gasic et al., 1998). Treatment of 3T3-L1 cells with trilostane or pioglitazone (but not finasteride) alone induced significant inhibition (p < 0.001, p < 0.03, respectively). There was no significant differ-

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Fig. 1. Effect of DHEA on preadipocyte cell-line proliferation. Box-and-whisker plots showing the effect on proliferation of 3T3-L1 (A) and PAZ6 (B) following 48 h exposure to varying concentrations of DHEA. Results are a combination of two experiments performed in quadruplicate and are representative of further experiments performed at least 3 times.

ence in the inhibitory effect of DHEA on 3T3-L1 proliferation in the presence of finasteride. We also tested estradiol and testosterone, at a range of doses (10−9 to 10−6 M) and neither hormone caused a significant reduction in the proliferation rate at any concentration (The concentration of these hormones present in conditioned medium from 3T3-L1 cells exposed to 10−6 M DHEA for 48 h was 30–50 nmol/L).

3.2. DHEA induces cell cycle blockade in preadipocyte cell lines In the absence of observed toxicity we postulated that the reduced proliferation in the presence of DHEA might be due to an increase in apoptosis or interruption of the cell cycle; we investigated these possibilities using flow cytometry of the cell lines following incubation with a single DHEA concentration (10−6 molar). The histograms in Fig. 2 indicate no increase in the pre-G1 peak; hence elevated apoptosis was unlikely to be the cause.

Fig. 3 illustrates that DHEA (10−6 molar) caused a block in the cell cycle of both 3T3-L1 (G1/S) and PAZ6 (G2 + M) cells but at different points within the cell cycle. 3.3. DHEA has minimal impact on adipogenesis in preadipocyte cell lines When cultured in DM, both preadipocyte cell lines undergo adipogenesis but 3T3-L1 possesses a greater ability to differentiate when compared to PAZ6. Also 3T3-L1 differentiation requires a shorter time period to achieve terminal differentiation (9–12 days) whereas PAZ6 cells required 15 days or longer to reach this stage. It was not possible to make accurate counts of differentiation foci in 3T3-L1 (because of the high level of adipogenesis) but there were significantly fewer foci (p < 0.001) in PAZ6 cells in the presence of DHEA 10−7 molar (mean 1.6 ± SD 1.96) compared with the DM plus 0.1% DMSO control (mean 4.53 ± SD 2.43). There were no significant differences in oil red O staining or in QPCR measurements

Fig. 2. Histograms of 3T3-L1 and PAZ6 with/without DHEA. Histograms for 3T3-L1 and PAZ6 cells exposed to DHEA 10−6 molar for 48 h, treated with propidium iodide and assessed by flow cytometry. Data presented are from one experiment (six replicates); similar results were obtained on repeat.

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Fig. 3. Cell cycle analyses of 3T3-L1 (A) and PAZ6 (B) with/without DHEA. Results are median values of percentages of gated controls for each cell cycle stage in 3T3-L1 and PAZ6 preadipocytes following 48 h exposure to DHEA 10−6 molar.

of terminal differentiation markers in either preadipocyte population when differentiated in the presence of DHEA (3T3-L1 GPDH p = 0.075, PAZ6 LPL p = 0.173). In contrast, transcript copy numbers of UCP-1 were significantly higher (325 ± 82%) in PAZ6 cells differentiated in the presence of DHEA (p = 0.028), albeit with the caveat that high Ct values were observed.

Attempts to investigate whether increased apoptosis or cell cycle blockade were responsible for the observed effects were not possible owing to the size heterogeneity of the primary preadipocytes.

3.4. DHEA inhibits the proliferation of human primary preadipocytes

Subcutaneous and omental preadipocytes were incubated in DM for 15–21 days supplemented with 0.1% DMSO (control) or DHEA 10−7 molar. The adipogenic potential of preadipocytes from different patients in the absence of DHEA was highly variable as illustrated by the QPCR measurement of LPL transcripts. Table 1 presents detailed results from two pairs of patient samples, which illustrate that similar levels of differentiation were observed when comparing subcutaneous and omental preadipocytes from the same individual. The in vitro induced adipogenesis was not associated with gender, age or BMI. Please note that there was no significant difference in the Ct values for the housekeeper gene APRT in the presence [mean 22.5 ± SD 0.8] and in the absence [mean 22.14 ± 0.4] of DHEA in either fat depot (n = 13). Several methods (not all applied to all samples) were then used to assess the impact of DHEA on the induced adipogenesis. Oil red O staining, although reduced in the presence of DHEA, was not significantly different in either subcutaneous (0.28 ± 0.04 without and

DHEA inhibited subcutaneous preadipocyte proliferation at both 72 (data not shown) and 168 h (Fig. 4) although no effects were observed for DHEA 10−8 molar at the longer time point, nor for concentrations below 10−5 molar at 72 h. Nevertheless our results demonstrate that following chronic exposure, DHEA inhibited proliferation at physiological, intra-adipose tissue concentrations. In omental preadipocytes there were again no effects apparent of DHEA on proliferation at concentrations below 10−5 molar at 72 h (data not shown) but inhibition was observed following more prolonged exposure at physiologically relevant concentrations (Fig. 4). Also the dose related effect is more pronounced in the omental relative to the subcutaneous preadipocytes. Overall the effects on primary cell proliferation are less dramatic than those observed in the cell lines.

3.5. DHEA inhibits adipogenesis of omental preadipocytes

Fig. 4. Effect of DHEA on human primary preadipocyte proliferation. Box-and-whisker plots demonstrating the effect of DHEA on proliferation of subcutaneous (A) and omental (B) preadipocytes (103 per well, 24 well plate) following 168 h exposure to varying concentrations of DHEA. Results are medians and IQRs for DHEA and control 0.1% DMSO exposed cultures. Pooled data are presented from four experiments performed in quadruplicate.

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Table 1 Lipoprotein lipase (LPL) transcript numbers following adipogenesis with/without DHEA. Ct value, copy number and fold increase in LPL copy number from day 0 to day 21 of differentiation protocol in control (DMSO 0.1%) and DHEA 10−7 molar treated subcutaneous (SC) and omental (OM) preadipocyte cultures. Patient number

8 (SC) 8 (OM) 1 (SC) 1 (OM)

Day 0

Day 21 DHEA 10−7 molar

Day 21 control (DMSO 0.1%)

Ct

Copy number

Ct

32.17 32.01 36.11 33.6

18.17 139.0 96.8 123

26.75 21.78 34.53 33.45

Copy number 558 70,130 246.9 134

Fold increase

Ct

31 504 2.6 1.1

24.00 22.42 34.16 33.92

Copy number 3163 47,500 306.7 103

Fold increase 174 341 3.2 0.83

Fig. 5. Foci of differentiation in omental preadipocytes cultured in differentiation medium for 15 days with added, 0.1% DMSO (control) (A) or 10−7 molar DHEA (B). Magnification 200×.

0.24 ± 0.04 with DHEA, n = 3) or omental (0.48 ± 0.1 without and 0.41 ± 0.06 with DHEA, n = 6) preadipocytes. When applying numbers of differentiation foci as a measure of effect, in subcutaneous cultures (n = 5) there was no reduction in foci number, conversely more foci were observed in DHEA exposed samples (mean 33.5 ± SD 37.47 compared with mean 25.21 ± SD 36.10 for DMSO controls) and the difference just achieved significance (Wilcoxon Signed Ranks test p = 0.034). In contrast, in omental preadipocyte cultures (n = 3) 10−7 molar DHEA induced a significant reduction (p < 0.0001) in the number of differentiation foci (mean 3.75 ± SD 3.33 compared with mean 6.44 ± SD 4.93) as illustrated in Fig. 5. We then measured transcripts for LPL, a marker of terminal differentiation, to assess the effect of DHEA on human primary preadipocyte adipogenesis. Subcutaneous preadipocytes (n = 7) differentiated in the presence of DHEA had significantly increased LPL transcripts, 270 ± 70% of those differentiated without DHEA (p < 0.025). In contrast, DHEA significantly reduced LPL transcripts in omental preadipocytes (n = 6), to 64 ± 16% of the control cells (p < 0.05). Furthermore there was a significant difference in the effect of DHEA on subcutaneous compared with omental preadipocytes (p < 0.01). There was no difference in the effects of DHEA on in vitro adipogenesis, on either adipose depot, when comparing preadipocytes from men and women.

3.6. DHEA does not alter adiponectin production by human primary preadipocytes When cultured in CM, the concentration of adiponectin in the culture supernatant of primary preadipocytes was 74.56 ± 47.16 ␮g/L, increasing to 1287.94 ± 1746.24 ␮g/L after 21

days incubation in DM. The omental preadipocytes (n = 4) tended to generate a greater fold increase in adiponectin concentration (19.54 ± 30.13) than the subcutaneous (n = 5) preadipocytes (8.55 ± 7.75) following differentiation, although this was not statistically significant. Furthermore there was no difference observed between the control and DHEA (10−7 molar) treated samples in either the omental or subcutaneous cell types.

4. Discussion We have compared the effect of DHEA on the proliferation and differentiation of two preadipocyte cell lines and human primary preadipocytes from two different fat depots. Our results, which indicate inhibition of proliferation using the well-characterized 3T3-L1 murine preadipocyte cell line, confirm the work of others (Gordon et al., 1986; Lea-Currie et al., 1998). Furthermore we report that the inhibition was associated with arrest in the G1 phase of the cell cycle, in agreement with results using various mesodermal cell types, e.g. human umbilical vein endothelial cells (Zapata et al., 2005). These authors also demonstrated that the effect was not apparent with DHEAS and did not depend on conversion to androgens or estrogens. In contrast, we were not able to demonstrate a clear negative impact on adipogenesis. Previous reports have used concentrations several orders of magnitude higher than those of our study (Shantz et al., 1989), which were chosen to correspond to the levels present in the human circulation, and more importantly in adipose tissue, where it is a major source of sex steroids especially after menopause (Szymczak et al., 1998). Of interest, significantly higher concentrations of DHEA were reported in the omental fat of obese men compared with subcutaneous adipose tissue (Belanger et al., 2006). The relevance of the DHEA effect on the 3T3-L1 cell

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line must be considered in the light of the very low concentration of this hormone in the rodent circulation. However, much of our knowledge concerning preadipocyte biology has been obtained using this cell line and its growth characteristics make it an obvious choice to dissect underlying mechanisms. Ideally we would have liked to demonstrate that the DHEA growth-retardant effect was not abolished in the presence of trilostane or pioglitazone, both inhibitors of 3-␤HSD, the next enzyme in the steroid synthesis pathway. Unfortunately both inhibitors inhibited proliferation directly although inhibition of 5-␣R using finasteride did not alter the DHEA anti-proliferative effects. Furthermore, we were able to exclude an inhibitory effect of estradiol or testosterone, the immediate downstream metabolites, at physiological concentrations found in culture medium of 3T3-L1 cells incubated with DHEA. A further interesting observation was the absence of detectable sulphotransferase activity in the 3T3-L1 preadipocytes as demonstrated by the lack of DHEAS production in culture conditions, this in the light of confirmed generation of terminal metabolites (testosterone, estradiol). This observation confirms a previous study (Dalla Valle et al., 2006) and may suggest that adipose tissue is, in terms of DHEA, pro-metabolic and does not possess the capacity to either inactivate it or add to the circulating pool. Previous studies have demonstrated that DHEA exhibits effects on bone, vascular smooth muscle and adipose tissue (Shantz et al., 1989; Williams et al., 2002; Gurnell et al., 2008) all of which have a common embryological origin being derived from mesenchymal stem cells. In order to evaluate the possibility of DHEA action in other (non-mesenchymal derived) cell types a differentiated thyroid cell line (FRTL5) and an undifferentiated skin keratinocyte cell line (HACAT) were employed. The absence of any effect suggests that DHEA inhibition of proliferation is specific for mesenchymal cells. The findings presented relating to the PAZ6 human BAT preadipocyte cell line are novel. The cell line was generated by SV40 transformation (which may explain why DHEA treatment of these cells is associated with arrest in G2) of peri-renal fat from a child, yet retains the ability to undergo differentiation (Zilberfarb et al., 1997). The demonstrated inhibitory effect on proliferation followed by the possible increase in BAT specific genes following DHEA exposure may indicate a pro-adipogenic effect. Studies in rats suggest that BAT may be a target of the hormone (Ryu et al., 2003) and although we are unaware of any relevant studies in humans, our findings would be consistent with the temporal association between high DHEA concentrations and BAT depots in the fetal state (Easterling et al., 1966). Subsequently we have investigated human primary preadipocytes and found that DHEA inhibited the proliferation of both subcutaneous and omental derived cells and at similar concentrations to those demonstrated to be effective in the cell lines. Limitations of sample size and cell-size heterogeneity precluded our investigating the mechanism responsible for the reduced growth. Various methods were used to assess the degree of induced adipogenesis and demonstrated that DHEA inhibits differentiation in omental but not subcutaneous preadipocytes, in which differentiation was enhanced. This is of potential importance given the adverse metabolic consequences associated with omental fat accumulations relative to subcutaneous adipose tissue deposits (Björntörp, 1991; Lapidus et al., 1984; Kaplan, 1989). Differentiation-induced adiponectin was produced in both omental and subcutaneous preadipocytes (a further marker of adipogenesis) but no difference was observed between DHEA treated and control samples. This contrasts with reports of increased adiponectin mRNA in the fat of obese humans and rats treated with DHEA (Hernandez-Morante et al., 2006; Kabowska and Kochan, 2005).

We are aware of just two previous studies which have examined the effect of DHEA on human preadipocytes in vitro. The first (McKintosh et al., 1999) reported inhibition of proliferation but enhancement of differentiation in a single subcutaneous sample treated with 10−4 to 10−6 M DHEA. High concentrations were also applied in the second study which compared the DHEA effect on subcutaneous and omental preadipocytes from 10 obese women (Sarac et al., 2006) and concluded that ‘DHEA predominantly influences the proliferation and differentiation of human omental adipose tissue’. Our experiments are in general agreement with both reports but are the first to have examined the effects of physiological levels of DHEA on the growth and adipogenesis of subcutaneous and omental preadipocytes from non-obese men and women. The anti-adipogenic effect of DHEA on human omental preadipoctyes in vitro accords with the significant decrease in abdominal fat in elderly subjects treated with 50 mg/dL DHEA for 6 months (Villareal and Holloszy, 2004). It is also of potential significance in the clinical setting, in particular in individuals with pathological DHEA deficiency in whom there is known to be an augmented cardiovascular risk (Rosén and Bengtsson, 1990; Tomlinson et al., 2001; Mills et al., 2004). In our recently reported clinical trial of DHEA replacement on vascular function in subjects with adrenal insufficiency (Rice et al., 2009) we did not find any changes in body weight or fat percentage, in keeping with many other human studies of DHEA therapy in a variety of disease states (Tchernof and Labrie, 2004). However, given the in vitro findings reported here, future trials of DHEA supplementation should incorporate methodologies which are sensitive enough to determine whether DHEA affects fat mass in a depot-specific manner, with a particular focus on the visceral compartment. Grants Supported by grants from the Wales Office for Research and Development (ref no ReF05/1/005), and Cardiff and Vale NHS Trust (ref 05/cmc/3294ES), and the Royal College of Physicians through the Lewis Thomas Gibbon Jenkins of Briton Ferry Fellowship awarded to S.R. Conflict of interest statement None of the authors have any conflict of interest to declare. Acknowledgments We are most grateful to the patients who provided tissue samples for study. We would like to thank Professor M.F. Scanlon for his continued support. References Belanger, C., Hould, F.-S., Lebel, S., Biron, S., Brochu, G., Tchernof, A., 2006. Omental and subcutaneous adipose tissue steroid levels in obese men. Steroids 71, 674–682. Bergthorsdottir, R., Leonsson-Zachrisson, M., Odén, A., Johannsson, G., 2006. Premature mortality in patients with Addison’s disease: a population based study. J. Clin. Endocrinol. Metab. 91, 4849–4853. Berr, C., Lafont, S., Deburie, B., Dartigues, J., Baulieu, E., 1996. Relationship of dehydroepiandrosterone sulfate in the elderly with functional, psychological and mental status and short term mortality: a French community-based study. Proc. Natl. Acad. Sci. U.S.A. 93, 13410–13415. Bleau, G., Roberts, K.D., Chapdelaine, A., 1974. The in vitro and in vivo uptake and metabolism of steroids in human adipose tissue. J. Clin. Endocrinol. Metab. 39, 236–246. Björntörp, P., 1991. Metabolic implications of body fat distribution. Diabetes Care 14, 1132–1143. Bonny, R., Scanlon, M.J., Jones, D.L., Beranek, P.A., Reed, M.J., James, V.H.T., 1984. The interrelationship between plasma 5-ene adrenal androgens in normal women. J. Steroid Biochem. 20, 1353–1355.

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