A novel selective 11β-hydroxysteroid dehydrogenase type 1 inhibitor prevents human adipogenesis

Glucocorticoid excess increases fat mass, preferentially within omental depots; yet circulating cortisol concentrations are normal in most patients with metabolic syndrome (MS). At a pre-receptor level, 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) activates cortisol from cortisone locally within adipose tissue, and inhibition of 11β-HSD1 in liver and adipose tissue has been proposed as a novel therapy to treat MS by reducing hepatic glucose output and adiposity. Using a transformed human subcutaneous preadipocyte cell line (Chub-S7) and human primary preadipocytes, we have defined the role of glucocorticoids and 11β-HSD1 in regulating adipose tissue differentiation. Human cells were differentiated with 1·0 μM cortisol (F), or cortisone (E) with or without 100 nM of a highly selective 11β-HSD1 inhibitor PF-877423. 11β-HSD1 mRNA expression increased across adipocyte differentiation (P<0·001, n=4), which was paralleled by an increase in 11β-HSD1 oxo-reductase activity (from nil on day 0 to 5·9±1.9 pmol/mg per h on day 16, P<0·01, n=7). Cortisone enhanced adipocyte differentiation; fatty acid-binding protein 4 expression increased 312-fold (P<0·001) and glycerol-3-phosphate dehydrogenase 47-fold (P<0·001) versus controls. This was abolished by co-incubation with PF-877423. In addition, cellular lipid content decreased significantly. These findings were confirmed in the primary cultures of human subcutaneous preadipocytes. The increase in 11β-HSD1 mRNA expression and activity is essential for the induction of human adipogenesis. Blocking adipogenesis with a novel and specific 11β-HSD1 inhibitor may represent a novel approach to treat obesity in patients with MS.


Introduction
Glucocorticoid excess (Cushing's syndrome) causes visceral obesity, insulin resistance, diabetes mellitus, dyslipidaemia, hypertension and premature vascular mortality; as such it represents an excellent paradigm for patients with the metabolic syndrome (MS). However, Cushing's syndrome is rare and circulating glucocorticoid levels are usually normal or even slightly reduced in obese patients (Fraser et al. 1999). At a cellular level, glucocorticoids within human adipose tissue, specifically in omental depots, can be generated from inactive circulating cortisone (in humans) or 11-dehydrocorticosterone (in rodents) through the oxo-reductase activity of 11b-hydroxysteroid dehydrogenase type 1 (11b-HSD1; Bujalska et al. 1997b). Transgenic mice with adipose tissue-targeted 11b-HSD1 overexpression develop visceral obesity, insulin resistance, hyperlipidaemia and hypertension without altering circulating glucocorticoids (Masuzaki et al. 2001(Masuzaki et al. , 2003, while mice with 11b-HSD1 overexpression in the liver develop MS without obesity . Conversely, global deletion of 11b-HSD1 caused reduced visceral fat accumulation and improved insulin sensitivity on a high fat diet (Kotelevtsev et al. 1997. At a molecular level, glucocorticoids exert potent effects upon adipose tissue; in mature rat adipocytes, dexamethasone decreases glucose uptake and oxidation (Olefsky 1975, De et al. 1981. Glucocorticoids increase lipolysis by up-regulating the expression of the ratelimiting enzyme hormone-sensitive lipase (Slavin et al. 1994), as well as lipogenic enzyme, lipoprotein lipase (Yang et al. 1993). In preadipocytes, glucocorticoids are essential for terminal adipogenesis (Hauner et al. 1987) and limit cell proliferation (Tomlinson et al. 2002). The process of cellular differentiation is a highly synchronized cascade of regulated differentiationdependent gene expression. Genes such as retinoblastoma proteins that regulate the cell cycle (Richon et al. 1997) are followed by adipogenic transcription factors, such as peroxisome proliferator-activated receptor g (PPARg) and CCAAT enhancer-binding protein (Rosen & MacDougald 2006). Mature adipocytes express late differentiation genes involved in lipid metabolism and lipid transport including glycerol-3-phosphate dehydrogenase (G3PD) and fatty acidbinding protein 4 (FABP4; Hotamisligil et al. 1996); many of these genes are regulated by glucocorticoids (Wu et al. 1996, Rosen & MacDougald 2006. Previously, we have shown that non-selective inhibition of 11b-HSD1 can prevent human adipocyte differentiation in vitro (Bujalska et al. 1999). The potential for therapeutic intervention has been tested in rodent models where selective 11b-HSD1 inhibitors lower plasma glucose, improve insulin sensitivity and in some studies reduce body weight in mice (Alberts et al. 2002, Kershaw et al. 2005. However, the potency of these inhibitors has been variable and there are no data on efficacy in human tissue. We report the development of a selective inhibitor against human 11b-HSD1, PF-877423 (Pfizer Global R&D, La Jolla, CA, USA) and the effect of this compound upon adipogenesis in a wellcharacterised differentiating human subcutaneous preadipocyte cell line (Darimont et al. 2003, Qiao et al. 2005 and in primary cultures of subcutaneous human preadipocytes.

Recombinant protein assay
Wild-type recombinant human 11b-HSD1 protein (24-292) was used for studying the inhibitor kinetics. Radio-labelled [1,2-3 H]-cortisone was purchased from American Radiolabeled Chemicals Inc (St Louis, MO, USA). NAD (reduced form; NADPH), glucose-6-phosphate (G6P) and G6P dehydrogenase (G6PD) were purchased from Sigma-Aldrich. All the concentrations reported in the following section are final in the assay buffer. In addition, the enzyme concentrations represent the active concentrations that were determined by active-site titration using a tight-binding inhibitor. The experimental data were fitted by using the non-linear regression analysis software, Grafit (Leatherbarrow (2001) GraFit Version 5, Erithacus Software Ltd, Horley, UK).
The measurement of the in vitro 11b-HSD1 activity was performed in a 100 mM triethanolamine buffer (pH 8 . 0), containing 200 mM NaCl, 0 . 02% n-dodecyl b-D-maltoside, 5% glycerol and 5 mM b-mercaptoethanol. A typical reaction for the determination of enzyme activity comprised the following: 5 nM enzyme pre-incubated for at least 30 minutes in the assay buffer in the presence of 500 mM NADPH in round-bottom 96-well plates (Costar cat # 3365). Next, the reaction was initiated by adding a regenerating system (consisting of 2 mM G6P, 1 U/ml G6PD and 6 mM MgCl 2 ) and labelled 3 H-cortisone as substrate. After an incubation period (30-40 min), 100 ml of the assay mixture were transferred to a second empty round-bottom 96-well plate and mixed with an equal volume of dimethylsulphoxide (DMSO) to quench the reaction. Then, a 15 ml aliquot of the assay solution was loaded into a C-18 column (Polaris C18-A, 50!4. 6 mm, 5 u, 180 Å , Varian; Polaris, Palo Alto, CA, USA connected to an automated High-Throughput Liquid Chromatography instrument (Cohesive Technologies, HTLC, Franklin, MA, USA). The radioactive material from the column was detected with a b-RAM model 3 Radio-HPLC detector (IN/US, Tampa, FL, USA). Substrate and product peaks were separated by using an isocratic mixture of 38:62 methanol to water (v/v) at a flow rate of 1. 0 ml/min. Under these experimental conditions, the retention time for cortisone and cortisol were 4 . 5 and 5 . 5 min respectively.
The initial reaction velocities recorded were in the linear range and were determined by measuring the peak area for cortisol formation with time.

Recombinant protein kinetic analysis
The inhibition of 11b-HSD1 by PF-877423 was analysed by fitting to the equation described below (Equation (1); Morrison 1969) and provided an accurate measurement for the value of Ki app at a fixed concentration of cortisone where where K m was the Michaelis-Menten constant for cortisone.
HEK293 and Chubb-S7 cell culture HEK293 cells stably transfected with human 11b-HSD1 (HEK293T1) or 11b-HSD2 (HEK293T2) cDNA as described previously (Bujalska et al. 1997a) were used to study the specificity of inhibitor PF-877423 upon 11b-HSDs. Cells were cultured in minimum essential medium (MEM) media supplemented with 10% fetal bovine serum (FBS) and 1% non-essential fatty acids; for experiments, cells were seeded into 24-well tissue culture dishes and maintained in MEM with 10% FBS until confluence. The Chub-S7 cell line was derived from human subcutaneous adipose tissue (Darimont et al. 2003) by co-expression of human telomerase reverse transcriptase and papillomavirus E7 oncoprotein (HPV-E7) genes. The cell line has an unlimited life span and the capacity to accumulate lipid without chromosomal alteration. Confluent Chub-S7 cells were cultured in 75 cm 2 TC flasks in DMEM/Ham's F-12 medium supplemented with 10% FBS. For experiments, cells were seeded in 24-well plates at density 10 5 cells/well. Chub-S7 were differentiated (up to 21 days) according to Hauner et al. (1987) with 166 nM human insulin (I-9278 Sigma), 1 mM PPARg agonist (GW1929, Camlab, Cambridge, UK) and, where specified, with the addition of 1 mM cortisol (F). For inhibition studies, Chub-S7 cells were differentiated with 0 . 5 mM cortisone (E) and an optimised concentration of the selective 11b-HSD1 inhibitor, PF-877423, at a final concentration of 100 nM.

Human subcutaneous stromal-vascular cell differentiation
Confluent human subcutaneous stromal-vascular (s-v) cells (96-well plates) were obtained from Zen-Bio Inc. (Research Triangle Park, NC, USA) and allowed to recover overnight at 37 8C 5% CO 2 . The following day, day 0, differentiation was initiated by incubating cells in differentiating media, DM2(E) (Zen-Bio Inc.), which consists of DMEM/Ham's F-12 medium containing FBS, L-glutamine, penicillin/streptomycin, insulin, indomethacin, 3-isobutyl-1-methylxanthine with 1 mM E and vehicle (DMSO not exceeding 0 . 1%). To define the effect of the PF-877423 inhibitor upon the differentiation of primary preadipocytes, cultures were supplemented with 300 nM PF-877423 (in DMSO). Control cells cultured without E. Media were replenished and PF-877423 or vehicle treatment repeated every 2-3 days, except when 11b-HSD1 activity was assessed.

Measuring lipid content in human subcutaneous s-v cells
Lipogenesis in human subcutaneous s-v cells was measured as triglyceride accumulation. Cells were carefully washed with PBS and lysed in situ by adding 50 ml/well of Hecameg (10% solution in water -Calbiochem, Nottingham, UK). After gentle shaking at room temperature for 10 min, 200 ml triglyceride (Infinity) reagent (Thermo DMA, Louisville, CO, USA) was added to each well. Plates were read after 10-20 min at 500 nm with correction at 660 nm (Spectra MAX PLUS -Molecular Devices Corporation, Sunnyvale, CA, USA). Results were expressed as optical density (OD) values.

HEK293 and Chubb-S7 11b-HSD assay
Cells were washed and incubated with 100 nM F (for dehydrogenase activity) or E (for oxo-reductase activity) with appropriate tritiated tracer -3 H F (Du Pont, Stevenage, UK) or During 11b-HSD1 activity assessment in human subcutaneous s-v cells, the medium was changed to basal medium (BM-1, Zen-Bio Inc.) containing only antibiotics, and cells were cultured for 24 h in the presence of PF-877423 with or without 0 . 5 mM E. At the end of the incubation, the cortisol signal was quantitatively determined in the 100 ml cell supernatant with the Correlate-Enzyme Immunoassay Cortisol kit (Assay Designs Inc. Ann Arbor, MI, USA) following the manufacturers' instructions. Plates were read on a plate reader (Spectra MAX PLUS -Molecular Devices Corporation) at 405 nm, with correction at 580 nm. Activity has been expressed as cortisol production in pg/ml per 24 h.

Total RNA extraction and RT reaction
Total RNA was extracted from cells using TriReagent (Sigma) according to the manufacturer's protocol. Integrity and concentration of RNA were assessed by electrophoresis and spectrophotometry respectively. Reverse transcription was carried out using AMV and random primers at 37 8C for 1 h. All RT reagents were purchased from Promega.

Real-time PCR
Quantitative mRNA expression levels of 11b-HSD1, H6PDH, GRa, FABP4, G3PD, GLUT-4 and PPARg2 were measured by real-time PCR using an ABI 7500 system (Perkin-Elmer, Biosystems, Warrington, UK). PCR was performed in 25 ml reactions on 96-well plates. Reactions contained TaqMan universal PCR master mix (Applied Biosystems, Foster City, CA, USA), 900 nmol primers, 100-200 nmol TaqMan probe and 25-50 ng cDNA. All reactions were correlated to expression of ribosomal 18S rRNA (provided as a pre-optimized mix; Perkin-Elmer) as an internal reference. All target gene probes were labelled with the fluorescent label FAM and the 18S probe with the fluorescent label VIC. Reactions were as follows: 50 8C for 2 min, 95 8C for 10 min, and then 40 cycles of 95 8C for 15 s and 60 8C for 1 min. Data were analysed according to the manufacturer's guidelines and were obtained as C t values (the cycle number at which logarithmic PCR plots cross a calculated threshold line) and used to determine dC t values (dC t ZC t of the target gene minus C t of the internal reference, 18S). Primers and probes for 11b-HSD1, H6PDH and G3PD were designed using PrimerExpress 1 . 0 software (Applied Biosystems). Sequences from 5 0 to 3 0 are shown in Table 2. Expression assay kits were purchased from Applied Biosystems to measure the gene expression of GRa, GLUT-4, PPARg2 and FABP4.

Statistical analysis
Where data were normally distributed, unpaired Student's t-test was used to compare single treatments with control. If normality tests failed, then non-parametric tests were used. One-way ANOVA on ranks was used to compare multiple treatments values and a P value of !0 . 05 was accepted as statistically significant. Statistical analysis on real-time PCR data was performed on mean DCt values and not on fold changes.

Kinetics of PF-877423 upon recombinant 11b-HSD1 protein
The potency for PF-877423 was strongly affected by the presence of the substrate in the assay buffer ( Fig. 1): K app i values increased at high cortisone concentration, suggesting that the inhibitor behaved as a reversible and competitive inhibitor against cortisone. Fitting the experimental data using equation (2)
The change in adipogenesis following incubation with the 11b-HSD1-specific inhibitor was confirmed visually through staining the cells with oil red O after 21 days of differentiation. A marked increase in the number of red-stained cells was observed in cells differentiated with E or F but not in the presence of PF-877423 (Fig. 6E).    Fig. 7B).
defined the permissive role of GCs in inducing adipocyte differentiation. Here, we extend those observations to a novel transformed human adipocyte cell line that should greatly enhance in vitro human-based adipocyte research studies. Within 5 days of incubation in chemically defined media comprising insulin, PPARg agonist and glucocorticoid, impressive differentiation was observed in Chub-S7 cells as assessed by markers including FABP4, G3PD and adipocyte-specific genes such as GLUT-4 and PPARg2. Adipogenesis was an ongoing process up to 16 days in culture with intracellular lipid stores confirmed by oil red O staining. No significant changes were observed in GRa expression during this differentiation phase; earlier studies had reported increased GR expression in omental versus subcutaneous adipose tissue and had argued that this may be the one factor explaining the predilection of glucocorticoids for visceral obesity (Bronnegard et al. 1990). However, more recently our array and real-time PCR analyses failed to demonstrate any difference in GR expression between human omental and subcutaneous preadipocytes (Bujalska et al. 2006) and adipose tissue (unpublished data). Together with the data in this study, it seems unlikely that changes in GR expression per se are important in the adipogenesis process. At a pre-receptor level our group has focussed on the role of 11b-HSD1 and the regeneration of cortisol from inactive cortisone in human adipose tissue. Previously we have demonstrated increased expression of 11b-HSD1 in omental compared with subcutaneous depots (Bujalska et al. 1997b), and importantly a switch in the directionality of enzyme activity from predominant dehydrogenase to oxo-reductase (Bujalska et al. 2002a), probably as a result of induction of the NADPH donor source for 11b-HSD1 within the endoplasmic reticulum (ER) lumen, H6PDH (Ozols 1993). In support of these observations, in this study, the rise in H6PDH mRNA levels probably explained the increase in 11b-HSD1-mediated oxoreductase activity between days 3 and 9 in addition to an increase in 11b-HSD1 mRNA levels. Furthermore, decreased H6PDH mRNA expression seen on day 16 might account for the decline in 11b-HSD1 oxo-reductase activity despite high mRNA. The impressive increase in 11b-HSD1 expression across adipocyte differentiation has been noted recently in mouse 3T3-L1 cells (Kim et al. 2007), as it has been done in other differentiating cell systems including osteoblasts (Eijken et al. 2005). In both cases cells have been incubated with glucocorticoids that themselves positively regulate 11b-HSD1 (Bujalska et al. 1997b); it is not possible from these or previous data sets to conclude whether or not the increased 11b-HSD1 expression is glucocorticoid mediated or a manifestation of the differentiation process per se.
Nevertheless, 11b-HSD1 expression, resulting in a functionally active oxo-reductase, was clearly linked to the differentiation phenotype. Incubation of Chub-S7 cells with inactive steroid, cortisone, induced a degree of adipocyte differentiation similar to that observed with cortisol. This could be explained on the basis of 11b-HSD1-mediated oxo-reductase activity with a K m for cortisone in Chubb-S7 cells of 100 nM which is similar to the reports in other 11b-HSD1-expressing cell systems (Monder & Lakshmi 1989, Ricketts et al. 1998 including studies on the recombinant enzyme (K m Z333 nM; Walker et al. 2001, Shafqat et al. 2003. Evaluation of the competitive 11b-HSD1 inhibitor, PF-877423, indicated selectivity for the type 1 11b-HSD isoform with a K i £0 . 2 nM in the recombinant assay and IC50w5 nM in Chubb-S7 cells. Selectivity of the inhibitor towards 11b-HSD1 isoform but not 11b-HSD2 is crucial as patients with impairment in the latter are presented with severe hypertension, (White et al. 1997). PF-877423 selectivity was undertaken using a transformed cell line expressing human 11b-HSD2 enzyme where no inhibitory effect was observed. Incubation of cells with 100 nM PF-877423 completely abolished the cortisone induction of FABP4, G3PDH and 11b-HSD1 itself in Chubb-S7 cells. We also demonstrated that the transformed cell line data were consistent with data in primary human subcutaneous preadipocytes. While incubation with cortisone yielded a less impressive effect on adipogenesis in human subcutaneous preadipocytes when compared with Chubb-S7 cells (this might reflect a more advanced adipogenic lineage of primary cultures), inhibition of 11b-HSD1 activity nevertheless reduced the ability of human subcutaneous preadipocytes to differentiate and accumulate lipid.
Whilst the cell line that we have used is subcutaneous in origin and the expression of 11b-HSD1 is higher in omental human preadipocytes (Bujalska et al. 1997b) and therefore we predict that the impact upon omental cells would be more pronounced. We anticipate that this would not lead to preferential loss of subcutaneous fat. Unfortunately, omental cell lines are not available for study and in vivo human clinical studies have not been performed.
Previously, non-selective 11b-HSD inhibitors have been shown to diminish human adipocyte differentiation in vitro (Bujalska et al. 1999) and increase insulin sensitivity in man (Walker et al. 1995), but a lack of isozyme selectivity can cause water retention and hypertension. Since then, patents have been filed on compounds that report to be selective 11b-HSD1 inhibitors. An aryl sulphonamide derivative has been shown to reduce insulin levels and improve glucose tolerance when administered to rodents for 7 days (Alberts et al. 2002). Similar data have been reported for an adamantyl triazole that also reduced body weight and the progression of atherosclerosis in mice (Hermanowski-Vosatka et al. 2005). Transgenic mouse models have established 11b-HSD1 as a novel therapeutic target in this regard; global deletion of 11b-HSD1 results in improved glucose tolerance through reduced gluconeogenesis and hepatic glucose output (Kotelevtsev et al. 1997, while targeted overexpression of 11b-HSD1 in adipose tissue recapitulates features of the MS including central adiposity (Masuzaki et al. 2001). In the liver of db/db mice (a model of type 2 diabetes), GR and 11b-HSD1 mRNA expression positively correlated with blood insulin and glucose (Liu et al. 2005). Inhibition of GR and 11b-HSD1 expression either with GR antagonist (RU486; Liu et al. 2005) or by chronic activation of liver X receptor (Liu et al. 2006) attenuated the phenotype of type 2 diabetes in mice.
In humans, the situation is less clear with regard to a role for 11b-HSD1 in the pathogenesis of MS. Some authors have argued for a primary overexpression of 11b-HSD1 in affected patients akin to that observed in some animal models (Paulmyer-Lacroix et al. 2002, Rask et al. 2002. However, while the expression of 11b-HSD1 might be increased, at least in subcutaneous adipose tissue in obese patients with MS and type 2 diabetes (Tomlinson et al. 2002, Alberti et al. 2007, no such increase was observed in omental adipose tissue. Furthermore, based on urinary cortisol/cortisone metabolite ratios and plasma cortisol generation curves following oral cortisone acetate that primarily reflects hepatic 11b-HSD1 expression, a reduction, not an increase, in 11b-HSD1 expression was observed at least in subjects with simple obesity . We have argued that obesity is not primarily a state of 11b-HSD1 overexpression, but that the fall in hepatic 11b-HSD1 activity with increased visceral adiposity might serve as a protective mechanism to offset hepatic glucose output and further adiposity (Valsamakis et al. 2004). Failure of such a switch off in 11b-HSD1 expression might be a factor that determines the onset and persistence of hyperglycaemia in obese patients with type 2 diabetes mellitus (Valsamakis et al. 2004). Further studies are indicated to define the exact role of 11b-HSD1 in the pathophysiology of human MS. Irrespective of the outcome of these studies, selective inhibitors such as PF-877423 offer a real advance in the prevention and treatment of diabetes in subjects with obesity with the potential added benefit of inhibiting adipocyte differentiation. A reduction in adipogenesis specifically within omental depots is likely to further improve the metabolic phenotype of these patients. Clinical studies characterising a novel, potent (K i 0 . 2 nM) and fully selective 11b-HSD1 inhibitor preventing lipogenesis in vitro will further our understanding of the role of local glucocorticoid metabolism in human adipose tissue.