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Endocrinology, doi:10.1210/en.2007-1028
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Endocrinology Vol. 149, No. 4 1861-1868
Copyright © 2008 by The Endocrine Society

Preadipocyte 11β-Hydroxysteroid Dehydrogenase Type 1 Is a Keto-Reductase and Contributes to Diet-Induced Visceral Obesity in Vivo

R. A. De Sousa Peixoto, S. Turban, J. H. Battle, K. E. Chapman, J. R. Seckl and N. M. Morton

Endocrinology Unit, Center for Cardiovascular Sciences, The Queen’s Medical Research Institute, University of Edinburgh, New Royal Infirmary, Edinburgh EH16 4TJ, United Kingdom

Address all correspondence and requests for reprints to: Dr. Nicholas M. Morton, C3.08, Endocrinology Unit, Center for Cardiovascular Sciences, The Queen’s Medical Research Institute, 47 Little France Crescent, University of Edinburgh, New Royal Infirmary, Edinburgh EH16 4TJ, United Kingdom. E-mail: nik.morton{at}ed.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Glucocorticoid excess promotes visceral obesity and cardiovascular disease. Similar features are found in the highly prevalent metabolic syndrome in the absence of high levels of systemic cortisol. Although elevated activity of the glucocorticoid-amplifying enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) within adipocytes might explain this paradox, the potential role of 11β-HSD1 in preadipocytes is less clear; human omental adipose stromal vascular (ASV) cells exhibit 11β-dehydrogenase activity (inactivation of glucocorticoids) probably due to the absence of cofactor provision by hexose-6-phosphate dehydrogenase. To clarify the depot-specific impact of 11β-HSD1, we assessed whether preadipocytes in ASV from mesenteric (as a representative of visceral adipose tissue) and sc tissue displayed 11β-HSD1 activity in mice. 11β-HSD1 was highly expressed in freshly isolated ASV cells, predominantly in preadipocytes. 11β-HSD1 mRNA and protein levels were comparable between ASV and adipocyte fractions in both depots. 11β-HSD1 was an 11β-reductase, thus reactivating glucocorticoids in ASV cells, consistent with hexose-6-phosphate dehydrogenase mRNA expression. Unexpectedly, glucocorticoid reactivation was higher in intact mesenteric ASV cells despite a lower expression of 11β-HSD1 mRNA and protein (homogenate activity) levels than sc ASV cells. This suggests a novel depot-specific control over 11β-HSD1 enzyme activity. In vivo, high-fat diet-induced obesity was accompanied by increased visceral fat preadipocyte differentiation in wild-type but not 11β-HSD1–/– mice. The results suggest that 11β-HSD1 reductase activity is augmented in mouse mesenteric preadipocytes where it promotes preadipocyte differentiation and contributes to visceral fat accumulation in obesity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
EXCESSIVE PLASMA glucocorticoids (GCs) cause Cushing’s syndrome and promote visceral obesity and metabolic disease (insulin resistance, type 2 diabetes, hypertension, and cardiovascular disease). Similarities between this rare condition and the highly prevalent metabolic syndrome are striking (1). However, because plasma cortisol levels are not raised in metabolic syndrome, a common underlying mechanism linking these disease processes was lacking until the finding that amplification of intracellular GC action, mediated by the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), was aberrantly elevated within the adipose tissue of obese humans (2, 3, 4, 5, 6, 7) and rodents (8, 9). 11β-HSD1, although bidirectional in homogenates, catalyzes 11β-reduction of inert cortisone (11-dehydrocorticosterone in mice) to active cortisol (corticosterone in mice) in intact adipocytes, hepatocytes, and neurons (10). A role for elevated 11β-HSD1 in the pathogenesis of metabolic syndrome was strongly supported by the phenotype of transgenic mice with adipocyte-specific 11β-HSD1 overexpression. These mice exhibited all the major features of the metabolic syndrome including visceral obesity, type 2 diabetes, and hypertension (9, 11). Conversely, 11β-HSD1 knockout mice showed preferential fat accumulation in peripheral depots and an improved cardiovascular risk profile, in part due to adipose insulin sensitization, whereas control mice accumulated fat in the mesenteric depot (12). Similar protective metabolic features were recapitulated in mice expressing 11β-HSD2 (a potent dehydrogenase, inactivating GCs) in adipose tissue (13).

Although elevated 11β-HSD1 drives lipid accumulation and adipocyte hypertrophy (7, 9, 14) and adipocyte insulin resistance (12), the role of 11β-HSD1 in preadipocytes remains unclear. This is due to the following factors. First, data from studies measuring 11β-HSD1 in clonal cell lines and primary cells is conflicting; although 11β-HSD1 expression is restricted to late stages of differentiation in 3T3-L1 and F442A mice (15, 16, 17), in rat (16) and human (18, 19, 20), adipose stromal vascular (ASV) cells express 11β-HSD1 at similar levels to adipocytes. Second, the ASV fraction is a heterogeneous mix of cells (21), and although 11β-HSD1 mRNA (6) and activity (18, 19) have been found, no studies have yet addressed the type of cells that express 11β-HSD1 in the ASV cells. Third, the function of 11β-HSD1 in ASV cells has been further obscured by the apparent switch in reaction direction from inactivation of GCs (11β-dehydrogenase) to activation of GCs (keto-reductase) upon differentiation of human omental, but not sc ASV cells (20). The switch has been linked with increased cofactor provision (17, 22) by hexose-6-phosphate dehydrogenase (H6PDH), which colocalizes with 11β-HSD1 in the lumen of the endoplasmic reticulum (23). GCs have an important role in promoting preadipocyte differentiation (24), and 11β-HSD1 reductase activity has been shown to promote adipocyte differentiation in cultured human ASV cells in vitro (25).

In this study, we tested the hypothesis that preadipocyte 11β-HSD1 promotes preadipocyte differentiation in vivo and that it influences regional fat distribution with consequent effects on the metabolic disease risk associated with mesenteric and sc fat accumulation (12, 26).

We have shown that 11β-HSD1 is predominantly expressed in ASV preadipocytes and investigated whether ASV 11β-HSD1 mRNA levels and activity direction were different between mesenteric and sc depots and the impact of 11β-HSD1 on preadipocyte differentiation in high-fat (HF)-fed 11β-HSD1–/– mice in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals
Male C57BL/6J and Lepob mice (Charles River, Margate, UK) were housed in standard conditions on a 12-h light, 12-h dark cycle (lights on at 0700 h) and fed with standard rodent laboratory chow (SDS, Edinburgh, UK) or with a diet consisting 58% of saturated fat as energy (D12331; Research Diets, New Brunswick, NJ). For the current studies, 2- or 6-month-old mice were used. Mice homozygous for a targeted disruption of the 11β-HSD1 gene have been previously described (27). The disrupted 11β-HSD allele (originally on a 129/OLA background) has been rederived onto a C57BL/6J background (eight backcrosses).

White adipose tissue was dissected postmortem from thigh and axillary (sc) and mesenteric depots then frozen rapidly on dry ice for RNA extraction or used fresh for intact cell preparation. All experiments conformed to ethical codes of the University of Edinburgh and Home Office (UK) regulations according to the Animals (Scientific Procedures) Act 1986.

Preadipocyte isolation and culture
Fat was pooled from four animals for each preadipocyte preparation. Fat pads were chopped with fine scissors and digested with 2 mg/ml collagenase type 1 (Worthington, Reading, UK) in Hanks’ buffered saline solution (Life Technologies, Inc., Paisley, UK) for 1 h at 37 C, then washed twice with Hanks’ buffered saline solution. Digested material was separated by centrifugation at 800 rpm for 8 min (Heraus, DJB Labcore, Buckinghamshire, UK). Freshly isolated ASV fraction (fASV) and cell supernatant (adipocyte fraction) were either added to Trizol (Invitrogen, Paisley, UK) for RNA extraction and snap frozen in liquid nitrogen for subsequent homogenization and determination of 11β-HSD1 dehydrogenase enzyme activity or used in intact cell suspension cultures for 11β-HSD1 dehydrogenase and reductase assays and immunofluorescence experiments. Culture of ASV cells was made in DMEM/F12 supplemented with 10% newborn calf serum (Life Technologies), 50 U/ml penicillin, and 50 µg/ml streptomycin (Life Technologies).

11β-HSD1 activity in intact cells
11β-HSD1 reductase and dehydrogenase activities were measured in intact cells by the conversion of [3H]corticosterone (PerkinElmer, Beaconsfield, UK) or [3H]11-dehydrocorticosterone, prepared as described (28), and 5 nM 3H-labeled tracer was added to 200 nM unlabeled steroid. Aliquots were taken at different times after addition of steroid to cells. Steroids were extracted with ethyl acetate, separated by thin-layer chromatography, and quantified with a phosphoimager tritium screen (FLA2000; Fujifilm, London, UK) using quantitative imaging software (Aida, Sheffield, UK). Activity was expressed per nanomole steroid converted per milligram protein per hour.

11β-HSD1 activity in homogenates
Under in vitro conditions in homogenates, 11β-HSD1 is bidirectional, with oxidative activity more stable (28), and hence, dehydrogenase activity was measured. Under these conditions, product formation was linear with respect to protein concentration (data not shown) and, therefore, accurately reflects absolute 11β-HSD1 protein levels. Preadipocytes were homogenized manually with a small pestle (Anachem, Luton, UK) in homogenization buffer (10% glycerol; 300 mM NaCl; 1 mM EDTA; 50 mM Tris, pH 7.7) and 11β-HSD1 activity measured as described before (28). Briefly, after total protein concentration determination for each homogenate by the Bradford method using BSA as standard (assay kit from Bio-Rad, Hemel Hampstead, UK), reactions (160 µl) containing 0.2 mg/ml protein, 10 nM [3H]corticosterone, and an excess (400 µM) of nicotinamide adenine dinucleotide phosphate with homogenization buffer were incubated in a shaking water bath at 37 C. After 1 h incubation, steroids were extracted with ethyl acetate, separated by thin-layer chromatography, identified by comparison with the migration of unlabeled corticosterone and 11-dehydrocorticosterone under UV light, and quantified with a phosphoimager tritium screen (Fujifilm, Tokyo, Japan). At this time point, product formation was linear with respect to time.

RNA extraction and analysis
RNA was extracted from frozen cells or tissues after homogenization in Trizol (Invitrogen). Total RNA was purified using an RNaid Plus kit BIO 101 (Anachem). RNA (5 µg) was eluted in diethylpyrocarbonate-pretreated water containing 400 U/ml RNAsin (Promega, Southampton, UK), 10 mM dithiothreitol and was resolved on denaturing 1% agarose MOPS/18% formaldehyde gels, blotted onto Hybond N+ membranes (Amersham Pharmacia Biotech, Amersham, UK), and hybridized to [32P]dCTP-labeled cDNA probes. Blots were washed at 65 C to a stringency of 0.5x standard saline citrate and 0.1% SDS and then exposed to a phosphoimager screen as described above or were subjected to autoradiography (Kodak Biomax-MR film; Sigma, Poole, UK). Probes were generated as previously described (11) from plasmid templates or by RT-PCR from ASV or adipocyte RNA using the following primer pairs: pref-1 forward 5'-TCT CAG GCA ACT TCT GTG-3' and reverse 5'-GGA TGG TGA AGC AGA TGG-3', aP2 forward 5'-TTT CTC ACC TGG AAG ACA-3' and reverse 5'-GAC TAT TGT AGT GTT TGA TGC-3', H6PDH forward 5'-CCA GCT TAG AGA TCA GCT CC-3' and reverse 5'-CTC TGT CYG ATT ACT ACG CC-3', 11β-HSD1 forward 5'-AGG ATC CAR AGC AAA CTT GCT TGC-3' and reverse 5'-AAA GCT TGT CAC GGG GCC AGC AAA-3', and 18S forward 5'-GGT TGA TCC TGC CAG TAG-3' and reverse 5'-TGA TCT GAT AAA TGC ACG-3'. For probes generated by PCR, identities were confirmed by sequencing. Hybridization to 18S RNA was used as an internal loading control.

Real-time PCR analysis
cDNA was synthesized from 1 µg RNA using a RT kit with random hexamer primers (Promega). Real-time PCR was performed using the TaqMan ABI Prism 7900 sequence detector (Applied Biosystems, Chester, UK). Briefly, after incubation at 50 C for 2 min and then 95 C for 10 min, 40 cycles of PCR (95 C for 15 sec and 60 C for 1 min) were performed. Data acquisition used Sequence Detector 1.6.3 software (Applied Biosystems). RNA levels were determined from standard curves generated for each primer-probe set using serial dilutions of cDNA from the tissue studied. TATA-binding protein (TBP) mRNA was used as the internal reference to normalize specific mRNA levels and had a cycle amplification profile similar to 11β-HSD1. Oligonucleotide primers and TaqMan probes were as follows: 11β-HSD1 primers, forward 5'-TGG TGC TCT TCC TGG CCT ACT-3' and reverse 5'-CCC AGT GAC AAT CAC TTT CTT TCC-3', and probe, 5'-AGA CCA GAA ATG CTC C-3'; H6PDH primers, forward 5'-CTT CCA GAG CCT GAC ACC AA-3' and reverse 5'-GCG CAG GTT GTC GAT GTG-3', and probe, 5'-CTT CGC AGG TGT CCT TG-3'; and pref-1 primers, forward 5'-ACG TGA CGG TAC CAA GGA A-3' and reverse 5'-AAT GTC TGC AGG TGC CAT GTT-3', and probe, 5'-CCC CTC TGT GAC AAG TGT GTA ACT GCC C-3'. TBP primer set was from Applied Biosystems, UK (lot no. Mm00446973_m1; probe 5'-ATC CCA AGC GAT TTG CTG CAG TCA-3').

Immunofluorescence staining protocol
Pellets of 105 cells from the ASV fraction were resuspended in PBS/10% fetal calf serum (vol/vol) and cytospun in a Shandon SP3 centrifuge (Shandon, Waltham, MA) at 300 rpm for 3 min. Slides were allowed to air dry and were incubated for 5 min in 90:10 dry acetone-methanol. Slides were washed with 1 x PBS and protein block added (Dakocytomation, Glostrup, Denmark) for 1 h. Primary antibodies were incubated overnight at 4 C followed by incubation with secondary antibodies for 4 h at room temperature. Monoclonal mouse antimouse p27kip1 antibody (BD Biosciences, Oxford, UK) was used at 2.5 µg/ml, and monoclonal rat anti-F4/80 antibody (Abcam, Cambridge, UK) was used at 1 µg/ml. Polyclonal anti-11β-HSD1 antibody raised in sheep by immunization with recombinant murine 11β-HSD1 protein and was a kind gift from Dr. Scott Webster (Endocrinology Unit, University of Edinburgh). Confirmation of the specificity of the antibody was made by Western blot on cell extracts and mouse liver proteins together with preabsorption of the antibody with the immunizing protein (data not shown). Further confirmation of its specificity was determined by immunohistochemistry against 11β-HSD1–/– mouse preadipocytes, as shown in Results (Fig. 2BGo). Anti-11β-HSD1 antibody was used at a concentration of 0.25 µg/ml. A mouse Alexa 488 staining kit (Molecular Probes, Cambridge, UK) was used as secondary staining for both p27kip1 and F4/80. An antisheep IgG coupled to rhodamine or fluorescein (Jackson ImmunoResearch, West Grove, PA) was used at a concentration of 3.75 µg/ml for 11β-HSD1 detection. Mounting media were from ProLong Gold antifade reagent with 4',6-diamidino-2-phenylindole (DAPI) (Invitrogen, Carlsbad, CA). For each experiment, negative controls were used both by staining cells that do not express the target protein and by omitting primary antibody. To ensure that cross-reactivity did not produce artifact, both primary and secondary antibodies were tested for cross-reactivity, with negative results (data not shown). Fluorescence microscopy was carried out on a Leica (Wetzlar, Germany) SP5 laser scanning confocal microscope using a x63 oil immersion objective with excitation at 405 nm (UV diode), 480 nm (argon), and 543 nm (HeNe). Images were acquired and treated with Leica LAS AF software (Wetzlar, Germany).


Figure 2
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  		FIG. 2. Validation of preadipocyte marker (p27kip1) and 11β-HSD1 antibodies. A, 3T3-L1 cells were used as a positive control for cells of the preadipocyte lineage and were identified by an anti-p27kip1 antibody. The control (top row) shows 3T3-L1 cells incubated with secondary antibody conjugated with a green fluorophore only. 3T3-L1 (middle row) and HEK-293-m11β1 (bottom row) cells were incubated with anti-p27kip1 antibodies as described in Materials and Methods. HEK-293-m11β1 cells were negative for p27kip1 staining. B, Stably transfected HEK-293-m11β1 cells were used as a positive control for the anti-11β-HSD1 antibody. The negative control (first row) shows HEK-293-m11β1 cells incubated with secondary antibody only. Similar negative control results were found in untransfected HEK-293 and ASV cells (not shown, see Fig. 3Go for ASV). HEK-293-m11β1 cells (second row), ASV cells isolated from sc adipose tissue of C57BL/6J mice (third row), and ASV cells from sc adipose tissue of 11β-HSD–/– mice (fourth row) were stained with anti-11β-HSD1 and secondary antibody conjugated with red fluorophore as described in Materials and Methods. The far right panel shows a merged image with nuclear DAPI staining.

 
Statistical analysis
Values are expressed as means ± SEM. Data were subjected to one- or two-way ANOVA using SigmaStat (Jandel Corp. San Rafael, CA). Unless otherwise stated, asterisks represent significant differences between depots (*, P < 0.05; **, P < 0.01) and daggers represent a significant difference between culture states within the same depot. Post hoc Student’s t tests were made to further evaluate differences. In all experiments n ≥ 4 was used. For ASV fractions, each n consisted of pooled adipose tissue, sc or mesenteric, from four animals.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
11β-HSD1 is expressed predominantly in ASV preadipocytes
11β-HSD1 is highly expressed in adipose tissue (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) and differentiated adipocytes (6, 16). Before testing for the expression levels and cellular localization of 11β-HSD1 in the ASV fraction, we wished to clarify the efficiency of our adipose tissue fractionation. We did this by Northern analysis of the RNA extracted from the ASV and adipocyte fractions by hybridization to cDNAs encoding pref-1, a specific preadipocyte marker (29), and aP2 (also known as FABP4), a marker for mature adipocytes (30) (Fig. 1Go). Pref-1 mRNA was confined to the ASV and aP2 mRNA to the adipocyte fraction (Fig. 1Go).


Figure 1
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  		FIG. 1. Representative qualitative Northern blot demonstrating the efficiency of ASV and adipocyte fractionation. A representative Northern blot of RNA from ASV and adipocyte (Ad) fractionation from whole adipose tissue showing pref-1 (preadipocyte specific) and aP2 (adipocyte specific) mRNA levels and indicating the efficiency of the fractionation process.

 
We then determined whether the ASV preadipocytes expressed 11β-HSD1 using double-immunofluorescence staining on fASV cells. Our efforts to use a commercially available pref-1 antibody in immunofluorescence experiments were unsuccessful due to the unsuitability of the antibodies for this method. As a result, we opted for the p27kip1 antibody, a marker of cells of the preadipocyte lineage that regulates the fate of the cell cycle in these cells (31, 32). As expected, anti-P27kip1 strongly stained 3T3-L1 fibroblastic preadipocytes (Fig. 2AGo, middle row, green) but not the control (see below) HEK-293-m11β-HSD1 cells (Fig. 2AGo, bottom row). The specificity of the 11β-HSD1 antibody was assessed using HEK-293 cells stably transfected with the mouse 11β-HSD1 (HEK-293-m11β1, a generous gift from Dr. Scott Webster, University of Edinburgh). The anti-11β-HSD1 antibody showed strong immunostaining in the HEK-293-m11β1 cells (Fig. 2BGo, second row, red) as well as in ASV cells from control C57BL/6J mice (Fig. 2BGo, third row, red). As an additional negative control, we stained ASV from 11β-HSD1–/– mice (12), which did not show any staining in the presence of the antibody (Fig. 2BGo, bottom row). DAPI nuclear staining is shown in blue.

P27kip1-positive cells were abundant in mouse ASV fraction (Fig. 3Go, top row, green) together with p27kip1-negative cells, reflecting the heterogeneous cellular nature of the ASV fraction from sc and mesenteric depots (when quantified, P27kip1-positive cells consisted of 43.6 ± 3.8% of the total). Merging the anti-11β-HSD1 signal (Fig. 3Go. middle row, red) with the p27kip1 signal revealed extensive colocalization of 11β-HSD1 and p27kip1 in both mesenteric and sc ASV fractions (Fig. 3Go, bottom row, yellow) and are shown with DAPI nuclear staining (blue). Negative controls without primary antibody (Fig. 3Go, left column) are shown for sc ASV compartment; similar results were found for mesenteric ASV compartment (not shown).


Figure 3
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  		FIG. 3. Colocalization of p27kip1 and 11β-HSD1 in ASV cells of sc and mesenteric adipose tissue. Control panels (first column, far left) show results where the relevant primary antibody has been omitted from staining of a sc ASV preparation and is representative of results in the ASV from both fat depots. The second and third columns (mesenteric ASV cells) represent the staining of cells from the mesenteric ASV fraction with anti-p27kip1 (top row, green) and anti-11β-HSD1 (middle row, red) antibodies and their secondary antibodies conjugated with green and redfluorophore, respectively. Colocalization of staining for both antibodies is shown merged with DAPI nuclear staining (blue) in the bottom row. The fourth and fifth columns (sc ASV cells) represent the same staining profile for cells from the sc depot.

 
Macrophages express 11β-HSD1 mRNA (33, 34), and recent evidence suggests that adipose tissue macrophage levels increase with obesity (35). To determine whether macrophages could account for the ASV 11β-HSD1 expression in our nonobese normal mice, we stained for both the macrophage-specific marker F4/80 and 11β-HSD1 in bone marrow-derived macrophages and ASV cells (Fig. 4AGo). Bone marrow-derived macrophages expressed abundant F4/80 (top left, green) but not 11β-HSD1 (middle left, red) as shown by the almost exclusively green signal from the merged picture (lower left). F4/80 and 11β-HSD1 colocalization in ASV (Fig. 4BGo) showed that the F4/80 signal was restricted predominantly to 11β-HSD1-negative cells (Fig. 4AGo, right, green; one such cell is highlighted with a yellow arrow in both the F4/80 and merged images). Note that HSD1 was detectable in F4/80-positive cells, but at very low levels (Fig. 4AGo). These data agree with our contention that in nonobese mice, 11β-HSD1 activity is not accounted for by macrophages but mainly by preadipocytes. This is likely due to both the low levels of macrophages in the adipose tissue of nonobese mice as shown by the low levels of emr1 mRNA (encoding F4/80) by quantitative real-time PCR compared with obese Lepob animals (5.2-fold, P < 0.001; Fig. 4BGo) and the absence of a proinflammatory environment that might activate the macrophages and induce 11β-HSD1 expression (35).


Figure 4
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  		FIG. 4. Macrophages do not predominantly account for the ASV 11β-HSD1 expression in nonobese mice. A, Bone marrow-derived macrophages (left column) and ASV cells (right column) were stained for the F4/80 macrophage marker with a green fluorescent secondary antibody (top row). A strong cell-specific staining for F4/80 in one representative cell of the ASV fraction is indicated by a long yellow arrow in both the F4/80 only (top, green) and merged images (bottom). Cells were costained for 11β-HSD1 with an anti-11β-HSD1 antibody and a red fluorescent secondary antibody (middle row). Strong cell-specific staining of 11β-HSD1 in one representative cell is indicated by a long white arrow in the 11β-HSD1 only (middle row, red) and merged images (bottom row). Colocalization is indicated in the merged picture image by yellow (bottom row) and shows nuclear DAPI staining. B, Quantitative realt-ime PCR of the macrophage-specific marker emr1 (encodes F4/80) in mesenteric adipose tissue of C57BL/6J and Lepob animals. ***, P < 0.001. A.U., Arbitrary units.

 
11β-HSD1 mRNA and protein levels are higher in sc than in mesenteric ASV fraction and adipocytes
11β-HSD1 is expressed at higher levels in sc compared with mesenteric depots in whole adipose tissue, suggesting that 11β-HSD1 contributes to altered regional fat accumulation in mature adipocytes (9, 12). To investigate whether 11β-HSD1 is expressed differentially in preadipocytes from distinct adipose depots, ASV cells and adipocytes from mesenteric and sc adipose tissue from adult (6 month old) mice were assessed for 11β-HSD1 mRNA and activity. Note that 11β-HSD1 dehydrogenase activity was measured in homogenates in this case as a surrogate marker for 11β-HSD1 protein levels (see Materials and Methods).11β-HSD1 mRNA (Fig. 5AGo) and activity (Fig. 5BGo) were similar in ASV and adipocyte fractions for each depot. 11β-HSD1 mRNA (4-fold, P < 0.05; Fig. 5AGo) and activity (2-fold, P < 0.05; Fig. 5BGo) levels were higher in sc compared with mesenteric ASV cells. However, in adipocytes, although higher 11β-HSD1 activity (2-fold, P < 0.05; Fig. 5BGo) was found in the sc compared with mesenteric depots, the difference in 11β-HSD1 mRNA did not reach statistical significance (1.8-fold, P = 0.08; Fig. 5AGo). Higher sc than mesenteric ASV 11β-HSD1 mRNA levels were also found in 2-month-old mice (data not shown), so the differences are not merely a reflection of aging.


Figure 5
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  		FIG. 5. Quantification of 11β-HSD1 mRNA and activity (protein equivalent) levels in sc and mesenteric ASV and adipocyte fractions. A and B, Quantitative real-time PCR levels (A) and dehydrogenase activity levels(B) (equivalent to protein levels in the linear range of product formation as described in Materials and Methods) of 11β-HSD1 in homogenates of ASV cells and adipocytes from mesenteric (black bars) and sc (white bars) fat depots. Levels of 11β-HSD1 mRNA were normalized to TBP mRNA levels as described in Materials and Methods. Dehydrogenase activity (as an indicator of protein levels) measured in homogenates is expressed as nanomoles of corticosterone (B) metabolized into 11-dehydrocorticosterone (A) per milligram of protein per hour. *, P < 0.05. Data are from four separate experiments (n = 4).

 
Preadipocyte 11β-HSD1 is exclusively keto-reductase with dissociation from mRNA and protein levels in mesenteric ASV cells
Although 11β-dehydrogenase activity (a reflection of the protein level) was readily detectable in ASV homogenates, closely paralleling 11β-HSD1 mRNA levels, the reaction direction was predominantly 11β-reductase in intact fASV cells from mesenteric and sc depots (Fig. 6AGo). Low levels of dehydrogenase activity were observed in intact fASV cells (Fig. 6AGo). We note that endothelial cells, a potential ASV cell contaminant, express 11β-HSD2 but not 11β-HSD1 (36) and could be responsible for the low levels of 11β-dehydrogenase observed in the fASV cells. However, the endothelial cell marker tie2 mRNA was barely detectable by RT-PCR when compared with the positive control human umbilical vein endothelial cells (data not shown). Alternatively, broken or damaged cells may account for dehydrogenase activity because the oxidative environment usually reveals 11β-dehydrogenase activity not seen in intact cells (37, 38). Cell viability after collagenase digestion was 90.4 ± 0.3% of total fASV cell number.


Figure 6
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  		FIG. 6. Quantification of 11β-HSD1 reductase activity and H6PDH mRNA levels in sc and mesenteric ASV. A, 11β-HSD1 reductase and dehydrogenase activity levels in freshly isolated mesenteric (mes, black bars) and sc (white bars) ASV cells. *, Significant difference in reductase activity between cells from different depots (P < 0.05; n = 5); {dagger}, significant difference between reductase and dehydrogenase levels within each depot (P < 0.05; n = 5). B, Quantitative real-time PCR determination of H6PDH mRNA levels in mesenteric (black bar) and sc (white bar) fASV cells. Data were normalized to TBP mRNA.

 
Surprisingly, mesenteric fASV cells had more 11β-reductase activity than intact fASV sc cells (Fig. 6AGo) despite the lower mRNA and dehydrogenase activity (protein) levels observed in homogenates (Fig. 5Go). The predominant reductase activity of 11β-HSD1 in mesenteric or sc fASV cells was consistent with abundant expression of H6PDH mRNA, which generates reduced nicotinamide adenine dinucleotide phosphate to drive the oxidoreductase direction (17, 22), in fASV cells from both depots (Fig. 6BGo).

Cell culture might alter 11β-HSD1 expression and/or reaction direction, and indeed 11β-HSD1 is induced during preadipocyte differentiation to the mature adipocyte-like phenotype in clonal preadipocyte cell lines 3T3-F442A and 3T3-L1 (15, 16). Therefore, we compared 11β-HSD1 mRNA levels in fASV and in primary cultured cells (6 d, cASV), as well as in undifferentiated 3T3-F442A preadipocytes (u3T3) (Fig. 7Go). 11β-HSD1 mRNA levels were markedly higher in both fASV and cASV primary cells than in undifferentiated 3T3-F442A preadipocytes (30-fold; undifferentiated 3T3-F442A vs. sc cASV, P > 0.001; Fig. 7AGo). Interestingly, after a 6-d culture of sc, but not mesenteric, ASV cells, 11β-HSD1 mRNA levels were markedly reduced (5-fold, P < 0.01; Fig. 7AGo). Notably, the decrease in 11β-HSD1 mRNA upon culture in sc cASV paralleled a loss in Pref-1 mRNA expression (5-fold, P = 0.008, Fig. 7BGo). There was no morphological evidence of spontaneous differentiation of our cASV into adipocytes, and aP2 mRNA levels did not differ between fASV and cASV (data not shown).


Figure 7
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  		FIG. 7. Effect of in vitro culture on ASV cell 11β-HSD1 and Pref-1 mRNA levels. A and B, Quantitative real-time PCR determination of 11β-HSD1 (A) and pref-1 (B) mRNA levels in fASV and 6-d cASV cells from mesenteric (black bars) and sc (white bars) fat depots. Undifferentiated 3T3-F442A (u3T3) cells were included as a control. §, P < 0.001, a significantly lower 11β-HSD1 mRNA level in the undifferentiated 3T3-F442A preadipocytes (u3T3) compared with sc cASV; *, P < 0.05; and **, P < 0.01, a significant difference for 11β-HSD1 or pref1 mRNA levels between depots (mesenteric vs. sc fASV or cASV); {dagger}, P < 0.05; and {dagger}{dagger}, P < 0.01, a significant difference for 11β-HSD1 or pref1 mRNA levels between fresh or cultured cells (fASV vs. cASV) from the same depot of origin. n = 5.

 
ASV 11β-HSD1 expression influences depot-specific differentiation in vivo
Our results suggested that 11β-HSD1 acts predominantly as an 11β-reductase and is highly expressed in ASV preadipocytes in normal mice. To address this in vivo, we exposed 11β-HSD1–/– and control C57BL/6J mice to HF feeding and assessed preadipocyte differentiation using Pref-1 mRNA diminution as a surrogate marker for this process (29). In mesenteric adipose tissue, C57BL/6J but not 11β-HSD1–/– mice showed a significant reduction of pref-1 mRNA after prolonged HF feeding (2.7-fold, P = 0.033; Fig. 8AGo), suggesting greater preadipocyte differentiation in control mice in this depot. In sc adipose tissue, HF feeding did not significantly reduce Pref-1 levels in either genotype (11β-HSD1–/–, 1.6-fold, P = 0.056; control, 1.3-fold, P = 0.08; Fig. 8BGo).


Figure 8
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  		FIG. 8. The effects of HF feeding on preadipocyte differentiation in C57BL/6J and 11β-HSD1–/– mice in vivo. Quantitative real-time PCR of Pref-1 mRNA levels in mesenteric (A) and sc (B) adipose tissue of chow-fed and HF-fed (18 wk) C57BL/6J (black bars) or 11β-HSD1–/– (white bars) mice. *, P < 0.05 (n = 4), significant effect of HF feeding in C57BL/6J mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
GCs have prodifferentiation effects on preadipocytes (24). Our results show that 11β-HSD1 is highly expressed in mouse ASV preadipocytes at levels comparable to those in adipocytes. 11β-HSD1 in mouse ASV preadipocytes is a keto-reductase (regenerating active GCs) and consistent with a level of H6PDH expression in ASV that is not limiting for this reaction direction. Despite lower 11β-HSD1 mRNA expression in mesenteric than sc preadipocytes, 11β-HSD1 keto-reductase activity is higher in intact mesenteric ASV cells. A HF diet stimulated preadipocyte differentiation in vivo selectively in mesenteric adipose depots in control mice. This effect was lost in 11β-HSD1–/– mice, consistent with HF-diet-induced mesenteric adipose expansion in control, but not in 11βHSD1–/–, mice (12). These data contrast with reports of 11β-dehydrogenase predominance in human preadipocytes and suggest a role for 11β-HSD1-generated GCs in murine preadipocyte differentiation.

To date, most studies have assumed that 11β-HSD1 in the ASV fraction comes from preadipocytes (19, 20, 21, 37). In our mice, preadipocytes formed about 43% of total cell number in the ASV fraction, in line with the literature (40, 41). In this study, we have shown that preadipocyte markers colocalize with 11β-HSD1 in ASV cells from both sc and mesenteric depots. Likewise, pref-1 mRNA levels were regulated by culture in a pattern that closely parallels the profile of 11β-HSD1 mRNA. Interestingly, we also found that only a minority of macrophages in nonobese mice express 11β-HSD1 mRNA. These are probably activated macrophages (33, 34) that are more likely to predominate in adipose tissue of obese mice, such as Lepob mice (35).

11β-HSD1 mRNA levels and homogenate dehydrogenase activity were higher in sc than mesenteric ASV cells, consistent with the fat depot-selective expression pattern of 11β-HSD1 in mice (i.e. higher in sc than mesenteric tissue) (12). 11β-HSD1 activity in intact fASV cells from both sc and mesenteric fat depots was predominantly 11β-reductase, consistent with the view that 11β-HSD1 regenerates GC in vivo in preadipocytes, as in adipocytes. These results differ from a previous report where predominant 11β-dehydrogenation was found in human omental cASV cells, a feature ascribed to the low levels of H6PDH in these cells (20). In contrast, human sc ASV cells are keto-reductive due to the presence of H6PDH expression (17, 20, 22). Mouse ASV cells express H6PDH in both depots, consistent with keto-reductase activity. Despite the predominance of keto-reductase activity, 11β-dehydrogenase was observed at low levels. This could be due to vasculature, because the endothelium expresses the potent dehydrogenase 11β-HSD2. However, there were low levels of endothelial cells (as defined by Tie2 endothelial cell marker expression, data not shown) in the ASV fraction, and this is unlikely to account for the modest levels of 11β-dehydrogenase present in fASV. Alternatively, ruptured cells may account for 11β-dehydrogenase activity observed in cultures, because this leads to 11β-dehydrogenase activity (28, 37, 38) and about 10% ruptured cells were found in the preparations after collagenase digestion.

Unexpectedly, a disproportionately higher level of GC reactivation was found in the intact mesenteric ASV than in the sc ASV fraction. These data suggest that an additional level of control of preadipocyte 11β-HSD1 activity exists that is dependent upon an intact plasma membrane. Steroids, including GCs, are thought to be actively transported into and from cells (39, 40, 41, 42). Therefore, the discrepancy observed between 11β-HSD1 activity in intact cells and homogenates may be due to another level of regulation of the protein activity and/or access of substrate. More studies are required to address this mechanism.

11β-HSD1 expression in undifferentiated 3T3-F442A (15, 16) suggested that 11β-HSD1 was a late-stage differentiation marker in mice adipocytes. However, 3T3-F442A had low levels of 11β-HSD1 when compared with mouse primary preadipocytes. In light of these results, we contend that these clonal cell lines do not reflect the true nature of mouse primary preadipocytes, at least as far as 11β-HSD1 expression is concerned (15, 16).

GCs promote preadipocyte differentiation in NIH/3T3 and primary cell cultures (24, 43). GC regeneration by 11β-HSD1 increases the differentiation potential of human preadipocytes in vitro (25). Furthermore, 11β-HSD1 affects regional fat distribution (12), promoting mesenteric over peripheral adipose depot expansion, thought due to adipocyte hypertrophy (9).

HF-fed C57BL/6J mice showed higher ASV differentiation in the mesenteric depot than in 11β-HSD1–/– mice, suggesting that this effect may account in part for the altered fat redistribution between these genotypes (12). Thus, 11β-HSD1 deficiency not only exerts beneficial effects on adipocyte biology (12, 27) but also attenuates preadipocyte differentiation stimulated by dietary challenge selectively in visceral (mesenteric) adipose tissue.


   Acknowledgments
 
We thank Dr. Scott Webster for kindly providing the HEK-11βHSD1 cells and the sheep anti-11β-HSD1 antibody.


   Footnotes
 
R.A.D.S.P. was funded by a Wellcome Trust Prize Doctoral Studentship, N.M.M. was holder of a Wellcome Trust Intermediate Fellowship and RCDF fellowships, and K.E.C. and J.R.S. were funded by a Wellcome Program grant.

Disclosure Statement: The authors have nothing to declare.

First Published Online January 3, 2008

Abbreviations: ASV, Adipose stromal vascular; cASV, cultured ASV; DAPI, 4',6-diamidino-2-phenylindole; fASV, freshly isolated ASV; GC, glucocorticoid; HF, high fat; H6PDH, hexose-6-phosphate dehydrogenase; 11β-HSD1, 11β-hydroxysteroid dehydrogenase type 1; TBP, TATA-binding protein.

Received July 26, 2007.

Accepted for publication December 26, 2007.


   References
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 Introduction
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 Results
 Discussion
 References
 

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