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Endocrinology, doi:10.1210/en.2007-0349
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Endocrinology Vol. 148, No. 11 5268-5277
Copyright © 2007 by The Endocrine Society

Ceramide and Adenosine 5'-Monophosphate-Activated Protein Kinase Are Two Novel Regulators of 11β-Hydroxysteroid Dehydrogenase Type 1 Expression and Activity in Cultured Preadipocytes

N. Arai, H. Masuzaki, T. Tanaka, T. Ishii, S. Yasue, N. Kobayashi, T. Tomita, M. Noguchi, T. Kusakabe, J. Fujikura, K. Ebihara, M. Hirata, K. Hosoda, T. Hayashi, H. Sawai, Y. Minokoshi and K. Nakao

Division of Endocrinology and Metabolism (N.A., H.M., T.Ta., T.I., S.Y., N.K., T.To., M.N., T.K., J.F., K.E., M.H., K.H., K.N.), Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto 606-8507, Japan; Department of Human Coexistence (T.H.), Kyoto University Graduate School of Human and Environmental Studies, Kyoto 606-8501, Japan; Department of Internal Medicine (H.S.), Osaka Dental University, Osaka 573-1121, Japan; and Department of Developmental Physiology (Y.M.), National Institute for Physiological Science, Aichi 444-8585, Japan

Address all correspondence and requests for reprints to: Hiroaki Masuzaki, M.D., Ph.D., Division of Endocrinology and Metabolism, Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, 54, Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. E-mail: hiroaki{at}kuhp.kyoto-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Increased activity of intracellular glucocorticoid reactivating enzyme, 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in obese adipose tissue contributes to adipose dysfunction. As recent studies have highlighted a potential role of preadipocytes in adipose dysfunction, we tested the hypothesis that a variety of metabolic stress mediated by ceramide or AMP-activated protein kinase (AMPK) would regulate 11β-HSD1 in preadipocytes. The present study is the first to show that 1) expression of 11β-HSD1 in 3T3-L1 preadipocytes was robustly induced when cells were treated with cell-permeable ceramide analogue C2 ceramide, bacterial sphingomyelinase, and sphingosine 1-phosphate, 2) 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR)-induced activation of AMPK augmented the expression and enzyme activity of 11β-HSD1, and 3) these results were reproduced in human preadipocytes. We demonstrate for the first time that C2 ceramide and AICAR markedly induced the expression of CCAAT/enhancer-binding protein (C/EBP) β and its binding to 11β-HSD1 promoter. Transient knockdown of C/EBPβ protein by small interfering RNA markedly attenuated the expression of 11β-HSD1 induced by C2 ceramide or AICAR. The present study provides novel evidence that ceramide- and AMPK-mediated signaling pathways augment the expression and activity of 11β-HSD1 in preadipocytes by way of C/EBPβ, thereby highlighting a novel, metabolic stress-related regulation of 11β-HSD1 in a cell-specific manner.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
METABOLIC SYNDROME IS characterized by a cluster of glucose intolerance, hypertension, and dyslipidemia on a basis of insulin resistance and excess in intraabdominal fat accumulation (1, 2, 3). Functional abnormalities of adipose tissue have been implicated in the pathophysiology of metabolic syndrome (2). A series of transgenic and knockout experiments in mouse models suggest that exaggerated reactivation of glucocorticoid in adipose tissue, mediated by enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), contributes to dysfunction of adipose tissue (3, 4, 5, 6, 7). 11β-HSD1 is a bidirectional (oxo-reductase and dehydrogenase) enzyme (8), expressing abundantly in adipose tissue, liver, and central nervous system (9, 10). Notably, 11β-HSD1 mainly acts as an oxo-reductase in vivo and reactivates inactive cortisone into active cortisol (8). Transgenic mice overexpressing 11β-HSD1 in adipose tissue exemplified major phenotype of metabolic syndrome (3, 4), whereas systemic 11β-HSD1 knockout mice were protected against diabetes and dyslipidemia on a high-fat diet (5, 6, 7). These data suggest that an increased level of adipose 11β-HSD1 considerably contributes to metabolic derangement. Consistent with this notion, selective 11β-HSD1 inhibitors are shown to ameliorate diabetes, dyslipidemia, and arteriosclerosis in experimental murine models (11, 12).

Obese adipose tissue is subjected to multiple cellular stresses such as endoplasmic reticulum stress and oxidative stress (13). Local hypoxia, tissue dysnutrition, and resultant cell death are potentially linked to macrophage recruitment and local inflammation in adipose tissue (13, 14). Recent studies also highlight a complexity of preadipocytes in controlling adipose tissue function (15, 16). Nevertheless mature adipocytes are the major component of adipose tissue and predominant source of 11β-HSD1 (10, 17); a considerable amount of 11β-HSD1 expression is also detected in stromal-vascular cells from adipose tissue (17). The underlying mechanism whereby 11β-HSD1 is elevated in obese adipose tissue still remains obscure, and the regulation of 11β-HSD1 in preadipocytes has been poorly understood.

In this context, we hypothesized that a variety of metabolic stresses would regulate the expression of 11β-HSD1 in preadipocytes. The sphingolipid ceramide serves as a bioactive lipid mediator in response to a variety of metabolic stresses including inflammation and oxidative stress (18). Ceramide is generated by de novo synthesis as well as hydrolysis of membrane sphingomyelin by sphingomyelinase (SMase) (18, 19). Ceramide and its metabolites, sphingosine and sphingosine 1-phosphate (S1P), mediate a variety of biological events such as apoptosis, cell growth, and the stress response (18, 19). In contrast, AMP-activated protein kinase (AMPK) is another mediator of metabolic stress that responds to the negative energy balance within cells (20, 21, 22). AMPK is activated by stresses that increase intracellular AMP level such as local hypoxia, glucose deprivation, and ischemia (21). Whereas the function of AMPK in liver and muscle has been well investigated, its role in adipose tissue still remains obscure (23, 24). In the present study, using murine 3T3-L1 preadipocytes and human preadipocytes, we investigated the effect of ceramide- and AMPK-mediated signaling pathways on the expression of 11β-HSD1.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and chemicals
5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) was obtained from Toronto Research Chemicals (Toronto, Canada). A selective AMPK inhibitor, Compound C, and C2 ceramide (N-acetyl-sphingosine) were purchased from Carbiochem (San Diego, CA). Bacterial SMase and S1P were from Sigma Chemicals (St. Louis, MO). Antibodies against phospho (Ser 79) acetyl-CoA carboxylase (ACC), CCAAT/enhancer-binding protein (C/EBP) β, and β-actin were from Upstate Biotechnology (Lake Placid, NY), Affinity BioReagents Inc. (Golden, CO), and Sigma Chemicals.

Cell culture and treatment
3T3-L1 fibrobrasts (kindly provided from Dr. H. Green and Dr. M. Morikawa, Harvard Medical School, Boston, MA) were maintained in DMEM containing 10% (vol/vol) calf serum at 37 C in 10% CO2. Human perirenal preadipocytes were purchased from Cambrex (Walkersville, MD) (25, 26) and maintained according to the manufacturer’s instructions. For differentiation of 3T3-L1 preadipocytes into mature adipocytes, cells (2 d postconfluence) were incubated with DMEM containing 10% (vol/vol) fetal bovine serum, 0.5 mM 3-isobutyl-1-methylxanthine, 0.25 µM dexamethasone, and 1 µg/ml insulin for 2 d, followed by another 2-d incubation with DMEM containing FBS and insulin (27). Additional incubation with DMEM containing FBS for 4 d completes the differentiation. Compound C and C2 ceramide were dissolved in DMSO and added to the media within 0.1% of volume. S1P was dissolved in water containing 0.4% BSA and added to the media within 1.0% of volume.

RNA preparation and quantitative real-time PCR
Total RNA was extracted from cultured cells using TRIzol Reagent (Invitrogen, Carlsbad, CA), and cDNA was then synthesized using iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. To determine the mRNA levels, probes and primers were employed as follows: probe (5'FAM-tccgagttcaaggcagcgagacactacc-TAMRA-3'), forward (5'-ccaggtcggaggaaggtctc-3'), and reverse (5'-ccagcaatgtagtgagcagagg-3') for murine 11β-HSD1; probe (5'FAM-ccccactcacctgctgctactcattca-TAMRA-3'), forward (5'-ttggctcagccagatgca-3'), and reverse (5'-ccagcctactcattgggatca-3') for murine monocyte chemoattractant protein-1 (MCP-1); probe (5'FAM-acattgtcagcctcgcagaatccatactg-TAMRA-3'), forward (5'-atgcgacccaccctgtgac-3'), and reverse (5'-gaccctcccagccaacatg-3') for murine preadipocyte factor-1 (Pref-1); and probe (5'-cattgttgtcgtctcctctctggctggg-3'), forward (5'-ttgcccatgctgaagcagag-3'), and reverse (5'-gcaaccattggataagccactt-3') for human 11β-HSD1. TaqMan PCR was performed using ABI Prism 7700 Sequence Detection System as instructed by the manufacturer (Applied Biosystems, Foster City, CA). Each value of mRNA level was normalized to that of 18S rRNA.

Western blot analysis
Cells were washed twice with ice-cold PBS, and harvested in lysis buffer [40 mM HEPES, 10 mM EDTA, 100 mM NaF, 10 mM sodium pyrophosphate, 1 mM Na3VO4, 0.1 mg/ml aprotinin, 1 mM PMSF, 50 nM okadaic acid, and 1% (vol/vol) Nonidet P-40 at pH 7.5]. For analysis of C/EBPβ, cells were harvested in lysis buffer [1% (wt/vol) SDS, 60 mM Tris-HCl, 1 mM Na3VO4, 0.1 mg/ml aprotinin, 1 mM PMSF and 50 nM okadaic acid at pH 6.8], and boiled at 100 C for 10 min. After centrifugation, supernatants were normalized for protein concentration via Bradford method and then equal amounts of protein were subjected to SDS-PAGE and immunoblot.

Measurement of AMPK activity
Activation of AMPK is assessed by the immunoblot of Thr172-phosphorylated AMPK{alpha}. Antibodies against AMPK{alpha} and phosphorylated (Thr172) AMPK (Cell Signaling Technology, Beverly, MA) can detect both {alpha}1 and {alpha}2 isoform of the catalytic subunit (28). In 3T3-L1 adipocytes, AMPK activity is largely attributable to the {alpha}1 isoform (29), and both {alpha}1 and {alpha}2 catalytic subunit isoforms are activated by AICAR (29). The phosphorylation at Thr172 parallels the degree of AMPK activation, thus making it possible to estimate the activity of both isoform complexes (28).

Measurement of 11β-HSD1 activity
Assays for 11β-HSD1 activity were performed by incubating viable cells with 250 nM corticosteroids with appropriate tritium-labeled tracer. In assays for oxo-reductase activity, cells were incubated in serum-free DMEM containing 250 nM cortisone that includes 6.4 µCi/ml tritium-labeled tracer [1,2-3H]2 cortisone (Muromachi Yakuhin LTD, Kyoto, Japan). In assays for dehydrogenase activity, cells were incubated in serum-free DMEM containing 250 nM cortisol with 6.4 µCi/ml tritium-labeled tracer [1,2,6,7-3H]4 cortisol (Muromachi Yakuhin LTD). After the incubation at 37 C for indicated time, corticosteroids were extracted by ethyl acetate, separated by thin-layer chromatography in chloroform:methanol (95:5), and quantified by autoradiography. As a control (indicated as ref.), serum-free DMEM with tritium-labeled cortisone or cortisol was incubated without cells.

Chromatin immunoprecipitation (ChIP) analysis
ChIP analysis was performed using an assay kit (Upstate Biotechnology) according to the manufacturer’s protocol. Anti-C/EBPβ antibody used for immunoprecipitation is from Santa Cruz Biotechnology (Santa Cruz, CA). Forward primer (5'-ctggaagttgcctcttactc-3') and reverse primer (5'-cctgtaggacacacgaagaa-3') were used to amplify the DNA fragment between –170 and +71 (relative to the transcription start site) of mouse 11β-HSD1 genomic DNA, which contains two putative C/EBP binding sites (30).

RNA interference (RNAi)
Two Stealth RNAi for mouse C/EBPβ were obtained (Invitrogen). Each small interfering RNA (siRNA), termed RNAi154 and RNAi168, was designed as a 25-bp duplex oligoribonucleotide with a sense strand corresponding to nucleotides 154–173 or 168–192 of the reported mouse C/EBPβ coding sequence (GenBank accession no. NM009883), respectively. The Stealth RNAi negative control duplex (Invitrogen) was used as a control oligoribonucleotide. According to the manufacturer’s protocol, 3T3-L1 preadipocytes were transfected with 10 nM siRNA in antibiotic-free medium using Lipofectamine RNAiMAX (Invitrogen). The transfected cells were cultured overnight and applied to the experiments.

Statistical analysis
The data are presented as means ± SEM from independent three or four experiments. Student’s t test was used to compare the data when the variances of two groups were regarded equal (within 5%). When variances of two groups were apparently different, Welch’s t test was used. Differences were accepted as significant at P < 0.05 level.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
When 3T3-L1 preadipocytes were differentiated into mature adipocytes, the level of 11β-HSD1 mRNA was increased by 150-fold (Fig. 1Go). Based on this result, we first analyzed the effect of ceramide signaling on the expression of 11β-HSD1 in both 3T3-L1 preadipocytes and differentiated adipocytes.


Figure 1
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  		FIG. 1. Expression profile of 11β-HSD1 mRNA during the course of differentiation in 3T3-L1 cells. Postconfluent 3T3-L1 preadipocytes were induced to differentiate as described in Materials and Methods. mRNA level of 11β-HSD1 was determined. Results are means ± SEM from three experiments. **, P < 0.01 compared with 2 d postconfluent preadipocytes (initial).

 
Effect of ceramide on the expression of 11β-HSD1 in 3T3-L1 preadipocytes
When 3T3-L1 preadipocytes were treated with a short chain ceramide analog, C2 ceramide (10–100 µM), for 24 h, mRNA of Pref-1, which is known to express abundantly in preadipocytes (31, 32), was not altered (Fig. 2AGo). In contrast, expression of MCP-1 and 11β-HSD1 was induced dose-dependently, reaching to 2.0 ± 0.1-fold (P < 0.01) and 2.2 ± 0.1-fold (P < 0.01), respectively (Fig. 2Go, B and C). To evaluate the effect of C2 ceramide on the 11β-HSD1 enzyme activity, cells were incubated with tritium-labeled cortisone or cortisol. C2 ceramide significantly increased the conversion of cortisone to cortisol by 1.7 ± 0.1-fold (P < 0.05) (Fig. 2DGo, left). This indicates that 11β-HSD1 oxo-reductase activity was increased in response to C2 ceramide. In contrast, 11β-HSD1 dehydrogenase activity was neither detected nor induced during a 24-h incubation period (Fig. 2DGo, right). These results indicate that 11β-HSD1 in 3T3-L1 preadipocytes acts solely as an oxo-reductase.


Figure 2
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  		FIG. 2. Ceramide induces the expression of 11β-HSD1 in 3T3-L1 preadipocytes. A–D, Cells were treated with C2 ceramide (10–100 µM) for 24 h. mRNA level of Pref-1 (A), MCP-1 (B), and 11β-HSD1 (C). D, Assay for 11β-HSD1 enzyme activity. Cells treated with C2 ceramide (100 µM) for 24 h were subsequently incubated in serum-free media containing 250 nM of cortisone or cortisol with tritium-labeled tracer (6.4 µCi/ml of [1,2-3H]2 cortisone or [1,2,6,7-3H]4 cortisol) for 12 h. Emerged spots of cortisol are indicated by arrows. E and F, mRNA level of 11β-HSD1 after treatment with bacterial SMase (25–200 mU/ml) (E) and S1P (1.0–10 µM) (F) for 24 h. Results are means ± SEM from three experiments. *, P < 0.05, **, P < 0.01 compared with vehicle-treated group. U.D., Under detectable.

 
Hydrolysis of membrane sphingomyelin by SMase provoke intracellular accumulation of ceramide (19). Ceramide is subsequently metabolized to sphingosine and S1P, both of which mediate biological events including cell growth and apoptosis (19). We thus treated 3T3-L1 preadipocytes with bacterial SMase and S1P. When cells were treated with bacterial SMase (25–200 mU/ml) and S1P (1.0–10 µM), 11β-HSD1 mRNA level was significantly induced, reaching to 1.7 ± 0.2-fold (P < 0.05) and 3.0 ± 0.5-fold (P < 0.05), respectively (Fig. 2Go, E and F). To further investigate whether ceramide signaling would be involved in the induction of 11β-HSD1 by inflammatory cytokines, inhibitors of ceramide [fumonisin B1, an inhibitor of ceramide synthase (33); myriocin, an inhibitor of serine palmitoyltransferase (34); desipramine, an inhibitor of SMase (35)] were cotreated with 3T3-L1 preadipocytes. Desipramine (10 and 20 µM) dose-dependently attenuated the expression of 11β-HSD1 mRNA induced by TNF{alpha} or IL-1β [46 ± 10% (P = 0.07) and 41 ± 2% (P < 0.01), respectively]. This result indicates that induction of 11β-HSD1 by TNF{alpha} or IL-1β, at least in part, is attributable to the activation of SMase. Such an effect was not observed when treated with fumonisin B1 or myriocin (data not shown). These results raise a possibility that ceramide-mediated signaling pathway is involved in the regulation of 11β-HSD1 in preadipocytes.

Effect of ceramide on the expression of 11β-HSD1 in 3T3-L1 differentiated adipocytes
Induction of 11β-HSD1 mRNA by C2 ceramide (10–100 µM) was not observed in 3T3-L1 differentiated adipocytes during 24-h incubation periods (Fig. 3AGo). Similarly, SMase (25–200 mU/ml) and S1P (1.0–10 µM) did not induce the expression of 11β-HSD1 (Fig. 3Go, B and C). These results are in agreement with our data that a robust induction of 11β-HSD1 expression by TNF{alpha} and IL-1β was observed only in 3T3-L1 preadipocytes but not in differentiated adipocytes (data not shown). Altogether, our data suggest that the effect of C2 ceramide, SMase, and S1P on 11β-HSD1 expression is restricted in preadipocytes.


Figure 3
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  		FIG. 3. Ceramide does not influence the expression of 11β-HSD1 in differentiated 3T3-L1 adipocytes. mRNA level of 11β-HSD1 after treatment with C2 ceramide (10–100 µM) (A), SMase (25–200 mU/ml) (B), and S1P (1.0–10 µM) (C) for 24 h. Results are means ± SEM from three independent experiments.

 
Effect of AICAR on the expression of 11β-HSD1 in 3T3-L1 preadipocytes
AMPK is activated by cellular stresses that interfere with ATP production (20, 21, 22). In contrast, 11β-HSD1 is a NADPH-dependent enzyme and its oxo-reductase activity requires NADPH supply from a glucose metabolizing pathway (36). This scheme tempted us to speculate that 11β-HSD1 would be involved in an AMPK-mediated fuel sensing mechanism. To test this hypothesis, we examined the effect of AMPK on the expression of 11β-HSD1 using a cell permeable AMP analog, AICAR (37).

When 3T3-L1 preadipocytes were treated with AICAR (0.1–0.5 mM) for 24 h, AMPK phosphorylation was increased pronouncedly (Fig. 4AGo). MCP-1 mRNA level was not altered even after the treatment with AICAR (Fig. 4BGo). In contrast, 11β-HSD1 mRNA level was increased in a dose-dependent manner, reaching to 8.7 ± 0.8-fold (P < 0.05) (Fig. 4CGo). Oxo-reductase activity of 11β-HSD1 was concomitantly increased by 3.0 ± 0.2-fold (P < 0.01) after the treatment with 0.5 mM AICAR (Fig. 4DGo).


Figure 4
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  		FIG. 4. AMPK activation enhances the expression of 11β-HSD1 in 3T3-L1 preadipocytes. Cells were treated with AICAR (0.1–0.5 mM) for 24 h. A, Western blot of AMPK protein and phosphorylated AMPK (p-AMPK). mRNA levels of MCP-1 (B) and 11β-HSD1 (C). D, Assay for 11β-HSD1 activity. Cells treated with AICAR (0.5 mM) for 24 h were incubated in serum-free media containing 250 nM of cortisone or cortisol with tritium-labeled tracer (6.4 µCi/ml of [1,2-3H]2 cortisone or [1,2,6,7-3H]4 cortisol) for 12 h. Emerged spots of cortisol were indicated by arrows. Results are means ± SEM from three or four experiments. *, P < 0.05, **, P < 0.01 compared with control (cells without AICAR treatment) group. U.D., Under detectable.

 
To further evaluate the involvement of AMPK in the regulation of 11β-HSD1, cells were cotreated with AICAR and a selective AMPK inhibitor, compound C (38). Western blot analyses showed that augmented phosphorylation of AMPK and ACC by the treatment with 0.5 mM AICAR were attenuated when cells were cotreated with 10 µM compound C (Fig. 5AGo). Compound C completely blocked the effect of 0.5 mM AICAR on its augmentation of 11β-HSD1 (80 ± 2.4% decrease compared with AICAR-treated group, P < 0.01) (Fig. 5BGo). These results indicate that augmented expression of 11β-HSD1 by AICAR largely is attributable to the activation of AMPK. In contrast, mRNA of glucocorticoid receptor, which is known to express considerably in preadipocytes (39), was not affected by AICAR or compound C (data not shown).


Figure 5
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  		FIG. 5. AICAR-induced expression of 11β-HSD1 is attenuated by an AMPK inhibitor. 3T3-L1 preadipocytes were cotreated with AICAR (0.5 mM) and indicated concentrations of compound C (Comp. C) for 24 h. A, Western blot of total AMPK protein, phosphorylated AMPK (p-AMPK) and phosphorylated ACC (p-ACC). B, mRNA level of 11β-HSD1. Results are means ± SEM from three experiments. **, P < 0.01 compared with vehicle-treated group. {dagger}{dagger}, P < 0.01 compared with AICAR-treated group.

 
Effects of ceramide and AMPK signaling on the expression of 11β-HSD1 in human preadipocytes
To explore whether potent effects of ceramide and AMPK on 11β-HSD1 expression are reproduced in human preadipocytes (25, 26), impact of C2 ceramide, S1P, and AICAR on 11β-HSD1 expression and enzyme activity were similarly analyzed. When cells were treated with C2 ceramide (25–100 µM) for 24 h, 11β-HSD1 mRNA level was increased dose dependently, reaching to 5.3 ± 0.3-fold (P < 0.01) (Fig. 6AGo). In contrast, the effect of S1P (2.5–10 µM) was marginal (Fig. 6AGo). AICAR (0.3–1.0 mM) induced the phosphorylation of AMPK (Fig. 6BGo) and augmented 11β-HSD1 mRNA level by 2.8 ± 0.1-fold (P < 0.01) (Fig. 6AGo). C2 ceramide (50 µM) and AICAR (1.0 mM) significantly increased the oxo-reductase activity of 11β-HSD1 by 2.9 ± 0.3-fold (P < 0.01) and 2.1 ± 0.04-fold (P < 0.05) (Fig. 6CGo).


Figure 6
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  		FIG. 6. Ceramide and AMPK signaling induces the expression of 11β-HSD1 in human preadipocytes. Human preadipocytes were treated with C2 ceramide (C2cer, 25–100 µM), S1P (2.5–10 µM) and AICAR (0.3–1.0 mM) for 24 h. A, mRNA levels of 11β-HSD1 were determined. B, Western blot of AMPK protein and phosphorylated AMPK (p-AMPK) after treatment with AICAR for 3 h. C, Assay for 11β-HSD1 oxo-reductase activity. Cells were treated with C2 ceramide (50 µM, left) or AICAR (1.0 mM, right) for 24 h, and subsequently incubated with tritium-labeled cortisone for 12 h. Results are means ± SEM from three experiments. *, P < 0.05, **, P < 0.01 compared with control or vehicle-treated group.

 
Induction of C/EBPβ by ceramide and AMPK signaling in 3T3-L1 preadipocytes
11β-HSD1 promoter contains a couple of C/EBP binding sites, and C/EBP mediated regulation of 11β-HSD1 has long been investigated exclusively in hepatocytes (40). Because C/EBP{alpha} is not expressed in preadipocytes (41), we tested a possibility that C/EBPβ would be involved in the regulation of 11β-HSD1 by ceramide or AICAR. Western blot analysis showed that C/EBPβ expression was substantially increased after the treatment with C2 ceramide (0.1 mM) or AICAR (0.5 mM), culminating in 1.8- and 6.5-fold increase within 3 h, respectively (Fig. 7Go, A and B). Expression of C/EBPβ by C2 ceramide or AICAR was increased in a dose-dependent manner (data not shown). In contrast, 11β-HSD1 expression was induced 12 h after the treatment with C2 ceramide or AICAR (data not shown).


Figure 7
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  		FIG. 7. Ceramide and AICAR induce the expression of C/EBPβ in 3T3-L1 preadipocytes. Western blot of C/EBPβ after the treatment with 0.1 mM C2 ceramide (A) or 0.5 mM AICAR (B). β-Actin was used as a loading control. ChIP analysis after the treatment with 0.1 mM C2 ceramide or 0.5 mM AICAR for 6 h (C). Chromatin-associated DNA was immunoprecipitated with an antibody against C/EBPβ. The immunoprecipitated DNA, samples processed without antibody (indicated as No antibody control), and 5% amount of sonicated DNA (indicated as 5% INPUT) were subjected to PCR using specific primers for 11β-HSD1 promoter region. Amplified DNA indicates the binding of C/EBPβ to the 11β-HSD1 promoter.

 
To explore a possible involvement of C/EBPβ in 11β-HSD1 regulation, ChIP analysis was performed with primers spanning putative C/EBP binding sites in mouse 11β-HSD1 promoter (30). No amplified band was observed in samples processed without antibody (No antibody control), excluding a possibility of nonspecific binding between DNA fragments and protein A-agarose (Fig. 7CGo). When treated with C2 ceramide or AICAR for 6 h, association of C/EBPβ with the promoter of 11β-HSD1 gene was substantially induced (Fig. 7CGo). Our data demonstrated that the activation of ceramide or AMPK pathways induced the expression of C/EBPβ and its binding to the 11β-HSD1 promoter.

Effect of C/EBPβ knockdown on the expression of 11β-HSD1 induced by ceramide or AMPK signaling in 3T3-L1 preadipocytes
To further validate a role of C/EBPβ in the control of 11β-HSD1 by ceramide or AICAR, C/EBPβ protein was transiently knocked down by siRNA. When 3T3-L1 preadipocytes were transfected with siRNA, C/EBPβ protein expression induced by C2 ceramide or AICAR was markedly attenuated, demonstrating effective silencing of C/EBPβ (Fig. 8Go, A and B). Notably, augmented expression of 11β-HSD1 induced by C2 ceramide or AICAR was significantly attenuated in cells transfected with C/EBPβ siRNA (Fig. 8Go, C and D). In contrast, negative control Stealth RNAi treatment had no impact on the expression of C/EBPβ or 11β-HSD1. These results suggest that C/EBPβ is involved critically in the induction of 11β-HSD1 by ceramide- or AMPK-mediated signaling pathways.


Figure 8
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  		FIG. 8. Effect of C/EBPβ knockdown on C2 ceramide- or AICAR-induced expression of 11β-HSD1 in 3T3-L1 preadipocytes. Cells were transfected with either Stealth RNAi negative control (N.C.) or C/EBPβ Stealth RNAi (RNAi154, RNAi168). After 12 h of incubation, cells were treated with C2 ceramide or AICAR. Western blot of C/EBPβ after the treatment with 0.1 mM C2 ceramide (A) or 0.5 mM AICAR (B) for 3 h. β-Actin was used as a loading control. mRNA level of 11β-HSD1 after the treatment with 0.1 mM C2 ceramide (C) or 0.5 mM AICAR (D) for 24 h. Results are means ± SEM from four experiments. *, P < 0.05, **, P < 0.01 compared with control group. {dagger}{dagger}, P < 0.01 compared with C2 ceramide or AICAR-treated group.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The major finding of the present study is that ceramide- and AMPK-mediated signaling pathways augment the expression and enzyme activity of 11β-HSD1 in both murine and human preadipocytes. We provide novel evidence that activation of ceramide and AMPK pathways induce the expression of C/EBPβ. ChIP analyses demonstrate the DNA binding of C/EBPβ to 11β-HSD1 promoter, and transient knockdown of C/EBPβ protein by siRNA further support the notion that C/EBPβ is critically involved in the expression of 11β-HSD1 induced by C2 ceramide or AICAR. Taken together, the present study highlights a novel mechanism that metabolic stress-related signaling pathways mediated by ceramide and AMPK regulate the expression of 11β-HSD1 in preadipocytes.

Ceramide acts as a lipid mediator of metabolic stress response (18, 42). Fatty acids, proinflammatory cytokines, glucocorticoids, and serum deprivation in cultured cells are known to induce intracellular accumulation of ceramide (43, 44). Importantly, aberrant accumulation of ceramide in insulin target tissues appreciably contributes to local insulin resistance and underlies, at least partly, the molecular mechanism of lipotoxicity (43, 44). A recent study demonstrated that mRNA level of enzymes involved in sphingolipid metabolism in adipose tissue as well as plasma level of SMase, ceramide, sphingosine, and S1P were increased in ob/ob mice (45). Thus we tested a possibility whether 11β-HSD1 would be induced by ceramide signals in preadipocytes. In this context, the present study is the first to demonstrate that the expression of 11β-HSD1 was induced by C2 ceramide, bacterial SMase and S1P in preadipocytes (Fig. 2Go). In contrast, induction of 11β-HSD1 expression by C2 ceramide, SMase, and S1P was not observed in differentiated adipocytes (Fig. 3Go), suggesting that such effects are restricted in preadipocytes.

SMase catalyzes the hydrolysis of sphingomyelin in outer side of the plasma membrane, leading to the production of ceramide (19). Ceramide is subsequently metabolized to S1P, which functions through S1P receptors (19). In the present study, we found that inhibition of SMase by desipramine attenuated the TNF{alpha}- or IL-1β-induced expression of 11β-HSD1. This observation is in agreement with previous reports that TNF{alpha} and IL-1β promptly induced the hydrolysis of sphingomyelin to generate ceramide (46). Assays in the present study are validated by the finding that expression of MCP-1 was induced by C2 ceramide (Fig. 2Go), consistent with the notion that NF{kappa}B and MAPK signaling pathways are involved in the regulation of MCP-1 (47) and that ceramide and S1P potently mediate NF{kappa}B and MAPK pathways (18, 48).

In the present study, C2 ceramide induced the expression of 11β-HSD1 at 50–100 µM (Fig. 2Go). It has been reported that the physiological concentration of ceramide within cells are approximately 1–5 µM (49). Accordingly, C2 ceramide does induce biological effects including differentiation and growth inhibition at 1–5 µM in serum-free media. However, higher concentration (50–100 µM) of C2 ceramide are required to induce equipotent effects in serum-containing medium, because serum proteins bind C2 ceramide and reduce its potency (50), and for this, 50–100 µM C2 ceramide has been commonly used in many previous studies (51, 52). It should also be noted that, because ceramide resides in "lipid rafts" in the membrane (53), local concentration of ceramide within cells must be much higher than 5 µM. In this context, the concentration of C2 ceramide used in the present study is appropriate for analyzing the responsiveness of ceramide in adipocytes.

AMPK, activated by the increase in intracellular AMP level, plays a crucial role in mediating cellular stress such as hypoxia, glucose deprivation, and ischemia (20, 21, 22). Recent studies highlighted a potential role of AMPK in regulating energy balance and mass of adipose tissue (23, 24). ATP level of adipose tissue is decreased in obese rodent models (54), supporting the notion that local hypoxia and inflammation is associated with defective energy metabolism in obese adipose tissue (14, 54). In this context, the present study demonstrates for the first time that AICAR markedly augmented the expression of 11β-HSD1 in preadipocytes (Fig. 4Go). Furthermore, a potent AMPK inhibitor compound C completely suppressed the induction of 11β-HSD1 (Fig. 5Go), verifying that AMPK signaling pathway is involved in the regulation of 11β-HSD1. Based on the present study in 3T3-L1 preadipocytes (Figs. 2Go, 4Go, and 5Go), a potential interaction between ceramide and AMPK in terms of the effect on 11β-HSD1 expression would be of considerable interest. In 3T3-L1 preadipocytes, AMPK was not activated when treated with C2 ceramide (data not shown). Although further studies are required, these results suggest that ceramide signal is not directly involved in AMPK-mediated induction of 11β-HSD1.

The present study is the first to demonstrate that C2 ceramide and AICAR augmented the expression of C/EBPβ in 3T3-L1 preadipocytes (Fig. 7Go). This result is consistent with a line of previous reports showing that S1P induced phosphorylation of cAMP-responsive-element-binding protein (CREB) (48), and CREB potentially controls the expression of C/EBPβ in adipocytes (55). Furthermore, the present study is the first demonstration that the activation of AMPK induced the expression of C/EBPβ in any kinds of cells. It is well-characterized that transient expression of C/EBPβ is essential for the induction of PPAR{gamma} and C/EBP{alpha} in the early phase of adipogenesis (41). A recent report raised a possibility that AICAR inhibited adipogenesis by interfering induction of PPAR{gamma} and C/EBP{alpha} (56). Therefore, it is tempting to speculate that AMPK-induced augmentation and sustainment of C/EBPβ and resultant suppression of adipogenesis may be a facet of adaptation to nutritional threat.

Compared with murine adipocytes, the mechanism responsible for adipogenesis and adipokine secretion is poorly understood in humans (57). For example, human preadipocytes do not require the process of clonal expansion in the course of adipogenesis (57). Therefore, we examined the effect of ceramide and AMPK signaling on the expression of 11β-HSD1 using human preadipocytes. The present study demonstrates that sphingolipids (C2 ceramide and S1P) and AICAR induced the expression of 11β-HSD1 also in human preadipocytes (Fig. 6Go). The effect of C2 ceramide was exaggerated in human preadipocytes compared with 3T3-L1 preadipocytes, whereas the effect of S1P was mild in human preadipocytes. Treatment of C2 ceramide did not affect MCP-1 mRNA level, but AICAR substantially reduced the expression in a dose-dependent manner (data not shown), representing a contrast to the data in 3T3-L1 preadipocytes (Figs. 2Go and 4Go). Even considering differences in cell types or species (58), our data provide novel evidence that ceramide and AMPK signals induce the expression of 11β-HSD1 in both rodent and human cultured preadipocytes. Recent works demonstrated that human adipose tissue contained a considerable amount of preadipocytes (59, 60), which may be involved in some aspects of adipose tissue function (15, 60). In this context, further in vivo studies are warranted to validate the possible involvement of ceramide and AMPK signals in 11β-HSD1 regulation in human preadipocytes.

C/EBP family of transcription factors (C/EBPs) in adipocytes serve as master regulators of a variety of cellular response (61), and expression of C/EBPs is regulated by a variety of hormones, cytokines, and nutrients (61, 62). 11β-HSD1 promoter contains a couple of C/EBP binding sites, and previous studies demonstrated that the expression of 11β-HSD1 was controlled by C/EBPs (40, 63). In this context, the present study demonstrates, for the first time, that induction of C/EBPβ in preadipocytes was observed around 3 h after the treatment with C2 ceramide or AICAR (Fig. 7Go), which preceded the robust induction of 11β-HSD1. Our data of ChIP analyses and C/EBPβ knockdown experiments further reinforced the notion that C/EBPβ is involved in ceramide- and AMPK-mediated augmentation of 11β-HSD1 in preadipocytes (Figs. 7Go and 8Go).

It should be noted that glucocorticoid is known to increase the expression and activity of SMase, resulting in intracellular ceramide accumulation and local insulin resistance (43). This notion prompts us to speculate a vicious cycle within cells where ceramide and 11β-HSD1-derived active glucocorticoid reciprocally aggravate preadipocyte dysfunction. Unexpected regulation of 11β-HSD1 by AMPK pathway may also provide a novel clue to better understand molecular pathophysiology of adipose dysfunction. Collectively, the present study is the first demonstration that ceramide and AMPK signaling pathways augment the expression and enzyme activity of 11β-HSD1 in human and rodent preadipocytes, thereby highlighting a metabolic stress-related regulation of 11β-HSD1 in a cell-specific manner.


   Acknowledgments
 
We thank Ms. S. Maki, K. Koyama, and M. Nagamoto for experimental assistance. We are grateful to Dr. S. Yokota and Mr. Y. Tominaga (Kaneka Corporation) for technical advice.


   Footnotes
 
This work was supported by Grant-in-Aid for Scientific Research (B2) (16390267); Grant-in-Aid for Scientific Research (S2) (16109007); Grant-in-Aid for Scientific Research on Priority Areas (Adipomics, 15081101); Grant-in-Aid for Research on Measures for Intractable Diseases (Health and Labor Science Research Grant); The Ministry of Education, Culture, Sports, Science and Technology of Japan; Research Grant from Special Coordination Funds for Promoting Science and Technology (Japan Science and Technology Agency); AstraZeneca International Research Grant; Takeda Medical Research Foundation; Smoking Research Foundation; Setsuro Fujii Memorial Osaka Foundation for Promotion of Fundamental Medical Research; Research Grant for Cardiovascular Diseases (National Cardiovascular Center); Mitsubishi Pharma Research Foundation; and Sankyo Research Foundation for Medical Research.

Disclosure Statement: The authors of this manuscript have nothing to disclose.

First Published Online August 16, 2007

Abbreviations: ACC, Acetyl-CoA carboxylase; AICAR, 5-aminoimidazole-4-carboxamide ribonucleosides; AMPK, AMP-activated protein kinase; C/EBP, CCAAT/enhancer-binding protein; ChIP, chromatin immunoprecipitation; 11β-HSD1, 11β-hydroxysteroid dehydronenase type 1; MCP-1, monocyte chemoattractant protein-1; Pref-1, preadipocyte factor-1; SMase, sphingomyelinase; S1P, sphingosine 1-phosphate; siRNA, small interfering RNA.

Received March 14, 2007.

Accepted for publication August 8, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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