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Contribution of glucocorticoid–mineralocorticoid receptor pathway on the obesity-related adipocyte dysfunction
Biochemical and Biophysical Research Communications 419 (2012) 182–187
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Contribution of glucocorticoid–mineralocorticoid receptor pathway on the obesity-related adipocyte dysfunction Ayumu Hirata ⇑, Norikazu Maeda ⇑, Hideaki Nakatsuji, Aki Hiuge-Shimizu, Takuya Okada, Tohru Funahashi, Iichiro Shimomura Department of Metabolic Medicine, Graduate School of Medicine, Osaka University, 2-2-B5 Yamada-oka, Suita, Osaka 565-0871, Japan
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Article history: Received 21 January 2012 Available online 3 February 2012 Keywords: Mineralocorticoid receptor Glucocorticoid Obesity Oxidative stress Adiponectin
a b s t r a c t Aims: Mineralocorticoid receptor (MR) blockade ameliorated insulin resistance with improvements in adipocytokine dysregulation, inflammation, and excess of reactive oxygen species (ROS) in obese adipose tissue and adipocytes, but its mechanism has not been clarified. The 11b-hydroxysteroid dehydrogenase type 1 (11b-HSD1), producing active glucocorticoids, is highly expressed in adipocytes and glucocorticoids bind to MR with higher affinity than to glucocorticoid receptor (GR). We investigated whether glucocorticoids effect on adipocytokines and ROS through MR in adipocytes. In addition, fat distributions of MR and GR were investigated in human subjects. Methods and Results: Corticoid receptors and their target genes were examined in adipose tissue of obese db/db mice. 3T3-L1 adipocytes were treated with glucocorticoids, H2O2, MR antagonist eplerenone (EP), GR antagonist RU486 (RU), MR-siRNA, and/or N-acetylcysteine. Human adipose tissues were obtained from seven patients who underwent abdominal surgery. The mRNA levels of MR and its target gene were higher in db/db mice than in control db/m + mice. In 3T3-L1 adipocytes, glucocorticoids, similar to H2O2, caused the dysregulation of mRNA levels of various genes related to adipocytokines and the increase of intracellular ROS. Such changes were rectified by MR blockade, not by GR antagonist. In human fat, MR mRNA level was increased in parallel with the increase of body mass index (BMI) and its increase was more significant in visceral fat, while there were no apparent correlations of GR mRNA level to BMI or fat distribution. Conclusion: Glucocorticoid-MR pathway may contribute to the obesity-related adipocytokine dysregulation and adipose ROS. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Accumulating clinical evidence indicates that excessive accumulation of visceral fat is closely associated with obesity-related metabolic disorders [1]. Our group demonstrated that overproduction of reactive oxygen species (ROS) in obese fat tissue leads to the dysregulation of adipocytokines and accelerates the development of metabolic disorder [2]. Several studies demonstrated that activation of MR promotes inflammation, proliferation, and fibrosis via ROS generation. As we and other group previously indicated [3,4], mineralocorticoid receptor (MR) blockade by MR antagonist eplerenone (EP) ameliorated insulin resistance and adipocytokine dysregulation, and reduced ROS and macrophage infiltration in adipose tissue of obese mice. In adipocytes, MR activation caused the increase of ROS ⇑ Corresponding authors. Fax: +81 6 6879 3739. E-mail addresses: [email protected] (A. Hirata), norikazu_ [email protected] (N. Maeda). 0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2012.01.139
and the adipocytokine dysregulation, while MR blockade improved such changes [3]. Collectively, adipose MR may play an important role in metabolic disorder in the development of obesity. The MR binds not only to aldosterone but also to glucocorticoid with equal affinity [5]. In addition, the affinities of glucocorticoids (cortisol in humans, corticosterone in rodents) for MR are 10-fold higher than those for glucocorticoid receptor (GR) [6]. 11bhydroxysteroid dehydrogenase type 1 (11b-HSD1), which converts cortisone (an inactive corticoid) into cortisol (an active corticoid), is highly expressed in adipocytes [7]. These evidences suggest that glucocorticoid action could be exhibited mainly through MR in adipocytes, but adipose glucocorticoid-MR pathway has not been fully elucidated. We here tested the glucocorticoid action in adipocytes by using antagonists for MR and GR. The effect of glucocorticoid on adipocytokines and adipose ROS was also examined. In addition, mRNA expressions for important proteins, including MR, GR, PPARc2, NADPH oxidase subunit p22, adiponectin, and 11bHSD1, were directly compared in subcutaneous and visceral fat of human subjects, according to their BMI.
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2. Methods
2.5. Analysis of human adipose tissue
2.1. Animals
Subcutaneous and visceral (omental) adipose tissues were obtained from the patients (three females and four males) who underwent abdominal surgery. The investigation conforms to the principles outlined in the Declaration of Helsinki. Ethics Committee approval from the Faculty of Medicine of the Osaka University and patient informed consent were obtained. Tissues were immediately frozen in liquid nitrogen and preserved in 80 °C freezer before analysis. Extraction of RNA and RT-PCR assays were conducted similar to mice and cell samples.
Male BKS.Cg-m+/+Leprdb/J (db/db) obese mice were purchased from Charles River Laboratories (Charles River Japan Inc., Yokohama, Japan). Their respective lean control male db/m + heterozygous littermates were purchased from the same supplier. Mice were sacrificed at 10 weeks of age by intraperitoneous administration of 5% pentobarbital in one shot. The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of Osaka University School of Medicine. This study also conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).
2.2. 3T3-L1 cell cultures 3T3-L1 cells were maintained and differentiated as previously described [8]. Introduction of siRNA was performed as follows. On day 6 after 3T3-L1 differentiation, the media of 3T3-L1 cells in 12-well plates were changed to OPTI-MEM (Invitrogen, San Diego, CA), and the cells were transfected with MR- and control-siRNA (Qiagen, Valencia, CA) using LipofectAMINE 2000 reagent (Invitrogen, San Diego, CA) according to the instructions provided by the manufacturer. The sequences of the MR-siRNA sense were as follows: 5’-GGUUCUCUGUACCAAUAAAdTdT-3’. On day 7, media of 3T3-L1 adipocytes was changed to Dulbecco’s modified Eagle medium (DMEM), and 3T3-L1 adipocytes were treated with the indicated concentrations of either 10 5 mol/L of eplerenone (EP) or RU486 (RU) dissolved in dimethyl sulfoxide (DMSO), 10 7 mol/L of corticosterone and cortisol dissolved in acetone, and 0.2 mmol/ L of H2O2 for 24 h. Concentrations of these reagents were referred to previous studies [9–11].
2.3. Measurement of intracellular TBARS On day 7, 3T3-L1 adipocytes were treated with 10 7 mol/L of corticosterone, 10 7 mol/L of cortisol, 10 5 mol/L of eplerenone (EP), 10 5 mol/L of RU486 (RU) or 10 m mol/L of N-acetylcysteine (NA), and cells were collected after 24 h from treatment. The intracellular levels of oxidative stress were measured as thiobarbituric acid reactive substance (TBARS) by using TBARS assay kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer protocol.
2.4. Quantification of mRNA levels Total RNA was isolated from mice tissues by using RNA STAT-60 (Tel-Test Inc., Friendswood, TX) according to the protocol supplied by the manufacturer. The quality and quantity of total RNA was determined by using ND-1000 Spectrophotometer (Nano Drop Technologies, Wilmington, DE). First-strand cDNA was synthesized from 200 ng of total RNA using Thermoscript RT (Invitrogen, San Diego, CA) and oligo dT primer. Real-time quantitative PCR amplification was conducted with the LightCycler 1.5 (Roche Diagnostics, Mannheim, Germany) using LightCycler-FastStart DNA Master SYBR Green I (Roche Diagnostics, Mannheim, Germany) according to the protocol recommended by manufacturer. Primers are listed in Supplemental Table. The final result for each sample was normalized to the respective 36B4 value in 3T3-L1 cell lysates and 18s in human adipose tissue.
2.6. Statistical analysis Results were expressed as the mean ± SEM of n separate experiments. Differences between groups were examined for statistical significance using Student’s t-test or ANOVA with Fisher’s protected least significant difference test. A P value less than 0.05 denoted the presence of a statistically significant difference.
3. Results 3.1. Expression of corticoid receptors and their target genes in obese model mice The mRNA levels of corticoid receptors and their target genes were examined in white adipose tissue (WAT) of db/db and db/ m + mice (Fig. 1). MR mRNA level of db/db mice was significantly higher than that of db/m + mice (Fig. 1A). In parallel with the change of MR, serum- and glucocorticoid-induced kinase 1 (Sgk1), a MR target gene [12], was also significantly increased in db/db mice (Fig. 1B). On the other hand, both GR and regulated in DNA damage and development 1 (REDD1), a GR target gene [13], had similar mRNA levels between db/db and db/m + mice (Fig. 1C and D).
3.2. Changes of mRNA levels relating to adipocytokines and ROS under glucocorticoid treatment in 3T3-L1 Adipocytes Next, effect of glucocorticoid, corticosterone and cortisol, on mRNA levels relating to adipocytokines and ROS was investigated by using MR antagonist eplerenone (EP) or GR antagonist RU486 (RU) in 3T3-L1 adipocytes (Fig. 2). Glucocorticoids significantly reduced adiponectin mRNA level while increased interleukin-6 (IL-6) mRNA level (Fig. 2A and B, lane 1 versus 4 and 7). Such dysregulations of adipocytokines were partially but significantly reversed by the treatment with EP (Fig. 2A and B, lane 4 versus 5, lane 7 versus 8). However, GR blockade with the RU treatment failed to reverse the glucocorticoids-induced dysregulations of adiponectin and IL-6 mRNA levels (Fig. 2A and B, lane 4 versus 6, lane 7 versus 9). Fig. 2C and D shows mRNA changes of NADPH oxidase subunit p22 promoting ROS production and catalase eliminating ROS. Treatment with glucocorticoids significantly increased mRNA level of NADPH oxidase subunit p22 while decreased catalase mRNA level (Fig. 2C and D, lane 1 versus 4 and 7). Such glucocorticoids-induced changes of ROS-relating mRNA levels were reversed by EP (Fig. 2C and D, lane 4 versus 5, lane 7 versus 8), but were not recovered by RU (Fig. 2C and D, lane 4 versus 6, lane 7 versus 9). These data suggest that glucocorticoids may induce the adipocytokine dysregulation and ROS partly through MR in adipocytes.
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Fig. 1. The mRNA levels of corticoid receptors and their target genes in fat tissue of db/m+ and db/db mice at 10 weeks of age. Total RNA was isolated from adipose tissue and first-strand cDNA was synthesized as described in Methods section. Real-time quantitative PCR was performed and values were normalized to the level of 36B4 mRNA. Data are mean ± SEM; n = 5–6 mice per group.
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Fig. 2. Effects of MR or GR antagonists on mRNA levels in 3T3-L1 adipocytes under glucocorticoid treatment. On day 7, 3T3-L1 adipocytes were treated with 10 7 mol/L of corticosterone, 10 7 mol/L of cortisol, 10 5 mol/L of eplerenone (EP) or 10 5 mol/L of RU486 (RU) for 24 h. Data are mean ± SEM; n = 6 per group. #P < 0.05; ##P < 0.01; ###P < 0.001 versus lane 1. ⁄P < 0.05.
3.3. Effect of MR blockade on mRNA levels relating to adipocytokines and ROS under glucocorticoid and H2O2 treatment in 3T3-L1 Adipocytes The significance of glucocorticoid-MR pathway was also confirmed by using siRNA for MR and the effect of ROS on its pathway was examined by administration of H2O2 (Supplemental Figure). Introduction of MR-siRNA caused 70% reduction of MR mRNA level
compared with control-siRNA (data not shown). As previously demonstrated [3], H2O2 treatment caused the changes of mRNA level mimicking obese adipocytes while such H2O2-induced dysregulations were significantly ameliorated by the introduction of MR-siRNA (Supplemental Figure A to D, lane 1 versus 3 versus 4). Glucocorticoids-induced dysregulation of adiponectin, IL-6, NADPH oxidase subunit p22, and catalase were reversed by MR-siRNA (Supplemental Figure A to D, lane 1 versus 5 versus 6,
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Fig. 3. Intracellular TBARS levels in 3T3-L1 adipocytes. On day 7, 3T3-L1 adipocytes were treated with 10 7 mol/L of corticosterone, 10 7 mol/L of cortisol, 10 5 mol/L of eplerenone (EP), 10 5 mol/L of RU486 (RU) or 10 mmol/L of N-acetylcysteine (NA), and cells were collected after 24 h from treatment. ⁄⁄P < 0.01. ###P < 0.001 versus lane 1.
lane 1 versus 9 versus 10), which results were consistent with Fig. 2. Interestingly, co-treatment with glucocorticoids and H2O2 resulted in the decease of adiponectin and catalase mRNA levels and the increase of IL-6 mRNA level in an additive manner compared to each treatment (Supplemental Figure A, B, and D, lane 1 versus 5 versus 7, lane 1 versus 9 versus 11), but such additional changes was not observed in mRNA level of NADPH oxidase subunit p22 (Supplemental Figure C). Dysregulations of adiponectin and catalase induced by the co-treatment with glucocorticoids and H2O2 were partly but significantly reversed by MR-siRNA, and especially such co-treatment-induced increase of IL-6 mRNA level was completely recovered by the MR blockade with MR-siRNA (Supplemental Figure A, B, and D, lane 7 versus 8, lane 11 versus 12).
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ROS is closely associated with chronic low-grade inflammation in adipocytes and development of metabolic syndrome, but effect of glucocorticoids on adipose ROS has not been examined. In the next series of experiments, intracellular levels of thiobarbituric acid reactive substance (TBARS), one of ROS indicators, was measured in 3T3-L1 adipocytes (Fig. 3). Glucocorticoids treatment significantly increased TBARS level and such increase was recovered by N-acetylcysteine (NA), one of anti-oxidants (Fig. 3, lane 1 versus 5 versus 8, lane 1 versus 9 versus 12). Interestingly, the glucocorticoid-induced elevations of TBARS were completely reversed by MR blockade with EP (Fig. 3, lane 5 versus 6, lane 9 versus 10), while were not significantly ameliorated by GR blockade with RU (Fig. 3, lane 5 versus 7, lane 9 versus 11). 3.5. Distributions of MR and GR mRNA levels in human adipose tissues Finally, adipose mRNA levels were investigated in human subcutaneous and visceral fat tissues (Fig. 4). Interestingly, MR mRNA level was increased in parallel with the increase of BMI and especially the visceral fat MR mRNA level was remarkably high according to the increase of BMI. However, there were no apparent correlations of GR mRNA level to BMI or fat distribution. PPARc2 mRNA level was significantly suppressed and NADPH oxidase subunit p22 mRNA level was remarkably elevated in the visceral fat compared to subcutaneous fat, according to increase of BMI. Adiponectin mRNA level was low in both subcutaneous and visceral fat tissues, and its decrease was more significant in visceral fat tissues. Importantly, 11b-HSD1 mRNA level was elevated in parallel with the augmentation of BMI and its level was also higher in visceral fat than in subcutaneous fat.
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Fig. 4. The mRNA expression levels in human subcutaneous and visceral adipose tissue. Both subcutaneous and visceral adipose tissues were obtained when the patients were underwent abdominal surgery. Real-time quantitative PCR was performed and values were normalized to the level of 18s mRNA.
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4. Discussion The main findings of the present study were: (1) MR and its target gene mRNA levels were significantly increased in obese adipose tissues. (2) Physiological concentration of glucocorticoid caused the increase of adipose ROS and the adipocytokine dysregulation. (3) Glucocorticoid-induced ROS and adipocytokine dysregulation were significantly reversed by blockade of MR, not by GR antagonism. (4) In human visceral adipose tissue, MR mRNA level was increased as the BMI increased, while no apparent correlation between GR mRNA level and BMI was observed. The binding affinities of glucocorticoids for MR are 10-fold higher than GR [5]. 11b-hydroxysteroid dehydrogenase type 1 (11bHSD1) converts cortisone into cortisol while 11b-HSD2 changes cortisol into cortisone. In the epithelial cells, cortisol is immediately inactivated by 11b-HSD2 and thus aldosterone exhibits its effect via MR. However, 11b-HSD1 was dominantly expressed in adipocytes [6] and its mRNA expression level is higher in obese subjects compared to non-obese subjects [14]. Adipose 11b-HSD1 mRNA level shows a positive correlation with waist circumference and insulin resistance [15,16]. Furthermore, fat-specific 11b-HSD1 transgenic mice revealed a phenotype of metabolic syndrome such as visceral obesity, hypertension, insulin resistance, and dyslipidemia [17,18], indicating that adipose glucocorticoids play an important role in the development of obesity. In adipocytes, it has been supposed that glucocorticoid action should be mediated by the cytosolic GR. However, in heart and kidney, several experiments demonstrated that glucocorticoids exhibit its effect mainly via MR [19,20]. We here showed for the first time that dysregulations of adipocytokine and increases of ROS were caused by the physiological concentration of glucocorticoids mainly via MR in adipocytes. Interestingly, co-treatment with glucocorticoids and H2O2 caused the additional suppression of adiponectin mRNA level (Fig. 4, lane 3 versus 5 versus 7 and lane 3 versus 9 versus 11), which was consistent with the previous reports demonstrating that the glucocorticoid-MR action was augmented by the excess of ROS [10]. Patients with excessive glucocorticoids, such as cushing syndrome and chronic administration with glucocorticoid, exhibit clinical phenotypes like a metabolic syndrome. Present study suggests that glucocorticoid-induced metabolic disorders might be completely or partly ameliorated by blockade of MR. Previous reports showed that MR blockade incompletely reversed the high dose cortisol-induced Na+ retention [21] and the ACTH stimulated high concentration glucocorticoid-induced hypertention [22]. The incomplete effect of MR blockade on such glucocorticoid-induced phenomena may be accounted for by extremely high concentration of glucocorticoid in these cases. High concentration of glucocorticoid exhibits its effect through both GR and MR [23]. In this case, glucocorticoid-GR pathway may be more dominant than glucocoid-MR pathway. In fact, the action of corticoids, both glucocorticoid and mineralocorticoid, is not fully clarified and there are some contradictory reports about corticoid actions, because physiological effect of these corticoids may depend on several intracellular conditions such as the balance of 11b-HSD1 and 11b-HSD2 activities and the redox states. Importantly, we for the first time showed that MR mRNA level of human fat was increased in parallel with the increase of BMI and especially its mRNA level of visceral fat was dramatically up-regulated according to the increase of BMI, while GR mRNA level of human adipose tissue was not related to BMI and fat distribution (Fig. 4). Furthermore, as the BMI increases, PPARc2 mRNA level was significantly suppressed and NADPH oxidase subunit p22 mRNA level was dramatically increased in visceral fat compared to subcutaneous fat. The activation of PPARc reduces the increased ROS in adipose tissues [24] and the increase of NADPH oxidase
subunits caused the excess of ROS [2]. Furthermore, more increase of 11b-HSD1 mRNA should supply active glucocorticoid for MR activation in accumulated visceral fat. Collectively, as the BMI increases, human visceral fat tissues may tend to fall into the excess of ROS and the visceral fat MR mRNA level was increased, resulting that the glucocorticoid-MR action may be augmented in visceral fat tissues. Our present and previous [3] studies may indicate that the glucocorticoid-MR pathway should be activated in obese adipocytes (especially in obese visceral adipocytes) through the increases of 11b-HSD1, MR, and ROS, resulting in adipocytokine dysregulation, adipose inflammation, and excess of adipose ROS. Blockade of adipose MR may cut off such vicious cycle and may be useful for the treatment of metabolic syndrome, although clinical trials about MR blockade therapy as treatment for the metabolic syndrome should be awaited in the future. Funding This work was supported by Grants-in-Aid for Scientific Research (C) No. 22590979 (to N. M.), and Scientific Research on Innovative Areas No. 22126008 (to T. F.). Acknowledgments We are grateful to Pfizer Inc (New York, NY) for providing eplerenone. We thank Miyuki Nakamura for the excellent technical assistance. We also thank all members of the III laboratory (Adiposcience laboratory), Department of Metabolic Medicine, Osaka University for helpful discussions on the project. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2012.01.139. References [1] Y. Matsuzawa, T. Funahashi, S. Kihara, I. Shimomura, Adiponectin and metabolic syndrome, Arterioscler. Thromb. Vasc. Biol. 24 (2004) 29–33. [2] S. Furukawa, T. Fujita, M. Shimabukuro, M. Iwaki, Y. Yamada, Y. Nakajima, O. Nakayama, M. Makishima, M. Matsuda, I. Shimomura, Increased oxidative stress in obesity and its impact on metabolic syndrome, J. Clin. Invest. 114 (2004) 1752–1761. [3] A. Hirata, N. Maeda, A. Hiuge, T. Hibuse, K. Fujita, T. Okada, S. Kihara, T. Funahashi, I. Shimomura, Blockade of mineralocorticoid receptor reverses adipocyte dysfunction and insulin resistance in obese mice, Cardiovasc. Res. 84 (2009) 164–172. [4] C. Guo, V. Ricchiuti, B.Q. Lian, T.M. Yao, P. Coutinho, J.R. Romero, J. Li, G.H. Williams, G.K. Adler, Mineralocorticoid receptor blockade reverses obesityrelated changes in expression of adiponectin, peroxisome proliferator-activated receptor-gamma, and proinflammatory adipokines, Circulation 117 (2008) 2253–2261. [5] J. Funder, K. Myles, Exclusion of corticosterone from epithelial mineralocorticoid receptors is insufficient for selectivity of aldosterone action: in vivo binding studies, Endocrinology 137 (1996) 5264–5268. [6] J.W. Funder, Mineralocorticoid receptors: distribution and activation, Heart Fail. Rev. 10 (2005) 15–22. [7] I.J. Bujalska, S. Kumar, P.M. Stewart, Does central obesity reflect ‘‘cushing’s disease of the omentum’’?, Lancet 349 (1997) 1210–1213 [8] N. Maeda, M. Takahashi, T. Funahashi, S. Kihara, H. Nishizawa, K. Kishida, H. Nagaretani, M. Matsuda, R. Komuro, N. Ouchi, H. Kuriyama, K. Hotta, T. Nakamura, I. Shimomura, Y. Matsuzawa, PPAR gamma ligands increase expression and plasma concentrations of adiponectin, an adipose-derived protein, Diabetes 50 (2001) 2094–2099. [9] A. Kurata, H. Nishizawa, S. Kihara, N. Maeda, M. Sonoda, T. Okada, K. Ohashi, T. Hibuse, K. Fujita, A. Yasui, A. Hiuge, M. Kumada, H. Kuriyama, I. Shimomura, T. Funahashi, Blockade of Angiotensin II type-1 receptor reduces oxidative stress in adipose tissue and ameliorates adipocytokine dysregulation, Kidney Int. 70 (2006) 1717–1724. [10] H. Hitomi, H. Kiyomoto, A. Nishiyama, T. Hara, K. Moriwaki, K. Kaifu, G. Ihara, Y. Fujita, T. Ugawa, M. Kohno, Aldosterone suppresses insulin signaling via the downregulation of insulin receptor substrate-1 in vascular smooth muscle cells, Hypertension 50 (2007) 750–755.
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Contribution of glucocorticoid–mineralocorticoid receptor pathway on the obesity-related adipocyte dysfunction Ayumu Hirata ⇑, Norikazu Maeda ⇑, Hideaki Nakatsuji, Aki Hiuge-Shimizu, Takuya Okada, Tohru Funahashi, Iichiro Shimomura Department of Metabolic Medicine, Graduate School of Medicine, Osaka University, 2-2-B5 Yamada-oka, Suita, Osaka 565-0871, Japan
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Article history: Received 21 January 2012 Available online 3 February 2012 Keywords: Mineralocorticoid receptor Glucocorticoid Obesity Oxidative stress Adiponectin
a b s t r a c t Aims: Mineralocorticoid receptor (MR) blockade ameliorated insulin resistance with improvements in adipocytokine dysregulation, inflammation, and excess of reactive oxygen species (ROS) in obese adipose tissue and adipocytes, but its mechanism has not been clarified. The 11b-hydroxysteroid dehydrogenase type 1 (11b-HSD1), producing active glucocorticoids, is highly expressed in adipocytes and glucocorticoids bind to MR with higher affinity than to glucocorticoid receptor (GR). We investigated whether glucocorticoids effect on adipocytokines and ROS through MR in adipocytes. In addition, fat distributions of MR and GR were investigated in human subjects. Methods and Results: Corticoid receptors and their target genes were examined in adipose tissue of obese db/db mice. 3T3-L1 adipocytes were treated with glucocorticoids, H2O2, MR antagonist eplerenone (EP), GR antagonist RU486 (RU), MR-siRNA, and/or N-acetylcysteine. Human adipose tissues were obtained from seven patients who underwent abdominal surgery. The mRNA levels of MR and its target gene were higher in db/db mice than in control db/m + mice. In 3T3-L1 adipocytes, glucocorticoids, similar to H2O2, caused the dysregulation of mRNA levels of various genes related to adipocytokines and the increase of intracellular ROS. Such changes were rectified by MR blockade, not by GR antagonist. In human fat, MR mRNA level was increased in parallel with the increase of body mass index (BMI) and its increase was more significant in visceral fat, while there were no apparent correlations of GR mRNA level to BMI or fat distribution. Conclusion: Glucocorticoid-MR pathway may contribute to the obesity-related adipocytokine dysregulation and adipose ROS. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Accumulating clinical evidence indicates that excessive accumulation of visceral fat is closely associated with obesity-related metabolic disorders [1]. Our group demonstrated that overproduction of reactive oxygen species (ROS) in obese fat tissue leads to the dysregulation of adipocytokines and accelerates the development of metabolic disorder [2]. Several studies demonstrated that activation of MR promotes inflammation, proliferation, and fibrosis via ROS generation. As we and other group previously indicated [3,4], mineralocorticoid receptor (MR) blockade by MR antagonist eplerenone (EP) ameliorated insulin resistance and adipocytokine dysregulation, and reduced ROS and macrophage infiltration in adipose tissue of obese mice. In adipocytes, MR activation caused the increase of ROS ⇑ Corresponding authors. Fax: +81 6 6879 3739. E-mail addresses: [email protected] (A. Hirata), norikazu_ [email protected] (N. Maeda). 0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2012.01.139
and the adipocytokine dysregulation, while MR blockade improved such changes [3]. Collectively, adipose MR may play an important role in metabolic disorder in the development of obesity. The MR binds not only to aldosterone but also to glucocorticoid with equal affinity [5]. In addition, the affinities of glucocorticoids (cortisol in humans, corticosterone in rodents) for MR are 10-fold higher than those for glucocorticoid receptor (GR) [6]. 11bhydroxysteroid dehydrogenase type 1 (11b-HSD1), which converts cortisone (an inactive corticoid) into cortisol (an active corticoid), is highly expressed in adipocytes [7]. These evidences suggest that glucocorticoid action could be exhibited mainly through MR in adipocytes, but adipose glucocorticoid-MR pathway has not been fully elucidated. We here tested the glucocorticoid action in adipocytes by using antagonists for MR and GR. The effect of glucocorticoid on adipocytokines and adipose ROS was also examined. In addition, mRNA expressions for important proteins, including MR, GR, PPARc2, NADPH oxidase subunit p22, adiponectin, and 11bHSD1, were directly compared in subcutaneous and visceral fat of human subjects, according to their BMI.
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2. Methods
2.5. Analysis of human adipose tissue
2.1. Animals
Subcutaneous and visceral (omental) adipose tissues were obtained from the patients (three females and four males) who underwent abdominal surgery. The investigation conforms to the principles outlined in the Declaration of Helsinki. Ethics Committee approval from the Faculty of Medicine of the Osaka University and patient informed consent were obtained. Tissues were immediately frozen in liquid nitrogen and preserved in 80 °C freezer before analysis. Extraction of RNA and RT-PCR assays were conducted similar to mice and cell samples.
Male BKS.Cg-m+/+Leprdb/J (db/db) obese mice were purchased from Charles River Laboratories (Charles River Japan Inc., Yokohama, Japan). Their respective lean control male db/m + heterozygous littermates were purchased from the same supplier. Mice were sacrificed at 10 weeks of age by intraperitoneous administration of 5% pentobarbital in one shot. The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of Osaka University School of Medicine. This study also conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).
2.2. 3T3-L1 cell cultures 3T3-L1 cells were maintained and differentiated as previously described [8]. Introduction of siRNA was performed as follows. On day 6 after 3T3-L1 differentiation, the media of 3T3-L1 cells in 12-well plates were changed to OPTI-MEM (Invitrogen, San Diego, CA), and the cells were transfected with MR- and control-siRNA (Qiagen, Valencia, CA) using LipofectAMINE 2000 reagent (Invitrogen, San Diego, CA) according to the instructions provided by the manufacturer. The sequences of the MR-siRNA sense were as follows: 5’-GGUUCUCUGUACCAAUAAAdTdT-3’. On day 7, media of 3T3-L1 adipocytes was changed to Dulbecco’s modified Eagle medium (DMEM), and 3T3-L1 adipocytes were treated with the indicated concentrations of either 10 5 mol/L of eplerenone (EP) or RU486 (RU) dissolved in dimethyl sulfoxide (DMSO), 10 7 mol/L of corticosterone and cortisol dissolved in acetone, and 0.2 mmol/ L of H2O2 for 24 h. Concentrations of these reagents were referred to previous studies [9–11].
2.3. Measurement of intracellular TBARS On day 7, 3T3-L1 adipocytes were treated with 10 7 mol/L of corticosterone, 10 7 mol/L of cortisol, 10 5 mol/L of eplerenone (EP), 10 5 mol/L of RU486 (RU) or 10 m mol/L of N-acetylcysteine (NA), and cells were collected after 24 h from treatment. The intracellular levels of oxidative stress were measured as thiobarbituric acid reactive substance (TBARS) by using TBARS assay kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer protocol.
2.4. Quantification of mRNA levels Total RNA was isolated from mice tissues by using RNA STAT-60 (Tel-Test Inc., Friendswood, TX) according to the protocol supplied by the manufacturer. The quality and quantity of total RNA was determined by using ND-1000 Spectrophotometer (Nano Drop Technologies, Wilmington, DE). First-strand cDNA was synthesized from 200 ng of total RNA using Thermoscript RT (Invitrogen, San Diego, CA) and oligo dT primer. Real-time quantitative PCR amplification was conducted with the LightCycler 1.5 (Roche Diagnostics, Mannheim, Germany) using LightCycler-FastStart DNA Master SYBR Green I (Roche Diagnostics, Mannheim, Germany) according to the protocol recommended by manufacturer. Primers are listed in Supplemental Table. The final result for each sample was normalized to the respective 36B4 value in 3T3-L1 cell lysates and 18s in human adipose tissue.
2.6. Statistical analysis Results were expressed as the mean ± SEM of n separate experiments. Differences between groups were examined for statistical significance using Student’s t-test or ANOVA with Fisher’s protected least significant difference test. A P value less than 0.05 denoted the presence of a statistically significant difference.
3. Results 3.1. Expression of corticoid receptors and their target genes in obese model mice The mRNA levels of corticoid receptors and their target genes were examined in white adipose tissue (WAT) of db/db and db/ m + mice (Fig. 1). MR mRNA level of db/db mice was significantly higher than that of db/m + mice (Fig. 1A). In parallel with the change of MR, serum- and glucocorticoid-induced kinase 1 (Sgk1), a MR target gene [12], was also significantly increased in db/db mice (Fig. 1B). On the other hand, both GR and regulated in DNA damage and development 1 (REDD1), a GR target gene [13], had similar mRNA levels between db/db and db/m + mice (Fig. 1C and D).
3.2. Changes of mRNA levels relating to adipocytokines and ROS under glucocorticoid treatment in 3T3-L1 Adipocytes Next, effect of glucocorticoid, corticosterone and cortisol, on mRNA levels relating to adipocytokines and ROS was investigated by using MR antagonist eplerenone (EP) or GR antagonist RU486 (RU) in 3T3-L1 adipocytes (Fig. 2). Glucocorticoids significantly reduced adiponectin mRNA level while increased interleukin-6 (IL-6) mRNA level (Fig. 2A and B, lane 1 versus 4 and 7). Such dysregulations of adipocytokines were partially but significantly reversed by the treatment with EP (Fig. 2A and B, lane 4 versus 5, lane 7 versus 8). However, GR blockade with the RU treatment failed to reverse the glucocorticoids-induced dysregulations of adiponectin and IL-6 mRNA levels (Fig. 2A and B, lane 4 versus 6, lane 7 versus 9). Fig. 2C and D shows mRNA changes of NADPH oxidase subunit p22 promoting ROS production and catalase eliminating ROS. Treatment with glucocorticoids significantly increased mRNA level of NADPH oxidase subunit p22 while decreased catalase mRNA level (Fig. 2C and D, lane 1 versus 4 and 7). Such glucocorticoids-induced changes of ROS-relating mRNA levels were reversed by EP (Fig. 2C and D, lane 4 versus 5, lane 7 versus 8), but were not recovered by RU (Fig. 2C and D, lane 4 versus 6, lane 7 versus 9). These data suggest that glucocorticoids may induce the adipocytokine dysregulation and ROS partly through MR in adipocytes.
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Fig. 1. The mRNA levels of corticoid receptors and their target genes in fat tissue of db/m+ and db/db mice at 10 weeks of age. Total RNA was isolated from adipose tissue and first-strand cDNA was synthesized as described in Methods section. Real-time quantitative PCR was performed and values were normalized to the level of 36B4 mRNA. Data are mean ± SEM; n = 5–6 mice per group.
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Fig. 2. Effects of MR or GR antagonists on mRNA levels in 3T3-L1 adipocytes under glucocorticoid treatment. On day 7, 3T3-L1 adipocytes were treated with 10 7 mol/L of corticosterone, 10 7 mol/L of cortisol, 10 5 mol/L of eplerenone (EP) or 10 5 mol/L of RU486 (RU) for 24 h. Data are mean ± SEM; n = 6 per group. #P < 0.05; ##P < 0.01; ###P < 0.001 versus lane 1. ⁄P < 0.05.
3.3. Effect of MR blockade on mRNA levels relating to adipocytokines and ROS under glucocorticoid and H2O2 treatment in 3T3-L1 Adipocytes The significance of glucocorticoid-MR pathway was also confirmed by using siRNA for MR and the effect of ROS on its pathway was examined by administration of H2O2 (Supplemental Figure). Introduction of MR-siRNA caused 70% reduction of MR mRNA level
compared with control-siRNA (data not shown). As previously demonstrated [3], H2O2 treatment caused the changes of mRNA level mimicking obese adipocytes while such H2O2-induced dysregulations were significantly ameliorated by the introduction of MR-siRNA (Supplemental Figure A to D, lane 1 versus 3 versus 4). Glucocorticoids-induced dysregulation of adiponectin, IL-6, NADPH oxidase subunit p22, and catalase were reversed by MR-siRNA (Supplemental Figure A to D, lane 1 versus 5 versus 6,
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Fig. 3. Intracellular TBARS levels in 3T3-L1 adipocytes. On day 7, 3T3-L1 adipocytes were treated with 10 7 mol/L of corticosterone, 10 7 mol/L of cortisol, 10 5 mol/L of eplerenone (EP), 10 5 mol/L of RU486 (RU) or 10 mmol/L of N-acetylcysteine (NA), and cells were collected after 24 h from treatment. ⁄⁄P < 0.01. ###P < 0.001 versus lane 1.
lane 1 versus 9 versus 10), which results were consistent with Fig. 2. Interestingly, co-treatment with glucocorticoids and H2O2 resulted in the decease of adiponectin and catalase mRNA levels and the increase of IL-6 mRNA level in an additive manner compared to each treatment (Supplemental Figure A, B, and D, lane 1 versus 5 versus 7, lane 1 versus 9 versus 11), but such additional changes was not observed in mRNA level of NADPH oxidase subunit p22 (Supplemental Figure C). Dysregulations of adiponectin and catalase induced by the co-treatment with glucocorticoids and H2O2 were partly but significantly reversed by MR-siRNA, and especially such co-treatment-induced increase of IL-6 mRNA level was completely recovered by the MR blockade with MR-siRNA (Supplemental Figure A, B, and D, lane 7 versus 8, lane 11 versus 12).
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ROS is closely associated with chronic low-grade inflammation in adipocytes and development of metabolic syndrome, but effect of glucocorticoids on adipose ROS has not been examined. In the next series of experiments, intracellular levels of thiobarbituric acid reactive substance (TBARS), one of ROS indicators, was measured in 3T3-L1 adipocytes (Fig. 3). Glucocorticoids treatment significantly increased TBARS level and such increase was recovered by N-acetylcysteine (NA), one of anti-oxidants (Fig. 3, lane 1 versus 5 versus 8, lane 1 versus 9 versus 12). Interestingly, the glucocorticoid-induced elevations of TBARS were completely reversed by MR blockade with EP (Fig. 3, lane 5 versus 6, lane 9 versus 10), while were not significantly ameliorated by GR blockade with RU (Fig. 3, lane 5 versus 7, lane 9 versus 11). 3.5. Distributions of MR and GR mRNA levels in human adipose tissues Finally, adipose mRNA levels were investigated in human subcutaneous and visceral fat tissues (Fig. 4). Interestingly, MR mRNA level was increased in parallel with the increase of BMI and especially the visceral fat MR mRNA level was remarkably high according to the increase of BMI. However, there were no apparent correlations of GR mRNA level to BMI or fat distribution. PPARc2 mRNA level was significantly suppressed and NADPH oxidase subunit p22 mRNA level was remarkably elevated in the visceral fat compared to subcutaneous fat, according to increase of BMI. Adiponectin mRNA level was low in both subcutaneous and visceral fat tissues, and its decrease was more significant in visceral fat tissues. Importantly, 11b-HSD1 mRNA level was elevated in parallel with the augmentation of BMI and its level was also higher in visceral fat than in subcutaneous fat.
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Fig. 4. The mRNA expression levels in human subcutaneous and visceral adipose tissue. Both subcutaneous and visceral adipose tissues were obtained when the patients were underwent abdominal surgery. Real-time quantitative PCR was performed and values were normalized to the level of 18s mRNA.
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4. Discussion The main findings of the present study were: (1) MR and its target gene mRNA levels were significantly increased in obese adipose tissues. (2) Physiological concentration of glucocorticoid caused the increase of adipose ROS and the adipocytokine dysregulation. (3) Glucocorticoid-induced ROS and adipocytokine dysregulation were significantly reversed by blockade of MR, not by GR antagonism. (4) In human visceral adipose tissue, MR mRNA level was increased as the BMI increased, while no apparent correlation between GR mRNA level and BMI was observed. The binding affinities of glucocorticoids for MR are 10-fold higher than GR [5]. 11b-hydroxysteroid dehydrogenase type 1 (11bHSD1) converts cortisone into cortisol while 11b-HSD2 changes cortisol into cortisone. In the epithelial cells, cortisol is immediately inactivated by 11b-HSD2 and thus aldosterone exhibits its effect via MR. However, 11b-HSD1 was dominantly expressed in adipocytes [6] and its mRNA expression level is higher in obese subjects compared to non-obese subjects [14]. Adipose 11b-HSD1 mRNA level shows a positive correlation with waist circumference and insulin resistance [15,16]. Furthermore, fat-specific 11b-HSD1 transgenic mice revealed a phenotype of metabolic syndrome such as visceral obesity, hypertension, insulin resistance, and dyslipidemia [17,18], indicating that adipose glucocorticoids play an important role in the development of obesity. In adipocytes, it has been supposed that glucocorticoid action should be mediated by the cytosolic GR. However, in heart and kidney, several experiments demonstrated that glucocorticoids exhibit its effect mainly via MR [19,20]. We here showed for the first time that dysregulations of adipocytokine and increases of ROS were caused by the physiological concentration of glucocorticoids mainly via MR in adipocytes. Interestingly, co-treatment with glucocorticoids and H2O2 caused the additional suppression of adiponectin mRNA level (Fig. 4, lane 3 versus 5 versus 7 and lane 3 versus 9 versus 11), which was consistent with the previous reports demonstrating that the glucocorticoid-MR action was augmented by the excess of ROS [10]. Patients with excessive glucocorticoids, such as cushing syndrome and chronic administration with glucocorticoid, exhibit clinical phenotypes like a metabolic syndrome. Present study suggests that glucocorticoid-induced metabolic disorders might be completely or partly ameliorated by blockade of MR. Previous reports showed that MR blockade incompletely reversed the high dose cortisol-induced Na+ retention [21] and the ACTH stimulated high concentration glucocorticoid-induced hypertention [22]. The incomplete effect of MR blockade on such glucocorticoid-induced phenomena may be accounted for by extremely high concentration of glucocorticoid in these cases. High concentration of glucocorticoid exhibits its effect through both GR and MR [23]. In this case, glucocorticoid-GR pathway may be more dominant than glucocoid-MR pathway. In fact, the action of corticoids, both glucocorticoid and mineralocorticoid, is not fully clarified and there are some contradictory reports about corticoid actions, because physiological effect of these corticoids may depend on several intracellular conditions such as the balance of 11b-HSD1 and 11b-HSD2 activities and the redox states. Importantly, we for the first time showed that MR mRNA level of human fat was increased in parallel with the increase of BMI and especially its mRNA level of visceral fat was dramatically up-regulated according to the increase of BMI, while GR mRNA level of human adipose tissue was not related to BMI and fat distribution (Fig. 4). Furthermore, as the BMI increases, PPARc2 mRNA level was significantly suppressed and NADPH oxidase subunit p22 mRNA level was dramatically increased in visceral fat compared to subcutaneous fat. The activation of PPARc reduces the increased ROS in adipose tissues [24] and the increase of NADPH oxidase
subunits caused the excess of ROS [2]. Furthermore, more increase of 11b-HSD1 mRNA should supply active glucocorticoid for MR activation in accumulated visceral fat. Collectively, as the BMI increases, human visceral fat tissues may tend to fall into the excess of ROS and the visceral fat MR mRNA level was increased, resulting that the glucocorticoid-MR action may be augmented in visceral fat tissues. Our present and previous [3] studies may indicate that the glucocorticoid-MR pathway should be activated in obese adipocytes (especially in obese visceral adipocytes) through the increases of 11b-HSD1, MR, and ROS, resulting in adipocytokine dysregulation, adipose inflammation, and excess of adipose ROS. Blockade of adipose MR may cut off such vicious cycle and may be useful for the treatment of metabolic syndrome, although clinical trials about MR blockade therapy as treatment for the metabolic syndrome should be awaited in the future. Funding This work was supported by Grants-in-Aid for Scientific Research (C) No. 22590979 (to N. M.), and Scientific Research on Innovative Areas No. 22126008 (to T. F.). Acknowledgments We are grateful to Pfizer Inc (New York, NY) for providing eplerenone. We thank Miyuki Nakamura for the excellent technical assistance. We also thank all members of the III laboratory (Adiposcience laboratory), Department of Metabolic Medicine, Osaka University for helpful discussions on the project. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2012.01.139. References [1] Y. Matsuzawa, T. Funahashi, S. Kihara, I. Shimomura, Adiponectin and metabolic syndrome, Arterioscler. Thromb. Vasc. Biol. 24 (2004) 29–33. [2] S. Furukawa, T. Fujita, M. Shimabukuro, M. Iwaki, Y. Yamada, Y. Nakajima, O. Nakayama, M. Makishima, M. Matsuda, I. Shimomura, Increased oxidative stress in obesity and its impact on metabolic syndrome, J. Clin. Invest. 114 (2004) 1752–1761. [3] A. Hirata, N. Maeda, A. Hiuge, T. Hibuse, K. Fujita, T. Okada, S. Kihara, T. Funahashi, I. Shimomura, Blockade of mineralocorticoid receptor reverses adipocyte dysfunction and insulin resistance in obese mice, Cardiovasc. Res. 84 (2009) 164–172. [4] C. Guo, V. Ricchiuti, B.Q. Lian, T.M. Yao, P. Coutinho, J.R. Romero, J. Li, G.H. Williams, G.K. Adler, Mineralocorticoid receptor blockade reverses obesityrelated changes in expression of adiponectin, peroxisome proliferator-activated receptor-gamma, and proinflammatory adipokines, Circulation 117 (2008) 2253–2261. [5] J. Funder, K. Myles, Exclusion of corticosterone from epithelial mineralocorticoid receptors is insufficient for selectivity of aldosterone action: in vivo binding studies, Endocrinology 137 (1996) 5264–5268. [6] J.W. Funder, Mineralocorticoid receptors: distribution and activation, Heart Fail. Rev. 10 (2005) 15–22. [7] I.J. Bujalska, S. Kumar, P.M. Stewart, Does central obesity reflect ‘‘cushing’s disease of the omentum’’?, Lancet 349 (1997) 1210–1213 [8] N. Maeda, M. Takahashi, T. Funahashi, S. Kihara, H. Nishizawa, K. Kishida, H. Nagaretani, M. Matsuda, R. Komuro, N. Ouchi, H. Kuriyama, K. Hotta, T. Nakamura, I. Shimomura, Y. Matsuzawa, PPAR gamma ligands increase expression and plasma concentrations of adiponectin, an adipose-derived protein, Diabetes 50 (2001) 2094–2099. [9] A. Kurata, H. Nishizawa, S. Kihara, N. Maeda, M. Sonoda, T. Okada, K. Ohashi, T. Hibuse, K. Fujita, A. Yasui, A. Hiuge, M. Kumada, H. Kuriyama, I. Shimomura, T. Funahashi, Blockade of Angiotensin II type-1 receptor reduces oxidative stress in adipose tissue and ameliorates adipocytokine dysregulation, Kidney Int. 70 (2006) 1717–1724. [10] H. Hitomi, H. Kiyomoto, A. Nishiyama, T. Hara, K. Moriwaki, K. Kaifu, G. Ihara, Y. Fujita, T. Ugawa, M. Kohno, Aldosterone suppresses insulin signaling via the downregulation of insulin receptor substrate-1 in vascular smooth muscle cells, Hypertension 50 (2007) 750–755.
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