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Resistin mRNA levels are downregulated by estrogen in vivo and in vitro
FEBS 29156
FEBS Letters 579 (2005) 449–454
Resistin mRNA levels are downregulated by estrogen in vivo and in vitro Seng-Wong Huanga,1, Kok-Min Seowb,d,g,i,1, Low-Tone Hoa,b,c,e,f, Yueh Chienc,e, Dong-Yue Chungc, Chih-Ling Changc, Ying-Hsiu Laic, Jiann-Loung Hwangd,h, Chi-Chang Juanb,c,e,* a School of Medicine, National Yang-Ming University, Taipei, Taiwan Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan c Institute of Physiology, No. 155, Sec. 2, Li-Nong St., National Yang-Ming University, Taipei, Taiwan d Department of Obstetrics and Gynecology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan e Department of Medical Research & Education, Taipei, Taiwan f Department of Internal Medicine, Taipei Veterans General Hospital, Taipei, Taiwan g School of Medicine, Fu-Jen Catholic University, Hsinchuang, Taipei county, Taiwan h Department of Obstetrics and Gynecology, Taipei Medical University, Taipei, Taiwan i Department of Obstetrics and Gynecology, E-Da Hospital/I-Shou University, Kaohsiung County, Taiwan b
Received 3 November 2004; revised 30 November 2004; accepted 4 December 2004 Available online 18 December 2004 Edited by Robert Barouki
Abstract Resistin, a hormone secreted by adipocytes, is suggested to be an important link between obesity and diabetes. The aim of this study was to evaluate the regulatory effect of estrogen on adipocyte resistin gene expression in ovariectomized (OVX) rats and in isolated rat adipocytes in vitro. Subcutaneous injection of estradiol benzoate reduced resistin mRNA levels in adipocytes isolated from the inguinal, parametrial, perirenal, retroperitoneal, or periovarian fat deposits of OVX rats, while an in vitro study showed that estradiol treatment decreased resistin mRNA levels in cultured rat periovarian fat adipocytes. Results of Western blotting analysis also showed that estrogen decreased adipose resistin contents in vivo and in vitro. These data suggest that estrogen is a pivotal negative regulator of resistin gene expression. 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Resistin; Estrogen; Adipocyte; Ovariectomy
1. Introduction Resistin, a 12.5 kDa cysteine-rich polypeptide specifically secreted by adipocytes [1], belongs to a multigene family termed resistin-like molecules (RELMs) or ‘‘found in inflammatory zone’’ (FIZZ) proteins [2,3]. Resistin gene expression increases markedly during the differentiation of 3T3-L1 cells and primary preadipocytes into adipocytes [1,4]. Serum resistin levels are significantly elevated in insulin-resistant mice and in mice which are genetically obese or obese due to a high fat diet (HFD) [1]. Immunoneutralization of endogenous resistin significantly improves insulin sensitivity in diet-induced obese mice, while administration of recombinant resistin impairs glucose tolerance in C57B1/6J mice in vivo [1]. In addition, resistin gene expression is downregulated in animals treated with * Corresponding author. Fax: +886 2 28264049. E-mail address: [email protected] (C.-C. Juan). 1
These authors contributed equally, as co-first authors, to this study.
thiazolidinedione compounds, a new class of antidiabetic drugs with an insulin-sensitizing action [1]. These findings suggest that resistin serves as a hormonal link between obesity and insulin resistance. In a human study, resistin mRNA levels in adipose tissue were increased in morbidly obese humans compared to lean control subjects [5]. A genetic epidemiological study of Caucasian families suggested that single nucleotide polymorphisms in the resistin gene promoter region may alter the negative correlation between the BMI and the insulin sensitivity index in non-diabetic family members of type 2 diabetics [6]. We recently developed a new method for production of bioactive recombinant resistin in Escherichia coli, carried out a structural characterization of the product, and found that it dose-dependently antagonized insulin-mediated glucose uptake in 3T3-L1 adipocytes [7]. Thus, resistin may play an important role in modulation of insulin sensitivity. To examine the possible pathogenic role of resistin in diabetes and obesity, it is important to study the endocrine mechanisms that regulate resistin expression and secretion. Several studies have shown that resistin expression is regulated by hormones, including glucocorticoids [8], endothelin-1 [9], growth hormone [10], thyroid hormone [11], and tumor necrosis factor-a [12]. Adipose resistin mRNA levels are higher in male rats than in female rats [11] and testosterone increases adipose tissue resistin mRNA levels in male rats [11,13]. In addition, pituitary resistin mRNA levels are also significantly higher in untreated male mice compared to females and testosterone treatment of male mice, but not females, increases resistin mRNA levels [14]. These observations suggest that another sex hormone(s) may contribute to the gender difference in resistin expression, estrogen being a possible candidate. Effects of estrogen on adipocytokine secretion have been demonstrated in several studies. Estrogen stimulates leptin secretion both in vitro and in vivo [15]. In addition, ovariectomy does not interfere with the pubertal rise in adiponectin in infant mice, but increases adiponectin mRNA and protein levels in adults, while estrogen decreases adiponectin mRNA and protein levels in mice in vivo and in 3T3-L1 adipocytes in vitro [16]. These findings indicate that estrogen may play an important role in the physiological modulation of adipocytokines. However, there are no data on the effect of estrogen on resistin gene
0014-5793/$22.00 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2004.12.010
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expression. The aim of the present study was: (1) to investigate, in the ovariectomized (OVX) rat model, the effect of in vivo estrogen administration on resistin mRNA levels in adipocytes from various fat deposits and (2) to determine whether in vitro estrogen treatment of adipocytes isolated from rat periovarian fat deposits could directly modulate resistin mRNA levels.
2. Materials and methods 2.1. Experimental animals and in vivo study of protocol Female Sprague–Dawley rats weighing 250–300 g were purchased from a local breeder and housed in a temperature-controlled room (22 ± 2 C) with 14 h of artificial illumination daily (6.00–20.00 h) with food and water available ad libitum. OVX rats were used to study the in vivo effects of estrogen (estradiol benzoate; EB). All animals were bilaterally ovariectomized under anesthesia. After a 2 week recovery period, the OVX rats were divided into two groups and injected subcutaneously with either EB (25 lg/kg BW/day; n = 7) or vehicle (n = 7) for three days, then decapitated the next day. A blood sample was collected from each rat after decapitation and the serum separated by centrifugation and stored at 20 C until assayed. Resistin mRNA levels in adipocytes from the inguinal (IG), perirenal (PR), parametrial (PM), retroperitoneal (RP), and periovarian (PO) adipose tissue were determined. All procedures were carried out in accordance with the Taiwan Government Guide for the Care and Use of Laboratory Animals, and the research protocol was approved by the animal welfare committee of the National Yang-Ming University. 2.2. In vitro culture of rat adipocytes Adipocytes isolated from the PO adipose tissue of untreated female rats (no OVX, no drug treatment) weighing 250–300 g were precultured for 5 h in DulbeccoÕs modified EagleÕs medium/HamÕs F12 containing 20 lg/ml of bovine serum albumin (BSA), 20 mM HEPES, 0.1 lg/l of sodium selenite and 4.88 mg/l of ethanolamine at 37 C in 95% air and 5% CO2 [17]. The concentration of the adipocyte suspension was then adjusted to 5 · 105 cell/ml in the same medium and 10 ml aliquots added to several flasks. The cells in one flask served as the untreated control, while the others were incubated either with 107 M EB for different time periods or with different concentrations of EB for 24 h, then the medium was removed from beneath the adipocyte layer and the cells transferred to tubes for a brief gentle centrifugation to remove any residual medium before total RNA was extracted. 2.3. Adipocyte isolation IG, PR, PM, RP, and PO adipose tissue from each individual rat was excised, weighed, and used to prepare adipocytes as described by Rodbell [18] with minor modifications. The adipose tissue was finely minced, then a cell suspension was prepared by gentle shaking for 60 min at 37 C in Krebs–Ringer bicarbonate (KRB) buffer containing 1 mmol/l of pyruvate, 1% BSA, and 0.1% collagenase. The cell suspension was filtered through a 400 lm nylon mesh and centrifuged at 100 · g for 1 min at room temperature, then the supernatant was harvested and washed three times with 50 ml of KRB buffer containing 1 mmol/l of pyruvate and 1% BSA. To count the cells, an aliquot of diluted cell suspension was fixed overnight in collidine buffer containing 2% osmium tetroxide prior to microscopic photography. 2.4. RNA extraction Total RNA was extracted from adipocytes isolated from each individual rat using a Tri Reagent kit (Sigma–Aldrich, Inc., St. Louis, MO). The integrity of the RNA was examined by 1% agarose gel electrophoresis and its concentration determined by the UV absorbance at 260 nm (Genequant RNA/DNA calculator; Pharmacia, LKB Biochrom, UK). 2.5. Northern blotting Northern blotting was carried out according to a standard procedure as described previously [19]. 32P-labeled full-length rat resistin cDNA and control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA (1.1 kb, from Clontech, Palo Alto, CA) hybridiza-
S.-W. Huang et al. / FEBS Letters 579 (2005) 449–454 tion probes were prepared using the random priming DNA labeling system. After hybridization, the signals were quantified by densitometry and the signals for the two resistin mRNAs (1.4 and 0.8 kb, due to different sizes of the 3 0 -untranslated region) summed and normalized to that of the corresponding GAPDH mRNA band. 2.6. Western blotting Whole cell lysate was made by sonication in lysis buffer (1% Triton X-100, 50 mM KCl, 25 mM HEPES, pH 7.8, 10 lg/ml leupeptin, 20 lg/ml aprotinin, 125 lM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride). Samples (100 lg of total protein) in 50 ll of Laemmli sample buffer were boiled for 10 min and resolved on 15% miniSDS–PAGE. The contents of the gel were then transferred onto polyvinylidene difluoride membrane. The membrane was pre-blotted in 5% skimmed milk in phosphate-buffered saline (PBS) for 30 min at room temperature and then immunoblotted with anti-resistin (Linco Research, Inc., St. Charles, MO) or anti-a-tubulin (Sigma–Aldrich) antibody for 24 h at 4 C followed by horseradish peroxidase-conjugated secondary antibody for 60 min at room temperature, followed by revelation with chemiluminescence reagent (Amersham Biosciences, Buckinghamshire, England). 2.7. Measurement of serum glucose, insulin, and estradiol Serum glucose concentrations were measured using a glucose analyzer (Model 23A, Yellow Springs Instrument, Yellow Springs, OH, USA). Serum insulin and estradiol concentrations were measured using commercial kits (Mercodia AB, Uppsala, Sweden; R&D systems Inc., Minneapolis, MN, USA). 2.8. Measurement of the insulin resistance indices The fasting glucose to insulin ratio (G0/I0) was measured as described previously [20]. The homeostasis model assessment of insulin resistance (HOMA-IR) value was calculated as: fasting glucose (mmol/l) · fasting insulin (lIU/l)/22.5 [21]. 2.9. Statistical analysis All results are expressed as means ± S.D. Differences between groups were analyzed using StudentÕs t test with a P value <0.05 being considered significant.
3. Results 3.1. Basic data and biochemical parameters No significant difference was observed in the body weight of OVX and EB-treated OVX (OVX + EB) rats (286 ± 39.6 vs. 278 ± 37.6 g). Serum estradiol levels were significantly higher in OVX + EB rats than in OVX rats (Table 1, P < 0.05), as was uterine weight (43 ± 0.8 vs. 19 ± 0.2 mg, P < 0.0001). There was no difference in fasting serum glucose levels between the two groups, but the fasting serum insulin concentration was significantly higher in OVX + EB rats than in OVX rats (Table 1), suggesting that EB administration induced a hyperinsulinemic status in OVX rats. The changes in the G0/I0 and HOMA-IR (Table 1) also indicated that OVX + EB rats were more insulin-resistant than OVX rats. 3.2. Estrogen decreases resistin mRNA levels in adipocytes from various anatomical sites in OVX rats The effect of estrogen on adipose resistin mRNA levels, evaluated by Northern blotting, was examined in vivo in OVX and OVX + EB rats. Fig. 1, a representative Northern blot of PM adipocytes using a resistin-specific probe, shows two transcripts of 0.8 and 1.4 kb in length, the same sizes reported previously for rat resistin [4], and that resistin mRNA levels were lower in PM adipocytes isolated from OVX + EB rats than in those isolated from OVX rats. Fig. 2 shows the results
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Table 1 Serum levels of estradiol, glucose, and insulin and insulin resistance indices in OVX and OVX + EB rats Group
OVX
OVX + EB
P
Estradiol (pg/ml) Glucose (mg/dl) Insulin (lU/ml) G0/I0 HOMA-IR
15 ± 3.1 128 ± 10.3 14 ± 6.9 11 ± 5.4 4 ± 2.3
202 ± 4.8 125 ± 12.9 28 ± 8.8 5 ± 1.2 9 ± 3.6
0.019 NS 0.006 0.019 0.018
G0/I0: fasting glucose-to-insulin ratio. HOMA-IR: Homeostasis model insulin resistance index. A P value <0.05 was taken as significant.
Fig. 2. Resistin gene expression as a function of the adipose tissue site. Total RNA was isolated from IG (A), PM (B), PR (C), RP (D), or PO (E) adipocytes and resistin mRNA levels measured by Northern blotting and densitometric analysis, as described in Section 2, and normalized to the GAPDH mRNA levels. The results are means ± S.D. for seven rats. *P < 0.05 compared to the OVX group. Fig. 1. Resistin mRNA levels are lower in PM adipocytes from OVX + EB rats than in those from OVX rats. Total RNA isolated from PM adipocytes of OVX and OVX + EB rats was subjected to Northern blotting (15 lg RNA per lane) using rat resistin and GAPDH probes. The figure shows a Northern blot and a picture of the ethidium bromide-stained gel to assess the amount and quality of the RNA; this result is representative of those of three separate experiments. Two rat resistin mRNAs with sizes of 1.4 and 0.8 kb are seen due to differences in the 3 0 -untranslated region.
of densitometric analysis of Northern blots of resistin mRNA levels in adipose tissue from the IG, PM, PR, RP, and PO fat deposits. In OVX rats, resistin mRNA levels in IG-isolated adipocytes were significantly lower than those isolated from the other fat deposits. In OVX + EB rats, resistin mRNA levels in adipocytes isolated from IG, PM, PR, RP, and PO adipose tissue were, respectively, 70%, 73%, 53%, 67% and 90% lower than the corresponding value in the OVX group (P < 0.05). 3.3. Resistin gene expression in cultured adipocytes is downregulated by estrogen in vitro When adipocytes isolated from the PO adipose tissue of untreated non-OVX female rats were used to test the in vitro effect of estradiol on resistin gene expression, the results showed that estradiol suppressed resistin gene expression in a timedependent manner (Fig. 3A). A significant 30% decrease in resistin mRNA levels was detected after 4 h exposure to 107 M estradiol, while the maximal decrease (50%) was seen after 24 h exposure (Fig. 3A). When adipocytes from PO fat deposits were cultured, then treated for 24 h with 1012, 1010, 108, or 106 M estradiol, resistin mRNA levels decreased in a dose-dependent manner, with a significant 20% decrease being
Fig. 3. Resistin mRNA levels in rat periovarian adipocytes are reduced by in vitro estradiol treatment. Resistin mRNA levels in adipocytes after in vitro exposure to 107 M estradiol for 4, 8, or 24 h (A), or to 1012, 1010, 108, 106 M of estradiol for 24 h (B). The results are means ± S.D. for four separate experiments, each using pooled adipocytes from six rats. *P < 0.05 compared to the control group.
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Fig. 4. Intracellular resistin contents are lower in PM adipocytes from OVX + EB rats than in those from OVX rats. Cell lysate from PM adipocytes of OVX and OVX + EB rats was subjected to Western blotting (100 lg protein per lane) using anti-resistin and anti-a-tubulin antibodies. This blot is representative of those of three separate experiments.
Fig. 5. Intracellular resistin contents in adipocytes are decreased by in vitro estradiol treatment. Adipocytes isolated from the PO adipose tissue of untreated non-OVX female rats were in vitro exposed to 107 M estradiol for 24 h. Intracellular resistin contents were measured by Western blotting and densitometric analysis, as described in Section 2, and normalized to the a-tubulin levels. The results are means ± S.D. for three separate experiments, each using pooled adipocytes from six rats. *P < 0.05 compared to the control group.
seen in adipocytes treated with 1010 M estradiol and the maximal decrease (50%) being seen with 108M estradiol (Fig. 3B). 3.4. Intracellular resistin contents in adipocytes are decreased by estrogen in vivo and in vitro The effect of estrogen on adipose resistin contents, evaluated by Western blotting, was also examined in vivo in OVX and OVX + EB rats. In OVX + EB rats, intracellular resistin contents in adipocytes isolated from PM adipose tissue were significantly lower than that in the OVX group (Fig. 4). In addition, rat adipocytes were treated in vitro with estradiol (107 M) or vehicle for 24 h and intracellular resistin contents were determined. Results showed that estradiol decreased intracellular resistin contents in cultured rat adipocytes (Fig. 5).
4. Discussion The present study demonstrated that in vivo treatment with estrogen suppressed resistin gene expression in adipocytes isolated from various white adipose tissues of OVX rats. In addition, in vitro estrogen treatment also decreased resistin gene expression in cultured adipocytes isolated from the PO adipose tissue of untreated non-OVX female rats. Resistin gene expression in OVX female rats was lower in adipocytes isolated from
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subcutaneous adipose tissue (IG) than in those isolated from abdominal adipose tissues (PM, PR, RP, and PO) (Fig. 2). In humans, more resistin is released by omental adipose tissue than by subcutaneous adipose tissue [22]. The increased expression of the resistin gene in central adipose tissue supports the hypothesis of a role for resistin in linking central obesity and type 2 diabetes and/or cardiovascular diseases [23]. Further studies are needed to investigate this possibility. The present study showed regional differences in the suppression of resistin gene expression by estrogen in vivo. Maximal inhibition of resistin gene expression was seen in PO adipocytes isolated from estrogen-treated OVX rats (Fig. 2E), showing that the effect of estrogen was greater on PO adipocytes than on adipocytes isolated from other anatomical sites in OVX rats. Site-specific regulation of estrogen has been discussed in several reports. An in vitro study showed that exposure to estrogen induces a 1.4-, 1.2-, or 1.75-fold increase in leptin mRNA levels in subcutaneous, PR, and PM adipocytes, respectively [15]. Pedersen et al. [24] also reported that estrogen lowers the lipolytic response in subcutaneous fat tissue, but seems not to affect lipolysis in adipocytes from the intraabdominal fat tissue. One possible explanation for this site-specific regulation by estrogen is the differential distribution of estrogen receptors (ERs) in various adipose tissues. Estrogens mediate their effects through two receptor isoforms, ER-a and ER-b, each of which is responsible for different biological functions as indicated by their tissue-specific expression patterns. Different roles have been assigned to ER-a and ER-b in mediating the effects of estrogen in adipose tissue. This was suggested by the observations that ER-a gene knockout mice develop obesity, whereas ER-b knockout mice have a normal amount of adipose tissue [25,26], and the fact that, in human adipocytes, ER-b mRNA levels are much lower than ER-a mRNA levels [27]. These findings indicated that the ER-a subtype may play a predominant role in mediating the effects of estrogens on adipocytes. In the present study, we found that in vivo treatment of OVX rats with EB resulted in lower resistin gene expression than in non-EB-treated rats (Fig. 2E) and such decreased expression was also observed after in vitro estradiol treatment of PO adipocytes (Fig. 3). However, the degree of the estrogeninduced decrease in adipocyte resistin mRNA levels was higher in vivo than in vitro, since plasma levels in the in vivo study were about 200 pg/ml (5 · 1010 M) and gave a 90% decrease, whereas the maximal effect in vitro was seen at concentrations of 108 and 106 M and consisted of only a 50% decrease. The difference in resistin suppression caused by in vivo and in vitro estrogen treatment may indicate that estrogen is not the only factor involved in the regulation of resistin expression in vivo in OVX + EB rats and that the decreased resistin gene expression may be due to a synergic effect of estrogen and other factors. As shown in Table 1, serum insulin levels were significantly higher in OVX + EB rats than in OVX rats. Haugen et al. [28] have shown that insulin has a marked suppressive effect on resistin mRNA levels in 3T3-L1 adipocytes. In addition to estrogen, insulin may be a possible candidate inhibitor of resistin expression in OVX + EB rats. On the other hand, study also demonstrated that estrogen increases adipose leptin expression [15]. Results of recent studies showed that leptin treatment decreases adipose resistin expression and circulating resistin level in mice [29,30]. It was also possible that
S.-W. Huang et al. / FEBS Letters 579 (2005) 449–454
estrogen suppressed adipose resistin expression through the estrogen-induced leptin overexpression in vivo. In our in vivo study, OVX + EB rats had higher insulin levels, a higher HOMA-IR, and a lower glucose/insulin ratio than OVX rats, showing that they developed hyperinsulinemia and insulin resistance. Several lines of evidence suggest an association between estrogen and insulin sensitivity. Estrogen has been proposed to cause insulin resistance in 3T3-L1 adipocytes by reducing the cellular content of insulin receptor substrate proteins [31]. However, there is considerable evidence against an adverse effect role of estrogen on glucose metabolism and for a beneficial action of estrogen. In male mice with complete aromatase deficiency, which are unable to catalyze the formation of estrogens from androgens, insulin resistance progressively develops [32]. OVX mice are more insulin-resistant than sham controls and treatment of these OVX mice with estrogen normalizes both glucose tolerance and insulin sensitivity [33]. The role of estrogen in hormone replacement therapy (HRT) also needs to be settled. Despite a recent review claiming that HRT reduces the incidence of type 2 diabetes [34], other data do not fit to this concept. For example, HRT is reported to be associated with attenuated insulin sensitivity [35] and with a worsening of coronary atherosclerosis and exacerbation of the profile of inflammatory markers in women with abnormal glucose tolerance [36]. Because of these conflicting results, we believe that insulin sensitivity in OVX rats or other experimental models and in postmenopausal women may be determined by multiple factors. HRT strategies should therefore be carefully designed and optimized to minimize possible adverse effects of estrogen in terms of producing metabolic disorders. Based on our in vivo and in vitro findings, we conclude that estrogen downregulates adipocyte resistin mRNA expression in a site-specific manner and that estrogen may be a pivotal negative regulator of resistin gene expression. Acknowledgments: This study was supported by grants from the National Science Council (NSC92-2312-B-010-005) and the Veterans General Hospitals University System of Taiwan (VGHUST93-P7-37).
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nectin but barely decreased plasma resistin of ob/ob mice. Am. J. Physiol. Endocrinol. Metab. 287, E446–E453. Collison, M., Campbell, I.W., Salt, I.P., Dominiczak, A.F., Connell, J.M., Lyall, H. and Gould, G.W. (2000) Sex hormones induce insulin resistance in 3T3-L1 adipocytes by reducing cellular content of IRS proteins. Diabetologia 43, 1374–1380. Takeda, K., Toda, K., Saibara, T., Nakagawa, M., Saika, K., Onishi, T., Sugiura, T. and Shizuta, Y. (2003) Progressive development of insulin resistance phenotype in male mice with complete aromatase (CYP19) deficiency. J. Endocrinol. 176, 237–246. Siri, P.W. and Ginsberg, H.N. (2003) Ovariectomy leads to increased insulin resistance in human apolipoprotein B transgenic mice lacking brown adipose tissue. Metabolism 52, 659–661. Louet, J.F., LeMay, C. and Mauvais-Jarvis, F. (2004) Antidiabetic actions of estrogen: insight from human and genetic mouse models. Curr. Atheroscler. Rep. 6, 180–185. Brown, M.D., Korytkowski, M.T., Zmuda, J.M., McCole, S.D., Moore, G.E. and Hagberg, J.M. (2000) Insulin sensitivity in postmenopausal women: independent and combined associations with hormone replacement, cardiovascular fitness, and body composition. Diabetes Care 23, 1731–1736. Howard, B.V., Hsia, J., Ouyang, P., Van Voorhees, L., Lindsay, J., Silverman, A., Alderman, E.L., Tripputi, M. and Waters, D.D. (2004) Postmenopausal hormone therapy is associated with atherosclerosis progression in women with abnormal glucose tolerance. Circulation 110, 201–206.
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Resistin mRNA levels are downregulated by estrogen in vivo and in vitro Seng-Wong Huanga,1, Kok-Min Seowb,d,g,i,1, Low-Tone Hoa,b,c,e,f, Yueh Chienc,e, Dong-Yue Chungc, Chih-Ling Changc, Ying-Hsiu Laic, Jiann-Loung Hwangd,h, Chi-Chang Juanb,c,e,* a School of Medicine, National Yang-Ming University, Taipei, Taiwan Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan c Institute of Physiology, No. 155, Sec. 2, Li-Nong St., National Yang-Ming University, Taipei, Taiwan d Department of Obstetrics and Gynecology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan e Department of Medical Research & Education, Taipei, Taiwan f Department of Internal Medicine, Taipei Veterans General Hospital, Taipei, Taiwan g School of Medicine, Fu-Jen Catholic University, Hsinchuang, Taipei county, Taiwan h Department of Obstetrics and Gynecology, Taipei Medical University, Taipei, Taiwan i Department of Obstetrics and Gynecology, E-Da Hospital/I-Shou University, Kaohsiung County, Taiwan b
Received 3 November 2004; revised 30 November 2004; accepted 4 December 2004 Available online 18 December 2004 Edited by Robert Barouki
Abstract Resistin, a hormone secreted by adipocytes, is suggested to be an important link between obesity and diabetes. The aim of this study was to evaluate the regulatory effect of estrogen on adipocyte resistin gene expression in ovariectomized (OVX) rats and in isolated rat adipocytes in vitro. Subcutaneous injection of estradiol benzoate reduced resistin mRNA levels in adipocytes isolated from the inguinal, parametrial, perirenal, retroperitoneal, or periovarian fat deposits of OVX rats, while an in vitro study showed that estradiol treatment decreased resistin mRNA levels in cultured rat periovarian fat adipocytes. Results of Western blotting analysis also showed that estrogen decreased adipose resistin contents in vivo and in vitro. These data suggest that estrogen is a pivotal negative regulator of resistin gene expression. 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Resistin; Estrogen; Adipocyte; Ovariectomy
1. Introduction Resistin, a 12.5 kDa cysteine-rich polypeptide specifically secreted by adipocytes [1], belongs to a multigene family termed resistin-like molecules (RELMs) or ‘‘found in inflammatory zone’’ (FIZZ) proteins [2,3]. Resistin gene expression increases markedly during the differentiation of 3T3-L1 cells and primary preadipocytes into adipocytes [1,4]. Serum resistin levels are significantly elevated in insulin-resistant mice and in mice which are genetically obese or obese due to a high fat diet (HFD) [1]. Immunoneutralization of endogenous resistin significantly improves insulin sensitivity in diet-induced obese mice, while administration of recombinant resistin impairs glucose tolerance in C57B1/6J mice in vivo [1]. In addition, resistin gene expression is downregulated in animals treated with * Corresponding author. Fax: +886 2 28264049. E-mail address: [email protected] (C.-C. Juan). 1
These authors contributed equally, as co-first authors, to this study.
thiazolidinedione compounds, a new class of antidiabetic drugs with an insulin-sensitizing action [1]. These findings suggest that resistin serves as a hormonal link between obesity and insulin resistance. In a human study, resistin mRNA levels in adipose tissue were increased in morbidly obese humans compared to lean control subjects [5]. A genetic epidemiological study of Caucasian families suggested that single nucleotide polymorphisms in the resistin gene promoter region may alter the negative correlation between the BMI and the insulin sensitivity index in non-diabetic family members of type 2 diabetics [6]. We recently developed a new method for production of bioactive recombinant resistin in Escherichia coli, carried out a structural characterization of the product, and found that it dose-dependently antagonized insulin-mediated glucose uptake in 3T3-L1 adipocytes [7]. Thus, resistin may play an important role in modulation of insulin sensitivity. To examine the possible pathogenic role of resistin in diabetes and obesity, it is important to study the endocrine mechanisms that regulate resistin expression and secretion. Several studies have shown that resistin expression is regulated by hormones, including glucocorticoids [8], endothelin-1 [9], growth hormone [10], thyroid hormone [11], and tumor necrosis factor-a [12]. Adipose resistin mRNA levels are higher in male rats than in female rats [11] and testosterone increases adipose tissue resistin mRNA levels in male rats [11,13]. In addition, pituitary resistin mRNA levels are also significantly higher in untreated male mice compared to females and testosterone treatment of male mice, but not females, increases resistin mRNA levels [14]. These observations suggest that another sex hormone(s) may contribute to the gender difference in resistin expression, estrogen being a possible candidate. Effects of estrogen on adipocytokine secretion have been demonstrated in several studies. Estrogen stimulates leptin secretion both in vitro and in vivo [15]. In addition, ovariectomy does not interfere with the pubertal rise in adiponectin in infant mice, but increases adiponectin mRNA and protein levels in adults, while estrogen decreases adiponectin mRNA and protein levels in mice in vivo and in 3T3-L1 adipocytes in vitro [16]. These findings indicate that estrogen may play an important role in the physiological modulation of adipocytokines. However, there are no data on the effect of estrogen on resistin gene
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expression. The aim of the present study was: (1) to investigate, in the ovariectomized (OVX) rat model, the effect of in vivo estrogen administration on resistin mRNA levels in adipocytes from various fat deposits and (2) to determine whether in vitro estrogen treatment of adipocytes isolated from rat periovarian fat deposits could directly modulate resistin mRNA levels.
2. Materials and methods 2.1. Experimental animals and in vivo study of protocol Female Sprague–Dawley rats weighing 250–300 g were purchased from a local breeder and housed in a temperature-controlled room (22 ± 2 C) with 14 h of artificial illumination daily (6.00–20.00 h) with food and water available ad libitum. OVX rats were used to study the in vivo effects of estrogen (estradiol benzoate; EB). All animals were bilaterally ovariectomized under anesthesia. After a 2 week recovery period, the OVX rats were divided into two groups and injected subcutaneously with either EB (25 lg/kg BW/day; n = 7) or vehicle (n = 7) for three days, then decapitated the next day. A blood sample was collected from each rat after decapitation and the serum separated by centrifugation and stored at 20 C until assayed. Resistin mRNA levels in adipocytes from the inguinal (IG), perirenal (PR), parametrial (PM), retroperitoneal (RP), and periovarian (PO) adipose tissue were determined. All procedures were carried out in accordance with the Taiwan Government Guide for the Care and Use of Laboratory Animals, and the research protocol was approved by the animal welfare committee of the National Yang-Ming University. 2.2. In vitro culture of rat adipocytes Adipocytes isolated from the PO adipose tissue of untreated female rats (no OVX, no drug treatment) weighing 250–300 g were precultured for 5 h in DulbeccoÕs modified EagleÕs medium/HamÕs F12 containing 20 lg/ml of bovine serum albumin (BSA), 20 mM HEPES, 0.1 lg/l of sodium selenite and 4.88 mg/l of ethanolamine at 37 C in 95% air and 5% CO2 [17]. The concentration of the adipocyte suspension was then adjusted to 5 · 105 cell/ml in the same medium and 10 ml aliquots added to several flasks. The cells in one flask served as the untreated control, while the others were incubated either with 107 M EB for different time periods or with different concentrations of EB for 24 h, then the medium was removed from beneath the adipocyte layer and the cells transferred to tubes for a brief gentle centrifugation to remove any residual medium before total RNA was extracted. 2.3. Adipocyte isolation IG, PR, PM, RP, and PO adipose tissue from each individual rat was excised, weighed, and used to prepare adipocytes as described by Rodbell [18] with minor modifications. The adipose tissue was finely minced, then a cell suspension was prepared by gentle shaking for 60 min at 37 C in Krebs–Ringer bicarbonate (KRB) buffer containing 1 mmol/l of pyruvate, 1% BSA, and 0.1% collagenase. The cell suspension was filtered through a 400 lm nylon mesh and centrifuged at 100 · g for 1 min at room temperature, then the supernatant was harvested and washed three times with 50 ml of KRB buffer containing 1 mmol/l of pyruvate and 1% BSA. To count the cells, an aliquot of diluted cell suspension was fixed overnight in collidine buffer containing 2% osmium tetroxide prior to microscopic photography. 2.4. RNA extraction Total RNA was extracted from adipocytes isolated from each individual rat using a Tri Reagent kit (Sigma–Aldrich, Inc., St. Louis, MO). The integrity of the RNA was examined by 1% agarose gel electrophoresis and its concentration determined by the UV absorbance at 260 nm (Genequant RNA/DNA calculator; Pharmacia, LKB Biochrom, UK). 2.5. Northern blotting Northern blotting was carried out according to a standard procedure as described previously [19]. 32P-labeled full-length rat resistin cDNA and control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA (1.1 kb, from Clontech, Palo Alto, CA) hybridiza-
S.-W. Huang et al. / FEBS Letters 579 (2005) 449–454 tion probes were prepared using the random priming DNA labeling system. After hybridization, the signals were quantified by densitometry and the signals for the two resistin mRNAs (1.4 and 0.8 kb, due to different sizes of the 3 0 -untranslated region) summed and normalized to that of the corresponding GAPDH mRNA band. 2.6. Western blotting Whole cell lysate was made by sonication in lysis buffer (1% Triton X-100, 50 mM KCl, 25 mM HEPES, pH 7.8, 10 lg/ml leupeptin, 20 lg/ml aprotinin, 125 lM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride). Samples (100 lg of total protein) in 50 ll of Laemmli sample buffer were boiled for 10 min and resolved on 15% miniSDS–PAGE. The contents of the gel were then transferred onto polyvinylidene difluoride membrane. The membrane was pre-blotted in 5% skimmed milk in phosphate-buffered saline (PBS) for 30 min at room temperature and then immunoblotted with anti-resistin (Linco Research, Inc., St. Charles, MO) or anti-a-tubulin (Sigma–Aldrich) antibody for 24 h at 4 C followed by horseradish peroxidase-conjugated secondary antibody for 60 min at room temperature, followed by revelation with chemiluminescence reagent (Amersham Biosciences, Buckinghamshire, England). 2.7. Measurement of serum glucose, insulin, and estradiol Serum glucose concentrations were measured using a glucose analyzer (Model 23A, Yellow Springs Instrument, Yellow Springs, OH, USA). Serum insulin and estradiol concentrations were measured using commercial kits (Mercodia AB, Uppsala, Sweden; R&D systems Inc., Minneapolis, MN, USA). 2.8. Measurement of the insulin resistance indices The fasting glucose to insulin ratio (G0/I0) was measured as described previously [20]. The homeostasis model assessment of insulin resistance (HOMA-IR) value was calculated as: fasting glucose (mmol/l) · fasting insulin (lIU/l)/22.5 [21]. 2.9. Statistical analysis All results are expressed as means ± S.D. Differences between groups were analyzed using StudentÕs t test with a P value <0.05 being considered significant.
3. Results 3.1. Basic data and biochemical parameters No significant difference was observed in the body weight of OVX and EB-treated OVX (OVX + EB) rats (286 ± 39.6 vs. 278 ± 37.6 g). Serum estradiol levels were significantly higher in OVX + EB rats than in OVX rats (Table 1, P < 0.05), as was uterine weight (43 ± 0.8 vs. 19 ± 0.2 mg, P < 0.0001). There was no difference in fasting serum glucose levels between the two groups, but the fasting serum insulin concentration was significantly higher in OVX + EB rats than in OVX rats (Table 1), suggesting that EB administration induced a hyperinsulinemic status in OVX rats. The changes in the G0/I0 and HOMA-IR (Table 1) also indicated that OVX + EB rats were more insulin-resistant than OVX rats. 3.2. Estrogen decreases resistin mRNA levels in adipocytes from various anatomical sites in OVX rats The effect of estrogen on adipose resistin mRNA levels, evaluated by Northern blotting, was examined in vivo in OVX and OVX + EB rats. Fig. 1, a representative Northern blot of PM adipocytes using a resistin-specific probe, shows two transcripts of 0.8 and 1.4 kb in length, the same sizes reported previously for rat resistin [4], and that resistin mRNA levels were lower in PM adipocytes isolated from OVX + EB rats than in those isolated from OVX rats. Fig. 2 shows the results
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Table 1 Serum levels of estradiol, glucose, and insulin and insulin resistance indices in OVX and OVX + EB rats Group
OVX
OVX + EB
P
Estradiol (pg/ml) Glucose (mg/dl) Insulin (lU/ml) G0/I0 HOMA-IR
15 ± 3.1 128 ± 10.3 14 ± 6.9 11 ± 5.4 4 ± 2.3
202 ± 4.8 125 ± 12.9 28 ± 8.8 5 ± 1.2 9 ± 3.6
0.019 NS 0.006 0.019 0.018
G0/I0: fasting glucose-to-insulin ratio. HOMA-IR: Homeostasis model insulin resistance index. A P value <0.05 was taken as significant.
Fig. 2. Resistin gene expression as a function of the adipose tissue site. Total RNA was isolated from IG (A), PM (B), PR (C), RP (D), or PO (E) adipocytes and resistin mRNA levels measured by Northern blotting and densitometric analysis, as described in Section 2, and normalized to the GAPDH mRNA levels. The results are means ± S.D. for seven rats. *P < 0.05 compared to the OVX group. Fig. 1. Resistin mRNA levels are lower in PM adipocytes from OVX + EB rats than in those from OVX rats. Total RNA isolated from PM adipocytes of OVX and OVX + EB rats was subjected to Northern blotting (15 lg RNA per lane) using rat resistin and GAPDH probes. The figure shows a Northern blot and a picture of the ethidium bromide-stained gel to assess the amount and quality of the RNA; this result is representative of those of three separate experiments. Two rat resistin mRNAs with sizes of 1.4 and 0.8 kb are seen due to differences in the 3 0 -untranslated region.
of densitometric analysis of Northern blots of resistin mRNA levels in adipose tissue from the IG, PM, PR, RP, and PO fat deposits. In OVX rats, resistin mRNA levels in IG-isolated adipocytes were significantly lower than those isolated from the other fat deposits. In OVX + EB rats, resistin mRNA levels in adipocytes isolated from IG, PM, PR, RP, and PO adipose tissue were, respectively, 70%, 73%, 53%, 67% and 90% lower than the corresponding value in the OVX group (P < 0.05). 3.3. Resistin gene expression in cultured adipocytes is downregulated by estrogen in vitro When adipocytes isolated from the PO adipose tissue of untreated non-OVX female rats were used to test the in vitro effect of estradiol on resistin gene expression, the results showed that estradiol suppressed resistin gene expression in a timedependent manner (Fig. 3A). A significant 30% decrease in resistin mRNA levels was detected after 4 h exposure to 107 M estradiol, while the maximal decrease (50%) was seen after 24 h exposure (Fig. 3A). When adipocytes from PO fat deposits were cultured, then treated for 24 h with 1012, 1010, 108, or 106 M estradiol, resistin mRNA levels decreased in a dose-dependent manner, with a significant 20% decrease being
Fig. 3. Resistin mRNA levels in rat periovarian adipocytes are reduced by in vitro estradiol treatment. Resistin mRNA levels in adipocytes after in vitro exposure to 107 M estradiol for 4, 8, or 24 h (A), or to 1012, 1010, 108, 106 M of estradiol for 24 h (B). The results are means ± S.D. for four separate experiments, each using pooled adipocytes from six rats. *P < 0.05 compared to the control group.
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Fig. 4. Intracellular resistin contents are lower in PM adipocytes from OVX + EB rats than in those from OVX rats. Cell lysate from PM adipocytes of OVX and OVX + EB rats was subjected to Western blotting (100 lg protein per lane) using anti-resistin and anti-a-tubulin antibodies. This blot is representative of those of three separate experiments.
Fig. 5. Intracellular resistin contents in adipocytes are decreased by in vitro estradiol treatment. Adipocytes isolated from the PO adipose tissue of untreated non-OVX female rats were in vitro exposed to 107 M estradiol for 24 h. Intracellular resistin contents were measured by Western blotting and densitometric analysis, as described in Section 2, and normalized to the a-tubulin levels. The results are means ± S.D. for three separate experiments, each using pooled adipocytes from six rats. *P < 0.05 compared to the control group.
seen in adipocytes treated with 1010 M estradiol and the maximal decrease (50%) being seen with 108M estradiol (Fig. 3B). 3.4. Intracellular resistin contents in adipocytes are decreased by estrogen in vivo and in vitro The effect of estrogen on adipose resistin contents, evaluated by Western blotting, was also examined in vivo in OVX and OVX + EB rats. In OVX + EB rats, intracellular resistin contents in adipocytes isolated from PM adipose tissue were significantly lower than that in the OVX group (Fig. 4). In addition, rat adipocytes were treated in vitro with estradiol (107 M) or vehicle for 24 h and intracellular resistin contents were determined. Results showed that estradiol decreased intracellular resistin contents in cultured rat adipocytes (Fig. 5).
4. Discussion The present study demonstrated that in vivo treatment with estrogen suppressed resistin gene expression in adipocytes isolated from various white adipose tissues of OVX rats. In addition, in vitro estrogen treatment also decreased resistin gene expression in cultured adipocytes isolated from the PO adipose tissue of untreated non-OVX female rats. Resistin gene expression in OVX female rats was lower in adipocytes isolated from
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subcutaneous adipose tissue (IG) than in those isolated from abdominal adipose tissues (PM, PR, RP, and PO) (Fig. 2). In humans, more resistin is released by omental adipose tissue than by subcutaneous adipose tissue [22]. The increased expression of the resistin gene in central adipose tissue supports the hypothesis of a role for resistin in linking central obesity and type 2 diabetes and/or cardiovascular diseases [23]. Further studies are needed to investigate this possibility. The present study showed regional differences in the suppression of resistin gene expression by estrogen in vivo. Maximal inhibition of resistin gene expression was seen in PO adipocytes isolated from estrogen-treated OVX rats (Fig. 2E), showing that the effect of estrogen was greater on PO adipocytes than on adipocytes isolated from other anatomical sites in OVX rats. Site-specific regulation of estrogen has been discussed in several reports. An in vitro study showed that exposure to estrogen induces a 1.4-, 1.2-, or 1.75-fold increase in leptin mRNA levels in subcutaneous, PR, and PM adipocytes, respectively [15]. Pedersen et al. [24] also reported that estrogen lowers the lipolytic response in subcutaneous fat tissue, but seems not to affect lipolysis in adipocytes from the intraabdominal fat tissue. One possible explanation for this site-specific regulation by estrogen is the differential distribution of estrogen receptors (ERs) in various adipose tissues. Estrogens mediate their effects through two receptor isoforms, ER-a and ER-b, each of which is responsible for different biological functions as indicated by their tissue-specific expression patterns. Different roles have been assigned to ER-a and ER-b in mediating the effects of estrogen in adipose tissue. This was suggested by the observations that ER-a gene knockout mice develop obesity, whereas ER-b knockout mice have a normal amount of adipose tissue [25,26], and the fact that, in human adipocytes, ER-b mRNA levels are much lower than ER-a mRNA levels [27]. These findings indicated that the ER-a subtype may play a predominant role in mediating the effects of estrogens on adipocytes. In the present study, we found that in vivo treatment of OVX rats with EB resulted in lower resistin gene expression than in non-EB-treated rats (Fig. 2E) and such decreased expression was also observed after in vitro estradiol treatment of PO adipocytes (Fig. 3). However, the degree of the estrogeninduced decrease in adipocyte resistin mRNA levels was higher in vivo than in vitro, since plasma levels in the in vivo study were about 200 pg/ml (5 · 1010 M) and gave a 90% decrease, whereas the maximal effect in vitro was seen at concentrations of 108 and 106 M and consisted of only a 50% decrease. The difference in resistin suppression caused by in vivo and in vitro estrogen treatment may indicate that estrogen is not the only factor involved in the regulation of resistin expression in vivo in OVX + EB rats and that the decreased resistin gene expression may be due to a synergic effect of estrogen and other factors. As shown in Table 1, serum insulin levels were significantly higher in OVX + EB rats than in OVX rats. Haugen et al. [28] have shown that insulin has a marked suppressive effect on resistin mRNA levels in 3T3-L1 adipocytes. In addition to estrogen, insulin may be a possible candidate inhibitor of resistin expression in OVX + EB rats. On the other hand, study also demonstrated that estrogen increases adipose leptin expression [15]. Results of recent studies showed that leptin treatment decreases adipose resistin expression and circulating resistin level in mice [29,30]. It was also possible that
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estrogen suppressed adipose resistin expression through the estrogen-induced leptin overexpression in vivo. In our in vivo study, OVX + EB rats had higher insulin levels, a higher HOMA-IR, and a lower glucose/insulin ratio than OVX rats, showing that they developed hyperinsulinemia and insulin resistance. Several lines of evidence suggest an association between estrogen and insulin sensitivity. Estrogen has been proposed to cause insulin resistance in 3T3-L1 adipocytes by reducing the cellular content of insulin receptor substrate proteins [31]. However, there is considerable evidence against an adverse effect role of estrogen on glucose metabolism and for a beneficial action of estrogen. In male mice with complete aromatase deficiency, which are unable to catalyze the formation of estrogens from androgens, insulin resistance progressively develops [32]. OVX mice are more insulin-resistant than sham controls and treatment of these OVX mice with estrogen normalizes both glucose tolerance and insulin sensitivity [33]. The role of estrogen in hormone replacement therapy (HRT) also needs to be settled. Despite a recent review claiming that HRT reduces the incidence of type 2 diabetes [34], other data do not fit to this concept. For example, HRT is reported to be associated with attenuated insulin sensitivity [35] and with a worsening of coronary atherosclerosis and exacerbation of the profile of inflammatory markers in women with abnormal glucose tolerance [36]. Because of these conflicting results, we believe that insulin sensitivity in OVX rats or other experimental models and in postmenopausal women may be determined by multiple factors. HRT strategies should therefore be carefully designed and optimized to minimize possible adverse effects of estrogen in terms of producing metabolic disorders. Based on our in vivo and in vitro findings, we conclude that estrogen downregulates adipocyte resistin mRNA expression in a site-specific manner and that estrogen may be a pivotal negative regulator of resistin gene expression. Acknowledgments: This study was supported by grants from the National Science Council (NSC92-2312-B-010-005) and the Veterans General Hospitals University System of Taiwan (VGHUST93-P7-37).
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