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Endocrinology, doi:10.1210/en.2006-0654
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Endocrinology Vol. 148, No. 4 1835-1842
Copyright © 2007 by The Endocrine Society

Endothelin-1 Regulates Adiponectin Gene Expression and Secretion in 3T3-L1 Adipocytes via Distinct Signaling Pathways

Chi-Chang Juan, Tung-Yueh Chuang, Chih-Ling Chang, Seng-Wong Huang and Low-Tone Ho

Departments of Physiology (C.-C.J., T.-Y.C., C.-L.C., L.-T.H.) and Surgery (S.-W.H.), School of Medicine, National Yang-Ming University, and Department of Medical Research and Education (C.-C.J., S.-W.H., L.-T.H.), Taipei Veterans General Hospital, Taipei, Taiwan

Address all correspondence and requests for reprints to: Chi-Chang Juan, Ph.D., Department of Physiology, National Yang-Ming University, No. 155, Section 2, Li-Nong Street, Taipei, Taiwan. E-mail: ccjuan{at}ym.edu.tw.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Adiponectin, which is specifically and highly expressed in adipose tissue, has pleiotropic insulin-sensitizing effects. Endothelin-1 (ET-1) is a potent vasoconstrictive peptide mainly produced by endothelial cells. We previously showed that ET-1 can induce insulin resistance in vitro and in vivo and proposed that it might regulate adiponectin expression and secretion, thus affecting the homeostasis of whole-body energy metabolism. In the present study, we explored the regulatory effects of ET-1 on adiponectin expression and secretion and the underlying mechanisms in 3T3-L1 adipocytes using Northern blotting and ELISA. ET-1 was found to cause a significant time- and dose-dependent decrease in adiponectin expression, and this effect was inhibited by the ET type A receptor (ETAR) antagonist BQ-610 but not by the ETBR antagonist BQ-788. To explore the underlying mechanism, we examined the involvement of the cAMP-dependent protein kinase A-, phospholipase A2-, protein kinase C-, and MAPK-mediated pathways using inhibitors and found that only PD98059 and U0126, inhibitors that blocked MAPK/ERK kinase’s ability to activate the ERKs, prevented ET-1-induced down-regulation of adiponectin. Furthermore, acute ET-1 treatment significantly stimulated adiponectin secretion by 3T3-L1 adipocytes, and this effect was inhibited by the ETAR antagonist BQ-610, the inositol-1,4,5-triphosphate receptor blocker 2-APB, and phospholipase C inhibitor U73122, showing that the release of adiponectin stimulated by ET-1 was mediated through the ETAR and the inositol-1,4,5-triphosphate pathway. In conclusion, ET-1 regulates adiponectin expression and secretion by two different signaling pathways in 3T3-L1 adipocytes. These findings suggested that the cardiovascular system affects adipocyte physiology by regulating the expression of adipocytokines and, consequently, energy homeostasis via vasoactive factors, such as ET-1.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ADIPONECTIN, THE PRODUCT of the ADIPOQ gene, is a 244-amino-acid protein homologous to collagen VIII and X and complement factor C1q (1). It is specifically and highly expressed in adipose tissue and is the most abundant secretory protein of adipose tissue in human plasma (1). Plasma adiponectin levels and adiponectin mRNA expression are decreased in obese humans (2) and in patients with type 2 diabetes with insulin resistance (3, 4). In addition, plasma adiponectin levels were negatively correlated with fasting plasma insulin, triglyceride, glucose, and postprandial glucose levels (4, 5). On the other hand, it was positively correlated with insulin-stimulated glucose utilization (5). In rhesus monkeys disposed to type 2 diabetes, circulating adiponectin levels decrease before the onset of hyperglycemia (6). A recent epidemiological study (7) suggested that high adiponectin levels predict increased insulin sensitivity and that this relationship affects not only insulin-stimulated glucose utilization, but also lipoprotein metabolism and insulin-mediated suppression of postprandial free fatty acid (FFA) release, suggesting pleiotropic insulin-sensitizing effects of adiponectin in humans.

Evidence indicates that adiponectin is not simply a factor passively regulated by insulin resistance and obesity, but it also actively influences metabolic processes. Fruebis et al. (8) showed that a proteolytic cleavage product of adiponectin increases fatty acid oxidation in muscle and causes weight loss in mice. In fact, adiponectin decreases insulin resistance by increasing fatty acid oxidation, thus reducing the tissue content of triglyceride in obese and diabetic mice (8, 9). Adiponectin also suppresses hepatic glucose production (10, 11). In addition, Yamauchi et al. (12) suggested that adiponectin stimulates glucose utilization and fatty acid oxidation by activating AMP-activated protein kinase. Furthermore, Kissebah et al. (13) demonstrated the presence of a quantitative-trait locus on human chromosome 3q27 (on which the adiponectin gene is located) that is strongly linked to insulin resistance syndrome in Europeans, and Vionnet et al. (14) mapped a diabetes susceptibility locus in a native French cohort to 3q27. Thus, both genetic and functional data suggest that adiponectin could be involved in the pathogenesis of insulin resistance and obesity.

Endothelin-1 (ET-1), a powerful vasoconstrictor primarily produced and secreted by endothelial cells, is released abluminally and acts on the underlying vascular smooth muscle and is the major form of endothelin in the circulation (15). ET-1 binds to two heptahelical transmembrane G protein-coupled receptors, ET type A receptor (ETAR) and ETBR, with a higher affinity for ETAR (16, 17). Several lines of evidence suggest that ET-1 may play a role in the regulation of metabolic events. Elevated plasma ET-1 levels have been reported in a number of clinical disorders associated with insulin resistance, including diabetes and obesity (18, 19). An epidemiological study showed that, in lean patients with essential hypertension, plasma ET-1 levels were higher in patients with glucose intolerance and/or hyperlipidemia than in those without these metabolic disorders or in lean normotensive subjects (20). In addition, studies also suggest that ET-1 inhibits adipocyte differentiation (21, 22). It was hypothesized that ET-1 may decrease the action of insulin by down-regulation of adiponectin expression.

The signal transduction pathways of ET-1 have been widely investigated. Binding of ET-1 to cell surface ET receptor results in the generation of a number of second messengers (23); these activate kinases or phosphatases, which, in turn, phosphorylate or dephosphorylate downstream signaling molecules, leading to a diverse range of biological responses. ET-stimulated effects can be classified into short-term and long-term responses, which are regulated through different signaling pathways. In short-term responses, ET-induced phospholipase C (PLC) activation leads to the generation of inositol-1,4,5-triphosphate (IP3), resulting in Ca2+ influx, and of diacylglycerol (DAG), resulting in protein kinase C (PKC) activation. ET-1 stimulates the translocation of cytosolic PKC to the plasma membrane, and PKC subsequently phosphorylates specific cellular proteins, resulting in multiple biological effects (24). ET-1 also activates phospholipase A2 (PLA2) in some tissues and leads to the formation of arachidonic acid metabolites, including leukotrienes, prostaglandins, and thromboxanes (25, 26). In long-term responses, ET-1 plays a significant role in the chronic modulation of cell proliferation and differentiation, and MAPK may serve as the key regulator in these nuclear and cytoplasmic events (25).

The purpose of this study was to explore the regulatory effects of ET-1 on adiponectin expression and secretion and the underlying mechanisms in 3T3-L1 adipocytes. The results showed that ET-1 down-regulated adiponectin gene expression in a dose- and time-dependent manner via the ETAR and activation of the ERK pathway. Furthermore, acute ET-1 treatment stimulated adiponectin secretion via the IP3/Ca2+ pathway. The information obtained may provide clues about the role of ET-1 in adipocytokine expression in adipose tissue and help to elucidate the physiological and pathological roles played by ET-1 and adiponectin in the cross-talk between the cardiovascular and metabolic systems.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
DMEM, penicillin, and streptomycin were obtained from Life Technologies, Inc. (Gaithersburg, MD). Fetal bovine serum was obtained from Biowest (Nuaillé, France). ET-1 was obtained from Peptide Institute, Inc. (Osaka, Japan). BQ-610 and BQ-788 were purchased from Phoenix Pharmaceutical, Inc. (Belmont, CA). D-myo-Inositol-1,4,5-trisphosphate, PD98059, SB203580, SQ22536, U0126, U73122, H7, and wortmannin were purchased from BIOMOL International, L.P. (Plymouth Meeting, PA). The Tri Reagent Kit was purchased from Molecular Research Center, Inc. (Cincinnati, OH). [{alpha}-32P]dCTP was purchased from Amersham (Aylesbury, UK). Diphenylboronic acid ethanolamine ester (also called 2-aminoethoxydiphenyl borate; 2-APB) was purchased from Aldrich Chemical (Milwaukee, WI). Isobutylmethylxanthine, dexamethasone, insulin, and all other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO).

Cell culture
3T3-L1 fibroblasts (American Type Culture Collection, Rockville, MD) were seeded onto 10-cm Petri dishes (for gene expression assay) or 12-well plates (for secretion assay) (Falcon, Becton Dickinson, NJ) and grown and maintained in high-glucose DMEM containing 100 U/ml penicillin and 100 µg/ml streptomycin (all from Life Technologies) and 10% fetal bovine serum (Biowest, Nuaillé, France) (complete medium) in 10% CO2. The differentiation procedure was that described previously (27) with minor modification. In brief, the cells were grown to 2 d post confluency, then induced to differentiate by incubation for 3 d in complete medium containing 0.5 mM isobutylmethylxanthine, 0.5 µM dexamethasone, and 1.7 µM insulin, then in complete medium containing insulin for another 3 d. The medium was then replaced with complete medium, which was changed every 3 d until the cells were fully differentiated. Typically, by d 10, more than 95% of the fibroblasts had differentiated into adipocytes as determined by staining for lipid accumulation using Oil Red O (28). Before each experiment, the cells were incubated for 6 h in the absence of serum using low-glucose DMEM containing 0.1% BSA (DMEM-BSA). This study protocol was used in all experiments.

To explore the effect of ET-1 on adiponectin secretion, 6-h serum-free 3T3-L1 adipocytes were washed three times with DMEM-BSA to remove any remaining adiponectin-containing media. Subsequently, 3T3-L1 adipocytes were treated for different times (1–24 h) with or without 10–7 M ET-1, then the culture media was aspirated and centrifuged in a microfuge to remove any nonadherent cells and the adiponectin in the supernatant was measured colorimetrically using ELISA kits. To gain an insight into the mechanism involved, the serum-free-treated adipocytes were preincubated for 1 h in the presence or absence of inhibitors or receptor blockers, washed three times with DMEM-BSA to remove any remaining adiponectin-containing media, then incubated with 10–8 M ET-1 in the continued presence or absence of these inhibitors or receptor blockers for another 4 h, and adiponectin secretion was determined.

RNA extraction
Total RNA was extracted from the treated 3T3-L1 adipocytes using a Tri Reagent Kit (Molecular Research Center). The integrity of the extracted total RNA was examined by 1% agarose gel electrophoresis and its concentration determined by UV light absorbance at 260 nm. All RNA samples were incubated for 30 min at 37 C with RNase-free DNase I, then for 10 min at 100 C to inactivate the DNase.

RT-PCR
Before RT, the RNA template was heated for 5 min at 70 C. RT was carried out for 1 h at 42 C in a final volume of 50 µl of 1x RT buffer containing 1 µg total RNA, 5 U SUPER RT reverse transcriptase (HT Biotechnology Ltd., Cambridge, UK), 200 nM poly (dT)12–18 primers (Promega, Madison, WI), 0.2 mM of each dNTP (HT Biotechnology), and 16 U human placental ribonuclease inhibitor (HT Biotechnology). The mixtures were then heated at 100 C for 10 min. For PCR, the final volume of 50 µl of 1x reaction buffer contained 10 µl of the RT template solution, 2 U Vent DNA polymerase (BioLabs Inc., Beverly, MA), 0.2 mM of each dNTP, and 0.2 µM of each of the primers adipoQ-5' (5'-TTGTC AGTGG ATCTG ACGAC-3') and adipoQ-3' (5'-CTGTT CCATG ATTCT CCTGG-3'). The PCR program used was 95 C for 4 min; 35 cycles of 94 C for 30 sec, 55 C for 30 sec, and 72 C for 1 min; and 72 C for 7 min. The PCR product was electrophoresed in a 1.5% agarose gel and the 0.83-kb cDNA of the adiponectin gene eluted from the gel using a NucleoSpin Expression kit (BD Biosciences Clontech, Palo Alto, CA).

cDNA probe preparation and Northern blot analysis
The adiponectin cDNA probes were prepared from the adiponectin PCR products. The PCR products were cloned into the SmaI site of the pGEM-3zf(+) vector and their identities confirmed by sequencing. For hybridization, the inserted cDNA probes were excised and 32P radiolabeled using a random primer labeling system with reagents purchased from Promega. Total RNA (15 µg per lane) was separated by electrophoresis on denaturing formaldehyde agarose gels and the separated RNA transferred to nylon membranes. Prehybridization and hybridization were carried out as described previously (29). The hybridization signals were expressed as arbitrary densitometric units and normalized to the signal from the corresponding GAPDH mRNA band, and then the normalized values were compared.

Analysis of adiponectin secretion
Adiponectin protein in the culture supernatant was measured using commercial mouse adiponectin ELISA kits (R&D Systems, Inc., Minneapolis, MN).

Statistical analysis
Experiments were repeated at least four times and the results expressed as the mean ± SD. Statistical significance was assessed by one-way ANOVA or Student’s t test, a value of P < 0.05 being considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ET-1 causes a decrease in adiponectin mRNA levels in a time- and dose-dependent manner
To determine the effect of ET-1 on adiponectin mRNA levels, fully differentiated 3T3-L1 adipocytes were incubated with 10–7 M ET-1 for various times, and then adiponectin mRNA was measured by Northern blotting. As shown in Fig. 1Go, adiponectin mRNA levels were decreased in a time-dependent manner, with a significant decrease (37%) being seen after 4 h of treatment and a maximal decrease (72%) being seen at 12 h and maintained for up to 24 h of treatment. To examine the dose effect of ET-1 on adiponectin mRNA levels, 3T3-L1 adipocytes were incubated for 24 h with 0~10–7 M ET-1. As shown in Fig. 2Go, incubation with 10–10 M ET-1 resulted in a significant decrease (30%) in adiponectin mRNA levels; this inhibitory effect was dose dependent, with maximal inhibition (~70%) being seen in the presence of 10–8 M ET-1, the IC50 (half-maximal inhibitory dose) being 2.27 nM.


Figure 1
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  		FIG. 1. Time-dependent effect of ET-1 on adiponectin mRNA levels in 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were treated with 10–7 M ET-1 for the indicated time, and then adiponectin mRNA levels were measured by Northern blotting as described in Materials and Methods. Top, Representative blot; bottom, densitometric results of the adiponectin mRNA levels normalized to the GAPDH mRNA levels and expressed as a percentage of the time zero result. Each bar represents the mean ± SD for four separate experiments. *, P < 0.05 compared with the time zero control.

 

Figure 2
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  		FIG. 2. Dose-dependent effect of ET-1 on adiponectin mRNA levels in 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were treated with 10–11 to 10–7 M ET-1 for 24 h, and then adiponectin mRNA levels were measured by Northern blots. Other details were as described in Fig. 1Go. *, P < 0.05 compared with in the absence of ET-1.

 
ET-1 acts through the ETAR to decrease adiponectin mRNA levels
We then used the ETAR antagonist BQ-610 and the ETBR antagonist BQ-788 to examine the contribution of the two ET receptors to the ET-1-induced decrease in adiponectin mRNA levels. 3T3-L1 adipocytes were pretreated for 1 h with 10–5 M BQ-610 or BQ-788 and then coincubated with inhibitor and 10–8 M ET-1 for another 24 h. As shown in Fig. 3Go, BQ-610 or BQ-788 alone had no effect on basal adiponectin mRNA expression, but BQ-610 completely prevented the ET-1-induced decrease in adiponectin mRNA levels, whereas BQ-788 had no effect, demonstrating that this effect was mediated through the ETAR.


Figure 3
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  		FIG. 3. Effect of ETAR and ETBR antagonists on the ET-1-mediated decrease in adiponectin mRNA levels in 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were preincubated for 1 h in the absence or presence of the ETAR antagonist BQ-610 (10–5 M) or the ETBR antagonist BQ-788 (10–5 M) and then incubated in the absence or presence of 10–8 M ET-1 in the continued presence or absence of the antagonist for another 24 h before adiponectin mRNA levels were measured by Northern blots. Other details were as described in Fig. 1Go. *, P < 0.05 compared with the untreated control.

 
ET-1 decreases adiponectin mRNA levels through the ERK-dependent pathway
We then examined whether signal molecules, such as cAMP-dependent PKA, PLA2, PKC, phosphatidylinositol 3-kinase (PI3-K), or MAPK, which have been implicated in ET-1 signaling, played a role in the ET-1-mediated decrease in adiponectin mRNA levels. The adipocytes were preincubated for 1 h in the presence or absence of various pharmacological inhibitors acting on different signaling pathways and then incubated for another 24 h with 10–8 M ET-1 in the continued presence or absence of these inhibitors. Pretreatment with inhibitors of adenylyl cyclase (SQ22536; 100 µM), PLA2 (dexamethasone; 100 nM), PKC (H7; 6 µM), or PI3-K (wortmannin; 100 nM) had no effect on the ET-1-mediated decrease in adiponectin mRNA levels (data not shown). When MAPK/ERK kinase (MEK) inhibitors were tested, pretreatment with PD098059 (75 µM), an inhibitor that blocked MEK’s ability to activate the ERKs, completely blocked the effect of ET-1, whereas SB203580 (20 µM), an inhibitor that blocked MEK’s ability to activate the p38MAPK, had no effect (Fig. 4AGo), showing that ERK activation, but not p38 activation, was necessary for ET-1 to cause the decrease in adiponectin mRNA levels in 3T3-L1 adipocytes. To further confirm the role of ERK activation on ET-1-down-regulated adiponectin gene expression, adipocytes were preincubated for 1 h in the presence or absence of another MEK inhibitor U0126 (2.5 x 10–5 µM) and then incubated for another 24 h with 10–8 M ET-1 in the continued presence or absence of this inhibitor. Results also showed that ET-1 down-regulated adiponectin mRNA levels, and this effect can be fully reversed by U0126 pretreatment (Fig. 4BGo). This result also suggested that ERK activation was necessary for ET-1 action on the regulation of adiponectin gene expression.


Figure 4
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  		FIG. 4. Effect of MAPK inhibitors on the ET-1-mediated decrease in adiponectin mRNA level in 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were preincubated for 1 h in the absence or presence of the ERK inhibitor PD98059 (7.5 x 10–5 M) or the p38MAPK inhibitor SB203580 (2 x 10–5 M) (A) or U0126 (2.5 x 10–5 µM) (B) and then incubated in the absence or presence of 10–8 M ET-1 in the continued presence or absence of the antagonist for another 24 h before adiponectin mRNA levels were measured by Northern blots. Other details were as described in Fig. 1Go. *, P < 0.05 compared with the untreated control.

 
The ET-1-mediated decrease in adiponectin mRNA levels is not due to degradation of adiponectin mRNA
To determine whether the ET-1-induced decrease in adiponectin mRNA levels was due to increased degradation of adiponectin mRNA, the stability of the adiponectin mRNA was measured. Serum-free 3T3-L1 adipocytes were pretreated with actinomycin D (5 µg/ml) for 30 min and then treated with or without 10–8 M ET-1 for 4, 8, or 16 h before adiponectin mRNA levels were measured by Northern blot analysis. As shown in Fig. 5Go, ET-1 had no effect on adiponectin mRNA stability, showing that the ET-1-mediated decrease was not due to degradation of adiponectin mRNA.


Figure 5
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  		FIG. 5. Effect of actinomycin D on the ET-1-mediated decrease in adiponectin mRNA levels. 3T3-L1 adipocytes were preincubated with actinomycin D (5 µg/ml) for 30 min and then incubated for 4, 8, or 16 h with (bullet) or without ({circ}) 10–8 M ET-1 before adiponectin mRNA levels were measured by Northern blots and expressed as a percentage of the levels in cells not treated with ET-1 or actinomycin D (time zero control). The data are the mean ± SD for four separate experiments.

 
Time course of ET-1-regulated adiponectin secretion
In contrast to a previous report that ET-1 stimulates adiponectin secretion (30), our results showed that ET-1 decreased adiponectin mRNA levels. To explore the reasons for this discrepancy, we studied the time course of the effect of ET-1 on adiponectin secretion. As shown in Fig. 6AGo, ET-1 caused a significant increase in adiponectin secretion at all time points up to 4 h. However, at later times (16 and 24 h), a reduction in adiponectin secretion was seen in ET-1-treated cells compared with controls. To further evaluate the intracellular adiponectin content after 24 h ET-1 treatment, we found that long-term ET-1 treatment caused a 70% decrease in adiponectin content of 3T3-L1 adipocytes, compared with the vehicle control (Fig. 6BGo).


Figure 6
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  		FIG. 6. A, Time course of ET-1-mediated adiponectin secretion from 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were treated with ({blacksquare}) or without ({square}) ET-1 (10–7 M) for the times indicated, and then adiponectin secreted into the medium was measured by ELISA and the secretion rate (ng/h) calculated. The results are the mean ± SD for four independent experiments. *, P < 0.05 compared with the corresponding time control. B, Effect of long-term ET-1 treatment on intracellular adiponectin contents in 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were incubated with or without 10–7 M ET-1 for 24 h. Intracellular adiponectin contents were measured by Western blotting. Top, Representative blot; bottom, densitometric results of the adiponectin protein levels normalized to the {alpha}-tubulin protein levels and expressed as a percentage of control result. Each bar represents the mean ± SD for three separate experiments. *, P < 0.05 compared with in the absence of ET-1.

 
Acute ET-1 treatment stimulates adiponectin secretion via the IP3/Ca2+ pathway
The PLC/IP3/Ca2+ cascade is reported to be involved in the secretagogue action of ET-1. Activation of the ET receptor causes a rapid increase in inositol lipid turnover, phosphatidylinositol 4,5-bisphosphate is cleavage by PLC to yield IP3 and DAG, which are linked to Ca2+ mobilization and PKC activation, and an increase in intracellular Ca2+ concentration often triggers the secretory processes (31). We therefore examined whether the IP3 pathway was involved in the release of adiponectin evoked by ET-1. As shown in Fig. 7Go, ET-1-stimulated adiponectin release was significantly inhibited by pretreatment with 2-APB (an IP3 receptor blocker, which inhibits IP3-induced calcium release) but not by the PKC inhibitor H7, whereas neither inhibitor affected basal release. In addition, PLC inhibitor U73122 and exogenous addition of D-myoinositol-1,4,5-trisphosphate (IP3) were used to further clarify the involvement of the IP3/Ca2+ pathway in the ET-1-stimulated adiponectin secretion. Results showed that pretreatment with U73122 can inhibit ET-1-stimulated adiponectin secretion in 3T3-L1 adipocytes. Exogenous addition of IP3 can directly stimulate adiponectin secretion that mimics ET-1 action in 3T3-L1 adipocytes (Fig. 8Go). Thus, the release of adiponectin stimulated by ET-1 is mediated by the IP3 pathway after PLC activation.


Figure 7
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  		FIG. 7. Effect of IP3 receptor blocker and PKC inhibitor on ET-1-stimulated adiponectin secretion by 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were preincubated for 1 h in the absence or presence of the IP3 receptor blocker 2-APB (2.5 x 10–5 M) or the PKC inhibitor H7 (6 x 10–6 M) and then incubated for another 4 h in the absence or presence of ET-1 (10–8 M) in the continued presence or absence of the antagonist. Adiponectin secreted into the medium was measured by ELISA. The results are the mean ± SD for four independent experiments. *, P < 0.05 compared with the untreated control.

 

Figure 8
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  		FIG. 8. Involvement of IP3/Ca2+ pathway in the ET-1-stimulated adiponectin secretion. Differentiated 3T3-L1 adipocytes were preincubated for 1 h in the absence or presence of the phospholipase C inhibitor U73122 (10–6 M) and then incubated for another 4 h in the absence or presence of ET-1 (10–8 M) in the continued presence or absence of the antagonist. Furthermore, differentiated 3T3-L1 adipocytes were directly treated with exogenous IP3 agonist D-myo-IP3 (IP3; 10–7 M) for 4 h. Adiponectin secreted into the medium was measured by ELISA. The results are the mean ± SD for four independent experiments. *, P < 0.05 compared with the untreated control.

 
Furthermore, pretreatment with BQ-610, but not BQ-788, completely prevented the ET-1-stimulated adiponectin release, demonstrating that this effect was also mediated through the ETAR (Fig. 9Go). To test whether ERK activation is also involved in the stimulatory effect of ET-1 on adiponectin release, 3T3-L1 adipocytes were pretreated with PD098059, an inhibitor that blocked MEK’s ability to activate the ERKs, and then the stimulatory effect of ET-1 was evaluated. As shown in Fig. 9Go, pretreatment with the PD098059 cannot block the stimulatory effect of ET-1 on adiponectin release, suggesting ERK activation was not involved in the ET-1-stimulated adiponectin release. In addition, we also tested whether the IP3/Ca2+ pathway is involved in the ET-1-down-regulated adiponectin mRNA expression. Differentiated 3T3-L1 adipocytes were preincubated for 1 h in the absence or presence of the IP3 receptor blocker, 2-APB (2.5 x 10–5 M), then incubated in the absence or presence of 10–8 M ET-1 in the continued presence or absence of the antagonist for another 24 h, and then adiponectin mRNA levels were measured by Northern blots. Results of three separate experiments showed that ET-1 significantly decreased adiponectin mRNA levels (P < 0.05), but ET-1-down-regulated adiponectin mRNA expression could not be prevented by pretreatment with 2-APB (data not show). These results suggested that the IP3/Ca2+ pathway was not involved in the ET-1-down-regulated adiponectin mRNA expression.


Figure 9
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  		FIG. 9. Effect of ETAR and ETBR antagonists and ERK inhibitor on ET-1-stimulated adiponectin secretion by 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were preincubated for 1 h in the absence or presence of the ETAR antagonist BQ-610 (10–5 M), the ETBR antagonist BQ-788 (10–5 M), or the ERK inhibitor, PD98059 (7.5 x 10–5 M) and then incubated for another 4 h in the absence or presence of ET-1 (10–8 M) in the continued presence or absence of the antagonist. Adiponectin secreted into the medium was measured by ELISA. The results are the mean ± SD for four independent experiments. *, P < 0.05 compared with the untreated control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The major finding of this study was that ET-1 has regulatory effects on adiponectin expression and secretion via two different signaling pathways. In terms of adiponectin gene expression, ET-1, acting via the ETAR, decreased adipocyte adiponectin mRNA levels in a time-dependent and dose-dependent manner by activation of the ERK pathway, because its effect was prevented by incubation of the cells with the PD98059, which blocked MEK’s ability to activate the ERKs. In contrast, the short-term stimulatory effect of ET-1 on adiponectin secretion was mediated through the IP3/Ca2+ pathway, because its effect was blocked by the IP3 receptor blocker, 2-APB. The adiponectin secretion data in the present study are compatible with those in a previous report (30). These authors reported that treatment with ET-1 for 1 or 4 h caused significant stimulation of adiponectin secretion, but this effect was lost after 8 h of treatment; however, the possible mechanism responsible for the action of ET-1 was not discussed. In the present study, we found that ET-1 decreased adiponectin mRNA levels in 3T3-L1 adipocytes. In the long term, ET-1-induced down-regulation of adiponectin mRNA may decrease the synthesis of adiponectin and consequently decrease intracellular adiponectin content (Fig. 6BGo), partially explaining why prolonged ET-1 exposure led to levels of adiponectin secretion lower than in the relative control (Fig. 6AGo).

Recently, Bedi et al. (32) proposed that ET-1 may regulate adiponectin secretion through phosphatidylinositol-4,5-bisphosphate modulation of actin cytoskeleton. Their data showed that ET-1 did not regulate adiponectin gene expression and that chronic ET-1 treatment significantly decreased adiponectin secretion through phosphatidylinositol-4,5-bisphosphate modulation of actin cytoskeleton. Both Bedi’s group and we conclude that long-term ET-1 treatment inhibited adiponectin secretion in 3T3-L1 adipocytes. However, there are some differences between our findings and Bedi’s study. In contrast to Bedi’s study, our results of Northern blot analysis showed that ET-1 down-regulated adiponectin mRNA levels in 3T3-L1 adipocytes. This difference may be due to the different mRNA measuring methods. To the best of our knowledge, however, Northern blot assay should provide more specific and solid data on mRNA expression. Furthermore, Bedi’s studies suggested that ET-1-caused inhibition on adiponectin secretion is through posttranscriptional regulation via interfering with adiponectin packaging and/or vesicular trafficking. It means that intracellular adiponectin accumulation should be observed after ET-1 treatment. However, our data showed that long-term ET-1 treatment caused a significant decrease in cellular adiponectin contents (Fig. 6BGo). Hence, ET-1-interfered adiponectin packaging and/or vesicular trafficking was not the only mechanism contributed to the ET-1-inhibited adiponectin secretion. On the other hand, the decreased adiponectin intracellular contents may partially explain why prolonged ET-1 treatment inhibited adiponectin secretion. Nevertheless, little evidence can exclude the possibility that ET-1 impaired the intracellular traffic system and then inhibited adiponectin secretion in 3T3-L1 adipocytes. This mechanism also possibly contributed to the regulatory effect of ET-1 on adiponectin secretion in 3T3-L1 adipocytes.

Many studies have shown that ET-1 interferes with several metabolic activities, including hepatic glucose production (33) and glucose uptake in skeletal muscle and adipocytes (34, 35). Our previous study showed that ET-1 impairs insulin-stimulated glucose uptake in rat adipocytes and that this effect was mediated through the ETAR (36). In vivo studies have also shown that ET-1 causes systemic insulin resistance in rats and human (37, 38, 39). Recently, we demonstrated that ET-1 stimulates lipolysis in 3T3-L1 adipocytes (40). In obesity and diabetes, circulating ET-1 levels are significantly increased (18, 19), suggesting that lipolysis may be accelerated and large amounts of FFAs released from adipose tissue. Because FFAs have been shown to induce insulin resistance in humans (41), the increased circulating FFA levels caused by ET-1-stimulated lipolysis may induce a more insulin-resistant status in diabetic and obese subjects with hyperendothelinemia. In addition, adiponectin is an insulin-sensitizing adipocytokine, and an increase in its levels results in increased insulin sensitivity (42, 43). In the present study, we demonstrated that ET-1 decreased adiponectin mRNA levels in 3T3-L1 adipocytes, so it is possible that hyperendothelinemia in obesity and type 2 diabetes may suppress adiponectin expression and continuously aggravate the insulin-resistant status.

Adipocytes are dispersed through the whole body and are widely distributed along and within major organs, such as the heart and blood vessels. Adipocyte-derived adiponectin may therefore have a paracrine effect on the cardiovascular system. It has been demonstrated to play a protective role in suppressing the development of cardiac hypertrophy (44) and atherosclerosis (45). In obesity and diabetes, plasma ET-1 levels are significantly increased (18, 19), and the cardiovascular system is highly infiltrated with adipose tissue. It is very likely that hyperendothelinemia suppresses adiponectin expression, attenuating its protective effects on the cardiovascular system and then indirectly accelerates the progression of cardiovascular diseases in obesity and type 2 diabetes.

ET-1, a potent endothelium-derived vasoconstricting factor, plays an important role in the regulation of vascular tone (15) and is involved in the pathogenesis of various cardiovascular diseases (18, 19, 20). ET-1 also acts on several peripheral tissues, including adipose tissue (21, 22, 35, 36, 40). Many studies suggest that ET-1 has a dramatic impact on adipocyte physiology. For example, ET-1 has been shown to inhibit adipocyte differentiation (21, 22), to reduce lipoprotein lipase activity in adipocytes (46), to inhibit insulin-stimulated glucose uptake (35, 36), and to stimulate lipolysis in adipocytes (40). In addition to its effects on adiponectin, ET-1 also stimulates leptin production (47) and inhibits resistin release (48) in 3T3-L1 adipocytes. These findings indicate that ET-1 can directly or indirectly modulate adipocyte functions. Dysregulation of the action of ET-1 on adipocytes may disrupt body energy homeostasis and progressively cause metabolic disorders, such as obesity and type 2 diabetes.

In the present study, we found that ET-1 suppressed adiponectin gene expression through the ERK-dependent pathway (Fig. 4Go). Our previous study also demonstrated that ET-1 stimulated ERK activation and consequently induced lipolysis in adipocytes (40). These results suggest that the signaling pathways leading to ERK activation are important in adipocyte physiology. Actually, studies performed on preadipocyte cell lines showed that the ERK pathway is implicated in adipocyte differentiation (49, 50). Bost et al. (51) also strongly suggested an essential and specific role of the ERK pathway at each step of adipogenesis from embryonic stem cells to adipocytes. Any abnormal modulation of the ERK pathway could affect adipogenesis and might lead to the development of obesity.

In conclusion, our results demonstrate that ET-1 has distinct regulatory effects on adiponectin expression and secretion via two different signaling pathways. Our data also support the pivotal pathophysiological role of ET-1 in adipocyte function. Dysregulation of the effect of ET-1 on adipose tissue may contribute to the pathogenesis of metabolic disorders, such as obesity and type 2 diabetes. Furthermore, our data suggest that the cardiovascular system affects adipocyte physiology by regulating the expression of adipocytokines and, consequently, energy homeostasis via vasoactive factors, such as ET-1.


   Acknowledgments
 
We thank Ms. Ying-Hsiu Lai for excellent technical assistance on the Northern blotting assay.


   Footnotes
 
These studies were supported by the National Science Council of Taiwan (NSC92-2312-B-010-005) and Ministry of Education, Aim for the Top University Plan.

Disclosure Summary: The authors have nothing to disclose.

First Published Online December 28, 2006

Abbreviations: 2-APB, 2-Aminoethoxydiphenyl borate; DAG, diacylglycerol; ET-1, endothelin-1; ETAR and ETBR, ET-type A and B receptors; FFA, free fatty acid; IP3, inositol-1,4,5-triphosphate; MEK, MAPK/ERK kinase; PI3-K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PLA2, phospholipase A2; PLC, phospholipase C.

Received May 15, 2006.

Accepted for publication December 15, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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