Effect of Endothelin-1 on Lipolysis in Rat Adipocytes

Authors

  • Chi-Chang Juan,

    1. Institutes of Physiology and Clinical Medicine, School of Medicine, National Yang-Ming University and the Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan.
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    • Co-corresponding author.

  • Li-Wei Chang,

    1. Institutes of Physiology and Clinical Medicine, School of Medicine, National Yang-Ming University and the Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan.
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  • Seng-Wong Huang,

    1. Institutes of Physiology and Clinical Medicine, School of Medicine, National Yang-Ming University and the Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan.
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  • Chih-Ling Chang,

    1. Institutes of Physiology and Clinical Medicine, School of Medicine, National Yang-Ming University and the Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan.
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  • Ching-Yin Lee,

    1. Institutes of Physiology and Clinical Medicine, School of Medicine, National Yang-Ming University and the Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan.
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  • Yueh Chien,

    1. Institutes of Physiology and Clinical Medicine, School of Medicine, National Yang-Ming University and the Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan.
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  • Yung-Pei Hsu,

    1. Institutes of Physiology and Clinical Medicine, School of Medicine, National Yang-Ming University and the Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan.
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  • Pei-Hsuan Ho,

    1. Institutes of Physiology and Clinical Medicine, School of Medicine, National Yang-Ming University and the Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan.
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  • Yu-Ching Chen,

    1. Institutes of Physiology and Clinical Medicine, School of Medicine, National Yang-Ming University and the Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan.
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  • Low-Tone Ho

    Corresponding author
    1. Institutes of Physiology and Clinical Medicine, School of Medicine, National Yang-Ming University and the Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan.
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  • The costs of publication of this article were defrayed, in part, by the payment of page charges. This article must, therefore, be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Department of Medical Research and Education, Taipei Veterans General Hospital, No. 201, Section 2, Shih-Pai Road, Taipei, Taiwan. E-mail: ltho@vghtpe.gov.tw

Abstract

Objective: To explore the role of endothelin-1 (ET-1) on lipid metabolism, we examined the effect of ET-1 on lipolysis in rat adipocytes.

Research Methods and Procedure: Adipocytes isolated from male Sprague-Dawley rats, weighing 400 to 450 grams, were incubated in Krebs-Ringer buffer with or without 10−7 M ET-1 for various times or with various concentrations of ET-1 for 4 hours; then glycerol release into the incubation medium was measured. In addition, selective ETAR and ETBR blockers were used to identify the ET receptor subtype involved. We also explored the involvement of cyclic adenosine monophosphate (cAMP) in ET-1-stimulated lipolysis using an adenylyl cyclase inhibitor and by measuring changes in intracellular cAMP levels in response to ET-1 treatment. To further explore the underlying mechanism of ET-1 action, we examined the involvement of the extracellular signal-regulated kinase (ERK)-mediated pathways.

Results: Our results showed that ET-1 caused lipolysis in rat adipocytes in a time- and dose-dependent manner. BQ610, a selective ETAR blocker, blocked this effect. The adenylyl cyclase inhibitor, 2′, 5′-dideoxyadenosine, had no effect on ET-1-stimulated lipolysis. ET-1 did not induce an increase in intracellular cAMP levels. In addition, ET-1-induced lipolysis was blocked by inhibition of ERK activation using PD98059. Coincubation of cells with ET-1 and insulin suppressed ET-1-stimulated lipolysis.

Discussion: These findings show that ET-1 stimulates lipolysis in rat adipocytes through the ETAR and activation of the ERK pathway. The underlying mechanism is cAMP-independent. However, this non-conventional lipolytic effect of ET-1 is inhibited by the anti-lipolytic effect of insulin.

Introduction

Endothelin-1 (ET-1),1 a potent vasoconstrictor synthesized by endothelial cells (1), exerts its main vascular effects by binding to two receptor subtypes, ET-type A and B receptors (ETAR and ETBR) (2, 3). In the blood vessels, the ETAR is mainly distributed in the smooth muscle layers, whereas the ETBR is mainly localized on endothelial cells (2, 3). ET-1 has dual vasoactive effects in vascular smooth muscle that are important in blood pressure regulation. Activation of the ETAR causes potent vasoconstriction by increasing intracellular calcium levels, whereas ETBR activation causes vasorelaxation by releasing nitric oxide and prostacyclin (2, 3). Although mainly expressed in vascular tissue, the ETAR and ETBR are also expressed in a variety of other tissues, suggesting that ET-1 is responsible for a diverse range of biological responses (4, 5, 6, 7, 8).

Our previous studies showed that ET-1 impairs insulin-stimulated glucose uptake (ISGU) in rat adipocytes (9) and that this effect is mainly mediated through the ETAR (10). We further explored the underlying mechanism and found that ET-1 suppresses the expression of the insulin receptor and insulin receptor substrate-1 in rat adipocytes, thus inhibiting ISGU (11). In addition, ET-1 has been shown to regulate hepatic glycogenolysis and glucose output (12, 13). These observations suggest that ET-1 might play a role in metabolism. Although studies on a possible role of ET-1 in lipid metabolism are very limited, its involvement is indirectly suggested by the observations that acute intraperitoneal injection of ET-1 significantly increases plasma free fatty acid (FFA) levels in pygmy goats (14), and chronic ET-1 infusion causes a significant increase in plasma FFA levels in rats (15). These findings strongly suggest that ET-1 is involved in lipolysis. There is also evidence that ET-1 stimulates sympathetic nerve activity (16, 17), and it is possible that ET-1 regulates lipolysis-activated sympathetic innervation of adipose tissue (18, 19). Therefore, ET-1 may regulate lipolysis directly or indirectly through regulation of sympathetic tone.

To address this issue, we used isolated rat adipocytes as the cell model to directly explore the role of ET-1 in lipid metabolism and hypothesized that ET-1 may have a direct regulatory effect on lipolysis in rat adipocytes. We first evaluated the effect of ET-1 on lipolysis by analyzing changes in glycerol release, then determined which ET-1 receptor was involved using specific receptor antagonists, and finally examined the relationship between cyclic adenosine monophosphate (cAMP) and ET-1-stimulated lipolysis.

Research Methods and Procedures

Design

Isolated rat adipocytes were incubated at 37 °C with 10−7 M ET-1 for different times (1 to 4 hours) or with different concentrations (0 to 10−6 M) of ET-1 for 4 hours; then glycerol in the culture supernatant was measured colorimetrically using glycerol assay kits. To determine the receptor subtype(s) mediating ET-1-induced lipolysis, rat adipocytes were incubated for 30 minutes in the presence or absence of 10−5 M BQ610 (an ETAR antagonist) or 10−5 BQ788 (an ETBR antagonist) and then incubated with 10−7 M ET-1 in the continued presence or absence of the antagonist for 4 hours. To explore the involvement of cAMP, the adipocytes were pre-incubated for 30 minutes in the presence or absence of the adenylyl cyclase inhibitor, 2′, 5′-dideoxyadenosine (3 × 10−5 M) and then incubated with 10−7 M ET-1 in the continued presence or absence of the inhibitors for 4 hours. We also directly evaluated the effect of ET-1 on adenylyl cyclase activity by measuring intracellular cAMP levels. To further explore the involvement of extracellular signal-regulated kinase (ERK) activation, the adipocytes were pre-incubated for 1 hour in the presence or absence of PD098059 (7.5 × 10−5 M) and then incubated with 10−7 M ET-1 in the continued presence or absence of these inhibitors for a further 4 hours. To test the anti-lipolytic effect of insulin on ET-stimulated lipolysis, adipocytes were incubated for 4 hours at 37 °C either alone or with 10−7 M ET-1 and/or 10−9 M insulin.

Animals

Male Sprague-Dawley rats weighing 400 to 450 grams from the Animal Center of the National Yang-Ming University were housed four to a cage in a temperature- and light-controlled room (22 ± 2 °C; 12-hour light/12-hour dark cycle, light on at 7 am) and were provided with regular diet chow and water ad libitum. The laboratory procedures used conformed to the guidelines of the Taiwan Government Guide for the Care and Use of Laboratory Animals.

Isolation of Adipocytes

After overnight fasting, the rats were sacrificed by decapitation and the epididymal fat pads from each group of rats (two to three rats) were pooled; then adipocytes were isolated using the Rodbell method (20) with minor modifications. In brief, the fat tissue was minced and incubated for 1 hour at 37 °C in Krebs-Ringer bicarbonate buffer containing 1% bovine serum albumin (KRBB) and 0.1% collagenase in an oxygen-rich shaking chamber (CO2/O2, 5:95; 75 strokes/min). The suspension was then filtered through nylon mesh (400 μm) and centrifuged at 100g for 1 minute. The supernatant containing the adipocytes was harvested, and the cells were washed twice with, then resuspended in, KRBB. The cell density in the adipocyte suspension was determined after fixation with 2% osmium tetroxide, and the lipocrit was measured before, during, and after each experiment to check cell viability.

Measurement of Lipolysis

The glycerol content of the incubation medium was used as an index for lipolysis and was measured colorimetrically using glycerol assay kits (Randox Laboratory, County Antrim, United Kingdom). The results were corrected for cell numbers.

Measurement of the Intracellular cAMP Concentration

The cells were pretreated for 30 minutes with KRBB containing 2 × 10−3 M 3-isobutyl-1-methylxanthine (IBMX) to inhibit phosphodiesterase (PDE) activity, then the ligand [ET-1 or isoproterenol (ISO)] in KRBB containing IBMX was added, and the cells were incubated at 37 °C for 4 hours. The medium was then rapidly removed, and 0.5 mL of 0.1 M HCl was added to the cells. After freezing and thawing, an aliquot was withdrawn, and the intracellular cAMP concentration was measured using an enzyme immunoassay kit from Cayman Chemical (Ann Arbor, MI).

Statistical Analysis

All data are presented as the mean ± standard error (SE) of four separate experiments. Statistical analyses were performed using Student's t test and the paired Student's t test. The level of probability was set at p < 0.05 for statistical significance.

Results

Time- and Dose-Dependent Effect of ET-1 on Lipolysis

Isolated rat adipocytes were incubated with 10−7 M ET-1 for 1 to 4 hours; then glycerol release was determined as a measure of lipolysis. As shown in Figure 1, a significant increase in lipolysis was seen in the ET-1-treated group after 1 hour of incubation (p < 0.05), and this increase continued for up to at least 4 hours. Adipocytes were then incubated for 4 hours alone or with various concentrations of ET-1 (10−10 to 10−6 M). Figure 2 shows that incubation with 10−10 M ET-1 resulted in a significant increase in lipolysis compared with the control group (10% increase, p < 0.05). The maximal lipolysis seen in the presence of ET-1 was ∼40% higher than that in controls (Figures 1 and 2).

Figure 1.

Time dependency of the effect of ET-1 on lipolysis in rat adipocytes. Isolated rat adipocytes were incubated in the presence (▪) or absence (□) of 10−7 M ET-1 for 1 to 4 hours; then glycerol in the culture supernatant was measured. The results are the mean ± SE for four separate experiments. * p < 0.05 compared with the vehicle control at the same time-point.

Figure 2.

Dose dependency of the effect of ET-1 on lipolysis in rat adipocytes. Isolated rat adipocytes were incubated alone or with 10−10 to 10−6 M ET-1 for 4 hours; then glycerol in the culture supernatant was measured. The results are the mean ± SE for four separate experiments. * p < 0.05 compared with the vehicle control.

Effect of ET Receptor Antagonists on ET-1-Stimulated Lipolysis

To examine the involvement of the ETAR or ETBR in ET-1-stimulated lipolysis, adipocytes were pretreated for 30 minutes with 10−5 M BQ610 or 10−5 M BQ788 and then incubated with inhibitor and 10−7 M ET-1 for a further 4 hours. BQ610 or BQ788 alone had no effect on basal lipolysis, whereas BQ610, but not BQ788, completely abolished ET-1-stimulated lipolysis (Figure 3), indicating that the effect of ET-1 on lipolysis was mediated through the ETAR.

Figure 3.

Effect of ET receptor antagonists on ET-1-induced lipolysis. Isolated rat adipocytes were pre-incubated for 30 minutes in the presence or absence of the ETAR antagonist, BQ610 (10−5 M), or the ETBR antagonist, BQ788 (10−5 M). Then the adipocytes were incubated in the presence or absence of ET-1 (10−7 M) in the continued presence or absence of the antagonist for a further 4 hours before the glycerol concentration in the culture supernatant was measured. The results are the mean ± SE for four separate experiments. * p < 0.05 compared with the vehicle control. + p < 0.05 compared with ET-1 alone.

Effect of an Adenylyl Cyclase Inhibitor on ET-1-Stimulated Lipolysis

As shown in Figure 4, treatment of adipocytes with ISO (10−8 M) caused a significant increase in glycerol release, and this effect was completely blocked by the adenylyl cyclase inhibitor, 2′, 5′-dideoxyadenosine (3 × 10−5 M), showing that cAMP was the intracellular mediator for ISO-induced lipolysis. In contrast, 2′, 5′-dideoxyadenosine pretreatment did not prevent ET-1-stimulated lipolysis. To further clarify the role of cAMP and adenylyl cyclase, we directly measured intracellular cAMP levels after ET-1 or ISO treatment and found that ISO, but not ET-1, caused a significant increase in cAMP levels (Figure 5). These data showed that ET-1-induced lipolysis does not involve the conventional cAMP-dependent pathway.

Figure 4.

Effect of an adenylyl cyclase inhibitor on ET-1induced lipolysis. Isolated rat adipocytes were pre-incubated for 30 minutes in the presence (▪) or absence (□) of the adenylyl cyclase inhibitor, 2′, 5′-dideoxyadenosine (3 × 10−5 M) and then incubated in the presence or absence of ET-1 (10−7 M) or ISO (10−8 M) in the continued presence or absence of the inhibitor for a further 4 hours before the glycerol concentration in the culture supernatant was measured; C, cells incubated without ET-1 or ISO. The results are the mean ± SE for four separate experiments. * p < 0.05 compared with the control without inhibitor. + p < 0.05 compared with ISO alone.

Figure 5.

Lack of effect of ET-1 on intracellular cAMP levels. Isolated rat adipocytes were pretreated for 30 minutes with IBMX (2 × 10−3 M) and then incubated with medium (C), ET-1 (10−7 M), or ISO (10−8 M) in the continued presence of IBMX for a further 4 hours. The cells were then rapidly lysed by 0.1 M HCl, and their cAMP content was measured. The results are the mean ± SE for four separate experiments. * p < 0.05 compared with the vehicle control.

Effect of ERK Inhibitor on ET-1-Stimulated Lipolysis

Our recently published data showed that ET-1 induced lipolysis in 3T3-L1 adipocytes through the activation of the ERK pathway (21). In the present study, we tested the possible role of ERK activation in ET-1-stimulated lipolysis in rat adipocytes. Isolated rat adipocytes were pre-incubated for 1 hour in the presence or absence of the ERK inhibitor PD98059 (7.5 × 10−5 M) and then the stimulatory effect of ET-1 on lipolysis was determined. As shown in Figure 6, pretreatment of adipocytes with PD98059 completely blocked ET-1-induced lipolysis (p < 0.05), showing that ERK activation was necessary for ET-1-stimulated lipolysis in isolated rat adipocytes.

Figure 6.

Effect of ERK inhibitor PD98059 on ET-1-induced lipolysis. Isolated rat adipocytes were pre-incubated for 1 hour in the absence or presence of the ERK inhibitor PD98059 (7.5 × 10−5 M) and then incubated in the absence or presence of ET-1 (10−7 M) in the continued absence or presence of the inhibitor for a further 4 hours before the glycerol concentration in the culture supernatant was measured. The results are the mean ± SE for four separate experiments. * p < 0.05 compared with the vehicle control. + p < 0.05 compared with ET-1 alone.

Anti-lipolytic Effect of Insulin on ET-1-Stimulated Lipolysis

Insulin plays an important role in the regulation of nutrient homeostasis. In addition to its stimulatory effect on glucose uptake, insulin is also associated with lipid metabolism, one important action being to inhibit lipolysis in adipocytes (22). To test the anti-lipolytic effect of insulin on ET-stimulated lipolysis, adipocytes were incubated alone or with 10−7 M ET-1 and/or 10−9 M insulin for 4 hours; then glycerol release was measured. As shown in Figure 7, insulin had no effect on basal lipolysis but significantly inhibited ET-1-stimulated lipolysis. To further explore the possible mechanism by which insulin inhibited the lipolytic effect of ET-1, adipocytes were pretreated with IBMX to inhibit PDE activity, and the anti-lipolytic effect of insulin on ET-1-stimulated lipolysis was determined. Result showed that inhibition of PDE blocked the anti-lipolytic effect of insulin on ET-stimulated lipolysis (Figure 7).

Figure 7.

Anti-lipolytic effect of insulin on ET-1-stimulated lipolysis. Isolated rat adipocytes were pretreated with or without IBMX (2 × 10−3 M) for 30 minutes and incubated alone or in the presence of ET-1 (10−7 M) and/or insulin (10−9 M) for 4 hours; then glycerol in the culture supernatant was measured. The results are the mean ± SE for four separate experiments. * p < 0.05 compared with the vehicle control. + p < 0.05 compared with ET-1 alone. ++ p < 0.05 compared with IBMX and insulin group.

Discussion

The aim of this study was to examine the metabolic effects of ET-1 in lipolysis in rat adipocytes. Our results clearly demonstrated that ET-1 had a marked time and dose effect (Figures 1 and 2) on adipocyte lipolysis and acted through the ETAR (Figure 3) and the ERK activation (Figure 6). These observations were compatible with our previous finding in 3T3-L1 adipocytes (21). In these two cell models, we demonstrated that ET-1 has an ERK-dependent lipolytic effect. However, the effect of ET-1 on lipolysis in rat adipocytes was smaller than that in 3T3-L1 adipocytes. One possible explanation for this phenomenon is the species difference in ETAR and hormone-sensitive lipase (HSL) contents of adipocytes. In addition, the characteristics of the cell line were essentially different from that of primary fat cells isolated from adipose tissue.

As shown in Figures 4 and 5, ET-1-stimulated lipolysis was cAMP/adenylyl cyclase-independent. It is very interesting that ET-1 evokes a non-conventional lipolysis response in adipocytes. Conventionally, adipocyte lipolysis is induced by activation of the cAMP-dependent protein kinase A pathway (23). Stimulation of G-protein-coupled receptors, such as the β-adrenergic receptor, causes activation of adenylyl cyclase, and the subsequent increase in intracellular cAMP levels leads to the dissociation of protein kinase A, the catalytic subunits of which then phosphorylate HSL. Phosphorylation of HSL increases its hydrolytic activity and the release of FFA and glycerol (23, 24). The present study showed that, in rat adipocytes, cAMP is not an essential mediator in ET-1-stimulated lipolysis.

As shown in Figure 7, ET-1-stimulated lipolysis was inhibited by insulin. Cyclic nucleotide PDE plays an important role in the anti-lipolytic action of insulin (25). The main type of PDE expressed in insulin-sensitive cells, such as adipocytes, is PDE3B (26). Insulin induces the activation of phosphatidylinositol 3-kinase and protein kinase B and, subsequently, the phosphorylation and activation of PDE3B (27, 28). Activated PDE3B then degrades cAMP, leading to a decrease in intracellular cAMP levels and halting lipolysis. Hence, inhibition of PDE may blunt the anti-lipolytic effect of insulin. In Figure 7, IBMX pretreatment inhibited PDE activity and blocked the anti-lipolytic effect of insulin on ET-1-stimulated lipolysis. This result suggested that insulin suppressed ET-1-stimulated lipolysis through the activation of PDE.

Our previous study demonstrated that ET-1 inhibited ISGU in rat adipocytes through decreased expression of insulin receptor and insulin receptor substrate-1 (11). In the present study, we showed that insulin has an anti-lipolytic action on ET-1-stimulated lipolysis in rat adipocytes. It was very interesting that if adipocytes were incubated with both insulin and ET-1, the ISGU was decreased, but the anti-lipolytic ability of insulin was still intact. Actually, the discrepancy between ISGU and insulin's anti-lipolytic action has been investigated. To compare the relationship between insulin levels and insulin actions, we can find that action of insulin on anti-lipolysis was more sensitive than that on glucose uptake (29). Hence, it was possible that insulin's anti-lipolytic action was still undamaged even though the ISGU had been impaired by ET-1 treatment.

However, ET-1 treatment did not cause an increase in intracellular cAMP levels in rat adipocytes, so the inhibitory action of insulin on ET-1-stimulated lipolysis is not mediated by manipulation of intracellular cAMP levels. In addition to PDE3B, another anti-lipolytic enzyme, PDE4, is regulated by insulin (30, 31) and is reported to be involved in the hydrolysis of intracellular cGMP (32). It is, therefore, possible that insulin inhibits ET-1-stimulated lipolysis by regulating intracellular cGMP levels. Cyclic GMP is an important mediator in the lipolytic activity caused by hormones, such as atrial natriuretic peptide (33, 34). Atrial natriuretic peptide-induced lipolysis in adipocytes is cGMP-dependent and induces the phosphorylation of HSL (34). However, there is little evidence that cGMP acts as a mediator in the effects of ET-1 on adipocytes. Further studies are required to elucidate the role of cGMP in ET-stimulated lipolysis in adipocytes.

ET-1 is reported to have several effects on adipocytes. For example, it inhibits the differentiation of preadipocytes to adipocytes (35) and suppresses lipoprotein lipase activity through the ETAR pathway in 3T3-L1 adipocytes (36). Recently, adipose tissue was reported to be a complex endocrine organ that expresses and secretes several cytokines, including leptin, adiponectin, and resistin (37), and the expression of these cytokines in adipose tissue is highly regulated by ET-1 (38, 39, 40). Taken together, these and our present findings show that ET-1 is a pivotal regulator in lipid metabolism in adipose tissue, and they provide a very good demonstration of the metabolic role of vasoactive peptides. In the present study, we demonstrated that ET-1 stimulated lipolysis in adipocytes. In some disease states, such as obesity and diabetes, circulating ET-1 levels are significantly elevated (41, 42), suggesting that lipolysis may be accelerated and large amount of FFAs released from adipose tissue. Because FFAs have been shown to induce insulin resistance in humans (43), the increased circulating FFA levels caused by ET-1-stimulated lipolysis may induce a more insulin-resistant status in diabetic patients and obese subjects with hyperendothelinemia.

In conclusion, our data show that, in rat adipocytes, ET-1 stimulates lipolysis through the ETAR and the ERK activation by a cAMP/adenylyl cyclase-independent pathway and that insulin blocks ET-1-stimulated lipolysis. These findings provide support for a role for ET-1 in the regulation of metabolism in adipose tissue. Further studies are needed to identify the molecules mediating ET-1-stimulated lipolysis and its regulatory mechanism in adipocytes.

Acknowledgement

This work was supported by the National Science Council of Taiwan (Research Grants NSC93-2314-B-075-039 and NSC94-2314-B-010-032) and by the Taipei Veterans General Hospital Research Foundation (Grant VGH 94-316).

Footnotes

  • 1

    Nonstandard abbreviations: ET-1, endothelin-1; ETAR and ETBR, ET-type A and B receptors; ISGU, insulin-stimulated glucose uptake; FFA, free fatty acid; cAMP, cyclic adenosine monophosphate; ERK, extracellular signal-regulated kinase; KRBB, Krebs-Ringer bicarbonate buffer containing 1% bovine serum albumin; IBMX, 3-isobutyl-1-methylxanthine; PDE, phosphodiesterase; ISO, isoproterenol; HSL, hormone-sensitive lipase; SE, standard error.

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