Endothelin Stimulates Glucose Uptake and GLUT4 Translocation via Activation of Endothelin ETA Receptor in 3T3-L1 Adipocytes*

Jinshyun R. Wu-WongDagger , Cathleen E. Berg, Jiahong Wang, William J. Chiou, and Brian Fissel

From the Pharmaceutical Products Division, Abbott Laboratories, Abbott Park, Illinois 60064-3500

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
REFERENCES

Endothelin-1 (ET-1) is a 21-amino acid peptide that binds to G-protein-coupled receptors to evoke biological responses. This report studies the effect of ET-1 on regulating glucose transport in 3T3-L1 adipocytes. ET-1, but not angiotensin II, stimulated glucose uptake in a dose-dependent manner with an EC50 value of 0.29 nM and a 2.47-fold stimulation at 100 nM. ET-1 stimulated glucose uptake in differentiated 3T3-L1 cells but had no effect in undifferentiated cells, although ET-1 stimulated phosphatidylinositol hydrolysis to a similar degree in both. The 3T3-L1 cells expressed ~560,000 sites/cell of ETA receptor, which was not altered during differentiation. Western blot analysis and immunofluorescence staining show that ET-1 stimulated the translocation of insulin-responsive aminopeptidase and GLUT4 to the plasma membrane. The effect of ET-1 on glucose uptake was blocked by A-216546, an antagonist selective for the ETA receptor. ET-1 treatment did not induce phosphorylation of insulin receptor beta -subunit, insulin receptor substrate-1, or Akt but stimulated the tyrosyl phosphorylation of a 75-kDa protein. Genistein (100 µM), an inhibitor of tyrosine kinases, inhibited ET-1-stimulated glucose uptake. Our results show that ET-1 stimulates GLUT4 translocation and glucose uptake in 3T3-L1 adipocytes via activation of ETA receptor.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES

One of the major problems in diabetes mellitus is the disruption of whole body glucose homeostasis. Glucose homeostasis is maintained by a balance of hepatic glucose production and cellular glucose uptake and metabolism (1). Glucose transport is the rate-limiting step in glucose metabolism. Among the six known facilitative glucose transporters (GLUT1 to -5 and GLUT7) (2), GLUT1 and GLUT4 are expressed in insulin-responsive tissues, such as adipose tissue and cardiac and skeletal muscle. GLUT4, which translocates from an intracellular membrane compartment to the plasma membrane after insulin stimulation, is particularly important in regulating postprandial glucose uptake. It is now known that, besides the insulin signaling pathway, other mechanisms also stimulate GLUT4 translocation and glucose uptake. For example, exercise induces GLUT4 translocation and glucose uptake in skeletal muscle through an insulin-independent pathway (3, 4). Also, introduction of GTP analogs, such as GTPgamma S,1 into 3T3-L1 adipocytes, and activation of alpha 1-adrenergic receptors, stimulate glucose uptake independent of insulin (5-7).

Endothelin (ET), originally isolated from cultured porcine aortic endothelial cells, is a peptide with 21 amino acid residues (8). Three distinct members of the ET family, namely, ET-1, ET-2, and ET-3, have been identified in humans through cloning (9). Binding of ETs to G-protein-coupled receptors (GPCRs) in tissues and cells activates various signaling molecules such as protein kinase C (PKC), PI 3-kinase, and extracellular signal-related kinases (10). Two types of mammalian ET receptors, ETA and ETB, have been characterized and purified (11, 12), and their cDNA have been cloned (13, 14). ETA receptor is selective for ET-1 and ET-2, while ETB receptor binds ET-1, ET-2, and ET-3 with equal affinity. ET-1 is thought to play important roles in various pathophysiological conditions.

Recently, several reports have shown that insulin stimulates ET-1 secretion from endothelial cells and also enhances ET-1 binding to its receptors (15). It has also been shown that the plasma ET-1 level is elevated in type II diabetes patients with microvascular complications, suggesting that ET-1 may be involved in diabetes-related complications such as microangiopathy (15, 16). Although there is an interest in investigating the role of ET-1 in the development of diabetic complications, very little is known about whether or not the ET system is involved in glucose metabolism. In our studies to examine whether ET-1 interacts with insulin in regulating glucose transport, we are surprised to find that ET-1 alone stimulates glucose uptake. This report shows for the first time in an unequivocal manner that the ETA receptor is expressed in 3T3-L1 adipocytes and that ET-1 stimulates GLUT4 translocation and glucose uptake in these cells via an insulin-independent pathway.

    EXPERIMENTAL PROCEDURES

Materials-- 3H-Labeled 2-deoxy-D-glucose (2-DOG) (26 Ci/mmol) was purchased from NEN Life Science Products. ET-1 and ET-3 were purchased from American Peptide Co. (Sunnyvale, CA). A-216546 and A-192621 were synthesized at Abbott Laboratories. Other reagents were of analytical grade.

Cell Culture-- The 3T3-L1 fibroblasts were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS). For experiments, cells were plated at 20,000 cells/well in 48-well plates or 10,000 cells/well in 96-well plates in DMEM containing 10% FBS. After 3 days, cells were fed with DMEM containing 10% FBS. On day 4, cells at 100% confluency were treated with induction medium (DMEM with 10% FBS, 400 nM insulin, 250 nM dexamethasone, and 0.5 mM isobutylmethylxanthine) for 3 days and then changed back to DMEM containing 10% FBS on day 7. Cells were fed again on day 10 with DMEM containing 10% FBS and used for studies between day 12 and day 14 (or as indicated) after plating. At that time, fat droplets were observed in ~70% of the cells.

3H-Labeled 2-DOG Uptake-- Cells in 48-well plates (or 96-well plates as indicated) were washed once with 500 µl/well (200 µl/well) of serum-free DMEM, and incubated in 500 µl/well (200 µl/well) of serum-free DMEM for 3 h at 37 °C. Cells were then washed twice with 500 µl/well (200 µl/well) of glucose-free serum-free DMEM, followed by incubating in 500 µl/well (200 µl/well) of glucose-free DMEM for 30 min at 37 °C. In experiments using kinase inhibitors, the agents were added at the beginning of this 30-min incubation period except for wortmannin, which was added 10 min before the addition of insulin or ET-1. Afterward, insulin and/or ET-1 (or others as indicated) were added, and cells were incubated for another 30 min at 37 °C. 3H-Labeled 2-DOG (final concentration of 50 µM, 0.33 µCi/ml) was added, and cells were incubated for 20 min at 37 °C. The incubation was stopped by washing cells with 500 µl/well (200 µl/well) of ice-cold phosphate-buffered saline (PBS) twice. Cells were dissolved in 0.1 N NaOH for scintillation counting.

[125I]ET-1 Binding to Cells-- Cells in 48-well culture plates were incubated with 125I-labeled ET-1 in 0.2 ml/well of buffer 1 (Earle's solution; 140 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO4, 5 mM glucose, buffered with 25 mM Hepes, pH 7.4) containing protease inhibitors (5 µg/ml pepstatin A, 0.01 mM phosphoramidon, 0.1 mM phenylmethylsulfonyl fluoride) for 4 h at 4 °C. After incubation, cells were washed twice with 0.5 ml/well of PBS, followed by solubilization in 0.5 ml of 0.1 N NaOH before counting. Nonspecific binding was determined in the presence of 1 µM ET-1.

Measurement of PI Hydrolysis-- PI hydrolysis was measured as described previously (17). Briefly, cells in 48-well culture plates were labeled with 1 µCi/well of myo-[3H]inositol for 24 h. Cells were washed with PBS and then incubated with buffer 1 containing protease inhibitors (as described above) and 10 mM LiCl for 60 min before being challenged with ET-1 for 30 min. ET challenge was terminated by the addition of 50 µl of N NaOH, and the mixture was immediately neutralized by adding 50 µl of 1 N HCl. The samples were treated with 1.5 ml of chloroform/methanol (1:2, v/v). Total inositol phosphates were extracted after adding chloroform and water to give final proportions of chloroform/methanol/water of 1:1:0.9 (v/v/v). The upper aqueous phase (1 ml) was retained and analyzed by batch chromatography using the anion exchange resin AG1-X8 (Bio-Rad). Total water-soluble inositol phosphates were eluted from the resin by 6 ml of 1 M ammonium formate with 0.1 N formic acid after the resin was washed with 6 ml of 60 mM sodium formate with 5 mM sodium tetraborate.

Immunofluorescence Staining and Confocal Microscopy-- The 3T3-L1 cells on day 12 after the induction medium treatment were treated with collagenase I (200 units/ml) and plated in two-chamber slides in DMEM containing 10% FBS 24 h before the experiment. Cells were put into serum-free medium for 3 h and then stimulated with or without ET-1 (10 nM) or insulin (100 nM) for 10 min (or as indicated). Afterward, cells were washed with PBS for 30 s, fixed with methanol at -20 °C for 20 min, rinsed with PBS three times, and incubated with PBS containing 10% rabbit serum for 30 min at 37 °C. The slides were then incubated with an anti-GLUT4 antibody (1:50 dilution) derived from goat (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in PBS with 10% rabbit serum for 2 h at 37 °C. After the incubation, slides were rinsed with PBS three times and then incubated with fluorescein-conjugated rabbit anti-goat IgG (1:50 dilution, Jackson ImmunoResearch Laboratories, West Grove, PA) for 30 min at 37 °C, followed by another three rinses with PBS. The slides were mounted, and pictures were taken using a Bio-Rad MRC1000 confocal microscope linked to an image analyzer.

Insulin-responsive Aminopeptidase (IRAP) Translocation-- Cells in 24-well plates were incubated in serum-free DMEM (1 ml/well) for 3 h at 37 °C and then incubated with insulin or ET-1 for 10 min. Cells were quickly chilled to 4 °C, washed briefly with 1 ml/well of PBS, washed once more with 1 ml/well of buffer 2 (PBS, 0.1 mM CaCl2, 1 mM MgCl2). Cells were then incubated with 0.4 ml/well of 1.5 mg/ml N-hydroxysuccinimide-SS-biotin (Pierce) in 10 mM HEPES, 2 mM CaCl2, 150 mM NaCl, pH 8.5, on ice with constant agitation for 20 min. The biotin cross-linking process was repeated once more. To stop the cross-linking reaction, cells were washed once with 1 ml/well of buffer 2 containing 100 mM glycine and followed by incubation in this buffer (1 ml/well) on ice for 20 min with constant agitation. Afterward, cells were washed once with PBS and lysed in 0.6 ml/well of lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 10% glycerol, 1% Triton X-100, 10 µg/ml aprotinin, and 10 µg/ml leupeptin). Cells were removed from the well into a microcentrifuge tube, homogenized by a microultrasonic cell disruptor (Kontes), and then centrifuged at 10,000 × g for 20 min. The supernatant (0.4 ml/sample) was incubated with 50 µl of a 50% slurry of streptavidin-agarose beads (Pierce) for 24 h at 4 °C with constant agitation. The beads were collected by centrifugation and washed three times with 1 ml/each of lysis buffer, followed by one wash with water. The proteins were released from the beads by incubating beads with 20 µl of 5× SDS-PAGE sample buffer containing 0.5% beta -mercaptoethanol. Samples were analyzed by SDS-PAGE using a 4-12% gradient gel (Novex, San Diego, CA).

SDS-PAGE and Western Blot Analysis-- Samples were resolved by SDS-PAGE using a gradient gel (as indicated; Novex, San Diego, CA) and proteins were electrophoretically transferred to a polyvinylidene difluoride membrane (Immobilon-P, 0.45-µm pore size, Millipore, Corp., Burlington, MA) for Western blotting. The membrane was blotted with 5% nonfat dry milk in PBS-T (10 mM Tris, pH 8.0, 0.15 M NaCl, 0.1% Tween 20) for at least 30 min and then incubated with primary antibodies in 5% bovine serum albumin (anti-IRAP, 5000-fold dilution; anti-phospho-Akt, 2000-fold dilution; both derived from rabbits) for 18 h at 4 °C. The anti-IRAP antibody was obtained from Metabolex (Hayward, CA), and the anti-phospho-Akt antibody was from New England Biolabs (Beverly, MA). For the detection of proteins with tyrosine phosphorylation, the membrane was blotted with PBS-T containing 1% nonfat dry milk and 1% bovine serum albumin, and the primary antibody was a mouse monoclonal antibody specific for phosphorylated tyrosine (from Santa Cruz Biotechnology; 10,000-fold dilution). The membrane was washed with PBS-T and incubated with a horseradish peroxidase-labeled anti-rabbit or anti-mouse antibody (Pierce) for at least 1 h at 25 °C. The paper was then incubated with detection reagent containing luminol in an alkaline buffer. The specific bands were visualized by exposing the paper to blue light-sensitive autoradiography films.

    RESULTS

Effects of ET-1 and Insulin on Glucose Uptake in 3T3-L1 Adipocytes-- To investigate whether ET-1 interacts with insulin in regulating cellular functions, we first examined glucose uptake stimulated by insulin in the presence or absence of ET-1. Fig. 1A shows that insulin-stimulated 2-DOG uptake in a dose-dependent manner, with a 5-fold stimulation at 100 nM. ET-1 (10 nM) alone stimulated 2-DOG uptake by 2.6-fold in the absence of insulin. An additive effect on stimulating 2-DOG uptake was observed when cells were treated with ET-1 and a low concentration (0.1 or 1 nM) of insulin. To further confirm the observation that ET-1 alone stimulates glucose uptake in the 3T3-L1 adipocytes, we compared the effects of ET-1 and angiotensin II (ANG II). Fig. 1B shows that, at three different concentrations examined (0.1, 1, and 10 nM), ET-1 stimulated 2-DOG by 2-2.5-fold. As a comparison, ANG II at the same concentrations, a GPCR (angiotensin II receptor) agonist and a potent vasoactive agent, which has been shown to stimulate lipogenesis in 3T3-L1 and human adipose cells (18), did not show a significant effect. Fig. 1C shows that the effect of ET-1 at 1 nM was completely blocked by 0.1 µM A-216546, an antagonist selective for ETA receptor (19). As a comparison, 0.1 µM A-192621, an antagonist selective for ETB receptor (20), had no effect in blocking ET-1-stimulated glucose uptake. These results show that ET-1 alone stimulates glucose uptake via activation of the ETA receptor.


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Fig. 1.   Effects of insulin, ET-1, and angiotensin II on 2-DOG uptake in the 3T3-L1 adipocytes. Cells in 96-well plates were incubated with increasing concentrations of insulin in the presence or absence of 10 nM ET-1 for 30 min (A) or increasing concentrations of ET-1 or angiotensin II for 30 min (B) and then assayed for the 2-DOG uptake as described under "Materials and Methods." C, cells were incubated with 1 nM ET-1 in the presence of A-216546 (0.1 µM) or A-192621 (0.1 µM). Each value represents the mean ± S.D. of four determinations. Statistical significance was determined by the unpaired Student's t test. *, p < 0.001; **, p < 0.05.

Effect of ET-1 Dependent on Adipocyte Differentiation-- Next, we examined whether the effect of ET-1 on glucose uptake is dependent on the differentiation state of the 3T3-L1 cells. Fig. 2 shows that, in undifferentiated 3T3-L1 cells, both insulin and ET-1 exhibited little or no effect on stimulating 2-DOG uptake. In comparison, insulin at 100 nM stimulated 2-DOG uptake in differentiated adipocytes by 9-fold (Fig. 2A), while ET-1 at 100 nM stimulated 2-DOG uptake in adipocytes by 3-fold (Fig. 2B). The results show that the effect of ET-1, like that of insulin, is dependent upon the differentiation state of the 3T3-L1 cells. From four independent experiments, the glucose uptake stimulation in 3T3-L1 adipocytes by 100 nM ET-1 in comparison with no treatment was 2.47 ± 0.23-fold (mean ± S.E.), and the EC50 value for ET-1 was determined to be 0.29 ± 0.13 nM. This effect is consistent with the EC50 values observed in other ET-1-mediated biological responses (21).


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Fig. 2.   Effects of insulin and ET-1 on 2-DOG uptake in differentiated versus undifferentiated 3T3-L1 cells. Cells in 48-well plates were incubated with increasing concentrations of insulin for 30 min (A) or increasing concentrations of ET-1 for 30 min (B) and then assayed for the 2-DOG uptake. The undifferentiated cells were maintained at ~80% confluency in DMEM containing 10% FBS. The differentiated cells were prepared as described under "Materials and Methods." Each value represents the mean ± S.D. of four determinations.

It is known that the expression of several proteins such as insulin receptor and GLUT4 increases during 3T3-L1 differentiation. We examined whether the expression of ET receptors was altered by differentiation. The number of ET binding sites was determined in saturation binding studies. As shown in Fig. 3A, ET-1 binding to undifferentiated cells reached a plateau when free ET-1 concentration in the buffer was 0.6 nM. The Scatchard plot (Fig. 3A, inset) resulted in a straight line and yielded a maximum binding (Bmax) value of 670 fmol/106 cells and an equilibrium dissociation constant (Kd) value of 0.52 nM. ET-1 saturation binding in differentiated cells yielded a similar result with a Bmax value of 640 fmol/106 cells and a Kd value of 1.3 nM (not shown). From three independent experiments, the Bmax and Kd values were determined to be 890 ± 124 fmol/106 cells and 0.99 ± 0.67 nM (mean ± S.E.) for the undifferentiated cells and 963 ± 191 fmol/106 cells and 1.65 ± 0.81 nM for the differentiated cells. The results show that the 3T3-L1 cells express a large number of ET receptors (~560,000 sites/cell), and the number of ET binding sites is not significantly altered after cell differentiation, although the affinity of ET-1 for the receptor seems to be lower in the differentiated cells.


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Fig. 3.   ET-1 binding studies and ET-1-evoked PI hydrolysis in 3T3-L1 cells. A, saturation binding studies. Undifferentiated 3T3-L1 cells in 48-well plates were incubated for 4 h at 4 °C with increasing concentrations of 125I-labeled ET-1 in the absence (open circle ) or presence (black-triangle) of 1 µM unlabeled ET-1. Specific binding (black-square) was determined by subtraction of nonspecific binding (black-triangle) from total binding (open circle ). Each value represents the mean ± S.D. of three determinations. Inset, Scatchard analysis of the data. B, competition binding studies. Differentiated 3T3-L1 cells in 48-well plates were incubated with 0.1 nM 125I-labeled ET-1 in the presence of increasing concentrations of unlabeled ligands for 4 h at 4 °C. Results are expressed as % of control (specific binding in the absence of unlabeled ligand). Nonspecific binding, determined in the presence of 1 µM of ET-1, was subtracted from total binding to give specific binding. Each value represents the mean ± S.D. of three determinations. C, PI hydrolysis. Cells in 48-well plates were prelabeled with myo-[3H]inositol (1 µCi/well) for 16 h. Cells were challenged with various concentrations of ET-1 in buffer 1 with 10 mM LiCl for 30 min at 37 °C. Results were calculated by normalizing AG1-X8-bound radioactivity at each point to that of control (no addition of ET). Each value represents the mean of two determinations.

To find out what subtypes of ET receptors are expressed in the differentiated 3T3-L1 cells, a competition binding study comparing ET-1 and ET-3 was conducted. Fig. 3B shows that unlabeled ET-1 effectively inhibited specific 125I-labeled ET-1 binding (IC50 = 0.03 nM). ETB-selective ligand ET-3 was much less effective in inhibiting specific 125I-labeled ET-1 binding with an IC50 value of 449 nM. These results show that ET receptor in 3T3-L1 adipocytes is predominantly the ETA subtype, which is consistent with the observation in Fig. 1C.

To investigate whether other biological responses stimulated by ET-1 are also dependent on cell differentiation, we examined ET-1-evoked PI hydrolysis in these cells. Fig. 3C shows that ET-1 stimulated PI hydrolysis in a dose-dependent manner. The EC50 values were 0.17 nM for undifferentiated cells versus 0.28 nM for differentiated cells. The maximal stimulation was reached at ~1 nM ET-1 in both cases, although the maximal stimulation was higher in the undifferentiated cells. The results suggest that ET-1 stimulated PI hydrolysis in both undifferentiated and differentiated cells in a similar manner, possibly mediated by a Gq protein known to be linked to PI hydrolysis.

Taken together, these results show that the differentiation of 3T3-L1 cells into adipocytes does not have a significant effect on the expression of the ET receptor or on ET-1-stimulated PI hydrolysis. However, differentiation specifically affects ET-1-stimulated glucose uptake, suggesting that ET-1-stimulated glucose uptake may be linked to GLUT4, which is expressed at a low level in the undifferentiated cells but is greatly up-regulated during differentiation.

Effect of ET-1 on the Translocation of IRAP and GLUT4-- To investigate whether ET-1 stimulated GLUT4 translocation, immunofluorescence staining and confocal microscopy were employed. In the control slides without ET-1 or insulin treatment, cells were found to have GLUT4 located in punctate structures distributed throughout the cytoplasm with intense staining concentrated on some spots and a low level of immunofluorescence in the plasma membrane; a typical example was shown in Fig. 4A, part a. As expected, cells treated with insulin (100 nM) exhibited a higher level of immunofluorescence in the plasma membrane (Fig. 4A, part f), indicating GLUT4 translocation to the plasma membrane. Cells treated with ET-1 (10 nM) also exhibited an increase in the level of immunofluorescence in the plasma membrane (Fig. 4A, parts b-d). The effect of ET-1 on GLUT4 translocation was time-dependent with the optimal effect observed at 20 min (Fig. 4A, part c), which was blocked by 10 µM A-216546 (Fig. 4A, part e). The results suggest that both insulin and ET-1 stimulate GLUT4 translocation, although the effect of ET-1 may be less than that of insulin. The results are consistent with the observation in the 2-DOG uptake studies.


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Fig. 4.   Effects of insulin and ET-1 on the translocation of GLUT4 and IRAP in 3T3-L1 adipocytes. A, immunofluorescence staining of GLUT4. Differentiated 3T3-L1 cells were treated with collagenase I and plated on chamber slides in DMEM containing 10% FBS for 24 h. Cells were put in serum-free DMEM for 3 h before treated with medium (a, control), or 10 nM ET-1 (b, 10 min; c, 20 min; d, 30 min), or 10 nM ET-1 in the presence of 10 µM A-216546 for 20 min (e), or 100 nM insulin for 10 min (f). Cells were then fixed and stained with an anti-GLUT4 antibody as described under "Materials and Methods." B, Western blot of membrane-associated IRAP. Differentiated 3T3-L1 cells in 24-well plates were treated with insulin or ET-1 (concentrations as indicated) for 10 min. Plasma membrane-associated proteins were cross-linked to biotin and isolated by streptavidin-agarose beads. The lysates were analyzed by SDS-PAGE and Western blotting as described under "Materials and Methods." The SDS-PAGE was also detected by silver staining to make sure that equal amounts of proteins were used for the samples. The molecular mass of IRAP is 165 kDa. The results are representative of three different experiments.

To further demonstrate that indeed ET-1 induces the translocation of GLUT4-containing vesicles in the 3T3-L1 adipocytes, we compared the effects of ET-1 and insulin on IRAP translocation using a sensitive cell surface biotinylation method (for details, see "Materials and Methods"). IRAP is an aminopeptidase that is one of the major polypeptides enriched in GLUT4-containing vesicles and is known to co-translocate with GLUT4 (22). Fig. 4B shows that both insulin and ET-1 caused an increase in membrane-associated IRAP in a dose-dependent manner, suggesting that ET-1, like insulin, indeed stimulates the translocation of GLUT4-containing vesicles from cytosol to the plasma membrane.

Effect of ET-1 on Proteins Involved in the Insulin Signaling Pathway-- To investigate whether ET-1 treatment affects insulin signaling molecules, we examined the effect of ET-1 on the phosphorylation of Akt, IRS-1, and the beta -subunit of the insulin receptor (IRbeta ). Fig. 5A (left) shows that insulin at 100 nM stimulated the tyrosyl phosphorylation of IRS-1 and IRbeta after 10 min of incubation, while ET-1 had no effect. The identities of IRS-1 and IRbeta were confirmed using anti-IRS-1 and anti-IRbeta antibodies in Western blot analysis (not shown). Interestingly, ET-1 stimulated the tyrosyl phosphorylation of a 75-kDa protein in a time-dependent manner (Fig. 5A). Fig. 5B shows that insulin stimulated Akt phosphorylation in a dose-dependent manner, while ET-1 had no effect. These results suggest that ET-1 treatment does not stimulate phosphorylation of IRbeta , IRS-1, or Akt. Probably, ET-1 stimulates glucose uptake via an insulin-independent pathway.


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Fig. 5.  Effects of insulin and ET-1 on the phosphorylation of IRS-1, ERbeta , and Akt in 3T3-L1 adipocytes. A, differentiated 3T3-L1 cells in 48-well plates were treated with 100 nM insulin for 10 min or ET-1 for 40 min (left) or 100 nM ET-1 for 10 or 30 min (right). Cells were lysed in a buffer containing 50 mMTris-HC1, 1% Nonidet P40, 1 mM sodium orthovanadate and then centrifuged at 10,000 × g to remove nuclei and debris. The supernatants were retained to be analyzed by SDS-PAGE and Western blotting. The samples were blotted using an anti-phosphotyrosine antibody as described. B, differentiated 3T3-L1 cells were treated with increasing concentrations of insulin or ET-1 FOR 10 min, and cells were lysed in the SDS-PAGE sample buffer. The samples were blotted using an anti-phospho-Akt antibody. The results ate representative of three different experiments.

Effects of Kinase Inhibitors on ET-1-stimulated Glucose Uptake in 3T3-L1 Adipocytes-- To investigate the mechanism of ET-1-stimulated glucose uptake, we examined the roles of PKC, PI 3-kinase, and MAPK, the three kinases known to be activated by ET-1 (10). Fig. 6, A-C, shows that bisindolylmaleimide (bisindo; an inhibitor of PKC), PD98059 (PD; an inhibitor of MEK1/2), and wortmannin (an inhibitor of PI 3-kinase) seemed to partially inhibit the effect of ET-1. However, these agents also inhibited the basal glucose uptake. The insets in Fig. 6, A-C, show that, after normalizing ET-1-stimulated glucose uptake by the basal glucose uptake in the presence of the inhibitor, none of the inhibitors had a significant effect. In control experiments, 1 µM wortmannin blocks insulin-stimulated glucose uptake in 3T3-L1 adipocytes by >80%, while 80 µM PD98059 inhibits ET-1-stimulated extracellular signal-related kinases 1/2 in human smooth muscle cells by >50% (23).2 We then tested the effect of genistein, a general tyrosine kinase inhibitor. Fig. 6D shows that genistein at 1 and 10 µM had no effect. However, genistein at 100 µM inhibited ET-1-stimulated glucose uptake by ~70%. These results suggest that ET-1-stimulated glucose uptake in the 3T3-L1 adipocytes is independent of PKC, PI 3-kinase, or the mitogen-activated protein kinase pathway but may be mediated by a genistein-sensitive tyrosine kinase.


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Fig. 6.   Effects of kinase inhibitors on insulin or ET-1 stimulated 2-DOG uptake in 3T3-L1 adipocytes. Cells in 48-well plates were treated with different concentrations of bisindolylmaleimide (bisindo) (A), wortmannin (B), PD98059 (C), or genistein (D) for 30 min before being treated with 10 nM ET-1 for another 30 min. The uptake of 2-DOG was then assayed. Each value represents the mean of four determinations. Statistical significance was determined by the unpaired Student's t test. *, p < 0.001.


    DISCUSSION

It is known that signal transduction mediated by certain GPCRs interacts with the insulin signaling pathway to regulate cellular functions in a complicated manner. In some cases, the interaction leads to insulin resistance and a decrease in insulin-stimulated intracellular signaling. In others, the GPCRs and insulin work in an additive manner to stimulate a cellular function. For example, it has been shown that angiotensin II causes an acute inhibition of both basal and insulin-stimulated PI 3-kinase activity in the rat heart and in rat aortic smooth muscle cells (24, 25). In cardiomyocytes isolated from adult rat hearts, the effect of insulin on glucose uptake can be partially blocked by modifying G-proteins with cholera toxin, yet isoprenaline alone, like insulin, increases glucose transport (26). Also, in both cardiomyocytes and brown adipocytes, adrenergic stimulation induces GLUT4 translocation and glucose uptake (27, 28). Furthermore, the introduction of GTP analogs such as GTPgamma S into 3T3-L1 adipocytes stimulates GLUT4 translocation and glucose uptake independent of insulin (5-7). Possibly, GPCRs play a role in the pathogenesis of insulin resistance and cardiovascular diseases by either modulating glucose uptake or directly interacting with insulin signaling.

In this report, we show for the first time, in an unequivocal manner, that ETA receptor is expressed in 3T3-L1 adipocytes and that ET-1 stimulates GLUT4 translocation and glucose uptake in these cells. In comparison with insulin, which stimulates glucose transport by a magnitude of 6-10-fold in 3T3-L1 adipocytes, the effect of ET-1 may seem modest (2-3-fold stimulation). However, an additive effect is observed when adipocytes are treated with low concentrations of insulin (<=  1 nM) and ET-1 simultaneously, suggesting that they may be acting through independent pathways. Our studies examining the phosphorylation of IRbeta , IRS-1, and Akt confirm that ET-1 has no effect on the early signaling molecules activated by insulin. Is the effect of ET-1 mediated by GLUT4 in the 3T3-L1 adipocytes? Results from studies comparing the differentiated versus undifferentiated 3T3-L1 cells show that the numbers of ET-1 binding sites are not significantly different in undifferentiated versus differentiated cells, but ET-1-stimulated glucose uptake is significantly higher in the differentiated cells. These results suggest that the effect of ET-1 on glucose uptake is probably linked to the presence of GLUT4 in one state versus the other. Indeed, we show that ET-1 directly stimulates the translocation of GLUT4 and IRAP. Our results demonstrate that immunofluorescence staining using the confocal microscopy is a sensitive method to examine GLUT4 translocation stimulated by ET-1 or insulin, although the effect of ET-1 on glucose uptake is ~25% of that of insulin. Furthermore, the cell surface biotinylation method, which has been used to detect IRAP translocation, is an extremely sensitive way to examine the increase of a protein in the plasma membrane (22). Fig. 4B shows that both insulin and ET-1 stimulate IRAP translocation in a dose-dependent manner. We do not have an explanation of why ET-1 stimulates the increase in membrane-associated IRAP to the same degree as insulin, because the effect of ET-1 on glucose uptake is clearly less than that of insulin.

The receptor binding studies show that 3T3-L1 cells express predominantly the ETA receptor. Consistent with the binding studies, A-216546, an antagonist selective for the ETA receptor, completely blocks the effect of ET-1 on glucose uptake, while A-192621, an antagonist selective for the ETB receptor, does not have an effect. Although in this report we did not address the issue of which G-protein is involved in ET-1-stimulated glucose uptake, ET-1 probably activates Gq and PLC-beta in these cells due to the observation that ET-1 stimulates PI hydrolysis in both undifferentiated and differentiated 3T3-L1 cells. Gq is coupled to phosphoinositide-specific phospholipase C-beta , which hydrolyzes phosphatidylinositol 4,5-bisphosphate to form inositol 1,4,5-triphosphate and 1,2-diacylglycerol. Interestingly, although PKC is the downstream target of phospholipase C-beta and PI hydrolysis, our results show that the PKC inhibitor does not have a significant effect on ET-1-stimulated glucose uptake, suggesting that PKC is not involved, consistent with the observation by Kishi et al. (6) on the PAF receptor and the alpha 1a-adrenergic receptor. Furthermore, wortmannin and PD98059 do not affect the 2.5-fold of stimulation in glucose uptake induced by ET-1, suggesting that PI 3-kinase and the mitogen-activated protein kinase pathway are not involved. Genistein, which has been shown to inhibit GTPgamma S-stimulated GLUT4 translocation (7), seems to inhibit ET-1-stimulated glucose uptake at a high concentration (100 µM). It is interesting to note that, in the Western blot analysis using the anti-phosphotyrosine antibody, we observe an increase in the tyrosyl phosphorylation of a 75-kDa protein after ET-1 treatment. Possibly, a genistein-sensitive tyrosine kinase plays a role in mediating ET-1-stimulated glucose uptake. We are in the process of investigating whether the 75-kDa protein is involved in ET-1-stimulated glucose uptake.

It is known that the skeletal muscle plays a central role in glucose metabolism, and impairment in glucose metabolism in the skeletal muscle often results in diabetes. Although this report mainly focuses on the 3T3-L1 adipocytes, we have found from both reverse transcription-PCR and receptor binding studies that human skeletal muscle cells express predominantly ETA receptor with Bmax and Kd values of 81.6 fmol/106 cells (or 49,000 sites/cell) and 0.14 nM for ET-1 binding. In membranes prepared from rat skeletal muscle (soleus), ET-1 binding is of high affinity with Bmax and Kd values of 58.4 fmol/mg of protein (or 3.5 × 1010 sites/mg of protein) and 0.15 nM. In addition, we have observed that ET-1 stimulates glucose uptake in neonatal rat cardiomyocytes (29). These results imply that the ET-1 system may play a role in glucose metabolism in both adipose and muscle tissues and is potentially a useful model to study the link between GPCRs and insulin signaling.

Does the finding that ET-1 stimulates GLUT4 translocation and glucose uptake have any physiological significance? We propose two possible scenarios. The first is that ET-1 is involved in exercise/hypoxia-induced glucose uptake, which occurs in an insulin-independent manner. It is now well accepted that the plasma ET-1 level is significantly elevated under hypoxic conditions (30). It has also been reported that exercise tends to elevate ET-1 in plasma and in major organs such as heart and kidney (31-34), although these results are not as conclusive as the hypoxia studies (35). So far, there is no report on whether hypoxia/exercise-induced increase in ET-1 is linked to glucose metabolism. A second completely different scenario is that chronic elevation of ET-1 localized in the skeletal muscle may cause a constant, albeit modest, increase in glucose influx into the muscle, which may result in insulin resistance from glucose toxicity. Infusion of ET-1 into rats has been shown to induce insulin resistance in one study (36) but to reduce the blood glucose level in another (37, 38). More studies will be needed to further examine whether ET-1 plays a physiological or pathophysiological role in glucose metabolism.

In conclusion, we have shown that 3T3-L1 adipocytes express ETA receptor. ET-1 alone stimulates glucose uptake in these cells. The effect of ET-1 on glucose uptake is dependent on the differentiation of the adipocytes, suggesting a link to the expression of GLUT4, and consistent with the observation that ET-1 activates IRAP and GLUT4 translocation in adipocytes.

    ACKNOWLEDGEMENTS

The anti-IRAP antibody was a gift from Metabolex (Hayward, CA). We thank Dr. E. Uli Frevert for setting up the IRAP translocation assay. We are indebted to Drs. Terry J. Opgenorth, E. Uli Frevert, Christine Collins, and Regina Brun for critical comments of this work.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: D47V, AP9A, Abbott Laboratories 100 Abbott Park Rd., Abbott Park, IL 60064-3500. Tel.: 847-937-8048; Fax: 847-935-3585; E-mail: ruth.r.wuwong{at}abbott.com.

2 J. R. Wu-Wong and T. J. Opgenorth, unpublished results.

    ABBREVIATIONS

The abbreviations used are: GTPgamma S, guanosine 5'-3-O-(thio)triphosphate; ET, endothelin; ETA, type-A endothelin receptor; ETB, type-B endothelin receptor; GPCRs, G-protein-coupled receptors; PKC, protein kinase C; PI, phosphatidylinositol; 2-DOG, 2-deoxy-D-glucose; IR, insulin receptor; IRS-1, insulin receptor substrate-1; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; IRAP, insulin-responsive aminopeptidase.

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