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Endocrinology Vol. 141, No. 12 4623-4628
Copyright © 2000 by The Endocrine Society

ARTICLES

The Acute and Chronic Stimulatory Effects of Endothelin-1 on Glucose Transport Are Mediated by Distinct Pathways in 3T3-L1 Adipocytes1

Ken-ichi Ishibashi, Takeshi Imamura, Prem M. Sharma, Satoshi Ugi and Jerrold M. Olefsky

Department of Medicine, Division of Endocrinology and Metabolism, University of California, San Diego, La Jolla, California 92093-0673

Address all correspondence and requests for reprints to: Jerrold M. Olefsky, M.D., Department of Medicine (0673), University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0673. E-mail: jolefsky{at}ucsd.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
We have recently shown that pretreatment with endothelin-1 (ET-1) for 20 min stimulates GLUT4 translocation in a PI3-kinase-dependent manner in 3T3-L1 adipocytes (Imamura, T. et al., J Biol Chem 274:33691–33695). This study presents another pathway by which ET-1 potentiates glucose transport in 3T3-L1 adipocytes. ET-1 treatment (10 nM) leads to approximately 2.5-fold stimulation of 2-deoxyglucose (2-DOG) uptake within 20 min, reaching a maximal effect of ~4-fold at ~6 h, and recovering almost to basal levels after 24 h. Insulin treatment (3 ng/ml) results in an approximately 5-fold increase in 2-DOG uptake at 1 h, and recovering to basal levels after 24 h. The ETA receptor antagonist, BQ 610, inhibited ET-1 induced glucose uptake both at 20 min and 6 h, whereas the ETB receptor antagonist, BQ 788, was without effect. Interestingly, ET-1 stimulated 2-DOG uptake at 6 h, not at 20 min, was almost completely blocked by the protein-synthesis inhibitor, cycloheximide and the RNA-synthesis inhibitor, actinomycin D, suggesting that the short-term (20 min) and long-term (6 h) effects of ET-1 involve distinct mechanisms. GLUT4 translocation assay showed that 20 min, but not 6 h, exposure to ET-1 led to GLUT4 translocation to the plasma membrane. In contrast, 6 h, but not 20 min, exposure to ET-1 increased expression of the GLUT1 protein, without affecting expression of GLUT4 protein. ET-1 induced 2-DOG uptake and GLUT1 expression at 6 h were completely inhibited by the MEK inhibitor, PD 98059, and partially inhibited by the PI3-kinase inhibitor, LY 294002, and the G{alpha}i inhibitor, pertussis toxin. The PLC inhibitor, U 73122, was without effect. These findings suggest that ET-1 induced GLUT1 protein expression is primarily mediated via MAPK, and partially via PI3K in 3T3-L1 adipocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
GLUCOSE TRANSPORT IS the rate-limiting step in glucose disposal, and, among the family of transporters (1), both the insulin-sensitive glucose transporter, GLUT4, and the constitutive glucose transporter, GLUT1, are expressed in insulin-responsive tissues, such as skeletal muscle, cardiac muscle, and adipose tissue, including 3T3-L1 adipocytes (2).

GLUT4 translocates from an intracellular membrane compartment to the plasma membrane after acute insulin stimulation, leading to increased glucose uptake (3, 4). Recent evidence shows that, in addition to insulin, there are several other stimulants that can cause GLUT4 translocation and glucose uptake. For example, exercise induces GLUT4 translocation and glucose uptake via an insulin-independent pathway (5, 6). In addition, osmotic shock and GTP{gamma}s can stimulate GLUT4 translocation and glucose uptake in the absence of insulin (7, 8). Recently, we, and others, have reported that endothelin-1 (ET-1) can also stimulate GLUT4 translocation in 3T3-L1 adipocytes (9, 10).

ET-1 is a vascular active polypeptide, mainly produced in cardiac myocytes and vascular endothelial cells (11). Elevated ET-1 levels in the plasma have been reported in patients with insulin resistance, such as type 2 diabetes (12, 13), obesity (14), and hypertension (15). Furthermore, ET-1 can inhibit insulin-stimulated glucose uptake in vitro and in vivo (16, 17, 18, 19, 20). ET-1 binds to G protein-coupled Endothelin type A (ETA) receptors and activates phospholipase Cß, which increases the formation of inositol triphosphate, and diacylglycerol, leading to an increase in cytosolic Ca2+ and activation of PKC (21). ET-1 also induces tyrosine phosphorylation of SHC and its association with GRB2, leading to MAPK activation (22). Further, we showed that ET-1 activates the p110{alpha} catalytic subunit of PI3K, leading to GLUT4 translocation in 3T3-L1 adipocytes (9). Because the kinases associated with mitogenic action are part of the ET-1 signaling pathway, we further examined their role in ET-1 mediated 2-DOG uptake at various time intervals and evaluated the relationship between glucose uptake and glucose transporter protein expression in 3T3-L1 adipocytes.

In this study, we found that ET-1 treatment not only stimulates GLUT4 translocation, but also stimulates GLUT1 protein expression, with a biphasic effect on 2-DOG uptake in 3T3-L1 adipocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Materials
Mouse monoclonal anti-GLUT4 antibody (1F 8) was from Biogenesis Inc. (Brentwood, NH), and rabbit polyclonal anti-GLUT1 antibody was from Chemicon International Inc. (Temecula, CA). Horseradish peroxidase-linked antirabbit and antimouse antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Sheep IgG, fluorescein isothiocynate (FITC)- and tetramethyl rhodamine isothiocyanate (TRITC)-conjugated antirabbit, -mouse, -goat, and -sheep IgG antibodies were from Jackson ImmmunoResearch Laboratories Inc. (West Grove, PA). Endothelin-1, ETA receptor antagonist (BQ-610) and ETB receptor antagonist (BQ-788) were from Peninsula Laboratories, Inc. (San Carlos, CA). Cycloheximide and PLC inhibitor (U73122) were from Calbiochem (San Diego, CA). DMEM and FBS were purchased from Life Technologies, Inc. (Grand Island, NY). All other reagents were purchased from Sigma (St. Louis, MO).

Cell treatment and 2-DOG uptake
3T3-L1 adipocytes were cultured and differentiated as previously described (23). The procedure for glucose uptake was a modification of the method described by Klip et al. (24). 3T3-L1 adipocytes were incubated at 37 C in the presence of either 10 nM ET-1 or 3 ng/ml insulin for 0–48 h. Glucose uptake was determined in duplicate or triplicate at each point after the addition of 10 µl substrate (2-[3H]deoxyglucose or L-[3H]glucose; 0.1 µCi, final concentration 0.01 mmol/liter) to provide a concentration at which cell membrane transport is rate limiting. The value for L-glucose was subtracted to correct each sample for the contributions of diffusion and trapping. For inhibitor treatments, 3T3-L1 adipocytes were incubated with 1 µM BQ-610, 1 µM BQ-788, 10 µM U73122, 25 µM LY 294002, 20 nM Rapamycin, or 50 µM PD 98059 for 30 min at 37 C before addition of ET-1 for 20 min or 6 h. Two micrgrams per ml actinomycin D or 10 µg/ml cycloheximide were added for 1 h, and 100 ng/ml pertussis toxin was added for 24 h at 37 C before addition of ET-1.

Immunostaining and immunofluorescence microscopy
Immunostaining of GLUT4 was performed essentially as described (25). The cells were fixed in 3.7% formaldehyde in PBS for 10 min at room temperature. Following PBS washing, the cells were permeabilized and blocked with 0.1% Triton X-100 and 2% FCS in PBS for 5 min. The cells were then incubated with anti-GLUT4 antibody in PBS with 2% FCS overnight at 4 C. After washing, GLUT4 and injected IgG were detected by incubation with TRITC-conjugated donkey antimouse IgG antibody and FITC-conjugated donkey antisheep antibody, respectively, followed by observation under immunofluorescence microscope. In all counting experiments, the observer was blinded to the experimental condition of each coverslip.

Western blotting
3T3-L1 adipocytes were stimulated with 10 nM ET-1 for 0–48 h at 37 C and lyzed in a solubilizing buffer containing 20 mM Tris, 1 mM EDTA, 140 mM NaCl, 1% Nonidet P-40 (NP-40), 50 U of aprotinin/ml, 1 mM Na3VO4, 1 mM PMSF, and 10 mM NaF, pH 7.5 for 30 min at 4 C. The cell lysates were centrifuged to remove insoluble materials. For Western blot analysis, whole cell lysates were denatured by boiling in Laemmli sample buffer containing 100 mM dithiothreitol and resolved by SDS-PAGE. For Western blot analysis using GLUT1 and GLUT4 antibodies, cell lysates were heated at 60 C in Laemmli sample buffer without dithiothreitol. Gels were transferred to polyvinylidene difluoride (PVDF) membrane (Immobilon-P, Millipore Corp., Bedford, MA), using Transblot apparatus (Bio-Rad Laboratories, Inc., Hercules, CA). For immunoblotting, membranes were blocked and probed with specified antibodies. Blots were then incubated with horseradish peroxidase-linked second antibody followed by chemiluminescence detection, according to the manufacturer’s instructions (Pierce Chemical Co., Rockford, IL). The experimental design for pretreatment with various inhibitors was identical with that described for the 2-DOG uptake assay.

Subcellular fractionation
3T3-L1 adipocytes were stimulated with or without 10 nM ET-1 for 6 h and then washed three times with ice-cold PBS. Cells were scraped into ice-cold HES buffer (225 mM Sucrose, 20 mM HEPES, pH 7.4, and 1 mM EDTA) supplemented with 100 mM sodium fluoride, 10 mM sodium pyrophosphate, 1 mM sodium vanadate and protease inhibitors (1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mg/ml benzamidine, 0.5 mM PMSF). Cells were then homogenized using an LSC homogenizer. Subcellular fractionation was carried out as described previously (26).


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Time course of ET-1 stimulated 2-DOG uptake and GLUT4 translocation in 3T3-L1 adipocytes
We compared the time course for ET-1 and insulin treatment on 2-DOG uptake over 48 h in 3T3-L1 adipocytes. Interestingly, treatment of cells with 10 nM ET-1 led to approximately 2.5-fold stimulation of 2-DOG uptake within 20 min, reaching a maximal effect approximately 4-fold at approximately 6 h, and recovering almost to basal levels after 24 h (Fig. 1AGo). In contrast, treatment of cells with 3 ng/ml insulin resulted in approximately 5-fold increase in 2-DOG uptake at 1 h, and recovering to basal levels after 24 h (Fig. 1AGo). To assess the correlation between 2-DOG uptake and GLUT4 translocation in response to ET-1 treatment, we measured GLUT4 translocation using immunofluorescent microscopy as previously described (9, 27). As shown previously (9), 20 min of ET-1 treatment led to GLUT4 translocation in 57% of cells, whereas, after 6 h exposure to ET-1, GLUT4 translocation was detected in only 23% of cells (Fig. 1BGo). These findings suggest that ET-1 induced 2-DOG uptake at 20 min depends on GLUT4 translocation, whereas the second peak of 2-DOG uptake at 6 h is independent of GLUT4 translocation.



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  		Figure 1. Time course of ET-1 stimulated 2-DOG uptake and GLUT4 translocation in 3T3-L1 adipocytes. A, 3T3-L1 adipocytes were treated with ET-1 (10 nM) or insulin (3 ng/ml) for various time points as indicated (0–48 h), followed by measurement of 2-DOG uptake as described in Materials and Methods. B, 3T3-L1 adipocytes on coverslips were treated with ET-1 (10 nM) for either 20 min or 6 h, and GLUT4 translocation was measured as described in Materials and Methods. The data are mean ± SE from three independent experiments.

 
Effect of ET receptor antagonists, and, RNA and protein synthesis inhibitors on ET-1 stimulated 2-DOG uptake
We (9), and others (10), have shown that short-term exposure to ET-1 stimulates 2-DOG uptake and GLUT4 translocation through the ETA receptor. To assess whether the longer-term effect of ET-1 to induce 2-DOG uptake at 6 h is mediated via the ETA or ETB receptor, we pretreated 3T3-L1 adipocytes with 1 µM BQ 610, an ETA receptor antagonist or with 1 µM BQ 788, an ETB receptor antagonist, and stimulated the cells with ET-1 for either 20 min or 6 h. BQ 610 completely prevented ET-1 induced 2-DOG uptake at both 20 min and 6 h (Fig. 2Go), whereas BQ 788 was without effect, indicating that both the long-term (6 h) as well as the short-term (20 min) effects of ET-1 are mediated through the ETA receptor. Because ET-1 induced 2-DOG uptake at 6 h appears to be independent of GLUT4 translocation, it seemed probable that RNA or protein synthesis are involved in this effect. To investigate this possibility, we pretreated cells with the protein-synthesis inhibitor, cycloheximide (10 µg/ml), or the RNA-synthesis inhibitor, actinomycin D (2 µg/ml), followed by ET-1 stimulation for 20 min or 6 h. Both cycloheximide and actinomycin D inhibited ET-1 induced 2-DOG uptake at 6 h, but they were without effect at 20 min (Fig. 2Go). These findings demonstrate that the short-term, and long-term effects of ET-1 involve distinct mechanisms and that ET-1 stimulated 2-DOG uptake at 6 h depends on new RNA and protein synthesis.



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  		Figure 2. Effect of ET receptor antagonist, and, RNA and protein synthesis inhibitors on ET-1 stimulated 2-DOG uptake. After treating with 1 µM BQ 610 or 1 µM BQ 788 for 30 min, or 10 µg/ml cycloheximide (CHX) or 2 µg/ml actinomycin D (AMD) for 1 h, 3T3-L1 adipocytes were treated with ET-1 (10 nM) for 20 min (open bar) or 6 h (closed bar), and 2-DOG uptake was measured as described in Materials and Methods. The data are mean ± SE from three independent experiments.

 
Time-course analysis of ET-1 treatment on GLUT1 and GLUT4 protein expression
Because protein synthesis is necessary for ET-1 induced 2-DOG uptake at 6 h, we examined the effects of ET-1 treatment on cellular GLUT1 and GLUT4 protein content. Cells were treated with ET-1 for various time intervals (0–48 h) as indicated in Fig. 3AGo. Cell lysates were then analyzed by SDS-PAGE, followed by Western blotting with either an anti-GLUT1 or an anti-GLUT4 antibody. Treatment of cells with ET-1 increased GLUT1 protein expression by approximately 3-fold, with a maximal effect at approximately 6 h, followed by a decline to basal level [Fig. 3Go, A (upper panel) and B]. In contrast, ET-1 treatment was without effect on GLUT4 protein expression (Fig. 3AGo, lower panel). These results are consistent with the finding that ET-1 stimulates 2-DOG uptake by approximately 4-fold at 6 h (Fig. 1AGo), and indicate that ET-1 stimulated 2-DOG uptake at 6 h depends on GLUT1 protein expression.



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  		Figure 3. Time course analysis of ET-1 treatment on GLUT1 and GLUT4 protein expression in 3T3-L1 adipocytes. A, 3T3-L1 adipocytes were incubated in the presence of ET-1 (10 nM) for various time points as indicated (0–48 h). Whole cell lysates were analyzed by Western blotting using anti-GLUT1 (top panel) or anti-GLUT4 (bottom panel) antibody as described in Materials and Methods. B, Graphic representation of the results shown in A (top panel) using a desk scanner. The data are mean ± SE from three independent experiments. C, Intracellular membranes (IM) or plasma membranes (PM) were isolated from 3T3-L1 adipocytes, that were incubated in the absence or presence of ET-1 (10 nM) for 6 h as described in Materials and Methods. These experiments were repeated twice with similar results.

 
It has been reported that chronic exposure to insulin also augments GLUT1 protein expression, whereas, unlike ET-1, chronic insulin treatment decreases GLUT4 protein levels (28, 29). Dexamethasone, arachidonic acid, and tumor necrosis factor-{alpha} (TNF-{alpha}) can also lead to enhanced GLUT1 protein expression in L6 skeletal muscle cells and in 3T3-L1 cells (30, 31, 32). Because plasma membrane GLUT1 is responsible for glucose uptake (31, 32), we isolated membrane fractions and analyzed them by Western blot analysis to examine the distribution of ET-1 induced GLUT1 protein expression. As shown in Fig. 3CGo, GLUT1 was detected in both intracellular membrane (IM) and plasma membrane (PM) fractions, and its levels were increased in both IM and PM upon 6 h ET-1 treatment. The increment of GLUT1 protein in PM was somewhat greater than that seen in the IM fraction.

Effect of inhibitors on ET-1 stimulated 2-DOG uptake and GLUT1 protein expression
ET-1 binds to the ETA receptor, which couples to G{alpha}q/11 and G{alpha}i (33), and activates phospholipase Cß (PLCß), which increases the formation of inositol triphosphate, and diacylglycerol, leading to an increase in cytosolic Ca2+ and activation of PKC (21). Further, ET-1 is reported to induce tyrosine phosphorylation of SHC and its association with GRB2, leading to MAPK phosphorylation (22). In addition, we have recently shown that ET-1 increases PI3K activity via G{alpha}q/11, leading to GLUT4 translocation (9).

To examine whether these proteins are involved in ET-1 stimulated 2-DOG uptake at 6 h and GLUT1 protein expression, we pretreated cells with various inhibitors. As shown in Fig. 4AGo, the G{alpha}i inhibitor, pertussis toxin (100 ng/ml) partially prevented ET-1 stimulated 2-DOG uptake, whereas the PLC inhibitor, U 73122 (10 µM), was without effect. Also, the PI3-kinase inhibitor, LY 294002 (25 µM) partially blocked ET-1 stimulated 2-DOG uptake. Rapamycin (20 nM), an inhibitor of p70 S6K, which is downstream of PI3-kinase, was without effect, whereas, the MEK inhibitor, PD 98059 (50 µM), completely blocked ET-1 stimulated 2-DOG uptake.



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  		Figure 4. Effect of inhibitors on ET-1 stimulated 2-DOG uptake and GLUT1 protein expression. A, After treating with 100 ng/ml pertussis toxin (PTX) for 24 h, 10 µM U 73122 for 10 min, 25 µM LY 294002 (LY), 20 nM Rapamycin (RM) or 50 µM PD 98059 (PD) for 30 min, 3T3-L1 adipocytes were treated with ET-1 (10 nM) for 6 h, and 2-DOG uptake was measured as described in Materials and Methods. The data are mean ± SE from three independent experiments. B, 3T3-L1 adipocytes were incubated in the absence or presence of ET-1 (10 nM) for 6 h, after treating with or without the inhibitors, as indicated. The experimental design for treatment with inhibitors was same as described in A, for the 2-DOG uptake assay. Whole cell lysates were subjected to SDS-PAGE and immunoblotted with anti-GLUT1 antibody as described in Materials and Methods. These experiments were repeated twice with similar results.

 
Similarly, PD 98059 completely blocked ET-1 stimulated GLUT1 protein expression, pertussis toxin and LY 294002 partially inhibited, and U 73122 and Rapamycin (data not shown) were without effect (Fig. 4BGo). Actinomycin D (2 µg/ml) inhibited ET-1 stimulated GLUT1 protein expression, consistent with the observation that actinomycin D inhibits ET-1 stimulated 2-DOG uptake at 6 h (Fig. 2Go). Because the pattern of 2-DOG uptake corresponds to that of GLUT1 protein expression, it seems reasonable to conclude that ET-1 stimulated 2-DOG uptake at 6 h is due to enhanced GLUT1 protein expression. Taken together, these findings suggest that MAPK activation and RNA synthesis are necessary for ET-1 stimulated GLUT1 protein expression, and that signaling via PI3K is at least partially involved.

It has been reported that a variety of hormones, drugs, and physiological manipulations alter the stability of the GLUT1 transcript in various cell lines and tissues (34). For example, treatment of 3T3-L1 fibroblasts with TNF-{alpha} increases the half-life of the GLUT1 messenger RNA, and the increased GLUT1 messenger RNA content results in the synthesis, and, plasma membrane localization of new GLUT1 protein, which leads to increased glucose transport activity (32). Recently, it has also been shown that cotransfection of myocytes with constitutively active versions of Ras, and MEK1, stimulate transcription from the GLUT1 promoter (35). These findings are consistent with our results and suggest that SHC/MAPK activation, followed by transcription of GLUT1 is necessary for ET-1 stimulated GLUT1 protein expression.

Although it has been shown that PI3K activity, and consequent activation of p70 S6K, both play important roles in the mitogenic signaling pathway (3), our inhibitor’s results suggest that PI3K, but not p70 S6K, is only involved in the mechanisms by which ET-1 stimulates GLUT1 protein expression. In contrast, insulin-stimulated GLUT1 protein expression is both PI3K and p70 S6K dependent in 3T3-L1 adipocytes (36), and, in L6 muscle cells (37, 38), unlike our results with ET-1, stimulated GLUT1 protein expression.

We also found that ET-1 stimulated GLUT1 protein expression and 2-DOG uptake were pertussis toxin sensitive, indicating the requirement for G{alpha}i. The ET-1 receptor can couple into G{alpha}i or G{alpha}q/11 (33), and we have previously shown that the acute effects of ET-1 on glucose transport were dependent on G{alpha}q/11, not G{alpha}i (9). Thus, it appears that the ET-1 receptor engages two distinct G proteins to mediate acute (G{alpha}q/11), and, chronic (G{alpha}i) stimulation of glucose transport. The acute effects feed through the PI3K pathway (9), whereas the chronic effects mainly require the Ras/MAPK signaling.

Effect of inhibitors on ET-1 stimulated p70 S6K and MAPK phosphorylation
To further assess the involvement of the MAPK and the PI3K pathway in ET-1 signaling, we examined ET-1 stimulated phosphorylation of MAPK, and p70 S6K, which is downstream of PI3K, after treating the cells with various inhibitors. As shown in Fig. 5Go, treatment with 10 nM ET-1 for 10 min rapidly led to p70 S6K and MAPK phosphorylation. BQ 610 (1 µM) completely inhibited ET-1 stimulated p70 S6K and MAPK phosphorylation, whereas BQ 788 (1 µM) had no effect, indicating that the ETA receptor rather than the ETB receptor conveys the ET-1 signal to PI3K and MAPK. Further, pertussis toxin (100 ng/ml) blocked ET-1 stimulated MAPK phosphorylation, and partially blocked ET-1 stimulated p70 S6K phosphorylation. Because ET-1 induces tyrosine phosphorylation of SHC and its association with GRB2, leading to MAPK phosphorylation (22), these findings suggest that in 3T3-L1 adipocytes, G{alpha}i transmits the ET-1 mediated signal to the SHC/MAPK pathway.



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  		Figure 5. Effect of inhibitors on ET-1 stimulated p70 S6K and MAPK phosphorylation. 3T3-L1 adipocytes were incubated in the absence or presence of ET-1 (10 nM) for 10 min, after treating with or without 1 µM BQ 610, 1 µM BQ 788, 25 µM LY 294002 (LY), 20 nM Rapamycin (RM) or 50 µM PD 98059 (PD) for 30 min or 100 ng/ml pertussis toxin (PTX) for 24 h. Whole cell lysates were subjected to SDS-PAGE, and immunoblotted with phospho specific p70 S6K or phospho specific MAPK antibody, as described in Materials and Methods. These experiments were repeated twice with similar results.

 
Because both MAPK and PI3K appear to be necessary for GLUT1 protein expression, we examined the existence of possible cross-talk between PI3K and MAPK in 3T3-L1 adipocytes pretreated with LY 294002 (25 µM), Rapamycin (20 nM) or PD 98059 (50 µM). The data in Fig. 5Go (lower panel) show that neither LY 294002 nor Rapamycin had any effect on ET-1 stimulated MAPK phosphorylation, although p70 S6K phosphorylation was completely blocked. However, PD 98059 was without effect on ET-1 stimulated p70 S6K phosphorylation (Fig. 5Go, upper panel), but it completely blocked MAPK phosphorylation. These results argue against cross-talk between the MAPK and the PI3K pathway in ET-1 signaling. However, further work is necessary to explain the mechanism of how both of these kinases are involved in ET-1 mediated GLUT1 protein expression.

In summary, our results indicate that ET-1 can stimulate glucose transport in 3T3-L1 adipocytes through a biphasic process. The acute stimulation involves GLUT4 translocation, whereas the more chronic (6 h) stimulatory effect is due to induction of GLUT1 protein synthesis. It is known that the G protein-coupled ETA receptor can use G{alpha}i or G{alpha}q/11 for its signaling functions, and we find that the acute and chronic effects of ET-1 on glucose transport are mediated by different G proteins. Thus, the acute effect of ET-1 to stimulate GLUT4 translocation involves coupling of the ETA receptor through G{alpha}q/11 to PI3K, and subsequent stimulation of glucose transport, and this effect does not involve G{alpha}i. Interestingly, the longer term effects of ET-1 to stimulate glucose transport involve coupling through G{alpha}i, which then leads to activation of the Ras/MAPK pathway, and enhanced GLUT1 protein expression.


   Acknowledgments
 
We thank Elizabeth Hansen for editorial assistance.


   Footnotes
 
1 This work was supported by NIH Grant DK-33651. Back

Received May 1, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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