Endothelin Stimulates Glucose Uptake and GLUT4 Translocation via Activation of Endothelin ETA Receptor in 3T3-L1 Adipocytes
1999; Elsevier BV; Volume: 274; Issue: 12 Linguagem: Inglês
10.1074/jbc.274.12.8103
ISSN1083-351X
AutoresJinshyun R. Wu‐Wong, Cathleen E. Berg, Jiahong Wang, William J. Chiou, Brian M. Fissel,
Tópico(s)Receptor Mechanisms and Signaling
ResumoEndothelin-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 β-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. 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 β-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. guanosine 5′-3-O-(thio)triphosphate endothelin type-A endothelin receptor type-B endothelin receptor G-protein-coupled receptors protein kinase C phosphatidylinositol 2-deoxy-d-glucose insulin receptor insulin receptor substrate-1 Dulbecco's modified Eagle's medium fetal bovine serum phosphate-buffered saline polyacrylamide gel electrophoresis insulin-responsive aminopeptidase 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 (1DeFronzo R.A. Diabetes. 1988; 37: 667-687Crossref PubMed Google Scholar). Glucose transport is the rate-limiting step in glucose metabolism. Among the six known facilitative glucose transporters (GLUT1 to -5 and GLUT7) (2Thorens B. Am. J. Physiol. 1996; 270: G541-G553PubMed Google Scholar), 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 (3Hansen P.A. Nolte L.A. Chen M.M. Holloszy J.O. J. Appl. Physiol. 1998; 85: 1218-1222Crossref PubMed Scopus (128) Google Scholar, 4Holloszy J.O. Kohrt W.M. Hansen P.A. Front. Biosci. 1998; 15: D1011-D1027Crossref Google Scholar). Also, introduction of GTP analogs, such as GTPγS,1 into 3T3-L1 adipocytes, and activation of α1-adrenergic receptors, stimulate glucose uptake independent of insulin (5Baldini G. Hohman R. Charron M.J. Lodish H.F. J. Biol. Chem. 1991; 266: 4037-4040Abstract Full Text PDF PubMed Google Scholar, 6Kishi K. Hayashi H. Wang L. Kamohara S. Tamaoka K. Shimizu T. Ushikubi F. Narumiya S. Ebina Y. J. Biol. Chem. 1996; 271: 26561-26568Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 7Elmendorf J.S. Chen D. Pressin J.E. J. Biol. Chem. 1998; 273: 13289-13296Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar).Endothelin (ET), originally isolated from cultured porcine aortic endothelial cells, is a peptide with 21 amino acid residues (8Yanagisawa M. Kurihara H. Kimura S. Tomobe Y. Kobayashi Y. Mitsui M. Yazaki Y. Goto K. Masaki T. Nature. 1988; 332: 411-415Crossref PubMed Scopus (10190) Google Scholar). Three distinct members of the ET family, namely, ET-1, ET-2, and ET-3, have been identified in humans through cloning (9Inoue A. Yanagisawa M. Kimura S. Kasuya Y. Miyauchi T. Goto K. Masaki T. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2863-2867Crossref PubMed Scopus (2557) Google Scholar). 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 (10Douglas S.A. Ohlstein E.H. J. Vasc. Res. 1997; 34: 152-164Crossref PubMed Scopus (108) Google Scholar). Two types of mammalian ET receptors, ETA and ETB, have been characterized and purified (11Kozuka M. Ito T. Hirose S. Lodhi K.M. Hagiwara H. J. Biol. Chem. 1991; 266: 16892-16896Abstract Full Text PDF PubMed Google Scholar, 12Wada K. Tabuchi H. Ohba R. Satoh M. Tachibana Y. Akiyama N. Hiraoka O. Asakura A. Miyamoto C. Furuichi Y. Biochem. Biophys. Res. Commun. 1990; 167: 251-257Crossref PubMed Scopus (73) Google Scholar), and their cDNA have been cloned (13Arai H. Hori S. Arimori I. Ohkubo H. Nakanishi S. Nature. 1990; 348: 730-732Crossref PubMed Scopus (2508) Google Scholar, 14Sakurai T. Yanagisawa M. Takuwa Y. Miyazaki H. Kimura S. Goto K. Masaki T. Nature. 1990; 348: 732-735Crossref PubMed Scopus (2359) Google Scholar). 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 (15Sármán B. Tóth M. Somogyi A. Diabetes Metab. Rev. 1998; 14: 171-175Crossref PubMed Scopus (43) Google Scholar). 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 (15Sármán B. Tóth M. Somogyi A. Diabetes Metab. Rev. 1998; 14: 171-175Crossref PubMed Scopus (43) Google Scholar, 16Kawamura M. Ohgawara H. Naruse M. Suzuki N. Iwasaki N. Naruse K. Hori S. Diabetes Care. 1992; 15: 1396-1397Crossref PubMed Scopus (72) Google Scholar). 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.DISCUSSIONIt 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 (24Velloso L.A. Folli F. Sun X.J. White M.F. Saad M.J. Kahn C.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12490-12495Crossref PubMed Scopus (352) Google Scholar, 25Folli F. Kahn C.R. Hansen H. Bouchie J.L. Feener E.P. J. Clin. Invest. 1997; 100: 2158-2169Crossref PubMed Scopus (404) Google Scholar). 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 (26Echel J. Gerlach-Eskuchen E. Reinauer H. Biochem. J. 1990; 272: 691-696Crossref PubMed Scopus (39) Google Scholar). Also, in both cardiomyocytes and brown adipocytes, adrenergic stimulation induces GLUT4 translocation and glucose uptake (27Fisher Y. Thomas J. Holman G.D. Rose H. Kammermeier H. Am. J. Physiol. 1996; 270: C1204-C1210Crossref PubMed Google Scholar, 28Shimizu Y. Shimazu T. Biochem. Biophys. Res. Commun. 1994; 202: 660-665Crossref PubMed Scopus (42) Google Scholar). Furthermore, the introduction of GTP analogs such as GTPγS into 3T3-L1 adipocytes stimulates GLUT4 translocation and glucose uptake independent of insulin (5Baldini G. Hohman R. Charron M.J. Lodish H.F. J. Biol. Chem. 1991; 266: 4037-4040Abstract Full Text PDF PubMed Google Scholar, 6Kishi K. Hayashi H. Wang L. Kamohara S. Tamaoka K. Shimizu T. Ushikubi F. Narumiya S. Ebina Y. J. Biol. Chem. 1996; 271: 26561-26568Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 7Elmendorf J.S. Chen D. Pressin J.E. J. Biol. Chem. 1998; 273: 13289-13296Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). 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 IRβ, 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 versusdifferentiated 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 (22Ross S.A. Scott H.M. Morris N.J. Leung W.-Y. Mao F. Lienhard G.E. Keller S.R. J. Biol. Chem. 1996; 271: 3328-3332Crossref PubMed Scopus (140) Google Scholar). Fig. 4 B 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 ETAreceptor, 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-β 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-β, 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-β 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. (6Kishi K. Hayashi H. Wang L. Kamohara S. Tamaoka K. Shimizu T. Ushikubi F. Narumiya S. Ebina Y. J. Biol. Chem. 1996; 271: 26561-26568Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) on the PAF receptor and the α1a-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 GTPγS-stimulated GLUT4 translocation (7Elmendorf J.S. Chen D. Pressin J.E. J. Biol. Chem. 1998; 273: 13289-13296Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), 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 B maxand K d 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 B max andK d 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.Wu-Wong, J. R., Polakowski, J., and Opgenorth, T. J. (1998)American Diabetes Association 58th Scientific Sessions, Chicago, Illinois, June 13–16, 1998, abstract.Google Scholar). 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 (30Rosendorff C. Cardiovasc. Drugs Ther. 1997; 10: 795-802Crossref PubMed Scopus (38) Google Scholar). It has also been reported that exercise tends to elevate ET-1 in plasma and in major organs such as heart and kidney (31Maeda S. Miyauchi T. Kobayashi T. Goto K. Matsuda M. J. Appl. Physiol. 1998; 85: 425-431Crossref PubMed Scopus (31) Google Scholar, 32Maeda S. Miyauchi T. Sakai S. Kobayashi T. Goto K. Sugishita Y. Matsuda M. J. Cardiovasc. Pharmacol. 1998; 31: S392-S394Crossref PubMed Scopus (4) Google Scholar, 33Maeda S. Miyauchi T. Goto K. Matsuda M. Life Sci. 1997; 61: 419-425Crossref PubMed Scopus (28) Google Scholar, 34Neri S.G.G. Cecioni I. Migliorini A. Vanni S. Galanti G. Modesti P.A. Eur. J. Clin. Invest. 1997; 27: 761-766Crossref PubMed Scopus (12) Google Scholar), although these results are not as conclusive as the hypoxia studies (35Lenz T. Nadansky M. Gossmann J. Oremek G. Geiger H. Am. J. Hypertens. 1998; 11: 1028-1031Crossref PubMed Scopus (14) Google Scholar). 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 (36Juan C.-C. Fang V.S. Huang Y.-J. Kwok C.-F. Hsu Y.-P. Ho L.-T. Biochem. Biophys. Res. Commun. 1996; 227: 694-699Crossref PubMed Scopus (55) Google Scholar) but to reduce the blood glucose level in another (37Zimmerman R.S. Maymind M. Metabolism. 1995; 44: 1321-1325Abstract Full Text PDF PubMed Scopus (12) Google Scholar, 38Zimmerman R.S. Maymind M. Metabolism. 1995; 44: 1532-1535Abstract Full Text PDF PubMed Scopus (8) Google Scholar). 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. 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 (1DeFronzo R.A. Diabetes. 1988; 37: 667-687Crossref PubMed Google Scholar). Glucose transport is the rate-limiting step in glucose metabolism. Among the six known facilitative glucose transporters (GLUT1 to -5 and GLUT7) (2Thorens B. Am. J. Physiol. 1996; 270: G541-G553PubMed Google Scholar), 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 (3Hansen P.A. Nolte L.A. Chen M.M. Holloszy J.O. J. Appl. Physiol. 1998; 85: 1218-1222Crossref PubMed Scopus (128) Google Scholar, 4Holloszy J.O. Kohrt W.M. Hansen P.A. Front. Biosci. 1998; 15: D1011-D1027Crossref Google Scholar). Also, introduction of GTP analogs, such as GTPγS,1 into 3T3-L1 adipocytes, and activation of α1-adrenergic receptors, stimulate glucose uptake independent of insulin (5Baldini G. Hohman R. Charron M.J. Lodish H.F. J. Biol. Chem. 1991; 266: 4037-4040Abstract Full Text PDF PubMed Google Scholar, 6Kishi K. Hayashi H. Wang L. Kamohara S. Tamaoka K. Shimizu T. Ushikubi F. Narumiya S. Ebina Y. J. Biol. Chem. 1996; 271: 26561-26568Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 7Elmendorf J.S. Chen D. Pressin J.E. J. Biol. Chem. 1998; 273: 13289-13296Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Endothelin (ET), originally isolated from cultured porcine aortic endothelial cells, is a peptide with 21 amino acid residues (8Yanagisawa M. Kurihara H. Kimura S. Tomobe Y. Kobayashi Y. Mitsui M. Yazaki Y. Goto K. Masaki T. Nature. 1988; 332: 411-415Crossref PubMed Scopus (10190) Google Scholar). Three distinct members of the ET family, namely, ET-1, ET-2, and ET-3, have been identified in humans through cloning (9Inoue A. Yanagisawa M. Kimura S. Kasuya Y. Miyauchi T. Goto K. Masaki T. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2863-2867Crossref PubMed Scopus (2557) Google Scholar). 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 (10Douglas S.A. Ohlstein E.H. J. Vasc. Res. 1997; 34: 152-164Crossref PubMed Scopus (108) Google Scholar). Two types of mammalian ET receptors, ETA and ETB, have been characterized and purified (11Kozuka M. Ito T. Hirose S. Lodhi K.M. Hagiwara H. J. Biol. Chem. 1991; 266: 16892-16896Abstract Full Text PDF PubMed Google Scholar, 12Wada K. Tabuchi H. Ohba R. Satoh M. Tachibana Y. Akiyama N. Hiraoka O. Asakura A. Miyamoto C. Furuichi Y. Biochem. Biophys. Res. Commun. 1990; 167: 251-257Crossref PubMed Scopus (73) Google Scholar), and their cDNA have been cloned (13Arai H. Hori S. Arimori I. Ohkubo H. Nakanishi S. Nature. 1990; 348: 730-732Crossref PubMed Scopus (2508) Google Scholar, 14Sakurai T. Yanagisawa M. Takuwa Y. Miyazaki H. Kimura S. Goto K. Masaki T. Nature. 1990; 348: 732-735Crossref PubMed Scopus (2359) Google Scholar). 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 (15Sármán B. Tóth M. Somogyi A. Diabetes Metab. Rev. 1998; 14: 171-175Crossref PubMed Scopus (43) Google Scholar). 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 (15Sármán B. Tóth M. Somogyi A. Diabetes Metab. Rev. 1998; 14: 171-175Crossref PubMed Scopus (43) Google Scholar, 16Kawamura M. Ohgawara H. Naruse M. Suzuki N. Iwasaki N. Naruse K. Hori S. Diabetes Care. 1992; 15: 1396-1397Crossref PubMed Scopus (72) Google Scholar). 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. DISCUSSIONIt 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 (24Velloso L.A. Folli F. Sun X.J. White M.F. Saad M.J. Kahn C.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12490-12495Crossref PubMed Scopus (352) Google Scholar, 25Folli F. Kahn C.R. Hansen H. Bouchie J.L. Feener E.P. J. Clin. Invest. 1997; 100: 2158-2169Crossref PubMed Scopus (404) Google Scholar). 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 (26Echel J. Gerlach-Eskuchen E. Reinauer H. Biochem. J. 1990; 272: 691-696Crossref PubMed Scopus (39) Google Scholar). Also, in both cardiomyocytes and brown adipocytes, adrenergic stimulation induces GLUT4 translocation and glucose uptake (27Fisher Y. Thomas J. Holman G.D. Rose H. Kammermeier H. Am. J. Physiol. 1996; 270: C1204-C1210Crossref PubMed Google Scholar, 28Shimizu Y. Shimazu T. Biochem. Biophys. Res. Commun. 1994; 202: 660-665Crossref PubMed Scopus (42) Google Scholar). Furthermore, the introduction of GTP analogs such as GTPγS into 3T3-L1 adipocytes stimulates GLUT4 translocation and glucose uptake independent of insulin (5Baldini G. Hohman R. Charron M.J. Lodish H.F. J. Biol. Chem. 1991; 266: 4037-4040Abstract Full Text PDF PubMed Google Scholar, 6Kishi K. Hayashi H. Wang L. Kamohara S. Tamaoka K. Shimizu T. Ushikubi F. Narumiya S. Ebina Y. J. Biol. Chem. 1996; 271: 26561-26568Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 7Elmendorf J.S. Chen D. Pressin J.E. J. Biol. Chem. 1998; 273: 13289-13296Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). 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 IRβ, 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 versusdifferentiated 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 (22Ross S.A. Scott H.M. Morris N.J. Leung W.-Y. Mao F. Lienhard G.E. Keller S.R. J. Biol. Chem. 1996; 271: 3328-3332Crossref PubMed Scopus (140) Google Scholar). Fig. 4 B 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 ETAreceptor, 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-β 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-β, 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-β 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. (6Kishi K. Hayashi H. Wang L. Kamohara S. Tamaoka K. Shimizu T. Ushikubi F. Narumiya S. Ebina Y. J. Biol. Chem. 1996; 271: 26561-26568Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) on the PAF receptor and the α1a-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 GTPγS-stimulated GLUT4 translocation (7Elmendorf J.S. Chen D. Pressin J.E. J. Biol. Chem. 1998; 273: 13289-13296Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), 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 B maxand K d 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 B max andK d 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.Wu-Wong, J. R., Polakowski, J., and Opgenorth, T. J. (1998)American Diabetes Association 58th Scientific Sessions, Chicago, Illinois, June 13–16, 1998, abstract.Google Scholar). 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 (30Rosendorff C. Cardiovasc. Drugs Ther. 1997; 10: 795-802Crossref PubMed Scopus (38) Google Scholar). It has also been reported that exercise tends to elevate ET-1 in plasma and in major organs such as heart and kidney (31Maeda S. Miyauchi T. Kobayashi T. Goto K. Matsuda M. J. Appl. Physiol. 1998; 85: 425-431Crossref PubMed Scopus (31) Google Scholar, 32Maeda S. Miyauchi T. Sakai S. Kobayashi T. Goto K. Sugishita Y. Matsuda M. J. Cardiovasc. Pharmacol. 1998; 31: S392-S394Crossref PubMed Scopus (4) Google Scholar, 33Maeda S. Miyauchi T. Goto K. Matsuda M. Life Sci. 1997; 61: 419-425Crossref PubMed Scopus (28) Google Scholar, 34Neri S.G.G. Cecioni I. Migliorini A. Vanni S. Galanti G. Modesti P.A. Eur. J. Clin. Invest. 1997; 27: 761-766Crossref PubMed Scopus (12) Google Scholar), although these results are not as conclusive as the hypoxia studies (35Lenz T. Nadansky M. Gossmann J. Oremek G. Geiger H. Am. J. Hypertens. 1998; 11: 1028-1031Crossref PubMed Scopus (14) Google Scholar). 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 (36Juan C.-C. Fang V.S. Huang Y.-J. Kwok C.-F. Hsu Y.-P. Ho L.-T. Biochem. Biophys. Res. Commun. 1996; 227: 694-699Crossref PubMed Scopus (55) Google Scholar) but to reduce the blood glucose level in another (37Zimmerman R.S. Maymind M. Metabolism. 1995; 44: 1321-1325Abstract Full Text PDF PubMed Scopus (12) Google Scholar, 38Zimmerman R.S. Maymind M. Metabolism. 1995; 44: 1532-1535Abstract Full Text PDF PubMed Scopus (8) Google Scholar). 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. 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 (24Velloso L.A. Folli F. Sun X.J. White M.F. Saad M.J. Kahn C.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12490-12495Crossref PubMed Scopus (352) Google Scholar, 25Folli F. Kahn C.R. Hansen H. Bouchie J.L. Feener E.P. J. Clin. Invest. 1997; 100: 2158-2169Crossref PubMed Scopus (404) Google Scholar). 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 (26Echel J. Gerlach-Eskuchen E. Reinauer H. Biochem. J. 1990; 272: 691-696Crossref PubMed Scopus (39) Google Scholar). Also, in both cardiomyocytes and brown adipocytes, adrenergic stimulation induces GLUT4 translocation and glucose uptake (27Fisher Y. Thomas J. Holman G.D. Rose H. Kammermeier H. Am. J. Physiol. 1996; 270: C1204-C1210Crossref PubMed Google Scholar, 28Shimizu Y. Shimazu T. Biochem. Biophys. Res. Commun. 1994; 202: 660-665Crossref PubMed Scopus (42) Google Scholar). Furthermore, the introduction of GTP analogs such as GTPγS into 3T3-L1 adipocytes stimulates GLUT4 translocation and glucose uptake independent of insulin (5Baldini G. Hohman R. Charron M.J. Lodish H.F. J. Biol. Chem. 1991; 266: 4037-4040Abstract Full Text PDF PubMed Google Scholar, 6Kishi K. Hayashi H. Wang L. Kamohara S. Tamaoka K. Shimizu T. Ushikubi F. Narumiya S. Ebina Y. J. Biol. Chem. 1996; 271: 26561-26568Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 7Elmendorf J.S. Chen D. Pressin J.E. J. Biol. Chem. 1998; 273: 13289-13296Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). 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 IRβ, 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 versusdifferentiated 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 (22Ross S.A. Scott H.M. Morris N.J. Leung W.-Y. Mao F. Lienhard G.E. Keller S.R. J. Biol. Chem. 1996; 271: 3328-3332Crossref PubMed Scopus (140) Google Scholar). Fig. 4 B 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 ETAreceptor, 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-β 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-β, 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-β 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. (6Kishi K. Hayashi H. Wang L. Kamohara S. Tamaoka K. Shimizu T. Ushikubi F. Narumiya S. Ebina Y. J. Biol. Chem. 1996; 271: 26561-26568Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) on the PAF receptor and the α1a-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 GTPγS-stimulated GLUT4 translocation (7Elmendorf J.S. Chen D. Pressin J.E. J. Biol. Chem. 1998; 273: 13289-13296Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), 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 B maxand K d 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 B max andK d 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.Wu-Wong, J. R., Polakowski, J., and Opgenorth, T. J. (1998)American Diabetes Association 58th Scientific Sessions, Chicago, Illinois, June 13–16, 1998, abstract.Google Scholar). 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 (30Rosendorff C. Cardiovasc. Drugs Ther. 1997; 10: 795-802Crossref PubMed Scopus (38) Google Scholar). It has also been reported that exercise tends to elevate ET-1 in plasma and in major organs such as heart and kidney (31Maeda S. Miyauchi T. Kobayashi T. Goto K. Matsuda M. J. Appl. Physiol. 1998; 85: 425-431Crossref PubMed Scopus (31) Google Scholar, 32Maeda S. Miyauchi T. Sakai S. Kobayashi T. Goto K. Sugishita Y. Matsuda M. J. Cardiovasc. Pharmacol. 1998; 31: S392-S394Crossref PubMed Scopus (4) Google Scholar, 33Maeda S. Miyauchi T. Goto K. Matsuda M. Life Sci. 1997; 61: 419-425Crossref PubMed Scopus (28) Google Scholar, 34Neri S.G.G. Cecioni I. Migliorini A. Vanni S. Galanti G. Modesti P.A. Eur. J. Clin. Invest. 1997; 27: 761-766Crossref PubMed Scopus (12) Google Scholar), although these results are not as conclusive as the hypoxia studies (35Lenz T. Nadansky M. Gossmann J. Oremek G. Geiger H. Am. J. Hypertens. 1998; 11: 1028-1031Crossref PubMed Scopus (14) Google Scholar). 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 (36Juan C.-C. Fang V.S. Huang Y.-J. Kwok C.-F. Hsu Y.-P. Ho L.-T. Biochem. Biophys. Res. Commun. 1996; 227: 694-699Crossref PubMed Scopus (55) Google Scholar) but to reduce the blood glucose level in another (37Zimmerman R.S. Maymind M. Metabolism. 1995; 44: 1321-1325Abstract Full Text PDF PubMed Scopus (12) Google Scholar, 38Zimmerman R.S. Maymind M. Metabolism. 1995; 44: 1532-1535Abstract Full Text PDF PubMed Scopus (8) Google Scholar). 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. 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.
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