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We showed previously that chronic exposure to both endothelin-1 (ET-1) and cAMP resulted in a synergistic increase in Glut1 transcription in 3T3-L1 adipocytes via a protein kinase C (PKC)-dependent mechanism. In the present study, we further examined the molecular mechanism involved. Employing transient transfections with Glut1 promoter/enhancer -luciferase reporter and several dominant negative or constitutively active PKC mutants, we identified PKCε as the responsible PKC. Investigation with deletion and mutation mutants of the promoter/enhancer reporter suggested that Sp1, CREB and AP-1 responsive elements on enhancer 2 were involved. Furthermore, chromatin immunoprecipitation and co-immunoprecipitation analysis were applied to characterize the interactions between these transcription factors and their bindings to enhancer 2 in vivo. The results indicate that there are both negative and positive interactions between ET-1 and cAMP signaling pathways. On the one hand, cAMP inhibits ET-1 induced NF-κB activation required for ET-1-stimulated Glut1 transcription; on the other hand, cAMP, via sustained CREB phosphorylation, may activate AP-1 and cooperate with ET-1-activated PKCε to enhance Sp1 expression and consequently to generate a stable enhancer 2-bound Sp1/pCREB/AP-1 complex, which can strongly facilitate Glut1 transcription more than the additive effect of ET-1 and cAMP alone., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We have shown previously that endothelin-1 (ET-1) induction of Glut1 transcription is mediated by ET-1 responsive elements on enhancer 2, via both protein kinase Cepsilon (PKCepsilon)- and p42/p44 mitogen-activated protein kinase (MAPK)-dependent pathways. In the present study, we further explored the molecular mechanism involved. By using mutation constructs of luciferase reporter driven by Glut1 promoter/enhancers, chromatin immunoprecipitation and co-immunoprecipitation experiments, we were able to demonstrate that cooperative interaction between NF-kappaB and Sp1 were required to enhance Glut1 transcription in response to ET-1. While ET-1 may induce Sp1 expression via both PKC-and MAPK-dependent pathways, activation of NF-kappaB by ET-1 is mediated by a PKCepsilon/reactive oxygen species (ROS) cascade. Taken together, these results suggest that by activating NF-kappaB via PKCepsilon/ROS cascade and increasing Sp1 expression through both PKCepsilon- and MAPK-dependent pathways, ET-1 may activate Glut1 transcription by enhancing interaction between nuclear NF-kappaB and Sp1 as well as their binding to enhancer 2.

We have shown previously that chronic exposure to endothelin-1 (ET-1) may stimulate GLUT1-mediated glucose transport in 3T3-L1 adipocytes via both protein kinase C (PKC)- and mitogen-activated protein kinase (p42/p44 MAPK)-dependent pathways. In the present study, by using a luciferase reporter driven by Glut1 promoter and enhancers (pLuc-GT1/E1/E2) and various constitutively active and dominant negative mutants of PKC isoforms, we identified PKCepsilon as the PKC isoform involved. In addition, we provide evidence that there is no direct interaction between ET-1 activated PKCepsilon and MAPK, at least at the kinase activity level. Furthermore, investigations employing deletion mutants of pLuc-GT1/E1/E2 to locate the putative ET-1 responsive sites and inhibitory agents to suppress the activities of putative transcription factors suggested that transcription factors CREB, Sp1 and NF-kappaB were involved. In summary, the results of this study indicate that ET-1 induction of Glut1 transcription involves distinct PKCepsilon- and MAPK-dependent pathways, as well as downstream transcription factors CREB, Sp1 and NF-kappaB.

We have demonstrated previously that chronic exposure to endothlin-1 enhances glucose transport in 3T3-L1 adipocytes via augmented GLUT1 mRNA and protein accumulation. In the present study, we further examined the combined effect of endothelin-1 (ET-1) and cAMP on glucose transport. In cells pretreated with ET-1 and 8-bromo cAMP for 8 h, a synergy between these two agents on glucose uptake was found. Insulin-stimulated glucose transport, on the other hand, was only slightly affected. The synergistic effect of these two agents was suppressed in the presence of cycloheximide and actinomycin D. Immunoblot and Northern blot analyses revealed that GLUT1 protein and mRNA levels were both increased in cells pretreated with both ET-1 and 8-bromo cAMP, greater than the additive effect of each agent alone. Further examination demonstrated that the stability of GLUT1 mRNA was markedly enhanced in the presence of both ET-1 and cAMP. To investigate the transcriptional activation of Glut1 gene, transient transfection of cells with luciferase reporter construct driven by Glut1 promoter was performed. We found that Glut1 transcription was also increased by ET-1 and cAMP in a synergistic fashion. In addition, similar synergy between ET-1 and beta-adrenergic agonists on glucose transport was found. The synergistic action of ET-1 with 8-bromo cAMP to enhance glucose transport was inhibited by GF109203X, a selective protein kinase C (PKC) inhibitor, and was mimicked by 4beta-phorbol 12beta-myristate 13alpha-acetate (PMA), a PKC activator. Furthermore, PMA was found to act synergistically with 8-bromo cAMP to induce Glut1 transcription and ET-1 was shown to activate novel PKCdelta and PKC. Taken together, these results indicate that ET-1 may act with cAMP in a synergistic way to increase glucose transport, probably through enhanced GLUT1 expression via a PKC-dependent mechanism.

We used nigericin, a K+/H+ exchanger, to test whether glucose transport in 3T3-L1 adipocytes was modulated by changes in intracellular pH. Our results showed that nigericin increased basal but decreased insulin-stimulated glucose uptake in a time- and dose-dependent manner. Whereas the basal translocation of GLUT1 was enhanced, insulin-stimulated GLUT4 translocation was inhibited by nigericin. On the other hand, the total amount of neither transporter protein was altered. The finding that insulin-stimulated phosphoinositide 3-kinase (PI 3-kinase) activity was not affected by nigericin implies that nigericin exerted its inhibition at a step downstream of PI 3-kinase activation. At maximal dose, nigericin rapidly lowered cytosolic pH to 6.7; however, this effect was transient and cytosolic pH was back to normal in 20 min. Removal of nigericin from the incubation medium after 20 min abolished its enhancing effect on basal but had little influence on its inhibition of insulin-stimulated glucose transport. Moreover, lowering cytosolic pH to 6.7 with an exogenously added HCl solution had no effect on glucose transport. Taken together, it appears that nigericin may inhibit insulin-stimulated glucose transport mainly by interfering with GLUT4 translocation, probably by a mechanism not related to changes in cytosolic pH.

The mechanism of enhancing glucose transport by prolonged endothelin-1 (ET-1) treatment of 3T3-L1 adipocytes was examined. Western and Northern blot analyses indicated that ET-1 increased the amount of both GLUT1 protein and mRNA. The degradation rate of GLUT1 mRNA as measured in the presence of actinomycin D, nevertheless, was not significantly altered by ET-1. Whereas various inhibitors for distinct signalling pathways were tested, only the mitogen-activated protein kinase (MAPK) kinase inhibitor, PD98059, was found to decrease significantly the enhancing effect of ET-1. Similar extent of inhibition was observed in cells pretreated with pertussis toxin (PT). Immunoblot analysis revealed that ET-1 may stimulate a transient phosphorylation of p42/p44 MAPK and both PT and PD98059 inhibited this stimulation. In addition, the effect of ET-1 on GLUT1 mRNA accumulation was inhibited by PD98059 and cycloheximide, implying that a trans-activation was involved. Taken together, these results suggest that ET-1 may induce GLUT1 gene expression by a MAPK-dependent mechanism.