c-Abl is required for oxidative stress-induced phosphorylation of caveolin-1 on tyrosine 14

Caveolin-1 is phosphorylated at tyrosine 14 in response to cellular stress. Tyrosine 14 is a consensus Abl phosphorylation site suggesting that caveolin-1 may be an Abl substrate. We report here that expression of c-Abl is required for oxidative stress-induced caveolin-1 phosphorylation. In contrast, c-Src expression is not required. Phosphocaveolin is one of only two phosphotyrosine signals missing in lysates from the Abl(-/-) cells, indicating that these cells still respond to oxidative stress. Oxidative stress-induced tyrosine phosphorylation of caveolin-1 occurs only at the Abl site, tyrosine 14. Caveolin-1 is also a major phosphotyrosine signal detected in cells over-expressing c-Abl. Our results show that Abl activation leads to phosphorylation of caveolin-1 on tyrosine 14. Both Abl and caveolin have been linked to the actin cytoskeleton, and oxidative stress-induced phosphocaveolin is enriched at focal contacts. This suggests that phosphocaveolin regulates these structures, perhaps through recruiting and activating SH2-domain proteins such as Csk.     

The role of caveolin-1 in PCB77-induced eNOS phosphorylation in human-derived endothelial cells

Polychlorinated biphenyls (PCBs) may contribute to the pathology of atherosclerosis by activating inflammatory responses in vascular endothelial cells. Endothelial nitric oxide synthase (eNOS) is colocalized with caveolae and is a critical regulator of vascular homeostasis. PCBs may be proatherogenic by causing dysfunctional eNOS signaling. The objective of this study was to investigate the role of caveolin-1 in PCB-induced endothelial dysfunction with a focus on mechanisms associated with eNOS signaling. Cells derived from an immortalized human vascular endothelial cell line were treated with PCB77 to study nitrotyrosine formation through eNOS signaling. Phosphorylation studies of eNOS, caveolin-1, and kinases, such as Src, phosphatidylinositol 3-kinase (PI3K), and Akt, were conducted in cells containing either functional or small-interfering RNA-silenced caveolin-1 protein. We also investigated caveolin-1-regulated mechanisms associated with PCB-induced markers of peroxynitrite formation and DNA binding of NF-kappaB. Cellular exposure to PCB77 increased eNOS phosphorylation and nitric oxide production, as well as peroxynitrite levels. A subsequent PCB-induced increase in NF-kappaB DNA binding may have implications in oxidative stress-mediated inflammatory mechanisms. The activation of eNOS by PCB77 treatment was blocked by inhibitors of the Src/PI3K/Akt pathway. PCB77 also increased phosphorylation of caveolin-1, indicating caveolae-dependent endocytosis. Caveolin-1 silencing abolished both the PCB-stimulated Akt and eNOS phosphorylation, suggesting a regulatory role of caveolae in PCB-induced eNOS signaling. These findings suggest that PCB77 induces eNOS phosphorylation in endothelial cells through a Src/PI3K/Akt-dependent mechanism, events regulated by functional caveolin-1. Our data provide evidence that caveolae may play a critical role in regulating vascular endothelial cell activation and toxicity induced by persistent environmental pollutants such as coplanar PCBs.     

Vesicle formation and trafficking in endothelial cells and regulation of endothelial barrier function

Endothelial barrier function is regulated in part by the transcellular transport of albumin and other macromolecules via endothelial caveolae (i.e., this process is defined as transcytosis). Using pulmonary microvascular endothelial cells, we have identified the specific interactions between a cell surface albumin-docking protein gp60 and caveolin-1 as well as components of the signaling machinery, heterotrimeric G protein (G(i))- and Src-family tyrosine kinase. Ligation of gp60 on the apical membrane induces the release of caveolae from the apical membrane and activation of endocytosis. The formed vesicles contain the gp60-bound albumin and also albumin and other solutes present in the fluid phase. Vesicles are transported in a polarized manner to the basolateral membrane, releasing their contents by exocytosis into the subendothelial space. The signaling functions of G(i) and Src are important in the release of caveolae from the plasma membrane. The Src-induced phosphorylation of caveolin-1 is crucial in regulating interactions of caveolin-1 with other components of the signaling machinery such as G(i), and key signaling entry of caveolae into the cytoplasm and endocytosis of albumin and other solutes. This review addresses the basis of transcytosis in endothelial cells, its central role as a determinant of endothelial barrier function, and signaling mechanisms involved in regulating fission of caveolae and trafficking of the formed vesicles from the luminal to abluminal side of the endothelial barrier.     

Role of Src-induced dynamin-2 phosphorylation in caveolae-mediated endocytosis in endothelial cells

Albumin transcytosis, a determinant of transendothelial permeability, is mediated by the release of caveolae from the plasma membrane. We addressed the role of Src phosphorylation of the GTPase dynamin-2 in the mechanism of caveolae release and albumin transport. Studies were made in microvascular endothelial cells in which the uptake of cholera toxin subunit B, a marker of caveolae, and (125)I-albumin was used to assess caveolae-mediated endocytosis. Albumin binding to the 60-kDa cell surface albumin-binding protein, gp60, induced Src activation (phosphorylation on Tyr(416)) within 1 min and resulted in Src-dependent tyrosine phosphorylation of dynamin-2, which increased its association with caveolin-1, the caveolae scaffold protein. Expression of kinase-defective Src mutant interfered with the association between dynamin-2, which caveolin-1 and prevented the uptake of albumin. Expression of non-Src-phosphorylatable dynamin (Y231F/Y597F) resulted in reduced association with caveolin-1, and in contrast to WT-dynamin-2, the mutant failed to translocate to the caveolin-rich membrane fraction. The Y231F/Y597F dynamin-2 mutant expression also resulted in impaired albumin and cholera toxin subunit B uptake and reduced transendothelial albumin transport. Thus, Src-mediated phosphorylation of dynamin-2 is an essential requirement for scission of caveolae and the resultant transendothelial transport of albumin.     

Gbetagamma activation of Src induces caveolae-mediated endocytosis in endothelial cells

Caveolae-mediated endocytosis in endothelial cells is stimulated by the binding of albumin to gp60, a specific albumin-binding protein localized in caveolae. The activation of gp60 induces its cell surface clustering and association with caveolin-1, the caveolar-scaffolding protein. This interaction leads to G(i)-induced Src kinase activation, which in turn signals dynamin-2-mediated fission and directed migration of caveolae-derived vesicles from apical to basal membrane. In this study, we investigated the possible role of the Gbetagamma heterodimer in signaling G(i)-induced Src activation and subsequent caveolae-mediated endocytosis. We observed using rat lung microvascular endothelial cells that expression of the C terminus of beta-adrenergic receptor kinase (ct-betaARK), an inhibitor Gbetagamma signaling, prevented gp60-dependent Src activation as well as caveolae-mediated endocytosis and transcellular transport of albumin and uptake of cholera toxin subunit B, a specific marker of caveolae internalization. Expression of ct-betaARK also prevented Src-mediated tyrosine phosphorylation of caveolin-1 and dynamin-2 and the resultant phosphorylation-dependent association of dynamin-2 and caveolin-1. Also, the direct activation of Gbetagamma using a specific cell-permeant activating peptide (myristoylated-SIRKALNILGYPDYD) simulated the effects of gp60 in inducing Src activation, caveolin-1, and dynamin-2 phosphorylation as well as caveolae-mediated endocytosis of cholera toxin subunit B. The myristoylated-SIRKALNILGYPDYD peptide-induced responses were inhibited by the expression of ct-betaARK. Taken together, our results demonstrate that Gbetagamma activation of Src signals caveolae-mediated endocytosis and transendothelial albumin transport via transcytosis.     

Agonist-modulated regulation of AMP-activated protein kinase (AMPK) in endothelial cells. Evidence for an AMPK -> Rac1 -> Akt -> endothelial nitric-oxide synthase pathway

The endothelial isoform of nitric-oxide synthase (eNOS), a key determinant of vascular homeostasis, is a calcium/calmodulin-dependent phosphoprotein regulated by diverse cell surface receptors. Vascular endothelial growth factor (VEGF) and sphingosine 1-phosphate (S1P) stimulate eNOS activity through Akt/phosphoinositide 3-kinase and calcium-dependent pathways. AMP-activated protein kinase (AMPK) also activates eNOS in endothelial cells; however, the molecular mechanisms linking agonist-mediated AMPK regulation with eNOS activation remain incompletely understood. We studied the role of AMPK in VEGF- and S1P-mediated eNOS activation and found that both agonists led to a striking increase in AMPK phosphorylation in pathways involving the calcium/calmodulin-dependent protein kinase kinase beta. Treatment with tyrosine kinase inhibitors or the phosphoinositide 3-kinase inhibitor wortmannin demonstrated differential effects of VEGF versus S1P. Small interfering RNA (siRNA)-mediated knockdown of AMPKalpha1or Akt1 impaired the stimulatory effects of both VEGF and S1P on eNOS activation. AMPKalpha1 knockdown impaired agonist-mediated Akt phosphorylation, whereas Akt1 knockdown did not affect AMPK activation, thus suggesting that AMPK lies upstream of Akt in the pathway leading from receptor activation to eNOS stimulation. Importantly, we found that siRNA-mediated knockdown of AMPKalpha1 abrogates agonist-mediated activation of the small GTPase Rac1. Conversely, siRNA-mediated knockdown of Rac1 decreased the agonist-mediated phosphorylation of AMPK substrates without affecting that of AMPK, implicating Rac1 as a molecular link between AMPK and Akt in agonist-mediated eNOS activation. Finally, siRNA-mediated knockdown of caveolin-1 significantly enhanced AMPK phosphorylation, suggesting that AMPK is negatively regulated by caveolin-1. Taken together, these results suggest that VEGF and S1P differentially regulate AMPK and establish a central role for an agonist-modulated AMPK --> Rac1 --> Akt axis in the control of eNOS in endothelial cells.     

Serine 23 and 36 phosphorylation of caveolin-2 is differentially regulated by targeting to lipid raft/caveolae and in mitotic endothelial cells

In the present study, using a combination of reconstituted systems and endothelial cells endogenously expressing caveolins, we show that phosphorylation of caveolin-2 at serines 23 and 36 can be differentially regulated by caveolin-1 mediated subcellular targeting to lipid raft/caveolae and in endothelial cells synchronized in mitosis. Detergent insolubility and sucrose flotation gradient experiments revealed that serine 23 phosphorylation of caveolin-2 preferably occurs in detergent-resistant membranes (DRMs), while serine 36 phosphorylation takes place in non-DRMs. Furthermore, immunofluorescence microscopy studies determined that in the presence of caveolin-1, serine 23-phosphorylated caveolin-2 mostly localizes to plasma membrane, while serine 36-phosphorylated caveolin-2 primarily resides in intracellular compartments. To directly address the role of caveolin-1 in regulating phosphorylation of endogenous caveolin-2, we have used the siRNA approach. The specific knockdown of caveolin-1 in endothelial cells decreases caveolin-2 phosphorylation at serine 23 but not at serine 36. Thus, upregulation of serine 23 phosphorylation of caveolin-2 depends on caveolin-1-driven targeting to plasma membrane lipid rafts and caveolae. Interestingly, although serine 36 phosphorylation does not seem to be regulated in endothelial cells by caveolin-1, it can be selectively upregulated in endothelial cells synchronized in mitosis. The latter data suggests a possible involvement of serine 36-phosphorylated caveolin-2 in modulating mitosis.     

Caveolae-deficient endothelial cells show defects in the uptake and transport of albumin in vivo.

The role of endothelial cell caveolae in the uptake and transport of macromolecules from the blood-space to the tissue-space remains controversial. To address this issue directly, we employed caveolin-1 gene knock-out mice that lack caveolin-1 protein expression and caveolae organelles. Here, we show that endothelial cell caveolae are required for the efficient uptake and transport of a known caveolar ligand, i.e. albumin, in vivo. Caveolin-1-null mice were perfused with 5-nm gold-conjugated albumin, and its uptake was followed by transmission electron microscopy. Our results indicate that gold-conjugated albumin is not endocytosed by Cav-1-deficient lung endothelial cells and remains in the blood vessel lumen; in contrast, gold-conjugated albumin was concentrated and internalized by lung endothelial cell caveolae in wild-type mice, as expected. To quantitate this defect in uptake, we next studied the endocytosis of radioiodinated albumin using aortic ring segments from wild-type and Cav-1-null mice. Interestingly, little or no uptake of radioiodinated albumin was observed in the aortic segments from Cav-1-deficient mice, whereas aortic segments from wild-type mice showed robust uptake that was time- and temperature-dependent and competed by unlabeled albumin. We conclude that endothelial cell caveolae are required for the efficient uptake and transport of albumin from the blood to the interstitium.     

Small interfering RNA-mediated down-regulation of caveolin-1 differentially modulates signaling pathways in endothelial cells

Caveolin-1 is a scaffolding/regulatory protein that interacts with diverse signaling molecules in endothelial cells. To explore the role of this protein in receptor-modulated signaling pathways, we transfected bovine aortic endothelial cells (BAEC) with small interfering RNA (siRNA) duplexes to down-regulate caveolin-1 expression. Transfection of BAEC with duplex siRNA targeted against caveolin-1 mRNA selectively "knocked-down" the expression of caveolin-1 by approximately 90%, as demonstrated by immunoblot analyses of BAEC lysates. We used discontinuous sucrose gradients to purify caveolin-containing lipid rafts from siRNA-treated endothelial cells. Despite the near-total down-regulation of caveolin-1 expression, the lipid raft targeting of diverse signaling proteins (including the endothelial isoform of nitric-oxide synthase, Src-family tyrosine kinases, Galphaq and the insulin receptor) was unchanged. We explored the consequences of caveolin-1 knockdown on kinase pathways modulated by the agonists sphingosine-1 phosphate (S1P) and vascular endothelial growth factor (VEGF). siRNA-mediated caveolin-1 knockdown enhanced basal as well as S1P- and VEGF-induced phosphorylation of the protein kinase Akt and did not modify the basal or agonist-induced phosphorylation of extracellular signal-regulated kinases 1/2. Caveolin-1 knock-down also significantly enhanced the basal and agonist-induced activity of the small GTPase Rac. We used siRNA to down-regulate Rac expression in BAEC, and we observed that Rac knockdown significantly reduced basal, S1P-, and VEGF-induced Akt phosphorylation, suggesting a role for Rac activation in the caveolin siRNA-mediated increase in Akt phosphorylation. By using siRNA to knockdown caveolin-1 and Rac expression in cultured endothelial cells, we have found that caveolin-1 does not seem to be required for the targeting of signaling molecules to caveolae/lipid rafts and that caveolin-1 differentially modulates specific kinase pathways in endothelial cells.     

Albumin endocytosis via megalin in astrocytes is caveola- and Dab-1 dependent and is required for the synthesis of the neurotrophic factor oleic acid

The synthesis and release of the neurotrophic factor oleic acid requires internalization of albumin into the astrocyte, which is mediated by megalin. In this study, we show that the binding and internalization of albumin involve its interaction with megalin, caveolin-1, caveolin-2 and cavin, but not with clathrin in astrocytes from primary culture. Electron microscopy analyses revealed albumin-gold complexes localized in caveolae, but not in clathrin-coated vesicles. Neither chlorpromazine nor silencing clathrin expression modified albumin uptake. Silencing caveolin-1 strongly reduced the binding and internalization of albumin and the distribution of megalin in the plasma membrane. However, silencing caveolin-2 only decreased albumin internalization, suggesting that caveolin-1 is responsible for megalin recruitment to the caveolae and that caveolin-2 participates in caveolae internalization. In most tissues, the cytosolic adaptor protein disabled (Dab)-2 connects megalin to clathrin, astrocytes lack Dab-2; instead, they express Dab-1, which interacts with caveolin-1 and megalin and is required for albumin internalization. The transcytosis of albumin in astrocytes, including the passage through the endoplasmic reticulum, which is a compulsory step for oleic acid synthesis, was confirmed by electron microscopy analyses. Thus, whereas silencing clathrin did not modify the synthesis and release of oleic acid, the knock-down of caveolin-1, caveolin-2 and Dab-1 strongly reduced the synthesis and release of this neurotrophic factor. In conclusion, caveola-mediated endocytosis of albumin requires megalin and the adaptor protein Dab-1 in cultured astrocytes. Albumin endocytosis may be a key step in brain development because it stimulates the synthesis of oleic acid, which in turn promotes neuronal differentiation.     

Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities.

Caveolin-1 is the principal structural protein of caveolae membranes in fibroblasts and endothelia. Recently, we have shown that the human CAV-1 gene is localized to a suspected tumor suppressor locus, and mutations in Cav-1 have been implicated in human cancer. Here, we created a caveolin-1 null (CAV-1 -/-) mouse model, using standard homologous recombination techniques, to assess the role of caveolin-1 in caveolae biogenesis, endocytosis, cell proliferation, and endothelial nitric-oxide synthase (eNOS) signaling. Surprisingly, Cav-1 null mice are viable. We show that these mice lack caveolin-1 protein expression and plasmalemmal caveolae. In addition, analysis of cultured fibroblasts from Cav-1 null embryos reveals the following: (i) a loss of caveolin-2 protein expression; (ii) defects in the endocytosis of a known caveolar ligand, i.e. fluorescein isothiocyanate-albumin; and (iii) a hyperproliferative phenotype. Importantly, these phenotypic changes are reversed by recombinant expression of the caveolin-1 cDNA. Furthermore, examination of the lung parenchyma (an endothelial-rich tissue) shows hypercellularity with thickened alveolar septa and an increase in the number of vascular endothelial growth factor receptor (Flk-1)-positive endothelial cells. As predicted, endothelial cells from Cav-1 null mice lack caveolae membranes. Finally, we examined eNOS signaling by measuring the physiological response of aortic rings to various stimuli. Our results indicate that eNOS activity is up-regulated in Cav-1 null animals, and this activity can be blunted by using a specific NOS inhibitor, nitro-l-arginine methyl ester. These findings are in accordance with previous in vitro studies showing that caveolin-1 is an endogenous inhibitor of eNOS. Thus, caveolin-1 expression is required to stabilize the caveolin-2 protein product, to mediate the caveolar endocytosis of specific ligands, to negatively regulate the proliferation of certain cell types, and to provide tonic inhibition of eNOS activity in endothelial cells.     

alpha2 But not alpha1 AMP-activated protein kinase mediates oxidative stress-induced inhibition of retinal pigment epithelium cell phagocytosis of photoreceptor outer segments

Oxidative stress causes retinal pigment epithelium (RPE) cell dysfunction and is a major risk factor leading to the development of dry-type age-related macular degeneration. Taking pharmacological and genetic approaches, we address the mechanisms by which sublethal oxidative stress inhibits RPE cell phagocytosis. Sublethal oxidative stress dose-dependently inhibited RPE cell phagocytosis of photoreceptor outer segments (POS) and activated AMP-activated protein kinase (AMPK) as determined by increased Thr172 and Ser79 phosphorylation of AMPKalpha and its substrate acetyl-CoA carboxylase, respectively. Similar to oxidative stress, 5-aminoimidazole-4-carboxamide riboside (AICAR), a pharmacological activator of AMPK, inhibited RPE cell phagocytosis of POS in a dose-dependent manner. Inhibition of RPE cell phagocytosis by AICAR was fully reversed by blockade of AICAR translocation into cells by dipyridamole or inhibition of AICAR conversion to ZMP by adenosine kinase inhibitor 5-iodotubercidin. In agreement, AICAR-induced activation of AMPK was abolished by preincubation with dipyridamole or 5-iodotubercidin. Knock-out experiments further revealed that alpha2 but not alpha1 AMPK was involved in RPE cell phagocytosis and that activation of alpha2 AMPK contributed to the inhibition of RPE cell phagocytosis by oxidative stress. Inhibition of RPE cell phagocytosis by activation of alpha2 AMPK was associated with a dramatic increase in acetyl-CoA carboxylase phosphorylation. In comparison, AMPK had no role in oxidative stress-induced breakdown of RPE barrier function. Taken together, reduction in POS load under oxidative stress might direct RPE cells to a self-protected status. Thus, activating AMPK could have therapeutic potential in treating dry macular degeneration.     

siRNA-induced caveolin-1 knockdown in mice increases lung vascular permeability via the junctional pathway

Caveolin-1, the principal integral membrane protein of caveolae, has been implicated in regulating the structural integrity of caveolae, vesicular trafficking, and signal transduction. Although the functions of caveolin-1 are beginning to be explored in caveolin-1-/- mice, these results are confounded by unknown compensatory mechanisms and the development of pulmonary hypertension, cardiomyopathy, and lung fibrosis. To address the role of caveolin-1 in regulating lung vascular permeability, in the present study we used small interfering RNA (siRNA) to knock down caveolin-1 expression in mouse lung endothelia in vivo. Intravenous injection of siRNA against caveolin-1 mRNA incorporated in liposomes selectively reduced the expression of caveolin-1 by approximately 90% within 96 h of injection compared with wild-type mice. We observed the concomitant disappearance of caveolae in lung vessel endothelia and dilated interendothelial junctions (IEJs) as well as increased lung vascular permeability to albumin via IEJs. The reduced caveolin-1 expression also resulted in increased plasma nitric oxide concentration. The nitric oxide synthase inhibitor L-NAME, in part, blocked the increased vascular albumin permeability. These morphological and functional effects of caveolin-1 knockdown were reversible within 168 h after siRNA injection, corresponding to the restoration of caveolin-1 expression. Thus our results demonstrate the essential requirement of caveolin-1 in mediating the formation of caveolae in endothelial cells in vivo and in negatively regulating IEJ permeability.     

Tyrosine phosphorylation of caveolin-1 in the endothelium

Caveolin-1, a scaffolding protein of caveolae, is known to be tyrosine-phosphorylated by Src kinases. Recently we generated a specific antibody to caveolin-1 phosphorylated at tyrosine-14 (PY14) (R. Nomura and T. Fujimoto, 1999, Mol. Biol. Cell 10, 975-986). In the present study, by applying PY14 to sections of normal rat tissues, we found that tyrosine phosphorylation of caveolin-1 occurred in limited locations, including the endothelium of the continuous capillaries and small venules. Cultured endothelial cells were not labeled by PY14 under a standard culture condition, but became positively labeled when exposed to oxidative stresses and/or tyrosine phosphatase inhibitors. The reaction was prohibited by pretreating the cells with herbimycin A or genistein. Vasoactive reagents or physical stimuli did not cause the phosphorylation. Concomitant with the tyrosine phosphorylation, the number of invaginated caveolae decreased drastically, and vesicles labeled intensely for caveolin-1 appeared in the cytoplasm; the average diameter of the vesicles was larger than that of caveolae. The result implies that tyrosine phosphorylation of caveolin-1 occurs at tyrosine-14 in the normal rat endothelium in vivo and may induce caveolar vesiculation and/or fusion.     

Stimulation of glucose transport in response to activation of distinct AMPK signaling pathways

AMP-activated protein kinase (AMPK) plays a critical role in the stimulation of glucose transport in response to hypoxia and inhibition of oxidative phosphorylation. In the present study, we examined the signaling pathway(s) mediating the glucose transport response following activation of AMPK. Using mouse fibroblasts of AMPK wild type and AMPK knockout, we documented that the expression of AMPK is essential for the glucose transport response to both azide and 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR). In Clone 9 cells, the stimulation of glucose transport by a combination of azide and AICAR was not additive, whereas there was an additive increase in the abundance of phosphorylated AMPK (p-AMPK). In Clone 9 cells, AMPK wild-type fibroblasts, and H9c2 heart cells, azide or hypoxia selectively increased p-ERK1/2, whereas, in contrast, AICAR selectively stimulated p-p38; phosphorylation of JNK was unaffected. Azide's effect on p-ERK1/2 abundance and glucose transport in Clone 9 cells was partially abolished by the MEK1/2 inhibitor U0126. SB 203580, an inhibitor of p38, prevented the phosphorylation of p38 and the glucose transport response to AICAR and, unexpectedly, to azide. Hypoxia, azide, and AICAR all led to increased phosphorylation of Akt substrate of 160 kDa (AS160) in Clone 9 cells. Employing small interference RNA directed against AS160 did not inhibit the glucose transport response to azide or AICAR, whereas the content of P-AS160 was reduced by approximately 80%. Finally, we found no evidence for coimmunoprecipitation of Glut1 and p-AS160. We conclude that although azide, hypoxia, and AICAR all activate AMPK, the downstream signaling pathways are distinct, with azide and hypoxia stimulating ERK1/2 and AICAR stimulating the p38 pathway.     

AMPK activation inhibits the expression of HIF-1alpha induced by insulin and IGF-1

Insulin, insulin like growth factor (IGF)-1, and AMP-activated protein kinase (AMPK) signaling regulate independently angiogenesis through vascular endothelial growth factor (VEGF) expression. In the present study, we investigated a potential cross-talk between these signaling pathways on hypoxia-inducible factor (HIF)-1alpha and VEGF expression. Retinal epithelial ARPE-19 cells were treated with AICAR, an AMPK activator, alone or in combination with insulin and IGF-1. AICAR stimulated VEGF mRNA expression, but did not modify the insulin- and IGF-1-induced VEGF expression. We have investigated the effect of AICAR on insulin and IGF-1 signaling pathways. We observed that AICAR increased insulin- and IGF-1-induced phosphorylation of PKB, whereas phosphorylation of S6K-1 was decreased. Moreover, AICAR and metformin inhibited the ability of insulin and IGF-1 to induce HIF-1alpha expression. These results show that AICAR and insulin/IGF-1 regulate VEGF expression through different mechanisms.     

Rapid Insulin-Dependent Endocytosis of the Insulin Receptor by Caveolae in Primary Adipocytes

Background

The insulin receptor is localized in caveolae and is dependent on caveolae or cholesterol for signaling in adipocytes. When stimulated with insulin, the receptor is internalized.

Methodology/Principal Findings

We examined primary rat adipocytes by subcellular fractionation to examine if the insulin receptor was internalized in a caveolae-mediated process. Insulin induced a rapid, t_1/2<3 min, endocytosis of the insulin receptor in parallel with receptor tyrosine autophosphorylation. Concomitantly, caveolin-1 was phosphorylated at tyrosine(14) and endocytosed. Vanadate increased the phosphorylation of caveolin-1 without affecting insulin receptor phosphorylation or endocytosis. Immunocapture of endosomal vesicles with antibodies against the insulin receptor co-captured caveolin-1 and immunocapture with antibodies against tyrosine(14)-phosphorylated caveolin-1 co-captured the insulin receptor, demonstrating that the insulin receptor was endocytosed together with tyrosine(14)-phosphorylated caveolin-1. By immunogold electron microscopy the insulin receptor and caveolin-1 were colocalized in endosome vesicles that resembled caveosomes. Clathrin was not endocytosed with the insulin receptor and the inhibitor of clathrin-coated pit-mediated endocytosis, chlorpromazine, did not inhibit internalization of the insulin receptor, while transferrin receptor internalization was inhibited.

Conclusion

It is concluded that in response to insulin stimulation the autophosphorylated insulin receptor in primary adipocytes is rapidly endocytosed in a caveolae-mediated process, involving tyrosine phosphorylation of caveolin-1.     

Regulation of Visceral and Subcutaneous Adipocyte Lipolysis by Acute AICAR‐induced AMPK Activation

This study investigated the role of adenosine monophosphate–activated protein kinase (AMPK) in the regulation of lipolysis in visceral (VC) and subcutaneous (SC) rat adipocytes and the molecular mechanisms involved in this process. VC (epididymal and retroperitoneal) and SC (inguinal) adipocytes were isolated from male Wistar rats (160–180 g). Adipocytes were incubated either in the absence or in the presence of the AMPK agonist 5‐aminoimidazole‐4‐carboxamide‐1‐β‐d‐ribofuranoside (AICAR, 0–500 µmol/l). AMPK and acetyl‐CoA carboxylase (ACC) phosphorylation, basal and epinephrine‐stimulated (100 nmol/l) glycerol release, and hormone‐sensitive lipase (HSL) phosphorylation and activity were determined. AICAR‐induced (500 µmol/l) AMPK activation inhibited basal glycerol release by ～42, 41, and 44% in epididymal, retroperitoneal, and inguinal adipocytes, respectively. Epinephrine‐stimulated glycerol release was almost completely prevented by AICAR treatment in adipocytes from all fat depots. The AMPK inhibitor compound C (20 µmol/l) prevented AICAR‐induced phosphorylation of AMPK and significantly increased basal (～1.3‐, 1.4‐, and 1.7‐fold) and epinephrine‐stimulated (～1.3‐, 1.2‐, 1.4‐fold) glycerol release in epididymal, retroperitoneal, and inguinal adipocytes, respectively. AICAR increased phosphorylation of HSL_Ser565 and inhibited epinephrine‐induced phosphorylation of HSL_Ser563 and HSL_Ser660. This was also accompanied by a 73% reduction in epinephrine‐stimulated HSL activity. Compound C prevented the phosphorylation of HSL_Ser565 induced by AICAR and partially prevented the inhibitory effect of this drug on basal and epinephrine‐stimulated lipolysis in adipocytes in VC and SC fat depots. In summary, despite different fat depots eliciting distinct rates of lipolysis, acute AICAR‐induced AMPK activation suppressed HSL phosphorylation/activation and exerted similar antilipolytic effects on both VC and SC adipocytes.     

In vitro antiglioma action of indomethacin is mediated via AMP-activated protein kinase/mTOR complex 1 signalling pathway

We investigated the role of the intracellular energy-sensing AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway in the in vitro antiglioma effect of the cyclooxygenase (COX) inhibitor indomethacin. Indomethacin was more potent than COX inhibitors diclofenac, naproxen, and ketoprofen in reducing the viability of U251 human glioma cells. Antiglioma effect of the drug was associated with p21 increase and G₂M cell cycle arrest, as well as with oxidative stress, mitochondrial depolarization, caspase activation, and the induction of apoptosis. Indomethacin increased the phosphorylation of AMPK and its targets Raptor and acetyl-CoA carboxylase (ACC), and reduced the phosphorylation of mTOR and mTOR complex 1 (mTORC1) substrates p70S6 kinase and PRAS40 (Ser183). AMPK knockdown by RNA interference, as well as the treatment with the mTORC1 activator leucine, prevented indomethacin-mediated mTORC1 inhibition and cytotoxic action, while AMPK activators metformin and AICAR mimicked the effects of the drug. AMPK activation by indomethacin correlated with intracellular ATP depletion and increase in AMP/ATP ratio, and was apparently independent of COX inhibition or the increase in intracellular calcium. Finally, the toxicity of indomethacin towards primary human glioma cells was associated with the activation of AMPK/Raptor/ACC and subsequent suppression of mTORC1/S6K. By demonstrating the involvement of AMPK/mTORC1 pathway in the antiglioma action of indomethacin, our results support its further exploration in glioma therapy.