Rosiglitazone stimulates nitric oxide synthesis in human aortic endothelial cells via AMP-activated protein kinase

The thiazolidinedione anti-diabetic drugs increase activation of endothelial nitric-oxide (NO) synthase by phosphorylation at Ser-1177 and increase NO bioavailability, yet the molecular mechanisms that underlie this remain poorly characterized. Several protein kinases, including AMP-activated protein kinase, have been demonstrated to phosphorylate endothelial NO synthase at Ser-1177. In the current study we determined the role of AMP-activated protein kinase in rosiglitazone-stimulated NO synthesis. Stimulation of human aortic endothelial cells with rosiglitazone resulted in the time- and dose-dependent stimulation of AMP-activated protein kinase activity and NO production with concomitant phosphorylation of endothelial NO synthase at Ser-1177. Rosiglitazone stimulated an increase in the ADP/ATP ratio in endothelial cells, and LKB1 was essential for rosiglitazone-stimulated AMPK activity in HeLa cells. Infection of endothelial cells with a virus encoding a dominant negative AMP-activated protein kinase mutant abrogated rosiglitazone-stimulated Ser-1177 phosphorylation and NO production. Furthermore, the stimulation of AMP-activated protein kinase and NO synthesis by rosiglitazone was unaffected by the peroxisome proliferator-activated receptor-gamma inhibitor GW9662. These studies demonstrate that rosiglitazone is able to acutely stimulate NO synthesis in cultured endothelial cells by an AMP-activated protein kinase-dependent mechanism, likely to be mediated by LKB1.     

Flow shear stress stimulates Gab1 tyrosine phosphorylation to mediate protein kinase B and endothelial nitric-oxide synthase activation in endothelial cells

Fluid shear stress generated by blood flow modulates endothelial cell function via specific intracellular signaling events. We showed previously that flow activated the phosphatidylinositol 3-kinase (PI3K), Akt, and endothelial nitric-oxide synthase (eNOS) via Src kinase-dependent transactivation of vascular endothelial growth factor receptor 2 (VEGFR2). The scaffold protein Gab1 plays an important role in receptor tyrosine kinase-mediated signal transduction. We found here that laminar flow (shear stress = 12 dynes/cm2) rapidly stimulated Gab1 tyrosine phosphorylation in both bovine aortic endothelial cells and human umbilical vein endothelial cells, which correlated with activation of Akt and eNOS. Gab1 phosphorylation as well as activation of Akt and eNOS by flow was inhibited by the Src kinase inhibitor PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) and VEGFR2 kinase inhibitors SU1498 and VTI, suggesting that flow-mediated Gab1 phosphorylation is Src kinase-dependent and VEGFR2-dependent. Tyrosine phosphorylation of Gab1 by flow was functionally important, because flow stimulated the association of Gab1 with the PI3K subunit p85 in a time-dependent manner. Furthermore, transfection of a Gab1 mutant lacking p85 binding sites inhibited flow-induced activation of Akt and eNOS. Finally, knockdown of endogenous Gab1 by small interference RNA abrogated flow activation of Akt and eNOS. These data demonstrate a critical role of Gab1 in flow-stimulated PI3K/Akt/eNOS signal pathway in endothelial cells.     

Isodihydrocapsiate stimulates plasma glucose uptake by activation of AMP-activated protein kinase

AMP-activated protein kinase (AMPK) is an energy-sensing enzyme that is implicated as a key factor in controlling whole body homeostasis, including fatty acid oxidation and glucose uptake. We report that a synthetic structural isomer of dihydrocapsiate, isodihydrocapsiate (8-methylnonanoic acid 3-hydroxy-4-methoxy benzyl ester) improves type 2 diabetes by activating AMPK through the LKB1 pathway. In L6 myotube cells, phosphorylation of AMPK and acetyl-CoA carboxylase (ACC) and glucose uptake were significantly increased, whereas these effects were attenuated by an AMPK inhibitor, compound C. In addition, increased phosphorylation of AMPK and ACC by isodihydrocapsiate was significantly reduced by radicicol, an LKB1 destabilizer, suggesting that increased glucose uptake in L6 cells with isodihydrocapsiate treatment is predominantly accomplished by a LKB1-mediated AMPK activation pathway. Oral administration of isodihydrocapsiate to diabetic (db/db) mice reduced blood glucose levels by 40% after a 4-week treatment period. Our results support the development of isodihydrocapsiate as a potential therapeutic agent to target AMPK in type 2 diabetes.     

Adiponectin stimulates angiogenesis by promoting cross-talk between AMP-activated protein kinase and Akt signaling in endothelial cells

Adiponectin is an adipocyte-specific adipocytokine with anti-atherogenic and anti-diabetic properties. Here, we investigated whether adiponectin regulates angiogenic processes in vitro and in vivo. Adiponectin stimulated the differentiation of human umbilical vein endothelium cells (HUVECs) into capillary-like structures in vitro and functioned as a chemoattractant in migration assays. Adiponectin promoted the phosphorylation of AMP-activated protein kinase (AMPK), protein kinase Akt/protein kinase B, and endothelial nitric oxide synthesis (eNOS) in HUVECs. Transduction with either dominant-negative AMPK or dominant-negative Akt abolished adiponectin-induced eNOS phosphorylation as well as adiponectin-stimulated HUVEC migration and differentiation. Dominant-negative AMPK also inhibited adiponectin-induced Akt phosphorylation, suggesting that AMPK is upstream of Akt. Dominant-negative Akt or the phosphatidylinositol 3-kinase inhibitor LY294002 blocked adiponectin-stimulated Akt and eNOS phosphorylation, migration, and differentiation without altering AMPK phosphorylation. Finally, adiponectin stimulated blood vessel growth in vivo in mouse Matrigel plug implantation and rabbit corneal models of angiogenesis. These data indicate that adiponectin can function to stimulate the new blood vessel growth by promoting cross-talk between AMP-activated protein kinase and Akt signaling within endothelial cells.     

Sustained activation of AMP-activated protein kinase induces c-Jun N-terminal kinase activation and apoptosis in liver cells

We report the first Fourier transform infrared analysis of prion protein (PrP) repeats and the first study of PrP repeats of marsupial origin. Large changes in the secondary structure and an increase in hydrogen bonding within the peptide groups were evident from a red shift of the amide I band by >7 cm(-1) and an approximately five-fold reduction in amide hydrogen-deuterium exchange for peptide interacting with Cu(2+) ions. Changes in the tertiary structure upon copper binding were also evident from the appearance of a new band at 1564 cm(-1), which arises from the ring vibration of histidine. The copper-induced conformational change is pH dependent, and occurs at pH >7.     

AMP-activated protein kinase phosphorylation of endothelial NO synthase

The AMP-activated protein kinase (AMPK) in rat skeletal and cardiac muscle is activated by vigorous exercise and ischaemic stress. Under these conditions AMPK phosphorylates and inhibits acetyl-coenzyme A carboxylase causing increased oxidation of fatty acids. Here we show that AMPK co-immunoprecipitates with cardiac endothelial NO synthase (eNOS) and phosphorylates Ser-1177 in the presence of Ca2+-calmodulin (CaM) to activate eNOS both in vitro and during ischaemia in rat hearts. In the absence of Ca2+-calmodulin, AMPK also phosphorylates eNOS at Thr-495 in the CaM-binding sequence, resulting in inhibition of eNOS activity but Thr-495 phosphorylation is unchanged during ischaemia. Phosphorylation of eNOS by the AMPK in endothelial cells and myocytes provides a further regulatory link between metabolic stress and cardiovascular function.     

2-Deoxy-D-glucose treatment of endothelial cells induces autophagy by reactive oxygen species-mediated activation of the AMP-activated protein kinase

Autophagy is a cellular self-digestion process activated in response to stresses such as energy deprivation and oxidative stress. However, the mechanisms by which energy deprivation and oxidative stress trigger autophagy remain undefined. Here, we report that activation of AMP-activated protein kinase (AMPK) by mitochondria-derived reactive oxygen species (ROS) is required for autophagy in cultured endothelial cells. AMPK activity, ROS levels, and the markers of autophagy were monitored in confluent bovine aortic endothelial cells (BAEC) treated with the glycolysis blocker 2-deoxy-D-glucose (2-DG). Treatment of BAEC with 2-DG (5 mM) for 24 hours or with low concentrations of H(2)O(2) (100 μM) induced autophagy, including increased conversion of microtubule-associated protein light chain 3 (LC3)-I to LC3-II, accumulation of GFP-tagged LC3 positive intracellular vacuoles, and increased fusion of autophagosomes with lysosomes. 2-DG-treatment also induced AMPK phosphorylation, which was blocked by either co-administration of two potent anti-oxidants (Tempol and N-Acetyl-L-cysteine) or overexpression of superoxide dismutase 1 or catalase in BAEC. Further, 2-DG-induced autophagy in BAEC was blocked by overexpressing catalase or siRNA-mediated knockdown of AMPK. Finally, pretreatment of BAEC with 2-DG increased endothelial cell viability after exposure to hypoxic stress. Thus, AMPK is required for ROS-triggered autophagy in endothelial cells, which increases endothelial cell survival in response to cell stress.     

Diphenylene iodonium stimulates glucose uptake in skeletal muscle cells through mitochondrial complex I inhibition and activation of AMP-activated protein kinase

NADPH oxidase inhibitors such as diphenylene iodonium (DPI) and apocynin lower whole body and blood glucose levels and improve diabetes when administered to rodents. Skeletal muscle has an important role in managing glucose homeostasis and we have used L6 cells, C(2)C(12) cells and primary muscle cells as model systems to investigate whether these drugs regulate glucose uptake in skeletal muscle cells. The data presented in this study show that apocynin does not affect glucose uptake in skeletal muscle cells in culture. Tat gp91ds, a chimeric peptide that inhibits NADPH oxidase activity, also failed to affect glucose uptake and we found no significant evidence of NADPH oxidase (subunits tested were Nox4, p22phox, gp91phox and p47phox mRNA) in skeletal muscle cells in culture. However, DPI increases basal and insulin-stimulated glucose uptake in L6 cells, C(2)C(12) cells and primary muscle cells. Detailed studies on L6 cells demonstrate that the increase of glucose uptake is via a mechanism independent of phosphoinositide-3 kinase (PI3K)/Akt but dependent on AMP-activated protein kinase (AMPK). We postulate that DPI through inhibition of mitochondrial complex 1 and decreases in oxygen consumption, leading to decreases of ATP and activation of AMPK, stimulates glucose uptake in skeletal muscle cells.     

AMP-activated protein kinase mediates VEGF-stimulated endothelial NO production

Vascular endothelial growth factor (VEGF) is an important regulator of endothelial cell function. VEGF stimulates NO production, proposed to be a result of phosphorylation and activation of endothelial NO synthase (eNOS) at Ser1177. Phosphorylation of eNOS at this site also occurs after activation of AMP-activated protein kinase (AMPK) in cultured endothelial cells. We therefore determined whether AMPK mediates VEGF-stimulated NO synthesis in endothelial cells. VEGF caused a rapid, dose-dependent stimulation of AMPK activity, with a concomitant increase in phosphorylation of eNOS at Ser1177. Infection of endothelial cells with an adenovirus expressing a dominant negative mutant AMPK partially inhibited both VEGF-stimulated eNOS Ser1177 phosphorylation and NO production. VEGF-stimulated AMPK activity was completely inhibited by the Ca(2+)/calmodulin-dependent protein kinase kinase inhibitor, STO-609. Stimulation of AMPK via Ca(2+)/calmodulin-dependent protein kinase kinase represents a novel signalling mechanism utilised by VEGF in endothelial cells that contributes to eNOS phosphorylation and NO production.     

Thrombin activates AMP-activated protein kinase in endothelial cells via a pathway involving Ca2+/calmodulin-dependent protein kinase kinase beta

AMP-activated protein kinase (AMPK) is a sensor of cellular energy state in response to metabolic stress and other regulatory signals. AMPK is controlled by upstream kinases which have recently been identified as LKB1 or Ca2+/calmodulin-dependent protein kinase kinase beta (CaMKKbeta). Our study of human endothelial cells shows that AMPK is activated by thrombin through a Ca2+-dependent mechanism involving the thrombin receptor protease-activated receptor 1 and Gq-protein-mediated phospholipase C activation. Inhibition of CaMKK with STO-609 or downregulation of CaMKKbeta using RNA interference decreased thrombin-induced AMPK activation significantly, indicating that CaMKKbeta was the responsible AMPK kinase. In contrast, downregulation of LKB1 did not affect thrombin-induced AMPK activation but abolished phosphorylation of AMPK with 5-aminoimidazole-4-carboxamide ribonucleoside. Thrombin stimulation led to phosphorylation of acetyl coenzyme A carboxylase (ACC) and endothelial nitric oxide synthase (eNOS), two downstream targets of AMPK. Inhibition or downregulation of CaMKKbeta or AMPK abolished phosphorylation of ACC in response to thrombin but had no effect on eNOS phosphorylation, indicating that thrombin-stimulated phosphorylation of eNOS is not mediated by AMPK. Our results underline the role of Ca2+ as a regulator of AMPK activation in response to a physiologic stimulation. We also demonstrate that endothelial cells possess two pathways to activate AMPK, one Ca2+/CaMKKbeta dependent and one AMP/LKB1 dependent.     

Stress stimulates AMP-activated protein kinase and meiotic resumption in mouse oocytes

This study examined the effects of three different cellular stresses on oocyte maturation in meiotically arrested mouse oocytes. Cumulus-cell enclosed oocytes (CEO) or denuded oocytes (DO) from immature, eCG-primed mice were cultured for 17-18 h in dbcAMP-containing medium plus increasing concentrations of the metabolic poison, sodium arsenite, or the free radical-generating agent, menadione. Alternatively, oocytes were exposed to osmotic stress by pulsing with sorbitol and returned to control inhibitory conditions for the duration of culture. Arsenite and menadione each dose-dependently induced germinal vesicle breakdown (GVB) in both DO and CEO. DO, but not CEO, pulsed for 60 min with 500 mM sorbitol were stimulated to resume maturation. The lack of effect in CEO suggests that the cumulus cells may be playing a protective role in osmotic stress-induced GVB. The AMP-activated protein kinase (PRKA; formerly known as AMPK) inhibitors, compound C and araA, completely blocked the meiosis-stimulating effects of all the tested stresses. Western blots showed that acetyl-CoA carboxylase, an important substrate of PRKA, was phosphorylated before GVB, supporting a role for PRKA in stress-induced maturation. Together, these data show that a variety of stresses stimulate GVB in meiotically arrested mouse oocytes in vitro and suggest that this effect is mediated through activation of PRKA.     

Ca2+ entry via TRPC channels is necessary for thrombin-induced NF-kappaB activation in endothelial cells through AMP-activated protein kinase and protein kinase Cdelta

The transient receptor potential canonical (TRPC) family channels are proposed to be essential for store-operated Ca2+ entry in endothelial cells. Ca2+ signaling is involved in NF-kappaB activation, but the role of store-operated Ca2+ entry is unclear. Here we show that thrombin-induced Ca2+ entry and the resultant AMP-activated protein kinase (AMPK) activation targets the Ca2+-independent protein kinase Cdelta (PKCdelta) to mediate NF-kappaB activation in endothelial cells. We observed that thrombin-induced p65/RelA, AMPK, and PKCdelta activation were markedly reduced by knockdown of the TRPC isoform TRPC1 expressed in human endothelial cells and in endothelial cells obtained from Trpc4 knock-out mice. Inhibition of Ca2+/calmodulin-dependent protein kinase kinase beta downstream of the Ca2+ influx or knockdown of the downstream Ca2+/calmodulin-dependent protein kinase kinase beta target kinase, AMPK, also prevented NF-kappaB activation. Further, we observed that AMPK interacted with PKCdelta and phosphorylated Thr505 in the activation loop of PKCdelta in thrombin-stimulated endothelial cells. Expression of a PKCdelta-T505A mutant suppressed the thrombin-induced but not the TNF-alpha-induced NF-kappaB activation. These findings demonstrate a novel mechanism for TRPC channels to mediate NF-kappaB activation in endothelial cells that involves the convergence of the TRPC-regulated signaling at AMPK and PKCdelta and that may be a target of interference of the inappropriate activation of NF-kappaB associated with thrombosis.     

Inorganic lead stimulates DNA synthesis in human astrocytoma cells: role of protein kinase Calpha

As lead has been shown to activate protein kinase C (PKC), and gliomas are reported to be highly dependent on PKC for their proliferation, this study was undertaken to investigate whether lead may act as a mitogen in human astrocytoma cells, and to determine the role of PKC in this effect. Lead acetate (from 100 nM to 100 microM) induced a concentration- and time-dependent increase in DNA synthesis, as measured by incorporation of [methyl-3H]thymidine into cell DNA, without causing any cytotoxicity. Flow cytometric analysis showed that lead was able to stimulate the cell cycle transition from the G0/G1 phase to the S/G2 phase, resulting in increased percentage of cells in the latter phase. Western blot analyses showed that lead induced translocation of PKCalpha, but not of PKCepsilon or PKCzeta, from the cytosolic to the particulate fraction, with a concomitant increase in PKC enzyme activity. Prolonged exposure to lead caused down-regulation of PKCalpha, but not of PKCepsilon. The effect of lead on DNA synthesis was mediated through PKC as evidenced by the finding that two PKC inhibitors, GF 109203X and staurosporine, as well as down-regulation of PKC through prolonged treatment with 12-O-tetradecanoylphorbol 13-acetate, blocked lead-induced DNA synthesis. Further experiments using a pseudosubstrate peptide targeting classical PKCs and selective down-regulation of specific PKC isoforms indicated that the effect of lead on DNA synthesis was mediated by PKCalpha. Altogether, these results suggest that lead stimulates DNA synthesis in human astrocytoma cells by a mechanism that involves activation of PKCalpha.     

Insulin rapidly stimulates L-arginine transport in human aortic endothelial cells via Akt

Insulin stimulates endothelial NO synthesis, at least in part mediated by phosphorylation and activation of endothelial NO synthase at Ser1177 and Ser615 by Akt. We have previously demonstrated that insulin-stimulated NO synthesis is inhibited under high culture glucose conditions, without altering Ca(2+)-stimulated NO synthesis or insulin-stimulated phosphorylation of eNOS. This indicates that stimulation of endothelial NO synthase phosphorylation may be required, yet not sufficient, for insulin-stimulated nitric oxide synthesis. In the current study we investigated the role of supply of the eNOS substrate, L-arginine as a candidate parallel mechanism underlying insulin-stimulated NO synthesis in cultured human aortic endothelial cells. Insulin rapidly stimulated L-arginine transport, an effect abrogated by incubation with inhibitors of phosphatidylinositol-3'-kinase or infection with adenoviruses expressing a dominant negative mutant Akt. Furthermore, supplementation of endothelial cells with extracellular L-arginine enhanced insulin-stimulated NO synthesis, an effect reversed by co-incubation with the L-arginine transport inhibitor, L-lysine. Basal L-arginine transport was significantly increased under high glucose culture conditions, yet insulin-stimulated L-arginine transport remained unaltered. The increase in L-arginine transport elicited by high glucose was independent of the expression of the cationic amino acid transporters, hCAT1 and hCAT2 and not associated with any changes in the activity of ERK1/2, Akt or protein kinase C (PKC). We propose that rapid stimulation of L-arginine transport contributes to insulin-stimulated NO synthesis in human endothelial cells, yet attenuation of this is unlikely to underlie the inhibition of insulin-stimulated NO synthesis under high glucose conditions.     

Activation of protein kinase C zeta by peroxynitrite regulates LKB1-dependent AMP-activated protein kinase in cultured endothelial cells

We previously reported the phosphoinositide 3-kinase-dependent activation of the 5'-AMP-activated kinase (AMPK) by peroxynitrite (ONOO-) and hypoxia-reoxygenation in cultured endothelial cells. Here we show the molecular mechanism of activation of this pathway. Exposure of bovine aortic endothelial cells to ONOO- significantly increased the phosphorylation of both Thr172 of AMPK and Ser1179 of endothelial nitric-oxide synthase, a known downstream enzyme of AMPK. In addition, activation of AMPK by ONOO- was accompanied by increased phosphorylation of protein kinase Czeta (PKCzeta) (Thr410/403) and translocation of cytosolic PKCzeta into the membrane. Further, inhibition of PKCzeta abrogated ONOO- -induced AMPK-Thr172 phosphorylation as that of endothelial nitric-oxide synthase. Furthermore, overexpression of a constitutively active PKCzeta mutant enhanced the phosphorylation of AMPK-Thr172, suggesting that PKCzeta is upstream of AMPK activation. In contrast, ONOO- activated PKCzeta in LKB1-deficient HeLa-S3 but affected neither AMPK-Thr172 nor AMPK activity. These data suggest that LKB1 is required for PKCzeta-enhanced AMPK activation. In vitro, recombinant PKCzeta phosphorylated LKB1 at Ser428, resulting in phosphorylation of AMPK at Thr172. Further, direct mutation of Ser428 of LKB1 into alanine, like the kinase-inactive LKB1 mutant, abolished ONOO- -induced AMPK activation. In several cell types originating from human, rat, and mouse, inhibition of PKCzeta significantly attenuated the phosphorylation of both LKB1-Ser428 and AMPK-Thr172 that were enhanced by ONOO-. Taken together, we conclude that PKCzeta can regulate AMPK activity by increasing the Ser428 phosphorylation of LKB1, resulting in association of LKB1 with AMPK and consequent AMPK Thr172 phosphorylation by LKB1.     

Endothelin-1 stimulates its own synthesis in human endothelial cells.

We studied whether endothelin-1 (ET-1) would affect its own synthesis. Human umbilical cord vein endothelial cells in methionine-poor culture medium containing [35S] methionine were treated with synthetic ET-1 or ET-3. Immunoprecipitation of 35 S-labeled ET-1 was performed with rabbit ET-1 antiserum. ET-1 caused an 40 +/- 4% (mean +/- SEM) increase of immunoprecipitable 35 S-labeled ET-1 as confirmed by its elution point in reversed phase high power liquid chromatography (HPLC). ET-3 caused a 23 +/- 2% increase in ET-1 concentration. Amplification of cDNA by PCR showed both ET-1 and ETB receptor mRNAs in human cord vein endothelial cells. We conclude that ET-1 increases its own synthesis in endothelial cells. This suggests a positive autocrine feed-back action of ET-1 on its own synthesis, an effect which is probably mediated by non-specific ETB receptors.     

Estrogen induces the Akt-dependent activation of endothelial nitric-oxide synthase in vascular endothelial cells

Although estrogen is known to activate endothelial nitric oxide synthase (eNOS) in the vascular endothelium, the molecular mechanism responsible for this effect remains to be elucidated. In studies of both human umbilical vein endothelial cells (HUVECs) and simian virus 40-transformed rat lung vascular endothelial cells (TRLECs), 17beta-estradiol (E2), but not 17alpha-E2, caused acute activation of eNOS that was unaffected by actinomycin D and was specifically blocked by the pure estrogen receptor antagonist ICI-182,780. Treatment of both TRLECs and HUVECs with 17beta-E2 stimulated the activation of Akt, and the PI3K inhibitor wortmannin blocked the 17beta-E2-induced activation of Akt. 17beta-E2-induced Akt activation was also inhibited by ICI-182,780, but not by actinomycin D. Either treatment with wortmannin or exogenous expression of a dominant negative Akt in TRLECs decreased the 17beta-E2-induced eNOS activation. Moreover, 17beta-E2-induced Akt activation actually enhances the phosphorylation of eNOS. 17beta-E2-induced Akt activation was dependent on both extracellular and intracellular Ca(2+). We further examined the 17beta-E2-induced Akt activity in Chinese hamster ovary (CHO) cells transiently transfected with cDNAs for estrogen receptor alpha (ERalpha) or estrogen receptor beta (ERbeta). 17beta-E2 stimulated the activation of Akt in CHO cells expressing ERalpha but not in CHO cells expressing ERbeta. Our findings suggest that 17beta-E2 induced eNOS activation through an Akt-dependent mechanism, which is mediated by ERalpha via a nongenomic mechanism.     

Histamine stimulates prostacyclin synthesis in cultured human umbilical vein endothelial cells

Human venous endothelial cells synthesize prostacyclin (PGI₂) in response to treatment with histamine. The amount of PGI₂ produced is proportional to the histamine concentration over the range of 10^?7 to 10^?5 M, with a maximal response at 2–5 × 10^?6 M. PGI₂ synthesis occurs as a burst lasting less than 3 minutes after histamine addition. The H1 histamine receptor antagonist pyrilamine causes an 87% inhibition of PGI₂ synthesis, whereas the H2 antagonist cimetidine gives no significant inhibition, suggesting that PGI₂ synthesis in response to histamine is mediated by an H1 receptor.