Angiotensin II (Ang II) induces the pathological process of vascular structures, including renal glomeruli by hemodynamic and nonhemodynamic direct effects. In kidneys, Ang II plays an important role in the development of proteinuria by the modification of podocyte molecules. We have previously found that Ang II suppressed podocyte AMP-activated protein kinase (AMPK) via Ang II type 1 receptor and MAPK signaling pathway. In the present study, we investigated the roles of AMPK on the changes of p130Cas of podocyte by Ang II. We cultured mouse podocytes and treated them with various concentrations of Ang II and AMPK-modulating agents and analyzed the changes of p130Cas by confocal imaging and western blotting. In immunofluorescence study, Ang II decreased the intensity of p130Cas and changed its localization from peripheral cytoplasm into peri-nuclear areas in a concentrated pattern in podocytes. Ang II also reduced the amount of p130Cas in time and dose-sensitive manners. AMPK activators, metformin and AICAR, restored the suppressed and mal-localized p130Cas significantly, whereas, compound C, an AMPK inhibitor, further aggravated the changes of p130Cas. Losartan, an Ang II type 1 receptor antagonist, recovered the abnormal changes of p130Cas suppressed by Ang II. These results suggest that Ang II induces the relocalization and suppression of podocyte p130Cas by the suppression of AMPK via Ang II type 1 receptor, which would contribute to Ang II-induced podocyte injury.
AMP-Activated Protein Kinases/antagonists & inhibitors/chemistry/*metabolism ; Aminoimidazole Carboxamide/analogs & derivatives/pharmacology ; Angiotensin II/*pharmacology ; Angiotensin II Type 1 Receptor Blockers/pharmacology ; Animals ; Blotting, Western ; Cell Line ; Cell Nucleus/metabolism ; Crk-Associated Substrate Protein/*metabolism ; Cytoplasm/metabolism ; Focal Adhesion Kinase 1/metabolism ; Losartan/pharmacology ; Metformin/pharmacology ; Mice ; Microscopy, Confocal ; Podocytes/cytology/drug effects/metabolism ; Protein Kinase Inhibitors/*pharmacology ; Ribonucleotides/pharmacology ; Signal Transduction/*drug effects

BACKGROUND/AIMS: 5'-Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a cellular energy sensor that monitors intracellular AMP/adenosine triphosphate (ATP) ratios and is a key regulator of the proliferation and survival of diverse malignant cell types. In the present study, we investigated the effect of activating AMPK by 5-aminoimidazole-4-carboxamide-ribonucleotide (AICAR) in thyroid cancer cells. METHODS: We used FRO thyroid cancer cells harboring the BRAF(V600E) mutation to examine the effect of AICAR on cell proliferation and cell survival. We also evaluated the involvement of mitogen-activated protein kinase (MAPK) pathways in this effect. RESULTS: We found that AICAR treatment promoted AMPK activation and suppressed cell proliferation and survival by inducing p21 accumulation and activating caspase-3. AICAR significantly induced activation of p38 MAPK, and pretreatment with SB203580, a specific inhibitor of the p38 MAPK pathway, partially but significantly rescued cell survival. Furthermore, small interfering RNA targeting AMPK-alpha1 abolished AICAR-induced activation of p38 MAPK, p21 accumulation, and activation of caspase-3. CONCLUSIONS: Our findings demonstrate that AMPK activation using AICAR inhibited cell proliferation and survival by activating p38 MAPK and proapoptotic molecules in FRO thyroid cancer cells. These results suggest that the AMPK and p38 MAPK signaling pathways may be useful therapeutic targets to treat thyroid cancer.
AMP-Activated Protein Kinases/genetics/metabolism ; Aminoimidazole Carboxamide/*analogs & derivatives/pharmacology ; Antineoplastic Agents/*pharmacology ; Caspase 3/metabolism ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Cell Survival/drug effects ; Cyclin-Dependent Kinase Inhibitor p21/metabolism ; Dose-Response Relationship, Drug ; Enzyme Activation ; Enzyme Activators/pharmacology ; Humans ; Mutation ; Protein Kinase Inhibitors/pharmacology ; Proto-Oncogene Proteins B-raf/genetics ; RNA Interference ; Ribonucleotides/*pharmacology ; Signal Transduction/*drug effects ; Thyroid Neoplasms/*enzymology/genetics/pathology ; Time Factors ; Transfection ; p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors/*metabolism

Down syndrome cell adhesion molecule (DSCAM) acts as a netrin-1 receptor and mediates attractive response of axons to netrin-1 in neural development. However, the signaling mechanisms of netrin-DSCAM remain unclear. Here we report that AMP-activated protein kinase (AMPK) interacts with DSCAM through its γ subunit, but does not interact with DCC (deleted in colorectal cancer), another major receptor for netrin-1. Netrin-treatment of cultured cortical neurons leads to increased phosphorylation of AMPK. Both AMPK mutant with dominant-negative effect and AMPK inhibitor can significantly suppress netrin-1 induced neurite outgrowth. Together, these findings demonstrate that AMPK interacts with DSCAM and plays an important role in netrin-1 induced neurite outgrowth. Our study uncovers a previously unknown component, AMPK, in netrin-DSCAM signaling pathway.
AMP-Activated Protein Kinases ; antagonists & inhibitors ; genetics ; metabolism ; Animals ; Cell Adhesion Molecules ; genetics ; metabolism ; Cells, Cultured ; HEK293 Cells ; Humans ; Mice ; Nerve Growth Factors ; pharmacology ; Netrin-1 ; Neurites ; physiology ; Neurons ; cytology ; drug effects ; metabolism ; Phosphorylation ; Protein Binding ; Protein Kinase Inhibitors ; pharmacology ; RNA Interference ; RNA, Small Interfering ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Signal Transduction ; drug effects ; Transfection ; Tumor Suppressor Proteins ; pharmacology

The AMP-activated protein kinase (AMPK) is a pivotal serine/threonine kinase participating in the regulation of glucose, lipid as well as protein metabolism and maintenance of energy homeostasis. Recent studies demonstrated that AMPK can also inhibit nuclear factor-κB, suppress the expression of inflammatory genes and attenuate inflammatory injury through phosphorylating its downstream targets including SIRT1, PGC-lα, p53 and FoxO3a. In addition, the widely used antidiabetic metformin also exerts its anti-inflammatory effects through activating AMPK. Therefore, AMPK is emerging as a promising novel target for the development of anti-inflammatory drugs. This review summarized the anti-inflammatory effects of AMPK and the underling molecular mechanisms.
AMP-Activated Protein Kinases ; metabolism ; Homeostasis ; Inflammation ; enzymology ; Metformin ; pharmacology ; NF-kappa B ; antagonists & inhibitors ; Phosphorylation

pH homeostasis is essential for development, proliferation and apoptosis of cells. Once the pH balances are broken, cell functions and survival will be affected. Nevertheless, moderate acidosis could result in adaptive responses for cell survival and increase tolerance to harmful stress. Here we found that acidic preconditioning (APC) could significantly increase the survival rate of Daphnia pulex, a freshwater invertebrate, during severe hypoxic insult. Meanwhile, the acidic treatment significantly increased the gene expression of hypoxia inducible factor (HIF). Both echinomycin, an inhibitor of HIF, and compound C, an inhibitor of AMP-activated protein kinase (AMPK), could effectively eliminate the acid-induced hypoxic tolerance and the enhanced transcription of HIF. Temsirolimus, an inhibitor of mammalian Target of Rapamycin (mTOR), though effectively abolished the increased transcription of HIF, improved the APC-mediated protection. This result suggests that the involvement of the HIF and AMPK and mTOR could signal the pathways in APC-induced protection against hypoxic insult.
AMP-Activated Protein Kinases ; antagonists & inhibitors ; Acids ; pharmacology ; Adaptation, Physiological ; Animals ; Daphnia ; genetics ; physiology ; Hypoxia ; physiopathology ; Hypoxia-Inducible Factor 1 ; genetics ; metabolism ; Ischemic Preconditioning ; methods ; Pyrazoles ; pharmacology ; Pyrimidines ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Sirolimus ; analogs & derivatives ; pharmacology ; TOR Serine-Threonine Kinases ; antagonists & inhibitors

BACKGROUNDEndothelial dysfunction is a key event in the onset and progression of atherosclerosis in diabetic patients. Apoptosis may lead to endothelial dysfunction and contribute to vascular complications. However, no study has addressed apoptosis in human umbilical vein endothelial cells (HUVECs) induced by an intermittent high-glucose media and its association with adiponectin receptor 1 (adipoR1), adipoR2, or adenosine monophosphate (AMP)-activated protein kinase (AMPK).

METHODSHUVECs were cultured in continuous normal glucose (5.5 mmol/L), continuous high glucose (25 mmol/L), alternating normal and high glucose and mannitol. In the alternating normal and high-glucose media, HUVECs were treated under different conditions. First, cells were transfected with the adipoR1-specific small-interfering RNA (siRNA) and then stimulated with globular adiponectin (gAD). Second, cells were cultured in both gAD and the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR). Third, cells were cultured in the AMPK inhibitor adenine-9-β-D-arabino-furanoside (araA), gAD, and in AICAR.

RESULTSHUVEC apoptosis increased more significantly in an intermittent high-glucose medium than in a constant high-glucose medium. HUVEC apoptosis induced by an intermittent high-glucose medium was inhibited when the cells were pretreated with 3 µg/ml gAD, which rapidly activated AMPK and adipoR1 in HUVECs. However, adipoR2 was not activated.

CONCLUSIONSWe found that adipoR1, not adipoR2, is involved in mediating intermittent high-concentration glucose-evoked apoptosis in endothelial cells. gAD activated AMPK through adipoR1, leads to the partial inhibition of HUVEC apoptosis. A fluctuating glucose medium is more harmful than a constant high-glucose medium to endothelial cells.

AMP-Activated Protein Kinases ; antagonists & inhibitors ; genetics ; metabolism ; Adiponectin ; pharmacology ; Aminoimidazole Carboxamide ; analogs & derivatives ; pharmacology ; Apoptosis ; drug effects ; Glucose ; pharmacology ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; metabolism ; Humans ; RNA, Small Interfering ; Receptors, Adiponectin ; genetics ; metabolism ; Ribonucleotides ; pharmacology ; Signal Transduction ; drug effects ; genetics

Chronic and heavy alcohol consumption is one of the causes of heart diseases. However, the effects of ethanol on insulin sensitivity in myocardium has been unclear. To investigate the effects of ethanol on the expression of AMP-activated protein kinase (AMPK), myocyte enhancer factor 2 (MEF2) and glucose transporter 4 (GLUT4), all of which are involved in the regulation of insulin sensitivity, in the myocardium, we performed three parts of experiments in vivo and in vitro. I: Rats were injected with 5-amino-4-imidazolecarboxamide ribonucleotide (AICAR, 0.8 mg.kg(-1)) for 2 h. II: Rats received different dose (0.5, 2.5 or 5 g.kg(-1).d(-1)) of ethanol for 22-week. III: Primary neonatal rat cardiomyocytes were isolated and treated with or without 100 mM ethanol or 1 mM AICAR for 4 h. The cardiac protein and mRNA expression of AMPKalpha subunits, MEF2 and GLUT4 were observed by western-blotting and RT-PCR, respectively. Serum TNFalpha levels were assessed by ELISA. The results showed chronic ethanol exposure induced insulin resistance. Ethanol decreased the mRNA levels of AMPKalpha1 and alpha2, the protein levels of total- and phospho-AMPKalpha in cardiomyocytes. Similarly, ethanol showed inhibitory effects on both the mRNA and protein levels of MEF2A and 2D, and GLUT4 in a dose-response-like fashion. Correlation analysis implied an association between phospho-AMPKalpha and MEF2A or MEF2D, and between the levels of MEF2 protein and GLUT4 transcription. In addition, ethanol elevated serum TNFalpha level. Taken together, chronic ethanol exposure decreases the expression of AMPKalpha and MEF2, and is associated with GLUT4 decline in rat myocardium.
AMP-Activated Protein Kinases/genetics/*metabolism ; Aminoimidazole Carboxamide/analogs & derivatives/pharmacology ; Animals ; Enzyme Activation/drug effects ; Ethanol/*administration & dosage/*pharmacology ; Feeding Behavior/*drug effects ; Gene Expression Regulation/drug effects ; Glucose Transporter Type 4/genetics/*metabolism ; Insulin/pharmacology ; Insulin Resistance ; Male ; Myocardium/*enzymology ; Myogenic Regulatory Factors/antagonists & inhibitors/genetics/*metabolism ; Protein Isoforms/antagonists & inhibitors/genetics/metabolism ; RNA, Messenger/genetics/metabolism ; Rats ; Rats, Wistar ; Ribonucleotides/pharmacology ; Time Factors ; Tumor Necrosis Factor-alpha/blood

AMPK (AMP-activated protein kinase) is highly conserved in eukaryotes, where it functions primarily as a sensor of cellular energy status. Recent studies indicate that AMPK activation strongly suppresses cell proliferation in non-malignant cells as well as in tumor cells. In this study, quercetin activated AMPK in MCF breast cancer cell lines and HT-29 colon cancer cells, and this activation of AMPK seemed to be closely related to a decrease in COX-2 expression. The application of a COX-2 inhibitor or cox-2(-/-) cells supported the idea that AMPK is an upstream signal of COX-2, and is required for the anti-proliferatory and pro-apoptotic effects of quercetin. The suppressive or growth inhibitory effects of quercetin on COX-2 were abolished by treating cancer cells with an AMPK inhibitor Compound C. These results suggest that AMPK is crucial to the anti-cancer effect of quercetin and that the AMPK-COX-2 signaling pathway is important in quercetin-mediated cancer control.
AMP-Activated Protein Kinases/antagonists & inhibitors/*physiology ; Anticarcinogenic Agents/*pharmacology ; Antioxidants/*pharmacology ; Apoptosis/*drug effects ; Cell Cycle/drug effects/physiology ; Cell Line, Tumor ; Cell Proliferation/*drug effects ; Cyclooxygenase 2/genetics/*physiology ; Cyclooxygenase 2 Inhibitors/pharmacology ; Enzyme Activation ; Humans ; Pyrazoles/pharmacology ; Pyrimidines/pharmacology ; Quercetin/*pharmacology

BACKGROUND/AIMS: We examined the effects of cilostazol on mitogen-activated protein kinase (MAPK) activity and its relationship with cilostazol-mediated protection against apoptosis in lipopolysaccharide (LPS)-treated endothelial cells. METHODS: Human umbilical vein endothelial cells (HUVECs) were exposed to LPS and cilostazol with and without specific inhibitors of MAPKs; changes in MAPK activity in association with cell viability and apoptotic signaling were investigated. RESULTS: Cilostazol protected HUVECs against LPS-induced apoptosis by suppressing the mitochondrial permeability transition, cytosolic release of cytochrome c, and subsequent activation of caspases, stimulating extracellullar signal-regulated kinase (ERK1/2) and p38 MAPK signaling, and increasing phosphorylated cAMPresponsive element-binding protein (CREB) and Bcl-2 expression, while suppressing Bax expression. These cilostazol-mediated cellular events were effectively blocked by MAPK/ERK kinase (MEK1/2) and p38 MAPK inhibitors. CONCLUSIONS: Cilostazol protects HUVECs against LPS-induced apoptosis by suppressing mitochondriadependent apoptotic signaling. Activation of ERK1/2 and p38 MAPKs, and subsequent stimulation of CREB phosphorylation and Bcl-2 expression, may be responsible for the cellular signaling mechanism of cilostazolmediated protection.
Apoptosis/*drug effects ; Caspases/metabolism ; Cell Line ; Cell Survival/drug effects ; Cyclic AMP Response Element-Binding Protein/metabolism ; Cytochromes c/metabolism ; Dose-Response Relationship, Drug ; Endothelial Cells/*drug effects/enzymology/pathology ; Humans ; Lipopolysaccharides/*toxicity ; Mitochondrial Membrane Transport Proteins/drug effects/metabolism ; Mitogen-Activated Protein Kinase 1/antagonists & inhibitors/*metabolism ; Mitogen-Activated Protein Kinase 3/antagonists & inhibitors/*metabolism ; Phosphodiesterase Inhibitors/*pharmacology ; Phosphorylation ; Protein Kinase Inhibitors/pharmacology ; Proto-Oncogene Proteins c-bcl-2/metabolism ; Signal Transduction/*drug effects ; Tetrazoles/*pharmacology ; Time Factors ; bcl-2-Associated X Protein/metabolism ; p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors/*metabolism

AMP-Activated Protein Kinases ; Animals ; Antioxidants ; pharmacology ; Apoptosis ; drug effects ; Humans ; Infant, Newborn ; Infection ; pathology ; Ischemia ; pathology ; Leukomalacia, Periventricular ; etiology ; Multienzyme Complexes ; physiology ; Nitric Oxide Synthase Type II ; antagonists & inhibitors ; Oligodendroglia ; cytology ; Protein-Serine-Threonine Kinases ; physiology ; Reactive Oxygen Species ; metabolism ; Stem Cells ; cytology