Advances on the anti-inflammatory and protective effect of AMPK activators / 生理学报

AMP-activated protein kinase (AMPK) is a key enzyme in the regulation of cellular energy homeostasis. Recent studies demonstrated that AMPK also plays an important role in the modulation of inflammation, an energy-intensive molecular response. The commonly used AMPK activators include 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and A-769662. In addition, the biological activities of metformin and adiponectin are closely related to activation of AMPK. Numerous studies have shown that these AMPK activators play an effectively protective role in animal models of acute lung injury, asthma, colitis, hepatitis, atherosclerosis and other inflammatory diseases. Therefore, AMPK activators may have promising potential for the prevention and treatment of inflammation related diseases.

Eficiência do fungicida do grupo químico das carboxamida + estrobilurina no controle da ferrugem asiática em diferentes estádios da soja / Efficiency of fungicide from the carboxamide + strobilurin chemical group in the control of asian rust in different soybean stages / Eficiencia del fungicida del grupo químico de carboxamida + estrobilurina en el control de la oxidación asiática en diferentes etapas de soya

Para estabelecer o momento adequado da aplicação do fungicida no controle da ferrugem asiática (Phakospsora pachyrhizi) da soja, avaliou-se o estádio de aplicação do fungicida do grupo químico carboxamida + estrobirulina (ELATUS®), sob condições de campo. O delineamento experimental foi em blocos casualizados com oito tratamentos e quatro repetições. Cada bloco foi formado por quatro linhas de cinco metros de comprimento com área total de 9 m². Os tratamentos foram: T1 - uma aplicação no estádio V5; T2 - uma aplicação no estádio R1; T3 - uma aplicação no estádio R3; T4 - uma aplicação no estádio R5; T5 - duas aplicações, uma no estádio V5 e uma 21 dias após a primeira (DAA1); T6 - duas aplicações, uma no estádio R1 e uma a 21 DAA1; T7 - três aplicações; uma no estádio V5, a segunda 21 DAA1 e a terceira 21 DAA2 e T8 - Testemunha (controle). A partir dos estudos realizados foi avaliado o índice de severidade da doença, o número de plantas m-1, a massa de mil grãos e o rendimento de grãos. As médias observadas foram submetidas à análise de variância (ANOVA) e teste de Duncan (p ≤ 0,05). Os resultados obtidos mostraram diferenças significativas na severidade da doença, peso de mil grãos e no rendimento. Os tratamentos que apresentaram a menor severidade e maior rendimento de grãos foram T5 e T6 com duas aplicações no estádio V5 e 21 DAA e R1 e 21 DAA. Recomenda-se a aplicação do fungicida ELATUS® em duas vezes, sendo uma no estádio V5 e 21 dias após ou uma no estádio R1 e outra 21 dias após, procedimento esses que mostrou uma boa eficiência no controle do fungo e aumento no rendimento de grãos.

The purpose of this study was to establish the best moment for applying fungicide in the control of Asian rust (Phakospsora pachyrhizi) in soybean. In order to do so, the application stage of the fungicide from the carboxamide + strobirulin chemical group (ELATUS®) was assessed under field conditions. A randomized block experimental design with eight treatments and four replications was used. Each block was formed by four lines of five meters in length with a total area of 9 m². The treatments were: T1 - one application at the V5 stage; T2 - on application at the R1 stage; T3 - one application at the R3 stage; T4 - one application at the R5 stage; T5 - two applications, one at V5 and the other, 21 days after the 1st application (DAA1); T6 - two applications, one at R1 and the other at 21 DAA1; T7 - three applications: one at V5, the second 21 DAA1, and the third 21 DAA2; and T8 ­ control. The disease severity index, number of plants m-1, the mass of one thousand grains and grain yield were assessed. The observed means were submitted to the analysis of variance (ANOVA) and Duncan's test (p ≤ 0.05). The results showed significant differences in disease severity, weight of a thousand grains, and yield. The treatments with the lowest severity and highest grain yield were T5 and T6 with two applications at V5 and 21 DAA, and R1 and 21 DAA, respectively. ELATUS® is recommended to be applied twice, one at V5 and the second application 21 days after the first one, or one at R1 and the other 21 days after it, which presented good efficiency in fungus control and an increase in grain yield.

Para determinar el momento apropiado de la aplicación de fungicidas en el control de la oxidación asiática (Phakospsora pachyrhizi) de la soya, se evaluó la etapa de aplicación de fungicidas del grupo químico carboxamida + estrobirulina (ELATUS®), en condiciones de campo. El diseño experimental fue de bloques al azar con ocho tratamientos y cuatro repeticiones. Cada bloque estaba formado por cuatro filas de cinco metros de largo con una superficie total de 9 m². Los tratamientos fueron: T1 - una aplicación en la etapa V5; T2 - una aplicación en la etapa R1; T3 - una aplicación en la etapa R3; T4 - una aplicación en la etapa R5; T5 - dos aplicaciones, una en la fase V5 y otra 21 días después de la primera (DAA1); T6 - dos aplicaciones, una en la fase de R1 y otra 21 DAA1; T7 - tres aplicaciones; uno en el estadio V5, la segunda 21 DAA1, y la tercera 21 DAA2 y T8 ­ Testigo (control). A partir de los estudios realizados se evaluó el índice de gravedad de la enfermedad, el número de plantas m-1, peso de mil granos y el rendimiento de granos. Los resultados obtenidos han sido sometidos al análisis de varianza (ANOVA) y a la prueba de Duncan (p ≤ 0,05). Los resultados mostraron diferencias significativas en la gravedad de la enfermedad, peso de mil granos y en el rendimiento. Los tratamientos que presentaron menor gravedad y el más alto rendimiento de granos fueron T5 y T6 con dos aplicaciones a la etapa V5, 21 DAA, R1 y 21 DAA. Se recomienda la aplicación del fungicida ELATUS® dos veces, una en la fase V5 y 21 días después, o una en la etapa R1 y otra después de 21 días, procedimiento esos que mostraron una buena eficiencia en el control de hongos y aumento del rendimiento de granos.

Acadesine Inhibits the Proliferation of K562 Cells and Enhances their Sensitivity to Imatinib / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects of AMPK agonist Acadesine (AICAR) on growth inhibition of K562 cells and their sensitivity to imatinib (IM).</p><p><b>METHODS</b>K562 cells were cultured with different concentrations of AICAR alone or its combination with IM for 48 hours, the CCK-8 assay was used to detect cell proliferation, the cell cycle distribution and apoptosis were analyzed by flow cytometry. The expression levels of Cyclin D1, Cyclin E1 and Caspase 3 protein were determined by Western blot.</p><p><b>RESULTS</b>AICAR inhibited the proliferation of K562 cells in dose-dependent manner, and their IC50 value was 0.45 mmol/L at 48 hours. AICAR could induce arrest of K562 cells in G1 phase and down-regulated the protein expression levels of Cyclin D1 and Cyclin E1; whereas it didn't influence the cell apoptosis. Additionally, the growth inhibition of cells induced by IM was enhanced by AICAR.</p><p><b>CONCLUSION</b>AICAR can inhibit the proliferation of K562 cells by arresting the cell cycle and enhancing the sensitivity of K562 cells to IM.</p>

AMPK activator down-regulates the expression of tissue factor in fulminant hepatitis mice / 生理学报

AMP activated protein kinase (AMPK) is a pivotal metabolic regulatory enzyme and novel target of controlling inflammation. Our previous studies had demonstrated that 5-amino-4-imidazolecarboxamide riboside (AICAR), an AMPK activator, attenuated lipopolysaccharide (LPS)/D-galactosamine (D-gal)-induced fulminant hepatitis via suppressing inflammatory response. Since inflammation usually activates the coagulation response and aggravates inflammation-induced tissue injury, the present study was to explore the effects of AICAR on inflammation-induced activation of coagulation. Male BALB/c mice received LPS/D-gal intraperitoneal injection were used as fulminant hepatitis model. Western blot was used to detect tissue factor (TF) and hypoxia-inducible factor 1α (HIF-1α) protein expressions in hepatic tissue, as well as nuclear factor kappa B (NF-κB) p65 translocation into the nucleus. Real-time quantitative PCR was used to analyze erythropoietin (EPO) mRNA expression level. Lactic acid (LA) level in hepatic tissue was detected by kit. The results showed that LPS/D-gal induced the enhanced expression of TF, elevation of NF-κB p65 nuclear translocation, up-regulation of HIF-1α and EPO expressions, and increased LA level. These above alterations could be suppressed by AICAR. These results suggest that AICAR may down-regulate LPS/D-gal-induced TF expression (coagulation activity), and relieve hepatic hypoxia and metabolic disorder via suppressing the activity of NF-κB, which may be a novel mechanism of the beneficial effect of AICAR on LPS/D-gal-induced fulminant hepatitis.

AMP-activated protein kinase activation regulates adhesion of monocytes to vascular endothelial cells and the underlying mechanism / 生理学报

The present study was aimed to explore the effect of AMP-activated protein kinase (AMPK) on monocyte adhesion to vascular endothelial cells and underlying molecular mechanism. Tumor necrosis factor α (TNFα)-activated human aortic endothelial cells (HAECs) were treated with different concentrations of AMPK agonist 5-Aminoimidazole-4-carboxamide-1-β-D-ribonucleotide (AICAR) or AMPK inhibitor compound C. And other HAECs were overexpressed with constitutive active or dominant negative AMPK protein and then treated with TNFα. The rates of monocytes adhering to endothelial cells were detected by fluorescent staining. Intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) mRNA levels and protein secretions were detected by quantitative PCR and ELISA, respectively. Acetylation of NF-κB p65 at lysine 221 site was assessed by Western blot. NF-κB p65 DNA binding activity was analyzed by an ELISA-based method. By using small interfering RNA based strategy, p300 expression in HAECs was down-regulated and then cells were incubated with TNFα. NF-κB p65 DNA binding activity, ICAM-1 and VCAM-1 expressions and adhesion rates were detected, respectively. The activity of p300 was also detected by ELISA. The results showed that AICAR treatment significantly reduced monocyte-endothelial adhesion rate, as well as ICAM-1 and VCAM-1 mRNA levels and protein secretions, in TNFα-activated HAECs. Moreover, transfection of constitutive active AMPKα but not dominant negative AMPKα strongly diminished TNFα-induced upregulation of ICAM-1 and VCAM-1 mRNA expressions and secretions, as well as monocyte-endothelial adhesion. Furthermore, AMPK activation decreased TNFα-mediated acetylation of NF-κB p65 at Lys221 site and reduced NF-κB p65 DNA binding activity. Silencing p300 by siRNA significantly abolished the effect of TNFα- induced adhesion molecules expression and monocyte-endothelial adhesion. Blocking AMPK activation by compound C almost completely reversed the effect of AICAR exerted on HAECs. These results suggest AMPK activation suppresses monocyte-endothelial adhesion, and the underlying mechanism is relevant to the inhibition of p300 activity and NF-κB p65 transcriptional activity.

Angiotensin II Modulates p130Cas of Podocytes by the Suppression of AMP-Activated Protein Kinase

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.

Vascular O-GlcNAcylation augments reactivity to constrictor stimuli by prolonging phosphorylated levels of the myosin light chain

O-GlcNAcylation is a modification that alters the function of numerous proteins. We hypothesized that augmented O-GlcNAcylation levels enhance myosin light chain kinase (MLCK) and reduce myosin light chain phosphatase (MLCP) activity, leading to increased vascular contractile responsiveness. The vascular responses were measured by isometric force displacement. Thoracic aorta and vascular smooth muscle cells (VSMCs) from rats were incubated with vehicle or with PugNAc, which increases O-GlcNAcylation. In addition, we determined whether proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation. PugNAc enhanced phenylephrine (PE) responses in rat aortas (maximal effect, 14.2±2 vs 7.9±1 mN for vehicle, n=7). Treatment with an MLCP inhibitor (calyculin A) augmented vascular responses to PE (13.4±2 mN) and abolished the differences in PE-response between the groups. The effect of PugNAc was not observed when vessels were preincubated with ML-9, an MLCK inhibitor (7.3±2 vs 7.5±2 mN for vehicle, n=5). Furthermore, our data showed that differences in the PE-induced contractile response between the groups were abolished by the activator of AMP-activated protein kinase (AICAR; 6.1±2 vs 7.4±2 mN for vehicle, n=5). PugNAc increased phosphorylation of myosin phosphatase target subunit 1 (MYPT-1) and protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), which are involved in RhoA/Rho-kinase-mediated inhibition of myosin phosphatase activity. PugNAc incubation produced a time-dependent increase in vascular phosphorylation of myosin light chain and decreased phosphorylation levels of AMP-activated protein kinase, which decreased the affinity of MLCK for Ca2+/calmodulin. Our data suggest that proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation, favoring vascular contraction.

The effect of 5-aminoimidazole-4-carboxamide-ribonucleoside was mediated by p38 mitogen activated protein kinase signaling pathway in FRO thyroid cancer cells

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.

Effect of AMPK agonist 5-aminoimidazole-4-carboxamide ribonucleoside on proliferation, differentiation and apoptosis in U937 cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of AMPK agonist 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) on proliferation, differentiation and apoptosis of U937 cells and explore its possible mechanism.</p><p><b>METHODS</b>U937 cells were cultured with different concentrations of AICAR for 24 h and 48 h. Cell proliferation was evaluated. Cell growth curve was analyzed by CCK-8; cell apoptosis was analyzed by cell morphology, Annexin V/7-AAD double labeling. The differentiation of U937 cells was evaluated by expression of CD11b. The Bcl-xL, Bax, Bim, caspase-3 mRNA expressions of U937 cells were determined by real time PCR.</p><p><b>RESULTS</b>AICAR significantly inhibited the growth of U937 cells in a time-and dose-dependent manner, with a 24 h IC50 value of 1.1 mmol/L and 48 h of 0.9 mmol/L. 1.0 mmol/L AICAR didn't induce differentiation of U937 cells with the increase of CD11b expression for 24 h (P > 0.05). The U937 cells apoptosis was confirmed by cell morphology and Annexin V/7-AAD labeling. AICAR induced apoptosis of U937 cells and the apoptosis rate was (6.81 ± 1.16)% at 1 mmol/L AICAR higher than control group (2.74 ± 0.32)% without AICAR for 24 h treatment (P < 0.05). The real time PCR assay revealed that as compared with control group, the expression of Bim and caspase-3 mRNA were increased, while Bcl-xL and Bax were unchanged on the AICAR treatment.</p><p><b>CONCLUSION</b>AICAR can effectively inhibit proliferation and induce apoptosis of U937 cells. However, it has no significant effect on differentiation of U937 cells. The mechanism may be related with up-regulating Bim and Caspase-3.</p>

Cilostazol Decreases Ethanol-Mediated TNFalpha Expression in RAW264.7 Murine Macrophage and in Liver from Binge Drinking Mice

Alcoholic hepatitis is a leading cause of liver failure in which the increased production of tumor necrosis factor alpha (TNFalpha) plays a critical role in progression of alcoholic liver disease. In the present study, we investigated the effects of cilostazol, a selective inhibitor of type III phosphodiesterase on ethanol-mediated TNFalpha production in vitro and in vivo, and the effect of cilostazol was compared with that of pentoxifylline, which is currently used in clinical trial. RAW264.7 murine macrophages were pretreated with ethanol in the presence or absence of cilostazol then, stimulated with lipopolysacchride (LPS). Cilostazol significantly suppressed the level of LPS-stimulated TNFalpha mRNA and protein with a similar degree to that by pentoxifylline. Cilostazol increased the basal AMP-activated protein kinase (AMPK) activity as well as normalized the decreased AMPK by LPS. AICAR, an AMPK activator and db-cAMP also significantly decreased TNFalpha production in RAW264.7 cells, but cilostazol did not affect the levels of intracellular cAMP and reactive oxygen species (ROS) production. The in vivo effect of cilostazol was examined using ethanol binge drinking (6 g/kg) mice model. TNFalpha mRNA and protein decreased in liver from ethanol gavaged mice compared to that from control mice. Pretreatment of mice with cilostazol or pentoxifylline further reduced the TNFalpha production in liver. These results demonstrated that cilostazol effectively decrease the ethanol-mediated TNFalpha production both in murine macrophage and in liver from binge drinking mice and AMPK may be responsible for the inhibition of TNFalpha production by cilostazol.

Beneficial effects of metformin on primary cardiomyocytes via activation of adenosine monophosphate-activated protein kinase / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Metformin has become a cornerstone in the treatment of patients with type-2 diabetes. Accumulated evidence suggests that metformin supports direct cardiovascular effects. The present study aimed to investigate if metformin has beneficial effects on primary cardiomyocytes damaged by H2O2, and reveal the potential mechanism of action of metformin.</p><p><b>METHODS</b>Cardiomyocytes were incubated in the presence of 100 µmol/L H2O2 for 12 hours. Cardiomyocytes were pretreated with metformin at different concentrations and time and with aminoimidazole carboxamide ribonucleotide (AICAR) (500 µmol/L), an adenosine monophophate (AMP)-activated protein kinase (AMPK) agonist for 60 minutes before the addition of H2O2. Other cells were preincubated with compound C (an AMPK antagonist, 20 µmol/L) for 4 hours. The viability and apoptosis of cells were analyzed. AMPK, endothelial nitric oxide synthase (eNOS), and transforming growth factor (TGF)-β1 were analyzed using immunblotting.</p><p><b>RESULTS</b>Metformin had antagonistic effects on the influences of H2O2 on cell viability and attenuated oxidative stress-induced apoptosis. Metformin also increased phosphorylation of AMPK and eNOS, and reduced the expression of TGF-β1, basic fibroblast growth factor (bFGF), and tumor necrosis factor (TNF)-α.</p><p><b>CONCLUSIONS</b>Metformin has beneficial effects on cardiomyocytes, and this effect involves activation of the AMPK-eNOS pathway. Metformin may be potentially beneficial for the treatment of heart disease.</p>

Globular adiponectin protects human umbilical vein endothelial cells against apoptosis through adiponectin receptor 1/adenosine monophosphate-activated protein kinase pathway / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Endothelial 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).</p><p><b>METHODS</b>HUVECs 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.</p><p><b>RESULTS</b>HUVEC 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.</p><p><b>CONCLUSIONS</b>We 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.</p>

Chronic ethanol feeding impairs AMPK and MEF2 expression and is associated with GLUT4 decrease in rat myocardium

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.

Effects of AMPK on the transcriptional activity of FOXO1 and ubiquitin ligase MuRF1 expression in rat cardiomyocytes / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effects of AICAR on the activity of transcription factor FOXO1 and expression of ubiquitin ligase MuRF1 in rat cardiomyocytes, and explore the possible role of AMP-activated protein kinase (AMPK) in proteolysis pathways.</p><p><b>METHODS</b>In vitro cultured neonatal rat cardiac myocytes were treated with AICAR, and Western blotting was used to detect the phosphorylation of FOXO1 and expression of MuRF1 in the cells.</p><p><b>RESULTS</b>AICAR activated AMPK in rat cardiac myocytes. Activated AMPK significantly inhibited the phosphorylation of FOXO1 and increased MuRF1 protein expression.</p><p><b>CONCLUSION</b>AMPK may regulate proteolysis by activating FOXO1 transcription factor and up-regulating MuRF1 expression.</p>

Identification of the Fungal Pathogen that Causes Strawberry Anthracnose in Bangladesh and Evaluation of In Vitro Fungicide Activity

This study was conducted to identify the Colletotrichum species causing anthracnose disease of strawberry in Balgladesh and to evaluate in vitro activity of commercial fungicides it. Based on morphological and cultural characteristics, all 22 isolates were identified as Colletotrichum gloeosporioides. They developed white or glittery colonies with grey to dark grey reverse colony colors and they produced cylindrical conidia. The efficacy of five commercial fungicides, Bavistin DF, Dithane M-45, Sulcox 50 WP, Corzim 50 WP and Rovral 50 WP, were tested against the fungus. Bavistin inhibited radial growth completely and was followed in efficacy by Dithane M-45. In Bavistin DF treated media, the fungus did not produce conidia. The percent inhibition of radial growth of the fungus was increased with the increasing concentrations of fungicide.

The Preconditioning with AICAR Protects Against Subsequent Renal Ischemia Reperfusion Injury / 대한신장학회지

PURPOSE:Preconditioning due to activation of AMPK might reduce ischemia-reperfusion (I/R) injury in the kidney, based on the key role of AMPK in preserving ATP. To evaluate this possibility, the effect of preconditioning with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), AMPK activator, before sustained ischemia was investigated. METHODS:Adult male Sprague-Dawley rats weighing approximately 220-250 g were used. To induce renal ischemia, a laparotomy was performed under ketamine and xylazine hydrochloride, and the blood supply to both kidneys was interrupted by placement of vessel clamps at the level of the renal pedicles. Reflow was initiated by removing the clamps. The following experimental groups were defined 1. Acute renal ischemia 0 sec, 10 min, 15 min, 2. AICAR treatment, 3. Sham group (S), 4. Ischemia/ Reperfusion group (I/R), 5. AICAR+I/R group (A+I/R), 6. AraA (Adenine-9-b-D-arabinofuranoside, an AMPK) inhibitor+AICAR+I/R group (AraA+A+I/R) RESULTS:There was only faint AMPK phosphorylation in the sham group. After 10 minutes of ischemia, or AICAR preconditioning however, Thr172 phosphorylation of AMPK was increased (p<0.05). The serum levels of BUN and creatinine were significantly decreased in AICAR preconditioning group (A+I/R). (128.0+/-7.33 mg/dL, 4.18+/-0.27 mg/dL vs. 90.2+/-11.13 mg/dL, 2.58+/-0.7 mg/dL, p<0.05), but these effects were attenuated by AMPK inhibitor, AraA (AraA+A+I/R group). In quantitative analysis of tubular injury, tubular injury score in AICAR preconditioning group significantly decreased (p<0.05). CONCLUSION:The AMPK activator AICAR has a protective effect against renal I/R injury.

Aging impairs insulin-stimulated glucose uptake in rat skeletal muscle via suppressing AMPKalpha

Insufficient intracellular fat oxidation is an important contributor to aging-related insulin resistance, while the precise mechanism underlying is unclear. AMP-activated protein kinase (AMPK) is an important regulator of intracellular fat oxidation and was evidenced to play a key role in high-glucose and high-fat induced glucose intolerance. In the present study, we investigated whether altered AMPK expression or activity was also involved in aging-related insulin resistance. Insulin sensitivity of rats' skeletal muscles was evaluated using in-vitro glucose uptake assay. Activity of alpha subunit of AMPK (AMPKalpha) was evaluated by measuring the phosphorylation of both AMPKalpha (P-AMPKalpha) and acetyl-CoA carboxylase (P-ACC), while expression of AMPKalpha was assessed by determining the mRNA levels of AMPKalpha1 and AMPKalpha2, and protein contents of AMPKalpha. Compared with 4-month old rats, 24-month old rats exhibited obviously impaired insulin sensitivity. At the same time, AMPKalpha activity significantly decreased, while AMPKalpha expression did not alter during aging. Glucose transporter 4 expression also decreased in old rats. Compared with 24-month old rats, administration of the specific activator of AMPK, 5-aminoimidazole-4-carboxamide riboside (AICAR), significantly elevated AMPKalpha activity and GluT4 expression. Also, aging-related insulin resistance was significantly ameliorated by AICAR treatment. In conclusion, aging-related insulin resistance is associated with impaired AMPKalpha activity and could be ameliorated by AICAR, thus indicating a possible role of AMPK in aging-induced insulin resistance.

Role of 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside in the Growth Regulation of Anaplastic Thyroid Cancer Cells Lines / 대한내분비학회지

BACKGROUND: Anaplastic thyroid carcinoma is one of the most aggressive human cancers with a median survival of only 6 months. Local surgical tumor debulking combined with radio-chemotherapy is generally used to treat this malady, but the low success rate has prompted the search for new therapeutic targets. We used 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) as an AMP-activated protein kinase (AMPK) activator to induce growth suppression and apoptosis in the anaplastic thyroid carcinoma cells. METHODS: We investigated the effect of AICAR on the proliferation of thyroid cancer cell lines (ARO, WRO and FRO) by performing methyl-thiazoletetrazolium bromide assay. We wanted to see the effect of AICAR on the apoptosis and cell cycle of the thyroid cancer cells, and we wanted to determine the mechanism of these changes. RESULTS: The proliferation of all thyroid cancer cell lines was significantly inhibited by administration of AICAR. FRO was the most susceptible cell line to AICAR treatment and so further studies were then performed with this cell line. The suppressive effect of AICAR on cell proliferation was related with phosphorylation of AMPK and the increased apoptosis. Also, cell cycle analysis revealed that progression to the G2-M phase was arrested (S-phase arrest) by AICAR treatment. S-phase arrest was associated with the increased protein expression of p21. CONCLUSION: In the anaplastic thyroid cancer cell lines, AICAR inhibited proliferation due to the arrest in the S-phase; this was accompanied with the increased expression of p21. Overall, AMPK activation by AICAR or any other pharmacological agent could be a tempting potential target for thyroid cancer therapy.

The antiandrogenic effect of dimethachlon and its mechanism / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To evaluate the antiandrogenic effect of heterocyclic fungicide dimethachlon and its mechanism.</p><p><b>METHODS</b>A combination of in vivo and in vitro assays was selected. Hershberger assay was used to determine the antiandrogenic potential of dimethachlon in vivo. Six-week-old castrated male SD rats were administrated once daily for 7 days with testosterone propionate (TP, 100 micro g/d, sc) plus gavage doses of dimethachlon (50, 100 or 200 mg x kg(-1) x d(-1)), or procymidone (150 or 300 mg x kg(-1) x d(-1), positive control), or iprodione (100 mg x kg(-1) x d(-1), positive control), or flutamide (50 mg x kg(-1) x d(-1), positive control). Transcriptional activation assay in vitro was employed to determine the mechanism of antiandrogenic activity of dimethachlon. Human hepatoma liver cells HepG2 were transiently cotransfected with human androgen receptor (AR) expression plasmid and AR-dependent luciferase report plasmid. Transfected cells were exposed to various concentrations of dimethachlon or flutamide with or without dihydrotestosterone to induce the expression of luciferase gene.</p><p><b>RESULTS</b>In Hershberger assay, dimethachlon, as well as other known antiandrogens, caused decrease in weight of androgen dependent organs or tissues. In 200 mg/kg group, the weight of seminal vesicle, ventral prostate, dorsolateral prostate, Cowper's gland, and levator ani plus bulbocavernosus muscles decreased by 57.8%, 44.8%, 43.9%, 30.1%, and 34.1% respectively, but did not decrease in the vehicle control group. The order of their antiandrogenic potencies was: flutamide > procymidone > dimethachlon > iprodione. In transcriptional activation assay, dimethachlon could inhibit dihydrotestosterone-dependent AR activity in transfected HepG2 cells in dose-effect relationship. The inhibiting potency of dimethachlon was about 1/100 of that of flutamide.</p><p><b>CONCLUSION</b>Dimethachlon has antiandrogenic effect, and acts as an AR antagonist. Its antiandrogenic potency is lower than flutamide and procymidone, but higher than iprodione.</p>