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1.
Nat Cell Biol ; 22(2): 225-234, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32029897

RESUMO

Energy stress depletes ATP and induces cell death. Here we identify an unexpected inhibitory role of energy stress on ferroptosis, a form of regulated cell death induced by iron-dependent lipid peroxidation. We found that ferroptotic cell death and lipid peroxidation can be inhibited by treatments that induce or mimic energy stress. Inactivation of AMP-activated protein kinase (AMPK), a sensor of cellular energy status, largely abolishes the protective effects of energy stress on ferroptosis in vitro and on ferroptosis-associated renal ischaemia-reperfusion injury in vivo. Cancer cells with high basal AMPK activation are resistant to ferroptosis and AMPK inactivation sensitizes these cells to ferroptosis. Functional and lipidomic analyses further link AMPK regulation of ferroptosis to AMPK-mediated phosphorylation of acetyl-CoA carboxylase and polyunsaturated fatty acid biosynthesis. Our study demonstrates that energy stress inhibits ferroptosis partly through AMPK and reveals an unexpected coupling between ferroptosis and AMPK-mediated energy-stress signalling.


Assuntos
Proteínas Quinases Ativadas por AMP/genética , Acetil-CoA Carboxilase/genética , Ferroptose/genética , Rim/enzimologia , Peroxidação de Lipídeos/genética , Traumatismo por Reperfusão/genética , Células A549 , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/metabolismo , Acetil-CoA Carboxilase/metabolismo , Animais , Linhagem Celular Tumoral , Cicloexilaminas/farmacologia , Embrião de Mamíferos , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/genética , Ácidos Graxos Insaturados/biossíntese , Ferroptose/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Glucose/deficiência , Glucose/farmacologia , Humanos , Ferro/metabolismo , Rim/efeitos dos fármacos , Rim/patologia , Peroxidação de Lipídeos/efeitos dos fármacos , Células MCF-7 , Camundongos , Camundongos Transgênicos , Fenilenodiaminas/farmacologia , Fosforilação , Piperazinas/antagonistas & inibidores , Piperazinas/farmacologia , Cultura Primária de Células , Pirazóis/farmacologia , Pirimidinas/farmacologia , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/patologia , Transdução de Sinais , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética
2.
Metabolism ; 105: 154182, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32061660

RESUMO

The impairment of podocyte protein filtration function caused by excessive mitochondrial calcium intake is a critical feature of diabetic nephropathy (DN). Ca2+ channel transient receptor potential cation channel subfamily V member 1 (TRPV1) has been reported to protect against ischemia-reperfusion induced acute renal injury, but there is no report about its role in DN. Here, we report that dietary capsaicin potently inhibits and reverses chronic renal structural and functional damages in db/db or streptozotocin (STZ)-induced diabetic mice in a TRPV1-dependent manner. Activation of TRPV1 by capsaicin alleviated hyperglycemia-induced mitochondrial dysfunction in podocytes, accompanied by reduced mitochondria-associated membranes (MAMs) formation and fewer Ca2+ transport from endoplasmic reticulum (ER) to mitochondria. Mechanistically, TRPV1-mediated transient Ca2+ influx activated 5' AMP-activated protein kinase (AMPK) that reduced the transcription of Fundc1, a key molecule participating in MAMs formation. Inhibition of AMPK or overexpression of Fundc1 obviously blocked the inhibitory effect of capsaicin on MAMs formation and functional decline in podocytes. These findings emphasize the critical role of mitochondrial Ca2+ homeostasis in the maintenance of normal renal function and suggest an effective intervention method to counteract DN.


Assuntos
Nefropatias Diabéticas/tratamento farmacológico , Retículo Endoplasmático/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Podócitos/efeitos dos fármacos , Canais de Cátion TRPV/metabolismo , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Canais de Cálcio/metabolismo , Capsaicina/uso terapêutico , Dieta , Inibidores Enzimáticos/farmacologia , Hiperglicemia/tratamento farmacológico , Hiperglicemia/microbiologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Membranas Mitocondriais/efeitos dos fármacos , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/biossíntese
3.
Phytomedicine ; 68: 153147, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32028184

RESUMO

BACKGROUND: Gomisin A (G.A), a lignan compound extracted from the fruits of Schisandra chinensis, is known to exert anti-tumor effects on hepatocarcinoma and colorectal cancer cells. Suppression of proliferation and metastatic abilities of cancer cells are some effective cancer treatment methods. PURPOSE: The objective of this study is to investigate the effects of G.A on metastatic melanoma, and the mechanism by which it affects metastatic melanoma. STUDY DESIGN: The anti-proliferative and anti-metastatic effects of G.A were observed in in vitro and in vivo. METHODS: WST assay and flow cytometry were conducted to investigate the effect of G.A on proliferation, cell cycle arrest, and apoptosis in metastatic melanoma cell lines. Migration and invasion abilities of G.A-treated melanoma cells were observed by wound healing and invasion assays. RESULTS: G.A (25-100 µM) decreased the viability of melanoma cells by inducing cell cycle arrest and apoptosis. These anti-proliferative effects of G.A were found to be mediated by AMPK, ERK, and JNK activation. G.A (5-20 µM) decreased the migration and invasion of melanoma cells by suppressing epithelial-mesenchymal transition (EMT). Consequently, G.A (2-50 mg/kg) inhibited lung metastasis by suppressing EMT and inducing cell cycle arrest and apoptosis in melanoma cells. CONCLUSION: These results conclude that G.A has the potential to reduce metastatic melanoma through its anti-proliferative and anti-metastatic effects.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Ciclo-Octanos/farmacologia , Dioxóis/farmacologia , Lignanas/farmacologia , Melanoma/tratamento farmacológico , Melanoma/patologia , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/secundário , MAP Quinase Quinase 4/metabolismo , Melanoma/metabolismo , Camundongos Endogâmicos C57BL , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Biochimie ; 168: 277-284, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31786229

RESUMO

Changes in brain energy metabolism in diabetes mellitus, including increased insulin resistance and mitochondrial dysfunction, are critically involved in diabetes-related neurodegeneration, and associate with early cognitive impairment as well. The aim of this study is to detect the specific phosphorylated-Thr485- AMP-activated protein kinase (AMPK-α2), regulated by cyclin-dependent kinase 5 (Cdk5) paly the inhibitory functional role of AMPK-α2, Which is maybe the link to the accelerated diabetic brain damage progression. Here, we used GK rats, the type 2 diabetic animal model for in vivo studies and performed In vitro kinase assay, high glucose treatment, -phosphorylated mutation and protein expression in both HEK-293T and HT-22 cell lines. In vitro, the results show that murine wild-type AMPK-α2 was phosphorylated by Cdk5 at a (S/T)PX(K/H/R) phosphorylation consensus sequence, which was associated with decreased AMPK-α2 activity. Surprisingly, mutation of Thr485 to alanine in AMPK-α2 results in the abolished Cdk5 effects, demonstrating that Thr485-phosphorylation is critical to AMPK-α2 inhibition by Cdk5. In addition, these alterations in AMPK-α2-phosphorylation and -activity induced by Cdk5 is specific at Thr485. Furthermore, in GK rats, the increased phosphorylated- Thr 485 of AMPK-α2 results in the decreased AMPK-α2 activity, which is correlated with the apoptosis of neurons in hippocamps. After high glucose treatment, the decreased survival showed in AMPK-α2T485A HT-22 cells compared to AMPK-α2WT. The down-regulated of p-CREB, SNAP25, synaptophysin as well as synapsin-1were shown in both GK rats and HT-22 cell line. Meanwhile, pre-treated with either the specific Cdk5-inhibitor (roscovitine) or the antidiabetic AMPK-α2-inhibitor (metformin) could restore the alterations in neuronal protein expression. Our results suggest that Cdk5-mediated phosphorylated- Thr485 in AMPK-α2 may be involved in the pathogenesis of diabetic brain damage.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Quinase 5 Dependente de Ciclina/fisiologia , Diabetes Mellitus , Hipocampo , Neurônios , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Animais , Linhagem Celular , Quinase 5 Dependente de Ciclina/antagonistas & inibidores , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Células HEK293 , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Masculino , Metformina/metabolismo , Camundongos , Neurônios/metabolismo , Neurônios/patologia , Ratos , Roscovitina/metabolismo
5.
Artigo em Inglês | MEDLINE | ID: mdl-31676444

RESUMO

LiCl is widely prescribed for bipolar disorder but adversely associated with a higher incidence of increased body weight. Here, we investigated effects and underlying mechanisms of LiCl on lipid accumulation. LiCl induced dose-dependent lipid accumulation in HepG2 and RAW264.7 cells under normal as well as high glucose conditions. LiCl exposure additionally promoted lipid accumulation in livers of zebrafish. SB216763, a specific GSK-3ß inhibitor, did not affect lipid accumulation in HepG2 cells. Expression of key lipogenic enzymes, such as FAS and aP2, as well as SR-B1 were increased in RAW264.7 cells. LiCl enhanced FAS, ACC and SCD-1 mRNA levels while suppressing CPT-1 in HepG2 cells. LiCl stimulated DNA binding activities of SREBP-1c and ChREBP. LiCl activated AMPK phosphorylation but the AMPK inhibitor, AICAR, did not suppress LiCl-induced lipid accumulation in RAW264.7. LiCl, but not SB216763, induced a significant increase in ROS in RAW264.7 and HepG2 cells. NOX activity was dose-dependently enhanced by LiCl. Furthermore, NOX-1, NOX-2 and DUOX-1 mRNA levels were upregulated at an early stage of LiCl stimulation. LiCl-induced lipid accumulation was suppressed by the antioxidant, NAC, and inhibitors of NOX, DPI and APO. Phosphorylation and transcriptional activity of CREB were enhanced by LiCl. The cell-permeable cAMP analog, di-butyryl cAMP, not only promoted lipid accumulation itself but also LiCl-induced lipid accumulation in RAW264.7 cells. H-89, a PKA inhibitor, suppressed CREB activation, lipid accumulation and NOX activity in RAW264.7 cells. Our results indicate that LiCl stimulates lipid accumulation in hepatocyte and macrophage cells potentially through increased PKA-dependent ROS production.


Assuntos
Antimaníacos/efeitos adversos , Metabolismo dos Lipídeos/efeitos dos fármacos , Cloreto de Lítio/efeitos adversos , Espécies Reativas de Oxigênio/metabolismo , Ganho de Peso/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Transtorno Bipolar/tratamento farmacológico , Glicogênio Sintase Quinase 3 beta/metabolismo , Células Hep G2 , Humanos , Indóis/farmacologia , Isoquinolinas/farmacologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Maleimidas/farmacologia , Camundongos , Células RAW 264.7 , Sulfonamidas/farmacologia , Peixe-Zebra
6.
Cell Prolif ; 53(1): e12714, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31696568

RESUMO

OBJECTIVES: Osteoclasts (OC) are unique terminally differentiated cells whose primary function is bone resorption. We previously showed that osteoprotegerin (OPG) inhibits OC differentiation in vitro by enhancing autophagy via the adenosine monophosphate-activated protein kinase (AMPK)/mTOR/p70S6K signalling pathway in vitro. Here, we aimed to elucidate the mechanism of AMPK mediated autophagy to regulate OPG-mediated inhibition of OC differentiation and identify potential therapeutic targets associated with bone loss. MATERIALS AND METHODS: We used the AMPK activator AICAR to determine the relationship between AMPK activation and OC differentiation, and studied the role of AMPK-mediated autophagy in OPG-mediated inhibition of OC differentiation by using autophagy inhibitors or AMPK knockdown. RESULTS: AMP-activated protein kinase activation caused LC3II accumulation and weakened OC differentiation activity. In contrast, inactivation of autophagy by 3-methyladenine or Bafilomycin A1 could attenuate OPG-mediated inhibition of OC differentiation via the AMPK/mTOR/p70S6K signalling pathway. Furthermore, the AMPK inhibitor compound C and knockdown of AMPK impaired OPG-mediated inhibition of OC differentiation by inducing autophagy. CONCLUSIONS: These results demonstrated that the AMPK signalling pathway functions as a critical regulator in the OPG-mediated inhibition of OC differentiation, by inducing autophagy. Our results provide a basis for future bone-related studies on the AMPK signalling pathway.


Assuntos
Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Aminoimidazol Carboxamida/análogos & derivados , Morte Celular Autofágica/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Osteoclastos/metabolismo , Osteoprotegerina/metabolismo , Ribonucleotídeos/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Aminoimidazol Carboxamida/farmacologia , Animais , Camundongos , Camundongos Endogâmicos BALB C , Osteoclastos/citologia , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo
7.
Environ Toxicol ; 35(2): 176-187, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31633292

RESUMO

Mesoporous silica is a drug carrier with strong targeting, large loading capacity, and easy modification of its surface while its toxicity draws increasing attention recently. In this study, we evaluated the impact of SBA-15 nanomaterials on hippocampal neurons. We found that SBA-15 induces oxidative damage to hippocampal neurons HT22, which further activates autophagy. Treatment with the mammalian target of rapamycin (mTOR) inhibitor AZD8055, the phosphorylation level of mTOR and P70S6K reduced and increased levels of p-AMPK meaning that the adenosine-activated protein kinase (AMPK)/mTOR/P70S6K pathway is involved in SBA-15 induced autophagy of HT22. These results suggested that mesoporous silica material SBA-15 might affect central nervous cells via oxidative stress activation of the AMPK/mTOR/P70S6K pathway, which provides a theoretical basis for safe administration of such materials in patients.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Autofagia/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Dióxido de Silício/toxicidade , Serina-Treonina Quinases TOR/metabolismo , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Animais , Linhagem Celular , Portadores de Fármacos/química , Portadores de Fármacos/toxicidade , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Camundongos , Nanoestruturas/química , Neurônios/metabolismo , Neurônios/patologia , Fosforilação , Transdução de Sinais , Dióxido de Silício/química
8.
Anticancer Res ; 39(12): 6635-6643, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31810928

RESUMO

BACKGROUND/AIM: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers apoptosis of cancer cells and, when used in combination with other anticancer drugs, is regarded as an effective strategy for anticancer treatment. In this study, we investigated the efficacy of combination treatment with TRAIL-secreting human mesenchymal stem cells (MSC-TRAIL) and compound C, an AMP-activated protein kinase (AMPK inhibitor), on glioblastoma. MATERIALS AND METHODS: The anticancer effect using MSC-TRAIL and compound C on glioma was evaluated in vitro and on in vivo models. RESULTS: Combination treatment of MSC-TRAIL and compound C increased apoptosis by enhancing expression of B-cell lymphoma 2 (BCL2)-associated X protein (BAX) and reducing that of anti-apoptotic proteins cellular FLICE-inhibitory protein (FLIP), X-linked inhibitor of apoptosis (XIAP), and BCL2 in glioma. In addition, MSC-TRAIL and compound C combination increased caspase-3 cleavage and apoptotic cells in a mouse glioma model compared with the group treated with the agents alone. CONCLUSION: Our results suggest that MSC-TRAIL and compound C are a novel combination for treatment of glioma.


Assuntos
Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Neoplasias Encefálicas/terapia , Glioblastoma/terapia , Transplante de Células-Tronco Mesenquimais , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Animais , Apoptose , Neoplasias Encefálicas/metabolismo , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspase 3/metabolismo , Terapia Combinada , Glioblastoma/metabolismo , Xenoenxertos , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Nus , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Proteína X Associada a bcl-2/metabolismo
9.
Cell Biochem Funct ; 37(8): 625-632, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31693217

RESUMO

The fat-derived protein adiponectin is known to reverse the effects of insulin resistance and to lower blood glucose levels. The AMP-activated protein kinase (AMPK) signalling pathway plays a central role in metabolism and energy homeostasis. Here, to investigate the role of AMPK in the protective effect of adiponectin against insulin resistance, we established the model of high-glucose (HG)- and high-lipid (HL)-induced insulin resistance in INS-1 pancreatic ß cells. We found that 25mM of glucose and 0.4mM of palmitic acid treatment significantly increased cell apoptosis and impaired insulin secretion in INS-1 cells. However, recombinant human adiponectin dramatically reduced HG- and/or HL-induced cell apoptosis and greatly improved insulin secretion. Interestingly, adiponectin treatment also activated AMPK signalling pathway by increasing the phosphorylation of Thr172 in the AMPK α subunit; 10µM of compound C, a potent AMPK inhibitor, blocked the protective effects of adiponectin against HG/HL-induced insulin resistance. Furthermore, knockout experiments by CRISPR/Cas9 technology showed that AMPK α1, but not AMPK α2, is involved in the protective effects of adiponectin. Taken together, adiponectin reversed the effects of insulin resistance via AMPK α1, which provides a novel insight into the protective mechanism of adiponectin and may be used as a new strategy for the treatment of type 2 diabetes. SIGNIFICANCE OF THE STUDY: Adiponectin can reverse the effects of insulin resistance and lower blood glucose levels. Here, adiponectin reduced HG/HL-induced cell apoptosis and greatly improved insulin secretion. These effects were blocked by AMPK inhibitor, compound C. Specifically, we found that AMPK α1, but not AMPK α2, mediates the protective effects of adiponectin, which provides a novel insight into the protective mechanism of adiponectin against insulin resistance.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Adiponectina/farmacologia , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/genética , Adiponectina/genética , Adiponectina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Glucose/farmacologia , Resistência à Insulina , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos
10.
Int J Mol Sci ; 20(22)2019 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-31744135

RESUMO

Atherosclerosis is one of the most reported diseases worldwide, and extensive research and trials are focused on the discovery and utilizing for novel therapeutics. Nitric oxide (NO) is produced mainly by endothelial nitric oxide synthase (eNOS) and it plays a key role in regulating vascular function including systemic blood pressure and vascular inflammation in vascular endothelium. In this study hypothesized that Impressic acid (IPA), a component isolated from Acanthopanax koreanum, acts as an enhancer of eNOS activity and NO production. IPA treatment induced eNOS phosphorylation and NO production, which was correlated with eNOS phosphorylation via the activation of JNK1/2, p38 MAPK, AMPK, and CaMKII. In addition, the induction of eNOS phosphorylation by IPA was attenuated by pharmacological inhibitor of MAPKs, AMPK, and CaMKII. Finally, IPA treatment prevented the adhesion of TNF-α-induced monocytes to endothelial cells and suppressed the TNF-α-stimulated ICAM-1 expression via activation of NF-κB, while treatment with L-NAME, the NOS inhibitor, reversed the inhibitory effect of IPA on TNF-α-induced ICAM-1 expression via activation of NF-κB. Taken together, these findings show that IPA protects against TNF-α-induced vascular endothelium dysfunction through attenuation of the NF-κB pathway by activating eNOS/NO pathway in endothelial cells.


Assuntos
Eleutherococcus/química , Transdução de Sinais/efeitos dos fármacos , Triterpenos/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Eleutherococcus/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , NF-kappa B/metabolismo , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Triterpenos/química
11.
Food Funct ; 10(10): 6858-6872, 2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31584590

RESUMO

Acetaminophen (APAP) overdose is a major cause of drug-induced liver injury worldwide. Our current study was performed to assess the potential protective effects of γ-oryzanol (ORY) on APAP-induced liver injury in mice and explore the underlying molecular mechanisms. We unveiled that ORY alleviated the APAP-induced death of HL-7702 hepatocytes in vitro and liver injury in mice. Moreover, ORY promoted the nuclear translocation of Nrf2, increased the expressions of Nrf2-downstream antioxidative enzymes, including HO-1, NQO1, GCLC, and GCLM, and thereby restrained APAP-induced oxidative stress in hepatocytes. Moreover, ORY modulated the AMPK/GSK3ß axis that acts upstream of Nrf2 in hepatocytes. Compound C, an inhibitor of AMPK, prevented the ORY-mediated activation of Nrf2 and protection against APAP toxicity in HL-7702 hepatocytes. Additionally, in the liver of mice receiving APAP, ORY suppressed the nuclear translocation of the NF-κB p65 subunit, downregulated the expressions of iNOS and COX-2, and reduced the levels of pro-inflammatory factors including TNF-α, IL-1ß, IL-6, and NO. Taken together, our findings revealed that ORY is capable of ameliorating APAP-induced liver injury. The modulation of AMPK/GSK3ß/Nrf2 and NF-κB signaling pathways is implicated in the hepatoprotective activity of ORY.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Acetaminofen/efeitos adversos , Doença Hepática Crônica Induzida por Substâncias e Drogas/tratamento farmacológico , Glicogênio Sintase Quinase 3 beta/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , Fenilpropionatos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Animais , Antioxidantes/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Glutamato-Cisteína Ligase/metabolismo , Heme Oxigenase-1/metabolismo , Hepatócitos , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Fígado/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , NAD(P)H Desidrogenase (Quinona)/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Oxazinas/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo
12.
Proc Natl Acad Sci U S A ; 116(40): 20210-20217, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31527268

RESUMO

Nitric oxide (NO) synthesized by the endothelial isoform of nitric oxide synthase (eNOS) is a critical determinant of vascular homeostasis. However, the real-time detection of intracellular NO-a free radical gas-has been difficult, and surrogate markers for eNOS activation are widely utilized. eNOS phosphorylation can be easily measured in cells by probing immunoblots with phosphospecific antibodies. Here, we pursued multispectral imaging approaches using biosensors to visualize intracellular NO and Ca2+ and exploited chemogenetic approaches to define the relationships between NO synthesis and eNOS phosphorylation in cultured endothelial cells. We found that the G protein-coupled receptor agonists adenosine triphosphate (ATP) and histamine promoted rapid increases in eNOS phosphorylation, as did the receptor tyrosine kinase agonists insulin and Vascular Endothelial Growth Factor (VEGF). Histamine and ATP also promoted robust NO formation and increased intracellular Ca2+ By contrast, neither insulin nor VEGF caused any increase whatsoever in intracellular NO or Ca2+-despite eliciting strong eNOS phosphorylation responses. Our findings demonstrate an unexpected and striking discordance between receptor-modulated eNOS phosphorylation and NO formation in endothelial cells. Previous reports in which phosphorylation of eNOS has been studied as a surrogate for enzyme activation may need to be reassessed.


Assuntos
Técnicas Biossensoriais , Imagem Molecular , Óxido Nítrico Sintase Tipo III/metabolismo , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/metabolismo , Cálcio/metabolismo , Células Cultivadas , Citosol , Células Endoteliais/metabolismo , Ativação Enzimática , Imagem Molecular/métodos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais
13.
Int J Mol Sci ; 20(18)2019 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-31540136

RESUMO

Insulin resistance and diabetes are both associated with chronic hepatitis C virus (HCV) infection, and the glucagon-like peptide-1(GLP-1) receptor agonist, liraglutide, is a common therapy for diabetes. Our aim was to investigate whether liraglutide treatment can inhibit HCV replication. A cell culture-produced HCV infectious system was generated by transfection of in vitro-transcribed genomic JFH-1 ribonucleic acid (RNA) into Huh-7.5 cells. Total RNA samples were extracted to determine the efficiency of HCV replication. The Ava5 cells were treated with liraglutide and cell viability was calculated. A Western blot analysis of the protein expression was performed. The immunoreactive blot signals were also detected. Liraglutide activated GLP-1 receptors in the HCV infectious system, and inhibited subgenomic HCV RNA replication in the HuH-7.5 cells. The Western blot analysis revealed both HCV protein and replicon RNA were reduced after treatment with liraglutide in a dose-dependent manner. Liraglutide decreased the cell viability of HCV RNA at an optimum concentration of 120 µg/mL, activated the 5' adenosine monophosphate-activated protein kinase (AMPK) and the phosphorylated- transducer of regulated cyclic adenosine monophosphate (CAMP) response element-binding protein 2 (TORC2), thereby decreasing the cell viability of phosphoenolpyruvate carboxykinase (PEPCK) and G6pase RNA Therefore, we conclude that liraglutide can inhibit HCV replication via an AMPK/TORC2-dependent pathway.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Hepacivirus/efeitos dos fármacos , Hepatócitos/enzimologia , Liraglutida/farmacologia , Replicação Viral/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Peptídeo 1 Semelhante ao Glucagon/agonistas , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Hepacivirus/fisiologia , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
14.
Nat Commun ; 10(1): 4363, 2019 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-31554794

RESUMO

The LKB1/AMPK pathway plays a major role in cellular homeostasis and tumor suppression. Down-regulation of LKB1/AMPK occurs in several human cancers and has been implicated in metabolic diseases. However, the precise upstream regulation of LKB1-AMPK pathway is largely unknown. Here, we report that AMPK activation by LKB1 is regulated by tankyrases. Tankyrases interact with and ribosylate LKB1, promoting its K63-linked ubiquitination by an E3 ligase RNF146, which blocks LKB1/STRAD/MO25 complex formation and LKB1 activation. LKB1 activation by tankyrase inhibitors induces AMPK activation and suppresses tumorigenesis. Similarly, the tankyrase inhibitor G007-LK effectively regulates liver metabolism and glycemic control in diabetic mice in a LKB1-dependent manner. In patients with lung cancer, tankyrase levels negatively correlate with p-AMPK levels and poor survival. Taken together, these findings suggest that tankyrase and RNF146 are major up-stream regulators of LKB1-AMPK pathway and provide another focus for cancer and metabolic disease therapies.


Assuntos
Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Metabolismo Energético/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Neoplasias/tratamento farmacológico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Tanquirases/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Linhagem Celular Tumoral , Células HeLa , Homeostase/efeitos dos fármacos , Humanos , Células MCF-7 , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Sulfonas/farmacologia , Tanquirases/metabolismo , Triazóis/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , para-Aminobenzoatos/farmacologia
15.
Environ Toxicol ; 34(12): 1340-1353, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31433112

RESUMO

This study investigated whether the apoptotic effect induced by cadmium chloride (CdCl2 ) in rat's hippocampi and neuroprotection afforded by resveratrol (RES) are mediated by modulation of ER stress and involve sirtuin 1 (SIRT1)/AMPK/Akt axis. Adult male Wistar rats were divided into four groups (n = 24/group) as control, control + RES (300 mg/kg), CdCl2 (5 mg/kg), and CdCl2 + RES. All treatments were conducted orally for 45 days. Also, cultured hippocampal cells were treated with CdCl2 in the presence or absence of RES and with or without preincubation with SIRT1, AMPK, or PI3K inhibitors. CdCl2 impaired retention and spatial memories of rats and reduced levels and activities of SIRT1 and inhibited AMPK/Akt axis in their hippocamapi where SIRT1 was the upstream regulator. It also enahnced hippocampal levels of reactive oxygen species (ROS) and expression of caspase-12 and caspase-3, depleted glutathione (GSH) levels, and activated GRP78, activating transcription factor-6, GAAD 153, X-box binding protein-1 arms of ER stress. On the contrary, RES coadminsitration completley abolished all these events. Interstingly and in control rats, RES not only increased levels of GSH, but also enhenced protein levels of B-cell lymphoma 2 (Bcl-2) and dwonregulated GAAD 153. In both control and CdCl2 -treated rats, pharmacological inhibtion of SIRT1, AMPK, and Akt compleltely abolished all effects afforded by RES. In conclusion, CdCl2 -induced hippocampal apopotis is associated with reduction of SIRT1/AMPK/Akt activity levels, ROS generation, downregulation of Bcl-2, and activities, activation of ER stress, and GAAD 153, whereas RES is able to reverse these effects through activation of SIRT1/AMPK/Akt.


Assuntos
Cloreto de Cádmio/toxicidade , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Resveratrol/farmacologia , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Apoptose/efeitos dos fármacos , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismo , Fator de Transcrição CHOP/metabolismo , Regulação para Cima/efeitos dos fármacos
16.
Biochimie ; 165: 210-218, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31401190

RESUMO

Dysfunction in lipid metabolism may result in a decrease in hepatic autophagy, which contributes to the pathogenesis of non-alcoholic steatohepatitis. ATP-binding cassette transporter A1 transports free cholesterol and phospholipids to apolipoprotein A-I (apoA-I) to form nascent high-density lipoprotein particles. Results from previous studies showed that the overexpression of apoA-I significantly reduced levels of hepatic lipids and endoplasmic reticulum stress by modifying lipid transport. Here, we investigated the effects of apoA-I overexpression on hepatic autophagy in cultured hepatocytes and mice. The overexpression of apoA-I in HepG2 cells resulted in an increase in the levels of autophagy as well as the phosphorylation of AMP-activated protein kinase α (AMPKα) and ULK1 and a decrease in the phosphorylation of mammalian target of rapamycin (mTOR). An AMPK inhibitor and siRNA eliminated this apoA-I effect. Consistently, apoA-I transgenic mice showed increased autophagy and AMPKα phosphorylation. These results suggest that apoA-I overexpression alleviates steatohepatitis by increasing hepatic autophagy through the AMPK-mTOR-ULK1 pathway.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Apolipoproteína A-I/fisiologia , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Fígado Gorduroso/metabolismo , Hepatócitos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fígado/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Animais , Autofagia , Células Hep G2 , Hepatócitos/citologia , Humanos , Metabolismo dos Lipídeos , Fígado/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
17.
Open Biol ; 9(7): 190099, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31288625

RESUMO

The AMP-activated protein kinase (AMPK) acts as a cellular energy sensor. Once switched on by increases in cellular AMP : ATP ratios, it acts to restore energy homeostasis by switching on catabolic pathways while switching off cell growth and proliferation. The canonical AMP-dependent mechanism of activation requires the upstream kinase LKB1, which was identified genetically to be a tumour suppressor. AMPK can also be switched on by increases in intracellular Ca2+, by glucose starvation and by DNA damage via non-canonical, AMP-independent pathways. Genetic studies of the role of AMPK in mouse cancer suggest that, before disease arises, AMPK acts as a tumour suppressor that protects against cancer, with this protection being further enhanced by AMPK activators such as the biguanide phenformin. However, once cancer has occurred, AMPK switches to being a tumour promoter instead, enhancing cancer cell survival by protecting against metabolic, oxidative and genotoxic stresses. Studies of genetic changes in human cancer also suggest diverging roles for genes encoding subunit isoforms, with some being frequently amplified, while others are mutated.


Assuntos
Proteínas Quinases Ativadas por AMP/fisiologia , Neoplasias/patologia , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Animais , Metabolismo Energético/genética , Genes Supressores de Tumor/fisiologia , Glucose/metabolismo , Humanos , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
18.
Mol Med Rep ; 20(2): 1279-1287, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31173269

RESUMO

Obesity is associated with increased sensitivity to pain, including neuropathic pain, but the precise mechanisms are not fully understood. Recent evidence has revealed that AMP­activated protein kinase (AMPK) in the central nervous system (CNS) regulates the neuropeptide calcitonin gene­related peptide (CGRP), a principal neurotransmitter of the class C nerve fiber, which serves an important role in initiating and maintaining neuropathic pain. AMPK has been demonstrated to be downregulated in the CNS in obesity. The present study hypothesized that obesity may lead to increased sensitivity to neuropathic pain by downregulating AMPK and upregulating CGRP expression levels in the CNS. Sprague­Dawley rats consuming a high­fat diet (HF) for 12 weeks developed obesity; they exhibited significantly decreased levels of phospho (p)­AMPK and increased CGRP expression levels in the spinal cord (SC) and dorsal root ganglion (DRG), respectively, compared with rats consuming a low­fat (LF) diet. HF­fed rats that underwent spared nerve injury (SNI) also exhibited lower p­AMPK and higher CGRP expression levels in the SC and DRG, compared with the corresponding LF­diet rats. The 50% paw withdrawal threshold (PWT; as measured by Von Frey testing) was significantly lower in HF­fed compared with LF­fed rats, with or without SNI. Through intrathecal treatment, the AMPK activator 5­aminoimidazole­4­carboxamide riboside (AICAR) or the CGRP antagonist CGRP8­37 decreased CGRP expression levels and increased the 50% PWT; however, the AMPK inhibitor dorsomorphin augmented CGRP expression levels and further reduced the 50% PWT in HF­fed rats, but not LF­fed rats, with or without SNI. The results indicated that blocking the AMPK­CGRP pathway may enhance neuropathic pain in HF­induced obesity, with or without nerve injury. Targeting AMPK in the CNS may be a novel strategy for the prevention and treatment of obesity­associated neuropathic pain.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Sistema Nervoso Central/metabolismo , Dieta Hiperlipídica , Tecido Nervoso/lesões , Neuralgia/metabolismo , Obesidade/metabolismo , Transdução de Sinais , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Animais , Peso Corporal , Sistema Nervoso Central/patologia , Hiperalgesia/complicações , Masculino , Camundongos Obesos , Tecido Nervoso/metabolismo , Tecido Nervoso/patologia , Neuralgia/complicações , Obesidade/complicações , Limiar da Dor , Fosforilação , Ratos Sprague-Dawley
19.
J Neuroinflammation ; 16(1): 110, 2019 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-31128596

RESUMO

BACKGROUND: Microglia-mediated neuroinflammation is important in Alzheimer's disease (AD) pathogenesis. Extracellular deposition of ß-amyloid (Aß), a major pathological hallmark of AD, can induce microglia activation. Adiponectin (APN), an adipocyte-derived adipokine, exerts anti-inflammatory effects in the periphery and brain. Chronic APN deficiency leads to cognitive impairment and AD-like pathologies in aged mice. Here, we aim to study the role of APN in regulating microglia-mediated neuroinflammation in AD. METHODS: Inflammatory response of cultured microglia (BV2 cells) to AßO and effects of APN were studied by measuring levels of proinflammatory cytokines (tumor necrosis factor α [TNFα] and interleukin-1ß [IL-1ß]) in cultured medium before and after exposure to AßO, with and without APN pretreatment. Adiponectin receptor 1 (AdipoR1) and receptor 2 (AdipoR2) were targeted by small interference RNA. To study the neuroprotective effect of APN, cultured HT-22 hippocampal cells were treated with conditioned medium of AßO-exposed BV2 cells or were co-cultured with BV2 cells in transwells. The cytotoxicity of HT-22 hippocampal cells was assessed by MTT reduction. We generated APN-deficient AD mice (APN-/-5xFAD) by crossing APN-knockout mice with 5xFAD mice to determine the effects of APN deficiency on microglia-mediated neuroinflammation in AD. RESULTS: AdipoR1 and AdipoR2 were expressed in BV2 cells and microglia of mice. Pretreatment with APN for 2 h suppressed TNFα and IL-1ß release induced by AßO in BV2 cells. Additionally, APN rescued the decrease of AMPK phosphorylation and suppressed nuclear translocation of nuclear factor kappa B (NF-κB) induced by AßO. Compound C, an inhibitor of AMPK, abolished these effects of APN. Knockdown of AdipoR1, but not AdipoR2 in BV2 cells, inhibited the ability of APN to suppress proinflammatory cytokine release induced by AßO. Moreover, pretreatment with APN inhibited the cytotoxicity of HT-22 cells co-cultured with AßO-exposed BV2 cells. Lastly, APN deficiency exacerbated microglia activation in 9-month-old APN-/-5xFAD mice associated with upregulation of TNFα and IL-1ß in the cortex and hippocampus. CONCLUSIONS: Our findings demonstrate that APN inhibits inflammatory response of microglia to AßO via AdipoR1-AMPK-NF-κB signaling, and APN deficiency aggravates microglia activation and neuroinflammation in AD mice. APN may be a novel therapeutic agent for inhibiting neuroinflammation in AD.


Assuntos
Proteínas Quinases Ativadas por AMP/biossíntese , Adiponectina/farmacologia , Peptídeos beta-Amiloides/toxicidade , Microglia/metabolismo , NF-kappa B/biossíntese , Fragmentos de Peptídeos/toxicidade , Receptores de Adiponectina/biossíntese , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Peptídeos beta-Amiloides/antagonistas & inibidores , Animais , Relação Dose-Resposta a Droga , Inflamação/induzido quimicamente , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/efeitos dos fármacos , NF-kappa B/antagonistas & inibidores , Fragmentos de Peptídeos/antagonistas & inibidores , Receptores de Adiponectina/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
20.
Neuropharmacology ; 155: 98-103, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-30986422

RESUMO

Oxidative stress and autophagy are involved in the pathogenesis of Parkinson's disease. The relationship between oxidative stress and autophagy is a hot spot of scientific research. Alpha-lipoic acid (ALA) is a natural antioxidant. ALA has been reported to reduce oxidative stress and apoptosis in PD models, but its role in autophagy regulation of PD has been reported very little. In this study, we investigated the protective effects of ALA on 6-OHDA induced neurotoxicity, and explored the potential mechanisms associated with the crosstalk between oxidative stress and autophagy. Our results showed that 6-OHDA induced accumulation of ROS in SH-SY5Y cells, accompanied by excessive autophagy and apoptosis. ALA protected against 6-OHDA induced neuronal death through inhibition of oxidative stress and autophagy. Furthermore, we found that ALA inhibited AMPK phosphorylation while activated mTOR phosphorylation thereby blocking AMPK/mTOR signaling pathway involved autophagy. In conclusion, ALA alleviated 6-OHDA induced cell injury possibly by inhibiting autophagy mediated by AMPK/mTOR pathway.


Assuntos
Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Antioxidantes/farmacologia , Autofagia/efeitos dos fármacos , Oxidopamina/toxicidade , Serina-Treonina Quinases TOR/antagonistas & inibidores , Ácido Tióctico/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Autofagia/fisiologia , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Linhagem Celular Tumoral , Humanos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Serina-Treonina Quinases TOR/metabolismo
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