RESUMO
Hypertension is a globally prevalent disease, but the pathogenesis remains largely unclear. AMP-activated protein kinase (AMPK) is a nutrition-sensitive signal of cellular energy metabolism, which has a certain influence on the development of hypertension. Previously, we found a down-regulation of the phosphorylated (p-) form of AMPK, and the up-regulation of the angiotensin II type 1 receptor (AT1-R) and that of p-ERK1/2 in the hypothalamic paraventricular nucleus (PVN) of hypertensive rats. However, the exact mechanism underlying the relationship between AMPK and AT1-R in the PVN during hypertension remains unclear. Thus, we hypothesized that AMPK modulates AT1-R through the ERK1/2-NF-κB pathway in the PVN, thereby inhibiting sympathetic nerve activity and improving hypertension. To examine this hypothesis, we employed a renovascular hypertensive animal model developed via two-kidney, one-clip (2K1C) and sham-operated (SHAM). Artificial cerebrospinal fluid (aCSF), used as vehicle, or 5-amino-1-ß-D-ribofuranosyl-imidazole-4-carboxamide (AICAR, an AMPK activator, 60 µg/day) was microinjected bilaterally in the PVN of these rats for 4 weeks. In 2K1C rats, there an increase in systolic blood pressure (SBP) and circulating norepinephrine (NE). Also, the hypertensive rats had lowered expression of p-AMPK and p-AMPK/AMPK, elevated expression of p-ERK1/2, p-ERK1/2/ERK1/2 and AT1-R, increased NF-κB p65 activity in the PVN compared with the levels of these biomarkers in SHAM rats. Four weeks of bilateral PVN injection of AMPK activator AICAR, attenuated the NE level and SBP, increased the expression of p-AMPK and p-AMPK/AMPK, lessened the NF-κB p65 activity, decreased the expression of p-ERK1/2, p-ERK1/2/ERK1/2 and AT1-R in the PVN of 2K1C rats. Data from this study imply that the activation of AMPK within the PVN suppressed AT1-R expression through inhibiting the ERK1/2-NF-κB pathway, decreased the activity of the sympathetic nervous system, improved hypertension.
Assuntos
Proteínas Quinases Ativadas por AMP , Modelos Animais de Doenças , Ativação Enzimática , Hipertensão Renovascular , Proteína Quinase 3 Ativada por Mitógeno , Núcleo Hipotalâmico Paraventricular , Ratos Sprague-Dawley , Receptor Tipo 1 de Angiotensina , Animais , Núcleo Hipotalâmico Paraventricular/metabolismo , Núcleo Hipotalâmico Paraventricular/enzimologia , Núcleo Hipotalâmico Paraventricular/efeitos dos fármacos , Núcleo Hipotalâmico Paraventricular/fisiopatologia , Hipertensão Renovascular/fisiopatologia , Hipertensão Renovascular/enzimologia , Hipertensão Renovascular/metabolismo , Hipertensão Renovascular/tratamento farmacológico , Masculino , Proteínas Quinases Ativadas por AMP/metabolismo , Fosforilação , Receptor Tipo 1 de Angiotensina/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fator de Transcrição RelA/metabolismo , Ribonucleotídeos/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Sistema Nervoso Simpático/fisiopatologia , Sistema Nervoso Simpático/efeitos dos fármacos , Sistema Nervoso Simpático/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , NF-kappa B/metabolismo , Transdução de Sinais , Anti-Hipertensivos/farmacologia , RatosRESUMO
Monopolar spindle 1 kinase (Mps1, also known as TTK protein kinase) inhibitors exert marked anticancer effects against triplenegative breast cancer (TNBC) by causing genomic instability and cell death. As aneuploid cells are vulnerable to compounds that induce energy stress through adenosine monophosphateactivated protein kinase (AMPK) activation, the synergistic effect of Mps1/TTK inhibition and AMPK activation was investigated in the present study. The combined effects of CFI402257, an Mps1/TTK inhibitor, and AICAR, an AMPK agonist, were evaluated in terms of cytotoxicity, cellcycle distribution, and in vivo xenograft models. Additional molecular mechanistic studies were conducted to elucidate the mechanisms underlying apoptosis and autophagic cell death. The combination of CFI402257 and AICAR showed selective cytotoxicity in a TNBC cell line. The formation of polyploid cells was attenuated, and apoptosis was increased by the combination treatment, which also induced autophagy through dual inhibition of the PI3K/Akt/mTOR and mitogenactivated protein kinase (MAPK) signaling pathways. Additionally, the combination therapy showed strongly improved efficacy in comparison with CFI402257 and AICAR monotherapy in the MDAMB231 xenograft model. The present study suggested that the combination of CFI402257 and AICAR is a promising therapeutic strategy for TNBC.
Assuntos
Proteínas Quinases Ativadas por AMP , Aminoimidazol Carboxamida , Apoptose , Autofagia , Proteínas de Ciclo Celular , Proteínas Serina-Treonina Quinases , Ribonucleotídeos , Neoplasias de Mama Triplo Negativas , Ensaios Antitumorais Modelo de Xenoenxerto , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/metabolismo , Humanos , Feminino , Animais , Camundongos , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Linhagem Celular Tumoral , Ribonucleotídeos/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Proteínas Tirosina Quinases/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sinergismo Farmacológico , Compostos de Bifenilo , Pironas , TiofenosRESUMO
Metabolic changes are critical in the regulation of Ca2+ influx in central and peripheral neuroendocrine cells. To study the regulation of L-type Ca2+ channels by AMPK we used biochemical reagents and ATP/glucose-concentration manipulations in rat chromaffin cells. AICAR and Compound-C, at low concentration, significantly induce changes in L-type Ca2+ channel-current amplitude and voltage dependence. Remarkably, an overlasting decrease in the channel-current density can be induced by lowering the intracellular level of ATP. Accordingly, Ca2+ channel-current density gradually diminishes by decreasing the extracellular glucose concentration. By using immunofluorescence, a decrease in the expression of CaV1.2 is observed while decreasing extracellular glucose, suggesting that AMPK reduces the number of functional Ca2+ channels into the plasma membrane. Together, these results support for the first time the dependence of metabolic changes in the maintenance of Ca2+ channel-current by AMPK. They reveal a key step in Ca2+ influx in secretory cells.
Assuntos
Proteínas Quinases Ativadas por AMP , Aminoimidazol Carboxamida , Canais de Cálcio Tipo L , Células Cromafins , Glucose , Animais , Células Cromafins/metabolismo , Células Cromafins/efeitos dos fármacos , Canais de Cálcio Tipo L/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Ratos , Glucose/metabolismo , Glucose/farmacologia , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Trifosfato de Adenosina/metabolismo , Ribonucleotídeos/farmacologia , Pirimidinas/farmacologia , Cálcio/metabolismo , Pirazóis/farmacologia , Células Cultivadas , Ratos Wistar , Ativação do Canal Iônico/efeitos dos fármacosRESUMO
In crustaceans, the steroid hormone 20-hydroxyecdysone (20E) initiates molting, and the molting process is also regulated by energy metabolism. AMPK is an energy sensor and plays a critical role in systemic energy balance. Here, the regulatory mechanism in the interaction between 20E and AMPK was investigated in Chinese mitten crab, Eriocheir sinensis. The results showed that the 20E concentration and the mRNA expression levels of 20E receptors in hepatopancreas were down-regulated post AMPK activator (AICAR) treatment, and were up-regulated after AMPK inhibitor (Compound C) injection in crabs. Besides, the molt-inhibiting hormone (MIH) gene expression in eyestalk showed the opposite patterns in response to the AICAR and Compound C treatment, respectively. Further investigation found that there was a significant reduction in 20E concentration post PI3K inhibitor (LY294002) treatment, and the phosphorylation level of PI3K was increased in hepatopancreas after AMPK inhibitor injection. On the other hand, the positive regulation of PI3K-mediated activation of AMPK was also observed, the phosphorylation levels of AMPKα, AMPKß and PI3K in hepatopancreas were significantly increased post 20E injection. In addition, the phosphorylation levels of AMPKα and AMPKß induced by 20E were decreased after the injection of PI3K inhibitor. Taken together, these results suggest that the regulatory cross-talk between 20E and AMPK is likely to act through PI3K pathway in E. sinensis, which appeared to be helpful for a better understanding in molting regulation.
Assuntos
Proteínas Quinases Ativadas por AMP , Braquiúros , Ecdisterona , Hepatopâncreas , Muda , Fosfatidilinositol 3-Quinases , Animais , Braquiúros/imunologia , Ecdisterona/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Hepatopâncreas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Hormônios de Invertebrado/metabolismo , Cromonas/farmacologia , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Ribonucleotídeos/farmacologia , Morfolinas/farmacologia , Proteínas de Artrópodes/metabolismo , Proteínas de Artrópodes/genética , Fosforilação , Metabolismo EnergéticoRESUMO
GDM, as a metabolic disease during pregnancy, regulates GLUT3 translocation by AMPK, thereby affecting glucose uptake in trophoblasts. It provides a new research idea and therapeutic target for alleviating intrauterine hyperglycemia in GDM. STZ was used to construct GDM mice, inject AICAR into pregnant mice, and observe fetal and placental weight; flow cytometry was employed for the detection of glucose uptake by primary trophoblast cells; immunofluorescence was applied to detect the localization of GLUT3 and AMPK in placental tissue; Cocofal microscope was used to detect the localization of GLUT3 in trophoblast cells;qRT-PCR and Western blot experiments were carried out to detect the expression levels of GLUT3 and AMPK in placental tissue; CO-IP was utilized to detect the interaction of GLUT3 and AMPK. Compared with the normal pregnancy group, the weight of the fetus and placenta of GDM mice increased (P < 0.001), and the ability of trophoblasts to take up glucose decreased (P < 0.001). In addition, AMPK activity in trophoblasts and membrane localization of GLUT3 in GDM mice were down-regulated compared with normal pregnant mice (P < 0.05). There is an interaction between GLUT3 and AMPK. Activating AMPK in trophoblasts can up-regulate the expression of GLUT3 membrane protein in trophoblasts of mice (P < 0.05) and increase the glucose uptake of trophoblasts (P < 0.05). We speculate that inhibition of AMPK activity in GDM mice results in aberrant localization of GLUT3, which in turn attenuates glucose uptake by placental trophoblast cells. AICAR activates AMPK to increase the membrane localization of GLUT3 and improve the glucose uptake capacity of trophoblasts.
Assuntos
Proteínas Quinases Ativadas por AMP , Diabetes Gestacional , Transportador de Glucose Tipo 3 , Glucose , Transdução de Sinais , Trofoblastos , Animais , Trofoblastos/metabolismo , Feminino , Gravidez , Glucose/metabolismo , Camundongos , Proteínas Quinases Ativadas por AMP/metabolismo , Transportador de Glucose Tipo 3/metabolismo , Transportador de Glucose Tipo 3/genética , Diabetes Gestacional/metabolismo , Placenta/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Ribonucleotídeos/farmacologiaRESUMO
BACKGROUND: Simvastatin (Sim), a hydroxy-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, has been widely used in prevention and treatment of cardiovascular diseases. Studies have suggested that Sim exerts anti-fibrotic effects by interfering fibroblast proliferation and collagen synthesis. This study was to determine whether Sim could alleviate silica-induced pulmonary fibrosis and explore the underlying mechanisms. METHODS: The rat model of silicosis was established by the tracheal perfusion method and treated with Sim (5 or 10 mg/kg), AICAR (an AMPK agonist), and apocynin (a NOX inhibitor) for 28 days. Lung tissues were collected for further analyses including pathological histology, inflammatory response, oxidative stress, epithelial mesenchymal transformation (EMT), and the AMPK-NOX pathway. RESULTS: Sim significantly reduced silica-induced pulmonary inflammation and fibrosis at 28 days after administration. Sim could reduce the levels of interleukin (IL)-1ß, IL-6, tumor necrosis factor-α and transforming growth factor-ß1 in lung tissues. The expressions of hydroxyproline, α-SMA and vimentin were down-regulated, while E-cad was increased in Sim-treated rats. In addition, NOX4, p22pox, p40phox, p-p47phox/p47phox expressions and ROS levels were all increased, whereas p-AMPK/AMPK was decreased in silica-induced rats. Sim or AICAR treatment could notably reverse the decrease of AMPK activity and increase of NOX activity induced by silica. Apocynin treatment exhibited similar protective effects to Sim, including down-regulating of oxidative stress and inhibition of the EMT process and inflammatory reactions. CONCLUSIONS: Sim attenuates silica-induced pulmonary inflammation and fibrosis by downregulating EMT and oxidative stress through the AMPK-NOX pathway.
Assuntos
Proteínas Quinases Ativadas por AMP , Fibrose Pulmonar , Dióxido de Silício , Sinvastatina , Animais , Masculino , Ratos , Acetofenonas/farmacologia , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Pulmão/patologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , NADPH Oxidase 4/metabolismo , NADPH Oxidases/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Pneumonia/induzido quimicamente , Pneumonia/prevenção & controle , Pneumonia/tratamento farmacológico , Pneumonia/metabolismo , Pneumonia/patologia , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/tratamento farmacológico , Ribonucleotídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Silicose/tratamento farmacológico , Silicose/patologia , Silicose/metabolismo , Sinvastatina/farmacologia , Fator de Crescimento Transformador beta1/metabolismoRESUMO
The bifunctional enzyme, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase/inosine monophosphate (IMP) cyclohydrolase (ATIC) is involved in catalyzing penultimate and final steps of purine de novo biosynthetic pathway crucial for the survival of organisms. The present study reports the characterization of ATIC from Candidatus Liberibacer asiaticus (CLasATIC) along with the identification of potential inhibitor molecules and evaluation of cell proliferative activity. CLasATIC showed both the AICAR Transformylase (AICAR TFase) activity for substrates, 10-f-THF (Km, 146.6 µM and Vmax, 0.95 µmol/min/mg) and AICAR (Km, 34.81 µM and Vmax, 0.56 µmol/min/mg) and IMP cyclohydrolase (IMPCHase) activitiy (Km, 1.81 µM and Vmax, 2.87 µmol/min/mg). The optimum pH and temperature were also identified for the enzyme activity. In-silico study has been conducted to identify potential inhibitor molecules through virtual screening and MD simulations. Out of many compounds, HNBSA, diosbulbin A and lepidine D emerged as lead compounds, exhibiting higher binding energy and stability for CLasATIC than AICAR. ITC study reports higher binding affinities for HNBSA and diosbulbin A (Kd, 12.3 µM and 34.2 µM, respectively) compared to AICAR (Kd, 83.4 µM). Likewise, DSC studies showed enhanced thermal stability for CLasATIC in the presence of inhibitors. CD and Fluorescence studies revealed significant conformational changes in CLasATIC upon binding of the inhibitors. CLasATIC demonstrated potent cell proliferative, wound healing and ROS scavenging properties evaluated by cell-based bioassays using CHO cells. This study highlights CLasATIC as a promising drug target with potential inhibitors for managing CLas and its unique cell protective, wound-healing properties for future biotechnological applications.
Assuntos
Aminoimidazol Carboxamida , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/química , Aminoimidazol Carboxamida/metabolismo , Aminoimidazol Carboxamida/farmacologia , Fosforribosilaminoimidazolcarboxamida Formiltransferase/metabolismo , Fosforribosilaminoimidazolcarboxamida Formiltransferase/química , Simulação de Acoplamento Molecular , Ribonucleotídeos/metabolismo , Ribonucleotídeos/química , Cinética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/antagonistas & inibidores , Nucleotídeo Desaminases/metabolismo , Nucleotídeo Desaminases/química , Nucleotídeo Desaminases/genética , Especificidade por Substrato , Proliferação de Células/efeitos dos fármacos , Hidroximetil e Formil Transferases/metabolismo , Hidroximetil e Formil Transferases/química , Hidroximetil e Formil Transferases/genética , Hidroximetil e Formil Transferases/antagonistas & inibidores , Complexos MultienzimáticosRESUMO
AMP-activated protein kinase (AMPK) is a cellular energy sensor regulating metabolic homeostasis. In this study, we investigated the role of AMPK in response to human herpesvirus 6A (HHV-6A) infection. We show that HHV-6A infection significantly downregulates the active phosphorylated state of AMPK in infected T cells. Pharmacological activation of AMPK highly attenuated HHV-6A propagation. Mechanistically, we found that the activation of AMPK by AICAR blocked HHV-6-induced glycolysis by inhibiting glucose metabolism and lactate secretion, as well as decreasing expressions of key glucose transporters and glycolytic enzymes. In addition, mTOR signaling has been inactivated in HHV-6A infected T cells by AICAR treatment. We also showed that HHV-6A infection of human umbilical cord blood mononuclear cells (CBMCs) reduced AMPK activity whereas the activation of AMPK by metformin drastically reduced HHV-6A DNA replication and virions production. Taken together, this study demonstrates that AMPK is a promising antiviral therapeutic target against HHV-6A infection.
Assuntos
Proteínas Quinases Ativadas por AMP , Glicólise , Herpesvirus Humano 6 , Transdução de Sinais , Serina-Treonina Quinases TOR , Replicação Viral , Herpesvirus Humano 6/fisiologia , Herpesvirus Humano 6/genética , Herpesvirus Humano 6/metabolismo , Humanos , Replicação Viral/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Infecções por Roseolovirus/virologia , Infecções por Roseolovirus/metabolismo , Metformina/farmacologia , Ribonucleotídeos/farmacologia , FosforilaçãoRESUMO
Doxorubicin (DOX) is a widely used chemotherapeutic agent that can cause serious cardiotoxic side effects, leading to heart failure (HF). Impaired mitochondrial function is thought to be key factor driving progression into HF. We have previously shown in a rat model of DOX-HF that heart failure with reduced ejection fraction correlates with mitochondrial loss and dysfunction. Adenosine monophosphate-dependent kinase (AMPK) is a cellular energy sensor, regulating mitochondrial biogenesis and energy metabolism, including fatty acid oxidation. We hypothesised that AMPK activation could restore mitochondrial function and therefore be a novel cardioprotective strategy for the prevention of DOX-HF. Consequently, we set out to assess whether 5-aminoimidazole-4-carboxamide 1-ß-D-ribofuranoside (AICAR), an activator of AMPK, could prevent cardiac functional decline in this chronic intravenous rat model of DOX-HF. In line with our hypothesis, AICAR improved cardiac systolic function. AICAR furthermore improved cardiac mitochondrial fatty acid oxidation, independent of mitochondrial number, and in the absence of observable AMPK-activation. In addition, we found that AICAR prevented loss of myocardial mass. RNAseq analysis showed that this may be driven by normalisation of pathways associated with ribosome function and protein synthesis, which are impaired in DOX-treated rat hearts. AICAR furthermore prevented dyslipidemia and excessive body-weight loss in DOX-treated rats, which may contribute to preservation of myocardial mass. Though it is unclear whether AICAR exerted its cardioprotective effect through cardiac or extra-cardiac AMPK-activation or via an AMPK-independent effect, these results show promise for the use of AICAR as a cardioprotective agent in DOX-HF to both preserve cardiac function and mass.
Assuntos
Proteínas Quinases Ativadas por AMP , Aminoimidazol Carboxamida , Cardiotônicos , Doxorrubicina , Insuficiência Cardíaca , Ribonucleotídeos , Animais , Doxorrubicina/efeitos adversos , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Insuficiência Cardíaca/induzido quimicamente , Insuficiência Cardíaca/prevenção & controle , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/tratamento farmacológico , Ribonucleotídeos/farmacologia , Masculino , Cardiotônicos/farmacologia , Ratos , Proteínas Quinases Ativadas por AMP/metabolismo , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/efeitos dos fármacos , Miocárdio/metabolismo , Miocárdio/patologia , Ácidos Graxos/metabolismo , Modelos Animais de DoençasRESUMO
BACKGROUND AND PURPOSE: The interleukin (IL)-36 pathway is a critical player in the pathogenesis of pustular psoriasis. However, therapies targeting this pathway are limited or unaffordable (e.g. the anti-IL-36 receptor antibody). AMP-activated protein kinase (AMPK), a regulator of cellular energy and metabolism, is known to participate in inflammatory diseases. However, its role in IL-36-induced skin inflammation remains unclear. Therefore, we sought to investigate the role of AMPK signals in regulating IL-36-induced responses in the skin. EXPERIMENTAL APPROACH: IL-36-stimulated primary normal human epidermal keratinocytes (NHEKs) and IL-36-injected (intradermally) BALB/c mice served as the cell and animal models, respectively. Additionally, 5-aminoimidazole-4-carboxamide riboside (AICAR) and A769662 served as AMPK activators. KEY RESULTS: AICAR and A769662 significantly suppressed the IL-36-induced IL-8 (CXCL8) and CCL20 production from NHEKs. IL-36-induced IκBζ protein expression was prominently reduced and IKK/IκBα phosphorylation was attenuated by AICAR and A769662. Conversely, AMPKα knockdown increased IκBζ protein expression and IKK/IκBα phosphorylation in IL-36-treated NHEKs. Furthermore, AICAR and A769662 enhanced IL-36-induced-IκBζ protein degradation via the proteasome-dependent but not the lysosome-dependent pathway. Pretreatment of NHEKs with IL-36 slightly suppressed the AICAR- and A769662-triggered phosphorylation of AMPK and acetyl-CoA carboxylase. In the mouse model, topical application of AICAR significantly reduced ear swelling, redness, epidermal thickening, neutrophil infiltration and inflammatory and antimicrobial peptide gene expression. CONCLUSION AND IMPLICATIONS: AMPK activation suppresses IL-36-induced IL-8 and CCL20 release by regulating IκBζ expression in keratinocytes and reduces IL-36-induced skin inflammation in mice, suggesting that AMPK activation is a potential strategy for treating patients with IL-36-mediated inflammatory skin disorders.
Assuntos
Proteínas Quinases Ativadas por AMP , Aminoimidazol Carboxamida , Pele , Animais , Humanos , Camundongos , Proteínas Adaptadoras de Transdução de Sinal , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Células Cultivadas , Quimiocina CCL20/metabolismo , Ativação Enzimática/efeitos dos fármacos , Inflamação/metabolismo , Inflamação/tratamento farmacológico , Interleucina-1/metabolismo , Interleucina-8/metabolismo , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Camundongos Endogâmicos BALB C , Ribonucleotídeos/farmacologia , Pele/efeitos dos fármacos , Pele/patologia , Pele/metabolismoRESUMO
BACKGROUND: Oxidative stress and inflammatory cytokines in the hypothalamus paraventricular nucleus (PVN) have been implicated in sympathetic nerve activity and the development of hypertension, but the specific mechanisms underlying their production in the PVN remains to be elucidated. Previous studies have demonstrated that activation of nuclear transcription related factor-2 (Nrf2) in the PVN reduced the production of reactive oxygen species (ROS) and inflammatory mediators. Moreover, AMP-activated protein kinase (AMPK), has been observed to decrease ROS and inflammatory cytokine production when activated in the periphery. 5-amino-1-ß-D-ribofuranosyl-imidazole-4-carboxamide (AICAR) is an AMPK agonist. However, little research has been conducted on the role of AMPK in the PVN during hypertension. Therefore, we hypothesized that AICAR in the PVN is involved in regulating AMPK/Nrf2 pathway, affecting ROS and inflammatory cytokine expression, influencing sympathetic nerve activity. METHODS: Adult male Sprague-Dawley rats were utilized to induce two-kidney, one-clip (2K1C) hypertension via constriction of the right renal artery. Bilateral PVN was microinjected with either artificial cerebrospinal fluid or AICAR once a day for 4 weeks. RESULTS: Compared to the SHAM group, the PVN of 2K1C hypertensive rats decreased p-AMPK and p-Nrf2 expression, increased Fra-Like, NAD(P)H oxidase (NOX)2, NOX4, tumor necrosis factor-α and interleukin (IL)-1ß expression, elevated ROS levels, decreased superoxide dismutase 1 and IL-10 expression, and elevated plasma norepinephrine levels. Bilateral PVN microinjection of AICAR significantly ameliorated these changes. CONCLUSION: These findings suggest that repeated injection of AICAR in the PVN suppresses ROS and inflammatory cytokine production through the AMPK/Nrf2 pathway, reducing sympathetic nerve activity and improving hypertension.
Assuntos
Proteínas Quinases Ativadas por AMP , Aminoimidazol Carboxamida , Hipertensão , Fator 2 Relacionado a NF-E2 , Núcleo Hipotalâmico Paraventricular , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio , Ribonucleotídeos , Transdução de Sinais , Animais , Núcleo Hipotalâmico Paraventricular/efeitos dos fármacos , Núcleo Hipotalâmico Paraventricular/metabolismo , Masculino , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Aminoimidazol Carboxamida/administração & dosagem , Ribonucleotídeos/farmacologia , Ribonucleotídeos/administração & dosagem , Proteínas Quinases Ativadas por AMP/metabolismo , Hipertensão/tratamento farmacológico , Hipertensão/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Sistema Nervoso Simpático/efeitos dos fármacos , Sistema Nervoso Simpático/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Citocinas/metabolismoRESUMO
Kimchi is a traditional fermented food that contains abundant nutrients and functional ingredients with various health benefits. We previously reported that kimchi active components suppress hepatic steatosis caused by endoplasmic reticulum (ER) stress in vitro and in vivo. Therefore, we assessed the effect of kimchi on the inhibition of hepatic steatosis caused by ER stress in HepG2 cells and C57BL/6N mice to verify the hypothesis that kimchi may potentially inhibit nonalcoholic fatty liver disease. We investigated the effect of kimchi on cell viability and triglyceride concentrations in cells and on lipid profile, lipid accumulation, and expression of related genes in cells and mice with hepatic steatosis. A mechanistic study was also performed using the liver X receptor α agonist T0901317 and the AMP-activated protein kinase agonist AICAR. Kimchi was noncytotoxic and effectively reduced triglyceride concentrations and suppressed hepatic steatosis-related gene expression in cells and mice. Additionally, kimchi recovered weight loss, lowered the serum and liver tissue lipid profiles, suppressed lipid accumulation, and reduced the effects of T0901317 and AICAR on lipogenic gene expression in tunicamycin-treated mice. Our results highlight that kimchi could prevent hepatic steatosis caused by ER stress in cells and mice.
Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Benzenossulfonamidas , Estresse do Retículo Endoplasmático , Alimentos Fermentados , Fluorocarbonos , Fígado , Camundongos Endogâmicos C57BL , Triglicerídeos , Animais , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Células Hep G2 , Triglicerídeos/sangue , Triglicerídeos/metabolismo , Masculino , Fígado/metabolismo , Fígado/efeitos dos fármacos , Camundongos , Aminoimidazol Carboxamida/farmacologia , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Hepatopatia Gordurosa não Alcoólica/etiologia , Sulfonamidas/farmacologia , Ribonucleotídeos/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Receptores X do Fígado/metabolismo , Tunicamicina/farmacologia , Lipogênese/efeitos dos fármacos , Fígado Gorduroso/tratamento farmacológico , Fígado Gorduroso/prevenção & controleRESUMO
We assessed the effects of two levels of calorie restriction (CR; eating either 15% or 35% less than ad libitum, AL, food intake for 8 weeks) by 24-month-old female and male rats on glucose uptake (GU) and phosphorylation of key signaling proteins (Akt; AMP-activated protein kinase, AMPK; Akt substrate of 160 kDa, AS160) measured in isolated skeletal muscles that underwent four incubation conditions (without either insulin or AICAR, an AMPK activator; with AICAR alone; with insulin alone; or with insulin and AICAR). Regardless of sex: (1) neither CR group versus the AL group had greater GU by insulin-stimulated muscles; (2) phosphorylation of Akt in insulin-stimulated muscles was increased in 35% CR versus AL rats; (3) prior AICAR treatment of muscle resulted in greater GU by insulin-stimulated muscles, regardless of diet; and (4) AICAR caused elevated phosphorylation of acetyl CoA carboxylase, an indicator of AMPK activation, in all diet groups. There was a sexually dimorphic diet effect on AS160 phosphorylation, with 35% CR exceeding AL for insulin-stimulated muscles in male rats, but not in female rats. Our working hypothesis is that the lack of a CR-effect on GU by insulin-stimulated muscles was related to the extended duration of the ex vivo incubation period (290 min compared to 40-50 min that was previously reported to be effective). The observed efficacy of prior treatment of muscles with AICAR to improve glucose uptake in insulin-stimulated muscles supports the strategy of targeting AMPK with the goal of improving insulin sensitivity in older females and males.
Assuntos
Proteínas Quinases Ativadas por AMP , Aminoimidazol Carboxamida , Restrição Calórica , Glucose , Insulina , Músculo Esquelético , Proteínas , Proteínas Proto-Oncogênicas c-akt , Ribonucleotídeos , Transdução de Sinais , Animais , Feminino , Masculino , Ratos , Acetil-CoA Carboxilase/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Glucose/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Hipoglicemiantes/farmacologia , Músculo Esquelético/metabolismo , Músculo Esquelético/efeitos dos fármacos , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ribonucleotídeos/farmacologia , Fatores Sexuais , Transdução de Sinais/efeitos dos fármacos , Fosforribosilaminoimidazolcarboxamida Formiltransferase/metabolismoRESUMO
Electroneutral NaCl transport by Na+/H+ exchanger 3 (NHE3, SLC9A3) is the major Na+ absorptive mechanism in the intestine and decreased NHE3 activity contributes to diarrhea. Patients with diabetes often experience gastrointestinal adverse effects and medications are often a culprit for chronic diarrhea in type 2 diabetes (T2D). We have shown previously that metformin, the most widely prescribed drug for the treatment of T2D, induces diarrhea by inhibition of Na+/H+ exchanger 3 (NHE3) in rodent models of T2D. Metformin was shown to activate AMP-activated protein kinase (AMPK), but AMPK-independent glycemic effects of metformin are also known. The current study is undertaken to determine whether metformin inhibits NHE3 by activation of AMPK and the mechanism by which NHE3 is inhibited by AMPK. Inhibition of NHE3 by metformin was abolished by knockdown of AMPK-α1 or AMPK-α2. AMPK activation by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) phosphorylated NHE3 at S555. S555 is the primary site of phosphorylation by protein kinase A (PKA), but AMPK phosphorylated S555 independently of PKA. Using Mass spectrometry, we found S563 as a newly recognized phosphorylation site in NHE3. Altering either S555 or S563 to Ala was sufficient to block the inhibition of NHE3 activity by AMPK. NHE3 inhibition is dependent on ubiquitination by the E3 ubiquitin ligase Nedd4-2 and metformin was shown to induce NHE3 internalization via Nedd4-2-mediated ubiquitination. AICAR did not increase NHE3 ubiquitination when S555 or S563 was mutated. We conclude that AMPK activation inhibits NHE3 activity and NHE3 inhibition is associated with phosphorylation of NHE3 at S555 and S563.NEW & NOTEWORTHY We show that AMP-activated protein kinase (AMPK) phosphorylates NHE3 at S555 and S563 to inhibit NHE3 activity in intestinal epithelial cells. Phosphorylation of NHE3 by AMPK is necessary for ubiquitination of NHE3.
Assuntos
Diabetes Mellitus Tipo 2 , Metformina , Humanos , Proteínas Quinases Ativadas por AMP/metabolismo , Trocador 3 de Sódio-Hidrogênio/metabolismo , Fosforilação , Diabetes Mellitus Tipo 2/tratamento farmacológico , Metformina/farmacologia , Intestinos , Diarreia , Aminoimidazol Carboxamida/farmacologiaRESUMO
Poly (ADP-ribose) polymerase-1 (PARP-1) inhibitors have been successfully applied in the clinical treatment of various cancer. Side effects and drug resistant cases were reported, and more effective PARP-1 inhibitors were required. However, studies on the AD site of PARP-1 inhibitors are currently incomplete. Therefore, to synthesize more potential candidate PARP-1 inhibitors and disclose some AD site SAR of the PARP-1 inhibitors, herein, a series of 2-phenyl-benzimidazole-4-carboxamide derivatives using different saturated nitrogen-contained heterocycles as linker group (6a-6t) have been designed, synthesized, and evaluated PARP-1 inhibitory activity and proliferation inhibitory against BRCA-1 mutant MDA-MB-436 cell line in vitro. The results showed 6b (IC50 = 8.65 nM) exhibited the most PARP-1 enzyme inhibitory activity comparable with Veliparib (IC50 = 15.54 nM) and Olaparib (IC50 = 2.77 nM); 6m exhibited the strongest MDA-MB-436 cell anti-proliferation activity (IC50 = 25.36 ± 6.06 µM) comparable with Olaparib (IC50 = 23.89 ± 3.81 µM). The compounds 6b, 6r, and 6m could be potential candidates for effective PARP-1 inhibitors and valuable for further optimization. The analysis of activity data also showed that the holistically anti-proliferation activity of the 1,4-diazepane group was about~twofold than that of the piperazine group. Meanwhile, the terminal 3-methyl-furanyl group exhibited the most robust PARP-1 inhibitory and anti-proliferation activity. It is hoped that the results could benefitable for further optimization of PARP-1 inhibitors. Furthermore, we note that some compounds (6d,6g,6n,6p,6s) showed poor PARP-1 inhibitory (>500 nM) but relatively good anti-proliferation activity, which indicates the proliferation inhibitory mechanism against MDA-MB-436 cell line was worth investigating in-depth.
Assuntos
Antineoplásicos , Inibidores de Poli(ADP-Ribose) Polimerases , Poli(ADP-Ribose) Polimerase-1 , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Relação Estrutura-Atividade , Aminoimidazol Carboxamida/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de CélulasRESUMO
Myostatin (Mstn) is a major negative regulator of skeletal muscle mass and initiates multiple metabolic changes. The deletion of the Mstn gene in mice leads to reduced mitochondrial functions. However, the underlying regulatory mechanisms remain unclear. In this study, we used CRISPR/Cas9 to generate myostatin-knockout (Mstn-KO) mice via pronuclear microinjection. Mstn-KO mice exhibited significantly larger skeletal muscles. Meanwhile, Mstn knockout regulated the organ weights of mice. Moreover, we found that Mstn knockout reduced the basal metabolic rate, muscle adenosine triphosphate (ATP) synthesis, activities of mitochondrial respiration chain complexes, tricarboxylic acid cycle (TCA) cycle, and thermogenesis. Mechanistically, expressions of silent information regulator 1 (SIRT1) and phosphorylated adenosine monophosphate-activated protein kinase (pAMPK) were down-regulated, while peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) acetylation modification increased in the Mstn-KO mice. Skeletal muscle cells from Mstn-KO and WT were treated with AMPK activator 5-aminoimidazole-4-carboxamide riboside (AICAR), and the AMPK inhibitor Compound C, respectively. Compared with the wild-type (WT) group, Compound C treatment further down-regulated the expression or activity of pAMPK, SIRT1, citrate synthase (CS), isocitrate dehydrogenase (ICDHm), and α-ketoglutarate acid dehydrogenase (α-KGDH) in Mstn-KO mice, while Mstn knockout inhibited the AICAR activation effect. Therefore, Mstn knockout affects mitochondrial function by inhibiting the AMPK/SIRT1/PGC1α signaling pathway. The present study reveals a new mechanism for Mstn knockout in regulating energy homeostasis.
Assuntos
Proteínas Quinases Ativadas por AMP , Miostatina , Animais , Camundongos , Aminoimidazol Carboxamida/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/metabolismo , Músculo Esquelético/metabolismo , Miostatina/genética , Miostatina/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismoRESUMO
The incidence of diastolic dysfunction increases with age in both humans and mice. This is characterized by increased passive stiffness and slower relaxation of the left ventricle. The stiffness arises at least partially from progressively increased interstitial collagen deposition because of highly secretory fibroblasts. In the past, we demonstrated that AMPK activation via the drug 5-aminoimidazole-4-carboxamide riboside (AICAR) in middle-aged mice reduced adverse remodeling after myocardial infarction. Therefore, as an attempt to normalize the fibroblast phenotype, we used 21-mo-old male and female mice and treated them with AICAR (0.166 mg/g body wt) where each mouse was followed in a functional study over a 3-mo period. We found sex-related differences in extracellular matrix (ECM) composition as well as heart function indices at baseline, which were further accentuated by AICAR treatment. AICAR attenuated the age-related increase in left atrial volume (LAV, an indicator of diastolic dysfunction) in female but not in male hearts, which was associated with reduced collagen deposition in the old female heart, and reduced the transcription factor Gli1 expression in cardiac fibroblasts. We further demonstrated that collagen synthesis was dependent on Gli1, which is a target of AMPK-mediated degradation. By contrast, AICAR had a minor impact on cardiac fibroblasts in the old male heart because of blunted AMPK phosphorylation. Hence, it did not significantly improve old male heart function indices. In conclusion, we demonstrated that male and female hearts are phenotypically different, and sex-specific differences need to be considered when analyzing the response to pharmacological intervention.NEW & NOTEWORTHY The aging heart develops diastolic dysfunction because of increased collagen deposition. We attempted to reduce collagen expression in the old heart by activating AMPK using AICAR. An improvement of diastolic function and reduction of cardiac fibrosis was found only in the female heart and correlated with decreased procollagen expression and increased degradation of the transcription factor Gli1. Male hearts display blunted AICAR-dependent AMPK activation and therefore this treatment had no benefits for the male mice.
Assuntos
Proteínas Quinases Ativadas por AMP , Cardiomiopatias , Proteínas Quinases Ativadas por AMP/metabolismo , Envelhecimento/metabolismo , Aminoimidazol Carboxamida/farmacologia , Animais , Colágeno/metabolismo , Feminino , Fibrose , Masculino , Camundongos , Fenótipo , Proteína GLI1 em Dedos de Zinco/genéticaRESUMO
OBJECTIVES: AICAR, an adenosine analog, has been shown to exhibit vascular protective effects through activation of AMP-activated protein kinase (AMPK). However, it remains unclear as to whether adenosine kinase-mediated ZMP formation or adenosine receptor activation contributes to AICAR-mediated AMPK activation and/or vasorelaxant response in vascular smooth muscle. METHODS AND RESULTS: In the present study using endothelium-denuded rat aortic ring preparations, isometric tension measurements revealed that exposure to 1 mM AICAR for 30 min resulted in inhibition of phenylephrine (1 µM)-induced smooth muscle contractility by â¼35%. Importantly, this vasorelaxant response by AICAR was prevented after pretreatment of aortic rings with an AMPK inhibitor (compound C, 40 µM) and adenosine kinase inhibitor (5-iodotubercidin, 1 µM), but not with an adenosine receptor blocker (8-sulfophenyltheophylline, 100 µM). Immunoblot analysis of respective aortic tissues showed that AMPK activation seen during vasorelaxant response by AICAR was abolished by compound C and 5-iodotubercidin, but not by 8-sulfophenyltheophylline, suggesting ZMP involvement in AMPK activation. Furthermore, LC-MS/MS MRM analysis revealed that exposure of aortic smooth muscle cells to 1 mM AICAR for 30 min enhanced ZMP level to 2014.9 ± 179.4 picomoles/mg protein (vs. control value of 8.5 ± 0.6; p<0.01), which was accompanied by a significant decrease in ATP/ADP ratio (1.08 ± 0.02 vs. 2.08 ± 0.06; p<0.01). CONCLUSIONS: Together, the present findings demonstrate that AICAR-mediated ZMP elevation and the resultant AMPK activation in vascular smooth muscle contribute to vasorelaxation.
Assuntos
Proteínas Quinases Ativadas por AMP , Vasodilatação , Ratos , Animais , Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Quinases Ativadas por AMP/farmacologia , Adenosina Quinase/farmacologia , Cromatografia Líquida , Espectrometria de Massas em Tandem , Aminoimidazol Carboxamida/farmacologia , Ribonucleotídeos/farmacologia , Endotélio/metabolismo , Vasodilatadores/farmacologia , Músculo Liso/metabolismo , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologiaRESUMO
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key glycolytic enzyme that participates in various cellular events, such as DNA repair and apoptosis. The functional diversity of GAPDH depends on its intracellular localization. Because AMP-activated protein kinase (AMPK) regulates the nuclear translocation of GAPDH in young cells and AMPK activity significantly increases during aging, we investigated whether altered AMPK activity is involved in the nuclear localization of GAPDH in senescent cells. Age-dependent nuclear translocation of GAPDH was confirmed by confocal laser scanning microscopy in human diploid fibroblasts (HDFs) and by immunohistochemical analysis in aged rat skin cells. Senescence-induced nuclear localization was reversed by lysophosphatidic acid but not by platelet-derived growth factor. The extracellular matrix from young cells also induced the nuclear export of GAPDH in senescent HDFs. An activator of AMPK, 5-Aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR), increased the level of nuclear GAPDH, whereas an inhibitor of AMPK, Compound C, decreased the level of nuclear GAPDH in senescent HDFs. Transfection with AMPKα siRNA prevented nuclear translocation of GAPDH in senescent HDFs. The stimulatory effect of AICAR and serum depletion on GAPDH nuclear translocation was reduced in AMPKα1/α2-knockout mouse embryonic fibroblasts. Overall, increased AMPK activity may play a role in the senescence-associated nuclear translocation of GAPDH.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Senescência Celular/fisiologia , Fibroblastos/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Matriz Extracelular , Regulação da Expressão Gênica/efeitos dos fármacos , Gliceraldeído-3-Fosfato Desidrogenases/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Lisofosfolipídeos/farmacologia , Ratos , Ribonucleotídeos/farmacologiaRESUMO
Fludioxonil and iprodione are effective fungicides widely used for crop protection and are essential for controlling plant pathogenic fungi. The emergence of fungicide-resistant strains of targeted pathogens is regularly monitored, and several cases have been reported. Non-targeted fungi may also be exposed to the fungicide residues in agricultural fields. However, there are no comprehensive reports on fungicide-resistant strains of non-targeted fungi. Here, we surveyed 99 strains, representing 12 Penicillium species, that were isolated from a variety of environments, including foods, dead bodies, and clinical samples. Among the Penicillium strains, including non-pathogenic P. chrysogenum and P. camembertii, as well as postharvest pathogens P. expansum and P. digitatum, 14 and 20 showed resistance to fludioxonil and iprodione, respectively, and 6 showed multi-drug resistance to the fungicides. Sequence analyses revealed that some strains of P. chrysogenum and Penicillium oxalicum had mutations in NikA, a group III histidine kinase of the high-osmolarity glycerol pathway, which is the mode of action for fludioxonil and iprodione. The single nucleotide polymorphisms of G693D and T1318P in P. chrysogenum and T960S in P. oxalicum were only present in the fludioxonil- or iprodione-resistant strains. These strains also exhibited resistance to pyrrolnitrin, which is the lead compound in fludioxonil and is naturally produced by some Pseudomonas species. This study demonstrated that non-targeted Penicillium strains distributed throughout the environment possess fungicide resistance.