RESUMO
Endometrial receptivity is essential for successful embryo implantation and pregnancy initiation and is regulated via various signaling pathways. Adiponectin, an important adipokine, may be a potential regulator of reproductive system functions. The aim of the present study was to elucidate the regulatory role of adiponectin receptor 1 (ADIPOR1) in endometrial receptivity. The endometrial receptivity between RL952 and AN3CA cell lines was confirmed using an in vitro JAr spheroid attachment model. 293T cells were transfected with control or short hairpin (sh)ADIPOR1 vectors and RL952 cells were transduced with lentiviral particles targeting ADIPOR1. Reverse transcriptionquantitative PCR and immunoblot assays were also performed. ADIPOR1 was consistently upregulated in the endometrium during the midsecretory phase compared with that in the proliferative phase and in receptive RL952 cells compared with that in nonreceptive AN3CA cells. Stable cell lines with diminished ADIPOR1 expression caused by shRNA showed reduced Ecadherin expression and attenuated in vitro endometrial receptivity. ADIPOR1 regulated AMPactivated protein kinase (AMPK) activity in endometrial epithelial cells. Regulation of AMPK activity via dorsomorphin and 5aminoimidazole4carboxamide ribonucleotide affected Ecadherin expression and in vitro endometrial receptivity. The ADIPOR1/AMPK/Ecadherin axis is vital to endometrial receptivity. These findings can help improve fertility treatments and outcomes.
Assuntos
Proteínas Quinases Ativadas por AMP , Caderinas , Endométrio , Receptores de Adiponectina , Transdução de Sinais , Receptores de Adiponectina/metabolismo , Receptores de Adiponectina/genética , Humanos , Feminino , Endométrio/metabolismo , Caderinas/metabolismo , Caderinas/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Linhagem Celular , Implantação do Embrião , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/genética , Adulto , Aminoimidazol Carboxamida/análogos & derivados , RibonucleotídeosRESUMO
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
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
Bronchial thermoplasty (BT), an effective treatment for severe asthma, requires heat to reach the airway to reduce the mass of airway smooth muscle cells (ASMCs). Autophagy is involved in the pathological process of airway remodeling in patients with asthma. However, it remains unclear whether autophagy participates in controlling airway remodeling induced by BT. In this study, we aim to elucidate the autophagy-mediated molecular mechanisms in BT. Our study reveal that the number of autophagosomes and the level of alpha-smooth muscle actin (α-SMA) fluorescence are significantly decreased in airway biopsy tissues after BT. As the temperature increased, BT causes a decrease in cell proliferation and a concomitant increase in the apoptosis of human airway smooth muscle cells (HASMCs). Furthermore, increase in temperature significantly downregulates cellular autophagy, autophagosome accumulation, the LC3II/LC3I ratio, and Beclin-1 expression, upregulates p62 expression, and inhibits the AMPK/mTOR pathway. Furthermore, cotreatment with AICAR (an AMPK agonist) or RAPA (an mTOR antagonist) abolishes the inhibition of autophagy and attenuates the increase in the apoptosis rate of HASMCs induced by the thermal effect. Therefore, we conclude that BT decreases airway remodeling by blocking autophagy induced by the AMPK/mTOR signaling pathway in HASMCs.
Assuntos
Proteínas Quinases Ativadas por AMP , Remodelação das Vias Aéreas , Apoptose , Autofagia , Termoplastia Brônquica , Miócitos de Músculo Liso , Transdução de Sinais , Serina-Treonina Quinases TOR , Serina-Treonina Quinases TOR/metabolismo , Humanos , Autofagia/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/metabolismo , Termoplastia Brônquica/métodos , Miócitos de Músculo Liso/metabolismo , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Asma/metabolismo , Asma/patologia , Masculino , Células Cultivadas , Brônquios/metabolismo , Brônquios/patologia , Aminoimidazol Carboxamida/análogos & derivados , RibonucleotídeosRESUMO
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: 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
Global cerebral ischemia (GCI) caused by clinical conditions such as cardiac arrest leads to delayed neuronal death in the hippocampus, resulting in physical and mental disability. However, the mechanism of delayed neuronal death following GCI remains unclear. To elucidate the mechanism, we performed a metabolome analysis using a mouse model in which hypothermia (HT) during GCI, which was induced by the transient occlusion of the bilateral common carotid arteries, markedly suppressed the development of delayed neuronal death in the hippocampus after reperfusion. Fifteen metabolites whose levels were significantly changed by GCI and 12 metabolites whose levels were significantly changed by HT were identified. Furthermore, the metabolites common for both changes were narrowed down to two, adenosine monophosphate (AMP) and xanthosine monophosphate (XMP). The levels of both AMP and XMP were found to be decreased by GCI, but increased by HT, thereby preventing their decrease. In contrast, the levels of adenosine, inosine, hypoxanthine, xanthine, and guanosine, the downstream metabolites of AMP and XMP, were increased by GCI, but were not affected by HT. Our results may provide a clue to understanding the mechanism by which HT during GCI suppresses the development of delayed neuronal death in the hippocampus.
Assuntos
Isquemia Encefálica , Hipotermia , Ribonucleotídeos , Humanos , Hipotermia/metabolismo , Isquemia Encefálica/metabolismo , Xantina/metabolismo , Infarto Cerebral/metabolismo , Hipocampo/metabolismo , Monofosfato de Adenosina/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
The savory or umami taste of the amino acid glutamate is synergistically enhanced by the addition of the purines inosine 5'-monophosphate (IMP) and guanosine 5'-monophosphate (GMP) disodium salt. We hypothesized that the addition of purinergic ribonucleotides, along with the pyrimidine ribonucleotides, would decrease the absolute detection threshold of (increase sensitivity to) l-glutamic acid potassium salt (MPG). To test this, we measured both the absolute detection threshold of MPG alone and with a background level (3 mM) of 5 different 5'-ribonucleotides. The addition of the 3 purines IMP, GMP, and adenosine 5'-monophosphate (AMP) lowered the MPG threshold in all participants (P < 0.001), indicating they are positive modulators or enhancers of glutamate taste. The average detection threshold of MPG was 2.08 mM, and with the addition of IMP, the threshold was decreased by approximately 1.5 orders of magnitude to 0.046 mM. In contrast to the purines, the pyrimidines uridine 5'-monophosphate (UMP) and cytidine 5'-monophosphate (CMP) yielded different results. CMP reliably raised glutamate thresholds in 10 of 17 subjects, suggesting it is a negative modulator or diminisher of glutamate taste for them. The rank order of effects on increasing sensitivity to glutamate was IMP > GMP> AMP >> UMP// CMP. These data confirm that ribonucleotides are modulators of glutamate taste, with purines enhancing sensitivity and pyrimidines displaying variable and even negative modulatory effects. Our ability to detect the co-occurrence of glutamate and purines is meaningful as both are relatively high in evolutionarily important sources of nutrition, such as insects and fermented foods.
Assuntos
Ácido Glutâmico , Ribonucleotídeos , Humanos , Ribonucleotídeos/farmacologia , Paladar , Guanosina Monofosfato/metabolismo , Uridina Monofosfato , Purinas , Inosina Monofosfato/metabolismo , Glutamato de SódioRESUMO
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
Under enzyme catalysis, adenosine triphosphate (ATP) transfers a phosphoryl group to canonical ribonucleotide diphosphates (NDPs) to form ribonucleotide triphosphates (NTPs), the direct biosynthetic precursors to RNA. However, it remains unclear whether the phosphorylation of NDPs could have occurred in water before enzymes existed and why an adenosine derivative, rather than another canonical NTP, typically performs this function. Here, we show that adenosine diphosphate (ADP) in the presence of Fe3+ or Al3+ promotes phosphoryl transfer from acetyl phosphate to all canonical NDPs to produce their corresponding NTP in water at room temperature and in the absence of enzymes. No other NDPs were found to promote phosphorylation, giving insight into why adenosine derivatives specifically became used for this purpose in biology. The metal-ADP complexes also promote phosphoryl transfer to ribonucleoside monophosphates (NMPs) to form a mixture of the corresponding NDPs and NTPs, albeit less efficiently. This work represents a rare example in which a single nucleotide carries out a function critical to biology without enzymes. ADP-metal complexes may have played an important role in nucleotide phosphorylation in prebiotic chemistry.
Assuntos
Complexos de Coordenação , Ribonucleotídeos , Fosforilação , Trifosfato de Adenosina/metabolismo , Difosfato de Adenosina/metabolismo , Adenosina , ÁguaRESUMO
Tumor cells generally require large amounts of nucleotides, and thus activate de novo purine synthesis (dnPS). In the dnPS reactions, 10-formyltetrahydorofolate (10-fTHF) supplied by one-carbon metabolism is utilized as a formyl group donor. We focused on aldehyde dehydrogenase 1 family member L1 (ALDH1L1), which metabolizes 10-fTHF to tetrahydrofolate and whose expression is often attenuated in hepatocellular carcinoma (HCC). We generated ALDH1L1-expressing HuH-7 cells to perform metabolome analysis and found that intracellular levels of serine were reduced and glycine was increased. In addition, 5-aminoimidazole-4-carboxamide ribonucleotide (ZMP), a dnPS intermediate, accumulated due to the consumption of 10-fTHF by ALDH1L1, which inhibited ZMP formylation. Importantly, ALDH1L1-expressing cells showed reduced ZMP sensitivity and higher mitochondrial activity. The suppression of mitochondrial serine catabolism by ALDH1L1 expression was speculated to be closely related to this phenotype. Gene set enrichment analysis utilizing The Cancer Genome Atlas data revealed that genes related to oxidative phosphorylation were enriched in HCC patients with high ALDH1L1 expression. Moreover, drug sensitivity data analysis demonstrated that HCC cell lines with low expression of ALDH1L1 were sensitive to ZMP and cordycepin, a structural analog of ZMP and AMP. Our study revealed that ZMP and AMP analogs might be effective in the pharmacotherapy of HCC patients with low expression of ALDH1L1.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/genética , Ribonucleotídeos/farmacologia , CarbonoRESUMO
Upper tract urothelial carcinoma (UTUC), including renal, pelvic, and ureteral carcinoma, has a high incidence rate in Taiwan, which is different from that in Western countries. Therefore, it is imperative to elucidate the mechanisms underlying UTUC growth and metastasis. To explore the function of miR-145-5p in UTUC, we transfected the BFTC909 cell line with miR-145-5p mimics and analyzed the differences in protein levels by performing two-dimensional polyacrylamide gel electrophoresis. Real-time polymerase chain reaction and Western blot analysis were used to analyze 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inositol monophosphate cyclohydrolase (ATIC) messenger RNA and protein levels. A dual-luciferase assay was performed to identify the target of miR-145-5p in ATIC. The effects of miR-145-5p and ATIC expression by cell transfection on cell proliferation, migration, and invasion were also assessed. miR-145-5p downregulated ATIC protein expression. High ATIC expression is associated with tumor stage, metastasis, recurrence, and a poor prognosis in patients with UTUC. Cell function assays revealed that ATIC knockdown inhibited the proliferation, migration, and invasive abilities of UTUC cells. In contrast, miR-145-5p affected the proliferation, migration, and invasive abilities of UTUC cells by directly targeting the 3'-untranslated regions of ATIC. Furthermore, we used RNA sequencing and Ingenuity Pathway Analysis to identify possible downstream genes regulated by ATIC and found that miR-145-5p regulated the protein levels of fibronectin 1, Slug, cyclin A2, cyclin B1, P57, and interferon-induced transmembrane 1 via ATIC. ATIC may be a valuable predictor of prognosis and a potential therapeutic target for UTUC.
Assuntos
Carcinoma de Células de Transição , Hidroximetil e Formil Transferases , MicroRNAs , Neoplasias da Bexiga Urinária , Humanos , MicroRNAs/genética , Carcinoma de Células de Transição/genética , Linhagem Celular Tumoral , Neoplasias da Bexiga Urinária/genética , Hidroximetil e Formil Transferases/genética , Proliferação de Células/genética , Ribonucleotídeos , Movimento Celular/genética , Regulação Neoplásica da Expressão GênicaRESUMO
Ribonucleotides, which widely exist in all living organisms and are essential to both physiological and pathological processes, can naturally appear as ribonucleoside mono-, di-, and triphosphates. Natural ribonucleotides can also dynamically switch between different phosphorylated forms, posing a great challenge for sensing. A specially engineered nanopore sensor is promising for full discrimination of all canonical ribonucleoside mono-, di-, and triphosphates. However, such a demonstration has never been reported, due to the lack of a suitable nanopore sensor that has a sufficient resolution. In this work, we utilized a phenylboronic acid (PBA) modified Mycobacterium smegmatis porin A (MspA) hetero-octamer for ribonucleotide sensing. Twelve types of ribonucleotides, including mono-, di-, and triphosphates of cytidine (CMP, CDP, CTP), uridine (UMP, UDP, UTP), adenosine (AMP, ADP, ATP), and guanosine (GMP, GDP, GTP) were simultaneously discriminated. A machine-learning algorithm was also developed, which achieved a general accuracy of 99.9% for ribonucleotide sensing. This strategy was also further applied to identify ribonucleotide components in ATP tablets and injections. This sensing strategy provides a direct, accurate, easy, and rapid solution to characterize ribonucleotide components in different phosphorylated forms.
Assuntos
Nanoporos , Ribonucleosídeos , Ribonucleotídeos , Trifosfato de AdenosinaRESUMO
AICAr (5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside, commonly referred to as AICAR) is an adenosine monophosphate-activated protein kinase agonist previously investigated for its therapeutic potential which has been shown to improve exercise performance in laboratory animals. For this reason, the World Anti-Doping Agency prohibits the use of AICAr in sports. AICAr can easily be detected by means of liquid chromatography-mass spectrometry, but being an endogenous metabolite, it cannot be discriminated from AICAr of a non-natural origin. Population-based concentration thresholds have been suggested as a means to identify suspicious samples that would require further analysis by carbon isotope ratio mass spectrometry (CIR); however, it remains at the discretion of the laboratory how to apply them. Here, the urinary ratio of AICAr to SAICA-riboside (SAICAr) that is a closely related purine metabolite was investigated. In an athlete population of 5517 samples, this ratio was relatively narrowly distributed with median values and 99th percentiles of 3.3 and 9.3, and 4.2 and 14 in male and female athletes, respectively. Analysis of urine samples obtained from an AICAr administration study demonstrated that the AICAr/SAICAr ratio can serve in addition to AICAr concentration as a valuable diagnostic trigger for follow-up analysis by CIR. Conceivably, this combination can offer better retrospectivity than AICAr concentration alone by allowing to decrease the AICAr concentration threshold without significantly increasing the number of suspicious samples.
Assuntos
Aminoimidazol Carboxamida , Ribonucleotídeos , Animais , Masculino , Feminino , Ribonucleotídeos/análise , Cromatografia LíquidaRESUMO
BACKGROUND: Postoperative abdominal adhesion is one of most common complications after abdominal operations. 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR) is an adenosine 5'-monophosphate activated protein kinase (AMPK) pathway agonist that inhibits inflammation, reduces cell fibrosis and cellular reactive oxygen species (ROS) injury, promotes autophagy and mitochondrial function. This study aimed to explore the mechanism of AICAR in inhibiting adhesion formation. MATERIALS AND METHODS: Forty rats were randomly divided into five groups. All of the rats except the sham group received cecal abrasion to establish an adhesion model. The rats in the sodium hyaluronate group were treated with 2 mL sodium hyaluronate before closing the peritoneal cavity. The AICAR 1 and 2 groups were treated with 100 mg/kg and 200 mg/kg AICAR, respectively. Seven days after the operation, all of the rats were euthanized, and the adhesion condition was evaluated by Nair's system. Inflammation was assessed by Eosin-hematoxylin (HE) staining and transforming growth factor-ß (TGF-ß1) detection. Oxidative stress effect was determined by ROS, nitric oxide (NO) level, superoxide dismutase (SOD), catalase, glutathione peroxidase (Gpx) and malondialdehyde (MDA) levels in adhesion tissue. Then, Sirius red picric acid staining was used to detect the fiber thickness. Immunohistochemical staining of cytokeratin-19 (CK-19), alpha-smooth muscle actin (α-SMA) and nuclear factor erythroid 2-related factor 2 (Nrf2) was also performed. Finally, HMrSV5 cells were treated with TGF-ß1 and AICAR, the mRNA expression of E-cadherin, α-SMA and vimentin was assessed by q-PCR and cellular immunofluorescent staining. RESULTS: The rats in the AICAR-treated group had fewer adhesion formation incidences and a reduced Nair's score. The inflammation was determined by HE staining and TGF-ß1 concentration. The ROS, SOD, Catalase, Gpx, MDA levels and fiber thickness were decreased by AICAR treatments compared to the control. However, the NO production, Nrf2 levels and peritoneal mesothelial cell integrity were promoted after AICAR treatments. In vitro work, AICAR treatments reduced E-cadherin, α-SMA and vimentin mRNA level compared to that in the TGF-ß1 group. CONCLUSION: AICAR can inhibit postoperative adhesion formation by reducing inflammation, decreasing oxidative stress response and promoting peritoneal mesothelial cell repair.
Assuntos
Ribonucleosídeos , Fator de Crescimento Transformador beta1 , Aminoimidazol Carboxamida/análogos & derivados , Animais , Caderinas/metabolismo , Catalase/metabolismo , Ácido Hialurônico , Inflamação , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , RNA Mensageiro/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Ribonucleosídeos/metabolismo , Ribonucleotídeos , Superóxido Dismutase/metabolismo , Aderências Teciduais/tratamento farmacológico , Aderências Teciduais/prevenção & controle , Fator de Crescimento Transformador beta1/metabolismo , Vimentina/metabolismoRESUMO
Human PAICS is a bifunctional enzyme that is involved in the de novo purine biosynthesis, catalyzing the conversion of aminoimidazole ribonucleotide (AIR) into N-succinylcarboxamide-5-aminoimidazole ribonucleotide (SAICAR). It comprises two distinct active sites, AIR carboxylase (AIRc) where the AIR is initially converted to carboxyaminoimidazole ribonucleotide (CAIR) by reaction with CO2 and SAICAR synthetase (SAICARs) in which CAIR then reacts with an aspartate to form SAICAR, in an ATP-dependent reaction. Human PAICS is a promising target for the treatment of various types of cancer, and it is therefore of high interest to develop a detailed understanding of its reaction mechanism. In the present work, density functional theory calculations are employed to investigate the PAICS reaction mechanism. Starting from the available crystal structures, two large models of the AIRc and SAICARs active sites are built and different mechanistic proposals for the carboxylation and phosphorylation-condensation mechanisms are examined. For the carboxylation reaction, it is demonstrated that it takes place in a two-step mechanism, involving a C-C bond formation followed by a deprotonation of the formed tetrahedral intermediate (known as isoCAIR) assisted by an active site histidine residue. For the phosphorylation-condensation reaction, it is shown that the phosphorylation of CAIR takes place before the condensation reaction with the aspartate. It is further demonstrated that the three active site magnesium ions are involved in binding the substrates and stabilizing the transition states and intermediates of the reaction. The calculated barriers are in good agreement with available experimental data.
Assuntos
Ácido Aspártico , Ribonucleotídeos , Domínio Catalítico , Humanos , Ribonucleotídeos/químicaRESUMO
The novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2 or COVID-19) has caused a global pandemic. The SARS-CoV-2 RNA genome is replicated by a conserved "core" replication-transcription complex (RTC) containing an error-prone RNA-dependent RNA polymerase holoenzyme (holo-RdRp, nsp12-nsp7-nsp8) and a RNA proofreading nuclease (nsp14-nsp10). Although structures and functions of SARS-CoV-2 holo-RdRp have been extensively studied and ribonucleotide-analog inhibitors, such as Remdesivir, have been treated for COVID-19 patients, the substrate and nucleotide specificity of SARS-CoV-2 holo-RdRp remain unknown. Here, our biochemical analysis of SARS-CoV-2 holo-RdRp reveals that it has a robust DNA-dependent RNA polymerase activity, in addition to its intrinsic RNA-dependent RNA polymerase activity. Strikingly, SARS-CoV-2 holo-RdRp fully extends RNAs with a low-fidelity even when only ATP and pyrimidine nucleotides, in particular CTP, are provided. This ATP-dependent error-prone ribonucleotide incorporation by SARS-CoV-2 holo-RdRp resists excision by the RNA proofreading nuclease in vitro. Our collective results suggest that a physiological concentration of ATP likely contributes to promoting the error-prone incorporation of ribonucleotides and ribonucleotide-analogs by SARS-CoV-2 holo-RdRp and provide a useful foundation to develop ribonucleotide analogs as an effective therapeutic strategy to combat coronavirus-mediated outbreak.