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1.
Nat Commun ; 10(1): 3668, 2019 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-31413316

RESUMO

Breast cancer stem cells (BCSCs) are unique in their ability to undergo unlimited self-renewal, an essential process in breast cancer recurrence following metastatic dormancy. Emergent metastatic lesions were subjected to microarray analysis, which identified 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (Pfkfb3) as a differentially expressed gene coupled to metastatic recurrence. Here, we report that elevated Pfkfb3 expression correlates with the appearance of aggressive breast cancers and reduces relapse-free survival, as well as enhances BCSC self-renewal and metastatic outgrowth. We observe an inverse relationship between Pfkfb3 expression and autophagy, which reduces Pfkfb3 expression and elicits cellular dormancy. Targeted depletion of Atg3, Atg7, or p62/sequestosome-1 to inactivate autophagy restores aberrant Pfkfb3 expression in dormant BCSCs, leading to their reactivation of proliferative programs and outgrowth. Moreover, Pfkfb3 interacts physically with autophagy machinery, specifically the UBA domain of p62/sequestosome-1. Importantly, disrupting autophagy and this event enables Pfkfb3 to drive dormant BCSCs and metastatic lesions to recur.


Assuntos
Autofagia/genética , Neoplasias da Mama/genética , Células-Tronco Neoplásicas/metabolismo , Fosfofrutoquinase-2/metabolismo , Animais , Proteína 7 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Autorrenovação Celular , Feminino , Humanos , Camundongos , Metástase Neoplásica , Proteína Sequestossoma-1/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo
2.
Int J Mol Sci ; 20(14)2019 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-31323770

RESUMO

The high abundance of mitochondria and the expression of mitochondrial uncoupling protein 1 (UCP1) confer upon brown adipose tissue (BAT) the unique capacity to convert chemical energy into heat at the expense of ATP synthesis. It was long believed that BAT is present only in infants, and so, it was not considered as a potential therapeutic target for metabolic syndrome; however, the discovery of metabolically active BAT in adult humans has re-stimulated interest in the contributions of BAT metabolic regulation and dysfunction to health and disease. Here we demonstrate that brown adipocyte autophagy plays a critical role in the regulation BAT activity and systemic energy metabolism. Mice deficient in brown adipocyte autophagy due to BAT-specific deletion of Atg7-a gene essential for autophagosome generation-maintained higher mitochondrial content due to suppression of mitochondrial clearance and exhibited improved insulin sensitivity and energy metabolism. Autophagy was upregulated in BAT of older mice compared to younger mice, suggesting its involvement in the age-dependent decline of BAT activity and metabolic rate. These findings suggest that brown adipocyte autophagy plays a crucial role in metabolism and that targeting this pathway may be a potential therapeutic strategy for metabolic syndrome.


Assuntos
Adipócitos Marrons/citologia , Adipócitos Marrons/metabolismo , Metabolismo Energético/fisiologia , Mitocôndrias/metabolismo , Envelhecimento/genética , Envelhecimento/fisiologia , Animais , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Camundongos , Camundongos Mutantes , /fisiologia
3.
Clin Interv Aging ; 14: 621-630, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30992660

RESUMO

Background: Disc degeneration (DD) is one of the common diseases worldwide, which deeply influences normal life and leads to excruciating pain. However, an effective treatment for DD is still not identified. Method: The present study systemically examined the effect of melatonin on annulus fibrosus (AF) cells of patients with DD. Results: Melatonin had the effect of promoting proliferation, inducing autophagy, and suppressing apoptosis on AF cells of patients with DD. Moreover, melatonin contributed to the translation and transcription of autophagy-related protein ATG7 and inhibited the function of miR-106a-5p in AF cells. In addition, the results suggested that miR-106a-5p mediated the expression of ATG7 by directly binding to its 3'UTR in AF cells. Conclusion: This research not only gained a deep insight of melatonin mode of action, but also indicated its potential target signaling pathway in AF cells.


Assuntos
Anel Fibroso , Proteína 7 Relacionada à Autofagia/metabolismo , Proliferação de Células , Degeneração do Disco Intervertebral , Melatonina , MicroRNAs/metabolismo , Anel Fibroso/efeitos dos fármacos , Anel Fibroso/metabolismo , Anel Fibroso/patologia , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Células Cultivadas , Humanos , Degeneração do Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/patologia , Melatonina/metabolismo , Melatonina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Resultado do Tratamento
4.
Nat Commun ; 10(1): 1567, 2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30952864

RESUMO

Selective autophagy ensures the removal of specific soluble proteins, protein aggregates, damaged mitochondria, and invasive bacteria from cells. Defective autophagy has been directly linked to metabolic disorders. However how selective autophagy regulates metabolism remains largely uncharacterized. Here we show that a deficiency in selective autophagy is associated with suppression of lipid oxidation. Hepatic loss of Atg7 or Atg5 significantly impairs the production of ketone bodies upon fasting, due to decreased expression of enzymes involved in ß-oxidation following suppression of transactivation by PPARα. Mechanistically, nuclear receptor co-repressor 1 (NCoR1), which interacts with PPARα to suppress its transactivation, binds to the autophagosomal GABARAP family proteins and is degraded by autophagy. Consequently, loss of autophagy causes accumulation of NCoR1, suppressing PPARα activity and resulting in impaired lipid oxidation. These results suggest that autophagy contributes to PPARα activation upon fasting by promoting degradation of NCoR1 and thus regulates ß-oxidation and ketone bodies production.


Assuntos
Autofagia , Metabolismo dos Lipídeos , Correpressor 1 de Receptor Nuclear/metabolismo , Animais , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Proteína 5 Relacionada à Autofagia/fisiologia , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Proteína 7 Relacionada à Autofagia/fisiologia , Jejum , Corpos Cetônicos/metabolismo , Fígado/metabolismo , Camundongos , Correpressor 1 de Receptor Nuclear/fisiologia , Oxirredução , PPAR alfa
5.
Nan Fang Yi Ke Da Xue Xue Bao ; 39(2): 134-143, 2019 02 28.
Artigo em Chinês | MEDLINE | ID: mdl-30890499

RESUMO

OBJECTIVE: To investigate the effect of estradiol (E2)/estrogen receptor 1 (ESR1) on the proliferation of human chondrocytes in vitro and explore the molecular mechanism. METHODS: The Ad-Easy adenovirus packaging system was used to construct and package the ESR1-overexpressing adenovirus Ad-ESR1. Western blotting and qPCR were used to detect the expression of ESR1 protein and mRNA in human chondrocyte C28I2 cells. In the cells treated with different adenoviruses, the effects of E2 were tested on the expressions of proteins related with cell autophagy and apoptosis and the phosphorylation of ERK signaling pathway using Western blotting. Immunofluorescence assay was used to observe the intracellular autophagic flow, flow cytometry was performed to analyze the cell apoptosis rate and the cell cycle changes, and qPCR was used to detect the expressions of PCNA, cyclin B1 and cyclin D1 mRNAs. The inhibitory effect of the specific inhibitor of ERK on the expressions of autophagy- and apoptosis-related genes at both the protein and mRNA levels were detected using Western blotting and qPCR. RESULTS: Transfection with the recombinant adenovirus overexpressing ESR1 and E2 treatment of C28I2 cells significantly enhanced the expressions of autophagy-related proteins LC3, ATG7, promoted the colocalization of LC3 and LAMP1 in the cytoplasm, increased the expressions of the proliferation-related marker genes PCNA, cyclin B1 and cyclin D1, and supressed the expressions of cleaved caspase-3, caspase-12 and pERK. RNA interference of ESR1 obviously lowered the expression levels of autophagy-related proteins in C28I2 cells, causing also suppression of the autophagic flow, increments of the expressions of apoptosis-related proteins and pERK, and down-regulated the expressions of the proliferation marker genes. Blocking ERK activation with the ERK inhibitor obviously inhibited the effects of E2/ESR1 on autophagy, proliferationrelated gene expressions and cell apoptosis. CONCLUSIONS: The targeted binding of E2 with ESR1 promotes the proliferation of human chondrocytes in vitro possibly by inhibiting the activation of ERK signaling pathway to promote cell autophagy and induce cell apoptosis.


Assuntos
Apoptose , Autofagia , Proliferação de Células , Condrócitos/citologia , Estradiol/metabolismo , Receptor alfa de Estrogênio/metabolismo , Sistema de Sinalização das MAP Quinases , Adenoviridae/metabolismo , Proteína 7 Relacionada à Autofagia/metabolismo , Linhagem Celular , Condrócitos/metabolismo , Humanos , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Transfecção
6.
Int J Biol Sci ; 15(3): 507-521, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30745838

RESUMO

Proliferative vitreoretinopathy (PVR) is the most serious fibrous complication that causes vision loss after intraocular surgery, and there is currently no effective treatment in clinical. Autophagy is an important cell biological mechanism in maintaining the homeostasis of tissues and cells, resisting the process of EMT. However, it is still unclear whether autophagy could resist intraocular fibrosis and prevent PVR progression. In this study, we investigated the expression of mesenchymal biomarkers in autophagy deficiency cells and found these proteins were increased. The mesenchymal protein transcription factor Twist can bind to autophagy related protein p62 and promote the degradation of Twist, which reduced the expression of mesenchymal markers. By constructing an EMT model of retinal pigment epithelial (RPE) cells in vitro, we found that autophagy was activated in the EMT process of RPE cells. Moreover, in autophagy deficient RPE cell line via knockdown autophagy related protein 7 (Atg7), the expression of epithelial marker claudin-1 was suppressed and the mesenchymal markers were increased, accompanied by an increase in cell migration and contractility. Importantly, RPE epithelial properties can be maintained by promoting autophagy and effectively reversing TFG-ß2-induced RPE fibrosis. These observations reveal that autophagy may be an effective way to treat PVR.


Assuntos
Transição Epitelial-Mesenquimal/fisiologia , Epitélio Pigmentado da Retina/metabolismo , Animais , Autofagia/fisiologia , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Biomarcadores/metabolismo , Western Blotting , Linhagem Celular , Claudina-1/genética , Claudina-1/metabolismo , Transição Epitelial-Mesenquimal/genética , Fibrose/genética , Fibrose/metabolismo , Homeostase/fisiologia , Imuno-Histoquímica , Imunoprecipitação , Camundongos , Camundongos Knockout
7.
Cells ; 8(2)2019 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-30736337

RESUMO

Autophagy, an intracellular degradation process, is essential for maintaining cell homeostasis by removing damaged organelles and proteins under various conditions of stress. In cancer, autophagy has conflicting functions. It plays a key role in protecting against cancerous transformation by maintaining genomic stability against genotoxic components, leading to cancerous transformation. It can also promote cancer cell survival by supplying minimal amounts of nutrients during cancer progression. However, the molecular mechanisms underlying how autophagy regulates the epithelial-to-mesenchymal transition (EMT) and cancer metastasis are unknown. Here, we show that starvation-induced autophagy promotes Snail (SNAI1) degradation and inhibits EMT and metastasis in cancer cells. Interestingly, SNAI1 proteins were physically associated and colocalized with LC3 and SQSTM1 in cancer cells. We also found a significant decrease in the levels of EMT and metastatic proteins under starvation conditions. Furthermore, ATG7 knockdown inhibited autophagy-induced SNAI1 degradation in the cytoplasm, which was associated with a decrease in SNAI1 nuclear translocation. Moreover, cancer cell invasion and migration were significantly inhibited by starvation-induced autophagy. These findings suggest that autophagy-dependent SNAI1 degradation could specifically regulate EMT and cancer metastasis during tumorigenesis.


Assuntos
Autofagia , Transição Epitelial-Mesenquimal , Proteólise , Fatores de Transcrição da Família Snail/metabolismo , Proteína 7 Relacionada à Autofagia/metabolismo , Movimento Celular , Núcleo Celular/metabolismo , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Biológicos , Invasividade Neoplásica , Metástase Neoplásica , Neoplasias/metabolismo , Neoplasias/patologia , Ligação Proteica , Proteína Sequestossoma-1/metabolismo
8.
Ecotoxicol Environ Saf ; 171: 484-492, 2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-30639875

RESUMO

Autophagy, a crucial process for maintaining cellular homeostasis, is under the control of several autophagy-related (ATG) proteins, and is highly conserved in most animals, but its response to adverse environmental conditions is poorly understood in crustaceans. Herein, we hypothesised that autophagy acts as a protective response to hypoxia, and Beclin 1, ATG7 and ATG8 in oriental river prawn (Macrobrachium nipponense) were chosen as potential biomarkers under hypoxia exposure; thus, their full-length cDNA sequences were cloned and characterised. Open reading frames (ORFs) of 1281, 2076 and 360 bp, encoding proteins of 427, 692 and 120 amino acid residues, respectively, were obtained. Phylogenetic analysis demonstrated the three M. nipponense proteins do not form a clade with vertebrate homologs. Protein and mRNA levels were investigated in different tissues and developmental stages, and all three were significantly upregulated in a time-dependent manner in the hepatopancreas following hypoxia stress. Biochemical and morphological analysis of hepatocytes revealed that hypoxia increased the abundance of hepatic autophagic vacuoles and stimulated anaerobic metabolism. RNA interference-mediated silencing of ATG8 significantly increased the death rate of M. nipponense juveniles under hypoxia stress conditions. Together, these results suggest that Beclin 1, ATG7 and ATG8 contribute to autophagy-based responses against hypoxia in M. nipponense. The findings also expand our understanding of the potential role of autophagy as an adaptive response against hypoxia toxicity in crustaceans. The results showed that hepatic ATG8 levels may be directly indicative of acute hypoxia in prawns, and provide insight into the time at which hypoxia exposure occurs. Autophagy-related genes expression pattern seems to be sensitive and good biomarkers of acute hypoxia exposure.


Assuntos
Autofagia/genética , Hipóxia/genética , Palaemonidae/genética , Animais , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Família da Proteína 8 Relacionada à Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Clonagem Molecular , Regulação da Expressão Gênica , Marcadores Genéticos , Hepatócitos/citologia , Hepatócitos/metabolismo , Hepatopâncreas/metabolismo , Hipóxia/diagnóstico , Palaemonidae/metabolismo , Filogenia , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Rios/química , Análise de Sequência de DNA , Estresse Fisiológico
9.
Oncogene ; 38(17): 3301-3315, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30635654

RESUMO

Over half a million US residents are suffering with bladder cancer (BC), which costs a total $4 billion in treatment annually. Although recent studies report that autophagy-related gene 7 (ATG7) is overexpressed in BCs, the regulatory effects of ATG7 on cancer stem-like phenotypes and invasion have not been explored yet. Current studies demonstrated that the deficiency of ATG7 by its shRNA dramatically reduced sphere formation and invasion in vitro, as well as lung metastasis in vivo in human invasive BC cells. Further studies indicated that the knockdown of ATG7 attenuated the expression of CD44 standard (CD44s), while ectopic introduction of CD44s, was capable of completely restoring sphere formation, invasion, and lung metastasis in T24T(shATG7) cells. Mechanistic studies revealed that ATG7 overexpression stabilized CD44s proteins accompanied with upregulating USP28 proteins. Upregulated USP28 was able to bind to CD44s and remove the ubiquitin group from CD44s' protein, resulting in the stabilization of CD44s protein. Moreover, ATG7 inhibition stabilized AUF1 protein and thereby reduced tet1 mRNA stability and expression, which was able to demethylate usp28 promoter, reduced USP28 expression, finally promoting CD44s degradation. In addition, CD44s was defined to inhibit degradation of RhoGDIß, which in turn promotes BC invasion. Our results demonstrate that CD44s is a key ATG7 downstream regulator of the sphere formation, invasion, and lung metastasis of BCs, providing significant insight into understanding the BC invasions, metastasis, and stem-like properties.


Assuntos
Proteína 7 Relacionada à Autofagia/genética , Receptores de Hialuronatos/genética , Neoplasias Pulmonares/secundário , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Proteína 7 Relacionada à Autofagia/metabolismo , Linhagem Celular Tumoral , Humanos , Receptores de Hialuronatos/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Metástase Neoplásica , Regiões Promotoras Genéticas , RNA Interferente Pequeno/genética , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Regulação para Cima , Neoplasias da Bexiga Urinária/metabolismo
10.
Nutrients ; 11(1)2019 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-30634545

RESUMO

Advanced glycation end products (AGE) play a causative role in the development of aberrant phenotypes of intraglomerular mesangial cells, contributing to acute/chronic glomerulonephritis. The aim of this study was to explore mechanistic effects of the flavonoid chrysin present in bee propolis and herbs on actin dynamics, focal adhesion, and the migration of AGE-exposed mesangial cells. The in vitro study cultured human mesangial cells exposed to 33 mM glucose and 100 µg/mL AGE-bovine serum albumin (AGE-BSA) for up to 5 days in the absence and presence of 1⁻20 µM chrysin. The in vivo study employed db/db mice orally administrated for 10 weeks with 10 mg/kg chrysin. The presence of ≥10 µM chrysin attenuated mesangial F-actin induction and bundle formation enhanced by AGE. Chrysin reduced the mesangial induction of α-smooth muscle actin (α-SMA) by glucose, and diminished the tissue α-SMA level in diabetic kidneys, indicating its blockade of mesangial proliferation. The treatment of chrysin inhibited the activation of vinculin and paxillin and the induction of cortactin, ARP2/3, fascin-1, and Ena/VASP-like protein in AGE-exposed mesangial cells. Oral administration of chrysin diminished tissue levels of cortactin and fascin-1 elevated in diabetic mouse kidneys. Mesangial cell motility was enhanced by AGE, which was markedly attenuated by adding chrysin to cells. On the other hand, chrysin dampened the induction of autophagy-related genes of beclin-1, LC3 I/II, Atg3, and Atg7 in mesangial cells exposed to AGE and in diabetic kidneys. Furthermore, chrysin reduced the mTOR activation in AGE-exposed mesangial cells and diabetic kidneys. The induction of mesangial F-actin, cortactin, and fascin-1 by AGE was deterred by the inhibition of autophagy and mTOR. Thus, chrysin may encumber diabetes-associated formation of actin bundling and focal adhesion and mesangial cell motility through disturbing autophagy and mTOR pathway.


Assuntos
Citoesqueleto de Actina/efeitos dos fármacos , Autofagia , Diabetes Mellitus Experimental , Flavonoides/administração & dosagem , Adesões Focais/efeitos dos fármacos , Produtos Finais de Glicação Avançada/farmacologia , Rim/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Cortactina/genética , Cortactina/metabolismo , Adesões Focais/metabolismo , Humanos , Masculino , Células Mesangiais/efeitos dos fármacos , Células Mesangiais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Vinculina/genética , Vinculina/metabolismo
11.
Oncol Rep ; 41(2): 819-828, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30483807

RESUMO

Aberrant expression of miR­154 is usually found in cancer studies; however, the role of miR­154 has seldom been reported in bladder cancer (BCa). In this study, we observed that miR­154 expression was significantly downregulated in BCa tissues and cell lines, and was associated with several clinicopathological characteristics, including advanced T stage, lymphatic invasion, and distant metastasis. Low expression level of miR­154 was associated with poor survival outcomes in BCa patients. Overexpression of miR­154 led to significant decrease in the proliferation, migration, and invasion of BCa cells, while knockdown of miR­154 yielded the opposite effect. ATG7 was identified as a direct target of miR­154. ATG7 expression was negatively correlated with miR­154 expression in BCa tissues. Silencing of ATG7 achieved a similar effect to miR­154 overexpression; overexpression of ATG7 reversed the inhibitory effect of miR­154 on BCa cell proliferation, migration and invasion. A xenograft study revealed that miR­154 inhibited BCa cell growth in vivo, and suppressed ATG7 expression. Altogether, this study demonstrated that miR­154 may function as a tumor suppressor in BCa and indicated that miR­154 may be a potential therapeutic target for BCa patients.


Assuntos
Proteína 7 Relacionada à Autofagia/genética , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , MicroRNAs/metabolismo , Neoplasias da Bexiga Urinária/genética , Animais , Apoptose/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação para Baixo , Feminino , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Pessoa de Meia-Idade , Estadiamento de Neoplasias , RNA Interferente Pequeno/metabolismo , Neoplasias da Bexiga Urinária/mortalidade , Neoplasias da Bexiga Urinária/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Andrologia ; 51(2): e13177, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30353556

RESUMO

Abnormal dilatation and tortuosity of the pampiniform plexus within the spermatic cord are termed varicocele which leads to impaired spermatogenesis due to heat-related oxidative stress and cell death. Previously, it was shown that both apoptosis and autophagy pathways were activated by heat in germ cells of mouse in vivo and in vitro. But, status of these pathways is not clear in chronic state of heat stress such as varicocele. Therefore, we aimed to access sperm apoptotic markers (active caspases 3/7 and DNA fragmentation), and autophagic markers (Atg7 and LC3 proteins) as primary outcomes, and also sperm parameters and protamine deficiency as secondary outcomes between 23 infertile men with varicocele and 16 fertile individuals. Sperm parameters were assessed according to World Health Organization 2010 protocol. Apoptotic markers (active caspases 3/7 and DNA fragmentation), autophagic markers (Atg7 and LC3 proteins), and protamine deficiency were evaluated by flow cytometry, fluorescence microscope, and western blotting techniques. Mean of autophagy and apoptosis markers, and also protamine deficiency have significantly increased in infertile men with varicocele compared to fertile individuals, but autophagy and apoptosis markers did not significantly correlate with each other. In conclusion, it seems that both apoptosis and autophagy pathways are independently active in spermatozoa of infertile men with varicocele.


Assuntos
Apoptose/fisiologia , Autofagia/fisiologia , Infertilidade Masculina/metabolismo , Espermatozoides/metabolismo , Varicocele/metabolismo , Adulto , Proteína 7 Relacionada à Autofagia/metabolismo , Biomarcadores/metabolismo , Caspase 3/metabolismo , Caspase 7/metabolismo , Forma Celular/fisiologia , Fragmentação do DNA , Fertilidade/fisiologia , Humanos , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Protaminas/metabolismo , Análise do Sêmen , Motilidade Espermática/fisiologia , Espermatozoides/citologia
13.
J Thorac Cardiovasc Surg ; 157(1): 185-193, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30195591

RESUMO

BACKGROUND: We sought to determine if endothelial autophagy affects myocardial energy metabolism. METHODS: We used isolated working mouse hearts to compare cardiac function, energy metabolism, and ischemic response of hearts from endothelial cell-specific ATG7 knockout (EC-ATG7-/-) mice to hearts from their wild-type littermates. We also conducted gene analyses on human umbilical vein endothelial cells incubated with scrambled small interfering RNA or small interfering ATG7. RESULTS: In the presence of insulin, working hearts from EC-ATG7-/- mice, relative to those from wild-type littermates, exhibited greater reductions in insulin-associated palmitate oxidation indicating a diminished reliance on fatty acids as a fuel source. Likewise, palmitate oxidation was markedly lower in the hearts of EC-ATG7-/- mice versus wild-type mice during reperfusion of ischemic hearts. Although hearts from EC-ATG7-/- mice revealed significantly lower triacylglycerol content compared with those from wild-type mice, ATG7-silenced human umbilical vein endothelial cells demonstrated appreciably lower fatty acid binding protein 4 and 5 expression relative to those treated with scrambled small interfering RNA. CONCLUSIONS: Disruption of endothelial autophagy reduces cardiac fatty acid storage and dampens reliance on fatty acid oxidation as a cardiac fuel source. The autophagy network represents a novel target for designing new strategies aimed at resetting perturbed myocardial bioenergetics.


Assuntos
Autofagia , Endotélio Vascular/metabolismo , Ácidos Graxos/metabolismo , Miocárdio/metabolismo , Oxirredução , Animais , Proteína 7 Relacionada à Autofagia/metabolismo , Endotélio Vascular/fisiologia , Metabolismo Energético , Masculino , Camundongos , Camundongos Knockout , Palmitatos/metabolismo , Triglicerídeos/metabolismo
14.
Mol Genet Genomic Med ; 6(6): 1209-1219, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30407747

RESUMO

BACKGROUND: Coronary artery disease including acute myocardial infarction (AMI) is mainly caused by atherosclerosis, an inflammatory and metabolic disease. Autophagy has been demonstrated to play critical roles in lipid metabolism and inflammation. Altered autophagic activity has been reported in AMI patients. However, molecular basis for dysfunctional autophagy in AMI remains unexplained. METHODS: In this study, the promoter of the ATG7 gene, encoding a core protein for autophagy, was genetically and functionally analyzed in large cohorts of AMI patients (n = 355) and ethnic-matched healthy controls (n = 363). Related molecular mechanisms were also explored. RESULTS: A total of 19 DNA sequence variants (DSVs) including single-nucleotide polymorphisms (SNPs) were found in the ATG7 gene promoter. Two novel DSVs and five SNPs were only identified in AMI patients group. These DSVs and SNPs, except one SNP, significantly altered the transcriptional activity of the ATG7 gene promoter in both HEK-293 and H9c2 cells (p < 0.05). Further electrophoretic mobility shift assay revealed that the DSVs and SNPs evidently affected the binding of transcription factors. CONCLUSIONS: ATG7 gene DSVs and SNPs identified in AMI patients may alter the transcriptional activity of the ATG7 gene promoter and change ATG7 level, contributing to the AMI development as a rare risk factor.


Assuntos
Proteína 7 Relacionada à Autofagia/genética , Infarto do Miocárdio/genética , Polimorfismo de Nucleotídeo Único , Proteína 7 Relacionada à Autofagia/metabolismo , Feminino , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas
15.
Nature ; 563(7732): 569-573, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30429607

RESUMO

Autophagy captures intracellular components and delivers them to lysosomes, where they are degraded and recycled to sustain metabolism and to enable survival during starvation1-5. Acute, whole-body deletion of the essential autophagy gene Atg7 in adult mice causes a systemic metabolic defect that manifests as starvation intolerance and gradual loss of white adipose tissue, liver glycogen and muscle mass1. Cancer cells also benefit from autophagy. Deletion of essential autophagy genes impairs the metabolism, proliferation, survival and malignancy of spontaneous tumours in models of autochthonous cancer6,7. Acute, systemic deletion of Atg7 or acute, systemic expression of a dominant-negative ATG4b in mice induces greater regression of KRAS-driven cancers than does tumour-specific autophagy deletion, which suggests that host autophagy promotes tumour growth1,8. Here we show that host-specific deletion of Atg7 impairs the growth of multiple allografted tumours, although not all tumour lines were sensitive to host autophagy status. Loss of autophagy in the host was associated with a reduction in circulating arginine, and the sensitive tumour cell lines were arginine auxotrophs owing to the lack of expression of the enzyme argininosuccinate synthase 1. Serum proteomic analysis identified the arginine-degrading enzyme arginase I (ARG1) in the circulation of Atg7-deficient hosts, and in vivo arginine metabolic tracing demonstrated that serum arginine was degraded to ornithine. ARG1 is predominantly expressed in the liver and can be released from hepatocytes into the circulation. Liver-specific deletion of Atg7 produced circulating ARG1, and reduced both serum arginine and tumour growth. Deletion of Atg5 in the host similarly regulated [corrected] circulating arginine and suppressed tumorigenesis, which demonstrates that this phenotype is specific to autophagy function rather than to deletion of Atg7. Dietary supplementation of Atg7-deficient hosts with arginine partially restored levels of circulating arginine and tumour growth. Thus, defective autophagy in the host leads to the release of ARG1 from the liver and the degradation of circulating arginine, which is essential for tumour growth; this identifies a metabolic vulnerability of cancer.


Assuntos
Arginina/sangue , Autofagia , Neoplasias/sangue , Neoplasias/patologia , Aloenxertos , Animais , Arginase/sangue , Arginase/metabolismo , Arginina/administração & dosagem , Arginina/farmacologia , Autofagia/genética , Proteína 5 Relacionada à Autofagia/deficiência , Proteína 5 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/deficiência , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Suplementos Nutricionais , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Fígado/enzimologia , Masculino , Camundongos , Transplante de Neoplasias , Neoplasias/genética , Ornitina/metabolismo
16.
Nat Commun ; 9(1): 4962, 2018 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-30470740

RESUMO

Activation of the Hippo pathway effector Yap underlies many liver cancers, however no germline or somatic mutations have been identified. Autophagy maintains essential metabolic functions of the liver, and autophagy-deficient murine models develop benign adenomas and hepatomegaly, which have been attributed to activation of the p62/Sqstm1-Nrf2 axis. Here, we show that Yap is an autophagy substrate and mediator of tissue remodeling and hepatocarcinogenesis independent of the p62/Sqstm1-Nrf2 axis. Hepatocyte-specific deletion of Atg7 promotes liver size, fibrosis, progenitor cell expansion, and hepatocarcinogenesis, which is rescued by concurrent deletion of Yap. Our results shed new light on mechanisms of Yap degradation and the sequence of events that follow disruption of autophagy, which is impaired in chronic liver disease.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagia , Hepatócitos/citologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/fisiopatologia , Fígado/metabolismo , Fosfoproteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Carcinogênese , Proteínas de Ciclo Celular , Diferenciação Celular , Feminino , Hepatócitos/metabolismo , Humanos , Fígado/citologia , Fígado/patologia , Neoplasias Hepáticas/genética , Masculino , Camundongos , Fosfoproteínas/genética , Proteólise , Fatores de Transcrição
17.
Anal Chim Acta ; 1037: 75-86, 2018 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-30292317

RESUMO

Autophagy is of great significance in maintaining cellular homeostasis. Aberrant autophagy has been reported to contribute to the disease aetiology of metabolic syndrome, especially several key lysosomal storage disorders. However, the molecular mechanisms and the correlation between autophagy and lipid metabolism remains unclear. This study was designed and aimed to reveal the alteration of lipid metabolism in response to the autophagy induced by nutrient stress to give new insights into the molecular mechanisms between autophagy and lipid metabolism. An online normal-phase/reversed-phase two-dimensional liquid chromatography-mass spectrometry (NP/RP 2D LC-MS) method was developed to perform the lipidomics analysis of Atg7-/- mouse embryonic fibroblast cells (MEFs) and wild-type MEFs under nutrient stress. 48 and 35 lipid species in wild-type and Atg7-/- MEFs respectively finally meet the screening criteria with p-value less than 0.05 and fold change more than 1.5 in response to nutrient stress. Their alterations indicated that autophagy participated lipid metabolism to generate energy and form autophagosomes with significantly increased free fatty acids and glycerophospholipids, which protected wild-type MEFs from serious damages and delayed cell death. However, in Atg7-/- MEFs, due to the inhibition of autophagy, lipids were continuously consumed and cells suffered from damages even death. These results illustrated the close relationship between autophagy and lipid metabolism comprehensively and revealed diverse lipid targets for the investigation of autophagy.


Assuntos
Autofagia/efeitos dos fármacos , Células-Tronco Embrionárias/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Nutrientes , Animais , Proteína 7 Relacionada à Autofagia/deficiência , Proteína 7 Relacionada à Autofagia/metabolismo , Células Cultivadas , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Camundongos , Camundongos Knockout , Estresse Oxidativo/efeitos dos fármacos
18.
Cell Host Microbe ; 24(5): 689-702.e7, 2018 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-30344003

RESUMO

Long-lived resting memory CD4+ T cells (TCM) are a major reservoir of latent HIV infection. We hypothesized that latent HIV-TCM cells are maintained by aberrant expression of cell survival factors, including XIAP, BIRC2/cIAP1, and beclin-1. DIABLO/SMAC mimetics are therapeutic agents that compromise cell survival by hijacking host apoptotic machinery. We found that DIABLO/SMAC mimetics (birinapant, GDC-0152, and embelin) selectively kill HIV-TCM without increasing virus production or targeting uninfected TCM. Treatment of HIV-TCM with DIABLO/SMAC mimetics promoted XIAP and BIRC2 degradation, which triggered autophagy and the formation of a cell death complex consisting of pro-apoptotic (FADD, RIPK1, RIPK3, and caspase 8) and autophagy (ATG5, ATG7, and SQSTM1) proteins. Genetic or pharmacological inhibition of autophagy induction, but not autophagy-mediated degradation, abrogated this interaction and subsequent cell death. Our findings identify a mechanism whereby DIABLO/SMAC mimetics exploit autophagy and apoptotic machinery to selectively induce killing of HIV-TCM without viral reactivation while sparing uninfected cells.


Assuntos
Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Autofagia/efeitos dos fármacos , Autofagia/fisiologia , Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/imunologia , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Proteínas Reguladoras de Apoptose , Proteína 5 Relacionada à Autofagia/metabolismo , Proteína 7 Relacionada à Autofagia/metabolismo , Proteína 3 com Repetições IAP de Baculovírus/metabolismo , Proteína Beclina-1/metabolismo , Benzoquinonas/farmacologia , Caspase 8/metabolismo , Morte Celular , Linhagem Celular , Cicloexanos/farmacologia , Dipeptídeos/farmacologia , Proteína de Domínio de Morte Associada a Fas/metabolismo , HIV-1/patogenicidade , Humanos , Indóis/farmacologia , Proteínas Inibidoras de Apoptose/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Mitocondriais/metabolismo , Pirróis/farmacologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteína Sequestossoma-1/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo
19.
Sci Rep ; 8(1): 14391, 2018 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-30258106

RESUMO

Autophagy is a degradation pathway important for cellular homeostasis. The E1-like enzyme ATG7 is a key component of the autophagy machinery, with the main function of mediating the lipidation of LC3/GABARAP during autophagosome formation. By analysing mRNA-sequencing data we found that in addition to the full-length ATG7 isoform, various tissues express a shorter isoform lacking an exon of 27 amino acids in the C-terminal part of the protein, termed ATG7(2). We further show that ATG7(2) does not bind LC3B and fails to mediate the lipidation of members of the LC3/GABARAP family. We have thus identified an isoform of ATG7 that is unable to carry out the best characterized function of the protein during the autophagic response. This short isoform will have to be taken into consideration when further studying the role of ATG7.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína 7 Relacionada à Autofagia/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Sequência de Aminoácidos , Animais , Autofagia , Proteína 7 Relacionada à Autofagia/química , Células HEK293 , Humanos , Metabolismo dos Lipídeos , Camundongos , Modelos Moleculares , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo
20.
Food Chem Toxicol ; 121: 283-296, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30208301

RESUMO

Non-alcoholic fatty liver disease (NAFLD) is often linked with impaired hepatic autophagy. Here, we studied the alterations in hepatocellular autophagy by high cholesterol and high-fat diet (HC-HF) diet in C57BL/6J mice, and by palmitic acid (PA), in AML-12 and HepG2 cells. Further, we analysed role of Trigonelline (TG), a plant alkaloid, in preventing NAFLD, by modulating autophagy. For this, C57BL/6J mice were fed with Standard Chow (SC) or HC-HF diet, with and without TG for 16 weeks. In-vitro; AML-12 cells and HepG2 cells, were exposed to PA with and without TG, for 24 h. Cellular events related to autophagy, lipogenesis, and lipo-toxicity were studied. The HC-HF diet fed mice showed hepatic autophagy blockade, increased triglycerides and steatosis. PA exposure to AML-12 cells and HepG2 cells induced impaired autophagy, ER stress, resulting in lipotoxicity. TG treatment in HC-HF fed mice, restored hepatic autophagy, and prevented steatosis. TG treated AML-12, and HepG2 cells exposed to PA showed autophagy restoration, and reduced lipotoxicity, however, these effects were diminished in Atg7-/- HepG2 cells, and in the presence of chloroquine. This study shows that HC-HF diet-induced impaired autophagy, and steatosis is prevented by TG, which attributes to its novel mechanism in treating NAFLD.


Assuntos
Alcaloides/farmacologia , Autofagia/efeitos dos fármacos , Colesterol/administração & dosagem , Dieta Hiperlipídica/efeitos adversos , Fígado Gorduroso/induzido quimicamente , Ração Animal/análise , Animais , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Fígado Gorduroso/prevenção & controle , Inativação Gênica , Humanos , Resistência à Insulina , Metabolismo dos Lipídeos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Regulação para Cima , Ganho de Peso
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