AIM: To clarify the relationship between autophagy and lipotoxicity-induced apoptosis, which is termed "lipoapoptosis," in non-alcoholic steatohepatitis. METHODS: Male C57BL/6J mice were fed a high-fat diet(...AIM: To clarify the relationship between autophagy and lipotoxicity-induced apoptosis, which is termed "lipoapoptosis," in non-alcoholic steatohepatitis. METHODS: Male C57BL/6J mice were fed a high-fat diet(HFD) for 12 wk, after which the liver histology and expression of proteins such as p62 or LC3 were evaluated. Alpha mouse liver 12(AML12) cells treated with palmitate(PA) were used as an in vitro model. RESULTS: LC3-Ⅱ, p62, and Run domain Beclin-1 interacting and cysteine-rich containing(Rubicon) proteins increased in both the HFD mice and in AML12 cells in response to PA treatment. Rubicon expression was decreased upon c-Jun N-terminal kinase(JNK) inhibition at both the m RNA and the protein level in AML12 cells. Rubicon knockdown in AML12 cells with PA decreased the protein levels of both LC3-Ⅱ and p62. Rubicon expression peaked at 4 h of PA treatment in AML12, and then decreased. Treatment with caspase-9 inhibitor ameliorated the decrease in Rubicon protein expression at 10 h of PA and resulted in enlarged AML12 cells under PA treatment. The enlargement of AML12 cells by PA with caspase-9 inhibition was canceled by Rubicon knockdown.CONCLUSION: The JNK-Rubicon axis enhanced lipoapoptosis, and caspase-9 inhibition and Rubicon had effects that were cytologically similar to hepatocyte ballooning. As ballooned hepatocytes secrete fibrogenic signals and thus might promote fibrosis in the liver, the inhibition of hepatocyte ballooning might provide antifibrosis in the NASH liver.展开更多
The RUN domain Beclin-1-interacting cysteine-rich-containing(Rubicon)protein is involved in the maturation step of autophagy and the endocytic pathway as a Beclin-1-binding partner,but little is known regarding the ro...The RUN domain Beclin-1-interacting cysteine-rich-containing(Rubicon)protein is involved in the maturation step of autophagy and the endocytic pathway as a Beclin-1-binding partner,but little is known regarding the role of Rubicon during viral infection.Here,we performed functional studies of the identified target in interferon(IFN)signaling pathways associated with Rubicon to elucidate the mechanisms of viral resistance to IFN.The Rubicon protein levels were elevated in peripheral blood mononuclear cells,sera and liver tissues from patients with hepatitis B virus(HBV)infection relative to those in healthy individuals.Assays of the overexpression and knockdown of Rubicon showed that Rubicon significantly promoted HBV replication.In addition,Rubicon knockdown resulted in the inhibition of enterovirus 71,influenza A virus and vesicular stomatitis virus.The expression o0f Rubicon led to the suppression of virus-induced type-I interferon(IFN-αand IFN-β)and type-III interferon(IFN-λ1).Translocation of activated IRF3 and IRF7 from the cytoplasm to the nucleus was involved in this process,and the NF-κB essential modulator(NEMO),a key factor in the IFN pathway,was the target with which Rubicon interacted.Our results reveal a previously unrecognized function of Rubicon as a virus-induced protein that binds to NEMO,leading to the inhibition of type-I interferon production.Rubicon thus functions as an important negative regulator of the innate immune response,enhances viral replication and may play a role in viral immune evasion.展开更多
Objective: Rubicon is an inhibitory interacting protein of the autophagy-related protein Uvrag. We previously showed thatRubicon deficiency promotes autophagic fluxin vivo and that autophagy can degrade lipid droplets...Objective: Rubicon is an inhibitory interacting protein of the autophagy-related protein Uvrag. We previously showed thatRubicon deficiency promotes autophagic fluxin vivo and that autophagy can degrade lipid droplets. This study aimed to investigate the effects of Rubicon deficiency on fasting-induced hepatic steatosis.Methods: Two-month-old wild-type (WT) andRubicon-deficient mice were subjected to feeding or fasting for 24 hours to induce hepatic steatosis. The distribution of liver lipid droplets was revealed by oil red O staining. Hepatic and plasma triglyceride, non-esterified fatty acid (NEFA), and cholesterol levels were detected using commercially available kits. Real-time reverse transcriptasepolymerase chain reaction was performed to analyze the mRNA expression of genes related to lipid metabolism in the liver. Western blot was conducted to assess autophagy-related protein levels in the liver. The animal experiments were approved by the Institutional Animal Care and Use Committee at Shanghai Jiao Tong University, China.Results: We showed that under fasting conditions,Rubicon-deficient mice had more lipid droplets in the liver than WT controls. Consistent with these results, the hepatic triglyceride, NEFA, and cholesterol levels in fastedRubicon-deficient mice were significantly higher than those of fasted WT controls. The levels ofSREBP-1, a key regulator of lipid synthesis, were significantly lower in livers from fasted WT mice than those of fed WT mice. However, the decrease inSREBP-1 in fasted mice was attenuated byRubicon deficiency. Western blot analysis demonstrated that the fasting-induced increase in autophagic flux was amplified byRubicon deficiency. Finally, we showed thatRubicon deficiency in mice led to elevated plasma triglyceride and NEFA acid levels under fasting conditions.Conclusion: Rubicon deficiency exacerbates fasting-induced hepatic steatosis in mice.展开更多
Macroautophagy(hereafter autophagy)is a catabolic process by which autophagosomes arising from an isolation membrane fuse with lysosomes to degrade components in the cytoplasm.Autophagosomelysosome fusion step is one ...Macroautophagy(hereafter autophagy)is a catabolic process by which autophagosomes arising from an isolation membrane fuse with lysosomes to degrade components in the cytoplasm.Autophagosomelysosome fusion step is one of the key steps during the process of macroautophagy.The step is extremely complicated and its detailed mechanisms remain unclear.It consists of two phases:first phase is autophagosome migration phase and second phase is fusion of autophagosomes and lysosomes phase.Recently,various molecules have been reported to be involved in each phase.In the first phase,microtubules and actin remodeling mechanism are involved.In the second phase,soluble N-ethylmaleimide-sensitive factor attachment protein receptor(SNARE)proteins,Rab family proteins,phosphoinositide 3-kinase(PI3K)complex and Rubicon are involved.In the present review,we introduce recent findings related to autophagosome-lysosome fusion step and discuss liver diseases possibly associated with autophagosome-lysosome fusion dysfunction.展开更多
文摘AIM: To clarify the relationship between autophagy and lipotoxicity-induced apoptosis, which is termed "lipoapoptosis," in non-alcoholic steatohepatitis. METHODS: Male C57BL/6J mice were fed a high-fat diet(HFD) for 12 wk, after which the liver histology and expression of proteins such as p62 or LC3 were evaluated. Alpha mouse liver 12(AML12) cells treated with palmitate(PA) were used as an in vitro model. RESULTS: LC3-Ⅱ, p62, and Run domain Beclin-1 interacting and cysteine-rich containing(Rubicon) proteins increased in both the HFD mice and in AML12 cells in response to PA treatment. Rubicon expression was decreased upon c-Jun N-terminal kinase(JNK) inhibition at both the m RNA and the protein level in AML12 cells. Rubicon knockdown in AML12 cells with PA decreased the protein levels of both LC3-Ⅱ and p62. Rubicon expression peaked at 4 h of PA treatment in AML12, and then decreased. Treatment with caspase-9 inhibitor ameliorated the decrease in Rubicon protein expression at 10 h of PA and resulted in enlarged AML12 cells under PA treatment. The enlargement of AML12 cells by PA with caspase-9 inhibition was canceled by Rubicon knockdown.CONCLUSION: The JNK-Rubicon axis enhanced lipoapoptosis, and caspase-9 inhibition and Rubicon had effects that were cytologically similar to hepatocyte ballooning. As ballooned hepatocytes secrete fibrogenic signals and thus might promote fibrosis in the liver, the inhibition of hepatocyte ballooning might provide antifibrosis in the NASH liver.
基金the supporting of special funding for the PhD students of Wuhan University short-term study abroadsupported by research grants from the Major State Basic Research Development Program of China(2013CB911102)the National Natural Science Foundation of China(81461130019,81271821 and 31570870).
文摘The RUN domain Beclin-1-interacting cysteine-rich-containing(Rubicon)protein is involved in the maturation step of autophagy and the endocytic pathway as a Beclin-1-binding partner,but little is known regarding the role of Rubicon during viral infection.Here,we performed functional studies of the identified target in interferon(IFN)signaling pathways associated with Rubicon to elucidate the mechanisms of viral resistance to IFN.The Rubicon protein levels were elevated in peripheral blood mononuclear cells,sera and liver tissues from patients with hepatitis B virus(HBV)infection relative to those in healthy individuals.Assays of the overexpression and knockdown of Rubicon showed that Rubicon significantly promoted HBV replication.In addition,Rubicon knockdown resulted in the inhibition of enterovirus 71,influenza A virus and vesicular stomatitis virus.The expression o0f Rubicon led to the suppression of virus-induced type-I interferon(IFN-αand IFN-β)and type-III interferon(IFN-λ1).Translocation of activated IRF3 and IRF7 from the cytoplasm to the nucleus was involved in this process,and the NF-κB essential modulator(NEMO),a key factor in the IFN pathway,was the target with which Rubicon interacted.Our results reveal a previously unrecognized function of Rubicon as a virus-induced protein that binds to NEMO,leading to the inhibition of type-I interferon production.Rubicon thus functions as an important negative regulator of the innate immune response,enhances viral replication and may play a role in viral immune evasion.
基金This study was supported by the National Natural Science Foundation of China(Nos.81974024 and 82171567)the Shanghai Natural Science Foundation(No.16ZR1418200).
文摘Objective: Rubicon is an inhibitory interacting protein of the autophagy-related protein Uvrag. We previously showed thatRubicon deficiency promotes autophagic fluxin vivo and that autophagy can degrade lipid droplets. This study aimed to investigate the effects of Rubicon deficiency on fasting-induced hepatic steatosis.Methods: Two-month-old wild-type (WT) andRubicon-deficient mice were subjected to feeding or fasting for 24 hours to induce hepatic steatosis. The distribution of liver lipid droplets was revealed by oil red O staining. Hepatic and plasma triglyceride, non-esterified fatty acid (NEFA), and cholesterol levels were detected using commercially available kits. Real-time reverse transcriptasepolymerase chain reaction was performed to analyze the mRNA expression of genes related to lipid metabolism in the liver. Western blot was conducted to assess autophagy-related protein levels in the liver. The animal experiments were approved by the Institutional Animal Care and Use Committee at Shanghai Jiao Tong University, China.Results: We showed that under fasting conditions,Rubicon-deficient mice had more lipid droplets in the liver than WT controls. Consistent with these results, the hepatic triglyceride, NEFA, and cholesterol levels in fastedRubicon-deficient mice were significantly higher than those of fasted WT controls. The levels ofSREBP-1, a key regulator of lipid synthesis, were significantly lower in livers from fasted WT mice than those of fed WT mice. However, the decrease inSREBP-1 in fasted mice was attenuated byRubicon deficiency. Western blot analysis demonstrated that the fasting-induced increase in autophagic flux was amplified byRubicon deficiency. Finally, we showed thatRubicon deficiency in mice led to elevated plasma triglyceride and NEFA acid levels under fasting conditions.Conclusion: Rubicon deficiency exacerbates fasting-induced hepatic steatosis in mice.
基金This study is partially supported by Japan MEXT KAKENHI to T.Takehara(18H02795)and by The Japanese Society of Gastroenterology to H.Hikita.
文摘Macroautophagy(hereafter autophagy)is a catabolic process by which autophagosomes arising from an isolation membrane fuse with lysosomes to degrade components in the cytoplasm.Autophagosomelysosome fusion step is one of the key steps during the process of macroautophagy.The step is extremely complicated and its detailed mechanisms remain unclear.It consists of two phases:first phase is autophagosome migration phase and second phase is fusion of autophagosomes and lysosomes phase.Recently,various molecules have been reported to be involved in each phase.In the first phase,microtubules and actin remodeling mechanism are involved.In the second phase,soluble N-ethylmaleimide-sensitive factor attachment protein receptor(SNARE)proteins,Rab family proteins,phosphoinositide 3-kinase(PI3K)complex and Rubicon are involved.In the present review,we introduce recent findings related to autophagosome-lysosome fusion step and discuss liver diseases possibly associated with autophagosome-lysosome fusion dysfunction.
