Ursodeoxycholic acid induces sarcopenia associated with decreased protein synthesis and autophagic flux

Background Skeletal muscle generates force and movements and maintains posture. Under pathological conditions, muscle fibers suffer an imbalance in protein synthesis/degradation. This event causes muscle mass loss and decreased strength and muscle function, a syndrome known as sarcopenia. Recently, our laboratory described secondary sarcopenia in a chronic cholestatic liver disease (CCLD) mouse model. Interestingly, the administration of ursodeoxycholic acid (UDCA), a hydrophilic bile acid, is an effective therapy for cholestatic hepatic alterations. However, the effect of UDCA on skeletal muscle mass and functionality has never been evaluated, nor the possible involved mechanisms. Methods We assessed the ability of UDCA to generate sarcopenia in C57BL6 mice and develop a sarcopenic-like phenotype in C2C12 myotubes and isolated muscle fibers. In mice, we measured muscle strength by a grip strength test, muscle mass by bioimpedance and mass for specific muscles, and physical function by a treadmill test. We also detected the fiber's diameter and content of sarcomeric proteins. In C2C12 myotubes and/or isolated muscle fibers, we determined the diameter and troponin I level to validate the cellular effect. Moreover, to evaluate possible mechanisms, we detected puromycin incorporation, p70S6K, and 4EBP1 to evaluate protein synthesis and ULK1, LC3 I, and II protein levels to determine autophagic flux. The mitophagosome-like structures were detected by transmission electron microscopy. Results UDCA induced sarcopenia in healthy mice, evidenced by decreased strength, muscle mass, and physical function, with a decline in the fiber's diameter and the troponin I protein levels. In the C2C12 myotubes, we observed that UDCA caused a reduction in the diameter and content of MHC, troponin I, puromycin incorporation, and phosphorylated forms of p70S6K and 4EBP1. Further, we detected increased levels of phosphorylated ULK1, the LC3II/LC3I ratio, and the number of mitophagosome-like structures. These data suggest that UDCA induces a sarcopenic-like phenotype with decreased protein synthesis and autophagic flux. Conclusions Our results indicate that UDCA induces sarcopenia in mice and sarcopenic-like features in C2C12 myotubes and/or isolated muscle fibers concomitantly with decreased protein synthesis and alterations in autophagic flux.

Research progress on autophagic flux regulation in cataract / 国际眼科杂志(Guoji Yanke Zazhi)

Autophagic flux refers to a series of dynamic process of autophagic bilayer membrane formation, autophagosome formation, autophagolysosomes formation and degradation. The etiology of cataract is complex, including congenital abnormalities in lens development due to genetic mutations, oxidative damage due to aging, abnormalities in glucose metabolism due to diabetes, and proliferation of lens epithelial cells(LECs)stimulated by postoperative inflammatory factor, all of which are associated with the development of cataracts. A growing number of research in recent years have discovered that altering the status of LECs can contribute to the pathophysiological process of cataract by regulating autophagic flux. This review summarized the impacts of autophagic flux regulation on cataract.

Nanosized copper particles induced mesangial cell toxicity via the autophagy pathway

Nanosized copper particles (nano Cu) have been incorporated into products in multiple industries, although studies have demonstrated that these particles are nephrotoxic. We investigated the cytotoxicity of nanosized copper particles on rat mesangial cells and measured rates of apoptosis, the expression of caspase-3, and generation of reactive oxygen species. We also measured autophagy through the acridine orange (AO) staining and expression of Beclin-1, microtubule-associated protein 1 light chain 3, and p62 to screen the underlying mechanism of toxicity. Nanosized copper particles inhibited mesangial cell viability, up-regulated the activity of caspase-3, and increased the rates of apoptosis and the generation of reactive oxygen species in a concentration-dependent manner. Exposure to nano Cu increased the formation of acidic vesicular organelles and the expression of Beclin-1, microtubule-associated protein 1 light chain 3, and p62, and treatment with an autophagy inhibitor reduced nephrotoxicity. This indicated that the autophagy pathway is involved in the toxicity induced by nanosized copper particles to mesangial cells. This finding can contribute to the development of safety guidelines for the evaluation of nanomaterials in the future.

IRE1α deficiency impairs autophagy in chondrocytes by upregulating calcium homeostasis endoplasmic reticulum protein / 南方医科大学学报

OBJECTIVE@#To explore the mechanism by which inositol-requiring enzyme-1α (IRE1α) regulates autophagy function of chondrocytes through calcium homeostasis endoplasmic reticulum protein (CHERP).@*METHODS@#Cultured human chondrocytes (C28/I2 cells) were treated with tunicamycin, 4μ8c, rapamycin, or both 4μ8c and rapamycin, and the expressions of endoplasmic reticulum (ER) stress- and autophagy-related proteins were detected with Western blotting. Primary chondrocytes from ERN1 knockout (ERN1 CKO) mice and wild-type mice were examined for ATG5 and ATG7 mRNA expressions, IRE1α and p-IRE1α protein expressions, and intracellular calcium ion content using qPCR, Western blotting and flow cytometry. The effect of bafilomycin A1 treatment on LC3 Ⅱ/LC3 Ⅰ ratio in the isolated chondrocytes was assessed with Western blotting. Changes in autophagic flux of the chondrocytes in response to rapamycin treatment were detected using autophagy dual fluorescent virus. The changes in autophagy level in C28/I2 cells overexpressing CHERP and IRE1α were detected using immunofluorescence assay.@*RESULTS@#Tunicamycin treatment significantly up-regulated ER stress-related proteins and LC3 Ⅱ/LC3 Ⅰ ratio and down-regulated the expression of p62 in C28/I2 cells (P < 0.05). Rapamycin obviously up-regulated LC3 Ⅱ/LC3 Ⅰ ratio (P < 0.001) in C28/I2 cells, but this effect was significantly attenuated by co-treatment with 4μ8c (P < 0.05). Compared with the cells from the wild-type mice, the primary chondrocytes from ERN1 knockout mice showed significantly down-regulated mRNA levels of ERN1 (P < 0.01), ATG5 (P < 0.001) and ATG7 (P < 0.001), lowered or even lost expressions of IRE1α and p-IRE1α proteins (PP < 0.01), and increased expression of CHERP (P < 0.05) and intracellular calcium ion content (P < 0.001). Bafilomycin A1 treatment obviously increased LC3 Ⅱ/ LC3 Ⅰ ratio in the chondrocytes from both wild-type and ERN1 knockout mice (P < 0.01 or 0.05), but the increment was more obvious in the wild-type chondrocytes (P < 0.05). Treatment with autophagy dual-fluorescence virus resulted in a significantly greater fluorescence intensity of LC3-GFP in rapamycin-treated ERN1 CKO chondrocytes than in wild-type chondrocytes (P < 0.05). In C28/I2 cells, overexpression of CHERP obviously decreased the fluorescence intensity of LC3, and overexpression of IRE1α enhanced the fluorescence intensity and partially rescued the fluorescence reduction of LC3 caused by CHERP.@*CONCLUSION@#IRE1α deficiency impairs autophagy in chondrocytes by upregulating CHERP and increasing intracellular calcium ion content.

Demethylzeylasteral (T-96) initiates extrinsic apoptosis against prostate cancer cells by inducing ROS-mediated ER stress and suppressing autophagic flux

BACKGROUND: Demethylzeylasteral (T-96) is a pharmacologically active triterpenoid monomer extracted from Tripterygium wilfordii Hook F (TWHF) that has been reported to exhibit anti-neoplastic effects against several types of cancer cells. However, the potential anti-tumour effects of T-96 against human Prostate cancer (CaP) cells and the possible underlying mechanisms have not been well studied. RESULTS: In the current study, T-96 exerted significant cytotoxicity to CaP cells in vitro and induced cell cycle arrest at S-phase in a dose-dependent manner. Mechanistically, T-96 promoted the initiation of autophagy but inhibited autophagic flux by inducing ROS-mediated endoplasmic reticulum (ER) stress which subsequently activated the extrinsic apoptosis pathway in CaP cells. These findings implied that T-96-induced ER stress activated the caspase-dependent apoptosis pathway to inhibit proliferation of CaP cells. Moreover, we observed that T-96 enhances the sensitivity of CaP cells to the chemotherapeutic drug, cisplatin. CONCLUSIONS: Taken together, our data demonstrated that T-96 is a novel modulator of ER stress and autophagy, and has potential therapeutic applications against CaP in the clinic.

Neuroprotective Autophagic Flux Induced by Hyperbaric Oxygen Preconditioning is Mediated by Cystatin C / 神经科学通报·英文版

We have previously reported that Cystatin C (CysC) is a pivotal mediator in the neuroprotection induced by hyperbaric oxygen (HBO) preconditioning; however, the underlying mechanism and how CysC changes after stroke are not clear. In the present study, we demonstrated that CysC expression was elevated as early as 3 h after reperfusion, and this was further enhanced by HBO preconditioning. Concurrently, LC3-II and Beclin-1, two positive-markers for autophagy induction, exhibited increases similar to CysC, while knockdown of CysC blocked these elevations. As a marker of autophagy inhibition, p62 was downregulated by HBO preconditioning and this was blocked by CysC knockdown. Besides, the beneficial effects of preserving lysosomal membrane integrity and enhancing autolysosome formation induced by HBO preconditioning were abolished in CysC rats. Furthermore, we demonstrated that exogenous CysC reduced the neurological deficits and infarct volume after brain ischemic injury, while 3-methyladenine partially reversed this neuroprotection. In the present study, we showed that CysC is biochemically and morphologically essential for promoting autophagic flux, and highlighted the translational potential of HBO preconditioning and CysC for stroke treatment.

Ginsenoside Rb1 Ameliorates Autophagy of Hypoxia Cardiomyocytes from Neonatal Rats via AMP-Activated Protein Kinase Pathway / 中国结合医学杂志

OBJECTIVE@#To investigate whether ginsenoside-Rb1 (Gs-Rb1) improves the CoCl-induced autophagy of cardiomyocytes via upregulation of adenosine 5'-monophosphate-activated protein kinase (AMPK) pathway.@*METHODS@#Ventricles from 1- to 3-day-old Wistar rats were sequentially digested, separated and incubated in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum for 3 days followed by synchronization. Neonatal rat cardiomyocytes were randomly divided into 7 groups: control group (normal level oxygen), hypoxia group (500 μmol/L CoCl), Gs-Rb1 group (200 μmol/L Gs-Rb1 + 500 μmol/L CoCl), Ara A group (500 μmol/L Ara A + 500 μmol/L CoCl), Ara A+ Gs-Rb1 group (500 μmol/L Ara A + 200 μmol/L Gs-Rb1 + 500 μmol/L CoCl), AICAR group [1 mmol/L 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) + 500 μmol/L CoCl], and AICAR+Gs-Rb1 group (1 mmol/L AICAR + 200 μmol/L Gs-Rb1 + 500 μmol/L CoCl). Cells were treated for 12 h and cell viability was determined by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and cardiac troponin I (cTnI) levels were detected by enzyme-linked immunosorbent assay (ELISA). AMPK activity was assessed by 2',7'-dichlorofluorescein diacetate (DCFH-DA) ELISA assay. The protein expressions of Atg4B, Atg5, Atg6, Atg7, microtubule-associated protein 1A/1B-light chain 3 (LC3), P62, and active-cathepsin B were measured by Western blot.@*RESULTS@#Gs-Rb1 significantly improved the cell viability of hypoxia cardiomyocytes (P0.05). Gs-Rb1 significantly down-regulated P62 levels of hypoxic cardiomyocytes (P<0.05). The P62 levels of hypoxic cardiomyocytes were inhibited by Ara A (P<0.05) and were not affected by AICAR (P=0.871).@*CONCLUSION@#Gs-Rb1 may improve the viability of hypoxia cardiomyocytes by ameliorating cell autophagy via the upregulation of AMPK pathway.

Role of tangeretin on autophagy in human gastric cancer AGS cells and its mechanism / 中国药理学通报

Aim To investigate the effect of tangeretin on autophagy of human gastric cancer AGS cells and its molecular mechanisms. Methods Western blot was used to detect the effect of tangeretin on expression of LC3, p62 and Akt phosphorylation, and the effects of autophagy inhibitor CQ and activator Rapa on tangere-tin-regulated autophagy. Annexin V/PI double staining was used to quantitatively detect the effect of CQ on apoptosis of cells induced by iangeretin. Results 24h after treatment, tangeretin enhanced the expression of LC3 II protein (P < 0. 05 , P < 0. 01) and p62 (P < 0. 05) in a dose-dependent manner. 48 h after treatment, tangeretin enhanced the expression of p62 protein in a dose-dependent manner (P < 0. 05, P < 0. 01) , and had no significant effect on expression of LC3II protein. At 24 h and 48 h, the expression of LC3II and p62 increased significantly in CQ + Tan group compared with that of Tan group (P < 0. 01). Compared with Tan group, the expression of LC3II and p62 protein decreased in Rapa + Tan group, and there was a statistical difference (P < 0. 05). The apoptotic rate of AGS cells in CQ + Tan group was significantly higher than that in Tan group (P <0. 01). The phosphorylation levels of Akt protein were reduced by tan-gerin for 24 and 48 h in a dose-dependent manner (P <0. 05 , P < 0. 01). Conclusions Tangeretin may promote cell apoptosis by inhibiting the autophagic flux in human gastric cancer AGS cells. Akt phosphorylation might be involved in the autophagy and apoptosis.

Loss of VAPB Regulates Autophagy in a Beclin 1-Dependent Manner / 神经科学通报·英文版

Autophagy is an evolutionarily-conserved self-degradative process that maintains cellular homeostasis by eliminating protein aggregates and damaged organelles. Recently, vesicle-associated membrane protein-associated protein B (VAPB), which is associated with the familial form of amyotrophic lateral sclerosis, has been shown to regulate autophagy. In the present study, we demonstrated that knockdown of VAPB induced the up-regulation of beclin 1 expression, which promoted LC3 (microtubule-associated protein light chain 3) conversion and the formation of LC3 puncta, whereas overexpression of VAPB inhibited these processes. The regulation of beclin 1 by VAPB was at the transcriptional level. Moreover, knockdown of VAPB increased autophagic flux, which promoted the degradation of the autophagy substrate p62 and neurodegenerative disease proteins. Our study provides evidence that the regulation of autophagy by VAPB is associated with the autophagy-initiating factor beclin 1.

Progress of autophagy screening systems / 药学学报

Autophagy is an active research area in the biomedical field as its role has been identified in many physiological and pathological processes. Accordingly, there is a growing demand to identify, quantify and manipulate the process accurately. Meanwhile, there is great interest in identifying compounds that modulate autophagy because they may have applications in the treatment of a variety of autophagy-related diseases. In this review, we summarize the current status of autophagy screening systems to facilitate identification of autophagy modulators.

New methods to detect autophagic flux / 药学学报

Autophagy is a crucial biological process of eukaryotes, which is involved in cell growth, survival and energy metabolism, while the premise of the autophagy function is activated autophagic flux. It has been confirmed that impaired autophagic flux promotes pathogenesis of many chronic inflammatory diseases, especially cancer, neurodegenerative disease and tissue fibrosis, therefore the analysis of autophagic flux state is important for revealing autophagy function and the mechanism of autophagy related diseases. Given that autophagy is a dynamic process with multiple steps, it is very hard to observe the real state of autophagic flux. Summarized here is the novel concept and current approach to detect autophagic flux. This knowledge is crucial for the researching of the biological function of autophagy, and may provide some strategies for developing autophagy-related drug.