Cytosolic LC3 Ratio as a Sensitive Index of Macroautophagy in Isolated Rat Hepatocytes and H4-II-E Cells

Karim, Md. Razaul; Kanazawa, Takumi; Daigaku, Yasuhiro; Fujimura, Shigefumi; Miotto, Giovanni; Kadowaki, Motoni

doi:10.4161/auto.4615

Cited by 114 publications

(83 citation statements)

References 30 publications

(43 reference statements)

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“…Tuni, tunicamycin; Rapa, rapamycin. (Karim et al, 2007;Zhou et al, 2011). Contrary to the expected results, a significant decrease in processing of LC3bI to LC3bII was observed in cells exposed to cART1 compared with vehicle, indicating a decrease in autophagic activity by this ARVd combination ( Fig.…”

Section: Resultscontrasting

confidence: 54%

Dysregulation of Endoplasmic Reticulum Stress and Autophagic Responses by the Antiretroviral Drug Efavirenz

Bertrand

Toborek

2015

Mol Pharmacol

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Increasing evidence demonstrates that the antiretroviral drugs (ARVds) used for human immunodeficiency virus (HIV) treatment have toxic effects that result in various cellular and tissue pathologies; however, their impact on the cells composing the blood-brain barrier is poorly understood. The current study focused on ARVds, used either in combination or alone, on the induction of endoplasmic reticulum (ER) stress responses in human brain endothelial cells. Among studied drugs (efavirenz, tenofovir, emtricitabine, lamivudine, and indinavir), only efavirenz increased ER stress via upregulation and activation of protein kinase-like ER kinase PERK and inositol requiring kinase 1a (IRE1a). At the same time, efavirenz diminished autophagic activity, a surprising result because typically the induction of ER stress is linked to enhanced autophagy. These results were confirmed in microvessels of HIV transgenic mice chronically administered with efavirenz. In a series of further experiments, we identified that efavirenz dysregulated ER stress and autophagy by blocking the activity of the Beclin-1/ Atg14/PI3KIII complex in regard to synthesis of phosphatidylinositol 3-phosphate, a process that is linked to the formation of autophagosomes. Because autophagy is a protective mechanism involved in the removal of dysfunctional proteins and organelles, its inhibition can contribute to the toxicity of efavirenz or the development of neurodegenerative disease in HIV patients treated with this drug.

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Section: Resultscontrasting

confidence: 54%

Dysregulation of Endoplasmic Reticulum Stress and Autophagic Responses by the Antiretroviral Drug Efavirenz

Bertrand

Toborek

2015

Mol Pharmacol

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“…The ratio of sarcoplasmic LC3-II to LC3-I is reported to correspond to the extent of autophagic-lysosomal activity (25). To determine whether the autophagic-lysosomal pathway is suppressed by administration of Lys, we measured the ratio of LC3-II to LC3-I by Western blotting.…”

Section: Methodsmentioning

confidence: 99%

Regulation of Skeletal Muscle Protein Degradation and Synthesis by Oral Administration of Lysine in Rats

Sato

Ito

Nagasawa

2013

J Nutr Sci Vitaminol

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Summary Several catabolic diseases and unloading induce muscle mass wasting, which causes severe pathological progression in various diseases and aging. Leucine is known to attenuate muscle loss via stimulation of protein synthesis and suppression of protein degradation in skeletal muscle. The aim of this study was to investigate the effects of lysine intake on protein degradation and synthesis in skeletal muscle. Fasted rats were administered 22.8-570 mg Lys/100 g body weight and the rates of myofibrillar protein degradation were assessed for 0-6 h after Lys administration. The rates of myofibrillar protein degradation evaluated by MeHis release from the isolated muscles were markedly suppressed after administration of 114 mg Lys/100 g body weight and of 570 mg Lys/100 g body weight. LC3-II, a marker of the autophagic-lysosomal pathway, tended to decrease (p50.05, 0.08) after Lys intake (114 mg/100 g body weight). However, expression of ubiquitin ligase E3 atrogin-1 mRNA and levels of ubiquitinated proteins were not suppressed by Lys intake. Phosphorylation levels of mTOR, S6K1 and 4E-BP1 in the gastrocnemius muscle were not altered after Lys intake. These results suggest that Lys is able to suppress myofibrillar protein degradation at least partially through the autophagic-lysosomal pathway, not the ubiquitin-proteasomal pathway, whereas Lys might be unable to stimulate protein synthesis within this time frame.

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“…35 GFP-LC3 fluorescence localization studies can be complemented by immunoblotting of LC3. 36 Lipidated LC3 (LC3-II) migrates at a different size by polyacrylamide gel electrophoresis and can be distinguished from unprocessed LC3 (LC3-I). 28 Immunoblotting of LC3 allows further discrimination of specific aspects in autophagy such as the determination of autophagic flux.…”

Section: Quantitation Of Autophagymentioning

confidence: 99%

Quantitation of autophagy by luciferase release assay

Ketteler

Seed²

2008

Autophagy

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Autophagy is a cellular process that has been defined and analyzed almost entirely by qualitative measures. In no small part, this is attributable to the absence of robust quantitative assays that can easily and reliably permit the progress of key steps in autophagy to be assessed. We have recently developed a cell-based assay that specifically measures proteolytic cleavage of a tripartite sensor protein by the autophagy protease ATG4B. Activation of ATG4B results in release of Gaussia luciferase from cells that can be noninvasively harvested from cellular supernatants. Here, we compare this technique to existing methods and propose that this type of assay will be suitable for genome-wide functional screens and in vivo analysis of autophagy.

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Cytosolic LC3 Ratio as a Sensitive Index of Macroautophagy in Isolated Rat Hepatocytes and H4-II-E Cells

Cited by 114 publications

References 30 publications

Dysregulation of Endoplasmic Reticulum Stress and Autophagic Responses by the Antiretroviral Drug Efavirenz

Dysregulation of Endoplasmic Reticulum Stress and Autophagic Responses by the Antiretroviral Drug Efavirenz

Regulation of Skeletal Muscle Protein Degradation and Synthesis by Oral Administration of Lysine in Rats

Quantitation of autophagy by luciferase release assay

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