Cytosolic LC3 ratio as a sensitive index of macroautophagy in isolated rat hepatocytes and H4-II-E cells.
Macroautophagy, an intracellular bulk degradation process in eukaryotes, is sensitive to nutrient supply and deprivation. Microtubule-associated protein 1 light chain 3 (LC3), a mammalian homologue of yeast Atg8, plays an indispensable role in macroautophagy formation and is a suitable marker for this process. Through analysis of the subcellular distribution of LC3, we determined that the cytosolic fraction contained not only a precursor form (LC3-I), but also an apparent active form (LC3-IIs). Both cytosolic LC3-I and LC3-IIs were more responsive to amino acids than those of total homogenate. Moreover, changes in the LC3-IIs/I ratio reflected those in the total proteolytic flux remarkably in both fresh rat hepatocytes and H4-II-E cell lines. Thus, in addition to a sensitive index of macroautophagy, calculating the cytosolic LC3 ratio became an easy and quick quantitative method for monitoring its regulation in hepatocytes and H4-II-E cells.
Karim
Md Razaul
MR
Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University, Niigata, Japan.
Kanazawa
Takumi
T
Daigaku
Yasuhiro
Y
Fujimura
Shinobu
S
Miotto
Giovanni
G
Kadowaki
Motoni
M
D000818:Animals; D001343:Autophagy; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000473:pathology; D045744:Cell Line, Tumor; D003600:Cytosol / Q000378:metabolism*; D022781:Hepatocytes / Q000378:metabolism*; D006868:Hydrolysis; D008113:Liver Neoplasms / Q000473:pathology; D008297:Male; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008954:Models, Biological; D051381:Rats; D017208:Rats, Wistar; D012680:Sensitivity and Specificity; D013347:Subcellular Fractions / Q000378:metabolism
D015415:Biomarkers; D008869:Microtubule-Associated Proteins; C526109:Anxa3 protein, rat