ABSTRACT
It has been recently reported that tea flavanols, including epigallocatechin gallate (EGCG), efficiently inhibit glucosidase II in liver microsomes. Since glucosidase II plays a central role in glycoprotein processing and quality control in the endoplasmic reticulum we investigated the possible contribution of endoplasmic reticulum stress and unfolded protein response (UPR) to the pro-apoptotic activity of EGCG in mouse hepatoma cells. The enzyme activity measurements using 4-methylumbelliferyl-alpha-d-glucopyranoside substrate confirmed the inhibition of glucosidase II in intact and alamethicin-permeabilized cells. EGCG treatment caused a progressive elevation of apoptotic activity as assessed by annexin staining. The induction of CHOP/GADD153, the cleavage of procaspase-12 and the increasing phosphorylation of eIF2alpha were revealed in these cells by Western blot analysis while the induction of endoplasmic reticulum chaperones and foldases was not observed. Time- and concentration-dependent depletion of the endoplasmic reticulum calcium stores was also demonstrated in the EGCG-treated cells by single-cell fluorescent detection. The massive alterations in the endoplasmic reticulum morphology revealed by fluorescent microscopy further supported the development of UPR. Collectively, our results indicate that EGCG interferes with protein processing in the endoplasmic reticulum presumably due to inhibition of glucosidase II and that the stress induces an incomplete unfolded protein response with dominantly pro-apoptotic components.
Subject(s)
Antineoplastic Agents/pharmacology , Catechin/analogs & derivatives , Glycoside Hydrolase Inhibitors , Liver Neoplasms, Experimental/enzymology , Transcription Factor CHOP/metabolism , Animals , Apoptosis/drug effects , Calcium/metabolism , Catechin/pharmacology , Dose-Response Relationship, Drug , Endoplasmic Reticulum/metabolism , Eukaryotic Initiation Factor-2/metabolism , Fluorescent Antibody Technique , Glucosides/metabolism , Hymecromone/analogs & derivatives , Hymecromone/metabolism , Liver Neoplasms, Experimental/pathology , Mice , Phosphorylation , Protein Folding , Stress, Physiological , Time Factors , Transcription Factor CHOP/genetics , alpha-GlucosidasesABSTRACT
11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) is a NADPH dependent oxidoreductase of the endoplasmic reticulum lumen which converts cortisone to cortisol and plays a role in the pathogenesis of metabolic syndrome and type 2 diabetes. The aim of our study was to investigate the correlation between the expression/activity of 11betaHSDI and obesity. Liver and adipose tissue microsomes of an obese (Zucker) and a non-obese (Goto-Kakizaki) type 2 diabetes model rat strains were used. 11betaHSDI expression was detected at mRNA, protein and activity level. The activity of 11betaHSD1 was increased in the adipose tissue and decreased in the liver of the obese Zucker rat, while its mRNA levels were significantly different only in the adipose tissue. In diabetic Goto-Kakizaki rat both the expression and the activity of 11betaHSD1 were elevated in liver, but not in adipose tissue. These results suggest that the prereceptorial glucocorticoid activation is different in the liver and adipose tissue of the two diabetes models. This phenomenon might be responsible for the obese and lean phenotypes in type 2 diabetes.
Subject(s)
11-beta-Hydroxysteroid Dehydrogenase Type 1/genetics , 11-beta-Hydroxysteroid Dehydrogenase Type 1/metabolism , Body Weight , Diabetes Mellitus, Type 2/metabolism , Gene Expression Regulation, Enzymologic , Obesity/metabolism , Adipose Tissue/enzymology , Animals , Diabetes Mellitus, Type 2/genetics , Diabetes Mellitus, Type 2/physiopathology , Disease Models, Animal , Enzyme Activation , Glucocorticoids/metabolism , Liver/enzymology , Obesity/genetics , Obesity/physiopathology , RNA, Messenger/metabolism , Rats , Rats, ZuckerABSTRACT
Addition of ascorbate or its generation from gulonolactone causes the oxidation of protein thiols and a simultaneous dehydroascorbate formation in rat liver microsomes. The participation of vitamin E in the phenomenon was studied. We measured ascorbate and protein thiol oxidation and lipid peroxidation in vitamin E deficient liver microsomes. Vitamin E deficiency partly uncoupled the two processes: ascorbate oxidation increased, while protein thiol oxidation decreased. These changes were accompanied with an accelerated lipid peroxidation in the vitamin E-deficient microsomes, which indicates the accumulation of reactive oxygen species. All these effects were reduced by the in vitro addition of vitamin E to the deficient microsomes, supporting its direct role in the process. The results demonstrate that vitamin E is a component of the protein thiol oxidizing machinery in the hepatic endoplasmic reticulum transferring electrons from the thiol groups towards oxygen.
