RESUMO
High fatty acid (FA) levels are deleterious to pancreatic ß-cells, largely due to the accumulation of biosynthetic lipid intermediates, such as ceramides and diglycerides, which induce ER stress and apoptosis. Toxicity of palmitate (16:0) and oleate (18:1 cis-Δ9) has been widely investigated, while very little data is available on the cell damages caused by elaidate (18:1 trans-Δ9) and vaccenate (18:1 trans-Δ11), although the potential health effects of these dietary trans fatty acids (TFAs) received great publicity. We compared the effects of these four FAs on cell viability, apoptosis, ER stress, JNK phosphorylation and autophagy as well as on ceramide and diglyceride contents in RINm5F insulinoma cells. Similarly to oleate and unlike palmitate, TFAs reduced cell viability only at higher concentration, and they had mild effects on ER stress, apoptosis and autophagy. Palmitate increased ceramide and diglyceride levels far more than any of the unsaturated fatty acids; however, incorporation of TFAs in ceramides and diglycerides was strikingly more pronounced than that of oleate. This indicates a correlation between the accumulation of lipid intermediates and the severity of cell damage. Our findings reveal important metabolic characteristics of TFAs that might underlie a long term toxicity and hence deserve further investigation.
Assuntos
Ceramidas/metabolismo , Gorduras Insaturadas na Dieta/toxicidade , Diglicerídeos/metabolismo , Ácido Oleico/toxicidade , Ácidos Oleicos/toxicidade , Ácidos Graxos trans/toxicidade , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Gorduras Insaturadas na Dieta/análise , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/patologia , MAP Quinase Quinase 4/metabolismo , Necrose/induzido quimicamente , Ácido Oleico/análise , Ácidos Oleicos/análise , Ácidos Palmíticos/análise , Ácidos Palmíticos/toxicidade , Fosforilação , Ratos , Ácidos Graxos trans/análiseRESUMO
The maintenance of cellular homeostasis is largely dependent on the ability of cells to give an adequate response to various internal and external stimuli. We have recently proposed that the life-and-death decision in endoplasmic reticulum (ER) stress response is defined by a crosstalk between autophagy, apoptosis, and mTOR-AMPK pathways, where the transient switch from autophagy-dependent survival to apoptotic cell death is controlled by GADD34. The aim of the present study was to investigate the role of epigallocatechin-3-gallate (EGCG), the major polyphenol of green tea, in promoting autophagy-dependent survival and to verify the key role in connecting GADD34 with mTOR-AMPK pathways upon prolonged ER stress. Our findings, obtained by using HEK293T cells, revealed that EGCG treatment is able to extend cell viability by inducing autophagy. We confirmed that EGCG-induced autophagy is mTOR-dependent and PKA-independent; furthermore, it also required ULK1. We show that pretreatment of cells with EGCG diminishes the negative effect of GADD34 inhibition (by guanabenz or siGADD34 treatment) on autophagy. EGCG was able to delay apoptotic cell death by upregulating autophagy-dependent survival even in the absence of GADD34. Our data suggest a novel role for EGCG in promoting cell survival via shifting the balance of mTOR-AMPK pathways in ER stress.
Assuntos
Catequina/análogos & derivados , Estresse do Retículo Endoplasmático/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Chá/química , Autofagia/efeitos dos fármacos , Catequina/farmacologia , Catequina/uso terapêutico , Sobrevivência Celular , HumanosRESUMO
Effect of 5-100 microM epigallocatechin gallate (EGCG) on hepatic glucose 6-phosphatase (G6Pase) system was investigated. EGCG inhibited G6Pase in intact but not in permeabilized rat liver microsomes, suggesting the interference with the transport. However, EGCG did not hinder microsomal glucose 6-phosphate (G6P) uptake. Instead, it increased the accumulation of radioactivity after the addition of [(14)C]G6P, presumably due to a slower release of [(14)C]glucose, the product of luminal hydrolysis. Indeed, EGCG was found to inhibit microsomal glucose efflux. Since G6Pase activity is depressed by glucose in a concentration-dependent manner, we concluded that EGCG inhibits G6Pase through an elevated luminal glucose level.
Assuntos
Catequina/análogos & derivados , Flavonóis/farmacologia , Glucose-6-Fosfatase/antagonistas & inibidores , Fígado/enzimologia , Chá/química , Animais , Catequina/isolamento & purificação , Catequina/farmacologia , Flavonóis/isolamento & purificação , Glucose/farmacologia , Glucose-6-Fosfatase/análise , Glucose-6-Fosfato/metabolismo , Fígado/efeitos dos fármacos , Fígado/ultraestrutura , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , RatosRESUMO
Toxic endogenous or exogenous compounds can be inactivated by various conjugation reactions. Glucuronidation (i.e. conjugation with glucuronate) is especially important due to the large number of drugs and chemical carcinogens that are detoxified through this pathway. Stable and harmless glucuronides can be reactivated by enzymatic hydrolysis thus inhibitors of glucuronidase activity reduce the risk of chemical carcinogenesis. The aim of this study was to reveal whether this mechanism contributes to the anti-cancer effect of green tea flavanols, which has been shown in various animal models. Therefore, we investigated the effect of these polyphenols on deglucuronidation in rat liver microsomes and in Hepa 1c1c7 mouse hepatoma cells, using 4-methylumbelliferyl glucuronide as model substrate. Tea flavanols inhibited beta-glucuronidase in intact vesicles, where glucuronide transport across the microsomal membrane is rate-limiting, but were almost ineffective in permeabilized vesicles. Epigallocatechin gallate, the major green tea flavanol was shown to have a concentration-dependent inhibitory effect on both beta-glucuronidase activity and glucuronide transport in native vesicles. Epigallocatechin gallate also inhibited beta-glucuronidase activity in native Hepa 1c1c7 mouse hepatoma cells, while failed to affect the enzyme in alamethicin-permeabilized cells, where the endoplasmic membrane barrier was eliminated. Our findings indicate that tea flavanols inhibit deglucuronidation in the endoplasmic reticulum at the glucuronide transport stage. This phenomenon might potentially contribute to the cancer-preventing dietary or pharmacological effect attributed to these catechins.
Assuntos
Catequina/análogos & derivados , Retículo Endoplasmático/metabolismo , Flavonoides/farmacologia , Glucuronídeos/metabolismo , Fenóis/farmacologia , Chá/química , Animais , Anticarcinógenos/farmacologia , Transporte Biológico/efeitos dos fármacos , Catequina/química , Catequina/farmacologia , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Retículo Endoplasmático/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Glucuronidase/metabolismo , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Polifenóis , Ratos , Ratos WistarRESUMO
Green tea is getting into the focus of scientific interest due to its beneficial health effects, most of which are attributed to its catechin content. Polyphenolic tea catechins have antioxidant, antiproliferative, antiangiogenic and proapoptotic effects, which makes them promising anticancer compounds. Other poly-hydroxy molecules have similar antitumor potentials through the inhibition of glucosidase II, which affects the glycoprotein maturation and quality control in the endoplasmic reticulum. We investigated the effect of tea catechins on glucosidase II activity in rat liver microsomes using 4-methylumbelliferyl glucoside and 4-nitrophenyl glucoside as substrates. A concentration-dependent inhibition with non-competitive kinetics was found. The IC50 and Ki values for certain tea catechins were comparable with those of N-butyldeoxynojirimycin, the widely used glucosidase inhibitor. The possible interference of tea catechins with the glycoprotein processing in the endoplasmic reticulum should be considered as a potential mechanism of their dietary or pharmacological effects.
Assuntos
Flavonóis/farmacologia , Inibidores de Glicosídeo Hidrolases , Chá/química , Animais , Catequina/análogos & derivados , Catequina/farmacologia , Cromatografia Líquida de Alta Pressão , Cinética , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Ratos , Ratos Wistar , alfa-GlucosidasesRESUMO
Insufficient ascorbate intake causes scurvy in certain species. Beyond its known functions, it has been suggested that ascorbate participates in oxidative protein folding in the endoplasmic reticulum (ER). Because redox imbalance in this organelle might cause ER stress and apoptosis, we hypothesized that this might contribute to the pathology of scurvy. Guinea pigs were divided into 7 groups: the control group was fed a commercial guinea pig food containing 0.1 g/100 g ascorbate for 4 wk, 5 groups consumed an ascorbate-free food for 0, 1, 2, 3, or 4 wk and 1 group was fed this scorbutic diet for 2 wk and then the commercial food plus 1 g/L ascorbate in drinking water for 2 wk. TBARS generation and the expression of some ER chaperones and foldases were determined in hepatic microsomes. The apoptotic index was assessed in histological sections. Although ascorbate, measured by HPLC, was undetectable in the livers of the guinea pigs after they had consumed the scorbutic diet for 2 wk, the microsomal TBARS level was elevated relative to the initial value only at wk 4. Western blot revealed the induction of GRP78, GRP94, and protein disulfide isomerase at wk 3 and 4. Apoptosis was greater than in the control, beginning at wk 3. None of the alterations occurred in the groups fed the commercial guinea pig food or ascorbate-free food followed by ascorbate supplementation. Therefore, persistent ascorbate deficiency leads to ER stress, unfolded protein response, and apoptosis in the liver, suggesting that insufficient protein processing participates in the pathology of scurvy.