74 research outputs found
Composition of the redox environment of the endoplasmic reticulum and sources of hydrogen peroxide
The endoplasmic reticulum (ER) is a metabolically active organelle, which has a central role in proteostasis by translating, modifying, folding, and occasionally degrading secretory and membrane proteins. The lumen of the ER represents a separate compartment of the eukaryotic cell, with a characteristic proteome and metabolome. Although the redox metabolome and proteome of the compartment have not been holistically explored, it is evident that proper redox conditions are necessary for the functioning of many luminal pathways. These redox conditions are defined by local oxidoreductases and the membrane transport of electron donors and acceptors. The main electron carriers of the compartment are identical with those of the other organelles: glutathione, pyridine and flavin nucleotides, ascorbate, and others. However, their composition, concentration, and redox state in the ER lumen can be different from those observed in other compartments. The terminal oxidases of oxidative protein folding generate and maintain an "oxidative environment" by oxidizing protein thiols and producing hydrogen peroxide. ER-specific mechanisms reutilize hydrogen peroxide as an electron acceptor of oxidative folding. These mechanisms, together with membrane and kinetic barriers, guarantee that redox systems in the reduced or oxidized state can be present simultaneously in the lumen. The present knowledge on the in vivo conditions of ER redox is rather limited; development of new genetically encoded targetable sensors for the measurement of the luminal state of redox systems other than thiol/disulfide will contribute to a better understanding of ER redox homeostasis
Mikroszómális glukóz-6-foszfát szerepe granulocita apoptózisában = Apoptotic role of microsomal glucose-6-phosphate in granulocytes
Az endoplazmás retikulum glukĂłz-6-foszfát transzporterĂ©re specifikus poliklonális antitestet termeltettĂĽnk, amely patkány Ă©s humán glukĂłz-6-foszfát transzportert ismer fel. Western blottal Ă©s immunhisztomkĂ©miai mĂłdszerekkel közvetlenĂĽl bizonyĂtottuk a transzporter jelenlĂ©tĂ©t humán májbĂłl, patkány májbĂłl Ă©s vesĂ©bĹ‘l származĂł mikroszĂłmákban; humán granulocitákban, patkány agyban Ă©s harántcsĂkolt izomszövetben. Kimutattuk, hogy humán granulociták Ă©s differenciálĂłdott HL60-as sejtekben megtalálhatĂł a hexĂłz-6-foszfát dehidrogenáz, amelynek a működĂ©se fĂĽgg a glukĂłz-6-foszfáttĂłl, aktĂv centruma az endoplazmás retikulum lumenĂ©ben találhatĂł Ă©s NADPH-t generál. A keletkezett 6-foszfoglukonátot a granulocita mikroszĂłmák nem metabolizálják tovább. mRNS Ă©s fehĂ©rje szinten kimutattuk 1-es tĂpusĂş 11-bĂ©ta-hidroxiszteriod dehidrogenáz jelenlĂ©tĂ©t granulociták mikroszĂłmáiban. Az enzim granulocitákban is oxidoreduktázkĂ©nt működik, működĂ©sĂ©hez intraluminális NADPH(NADP)-t használ fel. Az enzim NADPH-t generálĂł kortizol dehidrogenáz aktivitásával kivĂ©dtĂĽk a glukĂłz-6-foszfát transzport gátlásával lĂ©trehozott apoptĂłzist. EredmĂ©nyeink szerint a májhoz hasonlĂłan humán granulocitákban is megtalálhatĂł a glukĂłz-6-foszfát - hexĂłz-6-foszfát Ă©s 11-bĂ©ta-hidroxiszeriod dehidrogenáz 1 funkcionális egysĂ©ge. | We employed antibodies raised against the liver glucose-6-phoshphate transporter protein. With different immunodetection methods, we have shown that the protein is expressed in endoplasmic reticulum membranes derived from human and rat liver and rat kidney. The expression was also present in the following tissues: human granulocytes, rat brain and skeletal muscle. We have demonstrated the expression of hexose-6-phosphate dehydrogenase in human neutrophils and mature HL60-cells. Its catalytic subunit is intraluminal and activity of the enzyme was shown to be dependent on glucose-6-phosphate and produces NADPH. 6-phosphogluconate formed in this reaction was not metabolized further. The expression and activity of 11-beta-hydroxysteroid dehydrogenase type 1, another NADP(H)-dependent microsomal enzyme were also detected in human neutrophils. The NADPH-generating cortisol dehydrogenase activity of the enzyme prevented neutrophil apoptosis provoked by the inhibition of the glucose-6-phosphate transporter. The results show that granulocytes are equipped with a functional glucose-6-phosphate- hexose-6-phosphate dehydrogenase-11beta-hydroxysteroid dehydrogenase type 1 system
Analytical Approaches for the Quantitation of Redox-active Pyridine Dinucleotides in Biological Matrices
Some of the main electron carriers in the metabolism are mono- or dinucleotides and they play crucial roles in maintaining a balanced redox homeostasis of cells, and in coupling many anabolic and catabolic reactions. Altered cellular redox status can be an indicator of various metabolic disorders such as obesity, the metabolic syndrome, or type 2 diabetes and of other pathological conditions, which involve oxidative stress, such as cardiovascular diseases. Adequate NAD+/NADH and NADP+/NADPH ratios are fundamental for normal cellular functions, thus accurate measurement of these pyridine dinucleotides is essential in biochemical research. Liquid chromatography coupled to tandem mass spectrometry has become the leading analytical technology in (targeted) state-of-the-art metabolic profiling. Main difficulties that hamper quantification of metabolites are chemical similarities, high polarity, and chemical and biological instability of the molecules to be measured. In this review, some critical steps of studying cellular redox status are described, in particular, different techniques of sample preparation and challenges in chromatographic separation
A glukóz-6-foszfatáz rendszer extrahepatikus megjelenésének patofiziológiai szerepe = The pathophysiological role of the extrahepatic manifestations of glucose-6-phosphatase system
A mikroszĂłmális glukĂłz-6-foszfát transzporter extrahepatikus nem glukoneogenetikus sejt-tipusokban törtĂ©nĹ‘ manifesztáciĂłit vizsgálva kimutattuk a fehĂ©rje jelenlĂ©tĂ©t Ă©s funkciĂłját humán granulocitákban Ă©s patkány epididimális zsĂrszövetben. EredmĂ©nyeink alapján valĂłszĂnűsĂthetĹ‘, hogy a nem-glukoneogenetikus szövetekben a transzporter legfontosabb feladata a hexĂłz-6-foszfát dehidrogenáz szubsztrátellátása, s Ăgy az intraluminális pentĂłz foszfát ciklusban a NADPH generálása. A NADPH termelĂ©s cĂ©lja lehet egyrĂ©szt az antioxidáns homeosztázis fenntartása (granulocita), vagy a 11β-hidroxiszteroid dehidrogenáz 1-es tĂpusának kofaktor ellátása (máj, zsĂrszövet) s ennek rĂ©vĂ©n rĂ©szvĂ©tel a kortizon termelĂ©sben. Az eredmĂ©nyekbĹ‘l következĹ‘en a glukĂłz-6-foszfát transzporter Ă©s a hexĂłz-6-foszfát dehidrogenáz fontos terápiás cĂ©lpont lehet a metabolikus szindrĂłma gyĂłgyszeres kezelĂ©sĂ©ben. | The presence and function of the microsomal glucose-6-phosphate transporter was investigated in certain non gluconeogenic cell-types, such as human granulocytes and rat epidydymal adipose tissues. Based on our observations the substrate supply for the hexose-6-phosphate dehydrogenase and this way the generation of NADPH in the intralumenal pentose- phosphate pathway is one of the main tasks of the transporter. Thus NADPH production can participate in the maintenance of the antioxidant homeostasis in granulocytes, or in liver and adipose tissues it can contribute to the cofactor supply for the 11β-hydroxysteroid dehydrogenase type 1 and this way it is involved in cortisone production. It is suggested that glucose-6-phosphate transporter and hexose-6-phosphate dehydrogenase are potential therapeutic targets in treatment of the metabolic syndrome
Antioxidáns anyagcsere és transzportfolyamatok az endo/szarkoplazmás retikulumban. = Antioxidant metabolism and transport processes in the endo/sarcoplasmic reticulum.
Kimutattuk, hogy a glutation-transzport sebessĂ©ge jĂłl korrelál a rianodin receptor kalcium csatorna (RyR) egyik izoformája, a RyR1 mennyisĂ©gĂ©vel. KĂĽlönbözĹ‘ RyR gátlĂłszerek hatĂ©konyan gátolták, RyR agonisták pedig aktiválták a transzportot. Ă–sszehasonlĂtottuk a glutation transzport sajátosságait RyR1 csatornát expresszálĂł transzfektált, illetve vad tĂpusĂş HEK293 sejtekbĹ‘l izolált mikroszĂłmákon. EredmĂ©nyeink azt bizonyĂtják, hogy a RyR1 közvetlenĂĽl vesz rĂ©szt a glutation transzportjában. MegállapĂtottuk továbbá, hogy a RyR1 csatorna magas intraluminális glutation szint esetĂ©n rezisztenssĂ© válik egyes gátlĂłszereivel szemben. Kimutattuk, hogy az ER lumenben elhelyezkedĹ‘ hexĂłz-6-foszfát dehidrogenáz fenntartja a lokális redukált NADPH szintet. Igazoltuk, hogy a glutation reduktáz enzim, amely a sejt egyĂ©b rĂ©szein a tiol/diszulfid Ă©s a piridin nukleotid redox rendszerek közötti kapcsolatot biztosĂtja, nincs jelen az ER lumenben. Skorbutizált tengerimalacok májában a skorbut kialkulásával párhuzamosan az ER stressz jeleit Ă©s fokozott apoptĂłzist Ă©szleltĂĽk. A kimutatott ER-stressz Ă©s apoptĂłzis alátámasztja az aszkorbát szerepĂ©t a fehĂ©rje-foldingban. Kidolgoztunk egy Ăşj mĂłdszert, amely lehetĹ‘vĂ© teszi jelöletlen molekulák mikroszĂłmális transzportjának tanulmányozását. Az Ăşj mĂłdszer több vegyĂĽlet egyidejű transzportjának mĂ©rĂ©sĂ©re Ă©s a ligand stabilitásának nyomonkövetĂ©sĂ©re is alkalmas, Ă©rzĂ©kenysĂ©ge hasonlĂł a radioaktĂv detektálásĂ©hoz, viszont gazdaságosabb annál. | We found a correlation between the rate of glutathione transport and the abundance of an isoform of ryanodine receptor calcium channel (RyR1). The transport was inhibited or activated by various RyR antagonists or agonists, respectively. The characteristics of glutathione transport were compared in microsomes prepared from RyR1 expressing transfected and wild type HEK293 cells. Our results prove the direct involvement of RyR1 in glutathione transport. In addition, we found that RyR1 channel becomes resistant to some of its inhibitors at high luminal glutathione levels. We observed that hexose 6-phosphate dehydrogenase maintains the reduced NADPH pool in the ER lumen. We proved that glutathione reductase - that couples the thiol/disulfide and pyridine nucleotide redox systems in other cellular compartments - is not present in the ER. We detected the indicators of ER stress and enhanced apoptosis in the liver of scorbutic guinea pigs. These findings further support the hypothesis that ascorbate participates in the process of protein folding in the ER. We developed a new method that allows the investigation of the microsomal transport of unlabeled compounds. The novel technique is suitable for the detection of simultaneous multiple transport processes and for the monitoring of the ligand stability. Its sensitivity is similar to that of the transport measurements based on radiodetection
Epigallocatechin-3-Gallate (EGCG) Promotes Autophagy-Dependent Survival via Influencing the Balance of mTOR-AMPK Pathways upon Endoplasmic Reticulum Stress
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
Simultaneous Quantitative Determination of Different Ceramide and Diacylglycerol Species in Cultured Cells by Using Liquid Chromatography–Electrospray Tandem Mass Spectrometry
A sensitive, reproducible reverse-phased high performance liquid chromatography electrospray tandem mass spectrometry (HPLC-ESI-MS/MS) method with simple sample preparation was developed for the simultaneous determination of a wide range of ceramides, diacylglycerols (DAGs) in cultured cells. Chromatographic separation of the compounds was achieved in a 14-minute run using a C8 column with a gradient elution by methanol and 10 mM ammonium acetate buffer as mobile phase at a flow rate of 0.5 ml/min. Various ceramides, DAGs were detected with a triple quadrupol system in multiple reaction monitoring mode, which is based on a soft positive electrospray ionization. The usual sample preparation process was shortened by the application of pure methanol for the extraction instead of the widely used methanol/chloroform mixture. C17:0 ceramide which does not occur in the cell samples, was used as an internal standard. The sample preparation process was optimized and the methodology was tested on a human hepatocarcinoma cell culture. Our results clearly showed accumulation of some ceramides and DAGs in the cells treated with BSA-conjugated palmitate for 8 hours. Since both ceramides and DAGs are important lipid intermediates and signal messengers, alteration in their cellular levels have major impact on cell functions, and thus our novel analytic method can be widely used in lipotoxicity research. The presented technique can be further developed to measure other intermediates of ceramide synthesis and other derivatives of DAGs as well
Lipotoxicity in the liver
Obesity due to excessive food intake and the lack of physical activity is becoming one of the most serious public health problems of the 21st century. With the increasing prevalence of obesity, non-alcoholic fatty liver disease is also emerging as a pandemic. While previously this pathophysiological condition was mainly attributed to triglyceride accumulation in hepatocytes, recent data show that the development of oxidative stress, lipid peroxidation, cell death, inflammation and fibrosis are mostly due to accumulation of fatty acids, and the altered composition of membrane phospholipids. In fact, triglyceride accumulation might play a protective role, and the higher toxicity of saturated or trans fatty acids seems to be the consequence of a blockade in triglyceride synthesis. Increased membrane saturation can profoundly disturb cellular homeostasis by impairing the function of membrane receptors, channels and transporters. However, it also induces endoplasmic reticulum stress via novel sensing mechanisms of the organelle's stress receptors. The triggered signaling pathways in turn largely contribute to the development of insulin resistance and apoptosis. These findings have substantiated the lipotoxic liver injury hypothesis for the pathomechanism of hepatosteatosis. This minireview focuses on the metabolic and redox aspects of lipotoxicity and lipoapoptosis, with special regards on the involvement of endoplasmic reticulum stress responses
Application of Gas Chromatography – Flame Ionization Detection to Study Cellular Incorporation of Dietary Trans Fatty Acids of Medical Importance
Putative health effects of dietary trans fatty acids (TFAs) receive a growing attention; while very little is known about the metabolism of these special food components. In vitro studies carried out in cultured cells provide an efficient and standardizable approach to follow the metabolic fate of TFAs, but it requires suitable techniques for the quantitative measurement of FAs in cell samples. Here, the development and validation of a simple and reliable method for the quantification of a group of relevant FAs by gas chromatography and flame ionization detection is presented. Sample preparation used a fast one-step and chloroform-free process for simultaneous extraction and esterification, and chromatographic separation was achieved in 25 min using a Zebron ZB-88 capillary column. A linear calibration (of R2 >0.99) was obtained in the concentration range of 1-200 µg/mL for each FA. Recovery rate was 82 % for samples of non-esterified FAs and >95 % for complex lipids, such as ceramides, diglycerides and triglycerides. The LOD and LOQ were below 0.5 µg/mL, and a robust method precision was achieved (RSD % was below 6 % for each lipid classes). The present method was also tested on a cultured cell line with or without FA treatment at close to physiological concentration, and the observed changes in the metabolite concentration levels revealed characteristic differences between the metabolism of cis and trans unsaturated FAs
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