Autophagy is a major catabolic pathway by which eukaryotic cells degrade and recycle macromolecules and organelles. This pathway is activated under environmental stress conditions, during development and in various pathological situations. In this study, we describe the role of reactive oxygen species (ROS) as signaling molecules in starvation-induced autophagy. We show that starvation stimulates formation of ROS, specifically H2O2. These oxidative conditions are essential for autophagy, as treatment with antioxidative agents abolished the formation of autophagosomes and the consequent degradation of proteins. Furthermore, we identify the cysteine protease HsAtg4 as a direct target for oxidation by H2O2, and specify a cysteine residue located near the HsAtg4 catalytic site as a critical for this regulation. Expression of this regulatory mutant prevented the formation of autophagosomes in cells, thus providing a molecular mechanism for redox regulation of the autophagic process.

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Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4

Given their essential function in aerobic metabolism, mitochondria are intuitively of interest in regard to the pathophysiology of diabetes. Qualitative, quantitative, and functional perturbations in mitochondria have been identified and affect the cause and complications of diabetes. Moreover, as a consequence of fuel oxidation, mitochondria generate considerable reactive oxygen species (ROS). Evidence is accumulating that these radicals per se are important in the pathophysiology of diabetes and its complications. In this review, we first present basic concepts underlying mitochondrial physiology. We then address mitochondrial function and ROS as related to diabetes. We consider different forms of diabetes and address both insulin secretion and insulin sensitivity. We also address the role of mitochondrial uncoupling and coenzyme Q. Finally, we address the potential for targeting mitochondria in the therapy of diabetes. Antioxid. Redox Signal. 12, 537–577.

/pdf/mitochondrial-dysfunction-in-diabetes-from-molecular-23pa7wn6ys.pdf

Mitochondrial Dysfunction in Diabetes: From Molecular Mechanisms to Functional Significance and Therapeutic Opportunities

Free radical scavenging and antioxidant activities of flavonoids extracted from the radix of Scutellaria baicalensis Georgi.

Reactive oxygen species (ROS) are highly reactive molecules that are naturally generated in small amounts during the body’s metabolic reactions and can react with and damage complex cellular molecules such as lipids, proteins, or DNA Acute and chronic ethanol treatments increase the production of ROS, lower cellular antioxidant levels, and enhance oxidative stress in many tissues, especially the liver Ethanol-induced oxidative stress plays a major role in the mechanisms by which ethanol produces liver injury Many pathways play a key role in how ethanol induces oxidative stress This review summarizes some of the leading pathways and discusses the evidence for their contribution to alcohol-induced liver injury Special emphasis is placed on CYP2E1, which is induced by alcohol and is reactive in metabolizing and activating many hepatotoxins, including ethanol, to reactive products, and in generating ROS

Role of oxidative stress in alcohol-induced liver injury

This review describes some of the biochemical and toxicological properties of CYP2E1, especially as it relates to alcohol metabolism and toxicity and the establishment of human hepatoma HepG2 cell lines that overexpress human CYP2E1. Ethanol, polyunsaturated fatty acids, and iron were found to be cytotoxic in HepG2 cells that overexpress CYP2E1. GSH appears to be essential in protecting HepG2 cells against the CYP2E1-dependent cytotoxicity, and GSH levels were elevated owing to a twofold increase in activity and expression of glutamate cysteine ligase. We suggest that this up-regulation of GSH synthesis was an adaptive response to attenuate CYP2E1-dependent oxidative stress and toxicity. Induction of a state of oxidative stress appears to play a central role in the CYP2E1-dependent cytotoxicity. Mitochondrial membrane potential decreased in the CYP2E1-expressing HepG2 cells, and this decrease shared similar characteristics with the developing toxicity. Alcohol-dependent liver injury is likely to be a multifactorial process involving several mechanisms. We believe that the linkage between CYP2E1-dependent oxidative stress, mitochondrial injury, and GSH homeostasis contribute to the toxic actions of ethanol on the liver.

Oxidative Stress, Toxicology, and Pharmacology of CYP2E1*

Iron can potentiate the toxicity of ethanol. Ethanol increases the content of cytochrome P450 2E1 (CYP2E1), which generates reactive oxygen species, and transition metals such as iron are powerful catalysts of hydroxyl radical formation and lipid peroxidation. Experiments were carried out to attempt to link CYP2E1, iron, and oxidative stress as a potential mechanism by which iron increases ethanol toxicity. The addition of ferric-nitrilotriacetate (Fe-NTA) to a HepG2 cell line expressing CYP2E1 decreased cell viability, whereas little effect was observed in control cells not expressing CYP2E1. Toxicity in the CYP2E1-expressing cells was markedly enhanced after the depletion of glutathione. Lipid peroxidation was increased by Fe-NTA, especially in cell extracts and medium from the CYP2E1-expressing cells. Toxicity was completely prevented by vitamin E or by 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, which also decreased the lipid peroxidation. Levels of ATP were lowered by Fe-NTA, and this was associated with a decreased rate of oxygen consumption by permeabilized cells with substrates donating electrons to complexes I, II, and IV of the respiratory chain. This mitochondrial damage was prevented by vitamin E. Toxicity was accompanied by DNA fragmentation, and this fragmentation was prevented by antioxidants. Overexpression of bcl-2 decreased the toxicity and DNA fragmentation produced by the combination of CYP2E1 plus Fe-NTA, as did a peptide inhibitor of caspase 3. These results suggest that elevated generation of reactive oxygen species in HepG2 cells expressing CYP2E1 leads to lipid peroxidation in the presence of iron, and the ensuing prooxidative state damages mitochondria, releasing factors that activate caspase 3, leading to a loss in cell viability and DNA fragmentation.

Oxidative Stress and Cytotoxicity Induced by Ferric-Nitrilotriacetate in HepG2 Cells That Express Cytochrome P450 2E1

To evaluate the participation of mitochondrial damage, oxygen radicals and cell death in diabetes mellitus, we designed a way to investigate INS-1 cells, rat pancreatic β-cell line, to die by treatment with alloxan which generate reactive oxygen species (ROS). Incubation of INS-1 cells with alloxan for 24 h resulted in a decrease in viability of cells as well as inhibition of glucose-stimulated insulin release; this could be prevented by antioxidants, vitamin E and butylated hydroxyanisol (BHA). The formation of a DNA ladder and the distribution of phosphatidylserine at the external surface of plasma membrane were observed as indicators of apoptosis in the cells treated with alloxan at concentrations below 0.5 mM. The formation of DNA ladder was prevented by vitamin E, BHA and catalase, suggesting that the ROS is involved in the process of apoptosis in INS-1 cells treated with alloxan. Lower levels of intracellular ATP, collapse of mitochondrial membrane potential and release of cytochrome c from mitochondria were also observed in INS-1 cells treated with alloxan, suggesting that alloxan caused the damage of mitochondria in cells and was related to the process of apoptosis. In contrast, rat liver RLC-18 cells treated with alloxan were not observed in the decrease of viability. It follows from the present study that mitochondrial damages by ROS generated from alloxan is linked to apoptosis in INS-1 cells.

Apoptosis and mitochondrial damage in INS-1 cells treated with alloxan.

Baicalein decreased the production of thiobarbituric acid reactive substances, the rate of oxygen consumption and iron reduction in the reaction system of ascorbic acid with FeCl3. Superoxide dismutase, catalase and hydroxyl radical scavengers had no significant effect. Iron-chelators had an inhibitory effect similar to that of baicalein. The production of thiobarbituric acid reactive substances of baicalein-treated microsomes obtained by centrifugation after incubation with baicalein was not observed in the reaction system, but was stimulated by adding iron with increases in concentration. The amount of bound iron to microsomal membranes increased by increasing both the concentration of baicalein and iron. The amount of baicalein bound to microsomal membranes increased with increasing concentration of added baicalein. These results suggest that baicalein bound to microsomal membranes inhibits lipid peroxidation by formating an iron-baicalein complex.

https://iubmb.onlinelibrary.wiley.com/doi/pdf/10.1080/15216549600201221

Inhibition of microsomal lipid peroxidation by baicalein: A possible formation of an iron-baicalein complex

The effect of baicalein (5,6,7-trihydroxy-2-phenyl-4H-1-benzopyran-4-one), a flavonoid isolated from Scutellaria baicalensis Georgi, on lipid peroxidation in rat liver microsomes was studied. Ascorbic acid-induced lipid peroxidation in microsomes obtained from baicalein-treated rats was inhibited by treatment on different days and at different doses. Iron release induced by ascorbic acid from microsomes of baicalein-treated rats was markedly lower than from microsomes of control rats. However, no statistical differences in total, nonheme and nonprotein-bound (free iron) iron contents could be detected in the two microsomes. The degradation of calf thymus DNA, an indicator of free iron existence, was observed in the reactions of microsomes obtained from control and baicalein-treated rats with ascorbic acid in the presence of bleomycin. These results suggest that baicalein can inhibit lipid peroxidation in microsomes induced by ascorbic acid by forming an inert complex of iron.

Protection by baicalein against ascorbic acid-induced lipid peroxidation of rat liver microsomes.

Cell cycle arrest is associated with differentiation, senescence and apoptosis. We investigated alterations in the cell cycle during the development of hypertrophy induced by hydrogen peroxide (H(2)O(2)) in the H9c2 clonal myoblastic cell line. H(2)O(2) induced hypertrophy in H9c2 cells that was indicated by an increase in atrial natriuretic peptide (ANP) gene expression, a marker of cardiomyocyte hypertrophy, and a larger cell size. On induction of hypertrophy by H(2)O(2) in H9c2 cells, cell proliferation was arrested, indicated by the number of cells remaining constant during a 72-h incubation period. The cell cycle was arrested at the G1 and G2/M phases with an increase in p21 expression, a negative cell cycle regulator. Cell cycle arrest and increase in p21 expression were significantly inhibited by 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA-AM), an intracellular calcium chelator. Although ANP gene expression was induced significantly, H(2)O(2) failed to induce hypertrophy in the presence of BAPTA-AM, and the cell cycle progressed. We concluded that H(2)O(2) induced cell cycle arrest in H9c2 cells, which was related to cellular hypertrophy.

Koichi Sakurai

Papers

Oxidative Stress and Cytotoxicity Induced by Ferric-Nitrilotriacetate in HepG2 Cells That Express Cytochrome P450 2E1

Apoptosis and mitochondrial damage in INS-1 cells treated with alloxan.

Inhibition of microsomal lipid peroxidation by baicalein: A possible formation of an iron-baicalein complex

Protection by baicalein against ascorbic acid-induced lipid peroxidation of rat liver microsomes.

Hydrogen peroxide induces cell cycle arrest in cardiomyoblast H9c2 cells, which is related to hypertrophy.