Miklós Csala

Ascorbate metabolism and its regulation in animals

This article provides a comprehensive review on ascorbate metabolism in animal cells, especially in hepatocytes. The authors deal with the synthesis and the breakdown of ascorbate as a part of the antioxidant and carbohydrate metabolism. Hepatocellular and interorgan cycles with the participation of ascorbate are proposed, based on experiments with murine and human cells; reactions of hexuronic acid pathway, non-oxidative branch of the pentose phosphate cycle, glycolysis and gluconeogenesis are involved. Besides the well-known redox coupling between the two major water-soluble antioxidants (glutathione and ascorbate), their metabolic links have been also outlined. Glycogenolysis as a major source of UDP-glucuronic acid determines the rate of hexuronic acid pathway leading to ascorbate synthesis. Glycogenolysis is regulated by oxidized and reduced glutathione; therefore, glycogen, ascorbate and glutathione metabolism are related to each other. Hydrogen peroxide formation, due to the activity of gulonolactone oxidase catalyzing the last step of ascorbate synthesis, also affects the antioxidant status in hepatocytes. Based on new observations a complex metabolic regulation is supposed. Its element might be present also in humans who lost gulonolactone oxidase but they need and metabolize ascorbate. Finally, the obvious disadvantages and the possible advantages of the lost ascorbate synthesizing ability in humans are considered.

Endoplasmic reticulum: nutrient sensor in physiology and pathology

The endoplasmic reticulum (ER) is a metabolic organelle and an ideal nutrient sensor. In response to hypoglycemia, hyperglycemia or fatty acid overload, the ER triggers the unfolded protein response, which represses protein synthesis, alters insulin responsiveness and favors apoptosis. In addition, the ER affects steroid hormone activation and autophagy. The primary aim of these responses is to adjust the metabolism to environmental changes. Failure of the ER to adapt to changes in nutrient availability can result in a pathological transition in ER functions, as observed in cases of obesity-related diseases. This review highlights the recent evidence that the ER has a prominent role in cellular adaptation, as well as in the pathomechanism of type 2 diabetes.

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 21(st) 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 organelles 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.

Redox Control of Endoplasmic Reticulum Function

The lumen of the endoplasmic reticulum constitutes a separate intracellular compartment with a special proteome and metabolome. The redox conditions of the organelle are also characteristically different from those of the other subcellular compartments. The luminal environment has been considered more oxidizing than the cytosol due to the presence of oxidative protein folding. However, recent observations suggest that redox systems in reduced and oxidized states are present simultaneously. The concerted action of membrane transporters and oxidoreductase enzymes maintains the oxidized state of the thiol-disulfide and the reduced state of the pyridine nucleotide redox systems, which are prerequisites for the normal redox reactions localized in the organelle. The powerful thiol-oxidizing machinery of oxidative protein folding continuously challenges the local antioxidant defense. Alterations of the luminal redox conditions, either in oxidizing or reducing direction, affect protein processing, are sensed by the accumulation of misfolded/unfolded proteins, and may induce endoplasmic reticulum stress and unfolded protein response. The activated signaling pathways attempt to restore the balance between protein loading and processing and induce programmed cell death if these attempts fail. Recent findings strongly support the involvement of redox-based endoplasmic reticulum stress in a plethora of human diseases, either as causative agents or as complications.

Uncoupled Redox Systems in the Lumen of the Endoplasmic Reticulum PYRIDINE NUCLEOTIDES STAY REDUCED IN AN OXIDATIVE ENVIRONMENT

The redox state of the intraluminal pyridine nucleotide pool was investigated in rat liver microsomal vesicles. The vesicles showed cortisone reductase activity in the absence of added reductants, which was dependent on the integrity of the membrane. The intraluminal pyridine nucleotide pool could be oxidized by the addition of cortisone or metyrapone but not of glutathione. On the other hand, intraluminal pyridine nucleotides were slightly reduced by cortisol or glucose 6-phosphate, although glutathione was completely ineffective. Redox state of microsomal protein thiols/disulfides was not altered either by manipulations affecting the redox state of pyridine nucleotides or by the addition of NAD(P)+ or NAD(P)H. The uncoupling of the thiol/disulfide and NAD(P)+/NAD(P)H redox couples was not because of their subcompartmentation, because enzymes responsible for the intraluminal oxidoreduction of pyridine nucleotides were distributed equally in smooth and rough microsomal subfractions. Instead, the phenomenon can be explained by the negligible representation of glutathione reductase in the endoplasmic reticulum lumen. The results demonstrated the separate existence of two redox systems in the endoplasmic reticulum lumen, which explains the contemporary functioning of oxidative folding and of powerful reductive reactions.

On the role of 4-hydroxynonenal in health and disease

Polyunsaturated fatty acids are susceptible to peroxidation and they yield various degradation products, including the main α,β-unsaturated hydroxyalkenal, 4-hydroxy-2,3-trans-nonenal (HNE) in oxidative stress. Due to its high reactivity, HNE interacts with various macromolecules of the cell, and this general toxicity clearly contributes to a wide variety of pathological conditions. In addition, growing evidence suggests a more specific function of HNE in electrophilic signaling as a second messenger of oxidative/electrophilic stress. It can induce antioxidant defense mechanisms to restrain its own production and to enhance the cellular protection against oxidative stress. Moreover, HNE-mediated signaling can largely influence the fate of the cell through modulating major cellular processes, such as autophagy, proliferation and apoptosis. This review focuses on the molecular mechanisms underlying the signaling and regulatory functions of HNE. The role of HNE in the pathophysiology of cancer, cardiovascular and neurodegenerative diseases is also discussed.

Protein-disulfide Isomerase- and Protein Thiol-dependent Dehydroascorbate Reduction and Ascorbate Accumulation in the Lumen of the Endoplasmic Reticulum

The transport and intraluminal reduction of dehydroascorbate was investigated in microsomal vesicles from various tissues. The highest rates of transport and intraluminal isotope accumulation (using radiolabeled compound and a rapid filtration technique) were found in hepatic microsomes. These microsomes contain the highest amount of protein-disulfide isomerase, which is known to have a dehydroascorbate reductase activity. The steady-state level of intraluminal isotope accumulation was more than 2-fold higher in hepatic microsomes prepared from spontaneously diabetic BioBreeding/Worcester rats and was very low in fetal hepatic microsomes although the initial rate of transport was not changed. In these microsomes, the amount of protein-disulfide isomerase was similar, but the availability of protein thiols was different and correlated with dehydroascorbate uptake. The increased isotope accumulation was accompanied by a higher rate of dehydroascorbate reduction and increased protein thiol oxidation in microsomes from diabetic animals. The results suggest that both the activity of protein-disulfide isomerase and the availability of protein thiols as reducing equivalents can play a crucial role in the accumulation of ascorbate in the lumen of the endoplasmic reticulum. These findings also support the fact that dehydroascorbate can act as an oxidant in the protein-disulfide isomerase-catalyzed protein disulfide formation.

Ascorbate synthesis‐dependent glutathione consumption in mouse liver

Ascorbate synthesis causes glutathione consumption in the liver. Addition of gulonolactone resulted in an increase of ascorbate production in isolated murine hepatocytes. At the same time, a decrease in reduced glutathione (GSH) level was observed. In hepatic microsomal membranes, ascorbate synthesis stimulated by gulonolactone caused an almost equimolar consumption of GSH. This effect could be counteracted by the addition of catalase or mercaptosuccinate, indicating the role of hydrogen peroxide formed during ascorbate synthesis in the depletion of GSH. The observed phenomenon may be one of the reasons why the evolutionary loss of ascorbate synthesis could be advantageous.

Stress on redox

Redox imbalance in the endoplasmic reticulum lumen is the most frequent cause of endoplasmic reticulum stress and consequent apoptosis. The mechanism involves the impairment of oxidative protein folding, the accumulation of unfolded/misfolded proteins in the lumen and the initiation of the unfolded protein response. The participation of several redox systems (glutathione, ascorbate, FAD, tocopherol, vitamin K) has been demonstrated in the process. Recent findings have attracted attention to the possible mechanistic role of luminal pyridine nucleotides in the endoplasmic reticulum stress. The aim of this minireview is to summarize the luminal redox systems and the redox sensing mechanisms of the endoplasmic reticulum.

Metformin attenuates palmitate-induced endoplasmic reticulum stress, serine phosphorylation of IRS-1 and apoptosis in rat insulinoma cells

Lipotoxicity refers to cellular dysfunctions caused by elevated free fatty acid levels playing a central role in the development and progression of obesity related diseases. Saturated fatty acids cause insulin resistance and reduce insulin production in the pancreatic islets, thereby generating a vicious cycle, which potentially culminates in type 2 diabetes. The underlying endoplasmic reticulum (ER) stress response can lead to even β-cell death (lipoapoptosis). Since improvement of β-cell viability is a promising anti-diabetic strategy, the protective effect of metformin, a known insulin sensitizer was studied in rat insulinoma cells. Assessment of palmitate-induced lipoapoptosis by fluorescent microscopy and by detection of caspase-3 showed a significant decrease in metformin treated cells. Attenuation of β-cell lipotoxicity was also revealed by lower induction/activation of various ER stress markers, e.g. phosphorylation of eukaryotic initiation factor 2α (eIF2α), c-Jun N-terminal kinase (JNK), insulin receptor substrate-1 (IRS-1) and induction of CCAAT/enhancer binding protein homologous protein (CHOP). Our results indicate that the β-cell protective activity of metformin in lipotoxicity can be at least partly attributed to suppression of ER stress.