Gábor Bánhegyi

Vitamin C: update on physiology and pharmacology

Although ascorbic acid is an important water‐soluble antioxidant and enzyme cofactor in plants and animals, humans and some other species do not synthesize ascorbate due to the lack of the enzyme catalyzing the final step of the biosynthetic pathway, and for them it has become a vitamin. This review focuses on the role of ascorbate in various hydroxylation reactions and in the redox homeostasis of subcellular compartments including mitochondria and endoplasmic reticulum. Recently discovered functions of ascorbate in nucleic acid and histone dealkylation and proteoglycan deglycanation are also summarized. These new findings might delineate a role for ascorbate in the modulation of both pro‐ and anti‐carcinogenic mechanisms. Recent advances and perspectives in therapeutic applications are also reviewed. On the basis of new and earlier observations, the advantages of the lost ability to synthesize ascorbate are pondered. The increasing knowledge of the functions of ascorbate and of its molecular sites of action can mechanistically substantiate a place for ascorbate in the treatment of various diseases.

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 Stress

Abstract:  Stress is the imbalance of homeostasis, which can be sensed even at the subcellular level. The stress‐sensing capability of various organelles including the endoplasmic reticulum (ER) has been described. It has become evident that acute or prolonged ER stress plays an important role in many human diseases; especially those involving organs/tissues specialized in protein secretion. This article summarizes the emerging role of ER stress in diverse human pathophysiological conditions such as carcinogenesis and tumor progression, cerebral ischemia, plasma cell maturation and apoptosis, obesity, insulin resistance, and type 2 diabetes. Certain components of the ER stress response machinery are identified as biomarkers of the diseases or as possible targets for therapeutic intervention.

Preferential transport of glutathione versus glutathione disulfide in rat liver microsomal vesicles.

A bi-directional, saturable transport of glutathione (GSH) was found in rat liver microsomal vesicles. GSH transport could be inhibited by the anion transport blockers flufenamic acid and 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid. A part of GSH taken up by the vesicles was metabolized to glutathione disulfide (GSSG) in the lumen. Microsomal membrane was virtually nonpermeable toward GSSG; accordingly, GSSG generated in the microsomal lumen could hardly exit. Therefore, GSH transport, contrary to previous assumptions, is preferred in the endoplasmic reticulum, and GSSG entrapped and accumulated in the lumen creates the oxidized state of its redox buffer.

Endoplasmic reticulum: A metabolic compartment

Several biochemical reactions and processes of cell biology are compartmentalized in the endoplasmic reticulum (ER). The view that the ER membrane is basically a scaffold for ER proteins, which is permeable to small molecules, is inconsistent with recent findings. The luminal micro‐environment is characteristically different from the cytosol; its protein and glutathione thiols are remarkably more oxidized, and it contains a separate pyridine nucleotide pool. The substrate specificity and activity of certain luminal enzymes are dependent on selective transport of possible substrates and co‐factors from the cytosol. Abundant biochemical, pharmacological, clinical and genetic data indicate that the barrier function of the lipid bilayer and specific transport activities in the membrane make the ER a separate metabolic compartment.

The ascorbate-glutathione-α-tocopherol triad in abiotic stress response

The life of any living organism can be defined as a hurdle due to different kind of stresses. As with all living organisms, plants are exposed to various abiotic stresses, such as drought, salinity, extreme temperatures and chemical toxicity. These primary stresses are often interconnected, and lead to the overproduction of reactive oxygen species (ROS) in plants, which are highly reactive and toxic and cause damage to proteins, lipids, carbohydrates and DNA, which ultimately results in oxidative stress. Stress-induced ROS accumulation is counteracted by enzymatic antioxidant systems and non-enzymatic low molecular weight metabolites, such as ascorbate, glutathione and α-tocopherol. The above mentioned low molecular weight antioxidants are also capable of chelating metal ions, reducing thus their catalytic activity to form ROS and also scavenge them. Hence, in plant cells, this triad of low molecular weight antioxidants (ascorbate, glutathione and α-tocopherol) form an important part of abiotic stress response. In this work we are presenting a review of abiotic stress responses connected to these antioxidants.

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.