V. De Tata

Endogenous Oxidative Stress Induces Distinct Redox Forms of Tumor Necrosis Factor Receptor‐1 in Melanoma Cells

Abstract: Receptors of the TNFR superfamily possess abundant thiols in their extracellular domains, which makes them susceptible to redox modulation by prooxidant agents and processes. Previous studies from our laboratory have documented that membrane γ‐glutamyltransferase (GGT) activity can originate reactive oxygen species in the extracellular milieu, during the GGT‐mediated metabolism of extracellular glutathione. The present study was aimed thus to verify a possible redox‐modulating effect of GGT activity on TNFR1 receptors. The thiol‐specific probe maleimide‐polyethylene glycol was used to selectively label the reduced thiol groups in proteins of cell lysates; fractions corresponding to TNFR1 were then identified by immunoblot. In human melanoma Me665/2 cells, expressing varying GGT levels, at least five distinct forms of TNFR1 have been thus identified. The more oxidized forms appear to be prevalent in the 2/60 clone, expressing higher GGT levels, as compared to clone 2/21. Stimulation of GGT activity in the latter induced an increase of the oxidized TNFR1 forms. It is conceivable that different redox states of TNFR1 may correspond to different binding affinity and/or changes in the transducing function of the receptor. As GGT is frequently expressed by malignant tumors, the described phenomena might concur to alter the sensitivity of cancer cells to agents targeted on activation of TNF‐α‐dependent signaling pathways.

Transmural gradient of glycogen metabolism in the normal rat left ventricle.

The changes of glycogen metabolism with the location of tissue within the ventricle wall have been explored in the rat myocardium. The hearts were cut in 100 μm thick serial sections and all sections were analyzed for their content in glycogen, glucose-6-phosphate, UDPG and glycogen enzymes and for glucose incorporation into glycogen and for the 2-deoxyglucose uptake after the intravenous injection of the14C-labelled sugars.The rate of glycogen turnover was significantly higher in the subendocardial myocardium (P<0.01) and the levels of glucose-6-phosphate and the total (i.e. a+b) activity of glycogen phosphorylase were significantly higher in the subepicardial tissue (P<0.01 in both instances). No significant transmural gradient of UDPG was found and transmural mural changes of total (i.e. I+D) synthase activity were barely significant.These changes in glycogen metabolism may be related to regional differences in the cardiac work load and to a differentiation of the subendocardial and subepicardial heart fibers.

Increased degradation in rat liver induced by antilipolytic agents: a model for studying autophagy and protein degradation in liver?

A dramatic increase in the plasma glucagon/insulin ratio can be induced by treating fasted rats with antilipolytic drugs (e.g., with 3,5-dimethylpyrazole, 12 mg/kg body wt). These hormone changes are the physiologically appropriate response to a rapid decrease in free fatty acids and glucose plasma levels. Under this experimental condition, many vacuolated lysosomes can be observed at the electron microscopic level as early as 30 min and autophagic vacuoles are detectable in the liver cells 1 hr after the administration of the drug. By 1 hr and 45 min, vacuoles often contain recognizable peroxisomes. At the biochemical level, liver proteolysis in vitro is increased significantly. Very interestingly, changes in peroxisomal (but not mitochondrial or reticulum or cytosolic) enzyme activities are detected that are preventable by the administration of glutamine (i.e., of an inhibitor of proteolysis in vivo) but not by an isocaloric amount of glycine or alanine. It is concluded that the administration of antilipolytic agents to fasted animals may provide a convenient (i.e., an inexpensive, highly reproducible and timable) physiologic model to study hormone-induced autophagy in liver cells.

Age-dependent reduction in GLUT-2 levels is correlated with the impairment of the insulin secretory response in isolated islets of Sprague–Dawley rats

In this study we have investigated the insulin secretory response to glucose and other secretagogues (2-ketoisocaproate, 3-isobutyl-1-methyl-xanthine and arginine) of pancreatic islets isolated from Sprague-Dawley rats of various ages (from 2 to 28 months). Our results showed a significant decline in the glucose-stimulated insulin secretion, starting at 12 months of age. On the other hand, the response to non-glucose secretagogues (and mainly to 2-ketoisocaproate) was less impaired with advancing age than that to glucose. We also observed a progressive age-related decline of protein levels of the glucose transporter GLUT-2 in pancreatic islets, which was temporally concomitant and quantitatively comparable with the beta-cell alteration in glucose responsiveness (-40/50%). Finally, we observed a significant increase of the islets insulin content in older rats with respect to younger animals. We conclude that in the islet of older rats the impaired capability to respond to glucose could be dependent, at least in part, on the age-dependent reduction in GLUT-2 and could be compensated by mechanisms including a preserved responsiveness to non-glucose secretagogues and/or the development of islet hypertrophy.

Determination of glycated hemoglobins in the rat: comparison between two different chromatographic methods and application in experimental diabetology

Due to the unusual presence of several different hemoglobin components in the rat, determination of glycated hemoglobin (Hb) has been considered difficult and often unreliable in this animal species. In the present study, we compare a fully automated high-performance liquid chromatographic (HPLC) method of analysis of glycated hemoglobin that has been assessed for clinical use with an affinity chromatography technique using boronate micro-columns; we used blood samples taken from Sprague-Dawley rats of various ages and streptozotocin-diabetic rats. In nondiabetic rats, the sum of HbA1c and other minor glycated hemoglobins separated by the HPLC method is close to the total glycated hemoglobin obtained by affinity chromatography for each age group of animals. In diabetic rats, the glycated hemoglobins measured by whatever method show a linear increase during the first 3 weeks following streptozotcin administration, with the difference that glycated hemoglobin values obtained by affinity chromatography are markedly higher than those obtained by HPLC technique. Interestingly, a comparative determination of glycated hemoglobin in diabetic patients gives the same results with both methods. Therefore, it appears that in the rat, unlike man, at high glucose concentrations glycation occurs preferentially at the amino groups of hemoglobin components, which are not separated by the HPLC method. Our results indicate that while affinity chromatography should be used to detect the total extent of hemoglobin glycosylation in diabetic rats, the utilization of rapid and automatized HPLC procedures can be a very convenient alternative for the determination of glycated hemoglobin in both euglycemic and hyperglycemic rats.

Transmural differences of lipofuscin pigment accumulation in the left ventricle of rat heart during growth and aging1

In view of the higher metabolic rate in subendocardial heart tissue, the rate of age-related lipofuscin pigment accumulation was explored in different regions of the left ventricle heart wall of Sprague-Dawley rats. Hearts were removed from 2-, 6-, 12- and 24-month-old rats, and lipofuscin pigment accumulation was assessed in the subepicardial and subendocardial layers, either by measuring extractable fluorescent material, or by direct visualization with fluorescence microscopy. Findings showed that the amount of extractable fluorescent material and the number, size and brightness of the fluorescent lipofuscin granules increased with age in all the myocardial tissue layers. The rate of accumulation of extractable fluorescent material was higher in subendocardial compared to subepicardial tissue. At the microscope, fluorescent granules exhibited a different morphological appearance in the subendocardial and subepicardial tissue of the two older age-groups. These data support the hypothesis that liposoluble age-pigment deposition is linked to the rate of local oxidative metabolism. (Aging 3: 19-23, 1991)

The aryl receptor inhibitor epigallocatechin-3-gallate protects INS-1E beta-cell line against acute dioxin toxicity

The aim of this research was to investigate the mechanism(s) underlying the acute toxicity of dioxin in pancreatic beta cells and to evaluate the protective effects of epigallocatechin-3-gallate (EGCG), the most abundant of the green teas catechins and a powerful inhibitor of the aryl hydrocarbon receptor (AhR). Using the insulin-secreting INS-1E cell line we have explored the effect of 1h exposure to different concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), alone or in the presence of EGCG, on: (a) cell survival; (b) cellular ultrastructure; (c) intracellular calcium levels; (d) mitochondrial membrane potential; (e) glucose-stimulated insulin secretion and (f) activation of MAP kinases. Our results demonstrate that TCDD is highly toxic for INS-1E cells, suggesting that pancreatic beta cells should be considered a relevant and sensitive target for dioxin acute toxicity. EGCG significantly protects INS-1E cells against TCDD-induced toxicity in terms of both cell survival and preservation of cellular ultrastructure. The mechanism of this protective effect seems to be related to: (a) the ability of EGCG to preserve the mitochondrial function and thus to prevent the TCDD-induced inhibition of glucose-stimulated insulin secretion and (b) the ability of EGCG to inhibit the TCDD-induced activation of selected kinases, such as e.g. ERK 1/2 and JNK. Our results clearly show that EGCG is able to protect pancreatic beta cells against dioxin acute toxicity and indicate the mitochondrion as the most likely target for this beneficial effect.

Age-related changes in the urinary excretion of aldehydes in ad libitum fed and food-restricted rats

Age-related changes in the urinary excretion of aldehydes arising from lipid peroxidation have been investigated in male Sprague-Dawley rats aged 2, 4, 6, 12, 18, 24 and 27 months, fed ad libitum or subjected to two different regimens of calorie restriction (namely every-other-day ad libitum feeding--EOD--and 40% calorie restriction--40%DR). For only some age groups, results were compared with those obtained in ad libitum fed male Fisher 344 and Lewis rats. Results show that the urinary excretion of malondialdehyde (MDA) and formaldehyde (FA) significantly decreases, whereas that of propionaldehyde (PROP) progressively increases with age, and that urinary excretion of acetaldehyde (ACT) does not show any significant age-related variations. Dietary restriction significantly increases the urinary levels of MDA, FA and PROP without affecting their age-related modifications, and does not affect ACT urinary excretion. In conclusion, results indicate that the quantitative pattern of aldehyde production and urinary excretion may be altered by the process of aging.

Transmural distribution of glucose metabolizing enzymes across the left and the right ventricle heart walls in three different mammalian species.

The transmural distribution of five glucose metabolizing enzymes (hexokinase; glucose-6-phosphate dehydrogenase; phosphofructokinase; aldolase; and lactate dehydrogenase) were explored in the left and in the right ventricle wall of rat, ox and pig hearts. The levels of most of these enzyme activities were different in the different animal species and (within the same species) in the two ventricles. Most of these enzyme activities were found to be non-uniformly distributed across the left (but not across the right) ventricle wall. Differences in the transmural distribution of enzyme activities were detected among the three examined mammalian species.

Changes in the transmural distribution of glucose-metabolizing enzymes across the left and right ventricular wall of rat heart during growth and ageing

The age-related changes in the activities of five glucose-metabolizing enzymes (hexokinase, HK; glucose-6-phosphate dehydrogenase, G6P-DH; aldolase, ALD; phosphofructokinase, PFK; and lactate dehydrogenase, LDH) were investigated in the walls of left and right ventricles of rats of various age-groups (1-24 months). Age-related changes were found in the activities of all of the enzymes in both ventricles during growth (with significant decreases between 2 and 6 months of age) and in the levels of PFK and LDH in the left ventricle during ageing (with a significant increase between 12 and 24 months of age). The distribution of the enzyme activities across the wall of both ventricles was quite uniform in young, adult and mature rats (the distribution of G6P-DH activity in the left ventricle wall at 2 months of age was the only notable exception) but became non-uniform in the old rats with regard to G6P-DH, PFK, LDH and probably HK in the left ventricle and G6P-DH and HK in the right ventricle. These data support the hypothesis that alterations connected with ageing do not lead to a generalized decline of cardiac metabolic capacity, and that they are also the result of specific adaptive modifications, perhaps related to alteration in the distribution of the work load and/or of nutrition across the ventricular wall.