Jakub Drozak

Melatonin attenuates diabetes-induced oxidative stress in rabbits

Abstract:  Oxidative stress is considered to be the main cause of diabetic complications. As the role of antioxidants in diabetes therapy is still underestimated, the aim of the present investigation was to study the antioxidative action of melatonin in comparison with N‐acetylcysteine (NAC) under diabetic conditions. Alloxan‐diabetic rabbits were treated daily with either melatonin (1 mg/kg, i.p.), NAC (10 mg/kg, i.p.) or saline. Blood glutathione redox state and serum hydroxyl free radicals (HFR), creatinine and urea levels were monitored. After 3 wk of treatment animals were killed and HFR content, reduced glutathione/oxidized glutathione (GSH/GSSG) ratio as well as the activities of glutathione reductase, glutathione peroxidase and γ‐glutamylcysteine synthetase were estimated in both liver and kidney cortex. Diabetes evoked a several‐fold increase in HFR levels accompanied by a significant decline in GSH/GSSG ratio in serum and the examined organs. In contrast to NAC, melatonin (at 1/10 the dose of NAC) attenuated diabetes‐induced alterations in glutathione redox state and HFR levels, normalized creatinine concentration and diminished urea content in serum. Moreover, the indole resulted in an increase in glutathione reductase activity in both studied organs and in a rise in glutathione peroxidase and γ‐glutamylcysteine synthetase activities in the liver. In contrast to NAC, melatonin seems to be beneficial for diabetes therapy because of its potent antioxidative and nephroprotective action. The indole‐induced increase in the activities of the enzymes of glutathione metabolism might be of importance for antioxidative action of melatonin under diabetic conditions.

Molecular Identification of Carnosine Synthase as ATP-grasp Domain-containing Protein 1 (ATPGD1)

Carnosine (β-alanyl-l-histidine) and homocarnosine (γ-aminobutyryl-l-histidine) are abundant dipeptides in skeletal muscle and brain of most vertebrates and some invertebrates. The formation of both compounds is catalyzed by carnosine synthase, which is thought to convert ATP to AMP and inorganic pyrophosphate, and whose molecular identity is unknown. In the present work, we have purified carnosine synthase from chicken pectoral muscle about 1500-fold until only two major polypeptides of 100 and 90 kDa were present in the preparation. Mass spectrometry analysis of these polypeptides did not yield any meaningful candidate. Carnosine formation catalyzed by the purified enzyme was accompanied by a stoichiometric formation, not of AMP, but of ADP, suggesting that carnosine synthase belongs to the “ATP-grasp family” of ligases. A data base mining approach identified ATPGD1 as a likely candidate. As this protein was absent from chicken protein data bases, we reconstituted its sequence from a PCR-amplified cDNA and found it to fit with the 100-kDa polypeptide of the chicken carnosine synthase preparation. Mouse and human ATPGD1 were expressed in HEK293T cells, purified to homogeneity, and shown to catalyze the formation of carnosine, as confirmed by mass spectrometry, and of homocarnosine. Specificity studies carried out on all three enzymes were in agreement with published data. In particular, they acted with 15–25-fold higher catalytic efficiencies on β-alanine than on γ-aminobutyrate. The identification of the gene encoding carnosine synthase will help for a better understanding of the biological functions of carnosine and related dipeptides, which still remain largely unknown.

Diabetes-induced changes in glucose synthesis, intracellular glutathione status and hydroxyl free radical generation in rabbit kidney-cortex tubules

Diabetes-induced changes in glucose formation, intracellular and mitochondrial glutathione redox states as well as hydroxyl free radicals (HFR) generation have been investigated in rabbit kidney-cortex tubules. In contrast to renal tubules of control animals, diabetes-evoked increase in glucose formation in the presence of either aspartate + glycerol + octanoate or malate as gluconeogenic precursors (for about 50%) was accompanied by a diminished intracellular glutathione reduced form (GSH)/glutathione oxidised one (GSSG) ratio by about 30–40%, while the mitochondrial GSH/GSSG ratio was not altered. However, a relationship between the rate of gluconeogenesis and the intracellular glutathione redox state was maintained in renal tubules of both control and diabetic rabbits, as concluded from measurements in the presence of various gluconeogenic precursors. Moreover, diabetes resulted in both elevation of the glutathione reductase activity in rabbit kidney-cortex and acceleration of renal HFR generation (by about 2-fold). On the addition of melatonin, the hormone exhibiting antioxidative properties, the control values of HFR production were restored, suggesting that this compound might be beneficial during diabetes therapy. In view of the data, it seems likely that diabetes-induced increase in HFR formation in renal tubules might be responsible for a diminished intracellular glutathione redox state despite elevated glutathione reductase activity and accelerated rate of gluconeogenesis, providing glucose-6-phosphate for NADPH generation via pentose phosphate pathway. (Mol Cell Biochem 261: 91–98, 2004)

Differential effects of vanadium, tungsten and molybdenum on inhibition of glucose formation in renal tubules and hepatocytes of control and diabetic rabbits: Beneficial action of melatonin and N-acetylcysteine

Effect of vanadyl acetylacetonate (VAc), tungstate and molybdate on gluconeogenesis has been studied in isolated hepatocytes and kidney-cortex tubules. In renal tubules of control and alloxan-diabetic animals, the rank order of the metal-compounds-induced (i) inhibition of glucose formation from alanine + glycerol + octanoate or aspartate + glycerol + octanoate, (ii) decrease in the mitochondrial membrane potential (ΔΨm), (iii) increase in the hydroxyl free radicals (HFR) generation and (iv) decline in glucose-6-phosphatase activity was the following: VAc > tungstate > molybdate. Moreover, in contrast to VAc, both tungstate and molybdate at 100 μM concentration did not practically decrease glucose production in hepatocytes isolated from diabetic rabbits, and significantly increased the rate of lactate formation in renal tubules. N-acetylcysteine at 2 mM concentration partially attenuated vanadium-induced alterations in glucose formation, ΔΨm and the cellular glutathione redox state, whereas 0.1 mM melatonin did not abolish vanadium-induced changes in gluconeogenesis despite attenuation of vanadium effects on HFR formation and ΔΨm decline. However, similarly to control rabbits, following 6 days of intraperitoneal administration of both VAc (1.275 mg V/kg body weight daily) and melatonin (1 mg/kg body weight daily) to alloxan-diabetic animals, vanadium-induced elevated serum creatinine and urea levels were decreased, indicating the beneficial effect of melatonin on diabetes- and vanadium-induced nephrotoxicity in rabbits. As serum glucose levels were also significantly diminished by vanadium + melatonin treatment of diabetic animals, the combination therapy of vanadium compounds and melatonin needs a careful evaluation. (Mol Cell Biochem 261: 9–21, 2004)

Molecular Identification of Carnosine N-Methyltransferase as Chicken Histamine N-Methyltransferase-Like Protein (HNMT-Like)

Anserine (beta-alanyl-N(Pi)-methyl-L-histidine), a naturally occurring derivative of carnosine (beta-alanyl-L-histidine), is an abundant constituent of skeletal muscles and brain of many vertebrates. Although it has long been proposed to serve as a proton buffer, radicals scavenger and transglycating agent, its physiological function remains obscure. The formation of anserine is catalyzed by carnosine N-methyltransferase which exhibits unknown molecular identity. In the present investigation, we have purified carnosine N-methyltransferase from chicken pectoral muscle about 640-fold until three major polypeptides of about 23, 26 and 37 kDa coeluting with the enzyme were identified in the preparation. Mass spectrometry analysis of these polypeptides resulted in an identification of histamine N-methyltransferase-like (HNMT-like) protein as the only meaningful candidate. Analysis of GenBank database records indicated that the hnmt-like gene might be a paralogue of histamine N-methyltransferase gene, while comparison of their protein sequences suggested that HNMT-like protein might have acquired a new activity. Chicken HNMT-like protein was expressed in COS-7 cells, purified to homogeneity, and shown to catalyze the formation of anserine as confirmed by both chromatographic and mass spectrometry analysis. Both specificity and kinetic studies carried out on the native and recombinant enzyme were in agreement with published data. Particularly, several compounds structurally related to carnosine, including histamine and L-histidine, were tested as potential substrates for the enzyme, and carnosine was the only methyl group acceptor. The identification of the gene encoding carnosine N-methyltransferase might be beneficial for estimation of the biological functions of anserine.

Melatonin is more effective than taurine and 5-hydroxytryptophan against hyperglycemia-induced kidney-cortex tubules injury.

Abstract:  The antioxidative effects of melatonin (Mel), 5‐hydroxytryptophan (5‐HTP) and taurine (TAU) on hyperglycemia‐induced oxidative stress was investigated in primary cultures of kidney‐cortex tubule cells grown in metabolically and hormonally defined medium. In the presence of 30 mm glucose (hyperglycemic conditions), cell viability was decreased by about 35% in comparison with that estimated in the glucose‐depleted medium probably as a result of induction of apoptosis, as concluded from: (i) chromatin condensation and DNA fragmentation assays, (ii) a significant enhancement of reactive oxygen species (ROS) production, (iii) 8‐hydroxydeoxyguanosine (8‐OHdG) generation, (iv) an increased protein peroxidation and (v) a decline of reduced glutathione (GSH) levels leading to a disturbed glutathione redox state. The addition of 100 μm Mel to the hyperglycemic medium resulted in a twofold decrease in both 8‐OHdG accumulation and protein peroxidation as well as restoration of the control intracellular ROS levels accompanied by a substantial increase in GSH/oxidized glutathione (GSSG) ratio due to a decline in GSSG content. ROS elimination was also achieved in the presence of 1 mm TAU which diminished protein and DNA injuries by about 25% and 30%, respectively. On the contrary, the action of 100 μm 5‐HTP on ROS level, 8‐OHdG generation, protein peroxidation and GSH/GSSG ratio was negligible. Thus, in contrast to 5‐HTP and TAU, Mel might be considered as beneficial for diabetes therapy, particularly in terms of reduction of hyperglycemia‐induced kidney injury.

Arginine deprivation affects glioblastoma cell adhesion, invasiveness and actin cytoskeleton organization by impairment of β‑actin arginylation

A deficit of exogenous arginine affects growth and viability of numerous cancer cells. Although arginine deprivation-based strategy is currently undergoing clinical trials, molecular mechanisms of tumor cells’ response to arginine deprivation are not yet elucidated. We have examined effects of arginine starvation on cell motility, adhesion and invasiveness as well as on actin cytoskeleton organization of human glioblastoma cells. We observed for the first time that arginine, but not lysine, starvation affected cell morphology, significantly inhibited their motility and invasiveness, and impaired adhesion. No effects on glia cells were observed. Also, arginine deprivation in glioblastoma evoked specific changes in actin assembly, decreased β-actin filament content, and affected its N-terminal arginylation. We suggest that alterations in organization of β-actin resulted from a decrease of its arginylation could be responsible for the observed effects of arginine deprivation on cell invasiveness and migration. Our data indicate that arginine deprivation-based treatment strategies could inhibit, at least transiently, the invasion process of highly malignant brain tumors and may have a potential for combination therapy to extend overall patient survival.

UPF0586 Protein C9orf41 Homolog Is Anserine-Producing Methyltransferase

Background: Anserine is an abundant dipeptide in vertebrate skeletal muscles. Results: We identified UPF0586 protein C9orf41 homolog as a carnosine N-methyltransferase, responsible for anserine formation in rat muscle. Conclusion: Besides being a carnosine N-methyltransferase, UPF0586 protein is likely to be a novel peptide or protein methyltransferase in eukaryotes. Significance: This molecular identification will help to elucidate physiological functions of UPF0586 protein in eukaryotes. Anserine (β-alanyl-N(Pi)-methyl-l-histidine), a methylated derivative of carnosine (β-alanyl-l-histidine), is an abundant constituent of vertebrate skeletal muscles. Although it has been suggested to serve as a proton buffer and radical scavenger, its physiological function remains mysterious. The formation of anserine is catalyzed by carnosine N-methyltransferase, recently identified in chicken as histamine N-methyltransferase-like (HNMT-like) protein. Although the HNMT-like gene is absent in mammalian genomes, the activity of carnosine N-methyltransferase was reported in most mammalian species. In the present investigation, we purified carnosine N-methyltransferase from rat muscles about 2600-fold. Three polypeptides of ∼45, 50, and 70 kDa coeluting with the enzyme activity were identified in the preparation. Mass spectrometry analysis of these polypeptides resulted in the identification of UPF0586 protein C9orf41 homolog as the only meaningful candidate. Rat UPF0586 and its yeast, chicken, and human orthologs were expressed in COS-7 cells and purified to homogeneity. Although all recombinant proteins catalyzed the formation of anserine, as confirmed by chromatographic and mass spectrometry analysis, rat UPF0586 was more active on carnosine than other orthologs. Confocal microscopy of HeLa cells expressing recombinant UPF5086 proteins revealed their presence in both cytosol and nucleus. Carnosine and Gly-His were the best substrates for all UPF0586 orthologs studied, although the enzymes also methylated other l-histidine-containing di- and tripeptides. Finally, cotransfection of COS-7 cells with rat or human UPF0586 and carnosine synthase transformed the cells into efficient anserine producers. We conclude that UPF0586 is mammalian carnosine N-methyltransferase and hypothesize that it may also serve as a peptide or protein methyltransferase in eukaryotes.

The inhibition of gluconeogenesis by gatifloxacin may contribute to its hypoglycaemic action

The action of gatifloxacin, the broad-spectrum fluoroquinolone antibiotic commonly used in the therapy of various bacterial infections, was investigated in isolated rabbit hepatocytes and kidney-cortex tubules by measuring the activity of gluconeogenesis, a process that maintains whole body glucose homeostasis. The data show that in kidney-cortex tubules, application of gatifloxacin at up to 100 microM was followed by a marked accumulation of the drug in the intracellular milieu and a decrease in the rate of glucose formation from pyruvate by 20-50%. Gatifloxacin did not affect the rate of gluconeogenesis from either alanine + glycerol + octanoate or aspartate + glycerol + octanoate. At concentrations between 25 and 200 microM the drug decreased mitochondrial oxygen consumption by 20-45% with pyruvate + malate and ADP. As in the case of alpha-cyano-4-hydroxycinnamate, a well-established inhibitor of the mitochondrial pyruvate transporter, it diminished pyruvate uptake by both renal and hepatic mitochondria. The inhibitory action of gatifloxacin was less pronounced in hepatocytes where reduction in pyruvate-dependent glucose formation and mitochondrial respiration was by no more than 25%. The antibiotic did not influence mitochondrial oxygen consumption with glutamate + malate in either kidney-cortex or liver mitochondria. A differential substrate dependence of gatifloxacin action on gluconeogenesis and mitochondrial respiration combined with a decrease in pyruvate uptake by mitochondria suggest that the inhibitory action of this drug on gluconeogenesis might result from its impairment of pyruvate transport into mitochondria.

Proinsulin C-peptide potentiates the inhibitory action of insulin on glucose synthesis in primary cultured rabbit kidney-cortex tubules: metabolic studies.

Effects of equimolar concentrations of proinsulin C-peptide and insulin on glucose synthesis were studied in primary cultures of rabbit kidney-cortex tubules grown in the presence of alanine, glycerol, and octanoate. The rhodamine-labeled C-peptide entered renal tubular cells and localized in nuclei, both in the presence and absence of insulin; preincubations with the unlabeled compound inhibited internalization. C-peptide did not affect glucose formation when added alone but potentiated the inhibitory action of insulin by about 20% due to a decrease in flux through glucose-6-phosphate isomerase (GPI) and (or) glucose-6-phosphatase (G6Pase). GPI inhibition was caused by: (i) increased intracellular contents of fructose-1,6-bisphosphate and fructose-1-phosphate, inhibitors of the enzyme and (ii) reduced level of the phosphorylated GPI, which exhibits higher enzymatic activity in the presence of casein kinase 2. A decrease in flux through G6Pase, due to diminished import of G6P by G6P-transporter from the cytoplasm into endoplasmic reticulum lumen, is also suggested. The data show for the first time that in the presence of insulin and C-peptide, both GPI and G6P-ase may act as regulatory enzymes of renal gluconeogenic pathway.