Gergő A. Molnár

Accumulation of the hydroxyl free radical markers meta-, ortho-tyrosine and DOPA in cataractous lenses is accompanied by a lower protein and phenylalanine content of the water-soluble phase

Post-translational modifications of lens proteins play a crucial role in the formation of cataract during ageing. The aim of our study was to analyze protein composition of the cataractous lenses by electrophoretic and high-performance liquid chromatographic (HPLC) methods. Samples were obtained after extracapsular cataract surgery performed by phacoemulsification technique from cataract patients with type 2 diabetes mellitus (DM CAT, n = 22) and cataract patients without diabetes (non-DM CAT, n = 20), while non-diabetic non-cataractous lenses obtained from cadaver eyes served as controls (CONTR, n = 17). Lens fragments were derived from the surgical medium by centrifugation. Samples were homogenized in a buffered medium containing protease inhibitor. Soluble and insoluble protein fractions were separated by centrifugation. The electrophoretic studies were performed according to Laemmli on equal amounts of proteins and were followed by silver intensification. Oxidized amino acid and Phe content of the samples were also analyzed by HPLC following acid hydrolysis of proteins. Our results showed that soluble proteins represented a significantly lower portion of the total protein content in cataractous lenses in comparison with the control group (CONTR, 71.25%; non-DM CAT, 32.00%; DM CAT, 33.15%; p < 0.05 vs CONTR for both). Among the proteins, the crystallin-like proteins with low-molecular weight can be found both in the soluble and insoluble fractions, and high-molecular weight aggregates were found mainly in the total homogenates. In our HPLC analysis, oxidatively modified derivatives of phenylalanine were detected in cataractous samples. We found higher levels of m-Tyr, o-Tyr and DOPA in the total homogenates of cataractous samples compared to the supernatants. In all three groups, the median Phe/protein ratio of the total homogenates was also higher than that of the supernatants (total homogenates vs supernatants, in the CONTR group 1102 vs 633 μmol/g, in the DM CAT group 1187 vs 382 μmol/g and in the non-DM CAT group 967 vs 252 μmol/g; p < 0.05 for all). In our study we found that oxidized amino acids accumulate in cataractous lenses, regardless of the origin of the cataract. The accumulation of the oxidized amino acids probably results from oxidation of Phe residues of the non-water soluble lens proteins. We found the presence of high-molecular weight protein aggregates in cataractous total homogenates, and a decrease of protein concentration in the water-soluble phase of cataractous lenses. The oxidation of lens proteins and the oxidative modification of Phe residues in key positions may lead to an altered interaction between protein and water molecules and thus contribute to lens opacification.

Evidence of O-linked N-acetylglucosamine in diabetic nephropathy

AIMS There is increasing evidence that O-linked N-acetylglucosamine (O-GlcNAc) plays an important role in cell signaling pathways. It has also been reported that increases in O-GlcNAc contribute to the development of diabetes and diabetic complications; however, little is known about O-GlcNAc levels in diabetic nephropathy (DNP). Therefore the goal of this study was to determine whether O-GlcNAc could be detected in human kidney biopsy specimens, and if so to examine whether O-GlcNAc levels were increased in the kidneys of patients with DNP compared to the non-diabetic individuals. MAIN METHODS Kidney biopsy specimens were obtained from type-2 diabetic patients (n=6) and patients diagnosed with thin basement membrane nephropathy (n=7) were used as non-diabetic controls. O-GlcNAc levels were assessed by immunohistochemistry using the anti-O-GlcNAc antibody CTD110.6. KEY FINDINGS We show that O-GlcNAc modification of proteins can be detected in the human kidney biopsy specimens. Furthermore, in diabetic patients, we found significantly increased numbers of O-GlcNAc positive cells in the glomeruli and significantly elevated staining in the tubuli (both in the nucleus and in the cytosol). In addition we also observed an intense, granular O-GlcNAc staining specifically in diabetic tubuli. SIGNIFICANCE In light of the increase in O-GlcNAc staining in the diabetic patients, we propose that increased O-GlcNAc levels might contribute to the development of diabetic nephropathy.

Cigarette Smoke-Induced Alterations in Endothelial Nitric Oxide Synthase Phosphorylation: Role of Protein Kinase C

Endothelial nitric oxide synthase (eNOS) is regulated by phosphorylation of Ser(1177) and Thr(495), which affects NO bioavailability. Cigarette smoke disturbs the eNOS-cGMP-NO pathway and causes decreased NO production. Here the authors investigated the acute effects of cigarette smoke on eNOS phosphorylation, focusing on protein kinases (PKs). Endothelial cell culture was concentration- and time-dependently treated first with cigarette smoke buffer (CSB), then with reduced glutathione (GSH) or various PK inhibitors (H-89, LY-294002, Ro-318425, and ruboxistaurin). eNOS, phospho-Ser(1177)-eNOS, phospho-Thr(495)-eNOS, Akt(PKB), and phospho-Akt protein levels were determined by Western blot. CSB increased the phosphorylation of eNOS at Ser(1177) and more at Thr(495) in a concentration- and time-dependent manner (p < .01, p < .05 versus control, respectively) and resulted in the dissociation of the active dimeric form of eNOS (p < .05). GSH decreased the phosphorylation of eNOS at both sites (p < .05 versus CSB without GSH) and prevented the decrease of dimer eNOS level. CSB treatment also decreased the level of phospho-Ser(473)-Akt (p < .05 versus control). Inhibition of PKA by H-89 did not affect CSB-induced phosphorylation, whereas the PKB inhibitor LY-294002 enhanced it at Ser(1117). The PKC blockers Ro-318425 and ruboxistaurin augmented the CSB-induced phosphorylation at Ser(1177) but decreased phosphorylation at Thr(495) (p < .05 versus CSB). Cigarette smoke causes a disruption of the enzymatically active eNOS dimers and shifts the eNOS phosphorylation to an inhibitory state. Both effects might lead to reduced NO bioavailability. The shift of the eNOS phosphorylation pattern to an inhibitory state seems to be independent of the PKA and phosphoinositol 3-kinase (PI3-K)/Akt pathways, whereas PKC appears to play a key role.

Exenatide induces aortic vasodilation increasing hydrogen sulphide, carbon monoxide and nitric oxide production

BackgroundIt has been reported that GLP-1 agonist exenatide (exendin-4) decreases blood pressure. The dose-dependent vasodilator effect of exendin-4 has previously been demonstrated, although the precise mechanism is not thoroughly described. Here we have aimed to provide in vitro evidence for the hypothesis that exenatide may decrease central (aortic) blood pressure involving three gasotransmitters, namely nitric oxide (NO) carbon monoxide (CO), and hydrogen sulphide (H2S).MethodsWe determined the vasoactive effect of exenatide on isolated thoracic aortic rings of adult rats. Two millimetre-long vessel segments were placed in a wire myograph and preincubated with inhibitors of the enzymes producing the three gasotransmitters, with inhibitors of reactive oxygen species formation, prostaglandin synthesis, inhibitors of protein kinases, potassium channels or with an inhibitor of the Na+/Ca2+-exchanger.ResultsExenatide caused dose-dependent relaxation of rat thoracic aorta, which was evoked via the GLP-1 receptor and was mediated mainly by H2S but also by NO and CO. Prostaglandins and superoxide free radical also play a part in the relaxation. Inhibition of soluble guanylyl cyclase significantly diminished vasorelaxation. We found that ATP-sensitive-, voltage-gated- and calcium-activated large-conductance potassium channels are also involved in the vasodilation, but that seemingly the inhibition of the KCNQ-type voltage-gated potassium channels resulted in the most remarkable decrease in the rate of vasorelaxation. Inhibition of the Na+/Ca2+-exchanger abolished most of the vasodilation.ConclusionsExenatide induces vasodilation in rat thoracic aorta with the contribution of all three gasotransmitters. We provide in vitro evidence for the potential ability of exenatide to lower central (aortic) blood pressure, which could have relevant clinical importance.

Effects of erythropoietin on glucose metabolism

We purposed to determine the impact of erythropoietin on altering glucose metabolism in the settings of in vitro and in vivo experiments. The acute effect of erythropoietin on lowering blood glucose levels was studied in animal experiments. In [³H]-deoxy-D-glucose isotope studies we measured glucose uptake with insulin and erythropoietin using 3T3-L1 cells cultured under normal or high glucose conditions. Altered activation of Akt and ERK pathways was evaluated in immunoblot analyses. Immunocytochemistry was conducted to determine the glucose transporter 4 translocation to the plasma membrane. Addition of erythropoietin significantly lowered blood glucose levels in vivo in rats. The glucose uptake was markedly increased by erythropoietin treatment (at concentrations 0.15, 0.3, and 0.625 ng/ml) in adipocytes grown in high glucose medium (p<0.05), but it remained unaltered in cells under normal glucose conditions. Significant increase of phosphorylation of ERK and Akt was detected due to erythropoietin (p<0.05). Co-administration of erythropoietin and insulin resulted in higher phosphorylation of Akt and [³H]-deoxy-D-glucose uptake in adipocytes than insulin treatment alone. We found that erythropoietin induced the trafficking of glucose transporter 4 to the plasma membrane. Our data showed that erythropoietin significantly decreased blood glucose levels both in vivo and in vitro, in part, by increasing glucose uptake via the activation of Akt pathway. Preliminary data revealed that adipocytes most likely exhibit a specific receptor for erythropoietin.

Measurement of the modification and interference rate of urinary albumin detected by size-exclusion HPLC

The measurement of the excretion of urinary albumin (albuminuria) is an important and well-established method to assess clinical outcomes. A high-performance liquid chromatography (HPLC) method has been introduced to measure albuminuria. Using this method, it was found that commonly used immunological methods do not measure a fraction of urinary albumin. Some authors presumed that the reason of immuno-unreactivity is the modification of urinary albumin; some others presumed that the difference is merely because of interference. In order to decide this question, we established an HPLC method equipped with tandem UV and fluorescent detection to assess the changes in the detectability of albumin with the rate of modification. For this measurement, differently modified forms of albumin were used. Urine samples of diabetic patients were also measured to find a potential connection between the modification rate and clinical parameters. Secondly, we have established a reversed phase HPLC method to assess the interference rate. We conclude that albumin modification does not affect immunoreactivity. The modification rate of urinary albumin in diabetic patients showed a correlation with renal function. The interference rate of the albumin peak was found to be 12.7% on average, which does not explain the difference between the two methods.

Cigarette smoke and its formaldehyde component inhibit bradykinin-induced calcium increase in pig aortic endothelial cells

Bradykinin-induced increase in the intracellular concentration of free calcium evokes an activation of the endothelial nitric oxide synthase (eNOS) enzyme, producing nitric oxide (NO). Cigarette smoke inhibits the eNOS-NO-cGMP signaling pathway. The pathomechanism of this deleterious effect of smoke on NO production is unknown. The aim of this study was to investigate the effect of gas phase smoke trapped in a buffer (smoke buffer, SB) on the bradykinin-induced calcium increase in cultured endothelial cells. FURA-2-AM was used to detect bradykinin-induced calcium increase. A sensitive, fluorescent method using O-phthaldialdehyde was used for the determination of intracellular reduced glutathione (GSH) and protein-thiol levels. SB caused a time- and concentration-dependent inhibition of bradykinin-induced calcium increase. Formaldehyde, a component of SB, inhibited bradykinin-induced calcium increase in concentrations characteristic for SB. SB decreased both the intracellular GSH (0.22 +/- 0.06 vs. 2.23 +/- 0.32 mumol/g protein, SB vs. control, p < .001) and protein-thiol levels (4.98 +/- 0.54 vs. 7.31 +/- 0.97 microEqu GSH/g protein, SB vs. control, p < .05) in the endothelial cells. Intracellular GSH and protein-thiol levels were not changed by 80 microM formaldehyde. GSH (4 mM) prevented the effect of SB (p < .001) and formaldehyde (p < .05) on the bradykinin-induced calcium increase. Our data support the premise that SB inhibits bradykinin-induced calcium increase. This inhibition is partially due to protein-thiol oxidation but may also be caused by the formaldehyde content of SB, which inhibits calcium increase in a protein-thiol-independent manner.

Role of Tyrosine Isomers in Acute and Chronic Diseases Leading to Oxidative Stress - A Review

Oxidative stress plays a major role in the pathogenesis of a variety of acute and chronic diseases. Measurement of the oxidative stress-related end products may be performed, e.g. that of structural isomers of the physiological para-tyrosine, namely meta- and ortho-tyrosine, that are oxidized derivatives of phenylalanine. Recent data suggest that in sepsis, serum level of meta-tyrosine increases, which peaks on the 2nd and 3rd days (p<0.05 vs. controls), and the kinetics follows the intensity of the systemic inflammation correlating with serum procalcitonin levels. In a similar study subset, urinary meta-tyrosine excretion correlated with both need of daily insulin dose and the insulin-glucose product in non-diabetic septic cases (p<0.01 for both). Using linear regression model, meta-tyrosine excretion, urinary meta-tyrosine/para-tyrosine, urinary ortho-tyrosine/para-tyrosine and urinary (meta- + ortho-tyrosine)/para-tyrosine proved to be markers of carbohydrate homeostasis. In a chronic rodent model, we tried to compensate the abnormal tyrosine isomers using para-tyrosine, the physiological amino acid. Rats were fed a standard high cholesterol-diet, and were given para-tyrosine or vehicle orally. High-cholesterol feeding lead to a significant increase in aortic wall meta-tyrosine content and a decreased vasorelaxation of the aorta to insulin and the glucagon-like peptide-1 analogue, liraglutide, that both could be prevented by administration of para-tyrosine. Concluding, these data suggest that meta- and ortho-tyrosine are potential markers of oxidative stress in acute diseases related to oxidative stress, and may also interfere with insulin action in septic humans. Competition of meta- and ortho-tyrosine by supplementation of para-tyrosine may exert a protective role in oxidative stress-related diseases.

Elevated Vascular Level of ortho-Tyrosine Contributes to the Impairment of Insulin-Induced Arterial Relaxation

Previous studies have shown that in diabetes mellitus, insulin-induced relaxation of arteries is impaired and the level of ortho-tyrosine (o-Tyr), an oxidized amino acid is increased. Thus, we hypothesized that elevated vascular level of o-Tyr contributes to the impairment of insulin-induced vascular relaxation. Rats were fed with o-Tyr for 4 weeks. Insulin-induced vasomotor responses of isolated femoral artery were studied using wire myography. Vascular o-Tyr content was measured by HPLC, whereas immunoblot analyses were preformed to detect eNOS phosphorylation. Sustained oral supplementation of rats with o-Tyr increased the content of o-Tyr in the arterial wall and significantly reduced the relaxations to insulin. Sustained supplementation of cultured endothelial cells with o-Tyr increased the incorporation of o-Tyr and mitigated eNOS Ser (1 177) phosphorylation to insulin. Increasing arterial wall o-Tyr level attenuates insulin-induced relaxation - at least in part - by decreasing eNOS activation. Elevated level of o-Tyr could be an underlying mechanism for vasomotor dysfunction in diabetes mellitus.

Modeling long-term diabetes and related complications in rats.

INTRODUCTION Accurate preclinical modeling of diabetic complications such as retinopathy, nephropathy and neuropathy is crucial to enable the development of novel preventative therapies. The aims of this study were to establish a model of long-term diabetes with sustained medium scale hyperglycemia and characterize the pathological changes detectable after 4months, with particular respect to dependence on the degree of hyperglycemia. METHODS Streptozotocin-induced diabetic CFY rats were subjected to four different insulin substitution protocols to achieve different levels of glycemic control (Diabetic 1-4 groups). Eyes were investigated by ophthalmoscopy, kidney function by urine analysis, and neuropathy by functional tests. Retinal and renal morphological evaluations were performed by histology, immuno-histochemistry and electron microscopy. RESULTS Rats of the Diabetic 3 group showed massive hyperglycemia-dependent anterior segment neovascularization, enhanced total retinal score and retinal apoptotic cell number, degeneration of dopaminergic amacrine cells, increased glomerular PAS-positivity, altered excreted total protein/creatinine ratio and cold allodynia, parallel with medium scale hyperglycemia (blood glucose level between 22 and 25mmol/L) and satisfying state of health. DISCUSSION We established a treatment protocol in rats enabling complex investigation of diabetic retinopathy, nephropathy and neuropathy on a long-term period. Clearly hyperglycemic dependent parameters of these complications serve as good outcome measures for preclinical trials. Our results provide a useful basis for designing studies for testing preventative treatments as well as other translational medical research in this field.