Judit Cseh

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.

A polymorphism within the fructosamine-3-kinase gene is associated with HbA1c levels and the onset of type 2 diabetes mellitus

BACKGROUND Non-enzymatic glycation is a process, which leads to the formation of advanced glycation endproducts. These compounds are involved in the development of diabetic microvascular complications. Fructosamine-3-kinase (FN3K) is an intracellular enzyme that phosphorylates fructosamines resulting in fructosamine-3-phosphate, which subsequently decomposes to inorganic phosphate, 3-deoxyglucasone and the unmodified amine. Recently, the G900C (rs1056534) single nucleotide polymorpism (SNP) of the FN3K gene was found to be associated with the enzyme activity. OBJECTIVE/DESIGN The aim of the study was to investigate the impact of the SNP on clinical and biochemical features and microvascular complications of type 2 diabetes. PATIENTS A total of 859 type 2 diabetic subjects and 265 healthy controls were enrolled in the study and were genotyped with PCR-RFLP method. RESULTS Genotype frequencies were as follows, CC: 5%, GC: 54%, GG: 41% in subjects with type 2 diabetes and CC: 6%, GC: 51%, GG: 43% in the controls. Diabetic subjects with the CC variant had lower HbA (1c) levels compared with the others (CC: 6.48+/-0.05%; GC: 7.66+/-0.09%; GG: 7.68+/-0.09%; p<0.001). Furthermore, in case of the CC allelic variant type 2 diabetes was diagnosed at a later age than in case of GC or GG variants (CC: 56.0+/-1.90 years; GC: 52.0+/-0.62 years; GG: 50.1+/-0.71 years; p<0.05). Logistic regression analysis did not reveal association between CC genotype and diabetic complications, such as diabetic nephropathy, neuropathy and retinopathy (OR=1.036, CI 95% 0.652-1.647, p=0.880; OR=0.985, CI 95% 0.564-1.721 p=0.958; OR=1.213, CI 95% 0.470-3.132, p=0.690, respectively). CONCLUSION We conclude that the G900C polymorphism associates with the level of HbA (1c) and the onset of the disease, but not with either of the diabetic microvascular complications.

GCKR gene functional variants in type 2 diabetes and metabolic syndrome: do the rare variants associate with increased carotid intima-media thickness?

BackgroundRecent studies revealed that glucokinase regulatory protein (GCKR) variants (rs780094 and rs1260326) are associated with serum triglycerides and plasma glucose levels. Here we analyzed primarily the association of these two variants with the lipid profile and plasma glucose levels in Hungarian subjects with type 2 diabetes mellitus and metabolic syndrome; and also correlated the genotypes with the carotid intima-media thickness records.MethodsA total of 321 type 2 diabetic patients, 455 metabolic syndrome patients, and 172 healthy controls were genotyped by PCR-RFLP.ResultsBoth GCKR variants were found to associate with serum triglycerides and with fasting plasma glucose. However, significant association with the development of type 2 diabetes mellitus and metabolic syndrome could not be observed. Analyzing the records of the patients, a positive association of prevalence the GCKR homozygous functional variants and carotid intima-media thickness was found in the metabolic syndrome patients.ConclusionsOur results support that rs780094 and rs1260326 functional variants of the GCKR gene are inversely associated with serum triglycerides and fasting plasma glucose levels, as it was already reported for diabetic and metabolic syndrome patients in some other populations. Besides this positive replication, as a novel feature, our preliminary findings also suggest a cardiovascular risk role of the GCKR minor allele carriage based on the carotid intima-media thickness association.

Association of n -3 and n -6 long-chain polyunsaturated fatty acids in plasma lipid classes with inflammatory bowel diseases

In order to establish the biochemical basis for dietary interventions, we investigated the fatty acid composition of plasma lipid classes in patients with inactive inflammatory bowel disease. In this cross-sectional study thirty patients with ulcerative colitis (UC), twenty-one with Crohn disease (CD) and twenty-four controls were investigated (mean age: UC, 40.8 (sd 12.1); CD, 37.6 (sd 11.0); control, 31.5 (sd 8.4) years). Fatty acid composition of plasma lipids was determined by high-resolution capillary GLC. In plasma phospholipids, significantly higher values of eicosapentaenoic (20 : 5n-3), docosapentaenoic (22 : 5n-3) and gamma-linolenic (18 : 3n-6) acids were found in control patients and patients with UC as compared to patients with CD [median % (weight by weight), control v. UC v. CD : 20 : 5n-3, 0.09 (interquartile range (IQR) 0.05) v. 0.14 (IQR 0.10) v. 0.16 (IQR 0.10), P < 0.05; 22 : 5n-3, 0.14 (IQR 0.10) v. 0.27 (IQR 0.16) v. 0.31 (IQR 0.10), P < 0.001; 18 : 3n-6, 0.02 (IQR 0.02) v. 0.03 (IQR 0.02) v. 0.05 (IQR 0.03), P < 0.05]. When compared to the control, values of the principal n-3 and n-6 long-chain PUFA, arachidonic acid (20 : 4n-6) and DHA (22 : 6n-3) were significantly higher in patients with UC but not in patients with CD [median % (w/w), UC v. control: 20 : 4n-6, 8.43 (IQR 3.23) v. 6.92 (IQR 2.96), P < 0.05; 22 : 6n-3, 1.22 (IQR 0.56) v. 0.73 (IQR 0.39), P < 0.05]. As seen there are considerable differences between the long-chain PUFA status of patients suffering from UC or CD. The data obtained in the present study do not support the concept of eicosapentaenoic acid or DHA deficiency in patients with either UC or CD.

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.

Fatty acid composition of plasma lipid classes in chronic alcoholic pancreatitis.

Background/Aims: Supplementation of n–3 long-chain polyunsaturated fatty acids (LCPUFA) is considered as adjuvant therapy in acute pancreatitis. We investigated plasma fatty acid status in chronic pancreatitis (CP). Methods: Patients with alcoholic CP (n = 56, gender: 33/23 male/female, age: 60.0 [14.0] years (median [IQR]), who reported giving up alcohol consumption several years ago and 51 control subjects were included into the study. The fatty acid composition of plasma phospholipids (PL), triacylglycerols (TG) and sterol esters (STE) was analyzed. Results: The sum of monounsaturated fatty acids was significantly higher in patients with CP than in controls (PL: 12.83 [3.35] vs. 12.20 [1.95], TG: 40.51 [6.02] vs. 37.52 [5.80], STE: 20.58 [7.22] vs. 17.54 [3.48], CP vs. control, % weight/weight, median [IQR], p < 0.05). Values of arachidonic acid were significantly lower in patients with CP than in controls (PL: 10.57 [3.56] vs. 11.66 [3.25], STE: 8.14 [2.63] vs. 9.24 [2.86], p < 0.05). Values of eicosapentaenoic acid and docosahexaenoic acid did not differ and there was no difference in the ratio of n–3 to n–6 LCPUFA. Conclusion: Our present data do not furnish evidence for the supplementation of n–3 LCPUFA to the diet of CP patients in relatively good clinical condition.

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.

Selective association of endogenous ouabain with subclinical organ damage in treated hypertensive patients

According to previous studies endogenous ouabain (EO) closely correlates with high blood pressure, congestive heart failure and kidney disease in humans. Our aims were to analyse associations between plasma, urinary EO level and various markers of cardiovascular damage in treated hypertensive patients. Forty-one adult patients with hypertension and/or diabetes mellitus (DM) and/or chronic kidney disease (CKD) were studied. We assessed plasma and urinary EO, pro-brain natriuretic peptide and catecholamines, profile of ambulatory blood pressure monitor and cardiovascular status by echocardiography and echo-tracking. The highest level of plasma EO (19.7±9.5 pmol l–1) was measured in hypertensive patients with DM and CKD. The nighttime mean arterial blood pressure independently correlated with the level of plasma EO (P=0.004), while independent predictor of the β-stiffness of carotid artery was the urinary EO (P=0.011). Elevated level of EO was associated with nighttime blood pressure and subclinical organ damage in treated hypertensive patients, suggesting possible role of EO in the pathogenesis of impaired diurnal blood pressure rhythm and arterial stiffness.

Morphology of Glomerular Hematuria Is Reproduced in vitro by Carbonyl Stress

Abstract Background: In glomerulonephritides, dysmorphic red blood cells (RBCs) with membrane blebs can be found in the urine; this is referred to as glomerular hematuria. Glomerulonephritides are characterized by increased carbonyl stress and elevated methylglyoxal (MGO) levels. MGO causes oxidative stress and intracellular calcium accumulation. In the present study, we investigated whether the effect of MGO-induced calcium accumulation in RBCs develops through increased oxidative stress. Furthermore, we studied whether MGO can lead to RBC membrane blebbing. Methods: RBC suspensions from healthy volunteers were incubated with different concentrations of MGO at 37°C. We measured oxidative stress and intracellular calcium level using fluorescent indicators. We determined the frequency of dysmorphic RBCs, and also performed scanning electron microscopy. Results: MGO increased oxidative stress and caused accumulation of calcium in isolated RBCs. These effects could be prevented using antioxidants. In the presence of MGO, RBC membrane blebbing developed. Conclusion: According to our findings, MGO causes calcium accumulation through oxidative stress. Carbonyl and oxidative stress may play an important role in the formation of dysmorphic RBCs in glomerular hematuria.