Esztella Mikolás

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.

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.

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.

Tyrosine isomers and hormonal signaling: A possible role for the hydroxyl free radical in insulin resistance.

Oxidative stress processes play a major role in the development of the complications associated with diabetes and other diseases via non-enzymatic glycation, the hexosamine pathway, the polyol pathway and diacylglycerol-protein kinase C. Oxidative stress may lead to the production of hydroxyl free radicals, which can attack macromolecules, such as lipids, nucleic acids or amino acids. Phenylalanine (Phe) can be enzymatically converted to the physiological para-tyrosine (p-Tyr); however, a hydroxyl free radical attack on Phe may yield meta- and ortho-tyrosine (m- and o-Tyr, respectively) in addition to p-Tyr. Hence, m- and o-Tyr may be regarded as markers of hydroxyl free radical-induced damage. Their accumulation has been described; e.g., this accumulation has been found in the urine of patients with diabetes mellitus (DM) and/or chronic kidney disease, in cataract lenses, in vessel walls, in irradiated food and in amniotic fluid, and it may serve as an indicator of oxidative stress. The use of resveratrol to treat patients with type 2 DM led to a decrease in the urinary excretion of o-Tyr and concomitantly led to an improvement in insulin signaling and insulin sensitivity. Literature data also suggest that m- and o-Tyr may interfere with intracellular signaling. Our group has shown that erythropoietin (EPO) has insulin-like metabolic effects on fat cells in addition to its ability to promote the proliferation of erythroid precursor cells. We have shown that the supplementation of cell culture medium with m- and o-Tyr inhibits erythroblast cell proliferation, which could be ameliorated by p-Tyr. Additionally, in vivo, the o-Tyr/p-Tyr ratio is higher in patients with renal replacement therapy and a greater need for EPO. However, the o-Tyr/p-Tyr ratio was an independent determinant of EPO-resistance indices in our human study. The o-Tyr content of blood vessel walls inversely correlates with insulin- and acetylcholine-induced vasodilation, which could be further impaired by artificial oxidative stress and improved by the use of antioxidants. In rats that receive o-Tyr supplements, decreased vasorelaxation is detected in response to insulin. Additionally, o-Tyr supplementation led to the incorporation of the unnatural amino acid into cellular proteins and caused a decrease in the insulin-induced phosphorylation of endothelial nitric oxide synthase. Our data suggest that m- and o-Tyr may not only be markers of oxidative stress; instead, they may also be incorporated into cellular proteins, leading to resistance to insulin, EPO and acetylcholine.

Association of plasma ortho-tyrosine/para-tyrosine ratio with responsiveness of erythropoiesis-stimulating agent in dialyzed patients.

Abstract Objectives Patients with end-stage renal failure (ESRF) treated with erythropoiesis-stimulating agents (ESAs) are often ESA-hyporesponsive associated with free radical production. Hydroxyl free radical converts phenylalanine into ortho-tyrosine, while physiological isomer para-tyrosine is formed enzymatically, mainly in the kidney. Production of ‘para-tyrosine’ is decreased in ESRF and it can be replaced by ortho-tyrosine in proteins. Our aim was to study the role of tyrosines in ESA-responsiveness. Methods Four groups of volunteers were involved in our cross-sectional study: healthy volunteers (CONTR; n = 16), patients on hemodialysis without ESA-treatment (non-ESA-HD; n = 8), hemodialyzed patients with ESA-treatment (ESA-HD; n = 40), and patients on continuous peritoneal dialysis (CAPD; n = 21). Plasma ortho-, para-tyrosine, and phenylalanine levels were detected using a high performance liquid chromatography (HPLC)-method. ESA-demand was expressed by ESA-dose, ESA-dose/body weight, and erythropoietin resistance index1 (ERI1, weekly ESA-dose/body weight/hemoglobin). Results We found significantly lower para-tyrosine levels in all groups of dialyzed patients when compared with control subjects, while in contrast ortho-tyrosine levels and ortho-tyrosine/para-tyrosine ratio were comparatively significantly higher in dialyzed patients. Among groups of dialyzed patients the ortho-tyrosine level and ortho-tyrosine/para-tyrosine ratio were significantly higher in ESA-HD than in the non-ESA-HD and CAPD groups. There was a correlation between weekly ESA-dose/body weight, ERI1, and ortho-tyrosine/para-tyrosine ratio (r = 0.441, P = 0.001; r = 0.434, P = 0.001, respectively). Our most important finding was that the ortho-tyrosine/para-tyrosine ratio proved to be an independent predictor of ERI1 (β = 0.330, P = 0.016). In these multivariate regression models most of the known predictors of ESA-hyporesponsiveness were included. Discussion Our findings may suggest that elevation of the ratio of ortho-tyrosine/para-tyrosine could be responsible for decreased ESA-responsiveness in dialyzed patients.

Increase in insulin-induced relaxation of consecutive arterial segments toward the periphery: Role of vascular oxidative state

Abstract Rationale. The oxidative state has been implicated in the signaling of various vasomotor functions, yet its role regarding the vasomotor action of insulin is less known. Objective. To investigate the insulin-evoked relaxations of consecutive arterial segments of different oxidative state and the role of extracellular signal-regulated kinase (ERK) pathway. Methods and Results. The oxidative state, as assessed by the level of ortho-tyrosine, was higher in the thoracic aorta of rats than in the abdominal aorta, and was the lowest in the femoral artery. The vasomotor function of vessels of same origin was studied using a small-vessel myograph. Insulin-induced relaxations increased toward the periphery (i.e., thoracic < abdominal < femoral). Aortic banding and hydrogen peroxide/aminotriazole increased the oxidative state of the thoracic aorta that was accompanied by ERK activation and decreased relaxation to insulin, and vice versa, acutely lowered oxidative state by superoxide dismutase/catalase improved relaxation. In contrast, insulin-induced relaxation of the femoral artery could be enhanced with a higher oxidative state, and reduced with a lower state. Conclusions. Oxidative state of vessels modulates the magnitude of vasomotor responses to insulin, which appears to be mediated via the ERK signaling pathway.

Incorporation of ortho- and meta-tyrosine into cellular proteins leads to erythropoietin-resistance in an erythroid cell line.

Background/Aims: Erythropoietin-resistance is an unsolved concern in the treatment of renal anaemia. We aimed to investigate the possible role of ortho- and meta-tyrosine - the hydroxyl free radical products of L-phenylalanine - in the development of erythropoietin-resistance. Methods: TF-1 erythroblast cell line was used. Cell concentration was determined on day 1; 2 and 3 by two independent observers simultaneously in Bürker cell counting chambers. Protein concentration was determined with colorimetric method. Para-, ortho- and meta-tyrosine levels were measured using reverse phase-HPLC with fluorescence detection. Using Western blot method activating phosphorylation of STAT5 and ERK1/2 were investigated. Results: We found a time- and concentration-dependent decrease of erythropoietin-induced proliferative activity in case of ortho- and meta-tyrosine treated TF-1 erythroblasts, compared to the para-tyrosine cultured cells. Decreased erythropoietin-response could be regained with a competitive dose of para-tyrosine. Proteins of erythroblasts treated by ortho- or meta-tyrosine had lower para-tyrosine and higher ortho- or meta-tyrosine content. Activating phosphorylation of ERK and STAT5 due to erythropoietin was practically prevented by ortho- or meta-tyrosine treatment. Conclusion: According to this study elevated ortho- and meta-tyrosine content of erythroblasts may lead to the dysfunction of intracellular signaling, resulting in erythropoietin-hyporesponsiveness.