Katarína Kuricová

Evidence for altered thiamine metabolism in diabetes: Is there a potential to oppose gluco- and lipotoxicity by rational supplementation?

Growing prevalence of diabetes (type 2 as well as type 1) and its related morbidity due to vascular complications creates a large burden on medical care worldwide. Understanding the molecular pathogenesis of chronic micro-, macro- and avascular complications mediated by hyperglycemia is of crucial importance since novel therapeutic targets can be identified and tested. Thiamine (vitamin B1) is an essential cofactor of several enzymes involved in carbohydrate metabolism and published data suggest that thiamine metabolism in diabetes is deficient. This review aims to point out the physiological role of thiamine in metabolism of glucose and amino acids, to present overview of thiamine metabolism and to describe the consequences of thiamine deficiency (either clinically manifest or latent). Furthermore, we want to explain why thiamine demands are increased in diabetes and to summarise data indicating thiamine mishandling in diabetics (by review of the studies mapping the prevalence and the degree of thiamine deficiency in diabetics). Finally, we would like to summarise the evidence for the beneficial effect of thiamine supplementation in progression of hyperglycemia-related pathology and, therefore, to justify its importance in determining the harmful impact of hyperglycemia in diabetes. Based on the data presented it could be concluded that although experimental studies mostly resulted in beneficial effects, clinical studies of appropriate size and duration focusing on the effect of thiamine supplementation/therapy on hard endpoints are missing at present. Moreover, it is not currently clear which mechanisms contribute to the deficient action of thiamine in diabetes most. Experimental studies on the molecular mechanisms of thiamine deficiency in diabetes are critically needed before clear answer to diabetes community could be given.

Genetic variability in enzymes of metabolic pathways conferring protection against non-enzymatic glycation versus diabetes-related morbidity and mortality

Abstract Background: We hypothesized that genetic variability in genes encoding enzymes metabolizing glycolytic intermediates produced in excess under hyperglycemic conditions [i.e., transketolase (TKT), transaldolase, TKT-like protein 1, fructosamine 3-kinase (FN3K), glyoxalase 1 and glucose-6-phosphate dehydrogenase] could influence progression of diabetic nephropathy (DN) and diabetes-related morbidity and mortality. Methods: A total of 19 single nucleotide polymorphisms (SNPs) in six candidate genes were studied in 314 type 2 diabetic subjects with variable stage of kidney disease (normo- and microalbuminuria, proteinuria, end-stage renal disease). SNP selection criteria were based on known functional effect and gene coverage. SNPs were detected using polymerase chain reaction based methods. Subjects were followed up for median of 38 months. Time-to-event analysis considered three end-points: 1) DN progression by at least one stage; 2) major cardiovascular event; and 3) all-cause mortality. Results: We found combined effect of TKT SNP rs11130362 and FN3K SNP rs1056534 on DN progression (p<0.01). Additionally, TKT rs3736156 alone and also in combination with the previous two SNPs exhibited significant effect on incidence of major cardiovascular events (p<0.01 and p=0.01, respectively). Conclusions: Genetic variability in rate-limiting enzymes of pathways proposed to confer hypothetical protection against hyperglycemia might act as an important determinant of hyperglycemia toxicity in long-standing diabetes.

ADMA, SDMA and L-arginine/ADMA ratio but not DDAH genetic polymorphisms are reliable predictors of diabetic nephropathy progression as identified by competing risk analysis.

Background/Aims: Complex interplay of genetic and (patho)physiological factors influence availability of nitric oxide during the development and progression of diabetic complications. We assessed predictive value of commonly studied methylated asymmetric and symmetric dimethylarginines (ADMA and SDMA) and selected single nucleotide polymorphisms (SNPs) in dimethylarginine dimethylaminohydrolase (DDAH) 1 and 2 genes for the progression of diabetic nephropathy (DN). Methods: A total of 341 type 1 and type 2 diabetes patients with variable degree of kidney disease were included at baseline. Plasma levels of ADMA, SDMA and L-arginine were measured and six tagging SNPs in DDAH1 and 2 were determined. Progression of DN was defined as a transition from any given stage to a more advanced stage of albuminuria. Competing risk analysis was applied. Results: Plasma levels of ADMA and SDMA significantly correlated with GFR. No significant genotype-phenotype relationship was ascertained for ADMA and DDAH variants, but SNP rs805304 exhibited marginally significant association with DN. ADMA, SDMA and L-arginine/ADMA ratio standardised to GFR were identified as significant predictors of DN progression but not GFR decline using multivariate competing risk analysis. Conclusions: In our study we confirmed potentially significant role of ADMA and SDMA for the assessment of risk of DN progression in European diabetic population.

Hyperuricemia contributes to the faster progression of diabetic kidney disease in type 2 diabetes mellitus

AIMS The aims of the study were (i) to ascertain prognostic value of serum uric acid (SUA) for diabetic kidney disease (DKD) progression and major adverse cardiovascular event (MACE) in a cohort of T2DM patients, (ii) to ascertain eventual protective effect of allopurinol treatment, (iii) to determine the effect of genetic variability in UA transporters on DKD progression, and (iv) to define optimal cut-off values for SUA in patients with DKD. METHODS Study comprised 422 subjects with diabetes duration at least 15years followed-up for a median of 43 [IQR 22-77] months. Participants were categorized into stable or progressors according to their change in albuminuria or chronic kidney disease (CKD) stage. At baseline, 68% patients had hyperuricemia (SUA≥420μmol/l for men and ≥360μmol/l for women and/or allopurinol treatment). Five SNPs in the SLC2A9 and ABCG2 genes were determined by PCR. RESULTS Time-to-event analysis with subgroups defined by the presence/absence of initial hyperuricemia revealed significant differences in all three end-points (P<0.0001 for DKD progression, P=0.0022 for MACE and P=0.0002 for death, log-rank test). Subjects with normal SUA not requiring allopurinol had median time to DKD progression 49months compared with remaining subjects (32months, P=0.0002, log-rank test). Multivariate Cox regression model revealed hyperuricemia (i.e. high SUA and/or allopurinol treatment) significant predictor of DKD progression independent of baseline CKD stage. Optimal cut-off values identified by ROC analysis for T2DM subjects were ≤377.5μmol/l for men and ≤309.0μmol/l for women. We found no differences in allele or genotype frequencies in selected SNPs between patients with and without hyperuricemia (all P>0.05). CONCLUSIONS Our study demonstrated that initial hyperuricemia or need for allopurinol is an independent risk factor for DKD progression and that SUA levels in diabetic subjects conferring protection against DKD progression might be lower than current cut-offs for general population.

Vitamin D Status in Women with Gestational Diabetes Mellitus during Pregnancy and Postpartum.

Of many vitamin D extraskeletal functions, its modulatory role in insulin secretion and action is especially relevant for gestational diabetes mellitus (GDM). The aims of the present study were to determine midgestational and early postpartum vitamin D status in pregnant women with and without GDM and to describe the relationship between midgestational and postpartum vitamin D status and parallel changes of glucose tolerance. A total of 76 pregnant women (47 GDM and 29 healthy controls) were included in the study. Plasma levels of 25(OH)D were measured using an enzyme immunoassay. Vitamin D was not significantly decreased in GDM compared to controls during pregnancy; however, both groups of pregnant women exhibited high prevalence of vitamin D deficiency. Prevalence of postpartum 25(OH)D deficiency in post-GDM women remained significantly higher and their postpartum 25(OH)D levels were significantly lower compared to non-GDM counterparts. Finally, based on the oGTT repeated early postpartum persistent glucose abnormality was ascertained in 15% of post-GDM women; however, neither midgestational nor postpartum 25(OH)D levels significantly differed between subjects with GDM history and persistent postpartum glucose intolerance and those with normal glucose tolerance after delivery.

Effect of glucose variability on pathways associated with glucotoxicity in diabetes: Evaluation of a novel in vitro experimental approach

AIMS Glycaemic variability (GV) has been hypothesized to increase the risk of diabetes complications; however, results of clinical studies are contradictory. The effect of GV on cell phenotypes has been investigated in vitro showing that GV may have more deleterious effect on cells that high glucose itself. However, methodology used to study GV in vitro differs significantly between studies and does not reflect in vivo situation. Therefore we aimed to establish clinically relevant an in vitro experimental approach for the study of GV that reflects intra-day glucose fluctuations of subjects with type 1 diabetes mellitus (T1DM) and of healthy subjects and to test how low and high GV affect expression of genes that protects cells from hyperglycaemia-induced damage. METHODS Human umbilical vein endothelial cells (HUVEC) were cultured 24h in medium with different glucose profiles: high GV, low GV and GV of healthy subjects-profiles created according to CGM of T1DM patients and healthy subjects. These profiles were compared to commonly used 5.5 and 25mmol/l glucose concentrations. Gene expression was determined using quantitative PCR. RESULTS Our results showed general down-regulation of enzymes that are involved in the protection against hyperglycaemia-induced intracellular changes in both low and high GV compared to normal glycaemia similarly to the decrease induced by continuous hyperglycaemia. Gene expressions did not differ between high and low GV. CONCLUSION Our data indicate that GV may have similar or even greater effect than continuous hyperglycaemia on the expression of several genes relevant to pathogenesis of diabetes microvascular complications.

Association of the Arg72Pro Polymorphism in p53 with Progression of Diabetic Nephropathy in T2DM Subjects

Objective: In addition to its anticancer function, p53 (regulated by murine double minute 2 oncoprotein, MDM2) has recently been shown to control intracellular metabolic processes. It participates in the regulation of glucose, fatty and amino acid and purine metabolism, influences mitochondrial integrity and oxidative phosphorylation, insulin sensitivity, antioxidant response and autophagy. With respect to the possible impact of genetic variability in p53 and MDM2 on metabolic compensation the aim of the study was to analyse the effect of common germ line Single Nucleotide Polymorphisms (SNPs) - Arg72Pro in the TP53 and SNP309 in the MDM2 - on the progression of Diabetic Nephropathy (DN), cardiovascular morbidity and mortality and all-cause mortality in Type 2 Diabetes Mellitus (T2DM) subjects. Methods: The cross-sectional study comprised a total of 309 (a sum of 155 and 154) unrelated Caucasian diabetic patients with diabetes duration at least 10 years and variable renal function at baseline (309, mean age was 67.2 ± 10.8 years). The stage of diabetic nephropathy was defined according to the urinary albumin excretion and glomerular filtration rate. Patients were followed-up for median 37 (20-59) months. The following end-points were considered: (a) progression of DN, (b) major cardiovascular event (non-fatal or fatal myocardial infarction or stroke, limb amputation, revascularization), (c) all-cause mortality. Genotypes were determined by PCR-based methodology. Time-to-event analysis using Kaplan-Meier curves and log-rank test was used. Results: We found significant difference between CG+GG vs. CC genotypes of the p53 Arg72Pro SNP for DN progression (P=0.046, log-rank test). Carriers of genotypes containing G allele (previously associated with susceptibility to T2DM) had faster progression of DN than CC genotype carriers. We did not find any significant difference between genotypes of MDM2 SNP for any of the end-points studied. Conclusions: Presented findings in general support the role of p53 in the pathogenesis of metabolic diseases, namely progression of hyperglycemia-related morbidity. Nevertheless, further studies are warranted to elucidate the eventual causal involvement of p53 pathway in the development of diabetic complications.

Serum carboxymethyl-lysine, a dominant advanced glycation end product, is increased in women with gestational diabetes mellitus

AIMS The objective of the study was to measure one of the circulating Advanced Glycation End Products (AGEs) - Nε-(carboxymethyl)lysine (CML) - in a case-control study (n = 307) of pregnant women with gestational diabetes mellitus (GDM) and physiological pregnancies and to ascertain the factors contributing to CML levels and the potential relevance of CML for selected perinatal and postpartum outcomes. METHODS All subjects underwent oGTT between 24th and 30th week of gestation and GDM was diagnosed according to WHO criteria. CML was determined by ELISA using commercial kit. RESULTS Unadjusted and plasma protein adjusted CML levels were significantly higher in women with GDM compared to healthy controls (P = 0.00043 and P = 1x10(-5), respectively, Mann-Whitney). CML was significantly inversely correlated with both pre- and mid-gestational BMI, however, differences between GDM and control group remained significant even after adjustment for BMI. CML levels correlated with 1-h and 2-h post-load glycaemia during oGTT. CONCLUSION In conclusion, we found statistically significantly higher protein- and BMI-normalised CML levels measured during 24-30th week of gestation in women with GDM compared to healthy pregnant controls. Further studies are warranted to comprehensively asses the spectrum of AGEs in GDM and their relevance to future metabolic health of mother and offspring.

NOS3 894G>T Polymorphism is Associated With Progression of Kidney Disease and Cardiovascular Morbidity in Type 2 Diabetic Patients: NOS3 as a Modifier Gene for Diabetic Nephropathy?

Background/Aims: We have previously associated SNP 894G>T in the NOS3 gene with diabetic nephropathy (DN) using multi-locus analysis. Variant 894G>T has been widely studied as a DN susceptibility factor with contradictory results. In the present study we genotyped 894G>T in the cohort of prospectively followed type 2 diabetics with the aim to investigate its possible role in the progression of DN and development of morbidity and mortality associated with diabetes. Methods: 311 subjects with defined stage of DN were enrolled in the study and followed up for a median of 38 months. We considered three end-points: progression of DN, major cardiovascular event and all-cause mortality. Results: Considering baseline GFR, age at enrolment and diabetes duration as confounders, Cox regression analysis identified 894GT genotype as a risk factor for DN progression (HR = 1.843 [95% CI 1.088 - 3.119], P = 0.023) and 894TT genotype as a risk factor for major cardiovascular event (HR = 2.515 [95% CI 1.060 - 5.965], P = 0.036). Conclusion: We ascertained the significant effect of the NOS3 894G>T variant on DN progression and occurrence of major cardiovascular event in T2DM subjects. Based on these results NOS3 can be considered a modifier gene for DN.