Klára Rosta

Association between Estrogen Receptor α (ESR1) Gene Polymorphisms and Severe Preeclampsia

Associations have been reported between estrogen receptor α (ESR1) gene polymorphisms and various pathological conditions, including cardiovascular diseases. Our aim was to investigate whether two polymorphisms of the ESR1 gene (ESR1 c.454 −397T>C: PvuII restriction site and c.454 −351A>G: XbaI restriction site) are associated with preeclampsia. In a case-control study, we analyzed blood samples from 119 severely preeclamptic patients and 103 normotensive, healthy pregnant women using the polymerase chain reaction (PCR)−restriction fragment length polymorphism (RFLP) method. All of the women were Caucasian. There was no association between severe preeclampsia and the PvuII and XbaI ESR1 gene polymorphisms separately. However, with the simultaneous carriage of both polymorphisms, the TT/AA genotype combination was significantly more frequent in severely preeclamptic patients than in healthy control subjects (24.4% vs. 9.7%, p=0.003), whereas the TT/AG combination was significantly less frequent in the severely preeclamptic group than in the control group (5.0% vs. 18.4%, p=0.002). According to the haplotype estimation, the homozygous T-A haplotype carriers had an increased risk of severe preeclampsia independent of maternal age, prepregnancy BMI, primiparity and smoking status (adjusted odds ratio [OR]: 4.36, 95% confidence interval [CI]: 1.65–11.53). The GG genotype of the XbaI polymorphism was associated with a lower risk of fetal growth restriction in patients with severe preeclampsia (OR: 0.23, 95% CI: 0.07–0.73). In conclusion, the homozygous T-A haplotype carriers of ESR1 PvuII and XbaI polymorphisms showed an increased risk of severe preeclampsia. In addition, the GG genotype of the XbaI polymorphism decreased the risk of fetal growth restriction in severely preeclamptic patients.

Association of extracellular superoxide dismutase (SOD3) Ala40Thr gene polymorphism with pre-eclampsia complicated by severe fetal growth restriction.

OBJECTIVE Placental and systemic oxidative stress with an imbalance in the oxidant/antioxidant activity seems to play a central role in the pathogenesis of pre-eclampsia. The aim of our study was to examine whether two missense polymorphisms of the extracellular superoxide dismutase (SOD3) gene (Arg213Gly and Ala40Thr) are associated with pre-eclampsia in a Caucasian population from Hungary. STUDY DESIGN One hundred and fifty-nine pre-eclamptic patients and 114 normotensive, healthy pregnant women were involved in this case-control study. The SOD3 Arg213Gly and Ala40Thr genotypes were determined using the polymerase chain reaction-restriction length polymorphism (PCR-RFLP) and allele-specific amplification methods. RESULTS The Arg213Gly variant was not detected in our population. There were no significant differences in the genotype and allele frequencies of the SOD3 Ala40Thr polymorphism between pre-eclamptic patients and control subjects. However, the mutant allele carriers of this polymorphism showed an increased risk for severe fetal growth restriction-complicated pre-eclampsia, which was independent of maternal age, prepregnancy BMI, primiparity and smoking status (OR: 6.07, 95% CI: 1.33-27.8, p=0.020; adjusted OR: 4.89, 95% CI: 1.03-23.2, p=0.046). CONCLUSION Our results suggest a role of SOD3 Ala40Thr single nucleotide polymorphism in the risk of severe fetal growth restriction-complicated pre-eclampsia. However, further studies are needed with a larger sample size to confirm our findings and to explore the exact molecular basis of this observation.

Altered gene expression profiles in the hippocampus and prefrontal cortex of type 2 diabetic rats.

BackgroundThere has been an increasing body of epidemiologic and biochemical evidence implying the role of cerebral insulin resistance in Alzheimer-type dementia. For a better understanding of the insulin effect on the central nervous system, we performed microarray-based global gene expression profiling in the hippocampus, striatum and prefrontal cortex of streptozotocin-induced and spontaneously diabetic Goto-Kakizaki rats as model animals for type 1 and type 2 diabetes, respectively.ResultsFollowing pathway analysis and validation of gene lists by real-time polymerase chain reaction, 30 genes from the hippocampus, such as the inhibitory neuropeptide galanin, synuclein gamma and uncoupling protein 2, and 22 genes from the prefrontal cortex, e.g. galanin receptor 2, protein kinase C gamma and epsilon, ABCA1 (ATP-Binding Cassette A1), CD47 (Cluster of Differentiation 47) and the RET (Rearranged During Transfection) protooncogene, were found to exhibit altered expression levels in type 2 diabetic model animals in comparison to non-diabetic control animals. These gene lists proved to be partly overlapping and encompassed genes related to neurotransmission, lipid metabolism, neuronal development, insulin secretion, oxidative damage and DNA repair. On the other hand, no significant alterations were found in the transcriptomes of the corpus striatum in the same animals. Changes in the cerebral gene expression profiles seemed to be specific for the type 2 diabetic model, as no such alterations were found in streptozotocin-treated animals.ConclusionsAccording to our knowledge this is the first characterization of the whole-genome expression changes of specific brain regions in a diabetic model. Our findings shed light on the complex role of insulin signaling in fine-tuning brain functions, and provide further experimental evidence in support of the recently elaborated theory of type 3 diabetes.

Na+,K+‐ATPase is modulated by angiotensin II in diabetic rat kidney – another reason for diabetic nephropathy?

Angiotensin II (ANGII) plays a central role in the enhanced sodium reabsorption in early type 1 diabetes in man and in streptozotocin‐induced (STZ) diabetic rats. This study investigates the effect of untreated STZ‐diabetes leading to diabetic nephropathy in combination with ANGII treatment, on the abundance and localization of the renal Na+,K+‐ATPase (NKA), a major contributor of renal sodium handling. After 7 weeks of STZ‐diabetes (i.v. 65 mg kg−1) a subgroup of control (C) and diabetic (D7) Wistar rats were treated with ANGII (s.c. minipump 33 μg kg−1 h−1 for 24 h; CA and D7A). We measured renal function and mRNA expression, protein level, Serin23 phosphorylation, subcellular distribution, and enzyme activity of NKA α‐1 subunit in the kidney cortex. Diabetes increased serum creatinine and urea nitrogen levels (C versus D7), as did ANGII (C versus CA, D7 versus D7A). Both diabetes (C versus D7) and ANGII increased NKA α‐1 protein level and enzyme activity (C versus CA, D7 versus D7A). Furthermore, the combination led to an additive increase (D7 versus D7A, CA versus D7A). NKA α‐1 Ser23 phosphorylation was higher both in D7 and ANGII‐treated rats in the non‐cytoskeletal fraction, while no signal was detected in the cytoskeletal fraction. Control kidneys showed NKA α‐1 immunopositivity on the basolateral membrane of proximal tubular cells, while both D7 and ANGII broadened NKA immunopositivity towards the cytoplasm. Our study demonstrates that diabetes mellitus (DM) increases the mRNA expression, protein level, Ser23 phosphorylation and enzyme activity of renal NKA, which is further elevated by ANGII. Despite an increase in total NKA quantity in diabetic nephropathy, the redistribution to the cystosol suggests the Na+ pump is no longer functional. ANGII also caused translocation from the basolateral membrane, thus in diabetic states where ANGII level is acutely elevated, the loss of NKA will be exacerbated. This provides another mechanism by which ANGII blockade is likely to be protective.

Leptin receptor gene polymorphisms in severely pre-eclamptic women

Variants of the leptin receptor gene (LEPR) may modulate the effect of elevated serum leptin levels in pre-eclampsia. The aim of our study was to evaluate the LEPR gene polymorphisms Lys109Arg (A109G) and Gln223Arg (A223G) in severely pre-eclamptic women. In a case–control study, we analyzed blood samples from 124 severely pre-eclamptic patients and 107 healthy control women by the polymerase chain reaction–restriction fragment length polymorphism method. The Pearson χ2 test was used to estimate odds ratios (OR) and 95% confidence intervals (CI). The association was adjusted for maternal age, pre-pregnancy body mass index and primiparity with logistic regression analysis. Pregnant women with the LEPR 223G allele (223A/G or 223G/G genotype) had almost double the risk of developing severe pre-eclampsia compared with patients with the 223A/A genotype (adjusted OR = 1.92, 95% CI: 1.07–3.41). Genotype variants of LEPR A109G alone did not affect the risk of severe pre-eclampsia. Haplotype estimation of A109G and A223G polymorphisms of the LEPR gene revealed that the G-A haplotype versus other pooled haplotypes was significantly less common in the pre-eclamptic group (p < 0.01), while the G-G haplotype versus others was overrepresented among severely pre-eclamptic patients (p < 0.01), compared with controls. In conclusion, our data indicate that LEPR A223G polymorphism may individually modify the risk of severe pre-eclampsia.

Insulin induced translocation of Na+/K+ -ATPase is decreased in the heart of streptozotocin diabetic rats.

AbstractAim:To investigate the effect of acute insulin administration on the subcellular localization of Na+/K+-ATPase isoforms in cardiac muscle of healthy and streptozotocin-induced diabetic rats.Methods:Membrane fractions were isolated with subcellular fractionation and with cell surface biotinylation technique. Na+/K+-ATPase subunit isoforms were analysed with ouabain binding assay and Western blotting. Enzyme activity was measured using 3-O-methylfluorescein-phosphatase activity.Results:In control rat heart muscle α1 isoform of Na+/K+ ATPase resides mainly in the plasma membrane fraction, while α2 isoform in the intracellular membrane pool. Diabetes decreased the abundance of α1 isoform (25 %, P<0.05) in plasma membrane and α2 isoform (50%, P<0.01) in the intracellular membrane fraction. When plasma membrane fractions were isolated by discontinuous sucrose gradients, insulin-stimulated translocation of α2- but not α1-subunits was detected. α1-Subunit translocation was only detectable by cell surface biotinylation technique. After insulin administration protein level of α2 increased by 3.3-fold, α1 by 1.37-fold and β1 by 1.51-fold (P<0.02) in the plasma membrane of control, and less than 1.92-fold (P<0.02), 1.19-fold (not significant) and 1.34-fold (P<0.02) in diabetes. The insulin-induced translocation was wortmannin sensitive.Conclusion:This study demonstrate that insulin influences the plasma membrane localization of Na+/K+-ATPase isoforms in the heart. α2 isoform translocation is the most vulnerable to the reduced insulin response in diabetes. α1 isoform also translocates in response to insulin treatment in healthy rat. Insulin mediates Na+/K+-ATPase α1- and α2-subunit translocation to the cardiac muscle plasma membrane via a PI3-kinase-dependent mechanism.

Aldosterone antagonists in monotherapy are protective against streptozotocin-induced diabetic nephropathy in rats

Angiotensin converting enzyme inhibitors (ACEi) and angiotensin II receptor blockers (ARB) are the standard clinical therapy of diabetic nephropathy (DN), while aldosterone antagonists are only used as adjuncts. Previously in experimental DN we showed that Na/K ATPase (NKA) is mislocated and angiotensin II leads to superimposed renal progression. Here we investigated the monotherapeutic effect of aldosterone blockers on the progression of DN and renal NKA alteration in comparison to ACEi and ARBs. Streptozotocin-diabetic rats developing DN were treated with aldosterone antagonists; ACEi and ARB. Renal function, morphology, protein level and tubular localization of NKA were analyzed. To evaluate the effect of high glucose per se; HK-2 proximal tubular cells were cultured in normal or high concentration of glucose and treated with the same agents. Aldosterone antagonists were the most effective in ameliorating functional and structural kidney damage and they normalized diabetes induced bradycardia and weight loss. Aldosterone blockers also prevented hyperglycemia and diabetes induced increase in NKA protein level and enzyme mislocation. A monotherapy with aldosterone antagonists might be as, or more effective than ACEi or ARBs in the prevention of STZ-induced DN. Furthermore the alteration of the NKA could represent a novel pathophysiological feature of DN and might serve as an additional target of aldosterone blockers.

Association Study with 77 SNPs Confirms the Robust Role for the rs10830963/G of MTNR1B Variant and Identifies Two Novel Associations in Gestational Diabetes Mellitus Development

Context Genetic variation in human maternal DNA contributes to the susceptibility for development of gestational diabetes mellitus (GDM). Objective We assessed 77 maternal single nucleotide gene polymorphisms (SNPs) for associations with GDM or plasma glucose levels at OGTT in pregnancy. Methods 960 pregnant women (after dropouts 820: case/control: m99’WHO: 303/517, IADPSG: 287/533) were enrolled in two countries into this case-control study. After genomic DNA isolation the 820 samples were collected in a GDM biobank and assessed using KASP (LGC Genomics) genotyping assay. Logistic regression risk models were used to calculate ORs according to IADPSG/m’99WHO criteria based on standard OGTT values. Results The most important risk alleles associated with GDM were rs10830963/G of MTNR1B (OR = 1.84/1.64 [IADPSG/m’99WHO], p = 0.0007/0.006), rs7754840/C (OR = 1.51/NS, p = 0.016) of CDKAL1 and rs1799884/T (OR = 1.4/1.56, p = 0.04/0.006) of GCK. The rs13266634/T (SLC30A8, OR = 0.74/0.71, p = 0.05/0.02) and rs7578326/G (LOC646736/IRS1, OR = 0.62/0.60, p = 0.001/0.006) variants were associated with lower risk to develop GDM. Carrying a minor allele of rs10830963 (MTNR1B); rs7903146 (TCF7L2); rs1799884 (GCK) SNPs were associated with increased plasma glucose levels at routine OGTT. Conclusions We confirmed the robust association of MTNR1B rs10830963/G variant with GDM binary and glycemic traits in this Caucasian case-control study. As novel associations we report the minor, G allele of the rs7578326 SNP in the LOC646736/IRS1 region as a significant and the rs13266634/T SNP (SLC30A8) as a suggestive protective variant against GDM development. Genetic susceptibility appears to be more preponderant in individuals who meet both the modified 99’WHO and the IADPSG GDM diagnostic criteria.

Cord serum dipeptidyl-peptidase 4 activity in gestational diabetes

Tissue‐specific dipeptidyl‐peptidase 4 (DPP4) dysregulation has been described in adults with diabetes mellitus. The DPP4 ‐incretin system has not been studied in foetal life. In this study, DPP4 activity and glucagon‐like peptide‐1 (GLP‐1) levels were assessed in cord blood of neonates born to women with gestational diabetes mellitus (GDM) and nondiabetic controls.

Hearing impairment and tinnitus in patients with type 2 diabetes

INTRODUCTION Hearing impairment is one of the most frequent chronic health issue. The incidence of hearing impairment and tinnitus increases with age. AIM The aim of the authors was to determine the prevalence of hearing impairment and tinnitus in type 2 diabetic patients and to examine the possible associations between hearing impairment and/or tinnitus and increased HbA1c levels. METHODS 103 patients with type 2 diabetes (47 men, 56 women; age, 61.6±10.3 years, mean±SD; range, 33-88 years) evaluated at the 2nd Department of Medicine, Semmelweis University were enrolled in this study and the results were compared to those obtained from 589 type 2 diabetic (253 men, 336 women; age, 55.4±11.0 years, mean±SD; range, 26-97 years) and 15 622 non-diabetic patients (7002 men, 8620 women; age, 55.1±11.1 years, mean±SD; range, 26-98 years) who participated in a comprehensive health screening programme in Hungary. Hearing impairment was determined using the Interacoustics model AS608 screening audiometer in all patient groups. Tinnitus was evaluated with questionnaire. RESULTS It was found that hearing impairment and/or tinnitus occurred in a very high proportion of type 2 diabetic patients evaluated at the 2nd Department of Medicine, Semmelweis University (80% of cases) as compared to type 2 diabetic (34% of cases) and non-diabetic patients (14% of cases) enrolled in the national health screening programme. There was no significant correlation between increased HbA1c levels and hearing impairment or tinnitus in type 2 diabetic patients. CONCLUSION These results suggest that the prevalence of hearing impairment and tinnitus is higher and develop at an earlier age in patients with type 2 diabetes. The results indicate a high prevalence of hearing impairment and tinnitus in type 2 diabetic patients.