Effect of corticotropin releasing hormone and corticotropin releasing hormone antagonist on biosynthesis of gonadotropin relasing hormone and gonadotropin relasing hormone receptor in the hypothalamic-pituitary unit of follicular-phase ewes and contribution of kisspeptin.

This study aimed to determine the mechanisms governing Gonadotropin releasing hormone (GnRH) biosynthesis and luteinising hormone (LH) secretion in follicular-phase sheep after infusion of corticotropin releasing hormone (CRH) and/or CRH antagonist corticotropin releasing hormone nist (CRH-A) into the third cerebral ventricle. The study included two experimental approaches: first, we investigated the effect of CRH or CRH-A (α-helical CRH 9-41) on GnRH and GnRH receptor (GnRHR) biosynthesis in the preoptic area (POA), anterior (AH) and ventromedial hypothalamus (VMH), stalk/median eminence (SME), and on GnRHR in the anterior pituitary (AP) using an enzyme-linked immunosorbent assay (ELISA); second, we used real-time PCR to analyse the influence of CRH and CRH-A on the levels of kisspeptin (Kiss1) mRNA in POA and VMH including arcuate nucleus (VMH/ARC), and on Kiss1 receptor (Kiss1r) mRNA abundance in POA-hypothalamic structures. These analyses were supplemented by radioimmunoassay (RIA) and ELISA methods for measurement of LH and cortisol levels in the blood, respectively. Our results show that administration of CRH significantly decreased GnRH biosynthesis in the POA/hypothalamus. CRH also decreased GnRHR abundance in the hypothalamus and in the AP, but increased it in the POA. Furthermore, administration of CRH decreased plasma LH concentration and levels of Kiss1 mRNA in the POA and VMH/ARC as well as Kiss1r mRNA in these structures and in the SME. Significant increase in plasma cortisol concentration in the group treated with CRH was also observed. For CRH-A, all analysed effects were opposite to those induced by CRH. The study demonstrates that intracerebroventricular (i.c.v.) infusion of both CRH and CRH-A affects the GnRH/GnRHR biosynthesis and LH secretion in follicular-phase sheep conceivably via either central and peripheral mechanisms including Kiss1 neurons activity and cortisol signals. It has also been suggested that CRH and CRH-A infusion probably had effects directly at the AP.

The influence of dopaminergic system inhibition on biosynthesis of gonadotrophin-releasing hormone (GnRH) and GnRH receptor in anoestrous sheep; hierarchical role of kisspeptin and RFamide-related peptide-3 (RFRP-3).

This study aimed to explain how prolonged inhibition of central dopaminergic activity affects the cellular processes governing gonadotrophin-releasing hormone (GnRH) and LH secretion in anoestrous sheep. For this purpose, the study included two experimental approaches: first, we investigated the effect of infusion of sulpiride, a dopaminergic D2 receptor antagonist (D2R), on GnRH and GnRH receptor (GnRHR) biosynthesis in the hypothalamus and on GnRHR in the anterior pituitary using an immunoassay. This analysis was supplemented by analysis of plasma LH levels by radioimmunoassay. Second, we used real-time polymerase chain reaction to analyse the influence of sulpiride on the levels of kisspeptin (Kiss1) mRNA in the preoptic area and ventromedial hypothalamus including arcuate nucleus (VMH/ARC), and RFamide-related peptide-3 (RFRP-3) mRNA in the paraventricular nucleus (PVN) and dorsomedial hypothalamic nucleus. Sulpiride significantly increased plasma LH concentration and the levels of GnRH and GnRHR in the hypothalamic-pituitary unit. The abolition of dopaminergic activity resulted in a significant increase in transcript level of Kiss1 in VMH/ARC and a decrease of RFRP-3 in PVN. The study demonstrates that dopaminergic neurotransmission through D2R is involved in the regulatory pathways of GnRH and GnRHR biosynthesis in the hypothalamic-pituitary unit of anoestrous sheep, conceivably via mechanisms in which Kiss1 and RFRP-3 participate.

Biosynthesis of gonadotropin-releasing hormone (GnRH) and GnRH receptor (GnRHR) in hypothalamic-pituitary unit of anoestrous and cyclic ewes.

This study was performed to explain how the molecular processes governing the biosynthesis of gonadotropin-releasing hormone (GnRH) and GnRH receptor (GnRHR) in the hypothalamic-pituitary unit are reflected by luteinizing hormone (LH) secretion in sheep during anoestrous period and during luteal and follicular phases of the oestrous cycle. Using an enzyme-linked immunosorbent assay (ELISA), we analyzed the levels of GnRH and GnRHR in preoptic area (POA), anterior (AH) and ventromedial hypothalamus (VM), stalk-median eminence (SME), and GnRHR in the anterior pituitary gland (AP). Radioimmunoassay has also been used to define changes in plasma LH concentrations. The study provides evidence that the levels of GnRH in the whole hypothalamus of anoestrous ewes were lower than that in sheep during the follicular phase of the oestrous cycle (POA: p < 0.001, AH: p < 0.001, VM: p < 0.01, SME: p < 0.001) and not always than in luteal phase animals (POA: p < 0.05, SME: p < 0.05). It has also been demonstrated that the GnRHR amount in the hypothalamus-anterior pituitary unit, as well as LH level, in the blood in anoestrous ewes were significantly lower than those detected in animals of both cyclic groups. Our data suggest that decrease in LH secretion during the long photoperiod in sheep may be due to low translational activity of genes encoding both GnRH and GnRHR.

Effect of short-term and prolonged stress on the biosynthesis of gonadotropin-releasing hormone (GnRH) and GnRH receptor (GnRHR) in the hypothalamus and GnRHR in the pituitary of ewes during various physiological states.

Using an ELISA assay, the levels of GnRH and GnRHR were analysed in the preoptic area (POA), anterior (AH) and ventromedial hypothalamus (VM), stalk/median eminence (SME); and GnRHR in the anterior pituitary gland (AP) of non-breeding and breeding sheep subjected to short-term or prolonged stress. The ELISA study was supplemented with an analysis of plasma LH concentration. Short-term footshock stimulation significantly increased GnRH levels in hypothalamus in both seasons. Prolonged stress elevated or decreased GnRH concentrations in the POA and the VM, respectively during anoestrus, and lowered GnRH amount in the POA-hypothalamus of follicular-phase sheep. An up-regulation of GnRHR levels was noted in both, anoestrous and follicular-phase animals. In the non-breeding period, a prolonged stress procedure increased GnRHR biosynthesis in the VM and decreased it in the SME and AP, while in the breeding time the quantities of GnRHR were significantly lower in the whole hypothalamus. In follicular-phase ewes the fluctuations of GnRH and GnRHR levels under short-term and prolonged stress were reflected in the changes of LH secretion, suggesting the existence of a direct relationship between GnRH and GnRH-R biosynthesis and GnRH/LH release in this period. The study showed that stress was capable of modulating the biosynthesis of GnRH and GnRHR; the pattern of changes was dependent upon the animal's physiological state and on the time course of stressor application. The obtained results indicate that the disturbances of gonadotropin secretion under stress conditions in sheep may be due to a dysfunction of GnRH and GnRHR biosynthetic pathways.

The Central Effect of ß-Endorphin and Naloxone on The Biosynthesis of GnRH and GnRH Receptor (GnRHR) in The Hypothalamic-Pituitary Unit of Follicular-Phase Ewes.

The effects of prolonged, intermittent infusion of ß-endorphin or naloxone into the third cerebral ventricle of follicular-phase ewes on the expression of genes encoding GnRH and GnRHR in the hypothalamus and GnRHR in the anterior pituitary gland (AP) were examined by an enzyme-linked immunoabsorbent assay. Activation or blockade of µ-opioid receptors significantly decreased or increased the GnRH concentration and GnRHR abundance in the hypothalamus, respectively, and affected in the same way GnRHR quantity in the AP gland. The changes in the levels of GnRH and GnRHR after treatment with ß-endorphin as well as following action of naloxone were reflected in fluctuations of plasma LH concentrations. On the basis of these results, it is suggested that ß-endorphinergic system in the hypothalamus of follicular-phase ewes affects directly or via ß-endorphin-sensitive interneurons GnRH and GnRHR biosynthesis leading to suppression in secretory activity of the hypothalamic-pituitary axis.

Genetic evaluation of patients with Alström syndrome in the Polish population.

Alström syndrome (AS) is a rare syndromic form of obesity and type 2 diabetes (T2D) in children coexisting with retinal dystrophy and disorders of many organs caused by the mutations in ALMS1 gene. Aim of this study was to identify the causative mutations in ALMS1 in a group of 12 patients of Polish origin with clinical symptoms of AS, and their 21 first-degree relatives. Using DNA sequencing, nine different mutations including three novel were identified. These mutations were not present in 212 Polish individuals with no symptoms of AS, subjected to whole-exome sequencing and collected in a national registry. Looking for genotype-phenotype relationships, we confirmed a severe phenotype in a boy with homozygous mutation in exon 16, and a relationship between a presence of T2D and mutations in exon 19. Evaluation of the type of mutation and its clinical effects gives hope for earlier diagnosis of AS in future patients and more advanced therapeutic approaches for patients with already diagnosed AS.

Rapid increase in the incidence of type 1 diabetes in Polish children from 1989 to 2004, and predictions for 2010 to 2025.

AIMS/HYPOTHESIS: We analysed the temporal changes in the incidence of childhood type 1 diabetes and its demographic determinants in Poland from 1989 to 2004, validating the model with data from 1970 to 1989. We also estimated a predictive model of the trends in childhood diabetes incidence for the near future. METHODS: Children under 15 years with newly diagnosed type 1 diabetes mellitus and drawn from seven regional registries in Poland were ascertained prospectively using the Epidemiology and Prevention of Diabetes study (EURODIAB) criteria. The type 1 diabetes incidence rates (IRs) were analysed in dependency of age, sex, seasonality, geographical region and population density. Time trends in IR were modelled using several approaches. RESULTS: The average incidence, standardised by age and sex, for 1989 to 2004 was 10.2 per 100,000 persons per year and increased from 5.4 to 17.7. No difference was found between boys and girls, or between urban and rural regions. In children above 4 years, IR was significantly higher in the population of northern Poland than in that of the country's southern part, as well as in the autumn-winter season, this finding being independent of child sex. Based on the trend model obtained, almost 1,600 Polish children aged 0 to 14 years are expected to develop type 1 diabetes in 2010, rising to more than 4,800 in 2025. The estimates suggest at least a fourfold increase of IR between 2005 and 2025, with the highest dynamics of this increment in younger children. CONCLUSIONS/INTERPRETATION: These estimates show that Poland will have to face a twofold higher increase in childhood type 1 diabetes than predicted for the whole European population. The dramatic increase could have real downstream effects on Poland's healthcare system.

Implication of dopaminergic systems on GnRH and GnRHR genes expression in the hypothalamus and GnRH-R gene expression in the anterior pituitary gland of anestrous ewes.

We examined by Real-time PCR how prolonged inhibition of dopaminergic D-2 receptors (DA-2) in the hypothalamus of anestrous ewes by infusion of sulpiride into the third cerebral ventricle affected GnRH and GnRH-R gene expression in discrete parts of this structure and GnRH-R gene expression in the anterior pituitary. Blockaded DA-2 receptors significantly decreased GnRH mRNA levels in the ventromedial hypothalamus but did not evidently affect GnRH mRNA in the preoptic/ anteriorhypothalamicarea. Blockaded DA-2 receptors led to different responses in GnRH-R mRNA in various parts of the hypothalamus; increased GnRH-R mRNA levels in the preoptic/anterior hypothalamic area, and decreased GnRH-R mRNA amounts in the ventromedial hypothalamus stalk/median eminence. An infusion of sulpiride into the III-rd ventricle increased GnRH mRNA levels in the anterior pituitary gland and LH secretion. It is suggested that the increase of GnRH gene expression in the anterior pituitary gland and LH secretion in sulpiride-treated ewes are related with an increase of biosynthesis GnRH with concomitant decreased biosynthesis of GnRH-R protein in the ventromedial hypothalamus/stalk median eminence allowing to an increase of GnRH release.

The central effect of beta-endorphin and naloxone on the expression of GnRH Gene and GnRH receptor (GnRH-R) gene in the hypothalamus, and on GnRH-R gene in the anterior pituitary gland in follicular phase ewes.

The effect of prolonged intermittent infusion of beta-endorphin or naloxone into the third cerebral ventricle in ewes during the follicular phase of the estrous cycle on the expression of GnRH gene and GnRH-R gene in the hypothalamus and GnRH-R gene in the anterior pituitary gland was examined by Real time-PCR. Activation of micro opioid receptors decreased GnRH mRNA levels in the hypothalamus and led to complex changes in GnRH-R mRNA: an increase of GnRH-R mRNA in the preoptic area, no change in the anterior hypothalamus and decrease in the ventromedial hypothalamus and stalk/median eminence. In beta-endorphin treated ewes the levels of GnRH-R mRNA in the anterior pituitary gland also decreased significantly. These complex changes in the levels of GnRH mRNA and GnRH-R mRNA were reflected in the decrease of LH secretion. Blockade of micro opioid receptors affected neither GnRH mRNA and GnRH-R mRNA nor LH levels secretion. These results indicate that beta-endorphin displays a suppressive effect on the expression of the GnRH gene in the hypothalamus and GnRH-R gene in the anterior pituitary gland, but affects GnRH-R gene expression in a specific manner in the various parts of hypothalamus; altogether these events lead to the decrease in GnRH/LH secretion.

Increase in the incidence of type 1 diabetes mellitus in children in three cities in Poland, 1987-1999.

The aim of this study was to evaluate the trends in the incidence of type 1 diabetes mellitus (DM) in children aged 0-14 years between 1987 and 1999 in three cities in Poland. The study area comprised the provinces of Cracow and Wroclaw and the city of Warsaw. The data were collected prospectively on the basis of the register within the framework of the EURODIAB study up till 1997 and then within the project of the Ministry of Health. During the 13 years of the study period, 766 children (380 girls, 386 boys) with newly diagnosed type 1 DM were identified. The overall age-standardized incidence rates were 8.4/100,000 standardized population/year (95% CI 7.4-9.3) for Cracow province, 6.5/100,000/year (95% CI 5.6-7.4) for Wroclaw province and 7.9/100,000/year (95% CI 6.9-8.8) for Warsaw. A significant trend of increase for children aged 0-14 years was found in the three cities. The analysis of the trend in age subgroups showed a significant increase in incidence in all three age subgroups in Warsaw and Cracow province (0-4 year-old children, p <0.05; 5-9 year-olds, p <0.001 in Cracow province, p <0.05 in Warsaw, and in 10-14 year-olds, p <0.05 in Cracow province, p <0.005 in Warsaw). In the Wroclaw province a significant increase was observed in children aged 0-4 years (p <0.05) and 5-9 years (p <0.001). In children aged 10-14 years the increase was not statistically significant. The results of our study showed that the incidence of type 1 DM in children is rising. A similar phenomenon is occurring in many other countries. The greatest increase of incidence was observed in the 5-9 year-old subgroup of children in Cracow and Wroclaw provinces and in children aged 10-14 years in Warsaw. The incidence rates in excess of 9.0/100,000 per year observed since 1996 have placed Poland in the group of countries with low to medium incidence.

[Polish multicenter study on diabetes type I incidence in he age group 0-14 between 1998 and 1999]. / Wieloosrodkowe badania nad zapadalnoscia na cukrzyce typu 1 w grupie wiekowej 0-14 lat w Polsce w latach 1998-1999.

The increase in diabetes type 1 incidence observed in various centers in Poland and the need for a centralized study covering large population have resulted in the construction of a standardized registry of type 1 diabetes in 1998 within the Polish Multicenter Study in Diabetes Epidemiology. The aim of the study was to present the incidence rates of type 1 diabetes in the age group 0-14 in 7 distinct regions of Poland (Krakow, Wroclaw, Warsaw, Bialystok, Poznan, Rzeszow and Olsztyn centers) with over 30% of the Polish population at risk in 1998 and 1999. The data for the standardized registry were obtained prospectively from paediatric hospital wards and diabetes outpatient units. The incidence rates calculated in 1998 showed the highest value of 14.6 and 14.5/100,000 for Olsztyn and Warsaw, and the lowest (8.4/100,000) for Poznan center. In 1999 the highest value of 14.7/100,000 was noted in Krakow and the lowest (9.3/100,000) in Poznan center. The differences in diabetes type 1 incidence rates between age groups 0-4, 5-9 and 10-14 were found to be significant (p < 0.0005) and were also significant when incidence rates were compared between males and females in these age groups in the whole study area in 1998-1999 (p = 0.002 and p = 0.015 respectively).

[Risk of incidence of diabetes mellitus in relatives of patients with diabetes type I in retrospective questionnaire study]. / Ryzyko wystepowania cukrzycy u krewnych pacjentów z cukrzyca typu 1 w retrospektywnych badaniach ankietowych.

It is commonly known that there is a higher risk of diabetes type 1 in relatives of patients with diabetes type 1. According to some reports in families of these patients the incidence of diabetes type 2 is also higher. The aim of our study was the evaluation of incidence of diabetes type 1 and 2 in 1st and 2nd degree relatives of patients with diabetes type 1. Our study was conducted in the years 1993-2000 in the Department of Endocrinology for Children and Adolescents in Wroclaw and in the Department of Endocrinology of Jagiellonian University in Cracow among relatives of all the patients in the age of 0-19 years with newly diagnosed diabetes type 1. Special prepared questionnaires were used in which patients were asked about: number of relatives of 1st and 2nd degree, age, sex and diagnosis of diabetes in the relatives. Data from families of 332 patients were obtained. They concerned 4080 relatives. Diabetes occurred in 121 relatives (2.96%). 20 of them were 1st degree relatives and 101--2nd degree relatives. In 31 relatives (0.76%) diabetes type 1 was diagnosed and in 88 relatives (2.16%) diabetes type 2 was diagnosed. Except for one individual (patient's mother) diabetes type 2 occurred in 2nd degree relatives. Diabetes type 1 was diagnosed in 16 1st degree relatives and 15 2nd degree relatives. They were: 1st degree relatives: 9 fathers, 3 brothers, 2 mothers and 2 sisters. 2nd degree relatives: in 8 cases siblings of patient's parents, in 7 grandparents. In families of diabetic children and adolescents relatives with type 1 and type 2 diabetes were observed, with a dominance of relatives with diabetes type 2 in 2nd degree relatives.

[Analysis of changes in concentration of glycosylated hemoglobin and fructosamine during the first three weeks of treatment of children with newly diagnosed insulin-dependent diabetes]. / Analiza zmian stezenia hemoglobiny glikozylowanej i fruktozaminy w okresie trzech pierwszych tygodni leczenia dzieci ze swiezo rozpoznana cukrzyca insulinozalezna.

Blood serum fructosamine and blood glycosylated hemoglobin (HbA1) concentrations were determined in 47 children with newly diagnosed insulin-dependent diabetes during their first stay in the hospital. Three determinations at week's intervals were carried out in each patient. The mean values of concentration decrement after the first, second and third week of treatment were 0.47, 0.37 and 0.26 mmol/l, respectively, (median values 0.56, 0.33 and 0.17 mmol/l) for fructosamine concentration, and 1.8, 2.9 and 1.0% (median 1.5, 2.3 and 0.8%) for HbA1. No correlation was found between the weekly decrement values in fructosamine and HbA1 concentrations when all the values were taken into consideration, and only a slight positive correlation when the decrement after two weeks of treatment was considered (r = 0.57, p < 0.05). Very high rate of decrease in the concentration of glycosylated hemoglobin was noted in some children, reaching the values from 4 to even 8.6% per week.

[Level of glycosylated hemoglobin in children with newly diagnosed diabetes during their hospitalization]. / Stezenie hemoglobiny glukozylowanej u dzieci ze swiezo rozpoznana cukrzyca podczas ich pierwszej hospitalizacji.

Percent of HbA1 was determined in 60 children with newly diagnosed diabetes mellitus at the beginning and end of their first hospitalization. High levels of HbA1 (over 15%) were found in the majority of children at the admittance which means late diagnosis. A decrease in HbA1 by 0.5-7.5% was noted in 76% of all diabetic children treated at the hospital for 7-24 days. In 54% out of these children HbA1 levels decreased by 1.5-4.5%. Mean HbA1 value was 3.6% following a two-week hospitalization, and 3.7%--after 3 weeks. Mean decrease in HbA1 for all treated children was 0.18% per week. Positive correlation between HbA1 and its percentage and mean glycaemia at the beginning of hospitalization was seen. No correlation between HbA1 values measured at the end of hospitalization and blood glucose levels determined during the whole hospitalization period was found.

[Evaluation of the results of the treatment of children with insulin-dependent diabetes mellitus fully or partially trained in self care]. / Ocena wyników leczenia dzieci chorych na cukrzyce insulinozalezna, szkolonych w pelnym oraz niepelnym zakresie.

The study was aimed at assessing the results of diabetes mellitus therapy in 162 children, who underwent 1) the full programme of diabetes education (114 children) and 2) only the part of it (48 children). The first group was under care of Cracow Medical School Pediatrics Institute since the disease and had 2 weeks of formal diabetes mellitus teaching. The second group was treated in a less unified way in different hospitals and did not have the chance of regular teaching programme. The degree of metabolic control was assessed by series of Hb A1c determinations in whole 1987 year. The levels of Hb A1c were also compared with the children age, diabetes mellitus duration and the period elapsing from the time of last teaching. Children who were fully educated in the problems of their disease achieved significantly better results. In all group a deterioration of diabetes control during puberty was however observed. In children not fully educated such a deterioration was also noted in correlation with diabetes mellitus duration.