Ewa Wolińska-Witort

Molecular study of sex steroid receptor gene expression in human colon and in colorectal carcinomas.

Sex steroid hormones influence function of the human gastrointestinal tract. Although the specific receptor proteins have been identified in surgical specimens of both intestinal mucosa and colorectal carcinomas, it is still unknown whether they are expressed in intestinal epithelial cells.

Plasma leptin levels and free leptin index in women with Alzheimer's disease.

Alzheimers disease (AD) is a neurodegenerative disorder characterized by irreversible and progressive loss of memory and other cognitive functions. Controversies still exist on the precise mechanisms contributing to neurodegeneration. Obesity and disturbances in metabolic homeostasis are thought to be AD risk factors. Adipokine leptin has receptors in the brain, also in the regions related to AD. Leptin may protect against AD. The aim was to assess leptin and soluble leptin receptor levels in plasma as well as free leptin index (FLI) in correlation with metabolic status of women diagnosed with Alzheimers disease. Eighteen women with moderate to severe stage of AD, 40 women with AD at early stage, and 42 female controls, matched for age and body mass index, participated in the study. Leptin and soluble leptin receptor levels were measured with RIA and IRMA, respectively. Then, FLI was calculated. In addition, metabolic parameters (lipid profile, glucose and insulin concentrations, HOMA-IR) were estimated. Clinical and anthropometric data were collected. The Mini-Mental State Examination (MMSE) as a cognitive impairment measurement was performed. Correlations with both leptin and FLI, and MMSE, clinical and biochemical parameters were evaluated. Leptin levels and FLI were significantly lower and leptin receptor concentrations were higher in AD subjects when compared with the controls. In AD group leptin, soluble leptin receptor and FLI correlated with selected metabolic parameters but not with MMSE. We conclude that alterations in leptin, leptin receptor, and FLI were the most intensified in advanced AD. However, these results did not correlate with dementia stage measured with MMSE. Therefore, further intensive research is needed to explain the mechanisms involved in this phenomenon.

Effect of orexin A on the release of GnRH-stimulated gonadotrophins from cultured pituitary cells of immature and mature female rats

Orexin A (OxA), also known as hypocretin 1, is a regulatory neuropeptide involved in the control of various autonomic and neuroendocrine functions. It appears to have a significant impact on the regulation of trophic hormones secretion by influencing the hypothalamus and the pituitary. Orexin A acts through two types of receptor found in the pituitary. This suggests the possibility of direct action of OxA at the adenohypophysis level. The aim of this study was to investigate the direct effect of OxA on GnRH (gonadotrophin-releasing hormone)-stimulated LH and FSH secretion from cultured pituitary cells of sexually immature and mature female rats. Anterior pituitary cells obtained from immature and mature female rats (ovariectomized, and ovariectomized and treated with estradiol) were incubated with 10(-10)M or 10(-7)M orexin A for 1 hour and 4h and the effect on GnRH-stimulated (10(-9)M or 10(-6)M) LH and FSH release was examined. The concentrations of secreted gonadotrophins in the culture media were determined by RIA methods. Orexin A significantly inhibited GnRH-stimulated FSH release from pituitary cells isolated from immature female rats, whereas in cells of mature ovariectomized animals, the effect of OxA was dependent on the stimulatory dose of GnRH. When the cells were stimulated with a low dose of GnRH, orexin A inhibited the secretion of gonadotrophins, but when a high dose of GnRH was used, orexin A increased mainly the release of LH. In cultured pituitary cells from ovariectomized, estrogenized mature rats, orexin A inhibited the secretion of LH if the cells were stimulated with a high dose of GnRH. In conclusion, the results of this study revealed that orexin A may modify the sensitivity of gonadotrophic cells to GnRH, and its effect depends on the maturity and estrogen status of the rats from which the cells are isolated.

Intracellular mechanisms involved in copper-gonadotropin-releasing hormone (Cu-GnRH) complex-induced cAMP/PKA signaling in female rat anterior pituitary cells in vitro

The copper-gonadotropin-releasing hormone molecule (Cu-GnRH) is a GnRH analog, which preserves its amino acid sequence, but which contains a Cu(2+) ion stably bound to the nitrogen atoms including that of the imidazole ring of Histidine(2). A previous report indicated that Cu-GnRH was able to activate cAMP/PKA signaling in anterior pituitary cells in vitro, but raised the question of which intracellular mechanism(s) mediated the Cu-GnRH-induced cAMP synthesis in gonadotropes. To investigate this mechanism, in the present study, female rat anterior pituitary cells in vitro were pretreated with 0.1 μM antide, a GnRH antagonist; 0.1 μM cetrorelix, a GnRH receptor antagonist; 0.1 μM PACAP6-38, a PAC-1 receptor antagonist; 2 μM GF109203X, a protein kinase C inhibitor; 50 mM PMA, a protein kinase C activator; the protein kinase A inhibitors H89 (30 μM) and KT5720 (60 nM); factors affecting intracellular calcium activity: 2.5 mM EGTA; 2 μM thapsigargin; 5 μM A23187, a Ca(2+) ionophore; or 10 μg/ml cycloheximide, a protein synthesis inhibitor. After one of the above pretreatments, cells were incubated in the presence of 0.1 μM Cu-GnRH for 0.5, 1, and 3 h. Radioimmunoassay analysis of cAMP confirmed the functional link between Cu-GnRH stimulation and cAMP/PKA signal transduction in rat anterior pituitary cells, demonstrating increased intracellular cAMP, which was reduced in the presence of specific PKA inhibitors. The stimulatory effect of Cu-GnRH on cAMP production was partly dependent on GnRH receptor activation. In addition, an indirect and Ca(2+)-dependent mechanism might be involved in intracellular adenylate cyclase stimulation. Neither activation of protein kinase C nor new protein synthesis was involved in the Cu-GnRH-induced increase of cAMP in the rat anterior pituitary primary cultures. Presented data indicate that conformational changes of GnRH molecule resulting from cooper ion coordination affect specific pharmacological properties of Cu-GnRH molecule including specific pattern of intracellular activity induced by complex in anterior pituitary cells in vitro.

Decreased Endothelin-1 Plasma Levels in Multiple Sclerosis Patients: A Possible Factor of Vascular Dysregulation?

Background Multiple sclerosis (MS) is a demyelinating autoimmune disease of the central nervous system with possible involvement of vascular dysregulation secondary to endothelial dysfunction caused by destruction of the vessel wall. Vascular dysregulation leads to excessive vasoconstriction or insufficient vasodilatation, resulting in vasospasm mediated by endothelin-1 (ET-1), the most potent and long-lasting mediator. Vascular dysregulation can play an important role in the pathogenesis of some eye disorders and it has been hypothesized that it is a vascular risk factor for glaucomatous optic neuropathy. The aim of this study was to estimate endothelin-1 (ET-1) plasma levels in patients with MS. Material/Methods The MS group consisted of 39 patients (9 males, 30 females), mean age: 38.8±10.02 years, range: 22–62. The control group consisted of 27 healthy volunteers (3 males and 24 females), mean age: 37.4±10.88 years, range: 20–62; clinically, in a non-active stage of the disease. ET-1 plasma levels were measured using the Endothelin-1 ELISA Kit (Immuno-Biological Laboratories Co., Japan). Statistical analysis was performed with the nonparametric Mann-Whitney U test for independent groups. Results Endothelin-1 (ET-1) plasma levels were significantly lower in MS patients compared to healthy controls: mean value 0.55±0.44 pg/ml (146.05±118.27 fmol/ml) vs. 0.95±0.48 pg/ml (252.83±127.16 fmol/ml); P=0.012. Conclusions Significantly decreased ET-1 plasma levels in the MS patients could reflect the non-active disease at the time of ET-1 measurements or the effects of immunomodulatory treatment, but it cannot be excluded that decreased ET-1 plasma levels in these patients might result from vascular dysregulation.

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) in humans with multiple sclerosis.

Multiple sclerosis (MS) is a chronic neuroinflammatory disease of the central nervous system that leads to demyelination and neurodegeneration. VIP and PACAP are structurally related neuropeptides with neuroprotective and anti-inflammatory activities. To evaluate VIP and PACAP-38 in plasma and CSF in humans in correlation with IL-6, IL-10 and TNFα, we compared 20 MS individuals with 27 healthy controls. In MS, a decrease in PACAP-38 in CSF and a decrease in plasma IL-6 concentration were seen. A positive correlation between plasma VIP and plasma IL-6 was identified. We conclude that VIP and PACAP may influence the course of MS.

Vasoactive intestinal peptide modulates luteinizing hormone subunit gene expression in the anterior pituitary in female rat.

The direct monosynaptic pathway which exists between vasoactive intestinal peptide (VIP) and GnRH neurons in the hypothalamic preoptic area provides a neuroanatomical background for the modulatory effects of VIP exerted on GnRH neurons activity. Though central microinjection of VIP revealed its involvement in the modulation of LH release pattern, there is a lack of data concerning a possible VIP influence on the alpha and LHbeta subunit gene expression in the pituitary gland. Using a model based on intracerebroventricular pulsatile peptide(s) microinjections (1 pulse/h [10 microl/5 min] over 5 h) the effect of exogenous VIP (5 nM dose) microinjection on subunits mRNA content in ovariectomized/oestrogen-pretreated rats was studied. Subsequently, to obtain data concerning the involvement of GnRH and VIP receptor(s) in the regulation of alpha and LHbeta subunit mRNA expression, OVX/estrogen-primed rats received a pulsatile microinjections of 5 nM VIP with 3 nM antide (GnRH receptor antagonist) or 5 nM VIP with 15 nM VIP 6-28 (VIP receptor antagonist). In this case, substances were given separately with a 30 min lag according to which each antagonist pulse preceded a VIP pulse. Northern-blot analysis revealed that VIP microinjection resulted in a decreased alpha and LHbeta mRNA content in pituitary gland and this effect was dependent on GnRH receptor activity. Moreover, obtained results indicated that centrally administered VIP might operate through its own receptor(s) because a receptor antagonist, VIP 6-28, blocked the inhibitory effect of VIP exerted on both LH subunit mRNA content and LH release.

In vivo oestrogenic modulation of Egr1 and Pitx1 gene expression in female rat pituitary gland

EGR1 and PITX1 are transcription factors required for gonadotroph cell Lhb promoter activation. To determine changes in Egr1 and Pitx1 mRNA levels in central and peripheral pituitary stimulations, an in vivo model based on i.c.v. pulsatile (1 pulse/0.5 h over 2 h) GnRH agonist (1.5 nM buserelin) or antagonist (2 nM antide) microinjections was used. The microinjections were given to ovariectomised and 17β-oestradiol (E2) (3×20 μg), ERA (ESR1) agonist propyl pyrazole triol (PPT) (3×0.5 mg), ERB (ESR2) agonist diarylpropionitrile (DPN) (3×0.5 mg) s.c. pre-treated rats 30 min after last pulse anterior pituitaries were excised. Relative mRNA expression was determined by quantitative RT-PCR (qRT-PCR). Results revealed a gene-specific response for GnRH and/or oestrogenic stimulations in vivo. Buserelin pulses enhanced Egr1 expression by 66% in ovariectomised rats, whereas the oestradiol-supplemented+i.c.v. NaCl-microinjected group showed a 50% increase in Egr1 mRNA expression. The oestrogenic signal was transmitted via ERA (ESR1) and ERB (ESR2) activation as administration of PPT and DPN resulted in 97 and 62%, respectively, elevation in Egr1 mRNA expression. A synergistic action of GnRH agonist and 17β-oestradiol (E2) stimulation of the Egr1 gene transcription in vivo were found. GnRHR activity did not affect Pitx1 mRNA expression; regardless of NaCl, buserelin or antide i.c.v. pulses, s.c. oestrogenic supplementation (with E2, PPT or DPN) consistently decreased (by -46, -48 and -41% respectively) the Pitx1 mRNA in the anterior pituitary gland. Orchestrated Egr1 and Pitx1 activities depending on specific central and peripheral regulatory inputs could be responsible for physiologically variable Lhb gene promoter activation in vivo.

Plasma adiponectin array in women with Alzheimer’s disease

Introduction Alzheimers disease (AD) is a progressive and irreversible neurodegenerative disease. Typical features of AD include memory loss, social dysfunction and physical impairment. Although the pathological findings in the central nervous system are well established, the etiological factors are poorly known. Recent studies suggested the role of metabolic disturbances in the development of AD neurodegeneration. Adiponectin, an anti-inflammatory and metabolism regulating factor, was linked to AD. Aim The aim was to examine whether adiponectin fractions combined with insulin/insulin resistance-associated metabolic parameters correlate with AD progression. Material and methods The study comprised 98 women: 27 with moderate to severe AD, 31 with AD at early stage and 40 healthy controls, matched for age and BMI. To evaluate memory impairment, the MMSE was performed. Plasma total adiponectin and its high-, medium- and low molecular weights were measured with ELISA. Anthropometric, clinical and metabolic parameters were assessed. Correlations between adiponectin array and measured parameters were evaluated. Results In comparison to the controls, enhanced levels of total and medium molecular weight adiponectin characterized AD individuals. In AD, we found correlations between adiponectin array, and anthropometric and biochemical parameters. After adjustment to BMI, a significant increase of the total adiponectin and high- and medium molecular weight fractions was observed. A negative correlation between low molecular weight adiponectin and MMSE was found. Conclusions Our results indicate a possible link between adiponectin variations and AD. We hypothesize that changes in adiponectin profile observed in AD result from compensatory mechanism against neuropathological processes, as well as from adiponectin homeostasis impairment.

Vitamin D deficiency in obesity and its relationship to insulin resistance and plasma leptin levels – preliminary study**

Introduction. Obesity is a risk factor for vitamin D deficiency and there is some evidence that vitamin D may be involved in the pathogenesis of insulin resistance. Aim. The aim of the study was to evaluate whether vitamin D deficiency is associated with insulin resistance and leptin level in obese subjects. Material and methods. 43 obese (body mass index (BMI) ≥ 35 kg/m2) individuals before bariatric surgery and 16 healthy volunteers with normal body weight were enrolled in this study. In all subjects serum level of glucose, 25-hydroxyvitamin D (25(OH)D), calcium, insulin, leptin, leptin soluble receptor were evaluated, insulin resistance was estimated by the homeostasis model assessment (HOMA-IR), free leptin index was calculated. Results. In the obese group 90% of patients had lower than recommended level of 25(OH)D, in the control group – 75%. In all investigated groups significant negative correlations between 25(OH)D and insulin, HOMA-IR, leptin, and free leptin index were found but in obese subjects this correlations were not observed. There were significant negative correlations between serum calcium and BMI, insulin, HOMA-IR, leptin, and free leptin index and a significant positive correlation between serum calcium and leptin receptor in all investigated groups. In obese subjects there were correlations between calcium levels and soluble leptin receptor as well as free leptin index. Conclusions. Vitamin D deficiency and insufficiency are common in obese and normal weight subjects. There is a lack of correlation between 25(OH)D concentration and insulin resistance parameters and leptin in obese subjects. Vitamin D may impact leptin activity through calcium concentration.