Circulating Pro-Neurotensin in gestational diabetes mellitus.

BACKGROUND AND AIMS: Pro-Neurotensin (NT), a stable surrogate parameter of NT, has recently been introduced as a peptide predicting the development of obesity, diabetes mellitus, cardiovascular diseases, and cardiovascular mortality. However, regulation of Pro-NT in gestational diabetes mellitus (GDM) remains uninvestigated. METHODS AND RESULTS: Pro-NT was quantified in 74 women with GDM, 74 healthy, gestational age-matched, pregnant controls, as well as in a second cohort comprising of 74 healthy, non-pregnant control women, using a chemiluminometric sandwich immunoassay. Pro-NT was correlated to measures of obesity, hypertension, glucose and lipid metabolism, renal function, and inflammation. Mean ± standard deviation of circulating Pro-NT levels were not significantly different in women with GDM (100.2 ± 75.7 pmol/l) as compared to healthy, pregnant controls (103.2 ± 37.4 pmol/l) and healthy, non-pregnant female controls (105.9 ± 38.9 pmol/l) (p = 0.661). Homeostasis model assessment of insulin resistance (HOMA-IR) and creatinine positively correlated with serum Pro-NT in multivariate regression analysis. In contrast, free fatty acids (FFA) were inversely correlated with circulating Pro-NT. Results sustained adjustment for pregnancy status. CONCLUSIONS: Circulating Pro-NT is not independently associated with GDM, but is with HOMA-IR, creatinine, and FFA even after adjustment for pregnancy status.

New risk and protective factors for severe hypoglycaemia in people with type 1 diabetes.

AIMS: To evaluate risk factors for severe hypoglycaemia (SH) in patients with type 1 diabetes (T1DM). METHODS AND RESULTS: Retrospective observational and comparative study. All SH occurring between 2007 and 2014 in a German population (Lippe-Detmold) were captured. Characteristics of patients with T1DM and SH were compared with a control group being equivalent concerning age, diabetes duration, HbA1c, comorbidity, and ß-blocker treatment. SH was defined as a symptomatic event requiring treatment with intravenous glucose or glucagon administration and being confirmed by a blood glucose measurement of <2.8 mmol/l. Predictive factors for SH were analysed by a multivariable regression model. As many as 405 cases of SH in T1DM occurred in 206 subjects; 50% of episodes were related to 31 patients who experienced ≥3 SH. Need for nursing care (OR 4.88), treatment with NPH (OR 3.68), and impaired hypoglycaemia awareness (OR 2.06) were the strongest risk factors for SH (all p < 0.05, all pFDR-adjusted < 0.10; false discovery rate (FDR)). Depression (OR 0.14), treatment with CSII (OR 0.39) and short-acting insulin analogues (OR 0.31) appeared to be protective (all p < 0.10; FDR-adjusted). The probability of SH onset was significantly higher in patients who had previously experienced recurrent SH episodes. ß-Blocker treatment did not appear to be a risk factor. CONCLUSION: The complex risk for SH in people with T1DM can be reduced by treatment with CSII and short-acting analogues. Future structures of diabetes care will be challenged by the need of treating increasingly geriatric subjects with T1DM having a high risk of SH.

Association of metabolic parameters and rs726344 in FNDC5 with serum irisin concentrations.

BACKGROUND/OBJECTIVES: Irisin has been suggested as a novel myokine with beneficial effects in rodents. However, previous data in humans showed conflicting results regarding its association with metabolic phenotypes and regulation of secretion. Furthermore, although an association of rs726344 in FNDC5 (fibronectin type III domain containing 5) coding for irisin with insulin sensitivity was reported, the effects of genetic variation at this locus on irisin serum levels have not been investigated, so far. Therefore, we investigated circulating irisin and the associations with rs726344 in a cohort of >1000 subjects. SUBJECTS/METHODS: Irisin serum concentrations were measured with enzyme-linked immunosorbent assay. Associations with metabolic parameters including renal function, glucose and lipid metabolism, inflammation, as well as adipokine profiles, were assessed in regression models. Dynamic changes of serum irisin were investigated during oral glucose tolerance test (OGTT) in a subset of the cohort (n=136). rs726344 was genotyped in all subjects and analyzed for associations with serum irisin and traits of the metabolic syndrome. RESULTS: Irisin was negatively associated with fat mass, fasting glucose and dyslipidemia but not with other adipokines. Moreover, irisin decreased during an OGTT in a subcohort comprising subjects with normal glucose tolerance, impaired fasting glucose, impaired glucose tolerance and type 2 diabetes mellitus. rs726344 was not associated with serum irisin levels or with other anthropometric and biochemical parameters. CONCLUSIONS: Circulating irisin levels are associated with a beneficial metabolic profile but not with other adipokines and not with rs726344 in our cohort. Our data suggest a potential favorable role of irisin in the regulation of metabolism.

Novel genes on rat chromosome 10 are linked to body fat mass, preadipocyte number and adipocyte size.

BACKGROUND: The genetic architecture of obesity is multifactorial. We have previously identified a quantitative trait locus (QTL) on rat chromosome 10 in a F2 cross of Wistar Ottawa Karlsburg (WOKW) and Dark Agouti (DA) rats responsible for obesity-related traits. The QTL was confirmed in congenic DA.WOKW10 rats. To pinpoint the region carrying causal genes, we established two new subcongenic lines, L1 and L2, with smaller refined segments of chromosome 10 to identify novel candidate genes. METHODS: All lines were extensively characterized under different diet conditions. We employed transcriptome analysis in visceral adipose tissue (VAT) by RNA-Seq technology to identify potential underlying genes in the segregating regions. Three candidate genes were measured in human paired samples of VAT and subcutaneous (SC) AT (SAT) (N=304) individuals with a wide range of body weight and glucose homeostasis parameters. RESULTS: DA.WOKW and L1 subcongenic lines were protected against body fat gain under high-fat diet (HFD), whereas L2 and DA had significantly more body fat after high-fat feeding. Interestingly, adipocyte size distribution in SAT and epigonadal AT of L1 subcongenic rats did not undergo typical ballooning under HFD and the number of preadipocytes in AT was significantly elevated in L2 compared with L1 and parental rats. Transcriptome analysis identified three candidate genes in VAT on rat chromosome 10. In humans, these candidate genes were differentially expressed between SAT and VAT. Moreover, HID1 mRNA significantly correlates with parameters of obesity and glucose metabolism. CONCLUSIONS: Our data suggest novel candidate genes for obesity that map on rat chromosome 10 in an interval 102.2-104.7 Mb and are strongly associated with body fat mass regulation, preadipocyte number and adipocyte size in rats. Among those genes, AT head involution defective (HID1) mRNA expression may be relevant for human fat distribution and glucose homeostasis.

Fat depot-specific mRNA expression of novel loci associated with waist-hip ratio.

OBJECTIVE: We hypothesized that genes within recently identified loci associated with waist-hip ratio (WHR) exhibit fat depot-specific mRNA expression, which correlates with obesity-related traits. METHODS: Adipose tissue (AT) mRNA expression of 6 genes (TBX15/WARS2, STAB1, PIGC, ZNRF3 and GRB14) within these loci showing coincident cis-expression quantitative trait loci was measured in 222 paired samples of human visceral (vis) and subcutaneous (sc) AT. The relationship of mRNA expression levels with obesity-related quantitative traits was assessed by Pearson's correlation analyses. Multivariate linear relationships were assessed by generalized linear regression models. RESULTS: Whereas only PIGC, ZNFR3 and STAB1 mRNA expression in sc AT correlated nominally with WHR (P<0.05, adjusted for age and sex), mRNA expression of all studied genes in at least one of the fat depots correlated significantly with vis and/or sc fat area (P ranging from 0.05 to 4.0 × 10(6), adjusted for age and sex). Consistently, the transcript levels of WARS, PIGC and GRB14 were nominally associated with body mass index (BMI) (P ranging from 0.02 to 9.2 × 10(5), adjusted for age and sex). Moreover, independent of sex, obesity and diabetes status, differential expression between vis and sc AT was observed for all tested genes (P<0.01). Finally, the rs10195252 T-allele was nominally associated with increased GRB14 sc mRNA expression (P=0.025 after adjusting for age, sex and BMI). CONCLUSIONS: Our data including the inter-depot variability of mRNA expression suggests that genes within the WHR-associated loci might be involved in the regulation of fat distribution.

Functional relevance of genes implicated by obesity genome-wide association study signals for human adipocyte biology.

AIMS/HYPOTHESIS: Genome-wide association studies (GWAS) have identified numerous single-nucleotide polymorphisms associated with obesity, consequently implying a role in adipocyte biology for many closely residing genes. We investigated the functional relevance of such genes in human adipocytes. METHODS: We selected eight genes (BDNF, MAF, MTCH2, NEGR1, NPC1, PTER, SH2B1 and TMEM18) from obesity GWAS and analysed their effect in human adipogenesis using small interfering (si)RNA-mediated knockdown, their regulation by metabolic agents in adipocytes and pre-adipocytes, and gene expression in paired samples of human fat biopsies (68 non-obese, 165 obese) by quantitative real-time PCR. RESULTS: We show a two- to threefold upregulation of MAF, MTCH2 and NEGR1 and a two- to fourfold downregulation of BDNF and PTER during adipogenesis. Knockdown of BDNF (mean ± SEM; 83.8 ± 4.7% of control; p = 0.0002), MTCH2 (72.7 ± 9.5%; p = 0.0006), NEGR1 (70.2 ± 5.7%; p < 0.0001) and TMEM18 (70.8 ± 6.1%; p < 0.0001) significantly inhibited adipocyte maturation, while knockdown of the other proteins had no effect. Insulin slightly induced MAF (1.65-fold; p = 0.0009) and MTCH2 (1.72-fold; p < 0.0001), while it suppressed BDNF (59.6%; p = 0.0009), NEGR1 (58.0%; p = 0.0085) and TMEM18 (69.3%; p = 0.0377) in adipocytes. The synthetic glucocorticoid dexamethasone suppressed MAF (45.7%; p = 0.0022), BDNF (66.6%; p = 0.0012) and TMEM18 (63.5%; p = 0.0181), but induced NEGR1 (3.2-fold; p = 0.0117) expression. Furthermore, MTCH2, NEGR1 and TMEM18 were differentially expressed in subcutaneous and visceral adipose tissue. TMEM18 expression was decreased in the adipose tissue of obese patients, and negatively correlated with anthropometric variables and adipocyte size. CONCLUSIONS/INTERPRETATION: Our results imply a regulatory role for TMEM18, BDNF, MTCH2 and NEGR1 in adipocyte differentiation and biology. In addition, we show a variation of MAF expression during adipogenesis, while NPC1, PTER and SH2B1 were not regulated.

Genetic variation in the vaspin gene affects circulating serum vaspin concentrations.

OBJECTIVE: Visceral adipose tissue-derived serine protease inhibitor (vaspin) is an adipokine potentially linking obesity, insulin resistance and type 2 diabetes. Here, we searched for genetic determinants that could explain the variability in serum vaspin concentrations. RESEARCH DESIGN AND METHODS: First, we conducted a genome-wide association study (GWAS) for serum vaspin in the Sorbs cohort (N=826). Subsequently, 26 single-nucleotide polymorphisms (SNPs) covering genetic variation in the vaspin locus were genotyped in the Sorbs. In addition, we measured serum vaspin concentrations in 1806 samples from Augsburg/the Cooperative Health Research in the Region of Augsburg (KORA) for replication of the association signals. Finally, we conducted association analyses of vaspin SNPs with metabolic traits in the Sorbs (N=1013), KORA (N=1813) and a further cohort from Germany (Leipzig: N=1857). RESULTS: Six SNPs mapping between serpinA1 and serpinA4, including the vaspin locus, on chromosome 14 reached P-values < or = 10(-8) in the GWAS in the Sorbs. The fine mapping of variants within the vaspin locus in the Sorbs and subsequent replication in the KORA sample revealed several SNPs significantly associated with serum vaspin concentrations reaching P-values of up to 10(-35). However, no significant association with type 2 diabetes or related traits was found in either cohort after the Bonferroni correction for multiple comparisons. CONCLUSION: Our data show that the variability in serum vaspin concentrations might be explained by its genetic variants.

Metabolic and genetic predictors of circulating adipocyte fatty acid-binding protein.

BACKGROUND: Adipocyte fatty acid-binding protein (AFABP) was recently introduced as a novel adipokine playing an important role in glucose homeostasis. In this study, we investigated the relationship between serum AFABP levels and metabolic, as well as cardiovascular parameters, in the self-contained population of Sorbs. Furthermore, we conducted a genome-wide association study on serum AFABP concentrations in the Sorbs and we separately analyzed the effects of two common variants in the FABP4 gene on AFABP serum concentration. METHODS: Serum AFABP concentrations were quantified by enzyme-linked immunosorbent assay and correlated with metabolic and cardiovascular parameters, as well as inflammatory markers and renal function, in 868 well-characterized non-diabetic Sorbs from Germany. RESULTS: Median AFABP serum concentrations were 1.5-fold higher in female subjects (23.03 µg l(-1)) as compared to male subjects (15.86 µg l(-1)). Waist-to-height ratio and glomerular filtration rate were independently associated with AFABP concentrations in multiple regression analysis in both female and male subjects. The genome-wide scan for association of single-nucleotide polymorphisms with serum AFABP levels in the Sorbs revealed 39 loci reaching P-values <10(-4). Two single-nucleotide polymorphisms, rs16909187 and rs10808846, representing common genetic variation in FABP4 did not show any effect on serum AFABP concentrations in our study cohort. CONCLUSION: AFABP serum concentrations are determined by parameters of fat distribution, renal function and gender.

Number and sex ratio of children and impact of parental diabetes in individuals with Type 1 diabetes.

OBJECTIVE: To assess the number and sex ratio of children in individuals with Type 1 diabetes mellitus and the influence of parental diabetes on age at onset of Type 1 diabetes in our cohort. METHODS: In a cross-sectional study in a German region comprising 350,000 inhabitants, 697 subjects with Type 1 diabetes (364 women, 333 men) underwent a standardized assessment regarding the number and sex of their children and the family history of diabetes. RESULTS: Compared with 1.36 children per woman in the German background population, the total fertility rate in the calendar year of 2010 in our female cohort with Type 1 diabetes (age 18-49 years) was 0.88. Men with Type 1 diabetes had a fertility rate of 0.65. More men (51.1%) than women (35.7%; P < 0.0001) were childless. Twenty per cent of all women aged 41-45 years in the background population were childless compared with 36.2% of all women and 52% of all men in this specific age group from our cohort. The sex ratio of female vs. male offspring of individuals with Type 1 diabetes did not differ significantly from the expected 1:1 ratio. Maternal Type 1 or Type 2 diabetes increased the age at onset of Type 1 diabetes from 22.9 ± 13.7 (no maternal diabetes) to 28.6 ± 16.8 and 30.1 ± 15.1 years (p < 0.0001), respectively. CONCLUSIONS: Compared with the German reference population, individuals with Type 1 diabetes had significantly fewer children and were more often childless. The sex ratio female vs. male offspring of women and men with Type 1 diabetes was unaffected. Maternal history of Type 1 and Type 2 diabetes was associated with a significant later onset of Type 1 diabetes.

Central vaspin administration acutely reduces food intake and has sustained blood glucose-lowering effects.

AIMS/HYPOTHESIS: Vaspin (visceral adipose tissue-derived serpin) was first identified as an adipokine in a rat model of type 2 diabetes, in which it is predominantly secreted from visceral adipose tissue. Serum concentrations of vaspin show a food intake-related diurnal variation. We therefore tested the hypothesis that vaspin plays a role in the regulation of food intake. METHODS: Vaspin levels in the hypothalamus and human stomach were determined by western blotting. The cerebrospinal fluid concentration of vaspin was measured in five healthy volunteers using an ELISA. Fed 11-week-old female db/db mice were given intraperitoneal injections of 1 mg/kg body weight of vaspin (n = 6) or saline (n = 6) on experimental days 1, 3 and 4. Changes in food intake and fed plasma glucose concentrations were determined after one intracerebroventricular administration of either 1 µg vaspin or artificial cerebrospinal fluid into 11-week-old female db/db (n = 8) and C57BL/6 mice (n = 8) up to 6 days after injection. RESULTS: We detected vaspin in the hypothalamus of db/db and C57BL/6 mice and in the cerebrospinal fluid of healthy individuals. Both peripheral and central vaspin administration decrease food intake in obese db/db and lean C57BL/6 mice. In db/db mice, vaspin treatment is associated with sustained glucose-lowering effects for at least 6 days after injection. In addition, we demonstrated expression of the gene encoding vaspin in the gastric mucosa in humans, and found that this was subject to regional variations. CONCLUSIONS/INTERPRETATION: Our data suggest a previously unrecognised role of vaspin in the regulation of food intake. We postulate that vaspin inhibits a protease that degrades an anti-orexigenic factor.

Vaspin is related to gender, puberty and deteriorating insulin sensitivity in children.

BACKGROUND: Visceral adipose tissue-derived serine protease inhibitor (vaspin) has been suggested as a novel adipocytokine related to obesity and insulin sensitivity in adults. DESIGN: We quantified vaspin serum concentrations in 65 lean and 67 obese children and aimed to evaluate the relationship of vaspin with physical development, obesity, and metabolic and cardiovascular phenotypes in children. We further assessed the acute vaspin response to glucose provocation in 20 obese adolescents and evaluated tissue expression patterns of vaspin in humans. RESULTS: Vaspin levels were significantly higher in girls than in boys. In girls, vaspin increased with age and pubertal stage, whereas there was no change with development in boys. Obese girls had lower vaspin serum levels than those of lean controls, but there was no significant correlation with body mass index (BMI). Independent of sex, age and BMI, lower vaspin was associated with better insulin sensitivity, with higher systolic blood pressure and impaired endothelial function. In response to glucose provocation during an oral glucose tolerance test, vaspin serum levels declined by approximately 25% in adolescents with hyperinsulinemia, whereas there was no significant decline in normoinsulinemic patients. In support of our clinical data, we not only confirmed vaspin mRNA expression in adipose tissue but also found consistent expression of vaspin in the liver and indications for expression in the pancreas and the skin. CONCLUSION: We showed that gender differences in circulating vaspin levels develop during pubertal progression in girls. Although vaspin's association with obesity remains controversial, vaspin was increased with worsening insulin resistance already in children and was acutely down-regulated following glucose provocation in insulin-resistant adolescents independent of obesity. Besides adipose tissue, vaspin expression in the liver and the pancreas may potentially contribute to circulating vaspin levels and their regulation.

Association and evolutionary studies of the melatonin receptor 1B gene (MTNR1B) in the self-contained population of Sorbs from Germany.

AIMS: Several polymorphisms of the melatonin receptor 1B gene (MTNR1B) have been shown to be associated with elevated fasting plasma glucose and impaired early insulin release. The aim of this study was to assess the effects of MTNR1B variants on traits related to the metabolic syndrome in the self-contained population of Sorbs from Germany. As comprehensive studies concerning the conservation of MTNR1B are lacking, we also evaluated natural selection in vertebrates and human populations at this locus. METHODS: Five single nucleotide polymorphisms representing all blocks of linkage disequilibrium within and surrounding the MTNR1B locus were genotyped in 937 Sorbs for association analyses on metabolic traits related to Type 2 diabetes. The associations were assessed by regression analyses, the conservation between species was investigated with phylogenetic analysis by maximum likelihood (PAML). In addition, various tests of population genetic measures (e.g. fixation index, Tajima's D) were performed. RESULTS: Previously reported association between MTNR1B variants (rs10830963, rs4753426) and oral glucose tolerance test-derived indices of ß-cell function (homeostasis model assessment-B, P = 3.7 × 10⁻6 and P = 0.004, respectively), as well as insulin (fasting insulin: P=2×10⁻³ and P=0.02; 30-min insulin: P = 2.1 × 10⁻4 and P=0.03, respectively) and fasting glucose (rs10830963, P=1.2×10⁻6) parameters could be replicated in the present study. Phylogenetic analysis by maximum likelihood analyses showed that the gene was strongly conserved between species (ω=0.2583). Structures important for the receptor function are also conserved. On the lineage leading to human adaptive selection was present (ω=1.1030). Population genetic measures further indicated natural selection. CONCLUSIONS: Our data support the physiologic importance of MTNR1B in the context of glucose homeostasis and suggest evidence of selection at this locus.

Influence of food on the pharmacokinetics of oral alitretinoin (9-cis retinoic acid).

BACKGROUND: Previous studies have shown that concomitant administration of food may enhance the bioavailability of oral retinoids. AIM: To assess the influence of food on the pharmacokinetics (PK) of alitretinoin after a single oral dose. METHODS: This was a single-dose, open-label, randomized, crossover study, which enrolled 30 healthy men, aged 18-44 years. Subjects received sequential doses of alitretinoin 40 mg either after fasting (treatment A) or 5 min after completion of a standard breakfast (treatment B), with the dosing sequence randomized (A/B or B/A). The washout period between the two doses was 1 week. Plasma concentrations over time were plotted and standard PK variables [area under the curve (AUC) of plasma concentration vs. time, maximum plasma concentration (C(max)), time to maximum plasma concentration (t(max)) and elimination half-life (t(1/2)] were determined. RESULTS: Drug exposure was markedly increased when alitretinoin was taken with food compared with fasting, and there were significant increases in mean C(max) (82.8 vs.25.4 ng/mL, respectively) and AUC (220.2 vs. 55.7 ng · h/mL). The delaying effect of food on t(max) was less marked (median of 3.0 vs. 2.0 h). Administration with food also increased exposure to drug metabolites. Variability in exposure was markedly reduced if alitretinoin was taken with vs. without food (percentage coefficient of variation 40% vs. 74% for AUC; 49% vs. 85% for C(max)). Alitretinoin was generally well tolerated, with typical retinoid adverse reactions, mostly comprising headache. CONCLUSIONS: Intake of alitretinoin with food substantially increased the bioavailability of alitretinoin, but variability in exposure was reduced. Consequently, oral alitretinoin should be taken with food as outlined in the manufacturer's summary of product characteristics.

Lack of significant effects of the type 2 diabetes susceptibility loci JAZF1, CDC123/CAMK1D, NOTCH2, ADAMTS9, THADA, and TSPAN8/LGR5 on diabetes and quantitative metabolic traits.

Recently, several novel loci reaching genome-wide significance levels for type 2 diabetes (T2D) were identified through a meta-analysis of three genome-wide scans and large-scale follow-up. The aim of our study was to investigate the association of these loci with T2D and related subphenotypes in two cohorts from Germany. We performed an association study of 9 SNPs in or around JAZF1, CDC123/ CAMK1D, NOTCH2, BCL11A, ADAMTS9, VEGFA, DCD, THADA, and TSPAN8/ LGR5 with T2D and related quantitative traits (fasting insulin and glucose, indices derived from OGTT) in the isolated population of Sorbs (205 cases and 695 controls) and in a mixed German population (Leipzig) (938 subjects with and 918 without T2D). None of the variants was associated with T2D, but the meta-analysis of both cohorts revealed a modest trend of association of rs7578597 in THADA with T2D (p=0.055). Furthermore, Sorbian subjects homozygous for the rs7578597 T-allele had lower mean 30-minute plasma insulin when compared with carriers of the C-allele (p<0.05). The T-allele was also nominally associated with higher fasting plasma glucose in the Leipzig cohort (p<0.05). Although several other SNPs showed some evidence for association with T2D-related traits the effects were not replicated within our study. Associations of the T2D-risk alleles with T2D or related subphenotypes were overall very weak in the approximately 2 700 subjects studied. This is compatible with the modest effect size of these "second sweep" variants, which will require large-scale association studies on quantitative traits to clarify their role in the pathophysiology of T2D.

The role of apoptosis in the complex pathogenesis of the most common obstetrics and gynaecology diseases.

PURPOSE: We aimed to assess the etiological role of apoptotic genes Bcl-2 and Bax in the background of major obstetric and gynaecological diseases. METHODS: Placental tissue samples were collected from 101 pregnancies with intrauterine growth restriction and 104 pregnancies with premature birth with 140 controll samples from term, eutrophic newborns. In addition, gene expression assessment of the genes Bax and Bcl-2 was performed in 101 uterine leiomyoma tissue samples at our disposal with 110 control cases. Gene expression levels were assessed by PCR method. RESULTS: The expression of the Bcl-2 gene was decreased in placental samples with intrauterine growth restriction. Significant overexpression of the proapoptotic Bax gene was detected in samples from premature infants. Antiapoptotic Bcl-2 gene expression was found to be significantly increased in fibroid tissues. CONCLUSION: Apoptosis plays a crucial role in the development of the most common OB/GYN conditions. Decrease in the placental expression of the antiapoptotic gene Bcl-2 may upset the balance of programmed cell death.

Hormones in the nucleus of mast cells: confocal microscopic immunocytochemical observations.

In earlier experiments, the presence of histamine and serotonin in the nucleus of mast cells was demonstrated. At present we studied the presence or absence of four hormones: adrenocorticotropine (ACTH), growth hormone (GH), triiodothyronine (T(3)), and progesterone. Cells of the (adult female) rat peritoneal fluid were fixed by EDAC and studied by laser-scanning confocal microscope, after treatment with primary antibodies and FITC-labeled secondary antibody. Strong ACTH and growth hormone fluorescence can be seen in the nucleus. ACTH fluorescence is present also in the granules. The cytoplasm of lymphocytes contains both ACTH and GH. T(3) shows very pale fluorescence, and progesterone is negative. The conclusion is that the nucleus of mast cells contains two polypeptide hormones studied. This was demonstrated by using EDAC fixation as this is a cross-linking agent and does not allow the escape of the minute amounts of hormones from the nucleus. Earlier observations on peptide hormones' nuclear presence and their importance are discussed.

The E23K variant of KCNJ11 and the risk for severe sulfonylurea-induced hypoglycemia in patients with type 2 diabetes.

Severe sulfonylurea-induced hypoglycemia (SH) is a life-threatening and frequently misdiagnosed condition leading to a mortality of up to 10%. Pharmacogenetic factors could be of critical importance for the risk of SH. We investigated the effects of the E23K variant of KCNJ11 (potassium inwardly-rectifying channel, subfamily J, member 11) on risk for SH in patients with type 2 diabetes (T2D). In a case-control study, the frequency of the E23K KCNJ11 polymorphism of 43 diabetic patients with SH admitted to the emergency department was compared with a matched control group of 54 patients with T2D, but without a history of SH. All patients have been treated with the sulfonylureas glimepiride or glibenclamide. SH was defined as a symptomatic event requiring treatment with intravenous glucose and was confirmed by a blood glucose measurement of <50 mg/dl. The K variant was significantly more frequent in the control group (46%) than in cases with SH (31%) (p=0.04). However, in multivariate logistic regression analyses, age, HbA(1c) and sulfonylurea dose appeared to be the strongest predictors of SH. Nevertheless, in generalized linear model analyses, the E23K variant was significantly associated with increased HbA(1c) levels (adjusted p=0.04) independent of age, sex, body mass index, diabetes duration and sulfonylurea dose. Our data suggest that patients with T2D carrying the K variant of the E23K polymorphism in KCNJ11 have reduced response to sulfonylurea therapy, which results in increased HbA(1c) and consequently in lower risk for SH.

T lymphocytes are targets for platelet- and trophoblast-derived microvesicles during pregnancy.

Microvesicles (MVs) can derive from several cell types and their membranes contain cell surface elements. Their role is increasingly recognized in cell-to-cell communication, as they act as both paracrine and remote messengers, occurring in circulating form as well as in plasma. Successful pregnancy requires a series of interactions between the maternal immune system and the implanted fetus, such that the semi-allograft will not be rejected. These interactions occur at the materno-placental interface and/or at a systemic level. In the present study we identified for the first time the in vivo plasma pattern of the MVs of third-trimester, healthy pregnant women, their cellular origin, and their target cells using flow cytometry and confocal laser microscopy. We searched for the cellular target molecules of thrombocyte-derived MVs with the help of neutralizing antibodies. We examined the in vitro effects of MVs on STAT3 phosphorylation of primary lymphocytes and Jurkat cells. We found that both placental trophoblast-derived and maternal thrombocyte-derived MVs bind to circulating peripheral T lymphocytes, but not to B lymphocytes or NK cells. We were able to show that the P-selectin (CD62P)-PSGL-1 (CD162) interaction is one mechanism binding platelet-derived MVs to T cells. We were also able to demonstrate that MV-lymphocyte interactions induce STAT3 phosphorylation in T cells. Our findings indicate that both thrombocyte- and trophoblast-derived MVs may play an important role in the immunomodulation of pregnancy. We suggest that the transfer of different signals via MVs represents a novel form of communication between the placenta and the maternal immune system, and that MVs contribute to the establishment of stable immune tolerance to the semi-allograft fetus.

Effect of femtomolar concentrations of hormones on insulin binding by Tetrahymena, as a function of time.

The unicellular ciliate Tetrahymena, contains and binds hormones, characteristic of vertebrates. Earlier experiments demonstrated the effect of extremely low concentrations of hormones. In the present experiments, the effect of various hormones (endorphin, serotonin, histamine, insulin and epidermal growth factor [EGF]) in 10(-15) M, or oxytocin, gonadotropin at 0.001 IU concentrations) on the binding of FITC-insulin was studied by using flow cytometry and confocal microscopy, after 1, 5, 15, 30 and 60 min. Six of the seven hormones promptly decreased the cells' hormone binding capacity, the exception being EGF, and in four cases (endorphin, serotonin, insulin and oxytocin) the reduction was enormous. The decreased binding was durable. However, in the case of endorphin and oxytocin after 30 min, and in the case of serotonin after 60 min the binding returned to the control level. In the case of oxytocin after 60 min, binding significantly surpassed the control level. Histamine returned to the control level after 15 min, but after that the binding became even lower. EGF provoked special behaviour: it increased hormone binding after 30 and 60 min. The results call attention to the extreme sensitivity of Tetrahymena receptors to hormonal inductions and to its quick response ability.