Feeding and GLP-1 receptor activation stabilize ß-catenin in specific hypothalamic nuclei in male rats.

ß-catenin is a multifunctional protein that can act in the canonical Wnt/ß-catenin pathway to regulate gene expression but can also bind to cadherin proteins in adherens junctions where it plays a key role in regulating cytoskeleton linked with these junctions. Recently, evidence has been presented indicating an essential role for ß-catenin in regulating trafficking of insulin vesicles in ß-cells and showing that changes in nutrient levels rapidly alter levels of ß-catenin in these cells. Given the importance of neuroendocrine hormone secretion in the regulation of whole body glucose homeostasis, the objective of this study was to investigate whether ß-catenin signalling is regulated in the hypothalamus during the normal physiological response to food intake. Rats were subjected to a fasting/re-feeding paradigm, and then samples collected at specific timepoints for analysis of ß-catenin expression by immunohistochemistry and Western blotting. Changes in gene expression were assessed by RT-qPCR. Using immunohistochemistry, feeding acutely increased detectable cytoplasmic levels of ß-catenin ('stabilized ß-catenin') in neurons in specific regions of the hypothalamus involved in metabolic regulation, including the arcuate, dorsomedial and paraventricular nuclei of the hypothalamus. Feeding-induced elevations in ß-catenin in these nuclei were associated with increased transcription of several genes that are known to be responsive to Wnt/ß-catenin signalling. The effect of feeding was mimicked by administration of the GLP-1 agonist exendin-4, and was characterized by cAMP-dependent phosphorylation of ß-catenin at serine residues 552 and 675. The data suggest that ß-catenin/TCF signalling is involved in metabolic sensing in the hypothalamus. This article is protected by copyright. All rights reserved.

ß-catenin is important for the development of an insulin responsive pool of GLUT4 glucose transporters in 3T3-L1 adipocytes.

GLUT4 is unique among specialized glucose transporters in being exclusively expressed in muscle and adipocytes. In the absence of insulin the distribution of GLUT4 is preferentially intracellular and insulin stimulation results in the movement of GLUT4 containing vesicles to the plasma membrane. This process is responsible for the insulin stimulation of glucose uptake in muscle and fat. While signalling pathways triggering the translocation of GLUT4 are well understood, the mechanisms regulating the intracellular retention of GLUT4 are less well understood. Here we report a role for ß-catenin in this process. In 3T3-L1 adipocytes in which ß-catenin is depleted, the levels of GLUT4 at and near the plasma membrane rise in unstimulated cells while the subsequent increase in GLUT4 at the plasma membrane upon insulin stimulation is reduced. Small molecule approaches to acutely activate or inhibit ß-catenin give results that support the results obtained with siRNA and these changes are accompanied by matching changes in glucose transport into these cells. Together these results indicate that ß-catenin is a previously unrecognized regulator of the mechanisms that control the insulin sensitive pool of GLUT4 transporters inside these adipocyte cells.

Th2 responses are primed by skin dendritic cells with distinct transcriptional profiles.

The dendritic cell signals required for the in vivo priming of IL-4-producing T cells are unknown. We used RNA sequencing to characterize DCs from skin LN of mice exposed to two different Th2 stimuli: the helminth parasite Nippostrongylus brasiliensis (Nb) and the contact sensitizer dibutyl phthalate (DBP)-FITC. Both Nb and DBP-FITC induced extensive transcriptional changes that involved multiple DC subsets. Surprisingly, these transcriptional changes were highly distinct in the two models, with only a small number of genes being similarly regulated in both conditions. Pathway analysis of expressed genes identified no shared pathways between Nb and DBP-FITC, but revealed a type-I IFN (IFN-I) signature unique to DCs from Nb-primed mice. Blocking the IFN-I receptor at the time of Nb treatment had little effect on DC migration and antigen transport to the LN, but inhibited the up-regulation of IFN-I-induced markers on DCs and effectively blunted Th2 development. In contrast, the response to DBP-FITC was not affected by IFN-I receptor blockade, a finding consistent with the known dependence of this response on the innate cytokine TSLP. Thus, the priming of Th2 responses is associated with distinct transcriptional signatures in DCs in vivo, reflecting the diverse environments in which Th2 immune responses are initiated.

A Critical Role for ß-Catenin in Modulating Levels of Insulin Secretion from ß-Cells by Regulating Actin Cytoskeleton and Insulin Vesicle Localization.

The processes regulating glucose-stimulated insulin secretion (GSIS) and its modulation by incretins in pancreatic ß-cells are only partly understood. Here we investigate the involvement of ß-catenin in these processes. Reducing ß-catenin levels using siRNA knockdown attenuated GSIS in a range of ß-cell models and blocked the ability of GLP-1 agonists and the depolarizing agent KCl to potentiate this. This could be mimicked in both ß-cell models and isolated islets by short-term exposure to the ß-catenin inhibitory drug pyrvinium. In addition, short-term treatment with a drug that increases ß-catenin levels results in an increase in insulin secretion. The timing of these effects suggests that ß-catenin is required for the processes regulating trafficking and/or release of pre-existing insulin granules rather than for those regulated by gene expression. This was supported by the finding that the overexpression of the transcriptional co-activator of ß-catenin, transcription factor 7-like 2 (TCF7L2), attenuated insulin secretion, consistent with the extra TCF7L2 translocating ß-catenin from the plasma membrane pool to the nucleus. We show that ß-catenin depletion disrupts the intracellular actin cytoskeleton, and by using total internal reflectance fluorescence (TIRF) microscopy, we found that ß-catenin is required for the glucose- and incretin-induced depletion of insulin vesicles from near the plasma membrane. In conclusion, we find that ß-catenin levels modulate Ca2+-dependent insulin exocytosis under conditions of glucose, GLP-1, or KCl stimulation through a role in modulating insulin secretory vesicle localization and/or fusion via actin remodeling. These findings also provide insights as to how the overexpression of TCF7L2 may attenuate insulin secretion.

Glucagon phosphorylates serine 552 of ß-catenin leading to increased expression of cyclin D1 and c-Myc in the isolated rat liver.

In the last 20 years the prevalence of metabolic disorders, in particular type 2 diabetes (T2D), has more than doubled. Recently, a strong link between T2D and cancer, in particularly liver cancer has been reported. However, the mechanism connecting the development of type 2 diabetes and cancer remains unknown. One of the biggest drivers of liver cancer is alterations in the Wnt/ß-catenin pathway. In this study, we aimed to identify the effect of glucagon on ß-catenin in the isolated rat liver. We found glucagon, which is substantially raised in patients with T2D, rapidly phosphorylates ß-catenin on serine 552 that is associated with increased expression of genes cyclin D1 (CCND1) and c-Myc (MYC), which are known to be involved in liver cancer. This finding may explain the increased risk of liver cancer in people with T2D.

Aminoglycoside analysis in food of animal origin with a zwitterionic stationary phase and liquid chromatography-tandem mass spectrometry.

In this study, fourteen highly polar aminoglycoside (AGs) antibiotics were selected. Various stationary phases were tested, including Obelisc R, ZIC-HILIC, BEH amide and aminopropyl. The nature of the stationary phase, mobile phase (water or buffer solutions and acetonitrile), pH (percentage of formic acid), gradient conditions and injection solvents were systematically studied as relevant parameters for tuning retention selectivity and detectability of AGs in liquid chromatography electrospray tandem mass spectrometry (LC-(ESI)-MS/MS). Only the two zwitterionic phases (Obelisc R and ZIC-HILIC) achieved a proper chromatographic separation considering interferences due to the crosstalk effect in low resolution mass spectrometers. The water/acetonitrile mobile phase containing 1% formic acid used with Obelisc R provided more sensitivity than the highly concentrated buffered mobile phases required for ZIC-HILIC. A solid phase extraction (SPE) clean-up procedure with polymeric weak cation exchange (WCX) cartridges was optimized for honey, milk and liver samples. Different brands of cartridges and elution solvents were tested, and the Taurus WCX offered the best recovery rate with a buffer elution at pH 3. The final optimized method was validated in these matrices according to Decision 2002/657/EC. A monitoring campaign for sixty honey, milk and liver samples was carried out at the Food Authority Control in Geneva. The concentration of dihydrostreptomycin (DSTP) found in one ovine liver exceeded the established maximum residue levels (MRLs) within the European and Swiss legislations but it was compliant taking into account the validation data.

Comprehensive screening of veterinary drugs in honey by ultra-high-performance liquid chromatography coupled to mass spectrometry.

In the context of multi-residue screening in honey, a complete methodology was developed for 200 veterinary drugs comprising a sample preparation step and an ultra-high-performance liquid chromatography (UHPLC) coupled to time-of-flight (TOF) mass spectrometry analysis. In addition, specific analytical strategies were developed for two compounds, streptomycin and chloramphenicol, using UHPLC and tandem mass spectrometry (MS/MS). Methodologies were then applied to real honey samples obtained from the Swiss market.

Identification of a pathway by which glucose regulates ß-catenin signalling via the cAMP/protein kinase A pathway in ß-cell models.

Pancreatic ß-cells are highly responsive to changes in glucose, but the mechanisms involved are only partially understood. There is increasing evidence that the ß-catenin signalling pathway plays an important role in regulating ß-cell function, but the mechanisms regulating ß-catenin signalling in these cells is not well understood. In the present study we show that ß-catenin levels and downstream signalling are regulated by changes in glucose levels in INS-1E and ß-TC6-F7 ß-cell models. We found a glucose-dependent increase in levels of ß-catenin in the cytoplasm and nucleus of INS-1E cells. Expression of cyclin D1 also increased with glucose and required the presence of ß-catenin. This was associated with an increase in phosphorylation of ß-catenin on Ser552, which is known to stabilize the molecule and increase its transcriptional activity. In a search for possible signalling intermediates we found forskolin and cell-permeable cAMP analogues recapitulated the glucose effects, suggesting a role for cAMP and PKA (cAMP-dependent protein kinase/protein kinase A) downstream of glucose. Furthermore, glucose caused sustained increases in cAMP. Two different inhibitors of adenylate cyclase and PKA signalling blocked the effects of glucose, whereas siRNA (small interfering RNA) knockdown of PKA blocked the effects of glucose on ß-catenin signalling. Finally, reducing ß-catenin levels with either siRNA or pyrvinium impaired glucose- and KCl-stimulated insulin secretion. Taken together the results of the present study define a pathway by which changes in glucose levels can regulate ß-catenin using a mechanism which involves cAMP production and the activation of PKA. This identifies a pathway that may be important in glucose-dependent regulation of gene expression and insulin secretion in ß-cells.

Single-run separation of closely related cationic and anionic compounds by CE-ESI-MS: application to the simultaneous analysis of melamine and its analogs in milk.

In recent years, two adulteration incidents concerning the addition of melamine, a nitrogen-rich industrial small polar compound, to pet food and infant formula products have occurred in China. These issues prompted laboratories to develop methods for the analysis of melamine and related compounds in a wide variety of food products and ingredients. In this context, a CE-ESI-MS method was developed to simultaneously analyze melamine and its related products (ammeline, ammelide and cyanuric acid) that possess close physico-chemical properties. This method allows the simultaneous analysis of both cations and anions in a single run, using CE to divide the run into two time segments in normal polarity mode. For this purpose, ESI polarity was switched once during the run, increasing sensitivity and data quality. The method was applied to spiked powdered milk and melamine-contaminated powdered milk, with two sample preparation procedures.

Control strategies against grey mould (Botrytis cinerea Pers.: Fr) and corresponding fungicide residues in grapes and wines.

This study examines the most effective anti-Botrytis strategies leading to possible lower pesticides residues in wine. To provide wine growers with a number of high-quality solutions for protection against Botrytis for their vineyards while minimizing pesticide residues in the final product, various treatment approaches were tested. A total of 10 strategies with different specific fungicide treatments for controlling Botrytis cinerea were applied to grapes at different growing stages: flowering, bunch closure and colour change. The type of vine chosen was Gamay, as it is very sensitive to Botrytis cinerea. In each experimental plot, disease incidence and severity were assessed at harvest. In addition, pesticide residue analysis was carried out on grapes, musts and wines to monitor residue levels in each treatment and to follow changes at each stage of the wine-making process. A correlation was established between the efficiency of anti-Botrytis fungicide treatment and pesticide residues in wine. Several strategies using various fungicides showed good results in terms of treatment efficiency while minimizing pesticide residues in wine, thus providing interesting alternatives to limit the development of fungal resistance.

Clozapine and quetiapine acutely reduce glucagon-like peptide-1 production and increase glucagon release in obese rats: implications for glucose metabolism and food choice behaviour.

OBJECTIVE: Second generation antipsychotic drug (SGA) treatment is associated with detrimental effects on glucose metabolism which is often attributed to the development of obesity and insulin resistance. However, we have recently demonstrated that clozapine and quetiapine also have direct effects of glucose metabolism in animals. This study compares clozapine and quetiapine and investigates the effects of these on the development of obesity and the direct effects of these drugs on glucose metabolism compared with those caused by the obesity per se. RESEARCH DESIGN AND METHODS: Three groups of male Sprague-Dawley rats were fed a high fat/high sugar diet to induce obesity while another three groups were fed a chow diet. One group on each diet was injected daily with vehicle, clozapine or quetiapine and effects on glucose metabolism were monitored. RESULTS: Clozapine and quetiapine treatment did not directly cause obesity or potentiate diet induced obesity but did induce a preference for the high fat/high sugar diet. Neither drug caused a impairment in insulin tolerance over that caused by obesity but both drugs acutely induced impairments in glucose tolerance that were additive with the effects induced by the diet induced obesity. Both drugs caused increases in glucagon levels and a suppression of GLP-1. We investigated two strategies for restoring GLP-1 signalling. The DPP-IV inhibitor sitagliptin only partially restored GLP-1 levels and did not overcome the deleterious effects on glucose tolerance whereas the GLP-1 receptor agonist exendin-4 normalised both glucagon levels and glucose metabolism. CONCLUSIONS: Our findings indicate that the clozapine and quetiapine induced impairments in glucose tolerance in rats are independent of insulin resistance caused by obesity and that these defects are linked with a suppression of GLP-1 levels. These studies suggest the need to perform follow up studies in humans to determine whether clozapine and quetiapine induce acute derangements in glucose metabolism and whether GLP-1 replacement therapy might be the most appropriate therapeutic strategy for treating derangements in glucose metabolism in subjects taking these drugs.

Comprehensive fast multiresidue screening of 150 veterinary drugs in milk by ultra-performance liquid chromatography coupled to time of flight mass spectrometry.

This paper shows the use of ultra-performance liquid chromatography (UPLC) coupled to orthogonal acceleration time of flight mass spectrometry (TOF MS) for the comprehensive screening of 150 veterinary drugs residues in raw milk. An easy sample preparation based on protein precipitation associated with ultrafiltration was hyphenated to fast chromatography. This method enabled the screening for more than 50 samples per day and searched for 150 drugs and metabolites including avermectines, benzimidazoles, beta-agonists, beta-lactams, corticoides, macrolides, nitroimidazoles, quinolones, sulfonamides, tetracyclines and some others. Identification of contaminants is based on accurate mass measurement. UPLC-TOF also showed very good performances for quantitation and allowed the determination of majority of compounds below MRL. An in-house validation procedure was conducted based on European directive 2002/657/EC with measurement of response function, accuracy, repeatability, limits of detection (LOD), decision limit (CCalpha) and detection capability (CCbeta).

Fast screening and quantitation of microcystins in microalgae dietary supplement products and water by liquid chromatography coupled to time of flight mass spectrometry.

Cyanobacteria, commonly called "blue-green algae", may accumulate in surface water supplies as "blooms" and may concentrate on the surface as blue-green "scums". Some species of cyanobacteria produce toxins and are of relevance to water supplies and to microalgae dietary supplements. To ensure the safety of drinking water and blue-green algae products, analyses are the only way to determine the presence or absence of toxins. This paper shows the use of ultra performance liquid chromatography (UPLC) coupled to orthogonal acceleration time of flight (TOF) mass spectrometry for the detection and quantitation of microcystins. The method presented is very sensitive, simple, fast, robust and did not require fastidious clean-up step. Limits of detection of 0.1 microg L(-1) in water and 0.1-0.2 microg g(-1) in microalgae samples were achieved. Method performances were satisfactory and appropriate for monitoring of water and dietary supplements. The method was applied in routine to samples taken from Swiss market or buy on internet website. Among 19 samples, six showed the presence of microcystins LR and LA at harmful levels.

Validation of a gas chromatography--ion trap tandem mass spectrometry for simultaneous analyse of cocaine and its metabolites in saliva.

Cocaine (COC) is one of the most widely used drugs of abuse. Therefore numerous procedures are published in the literature to propose an analysis of this substance and related compounds in different matrixes. In the same way, the authors have described, in a previous work, the simultaneous analysis of COC and three of its metabolites in hair by gas chromatography-ion-trap tandem mass spectrometry (GC-MS/MS) using chemical ionization with isobutane. The present paper investigated the ability to transfer this convenient existing method for hair to another matrix, in occurrence saliva. The aim of this work was then to verify that the whole procedure (solid phase extraction (SPE) and analytical method) was also convenient to analyse simultaneously COC and three of its metabolites in this matrix. Therefore this sensitive GC-MS/MS method has been studied for the simultaneous analysis of COC, anhydroecgonine methylester (AEME), ecgonine methylester (EME) and cocaethylene (COET) in saliva samples. The method has been validated and its performances were evaluated in terms of trueness and precision using quality control (QC) samples. For quantification, the following ranges were found appropriate: 5-500 ng/ml for EME, 2-500 ng/ml for COC and COET; AEME could only be determined "semi-quantitatively" between 2 and 200 ng/ml according to our chosen acceptance criteria. Suggested dissociation pathways have also been proposed to interpret the obtained spectra.

Analysis of cocaine and three of its metabolites in hair by gas chromatography-mass spectrometry using ion-trap detection for CI/MS/MS.

A sensitive GC/CI/MS/MS method was developed for the simultaneous determination of cocaine (COC), anhydroecgonine methylester (cocaine pyrolysis product, AEME), ecgonine methylester (cocaine enzymatic hydrolysis product, EME) and cocaethylene (cocaine with ethanol trans-esterification product, COET) in human hair samples. After acid hydrolysis, hair samples were extracted with an automated solid phase extraction (SPE). The analysis of cocaine and its three metabolites was performed using an ion-trap spectrometer in positive chemical ionization with isobutane as gas reagent. The procedure was validated. Weighted linear regression was found appropriate in a concentration range of 0.10-5.00 ng/mg for AEME, 0.05-5.00 ng/mg for COC, EME and COET. The limit of detection was estimated at 0.005 ng/mg for COC and COET, at 0.025 ng/mg for EME, and at 0.050 ng/mg for AEME. Method performance was evaluated in terms of trueness and precision using quality control (QC) samples over the investigated ranges. Method selectivity and robustness were also demonstrated.

Determination of nicotine and its major metabolite cotinine in plasma or serum by gas chromatography-mass spectrometry using ion-trap detection.

A specific method has been developed for the quantitative determination of nicotine and its major metabolite cotinine in plasma or serum of active and passive smokers. Deuterium-labelled nicotine and cotinine were used as internal standards. The amounts of nicotine and cotinine present in a sample of plasma or serum were extracted with a simple extraction procedure (liquid-liquid or solid-phase extraction). The extracts were analysed by gas chromatography coupled with mass spectrometry using ion-trap detection. The analysis was done in positive chemical ionisation with methanol as the liquid reagent. The method has been demonstrated to be linear up to 1000 microg/l. Limits of quantification for nicotine and cotinine are 10 and 5 microg/l, respectively with liquid-liquid extraction, and 1 microg/l for each of the compounds with solid-phase extraction. The present method has been applied to several real cases.