Project description:Transcriptional profiling of insulin exposed H4IIE cells at different time points (6h-24h) to 100 nM insulin, with untreated solvent control cells (blanks). The central role of hepatic insulin resistance in pandemic metabolic diseases urges investigation of the underlying mechanisms of its development and pathogenesis. Besides genetic susceptibility and lifestyle factors, environmental pollutants are suggested risk factors. In order to appraise the role of the thousands of pollutants we are daily exposed to, stable, robust and high throughput cell-based systems should be developed. In this context, it is of paramount importance to select an in vitro system which mimics in vivo insulin responses as close as possible. Therefore, this study was designed to evaluate if the rat H4IIE hepatoma cell line is a physiologically relevant model to study hepatic insulin responses. DNA microarray analysis, real-time PCR and flow cytometric cell cycle analysis were used to assess the relevance of the insulin response in H4IIE cells. Insulin dose dependently stimulated H4IIE growth. Time dependency of the insulin response was shown with real-time PCR for the known insulin responsive genes: Fasn, Pck1 and Irs2. Based on these results, microarray analysis was performed on H4IIE cells exposed to insulin (100 nM) for 6h and 24h. Genes related to carbohydrate (glycolysis and gluconeogenesis) and lipid metabolism (glycerolipid metabolism, cholesterol synthesis and fatty acid oxidation) were most profoundly afflicted, in accordance with in vivo insulin action in liver. Since changes in carbohydrate and lipid metabolism are pivotal in the pathogenesis of insulin resistance, the presence of a physiological relevant insulin response in H4IIE cells pleads for further testing of its potential use in research on pollutant-driven insulin resistance. Microarray analysis was performed on H4IIE cells exposed to insulin (100 nM) for 6h and 24h. A separate v+2 A-optimal hybridization design was used for each time-point. Using this design each exposure condition is represented by three biological replicates, with each biological replicate analysed in technical duplicate while applying the dye swap principle to correct for dye bias.

Project description:The obesogen hypothesis states that exposure to environmental compounds early in life or throughout lifetime might have an influence on obesity development. In this paper a new approach for obesogen screening is proposed, namely the use of transcriptomics in the 3T3-L1 pre-adipocyte cell line. Based on the data from a previous study of our group using a lipid accumulation based adipocyte differentiation assay, several humanly relevant obesogenic compounds were selected: reference obesogens [Rosiglitazone (ROSI), Tributyltin (TBT)], test obesogens [Butylbenzyl phthalate (BBP), butylparaben (BP), propylparaben (PP), Bisphenol A (BPA)] and non-obesogens [Ethylene Brassylate (EB), Bis (2-ethylhexyl)phthalate (DEHP)]. In this paper, the high stability and reproducibility of the 3T3-L1 gene transcription patterns over different experiments and cell batches was shown. Obesogens and non-obesogen gene transcription profiles were clearly distinguished using hierarchical clustering. Furthermore, a gradual distinction corresponding to differences in induction of lipid accumulation could be made between test and reference obesogens based on transcription patterns, indicating the potential use of this strategy for classification of obesogens. Marker genes that are able to distinguish between non, test and reference obesogens were identified. Well known genes involved in adipocyte differentiation, as well as genes with unknown functions were selected, implying a potential adipocyte related function of the latter. Cell physiological lipid accumulation was well predicted based on transcription levels of the marker genes, indicating the biological relevance of omics data. In conclusion, this study shows the high relevance and reproducibility of this 3T3-L1 based in vitro toxicogenomics tool for classification of obesogens and biomarker discovery. The hybridization design was a reference design (reference sample was a mixture of equal aliquots from control and exposed samples), recommended for class discovery purposes (Knapen et al. 2009). The reference sample was labeled with Cy5, and the exposed samples with Cy3. Three biological replicates were measured for every condition.

Project description:We used microarrays to investigate the transcriptome of 6 days old male flies exposed to either 15 or 25 C development at either constant or fluctuating temperatures. Further, we investigated gene expression at benign (20C) and high (35C) temperatures With global climate change temperature means and variability are expected to increase. Thus, exposures to elevated temperatures are expected to become an increasing challenge for terrestrial ectotherm populations. While evolutionary adaptation seems to be constrained or proceed at an insufficient pace, many populations are expected to rely on phenotypic plasticity (thermal acclimation) for coping with the predicted changes. However, the effects of fluctuating temperature on the molecular mechanisms and the implications for heat tolerance are not well understood. To understand and predict consequences of climate change it is important to investigate how different components of the thermal environment, including fluctuating thermal conditions, contribute to changes in thermal acclimation. In this study we investigated the impact of mean and diurnal fluctuation of temperature on heat tolerance in Drosophila melanogaster and on the underlying molecular mechanisms in adult male flies. Flies from two constant and two ecologically relevant fluctuating temperature regimes were tested for their critical thermal maxima (CTmax) and associated global gene expression profiles at benign and thermally stressful conditions. Both temperature parameters contributed independently to the thermal acclimation, with regard to heat tolerance as well as the global gene expression profile. Although the independent transcriptional effects caused by fluctuations were relatively small, they are likely to be essential for our understanding of thermal adaptation. Thus, high temperature acclimation ability might not be measured correctly and might even be underestimated at constant temperatures. Our data suggests that the particular mechanisms affected by thermal fluctuations are related to phototransduction and environmental sensing. Thus genes and pathways involved in those processes are likely to be of major importance in a future warmer and more fluctuating climate. Eight experimental groups were analyzed in triplicate, in total 24 Affymetrix GeneChip Drosophila Genome 2.0 Arrays

Project description:Genome-wide transcriptional profiling of Arabidopsis thaliana to a combination of heatwave and drought under ambient and elevated CO2. Goal of this study was elucidate the transcriptional responses to a combination of heat wave and drought, and to see how these responses are modifed under future climate (high) CO2. Climate changes increasingly threaten plant growth and productivity. Such changes are complex and involve multiple environmental factors, including rising CO2 levels and climate extreme events. As the molecular and physiological mechanisms underlying plant responses to realistic future climate extreme conditions are still poorly understood, a multiple organizational level-analysis (i.e. eco-physiological, biochemical and transcriptional) was performed, using Arabidopsis exposed to incremental heat wave and water deficit under elevated CO2.The climate extreme resulted in biomass reduction, photosynthesis inhibition, and considerable increases in stress parameters. Photosynthesis was a major target as demonstrated at the physiological and transcriptional levels. In contrast, the climate extreme treatment induced a protective effect on oxidative membrane damage, most likely as a result of strongly increased lipophilic antioxidants and membrane-protecting enzymes. Elevated CO2 significantly mitigated the negative impact of a combined heat and drought, as apparent in biomass reduction, photosynthesis inhibition, chlorophyll fluorescence decline, H2O2 production and protein oxidation. Analysis of enzymatic and molecular antioxidants revealed that the stress-mitigating CO2 effect operates through up-regulation of antioxidant defense metabolism, as well as by reduced photorespiration resulting in lowered oxidative pressure. Therefore, exposure to future climate extreme episodes will negatively impact plant growth and production, but elevated CO2 is likely to mitigate this effect. Transcriptome analysis was performed by Agilent Arabidopsis (V4) 4x44K platform which represented all known genes in the Arabidopsisgenome. Experiments were performed using a modified loop design (Knapen et al., 2009). This design consisted of total 8 arrays; sample from each treatment was labelled once and has 4 biological replicates, two of which were labelled in red and two in green

Project description:We used microarrays to investigate the transcriptome of 6 days old male flies exposed to either 15 or 25 C development at either constant or fluctuating temperatures. Further, we investigated gene expression at benign (20C) and high (35C) temperatures With global climate change temperature means and variability are expected to increase. Thus, exposures to elevated temperatures are expected to become an increasing challenge for terrestrial ectotherm populations. While evolutionary adaptation seems to be constrained or proceed at an insufficient pace, many populations are expected to rely on phenotypic plasticity (thermal acclimation) for coping with the predicted changes. However, the effects of fluctuating temperature on the molecular mechanisms and the implications for heat tolerance are not well understood. To understand and predict consequences of climate change it is important to investigate how different components of the thermal environment, including fluctuating thermal conditions, contribute to changes in thermal acclimation. In this study we investigated the impact of mean and diurnal fluctuation of temperature on heat tolerance in Drosophila melanogaster and on the underlying molecular mechanisms in adult male flies. Flies from two constant and two ecologically relevant fluctuating temperature regimes were tested for their critical thermal maxima (CTmax) and associated global gene expression profiles at benign and thermally stressful conditions. Both temperature parameters contributed independently to the thermal acclimation, with regard to heat tolerance as well as the global gene expression profile. Although the independent transcriptional effects caused by fluctuations were relatively small, they are likely to be essential for our understanding of thermal adaptation. Thus, high temperature acclimation ability might not be measured correctly and might even be underestimated at constant temperatures. Our data suggests that the particular mechanisms affected by thermal fluctuations are related to phototransduction and environmental sensing. Thus genes and pathways involved in those processes are likely to be of major importance in a future warmer and more fluctuating climate.

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An integrated study to the effects of thermal acclimation in zebrafish

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