Dataset Information

Phosphoprotoemics analysis of FAO cells stimulated with insulin

ABSTRACT: FAO cells, rat hepatoma cells, were stimulated with 1 nM insulin and collected at seven time points for ~60 minutes. Ion metal affinity chromatography (IMAC) was performed to enrich the phosphopeptides. As we used two sets of 4-plex iTRAQ to quantify the peptides from seven samples, we set the internal control to calibrate two sets of phosphoproteomics data

ORGANISM(S): Rattus Rattus (black Rat)

SUBMITTER: Sinya Kuroda

PROVIDER: PXD005900 | JPOST Repository | Fri Nov 30 00:00:00 GMT 2018

REPOSITORIES: jPOST

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Publications

Reconstruction of insulin signal flow from phosphoproteome and metabolome data.

Yugi Katsuyuki K Kubota Hiroyuki H Toyoshima Yu Y Noguchi Rei R Kawata Kentaro K Komori Yasunori Y Uda Shinsuke S Kunida Katsuyuki K Tomizawa Yoko Y Funato Yosuke Y Miki Hiroaki H Matsumoto Masaki M Nakayama Keiichi I KI Kashikura Kasumi K Endo Keiko K Ikeda Kazutaka K Soga Tomoyoshi T Kuroda Shinya S

Cell reports 20140814 4

Cellular homeostasis is regulated by signals through multiple molecular networks that include protein phosphorylation and metabolites. However, where and when the signal flows through a network and regulates homeostasis has not been explored. We have developed a reconstruction method for the signal flow based on time-course phosphoproteome and metabolome data, using multiple databases, and have applied it to acute action of insulin, an important hormone for metabolic homeostasis. An insulin sign ...[more]

PMID: 25131207

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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.

Dataset Information

Phosphoprotoemics analysis of FAO cells stimulated with insulin

Publications

Reconstruction of insulin signal flow from phosphoproteome and metabolome data.

Similar Datasets

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