LXR activation induces insulin resistance in primary human adipocytes

The effects of LXR stimulation by GW3965 treatment on global mRNA and miRNA expression in primary human in vitro differentiated adipocytes was investigated using microarray profiling. Preadipocytes were isolated from human adipose tissue obtained from healthy subjects and differentiated in vitro. Around day 11 of differentiation cells were treated with the Liver X Receptor agonist GW3865 for 24 h and lysed. Total RNA as prepared and hybridised biotinylated complementary RNA to Gene 1.0 ST Arrays using standardized protocols (Affymetrix Inc., Santa Clara, CA). The GeneChip Operating Software Version 1.4 was used for analysis. Arrays were normalized using PLIER and all samples were subjected to an all-probeset scaling-to-target signal of 100. Preadipocytes were isolated from human adipose tissue obtained from healthy subjects and differentiated in vitro. Around day 11 of differentiation cells were treated with the Liver X Receptor agonist GW3865 for 12 h and lysed. Total RNA was prepared and and labelled using the FlashTag biotin HSR labeling kit (Genisphere Inc., Hatfield, PA) according to the supplier's protocol. The labelled samples were placed in a hybridization cocktail mix containing 4% formamide and hybridised overnight to Affymetrix miRNA Arrays (Affymetrix Inc.) following the indicated Genisphere protocol. The arrays were washed, stained and scanned in an Affymetrix GCS 3000 scanner. Signal intensities and present calls were generated by using the microRNA QC tool by Affymetrix using default settings i.e. Background Adjustment: BC-CG adjust, Normalization: Quantile: Summarization: Median Polish.