Application of bioinformatics to the analysis of a candidate gene for insulin resistance in skeletal muscle.
An experiment was set up to analyze and compare the expression of genes in skeletal muscle of obese individuals, showing a high level of circulating fatty acids and insulin resistance, with a control group. Skeletal muscle biopsies were taken from participants within each group and total RNA was extracted and converted to cDNA. The cDNA from each group was labelled with different fluorophores and a microarray was conducted to allow the comparison of global gene expression within the two groups. The following candidate gene was identified as being differentially expressed between the two groups; therefore, further laboratory analysis was conducted to further investigate the regulation of this gene in vitro.
Promoter deletion analysis
An experiment was conducted in C2C12 murine muscle cells to assess the direct impact of palmitate +/- insulin on the promoter activity for this particular candidate gene.
A series of 5’ promoter deletions were generated from the sequence analysed above using restriction enzymes, all of the sequences contained the first 22 nucleotides of the transcribed gene and variable lengths of promoter sequence (see figure 1 below). The promoter DNA was inserted into a vector containing the reporter gene luciferase.
Candidate gene 5’ genomic DNA
Figure 1: Promoter deletion constructs of the candidate gene.
Experiments were conducted to look at the impact of both palmitate (saturated fatty acid) and insulin on the promoter activity of the candidate gene. The overall aim of the experiment was to assess the impact of these factors individually and to determine if palmitate influenced the insulin responsiveness of this gene.
The constructs were individually transfected into C2C12 cells and were subjected to different experimental conditions. Cells were seeded into a 96-well plate, where some wells remained as controls and others were subjected to treatment with palmitate (0.6mM) for a 24-hour period. Selected wells were then stimulated with insulin (100nM) for 15 minutes before fluorescence was detected in all wells. Presence of the luciferase protein (detected by fluorescence) indicates that the DNA construct has promoter activity. A positive control was included in the experiment, which has strong promoter activity, alongside a negative control which is known not to initiate transcription; these were treated the same as control unstimulated cells. Fluorescence data for each sample was normalised to the positive control, which was set at 100% and is shown in figure 2.
Figure 2: Promoter activity of the 5’ deletion constructs under different treatment conditions. Positive and negative control represent strong and weak promoters respectively and were not included in the statistical analysis. Different lowercase letters above the bars indicate significant differences (P<0.05)