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
| -459 |
| +22 |
| -92 |
| -253 |
| +22 |
| Luciferase |
| -459 |
| Luciferase |
| -92 |
| Luciferase |
| -253 |
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)
- a) Using the information that you gathered from the bioinformatics analysis of the core promoter and proximal promoter elements, what is your interpretation of the 5’ promoter deletion analysis in control cells +/- insulin stimulation? (20 marks) – 700words
- b) Outline the step by step changes in the insulin signalling pathway that would lead to an alteration in expression of this particular gene.(10 marks) – 400 words
- c) What impact does palmitate have on the candidate gene and how does it influence the genes responsiveness to insulin? (5 marks) – 300words
- d) What experiment would you conduct to confirm that it is transcription factors binding to the particular elements identified that are responsible for changes in promoter activity? (5 marks) – 300 words
- e) Investigators would like to continue to look at the function of the protein product produced by this candidate gene, as a potential drug target. Briefly outline an experiment you could conduct in C2C12 cells to investigate the functionality of the protein (Hint: You will want to over express your protein within cells and look at particular downstream targets). (10 marks) – 500words
- Background information on obesity and insulin resistance its link to type 2 diabetes, considering the role of saturated fatty acids (circulating fatty acid levels) in the development of insulin resistance in skeletal muscle. Consider cell signalling mechanisms (the insulin signalling pathway) in muscle and the use of AKT as a marker of insulin signalling pathway. Introduction to the Western Blotting technique for the analysis of phosphorylated proteins within cell signalling pathways, particularly AKT.4: Aim and objectives of lab (600words)
