Gene expression in skeletal muscle biopsies from people with type 2 diabetes and relatives: differential regulation of insulin signaling pathways

Abstract

Background: Gene expression alterations have previously been associated with type 2 diabetes, however whether these changes are primary causes or secondary effects of type 2 diabetes is not known. As healthy first degree relatives of people with type 2 diabetes have an increased risk of developing type 2 diabetes, they provide a good model in the search for primary causes of the disease.

Methods/principal findings: We determined gene expression profiles in skeletal muscle biopsies from Caucasian males with type 2 diabetes, healthy first degree relatives, and healthy controls. Gene expression was measured using Affymetrix Human Genome U133 Plus 2.0 Arrays covering the entire human genome. These arrays have not previously been used for this type of study. We show for the first time that genes involved in insulin signaling are significantly upregulated in first degree relatives and significantly downregulated in people with type 2 diabetes. On the individual gene level, 11 genes showed altered expression levels in first degree relatives compared to controls, among others KIF1B and GDF8 (myostatin). LDHB was found to have a decreased expression in both groups compared to controls.

Conclusions/significance: We hypothesize that increased expression of insulin signaling molecules in first degree relatives of people with type 2 diabetes, work in concert with increased levels of insulin as a compensatory mechanism, counter-acting otherwise reduced insulin signaling activity, protecting these individuals from severe insulin resistance. This compensation is lost in people with type 2 diabetes where expression of insulin signaling molecules is reduced.

Figure 1. Number of differentially expressed genes.

Diagram showing the number of genes found to be differentially expressed in skeletal muscle biopsies from people with type 2 diabetes and first degree relatives compared to healthy controls in a dChip analysis of the generated microarray data. Criteria used in the analysis were set as described in the Methods section. The number of up- and downregulated genes in each group is indicated. Only 2 genes were found to have an altered expression in both groups, namely TncRNA and LDHB. LDHB is a key-enzyme in anaerobic glycolysis and had a reduced expression in both the ‘R’ and the ‘D’ group compared to controls.

Figure 2. Regulation of insulin signaling in people with type 2 diabetes.

The insulin signaling pathways were found to be significantly downregulated on the gene expression level using the program GenMAPP/MAPPFinder. Analysis criteria were set as described in the Methods section. Underneath each section of the pathway, genes found to have an increased expression are depicted in red, and genes found to have a decreased expression are depicted in green. Figure adapted from GenMAPP.

Figure 3. Regulation of insulin signaling in first degree relatives of people with type 2 diabetes.

The insulin signaling pathways were found to be significantly upregulated on the gene expression level using the program GenMAPP/MAPPFinder. Analysis criteria were set as described in the Methods section. Underneath each section of the pathway, genes found to have an increased expression are depicted in red, and genes found to have a decreased expression are depicted in green. Figure adapted from GenMAPP.

Figure 4. Protein expression of IR and PGC1α.

Western blot (WB) analyses were performed for the IR and PGC1α for all samples used in the microarray study in order to verify mRNA results on protein level. Band intensities were determined and the average result for each group is shown in this figure. The only significant difference between groups was found to be a downregulation of the IR in the diabetic group compared to controls (unpaired t-test, p-value <0.05). This result fits with what was observed in the microarray study.