Genetic and epigenetic factors are associated with expression of respiratory chain component NDUFB6 in human skeletal muscle

Abstract

Insulin resistance and type 2 diabetes are associated with decreased expression of genes that regulate oxidative phosphorylation in skeletal muscle. To determine whether this defect might be inherited or acquired, we investigated the association of genetic, epigenetic, and nongenetic factors with expression of NDUFB6, a component of the respiratory chain that is decreased in muscle from diabetic patients. Expression of NDUFB6 was influenced by age, with lower gene expression in muscle of elderly subjects. Heritability of NDUFB6 expression in muscle was estimated to be approximately 60% in twins. A polymorphism in the NDUFB6 promoter region that creates a possible DNA methylation site (rs629566, A/G) was associated with a decline in muscle NDUFB6 expression with age. Although young subjects with the rs629566 G/G genotype exhibited higher muscle NDUFB6 expression, this genotype was associated with reduced expression in elderly subjects. This was subsequently explained by the finding of increased DNA methylation in the promoter of elderly, but not young, subjects carrying the rs629566 G/G genotype. Furthermore, the degree of DNA methylation correlated negatively with muscle NDUFB6 expression, which in turn was associated with insulin sensitivity. Our results demonstrate that genetic, epigenetic, and nongenetic factors associate with NDUFB6 expression in human muscle and suggest that genetic and epigenetic factors may interact to increase age-dependent susceptibility to insulin resistance.

Figure 1. Effects of age and insulin on human skeletal muscle NDUFB6 levels.

(A) Skeletal muscle biopsies were taken from young (n = 91) and elderly (n = 70) twins, before and after a hyperinsulinemic clamp. RNA was analyzed for NDUFB6 mRNA expression together with the internal standard cyclophilin A. The NDUFB6/cyclophilin A ratio was calculated for each sample. In order to adjust for the lack of independence between monozygotic and dizygotic twins, mean differences between groups were compared using GEE methodology. (B and C) The effect of age on NDUFB6 protein levels before (B) and after (C) insulin stimulation was analyzed by Western blot in 8 young and 8 elderly twins. (D and E) Correlations between muscle NDUFB6 mRNA and protein levels in muscle biopsies from 8 young and 8 elderly twins before (D) and after (E) insulin stimulation. Results are mean ± SEM. *P < 0.05.

Figure 2. Association among the NDUFB6 polymorphism rs629566 (A/G), DNA methylation, and mRNA expression in muscle.

(A and B) Association between skeletal muscle NDUFB6 mRNA levels and rs629566 (A/G) in young (A; n = 91) and elderly (B; n = 70) twins after a hyperinsulinemic clamp. The level of NDUFB6 transcripts were normalized to the mRNA level of endogenous cyclophilin A, and the NDUFB6/cyclophilin A ratio was calculated for each sample. In order to adjust for the lack of independence between monozygotic and dizygotic twins, all mean differences between groups were compared using GEE methodology. Results are mean ± SEM. *P < 0.05. (C) The NDUFB6 promoter sequence investigated, showing rs629566 (–544) and 3 additional DNA methylation target sites: –634, –663 and –676. (D and E) Correlation between percent DNA methylation and basal (D) and insulin-stimulated (E) NDUFB6 expression in muscle (n = 14; correlations adjusted for age).

Figure 3. Schematic presentation of the NDUFB6 gene with the analyzed tagSNPs and LD structure.

LD between the NDUFB6 SNPs was analyzed using Haploview (50). D′ values with 95% confidence intervals were calculated as a measure of LD (51). The analysis was performed based on the genotyping results from the Botnia cohort. The figure was prepared using LocusView 2.0 (http://www.broad.mit.edu/mpg/locusview/). The following SNPs were in LD: rs540467 and rs2376223 (D′ = 1); rs2050456, rs12003093, and rs920029 (D′ > 0.83); and rs629566, rs829535, rs653790, rs17218354, rs10813831, rs3824456, and rs4013845 (D′ > 0.94).