Gene Expression Pattern in Transmitochondrial Cytoplasmic Hybrid Cells Harboring Type 2 Diabetes-Associated Mitochondrial DNA Haplogroups

Decreased mitochondrial function plays a pivotal role in the pathogenesis of type 2 diabetes mellitus (T2DM). Recently, it was reported that mitochondrial DNA (mtDNA) haplogroups confer genetic susceptibility to T2DM in Koreans and Japanese. Particularly, mtDNA haplogroup N9a is associated with a decreased risk of T2DM, whereas haplogroups D5 and F are associated with an increased risk. To examine functional consequences of these haplogroups without being confounded by the heterogeneous nuclear genomic backgrounds of different subjects, we constructed transmitochondrial cytoplasmic hybrid (cybrid) cells harboring each of the three haplogroups (N9a, D5, and F) in a background of a shared nuclear genome. We compared the functional consequences of the three haplogroups using cell-based assays and gene expression microarrays. Cell-based assays did not detect differences in mitochondrial functions among the haplogroups in terms of ATP generation, reactive oxygen species production, mitochondrial membrane potential, and cellular dehydrogenase activity. However, differential expression and clustering analyses of microarray data revealed that the three haplogroups exhibit a distinctive nuclear gene expression pattern that correlates with their susceptibility to T2DM. Pathway analysis of microarray data identified several differentially regulated metabolic pathways. Notably, compared to the T2DM-resistant haplogroup N9a, the T2DM-susceptible haplogroup F showed down-regulation of oxidative phosphorylation and up-regulation of glycolysis. These results suggest that variations in mtDNA can affect the expression of nuclear genes regulating mitochondrial functions or cellular energetics. Given that impaired mitochondrial function caused by T2DM-associated mtDNA haplogroups is compensated by the nuclear genome, we speculate that defective nuclear compensation, under certain circumstances, might lead to the development of T2DM

Mitochondrial DNA variability modulates mRNA and intra-mitochondrial protein levels of HSP60 and HSP75: experimental evidence from cybrid lines

To explore possible relationships between mitochondrial DNA (mtDNA) polymorphism and the expression levels of stress-responder nuclear genes we assembled five cybrid cell lines by repopulating 143B.TK− cells, depleted of their own mtDNA (Rho0 cells), with foreign mitochondria with different mtDNA sequences (lines H, J, T, U, X). We evaluated, at both basal and under heat stress conditions, gene expression (mRNA) and intra-mitochondrial protein levels of HSP60 and HSP75, two key components in cellular stress response. At basal conditions, the levels of HSP60 and HSP75 mRNA were lower in one cybrid (H) than in the others (p = 0.005 and p = 0.001, respectively). Under stress conditions, the H line over-expressed both genes, so that the inter-cybrid difference was abolished. Moreover, the HSP60 intra-mitochondrial protein levels differed among the cybrid lines (p = 0.001), with levels higher in H than in the other cybrid lines. On the whole, our results provide further experimental evidence that mtDNA variability influences the cell response to stressful conditions by modulating components involved in this response. Sentence summary of the article: the results reported in the present study provide important experimental evidence that in human cells mtDNA variability is able to influence the cellular response to heat stress by modulating both the transcription of genes involved in this response and their intra-mitochondrial protein levels