Abstract
Metformin is a widely-used treatment for type 2 diabetes and is reported to extend health and lifespan as a caloric restriction (CR) mimetic. Although the benefits of metformin are well documented, the impact of this compound on the function and organization of the genome in normal tissues is unclear. To explore this impact, primary human fibroblasts were treated in culture with metformin resulting in a significant decrease in cell proliferation without evidence of cell death. Furthermore, metformin induced repositioning of chromosomes 10 and 18 within the nuclear volume indicating altered genome organization. Transcriptome analyses from RNA sequencing datasets revealed that alteration in growth profiles and chromosome positioning occurred concomitantly with changes in gene expression profiles. We further identified that different concentrations of metformin induced different transcript profiles; however, significant enrichment in the activator protein 1 (AP-1) transcription factor network was common between the different treatments. Comparative analyses revealed that metformin induced divergent changes in the transcriptome than that of rapamycin, another proposed mimetic of CR. Promoter analysis and chromatin immunoprecipitation assays of genes that changed expression in response to metformin revealed enrichment of the transcriptional regulator forkhead box O3a (FOXO3a) in normal human fibroblasts, but not of the predicted serum response factor (SRF). Therefore, we have demonstrated that metformin has significant impacts on genome organization and function in normal human fibroblasts, different from those of rapamycin, with FOXO3a likely playing a role in this response.
Highlights
Metformin (MET, N,N- dimethylimidodicarbonimidic diamide) is a well-established and commonly used treatment for type 2 diabetes (T2D)[1]
Cultures were confirmed to be mostly proliferative by β-galactosidase assays and neither concentration of metformin induced an increase in cell death as determined by Trypan Blue, with proliferative samples exhibiting 95.9% cell survival, 0.5 mM metformin-treated samples had 96.6% survival, and 1.0 mM metformin treatment had 98.6% survival (Supplemental Fig. 3)
Intergenic region of chromosome 18 was analysed as a binding control and demonstrated no enrichment. These results implicate forkhead box O3a (FOXO3a) in regulating cellular response to metformin treatments and because of its role in the regulation in cytokine expression they suggest that cytokines may be important in fibroblast response to dietary restriction mimetics. We have identified both changes in genome organization and function in response to metformin treatment in primary human fibroblasts
Summary
Metformin (MET, N,N- dimethylimidodicarbonimidic diamide) is a well-established and commonly used treatment for type 2 diabetes (T2D)[1]. Rapamycin was shown to decrease neuronal progenitors whereas metformin had no effect whilst still inhibiting mTOR21, and both compounds were demonstrated to differentially influence cardiac markers in rats[22] Despite these findings, the impact of metformin at the transcriptomic level of normal fibroblasts is unknown, as is the extent to which these proposed mimetics differ in influencing the transcriptome. Metformin has numerous reported beneficial effects on health and lifespan, with some previous analyses in Mus musculus demonstrating changes in gene expression and a shift towards a CR-like expression profile[13] These CR-like states function most commonly through inhibition of the mTOR pathway[23] and mTOR inhibition and some biochemical impacts of metformin treatment are well characterized, the effects of metformin on genome function (gene expression) and organization (positioning of chromosome territories with the nucleus) in normal human fibroblasts are currently unknown. Understanding genome function and organization in response to metformin (a method of altering nutrient sensing) could provide insight into how altering nutrient sensing can influence three-dimensional folding of the genome
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