Abstract
Forkhead box O 3 (FOXO3) is a transcription factor involved in cell metabolism, inflammation and longevity. Here, we investigated if metformin can activate FOXO3 in human immune cells and affects the subsequent level of reactive oxygen/nitrogen species (ROS/RNS) in immune cells. AMP-activated protein kinase (AMPK) and FOXO3 activation were investigated by immunoblot or flow cytometry (FC) analysis, respectively. FOXO3 target gene expression was quantified by real-time PCR. ROS/RNS measurement using dichlorodihydrofluorescein diacetate (DCFH-DA) dye was investigated by FC. The role of the FOXO3 single nucleotide polymorphisms (SNPs) rs12212067, rs2802292 and rs12206094 on ROS/RNS production was studied using allelic discrimination PCR. Metformin induced activation of AMPK (pT172) and FOXO3 (pS413). ROS/RNS level was reduced in immune cells after metformin stimulation accompanied by induction of the FOXO3 targets mitochondrial superoxide dismutase and cytochrome c. Studies in Foxo3 deficient (Foxo3-/-) mouse splenocytes confirmed that metformin mediates its effects via Foxo3 as it attenuates ROS/RNS in myeloid cells of wildtype (WT) but not of Foxo3-/- mice. Our results suggest that FOXO3 can be activated by metformin leading to reduced ROS/RNS level in immune cells. This may add to the beneficial clinical effects of metformin observed in large cohort studies on longevity, cardiovascular and cancer risk.
Highlights
Forkhead box O 3 (FOXO3) belongs to the family of FoxO transcription factors and is crucial in the regulation of cellular processes such as cell cycle regulation, apoptosis, cell metabolism, stress resistance and immunity [1]
The inhibition of AMPactivated protein kinase (AMPK) resulted in a significant reduction of metformin induced phosphorylation of FOXO3 in all immune cells (Figure 2B)
We provide evidence that metformin activates FOXO3 via the AMPK signalling pathway which subsequently leads to the induction of FOXO3 antioxidative target genes SOD2 and CYCS as well as the induction of autophagic gene expression
Summary
Forkhead box O 3 (FOXO3) belongs to the family of FoxO transcription factors and is crucial in the regulation of cellular processes such as cell cycle regulation, apoptosis, cell metabolism, stress resistance and immunity [1]. Single-nucleotide polymorphisms (SNPs) for FOXO3 have been found to be associated with longevity [2, 3] and favorable outcomes in inflammatory disease [4]. A role of FOXO3 in dampening immune reactions was discussed in several studies [5, 6]. We showed that Foxo plays an important role in acute viral infection as its activation attenuated natural killer (NK) cell responses in a viral myocarditis model [7]. FOXO3 modulation would have therapeutic potential in chronic and autoimmune diseases.
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