Metformin activates KDM2A to reduce rRNA transcription and cell proliferation by dual regulation of AMPK activity and intracellular succinate level

Abstract

Metformin is used to treat type 2 diabetes. Metformin activates AMP-activated kinase (AMPK), which may contribute to the action of metformin. Metformin also shows anti-proliferation activity. However, the mechanism is remained unknown. We found that treatment of MCF-7 cells with metformin induced the demethylase activity of KDM2A in the rDNA promoter, which resulted in reductions of rRNA transcription and cell proliferation. AMPK activity was required for activation of KDM2A by metformin. Because demethylase activities of JmjC-type enzymes require a side reaction converting α-ketoglutarate to succinate, these organic acids may affect their demethylase activities. We found that metformin did not induce KDM2A demethylase activity in conditions of a reduced level of α-ketoglutarate. A four-hour treatment of metformin specifically reduced succinate, and the replenishment of succinate inhibited the activation of KDM2A by metformin, but did not inhibit the activation of AMPK. Metformin reduced succinate even in the conditions suppressing AMPK activity. These results indicate that metformin activates AMPK and reduces the intracellular succinate level, both of which are required for the activation of KDM2A to reduce rRNA transcription. The results presented here uncover a novel factor of metformin actions, reduction of the intracellular succinate, which contributes to the anti-proliferation activity of metformin.

Production of ROS by Gallic Acid Activates KDM2A to Reduce rRNA Transcription

“…To evaluate levels of rRNA transcription, the amounts of pre-rRNA were measured. The sets of PCR primers for amplification of the pre-rRNA were 5′-GCTGACACGCTGTCCTCTG-3′ and 5′-TCGGACGCGCGAGAGAAC-3′ (a sequence of 1–155 in pre-rRNA); for KDM2A, the primers were 5′-TCCCCACACACATTTTGACATC-3′ and 5′-GGGGTGGCTTGAGAGATCCT-3′; for β-actin, the primers were 5′-CGTCTTCCCCTCCATCGT-3′ and 5′-GAAGGTGTGGTGCCAGATTT-3′, as previously described [ 14 ].…”

“…The chromatin immunoprecipitation (ChIP) assay was performed as described previously [ 14 ] using Dynabeads ® protein G (ThermoFisher; #10003D). In brief, the cells were fixed with 1% formaldehyde for 10 min at 37 °C.…”

“…Compound C (IN Solution™ AMPK Inhibitor, compound C, Calbiochem, St. Louis, MO, USA; #171261) and SBI-0206965 (Sigma; #SML1540) [ 14 ] were purchased. The food-additive compound library was purchased (Sigma #S990043-FDS1).…”

“…Further, we found that glucose depletion activates AMP-activated protein kinase (AMPK), which is required for KDM2A to reduce rRNA transcription and cell proliferation of breast cancer cells [ 12 ]. We also found that metformin, known as a treatment for type 2 diabetes, activates KDM2A to reduce rRNA transcription and cell proliferation [ 14 ]. A 4-h treatment with metformin not only activated AMPK, but also independently decreased the intracellular succinate level [ 14 ].…”

“…We also found that metformin, known as a treatment for type 2 diabetes, activates KDM2A to reduce rRNA transcription and cell proliferation [ 14 ]. A 4-h treatment with metformin not only activated AMPK, but also independently decreased the intracellular succinate level [ 14 ]. The activation of KDM2A by metformin required both AMPK activity and a decrease of the intracellular succinate level [ 14 ], suggesting that reduction of intracellular succinate, in addition to AMPK activation, is a mechanism by which KDM2A is activated to reduce rRNA transcription.…”