METTL3 mediates atheroprone flow-induced glycolysis in endothelial cells

Abstract

Atheroprone flow-increased glycolysis in vascular endothelial cells (ECs) is pivotal in EC dysfunction and the initiation of atherosclerosis. Methyltransferase 3 (METTL3) is a major m⁶A methyltransferase for RNA N6-mehtyladenosine (m⁶A) modifications to regulate epitranscriptome and cellular functions. With the atheroprone flow upregulating METTL3 and m⁶A RNA hypermethylation, we investigate the role of METTL3 in atheroprone flow-induced glycolysis in ECs in vitro and in vivo. Compared to pulsatile shear stress (PS, atheroprotective flow), oscillatory shear stress (OS, atheroprone flow) increases METTL3 expression to enhance the m⁶A modifications of mRNAs encoding HK1, PFKFB3, and GCKR, which are rate-limiting enzymes of glycolysis. These augmented m⁶A modifications increase the expressions of HK1 and PFKFB3 while decreasing GCKR, resulting in elevated EC glycolysis, as revealed by seahorse analysis. Moreover, a stimulated Raman scattering (SRS) imaging study demonstrates the elevation of glucose incorporation into de novo synthesized lipids in ECs under atheroprone flow in vitro and in vivo. Empagliflozin, a sodium-glucose cotransporter-2 inhibitor (SGLT2i) drug, represses METTL3 expression, thereby mitigating OS-induced glycolysis in ECs. These data suggest mechanisms by which METTL3 links EC mechanotransduction with metabolic reprogramming under atherogenic conditions.

Keywords: atherosclerosis; endothelial cell; glycolysis; shear stress.

Fig. 1.

OS increases glycolysis via METTL3. (A) Heatmap of glycolytic gene expression based on RNA-seq analysis of ECs subjected to OS (0.5 ± 4 dyn/cm²) or PS (12 ± 4 dyn/cm²) for 48 h. The color bars represent OS/PS log2 (fold change). (B) scRNA-seq analysis demonstrating upregulation of glycolytic genes PFKFB3, HK2, ENO1, PFKP, and LDHB in the partially ligated left carotid artery (LCA) compared to the control right carotid artery (RCA). (C) Seahorse assay demonstrating the extracellular acidification rate (ECAR) in the indicated groups (n = 9/group). (D) Measurement of relative lactate levels in ECs under PS, OS, and OS with METTL3 knockdown (n = 6/group). (E) qPCR analysis of the indicated glycolytic genes (n = 5/group). (F) Western blot and quantification of METTL3, HK1, PFKFB3, and GCKR expressions in ECs subjected to PS or OS, with or without METTL3 knockdown (n = 4/group). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01.

Fig. 2.

Shear stress regulates glycolytic genes via m⁶A modifications. (A) Prediction of the m⁶A methylation sites in HK1, PRKFB3, and GCKR mRNA. (B and C) m⁶A-RIP-qPCR analysis of m⁶A modified HK1, PFKFB3, and GCKR mRNA in ECs under the indicated conditions (n = 4/group). (D) qPCR analysis of the indicated mRNA levels in ECs overexpressing METTL3 or METTL3(APPA) under PS (n = 5/group). (E) Western blot and quantification of METTL3, HK1, PFKFB3, and GCKR in ECs overexpressing METTL3 or METTL3(APPA) under PS (n = 4/group). (F) qPCR analysis of HK1, PFKFB3, and GCKR in ECs overexpressing FTO or control vector under PS or OS (n = 4/group). (G) Western blot and quantification of METTL3, HK1, PFKFB3, and GCKR levels in the indicated groups (n = 4/group). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01.

Fig. 3.

Empagliflozin (EMPA) reduces EC glycolysis via METTL3. (A) qPCR analysis of HK1, PFKFB3, and GCKR mRNA expressions in EMPA treated-ECs with/without METTL3 overexpression for 24 h (n = 5/group). (B) Western blot and quantification of METTL3, HK1, PFKFB3, and GCKR expressions in EMPA-treated ECs with/without METTL3 overexpression (n = 4/group). (C) Seahorse assay measuring ECAR changes in the indicated groups (n = 4/group). (D) Measurement of relative lactate content in ECs in the indicated groups (n = 6/group). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01.

Fig. 4.

Atheroprone flow increases glycolysis via METTL3 in vivo. (A) Schematic diagram of vascular intima isolated from the mouse TA and AA; (B) qPCR analysis of Mettl3 mRNA expression in the intima from TA and AA of C57BL/6 J WT mice; (C) qPCR analysis of Hk1, Pfkfb3, and Gckr mRNA expressions in the intima of the TA or AA from WT (Mettl3-floxed) and Mettl3 KO mice; (D) ECs were treated with 25 mM D7-glu for 72 h and then subjected to OS for 48 h, followed by SRS imaging analysis for CD/CH and NADH/Flavin for the assessment of lipid turnover and redox ratio, respectively. (Scale bar, 10 µm.) n = 9; data are presented as mean ± SEM. *P <0.05; **P <0.01 between indicated groups. (E) Mettl3 KO and WT (Mettl3-floxed) mice were fed D7-glu water for 2 wk. The TA and AA regions of the arterial tree were subjected to SRS imaging analysis ex vivo for CD/CH lipid turnover rate and NADH/Flavin redox ratio. (Scale bar, 10 µm.) n = 7 for METTL3 KO mice and n = 9 for WT mice; data are presented as mean ± SEM. *P <0.05, **P <0.01 between indicated groups.

Fig. 5.

Schematic illustration showing that atheroprone flow increases glycolysis in ECs via METTL3. Atheroprotective flow such as PS suppresses the expression of METTL3, resulting in low levels of m⁶A modification of the glycolysis genes HK1, PFKFB3, and GCKR. Such regulation of glycolysis maintains EC homeostasis. Under atheroprone flow such as OS, METTL3 expression is increased, leading to enhanced m⁶A modifications of HK1, PFKFB3, and GCKR. These augmented m6A modifications upregulate HK1 and PFKFB3 expressions while inhibiting GCKR expression, contributing to elevated glycolysis and EC dysfunction.