METTL3 Mediates Atheroprone Flow–Induced Glycolysis in Endothelial Cells
1. Research Background
Atherosclerosis is the primary pathological basis of cardiovascular diseases, closely associated with endothelial cell (EC) dysfunction. Hemodynamic factors play a critical role in the regional selectivity of atherosclerosis: oscillatory shear stress (OS) (e.g., at vascular bifurcations) promotes plaque formation, while pulsatile shear stress (PS) (e.g., in straight vessel segments) exerts protective effects. Recent studies have revealed that ECs undergo metabolic reprogramming under OS, characterized by enhanced glycolysis, but the precise molecular mechanisms remain unclear.
In the field of epitranscriptomics, RNA m6A methylation (N6-methyladenosine modification) has been shown to influence cellular functions by regulating mRNA stability, splicing, and translation efficiency. METTL3, the core m6A methyltransferase (“writer”), has been implicated in inflammatory responses, but its role in EC metabolic regulation remains unexplored. This study is the first to demonstrate how METTL3 mediates OS-induced glycolytic hyperactivity in ECs via m6A modification of glycolysis-related genes (HK1/PFKFB3/GCKR). Additionally, it reveals that the antidiabetic drug SGLT2 inhibitor (empagliflozin) exerts cardiovascular protective effects by suppressing METTL3.
2. Paper Source
This collaborative study between Chinese and American institutions was led by corresponding authors Prof. Shu Chien and Prof. John Y-J Shyy from the University of California, San Diego, with first authors Guo-Jun Zhao from Zhengzhou University and So Yun Han from UC San Diego. Published on May 6, 2025, in PNAS (Volume 122, Issue 19), the paper is titled “METTL3 mediates atheroprone flow–induced glycolysis in endothelial cells.”
3. Research Workflow and Results
1. Shear Stress Regulation of Glycolytic Gene Expression Profiles
Experimental Design:
- Analyzed GSE103672 dataset (RNA-seq of HUVECs exposed to OS/PS for 48 hours)
- Single-cell RNA-seq (scRNA-seq) data from a mouse partial carotid ligation model (n=6/group)
Key Findings:
- OS significantly upregulated glycolytic genes (HK1, PFKFB3, ENO1, etc.; log2FC>1.5, P<0.01)
- HK2/PFKFB3 expression was 2-3 times higher in ECs from ligated carotid arteries (low-shear regions)
Technical Highlight:
Employed a bioinformatics cross-validation strategy integrating in vitro fluid shear systems and in vivo arterial ligation models.
2. Functional Validation of METTL3 in Glycolysis Regulation
Experimental Systems:
- Genetic interventions: siRNA-mediated METTL3 knockdown vs. wild-type/catalytic mutant (APPA) overexpression
- Functional assays: Seahorse extracellular flux analyzer (ECAR measurements), lactate quantification
Results:
- METTL3 knockdown reduced OS-induced glycolysis rates (ECAR) by 40% (P<0.01)
- Lactate production decreased by 35% (n=6, P<0.05)
- Western blot showed METTL3 knockdown reduced HK1/PFKFB3 protein by 50-60% and increased GCKR 2-fold
Mechanistic Validation:
- m6A-RIP-qPCR confirmed OS-enhanced m6A modifications in 3’UTRs of HK1/PFKFB3/GCKR mRNAs
- Overexpression of m6A demethylase FTO reversed OS-induced metabolic effects
3. Exploration of SGLT2 Inhibitor Mechanisms
Pharmacological Experiments:
- Treated with empagliflozin (10 μM, 24 hours)
- Conducted METTL3 overexpression rescue experiments
Discoveries:
- Empagliflozin reduced METTL3 protein (but not mRNA) levels by 60%
- Changes in glycolytic enzyme expression mirrored METTL3 knockdown phenotypes
- METTL3 overexpression counteracted empagliflozin’s inhibitory effects
Technical Innovation:
First established a cross-scale model linking shear stress, epitranscriptomics, and pharmacological intervention.
4. In Vivo Metabolic Imaging Validation
Methodological Breakthrough:
- Developed stimulated Raman scattering (SRS)-based glucose metabolic tracing
- Used deuterated glucose (D7-glucose) to label de novo lipid synthesis
Animal Experiments:
- EC-specific METTL3 knockout mice (n=7) vs. wild-type (n=9)
- Fed 3% D7-glucose water for 2 weeks, then analyzed aortic arch (atheroprone) and thoracic aorta (atheroprotective) regions
Key Data:
- Wild-type mice showed 2.1-fold higher CD/CH ratios (lipid synthesis index) in aortic arch vs. thoracic aorta (P<0.01)
- METTL3 knockout abolished this difference
- NADH/flavin ratios (redox indicator) normalized concomitantly
4. Conclusions and Impact
Scientific Discoveries:
1. Identified the OS-METTL3-m6A-HK1/PFKFB3/GCKR axis as the core mechanism regulating EC glycolysis
2. Revealed a novel epigenetic pathway for SGLT2 inhibitor cardiovascular benefits
Clinical Implications:
- Provides a mechano-metabolic explanation for regional atherosclerosis susceptibility
- Proposes METTL3 as a potential therapeutic target for cardio-metabolic syndromes
Methodological Contributions:
- Established SRS imaging for vascular metabolism research
- Pioneered biological applications of m6A site prediction algorithm (SRAMP)
5. Research Highlights
- Novel Mechanism: First to couple mechanical shear stress, m6A epitranscriptomic regulation, and metabolic reprogramming
- Translational Value: Discovered empagliflozin’s non-canonical METTL3-dependent pathway
- Technology Integration: Combined scRNA-seq, SRS live imaging, and multi-omics approaches
- Animal Model: Generated EC-specific METTL3 knockout mice to validate clinical relevance
6. Future Directions
The authors suggest further investigation into:
- Molecular mechanisms regulating METTL3 protein stability
- Involvement of other shear-sensitive m6A “reader” proteins
- Preventive potential of SGLT2 inhibitors in non-diabetic atherosclerosis