Natural Flavonoid Acacetin Targets LAMTOR1 to Boost Autophagy and Reverse Fatty Liver
Researchers identify LAMTOR1 as a druggable target of acacetin, linking lysosomal signaling to autophagy induction and MAFLD reversal.
Summary
Metabolic dysfunction-associated fatty liver disease (MAFLD) lacks approved treatments. This study shows the natural flavonoid acacetin (ACA) reduces hepatic lipid accumulation and liver injury in a mouse model of steatohepatitis by binding directly to LAMTOR1, a lysosomal membrane protein. This binding disrupts the LAMTOR complex, suppresses MTORC1 signaling, and activates autophagy. The MTORC1-AMPK axis is rebalanced, promoting lipid clearance. Genetic knockdown of LAMTOR1 replicated ACA's beneficial effects, confirming LAMTOR1 as ACA's functional target. These findings reveal a previously uncharacterized therapeutic mechanism and nominate LAMTOR1 as a novel drug target for metabolic liver disease.
Detailed Summary
Metabolic dysfunction-associated fatty liver disease (MAFLD) affects hundreds of millions worldwide and can progress to steatohepatitis, cirrhosis, and hepatocellular carcinoma. Despite its enormous burden, no FDA-approved therapies exist, creating urgent demand for novel molecular targets and agents. Autophagy—the cellular recycling process that clears damaged organelles and lipid droplets—is impaired in MAFLD, and restoring it has emerged as a promising therapeutic strategy.
This study investigated acacetin (ACA), a 5,7-dihydroxy-4′-methoxyflavone isolated from Korean mint (Agastache rugosa), in a choline-deficient, amino acid-defined high-fat diet (CDAHFD) mouse model that rapidly recapitulates human MASH pathology. Mice treated with ACA (10 mg/kg i.p. every other day for 4 weeks) showed reduced serum liver enzymes (GOT1/AST down ~35%; GPT/ALT significantly reduced), less Oil Red O-stained hepatic lipid, markedly decreased Masson's trichrome-positive fibrotic area, and lower ADGRE1/F4/80 macrophage infiltration. ACA also elevated VMP1, an early autophagy marker, in liver tissue, and reduced lipid accumulation in 3T3-L1 adipocytes in vitro through autophagy induction confirmed by LC3 flux assays.
To pinpoint ACA's molecular target without chemical labeling, the team combined Drug Affinity Responsive Target Stability (DARTS) with LC-MS/MS proteomics. Among proteins showing ACA-dependent protection from pronase digestion, LAMTOR1—a lysosomal membrane scaffold that anchors the Ragulator/LAMTOR complex—was identified as the top hit. Cellular Thermal Shift Assay (CETSA) and Proximity Ligation Assay (PLA) confirmed direct ACA-LAMTOR1 binding in intact cells. ACA treatment caused LAMTOR1 to dissociate from its LAMTOR complex partners (LAMTOR2–5) and from RRAGA/B GTPases, which are obligatory for recruiting MTORC1 to the lysosomal surface. Consequently, MTORC1 activity (assessed by RPS6KB1/p70S6K and ULK1 phosphorylation) was suppressed, AMPK was activated, and autophagic flux increased—evidenced by LC3-II accumulation, SQSTM1/p62 degradation, tandem mRFP-GFP-LC3 reporter shifts, DQ-BSA lysosomal activity, and TFEB nuclear translocation. LAMTOR1 siRNA knockdown phenocopied all these effects, and ACA showed no additive benefit on top of knockdown, confirming on-target action.
Functionally, autophagy induction by ACA required intact lysosomal function: co-treatment with chloroquine blocked lipid clearance. The MTORC1-AMPK rebalancing also restored metabolic signaling impaired in fatty liver. In the CDAHFD model, ACA treatment increased hepatic LC3-II and reduced lipid droplet burden in a manner consistent with enhanced lipophagy. These data collectively position the LAMTOR1→MTORC1→AMPK→autophagy axis as a coherent therapeutic pathway modulated by ACA.
The study is notable for using label-free target identification (DARTS-LC-MS/MS) to move from phenotype to mechanism, and for genetic validation that strengthens the causal link between LAMTOR1 inhibition and metabolic benefit. LAMTOR1 emerges as a previously underappreciated but tractable target for MAFLD drug development.
Key Findings
- ACA reduced hepatic fibrosis, macrophage infiltration, and liver enzyme levels in CDAHFD-induced MASH mice.
- DARTS-LC-MS/MS identified LAMTOR1 as a direct binding target of ACA on the lysosomal membrane.
- ACA binding disrupts the LAMTOR complex and RRAGA/B interaction, suppressing MTORC1 and activating autophagy.
- LAMTOR1 siRNA knockdown fully phenocopied ACA's autophagy-inducing and lipid-clearing effects.
- ACA activates AMPK and promotes TFEB nuclear translocation, reinforcing lysosomal biogenesis and lipid catabolism.
Methodology
Researchers used a CDAHFD mouse model (4-week i.p. ACA treatment) for in vivo validation, 3T3-L1 adipocytes and HepG2 hepatocytes for in vitro studies, and label-free DARTS coupled with LC-MS/MS proteomics to identify ACA's protein target. Target engagement was confirmed by CETSA, PLA, co-immunoprecipitation, and siRNA knockdown experiments.
Study Limitations
The in vivo work relies on a single CDAHFD mouse model, which does not fully capture the heterogeneity of human MAFLD progression. ACA bioavailability and pharmacokinetics in humans are not characterized in this study. The precise binding site of ACA on LAMTOR1 and the structural basis for complex disruption remain to be elucidated by crystallography or cryo-EM.
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