Inhibition of lysosomal LAMTOR1 increases autophagy by suppressing the MTORC1 pathway to ameliorate lipid accumulations in MAFLD

Abstract

Metabolic dysfunction-associated fatty liver disease (MAFLD) is a serious metabolic disorder characterized by fat accumulation in the liver, which can trigger liver inflammation and fibrosis, potentially leading to cirrhosis or liver cancer. Despite many studies, effective treatments for MAFLD remain elusive due to its complex etiology. In this study, we have focused on the discovery of therapeutic agents and molecular targets for MAFLD treatment. We demonstrated that the natural compound acacetin (ACA) alleviates MAFLD by regulating macroautophagy/autophagy in a CDAHFD mouse model of rapidly induced steatohepatitis. In addition, ACA inhibits lipid accumulation in 3T3-L1 adipocytes through autophagy induction. To identify the target responsible for the autophagy activity induced by ACA, we performed drug affinity responsive target stability (DARTS) combined with LC-MS/MS proteomic analysis. This led to the identification of LAMTOR1 (late endosomal/lysosomal adaptor, MAPK and MTOR activator 1), a lysosomal membrane adaptor protein. We found that binding of ACA to LAMTOR1 induces its release from the LAMTOR complex, leading to inhibition of MTOR (mechanistic target of rapamycin kinase) complex 1 (MTORC1), thereby increasing autophagy. This process helps ameliorate metabolic disorders by modulating the MTORC1-AMPK axis. Genetic knockdown of LAMTOR1 phenocopies the effects of ACA treatment, further supporting the role of LAMTOR1 as a target of ACA. These findings suggest LAMTOR1 plays a crucial role in ACA's therapeutic effects on MAFLD. In summary, our study identifies LAMTOR1 as a key protein target of ACA, revealing a potential therapeutic avenue for MAFLD by modulating autophagy via the LAMTOR1-MTORC1-AMPK signaling pathway.Abbreviations: ACA: acacetin; ADGRE1/EMR1/F4/80: adhesion G protein-coupled receptor E1; AMPK: AMP-activated protein kinase; CDAHFD: choline-deficient amino acid-defined, high-fat diet; CETSA: cellular thermal shift assay; CQ: chloroquine; DARTS: drug affinity responsive target stability; DQ-BSA: dye quenched-bovine serum albumin; GOT1/AST: glutamic-oxaloacetic transaminase 1; GPT/ALT: glutamic-pyruvic transaminase; LAMP2: lysosomal associated membrane protein 2; LAMTOR1: late endosomal/lysosomal adaptor, MAPK and MTOR activator 1; LC-MS/MS: liquid chromatography-tandem mass spectrometry; MAFLD: metabolic dysfunction-associated fatty liver disease; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MASH: metabolic dysfunction-associated steatohepatitis; mRFP-GFP-MAP1LC3B: tandem fluorescent-tagged MAP1LC3B; MTORC1: mechanistic target of rapamycin complex 1; PA: palmitic acid; PRKAA: protein kinase AMP-activated catalytic subunit alpha; PLA: proximity ligation assay; Rapa: rapamycin; RPS6KB1/p70S6K: ribosomal protein S6 kinase B1; RRAG: Ras-related GTP-binding; SQSTM1: sequestosome 1; TFEB: transcription factor EB; VMP1: vacuole membrane protein 1.

Keywords: Acacetin; DARTS; LAMTOR1; MAFLD; MTORC1; autophagy.

Figure 1.

Acacetin ameliorates pathological liver damage in the CDAHFD-induced MASH mouse model. (A) Overview of the CDAHFD-induced mouse model experiment. (B) Overall scheme of the experiment. (C) External photographs of representative livers from chow-fed mice (n = 4), CDAHFD-fed mice (n = 10), and CDAHFD-fed mice treated with 10 mg/kg ACA (n = 10). Mice were injected intraperitoneally with ACA for 4 weeks. Scale bar: 10 mm. (D) Body weight of the mice. (E) Liver weight of the mice. ns: not significant (F and G) Serum levels of GOT1/AST and GPT/ALT in each group of mice. Values are means ± SEM; n = 10, *p < 0.05, ****p < 0.0001. (H) Representative images of neutral triglycerides and lipids in mouse liver tissues examined by light microscopy after Oil red O staining. Scale bar: 10 µm. (I) Representative images of Masson’s trichrome staining in liver sections from each group. Scale bar: 100 µm. (J) Quantitative data of Masson’s trichrome staining of relative fibrotic area (red arrow: fibrosis, black arrow: vacuolation). Values are means ± SEM; ****p < 0.0001. (K) Representative images of immunostaining for ADGRE1/EMR1 macrophage antigens (red) in liver. Scale bar: 20 µm. (L) Representative images of immunostaining for VMP1 expression (yellow) in mouse liver tissues. Scale bar: 20 µm. (M) Quantitative data of ADGRE1/EMR1 expression. Values are means ± SEM; **p < 0.01, ***p < 0.001. (N) Quantitative data of VMP1 expression. Values are means ± SEM; **p < 0.01.

Figure 2.

Acacetin inhibits lipid accumulation in an autophagy-dependent manner. (A) Effect of ACA on cell proliferation using MTT assay in 3T3-L1 cells. (B) Effect of ACA on cell cytotoxicity using trypan blue assay in 3T3-L1 cells. Values are means ± SEM; n > 30 cells. *p < 0.05, ns: not significant. (C) Effect of ACA on cell proliferation using MTT assay in HepG2 cells. (D) Effect of ACA on cell cytotoxicity using trypan blue assay in HepG2 cells. Values are means ± SEM; n > 30 cells. *p < 0.05, **p < 0.01, ***p < 0.001, ns: not significant. (E) Representative image of Oil red O staining. 3T3-L1 cells were differentiated for 8 days and treated with ACA (50 μM) every 2 days from day 4 to day 8. Cells were stained with Oil red O and examined microscopically. Scale bar: 50 µm. (F) Oil red O dye was extracted, and the extracted dye solution was measured for absorbance at 490 nm. Values are means ± SEM; ****p < 0.0001. (G) 3T3-L1 cells were incubated in DMEM containing DQ-BSA (10 μg/mL) for 2 h. The cells were then washed with PBS, treated with Rapa (10 μM), bafilomycin A₁ (10 nM) or ACA (50 μM) for 6 h, and fixed, and the fluorescence of DQ-BSA (red) was imaged using a confocal microscope. Scale bar: 20 µm. (H) Quantitative data of lysosomal puncta of DQ-BSA. Values are means ± SEM; ****p < 0.0001. (I) Representative images of autophagic flux evaluation using mRFP-GFP-MAP1LC3B/LC3 in the presence of each compound. HepG2 cells were treated with Rapa (10 μM), bafilomycin A₁ (10 nM) or ACA (50 μM) for 24 h. Scale bar: 10 µm. (J) Quantitative data of mRFP-GFP-MAP1LC3B/LC3. Values are means ± SEM; n > 10 cells, *p < 0.05, ****p < 0.0001. (K) Measurement of time-dependent expression of autophagy markers (LC3 and SQSTM1) using western blot. The differentiated 3T3-L1 cells were treated with Rapa (10 μM) or ACA (30 μM or 50 μM) for 24 h, 48 h, or 72 h. (L) The differentiated 3T3-L1 cells were co-treated with ACA (50 μM) for 24 h in the absence or presence of the autophagy inhibitor CQ (10 μM). (M) The differentiated 3T3-L1 cells were treated with ACA (50 μM) in the absence or presence of the autophagy inhibitor CQ (10 μM). Cells were subjected to immunostaining with an anti-LC3 antibody (red) and stained with BODIPY 493/503 (green) to visualize lipid droplets. Confocal microscopy was used to examine the colocalization of LC3 and BODIPY 493/503 (yellow). Scale bar: 20 µm. (N) Quantitative data of lipid droplet intensity. Values are means ± SEM; **p < 0.01. (O) HeLa WT cells were treated with ACA (50 μM) for 24 h, followed by staining with BODIPY 493/503 (green) to visualize lipid droplets. Lipid droplets were analyzed using confocal microscopy. Scale bar: 20 µm. (P) Quantitative data of lipid droplet intensity. Values are means ± SEM; ****p < 0.0001. (Q) HeLa ATG3 KO cells were treated with ACA (50 μM) for 24 h, followed by staining with BODIPY 493/503 (green) to visualize lipid droplets. Lipid droplets were analyzed using confocal microscopy. Scale bar: 20 µm. (R) Quantitative data of lipid droplet intensity. Values are means ± SEM; ns: not significant.

Figure 3.

Identification of the pharmacological target of acacetin by DARTS LC-MS/MS analysis. (A) Flowchart of DARTS LC-MS/MS to identify the protein target of ACA. First, pronase digestion was performed and DARTS analysis was used to identify the target proteins of ACA in the cell lysate proteome pool. Second, DARTS analysis was used to stabilize target proteins bound to ACA by structural conformational change, and proteins that became resistant to pronase degradation were analyzed and identified by LC-MS/MS. Finally, LAMTOR1 was selected from the 18 protein candidates with pronase resistance, an increase in binding stability of more than 15%, and reasonable sequence coverage. (B) Functional enrichment heatmap analysis of the 18 candidate proteins. (C) STRING analysis of protein candidates selected by DARTS LC-MS/MS analysis, which is a label-free method. (D) Pronase-dependent DARTS analysis was performed for target validation. 3T3-L1 cell lysate was treated with vehicle control or ACA (50 μM). Compound binding was measured for 3 h at 4°C, followed by treatment with 1 μg/mL or 2.5 μg/mL for 10 min. (E) Quantitative data of the pronase-dependent DARTS analysis. (F) Dose-dependent DARTS analysis was conducted for target validation. 3T3-L1 cell lysate was treated with vehicle control or ACA at concentrations ranging from 10 μM to 200 μM. Binding of the compound was performed at 4°C for 3 h, followed by pronase treatment with 1 μg/mL for 10 min. (G) Quantitative data of the dose-dependent DARTS analysis.

Figure 4.

Analysis of the binding site of acacetin to LAMTOR1 by in silico docking. (A) in silico docking analysis of ACA directly interacting with LAMTOR1. (B) Two-dimensional diagram of the binding interaction and predicted amino acid binding sites of LAMTOR1 with ACA. (C) HEK293 cells were transfected with WT MYC-LAMTOR1, MYC-LAMTOR1^L22A, MYC-LAMTOR1^D24G, or MYC-LAMTOR1^D49A vectors for 48 h. Cell lysate was treated with vehicle control or ACA (50 μM), and compound binding was assessed at 4°C for 3 h, followed by pronase treatment with 1 μg/mL for 10 min. (D) Quantitative data of the point mutation DARTS analysis. Values are means ± SEM; *p < 0.05, **p < 0.01, ns: not significant. (E) HEK293 cells were transfected with WT MYC-LAMTOR1, MYC-LAMTOR1^L22A, MYC-LAMTOR1^D24G, or MYC-LAMTOR1^D49A vectors for 48 h, treated with vehicle control or ACA (50 μM), fixed, and immunostained with MYC (red) and TFEB (green). Images were acquired by confocal microscopy. Scale bar: 20 µm. (F) HEK293 cells were transfected with WT MYC-LAMTOR1, MYC-LAMTOR1^L22A, MYC-LAMTOR1^D24G, or MYC-LAMTOR1^D49A vectors for 48 h, treated with vehicle control or ACA (50 μM), fixed, and immunostained with MYC (red) and LC3 (green); Images were acquired by confocal microscopy. Scale bar: 20 µm. (G) Quantification of relative nuclear TFEB intensity per cell. Values are means ± SEM; **p < 0.01, ****p < 0.0001, ns: not significant. (H) Quantification of the number of LC3 puncta per cell. Values are means ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5.

Acacetin induces TFEB nuclear translocation by modulating the MTORC1/AMPK axis. (A) Schematic illustration of the regulation of MTORC1 by the LAMTOR complex. (B) 3T3-L1 cells were treated with vehicle control or ACA (50 μM) for 4 h. PLA analysis was performed to assess the interaction between MTOR and LAMTOR1. Scale bar: 20 µm. (C) Quantitative data of the PLA analysis representing the interaction between MTOR and LAMTOR1. Values are means ± SEM; n > 30 cells. ****p < 0.0001. (D) The differentiated 3T3-L1 cells were treated with vehicle control or ACA (50 μM) for 4 h. Immunostaining was performed for MTOR (red) and LAMP2 (green), followed by confocal microscopy. Scale bar: 10 µm. (E) Manders’ coefficient values for the colocalization of MTOR (red) and LAMP2 (green) in 3T3-L1 cells [M1: LAMP2 (green) overlapping with MTOR (red), M2: MTOR (red) overlapping with LAMP2 (green)]. Values are means ± SEM; **p < 0.01. (F) The differentiated 3T3-L1 cells were treated with vehicle control or ACA (50 μM) for 24 h. The protein expression levels of p-MTOR and MTOR were analyzed by western blot analysis. (G) The differentiated 3T3-L1 cells were treated with vehicle control or ACA (30 μM, 50 μM) in the absence or presence of the AMPK inhibitor compound C (20 μM) for 24 h. The protein expression levels of p-PRKAA/AMPK and AMPK were analyzed by western blot analysis. (H) 3T3-L1 cells were treated with vehicle control or ACA (50 μM) for 3 h, and nuclear fractionation was followed by western blot analysis.

Figure 6.

Acacetin restores autophagy in the MASH mouse model. (A) PLA analysis of MTOR and LAMTOR1 in liver tissues of mice treated with vehicle or ACA (10 mg/kg). Scale bar: 20 µm. (B) Representative images of immunostaining for p-PRKAA/AMPK expression (green) in mouse liver tissues. Scale bar: 50 µm. (C) Quantitative data of p-PRKAA/AMPK expression. Values are means ± SEM; ***p < 0.001. (D) Representative images of TFEB (green) immunostaining from the cytosol to the nucleus in liver tissues of mice treated with vehicle or ACA (10 mg/kg). Arrows indicate the translocation of TFEB from the cytosol to the nucleus. Scale bar: 20 µm. (E) Representative images of immunostaining for SQSTM1 expression (red) in mouse liver tissues. Scale bar: 20 µm. (F) Quantitative data of SQSTM1 expression. Values are means ± SEM; ****p < 0.0001. (G) Representative images of immunostaining for LC3 (red) and BODIPY 493/503 (green). Confocal images were examined for colocalization of LC3 and BODIPY 493/503 (yellow). Scale bar: 20 µm.

Figure 7.

LAMTOR1 inhibition contributes to lipid reduction via autophagy and is as an important factor in MAFLD and MASH patients. (A) 3T3-L1 cells were either untreated or transfected with Lamtor1-targeting shRNA for 48 h. Protein levels of p-mtor, mtor, p-RPS6KB, and RPS6KB were analyzed by western blot analysis. (B) 3T3-L1 cells were either untreated or transfected with Lamtor1-targeting shRNA for 48 h, followed by nuclear fractionation and western blot analysis. (C) 3T3-L1 cells were either untreated or transfected with Lamtor1-targeting shRNA for 48 h. LysoTracker staining (red) was performed and confirmed by confocal microscopy. Scale bar: 20 µm. (D) Quantitative data of lysosomal fluorescence intensity from LysoTracker per cell. Values are means ± SEM; n > 30 cells. ****p < 0.0001. (E) Representative images of autophagic flux evaluation using mRFP-GFP-MAP1LC3B/LC3 in the presence or absence of LAMTOR1-targeting shRNA transfection for 48 h in HepG2 cells. Scale bar: 10 µm. (F) Quantitative data of mRFP-GFP-MAP1LC3B/LC3. Values are means ± SEM; n > 10 cells, **p < 0.01, ****p < 0.0001. (G) Representative image of Oil red O staining. 3T3-L1 cells were either untreated or transfected with LAMTOR1-targeting shRNA for 48 h, followed by differentiation for 8 days. Scale bar: 2 µm. (H) RNA sequencing analysis of LAMTOR1 expression in MAFLD and MASH patient cohorts from the Gene Expression Omnibus (GEO: GSE126848). Values are means ± SEM; *p < 0.05, ***p < 0.001. (I and J) Significant positive correlations between LAMTOR1 and MAFLD-related factors (SREBF2, FABP1) in the GEO dataset (GSE126848). (K) Summary of the study on LAMTOR1-mediated autophagy regulation by ACA treatment: In metabolic dysfunction-associated fatty liver disease, abnormal autophagy operation causes autophagy dysfunction. ACA binds to the target protein LAMTOR1 and activates autophagy through the inhibition of LAMTOR1. ACA induces the translocation of TFEB to the nucleus by inhibiting MTORC1, that ameliorates autophagy dysfunction in metabolic disease.