m6A mRNA methylation controls autophagy and adipogenesis by targeting Atg5 and Atg7

Abstract

N: ⁶-methyladenosine (m⁶A), the most abundant internal modification on mRNAs in eukaryotes, play roles in adipogenesis. However, the underlying mechanism remains largely unclear. Here, we show that m⁶A plays a critical role in regulating macroautophagy/autophagy and adipogenesis through targeting Atg5 and Atg7. Mechanistically, knockdown of FTO, a well-known m⁶A demethylase, decreased the expression of ATG5 and ATG7, leading to attenuation of autophagosome formation, thereby inhibiting autophagy and adipogenesis. We proved that FTO directly targeted Atg5 and Atg7 transcripts and mediated their expression in an m⁶A-dependent manner. Further study identified that Atg5 and Atg7 were the targets of YTHDF2 (YTH N6-methyladenosine RNA binding protein 2). Upon FTO silencing, Atg5 and Atg7 transcripts with higher m⁶A levels were captured by YTHDF2, which resulted in mRNA degradation and reduction of protein expression, thus alleviating autophagy and adipogenesis. Furthermore, we generated an adipose-selective fto knockout mouse and find that FTO deficiency decreased white fat mass and impairs ATG5- and ATG7-dependent autophagy in vivo. Together, these findings unveil the functional importance of the m⁶A methylation machinery in autophagy and adipogenesis regulation, which expands our understanding of such interplay that is essential for development of therapeutic strategies in the prevention and treatment of obesity.

Abbreviations: 3-MA: 3-methyladenine; ACTB: actin, beta; ATG: autophagy-related; Baf A1: bafilomycin A₁; CEBPA: CCAAT/enhancer binding protein (C/EBP), alpha; CEBPB: CCAAT/enhancer binding protein (C/EBP), beta; FABP4: fatty acid binding protein 4, adipocyte; FTO: fat mass and obesity associated; HFD: high-fat diet; LC-MS/MS: liquid chromatography-tandem mass spectrometry; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; m⁶A: N⁶-methyladenosine; MEFs: mouse embryo fibroblasts; MeRIP-qPCR: methylated RNA immunoprecipitation-qPCR; PPARG: peroxisome proliferator activated receptor gamma; RIP: RNA-immunoprecipitation; SAT: subcutaneous adipose tissue; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; ULK1: unc-51 like kinase 1; VAT: visceral adipose tissue; WAT: white adipose tissue; YTHDF: YTH N6-methyladenosine RNA binding protein.

Keywords: ATG5; ATG7; Adipogenesis; FTO; autophagy; m6A.

Figure 1.

FTO promotes adipogenesis via facilitating autophagy in 3T3-L1 preadipocytes. (A) Western blotting analysis of FTO, LC3 and SQSTM1 in 3T3-L1 preadipocytes with or without FTO knockdown, control or overexpressing FTO. ACTB was used as loading control. (B) Immunofluorescence assays of LC3 and HA in control and FTO-overexpressing cells. Scale bar: 20 μm. (C) Statistical analysis of the number of LC3 puncta per cell. (D) TEM analysis of autophagosomes in control, Fto-depleted or FTO-overexpressing cells. Arrows indicate autophagosomes. Scale bar: 0.5 μm. (E) Quantification of autophagosomes in cells. (F) Western blotting analysis of LC3 in control and FTO-overexpressing cells treated with or without 10 mM 3-MA for 4 h. (G) Western blotting analysis of LC3 in control and FTO-overexpressing cells treated with or without 10 nM Baf A1 for 4 h. (H) Oil Red O staining of control and FTO-overexpressing cells induced to differentiate in the presence or absence of 5 mM 3-MA or 4 nM Baf A1 for 8 d. Scale bar: 100 μm. (I) Relative lipid accumulation was quantified with a microplate spectrophotometer at 500 nm. (J) Relative triglyceride accumulation was measured using a triglyceride assay kit. (K) Real-time quantitative PCR (qPCR) analysis of Pparg, Fabp4 and Cebpa expression in control and FTO-overexpressing cells induced to differentiate in the presence or absence of 5 mM 3-MA or 4 nM Baf A1 for 8 d. Actb was used as an internal control. (L) Western blotting analysis of PPARG, FABP4 and CEBPA expression in control and FTO-overexpressing cells induced to differentiate in the presence or absence of 5 mM 3-MA or 4 nM Baf A1 for 8 d. The data were presented as the mean ± SD of triplicate tests. ***P < 0.001 compared to control group.

Figure 1.

FTO promotes adipogenesis via facilitating autophagy in 3T3-L1 preadipocytes. (A) Western blotting analysis of FTO, LC3 and SQSTM1 in 3T3-L1 preadipocytes with or without FTO knockdown, control or overexpressing FTO. ACTB was used as loading control. (B) Immunofluorescence assays of LC3 and HA in control and FTO-overexpressing cells. Scale bar: 20 μm. (C) Statistical analysis of the number of LC3 puncta per cell. (D) TEM analysis of autophagosomes in control, Fto-depleted or FTO-overexpressing cells. Arrows indicate autophagosomes. Scale bar: 0.5 μm. (E) Quantification of autophagosomes in cells. (F) Western blotting analysis of LC3 in control and FTO-overexpressing cells treated with or without 10 mM 3-MA for 4 h. (G) Western blotting analysis of LC3 in control and FTO-overexpressing cells treated with or without 10 nM Baf A1 for 4 h. (H) Oil Red O staining of control and FTO-overexpressing cells induced to differentiate in the presence or absence of 5 mM 3-MA or 4 nM Baf A1 for 8 d. Scale bar: 100 μm. (I) Relative lipid accumulation was quantified with a microplate spectrophotometer at 500 nm. (J) Relative triglyceride accumulation was measured using a triglyceride assay kit. (K) Real-time quantitative PCR (qPCR) analysis of Pparg, Fabp4 and Cebpa expression in control and FTO-overexpressing cells induced to differentiate in the presence or absence of 5 mM 3-MA or 4 nM Baf A1 for 8 d. Actb was used as an internal control. (L) Western blotting analysis of PPARG, FABP4 and CEBPA expression in control and FTO-overexpressing cells induced to differentiate in the presence or absence of 5 mM 3-MA or 4 nM Baf A1 for 8 d. The data were presented as the mean ± SD of triplicate tests. ***P < 0.001 compared to control group.

Figure 2.

Loss of FTO attenuates the expression of ATG5 and ATG7, rather than ULK1. (A) qPCR analysis of expression levels of ATG (autophagy-related) proteins in control and FTO knockdown cells. Actb was used as an internal control. (B) Western blotting analysis of FTO, ATG5, ATG7, ULK1, ATG12 and ATG16L1 in control and FTO knockdown cells. ACTB was used as a loading control. (C) Western blotting analysis of ATG5 and ATG7 expression profiles during adipogenesis. (D) Western blotting analysis of ATG5, FTO and LC3 in control and ATG5 knockdown cells. (E) Western blotting analysis of ATG7, FTO and LC3 in control and ATG7 knockdown cells. (F) Oil Red O staining of control, FTO-depleted, ATG5-depleted and ATG7-depleted cells on day 6 of differentiation. Scale bar: 100 μm. (G) Relative lipid accumulation was quantified with a microplate spectrophotometer at 500 nm. (H) Relative triglyceride accumulation was measured using a triglyceride assay kit. (I) qPCR analysis of Pparg, Fabp4 and Cebpa expression in control, FTO-depleted, ATG5-depleted and ATG7-depleted cells on day 6 of differentiation. Actb was used as an internal control. (J) Western blotting analysis of PPARG, FABP4 and CEBPA expression in control, FTO-depleted, ATG5-depleted and ATG7-depleted cells on day 6 of differentiation. The data were presented as the mean ± SD of triplicate tests. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control group.

Figure 2.

Loss of FTO attenuates the expression of ATG5 and ATG7, rather than ULK1. (A) qPCR analysis of expression levels of ATG (autophagy-related) proteins in control and FTO knockdown cells. Actb was used as an internal control. (B) Western blotting analysis of FTO, ATG5, ATG7, ULK1, ATG12 and ATG16L1 in control and FTO knockdown cells. ACTB was used as a loading control. (C) Western blotting analysis of ATG5 and ATG7 expression profiles during adipogenesis. (D) Western blotting analysis of ATG5, FTO and LC3 in control and ATG5 knockdown cells. (E) Western blotting analysis of ATG7, FTO and LC3 in control and ATG7 knockdown cells. (F) Oil Red O staining of control, FTO-depleted, ATG5-depleted and ATG7-depleted cells on day 6 of differentiation. Scale bar: 100 μm. (G) Relative lipid accumulation was quantified with a microplate spectrophotometer at 500 nm. (H) Relative triglyceride accumulation was measured using a triglyceride assay kit. (I) qPCR analysis of Pparg, Fabp4 and Cebpa expression in control, FTO-depleted, ATG5-depleted and ATG7-depleted cells on day 6 of differentiation. Actb was used as an internal control. (J) Western blotting analysis of PPARG, FABP4 and CEBPA expression in control, FTO-depleted, ATG5-depleted and ATG7-depleted cells on day 6 of differentiation. The data were presented as the mean ± SD of triplicate tests. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control group.

Figure 3.

FTO regulates autophagy and adipogenesis through targeting Atg5 and Atg7. (A) qPCR analysis of Atg5 and Atg7 expression in control and FTO-overexpressing cells. Actb was used as an internal control. (B) Western blotting analysis of FTO, ATG5 and LC3 in control and FTO-overexpressing cells transfected with or without Atg5 siRNA. ACTB was used as a loading control. (C) Western blotting analysis of FTO, ATG7 and LC3 in control and FTO-overexpressing cells transfected with or without Atg7 siRNA. (D) Western blotting analysis of ATG5, ATG7 and CEBPB in control and FTO-overexpressing cells transfected with or without Atg5 and Atg7 siRNA. (E) Oil Red O staining of control and FTO-overexpressing cells transfected with or without Atg5 and Atg7 siRNA on day 6 of differentiation. Scale bar: 100 μm. (F) Relative lipid accumulation was quantified with a microplate spectrophotometer at 500 nm. (G) Relative triglyceride accumulation was measured using a triglyceride assay kit. (H) qPCR analysis of Pparg, Fabp4 and Cebpa expression in control and FTO-overexpressing cells transfected with or without Atg5 and Atg7 siRNA on day 6 of differentiation. (I) Western blotting analysis of PPARG, FABP4 and CEBPA expression in control and FTO-overexpressing cells transfected with or without Atg5 and Atg7 siRNA on day 6 of differentiation. The data were presented as the mean ± SD of triplicate tests. ***P < 0.001 compared to control group.

Figure 3.

FTO regulates autophagy and adipogenesis through targeting Atg5 and Atg7. (A) qPCR analysis of Atg5 and Atg7 expression in control and FTO-overexpressing cells. Actb was used as an internal control. (B) Western blotting analysis of FTO, ATG5 and LC3 in control and FTO-overexpressing cells transfected with or without Atg5 siRNA. ACTB was used as a loading control. (C) Western blotting analysis of FTO, ATG7 and LC3 in control and FTO-overexpressing cells transfected with or without Atg7 siRNA. (D) Western blotting analysis of ATG5, ATG7 and CEBPB in control and FTO-overexpressing cells transfected with or without Atg5 and Atg7 siRNA. (E) Oil Red O staining of control and FTO-overexpressing cells transfected with or without Atg5 and Atg7 siRNA on day 6 of differentiation. Scale bar: 100 μm. (F) Relative lipid accumulation was quantified with a microplate spectrophotometer at 500 nm. (G) Relative triglyceride accumulation was measured using a triglyceride assay kit. (H) qPCR analysis of Pparg, Fabp4 and Cebpa expression in control and FTO-overexpressing cells transfected with or without Atg5 and Atg7 siRNA on day 6 of differentiation. (I) Western blotting analysis of PPARG, FABP4 and CEBPA expression in control and FTO-overexpressing cells transfected with or without Atg5 and Atg7 siRNA on day 6 of differentiation. The data were presented as the mean ± SD of triplicate tests. ***P < 0.001 compared to control group.

Figure 4.

FTO regulates the expression of ATG5 and ATG7 in an m⁶A-dependent manner. (A) LC-MS/MS quantification of the m⁶A/A in mRNA of control, wild-type (WT)- and mutant (MUT)-FTO overexpressing cells. (B) Western blotting analysis of FTO and LC3 in control, WT- and MUT-FTO overexpressing cells. ACTB was used as a loading control. (C) Immunofluorescence assays of LC3 and HA in control, WT- and MUT-FTO overexpressing cells. Scale bar: 20 μm. (D) Statistical analysis of the number of LC3 puncta per cell. (E) TEM analysis of autophagosomes in control, WT- and MUT-FTO overexpressing cells. Arrows indicate autophagosomes. Scale bar: 0.5 μm. (F) Quantification of autophagosomes in cells. (G) Western blotting analysis of ATG5, ATG7 and ULK1 in control, WT- and MUT-FTO overexpressing cells. (H) qPCR analysis of Atg5 and Atg7 expression in control, WT- and MUT-FTO overexpressing cells. Actb was used as an internal control. (I) Integrative genomics viewer (IGV) plots of m⁶A peaks at Atg5 and Atg7 mRNAs. The y-axis shows sequence read number, blue boxes represent exons, and blue lines represent introns. (J) LC-MS/MS quantification of the m⁶A/A in mRNA of control and Fto-depleted cells. (K) Methylated RNA immunoprecipitation (MeRIP)-qPCR analysis of m⁶A levels of Atg5, Atg7 and Ulk1 mRNA in control and Fto-depleted cells. (L) RNA immunoprecipitation-qPCR (RIP-qPCR) analysis of the interaction of Atg5 and Atg7 with HA in cells overexpressing HA-tagged FTO. Enrichment of Atg5 and Atg7 with HA was measured by qPCR and normalized to input. (M) Left panel: Schematic diagram of dual-luciferase reporter constructs. Right panel: Relative luciferase activity of WT or MUT (A-to-T mutation) Atg5-3ʹUTR (or Atg7-3ʹUTR) luciferase reporter in control, WT- and MUT-FTO overexpressing cells. Firefly luciferase activity was measured and normalized to Renilla luciferase activity. (N) Oil Red O staining of control, WT- and MUT-FTO overexpressing cells on day 8 of differentiation. Scale bar: 100 μm. (O) Relative lipid accumulation was quantified with a microplate spectrophotometer at 500 nm. (P) Relative triglyceride accumulation was measured using a triglyceride assay kit. The data were presented as the mean ± SD of triplicate tests. **P < 0.01, ***P < 0.001 compared to control group.

Figure 4.

FTO regulates the expression of ATG5 and ATG7 in an m⁶A-dependent manner. (A) LC-MS/MS quantification of the m⁶A/A in mRNA of control, wild-type (WT)- and mutant (MUT)-FTO overexpressing cells. (B) Western blotting analysis of FTO and LC3 in control, WT- and MUT-FTO overexpressing cells. ACTB was used as a loading control. (C) Immunofluorescence assays of LC3 and HA in control, WT- and MUT-FTO overexpressing cells. Scale bar: 20 μm. (D) Statistical analysis of the number of LC3 puncta per cell. (E) TEM analysis of autophagosomes in control, WT- and MUT-FTO overexpressing cells. Arrows indicate autophagosomes. Scale bar: 0.5 μm. (F) Quantification of autophagosomes in cells. (G) Western blotting analysis of ATG5, ATG7 and ULK1 in control, WT- and MUT-FTO overexpressing cells. (H) qPCR analysis of Atg5 and Atg7 expression in control, WT- and MUT-FTO overexpressing cells. Actb was used as an internal control. (I) Integrative genomics viewer (IGV) plots of m⁶A peaks at Atg5 and Atg7 mRNAs. The y-axis shows sequence read number, blue boxes represent exons, and blue lines represent introns. (J) LC-MS/MS quantification of the m⁶A/A in mRNA of control and Fto-depleted cells. (K) Methylated RNA immunoprecipitation (MeRIP)-qPCR analysis of m⁶A levels of Atg5, Atg7 and Ulk1 mRNA in control and Fto-depleted cells. (L) RNA immunoprecipitation-qPCR (RIP-qPCR) analysis of the interaction of Atg5 and Atg7 with HA in cells overexpressing HA-tagged FTO. Enrichment of Atg5 and Atg7 with HA was measured by qPCR and normalized to input. (M) Left panel: Schematic diagram of dual-luciferase reporter constructs. Right panel: Relative luciferase activity of WT or MUT (A-to-T mutation) Atg5-3ʹUTR (or Atg7-3ʹUTR) luciferase reporter in control, WT- and MUT-FTO overexpressing cells. Firefly luciferase activity was measured and normalized to Renilla luciferase activity. (N) Oil Red O staining of control, WT- and MUT-FTO overexpressing cells on day 8 of differentiation. Scale bar: 100 μm. (O) Relative lipid accumulation was quantified with a microplate spectrophotometer at 500 nm. (P) Relative triglyceride accumulation was measured using a triglyceride assay kit. The data were presented as the mean ± SD of triplicate tests. **P < 0.01, ***P < 0.001 compared to control group.

Figure 5.

FTO modulates ATG5 and ATG7 expression levels by YTHDF2-mediated mRNA decay. (A) Western blotting analysis of YTHDF2, ATG5, ATG7, ULK1 and ATG12 in control and YTHDF2-overexpressed cells. ACTB was used as a loading control. (B) Western blotting analysis of YTHDF1, ATG5 and ATG7 in control and YTHDF1-overexpressed cells. (C) RIP-qPCR analysis of the interaction of Atg5 and Atg7 with FLAG in cells overexpressing FLAG-YTHDF2. Enrichment of Atg5 and Atg7 with FLAG was measured by qPCR and normalized to input. (D) Relative luciferase activity of WT or MUT Atg5-3ʹUTR (or Atg7-3ʹUTR) luciferase reporter in cells transfected with control or YTHDF2 plasmid. Firefly luciferase activity was measured and normalized to Renilla luciferase activity. (E) qPCR analysis of Ythdf2, Atg5, Atg7 and Ulk1 in control and FTO knockdown cells. Actb was used as internal control. (F) Lifetime of Atg5 and Atg7 mRNA in control, FTO knockdown or YTHDF2 knockdown cells. Relative mRNA levels were quantified by qPCR. (G) Western blotting analysis of YTHDF2, ATG5, ATG7 and LC3 in control and FTO-overexpressing cells transfected with or without YTHDF2 plasmid. (H) Oil Red O staining of control and FTO-overexpressing cells transfected with or without YTHDF2 plasmid on day 8 of differentiation. Scale bar: 100 μm. (I) Relative lipid accumulation was quantified with a microplate spectrophotometer at 500 nm. (J) Relative triglyceride accumulation was measured using a triglyceride assay kit. The data were presented as the mean ± SD of triplicate tests. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control group. ^###P < 0.001 compared to OE-FTO group.

Figure 5.

FTO modulates ATG5 and ATG7 expression levels by YTHDF2-mediated mRNA decay. (A) Western blotting analysis of YTHDF2, ATG5, ATG7, ULK1 and ATG12 in control and YTHDF2-overexpressed cells. ACTB was used as a loading control. (B) Western blotting analysis of YTHDF1, ATG5 and ATG7 in control and YTHDF1-overexpressed cells. (C) RIP-qPCR analysis of the interaction of Atg5 and Atg7 with FLAG in cells overexpressing FLAG-YTHDF2. Enrichment of Atg5 and Atg7 with FLAG was measured by qPCR and normalized to input. (D) Relative luciferase activity of WT or MUT Atg5-3ʹUTR (or Atg7-3ʹUTR) luciferase reporter in cells transfected with control or YTHDF2 plasmid. Firefly luciferase activity was measured and normalized to Renilla luciferase activity. (E) qPCR analysis of Ythdf2, Atg5, Atg7 and Ulk1 in control and FTO knockdown cells. Actb was used as internal control. (F) Lifetime of Atg5 and Atg7 mRNA in control, FTO knockdown or YTHDF2 knockdown cells. Relative mRNA levels were quantified by qPCR. (G) Western blotting analysis of YTHDF2, ATG5, ATG7 and LC3 in control and FTO-overexpressing cells transfected with or without YTHDF2 plasmid. (H) Oil Red O staining of control and FTO-overexpressing cells transfected with or without YTHDF2 plasmid on day 8 of differentiation. Scale bar: 100 μm. (I) Relative lipid accumulation was quantified with a microplate spectrophotometer at 500 nm. (J) Relative triglyceride accumulation was measured using a triglyceride assay kit. The data were presented as the mean ± SD of triplicate tests. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control group. ^###P < 0.001 compared to OE-FTO group.

Figure 6.

Adipose-specific fto knockout mice exhibit deceased fat mass and inactivated ATG5 and ATG7-dependent autophagy during high-fat diet feeding. (A) Western blotting analysis of FTO in white adipose tissue (WAT) from control (Fto^flox/flox) and adipose-specific fto knockout (Fabp4-Cre Fto^flox/flox, fto-AKO) mice. ACTB was used as a loading control. (B) Body weights of control (female, n = 10) and fto-AKO (female, n = 8) mice on high-fat diet (HFD). (C) Food intake of control and fto-AKO mice during HFD feeding. (D) Representative pictures of control and fto-AKO mice. (E) Representative pictures of subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) from control and fto-AKO mice. (F) Weights of SAT and VAT from control and fto-AKO mice. (G) Representative hematoxylin and eosin–stained sections of SAT and VAT isolated from control and fto-AKO mice. Scale bars: 100 μm. (H) Adipocyte area size quantification for SAT and VAT isolated from control and fto-AKO mice. (I) Blood glucose levels of control and fto-AKO mice. (J) Serum triglyceride levels of control and fto-AKO mice. (K) Western blotting analysis of LC3, SQSTM1, ATG5 and ATG7 in WAT isolated from control and fto-AKO mice. (L) qPCR analysis of Atg5, Atg7 and Cebpb in WAT isolated from control and fto-AKO mice. Actb was used as an internal control. The data were presented as the mean ± SD of triplicate tests. **P < 0.01, ***P < 0.001 compared to control group.

Figure 6.

Adipose-specific fto knockout mice exhibit deceased fat mass and inactivated ATG5 and ATG7-dependent autophagy during high-fat diet feeding. (A) Western blotting analysis of FTO in white adipose tissue (WAT) from control (Fto^flox/flox) and adipose-specific fto knockout (Fabp4-Cre Fto^flox/flox, fto-AKO) mice. ACTB was used as a loading control. (B) Body weights of control (female, n = 10) and fto-AKO (female, n = 8) mice on high-fat diet (HFD). (C) Food intake of control and fto-AKO mice during HFD feeding. (D) Representative pictures of control and fto-AKO mice. (E) Representative pictures of subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) from control and fto-AKO mice. (F) Weights of SAT and VAT from control and fto-AKO mice. (G) Representative hematoxylin and eosin–stained sections of SAT and VAT isolated from control and fto-AKO mice. Scale bars: 100 μm. (H) Adipocyte area size quantification for SAT and VAT isolated from control and fto-AKO mice. (I) Blood glucose levels of control and fto-AKO mice. (J) Serum triglyceride levels of control and fto-AKO mice. (K) Western blotting analysis of LC3, SQSTM1, ATG5 and ATG7 in WAT isolated from control and fto-AKO mice. (L) qPCR analysis of Atg5, Atg7 and Cebpb in WAT isolated from control and fto-AKO mice. Actb was used as an internal control. The data were presented as the mean ± SD of triplicate tests. **P < 0.01, ***P < 0.001 compared to control group.

Figure 7.

Working model of the mechanism of FTO regulates autophagy and adipogenesis in an m⁶A-dependent manner. In this model, FTO demethylases the m⁶A modification of Atg5 and Atg7 mRNAs, which in turn prevents YTHDF2-mediated mRNA decay, thereby increasing their mRNA stability and protein expression, leading to promoting autophagy and adipogenesis. Loss of FTO increases the m⁶A levels of Atg5 and Atg7 mRNAs, which are specifically recognized and destabilized by YTHDF2, resulting in the decreased protein expression, thereby inhibiting autophagy and adipogenesis.

Figure 7.

Working model of the mechanism of FTO regulates autophagy and adipogenesis in an m⁶A-dependent manner. In this model, FTO demethylases the m⁶A modification of Atg5 and Atg7 mRNAs, which in turn prevents YTHDF2-mediated mRNA decay, thereby increasing their mRNA stability and protein expression, leading to promoting autophagy and adipogenesis. Loss of FTO increases the m⁶A levels of Atg5 and Atg7 mRNAs, which are specifically recognized and destabilized by YTHDF2, resulting in the decreased protein expression, thereby inhibiting autophagy and adipogenesis.