AMPK regulates lipid accumulation in skeletal muscle cells through FTO-dependent demethylation of N6-methyladenosine - PubMed (original) (raw)
AMPK regulates lipid accumulation in skeletal muscle cells through FTO-dependent demethylation of N6-methyladenosine
Weiche Wu et al. Sci Rep. 2017.
Abstract
Skeletal muscle plays important roles in whole-body energy homeostasis. Excessive skeletal muscle lipid accumulation is associated with some metabolic diseases such as obesity and Type 2 Diabetes. The energy sensor AMPK (AMP-activated protein kinase) is a key regulator of skeletal muscle lipid metabolism, but the precise regulatory mechanism remains to be elucidated. Here, we provide a novel mechanism by which AMPK regulates skeletal muscle lipid accumulation through fat mass and obesity-associated protein (FTO)-dependent demethylation of N6-methyladenosine (m6A). We confirmed an inverse correlation between AMPK and skeletal muscle lipid content. Moreover, inhibition of AMPK enhanced lipid accumulation, while activation of AMPK reduced lipid accumulation in skeletal muscle cells. Notably, we found that mRNA m6A methylation levels were inversely correlated with lipid content in skeletal muscle. Furthermore, AMPK positively regulated the m6A methylation levels of mRNA, which could negatively regulate lipid accumulation in C2C12. At the molecular level, we demonstrated that AMPK regulated lipid accumulation in skeletal muscle cells by regulating FTO expression and FTO-dependent demethylation of m6A. Together, these results provide a novel regulatory mechanism of AMPK on lipid metabolism in skeletal muscle cells and suggest the possibility of controlling skeletal muscle lipid deposition by targeting AMPK or using m6A related drugs.
Conflict of interest statement
The authors declare no competing financial interests.
Figures
Figure 1. The AMPK activity in skeletal muscle is inversely correlated with lipid content.
(A,B) The triglyceride content in SOL was much higher than EDL (A) while the p-AMPKα2 level in SOL was lower than EDL (B). (C,D) The EDL of OB mice contained more triglyceride (C) but a lower p-AMPKα2 level compared to WT mice (D). (E,F) The triglyceride content (E) and the p-AMPKα2 levels (F) in SOL of OB mice (F). The results are presented as the mean ± SEM. n = 4–6. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 2. AMPK negatively regulates lipid accumulation in C2C12 cells.
(A) The p-AMPKα2 level was significantly inhibited by Compound C. (B,C) ORO staining (B) and triglyceride content (C) in C2C12 cells treated withor without Compound C. (D) AICAR upregulates p-AMPKα2 expression in C2C12 cells. (E,F) ORO staining (E) and triglyceride content (F) in C2C12 cells treated withor without AICAR. Data are presented as the mean ± SEM. n = 3. *p < 0.05, **p < 0.01.
Figure 3. The m6A methylation level is inversely correlated with skeletal muscle lipid content.
(A) The m6A level in the EDL and SOL. (B,C) The m6A level in the EDL (B) and SOL (C) of WT and OB mice. (D,E) The m6A level in the EDL (D) and SOL (E) of the ND and HFD group. The results are presented as the mean ± SEM. n = 4–6. **p < 0.01, ***p < 0.001.
Figure 4. Inhibition of m6A methylationin C2C12 cells reduces cellular lipid accumulation.
(A) Cycloleucine treatment decreased the m6A level in C2C12 cells. (B) ORO staining of C2C12 cells treated with vehicle or cycloleucine. (C) Cycloleucine treatment increased cellular triglyceride content. (D,E) Cycloleucine treatment increased the mRNA levels of Cebpα but decreased the expression of pnpla2, Lipe, and Pgc1α (D), as well as the protein levels (E). CL, cycloleucine. The results are presented as the mean ± SEM. n = 3. *p < 0.05, ** p < 0.01. Scale bars: 100 μm.
Figure 5. AMPK affects cellular lipid accumulation by regulating m6A methylation in C2C12.
(A) The mRNA levels of AMPKα2 in C2C12 cells treated with AMPKα2 siRNA (siAMPKα2). (B–D) m6A methylation in C2C12 cells treated with siAMPKα2 (B), CC (C) or AICAR (D). (E) The m6A levelsin C2C12 cells treated with CL, AICAR or their combination. (F,G) ORO staining (F) and TG content (G) in C2C12 cells treated with CL, AICAR or their combination. (H,I) The mRNA and protein levels of the adipogenic and fatty acid lipolysis related genesin C2C12 cells treated with CL, AICAR or their combination. CC, Compound C; CL, cycloleucine. The results are presented as the mean ± SEM. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001. Scale bars: 100 μm.
Figure 6. The expression of FTO is upregulated in the skeletal muscle of OB mice and HFD group mice.
(A,B) The protein levels of FTO, METTL3 and METTL14 (A) and the relative mRNA levels of WTAP and ALKBH5 (B) in the EDL and SOL of WT mice. (C,D) The protein expression of FTO, METTL3 and METTL14 (C) and the mRNA levels of WTAP and ALKBH5 (D) in the SOL of WT and OB mice. (E,F) The protein levels of FTO, METTL3 and METTL14 (E) and the mRNA levels of WTAP and ALKBH5 (F) in the EDL of WT and OB mice. (G,H) The protein expression levels of FTO, METTL3 and METTL14 (G) and the relative expression of WTAP and ALKBH5 (H) in the SOL of the ND and HFD group. (I,J) The protein expression of FTO, METTL3 and METTL14 (I) and the relative expression of WTAP and ALKBH5 (J) in the EDL of the ND and HFD groups. The results are presented as the mean ± SEM. n = 4–6. *p < 0.05, ** p < 0.01, ***p < 0.001.
Figure 7. FTO is essential for m6A methylation and lipid accumulation in skeletal muscle cells.
(A,B) The protein (A) and mRNA (B) levels of FTO in C2C12 cells after knockdown of FTO with siRNA (siFTO). (C) The level of mRNA m6A methylation was increased after FTO knockdown. (D) ORO staining of C2C12 cells after knockdown of FTO. (E) The expression of lipid metabolism related genes after FTO knockdown. The results are presented as the mean ± SEM. n = 3–5. *p < 0.05, ** p < 0.01. Scale bars: 100 μm.
Figure 8. AMPK regulates lipid accumulation and m6A methylation through FTO-dependent demethylation in C2C12 cells.
(A,B) The protein levels of AMPKα2, FTO, METTL3 and METTL14 in C2C12 cells after knockdown of AMPKα2. The relative expression levels of AMPKα2, METTL3, METTL14 and FTO were normalized to GAPDH. (C,D) The protein levels of AMPKα2, p-AMPKα2, FTO, METTL3 and METTL14 in C2C12 cells after AICAR treatment. The relative protein levels of p-AMPKα2, METTL3, METTL14 and FTO were normalized to GAPDH in C2C12 cells after AICAR treatment. (E) The m6A methylation levels after treatment with siAMPKα2, siFTO or their combination. (F) The triglyceride content after treatment with siAMPKα2, siFTO or their combination. (G) A model depicting the role of AMPK in the regulation of lipid accumulation and m6A methylation through FTO-dependent demethylation in skeletal muscle cells. CC, Compound C. Data are presented as the mean ± SEM. n = 3. *p < 0.05, **p < 0.01.
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