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PNAS 110 (7): 2557-2562

Copyright © 2013 by the National Academy of Sciences.

Role for the obesity-related FTO gene in the cellular sensing of amino acids

Pawan Gulatia,b,1, Man Ka Cheunga,b, Robin Antrobusc, Chris D. Churchd, Heather P. Hardinga,b, Yi-Chun Loraine Tunga,b, Debra Rimmingtona,b, Marcella Maa,b, David Rona,b, Paul J. Lehnerc, Frances M. Ashcrofte, Roger D. Coxd, Anthony P. Colla,b, Stephen O’Rahillya,b,1, and Giles S. H. Yeoa,b,1

aUniversity of Cambridge Metabolic Research Laboratories, Institute of Metabolic Science, and bNational Institute for Health Research Cambridge Biomedical Research Centre, Addenbrooke’s Hospital, Cambridge CB2 0QQ, United Kingdom; cDepartment of Medicine, Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, United Kingdom; dMedical Research Council Harwell, Oxfordshire OX11 0RD, United Kingdom; and eDepartment of Physiology, Anatomy, and Genetics, Henry Wellcome Centre for Gene Function, University of Oxford, Oxford OX1 3PT, United Kingdom


Figure 01
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Fig. 1. Fto–/– MEFs have reduced rates of cell growth, mRNA translation, and low levels of MSC. (A) For measurement of growth rate, Fto+/+ and Fto–/– MEFs were allowed to grow for different time periods. After each time point, 250 µL of phenol red-free DMEM with 10 µM phenazine methosulfate and 50 µM WST-1 was added for 2 h. Absorbance of media samples was measured as described in Materials and Methods. All data are expressed as mean ± SEM. (B) Fto+/+ and Fto–/– MEFs were treated with 2 µM puromycin for different time periods. MEFs were then harvested, and the resulting cell extracts analyzed by Western blotting utilizing the indicated antibodies. (C) Fto–/– MEFs were transfected with GFP alone or GFP-WT FTO using a Neon transfection system. At 48 h after transfection, cells were labeled with 2 µM puromycin for 15 min and the mRNA translation rate was measured as described in B. (D) Equal amounts of total protein containing cell extracts from Fto+/+ and Fto–/– MEFs were subjected to 10% to 45% glycerol gradients fractionation as described in Materials and Methods. Different fractions obtained were analyzed by Western blotting using the indicated antibodies. (E) Tissue extracts of mouse hypothalamus, obtained from different Fto+/+ and Fto–/– mice, were analyzed for components of the MSC by Western blotting using the indicated antibodies. (F) Fto–/– MEFs were transfected with GFP or GFP-WT FTO as in C. At 48 h after transfection, cells were harvested, and MRS protein was immunoprecipitated as described in Materials and Methods. Cell extracts and MRS immunoprecipitates were analyzed for components of the MSC by Western blotting using the indicated antibodies.

 

Figure 02
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Fig. 2. FTO is necessary for the normal responsiveness of mTORC1 signaling to amino acid status. (A) Fto+/+ and Fto–/– MEFs grown for 24 h were subjected to total amino acid deprivation for a further 24 h in the presence of dialyzed serum. After amino acid deprivation, cells were stained with trypan blue and the number of live cells was counted. (B) Cell extracts from Fto+/+ and Fto–/– MEFs grown for 24 h were analyzed for mTORC1 signaling by Western blotting using indicated antibodies. (C) Fto+/+ and Fto–/– MEFs grown for 24 h were amino acid-deprived and/or treated with 400 µM bafilomycin for 4 h in the presence of dialyzed serum. After 4 h treatment, cells were harvested, and the resulting cell extracts were analyzed by Western blotting using indicated antibodies. (D) Tissue extracts of mouse liver obtained from different Fto+/+ and Fto–/– mice were analyzed by Western blotting using indicated antibodies. (E) HEK cells were transfected with 2 µg or 4 µg of vector alone or GFP-WT FTO and cultured for 48 h as described in Materials and Methods. At 48 h after transfection, cells were harvested and cell extracts were analyzed for mTORC1 signaling by Western blotting using indicated antibodies. (F) HEK cells were transfected with 4 µg of vector alone or GFP-WT FTO and cultured for 48 h as described in Materials and Methods. After 48 h of transfection, cells were deprived of all amino acids for 4 h in the presence of dialyzed serum. Cells were then harvested, and cell extracts analyzed by Western blotting. Western blots were quantitated by densitometry and data normalized to actin levels. Data are shown as mean ± SEM. (G) HEK cells were transfected with 4 µg of vector alone or GFP-FTO R316Q mutant construct and were cultured for 48 h. Afterward, cells were treated and harvested and lysates were analyzed as in F. Quantification of Western blotting results obtained from separate experiments was performed and plotted as in F.

 

Figure 03
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Fig. 3. FTO physically interacts with multiple members of MSC. (A) HEK cells overexpressing vector alone or Flag-WT FTO were cultured for 48 h. Cells were then harvested and cell extracts subjected to Flag immunoprecipitation using an antibody against Flag protein. Flag immunoprecipitates were analyzed by Western blotting using the indicated antibodies. (B) FTO was immunoprecipitated from cell extracts of Fto+/+ and Fto–/– MEFs (grown for 24 h) using an anti-FTO antibody, as described in Materials and Methods. FTO immunoprecipitates were analyzed by Western blotting using the indicated antibodies. (C) Fto+/+ MEF cell extracts were loaded onto 10% to 45% glycerol gradients prepared as described for Fig. 1D. Different fractions were subjected to Western blotting using the indicated antibodies. (D) Part of the glycerol gradient fractions 2 and 3 prepared from the extracts of Fto+/+ and Fto–/– MEFs in C were used for the MRS IP as in B. (E) Extracts derived from Fto+/+ MEFs were subjected to subcellular fractionation using the compartmental protein extraction kit from Millipore per manufacturer protocol.

 

Figure 04
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Fig. 4. Levels of LRS, known to link amino acid signaling to mTORC1, are reduced in Fto–/– MEFs. (A) Cell extracts from Fto+/+ and Fto–/– MEFs were analyzed for the mTORC1 signaling pathway as in Fig. 2B using the indicated antibodies. (B) Fto+/+ and Fto–/– MEFs were grown for 24 h, after which mRNAs were extracted and used for quantitative RT-PCR analysis of LRS mRNA levels. (C) Working model for the cellular role of FTO based on our current data.

 


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