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mTORC1 regulates a lysosome-dependent adaptive shift in intracellular lipid species

Nature Metabolism volume 4, pages 1792–1811 (2022)Cite this article

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Abstract

The mechanistic target of rapamycin complex 1 (mTORC1) senses and relays environmental signals from growth factors and nutrients to metabolic networks and adaptive cellular systems to control the synthesis and breakdown of macromolecules; however, beyond inducing de novo lipid synthesis, the role of mTORC1 in controlling cellular lipid content remains poorly understood. Here we show that inhibition of mTORC1 via small molecule inhibitors or nutrient deprivation leads to the accumulation of intracellular triglycerides in both cultured cells and a mouse tumor model. The elevated triglyceride pool following mTORC1 inhibition stems from the lysosome-dependent, but autophagy-independent, hydrolysis of phospholipid fatty acids. The liberated fatty acids are available for either triglyceride synthesis or β-oxidation. Distinct from the established role of mTORC1 activation in promoting de novo lipid synthesis, our data indicate that mTORC1 inhibition triggers membrane phospholipid trafficking to the lysosome for catabolism and an adaptive shift in the use of constituent fatty acids for storage or energy production.

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Fig. 1: Lipidomic analysis of cells treated with mTORC1 inhibitors.
Fig. 2: Cells accumulate TGs following mTORC1 inhibition.
Fig. 3: mTORC1 suppresses endogenous lipid remodeling.
Fig. 4: TG accumulation is linked to other changes in lipid metabolism.
Fig. 5: Changes in intracellular lipid species following mTORC1 inhibition require lysosomal function.
Fig. 6: TG and other lipid species accumulate independently of autophagy following mTORC1 inhibition.
Fig. 7: Induction of endosomal delivery to the lysosome is required for the changes in cellular lipid species following mTORC1 inhibition.
Fig. 8: Model of the differential effects of mTORC1 activation and inhibition on cellular lipid metabolism.

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All data generated or analyzed during this study are included in this published article (and its supplementary information files). Source data are provided with this paper.

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Acknowledgements

We thank members of the Manning laboratory for helpful input, in particular S. Schrötter for her guidance in microscopy studies. We thank D. Kwiatkowski (Brigham and Women’s Hospital), E. Henske (Brigham and Women’s Hospital), D. Sabatini and W. Harper (Harvard Medical School) for providing cell lines and other reagents. We thank A. Kreutzberger, A. Sanyal and T. Kirchausen for helpful conversations and initial guidance in studying endocytosis and Z. W. Lai and Y. Ambaw from the Harvard-Chan Advanced Multi-omics Platform for guidance in piloting the Lyso-IP lipidomics experiments. Cell sorting was performed with the Harvard Medical School Systems Biology Flow Cytometry Core. A.M.H. was supported by a postdoctoral fellowship from the Jane Coffin Childs Memorial Fund for Medical Research. This study was funded by National Institutes of Health grants R35-CA197459 (B.D.M) and P01-CA120964 (B.D.M. and J.M.A.).

Author information

Author notes

  1. Margaret E. Torrence

    Present address: David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA

Authors and Affiliations

  1. Department of Molecular Metabolism, Harvard T.H. Chan School of Public Health, Boston, MA, USA

    Aaron M. Hosios, Meghan E. Wilkinson, Molly C. McNamara, Krystle C. Kalafut, Margaret E. Torrence & Brendan D. Manning

  2. Department of Cell Biology, Harvard Medical School, Boston, MA, USA

    Aaron M. Hosios, Meghan E. Wilkinson, Molly C. McNamara, Krystle C. Kalafut, Margaret E. Torrence & Brendan D. Manning

  3. Mass Spectrometry Core, Beth Israel Deaconess Medical Center, Department of Medicine, Harvard Medical School, Boston, MA, USA

    John M. Asara

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Contributions

Conceptualization was the responsibility of A.M.H. and B.D.M. Investigation was the responsibility of A.M.H., M.E.W, M.C.M., K.C.K., M.E.T. and J.M.A. Writing of the manuscript was conducted by A.M.H. and B.D.M. Funding was acquired by A.M.H., J.M.A. and B.D.M. Resources were the responsibility of B.D.M. Supervision was carried out by B.D.M.

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Correspondence to Brendan D. Manning.

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B.D.M. is a member of the scientific advisory board and a shareholder of Navitor Pharmaceuticals. All other authors declare no competing interests.

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Nature Metabolism thanks Martin Giera, Mathieu Laplante and the other, anonymous, reviewers for their contribution to the peer review of this work. Primary Handling Editor: Yanina-Yasmin Pesch, in collaboration with the Nature Metabolism team.

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Extended data

Extended Data Fig. 1 Lipidomic analysis of cells treated with mTORC1 inhibitors.

(a–c) Immunoblots for AKT and pAKT corresponding to Fig. 1a–c. (d, e) Fold changes of individual lipids grouped by class from the lipidomics experiment shown in Fig. 1d. (d) Non-ether and (e) ether lipids are separated. Dots represent the fold change of each individual lipid species relative its abundance in vehicle-treated cells. Bars represent the mean fold change for that class, graphed ± SD for n = 3 replicates. (f, g) Triglyceride species from the results in Fig. 1c stratified by the total number of double bonds (f) or carbon atoms (g) in their side chains. Each point represents an individual triglyceride. Group mean ± SD is shown in (f). Pearson’s r and line of best fit are shown in (g). Data represent n = 3 replicates. (h, i) Immunoblots for AKT and pAKT corresponding to Fig. 1g–j. (j) Changes in the abundance of individual lipid species from the experiment in Fig. 1h, with neutral lipids color coded, for mouse ES cells treated with torin1. Dotted lines indicate a 2-fold change (FC) and an adjusted p-value of 0.1. Statistical analysis by one-way ANOVA (f); n.s., not significant.

Source data

Extended Data Fig. 2 Intracellular triglycerides accumulate following mTORC1 inhibition.

(a) Triglyceride accumulation measured by enzyme assay and normalized to protein content from the same sample in Tsc2−/− MEFs serum-starved for 16 hrs, followed by 0–8 hrs of vehicle, rapamycin (20 nM), or torin1 (250 nM) treatment, graphed as mean ± SD relative to the 0 hr time point, n = 3. (b) Immunoblot for AKT and pAKT corresponding to Fig. 2m. (c) Immunoblot for the tumors shown in Fig. 2o. (d) Tumor TG versus plasma TG for individual mice shown in Fig. 2o, p. (e) Plasma non-esterified fatty acids (NEFA) measured for the mice shown in Fig. 2o, graphed as mean ± SD, n = 5 mice. (f) Immunoblot corresponding to the experiment shown in Fig. 2q. (g, h) Cell cycle profile for PC3 (g) and H1299 (H) cells treated for 16 hrs with vehicle, palbociclib (CDK4/6i, 100 nM), and/or torin1 (250 nM), n = 1. (i,j) Triglyceride levels quantified by enzyme assay and normalized to protein content from the same sample for cells treated as in (g, h), graphed as mean ± SD relative to vehicle-treated cells, n = 3. Statistical analysis by two-way ANOVA (i, j) and two-tailed Student’s t-test (e); n.s., not significant.

Source data

Extended Data Fig. 3 mTORC1 suppresses endogenous lipid remodeling and activates de novo lipid synthesis.

(a) SREBP1 target gene expression in Tsc2+/+ and Tsc2−/− MEFs treated for 16 hrs with vehicle, rapamycin (20 nM), or torin1 (250 nM), graphed as mean ± SD relative to vehicle-treated cells, n = 3. (B) Representative thin layer chromatogram for Tsc2−/− MEFs labeled with [1-14C]-oleate and treated with vehicle, rapamycin (20 nM), or torin1 (250 nM) for 16 hrs, developed to separate neutral lipids. ChE, cholesteryl-esters; TG, triglycerides; FA, fatty acids; DG, diglycerides; MG, monoglycerides. (c,d) Immunoblot (c) and TGs (d) assayed by pulse-chase with [1-14C]-oleate tracer (6 h) in Tsc2+/+ MEFs followed by chase in cold medium for 16 h with vehicle, 20 nM rapamycin, 250 nM torin1, 500 nM AZD2014, or 5 nM RapaLink-1, graphed as mean ± SD relative to vehicle-treated cells, n = 3. (e, f) Immunoblot (e) and TGs (f) assayed by pulse-chase with [1-14C]-oleate tracer (6 h) in MCF7 cells followed by chase in cold medium for 16 h with vehicle, 250 nM torin1, or 1 µM BYL719 treatment, graphed as mean ± SD relative to vehicle-treated cells, n = 3. NS denotes a non-specific band. (g–j) Immunoblot (g), de novo lipogenesis of all lipids (h), TG labeled from de novo lipogenesis (i), and lipid remodeling (j), assayed as in Fig. 3h,i in primary mouse hepatocytes serum-starved for 16 hrs, pretreated for 30 min with vehicle, rapamycin (20 nM), or torin1 (250 nM), and stimulated with insulin (100 nM) for 6 hrs, graphed as mean ± SD relative to vehicle-treated unstimulated cells, n = 3. Statistical analysis by one-way ANOVA (a, d, f, h–j); n.s., not significant.

Source data

Extended Data Fig. 4 TG accumulation is linked to other changes in lipid metabolism.

(a) 14C-TG turnover in PC3 cells treated with vehicle, torin1, or ATGLi, measured as in Fig. 4c. 14C-TG abundance is graphed over time as mean ± SD relative to 0 hrs, n = 3. (b) Immunoblots for ATGL and DGAT1 levels from two separate experiments in cell lines treated with vehicle, rapamycin, or torin1 for 16 hrs. NS denotes a non-specific band. (c) 14C-Fatty acid levels in Tsc2−/− MEFs labeled with [1-14C]-palmitate, treated with vehicle, rapamycin, or torin1 for 16 hrs, graphed as mean ± SD relative to vehicle, n = 3. (d) Acyl-carnitine species in Tsc2−/− MEFs in the experiment in Fig. 1d, graphed as mean ± SD relative to vehicle, n = 3. (e, f) Acyl-carnitine species detected in mouse embryonic stem (ES) cells (e) and wild-type, primary MEFs (f) in the experiments in Fig. 1h, j, graphed as mean ± SD relative to vehicle, n = 3. (g) Representative thin layer chromatogram for Tsc2−/− MEFs traced with [methyl-14C]-choline and treated with vehicle, rapamycin, or torin1 for 16 hrs, developed to separate phospholipids. PC, phosphatidylcholine; SM, sphingomyelin; LPC, lysophosphatidylcholine. (h) Ratio of lysophosphatidylcholine to phosphatidylcholine in PC3 cells pulse-labeled with [methyl-14C]-choline as in (g); mean ± SD relative to vehicle, n = 3. (i,j) Glycerophosphocholine levels in HEK-293T (i) and PC3 (j) cells treated for 16 hrs with vehicle, rapamycin, or torin1, graphed as mean ± SD relative to vehicle, n = 3. (k, l) 14C-TG (k) and 14C-fatty acids (L) in PC3 cells measured as in Fig. 4i, j and graphed as mean ± SD, n = 3. (m) Fatty acid accumulation in Tsc2−/− MEFs labeled with [1-14C]-oleate following vehicle (DMSO) treatment with 1 hr pretreatment with DGAT inhibitors and/or etomoxir; mean ± SD relative to 0 hrs, n = 3. (n) Triglyceride ion counts in the experiment shown in Fig. 4l, m; mean ± SD relative to vehicle, n = 3. (o) Carnitine palmitoyl transferase (CPT) gene expression in MEFs treated for 16 hrs with inhibitors, graphed as mean ± SD relative to vehicle, n = 6. Statistics: by one-way ANOVA (c, h–j, n), two-way ANOVA (d–f, k–m, o), and extra sum-of-squares F-test (A). n.s., not significant.

Source data

Extended Data Fig. 5 Changes in intracellular lipid species following mTORC1 inhibition require lysosomal function.

(a, b) Immunoblot (a) and fatty acids (b) corresponding from the experiment shown in Fig. 5a. Fatty acids quantification is graphed as mean ± SD relative to vehicle-treated cells, n = 3. (c, d) Immunoblots corresponding to the experiments shown in Fig. 5b, c. (e–g) Triglyceride and acyl-carnitine levels from the experiment shown in Fig. 5d–g. (h) Immunoblot for organelle markers in whole cell lysates or in HA-tag immunopurified lysosomes (IP) from HEK-293T cells expressing TMEM192-2xFLAG or 3xHA. (i) Immunoblot corresponding to the experiment shown in Fig. 5k, l. (j) Immunoblot corresponding to the experiment shown in Fig. 5m, n. LC3B is indicated in its unmodified (LC3B-I) and lipidated (LC3B-II) forms. Statistical analysis by two-way ANOVA (b, e–g). n.s., not significant.

Source data

Extended Data Fig. 6 Regulation of intracellular lipids by mTORC1 is inhibited by MAFP but not by loss of LPLA2.

(a–f) Lipid class sums (a–e) and glycerophosphocholine levels (f) detected in Tsc2−/− MEFs treated with vehicle, MAFP (a non-specific serine hydrolase inhibitor, 10 µM), and/or torin1 (250 nM) for 16 hrs, graphed as mean ± SD relative to vehicle-treated cells, n = 3. (g) Phospholipase A1 (PLA1) and A2 (PLA2) gene expression in Tsc2+/+ and Tsc2−/− MEFs treated for 16 hrs with vehicle, rapamycin (20 nM), or torin1 (250 nM), calculated relative to vehicle-treated Tsc2−/− MEFs, n = 3. cPLA2, cytosolic PLA2; iPLA2, calcium-independent PLA2; PA-PLA1, phosphatidic acid-preferring PLA1; PLB, phospholipase B. (h, i) Immunoblot (h) and TGs measured by pulse-chase with [1-14C]-oleate tracer (i) in parental and Pla2g15 knockout Tsc2−/− MEFs expressing enhanced green fluorescent protein (EGFP) or wild-type LPLA2 (PLA2G15) treated for 16 hrs with vehicle or torin1 (250 nM), graphed as mean ± SD relative to vehicle-treated cells, n = 3. Statistical analysis by two-way ANOVA (A-F,I). n.s., not significant.

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Extended Data Fig. 7 TG and other lipid species accumulate independently of autophagy following mTORC1 inhibition.

(a, b) Immunoblot (a) and TGs measured by pulse-chase with [1-14C]-oleate tracer (b) in Atg5+/+ and −/− MEFs treated for 16 hrs with vehicle or BafA1 (250 nM), graphed as mean ± SD relative to vehicle-treated cells, n = 3. (c,d) Immunoblot (c) and triglyceride levels measured by enzyme assay (D) and normalized to protein content from the same sample for parental, ATG7 knockout, and FIP200 knockout HEK-293T cells treated with vehicle or bafilomycin A1 (250 nM) for 16 hrs, graphed as mean ± SD relative to vehicle-treated cells, n = 3. (e, f) Immunoblot (e) and triglyceride levels (f) corresponding to the results shown in Fig. 6e, f. Triglyceride levels are graphed as mean ± SD relative to vehicle-treated cells, n = 3. (g, h) Immunoblot (g) and triglyceride levels measured by enzyme assay (h) and normalized to protein content from the same sample for parental and FIP200 knockout HEK-293T cells treated with vehicle, torin1 (250 nM), and/or bafilomycin A1 (250 nM) for 16 hrs, graphed as mean ± SD relative to vehicle-treated cells, n = 3. (i) EGFR degradation assay in parental Tsc2−/− MEFs and sgUvrag or sgAtg14 clones. Cells were serum-starved for 16 hrs and stimulated with EGF (10 ng/mL) for the indicated times. (j) Immunoblot for autophagy markers in parental Tsc2−/− MEFs and sgAtg14 clone after 4 hrs in amino acid replete or free medium. Statistical analysis by two-way ANOVA (B,D,F,H). n.s., not significant.

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Extended Data Fig. 8 TGs accumulate independently of TFEB and TFE3 following mTORC1 inhibition.

(a) Immunoblot for TFEB, TFE3, and proteins corresponding to their transcriptional target genes in Tsc2−/− MEFs treated for two days with control siRNA, siTfeb, or siTfe3. (b,c) 14C-TGs assayed by pulse-chase with [1-14C]-oleate tracer (6 h) in Tsc2−/− MEFs treated as in (A) followed by chase in cold medium for 16 hrs with vehicle or 250 nM torin1, graphed as mean ± SD relative to control siRNA-treated cells (b) and relative to vehicle-treated cells (C), n = 3. Statistical analysis by one-way ANOVA (b) and two-way ANOVA (c). n.s., not significant.

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Extended Data Fig. 9 Induction of endosomal delivery to the lysosome is required for the changes in cellular lipid species following mTORC1 inhibition.

(a) Immunoblot corresponding to the experiment shown in Fig. 7a, b. (b–d) Immunoblots (b) and 14C-TGs (c, d) assayed by pulse-chase with [1-14C]-oleate tracer (6 h) in Tsc2−/− MEFs followed by chase in cold medium for 16 hrs with vehicle, 250 nM torin1, 5 µM VPS34-IN1, or 80 µM Dynasore, graphed as mean ± SD relative to vehicle-treated cells (c) and relative to no-torin1 controls (d), n = 3. (e) Immunoblot corresponding to the experiment shown in Fig. 7c. (f) Fatty acid accumulation measured by pulse-chase with [1-14C]-oleate tracer in the presence of DGAT inhibitors (3 µM each) and etomoxir (20 µM) in Tsc2−/− MEFs following a 4 hr treatment with endocytosis inhibitors (5 µM VPS34-IN1, 1 µM PIK-FYVEi, 80 µM Dynasore, or 20 µM PitStop 2). Graphed as mean ± SD relative to vehicle-treated cells at time 0, n = 3. (g, h) Binding of transferrin to Tsc2−/− MEFs (g) and PC3 (h) cells treated for 4 hrs with vehicle or torin1 (250 nM) and kept on ice while labeling with transferrin, confirming that the amount of plasma membrane transferrin receptor is not regulated by mTOR and that surface-bound transferrin is eliminated following an acid wash. Graphed as mean ± SD, n = 3. (i, j) Endocytosis assays in PC3 cells, measuring the uptake of transferrin (i) or 10 kDa dextran (j), following 4 hrs of vehicle, rapamycin (20 nM), or torin1 (250 nM) treatment, graphed as mean ± SD, n = 3. a.u., Arbitrary Units. Statistical analysis by one-way ANOVA (c, f, j), and two-way ANOVA (d, g–i). n.s., not significant.

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Extended Data Fig. 10 EIPA and protease inhibitors serve as controls for BSA uptake and lysosomal cleavage assays.

(a) BSA uptake in Tsc2−/− MEFs treated with torin1 (250 nM) and vehicle or EIPA (20 µM) for 1 hr prior to labeling with 10 µg/mL BSA Alexa Fluor 647 (uptake, red) for 3 hrs. Nuclei are shown in blue. (b) BSA cleavage in Tsc2−/− MEFs treated with torin1 (250 nM) and vehicle or protease inhibitors (2 µM E64d, 2 µM Pepstatin A, and 10 µM Leupeptin) for 1 hr prior to labeling with 10 µg/mL each of BSA Alexa Fluor 647 (red, uptake) and DG-Green BSA (green, cleavage) for 3 hrs. Nuclei are shown in blue. (c, d) Quantification of the effect of EIPA on BSA uptake (c) and protease inhibitors on BSA cleavage (d) for cells treated as in (a, b), graphed as mean ± SD relative to vehicle-treated cells, n = 57–84 cells. (e) Co-localization of hydrolyzed DQ-Green BSA (green) and LysoTracker Deep Red (red) in Tsc2−/− MEFs pretreated with torin1 (250 nM) for 1 hr prior to labeling with 10 µg/mL DQ-Green BSA for 3 hrs. Nuclei are shown in blue. These images are representative of two replicate experiments in which similar results were obtained.

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Supplementary information

Supplementary Information

Supplementary Methods, Supplementary Tables 1–4 and Supplementary Fig. 1.

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Hosios, A.M., Wilkinson, M.E., McNamara, M.C. et al. mTORC1 regulates a lysosome-dependent adaptive shift in intracellular lipid species. Nat Metab 4, 1792–1811 (2022). https://doi.org/10.1038/s42255-022-00706-6

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