Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Dihydroxyacetone phosphate signals glucose availability to mTORC1

Abstract

The mechanistic target of rapamycin complex 1 (mTORC1) kinase regulates cell growth by setting the balance between anabolic and catabolic processes. To be active, mTORC1 requires the environmental presence of amino acids and glucose. While a mechanistic understanding of amino acid sensing by mTORC1 is emerging, how glucose activates mTORC1 remains mysterious. Here, we used metabolically engineered human cells lacking the canonical energy sensor AMP-activated protein kinase to identify glucose-derived metabolites required to activate mTORC1 independent of energetic stress. We show that mTORC1 senses a metabolite downstream of the aldolase and upstream of the GAPDH-catalysed steps of glycolysis and pinpoint dihydroxyacetone phosphate (DHAP) as the key molecule. In cells expressing a triose kinase, the synthesis of DHAP from DHA is sufficient to activate mTORC1 even in the absence of glucose. DHAP is a precursor for lipid synthesis, a process under the control of mTORC1, which provides a potential rationale for the sensing of DHAP by mTORC1.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Various sugars can activate mTORC1 independently of AMPK.
Fig. 2: GPI and ALDO are upstream of the metabolite that signals glucose availability to mTORC1.
Fig. 3: GAPDH is downstream of the metabolite that signals glucose availability to mTORC1.
Fig. 4: TPI is downstream of the metabolite that signals glucose availability to mTORC1, and DHAP synthesis is sufficient to activate mTORC1.

Data availability

The data supporting the plots within this paper are available from the corresponding author upon reasonable request. Source data are provided with this paper.

References

  1. 1.

    Saxton, R. A. & Sabatini, D. M. mTOR signaling in growth, metabolism and disease. Cell 168, 960–976 (2017).

    CAS  Article  Google Scholar 

  2. 2.

    Kubrusly, M. S. et al. A role for mammalian target of rapamycin (mTOR) pathway in non-alcoholic steatohepatitis-related cirrhosis. Histol. Histopathol. 25, 1123–1131 (2010).

    CAS  PubMed  Google Scholar 

  3. 3.

    Guillén, C. & Benito, M. mTORC1 overactivation as a key aging factor in the progression to type 2 diabetes mellitus. Front. Endocrinol. 9, 621 (2018).

  4. 4.

    Ardestani, A., Lupse, B., Kido, Y., Leibowitz, G. & Maedler, K. mTORC1 signaling: a double-edged sword in diabetic β cells. Cell Metab. 27, 314–331 (2018).

    CAS  Article  Google Scholar 

  5. 5.

    Lee, P. L., Tang, Y., Li, H. & Guertin, D. A. Raptor/mTORC1 loss in adipocytes causes progressive lipodystrophy and fatty liver disease. Mol. Metab. 5, 422–432 (2016).

    CAS  Article  Google Scholar 

  6. 6.

    Inoki, K., Zhu, T. & Guan, K.-L. TSC2 mediates cellular energy response to control cell growth and survival. Cell 115, 577–590 (2003).

    CAS  Article  Google Scholar 

  7. 7.

    Shaw, R. J. et al. The LKB1 tumor suppressor negatively regulates mTOR signaling. Cancer Cell 6, 91–99 (2004).

    CAS  Article  Google Scholar 

  8. 8.

    Gwinn, D. M. & Shaw, R. J. AMPK control of mTOR signaling and growth. in The Enzymes Vol. 28 (eds Tamanoi, F. & Hall, M. N.) 49–75 (Academic Press, 2010).

  9. 9.

    Efeyan, A. et al. Regulation of mTORC1 by the Rag GTPases is necessary for neonatal autophagy and survival. Nature 493, 679–683 (2013).

    CAS  Article  Google Scholar 

  10. 10.

    Wolfson, R. L. et al. KICSTOR recruits GATOR1 to the lysosome and is necessary for nutrients to regulate mTORC1. Nature 543, 438–442 (2017).

    CAS  Article  Google Scholar 

  11. 11.

    Kalender, A. et al. Metformin, independent of AMPK, inhibits mTORC1 in a Rag GTPase-dependent manner. Cell Metab. 11, 390–401 (2010).

    CAS  Article  Google Scholar 

  12. 12.

    Buller, C. L., Heilig, C. W. & Brosius, F. C. GLUT1 enhances mTOR activity independently of TSC2 and AMPK. Am. J. Physiol. Renal Physiol. 301, F588–F596 (2011).

    CAS  Article  Google Scholar 

  13. 13.

    Almacellas, E. et al. Phosphofructokinases axis controls glucose-dependent mTORC1 activation driven by E2F1. iScience 20, 434–448 (2019).

    CAS  Article  Google Scholar 

  14. 14.

    Roberts, D. J., Tan-Sah, V. P., Ding, E. Y., Smith, J. M. & Miyamoto, S. Hexokinase-II positively regulates glucose starvation induced autophagy through TORC1 inhibition. Mol. Cell 53, 521–533 (2014).

    CAS  Article  Google Scholar 

  15. 15.

    Zhang, C.-S. et al. Fructose-1,6-bisphosphate and aldolase mediate glucose sensing by AMPK. Nature 548, 112–116 (2017).

    CAS  Article  Google Scholar 

  16. 16.

    Lee, M. N. et al. Glycolytic flux signals to mTOR through glyceraldehyde-3-phosphate dehydrogenase-mediated regulation of Rheb. Mol. Cell. Biol. 29, 3991–4001 (2009).

    CAS  Article  Google Scholar 

  17. 17.

    Sharma, S., Guthrie, P. H., Chan, S. S., Haq, S. & Taegtmeyer, H. Glucose phosphorylation is required for insulin-dependent mTOR signalling in the heart. Cardiovasc. Res. 76, 71–80 (2007).

    CAS  Article  Google Scholar 

  18. 18.

    Karlstaedt, A., Khanna, R., Thangam, M. & Taegtmeyer, H. Glucose 6-phosphate accumulates via phosphoglucose isomerase inhibition in heart muscle. Circ. Res. 126, 60–74 (2020).

    CAS  Article  Google Scholar 

  19. 19.

    Shiraj, S. et al. Glucose regulation of load‐induced mTOR signaling and ER stress in mammalian heart. J. Am. Heart Assoc. 2, e004796 (2013).

  20. 20.

    Hardie, D. G., Ross, F. A. & Hawley, S. A. AMPK: a nutrient and energy sensor that maintains energy homeostasis. Nat. Rev. Mol. Cell Biol. 13, 251–262 (2012).

    CAS  Article  Google Scholar 

  21. 21.

    Kanehisa, M. & Goto, S. KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res. 28, 27–30 (2000).

  22. 22.

    Sakai, K., Hasumi, K. & Endo, A. Two glyceraldehyde-3-phosphate dehydrogenase isozymes from the koningic acid producer Trichoderma koningii. Eur. J. Biochem. 193, 195–202 (1990).

    CAS  Article  Google Scholar 

  23. 23.

    Liberti, M. V. et al. A predictive model for selective targeting of the warburg effect through GAPDH inhibition with a natural product. Cell Metab. 26, 648–659 (2017).

    CAS  Article  Google Scholar 

  24. 24.

    Rieder, S. V. & Rose, I. A. The mechanism of the triosephosphate isomerase reaction. J. Biol. Chem. 234, 1007–1010 (1959).

    CAS  Article  Google Scholar 

  25. 25.

    Fang, Y., Vilella-Bach, M., Bachmann, R., Flanigan, A. & Chen, J. Phosphatidic acid-mediated mitogenic activation of mTOR signaling. Science 294, 1942–1945 (2001).

    CAS  Article  Google Scholar 

  26. 26.

    Menon, D. et al. Lipid sensing by mTOR complexes via de novo synthesis of phosphatidic acid. J. Biol. Chem. 292, 6303–6311 (2017).

    CAS  Article  Google Scholar 

  27. 27.

    Ye, J. et al. GCN2 sustains mTORC1 suppression upon amino acid deprivation by inducing Sestrin2. Genes Dev. 29, 2331–2336 (2015).

    CAS  Article  Google Scholar 

  28. 28.

    Wolfson, R. L. et al. Sestrin2 is a leucine sensor for the mTORC1 pathway. Science 351, 43–48 (2016).

    CAS  Article  Google Scholar 

  29. 29.

    Chantranupong, L. et al. The CASTOR proteins are arginine sensors for the mTORC1 Pathway. Cell 165, 153–164 (2016).

    CAS  Article  Google Scholar 

  30. 30.

    Huttlin, E. L. et al. The BioPlex Network: a systematic exploration of the human interactome. Cell 162, 425–440 (2015).

    CAS  Article  Google Scholar 

  31. 31.

    Huttlin, E. L. et al. Architecture of the human interactome defines protein communities and disease networks. Nature 545, 505–509 (2017).

    CAS  Article  Google Scholar 

  32. 32.

    Düvel, K. et al. Activation of a metabolic gene regulatory network downstream of mTOR complex 1. Mol. Cell 39, 171–183 (2010).

    Article  Google Scholar 

  33. 33.

    Peterson, T. R. et al. mTOR complex 1 regulates lipin 1 localization to control the SREBP pathway. Cell 146, 408–420 (2011).

    CAS  Article  Google Scholar 

  34. 34.

    Herman, M. A., She, P., Peroni, O. D., Lynch, C. J. & Kahn, B. B. Adipose tissue branched chain amino acid metabolism (BCAA) modulates circulating BCAA levels. J. Biol. Chem. 285, 11348–11356 (2010).

    CAS  Article  Google Scholar 

  35. 35.

    Nelson, D. L. & Cox, M. M. Lehninger Principles of Biochemistry (W. H. Freeman, 2012).

  36. 36.

    Jang, C. et al. The small intestine converts dietary fructose into glucose and organic acids. Cell Metab. 27, 351–361 (2018).

    CAS  Article  Google Scholar 

  37. 37.

    Herman, M. A. & Samuel, V. T. The sweet path to metabolic demise: fructose and lipid synthesis. Trends Endocrinol. Metab. 27, 719–730 (2016).

    CAS  Article  Google Scholar 

  38. 38.

    Frias, M. A. et al. Phosphatidic acid drives mTORC1 lysosomal translocation in the absence of amino acids. J. Biol. Chem. 295, 263–274 (2020).

    CAS  Article  Google Scholar 

  39. 39.

    Joy, J. M. et al. Phosphatidic acid enhances mTOR signaling and resistance exercise induced hypertrophy. Nutr. Metab. 11, 29 (2014).

    Article  Google Scholar 

  40. 40.

    Dennis, P. B. et al. Mammalian TOR: a homeostatic ATP sensor. Science 294, 1102–1105 (2001).

    CAS  Article  Google Scholar 

  41. 41.

    Yang, W.-C. et al. Simultaneous quantification of metabolites involved in central carbon and energy metabolism using reversed-phase liquid chromatography−mass spectrometry and in vitro 13C labeling. Anal. Chem. 80, 9508–9516 (2008).

    CAS  Article  Google Scholar 

  42. 42.

    Jannasch, A., Sedlak, M. & Adamec, J. Quantification of pentose phosphate pathway (PPP) metabolites by LC–MS. in Metabolic Profiling: Methods and Protocols (ed. Metz, T. O.) 159–171 (Humana Press, 2011).

Download references

Acknowledgements

The authors would like to thank M. Valenstein, J. Spinelli and all the current members of the Sabatini laboratory for helpful insights. This work was supported by grants from the the National Institutes of Health (NIH; R01 CA103866, R01 CA129105 and R37 AI047389). J.M.O. was supported by a fellowship grant (no. F30CA210373) from the National Cancer Institute and the Harvard–MIT MSTP training grant (no. T32GM007753) from the National Institute of General Medical Sciences. P.A.K. was supported by a scholarship from Santander Universidades mobility fund granted by A. Mickiewicz University in Poznan. A.L.C. was supported by a fellowship grant (no. F31DK113665) from the National Institute of Diabetes and Digestive and Kidney Diseases. D.M.S. is an investigator of the Howard Hughes Medical Institute and an American Cancer Society research professor.

Author information

Affiliations

Authors

Contributions

J.M.O. and D.M.S. initiated the project and designed the research plan with input from P.A.K. J.M.O. and P.A.K. conducted experiments and analysed data with technical assistance from S.M.S. A.L.C. assisted with experiments. S.H.C., T.K. and C.A.L. operated the LC–MS platform, analysed the metabolomics data and assisted in the method development to measure GAP–aniline. J.M.O. and D.M.S. wrote the manuscript with assistance from P.A.K. and C.A.L.

Corresponding author

Correspondence to David M. Sabatini.

Ethics declarations

Competing interests

D.M.S. is a founder, shareholder and member of the scientific advisory board for Navitor Pharmaceuticals, which is targeting the mTORC1 pathway for therapeutic benefit. J.M.O. is a shareholder of Navitor Pharmaceuticals. The other authors declare no competing interests.

Additional information

Peer review information Primary Handling Editor: Christoph Schmitt.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Glucose metabolism via the pentose phosphate pathway (PPP) is not required for the activation of mTORC1.

a, AMPKα DKO cells were incubated for 1 hour without glucose, then either maintained without an added sugar or 10 mM of the indicated sugar or sugar analog was added for 15 minutes prior to cell lysis. Whole cell lysates were analyzed for the phosphorylation state or level of S6K1 by immunoblotting. b, 2-Deoxyglucose inhibits mTORC1 activity in a glucose-competitive manner. Wildtype HEK-293T cells were incubated in the given concentration of glucose for 1 hour and the indicated amount of 2-DG was added to them for 15 minutes. Whole cell lysates were analyzed for the phosphorylation state or level of S6K1 by immunoblotting. c, Loss of G6PD, the rate limiting enzyme for entry into the PPP, does not prevent the mTORC1 pathway from sensing glucose. Cells targeted with CRISPR/Cas9 using a control guide (AAVS1) or one against G6PD were incubated in the given glucose condition for 2 hours. Whole cell lysates were analyzed as in (a). d, Loss of G6PD causes a predictable decrease in pentose phosphate intermediates but had no impact on most glycolytic intermediates. Cells were treated the same as in (c) and metabolite extracts were analyzed by LC/MS for the indicated metabolites. Data are shown as mean ± s.e.m. for n = 3 biologically independent replicates. P-values were determined for two-sided Student’s t-test. An asterisk denoted The peak used to quantify fructose 6-phosphate (F6P) levels may also contain glucose 1-phosphate. Source data

Extended Data Fig. 2 Metabolite profiling of cells lacking GAPDH activity.

a, In a dose-dependent manner, Koningic Acid (KA) leads to an increase in the metabolites upstream of GAPDH despite the removal of glucose from the cell media. Metabolites downstream of GAPDH are predictably depleted upon KA treatment. NAD levels fall slightly upon glucose starvation and are not affected by KA treatment. NADH became undetectable in the absence of glucose, consistent with the role of glucose metabolism in NADH production. The effect of glucose deprivation on ATP/ADP/AMP levels was not reversed by KA treatment. HEK-293T cells were treated with the indicated concentrations of KA at the beginning of a glucose starvation period, and metabolites were extracted and analyzed by LC/MS. Data are shown as mean ± s.e.m. for n = 3 biologically independent replicates. * The peak used to quantify fructose 6-phosphate (F6P) levels may also contain glucose 1-phosphate. b, Overexpression of TK-GAPDH prevents all the effects on glycolytic metabolites caused by KA. Cells stably expressing FLAG-metap2 or FLAG-TK-GAPDH were incubated under the indicated conditions, and metabolites were extracted and analyzed by LC/MS. KA was added at the beginning of starvation. This panel is an extension of the data shown in Fig. 3e. Data are shown as mean ± s.e.m. for n = 3 biologically independent replicates. * The peak used to quantify fructose 6-phosphate (F6P) levels may also contain glucose 1-phosphate. c, Consistent with pharmacological inhibition of GAPDH, loss of GAPDH expression increases levels of GAP and DHAP and decreases those of phosphoenolpyruvate (PEP). GAPDH Dox-off cells were exposed to the indicated glucose concentrations in the absence or presence of dox. Data are shown as mean ± s.e.m. for n = 3 biologically independent replicates. P-values were determined for two-sided Student’s t-test. Source data

Extended Data Fig. 3 GAP can be measured as its aniline adduct as it has a unique mass.

a, In cells with normal TPI activity, the DHAP and GAP metabolites elute at similar retention times (11 mins vs 11.5 mins, respectively) but the peaks are separate enough to integrate independently. However, when cells lose TPI expression, the DHAP levels increase by an order of magnitude, and the peak normally integrated to measure GAP is obscured by the broader DHAP peak, which has a shoulder that extends into the GAP peak at 11.5 mins. Because the mass of DHAP and GAP are the same, quantifying them accurately in the context of TPI loss is an analytical challenge. HEK-293T AMPKα DKO TPI dox-off cells were treated with (+Dox) or without dox (-Dox) for 10 days. The media was then replaced with fresh RPMI media containing 5 mM glucose for thirty minutes. Metabolites were extracted and analyzed by HILIC LC/MS. Peak traces are shown for the +TPI (-Dox) condition in black and -TPI (+Dox) in blue, both separately with different y-axis scales, or overlaid to highlight the observed changes in DHAP. b, DHAP and GAP can form aniline adducts that are singly labeled with an m/z value of 244.03803 but only GAP can form an aniline adduct that is doubly labeled with an m/z value of 319.08532. c, As expected, only GAP forms a product with m/z value of 319.08532 when reacted with aniline. 10 μM GAP or DHAP standards were reacted with aniline in the presence of EDC in 80% methanol to yield their respective aniline adducts. The reaction was analyzed by reverse phase LC/MS. d, Metabolites upstream of TPI increase and metabolites downstream of TPI decrease in the absence of TPI expression. HEK-293T AMPKα DKO TPI dox-off cells were incubated in full media (5mM glucose) following 10 days of doxycycline. Half of the extract was used in an aniline labeling reaction and analyzed by reverse phase LC/MS and the other half was analyzed by HILIC LC/MS. These data are an extension of the data shown in Fig. 4b, showing only the unstarved samples. Data are shown as mean ± s.e.m. for n = 3 biologically independent replicates. Source data

Extended Data Fig. 4 Glycerol Kinase-mediated glycerol 3-phosphate (G3P) synthesis partially activates mTORC1 in glucose starved cells.

a, Diagram detailing the metabolism of glycerol, and its connection to the G3P-shuttle and the glycolytic metabolite dihydroxyacetone phosphate (DHAP). The role of GPD1 in coupling DHAP reduction to NADH oxidation is emphasized. b, HEK-293T AMPKα DKO cells stably overexpressing either a FLAG-tagged control cDNA (metap2) or glycerol kinase (GK) were incubated with the indicated glucose or glycerol concentrations for 1 hour. Whole cell protein lysates were analyzed by immunoblotting for the phosphorylation state and levels of the indicated proteins. Dashed line in panel B marks the removal of irrelevant intervening lanes in the original western blot. c, Cells were treated as in (B) and metabolite extracts were analyzed by LC/MS. Notably, glycerol addition to cells expressing GK, but not the control protein, generated supraphysiological levels of G3P but only partially restored DHAP levels. Data are shown as mean ± s.e.m. for n = 3 biologically independent replicates. P-values were determined for two-sided Student’s t-test. d, Diagram describing the position of phosphatidic acid (PA) in relation to its precursors DHAP and G3P. e, Addition of 100 uM PA does not activate mTORC1 in cells starved of either glucose or leucine. HEK-293T AMPKα DKO cells were starved of either glucose or leucine for 2.5 h and were restimulated for 30 mins with either glucose or leucine in the absence or presence of 100 uM PA. Source data

Extended Data Fig. 5 DHAP plays a key role in the activation of mTORC1 by glucose.

a, GPD1 overexpression decreases the levels of several glycolytic intermediates (G6P and DHAP are shown) but increases those of glycerol 3-phosphate. HEK-293T AMPKα DKO cells expressing the indicated cDNAs were incubated with the indicated glucose concentration for 15 mins following a 3-hour glucose starvation. Metabolite extracts were analyzed by HILIC LC/MS and by reverse phase LC/MS for GAP-aniline. Data are shown as mean ± s.e.m. for n = 3 biologically independent replicates. P-values were determined for two-sided Student’s t-test. b, Dihydroxyacetone (DHA) makes DHAP only in TKFC-expressing cells, restoring levels to nearly those seen upon glucose restimulation. DHA treatment has little to no effect on glucose 6-phosphate (G6P) or fructose 1,6-bisphosphate (FBP) levels and only partially rescues glyceraldehyde 3-phosphate (GAP) levels. HEK-293T AMPKα DKO cells expressing a FLAG-tagged control cDNA (metap2) or triose kinase (TKFC) were incubated with the indicated concentrations of glucose or DHA for 1 hour. Metabolite extracts were analyzed by LC/MS. Data are shown as mean ± s.e.m. for n = 3 biologically independent replicates. P-values were determined for two-sided Student’s t-test. c, Glyceraldehyde (GA partially rescues mTORC1 signaling in the absence of glucose. HEK-293T AMPKα DKO cells expressing the indicated cDNAs were incubated with the indicated concentrations of glucose or GA for one hour. d, The concentrations required to obtain partial rescue of mTORC1 signaling with GA addition are also capable of leading to generation of DHAP along with GAP, here measured as the derivative GAP-aniline. Cells were treated as in (c). Metabolite extracts were analyzed by HILIC LC/MS for DHAP or reacted with aniline and analyzed by reverse phase LC/MS for GAP-aniline. Data are shown as mean ± s.e.m. for n = 3 biologically independent replicates. e, DHA does not require TPI or ALDO expression in order to activate mTORC1. HEK-293T AMPKα DKO TPI Dox-off and AMPKα DKO ALDO Dox-off cells were treated with either 10mM glucose or 0.2 mM DHA in the absence or presence of doxycycline, and cell lysates were analyzed by immunoblotting for the phosphorylation state and levels of the indicated proteins. Source data

Extended Data Fig. 6 Glucose sensing does not require canonical stress pathway components.

a, GCN2 loss is associated with increased mTORC1 activity but the pathway remains capable of sensing glucose and leucine in GCN2 KO cells. HEK-293T AMPKα DKO or AMPKα/GNC2 triple KOs (TKO) were starved of glucose or leucine for 3 hours or starved and restimulated with glucose or leucine for 15 minutes. Whole cell lysates were analyzed for the phosphorylation states and levels of the indicated proteins by immunoblot. b, ATF4 loss is associated with increased mTORC1 activity but the pathway remains capable of sensing glucose and leucine in ATF4 KO cells. HEK-293T AMPKα DKO stably expressing Cas9 with either a control guide (AAVS1) or guides against ATF4 were starved of glucose or leucine for 3 hours or starved and restimulated with glucose or leucine for 15 minutes. Whole cell lysates were analyzed for the phosphorylation states and levels of the indicated proteins by immunoblotting. c, Loss of Axin1 does not affect glucose sensing by the mTORC1 pathway or that of AMPK, measured by phosphorylation of Raptor and ACC. Wildtype or AMPKα DKO HEK-293T cells stably expressing Cas9 and either a guide against a control locus (AAVS1) or Axin1 were starved of glucose for 1 hour or starved and restimulated with glucose for 10 minutes. Whole cell lysates were analyzed for the phosphorylation states and levels of the indicated proteins by immunoblotting. d, The RagC-RagB heterodimer but not Axin1 is able to co-immunoprecipitate the Ragulator components c7orf59 and p18. None of the tested proteins were able to co-immunoprecipitate AMPK. Wildtype HEK-293T cells transfected with cDNAs for either HA-tagged metap2, Axin1(N-terminal or C-terminal tags), or HA-RagB/Flag-RagC were starved of glucose for 1 hour or starved and restimulated with glucose for 15 minutes. HA-immunopreciptates and whole cell lysates were analyzed for the levels of the indicated proteins by immunoblotting. Source data

Extended Data Fig. 7 Glucose sensing by mTORC1 requires an intact GATOR/KICSTOR pathway.

a, mTORC1 is insensitive to glucose starvation in Nprl3 KO but not AMPK DKO cells. HEK-293T cells of the indicated genotypes were tested starved of or restimulated with glucose. Protein lysates were analyzed for the phosphorylation states or levels of the indicated proteins. b-c, KICSTOR is required for mTORC1 to sense glucose and leucine. Wildtype HEK-293T cells or with the indicated genotypes were starved of either glucose or leucine for one hour or starved and restimulated with glucose or leucine. d, FLCN is not required for mTORC1 sensing of glucose or leucine starvation. HEK-293T AMPKα DKO or AMPKα/FLCN TKO cells were starved of either glucose or leucine for one hour or starved and restimulated with glucose or leucine for 15 minutes. e, A decrease in Mios expression leads to a concomitant decrease in the activation by glucose of mTORC1. HEK-293T AMPKα DKO stably expressing Cas9 and either a control guide or one targeting Mios were starved of glucose for one hour or starved and restimulated with glucose for 15 minutes. f, Overexpression of the components of GATOR2 prevents glucose or leucine, but not amino acid, starvation from regulating mTORC1. HEK-293T AMPKα DKO co-transfected with cDNAs for FLAG-S6K and either metap2 or all five components of the GATOR2 complex (Mios, WDR24, WDR59, SEC13, SEH1L) were starved of glucose, leucine, or amino acids or were starved and restimulated with glucose, leucine, or amino acids. FLAG-IP were analyzed for the phosphorylation state or levels of the indicated proteins. g-h, Glucose and amino acids regulate mTOR localization to the lysosome consistent with glucose regulating the Rag-GTPase pathway upstream of mTORC1. HEK-293T AMPK DKO cells were starved of amino acids or glucose for 50 minutes or starved for 50 minutes and restimulated with amino acids or glucose for 10 minutes. Cells were fixed and permeabilized and analyzed in an immunofluorescence assay with mTOR and LAMP2 antibodies. Representative images are shown in (g) and quantification of mTOR/LAMP2 colocalization is shown in (h), mean ± s.d. are represented for the indicated number of lysosomes obtained from 3 biologically independent replicates. P-values were determined for two-sided Student’s t-test. Source data

Extended Data Fig. 8 Testing of the role of GPD1/1L/2 in glucose sensing and the reported GAPDH-Rheb and ALDOA-v-ATPase interactions.

a, Loss of GPD1, GPD1L, or GPD2 does not affect glucose sensing by mTORC1. HEK-293T AMPKα DKO stably expressing Cas9 with either a control guide (AAVS1) or guides against GPD1, GPD1L, or GPD2 were starved of glucose for 3 hours or starved for 3 hours and restimulated with glucose for 15 minutes. Whole cell lysates were analyzed for the phosphorylation state and levels of the indicated proteins by immunoblot. b, ALDOA does not co-immunopreciptate the v-ATPase component ATP6V1B2 nor did GAPDH co-immunopreciptate RHEB. HEK-293T AMPKα DKO stably expressing FLAG-metap2, FLAG-ALDOA, or FLAG-GAPDH were starved of glucose or starved and restimulated with glucose. FLAG-IPs or whole cell lysates were analyzed for the levels of the indicated proteins by immunoblotting. Source data

Supplementary information

Source data

Source Data Fig. 1

Unprocessed western blots.

Source Data Fig. 2

Unprocessed western blots.

Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

Unprocessed western blots.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Unprocessed western blots.

Source Data Fig. 4

Statistical source data.

Source Data Extended Data Fig. 1

Unprocessed western blots.

Source Data Extended Data Fig. 1

Statistical source data.

Source Data Extended Data Fig. 2

Statistical source data.

Source Data Extended Data Fig. 3

Statistical source data.

Source Data Extended Data Fig. 4

Unprocessed western blots.

Source Data Extended Data Fig. 4

Statistical source data.

Source Data Extended Data Fig. 5

Unprocessed western blots.

Source Data Extended Data Fig. 5

Statistical source data.

Source Data Extended Data Fig. 6

Unprocessed western blots.

Source Data Extended Data Fig. 7

Unprocessed western blots.

Source Data Extended Data Fig. 7

Statistical source data.

Source Data Extended Data Fig. 8

Unprocessed western blots.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Orozco, J.M., Krawczyk, P.A., Scaria, S.M. et al. Dihydroxyacetone phosphate signals glucose availability to mTORC1. Nat Metab 2, 893–901 (2020). https://doi.org/10.1038/s42255-020-0250-5

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing