Extended Data Fig. 5: DHAP plays a key role in the activation of mTORC1 by glucose.
From: Dihydroxyacetone phosphate signals glucose availability to mTORC1
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.
