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Ammonia stimulates SCAP/Insig dissociation and SREBP-1 activation to promote lipogenesis and tumour growth

Abstract

Tumorigenesis is associated with elevated glucose and glutamine consumption, but how cancer cells can sense their levels to activate lipid synthesis is unknown. Here, we reveal that ammonia, released from glutamine, promotes lipogenesis via activation of sterol regulatory element-binding proteins (SREBPs), endoplasmic reticulum-bound transcription factors that play a central role in lipid metabolism. Ammonia activates the dissociation of glucose-regulated, N-glycosylated SREBP-cleavage-activating protein (SCAP) from insulin-inducible gene protein (Insig), an endoplasmic reticulum-retention protein, leading to SREBP translocation and lipogenic gene expression. Notably, 25-hydroxycholesterol blocks ammonia to access its binding site on SCAP. Mutating aspartate D428 to alanine prevents ammonia binding to SCAP, abolishes SREBP-1 activation and suppresses tumour growth. Our study characterizes the unknown role, opposite to sterols, of ammonia as a key activator that stimulates SCAP–Insig dissociation and SREBP-1 activation to promote tumour growth and demonstrates that SCAP is a critical sensor of glutamine, glucose and sterol levels to precisely control lipid synthesis.

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Fig. 1: Glutamine is necessary for SREBP activation and lipogenesis.
Fig. 2: Glutamine-released ammonia activates SREBPs and lipogenesis.
Fig. 3: Inhibiting glutaminolysis abolishes SREBP activation.
Fig. 4: GLS and SREBP-1 are highly correlated in human tumours.
Fig. 5: Ammonia binds SCAP to activate its dissociation from Insig.
Fig. 6: Disrupting ammonia–SCAP interaction suppresses tumour growth.
Fig. 7: Model of ammonia activating SCAP–SREBP and lipogenesis.

Data availability

All data that support the findings of this study are available within the paper and its supplementary information files. RNA-seq data for Figs. 1a and 2f and Extended Data Fig. 1a are provided in Supplementary Data and the raw data are deposited in the Gene Expression Omnibus (accession no. GSE199089). Source data are provided with this paper.

Code availability

No custom codes were used during this study.

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Acknowledgements

This work was supported by the National Institute of Neurological Disorders and Stroke and the National Cancer Institute (USA) grants NS104332, NS112935 and R01CA240726 to D.G., CA227874 to D.G. and A.C. and an American Cancer Society Research Scholar Grant RSG-14-228-01–CSM to D.G. We also appreciate the support from the OSU Comprehensive Cancer Center–Pelotonia Idea grant and Urban & Shelly Meyer Fund for Cancer Research to D.G. The authors thank M. Torres for editorial assistance.

Author information

Authors and Affiliations

Authors

Contributions

D.G. conceived the ideas. C.C. and D.G. designed the experiments. C.C., F.G., Y. Zhong, H.W. and X.C. performed the experiments. Z.L. and X.-l.C. conducted computational simulations, Y. Zhao and X.M. performed RNA-seq data and pathway analyses. C.C., F.G., A.C. and D.G. analyzed the data. C.H. and W.D. provided glioma and lung cancer TMA. C.C., Z.L., X.-l.C. and D.G. wrote the manuscript and all authors reviewed and approved the manuscript for publication.

Corresponding author

Correspondence to Deliang Guo.

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The authors declare no competing interests.

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Nature Metabolism thanks Jiyeon Kim, Evripidis Gavathiotis and the other, anonymous, reviewers for their contribution to the peer review of this work. Primary handling editor: Alfredo Giménez-Cassina

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Extended data

Extended Data Fig. 1 Glutamine activates SREBP-1 to promote cell proliferation.

a, Heat map comparison of metabolic and overall pathways based on RNA-seq data from H1299 cells under glucose, glutamine or a combination of glucose and glutamine vs. both free conditions (12 h) using the bioinformatics Ingenuity Pathway Analysis (IPA). #NUM, no activity pattern available. b, c, Western blot analysis of cell lysates of cells stimulated with glutamine for 12 h (b) or with 4 mM glutamine at the indicated times (c) under serum-free conditions (glucose 5 mM). d, Lipids derived from 14C-labeled glucose (0.5 μCi, 2 h) in cells after culturing cells with/without glutamine (4 mM) for 12 h in serum-free medium containing 5 mM non-labeled glucose. The results are presented as mean ± SEM (n = 3). e, Proliferation of cancer cells cultured in medium supplemented with 1% dialyzed FBS with/without glutamine (4 mM) or glucose (5 mM) (mean ± SD, n = 3). f, g, Western blot analysis of cells after infection with shRNA-expressing lentivirus for 48 h and then placed in fresh medium (5 mM glucose) with/without glutamine (4 mM) for another 12 h (left panels). Cell proliferation was determined under 1% dialyzed FBS (right panels). The results are shown as mean ± SD (n = 3). h, Western blot analysis of cells after treatment with atorvastatin (5 μM) for 12 h in 5% lipoprotein-deficient serum (LPDS) containing 5 mM glucose with/without glutamine (4 mM). i, Western blot analysis of cells after stimulation with EGF (20 ng/ml) for 12 h in serum-free medium (5 mM glucose) with/without glutamine (4 mM). j, Western blot analysis of cells after incubation with/without aspartate (0.15 mM), asparagine (0.38 mM), leucine (0.38 mM), methionine (0.1 mM), threonine (0.17 mM) or glutamine (2 mM) for 12 h in HBSS buffer (containing 5.6 mM glucose) supplemented with essential amino acids. The dose selected for each amino acid is same as their concentration included in RPMI 1640 medium. Significance was determined by unpaired and two-tailed Student’s t-test (d) or two-way ANOVA with Dunnett’s (e) or Tukey’s (g) multiple comparisons adjustment.

Source data

Extended Data Fig. 2 Ammonia activates SREBPs and lipogenesis.

a, Western blot analysis of cells stimulated with glutamine, NH4Cl, NH3·H2O, NaCl, NaOH or NaNO3 (all 4 mM) under serum-free medium (5 mM glucose) for 12 hr. b, Western blot analysis of cells stimulated with NaCl (12 hr) in the absence of glutamine under serum-free culture conditions containing 5 mM glucose. c, Representative IF images of cells after stimulation with glutamate (4 mM), α-KG (4 mM), lactate (10 mM) or glutamine (4 mM) for 12 hr under serum-free culture conditions (5 mM glucose). Scale bars, 10 μm. The nuclear intensity of SREBP-1 (bottom panel) was quantified over 30 cells by ImageJ (mean ± SEM, n ≥ 30). df, Western blot analysis of H1299 cells stimulated with glutamine (4 mM), glutamate (Glu, 4 mM), α-KG (4 mM), octyl-α-KG (OA-KG) (2 mM) or NH4Cl (4 mM) for 12 hr under serum-free culture conditions (5 mM glucose) (d). The levels of glutamate (e) and α-KG (f) in the cells were measured using the appropriate assay kits. The results (e and f) are presented as mean ± SEM (n = 3). g, Western blot analysis of H1299 cells stimulated with glutamine (4 mM) or NH4Cl (4 mM) for 12 hr in the presence of glucose (5 mM) after ATG5 siRNA knockdown for 24 hr. h, Western blot analysis of cells stimulated with NH4Cl at the indicated doses for 12 hr under serum-free culture conditions (5 mM glucose). ik, Western blot analysis of membranes (for GFP-SCAP, PDI and SREBP precursors) and nuclear extracts (for N-terminal SREBPs and Lamin A) from HEK293T cells transfected with GFP (2 μg), GFP-SCAP wild-type (NNN) (2 μg) or its mutant QQQ (5 μg), obtained by replacing all three N-glycosylation residues asparagine (N) to glutamine (Q), together with full length Flag-SREBP-1a (i), -1c (j), or HA-SREBP -2 (k) for 24 hr and then stimulated with glutamine or NH4Cl (all 4 mM) for another 12 hr under serum-free culture conditions (5 mM glucose). Significance was determined by unpaired and two-tailed Student t-test or one-way ANOVA with Dunnett’s multiple comparisons adjustment.

Source data

Extended Data Fig. 3 Suppressing ammonia release from glutamine inhibits SREBPs.

a, Relative metabolite levels in H1299 cells after treatment with GPNA (5 mM) or CB-839 (100 nM) for 12 hr under serum-free medium containing glutamine (4 mM) and glucose (5 mM) via using appropriate assay kits (mean ± SEM, n = 3). Cell culture conditions upon treatment are the same for the subsequent panels. b, Relative glutamine consumption of cells treating with GPNA (5 mM) or CB-839 (100 nM) for 12 hr (mean ± SEM, n = 3). c, d, Western blot analysis of cells treated with GPNA or CB-839 (48 hr). e, Western blot analysis of GBM30 cells treated with CB-839 (200 nM) for 12 hr with/without glutamine, glutamate or NH4Cl (all 4 mM). f, Ammonia measurement (left panel) in tumour tissues from H1299 cells (4 × 106) derived xenograft model treated with CB-839 (30 mg/kg/mouse, i.p., twice per day for 3 days) when tumour size reached 200 mm3 (mean ± SEM, n = 6). Middle panel shows representative IHC images. Scale bars, 50 μm. The expression levels were quantified by using ImageJ to analyze 4 images per tumour (3 tumours/group) (mean ± SEM, n ≥ 2441 cells) (right panel). g, Relative glutamine consumption (12 hr) of cells in culture condition as (a) after infection with shRNA-expressing lentiviruses (48 hr) (mean ± SEM, n = 3). h, Relative metabolite levels (12 hr) in cells in culture condition as (a) measured by the appropriate assay kit after infection with shRNA-expressing lentiviruses (48 hr) (mean ± SD, (n = 3). i, j, Western blot analysis of cells after infection with shRNA-expressing lentiviruses for 48 hr and then stimulated with 4 mM glutamate, α-KG or NH4Cl for 12 hr. k, l, Real-time qPCR (k) and Western blot (l) analysis of cells under serum-free medium containing 5 mM glucose and 4 mM glutamine for 12 hr after siRNA knockdown of glutamate dehydrogenase (GDH1/2), asparaginase (ASPG) or serine deaminase (SDS) (24 hr). The results (k) are presented as mean ± SEM (n = 3). Significance was determined by unpaired and two-tailed Student’s t-test or one-way ANOVA with Dunnett’s multiple comparisons adjustment.

Source data

Extended Data Fig. 4 GLS is correlated with SREBP-1 in human tumour tissues.

a, Representative IHC images of anti-GLS and -SREBP-1 staining in tumour vs. adjacent normal tissues from individuals with adenocarcinoma (Adeno) or squamous lung cancer. Scale bars, 50 μm. b, c, Representative IHC images of anti-GLS and anti-SREBP-1 staining from lung cancer TMA (b). Representative images of different levels of anti-GLS or anti-SREBP-1 staining and scoring are shown in (c). d, Comparison of GLS expression and SREBP-1 levels in 50 paired tumours vs. adjacent normal lung tissues from the lung cancer TMA based on H score. Significance was determined by an unpaired Student’s t-test. e, Genetic inhibition of GLS or SREBP-1 dramatically suppressed lung tumour growth in vivo. NSCLC H1299 cells were infected with shGLS- or shSREBP-1-expressing lentivirus for 48 hr and then were implanted (2 ×106 cells/mouse) into the flank of nude mice. The tumours were isolated from mice at 53 days post-implantation and were imaged (left panel) and weighed (right panel) for comparison. Data are shown as mean ± SEM (n = 6). Significance was determined by one-way ANOVA with Dunnett’s multiple comparisons adjustment. f, Representative IHC images of anti-GLS, anti-SREBP-1, anti-ASPG and anti-SDS staining in tumour tissues from patients with GBM. Scale bars, 50 μm. g, h, Representative images of anti-GLS and anti-SREBP-1 staining from glioma TMA (g). Representative images of different levels of anti-GLS or anti-SREBP-1 staining and scoring are shown in (h).

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Extended Data Fig. 5 Ammonia binds to SCAP stimulating SCAP/SREBP activation.

a, Representative confocal images of U87 cells in response to glutamine (4 mM), glucose (5 mM) or NH4Cl (4 mM) stimulation for 12 hr with/without CB-839 (100 nM) under serum-free culture conditions. Scale bars, 10 μm. b, In vitro SCAP ER-budding assay. H1299 cells were stimulated with/without glutamine (4 mM) or NH4Cl (4 mM) for 4 hr under serum-free medium (5 mM glucose). Microsomes were purified and incubated at 37 °C for 15 min or on ice (as time 0) with cytosol extracts from rat liver in the presence of ATP and GTP (left panel). Alternatively, microsomes purified from H1299 cells cultured with glucose (5 mM) alone (2 hr) were incubated with NH4Cl (1 mM) or NaCl (1 mM) at 37 °C or on ice together with liver extracts as above (right panel). The mixtures were centrifuged to separate budded vesicles from the ER membrane fractions, which were then analyzed by Western blot by using indicated antibodies. c, Co-solvent NH3 computational mapping of SCAP. d, Alignment of the SCAP protein fragment. e, A schematic model for the sequential binding of NH4+ to SCAP obtained from the co-solvent ammonia mapping and NH4+-bound SCAP simulations. f, Western blot analysis of HEK293T cells transfected with GFP, wild-type or different GFP-SCAP mutants together with full-length Flag-SREBP-1c for 24 hr and then stimulated with glutamine (4 mM) for 12 hr under serum-free conditions (5 mM glucose). g. Co-solvent ammonia mapping for SCAP bound with 25-HC. Right panel shows the biochemical analysis of GFP-SCAP-bound ammonia in HEK293T cells stimulated with NH4Cl (4 mM) for 2 hr with/without pretreatment with 25-HC (10 µg/ml, 1 hr) using an ammonia assay kit. Top panel shows by western blot that equal amounts of proteins were purified. The results are presented as mean ± SEM (n = 3). Significance was determined by unpaired and two-tailed Student’s t-test. h, i, Western blot analysis of H1299 cells cultured with NH4Cl (4 mM) (h) or glutamine (4 mM) (i) for 12 hr in serum-free medium (5 mM glucose) together with a cholesterol/25-hydroxycholesterol mixture (sterols).

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Extended Data Fig. 6 Ammonia binding induces SCAP dissociation from Insig.

a–d, Comparison of the coupling, tilting and kink angles of S3, S5 and S6 helices during the 1 μs simulations of SCAP bound with NH4+ vs. SCAP without bound NH4+. In panel (a), S3 and S6 helices from the NH4+ unbound simulation (in light gray) is aligned with the NH4+ bound simulation (in dark gray). NH4+, D428, S326 and S330 are shown in the stick representation. The coupling of the S3 and S6 helices was altered by the binding of NH4+ (a). In the NH4+ bound simulation, the S3 helix had a smaller tilting angle (b) and S5 and S6 helix had a larger tilting angle (c and d). Inset in panel (b) illustrates a helix titling angle. Insets in panel (c) and (d) illustrate a helix kink conformation with the lower part of the helix aligned (white), and the top part of the helix showing a difference between NH4+ bound and NH4+ unbound SCAP. Only converged data from the last 500 ns of each simulation were used for the histogram analysis. e, Comparison of the interface contact area between SCAP and Insig during the simulations of the NH4+ bound SCAP vs. the NH4+ unbound SCAP. f, A schematic model for NH4+ regulated SCAP activation. Left: Insig-SCAP binding in the absence of 25-HC and NH4+. Top: Binding of 25-HC blocks NH4+ binding to prevent SCAP activation (orange). Middle: Absence of 25-HC opens the channel, which permits the entry of NH4+ to bind to D428 first, then to S326/S330 to form a stable binding site, leading to significant conformational changes of SCAP (red) and its dissociation from Insig for subsequent translocation and SREBP activation. Bottom: D428A mutant is unable to bind NH4+, preventing NH4+ from inducing conformational changes required for SCAP dissociation from Insig in the absence of 25-HC; thus, it cannot be activated by NH4+.

Extended Data Fig. 7 SCAP D428A mutation completely abolishes ammonia function.

a, Representative confocal microscopy images of wild-type or mutant (D428A) GFP-SCAP in U87 cells compared to the Golgi marker Giantin (red) in response to glutamine or NH4Cl stimulation in the presence of glucose. U87 cells were cultured on coverslips in DMEM medium supplemented with 5% FBS for 24 hr, followed by transfection with wild-type or mutant (D428A) GFP-SCAP plasmids for 24 hr. The transfected cells were washed with PBS once and incubated with glutamine (4 mM) or NH4Cl (4 mM) for 12 hr in fresh serum-free DMEM medium with the presence of glucose (5 mM). Cell culture conditions prior to treatment are the same for subsequent panels. Scale bars, 10 μm. b, Western blot analysis of membrane and nuclear extracts from HEK293T cells transfected with GFP, GFP-SCAP wild-type or D428A mutant plasmids at the indicated doses together with full-length Flag-SREBP-1c for 24 hr and then placed in fresh serum-free DMEM medium containing glutamine (4 mM) and glucose (5 mM) for another 12 hr. c, Western blot analysis of membrane and nuclear extracts from HEK293T cells transfected with GFP, GFP-SCAP wild-type or mutant D428A, D428E (glutamate), D428N (asparagine), D428K (lysine) together with full-length Flag-SREBP-1c for 24 hr and then placed in fresh serum-free medium in the presence of glucose (5 mM) and glutamine (4 mM) for another 12 hr. d, e, Western blot analysis of membrane (for GFP-SCAP and SREBP precursors) and nuclear extracts (for N-terminal SREBPs) from HEK293T cells transfected with GFP, wild-type or mutant GFP-SCAP (D428A) together with full-length Flag-SREBP-1a (d) or HA-SREBP-2 (e) for 24 hr and then stimulated with glutamine (4 mM) or NH4Cl (4 mM) in the presence of glucose (5 mM) under fresh serum-free medium.

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Extended Data Fig. 8 D428A mutation abolishes SCAP-promoted tumour growth.

a, b, Gross and macroscopic images of mouse lungs (a) and H&E staining of lung sections (b) at day 50 after mouse implantation with H1299 cells expressing GFP, wild-type (WT) or mutant GFP-SCAP D428A. Framed images in red were presented in Fig. 6d as representatives. Scale bars, 2 mm. The number of nodules on mice lung sections was quantified by ImageJ (b, lower panel). Data are shown as mean ± SEM (n = 5). Significance was determined by one-way ANOVA with Dunnett’s multiple comparisons adjustment. c, MRI scans of mouse brain at day 12 after implantation of GBM30 cells stably transfected with GFP, wild-type or mutant (D428A) GFP-SCAP (3.5 × 103 cells/mouse). Yellow circles indicate tumour location. White arrows indicate injection site. Scatter plot shows tumour volume from MRI scans quantified from the outlined region-of-interest (ROIs) (right panel). The results are presented as mean ± SEM (n = 5). Significance was determined by unpaired and two-tailed Student’s t-test. d, H&E staining of mouse brain sections excised at day 17 after implantation of GBM30 cells as described in (c). Rectangle-framed images were used in Fig. 6h as representatives. Scale bars, 1 mm.

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Cheng, C., Geng, F., Li, Z. et al. Ammonia stimulates SCAP/Insig dissociation and SREBP-1 activation to promote lipogenesis and tumour growth. Nat Metab 4, 575–588 (2022). https://doi.org/10.1038/s42255-022-00568-y

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