The ability to utilise ammonia as nitrogen source is cell type specific and intricately linked to GDH, AMPK and mTORC1

Abstract

Ammonia can be utilised as an alternative nitrogen source to glutamine to support cell proliferation. However, the underlying molecular mechanisms and whether all cells have this ability is not fully understood. We find that eleven cancer and non-cancerous cell lines have opposite abilities to tolerate and utilise ammonia to support proliferation in a glutamine-depleted environment. HEK293, Huh7, T47D and MCF7 cells can use ammonia, when starved of glutamine, to support proliferation to varying degrees. Glutamine depletion reduced mTORC1 activity, while additional ammonia supplementation diminished this mTORC1 inhibition. Depletion of glutamine promoted a rapid and transient activation of AMPK, whereas, additional ammonia supplementation blocked this starvation-induced AMPK activation. As expected, drug-induced AMPK activation reduced cell proliferation in glutamine-depleted cells supplemented with ammonia. Surprisingly, mTORC1 activity was largely unchanged despite the enhanced AMPK activity, suggesting that AMPK does not inhibit mTORC1 signalling under these conditions. Finally, glutamate dehydrogenase (GDH) inhibition, a key enzyme regulating ammonia assimilation, leads to AMPK activation, mTORC1 inhibition and reduced proliferation. Ammonia provides an alternative nitrogen source that aids certain cancer cells ability to thrive in nutrient-deprived environment. The ability of cells to utilise ammonia as a nitrogen source is intricately linked to AMPK, mTORC1 and GDH.

Figure 1

Tolerance of varying cell types of glutamine depletion and ammonia utilisation as an alternative nitrogen source. Cells in the control group were fixed in 4% formaldehyde 24 h after plating (n = 6). This represents the starting number of cells grown in normal media (DMEM containing high glucose, 10% FBS and 8 mM glutamine). Cells in the other groups (n = 6) were washed with DPBS and fresh media with or without glutamine or 0.8 mM NH₄Cl was added. After 3 days, cells in all groups were fixed and stained with 0.5% crystal violet solution in 4% formaldehyde and colorimetric (OD) measurement was quantified using spectrophotometer. SH-SY5Y, HCT116, HT29, HACAT and A549 cells can survive in glutamine-depleted culture, but cannot utilise NH₄Cl to proliferate (A). LNCAP and MCF10A cells displayed cell loss in glutamine-depleted media that was worsen by NH₄Cl supplementation (B). MCF7 and T47D cells can survive in glutamine-depleted culture and they can utilise NH₄Cl as alternative nitrogen source to support proliferation (C). Huh7 and HEK293 cells can proliferate in glutamine-depleted culture and proliferation was enhanced in the presence of NH₄Cl (D). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Figure 2

Glutamine synthetase and glutamate dehydrogenase protein abundance varies in different cell lines. Cells (HCT116, MCF10A, T47D, MCF7, HEK293 and Huh7) were plated in complete medium containing glutamine for 24 h. Cultures were lysed and collected for western blotting 24 h later. Levels of glutamine synthetase (GS) (A) and glutamate dehydrogenase (GDH) (B) relative to total protein (ponceau) were quantified using western blot. Note that MCF10A yielded very low amount of protein (total protein loaded ~4–16x more than other samples). (n = 3) One-way ANOVA p ≤ 0.001 demonstrates significantly different protein levels across samples, letters represent statistically distinct groups. Full-length blots are presented in Supplementary information Fig. 4

Figure 3

Proliferation in the presence or absence of ammonia in glutamine-depleted medium. HEK293 (A), Huh7 (B), T47D (C) and MCF7 (D) cells were plated and 24 h later they were washed with DPBS and cultured in fresh glutamine-depleted medium or glutamine-depleted medium with NH₄Cl (n = 6). Cell proliferation was then measured based on live imaging using the Incucyte FLR assay over 7 days. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Figure 4

Acute glutamine depletion and ammonia regulate AMPK and mTORC1 signalling. Cells (HEK293, Huh7, T47D and MCF7) were plated in complete medium containing glutamine for 24 h. One culture was lysed and collected for western blotting 24 h later (T0). The remainder of the cultures were washed with DPBS and fresh glutamine-depleted media with or without 0.8 mM NH₄Cl was added. Cultures were collected and lysed at 15 min for western blotting. Levels of phosphorylated S6K (T389) relative to total S6K protein levels (A) and phosphorylated ACC (S79) relative to total ACC protein levels (B) were quantified using western blot. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Figure 5

Acute glutamine depletion and ammonia lead to dynamic changes in mTORC1 and AMPK signalling in HEK293 and Huh7 cells. HEK293 and Huh7 cells were plated in complete media containing glutamine for 24 h. One culture was collected for western blotting 24 h later (T0). The remainder of the cultures were washed with DPBS and fresh glutamine-depleted media with or without 0.8 mM NH₄Cl was added. Cultures were collected at different time points and protein was extracted for western blotting. Protein abundance of phosphorylated S6K (T389) relative to total S6K protein levels (A,C) and phosphorylated ACC (S79) relative to total ACC protein levels (B,D) at 15 min, 30 min, 1 h, 2 h, 4 h and 6 h post-incubation was quantified using western blot. Blots shown were representative from 3 independent experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Full length blots are presented in Supplementary information Fig. 4.

Figure 6

Acute glutamine depletion and ammonia lead to dynamic changes in mTORC1 and AMPK signalling in T47D and MCF7 cells. T47D and MCF7 cells were plated in complete media containing glutamine for 24 h. One culture was collected for western blotting 24 h later (T0). The remainder of the cultures were washed with DPBS and fresh glutamine-depleted media with or without 0.8 mM NH₄Cl was added. Cultures were collected at different time points and protein was extracted for western blotting. Protein abundance of phosphorylated S6K (T389) relative to total S6K protein levels (A,C) and phosphorylated ACC (S79) relative to total ACC protein levels (B,D) at 15 min, 30 min, 1 h, 2 h, 4 h and 6 h post-incubation was quantified using western blot. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Full length blots are presented in Supplementary information Fig. 4.

Figure 7

Chronic glutamine depletion and ammonia regulate AMPK and mTORC1 signalling in HEK293 and T47D cells. HEK293 and T47D cells were plated in complete medium containing glutamine for 24 h. Cultures were washed with DPBS and fresh glutamine-depleted media with or without NH₄Cl was added. Cultures were collected 3 days later and protein was extracted for western blotting. Protein abundance of phosphorylated ACC (S79) or S6K (T389) relative to total ACC or S6K in HEK293 (A) and T47D (B) cells 3 days post-incubation was quantified using western blot. Blots shown were representative from 4 independent experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Full length blots are presented in Supplementary information Fig. 4.

Figure 8

Effects of AMPK activation using A769662 on mTORC1 signalling and proliferation in HEK293 cells. HEK293 cells were plated in full media containing glutamine for 24 h. Cultures were washed with DPBS and fresh glutamine-depleted media with or without NH₄Cl or A769662 (200 µM) was added. Cells cultured in glutamine-depleted media with or without NH₄Cl or DMSO served as vehicle controls. Cultures were collected at 4 h and 3 days post-incubation for protein extraction or subjected to live imaging using IncuCyte FLR assay (n = 3) to measure cell proliferation rate over 7 days (C). Protein abundance of phosphorylated ACC (S79) (A) or S6K (T389) (B) relative to total ACC or S6K was quantified using western blot. Blots shown were representative from 3 independent experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Full length blots are presented in Supplementary information Fig. 4.

Figure 9

Effects of GDH inhibition on AMPK and mTORC1 activity and proliferation in HEK293 and T47D cells. HEK293 and T47D cells were plated in full media containing glutamine for 24 h. Cultures were washed with DPBS and fresh glutamine-depleted media with NH₄Cl with or without hexachlorophene (Hex – 2.5 µM) was added. Cells cultured in glutamine-depleted media with or without NH₄Cl or DMSO served as vehicle controls. Cultures were collected at 4 h and 3 days post-incubation for protein extraction or subjected to live imaging using IncuCyte FLR assay (n = 3) to measure cell proliferation rate over 7 days (A,D). Protein abundance of phosphorylated ACC (S79) (B,E) or S6K (T389) (C,F) relative to total ACC or S6K was quantified using western blot. Blots shown were representative from 3 independent experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Full length blots are presented in Supplementary information Fig. 4.

Figure 10

Effect of Ulk1 inhibition on proliferation in HEK293 and T47D cells. HEK293 and T47D cells were plated in full media containing glutamine for 24 h. Cultures were washed with DPBS and fresh glutamine-depleted media with or without NH₄Cl or SBI0206965 (6965–10 µM) was added. Cells cultured in glutamine-depleted media with or without NH₄Cl or DMSO served as vehicle controls. Cultures were subjected to live imaging using Incucyte FLR assay (n = 3) to measure cell proliferation rate over 7 days (A,B). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.