Glutamate production from ammonia via glutamate dehydrogenase 2 activity supports cancer cell proliferation under glutamine depletion

https://doi.org/10.1016/j.bbrc.2017.11.088Get rights and content

Highlights

  • Ammonia allows cell growth under glutamine depleted conditions.

  • GDH2 synthesizes glutamate from ammonia and α-KG.

  • Cancer cells proliferate depending on GDH2 in response to nutrient limitation.

Abstract

Cancer cells rapidly consume glutamine as a carbon and nitrogen source to support proliferation, but the cell number continues to increase exponentially after glutamine is nearly depleted from the medium. In contrast, cell proliferation rates are strongly depressed when cells are cultured in glutamine-free medium. How cancer cells survive in response to nutrient limitation and cellular stress remains poorly understood. In addition, rapid glutamine catabolism yields ammonia, which is a potentially toxic metabolite that is secreted into the extracellular space. Here, we show that ammonia can be utilized for glutamate production, leading to cell proliferation under glutamine-depleted conditions. This proliferation requires glutamate dehydrogenase 2, which synthesizes glutamate from ammonia and α-ketoglutarate and is expressed in MCF7 and T47D cells. Our findings provide insight into how cancer cells survive under glutamine deprivation conditions and thus contribute to elucidating the mechanisms of tumor growth.

Introduction

Glutamine is the major nitrogen source for nonessential amino acids, nucleotides, and hexosamines [1], [2], [3]. Although glutamine can be synthesized in most tissues, demand often outpaces supply, and glutamine typically becomes an essential nutrient for proliferating cells [4], [5]. Glutamine is metabolized to glutamate by glutaminase (GLS), which releases the amide nitrogen of glutamine as ammonia [6]. Then, glutamate is converted to α-ketoglutarate (α-KG), an intermediate metabolite in the TCA cycle, by two types of reactions. In one, glutamate is deaminated by glutamate dehydrogenase (GDH), releasing ammonia in the mitochondria. In the other, glutamate is transaminated to produce nonessential amino acids by transaminases in either the mitochondria or cytosol [7].

GDH is a housekeeping gene that is widely conserved in many species. GDH normally catalyzes glutamate to α-KG and ammonia, employing nicotinamide adenine dinucleotide phosphate (NADP+) and nicotinamide adenine dinucleotide (NAD+) as cofactors. However, GDH can also catalyze reductive amination to produce glutamate from α-KG and ammonia, employing NADPH and NADH as cofactors, depending on the environment [8], [9]. Humans possess two GDH isoforms —GDH1 and GDH2 (encoded by the GLUD1 and GLUD2 genes, respectively)—that have high sequence similarity. GDH1 is widely expressed in many tissues, and GDH2 is expressed in the brain, testis, embryonic tissue and various cancers [10], [11]. GDH1 is upregulated in human cancers and plays an essential role in redox homeostasis by controlling intracellular α-KG levels [12]. In contrast, the roles of GDH2 in cancer cells remain elusive.

Ammonia is produced during glutamine catabolism, which is the conversion of glutamine to α-KG in mitochondria, and is actively or passively exported from the cells [13]. Ammonia is also an important nitrogen source that is involved in amino acid metabolism, protein synthesis and pH homeostasis [14], [15]. However, the relationship between glutamine metabolism and GDH functions remains unknown.

Here, we show that cancer cells rapidly consume glutamine and secrete ammonia during the growth phase and that glutamine limitation suppresses cancer cell proliferation. Strikingly, this proliferation impairment can be partially overcome by supplementing cells with ammonia, depending on the cellular level of GDH2, which synthesizes glutamate from ammonia and α-KG. Our findings provide insight into how cancer cells survive under glutamine deprivation conditions and contribute to elucidating the mechanisms of cancer cell proliferation.

Section snippets

Metabolite extraction and derivatization

Cells were quenched with chilled 80% methanol supplemented with 5 μg of sinapic acid as an internal standard at −80 °C for 15 min after washing with ice-cold phosphate-buffered saline (PBS). Then, the cells were harvested. After centrifugation to remove cell debris, the supernatant was freeze-dried. The culture medium was also collected and centrifuged, and the supernatant was freeze-dried. The metabolite levels in the medium were compared with those measured in the control medium not exposed

Ammonia utilization stimulates glutamate synthesis and enables cells to proliferate under glutamine-depleted conditions

To understand the role of cellular metabolism during cancer cell proliferation, we initially cultured MCF7 cells and quantified the changes in metabolite levels in the culture medium. We confirmed that as glutamine and glucose levels decreased, lactate production increased in a cell number-dependent manner, consistent with previous reports and with the Warburg effect (Fig. S1). Because MCF7 cells grew even when most of the glutamine was consumed after 72 h of incubation, we investigated whether

Discussion

In this study, we showed that cancer cells can survive even under glutamine-limited conditions and that the ability to utilize ammonia enables specific types of cancer cells to partially restore proliferation under glutamine starvation. In such cells, GDH2 can induce increased intracellular levels of glutamate, which is synthesized from ammonia and α-KG. In contrast, in cells expressing low levels of GDH2, ammonia supplementation had no effect on proliferation in the absence of glutamine.

Acknowledgments

This research was supported by a Grant-in-Aid for Scientific Research (C) 16K07115 to Y.I. and partially supported by AMED-CREST from the Japan Agency for Medical Research and Development to E.F. The authors declare that there are no conflicts of interest.

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