http://utsc.utoronto.ca/%7Ebritto/publications/amtox.pdf

NH4+ toxicity in higher plants: a critical review

The inhibition of the Anammox process: A review

Textbook of biochemistry with clinical correlations , 4th edn TM Devlin, ed pp xvii + 1186, illustrated Wiley-Liss, New York, 1997 £2995, hardback

This beautifully illustrated and well-written book, with an impressive array of authors, is aimed at both undergraduate and postgraduate level As the editor states in the preface, it is not intended to be a compendium of biochemistry but rather emphasises the biochemistry of mammalian cells The first 22 chapters cover …

Textbook of biochemistry with clinical correlations

Cancer cells are frequently confronted with metabolic stress in tumor microenvironments due to their rapid growth and limited nutrient supply. Metabolic stress induces cell death through ROS-induced apoptosis. However, cancer cells can adapt to it by altering the metabolic pathways. AMPK and AKT are two primary effectors in response to metabolic stress: AMPK acts as an energy-sensing factor which rewires metabolism and maintains redox balance. AKT broadly promotes energy production in the nutrient abundance milieu, but the role of AKT under metabolic stress is in dispute. Recent studies show that AMPK and AKT display antagonistic roles under metabolic stress. Metabolic stress-induced ROS signaling lies in the hub between metabolic reprogramming and redox homeostasis. Here, we highlight the cross-talk between AMPK and AKT and their regulation on ROS production and elimination, which summarizes the mechanism of cancer cell adaptability under ROS stress and suggests potential options for cancer therapeutics.

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ROS signaling under metabolic stress: cross-talk between AMPK and AKT pathway.

Oncogenes influence nutrient metabolism and nutrient dependence. The oncogene c-Myc stimulates glutamine metabolism and renders cells dependent on glutamine to sustain viability ("glutamine addiction"), suggesting that treatments targeting glutamine metabolism might selectively kill c-Myc-transformed tumor cells. However, many current or proposed cancer therapies interfere with the metabolism of glucose, not glutamine. Here, we studied how c-Myc-transformed cells maintained viability when glucose metabolism was impaired. In SF188 glioblastoma cells, glucose deprivation did not affect net glutamine utilization but elicited a switch in the pathways used to deliver glutamine carbon to the tricarboxylic acid cycle, with a large increase in the activity of glutamate dehydrogenase (GDH). The effect on GDH resulted from the loss of glycolysis because it could be mimicked with the glycolytic inhibitor 2-deoxyglucose and reversed with a pyruvate analogue. Furthermore, inhibition of Akt signaling, which facilitates glycolysis, increased GDH activity whereas overexpression of Akt suppressed it, suggesting that Akt indirectly regulates GDH through its effects on glucose metabolism. Suppression of GDH activity with RNA interference or an inhibitor showed that the enzyme is dispensable in cells able to metabolize glucose but is required for cells to survive impairments of glycolysis brought about by glucose deprivation, 2-deoxyglucose, or Akt inhibition. Thus, inhibition of GDH converted these glutamine-addicted cells to glucose-addicted cells. The findings emphasize the integration of glucose metabolism, glutamine metabolism, and oncogenic signaling in glioblastoma cells and suggest that exploiting compensatory pathways of glutamine metabolism can improve the efficacy of cancer treatments that impair glucose utilization.

https://cancerres.aacrjournals.org/content/canres/69/20/7986.full.pdf

Glioblastoma Cells Require Glutamate Dehydrogenase to Survive Impairments of Glucose Metabolism or Akt Signaling

Mechanisms of ammonia and ammonium ion toxicity in animal cells: transport across cell membranes.

The glutamine metabolism was studied in glucose-starved and glucose-sufficient hybridoma and Sp2/0-Ag14 myeloma cells. Glucose starvation was attained by cultivating the hybridoma cells with fructose instead of glucose, and the myeloma cells with a low initial glucose concentration which was rapidly exhausted. Glutamine used in the experiments was labeled with 15N, either in the amine or in the amide position. The fate of the label was monitored by 1H/15N NMR analysis of released 15NH and 15N-alanine. Thus, NH formed via glutaminase (GLNase) could be distinguished from NH formed via glutamate dehydrogenase (GDH). In the glucose-sufficient cells a small but measurable amount of 15NH released by GDH could be detected in both cell lines (0.75 and 0.31 μmole/106 cells for hybridoma and myeloma cells, respectively). The uptake of glutamine and the total production of NH was significantly increased in both fructose-grown hybridoma and glucose-starved myeloma cells, as compared to the glucose-sufficient cells. The increased NH production was due to an increased throughput via GLNase (1.6 –1.9-fold in the hybridoma, and 2.7-fold in the myeloma cell line) and an even further increased metabolism via GDH (4.8–7.9-fold in the hybridoma cells, and 3.1-fold in the myeloma cells). The data indicate that both GLNase and GDH are down-regulated when glucose is in excess, but up-regulated in glucose-starved cells. It was calculated that the maximum potential ATP production from glutamine could increase by 35–40 % in the fructose-grown hybridoma cells, mainly due to the increased metabolism via GDH. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 60: 508–517, 1998.

Elevated glutamate dehydrogenase flux in glucose-deprived hybridoma and myeloma cells: Evidence from 1H/15N NMR

Ammonium can be transported into the cell by ion pumps in the cytoplasmic membrane. Ammonia then diffuse out through the cell membrane. A futile cycle is created that results in cytoplasmic acidification and extracellular alkalinisation. Ammonium transport can be quantified by measuring the extracellular pH changes occurring in a cell suspension (in PBS) after addition of ammonium. By using this technique, in combination with specific inhibitors of various ion pumps, it was shown that ammonium ions are transported across the cytoplasmic membrane by the Na+K+2Cl--cotransporter in both hybridoma and myeloma cells. Further, the Na+/H+ exchanger, which regulates intracellular pH by pumping out protons, was shown to be active during ammonium exposure. The viability of hybridoma cells suspended in PBS and exposed to NH
 inf4
 sup+
 for only 90 min, was reduced by 11% (50% necrosis and 50% apoptosis). A control cell suspension did not loose viability during this time. Turning off the activity of the Na+/H+ exchanger (by amiloride) during ammonium exposure decreased viability further, while inhibiting transport itself (by bumetanide) restored viability to the same level as for the control experiment with bumetanide alone. These results show that one effect of ammonia/ammonium on cell physiology is specifically related to the inward transport of ammonium ions by membrane bound ion pumps.

Ammonium ion transport-a cause of cell death.

The dissociation constant of NH 4 + , pK a NH 4 + , was used to calculate the ammonia concentration in cell cultures. pK a NH 4 + is 8.95 at 35°C. As an incorrect pK a (e.g. 9.27-9.3 at 37°C) is often used, this results in errors. The probable origin for this appears to be a combination of pK w at 24°C and pK b at 35°C. At 35°C and any pH, the ammonia concen-tration calculated from pK a = 8.95 is twice the ammonia concentration calculated from pK a = 9.27, but the differences in ammonium concentration are negligible.

On the dissociation constant of ammonium: effects of using an incorrect pK a in calculations of the ammonia concentration in animal cell cultures

Potassium ions decrease the transport rate of ammonium ions into myeloma and hybridoma cells, one effect of the involved transport processes being an increased energy demand (Martinelle and Haggstrom, 1993; Martinelle et al., 1998b). Therefore, the effects of K+ and NH4+ on the energy metabolism of the murine myeloma cell line, Sp2/0-Ag14, were investigated. Addition of NH4Cl (10 mM) increased the metabolism via the alanine transaminase (alaTA) pathway, without increasing the consumption of glutamine. As judged by the alanine production, the energy formation from glutamine increased by 155%. The presence of elevated concentrations of KCl (10 mM) was positive, resulting in a decreased uptake of glutamine (45%), and an even larger suppression of ammonium ion formation (70%), while the same throughput via the alaTA pathway (and energy production from glutamine) was retained as in the control culture. However, the simultaneous presence of 10 mM K+ and 10 mM NH4+ was more inhibitory than NH4Cl alone; an effect that could not be ascribed to increased osmolarity. Although the culture with both K+ and NH4+ consumed 60% more glutamine than the culture with NH4+ alone, the energy generation from glutamine could not be increased further, due to the suppression of the glutamate dehydrogenase pathway. Furthermore, the data highlighted the importance of evaluating the metabolism via different energy yielding pathways, rather than solely considering the glutamine consumption for estimating energy formation from glutamine.

Kristina Martinelle

Papers

Mechanisms of ammonia and ammonium ion toxicity in animal cells: transport across cell membranes.

Elevated glutamate dehydrogenase flux in glucose-deprived hybridoma and myeloma cells: Evidence from 1H/15N NMR

Ammonium ion transport-a cause of cell death.

On the dissociation constant of ammonium: effects of using an incorrect pK a in calculations of the ammonia concentration in animal cell cultures

Effects of NH4+ and K+ on the energy metabolism in Sp2/0-Ag14 myeloma cells