Elevated glutamate dehydrogenase flux in glucose-deprived hybridoma and myeloma cells: Evidence from1H/15N NMR

Abstract

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 4+ and 15N‐alanine. Thus, NH 4+ formed via glutaminase (GLNase) could be distinguished from NH 4+ formed via glutamate dehydrogenase (GDH). In the glucose‐sufficient cells a small but measurable amount of 15NH 4+ 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 4+ was significantly increased in both fructose‐grown hybridoma and glucose‐starved myeloma cells, as compared to the glucose‐sufficient cells. The increased NH 4+ 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.

Growth and metabolism of marine fish Chinook salmon embryo cells: response to lack of glucose and glutamine

“…The unchanged activities of GOT and GPT and increased activity of GDH by 24% meant an increased flux via the GDH route, which matched to the increase of Y Amm/Gln above mentioned. A similar result was reported (Martinelle et al 1998) that GDH flux increased under glucose-limited conditions in hybridoma cell culture.…”

“…In the glutamine-free culture, Q Glc kept constant, Q Lac and Y Lac/Glc decreased by 17% and 20% respectively, showing that lack of glutamine did not expedite glucose consumption but make it shift to lower lactate production and more efficient energy metabolism. This result differed from pulse culture of baby hamster kidney (BHK) cells, whose Q Glc reduced with the decrease of glutamine concentration (Martinelle et al 1998). …”

Expression of anti‐apoptosis genes alters lactate metabolism of Chinese Hamster Ovary cells in culture

“…These results are indicative of heightened TCA cycle energetics in cell lines expressing anti-apoptotic genes although an exact interpretation of the cellular reactions awaits more detailed metabolic flux analysis (Altamirano et al, 2001;Martinelle et al, 1998). Irrespective of the fate of the consumed lactate, this is the first time it has been demonstrated that apoptotic R cell lines consume accumulated lactate, which in turn, presumably aids in their increased longevity and IVCC.…”

“…Numerous strategies have been devised to address the accumulation of excessive lactate build-up including (1) maintaining low medium glucose concentrations (Kurokawa et al, 1994;Xie and Wang, 1993;Zhang et al, 2004;Zhou et al, 1995), (2) feeding alternative sugars, including fructose (Martinelle et al, 1998, Altamirano et al, 2004Wlaschin and Hu, 2007), (3) partially knocking out lactate dehydrogenase (LDH) expression by homologous recombination or siRNA technology (Chen et al, 2001;Kim and Lee, 2007a); (4) over-expression of pyruvate carboxylase (Kim and Lee, 2007b); (5) use of dichloracetate (DCA), a pyruvate dehydrogenase (PDH) activator (via PDH kinase inhibition) (Stacpoole et al, 2003) and (6) oxamic acid, an LDH competitive inhibitor (Mothersill and Seymour, 1986). While the above strategies have been partially successful in reducing the lactate concentration, an alternative approach to consider is stimulating mitochondrial respiration in order to enhance culture performance.…”

Metabolomics and Mammalian Cell Culture

“…The accumulation of fermentation by-products causes a reduction of the culture density and product titer that can be realized (Martinelle et al, 1998).…”