Multiplicity of steady states in glycolysis and shift of metabolic state in cultured mammalian cells.

Bhanu Chandra Mulukutla,Wei-Shou Hu,Prodromos Daoutidis,Andrew Yongky,Simon Grimm,Jonathan A Coles

doi:10.1371/journal.pone.0121561

Abstract

Cultured mammalian cells exhibit elevated glycolysis flux and high lactate production. In the industrial bioprocesses for biotherapeutic protein production, glucose is supplemented to the culture medium to sustain continued cell growth resulting in the accumulation of lactate to high levels. In such fed-batch cultures, sometimes a metabolic shift from a state of high glycolysis flux and high lactate production to a state of low glycolysis flux and low lactate production or even lactate consumption is observed. While in other cases with very similar culture conditions, the same cell line and medium, cells continue to produce lactate. A metabolic shift to lactate consumption has been correlated to the productivity of the process. Cultures that exhibited the metabolic shift to lactate consumption had higher titers than those which didn’t. However, the cues that trigger the metabolic shift to lactate consumption state (or low lactate production state) are yet to be identified. Metabolic control of cells is tightly linked to growth control through signaling pathways such as the AKT pathway. We have previously shown that the glycolysis of proliferating cells can exhibit bistability with well-segregated high flux and low flux states. Low lactate production (or lactate consumption) is possible only at a low glycolysis flux state. In this study, we use mathematical modeling to demonstrate that lactate inhibition together with AKT regulation on glycolysis enzymes can profoundly influence the bistable behavior, resulting in a complex steady-state topology. The transition from the high flux state to the low flux state can only occur in certain regions of the steady state topology, and therefore the metabolic fate of the cells depends on their metabolic trajectory encountering the region that allows such a metabolic state switch. Insights from such switch behavior present us with new means to control the metabolism of mammalian cells in fed-batch cultures.

Highlights

Glucose metabolism plays a central role in supplying carbon precursors for cellular energy and biosynthetic needs
Only one steady state is observed for a given glucose concentration; below 0.5 mM, glycolysis has only the low flux state, whereas above 2.2 mM, only the high flux state exists
Using a mechanistic model we have shown previously that with the combination of isozymes typically seen in proliferating cells, the glycolysis flux exhibits classical steady state multiplicity [5]

Summary

Introduction

Glucose metabolism plays a central role in supplying carbon precursors for cellular energy and biosynthetic needs. Cancer cells have elevated glucose consumption and glycolytic flux in ways similar to the response of tissues to growth promoting signals [1]. Cellular glucose metabolism is subjected to vast interacting regulations exerted at various levels [2,3,4]. Many enzymatic steps are controlled through feedback and feed-forward allosteric regulation by metabolic intermediates [5]. The regulatory effectors and control action on the enzyme kinetics differ for different isozymes catalyzing the same reaction step. Cells in different tissues and even cells at different disease or development stages, may express different isozymes to meet their cellular demands [6, 7]. Through signaling pathways, glycolysis activity is tied to growth control [2, 3]. In the past decade there has been an increasing interest in controlling a cell’s disease state, for instance to minimize uncontrolled proliferation through modulation of cellular metabolism

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Conclusion