Examination of primary metabolic pathways in a murine hybridoma with carbon‐13 nuclear magnetic resonance spectroscopy

Abstract

Primary metabolism of a murine hybridoma was probed with (13)C nuclear magnetic resonance (NMR) spectroscopy. Cells cultured in a hollow fiber bioreactor were serially infused with [1-(13)C] glucose, [2-(13)C] glucose, and [3-(13)C] glutamine. In vivo spectroscopy of the culture was used in conjunction with off-line spectroscopy of the medium to determine the intracellular concentration of several metabolic intermediates and to determine fluxes for primary metabolic pathways. Intracellular concentrations of pyruvate and alanine were very high relative to levels observed in normal quiescent mammalian cells. Estimates made from labeling patterns in lactate indicate that 76% of pyruvate is derived directly from glycolysis; some is also derived from the malate shunt, the pyruvate/melate shuttle associated with lipid synthesis and the pentose phosphate pathway. The rate of formation of pyruvate from the pentose phosphate pathway was estimated to be 4% of that from glycolysis; This value is a lower limit and the actual value may be higher. Incorporation of pyruvate into the tricarboxylic acid (TCA) cycle appears to occur through only pyruvate dehydrogenase; no pyruvate carboxylase activity was detected. The malate shunt rate was approximately equal to the rate of glutamine uptake. The rate of incorporation of glucosederived acetyl-CoA into lipids was 4% of the glucose uptake rate. The TCA cycle rate between isocitrate and alpha-ketoglutarate was 110% of the glutamine uptake rate.

Enhanced glycoprotein production in HEK-293 cells expressing pyruvate carboxylase

“…The analysis revealed that, at the pyruvate branch point, the parental cells converted 51% of the pyruvate pool into acetyl-CoA via the pyruvate dehydrogenase, 48% to lactate via lactate dehydrogenase and the remaining fraction to amino acids (mainly alanine and glycine). Our data suggested little to no significant flux through the pyruvate carboxylase, confirming an observation made by previous studies on several mammalian cells in culture (Bonarius et al, 2001;Mancuso et al, 1994;Metallo et al, 2009). In contrast, the PYC-expressing cells converted 56% of the pyruvate into acetyl-CoA, 26% into oxaloacetate via the pyruvate carboxylase and only 9% into lactate.…”

“…Another approach consisted of the expression of a yeast pyruvate carboxylase (PYC) gene in mammalian cells to compensate for the apparent lack of enzymatic activities linking glycolysis and the TCA cycle. This enzyme, catalyzing the anaplerotic conversion of pyruvate to oxaloacetate, is naturally present in mammalian cells, but several isotopic tracer studies revealed that pyruvate incorporation into the TCA mainly occurs via the pyruvate dehydrogenase reaction (Bonarius et al, 2001;Mancuso et al, 1994;Metallo et al, 2009). PYC expression was shown to have a profound effect on the metabolism of several relevant cell lines, including BHK-21 cells (Irani et al, 1999), HEK-293 cells (Elias et al, 2003) and CHO cells (Kim and Lee, 2007).…”

ACSS2-mediated acetyl-CoA synthesis from acetate is necessary for human cytomegalovirus infection

“…6A). PDH E1 is the rate-limiting multimeric subunit in the PDH complex; depletion of its E1α subunit causes loss of activity (31). As expected, PDH inactivation increased the levels of [3][4][5][6][7][8][9][10][11][12][13] C]-pyruvate derived from [1,[6][7][8][9][10][11][12][13] C2]-D-glucose in both mock-and, to a greater extent, HCMV-infected cells (Fig.…”

Reduction of Proteinuria through Podocyte Alkalinization

“…Higher lactate production rates in disease models are the results of higher use of glucose than the control model based on the assumption that lactate generated by cultured cells was only derived from glucose. Our assumption was largely supported by NMR experiments with hybridoma cells (43,44). The amount of lactate derived from glutamine was not determined in our studies.…”