Metabolic characterization of cultured mammalian cells by mass balance analysis, tracer labeling experiments and computer-aided simulations.
Studying metabolic directions and flow rates in cultured mammalian cells can provide key information for understanding metabolic function in the fields of cancer research, drug discovery, stem cell biology, and antibody production. In this work, metabolic engineering methodologies including medium component analysis, (13)C-labeling experiments, and computer-aided simulation analysis were applied to characterize the metabolic phenotype of soft tissue sarcoma cells derived from p53-null mice. Cells were cultured in medium containing [1-(13)C] glutamine to assess the level of reductive glutamine metabolism via the reverse reaction of isocitrate dehydrogenase (IDH). The specific uptake and production rates of glucose, organic acids, and the 20 amino acids were determined by time-course analysis of cultured media. Gas chromatography-mass spectrometry analysis of the (13)C-labeling of citrate, succinate, fumarate, malate, and aspartate confirmed an isotopically steady state of the cultured cells. After removing the effect of naturally occurring isotopes, the direction of the IDH reaction was determined by computer-aided analysis. The results validated that metabolic engineering methodologies are applicable to soft tissue sarcoma cells derived from p53-null mice, and also demonstrated that reductive glutamine metabolism is active in p53-null soft tissue sarcoma cells under normoxia.
Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address: n-okahashi@ist.osaka-u.ac.jp.
Nobuyuki
N
Okahashi
Division of Oncology and Molecular Biology, Cancer Research Institute, Kanazawa University, Kakuma, Kanazawa, Ishikawa 920-1192, Japan. Electronic address: skohno@staff.kanazawa-u.ac.jp.
Susumu
S
Kohno
Division of Oncology and Molecular Biology, Cancer Research Institute, Kanazawa University, Kakuma, Kanazawa, Ishikawa 920-1192, Japan. Electronic address: skitajim@staff.kanazawa-u.ac.jp.
Shunsuke
S
Kitajima
Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address: fmatsuda@ist.osaka-u.ac.jp.
Fumio
F
Matsuda
Division of Oncology and Molecular Biology, Cancer Research Institute, Kanazawa University, Kakuma, Kanazawa, Ishikawa 920-1192, Japan. Electronic address: chtakaha@staff.kanazawa-u.ac.jp.
Chiaki
C
Takahashi
Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address: shimizu@ist.osaka-u.ac.jp.
Hiroshi
H
Shimizu
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D045744:Cell Line, Tumor; D003198:Computer Simulation; D008401:Gas Chromatography-Mass Spectrometry; D005947:Glucose / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism*; D007521:Isocitrate Dehydrogenase / Q000378:metabolism; D008297:Male; D060847:Metabolic Engineering; D051379:Mice; D010100:Oxygen / Q000378:metabolism; D012509:Sarcoma / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology*; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000172:deficiency
D000596:Amino Acids; D016159:Tumor Suppressor Protein p53; D005973:Glutamine; D007521:Isocitrate Dehydrogenase; D005947:Glucose; D010100:Oxygen
(13)C-tracer experiment; Cultured mammalian cell; Metabolic engineering; Reductive glutamine metabolism; Soft tissue sarcoma; p53