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13 C and 31 P NMR investigation of pyruvate dehydrogenase complex (PDC)‐deficient human fibroblasts
Author(s) -
Simpson Nicholas,
Han Zongchao,
Berendzen Kristen,
Sweeney Carol,
OcaCossio Jose,
Constantinidis Ioannis,
Stacpoole Peter
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a921-a
Subject(s) - glycolysis , pyruvate dehydrogenase complex , metabolism , pyruvate carboxylase , citric acid cycle , biochemistry , lactate dehydrogenase , lactic acidosis , lactic acid , carbohydrate metabolism , mitochondrion , chemistry , biology , enzyme , bacteria , genetics
Mitochondrial PDC is integral to cellular metabolism and energetics. PDC deficiency is a common cause of congenital lactic acidosis, neurological degeneration and early death. Dichloroacetate (DCA) activates PDC and is an investigational drug for this disease. We used NMR spectroscopy and isotopomeric analysis to characterize the independent effects of PDC deficiency on glucose metabolism and energetics in primary cultures of human fibroblasts from 5 healthy subjects and 5 patients with mutations in the E1 component of PDC. Cells were grown in media ± DCA, then in media containing uniformly labeled 13 C‐glucose. Rates of glucose consumption (GCR) and lactate production (LPR) were measured. Fluxes through the PDC, pyruvate carboxylase (PC) and the Krebs cycle were estimated by NMR analysis of cell extracts and computer analysis of the isotopomeric patterns of glutamate. PDC deficient cells had significantly higher GCR, LPR and fraction of acetyl‐CoA derived from the label, indicating increased glycolysis vs. controls. In both healthy and defective cells, DCA caused major (~ 40%) decreases in anaplerotic‐related pathways (e.g., PC), and an increase in flux through PDC. DCA decreased glycolysis (29%) in patient cells but had no effect in control cells and did not change LPR or ATP/ADP in either cell type. Maintenance of ATP despite reduced glycolysis indicates that DCA improves metabolic efficiency by increasing glucose oxidation. We conclude that NMR provides novel insight into the biochemical consequences of PDC deficiency and putative therapeutic agents. Supported by NIH DK56890 and DK47858