Abstract
Cardiolipin (CL) is the signature phospholipid of mitochondrial membranes. Although it has long been known that CL plays an important role in mitochondrial bioenergetics, recent evidence in the yeast model indicates that CL is also essential for intermediary metabolism. To gain insight into the function of CL in energy metabolism in mammalian cells, here we analyzed the metabolic flux of [U-13C]glucose in a mouse C2C12 myoblast cell line, TAZ-KO, which is CL-deficient because of CRISPR/Cas9-mediated knockout of the CL-remodeling enzyme tafazzin (TAZ). TAZ-KO cells exhibited decreased flux of [U-13C]glucose to [13C]acetyl-CoA and M2 and M4 isotopomers of tricarboxylic acid (TCA) cycle intermediates. The activity of pyruvate carboxylase, the predominant enzyme for anaplerotic replenishing of the TCA cycle, was elevated in TAZ-KO cells, which also exhibited increased sensitivity to the pyruvate carboxylase inhibitor phenylacetate. We attributed a decreased carbon flux from glucose to acetyl-CoA in the TAZ-KO cells to a ∼50% decrease in pyruvate dehydrogenase (PDH) activity, which was observed in both TAZ-KO cells and cardiac tissue from TAZ-KO mice. Protein-lipid overlay experiments revealed that PDH binds to CL, and supplementing digitonin-solubilized TAZ-KO mitochondria with CL restored PDH activity to WT levels. Mitochondria from TAZ-KO cells exhibited an increase in phosphorylated PDH, levels of which were reduced in the presence of supplemented CL. These findings indicate that CL is required for optimal PDH activation, generation of acetyl-CoA, and TCA cycle function, findings that link the key mitochondrial lipid CL to TCA cycle function and energy metabolism.
Highlights
Cardiolipin (CL) is the signature phospholipid of mitochondrial membranes
To investigate metabolic changes resulting from tafazzin deficiency, TAZ-KO and isogenic WT cells were cultured in medium containing [U-13C]glucose, and glycolytic and tricarboxylic acid (TCA) pathway metabolites in cell extracts were analyzed by LC-MS and GC-MS. [U-13C]glucose was metabolized quickly to glycolytic intermediates during fermentation in both WT and TAZ-KO cells
To investigate the possibility that pyruvate dehydrogenase (PDH) is activated by CL, levels of which are reduced in TAZ-KO cells, digitonin-solubilized mitochondria from TAZ-KO and WT cells were incubated with exogenous CL
Summary
To investigate metabolic changes resulting from tafazzin deficiency, TAZ-KO and isogenic WT cells were cultured in medium containing [U-13C]glucose, and glycolytic and TCA pathway metabolites in cell extracts were analyzed by LC-MS and GC-MS. [U-13C]glucose was metabolized quickly to glycolytic intermediates during fermentation in both WT and TAZ-KO cells. Activity of the TCA cycle enzyme MDH was not affected in mutant mitochondria (Fig. 5B) Taken together, these experiments indicate that carbon flux from glucose to TCA cycle intermediates is reduced in TAZ-KO cells. These experiments indicate that carbon flux from glucose to TCA cycle intermediates is reduced in TAZ-KO cells This defect is partially compensated by up-regulation of PC, but compensation is limited by decreased SDH activity in the mutant. As glucose-derived pyruvate is the primary source of carbon for the generation of acetyl-CoA by PDH, decreased [13C]acetyl-CoA in TAZ-KO cells suggested that PDH activity was decreased in the mutant To test this possibility, PDH was assayed in mitochondrial extracts from TAZ-KO and WT cells. When treated with the PDH kinase inhibitor dichloroacetate (DCA), which activates PDH flux, [14C]CO2 production in TAZ-KO cells was restored to WT levels (Fig. 6D), suggesting that the reduced PDH flux in TAZ-KO cells is due to active inhibition rather than impaired capacity
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