Abstract
Reports suggest that excessive ceramide accumulation in mitochondria is required to initiate the intrinsic apoptotic pathway and subsequent cell death, but how ceramide accumulates is unclear. Here we report that liver mitochondria exhibit ceramide formation from sphingosine and palmitoyl-CoA and from sphingosine and palmitate. Importantly, this activity was markedly decreased in liver from neutral ceramidase (NCDase)-deficient mice. Moreover, the levels of ceramide were dissimilar in liver mitochondria of WT and NCDase KO mice. These results suggest that NCDase is a key participant of ceramide formation in liver mitochondria. We also report that highly purified liver mitochondria have ceramidase, reverse ceramidase, and thioesterase activities. Increased accessibility of palmitoyl-CoA to the mitochondrial matrix with the pore-forming peptide zervamicin IIB resulted in 2-fold increases in palmitoyl-CoA hydrolysis by thioesterase. This increased hydrolysis was accompanied by an increase in ceramide formation, demonstrating that both outer membrane and matrix localized thioesterases can regulate ceramide formation. Also, ceramide formation might occur both in the outer mitochondrial membrane and in the mitochondrial matrix, suggesting the existence of distinct ceramide pools. Taken together, these results suggest that the reverse activity of NCDase contributes to sphingolipid homeostasis in this organelle in vivo.
Highlights
Reports suggest that excessive ceramide accumulation in mitochondria is required to initiate the intrinsic apoptotic pathway and subsequent cell death, but how ceramide accumulates is unclear
Mitochondria from neutral ceramidase (NCDase)-deficient mice have significantly decreased formation of ceramide from sphingosine and palmitoyl-CoA compared with mitochondria from WT mice, indicating that NCDase participates in ceramide formation in liver mitochondria and that ceramide formation may occur from sphingosine and palmitoyl-CoA from coupled activities of a mitochondrial thioesterase and NCDase catalyzing the reverse reaction
Ceramide is considered to be the central hub of sphingolipid metabolism [1], a trigger of cell death pathways associated with mitochondrial dysfunction [2, 3, 5, 70, 71]
Summary
THIOESTERASE AND NEUTRAL CERAMIDASE PRODUCE CERAMIDE FROM SPHINGOSINE AND ACYL-CoA*□S. We report that liver mitochondria exhibit ceramide formation from sphingosine and palmitoyl-CoA and from sphingosine and palmitate This activity was markedly decreased in liver from neutral ceramidase (NCDase)-deficient mice. Ceramide formation might occur both in the outer mitochondrial membrane and in the mitochondrial matrix, suggesting the existence of distinct ceramide pools Taken together, these results suggest that the reverse activity of NCDase contributes to sphingolipid homeostasis in this organelle in vivo. We report that highly purified rat/mice liver mitochondria have ceramidase, reverse ceramidase, and thioesterase (which hydrolyzes palmitoyl-CoA to CoA and fatty acid) activities. Mitochondria from NCDase-deficient mice have significantly decreased formation of ceramide from sphingosine and palmitoyl-CoA (or palmitate) compared with mitochondria from WT mice, indicating that NCDase participates in ceramide formation in liver mitochondria and that ceramide formation may occur from sphingosine and palmitoyl-CoA from coupled activities of a mitochondrial thioesterase and NCDase catalyzing the reverse reaction
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