Novel pathway of ceramide production in mitochondria: thioesterase and neutral ceramidase produce ceramide from sphingosine and acyl-CoA

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.

FIGURE 1.

Percoll-purified mitochondria display minimal contamination with other cellular membrane compartments. Panel A, shown are rat liver fractions. Panel B, shown are mouse liver fractions. A Western blot for Na⁺, K⁺ATPase (plasma membrane marker), LAMP-2 (lysosomal marker), calnexin (ER marker), and VDAC (mitochondrial marker) was performed by loading an equal amount (30 μg) of homogenate (H) or mitochondrial (M) protein per lane, except rat VDAC was assessed using protein loading of 15 μg per lane. The blot is representative of three independent experiments.

FIGURE 2.

Ceramide is produced from sphingosine and palmitoyl-CoA by purified rat liver mitochondria at isotonic conditions. Panel A, ceramide formation as a function of protein concentration is shown. Mitochondria at the indicated amounts were incubated as described under “Experimental Procedures” in the presence of 50 μm palmitoyl-CoA and 15 μm sphingosine for 15 min before termination of the reaction by addition of ethyl acetate/isopropyl alcohol extraction mixture. Where indicated, Triton X-100 (final concentration 0.5%) was added simultaneously with mitochondria. Panel B, absorption of mitochondrial suspension as a function of protein concentration in the absence or in the presence of 15 μm sphingosine is shown. Absorption was measured 15 min after sphingosine addition. Panel C, time course of ceramide formation is shown. Mitochondria (1 mg/ml) were incubated as described under “Experimental Procedures” in the presence of 50 μm palmitoyl-CoA and 15 μm sphingosine for the indicated times before termination of the reaction by the addition of ethyl acetate/isopropyl alcohol extraction mixture. Results represent the average ±S.D. (n = 3).

FIGURE 3.

Effect of inhibitors of sphingolipid metabolism on ceramide production from sphingosine and palmitoyl-CoA by rat liver mitochondria. Mitochondria (1 mg/ml) were incubated as described under “Experimental Procedures” in the presence of 50 μm palmitoyl-CoA and 15 μm sphingosine, except that in column 2 palmitoyl-CoA was omitted. After 15 min the reaction was terminated by the addition of ethyl acetate/isopropyl alcohol extraction mixture. CerS inhibitor FB1 (50 μm), NCDase inhibitor urea-C₆-ceramide (50 μm), acid CDase inhibitor N-oleoylethanolamine (NOE, 100 μm), and the inhibitor of alkaline CDase d-MAPP (20 μm) were added simultaneously with mitochondria and incubated for 5 min before substrates addition. Results are expressed as a % of the ceramide formed in the presence of palmitoyl-CoA and sphingosine and represent the average ± S.D. *, p < 0.05, n = 3.

FIGURE 4.

Effect of thioesterase inhibitors on ceramide production from sphingosine and palmitoyl-CoA by rat liver mitochondria. Mitochondria (1 mg/ml) were incubated as described under “Experimental Procedures” in the presence of 50 μm palmitoyl-CoA and 15 μm sphingosine for 15 min before the reaction was terminated by the addition of ethyl acetate/isopropyl alcohol extraction mixture. Where indicated, NDGA (100 μm), p-chloromercuribenzoic acid (PCMB; 40 μm), MnCl₂ (1.5 mm), and NiCl₂ (1.5 mm) were added simultaneously with mitochondria and incubated for 5 min before substrate addition. In experiments with MnCl₂ and NiCl₂, EDTA was omitted from incubation medium. Results are expressed as a % of ceramide formed in the presence of palmitoyl-CoA and sphingosine and represent the average ±S.D. *, p < 0.05, n = 3.

FIGURE 5.

Liver mitochondria from WT mouse and rat display ceramidase activity at neutral pH and contain NCDase. Panel A, mitochondria from WT mouse, NCDase KO mouse, and rat were treated and subjected to neutral ceramidase activity assay as described under “Experimental Procedures” using d-erythro-C₁₂-ceramide as a substrate. The results represent the average ±S.D. *, p < 0.05, n = 3. Panel B, Western blot (IB) analysis shows the presence of NCDase in WT mouse and rat mitochondria but not in mitochondria from NCD KO mouse. VDAC was used as a loading control. The blot is representative of three independent experiments.

FIGURE 6.

Dose-response curve (A) and FB1 sensitivity (B) of ceramide formation from sphingosine and palmitate by rat liver mitochondria. Panel A, mitochondria (1 mg/ml) were incubated with 15 μm sphingosine and the indicated amounts of palmitate at the conditions described in “Experimental Procedures” for 15 min before the reaction was terminated by addition of ethyl acetate/ isopropyl alcohol extraction mixture. Results represent the average ±S.D., n = 3. In panel B, where indicated, 50 μm palmitate, 15 μm sphingosine, and 50 μm FB1 (simultaneously with mitochondria) were added to incubation medium. Results are expressed as a % of the ceramide formed in the presence of palmitate and sphingosine (Sph) and represent the average ±S.D., n = 3.

FIGURE 7.

Knock-out of NCDase suppresses formation of ceramide from sphingosine and palmitoyl-CoA (panel A) or palmitate (panel B) in mouse liver mitochondria. Mitochondria (1 mg/ml) were incubated as indicated with 50 μm palmitoyl-CoA, 50 μm palmitate (Palm), and 15 μm sphingosine (Sph) under conditions described under “Experimental Procedures” for 15 min before the reaction was terminated by addition of ethyl acetate/isopropyl alcohol extraction mixture. The results represent the average ± S.D. *, p < 0.05, n = 3.

FIGURE 8.

Liver mitochondria from rat display thioesterase activity, which is enhanced in the presence of pore-forming peptide zervamicin IIB. Rat mitochondrial thioesterase activity was assayed as described under “Experimental Procedures.” Traces are corrected for the initial rapid drop in absorbance caused by the addition of palmitoyl-CoA, which reflects interaction of this compound with the mitochondrial membranes (96). Trace 1, thioesterase activity in the presence of pore-forming peptide zervamicin IIB (20 μg/ml); trace 2, no pretreatment; trace 3, suppression of thioesterase activity by 100 μm of NDGA. The trace was corrected for the time-dependant change in absorbance induced by simultaneous presence of NDGA and palmitoyl-CoA; trace 4, control for absorbance changes for traces 1 and 2 induced by palmitoyl-CoA in the absence of DTNB. The plot is representative of three independent experiments.

FIGURE 9.

Permeabilization in the mitochondrial inner membrane with pore-forming peptide zervamicin IIB increases ceramide production from palmitoyl-CoA and sphingosine in rat liver mitochondria. Mitochondria (RLM, 1 mg/ml) were incubated as described under “Experimental Procedures” in the presence of 50 μm palmitoyl-CoA (Palm-CoA) and 15 μm sphingosine (Sph) for 15 min before the reaction was terminated by addition of ethyl acetate/isopropyl alcohol extraction mixture. Where indicated, zervamicin IIB (20 μg/ml) was added simultaneously with mitochondria. In experiments where mitochondrial swelling was induced by hypotonic treatment, the medium consisted of 10 mm HEPES, 5 μm rotenone, and 500 μm EDTA (pH 7.4 adjusted by KOH). Panel A, ceramide production by mitochondria is shown. Panel B, absorbance of mitochondrial suspension at 520 nm is shown.