A critical role for ceramide synthase 2 in liver homeostasis: I. alterations in lipid metabolic pathways - PubMed (original) (raw)

. 2010 Apr 2;285(14):10902-10.

doi: 10.1074/jbc.M109.077594. Epub 2010 Jan 28.

Hyejung Park, Elad L Laviad, Liana C Silva, Sujoy Lahiri, Johnny Stiban, Racheli Erez-Roman, Britta Brügger, Timo Sachsenheimer, Felix Wieland, Manuel Prieto, Alfred H Merrill Jr, Anthony H Futerman

Affiliations

A critical role for ceramide synthase 2 in liver homeostasis: I. alterations in lipid metabolic pathways

Yael Pewzner-Jung et al. J Biol Chem. 2010.

Abstract

Ceramide is an important lipid signaling molecule that plays critical roles in regulating cell behavior. Ceramide synthesis is surprisingly complex and is orchestrated by six mammalian ceramide synthases, each of which produces ceramides with restricted acyl chain lengths. We have generated a CerS2 null mouse and characterized the changes in the long chain base and sphingolipid composition of livers from these mice. Ceramide and downstream sphingolipids were devoid of very long (C22-C24) acyl chains, consistent with the substrate specificity of CerS2 toward acyl-CoAs. Unexpectedly, C16-ceramide levels were elevated, and as a result, total ceramide levels were unaltered; however, C16-ceramide synthesis in vitro was not increased. Levels of sphinganine were also significantly elevated, by up to 50-fold, reminiscent of the effect of the ceramide synthase inhibitor, fumonisin B1. With the exceptions of glucosylceramide synthase and neutral sphingomyelinase 2, none of the other enzymes tested in either the sphingolipid biosynthetic or degradative pathways were significantly changed. Total glycerophospholipid and cholesterol levels were unaltered, although there was a marked elevation in C18:1 and C18:2 fatty acids in phosphatidylethanolamine, concomitant with a reduction in C18:0 and C20:4 fatty acids. Finally, differences were observed in the biophysical properties of lipid extracts isolated from liver microsomes, with membranes from CerS2 null mice displaying higher membrane fluidity and showing morphological changes. Together, these results demonstrate novel modes of cross-talk and regulation between the various branches of lipid metabolic pathways upon inhibition of very long acyl chain ceramide synthesis.

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Figures

FIGURE 1.

FIGURE 1.

Generation and characterization of CerS2 null mice. A, CerS2 null mice were created by crossing CerS2GT/+ mice generated from a gene trap embryonic stem cell line (stock number 013236-UCD, Mutant Mouse Regional Resource Center). The upper panel shows the Rosafary gene trap retroviral vector used to generate CerS2 gene trap embryonic stem cells (35). A splice acceptor (SA) is located upstream to a β-geo gene (neo resistance + lacZ genes), which is followed by a polyadenylation sequence (pA). A selectable marker for hygromycin (hyg) resistance is expressed under the phosphoglycerate kinase (PGK) promoter, followed by a splice donor (SD) sequence, which is within two frt sites that are recognized by Flp recombinase. Elements of this vector are marked as gray boxes. The vector was inserted between exons 1 and 2 of the CerS2 gene (dashed lines). Exons are marked as black boxes, and translational start and stop sites are shown as arrows. A spliced transcript of the WT locus is also shown. The lower panel shows the integration of the retroviral vector into the CerS2 gene, leading to generation of two transcripts. Transcript 1 is generated by splicing exon 1 of CerS2 (5′-untranslated region) into the Rosafary splice acceptor, resulting in expression of lacZ under the CerS2 promoter. The second transcript is driven by the PGK promoter of the hygromycin gene, which is spliced into the second exon of CerS2. B, CerS2 null mice do not express full-length transcript 2, demonstrated using primers that amplify the full-length CerS2 transcript. C, weight of CerS2 null mice. *, p < 0.01; n = 5.

FIGURE 2.

FIGURE 2.

Ceramide synthesis in CerS2 null mouse liver. The synthesis of C22- or C24-ceramides is highly reduced in the CerS2 null mouse; the inset shows C16-ceramide synthesis, which is unchanged. *, p < 0.05; n = 3.

FIGURE 3.

FIGURE 3.

Ceramide and long chain base levels in CerS2 null mouse liver. Ceramide and sphinganine levels in CerS2 null mice versus age of the mice, measured by ESI-MS/MS. C24:1-ceramide is shown as an example of a very long acyl chain ceramide; data for the other very long acyl chain ceramides are in Table 1. For day 0, n = 4; for other time points, n = 2. *, p < 0.05; **, p < 0.01.

FIGURE 4.

FIGURE 4.

Levels of acyl-CoAs. Levels of fatty acyl-CoAs were analyzed by ESI-MS/MS in 30-day-old WT and CerS2 null mouse livers and are shown as a ratio. n = 2. There were no statistically significant differences between WT and the CerS2 null mouse.

FIGURE 5.

FIGURE 5.

Levels of CerS mRNA expression. mRNA was isolated from 1-month-old WT and CerS2 null mice. n = 5; *, p < 0.005.

FIGURE 6.

FIGURE 6.

Activity of enzymes of ceramide metabolism. Activity was measured in liver homogenates (15–200 μg of protein) using fluorescent (NBD) lipid analogs. n = 3–4; *, p < 0.01.

FIGURE 7.

FIGURE 7.

N-SMase activity. The left panel shows reverse transcription-PCR of N-SMase 1 and 2 in WT versus CerS2 null mice at 1 month of age; hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT) was used as a control for RNA isolation and cDNA synthesis. The right panel shows N-SMase activity. n = 3; *, p < 0.05.

FIGURE 8.

FIGURE 8.

Glycerophospholipid levels. Glycerophospholipid levels were analyzed by nano-ESI-MS/MS in 30-day-old WT and CerS2 null mouse livers. A, glycerophospholipid levels are shown as a percent of total glycerophospholipids. n = 4 ± S.D. B, distribution of fatty acid chains in phosphatidylethanolamine is shown. n = 2. aPC, phosphatidylcholine (acyl chains); ePC, phosphatidylcholine (ethyl chains); PE, phosphatidylethanolamine; PS, phosphatidylserine; PI, phosphatidylinositol; PG, phosphatidylglycerol. n = 3 ± S.D.; *, p < 0.05.

FIGURE 9.

FIGURE 9.

Cholesterol levels. A, total cholesterol levels were measured by the ferric chloride method for 14- and 30-day-old mouse liver (n = 3 ± S.D.). B, free (unesterified) cholesterol was measured by nano-ESI-MS/MS for 30-day-old liver (n = 4).

FIGURE 10.

FIGURE 10.

Membrane biophysical properties and morphology of microsomal lipids. A, fluorescence anisotropy of three different probes in microsomal lipid extracts obtained from membranes of CerS2 null and WT mice at 30 days of age. n = 3; *, p < 0.001. B, confocal fluorescence microscopy showing the morphological features of microsomal lipid extracts from WT (upper panel) and CerS2 null mice (lower panel). Scale bar, 10 μm.

FIGURE 11.

FIGURE 11.

Protein phosphatase 2A activity. Protein phosphatase 2A activity was measured in homogenates from WT and CerS2 null mouse liver. n = 3.

FIGURE 12.

FIGURE 12.

Pathway relational map showing differences in the relative amounts of sphingolipids in 60-day-old CerS2 null versus WT mouse liver. The upper scheme (diagonal from lower left to upper right) summarizes the biosynthetic pathway of SLs beginning with condensation of serine and palmitoyl-CoA to 3-ketosphinganine (3-KetoSa) and then sphinganine (Sa), which is either _N_-acylated (to N-AcylSa, dihydroceramide (DHCer)) or phosphorylated (Sa1P). _N_-Acylated sphinganine can be desaturated to _N_-acylsphingosine (N-AcylSo, Cer), and both dihydroceramide and Cer can be converted into SM or glycosylated to ceramide monohexoses (CMH). Also shown is the hydrolysis of Cer to sphingosine (upper right), which can undergo phosphorylation to sphingosine 1-phosphate. Other intermediates and products of this scheme can also undergo turnover; some additional reactions (such as ceramide phosphate formation) are not shown because their amounts are small. The lower part of the figure depicts this pathway with all of the measured individual molecular subspecies as nodes that have been colored in the style of a heat map. The colors display the fold difference in the amounts of each compounds in CerS2 null versus WT mouse liver (i.e. CerS2/WT) using the color scale shown at the lower right. Thus, the light blue circle at the bottom left is for palmitoyl-CoA, followed by 3-ketosphinganine (3-keto-Sa) (which is shown smaller and faded to reflect that the amounts in both samples were too low for detection, but there was no evidence for accumulation of this intermediate in CerS2 null mouse), then by the node for sphinganine, which is deep red because sphinganine was substantially higher in the CerS2 null mice. Radiating from this hub are each _N_-acyl chain length metabolite of sphinganine (for examples, _N_-palmitoylsphinganine is labeled 16, and _N_-nervonoylsphinganine is labeled 24:1) (note that the former is elevated and the latter reduced), followed by the respective dihydrosphingomyelins (DHSM) (left nodes) and dihydroceramide monohexoses (DHCMH) (right nodes). Dashed lines relate each _N_-acyl chain length dihydroceramide to the respective Cer, which can be converted to SM and ceramide monohexose (outer nodes) or hydrolyzed to sphinganine (at the hub of this diagram). The phosphorylation products of sphinganine and So are also shown, labeled Sa1P and sphingosine 1-phosphate (S1P). The data used for this figure are from Tables 1 to 3. The layout of this scheme is from Ref. .

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