Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 May 10;288(19):13397-409.
doi: 10.1074/jbc.M112.428185. Epub 2013 Mar 25.

Myristate-derived d16:0 sphingolipids constitute a cardiac sphingolipid pool with distinct synthetic routes and functional properties

Sarah Brice Russo  1 , Rotem Tidhar, Anthony H Futerman, L Ashley Cowart
Affiliations

Myristate-derived d16:0 sphingolipids constitute a cardiac sphingolipid pool with distinct synthetic routes and functional properties

Sarah Brice Russo et al. J Biol Chem. .

Abstract

Background: Myristate is a novel potential substrate for sphingoid base synthesis.

Results: Myocardial sphingoid base synthesis utilizes myristate; these sphingolipids are functionally non-redundant with canonical sphingoid bases.

Conclusion: d16:0 and d16:1 sphingolipids constitute an appreciable proportion of cardiac dihydrosphingosine and dihydroceramide, with distinct biological roles.

Significance: This pool of sphingolipids may play a heretofore unsuspected role in myocardial pathology or protection. The enzyme serine palmitoyltransferase (SPT) catalyzes the formation of the sphingoid base "backbone" from which all sphingolipids are derived. Previous studies have shown that inhibition of SPT ameliorates pathological cardiac outcomes in models of lipid overload, but the metabolites responsible for these phenotypes remain unidentified. Recent in vitro studies have shown that incorporation of the novel subunit SPTLC3 broadens the substrate specificity of SPT, allowing utilization of myristoyl-coenzyme A (CoA) in addition to its canonical substrate palmitoyl-CoA. However, the relevance of these findings in vivo has yet to be determined. The present study sought to determine whether myristate-derived d16 sphingolipids are represented among myocardial sphingolipids and, if so, whether their function and metabolic routes were distinct from those of palmitate-derived d18 sphingolipids. Data showed that d16:0 sphingoid bases occurred in more than one-third of total dihydrosphingosine and dihydroceramides in myocardium, and a diet high in saturated fat promoted their de novo production. Intriguingly, d16-ceramides demonstrated highly limited N-acyl chain diversity, and in vitro enzyme activity assays showed that these bases were utilized preferentially to canonical bases by CerS1. Functional differences between myristate- and palmitate-derived sphingolipids were observed in that, unlike d18 sphingolipids and SPTLC2, d16 sphingolipids and SPTLC3 did not appear to contribute to myristate-induced autophagy, whereas only d16 sphingolipids promoted cell death and cleavage of poly(ADP-ribose) polymerase in cardiomyocytes. Thus, these results reveal a previously unappreciated component of cardiac sphingolipids with functional differences from canonical sphingolipids.

Keywords: Cardiac Muscle; Cell Metabolism; Ceramide; Ceramide Synthase; Heart; Lipid Metabolism; Obesity; Serine Palmitoyltransferase; Sphingolipid.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
SPTLC3 is highly expressed in the heart. Hearts and skeletal muscle were harvested from three 8-week-old C57Bl/6J mice fed standard laboratory chow, and whole tissues were subjected to RNA extraction, followed by cDNA synthesis and qRT-PCR, as described under “Materials and Methods.” SPTLC3 and SPTLC1 expression levels were normalized to GAPDH. Expression levels of SPTLC3 are presented both absolutely and relative to expression of the SPTLC1 subunit, which dimerizes with one SPTLC2 or SPTLC3 subunit to form the minimal functional unit of SPT. A.U., arbitrary units. Results are expressed as mean ± S.E. (error bars). ***, p < 0.01.
FIGURE 2.
FIGURE 2.
d16-base sphingolipids comprise a significant part of the cardiac sphingolipidome. d16:0-base sphingolipids make up approximately one-third of total dihydrosphingosine (A) and total dihydroceramide (B), whereas d16:1-ceramide (C) and d16:1-sphingosine (D) comprise a smaller proportion of total ceramide and sphingosine, respectively. Hearts were harvested from six 16-week-old adult male C57Bl6/J mice, homogenized, and subjected to LC-MS analysis as described under “Materials and Methods.” Results were normalized to protein content, and relative base composition was calculated for each sphingolipid species in each mouse. Results were aggregated and are presented as mean ± S.E. (error bars). *, p < 0.05; n.s., not significant (p ≥ 0.05).
FIGURE 3.
FIGURE 3.
Myocardial SPT utilizes myristoyl-CoA and palmitoyl-CoA similarly. A, reaction velocities were similar between palmitoyl-CoA and myristoyl-CoA across substrate concentrations of 1.56–800 μm fatty acyl-CoA. Activities with concentrations of 1.56–100 μm fatty acyl-CoA are shown in detail in the inset (logarithmic scale). B, the Hanes-Woolf representation of these data revealed an inverse linear correlation between reaction velocity (v) and substrate concentration. Microsomal SPT was isolated from whole hearts of 12-week-old male C57Bl/6J mice and assayed with palmitoyl-CoA or myristoyl-CoA, as described under “Materials and Methods.” Microsomes were generated from pooled homogenate from nine mice per assay. The assay was performed three times, with a total of 4–10 replicates/point. To allow robust comparisons between assays, data are presented as relative SPT activity, as described under “Materials and Methods.” There were no statistically significant differences between palmitoyl-CoA and myristoyl-CoA utilization for the points tested (p < 0.05). Data are presented as mean ± S.E. (error bars).
FIGURE 4.
FIGURE 4.
N-Acyl chain length profile of myocardial d16:1-base ceramide. Most d16:1 sphingoid bases partition into only a few N-acyl chain lengths of ceramide, particularly d16:1/C18:1- and d16:1/C20:0-ceramide. Hearts were harvested from six 16-week-old male C57Bl/6J mice, and lipids were extracted, analyzed by LC-MS, and normalized to protein as described under “Materials and Methods.” Results are presented as mean ± S.E. (error bars).
FIGURE 5.
FIGURE 5.
CerS isoforms differentially utilize d16:0-dihydrosphingosine for dihydroceramide synthesis. Cell extracts were prepared from cells overexpressing individual CerS or empty vector. CerS activity was assayed with d16:0- or d18:0-DHS and either [14C]stearoyl-CoA (A) or [14C]palmitoyl-CoA (B). Controls for CerS1, CerS5, and CerS6 were transfected with 50 μg of the pCMV vector, whereas controls for CerS4 were transfected with 100 μg of the vector, as indicated. Results are means ± S.E. (error bars). *, p < 0.05 versus d18:0-DHS.
FIGURE 6.
FIGURE 6.
A diet high in myristate stimulates d16:0 and d16:1 sphingolipid production in whole hearts. Feeding with a milk fat-based high fat diet (MFBD) (60% kcal from fat), which is high in myristate and total saturated fat, increased total levels of d16 simple sphingolipids (i.e. dihydrosphingosine, sphingosine, dihydroceramide, and ceramide) (A) and total levels of d16:1-ceramide (B) compared with a control diet (16.8% kcal from fat). C, in contrast, the milk fat-based diet did not stimulate an increase in levels of total d18 simple sphingolipids (i.e. dihydrosphingosine, sphingosine, dihydroceramide, and ceramide) or d18:1-ceramide compared with control. Hearts were harvested from adult male C57Bl6/J mice after 8 or 16 weeks of high fat feeding, homogenized, and subjected to LC-MS analysis as described under “Materials and Methods.” Data are presented for nine control mice and 12 milk fat-based high fat diet mice (i.e. all surviving animals from the original cohort). Results were normalized to protein content. Results were similar between time points and were pooled for this analysis. Individual data points are shown; the mean is represented by a dotted line. *, p < 0.05 versus control.
FIGURE 7.
FIGURE 7.
Feeding with a high fat diet shifted N-acyl chain length profiles of d16:1-Cer and promoted CerS4 expression in the murine myocardium. A, six mice per group were fed a milk fat-based high fat diet (MFBD) for 8 weeks. Hearts were harvested and subjected to lipidomic analysis, as described under “Materials and Methods.” Results were normalized to protein, and the percentage of the total was calculated for each mouse. Results are presented as mean ± S.E. (error bars) for all species comprising at least 2% of total d16:1-Cer. *, p < 0.05 versus control diet. Changes were more pronounced after 16 weeks on the diet (not shown). B, CerS expression was measured by qRT-PCR in hearts of mice fed the milk fat-based high fat diet for 8 weeks, as described under “Materials and Methods.” Results are presented as mean ± S.E. *, p < 0.05 versus control diet.
FIGURE 8.
FIGURE 8.
A diet high in myristate stimulates d16 sphingolipid production in the left ventricle. Feeding with a milk fat-based high fat diet (MFBD) (60% kcal from fat), which is high in myristate and total saturated fat, increased total levels of d16:0-DHC and d16:1-ceramide. This was inhibited by treatment with myriocin. In contrast, feeding with a lard-based high fat diet (LBD; 60% kcal from fat), which is high in unsaturated fat but not saturated fat or myristate, did not stimulate production of d16:0-DHC and d16:1-ceramide. Hearts were harvested from six adult male C57Bl6/J mice per group after 18 weeks of high fat feeding. Left ventricles were isolated, homogenized, and subjected to LC-MS analysis as described under “Materials and Methods.” Results are normalized to protein content. *, p < 0.05 versus control; #, p < 0.05 versus lard; @, p < 0.05 versus milk plus myriocin. Error bars, S.E.
FIGURE 9.
FIGURE 9.
Control of d16 sphingolipid levels and SPTLC3 expression by fatty acids. A, treatment with the saturated fatty acid myristate (Myr; C14:0), but not palmitate (Pal; C16:0), stimulated production of d16:1-ceramide (Cer) and d16:0-dihydroceramide (DHC). B, myristate, but not palmitate, treatment also stimulated an increase in SPTLC3 mRNA levels. Primary adult cardiomyocytes were treated with BSA or fatty acid conjugated to BSA, as indicated, for 16 h. Lipids and RNA were extracted and prepared for analysis by LC-MS or qRT-PCR, respectively, as indicated under “Materials and Methods.” Results are presented as mean ± S.E. (error bars). *, p < 0.05 versus BSA; #, p < 0.05 versus palmitate.
FIGURE 10.
FIGURE 10.
SPTLC3 and d16:0-DHS did not contribute to myristate-induced sphingolipid-dependent autophagy. A, treatment with d18:0-DHS induced expression of the autophagy markers Beclin 1 and Atg7. In contrast, treatment with d16:0-DHS did not induce Beclin 1 or Atg7 expression. Primary adult cardiomyocytes were treated with vehicle or with 2.5 μm d18:0-DHS or d16:0-DHS, as indicated, for 3 h. RNA was extracted and analyzed by qRT-PCR as indicated under “Materials and Methods.” B, knockdown of SPTLC2, but not SPTLC3, protected isolated cardiomyocytes from overexpression of the autophagy marker Atg7. Cells were transfected with siRNA for 24 h and then treated with BSA or 0.l mm myristate for 16 h, as described under “Materials and Methods.” Quantifications of immunoblots are presented with representative images; non-contiguous lanes, separated by white lines, are shown from the same gel. All results are presented as mean ± S.E. (error bars). *, p < 0.05 versus vehicle or BSA plus control siRNA.
FIGURE 11.
FIGURE 11.
d16:0-DHS treatment diminished cell viability in cardiomyocytes. Treatment with 2.5 μm d16:0-DHS, but not d18:0-DHS, diminished cell viability of H9c2 immortalized cardiomyocytes, as determined by the MTT assay. A, cells were treated for 3 h and subjected to the MTT assay, as described under “Materials and Methods.” B, even over an extended time course of 4.5 and 9 h, cell viability was reduced selectively by d16:0-DHS but not d18:0-DHS. There was no significant difference between vehicle groups at 4.5 and 9 h of sphingoid base treatment (p = 0.6). Results are presented as a percentage of vehicle for each time point and given as mean ± S.E. (error bars). *, p < 0.05 versus vehicle; @, p < 0.05 versus d18:0-DHS.
FIGURE 12.
FIGURE 12.
d16:0-DHS treatment promoted PARP cleavage in cardiomyocytes. Treatment with d18:0-DHS resulted in increased expression of the intact PARP enzyme (116 kDa) but did not increase levels of its cleaved form (89 kDa). In contrast, d16:0-DHS treatment increased levels of the cleaved form of PARP but not of its intact form. Primary adult cardiomyocytes were treated with vehicle or with 2.5 μm d18:0-DHS or d16:0-DHS, as indicated, for 3 h. Cells were subject to immunoblotting, as described under “Materials and Methods.” Quantifications are shown with images from a representative immunoblot. Top and bottom bands of PARP are shown from different exposures of the same lanes in order to prevent image oversaturation. In our hands under the present culture conditions, primary cardiomyocytes displayed a low but detectable basal level of PARP cleavage and cell death; however, this is normal for this cell type under these conditions. Quantifications of immunoblots were normalized to actin and are presented as mean ± S.E. (error bars). *, p < 0.05 versus vehicle.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Cowart L. A. (2009) Sphingolipids. Players in the pathology of metabolic disease. Trends Endocrinol. Metab. 20, 34–42 - PubMed
    1. Bartke N., Hannun Y. A. (2009) Bioactive sphingolipids. Metabolism and function. J. Lipid Res. 50, S91–S96 - PMC - PubMed
    1. Brice S. E., Cowart L. A. (2011) Sphingolipid metabolism and analysis in metabolic disease. Adv. Exp. Med. Biol. 721, 1–17 - PubMed
    1. Hanada K. (2003) Serine palmitoyltransferase, a key enzyme of sphingolipid metabolism. Biochim. Biophys. Acta 1632, 16–30 - PubMed
    1. Gault C. R., Obeid L. M., Hannun Y. A. (2010) An overview of sphingolipid metabolism. From synthesis to breakdown. Adv. Exp. Med. Biol. 688, 1–23 - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources

  NODES
twitter 2