Regulation of mitochondrial ceramide distribution by members of the BCL-2 family

Article abstract of DOI:10.1194/jlr.M058750

Apoptosis is an intricately regulated cellular process that proceeds through different cell type- and signaldependent pathways. In the mitochondrial apoptotic program, mitochondrial outer membrane permeabilization by BCL-2 proteins leads to the release of apoptogenic factors, caspase activation, and cell death. In addition to protein components of the mitochondrial apoptotic machinery, an interesting role for lipids and lipid metabolism in BCL-2 family-regulated apoptosis is also emerging. We used a comparative lipidomics approach to uncover alterations in lipid profi le in the absence of the proapoptotic proteins BAX and BAK in mouse embryonic fi broblasts (MEFs). We detected over 1,000 ions in these experiments and found changes in an ion with an m/z of 534.49. Structural elucidation of this ion through tandem mass spectrometry revealed that this molecule is a ceramide with a 16-carbon N-acyl chain and sphingadiene backbone (d18:2/16:0 ceramide). Targeted LC/MS analysis revealed elevated levels of additional sphingadiene-containing ceramides (d18:2-Cers) in BAX, BAK-double knockout MEFs. Elevated d18:2-Cers are also found in immortalized baby mouse kidney epithelial cells lacking BAX and BAK. These results support the existence of a distinct biochemical pathway for regulating ceramides with different backbone structures and suggest that sphingadiene-containing ceramides may have functions that are distinct from the more common sphingosinecontaining species .

Full text of DOI:10.1194/jlr.M058750

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Regulation of mitochondrial ceramide distribution by
members of the BCL-2 family
Tejia Zhang,* Lauren Barclay,† Loren D. Walensky,†,§ and Alan Saghatelian1,*
Clayton Foundation Laboratories for Peptide Biology,* Helmsley Center for Genomic Medicine, Salk
Institute for Biological Studies, San Diego, CA 92037; Department of Pediatric Oncology,† Dana-Farber
Cancer Institute and Children’s Hospital Boston, and Linde Program in Cancer Chemical Biology,§ Dana-
Farber Cancer Institute, Harvard Medical School, Boston, MA 02215
Abstract Apoptosis is an intricately regulated cellular
process that proceeds through different cell type- and signal-
dependent pathways. In the mitochondrial apoptotic pro-
gram, mitochondrial outer membrane permeabilization
by BCL-2 proteins leads to the release of apoptogenic fac-
tors, caspase activation, and cell death. In addition to pro-
tein components of the mitochondrial apoptotic machinery,
an interesting role for lipids and lipid metabolism in BCL-2
family-regulated apoptosis is also emerging. We used a com-
parative lipidomics approach to uncover alterations in lipid
profile in the absence of the proapoptotic proteins BAX and
BAK in mouse embryonic fibroblasts (MEFs). We detected
over 1,000 ions in these experiments and found changes
in an ion with an m/z of 534.49. Structural elucidation of
this ion through tandem mass spectrometry revealed that
this molecule is a ceramide with a 16-carbon N-acyl chain
and sphingadiene backbone (d18:2/16:0 ceramide). Tar-
geted LC/MS analysis revealed elevated levels of addi-
tional sphingadiene-containing ceramides (d18:2-Cers) in
BAX, BAK-double knockout MEFs. Elevated d18:2-Cers are
also found in immortalized baby mouse kidney epithelial
cells lacking BAX and BAK. These results support the
existence of a distinct biochemical pathway for regulating
ceramides with different backbone structures and suggest
that sphingadiene-containing ceramides may have func-
tions that are distinct from the more common sphingosine-
containing species.—Zhang, T., L. Barclay, L. D. Walensky,
and A. Saghatelian. Regulation of mitochondrial ceramide
distribution by members of the BCL-2 family. J. Lipid Res.
2015. 56: 1501–1510.
antiapoptotic B-cell lymphoma-2 (2–4), the BCL-2 family
includes pro- and antiapoptotic members whose interac-
tions regulate the balance between cell death and survival
(1). Oligomerization of the proapoptotic executioner pro-
teins BAX and BAK in the mitochondrial outer membrane
leads to mitochondrial outer membrane permeabiliza-
tion. The permeabilized mitochondria release cytochrome
c (cyt c) and additional apoptogenic factors to promote
caspase activation and proteolysis (1, 5, 6). Dysregulated
expression of BCL-2 proteins is associated with cancer
(7) and with autoimmune and neurodegenerative dis-
eases (8, 9).
Murine knockout models of BCL-2 members have
proven useful for understanding the cellular functions of
these proteins (9, 10). Lymphocytes and fibroblasts from
BAX and BAK double-knockout (DKO) mice are resistant
to apoptosis induced by a range of death signals, supporting
an indispensable role for these proteins in mitochondrial
apoptosis (9, 11, 12). Cells from BakϪ/Ϫ mice could activate
both extrinsic and intrinsic apoptotic pathways (11). BAX
or BAK is sufficient for anoikis- or serum deprivation-
induced cell death (12), implying some degree of func-
tional redundancy between these two proteins.
Although much of apoptosis research has focused on its
protein components, evidence supports equally important
functions for lipids in promoting or inhibiting apoptosis at
the mitochondria (13–17). Execution of the mitochon-
drial apoptotic program requires mobilization of critical
protein factors, such as BCL-2 members and cyt c, which
requires structural reorganization within the lipid bilayer
(14, 17). The mitochondrial lipid cardiolipin tethers cyt c
Supplementary key words apoptosis • ceramides • lipidomics • mass
spectrometry • sphingolipids
The mitochondrial apoptotic pathway follows a highly
regulated sequence of events and is dependent on BCL-2
proteins (1). Named after its founding member, the
Abbreviations: CE, collision energy; cyt c, cytochrome c; d18:1-
Cer, sphingosine-containing ceramide; d18:2-Cer, sphingadiene-
containing ceramide; DKO, double-knockout; FA, fatty acid; iBMK,
immortalized baby mouse kidney epithelial cell; MEF, mouse embry-
onic fibroblast; MRM, multiple reaction monitoring; rt, room tempera-
ture; SKO, single-knockout; THF, tetrahydrofuran.
This research is supported by an Eli Lilly Fellowship (T.Z.), a Burroughs Well-
come Fund Award (A.S.), a Searle Scholars Award (A.S.), and NIH grant
5R01CA050239 (L.D.W.).
1 To whom correspondence should be addressed.
Manuscript received 21 February 2015 and in revised form 6 June 2015.
e-mail: asaghatelian@salk.edu
Published, JLR Papers in Press, June 9, 2015
DOI 10.1194/jlr.M058750
The online version of this article (available at http://www.jlr.org)
contains supplementary data in the form of two tables.
Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.
This article is available online at http://www.jlr.org
Journal of Lipid Research Volume 56, 2015
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107 cells) pellets were shaken for 30 s in glass vials (with PTFE-
to the inner mitochondrial membrane, and cardiolipin
peroxidation during apoptosis is a crucial step in releas-
ing cyt c (18, 19). Previous studies also demonstrate that
sphingolipids cooperate with BAX and BAK in promoting
membrane permeabilization (20–23). The interplay be-
tween lipids and apoptosis led us to speculate that BAX
and BAK might regulate mitochondrial lipids.
lined caps) with 2 ml:1 ml:1 ml chloroform-methanol-citric acid
buffer (100 mM trisodium citrate, 1 M NaCl [pH 3.6]). The vials
were vortexed for 15 s and centrifuged at 2,200 g at 4°C for 6 min.
The organic (bottom) layer containing lipids was carefully taken
up with a glass Pasteur pipet, transferred to a new glass vial, and
concentrated under nitrogen. Lipids were reconstituted in chlo-
roform and 1/4–1/8 of sample used for LC/MS.
We reasoned that an LC/MS analysis of the BAX and
BAK-regulated lipidome could provide an unbiased view
into potential lipid metabolic pathways regulated by BAX
and BAK. Our lipidomic analysis of WT and DKO mouse
embryonic fibroblasts (MEFs) identified distinct differ-
ences in mitochondrial lipid composition between DKO
and WT cells, revealing a previously unknown function for
BAX and BAK in cellular sphingolipid metabolism.
Lipidomic analysis of WT, DKO, and
single-knockout MEFs
Lipidomic analysis was performed with an Agilent 1200 Series
HPLC online with an Agilent 6220 ESI-TOF (Agilent Technolo-
gies). Data were acquired in positive and negative ionization
modes. For negative mode, a Gemini (Phenomenex) or Inspire
(Dikma Technologies) C18 column (5 µm, 4.6 mm × 50 mm) was
used with a guard column (C18, 2 µm frit, 2 mm × 20 mm). Sol-
vent A was 95:5 water-methanol with 0.1% ammonium hydrox-
ide, and solvent B was 60:35:5 isopropanol-methanol-water with
0.1% ammonium hydroxide. For positive mode, a Luna (Phe-
nomenex) C5 or Bio-Bond (Dikma Technologies) C4 column
(5 µm, 4.6 mm × 50 mm) was used with a guard column (C4, 2 µm
frit, 2 mm × 20 mm). Solvent A was 95:5 water-methanol with
0.1% formic acid and 5 mM ammonium formate, and solvent B
was 60:35:5 isopropanol-methanol-water with 0.1% formic acid
and 5 mM ammonium formate. Identical gradient was used for
both modes. The gradient was held at 0% B between 0 and 5 min,
changed to 20% B at 5.1 min, increased linearly from 20% B to
100% B between 5.1 min and 45 min, held at 100% B between
45.1 min and 53 min, and returned to 0% B at 53.1 min and held
at 0% B between 53.1 min and 60 min to allow column reequili-
bration. Flow rate was maintained at 0.1 ml/min between 0 and
5 min to counter the pressure increase caused by chloroform
injection. The flow rates were 0.4 ml/min between 5.1 min and
45 min and 0.5 ml/min between 45.1 min and 60 min. Injection
volume was 10–30 µl. Capillary, fragmentor, and skimmer volt-
ages were 3.5 kV, 100 V, and 60 V, respectively. Drying gas tem-
perature was 350°C, drying gas flow rate was 10 l/min, and
nebulizer pressure was 45 psi. Data were collected in both profile
and centroid modes using a mass range of 100–1,500 Da.
Data were analyzed via targeted and untargeted approaches.
Targeted analysis of known lipids was performed with manual
integration in MassHunter Qualitative Analysis (Agilent Tech-
nologies) using a symmetric m/z expansion of either 50 or
100 ppm (m/z expansions of 20, 50, and 100 ppm were compared
and there were no significant differences in peak integration).
For untargeted analysis, raw data were converted to .mzXML for-
mat using trapper (27) and analyzed by XCMS (28) operated in R
with the following parameters: family = “s”, plottype = “m”, bw = 10,
and metlin = 0.15; retention correction (retcor) was iterated
at least three times to maximize peak alignment across samples.
XCMS output files were filtered by statistical significance (P р
0.05), fold change (у 3), and reproducibility across four inde-
pendent data sets, and the remaining ions were further verified
by manual integration in Qualitative Analysis. Database searches
were performed in LIPID MAPS (29) and METLIN (30). A neu-
tral mass of 535.50 was used to search the LIPID MAPS Structural
Database (29) using the text/ontology-based search option with
a mass expansion of 0.01 and the METLIN (30) database using
the Simple search option with a mass tolerance of 20 ppm.
For construction of volcano plot (see Fig. 2B), ions between 0
and 5 min and 50 and 60 min were removed because these ions
fall within the aqueous portions of the LC method and are un-
likely to be lipids; the remaining ions between 5 and 50 min
were examined in qualitative analysis. Isotopes were manually
removed, and the resulting parent ions were graphed by their
MATERIALS AND METHODS
Materials
d31-d18:1/16:0 (868516) and d18:1/16:0 (860516) ceramides
were from Avanti Polar Lipids; d14-palmitoleic acid (9000431)
was from Cayman Chemical; ammonium formate (516961),
ammonium hydroxide (338818), and formic acid (06440) were
from Sigma-Aldrich; water (BJ365-4), methanol (BJ230-4), iso-
propanol (BJ323-4), acetonitrile (BJ017-4), chloroform (BJ049-
1L), guard column kit (21511-492), and C18 silica (53501-270)
were from VWR. C4 silica (214TPB1520) was from Western Ana-
lytical Products. C4 and C18 analytical columns with the reported
dimensions were from Phenomenex or Dikma Technologies.
Chemicals for ceramide synthesis were as follows: Grubbs second-
generation catalyst, N-succinimidyl palmitate, palmitoleic acid,
triethylamine, and vinyl magnesium bromide (Sigma-Aldrich);
(S)-Garner aldehyde (TCI America); oct-7-enal (Novel Chemical
Solutions); heptyltriphenylphosphonium bromide, sodium
bis(trimethylsilyl)amide, and oxalyl chloride (Alfa Aesar); d18:1
sphingosine (Avanti Polar Lipids); tetrahydrofuran (Acros Organics);
and pyridine (Mallinckrodt Chemicals). Additional solvents were
from EMD Chemicals.
Tissue culture and harvest
MEFs were maintained at 37°C and 5% CO2 in DMEM (11965,
Life Technologies) with 10% FBS (HyClone or Seradigm), peni-
cillin, streptomycin, nonessential amino acids, and L-glutamine
(final concentration, 6 mM). Immortalized baby mouse kidney
epithelial cells (iBMKs) were maintained in the same medium
with 5% FBS, penicillin, streptomycin, and L-glutamine (6 mM).
All cells were passaged (1:4–1:6) at least three times and har-
vested at confluency (48–52 h after last passage) by scraping into
cold PBS on ice. Cell pellets for lipid extraction were used im-
mediately for extraction or frozen at Ϫ80°C until further use.
Mitochondria isolation was performed immediately after cell har-
vesting without freezing.
Mitochondria isolation and lipid extraction
Mitochondria were isolated from MEFs (ف2.5 × 108 cells) fol-
lowing a known protocol (24) using a sucrose buffer (250 mM
sucrose, 10 mM Tris-HCl, 0.1 mM EGTA·4 Na [pH 7.4]), and
isolated mitochondria were used immediately for lipid extraction
or stored at Ϫ80°C until further use. Lipid extraction was per-
formed with modifications on the Bligh and Dyer procedure (25,
26). Mitochondrial (from ف2.5 × 108 cells) or whole cell (ف1.5 ×
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XCMS-calculated DKO/WT fold changes (average of three bio-
logical replicates) and associated P values. Statistical significance
was determined by two-tailed Student’s t-test.
Measurement of d18:1/X:0, d18:2/X:0, and d18:1/X:1
ceramides by MRM
Differently saturated ceramide isomers were quantified on the
Triple Quad instrument using identical conditions as coinjection.
A list of targeted ceramides and associated parameters is pro-
vided in supplementary Table 1. Mitochondrial sample (1/4–1/8
from ف2.5 × 108 cells) or 1/3–1/6 of whole cell sample (from ف1.5 ×
107 cells) was used for analysis. For each ceramide N-acyl chain, the
relative ion abundances of d18:2/X:0 and d18:1/X:0 species were
taken as an estimation of the d18:2 to d18:1-Cers ratio.
Ceramide quantification, coinjection, and tandem MS
A d31-d18:1/16:0 ceramide standard was used for quantification
of endogenous C16-C24 ceramides on the TOF instrument using
general profiling conditions. The standard was added to chloroform
before lipid extraction to account for metabolite loss during extrac-
tion. Tandem MS in positive mode was performed with an Agilent
1200 Series HPLC online with an Agilent 6410 Triple Quad MS
(Agilent Technologies) in Product Ion mode. Precursor ions were
m/z 538.5 (d18:1/16:0 ceramide) and m/z 536.5 (d18:2/16:0 and
d18:1/16:1 ceramides). Time filter width was 0.07 min. Fragmentor
voltage, collision energy (CE), skimmer voltage, and ⌬EMV were
132, 26, 15, and 400 V, respectively. Capillary voltage was 4.0 kV,
skimmer voltage was 15 V, drying gas temperature was 350°C, drying
gas flow rate was 8 l/min, and nebulizer pressure was 35 psi for all
Triple Quad measurements. Injection volume was 30 µl.
Syntheses of N-[(2S, 3R, 4E, 11Z)-1, 3-dihydroxyoctadeca-4,
11-dien-2-yl]palmitamide (d18:2/16:0 ceramide)
Step 1. Stereoselective addition of vinyl magnesium bromide
to (S)-Garner aldehyde was carried out in tetrahydrofuran (THF),
and the crude product was purified by silica gel chromatography
based on known procedures (31, 32) to yield (S)-tert-butyl 4-[(R)-
1-hydroxyallyl]-2,2-dimethyloxazolidine-3-carboxylate (1) for syn-
thesis of d18:2/16:0 ceramide (Fig. 1A, 1).
Tandem MS in negative mode was performed with an Agilent
6500 Series Q-TOF LC/MS system. Precursor ions were m/z
534.4892 (d18:2/16:0 and d18:1/16:1 ceramides) and m/z 536.5048
(d18:1/16:0 ceramide). Injection volume was 15 µl. VCap, nozzle,
skimmer1, and fragmentor voltages were 4,500, 2,000, 0, and 365 V,
respectively. CE was 26 V. Octopole RF peak voltage was 750 V.
Isolation width was set to Medium. Gas and sheath gas tempera-
tures were 250°C; gas and sheath gas flow rates were 13 l/min and
12 l/min, respectively; and nebulizer pressure was 55 psi. Positive-
and negative-mode tandem MS was recorded for mitochondrial
and whole cell lipids from WT and DKO MEFs to verify the pres-
ence of the same ceramide species in all sample fractions.
The Triple Quad instrument operated in multiple reaction moni-
toring (MRM) mode was used for coinjection. MEF whole cell lipid
extract was used as sample, synthetic ⌬4, 11-d18:2/16:0 ceramide
was used as standard, and a combination of sample and standard
used for coinjection. All samples were dissolved in chloroform
such that the final volume was 10 µl. The fragmentor and CE
were the same as Product Ion. The transition in negative mode
was m/z 534.5 → m/z 280.3, ⌬EMV was 400 V, and MS1 resolution
was set to Wide and MS2 resolution to Unit. LC parameters were
identical to comparative lipidomics. d12-d18:2/16:0 ceramide was
detected by dynamic MRM using the transition m/z 548.6 → 274.3
in positive mode. Retention time was 42.55 min, and the reten-
tion window was 10 min. All other parameters were identical to
coinjection.
Step 2. (S)-tert-BUTYL 4-[(R, E)-1-HYDROXY-9-OXONON-2-EN-1-YL]-2,
2-DIMETHYLOXAZOLIDINE-3-CARBOXYLATE. To a solution of 1 (280 mg,
1.1 mmol, 1 eq.) in dry dichloromethane (10 ml) were added
oct-7-enal (549 mg, 660 ␮l, 4.4 mmol, 4 eq.) and Grubbs second-
generation catalyst (23 mg, 0.027 mmol, 0.025 eq.). The solution
was refluxed overnight (ف16 h), and completion of reaction was
confirmed by TLC (40:60 ethyl acetate-hexanes, PMA stain, Rf =
0.35–0.4). The reaction was concentrated and purified by silica
gel chromatography (35:65 ethyl acetate-hexanes). Fractions
containing product were concentrated to afford 2 (190 mg, 49%
1
yield) as a colorless oil. H-NMR (500 MHz, CDCl3): 1.19–1.54
(m, 21H), 1.94 (dt, 2H), 2.31 (t, 2H), 3.73–4.07 (m, 5H including
OH), 5.35 (dd, J = 15.3, 6.3, 1H), 5.9 (m, 1H), 9.64 (s, 1H). ESI-MS:
theoretical m/z for C19H33NO5Na+ (sodium adduct): 378.2251,
detected m/z: 378.2224, ppm difference: 7.14 (Fig. 1A, 2).
Step 3. (S)-tert-BUTYL 4-[(R, 2E, 9Z]-1-HYDROXYHEXADECA-2,
9-DIEN-1-YL)-2, 2-DIMETHYLOXAZOLIDINE-3-CARBOXYLATE. To a sus-
pension of heptyltriphenylphosphonium bromide (330 mg,
0.75 mmol, 3 eq.) in dry THF (10 ml) at 0°C was added sodium
bis(trimethylsilyl)amide (725 ␮l of 1 M solution in dry THF,
2.9 mmol, 2.9 eq.). The solution immediately turned orange and
was stirred at 0°C for 30 min. The resulting solution was cooled to
Ϫ78°C, and a THF solution (2 ml) of 2 (90 mg, 0.25 mmol, 1 eq.)
Fig. 1. Chemical synthesis. A: Synthesis of d18:2/16:0 ceramide. B: Synthesis of d18:1/16:1 ceramide.
Ceramide regulation by BCL-2 proteins
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was added. The reaction was allowed to proceed overnight where
RESULTS
it warmed from Ϫ78°C to room temperature (rt) and the color
dissipated until white. Saturated aqueous sodium bicarbonate
was added, and the aqueous phase was extracted three times with
diethyl ether (10 ml). The combined organic phases were washed
with brine, dried over anhydrous sodium sulfate, concentrated,
and purified by silica gel chromatography (15:85 ethyl acetate-
hexanes, Rf = 0.3) to yield 3 (36 mg, 32% yield) as a colorless oil.
1H-NMR (400 MHz, CDCl3): 0.88 (t, 3H), 1.25–1.62 (m, 29H),
2.02 (m, 6H), 3.83 (m, 1H), 4.02 (m, 1H), 4.14 (m, 2H), 5.34 (m, 2H),
5.44 (dd, J = 15.3, 5.2, 1H), 5.73 (m, 1H). ESI-MS: theoretical m/z
for C26H47NO4Na+ (sodium adduct): 460.3397, detected m/z:
460.3388, ppm difference: 1.96 (Fig. 1A, 3).
Comparative metabolite profiling reveals elevated levels
of unsaturated C16 ceramide in DKO mitochondria
Lipids were extracted (25, 26) from WT and DKO MEF
mitochondria (24) and subjected to LC/MS to identify
lipidomic changes in the absence of BAX and BAK (Fig.
2A). Major lipid classes (free fatty acids, phospholipids,
sphingolipids, acylglycerols, acylcarnitines, ether lipids,
Step 4. N-([2S, 3R, 4E, 11Z]-1, 3-DIHYDROXYOCTADECA-4,
11-DIEN-2-YL)PALMITAMIDE. A solution of 3 (33 mg, 0.076 mmol,
1 eq.) in 1 M HCl (0.75 ml) and dioxane (0.75 ml) was heated at
100°C for 1 h. The mixture was cooled to rt, neutralized with 1 M
NaOH (1 ml), and extracted twice with ethyl acetate (3 ml). The
combined organic phases were sent through a plug of sodium
sulfate and concentrated to yield crude sphingadiene. The yel-
lowish solid was dissolved in THF (0.5 ml), and N-succinimidyl
palmitate (40 mg, 0.11 mmol, 1.5 eq.) was added, followed by
triethylamine (22 ␮l). After stirring overnight, 1 M HCl (1 ml)
was added and the mixture extracted twice with ethyl acetate
(2 ml). The combined organic phases were dried over anhydrous
sodium sulfate, concentrated, and purified by silica gel chroma-
tography (66:34 ethyl acetate-hexanes, Rf = 0.5) to yield 4 (17 mg,
1
41% yield) as a white solid. H-NMR (500 MHz, CDCl3): 0.88
(m, 6H), 1.25–1.35 (m, 46H), 1.63 (m, 2H), 2.00–2.08 (m, 6H), 2.25
(t, J = 7.5, 2H), 2.88 (s, 1H), 3.70 (m, 1H), 3.88–3.96 (m, 2H), 4.3
(m, 1H), 5.34 (m, 1H), 5.53 (dd, J = 15.5, 6.5, 1H), 5.78 (m, 1H),
6.29 (d, J = 7.5, 1H). ESI-MS: theoretical m/z for C34H66NO3+ (hy-
drogen adduct): 536.5037, detected m/z: 536.5029; ppm differ-
ence: 1.49 (Fig. 1A, 4).
Synthesis of (Z)-N-([2S, 3R, E]-1, 3-dihydroxyoctadec-4-
en-2-yl)hexadec-9-enamide (d18:1/16:1 ceramide)
To a stirring solution of (Z)-hexadec-9-enoic acid (palmitoleic
acid, 9.7 mg, 0.041 mmol, 1 eq.) at rt was added oxalyl dichloride
(1 ml, excess). Bubble formation was observed over the next
30 min, and the reaction was stirred overnight at rt before being
concentrated to afford the crude (Z)-hexadec-9-enoyl chloride (1).
Compound 1 was dried under vacuum for 3 h, dissolved in a mix-
ture of pyridine (15.1 ␮l, 0.195 mmol, 4.8 eq. assuming 100% yield
for previous reaction) and chloroform (0.5 ml) and added drop-
wise to a stirring solution of (2S, 3R, E)-2-aminooctadec-4-ene-1,3-
diol (d18:1 sphingosine, 14.7 mg, 0.049 mmol, 1.2 eq. assuming
100% yield for previous reaction) in chloroform (4 ml). The reac-
tion was stirred at rt for 5 h, water was added, and the resulting
mixture was extracted three times with chloroform. The combined
organic layers were washed with brine, dried with sodium sul-
fate, filtered, and concentrated to afford the crude (Z)-N-([2S,
3R, E]-1, 3-dihydroxyoctadec-4-en-2-yl)hexadec-9-enamide (2),
which was used directly for tandem MS. ESI-MS: theoretical m/z
Fig. 2. Comparative lipidomics reveals selective upregulation of
diunsaturated ceramides in DKO MEF mitochondria. A: Schematic
of comparative lipidomics enabling identification of lipid changes
between WT and DKO samples. B: Volcano plot of detected parent
ions plotted as DKO/WT fold change versus P value (n = 3); the
DKO-elevated m/z 534 metabolite and its chlorine adduct are in
green. C: Structure of d18:1/16:0 ceramide. The trans double bond
is found at C4-C5 of the sphingosine backbone. D: DKO-elevated
ceramide elutes ف1 min before d18:1/16:0 ceramide, in agree-
ment with the commonly observed phenomenon of decreasing
retention time with increasing unsaturation in reversed-phase
chromatography (37).
Ϫ
for C34H64NO3 (loss of hydrogen): 534.4892; detected m/z:
534.4903; ppm difference: 2.06.
Cellular production of d12-d18:2/16:0 ceramide
For each sample, 20 µl 83 mM d14-palmitoleic acid in DMSO
was mixed with 10.5 ml MEF media, and 10 ml of the media intro-
duced to DKO MEFs in a 10 cm tissue culture dish. 20 µl DMSO
was used as negative control. Treated cells were incubated at
37°C for 2 h or 21 h before harvest by scraping into cold PBS on
ice and lipid extraction.
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cardiolipins, cholesterol, and cholesterol esters) from four
(i.e., sphingadiene-containing ceramide) or the N-acyl
chain. We refer to ceramides with two double bonds as
“diunsaturated ceramides” and ceramides with one dou-
ble bond as “monounsaturated ceramides.”
independent data sets were analyzed to ensure data quality
and reproducibility. Levels of most lipids were unaltered
between WT and DKO MEFs (supplementary Table 2),
demonstrating that the majority of lipid pathways were un-
affected by the absence of BAX and BAK.
Data analysis was also performed using the XCMS software
package (28), which aligns, quantifies, and statistically
ranks changes in detected ions between WT and DKO
groups. XCMS uncovered a metabolite with a detected m/z
of 534.4886 in negative ionization mode that was consis-
tently elevated in DKO mitochondria relative to WT (Fig.
2B). Our finding indicates that the loss of BAX and BAK
can influence cellular metabolism, and the observation of
one change in a background of mostly unaltered metabo-
lites demonstrates specificity in the metabolic pathway
regulated by BAX and BAK.
A database search [LIPIDMAPS (29) and METLIN
(30)] with the exact neutral mass of the DKO-elevated
metabolite afforded the molecular formula C34H65NO3,
corresponding to the sphingolipid ceramide. Mamma-
lian ceramides are typically comprised of a sphingosine,
with a trans double bond at C4-C5 (Fig. 2C, red) and an
N-acyl fatty acid (Fig. 2C, blue) (33–35). One of the
major mammalian ceramides is d18:1/16:0 (Fig. 2C),
which has the molecular formula C34H67NO3 (33, 36).
The DKO-elevated ceramide (C34H65NO3) contains two
fewer hydrogens, or one more double bond, than the
d18:1/16:0 species. This is consistent with the earlier ob-
served LC retention time for the DKO-elevated ceramide
(Fig. 2D) (37). The structure of the DKO-elevated ce-
ramide is, therefore, d18:2/16:0 or d18:1/16:1, and the
second double bond is present in either the backbone
Diunsaturated ceramides are upregulated
in DKO MEF mitochondria
To account for less abundant diunsaturated ceramides
not identified during untargeted XCMS analysis, monoun-
saturated and diunsaturated C16–C24 ceramide levels were
measured by isotope-dilution MS (26, 38). A d31-d18:1/16:0
ceramide standard was added to biological samples dur-
ing lipid extraction, allowing ratiometric quantification
of endogenous ceramides. For each N-acyl chain, both
the monounsaturated and diunsaturated species were
considered.
Of all detectable ceramides (Fig. 3A), ceramides with
C16 acyl chains occurred at the highest level (ف300–
1,400 pmol/mg protein). The next highest level be-
longed to ceramides, with C24 acyl chains (ف150–600
pmol/mg protein) (Fig. 3B). A decrease in cellular con-
centration was observed for C18 and C20 ceramides
(Fig. 3B), corresponding to an ف100-fold decrease rela-
tive to C16 ceramides (Fig. 3B). Comparison of WT
and DKO MEFs revealed a unique change in the ceramide
profile. Levels of most monounsaturated ceramides
(i.e., d18:1/16:0) were unaltered between WT and DKO
MEF mitochondria. By contrast, a significant increase
(ف2–4.5-fold) in most diunsaturated ceramides was ob-
served in the absence of BAX and BAK (Fig. 3B). This
increase led to an ف2-fold rise in the total diunsatu-
rated-to-monounsaturated mitochondrial ceramide ra-
tio (Fig. 3C, D).
Fig. 3. Selective elevation of diunsaturated cerami-
des requires BAX and BAK. A: Extracted ion chro-
matograms of C16-C24 ceramides. m/z expansion:
50 ppm. B: Quantification of C16-C24 ceramides
with d31-d18:1/16:0 ceramide as internal standard. C:
Total mitochondrial di/monounsaturated ceramide
ratios for WT and DKO MEFs. D: WT and DKO MEF
mitochondrial di/monounsaturated ceramide ratio
by N-acyl chain. * P р 0.05, ** P р 0.01, and *** P р
0.001, two-tailed Student’s t-test (SEM; n = 3 for all
sections).
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We also monitored the levels of additional sphingolip-
synthetic ⌬4, 11-d18:2/16:0 ceramide standard overlapped
with the DKO-elevated ceramide (Fig. 5D). Thus, ⌬4, 11-
d18:2/16:0 ceramide could be contributing toward the BAX,
BAK-regulated d18:2 ceramide pool in cells. However, we
cannot rule out the presence of multiple d18:2/16:0 ce-
ramide double-bond isomers (35, 43–45) coeluting with
this peak. We designate sphingadiene-containing diunsat-
urated ceramides as d18:2-Cers.
ids. No differences in additional sphingolipids or in pal-
mitic or palmitoleic acids were observed between WT and
DKO MEF mitochondria (Fig. 4A, B; supplementary Table
2), indicating that changes in ceramides are not the direct
consequence of an alteration in the fatty acid composition
or in other sphingolipids. Most ceramides were also un-
changed between WT and BAX or BAK single-knockout
(SKO) MEFs (Fig. 4C, D), suggesting that elevation of
these diunsaturated ceramides required both BAX and
BAK.
BAX and BAK regulate d18:2-Cers in
two different cell types
We next used a targeted MRM detection method to
measure all detectable d18:2- and d18:1-ceramides (sup-
plementary Table 1) in MEF and iBMKs. The absence of
BAX and BAK raised the levels of C16-C24 d18:2-Cers
relative to monounsaturated ceramides in both cell lines
(Fig. 6A, B). Thus, BAX and BAK regulate d18:2-Cers in
at least two distinct cell lines. BCL-2 regulation of these
ceramides is also highly specific; d18:2-Cers were up-
regulated, whereas other diunsaturated ceramides (e.g.,
d18:1/16:1) were unchanged (Fig. 6C). Therefore, the
location of the additional double bond in the backbone
sphingosine is essential for ceramide regulation by BAX
and BAK.
DKO-elevated metabolite is d18:2/16:0 ceramide
To locate the double bonds in the DKO-regulated diun-
saturated ceramide, we compared the fragmentation pro-
file of d18:1/16:0 ceramide with the diunsaturated C16
ceramide. Positive-mode tandem MS of d18:1/16:0 ce-
ramide yielded the sphingoid backbone-derived product
ions m/z 282, 264, and 252 (Fig. 5A) (39–41). Tandem MS
of the DKO-regulated diunsaturated C16 ceramide yielded
the product ions m/z 280, 262, and 250; each of these ions
is 2 Da lower in mass than the corresponding product ion
from the d18:1/16:0 ceramide (Fig. 5A). Thus, the struc-
ture of the DKO-elevated diunsaturated C16 ceramide is
d18:2/16:0. Negative-mode tandem MS was also consistent
with this structural assignment, as the 2-Da mass shift was
observed only for product ions from the sphingoid back-
bone (Fig. 5B).
Identical fragmentation patterns were observed be-
tween the DKO-elevated ceramide and the synthetic
d18:2/16:0 ceramide standard (Fig. 5C). For the initial
synthesis, weinstalledthecisdoublebondinthed18:2/16:0
ceramide at C11-C12 (Fig. 1A). A double bond at this posi-
tion would arise from potential incorporation of palmitoleyl
(⌬9-cis-16:1)-CoA during de novo ceramide biosynthesis
(34, 42) (also see DISCUSSION). In a coelution study, this
DISCUSSION
Several BCL-2 proteins affect cellular metabolism,
including glucose and fatty acid metabolism (46–48),
calcium homeostasis (49), and insulin secretion (50).
Similar to these findings, our data demonstrate a novel
metabolic function for BAX and BAK in the regulation
of endogenous ceramides. Specifically, we find that
BAX and BAK preferentially regulate diunsaturated,
sphingadiene-containing ceramides (d18:2-Cers). The
presence of the second double bond in the sphingoid
Fig. 4. Additional sphingolipids and ceramide lev-
els in WT, DKO, and SKO MEFs. A: Relative sphingo-
myelin levels in WT and DKO MEF mitochondria. No
statistically significant changes were observed. B: Rel-
ative sphingadiene, sphingosine, and sphinganine
levels in WT and DKO MEF mitochondria. No statisti-
cally significant changes were observed. C: Relative
C16-C24 ceramide levels in WT and BaxϪ/Ϫ MEF mi-
tochondria. D: Relative C16-C24 ceramide levels in
WT and BakϪ/Ϫ MEF mitochondria. * P р 0.05 and
** P р 0.01, two-tailed Student’s t-test (SEM; n = 3 for
C and D).
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Fig. 5. Structural identification of C16 diunsaturated ceramide. A: Positive ionization mode tandem MS of synthetic and endogenous
d18:1/16:0 ceramide and the DKO-elevated ceramide. B: Negative-mode tandem MS of synthetic and endogenous d18:1/16:0 ceramide
and the DKO-elevated ceramide. N-acyl chain-derived fragments are in blue. C: Tandem MS of the DKO-elevated ceramide, and synthetic
d18:1/16:1, d18:2/d16:0 and d18:1/16:0 ceramides in positive ionization mode. D: Coinjection with synthetic and natural d18:2/16:0 ce-
ramides. All illustrated fragmentations are based on published predictions (39, 41, 59–63).
Ceramide regulation by BCL-2 proteins
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not d18:1-Cers, are regulated by both BAX and BAK
supports the existence of subpopulations of structurally
distinct ceramides that could be selectively modulated
by different BCL-2 proteins. To our knowledge, this is
the first example of ceramide regulation based on back-
bone structure, and this result suggests that the back-
bone specificity in ceramide biosynthetic enzymes requires
exploration.
This metabolic function of BAX and BAK is likely
connected to their apoptotic function because of the
proapoptotic activity of ceramides (15). Ceramides are
bioactive lipids involved in multiple cellular processes,
including apoptosis, proliferation, and autophagy (17).
Cellular ceramide levels rise in response to a range of
apoptotic signals (22), and ceramide macrodomains in the
outer mitochondrial membrane act as sites of BAX activa-
tion during irradiation-induced apoptosis (52). Although
numerous studies have demonstrated a proapoptotic role
for ceramides, the apoptotic functions of ceramides also
depend on lipid structure. Dihydroceramide inhibits
ceramide channel formation in isolated mitochondria
(53), and long-chain and very long-chain ceramides have
been shown to exhibit opposite effects toward cancer cell
survival (54). Our discovery of preferential regulation of
d18:2-Cers by BAX and BAK suggests that there could
also exist distinct cellular ceramide pools with different
influences over cellular viability, which we will investigate
in future studies.
Sphingadiene-containing sphingolipids are not new.
Sphingadienes with a trans double bond at C4 and a cis
double bond at C14 are a component of human serum
sphingomyelins (43, 44). Sphingadienes with 14 or 16 car-
bons and conjugated cis double bonds at C4, 6 are present
in Drosophila (55), and cis-C4, 8-sphingadienes are found
in plants (56). C16 sphingoid backbones are also present
in human plasma due to the activity of an additional serine
palmitoyltransferase isoform (SPTLC3) that favors my-
ristoyl (C14:0)-CoA (35, 45). Although there could be
multiple biochemical pathways or dietary sources for
generating mammalian sphingadiene-containing cerami-
des (Fig. 7A), we have examined the incorporation of un-
saturated palmitoleic acid (d14-C16:1 fatty acid [FA]) into
the sphingoid backbone. d14-16:1 FA-treated cells pro-
duced labeled ceramides to suggest that unsaturated FA
incorporation could be one of the routes toward d18:2-
Cers production (Fig. 7B). Additional questions, such as
the known preference of serine palmitoyltransferases
for palmitoyl-CoA over cis-unsaturated fatty acyl-CoA sub-
strates (57, 58) and how differently saturated cellular FA
pools could be directed toward biosynthesis of sphingosine-
or sphingadiene-containing ceramides, will need to be fur-
ther addressed.
Fig. 6. BAX and BAK selectively regulate d18:2-Cers in MEFs and
iBMKs. A: WT and DKO MEF mitochondrial and whole cell d18:2-
Cer/d18:1-Cer ratio by N-acyl chain. B: WT and DKO iBMK mito-
chondrial and whole cell d18:2-Cer/d18:1-Cer ratio by N-acyl
chain. C: Total d18:1/X:0, d18:2/X:0, and d18:1/X:1 ceramide
levels in WT and DKO MEFs; data are normalized to WT d18:1/X:0
value. * P р 0.05, ** P р 0.01, and *** P р 0.001, two-tailed Stu-
dent’s t-test (SEM; n = 4 for all sections).
backbone (i.e., d18:2-Cers) is essential for BAX and
BAK regulation. Diunsaturated ceramides with the sec-
ond double bond in the N-acyl chain were not regulated
by BAX and BAK. Ceramide levels were also unaltered
in BAX or BAK SKO MEFs, suggesting that BAX and
BAK compensate for each other in this metabolic func-
tion. Whereas mitochondrial morphogenesis depends on
both BAX and BAK (51), only BAK is required for long-
chain d18:1-Cers release in response to a range of apo-
ptotic agents (21, 22). Our observation that d18:2-Cers, but
The discovery of the DKO-elevated d18:2-Cers relied on
tandem MS. Most research into the role of ceramides in
apoptosis focuses on the levels and functions of d18:1-Cers
(15). Our work indicates that it would be of interest to
revisit these studies using tandem MS and targeted LC/
MS approaches to evaluate whether d18:2-Cers were over-
looked.
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Fig. 7. De novo biosynthesis of d18:2/16:0 ceramide via incorporation of unsaturated fatty acid precursor. A: De novo ceramide biosyn-
thetic pathway condenses a free fatty acyl-CoA with serine to produce the unique sphingosine backbone (34). B: Generation of d12-
d18:2/16:0 ceramide by d14-palmitoleic acid-treated MEFs.
and bax are essential for normal development of multiple tissues.
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Thompson. 2001. Bh3-only proteins that bind pro-survival bcl-2
family members fail to induce apoptosis in the absence of bax and
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The authors thank Prof. Eileen White for the generous gift of
WT and DKO iBMK cells; Dr. Sunia A. Trauger at the Harvard
FAS Small Molecule Mass Spectrometry core for acquisition of
the negative mode ceramide tandem MS spectra; Dr. Qian Chu
and Dr. Christopher Vickers for manuscript reviews; and
members of the Saghatelian and Walensky research groups for
general technical guidance and discussions.
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