TNF-α is a pleiotropic cytokine that in addition to its
role in infection and immunity induces apoptosis in
many cell types. The cellular signaling network used by
TNF-αto cause apoptosis is complex and involves a vari-
ety of intermediates and protein-protein interactions
(1–4). In hepatocytes as in other type II cells, mito-
chondria play a pivotal role in apoptosis, and TNF-α–
mediated hepatocellular apoptosis contributes to
many forms of liver injury (3–8). The specific interac-
tion of proapoptotic proteins — including Bcl-2 family
members, signaling enzymes, transcription factors, or
viral-encoded proteins — with mitochondrial compo-
nents stimulates the release of mitochondrial factors
that set in motion apoptotic pathways leading to the
demise of the cell (3, 5–7, 9, 10). The mitochondrial
downstream process that activates the caspases respon-
sible for cell death involves the interaction of
cytochrome c with apaf-1, resulting in the proteolytic
activation of procaspase 9, which then triggers caspase
3 activation (11, 12). In addition, mitochondria can
also regulate cell death by releasing several other
proapoptotic factors, including caspases, the apopto-
sis-inducing factor, and Smac/Diablo (13–16).
Sphingolipids — in particular, ceramide — have
emerged as putative signaling lipid intermediates that
play a role in the stress response and cell death (17).
The production of ceramide can occur by de novo syn-
thesis through activation of serine-palmitoyl trans-
ferase, the rate-limiting enzyme in ceramide synthesis,
The Journal of Clinical Investigation| January 2003| Volume 111| Number 2
Defective TNF-α–mediated hepatocellular apoptosis and
liver damage in acidic sphingomyelinase knockout mice
Carmen García-Ruiz,1,2Anna Colell,1Montserrat Marí,1Albert Morales,1María Calvo,3
Carlos Enrich,3and José C. Fernández-Checa1,2
1Liver Unit, Instituto de Malalties Digestives, Hospital Clinic i Provincial, Instituto de Investigaciones Biomédicas
August Pi Suñer, Barcelona, Spain
2Department of Experimental Pathology, Instituto de Investigaciones Biomédicas de Barcelona, Consejo Superior
Investigaciones Científicas, Barcelona, Spain
3Department of Cell Biology, School of Medicine, University of Barcelona, Instituto de Investigaciones Biomédicas
August Pi Suñer, Barcelona, Spain
This study addressed the contribution of acidic sphingomyelinase (ASMase) in TNF-α–mediated
hepatocellular apoptosis. Cultured hepatocytes depleted of mitochondrial glutathione (mGSH)
became sensitive to TNF-α, undergoing a time-dependent apoptotic cell death preceded by mito-
chondrial membrane depolarization, cytochrome c release, and caspase activation. Cyclosporin A
treatment rescued mGSH-depleted hepatocytes from TNF-α–induced cell death. In contrast, mGSH-
depleted hepatocytes deficient in ASMase were resistant to TNF-α–mediated cell death but sensi-
tive to exogenous ASMase. Furthermore, although in vivo administration of TNF-αor LPS to galac-
tosamine-pretreated ASMase+/+mice caused liver damage, ASMase–/–mice exhibited minimal
hepatocellular injury. To analyze the requirement of ASMase, we assessed the effect of glucosylce-
ramide synthetase inhibition on TNF-α–mediated apoptosis. This approach, which blunted gly-
cosphingolipid generation by TNF-α, protected mGSH-depleted ASMase+/+hepatocytes from
TNF-αdespite enhancement of TNF-α–stimulated ceramide formation. To further test the involve-
ment of glycosphingolipids, we focused on ganglioside GD3 (GD3) because of its emerging role in
apoptosis through interaction with mitochondria. Analysis of the cellular redistribution of GD3 by
laser scanning confocal microscopy revealed the targeting of GD3 to mitochondria in ASMase+/+but
not in ASMase–/–hepatocytes. However, treatment of ASMase–/–hepatocytes with exogenous ASMase
induced the colocalization of GD3 and mitochondria. Thus, ASMase contributes to TNF-α–induced
hepatocellular apoptosis by promoting the mitochondrial targeting of glycosphingolipids.
J. Clin. Invest.111:197–208 (2003). doi:10.1172/JCI200316010.
Received for publication May 28, 2002, and accepted in revised form
November 26, 2002.
Address correspondence to: José C. Fernández-Checa, Liver Unit,
Hospital Clinic i Provincial, Villarroel, 170, 08036-Barcelona,
Spain. Phone: 34-93-227-5709; Fax: 34-93-451-5272;
Carmen García-Ruiz and Anna Colell contributed equally to this
Conflict of interest: The authors have declared that no conflict of
Nonstandard abbreviations used: acidic sphingomyelinase
(ASMase); mitochondrial glutathione (mGSH); ganglioside GD3
(GD3); sphingolmyelinases (SMases); neutral SMase (NSMase);
acidic SMase (ASMase); factor associated with NSMase activation
(FAN); (R,S)-3-hydroxy-4-pentenoate (HP); 2′-7′-
dichlorofuorescein diacetate (DCFDA); tetramethylrhodamine
methylester (TMRM); alanine aminotransferase (ALT);
mitochondrial permeability transition (MPT); d-threo-1-phenyl-2-
decanoylamino-3-morpholino-propanol HCl (d-threo-PDMP);
reactive oxygen species (ROS).
or ceramide synthetase (17–19). Ceramide can also arise
from hydrolysis of sphingomyelin-engaging sphin-
golmyelinases (SMases) (20). TNF-αbinding to its plas-
ma membrane receptor results in the activation of two
major known types of SMases. The first, neutral SMase
(NSMase), with an optimum pH of approximately 7.5,
is membrane bound and Mg2+dependent. The second,
acidic SMase (ASMase), with an optimum pH of
approximately 4.8, is further subclassified into two iso-
forms, an endosomal/lysosomal ASMase and a secre-
tory Zn2+-dependent ASMase (21–24). These enzymes
are responsible for the ability of TNF-α to generate
ceramide with various kinetics and, most importantly,
at different intracellular locations (17, 23).
The role of individual SMases in apoptosis is contro-
versial and not well established, and their engagement
in apoptotic pathways depends on several conditions,
such as the kind of apoptotic stimuli used and the cell
type studied. For instance, NSMase has been shown to
play a role in chemotherapy-mediated cell death (25).
Furthermore, the ability of NSMase to signal apopto-
sis depends on the site within the plasma membrane
where ceramide is released (26). Moreover, the factor
associated with NSMase activation (FAN), an adaptor
protein essential for NSMase activation, has been
shown to contribute to TNF-α–mediated fibroblast
apoptosis (27). On the other hand, the deficiency in
ASMase has been reported to impair oocyte maturation
and apoptosis caused by ionizing radiation (28, 29). In
addition, while overexpression of acid ceramidase,
which diminishes ceramide production by ASMase,
protects L929 cells from TNF-α (30), inhibition of
ASMase early but not late during TNF-αsignaling pro-
tects ML-1a cells against TNF-α(31). Finally, although
some reports have indicated a role for ASMase in Fas-
mediated apoptosis (32–35), others have not (36), thus
establishing a conundrum whether or not ASMase
plays a role in Fas-mediated cell death.
In addition to its involvement in apoptotic signaling,
ceramide also provides the carbon source for gly-
cosphingolipid synthesis in the Golgi network. Gan-
glioside GD3 (GD3), a sialic acid–containing gly-
cosphingolipid, has been identified as a lipid death
effector because of its ability to interact and recruit
mitochondria to apoptotic pathways, contributing to
the mitochondrial-dependent apoptosome activation
triggered by death ligands (37–42). On the other hand,
it has been shown that prevention of glycolipid syn-
thesis enhances apoptosis, reversing multidrug resist-
ance in cancer cells (43, 44).
Information on signaling of hepatocellular apopto-
sis by TNF-α may be of therapeutic relevance for the
treatment of liver diseases. Although several interme-
diates have been described to contribute to TNF-α–
induced hepatocellular cell death — including FAN
(27), cathepsin B (45) or Bid, a BH3-only member of
the Bcl-2 family (46) — the role of ASMase in TNF-α–
mediated apoptosis in hepatocytes has not been previ-
ously reported. Thus, the aims of the present study
were to determine the role of ASMase in TNF-α–medi-
ated hepatocellular cell death and to estimate the rel-
ative contribution of ceramide versus glycosphingo-
lipids in this process.
ASMase knockout mice and hepatocyte isolation. ASMase
knockout mice (ASMase–/–), maintained in C57BL/6
background (kindly provided by E. Gulbins and R.
Kolesnick), were propagated using heterozygous breed-
ing pairs and genotyped by polymerase chain reaction
of tail DNA as described previously (35, 47). Primers 5′-
AGCCGTGTCCTCTTCCTTAC-3′ and 5′-CGAGACTGTT-
GCCAGACATC-3′ were used to amplify a 269-bp prod-
uct that is specific for exon 2 of the wild-type ASMase
gene in conditions that have been described previously
(33). Hepatocytes from male mice (8–12 weeks old)
were prepared by collagenase perfusion with a flow rate
of 7–9 ml per minute. Hepatocytes were cultured on
rat-tail collagen–coated dishes as indicated for rat
hepatocytes (48, 49).
Cell culture, incubations, and antibodies. Rat or mouse
hepatocytes were incubated with recombinant human
TNF-α(15–280 ng/ml; Promega, Madison, Wisconsin,
USA) or human placental ASMase (in 50% glycerol, 25
mM potassium phosphate [pH 4.5], 0.1% Triton X-100,
and 0.05 mM phenylmethylsulfonylfluoride; Sigma
Chemical Co., Madrid, Spain) as described previously
(49). Mouse anti-GD3 monoclonal antibody, clone
R24, produced by Matreya (Pleasant Gap, Pennsylva-
nia, USA) as described originally (41), displays a speci-
ficity toward GD3 characterized previously in detail by
compositional, partial structural analyses, immunos-
taining in thin-layer chromatography plates, and
immunoelectron microscopy (34, 41, 50). The human
anti-mitochondrial antibodies, a gift from A. Serrano
(CNB, CSIC, Madrid, Spain), recognize the E2 polypep-
tide of the mammalian mitochondrial pyruvate dehy-
drogenase complex (51, 52).
Selective depletion of mitochondrial GSH. (R,S)-3-hydroxy-
4-pentenoate (HP) was synthesized as described previ-
ously (53) and used to selectively deplete hepatocellu-
lar mitochondrial glutathione (mGSH) levels (54).
Hepatocytes were incubated with 1 mM HP for 5 min-
utes and then washed and fractionated into cytosol and
mitochondria by Percoll centrifugation (54). GSH lev-
els were determined in each fraction by high-perform-
ance liquid chromatography (48).
Determination of reactive oxygen species and mitochondrial
membrane potential by flow cytometry. Hydrogen peroxide
and mitochondrial membrane potential were determined
using 2′-7′-dichlorofuorescein diacetate (DCFDA) and
tetramethylrhodamine methylester (TMRM; Molecular
Probes, Eugene, Oregon, USA), respectively, in a FACStar
flow cytometer (Becton Dickinson, San Jose, California,
USA) as described previously (40, 55).
Cytochrome c release. The release of cytochrome cfrom
mitochondria after TNF-α exposure with or without
HP preincubation was analyzed upon permeabilization
The Journal of Clinical Investigation|January 2003| Volume 111|Number 2
of the plasma membrane of hepatocytes with digitonin
(80 µg/ml) in permeabilization buffer (210 mM man-
nitol, 70 mM sucrose, 5 mM succinate, 0.2 mM EGTA,
0.15% BSA, 10 mM HEPES [pH 7.2]). Cell plates were
shaken at 4°C for 5 minutes, after which the perme-
abilization buffer was removed, and cells were cen-
trifuged at 13,000 g for 10 min. The presence of
cytochrome c and cytochrome c oxidase was analyzed
by immunoblotting as described previously (40) using
antibody (clone 7H8.2C12;
PharMingen, San Diego, California, USA) or mouse
monoclonal anti–cytochrome c oxidase (subunit IV;
Caspase processing and activation. The activation of cas-
pase 3 in hepatocytes treated with TNF-α was deter-
mined as described previously (49) using a polyclonal
anti–caspase 3 antibody (Santa Cruz Biotechnology,
Santa Cruz, California, USA) after enhanced chemilu-
minescence detection. Caspase activity was determined
by release of 7-amino-4-trifluoromethyl coumarin
from Ac-DEVD-AMC (Bachem, Budendorf, Switzer-
land), and fluorescence was continuously recorded
with emission at 460 nm and excitation at 355 nm.
In vivo liver damage. C57BL6 mice were used at the age
of 8–10 weeks (25–30 g). A single dose of recombinant
murine TNF-α (10 mg/kg intravenously; Peprotech,
London, United Kingdom) or LPS (50 µg/kg intraperi-
toneally; Escherichia coli serotype 0128:B12; Sigma-
Aldrich, St. Louis, Missouri, USA) in a total volume of
150 µl of pyrogen-free saline containing 0.1% human
serum was injected 10 minutes after intraperitoneal
administration of galactosamine (700 mg/kg) in 200 µl
of pyrogen-free saline. Samples were taken at 2 and 4
hours after the challenge at the moment when the con-
dition of the animals began to deteriorate (56, 57).
Serum alanine aminotransferase (ALT) level was deter-
mined by a colorimetric test (Sigma-Aldrich). For his-
tology, liver specimens were washed with PBS and paraf-
fin embedded. Sections of 7 µm were made and stained
with hematoxylin and eosin using standard methods.
Inactivation of ASMase. ASMase from rat hepatocytes
was inactivated before treatment with TNF-α by prior
incubation of cells with imipramine (50 µM) (58, 59).
ASMase activity in cellular extracts was determined by
monitoring N-methyl-[14C]sphingomyelin (56.6 mCi/
mmol) hydrolysis as described previously (49).
Measurement of GD3 and ceramide levels. Ganglioside
(including GM1 and GD3) levels in hepatocytes
(3–4 × 106) were determined by high-performance
thin-layer chromatography as described previously
(40). Ceramide levels were quantified by high-per-
formance liquid chromatography (49).
Assessment of cell death. To determine the incidence of
cell death in hepatocytes treated with TNF-α, cell cul-
tures were doubly stained with propidium iodide and
Hoechst 33258. Propidium iodide is a vital nucleic
acid–staining dye that penetrates cells with compro-
mised plasma membrane (necrotic cells). Morphologi-
cal changes in the nuclei of cells undergoing apoptotic
cell death were determined by staining with the DNA-
binding fluorochrome Hoechst 33258. Apoptotic
nuclear changes include condensation, margination,
and segmentation of the nuclei into several fragments.
After TNF-α incubation with or without HP pretreat-
ment, cells were stained with both dyes (10 µM final
concentration) for 10–15 minutes at 37°C. Afterwards,
cells were fixed with paraformaldehyde and mounted
on a fluorescence microscope. Quantitation of apop-
totic and necrotic cells was performed by counting at
least 250 cells in six different high-power fields and was
expressed as a percentage of total cells counted.
NF-κB activation and translocation of Bax to mitochondria.
NF-κB DNA binding activity in nuclear extracts was
assessed by electrophoretic mobility shift assays using
an NF-κB consensus oligonucleotide (5′-AGTTGAGG-
GGACTTTCCCAGGC-3′) as described previously (42).
Supershift assays were conducted by using the anti-
bodies anti-RelA, p50, and p52 as described (42). The
mitochondrial translocation of Bax was assessed by
Western blot analyses of mitochondria prepared from
cultured mouse hepatocytes upon incubation with
TNF-α as described previously (60). Cells (2–3 × 106)
were collected, washed twice with PBS, and resuspend-
ed in 500 µl of isolation buffer (200 mM mannitol,
70 mM sucrose, 10 mM HEPES-KOH, 1 mM EGTA,
0.2% BSA [pH 7.5]) supplemented with a protease
inhibitor cocktail (Roche, Manheim, Germany). After
chilling on ice for 10 minutes, cells were disrupted by
20 passages through a 25G syringe. The homogenate
was centrifuged at 4°C for 10 minutes at 600 g, and the
supernatant obtained was centrifuged at 10,000 g
for 10 minutes. The mitochondrial pellet was resus-
pended in 250 mM sucrose, 10 mM HEPES-KOH, 1
mM ATP, 0.08 mM ADP, and 2 mM K2HPO4(pH 7.5).
Mouse hepatocytes were treated with TNF-α (0.28
µg/ml) plus galactosamine (1 mM) for 16 hours. West-
ern blots (40 µg of protein per lane) were performed
using Bax mouse monoclonal antibody (SC-7480,
1:200; Santa Cruz Biotechnology) and developed as
described above for cytochrome c.
Immunocytochemical staining.Mouse hepatocytes were
fixed for 10 minutes in 3.7% paraformaldehyde in 0.1
M phosphate buffer before permeabilization with 0.1%
saponin in 0.5% BSA/PBS buffer for 15 minutes. The
immunolocalization of GD3 relative to mitochondria
was performed as described before for rat hepatocytes
(61) using mouse anti-GD3 antibody (1:500) and
human anti-mitochondrial antibody (1:2000). Confo-
cal images were collected using a Leica TCS-NT laser
scanning confocal microscope equipped with an
argon-krypton laser and a 63× Leitz Plan-Apo objec-
tive (numerical aperture, 1.4). The number of cells
observed per field was at least 100 for each treatment.
Statistical analyses. Results are expressed as means ±SD
and are averages of three to five values per experiment
and condition. Statistical analyses of mean values for
multiple comparisons were made by one-way analysis
of variance followed by Fisher’s exact test.
The Journal of Clinical Investigation|January 2003|Volume 111|Number 2
Selective mGSH depletion sensitizes hepatocytes to TNF-α.
Hepatocytes are known to be resistant to TNF-α.
Blocking protein or total RNA synthesis or NF-κB
activation has been commonly used to unmask the
ability of TNF-α to induce hepatocellular apoptosis
(4, 8). We have used HP to generate a Michael accep-
tor within mitochondria as a strategy to selectively
deplete the mGSH (53, 54). These mGSH-depleted
hepatocytes were then challenged with TNF-α
(28–280 ng/ml). HP caused a dramatic and selective
mGSH depletion but spared cytosolic GSH in rat
hepatocytes (Figure 1a). This approach by itself did
not impair mitochondrial function, as shown by
maintenance of mitochondrial membrane potential
estimated from TMRM fluorescence by flow cytome-
try. However, when mGSH-depleted hepatocytes were
exposed to TNF-α(280 ng/ml), there was a significant
and early generation of peroxides that preceded the
loss of mitochondrial membrane potential (Figure
1b), indicating the onset of mitochondrial permeabil-
ity transition (MPT). Since one of the critical compo-
nents of apoptotic pathways is the release of
cytochrome c from mitochondria, we next character-
ized the kinetics of this event in hepatocytes after
TNF-α exposure and the effect of HP preincubation.
The release of cytochrome c was detectable after 2
hours of incubation with TNF-α only in hepatocytes
that were pretreated with HP (Figure 1c). Further-
more, this event preceded the activation of caspase 3
as assessed by the time-dependent increase of active
caspase 3 fragments (Figure 1d) and activity deter-
mined from the fluorescence of AMC released from
the synthetic peptide DEVD-AMC (Figure 1e).
Previous studies have shown that various strategies
— for example, cycloheximide, galactosamine, or acet-
aminophen — rendered hepatocytes sensitive to
TNF-αexposure which induced apoptotic and necrot-
ic cell death (62–64). To determine the extent of cell
death induced by TNF-α under these conditions, cell
cultures were labeled with propidium iodide, a vital
stain, and the permeable DNA-binding fluorochrome
Hoechst 33258. HP pretreatment sensitized hepato-
cytes to TNF-α exposure with increasing incidence of
both apoptosis and necrosis (Figure 2a). Although
apoptosis increased gradually during the initial 8
hours of incubation with TNF-α (declining there-
after), necrosis increased continually over time. By 8
hours after TNF-α incubation, the magnitude of
apoptosis and necrosis was comparable and seen only
in HP-pretreated cells (Figure 2a). Consistent with the
critical role of MPT in TNF-α–mediated cell death (8),
cyclosporin A treatment protected HP-exposed hepa-
tocytes from TNF-α (Figure 2a). The sensitivity of
mGSH-depleted hepatocytes to TNF-α was also
observed at lower doses of TNF-α. HP-pretreated
hepatocytes underwent a time-dependent cell death
after incubation with 25 ng/ml of TNF-α (Figure 2a).
Similar findings of cell death were observed when
hepatocytes were sensitized to TNF-α by actinomycin
The Journal of Clinical Investigation| January 2003| Volume 111| Number 2
Mitochondrial GSH depletion facilitates TNF-α–mediated mitochondrial depolarization. (a) Cultured rat hepatocytes were pretreated with HP
(1 mM) for 5 minutes and then fractionated to obtain cytosol and mitochondria for GSH determination by high-performance liquid chro-
matography. Results are expressed as means ±SD (n= 4 or 5). *P< 0.05 versus control. (b) After HP treatment, hepatocytes were then exposed
to TNF-α (280 ng/ml) for 15–60 minutes. Peroxide formation and mitochondrial membrane potential were determined upon staining of cells
with DFCDA and TMRM, respectively, and fluorescence of both fluorochromes was determined by flow cytometry. A representative profile of
four independent experiments is shown. (c) Hepatocytes treated with TNF-α(280 ng/ml) over time with or without HP pretreatment were per-
meabilized with digitonin, and cell extracts were analyzed for cytochrome c or cytochrome c oxidase as indicated in the Methods. (d and e)
Aliquots of cell extracts were used for the detection of active caspase 3 fragments by immunoblotting (d) or activity determination using a flu-
orescent peptide (e). Results are expressed as means ± SD (n = 3 or 4 independent experiments).
D preincubation in agreement with previous reports
(45). These findings indicate that the selective deple-
tion of mGSH sensitizes hepatocytes to TNF-α by
facilitating the onset of MPT.
Inhibition of ASMase activity protects sensitized hepato-
cytes from TNF-α. To examine the role of ASMase in
TNF-α–mediated hepatocellular apoptosis, we ana-
lyzed the effects of ASMase inactivation on the sur-
vival of mGSH-sensitized hepatocytes after TNF-α
treatment. The activity of ASMase in response to
TNF-α increased twofold as compared with control
hepatocytes, an effect that was observed within 10–20
minutes after TNF-α incubation (Figure 3, a and b).
The pretreatment of hepatocytes with HP did not
modify the activity of ASMase in response to TNF-α
(data not shown). ASMase has been shown to be
processed from its precursor form (75 kDa) to the
active 72 kDa ASMase form (65). Imipramine, a tri-
cyclic antidepressant, induces the proteolysis of the
active 72 kDa ASMase form (58, 59), and hence it is
expected to inhibit the stimulated ASMase activity, as
shown previously (59, 66). Hepatocytes were preincu-
bated with imipramine before exposure to TNF-α,
attenuating the ASMase activity induced by TNF-α
(Figure 3a). To test the role of imipramine on cell sur-
vival, hepatocytes were first treated with HP and then
exposed to TNF-α. Although HP-pretreated hepato-
cytes were sensitive to TNF-α–induced apoptosis,
imipramine pretreatment protected HP-sensitized
hepatocytes against TNF-α exposure (Figure 3c). This
protective effect of imipramine was accompanied by a
decrease in the activation of caspase 3 (Figure 3d).
The Journal of Clinical Investigation| January 2003| Volume 111| Number 2
Mitochondrial GSH depletion sensitizes hepatocytes to TNF-α. (a)
Cultured hepatocytes were exposed to TNF-α (280 ng/ml, solid line;
25 ng/ml, dashed line) for various periods of time with or without
HP preincubation to deplete the mGSH levels. Cell death was deter-
mined by double staining with Hoechst 33258 and propidium iodide
to detect apoptotic and necrotic cells, respectively. At least 250 cells
in six different high-power fields were counted and expressed as a
percentage of total cells (please note the scale difference). HP alone
did not affect cell survival. Results are expressed as means ± SD
(n = 4 experiments). *P < 0.05 versus control. (b) Representative flu-
orescent micrographs of control hepatocytes exposed to TNF-α
(panels 1 and 3) or HP-pretreated hepatocytes (panels 2 and 4). Blue
fragmented nuclei (panel 2) represent apoptotic cells, whereas red
nuclei (panel 4) are indicative of necrotic cells.
Imipramine inactivates ASMase in hepatocytes. (a) Hepatocytes were
treated with TNF-α (280 ng/ml) for 2 hours with or without prein-
cubation with imipramine (50 µM). Cellular extracts were used for
ASMase activity determined from N-methyl-[14C]sphingomyelin
(56.6 mCi/mmol) hydrolysis. Levels of phosphorylcholine produced
from sphingomyelin were determined in the aqueous phase by scin-
tillation counting. Results are expressed as means ± SD (n = 5 inde-
pendent experiments). *P < 0.05 versus control, and **P < 0.05 ver-
sus TNF-α. (b) The activity of ASMase with or without imipramine
pretreatment was determined at different times after TNF-α expo-
sure as in a. (c) To test the role of imipramine in cell survival, hepa-
tocytes were first treated with HP and then incubated with TNF-α
(280 ng/ml) for 12 hours. Cell death was determined by Hoechst
33258 and propidium iodide staining. Results are expressed as
means ± SD (n = 4 independent experiments). *P < 0.05 versus con-
trol, and **P < 0.05 versus HP plus TNF-α. (d) The activity of cas-
pase 3 was determined under these various conditions with or with-
out imipramine preincubation from the fluorescence of released
AMC fragments. Results are expressed as means ± SD (n = 4 inde-
pendent experiments). *P < 0.05 versus control, and **P < 0.05 ver-
sus HP plus TNF-α.
Thus, these findings reveal that ASMase contributes
to TNF-α apoptosis in sensitized hepatocytes and
that prevention of stimulated ASMase activity pro-
tects hepatocytes from TNF-α–induced cell death.
ASMase-deficient hepatocytes are resistant to TNF-α–
induced cell death. In order to establish the role of
ASMase in TNF-α–mediated apoptosis in a more
definitive fashion, we used hepatocytes from
ASMase+/+and ASMase–/–mice. Treatment of mouse
hepatocytes with HP resulted in selective depletion of
mGSH levels by 68% ± 6% (P < 0.05) without affecting
the cytosolic pool of GSH (88% ± 8% of control hepa-
tocytes). Under these conditions, and similar to the
outcome seen with rat hepatocytes, HP pretreatment
sensitized mouse hepatocytes to TNF-α–induced
apoptotic and necrotic cell death (Figure 4a).
Cyclosporin A pretreatment protected HP-treated
ASMase+/+hepatocytes against TNF-α–mediated cell
death (data not shown). Furthermore, HP pretreat-
ment sensitized hepatocytes to human placental
ASMase (Figure 4a). Monensin pretreatment pre-
vented TNF-α or exogenous human placental
ASMase-induced apoptosis in HP-pretreated mouse
hepatocytes (data not shown).
In contrast with these findings, ASMase–/–hepato-
cytes were resistant to TNF-α exposure, despite HP
pretreatment (Figure 4b). The levels of GSH in the
mitochondrial fraction after HP exposure were deplet-
ed down to 26% ± 8% of those of control hepato-
cytes, with the sparing of cytosolic GSH. Despite the
The Journal of Clinical Investigation| January 2003| Volume 111| Number 2
Resistance of hepatocytes from ASMase knockout
mice to TNF-α–induced apoptosis. ASMase+/+(a) or
ASMase–/–(b) mouse hepatocytes were first pretreat-
ed with HP to deplete the mGSH pool and then treat-
ed with either TNF-α (280 ng/ml) or ASMase (0.4
U/ml) for 12 hours. Cell death was determined by
Hoechst 33258 and propidium iodide staining.
Results are expressed as means ± SD (n = 5 independ-
ent experiments). *P < 0.05 versus control.
In vivo TNF-α–induced liver damage in ASMase knockout mice. (a) ASMase+/+and ASMase–/–mice were first administered galactosamine (700
mg/kg) 10 minutes before the intravenous injection of TNF-α (10 mg/kg) or saline (controls). At 2 and 4 hours, mice were sacrificed, and
blood was drawn to determine ALT concentrations. ALT concentrations in control samples were less than 20 IU/ml. Results are expressed as
means ± SD (n = 5 independent experiments). *P < 0.05 versus ASMase+/+. (b) Serum ALT levels were determined as in (a), except that mice
were injected with LPS (50 µg/kg) after galactosamine pretreatment. Results are expressed as means ± SD (n = 5 independent experiments).
*P < 0.05 versus ASMase+/+. (c) Representative hematoxylin and eosin staining of ASMase+/+and ASMase–/–mouse liver specimens harvested 4
hours after administration of TNF-αplus galactosamine. (d) Representative hematoxylin and eosin staining of ASMase+/+and ASMase–/–mouse
liver specimens harvested 4 hours after administration of LPS plus galactosamine.
resistance of ASMase–/–hepatocytes to TNF-α, these
cells were susceptible to exogenous human placental
ASMase exposure (Figure 4b). Addition of exogenous
ASMase induced cytochrome c release and caspase 3
activation in both ASMase+/+and ASMase–/–hepato-
cytes (data not shown). Moreover, ASMase+/+and
ASMase–/–hepatocytes were resistant to bacterial
NSMase (0.4–1.0 U/ml) treatment with or without
pretreatment with HP. Similar findings were observed
when hepatocytes were sensitized with actinomycin D
pretreatment (62 ± 7 vs. 19 ± 6 apoptosis in ASMase+/+
as compared with ASMase–/–cells, P < 0.05), indicating
that ASMase is involved after sensitization of hepato-
cytes by means other than mGSH depletion. Thus,
taken together, these findings indicate that selective
mGSH depletion sensitizes mouse hepatocytes to
both TNF-α and exogenous ASMase.
Resistance of ASMase–/–mice to endogenous or exogenous
TNF-α–induced liver damage in vivo. To examine the role
of ASMase in TNF-α–mediated liver injury in vivo,
ASMase+/+and ASMase–/–mice were sensitized by galac-
tosamine treatment to endogenous TNF-α secretion
or exposure to exogen-
ous TNF-α (56, 57). At 2
and 4 hours after TNF-α
treatment, serum ALT con-
centrations were signifi-
cantly higher in ASMase+/+
mice than in ASMase–/–mice
(1545 ± 234 IU/ml vs.
257 ± 67 IU/ml at 2 hours
and 8157 IU/ml ± 783 vs.
1787 ± 324 IU/ml at 4
hours) (Figure 5a). Livers
from the ASMase+/+mice dis-
played extensive hemorrhag-
ic lesions and clusters of
damaged cells. In contrast,
liver specimens from
ASMase–/–mice showed min-
imal damage (Figure 5c).
Further studies revealed that
TNF-αkilled 91% of mice at
5–6 hours after treatment
(n = 5), whereas 85% of
ASMase–/–mice survived at
least 48 hours after TNF-α
treatment (n = 4). Similar
findings were observed
when mice were injected
with a single dose of LPS
to stimulate endogenous
TNF-αsecretion in terms of
se-rum ALT levels and histo-
logical findings (Figure 5, b
and d). Thus, these data
clearly show that ASMase
plays a key role in TNF-α–
mediated liver damage.
Glycosphingolipids rather than ceramide contribute to
TNF-α–mediated hepatocellular apoptosis. The preceding
data indicate that ASMase is required for efficient
TNF-α–induced hepatocellular apoptosis. Since ASMase
generates ceramide — which, in turn, fuels glycosphin-
golipid formation — we next estimated the relative con-
tribution of ceramides versus glycosphingolipids in
TNF-α–induced apoptosis. This question is of particu-
lar relevance since both types of lipids have been involved
in apoptosis. To address this issue, the generation of
ceramide and glycosphingolipids by TNF-α was deter-
mined with or without glucosylceramide synthase inhi-
bition. Treatment of hepatocytes with TNF-α signifi-
cantly increased the levels of ceramide (296% ± 34% of
control levels, P < 0.05) and glycosphingolipids, includ-
ing gangliosides GD3 and GM1 (367% ± 47% and
338% ±54% of control levels, respectively; P< 0.05) (Fig-
ure 6, a and b), at 2 hours after incubation, with the
synthesis of GD3 being significantly higher than
basal levels by 40–60 minutes after TNF-α incu-
bation. Pretreatment of hepatocytes with d-threo-1-
The Journal of Clinical Investigation| January 2003| Volume 111| Number 2
Effect of glucosylceramide synthetase inhibition on sphingolipids and hepatocellular survival. (a)
Cultured hepatocytes were treated with TNF-α (280 ng/ml) for 2 hours with or without d-threo-
PDMP (d-t-PDMP) pretreatment to inhibit glucosylceramide synthase. Cellular lipids were extract-
ed by chloroform/methanol, and ceramide levels were determined by high-performance liquid chro-
matography. Results are expressed as means ± SD (n = 4 independent experiments). *P < 0.05 versus
control, and **P < 0.05 versus HP plus TNF-α. (b) Lipid extracts from hepatocytes treated with
TNF-α with or without d-threo-PDMP were applied to high-performance thin-layer chromatography
plates to resolve glycosphingolipids along with authentic ganglioside standards (GD3 and GM1).
The level of GD3 was calculated by densitometric analyses of high-performance thin-layer chro-
matography plates and compared with a standard curve generated using known amounts of GD3.
(c) To determine the role of glycosphingolipid synthesis inhibition on hepatocellular survival, cells
were first treated with HP and then exposed to TNF-α for 12 hours with or without d-threo-PDMP
pretreatment. Cell death was determined by Hoechst 33258 and propidium iodide staining after 12
hours of TNF-α incubation. Results are expressed as means ± SD (n = 6 independent experiments).
*P < 0.05 versus control, and **P < 0.05 versus HP plus TNF-α.
(d-threo-PDMP), an inhibitor of glucosylceramide syn-
thase, abolished the stimulation of glycosphingolipid
levels by TNF-α, whereas it enhanced ceramide genera-
tion as compared with the stimulation induced by
TNF-α itself (Figure 6, a and b). The inactive isomer d-
erythro-PDMP did not have any effect (data not shown).
In order to test the effect of d-threo-PDMP on hepatocyte
survival, cells were first sensitized with HP treatment.
The exposure of HP-treated hepatocytes to TNF-α
increased the levels of ceramide and gangliosides to
245% ± 34% and 324% ± 54%, respectively (P = 0.05 vs.
percentage of unstimulated controls), in the absence of
d-threo-PDMP and to 623% ± 76% and 113% ± 23%,
respectively, in the presence of d-threo-PDMP. The gen-
eration of ceramide and gangliosides by TNF-αwas sim-
ilar in the presence or absence of HP treatment. Despite
the divergent TNF-α–induced generation of ceramide
and glycosphingolipids in the presence of d-threo-PDMP,
this agent protected HP-treated hepatocytes against
TNF-α–induced apoptotic and necrotic cell death (Fig-
ure 6c). Similar findings were observed when glucosyl-
ceramide synthase was inhibited by N-butyldeoxynojir-
imycin (data not shown). Thus, these findings suggest
the involvement of glycosphingolipids generated from
ASMase-induced ceramide formation as key intermedi-
ates of TNF-α–induced hepatocellular apoptosis.
TNF-α stimulates the targeting of ganglioside GD3 to mito-
chondria. Out of the repertoire of glycosphingolipids syn-
thesized from ceramide, we focused on GD3, since this
glycosphingolipid has emerged as a proapoptotic lipid
effector because of its interaction with mitochondria
(38–42). To examine if GD3 is targeted to mitochondria
during TNF-α signaling, we examined the localization
of GD3 and mitochondria by confocal microscopy using
an anti-GD3 antibody, whose specificity toward GD3
has been documented previously (41, 50), and an anti-
mitochondrial antibody that recognizes the E2 polypep-
tide of the mammalian pyruvate dehydrogenase complex
(51, 52). The merged fluorescence of both antibodies is
shown in Figure 7. In control ASMase+/+mouse hepato-
cytes, most of the GD3 was present at the cell surface
and in intracellular structures corresponding to the
Golgi network as verified by the colocalization of GD3
with the asialoglycoprotein receptor, as shown recently
in cultured rat hepatocytes (61). After TNF-αtreatment,
GD3 disappeared from the plasma membrane and was
targeted to mitochondria, as estimated by the presence
of merged red and green fluorescence (Figure 7). In con-
trast, TNF-αdid not induce the mitochondrial targeting
of GD3 in ASMase−/−hepatocytes (Figure 7). These find-
ings, therefore, correlate with the resistance of hepato-
cytes deficient in ASMase to TNF-α–mediated cell death
(Figure 4). Since these cells were sensitive to exogenous
ASMase treatment, we examined the redistribution of
GD3 in response to exogenous human placental
ASMase. Both ASMase+/+and ASMase–/–hepatocytes
showed the colocalization of GD3 with mitochondria
(Figure 7), in agreement with previous results (61). More-
over, to test the role of ASMase in the cytotoxicity of
GD3, ASMase+/+and ASMase–/–were exposed to GD3
after HP pretreatment. As seen, both types of cells were
equally sensitive to GD3 treatment, and this effect was
The Journal of Clinical Investigation| January 2003| Volume 111|Number 2
Immunocytochemical localization of GD3 and mitochondria in
response to TNF-α and exogenous ASMase. ASMase+/+and ASM–/–
hepatocytes were incubated with TNF-α (280 ng/ml), human pla-
cental ASMase (0.4 U/ml), or vehicle (control) for 4 hours and then
doubled immunostained with anti-GD3 and anti-mitochondrial anti-
bodies followed by appropriate secondary antibodies. Merged images
of red (GD3) and green (mitochondria) fluorescence were collected
by confocal microscopy. Colocalization of GD3 with mitochondria
appears as yellow spots reflecting the merged red and green fluores-
cence. The bar represents 10 µm. At least 100 cells per condition were
examined. Less than 10% of control cells displayed merged fluores-
cence, whereas colocalization was observed in more than 85% of the
cells examined following TNF-α. Representative images of four inde-
pendent experiments with similar results are shown.
Cytotoxicity of GD3 in mouse hepatocytes. ASMase+/+(a) and ASM–/–
(b) hepatocytes were treated with GD3 (10 µM) with or without HP
preincubation, and cell death was determined by Hoechst 33258 and
propidium iodide staining to quantify apoptosis and necrosis after
8 hours. Caspase 3 activity was determined in cell extracts from the
fluorescence of AMC fragments. Results are expressed as means ± SD
(n = 4 independent experiments). *P < 0.05 versus control, and
** P < 0.05 versus GD3 without HP preincubation.
potentiated upon depletion of mGSH by HP pre-expo-
sure (Figure 8). Furthermore, these findings were accom-
panied by increased caspase 3 activity, which was poten-
tiated by HP pretreatment in agreement with previous
results in cultured rat hepatocytes (40, 42). Taken
together, these findings indicate that TNF-αsignals the
mitochondrial targeting of GD3 and that this process is
likely involved in cell death.
NF-κB activation and mitochondrial Bax translocation in
ASMase-deficient hepatocytes. To examine whether the resist-
ance of ASMase–/–hepatocytes to TNF-αis related to defi-
cient signaling upstream of mitochondria, we examined
the activation of NF-κB and the translocation of Bax to
mitochondria. TNF-αenhanced the DNA binding activ-
ity of NF-κB in nuclear extracts in both ASMase+/+and
ASMase–/–hepatocytes (Figure 9a). Furthermore, the lev-
els of Bax detected in the mitochondrial fraction after
TNF-αadministration was similar in both wild-type and
ASMase-deficient hepatocytes (Figure 9b). Moreover,
similar results were observed in rat hepatocytes in which
endogenous ASMase was inactivated by imipramine
without impairment in the activation of NF-κB nor mito-
chondrial translocation of Bax induced by TNF-α (data
not shown). Hence, these findings indicate that signaling
events upstream of mitochondria induced by TNF-αare
preserved in hepatocytes lacking ASMase.
The present study was undertaken to specifically
address the role of ASMase in the apoptotic signaling
of TNF-αin hepatocytes. TNF-αapoptotic signaling is
a complex process that involves protein-protein inter-
actions and the participation of several intermediates.
Sphingolipid generation through SMase activation has
been involved in apoptotic pathways induced by death
ligands (17–20, 23). We provide compelling evidence
that ASMase plays an important contributory role in
TNF-α–mediated hepatocellular apoptosis. Because of
the intrinsic resistance of primary hepatocytes to
TNF-α, survival pathways must be antagonized to
unmask the efficient apoptotic ability of TNF-α. Hepa-
tocytes selectively depleted of mGSH levels using HP,
which generates a Michael acceptor within mitochon-
dria that is conjugated with the endogenous mGSH
pool (53, 54), become susceptible to induction by
TNF-αof both apoptotic and necrotic cell death. Both
forms of cell death, however, were prevented by
cyclosporin A, indicating the relevance of MPT in initi-
ating either outcome of cell demise (67).
ASMase contributes to TNF-α–induced hepatocellular apop-
tosis. To address the role of ASMase in apoptotic sig-
naling by TNF-α, we used two distinct approaches. The
first approach was the inactivation of endogenous
hepatocellular ASMase, and the second was the use of
hepatocytes from ASMase knockout mice. Both strate-
gies indicated resistance to TNF-α–mediated cell death.
The role of individual SMases — namely, NSMase and
ASMase — in death ligand–induced apoptosis is con-
troversial and not well established. In focusing on
TNF-α–mediated apoptosis, previous studies have
shown that FAN, an adapter factor associated with
NSMase activation (68), is involved in TNF-α–induced
apoptosis in fibroblasts (27). Although these data clear-
ly show an important role of NSMase in TNF-α–
induced apoptosis in fibroblasts, the fate of FAN-defi-
cient hepatocytes in TNF-α–mediated cell death was
not examined. Consistent with the ability of TNF-α to
activate both NSMase and ASMase (17, 23), we observed
that TNF-αstimulated NSMase activity in hepatocytes
deficient in ASMase. Thus, although our present data
argue for a critical role of ASMase in TNF-α–mediated
hepatocellular cell death, at present we cannot rule out
an indirect contributory role of NSMase. Whether or
not TNF-αrequires both SMases for optimal cell-death
induction requires further investigation.
ASMase needs an acidic pH environment for optimal
function, and according to this functional require-
ment, ASMase is assumed to reside in acidic vesicles or
compartments (22, 23). In studies that exploit this fea-
ture, the role of acidic compartments in TNF-α–medi-
ated cell death has been recognized in U937 and L929
cells (69). Moreover, we have previously reported that
monensin prevents the burst of mitochondrial reactive
oxygen species (ROS) generation and hepatocellular
cell death by human placental ASMase (49). Recently,
it has been shown that cathepsin B, a lysosomal
The Journal of Clinical Investigation| January 2003| Volume 111|Number 2
NF-κB activation and mitochondrial Bax translocation in mouse
hepatocytes. (a) Nuclear extracts from ASMase+/+and ASMase–/–hepa-
tocytes after TNF-αexposure were prepared and used for NF-κB acti-
vation using a consensus oligonucleotide. (b) Hepatocytes were treat-
ed with galactosamine plus TNF-α (280 ng/ml) for 16 hours. Cells
were then fractionated to prepare mitochondria as described in the
Methods. Bax levels in the mitochondrial fraction were analyzed by
Western blotting and quantitated by densitometry. Results are
expressed as means ± SD (n = 4 independent experiments). *P < 0.05
versus control. Gal + TNF-α, galactosamine plus TNF-α.
enzyme, plays an important role in TNF-α–mediated
apoptosis in hepatocytes (45). Our current study adds
to these reports, indicating an important function of
acidic components for efficient TNF-α–induced hepa-
tocellular apoptosis. In addition to the participation of
these components, hepatocytes deficient in Bid, a BH3-
only member of the Bcl-2 family, exhibited resistance
to TNF-α and Fas-induced hepatocellular injury (46).
Taken together, these observations indicate the partic-
ipation of several partners (including at least Bid,
cathepsin B, and ASMase) for optimal TNF-α–mediat-
ed cell death in hepatocytes. The functional interplay
between these players in TNF-α apoptotic signaling
remains to be elucidated.
Signaling events upstream of mitochondria are preserved in
ASMase-deficient hepatocytes. Having observed that
ASMase is necessary for efficient TNF-α–mediated
hepatocellular damage, we tested whether the absence
of ASMase interfered with known effectors of TNF-α
action. Our results show that the activation of NF-κB,
which occurs early during TNF-α signaling, is compa-
rable between ASMase+/+and ASMase–/–hepatocytes. In
agreement with these findings, previous studies report-
ed that the activation pattern of p38, JNK, and extra-
cellular signal-regulated kinases in response to epider-
mal growth factor or TNF-α were similar in ASMase+/+
and ASMase–/–mice (35). Moreover, a requirement has
been shown for BH1-3 multidomain Bcl-2 family mem-
bers, Bax or Bak, for the efficient apoptosis induced by
a variety of inducing signals (70). Bax is predominant-
ly localized in the cytosol of healthy cells and translo-
cates to mitochondria after apoptotic stimulation
because of a conformational change in the N and C ter-
mini of Bax that results in its translocation, oligomer-
ization or cluster formation, and integration into the
mitochondrial membranes (71). We determined the lev-
els of Bax in the mitochondrial fraction of ASMase–/–in
response to TNF-α to ascertain whether this critical
step was missing or defective. Unexpectedly, our find-
ings indicate that the translocation of Bax to mito-
chondria was unimpaired in hepatocytes deficient in
ASMase. These results therefore indicate that the
translocation of BH1-3 multidomain Bcl-2 family
members — for example, Bax — appears to be inde-
pendent of ASMase, yet downstream steps of mito-
chondria, including cytochrome c release and caspase
activation, seem to require ASMase. Further work will
be needed to address these fundamental questions. Per-
haps through altered ceramide generation and/or
sphingomyelin metabolism, ASMase may contribute to
the maintenance of a mitochondrial lipid environment
adequate for the proper mitochondrial docking of Bax
to facilitate the apoptosome.
Glycosphingolipids contribute to TNF-α–induced apoptosis.
In pursuing the mechanisms involved in linking
ASMase to mitochondria during TNF-αapoptotic sig-
naling in hepatocytes, we addressed the relative contri-
bution of ceramide versus glycosphingolipids, two
major products of ASMase. Previous studies have
shown that ceramide interacts with isolated mito-
chondria, impairing mitochondrial respiration and
stimulating ROS formation and MPT onset (55,
72–74). On the other hand, disialoganglioside GD3 has
been shown to interact with mitochondria in vitro,
leading to MPT, cytochrome crelease, and caspase acti-
vation (38–42). To discern the participation of these
putative mitochondria-interacting lipids in TNF-α
apoptotic signaling, we blocked the synthesis of gan-
gliosides and analyzed the survival of hepatocytes dur-
ing TNF-α exposure. Inhibition of glucosylceramide
synthase, a resident Golgi enzyme that forms glucosyl-
ceramide, the precursor for complex glycosphingolipid
synthesis (75), results in depressed basal and TNF-α–
stimulated glycolipid levels, while, consequently, lead-
ing to increased basal and enhanced TNF-α–stimulat-
ed ceramide levels. Unexpectedly, HP-sensitized hepa-
tocytes become protected against TNF-α in the
presence of d-threo-PDMP despite the fact that
ceramide accumulated twofold with respect to the
stimulation of ceramide induced by TNF-α itself.
Hence, although these results minimize the participa-
tion of ceramide itself in the targeting and subsequent
engagement of mitochondria in the TNF-α cell-death
pathway, it may be conceivable that both lipids
(ceramide and glycosphingolipids, e.g., GD3) may be
required for optimal cytotoxic activity of TNF-α. The
relative contribution of ceramide versus glycosphin-
golipids in apoptosis and cell death may be dependent
on the cellular context in which these sphingolipids are
generated (76). Furthermore, the monitoring of the cel-
lular distribution of GD3 during TNF-αsignaling indi-
cated its mitochondrial targeting in ASMase+/+but not
in ASMase–/–hepatocytes, suggesting that ASMase is
necessary for this event. In further support for this
interpretation, exogenous ASMase induced both the
mitochondrial targeting of GD3 in ASMase–/–hepato-
cytes and the sensitization of these cells to exogenous
ASMase exposure, and, as expected, ASMase–/–mice
were sensitive to GD3 in agreement with previous
results (38, 40, 42). Moreover, recent findings in cul-
tured rat hepatocytes showed that the targeting of GD3
to mitochondria induced by TNF-α occurred within 1
hour after incubation (61), well before the activation of
caspase 3, indicating that the formation of GD3 and its
subsequent translocation to mitochondria initiated the
molecular mechanisms of cell death. In line with this
outcome in hepatocytes, a recent study has shown that
GD3 contributes to Fas-mediated apoptosis, reporting
the colocalization of GD3 with mitochondria in lym-
phoid cells (77). Thus, although our data support a role
for GD3 synthesized from ASMase in the apoptotic sig-
naling by TNF-α, at present we cannot discard the
involvement of other glycosphingolipids derived from
ASMase in this process. Other related glycosphin-
golipids share the structural features of GD3 for its
mitochondria-interacting function (42). However, as
shown previously, the downregulation of targeted GD3
synthase, the enzyme responsible for GD3 synthesis
The Journal of Clinical Investigation| January 2003| Volume 111| Number 2
from GM3, protected lymphoid cells against Fas-medi-
ated apoptosis (37) or human colon HT-29 cells against
TNF-α–induced apoptosis (78).
Hence, our findings indicate a sequence of events ini-
tiated by the mitochondrial targeting of GD3 generat-
ed from ASMase-induced ceramide by TNF-α. GD3
then permeabilizes mitochondria, setting in motion
the molecular pathways leading to apoptotic and
necrotic cell death.
As referred to above, our data indicate that although
GD3 reaches mitochondria in hepatocytes in response
to TNF-α, the consequences of this interaction are con-
trolled by mGSH levels. The sensitization of mGSH-
depleted hepatocytes to either TNF-α or exogenous
human placental ASMase indicates that mGSH regu-
lates cell survival by modulating MPT through control
of an appropriate redox environment. Increased mito-
chondrial ROS formation may induce MPT opening
through the targeting of susceptible sulfhydryl groups
of MPT components (79). The relevance of mGSH for
hepatocyte survival in response to TNF-αmay be of ther-
apeutic significance for the treatment of alcohol-
induced liver disease. Increased TNF-αlevels are impor-
tant for the progression of alcohol-induced liver damage
(80), and hepatocytes from chronically alcohol-fed rats,
which exhibit depleted mGSH, are susceptible to
TNF-α–mediated cell death (54, 81–83). According to
our present data, strategies aimed to reduce or antago-
nize TNF-α–stimulated glycosphingolipid generation
and/or to replenish mGSH levels may prevent or dimin-
ish the susceptibility of the liver to alcohol consumption.
We want to thank E. Gulbins and R. Kolesnick for the
generous gift of ASMase–/–mice and A. Serrano for anti-
mitochondrial serum. The technical assistance of A.
Bosch and M. Taulés from Serveis Cientifics Tecnics in
confocal and immunoelectron microscopy studies, as
well as S. Nuñez and O. Coll for technical help with cell
cultures and mouse genotyping is recognized. This work
was supported in part by the Research Center for Liver
and Pancreatic Diseases (P50 AA11999) and grant 1R21
AA014135-01 funded by the U.S. National Institute on
Alcohol Abuse and Alcoholism; Plan Nacional de I+D
grants SAF 99-0138, 2FD97-0988, and PM99-0166; and
Fondo Investigaciones Sanitarias grant FISS 00-907. C.
García-Ruiz is a Sistema Nacional de Salud investigator
from the Fondo Investigaciones Sanitarias.
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The Journal of Clinical Investigation| January 2003|Volume 111|Number 2