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INTRODUCTION |
Intracellular accumulation of long chain fatty acids in nonadipose
tissues is associated with cellular dysfunction and cell death and may
ultimately contribute to the pathogenesis of disease. For example,
lipotoxic accumulation of long chain fatty acids in the pancreatic
-cells of the Zucker diabetic fatty
(ZDF)1 rat leads to the
development of diabetes caused by
-cell death (1). The ZDF rat also
develops cardiomyopathy secondary to cardiomyocyte lipid accumulation
(2). Similarly, human diabetic cardiomyopathy is associated with
increased myocardial triglyceride content, which has been proposed to
contribute to susceptibility to arrhythmia and reduced contractile
function (3). Patients with inherited defects of the mitochondrial
fatty acid oxidation pathway also show signs of lipid accumulation in
the heart. This may contribute to the development of cardiomyopathy or
sudden death in these patients (4). Lastly, triglyceride accumulation in liver and muscle of the A-ZIP/F-1 "fatless" mice has been
proposed to induce the insulin resistance of these peripheral tissues
(5). Although intracellular long chain fatty acid accumulation is
associated with numerous pathophysiologic states, the mechanism of this
lipotoxicity is not fully understood.
In mouse and human fibroblasts and in cultured human endothelial cell
monolayers, high concentrations (70-300 µM) of long chain saturated fatty acids inhibit cell proliferation and lead to cell
death (6-9). Evidence is emerging that long chain fatty acids induce
cell death through apoptosis. Cultured neonatal rat cardiomyocytes,
pancreatic
cells of the ZDF rat, and the hematopoietic precursor
cell lines LyD9 and WEHI-231 demonstrate signs of apoptosis, including
DNA laddering and caspase activation, following fatty acid
supplementation (1, 10-12). Notably, fatty acid-induced apoptosis is
specific for the saturated fatty acids palmitate (C16:0) and stearate
(C18:0) and does not occur with saturated fatty acids of carbon chain
length ranging from C4-C14 or with unsaturated fatty acids (10,
12).
Because palmitate and stearate, but not unsaturated fatty acids, are
precursors for de novo ceramide synthesis, it has been hypothesized that fatty acid-induced apoptosis occurs through this
pathway. Ceramide is a lipid second messenger involved in the apoptotic
response induced by tumor necrosis factor
, ionizing radiation, and
heat shock (13). These stimuli are thought to increase ceramide by
hydrolysis of sphingomyelin rather than de novo
biosynthesis. The downstream signaling pathways through which ceramide
initiates apoptosis remain unclear, but several possible components
have been identified. Direct targets of ceramide include ceramide-activated protein kinase (CAPK, KSR), protein kinase C
, and ceramide-activated protein phosphatase (14). Further downstream, ceramide signaling can affect the mitogen-activated protein
kinase and c-Jun N-terminal kinase signaling cascades or the activation
of NF-KB and ultimately lead to growth arrest and apoptosis (14,
15).
In the present study, we explored the role of de novo
ceramide synthesis in fatty acid-induced lipotoxicity. Specifically, we
utilized Chinese hamster ovary cells (CHO), a cell line amenable to
genetic manipulation, to determine the mechanism whereby palmitate causes cell death. Lipotoxicity in CHO cells is specific for the saturated fatty acid palmitate and does not occur with the
monounsaturated fatty acid oleate. We demonstrate that CHO cells do not
require de novo ceramide synthesis for palmitate-induced
apoptosis. Rather, our studies suggest that palmitate supplementation
leads to the generation of reactive intermediates that initiate
apoptosis. Cellular damage and death from reactive intermediates
generated by saturated fatty acids may contribute to the pathogenesis
of diseases such as diabetes mellitus.
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EXPERIMENTAL PROCEDURES |
Materials--
Palmitic acid, oleic acid, and cholesteryl oleate
were purchased from Nu-Check Prep, BOC-asp(OMe)-fluoromethylketone
(BAF) was from Enzyme Systems Products, fumonisin B1 from Biomol,
[3H]serine was from Amersham Pharmacia Biotech,
[14C]cholesteryl oleate from PerkinElmer Life Sciences,
and egg ceramide was from Avanti Polar Lipids.
L-Cycloserine, fatty acid-free bovine serum albumin (BSA),
propidium iodide (PI), pyrrolidine dithiocarbamate (PDTC), and
4,5-dihydroxy-1,3-benzene-disulfonic acid (DBDA) were purchased from
Sigma. 6-carboxy-2',7'-Dicholorodihydrofluorescein diacetate,
di(acetoxymethyl ester) (C-2938) was from Molecular Probes, and
H2O2 was from Fisher Scientific.
Cell Culture--
Chinese hamster ovary cells (American Type
Culture Collection) and LY-B cells (gift from K. Hanada, National
Institute of Infectious Diseases, Tokyo, Japan) were cultured in
Dulbecco's modified Eagle's medium and Ham's F-12 medium (1:1) with
5% fetal bovine serum supplemented with 2 mM
L-glutamine, 50 units/ml penicillin G sodium, 50 units/ml
streptomycin sulfate, and 1 mM sodium pyruvate. Where
indicated, medium was supplemented with 500 µM palmitate or oleate. Fatty acid supplemented medium was prepared by modification of the method of Spector (16). Briefly, a 20 mM solution of fatty acid in 0.01 M NaOH was incubated at 70 °C for 30 min. Dropwise addition of 1 N NaOH facilitated
solubilization of the fatty acid. Fatty acid soaps were complexed with
5% fatty acid-free BSA in phosphate-buffered saline at an 8:1 fatty
acid to BSA molar ratio. The complexed fatty acid was added to the
serum-containing cell culture medium to achieve a fatty acid
concentration of 500 µM. The final fatty acid
concentration in the medium was measured using a semimicroanalysis kit
(Wako Chemicals). The final BSA concentration was measured using the
Albumin Reagent (BCG, Sigma). The pH of the medium did not differ
significantly with the addition of complexed fatty acid. PDTC, DBDA,
BAF (40 mM stock in dimethyl sulfoxide), fumonisin B1 (10 mM stock in water), or L-cycloserine (0.5 M stock in phosphate-buffered saline) was added to the cell culture medium where indicated. The pH was corrected when addition of
the compound significantly altered the pH of the medium.
Apoptosis Assays--
Annexin V-FITC (Pharmingen 65874X) binding
and PI staining were performed according to the recommended protocol
and the cells were analyzed by flow cytometry (Becton Dickinson
FACScan). Apoptotic cells were defined as 1) PI negative (indicating an
intact plasma membrane) and 2) annexin V-FITC positive relative to
cells incubated in the absence of palmitate. Each data point represents
fluorescence analysis from 104 cells. Activity of the
caspase 3 class of cysteine proteases was determined with the
Colorimetric Caspase 3 activation assay (R&D Systems) according to the
manufacturer's protocol. Ability of the cell lysate to cleave the
reporter molecule was quantified spectrophotometrically at a wavelength
of 415 nm using a microplate reader (Bio-Rad). The level of caspase
enzymatic activity was normalized to cell lysate protein concentration
(BCA assay, Pierce). DNA Laddering was assessed by modification of the
protocol of Bialik et al. (17). Briefly, 5 × 106 cells/sample were resuspended in 425 µl of lysis
buffer (10 mM Tris, pH 8.0, 100 mM NaCl, 25 mM EDTA, 0.5% SDS). Proteinase K was added (25 µl of 20 mg/ml solution), and the samples were incubated overnight at room
temperature. Protein was precipitated by the dropwise addition of 200 µl of 4 M NaCl. The samples were centrifuged at
10,000 × g for 30 min at 4 °C, and the supernatant
was extracted with phenol:chloroform (1:1) and
phenol:chloroform:isoamyl alcohol (25:24:1). The DNA was precipitated
with EtOH and resuspended in Tris-EDTA buffer containing 70 µg/ml RNase. Equal quantities (5-30 µg) of each DNA sample were
run on 1.4% agarose gels in TBE buffer (45 mM Tris, 45 mM boric acid, 1 mM EDTA, pH 8.0). Bands were
detected by ethidium bromide staining.
Ceramide Synthesis--
To measure ceramide synthesis, cells
were plated at 2 × 104 cells/35-mm well. The
following day, the cells were incubated with serine-free medium
supplemented with 2.5-3 µCi of [3H]serine for 20 h to facilitate the incorporation of the tritium label into cellular
serine and sphingomyelin pools (18, 19). Following overnight labeling,
the cells were fed serine-free medium with 2.5 µCi of
[3H]serine ± 500 µM palmitate ± fumonisin or L-cycloserine for 4.25-5 h. Lipids were
extracted (20) and resuspended in chloroform containing 60 µg of egg
ceramide and 40 µg of cholesteryl oleate. The lipids were separated
by thin layer chromatography on silica gel plates (Whatman 4410 221)
using CHCl3:MeOH:NH4OH (200:25:2.5) as the
solvent. Samples were visualized by iodine vapor staining and
radioactivity incorporated into the ceramide or cholesteryl oleate
spots was determined by scintillation counting. The presence of
radiolabeled ceramide was normalized to protein concentration (BCA
assay, Pierce) and corrected for lipid recovery during extraction using
0.01 µCi of [14C]cholesteryl oleate as a recovery standard.
Detection of Reactive Intermediates--
Cells were plated at
1.4 × 105 cells/35-mm well. The following day, cells
were supplemented with fatty acid media for 14 h. Prior to C-2938
loading, control cells were supplemented with medium containing 5 mM H2O2 for 1 h at 37 °C.
Cells were washed with phosphate-buffered saline and incubated with 0.5 µM C-2938 in phosphate-buffered saline supplemented with
0.5 mM MgCl2 and 0.92 mM
CaCl2 for 1 h at 37 °C. Cells were collected and
resuspended in media containing 1 µM PI. C-2938
fluorescence was measured by flow cytometry on 104
cells/sample (Becton Dickinson FACScan). Cells were gated for cell size
and intact plasma membranes (PI negative). The fold increase in median
fluorescence over unsupplemented cells was determined. The values
reported are the average fold increase for three independent experiments.
Statistics--
Differences among groups were compared by
one-way analysis of variance in conjunction with the post hoc Scheffe test.
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RESULTS |
Palmitate Induces Apoptosis in CHO Cells--
To determine whether
long chain saturated fatty acids induce cell death in CHO cells, cells
were incubated in medium supplemented with 500 µM
palmitate complexed to BSA. The final fatty acid and albumin
concentrations in the media were measured and yielded a molar ratio of
fatty acid to albumin of 6.6:1. Although the normal physiologic ratio
of fatty acid to albumin is ~2:1, serum fatty acid levels in disease
states (e.g. acute coronary syndromes) are elevated yielding
ratios as high as 7.5:1 (21). Thus, our experimental system was
designed to evaluate mechanisms of palmitate toxicity relevant to
pathophysiologic states.
CHO cells incubated with palmitate supplemented medium showed signs of
growth arrest and cell death by 5 h. By 11 h ~80% of CHO
cells displayed cell shrinkage. The cells began to detach from the
plate after 16 h in palmitate. Cytotoxicity was observed using
medium supplemented with as little as 100 µM palmitate, and the degree of cell death correlated with the amount of palmitate supplementation from 100 to 500 µM (data not shown).
We assayed palmitate supplemented CHO cells for annexin V binding,
caspase 3 activity, and DNA laddering to determine whether cell death
was occurring through apoptosis (Fig. 1).
Early in apoptosis, phosphatidylserine is translocated from the inner
to the outer leaflet of the plasma membrane. Annexin V, a
membrane-impermeable protein, binds phosphatidylserine on intact cells
only if phosphatidylserine is present on the outer leaflet. Flow
cytometry was used to measure the binding of FITC-labeled annexin V to
the surface of CHO cells after incubation in medium supplemented with
500 µM palmitate (Fig. 1A). Cells with
permeabilized plasma membranes were excluded from measurements of
annexin V positivity with PI, a fluorescent DNA binding dye. CHO cells
began to show annexin V binding after 10 h in palmitate. The
percentage of cells binding annexin V increased until 70% of CHO cells
were annexin V-positive after 16 h in palmitate. Notably, PI
staining, an indication of cell death, followed the appearance of
annexin V positivity and steadily increased after 16 h in
palmitate supplemented medium (Fig. 1A). Because activation of the cysteine protease, caspase 3, has been implicated as a common
downstream effector of diverse apoptotic pathways, we measured cleavage
of a colorimetric substrate specific to the caspase 3 class of cysteine
proteases following 25 h of palmitate feeding. Caspase activity
increased 9.2-fold with palmitate supplementation and was inhibited by
BAF, a pan-caspase inhibitor (Fig. 1B). DNA laddering, an
end stage apoptotic event, was evident after 28 h in 500 µM palmitate and was inhibited when caspases were
inhibited with BAF (Fig. 1C). Taken together, the detection
of phosphatidylserine externalization, membrane permeabilization,
caspase activation, and DNA laddering following palmitate
supplementation supports the hypothesis that the saturated fatty acid
palmitate induces programmed cell death in CHO cells.

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Fig. 1.
Palmitate but not oleate induces apoptosis in
CHO cells. A, CHO cells were incubated in medium
supplemented with 500 µM palmitate or oleate complexed to
BSA and stained with annexin V-FITC (measure of phosphatidylserine
externalization) and propidium iodide (measure of plasma membrane
integrity). The graph shows flow cytometric analysis of annexin V-FITC
and propidium iodide staining at various times after fatty acid
supplementation. Data are displayed as the percentages of
104 cells stained with annexin V-FITC or propidium iodide.
Apoptotic cells are annexin V positive (annV+)
and propidium iodide negative (PI ).
B, caspase 3 activity in cell lysate from cells incubated
with palmitate, oleate, or palmitate and a pan caspase inhibitor (BAF)
for 25 h was detected by cleavage of a colorimetric substrate.
Graph displays caspase activity normalized to untreated CHO cells. Data
are expressed as the averages of nine samples ± S.E. from three
independent experiments. *, p < 0.001 relative to
untreated, palmitate + BAF or oleate supplemented cells. C,
DNA was extracted following incubation with palmitate, oleate, or
palmitate plus the pan caspase inhibitor BAF for 28 h. DNA
laddering was visualized with agarose gel electrophoresis and ethidium
bromide staining and is representative of three independent
experiments.
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To demonstrate the specificity of fatty acid-induced apoptosis in CHO
cells, CHO cells were also incubated with medium containing 500 µM oleate. In contrast to the effects of palmitate,
oleate, an 18-carbon monounsaturated fatty acid did not cause CHO cell death. In addition to the absence of morphological changes associated with palmitate feeding, oleate supplementation did not induce phosphatidylserine externalization (Fig. 1A), caspase 3 activity (Fig. 1B), or DNA laddering (Fig. 1C).
The inability of oleate to induce cell death is consistent with the
hypothesis that this response is specific to saturated fatty acids as
reported previously (10, 12). Thus, these results demonstrate that CHO
cells are an appropriate model system in which to study the mechanism
through which saturated fatty acids specifically induce programmed cell death.
Palmitate Supplementation Is Associated with Increased de Novo
Ceramide Synthesis--
Prior studies have implicated that de
novo synthesis of ceramide, a known inducer of apoptosis, is
critical for palmitate-induced apoptosis (10, 22). Serine
palmitoyltransferase catalyzes the first and rate-limiting step of
de novo ceramide synthesis (Fig.
2A, adapted from Ref. 23). This
enzyme has high specificity for palmitoyl CoA, the activated form of
palmitate, whereas saturated fatty acids such as stearate are the
preferred substrates for ceramide synthase (24). Serine
palmitoyltransferase and ceramide synthase are specifically inhibited
by L-cycloserine and fumonisin B1, respectively. To
determine whether palmitate supplementation induces ceramide synthesis
in CHO cells, we labeled cells to equilibrium with
[3H]serine to incorporate the label into the cellular
serine and sphingomyelin pools (19, 25). Then, the cells were
supplemented with [3H]serine and palmitate for 4.25 h before measuring the production of radiolabeled ceramide. Palmitate
feeding for 4.25 h increased the synthesis of labeled ceramide by
2.4-fold (Fig. 2B). This increase in ceramide production was
attributable to de novo ceramide synthesis and not cleavage
of sphingomyelin because it was completely inhibited by the inclusion
of fumonisin B1 or L-cycloserine.

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Fig. 2.
Palmitate supplementation induces de
novo ceramide synthesis. A, metabolic
pathway for de novo ceramide synthesis from palmitoyl CoA.
B, CHO cells were labeled to equilibrium with
[3H]serine followed by palmitate supplementation (in the
presence of [3H]serine) for 4.25 h. Lipids were
extracted, ceramide was isolated by thin layer chromatography, and
radiolabel incorporation was determined. As controls, 1 mM
L-cycloserine and 100 µM fumonisin B1,
inhibitors of the pathway of de novo ceramide synthesis,
were included during fatty acid supplementation. The bar
graph displays the amount of radiolabeled ceramide (per mg of
protein) detected relative to CHO cells incubated in the absence of
fatty acid and inhibitors. Data was corrected for lipid recovery and
are expressed as the means of triplicate samples ± S.E. *,
p < 0.001 relative to untreated, palmitate + L-cycloserine or palmitate + fumonisin. Data are
representative of two independent experiments. On average, untreated
CHO cells synthesized 52 pmol of labeled ceramide/gram of
protein.
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De Novo Ceramide Synthesis Is Not Required for Palmitate-induced
Apoptosis--
To determine whether the increase in de novo
ceramide synthesis is critical for palmitate-induced apoptosis, CHO
cells were incubated with medium supplemented with 500 µM
palmitate and 100 µM fumonisin B1 or 1 mM
L-cycloserine. These concentrations of inhibitors
completely blocked the increase in ceramide synthesis associated with
palmitate supplementation (Fig. 2B). Apoptosis was assessed
by caspase 3 activity and DNA laddering. Surprisingly, inhibition of
de novo ceramide synthesis did not rescue the morphological changes (cell shrinkage and detachment) associated with palmitate feeding. Inhibition of de novo ceramide synthesis blunted
but did not completely prevent caspase activity (Fig.
3A), with reduced relative levels
of caspase activity at each time point measured. Biochemical inhibition
of de novo ceramide synthesis also did not prevent DNA
laddering induced by 28 h of palmitate supplementation (Fig.
3B).

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Fig. 3.
Inhibitors of de novo
ceramide synthesis do not block palmitate-induced apoptosis in
CHO cells. A, caspase 3 activity was assessed following
incubation with palmitate or palmitate plus L-cycloserine
(1 mM) or fumonisin B1 (100 µM) for 0-28 h.
Caspase activity is normalized to the value for time 0 (CHO cells
incubated in the absence of palmitate and without inhibitor
supplementation). Caspase activity is expressed as the average of
duplicate samples ± S.E. *, p < 0.001 comparing
palmitate to palmitate + L-cycloserine or palmitate + fumonisin at 28 h. The absence of error bars indicates the errors
were too small to appear on the graph. The data are representative of
three independent experiments. B, DNA laddering was assessed
by agarose gel electrophoresis and ethidium bromide staining following
incubation with palmitate or palmitate plus L-cycloserine
(1 mM) or fumonisin B1 (100 µM) for 28 h. Data are representative of three independent experiments.
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To verify that de novo ceramide synthesis is not required
for cell death, we assayed for palmitate-induced apoptosis in a mutant
CHO cell line incapable of de novo ceramide synthesis. LY-B
cells lack serine palmitoyltransferase activity, the rate-limiting step
in de novo ceramide synthesis (18). We verified that LY-B cells failed to stimulate ceramide synthesis in response to palmitate supplementation (Fig. 4A). Despite
the absence of de novo ceramide synthesis in these cells,
palmitate supplementation induced the morphological changes associated
with cell death. Additionally, caspase 3 was activated in LY-B cells
with palmitate supplementation, although the magnitude of this increase
was diminished compared with wild-type CHO cells (Fig. 4B).
DNA laddering occurred in response to palmitate feeding in a manner
indistinguishable from wild-type cells (Fig. 4C). Taken
together, the studies with L-cycloserine, fumonisin B1, and
LY-B cells demonstrate by both biochemical and genetic means that
de novo ceramide synthesis is not required for
palmitate-induced apoptosis in CHO cells.

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Fig. 4.
Palmitate-induced apoptosis occurs in mutant
CHO cells deficient in de novo ceramide
synthesis. LY-B cells are a mutant CHO cell line lacking serine
palmitolytransferase activity, the rate-limiting step in de
novo ceramide synthesis. Markers of palmitate-induced apoptosis
were assessed in LY-B cells and compared with CHO cells. A,
ceramide synthesis was measured in CHO and LY-B cells by labeling the
cells to equilibrium with [3H]serine followed by 5 h
of palmitate supplementation (still in the presence of
[3H]serine). Lipids were extracted, ceramide was isolated
by thin layer chromatography, and radiolabel incorporation was
determined. Data are expressed as the amount of radiolabeled
ceramide/mg protein and normalized to untreated CHO cells. Data are
expressed as the means of triplicate samples ± S.E. and are
representative of two independent experiments. *, p < 0.001 relative to CHO cells without palmitate or LY-B cells with
palmitate. On average, untreated CHO cells synthesized 52 pmol of
labeled ceramide per gram of protein. B, caspase 3 activity
was measured by the ability of isolated cell lysate to cleave a
colorimetric substrate following 25 h of palmitate
supplementation. Data are normalized to the value in untreated CHO or
untreated LY-B cells and are expressed as the averages of nine samples
from three independent experiments ± S.E. *, p < 0.001 for the comparison of CHO cells with and without palmitate, LY-B
cells with and without palmitate, and CHO and LY-B cells supplemented
with palmitate. C, DNA laddering in CHO and LY-B cells was
assessed by agarose gel electrophoresis following 28 h of
palmitate supplementation. Laddering is representative of three
independent experiments.
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Palmitate Supplementation Induces the Generation of Reactive
Intermediates--
We next attempted to identify the mechanism whereby
palmitate supplementation induces apoptosis. Evidence is emerging that free fatty acids can stimulate the production of reactive oxygen species (ROS) to a level that exceeds the intrinsic capacity of the
cell to detoxify these molecules (26, 27). Moreover, ROS have been
implicated as important regulators of apoptotic pathways (28) and thus
may play a role in palmitate-induced apoptosis. To determine whether
reactive intermediates were generated with palmitate supplementation,
we measured cell fluorescence following C-2938 loading as a marker for
oxidative intermediates. C-2938 is a nonfluorescent, membrane permeable
probe that becomes fluorescent upon reaction with ROS within the cell
and can be detected by flow cytometry. Although there is some debate
regarding the specificity of this assay (29), fluorescence of C-2938
has been widely used as a measure of oxidative stress and as a marker
for ROS in cells (30-32).
Supplementation of CHO cells with palmitate resulted in an increase in
C-2938 fluorescence. Increased C-2938 fluorescence was observed as
early as 5 h, and the levels of fluorescence increased with
increasing periods of palmitate supplementation. Fig.
5A shows that following 14 h
of palmitate supplementation, the level of fluorescence was 3.5-fold
higher than the level in unsupplemented cells. Notably, the level of
fluorescence with 14 h of palmitate supplementation was similar to
that detected when CHO cells were supplemented with 5 mM
H2O2 for 1 h prior to C-2938 loading.
Including 5 mM of the antioxidant PDTC (28, 33) with
palmitate supplementation decreased the fluorescence to 1.3-fold the
level detected in unsupplemented cells. Similarly, 20 mM
DBDA, a membrane-permeable nonenzymatic superoxide scavenger (34, 35),
reduced fluorescence to 1.6-fold that detected in unsupplemented cells
(Fig. 5A). Importantly, the oxidative stress observed in
palmitate supplemented CHO cells was not dependent on de
novo ceramide synthesis. LY-B cells, similar to CHO cells, showed
increased C-2938 fluorescence following palmitate supplementation (Fig.
5A). Additionally, supplementation with 500 µM
oleate did not induce C-2938 fluorescence, indicating reactive intermediates were not produced (Fig. 5A). This is
consistent with the inability of oleate to induce cell death. Taken
together, these findings suggest that palmitate supplementation leads
to accumulation of reactive oxygen intermediates.

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Fig. 5.
Palmitate-induced apoptosis causes generation
of reactive intermediates and is inhibited by antioxidants.
A, cells were supplemented with oleate, palmitate, or
palmitate plus 5 mM PDTC or 20 mM DBDA for
14 h (or with 5 mM H2O2 for
1 h) followed by C-2938 loading. C-2938 fluorescence was
determined by flow cytometry and is indicative of the oxidation of
C-2938 by reactive intermediates. The bar graph displays
median C-2938 fluorescence of 104 cells normalized to
unsupplemented CHO or LY-B cells. Each bar represents the
average median fluorescence of three independent experiments ± S.E. *, p < 0.001 for H2O2 or
palmitate supplemented CHO cells compared with palmitate + PDTC,
palmitate + DBDA or oleate supplemented cells or p < 0.001 for palmitate supplemented LY-B cells verses untreated LY-B
cells; The difference between palmitate supplemented CHO and palmitate
supplemented LY-B cells was not statistically significant. Caspase 3 activity (B) and DNA laddering (C) were measured
after palmitate or palmitate plus 5 mM PDTC or 20 mM DBDA supplementation. Data in B are expressed
as the averages of nine samples from three independent experiments ± S.E. *, p < 0.001 relative to untreated, palmitate + PDTC or palmitate + DBDA. Laddering in C is representative
of three independent experiments.
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Palmitate-induced Apoptosis Requires the Generation of Reactive
Intermediates--
To determine whether the generation of reactive
intermediates is essential for palmitate-induced apoptosis, we
measured the ability of the antioxidants PDTC and DBDA to inhibit
caspase activation and DNA laddering. PDTC (5 mM)
effectively blocked caspase 3 activity after 24 h of palmitate
supplementation (Fig. 5B). Similarly, 20 mM DBDA
significantly reduced caspase 3 activity from 10.0- to 2.2-fold over
untreated cells. The failure of DBDA to completely inhibit caspase 3 activation may be due to the low level of reactive intermediates that
remained with 20 mM DBDA as shown in Fig. 5A, or
the observation that 20 mM DBDA alone caused an increase in caspase 3 activity (2.1 ± 0.2-fold increase over untreated cells, n = 7). In addition to the effect on caspase
activation, PDTC and DBDA both effectively blocked DNA laddering (Fig.
5C). Thus, antioxidants prevent both the generation of
reactive intermediates following palmitate supplementation and the
induction of apoptosis.
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DISCUSSION |
Our studies implicate a novel mechanism through which palmitate
supplementation leads to apoptosis. Specifically, we propose that CHO
cells do not require de novo ceramide synthesis for
palmitate-induced cell death. This conclusion is supported by the
observations that CHO cells treated with biochemical inhibitors of
de novo ceramide synthesis and CHO cells with a mutation in
serine palmitoyltransferase continue to undergo apoptosis in response
to palmitate supplementation. In contrast to the hypothesis that
de novo ceramide synthesis is required, our data suggest
that palmitate-induced apoptosis occurs through oxidative stress. We
observed an increase in reactive intermediates with palmitate
supplementation that is independent of de novo ceramide
synthesis. Antioxidants inhibited both C-2938 fluorescence and
palmitate-induced caspase activation and DNA laddering. Thus, our data
support an integral role for the generation of reactive intermediates
in palmitate-induced lipotoxicity.
Ceramide is generated by de novo biosynthesis following
palmitate supplementation and may serve to amplify the apoptotic
response in CHO cells. We observed a decrease in the magnitude of
caspase 3 activity when de novo ceramide synthesis was
inhibited. This reduction was evident when ceramide synthesis was
blocked by either the mutation in serine palmitoyltransferase or with
the biochemical inhibitors. The reduction in caspase 3 activity
occurred at every time point measured (from 17-28 h), indicating that
we did not simply observe a delay in caspase 3 activation. Despite this
decrease in caspase 3 activity, we continued to observe DNA laddering
and cell death, indicating that the remaining caspase 3 activity was sufficient for the induction of apoptosis. These findings are most
consistent with a model in which ceramide serves to amplify but not
induce the apoptotic response to palmitate supplementation. Recent data
showing that cytochrome C release and caspase 3 activation precedes
ceramide accumulation in palmitate-treated cardiac myocytes also
support a nonessential role for ceramide synthesis (36).
The mechanism of cellular lipotoxicity likely depends on cell
type-specific processes for channeling fatty acids to particular metabolic fates. Our observation that ceramide synthesis is not required for palmitate-induced apoptosis is in contrast to published data showing palmitate-induced apoptosis in hematopoietic precursor cell lines (LyD9 and WEHI-231 cells) and in pancreatic
-cells of the
ZDF rat is blocked by inhibitors of de novo ceramide
synthesis (1, 10, 22). Our results suggest that fatty acids may be targeted to different metabolic fates in CHO cells as compared with
LyD9 and WEHI-231 cells and cells from the ZDF rat. Consistent with
this notion, the ZDF rat harbors a mutation in the leptin receptor that
is associated with alterations in handling of intracellular fatty acids
as shown by an increased capacity to accumulate triglycerides in
nonadipose tissues (37). Future studies into the mechanisms that
control channeling of fatty acids to specific metabolic fates will
provide insight into these cell-specific differences.
Our studies with an oxidant-sensitive probe and agents that scavenge
oxidants suggest that generation of ROS is essential for the induction
of apoptosis in response to palmitate supplementation. Palmitate-induced caspase 3 activity and DNA laddering are inhibited by
both PDTC and DBDA. Depending on concentration and cell background, PDTC may function to increase cellular glutathione or directly inhibit
the NF-
B pathway, both of which actions may protect against oxidative stress (38). DBDA has been used as a scavenger of superoxide
but has no known effects on NF-
B signaling (34, 35). Therefore, we
believe the observed inhibition of palmitate-induced apoptosis is due
to the ability of both PDTC and DBDA to decrease ROS. Although
production of ROS can be a coincident finding in cell death, evidence
from this and other studies suggest that reactive intermediates play a
primary role in the activation stage of apoptosis (reviewed in Refs.
39-41). ROS can initiate signaling pathways that affect protein
phosphorylation or activate nuclear transcription factors such as
NF-
B (30, 42, 43). In our studies, two observations support a role
for ROS in the induction rather than execution of palmitate-induced
apoptosis. First, we observed a 2.2-fold increase in reactive
intermediates following 5 h of palmitate supplementation. This
early time point corresponds to the time at which we began to see
morphological changes caused by palmitate supplementation but before
caspase activation and DNA laddering. Secondly, antioxidants inhibited
both caspase activation and DNA laddering, suggesting that ROS are
acting upstream of these events. Our data is most consistent with a
primary role for ROS in the induction of apoptosis following palmitate supplementation.
Studies are underway in our laboratory to further characterize the
mechanism whereby palmitate supplementation leads to the generation of
reactive intermediates. Our observation of palmitate-induced C-2938
fluorescence in LY-B cells suggests that reactive intermediates can be
generated independent of de novo ceramide synthesis. ROS may
be generated from lipid peroxidation, but this mechanism would require
fatty acid desaturation and is unlikely to occur directly from
supplementation of a saturated fatty acid. Alternatively, excess
palmitate may lead to increased cycling through mitochondrial
-oxidation pathways generating ROS in excess of endogenous cellular antioxidants. However, it is unlikely that this effect would be specific for the saturated fatty acid palmitate and not occur with the
unsaturated fatty acid oleate. Finally, evidence is emerging that
palmitate can induce the formation of reactive oxygen species through
protein kinase C-dependent activation of NAD(P)H oxidase (27). Furthermore, the generation of ROS may cause further cell damage
through the production of reactive nitrogen species by the reaction of
ROS with nitric oxide, a compound that has been shown to increase with
palmitate supplementation (44). We are currently exploring whether the
toxicity associated with palmitate supplementation is affected by
independent perturbation of fatty acid metabolism, NAD(P)H oxidase or
NO synthase.
In conclusion, our studies indicate that the saturated free fatty acid
palmitate induces the formation of reactive intermediates and leads to
programmed cell death. Fatty acid-induced apoptosis may contribute to
cardiac myocyte death in diabetic cardiomyopathy, cardiomyopathy
associated with inherited disorders of mitochondrial fatty acid
oxidation, and pancreatic
cell loss in diabetes. Additionally,
palmitate-mediated production of ROS may cause significant cellular
dysfunction that contributes to the pathogenesis of these diseases
prior to cell death. Our findings suggest novel approaches to
pharmacologic and genetic rescue strategies in animal models of human
heart disease and diabetes.