palmitate-induced apoptosis can occur through a ceramide

1

Palmitate-induced Apoptosis Can Occur Through a Ceramide-Independent

Pathway

Laura L. Listenberger, Daniel S. Ory and Jean E. Schaffer*

From the Center for Cardiovascular Research, Departments of Internal Medicine,

Molecular Biology and Pharmacology, Washington University School ofMedicine, St. Louis Missouri 63110-1010

*To whom correspondence should be addressed:Center for Cardiovascular ResearchWashington University School of Medicine660 South Euclid Avenue, Box 8086St. Louis, MO 63110-1010tel: 314-362-8717fax: 314-362-0186email:[email protected]

Running title:Palmitate-induced Apoptosis

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on February 13, 2001 as Manuscript M010286200 by guest on February 11, 2018

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SUMMARY

Cytotoxic accumulation of long-chain fatty acids has been proposed to play an important role in

the pathogenesis of diabetes mellitus and heart disease. To explore the mechanism of cellular

lipotoxicity, we cultured Chinese hamster ovary (CHO) cells in the presence of media

supplemented with fatty acid. The saturated fatty acid palmitate, but not the mono-unsaturated

fatty acid oleate, induced programmed cell death as determined by annexin V positivity, caspase

3 activity, and DNA laddering. De novo ceramide synthesis increased 2.4 fold with palmitate

supplementation, however this was not required for palmitate-induced apoptosis. Neither

biochemical nor genetic inhibitors of de novo ceramide synthesis arrested apoptosis in CHO cells

in response to palmitate supplementation. Rather, our data suggest that palmitate-induced

apoptosis occurs through the generation of reactive oxygen species. Fluorescence of an oxidant-

sensitive probe was increased 3.5 fold with palmitate supplementation indicating that production

of reactive intermediates increased. In addition, palmitate-induced apoptosis was blocked by

pyrrolidine dithiocarbamate and 4,5-dihydroxy-1,3-benzene-disulfonic acid, two compounds that

scavenge reactive intermediates. These studies suggest that generation of reactive oxygen

species, independent of ceramide synthesis, is important for the lipotoxic response and may

contribute to the pathogenesis of diseases involving intracellular lipid accumulation.

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INTRODUCTION

Intracellular accumulation of long-chain fatty acids (LCFAs)1 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 LCFAs in the pancreatic β-cells of the Zucker

diabetic fatty (ZDF) rat leads to the development of diabetes due to β-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 LCFA accumulation is associated with

numerous pathophysiologic states, the mechanism of this lipotoxicity is not fully understood.

1 The abbreviations used are: LCFAs, long-chain fatty acids; ZDF, Zucker diabetic fatty; CHO, Chinese hamsterovary; BSA, bovine serum albumin; PI , propidium iodide; PS, phosphatidylserine; BAF, BOC-asp(OMe)-

fluoromethylketone; PBS, phosphate buffered saline; ROS, reactive oxygen species; C-2938, 6-carboxy-2'.7'-dichlorodihydrofluorecein diacetate, di(acetoxymethyl ester); PDTC, pyrrolidine dithiocarbamate; DBDA, 4,5-

dihydroxy-1,3-benzene-disulfonic acid; S.E., standard error.


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In mouse and human fibroblasts and in cultured human endothelial cell monolayers, high

concentrations (70 µM - 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 β 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 α, 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 Cζ, and ceramide -activated protein

phosphatase (14). Further downstream, ceramide signaling can affect the MAPK and JNK


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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 mono-unsaturated 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 which 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) from Enzyme Systems Products, fumonisin B1 from

Biomol, [3H]serine from Amersham, [14C]cholesteryl oleate from NEN Life Sciences and egg

ceramide 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 from Fisher Scientific.

Cell culture

Chinese hamster ovary cells (ATCC) and LY-B cells (gift from K. Hanada, National Institute of

Infectious Diseases, Tokyo, Japan) were cultured in Dulbecco's modified Eagle's media

(DMEM) and F-12 media (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, media was supplemented with 500 µM palmitate or oleate. Fatty

acid supplemented media 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 minutes. Dropwise

addition of 1N NaOH facilitated solubilization of the fatty acid. Fatty acid soaps were


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complexed with 5% fatty acid-free BSA in phosphate buffered saline (PBS) at an 8:1 fatty acid

to BSA molar ratio. The complexed fatty acid was added to the serum-containing cell culture

media to achieve a fatty acid concentration of 500 µM. The final fatty acid concentration in the

media was measured using a semimicroanalysis kit (Wako Chemicals). The final BSA

concentration was measured using the Albumin Reagent (BCG, Sigma). The pH of the media

did not differ significantly with the addition of complexed fatty acid. PDTC, DBDA, BAF (40

mM stock in dimethylsulfoxide), fumonisin B1 (10 mM stock in water) or L-cycloserine (0.5 M

stock in PBS) was added to the cell culture media where indicated. The pH was corrected when

addition of the compound significantly altered the pH of the media.

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 105 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


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to cell lysate protein concentration (BCA assay, Pierce). DNA Laddering was assessed by

modification of the protocol of Bialik et al (17). Briefly, 5x106 cells per sample were

resuspended in 425 µl 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 4 M

NaCl. The samples were centrifuged at 10,000 x g for 30 minutes 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 TE 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 2x104 cells/35 mm well. The following day,

the cells were incubated with serine-free media supplemented with 2.5-3 µCi [3H]serine for 20

hours 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 media with 2.5 µCi

[3H]serine ± 500 µM palmitate ± fumonisin or L-cycloserine for 4.25-5 hours. Lipids were

extracted (20) and resuspended in chloroform containing 60 µg egg ceramide and 40 µg

cholesteryl oleate. The lipids were separated by thin layer chromatography on silica gel plates


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(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 [14C]cholesteryl oleate as a recovery standard.

Detection of reactive intermediates

Cells were plated at 1.4x105 cells per 35 mm well. The following day, cells were supplemented

with fatty acid media for 14 hours. Prior to C-2938 loading, control cells were supplemented

with media containing 5 mM H2O2 for one hour at 37°C. Cells were washed with PBS and

incubated with 0.5 µM C-2938 in PBS supplemented with 0.5 mM MgCl2 and 0.92 mM CaCl2

for one hour at 37°C. Cells were collected and resuspended in media containing 1 µM PI. C-

2938 fluorescence was measured by flow cytometry on 105 cells per 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 ANOVA in conjunction with the post hoc

Scheffe test.


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RESULTS

Palmitate induces apoptosis in CHO cells

To determine if long-chain saturated fatty acids induce cell death in CHO cells, cells were

incubated in media 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. While the normal physiologic ratio of fatty acid to albumin is

approximately 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 media showed signs of growth arrest and cell

death by 5 hours. By 11 hours approximately 80% of CHO cells displayed cell shrinkage. The

cells began to detach from the plate after 16 hours in palmitate. Cytotoxicity was observed using

media supplemented with as little as 100 µM palmitate, and the degree of cell death correlated

with the amount of palmitate supplementation from 100-500 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 (Figure 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 (PS)


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on intact cells only if PS 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 media

supplemented with 500 µM palmitate (Figure 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 hours in palmitate. The percentage of

cells binding annexin V increased until 70% of CHO cells were annexin V-positive after 16

hours in palmitate. Notably, PI staining, an indication of cell death, followed the appearance of

annexin V positivity and steadily increased after 16 hours in palmitate supplemented media

(Figure 1A). Since 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 hours of

palmitate feeding. Caspase activity increased 9.2 fold with palmitate supplementation and was

inhibited by BAF, a pan-caspase inhibitor (Figure 1B). DNA laddering, an end stage apoptotic

event, was evident after 28 hours in 500 µM palmitate and was inhibited when caspases were

inhibited with BAF (Figure 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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To demonstrate the specificity of fatty acid-induced apoptosis in CHO cells, CHO cells were also

incubated with media containing 500 µM oleate. In contrast to the effects of palmitate, oleate, an

18-carbon mono-unsaturated 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 (Figure 1A), caspase 3 activity (Figure 1B), or DNA

laddering (Figure 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 (Figure 2A, adapted from (23)). This

enzyme has high specificity for palmitoyl CoA, the activated form of palmitate, while 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 if palmitate supplementation induces ceramide

synthesis in CHO cells, we labeled cells to equilibrium with [3H]serine to incorporate the label


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into the cellular serine and sphingomyelin pools (19,25). Then, the cells were supplemented

with [3H]serine and palmitate for 4.25 hours before measuring the production of radiolabeled

ceramide. Palmitate feeding for 4.25 hours increased the synthesis of labeled ceramide by 2.4

fold (Figure 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.

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 media 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 (Figure

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 (Figure 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 hours of palmitate supplementation

(Figure 3B).


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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 (Figure 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 to wild-type CHO cells

(Figure 4B). DNA laddering occurred in response to palmitate feeding in a manner

indistinguishable from wild-type cells (Figure 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.

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 if reactive

intermediates were generated with palmitate supplementation, we measured cell fluorescence


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following C-2938 loading as a marker for oxidative intermediates. C-2938 is a non-fluorescent,

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 hours and the levels of fluorescence

increased with increasing periods of palmitate supplementation. Figure 5A shows that following

14 hours 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 hours of palmitate

supplementation was similar to that detected when CHO cells were supplemented with 5 mM

H2O2 for 1 hour prior to C-2938 loading. Including 5 mM of the antioxidant pyrrolidine

dithiocarbamate (PDTC) (28,33) with palmitate supplementation decreased the fluorescence to

1.3 fold the level detected in unsupplemented cells. Similarly, 20 mM 4,5-dihydroxy-1,3-

benzene-disulfonic acid (DBDA), a membrane permeable nonenzymatic superoxide scavenger

(34,35), reduced fluorescence to 1.6 fold that detected in unsupplemented cells (Figure 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 (Figure 5A). Additionally,


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supplementation with 500 µM oleate did not induce C-2938 fluorescence, indicating reactive

intermediates were not produced (Figure 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.

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

hours of palmitate supplementation (Figure 5B). Similarly, 20 mM DBDA significantly reduced

caspase 3 activity from 10.0 fold 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 Figure 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 (Figure 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 hours) indicating that we did not simply observe a delay in caspase 3


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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 non-essential 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 β-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 to 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 which

control channeling of fatty acids to specific metabolic fates will provide insight into these cell-

specific differences.


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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-ΚB 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-ΚB 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.

While 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 (39-41)). ROS can initiate signaling pathways that affect protein phosphorylation

or activate nuclear transcription factors such as NF-ΚB (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 hours of

palmitate supplementation. This early time point corresponds to the time at which we began to

see morphological changes due to 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.


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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 β-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 which 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


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apoptosis may contribute to cardiac myocyte death in diabetic cardiomyopathy, cardiomyopathy

associated with inherited disorders of mitochondrial fatty acid oxidation, and pancreatic β 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.


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ACKNOWLEDGMENTS

We thank K. Hanada for the gift of the LY-B cells and members of the Schaffer and Ory labs and

SSSG for helpful discussions. We are grateful to D. Kelly, M. Linder, and J. Heinecke for

critical evaluation of this manuscript. This work is supported by grants from the NSF (graduate

research fellowship, LL), NIH (#DK54268, JS) and AHA (#0040040N, JS).


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REFERENCES

1. Shimabukuro, M., Zhou, Y.-T., Levi, M., and Unger, R. H. (1998) Proc. Natl. Acad. Sci

USA 95, 2498-2502.

2. Zhou, Y., Grayburn, P., Karim, A., Shimabukuro, M., Higa, M., Baetens, D., Orci, L.,and Unger, R. (2000) PNAS 97(4), 1784-1789.

3. Rodrigues, B., Cam, M. C., and McNeill, J. H. (1995) J Mol Cell Cardiol 27(1), 169-79.

4. Kelly, D. P., and Strauss, A. W. (1994) N Engl J Med 330(13), 913-9.

5. Kim, J. K., Gavrilova, O., Chen, Y., Reitman, M. L., and Shulman, G. I. (2000) J Biol

Chem 275(12), 8456-60.

6. Chun-Lan, Z., Lyngmo, V., and Nordoy, A. (1992) Thrombosis Research 65, 65-75.

7. Gordon, G. B. (1977) Experimental and Molecular Pathology 27, 262-276.

8. Doi, O., Doi, F., Schroeder, F., Alberts, A. W., and Vagelos, P. R. (1978) Biochimica et

Biophysica Acta 509, 239-250.

9. Rosenthal, M. D. (1981) Lipids 16(3), 173-182.

10. Paumen, M. B., Ishida, Y., Muramatsu, M., Yamamoto, M., and Honjo, T. (1997) J. of

Biol. Chem. 272(February 7), 3324-3329.

11. Hickson-Bick, D. L., Buja, M. L., and McMillin, J. B. (2000) J Mol Cell Cardiol 32(3),511-9.

12. de Vries, J. E., Vork, M. M., Roemen, T. H. M., de Jong, Y. F., Cleutjens, J. P. M., vander Vusse, G. J., and van Bilsen, M. (1997) Journal of Lipid Research 38, 1384-1394.

13. Kolesnick, R. N., and Kronke, M. (1998) Ann. Rev. Physiol. 60, 643-65.

14. Mathias, S., Pena, L. A., and Kolesnick, R. N. (1998) Biochem. J. 335, 465-480.

15. Obeid, L. M., and Hannun, Y. A. (1995) J. Of Cell. Biochem. 58, 191-198.

16. Spector, A. A. (1986) Methods Enzymol 128, 320-39.

17. Bialik, S., Geenen, D. L., Sasson, I. E., Cheng, R., Horner, J. W., Evans, S. M., Lord, E.M., Koch, C. J., and Kitsis, R. N. (1997) J Clin Invest 100(6), 1363-72.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

24

18. Hanada, K., Hara, T., Fukasawa, M., Yamaji, A., Umeda, M., and Nishijima, M. (1998)J. Biol. Chem. 273(50), 33787-33794.

19. Fukasawa, M., Nishijima, M., and Hanada, K. (1999) J Cell Biol 144(4), 673-85.

20. Morand, O., Fibach, E., Dagan, A., and Gatt, S. (1982) Biochim Biophys Acta 711(3),539-50.

21. Kleinfeld, A. M., Prothro, D., Brown, D. L., Davis, R. C., Richieri, G. V., and DeMaria,A. (1996) Am J Cardiol 78(12), 1350-4.

22. Shimabukuro, M., Higa, M., Zhou, Y.-T., Wang, M.-Y., Newgard, C. B., and Unger, R.H. (1998) J. of Biol. Chem. 273, 32487-32490.

23. Hannun, Y. A., and Luberto, C. (2000) Trends Cell Biol 10(2), 73-80.

24. Riboni, L., Viani, P., Bassi, R., Prinetti, A., and Tettamanti, G. (1997) Prog. Lipid Res.

36(2/3), 153-195.

25. Tepper, A., Boesen-de Cock, J., de Vries, E., Borst, J., and van Blitterswijk, W. (1997) J.

Biol. Chem 1997(39), 24308-24312.

26. Carlsson, C., Borg, L. A., and Welsh, N. (1999) Endocrinology 140(8), 3422-8.

27. Inoguchi, T., Li, P., Umeda, F., Yu, H. Y., Kakimoto, M., Imamura, M., Aoki, T., Etoh,T., Hashimoto, T., Naruse, M., Sano, H., Utsumi, H., and Nawata, H. (2000) Diabetes

49(11), 1939-45.

28. Gottlieb, E., Vander Heiden, M. G., and Thompson, C. B. (2000) Mol Cell Biol 20(15),5680-9.

29. Rota, C., Fann, Y. C., and Mason, R. P. (1999) J Biol Chem 274(40), 28161-8.

30. Nishikawa, T., Edelstein, D., Du, X. L., Yamagishi, S., Matsumura, T., Kaneda, Y.,

Yorek, M. A., Beebe, D., Oates, P. J., Hammes, H. P., Giardino, I., and Brownlee, M.(2000) Nature 404(6779), 787-90.

31. Hockenbery, D. M., Oltvai, Z. N., Yin, X. M., Milliman, C. L., and Korsmeyer, S. J.

(1993) Cell 75(2), 241-51.

32. Quillet-Mary, A., Jaffrezou, J. P., Mansat, V., Bordier, C., Naval, J., and Laurent, G.(1997) J Biol Chem 272(34), 21388-95.

33. Joshi, B., Li, L., Taffe, B. G., Zhu, Z., Wahl, S., Tian, H., Ben-Josef, E., Taylor, J. D.,Porter, A. T., and Tang, D. G. (1999) Cancer Res 59(17), 4343-55.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

25

34. Liao, D. F., Jin, Z. G., Baas, A. S., Daum, G., Gygi, S. P., Aebersold, R., and Berk, B. C.(2000) J Biol Chem 275(1), 189-96.

35. Moreno-Manzano, V., Ishikawa, Y., Lucio-Cazana, J., and Kitamura, M. (2000) J Biol

Chem 275(17), 12684-91.

36. Sparagna, G. C., Hickson-Bick, D. L., Buja, L. M., and McMillin, J. B. (2000) Am J

Physiol Heart Circ Physiol 279(5), H2124-H2132.

37. Unger, R. H., Zhou, Y. T., and Orci, L. (1999) Proc Natl Acad Sci U S A 96(5), 2327-32.

38. Moellering, D., McAndrew, J., Jo, H., and Darley-Usmar, V. M. (1999) Free Radic Biol

Med 26(9-10), 1138-45.

39. Buttke, T., and Sandstrom, P. (1995) Free Rad Res 22(5), 389-397.

40. Jacobson, M. (1996) TIBS (March), 83-86.

41. Mignotte, B., and Vayssiere, J. (1998) Eur J Biochem 252, 1-15.

42. Schreck, R., Albermann, K., and Baeuerle, P. A. (1992) Free Radic Res Commun 17(4),221-37.

43. Staal, F. J., Anderson, M. T., Staal, G. E., Herzenberg, L. A., and Gitler, C. (1994) Proc

Natl Acad Sci U S A 91(9), 3619-22.

44. Shimabukuro, M., Ohneda, M., Lee, Y., and Unger, R. H. (1997) J Clin Invest

100(2),290-5.


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FIGURE LEGENDS

FIG. 1. Palmitate but not oleate induces apoptosis in Chinese hamster ovary (CHO) cells.

A, CHO cells were incubated in media 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 percent of 105 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 hours was detected by cleavage of a colorimetric substrate.

Graph displays caspase activity normalized to untreated CHO cells. Data are expressed as the

average of nine samples ± standard error (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 hours. DNA laddering was visualized with agarose gel

electrophoresis and ethidium bromide staining and is representative of three independent

experiments.


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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 hours. Lipids were extracted, ceramide isolated by thin

layer chromatography, and radiolabel incorporation 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 mean of triplicate samples ± S.E. (*p<0.001 relative to untreated, palmitate + L-cycloserine

or palmitate + fumonisin). Data are representative of 2 independent experiments. On average,

untreated CHO cells synthesized 52 pmol of labeled ceramide per gram of protein.


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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 hours. 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

hours). 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 hours. Data are representative of three independent experiments.


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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 to 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 hours of palmitate supplementation (still in the presence of

[3H]serine). Lipids were extracted, ceramide isolated by thin layer chromatography and

radiolabel incorporation determined. Data are expressed as the amount of radiolabeled ceramide

per mg protein and normalized to untreated CHO cells. Data are expressed as the mean 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 hours of palmitate supplementation. Data are normalized to the value in

untreated CHO or untreated LY-B cells and are expressed as the average 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).


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C, DNA Laddering in CHO and LY-B cells was assessed by agarose gel electrophoresis

following 28 hours of palmitate supplementation. Laddering is representative of three

independent experiments.


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31

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 hours (or with 5 mM H2O2 for 1 hour) 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 105 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 to palmitate + PDTC, palmitate +

DBDA or oleate supplemented cells. 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) was measured after palmitate or palmitate plus 5 mM PDTC or 20 mM DBDA

supplementation. Data in (B) are expressed as the average 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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MW

mar

kers

0.5Kb

1.0Kb

palmitateBAFoleate

+ ++

+---

-----1C

time in supplemented media (hours)

po

siti

vely

sta

ined

cel

ls (

%)

1AannV+PI- cells, palmitate fedPI+ cells, palmitate fedannV+PI- cells, oleate fedPI+cells, oleate fed

0

20

40

60

80

0 5 10 15 20 250 5 10 15 20 25

palmitateBAF

oleate

+ ++

+-- -

- -- - -

casp

ase

3 ac

tivi

ty

(r

elat

ive

un

its)

1B

0

2

4

6

8

10 *


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serine + palmitoyl CoA

3-ketosphinganine

sphinganine + fatty acyl CoA

dihydroceramide

ceramide

serine palmitoyltransferase

fumonisin B1ceramide synthase

L-cycloserine

2A

rela

tive

cer

amid

e sy

nth

esis

(

per

mg

pro

tein

)

palmitateL-cycloserine

fumonisin

+-- -- - - -- - - -

+ ++ +

+ +

2B

0

1

2

3 *


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MW

mar

kers

1.0Kb

0.5Kb

palmitatefumonisincycloserine+

++++-

-

-----3B

time in palmitate media (hours)

casp

ase

3 ac

tivi

ty (

rela

tive

un

its)

3A palmitatepalmitate + fumonisinpalmitate + L-cycloserine

0

1

2

3

4

5

6

7

0 5 10 15 20 25 30

**


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palmitateMW

mar

kers

0.5Kb1.0Kb

+ +- -CHO cells LY-B cells4C

0

1

2

3

4

rela

tive

cer

amid

e sy

nth

esis

(p

er m

g p

rote

in)

palmitate + +--

4A CHO cells

LY-B cells*

lyb-palm/baf

cho-unfed

CHO cells

LY-B cells

casp

ase

3 ac

tivi

ty (

rela

tive

un

its)

palmitate + +- -

4B *

*

0

2

4

6

8

10


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1.6Kb

MW

mar

kers

1.0Kb

0.5Kb

palmitatePDTCDBDA

+ +++

+

-

--

-- -

-5C

casp

ase

3 ac

tivi

ty (

rela

tive

un

its)

palmitatePDTCDBDA

+

++

++-

------

5B

0

2

4

6

8

10*

C-2

938

flu

ore

sen

ce

(

rela

tive

un

its)

palmitate

5ACHO cells LY-B

*

0

1

2

3

4

H2O2 -- + -- - +

PDTCDBDA +

+-

-- -

-+-

oleate -

-+-----

----

- +

-+---

-

---

-

* *


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Laura L. Listenberger, Daniel S. Ory and Jean E. SchafferPalmitate-induced apoptosis can occur through a ceramide-independent pathway

published online February 13, 2001J. Biol. Chem.

10.1074/jbc.M010286200Access the most updated version of this article at doi:

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palmitate-induced apoptosis can occur through a ceramide

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