Original Article
Saturated, but Not Unsaturated, Fatty Acids Induce
Apoptosis of Human Coronary Artery Endothelial Cells
via Nuclear Factor-␬B Activation
Katrin Staiger,1 Harald Staiger,1 Cora Weigert,1 Carina Haas,1 Hans-Ulrich Häring,1
and Monika Kellerer1,2
High nonesterified fatty acid (NEFA) concentrations, as
observed in the metabolic syndrome, trigger apoptosis of
human umbilical vein endothelial cells. Since endothelial
apoptosis may contribute to atherothrombosis, we studied
the apoptotic susceptibility of human coronary artery endothelial cells (HCAECs) toward selected NEFAs and the
underlying mechanisms. HCAECs were treated with single
or combined NEFAs. Apoptosis was quantified by flow
cytometry, nuclear factor ␬B (NF␬B) activation by electrophoretic mobility shift assay, and secreted cytokines by
enzyme-linked immunosorbent assay. Treatment of
HCAECs with saturated NEFAs (palmitate and stearate)
increased apoptosis up to fivefold (P < 0.05; n ⴝ 4).
Unsaturated NEFAs (palmitoleate, oleate, and linoleate)
did not promote apoptosis but prevented stearate-induced
apoptosis (P < 0.05; n ⴝ 4). Saturated NEFA-induced
apoptosis neither depended on ceramide formation nor on
oxidative NEFA catabolism. However, NEFA activation via
acyl-CoA formation was essential. Stearate activated NF␬B
and linoleate impaired stearate-induced NF␬B activation.
Pharmacological inhibition of NF␬B and inhibitor of ␬B
kinase (IKK) also blocked stearate-induced apoptosis. Finally, the saturated NEFA effect on NF␬B was not attributable to NEFA-induced cytokine production. In
conclusion, NEFAs display differential effects on HCAEC
survival; saturated NEFAs (palmitate and stearate) are
proapoptotic, and unsaturated NEFAs (palmitoleate,
oleate, and linoleate) are antilipoapoptotic. Mechanistically, promotion of HCAEC apoptosis by saturated NEFA
requires acyl-CoA formation, IKK, and NF␬B activation.
Diabetes 55:3121–3126, 2006
From the 1Division of Endocrinology, Metabolism, and Pathobiochemistry,
Department of Internal Medicine, Eberhard-Karls University Tübingen, Tübingen, Germany; and the 2Clinic of Diabetology, Endocrinology, Intensive
Care Medicine, Vascular Medicine, and Cardiology, Center for Internal Medicine I , Marienhospital Stuttgart, Stuttgart, Germany.
Address correspondence and reprint requests to Dr. Monika Kellerer,
Internal Medicine IV, Medical Clinic, University of Tübingen, Otfried-MüllerStr. 10, D-72076 Tübingen, Germany. E-mail: [email protected].
Received for publication 9 February 2006 and accepted in revised form 15
August 2006.
K.S. and H.S. contributed equally to this work.
HCAEC, human coronary artery endothelial cell; HUVEC, human umbilical
vein endothelial cell; IKK, inhibitor of ␬B kinase; IL, interleukin; NEFA,
nonesterified fatty acid; NF␬B, nuclear factor ␬B; TNF, tumor necrosis factor.
DOI: 10.2337/db06-0188
© 2006 by the American Diabetes Association.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement” in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
DIABETES, VOL. 55, NOVEMBER 2006
E
levated plasma concentrations of nonesterified
fatty acids (NEFAs) are a hallmark of visceral
obesity and are frequently observed in patients
suffering from the metabolic syndrome (1). In
these patients who are at an increased risk to develop
diseases, such as type 2 diabetes, peripheral vascular
disease, and coronary heart disease, the abnormally high
NEFA levels derive from excessive dietary fat intake
and/or increased adipose tissue lipolysis. From animal and
in vitro data, there is ample evidence that chronically
elevated NEFA levels exert detrimental effects on muscular and hepatic insulin sensitivity and on pancreatic insulin
secretion (summarized as lipotoxicity) (rev. in 2). Moreover, high NEFA levels are thought to contribute to
atherogenesis via promotion of hepatic VLDL synthesis
(1,3). In addition to these indirect proatherogenic properties, multiple direct effects of NEFA on cell types involved
in atherogenesis are described: in endothelial cells, NEFAs
induce the expression of cell adhesion molecules and
inflammatory cytokines; in macrophages, NEFAs increase
cholesterol uptake and reduce cholesterol efflux; and in
arterial smooth muscle cells, NEFAs increase proliferation
and migration (4). Interestingly, NEFAs also reveal proapoptotic effects in human umbilical vein endothelial cells
(HUVECs) (5,6). Since increasing evidence, mainly from
histological examinations, suggests endothelial cell apoptosis to play an important role in atherogenesis, plaque
erosion, and acute coronary syndromes (7,8), we studied
the susceptibility of endothelial and smooth muscle cells
from human coronary arteries toward NEFA-induced apoptosis (lipoapoptosis). As we recently reported, the saturated NEFAs palmitate and stearate, at high physiological
concentrations, provoke substantial apoptotic events in
human coronary artery endothelial cells (HCAECs) and a
mixed form of apoptosis and necrosis in human coronary
artery smooth muscle cells (9). To further characterize
these findings, we investigated in this study the role of
selected saturated versus unsaturated NEFAs in HCAEC
lipoapoptosis and the underlying molecular mechanisms.
RESEARCH DESIGN AND METHODS
Cell culture. HCAECs were purchased from Clonetics/BioWhittaker (Verviers, Belgium) and cultured in the commercially available endothelial cell
growth medium EGM-2-MV (EBM-2 supplemented with EGM-2-MV SingleQuots; Clonetics/BioWhittaker). This medium contained 5% (vol/vol) FCS.
Cells were kept in this medium during all experiments and were not serum
starved. Only cells from passages 2 and 3 were used for experiments. Cells
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ROLE OF NF␬B IN HCAEC LIPOAPOPTOSIS
were obtained from healthy donors (aged 16 –56 years) who had given their
informed consent. In most experiments, cells were incubated with NEFA.
NEFAs (Sigma-Aldrich, Taufkirchen, Germany) were bound to fatty acid–free
BSA, as previously described (10). In brief, NEFAs (200 mmol/l in ethanol
except for stearate, which was dissolved to 100 mmol/l) were diluted 1:25 into
Krebs-Ringer HEPES buffer containing 20% (wt/vol) BSA. This mixture was
gently agitated at 37°C under nitrogen overnight. Control medium containing
ethanol and BSA was prepared analogously. These stock solutions were
stored in aliquots under nitrogen at ⫺20°C. At the NEFA concentration used,
BSA reached a concentration of 2.5 or 5% (wt/vol) in the medium. The SN50
inhibitor peptide and hypoestoxide were obtained from Calbiochem (Schwalbach, Germany), trichodion from Alexis (Grünberg, Germany), and human
tumor necrosis factor (TNF)-␣ from R&D Systems (Wiesbaden, Germany).
Triacsin C, cycloserine, fumonisin B1, etomoxir, cycloheximide, actinomycin
D, and the neutralizing anti–TNF-␣ antibody were obtained from SigmaAldrich.
Cell cycle analysis. Confluent cells were treated as indicated. Detached cells
were harvested from the supernatant by centrifugation and added to the
adherent cells harvested by trypsinization. Cells were washed with PBS, fixed
in 70% ice-cold ethanol, centrifuged, and washed again with PBS. After
staining with propidium iodide (50 ␮g/ml) diluted in PBS containing ribonuclease A (100 ␮g/ml), cells were subjected to flow cytometric analysis of DNA
content using a Becton Dickinson FACScalibur cytometer. Percentages of
cells in the different cell cycle phases were calculated by CellQuest software
(Becton Dickinson, Heidelberg, Germany).
Electrophoretic mobility shift assay. Nuclear proteins were prepared as
described previously (11). Synthetic oligonucleotides containing a high-affinity
binding site for nuclear factor ␬B (NF␬B), 5⬘-GTTAGTTGAGGGGACTTTC
CCAGGC-3⬘, were end-labeled with [␣-32P]dATP (3,000 Ci/mmol) and Klenow
enzyme and incubated with up to 10 ␮g nuclear protein in 20 ␮l of 22 mmol/l
HEPES-KOH, pH 7.9, 70 mmol/l KCl, 2.2 mmol/l dithiothreitol, and 10%
glycerol on ice for 20 min. Polydeoxyinosinic-deoxycytidylic acid (0.05 mg/ml)
was added as unspecific competitor. The samples were run on a 5% nondenaturing polyacrylamide gel in a buffer containing 25 mmol/l Tris-HCl, pH 8.0,
190 mmol/l glycine, and 1 mmol/l EDTA. Gels were dried and analyzed by
autoradiography.
Cytokine quantification. Intracellular and secreted proteins (TNF-␣ and
interleukin [IL]-1␤) were quantified with Quantikine enzyme-linked immunosorbent assays from R&D Systems. To measure cytokines in cell lysates,
cell monolayers were washed with PBS and scraped off in PBS supplemented
with 1 mmol/l EDTA, 1 mmol/l phenylmethylsulfonylfluoride, 10 ␮g/ml aprotinin, 0.5 ␮g/ml leupeptin, and 0.7 ␮g/ml pepstatin. After cell lysis by
sonication, lysates were cleared by centrifugation. Measurements were performed in duplicate. For standardization, cellular protein of the cell lysates
was determined with the Bradford method. Cytokines in cell culture supernatants were measured after centrifugation.
Statistics. Data were analyzed by ANOVA with Bonferroni’s post hoc test. A
P value ⬍0.05 was considered statistically significant. For these tests, the
statistical software package SigmaStat for Windows 1.0 (Jandel, San Rafael,
CA) was used.
RESULTS
Different effects of saturated versus unsaturated
NEFAs on HCAECs. HCAECs were treated for 24 h with
different saturated and unsaturated NEFAs (1 mmol/l
each), and apoptosis was measured by flow cytometric
cell cycle analysis (quantification of cells with subG1 DNA
content). The saturated NEFAs stearate (C18:0) and, to a
lesser extent, palmitate (C16:0) significantly induced apoptosis in HCAECs (Fig. 1A). The apoptotic mechanism was
recently confirmed by demonstration of caspase-3 activation after palmitate and stearate treatment (9). We were
not able to detect saturated NEFA-induced cytochrome c
release, which is seen in most, but not all, forms of
apoptosis (data not shown). Compared with these saturated NEFAs, the carrier BSA (2.5 and 5%, data not shown)
and the monounsaturated NEFA palmitoleate (C16:1 ␻7)
and oleate (C18:1 ␻9) and the polyunsaturated NEFA
linoleate (C18:2 ␻6) had no proapoptotic effect (Fig. 1A).
Moreover, all unsaturated NEFAs tested significantly prevented stearate-induced apoptosis (Fig. 1B). In palmitateinduced apoptosis, unsaturated NEFAs also tended to be
3122
FIG. 1. A: Effects of individual NEFAs on HCAEC apoptosis. HCAECs
were left untreated (䡺) or were treated with 1 mmol/l NEFA for 24 h
(f, saturated NEFAs; o, unsaturated NEFAs). Apoptosis was determined as described in RESEARCH DESIGN AND METHODS. Data are given as
means ⴞ SE. *Significantly different from control (P < 0.05; n ⴝ 4). B:
Effects of unsaturated NEFAs on stearate- and palmitate-induced
HCAEC lipoapoptosis. HCAECs were treated with 1 mmol/l palmitate
(o) or stearate (f) for 24 h. Unsaturated NEFAs (1 mmol/l) were
added 30 min before and during the 24-h treatment with saturated
NEFAs. Apoptosis was determined as described in RESEARCH DESIGN AND
METHODS. Data are given as means ⴞ SE. *Significantly different from
cells treated with stearate alone (P < 0.05; n ⴝ 4).
protective. Due to the weak proapoptotic effect of palmitate, protection by unsaturated NEFAs, however, did not
reach the level of significance (Fig. 1B). Thus, saturated
and unsaturated NEFAs display differential effects on
HCAEC survival; saturated NEFAs trigger apoptosis,
whereas unsaturated NEFAs prevent this kind of lipoapoptosis.
Acyl-CoA formation, but not mitochondrial ␤-oxidation, is required for lipoapoptosis. As palmitate induces apoptosis in other cell systems via ceramide
biosynthesis (rev. in 12), we investigated the role of
ceramide formation in lipoapoptosis of HCAECs. To this
end, we treated HCAECs with specific inhibitors of several
steps of the ceramide de novo synthesis pathway. Triacsin
C was recently shown to efficiently inhibit the long-chain
acyl-CoA synthetase activity (palmitoyl-CoA formation) of
HCAECs at 5–10 ␮mol/l (13). In our experimental setting,
triacsin C (10 ␮mol/l) revealed cytotoxic effects in the
absence of NEFA (Fig. 2). This might be due to the
reduction of basal intracellular acyl-CoA concentrations
below a critical level necessary for the maintainance of
cellular viability. Apart from this, triacsin C significantly
prevented palmitate- and stearate-induced apoptosis (Fig.
2). In contrast, inhibitors of enzymes more downstream in
the ceramide biosynthesis pathway, i.e., cycloserine (1
mmol/l) and fumonisin B1 (50 ␮mol/l), had no protective
DIABETES, VOL. 55, NOVEMBER 2006
K. STAIGER AND ASSOCIATES
FIG. 2. Acyl-CoA formation is required for HCAEC lipoapoptosis. HCAECs were left untreated (䡺) or were treated with 1 mmol/l palmitate (o)
or 1 mmol/l stearate (f) for 24 h. Triacsin C (10 ␮mol/l), cycloserine (cycloSer; 1 mmol/l), fumonisin B1 (fumo B1; 50 ␮mol/l), or etomoxir (100
␮mol/l) were added 30 min before and during NEFA treatment. Apoptosis was determined as described in RESEARCH DESIGN AND METHODS. Data are
given as means ⴞ SE. *Significantly different from the corresponding inhibitor-free NEFA-treated controls (P < 0.01; n ⴝ 5).
effect (Fig. 2). In addition, treatment of cells with exogenous C2-ceramide (10 ␮mol/l) did not induce apoptosis in
HCAECs (data not shown). Therefore, NEFA activation via
fatty acyl-CoA formation, the first step of NEFA metabolism, but not ceramide de novo synthesis, seems to be
required for lipoapoptosis in HCAECs. Treating HCAECs
with etomoxir (100 ␮mol/l), an inhibitor of carnitine
palmitoyltransferase I that catalyzes the rate-limiting step
of the ␤-oxidation, had no significant effect on lipoapoptosis (Fig. 2). This suggests that ␤-oxidation is not involved
in the mechanism used by saturated NEFAs to induce
lipoapoptosis in these cells.
Saturated NEFAs induce lipoapoptosis via inhibitor
of ␬B kinase and NF␬B. As treatment of HCAECs with
the inhibitor of gene transcription actinomycin D (10
␮g/ml) or the inhibitor of protein biosynthesis cycloheximide (20 ␮g/ml) significantly prevented stearate-induced
apoptosis (Fig. 3), de novo synthesis of protein(s) seems
to be required for HCAEC lipoapoptosis. Therefore, we
investigated potential candidate transcription factors for
apoptotic gene expression. One transcription factor
known to be activated by NEFA is NF␬B (14 –16). Figure 4
shows that the proapoptotic saturated NEFA stearate
promotes activation of NF␬B. In agreement with its minor
proapoptotic properties, the saturated NEFA palmitate
had only a weak effect on NF␬B activity (data not shown).
Linoleate, which was chosen as a representative unsatur-
FIG. 3. De novo protein synthesis is required for stearate-induced
apoptosis. HCAECs were left untreated (䡺) or were treated with 1
mmol/l stearate (f) for 24 h. Cycloheximide (CHX; 20 ␮g/ml) or
actinomycin D (Act D; 10 ␮g/ml) were added 30 min before and during
stearate treatment. Apoptosis was determined as described in RESEARCH
DESIGN AND METHODS. Data are given as means ⴞ SE. *Significantly
different from cells treated with stearate alone (P < 0.05; n ⴝ 5).
DIABETES, VOL. 55, NOVEMBER 2006
ated NEFA shown above to lack proapoptotic effects in
HCAECs, is not able to induce NF␬B activation (Fig. 4).
Thus, activation of NF␬B appears to be restricted to the
proapoptotic saturated NEFA. Consistent with its antilipoapoptotic effect, linoleate reduced stearate-induced NF␬B
activation to the level of BSA (Fig. 5B). Furthermore,
treatment of HCAECs with the NF␬B inhibitors SN50 (18
␮mol/l) and trichodion (50 ␮mol/l) protected HCAECs
from lipoapoptosis (Fig. 5A) and reduced stearate-induced
NF␬B activation even below the level of BSA (Fig. 5B).
Inhibitor of ␬B kinase (IKK) is the best characterized
upstream kinase regulating NF␬B activity via phosphorylation of inhibitor of ␬B. Treatment of HCAECs with
hypoestoxide (100 ␮mol/l), a selective and direct inhibitor
of IKK, prevented lipoapoptosis (Fig. 6). Taken together,
these data suggest that palmitate and stearate exert their
lipoapoptotic effects via activation of IKK and NF␬B.
Saturated NEFAs do not activate NF␬B via induction
of TNF-␣ or IL-1␤. To investigate whether saturated
NEFAs activate NF␬B via induction of NF␬B-activating
cytokines, such as TNF-␣ or IL-1␤, we determined TNF-␣
and IL-1␤ production after treament of HCAECs with
palmitate or stearate (1 mmol/l each). Under these conditions, HCAECs did not produce measurable amounts of
intracellular or secreted IL-1␤ protein (data not shown).
TNF-␣ was detected in minor amounts in the supernatant
(2.5% BSA: 12.5 ⫾ 1.8 pg/ml; 5% BSA: 13.5 ⫾ 2.7 pg/ml; n ⫽
3). However, neither palmitate nor stearate was able to
significantly increase the amount of secreted TNF-␣ protein over the respective BSA control (1 mmol/l palmitate:
12.4 ⫾ 2.2 pg/ml; 1 mmol/l stearate: 23.1 ⫾ 10.5 pg/ml; n ⫽
3). In addition, the detected TNF-␣ concentrations (in the
picograms per milliliter range) are considered too low to
significantly induce apoptosis in HCAECs, as exogenously
applied TNF-␣ concentrations up to 500 ng/ml were not
effective in triggering substantial apoptosis (data not
shown). Furthermore, a neutralizing anti–TNF-␣ antibody
was not able to significantly reduce palmitate- or stearateinduced apoptosis in HCAECs (data not shown). Taken
together, these data suggest that saturated NEFAs do not
activate NF␬B via stimulation of TNF-␣ or IL-1␤ synthesis/
secretion.
DISCUSSION
Consistent with our recent report (9), the common plasma
saturated NEFAs palmitate and stearate, at 1 mmol/l,
clearly reveal proapoptotic effects on HCAECs. Taking
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ROLE OF NF␬B IN HCAEC LIPOAPOPTOSIS
FIG. 4. Different effects of stearate and linoleate on NF␬B activation in HCAECs. HCAECs were incubated with 5% BSA for control or with 1
mmol/l stearate (S) or 1 mmol/l linoleate (L) for the indicated time intervals. As control for NF␬B activation, cells were treated for 1 h with TNF-␣
(100 ng/ml). Electrophoretic mobility shift assay was performed as described in RESEARCH DESIGN AND METHODS. Arrows indicate protein complexes
formed with NF␬B binding site– containing oligonucleotides.
into account that 1) the total plasma NEFA concentration
in the fasting state can reach 1.6 mmol/l, as detected in
some healthy participants of the TÜF (Tübingen family
study for type 2 diabetes) (A. Fritsche, N. Stefan, unpublished data) and that 2) palmitate and stearate comprise
⬃28 and 12%, respectively, of total NEFA, as measured in
54 TÜF participants (17), the concentrations used here
are, at least in the case of palmitate, not too far from the
physiological situation. As for stearate, it can be estimated
that 1 mmol/l is about fivefold higher than its plasma
concentration in healthy subjects. Even though respective
data are lacking, it is conceivable that this stearate concentration can be reached in pathological situations associated with markedly increased lipolytic rates, such as
morbid obesity and type 2 diabetes. The proapoptotic
effects of saturated NEFAs on HCAECs is in agreement
with earlier studies in HUVECs (5,6). In these cells,
however, Artwohl et al. (5) additionally demonstrated that
unsaturated NEFAs, at a concentration of 300 ␮mol/l, act
proapoptotically as well. Moreover, these authors point
out that the proapoptotic properties of unsaturated NEFAs
are a function of the degree of desaturation with linoleate
being comparably effective as stearate. In HCAECs, we
were not able to detect any proapoptotic effects of unsaturated NEFAs (linoleate included), even at a concentration of 1 mmol/l. Moreover, we provide data demonstrating
potent antilipoapoptotic effects of these NEFA species. As
to these discrepancies, we speculate that endothelial cells
FIG. 5. Saturated NEFAs induce HCAEC apoptosis via activation of NF␬B. A: HCAECs were left untreated (䡺) or were treated with 1 mmol/l
palmitate (o) or 1 mmol/l stearate (f) for 24 h. The NF␬B inhibitors SN50 (18 ␮mol/l) or trichodion (50 ␮mol/l) were added 30 min before and
during NEFA treatment. Apoptosis was determined as described in RESEARCH DESIGN AND METHODS. Data are given as means ⴞ SE. *Significantly
different from the corresponding inhibitor-free NEFA-treated controls (P < 0.05; n ⴝ 8). B: HCAECs were incubated with 2.5 and 5% BSA for
control or 0.5 and 1 mmol/l stearate for 4 h. The NF␬B inhibitors SN50 (SN; 18 ␮mol/l) and trichodion (Trich; 50 ␮mol/l) or the unsaturated NEFA
linoleate (L; 1 mmol/l) were added 30 min before and during stearate (S; 1 mmol/l) treatment. Electrophoretic mobility shift assay was performed
as described in RESEARCH DESIGN AND METHODS. Black triangles mark increasing concentrations. Arrows indicate protein complexes formed with
NF␬B binding site– containing oligonucleotides.
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DIABETES, VOL. 55, NOVEMBER 2006
K. STAIGER AND ASSOCIATES
FIG. 6. I␬B kinase activity is required for saturated NEFA-induced
HCAEC apoptosis. HCAECs were left untreated (䡺) or were treated
with 1 mmol/l palmitate (o) or 1 mmol/l stearate (f) for 24 h.
Hypoestoxide (100 ␮mol/l) was added 30 min before and during NEFA
treatment. Apoptosis was determined as described in RESEARCH DESIGN
AND METHODS. Data are given as means ⴞ SE. *Significantly different
from cells treated with palmitate or stearate alone (P < 0.05; n ⴝ 5).
from different sources, i.e., vein versus artery and umbilical versus coronary vessels, diverge in their apoptotic
susceptibility toward unsaturated NEFAs. With regard to
the importance of endothelial cell apoptosis in atherogenesis, plaque erosion, and acute coronary syndromes (7,8),
our findings, which clearly demonstrate antilipoapoptotic
properties of palmitoleate, oleate, and linoleate in
HCAECs,
could
provide a mechanistic explanation for the previously
observed cardioprotective effects of these non-␻3 unsaturated NEFAs (18 –20).
Furthermore, we provide evidence that saturated
NEFAs, after being activated to fatty acyl-CoA, induce
HCAEC apoptosis via activation of IKK and NF␬B. We
show that this signaling pathway does neither involve
conversion of NEFA to ceramide nor NEFA degradation
via ␤-oxidation. In addition, we tend to exclude nitric
oxide as a mediator of saturated NEFA-induced apoptosis
since recent attempts to inhibit lipoapoptosis by blocking
nitric oxide synthase isoforms using pharmacological
inhibitors, i.e., NG-monomethyl-L-arginine, NG-nitro-L-arginine methyl ester, and NG-(1-iminoethyl)-L-lysine, were not
successful (data not shown). Further studies are, however,
needed to substantiate this finding. It was recently demonstrated that saturated NEFAs are able to activate NF␬B
in several cell systems, such as murine and human skeletal
muscle cells (14,21), bovine eye pericytes (22), bovine
aortic endothelial cells (16), and HUVECs (23). Since
NF␬B activation by various physiological stimuli, e.g.,
IL-18 (24), high glucose (25), hypoxia (26), and TNF-␣ (27),
is reported to trigger apoptosis in endothelial cells, the
mechanism of lipoapoptosis described in this study appears plausible. Consistent with the antiapoptotic effect of
unsaturated NEFAs observed in this study, unsaturated
NEFAs are reported to inhibit NF␬B activation in endothelial cells (28) and macrophages (29) and to prevent
saturated NEFA-induced apoptosis in rat insulinoma and
human islet cells (30). In addition, our findings point to
NEFA-stimulated and NF␬B-directed induction of proapoptotic genes. Preliminary data using the Apoptosis
Oligo GEArray from SuperArray Bioscience support this
suggestion: among 112 spotted genes, 7 were found regulated by stearate in HCAECs. Increased were, for example,
the proapoptotic genes Bcl-2–like protein 13, lymphotoxin-␤ receptor, TWEAK (TNF-related weak inducer of
DIABETES, VOL. 55, NOVEMBER 2006
apoptosis) receptor, and TRAF1 (TNF receptor–associated factor 1). Studies further evaluating the role of these
genes in HCAEC lipoapoptosis are currently on the way.
As to the molecular mechanisms upstream of IKK and
NF␬B that constitute the divergent behavior of saturated
and unsaturated NEFA in HCAECs, we can at the moment
only speculate. However, the very recently reported findings on the differential potential of these NEFA classes to
provoke an endoplasmic reticulum stress response (31) or
to stimulate formation of diacylglycerol (32,33), a wellknown activator of classical and novel protein kinase C
isoforms, represent good starting points for further investigations. Finally, we show that the effect of saturated
NEFAs is not mediated by NF␬B-dependent induction of
proapoptotic cytokines, such as TNF-␣ and IL-1␤; 1) IL-1␤
was not expressed by HCAECs, as was previously demonstrated (17,34), and 2) we detected only low amounts of
secreted TNF-␣ protein, which were found to be ineffective in provoking apoptosis and, in addition, were not
regulated by saturated NEFAs. Very recently, it was reported that saturated, but not unsaturated, NEFAs induce
insulin receptor and insulin receptor substrate 1 expression in human aortic endothelial cells and confer insulin
sensitivity to these cells (35). This finding could also imply
enhanced survival factor signaling in these cells in response to saturated NEFAs. Whether such a protective
mechanism is absent or counterregulated in HCAECs
cannot be answered at the moment and awaits further
studies.
In conclusion, we show here that saturated and unsaturated NEFAs display differential effects on HCAEC survival; saturated NEFAs, such as palmitate and stearate, are
proapoptotic, whereas unsaturated NEFAs, such as palmitoleate, oleate, and linoleate, are antilipoapoptotic. Mechanistically, promotion of HCAEC apoptosis by saturated
NEFAs requires fatty acyl-CoA formation and subsequent
NF␬B activation. As endothelial cell apoptosis is supposed
to play an important role in plaque erosion and acute
coronary syndromes, our findings could provide a theoretical explanation for the increased incidence of such lifethreatening events in medical disorders associated with
chronically elevated plasma NEFA concentrations.
ACKNOWLEDGMENTS
This study was supported in part by a grant from the
German Research Foundation (KFO 114/1-1) and the European Community’s FP6 EUGENE 2 (LSHM-CT-2004512013).
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DIABETES, VOL. 55, NOVEMBER 2006