Báo cáo khoa học: Peroxisome proliferator-activated receptor a–retinoid X receptor agonists induce beta-cell protection against palmitate toxicity doc

Peroxisome proliferator-activated receptor a–retinoid X
receptor agonists induce beta-cell protection against
palmitate toxicity
Karine Hellemans
1
, Karen Kerckhofs
1
, Jean-Claude Hannaert
1
, Geert Martens
1
,
Paul Van Veldhoven
2
and Daniel Pipeleers
1
1 Diabetes Research Center (DRC), Brussels Free University-VUB, Belgium
2 Afdeling Farmakologie, Departement Moleculaire Celbiologie, K. U. Leuven, Belgium
Under normal circumstances, long-chain fatty acids
serve as regulators of beta-cell function [1,2]. At sus-
tained, elevated concentrations, they can exert cyto-
toxic actions on beta-cells, and this has led to the view
that they could be, at least in part, responsible for the
loss of beta-cells in diabetes [3,4]. Several in vitro and
in vivo studies have supported this lipotoxicity concept
and extended into experiments aimed at pharmacologic
prevention of this process [3,5]. We previously reported
that pancreatic beta-cells possess defense mechanisms
against oxidative damage [6] and could be induced to
provide cytoprotection [7]. It is still uncertain whether
they also exhibit such properties when exposed to cyto-

toxic fatty acid concentrations, and if so, whether they
can be activated through similar or other mechanisms.
Indirect evidence for the presence of such a protective
mechanism comes from the observation that fatty
acid-induced toxicity was limited to a subpopulation of
beta-cells, and apparently related to the cellular ability
to accumulate neutral lipids in the cytoplasm [7]. We
thus proposed that the formation of cytoplasmatic
lipids could reduce fatty acid-induced toxicity by
Keywords
cytotoxicity; fibrate; free fatty acid;
pancreatic beta-cells; peroxisome
proliferator-activated receptor a
Correspondence
K. Hellemans, Diabetes Research Center,
Brussels Free University-VUB,
Laarbeeklaan 103, 1090 Brussels, Belgium
Fax: +32 2 4774545
Tel: +32 2 4774541
E-mail:
(Received 11 July 2007, revised 1 October
2007, accepted 8 October 2007)
doi:10.1111/j.1742-4658.2007.06131.x
Fatty acids can stimulate the secretory activity of insulin-producing beta-
cells. At elevated concentrations, they can also be toxic to isolated beta-
cells. This toxicity varies inversely with the cellular ability to accumulate
neutral lipids in the cytoplasm. To further examine whether cytoprotection
can be achieved by decreasing cytoplasmic levels of free acyl moieties, we
investigated whether palmitate toxicity is also lowered by stimulating its
b-oxidation. Lower rates of palmitate-induced beta-cell death were mea-

sured in the presence of l-carnitine as well as after addition of peroxisome
proliferator-activated receptor a (PPARa) agonists, conditions leading to
increased palmitate oxidation. In contrast, inhibition of mitochondrial
b-oxidation by etomoxir increased palmitate toxicity. A combination of
PPARa and retinoid X receptor (RXR) agonists acted synergistically and
led to complete protection; this was associated with enhanced expression
levels of genes involved in mitochondrial and peroxisomal b-oxidation,
lipid metabolism, and peroxisome proliferation. PPARa–RXR protection
was abolished by the carnitine palmitoyl transferase 1 inhibitor etomoxir.
These observations indicate that PPARa and RXR regulate beta-cell
susceptibility to long-chain fatty acid toxicity by increasing the rates of
b-oxidation and by involving peroxisomes in fatty acid metabolism.
Abbreviations
CPT1, carnitine palmitoyl transferase 1, liver; GPAT, glycerol-3-phosphate acyltransferase, mitochondrial; Pex2, peroxisomal biogenesis
factor 2; Pex3, peroxisomal biogenesis factor 3; Pex11a, peroxisomal biogenesis factor 11a; Pex14, peroxisomal biogenesis factor 14;
Pex16, peroxisomal biogenesis factor 16; PPARa, peroxisome proliferator-activated receptor a; PMP70, peroxisomal membrane protein 70;
RA, retinoic acid; RXRa, retinoid X receptor; SCD1, stearoyl-CoA desaturase 1; SCD2, stearoyl-CoA desaturase 2.
6094 FEBS Journal 274 (2007) 6094–6105 ª 2007 The Authors Journal compilation ª 2007 FEBS
preventing a rise in toxic free acyl moieties [7] and ⁄ or
fatty acid metabolites such as ceramides [8,9]. Along
this line, one can further hypothesize that an increased
rate of fatty acid oxidation also lowers the formation
of these cytotoxic mediators and could thus also act as
a cytoprotective mechanism. To test this hypothesis,
we examined whether palmitate toxicity can be reduced
by increasing its oxidation rates. We first assessed
whether protection could be conferred by l-carnitine, a
rate-limiting component for long-chain fatty acid
transport into the mitochondria, or suppressed by
etomoxir, an irreversible carnitine palmitoyl transfer-

ase 1 (CPT1) inhibitor [10]. In a second set of experi-
ments, we assessed the effects of agonists for
peroxisome proliferator-activated receptor a (PPARa)
and retinoid X receptor (RXR). PPARa–RXR dimers
can be activated by both PPARa and RXR agonists.
PPARa–RXR dimers typically regulate the expression
of multiple genes involved in mitochondrial and perox-
isomal b-oxidation as well as lipoprotein metabolism
[11]. PPARa is expressed in primary rat beta-cells [12],
and has been shown to activate fatty acid oxidation
[13,14]. PPARa agonists have been reported to prevent
fatty acid-induced beta-cell dysfunction and apoptosis
in human islets [15], and improve beta-cell function in
insulin-resistant rodent models [16].
Results
Specificity of palmitate toxicity
When rat beta-cells were cultured with palmitate, time-
and concentration-dependent cytotoxicity was mea-
sured. At 50 and 100 lm, no toxic effect was detected
after 2 days, and only 10–20% cells were damaged
after 8 days (data not shown). At 250 and 500 lm,
cytotoxicity was noticed after 2 days and resulted,
after 8 days, in, respectively, 38 ± 2% and 75 ± 4%
dead cells (Fig. 1). It was noticed that with exposure
longer than 3 days at 500 lm, the slope of the toxicity
curve declined. The percentage of surviving cells
tended to stabilize around 25% after 6 days, despite
administration of a new bolus of fatty acid every 48 h.
With 250 lm, the fraction of cell survival stabilized
around 60%. The cytotoxic effect of palmitate did not

vary with the glucose concentration in the medium
(comparison of 5, 10 and 20 mm glucose, data not
shown). Subsequent studies were conducted at 10 mm
glucose for 2 days with 500 lm palmitate (acute toxic-
ity) and for 8 days at 250 lm (chronic toxicity).
Islet endocrine nonbeta-cells exhibited lower suscep-
tibility to palmitate toxicity: cytotoxicity was
9 ± 0.5% after 2 days at 500 lm, and 16 ± 3% after
8 days at 250 lm. After 8 days at 500 lm, the cytotox-
icity increased to 46 ± 8% (results not shown). In the
latter condition, more than 95% of purified beta-cells
died, which means that approximately 20% of the
dead cells in the nonbeta-cell fraction correspond to
beta-cells, as the nonbeta-cell fraction is contaminated
to this extent by beta-cells; consequently, the palmitate
toxicity for the islet nonbeta-cells is calculated to be
about 25% after 8 days at 500 lm.
Palmitate toxicity in beta-cells was compared with
that of equimolar concentrations of other fatty acids
in the presence of 1% BSA (Table 1). Oleate, an unsat-
urated long-chain fatty acid, was less toxic, and the
shorter-chain molecules butyrate (C4), hexanoate (C6)
and octanoate (C8) were only marginally toxic. The
2-methyl and 3-methyl derivatives of palmitate were
virtually nontoxic: < 5% after 2 days and < 10%
after 8 days, both at 250 and at 500 lm, whereas
2 4 6 8 10
0
25
50

75
100
250
M C16:0
500 M C16:0
Days of exposure
Cytotoxicity (%)
Fig. 1. Time course analysis for palmitate cytotoxicity. Primary rat
beta-cells were exposed to 250 or 500 l
M palmitate. Cytotoxicity
was measured on a daily basis (n ¼ 4, vertical bars represent
SEM).
Table 1. Cytotoxicity of fatty acids for rat beta-cells. Beta-cells
were exposed to the following fatty acids at 500 l
M for 2 days or
at 250 l
M for 8 days: palmitate (C16:0), oleate (C18:1), butyrate
(C4:0), hexanoate (C6:0), octanoate (C8:0), 2-methylhexadecanoic
acid (2-Me-C16:0), 3-methylhexadecanoic acid (3-Me-C16:0),
2-bromopalmitate (Br-C16:0). Data represent means ± SD (n ¼
3–6). *P < 0.001 as compared to C16:0 (student’s t-test).
Fatty acid
Percentage cytotoxicity
500 l
M for 2 days 250 lM for 8 days
C16:0 24 ± 2 38 ± 2
C18:1 11 ± 2* 28 ± 3*
C4:0 5 ± 2* 4 ± 1*
C6:0 1 ± 1* 5 ± 1*
C8:0 9 ± 1* 11 ± 1*

2-Me-C16:0 4 ± 1* 4 ± 4*
3-Me-C16:0 5 ± 4* 6 ± 4*
Br-C16:0 58 ± 6* 92 ± 3*
K. Hellemans et al. Beta-cell protection against palmitate toxicity
FEBS Journal 274 (2007) 6094–6105 ª 2007 The Authors Journal compilation ª 2007 FEBS 6095
2-bromopalmitate was markedly more toxic than palm-
itate. There was no difference in the percentage of liv-
ing cells following vehicle treatment when compared to
the standard control.
Palmitate and glucose metabolism by beta-cells
and islet nonbeta-cells
Palmitate and glucose metabolism was measured in
freshly isolated beta-cells and in islet nonbeta-cells that
were incubated for 2 h with 50 lm [
14
C]palmitate or
5 lCi of d-[U-
14
C]glucose.
14
C incorporation was mea-
sured in CO
2
and small metabolic intermediates, as well
as in the lipid and protein fractions (Table 2). After
incubation with [
14
C]palmitate at 3.3 mm glucose, the
largest amount was recovered in the lipid-soluble frac-
tion (55%) and the lowest in the protein fraction,

whereas comparable amounts were converted to CO
2
(17%) and to small metabolic intermediates (20%); at
20 mm glucose, the total amounts in the lipid fraction
(representing 82%) and protein fraction were not sig-
nificantly different from those at 3 mm glucose, but the
production of
14
CO
2
and of
14
C intermediates was,
respectively, five- and 10-fold lower (Table 2). Analysis
of the fate of [
14
C]glucose indicated that, at 3.3 mm
glucose, the tracer was converted to CO
2
and to small
intermediates, and that high glucose increased this rate
five-fold, and also increased seven-fold the
14
C incorpo-
ration into the lipid and protein fraction.
Islet endocrine nonbeta-cells exhibit much lower
rates of glucose oxidation and utilization than islet
beta-cells; the values shown in Table 2 are an overesti-
mation, in view of the contamination of this fraction
by 20–25% beta-cells; the CO

2
production from glu-
cose that is calculated for islet nonbeta-cells is thus less
than 10% of that in beta-cells. On the other hand,
their level of CO
2
production from palmitate is higher,
in particular at 20 mm glucose, where four-fold higher
rates were measured than in beta-cells (Table 2). In
contrast to the situation in beta-cells, 30% of the
labeled palmitate was converted to CO
2
independently
of the glucose concentration.
Effect of regulators of palmitate metabolism on
palmitate toxicity in beta-cells
When beta-cells were precultured for 24 h with the
CPT1 activator l-carnitine before measurement of
their palmitate oxidation during 2 h of incubation in
the further presence of l-carnitine,
14
CO
2
formation
was six-fold higher (to 0.83 ± 0.14 pmol per 2 h per
10
3
cells, n ¼ 4, P < 0.05) than in control cells
cultured and incubated with the solvent
(0.12 ± 0.03). This stimulatory effect was preserved

when 250 lm palmitate was added to the preculture
medium (0.78 ± 0.19 pmol per 2 h per 10
3
cells,
n ¼ 4, P < 0.01) (Fig. 2A). It was associated with
an eight-fold elevation of
14
C incorporation into
metabolic intermediates (from 0.15 ± 0.05 to
0.95 ± 0.07 pmol per 2 h per 10
3
cells, P < 0.05)
(results not shown). Preculture with 1 mml-carnitine
protected beta-cells from palmitate toxicity during a
Table 2. Metabolism of [
14
C]palmitate and [
14
C]glucose in beta-cells and islet nonbeta-cells at 3.3 and 20 mM glucose. Palmitate and glucose
metabolism was measured in freshly isolated beta-cells and islet nonbeta-cells incubated for 2 h with 50 l
M [
14
C]palmitate or 5 lCi of
D-[U-
14
C]glucose.
14
C incorporation was measured in CO
2
and small metabolic intermediates, as well as in the lipid and protein fractions and

expressed as (pmol per 2 h per 10
3
cells). Data are indicated as mean ± SD (n ¼ 5). Italic: P < 0.05 for 20 mM glucose as compared to 3.3 mM
glucose. *P < 0.05 for nonbeta-cells as compared to beta-cells, **P < 0.001 for nonbeta-cells as compared to beta-cells (Student’s t-test).
14
C recovery as:
CO
2
Intermediates Lipid Protein
From [
14
C]palmitate
Beta-cells
Glucose 3.3 m
M 0.52 ± 0.04 0.62 ± 0.05 1.71 ± 0.11 0.21 ± 0.06
Glucose 20 m
M 0.10 ± 0.01 0.06 ± 0.01 1.73 ± 0.04 0.13 ± 0.08
Nonbeta-cells
Glucose 3.3 m
M 0.63 ± 0.04* 0.18 ± 0.003* 1.18 ± 0.04* 0.13 ± 0.08
Glucose 20 m
M 0.46 ± 0.04* 0.10 ± 0.01 1.06 ± 0.07* 0.07 ± 0.03
From [
14
C]glucose
Beta-cells
Glucose 3.3 m
M 5.49 ± 0.31 4.41 ± 0.17 0.29 ± 0.08 0.40 ± 0.02
Glucose 20 m
M 31.15 ± 1.86 19.90 ± 1.96 2.16 ± 0.72 3.16 ± 0.99

Nonbeta-cells
Glucose 3.3 m
M 0.96 ± 0.22** 1.20 ± 0.11** 0.13 ± 0.03* 0.19 ± 0.04*
Glucose 20 m
M 3.82 ± 0.95** 3.78 ± 0.48** 1.13 ± 0.55 0.45 ± 0.07**
Beta-cell protection against palmitate toxicity K. Hellemans et al.
6096 FEBS Journal 274 (2007) 6094–6105 ª 2007 The Authors Journal compilation ª 2007 FEBS
subsequent exposure to 500 lm palmitate for 2 days
or 250 lm for 8 days by, respectively, 70% and 40%
(Fig. 2B).
On the other hand, when beta-cells were exposed to
palmitate in the presence of the CPT1 inhibitor etom-
oxir (200 lm) (Fig. 2C), their survival was further
decreased. Lower concentrations of etomoxir (1, 5 and
50 lm), which are known to stimulate PPARa, failed
to show an effect on palmitate toxicity. Etomoxir
(200 lm) did not affect beta-cell survival in the pres-
ence of butyrate (C4), which is known to enter mito-
chondria independently of CPT1.
Addition of l-cycloserine (100 lm), an inhibitor of
serine palmitoyl transferase, was also found to reduce
palmitate toxicity, both after 2 days at 500 lm (from
24 ± 2% to 10 ± 1%), and after 8 days at 250 lm
(from 38 ± 2% to 20 ± 4%), but not that of oleate
(results not shown).
Effect of PPARa–RXR agonists on palmitate
cytotoxicity
The effects of PPARa–RXR agonists clofibrate, cipro-
fibrate and 9-cis-retinoic acid (9-cis-RA) were examined
by adding these compounds to the 2 and 8 day culture

media. Clofibrate alone (tested at 100, 250 and
500 lm, two-way ANOVA, P < 0.001) reduced palmi-
tate toxicity at both time points, with a maximal effect
at 250 lm (Fig. 3A). Protection from palmitate toxicity
was also observed after treatment with 9-cis-RA alone
at all tested concentrations (0.5, 2 or 5 lm, two-way
ANOVA, P < 0.001); at 5 lm, the effect was compa-
rable to that of 250 lm clofibrate, namely 60% protec-
tion after 2 days at 500 lm and 70% after 8 days at
250 lm (Fig. 3A). Combinations of different con-
centrations of both agents further reduced palmitate
toxicity; the maximal level of protection, reducing
palmitate-induced cell death to only 5%, was reached
using a combination of 5 lm 9-cis-RA and 100 lm clo-
fibrate. Higher clofibrate concentrations in the pres-
ence of 9-cis-RA did not lower palmitate toxicity any
further (Fig. 3A). For all further experiments, a combi-
nation of 250 lm clofibrate and 2 lm 9-cis-RA was
used, resulting in a reduction of palmitate toxicity by
90% (Fig. 3B). The efficacy of this combination did
not significantly differ from that of 250 lm clofibrate
plus 5 lm 9-cis-RA. The same level of protection as
found in the presence of 10 mm glucose was found at
low (5 mm) and high (20 mm) glucose concentrations
(results not shown). Under the same conditions, clofi-
brate and 9-cis-RA were also found to protect against
oleate toxicity (from 25 ± 3% to 13 ± 2% for
500 lm after 2 days, and from 32 ± 3% to 14 ± 4%
for 250 lm after 8 days, P < 0.01, results not shown).
Ciprofibrate (10, 50 and 100 lm) mimicked the effect

of clofibrate, with a maximal effect at 100 lm, and
similar additive protection by 9-cis-RA (Fig. 3B).
Addition of fibrate and ⁄ or 9-cis-RA to palmitate-free
control medium did not influence cell survival during
culture (data not shown).
When endocrine nonbeta-cells were exposed to
500 lm palmitate for 8 days with or without 250 lm
clofibrate plus 2 lm 9-cis-RA, no differences in toxicity
were noticed, indicating the absence of a protective
effect of the supplement (results not shown).
Effect of PPARa–RXR agonists on palmitate
metabolism
Preculture (24 h) of beta-cells with 250 lm clofibrate
plus 2 lm 9-cis-RA increased palmitate oxidation
during a subsequent 2 h incubation with 50 lm
C16:0 + L-carnitine
Palmitate oxidation (pmol/Kc/2h)
L-carnitine
vehicle
0.00
0.50
1.00
** **
1.50
0
10
20
30
40
50

500 M - 2d
% Cytotoxicity
C16:0
C16:0 +
L-carnitine
***
***
% Cytotoxicity
Fatty acid
Fatty acid + etomoxir
0
40
60
500 M - 2d
250 M - 8d
C16:0
500 M - 2d 250 M - 8d
C4:0
20
***
**
250 M - 8d
AB
C
Fig. 2. Effect of regulators of fatty acid metabolism on fatty acid
toxicity. (A) Effect of
L-carnitine on palmitate oxidation. Beta-cells
were precultured for 24 h with the CPT1 activator
L-carnitine
(1 m

M) in the absence or presence of 250 lM palmitate before
measurement of [
14
C]palmitate oxidation during a 2 h incubation in
the further presence of
L-carnitine. (B) Effect of L-carnitine on palmi-
tate cytotoxicity. Cells were exposed for 2 or 8 days to 500 or
250 l
M palmitate in the presence of L-carnitine following 24 h of
preculture with 1 m
ML-carnitine. (C) Effect of a CPT1 inhibitor on
fatty acid toxicity. Cells were exposed for 2 or 8 days to 500 or
250 l
M butyrate or palmitate alone, or in combination with 200 lM
etomoxir. Data are presented as mean ± SEM (n > 4); **P < 0.01,
***P < 0.001 (Student’s t-test).
K. Hellemans et al. Beta-cell protection against palmitate toxicity
FEBS Journal 274 (2007) 6094–6105 ª 2007 The Authors Journal compilation ª 2007 FEBS 6097
[
14
C]palmitate by 80% (P<0.001) (Table 3). This
effect was also seen when palmitate (250 lm) was
present during preculture (Table 3). The lower
14
CO
2
values measured in this condition when compared with
the control reflect isotopic dilution in palmitate-pre-
treated cells. No differences were found for the incor-
poration of label in lipids, intermediates or proteins.

Effect of PPARa–RXR agonists on gene
expression of enzymes involved in fatty acid
metabolism and peroxisomal membrane proteins
RT-PCR analysis was performed to investigate
whether the agonist combination (250 lm clofibrate
plus 2 lm 9-cis-RA) increased expression of genes cod-
ing for enzymes involved in peroxisomal or mitochon-
drial lipid metabolism or of peroxisomal membrane
proteins (Table 4). Palmitate 250 lm alone induced the
mRNA expression of CPT1 (two-fold increase over
control, P < 0.05), and decreased that of PPARa, ste-
aroyl-CoA desaturase 1 (SCD1), stearoyl-CoA desatur-
ase 2 (SCD2), and the peroxisomal membrane proteins
peroxisomal biogenesis factor 2 (Pex2) and peroxi-
somal biogenesis factor 14 (Pex14) (P<0.05). Addi-
tion of clofibrate plus 9-cis-RA further increased
mRNA levels of CPT1 (1.7-fold, P < 0.001) and
induced the expression of the mitochondrial enzymes
acyl-CoA dehydrogenase (medium chain), acyl-CoA
dehydrogenase (long chain), and mitochondrial acetyl-
CoA acetyltransferase 2 (P<0.01), as well as of the
peroxisomal enzymes peroxisomal acetyl-CoA acetyl-
transferase 1, palmitoyl-CoA oxidase 1, and prista-
noyl-CoA oxidase (P<0.01), and the prolipogenic
endoplasmatic reticulum enzymes glycerol-3-phosphate
acyltransferase, mitochondrial (GPAT), SCD1 and
SCD2 (P<0.01). This combination was also found to
increase the mRNA levels of peroxisomal biogenesis
factor 3 (Pex3), peroxisomal biogenesis factor 16
(Pex16), and peroxisomal biogenesis factor 11a

(Pex11a), as well as of Pex2, Pex14 and peroxisomal
membrane protein 70 (PMP70). Comparable changes
in expression were also found after stimulation with
clofibrate and 9-cis-RA in the absence of palmitate.
% cytotoxicity
500
0
10
20
500
0 ( M)
0.5
2
5
9-cis RA
250 M–8 days
M–2 days
0 250
0
10
20
30
40
clofibrate (
M)
100
% cytotoxicity
10
20
30

40
500
M - 2 days
C16:0
+ Clofibrate
+Ciprofibrate
+9-cisRA
+ Clofibrate / 9-cis RA
+ Ciprofib. / 9-cis RA
#
#
#
#
#
#
% cytotoxicity
#$
#$
#$
#$
250 M - 8 days
A
B
Fig. 3. Effect of PPARa and RXR agonists on palmitate toxicity. (A) Effect of clofibrate and 9-cis-RA on palmitate toxicity. Primary rat beta-
cells were exposed to 500 l
M palmitate for 2 days, or to 250 lM palmitate for 8 days, in the presence or absence of clofibrate (100, 250,
500 l
M) and ⁄ or 9-cis-RA (0.5, 2, 5 lM). (n ¼ 4–8). Vertical bars represent SEM. (B) Comparison between the protective effect of clofibrate
(250 l
M) and of ciprofibrate (100 lM) against palmitate toxicity, alone or in combination with 2 lM 9-cis-RA. Data are indicated as mean ±

SEM (n > 5).
#
P < 0.001 as compared to palmitate;
$
P < 0.001 as compared to single agonist treatment (clofibrate, ciprofibrate or 9-cis-RA)
(two-way ANOVA).
Table 3. Effect of palmitate and PPARa–RXR agonists on
[
14
C]palmitate metabolism. Beta-cells were precultured for 24 h in
the presence or absence of 250 l
M palmitate and ⁄ or 250 lM clofi-
brate (Clof) plus 2 l
M 9-cis-RA (RA). Incorporation of the
14
C label
into CO
2
, lipid intermediates, lipids and proteins was measured dur-
ing a 2 h incubation with 50 l
M [
14
C]palmitate, and expressed as a
percentage of the control condition (vehicle only, 10 m
M glucose).
*P < 0.001 as compared to control, **P < 0.001 as compared to
cytochrome P250, Student’s t-test (n ¼ 5).
Treatment
14
C-recovery as

CO
2
Intermediates Lipid Protein
C16:0 59 ± 13* 80 ± 18 101 ± 9 93 ± 12
C16:0 + Clof ⁄ RA 94 ± 12** 78 ± 10 94 ± 7 77 ± 27
Clof ⁄ RA 181 ± 27* 94 ± 27 108 ± 9 83 ± 24
Beta-cell protection against palmitate toxicity K. Hellemans et al.
6098 FEBS Journal 274 (2007) 6094–6105 ª 2007 The Authors Journal compilation ª 2007 FEBS
Effect of etomoxir on palmitate toxicity in the
presence of PPARa–RXR agonists
In the presence of etomoxir, the protective effect of
clofibrate and 9-cis-RA was abolished, and the
cytotoxicity of palmitate 500 lm for 2 days increased
four-fold, i.e. from 4 ± 3% to 17 ± 5%. (Fig. 4).
This toxicity was also increased when the cells were
precultured for 24 h with 200 lm etomoxir prior to
their incubation with the palmitate ⁄ clofibrate ⁄ RA mix-
ture for 2 days (19 ± 3%, P < 0.05 versus 5 ± 2%).
The effect of preculture with etomoxir was lost after
prolonged subsequent culture in its absence (250 lm
palmitate for 8 days).
Discussion
This study confirms that sustained exposure to palmi-
tate causes time- and dose-dependent toxicity on rat
beta-cells. Over an 8 day culture period, palmitate, at
250 or 500 lm, progressively reduced the number of
surviving cells, involving both necrotic and apoptotic
pathways [7], but a significant fraction remained resis-
tant. These survival curves show that primary beta-
cells can be less susceptible or even resistant to fatty

acid toxicity, and that this property is heterogeneously
expressed, like other cell functions [17,18]. We previ-
ously reported that addition of oleate increases the
Table 4. Effect of palmitate and PPARa–RXR agonists on mRNA expression levels. Beta-cells were exposed for 2 days to 250 lM palmitate
and ⁄ or 250 l
M clofibrate plus 2 lM 9-cis-RA or vehicle (control). qPCR values were normalized to actin and calculated as DDCt values relative
to the indicated control conditions. Unpaired student t-test, two-tailed, mean ± SD, n ¼ 4–6, *P < 0.05, **P < 0.01, ***P < 0.001.
Protein
C16:0, compared
to control
Clofibrate +9-cis-RA,
compared to control
C16:0 + clofibrate + 9-cis-RA,
compared to C16:0
Gene transcription
Peroxisome proliferator-activated receptor a 0.7 ± 0.2 1.6 ± 0.2*** 1.9 ± 0.4 ***
Mitochondrial b-oxidation
Carnitine palmitoyl transferase 1 1.9 ± 0.6** 2.6 ± 0.4*** 1.9 ± 0.2***
Acyl-CoA dehydrogenase, long chain 1.4 ± 0.7 1.3 ± 0.2 1.5 ± 0.2**
Acyl-CoA dehydrogenase, medium chain 0.8 ± 0.2 1.3 ± 0.3 1.6 ± 0.3**
Acyl-CoA dehydrogenase, short chain 1.0 ± 0.1 1.8 ± 0.6* 1.1 ± 0.2
Mitochondrial acetyl-CoA acetyltransferase 2 1.3 ± 0.4 2.5 ± 1.0** 1.6 ± 0.1***
Peroxisomal fatty acid oxidation
Peroxisomal acetyl-CoA acetyltransferase 1 0.8 ± 0.2 0.9 ± 0.5 1.6 ± 0.1***
Palmitoyl-CoA oxidase 1 1.0 ± 0.3 1.6 ± 0.2*** 1.6 ± 0.4**
Pristanoyl-CoA oxidase 1.4 ± 0.9 2.0 ± 0.8* 1.5 ± 0.2***
a-Methylacyl-CoA racemase 1.1 ± 0.2 0.9 ± 0.1 1.2 ± 0.2
Lipid synthesis
Stearoyl-CoA desaturase 1 0.5 ± 0.2*** 4.5 ± 2.5** 5.8 ± 2.7***
Stearoyl-CoA desaturase 2 0.7 ± 0.1*** 1.8 ± 0.3*** 2.6 ± 0.6***

Glycerol-3-phosphate acyltransferase 1.1 ± 0.3 1.5 ± 0.2** 1.5 ± 0.2**
Peroxisomal membrane proteins
Peroxisomal biogenesis factor 3 1.2 ± 0.5 1.2 ± 0.6 1.5 ± 0.3*
Peroxisomal biogenesis factor 16 0.9 ± 0.1 1.7 ± 0.5** 1.4 ± 0.2**
Peroxisomal biogenesis factor 11a 1.0 ± 0.2 1.3 ± 0.2* 1.6 ± 0.3**
Peroxisomal membrane protein 70 0.9 ± 0.3 1.4 ± 0.3** 2.0 ± 0.6**
Peroxisomal biogenesis factor 14 0.7 ± 0.1** 2.6 ± 1.0* 1.4 ± 0.2**
Peroxisomal biogenesis factor 2 0.7 ± 0.2* 1.4 ± 0.5 1.4 ± 0.2*
***
***
% Cytotoxicity
500 M - 2d
250
M - 8d
0
10
20
30
40
***
$
$
C16:0 + Clofibrate / 9-cis RA
C16:0 + Clof / 9RA + etomoxir
C16:0
Fig. 4. Effect of etomoxir on PPARa–RXR protection against palmi-
tate toxicity. Primary beta-cells were cultured with 500 l
M palmi-
tate for 2 days, or 250 l
M palmitate for 8 days, in the absence or

presence of 250 l
M clofibrate plus 2 lM 9-cis-RA, or in combination
with 200 l
M etomoxir. Data are presented as mean ± SEM (n ¼ 4).
***P < 0.001 as compared to palmitate;
$
P < 0.01 for etomoxir as
compared to clofibrate plus 9-cis-RA (Student’s t-test).
K. Hellemans et al. Beta-cell protection against palmitate toxicity
FEBS Journal 274 (2007) 6094–6105 ª 2007 The Authors Journal compilation ª 2007 FEBS 6099
resistance of rat beta-cells to palmitate-induced cell
death [7]. This oleate effect was also observed in
human beta-cells [19] and in other cell types [20–23]. It
appeared to be correlated with the formation of trigly-
cerides, supporting the view that fatty acid incorpora-
tion into neutral lipids prevents the accumulation of
toxic free palmitoyl acyl moieties [22], and ⁄ or ceramide
derivatives [24,25]. A role of ceramides in palmitate
toxicity is supported by the observed protection by
l-cycloserine, an inhibitor of serine palmitoyl transfer-
ase and thus of ceramide synthesis.
At variance with other cytotoxic conditions [26], no
protective effect could be attributed to glucose, as simi-
lar palmitate toxicities were measured following culture
at 5 or 10 mm glucose. The percentage of dead cells was
not increased when palmitate exposure was assessed at
excessive glucose levels (20 mm), which contrasts with
observations in beta-cell lines [27–30]. The latter dis-
crepancy might be related to differences in experimental
protocols, such as free fatty acid concentrations, free

fatty acid ⁄ BSA ratios [7], or the use of serum, but could
also result from differences between primary beta-cells
and cell lines. Glucose cytotoxicity has also been seen in
cell lines [27,31], whereas increased glucose exerts cyto-
protective effects in primary beta-cells, at least in condi-
tions that cause an oxidative shift in their metabolic
state [32–35]. Our data suggest that glucose-induced
changes in the cellular metabolic redox state do not alter
cellular susceptibility to palmitate toxicity. At a
nontoxic palmitate concentration (50 lm), glucose
suppresses its oxidation, probably due to dynamic
regulation of the malonyl-CoA ⁄ CPT1 axis [3], but this
seems not to be accompanied by a toxic effect; in fact,
this may lead to the formation of fatty acid derivatives
with physiologic action in the presence of glucose [36–
38], or in protective accumulation in the form of neutral
lipids [7,39]. Furthermore, palmitate induced the expres-
sion of CPT1 two-fold, suggesting that beta-cells have
the inherent capacity to adapt their oxidation rate to
elevated fatty acid levels, independently of the suppres-
sive effect of glucose.
Our data indicate that palmitate toxicity can be
reduced by increasing its oxidation through mitochon-
drial and ⁄ or peroxisomal pathways. Mitochondrial
oxidation of long-chain fatty acids is known to be rate
limited at the level of CPT1 [10]. Viral overexpression
of CPT1 has been shown to enhance palmitate oxida-
tion in INS-1 cells and islets [40,41]. In our study, cul-
ture with l-carnitine, an essential component of CPT1,
stimulated palmitate oxidation and reduced its toxicity,

while etomoxir, an inhibitor of CPT1, increased the
toxicity of palmitate (C16:0) but not of butyrate
(C4:0), which enters mitochondria independently of
CPT1. In fact, no toxicity was measured for any of the
tested shorter-chain fatty acids, raising the possibility
that shortening the palmitate chain represents another
and perhaps more important mechanism for inducing
cytoprotection.
It is so far unclear to what extent peroxisomes in
beta-cells contribute to fatty acid metabolism. That
they could be involved is suggested by the absence of
cytotoxicity for the 2-methyl and 3-methyl derivatives
of palmitate. Like other branched fatty acids, these
compounds are known to be preferentially transported
to the peroxisomes, where 2-methyl-C16 undergoes
b-oxidation and 3-methyl-C16 will be a-oxidized before
being transported to the mitochondria [42].
Fibrates are known to regulate genes involved in
mitochondrial, as well as peroxisomal, fatty acid oxi-
dation and to induce peroxisome proliferation and
maturation in multiple cell types [11]. Both clofibrate
and ciprofibrate were found to increase palmitate
breakdown and to reduce its toxicity; their combina-
tion with 9-cis-RA resulted in complete protection.
This protective action of 9-cis-RA against palmitate
toxicity contrasts with the proapoptotic effect observed
in MIN6 cells at a 10-fold higher concentration [43].
Clofibrate plus 9-cis-RA was found to provide the
same level of protection at all examined glucose con-
centrations. This effect correlated with induced expres-

sion of CPT1 and mitochondrial and peroxisomal
b-oxidation enzymes, and resulted in normalization of
palmitate oxidation. In further support of this, inhibi-
tion of CPT1 by etomoxir was found to abolish
the protective action of clofibrate plus 9-cis-RA.
PPARa:RXR agonists also induced expression of
GPAT, SCD1 and SCD2 mRNA, which might medi-
ate incorporation of the fatty acid into (phospho)lipids
[44]. Increased SCD1 expression has been previously
noticed in palmitate-resistant MIN6 cells but has not
yet been directly correlated with a cytoprotective diver-
sion of palmitate into lipid formation [39]; esterifica-
tion of palmitate was shown to result in accumulation
of insoluble tripalmitin and to correlate with endoplas-
mic reticulum stress and apoptosis [45,46].
The PPARa–RXR agonists were also found to act at
a third level of potential relevance, namely the expres-
sion of proteins involved in peroxisome biogenesis
(Pex3 and Pex16), proliferation (Pex11a) and matura-
tion (PMP70, Pex2 and Pex14) [47]. By inducing the
peroxisomal compartment, they might indeed increase
the channeling of palmitate through the first cycles of
chain shortening before further breakdown in mito-
chondria. This view is consistent with the virtual
absence of toxicities for short-chain fatty acids. Peroxi-
somes might thus represent a key site for reducing the
Beta-cell protection against palmitate toxicity K. Hellemans et al.
6100 FEBS Journal 274 (2007) 6094–6105 ª 2007 The Authors Journal compilation ª 2007 FEBS
toxicity of palmitate in primary beta-cells. Their activ-
ity might be low in normal circumstances, as judged by

the low catalase activity in islet beta-cells [48,49]. Addi-
tion of clofibrate was shown to increase catalase activ-
ity in INS-1 cells together with palmitate oxidation
[50]. Clofibrate plus 9-cis-RA did not provide protec-
tion in the nonbeta-cells where, independently of glu-
cose, a substantially higher proportion of palmitate was
converted to CO
2
.
Our data supplement previous in vivo findings or
observations in cell lines, and more specifically indicate
to what extent they reflect effects on the survival of pri-
mary beta-cells and, if so, through which mechanism
these can then be explained. Although fatty acids, and
particularly palmitate, are classically seen as mediators
of lipotoxicity at the level of the beta-cells, it is often
not clear whether the reported derangements are the
result of beta-cell death and ⁄ or dysfunction. Conse-
quently, the protective action of PPARa agonists with
or without RXR agonists was not always well specified
in these terms [51]. Adenoviral coexpression of PPARa
and RXRa synergistically – and in a dose- and ligand-
dependent manner ) potentiated glucose-stimulated
insulin secretion from INS-1E cells while increasing
their expression of genes involved in free fatty acid
uptake and b-oxidation [52,53]. An increase of PPARa-
driven b-oxidation in response to topiramate was also
found to protect INS-1E cells from oleate toxicity [54].
When administered in vivo , PPAR–RXR ligands
induced expression of b-oxidation enzymes and stimu-

lated palmitate oxidation in isolated islets [13]. Fibrate
treatment restored the coupling between insulin secre-
tion and action in glucose-intolerant rats on a high-fat
diet [55] and prevented diabetes in obese OLETF rats
[56,57]. Combination therapy with PPARa and a-agon-
ists, or dual agonists, ameliorated insulin secretion and
increased insulin stores in genetically obese diabetic
db ⁄ db mice [58]. The present work has shown that
PPARa–RXR agonists can protect primary beta-cells
against the cytodestructive effects of palmitate. It has
provided evidence that this protection is achieved by
stimulating mitochondrial and peroxisomal pathways
for palmitate breakdown. Further work is needed to
assess the functional properties of these protected
beta-cells and to evaluate the influence of the agonists
at nontoxic palmitate concentrations.
Experimental procedures
Materials
Palmitate, oleate, butyrate, hexanoate, octanoate (sodium
salts), 2-bromohexadecanoic acid, clofibrate, ciprofibrate,
9-cis-RA, l-carnitine and l-cycloserine were purchased
from Sigma-Aldrich (Bornem, Belgium). Branched 2-meth-
ylhexadecanoic acid and 3-methylhexadecanoic acid were
prepared as described previously [59,60]. Stock solutions of
fatty acids (25 mm,50mm) were made in 90% ethanol by
heating to 60 °C, except for 2-bromopalmitate, which was
dissolved at room temperature. Stock solutions of clofibrate
(200 mm), ciprofibrate (20 mm) and 9-cis-RA (10 mm) were
dissolved in absolute ethanol. Etomoxir was a gift from
V. Grill (Trondheim University, Norway) and dissolved in

saline. d-[U-
14
C]glucose (287–311 mCiÆmmol
)1
; 1 mCi per
5 mL) was purchased from Amersham Biosciences (Roose-
ndaal, Belgium), and [U-
14
C]palmitic acid (850 mCiÆ
mmol
)1
; 0.1 mCiÆ mL
)1
) from Perkin Elmer Life Sciences
(Zaventem, Belgium).
Preparation and culture of rat beta-cells
Adult male Wistar rats were bred according to Belgian reg-
ulations on animal welfare. Experiments were carried out in
accordance with the European Communities Council Direc-
tive (86/609/EEC). Pancreatic islets were isolated, dissoci-
ated and purified into single beta-cells (purity 88 ± 4%
insulin-positive cells) and endocrine nonbeta-cells
(70 ± 11% alpha-cells, 23 ± 3% beta-cells) by autofluores-
cence-activated cell sorting [61]. For studies on cytoxicity,
isolated cells were cultured in polylysine-coated microtiter
plates (2500–3000 cells per well) with Ham’s F10 medium
containing 10 mm glucose (unless stated otherwise), 1%
charcoal-extracted BSA (fraction V, radioimmunoassay
grade; Sigma-Aldrich), 2 mml-glutamine, 50 mm 3-iso-
butyl-1-methylxanthine, 0.075 mgÆmL

)1
penicillin and
0.1 mgÆmL
)1
streptomycin [7]. Test reagents were added to
the culture medium, with control conditions receiving simi-
lar dilutions of solvent. After 2 and 5 days of culture, the
medium was changed and fresh reagents were added. Per-
centages of living and dead cells were determined by vital
staining using neutral red [7]. For metabolic and gene
expression studies, freshly isolated cells were reaggregated
and cultured in suspension as previously described [62].
Measurement of glucose and palmitate
metabolism
Duplicate samples of 5 · 10
4
rat beta-cells were incubated
for 2 h at 37 °C using Ham’s F10 medium containing 0.5%
BSA, 2 mml-glutamine and 10 mm Hepes for measuring
glucose metabolism (5 lCi of d-[U-
14
C]glucose with
different concentrations of unlabeled d-glucose) [63]. Palmi-
tate metabolism was measured using KRBH medium,
containing 0.2% BSA (fraction V), 2 mm calcium chloride,
and 10 mm Hepes (0.5 lCi of [U-
14
C]palmitic acid, with
unlabeled palmitate up to 50 lm in order to achieve the
same ratio of free fatty acid over BSA as in the cytotoxicity

experiments with 250 lm palmitate). The rate of
K. Hellemans et al. Beta-cell protection against palmitate toxicity
FEBS Journal 274 (2007) 6094–6105 ª 2007 The Authors Journal compilation ª 2007 FEBS 6101
d-[U-
14
C]glucose or [U-
14
C]palmitic acid oxidation was
assessed through the formation of
14
CO
2
[63]. Cells were
incubated in a siliconized tube trapped in an airtight glass
vial. After 2 h, 20 lLof1m HCl was injected, and 250 lL
of Hyamine (Packard Bioscience, Groningen, the Nether-
lands) added to capture
14
CO
2
for 1 h at room temperature.
The
14
C incorporation into lipids, proteins and metabolic
intermediates was measured as previously described [64].
Gene expression analysis
Total beta-cell RNA was extracted with TRIzol Reagent
(Gibco BRL, Carlsbad, CA, USA) and its quality was
assessed on a 2100 Bioanalyzer (Agilent, Waldbronn, Ger-
many), taking a minimal cutoff RNA integrity number of

8. RNA clean-up was performed with the Turbo DNA Free
Kit (Ambion, Austin, TX, USA) and cDNA prepared with
the High-Capacity cDNA Archive Kit (Applied Biosystems,
Foster City, CA, USA). Real-time PCR was performed
using an ABI Prism Sequence Detector (Applied Biosys-
tems). Primers were obtained from Applied Biosystems
(Table 4). For each RT-PCR reaction, the cycle threshold
(Ct) was determined with sds 1.9.1 software. DDCt values
were calculated versus b-actin. Fold changes were calcu-
lated starting from DDCt values of a minimum of four
independent experiments performed in duplicate.
Data analysis
Data are presented as mean ± SEM, or as mean ± SD
of n independent experiments. Statistical analysis was
performed using Student’s t-test, unless stated otherwise.
Differences were considered significant for P < 0.05.
Acknowledgements
This work was supported by the Research Foundation
Flanders (Fonds Voor Wetenschappelijk Onderzoek-
Vlaanderen, Grant FWO-G.0357.03, Grant
FWO-1.5.195.05 and PhD grant FWOTM277 to K.
Kerckhofs) and by the Inter-University Poles of
Attraction Program (IUAP P5 ⁄ 17) from the Belgian
Science Policy. The Diabetes Research Center is a
partner of the Juvenile Diabetes Research Center for
Beta Cell Therapy in Diabetes.
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