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
Trang 1Peroxisome proliferator-activated receptor a–retinoid X receptor agonists induce beta-cell protection against
palmitate toxicity
Karine Hellemans1, Karen Kerckhofs1, Jean-Claude Hannaert1, Geert Martens1,
Paul Van Veldhoven2and Daniel Pipeleers1
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 vivostudies 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: karine.hellemans@vub.ac.be
(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 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.
Trang 2preventing 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 toxictoxic-ity)
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
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 l M for 8 days
Trang 32-bromopalmitate was markedly more toxic than
palm-itate There was no difference in the percentage of
liv-ing cells followliv-ing 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 [14C]palmitate or
5 lCi of d-[U-14C]glucose.14C incorporation was
mea-sured in CO2and small metabolic intermediates, as well
as in the lipid and protein fractions (Table 2) After
incubation with [14C]palmitate at 3.3 mm glucose, the
largest amount was recovered in the lipid-soluble
frac-tion (55%) and the lowest in the protein fracfrac-tion,
whereas comparable amounts were converted to CO2
(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 14CO2 and of 14C intermediates was,
respectively, five- and 10-fold lower (Table 2) Analysis
of the fate of [14C]glucose indicated that, at 3.3 mm
glucose, the tracer was converted to CO2 and to small
intermediates, and that high glucose increased this rate
five-fold, and also increased seven-fold the14C
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 CO2 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 CO2production 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 CO2 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, 14CO2 formation was six-fold higher (to 0.83 ± 0.14 pmol per 2 h per
103cells, n¼ 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 103cells,
n¼ 4, P < 0.01) (Fig 2A) It was associated with
an eight-fold elevation of 14C incorporation into metabolic intermediates (from 0.15 ± 0.05 to 0.95 ± 0.07 pmol per 2 h per 103cells, P < 0.05) (results not shown) Preculture with 1 mm l-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 m M glucose Palmitate and glucose metabolism was measured in freshly isolated beta-cells and islet nonbeta-cells incubated for 2 h with 50 l M [14C]palmitate or 5 lCi of
D -[U- 14 C]glucose 14 C incorporation was measured in CO2and 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 m M glucose as compared to 3.3 m M 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:
From [ 14 C]palmitate
Beta-cells
Nonbeta-cells
From [ 14 C]glucose
Beta-cells
Nonbeta-cells
Trang 4subsequent 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
vehicle
0.00
0.50
1.50
0 10 20 30 40 50
C16:0
***
***
Fatty acid
Fatty acid + etomoxir
0
40
60
C16:0
C4:0 20
***
**
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 l M 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 M L -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 l M
etomoxir Data are presented as mean ± SEM (n > 4); **P < 0.01,
***P < 0.001 (Student’s t-test).
Trang 5[14C]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 14CO2
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), steste-aroyl-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 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
500
0 10 20
500
0.5 2 5 9-cis RA
0 10 20 30 40
100
10 20 30 40
C16:0 + Clofibrate + Ciprofibrate + 9-cis RA + Clofibrate / 9-cis RA + Ciprofib / 9-cis RA
#
#
#
#
#
#
# $
# $
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 l M 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 l M ) (n ¼ 4–8) Vertical bars represent SEM (B) Comparison between the protective effect of clofibrate (250 l M ) and of ciprofibrate (100 l M ) against palmitate toxicity, alone or in combination with 2 l M 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 l M
clofi-brate (Clof) plus 2 l M 9-cis-RA (RA) Incorporation of the 14 C label
into CO2, lipid intermediates, lipids and proteins was measured
dur-ing a 2 h incubation with 50 l M [14C]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
CO2 Intermediates Lipid Protein
C16:0 + Clof ⁄ RA 94 ± 12** 78 ± 10 94 ± 7 77 ± 27
Trang 6Effect 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 l M palmitate and ⁄ or 250 l M clofibrate plus 2 l M 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
Mitochondrial b-oxidation
Peroxisomal fatty acid oxidation
Lipid synthesis
Peroxisomal membrane proteins
***
***
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 l M 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).
Trang 7resistance 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 accumulaincorpora-tion 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 proteccombina-tion 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 accumulaesterifica-tion
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
Trang 8toxicity 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 CO2
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 isostimu-lated islets [13] Fibrate
treatment restored the coupling between insulin
secre-tion and acsecre-tion 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 agona-agon-ists, 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-meth2-meth-ylhexadecanoic acid were prepared as described previously [59,60] Stock solutions of fatty acids (25 mm, 50 mm) were made in 90% ethanol by heating to 60C, 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-14C]glucose (287–311 mCiÆmmol)1; 1 mCi per
5 mL) was purchased from Amersham Biosciences (Roose-ndaal, Belgium), and [U-14C]palmitic acid (850 mCiÆ mmol)1; 0.1 mCiÆmL)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 mm l-glutamine, 50 mm 3-iso-butyl-1-methylxanthine, 0.075 mgÆmL)1 penicillin and 0.1 mgÆmL)1streptomycin [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· 104rat beta-cells were incubated for 2 h at 37C using Ham’s F10 medium containing 0.5% BSA, 2 mm l-glutamine and 10 mm Hepes for measuring glucose metabolism (5 lCi of d-[U-14C]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-14C]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
Trang 9d-[U-14C]glucose or [U-14C]palmitic acid oxidation was
assessed through the formation of 14CO2 [63] Cells were
incubated in a siliconized tube trapped in an airtight glass
vial After 2 h, 20 lL of 1 m HCl was injected, and 250 lL
of Hyamine (Packard Bioscience, Groningen, the
Nether-lands) added to capture14CO2for 1 h at room temperature
The 14C 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
References
1 Haber EP, Procopio J, Carvalho CR, Carpinelli AR,
Newsholme P & Curi R (2006) New insights into fatty
acid modulation of pancreatic beta-cell function Int
Rev Cytol 248, 1–41
2 Prentki M & Nolan CJ (2006) Islet beta cell failure in
type 2 diabetes J Clin Invest 116, 1802–1812
3 Prentki M, Joly E, El Assaad W & Roduit R (2002) Malonyl-CoA signaling, lipid partitioning, and gluco-lipotoxicity: role in beta-cell adaptation and failure in the etiology of diabetes Diabetes 51 (Suppl 3), S405– S413
4 Unger RH & Zhou YT (2001) Lipotoxicity of beta-cells
in obesity and in other causes of fatty acid spillover Diabetes 50 (Suppl 1), S118–S121
5 Robertson RP, Harmon J, Tran PO & Poitout V (2004) Beta-cell glucose toxicity, lipotoxicity, and chronic oxi-dative stress in type 2 diabetes Diabetes 53 (Suppl 1), S119–S124
6 Martens G, Cai Y, Hinke S, Stange G, Van De CM & Pipeleers D (2005) Nutrient sensing in pancreatic beta cells suppresses mitochondrial superoxide generation and its contribution to apoptosis Biochem Soc Trans
33, 300–301
7 Cnop M, Hannaert JC, Hoorens A, Eizirik DL & Pipeleers DG (2001) Inverse relationship between cyto-toxicity of free fatty acids in pancreatic islet cells and cellular triglyceride accumulation Diabetes 50, 1771– 1777
8 Kharroubi I, Ladriere L, Cardozo AK, Dogusan Z, Cnop M & Eizirik DL (2004) Free fatty acids and cyto-kines induce pancreatic beta-cell apoptosis by different mechanisms: role of nuclear factor-kappaB and endo-plasmic reticulum stress Endocrinology 145, 5087–5096
9 Kelpe CL, Moore PC, Parazzoli SD, Wicksteed B, Rhodes CJ & Poitout V (2003) Palmitate inhibition of insulin gene expression is mediated at the transcriptional level via ceramide synthesis J Biol Chem 278, 30015– 30021
10 Kerner J & Hoppel C (2000) Fatty acid import into mitochondria Biochim Biophys Acta 1486, 1–17
11 Desvergne B & Wahli W (1999) Peroxisome prolifera-tor-activated receptors: nuclear control of metabolism Endocr Rev 20, 649–688
12 Schuit F, Flamez D, De Vos A & Pipeleers D (2002) Glucose-regulated gene expression maintaining the glu-cose-responsive state of beta-cells Diabetes 51 (Suppl 3), S326–S332
13 Zhou YT, Shimabukuro M, Wang MY, Lee Y, Higa
M, Milburn JL, Newgard CB & Unger RH (1998) Role
of peroxisome proliferator-activated receptor alpha in disease of pancreatic beta cells Proc Natl Acad Sci USA 95, 8898–8903
14 Gremlich S, Nolan C, Roduit R, Burcelin R, Peyot ML, Delghingaro-Augusto V, Desvergne B, Michalik L, Prentki M & Wahli W (2005) Pancreatic islet adaptation
to fasting is dependent on peroxisome proliferator-acti-vated receptor alpha transcriptional up-regulation of fatty acid oxidation Endocrinology 146, 375–382
15 Lalloyer F, Vandewalle B, Percevault F, Torpier G, Kerr-Conte J, Oosterveer M, Paumelle R, Fruchart JC, Kuipers F, Pattou F et al (2006) Peroxisome
Trang 10prolifera-tor-activated receptor {alpha} improves pancreatic
adaptation to insulin resistance in obese mice and
reduces lipotoxicity in human islets Diabetes 55, 1605–
1613
16 Koh EH, Kim MS, Park JY, Kim HS, Youn JY, Park
HS, Youn JH & Lee KU (2003) Peroxisome
prolifera-tor-activated receptor (PPAR)-alpha activation prevents
diabetes in OLETF rats: comparison with
PPAR-gamma activation Diabetes 52, 2331–2337
17 Pipeleers D, Kiekens R, Ling Z, Wilikens A & Schuit F
(1994) Physiologic relevance of heterogeneity in the
pan-creatic beta-cell population Diabetologia 37 (Suppl 2),
S57–S64
18 Ling Z, Wang Q, Stange G, In’t Veld P & Pipeleers D
(2006) Glibenclamide treatment recruits beta-cell
sub-population into elevated and sustained basal insulin
synthetic activity Diabetes 55, 78–85
19 Maedler K, Oberholzer J, Bucher P, Spinas G &
Do-nath MY (2003) Monounsaturated fatty acids prevent
the deleterious effects of palmitate and high glucose on
human beta-cell turnover and function Diabetes 52,
726–733
20 Welters HJ, Tadayyon M, Scarpello JH, Smith SA &
Morgan NG (2004) Mono-unsaturated fatty acids
pro-tect against beta-cell apoptosis induced by saturated
fatty acids, serum withdrawal or cytokine exposure
FEBS Lett 560, 103–108
21 Eitel K, Staiger H, Brendel MD, Brandhorst D,
Bretzel RG, Haring HU & Kellerer M (2002) Different
role of saturated and unsaturated fatty acids in beta-cell
apoptosis Biochem Biophys Res Commun 299, 853–
856
22 Listenberger LL, Han X, Lewis SE, Cases S, Farese RV
Jr, Ory DS & Schaffer JE (2003) Triglyceride
accumula-tion protects against fatty acid-induced lipotoxicity
Proc Natl Acad Sci USA 100, 3077–3082
23 Beeharry N, Chambers JA & Green IC (2004) Fatty
acid protection from palmitic acid-induced apoptosis is
lost following PI3-kinase inhibition Apoptosis 9, 599–
607
24 Shimabukuro M, Higa M, Zhou YT, Wang MY,
New-gard CB & Unger RH (1998) Lipoapoptosis in beta-cells
of obese prediabetic fa⁄ fa rats Role of serine
palmitoyl-transferase overexpression J Biol Chem 273, 32487–
32490
25 Lupi R, Dotta F, Marselli L, Del Guerra S, Masini M,
Santangelo C, Patane G, Boggi U, Piro S, Anello M
et al.(2002) Prolonged exposure to free fatty acids has
cytostatic and pro-apoptotic effects on human
pancre-atic islets: evidence that beta-cell death is caspase
medi-ated, partially dependent on ceramide pathway, and
Bcl-2 regulated Diabetes 51, 1437–1442
26 Pipeleers D & Van De Winkel M (1986) Pancreatic B
cells possess defense mechanisms against cell-specific
toxicity Proc Natl Acad Sci USA 83, 5267–5271
27 El Assaad W, Buteau J, Peyot ML, Nolan C, Roduit R, Hardy S, Joly E, Dbaibo G, Rosenberg L & Prentki M (2003) Saturated fatty acids synergize with elevated glucose to cause pancreatic beta-cell death Endocrinol-ogy 144, 4154–4163
28 Maestre I, Jordan J, Calvo S, Reig JA, Cena V, Soria
B, Prentki M & Roche E (2003) Mitochondrial dysfunc-tion is involved in apoptosis induced by serum with-drawal and fatty acids in the beta-cell line INS-1 Endocrinology 144, 335–345
29 Briaud I, Harmon JS, Kelpe CL, Segu VB & Poitout V (2001) Lipotoxicity of the pancreatic beta-cell is associ-ated with glucose-dependent esterification of fatty acids into neutral lipids Diabetes 50, 315–321
30 Zhou YP & Grill VE (1994) Long-term exposure of rat pancreatic islets to fatty acids inhibits glucose-induced insulin secretion and biosynthesis through a glucose fatty acid cycle J Clin Invest 93, 870–876
31 Buteau J, El Assaad W, Rhodes CJ, Rosenberg L, Joly
E & Prentki M (2004) Glucagon-like peptide-1 prevents beta cell glucolipotoxicity Diabetologia 47, 806–815
32 Ling Z, Kiekens R, Mahler T, Schuit FC, Pipeleers-Marichal M, Sener A, Kloppel G, Malaisse WJ & Pipeleers DG (1996) Effects of chronically elevated glucose levels on the functional properties of rat pancre-atic beta-cells Diabetes 45, 1774–1782
33 Hoorens A, Van De Casteele M, Kloppel G & Pipeleers
D (1996) Glucose promotes survival of rat pancreatic beta cells by activating synthesis of proteins which sup-press a constitutive apoptotic program J Clin Invest 98, 1568–1574
34 Khaldi MZ, Guiot Y, Gilon P, Henquin JC & Jonas JC (2004) Increased glucose sensitivity of both triggering and amplifying pathways of insulin secretion in rat islets cultured for 1 wk in high glucose Am J Physiol Endo-crinol Metab 287, E207–E217
35 Martens GA, Cai Y, Hinke S, Stange G, Van De Caste-ele M & PipCaste-eleers D (2005) Glucose suppresses super-oxide generation in metabolically responsive pancreatic beta cells J Biol Chem 280, 20389–20396
36 Nolan CJ, Madiraju MS, Delghingaro-Augusto V, Peyot ML & Prentki M (2006) Fatty acid signaling in the {beta}-cell and insulin secretion Diabetes 55 (Suppl 2), S16–S23
37 Yaney GC & Corkey BE (2003) Fatty acid metabolism and insulin secretion in pancreatic beta cells Diabetolo-gia 46, 1297–1312
38 Warnotte C, Nenquin M & Henquin JC (1999) Unbound rather than total concentration and saturation rather than unsaturation determine the potency of fatty acids on insulin secretion Mol Cell Endocrinol 153, 147–153
39 Busch AK, Gurisik E, Cordery DV, Sudlow M, Denyer
GS, Laybutt DR, Hughes WE & Biden TJ (2005) Increased fatty acid desaturation and enhanced