From the INSERM U449, Faculté de Médecine René Laennec, Université Claude Bernard Lyon-1, and § CRNHL Faculté de Médecine René Laennec, Université Claude Bernard Lyon-1, 69372 Lyon, France
Received for publication, September 1, 2000, and in revised form, November 29, 2000
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ABSTRACT |
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Fatty acids have been postulated to
regulate uncoupling protein (UCP) gene expression in skeletal muscle
in vivo. We have identified, at least in part, the
mechanism by which polyunsaturated fatty acids increase UCP-2
expression in primary culture of human muscle cells. Uncoupling protein-2
(UCP-2)1 and uncoupling
protein-3 (UCP-3) are members of the mitochondrial carrier family that
show sequence identity with UCP-1 (1-6), the brown adipose
tissue-specific uncoupling protein involved in the control of
cold-induced nonshivering thermogenesis (7, 8). UCP-1 mediates the
dissipation of the mitochondrial proton gradient generated by the
respiratory chain resulting in the production of heat instead of ATP
(7, 8). Because there is little brown fat in adult, UCP-1 activity may
not contribute to a large extent to energy expenditure in humans. UCP-2
mRNA is present in many tissues including adipose tissue and
skeletal muscle (1, 2), whereas UCP-3 mRNA is preferentially
expressed in skeletal muscle (3, 4). Biochemical studies have
demonstrated that UCP-2 and UCP-3, like UCP-1, are located in the inner
mitochondrial membrane and have uncoupling activity (5, 6). Therefore,
they are candidates to explain the mitochondrial proton leak in tissues
devoid of UCP-1. To date, the proposed functions for UCP-2 and UCP-3
include the control of different components of energy expenditure, the
control of reactive oxygen species (ROS) production generated by the
mitochondrial electron transport chain, the regulation of ATP
synthesis, and the regulation of fatty acid oxidation (5, 6).
Several lines of evidence implicate fatty acids in the regulation of
ucp-2 and ucp-3 gene expression. For example,
high fat diet for 2 weeks produces a marked increase in UCP-2 mRNA
expression in white adipose tissue in mice (9). The induction of
ucp-3 gene expression in mouse muscle just after birth
correlates with the increase in plasma levels of NEFAs due to the
initiation of suckling (10). In rats, an increase in plasma NEFA level
induced by intralipid plus heparin infusion causes a rise in skeletal muscle UCP-3 mRNA level (11). In humans also, data support a role
for fatty acids in the regulation of ucp-2 and
ucp-3 gene expression in vivo. A positive
correlation was reported between plasma NEFA levels and total UCP-3
mRNA levels in skeletal muscle of obese subjects (12). We have
previously shown that UCP-2 and UCP-3 mRNA expression is
up-regulated during severe calorie restriction (13, 14), a condition
associated with increased adipose tissue lipolysis and plasma
nonesterified fatty acid (NEFA) concentrations. In addition, we have
observed positive relationships between variations in plasma NEFA
levels during calorie restriction and changes in UCP-3 mRNA
abundance in the muscle of obese nondiabetic and type 2 diabetic
patients (15). Finally, we have demonstrated that the rise in NEFA
plasma levels and in lipid oxidation during triglyceride infusion is
associated with increased expression of UCP-3 in skeletal muscle of
healthy lean subjects (16).
Little data are available regarding the nature of the fatty acids
involved in the regulation of UCP genes expression in muscle and in
adipose tissue, and thus their mechanisms of action are not yet clearly
defined. So far, in vitro studies have mainly tested the
role of the peroxisome proliferator-activated receptors (PPARs) because
these nuclear receptors have been proposed to mediate the
transcriptional effect of polyunsaturated fatty acids (PUFAs) or their
derivatives on a number of lipid-related genes (17, 18). In white
adipocytes, PPAR Additional pathways, distinct from PPAR activation, participate in the
regulation of gene expression by fatty acids (25). Fatty acids or their
derivatives (acyl-coenzyme A, oxidized fatty acids) may directly bind
to and modify the transcriptional activity of other transcription
factors (25). In addition, eicosanoids can bind cell surface receptors
that are linked to G proteins and then activate several signaling
cascades (MAP kinase, protein kinase A, protein kinase C) (25). The
concomitant participation of multiple pathways in the transcriptional
action of fatty acids has been recently illustrated in a study of the
regulation of Glut 4 gene expression by arachidonic acid (26).
Therefore, in the present work we attempted to identify the nature and
the mechanism of action of the fatty acids that may be involved in the
previously observed induction of UCP expression in human skeletal
muscle in vivo (14, 15, 16). To this end, we used a model of
human differentiated myotubes in primary culture (27). We found that
Materials--
Culture media were from Life Technologies, Inc.,
or from Biomedia (Boussens, France). Fetal calf serum was purchased
from Biomedia. All fatty acids and prostaglandins were obtained from Sigma and Biomol Research Laboratories (Plymouth Meeting, PA). Indomethacin, nordihydroguaiaretic acid (NDGA), and forskolin were
purchased from Sigma. H-89 (protein kinase A inhibitor) and PD-98059
(MAP kinase inhibitor) were from Calbiochem. Wy-14643 was obtained from
Biomol Research Laboratories. Rosiglitazone was kindly provided by
SmithKline Beecham (Harlow, UK). L-165041 was a gift from Drs. J. Berger and D. Moller from Merck.
Primary Culture of Human Skeletal Muscle Cells--
Biopsies of
the lumbar mass (erector spinae) muscle (about 1 g) were taken,
with the consent of the patient, during surgical procedure. The Ethics
Committee of Lyon Hospitals approved the experimental protocol. In the
present study, biopsies were taken from healthy lean subjects (49 ± 5 years) with no familial or personal history of diabetes,
dyslipidemia, or hypertension. The satellite cells were isolated from
the muscle biopsy by trypsin digestion and were grown in Ham's F-10
medium supplemented with 20% fetal calf serum, 1% chicken embryo
extract, and 1% antibiotics (100 units/ml penicillin and 100 µg/ml
streptomycin) as described previously in detail (27). Confluent
myoblast were allowed to differentiate into myotubes in Cell Treatments--
Stock solutions (50 mM) of
fatty acid were prepared in absolute ethanol (EtOH) and stored at
Quantification of Target mRNAs--
At the end of the
incubation periods, cells were scraped in the presence of 350 µl of
the lysis buffer from the RNeasy kit for total RNA preparation (Qiagen,
Courtaboeuf, France). Total RNA was purified following the instructions
of the manufacturer, resuspended in 40 µl of RNase-free water, and
stored at
The mRNA levels UCP-2, UCP-3, and the different isoforms of PPARs
were determined using the RT-competitive PCR (RT-cPCR) assays that were
described previously and validated (14, 28). The RT reactions were
performed with 0.2 µg of total RNA in the presence of a specific
antisense primer and a thermostable reverse transcriptase enzyme to
warrant optimal synthesis of first strand cDNA (29). Cy-5
5'-end-labeled sense primers were used during the cPCR to generate
fluorescent PCR products that were analyzed using an automated laser
fluorescence DNA sequencer (ALFexpress, Amersham Pharmacia Biotech) in
4% denaturating polyacrylamide gels. The sequences of the primers were
identical to those reported previously (14, 28). The initial
concentration of the target mRNA was determined at the competition
equivalence point, as described in detail (29).
Determination of Prostaglandin Production--
Myotubes were
incubated with 100 µM arachidonic acid under the
conditions described above. Incubation medium (3 ml) was collected from
the well and filtrated to remove cells and cellular debris using
0.45-µm Minisart filter units (Sartorius AG, Goettingen, Germany).
The clarified medium was kept at 4 °C in the presence of 40 µl of
butylated hydroxytoluene as antioxidant. Extraction of the lipid
fraction was performed with chloroform/methanol (2:1 v/v) (4.5 ml at pH
3). After centrifugation, the organic phase was removed and dried under
nitrogen. Prostaglandins E2, F2 Determination of Cyclic AMP Production--
The production of
cAMP was estimated according to the procedure described by Kasagi
et al. (31), by measuring the amount of cAMP released from
the cultured myotubes into 1.5 ml of medium (Hanks' solution without
NaCl supplemented with 220 mM sucrose and 1 mM
of 3-isobutyl-1-methylxanthine). Cyclic AMP was directly quantified in
5 µl of medium using the Biotrack cAMP 125I assay system
from Amersham Pharmacia Biotech.
Determination of Phosphorylated ERK-1 and ERK-2 by Western
Blotting--
After incubation, differentiated myotubes were washed
twice with ice-cold phosphate-buffered saline buffer and then lysed in
350 µl of ice-cold lysis buffer (150 mM NaCl,
20mM NaH2PO4, 1% Triton
X-100, 10 mM EDTA, 20 mM 1% glycerol, 50 mM HEPES, pH 7.4) containing 200 mM NaF and 4 mM NaVO4. After 1 h on ice, cells were
scraped and the lysates were cleared of nuclei and detergent-insoluble materials by centrifugation (14,000 rpm) for 10 min at 4 °C.
Proteins (35 µg) were resolved by electrophoresis through 9%
SDS-polyacrylamide gel and electrophoretically transferred to
polyvinylidine difluoride membrane. Phosphorylated MAP kinase (ERK-1
and ERK-2) were detected using a human anti-phospho-MAP kinase antibody
(Upstate Biotechnology Inc.) as described by Sarbassov et
al. (32). The phosphorylated MAP kinases were visualized using the
Enzyme-catalyzed Fluorescence Substrate for Western blotting from
Amersham Pharmacia Biotech and quantified using a FluorImager SI and
ImageQuant software (Molecular Dynamics, Sunnyvale, CA).
Expression of UCP-2 and UCP-3 in Human Myotubes and Effects of
Fatty Acids--
UCP-2 and UCP-3 mRNA levels was determined by
RT-competitive PCR in human skeletal muscle cells after an overnight
incubation in the absence of serum in the culture medium. Fig.
1 shows the comparison of the results
obtained in myotubes with UCP-2 and UCP-3 mRNA levels measured in
human skeletal muscle biopsies. UCP-2 mRNA concentration was
approximately two times lower in cultured cells than in muscle
biopsies. However, the expression level was readily measurable, and the
values were within the lower range of the individual data in muscle
biopsies (ranging from 1.6 to 7 amol/µg total RNA) (14). In contrast,
UCP-3 mRNA levels were dramatically reduced in cultured myotubes
when compared with muscle biopsies (0.05 ± 0.08 versus
12 ± 2 amol/µg total RNA). The measured values were close to
the detection limit of the RT-cPCR methodology when using 0.2 µg of
total RNA in the reverse transcription reaction (29). Extremely low
level of expression of UCP-3 is a classical feature of skeletal muscle
cells in culture. Hwang and Lane (22) have indicated that detection of
UCP-3 mRNA in mice C2C12 cells required the use of 5 µg of
poly(A)+ mRNA. Under such conditions, they have
obtained a signal, using Northern blot, which was about 104
less important than the glyceraldehyde-3-phosphate dehydrogenase signal
(22). In the same study, in contrast, UCP-3 mRNA was readily
measurable using total RNA preparations from mice skeletal muscle (22).
The reasons for the down-regulation of ucp-3 gene expression
in cultured muscle cells are unclear but may be related to the lack of
signals arising from the innervation. A recent report demonstrates
indeed that muscle denervation in mice led to a marked decrease in
UCP-3 mRNA levels in gastrocnemius muscle (33).
To study the effects of fatty acids on UCP expression, 100 µM of C-18 and C-20 fatty acids that were representative
of the different families (saturated,
Fig. 2 shows the effects of various fatty
acids on UCP-2 mRNA expression. Stearic acid (C18:0) reduced UCP-2
mRNA levels. In contrast, UCP-2 mRNA levels were significantly
increased by Mechanism of Action of
To identify which prostanoids were involved in the induction of
ucp-2 gene expression, we verified whether the main
prostaglandins derived from arachidonic acid elicited the same effect
when added directly into the culture medium. Because PGI2
(prostacyclin) is known to be highly labile, we used its
nonmetabolizable analog cPGI2 (carbaprostacyclin). Fig.
4 (upper panel) shows that
both PGE2 and cPGI2 induced a robust increase
in UCP-2 mRNA levels after 24 h of incubation. In contrast,
neither PGD2 nor PGF2
To our knowledge, the nature of the major prostaglandins synthesized
from arachidonic acid in human skeletal muscle cells is not known. In
chicken and rat muscle cells in culture, it has been reported that
PGE2 and PGF2 Signal Transduction Pathways Involved in the Effect of
Prostaglandins on ucp-2 Gene Expression--
Several high affinity
specific receptors for prostaglandins have been described in various
cell models (39). In human myotubes, the induction of UCP-2 mRNA
expression by PGE2 and cPGI2 was dependent upon
the concentrations of prostaglandins, with half-maximal effect occurring with concentrations of about 5 nM for
PGE2 and about 10 nM for cPGI2
(data not shown, n = 3). PGE2 and
PGI2 are known to stimulate adenylate cyclase through
activation of G protein-coupled membrane receptors (39). Fig.
5 (upper panel) shows that
both PGE2 and cPGI2 (1 µM)
produced a time-dependent increase in cAMP levels in human
myotubes. In addition, incubation of human myotubes with forskolin, a
potent inducer of adenylate cyclase activity, produced a significant
increase in UCP-2 mRNA (Fig. 5, lower panel). Carbaprostacyclin (cPGI2) induced a more important increase
in UCP-2 mRNA concentration than PGE2 did
(p < 0.05). This could be related to the more
important production of cAMP in the presence of cPGI2 (Fig.
5, upper panel). All these results suggest thus that the
production of cAMP and the activation of the protein kinase
A-dependent pathway are likely to be involved in the effect of prostaglandins and arachidonic acid on ucp-2 gene
expression. To confirm this hypothesis further, we tested the effect of
H-89, a specific inhibitor of protein kinase A (40). Fig.
6 clearly shows that addition of H-89 (10 µM) completely suppressed the effect of PGE2.
In contrast, both cPGI2 and arachidonic acid were still
able to increase significantly UCP-2 mRNA expression in the
presence of the inhibitor. It should be noted, however, that the
magnitude of the effect of cPGI2 on UCP-2 mRNA level
was reduced in the presence of H-89 (+132 ± 42%
versus +241 ± 23% with versus without
H-89, n = 4). The effect of arachidonic acid appeared to be less affected by H-89 (+154 ± 20% versus
+199 ± 64% with versus without H-89,
n = 4). Taken together, these results suggest that
elevation of cAMP level and activation of the protein kinase A pathway
are involved in the regulation of ucp-2 gene expression by
Prostaglandins activate the MAP kinase pathways in various cell models
(39). To verify whether a stimulation of the MAP kinases is implicated
in the regulation of ucp-2 gene expression, we studied the
effect of PD-98059, a classically used MAP kinase kinase inhibitor. We
found that addition of PD-98059 (50 µM) to human myotubes
did not prevent the induction of UCP-2 mRNA by arachidonic acid,
PGE2, or cPGI2 (data not shown). In addition, whereas PGF2 Involvement of Peroxisome Proliferator-activated
Receptors--
Prostanoids are also potent activators of the nuclear
receptors of the PPAR family (41, 42). cPGI2 has been
demonstrated to bind and activate the isoforms
A role of PPAR
UCP-2 and PPAR
As for UCP-2, the functions of PPAR
In summary, our study demonstrates that -6 fatty acids
and arachidonic acid induced a 3-fold rise in UCP-2 mRNA levels
possibly through transcriptional activation. This effect was prevented
by indomethacin and mimicked by prostaglandin (PG) E2 and
carbaprostacyclin PGI2, consistent with a
cyclooxygenase-mediated process. Incubation of myotubes for 6 h
with 100 µM arachidonic acid resulted in a 150-fold
increase in PGE2 and a 15-fold increase in PGI2
in the culture medium. Consistent with a role of cAMP and protein
kinase A, both prostaglandins induced a marked accumulation of cAMP in
human myotubes, and forskolin reproduced the effect of arachidonic acid
on UCP-2 mRNA expression. Inhibition of protein kinase A with H-89
suppressed the effect of PGE2, whereas cPGI2 and arachidonic acid were still able to increase
ucp-2 gene expression, suggesting additional
mechanisms. We found, however, that the MAP kinase pathway was not
involved. Prostaglandins, particularly PGI2, are potent
activators of the peroxisome proliferator-activated receptors. A
specific agonist of peroxisome proliferator-activated receptor (PPAR)
(L165041) increased UCP-2 mRNA levels in myotubes, whereas
activation of PPAR
or PPAR
was ineffective. These results suggest
thus that ucp-2 gene expression is regulated by
-6 fatty acids in human muscle cells through mechanisms involving at least protein kinase A and the nuclear receptor PPAR
.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
seems to be involved in the regulation of
ucp-2 gene expression in undifferentiated cells (19),
whereas PPAR
is likely to play a major role in differentiated
adipocytes (19 -21). In muscle, depending upon the cell line used, the
data are less clear. Oleic acid was found to induce ucp-3
gene expression in several models (22, 23). This effect can be
reproduced by a specific agonist of PPAR
in mouse C2C12 cells (22).
In rat L6 myotubes, however, PPAR
activation does not modify
ucp-3 gene expression, and PPAR
was postulated to play a
role in this process (23). Nevertheless, in the L6 cell model, UCP-2
expression was reported to be induced by thiazolidine diones and
PPAR
activation (21). Finally, in cardiomyocytes, activation of
PPAR
, and not of PPAR
, promoted a strong induction of UCP-2
expression (24).
-6 PUFAs, but not the
-3, up-regulate ucp-2 gene
expression in human muscle cells through the production of
prostaglandins and multiple intracellular pathways including protein
kinase A-dependent activation and involvement of the
nuclear receptor PPAR
.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-minimal
essential medium containing 2% fetal calf serum and 1% antibiotics.
Twelve to 16 days after induction of the differentiation process, most
of the cells showed a multinuclear status that characterized mature
myotubes (27). In addition, the differentiated cells expressed
muscle-specific markers (myosin, sarcomeric
-actin, and creatine
kinase), and they were characterized by an insulin-sensitive glucose
transport (27). The human myotubes were serum-starved overnight before incubations with fatty acids or other products.
20 °C in glass tubes, away from light. To obtain the desired
concentrations, small amounts of stock solution were added, while
stirring gently, to pre-warmed (37 °C)
-minimal essential medium
containing 1% antibiotics and 1% free fatty acid bovine serum albumin
(Roche Molecular Biochemicals). Dilutions were held at 37 °C for at
least 1 h before addition to the cells. The molar ratio of fatty
acid to albumin was 0.8, and the final concentration of EtOH was always
kept below 0.2%. Solutions of prostaglandins, indomethacin, and NDGA
were prepared in EtOH and kept at
20 °C under the same conditions
as for the fatty acids. Proper amounts were directly added to the cell
medium. Concentrated (10
2 M)
stock solutions of the different PPAR agonists, PD-98059 and forskolin,
were done in dimethyl sulfoxide (Me2SO) and stored at
20 °C away from the light. The final concentration was made by
dilution with culture media. The final concentration of
Me2SO was always kept less than 0.1%. All experiments with
differentiated myotubes were done in 6-well culture plates, and
incubations with vehicle (EtOH or Me2SO) alone were
systematically performed as controls.
80 °C until quantification of mRNAs. Concentration
and purity of each sample were assessed by absorbance measurement at
260 nm and by the 260:280 nm ratio, respectively. The yield of total
RNA averaged 1.3 µg/well from a 6-well culture plate, corresponding
to ~250,000 myoblasts per well at confluence, before induction of the
differentiation process.
, and
PGI2 (measured as its stable metabolite
6-oxo-PGF1
) concentrations were determined by gas
chromatography-mass spectrometry using the protocol described by Tsikas
(30). Briefly, lipids in the dried organic phase were first derivatized
in 100 µl of 10% pentafluorobenzyl bromide and 30 µl of 10%
ethyldiisopropylamine (both in acetonitrile) for 30 min at 40 °C.
The medium was then dried under nitrogen. Next, the oxo groups of the
prostaglandins were methoximated with methoxamine hydrochloride (30 µl) in pyridine (30 µl) for 60 min at 60 °C. After drying under
nitrogen, the hydroxy groups were esterified in 100 µl of pure
N,O-bis(trimethylsilyl)-trifluoroacetamide for 30 min at
60 °C. Analysis was performed on a Hewlett-Packard 5973 gas
chromatography-mass spectrometer (Les Ulis, France) in negative ions
chemical ionization using CH4 as reagent gas.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Comparison of UCP-2 and UCP-3 mRNA levels
in cultured human myotubes and in human skeletal muscle biopsies.
UCP mRNA levels were determined by RT-competitive PCR in primary
culture of myotubes from five subjects (closed bars) and in
vastus lateralis muscle biopsies from eight subjects (open
bars). Data are means ± S.E.
-3,
-6 and
-9), were
added for 24 h to human myotubes, and the changes in UCP-2 and
UCP-3 mRNA levels were monitored by RT-cPCR. Regarding UCP-3, oleic
acid (C18:1,
-9) produced an ~3-fold increase in UCP-3 mRNA
levels (data not shown). This result is in agreement with preceding
reports (22, 23). However, the amplitude of the effect showed large variations (0.2-18-fold increase, n = 6). This was
probably due to the difficulty to measure accurately the low levels of
UCP-3 mRNA by RT-cPCR. Therefore, we decided not to investigate the regulation of UCP-3 in this cell model in more detail, and we focused
our attention on the effects of fatty acids on ucp-2 gene expression. Because a number of data show coordinate variations in
UCP-2 and UCP-3 mRNA levels during metabolic changes in rodents and
in humans (6, 14), it could be hypothesized that similar mechanisms are
involved in the regulation of both genes by fatty acids.
-linolenic acid (C18:3,
-6), di-homo-
-linolenic
acid (C20:3,
-6), and arachidonic acid (C20:4,
-6). In rat L6
muscle cells, an induction of UCP-2 mRNA expression by linoleic
acid (C18:2,
-6) has been observed previously (21). Under our
experimental conditions, incubation of human primary muscle cells with
linoleic acid did not affect UCP-2 expression. However, all the
-6
PUFAs that are directly derived from linoleic acid induced a robust induction of UCP-2 mRNA (Fig. 2). This suggests that either longer incubations with linoleic acid are required to allow its metabolism or
that enzymes metabolizing linoleic acid are lacking in the human
myotube model. In contrast to the
-6 PUFAs, fatty acids of the
-3
series, as well as oleic acid (C18:1,
-9), did not modify the
expression of UCP-2 in human muscle cells in culture. Therefore, these
results suggest that the up-regulation of UCP-2 mRNA expression by
fatty acids is mainly dependent on the PUFAs from the
-6 series.
This effect was dependent on the concentration of fatty acid added into
the culture medium. The half-maximal effect of
-linolenic acid or
arachidonic acid was observed with concentrations between 1 and 5 µM (data not shown). To verify whether the induction of
UCP-2 mRNA reflected a transcriptional activation of the
ucp-2 gene or a post-transcriptional stabilization of the
mRNA, myotubes were preincubated for 24 h with or without 100 µM arachidonic acid before addition of
10
6 M actinomycin D, a potent
inhibitor of RNA polymerase II. The kinetics of the decrease in UCP-2
mRNA concentration were similar under the two conditions (data not
shown). The results from three independent preparations of human
myotubes demonstrated that arachidonic acid does not significantly
affect the half-life of UCP-2 mRNA (14 ± 2 versus
11 ± 1 h, without versus with 100 µM arachidonic acid, respectively). This result suggests
that
-6 PUFAs and arachidonic acid affect the transcriptional
regulation of the ucp-2 gene.
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Fig. 2.
Effect of fatty acids on UCP-2 mRNA
levels. Myotubes were incubated for 24 h with 100 µM of the indicated fatty acids, as described under
"Experimental Procedures." UCP-2 mRNA levels were determined by
RT-cPCR. Results are the means ± S.E. with cell preparations from
at least four different subjects. Effect of -6 PUFAs are indicated
in closed bars. *, p
0.01 versus vehicle (ethanol) with six different preparations of
myotubes (Student's t test for paired data).
-6 PUFAs on ucp-2 Gene
Expression--
When human myotubes were incubated for 6 h
instead of 24 h,
-linolenic acid (100 µM) had no
effect, whereas arachidonic acid significantly increased UCP-2 mRNA
levels (Fig. 3, upper panel). Because it is well known that C-18 and C-20
-6-PUFAs can
be rapidly transformed into arachidonic acid which is the major
metabolically active fatty acid of the
-6 series, these results
suggested that the regulation of ucp-2 gene expression by
-6 PUFAs could be mediated by arachidonic acid. To address the
mechanism by which arachidonic acid induces UCP-2 expression, we have
investigated its metabolism in the human myotubes. Arachidonic acid is
rapidly converted to a number of eicosanoids that have potent
physiological properties (34). To identify the involvement of either
the lipo- or cyclooxygenase pathway in the generation of active
metabolites that can induce ucp-2 gene expression,
inhibitors of the two enzyme systems were used (Fig. 3, lower
panel). Myotubes were pretreated with nordihydroguaiaretic acid
(NDGA), an inhibitor of lipoxygenases or with indomethacin, an
inhibitor of cyclooxygenases, before addition of arachidonic acid. The
effect of arachidonic acid was completely prevented by indomethacin,
whereas treatment with NDGA did not affect the induction of UCP-2
mRNA (Fig. 3). In addition to lipo- and cyclooxygenases, the
cytochrome P-450 epoxygenase pathway may eventually be involved in the
metabolism of arachidonic acid. It has been demonstrated that NDGA is
also an inhibitor of arachidonic acid epoxygenase activity at
concentrations higher than 50 µM (35). Since NDGA did not
prevent the effect of arachidonic acid on UCP-2 expression under our
experimental conditions (Fig. 3), the contribution of the epoxygenase
pathway was not likely to be involved. Therefore, these results are
consistent with a major role of the cyclooxygenases to convert
arachidonic acid to prostanoids which in turn mediate the induction of
ucp-2 gene expression. Of note, indomethacin also prevented
the effect of
-linolenic acid in cells incubated for 24 h (data
not shown), indicating that the
-6 PUFAs do not act directly on
ucp-2 gene expression but rather that their effect is
mediated by products of the cyclooxygenase pathway.
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Fig. 3.
Induction of UCP-2 expression is dependent on
arachidonic acid metabolism. Upper panel, kinetics of
-6 action on PUFAs. Cells were incubated with 100 µM
of either
-linolenic acid (hatched bars) or arachidonic
acid (closed bars) for the indicated times. Data are
means ± S.E. for 5 different experiments. *, p < 0.01 versus vehicle (ethanol) (open bars).
Lower panel, effect of inhibitors of arachidonic acid
metabolism. Myotubes were preincubated for 30 min with 50 µM indomethacin or 200 µM NDGA before
addition of 100 µM arachidonic acid (A.A.) for
6 h (closed bars). Results are means ± S.E. with
five different cells preparations. *, p < 0.02 in the
presence versus in the absence of arachidonic acid
(open bars).
affected the mRNA
levels of UCP-2 in human myotubes in culture. The magnitude of the
effect of PGE2 was slightly lower, albeit not significantly
different, than the effect of arachidonic acid. On the other hand,
cPGI2 produced a significantly more important increase in
UCP-2 mRNA levels than arachidonic acid or PGE2 did (Fig. 4). Cyclooxygenase can also directly convert dihomo-
-linolenic acid into prostaglandins (34) that might be active on UCP2 expression. In agreement, PGE1 increased UCP2 mRNA levels to a
similar extend as PGE2 did (2.6 ± 0.9 versus 1.1 ± 0.3 amol/µg total RNA in the presence
of 1 µM PGE1 versus vehicle,
n = 3).
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Fig. 4.
Involvement of prostaglandins in the
regulation of ucp-2 gene expression. Upper
panel, effects of prostaglandins on UCP-2 mRNA levels.
Myotubes were incubated for 24 h with either 100 µM
arachidonic acid (A.A.) or 1 µM of the
indicated prostaglandin. Data are means ± S.E. for six different
experiments. *, p < 0.01 versus vehicle
(ethanol), and $, p < 0.05 versus
incubation with arachidonic acid and with PGE2. Lower
panel, production of prostaglandins. Myotubes were incubated with
100 µM arachidonic acid for 6 h, and the amounts of
PGE2 and PGI2 were determined in the incubation
medium by gas chromatography-mass spectrometry according to the method
described under "Experimental Procedures." PGI2 was
measured in the form of its stable metabolite
6-oxo-PGF1 . The data are presented as arbitrary units
corresponding to the area under the curves of the mass spectrometry
analysis. Triangles show the data obtained in the presence
of 50 µM indomethacin. There was no change in the level
of PGE2 or PGI2 when cells were incubated in the absence of
arachidonic acid.
are produced in noticeable amounts (36). In addition, several studies have demonstrated that
PGI2 is generally one of the major prostaglandins derived from arachidonic acid in various cell systems (34, 37). In human
adipocytes, for example, the production of PGI2 was found to be about 5-fold more important than the production of
PGE2 (38). We determined, in the culture medium of human
myotubes, the productions of the prostaglandins that have been found to induce ucp-2 gene expression. In the absence of arachidonic
acid, using a C-19 saturated fatty acid as internal standard to
estimate the basal concentrations, we found that the amounts of
PGE2, PGF2
, and PGI2 (measured
as its stable metabolite 6-oxo-PGF1
) were very low
(5-10 fmol/µg of protein) and similar for the three prostaglandins. These levels did not change during 6 h of incubation without
arachidonic acid (data not shown). In contrast, addition of 100 µM arachidonic acid produced an ~150-fold increase in
PGE2 levels after 6 h of incubation (Fig. 4,
lower panel). Under the same conditions, the levels of
PGI2 increased about 15-fold (Fig. 4) although there was no
change in the amounts of PGF2
(data not shown).
Moreover, the production of both PGE2 and PGI2
was strongly reduced in the presence of 50 µM
indomethacin (Fig. 4) which prevented the induction of UCP-2 mRNA
expression by arachidonic acid (Fig. 3). It should be also indicated
that there was no significant production of PGE1 in
myotubes incubated with 100 µM of
-linolenic acid for 6 h (data not shown). Taken together these results strongly
suggest that the effect of
-6 PUFAs on UCP-2 mRNA expression is
probably mediated by arachidonic acid-derived PGE2 and
PGI2 that are both produced rapidly and in large amounts in
human myotubes.
-6 PUFAs and prostaglandins. However, it seemed that additional mechanisms could also play a role.
View larger version (11K):
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Fig. 5.
Effect of forskolin on UCP-2 mRNA
expression. Upper panel, production of cAMP in human
myotubes. Cells were incubated for the indicated times with 1 µM of either PGE2 (closed symbols)
or PGI2 (open symbols) under the conditions
described under "Experimental Procedures." Data are means ± S.E. for three different experiments. Cyclic AMP concentration remained
at basal level in the absence of prostaglandins (data not shown).
Lower panel, effect of forskolin on UCP-2 mRNA
expression. Myotubes were incubated for 24 h with 1 µM of forskolin or of the indicated prostaglandin. Data
are means ± S.E. for five different experiments. *,
p < 0.01 versus vehicle (ethanol), and $,
p < 0.05 versus incubation with forskolin
and with PGE2.
View larger version (57K):
[in a new window]
Fig. 6.
Effect of H-89, an inhibitor of protein
kinase A, on UCP-2 mRNA expression. Cells were incubated for
24 h in the presence H-89 (10 µM) and the indicated
inducers of UCP-2 expression. PGE2 and PGI2
were used at a concentration of 1 µM and arachidonic acid
(A.A.) at a concentration of 100 µM. Data are
means ± S.E. with four different experiments. *,
p < 0.01 versus control condition (ethanol + H-89).
, which is known to stimulate the MAP kinase pathway in other cell models (39), increased the phosphorylation of
ERK-1 and ERK-2 (p44 and p42 MAP kinases), we found that these kinases
were not modified by either PGE2 or cPGI2 (data
not shown), indicating that these prostaglandins did not activate the
MAP kinase pathway in human myotubes. Because PGF2
did
not affect UCP-2 mRNA levels (Fig. 3), these results clearly
demonstrate that activation of the MAP kinase pathway is not involved
in the regulation of ucp-2 gene expression by
-6 PUFAs in
human myotubes.
and
of the
PPARs, whereas PGE2 is a less potent agonist (41). We have
previously reported the expression profile of the three PPAR mRNAs
in human skeletal muscle (28). Muscles are characterized by extremely
low mRNA concentrations of PPAR
, whereas PPAR
and PPAR
mRNAs are expressed at a higher level. In cultured human myotubes,
we found that the average mRNA levels of the three PPARs were
grossly similar to what was previously determined in muscle biopsies
(28). We found low levels of PPAR
mRNA (0.2 ± 0.1 amol/µg total RNA, n = 5). PPAR
mRNA (2.1 ± 0.3 amol/µg total RNA, n = 5, p < 0.001) was about 10-fold more abundant than PPAR
. There were
intermediate levels of PPAR
mRNA (0.7 ± 0.1 amol/µg
total RNA, n = 5). By using selective agonists of the
different PPAR isoforms, we then tested their possible involvement in
the effect of arachidonic acid on ucp-2 gene expression.
Fig. 7 shows that WY-14643 (PPAR
agonist) and Rosiglitazone (PPAR
agonist) did not alter UCP-2 mRNA levels in human myotubes.
15-Deoxy-
12,14-PGJ2, the proposed natural
ligand of PPAR
(43), also did not affect UCP-2 mRNA expression
(data not shown), further confirming that PPAR
is not involved in
the regulation of ucp-2 gene in human myotubes. In contrast,
incubation with L-165041, a recently developed agonist of PPAR
(44),
produced a robust induction of UCP-2 mRNA levels (Fig. 10). Because
PGI2 is a potent activator of PPAR
(41, 42) and because
we found that only the activation of this nuclear receptor resulted in
an activation of ucp-2 gene expression, it is thus likely
that part of the action of PGI2 can be mediated by a direct
activation of PPAR
, in addition to the stimulation of the protein
kinase A pathway.
View larger version (12K):
[in a new window]
Fig. 7.
Effect of PPAR agonists on UCP-2
expression. Myotubes were incubated for 24 h with 1 µM of the indicated PPAR agonists. Wy-14643 is a PPAR
agonist, Rosiglitazone a PPAR
agonist, and L-165041 a PPAR
agonist. Results are means ± S.E. with six different preparations
of myotubes. *, p < 0.01 versus vehicle
(DMSO).
in the regulation of ucp-2 gene expression
has already been postulated in preadipocyte cells (19). In contrast, in
fully differentiated adipocytes it has been clearly demonstrated that
activation of PPAR
can induce ucp-2 gene expression (19, 20, 45). PPAR
is expressed at very high levels in mature adipocytes
but is virtually absent in preadipocytes, whereas PPAR
is
constitutively expressed (46). This suggests that the regulation of
ucp-2 gene may implicate either PPAR
or PPAR
,
depending on the relative abundance of the two nuclear receptors. In
human skeletal muscle (28) and in cultured muscle cells (Ref. 23 and
this work), the expression pattern of PPAR
and PPAR
is rather similar to what has been reported in preadipocytes. PPAR
mRNA levels are extremely low, and PPAR
is the major isoform of the PPARs. Under such conditions, the regulation of ucp-2 gene
expression by PPAR agonists, and probably also by fatty acid
derivatives, implicates PPAR
. Recently, a similar conclusion has
been proposed, but not tested directly, for the induction of UCP-3
expression by fatty acids in rat L6 myotubes (23).
are expressed ubiquitously (6, 47). The highest
levels of UCP-2 mRNA have been found in intestine, lung, spleen,
and monocytes/macrophages (6, 5), tissues that are also characterized
by high expression levels of PPAR
(47, 48) and by an important
metabolism of prostanoids. Therefore, the observed up-regulation of
ucp-2 gene expression by
-6 PUFAs and PGI2
may occur also in other cell types than in myotubes and in preadipocytes. To date, the exact role of UCP-2 is not clearly defined
(5, 6), but increasing body of evidence suggests a participation of
UCP-2 in the regulation of the production of reactive oxygen species
(ROS) (6, 49). An excess of ROS can produce cell damage, and it has
been demonstrated that respiration uncoupling limits ROS synthesis (6).
It might thus be of interest to verify whether the regulatory mechanism
of ucp-2 gene expression described in the present work
participates in an antioxidant defense process of the cells (6,
49).
are poorly known, in contrast to
the well established functions of PPAR
in adipocyte differentiation
and of PPAR
in the
-oxidation of fatty acids and other aspects of
lipid metabolism in the liver (17, 18). It has been proposed that
PPAR
plays a role in the initiation of the adipocyte differentiation
process and in the effect of long chain fatty acids on post-confluent
preadipocyte proliferation (46, 50, 51). However, PPAR
is expressed
in almost all tissues or cells both in rodents (47) and in humans (28,
48), suggesting additional functions. The availability of a specific agonist of PPAR
(44) will probably help to rapidly elucidate the
role of this nuclear receptor. To date, only few studies have investigated the consequences of an activation PPAR
in
vivo in rodents. Treatment for 2 weeks with L-165041 did not
affect plasma glucose and triglyceride concentrations in the insulin
resistant db/db mice (44, 52), in contrast to
what has been observed during treatments with a PPAR
agonist (44).
On the other hand, mice treated with L-165041 showed increased plasma
cholesterol concentrations and elevation of circulating high density
lipoprotein levels, suggesting that PPAR
may be involved in the
regulation of cholesterol metabolism in vivo (52). The
target genes of PPAR
involved in this regulation have yet to be
identified. At present, only a limited number of target genes of the
nuclear receptor PPAR
have been described. Activation of PPAR
by
fatty acids has been shown to induce the transcription of the genes encoding fatty acid transporter and adipocyte lipid-binding protein both in preadipocytes and in fibroblasts overexpressing PPAR
(46,
50). Our work suggests that UCP-2 may be an additional target gene of
PPAR
. Consensus sequences for peroxisome proliferator-responsive elements have been identified in the 3.3-kilobase pair sequence of the
promoter region of the human UCP-2 gene recently published by Tu et al. (53). However, the confirmation that the
UCP-2 gene is a genuine target gene of PPAR
will now
require the direct study of the regulation of the promoter.
-6 PUFAs up-regulate
ucp-2 gene expression in human skeletal muscle cells in
primary culture. We identified two potential mechanisms, both requiring the oxidative metabolism of arachidonic acid by cyclooxygenases, as
follows: 1) the increase of intracellular cAMP levels and the activation of the protein kinase A pathway in response to the production of PGE2 and PGI2, and 2) the direct
activation of the nuclear receptor PPAR
, probably by
PGI2. In addition, we demonstrate that the MAP kinase
pathway is not involved in the regulation of ucp-2 gene
expression by
-6 PUFAs. This work thus provides potential mechanisms
for the regulation of ucp-2 gene expression by fatty acids,
a process initially proposed on the basis of observations made in human
skeletal muscle in vivo (13). In addition, the involvement
of PPAR
in the regulation of ucp-2 gene expression suggests a possible role of this nuclear receptor in the control of
mitochondrial uncoupling activity and its related consequences.
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ACKNOWLEDGEMENTS |
---|
We are grateful to M. Odeon and A. Stefanutti for technical assistance with the assay of prostaglandins and cAMP and to K. Bouzakri for assistance in setting up the Western blots of the phosphorylated MAP kinases. We thank Prof. M. Laville, Dr. D. Langin, and Dr. P.A. Grimaldi for discussions.
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FOOTNOTES |
---|
* This work was supported in part by grants from ALFEDIAM-Novo Nordisc, from Institut de Recherche Servier, and from INSERM Progres 4P020D.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a Ph.D. grant from the French Ministère de la
Recherche et de l'Enseignement Supérieur. To whom correspondence should be addressed: INSERM U449, Faculté de Médecine
René Laennec, Rue G. Paradin, F-69372 Lyon Cedex 08, France.
Tel.: 33 478 77 86 29; Fax: 33 478 77 87 62; E-mail:
vidal@laennec.univ-lyon1.fr.
Published, JBC Papers in Press, January 12, 2001, DOI 10.1074/jbc.M008010200
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ABBREVIATIONS |
---|
The abbreviations used are: UCP, uncoupling protein; PUFAs, polyunsaturated fatty acids; NEFAs, nonesterified fatty acids; PPAR, peroxisome proliferator-activated receptor; NDGA, nordihydroguaiaretic acid; EtOH, ethanol; PG, prostaglandin; ROS, reactive oxygen species; MAP, mitogen-activated protein; PCR, polymerase chain reaction; cPCR, competitive PCR; RT, reverse transcriptase; ERK, extracellular signal-regulated kinase; cPGI2, carbaprostacyclin PGI2.
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