|
|
|
|||||||
|
|||||||||
(Received for publication, July 24,
1995; and in revised form, November 3, 1995) From the
We have previously described the ability of arachidonic acid
(AA) to regulate GLUT4 gene expression (Tebbey, P. W., McGowan, K. M.,
Stephens, J. M., Buttke, T. M., and Pekala, P. H.(1994) J. Biol.
Chem. 269, 639-644). Chronic exposure (48 h) of fully
differentiated 3T3-L1 cells to AA resulted in an
Glucose transport across the plasma membrane represents the
rate-limiting step in glucose metabolism and is a highly regulated
process in the animal cell. Facilitated diffusion of glucose into the
cell is carried out by a family of stereospecific transport proteins
known as the glucose transporters (GLUT1 through GLUT5 and GLUT7).
These integral membrane proteins are members of a gene family in which
tissue-specific expression of one or more members will in part
determine the net rate of glucose entry into the cell. In addition to
tissue-specific expression, hormones, growth factors, and fatty acids
can influence the net flux of glucose across the plasma membrane (Kahn,
1992; James et al., 1989; Birnbaum, 1989; Corneilus et
al., 1990; Stephens and Pekala, 1992; Tebbey et al., 1994). ( In addition to roles in the biosynthesis of
lipids and energy production, fatty acids are involved in the
regulation of glucose metabolism. Fatty acids in general and
arachidonic acid (AA) ( These in vitro results
are supported by dietary experiments demonstrating that high safflower
oil content diets (rich in Previous studies from
our laboratory (Tebbey et al., 1994) examined the mechanisms
by which AA can modulate glucose homeostasis in fully differentiated
3T3-L1 adipocytes. Chronic exposure of the adipocytes to 50 µM AA was demonstrated to alter both basal- and insulin-stimulated
glucose uptake and render the cells insulin resistant. The AA treatment
specifically reduced the GLUT4 content of both the plasma and
intracellular membranes, while GLUT1 content increased slightly.
Consistent with the down-regulation of the protein, GLUT4 mRNA levels
decreased to 10% of the initial content. Mechanistically the decrease
was determined to be the result of both transcriptional down-regulation
(50% of control) and a destabilization of the message (GLUT4 mRNA t The oxidation of AA to very potent biological molecules is mediated
by three different enzyme systems to include: the cyclooxygenase,
lipoxygenase, and cytochrome P-450 epoxygenase. Thus, it became
important to determine if oxidation of AA was necessary for its effect
on GLUT4 or whether the observed effects were mediated by alternative
pathways. In the current report we demonstrate that mechanistically
fatty acids may act via two independent mechanisms, only one of which
requires an oxidized metabolite of AA.
Figure 1:
Effect of lipo- and cyclooxygenase
inhibitors on arachidonic acid-induced decrease of GLUT4 mRNA levels.
Fully differentiated 3T3-L1 cells were treated for 30 min with 50
µM indomethacin (Indo) and nordihydroguaiaretic
acid (NDGA) and then treated for an additional 12 h with 100
µM arachidonic acid (AA). Total cellular RNA was
isolated and 20 µg/lane were subjected to electrophoresis and
Northern blot analysis. A, graphical representation of three
independent experiments. In each case the levels of GLUT4 mRNA were
normalized to
Figure 2:
Effect of prostaglandin E
To further establish the role of PGE
Figure 3:
Effect of 5(S)-, 12(S)-,
and 15(S)-HPETEs on GLUT4 mRNA expression. Fully
differentiated 3T3-L1 cells were treated for 12 h with 5 nM TNF-
To determine if
AA was being converted to PGE
Figure 4:
Arachidonic acid induction of PGE
Figure 5:
Arachidonic Acid induction of cAMP as
detected by ELISA. Fully differentiated 3T3-L1 adipocytes were treated
with 100 µM AA, and at the indicated times the cells were
harvested and cAMP was extracted as described under ``Experimental
Procedures.'' The content of cAMP was then determined by an ELISA.
The data are plotted as the mean picomoles of cAMP/ml ± S.D. The
graph represents two independent determinations. Where not indicated
with an error bar, the error of the data lies within the data point
itself.
Figure 6:
Effect of
Figure 7:
Effect of AA on SM hydrolysis.
[
Figure 8:
Effect of clofibrate on GLUT4 mRNA
expression. Fully differentiated 3T3-L1 cells were treated for 12 h
with 0.5 and 1.0 mM clofibrate as indicated. CTL, no
treatment; CTL (Etoh), cells were treated with the same volume
of EtOH as clofibrate to rule out effects of EtOH as clofibrate was
administered as an EtOH stock. Total cellular RNA was then isolated and
20 µg/lane were subjected to electrophoresis and Northern blot
analysis. The results dispalyed are representative of an experiment
performed twice with identical results. For quantification purposes the
data were normalized to the 18 S ribosomal band (not
shown).
Figure 9:
EMSA
of nuclear extracts prepared from 3T3-L1 adipocytes. A,
nuclear extracts prepared from fully differentiated 3T3-L1 adipocytes
were treated with 5 nM TNF (lane 2), 50 µM AA (lane 3), 50 µM ETYA (lane 4),
and 10 µM retinoic acid (lane 5), untreated (lane 1) followed by incubation with radiolabeled
oligonucleotide containing the PPRE (TGACCTTTGTCCT). The binding
reactions were then subjected to EMSA analysis. B, fully
differentiated 3T3-L1 adipocytes were treated with 100 µM AA for 2 h (lane 2) and 6 h (lane 3) and 100
µM ETYA for 12 h (lane 4), untreated (lane
1). Nuclear extracts were then prepared as described under
``Experimental Procedures,'' and 10 µg of protein from
each sample were incubated with radiolabeled PPRE and then subjected to
EMSA analysis.
To address the mechanism by which AA can suppress GLUT4 gene
expression, we have investigated the metabolism and the signal
transduction properties of AA in the 3T3-L1 adipocytes. Our studies
have demonstrated that the major metabolic fate of exogenously added AA
is the oxidative metabolism through the cyclooxygenase pathway to a
prostanoid. These conclusions are based on the data demonstrating that
the effect of AA on GLUT4 can be blocked by indomethacin, a
cyclooxygenase inhibitor, while NDGA, a lipoxygenase inhibitor, cannot
inhibit the effect of AA. Of the prostanoids derived from AA, isolated
adipocytes have been demonstrated to produce only the prostaglandins,
PGE In a number of systems, the oxidative
metabolism of AA appears to be a requirement to derive the metabolite
capable of regulating specific gene expression. In Swiss 3T3
fibroblasts, AA was shown to induce c-fos and Egr-1 mRNA through formation of PGE Prostanoids are local hormones that once released
from the cell bind to cell surface receptors to act in an autocrine or
paracrine fashion. Depending on the cell type, there are three distinct
receptors for PGE We (Stephens
and Pekala, 1992) and others (Ezaki et al., 1993;
Flores-Riveros, et al., 1993b) have demonstrated the ability
of cAMP to suppress transcription of the GLUT4 gene, leading to a
decrease in the content of GLUT4 mRNA. Stephens and Pekala(1992)
demonstrated that the effect of 8-bromo-cAMP on GLUT4 transcription was
rapid, with a 40% decrease in rate detected within 1 h and a 52%
decrease within 4 h. With respect to both magnitude and temporal
considerations, these results were very similar to those we have
reported for AA, where a 50% decrease in GLUT4 gene transcription
occurred in response to AA treatment (Tebbey et al., 1994).
Studies by Flores-Riveros et al. (1993b) indicated that the
regulatory element mediating transcriptional repression by cAMP resides
in the proximal promoter of the GLUT4 gene between positions -469
and -78. Thus, based on these observations it is likely that a
cAMP-dependent protein kinase is involved in the transcriptional
repression of the GLUT4 gene where an interaction between the
cis-regulatory element and the activity of a trans-acting factor is
regulated by cAMP-mediated phosphorylation. Examination of a diverse
family of fatty acids for the specificity of the AA effect demonstrated
that suppression of GLUT4 was not limited to AA (Fig. 6).
Perhaps the strongest evidence for an alternative pathway is that ETYA,
a nonmetabolizable AA analog reported to be a potent inhibitor of AA
transport and metabolism (Tobias and Hamilton, 1978), is nearly as
potent as AA in its ability to suppress GLUT4 mRNA accumulation. These
data would suggest that the fatty acids can function as a regulator of
GLUT4 mRNA expression by a pathway that does not involve oxidative
metabolism. Our previous studies with AA demonstrated that in
addition to a suppression of transcription, exposure of the adipocytes
to AA resulted in a destabilization of the GLUT4 mRNA (t Fatty acid levels fluctuate
in normal metabolism as well as in certain pathological diseases and
are appropriately considered to serve as biological effectors as well
as metabolic substrates. Arachidonic acid has become recognized as a
novel second messenger regulating cell growth through activation of
various enzyme systems such as neutral sphingomyelinase (Jayadev et
al., 1994), protein kinase A (Doolan and Keenan, 1994), protein
kinase C (Bell and Burns, 1991), and the platelet-derived growth factor
receptor (Tomaska and Resnick, 1993). In addition, AA as well as its
metabolites have been shown to regulate gene expression either directly
(Danesch et al., 1994) or as mediators of various stimuli
including tumor necrosis factor- In addition to regulation
of GLUT4 gene expression by a direct mechanism, the PPAR may
potentially regulate indirectly by altering expression or activation of
another transcription factor. The CCAAT/enhancer-binding protein
(C/EBP In
summary, these studies demonstrate that in 3T3-L1 adipocytes AA/fatty
acids regulate GLUT4 gene expression by potentially two independent
mechanisms: 1) via oxidative metabolism to the cyclooxygenase
metabolite PGE
Volume 271,
Number 2,
Issue of January 12, 1996 pp. 1138-1144
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
EVIDENCE FOR MULTIPLE PATHWAYS, ONE OF WHICH REQUIRES OXIDATION TO
PROSTAGLANDIN E
(*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
90% suppression
of GLUT4 mRNA accumulation. This decrease was demonstrated to be due to
a 50% decrease in GLUT4 gene transcription as well as a destabilization
of the GLUT4 message (t
decreased from 8.0 to
4.6 h). In the current study we have identified, at least in part, the
mechanism by which AA exerts its effects on GLUT4 expression.
Compatible with a cyclooxygenase mediated event, the AA-induced
suppression of GLUT4 mRNA was abolished by pretreating the cells with
the inhibitor, indomethacin. Consistent with this observation, exposure
of the cells to 10 µM PGE
mimicked the effect
of AA, in contrast to products of the lipoxygenase pathway which were
unable to suppress GLUT4 mRNA content. Quantification of the conversion
of AA to PGE demonstrated a 50-fold increase in
PGE released into the media within 7 h of AA addition.
Cyclic AMP levels were also increased 50-fold with AA treatment
consistent with PGE activation of adenylate cyclase.
Various long chain fatty acids, including the nonmetabolizable analog
of AA, eicosatetraenoic acid (ETYA), also decreased GLUT4 mRNA levels.
The effect of ETYA, a potent inhibitor of both lipo- and
cyclooxygenases and a potent activator of peroxisome proliferator
activated receptors (PPARs), suggested the presence of a second pathway
where nonmetabolized fatty acid functioned to suppress GLUT4 mRNA
levels. Further support for a PPAR-mediated mechanism was obtained by
exposure of the cells to the classic PPAR activator, clofibrate, which
resulted in a 75% decrease in GLUT4 mRNA content. Nuclear extracts
prepared from the adipocytes contained a protein complex that bound to
the PPAR responsive element (PPRE) found in the promoter of the fatty
acyl-CoA oxidase gene. When the adipocytes were treated with either AA
or ETYA, binding to the PPRE was disrupted, consistent with an ability
of these fatty acids to control gene expression by altering the
occupation of a PPRE. However, a perfect PPRE has yet to be identified
in the GLUT4 promoter, but this does not rule the possibility of a PPAR
playing an indirect role in the AA-induced GLUT4 mRNA suppression.
))specifically have been demonstrated
to be physiological regulators of the adipocyte glucose transport
system (Hardy et al., 1991; Murer et al., 1992;
Hunnicut et al., 1994; Tebbey et al., 1994).
Arachidonic acid (20:4), a major metabolically important
polyunsaturated fatty acid present in mammalian cells, is synthesized
in the liver from dietary linoleic acid (18:2) and then transported via
serum albumin or lipoproteins to various tissues. Serum levels of AA
are low relative to other fatty acids except in obesity and diabetes
where levels can be significantly elevated over normal matched controls
(Distel et al., 1992; Svedberg et al., 1990; Grunfeld et al., 1981). In addition, many cells possess a high affinity
arachidonyl-CoA synthetase which facilitates selective accumulation of
AA even when other fatty acid species are in excess (Neufeld et
al., 1983; Taylor et al., 1985). Such characteristics
imply an important role for AA in cellular growth and function and as
evidence for such, AA has been implicated in the regulation of gene
expression (Tebbey and Buttke, 1992; Glaslow et al., 1992;
Ntambi, 1992; Clarke and Abraham, 1992; Tebbey et al., 1994).
Specifically, AA was found to suppress the transcription rate of a
number of genes, including: the stearoyl-CoA desaturase 2 in T-cells
(Tebbey and Buttke, 1992), the hepatic fatty acid synthase (Clarke and
Abraham, 1992), and GLUT4 in 3T3-L1 adipocytes (Tebbey et al., 1994). Interestingly, stearoyl-CoA desaturase 2 and GLUT4 are
coordinately expressed during the differentiation process in the 3T3-L1
adipocytes (Kaestner et al., 1989, 1990) and results obtained
in transient co-transfection assays suggest the possibility of
identical mechanisms of activation of the promoters for both the GLUT4
and stearoyl-CoA desaturase 2 genes (Kaestner et al., 1990;
Christy et al., 1989). Thus, control of the expression of
these genes by AA may be part of a generalized regulation of
adipose-specific gene expression.-6 fatty acids) resulted in decreased
cellular content and distribution of GLUT4 in rat adipocytes (Ezaki et al., 1992). Insulin-stimulated glucose transport activity
was decreased to 51% of controls, and GLUT4 protein content of both
plasma and microsomal membranes decreased by 35%. Fish oil feeding
(increased
-3 fatty acids) transiently improved the safflower
oil-mediated decrease of insulin-stimulated glucose transport activity
by increasing the amount of both GLUT1 and GLUT4 proteins. These data
suggest that the regulatory properties of fatty acids may be determined
by specific structure-function relationships.
decreased by
43%). These data suggested
that AA, in a manner similar to insulin (Flores-Riveros et al., 1993a) and TNF (Stephens and Pekala, 1991, 1992), down-regulates
expression of the insulin-responsive glucose transporter in adipocytes.
Materials
Dulbecco's modified
Eagle's medium was purchased from Life Technologies, Inc. Fetal
bovine serum was purchased from HyClone (Logan, UT) and used at a 1:10
dilution in Dulbecco's modified Eagle's medium. Based on
specifications provided by HyClone, the mean AA content of culture
medium containing 10% fetal bovine serum would be 3
µM. Radiolabeled compounds were obtained from DuPont NEN.
Hybond-N blotting membrane was purchased from Amersham Corp.
Deoxyribonucleotides were obtained from Pharmacia Biotech Inc. Klenow
fragment and TRIzol Reagent was obtained from Life Technologies, Inc.
The polyclonal antiserum against PPAR
2 was developed against a
peptide containing amino acids 284-298 as described by the clone;
it was obtained from James Stiehr of Affinity Bioreagents Inc. Tumor
necrosis factor-
was the generous gift of Biogen (Cambridge, MA).
The specific activity was 9.6 
10
units/mg protein,
based on a cytotoxicity assay using L929 cells. Prostaglandin
E-Monoclonal and cAMP Enzyme Immunoassay Kits were
purchased from Cayman Chemical. All fatty acids and prostaglandins were
purchased from Cayman Chemical. All other chemicals, unless otherwise
stated, were of molecular biology grade and purchased from Sigma.3T3-L1 Cell Culture
The murine 3T3-L1 cells used
in this study were originally obtained from Dr. Howard Greene, Harvard
University, Boston, MA. Cells were cultured, maintained, and
differentiated as described previously (Tebbey et al., 1994).
The cells were maintained for 10 days post-differentiation and then
treated with the indicated agents for various times prior to RNA
isolation.Fatty Acids
All fatty acids used were absorbed
onto diatomaceous earth and subsequently complexed to essentially fatty
acid-free bovine serum albumin (BSA) to yield a FA:BSA ratio of
1.4:1 (Tebbey and Buttke, 1992; Buttke et al.,1989).
FA/BSA was added to cells from a 2.5 mM stock solution to
yield a final concentration of 100 µM.
RNA Isolation and Northern Blot Analyses
Total RNA
was isolated by extraction with guanidine isothiocyanate and
centrifugation through 5.7 M cesium chloride (Chirgwin et
al., 1979) or by the TRIzol Method (Life Technologies, Inc.).
Northern analyses were performed as described previously (Stephens and
Pekala, 1991).DNA Probes
GLUT4, a 2.8-kilobase pair EcoRI fragment encoding the 3T3-L1 homolog of the
adipose/muscle (insulin-responsive) glucose transporter (Kaestner et al., 1989); 
-actin, a 1.9-kilobase pair HindIII fragment obtained from Dr. D. W. Cleveland (Cleveland et al., 1980).Enzyme Immunoassay for PGE
Cells were
treated with 100 µM AA/BSA for various times. At the
indicated times 100-µl aliquots of medium were taken and analyzed
for PGE content using an enzyme immunoassay kit for
PGE (Cayman Chemical) based on the ELISA method.Enzyme Immunoassay for cAMP
Cells were treated
with 100 µM AA/BSA for various times and at the indicated
times the cells were washed in phosphate-buffered saline and then
scraped in 1 ml of 10% trichloroacetic acid. Cell debris was pelleted
and cAMP was then extracted from the supernatant with an equal volume
of 3:1 mixture of Freon/tri-n-octylamine. The upper aqueous
phase was collected (500 µl) and then assayed for cAMP content
using an enzyme immunoassay kit for cAMP (Cayman Chemical) based on the
ELISA method.
Sphingomyelin Assay
Cells were labeled with
[
H] choline chloride (specific activity of 1
mCi/ml) at 0.5 µCi/ml for 6 days after initiation of
differentiation, and the cells were grown in normal media for the
remaining 3 days. On day 9 the cells were treated with either 100
µM AA or 5 nM TNF for the indicated times, the
lipids extracted, and labeled sphingomyelin quantified by the bacterial
sphingomyelinase method as described by Jayadev et al. (1994). Preparation of Nuclear Extract
After the indicated
treatments for various times, the cells were pelleted in
phosphate-buffered saline for 10 min at 3000 rpm. The pellet was then
resuspended in 3 ml of M3 lysis buffer (250 mM sucrose, 25
mM Tris, pH 7.8, 1.1 mM MgCl, 0.2% Triton
X-100, 0.5 mM phenylmethylsulfonyl fluoride, 10 mM EDTA, 10 mg/ml leupeptin, and 1 mg/ml aprotinin) and centrifuged
at 10,000 rpm for 15 min. The nuclei were extracted using 250 µl of
cold M4 extraction buffer (20 mM HEPES, pH 7.8, 0.4 M
KCl, 2 mM dithiothreitol, 20% glycerol, 0.5 mM phenylmethylsulfonyl fluoride, 10 mM EDTA, 10 mg/ml
leupeptin, and 1 mg/ml aprotinin) for 30 min and then the nuclear
debris and DNA pelleted at 12,000 rpm for 15 min. Nuclear extracts were
assayed for protein concentrations by the method of Bradford(1976).Electrophoretic Mobility Shift Assay (EMSA)
EMSAs
were performed using the direct repeat half-site PPRE
(5`-AATTTCGAGAACGTGACCTTTGTCCTGGTCCAGCT-3`). Briefly, 100 ng
(60,000 cpm) of a
P-labeled double-stranded DNA oligomer
was incubated with 5 µg of nuclear extract for 15 min at room
temperature in a total volume of 20 µl of reaction buffer (25
mM Tris, pH 7.8, 0.5 mM EDTA, 88 mM KCl, 1
mM dithiothreitol, 150 µg of
poly(dI
dC)-poly(dIdC), 0.05% Triton X-100, and 12.5
µg/ml salmon sperm DNA). Reaction mixtures were subjected to
electrophoresis on a 5% nondenaturing polyacrylamide gel at 4 °C,
40 amps (200 V), for 2 h. The gels were dried and exposed to x-ray
film for 12 h at -80 °C.
Statistical Analyses
The results of these
experiments were analyzed using the Sigma-Stat package (Jandel
Scientific Software, San Rafael, CA). Comparisons of means among groups
were tested for significant differences by a Newman-Keuls procedure
following one-way analysis of variance. The level of statistical
significance chosen for these experiments was p < 0.05.
Inhibition of Cyclooxygenase Abolishes Arachidonic
Acid-induced GLUT4 mRNA Suppression
AA is rapidly converted to a
number of eicosanoids which express potent physiological properties. In
order to identify involvement of either the lipo- or cyclooxygenase in
the conversion of AA to a metabolite active in the suppression of GLUT4
gene expression, inhibitors of the two enzyme systems were used.
Pretreatment of the 3T3-L1 adipocytes with nordihydroguaiaretic acid
(NDGA), a selective inhibitor of the lipoxygenases (IC = 0.2, 30, and 30 µM for 5-, 12-, and 15-LO,
respectively, as opposed to 100 µM for the cyclooxygenase;
Tobias and Hamilton, 1978) for 1 h at 50 µM did not
significantly alter the AA-induced suppression of GLUT4 mRNA content (Fig. 1). In contrast, pretreatment with 50 µM indomethacin, which inhibits the cyclooxygenase (IC
= 0.1 µM) selectively over the lipoxygenases
(IC
> 100 µM; Tobias and Hamilton, 1978),
for 1 h completely abolished the AA-induced decrease of GLUT4 mRNA
content, consistent with conversion of AA to a prostanoid which in turn
mediated suppression. Similar results were observed using the
cyclooxygenase inhibitor ibuprofen (data not shown). While these
results were rather compelling, a third oxidative pathway was
considered. The cytochrome P-450 epoxygenase pathway has only been
considered a major pathway in liver and kidney and a minor pathway in
occular and pituitary tissues (Fitzpatrick and Murphy, 1988). Its
existence in adipose tissue, to our knowledge, has not been described.
However, we attempted to block any potential metabolism through this
enzyme system using the inhibitor SKF-541. At concentrations of up to
50 µM, this inhibitor had no effect on the AA-mediated
suppression of GLUT4 mRNA. In addition, NDGA exhibits an IC
value of 15 µM for AA-specific epoxygenase
(Capdevila et al., 1988) and should have blocked the
AA-induced GLUT4 down-regulation had an epoxygenase metabolite been
responsible for the regulation; as seen from the data in Fig. 1,
this did not occur. These results strongly indicate a cyclooxygenase
metabolite rather than AA itself as the suppressive agent in the
down-regulation of GLUT4 message.
-actin. Data are plotted as the mean percent of mRNA
remaining ± S.D. B, representative Northern blot
analysis. The same blot was sequentially hybridized with a GLUT4 cDNA
probe, stripped, and rehybridized with an actin cDNA probe. The size of
the relevant mRNA species is shown in kilobases (Kb) to the right of each panel. Concentrations of inhibitors used in this
study were based on preliminary experiments where maximum nonlethal
doses were determined.
PGE
Of the cyclooxygenase metabolites, PGESuppresses GLUT4 mRNA
Levels and prostacyclin (PGI) are the major prostaglandins
formed in the fat cell (Axelrod and Levine, 1981; Richelsen, 1987,
1992). Given the inhibition of indomethacin and ibuprofen on AA-induced
GLUT4 mRNA suppression, we examined whether PGE could
elicit the same effect as AA. The results shown in Fig. 2,
indicate that exposure of the cells for 12 h to 10 µM PGE resulted in a marked decrease (70%) in GLUT4
mRNA content. The effect was significantly (p < 0.05%) more
potent than either AA (
55% decrease) or tumor necrosis
factor-
(TNF) (60% decrease). Treatment of the cells with the
nonmetabolizable analog of prostacyclin, carbaprostacyclin, for 12 h at
5, 10, or 25 µM had little or no effect on GLUT4 mRNA
levels (data not shown), suggesting the specificity of PGE
.
on
GLUT4 mRNA expression. Fully differentiated 3T3-L1 cells were treated
for 12 h with 5 nM tumor necrosis factor- (TNF),
100 µM arachidonic acid (AA), and 10 µM prostaglandin E (PGE).
Total cellular RNA was isolated, and 20 µg/lane were subjected to
electrophoresis and Northern blot analysis. A, graphical
representation of three independent experiments. In each case the
levels of GLUT4 mRNA were normalized to
-actin. Data are plotted
as the mean percent of mRNA remaining ± S.D. B,
representative Northern blot analysis. The same blot was sequentially
hybridized with a GLUT4 cDNA probe, stripped, and rehybridized with an
actin cDNA probe. The size of the relevant mRNA species are shown in
kilobases (Kb) to the right of each
panel.
in the regulation
of GLUT4, as well as obtain support for the NDGA inhibitor data, we
treated the cells with the hydroperoxy products of the lipoxygenase
pathway, the 5(S)-, 12(S)-, and 15(S)-HPETEs
for 12 h at 5 and 10 µM (Fig. 3). The data clearly
demonstrated that, in contrast to PGE, these lipoxygenase
metabolites had no effect on GLUT4 mRNA expression.
(lane 2), 100 µM AA (lane
3), 10 µM PGE (lane 4), 5
µM 5(S), 12(S), and
15(S)-HPETEs (lanes 5, 6, 7), and 10 µM 5, 12, and 15 (S) HPETEs (lanes 8, 9, 10). Lane
1, No treatment. Total cellular RNA was isolated, and 20 µg of
each RNA sample was incubated with complementary radiolabeled GLUT4 RNA
transcript and then subjected to Ribo-nuclease Protection analysis. The
182-base pair protected fragment is indicated by the arrow.
by the adipocytes, we
measured PGE levels present in the media at various times
after the cells were treated with AA, using an ELISA. As shown in Fig. 4, within 1 h of AA addition to the cells, PGE content increased 10-fold above basal levels. A maximum 50-fold
increase above basal was observed 7 h after AA addition (Fig. 4). The duration of the PGE peak was
relatively short, lasting only 1 h and then returning slowly to
basal levels over the next 15 h. These data suggest that AA is being
converted via the cyclooxygenase pathway to PGE
, the
apparent active metabolite mediating GLUT4 suppression.
as detected by ELISA. Fully differentiated 3T3-L1 adipocytes were
treated with 100 µM AA, and aliquots of the media were
removed at various times and tested for the presence of
PGE. The data are plotted as the mean picomoles of
PGE/ml ± S.D. The graph represents two independent
determinations. Where not indicated with an error bar, the error of the
data lies within the data point itself.
Cyclic AMP Increases in the 3T3-L1 Adipocytes with AA
Treatment
PGE functions to increase intracellular
cAMP levels in various cells by stimulation of adenylyl cyclase in an
autocrine fashion (Davies and MacIntyre, 1992). In adipocytes,
PGE has been demonstrated to exhibit a concentration
dependence with respect to its inhibitory or stimulatory effects on
adenylate cyclase (Kather and Simon, 1977, 1979; Kather, 1982). In
intact adipose cells PGE inhibited isoproternol-stimulated
lipolysis at nanomolar concentrations, while exposure of cells to
concentrations in excess of 1 µM resulted in cAMP
accumulation and stimulation of lipolysis (Kather, 1981). To determine
if cAMP content increases with AA treatment, we measured cAMP levels
present in the cytosol at various times after the cells were treated
with AA using an ELISA. As shown in Fig. 5, cAMP levels
increased 50-fold above the basal level within 3 h of AA treatment. The
increase in cAMP occurred consistent with stimulation of adenylate
cyclase by PGE (compare Fig. 4and Fig. 5).
Unlike the increase in PGE, the rise in cAMP was relatively
sharp, and the levels remained high for less than 30 min, potentially
due to the desensitization of the enzyme. These data are consistent
with AA metabolism to PGE which acts in an autocrine
fashion to increase cAMP levels.
Specificity of the AA Regulatory
Effect
Specificity of AA in suppressing GLUT4 mRNA levels was
investigated using a series of fatty acids as well as a
nonmetabolizable analog of AA. To obtain a maximal response, based on
preliminary studies, the cells were exposed to the fatty acid-albumin
complex at a final concentration of 100 µM for 24 h. The
results, displayed in Fig. 6, indicate that all fatty acids
examined, stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2),
linolenic acid (18:3), -linolenic acid (
18:3), mead fatty
acid (20:3), AA (20:4), eicosapentaenoic acid (20:5), and the
acetylenic analog of AA, ETYA, were able to suppress GLUT4 mRNA levels
by at least 50% with AA providing the maximum suppression of 80%. While
18:2 and
18:3 can be converted to AA and potentially lead to
increased cAMP, none of the other fatty acids tested exhibit this
potential. To confirm this, we examined the potential for 18:0, 18:1,
and ETYA to increase cAMP levels; our data indicated that cAMP did not
increase over the 5-h time frame of the experiment (mean ± S.D.
of the [cAMP] over the 5-h time course, control: 178 ±
32 pmol/ml; 18:0-, 18:1-, and ETYA-treated: 149 ± 28, 139
± 19, and 159 ± 21 pmol/ml, respectively). Interestingly
ETYA, which inhibits AA uptake, AA-specific and nonspecific acyl-CoA
synthetases, the cyclooxygenase, and all lipoxygenases, PLA
as well as the cytochrome P-450 epoxygenase, and thus cannot be
converted to PGE, was as effective as AA in suppressing
GLUT4 mRNA accumulation. In addition, the suppression of GLUT4 mRNA
accumulation could not be blocked by addition of indomethacin to the
cultures. These studies are consistent with the premise that fatty
acids may regulate GLUT4 mRNA content without the requirement for
further metabolism and are consistent with the presence of a second
signal transduction pathway.
-3 and
-6 mono- and
polyunsaturated fatty acids on GLUT4 mRNA expression. Fully
differentiated 3T3-L1 cells were treated for 24 h with 100 µM of each fatty acid. Total cellular RNA was isolated, and 20
µg/lane were subjected to electrophoresis and Northern blot
analysis. A, graphical representation of three independent
experiments. In each case the levels of GLUT4 mRNA were normalized to
-actin. Data are plotted as the mean percent of mRNA remaining
± S.D. B, representative Northern blot analysis. The
same blot was sequentially hybridized with a GLUT4 cDNA probe,
stripped, and rehybridized with an actin cDNA probe. The size of the
relevant mRNA species are shown in kilobases (Kb) to the right of each panel.
AA Does Not Induce Sphingomyelin Turnover
Jayadev et al.(1994) have identified AA as one of several fatty acids
that can activate the sphingomyelinase in HL-60 leukemia cells. This
results in generation of free ceramide and initiation of a signal
transduction cascade that potentially could control GLUT4 gene
expression. To determine if this regulatory pathway was viable in the
adipocytes, we examined the ability of AA to activate the
sphingomyelinase and initiate sphingomyelin turnover. As shown in Fig. 7, after addition of AA to the cells, no significant
hydrolysis of sphingomyelin was detected. TNF activation of the
sphingomyelinase at a single 40-min time point resulted in a 50%
decrease in sphingomyelin and was utilized as a positive control. The
data suggest that in the 3T3-L1 adipocytes, AA is not mediating
activation of the sphingomyelinase and generation of free ceramide.
H]Choline-labeled adipocytes were treated at
time 0 with 100 µM AA or 5 nM TNF. At the
indicated times cells were harvested, lipids were extracted, and SM was
quantitated as described under ``Experimental Procedures.''
The data are plotted as the mean percent of sphingomyelin remaining
± S.D. and are representative of results from three separate
experiments.
Involvement of Peroxisome Proliferator-activated
Receptors (PPARs)
The newest members of the nuclear hormone
receptor superfamily are the PPARs, which are known to be activated, in
addition to the classic peroxisome activators such as clofibrate, by
long chain saturated (Gottlicher et al., 1992; Gulick et
al., 1994) and polyunsaturated fatty acids (Tontonoz et al., 1994a; Keller et al., 1993; Gottlicher et al., 1992). Thus, a predicted activity profile for an effect mediated
by fatty acid activation of a PPAR would be similar to that displayed
in Fig. 6and discussed in the previous section. To establish a
role for PPARs in the regulation of GLUT4 mRNA expression, fully
differentiated 3T3-L1 cells were exposed to 0.5 and 1.0 mM clofibric acid for 12 h and GLUT4 mRNA levels were analyzed (Fig. 8). In a manner similar to AA and ETYA, 0.5 and 1.0 mM clofibric acid decreased GLUT4 mRNA content 35 and 75%,
respectively (the average area by quantification of phosphorimage scans
± experimental variation for: control, 4450 ± 273; 0.5
mM clofibrate, 2850 ± 140; 1.0 mM clofibrate,
1187 ± 112). These data suggest that PPAR activation may result
in GLUT4 mRNA suppression. PPAR
2, the predominant isoform
expressed in adipose tissue (Tontonoz et al., 1994a; 1994b)
has been shown to regulate the expression of adipocyte-specific genes
and is activated by a diverse group of compounds including ETYA and
fatty acids. In order to determine the potential for PPAR
2
involvement in the fatty acid-induced GLUT4 mRNA suppression, we
performed two experiments using a DNA gel mobility shift assay (Fig. 9). Oligonucleotides containing the classic PPRE
(TGACCTTTGTCCT) from the promoter of the fatty acyl-CoA oxidase gene
were end-labeled with [
-P]dATP and then
incubated with 10 µg of nuclear extracts. In the first experiment (Fig. 9A), nuclear extracts were prepared from the
3T3-L1 adipocytes and then treated with 50 µM AA, ETYA,
and 10 µM retinoic acid. Separation of the complexes on a
5% nondenaturing polyacrylamide gel demonstrated that addition of AA or
ETYA to the nuclear extracts results in a loss of binding of the PPAR
to its responsive element. In the second experiment, nuclear extracts
were prepared from cells that had been treated with 100 µM AA for 2 and 6 h or 100 µM ETYA for 12 h. Binding
reactions were performed as described above. The results (Fig. 9B) demonstrate that treatment of the intact
cells with these fatty acids markedly diminished protein binding to the
PPRE. A partial supershift, performed with an antibody to PPAR
2,
demonstrated that the protein-DNA complex was a heterodimer with one
partner being PPAR
2 (data not shown). The data from the two
experiments suggest that both AA and its nonmetabolizable analog are
capable of disrupting the occupancy of a PPAR and that neither protein
synthesis nor oxidation to PGE
is necessary for the
alteration of binding.
and PGI (prostacyclin) in considerable
amounts (Axelrod and Levine, 1981; Richelsen, 1987). To determine if
either of these compounds contributed to the suppression of GLUT4, the
fully differentiated 3T3-L1 adipocytes were exposed to PGE and carbaprostacyclin, the chemically stable analog of
prostacyclin. Prostaglandin E treatment resulted in an 70%
decrease in GLUT4 mRNA levels, while carbaprostacyclin had no effect on
GLUT4 mRNA content. Quantification of the conversion of AA to PGE demonstrated a 50-fold increase in PGE released into
the media within 3 h of AA addition (Fig. 3). These data,
demonstrating the time course and magnitude of PGE formation, are consistent with PGE synthesis as being
the major metabolic fate of exogenously added AA. Moreover, they
suggest that PGE may be an intermediate in regulation of
GLUT4 gene expression. and subsequent
activation of protein kinase C (Danesch et al., 1994).
Lipoxygenase metabolites have also been shown to stimulate the
accumulation of c-fos and c-jun mRNA in human
monocytes while they increase c-fos accumulation in quiescent
TA1 cells (Haliday et al., 1991). In both cases, message
accumulation occurs through increased gene transcription (Stankova and
Rola-Pleszczynski, 1992; Haliday et al., 1991). While
underlying mechanisms have not as of yet been identified, these reports
demonstrate a role for AA and its metabolites in the regulation of
specific genes.. Ligand occupation of the appropriate
receptor can lead to activation or inhibition of adenylate cyclase,
each mediated through a specific G-protein, while occupation of the
third class of receptors can stimulate Ca
mobilization and subsequent activation of protein kinase C (Davies and
MacIntyre, 1992). Interestingly, the 3T3-L1 adipocytes have recently
been shown to lack the Ca
-activated protein kinase C
isoforms (McGowan et al., 1996) and thus a mechanism dependent
on PGE
activation of protein kinase C is ruled out. Our
data are consistent with a cAMP-mediated mechanism with cAMP levels
rising 50-fold above basal (10 µM) within 3 h of AA
addition. The decrease in cAMP content, with levels returning to basal
within
30 min while PGE
levels remained markedly
elevated, suggest a rapid desensitization of the PGE receptor to adenylate cyclase coupling mechanism. decreased from 9.3 to 4.5 h). However, in the
studies described above (Stephens and Pekala, 1992; Ezaki et al., 1993) no effect was observed on the stability of the GLUT4 mRNA
when the cells were exposed to 8-bromo-cAMP, thereby localizing the
effect on transcription to cAMP. These data taken collectively suggest
that AA must initiate a second signal transduction cascade that is
responsible for the alteration of GLUT4 mRNA stability. In an attempt
to further define this issue, we examined whether ETYA or 18:1 could
alter GLUT4 mRNA stability. After exposure of the cells to ETYA for 24
h, the half-life of the GLUT4 mRNA was determined to be 8.1 ±
1.1 h (n = 3). This did not test significantly
different than control. However, incubation of the cells in the
presence of 18:1 resulted in a stability decrease similar to that
observed for AA (5.5 ± 0.5 h and 4.2 ± 0.3 h,
respectively). Thus, while ETYA appears to exert its effect solely by
means of transcriptional mechanisms, the metabolizable fatty acids
appear to also influence mRNA stability.
and growth factors (Jayadev et al., 1994). Long chain fatty acids have also been shown to
activate the recently cloned PPARs, the newest members of the nuclear
hormone receptor superfamily. Interestingly, ETYA, which suppressed
GLUT4 mRNA as strongly as AA, is one of the most potent activators of
this class of nuclear receptors. Signaling through these receptors may
provide the second mechanism by which AA can function to suppress GLUT4
gene expression. The fact that the classic PPAR activator, clofibrate,
can also suppress GLUT4 mRNA levels further supports a potential role
for activation of PPARs in the regulation of GLUT4 mRNA accumulation.
The observation that AA and ETYA can disrupt PPAR binding to its
response element indicates that these fatty acids are capable of
regulating occupation of the PPARs in the 3T3-L1 adipocytes. Whether or
not this AA-induced disruption of PPAR binding to its response element
is involved in GLUT4 mRNA suppression has yet to be elucidated. To
date, a perfect PPRE has not been found in the GLUT4 promoter, and as
such, activation of a PPAR by a fatty acid or clofibrate may play an
indirect role in GLUT4 mRNA suppression.) (Christy et al., 1989; Herrera et al.,
1989), a transcription factor demonstrated to bind to and activate the
GLUT4 gene promoter upon differentiation of the 3T3-L1 adipocytes
(Christy et al., 1989), has been demonstrated to act
synergistically with PPAR2 in the development of adipose cells
from uncommitted mesodermal precursers (Tontonoz et al., 1994b). Thus, perhaps an essential interaction between the
PPAR
2 and C/EBP
necessary for GLUT4 expression is altered on
exposure to the fatty acids. This remains to be resolved. and the subsequent elevation of cAMP levels,
based on our previous studies it is likely that the increase in cAMP
represents the potential mechanism for suppression of GLUT4
transcription. We note, however, that as of yet we have not ruled out a
mechanism by which PGE functions to suppress GLUT4
independent of the increase in cAMP and 2) via nonoxidized fatty acid
with the potential involvement of a PPAR. In addition, the
demonstration that AA did not activate sphingomyelinase confirms the
absence of another potential fatty acid activated signaling pathway in
the adipocytes.
)), prostaglandin E; ETYA,
eicosatetraenoic acid; BSA, bovine serum albumin; ELISA, enzyme-linked
immunosorbent assay; EMSA, electrophoretic mobility shift assay; NDGA,
nordihydroguaiaretic acid; HPETE, hydroperoxyeicosatetraenoic acid.
We thank Kimberly Seurynck and Ashlie Pruett for
expert technical assistance, Mary Peace Datillo for initiating the
studies with the various fatty acids, and Dr. Kevin McGowan for the
discussion that led to the experiment described in the legend to Fig. 3. We are grateful to Drs. Yusuf A. Hannun and Rick T.
Dobrowsky for teaching us the sphingomyelin turnover assay and to Dr.
Dean Londos for discussions of PGE action in adipocytes. In
addition we thank Drs. Bernlohr, Cornelius, Dohm, Kasperek, Tebbey,
Stephens, and Ways for their critical comments on the manuscript.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  S. Kehlenbrink, J. Tonelli, S. Koppaka, V. Chandramouli, M. Hawkins, and P. Kishore Inhibiting gluconeogenesis prevents fatty acid-induced increases in endogenous glucose production Am J Physiol Endocrinol Metab, July 1, 2009; 297(1): E165 - E173. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Kishore, J. Tonelli, S. Koppaka, C. Fratila, A. Bose, D.-E. Lee, K. Reddy, and M. Hawkins Time-Dependent Effects of Free Fatty Acids on Glucose Effectiveness in Type 2 Diabetes Diabetes, June 1, 2006; 55(6): 1761 - 1768. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Armoni, C. Harel, F. Bar-Yoseph, S. Milo, and E. Karnieli Free Fatty Acids Repress the GLUT4 Gene Expression in Cardiac Muscle via Novel Response Elements J. Biol. Chem., October 14, 2005; 280(41): 34786 - 34795. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. H. Liu, C. F. Kuo, Y. C. Wang, and S. T. Ding Effect of docosahexaenoic acid and arachidonic acid on the expression of adipocyte determination and differentiation-dependent factor 1 in differentiating porcine adipocytes J Anim Sci, July 1, 2005; 83(7): 1516 - 1525. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Haugen, N. Zahid, K. T. Dalen, K. Hollung, H. I. Nebb, and C. A. Drevon Resistin expression in 3T3-L1 adipocytes is reduced by arachidonic acid J. Lipid Res., January 1, 2005; 46(1): 143 - 153. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Gertow, M. Bellanda, P. Eriksson, S. Boquist, A. Hamsten, M. Sunnerhagen, and R. M. Fisher Genetic and Structural Evaluation of Fatty Acid Transport Protein-4 in Relation to Markers of the Insulin Resistance Syndrome J. Clin. Endocrinol. Metab., January 1, 2004; 89(1): 392 - 399. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Hawkins, J. Tonelli, P. Kishore, D. Stein, E. Ragucci, A. Gitig, and K. Reddy Contribution of Elevated Free Fatty Acid Levels to the Lack of Glucose Effectiveness in Type 2 Diabetes Diabetes, November 1, 2003; 52(11): 2748 - 2758. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Armoni, N. Kritz, C. Harel, F. Bar-Yoseph, H. Chen, M. J. Quon, and E. Karnieli Peroxisome Proliferator-activated Receptor-{gamma} Represses GLUT4 Promoter Activity in Primary Adipocytes, and Rosiglitazone Alleviates This Effect J. Biol. Chem., August 15, 2003; 278(33): 30614 - 30623. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Alaoui-El-Azher, Y. Wu, N. Havet, A. Israel, A. Lilienbaum, and L. Touqui Arachidonic Acid Differentially Affects Basal and Lipopolysaccharide-Induced sPLA2-IIA Expression in Alveolar Macrophages through NF-kappa B and PPAR-gamma -Dependent Pathways Mol. Pharmacol., April 1, 2002; 61(4): 786 - 794. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. J. Sidell, M. A. Cole, N. J. Draper, M. Desrois, R. E. Buckingham, and K. Clarke Thiazolidinedione Treatment Normalizes Insulin Resistance and Ischemic Injury in the Zucker Fatty Rat Heart Diabetes, April 1, 2002; 51(4): 1110 - 1117. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Han, A. J. Demetris, G. Michalopoulos, J. H. Shelhamer, and T. Wu 85-kDa cPLA2 plays a critical role in PPAR-mediated gene transcription in human hepatoma cells Am J Physiol Gastrointest Liver Physiol, April 1, 2002; 282(4): G586 - G597. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. M. Campbell, R. Kozak, A. Wagner, J. Y. Altarejos, J. R. B. Dyck, D. D. Belke, D. L. Severson, D. P. Kelly, and G. D. Lopaschuk A Role for Peroxisome Proliferator-activated Receptor alpha (PPARalpha ) in the Control of Cardiac Malonyl-CoA Levels. REDUCED FATTY ACID OXIDATION RATES AND INCREASED GLUCOSE OXIDATION RATES IN THE HEARTS OF MICE LACKING PPARalpha ARE ASSOCIATED WITH HIGHER CONCENTRATIONS OF MALONYL-CoA AND REDUCED EXPRESSION OF MALONYL-CoA DECARBOXYLASE J. Biol. Chem., February 1, 2002; 277(6): 4098 - 4103. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Brydon, L. Petit, P. Delagrange, A. D. Strosberg, and R. Jockers Functional Expression of MT2 (Mel1b) Melatonin Receptors in Human PAZ6 Adipocytes Endocrinology, October 1, 2001; 142(10): 4264 - 4271. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Krebs, M. Krssak, P. Nowotny, D. Weghuber, S. Gruber, V. Mlynarik, M. Bischof, H. Stingl, C. Fürnsinn, W. Waldhäusl, et al. Free Fatty Acids Inhibit the Glucose-Stimulated Increase of Intramuscular Glucose-6-Phosphate Concentration in Humans J. Clin. Endocrinol. Metab., May 1, 2001; 86(5): 2153 - 2160. [Abstract] [Full Text]
|
|
|
|
|
|
J. M. Ntambi Regulation of stearoyl-CoA desaturase by polyunsaturated fatty acids and cholesterol J. Lipid Res., September 1, 1999; 40(9): 1549 - 1558. [Abstract] [Full Text]
|
|
|
|
|
|
A. M. Lombardi, R. Fabris, F. Bassetto, R. Serra, A. Leturque, G. Federspil, J. Girard, and R. Vettor Hyperlactatemia reduces muscle glucose uptake and GLUT-4 mRNA while increasing (E1alpha )PDH gene expression in rat Am J Physiol Endocrinol Metab, May 1, 1999; 276(5): E922 - E929. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. P. Barry, M. G. Kazanietz, D. Pratico, and G. A. FitzGerald Arachidonic Acid in Platelet Microparticles Up-regulates Cyclooxygenase-2-dependent Prostaglandin Formation via a Protein Kinase C/Mitogen-activated Protein Kinase-dependent Pathway J. Biol. Chem., March 12, 1999; 274(11): 7545 - 7556. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. H. Lang, G. J. Nystrom, and R. A. Frost Regulation of IGF binding protein-1 in Hep G2 cells by cytokines and reactive oxygen species Am J Physiol Gastrointest Liver Physiol, March 1, 1999; 276(3): G719 - G727. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. K. Mater, D. Pan, W. G. Bergen, and D. B. Jump Arachidonic acid inhibits lipogenic gene expression in 3T3-L1 adipocytes through a prostanoid pathway J. Lipid Res., July 1, 1998; 39(7): 1327 - 1334. [Abstract] [Full Text]
|
|
|
|
 |
|
 F. M. GREGOIRE, C. M. SMAS, and H. S. SUL Understanding Adipocyte Differentiation Physiol Rev, July 1, 1998; 78(3): 783 - 809. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. M. Sessler and J. M. Ntambi Polyunsaturated Fatty Acid Regulation of Gene Expression J. Nutr., June 1, 1998; 128(6): 923 - 926. [Abstract] [Full Text]
|
|
|
|
|
|
M. J. Reginato, S. L. Krakow, S. T. Bailey, and M. A. Lazar Prostaglandins Promote and Block Adipogenesis through Opposing Effects on Peroxisome Proliferator-activated Receptor gamma J. Biol. Chem., January 23, 1998; 273(4): 1855 - 1858. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. M. Stuhlmeier, J. J. Kao, and F. H. Bach Arachidonic Acid Influences Proinflammatory Gene Induction by Stabilizing the Inhibitor-kappa Balpha /Nuclear Factor-kappa B (NF-kappa B) Complex, thus Suppressing the Nuclear Translocation of NF-kappa B J. Biol. Chem., September 26, 1997; 272(39): 24679 - 24683. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Chevillotte, J. Rieusset, M. Roques, M. Desage, and H. Vidal The Regulation of Uncoupling Protein-2 Gene Expression by omega -6 Polyunsaturated Fatty Acids in Human Skeletal Muscle Cells Involves Multiple Pathways, Including the Nuclear Receptor Peroxisome Proliferator-activated Receptor beta J. Biol. Chem., March 30, 2001; 276(14): 10853 - 10860. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Nugent, J. B. Prins, J. P. Whitehead, J. M. Wentworth, V. K. K. Chatterjee, and S. O'Rahilly Arachidonic Acid Stimulates Glucose Uptake in 3T3-L1 Adipocytes by Increasing GLUT1 and GLUT4 Levels at the Plasma Membrane. EVIDENCE FOR INVOLVEMENT OF LIPOXYGENASE METABOLITES AND PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR gamma J. Biol. Chem., March 16, 2001; 276(12): 9149 - 9157. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |