(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 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.
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) ()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.
These in vitro results
are supported by dietary experiments demonstrating that high safflower
oil content diets (rich in -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.
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 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.
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 -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.
Figure 2:
Effect of prostaglandin E 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.
To further establish the role of PGE 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.
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- (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.
To determine if
AA was being converted to PGE 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.
Figure 4:
Arachidonic acid induction of PGE 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.
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 -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.
Figure 7:
Effect of AA on SM hydrolysis.
[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.
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 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.
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 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.
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. 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.
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 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.
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- 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.
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) (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 PPAR
2 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.
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 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.