Download Polyunsaturated fatty acids stimulate hepatic UCP

Am J Physiol Endocrinol Metab
281: E1197–E1204, 2001.
Polyunsaturated fatty acids stimulate hepatic UCP-2
expression via a PPAR␣-mediated pathway
MICHAEL B. ARMSTRONG AND HOWARD C. TOWLE
Department of Biochemistry, Molecular Biology, and Biophysics,
University of Minnesota, Minneapolis, Minnesota 55455
Received 7 February 2001; accepted in final form 31 July 2001
uncoupling protein; prostaglandins; energy metabolism; peroxisome proliferator-activated receptor-␣
THE DISCOVERY OF TWO HOMOLOGS of the brown fat uncoupling protein (UCP) now called UCP-1 has rekindled
interest in the possibility that UCPs play a role in the
regulation of energy metabolism (13, 16). UCP-1, as
well as its homologs UCP-2 and UCP-3, is able to
deplete the mitochondrial proton gradient by allowing
the protons to pass through the inner mitochondrial
membrane without the production of ATP (19). Instead, the stored energy from the gradient intended for
ATP synthesis is converted to heat. When first described in the 1970s, UCP-1 was believed to play a role
in the regulation of weight in mammals, because it
could potentially reduce excess fuel supplies by dietinduced thermogenesis (31). In fact, selectively destroying brown adipose tissue, the only tissue in which
UCP-1 is expressed, led to obesity in mice (27). How-
Address for reprint requests and other correspondence: H. C.
Towle, 6–155 Jackson Hall, 321 Church St. SE, Minneapolis, MN
55455 (E-mail: [email protected]).
http://www.ajpendo.org
ever, adult humans have very little brown adipose
tissue, making it unlikely to be a major contributor to
human weight regulation. The discovery of UCP-2 and
UCP-3, which are more widely expressed, has revived
this hypothesis. UCP-2, like UCP-1, has been shown to
be able to dissipate the proton gradient when overexpressed in yeast or reconstituted in vesicles with coenzyme Q (12, 13, 16). Also, there is indirect evidence in
primary hepatocytes suggesting that UCP-2 is capable
of dissipating the mitochondrial proton gradient in
mammalian cells as well (8, 24). Additionally, obesityprone C57Bl/6 mice fail to upregulate UCP-2 message
levels in response to a high-fat diet, whereas an obesity-resistant strain exhibits a twofold increase (13,
35). These findings support UCP-2 as a potential candidate in the regulation of mammalian energy stores.
However, a recent report demonstrated that UCP-2 is
not capable of uncoupling when expressed at physiological levels in yeast (34), raising a question as to the
true role of UCP-2 in mammals.
The liver plays a major role in the regulation of
intermediary metabolism. It is the first organ to receive most of the absorbed nutrients from the gut.
Because the liver receives the bulk of these compounds,
it is a major source of pathways, such as glycolysis,
gluconeogenesis, lipolysis, and lipogenesis, central to
breakdown and processing of ingested nutrients. The
liver is also responsible for the maintenance of energy
stores via glycogen production and the export of fatty
acids and cholesterol. Because the liver plays such a
central role in the maintenance of overall energy homeostasis, it is under tight regulation by both hormonal and metabolic factors. Although some of these
regulatory pathways, such as the effects of glucagon on
glucose handling, have been well characterized, many
mechanisms of regulation have yet to be defined.
One of the major classes of substrate processed by
the liver is the fatty acids, which are known to play a
regulatory role in hepatic metabolism. They have been
demonstrated to upregulate expression of certain enzymes of ␤-oxidation, such as acyl-CoA oxidase, an
effect that can be elicited by saturated, monounsaturated, and polyunsaturated fatty acids (PUFAs) (22).
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.
0193-1849/01 $5.00 Copyright © 2001 the American Physiological Society
E1197
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on May 10, 2017
Armstrong, Michael B., and Howard C. Towle. Polyunsaturated fatty acids stimulate hepatic UCP-2 expression
via a PPAR␣-mediated pathway. Am J Physiol Endocrinol
Metab 281: E1197–E1204, 2001.—The discovery of homologs
of the brown fat uncoupling protein(s) (UCP) UCP-2 and
UCP-3 revived the hypothesis of uncoupling protein involvement in the regulation of energy metabolism. Thus we hypothesized that UCP-2 would be regulated in the hepatocyte
by fatty acids, which are known to control other energyrelated metabolic processes. Treatment with 250 ␮M
palmitic acid was without effect on UCP-2 expression,
whereas 250 ␮M oleic acid exhibited a modest eightfold
increase. Eicosapentaenoic acid (EPA), a polyunsaturated
fatty acid, exerted a 50-fold upregulation of UCP-2 that was
concentration dependent. This effect was seen within 12 h
and was maximal by 36 h. Aspirin blocked the induction of
UCP-2 by EPA, indicating involvement of the prostaglandin
pathway. Hepatocytes treated with arachidonic acid, the
immediate precursor to the prostaglandins, also exhibited an
aspirin-inhibitable increase in UCP-2 levels, further supporting the involvement of prostaglandins in regulating hepatic
UCP-2. The peroxisome proliferator-activated receptor-␣
(PPAR␣) agonist Wy-14643 stimulated UCP-2 mRNA levels
as effectively as EPA. These data indicate that UCP-2 is
upregulated by polyunsaturated fatty acids, potentially
through a prostaglandin/PPAR␣-mediated pathway.
E1198
REGULATION OF HEPATIC UCP-2 BY FATTY ACIDS
MATERIALS AND METHODS
Animals. Hepatocytes were obtained from Sprague-Dawley rats of 180–240 g maintained on a 12:12-h dark-light
cycle with free access to food and water. All animals were
handled in accordance with experimental protocols approved
by the University of Minnesota Institutional Committee on
the Care and Use of Animals.
Isolation of spleen and white adipose tissue. Sprague-Dawley rats were anesthetized, and the abdominal cavity was
opened. The epididymal fat pads and spleen were then removed. The entire fat pads and ⬃1 g of spleen tissue were
immediately rinsed twice in ice-cold PBS to remove residual
blood and were homogenized with a Teflon-on-glass homogenizer in TRIzol reagent (Life Technologies, Gaithersburg,
MD). Total RNA was then isolated according to the manufacturer’s protocol.
Primary hepatocyte isolation and culture. Primary hepatocytes were isolated on the basis of the procedure described
previously (4) and modified by Shih and Towle (32). Briefly,
the portal vein was cannulated, and the liver was perfused
AJP-Endocrinol Metab • VOL
with buffer (142 mM NaCl, 6.7 mM KCl, and 10 mM HEPES,
pH 7.4) to flush out the residual blood. The liver was then
perfused with collagenase (0.5 mg/ml in 66.7 mM NaCl, 6.7
mM KCl, 1 mM HEPES, 4.8 mM CaCl2, and 10 mg/ml
fatty-acid free BSA, pH 7.6; Wako BioProducts, Richmond,
VA) to disperse the cells in the liver. The hepatocytes were
purified by differential centrifugation on a 45% Percoll cushion. Cell viability was assessed by trypan blue staining and
was ⬎95%. Hepatocytes were plated on 35-mm Primeria
plates (Becton-Dickinson, Franklin, NJ) at a density of 1.2
million cells per plate. In all experiments, cells were incubated overnight in modified Williams E media (lacking
methyl linoleate and glucose) supplemented with 23 mM
HEPES, 26 mM NaHCO3, 0.1 ␮M dexamethasone, 0.1 U/ml
insulin, 1 U/ml penicillin, 1 ␮g/ml streptomycin, 2 mM Lglutamine, and 5.5 mM glucose before experimental treatment. After the plating incubation, 667 ␮g of Matrigel (Collaborative Biomedical Products, Bedford, MA) were added to
each plate to provide an extracellular matrix overlay (33).
Determination of the purity of hepatocyte preparation. To
determine the extent of Kupffer cell contamination in the
hepatocyte preparation, hepatocytes were taken after purification or after 36 h in culture and stained for macrophages.
The cells were washed in PBS and then fixed in 2% formaldehyde in PBS for 15 min at room temperature. The formaldehyde was removed, and the cells were washed in PBS and
pelleted. The cell pellet was resuspended in PBS ⫹ 1 mg/ml
BSA. FITC-tagged anti-rat macrophage antibody (Accurate
Chemical and Scientific, Westbury, NY) was added at a 1:16
dilution and incubated at room temperature in the dark for
15 min. Cells were washed in PBS, pelleted, and resuspended
in 50 ␮l of PBS. An aliquot of the sample was placed on a
microscope slide and viewed under fluorescence and bright
field. The number of cells that fluoresced from a total of
⬃2,000 cells of the freshly isolated cells or of 7,600 of the
cultured cells was counted using several random fields.
Stock solutions. Stock solutions of the fatty acids were
prepared in modified Williams E media with 5.5 mM glucose
and 2 mM BSA. Palmitic acid was made at a concentration of
10 mM, and oleic acid, arachidonic acid, and eicosapentaenoic acid (EPA) were prepared at a concentration of 100
mM. To prevent oxidation of the fatty acids, 1.1 mM butylated hydroxytoluene and 400 ␮M vitamin E were added to
the stocks. All fatty acid stocks were stored at ⫺70°C under
nitrogen. Wy-14643 and troglitazone were prepared as 10
mM stocks in DMSO. The 250 mM stock of aspirin (ASA) was
made in absolute ethanol.
Isolation of mRNA and synthesis of cDNA. Total RNA was
extracted from hepatocytes, homogenized white adipose tissue, and homogenized spleen tissue by use of the TRIzol
Reagent System (Life Technologies). First-strand cDNA was
generated from 1.5 ␮g of total RNA in a 30-␮l final volume
with 750 ng of oligo (dT) as a primer and Superscript II (Life
Technologies) as reverse transcriptase, according to the manufacturer’s protocol. Quality of the cDNA preparations was
routinely checked by amplifying aliquots with primers to
␤-actin cDNA.
Quantitation of mRNA by competitive RT-PCR. A competitive template was created consisting of the wild-type UCP-2
sequence with a 72-bp deletion interior to the primer sites, as
previously described (7). Increasing known amounts of competitive template were added to each experimental sample to
titrate the amount of wild-type cDNA present. The following
primers, which amplify a 218-bp product, were used: forward-5⬘-tta agt gtt tcg tct ccc agc; reverse-5⬘-gct cag cac agt
tga caa tgg. The resulting product from these primers was
sequenced and verified to be UCP-2. The mixed samples were
281 • DECEMBER 2001 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on May 10, 2017
Also, most enzymes involved in lipogenesis, including
fatty acid synthase, S14, and liver-type pyruvate kinase, are downregulated in the presence of fatty acids
(1, 9, 20, 21). However, unlike the effect seen with the
enzymes of ␤-oxidation, the inhibition of these genes
appears to be mediated only by the PUFAs (1, 20, 21).
These examples demonstrate the wide range of effects
and different specificities that fatty acids can have on
regulation in the hepatocyte.
One family of transcriptional regulators implicated
in mediating metabolic control of fatty acids in the liver
is the peroxisome proliferator-activated receptors
(PPAR). The PPAR family of transcription factors is
comprised of three subtypes: PPAR␣, PPAR␦, and
PPAR␥. PPAR␣ is expressed mainly in liver, kidney,
intestinal mucosa, and brown adipose tissue, all tissues that are known to have high catabolic rates for
fatty acids (5, 18). In the liver, PPAR␣ has been shown
to increase the expression of acyl-CoA oxidase in response to binding of various fatty acids (22). PPAR␦ is
ubiquitously expressed. Expression of PPAR␥2, one of
the two splice variants in the mouse, is limited to
adipose tissue and macrophages, whereas PPAR␥1 is
found in a fairly wide range of tissues (36, 37). PPAR␥
has been shown to modulate various genes involved in
the differentiation of the adipocyte, such as aP2 and
adipsin, and to be activated by the prostaglandin 15deoxy-⌬12,14-prostaglandin J2 in the adipocyte (15). Additionally, some groups have shown that PPAR␥ agonists are able to increase the expression of UCP-2
mRNA in adipocyte and muscle cells (3, 6).
In light of the hypothesized role of UCP-2 in energy
regulation and the central role of the hepatocyte in
overall energy metabolism, we proposed that UCP-2
would be regulated in the hepatocyte by fatty acids. In
these studies, we examine the effects of different fatty
acids on the expression of UCP-2 mRNA in primary rat
hepatocytes. Additionally, we search for possible mediators and pathways involved in exerting the effects
seen in response of UCP-2 gene expression to fatty
acids.
REGULATION OF HEPATIC UCP-2 BY FATTY ACIDS
amplified using 0.5 units of Biolase Taq polymerase (Bioline,
Reno, NV) for 35 cycles, as follows: 1.5-min denaturation at
95°C, 1-min annealing at 56°C, and 1.5-min extension at
72°C. Final products were separated on 2% agarose gels. The
ethidium bromide-stained bands were then quantified by
densitometry. The quantity of wild-type template was determined by plotting the competitive product-to-wild-type product ratio vs. the starting quantity of competitive template. At
the point on the linear curve where the ratio is one, the
amount of starting wild-type template is equal to the starting
amount of competitive template. Correlation coefficients of
the PCR curves were typically ⬎0.98.
Statistics. All results are reported as averages ⫾ SE unless
otherwise noted. Statistical significance is determined by
Student’s t-test.
RESULTS
Fig. 1. Effect of fatty acid treatment on hepatic uncoupling-protein 2
(UCP-2) expression. Hepatocytes were incubated for 36 h in 5.5 mM
(low) glucose in the absence or presence of 250 ␮M palmitic acid, 250
␮M oleic acid, or 100 ␮M eicosapentaenoic acid (EPA). After the
incubation period, cells were lysed and total RNA was isolated. The
RNA was reverse transcribed, and UCP-2 cDNA levels were measured by competitive PCR. Values are means ⫾ SE; n ⫽ 6. *P ⬍ 0.05
vs. low-glucose condition.
AJP-Endocrinol Metab • VOL
acid, with no response to palmitic acid, indicates that
regulation of UCP-2 by fatty acid is specific to the
unsaturated fatty acids, with a larger effect elicited by
the PUFAs. Additionally, it appears that the upregulation of UCP-2 by the fatty acids is a specific regulatory effect and not due to a general increase in the
availability of energy substrates, as evidenced by the
lack of a response to palmitic acid.
Because it has been shown that Kupffer cells contribute a major portion of the UCP-2 expression in the
liver of normal rats (26), it was important to assess the
extent of Kupffer cell contamination of our hepatocyte
cultures. A significant amount of Kupffer cells mixed in
with the hepatocytes could mask effects of treatment
by increasing background levels or exhibiting regulation patterns different from the hepatocyte, as has
been shown previously with lipopolysaccharide treatment (11). After purification of the hepatocytes or after
36 h of incubation in low-glucose media on culture
plates, cell aliquots were treated with an FITC-tagged
anti-macrophage antibody that binds Kupffer cells.
Cells were then viewed under fluorescence microscopy.
A sample photomicrograph in which a single Kupffer
cell is observed is shown in Fig. 2. In the freshly
purified hepatocyte preparation, Kupffer cells made up
0.32 ⫾ 0.03% (average and range of duplicate samples)
of the total cell population. However, after 36 h in
culture, the prevalence of Kupffer cells dropped to
0.053 ⫾ 0.004% (average and range of duplicate samples). UCP-2 measured in a purified hepatocyte preparation treated with oleic acid was 511 ⫾ 200 fg/␮g
total RNA, whereas UCP-2 levels measured in RNA
isolated from whole liver were 12,360 ⫾ 1,854 fg/␮g
total RNA (n ⫽ 3). Although the oleic acid condition
does not necessarily represent the physiological state
of a fed rat from which the liver samples were obtained,
the amount of cDNA seen is midrange of the states
encountered in these experiments. Thus, we utilized
that value as an estimation of baseline quantities of
UCP-2 in the hepatocytes of the whole animal. If we
assume that Kupffer cells make up 25% of the liver cell
population, for all of the UCP-2 to be produced by the
Kupffer cells in the purified hepatocyte population, the
calculated amount of UCP-2 in whole liver would be
2.56 ⫻ 105 fg/␮g total RNA. The major discord between
the calculated and measured levels of UCP-2 in the
whole liver is an indication that Kupffer cells are not
the sole source of UCP-2 cDNA. Also, the large changes
in expression of UCP-2 seen in these experiments are
unlikely to be caused solely by such a small population
of cells in our cultures. Thus the hepatocyte isolation
procedure provides us with the ability to measure
UCP-2 in the hepatocyte, enabling us to pursue physiological studies.
To better define the relative levels of UCP-2 expression, we compared the levels of expression found in
EPA-treated hepatocytes to the level in other tissues
known to express the gene. UCP-2 mRNA expression
in white adipose tissue and spleen has been characterized previously in other studies (14). RNA from these
tissues was isolated, and levels of expression were
281 • DECEMBER 2001 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on May 10, 2017
Given the central role of the hepatocyte in energy
fuel homeostasis, we were interested in determining
whether UCP-2 expression is regulated by fatty acids
in the hepatocyte. For this purpose, primary rat hepatocytes were purified and cultured in the presence of
various fatty acids. One representative from each major class of fatty acid, i.e., saturated, monounsaturated,
and polyunsaturated, was selected. Hepatocytes were
treated for 36 h in low-glucose media without or with
one of the selected fatty acids, and UCP-2 mRNA levels
were measured. As demonstrated in Fig. 1, 250 ␮M
palmitic acid, a saturated fatty acid, did not alter the
levels of UCP-2 mRNA vs. control. The monounsaturated fatty acid oleic acid did elicit a moderate effect at
a concentration of 250 ␮M. However, treating cells
with 100 ␮M EPA, a PUFA, resulted in a large increase
in UCP-2 expression. This response to EPA and oleic
E1199
E1200
REGULATION OF HEPATIC UCP-2 BY FATTY ACIDS
Fig. 2. Quantitation of Kupffer cells. Aliquots of purified hepatocytes were treated with an FITC-conjugated
anti-rat macrophage antibody at a 1:16 dilution. After
incubation with the antibody, cells were placed on a
glass microscope slide and viewed under fluorescence.
Cells detected under fluorescence were counted, as well
as the total no. of cells present under bright field. A
sample field is shown under bright field (A) and fluorescence (B). The Kupffer cell seen under fluorescence
is marked with the arrow in A.
Fig. 3. Concentration-dependent effects of EPA on UCP-2 expression. Hepatocytes were incubated for 36 h in low-glucose media with
varying concentrations of EPA. Total RNA was isolated, and UCP-2
cDNA levels were measured by competitive RT-PCR. Values are
means ⫾ SE; n ⫽ 3. *P ⬍ 0.05, **P ⬍ 0.025, †P ⬍ 0.005 vs. no EPA
control.
AJP-Endocrinol Metab • VOL
tration-dependent response with a maximal effect at
200 ␮M and a half-maximal effect occurring at ⬃100
␮M. This concentration dependence curve mirrors that
of the effects of EPA on S14, although EPA inhibits S14
expression (20). Next, we examined the time course of
the effect of EPA on UCP-2 expression. Hepatocytes
were treated with a maximal concentration of EPA for
varying amounts of time, after which UCP-2 levels
were quantified (Fig. 4). The effect of EPA on UCP-2
levels was seen within 12 h and appears to plateau by
36 h. To verify that the increases in UCP-2 expression
are not due to phenotypic changes of the primary
hepatocytes in culture, as noted previously by Kimura
et al. (24), we also incubated the cells in high-glucose
media without EPA (Fig. 4). The lack of upregulation of
UCP-2 in response to high-glucose media indicates
that the effect seen with EPA is not due to phenotypic
Fig. 4. Time course of EPA upregulation of UCP-2. Hepatocytes were
incubated in low-glucose medium with 200 ␮M EPA or in highglucose medium (27.5 mM) without EPA for various amounts of time.
After the appropriate incubation time, cells were lysed, and total
RNA was isolated. UCP-2 cDNA levels were measured as described.
Values are means ⫾ SE; n ⫽ 4, Low ⫹ EPA, or n ⫽ 6, High Glc. *P ⬍
0.005 vs. 0 h time point.
281 • DECEMBER 2001 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on May 10, 2017
measured. The amount of UCP-2 mRNA measured in
adipose tissue was 2,360 fg/␮g RNA (average of 3
animals; range 2,080–2,580). For spleen, a value of
11,720 fg/␮g RNA (n ⫽ 3; range 6,300–25,760) was
observed. Hence, the EPA-treated hepatocytes had
UCP-2 mRNA levels that were comparable to those
observed in adipose tissue, and, as would be anticipated from previous Northern analysis data, substantially higher levels were detected in spleen RNA (14).
In untreated hepatocytes, however, UCP-2 mRNA levels are substantially lower than those observed in the
other tissues.
Having established an upregulation of UCP-2 mRNA
expression to the treatment with EPA, we further
characterized this response. We first tested the concentration dependence of this effect. Hepatocytes were
treated with varying concentrations of EPA for 36 h,
and the resulting UCP-2 mRNA levels were measured
by competitive RT-PCR (Fig. 3). EPA elicited a concen-
REGULATION OF HEPATIC UCP-2 BY FATTY ACIDS
Fig. 5. Effect of peroxisome proliferator-activating receptor (PPAR)
agonists on UCP-2 expression in the hepatocyte. Hepatocytes were
incubated for 36 h in low-glucose media in the absence or presence of
200 ␮M EPA, 1 ␮M Wy-14643, or 2 ␮M troglitazone. After the
incubation, total RNA was isolated and the UCP-2 cDNA levels were
quantitated by competitive RT-PCR. Values are means ⫾ SE; n ⫽ 4.
*P ⬍ 0.005 vs. low-glucose control.
AJP-Endocrinol Metab • VOL
mit the effects of EPA on various genes in the liver. It
has been demonstrated that indomethacin, a potent
inhibitor of the prostaglandin pathway, is able to block
the EPA-mediated inhibition of apoB synthesis in the
liver (2). To test for the possibility of prostaglandin
involvement in the EPA stimulation of UCP-2 expression, hepatocytes were treated with arachidonic acid,
the immediate precursor to prostaglandin synthesis,
EPA, or Wy-14643 (Fig. 6). Additionally, aspirin was
added to the media to block prostaglandin synthesis in
these cells. As can be seen, aspirin is able to block the
stimulatory effects of both EPA and arachidonic acid.
However, the effect of Wy-14643, which works downstream of the prostaglandin pathway, was not affected
by the aspirin treatment. Additionally, treatment of
control cells with aspirin did not decrease UCP-2
mRNA levels, which along with the Wy-14643 data
indicate that the aspirin does not inhibit UCP-2 expression through a toxic effect. Overall, these data
suggest that the EPA-mediated stimulation of UCP-2
in the hepatocyte is exerted through the prostaglandin
signaling pathway.
DISCUSSION
Despite much work to define the regulation and
function of UCP-2 in various tissues, the true physiological function of UCP-2 remains controversial. Our
data demonstrate that UCP-2 mRNA is upregulated by
EPA in the hepatocyte. A modest effect is seen in
response to treatment with oleic acid, which was consistent with that seen by Cortez-Pinto et al. (10). However, in light of the magnitude of the EPA and arachidonic acid responses, the PUFAs elicit a much stronger
effect. The concentration curve of the upregulation of
UCP-2 by EPA mirrors that of the EPA effect on S14
gene expression (20). The time course of this effect
reveals that the increase in UCP-2 mRNA is delayed,
which could be an indication that the upregulation of
UCP-2 by EPA is a secondary response that requires
the transcription of another gene, such as a transcription factor, before activation of the UCP-2 promoter.
Because a PPAR response element has not been identified in the UCP-2 promoter to date (28), this could be
a likely explanation. Similar results with EPA have
been seen in vivo. Feeding mice with fish oil, which is
rich in PUFAs such as EPA, resulted in a significant
increase in liver UCP-2, whereas the monounsaturated
fatty acid-rich safflower oil group demonstrated only a
modest increase in UCP-2 expression (38). However, in
the whole animal experiment, it is not possible to
discern whether the increased UCP-2 cDNA is due to
changes in the hepatocyte or changes in other liver cell
types. Also, although it will be important to confirm
that UCP-2 protein levels increase in response to fatty
acid treatment, the large increases in mRNA levels
seen in these experiments clearly are suggestive of an
increase in protein levels as well.
After defining the effects of the various fatty acids on
UCP-2 expression, we identified potential mediators
for these effects. The ability of aspirin to block the
281 • DECEMBER 2001 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on May 10, 2017
changes of the hepatocyte and also supports the hypothesis that this effect is specific and not due to a
general increase in energy availability. This delayed
effect could indicate that the upregulation of UCP-2 by
EPA is by a secondary response rather than a primary
one, an indication that is supported by the observations of Medvedev et al. (28), which indicate that there
is no PPAR response element present on the UCP-2
promoter.
After defining the regulation of UCP-2 by EPA, we
sought to identify potential pathways involved in mediating this effect. The transcription factor PPAR␣ is
known to be involved in the regulation of genes in the
hepatocyte by fatty acids. Because of its involvement in
hepatic gene regulation, we tested various PPAR-specific agonists and measured their effect on UCP-2 expression in the hepatocyte. Cells were treated with
EPA, Wy-14643, or troglitazone for 36 h (Fig. 5). Wy14643, a PPAR␣ agonist, elicited a large increase in
UCP-2 mRNA levels, which was of similar magnitude
to that seen with EPA, although the response to EPA in
this experiment was somewhat less than seen previously. Troglitazone, a PPAR␥ agonist, also caused a
moderate increase in UCP-2. Because PPAR␥ is undetectable in the hepatocyte by Northern analysis (5), the
effect seen with troglitazone may be due to crossreactivity of the PPAR agonists among the different
subtypes. However, a direct effect of PPAR␥ certainly
cannot be ruled out. These data indicate that the effect
of EPA on UCP-2 expression may be largely mediated
by PPAR␣.
Having identified a possible mediator for the effect of
EPA on UCP-2 expression in the hepatocyte, we then
sought a potential signaling pathway that activates
PPAR␣. It is suspected that the prostaglandins trans-
E1201
E1202
REGULATION OF HEPATIC UCP-2 BY FATTY ACIDS
stimulatory effect of arachidonic acid and EPA indicates that the prostaglandin pathway is likely involved
as the signaling pathway. The blocking effect of aspirin
is not due to general cellular toxicity, as evidenced by
the full response to Wy-14643 in the presence of aspirin. These results are consistent with those seen in
other hepatic genes regulated by EPA. EPA-mediated
inhibition of apoB synthesis for VLDL production also
appears to be mediated through a prostaglandin signaling pathway (2). Treatment with indomethacin,
which blocks prostaglandin synthesis at the initial
step, alleviates the inhibition by EPA on these genes
(2). Together, these reports indicate that prostaglandins play a role in transmitting the effects of EPA in
the hepatocyte.
The transcription factor PPAR␣ plays a major role in
exerting the regulation of genes in the liver by fatty
acids. It appears to be able to respond to a wide range
of fatty acids and to inhibit or upregulate gene expression appropriately. As with other genes in the hepatocyte, UCP-2 appears to be activated through a PPAR␣mediated pathway. Treatment of hepatocytes with
Wy-14643 resulted in a significant increase in UCP-2
expression similar to that of EPA. This effect has also
been seen in vivo. Mice fed Wy-14643 in their diet
showed a fourfold increase in UCP-2 expression in the
liver (23). A ninefold increase was seen in response to
AJP-Endocrinol Metab • VOL
fenofibrate, another activator of PPAR␣ (38). The
smaller increase in UCP-2 expression seen in these
studies than we see in vitro could be due to measuring
UCP-2 from whole liver samples, because a large portion of UCP-2 comes from the Kupffer cells. A less
robust response from the Kupffer cells to the PPAR␣
agonists would blunt the overall measured response in
whole liver samples. The involvement of a PPAR␣mediated pathway is further supported by the fact that
PPAR␣ binds unsaturated fatty acids with a higher
affinity than saturated fatty acids (25). We saw no
response of UCP-2 expression to treatment with a
saturated fatty acid, whereas the unsaturated fatty
acids significantly upregulated UCP-2 expression.
Other members of the PPAR family have also been
implicated in the regulation of UCP-2. In white and
brown adipose tissue, activators of PPAR␥ increase
expression of UCP-2 (3, 6). Also, PPAR␦ agonists have
been shown to upregulate UCP-2 in preadipocytes (3).
These studies further support the results we see in the
hepatocyte, in which PPAR␣ is the predominant receptor subtype present.
Much of the work done on understanding UCP-2
expression has been undertaken with the goal of elucidating the physiological role of UCP-2. Although we
have a greater understanding of how the UCP-2 gene is
regulated, its role in physiology remains unclear. One
281 • DECEMBER 2001 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on May 10, 2017
Fig. 6. Effect of aspirin on the stimulation of UCP-2 by fatty acids and Wy-14643. Hepatocytes in low-glucose
media were treated with 200 ␮M EPA, 200 ␮M arachidonic acid (AA), or 1 ␮M Wy-14643. The effect of aspirin
(ASA) on UCP-2 regulation was assessed by the addition of 2.5 mM ASA to the media during the incubation. After
36 h, cells were lysed and total RNA was harvested. UCP-2 cDNA levels were quantitated as described. Values are
means ⫾ SE; n ⫽ 4. *P ⬍ 0.0005, **P ⬍ 0.01, †P ⫽ not significant.
REGULATION OF HEPATIC UCP-2 BY FATTY ACIDS
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
We thank Angela Dutcher and Dr. Seung-Hoi Koo for preparing
hepatocytes and for helpful discussions.
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-26919.
19.
REFERENCES
20.
1. Allmann DW and Gibson DW. Fatty acid synthesis during
early linoleic acid deficiency in the mouse. J Lipid Res 6: 51–60,
1965.
2. Anil K, Jayadeep A, and Sudhakaran PR. Effect of n-3 fatty
acids on VLDL production by hepatocytes is mediated through
prostaglandins. Biochem Mol Biol Int 43: 1071–1080, 1997.
3. Aubert J, Champigny O, Saint-Marc P, Negrel R, Collins S,
Ricquier D, and Aihaund G. Up-regulation of UCP-2 gene
expression by PPAR agonists in preadipose and adipose cells.
Biochem Biophys Res Commun 238: 606–611, 1997.
4. Berry MN and Friend DS. High-yield preparation of isolated
rat liver parenchymal cells: a biochemical and fine structural
study. J Cell Biol 43: 506–520, 1969.
5. Braissant O, Foufelle F, Scotto C, Dauca M, and Wahli W.
Differential expression of peroxisome proliferator-activated reAJP-Endocrinol Metab • VOL
21.
22.
23.
ceptors (PPARs): tissue distribution of PPAR-␣, -␤, and -␥ in the
adult rat. Endocrinology 137: 354–366, 1996.
Camirand A, Marie V, Rabelo R, and Silva JE. Thiazolidinediones stimulate uncoupling protein-2 expression in cell
lines representing white and brown adipose tissues and skeletal
muscle. Endocrinology 139: 428–431, 1998.
Celi FS, Zenilman ME, and Shuldiner AR. A rapid and
versatile method to synthesize internal standards for competitive PCR. Nucleic Acids Res 21: 1047, 1993.
Chavin KD, Yang S, Lin HZ, Chatham J, Chacko VP, Hoek
JB, Walajtys-Rode E, Rashid A, Chen CH, Huang CC, Wu
TC, Lane MD, and Diehl AM. Obesity induces expression of
uncoupling protein-2 in hepatocytes and promotes liver ATP
depletion. J Biol Chem 274: 5692–5700, 1999.
Clarke SD and Jump DB. Polyunsaturated fatty acid regulation of hepatic gene transcription. J Nutr 126: 1105S–1109S,
1996.
Cortez-Pinto H, Lin HZ, Yang SQ, Da Costa SO, and Diehl
AM. Lipids up-regulate uncoupling protein 2 expression in rat
hepatocytes. Gastroenterology 116: 1184–1193, 1999.
Cortez-Pinto H, Yang SQ, Lin HZ, Costa S, Hwang CS,
Lane MD, Bagby G, and Diehl AM. Bacterial lipopolysaccharide induces uncoupling protein-2 expression in hepatocytes by a
tumor necrosis factor-␣-dependent mechanism. Biochem Biophys Res Commun 251: 313–319, 1998.
Echtay KS, Winkler E, Frischmuth K, and Klingenberg M.
Uncoupling proteins 2 and 3 are highly active H⫹ transporters
and highly nucleotide sensitive when activated by coenzyme Q
(ubiquinone). Proc Natl Acad Sci USA 98: 1416–1421, 2001.
Fleury C, Neverova M, Collins S, Raimbault S, Champigny
O, Levi-Meyrueis C, Bouillaud F, Seldin MF, Surwit RS,
Ricquier D, and Warden CH. Uncoupling protein-2: a novel
gene linked to obesity and hyperinsulinemia. Nature Genet 15:
269–272, 1997.
Fleury C and Sanchis D. The mitochondrial uncoupling protein-2: current status. Int J Biochem Cell Biol 31: 1261–1278,
1999.
Forman BM, Tontonoz P, Chen J, Brun RP, Spiegelman
BM, and Evans RM. 15-Deoxy-delta 12, 14 prostaglandin J2 is
a ligand for the adipocyte determination factor PPAR gamma.
Cell 83: 803–812, 1995.
Gimeno RE, Dembski M, Weng X, Deng N, Shyjan AW,
Gimeno CJ, Iris F, Ellis SJ, Woolf EA, and Tartaglia LA.
Cloning and characterization of an uncoupling protein homolog:
a potential molecular mediator of human thermogenesis. Diabetes 46: 900–906, 1997.
Innoe I, Noji S, Shen MZ, Takahashi K, and Katayama S.
The peroxisome proliferator-activated receptor ␣ (PPAR␣) regulates the plasma thiobarbituric acid-reactive substance (TBARS)
level. Biochem Biophys Res Commun 237: 606–610, 1997.
Issemann I and Green S. Activation of a member of the steroid
hormone receptor superfamily by peroxisome proliferators. Nature 347: 645–650, 1990.
Jaburek M, VM, Gimeno RE, Dembski M, Jezek P, Zhang
M, Burn P, Tartaglia LA, and Garlid KD. Transport function
and regulation of mitochondrial uncoupling proteins 2 and 3.
J Biol Chem 2274: 26003–26007, 1999.
Jump DB, Clarke SD, MacDougald O, and Thelen A. Polyunsaturated fatty acids inhibit S14 gene transcription in rat liver
and cultured hepatocytes. Proc Natl Acad Sci USA 90: 8454–
8458, 1993.
Jump DB, Clarke SD, Thelen A, and Liimatta M. Coordinate
regulation of glycolytic and lipogenic gene expression by polyunsaturated fatty acids. J Lipid Res 35: 1076–1084, 1994.
Keller H, Dreyer C, Medin J, Mahfoudi A, Ozato K, and
Wahli W. Fatty acids and retinoids control lipid metabolism
through activation of peroxisome proliferator-activated receptorretinoid X receptor heterodimers. Proc Natl Acad Sci USA 90:
2160–2164, 1993.
Kelly LJ, Vicario PP, Thompson GM, Candelore MR,
Doebber TW, Ventre J, Wu MS, Meurer R, Forrest MJ,
Conner MW, Cascieri MA, and Moller DE. Peroxisome proliferator-activated receptors ␥ and ␣ mediate in vivo regulation
281 • DECEMBER 2001 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on May 10, 2017
hypothesis suggests that UCP-2 is capable of eliminating excess energy and hence plays a role in the regulation of energy stores. This hypothesis is supported by
studies demonstrating that UCP-2 is upregulated in
pancreatic islets by leptin, a protein known to be involved in energy regulation (39). Also, an obesity-prone
strain of mice, C57Bl/6J, fails to show an increase in
UCP-2 expression in response to a high-fat diet,
whereas an obesity-resistant strain increases expression twofold (13). Finally, the fibrates, which activate
PPAR␣, decrease serum triglyceride levels and body
weight in humans, indicating a potential role of PPAR␣
in body weight regulation (29). Our data indicate that
UCP-2 could potentially be involved, in that PPAR␣
agonists result in a large increase in UCP-2 expression.
Overall, these studies indicate a possible role of UCP-2
in the control of energy stores.
The second hypothesized physiological role for
UCP-2 is to reduce free radical formation within the
cell. The electron transport chain, which ultimately
transfers electrons onto oxygen in the process of forming water, is known to create superoxide radicals and
hydrogen peroxide (30). Induction of UCP-2 decreases
the redox pressure on the electron transport chain and
may reduce reactive oxygen species (8, 30). Additionally, it has been shown that the level of plasma thiobarbituric acid-reactive substance, a marker of lipid
peroxidation, is inversely proportional to liver PPAR␣
mRNA levels and was reduced by treatment of the
animals with EPA (17). This observation provides indirect evidence for UCP-2 playing a role in management of reactive oxygen species. Finally, macrophages,
which create a significant amount of free radicals, also
express high levels of UCP-2 (13, 16, 30). Because
macrophages are not thought to be active in controlling
overall metabolism, it is likely that they use UCP-2 to
protect themselves from free-radical damage. Hence,
the induction of UCP-2 in hepatocytes by EPA could
reflect a protective role against formation of free radicals in these cells. Further experimentation will be
necessary to distinguish between these potential physiological roles of UCP-2.
E1203
E1204
24.
25.
26.
27.
29.
30.
of uncoupling protein (UCP-1, UCP-2, UCP-3) gene expression.
Endocrinology 139: 4920–4927, 1998.
Kimura K, Jung BD, Kanehira K, Irie Y, Cañas X, and
Saito M. Induction of uncoupling protein (UCP) 2 in primary
cultured hepatocytes. FEBS Lett 457: 75–79, 1999.
Kliewer SA, Sundseth SS, Jones SA, Brown PJ, Wisely GB,
Koble CS, Devchand P, Wahli W, Willson TM, Lenhard JM,
and Lehmann JM. Fatty acids and eicosanoids regulate gene
expression through direct interactions with peroxisome proliferator-activated receptors alpha and gamma. Proc Natl Acad Sci
USA 94: 4318–4323, 1997.
Larrouy D, Lagarrague P, Carrera G, Viguerie-Bascands
N, Levi-Meyrueis C, Fleury C, Pecqueur C, Nibbelink M,
André M, Casteilla L, and Ricquier D. Kupffer cells are a
dominant site of uncoupling protein-2 expression in rat liver.
Biochem Biophys Res Commun 253: 760–764, 1997.
Lowell BB, Susulic VS, Hamann A, Lawitts JA, HimmsHagen J, Boyer BB, Kozak LP, and Flier JS. Development of
obesity in transgenic mice after genetic ablation of brown adipose tissue. Nature 366: 740–742, 1993.
Medvedev AV, Snedden SK, Raimbault S, Ricquier D, and
Collins S. Transcriptional regulation of the mouse uncoupling
protein-2 gene. J Biol Chem 276: 10817–10823, 2001.
Muls E, Van Gaal L, Autier P, and Vansant G. Effects of
initial BMI and on-treatment weight change on the lipid-lowering efficacy of fibrates. Int J Obes 21: 155–158, 1997.
Negré-Salvayre A, Hirtz C, Carrera G, Cazenave R, Troly
M, Salvayre R, Pénicaud L, and Casteilla L. A role for
uncoupling protein-2 as a regulator of mitochondrial hydrogen
peroxide generation. FASEB J 11: 809–815, 1997.
AJP-Endocrinol Metab • VOL
31. Rothwell NJ and Stock MJ. A role for brown adipose tissue in
diet-induced thermogenesis. Nature 281: 31–35, 1979.
32. Shih HM, Liu Z, and Towle HC. Two CACGTG motifs with
proper spacing dictate the carbohydrate regulation of hepatic
gene transcription. J Biol Chem 270: 21991–21997, 1995.
33. Shih HM and Towle HC. Matrigel treatment of primary
hepatocytes following DNA transfection enhances responsiveness to extracellular stimuli. Biotechniques 18: 813–814,
1995.
34. Stuart JA, Harper JA, Brindle KM, Jekabsons MB, and
Brand MD. Physiological levels of mammalian uncoupling protein 2 do not uncouple yeast mitochondria. J Biol Chem 276:
18633–18639, 2001.
35. Surwit RS, Wang S, Petro AE, Sanchis D, Raimbault S,
Ricquier D, and Collins S. Diet-induced changes in uncoupling proteins in obesity-prone and obesity-resistant strains of
mice. Proc Natl Acad Sci USA 95: 4061–4065, 1998.
36. Tontonoz P, Hu E, Graves RA, Budavari AI, and
Spiegelman BM. mPPAR␥2: tissue-specific regulator of an adipocyte enhancer. Gene Dev 8: 1224–1234, 1994.
37. Tontonoz P, Hu E, and Spiegelman BM. Stimulation of
adipogenesis in fibroblasts by PPAR␥2. Cell 79: 1147–1156,
1994.
38. Tsuboyama-Kasaoka N, Takahashi M, Kim H, and Ezaki
O. Up-regulation of liver uncoupling protein-2 mRNA by either
fish oil feeding or fibrate administration in mice. Biochem Biophys Res Commun 257: 879–885, 1999.
39. Zhou Y-T, Shimabukuro M, Koyama K, Lee Y, Wang M-Y,
Trieu F, Newgard CB, and Unger RH. Induction by leptin of
uncoupling protein-2 and enzymes of fatty acid oxidation. Proc
Natl Acad Sci USA 94: 6386–6390, 1997.
281 • DECEMBER 2001 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on May 10, 2017
28.
REGULATION OF HEPATIC UCP-2 BY FATTY ACIDS