Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455
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ABSTRACT |
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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
energy-related metabolic processes. Treatment with 250 µM palmitic
acid was without effect on UCP-2 expression, whereas 250 µM 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.
uncoupling protein; prostaglandins; energy metabolism; peroxisome
proliferator-activated receptor-
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INTRODUCTION |
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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 diet-induced thermogenesis (31). In fact, selectively destroying brown adipose tissue, the only tissue in which UCP-1 is expressed, led to obesity in mice (27). However, 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, obesity-prone 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). 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 15-deoxy-
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.
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MATERIALS AND METHODS |
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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 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 µM dexamethasone, 0.1 U/ml insulin, 1 U/ml penicillin, 1 µg/ml streptomycin, 2 mM L-glutamine, and 5.5 mM glucose before experimental treatment. After the plating incubation, 667 µg 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 µl 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 µM 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 µg of total RNA in a 30-µl 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 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.
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RESULTS |
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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 µM 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 µM.
However, treating cells with 100 µM EPA, a PUFA, resulted in a large
increase in UCP-2 expression. This response to EPA and oleic 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.
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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/µg total RNA, whereas UCP-2 levels measured in RNA
isolated from whole liver were 12,360 ± 1,854 fg/µg 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/µg 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.
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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 measured. The amount of UCP-2 mRNA measured in adipose tissue was 2,360 fg/µg RNA (average of 3 animals; range 2,080-2,580). For spleen, a value of 11,720 fg/µg 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
concentration-dependent response with a maximal effect at 200 µM and
a half-maximal effect occurring at ~100 µM. 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 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.
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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). Wy-14643, 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 cross-reactivity 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
.
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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
transmit 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.
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DISCUSSION |
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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 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 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 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.
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ACKNOWLEDGEMENTS |
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We thank Angela Dutcher and Dr. Seung-Hoi Koo for preparing hepatocytes and for helpful discussions.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-26919.
Address for reprint requests and other correspondence: H. C. Towle, 6-155 Jackson Hall, 321 Church St. SE, Minneapolis, MN 55455 (E-mail: towle{at}mail.ahc.umn.edu).
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.
Received 7 February 2001; accepted in final form 31 July 2001.
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