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and 
Mediate in Vivo Regulation of Uncoupling Protein (UCP-1, UCP-2, UCP-3) Gene Expression
Departments of Molecular Endocrinology (L.J.K., G.M.T., T.W.D., J.V., M.S.W., D.E.M.), Molecular Pharmacology and Biochemistry (P.P.V., M.R.C., M.A.C.), and Cellular and Molecular Pharmacology (R.M., M.J.F.), Merck Research Laboratories, Rahway, New Jersey 07065; and Department of Safety Assessment (M.W.C.), Merck Research Laboratories, West Point, Pennsylvania 19486
Address all correspondence and requests for reprints to: Dr. Linda Kelly, Merck Research Laboratories, 80W-207 P.O. Box 2000, Rahway, New Jersey 07065. E-mail: kelly_linda{at}merck.com
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and
PPAR
, as regulators of energy homeostasis and lipid metabolism, has
been suggested. Recently, three distinct uncoupling protein isoforms,
UCP-1, UCP-2, and UCP-3, have also been identified and implicated as
mediators of thermogenesis. Here, we examined whether in
vivo PPAR or PPAR activation regulates the expression of
all three UCP isoforms. Rats or lean and db/db mice were
treated with PPAR [thiazolidinedione (TZD)] or PPAR (WY-14643)
agonists, followed by measurement of messenger RNAs (mRNAs) for
UCP-1, UCP-2, and UCP-3 in selected tissues where they are expressed.
TZD treatment (AD 5075 at 5 mg/kg·day) of rats (14 days) increased
brown adipose tissue (BAT) depot size and induced the expression of
each UCP mRNA (3x control levels for UCP-1 and UCP-2, 2.5x control
for UCP-3). In contrast, UCP-2 and UCP-3 mRNA levels were not affected
in white adipose tissue or skeletal muscle. Chronic (30 days) low-dose
(0.3 mg/kg·day) TZD treatment induced UCP-1 mRNA and protein in BAT
(2.5x control). In contrast, chronic TZD treatment (30 mg/kg·day)
suppressed UCP-1 mRNA (>80%) and protein (50%) expression in BAT.
This was associated with further induction of UCP-2 expression
(>10-fold) and an increase in the size of lipid vacuoles, a decrease
in the number of lipid vacuoles in each adipocyte, and an increase in
the size of the adipocytes. TZD treatment of db/db mice
(BRL 49653 at 10 mg/kg·day for 10 days) also induced
UCP-1 and UCP-3 (but not UCP-2) expression in BAT. PPAR is present
in BAT, as well as liver. Treatment of rats or db/db
mice with WY-14643 did not affect expression of UCP-1, -2, or -3 in
BAT. Hepatic UCP-2 mRNA was increased (4x control level) in
db/db and lean mice, although this effect was not
observed in rats. Thus, in vivo PPAR activation can
induce expression of UCP-1, -2, and -3 in BAT; whereas chronic-intense
PPAR activation may cause BAT to assume white adipose tissue-like
phenotype with increased UCP-2 levels. PPAR activation in mice is
sufficient to induce liver UCP-2 expression. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An elegant demonstration of the importance of BAT in rodents was achieved by Lowell et al., who generated BAT-deficient transgenic mice via expression of diphtheria toxin under the control of the UCP-1 promoter (6). These mice developed marked obesity, cold intolerance, and a surprising increase in food intake (6). In contrast to the striking effects of in vivo BAT ablation, targeted disruption of the mouse of UCP-1 gene did not result in an obese phenotype, although the mice were modestly more sensitive to cold exposure (7), suggesting the presence of other thermogenic regulatory proteins in BAT.
Recently, two other mammalian UCP isoforms (UCP-2 and UCP-3) have been identified. UCP-2 has 59% amino acid identity to UCP-1; it is expressed in a variety of tissues, including BAT, white adipose tissue (WAT), and liver (8). UCP-2 has been implicated as a mediator of thermogenesis, because its expression in WAT is induced by high-fat feeding (8) and in ob/ob vs. lean mice (9). UCP-3, which is 57% identical to UCP-1 (73% vs. UCP-2) is expressed predominantly in BAT and in skeletal muscle (10, 11). Because its expression is induced by a ß3-adrenergic receptor agonist (in WAT) or by thyroid hormone (in BAT, muscle), UCP-3 is also likely to serve a role in thermoregulation (12). Importantly, both UCP-2 (8, 9) and UCP-3 (12) affected mitochondrial membrane potential when expressed in yeast, suggesting that, like UCP-1, either new isoform can function to uncouple oxidative phosphorylation.
The function of UCP-1 in BAT is predominantly regulated by
transcriptional control (13). In addition to regulation by thyroid
hormone and catecholamines (via cAMP), expression and activation of a
specific nuclear receptor [peroxisome proliferator-activated receptor
(PPAR)] has been proposed as a mechanism for induction of BAT
differentiation and UCP-1 gene expression (13). Indeed, in
vivo treatment with thiazolidinedione (TZD) compounds, which are
selective PPAR agonists (14, 15), has been reported to stimulate an
increase in BAT mass (16, 17) and to induce UCP-1 messenger RNA (mRNA)
expression in this tissue (16). PPAR is expressed at high levels in
both BAT and WAT (18, 19). A related nuclear receptor, PPAR, is
predominantly expressed in liver (20) but is also present in BAT
(21).
Given the potential for regulation of UCP-1 gene expression by PPAR,
and that both PPAR and PPAR are implicated as regulators of lipid
metabolism and energy homeostasis, we sought to assess the effect of
in vivo activation of either receptor on the expression of
all three UCP isoforms in rats and mice. Animals were treated with
potent TZD PPAR agonists or with a specific PPAR agonist,
followed by the determination of UCP-1, UCP-2, and UCP-3 levels in
tissues where they are known to be expressed. We confirmed that PPAR
activation stimulates UCP-1 mRNA expression in BAT, and we showed that
UCP-1 protein levels are also affected. In addition, we found that
in vivo activation of PPAR modulates both UCP-2 and UCP-3
mRNA expression. Finally, we present evidence which suggests that
activation of PPAR can induce hepatic UCP-2 expression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials
Two TZD PPAR agonist compounds (14) were studied: AD-5075
(5-[4-[2-(5
methyl-2-phenyl-4-oxazolyl)-2-hydroxyethoxy]benzyl]-2,4-TZD) and
BRL 49653
(5-(4-[2-[methyl-(2-pyridyl)amino]ethoxy]benzyl)thiazolidine-2,4-dione);
these were kindly provided by Gerard Kieczykowski, Philip Eskola,
Conrad Santini, Joseph Leone, and Peter Cicala (Merck Research
Laboratories). A known PPAR-selective agonist (22) (WY14643
[4-chloro-6-(2, 3-xylidino)-2-pyrimidinylthio]acetic acid) was
purchased from Chemson Science Laboratory (Lomexa, KS).
[32P]deoxy-cytosine triphosphate was obtained from
DuPont-New England Nuclear. Taq polymerase was purchased
from Perkin Elmer Cetus (Emeryville, CA). A polyclonal
antibody, specific for UCP-1 (hamster), was kindly provided by Dr. Jean
Himms-Hagen (University of Ottawa, Ottawa, Ontario, Canada). All other
chemicals were obtained from Sigma Chemical Co. (St.
Louis, MO).
RNA isolation
Frozen tissues were pulverized and then homogenized in 10 vol
ULTRA SPEC buffer (Biotecx Laboratories, Inc., Houston, TX), in
accordance with the manufacturer’s specifications, followed by
extraction of total RNA, as described (23). Aliquots of total RNA
(10–20 µg) were denatured in a solution of 2.2 M
formaldehyde and 50% formamide, 1x 3-[N-morpholino]
propanesulfonic acid/EDTA buffer (Digene Diagnostics, Inc., Beltsville,
MD), and 0.01 mg/ml ethidium bromide, by heating at 70 C for 10 min.
The samples were cooled, and gel loading buffer (0.5% xylene cyanol,
0.5% bromophenol blue, 40% sucrose 2.2 M formaldehyde and
50% formamide) was added. The samples were then loaded onto 1.2%
SeaKem Gold agarose (FMC BioProducts, Rockland, ME) gels in 1x
3-[N-morpholino]propanesulfonic acid. After
electrophoresis, RNA was transferred to Duralon-UV membranes
(Stratagene, La Jolla, CA) overnight in 20x saline-sodium
phosphate EDTA (SSPE; 3.0 M NaCl, 0.2 M
NaH2PO4, 0.02 M EDTA sodium, pH
7.4) (Digene Diagnostics, Inc.) (24). After UV cross-linking with a UV
Crosslinker 1800 (Stratagene), Northern blots were
prehybridized in Express-Hyb (CLONTECH Laboratories, Inc.,
Palo Alto, CA) at 60 C for 1 h, then hybridized to specific
32P-labeled complementary DNA (cDNA) probes at a
concentration of 1–4 x 106 cpm/ml. The hybridization
was carried out for at least 16 h at 60 C. The blots were washed
twice in 2x SSPE, 0.1% SDS at 60 C for 30 min each and then twice in
0.1x SSPE, 0.1% SDS at 50 C for 10 min. The membranes were sealed in
plastic and exposed to a PhosphorImager screen. The screens were
analyzed on a Molecular Dynamics, Inc. PhosphorImager
(Sunnyvale, CA) with the ImageQuant program.
Generation of cDNA probes
First-strand cDNA synthesis was achieved using reverse
transcriptase and other reagents from Boehringer Mannheim
(Indianapolis, IN). The specific cDNA fragments corresponding to
individual UCP isoforms were generated by RT-PCR from total RNA
isolated from BAT of rat. A 287-bp rat UCP-1 cDNA fragment was
amplified using the following PCR primer pair:
5'-AACACTGTGGAAAGGGACGAC-3' (coding strand), 5'-CATGGTCATTGCACAGCTG-3'
(noncoding strand). A 310-bp rat UCP-2 fragment was amplified from rat
skeletal muscle cDNA using the following PCR primer pair:
5'-CTGAGCTGGTGACCTATGAC-3' (coding strand) and
5'-CAAGCTGCTCAATAGGTGAC-3' (noncoding strand). The noncoding strand
primer was then used to synthesize a 32P-labeled
single-stranded antisense probe. An 813-bp rat UCP-3 cDNA probe was
amplified from rat adipose tissue RNA using a coding strand primer,
5'-ATGGTTGGACTGAAGCCTTCA-3', and a noncoding strand primer,
5'-TGTGGGGCCCTCCTGGGCCAC-3'. These primer sequences were based on
published rodent sequences. The PCR products for UCP-1 and
UCP-3 were gel-purified and used as a template for random prime
labeling (Gibco BRL, Gaithersburg, MD). Northern blots
were stripped and rehybridized with a glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) cDNA probe to control for small differences in
RNA loading and transferring.
Western analysis
Frozen tissue was pulverized and then homogenized in a buffer
containing 50 mM Tris (pH 7.2), 10% glycerol, 0.2
mM EDTA, 0.5 mM dithiothreitol,
phenylmethlysulfonylflouride (75 µg/ml), and leupeptin (1 µg/ml).
The solution was adjusted to contain 150 mM NaCl and 1%
Triton X-100. The protein lysate was centrifuged, followed by removal
of the lipid layer; aliquots were stored at -80 C. Ten-microgram
aliquots of protein from the tissue homogenates were separated by
SDS-PAGE (8–16% Tris-glycine gel; Novex, San Diego, CA); the protein was transferred overnight at 20 V to
nitrocellulose (Hybond ECL, Amersham, Arlington Heights, IL).
Before incubation with a hamster UCP-1 antibody, filters were blocked
in Tris-buffered saline (Novex) containing 0.1% Tween and
5% low fat milk. After washing in Tris-buffered saline containing
0.1% Tween, blots were incubated with an antirabbit IgG antibody
conjugated to horseradish peroxidase. The UCP-1 band was detected using
the fluorescent substrate ATTOPHOS (JBL Scientific Inc., San Luis
Obispo, CA) and the signal was quantitated on a FluorImager
(Molecular Dynamics, Inc.) with the ImageQuant program.
Western analysis, using increasing concentrations of homogenized
tissue, produced a linear response. Analysis of each sample was
performed on two separate gels. Data obtained from each gel was
normalized to one vehicle sample before averaging the two values for
each sample. Protein concentrations were determined using the Bradford
reagent.
Microscopic examination
At necropsy, interscapular BAT and epididymal fat were collected
from the Sprague-Dawley rats and fixed in 10% neutral buffered
formalin. Tissues were processed by routine methods and embedded in
paraffin. Sections of approximately 5-µm thickness were stained with
hematozylin and eosin for microscopic examination.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in lean
rats ligand (AD 5075) for
14 days. Treatment with this dose (5 mg/kg·day) of TZD was previously
shown to result in marked lowering of elevated glucose and
triglycerides in insulin-resistant diabetic mice and rats (14, 25).
After treatment, the animals were euthanized, and the tissues were
removed. There was a noticeable increase in size of the interscapular
BAT depot (2- to 4-fold increase in weight, data not shown), as
previously reported (17). Quantization of UCP-1 mRNA expression in BAT,
by Northern analysis, revealed that TZD treatment increased expression
300% of control levels (Fig. 1
). In
contrast, GAPDH mRNA levels were not significantly affected (Fig. 1A),
allowing for normalization of the data, using this marker. It is
important to note that the increase in BAT depot weight would
contribute to an even greater increase in net UCP-1 mRNA, expressed per
individual animal’s interscapular depot. Thus, we calculated that a
mean increase of 9-fold occurred in the treated rats, compared with
controls.
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).
Thus, treatment of lean rats for 14 days with TZD resulted in an
increase in BAT depot weight, as well as an increase in the expression
of all three UCP genes in this tissue. In contrast to the effect of TZD
treatment on the expression of UCP isoforms in BAT, the expression of
mRNAs for UCP-2 or UCP-3, in both hind-leg muscle and WAT (epididymal
fat pad), did not change in response to this regimen of TZD treatment.
In addition, the expression of UCP-2 in heart was unaffected.
Effect of PPAR activation on UCP expression in BAT from db/db
mice
PPAR agonists have been shown to be effective
insulin-sensitizers when administered to obese diabetic rodents, such
as the db/db mouse model. Selective TZD PPAR ligands, AD
5075 and BRL 49653 (at oral doses of 
1.7 mg/kg·day and
3 mg/kg·day, respectively), were previously shown to result in
normalization of hyperglycemia and hypertriglyceridemia when
administered to db/db mice for more than 10 days (14).
Concomitantly, the mass of the interscapular BAT depot was increased
approximately 100–150% in these mice treated with the PPAR
ligands. In the present study, BRL 49653 was administered
to db/db mice at 10 mg/kg·day for 10 days, which resulted
in 70–80% correction of hyperglycemia and normalization of increased
triglycerides (not shown). As depicted in Fig. 2, mean UCP-1 mRNA levels in BAT were
increased to 200% of the control. However, the expression of UCP-3
mRNA was only 150% of control, and there was no significant effect on
the mean level of UCP-2 mRNA expression. A group of db/db
mice (n = 6) were also treated with either vehicle or AD 5075 (2
mg/kg·day) for 10 days. At this dose of TZD, there was a
normalization of plasma glucose and triglyceride levels. Again, UCP2
mRNA levels were unchanged after AD 5075 treatment, and UCP1 mRNA
levels were increased 2.7-fold (P = 0.02, data not
shown). These results reflect those seen in db/db mice
treated with BRL 49653 (Fig. 2).
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, only the very lowest dose of
TZD (0.3 mg/kg·day) was associated with an increase (2.5 times that
of control) in UCP-1 mRNA expression. As the dose of TZD increased, the
level of UCP-1 mRNA expression decreased; in contrast to the increase
in UCP-1 mRNA observed after 10 days treatment with 5 mg/kg·day, the
3-mg/kg·day dose was not associated with a net change in UCP-1
expression after 30 days; the highest dose, 30 mg/kg·day, resulted in
a dramatic decrease in expression. Changes in UCP-1 mRNA levels were
also reflected by parallel effects on UCP-1 protein content, as
measured by Western blot analysis (Fig. 3, B and D). The
0.3-mg/kg·day dose resulted in levels that were 2.5-fold of control
mean protein levels; whereas treatment with 30 mg/kg·day caused a
50% decrease in mean protein levels. Although relative levels of UCP-1
were lower in BAT from rats treated with the 30-mg/kg·day dose, as
were both total RNA and protein/g tissue, there was a substantial
increase in BAT depot weight, such that we calculated a net increase of
6-fold in the amount of UCP-1 per individual animal’s interscapular
depot.
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agonists. to
increase BAT depot size in normal rats, is not solely caused by
stimulation of BAT differentiation, because the greatest increase in
interscapular depot weight was associated with decreased UCP-1
expression (per µg total RNA). In conjunction with the marked
increase in interscapular fat depot weight, we also observed in this
study that chronic high-dose TZD treatment was associated with striking
morphologic changes, including increased BAT cell size and decreased
lipid droplet number with an increase in droplet size (Fig. 4). Given that UCP-2 is expressed in both
BAT and WAT (10), one might expect that transdifferentiation from BAT
to WAT would be associated with reduced UCP-1, but sustained UCP-2
expression. In fact, mean UCP-2 mRNA levels in the interscapular fat
depot were progressively increased with increasing TZD doses after 30
days of treatment (Fig. 3C). At the 30-mg/kg·day dose, where UCP-1
mRNA levels were suppressed by 90%, mean UCP-2 mRNA levels were
increased by 13-fold.
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activation on UCP-2 expression, which is highly expressed in WAT and BAT, PPAR
is predominantly expressed in liver; and it is also present in other
tissues, including BAT, heart, and kidney (21). Because the expression
pattern of UCP-2 overlaps with that of PPAR (8, 9), we treated rats
and mice with a potent and selective PPAR agonist, WY14643, followed
by assessment of UCP-2 mRNA expression in selected tissues. As depicted
in Fig. 5, treatment of either obese
db/db or lean db/+ mice, with WY14643 at 10
mg/kg·day for 10 days, resulted in mean UCP-2 mRNA levels that were
400% of mean values for vehicle-treated controls. We also verified
that this dose of WY14643 caused other expected consequences of
in vivo PPAR activation: in livers from treated
db/db mice, mean mRNA levels of acyl Co-A oxidase and fatty
acid-binding protein were increased 2.6- (P = 0.01) and
2.8-fold (P = 0.015), respectively (data not shown). In
addition, elevated plasma triglyceride levels were reduced by 50%
(data not shown). In contrast to the effect of WY14643 on mouse liver
UCP-2, the same treatment protocol did not affect mean UCP-2 mRNA
levels in livers from either lean normal or obese ZDF rats (not shown).
WY14643 treatment also had no effect on the expression of UCP-2 in
heart or on expression of either UCP-1, UCP-2, or UCP-3 in BAT from
both db/db mice and lean normal rats.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is highly expressed in both BAT and WAT. Although
treatment with TZD PPAR agonists is known to suppress circulating
FFA levels, older evidence suggested that treatment of obese
ob/ob mice with ciglitazone (a weak TZD PPAR ligand)
could increase BAT-mediated thermogenesis, as assessed by an increase
in GDP binding to mitochondria after cold exposure (26). In addition,
Rothwell et al. (27) noted that ciglitazone treatment of
normal rats caused an increase in BAT mitochondrial GDP binding, along
with a modest increase in resting energy expenditure. In a subsequent
study, Foellmi-Adams et al. (16) observed that treatment of
both lean and obese (ob/ob) mice with
Pioglitazone resulted in an increase in interscapular BAT
mass and UCP-1 mRNA expression in this depot. Our results confirm these
findings in both lean rats and db/db mice. Furthermore, we
have established that in vivo PPAR activation is
sufficient to induce UCP-1 protein expression in BAT. In separate
experiments, using a model system (ZDF rats), where there is little
sympathetic signaling to BAT to maintain UCP-1 mRNA levels, we recently
documented an even greater induction of BAT mass and UCP-1 expression
(>5-fold for both protein and mRNA) after in vivo TZD
treatment (Kelly and Doebber, unpublished results).
PPAR is well described as an important mediator of
white adipocyte differentiation (28). The results of published in
vitro studies also indicate that stimulation with PPAR agonists
is sufficient to induce differentiation of cultured brown
preadipocytes, along with up-regulation of UCP-1 gene expression (16, 17, 29). Sears et al. (30) identified an enhancer element
within the UCP-1 promoter that seems to contain a PPAR response
element. In contrast, Rabelo et al. (29) suggest that the
effect of PPAR on the UCP-1 promoter may be indirect (via increasing
sensitivity to adrenergic stimulation mediated by multiple
cAMP-response elements).
In addition to the effect of TZD treatment on UCP-1, our results
demonstrate, for the first time, that in vivo PPAR
activation is sufficient to increase the expression of both UCP-2 and
UCP-3 mRNAs in BAT. Of interest, a very recent report by Camirand
et al. (31) showed that in vitro incubation of a
cultured brown adipocyte cell line (HIB-1B) with TZDs also resulted in
stimulation of UCP-2 mRNA expression. Despite the effects on UCP-2 and
UCP-3 that we observed in BAT, we did not detect an effect of TZD
treatment on UCP-2 and UCP-3 mRNA levels in other tissues (WAT,
muscle), which are known to express both of these UCP isoforms (8, 10, 11) and PPAR (18). These results seem to differ from an apparent
PPAR-mediated increase in UCP-2 expression noted in cultured 3T3-L1
adipocytes (31, 32) or cultured L6 myocytes (31). Several additional
recent studies show that in vivo expression of both UCP-2
and UCP-3 can be modulated by other physiologic perturbations that
affect energy homeostasis. The fact that UCP-2 is constitutively
up-regulated in WAT from ob/ob and db/db mice
suggests a compensatory role for this isoform in the context of severe
genetic obesity (9). Rats subjected to cold exposure or fasting were
also shown to have higher UCP-2 expression in BAT and muscle (33).
UCP-3 gene expression in rats is apparently induced by fasting and
thyroid hormone (in muscle) or by stimulation with a ß3 adrenergic
agonist (in WAT) but is unaffected by cold exposure (12, 34).
Furthermore, recently reported RT-competitive PCR experiments seem to
suggest that both UCP-2 and UCP-3 mRNA levels are up-regulated in human
WAT and skeletal muscle during caloric restriction (35). Although
induction of UCP-2 and UCP-3 gene expression seems to be a consistent
metabolic adaptation to fasting, it is unclear how this relates to
their putative function as mediators of thermogenesis.
Given that PPAR promotes differentiation of both brown and white
adipocytes, it is likely that additional trans-acting
factors contribute to terminal differentiation that is specific to
either lineage. Indeed, results we obtained after administration of
increasing TZD doses to lean rats for 30 days suggest that different
degrees of PPAR activation can promote either increased UCP-1
expression (a characteristic of BAT) or a relative decrease in UCP-1
expression coincident with a WAT-like appearance and further induction
of UCP-2. Interestingly, Enerback et al. (7) not only noted
an increase in BAT UCP-2 expression in UCP-1 null mice but also in
transgenic mice with overexpression of glycerol-3-phosphate
dehydrogenase. In this context, BAT morphology was also clearly
modified by an increase in lipid accumulation (36). This finding
suggests that TZD-mediated regulation of UCP-2 may have occurred as a
secondary consequence of changes in intracellular lipid content, rather
than as a direct effect of PPAR on the gene promoter. Perhaps the
effect of fasting on UCP-2 (and UCP-3) is also mediated by changes in
lipid metabolism (e.g. increased FFA levels).
Having demonstrated that in vivo activation of PPAR by
potent and selective TZDs can modulate the expression of UCP isoforms
in BAT and BAT morphology, we sought to also test whether in
vivo treatment with a PPAR agonist might affect UCP gene
expression. Although PPAR is expressed in BAT (21), treatment with a
dose of WY14643 that is sufficient to lower triglycerides and induce
hepatic expression of known target genes (liver fatty acid-binding
protein and acyl Co-A oxidase) did not seem to change the mRNA levels
for all three UCPs. Liver is a predominant site of PPAR expression
where UCP-2 is also expressed. Here, we noted a reproducible effect of
WY14643, to significantly induce UCP-2 mRNA levels in both lean and
obese mice. Of interest, one previously published report showed that
in vivo treatment of Zucker fa/fa rats with nafenopin, a
known peroxisome proliferator, was associated with a relative increase
in metabolic rate and decreased body fat content, when compared with
untreated, but pair-fed, obese rats (37). Although we did not detect a
significant effect of WY14643 treatment on hepatic UCP-2 expression in
lean or ZDF rats, it is plausible that the effects noted by
Assimacopoulos-Jeannet, et al. (37) were, in part, mediated
by increased expression of UCP-2. Further studies will be required to
explore this question.
In summary, we demonstrated that in vivo administration of a
potent and selective TZD PPAR agonist to normal rats for 14 days
resulted in up-regulation of all three UCP isoforms in BAT without
affecting UCP-2 or UCP-3 in either WAT or muscle. Similar treatment of
obese (db/db) mice was associated with induction of UCP-1
and UCP-3 in BAT. In contrast to the effects seen in BAT of rats after
14 days, longer-term treatment of normal rats with higher-dose TZD
caused marked down-regulation of UCP-1 expression in BAT (with a
relative increase in UCP-2), along with histologic changes consistent
with a shift toward a WAT phenotype. In addition, we showed that
in vivo treatment of mice with a selective PPAR agonist
was sufficient to induce an increase in hepatic UCP-2 gene expression.
These data suggest that in vivo activation of either PPAR
or PPAR can function to modulate the expression of one or more UCP
isoforms. This may underlie the known (and now emerging) effects of
these receptors as important regulators of energy balance.
Received March 30, 1998.
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 L. S. Szczepaniak, R. G. Victor, L. Orci, and R. H. Unger Forgotten but Not Gone: The Rediscovery of Fatty Heart, the Most Common Unrecognized Disease in America Circ. Res., October 12, 2007; 101(8): 759 - 767. [Abstract] [Full Text] [PDF]
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 S. Ghosh, N. Patel, D. Rahn, J. McAllister, S. Sadeghi, G. Horwitz, D. Berry, K. X. Wang, and R. H. Swerdlow The Thiazolidinedione Pioglitazone Alters Mitochondrial Function in Human Neuron-Like Cells Mol. Pharmacol., June 1, 2007; 71(6): 1695 - 1702. [Abstract] [Full Text] [PDF]
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 A. G. Smith and G. E. O. Muscat Orphan nuclear receptors: therapeutic opportunities in skeletal muscle Am J Physiol Cell Physiol, August 1, 2006; 291(2): C203 - C217. [Abstract] [Full Text] [PDF]
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S. Franckhauser, S. Munoz, I. Elias, T. Ferre, and F. Bosch Adipose Overexpression of Phosphoenolpyruvate Carboxykinase Leads to High Susceptibility to Diet-Induced Insulin Resistance and Obesity Diabetes, February 1, 2006; 55(2): 273 - 280. [Abstract] [Full Text] [PDF]
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A. Tsuchida, T. Yamauchi, S. Takekawa, Y. Hada, Y. Ito, T. Maki, and T. Kadowaki Peroxisome Proliferator-Activated Receptor (PPAR){alpha} Activation Increases Adiponectin Receptors and Reduces Obesity-Related Inflammation in Adipose Tissue: Comparison of Activation of PPAR{alpha}, PPAR{gamma}, and Their Combination Diabetes, December 1, 2005; 54(12): 3358 - 3370. [Abstract] [Full Text] [PDF]
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 J. Bispham, D. S. Gardner, M. G. Gnanalingham, T. Stephenson, M. E. Symonds, and H. Budge Maternal Nutritional Programming of Fetal Adipose Tissue Development: Differential Effects on Messenger Ribonucleic Acid Abundance for Uncoupling Proteins and Peroxisome Proliferator-Activated and Prolactin Receptors Endocrinology, September 1, 2005; 146(9): 3943 - 3949. [Abstract] [Full Text] [PDF]
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 C L. Kien, J. Y Bunn, and F. Ugrasbul Increasing dietary palmitic acid decreases fat oxidation and daily energy expenditure Am. J. Clinical Nutrition, August 1, 2005; 82(2): 320 - 326. [Abstract] [Full Text] [PDF]
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K. Ravnskjaer, M. Boergesen, B. Rubi, J. K. Larsen, T. Nielsen, J. Fridriksson, P. Maechler, and S. Mandrup Peroxisome Proliferator-Activated Receptor {alpha} (PPAR{alpha}) Potentiates, whereas PPAR{gamma} Attenuates, Glucose-Stimulated Insulin Secretion in Pancreatic {beta}-Cells Endocrinology, August 1, 2005; 146(8): 3266 - 3276. [Abstract] [Full Text] [PDF]
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 I. Valle, A. Alvarez-Barrientos, E. Arza, S. Lamas, and M. Monsalve PGC-1{alpha} regulates the mitochondrial antioxidant defense system in vascular endothelial cells Cardiovasc Res, June 1, 2005; 66(3): 562 - 573. [Abstract] [Full Text] [PDF]
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 X. Sun and M. B. Zemel Calcium and Dairy Products Inhibit Weight and Fat Regain during Ad Libitum Consumption Following Energy Restriction in Ap2-Agouti Transgenic Mice J. Nutr., November 1, 2004; 134(11): 3054 - 3060. [Abstract] [Full Text] [PDF]
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M. Berthiaume, H. Sell, J. Lalonde, Y. Gelinas, A. Tchernof, D. Richard, and Y. Deshaies Actions of PPAR{gamma} agonism on adipose tissue remodeling, insulin sensitivity, and lipemia in absence of glucocorticoids Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2004; 287(5): R1116 - R1123. [Abstract] [Full Text] [PDF]
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 K. E. Davis, M. Moldes, and S. R. Farmer The Forkhead Transcription Factor FoxC2 Inhibits White Adipocyte Differentiation J. Biol. Chem., October 8, 2004; 279(41): 42453 - 42461. [Abstract] [Full Text] [PDF]
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M. Miyazaki, A. Dobrzyn, H. Sampath, S.-H. Lee, W. C. Man, K. Chu, J. M. Peters, F. J. Gonzalez, and J. M. Ntambi Reduced Adiposity and Liver Steatosis by Stearoyl-CoA Desaturase Deficiency Are Independent of Peroxisome Proliferator-activated Receptor-{alpha} J. Biol. Chem., August 13, 2004; 279(33): 35017 - 35024. [Abstract] [Full Text] [PDF]
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H. Sell, J. P. Berger, P. Samson, G. Castriota, J. Lalonde, Y. Deshaies, and D. Richard Peroxisome Proliferator-Activated Receptor {gamma} Agonism Increases the Capacity for Sympathetically Mediated Thermogenesis in Lean and ob/ob Mice Endocrinology, August 1, 2004; 145(8): 3925 - 3934. [Abstract] [Full Text] [PDF]
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N. M. Morton, J. M. Paterson, H. Masuzaki, M. C. Holmes, B. Staels, C. Fievet, B. R. Walker, J. S. Flier, J. J. Mullins, and J. R. Seckl Novel Adipose Tissue-Mediated Resistance to Diet-Induced Visceral Obesity in 11{beta}-Hydroxysteroid Dehydrogenase Type 1-Deficient Mice Diabetes, April 1, 2004; 53(4): 931 - 938. [Abstract] [Full Text] [PDF]
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 J. R. Seckl, N. M. Morton, K. E. Chapman, and B. R. Walker Glucocorticoids and 11beta-Hydroxysteroid Dehydrogenase in Adipose Tissue Recent Prog. Horm. Res., January 1, 2004; 59(1): 359 - 393. [Abstract] [Full Text]
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 W. He, Y. Barak, A. Hevener, P. Olson, D. Liao, J. Le, M. Nelson, E. Ong, J. M. Olefsky, and R. M. Evans Adipose-specific peroxisome proliferator-activated receptor {gamma} knockout causes insulin resistance in fat and liver but not in muscle PNAS, December 23, 2003; 100(26): 15712 - 15717. [Abstract] [Full Text] [PDF]
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 U. Dressel, T. L. Allen, J. B. Pippal, P. R. Rohde, P. Lau, and G. E. O. Muscat The Peroxisome Proliferator-Activated Receptor {beta}/{delta} Agonist, GW501516, Regulates the Expression of Genes Involved in Lipid Catabolism and Energy Uncoupling in Skeletal Muscle Cells Mol. Endocrinol., December 1, 2003; 17(12): 2477 - 2493. [Abstract] [Full Text] [PDF]
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 T. Kadowaki, K. Hara, T. Yamauchi, Y. Terauchi, K. Tobe, and R. Nagai Molecular Mechanism of Insulin Resistance and Obesity Experimental Biology and Medicine, November 1, 2003; 228(10): 1111 - 1117. [Abstract] [Full Text] [PDF]
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G. Solanes, N. Pedraza, R. Iglesias, M. Giralt, and F. Villarroya Functional Relationship between MyoD and Peroxisome Proliferator-Activated Receptor-Dependent Regulatory Pathways in the Control of the Human Uncoupling Protein-3 Gene Transcription Mol. Endocrinol., October 1, 2003; 17(10): 1944 - 1958. [Abstract] [Full Text] [PDF]
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C. Tiraby, G. Tavernier, C. Lefort, D. Larrouy, F. Bouillaud, D. Ricquier, and D. Langin Acquirement of Brown Fat Cell Features by Human White Adipocytes J. Biol. Chem., August 29, 2003; 278(35): 33370 - 33376. [Abstract] [Full Text] [PDF]
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H. J. Grav, K. J. Tronstad, O. A. Gudbrandsen, K. Berge, K. E. Fladmark, T. C. Martinsen, H. Waldum, H. Wergedahl, and R. K. Berge Changed Energy State and Increased Mitochondrial {beta}-Oxidation Rate in Liver of Rats Associated with Lowered Proton Electrochemical Potential and Stimulated Uncoupling Protein 2 (UCP-2) Expression: EVIDENCE FOR PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR-{alpha} INDEPENDENT INDUCTION OF UCP-2 EXPRESSION J. Biol. Chem., August 15, 2003; 278(33): 30525 - 30533. [Abstract] [Full Text] [PDF]
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M. Laplante, H. Sell, K. L. MacNaul, D. Richard, J. P. Berger, and Y. Deshaies PPAR-{gamma} Activation Mediates Adipose Depot-Specific Effects on Gene Expression and Lipoprotein Lipase Activity: Mechanisms for Modulation of Postprandial Lipemia and Differential Adipose Accretion Diabetes, February 1, 2003; 52(2): 291 - 299. [Abstract] [Full Text] [PDF]
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T. Murase, A. Nagasawa, J. Suzuki, T. Wakisaka, T. Hase, and I. Tokimitsu Dietary {alpha}-Linolenic Acid-Rich Diacylglycerols Reduce Body Weight Gain Accompanying the Stimulation of Intestinal {beta}-Oxidation and Related Gene Expressions in C57BL/KsJ-db/db Mice J. Nutr., October 1, 2002; 132(10): 3018 - 3022. [Abstract] [Full Text] [PDF]
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G. Patane, M. Anello, S. Piro, R. Vigneri, F. Purrello, and A. M. Rabuazzo Role of ATP Production and Uncoupling Protein-2 in the Insulin Secretory Defect Induced by Chronic Exposure to High Glucose or Free Fatty Acids and Effects of Peroxisome Proliferator-Activated Receptor-{gamma} Inhibition Diabetes, September 1, 2002; 51(9): 2749 - 2756. [Abstract] [Full Text] [PDF]
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 T. Murase, M. Aoki, T. Wakisaka, T. Hase, and I. Tokimitsu Anti-obesity effect of dietary diacylglycerol in C57BL/6J mice: dietary diacylglycerol stimulates intestinal lipid metabolism J. Lipid Res., August 1, 2002; 43(8): 1312 - 1319. [Abstract] [Full Text] [PDF]
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D. M. Muoio, P. S. MacLean, D. B. Lang, S. Li, J. A. Houmard, J. M. Way, D. A. Winegar, J. C. Corton, G. L. Dohm, and W. E. Kraus Fatty Acid Homeostasis and Induction of Lipid Regulatory Genes in Skeletal Muscles of Peroxisome Proliferator-activated Receptor (PPAR) alpha Knock-out Mice. EVIDENCE FOR COMPENSATORY REGULATION BY PPARdelta J. Biol. Chem., July 12, 2002; 277(29): 26089 - 26097. [Abstract] [Full Text] [PDF]
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L.-X. Li, F. Skorpen, K. Egeberg, I. H. Jorgensen, and V. Grill Induction of Uncoupling Protein 2 mRNA in {beta}-Cells Is Stimulated by Oxidation of Fatty Acids But Not by Nutrient Oversupply Endocrinology, April 1, 2002; 143(4): 1371 - 1377. [Abstract] [Full Text] [PDF]
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T. Nakatani, N. Tsuboyama-Kasaoka, M. Takahashi, S. Miura, and O. Ezaki Mechanism for Peroxisome Proliferator-activated Receptor-alpha Activator-induced Up-regulation of UCP2 mRNA in Rodent Hepatocytes J. Biol. Chem., March 8, 2002; 277(11): 9562 - 9569. [Abstract] [Full Text] [PDF]
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V. M Rodriguez, M. P Portillo, C. Pico, M T. Macarulla, and A. Palou Olive oil feeding up-regulates uncoupling protein genes in rat brown adipose tissue and skeletal muscle Am. J. Clinical Nutrition, February 1, 2002; 75(2): 213 - 220. [Abstract] [Full Text] [PDF]
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M. B. Armstrong and H. C. Towle Polyunsaturated fatty acids stimulate hepatic UCP-2 expression via a PPARalpha -mediated pathway Am J Physiol Endocrinol Metab, December 1, 2001; 281(6): E1197 - E1204. [Abstract] [Full Text] [PDF]
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C. Son, K. Hosoda, J. Matsuda, J. Fujikura, S. Yonemitsu, H. Iwakura, H. Masuzaki, Y. Ogawa, T. Hayashi, H. Itoh, et al. Up-Regulation of Uncoupling Protein 3 Gene Expression by Fatty Acids and Agonists for PPARs in L6 Myotubes Endocrinology, October 1, 2001; 142(10): 4189 - 4194. [Abstract] [Full Text] [PDF]
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S. H. Cha, A. Fukushima, K. Sakuma, and Y. Kagawa Chronic Docosahexaenoic Acid Intake Enhances Expression of the Gene for Uncoupling Protein 3 and Affects Pleiotropic mRNA Levels in Skeletal Muscle of Aged C57BL/6NJcl Mice J. Nutr., October 1, 2001; 131(10): 2636 - 2642. [Abstract] [Full Text] [PDF]
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N. Lameloise, P. Muzzin, M. Prentki, and F. Assimacopoulos-Jeannet Uncoupling Protein 2: A Possible Link Between Fatty Acid Excess and Impaired Glucose-Induced Insulin Secretion? Diabetes, April 1, 2001; 50(4): 803 - 809. [Abstract] [Full Text]
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S. Viengchareun, P. Penfornis, M.-C. Zennaro, and M. Lombes Mineralocorticoid and glucocorticoid receptors inhibit UCP expression and function in brown adipocytes Am J Physiol Endocrinol Metab, April 1, 2001; 280(4): E640 - E649. [Abstract] [Full Text] [PDF]
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T. Albrektsen and J. Fleckner The Transcription Factor Fos-Related Antigen 1 Is Induced by Thiazolidinediones During Differentiation of 3T3-L1 Cells Mol. Pharmacol., March 1, 2001; 59(3): 567 - 575. [Abstract] [Full Text]
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 C. S. Elangbam, R. D. Tyler, and R. M. Lightfoot Peroxisome Proliferator-activated Receptors in Atherosclerosis and Inflammation--An Update Toxicol Pathol, February 1, 2001; 29(2): 224 - 231. [Abstract] [PDF]
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 H. Tilg and A. M. Diehl Cytokines in Alcoholic and Nonalcoholic Steatohepatitis N. Engl. J. Med., November 16, 2000; 343(20): 1467 - 1476. [Full Text] [PDF]
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R. A. Memon, L. H. Tecott, K. Nonogaki, A. Beigneux, A. H. Moser, C. Grunfeld, and K. R. Feingold Up-Regulation of Peroxisome Proliferator-Activated Receptors (PPAR-{alpha}) and PPAR-{gamma} Messenger Ribonucleic Acid Expression in the Liver in Murine Obesity: Troglitazone Induces Expression of PPAR-{gamma}-Responsive Adipose Tissue-Specific Genes in the Liver of Obese Diabetic Mice Endocrinology, November 1, 2000; 141(11): 4021 - 4031. [Abstract] [Full Text] [PDF]
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X. X. Yu, J. L. Barger, B. B. Boyer, M. D. Brand, G. Pan, and S. H. Adams Impact of endotoxin on UCP homolog mRNA abundance, thermoregulation, and mitochondrial proton leak kinetics Am J Physiol Endocrinol Metab, August 1, 2000; 279(2): E433 - E446. [Abstract] [Full Text] [PDF]
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 K. A. J. M. VAN DER LEE, P. H. M. WILLEMSEN, G. J. VAN DER VUSSE, and M. VAN BILSEN Effects of fatty acids on uncoupling protein-2 expression in the rat heart FASEB J, March 1, 2000; 14(3): 495 - 502. [Abstract] [Full Text]
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 B. Desvergne and W. Wahli Peroxisome Proliferator-Activated Receptors: Nuclear Control of Metabolism Endocr. Rev., October 1, 1999; 20(5): 649 - 688. [Abstract] [Full Text]
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A. Matthias, A. Jacobsson, B. Cannon, and J. Nedergaard The Bioenergetics of Brown Fat Mitochondria from UCP1-ablated Mice. UCP1 IS NOT INVOLVED IN FATTY ACID-INDUCED DE-ENERGIZATION ("UNCOUPLING") J. Biol. Chem., October 1, 1999; 274(40): 28150 - 28160. [Abstract] [Full Text] [PDF]
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L. Poretsky, N. A. Cataldo, Z. Rosenwaks, and L. C. Giudice The Insulin-Related Ovarian Regulatory System in Health and Disease Endocr. Rev., August 1, 1999; 20(4): 535 - 582. [Abstract] [Full Text] [PDF]
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M. J. Barbera, A. Schluter, N. Pedraza, R. Iglesias, F. Villarroya, and M. Giralt Peroxisome Proliferator-activated Receptor alpha Activates Transcription of the Brown Fat Uncoupling Protein-1 Gene. A LINK BETWEEN REGULATION OF THE THERMOGENIC AND LIPID OXIDATION PATHWAYS IN THE BROWN FAT CELL J. Biol. Chem., January 5, 2001; 276(2): 1486 - 1493. [Abstract] [Full Text] [PDF]
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A. V. Medvedev, S. K. Snedden, S. Raimbault, D. Ricquier, and S. Collins Transcriptional Regulation of the Mouse Uncoupling Protein-2 Gene. DOUBLE E-BOX MOTIF IS REQUIRED FOR PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR-gamma -DEPENDENT ACTIVATION J. Biol. Chem., March 30, 2001; 276(14): 10817 - 10823. [Abstract] [Full Text] [PDF]
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T. Yamauchi, J. Kamon, H. Waki, K. Murakami, K. Motojima, K. Komeda, T. Ide, N. Kubota, Y. Terauchi, K. Tobe, et al. The Mechanisms by Which Both Heterozygous Peroxisome Proliferator-activated Receptor gamma (PPARgamma ) Deficiency and PPARgamma Agonist Improve Insulin Resistance J. Biol. Chem., October 26, 2001; 276(44): 41245 - 41254. [Abstract] [Full Text] [PDF]
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N. M. Morton, M. C. Holmes, C. Fievet, B. Staels, A. Tailleux, J. J. Mullins, and J. R. Seckl Improved Lipid and Lipoprotein Profile, Hepatic Insulin Sensitivity, and Glucose Tolerance in 11beta -Hydroxysteroid Dehydrogenase Type 1 Null Mice J. Biol. Chem., October 26, 2001; 276(44): 41293 - 41300. [Abstract] [Full Text] [PDF]
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Y. Nagai, Y. Nishio, T. Nakamura, H. Maegawa, R. Kikkawa, and A. Kashiwagi Amelioration of high fructose-induced metabolic derangements by activation of PPARalpha Am J Physiol Endocrinol Metab, May 1, 2002; 282(5): E1180 - E1190. [Abstract] [Full Text] [PDF]
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