Impact of endotoxin on UCP homolog mRNA abundance,
thermoregulation, and mitochondrial proton leak kinetics
Xing Xian
Yu1,
Jamie L.
Barger2,
Bert B.
Boyer2,
Martin D.
Brand3,
Guohua
Pan1, and
Sean H.
Adams1
1 Department of Endocrinology, Genentech, Inc., South San
Francisco, California 94080; 2 Institute of Arctic Biology,
University of Alaska, Fairbanks, Alaska 99775; and 3 Medical
Research Council Dunn Human Nutrition Unit, Cambridge CB2 2XY,
United Kingdom
 |
ABSTRACT |
Linking tissue
uncoupling protein (UCP) homolog abundance with functional metabolic
outcomes and with expression of putative genetic regulators promises to
better clarify UCP homolog physiological function. A murine endotoxemia
model characterized by marked alterations in thermoregulation was
employed to examine the association between heat production, UCP
homolog expression, and mitochondrial proton leak ("uncoupling").
After intraperitoneal lipopolysaccharide (LPS, ~6 mg/kg) injection,
colonic temperature (Tc) in adult female C57BL6/J mice
dropped to a nadir of ~30°C by 8 h, preceded by a four- to
fivefold drop in liver UCP2 and UCP5/brain mitochondrial carrier
protein 1 mRNA levels, with no change in their hindlimb skeletal muscle
(SKM) expression. SKM UCP3 mRNA rose fivefold during development of
hypothermia and was correlated with an LPS-induced increase in plasma
free fatty acid concentration. UCP2 and UCP5 transcripts
recovered about three- to sixfold in both tissues starting at 6-8
h, preceding a recovery of Tc between 16 and 24 h. SKM
UCP3 followed an opposite pattern. Such results are not consistent with
an important influence of UCP3 in driving heat production but do not
preclude a role for UCP2 or UCP5 in this process. The transcription
coactivator PGC-1 displayed a transient LPS-evoked rise (threefold) or
drop (two- to fivefold) in SKM and liver expression, respectively. No
differences between control and LPS-treated mouse liver or SKM in vitro
mitochondrial proton leak were evident at time points corresponding to
large differences in UCP homolog expression.
uncoupling proteins; lipopolysaccharide; metabolic rate; hypothermia; peroxisome proliferator-activated receptor
 |
INTRODUCTION |
MAINTENANCE OF A
STABLE body temperature involves a precise balance between heat
acquisition and loss, driven by a complex interaction of physiological,
behavioral, and environmental processes. The ability to generate and
regulate metabolic heat is a hallmark of endothermy and a critical
adjunct to other events that contribute to efficient thermoregulation
(physiological control of convective and conductive heat loss, heat- or
cold-seeking behavior, etc.). Heat production in endotherms is in part
ascribed to a global energetic inefficiency inherent to cellular
biochemical reactions augmented by additional energy-consuming
mechanisms, including protein turnover and futile cycling, which drive
ATP consumption (9). The relative contribution of these
and other mechanisms toward establishment of metabolic rate is the
subject of active research. Identification of the molecular and
biochemical components underlying regulation of heat balance has
important ramifications for the development of pharmaceutical
intervention strategies to treat metabolic disorders, and for
clarifying regulation of adaptational thermoregulation in nature.
One potential locus of thermoregulatory control is proton flux
across the inner mitochondrial membrane. It is now apparent that the
inward flow of protons via mechanisms independent of F1F0ATP synthase (termed "proton
leak") is not insignificant and would act to dissipate fuel-derived
energy as heat (9, 37). In rodents, this
process is modified by changes in thyroid status (18,
24) and in some forms of obesity
(11), and it may account for ~20 to 40% of tissue
metabolic rate under normal conditions (9). The
underpinnings of proton leak remain to be established. The
characterization and cloning of UCP1 (8, 19),
a specific uncoupling protein (UCP) that facilitates accelerated proton
leak in stimulated rodent brown adipose tissue (BAT), have supported the notion that bodywide proton leak may be regulated by specific mitochondrial proteins. Recent descriptions of putative UCP homologs residing in various tissues (7, 15-17,
28, 40, 42, 46) have sparked interest in exploring whether these proteins influence tissue-specific or whole animal heat production. Some studies examining
homolog mRNA abundance are consistent with a role for UCP homologs in
modifying proton leak and metabolic rate, whereas other results may
point to additional metabolic roles for these proteins.
Supportive of an uncoupling role for UCP homologs, there are certain
conditions in which UCP2 or UCP3 mRNAs appear to correlate with in
vitro determinations of mitochondrial proton leak (11, 24). Furthermore, expression of UCP2 and UCP3 in rodent
BAT rises in response to cold exposure (4), concurrent
with increased proton leak in this tissue. Thyroid hormone
administration to rodents, a condition in which tissue proton leak has
been shown to rise (18, 24), increases UCP3
mRNA in muscle (17, 21, 24) and
upregulates UCP2 in a tissue-specific manner (21, 25). UCP5 [also termed brain mitochondrial carrier
protein 1 (BMCP1) (40)] is widely expressed, and its
liver mRNA level is altered in parallel with metabolism during fasting,
cold challenge, and a high-fat diet in obesity-resistant mice; cold
also induces its expression in the brain (46).
Brain-specific UCP4 mRNA is upregulated in this organ upon exposure of
mice to cold (46), consistent with the hypothesis that
UCP4 could be involved in localized adaptational thermoregulation
(27). Results that do not support a classic thermogenic
uncoupling role for UCP2 and UCP3 include reports of a rise in their
skeletal muscle (SKM) transcript levels during fasting or food
restriction (5, 6, 9a, 17, 20, 31, 32, 38, 44), despite a lack of
change in in vitro SKM proton leak (9a), the higher abundance of UCP2
mRNA sometimes observed in obesity (11, 16,
31), and the lack of a consistent demonstration for a
robust rise in SKM mRNAs upon cold exposure (6, 7, 15; but see also
Ref. 5 for induction of UCP2 by cold).
Divergent tissue expression patterns (UCP2 is widely expressed, whereas
UCP3 is most abundant in SKM and BAT, but without expression in liver)
and differential modification after certain experimental manipulations
(17, 44, 45) indicate that some differences in gene regulation exist when UCP2 and UCP3 are compared. Often, however, patterns of expression for these homologs converge (4). The association between their expression and that of
UCP5 and the differential expression of UCP5 in SKM have not been reported.
Animal models that display broad alterations in heat production serve
as valuable tools to better understand cellular mechanisms that drive
metabolic rate, including the role of UCP homologs. The murine model of
endotoxemia provides an interesting system in this regard, because
administration of lipopolysaccharide (LPS) elicits a range of
thermoregulatory responses, including acute hypothermia usually
followed by temperature recovery (23, 33). It
is plausible that LPS-induced changes in UCP homolog expression and
uncoupling activity in metabolically relevant tissues underlie the
decline and/or rebound of body temperature in this model. Indeed, it
has been reported that liver UCP2 mRNA is increased at 12-24 h
after LPS administration in rodents (11, 12,
14), leading to speculation that this rise may signal an
increase in active uncoupling and thermogenesis in that organ
(14). However, no physiological or biochemical correlates
were presented, and studies investigating the temporal effects of LPS
on UCP homolog expression in SKM have not been reported. In this study,
we examined the degree to which UCP homolog mRNA abundance correlates
with observed changes in functional metabolic outcomes (body
temperature, mitochondrial proton leak, and metabolic rate) after a
hypothermia-inducing dose of LPS, focusing on the liver and SKM, which
are estimated to contribute over one-half of the metabolic rate under
normal conditions (9). In an effort to better understand
the regulatory factors driving observed UCP homolog expression changes,
the association between such changes with those of the recently
characterized peroxisome proliferator-activated receptor-
(PPAR
)
coactivator 1 [PGC-1 (34, 45)] was
assessed. PGC-1 has been implicated in the induction of genes encoding
UCP1, UCP2, and other metabolically relevant proteins (34,
45). In addition, the idea was explored that core body
temperature-sensing pathways trigger alterations in UCP homolog gene
expression in LPS-treated mice. Despite large changes in liver and SKM
UCP homolog mRNA abundance, PGC-1 expression, and evidence for
remarkable metabolic shifts after LPS, no association of these
parameters with mitochondrial proton leak could be discerned. A
disconnect between PGC-1 and UCP2 expression (particularly evident in
SKM) after LPS indicated that under these conditions regulatory factors
distinct from PGC-1 modulated UCP2 transcription.
 |
MATERIALS AND METHODS |
Animals.
All animal studies conformed to the "Guiding Principles for Research
Involving Animals and Human Beings" and were done in accordance with
guidelines set forth by the Institutional Animal Care and Use Committee
at Genentech. Female C57BL6/J mice (Jackson Labs, Bar Harbor, ME) aged
54-63 days and weighing 16-18 g were used for all studies.
Mice were received 1 wk before experimentation and, unless otherwise
noted, were housed on a 12:12-h light-dark cycle (lights on at 0600) at
22°C and were fed normal rodent chow (Ralston Purina Chow 5010, St.
Louis, MO). Where indicated, heparinized blood was obtained by heart
puncture from CO2-anesthetized mice and was promptly
centrifuged to obtain plasma, after which free fatty acid (FFA)
concentrations were measured (NEFA C kit, Wako Chemicals, Richmond, VA).
Reagents.
LPS was a Westphal preparation from Escherichia coli 055:B5
(Lot 121379JD, Difco Laboratories, Detroit, MI).
Methyltriphenylphosphonium bromide (TPMP) was purchased from Aldrich
Chemicals (Milwaukee, WI). Collagenase Type IV was obtained from
Worthington Biochemical (Lakewood, NJ). Other chemicals were from Sigma
(St. Louis, MO).
LPS administration.
To best compare results with those reported by Faggioni et al.
(14), conditions used herein were generally similar to
those used by that group. A working stock of LPS was prepared in
sterile PBS, and aliquots were frozen at
20°C and thawed once on
the day of the experiment. For all LPS experiments, LPS was injected intraperitoneally at 100 µg/mouse (5.6-6.2 mg/kg) in a volume of
100 µl between 1430 and 1630. Preliminary experiments indicated that
this dosage elicited a marked hypothermia compared with doses 10- to
100-fold lower (not shown) and was similar in magnitude to the amount
given by Faggioni et al. Control mice received 100 µl PBS
intraperitoneally. After injection, mice were given access to water but
were fasted to account for the effects of LPS-induced cachexia
(14). Various parameters hypothesized to be influenced by
endotoxemia were monitored for up to 48 h (see below). Unless otherwise noted (see study 2), mice were housed at 22°C
for the duration of each experiment.
Body temperature and UCP homolog mRNA after LPS (studies 1 and
2).
An initial experiment (study 1) was designed to ascertain
whether mRNAs encoding UCP homologs track metabolism after LPS. At
intervals of 0, 2, 4, 6, 8, 16, 24, and 48 h after injection, body
temperatures in groups of control or LPS-treated mice
(n = 3-5 per treatment per time point) were
determined as follows. Mice were removed from the cage with minimal
disturbance, and colonic temperature (Tc) was rapidly
measured with a mouse rectal thermocouple (Physitemp BAT-10
recorder/RET-3 thermocouple, Clifton, NJ). Mice were then killed under
CO2, and tissues were harvested and snap-frozen. For
studies of SKM, whole hindlimb SKM was excised, and visible fat and
nervous tissue were removed before freezing. In study 2, a
new group of mice were subjected to the same protocol but were placed
in a warm (34°C) room after injection to test the effects of core
temperature maintenance on LPS-altered parameters.
Total RNA preparations from liver or pulverized SKM of individual mice
were made (Ultraspec reagent, Biotecx Laboratories, Houston, TX) and
were assayed for mRNA abundance with quantitative real time RT-PCR
after digestion of samples with DNAse per manufacturer's instructions
(GIBCO BRL, Grand Island, NY). This system employed primers and probes
specific to murine UCP2, total UCP5, UCP3, PGC-1, and
macrophage-specific marker F4/80 (Table
1). 18S primers/probes were
purchased from Perkin-Elmer Applied Biosystems (Foster City, CA).
Specificity of UCP primers/probes was confirmed by testing against a
panel of plasmids containing cDNAs encoding UCPs 2-5, and the
amount of RNA analyzed was in the linear range of the assay (not
shown). Reactions and detection were carried out with the use of a
model 7700 sequence detector and TaqMan reagents (PE Applied
Biosystems) in a volume of 50 µl and containing 100 ng RNA, 3 mM
MgCl2, reaction buffer A (1×), 12.5 U MuLV reverse transcriptase, 1.25 U TaqGold, forward and reverse primers (0.01 OD
each), and 0.1 µM probe (18S analyses utilized 240 pg RNA, 5.5 mM
MgCl2, and 0.05 µM probe/primer). Cycling conditions were 50°C for 15 min and 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Putative housekeeping gene mRNAs (
-actin, GAPDH, RPL19) tested in a subset of LPS liver samples indicated that their levels declined over time after injection (14); thus 18S mRNA abundance was used as a loading
control, and all values reported herein represent 18S-corrected values. Based on established tissue distribution patterns for the UCP homologs
(see the introductory section of this paper), analyses focused on UCP3
in SKM and UCP2/UCP5 in SKM and liver.
Mitochondrial proton leak measurements (study 3).
In study 3, examining the correlation between UCP homolog
mRNA and mitochondrial proton leak in liver and SKM, a new set of mice
was used (injection protocol identical to that of study 1). At certain times after injection, mitochondria were prepared from control or LPS-injected animals and assayed in parallel. Protocols for
mitochondria isolation and measurement of proton leak were patterned
after those reported elsewhere (37). After measuring Tc, mice were killed under CO2, livers were
removed, a fraction was snap-frozen for mRNA analysis, and the
remainder was placed in ice-cold STE buffer (250 mM sucrose, 5 mM Tris,
2 mM EGTA, pH 7.4). For mitochondria, livers from 3-5 mice per
treatment were pooled and minced on ice in a small volume of STE.
Samples were then processed by standard homogenization and
centrifugation methods (37), with the final mitochondrial
pellet resuspended in 500 µl STE and kept on ice until analysis.
Hindlimb SKM was obtained from a separate set of mice. For these
studies, muscle from a single mouse per treatment was obtained and
snap-frozen for RNA, after which hindlimb muscles from each of 5-6
mice per treatment were pooled to obtain mitochondria. Samples were
processed by use of a slight modification of the protocols employed by
Rolfe et al. (37): after removal of visible fat and
nervous tissue, samples in the current study were placed in ice-cold
CP1 buffer (100 mM KCl, 50 mM Tris · HCl, 2 mM EGTA, pH 7.4),
minced on an ice-cold glass plate, and added to 50 ml of pH 7.4 CP2
buffer (100 mM KCl, 50 mM Tris · HCl, 2 mM EGTA, 0.5% FFA-free
BSA, 5 mM MgCl2, 1 mM ATP, 2.1 U/ml nagarse; ATP/nagarse
added on day of experiment). Samples were kept on ice for 10 min with
occasional agitation and then subjected to a brief (10 s, 20,000 rpm)
polytron on ice (PowerGen 700, Fisher Scientific, Santa Clara, CA) and an additional 10-min cold incubation before differential centrifugation and washes at 4°C (37). Supernatants from the initial
10-min/500-g centrifugation were poured through two layers
of gauze before the high-speed spins/washes. Resulting pellets were
resuspended in 200-300 µl CP1 and kept on ice until assay.
Mitochondria prepared in this way yielded respiratory control ratios
(state 3/state 4 respiration with succinate as substrate) of >5
(liver) or >3 (SKM). Protein concentrations were determined by the
bicinchoninic acid assay (BCA kit, Pierce, Rockford IL).
For proton leak assays, mitochondria were introduced at ~1 mg
protein/ml to a water-jacketed chamber containing 3.5 ml pH 7.2 KHE
buffer (120 mM KCl, 5 mM KH2PO4, 3 mM HEPES, 1 mM EGTA, 0.3% fatty acid-free BSA) containing rotenone (5 µM),
oligomycin (1 µg/ml), and nigericin (80 ng/ml). Oxygen consumption
(
O2) of mitochondria was monitored by a
Clark-type model 300 oxygen electrode/Type 1 electronic stirring head
(Rank Brothers, Cambridge, UK) interfaced with a Unit DW4 oxygen
back-off system (Gritech Engineering, UK) and a chart recorder (Kipp
and Zonen). Oxygen saturation values of 471 nmol O/ml and 406 nmol O/ml were used to calculate
O2 of
preparations assayed at 26 and 37°C, respectively (35).
Measurements of mitochondrial TPMP+ uptake were made with a
TPMP+-sensitive electrode (37) referenced with
a semimicro CE2 pH electrode (Unicam, Cambridge, UK) and interfaced
with a back-off box, pH meter, and chart recorder. Within each
individual run, a standard curve of recorder distance vs.
[TPMP+] was calculated using 1-µM additions of
TPMP+ to 5 µM (liver) or 0.5-µM additions to 2 µM
(SKM). After addition of Na2-succinate (4 mM), respiration
was titrated by additions of Na2-malonate (
7.9 µM and
4.5 µM in liver and SKM, respectively). Drift was determined by
addition of 2 µM carbonyl cyanide
p-(trifluoromethoxy)phenylhydrazone to abolish membrane
potential at the end of each assay run. Mitochondrial membrane
potential (
, in mV) was calculated with the equation
where the TPMP binding corrections are 0.4 and 0.35 for liver
and SKM, respectively (37).
Hepatocyte isolation (study 4).
Changes in UCP homolog and PGC-1 expression in hepatocytes
isolated from control and LPS-treated mice (injection protocols identical to that in study 1) were assessed with the
following procedure. At certain times after injection, mice were
anesthetized by intraperitoneal injection of 100 µl
ketamine-xylazine-saline (2:1:10) and immobilized, and the inferior
vena cava was exposed. With introduction of air bubbles avoided at all
steps, a 22-gauge Angiocath catheter (Becton-Dickinson, Rutherford, NJ)
was placed below the liver, with clotting avoided by injection of 200 µl 1,000 U/ml heparin. An infusion line containing 37°C
buffer I (142 mM NaCl, 6.7 mM KCl, 100 mM HEPES, 5.3 mM
EGTA, pH 7.4) was attached, flow was initiated at 4 ml/min, the portal
vein was severed, and the inferior vena cava below the heart was
ligated. After 5 min, flow was switched to 37°C buffer II
(66.7 mM NaCl, 6.7 mM KCl, 100 mM HEPES, 4.8 mM CaCl2, 1%
FFA-free BSA, 77 U/ml Type IV collagenase, pH 7.6) for 20-30 min.
Perfused livers were excised, the gall bladder was removed, and cells
were dissociated by cutting the liver capsule and agitating the tissue
in a small volume of buffer II with mincing. The preparation
was poured into a 50-ml conical tube through a 250 µM filter and then
a 40 µM filter. The preparation was brought to ~20 ml with cold
HBSS and then centrifuged at ~50 g for 3 min. The
supernatant containing nonparenchymal cells (NPCs) with nonpelleted
hepatocytes was withdrawn and saved on ice (cells in supernatants from
this and subsequent washes are termed the "NPC-enriched fraction").
Cells were resuspended in 20 ml cold HBSS via gentle rocking and
recentrifuged at 50 g for 1 min, and the supernatant was
withdrawn. This step was repeated, and the resulting
hepatocyte-enriched pellet was snap-frozen. The pooled supernatants
were centrifuged at 5,000 g for 10 min, and the NPC-enriched
pellet was snap-frozen. This protocol using repeated washes yields a
low-speed pellet containing >95% hepatocytes (1).
Indirect calorimetry (study 5).
Twelve ad libitum-fed mice were acclimated for 24 h to respiration
chambers (Oxymax System, Columbus Instruments, Columbus, OH). After
acclimation, one-half of the mice were injected with LPS, and one-half
served as PBS-injected controls (injection protocol as in study
1). The first postinjection measurement of
O2 occurred ~1 h after injection and
was followed hourly thereafter. Tc was determined
immediately after the 24-h postinjection chamber measurement. Mice were
fasted after injection but were allowed free access to water.
Statistics.
Changes in mRNA abundance and Tc over time after
injection were assessed by use of the general linear models procedure
of SAS (SAS Institute, Cary, NC) as a 2 × 8 factorial design
analyzing the effects of treatment (LPS vs. controls), time, and
treatment × time interactions. Significant (P < 0.05) time or treatment × time interactions were observed for all
parameters; however, individual comparisons between time-matched
control vs. LPS-treated mice (see figure legends) were made only if
treatment × time effects were significant. Means ± SE are reported.
 |
RESULTS |
Study 1: LPS-evoked temperature, UCP homolog, and PGC-1 mRNA
changes.
The administration of a ~6 mg/kg dose of endotoxin induced a profound
hypothermia in mice (Fig.
1A).
Tc fell to 34°C by 4 h after injection, dropping
further to a nadir of ~30°C by 8 h after injection. Although
there was a decline in Tc by 2 h, this change was not
statistically significant. Individuals sampled at 24 and 48 h
after injection displayed a robust recovery of Tc, with a
slight overshoot above time-matched controls by 48 h (Fig.
1A). Both control and LPS-treated mice were fasted after injection, and this led to a significant drop in Tc in
controls by 16 h, remaining low through 48 h.

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Fig. 1.
Marked hypothermia and large changes in whole
liver uncoupling protein (UCP) homolog mRNA abundance in mice treated
with lipopolysaccharide (LPS). Adult female C57BL6/J mice were treated
with a single ip dose of PBS (controls, ) or LPS (~6 mg/kg,
), after which colonic temperature (A), whole liver UCP2
mRNA abundance (B), and whole liver UCP5 mRNA abundance
(C) were determined over 48 h postinjection
(study 1, see MATERIALS AND
METHODS). Animals were injected (0 h) between 1530 and
1630, fasted after treatment, and housed at 22°C for the duration of
the study (see MATERIALS AND METHODS for
details). Each point represents the mean ± SE of 3-5
independent measurements (* P 0.05,
** P 0.01 vs. time 0 values); some error bars
are within the symbol. Not indicated are significant differences
(P < 0.05) between LPS mice and time-matched controls,
occurring at 4-8, 16, and 48 h (temperature), 2-6, 24, and 48 h (UCP2), and 2, 4, and 8 h (UCP5).
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UCP homolog mRNA abundance in the liver was blunted sharply and
rapidly after LPS administration (Fig. 1, B and
C), with 2 h postinjection values only ~20-25%
of controls. Transcript amount for each began to rise between 6 and
8 h after injection and were no different from time-matched
controls at the 16-h time point. Hepatic UCP2 mRNA levels rose further
between 16 and 24 h, remaining elevated compared with time-matched
controls (Fig. 1B). In the latter, fasting elicited a
significant drop in UCP2 and UCP5 mRNA by 16-24 h (Fig. 1,
B and C). Based on real-time RT-PCR analysis of
time-zero control samples, whole liver mRNA expression of UCP2 was
about twofold higher than UCP5 (not shown). The magnitude of change in
rodent liver UCP2 mRNA after LPS in the current study using
quantitative RT-PCR is less than that reported by researchers who used
Northern blot analysis (11, 12,
14), likely caused by differences in experimental regimens
and our use of a more sensitive analytical methodology.
Skeletal muscle patterns of UCP2 and UCP5 mRNA abundance after
LPS administration differed substantially compared with liver (Fig.
2, A-C). For
instance, the decline in UCP5 mRNA over the first 4 h after
injection was not statistically significant (Fig. 2C), and
an LPS-induced decline in UCP2 expression was not apparent (Fig.
2A). In contrast to the liver pattern, SKM UCP5 mRNA in LPS-treated mice rose significantly above controls, reaching levels more than threefold higher than time-zero amounts by 48 h after injection (Fig. 2C). Furthermore, a fasting-induced drop in
UCP2 or UCP5 transcript was not seen in SKM from control mice (Fig. 2,
A and C). Interestingly, however, the mRNA levels
for UCP2 and UCP5 began to rise between 6 and 8 h after LPS (Fig.
2, A and C), an induction reminiscent of that
seen in liver (Fig. 1, B and C). As in liver, SKM
UCP2 mRNA increased significantly above control values between 16 and
48 h in LPS-treated mice (Fig. 2A).

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Fig. 2.
Time-dependent stimulation of UCP homolog
expression in hindlimb skeletal muscle (SKM) of LPS-treated mice.
Determinations of UCP2 (A), UCP3 (B), and UCP5
mRNA abundance (C) were made on SKM derived from control
( ) or LPS-treated ( ) mice housed at 22°C (see
Fig. 1 legend). Each point represents the mean ± SE of 3-5
independent measurements, derived from mice depicted in Fig. 1
(* P 0.05, ** P 0.01 vs.
time 0 values); some error bars are within the symbol.
Values for UCP2 mRNA at 8 h tended to be higher relative to
time 0 (P < 0.1). Not indicated are
significant differences (P < 0.05) between LPS mice
and time-matched controls, occurring at 2-6 and 16 h (UCP3)
and at 48 h (UCP5) (no significant treatment × time
interaction for UCP2).
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The UCP3 mRNA changes observed in SKM after LPS were markedly
different relative to those observed for UCP2 and UCP5 (Fig. 2,
A-C). After injection, there was an immediate and
substantial induction of the UCP3 gene as judged by mRNA abundance,
reaching levels more than fivefold above time-zero controls by 6 h
(Fig. 2B). Levels tapered off over the remaining hours to
equal those of time-matched controls by 24 h after injection, an
event concurrent with time-dependent increases in UCP2 and UCP5 mRNA
(see above). The rise in UCP3 mRNA in fasted controls was transient. In
data not shown, RT-PCR detection values in SKM from time-zero controls revealed that expression of UCP3 exceeded that of UCP2 and UCP5 by
~4- and >30-fold, respectively. UCP2 mRNA abundance was about fourfold higher in SKM than liver, whereas UCP5 was similar between tissues.
Exploration of the molecular basis of the remarkable UCP homolog
expression changes noted over the first 24 h after injection of LPS (see above) prompted analysis of PGC-1 mRNA abundance in a
subset of samples from these same mice. In liver, LPS significantly depressed PGC-1 up to fivefold in a time-dependent manner, with eventual recovery of transcript levels between 8 and 16 h after injection (Fig. 3). In contrast, PGC-1
mRNA abundance had risen more than threefold by 2 h after
injection in SKM from LPS-treated mice, falling significantly to reach
levels not statistically different from time-zero controls between 4 and 16 h (Fig. 3). In time-zero control tissues, mRNA abundance
for PGC-1 in whole liver was ~2- and ~10-fold lower than that
determined in SKM and BAT, respectively (not shown).

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Fig. 3.
Tissue-dependent stimulation or repression of peroxisome
proliferator-activated receptor- coactivator 1 (PGC-1) expression in
mice administered LPS. PGC-1 mRNA abundance was determined in a subset
of SKM ( ) and whole liver ( ) samples from mice
treated with LPS (samples are the same as those whose data are
presented in Figs. 1 and 2). Three independent samples were used at
each time point (* P 0.05,
** P 0.01 vs. time 0 values); some error
bars are within the symbol. The drop in liver mRNA was apparent by
2 h (P = 0.06). Not shown are PBS-injected control
values, which were not altered significantly relative to time
0.
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Study 2: effect of core body temperature clamp on LPS-induced
eexpression changes.
It was intriguing to note that induction of UCP homolog
expression in liver and SKM in study 1, clear by 6-8 h
after injection of LPS, occurred when core body temperature had
approached or reached its nadir (see Figs. 1 and 2). Although these
events may simply be coincidental, one may consider a paradigm in which
signals emanating from core body temperature
(Tcore)-sensitive neurons in the brain act to modulate
peripheral cellular heat production. Were the delayed UCP homolog gene
expression increases observed after LPS "triggered" by low
Tcore, and would such increases be blunted should
Tcore be clamped to near-control temperature? As an initial
test of this hypothesis, a second set of LPS and control mice was
placed in a warm room (34°C) after injection, thus preventing the
large drop in Tc displayed in study 1 (compare
Figs. 1A and 4A).
This treatment did not alter most patterns of UCP homolog mRNA
changes (Figs. 4 and
5). For example, liver
UCP2 and UCP5 mRNAs in LPS-treated mice dropped and then recovered to
exceed or equal control values, respectively (Fig. 4, B and
C), similar to what was observed at 22°C (Fig. 1,
B and C). As in studies performed at 22°C (Fig.
2B), SKM UCP3 transcript in temperature-clamped LPS-treated
mice increased significantly (albeit with more of a delay) and then
fell in a time-dependent manner (Fig. 5B). Generally, control patterns of UCP homolog expression were similar in both studies, although the increase in UCP3 mRNA in fasted control SKM
appeared to be higher and of longer duration at 34°C (compare Figs.
2B and 5B). Despite such similarities, there were
notable differences in expression between the studies. Although a
significant and delayed induction of UCP2 mRNA in SKM in LPS-treated
mice was seen in both 22°C and 34°C studies (compare Figs.
2A and 5A), a substantial transient fall in UCP2
expression was observed only at 34°C (Fig. 5A). The
pattern for UCP5 mRNA in the mice given LPS was generally similar
(compare Figs. 2C and 5C); however, the magnitude
of the delayed induction in UCP5 mRNA abundance was blunted at 34°C
(Fig. 5C).

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Fig. 4.
Whole liver patterns of UCP homolog mRNA
abundance after prevention of hypothermia in LPS-treated mice. Adult
female C57BL6/J mice were treated between 1530 and 1630 with a single
ip dose of PBS (controls, ) or LPS ( ) and
housed at an ambient temperature of 34°C with fasting (study
2, see MATERIALS AND METHODS). Colonic
temperature (A), liver UCP2 mRNA abundance (B),
and liver UCP5 mRNA abundance (C) were determined in groups
of mice through 48 h postinjection. Each point represents the
mean ± SE of 3-4 independent measurements
(* P 0.05, ** P 0.01 vs.
time 0 values); some error bars are within the symbol. The
lower values for UCP2 at 2 and 6 h after LPS (P < 0.1) did not achieve statistical significance. Not indicated are
significant differences (P < 0.05) between LPS mice
and time-matched controls, occurring at 4-16 h (body temperature),
16-48 h (UCP2), and 2 h (UCP5).
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Fig. 5.
Hindlimb SKM patterns of UCP homolog mRNA abundance
after prevention of hypothermia in LPS-treated mice. Determinations of
UCP2 (A), UCP3 (B), and UCP5 mRNA abundance
(C) were made on SKM derived from control ( ) or
LPS-treated ( ) mice housed at 34°C (see Fig. 4 legend
for details). Each point represents the mean ± SE of 3-4
independent measurements (* P 0.05,
** P 0.01 vs. time 0 values); some error
bars are within the symbol. Values for UCP2 at 2 and 6 h
(P < 0.1), UCP5 at 8 h (P = 0.06), and increased UCP3 at 4 h (P = 0.06)
post-LPS clearly differed from time 0 values but did not
achieve statistical significance at P < 0.05. Not
indicated are significant differences (P < 0.05)
between LPS mice and time-matched controls, occurring at 6-8 and
24-48 h (UCP2), 2-4 and 8 h (UCP3), and 2-6 h
(UCP5).
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Study 3: mitochondrial proton leak after LPS treatment.
Marked alterations in body temperature and UCP homolog gene
expression after LPS treatment (Figs. 1 and 2) suggested that significant time-dependent changes in mitochondrial uncoupling kinetics, an expected functional correlate of UCP activity, could have
occurred in endotoxin-challenged mice. To assess this possibility, mitochondrial proton leak in LPS-injected mouse liver was assayed in
parallel with control liver mitochondria at time points corresponding to diminution and recovery of UCP2 and UCP5 expression (4 and 16 h, respectively; Fig. 1, B and C). Liver samples
used for proton leak measurements yielded an expression pattern
matching that of the initial study (significant drop in UCP2 and UCP5
mRNA at 4 h post-LPS, recovery to control levels by 16 h; not
shown). An LPS-induced Tc decline was again observed at
4 h (controls, 37.0 ± 0.18°C; LPS, 34.9 ± 0.34°C;
n = 9/treatment) and 16 h (controls, 34.5 ± 0.32°C; LPS, 25.8 ± 0.41°C; n = 24/treatment)
resembling that of the initial study (Fig. 1A). Despite
large changes in mRNA abundance and Tc in this group of
mice (similar to mice in study 1), no significant difference
in liver mitochondrial respiration due to proton leak between
treatments was discernible under our assay conditions (Fig.
6, A and
B). Similarly, no suggestion of proton leak differences was
evident in SKM mitochondria at 16 h after injection (not shown).

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Fig. 6.
Lack of effect of in vivo LPS treatment on liver
mitochondrial proton leak kinetics in mice. Respiration attributable to
liver mitochondrial proton leak was measured at various titrations of
mitochondrial membrane potential in isolated organelles derived from
adult female C57BL6/J mice injected ip with PBS (controls, ) or
LPS ( ) between 1530 and 1630 (study 3, see
MATERIALS AND METHODS). Graphs correspond to
measurements taken beginning at 4 h (A) or at 16 h
(B and C) postinjection. Data in (C)
illustrate the alteration of proton leak that occurred in a subset
(n = 3) of 16-h LPS-treated mouse preparations assayed
at 26°C ( ) vs. kinetics of mitochondria assayed at
the typical 37°C [data from (B) are reproduced in
(C) for comparison]. Symbols represent mean values for
independent experiments at any given titration point in the assay
(n = 3/treatment at 4 h, n = 7/treatment at 16 h), with SEs for respiration and membrane
potentials indicated by vertical and horizontal error bars,
respectively; some error bars are within the symbol.
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To gather additional insight into the physiological relevance of proton
leak in hypothermic mice, a subset of LPS mitochondria used for the
16-h analyses described above was also assayed at 26°C. This
temperature corresponds to the average Tc of LPS-treated mice (see above) and is therefore more likely to reflect the kinetics of proton leak in mitochondria from these animals in situ compared with
assays performed at 37°C. At this cooler temperature, oxygen consumption due to proton leak was a fraction of that observed in
control or LPS mitochondria assayed at 37°C, despite increased estimated membrane potentials overall (Fig. 6C).
Study 4: hepatocyte expression of UCP homologs.
It has been reported that, in rodent liver, UCP2 expression in
hepatocytes is minor compared with that in the far less abundant Kupffer cells (11, 12, 26). This
issue is relevant to interpretation of correlations between proton leak
and whole liver UCP homolog mRNA abundance, because the contribution of
NPC mitochondria to preparations used for proton leak is vanishingly
small compared with the contribution from hepatocytes (2).
In addition, the cell-specific expression of UCP5 or PGC-1 in liver has
not been reported. To address these issues, mRNA was analyzed from
hepatocyte-enriched preparations1 derived from a
separate set of control or LPS-treated mice at time points
corresponding to proton leak measurements made in mice used for
study 3. As seen for whole liver (Fig. 1, B and C), hepatocyte UCP2 and UCP5 mRNA dropped substantially by
4 h after injection of LPS, with UCP5 mRNA recovering to control
values by 16 h (Fig. 7, A
and B). However, hepatocyte UCP2 expression was not
increased by 16 h in this group of mice. Hepatocytes expressed PGC-1 (Fig. 7C), with the drop in mRNA after LPS
administration similar to the pattern observed in whole liver (Fig. 3).

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Fig. 7.
Depression of UCP homolog and PGC-1 mRNA in hepatocytes
isolated from LPS-treated mice. At each time point depicted,
hepatocyte-enriched cell fractions were obtained from adult female
C57BL6/J mice injected with PBS (controls, ) or LPS
( )(study 4, see MATERIALS AND
METHODS). Values at each time point represent the mean mRNA
abundance of UCP2 (A), UCP5 (B), or PGC-1
(C) from 2 independent preparations/treatment.
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Study 5: oxygen consumption after LPS administration.
The profound hypothermic response and subsequent body temperature
recovery in LPS-treated mice (Fig. 1A) indicated that
substantial changes in metabolic heat production occur in these animals
over the first 24 h after LPS. As a functional corollary to in
vitro observations of UCP homolog expression and mitochondrial proton leak, metabolic rate was assessed by indirect calorimetry in mice injected with saline or LPS. Compared with mice from study 1 (e.g., Fig. 1A), animals from this group displayed
substantial variation in their metabolic response to endotoxin (Fig.
8). In all but one animal (cage
2, see Fig. 8 legend),
O2 began to
diverge from control mice between 3 and 4 h after injection with a
progressive and marked hypometabolism occurring from 4 h onward.
One animal (cage 12) exhibited a partial recovery of
O2 beginning by ~16 h (Fig. 8).
Importantly, differences in
O2 were
reflected by Tc measures taken at 24 h after
injection: controls (33.9 ± 0.6°C, n = 6),
hypometabolic mice (24.6 ± 0.22°C, n = 4), the
cage 2 mouse (34.2°C), and the cage 12 mouse
(28.6°C).

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Fig. 8.
Metabolic depression in mice injected with endotoxin.
Metabolic rate was determined via indirect calorimetry in adult female
C57BL6/J mice before and after ip injection of PBS (controls, )
or LPS (other symbols) between 1430 and 1530 (0 h), after which animals
were fasted (study 5, see MATERIALS AND
METHODS). Control values represent the mean ± SE of
all mice at 8 to 0 h (n = 12) or those injected
with PBS at 1-h postinjection onward (n = 6). Data on
individual LPS-treated mice are shown separately to highlight
individual variation observed after treatment in this particular group
of animals. Not shown is a single LPS-treated mouse (cage 2)
that for unclear reasons did not display a drop in metabolic rate after
injection (see RESULTS).
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 |
DISCUSSION |
Association of UCP homolog expression and metabolic outcomes.
Whether newly described or still undiscovered mitochondrial carrier
proteins act to uncouple mitochondrial respiration analogous to UCP1 is
actively being investigated. There is an abundance of information
regarding UCP homolog expression changes under various metabolic
conditions, but few studies have correlated such changes with
functional metabolic outcomes in rodents (11, 21, 24, 38, 39).
The current set of experiments was centered on the premise that, should
a particular UCP homolog act as an uncoupler in situ, robust modulation
of its expression in vivo should elicit concurrent detectable changes
in functional outcomes, including metabolic heat production and
mitochondrial proton leak. Analyses described herein focused on liver
and SKM, which are believed to account for over one-half of the
metabolic rate of rodents (9).
Based on mRNA patterns alone, certain aspects of UCP2 and UCP5
expression in the current model are consistent with the view that these
homologs contribute to uncoupling in vivo. First, development of
hypothermia in LPS-treated mice was preceded by a drop in liver UCP2
and UCP5 mRNA abundance (Fig. 1), and under normal conditions the
liver's contribution to metabolic rate is significant
(9). The failure of LPS to diminish UCP2 or UCP5
expression in SKM during hypothermia (Fig. 2), however, suggests that
any putative impact of these homologs on diminution of heat production
would not have been manifested in SKM. Second, recovery of
Tc was preceded by significant increases in UCP2 and UCP5
expression in liver and SKM (Figs. 1 and 2), consistent with the
hypothesis that increased activity of these homologs contributed to
reestablishment of Tc after LPS. The significant lag
between UCP2 and UCP5 expression changes (beginning at ~6-8 h
after injection, Figs. 1 and 2) and the recovery of Tc to
control levels (between 16 and 24 h after injection, Fig. 1) do
not preclude the possibility that these proteins are involved in
Tc recovery after LPS, because a sustained rise in energy
expenditure would have been required to normalize the Tc of
hypothermic mice. For example, an increase of 4°C in a 17-g mouse
over a period of 8 h (Fig. 1) would have required the generation
and full sequestration of ~60 calories just to account for the
temperature rise.2 This is an
underestimate of the actual energy needs to accomplish this feat,
because it does not take into account the loss of metabolic heat via
respiration and other routes. Furthermore, mitochondrial proton leak
kinetics slow considerably at cooler temperatures (Fig. 6C).
Thus truly effective activities of putative UCP homologs are not likely
to parallel expression changes in hypothermic animals, and would
manifest themselves fully only as mice begin to warm.
Expression patterns of UCP3 in SKM (Fig. 2B) raise questions
about the relevance of UCP3 toward driving truly meaningful heat production changes in LPS-treated mice, and they lend support to the
idea that this homolog has other primary functions in vivo (e.g., Refs.
38, 39, 44). For instance, UCP3 transcript levels in SKM were rapidly
triggered fivefold by LPS administration (Fig. 2), rising concurrently
with the drop in body temperature (the latter tracked metabolic rate,
see RESULTS). On the other hand, Tc recovery
was accompanied by diminishing UCP3 mRNA levels.
Regardless of the roles of UCP homologs in modifying in situ
uncoupling, tissue proton leak could not have been the only factor contributing to hypometabolism after LPS. The four- to eightfold drop
in
O2 (Fig. 8) and the magnitude of
decline in Tc (Fig. 1) caused by LPS appear too large to be
accounted for by changes in proton leak alone, which under basal
conditions may represent up to ~20-40% of tissue
O2 in rodents (9). Global
depression of metabolism, including a diminution of reactions
generating and consuming the mitochondrial electrochemical gradient,
likely occurs after high-dose LPS administration to mice. For example, although not measured in the current study, endotoxin has been shown to
increase nitric oxide (NO) production (27,
41), and NO or its derivatives powerfully inhibit the
electron transport chain (36). Furthermore, mice
developing hypothermia after LPS (Fig. 1) displayed a marked reduction
in motor activity (23). Thus, regardless of any possible
changes in proton leak, LPS-induced diminution of ATP consumption or
depression of the 
generating pathways would contribute to
alterations of metabolic rate and body temperature.
Despite large alterations of Tc (Fig. 1), metabolic rate
(Fig. 8), and UCP homolog mRNA in liver (Figs. 1 and 7) and SKM (Fig. 2), no discernible difference in proton leak could be detected in
mitochondria prepared from liver (Fig. 6) or SKM (see
RESULTS). Recently, a lack of correlation was reported
between leak and liver/SKM UCP2 and SKM UCP3 mRNA in mice administered
thyroid hormone or after a fast (9a, 21). A lack of correlation between in vitro proton leak assays and UCP homolog mRNA abundance could signal
that the primary physiological role of UCP2, UCP3, and UCP5 is not to
catalyze mitochondrial proton leak per se but rather to serve as
carriers for fatty acids or other moieties. Alternatively, it is
possible that the commonly used assay conditions for measurement of
mitochondrial proton leak employed in the current study do not
adequately reflect leak kinetics in vivo under every condition and may
therefore lead to negative interpretations of UCP homolog activity. In
a recent study, Lanni et al. (24) noted that proton leak
differences in SKM mitochondria derived from rats differing in thyroid
status were observed only when assays were carried out free of BSA,
suggesting involvement of fatty acids with proton leak. As noted by
Porter et al. (32a), higher proton leak was observed in hepatocytes
from aged rats compared with young rats, but such a difference was not
apparent with the use of isolated mitochondria. Thus various factors,
including fatty acids or purine nucleotides (e.g., Refs. 4, 19, 40),
and perhaps protein modulators, may regulate UCPs in the context of the
cell in situ. In addition, it is possible that changes in protein
levels in the tissues examined herein were too small to enable
detection of differences in proton leak despite alterations of mRNA.
Finally, the possibility cannot be discounted that still
uncharacterized mitochondrial carriers exist and importantly influenced
proton leak in these tissues.
The variable results to date regarding UCP homolog expression and
functional outcomes highlight the necessity for research to clarify
further the metabolic roles of these proteins. Although we observed no
differences in mitochondrial proton leak, despite remarkable
alterations of UCP homolog gene expression in LPS-treated mice,
increased proton leak was observed in mitochondria prepared from
ob/ob mouse liver and hyperthyroid rat SKM that
displayed increased hepatocyte UCP2 and SKM UCP3 expression,
respectively (11, 24). Samec et al.
(39) did not observe a correlation between UCP homolog
mRNA abundance and whole animal
O2 in a high-fat/low-fat model, whereas Jekabsons et al. (21)
presented evidence of a positive association between muscle UCP2 and
UCP3 expression changes and whole animal
O2. Our correlative data on
Tc recovery and UCP2/UCP5 mRNA (Figs. 1 and 2) concur with this latter finding, because Tc tracks changes in
O2 (see RESULTS), but they
differ in that UCP3 mRNA changes generally followed a pattern opposite
that of Tc (Fig. 2). Finally, others have reported increased rodent SKM UCP2 and UCP3 mRNA with fasting or food
restriction (5, 6, 9a, 17, 20, 38, 44), but our results in fasting
control mice indicated that UCP2 and UCP3 mRNA changes were transient and varied between studies 1 and 2 (Figs. 2 and
5). Differences between our study and those of others could be related
to our use of analytical mRNA methodology or to the fact that we
initiated the injection and fasting regimen in late afternoon, a time
point preceded by low food intake relative to the dark cycle.
Gene regulation of UCP homologs.
LPS administration initiates a complex cascade of events in which an
array of cytokines, prostaglandins, and other factors change temporally
to influence metabolism (28). With respect to humoral,
paracrine, or autocrine components that influenced UCP homolog
expression in the first hours after LPS (Figs. 1 and 2), factors such
as tumor necrosis factor-
(TNF-
) and interleukin-1
that emerge
early in the cascade are good candidates. Faggioni et al.
(14) reported that a single injection of TNF-
to mice caused a two- to threefold increase in liver, SKM, and WAT UCP2 mRNA
levels at 12 h, but changes at earlier time points were not reported. Indeed, a delay in liver UCP2 induction after LPS or TNF-
treatment has been consistently observed (11, 12, 14, this study). Thus
a direct stimulation of UCP2 or UCP5 genes in mouse liver or SKM by
TNF-
does not appear possible, because the transient nature of this
cytokine dictates that its direct effects must occur in the first ~2
h after LPS (47), when UCP2 and UCP5 mRNA levels were
stable or falling (Figs. 1 and 2). Cytokines induced later in the
LPS-induced cascade (28) or other humoral factors, such as
the newly described high mobility group 1 (43), might have
influenced the delayed induction of UCP2 and UCP5 in liver and SKM
after LPS (Figs. 1 and 2).
The current study illustrates that, under certain conditions, the
mechanisms controlling UCP2 and UCP5 genes may converge and that
genetic regulation of UCP3 is markedly different from that of UCP2 or
UCP5 after high-dose endotoxin in mice. For instance, LPS evoked a
similar repression and recovery of UCP2 and UCP5 expression in liver
(Figs. 1 and 4) plus a delayed induction in SKM (Figs. 2 and 5),
contrasting with the early and transient induction of SKM UCP3 (Figs. 2
and 5). There is evidence that the availability of fatty
acid-associated moieties (20, 44) stimulates
expression of UCPs. Indeed, the availability of fatty acids likely
influenced the expression of SKM UCP3 in the current study, as
evidenced by a separate experiment in which plasma FFA levels plus UCP3
mRNA were determined at 0, 2, and 6 h after injection of LPS or
PBS. A strong correlation between plasma FFA concentration and UCP3
expression was observed in LPS-treated mice (Fig.
9). Compared with time-matched controls,
the 6-h postinjection FFA concentration (1.20 ± 0.12 mM) and SKM
UCP3 expression (600 ± 32% of time 0) in LPS-treated
mice was approximately two- and threefold greater. The mechanisms
underlying the LPS-induced rise in circulating FFA levels and their
association with UCP3 gene expression remain unknown.

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Fig. 9.
UCP3 expression in SKM of LPS-treated mice correlates
with a rise in plasma free fatty acid (FFA) concentration.
Determinations of circulating FFA and hindlimb SKM UCP3 mRNA abundance
were made in samples derived from adult female C57BL6/J mice injected
ip with PBS (controls, ) or LPS ( ). UCP3 mRNA was strongly
correlated with plasma FFA in LPS mice (r2 = 0.75, P < 0.001/slope different from zero), whereas
this relationship was not as apparent in controls
(r2 = 0.33, P = 0.05/slope
different from zero). Data points represent matched samples across all
time points measured (0, 2, and 6 h postinjection; see
DISCUSSION).
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|
Activation of PPAR
and/or PPAR
(10, 20,
22), and tissue-specific upregulation of the coactivator
PGC-1 (34, 45), could influence UCP homolog
expression. Studies examining the role of ectopic PGC-1 in
C2C12 cells and other in vitro preparations suggested that this coactivator is critical for the activation of the
UCP1 and UCP2 genes, with little to no impact on UCP3 gene expression
(34, 45). Interestingly, LPS sparked a
significant increase in SKM PGC-1 expression (Fig. 3), which correlated
well with initiation of UCP3 expression but was not associated with a
change in UCP2 or UCP5 mRNA (Fig. 2). Thus it is intriguing to
postulate that SKM PGC-1 activity may influence the UCP3 gene in the
context of the whole animal and may have influenced changes in UCP3
gene expression in LPS-treated mice. There was some disconnect between
PGC-1 and UCP2 or UCP5 expression in whole liver, such that UCP homolog
mRNA levels rose well before recovery of PGC-1 expression (Figs. 1 and
3). Recently, Boss et al. (3) reported that administration
of
-adrenergic agonists or exposure to cold in wild-type or
3-receptor knock-out mice could lead to increases in SKM
UCP2 and UCP3 expression without concomitant changes in PGC-1 mRNA.
Such examples of minimal correlation are not consistent with the idea
that PGC-1 changes alone drive UCP2 or UCP5 expression in vivo, with
the caveat that PGC-1 protein abundance was not measured (Ref. 3, this
study). Using quantitative PCR, we found PGC-1 to be expressed in whole
liver, hepatocytes, and SKM (Figs. 3 and 7), at odds with the data of
Puigserver et al. (34), which indicated nominal expression
in liver or SKM (PGC-1 could be induced by cold in the latter tissue)
using Northern analysis. These discrepancies are likely explained by
technical differences in sensitivity, because PGC-1 expression using
real time RT-PCR was far greater (10-fold) in mouse BAT than liver (see
RESULTS), consistent with Puigserver et al. Results to date
(3, 34, 45, this study) indicate that the relative impact of PGC-1
activity on genes encoding UCP family members largely depends on the
specific cell type, UCP homolog, and biological context of the system
in study (i.e., whether or not PPAR
is activated by ligand). It is
clear that additional regulatory mechanisms exist that modify UCP genes
independently of changes in PGC-1 expression alone.
Exposure to cold ambient temperature (Ta) is a powerful
stimulus that engages the metabolic machinery of mammals, including
-adrenergic stimulation of BAT thermogenic activity
(4). There is some evidence that a cold Ta
induces UCP homolog expression in a tissue-dependent manner (reviewed
in Ref. 4; see also Ref. 46). We are unaware of any reports that have
studied the effects of core body temperature (Tcore) on UCP
homologs, and we wondered whether experimental modulation of
Tcore would elicit changes in their expression levels. One
hypothesis, for instance, is that the drop in Tcore after
LPS administration in mice (Fig. 1) may have stimulated the genes
encoding UCP2 and UCP5 (Figs. 1 and 2) via Tcore sensors
that communicate with the brain. Teleologically, a hypothermia-induced
enhancement of thermogenic uncoupling activity via UCP homologs could
help counteract an excessive Tcore drop. Artificial
maintenance of Tcore after LPS would be expected to dampen
any cold-stimulated rise in UCP homolog expression. In an initial test
of this hypothesis, clamping of Tcore after LPS challenge
generally failed to blunt the delayed stimulation of UCP2 and UCP5 gene
expression, with the possible exception of SKM UCP5, whose induction
was lower compared with that seen at 22°C (Figs. 3 and 4). These
findings indicate that regulatory factors independent of
Tcore were at play in our LPS model.
In summary, some aspects of the current set of experiments are
supportive of the idea that UCP2 and UCP5 are involved in metabolic changes occurring in LPS-treated mice. mRNA for these homologs was
induced in whole liver and SKM during recovery from LPS-induced hypothermia, and in liver, their transcript levels dropped during the
onset of hypothermia. Despite these patterns, no difference was
observed in in vitro mitochondrial proton leak. Alterations in SKM UCP3
mRNA after endotoxin were distinctly different from those observed for
UCP2 or UCP5, changing in the opposite direction from body temperature.
These data point to different mechanisms of gene regulation and suggest
that UCP3 does not serve in a thermogenic capacity under these
conditions. Changes in expression of the transcription coactivator
PGC-1 in muscle appeared to correlate with UCP3 expression, whereas the
association between PGC-1 and UCP2 was less compelling. Further study
is warranted to assess the possibility that LPS-induced increases in
UCP homolog expression/activity help minimize LPS-associated reactive
oxygen species production and damage. Ultimately, titration of UCP
homolog abundance through gene delivery, transgenic construction, and
knock-out technologies promises to clarify further the physiological
roles of these proteins.
 |
ACKNOWLEDGEMENTS |
The authors thank E. Filvaroff and T. A. Stewart for helpful
discussions of the manuscript, M. Renz for significant technical input,
and C. Galindo and M. Ostland for statistical assistance.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: S. H. Adams, Dept. of Endocrinology, Genentech, Inc., 1 DNA Way, South San
Francisco, CA 94080 (E-mail: shadams{at}gene.com).
1
Compared with the NPC fraction (see MATERIALS
AND METHODS), the hepatocyte-enriched fraction
contained >50-fold less transcript for the macrophage-specific marker
F4/80 (30), indicating that Kupffer contamination was
negligible. The focus of this study was to assess expression changes in
purified hepatocytes and was not designed as a formal examination of
UCPs in NPCs. Therefore, cells in the NPC-enriched fraction were not
separated further. Nonetheless, it is notable that the NPC-enriched
fractions consistently contained at least two- to fourfold higher UCP2
mRNA abundance than the hepatocyte-enriched fraction (not shown). These
findings are consistent with far greater expression of UCP2 in NPCs
relative to hepatocytes (11, 12,
26), because the contribution of NPC mRNA in the
NPC-enriched fraction was largely diluted by significant contaminating
hepatocyte mRNA (albumin transcript was about equal between fractions;
not shown). UCP5 mRNA was detected about equally in both fractions;
thus the UCP5 mRNA in the NPC-enriched fraction was largely due to the
contribution of hepatocyte mRNA (see above). However, such preliminary
findings cannot rule out the possibility that UCP5 is also expressed in NPCs.
2
Calculation of energy requirements to raise body
temperature assumed a heat capacity of 3.66 kJ · kg
1 · °C
1 for normal mice
(13) and a conversion factor of 4.184 J/cal.
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. §1734 solely to indicate this fact.
Received 20 December 1999; accepted in final form 16 March 2000.
 |
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