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INTRODUCTION |
Obesity results from an imbalance between energy intake and energy
expenditure (1). Uncoupling protein
(UCP)1 homologues uncouple
mitochondrial respiration from oxidative phosphorylation, increasing
thermogenesis while reducing the efficiency of ATP synthesis (2).
While UCP1 is expressed exclusively in brown fat, UCP2 and UCP3
are also expressed in white fat and skeletal muscle (3). The tissue
distribution of UCP2 and UCP3 has provoked speculation that these two
proteins may be important regulators of energy homeostasis in adults
(4), a possibility that is supported by evidence that the UCP2-UCP3
gene cluster maps to regions of human and murine chromosomes that have
been linked to obesity (5).
Because net energy expenditure is reduced in obese subjects, UCP2
and/or UCP3 expression or activity are predicted to be decreased. However, experimental evidence for this is relatively limited. A recent
study of 6 lean and 6 obese, but otherwise healthy, men demonstrated a
slight, but consistent, reduction in UCP2 mRNA levels in the
abdominal muscle of the obese subjects (6). Polymorphisms of UCP2, but
not UCP3, have been associated with decreased basal metabolic rate in
young Pima Indian men, although UCP2 mRNA levels in skeletal muscle
were not influenced (5). In mice, resistance to obesity induced by
feeding high fat diets has been associated with an early, selective
induction of UCP1 and UCP2 in brown and white fat, respectively, but
not with changes in UCP3 expression (7).
On the other hand, this evidence that decreased UCP2 may promote
obesity is difficult to reconcile with observations that ob/ob and
db/db obese mice have increased UCP2 mRNA levels in white adipose
tissue (8), and that UCP2 mRNA levels in white fat are positively
correlated with body mass index in humans (9). Also confusing are
reports that caloric restriction, a situation that decreases resting
energy expenditure, leads to increased UCP2-UCP3 mRNA expression in
white fat and skeletal muscle in both obese and lean human subjects (9)
and experimental animals (4, 10). Some explanation for these
paradoxical findings may be provided by recent data that correlate
circulating free fatty acid concentrations with UCP2 and UCP3 induction
in white fat and skeletal muscle, respectively, suggesting that
increased UCP2-UCP3 may represent a metabolic adaptation of these
tissues to increased fatty acid supply (11).
Of the UCP isoforms that have been identified, UCP2 has the widest
tissue distribution. Low levels of UCP2 mRNA have been detected by
Northern blot analysis of many organs, including the liver (8). Tissues
that express low levels of UCP2 transcripts constitutively are rich in
macrophages, leading to speculation that the resident macrophage
populations account for basal expression of UCP2 in these organs. This
concept is supported by evidence that UCP2 mRNA was identified in
macrophages (Kupffer cells), but not hepatocytes, that were isolated
from the livers of healthy, lean rats (12). However, similar to
adipocytes and myocytes, hepatocytes play a major role in regulating
intermediary metabolism and energy homeostasis (13-15). Thus, UCP2
expression may be induced in these cells during obesity. The aim of the
present study was to test this hypothesis and to evaluate the
implications of altered UCP2 expression on hepatic mitochondrial function.
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MATERIALS AND METHODS |
In total, 38 adult (10-12 week), male ob/ob mice and 38 of
their ?/ob (lean control) litter mates were obtained from Jackson Laboratories (Bar Harbor, MI). Mice were housed in a temperature and
light-controlled environment with free access to food and water. All
experiments were conducted in accordance with NIH and Johns Hopkins
University guidelines for the humane use of laboratory animals.
Evaluation of Liver UCP2 Expression
Northern Blot Analysis--
Using the method of Chomczynski and
Sacchi (16), total RNA was isolated from aliquots of freeze-clamped
livers obtained from 14 ob/ob mice and 15 lean control mice, as
described (17). After RNA concentration and quality were confirmed,
UCP2 mRNA expression from each mouse was evaluated by Northern blot
analysis. Briefly, RNA (20 µg/lane) was fractionated by
electrophoresis on agarose gels under nondenaturing conditions,
transferred to nylon membranes by capillary blotting, and hybridized
overnight at 42 °C with 32P-UCP2 cDNA (provided by
M. D. Lane). After staining the membranes with methylene blue to
demonstrate lane-lane variations in 18 S RNA, blots were washed at
55 °C under stringent conditions, examined by PhosphorImager and
exposed to Kodak XAR film in cassettes with intensifying screens (17).
For each sample, the UCP2 signal intensity was normalized to the 18 S
RNA expression in the same sample and expressed as a percentage of the
lean control on the same blot.
In Situ Hybridization--
Formalin-fixed, paraffin-embedded
sections from some of these livers (n = 3 ob/ob mice
and 3 lean control mice) were also evaluated for UCP2 by in
situ hybridization and immunohistochemistry. Liver sections were
fixed overnight in 3% buffered formalin, embedded in paraffin,
sectioned, and placed on polylysine-coated glass slides. For in
situ hybridization, the slides were put in a 60 °C incubator
for 30 min, deparaffinized in xylene twice for 5 min each, rehydrated
in a series of graded ethanol washes of 100, 95, and 70%, and then
rinsed in distilled water. The sections were digested with 20 µg/ml
proteinase K (Boehringer-Mannheim, Indianapolis, IN) in 50 mM Tris-HCl (pH 7.6) at 55 °C for 10 min, then the
proteinase K was inactivated by washing 3 times in distilled water with
agitation. The digestion procedure was followed by quenching of the
endogenous peroxidase with 3% hydrogen peroxide at room temperature
for 10 min. The biotin-labeled RNA probe for UCP2 was denaturated at
95 °C for 3 min and added to the slides, which were then covered
with coverslips and incubated at 45 °C overnight in a moisture chamber.
UCP2 signal amplification and development was done according to the
manufacturer's recommendations using the catalyzed signal amplification system from the DAKO Corp. (Carpinteria, CA). Briefly, after hybridization, the coverslips were removed by soaking in TBST
solution (50 mM Tris-HCl, pH 7.6, 300 mM NaCl,
0.1% Tween 20). The slides were washed under stringent conditions by
incubating in 0.1 × SSC (containing 15 mM NaCl and
1.5 mM sodium citrate) at 55 °C for 20 min. Signal was
amplified by applying streptavidin-horseradish peroxidase (1:500
dilution in the diluent) for 15 min, washing with TBST for 5 min 3 times, applying biotinyl-tyramide solution for 15 min, and again
washing with TBST for 5 min 3 times. After these amplification cycles,
secondary streptavidin-horseradish peroxidase was applied for 15 min.
After three 5-min washes in TBST, UCP2 mRNAs were demonstrated by
adding diaminobenzidine for 5 min. Control experiments were done with
UCP-2 sense and with an unrelated oligonucleotide. Mayer's hematoxylin
was used as counterstain before mounting.
Immunohistochemistry--
Formalin-fixed,
paraffin-embedded sections from the same livers that had been
studied by in situ hybridization were incubated with primary
goat antisera (1:50, v/v) to a conserved peptide sequence in murine and
human UCP2 (Santa Cruz, CA) for 5-10 min after antigen retrieval using
a heat-induced epitope retrieval method (18). Immunoperoxidase staining
using diaminobenzidine as chromogen was performed with the TechMate
automatic staining system on ChemMate slides (Bio Tek Solutions). As
negative controls, sections exposed to nonimmune antisera were included
in each assay to assure specificity.
Analysis of Hepatic Mitochondrial Function
Mitochondria from an additional 6 ob/ob mice and 6 lean controls
(n = 2 ob/ob mice or 2 lean mice/experiment × 3 experiments) were isolated in parallel using a standard differential
centrifugation protocol (19) in a medium containing 0.25 M
sucrose, 10 mM HEPES (K-salt) (pH 7.4), 0.1 mM
EGTA, and 5 mg/ml fatty acid-free BSA (ob/ob animals) or 2 mg/ml fatty
acid-free BSA (lean litter mates). After a final wash in BSA-free
medium, the pooled mitochondria from each group of mice were suspended
at a concentration of 30 mg/ml and kept on ice. Incubations were
carried out at 37 °C in a glass reaction vessel equipped with a
Clark electrode (Rank Brothers) and a TPP+ electrode
(purchased from Dr. A. Zimkius, Vilnius University, Lithuania). The
incubation medium (final volume 1.5 ml) contained 100 mM
KCl, 5 mM KH2PO4, 20 mM
HEPES (K-salt) (pH 7.4). The final pH was adjusted to 7.2 and
TPP+ was added to a final concentration of 0.33 µM. Other additions were as indicated. Mitochondrial
protein concentrations were 1 mg/ml. Oxygen uptake was measured
polarographically using a Rank Brothers oxygen electrode and membrane
potentials were measured using a TPP+ electrode connected
to a Cole-Parmer pH meter/ion detector. Membrane potential calculations
were made assuming a mitochondrial volume of 1 µl/mg of protein and a
mitochondrial TPP+ binding ratio (free
TPP+/total TPP+) of 0.2 (20).2 Previous studies had
demonstrated that these parameters were not significantly different
between liver mitochondria from ob/ob and lean litter mates (21-23).
The determination of membrane potential dependence of the proton leak
activity in isolated mitochondria is based on the protocol described by
Porter and Brand (24). The dependence of the proton leak rate on the
membrane potential (detected simultaneously using a
TPP+-sensitive electrode) shows a marked nonlinearity at
high membrane potential, but is close to linear at lower membrane
potential, giving a force-flow relationship that reflects the coupling
efficiency of the mitochondrial membrane. Mitochondria were incubated
at 37 °C in the standard incubation medium in the presence of
succinate (5 mM), rotenone (2 µM), and
oligomycin (5 µM). The respiration rate was titrated with
malonate, an inhibitor of succinate dehydrogenase, to generate a
gradually decreasing energy supply. Malonate was added from a stock
solution of 0.5 M in steps of 0.3-0.5 mM
(final concentration) with full inhibition being obtained at 3.3 mM. The rate of O2 uptake at different malonate
concentrations was compared with the change in membrane potential
determined in the same incubation from the distribution of
TPP+, using an ion-selective electrode. The force-flow
relationship was determined from the linear portion of the plot of
respiration rate to membrane potential. Essentially similar results are
obtained when other substrates or other methods to inhibit electron
transport activity are used (see Ref. 24 and references therein).
Evaluation of Hepatic ATP Stores
In 9 ob/ob mice and 9 controls, liver ATP stores were evaluated
in mid-morning (9-11 a.m.) either by phosphorous-NMR
(n = 6 ob/ob mice and 6 controls) or by biochemical
assay of freeze-clamped liver tissues obtained immediately after
sacrifice (n = 3 ob/ob mice and 3 control mice).
Biochemical Assays--
Frozen aliquots of liver were
homogenized on ice in chilled perchloric acid and ATP concentration was
measured by luminometer using commercial kits (from Sigma) according to
the manufacturer's instructions. ATP results (micrograms of ATP/mg of
liver wet weight) were normalized for variations in the DNA
concentrations in an equivalent amount of liver. Liver DNA was
quantitated according to the method of Blin and Stafford (25).
31P NMR Studies--
Ob/ob mice (n = 6) or lean controls (n = 6) were lightly anesthetized
with nembutal (50 µg/g of body weight) and the right lobe of the
liver was exteriorized through a midline laparotomy incision. A vessel
loop was loosely positioned around the porta hepatis and a two-turn
dual-tuned (to both 1H and 31P NMR frequency),
1-cm diameter, NMR surface coil was placed on the surface of the liver.
A sealed vial containing a known concentration of phenylphosphonic
acid, a 31P standard, was glued to the top of the surface
coil to provide an intensity reference. The animal was then placed in
the center of a GE Omega CSI 4.7 Tesla NMR magnet and the magnetic
field homogeneity was optimized using the 1H NMR signal.
Baseline 31P NMR spectra were recorded from the liver for
20 min; each spectrum consisted of an average of 400 acquisitions and
took approximately 5 min to record. Following baseline spectra, the
vessel loop was tightened to occlude vascular in-flow for 15 min, this
ligature was then released for 60 min of reperfusion. 31P
NMR spectra were recorded continuously throughout the ischemic and
reperfusion periods normalized to readings from the adjacent standard
in the same animal. The means (and standard deviations) of the
normalized results from the entire groups of lean or ob/ob mice were
calculated and analyzed by analysis of variance using computerized
statistical programs.
Evaluation of Hepatic Injury
The response to two different, sublethal, acute stresses,
i.e. hepatic ischemia/reperfusion and intraperitoneal
injection of LPS injection, were compared in ob/ob mice and lean
controls. The response to ischemia/reperfusion was evaluated in the
same 6 ob/ob mice and 6 lean controls that had been studied by
phosphorous-NMR. An additional 8 ob/ob mice and 8 lean control mice
were injected intraperitoneal with 10 µg of LPS from
Escherichia coli serotype B:01114 (Sigma). In both studies,
serum activities of the liver-associated enzyme, alanine
aminotransferase (ALT), and liver histology were obtained 24 h
after the stress and used to measure liver injury. ALT determinations
were performed by an autoanalyzer in the Clinical Chemistry Laboratory
of The Johns Hopkins Hospital. Coded, formalin-fixed, paraffin-embedded
liver sections were stained with hematoxylin and eosin and inspected
for hepatic necrosis and inflammation.
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RESULTS |
Body and Liver Weights and Liver Histology in Obese and Lean
Mice--
Genetically obese ob/ob mice have a spontaneous mutation in
the OB gene that prevents synthesis of the appetite suppressing hormone, leptin (26). The resultant hyperphagia contributes to obesity.
Thus, the ob/ob mice used in the subsequent studies weighed about twice
as much as the lean controls (48 ± 3 versus 27 ± 6 g, p < 0.001). At baseline, these ob/ob mice
exhibited no overt evidence of liver disease, such as jaundice,
ascites, coagulopathy, or splenic congestion. However, the livers of
ob/ob mice were heavier than those of lean mice (2.1 ± 0.4 versus 1.1 ± 0.1 g, p < 0.01)
and histologic evaluation demonstrated that hepatocytes in adult ob/ob
mice were engorged with large and small droplets of lipid, as reported
previously (17). In contrast, the hepatocytes of the lean mice
contained little lipid. Thus, like peripheral adiposity, fatty liver
may be an adaptive response to overeating.
Interestingly, although ob/ob mice livers were larger and fattier than
those of lean mice, liver weight normalized to body weight was similar
in the two groups. The liver weight was 4.4% of the body weight in
ob/ob mice and 4.1% of the body weight in lean mice. In addition,
evaluation of hepatic DNA content in ob/ob and lean livers demonstrated
that, when these results were expressed per g of liver wet weight, DNA
concentrations in ob/ob mice (6.9 ± 0.6 mg of DNA/g of liver)
were only 15% lower than in lean controls (8.1 ± 0.1 mg of DNA/g
of liver). Furthermore, since ob/ob livers weighed almost twice as much
as lean livers, the total amount of liver DNA and protein per animal
was actually greater in ob/ob mice than in the lean controls. These
findings suggested that ob/ob livers had adapted to the animal's obese
state, at least in part, by hyperplasia.
Effect of Obesity on Uncoupling Protein Expression in the
Liver--
Northern blot analysis was performed to compare the hepatic
expression of UCP2 mRNAs in ob/ob mice and lean controls. As shown in Fig. 1A, UCP2 was barely
detectable in "resting" livers obtained from 3 lean mice. In
contrast, the livers of 3 ob/ob mice expressed more UCP-2 mRNA at
baseline. Results of the UCP-2 mRNA expression in 6 ob/ob mice and
6 lean controls is graphed in Fig. 1B and indicate that
UCP-2 mRNA levels are 500-600% greater in ob/ob mice than lean
controls.

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Fig. 1.
Northern blot analysis of UCP2 mRNAs in
the livers of lean and ob/ob mice. A, a representative
autoradiograph with RNA (20 µg/lane) from 3 mice/group is shown.
Top panel, uncoupling protein 2 (UCP2). Bottom
panel, a constitutively expressed RNA (18 S) on the same blot.
B, PhosphorImage data from three Northern blots. On each
blot, UCP2 signal was normalized for 18 S RNA expression in the same
sample. In total, UCP2 expression was evaluated in 6 ob/ob mice and 7 controls. Results (mean ± S.E.) of data. p < 0.01 ob/ob versus control.
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In situ hybridization confirmed these differences in UCP2
expression. No UCP2 transcripts were identified in hepatocytes in lean
livers (Fig. 2, A and
C). In contrast, in 3/3 ob/ob mice, expression of UCP2
mRNA was apparent in all acinar zones of the liver. A
representative photomicrograph illustrating UCP2 transcripts in ob/ob
liver is shown (Fig. 2B). Inspection of the sections at
higher magnification indicated that the UCP2 signal was distributed in
the cytoplasm of hepatocytes in ob/ob liver (Fig. 2D).

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Fig. 2.
In situ hybridization of UCP2
mRNA in the livers of lean mice (A and
C) and ob/ob mice (B and
D). Formalin-fixed liver sections were incubated
with antisense probes for UCP2 as described under "Materials and
Methods." No signal was obtained when sections from control livers
were incubated with UCP2 antisense probes (A and
C) or when ob/ob sections were incubated in parallel with
sense UCP2 probes or with unrelated oligonucleotide fragments (data not
shown). However, in ob/ob livers, UCP2 transcripts are expressed
throughout the lobule (B) and the speckle-typed expression
of UCP2 mRNA is localized in hepatocytes (solid arrowheads)
(D). Final magnifications = 200 × (A
and B); 400 × (C and D).
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Immunohistochemistry confirmed that, similar to UCP2 mRNA levels,
hepatocyte UCP2 protein expression was increased in all 3 ob/ob livers
but could not be detected in any of the 3 lean livers that were
examined. Representative photomicrographs that demonstrate the
differences in UCP2 protein expression in lean (Fig.
3A) and ob/ob (Fig.
3B) mice are shown. Unlike UCP2, UCP1 and UCP3 could not be
detected in the livers of ob/ob or lean mice by RNA analysis (data not
shown). Therefore, an immunohistochemical evaluation of these UCP
isoforms was not done.

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Fig. 3.
Immunohistochemical evaluation of UCP2
protein in lean (A) and ob/ob (B)
liver. The photomicrographs shown are representative of results in
3/3 ob/ob livers and 3/3 livers from lean controls. UCP2 appears as
brown staining in hepatocytes of ob/ob mice at low (× 220) and high
(× 480) magnification but is notdetected in livers of lean mice (× 220).
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Effect of Obesity on Liver Mitochondrial Function--
Liver
mitochondria were isolated from ob/ob and lean mice and studied to
determine if obesity-related differences in hepatocyte UCP2 expression
influenced mitochondrial function. Table
I summarizes the respiratory activities
and membrane potentials with two different substrate conditions
(glutamate + malate or succinate + rotenone (a complex I inhibitor)),
that activate the respiratory chain either at the level of NADH or at
ubiquinone. As has been reported in previous studies (22), the ob/ob
mitochondria had a markedly higher rate of succinate oxidation, both in
State 3 (with ADP) and State 4 (no ADP). However, they maintained a
normal membrane potential and the decrease in membrane potential after
the addition of ADP was similar in preparations from ob/ob mice and
lean controls. Furthermore, the RCI of ob/ob mitochondria was not
decreased. These results indicate that ob/ob liver mitochondria
generally exhibit an increased oxidation capacity, but maintain a
similar degree of respiratory control.
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Table I
Conditions used were: mitochondria were isolated in a medium containing
sucrose (250 mM), K-Hepes (10 mM), EGTA (0.1 mM), and fatty acid-free BSA (5 mg/ml for ob/ob mice, 2 mg/ml for lean littermates). All incubations were carried out at
37 °C in medium containing KCl (100 mM), sucrose (50 mM), K-Hepes (20 mM), MgCl2 (1 mM), K-Pi (5 mM), TPP+ (0.33 µM), and the final pH was 7.2. Mitochondria were added to
a protein concentration of 1 mg/ml, BSA was carried over with the
mitochondrial addition to a final concentration of 50 or 125 µg/ml
for the lean litter mates and ob/ob mice, respectively. Substrate
concentrations: glutamate, 5 mM; malate, 5 mM;
succinate, 2 mM; rotenone, 2.5 µM; ADP was
added at 0.33 mM (with glutamate/malate as substrate) or
0.17 mM (with succinate/rotenone as substrate) to initiate
the State 4-State 3 transition. RCI, respiratory control index, ratio
of O2 uptake in State 3 and State 4. Data are mean ± S.E.
obtained from three or four separate preparations.
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However, very different results were obtained when mitochondria were
incubated with substrate (succinate) and complex I inhibitor (rotenone)
in the presence of an agent (oligomycin) which inhibits ATP synthase.
Under these conditions, the O2 uptake is entirely due to
the proton leak across the mitochondrial membrane (since the ATPase is
inhibited and there is no other ion transport), which is driven by the
proton motive force (existing predominantly in the form of a membrane
potential). A titration with increasing concentrations of malonate (an
inhibitor of succinate dehydrogenase) suppresses the supply of
electrons to the respiratory chain and decreases the membrane potential
that the mitochondria can maintain. Since the dissipation of the
membrane potential (measured as the O2 uptake) is entirely
due to the proton leak, this experiment allows measurement of the
proton leak rates as a function of the membrane potential.
Under these experimental conditions, the lean mitochondria exhibited
the standard relationship between membrane potential and proton leak
(Fig. 4, left panel), at low
membrane potential there was very little proton leak and the increase
in proton leak with increasing membrane potential was very modest. Only
when the membrane potential reached about 150 mV was there a dramatic rise in the rate of proton leak. As shown in Fig. 4 (right
panel), the steepness of the plot in the membrane potential range
of 100-160 mV was dramatically increased by 4-5-fold when ob/ob
mitochondria were studied. However, the maximal potential was
approximately similar to normal mitochondria. These findings suggest
that ob/ob mitochondria have an increased rate of proton leak which
partially dissipates their mitochondrial membrane potential when the
rate of electron transport is suppressed. These results are consistent with the presence of an uncoupling agent in ob/ob mitochondria. However, the increased uncoupling is not due to the presence of excess
free fatty acids in the mitochondrial membrane, since pretreatment of
the ob/ob mitochondria with fatty acid-free BSA did not affect the
proton leak rate under the conditions of Fig. 4. Further studies (not
shown) demonstrated that titration of oligomycin-inhibited mitochondria
with palmitate (2-20 µM) induced a similar % increase in O2 uptake in both preparations, suggesting that the
differences detected here are not due to selective uncoupling by
endogenous free fatty acids retained in the ob/ob mitochondria. The
rate of uncoupling in ob/ob mitochondria is insufficient to prevent mitochondria from achieving the maximally tolerated membrane potential, in part due to the enhanced rate of electron transport. Taken together,
the isolated liver mitochondria studies demonstrate that the degree of
functional uncoupling in ob/ob liver mitochondria is sensitive to
substrate availability. When substrate is not limiting, increased
mitochondrial electron transport activity compensates for the proton
leak and preserves a sufficient electrochemical gradient for ATP
synthesis. However, when the supply of electrons to the respiratory
chain is suppressed, the proton leak is uncompensated and the
mitochondrial membrane potential declines, inhibiting ATP
synthesis.

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Fig. 4.
H+ permeability of isolated liver
mitochondria from lean and ob/ob mice. The membrane potential
dependence of the proton leak rate was determined in mitochondria
oxidizing succinate (5 mM) in the presence of rotenone (2 µM). Oligomycin (5 µM) was added to inhibit
ATP synthase activity. Under these conditions, O2 uptake is
attributable entirely to the proton leak across the inner membrane (20,
30, 48, 49). Simultaneous measurements of membrane potential and
O2 uptake were made (see "Materials and Methods") as
the membrane potential was varied by inhibition of electron transport
activity by titration of succinate oxidation with malonate (to a
maximal concentration of 3.3 mM). Under the conditions used
(i.e. in the presence of excess phosphate), there is no
significant variation of pH and  was taken as an indicator of
the proton motive force. Data points from three different pairs of
ob/ob mice (open symbols) and lean controls are shown
(closed symbols). In one experiment, mitochondria from ob/ob
mice received an additional 30-min pretreatment with fatty acid-free
BSA (10 mg/ml) on ice to remove free fatty acids, as recommended by
Garlid et al. (35). This treatment did not significantly
affect the relationship between membrane potential and proton leak rate
(as reflected in O2 uptake). The increased slope of the
force-flow relationship at lower membrane potentials in mitochondria
from ob/ob livers is consistent with uncoupling.
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Effect of Obesity on Liver ATP Stores--
Because hepatocytes may
adapt to obesity-related increases in UCP2 expression by increasing
mitochondrial electron transport activity, it was uncertain if ob/ob
liver ATP stores would be compromised either basally, or in response to
an ATP-depleting interference (i.e. transient ischemia). Two
strategies were used to evaluate liver ATP content. Basal ATP
concentrations were measured biochemically in equivalent masses of
freeze-clamped liver and normalized for DNA content in the same tissues
to correct for intrinsic differences in hepatic lipid accumulation that
could complicate comparison of liver ATP stores between ob/ob and lean mice. For basal ATP measurements, all livers (n = 3 ob/ob and 3 lean) were harvested from ether-anesthetized mice while the animals hearts were beating. The ATP levels in these ob/ob mice (208 ± 3 µmol of ATP/mg of DNA) were slightly (i.e.
about 15%), but consistently, lower than in the lean controls
(253 ± 6 µmol of ATP/mg of DNA) when ATP results were expressed
relative to liver DNA to correct for differences in hepatic steatosis
(p < 0.05).
Hepatic 31P NMR spectroscopy was used to monitor changes in
the ATP levels of each individual animal over time. To assure
comparability among spectra obtained in 6 different ob/ob mice and 6 lean controls, all spectra were scaled to the intensity of the
resonance from the phosphate standard, which was the same in all
experiments. Baseline 31P NMR spectra from lean and ob/ob
mice are shown in Fig. 5A. The three resonances from the
-,
-, and
- phosphates of ATP are clearly visible in both spectra as are resonances from sugar
phosphates, inorganic phosphate, and phosphodiesters. The
- and
-phosphate resonances of ATP may contain significant contributions
from nucleotide diphosphates; however, the
-phosphate resonance is
unique to nucleotide triphosphates of which ATP is by far the most
prominent. Therefore, intensity of the
-ATP resonance is directly
proportional to the ATP content of the liver.

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Fig. 5.
A, 31P NMR spectra from the
liver of a single, representative lean mouse (left) and the
liver of one, representative ob/ob mouse (right) during the
basal state (top panel), at the end of the ischemic period
(middle panel), and at the end of reperfusion (bottom
panel). The sharp signal at the beginning of each spectra
(S) is from the phenylphosphonic acid reference in the
sealed glass bulb. The next cluster of peaks contains sugar and
inorganic phosphates (Pi). The -, -, and
-phosphate peaks of ATP follow. The intensity of the -ATP
resonance is directly proportional to the ATP content of the liver and
is indicated by the large arrow above each spectrum.
B, hepatic ATP stores in lean and ob/ob mice before
(baseline), after 15 min occlusion of portal venous blood (end
ischemia), and after 60 min of reperfusion (end reflow). ATP stores
were evaluated by 31P NMR spectroscopy. The intensity of
the -ATP resonance has been normalized to the phosphorous spectra of
an adjacent phenylphosphonic acid reference standard in each
animal. Results shown are the mean ± S.E. in 6 lean, control mice
and the all surviving ob/ob mice at each end point (n = 4-6). *, p < 0.0001 compared with lean; ,
p = 0.003 compared with lean; , p = 0.04 compared with lean. Note that in the obese group, end reflow is
still significantly depressed compared with baseline (p = 0.01); in the lean group this is not significantly different
(p = 0.2).
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It is evident from the spectra that the baseline ATP content in the
liver from the ob/ob mouse is markedly lower than that in the lean.
This difference is also clear in the mean data obtained from 6 ob/ob
and 6 lean mice shown in Fig. 5B. It is not certain why
ob/ob and lean ATP stores differed by more than 50% when evaluated by
phosphorous NMR and only by 15% when measured biochemically. However,
at least two factors may have contributed. First, the NMR data cannot
be corrected for differences in liver fat content, and second, NMR
analysis was performed on anesthetized mice after a small laparotomy
incision was done to expose the liver. Thus, differences in the
response to acute surgical stress may have contributed to the liver ATP differences.
In any case, portal vein occlusion rapidly decreased liver ATP in both
groups; however, the degree of ATP depletion was much greater in the
ob/ob mice. As shown in Fig. 5B, at the end of the 15-min
ischemic period, ATP levels had fallen about 30% from baseline values
in the lean group (n = 6). In contrast, the same ischemic insult caused more than a 60% reduction in ATP levels in the
ob/ob group (n = 6). Furthermore, after 60 min of
reperfusion (reflow), liver ATP levels in the 6 lean controls were not
significantly different from their baseline ATP values
(p = 0.2 for end reflow versus baseline),
while ATP stores in the 4 surviving mice in the ob/ob group remained
significantly depressed compared with the baseline values of that group
(p = 0.01 for end reflow versus baseline).
End of reflow data are not available for 2 other ob/ob mice that died
during the reperfusion period.
Differences in liver ATP stores after hepatic ischemia/reperfusion are
illustrated in the spectra obtained from representative lean and ob/ob
mice (Fig. 5A). In the lean mouse (left panels), liver ATP decreased at the end of ischemia and was near normal after
reflow. Consistent with these results, in the lean mouse, the
Pi peak increased by the end of ischemia and then returned toward baseline by the end of reflow. In contrast, in the ob/ob mouse
(right panels), the ATP peak virtually disappeared by the end of ischemia and remained almost undetectable after reflow. In this
animal, Pi increased during ischemia but did not fall appreciably after reflow. Thus, when animals are used as their own
controls, it is even more apparent that lean and obese mice exhibited
very different responses to ischemia/reperfusion. The lean mice
tolerated this insult with minimal depletion of their hepatic ATP
stores; the ob/ob mice became profoundly ATP depleted by the same brief
ischemic episode. In fact, one-third of the ob/ob animals did not
survive and in survivors, hepatic ATP levels remained consistently
lower than pre-ischemic values after reperfusion.
Effect of Obesity on Hepatic Sensitivity to Acute Stress--
The
laparotomy wound was closed in all surviving mice; the animals were
permitted to recover for 24 h and then sacrificed to evaluate the
sequella of the hepatic ischemic episode. Serum activity of the
liver-associated enzyme, ALT, was almost 10-fold greater in ob/ob mice
than in lean controls (1598 ± 207 versus 163 ± 70 IU/liter, p < 0.001). Histologic assessment of the
livers from the two groups of mice confirmed that this ALT elevation reflected massive hepatic necrosis in the ob/ob group.
We previously reported that obesity increases vulnerability to
endotoxin (LPS)-mediated liver injury (17). Other groups have suggested
that occluding the porta hepatitis to cause hepatic ischemia leads to
local increases in several LPS-inducible cytokines (27). Therefore, it
is conceivable that endotoxemia or endotoxin-related cytokines may have
contributed to the differences in liver injury that were observed after
ischemia-reperfusion. Recently, Faggioni and colleagues (28) reported
that LPS injection increased UCP2 expression in the livers of normal
rats. Subsequently, we showed that hepatocytes are predominately
responsible for the increase in liver UCP2 expression that follows LPS
injection in rats (29). Because obesity increases hepatic sensitivity
to LPS-mediated injury, LPS treatment may result in a greater induction
of UCP-2 in ob/ob mice than lean controls. To evaluate this
possibility, 8 ob/ob mice and 8 lean controls were injected
intraperitoneal with a dose of LPS (10 µg/mouse) that produces little
liver injury in healthy, lean mice. As previously reported (17), ob/ob
mice experienced significantly more liver injury after this treatment than did lean controls, as evidenced by 10-fold higher ALT activities at 24 h after LPS exposure (data not shown). Notably, at this time
point, UCP2 mRNA levels in the livers of ob/ob mice were 6 times
greater than lean control mice that had been treated with an identical
dose of LPS (Fig. 6).

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Fig. 6.
Northern blot analysis of hepatic UCP2
mRNAs in lean and ob/ob mice before (0) an 24 h after
intraperitoneal injection of LPS (10 µg/mouse).
A, a representative blot demonstrating UCP2 expression in 2 mice/group at each time point. Top panel, UCP2 mRNA.
Bottom panel, the constitutively expressed 18 S RNA on the
same blot. All lanes contain 20 µg of total RNA. B,
PhosphorImager results from duplicate Northern blots (n = 8 ob/ob and 8 lean control mice). On each blot, UCP2 signal intensity
was normalized to 18 S RNA expression in the same sample and expressed
as a percentage of the untreated (time zero) lean control UCP2
expression on the same blot. Data are expressed as mean ± S.E. *,
p < 0.05 for lean LPS(+) versus lean
LPS( ); +, p < 0.001 for obese LPS( )
versus lean LPS( ); **, p < 0.01 for obese
LPS(+) versus lean LPS(+).
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DISCUSSION |
Obesity is a complex syndrome that results from imbalanced intake
of energy substrates and energy utilization (1). The liver plays a
major role in the regulation of energy homeostasis in mammals (13, 14)
and, thus, is an important target organ in obesity. The combination of
increased food (energy) consumption and hyperinsulinemia produce a
state in which the supply of oxidizable substrates to hepatocytes is
excessive. However, since this hormonal milieu also promotes the
shunting of substrates into anabolic pathways (i.e. the
synthesis and storage of fat), it is difficult to predict whether
obesity increases or decreases the supply of substrate that is
available for hepatic mitochondrial oxidation. Previous studies on
mitochondrial function in livers of ob/ob mice demonstrated an
increased electron transport activity in these animals (21-23),
suggesting that the mitochondria experience an adaptive response that
enhances the capacity for substrate oxidation. However, the
implications of this adaptation for hepatic energy metabolism are
poorly understood.
Mitochondria are the major sites of ATP production in the cell. During
mitochondrial respiration, electrons derived from cellular substrates
are transferred serially through the electron transport chain leading
to the reduction of molecular O2. The energy derived from
these redox reactions is conserved in the form of the proton electrochemical gradient, which drives the synthesis of ATP through the
transmembrane ATP synthase complex (30). In the presence of an abundant
energy supply, the rate of mitochondrial electron transport, and hence
the rate of substrate oxidation, is generally matched to the cell's
need for energy. Many of the mechanisms responsible for matching these
processes in the intact cell are not yet well characterized. However,
experimental conditions where the supply of mitochondrial ADP is
restricted in the presence of saturating substrate supply (State 4) are
characterized by a limited rate of electron transport, accompanied by a
highly reduced state of NAD and other electron carriers of the electron transport chain, as well as a maximal proton electrochemical gradient (30).
The mitochondrial electron transport chain is also one of the
predominant sites of production of reactive oxygen species (ROS), such
as superoxide (O
2), which is generated by the one-electron reduction of O-2 under conditions of highly reduced electron transport intermediates (31). The State 4 condition of high redox pressure combined with a limited rate of utilization of the proton
electrochemical gradient in an oxygen-rich environment promotes the
formation of O
2. Mitochondrially generated O
2 reacts
with water to generate hydrogen peroxide (H2O2)
in a reaction catalyzed by the manganese-dependent superoxide dismutase in the matrix. H2O2 may
itself be a source of more highly reactive intermediates, such as the
hydroxyl radical (OH
), which may damage cellular
constituents, or it may affect extramitochondrial redox-dependent signaling processes. Alternatively,
mitochondrially generated O
2 can react with nitric oxide (NO)
to generate peroxynitrite, which may cause covalent modification of
proteins (32). Hence, a careful regulation of the formation of ROS
through the mitochondrial electron transport chain is important for
minimizing these and other potentially damaging consequences.
Among the mechanisms ostensibly developed by a variety of organisms to
control unwanted generation of ROS under conditions of high substrate
supply that exceeds its energy requirements, is the activation of
alternative substrate oxidation pathways that are not efficiently
coupled to ATP synthesis (33). Such alternative electron transport
pathways are prominent in a variety of microorganisms and plants.
During the past few years it has become apparent that a similar
function may be fulfilled by uncoupling proteins in the inner
mitochondrial membrane. Uncoupling proteins were first described in
mitochondria from brown adipose tissue, where they are activated to
meet the need for heat generation (2, 34). The brown adipose tissue
uncoupling protein (UCP1) catalyzes the net transfer of H+
across the mitochondrial membrane in the presence of appropriate activators (e.g. free fatty acids) leading to the
dissipation of the electron transport-driven proton electrochemical
gradient, at the expense of ATP synthesis (35). In recent years, other isoforms of uncoupling protein (UCP2 and UCP3) have been described that
have a more widespread tissue distribution (8, 36-38). Interestingly,
recent studies have linked the activation of the UCP2 protein in
mitochondria isolated from rat liver to the suppression of
H2O2, presumably reflecting the suppression of
O
2 formation through the electron transport chain (39).
However, these authors demonstrate that this effect of UCP2 protein was
limited entirely to mitochondria derived from non-parenchymal cells of
the liver and that mitochondria obtained from hepatocytes did not
contain the UCP2 protein. In agreement with this report, others have
demonstrated that UCP2 is expressed in Kupffer cells in the normal
liver, but not in hepatocytes (12).
The present study provides in vivo evidence that hepatocytes
induce UCP2 mRNA and protein expression during obesity. Thus, when
confronted with an overly abundant substrate supply that exceeds
cellular energy requirements, hepatocytes, similar to many other cells
(2, 33), activate pathways that are not efficiently coupled to ATP
synthesis. By decreasing the efficiency with which substrate oxidation
generates ATP, these responses permit the utilization of excess
substrate while helping to balance ATP supply with cellular energy
requirements. Furthermore, if the model is correct that excess
substrate supply to the mitochondrial electron transport chain
increases the probability of ROS formation, induction of UCP2 to affect
uncoupling of oxidative phosphorylation may diminish the redox pressure
on the mitochondrial electron transport carriers and provide an added
advantage by constraining mitochondrial ROS production (40). At this
point, the molecular mechanisms that are involved in this
obesity-related induction of UCP2 are unknown. However, it is
intriguing that hepatocytes also up-regulate UCP2 after exposure to LPS
(29), a situation that is known to induce the proinflammatory cytokine,
tumor necrosis factor (TNF)-
, and increase hepatocyte ROS production
(41, 42). Many of the hepatic responses to LPS, including the induction of mitochondrial oxidant production (43), are mediated by TNF-
and
TNF-inducible cytokines. Indeed, recent evidence demonstrates that
overnight exposure of primary hepatocyte cultures to recombinant TNF-
induces UCP2 mRNA expression by 200-300% (29). Adipose expression of TNF-
mRNA and circulating levels of TNF-
protein are increased in obese humans (44) and in two strains (ob/ob and db/db) of genetically obese mice (17, 45). The latter are known to
express increased UCP2 mRNA in adipocytes (46). Thus, there is a
growing body of evidence that UCP2 may be a TNF-inducible gene.
Up-regulation of UCP2 activity is predicted to decrease the efficiency
of energy trapping (3) and, thus, has the potential for compromising
the capacity to respond to acute energy needs of the cell in conditions
of stress. The baseline 5-fold increase in UCP2 mRNA levels appears
to be reasonably well tolerated by hepatocytes in obese mice because
this is not accompanied by release of liver enzymes or histologic
evidence of liver injury. It is conceivable that this benign outcome
reflects the fact that UCP2 mRNA induction does not result in
increased levels of UCP2 protein under normal conditions. However, the
latter seems unlikely in the hepatocytes of ob/ob mice given the
present hepatic immunohistochemistry results. On the other hand,
studies with mitochondria isolated from ob/ob livers suggest that the
consequences of increased UCP2 expression are influenced strongly by
the cellular context in which this occurs. For example, changes in the
proton motive force of mitochondria from ob/ob livers could only be
demonstrated under experimental conditions that limited the supply of
electrons to the respiratory chain. Similarly, in living ob/ob mice,
the physiological implications of hepatic mitochondrial uncoupling
appeared to depend on the balance between the availability of energy
substrates and cellular energy requirements. The largest differences in
the hepatic ATP stores of ob/ob mice and lean controls were observed
after liver blood flow was interrupted transiently. This finding is consistent either with stress-related decreases in the efficiency of
mitochondrial ATP synthesis or collapse of the mitochondrial membrane
potential both of which may reflect increased uncoupling protein
activity. However, because the effect of UCP2 on these parameters was
not tested directly, potential contributions from other factors cannot
be excluded. Intrinsic differences in hepatocyte vulnerability to
injury induced by endotoxin or endotoxin-induced cytokines are
particularly important to consider, because obstruction of portal
perfusion increases hepatic exposure to these factors (27). Of
interest, the present results confirm other reports that LPS induces
UCP2 expression in normal liver (28) and indicate that ob/ob livers
express more UCP2 mRNA than controls when challenged with
endotoxin. Thus, relative overexpression of UCP2 (and consequent ATP
depletion after exposure to an endotoxin challenge that acutely consumes liver ATP (47)) may also contribute to the obesity-related vulnerability to endotoxin liver injury that has been reported previously (17).
In summary, modulation of UCP2 activity may be a mechanism that
hepatocytes employ to titrate the rates of mitochondrial ATP or oxidant
production. Presumably, induction of UCP2 in hepatocytes conveys some
adaptative advantage during obesity. However, the up-regulation of UCP2
may become maladaptive when the hepatic environment changes abruptly,
emphasizing the importance of environmental conditions in dictating the
ultimate fate of metabolically active cells. Adaptive responses,
including the up-regulation of uncoupling proteins, that are beneficial
when the supply of exogenous substrates is overly abundant, compromise
cellular viability when substrate availability becomes limited acutely.